Synthesis, Characterization, and Biological

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Oct 18, 2017 - Metformin Cyclization with Berberine and Magnolol .... the cyclization reaction, the ester groups of product 7 were hydrolyzed by sodium ...

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Synthesis, Characterization, and Biological Evaluations of 1,3,5-Triazine Derivatives of Metformin Cyclization with Berberine and Magnolol in the Presence of Sodium Methylate Han Cao 1 , Shili Liao 1 , Wenjing Zhong 1 , Xuerong Xiao 1 , Jiancheng Zhu 1 , Weimin Li 2, *, Xia Wu 1, * and Yifan Feng 1, * 1

2

*

Central Laboratory of Guangdong, Pharmaceutical University, Guangzhou 510000, China; [email protected] (H.C.); [email protected] (S.L.); [email protected] (W.Z.); [email protected] (X.X.); [email protected] (J.Z.) College of Chinese Materia Medica, Guangzhou University of Chinese Medicine, Guangzhou 510000, China Correspondence: [email protected] (W.L.); [email protected] (X.W.); [email protected] (Y.F.); Tel.: +86-20-39352522 (Y.F.)

Received: 29 September 2017; Accepted: 16 October 2017; Published: 18 October 2017

Abstract: The novel target products were synthesized in the formation of a triazine ring from berberine, magnolol, and metformin catalyzed by sodium methylate. The structures of products 1–3 were firstly confirmed by extensive spectroscopic analyses and single-crystal X-ray diffraction. The crystal structures of the target product 2 and the intermediate product 7b were reported for the first time. All target products were evaluated for their anti-inflammatory and antidiabetic activities against INS-1 and RAW264.1 cells in vitro and all products showed excellent anti-inflammatory effects and anti-insulin resistance effects. Our studies indicated that new compounds 1–3 were found to be active against inflammation and insulin resistance. Keywords: cyclization triazine; anti-inflammatory activity; anti-insulin resistance

1. Introduction Berberine is an isoquinoline alkaloid originally isolated from the Chinese herb Coