Synthesis and Antiproliferative Activity of Marine

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Synthesis and Antiproliferative Activity of Marine Bromotyrosine Purpurealidin I and Its Derivatives Chinmay Bhat 1,† , Polina Ilina 2 , Irene Tilli 1 , Manuela Voráˇcová 1 , Tanja Bruun 1 , Victoria Barba 1 , Nives Hribernik 1 , Katja-Emilia Lillsunde 2 , Eero Mäki-Lohiluoma 1 , Tobias Rüffer 3 , Heinrich Lang 3 , Jari Yli-Kauhaluoma 1 , Paula Kiuru 1 and Päivi Tammela 2, * 1

2

3

* †

Drug Research Program, Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, Viikinkaari 5 E (P.O. Box 56), University of Helsinki, FI-00014 Helsinki, Finland; [email protected] (C.B.); [email protected] (I.T.); [email protected] (M.V.); [email protected] (T.B.); [email protected] (V.B.); [email protected] (N.H.); [email protected] (E.M.-L.); [email protected] (J.Y.-K.); [email protected] (P.K.) Drug Research Program, Division of Pharmaceutical Biosciences, Faculty of Pharmacy, Viikinkaari 5 E (P.O. Box 56), University of Helsinki, FI-00014 Helsinki, Finland; [email protected]helsinki.fi (P.I.); [email protected] (K.-E.L.) Institute of Chemistry, Technische Universität Chemnitz, 09107 Chemnitz, Germany; [email protected] (T.R.); [email protected] (H.L.) Correspondence: [email protected]; Tel.: +358-2941-59628 Current address: Government First Grade College, Chamarajanagar 571313 (Affiliated to University of Mysore), India.

Received: 6 November 2018; Accepted: 27 November 2018; Published: 3 December 2018

 

Abstract: The first total synthesis of the marine bromotyrosine purpurealidin I (1) using trifluoroacetoxy protection group and its dimethylated analog (29) is reported along with 16 simplified bromotyrosine derivatives lacking the tyramine moiety. Their cytotoxicity was evaluated against the human malignant melanoma cell line (A-375) and normal skin fibroblast cells (Hs27) together with 33 purpurealidin-inspired simplified amides, and the structure–activity relationships were investigated. The synthesized simplified analogs without the tyramine part retained the cytotoxic activity. Purpurealidin I (1) showed no selectivity but its simplified pyridin-2-yl derivative (36) had the best improvement in selectivity (Selectivity index 4.1). This shows that the marine bromotyrosines are promising scaffolds for developing cytotoxic agents and the full understanding of the elements of their SAR and improving the selectivity requires further optimization of simplified bromotyrosine derivatives. Keywords: Purpurealidin I; bromotyrosines; Pseudoceratina purpurea; synthesis; cytotoxicity; selectivity to cancer cells

1. Introduction Globally, cancer is the second leading cause of death and in 2018 it is estimated to lead to 9.6 million deaths [1] and malignant melanoma is one of the most life-threatening cancers due to resistance to most therapies [2]. While prevention is important, there is a continuous need for novel treatments. The marine environment provides a potential source for discovering new drug lead molecules, especially against cancer. So far, four medicinal products originating from marine ecosystems have been registered for the treatment of different kinds of cancer such as leukemia, metastatic breast cancer, and ovarian cancer [3,4].

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Bromotyrosines are a large and structurally diverse group of bromine-containing marine Bromotyrosines areshown a large aand structurally diverse functions group of bromine-containing marine antiviral, alkaloids alkaloids which have variety of biological including antimicrobial, which have shown a variety of biological functions including antimicrobial, antiviral, antifungal antifungal and in particular, anticancer activity [5–8] Bromotyrosines are mainly isolated fromand the in particular, anticancer activity [5–8] Bromotyrosines are mainly isolated from the marine sponges of of marine sponges of the order Verongida. For representative articles about secondary metabolites the order Verongida. For representative articles about secondary metabolites of Verongida order, see, Verongida order, see, for example, Fattorusso group’s work [9,10]. for example, Fattorusso group’s1)work [9,10]. Purpurealidin I (1; Figure together with several other bromotyrosines have been isolated from Purpurealidin I (1; Figure 1) together with severalpurpurea other bromotyrosines have been aplysamine isolated from2 the Indian sea sponge Pseudoceratina (Psammaplysilla) [11]. Structurally similar the Indian sea sponge Pseudoceratina (Psammaplysilla) purpurea [11]. Structurally similar aplysamine (2) was isolated from the Australian marine sponge, Aplysina sp. [12]. Purpurealidin I (1) has been 2 (2) was isolated fromwhen the Australian marine sponge, Aplysina sp. [12]. Purpurealidin I (1) and has found to be cytotoxic tested against various cancer cell lines (ovarian cancer A2780S been found to be cytotoxic when tested against various cancer cell lines (ovarian cancer A2780S and cisplatin-resistant variant A2780CP (SCP5), non-small cell lung cancer A549, human breast cancer cisplatin-resistant variant A2780CP non-small cell cancer A549, human breast cancer MCF7 and glioma U251MG cells), as (SCP5), well as non-cancer cell lung line NIH3T3 (normal mouse fibroblasts) MCF7 andother glioma U251MG cells),aplysamine as well as non-cancer lineJBIR-44 NIH3T3 mouse fibroblasts) [6]. Two bromotyrosines 4 (3) [13]cell and (4)(normal [14] were isolated from [6]. P. Two other bromotyrosines aplysamine 4 (3) [13] and JBIR-44 (4) [14] were isolated from P. purpurea purpurea and have been tested against human cervical carcinoma HeLa cells [5]. A comparably strong and have been against human cervical HeLa cells [5]. comparably with strong cytotoxic cytotoxic effecttested was observed and there was carcinoma no difference between theAcompounds a longer or effect was observed and there was no difference between the compounds with a longer or shorter alkyl shorter alkyl chain attached to the tyramine part. This presents the opportunity for the design of chain attached to the part. This presents thelong opportunity fordoes the design of simplified analogs simplified analogs oftyramine marine bromotyrosines as the alkyl chain not seem to be essential for of marine bromotyrosines as the long alkyl chain does not seem to be essential for cytotoxicity. In our cytotoxicity. In our previous studies, simplified amide-linked bromotyrosines inspired by previous studies, simplified amide-linked bromotyrosines inspired by purpurealidin I (1) displayed purpurealidin I (1) displayed good Kv10.1 channel inhibition [15]. Kv10.1 potassium channel regulates good inhibitionin[15]. Kv 10.1 potassiumcell channel many functions v 10.1 channelfunctions manyKfundamental a cell, for example cycle regulates progression andfundamental cellular proliferation in a cell, for example cell cycle progression cellular proliferation [16]. We report total [16]. We report here the total synthesis of the and marine natural product purpurealidin I (1)here and athe related synthesis of the marine natural product purpurealidin I (1) and a related tetrabrominated analog tetrabrominated analog of aplysamine 2 (2; also, a dimethyl analog of 1). Medicinal chemistry of aplysamine 2 (2; also, a dimethyl analog of 1).outlined. Medicinal chemistry strategies simplify these their strategies to simplify their structures are also Furthermore, we have to evaluated structures are also outlined. Furthermore, we have evaluated these compounds for selective cytotoxic compounds for selective cytotoxic effects to skin cancer cells and discussed their structure-activity effects to skin cancer cells and discussed their structure-activity relationships. relationships.

Figure 1. Bromotyrosines Bromotyrosines purpurealidin I (1), aplysamine 2 (2), aplysamine 4 (3) and JBIR-44 (4).

2. Results 2. Results 2.1. Chemistry 2.1. Chemistry The purpurealidin I (1) skeleton can be viewed as a secondary amide. The retrosynthetic pathway The purpurealidin I (1) skeleton can be viewed as a secondary amide. The retrosynthetic (Scheme 1) illustrated that the synthesis of the bromotyrosine carboxylic acid part could be initiated pathway (Scheme 1) illustrated that the synthesis of the bromotyrosine carboxylic acid part could be from O-methyltyrosine (7) and the corresponding amine part from tyramine (8). initiated from O-methyltyrosine (7) and the corresponding amine part from tyramine (8).

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Scheme1. 1.Retrosynthetic Retrosyntheticroute routeto topurpurealidin purpurealidinIII(1). (1). Scheme 1. Retrosynthetic route to purpurealidin Scheme (1).

For the synthesis of ofthe thebromotyrosine bromotyrosinecarboxylic carboxylicacid acid moiety, commercially available For the the synthesis of the bromotyrosine carboxylic acid moiety, commercially available OFor moiety, commercially available OO-methyltyrosine (7) was brominated [17] and subjected to an esterification reaction with SOCl methyltyrosine (7) was brominated [17] and subjected to an esterification reaction with SOCl 2 in methyltyrosine (7) was brominated [17] and subjected to an esterification reaction with SOCl2 in2 in MeOH. The ester (10) was converted oxime(11) (11)using usingsodium sodiumtungstate tungstateand andhydrogen peroxide, MeOH. The ester (10) was converted totooxime oxime (11) using sodium tungstate and hydrogenperoxide, peroxide, MeOH. The ester (10) was converted to following a literature procedure (Scheme 2) [17–20]. The corresponding carboxylic acid subunit (5) following aa literature literature procedure procedure (Scheme (Scheme 2) 2) [17–20]. [17–20]. The The corresponding corresponding carboxylic carboxylic acid acid subunit subunit (5) (5) following was then synthesized via the LiOH-mediated hydrolysis of methyl ester (11) in 90% yield (See the was then synthesized via the LiOH-mediated hydrolysis of methyl ester (11) in 90% yield (See the was then synthesized via the LiOH-mediated hydrolysis of methyl ester (11) in 90% yield (See the Supplementary SupplementaryInformation Informationfor forthe theexperimental experimentaldetails). details). Supplementary Information for the experimental details).

Scheme 2. 2. Synthesis Synthesis of the thethe bromotyrosine carboxylic acid part part (5) [17](5) for[17] the first firstthe amide coupling Scheme of bromotyrosine carboxylic acid [17] for the amide coupling 2. Synthesis of bromotyrosine carboxylic acid(5) part for first amide attempts. attempts. coupling attempts.

The first first attempts attempts of of amide amide coupling coupling were were made made using using the the free free primary primary amine amine (6) (6) (Scheme (Scheme 3). 3). coupling were made using the free primary amine (6) (Scheme The Tyramine(8) (8)underwent underwentbromination brominationfollowed followed by by N-Boc N-Boc protection protection to to give give (13) (13) [19–23] [19–23]which which was was Tyramine then subjected to O-alkylation with Boc-protected 3-chloro-N-methylpropan-1-amine (15) [24] to give 3-chloro-N-methylpropan-1-amine (15) [24] to give then subjected to O-alkylation with Boc-protected 3-chloro-N-methylpropan-1-amine (14)in in83% 83%yield. yield.The Thetrifluoroacetic trifluoroaceticacid acid(TFA)-mediated (TFA)-mediatedBoc Bocdeprotection deprotectionof of(14) (14)gave gavethe thediamine diamine (14) (6)in inquantitative quantitativeyield. yield. (6) The direct coupling of compounds (5) and (6) with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was unsuccessful. This was likely due to the interfering free hydroxy group within (5). A condensation reaction of ester (11) with compound (6) also proved unsuccessful. Several alternative coupling conditions were tried (Supplementary Information, Table S1) Unfortunately, each resulted in the formation of inseparable mixtures with poor yields of the desired product, purpurealidin I (1), as determined by 1 H NMR and LC-MS analyses. After several additional approaches proved unsuccessful (data not shown), trifluoroacetyl was found to be a suitable protecting group for the secondary amine (Scheme 4). The Boc-protected bromo tyramine (13) was O-alkylated with (18) to produce (16). Treatment of (16) with TFA led to the selective Boc deprotection and led to the formation of the desired target amine (17) in 77% overall yield [15].

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Scheme 3. Synthesis of the tyramine derivative (6) for use in the first amide coupling approach.

The direct coupling of compounds (5) and (6) with 1-ethyl-3-(3dimethylaminopropyl)carbodiimide (EDC) was unsuccessful. This was likely due to the interfering free hydroxy group within (5). A condensation reaction of ester (11) with compound (6) also proved unsuccessful. Several alternative coupling conditions were tried (Supplementary Information, Table S1) Unfortunately, each resulted in the formation of inseparable mixtures with poor yields of the desired product, purpurealidin I (1), as determined by 1H NMR and LC-MS analyses. After several additional approaches proved unsuccessful (data not shown), trifluoroacetyl was found to be a suitable protecting group for the secondary amine (Scheme 4.). The Boc-protected bromo tyramine (13) was O-alkylated with (18) to produce (16). Treatment of (16) with TFA led to the selective Boc deprotection and led to theofformation of derivative the desired (17)amide in 77% overallapproach. yield [15]. Scheme the tyramine approach. 3. Synthesis (6) target for useamine in the first coupling The direct coupling of compounds (5) and (6) with 1-ethyl-3-(3dimethylaminopropyl)carbodiimide (EDC) was unsuccessful. This was likely due to the interfering free hydroxy group within (5). A condensation reaction of ester (11) with compound (6) also proved unsuccessful. Several alternative coupling conditions were tried (Supplementary Information, Table S1) Unfortunately, each resulted in the formation of inseparable mixtures with poor yields of the desired product, purpurealidin I (1), as determined by 1H NMR and LC-MS analyses. After several additional approaches proved unsuccessful (data not shown), trifluoroacetyl was found to be a suitable protecting group for the secondary amine (Scheme 4.). The Boc-protected bromo tyramine (13) was O-alkylated with (18) to produce (16). Treatment of (16) with TFA led to the selective Boc Scheme 4. Synthesis Synthesisofoftrifluoroacetyl trifluoroacetylprotected protectedtyramine tyramine part (17) [15] purpurealidin part (17) [15] forfor thethe purpurealidin I (1)I deprotection and led to the formation of the desired target amine (17) in 77% overall yield [15]. (1) synthesis. synthesis. An An initial initial attempt attempt to to synthesize synthesize the the immediate immediate precursor precursor of of purpurealidin purpurealidin II (1) (1) by by direct direct condensation This was was likely likely due condensation of of (17) (17) with with hydroxylamine hydroxylamine ester ester (11) (11) was was unsuccessful. unsuccessful. This due to to the the presence of the hydroxylamino moiety. We then decided to introduce the free hydroxylamino group presence of the hydroxylamino moiety. We then decided to introduce the free hydroxylamino group after The bromotyrosine bromotyrosine carboxylic carboxylic acid acid fragment fragment (22a) (22a) was after the the amide amide coupling. coupling. The was prepared prepared via via the the Erlenmeyer–Plöchl Erlenmeyer–Plöchl azlactone azlactone method method (Scheme (Scheme 5) 5) in in 84% 84% overall overall yield yield [25,26]. [25,26]. This This procedure procedure was was also also used used for for the the preparation preparation of of the the carboxylic carboxylic acid acid fragments fragments (22b–d) (22b–d) in in the the mono-brominated, mono-brominated, mono-chlorinated derivatives (41–45). Reddy et al. reported the mono-chlorinated and andnon-halogenated non-halogenatedsimplified simplified derivatives (41–45). Reddy et have al. have reported synthesis of methyl carbamate containing bromotyrosine purpuramine K leaving the tetrahydropyranyl the synthesis of methyl carbamate containing bromotyrosine purpuramine K leaving the (THP) group 4. toSynthesis the (THP) molecule [22].to the molecule tetrahydropyranyl Scheme ofgroup trifluoroacetyl protected[22]. tyramine part (17) [15] for the purpurealidin I (1) The synthesis of purpurealidin I (1), began with the bromination of p-hydroxybenzaldehyde, synthesis. followed by methylation to give (19a). The requisite azlactone (20a) was then prepared by the condensation (19a) with N-acetylglycine (Scheme 5) and subjecting the resulting to An initialofattempt to synthesize the immediate precursor of purpurealidin I (1)product by direct hydrolysis using a 10% solutionester of HCl give pyruvic acid (21a). condensation of (17) withaqueous hydroxylamine (11)towas unsuccessful. This was Compound likely due to(21a) the was then of converted into the THP-protected oxime (22a) by reaction with O-(tetrahydropyran-2-yl)presence the hydroxylamino moiety. We then decided to introduce the free hydroxylamino group hydroxylamine crude oximecarboxylic was then subjected to EDC coupling with (17)via under after the amide (THPONH coupling. The bromotyrosine acid fragment (22a) was prepared the 2 ). The microwave conditions to produce (26)(Scheme in a moderate yield (56%;yield Scheme 6). The group was Erlenmeyer–Plöchl azlactone method 5) in 84% overall [25,26]. ThisTHP procedure removed a 2 preparation M solution of Et2 O to give free oxime (28). in Trifluoroacetyl mediated also usedwith for the ofHCl the in carboxylic acid the fragments (22b–d) the mono-brominated, deprotection of (28)and using MeOH/K2 COsimplified purpurealidin I (1)Reddy in an overall yield of 12% mono-chlorinated non-halogenated derivatives (41–45). et al. have reported 3 resulted in (11 The aplysamine with a dimethylamino moietyKat the tyramine the steps). synthesis of methyl2 tetrabromo carbamate derivative containing(29), bromotyrosine purpuramine leaving the fragment, was synthesized usingto purpurealidin (25) [15,27] in the coupling with (22a) (Scheme 6). tetrahydropyranyl (THP) group the moleculeE[22].

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Table 1. Structures of final hydroxyimino propanamides (30–45) from the Scheme 5. The Table 1.Table Structures of of final hydroxyimino propanamides (30–45) from the Scheme 5. 5. The Table 1. Structures of final hydroxyimino propanamides (30–45) from the Scheme 5. The Table 1. hydroxyimino propanamides (30–45) from the Scheme The Table 1. Structures of final hydroxyimino propanamides (30–45) from the Scheme 5. The 1. Structures of final hydroxyimino propanamides (30–45) from the Scheme 5. The Table 1. Structures of final hydroxyimino propanamides (30–45) from the Scheme 5. The Table 1. of final hydroxyimino propanamides (30–45) from the 5. Table 1.Structures Structures of final hydroxyimino propanamides (30–45) from theScheme Scheme 5.The The Table 1. Structures of final hydroxyimino (30–45) from the Scheme 5. The Table 1. Structures Structures of final final hydroxyimino propanamides (30–45) from the Scheme 5.as The corresponding THP-ethers (23a–d) are found in propanamides the Experimental part (Section 4.1.2) numbered 30corresponding THP-ethers (23a–d) are found in the Experimental part (Section 4.1.2) numbered as 30corresponding THP-ethers (23a–d) are found in the Experimental part (Section 4.1.2) numbered as corresponding THP-ethers (23a–d) are found in the Experimental part (Section 4.1.2) numbered as 30corresponding corresponding THP-ethers THP-ethers (23a–d) (23a–d) are found are found in the in Experimental the Experimental part (Section part (Section 4.1.2) 4.1.2) numbered numbered as 30as 30corresponding THP-ethers (23a–d) are found in the Experimental part (Section 4.1.2) numbered as 30corresponding THP-ethers (23a–d) are found in the Experimental part (Section 4.1.2) numbered as 30corresponding THP-ethers (23a–d) are found in the Experimental part (Section 4.1.2) numbered as 30corresponding THP-ethers (23a–d) are found in the Experimental part (Section 4.1.2) numbered as 30corresponding THP-ethers (23a–d) are found in the Experimental part (Section 4.1.2) numbered as 30corresponding THP-ethers (23a–d) are found in the Experimental part (Section 4.1.2) numbered as 30Table 1. Structures of final hydroxyimino propanamides (30–45) from the Scheme 5. The corresponding 30THP–45-THP. THP–45-THP. THP–45-THP. THP–45-THP. THP–45-THP. THP–45-THP. THP–45-THP. THP–45-THP. THP–45-THP. THP–45-THP. THP–45-THP. THP–45-THP. THP-ethers (23a–d) are found in the Experimental part (Section 4.1.2) numbered as 30-THP–45-THP.

Cmpd R1 R2 R3 Cmpd R1 R2 R3 Cmpd R1 R2 R3 22 R 22 R 22 R Cmpd R1111 R R22R R3333 R Cmpd R1111 R R212R R3333 R R11111 R R222R R33333 R Cmpd R R Cmpd R R Cmpd R R R R R R Cmpd R Cmpd Cmpd R R Cmpd R Cmpd Cmpd 111R 12211212 RR 222R 3333 RR 333Cmpd Cmpd Cmpd 111R 1221121R 222R 333Cmpd Cmpd Cmpd 111R 12211212 RR 222R 3333 RR 3333 Cmpd R R Cmpd R Cmpd R R Cmpd R R R Cmpd 1R 2R 33R Cmpd 1R 2R 3R Cmpd 1R 2R 3R Cmpd 3Cmpd Cmpd R 2R 3Cmpd Cmpd R 2R Cmpd RR RR Cmpd RR RR Cmpd RR RR 2R 33333 RR Cmpd 1R R Cmpd 1R R 2 2RR Cmpd 1R R 30 Br Br 30 30 Br BrBr Br 30 Br Br 30 Br Br 30 Br Br Br 30 Br 30 30 30 Br Br 30 Br 30 Br BrBr Br Br

35 Br Br 35 35 Br BrBr Br 35 Br Br 35 Br Br 35 Br Br Br 35 Br 35 35 35 Br Br 35 Br 3535Br BrBr BrBrBr Br

40 Br Br 40 40 Br BrBr Br 40 Br Br 40 Br Br 40 Br Br Br 40 Br 40 40 40 Br Br 40 Br 40 Br BrBr Br Br 40 Br Br

31 Br Br 31 31 Br BrBr Br 31 Br Br 31 Br Br 31 Br Br Br 31 Br 31 31 31 Br Br 31 Br 31 Br BrBr Br Br

36 Br Br 36 36 Br BrBr Br 36 Br Br 36 Br Br 36 Br Br Br 36 Br 36 36 36 Br Br 36 Br 3636Br BrBr BrBrBr Br

41 Br H 41 41 Br HBr 41 Br H 41 Br H 41 Br Br H 41 Br H 41 41 41 Br H 41 Br H 41 Br BrH H HH 41 Br H

32 Br Br 32 32 Br BrBr Br 32 Br Br 32 Br Br 32 Br Br Br 32 Br 32 32 32 Br Br 32 Br 32 Br BrBr Br Br

37 Br Br 37 37 Br BrBr Br 37 Br Br 37 Br Br 37 Br Br Br 37 Br 37 37 37 Br Br 37 Br Br 3737Br BrBr BrBrBr

42 Br H 42 42 Br HBr 42 Br H 42 Br H 42 Br Br H 42 Br H 42 42 42 Br H 42 Br H 42 Br H 42 Br BrH H HH

33 Br Br 33 33 Br BrBr Br 33 Br Br 33 Br Br 33 Br Br Br 33 Br 33 33 33 Br Br 33 Br 33 Br BrBr Br Br

38 Br Br BrBr 38 38 Br Br 38 Br Br 38 Br 38 Br Br Br 38 Br 38 38 Br 38 Br Br 38 Br 3838Br BrBr BrBrBr

43 H H 43 43 H HHH H 43 H H 43 H H 43 43 H 43 H 43H H H 43 H 43 43 HH H HH

34 Br Br 34 34 Br BrBr Br 34 Br Br 34 Br Br 34 Br Br Br 34 Br 34 34 34 Br Br 34 Br 34 Br BrBr Br Br

39 Br Br BrBr 39 39 Br Br 39 Br Br 39 Br 39 Br Br Br 39 Br 39 39 39 Br Br 39 Br Br 3939Br BrBr BrBrBr

44 H H 44 44 H HHH H 44 H H 44 H H 44 44 H 44 H 44H H H 44 H 44 44 44 HH H HH 45 Cl H 45 45 Cl HCl 45 Cl H 45 Cl H 45 Cl Cl H 45 Cl H 45 45 45 Cl H 45 Cl H 45 Cl ClH H HH

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Before finalizing the purpurealidin I (1) synthesis, several synthetically simpler amide analogs Before finalizing thethe purpurealidin (1) synthesis, several synthetically simpler amide analogs Before finalizing the purpurealidin I(1) (1) synthesis, several synthetically simpler amide analogs Before finalizing the purpurealidin (1) synthesis, several synthetically simpler amide analogs Before Before finalizing finalizing purpurealidin the purpurealidin II (1) Isynthesis, (1) synthesis, several several synthetically synthetically simpler simpler amide amide analogs analogs Before finalizing the purpurealidin (1) several synthetically simpler amide analogs Before finalizing the purpurealidin III (1) synthesis, several synthetically simpler amide analogs Before finalizing the Isynthesis, synthesis, several synthetically simpler amide analogs Before finalizing thepurpurealidin purpurealidin Isynthesis, (1) several synthetically simpler amide Before finalizing the purpurealidin synthesis, several synthetically simpler amide analogs Before finalizing the purpurealidin (1) several synthetically simpler amide analogs containing the tyramine fragment and aII (1) series of synthesis, compounds with substituted phenyl rings (Aranalogs in containing the tyramine fragment and a series of compounds with substituted phenyl rings (Ar in containing the tyramine fragment and a series of compounds with substituted phenyl rings (Ar in containing the tyramine fragment and a series of compounds with substituted phenyl rings (Ar in containing containing the tyramine the tyramine fragment fragment and a and series a series of compounds of compounds with with substituted substituted phenyl phenyl rings rings (Ar in (Ar containing the tyramine fragment and a series of compounds with substituted phenyl rings (Ar in containing the tyramine fragment and a aseries ofofcompounds with substituted phenyl rings (Ar containing the tyramine fragment and series compounds with substituted phenyl rings (Arinin in containing the tyramine fragment and a series of compounds with substituted phenyl rings (Ar in containing the tyramine fragment and a series of compounds with substituted phenyl rings (Ar in Table 2) instead of the bromotyrosine part were synthesized and screened. The synthesis of these Table 2) instead of the bromotyrosine part were synthesized and screened. The synthesis of these these Table 2) instead of the bromotyrosine part were synthesized and screened. The synthesis of these Table 2) instead of the bromotyrosine part were synthesized and screened. The synthesis of these Table Table 2) instead instead of the of bromotyrosine the bromotyrosine part were part were synthesized synthesized and screened. and screened. The synthesis The synthesis of these these Table 2) instead of the bromotyrosine part were synthesized and screened. The synthesis of these Table 2) instead of the bromotyrosine part were synthesized and screened. The synthesis of Table 2)2) instead of the bromotyrosine part were synthesized and screened. The synthesis ofof these Table 2) instead of the bromotyrosine part were synthesized and screened. The synthesis of these Table 2) instead of the bromotyrosine part were synthesized and screened. The synthesis of these Table 2) instead of the bromotyrosine part were synthesized and screened. The synthesis of these simplified amide derivatives (46–78) (Table 2) have previously been reported by our group [15]. simplified amide derivatives (46–78) (Table 2) have previously been reported byby our group [15]. simplified amide derivatives (46–78) (Table 2) have previously been reported by our group [15]. simplified amide derivatives (46–78) (Table 2) have previously been reported by our group [15]. simplified simplified amide amide derivatives derivatives (46–78) (46–78) (Table (Table 2) have previously previously been been reported reported by group our group [15]. [15]. simplified amide derivatives (46–78) (Table 2) have previously been reported by our group [15]. simplified amide derivatives (46–78) (Table 2) have previously been reported by our group [15]. simplified amide derivatives (46–78) (Table 2)2) have previously been reported by our group [15]. simplified amide derivatives (46–78) (Table 2) have previously been reported by our group [15]. simplified amide derivatives (46–78) (Table 2) have previously been reported by our group [15]. simplified amide derivatives (46–78) (Table 2)have have previously been reported byour our group [15]. Table 2. Structures of final amide compounds (46–78) [15]. Table 2. Structures of final final amide compounds (46–78) [15]. Table 2. Structures of final amide compounds (46–78) [15]. Table 2. Structures of amide compounds (46–78) [15]. Table Table 2. Structures of of amide final amide compounds compounds (46–78) (46–78) [15]. [15]. Table 2. Structures of final amide compounds (46–78) [15]. Table 2.2. Structures of final amide compounds (46–78) Table 2. Structures of final amide compounds (46–78) [15]. Table 2.2.Structures Structures of final amide compounds (46–78) [15]. Table Structures offinal final amide compounds (46–78) [15].[15].

Cmpd Ar R1 R2 Cmpd Ar R1 R2 Cmpd Ar R1 R2 Cmpd Ar Ar R R12121221 R Cmpd Ar Ar R1111 R R12121221 R Cmpd Ar Ar RAr 1Ar R Cmpd Ar Cmpd Ar Cmpd Cmpd Ar R Cmpd Ar R Cmpd Ar R Cmpd Cmpd Ar 1111 RR 2222 R R Cmpd Ar 1111 RR 2222 RR Cmpd R 1111 R R 2222 RR Cmpd Ar R R Cmpd Ar R R Cmpd Ar R R Cmpd Ar R 1111 R R Cmpd Ar R R Cmpd Ar 111Ar R 22122111R Cmpd 1R 2R Cmpd 1R 2R Cmpd Ar 1R 2R Cmpd Ar R2222Cmpd Cmpd Ar R2222 Cmpd Cmpd R R2222 Cmpd Ar RR RR Cmpd Ar RR RR Cmpd Ar RR RR Cmpd Ar Ar Cmpd Ar *Ar Cmpd Ar R 46 Br N(CH3)2 57 N(CH3)2 68 Br N(CH3)2 **** * ***** * **** Br 46 46 Br Br N(CH 57 Br Br N(CH 68 Br Br N(CH 257 46 Br 57 Br 68 Br 33)22 3N(CH 3N(CH 3N(CH 46 Br N(CH Br N(CH Br N(CH N(CH 3N(CH N(CH 3N(CH 3233)3)2)22268 N(CH 3N(CH 46 Br N(CH Br N(CH 68 Br N(CH 46 Br 3333)))2222N(CH 3333)))2222N(CH Br 3333)))2222N(CH BrBr 357 )3233)3)2)257 BrBr )68 BrBr 46 Br 257 57 F F F Br Br 68 Br 46 Br N(CH 3))N(CH Br N(CH 3))N(CH 68 Br N(CH 3))N(CH 46 4646 BrN(CH N(CH 3)22)22 57 57 5757 BrN(CH N(CH 3)22)22 368 68 6868 BrN(CH N(CH 3)22)22 3)32)32)2 FF FF F FFF F

FF

F

47 Br N(CH3)2 58 Br N(CH3)2 69 Br N(CH3)2 47 47 Br5.Br N(CH 2of Br Br N(CH Br N(CH 258 47 Br 58 Br 33)3)2)22269 69 Br 33)22 3N(CH 2)22 58 3N(CH 3N(CH Synthesis purpurealidin (1) carboxylic carboxylic part a69 route simplified 47 Br N(CH N(CH 3N(CH N(CH 3N(CH 32and 69 Br N(CH 3N(CH 47 Br N(CH Br N(CH 69 Br N(CH 3333))2222(22a) 3333))2222N(CH 4747 BrBr N(CH 358 )3233)3)2)258 BrBr N(CH 369 )69 BrBr 47 Scheme Br 258 58 II (1) Br 69to the Br 47 Br N(CH 3))N(CH Br N(CH 3))N(CH Br N(CH 3))N(CH 47 Scheme Br N(CH N(CH 3)2the 58 5858 Br N(CH N(CH 3)22)22 and 69 Br N(CH 3)22)22 3)32)32)2 5. Br Synthesis of3333))222the purpurealidin part (22a) a 69 route to Br the simplified 3 substituents are given in Table 1. hydroxyimino propanamides (24a–d), R hydroxyimino propanamides (24a–d), R substituents are given in Table 1. 3

48 Br N(CH3)2 59 Br N(CH3)2 70 Br N(CH3)2 48 Br N(CH 59 Br N(CH 70 Br N(CH 48 48 Br Br N(CH 59 Br Br N(CH 70 Br Br N(CH 259 48 Br 59 Br 70 Br 33)22 3N(CH 3N(CH 3N(CH Br N(CH 3N(CH N(CH 3N(CH 3233)3)2)222of 70 N(CH 3N(CH 48 Br N(CH Br N(CH Br N(CH 48 Br N(CH 3333))))2222N(CH N(CH 3333))))2222N(CH Br 3333))))2222N(CH 4848 Br )3233)3)2)259 BrBr 7070 BrBr 48 Br N(CH Br N(CH Br N(CH 48 The synthesis Br 259 59 began Br Br 70 Br 48 Br N(CH 3))N(CH Br N(CH 3))N(CH 70 Br N(CH 3))N(CH of purpurealidin (1), bromination 48 The Br N(CH 3)22)22 59 59 I5959 Brthe N(CH 3)22)22 370 70p-hydroxybenzaldehyde, BrN(CH N(CH 3)22)22 3)32)32)2 simplified derivatives of359 purpurealidin I (1)with were prepared in)70 an70 analogous manner (Scheme 5)

followed by methylation to give (19a). The requisite azlactone (20a) was then prepared by the

with the appropriate anilines and benzylamines (Table 1) followed by THP deprotection. The yields 3)2 N-acetylglycine 60 NH(CH 3) 71 Br N(CH 49 condensation ofBr(19a)C(CH with (SchemeBr5) and subjecting the resulting product to 3)2 the4949 amide couplings ranged deprotection was achieved using for33)22 Br Br C(CH 60 BrTHP NH(CH 3)))NH(CH 71 Br Br N(CH 49of49 Br 60 Br NH(CH 71 Br 49 3N(CH Br C(CH 60 Br NH(CH 71 Br N(CH 49 333)))2222N(CH C(CH 33C(CH 22)22 60 Br Br NH(CH 333))3)) 71 N(CH 3N(CH Br C(CH )3)C(CH Br NH(CH Br N(CH 49 Br 3333)))2222C(CH NH(CH 333NH(CH Br 3TFA 49 BrBr 3from )3233)3)2)2260 6060 Br 3)333)3)71 7171 BrBr Br 260 60of HClThe Br NH(CH )71 71 Br 49 Br C(CH 3)C(CH 60 Br NH(CH 71 Br N(CH 3))N(CH 49 BraC(CH C(CH 60 19–87%. Brpyruvic NH(CH 71 BrN(CH N(CH 3)22)22 3)32)32)2 49 hydrolysis using 10% aqueous solution toBr give acid (21a). Compound (21a) was then the various simplified derivatives of (1), as heating with 2 M HCl in Et O proved sluggish. Several converted into the THP-protected oxime (22a) by reaction with 2O-(tetrahydropyran-2-yl)50 hydroxylamine N(CH 3)2 H NH(CH(31) 3) 72ExperimentalBrSection NH(CH 3) different conditions Br were attempted in61the synthesis of compound (see 4.1.2). (THPONH 2). The crude oxime was then subjected to EDC coupling with (17) under 50 50 Br Br N(CH 61 H H NH(CH 3)))NH(CH 72 Br Br NH(CH 3)))NH(CH 261 50 Br 61 H NH(CH 72 Br NH(CH 3N(CH 50 Br N(CH H NH(CH 72 Br NH(CH N(CH 3N(CH NH(CH 333))3)) 72 NH(CH Br 333))3)) 50 Br N(CH H NH(CH Br NH(CH 50 Br 3333)))2222N(CH H 333NH(CH Br NH(CH BrBr 361 )3233)3)2)261 HH 3)333)3)72 Br 3)333)3)) 50 Br 261 61 H NH(CH )72 72 Br 333NH(CH NH(CH 50 Br N(CH 3))N(CH H NH(CH 72 Br NH(CH 50 5050 BrN(CH N(CH 3)22)22 61 61 6161 HNH(CH NH(CH 72 7272 Br NH(CH After microwave purificationconditions on silica gel, the yields final hydroxyimino ranged to produce (26)ofinthe a moderate yield (56%; propanamides Scheme 6). The (30–45) THP group wasfrom solution the free oxime (28).73TrifluoroacetylBr mediated 13–50%. 3)2 of HCl 62 in Et2O to give Br N(CH 3)2 N(CH3)2 51 removed with aBr2 M N(CH Br N(CH 62 Br N(CH 73 Br N(CH 51 Brof N(CH 62 Brpurpurealidin N(CH 73 Br Br N(CH 51 51 262 Br 62 Br 22273 73 Br 33)22 51 3N(CH 3N(CH 3N(CH Br Br N(CH 3N(CH 22)22 62 3233)3)2)262 62 Br Br N(CH 3N(CH 22)2I 3233)3)2in 73 N(CH 3N(CH Br N(CH Br N(CH 73 Br N(CH 51 Br N(CH 3333))))2222N(CH Br N(CH 3333))))2222N(CH Br N(CH 3333)(11 51 Br 362 ) 62 Br 373 ) 73 BrBr 5151 Br N(CH Br N(CH 73 Br N(CH )))2222N(CH 51 Br 262 62 Br ) 73 Br 51 Br N(CH 3))N(CH ) Br N(CH 3))N(CH ) 73 Br N(CH 3))N(CH 51 Br N(CH 3 62 Br N(CH 3 2 73 Br N(CH 3)22)22 3)32)32)2 51 Before deprotection (28) using MeOH/K 2 CO 3 resulted in (1) an overall yield of 12% finalizing the purpurealidin I (1) synthesis, several synthetically simpler amide analogs steps). The aplysamine 2 tetrabromo derivative (29), with a dimethylamino moiety at the tyramine containing the tyramine fragment and a series of compounds with substituted phenyl rings (Ar in fragment, was synthesized using purpurealidin E (25) [15,27] in the coupling with (22a) (Scheme 6).

followed by methylation to give (19a). The requisite azlactone (20a) was then prepared by the condensation of (19a) with N-acetylglycine (Scheme 5) and subjecting the resulting product to Cmpd R R R Cmpd R R R Cmpd R R R Cmpd R 11 11R R 22 R R 33 aqueous Cmpd R 11 11HCl R 2 to R 33 33R Cmpd R 11 11R R 22 R R 33 33R 3 R 2 R Cmpd 111R 222 a 10% 333 Cmpd R 111R 222 333 acid Cmpd 111R 222 333 hydrolysis solution of pyruvic (21a). Compound was then Cmpd R R R Cmpd R R Cmpd R R Cmpd Cmpd 2R Cmpd 3R Cmpd R R R Cmpd R R R Cmpd R R R Cmpd R1R R222R R3R Cmpd R1R Rgive R3R Cmpd R1R R222(21a) R Cmpd R 11 R R 333R Cmpd R 11R R 33 R Cmpd R 11 R R 33 R Cmpd 1222222R 2 3 Cmpd 1222222R 2 3 Cmpd 1222222R R 2 3 Cmpd R R R Cmpd R R R Cmpd R R R Cmpd R 1R Rusing R 3R Cmpd R 1R RR R 3R Cmpd R 1R RR R 3R Cmpd 2R Cmpd 2R Cmpd 2R Cmpd R R Cmpd R R Cmpd R R Cmpd Cmpd Cmpd 22 11 1R11 R2R 33 3R33 11 1R11 R2R 33 3R33 11 1R11 R2R 33 3R33 converted into the THP-protected oxime (22a) by reaction with O-(tetrahydropyran-2-yl)hydroxylamine (THPONH2). The35 crude oxime was then subjected to40 EDC coupling with (17) under 40 Br Br 30 Br Br 35 Br Br Br 30 Br Br 40 Br Br 30 Br Br 35 Br Br 40 Br Br 30 Br Br 35 Br Br 40 Br Br 30 Br Br 35 Br Br 40 Br Br 30 Br Br 35 Br Br 40 Br Br 30 Br Br 35 Br Br 40 Br Br 30 Br Br 35 Br Br 40 Br Br 30 Br Br 35 Br Br 40Br Br Br 30Br Br Br 35Br Br Br 40 Br Br 30 Br Br 35 Br Br 4040 BrBr BrBr 3030 BrBr BrBr 3535 BrBr BrBr 40 Br Br 30 Br Br 35 Br Br Mar. Drugs 2018, 16, 481 6 of 26 microwave conditions to produce (26) in a moderate yield (56%; Scheme 6). The THP group was removed with a 2 M solution of HCl in Et2O to give the free oxime (28). Trifluoroacetyl mediated deprotection (28) using MeOH/K 3Br resulted in purpurealidin I (1) anBr overall yield (11 31 Br Br 36 Br Br 41 Br H 31 Br Br 36 Br 41 H 31 BrBr Brof 362CO BrBr BrBr 41in Br HH 31 Br Br 36 Br Br 41 Br H 31 Br 36 41 Br 31 Br Br 36 Br Br H 31 Br Br 36 Br Br 41 Br HH of 12% 31 Br Br 36 Br Br 41 Br H 312) Br Br 36 Br Br 41Br Br 31 Br Br 36 Br Br 41 Br H 31 Br Br 36 Br Br 41 Br H 3131 Br BrBr 3636 BrBr BrBr 4141 BrBr HH Table instead of the bromotyrosine part were synthesized and 41 screened. The synthesis of these Br 31 Br Br 36 Br Br 41 Br H steps). The aplysamine 2 tetrabromo derivative (29), with a dimethylamino moiety at the tyramine simplified derivativesusing (46–78) (Table 2) have previously reported by (22a) our group [15]. fragment,amide was synthesized purpurealidin E (25) [15,27] in been the coupling with (Scheme 6). 32 Br Br 32 Br 3232 BrBr BrBr 32 Br Br 32 Br Br 32 Br Br 32 Br Br 32Br Br Br 32 Br Br 32 Br Br 3232 BrBr BrBr 32 Br Br

37 Br Br 37 Br 3737 BrBr BrBr 37 Br Br 37 Br Br 37 Br Br 37 Br Br 37Br Br Br 37 Br Br 37 Br Br 3737 BrBr BrBr 37 Br Br

42 Br H 42 H 4242 BrBr HH 42 Br H 42 Br H 42 Br HH 42 Br H 42Br Br 42 Br H 42 Br H 4242 BrBr HH 42 Br H

33 Br Br 33 Br 3333 BrBr BrBr 33 Br Br 33 Br Br 33 Br Br 33 Br Br 33Br Br Br 33 Br Br 33 Br Br 3333 BrBr BrBr 33 Br Br

38 Br Br 38 Br 3838 BrBr BrBr 38 Br Br 38 Br Br 38 Br Br 38 Br Br 38Br Br Br 38 Br Br 38 Br Br 3838 BrBr BrBr 38 Br Br

43 H H 43 H 4343 HH HH 43 H H 43 H H 43 HH HH 43 H H 43 H 43 H H 43 H H 4343 HH HH 43 H H

34 Br Br 34 Br 3434 BrBr BrBr 34 Br Br 34 Br Br 34 Br Br 34 Br Br 34Br Br Br 34 Br Br 34 Br Br 3434 BrBr BrBr 34 Br Br

39 Br Br 39 Br 3939 BrBr BrBr 39 Br Br 39 Br Br 39 Br Br 39 Br Br 39Br Br Br 39 Br Br 39 Br Br 3939 BrBr BrBr 39 Br Br

44 H H 44 H 4444 HH HH 44 H H 44 H H 44 HH HH 44 H H 44 H 44 H H 44 H H 4444 HH HH 44 H H 45 Cl H 45 H 45 Cl H 45 Cl H 45 Cl H 45 Cl H 45 Cl HH 45 Cl H 45 Cl H 45Cl Cl 45 Cl H 4545 ClCl HH 45 Cl H

Before finalizing the purpurealidin (1) synthesis, several synthetically simpler amide analogs Before finalizing the purpurealidin IIIIII(1) synthesis, several synthetically simpler amide analogs Before finalizing the purpurealidin (1) synthesis, several synthetically simpler amide analogs Before finalizing the purpurealidin III(1) synthesis, several synthetically simpler amide analogs Before finalizing the purpurealidin (1) synthesis, several synthetically simpler amide analogs Before finalizing the purpurealidin (1) synthesis, several synthetically simpler amide analogs Before finalizing the purpurealidin synthesis, several synthetically simpler amide analogs Before finalizing the purpurealidin Isynthesis, (1) synthesis, several synthetically simpler amide analogs Before finalizing the purpurealidin (1) synthesis, several synthetically simpler amide analogs Before finalizing the purpurealidin several synthetically simpler amide analogs Before finalizing the purpurealidin I(1) synthesis, several synthetically simpler amide analogs Before finalizing the purpurealidin I(1) (1) synthesis, several synthetically simpler amide analogs Before finalizing the purpurealidin I (1) (1) synthesis, several synthetically simpler amide analogs containing the tyramine fragment and a series of compounds with substituted phenyl rings (Ar in containing the tyramine fragment and a series of compounds with substituted phenyl rings (Ar in containing the tyramine fragment and a series of compounds with substituted phenyl rings (Ar in containing the tyramine fragment and a series of compounds with substituted phenyl rings (Ar containing the tyramine fragment and a series of compounds with substituted phenyl rings (Ar in containing the tyramine fragment and aaseries series of compounds with substituted phenyl rings (Ar inin containing the tyramine fragment and series of compounds with substituted phenyl rings (Ar in containing the tyramine fragment series ofcompounds compounds with substituted phenyl rings (Ar containing the tyramine fragment and series of compounds with substituted phenyl rings (Ar in containing the tyramine fragment and aand of with substituted phenyl rings (Ar in containing the tyramine fragment and ofcompounds compounds with substituted phenyl rings (Ar inin containing the tyramine fragment and aseries of with substituted phenyl rings (Ar in containing the tyramine fragment and aaaaseries series of compounds with substituted phenyl rings (Ar in Table 2) instead of the bromotyrosine part were synthesized and screened. The synthesis of these Table 2) instead of the bromotyrosine part were synthesized and screened. The synthesis of these Table 2) instead of the bromotyrosine part were synthesized and screened. The synthesis of these Table 2) instead of the bromotyrosine part were synthesized and screened. The synthesis of these Table 2) instead of the bromotyrosine part were synthesized and screened. The synthesis of these Table 2) instead of the bromotyrosine part were synthesized and screened. The synthesis of these Table 2) instead of the bromotyrosine part were synthesized and screened. The synthesis of these Table 2) instead of the bromotyrosine part were synthesized and screened. The synthesis of these Table 2) instead of the bromotyrosine part were synthesized and screened. The synthesis of these Table 2) instead of the bromotyrosine part were synthesized and screened. The synthesis of these Table 2) instead of the bromotyrosine part were synthesized and screened. The synthesis of these Table 2) instead bromotyrosine part were synthesized screened. synthesis these Table 2) instead of of thethe bromotyrosine part were synthesized andand screened. TheThe synthesis of of these simplified amide derivatives (46–78) (Table 2) have previously been reported by our group [15]. simplified amide derivatives (46–78) (Table 2) have previously been reported by our group [15]. simplified amide derivatives (46–78) (Table 2) have previously been reported by our group [15]. simplified amide derivatives (46–78) (Table 2) have been reported by our group [15]. simplified amide derivatives (46–78) (Table 2) previously been reported by our group [15]. Scheme 6. Synthesis of purpurealidin Ipreviously and 2reported analog (29). simplified amide derivatives (46–78) (Table 2) have previously been by our group [15]. simplified amide derivatives (46–78) (Table 2) have previously been reported by our group [15]. simplified amide derivatives (46–78) (Table 2) have previously been reported by our group [15]. simplified amide derivatives (46–78) (Table 2) have previously been reported by our group [15]. simplified amide derivatives (46–78) (Table 2) have previously been reported by our group [15]. Scheme 6. Synthesis of purpurealidin I(1) (1) andaplysamine aplysamine 2 analog (29). simplified amide derivatives (46–78) (Table 2)have have previously been reported by our group [15]. simplified amide derivatives (46–78) (Table 2) have previously been reported by our group [15]. simplified amide derivatives (46–78) (Table 2) have previously been reported by our group [15]. Table 2. Structures of final amide compounds (46–78) [15]. Table 2. Structures of final amide compounds (46–78) [15]. Table 2. Structures of final amide compounds (46–78) [15]. The simplified derivatives of purpurealidin Iamide (1) were prepared in an analogous manner (Scheme Table 2. Structures of final amide compounds (46–78) [15]. Table 2. Structures of final compounds (46–78) [15]. Table 2. Structures of final amide compounds (46–78) [15]. Table 2.2. Structures of final amide compounds (46–78) [15]. Table 2. Structures of final amide compounds (46–78) [15]. Table Structures of final amide compounds (46–78) [15]. Table 2.2. Structures of final amide compounds (46–78) [15]. Table 2. Structures of final amide compounds (46–78) [15]. Table of final amide compounds (46–78) [15]. Table 2. Structures of final amide compounds (46–78) [15]. Table 2.Structures Structures of final amide compounds (46–78) [15]. 2. Structures of final amide compounds (46–78)by [15]. 5) with the appropriateTable anilines and benzylamines (Table 1) followed THP deprotection. The yields of the amide couplings ranged from 19–87%. The THP deprotection was achieved using TFA for the various simplified derivatives of (1), as heating with 2 M HCl in Et2O proved sluggish. Several different conditions were attempted in the synthesis of compound (31) (see Experimental Section 4.1.2). After purification on silica gel, the yields of the final hydroxyimino propanamides (30–45) Cmpd Ar R1 111 R R2 222 Cmpd Cmpd Ar R1 111 R R2 222 Cmpd Cmpd Ar R1 111 R R2 222 Cmpd Ar R 11 11 R 22 22 R Ar RR 11 11 R 22 22 R Ar RR 11 11 R 22 22 R Cmpd ArAr R R Cmpd ArAr R R Cmpd ArAr R R ranged from 13–50%. Cmpd R R Cmpd R R Cmpd R R Cmpd Ar R R Cmpd Ar R Cmpd Ar R Cmpd Cmpd Cmpd Cmpd R Cmpd Ar Cmpd Ar Cmpd Ar Cmpd Ar Cmpd Ar Cmpd Ar Cmpd Ar Ar 1 R 2 Cmpd Cmpd Ar Ar R 1 R 2 Cmpd CmpdAr ArR 1 R 2 Cmpd Ar Ar R R Cmpd Ar Ar R R Cmpd Ar Ar R R Cmpd Ar RR 111 RR RR 222 RR Cmpd Ar RR 111 RR RR 222 RR Cmpd Ar RR 111 RR RR 222 RR Cmpd ArArAr Cmpd Cmpd Ar Cmpd Ar Cmpd

1R R111 R11RR

R2R R 222 R22R

Cmpd Cmpd Cmpd Cmpd Cmpd

46 46 46 46 46 46 46 46 46 46 46 46 4646 46 46

Br N(CH 57 Br N(CH 33) Br N(CH 57 Br N(CH 57 Br N(CH 57 Br N(CH 57 Br N(CH )3322))222 3)3332)))3222)2 57 57 Br N(CH 57 Br N(CH 57 Br N(CH 57 Br N(CH 57 Br N(CH 333)3)223)2) 57 BrBr N(CH 5757 Br N(CH 57 Br N(CH Br N(CH 3)2 233))2)2 57 N(CH 57

47 47 47 47 47 47 47 47 47 47 47 47 4747 47 47 47

Ar Ar ArArAr * ** *** ** * * * F FF FFF FF F F F

* * ** * ** F F FF F F F

R11R R11 1 1R R

R22 RR22R2 2R

Cmpd Cmpd Cmpd Ar Ar RR11R11R R11 Cmpd Cmpd ArArAr

R222 R22RR2R

Br N(CH 68 Br N(CH 33) Br N(CH 68 Br N(CH 68 Br N(CH 68 N(CH 68 Br N(CH )3322))22223)3332)))3222)2 68 68 Br N(CH 68 Br N(CH 68 Br Br N(CH 68 Br N(CH 68 Br N(CH 333)3)223)2) 68 N(CH 6868 Br N(CH 3)2 68 N(CH Br N(CH 3)2 ) 68 BrBrBr N(CH 3 32)2 68

Br N(CH Br N(CH 33) Br N(CH Br N(CH Br N(CH Br N(CH Br N(CH )3322))222 3)3332)))3222)2 Br N(CH Br N(CH Br N(CH Br N(CH Br N(CH 333)3)223)2) BrBr N(CH Br N(CH N(CH ))22 Br N(CH 3)2 233) Br N(CH

Br N(CH 58 Br N(CH 33) Br Br N(CH 58 58 Br N(CH 58 Br N(CH 58 N(CH Br N(CH )3322))22 3)332))22 58 58 Br N(CH 58 Br N(CH 58 Br N(CH 58 Br N(CH 58 Br N(CH 333))3)22)322)23)32)32)2 58 BrBr N(CH 5858 Br N(CH Br N(CH 58 Br N(CH N(CH 58 3 2 33)2)2 58

Br N(CH 69 Br N(CH 33) Br Br N(CH 69 69 Br N(CH 69 Br N(CH 69 N(CH Br N(CH )3322))22 3)332))22 69 69 Br N(CH 69 Br N(CH 69 Br N(CH 69 Br N(CH 69 Br N(CH 333))3)22)322)23)32)32)2 69 BrBr N(CH 6969 Br N(CH 69 Br N(CH 69 N(CH )2 69 Br N(CH 3 23 )32

Br N(CH Br N(CH 33) Br Br N(CH Br N(CH Br N(CH N(CH Br N(CH )3322))22 3)332))22 Br N(CH Br N(CH Br N(CH Br N(CH Br N(CH 333))3)22)322)23)32)32)2 BrBr N(CH Br N(CH Br N(CH N(CH Br N(CH 3 2 33)22

48 48 48 48 48 48 48 48 48 48 48 48 4848 48 48 48

Br N(CH 59 Br N(CH 33) Br Br N(CH 59 59 Br N(CH 59 Br N(CH 59 N(CH Br N(CH )3322))22 3)332))22 59 59 Br N(CH 59 Br N(CH 59 Br N(CH 59 Br N(CH 59 Br N(CH 333)3)22)322)23)3)2)32)2 59 BrBr N(CH 5959 N(CH 59 Br N(CH 59 Br N(CH Br N(CH 3)2 33)2 2 59

Br N(CH 70 Br N(CH 33) Br Br N(CH 70 70 Br N(CH 70 Br N(CH 70 N(CH Br N(CH )3322))22 3)3332)))3222)2 70 70 Br N(CH 70 Br N(CH 70 Br N(CH 70 Br N(CH 70 Br N(CH 333)3)22)322)) 70 BrBr N(CH 7070 Br N(CH Br N(CH 70 N(CH ))22 70 Br N(CH 3)23 2332 70

Br N(CH Br N(CH 33) Br Br N(CH Br N(CH Br N(CH N(CH Br N(CH )3322))22 3)332))22 Br N(CH Br N(CH Br N(CH Br N(CH Br N(CH 333)3)22)322)23))32)32)2 BrBr N(CH Br N(CH Br N(CH N(CH Br N(CH 3)2 33)22

49 49 49 49 49 49 49 49 49 49 49 49 49 4949 49 49

Br C(CH 60 Br C(CH 33) Br Br C(CH 60 60 Br C(CH 60 Br C(CH 60 C(CH Br )3322))22 3)333)2)))3222)2 60 60 Br C(CH 60 Br C(CH 60 BrC(CH C(CH 60 Br C(CH 60 Br C(CH 333)3)22)322)23 60 C(CH 6060 BrBr C(CH Br C(CH Br C(CH Br C(CH 3)2 33))222 606060

Br NH(CH 71 Br NH(CH 33) Br Br NH(CH 71 71 Br NH(CH 71 Br NH(CH 71 NH(CH Br NH(CH )33)) 3)33)) 71 71 Br NH(CH 71 Br NH(CH 71 Br NH(CH 71 Br NH(CH 71 Br NH(CH 333)3))3)33))3)3)71 71 NH(CH BrBr NH(CH 7171 Br NH(CH 71 NH(CH Br NH(CH 3) 3) 71

Br N(CH Br N(CH 33) Br Br N(CH Br N(CH Br N(CH N(CH Br N(CH )3322))22 3)332)))3222)2 Br N(CH Br N(CH Br N(CH Br N(CH Br N(CH 333)3)22)322)323))32 N(CH BrBr N(CH Br N(CH N(CH Br N(CH 3)2 3)22

50 50 50 50 50 50 50 50 50 50 50 50 50 5050 50 50

Br N(CH 61 Br N(CH 33) Br N(CH 61 Br N(CH 61 Br N(CH 61 Br N(CH 61 Br N(CH )3322))222 33)3332))))32222)2 61 61 N(CH 61 Br N(CH 61 Br N(CH 61 Br N(CH 61 Br N(CH 61 Br N(CH 333)3)223)2) 61 BrBr N(CH 6161 Br N(CH 61 Br N(CH Br N(CH 3)2 233))22 61

HNH(CH NH(CH 72 H 33) H HH NH(CH 72 NH(CH 72 H NH(CH 72 NH(CH 72 H NH(CH )33)) 3 33)) 72 72 H NH(CH NH(CH 72 NH(CH 72 NH(CH 72 NH(CH 72 H NH(CH 333)3)3))33))3)3)72 72 HH NH(CH 7272 HHH NH(CH 72 NH(CH HH NH(CH 3) 3) 72

Br NH(CH Br NH(CH 33) Br Br NH(CH Br NH(CH Br NH(CH NH(CH Br NH(CH )33)) 3)333)))3) Br NH(CH Br NH(CH Br NH(CH NH(CH Br NH(CH Br Br NH(CH 333)3)3))33) BrBr NH(CH Br NH(CH NH(CH Br NH(CH 3) 3))

Mar. Drugs 2018, 16, x16, FOR PEER REVIEW Mar. Drugs 2018, xxFOR FOR PEER REVIEW Mar. Drugs 2018, 16, x16, FOR PEER REVIEW Mar. Drugs 2018, FOR PEER REVIEW Mar. Drugs 2018, 16, FOR PEER REVIEW Mar. Drugs 2018, 16, xFOR PEER REVIEW Mar. Drugs 2018, 16, FOR PEER REVIEW Mar. Drugs 2018, 16, FOR PEER REVIEW Mar. Drugs 2018, 16, xxx PEER REVIEW Mar. Drugs 2018, x FOR PEER REVIEW Mar. Drugs 2018, 16, xx16, FOR PEER REVIEW Br N(CH 62 51 Br N(CH 3 51 Br N(CH 62 51 51 Br N(CH 6262 Br N(CH 62 51 Br N(CH 62 51 Br N(CH 51 Br N(CH 3) )3)3332)22)))32222)233)3332)))32222)2 62 62 51 Br N(CH 62 51 Br N(CH 62 51 Br N(CH 62 51 Br N(CH 62 51 Br N(CH 3 62 51 Br N(CH 6262 51 Br N(CH N(CH 62 51 N(CH Br Br 33)22 33))22 62 51 51

Br N(CH 73 Br N(CH 33) 73 Br Br N(CH 73 73 Br N(CH Br N(CH 73 Br N(CH 73 N(CH Br N(CH )3322))22)3)332))22 73 73 Br N(CH 73 Br N(CH 73 Br N(CH 73 Br N(CH 73 Br N(CH 333)3)22)3232)232)32)32)2 73 BrBr N(CH 7373 Br N(CH 73 N(CH Br N(CH 3)2 3)2 73

25 7725 of 25 7777of of25 25 of 25 7of of of 25 of 25 7of of 25 725 of 25 77 N(CH Br 3 ) 2 Br N(CH 3 ) 2 N(CH 3 ) 2 Br N(CH ) Br N(CH 3 ) 2 Br N(CH 3 ) 2 Br N(CH 3 2 Br N(CH 3) 22 3 32)32)2 Br N(CH Br N(CH 3)3) Br N(CH Br N(CH ) 2 Br N(CH 3 2 Br N(CH 3 ) 2 Br N(CH N(CH N(CH Br Br 33)22 33))22

46

3 2

3 2

52 52 52 52 5252 52 52 52 52 52 52

Br C(CH 63 Br C(CH 63 Br 33)3223))223)32)32)2 63 Br C(CH C(CH 63 BrBr C(CH 6363 C(CH 3 2 63 Br C(CH 63 Br C(CH Br C(CH 63 Br C(CH C(CH Br 333))23322))22 33))22 63 Br C(CH 63 63 3 2

Br NH(CH 74 Br NH(CH 74 Br 33)33)) 3)3)3) 74 BrNH(CH NH(CH 74 BrBr NH(CH 7474 NH(CH 3 Br NH(CH 74 Br NH(CH 74 Br NH(CH 74 BrNH(CH NH(CH 74 Br 333))33)) )33) 74 74 Br NH(CH 3

Br NH(CH Br NH(CH Br NH(CH 33)33)) 3)3)3) Br NH(CH BrBr NH(CH NH(CH Br NH(CH Br NH(CH 33 Br NH(CH Br NH(CH Br NH(CH 333))33)) 3) Br NH(CH 3)

53 53 53 53 5353 53 53 53 53 53 53

N(CH 64 N(CH 64 H 33)3223))223)32)32)2 64 H N(CH N(CH 64 HHH N(CH 6464 N(CH 3 2 64 H N(CH 64 HH N(CH H N(CH N(CH H 333))23322))22 33))22 64 HH N(CH N(CH 64 64 3 2 64

Br N(CH 75 Br N(CH 75 Br 33)3223))223)32)32)2 75 Br N(CH N(CH 75 BrBr N(CH 7575 N(CH 3 2 Br N(CH 75 Br N(CH 75 Br N(CH 75 Br N(CH N(CH 75 Br 333))23322))22) 33)22 75 75 Br N(CH 3 2

N(CH N(CH H 33)3223))223)32)32)2 HN(CH N(CH HHH N(CH N(CH 3 2 H N(CH HH N(CH H N(CH HN(CH N(CH H 333))23322))22 )33)22 H N(CH 3 2

54 54 54 54 5454 54 54 54 54 54 54

NH(CH 65 HHNH(CH NH(CH 65 H 33)33)) 3)3)3) 65 NH(CH 65 HH NH(CH 65 NH(CH 3 65 H NH(CH 65 HH NH(CH HH NH(CH 656565 H NH(CH 65 NH(CH H NH(CH 333))33)) 333))65

Br NH(CH 76 Br NH(CH 76 Br 33)33)) 3)3)3) 76 BrNH(CH NH(CH 76 BrBr NH(CH 7676 NH(CH 3 Br NH(CH 76 Br NH(CH 76 Br NH(CH Br NH(CH 76 BrNH(CH NH(CH 76 Br 333))33)) 3 )33) 76 76

NH(CH NH(CH H 33)33)) 3)3)3) HNH(CH NH(CH HHH NH(CH NH(CH H NH(CH HH NH(CH H NH(CH 33) H NH(CH HNH(CH NH(CH H 333))33)) 33)

55 55 55 55 5555 55 55 55 55 55 55

Br Br Br Br BrBr Br Br Br Br Br Br

66 66 66 66 66 6666 66 66 66 66 66

H N(CH 77 N(CH 77 H 33)33)) 3)3)3) 77 H N(CH N(CH 77 HHH N(CH 7777 N(CH H N(CH H N(CH 77 H N(CH 77 H N(CH )) ) 333) 77 77 H N(CH N(CH 77 H 333))333 77

Br N(CH Br N(CH Br 33)3223))223)32)32)2 BrN(CH N(CH BrBr N(CH N(CH Br N(CH )3)222 Br N(CH Br N(CH Br N(CH BrN(CH N(CH Br 333))23322))223 332

56 56 56 56 56 5656 56 56 56 56 56

Br N(CH 67 Br N(CH 67 Br 33)3223))223)32)3)2 2 67 BrN(CH N(CH 67 Br N(CH 6767 BrBr N(CH 67 N(CH 33)22 67 Br N(CH 67 Br N(CH Br N(CH 67 BrN(CH N(CH 67 Br 333))23322))22 33)22 67

Br N(CH 78 Br N(CH 78 Br 33)3223))223))32)32)2 78 Br N(CH N(CH 78 Br N(CH BrBr N(CH 7878 N(CH 3 23 2 78 Br N(CH 78 Br N(CH 78 Br N(CH 78 Br N(CH N(CH 78 Br 333))23322))22 33)22 78

Br N(CH Br N(CH Br 33)3223))223)3)2)32)2 BrN(CH N(CH Br N(CH BrBr N(CH N(CH 3 32 2 Br N(CH Br N(CH Br N(CH BrN(CH N(CH Br 333))23322))22 33)22

2.2. Stereochemistry 2.2. Stereochemistry 2.2. Stereochemistry 2.2. Stereochemistry 2.2. Stereochemistry 2.2. Stereochemistry 2.2. Stereochemistry 2.2. Stereochemistry 2.2. Stereochemistry 2.2. Stereochemistry has previously been reported that the configuration of the N-oxime can be determined by It has previously been reported that the configuration of the N-oxime can be determined by It has previously been reported that the configuration of the N-oxime can be determined by It has previously been reported that the configuration of the N-oxime can be determined by It previously been reported that the configuration the N-oxime can determined by Ithas has previously been reported that the configuration ofof the N-oxime can bebe determined by ItIt has previously been reported that the configuration of the N-oxime can be determined by Ithas has previously been reported that the configuration of the N-oxime can be determined by It has previously been reported that the configuration of the N-oxime can be determined by It previously been reported that the configuration of the N-oxime can be determined by 13 13 13 13 13 13 13 analysis of the C NMR C-8 carbon shifts, which are known to be approximately 26–30 ppm (E analysis of the C NMR C-8 carbon shifts, which are known to be approximately 26–30 ppm (E analysis of the C NMR C-8 carbon shifts, which are known to be approximately 26–30 ppm (E 13 13 13 analysis of the C NMR C-8 carbon shifts, which are known to be approximately 26–30 ppm (E analysis of the C NMR C-8 carbon shifts, which are known to be approximately 26–30 ppm (E 13 13 13 13 13 analysis of the C NMR C-8 carbon shifts, which are known to be approximately 26–30 ppm (E analysis of the C NMR C-8 carbon shifts, which are known to be approximately 26–30 ppm (E analysis thethe NMR C-8C-8 carbon shifts, which areare known beapproximately approximately 26–30 ppm analysis of C NMR carbon shifts, which known to be approximately 26–30 ppm analysis ofofthe CCNMR C-8 carbon shifts, which are known totobe 26–30 ppm (E(E (E configuration) or over 35 ppm (Z configuration) [28]. The reported X-ray structure of a disulfideconfiguration) or over 35 ppm (Z configuration) [28]. The reported X-ray structure of a disulfideconfiguration) or over 35 ppm (Z configuration) [28]. The reported X-ray structure of a disulfideconfiguration) or over 35 ppm (Z configuration) [28]. The reported X-ray structure of a disulfideconfiguration) or over 35 ppm (Z configuration) [28]. The reported X-ray structure of a disulfideconfiguration) or over 35 ppm (Z configuration) [28]. The reported X-ray structure of a disulfideconfiguration) or over 35 ppm (Z configuration) [28]. The reported X-ray structure ofofaaof disulfideconfiguration) orover over 35ppm ppm (Zconfiguration) configuration) [28]. The reported X-ray structure adisulfidedisulfideconfiguration) or over 35 ppm (Z configuration) [28]. The reported X-ray structure a disulfideconfiguration) or 35 (Z [28]. The reported X-ray structure of bridged psammaplin analog supported this observation [29]. We, therefore, expect the bridged psammaplin analog supported this observation [29]. We, therefore, expect the bridged psammaplin AA analog supported this observation [29]. We, therefore, expect the bridged psammaplin A analog supported this observation [29]. We, therefore, expect the bridged psammaplin AAA analog supported this observation [29]. We, therefore, expect the bridged psammaplin analog supported this observation [29]. We, therefore, expect the

54

H

55

Br

Mar. Drugs 2018, 16, 481 Br 56

NH(CH3)

N(CH3)2

65

Br

NH(CH3)

76

H

NH(CH3)

66

H

N(CH3)

77

Br

N(CH3)2

67

Br

N(CH3)2

78

Br

7 of 26 N(CH3)2

2.2. Stereochemistry 2.2. Stereochemistry It has previously been reported that the configuration of the N-oxime can be determined by It has previously been reported that the configuration of the N-oxime can be determined by analysis of the 1313 C NMR C-8 carbon shifts, which are known to be approximately 26–30 ppm analysis of the C NMR C-8 carbon shifts, which are known to be approximately 26–30 ppm (E (E configuration) or over 35 ppm (Z configuration) [28]. The reported X-ray structure of a configuration) or over 35 ppm (Z configuration) [28]. The reported X-ray structure of a disulfidedisulfide-bridged psammaplin A analog supported this observation [29]. We, therefore, expect the bridged psammaplin A analog supported this observation [29]. We, therefore, expect the stereochemistry of all bromotyrosines synthesized herein to be E, since the benzylic C-8 shifts in 13 C stereochemistry of all bromotyrosines synthesized herein to be E, since the benzylic C-8 shifts in 13C NMR were around 28 ppm. This was supported by the crystal structure of pyridin-2-yl analog (36), NMR were around 28 ppm. This was supported by the crystal structure of pyridin-2-yl analog (36), which was determined by the single crystal X-ray diffraction (Figure 2; see Supplementary Information which was determined by the single crystal X-ray diffraction (Figure 2; see Supplementary for experimental details and crystallographic data). An intramolecular hydrogen bond between the Information for experimental details and crystallographic data). An intramolecular hydrogen bond secondary amide hydrogen atom H2 and the lone electron pair of oxime nitrogen atom N1 could between the secondary amide hydrogen atom H2 and the lone electron pair of oxime nitrogen atom explain the observed E geometry of the oxime. N1 could explain the observed E geometry of the oxime.

Figure 2. probability ellipsoids) of the molecular structure of (36). The Figure 2. ORTEP ORTEPrepresentation representation(50% (50% probability ellipsoids) of the molecular structure of (36). 3 molecule as a packing solvent has been omitted for clarity. CHCl The CHCl3 molecule as a packing solvent has been omitted for clarity.

2.3. Biological Activity The cytotoxicity of the synthetic purpurealidin I (1) and compounds (29–78) against cancer cells was primarily evaluated in human malignant melanoma A-375 cell line at the single concentration of 50 µM µM (Table (Table 3). The compounds demonstrating over 80% cytotoxicity were selected for confirmatory dose-response CC that caused death of dose-responseexperiments experimentsininthe thesame samecell cellline, line,and and CC (cytotoxicconcentration concentration that caused death 5050(cytotoxic 50% cells) was calculated (Table 3). We furthermore aimed to evaluate the potential of the compounds of 50% cells) was calculated (Table 3). We furthermore aimed to evaluate the potential of the to selectivelyto perturb the growth of cancer cells. of Therefore, the compounds the highest cytotoxic compounds selectively perturb the growth cancer cells. Therefore, with the compounds with the activities (CC50 below 15 µM)(CC were studied15for cytotoxicity in normal human fibroblast cell line Hs27 highest cytotoxic activities 50 below µM) were studied for cytotoxicity in normal human (Table 3). The degree of selectivity towards cancer cells can be expressed by selectivity index fibroblast cell line Hs27 (Table 3). The degree of selectivity towards cancer cells can be expressed(SI). by High valuesindex show(SI). selectivity towards cancer cells, while valuescancer pyridin-4-yl (38). Furthermore, pyridin-3-yl methyl amide (39) retained the activity. Two bromine atoms in the tyrosine part seem to be essential for the cytotoxicity since the non-halogenated (44) or mono-halogenated (42 and 45) pyridin-2-yls were inactive. However, compound (41) with two mono-brominated p-methoxyphenyl rings showed activity even though the mono-brominated (42), with pyridin-2-yl group, was inactive. This may imply a different binding mode exists for this bis mono-brominated p-methoxyphenyl compound. However, both structural data of the binding sites and the mechanism of action are currently unknown, and cytotoxicity of these compounds cannot be accounted for. The synthesis of the simplified amides with the tyramine end allowed for the more feasibly exploration of the aromatic substituents at the tyrosine part of the molecule. The CC50 (A-375 cells) values were retained with the best compounds, monomethylamino m-dichloro-p-methoxy (65) (6.4 µM) and m-iodo-p-methoxy (74) (6.2 µM). Non-halogenated amides in the tyrosine part (59) and (61) were also inactive, and o-bromo (46) or o-fluoro (57) substitution resulted in low activity. The CC50 values did not show a significant difference when compared the monomethylated amines at the end of the tyramine part to the dimethylated ones (e.g., CC50 in A-375 cells 6.2 µM for (74) and 8.4 µM for (73)). Replacement of the amino group in the tyramine end to isopropyl in compounds (49) and (52), as well as the addition of the morpholine moiety in (55) resulted in the loss of the activity. Purpurealidin I (1) and its dimethylated analog (29) showed no selectivity in cytotoxicity between melanoma A-375 cell line and normal human fibroblast cell line Hs27 (SI 1.2 and 0.7, respectively, Table 3). Changing the linker from hydroxyimino amide to amide did not improve the selectivity, and different aromatic substitution on the tyrosine fragment also had no effect (SI’s varied between 0.5–1.3) However, when the longer tyramine part was replaced with directly attached aniline, some improvement in the selectivity was observed (1 SI 1.2 compared to 33 SI 2.0 or 36 SI 4.1). The pyridin-2-yl compound 36 displayed the best, albeit only moderate, selectivity (SI 4.1, Table 3). 4. Materials and Methods 4.1. Synthesis Experimental 4.1.1. General All reactions were carried out using commercially available starting materials unless otherwise stated. The melting points were measured using a Stuart SMP40 automated melting point apparatus and are uncorrected. 1 H NMR (300 MHz) and 13 C NMR (75 MHz) spectra were measured in CDCl3 , d6 -DMSO, CD3 OD, or d6 -acetone at room temperature and were recorded on a Varian Mercury Plus 300 spectrometer or Bruker AV400 MHz NMR with smart probe. Chemical shifts (δ) are given in parts per million (ppm) relative to the 1 H and 13 C NMR reference solvent signals (CDCl3 : 7.26 and 77.16 ppm; CD3 OD: 3.31 and 49.00 ppm; d6 -DMSO: 2.50 ppm and 39.52; d6 -acetone: 2.05 and 29.84 ppm). Multiplicities are indicated by s (singlet), br s (broad singlet), d (doublet), dd (doublet of doublet), ddd (doublet of doublet of doublets), t (triplet), dt (doublet of triplets), q (quartet) and m (multiplet). The coupling constants J are quoted in Hertz (Hz). LC-MS and HRMS-spectra were recorded using a Waters Acquity UPLC® -system (Milford MA, USA) with Acquity UPLC® BEH C18

300 spectrometer or Bruker AV400 MHz NMR with smart probe. Chemical shifts (δ) are given in parts Mar. Drugs 2018, 16, x FOR PEER REVIEW1 of 25 per million (ppm) relative to the H and 13C NMR reference solvent signals (CDCl3: 7.26 and1077.16 Mar. Drugs 2018, 16, x FOR PEER REVIEW 10 of 25 ppm; CD3OD: 3.31 and 49.00 ppm; d6-DMSO: 2.50 ppm and 39.52; d6-acetone: 2.05 and 29.84 ppm). Mar. Drugs 2018, 16, FOR PEER REVIEW 10 of 25 Multiplicities arex indicated by s (singlet), br s (broad singlet), d (doublet), dd (doublet of doublet), Multiplicities are indicated by s (singlet), br s (broad singlet), d (doublet), dd (doublet of doublet), ddd (doublet of of doublets), t (triplet), dt (doublet of triplets), q (quartet) and m of (multiplet). Multiplicities aredoublet indicated by s (singlet), br s (broad singlet), d (doublet), dd (doublet doublet), ddd (doublet ofare doublet of doublets), t (triplet), dt (doublet of triplets), q (quartet) and m (multiplet). Multiplicities indicated by s (singlet), br s (broad singlet), d (doublet), dd (doublet of doublet), Mar. Drugs 2018, 16, 481 10 of 26 The aredoublets), quoted int Hertz (Hz). LC-MS and HRMS-spectra wereand recorded using a dddcoupling (doublet constants of doubletJ of (triplet), dt (doublet of triplets), q (quartet) m (multiplet). The coupling constants J are quoted in tHertz (Hz). LC-MS and HRMS-spectra wereand recorded using a ddd (doublet of doublet of doublets), (triplet), dt (doublet of triplets), q (quartet) m (multiplet). ® ® Waters Acquity UPLC® -system (Milford MA,(Hz). USA)LC-MS with Acquity UPLC® BEHwere C18 recorded column (1.7 µm,a The coupling constants J are quoted in Hertz and HRMS-spectra using Waters Acquity UPLC -system (Milford MA, (Hz). USA)LC-MS with Acquity UPLC BEH were C18 column (1.7 µ m,a The constants J are quoted in Hertz and recorded using ®-system ® BEH C18 50 × coupling 2.1 Acquity mm, Waters, Wexford, Ireland) with Synapt G2HRMS-spectra HDMS MA, USA) with the Waters UPLC (Milford MA,Waters USA) with Acquity UPLC(Milford column (1.7 µm, 50 × 2.1 mm, Waters, Wexford, Ireland) with Waters Synapt G2 HDMS (Milford MA, USA) with the column (1.7 µm, 50 × 2.1 mm, Waters, Wexford, Ireland) with Waters Synapt G2 HDMS (Milford MA, ® ® Waters Acquity UPLC -system (Milford MA, USA) with Acquity UPLC BEH C18 column (1.7 µm, ESI high Waters, resolution mode. Ireland) The mobile ofHDMS H2O (A) and acetonitrile 50 × (+), 2.1 mm, Wexford, with phase Watersconsisted Synapt G2 (Milford MA, USA) (B) withboth the ESI (+), high resolution mode. The mobile phase consisted ofconsisted H2O (A) acetonitrile (B) both USA) with theWaters, ESI (+),Wexford, high resolution mode. The mobile phase ofand H2 O (A) acetonitrile 50 2.1 mm, Ireland) with Waters Synapt G2 (Milford MA,and USA) with the containing 0.1% HCOOH. Microwave synthesis were performed sealed using (B) Biotage ESI× (+), high resolution mode. The mobile phase consisted ofHDMS H2Oin(A) and tubes acetonitrile both containing 0.1% HCOOH. Microwave synthesis were performed in (A) sealed Biotage (B) both 0.1% HCOOH. Microwave synthesis were performed in sealed tubesusing using(B) Biotage ESI (+), containing high resolution mode. The mobile phase consisted H2chromatography O and tubes acetonitrile both Initiator+ instrument equipped with an external IR sensor. The of flash was performed containing 0.1% HCOOH. Microwave synthesis were performed in sealed tubes using Biotage Initiator+ instrument equipped with an external IR sensor. The flash chromatography was performed Initiator+ instrument equipped with an external IR sensor. The flash chromatography was performed containing 0.1% HCOOH. Microwave synthesis were performed in sealed tubes using Biotage with Biotage SP1 flash chromatography purification system nm UV-detector Biotage Initiator+ instrument equipped with an external IR sensor. The with flash 254 chromatography was or performed with Biotage SP1 chromatography purification system with 254 or Biotage with Biotage SP1 flash flash chromatography purification system with 254 nm nm UV-detector UV-detector or Biotage Initiator+ instrument equipped with an external IR sensor. The flash chromatography was performed Isolera™ Spektra Systems with 200–800 nm UV-detector using SNAP 10, 25, 50 or 100 g cartridges with Biotage SP1 flash chromatography purification system with 254 nm UV-detector or Biotage Isolera™ Spektra with UV-detector using SNAP 25, 50 Isolera™ Spektra Systems with200–800 200–800nm nm UV-detector usingwith SNAP 10, 25,UV-detector 50 or or 100 100 gg cartridges cartridges with Biotage SP1Systems flash purification 25410, nm Biotage (Uppsala, Sweden). Thechromatography TLC-plates werenm provided bysystem Merck Germany, gel 60Isolera™ Spektra Systems with 200–800 UV-detector using(Darmstadt, SNAP 10, 25, 50 or 100Silica g or cartridges (Uppsala, The TLC-plates were provided by Merck (Darmstadt, Germany, Silica gel 60(Uppsala, Sweden). The TLC-plates were provided by Merck (Darmstadt, Germany, Silica gel 60-F254) Isolera™ Spektra Systems with 200–800 nm UV-detector using SNAP 10, 25, 50 or 100 g cartridges F254) and visualization the amine compounds was by done using(Darmstadt, ninhydrin staining andSilica THPgel ethers (Uppsala, Sweden). TheofTLC-plates were provided Merck Germany, 60F254) and visualization ofamine the amine compounds was done using ninhydrin staining andSilica THP ethers and visualization of the compounds was done using ninhydrin staining and THP ethers with (Uppsala, Sweden). The TLC-plates were provided by Merck (Darmstadt, Germany, gel 60with vanillin staining. F254) and visualization of the amine compounds was done using ninhydrin staining and THP ethers with vanillin staining. of the amine compounds was done using ninhydrin staining and THP ethers vanillin staining. F254) and visualization with vanillin staining. with vanillin staining. 4.1.2. Experimental Procedures 4.1.2. Experimental ExperimentalProcedures Procedures 4.1.2. 4.1.2. Experimental Procedures 4.1.2. Experimental Procedures General Procedure for for the Formation Formation of of Azlactones Azlactones General Procedure the General Procedure for the Formation of Azlactones General Procedure for acetylglycine the Formation(1.5 of Azlactones Aldehyde 19a–d, equiv.) and and anhyd. anhyd. NaOAc (1.5 equiv.) were were dissolved dissolved in in Aldehyde 19a–d, acetylglycine (1.5 equiv.) NaOAc(1.5 (1.5 equiv.) equiv.) General Procedure foracetylglycine the Formation(1.5 of Azlactones Aldehyde 19a–d, equiv.) and anhyd. NaOAc were dissolved in ◦ Ac 2 O (10–15 mL) and the reaction mixture was stirred at 80 °C overnight. Afterwards, the reaction 19a–d, acetylglycine (1.5 equiv.) and anhyd. NaOAc (1.5 equiv.) were dissolved in Ac22OOAldehyde (10–15mL) mL) andthe the reaction mixture mixture was stirred stirred at 80 80 °C C overnight. overnight. Afterwards, the reaction reaction Ac (10–15 and reaction was at Afterwards, the 19a–d, acetylglycine (1.5 and equiv.) and into anhyd. NaOAc (1.5The equiv.) were dissolved in mixture was cooled cooled to room room temperature poured water (50 mL). formed precipitate was Ac2OAldehyde (10–15 mL) and the reaction mixture was stirred atwater 80 °C overnight. Afterwards, the reaction mixture was to temperature and poured into (50 mL). The formed precipitate was mixture was cooled to room temperature and poured into water (50 mL). The formed precipitate was Ac 2O (10–15 mL) and the reaction mixture was stirred at 80 °C overnight. Afterwards, the reaction filtered, washed with water (4 × 20 mL) and in vacuo. The obtained crude product 20a–d was was mixture was cooled towater room (4 temperature anddried poured into water mL). The formed precipitate filtered, washed with 20mL) mL)and and dried invacuo. vacuo. The(50 obtained crude product 20a–d filtered, washed with water (4 ××20 dried in The obtained crude product 20a–d was mixture was cooled to room temperature and poured into water (50 mL). The formed precipitate used in the subsequent step without further purification. filtered, washed with water (4 × 20 mL) andpurification. dried in vacuo. The obtained crude product 20a–d was was used in the the subsequent step without without further used in subsequent step further purification. filtered, washed with water (4 × 20 mL) and purification. dried in vacuo. The obtained crude product 20a–d was used in the subsequent step without further used in the subsequent step without further purification.

4-(3,5-Dibromo-4-methoxybenzylidene)-2-methyloxazol-5(4H)-one (20a). 4-(3,5-Dibromo-4-methoxybenzylidene)-2-methyloxazol-5(4H)-one (20a). 4-(3,5-Dibromo-4-methoxybenzylidene)-2-methyloxazol-5(4H)-one 3,5-Dibromo-4-methoxybenzaldehyde 19a (1.57 g, 5.33 mmol)(20a). gave 20a as a grey solid (1.94 g, 97%). 4-(3,5-Dibromo-4-methoxybenzylidene)-2-methyloxazol-5(4H)-one (20a). 3,5-Dibromo-4-methoxybenzaldehyde 19a (1.57 g, 5.33 mmol) gave 20a as a grey solid1(1.94 g, 97%). 1H 4-(3,5-Dibromo-4-methoxybenzylidene)-2-methyloxazol-5(4H)-one (20a). 1H NMR (400 MHz, CDCl3) δ 8.26 (s, 3,5-Dibromo-4-methoxybenzaldehyde 19a (1.57 g, 5.33 mmol) gave as aas grey solid (1.94 97%). 2H), 6.92 (s, 1H), 3.93 (s,20a 3H), 2.43 (s, 3H). H g, NMR is in 3,5-Dibromo-4-methoxybenzaldehyde 19a (1.57 g, 5.33 mmol) gave 20a a grey solid g, 97%). 1H 1H(1.94 NMR (400 MHz, CDCl 3) δ 8.26 (s, 2H), 6.92 (s, 1H), 3.93 (s, 3H), 2.43 (s, 3H). NMR is in 1 3,5-Dibromo-4-methoxybenzaldehyde 19a (1.57 g, (s, 5.331H), mmol) 20a as aH grey solid (1.94 g, 97%). NMR (400 (400 MHz, CDCl (s,8.26 2H),(s,6.92 (s, 6.92 1H), 3.93 (s, 3H), 2.43 3H). NMR is1H in accordance 1H NMR accordance withMHz, the literature [26]. 3 ) δ 8.26 CDCl 3) δ 2H), 3.93gave (s, (s, 3H), 2.43 (s, 3H). NMR is in accordance withMHz, the literature [26]. 1H NMR (400 1 CDCl3) δ[26]. 8.26 (s, 2H), 6.92 (s, 1H), 3.93 (s, 3H), 2.43 (s, 3H). H NMR is in with the literature [26]. accordance with the literature accordance with the literature [26].

4-(3-Bromo-4-methoxybenzylidene)-2-methyloxazol-5(4H)-one (20b). 4-(3-Bromo-4-methoxybenzylidene)-2-methyloxazol-5(4H)-one (20b). 3-Bromo-4-methoxybenzaldehyde 19b (2.00 g, 9.30 mmol) gave 4-(3-Bromo-4-methoxybenzylidene)-2-methyloxazol-5(4H)-one (20b).20b as a yellow solid (2.70 g, 98%)11H 4-(3-Bromo-4-methoxybenzylidene)-2-methyloxazol-5(4H)-one (20b). 3-Bromo-4-methoxybenzaldehyde 19b (2.00 g, 9.30 mmol) gave 20b as a yellow solid (2.70 g, 98%) H 4-(3-Bromo-4-methoxybenzylidene)-2-methyloxazol-5(4H)-one (20b). NMR (400 MHz, CDCl3) δ 8.43 (d, J19b = 2.1 Hz,g,1H), 7.98–7.94 (m,20b 1H),as7.02 (s, 1H), 6.95 (d, Jg,=98%) 8.6 Hz, H 3-Bromo-4-methoxybenzaldehyde (2.00 9.30mmol) mmol)gave gave yellow solid (2.70 11H 3-Bromo-4-methoxybenzaldehyde 19b (2.00 g, 9.30 20b aaayellow solid NMR (400 MHz, CDCl 3) δ 8.43 (d, J = 2.1 Hz, 1H), 7.98–7.94 (m, 1H),as 7.02 (s, 1H), 6.95(2.70 (d, Jg, =98%) 8.6 Hz, 1H 3-Bromo-4-methoxybenzaldehyde 19b (2.00 g, 9.30 mmol) gave 20b as yellow solid (2.70 g, 98%) 1 1H), 3.96 (s,MHz, 3H), 2.41 (d, 0.7 Hz, 3H). H NMR is in accordance with the literature [25]. NMR (400 CDCl 3) Jδ=8.43 (d, J = 2.1 Hz, 1H), 7.98–7.94 (m, 1H), 7.02 (s, 1H), 6.95 (d, J = 8.6 Hz, 1H NMR (400 CDCl (d, = 2.1 Hz, 1H), (m, 1H), 1H), 7.02 1H), (d, 8.6 Hz, 1H), 3.96 (s,MHz, 3H), 2.41 (d,33))J δδ= 8.43 0.7 Hz, NMR in accordance with7.02 the (s, literature [25]. NMR (400 MHz, CDCl 8.43 (d, J3H). J3H). = 2.1 Hz, 1H),isis7.98–7.94 7.98–7.94 (m, (s, 1H), 6.95 6.95 (d, JJ = = 8.6 Hz, 1H 1H), 3.96 (s, 3H), 2.41 (d, J = 0.7 Hz, NMR in accordance with the literature [25]. 1 1H), 3.96 (s, 3H), 2.41 (d, J = 0.7 Hz, 3H). H NMR is in accordance with the literature [25]. 1 1H), 3.96 (s, 3H), 2.41 (d, J = 0.7 Hz, 3H). H NMR is in accordance with the literature [25].

4-(3-Chloro-4-methoxybenzylidene)-2-methyloxazol-5(4H)-one (20c). 3-Chloro-4-methoxybenzaldehyde 19c (2.43 g, 14.3 mmol) gave 4-(3-Chloro-4-methoxybenzylidene)-2-methyloxazol-5(4H)-one (20c).20c as a yellow solid (2.66 g, 74%). 1H 4-(3-Chloro-4-methoxybenzylidene)-2-methyloxazol-5(4H)-one (20c). 4-(3-Chloro-4-methoxybenzylidene)-2-methyloxazol-5(4H)-one (20c). NMR (400 MHz, CDCl3) δ 8.28 (d, J19c = 2.1 Hz,g,1H), (dd,gave J = 8.6, 1H), solid 7.02 (s, 1H), (d,1HJ 3-Chloro-4-methoxybenzaldehyde (2.43 14.37.89 mmol) 20c2.1 as aHz, yellow (2.66 g, 6.98 74%). 11H 3-Chloro-4-methoxybenzaldehyde 19c (2.43 g, 14.3 mmol) gave 20c as a yellow solid (2.66 g, 74%). 3-Chloro-4-methoxybenzaldehyde 19c (2.43 g, 1H), 14.3 mmol) gave yellow g, 74%). =NMR 8.6 Hz, 1H), 3.97CDCl (s, 3H), (400 MHz, 3) δ 2.41 8.28 (s, (d,3H). J = 2.1 Hz, 7.89 (dd, J = 20c 8.6, as 2.1aHz, 1H),solid 7.02 (2.66 (s, 1H), 6.98 (d,H J NMR (400 MHz, CDCl 3)) δδ8.28 8.28 (d, J = 2.1 Hz, 1H), 7.89 (dd, J = 8.6, 2.1 Hz, 1H), 7.02 (s, 1H), 6.98 (d, J NMR (400 MHz, CDCl (d, J = 2.1 Hz, 1H), 7.89 (dd, J = 8.6, 2.1 Hz, 1H), 7.02 (s, 1H), 6.98 (d, J = 3 = 8.6 Hz, 1H), 3.97 (s, 3H), 2.41 (s, 3H). = 8.6 Hz, 1H), 3.97 3H), 2.41 3H). 8.6 Hz, 1H), 3.97 (s,(s, 3H), 2.41 (s,(s, 3H). 4-(4-Methoxybenzylidene)-2-methyloxazol-5(4H)-one (20d). 4-Methoxybenzaldehyde 19d (3.03 g, 22.3 mmol) gave 4-(4-Methoxybenzylidene)-2-methyloxazol-5(4H)-one (20d).20d as a yellow solid (2.21 g, 46%). 1H NMR 4-(4-Methoxybenzylidene)-2-methyloxazol-5(4H)-one (20d). (400 MHz, CDCl3) δ 8.06 (d, 8.5 Hz, 2H),mmol) 7.11 (s, 1H),20d 6.96as(d, J = 8.9 Hz, 3.87 3H),1H2.39 (s, NMR 4-Methoxybenzaldehyde 19dJ =(3.03 g, 22.3 gave a yellow solid2H), (2.21 g, (s, 46%). 4-(4-Methoxybenzylidene)-2-methyloxazol-5(4H)-one (20d). 1H NMR 4-Methoxybenzaldehyde 19d (3.03 g, 22.3 mmol) gave 20d as a yellow solid (2.21 g, 46%). 3H). (400 MHz, CDCl3) δ 8.06 (d, = 8.5 g, Hz, 2H), 7.11 (s, 1H), 6.96 (d, J = 8.9 Hz, 2H), 3.87 (s, 3H), (s, 1 2.39 4-Methoxybenzaldehyde 19dJJ (3.03 22.3 mmol) gave 20d as (d, a yellow (2.213.87 g, 46%). NMR (400 MHz, CDCl3) δ 8.06 (d, = 8.5 Hz, 2H), 7.11 (s, 1H), 6.96 J = 8.9 solid Hz, 2H), (s, 3H),H2.39 (s, 3H). (400 3H). MHz, CDCl3 ) δ 8.06 (d, J = 8.5 Hz, 2H), 7.11 (s, 1H), 6.96 (d, J = 8.9 Hz, 2H), 3.87 (s, 3H), 2.39 (s, 3H).

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General Method Method for for the the Hydrolysis Hydrolysis of of Azlactones Azlactones General General Method for the Hydrolysis of Azlactones General Method for the Hydrolysis of Azlactones Azlactone 20a–d 20a–d was was dissolved dissolved in in aa 10% 10% solution solution of of HCl HCl in in H H22O O (30 (30 mL). mL). A A capillary capillary tube tube was was Azlactone Azlactone 20a–d was dissolved in a 10% solution of HCl in H 22O (30 mL). A capillary tube was Azlactone 20a–d was dissolved in a 10% solution of HCl in H 2 O (30 mL). A capillary tube was introduced in the flask to allow the reflux despite a solid layer forming while heating. The reaction introduced in in the the flask flask to to allow allow the the reflux reflux despite despite aa solid solid layer layer forming forming while while heating. heating. The The reaction reaction introduced solid layer forming introduced the flaskovernight. to allow the despite a solid forming while heating. The mixture was wasinrefluxed refluxed overnight. Thereflux reaction mixture waslayer cooled to room room temperature andreaction poured mixture The reaction mixture was cooled to temperature and poured mixture was refluxed overnight. The reaction mixture was cooled to room temperature and poured mixture was refluxed overnight. The reaction mixture was cooled to room temperature and poured into cold water (2 × 10 mL). The resulting precipitate was filtered, washed with water (4 × 20 mL) and into cold cold water (2 1010mL). mL). The resulting precipitate was filtered, washed with water (4 ××(4 20×mL) mL) and into The resulting precipitate was filtered, washed with water (4 20 and cold water water(2 (2×××10 mL). The resulting precipitate was filtered, washed with water 20 mL) into cold water (2 × 10 mL). The resulting precipitate was filtered, washed with water (4 × 20 mL) and dried in in vacuo. vacuo. The The obtained obtained crude crude product product 21a–d 21a–d was was used used in in the the subsequent subsequent step step without without further further dried dried in vacuo. The obtained crude crude product 21a–d 21a–d was used the subsequent step without further and dried in vacuo. The obtained product wasinused in the subsequent step without dried in vacuo. The obtained crude product 21a–d was used in the subsequent step without further purification. purification. purification. further purification. purification.

3-(3,5-Dibromo-4-methoxyphenyl)-2-oxopropanoic acid acid (21a) (21a) 3-(3,5-Dibromo-4-methoxyphenyl)-2-oxopropanoic 3-(3,5-Dibromo-4-methoxyphenyl)-2-oxopropanoic acid (21a) (21a) 3-(3,5-Dibromo-4-methoxyphenyl)-2-oxopropanoic acid 3-(3,5-Dibromo-4-methoxyphenyl)-2-oxopropanoic acid (21a) Azlactone 20a 20a (1.74 (1.74 g, g, 4.63 4.63 mmol) mmol) gave gave acid acid 21a 21a as as aa yellowish yellowish solid solid (1.46 (1.46 g, g, 89%). 89%). 11H H NMR NMR (400 (400 Azlactone Azlactone 20a (1.74 g, g, 4.63 4.63 mmol) mmol) gave gave acid acid 21a 21a as as a13 a yellowish yellowish solid solid (1.46 (1.46 g, g, 89%). 89%). 111H NMR (400 Azlactone 20a (1.74 H165.7, NMR151.8, (400 Azlactone 20a (1.74 g, 4.63 mmol) gave acid 21a as a yellowish solid (1.46 g, 89%). H NMR (400 13 MHz, d 6 -DMSO) δ 8.06 (s, 2H), 3.80 (s, 3H), 3.32 (s, 2H); C NMR (101 MHz, d 6 -DMSO) δ MHz, dd66-DMSO) -DMSO) δδ 8.06 8.06 (s, (s, 2H), 2H), 3.80 3.80 (s, (s, 3H), 3H), 3.32 3.32 (s, 2H); 2H); 13 C NMR NMR (101 (101 MHz, MHz, dd66-DMSO) -DMSO) δδ 165.7, 165.7, 151.8, 151.8, 13C MHz, MHz, -DMSO) 8.06 (s, (s,106.1, 2H), 3.80 3.80 (s, 3H),showed 3.32 (s, (s, C NMR NMR (101 MHz, -DMSO) 165.7, 151.8, 151.8, 13C 66-DMSO) MHz, δδ 8.06 2H), 3H), 3.32 (s, 2H); 2H); (101 dd66-DMSO) δδ 165.7, 11MHz, 143.3, dd134.5, 134.5, 132.9, 117.3, 60.5.(s, NMR the enol enol tautomer. H NMR NMR is in in accordance accordance with 143.3, 132.9, 117.3, 106.1, 60.5. NMR showed the tautomer. H is with 1 143.3, 134.5, 134.5, 132.9, 132.9, 117.3, 117.3, 106.1, 106.1, 60.5. 60.5. NMR showed the the enol enol tautomer. tautomer. 11H NMR is in accordance with 143.3, NMR showed H NMR is in accordance with 143.3, 134.5, 132.9, the literature literature [25]. 117.3, 106.1, 60.5. NMR showed the enol tautomer. H NMR is in accordance with the [25]. the the literature literature [25]. [25]. the literature [25].

3-(3-Bromo-4-methoxyphenyl)-2-oxopropanoic acid acid (21b) (21b) 3-(3-Bromo-4-methoxyphenyl)-2-oxopropanoic 3-(3-Bromo-4-methoxyphenyl)-2-oxopropanoic acid (21b) (21b) 3-(3-Bromo-4-methoxyphenyl)-2-oxopropanoic acid 3-(3-Bromo-4-methoxyphenyl)-2-oxopropanoic acid (21b) Azlactone 20b 20b (3.33 (3.33 g, g, 9.11 9.11 mmol) mmol) gave gave 21b 21b as as aa dark dark red red solid solid (1.89 (1.89 g, g, 79%). 79%). 1111H H NMR NMR (400 (400 MHz, MHz, Azlactone Azlactone 20b (3.33 g, 9.11 mmol) gave 21b as a dark red solid (1.89 g, 79%). H H NMR NMR (400 (400 MHz, MHz, 1H Azlactone 20b (3.33 g, 9.11 mmol) gave 21b as a dark red solid (1.89 g, 79%). NMR (400 MHz, d 6 -DMSO) δ 9.26 (bs, 1H), 8.10 (d, J = 2.1 Hz, 1H), 7.69–7.64 (m, 1H), 7.10 (d, J = 8.7 Hz, 1H), 6.35 (s, -DMSO) δδ 9.26 9.26 (bs, 1H), 1H), 8.10 (d, (d, J = 2.1 Hz, Hz, 1H), 7.69–7.64 7.69–7.64 (m, 1H), 1H), 7.10 (d, (d, J = 8.7 Hz, Hz, 1H), 6.35 6.35 (s, dd666-DMSO) -DMSO) δ 9.26 (bs, (bs, 1H), 8.10 8.10 (d, J == 2.1 2.1 Hz, 1H), 1H), 17.69–7.64 (m, (m, 1H), 7.10 7.10 (d, JJ == 8.7 8.7 Hz, 1H), 1H), 6.35 (s, (s, d1H), 6-DMSO) δ 9.26 (bs, 1H), 8.10 (d, J = 2.1 Hz, 1H), 7.69–7.64 (m, 1H), 7.10 (d, J = 8.7 Hz, 1H), 6.35 (s, 1 1H), 3.85 (s, 3H). NMR showed the enol tautomer. H NMR is in accordance with the literature [25]. 3.85 (s, 3H). NMR showed the enol tautomer. tautomer. 11H H NMR NMR is in accordance with the literature [25]. [25]. 1H), 3.85 (s, 3H). NMR showed the enol tautomer. is in accordance with the literature [25]. 1H), 3.85 (s, 3H). NMR showed the enol tautomer. 1H NMR is in accordance with the literature [25].

3-(3-Chloro-4-methoxyphenyl)-2-oxopropanoic acid acid (21c) (21c) 3-(3-Chloro-4-methoxyphenyl)-2-oxopropanoic 3-(3-Chloro-4-methoxyphenyl)-2-oxopropanoic acid (21c) 3-(3-Chloro-4-methoxyphenyl)-2-oxopropanoic acid (21c) Azlactone 20c 20c (2.66 (2.66 g, g, 10.6 10.6 mmol) mmol) gave gaveacid 21c(21c) as aa brown brown solid solid (1.92 (1.92 g, g, 79%). 79%). 11H H NMR NMR (400 (400 MHz, MHz, Azlactone 21c as 3-(3-Chloro-4-methoxyphenyl)-2-oxopropanoic Azlactone 20c (2.66 g, 10.6 mmol) gave 21c as a brown solid (1.92 g, 79%). 11H NMR (400 MHz, Azlactone 20c (2.66 g, 10.6 mmol) gave 21c as a brown solid (1.92 g, 79%). H NMR (400 MHz, CD 3 OD) δ 7.91 (d, J = 2.1 Hz, 1H), 7.59 (ddd, J = 8.6, 2.1, 0.5 Hz, 1H), 7.03 (d, J = 8.7 Hz, 1H), 6.40 (s, CD33OD) OD) 7.91 (d, (d, 2.1 g, Hz, 1H), 7.59 (ddd, (ddd, 8.6, 2.1, 0.5 Hz, Hz, 1H), 7.03 (d, JJ == 18.7 8.7 Hz, 1H), 1H), 6.40 (s, Azlactone 20cJJ(2.66 10.6 mmol) gave 21c as a2.1, brown solid1H), (1.92 g, 79%). H NMR (4006.40 MHz, CD δδ 7.91 == 2.1 Hz, 1H), 7.59 JJ == 8.6, 0.5 7.03 (d, Hz, (s, CD (d,NMR J = 2.1 Hz, 1H), J = 8.6, 2.1, 0.5 Hz, 1H), 7.03 (d, J = 8.7 Hz, 1H), 6.40 (s, 1H),3OD) 3.89δδ(s, (s,7.91 3H). NMR showed the7.59 enol(ddd, tautomer. 1H), 3.89 3H). showed the enol tautomer. CD (d, J = 2.1 Hz, 1H), 7.59 (ddd, J = 8.6, 2.1, 0.5 Hz, 1H), 7.03 (d, J = 8.7 Hz, 1H), 6.40 (s, 3 OD) 1H), 3.89 (s,7.91 3H). NMR showed the enol tautomer. 1H), 3.89 (s, 3H). NMR showed the enol tautomer. 1H), 3.89 (s, 3H). NMR showed the enol tautomer.

3-(4-Methoxyphenyl)-2-oxopropanoic acid acid (21d) (21d) 3-(4-Methoxyphenyl)-2-oxopropanoic 3-(4-Methoxyphenyl)-2-oxopropanoic acid (21d) 3-(4-Methoxyphenyl)-2-oxopropanoic acid (21d) Azlactone 20d 20d (2.21 (2.21 g, g, 10.2 10.2 mmol) mmol) gave gave product product 21d 21d as as a brown brown solid solid (1.78 (1.78 g, g, 90%). 90%). 111H H NMR NMR (400 (400 Azlactone 3-(4-Methoxyphenyl)-2-oxopropanoic acid gave (21d)product 21d Azlactone 20d (2.21 g, 10.2 mmol) as aa brown solid (1.78 g, 90%). H NMR (400 1H NMR (400 10.2 mmol) product a brown g, MHz,Azlactone CDCl33)) δδ 20d 7.75(2.21 (d, JJ ==g,8.7 8.7 Hz, 2H), gave 6.92 (d, (d, J == 8.9 8.9 21d Hz, as 2H), 6.64 (s, (s,solid 1H),(1.78 3.84 (s, (s,90%). 3H). NMR NMR showed 1 MHz, CDCl 7.75 (d, Hz, 2H), 6.92 J Hz, 2H), 6.64 1H), 3.84 3H). showed 10.2 mmol) product a brown H NMR (400 MHz,Azlactone CDCl3) δ 20d 7.75(2.21 (d, J =g,8.7 Hz, 2H), gave 6.92 (d, J = 8.921d Hz, as 2H), 6.64 (s,solid 1H),(1.78 3.84 g, (s,90%). 3H). NMR showed MHz, CDCl 3) δ 7.7511H (d,NMR J = 8.7is 2H), 6.92 (d, J = 8.9 2H), 6.64 (s, 1H), 3.84 (s, 3H). NMR showed the enol enol tautomer. H NMR isHz, in accordance accordance with theHz, literature [31]. the tautomer. in with the literature [31]. 1 MHz, CDCl (d,NMR J = 8.7 2H), 6.92 (d, J = the 8.9 Hz, 2H), 6.64 the enol tautomer. is Hz, in accordance with literature [31].(s, 1H), 3.84 (s, 3H). NMR showed 3 ) δ 7.75H the enol tautomer. 11H NMR is in accordance with the literature [31]. the enol tautomer. H NMR is in accordance with the literature [31]. General Procedure Procedure for for THP-Protection THP-Protection General General Procedure for THP-Protection General Procedure for THP-Protection General Procedure for21a–d THP-Protection Carboxylic acid 21a–d and THPONH THPONH2 (2 (2 equiv.) equiv.) were were dissolved dissolved in in dry dry ethanol ethanol (15–20 (15–20 mL). mL). The The Carboxylic acid and Carboxylic acid 21a–d and THPONH22 (2 equiv.) were dissolved in dry ethanol (15–20 mL). The Carboxylic acid 21a–d and THPONH 2 (2 equiv.) were dissolved in dry ethanol (15–20 mL). The reaction mixture was stirred at room temperature for 18–48 h under argon atmosphere. The reaction reaction mixture was stirred at room temperature for 18–48 h under argon atmosphere. The reaction Carboxylic THPONH were dissolved in dryatmosphere. ethanol (15–20 The 2 (2 equiv.) reaction mixtureacid was21a–d stirredand at room temperature for 18–48 h under argon ThemL). reaction reaction mixture was stirred stirred at room room temperature for 18–48 h under under(20 argon atmosphere. mixture was was concentrated under reduced pressurefor and18–48 then EtOAc EtOAc (20 mL) atmosphere. was added added to toThe the reaction residue. mixture concentrated under reduced pressure and then mL) was the residue. reaction mixture was at temperature h argon The reaction mixture was concentrated under reduced pressure and then EtOAc (20 mL) was added to the residue. mixture was concentrated under reduced and then mL) was added to residue. The organic organic layer was washed washed with a2M Mpressure solution and of HCl HCl inEtOAc H22O O (2 (2(20 20 mL). The aqueous layer was The layer was with solution of in H ×× 20 mL). The aqueous layer was mixture waslayer concentrated under reduced pressure then EtOAc (20 mL) was added to the the residue. The organic was washed with aa 22 M solution of HCl in H 2O (2 × 20 mL). The aqueous layer was The organic layer was washed with a 2 M solution of HCl in H 2 O (2 × 20 mL). The aqueous layer was extracted back with EtOAc (2 × 10 mL). The combined organic layers were dried over Na 2 SO 4 , filtered extracted back withwas EtOAc (2 ×× 10 10 mL). The combined organic layers were dried over Na 2SO4, filtered The organic layer washed with a 2The M combined solution oforganic HCl inlayers H2 O (2 × 20 mL). TheNa aqueous layer extracted back with EtOAc (2 mL). were dried over 2SO4, filtered extracted back with EtOAc (2 × 10 mL). The combined organic layers were dried over Na2SO4, filtered

Mar. Drugs 2018, 16, 481

Mar. Drugs 2018, 16, x FOR PEER REVIEW Mar. Drugs 2018, 16, x FOR PEER REVIEW Mar. Drugs 2018, 16, x FOR PEER REVIEW was extracted back with EtOAc (2 × Mar. Drugs 2018, 16, x FOR PEER REVIEW

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12 of 25 12 of 25

12 SO of 25, 10 mL). The combined organic layers were dried over Na 2 4 12 of 25 and concentrated in vacuo. The obtained crude product 22a–d 22a–d was used in theinsubsequent step filtered and concentrated in vacuo. The obtained crude product was used the subsequent and concentrated in vacuo. The Mar. 2018, 16, x FOR PEER REVIEW 12 step of 25 and Drugs concentrated inpurification. vacuo. The obtained obtained crude crude product product 22a–d 22a–d was was used used in in the the subsequent subsequent step without further purification. step without further without further purification. and concentrated in vacuo. The obtained crude product 22a–d was used in the subsequent step without further purification. and concentrated in vacuo. The obtained crude product 22a–d was used in the subsequent step without further purification. without further purification.

(E)-3-(3,5-Dibromo-4-methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanoic acid (22a) (E)-3-(3,5-Dibromo-4-methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanoic acid (22a) (E)-3-(3,5-Dibromo-4-methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanoic acid (E)-3-(3,5-Dibromo-4-methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanoic acid(22a) (22a) Carboxylic acid 21a (1.16 g, 3.30 mmol) and THPONH2 (0.97 g, 8.3 mmol, 2 equiv.) were used. (E)-3-(3,5-Dibromo-4-methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanoic acid (22a) Carboxylic acid 21a (1.16 g, 3.30 mmol) and THPONH 2 (0.97 g, 8.3 mmol, 2 equiv.) were used. Carboxylic acid 21a (1.16 g, 3.30 mmol) and THPONH (0.97 g, 8.3 mmol, 2 equiv.) were used. 2 Carboxylic 21a (1.16 g, 3.30chromatography, mmol) and THPONH 2 (0.97 g, 8.3 mmol, KP-Sil 2 equiv.) were used. The product wasacid purified by column Biotage SNAP Cartridge 25 g, gradient (E)-3-(3,5-Dibromo-4-methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanoic acid (22a) The product was purified by column chromatography, Biotage SNAP Cartridge KP-Sil 25 g, gradient Carboxylic acid 21a (1.16 g, 3.30 mmol) and THPONH 2 (0.97 g, 8.3 mmol, 2 equiv.) were used. The product was purified by column chromatography, Biotage SNAP Cartridge KP-Sil 25 g, gradient 1H NMR The product was purified by column chromatography, Cartridge KP-Sil 25 g, gradient elution: (DCM/MeOH, 0→10%) to give 22a as a yellow Biotage oil (1.44SNAP g, 97%). (400 MHz, CDCl3) 11H NMR (400 MHz, CDCl3) elution: (DCM/MeOH, 0→10%) give 22a as aaa yellow oil (1.44 g, 97%). Carboxylic 21a (1.16 g,to 3.30 mmol) and THPONH 2 (0.97 g, 8.3 mmol, 2KP-Sil equiv.) were used. The product wasacid purified by10%) column chromatography, Biotage SNAP Cartridge 25 g, gradient 1H elution: (DCM/MeOH, 0 → to give 22a as yellow oil (1.44 g, 97%). H NMR (400 MHz, CDCl 33)) elution: (DCM/MeOH, 0→10%) to give 22a as yellow oil (1.44 g, 97%). NMR (400 MHz, CDCl δ 7.48 (s, 2H), 5.50–5.41 (m, 1H), 3.95–3.88 (m, 2H), 3.84 (s, 3H), 3.69–3.54 1(m, 2H), 1.93–1.83 (m, 2H), δelution: 7.48 (s, 2H), 5.50–5.41 (m, 1H), 3.95–3.88 (m, 2H), 3.84 (s, 3H), 3.69–3.54 (m, 2H), 1.93–1.83 (m, 2H), The product was purified by column chromatography, Biotage SNAP Cartridge KP-Sil 25 g, gradient (DCM/MeOH, 0→10%) to give 22a as a yellow oil (1.44 g, 97%). H NMR (400 MHz, CDCl 3) δ 7.48 (s, 2H), 5.50–5.41 (m, 1H), 3.95–3.88 (m, 2H), 3.84 (s, 3H), 3.69–3.54 (m, 2H), 1.93–1.83 (m, 2H), 13C NMR δ 7.48 (s, 2H), 5.50–5.41 (m, 1H), 3.95–3.88 (m, 2H), (s, 3H), (m, 2H),153.2, 1.93–1.83 (m,133.6, 2H), 1.77–1.65 (m, 2H), 1.64–1.58 (m, 2H). (1013.84 MHz, CDCl3.69–3.54 3) δ 163.7, 163.3, 149.9, 13C NMR (101 MHz, CDCl3) δ 163.7, 1(m, 13 1.77–1.65 (m, 2H), 1.64–1.58 (m, 2H). 163.3, 153.2, 149.9, 133.6, (DCM/MeOH, 0→10%) to give 22a as a yellow oil (1.44 g, 97%). H NMR (400 MHz, CDCl 3) δelution: 7.48 (s, 2H), 5.50–5.41 (m, 1H), 3.95–3.88 (m, 2H), 3.84 (s, 3H), 3.69–3.54 2H), 1.93–1.83 (m, 2H), 13 1.77–1.65 (m, 2H), 1.64–1.58 (m, 2H). C NMR (101 MHz, CDCl ) δ 163.7, 163.3, 153.2, 149.9, 133.6, 1.77–1.65 (m,62.4, 2H),60.8, 1.64–1.58 (m, 24.9, 2H). 18.6. C NMR (101 MHz, CDCl33) δ 163.7, 163.3, 153.2, 149.9, 133.6, 118.2, 102.2, 29.6, 28.1, 13C NMR 118.2, 102.2, 62.4, 60.8, 29.6, 28.1, 24.9, 18.6. δ 7.48 (s, 2H), 5.50–5.41 (m, 1H), 3.95–3.88 (m, 2H), 3.84 (s, 3H), 3.69–3.54 (m, 2H), 1.93–1.83 (m, 2H), 1.77–1.65 (m, 2H), 1.64–1.58 (m, 2H). (101 MHz, CDCl 3 ) δ 163.7, 163.3, 153.2, 149.9, 133.6, 118.2, 118.2, 102.2, 102.2, 62.4, 62.4, 60.8, 60.8, 29.6, 29.6, 28.1, 28.1, 24.9, 24.9, 18.6. 18.6. 13C NMR (101 MHz, CDCl3) δ 163.7, 163.3, 153.2, 149.9, 133.6, 118.2, 102.2, 29.6, 28.1, 1.77–1.65 (m,62.4, 2H),60.8, 1.64–1.58 (m, 24.9, 2H). 18.6. 118.2, 102.2, 62.4, 60.8, 29.6, 28.1, 24.9, 18.6.

(E)-3-(3-Bromo-4-methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanoic acid (22b) (E)-3-(3-Bromo-4-methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanoic acid (22b) (E)-3-(3-Bromo-4-methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanoic acid (22b) Carboxylic acid 21b (2.00 g, 7.32 mmol) and THPONH2 (1.71 g, 14.6 mmol, 2acid equiv.) were used. (E)-3-(3-Bromo-4-methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanoic (22b) (E)-3-(3-Bromo-4-methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanoic (22b)were used. Carboxylic acid 21b (2.00 g, 7.32 mmol) and THPONH 2 (1.71 g, 14.6 mmol, 2acid equiv.) 1 Carboxylic acid 21b (2.00 g, 7.32 mmol) and THPONH 2 (1.71 g, 14.6 mmol, 2 equiv.) 22b was obtainedacid as an (2.74g,g,7.32 quant.). H NMR (400 MHz, CDCl 3) δ 7.54 (d, J2 =equiv.) 2.2 Hz,were 1H),used. 7.23 Carboxylic 21boil (2.00 mmol) THPONH g, 14.6 mmol, were used. 2 (1.71 1Hand (E)-3-(3-Bromo-4-methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanoic (22b) 22b was obtained as an oil (2.74 quant.). NMR (400 MHz, CDCl 3) δ 7.54 (d, Jacid == 2.2 Hz, 1H), 7.23 Carboxylic acid 21b (2.00 g,g, 7.32 mmol) and THPONH 2 (1.71 g, 14.6 mmol, 2 equiv.) were used. 1 22b was obtained as an oil (2.74 g, quant.). H NMR (400 MHz, CDCl 3 ) δ 7.54 (d, J 2.2 Hz, 1H), 7.23 1 H NMR (dd, J = 2.2, 8.5 Hz,as 1H), 6.81(2.74 (d, Jg,= 8.5 Hz, 1H), 5.46 (d, J = 3.1 Hz, 1H), 3.94–3.87 (m, 2H), 3.86 (s, 3H), 22b was obtained an oil quant.). (400 MHz, CDCl ) δ 7.54 (d, J = 2.2 Hz, 1H), 7.23 3 1 (dd, J = 2.2, 8.5 Hz, 1H), 6.81 (d, J = 8.5 Hz, 1H), 5.46 (d, J = 3.1 Hz, 1H), (m, 2H), 3.86 (s, 3H), Carboxylic acid 21b (2.00 g, 7.32 mmol) and THPONH 2 (1.71 g, 14.6 mmol, 2 equiv.) were used. 22b was obtained as an oil (2.74 g, quant.). H NMR (400 MHz, CDCl 3)3.94–3.87 δ 7.54 (d, J = 2.2 Hz, 1H), 7.23 (dd, JJ = = 2.2, Hz, 1H), 6.81(m, (d, 2H), Hz, 5.46 (d, 1H), 3.94–3.87 3.69–3.65 (m,8.5 2H), 1.76–1.66 (m, 2H), 2H). (dd, 2.2, 8.5 Hz,1.91–1.81 1H), 6.81 (d, JJ == 8.5 8.5 Hz, 1H), 1H), 5.46 (d, J1.65–1.56 J == 3.1 3.1 Hz, Hz,(m, 1H), 3.94–3.87 (m, (m, 2H), 2H), 3.86 3.86 (s, (s, 3H), 3H), 1H NMR 3.69–3.65 (m, 1.91–1.81 (m, 1.76–1.66 (m, (m, 22b was obtained an 6.81 oil (2.74 g, quant.). (400 CDCl 3)3.94–3.87 δ 7.54 (d,(m, J = 2H), 2.2 Hz, 7.23 (dd, J = 2.2, 8.52H), Hz,as 1H), (d, J2H), = 8.5 Hz, 1H), 5.462H), (d, J1.65–1.56 =MHz, 3.1 Hz, 1H),2H). 3.861H), (s, 3H), 3.69–3.65 (m, 2H), 1.91–1.81 (m, 2H), 1.76–1.66 (m, 2H), 1.65–1.56 (m, 2H). 3.69–3.65 (m, 2H), 1.91–1.81 (m, 2H), 1.76–1.66 (m, 2H), 1.65–1.56 (m, 2H). 3.69–3.65 (m, (dd, J = 2.2, 8.52H), Hz,1.91–1.81 1H), 6.81 (m, (d, J2H), = 8.51.76–1.66 Hz, 1H),(m, 5.462H), (d, J1.65–1.56 = 3.1 Hz, (m, 1H),2H). 3.94–3.87 (m, 2H), 3.86 (s, 3H), 3.69–3.65 (m, 2H), 1.91–1.81 (m, 2H), 1.76–1.66 (m, 2H), 1.65–1.56 (m, 2H).

(E)-3-(3-Chloro-4-methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanoic acid (22c) (E)-3-(3-Chloro-4-methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanoic acid (22c) (E)-3-(3-Chloro-4-methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanoic (22c)were used. Carboxylic acid 21c (1.86 g, 8.13 mmol) and THPONH2 (1.90 g, 16.3 mmol, 2 acid equiv.) (E)-3-(3-Chloro-4-methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanoic acid (22c)were used. Carboxylic acid 21c (1.86 g, 8.13 mmol) and THPONH 2 (1.90 g, 16.3 mmol, 2 equiv.) (E)-3-(3-Chloro-4-methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanoic acid (22c) Carboxylic (1.86oil g, (3.05 8.13 mmol) and1H THPONH 2 (1.90 g, CDCl 16.3 mmol, 2 equiv.) were 22c was obtainedacid as a21c yellow g, quant). NMR (400 MHz, 3) δ 7.36 (d, J = 2.2 Hz,used. 1H), (E)-3-(3-Chloro-4-methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanoic (22c) 22c was obtained as aa21c yellow g, quant). NMR (400 MHz, 3) δ 7.36 (d, JJ == 2.2 Hz, 1H), (1.90 g,CDCl 16.3 mmol, mmol, equiv.) were used. Carboxylic acid (1.86oil g, (3.05 8.13 mmol) and11H THPONH 22 (1.90 g, 16.3 22acid equiv.) were used. 22c was obtained as yellow oil (3.05 g, quant). H NMR (400 MHz, CDCl 3 ) δ 7.36 (d, 2.2 Hz, 1H), 7.18 (dd, J = 8.4, 2.2 Hz, 1H), 6.84 (d, J = 8.4 Hz, 1H), 5.47 (d, J = 3.1 Hz, 1H), 3.87 (s, 3H), 3.86 (s, 2H), 11 H NMR 7.18 (dd, J = 8.4, 2.2 Hz, 1H), 6.84 (d, J = 8.4 Hz, 1H), 5.47 (d, J = 3.1 Hz, 1H), 3.87 (s, 3H), 3.86 (s, 2H), 22c was obtained as a yellow oil (3.05 g, quant). (400 MHz, CDCl ) δ 7.36 (d, J = 2.2 Hz, 1H), Carboxylic acid 21c (1.86 g, 8.13 mmol) and THPONH 2 (1.90 g, 16.3 mmol, 2 equiv.) were used. (3.05 g, quant). 3 δ = 2.2 Hz, 1H), 3 7.18 (dd, (dd, JJ == 7.6, 8.4,3.4 2.2Hz, Hz,2H), 1H),1.91–1.82 6.84 (d, J (m, = 8.42H), Hz,1.77–1.66 1H), 5.47(m, (d, 2H), J = 3.1 Hz, 1H),(m, 3.87 (s, 3H), 3.86 (s, 2H), 3.63 1.65–1.52 2H). 1 3.63 (dd, JJ == 2H), 2H), 1.65–1.52 2H). 8.4, 3.4 2.2 = 8.4 Hz,1.77–1.66 1H), 5.47(m, (d, J2H), 3.1 Hz, 1H), 3.87 (s,(d, 3H), 3.86Hz, (s, 2H), 2H), 22c asHz, a yellow oil (3.05 quant). H NMR (400 MHz, CDCl 3)(m, δ3.87 7.36 J = 2.2 1H), 7.18 8.4, 2.2 Hz, 1H), 1.91–1.82 6.84 (d, J g, =(m, 8.4 Hz, 1H), 5.47 (d, == 3.1 Hz, 1H), (s, 3H), 3.86 (s, 3.63was (dd,obtained = 7.6, 7.6, 3.4 Hz, 2H), 1.91–1.82 (m, 2H), 1.77–1.66 (m, 2H), 1.65–1.52 (m, 2H). 7.6, 3.4 3.4 Hz, Hz, 2H), 2H), 7.18 (dd, J == 7.6, 8.4, 2.2 1H), 1.91–1.82 6.84 (d, J =(m, 8.42H), Hz,1.77–1.66 1H), 5.47 (m, (d, J2H), = 3.11.65–1.52 Hz, 1H),(m, 3.872H). (s, 3H), 3.86 (s, 2H), 3.63 3.63 (dd, J = 7.6, 3.4 Hz, 2H), 1.91–1.82 (m, 2H), 1.77–1.66 (m, 2H), 1.65–1.52 (m, 2H). (E)-3-(4-Methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanoic acid (22d) (E)-3-(4-Methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanoic acid (22d) (E)-3-(4-Methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanoic (22d) mmol, 2 equiv.) were used. Carboxylic acid 21d (1.73 g, 7.98 mmol) and THPONH2 (1.87 g, 16.0 acid 2 (1.87 g, 16.0 acid mmol, 22 equiv.) were used. Carboxylic acid 21d (1.73 g, 7.98 mmol) and THPONH (E)-3-(4-Methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanoic (22d) 2 (1.87 g, CDCl 16.0 mmol, equiv.) were Carboxylic (1.73oil g,(2.78 7.98 mmol) and1H THPONH 22d was obtainedacid as a21d yellow g, quant). NMR (400 MHz, 3) δ 7.23 (d, J = 8.7 Hz,used. 2H), 1H NMR (400 MHz, CDCl3) δ 7.23 (d, J = 8.7 Hz, 2H), 22d was obtained as a yellow oil (2.78 g, quant). (E)-3-(4-Methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanoic acid (22d) 2 (1.87 g, 16.0acid mmol, 2 equiv.) were used. Carboxylic acid 21d (1.73 g, 7.98 mmol) and THPONH (E)-3-(4-Methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanoic (22d) 1 22d was obtained as a yellow oil (2.78 g, quant). H NMR (400 MHz, CDCl 3 ) δ 7.23 (d, J = 8.7 Hz, 2H), 6.81 (d, J = 8.7 Hz, 2H), 5.47–5.43 (t, 1H), 3.87 (s, 2H), 3.76 (s, 3H), 3.67–3.59 (m, 2H), 1.93–1.80 (m, 2H), 1H 6.81 (d, J = 8.7 Hz, 2H), 5.47–5.43 (t, 1H), 3.87 (s, 2H), 3.76 (s, 3H), 3.67–3.59 (m, 1.93–1.80 (m, 2H), 22d was obtained as a yellow oil (2.78 g, quant). NMR (400 MHz, CDCl 3) δ 2H), 7.23 (d, J = 8.7 Hz, 22 (1.87 g, 16.0 mmol, 2 equiv.) were used. acid 21d g, 7.98 and THPONH Carboxylic (1.73 mmol) (1.87 g, 16.0 mmol, 2 equiv.) were used. 6.81 (d, J =(m, 8.72H), Hz, 1.60–1.51 2H), 5.47–5.43 (t, 1H), 3.87 (s, 2H), 3.76 (s, 3H), 3.67–3.59 (m, 2H), 1.93–1.80 (m, 2H), 1.72–1.65 (m, 2H). 11H NMR 1.72–1.65 1.60–1.51 (m, yellow oil2H). (2.78 g,3.87 quant). 7.23 (d, J == 8.7 6.81 (d, J obtained =(m, 8.72H), Hz, as 2H), 5.47–5.43 (t, 1H), (s, 2H), 3.76 (s, 3H), 3.67–3.59 1.93–1.80 (m, 22d was a yellow oil (2.78 g, quant). (400 MHz, CDCl(m, δ 2H), 8.7 Hz, Hz, 2H), 2H), 33) δ 1.72–1.65 (m, 2H), 1.60–1.51 (m, 2H). 6.81 (d, J ==(m, 8.7 Hz, 2H), 5.47–5.43 (t, 1H), 3.87 (s, 2H), 3.76 (s, 3H), 3.67–3.59 (m, 2H), 1.93–1.80 (m, 2H), 1.72–1.65 2H), 1.60–1.51 (m, 2H). 8.7 Hz, 2H), 5.47–5.43 (t, 1H), 3.87 (s, 2H), 3.76 (s, 3H), 3.67–3.59 (m, 2H), 1.93–1.80 (m, 2H), 1.60–1.51 (m, (m, 2H). 2H). 1.72–1.65 (m, 2H), 1.60–1.51

(E)-N-[3,5-Dibromo-4-[3-(2,2,2-trifluoro-N-methylacetamido)propoxy]phenethyl]-3-(3,5-dibromo-4(E)-N-[3,5-Dibromo-4-[3-(2,2,2-trifluoro-N-methylacetamido)propoxy]phenethyl]-3-(3,5-dibromo-4(E)-N-[3,5-Dibromo-4-[3-(2,2,2-trifluoro-N-methylacetamido)propoxy]phenethyl]-3-(3,5-dibromo-4methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanamide (26) methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanamide (26) (E)-N-[3,5-Dibromo-4-[3-(2,2,2-trifluoro-N-methylacetamido)propoxy]phenethyl]-3-(3,5-dibromo-4methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanamide (26)(0.24 g, 0.53 mmol), amine 17 A 20 mL Biotage MW tube was charged with carboxylic acid 22a A 20 mL Biotage MW tube was charged with carboxylic acid 22a (0.24 g, 0.53 mmol), amine 17 (E)-N-[3,5-Dibromo-4-[3-(2,2,2-trifluoro-N-methylacetamido)propoxy]phenethyl]-3-(3,5-dibromo-4methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanamide (26) A 20 mL Biotage MW tube was charged with carboxylic acid 22a(0.11 (0.24g, g,0.79 0.53mmol, mmol), 17 [15] (0.25 g, 0.53 mmol), EDC·HCl (0.15 g, 0.79 mmol, 1.5 equiv.), HOBt 1.5amine equiv.), [15] (0.25 g, 0.53 mmol), EDC·HCl (0.15 g, 0.79 mmol, 1.5 equiv.), HOBt (0.11 g, 0.79 mmol, 1.5 equiv.), methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanamide (26) A 20 mL Biotage MW tube was charged with carboxylic acid 22a (0.24 g, 0.53 mmol), amine 17 [15] (0.25 g, 0.53 mmol), EDC·HCl (0.15 g, 0.79 mmol, 1.5 equiv.), HOBt (0.11 g, 0.79 mmol, 1.5 equiv.), DIPEA (0.10 g, 0.79 mmol, 1.5 equiv.) and dry DCM (15 mL). The reaction was MW irradiated for 2 h DIPEA (0.10 g, 0.79 mmol, 1.5 equiv.) and dry DCM (15 The reaction was MW irradiated for hh A 20 MW tube was charged acid 22a (0.11 (0.24 g,0.79 0.53mmol, mmol), [15] (0.25 g,mL 0.53 mmol), EDC·HCl (0.15 g, 0.79 mmol, 1.5mL). equiv.), HOBt g, 1.5amine equiv.), DIPEA (0.10 g,Biotage 0.79 mmol, 1.5 equiv.) and dry with DCMcarboxylic (15 mL). The reaction was MW irradiated for 2217 [15] (0.25 g, 0.53 mmol), EDC·HCl (0.15and g, 0.79 1.5mL). equiv.), (0.11 g, 0.79 1.5 equiv.), DIPEA (0.10 g, 0.79 mmol, 1.5 equiv.) dry mmol, DCM (15 TheHOBt reaction was MWmmol, irradiated for 2 h DIPEA (0.10 g, 0.79 mmol, 1.5 equiv.) and dry DCM (15 mL). The reaction was MW irradiated for 2 h

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(E)-N-[3,5-Dibromo-4-[3-(2,2,2-trifluoro-N-methylacetamido)propoxy]phenethyl]-3-(3,5-dibromo-4methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanamide (26) A 20 mL Biotage MW tube was charged with carboxylic acid 22a (0.24 g, 0.53 mmol), amine 17 [15] (0.25 g, 0.53 EDC ·HCl (0.15 g, 0.79 mmol, 1.5 equiv.), HOBt (0.11 g, 0.79 mmol, 1.5 equiv.), Mar. Drugs 2018,mmol), 16, x FOR PEER REVIEW 13 of 25 DIPEA (0.10 g, 0.79 mmol, 1.5 equiv.) and dry DCM (15 mL). The reaction was MW irradiated for Mar. Drugs 2018, 16, x FOR PEER REVIEW 13 of 25 ◦ 2ath60at°C 60(5 C (5 bar). The reaction mixture was diluted with DCM (25washed mL), washed with (2 water × bar). The reaction mixture was diluted with DCM (25 mL), with water × 15 (2 mL) 15 mL) and a 1 M solution of HCl in H O (15 mL). The organic layer was dried over Na SO and and a 1 M solution of HCl in H 2 O (15 mL). The organic layer was dried over Na 2 SO 4 and concentrated at 60Drugs °C (52018, bar). reaction mixture was2 diluted with DCM (25 mL), washed with water (22× 15 mL) Mar. 16,The x FOR PEER REVIEW 134 of 25 concentrated vacuo. Theincrude product purified by flash twice, 25 g, gradient in vacuo. Theincrude product purified byorganic flash chromatography twice, g,4 and gradient elution: and a 1 M solution of HCl H2was O (15 mL). was The layer waschromatography dried over Na25 2SO concentrated 1 H (400 elution: (heptane/EtOAc, 0to→ 40%) 23 as aby pale yellow viscous liquid g, 134%). NMR (400 (heptane/EtOAc, 0→40%) 23 as purified atowas pale yellow viscous liquid (0.16 g,(0.16 34%). H NMR at 60 °C (5 bar).crude The reaction mixture diluted with DCM (25 mL), washed with (2 ×elution: 15MHz, mL) in vacuo. The product was flash chromatography twice, 25 g, water gradient 1 MHz, CDCl ) δ 7.50 (s, 2H), 7.34 and 7.33 (2 s, 2H, rotamers ratio 2:1), 6.92 (t, J = 6.2 Hz, 1H), 5.38 (t, J= CDCl 3 ) δ 7.50 (s, 2H), 7.34 and 7.33 (2 s, 2H, rotamers ratio 2:1), 6.92 (t, J = 6.2 Hz, 1H), 5.38 (t, J = 2.8 and a 1 M solution of HCl in H 2 O (15 mL). The organic layer was dried over Na 2 SO 4 and concentrated 3 (heptane/EtOAc, 0→40%) to 23 as a pale yellow viscous liquid (0.16 g, 34%). H NMR (400 MHz, 2.8 Hz, 4.06–4.00 (m, 2H), 3.93 J 13.1 =2H, 13.1 Hz, 1H), 3.84(s, (s,3H), 3H), 3.83 J ==13.1 13.1 Hz, Hz,vacuo. 1H), 4.06–4.00 (m,product 2H), 3.93 (d,(d, J =s, Hz, 1H), 3.84 3.83 1H), in The crude was purified by flash chromatography twice, 25 g,Hz, gradient CDCl 3) 1H), δ 7.50 (s, 2H), 7.34 and 7.33 (2 rotamers ratio 2:1), 6.92 (t, (d, J(d, = J6.2 Hz, 1H), 5.38 3.80–3.72 (t,elution: J = 2.8 1(m) (m, 2H), 3.65–3.54 (m, 3H), 3.43 (ddt, J = 12.9, 8.2, 6.6 Hz, 1H), 3.23 (m) and 3.11 (m) N-CH rotamers 3.65–3.54 (m, 3H), 3.43 (ddt, J = 3.23 (m) and 3.11 N-CH 3 (heptane/EtOAc, 0→40%) to 23 as a pale yellow viscous liquid (0.16 g, 34%). H NMR (400 MHz, Hz, 1H), 4.06–4.00 (m, 2H), 3.93 (d, J = 13.1 Hz, 1H), 3.84 (s, 3H), 3.83 (d, J = 13.1 Hz, 1H),3 3.80–3.72 2:1, 2.77 (td, J = 1.89–1.54 (m, 6H). 7.3, 1.7 Hz, 2H), 2.23–2.09 (m, 2H), CDCl 3) δ3.65–3.54 7.50 (s, 2H), (2Js,= 2H, ratio 2:1), 6.92 (t, and J = 6.2 Hz, 1H), 5.383 rotamers (t, J = 2.8 (m, 2H), (m,7.34 3H),and 3.437.33 (ddt, 12.9,rotamers 8.2, 6.6 Hz, 1H), 3.23 (m) 3.11 (m) N-CH Hz, 2.77 1H),(td, 4.06–4.00 2H),2H), 3.932.23–2.09 (d, J = 13.1 1H), 3.84 (s,(m, 3H), 3.83 (d, J = 13.1 Hz, 1H), 3.80–3.72 2:1, J = 7.3, (m, 1.7 Hz, (m,Hz, 2H), 1.89–1.54 6H). (m, 2H), 3.65–3.54 (m, 3H), 3.43 (ddt, J = 12.9, 8.2, 6.6 Hz, 1H), 3.23 (m) and 3.11 (m) N-CH3 rotamers 2:1, 2.77 (td, J = 7.3, 1.7 Hz, 2H), 2.23–2.09 (m, 2H), 1.89–1.54 (m, 6H).

(E)-N-[3,5-Dibromo-4-[3-(2,2,2-trifluoro-N-methylacetamido)propoxy]phenethyl]-3-(3,5-dibromo-4methoxyphenyl)-2-(hydroxyimino)propanamide (28) (E)-N-[3,5-Dibromo-4-[3-(2,2,2-trifluoro-N-methylacetamido)propoxy]phenethyl]-3-(3,5-dibromo-4(E)-N-[3,5-Dibromo-4-[3-(2,2,2-trifluoro-N-methylacetamido)propoxy]phenethyl]-3-(3,5-dibromo-4THP ether 26 (0.14 g, 0.16 mmol), a 2 M (28) solution of HCl in Et2O (4 mL), dry DCM (4 mL) and dry methoxyphenyl)-2-(hydroxyimino)propanamide MeOH (0.3 mL) were added to a 20-mL sealed tubeof and heated in2(4 anmL), bath at 70 °C for 3and h. and The (E)-N-[3,5-Dibromo-4-[3-(2,2,2-trifluoro-N-methylacetamido)propoxy]phenethyl]-3-(3,5-dibromo-4THP ether solution HCl in in Et2Et O drydry DCM (4 mL) dry ether 26 26 (0.14 (0.14g,g,0.16 0.16mmol), mmol),a a2 2MM solution of HCl O (4oilmL), DCM (4 mL) ◦ C3column reaction mixture was thenadded concentrated in(28) vacuo. The and crude product was purified by methoxyphenyl)-2-(hydroxyimino)propanamide MeOH (0.3 mL) were added to a to 20-mL sealed tubetube and heated in aninoil at 70at °C70for h. The dry MeOH (0.3 mL) were a 20-mL sealed heated anbath oil bath for 3 h. chromatography, Biotage SNAP Cartridge KP-Sil 10 g, gradient elution: (heptane/EtOAc, 0→30%) to THP ether 26 (0.14 g, 0.16 mmol), a 2 M solution of HCl in Et 2 O (4 mL), dry DCM (4 mL) and dry reaction mixture waswas thenthen concentrated in in vacuo. The crude The reaction mixture concentrated vacuo. The crudeproduct productwas waspurified purified by by column 1 give 28 (0.3 as a mL) pale were yellow viscous liquid (0.068 g, 54%). H NMR (400 CD 3OD) δ 7.48 (s, 2H), MeOH added to Cartridge aCartridge 20-mL sealed tube heated inMHz, an oil bath at 70 °C for 330%) h. 7.43 The chromatography, Biotage SNAP KP-Sil 10g, g,and gradient elution: (heptane/EtOAc, to chromatography, Biotage SNAP KP-Sil 10 gradient elution: (heptane/EtOAc, 00→30%) → 1 1 and 7.42 (2 s, 2H, rotamers ratio 2:1), 4.00 (t, J = 6.0 Hz, 2H), 3.82 (m, 5H), 3.78–3.70 (m, 2H), 3.44 (t, reaction mixture was then concentrated in vacuo. The crude product was purified by column give 28 as a pale yellow viscous liquid liquid (0.068 (0.068 g, g, 54%). 54%). H NMR (400 MHz, CD33OD) δδ 7.48 (s, 2H), 7.43J = 7.1 Hz, 2H), 3.24 (q) and 3.10 (m) N-CH 3 rotamers 2:1, 2.763.82 (t, J(m, =(m, 7.1 Hz, 2H), 2.25–2.08 (m, 2H). chromatography, Biotage SNAP Cartridge KP-Sil g,2H), gradient elution: (heptane/EtOAc, 0→30%) and 7.42 (2 s, s, 2H, 2H, rotamers rotamersratio ratio2:1), 2:1),4.00 4.00(t, (t,J J==6.0 6.010 Hz, 2H), 3.82 5H), 3.78–3.70 (m,2H), 2H), 3.44 Hz, 5H), 3.78–3.70 (m, 3.44 (t,(t, Jto =J 1 28 as2H), a pale yellow viscous liquid (0.068 g, 54%). H NMR (400 MHz, CD 32.25–2.08 OD) δ 7.48 (s, 2H), 7.43 =give 7.1Hz, Hz, 3.24 and 3.10 (m) N-CH 3 rotamers 2:1, 2.76 (t, J = 7.1 Hz, 2H), 2.25–2.08 (m, 2H). 7.1 2H), 3.24 (q)(q) and 3.10 (m) N-CH rotamers 2:1, 2.76 (t, J = 7.1 Hz, 2H), (m, 2H). 3 and 7.42 (2 s, 2H, rotamers ratio 2:1), 4.00 (t, J = 6.0 Hz, 2H), 3.82 (m, 5H), 3.78–3.70 (m, 2H), 3.44 (t, J = 7.1 Hz, 2H), 3.24 (q) and 3.10 (m) N-CH3 rotamers 2:1, 2.76 (t, J = 7.1 Hz, 2H), 2.25–2.08 (m, 2H).

(E)-N-[3,5-Dibromo-4-[3-(methylamino)propoxy]phenethyl]-3-(3,5-dibromo-4-methoxyphenyl)-2(hydroxyimino)propenamide, purpurealidin I, (1) (E)-N-[3,5-Dibromo-4-[3-(methylamino)propoxy]phenethyl]-3-(3,5-dibromo-4-methoxyphenyl)-2Compound 28 (0.050 g,purpurealidin 0.062 mmol)I,and (hydroxyimino)propenamide, (1) K2CO3 (0.018 g, 0.12 mmol, 2.0 equiv.) in MeOH (5 (E)-N-[3,5-Dibromo-4-[3-(methylamino)propoxy]phenethyl]-3-(3,5-dibromo-4-methoxyphenyl)-2mL) and H 2 O (0.5 mL) were refluxed for 2.5 mixture was concentrated vacuo(5and (E)-N-[3,5-Dibromo-4-[3-(methylamino)propoxy]phenethyl]-3-(3,5-dibromo-4-methoxyphenyl)-2Compound 28 (0.050 g,purpurealidin 0.062 mmol)I,and 2COreaction 3 (0.018 g, 0.12 mmol, 2.0 equiv.) in in MeOH (hydroxyimino)propenamide, (1)h.KThe partitioned between EtOAc (10 mL) and water (4 mL). The aqueous layerconcentrated was back-extracted with (hydroxyimino)propenamide, (1)h.KThe mL) and H2O (0.528mL) were refluxed for I,2.5 mixture was vacuo Compound (0.050 g,purpurealidin 0.062 mmol) and 0.12 mmol, 2.0 equiv.) in in MeOH (5and mL) 2 COreaction 3 (0.018 g, EtOAc (10 mL). The combined organic layers ware dried over Na 2 SO 4 , filtered and the solvent was Compound 28 (0.050 g, 0.062 mmol) and K 2 CO 3 (0.018 g, 0.12 mmol, 2.0 equiv.) in MeOH (5 partitioned between EtOAc (10 mL)for and (4 mL). The mixture aqueous was layerconcentrated was back-extracted with and H2 O (0.5 mL) were refluxed 2.5water h. The reaction in vacuo and 1H NMR (300 removed inmL). vacuo tocombined give 1, refluxed as amL) paleand yellow liquid (0.040 -g,was MHz,was mL) and 2O (0.5 mL) were for 2.5 h.viscous The reaction mixture concentrated vacuo and EtOAc (10H The layers ware dried over Na2SO 491%). ,layer filtered the in solvent partitioned between EtOAc (10organic water (4 mL). The aqueous wasand back-extracted with 1H NMR CDCl 3) δ 7.49 (s, 2H), 7.26 (s, 2H), 4.16–4.11 (m, 2H), 3.84–3.83 (m, 5H), 3.42–3.35 (m, 2H), 2.93 (t, J= partitioned between EtOAc (10 mL) and water (4 mL). The aqueous layer was back-extracted with removed in vacuo to give 1, as a pale yellow viscous liquid (0.040 -g, 91%). (300 MHz, EtOAc (10 mL). The combined organic layers ware dried over Na2 SO4 , filtered and the solvent was 13 1 7.2 Hz, 2H), 2.71 (t, J = 6.9 Hz, 2H), 2.53 (s, 3H), 2.09–2.00 (m, 2H). C NMR (75 MHz, CDCl 3 ) δ EtOAc (10 mL). The combined organic layers ware dried over Na 2 SO 4 , filtered and the solvent was CDCl 3) δ in 7.49 (s, 2H), 7.261,(s,as2H), 4.16–4.11 2H),liquid 3.84–3.83 (m, 3.42–3.35 (m, 2H), 2.93 CDCl (t, J =3 ) removed vacuo to give a pale yellow (m, viscous (0.040 -g,5H), 91%). H NMR (300 MHz, 1H 40.0, 13 152.5, 151.3, 150.4, 133.5, 133.0, 118.3, 117.8, 71.8, 60.7, 49.0, 35.5, 34.3, removed in vacuo 1,4.16–4.11 as2H), a135.9, pale yellow viscous liquid (0.040 -g, 91%). NMR (300 MHz, 7.2 Hz,(s, 2H), 2.71 (t,to J =give 6.9138.2, Hz, 2.53 (s, 3H), 2.09–2.00 (m, 2H). C NMR (752H), MHz, CDCl ) δ29.0, δ163.6, 7.49 2H), 7.26 (s, 2H), (m, 2H), 3.84–3.83 (m, 5H), 3.42–3.35 (m, 2.93 (t, J3= 7.2 Hz, +2H), + 711.8657; 13 C(m, 28.0. 2.71 HRMS (ESI ):150.4, calcd. for C 22 H N 3133.5, O4Br 4 (m, [M +2H), H] found, 711.8660. CDCl 3152.5, ) δ 7.49 7.26 (s,2.53 2H), 4.16–4.11 5H), 3.42–3.35 (m,35.5, 2H), 2.9329.0, (t,152.5, J= 163.6, 138.2, 135.9, 133.0, 118.3, 117.8, 71.8, 60.7, 49.0, 2H), (t, J151.3, =(s,6.9 Hz, 2H), (s,26 3H), 2.09–2.00 (m,,3.84–3.83 2H). NMR (75 MHz,40.0, CDCl 163.6, 3 ) δ34.3, 13C NMR (75 MHz, CDCl3) δ + + 7.2 Hz, 2H), 2.71 (t, J = 6.9 Hz, 2H), 2.53 (s, 3H), 2.09–2.00 (m, 2H). 28.0. HRMS ): calcd. C22H 26N3O 4Br4 [M + H]71.8, , 711.8657; found, 711.8660. 151.3, 150.4, (ESI 138.2, 135.9, for 133.5, 133.0, 118.3, 117.8, 60.7, 49.0, 40.0, 35.5, 34.3, 29.0, 28.0. HRMS + + 163.6, 152.5, 151.3, 150.4, 138.2, 135.9, 133.5, 133.0, 118.3, 117.8, 71.8, 60.7, (ESI ): calcd. for C22 H26 N3 O4 Br4 [M + H] , 711.8657; found, 711.8660. 49.0, 40.0, 35.5, 34.3, 29.0, 28.0. HRMS (ESI+): calcd. for C22H26N3O4Br4 [M + H]+, 711.8657; found, 711.8660. (E)-N-[3,5-Dibromo-4-[3-(dimethylamino)propoxy]phenethyl]-3-(3,5-dibromo-4-methoxyphenyl)-2[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanamide (27) (E)-N-[3,5-Dibromo-4-[3-(dimethylamino)propoxy]phenethyl]-3-(3,5-dibromo-4-methoxyphenyl)-2A 20-mL Biotage MW tube was charged with [[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanamide (27)purpurealidin E 25 [15,27] (0.17 g, 0.44 mmol), carboxylic acid 22a (0.20 g, 0.44 mmol), EDC·HCl g, 0.66 mmol, 1.5[15,27] equiv.), HOBt (0.10mmol), g, 0.66 (E)-N-[3,5-Dibromo-4-[3-(dimethylamino)propoxy]phenethyl]-3-(3,5-dibromo-4-methoxyphenyl)-2A 20-mL Biotage MW tube was charged with(0.13 purpurealidin E 25 (0.17 g, 0.44 mmol, 1.2 equiv.), (0.12 0.66 mmol, 1.2(27) equiv.) DCM (15 mL). The tube(0.10 was g, sealed [[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanamide carboxylic acid 22aDIPEA (0.20 g, 0.44mL, mmol), EDC·HCl (0.13 g,and 0.66dry mmol, 1.5 equiv.), HOBt 0.66 and microwave irradiated at 60 °C for 5 h. The reaction mixture was diluted with DCM (10 mL) and A 20-mL Biotage MW tube was charged with purpurealidin E 25 [15,27] (0.17 g, 0.44 mmol), mmol, 1.2 equiv.), DIPEA (0.12 mL, 0.66 mmol, 1.2 equiv.) and dry DCM (15 mL). The tube was sealed washed with water (2 × 15g,at mL), 1 for M solution HCl inmixture H20.66 O (15 mL)diluted and brine (15HOBt mL). (10 ThemL) organic carboxylic acid 22a (0.20 0.44 mmol), (0.13 g, mmol, 1.5 equiv.), (0.10 g, 0.66 and microwave irradiated 60 a°C 5 EDC·HCl h. The of reaction was with DCM and phase was dried over Na 2 SO 4 (anhyd.), filtered and volatiles were removed in vacuo. The crude mmol, 1.2 equiv.), DIPEA (0.12 mL, 0.66 mmol, 1.2 equiv.) and dry DCM (15 mL). The tube was sealed washed with water (2 × 15 mL), a 1 M solution of HCl in H2O (15 mL) and brine (15 mL). The organic

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(E)-N-[3,5-Dibromo-4-[3-(dimethylamino)propoxy]phenethyl]-3-(3,5-dibromo-4-methoxyphenyl)-2[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanamide (27) A 20-mL Biotage MW tube was charged with purpurealidin E 25 [15,27] (0.17 g, 0.44 mmol), carboxylic acid 22a (0.20 g, 0.44 mmol), EDC·HCl (0.13 g, 0.66 mmol, 1.5 equiv.), HOBt (0.10 g, 0.66 mmol, 1.2 equiv.), DIPEA (0.12 mL, 0.66 mmol, 1.2 equiv.) and dry DCM (15 mL). The tube was sealed and microwave irradiated at 60 ◦ C for 5 h. The reaction mixture was diluted with DCM (10 mL) and washed with water (2 × 15 mL), a 1 M solution of HCl in H2 O (15 mL) and brine (15 mL). The organic phase was dried over Na2 SO4 (anhyd.), filtered and volatiles were removed in vacuo. The Mar.Drugs Drugs 2018,16, 16, FOR PEERREVIEW REVIEW 14 of of 25 25 Mar. 2018, xxFOR PEER 14 crude product was purified by column chromatography, Biotage SNAP Cartridge KP-Sil 25 g, isocratic 1 elution: (DCM/MeOH, 7:3) to give 27 as a yellow oil (0.30 g, 83%). H NMR (400 MHz, CDCl3 ) δ 7.48 (s, 2H), 7.31 7.31 (s, (s, 2H), 2H), 6.94 6.94 (t, (t, J == 6.2 6.2 Hz, 1H), 1H), 5.36 (t, (t, J = 2.8 Hz, Hz, 1H), 4.03 4.03 (t, JJ == 5.7 5.7 Hz, 2H), 2H), 3.89 (s, (s, 1H), (s, (s, 2H), 2H), 7.31 (s, 2H), 6.94 (t, JJ = 6.2 Hz, Hz, 1H), 5.36 5.36 (t, JJ == 2.8 2.8 Hz, 1H), 1H), 4.03 (t, (t, J = 5.7 Hz, Hz, 2H), 3.89 3.89 (s, 1H), 1H), 3.82 (s, (s, 3H), 3H), 3.58 3.58 (dd, (dd, J = 4.9, 4.9, 8.1 8.1 Hz, Hz, 3H), 3H), 3.42 3.42 (m, (m, 1H), 1H), 3.23–3.10 3.23–3.10 (m, (m, 2H), 2H), 2.75 2.75 (t, (t, J == 7.3 7.3 Hz, Hz, 2H), 2H), 2.69 2.69 3.82 3.82 (s, 3H), 3.58 (dd, JJ== 4.9, 8.1 Hz, 3H), 3.42 (m, 1H), 3.23–3.10 (m, 2H), 2.75 (t, J =J 7.3 Hz, 2H), 2.69 (s, (s, 6H), 2.29 (m, 2H), 1.89–1.59 (m, 6H). (s, 6H), 2.29 2H), 1.89–1.59 6H). 6H), 2.29 (m,(m, 2H), 1.89–1.59 (m,(m, 6H).

(E)-N-[3,5-Dibromo-4-[3-(dimethylamino)propoxy]phenethyl]-3-(3,5-dibromo-4-methoxyphenyl)-2(E)-N-[3,5-Dibromo-4-[3-(dimethylamino)propoxy]phenethyl]-3-(3,5-dibromo-4-methoxyphenyl)-2(hydroxyimino)propanamide (29) (29) (hydroxyimino)propanamide (E)-N-[3,5-Dibromo-4-[3-(dimethylamino)propoxy]phenethyl]-3-(3,5-dibromo-4-methoxyphenyl)-2THP ether 27 (0.30 g, 0.36 mmol) was was dissolved dissolved to to dry dry DCM DCM (7 (7 mL), mL), and and TFA TFA (3 (3 mL) mL) was was added added THP ether 27 (0.30 g, 0.36 (hydroxyimino)propanamide (29) mmol) under argon atmosphere. The reaction reaction mixture was stirred stirred at room room temperature for 17 h. It It was was then under argon atmosphere. mixture was for h. then THP ether 27 (0.30 g, The 0.36 mmol) was dissolved to dry at DCM (7 temperature mL), and TFA (317 mL) was added diluted by adding DCM (10 mL) and washed with a 2 M solution of NaOH in H 2O (15 mL) and water diluted by adding DCM (10 mL) and washed with M solution of NaOH in H2Ofor (1517mL) under argon atmosphere. The reaction mixture wasa 2stirred at room temperature h. Itand waswater then (15 mL) mL)by until pH was was neutral. neutral. The aqueous phase back extracted extracted with DCM (10 mL) and the (15 until pH phase back with DCM (10 mL) and the diluted adding DCM (10 mL)The andaqueous washed with a was 2was M solution of NaOH in H O (15 mL) and water 2 combined organic phase was dried dried over Na Na22phase SO44,, filtered filtered andextracted concentrated in vacuo to give crude combined organic phase was over SO and concentrated vacuo give crude (15 mL) until pH was neutral. The aqueous was back within DCM (10to mL) and the product (0.21 g, 80%). The crude product was purified by column chromatography, Biotage SNAP product (0.21 g, 80%). Thewas crude product was purified by column chromatography, Biotage SNAP combined organic phase dried over Na SO , filtered and concentrated in vacuo to give crude 2 4 Cartridge KP-Sil 25 g, g, The gradient elution: (DCM/MeOH, 2→20%) to chromatography, give 29 29 as as aa yellow yellow oil (0.074 (0.074 g, Cartridge KP-Sil 25 gradient (DCM/MeOH, to give oil g, product (0.21 g, 80%). crudeelution: product was purified 2→20%) by column Biotage SNAP 1 28%). 1H H NMR NMR (400 MHz, CD33OD) OD) 7.48(DCM/MeOH, (s, 2H), 2H), 7.43 7.43 (s, (s, 2H), 2H), 4.02 (t, (t, Jgive J == 5.9 5.929 Hz, 2H), 3.82 (s, (s, 3H), 3H), 3.44 28%). (400 CD δδ 7.48 (s, 4.02 Hz, 3.82 3.44 Cartridge KP-Sil 25 MHz, g, gradient elution: 2→20%) to as 2H), a yellow oil (0.074 g, (dd, J = 6.7, 7.6 Hz, 2H), 3.35 (s, 2H), 3.15–2.98 (m, 2H), 2.85–2.69 (m, 2H), 2.64 (s, 6H), 2.15 (dq, J = 5.8, 1 (dd, 6.7, 7.6 Hz, 3.35 (s,3 OD) 2H), δ3.15–2.98 (m, 7.43 2H), (s, 2.85–2.69 (m, 2.64 (s,2H), 6H),3.82 2.15(s, (dq, J = 3.44 5.8, 28%).J = H NMR (4002H), MHz, CD 7.48 (s, 2H), 2H), 4.02 (t,2H), J = 5.9 Hz, 3H), 13C NMR (101 MHz, CD3OD) δ 165.4, 153.8, 152.5, 152.1, 140.0, 137.4, 134.5, 134.4, 118.8, 7.7 Hz, Hz, 2H). 13C 7.7 NMR (101 MHz, CD3OD) δ 165.4, 152.5, 152.1, 137.4,(s,134.5, 134.4,(dq, 118.8, (dd, J =2H). 6.7, 7.6 Hz, 2H), 3.35 (s, 2H), 3.15–2.98 (m,153.8, 2H), 2.85–2.69 (m,140.0, 2H), 2.64 6H), 2.15 J= +): calcd. for C23H27Br4N3O4 [M+H]+ 118.6, 71.8, 61.0, 57.3, 44.6, 41.4, 35.2, 28.8, 27.7. HRMS (ESI 13 + 118.6, 61.0, 57.3, 44.6,(101 41.4, 35.2,CD 28.8, 27.7. HRMS (ESI152.5, ): calcd. for140.0, C23H137.4, 27Br4N134.5, 3O4 [M+H] 5.8, 7.771.8, Hz, 2H). C NMR MHz, δ 165.4, 153.8, 152.1, 134.4,+ 3 OD) 725.8813, found: 725.8809. 725.8813, found: 118.8, 118.6, 71.8, 725.8809. 61.0, 57.3, 44.6, 41.4, 35.2, 28.8, 27.7. HRMS (ESI+ ): calcd. for C23 H27 Br4 N3 O4 [M+H]+ 725.8813, found: 725.8809. General Method Method for for the the Amide Amide Coupling Coupling General General Method acid for the Amide Coupling Carboxylic (0.20 g),aniline aniline oramine amine(1–1.5 (1–1.5equiv.), equiv.),EDC⋅HCl EDC⋅HCl(1.5 (1.5equiv.), equiv.),HOBt HOBt(1.5 (1.5equiv.), equiv.), Carboxylic acid (0.20 g), or and DIPEA (1.5 equiv.) were dissolved in dry DCM (5 mL). The mixture was irradiated by Carboxylic acid (0.20 g), aniline or amine (1–1.5 equiv.), EDC · HCl (1.5 equiv.), HOBt (1.5 equiv.), and DIPEA (1.5 equiv.) were dissolved in dry DCM (5 mL). The mixture was irradiated by microwaves for 2 h at 60 °C. The TLC indicated the completion of the reaction using vanillin as and DIPEA (1.5 dry DCMthe (5 mL). The mixture irradiated byvanillin microwaves microwaves for equiv.) 2 h at were 60 °C.dissolved The TLCinindicated completion of thewas reaction using as aa ◦ visualization reagent. Theindicated reaction mixture mixture was diluted diluted with DCM using (20 mL) mL) and washed washed with water water for 2 h at 60 reagent. C. The TLC the completion of the reaction vanillin as a visualization visualization The reaction was with DCM (20 and with (2 × 15 mL) and a 2 M solution of HCl in H 2 O (2 × 15 mL). The organic layer was dried over anhyd. reagent. Theand reaction was with DCM mL)The andorganic washedlayer withwas water (2 ×over 15 mL) and (2 × 15 mL) a 2 Mmixture solution of diluted HCl in H 2O (2 × 15(20 mL). dried anhyd. SOsolution 4, filtered andinconcentrated concentrated in vacuo. vacuo. The products products were purified with flash column aNa 2 22M of HCl H2 O (2 × 15 in mL). The organic layer waswere driedpurified over anhyd. Naflash filtered Na SO 4, filtered and The with 2 SO4 ,column chromatography. and concentrated in vacuo. The products were purified with flash column chromatography. chromatography.

(E)-3-(3,5-Dibromo-4-methoxyphenyl)-N-(3,4-dichlorophenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy](E)-3-(3,5-Dibromo-4-methoxyphenyl)-N-(3,4-dichlorophenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy](E)-3-(3,5-Dibromo-4-methoxyphenyl)-N-(3,4-dichlorophenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]-imino] imino]propanamide (30-THP) (30-THP) imino]propanamide propanamide (30-THP) Carboxylic acid 22a 22a (0.20 g, g, 0.44 mmol) mmol) and 3,4-dichloroaniline 3,4-dichloroaniline (0.11 g, g, 0.67 mmol, mmol, 1.5 equiv.) equiv.) Carboxylic Carboxylic acid acid 22a (0.20 (0.20 g, 0.44 0.44 mmol) and and 3,4-dichloroaniline (0.11 (0.11 g, 0.67 0.67 mmol, 1.5 1.5 equiv.) were reacted according to the general procedure for amide coupling. The product was purified by were TheThe product waswas purified by were reacted reacted according according to to the thegeneral generalprocedure procedurefor foramide amidecoupling. coupling. product purified column chromatography, Biotage SNAP Cartridge KP-Sil 25 g and Ultra 25 g, gradient elution: column chromatography, Biotage SNAP Cartridge KP-Sil 25 g and Ultra 25 g, gradient elution: (heptane/EtOAc, 12→100%) 12→100%) to to give give 30-THP 30-THP as as aa crude crude product product (0.19 (0.19 g, g, 73%). 73%). 11H H NMR NMR (400 (400 MHz, MHz, (heptane/EtOAc, CDCl 3) δ 8.64 (s, 1H), 7.87 (d, J = 1.9 Hz, 1H), 7.52 (s, 2H), 7.41–7.38 (m, 2H), 5.49 (t, J = 2.6 Hz, 1H), CDCl3) δ 8.64 (s, 1H), 7.87 (d, J = 1.9 Hz, 1H), 7.52 (s, 2H), 7.41–7.38 (m, 2H), 5.49 (t, J = 2.6 Hz, 1H), 3.99 (d, (d, JJ == 13.1 13.1 Hz, Hz, 1H), 1H), 3.89 3.89 (d, (d, JJ == 13.1 13.1 Hz, Hz, 1H), 1H), 3.84 3.84 (s, (s, 3H), 3H), 3.65–3.59 3.65–3.59 (m, (m, 2H), 2H), 1.93–1.59 1.93–1.59 (m, (m, 6H). 6H). 3.99

(E)-3-(3,5-Dibromo-4-methoxyphenyl)-N-(3,4-dichlorophenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanamide (30-THP) 15 of 26 22a (0.20 g, 0.44 mmol) and 3,4-dichloroaniline (0.11 g, 0.67 mmol, 1.5 equiv.) were reacted according to the general procedure for amide coupling. The product was purified by column chromatography, Biotage SNAP Cartridge KP-Sil 25 g and Ultra 25 g, gradient elution: by column chromatography, Biotage SNAP Cartridge KP-Sil 25 g and Ultra 25 g, gradient elution: (heptane/EtOAc, 12→100%) to give 30-THP as a crude product (0.19 g, 73%). 1H NMR (400 MHz, (heptane/EtOAc, 12→100%) to give 30-THP as a crude product (0.19 g, 73%). 1 H NMR (400 MHz, CDCl3) δ 8.64 (s, 1H), 7.87 (d, J = 1.9 Hz, 1H), 7.52 (s, 2H), 7.41–7.38 (m, 2H), 5.49 (t, J = 2.6 Hz, 1H), CDCl3 ) δ 8.64 (s, 1H), 7.87 (d, J = 1.9 Hz, 1H), 7.52 (s, 2H), 7.41–7.38 (m, 2H), 5.49 (t, J = 2.6 Hz, 1H), 3.99 (d, J = 13.1 Hz, 1H), 3.89 (d, J = 13.1 Hz, 1H), 3.84 (s, 3H), 3.65–3.59 (m, 2H), 1.93–1.59 (m, 6H). 3.99 (d, J = 13.1 Hz, 1H), 3.89 (d, J = 13.1 Hz, 1H), 3.84 (s, 3H), 3.65–3.59 (m, 2H), 1.93–1.59 (m, 6H). Mar. Drugs 2018, 16, 481 Carboxylic acid

(E)-3-(3,5-Dibromo-4-methoxyphenyl)-N-(3,4-dichlorobenzyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]Mar. Drugs 2018, 16, x FOR PEER REVIEW 15 of 25 (E)-3-(3,5-Dibromo-4-methoxyphenyl)-N-(3,4-dichlorobenzyl)-2-[[(tetrahydro-2H-pyran2-yl)oxy]-imino] Mar. Drugs Drugs 2018, 2018, 16, 16, x x FOR FOR PEER PEER REVIEW REVIEW 15 of of 25 25 Mar. 15 imino]propanamide (31-THP) propanamide (31-THP) Carboxylic acid 22a (0.20 g, g, 0.44 mmol) mmol) and 3,4-dichlorobenzylamine 3,4-dichlorobenzylamine (0.090 mL, 0.67 mmol, 1.5 Carboxylic Carboxylic acid acid 22a 22a (0.20 (0.20 g, 0.44 0.44 mmol) and and 3,4-dichlorobenzylamine (0.090 (0.090 mL, mL, 0.67 0.67 mmol, mmol, 1.5 1.5 equiv.) were reacted according to the general procedure for amide coupling. The product was equiv.) were reacted according to the general procedure for amide coupling. The product was purified equiv.) were reacted according to the general procedure for amide coupling. The product was equiv.) were reacted according to the general procedure for amide coupling. The product was purified by column Biotage SNAP Cartridge KP-Sil 25 elution: by column Biotage SNAP Cartridge KP-Sil 25 g, gradient (heptane/EtOAc, purified bychromatography, column chromatography, chromatography, Biotage SNAP Cartridge KP-Sil elution: 25 g, g, gradient gradient elution: purified by column chromatography, Biotage SNAP Cartridge KP-Sil 25 g, elution: 1H gradient 1 (heptane/EtOAc, 7→60%) to give 31-THP as a light yellow solid (0.23 g, 84%). NMR (400 MHz, 1H NMR 7(heptane/EtOAc, →60%) to give 31-THP as a light yellow solid (0.23 g, 84%). H NMR (400 MHz, CDCl ) δ 7.50 2H), (heptane/EtOAc, 7→60%) to give 31-THP as a light yellow solid (0.23 g, 84%). (400 MHz, 3 7→60%) to give 31-THP as a light yellow solid (0.23 g, 84%). 1H NMR (400(s, MHz, CDCl 3) δ 7.50 (s, 2H), 7.39 (d, J = 8.2 Hz, 1H), 7.37 (d, J = 2.0 Hz, 1H), 7.19 (t, J = 5.9 Hz, 1H), 7.12 (dd, 7.39 J= 8.2(s, Hz, 1H), 7.37 = 2.0 1H), = 5.9 1H), 7.12 2.1,1H), 8.2 7.12 Hz, 1H), CDCl(d, 3) δ 7.50 2H), 7.39 (d,(d, J =J8.2 Hz,Hz, 1H), 7.377.19 (d, (t, J =J2.0 Hz,Hz, 1H), 7.19 (t, J(dd, = 5.9J = Hz, (dd, CDCl 3) δ 7.50 (s, 2H), 7.39 (d, J = 8.2 Hz, 1H), 7.37 (d, J = 2.0 Hz, 1H), 7.19 (t, J = 5.9 Hz, 1H), 7.12 (dd, J5.38 8.2 Hz, 1H), 5.38 (s, 1H), 4.54 (dd, JJ 4.35 == 6.7, 15.2 Hz, 1H), 4.35 (dd, JJ3.95 == 5.8, 15.2 Hz, 1H), 3.95 (d, (s, 1H), 4.54 (dd, J = 6.7, 15.2 Hz, 1H), (dd, J = 5.8, 15.2 Hz, 1H), (d, J = 13.1 Hz, 1H), 3.85JJJ JJ === 2.1, 2.1, 8.2 Hz, 1H), 5.38 (s, 1H), 4.54 (dd, 6.7, 15.2 Hz, 1H), 4.35 (dd, 5.8, 15.2 Hz, 1H), 3.95 (d, 2.1, 8.2 Hz, 1H), 5.38 (s, 1H), 4.54 (dd, J = 6.7, 15.2 Hz, 1H), 4.35 (dd, J = 5.8, 15.2 Hz, 1H), 3.95 (d, = 13.1 Hz, 1H), 3.85 (d, 13.1 Hz, 1H), 3.84 (s, 1H), 3.61–3.55 (m, 2H), 1.87–1.56 (m, 6H). (d, 13.1Hz, Hz,1H), 1H),3.85 3.84(d, (s,13.1 1H),Hz, 3.61–3.55 (m,(s, 1.87–1.56 (m, 6H). 13.1 Hz, 1H), 3.85 (d, 13.1 Hz, 1H), 3.84 3.84 (s,2H), 1H), 3.61–3.55 (m, 2H), 1.87–1.56 1.87–1.56 (m, (m, 6H). 6H). == 13.1 1H), 1H), 3.61–3.55 (m, 2H),

(E)-N-[4-Chloro-3-(trifluoromethyl)phenyl]-3-(3,5-dibromo-4-methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2(E)-N-[4-Chloro-3-(trifluoromethyl)phenyl]-3-(3,5-dibromo-4-methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2(E)-N-[4-Chloro-3-(trifluoromethyl)phenyl]-3-(3,5-dibromo-4-methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2(E)-N-[4-Chloro-3-(trifluoromethyl)phenyl]-3-(3,5-dibromo-4-methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanami yl)oxy]imino]propanamide yl)oxy]imino]propanamide (32-THP) (32-THP) (32-THP) yl)oxy]imino]propanamide (32-THP) Carboxylic acid 22a (0.19 g, 0.42 mmol) and 4-chloro-3-(trifluoromethyl)aniline Carboxylic acid acid 22a 22a (0.19 (0.19 g, g, 0.42 0.42 mmol) mmol) and and 4-chloro-3-(trifluoromethyl)aniline 4-chloro-3-(trifluoromethyl)aniline (0.12 (0.12 g, g, 0.63 0.63 Carboxylic (0.12 g, 0.63 0.42 mmol) and 4-chloro-3-(trifluoromethyl)aniline mmol, 1.5 equiv.) were reacted according to the general procedure for amide coupling. The product mmol, 1.5 1.5 equiv.) equiv.) were were reacted reacted according according to to the the general general procedure procedure for for amide amide coupling. coupling. The The product product mmol, general for amide coupling. was purified by column chromatography, Biotage SNAP 25 g, gradient elution: was purified purified by by column column chromatography, chromatography, Biotage Biotage SNAP SNAP Cartridge Cartridge KP-Sil KP-Sil 25 g, gradient elution: was Cartridge KP-Sil 25 g, gradient elution: 1H NMR (400 MHz, CDCl3elution: (heptane/EtOAc, 7→60%). to give 32-THP as an oil (0.19 g, 72%). 8.73 1H NMR (400 MHz, CDCl3)) δ 1 (heptane/EtOAc, 7→60%). to give 32-THP as an oil (0.19 g, 72%). 8.73 (heptane/EtOAc, 7→60%).to togive give 32-THP 32-THP as as an an oil (0.19 g, 72%). 1H H NMR (400 MHz, CDCl3) δδ 8.73 (heptane/EtOAc, 7→60%). (s, 1H), 7.95 (d, J = 2.6 Hz, 1H), 7.79 (dd, J = 2.6, 8.7 Hz, 1H), 7.52 (s, 2H), 7.46 (d, J = 8.8 Hz, 1H), 5.50 (s, 1H), 1H), 7.95 7.95 (d, (d, JJJ = = 2.6 2.6 Hz, Hz, 1H), 1H), 7.79 7.79 (dd, (dd, JJ = = 2.6, 1H), 7.52 7.52 (s, (s, 2H), 2H), 7.46 7.46 (d, (d, JJ = = 8.8 Hz, 1H), 1H), 5.50 5.50 (s, 2.6, 8.7 8.7 Hz, Hz, 1H), 8.8 Hz, (t, JJ == 2.7 Hz, 1H), 4.00 (d, JJ == 13.2 Hz, 1H), 3.90 (d, JJ == 13.2 Hz, 1H), 3.84 (s, 3H), 3.66–3.59 (m, 2H), (t, 2.7 Hz, 1H), 4.00 (d, 13.2 Hz, 1H), 3.90 (d, 13.2 Hz, 1H), 3.84 (s, 3H), 3.66–3.59 (m, 2H), (t, J = 2.7 Hz, 1H), 4.00 (d, J = 13.2 Hz, 1H), 3.90 (d, J == 13.2 13.2 Hz, Hz, 1H), 1H), 3.84 3.84 (s, 3H), 3.66–3.59 3.66–3.59 (m, (m, 2H), 2H), 1.95–1.66 (m, 6H). 1.95–1.66 (m, (m, 6H). 6H). 1.95–1.66

(E)-N-(3-Chloro-4-methoxyphenyl)-3-(3,5-dibromo-4-methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2(E)-N-(3-Chloro-4-methoxyphenyl)-3-(3,5-dibromo-4-methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2(E)-N-(3-Chloro-4-methoxyphenyl)-3-(3,5-dibromo-4-methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2(E)-N-(3-Chloro-4-methoxyphenyl)-3-(3,5-dibromo-4-methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy] yl)oxy]imino]propanamide yl)oxy]imino]propanamide (33-THP) (33-THP) yl)oxy]imino]propanamide (33-THP) imino]propanamide (33-THP) Carboxylic acid 22a Carboxylic acid acid 22a 22a (0.20 (0.20 g, g, 0.44 0.44 mmol) mmol) and and 3-chloro-4-methoxyaniline 3-chloro-4-methoxyaniline (0.11 (0.11 g, g, 0.67 0.67 mmol, mmol, 1.5 1.5 Carboxylic (0.20 g, 0.44 mmol) and 3-chloro-4-methoxyaniline (0.11 g, 0.67 mmol, 1.5 Carboxylic acid 22a (0.20 g, 0.44 mmol) and 3-chloro-4-methoxyaniline (0.11 g, 0.67 mmol, 1.5 equiv.) were reacted according to the general procedure for amide coupling. The product was equiv.) were were reacted reacted according according to to the general general procedure procedure for for amide coupling. coupling. The The product product was was equiv.) equiv.) were reacted according to the the general procedure forCartridge amideamide coupling. The product was purified purified by column chromatography, Biotage SNAP KP-Sil 25 g, gradient elution: purified by column chromatography, Biotage SNAP Cartridge KP-Sil 25 g, gradient elution: purified bychromatography, column chromatography, Biotage SNAP Cartridge KP-Sil elution: 25 g, gradient elution: 1 by column Biotage SNAP Cartridge KP-Sil 25 g, gradient (hexane/EtOAc, (hexane/EtOAc, (hexane/EtOAc, 7→60%) 7→60%) to to give give 33-THP 33-THP as as aaa light light yellow yellow 1solid solid (0.20 (0.20 g, g, 77%). 77%). 11H H NMR NMR (400 (400 MHz, MHz, (hexane/EtOAc, 7→60%) to give 33-THP as light yellow solid (0.20 g, 77%). H NMR (400 MHz, 7→60%) give 33-THP as(d, a light yellow solid7.53 (0.20(s,g,2H), 77%). H(dd, NMR (400 MHz, CDCl δ 8.51 (s, 1H), CDCl 3) δ to 8.51 (s, 1H), 7.70 J = 2.6 Hz, 1H), 7.44 J = 2.6, 8.9 Hz, 1H), (d, J 3 )6.89 CDCl33)) δδ 8.51 8.51 (s, (s, 1H), 1H), 7.70 7.70 (d, (d, JJ == 2.6 2.6 Hz, Hz, 1H), 1H), 7.53 7.53 (s, (s, 2H), 2H), 7.44 7.44 (dd, (dd, JJ == 2.6, 2.6, 8.9 8.9 Hz, Hz, 1H), 1H), 6.89 6.89 (d, (d, JJ === 8.9 8.9 CDCl 8.9 7.70 1H), (d, J 5.50–5.46 = 2.6 Hz,(m, 1H), 7.533.99 (s, 2H), (dd, J1H), = 2.6, 8.9(d, Hz, 6.89 (d, J = 8.9(s, Hz, 1H), 5.50–5.46 Hz, 1H), (d, JJ ==7.44 13.2 Hz, 3.89 JJ ==1H), 13.2 Hz, 1H), 3.89 3H), 3.84 (s, 3H), Hz, 1H), 5.50–5.46 (m, 1H), 3.99 (d, 13.2 Hz, 1H), 3.89 (d, 13.2 Hz, 1H), 3.89 (s, 3H), 3.84 (s, 3H), Hz, 1H), 1H), 3.99 5.50–5.46 (m, 1H), J = (d, 13.2J =Hz, 1H), J = (s, 13.2 Hz,3.84 1H),(s,3.89 (s,3.65–3.60 3H), 3.84(m, (s, 3H), (m, (d, J =1.92–1.66 13.2 Hz,3.99 1H),(d, 3.89 13.2 Hz,3.89 1H),(d,3.89 3H), 3H), 2H), 3.65–3.60 3.65–3.60 (m, (m, 2H), 2H), 1.92–1.66 1.92–1.66 (m, (m, 6H). 6H). 3.65–3.60 (m, 2H), (m, 6H). 1.92–1.66 (m, 6H).

(E)-N-(3-Bromo-4-methoxyphenyl)-3-(3,5-dibromo-4-methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2(E)-N-(3-Bromo-4-methoxyphenyl)-3-(3,5-dibromo-4-methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2(E)-N-(3-Bromo-4-methoxyphenyl)-3-(3,5-dibromo-4-methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2yl)oxy]imino]propanamide yl)oxy]imino]propanamide (34-THP) (34-THP) yl)oxy]imino]propanamide (34-THP) Carboxylic acid 22a Carboxylic acid acid 22a 22a (0.20 (0.20 g, g, 0.44 0.44 mmol) mmol) and and 3-bromo-4-methoxyaniline 3-bromo-4-methoxyaniline (0.13 (0.13 g, g, 0.67 0.67 mmol, mmol, 1.5 1.5 Carboxylic (0.20 g, 0.44 mmol) and 3-bromo-4-methoxyaniline (0.13 g, 0.67 mmol, 1.5 equiv.) were reacted according to the general procedure for amide coupling. The product was equiv.) were were reacted reacted according according to to the the general general procedure procedure for for amide amide coupling. coupling. The The product product was was equiv.) purified by column chromatography, Biotage SNAP Cartridge KP-Sil 25 g, gradient elution: purified by column chromatography, Biotage SNAP Cartridge KP-Sil 25 g, gradient elution: purified by column chromatography, Biotage SNAP Cartridge KP-Sil 25 g, gradient elution: 1

Mar. Drugs 2018, 16, 481

16 of 26

(E)-N-(3-Bromo-4-methoxyphenyl)-3-(3,5-dibromo-4-methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy] imino]propanamide (34-THP) Carboxylic acid 22a (0.20 g, 0.44 mmol) and 3-bromo-4-methoxyaniline (0.13 g, 0.67 mmol, 1.5 equiv.) were reacted according to the general procedure for amide coupling. The product was purified by column chromatography, Biotage SNAP Cartridge KP-Sil 25 g, gradient elution: (heptane/EtOAc, 7→60%) to give 34-THP as a light yellow solid (0.20 g, 71%). 1 H NMR (400 MHz, CDCl3 ) δ 8.51 (s, 1H), 7.85 (d, J =2018, 2.6 16, Hz, FOR 1H),PEER 7.53 REVIEW (s, 2H), 7.51 (dd, J = 2.6, 8.9 Hz, 1H), 6.86 (d, J = 8.9 Hz, 1H), 5.47 (d,16 J =of2.9 Mar. 25 Mar. Drugs Drugs 2018, 16, xx FOR PEER REVIEW 16 of 25 Hz, 1H), 3.99 (d, J = 13.1 Hz, 1H), 3.92–3.87 (m, 4H), 3.84 (s, 3H), 3.65–3.60 (m, 2H), 1.91–1.66 (m, 6H).

(E)-3-(3,5-Dibromo-4-methoxyphenyl)-N-(3,5-dichlorophenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy](E)-3-(3,5-Dibromo-4-methoxyphenyl)-N-(3,5-dichlorophenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy](E)-3-(3,5-Dibromo-4-methoxyphenyl)-N-(3,5-dichlorophenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]-imino] imino]propenamide (35-THP) imino]propenamide (35-THP) propenamide (35-THP) Carboxylic acid (0.11 g, 0.67 0.67 mmol, 1.5 equiv.) Carboxylic acid 22a 22a (0.20 (0.20 g, g, 0.44 0.44 mmol) mmol) and and 3,5-dichloroaniline 3,5-dichloroaniline (0.11 (0.11 g, 0.67 mmol, mmol, 1.5 1.5 equiv.) were reacted according to the general procedure for amide coupling. The product was purified were reacted reacted according according to to the the general general procedure procedure for for amide amide coupling. coupling. The product was purified by by column chromatography, Biotage SNAP Cartridge KP-Sil 25 g and SNAP KP-NH 11g, gradient column chromatography, chromatography,Biotage Biotage SNAP Cartridge KP-Sil and SNAP 11g, gradient column SNAP Cartridge KP-Sil 25 g 25 andgSNAP KP-NHKP-NH 11g, gradient elution: 1H NMR (400 MHz, elution: to as yellow oil (0.18 elution: (heptane/EtOAc, (heptane/EtOAc, 7→60%). to give give 35-THP 35-THP as aaoil yellow oil69%). (0.181g, g, 69%). H NMR MHz, (heptane/EtOAc, 7→60%).7→60%). to give 35-THP as a yellow (0.18 g, H 69%). NMR 1(400 MHz,(400 CDCl 3) δ CDCl 3) δ 8.64 (s, 1H), 7.58 (d, J = 1.8 Hz, 2H), 7.52 (s, 2H), 7.12 (t, J = 1.8 Hz, 1H), 5.48 (t, J = 2.7 Hz, 1H), CDCl δ 8.64 (s, (d, 1H), = 1.8 7.52 Hz, (s, 2H), 7.52 (s, (t, 2H), (t, J 1H), = 1.8 5.48 Hz, 1H), (t, J =1H), 2.7 3.98 Hz, 1H), 8.64 (s,3)1H), 7.58 J =7.58 1.8 (d, Hz,J 2H), 2H), 7.12 J =7.12 1.8 Hz, (t, J =5.48 2.7 Hz, (d, J 3.98 (d, JJ == 1H), 13.2 (d, JJ == 1H) 13.2 Hz, 3.84 (s, (m, 3.98 (d,Hz, 13.2 Hz, Hz, 1H), 1H), 3.88 (d,Hz, 13.2 3.84 Hz, 1H) 1H)3H), 3.843.65–3.59 (s, 3H), 3H), 3.65–3.59 3.65–3.59 (m, 2H), 2H), 1.92–1.66 1.92–1.66 (m, 6H). 6H). = 13.2 3.88 (d, J 3.88 = 13.2 (s, (m, 2H), 1.92–1.66 (m, 6H). (m, MeO MeO Br Br

Br Br THPO THPO N N

H H N N

N N

O O

(E)-3-(3,5-Dibromo-4-methoxyphenyl)-N-(pyridin-2-yl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino] (E)-3-(3,5-Dibromo-4-methoxyphenyl)-N-(pyridin-2-yl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino] (E)-3-(3,5-Dibromo-4-methoxyphenyl)-N-(pyridin-2-yl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino] propanamide (36-THP) propanamide (36-THP) (36-THP) propanamide Carboxylic acid 22a (0.20 0.44 mmol) and 2-aminopyridine (0.063 g, mmol, 1.5 Carboxylic acid acid22a 22a(0.20 (0.20g,g, g,0.44 0.44 mmol) and 2-aminopyridine (0.063 g, 0.67 0.67 mmol, 1.5 equiv.) equiv.) Carboxylic mmol) and 2-aminopyridine (0.063 g, 0.67 mmol, 1.5 equiv.) were were reacted according to the general procedure for amide coupling. The product was purified by were reacted according to the general procedure for amide coupling. The product was purified by reacted according to the general procedure for amide coupling. The product was purified by column column chromatography, Biotage SNAP Cartridge KP-Sil 25 g, gradient elution: (hexane/EtOAc, 12 column chromatography, Biotage SNAP Cartridge KP-Sil 25 g, gradient elution: (hexane/EtOAc, chromatography, Biotage SNAP Cartridge KP-Sil 25 g, gradient elution: (hexane/EtOAc, 12→60%) 12 to 1 →60%) to 36-THP as yellow solid (0.11 g, 45%). (400 MHz, CDCl 3) δ 9.26 (s, 1H), 1H 1H →60%) to give give as aa light light yellow (0.11 g,NMR 45%).(400 H NMR NMR MHz, CDCl 3)1H), δ 9.26 (s, 1H), give 36-THP as36-THP a light yellow solid (0.11 solid g, 45%). MHz,(400 CDCl ) δ 9.26 (s, 8.32–8.28 3 8.32–8.28 (m, 8.24 (d, JJ == 8.4 1H), (m, 1H), 7.53 (s, 2H), 7.06 J = 0.8, 4.9, 7.3 Hz, 8.32–8.28 (m, 1H), 1H), 8.4 Hz, Hz, 1H), 7.76–7.70 7.76–7.70 1H), 2H), 7.06 J(ddd, (ddd, 0.8,7.3 4.9,Hz, 7.31H), Hz, (m, 1H), 8.24 (d, J 8.24 = 8.4(d, Hz, 1H), 7.76–7.70 (m, 1H),(m, 7.53 (s, 7.53 2H),(s, 7.06 (ddd, = 0.8,J =4.9, 1H), 5.47 (t, JJ == 2.9 Hz, 1H), 3.99 (d, JJ == 13.2 Hz, 1H), 3.89 (d, JJ == 13.2 Hz, 1H), 3.83 (s, 3H), 3.64–3,61 1H), 5.47 (t, 2.9 Hz, 1H), 3.99 (d, 13.2 Hz, 1H), 3.89 (d, 13.2 Hz, 1H), 3.83 (s, 3H), 3.64–3,61 5.47 (t, J = 2.9 Hz, 1H), 3.9913(d, J = 13.2 Hz, 1H), 3.89 (d, J = 13.2 Hz, 1H), 3.83 (s, 3H), 3.64–3,61 (m, (m, 1.91–1.64 (m, 6H). C (100 MHz, CDCl 3) δ 160.5, 153.0, 151.8, 150.7, 148.1, 138.6, 134.6, 13 C13NMR (m, 2H), 2H), 1.91–1.64 C NMR NMR (100 MHz, CDCl 3) δ 160.5, 153.0, 151.8, 150.7, 148.1, 148.1, 138.6, 138.6, 134.6, 2H), 1.91–1.64 (m, (m, 6H).6H). (100 MHz, CDCl 134.6, 3 ) δ 160.5, 153.0, 151.8, 150.7, +): calculated 525.9977 133.7, 120.2, 118.0, 114.2, 102.3, 62.5, 60.7, 28.8, 28.5, 25.1, 19.0. HRMS (ESI +): calculated 525.9977 + 133.7, 120.2, 118.0, 114.2, 102.3, 62.5, 60.7, 28.8, 28.5, 25.1, 19.0. HRMS (ESI 133.7, 120.2, 118.0, 114.2, 102.3, 62.5, 60.7, 28.8, 28.5, 25.1, 19.0. HRMS (ESI ): calculated 525.9977 (C 20H22Br2N3O4), found 525.9972. (C20 20H 3O4), found 525.9972. (C H2222Br Br2N 2 N3 O4 ), found 525.9972.

(E)-3-(3,5-Dibromo-4-methoxyphenyl)-N-(pyridin-3-yl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino] (E)-3-(3,5-Dibromo-4-methoxyphenyl)-N-(pyridin-3-yl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino] (E)-3-(3,5-Dibromo-4-methoxyphenyl)-N-(pyridin-3-yl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino] propanamide propanamide (37-THP) (37-THP) propanamide (37-THP)22a (0.42 g, 0.94 mmol), 3-aminopyridine (0.11 g, 1.12 mmol, 1.2 equiv.) were Carboxylic Carboxylic acid acid 22a (0.42 g, 0.94 mmol), 3-aminopyridine (0.11 g, 1.12 mmol, 1.2 equiv.) were Carboxylic acid 22a (0.42 g,procedure 0.94 mmol), 3-aminopyridine (0.11 g, 1.12 was mmol, 1.2 equiv.) were reacted according reacted according to to the the general general procedure for for amide amide coupling. coupling. The The product product was purified purified by by column column reacted according to the general procedure for amide coupling. The product was purified by column chromatography chromatography Biotage Biotage SNAP SNAP Cartridge Cartridge KP-Sil KP-Sil 25 25 g g eluent eluent (DCM/MeOH (DCM/MeOH (0→10%) (0→10%) gradient gradient to to give give chromatography Biotage SNAP Cartridge KP-Sil 25 g eluent (DCM/MeOH (0 → 10%) gradient to give 1 the the product product 37-THP 37-THP as as an an oil oil (0.34 (0.34 g, g, 68%). 68%). 11H H NMR NMR (400 (400 MHz, MHz, CDCl CDCl33)) δδ 8.71 8.71 (d, (d, JJ == 10.1 10.1 Hz, Hz, 1H), 1H), 8.38 8.38 the product 37-THP as1.4, an 2.7, oil (0.34 g, 68%). H NMR (400 MHz, CDCl 8.71 (d, J = 5.51 10.1 Hz, 1H), 8.38 3) δ (s, 1H), 8.20 (ddd, J = 8.4 Hz, 1H), 7.53 (s, 2H), 7.30 (dd, J = 4.6, 8.4 Hz, 1H), (t, J = 2.9 (s, 1H), 8.20 (ddd, J = 1.4, 2.7, 8.4 Hz, 1H), 7.53 (s, 2H), 7.30 (dd, J = 4.6, 8.4 Hz, 1H), 5.51 (t, J = 2.9 Hz, Hz, (s, 1H), 8.20 (ddd, J = 1.4, 2.7, 8.4 Hz, 1H), 7.53(m, (s, 2H), 1.90–1.59 7.30 (dd, J = 4.6, 8.4 Hz, 1H), 5.51 (t, J = 2.9 Hz, 1H), 1H), 4.06–3.87 4.06–3.87 (m, (m, 2H), 2H), 3.84 3.84 (s, (s, 3H), 3H), 3.67–3.59 3.67–3.59 (m, 2H), 2H), 1.90–1.59 (m, (m, 6H). 6H). 1H), 4.06–3.87 (m, 2H), 3.84 (s, 3H), 3.67–3.59 (m, 2H), 1.90–1.59 (m, 6H).

(E)-3-(3,5-Dibromo-4-methoxyphenyl)-N-(pyridin-4-yl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino] (E)-3-(3,5-Dibromo-4-methoxyphenyl)-N-(pyridin-4-yl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]

Carboxylic acid 22a (0.42 g, 0.94 mmol), 3-aminopyridine (0.11 g, 1.12 mmol, 1.2 equiv.) were reacted according to the general procedure for amide coupling. The product was purified by column chromatography Biotage SNAP Cartridge KP-Sil 25 g eluent (DCM/MeOH (0→10%) gradient to give the product 37-THP as an oil (0.34 g, 68%). 1H NMR (400 MHz, CDCl3) δ 8.71 (d, J = 10.1 Hz, 1H), 8.38 (s, 1H), Hz, Mar. Drugs8.20 2018,(ddd, 16, 481J = 1.4, 2.7, 8.4 Hz, 1H), 7.53 (s, 2H), 7.30 (dd, J = 4.6, 8.4 Hz, 1H), 5.51 (t, J = 2.9 17 of 26 1H), 4.06–3.87 (m, 2H), 3.84 (s, 3H), 3.67–3.59 (m, 2H), 1.90–1.59 (m, 6H).

(E)-3-(3,5-Dibromo-4-methoxyphenyl)-N-(pyridin-4-yl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino] (E)-3-(3,5-Dibromo-4-methoxyphenyl)-N-(pyridin-4-yl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino] propanamide (38-THP) propanamide (38-THP) Carboxylic acid 22a (0.20 g, 0.45 mmol) and 4-aminopyridine (0.040 g, 0.45 mmol) were reacted Carboxylic acid 22a (0.20 g, 0.45 mmol) and 4-aminopyridine (0.040 g, 0.45 mmol) were reacted according2018, to thex FOR general procedure for amide coupling. The product was purified by column Mar. PEER REVIEW 17 Mar. Drugs Drugs 2018, 16, PEER procedure REVIEW 17 of of 25 25 according to 16, thex FOR general for amide coupling. The product was purified by column chromatography Biotage SNAP Cartridge KP-Sil 10 g, eluent (DCM/MeOH, 0→10%) gradient to give chromatography Biotage SNAP Cartridge KP-Sil 10 g, eluent (DCM/MeOH, 0→10%) gradient to give 1 the 38-THP as an oil (0.049 g, 21%). CDCl Hz, 2H), 7.64 38-THP as as an an oil oil (0.049 MHz, CDCl (d, JJJ === 5.6 the product product 38-THP (0.049 g, g, 21%). 21%). 111H H NMR NMR (400 (400 MHz, CDCl3333)) δδδ 8.55 8.55 (d, 5.6 MHz, (d, 5.6 Hz, Hz, 2H), 2H), 7.64 (d, J = 5.8 Hz, 2H), 7.52 (s, 2H), 5.51 (t, 1H), 4.04–3.86 (m, 2H), 3.84 (s, 3H), 3.66–3.59 (m, 2H), 1.85–1.60 (d, J == 5.8 5.8 Hz, Hz, 2H), 2H), 7.52 7.52 (s, (s, 2H), 2H), 5.51 5.51 (t, (t, 1H), 1H), 4.04–3.86 4.04–3.86 (m, (m, 2H), 2H), 3.84 3.84 (s, (s, 3H), 3H), 3.66–3.59 3.66–3.59 (m, (m, 2H), 2H), 1.85–1.60 1.85–1.60 (m, 6H). (m, 6H). (m, 6H).

(E)-3-(3,5-Dibromo-4-methoxyphenyl)-N-(pyridin-3-ylmethyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino) (E)-3-(3,5-Dibromo-4-methoxyphenyl)-N-(pyridin-3-ylmethyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino) (E)-3-(3,5-Dibromo-4-methoxyphenyl)-N-(pyridin-3-ylmethyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino) propanamide propanamide (39-THP) (39-THP) propanamide (39-THP) Carboxylic Carboxylic acid acid 22a 22a (0.20 (0.20 g, g, 0.44 0.44 mmol) mmol) and and 3-picolylamine 3-picolylamine (0.068 (0.068 mL, mL, 0.67 0.67 mmol, mmol, 1.5 1.5 equiv.) equiv.) Carboxylic acid 22a (0.20 g, 0.44 mmol) and 3-picolylamine (0.068 mL, 0.67 mmol, 1.5 equiv.) were were by were reacted reacted according according to to the the general general procedure procedure for for amide amide coupling. coupling. The The product product was was purified purified by reacted according to the general procedure for amide coupling. The product was purified by column column chromatography, Biotage SNAP Cartridge KP-Sil 25 g, gradient elution: (DCM/MeOH, 0→ column chromatography, Biotage SNAP Cartridge KP-Sil 25 g, gradient elution: (DCM/MeOH, 0→ 11H NMR chromatography, Biotage Cartridge 25 (400 g, gradient elution: (DCM/MeOH, 0→ to 10%) as oil g, MHz, 33)) δ 8.55 (br, 2H), 7.63 (d,10%) J = 7.8 1 H NMR (400 10%) to to give give 39-THP 39-THP as an anSNAP oil (0.20 (0.20 g, 82%). 82%). KP-Sil MHz, CDCl CDCl 3 δ 8.55 (br, 2H), 7.63 (d, J = 7.8 1 H NMR (400 MHz, CDCl ) δ 8.55 (br, 2H), 7.63 (d, J = 7.8 Hz, 1H), give 39-THP as an oil (0.20 g, 82%). Hz, Hz, 1H), 1H), 7.51 7.51 (s, (s, 2H), 2H), 7.30–7.25 7.30–7.25 (m, (m, 1H), 1H), 7.22 7.22 (t, (t, JJ == 6.1 6.1 Hz, Hz, 1H), 1H),3 5.36 5.36 (t, (t, JJ == 2.4 2.4 Hz, Hz, 1H), 1H), 4.61 4.61 (dd, (dd, JJ == 6.6, 6.6, 7.51 Hz, (s, 2H), 7.30–7.25 (m, 1H),15.1 7.22Hz, (t, 1H), J = 6.1 Hz, 1H), 5.36Hz, (t, J1H), = 2.43.88–3.81 Hz, 1H),(m, 4.61 (dd,3.60–3.55 J = 6.6, 15.1 15.1 1H), 4.44 (dd, J = 5.8, 3.94 (d, J = 13.2 4H), 15.1 Hz, 1H), 4.44 (dd, J = 5.8, 15.1 Hz, 1H), 3.94 (d, J = 13.2 Hz, 1H), 3.88–3.81 (m, 4H), 3.60–3.55 (m, (m, Hz, 4.44 (dd, J6H). = 5.8, 15.1 Hz, 1H), 3.94 (d, J = 13.2 Hz, 1H), 3.88–3.81 (m, 4H), 3.60–3.55 (m, 2H), 2H), 1.88–1.62 (m, 2H),1H), 1.88–1.62 (m, 6H). 1.88–1.62 (m, 6H).

(E)-3-(3,5-Dibromo-4-methoxyphenyl)-N-(4-(dimethylamino)phenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy] (E)-3-(3,5-Dibromo-4-methoxyphenyl)-N-(4-(dimethylamino)phenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy] (E)-3-(3,5-Dibromo-4-methoxyphenyl)-N-(4-(dimethylamino)phenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy] imino]propanamide imino]propanamide (40-THP) (40-THP) imino]propanamide (40-THP) Carboxylic acid 22a (0.22 g, 0.49 mmol and 4-(dimethylamino)aniline (0.060 g, 0.49 mmol) were Carboxylic Carboxylic acid acid 22a 22a (0.22 (0.22 g, g, 0.49 0.49 mmol mmol and and 4-(dimethylamino)aniline 4-(dimethylamino)aniline (0.060 (0.060 g, g, 0.49 0.49 mmol) mmol) were were reacted according to the general procedure for amide coupling. The product was purified by column reacted according to the general procedure for amide coupling. The product was purified by column reacted according to the general procedure for amide coupling. The product was purified by column chromatography, Biotage SNAP Cartridge KP-Sil 25 g, gradient elution: (EtOAc/MeOH, to chromatography, Biotage SNAP SNAP Cartridge Cartridge KP-Sil KP-Sil 25 25 g, g,gradient gradientelution: elution:(EtOAc/MeOH, (EtOAc/MeOH, 0→20%) 0→20%) chromatography, Biotage 0→20%) to to 1 give 40-THP as an oil (0.16 g, 56%). NMR (400 MHz, CDCl 33)) δ 7.55 (s, 2H), 7.45 (d, J = 9.0 Hz, 2H), 11H give 40-THP as an oil (0.16 g, 56%). H NMR (400 MHz, CDCl 3 δ 7.55 (s, 2H), 7.45 (d, J = 9.0 Hz, 2H), 1 give 40-THP as an oil (0.16 g, 56%). H NMR (400 MHz, CDCl3 ) δ 7.55 (s, 2H), 7.45 (d, J = 9.0 Hz, 6.72 (d, JJ == 8.5 2H), 5.47 (t, 1H), 4.03-3.89 (m, 3.83 (s, 3H), 3.65–3.60 (m, 2.93 (s, 6H), 1.91– 6.72 8.5J Hz, Hz, 1H), (m, 2H), 2H), 3H), (m, 2H), 2H), 6H), 2H),(d, 6.72 (d, = 8.52H), Hz,5.47 2H),(t,5.47 (t,4.03-3.89 1H), 4.03-3.89 (m,3.83 2H),(s,3.83 (s,3.65–3.60 3H), 3.65–3.60 (m,2.93 2H),(s,2.93 (s,1.91– 6H), 1.57 (m, 6H) 1.57 (m, 6H) 1.91–1.57 (m, 6H)

(E)-N,3-Bis(3-bromo-4-methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propenamide (E)-N,3-Bis(3-bromo-4-methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propenamide (41-THP) (41-THP) (E)-N,3-Bis(3-bromo-4-methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propenamide (41-THP) Carboxylic acid 22b (0.40 g, 1.07 mmol) and 3-bromo-4-methoxyaniline (0.12 g, 1.29 mmol, 1.2 Carboxylic g, 1.07 1.07 mmol) mmol) and and 3-bromo-4-methoxyaniline 3-bromo-4-methoxyaniline (0.12 Carboxylic acid acid 22b 22b (0.40 (0.40 g, (0.12 g, g, 1.29 1.29 mmol, mmol, 1.2 1.2 equiv.) were reacted according to the general procedure for amide coupling. The product was equiv.) were reacted according to the general procedure for amide coupling. The product was equiv.) were reacted according to the general procedure for amide coupling. The product was purified purified by chromatography Biotage SNAP KP-Sil 25 (DCM/MeOH (0→ purified by column column chromatography SNAP Cartridge Cartridge 25 g g eluent eluent (DCM/MeOH (0→ by column chromatography Biotage Biotage SNAP Cartridge KP-Sil KP-Sil 25 g eluent (DCM/MeOH (0→10%) 11H NMR (400 MHz, CDCl3) δ 8.54 (s, 1H), 7.86 10%) gradient to give 41-THP as an oil (0.43 g, 72%). 10%) gradient to give 41-THP as an oil (0.43 g, 72%). 1H NMR (400 MHz, CDCl33) δ 8.54 (s, 1H), 7.86 (d, (d, JJ == 2.6 2.6 Hz, Hz, 1H), 1H), 7.58 7.58 (d, (d, JJ == 2.2 2.2 Hz, Hz, 1H), 1H), 7.49 7.49 (dd, (dd, JJ == 2.6, 2.6, 8.9 8.9 Hz, Hz, 1H), 1H), 7.29 7.29 (dd, (dd, JJ == 2.2, 2.2, 8.4 8.4 Hz, Hz, 1H), 1H), 6.85 (d, J = 8.9 Hz, 1H), 6.80 (d, J = 8.5 Hz, 1H), 5.46 (t, J = 2.8 Hz, 1H), 3.99 (d, J = 13.0 Hz, 1H), 3.91 6.85 (d, J = 8.9 Hz, 1H), 6.80 (d, J = 8.5 Hz, 1H), 5.46 (t, J = 2.8 Hz, 1H), 3.99 (d, J = 13.0 Hz, 1H), 3.91 (d, (d, JJ == 13.0 13.0 Hz, Hz, 1H), 1H), 3.87 3.87 (s, (s, 3H), 3H), 3.85 3.85 (s, (s, 3H), 3H), 3.71–3.58 3.71–3.58 (m, (m, 2H), 2H), 1.92–1.60 1.92–1.60 (m, (m, 6H). 6H).

(E)-N,3-Bis(3-bromo-4-methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propenamide (41-THP) Carboxylic acid 22b (0.40 g, 1.07 mmol) and 3-bromo-4-methoxyaniline (0.12 g, 1.29 mmol, 1.2 Mar. Drugs 2018, 16, 481 18 of 26 equiv.) were reacted according to the general procedure for amide coupling. The product was purified by column chromatography Biotage SNAP Cartridge KP-Sil 25 g eluent (DCM/MeOH (0→ 1H NMR (400 MHz, CDCl3) δ 8.54 (s, 1H), 7.86 10%) gradient to41-THP give 41-THP as an oilg,(0.43 g, 172%). gradient to give as an oil (0.43 72%). H NMR (400 MHz, CDCl3 ) δ 8.54 (s, 1H), 7.86 (d, J = (d, = 2.6 Hz, 1H), (d, Hz, J = 2.2 Hz, 1H), J = Hz, 2.6, 1H), 8.9 Hz, (dd,8.4 J =Hz, 2.2,1H), 8.4 Hz, 2.6 JHz, 1H), 7.58 (d,7.58 J = 2.2 1H), 7.49 (dd,7.49 J = (dd, 2.6, 8.9 7.291H), (dd,7.29 J = 2.2, 6.85 1H), (d, J 6.85 (d, J = 8.9 Hz, 1H), 6.80 (d, J = 8.5 Hz, 1H), 5.46 (t, J = 2.8 Hz, 1H), 3.99 (d, J = 13.0 Hz, 1H), 3.91 (d, = 8.9 Hz, 1H), 6.80 (d, J = 8.5 Hz, 1H), 5.46 (t, J = 2.8 Hz, 1H), 3.99 (d, J = 13.0 Hz, 1H), 3.91 (d, J = 13.0 JHz, = 13.0 1H), 3.87 (s, 3H), 3.85 (s, 3H), 3.71–3.58 2H), 1.92–1.60 1H),Hz, 3.87 (s, 3H), 3.85 (s, 3H), 3.71–3.58 (m, 2H),(m, 1.92–1.60 (m, 6H).(m, 6H).

(E)-3-(3-Bromo-4-methoxyphenyl)-N-(pyridin-2-yl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanamide Mar. Drugs 2018, 16, x FOR PEER REVIEW 18 of 25 (E)-3-(3-Bromo-4-methoxyphenyl)-N-(pyridin-2-yl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino] propanamide Mar. Drugs 2018, 16, x FOR PEER REVIEW 18 of 25 (42-THP) Mar. Drugs 2018, 16, x FOR PEER REVIEW 18 of 25 (42-THP) Carboxylic acid 22b (0.40 g, 1.07 mmol) and 2-aminopyridine (0.12 g, 1.29 mmol, 1.2 equiv.) were Carboxylic acid acid 22b 22b (0.40 1.07 mmol) mmol) and and 2-aminopyridine 2-aminopyridine (0.12 1.29 mmol, mmol, 1.2 1.2 equiv.) equiv.) were were Carboxylic acid 22b (0.40 g, g, 1.07 (0.12 g, g, 1.29 Carboxylic reacted according to the general procedure for amide coupling. The product was purified by column reacted according to the general procedure for amide coupling. The product was purified by column procedure for amide coupling. The product was purified by column reacted accordingBiotage to the general procedure chromatography SNAP Cartridge KP-Sil 25 gg eluent (DCM/MeOH (0→10%) gradient to give chromatography Biotage SNAP Cartridge KP-Sil25 25gg eluent(DCM/MeOH (DCM/MeOH (0→10%) (0→10%) gradient to give give chromatography Biotage SNAP Cartridge eluent (0 →10%) gradient gradient to to give chromatography Biotage SNAP1 Cartridge KP-Sil KP-Sil 25 eluent (DCM/MeOH 42-THP as an oil (0.31 g, 64%). H NMR (400 MHz, CDCl 3) δ 9.27 (s, 1H), 8.29 (ddd, J = 0.9, 2.0, 4.9 Hz, 1 42-THP as an oil (0.31 g, 64%). H NMR (400 MHz, CDCl 3 ) δ 9.27 (s, 1H), 8.29 (ddd, J = 0.9, 2.0, 4.9 Hz, 1 42-THP as as an an oil oil (0.31 (0.31 g, g, 64%). 64%). H H NMR NMR (400 (400 MHz, MHz, CDCl CDCl33)) δδ 9.27 9.27 (s, (s, 1H), 1H), 8.29 8.29 (ddd, (ddd, J ==0.9, 0.9,2.0, 2.0,4.9 4.9 Hz, Hz, 42-THP 1H), 8.23 (dt, JJ == 1.0, 8.4 Hz, 1H), 7.70 (ddd, JJ == 1.9, 7.4, 8.5 Hz, 1H), 7.57 (d, JJ == 2.2 Hz, 1H), 7.07–7.01 1H), 8.23 (dt, 1.0, 8.4 Hz, 1H), 7.70 (ddd, 1.9, 7.4, 8.5 Hz, 1H), 7.57 (d, 2.2 Hz, 1H), 7.07–7.01 1H), 8.23 (dt, J = 1.0, 8.4 Hz, 1H), 7.70 (ddd, = 1.9, 7.4, 8.5 Hz, 1H), 7.57 (d, = 2.2 Hz, 1H), 7.07–7.01 1H), 8.23 (dt, J = 1.0, 8.4 Hz, 1H), 7.70 (ddd, J = 1.9, 7.4, 8.5 Hz, 1H), 7.57 (d, J = 2.2 Hz, 1H), 7.07–7.01 (m, 1H), 6.80 (d, = 8.4 Hz, 1H), 5.47–5.44 (m, 1H), 3.99 (d, == 13.1 Hz, 1H), 3.91 (d, J == 13.1 Hz, 1H), (m, 1H), 1H), 6.80 6.80 (d, (d, JJJJ = 8.4 Hz, Hz, 1H), 1H), 5.47–5.44 5.47–5.44 (m, (m, 1H), 1H), 3.99 3.99 (d, (d, JJJ == 13.1 Hz, 1H), 3.91 (d, 13.1 Hz, 1H), (m, 1H), 8.4 Hz, 1H), 5.47–5.44 (m, 1H), 3.99 (d, 13.1 Hz, Hz, 1H), 1H), 3.91 3.91 (d, (d, JJ == 13.1 Hz, Hz, 1H), 1H), (m, 6.80 (d, == 8.4 13.1 13.1 3.84 (s, 3H), 3.68–3.60 (m, 2H), 1.90–-1.61 (m, 6H). 3.84 (s, 3H), 3.68–3.60 (m, 2H), 1.90–-1.61 (m, 6H). 3.84 (s, (s, 3H), 3H), 3.68–3.60 3.68–3.60 (m, (m, 2H), 2H), 1.90—1.61 (m, 6H). 6H). 3.84 1.90–-1.61 (m,

(E)-N-(3,4-Dichlorobenzyl)-3-(4-methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanamide (E)-N-(3,4-Dichlorobenzyl)-3-(4-methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanamide (E)-N-(3,4-Dichlorobenzyl)-3-(4-methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanamide (E)-N-(3,4-Dichlorobenzyl)-3-(4-methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanamide (43-THP) (43-THP) (43-THP) (43-THP) Carboxylic acid 22d g, 1.36 mmol) and 3,4-dichlorobenzylamine (0.11 g, mmol, 1.2 Carboxylic acid acid 22d 22d (0.40 (0.40 g, g, 1.36 1.36 mmol) mmol) and and 3,4-dichlorobenzylamine 3,4-dichlorobenzylamine (0.11 (0.11 g, g, 1.12 1.12 mmol, mmol, 1.2 1.2 Carboxylic Carboxylic acid according 22d (0.40 (0.40 g, 1.36 mmol) and 3,4-dichlorobenzylamine (0.11 g, 1.12 1.12 mmol, 1.2 equiv.) were reacted to the general procedure for amide coupling. 43-THP was obtained equiv.) were reacted according to the general procedure for amide coupling. 43-THP was obtained equiv.) were reacted reactedaccording accordingtotothe generalprocedure procedureforfor amide coupling. 43-THP was obtained equiv.) were general amide coupling. was obtained as 1the as light brown solid (0.54 g, 87%). NMR (400 MHz CDCl 3) δ 7.37 (d, J = 8.243-THP Hz, 1H), 7.34 (d, J = 2.1 1H as light brown solid (0.54 g, 87%). H NMR (400 MHz CDCl 3) δ 7.37 (d, J = 8.2 Hz, 1H), 7.34 (d, J = 2.1 1 1 as light brown solid (0.54 g, 87%). H NMR (400 MHz CDCl 37.37 ) δ 7.37 (d, J = 8.2 Hz, 1H), 7.34 (d, J = 2.1 light brown solid (0.54 g, 87%). H NMR (400 MHz CDCl ) δ (d, J = 8.2 Hz, 1H), 7.34 (d, J = 2.1 Hz, 3 5.38–5.35 (m, 1H), 4.52 (dd, J = 6.7, 15.3 Hz, Hz, 1H), 7.24 (s, 1H), 7.11–7.06 (m, 1H), 6.83–6.79 (m, 2H), Hz, 1H), 1H), 7.24 7.24 (s, (s, 1H), 1H), 7.11–7.06 7.11–7.06 (m, (m, 1H), 1H), 6.83–6.79 6.83–6.79 (m, (m, 2H), 2H), 5.38–5.35 5.38–5.35 (m, (m, 1H), 1H), 4.52 4.52 (dd, (dd, JJ == 6.7, 6.7, 15.3 15.3 Hz, Hz, Hz, 1H), 7.24 (s, 1H), 7.11–7.06 (m,1H), 1H),3.95 6.83–6.79 (m, 2H), 5.38–5.35 (m, 4.52 (dd, J =3.77 6.7, (s, 15.3 Hz,3.69– 1H), 1H), 4.34 (dd, J = 5.8, 15.2 Hz, (d, J = 13.1 Hz, 1H), 3.85 (d, JJ1H), == 13.1 Hz, 1H), 3H), 1H), 4.34 (dd, J = 5.8, 15.2 Hz, 1H), 3.95 (d, J = 13.1 Hz, 1H), 3.85 (d, 13.1 Hz, 1H), 3.77 (s, 3H), 3.69– 1H), 4.34 J(dd, J =15.2 5.8, 15.2 Hz, 1H), J = 13.1 Hz, 1H), J = 13.1 (s, 3H), 3.69– 4.34 (m, (dd, = 5.8, Hz, 1H), 3.95 3.95 (d, J (d, = 13.1 Hz, 1H), 3.85 3.85 (d, J (d, = 13.1 Hz, Hz, 1H),1H), 3.773.77 (s, 3H), 3.69–3.55 3.55 2H), 1.88–1.54 (m, 6H). 3.55 (m, 2H), 1.88–1.54 (m, 6H). 3.55 (m, 2H), 1.88–1.54 (m, 6H). (m, 2H), 1.88–1.54 (m, 6H).

(E)-3-(4-Methoxyphenyl)-N-(pyridin-2-yl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanamide (44(E)-3-(4-Methoxyphenyl)-N-(pyridin-2-yl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanamide (44(44(E)-3-(4-Methoxyphenyl)-N-(pyridin-2-yl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanamide THP) (E)-3-(4-Methoxyphenyl)-N-(pyridin-2-yl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanamide (44-THP) THP) THP) Carboxylic acid 22d (0.40 g, 1.37 mmol), 2-aminopyridine (0.13 g, 1.37 mmol) were reacted Carboxylic acid acid 22d 22d (0.40 (0.40 g, g, 1.37 1.37 mmol), mmol), 2-aminopyridine 2-aminopyridine (0.13 (0.13 g, g, 1.37 1.37 mmol) mmol) were were reacted reacted Carboxylic according general procedure for amide coupling. The by to the general procedure for amide coupling. The product product was was purified column according to the general procedure for amide coupling. The product was purified by column according to the general procedure for amide coupling. The product was purified by column chromatography Biotage SNAP Cartridge KP-Sil 25 gg and 10 g, eluent (EtOAc/MeOH, 0→10%) chromatography Biotage 0→10%) chromatography Biotage Biotage SNAP SNAP Cartridge Cartridge KP-Sil KP-Sil 25 25 g and 10 10 g, g, eluent eluent (EtOAc/MeOH, (EtOAc/MeOH, 0→10%) 0→10%) chromatography and 10 g, eluent (EtOAc/MeOH, 1 gradient (0.095 g, 19%). H NMR (400 MHz, 3OD) δ 8.28 (ddd, J = 5.0, to give 44-THP as an oil (0.095 g, 19%). CD OD) δ = 5.0, 1.9, 1.9, 1 3 gradient to to give give 44-THP 44-THP as as an an oil oil (0.095 (0.095 g, g, 19%). 19%). 1H H NMR NMR (400 (400 MHz, MHz, CD CD33OD) OD) δδ 8.28 8.28 (ddd, (ddd, JJ == 5.0, 5.0, 1.9, gradient 1.9, 0.9 Hz, 1H), 8.18 (dt, J = 8.4, 1.0 Hz, 1H), 7.81 (ddd, J = 8.4, 7.4, 1.9 Hz, 1H), 7.25 (d, J = 8.8 Hz, 2H), Hz, 1H), 8.18 (dt, J = 8.4, 1.0 Hz, 1H), 7.81 (ddd, J = 8.4, 7.4, 1.9 Hz, 1H), 7.25 (d, J = 8.8 Hz, 2H), 7.13 0.9 Hz, Hz, 1H), 1H), 8.18 8.18 (dt, (dt, JJ == 8.4, 8.4, 1.0 1.0 Hz, Hz, 1H), 1H), 7.81 7.81 (ddd, (ddd, JJ == 8.4, 8.4, 7.4, 7.4, 1.9 1.9 Hz, Hz, 1H), 1H), 7.25 7.25 (d, (d, JJ == 8.8 8.8 Hz, Hz, 2H), 2H), 0.9 7.13 (ddd, JJ ==5.0, 7.4, 5.0, 1.0 Hz, 1H), JJ ==Hz, 8.8 Hz, 2H), 3.96-3.79 (m, 2H), (s, 3H), 3.66–3.48 (m, (ddd, J = 7.4, 1.0 Hz, 1H), 6.83 6.83 (d, J (d, = 8.8 2H), 3.96-3.79 (m, 2H), 3.73 3.73 (s, 3H), 3.66–3.48 (m, 2H), 7.13 (ddd, 7.4, 5.0, 1.0 Hz, 1H), 6.83 (d, 8.8 Hz, 2H), 3.96-3.79 (m, 2H), 3.73 (s, 3H), 3.66–3.48 (m, 7.13 (ddd, J = 7.4, 5.0, 1.0 Hz, 1H), 6.83 (d, J = 8.8 Hz, 2H), 3.96-3.79 (m, 2H), 3.73 (s, 3H), 3.66–3.48 (m, 2H), 1.91–1.46 (m, 6H). 1.91–1.46 (m, 6H). 2H), 1.91–1.46 1.91–1.46 (m, (m, 6H). 6H). 2H),

(E)-3-(3-Chloro-4-methoxyphenyl)-N-(pyridin-2-yl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanamide (E)-3-(3-Chloro-4-methoxyphenyl)-N-(pyridin-2-yl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanamide (E)-3-(3-Chloro-4-methoxyphenyl)-N-(pyridin-2-yl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanamide (45-THP) (45-THP) (45-THP) Carboxylic acid 22c (0.32 g, 0.98 mmol) and 2-aminopyridine (0.093 g, 0.98 mmol) were reacted Carboxylic acid acid 22c 22c (0.32 (0.32 g, g, 0.98 0.98 mmol) mmol) and and 2-aminopyridine 2-aminopyridine (0.093 (0.093 g, g, 0.98 0.98 mmol) mmol) were were reacted reacted Carboxylic according to the general procedure for amide coupling. The product was purified by column according to the general procedure for amide coupling. The product was purified by column according to theBiotage generalSNAP procedure for KP-Sil amide 10 coupling. The product was purified by column chromatography Cartridge g, eluent (DCM/MeOH, 0→10%) gradient to give chromatography Biotage Biotage SNAP SNAP Cartridge Cartridge KP-Sil KP-Sil 10 10 g, g, eluent eluent (DCM/MeOH, (DCM/MeOH, 0→10%) 0→10%) gradient gradient to to give give chromatography 1 45-THP as an oil (0.29 g, 74%). H NMR (400 MHz, CDCl 3) δ 8.28 (ddd, J = 4.9, 1.9, 0.9 Hz, 1H), 8.24 1 45-THP as an oil (0.29 g, 74%). H NMR (400 MHz, CDCl3) δ 8.28 (ddd, J = 4.9, 1.9, 0.9 Hz, 1H), 8.24

Mar. Drugs 2018, 16, 481

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(E)-3-(3-Chloro-4-methoxyphenyl)-N-(pyridin-2-yl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanamide (45-THP) Carboxylic acid 22c (0.32 g, 0.98 mmol) and 2-aminopyridine (0.093 g, 0.98 mmol) were reacted according to the general procedure for amide coupling. The product was purified by column chromatography Biotage SNAP Cartridge KP-Sil 10 g, eluent (DCM/MeOH, 0→10%) gradient to give 45-THP as an oil (0.29 g, 74%). 1 H NMR (400 MHz, CDCl3 ) δ 8.28 (ddd, J = 4.9, 1.9, 0.9 Hz, 1H), 8.24 (dt, J = 8.4, 1.0 Hz, 1H), 7.71 (ddd, J = 8.4, 7.4, 1.9 Hz, 1H), 7.40 (d, J = 2.2 Hz, 1H), 7.22 (dd, J = 8.4, 2.2 Hz, 1H), 7.04 (ddd, J = 7.4, 4.9, 1.0 Hz, 1H), 6.82 (d, J = 8.5 Hz, 1H), 5.46 (t, 1H), 3.99 (d, J = 13.1 Hz, 1H), 3.91 (d, J = 13.1 Hz, 1H), 3.85 (s, 3H), 3.68–3.56 (m, 2H), 1.90–1.57 (m, 6H). General Method for THP Deprotection Mar. Drugs 2018, 16, x FOR PEER REVIEW (0.16 THP2018, ethers Mar. Drugs 16, x30-THP–45-THP FOR PEER REVIEW Mar. Drugs 2018, 16, x FOR PEER REVIEW

19DCM of 25 g, 0.27 mmol) and TFA (3 mL) were dissolved in dry19 of 25 19 of 25 (7 mL). The reaction mixture was stirred under argon atmosphere for 3 d. Subsequently, the reaction mixture was quenched quenched with aa 22 M M solution of of NaOH in in H22O O (15 (15 mL) mL) and and extracted extracted with with DCM DCM (2 (2 ×× 15 15 mixture mixture was was quenchedwith with a 2 Msolution solution ofNaOH NaOH inHH (15 mL) and extracted with DCM (2 × 2 O(15 mixture was quenched with a layers 2 M solution of NaOH inanhyd. H2O mL) extracted with DCM (2 × 15 mL). The combined organic were dried over Na 2SOand 4, filtered and concentrated in mL). TheThe combined organic layers were 4, ,filtered 15 mL). combined organic layers weredried driedover overanhyd. anhyd.Na Na2SO SO filteredand and concentrated concentrated in in 2SO 4 filtered mL). The combined organic layers were dried over anhyd. Na 2 4 , and concentrated in vacuo. The crude crude products were were purified purified using using flash flash column column chromatography. chromatography. vacuo. vacuo. The The crude products products chromatography. vacuo. The crude products were purified using flash column chromatography.

(E)-3-(3,5-Dibromo-4-methoxyphenyl)-N-(3,4-dichlorophenyl)-2-(hydroxyimino)propanamide (30) (30) (E)-3-(3,5-Dibromo-4-methoxyphenyl)-N-(3,4-dichlorophenyl)-2-(hydroxyimino)propanamide (E)-3-(3,5-Dibromo-4-methoxyphenyl)-N-(3,4-dichlorophenyl)-2-(hydroxyimino)propanamide (30) The general procedure for THP deprotection was used, starting from ether 30 (0.16 g, 0.27mmol). mmol). (E)-3-(3,5-Dibromo-4-methoxyphenyl)-N-(3,4-dichlorophenyl)-2-(hydroxyimino)propanamide The general procedure for THP deprotection was used, starting from ether 30 (0.16 (30) g, 0.27 The general procedure for THP deprotection was used, starting from ether 30 (0.16 g, 0.27 mmol). The worked-up worked-up reaction mixture mixture was attempted toused, be purified purified usingether column chromatography The general reaction procedure for THPwas deprotection was starting using from 30 (0.16 g, 0.27 mmol). The attempted to be column chromatography The worked-up reaction mixture was attempted to be purified using column chromatography Biotage SNAP Cartridge KP-Sil 25 g and SNAP Ultra 10 g without success. A successful purification The worked-up reaction mixture was attempted to be purified usingsuccess. columnAchromatography Biotage Biotage SNAP Cartridge KP-Sil 25 g and SNAP Ultra 10 g without successful purification Biotage SNAP Cartridge KP-Sil 25 g and SNAP Ultra 10 g without success. A successful purification was achieved by using Biotage SNAP Cartridge KP-NH 11 g, gradient elution: (heptane/EtOAc, 12→ SNAP Cartridge KP-Sil 25 g and SNAP Ultra 10 g without success. A successful purification was was achieved by using Biotage SNAP Cartridge KP-NH 11 g, gradient elution: (heptane/EtOAc, 12→ was achieved by using Biotage SNAP Cartridge KP-NH 11 g, gradient elution: (heptane/EtOAc, 12→ 100%). The obtained oil was recrystallized from CHCl 3 gradient to give 30 as a white solid (0.052 g, 36%). Mp: achieved by using Biotage SNAP Cartridge KP-NH 11 g, elution: (heptane/EtOAc, 12 → 100%). 100%). The obtained oil was recrystallized from CHCl3 to give 30 as a white solid (0.052 g, 36%). Mp: 1Hwas 100%). The obtained oil recrystallized from CHCl 3 12.38 to give 30 as a white solid (0.052 g, 36%). Mp: ◦C 198 °C (decomposed). NMR (400 MHz, d 6 -DMSO) δ (s, 1H), 10.30 (s, 1H), 8.08 (d, J = 2.4 Hz, 1H NMR (400 from The°C obtained oil was recrystallized 30 as (s, a white solid (0.052 36%). 198Hz, 3 to give 198 (decomposed). MHz,CHCl d6-DMSO) δ 12.38 1H), 10.30 (s, 1H),g,8.08 (d,Mp: J = 2.4 1H NMR (400 MHz, d6-DMSO) δ 12.38 (s, 1H), 10.30 (s, 1H), 8.08 (d, J = 2.4 Hz, 13 198 °C (decomposed). 1 1H), 7.71 7.71 (dd, (dd, JJH 2.5, 8.9 8.9(400 Hz,MHz, 1H), 7.57 (d, JJ ==δ8.8 8.8 Hz,(s,1H), 1H), 7.50 (s,(s, 2H), 3.86 (s,(d, 2H), 3.76Hz, (s,1H), 3H).7.71 C 13C (decomposed). NMR d7.57 12.38 1H),7.50 10.30 1H), 8.08(s, J = 3.76 2.4 6 -DMSO) 1H), == 2.5, Hz, 1H), (d, Hz, (s, 2H), 3.86 2H), (s, 3H). 13C 1H), 7.71 (dd, J = 2.5, 8.9 Hz, 1H), 7.57 (d, J = 8.8 Hz, 1H), 7.50 (s, 2H), 3.86 (s, 2H), 3.76 (s, 3H). 13 NMR (101 MHz, d 6 -DMSO) δ 162.2, 151.9, 151.3, 138.4, 136.0, 132.9, 130.8, 130.5, 125.3, 121.4, 120.3, (dd, J (101 = 2.5,MHz, 8.9 Hz, 1H), 7.57δ(d, J = 8.8 Hz,151.3, 1H), 7.50 (s, 136.0, 2H), 3.86 (s, 2H), (s, 3H). NMR (101 NMR d6-DMSO) 162.2, 151.9, 138.4, 132.9, 130.8,3.76 130.5, 125.3, C 121.4, 120.3, +): NMR (101 MHz, -DMSO) δ calculated 162.2, 151.3, 136.0, 117.2, 60.4, 60.4, 27.9. HRMS HRMS (ESI 508.8670 (C 16H13 Br 2Cl2132.9, N22O O33),),130.8, found 508.8670. +): MHz, d6 -DMSO) δd6162.2, 151.9, 151.3,151.9, 138.4, 136.0,138.4, 132.9, 130.8, 125.3,130.5, 121.4,125.3, 120.3,121.4, 117.2,120.3, 60.4, 117.2, 27.9. (ESI calculated 508.8670 (C 16H13Br 2Cl2N130.5, found 508.8670. 117.2,HRMS 60.4, 27.9. (ESI+): 508.8670 calculated H13O Br), 2Cl2N2O3), found 508.8670. + ): calculated 27.9. (ESIHRMS (C508.8670 H Br (C Cl16N found 508.8670. 16

13

2

2

2

3

(E)-3-(3,5-Dibromo-4-methoxyphenyl)-N-(3,4-dichlorobenzyl)-2-(hydroxyimino)propanamide (31) (31) (E)-3-(3,5-Dibromo-4-methoxyphenyl)-N-(3,4-dichlorobenzyl)-2-(hydroxyimino)propanamide (E)-3-(3,5-Dibromo-4-methoxyphenyl)-N-(3,4-dichlorobenzyl)-2-(hydroxyimino)propanamide (31) Unlike in in the the general general procedure, procedure, compound compound 31-THP 31-THP (0.20 (0.20 g, g, 0.33 0.33 mmol) mmol) was was deprotected deprotected using using Unlike (E)-3-(3,5-Dibromo-4-methoxyphenyl)-N-(3,4-dichlorobenzyl)-2-(hydroxyimino)propanamide (31) Unlike in the general procedure, compound 31-THP (0.20 g,various 0.33 mmol) was deprotected using a 2 M solution of HCl in Et 2O (5 mL) in dry DCM (5 mL) under conditions (sealed tube, h, a 2 MUnlike solution of HCl in Etprocedure, 2O (5 mL) in dry DCM31-THP (5 mL) (0.20 underg,various conditions (sealed tube, 22 h, in the general compound 0.33 mmol) was deprotected using a a602°C, M solution of HCl in Et 2 O (5 mL) in dry DCM (5 mL) under various conditions (sealed tube, 2 h, MW; sealed sealed tube, tube, 22 h, h, 70 °C, °C, oil bath; bath; sealed sealed tube, tube, 12 h, h, 30 °C, °C, oil oil bath; bath; reflux reflux under under argon, 60 60 60 °C,solution MW; 2M of HCl in Et (5 mL)oil in dry DCM (5 mL) 12 under30various conditions (sealed argon, tube, 2 h, 2 O 70 60 sealed tube, 2 h, mixture 70◦°C, oilwas bath; sealed twice tube, by 12 h, 30 °C, chromatography, oil bath; reflux under argon, 60 h). °C, TheMW; worked-up reaction purified column using Biotage h). worked-up reaction mixture twice12by chromatography, using Biotage 60 ◦The C, MW; sealed tube, 2 h, 70 C, oilwas bath;purified sealed tube, h,column 30 ◦ C, oil bath; reflux under argon, 60 h). h). The worked-up reaction mixture was purified twice by column chromatography, using Biotage SNAP KP-Sil KP-Sil 25 25 g, g, gradient gradient elution: elution: (heptane/EtOAc, (heptane/EtOAc, 12→100%) 12→100%) to to give give 31 31 as as aa light light yellow yellow solid solid SNAP The worked-up reaction mixture was purified twice by column chromatography, using Biotage SNAP 1Hg, SNAP KP-Sil 25 gradient elution: (heptane/EtOAc, 12→100%) to give 31 Jas= a8.2light yellow solid (0.038 g, 22%). NMR (400 MHz, CDCl 3) δ 7.79 (s, 1H), 7.49 (s, 2H), 7.39 (d, Hz, 1H), 7.35 (d, 1 (0.038 H NMR (400 MHz, CDCl3) δ 7.7912 (s,→1H), 7.49 2H), J =yellow 8.2 Hz,solid 1H), (0.038 7.35 (d, KP-Silg,2522%). g, gradient elution: (heptane/EtOAc, 100%) to(s, give 31 7.39 as a (d, light g, 1H NMR (400 MHz, CDCl3) δ 7.79 (s, 1H), 7.49 (s, 2H), 7.39 (d, J = 8.2 Hz, 1H), 7.35 (d, (0.038 g, 22%). J = 2.0 Hz, 1H), 7.10 (dd, J = 2.0, 8.2 Hz, 1H), 7.02 (t, J = 5.5 Hz, 1H), 4.45 (d, J = 6.2 Hz, 2H), 3.90 (s, 2H), 1 H NMR J22%). = 2.0 Hz, 1H), 7.10 (dd, J = 2.0, 8.2 Hz, 1H), 7.02 (t, J = 5.5 Hz, 1H), 4.45 (d, J = 6.2 Hz, 2H), 3.90 (s, 2H), (400 MHz, CDCl3 ) δ 7.79 (s, 1H), 7.49 (s, 2H), 7.39 (d, J = 8.2 Hz, 1H), 7.35 (d, J = 2.0 13C7.10 J3.85 = 2.0(s,Hz, 1H), (dd,(101 J = 2.0, 8.2CDCl Hz, 1H), (t,153.0, J = 5.5 152.6, Hz, 1H), 4.45134.6, (d, J =133.6, 6.2 Hz, 2H), 131.9, 3.90 (s,130.9, 2H), 3H). NMR MHz, 3) δ 7.02 162.5, 138.1, 132.9, 3.85 (s, 3H). C NMR (1018.2MHz, CDCl 3) δ(t, 162.5, 153.0, 152.6,4.45 138.1, 133.6, 132.9, 130.9, Hz, 1H), 7.101313(dd, J = 2.0, Hz, 1H), 7.02 J = 5.5 Hz, 1H), (d, J134.6, = 6.2 Hz, 2H), 3.90131.9, (s, 2H), 3.85 + 3.85 (s, 3H). C NMR (101 MHz, CDCl 3 ) δ 162.5, 153.0, 152.6, 138.1, 134.6, 133.6, 132.9, 131.9, 130.9, 129.7, 127.1, 118.1, 60.7, 60.7, 42.6, 42.6, 28.1. 28.1. HRMS (ESI (ESI+):): calculated calculated 522.8825 522.8825 (C (C1717H H1515Br Br22Cl Cl22N N22O O33),), found found 13 C NMR 129.7, 118.1, (s, 3H).127.1, (101 MHz, CDCl3 ) δHRMS 162.5, 153.0, 152.6, 138.1, 134.6, 133.6, 132.9, 131.9, 130.9, 129.7, 129.7, 127.1, 118.1, 60.7, 42.6, 28.1. HRMS (ESI+): calculated 522.8825 (C17H15Br2Cl2N2O3), found 522.8827. 522.8827. 127.1, 118.1, 60.7, 42.6, 28.1. HRMS (ESI+ ): calculated 522.8825 (C17 H15 Br2 Cl2 N2 O3 ), found 522.8827. 522.8827.

(E)-N-[4-Chloro-3-(trifluoromethyl)phenyl]-3-(3,5-dibromo-4-methoxyphenyl)-2-(hydroxyimino) (E)-N-[4-Chloro-3-(trifluoromethyl)phenyl]-3-(3,5-dibromo-4-methoxyphenyl)-2-(hydroxyimino) (E)-N-[4-Chloro-3-(trifluoromethyl)phenyl]-3-(3,5-dibromo-4-methoxyphenyl)-2-(hydroxyimino) propanamide (32) (32) propanamide propanamide (32) The general general procedure procedure for for THP THP deprotection deprotection was was employed employed to to deprotect deprotect 32-THP 32-THP (0.19 (0.19 g, g, 0.30 0.30 The TheThe general procedure for THP deprotection was employed tochromatography, deprotect 32-THP (0.19 g, 0.30 mmol). worked-up reaction mixture was purified by column Biotage SNAP mmol). The worked-up reaction mixture was purified by column chromatography, Biotage SNAP mmol). The worked-up reactionelution: mixture(heptane/EtOAc, was purified by12→100%) column chromatography, Biotage Cartridge KP-NH 11 g, gradient to give 32 as a light yellowSNAP solid

129.7, 127.1, 118.1, 60.7, 42.6, 28.1. HRMS (ESI+): calculated 522.8825 (C17H15Br2Cl2N2O3), found 522.8827.

Mar. Drugs 2018, 16, 481

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(E)-N-[4-Chloro-3-(trifluoromethyl)phenyl]-3-(3,5-dibromo-4-methoxyphenyl)-2-(hydroxyimino) (E)-N-[4-Chloro-3-(trifluoromethyl)phenyl]-3-(3,5-dibromo-4-methoxyphenyl)-2-(hydroxyimino) propanamide (32) propanamide (32) The general procedure for THP deprotection was employed to deprotect 32-THP (0.19 g, The general procedure for THP deprotection was employed to deprotect 32-THP (0.19 g, 0.30 0.30 mmol). The worked-up reaction mixture was purified by column chromatography, Biotage mmol). The worked-up reaction mixture was purified by column chromatography, Biotage SNAP SNAP Cartridge KP-NH 11 g, gradient elution: (heptane/EtOAc, 12→100%) to give 32 as a light Cartridge KP-NH 11 g, gradient elution: (heptane/EtOAc, 12→100%) to give 32 as a light yellow solid yellow solid (0.055 g, 33%). 1 H NMR (400 MHz, CDCl3 ) δ 8.63 (s, 1H), 8.13 (s, 1H), 7.90 (d, J = 2.6 Hz, 1 (0.055 g, 33%). H NMR (400 MHz, CDCl3) δ 8.63 (s, 1H), 8.13 (s, 1H), 7.90 (d, J = 2.6 Hz, 1H), 7.77 (dd, 1H), 7.77 (dd, J = 2.6, 8.7 Hz, 1H), 7.51 (s, 2H), 7.46 (d, J = 8.7 Hz, 1H), 3.95 (s, 2H), 3.84 (s, 3H). 13 C J = 2.6, 8.7 Hz, 1H), 7.51 (s, 2H), 7.46 (d, J = 8.7 Hz, 1H), 3.95 (s, 2H), 3.84 (s, 3H). 13C NMR (101 MHz, NMR (101 MHz, CDCl3) δ 160.2, 153.0, 152.4, 135.9, 134.1, 133.5, 132.1, 129.1, 128.8, 127.2 (m), 123.6, CDCl3) δ 160.2, 153.0, 152.4, 135.9, 134.1, 133.5, 132.1, 129.1, 128.8, 127.2 (m), 123.6, 118.7 (q, JCF = 5.7), 118.7 (q, JCF = 5.7), 118.0, 60.6, 27.7. HRMS (ESI+ ): calculated 542.8934 (C17 H13 Br2 ClF3 N2 O3 ), found + 118.0, 60.6, 27.7. HRMS (ESI ): calculated 542.8934 (C17H13Br2ClF3N2O3), found 542.8937. 542.8937.

Mar. Mar. Drugs Drugs 2018, 2018, 16, 16, xx FOR FOR PEER PEER REVIEW REVIEW

20 20 of of 25 25

(E)-N-(3-Chloro-4-methoxyphenyl)-3-(3,5-dibromo-4-methoxyphenyl)-2-(hydroxyimino)propanamide (33) (E)-N-(3-Chloro-4-methoxyphenyl)-3-(3,5-dibromo-4-methoxyphenyl)-2-(hydroxyimino)propanamide (33) The was used, starting from ether 33-THP (0.20(0.20 g, 0.34 The general generalprocedure procedurefor forTHP THPdeprotection deprotection was used, starting from ether 33-THP g, mmol). The product was was purified by column chromatography, Biotage SNAP Cartridge KP-NH 11 11 g, 0.34 mmol). The product purified by column chromatography, Biotage SNAP Cartridge KP-NH 11H gradient elution: (cyclohexane/EtOAc, 12→100%) to give 33 as light orange solid (0.086 g, 50%). g, gradient elution: (cyclohexane/EtOAc, 12→100%) to give 33aas a light orange solid (0.086 g, 50%). (400(400 MHz, CDCl 33) δ 8.41 (s, 1H), 8.07 (s, 1H), 7.64 (d, J = 2.6 Hz, 1H), 7.52 (s, 2H), 7.42 (dd, J = 1NMR H NMR MHz, CDCl 3 ) δ 8.41 (s, 1H), 8.07 (s, 1H), 7.64 (d, J = 2.6 Hz, 1H), 7.52 (s, 2H), 7.42 (dd, J 13 13 2.6, 8.9 Hz, 1H), 6.88 (d, = 2.6, 8.9 Hz, 1H), 6.88 (d,J J= =8.9 8.9Hz, Hz,1H), 1H),3.94 3.94(s, (s,2H), 2H),3.88 3.88(s, (s,3H), 3H), 3.84 3.84 (s, (s, 3H). 3H). 13CC NMR NMR (101 (101 MHz, MHz, CDCl 33) δ 160.0, 153.0, 152.7, 152.2, 134.5, 133.6, 130.8, 122.8, 122.4, 119.6, 118.1, 112.4, 60.7, 56.6, 27.9. CDCl3 ) δ 160.0, 153.0, 152.7, 152.2, 134.5, 133.6, 130.8, 122.8, 122.4, 119.6, 118.1, 112.4, 60.7, 56.6, 27.9. ++ HRMS 22ClN22O44), found 504.9164. 17H16 HRMS (ESI (ESI+):):calculated calculated504.9165 504.9165(C (C17 H16BrBr ClN O ), found 504.9164. 17

16

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4

(E)-N-(3-Bromo-4-methoxyphenyl)-3-(3,5-dibromo-4-methoxyphenyl)-2-(hydroxyimino)propanamide (34) (E)-N-(3-Bromo-4-methoxyphenyl)-3-(3,5-dibromo-4-methoxyphenyl)-2-(hydroxyimino)propanamide The general procedure for THP deprotection was used, starting from ether 34-THP (0.20(34) g, 0.31 The general procedure for THP deprotection was used, starting from ether 34-THP (0.20 g, mmol). The worked-up reaction mixture was attempted to be purified using Biotage SNAP Cartridge 0.31 mmol). The worked-up reaction mixture was attempted to be purified using Biotage SNAP KP-Sil 25 g and SNAP Ultra 10 g. A successful purification was achieved by column chromatography Cartridge KP-Sil 25 Cartridge g and SNAP Ultra1110 A successful was achieved using Biotage SNAP KP-NH g, g. gradient elution: purification (heptane/EtOAc, 12→100%)by to column give 34 chromatography using Biotage SNAP Cartridge KP-NH 11 g, gradient elution: (heptane/EtOAc, 1 as a yellow oily solid (0.087 g, 50%). 1H NMR (400 MHz, CDCl33) δ 8.45 (s, 1H), 8.42 (s, 1H), 7.78 (d, J 1 H NMR (400 MHz, CDCl ) δ 8.45 (s, 1H), 12 →100%) to give a yellow oilyJ solid g,1H), 50%).6.85 = 2.6 Hz, 1H), 7.52 34 (s, as 2H), 7.48 (dd, = 2.6, (0.087 8.9 Hz, (d, J = 8.9 Hz, 1H), 3.94 3(s, 2H), 3.86 (s, 8.42 (s, 1H), 7.78 (d, J = 2.6 Hz, 1H), 7.52 (s, 2H), 7.48 (dd, J 2.6, 8.9 Hz,134.6, 1H), 6.85 J = 8.9 Hz, 13 13 3H), 3.83 (s, 3H). C NMR (101 MHz, CDCl33) δ 160.1, 153.2, = 152.9, 152.6, 133.6,(d,131.1, 125.4, 13 C NMR (101 MHz, CDCl ) δ 160.1, 153.2, 152.9, 152.6, 1H), 3.94 (s, 2H), 3.86 (s, 3H), 3.83 (s, 3H). + 3 120.4, 118.1, 112.2, 111.8, 60.7, 56.6, 27.9. HRMS (ESI +): calculated 548.8660 (C17 17H16 16Br33N22O44), found 134.6, 133.6, 131.1, 125.4, 120.4, 118.1, 112.2, 111.8, 60.7, 56.6, 27.9. HRMS (ESI+ ): calculated 548.8660 548.8660. (C17 H16 Br3 N2 O4 ), found 548.8660.

(E)-3-(3,5-Dibromo-4-methxyphenyl)-N-(3,5-dichlorophenyl)-2-(hydroxyimino)propanamide (35) (E)-3-(3,5-Dibromo-4-methxyphenyl)-N-(3,5-dichlorophenyl)-2-(hydroxyimino)propanamide (35) The general procedure for THP deprotection was used, starting from ether 35-THP (0.18 g, 0.30 The general procedure for THP deprotection was used, starting from ether 35-THP (0.18 g, mmol). The worked-up reaction mixture was purified by column chromatography, Biotage SNAP 0.30 mmol). The worked-up reaction mixture was purified by column chromatography, Biotage SNAP Cartridge KP-NH 11 g, gradient elution: (heptane/EtOAc, 12→100%) to give 35 as a white solid (0.021 Cartridge KP-NH 11 g, gradient elution: (heptane/EtOAc, 12→100%) to give 35 as a white solid (0.021 mg, 14%). 111H NMR (400 MHz, CD33OD) δ 7.71 (d, J = 1.8 Hz, 2H), 7.53 (s, 2H), 7.14 (t, J = 1.9 Hz, 1H), mg, 14%). H NMR (400 MHz, CD3 OD) δ 7.71 (d, J = 1.8 Hz, 2H), 7.53 (s, 2H), 7.14 (t, J = 1.9 Hz, 1H), 13 13C NMR (101 MHz, CD33OD) δ 162.2, 152.6, 150.9, 140.2, 135.7, 134.7, 133.2, 3.90 (s, 2H), 3.81 (s, 3H). 13 3.90 (s, 2H), 3.81 (s, 3H). C NMR (101 MHz, CD3 OD) δ 162.2, 152.6, 150.9, 140.2, 135.7, 134.7, 133.2, 123.2, 118.0, 117.3, 59.6, 27.3. HRMS (ESI+++): calculated 508.8670 (C16 16H13 13Br22Cl22N22O33), found 508.8659. 123.2, 118.0, 117.3, 59.6, 27.3. HRMS (ESI ): calculated 508.8670 (C16 H13 Br2 Cl2 N2 O3 ), found 508.8659.

(E)-3-(3,5-Dibromo-4-methoxyphenyl)-2-(hydroxyimino)-N-(pyridin-2-yl)propanamide (36)

The general procedure for THP deprotection was used, starting from ether 35-THP (0.18 g, 0.30 mmol). The worked-up reaction mixture was purified by column chromatography, Biotage SNAP Cartridge KP-NH 11 g, gradient elution: (heptane/EtOAc, 12→100%) to give 35 as a white solid (0.021 mg, 14%). 1H NMR (400 MHz, CD3OD) δ 7.71 (d, J = 1.8 Hz, 2H), 7.53 (s, 2H), 7.14 (t, J = 1.9 Hz, 1H), 3.90 (s, 2H), 3.81 (s, 3H). 13C NMR (101 MHz, CD3OD) δ 162.2, 152.6, 150.9, 140.2, 135.7, 134.7, 133.2, Mar. Drugs 2018, 16, 481 21 of 26 123.2, 118.0, 117.3, 59.6, 27.3. HRMS (ESI+): calculated 508.8670 (C16H13Br2Cl2N2O3), found 508.8659.

(E)-3-(3,5-Dibromo-4-methoxyphenyl)-2-(hydroxyimino)-N-(pyridin-2-yl)propanamide (36) (E)-3-(3,5-Dibromo-4-methoxyphenyl)-2-(hydroxyimino)-N-(pyridin-2-yl)propanamide (36) The general procedure for THP deprotection was used, starting from ether 36-THP (0.087 g, 0.17 The general procedure for THP deprotection was used, starting from ether 36-THP (0.087 g, mmol). The product was purified by column chromatography, Biotage SNAP Cartridge KP-NH 11 g, 0.17 mmol). The product was purified by column chromatography, Biotage SNAP Cartridge KP-NH gradient elution: (hexane/EtOAc, 12→100%). The obtained 36 as a white powder was further re11 g, gradient elution: (hexane/EtOAc, 12→100%). The obtained 36 as a white powder was further crystallized from CHCl3 for the X-ray to give transparent crystals (0.037 g, 51%). M.p.: 192–196 ◦°C re-crystallized from CHCl3 for the X-ray to give transparent crystals (0.037 g, 51%). M.p.: 192–196 C (decomposed). 11H NMR (400 MHz, CDCl3) δ 13.85 (s, 1H), 9.41 (s, 1H), 8.39 (d, J = 8.5 Hz, 1H), 8.32– (decomposed). H NMR (400 MHz, CDCl3 ) δ 13.85 (s, 1H), 9.41 (s, 1H), 8.39 (d, J = 8.5 Hz, 1H), 8.32–8.27 8.27 (m, 1H), 7.89–7.84 (m, 1H), 7.57 (s, 2H), 7.19 (ddd, J = 0.8, 5.2, 7.3 Hz, 1H), 4.02 (s, 2H), 3.84 (s, 3H. (m, 1H), 7.89–7.84 (m, 1H), 7.57 (s, 2H), 7.19 (ddd, J = 0.8, 5.2, 7.3 Hz, 1H), 4.02 (s, 2H), 3.84 (s, 3H. 13 C 13C NMR (101 MHz, CDCl3) δ 161.7, 152.8, 151.7, 150.1, 146.4, 140.2, 135.2, 133.7, 120.4, 118.0, 115.7, NMR (101 MHz, CDCl+3 ) δ 161.7, 152.8, 151.7, 150.1, 146.4, 140.2, 135.2, 133.7, 120.4, 118.0, 115.7,+60.7, 60.7, 28.2. HRMS+ (ESI ): calculated 441.9402 (C15H14Br2N3O3), found 441.9401. LC-MS: [M ++ H] m/z 28.2. HRMS (ESI ): calculated 441.9402 (C15 H14 Br2 N3 O3 ), found 441.9401. LC-MS: [M + H] m/z 442 442 (tR = 5.39 min), >99%. Mar. Drugs 2018, 16, x FOR PEER REVIEW 21 of 25 (t R = 5.39 min), >99%. Mar. Drugs 2018, 16, x FOR PEER REVIEW Mar. Drugs 2018, 16, x FOR PEER REVIEW

21 of 25 21 of 25

(E)-3-(3,5-Dibromo-4-methoxyphenyl)-2-(hydroxyimino)-N-(pyridin-3-yl)propanamide (37) (E)-3-(3,5-Dibromo-4-methoxyphenyl)-2-(hydroxyimino)-N-(pyridin-3-yl)propanamide(37) (37) (E)-3-(3,5-Dibromo-4-methoxyphenyl)-2-(hydroxyimino)-N-(pyridin-3-yl)propanamide (E)-3-(3,5-Dibromo-4-methoxyphenyl)-2-(hydroxyimino)-N-(pyridin-3-yl)propanamide (37) The general procedure for THP deprotection was used, starting from ether 37-THP (0.34 g, 0.64 The general procedure for THP deprotection was used, starting from ether 37-THP (0.34(0.34 g, 0.64 The general procedure for THP deprotection was used, starting from ether 37-THP g, TheThe general procedure for THP was used, starting from etherCartridge 37-THP (0.34 g,25 0.64 mmol). product was purified bydeprotection column chromatography, Biotage SNAP KP-Sil g, mmol). The product was purified by column chromatography, Biotage SNAP Cartridge KP-Sil 25 g, 0.64 mmol). The product was purified by column Biotage Cartridge 1HKP-Sil mmol). The product was purified by column chromatography, SNAPSNAP Cartridge KP-Sil 25 g, gradient elution: (hexane/acetone, 0→100%) to givechromatography, 37 as a paleBiotage yellow solid (0.052 g, 18%). NMR 1H NMR gradient elution: (hexane/acetone, 0→100%) to100%) give 37 as a pale yellow solid (0.052 g, 18%). 25 g, gradient elution: (hexane/acetone, 0 → to give 37 as a pale yellow solid (0.052 g, 18%). 1 gradient elution: (hexane/acetone, to give as a pale(m, yellow (0.052 g, 18%). H NMR (400 MHz, CD3OD) δ 8.84 (dd, J = 0→100%) 2.5, 19.4 Hz, 1H),37 8.30–8.16 2H), solid 7.53 (d, J = 8.4 Hz, 2H), 7.44– 1(400 MHz,(400 CD3MHz, OD) δCD 8.84 (dd,δJ8.84 = 2.5, 19.4J = Hz, (m, 2H), 7.53 J = 7.53 8.4 Hz, 7.44– H NMR (dd, 2.5,1H), 19.48.30–8.16 Hz, 1H), 8.30–8.16 (m,(d, 2H), (d,152.3, J2H), = 8.4 Hz, 13 3 OD) (400 MHz, CD3.92 3OD) 8.84 (dd,(s, J =3H). 2.5, 19.4 Hz, 1H), 8.30–8.16 2H), 7.53 (d, J = 153.9, 8.4 Hz, 2H), 7.44– 7.35 (m, 1H), (s,δ2H), 3.80 C NMR (101 MHz, CD(m, 3OD) δ 163.8, 154.4, 145.3, 13C NMR (101 13 7.35 (m, 1H), 3.92 (s, 2H), 3.80 (s, 3H). MHz, CD 3 OD) δ 163.8, 154.4, 153.9, 152.3, 145.3, 2H), 7.44–7.35 (m,(s, 1H), 3.92 (s,(s,2H), 3.80 (s, 3H). CMHz, NMR (101 MHz, CD3441.9402 OD) δ 163.8, 154.4, 153.9, 13C +3): 7.35 (m, 1H),134.44, 3.92 2H), 3.80 3H). NMR (101 CD OD) δ 163.8, 154.4, 153.9, 152.3, 145.3, 142.3, 137.1, 129.41, 125.2, 118.7, 61.0, 28.7. HRMS (ESI calculated (C15H 14N3O 3Br2), +): calculated 441.9402 + ): calculated 142.3, 137.1, 134.44, 129.41,134.44, 125.2, 118.7, 61.0, 28.7. HRMS (ESI (C15H14N 3O3Br2), 152.3, 145.3, 142.3, 137.1, 129.41, 125.2, 118.7, 61.0, 28.7. HRMS (ESI 441.9402 + 142.3, 134.44, 129.41, 125.2, 118.7, 61.0, 28.7. HRMS (ESI ): calculated 441.9402 (C15H14N3O3Br2), found137.1, 441.9402. found 441.9402. (C N3 O3 Br2 ), found 441.9402. 15 H14 found 441.9402.

(E)-3-(3,5-Dibromo-4-methoxyphenyl)2-(hydroxyimino)-N-(pyridin-4-yl)propanamide (38) (E)-3-(3,5-Dibromo-4-methoxyphenyl)2-(hydroxyimino)-N-(pyridin-4-yl)propanamide (38) (E)-3-(3,5-Dibromo-4-methoxyphenyl)2-(hydroxyimino)-N-(pyridin-4-yl)propanamide (38) The general procedure for THP deprotection was used, starting from ether 38-THP (0.49 g, 0.093 (E)-3-(3,5-Dibromo-4-methoxyphenyl)2-(hydroxyimino)-N-(pyridin-4-yl)propanamide (38) (0.49 The general procedure for THP deprotection was used, starting from ether 38-THP g, 0.093 The general procedure for THP deprotection was used, starting from ether 38-THP (0.49 g, 0.093 mmol). The product was purified by column chromatography, Biotage SNAP Cartridge KP-Sil 10 g, The general procedure for THP deprotection was used, starting from ether 38-THP (0.49 g, mmol). The product was purified by column chromatography, Biotage SNAP Cartridge1 KP-Sil 10 g, mmol). The product was purified by column chromatography, Biotage SNAP Cartridge KP-Sil 10 g, gradient elution: (DCM/MeOH, 0→10%) to give 38 as a brownish solid (0.0099 g, 24%). H NMR (400 0.093 mmol). The product was purified by column chromatography, Biotage SNAP Cartridge KP-Sil 10 gradient elution: (DCM/MeOH, 0→10%) to give 38 as a brownish solid (0.0099 g, 24%). 11H NMR (400 gradient elution: (DCM/MeOH, 0→10%) give 382H), as38a7.59 brownish (0.0099 g, 24%). H NMR (400 MHz, d6-acetone) δ 8.47 (s, 2H), 7.73 (d, J =to5.5 Hz, (s, 2H),solid 3.98 (s, 2H), 3.82 (s,24%). 3H). C NMR NMR 113H g, gradient elution: (DCM/MeOH, 0 → 10%) to give as a brownish solid (0.0099 g, 13 MHz, d6-acetone) δ 8.47 (s, 2H), 7.73 (d, J = 5.5 Hz, 2H), 7.59 (s, 2H), 3.98 (s, 2H), 3.82 (s, 3H). 13C NMR MHz, d 6-acetone) δ 8.47 (s, 2H), 7.73 (d, J = 5.5 Hz, 2H), 7.59 (s, 2H), 3.98 (s, 2H), 3.82 (s, 3H). C NMR (101 MHz, d 6-acetone) δ 162.9, 153.5, 152.2, 151.3, 145.8, 136.7, 134.3, 118.3, 114.5, 60.9, 28.4. HRMS (400 MHz, -acetone) δδ 162.9, 8.47 (s,153.5, 2H), 152.2, 7.73 (d, J = 5.5 Hz, 136.7, 2H), 7.59 (s, 118.3, 2H), 3.98 (s, 60.9, 2H), 28.4. 3.82 (s, 3H). (101 MHz, dd66-acetone) 151.3, 145.8, 134.3, 114.5, HRMS +): (101 MHz, d6-acetone) δ 162.9, 153.5, 151.3, 441.9400. 145.8, 136.7, 134.3, 118.3, 114.5, 60.9, 28.4. HRMS (ESI calculated 441.9402 (C 15H 14N3O152.2, 3Br2), found 13 C NMR (101 MHz, d6 -acetone) 152.2, 151.3, 145.8, 136.7, 134.3, 118.3, 114.5, 60.9, 28.4. +): calculated (ESI 441.9402 (C15H14δN162.9, 3O3Br2153.5, ), found 441.9400. (ESI+): calculated 441.9402 (C15H14N3O3Br2), found 441.9400. HRMS (ESI+ ): calculated 441.9402 (C15 H14 N3 O3 Br2 ), found 441.9400.

(E)-3-(3,5-Dibromo-4-methoxyphenyl)-2-(hydroxyimino)-N-(pyridin-3-ylmethyl)propanamide (39) (E)-3-(3,5-Dibromo-4-methoxyphenyl)-2-(hydroxyimino)-N-(pyridin-3-ylmethyl)propanamide (39) (E)-3-(3,5-Dibromo-4-methoxyphenyl)-2-(hydroxyimino)-N-(pyridin-3-ylmethyl)propanamide The general procedure for THP deprotection was used, starting from ether 39-THP(39) (0.17 g, 0.32 (E)-3-(3,5-Dibromo-4-methoxyphenyl)-2-(hydroxyimino)-N-(pyridin-3-ylmethyl)propanamide (39) The general procedure for THP deprotection was used, starting from ether 39-THP (0.17 g, 0.32 TheThe general procedure for THP was used, starting from ether 39-THPKP-NH (0.17 g, 11 0.32 mmol). product was purified by deprotection column chromatography, Biotage SNAP Cartridge g, TheThe general procedure for THP deprotection was used,Biotage startingSNAP fromCartridge ether 39-THP (0.17 g, mmol). product was purified by column chromatography, KP-NH 11 g, 1 mmol). product was purified0→10%) by column chromatography, Biotage g, gradientThe elution: (DCM/MeOH, to give 39 as a yellowish solidSNAP (0.039Cartridge g, 26%). KP-NH H NMR11 (400 1 0.32 mmol). The product was purified by column SNAP KP-NH 11 gradient elution: (DCM/MeOH, 0→10%) to give chromatography, 39 as a yellowish Biotage solid (0.039 g, Cartridge 26%). 1H NMR (400 gradient elution: (DCM/MeOH, 0→10%) give 391H), as a 7.52 yellowish (0.039 (m, g, 26%). H NMR MHz, CDCl 3) δ 8.52 (br s, 2H), 7.75 (d, J =to7.8 Hz, (s, 2H),solid 7.39–7.33 1H), 7.16 (t, J =(400 6.1 MHz, CDClelution: 3) δ 8.52 (DCM/MeOH, (br s, 2H), 7.75 (d, = 7.8toHz, 1H), (s, 2H), 7.39–7.33 (m, 1H), 7.16 1(t, = 6.1 g, gradient 0→J10%) give 39 7.52 as 13 a yellowish solid (0.039 g, 26%). HJNMR MHz, CDCl 3) (d, δ 8.52 (br Hz, s, 2H), = 7.8 3.84 Hz, (s, 1H), 7.52C(s,NMR 2H), (101 7.39–7.33 1H), 7.16 (t, J152.7, = 6.1 Hz, 1H), 4.54 J = 6.1 2H),7.75 3.91(d,(s,J 2H), 3H). MHz,(m, CDCl 3) δ 163.3, 13C7.52 Hz, 4.54 (d, 3J )=δ6.1 Hz, 3.917.75 (s, 2H), 3H). NMR CDCl 3) δ1H), 163.3, 152.7, (4001H), MHz, CDCl 8.52 (br2H), s, 2H), (d, J 3.84 = 7.8(s,Hz, 1H), (s,(101 2H),MHz, 7.39–7.33 (m, 7.16 (t, J 13 + Hz, 1H), 4.54 147.8, (d, J = 137.5, 6.1 Hz,135.4, 2H), 3.91 2H), 3.84 (s, 118.0, 3H). 60.7, C NMR (101 MHz, CDCl 3) δ ): 163.3, 152.7, 151.1, 148.0, 134.7,(s,133.6, 124.4, 41.0, 28.2. HRMS (ESI calculated 13 + 151.1, 148.0, 137.5, 135.4, 133.6, 118.0, 60.7, 41.0, 28.2.(101 HRMS (ESI ): calculated = 6.1 Hz, 1H),147.8, 4.54 (d, J = 6.1 Hz,134.7, 2H), 3.91 (s, 124.4, 2H), 3.84 (s, 3H). C NMR MHz, CDCl 3 ) δ 163.3, 151.1, 148.0, 147.8, 137.5, 135.4, 455.9563. 134.7, 133.6, 124.4, 118.0, 60.7, 41.0, 28.2. HRMS (ESI+): calculated 455.9558 (C16H 16Br2N 3O3), found 455.9558 (C16H16Br2N3O3), found 455.9563. 455.9558 (C16H16Br2N3O3), found 455.9563.

(E)-3-(3,5-Dibromo-4-methoxyphenyl)-2-(hydroxyimino)-N-(pyridin-3-ylmethyl)propanamide (39) The general procedure for THP deprotection was used, starting from ether 39-THP (0.17 g, 0.32 mmol). The product was purified by column chromatography, Biotage SNAP Cartridge KP-NH 11 g, gradient elution: (DCM/MeOH, 0→10%) to give 39 as a yellowish solid (0.039 g, 26%). 1H NMR (400 Mar. Drugs 2018, 481 (br s, 2H), 7.75 (d, J = 7.8 Hz, 1H), 7.52 (s, 2H), 7.39–7.33 (m, 1H), 7.16 (t, 22 26 MHz, CDCl 3) 16, δ 8.52 J =of6.1 Hz, 1H), 4.54 (d, J = 6.1 Hz, 2H), 3.91 (s, 2H), 3.84 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 163.3, 152.7, 151.1, 148.0, 147.8, 137.5, 135.4, 134.7, 133.6, 124.4, 118.0, 60.7, 41.0, 28.2. HRMS (ESI+): calculated 152.7, 151.1, 148.0, 147.8, 137.5, 135.4, 134.7, 133.6, 124.4, 118.0, 60.7, 41.0, 28.2. HRMS (ESI+ ): calculated 455.9558 (C16H16Br2N3O3), found 455.9563. 455.9558 (C16 H16 Br2 N3 O3 ), found 455.9563.

(E)-3-(3,5-Dibromo-4-methoxyphenyl)-N-[4-(dimethylamino)phenyl]-2-(hydroxyimino)propanamide (40) (E)-3-(3,5-Dibromo-4-methoxyphenyl)-N-[4-(dimethylamino)phenyl]-2-(hydroxyimino)propanamide (40) The general procedure for THP deprotection was used, starting from ether 40-THP (0.16 g, 0.27 The general procedure for THP deprotection was used, starting from ether 40-THP (0.16 g, mmol). The product was purified by column chromatography, Biotage SNAP Cartridge KP-Sil 25 g, 0.27 mmol). The product was purified by column chromatography, Biotage SNAP Cartridge KP-Sil 25 gradient elution: (hexane/EtOAc, 7→60%). The obtained product was further re-crystallized from g, gradient elution: (hexane/EtOAc, 7→60%). The obtained product was further re-crystallized from 1H NMR (400 MHz, d6-acetone) δ 7.61 hexane and acetone to give 40 as a yellow solid (0.07 g, 50%). 1 hexane and acetone to give 40 as a yellow solid (0.07 g, 50%). H NMR (400 MHz, d6 -acetone)13δ 7.61 (s, (s, 2H), 7.56 (d, J = 9.1 Hz, 2H), 6.72 (d, J = 9.2 Hz, 2H), 3.96 (s, 2H), 3.82 (s, 3H), 2.90 (s, 6H). C NMR 2H), 7.56 (d, J = 9.1 Hz, 2H), 6.72 (d, J = 9.2 Hz, 2H), 3.96 (s, 2H), 3.82 (s, 3H), 2.90 (s, 6H). 13 C NMR (101 MHz, d6-acetone) δ 161.2, 153.4, 153.0, 148.8, 137.2, 134.4, 129.1, 122.0, 118.2, 113.5, 60.9, 40.9, 28.5. (101 MHz, d+6 -acetone) δ 161.2, 153.4, 153.0, 148.8, 137.2, 134.4, 129.1, 122.0, 118.2, 113.5, 60.9, 40.9, 28.5. HRMS (ESI+): calculated 483.9871 (C18H20N3O3Br2), found 483.9876. Mar. Drugs 2018, x FOR PEER REVIEW(C18 H20 N3 O3 Br2 ), found 483.9876. 22 of 25 HRMS (ESI ): 16, calculated 483.9871 Mar. Drugs 2018, 16, x FOR PEER REVIEW Mar. Drugs 2018, 16, x FOR PEER REVIEW

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(E)-N,3-Bis(3-bromo-4-methoxyphenyl)-2-(hydroxyimino)propanamide (41) (E)-N,3-Bis(3-bromo-4-methoxyphenyl)-2-(hydroxyimino)propanamide (41) (E)-N,3-Bis(3-bromo-4-methoxyphenyl)-2-(hydroxyimino)propanamide (41) The general procedure for THP deprotection was used, starting (E)-N,3-Bis(3-bromo-4-methoxyphenyl)-2-(hydroxyimino)propanamide (41)from ether 41-THP (0.43 g, 0.78 The general procedure for THP deprotection was used, starting from ether 41-THP (0.43 g, 25 0.78 TheThe general procedure for THP deprotection was used, Biotage starting from ether 41-THP (0.43 mmol). product was purified by column chromatography, SNAP Cartridge KP-Sil g, The general procedure for THP deprotection was used, starting from ether 41-THP (0.43 g, 0.78 mmol). The product was purified by column chromatography, Biotage SNAP Cartridge KP-Sil 25 g, 1 0.78 mmol). The product was purified by column chromatography, Biotage SNAP Cartridge KP-Sil 25 gradientThe elution: (hexane/EtOAc, to chromatography, give 41 as a yellow solid (0.087 24%). HKP-Sil NMR (400 mmol). product was purified 0→60%) by column Biotage SNAP g, Cartridge g, 11H NMR 25 gradient elution: (hexane/EtOAc, 0→60%) to to give 41412H), asasa ayellow solid (0.087 g, g, gradient elution: (hexane/EtOAc, 0→60%) give yellow solid (0.087 g, 24%). 24%). H NMR (400 MHz, CD 3OD) δ 7.85 (d, J = 2.5 Hz, 1H), 7.51–7.45 (m, 7.24 (dd, J = 2.2, 8.5 Hz, 1H), 6.92 (d, J =(400 8.9 1 gradient elution: (hexane/EtOAc, 0→60%) to give 41 as a yellow solid (0.087 g, 24%). H NMR (400 MHz, CD 3OD) (d, JJ == 2.5 1H), 2H), 7.24(s, (dd, =132.2, 8.5 1H), 6.92CD (d, JJ = = 8.9 MHz, CD6.86 OD) δδ 7.85 7.85 (d,Hz, 2.5 Hz, Hz, 1H), 7.51–7.45 (m,3H), 2H), 7.24 (dd, JJ = 2.2, 8.5 Hz, Hz, 1H), 6.92 (d, 8.9 Hz, 1H), J = 8.5 1H), 3.88 (s, 7.51–7.45 2H), 3.81 (m, (s, 3H). C NMR (101 MHz, MHz, CD33OD)(d, δ 7.85 (d, J = 2.5 Hz, 1H), 7.51–7.45 (m, 2H),3.78 7.24 (dd, J =13 2.2, 8.5 Hz, 1H), 6.92 (d,3OD) J = 8.9δ 13 Hz, 1H), 6.86 (d, J = 8.5 Hz, 1H), 3.88 (s, 2H), 3.81 (s, 3H), 3.78 (s, 3H). C NMR (101 MHz, CD 3OD) δδ Hz, 1H), 6.86 (d, J = 8.5 Hz, 1H), 3.88 (s, 2H), 3.81 (s, 3H), 3.78 (s, 3H). C NMR (101 MHz, CD OD) 163.4, 155.9, 134.8, 131.6, 126.6, 112.1, CD 56.8, 56.6, 33OD) Hz, 1H), 6.86154.1, (d, J =153.3, 8.5 Hz, 1H),133.0, 3.88 (s, 2H),130.5, 3.81 (s, 3H), 121.9, 3.78 (s,113.11, 3H). 13113.09, C NMR112.2, (101 MHz, δ 163.4, 155.9, 154.1, 153.3, 134.8, 133.0, 131.6, 130.5, 126.6, 121.9, 113.11, 113.09, 112.2, 112.1, 56.8, 56.6, +): 153.3, 163.4, 155.9, (ESI 154.1, 134.8,470.9555 133.0, 131.6, 121.9,470.9554. 113.11, 113.09, 112.2, 112.1, 56.8, 56.6, 28.7. HRMS calculated (C17H130.5, 17N2O4126.6, Br2), found 163.4, 155.9, 154.1, 153.3, 134.8, 133.0, 131.6, 130.5, 126.6, 121.9, 113.11, 113.09, 112.2, 112.1, 56.8, 56.6, + 28.7. 17NN 2O4O Br4 Br 2), found 470.9554. 28.7. HRMS HRMS (ESI (ESI+):):calculated calculated470.9555 470.9555(C (C17HH 2 ), found 470.9554. 28.7. HRMS (ESI+): calculated 470.9555 (C1717H1717 N2O2 4Br 2), found 470.9554.

(E)-3-(3-Bromo-4-methoxyphenyl)-2-(hydroxyimino)-N-(pyridin-2-yl)propanamide (42) (E)-3-(3-Bromo-4-methoxyphenyl)-2-(hydroxyimino)-N-(pyridin-2-yl)propanamide (42) The general procedure for THP deprotection was used, starting from ether (E)-3-(3-Bromo-4-methoxyphenyl)-2-(hydroxyimino)-N-(pyridin-2-yl)propanamide (42)42-THP (0.31 g, 0.68 (E)-3-(3-Bromo-4-methoxyphenyl)-2-(hydroxyimino)-N-(pyridin-2-yl)propanamide (42) TheThe general procedure for THP deprotection was used, starting from ether 42-THP (0.31 g, 25 0.68 mmol). product was purified by column chromatography, Biotage SNAP Cartridge KP-Sil g, The general procedure for THP deprotection was used, starting from ether 42-THP (0.31 g, 0.68 The general procedure for THP deprotection was used, starting from ether 42-THP (0.31 g, mmol). The product was purified by column chromatography, Biotage SNAP Cartridge KP-Sil 25 g, gradientThe elution: (DCM/MeOH, 0→10%). Thechromatography, obtained solid was furtherSNAP re-crystallized from hexane mmol). product was purified by column Biotage Cartridge KP-Sil 25 g, 0.68 mmol). The product was purified by column chromatography, Biotage SNAP Cartridge KP-Sil gradient (DCM/MeOH, 0→10%). The obtained solid was further from hexane 1Hre-crystallized to give 42elution: as a white solid (0.062 g, 25%). M.p.: 214–7 °C (decomposed). NMR (400 MHz, d6-DMSO) gradient elution: (DCM/MeOH, 0→10%). The obtained solid was further re-crystallized from hexane 1Hfurther 25 g, gradient elution: (DCM/MeOH, 0→ 10%). The°C obtained solid was re-crystallized from to give 42 as a white solid (0.062 g, 25%). M.p.: 214–7 (decomposed). NMR (400 MHz, d 6-DMSO) δ 12.47 (s, as 1H), 9.55 (s, 1H), 8.33 g, (ddd, J =M.p.: 4.9, 1.9, 0.9°C Hz, 1H), ◦8.07 (dt, 1JH= NMR 8.3, 1.0 Hz,MHz, 1H), d7.83 (ddd, 1 to give 42 a white solid (0.062 25%). 214–7 (decomposed). (400 6-DMSO) hexane to give 42 as a white solid (0.062 g, 25%). M.p.: 214–7 C (decomposed). H NMR (400 MHz, δJ =12.47 (s, 1H), 9.551H), (s, 1H), J = 4.9, 1.9, 0.9(dd, Hz,J1H), = 8.3,7.16 1.0(ddd, Hz, 1H), 7.834.9, (ddd, 8.5, 7.3, 1.9 Hz, 7.458.33 (d, J(ddd, = 2.1 Hz, 1H), 7.23 = 8.5,8.07 2.2 (dt, Hz,JJ1H), J = 7.3, 1.0 δ612.47 (s, 1H), 9.55(s,(s,1H), 1H), 8.33 (ddd, J8.33 = 4.9, 1.9, Hz,1.9, 1H), 8.07 (dt, =8.07 8.3, (dt, 1.0 Hz, 1H), 7.83 (ddd, δ 12.47 9.55 (s, 1H), (ddd, J0.9 =(dd, 4.9, 0.9 Hz,Hz, 1H), J = 8.3, 1.0 Hz, 1H), JdHz, =-DMSO) 8.5, 7.3, 1.9 Hz, 1H), 7.45 (d, J = 2.1 Hz, 1H), 7.23 J = 8.5, 2.2 1H), 7.16 (ddd, J = 7.3, 4.9, 1.0 13 1H), 7.03 (d, J = 8.5 Hz, 1H), 3.84 (s, 2H), 3.80 (s, 3H). C NMR (101 MHz, d 6-DMSO) δ 161.6, 153.9, J = 8.5, 7.3, J1.9 Hz,7.3, 1H), 7.45 (d, J = 7.45 2.1 Hz, 7.23 (dd, J =7.23 8.5,(dd, 2.2 Hz, 1H),2.2 7.16 = 7.3, 4.9, 1.0 13 7.83 (ddd, 8.5, 1.9 1H), (d, 1H), J= 2.1 Hz, 1H), J =MHz, 8.5, Hz,(ddd, 1H), Jδ7.16 (ddd, J+= Hz, 1H), 7.03=(d, J = 8.5 Hz,Hz, 1H), 3.84 (s, 2H), 3.80 (s, 3H). C NMR (101 d6-DMSO) 161.6, 153.9, 151.1, 150.6, 148.3, 138.5, 133.0, 130.0, 129.3, 120.0, 113.4, 112.7, 110.3, 56.2, 27.4. HRMS (ESI ): 13C NMR 13 Hz, 1H), 7.03 (d, J = 8.5 Hz, 1H), 3.84 (s, 2H), 3.80 (s, 3H). (101 MHz, d 6-DMSO) δ 161.6, 153.9, +): 7.3, 4.9, 1.0 Hz, 1H), 7.03 (d, J = 8.5 Hz, 1H), 3.84 (s, 2H), 3.80 (s, 3H). C NMR (101 MHz, d -DMSO) δ 6 151.1, 150.6, 148.3, 138.5, 133.0, 130.0, 129.3, 120.0, 113.4, 112.7, 110.3, 56.2, 27.4. HRMS (ESI calculated 364.0297 (C 15H15N3O3Br), found 364.0299. +): 151.1, 150.6, 148.3, 138.5, 133.0, 130.0, 129.3, 120.0, 113.4, 112.7, 110.3, 56.2, 27.4. HRMS (ESI 161.6, 153.9, 151.1, 150.6, 148.3, 138.5, 133.0, 130.0, 129.3, 120.0, 113.4, 112.7, 110.3, 56.2, 27.4. HRMS calculated 364.0297 (C15H15 N3O3Br), found 364.0299. + ): calculated calculated 364.0297 (C15H15N OH 3Br), found 364.0299. (ESI 364.0297 (C315 15 N3 O3 Br), found 364.0299.

(E)-N-(3,4-Dichlorobenzyl)-2-(hydroxyimino)-3-(4-methoxyphenyl)propanamide (43) (E)-N-(3,4-Dichlorobenzyl)-2-(hydroxyimino)-3-(4-methoxyphenyl)propanamide (43) The general procedure for THP deprotection was used, starting from ether (E)-N-(3,4-Dichlorobenzyl)-2-(hydroxyimino)-3-(4-methoxyphenyl)propanamide (43) 43-THP (0.53 g, 1.18 TheThe general procedure for THP deprotection was used, starting from etherCartridge 43-THP (0.53 g, 25 1.18 mmol). product was purified by column chromatography, Biotage SNAP KP-Sil g, The general procedure for THP deprotection was used, starting from ether 43-THP (0.53 g, 1.18 mmol). The product was purified by column chromatography, Biotage SNAP Cartridge KP-Sil 25 g, 1H NMR (400 gradient elution: (heptane/EtOAc, 0→100%) to give 43 as a yellow solid (0.057 g, 13%). mmol). The product was purified by column chromatography, Biotage SNAP Cartridge KP-Sil 25 g, gradient elution: (heptane/EtOAc, 0→100%) to give 43 as a yellow solid (0.057 g, 13%). 1H NMR MHz, CD 3OD) δ 7.39 (d, J = 8.3 Hz, 1H), 7.35 (d, J = 2.0 Hz, 1H), 7.22–7.15 (m, 2H), 7.13–7.07 (m, (400 1H), 1 gradient elution: (heptane/EtOAc, 0→100%) to give 43 as a yellow solid (0.057 g, 13%). H NMR (400 MHz, CD 3OD) δ 7.39 (d, J = 8.3 Hz, 1H), 7.35 (d, J = 2.0 Hz, 1H), 7.22–7.15 (m, 2H), 7.13–7.07 (m, 1H), 13 6.82–6.72 (m, 2H), 4.36 (s, 2H), 3.87 (s, 2H), 3.74 (s, 3H). C NMR (101 MHz, CD 3OD) δ 166.3, 159.7, MHz, CD3OD) δ 7.39 (d, J = 8.3 Hz, 1H), 7.35 (d, J = 2.0 Hz, 1H), 7.22–7.15 (m, 2H), 7.13–7.07 (m, 1H), 13C NMR (101 MHz, CD3OD) δ 166.3, 159.7, 6.82–6.72 (m,133.2, 2H), 4.36 (s,131.5, 2H), 3.87 (s, 2H),129.9, 3.74 (s, 3H).114.8, +): calculated 153.9, 141.1, 130.3, 128.1, 55.6, (101 42.7, MHz, 29.2. HRMS (ESI 6.82–6.72 (m, 2H), 131.7, 4.36 (s, 2H),131.1, 3.87 (s, 2H), 3.74 (s, 3H). 13C NMR CD3OD) δ+ 166.3, 159.7, 153.9, 141.1, 133.2, 131.7, 131.5, 131.1, 130.3, 129.9, 128.1, 114.8, 55.6, 42.7, 29.2. HRMS (ESI ): calculated 367.0616 (C17H17N2O3Cl2), found 367.0615.

calculated 364.0297 (C15H15N3O3Br), found 364.0299.

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(E)-N-(3,4-Dichlorobenzyl)-2-(hydroxyimino)-3-(4-methoxyphenyl)propanamide (43) (E)-N-(3,4-Dichlorobenzyl)-2-(hydroxyimino)-3-(4-methoxyphenyl)propanamide (43) The general procedure for THP deprotection was used, starting from ether 43-THP (0.53 g, 1.18 The general procedure for THP deprotection was used, starting from ether 43-THP (0.53 g, mmol). The product was purified by column chromatography, Biotage SNAP Cartridge KP-Sil 25 g, 1.18 mmol). The product was purified by column chromatography, Biotage SNAP Cartridge KP-Sil 25 gradient elution: (heptane/EtOAc, 0→100%) to give 43 as a yellow solid (0.057 g, 13%). 1H NMR (400 1 H NMR g, gradient elution: (heptane/EtOAc, 0 → 100%) to give 43 as a yellow solid (0.057 g, 13%). MHz, CD3OD) δ 7.39 (d, J = 8.3 Hz, 1H), 7.35 (d, J = 2.0 Hz, 1H), 7.22–7.15 (m, 2H), 7.13–7.07 (m, 1H), (400 MHz,(m, CD2H), δ 7.39 (d, J =3.87 8.3 (s, Hz,2H), 1H),3.74 7.35(s,(d, J = 132.0 Hz, 1H), 7.22–7.15 (m, 2H), 7.13–7.07 (m, 3 OD) 6.82–6.72 4.36 (s, 2H), 3H). C NMR (101 MHz, CD 3OD) δ 166.3, 159.7, 13 1H), (m, 131.7, 2H), 4.36 (s,131.1, 2H), 3.87 (s,129.9, 2H), 128.1, 3.74 (s, 3H).55.6, C NMR (101HRMS MHz, (ESI CD3+OD) δ 166.3, 153.9,6.82–6.72 141.1, 133.2, 131.5, 130.3, 114.8, 42.7, 29.2. ): calculated 159.7, 153.9, 141.1, 133.2, 131.7, 131.5, 131.1, 130.3, 129.9, 128.1, 114.8, 55.6, 42.7, 29.2. HRMS (ESI+ ): 367.0616 (C17H17N2O3Cl2), found 367.0615. calculated 367.0616 (C17 H17 N2 O3 Cl2 ), found 367.0615.

(E)-2-(Hydroxyimino)-3-(4-methoxyphenyl)-N-(pyridin-2-yl)propanamide (44) (44) (E)-2-(Hydroxyimino)-3-(4-methoxyphenyl)-N-(pyridin-2-yl)propanamide The was used, starting from ether 44-THP (0.095 g, 0.26 The general generalprocedure procedurefor forTHP THPdeprotection deprotection was used, starting from ether 44-THP (0.095 g, mmol). The product was was purified by column chromatography, Biotage SNAP Cartridge KP-Sil 10 10 g, 0.26 mmol). The product purified by column chromatography, Biotage SNAP Cartridge KP-Sil gradient elution: (DCM/MeOH, 2→10%). The obtained g, gradient elution: (DCM/MeOH, 2→10%). The obtainedproduct productwas wasfurther further re-crystallized re-crystallized from from 11H NMR (400 MHz, CD3OD) δ 8.26 (ddd, hexane and acetone to give 44 as a white solid (0.02 g, 28%). hexane and acetone to give 44 as a white solid (0.02 g, 28%). H NMR (400 MHz, CD3 OD) δ 8.26 (ddd, JJMar. == 5.0, 1.9, 0.9 Hz, 1H), 8.19 (dt, Drugs 2018, x FOR PEER 23 of(m, 25 5.0, 1.9, 0.916, Hz, 1H), 8.19REVIEW (dt, JJ == 8.4, 8.4, 1.0 1.0 Hz, Hz, 1H), 1H), 7.81 7.81 (ddd, (ddd, JJ == 8.4, 8.4, 7.4, 7.4, 1.9 1.9 Hz, Hz, 1H), 1H), 7.30–7.21 7.30–7.21 (m, 13 13 2H), 7.12 (ddd, J = 7.4, 5.0, 1.1 Hz, 1H), 6.85–6.76 (m, 2H), 3.92 (s, 2H), 3.74 (s, 3H). C NMR (101 MHz, 2H), 7.12 (ddd, J = 7.4, 5.0, 1.1 Hz, 1H), 6.85–6.76 (m, 2H), 3.92 (s, 2H), 3.74 (s, 3H). C NMR (101 MHz, CD3OD) δ 163.4, 159.7, 153.2, 152.3, 149.0, 140.0, 131.2, 129.9, 121.1, 115.1, 114.8, 55.6, 28.6. HRMS CD 3 OD) δ 163.4, 159.7, 153.2, 152.3, 149.0, 140.0, 131.2, 129.9, 121.1, 115.1, 114.8, 55.6, 28.6. HRMS + (ESI+):): calculated 3O3), found 286.1195. (ESI calculated 286.1192 286.1192 (C (C15HH16NN O ), found 286.1195. 15

16

3

3

(E)-3-(3-Chloro-4-methoxyphenyl)-2-(hydroxyimino)-N-(pyridin-2-yl)propanamide (45) (E)-3-(3-Chloro-4-methoxyphenyl)-2-(hydroxyimino)-N-(pyridin-2-yl)propanamide (45)45-THP (0.29 g, 0.72 The general procedure for THP deprotection was used, starting from ether The general procedure for THP deprotection was used, starting from ether 45-THP (0.29 g, mmol). The obtained product was further re-crystallized from acetone to give 45 as a white solid (0.05 0.72 mmol). The obtained was further re-crystallized acetoneδto give 45 asJ a= white solid 1H NMR g, 20%). M.p.: 218–219 °C product (decomposed) (400 MHz,from d6-acetone) 8.29 (ddd, 4.9, 1.9, 0.9 ◦ C (decomposed) 1 H NMR (400 MHz, d -acetone) δ 8.29 (ddd, J = 4.9, 1.9, (0.05 g, 20%). M.p.: 218–219 6 Hz, 1H), 8.20 (d, J = 8.3 Hz, 1H), 7.84–7.78 (m, 1H), 7.42–7.40 (m, 1H), 7.29 (ddt, J = 8.4, 2.2, 0.6 Hz, 0.9 1H), 8.20J =(d,7.4, J =4.9, 8.3 1.0 Hz,Hz, 1H), 7.84–7.78 7.42–7.40 (m,(s, 1H), 7.29 (ddt, J = 8.4, 0.6(101 Hz, 13C 2.2, 1H),Hz, 7.12 (ddd, 1H), 7.02 (d,(m, J = 1H), 8.5 Hz, 1H), 3.96 2H), 3.85 (s, 3H). NMR 13 C NMR (101 1H), 7.12 (ddd, J = 7.4, 4.9, 1.0 Hz, 1H), 7.02 (d, J = 8.5 Hz, 1H), 3.96 (s, 2H), 3.85 (s, 3H). MHz, d6-acetone) δ 162.0, 154.7, 152.8, 151.9, 149.2, 139.1, 131.5, 130.6, 129.7, 122.4, 120.7, 114.0, 113.3, MHz, d6 -acetone) δ 162.0, 154.7, 152.8, 151.9,(C 149.2, 139.1, 131.5, 130.6, 129.7, 122.4, 120.7, 114.0, 113.3, +): calculated 56.4, 28.2. HRMS (ESI 320.0802 15H15N3O3Cl), found 320.0802. + 56.4, 28.2. HRMS (ESI ): calculated 320.0802 (C15 H15 N3 O3 Cl), found 320.0802. 4.2. Cell Lines 4.2. Cell Lines Human malignant melanoma cell line A-375 was kindly provided by Prof. Marikki Laiho Human malignant melanoma cell line A-375 was kindly provided by Prof. Marikki Laiho (University of Helsinki, Finland) and Hs27 human skin fibroblast cell line was kindly provided by (University of Helsinki, Finland) and Hs27 human skin fibroblast cell line was kindly provided Dr. Carmen Escobedo-Lucea (University of Helsinki, Finland). The cells were maintained in by Dr. Carmen Escobedo-Lucea (University of Helsinki, Finland). The cells were maintained in Glutamax high glucose Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco), supplemented with Glutamax high glucose Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco), supplemented with 10% fetal bovine serum (FBS, Gibco), at 37 °C, 5% CO2. 10% fetal bovine serum (FBS, Gibco), at 37 ◦ C, 5% CO2 . 4.3. Analysis Cells 4.3. Analysis of of Selectivity Selectivity to to Cancer Cancer Cells The cells cells were were seeded seeded to to white white frame frame and and clear clear bottom bottom 96-well 96-well plates plates (Perkin (Perkin Elmer) Elmer) at at the the The density of 10,000 cells/well for A-375 cell line and 7500 cells/well for Hs27 cell line. The cells were density of 10,000 cells/well for A-375 cell line and 7500 cells/well for Hs27 cell line. The cells were grown at at 37 37 ◦°C, 5% CO CO2 until they reached 70–80% confluence (approximately 24 h). Stock solutions grown C, 5% 2 until they reached 70–80% confluence (approximately 24 h). Stock solutions of test control (camptothecin, Sigma-Aldrich, SaintSaint Louis, MO, USA) were of test compounds compoundsand anda positive a positive control (camptothecin, Sigma-Aldrich, Louis, MO, USA) prepared in DMSO and diluted into assay medium (growth medium with 5% FBS) to the final were prepared in DMSO and diluted into assay medium (growth medium with 5% FBS) to the final concentration. Final DMSO concentration was 0.5% in all samples. The culture medium was removed concentration. Final DMSO concentration was 0.5% in all samples. The culture medium was removed from the the plate plate and and compounds compounds added, added, 200 200 µL/well. µL/well. After from After48-h 48-hincubation, incubation, the the amount amount of of ATP, ATP, which which is directly proportional to the number of cells present in culture, was quantified using CellTiter-Glo® Luminescent Cell Viability kit (Promega, Madison, WI, USA), according to manufacturer’s instructions. Origin Graphing and Analysis, version 8.6 (OriginLab, Northampton, MA, USA) was used for determination of CC50 values. The cancer cell selectivity index (SI) was calculated as a ratio of CC50

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is directly proportional to the number of cells present in culture, was quantified using CellTiter-Glo® Luminescent Cell Viability kit (Promega, Madison, WI, USA), according to manufacturer’s instructions. Origin Graphing and Analysis, version 8.6 (OriginLab, Northampton, MA, USA) was used for determination of CC50 values. The cancer cell selectivity index (SI) was calculated as a ratio of CC50 values between Hs27 fibroblasts and A-375 melanoma cells. 5. Conclusions Several syntheses of bromotyrosines have been reported but the synthesis of bromotyrosines with monomethylated tyramine part have not been reported before. The selective removal of the protective groups from the tyramine fragment before the coupling reaction is a challenging step in the total synthesis of purpurealidin I (1). We succeeded at this by using trifluoroacetyl protection. This route can be utilized further for the synthesis of additional bromotyrosine derivatives possessing the monomethylated tyramine fragment. The synthesized simplified analogs without the tyramine fragment retained the cytotoxic activity. The selectivity towards melanoma cell line was generally low. The highest selectivity (SI 4.1) was demonstrated in the case of pyridin-2-yl compound (36). This shows that the marine cytotoxic bromotyrosines are promising scaffolds for developing cytotoxic agents and the full understanding of the elements of their SAR is still in very early stage. Further optimization of simplified bromotyrosine derivatives is needed to attain high selectivity. Supplementary Materials: The following are available online at http://www.mdpi.com/1660-3397/16/12/ 481/s1, Appendix.pdf (synthesis, NMR spectra of compounds 1 and 36, and single crystal X-ray diffraction measurements), Compound36.cif and checkcif-Compound36.pdf Author Contributions: Synthesis, C.B., I.T., M.V., V.B., N.H., T.B. and E.M.-L.; X-ray analysis, T.R. and H.L.; Conceptualization, P.K., and P.T.; Biological screening, P.I., I.T., K.-E.L., T.B.; Writing—original draft preparation, P.K., C.B., I.T., M.V., P.I.; Writing—review and editing, P.K., J.Y.-K. and P.T.; Supervision, P.K., C.B., P.I. and P.T.; Resources, J.Y.-K., P.T. and H.L.; Funding acquisition J.Y.-K. and P.T. Funding: This research was funded by European Union Seventh Framework Programme grant agreement No. FP7-KBBE-2009-3-245137 (MAREX) Exploring Marine Resources for Bioactive Compounds: From Discovery to Sustainable Production and Industrial Applications 2010–2014. J.Y.-K. thanks Academy of Finland for the project No. 285103 and P.K. thanks Academy of Finland for the project No. 315937. Acknowledgments: We thank Paul Flemmich for the synthesis assistance, Heidi Mäkkylä for her excellent technical assistance in the biological experiments and Andrew Neal for checking the language. We also wish to thank the DDCB core facility supported by the University of Helsinki and Biocenter Finland. Conflicts of Interest: The authors declare no conflict of interest.

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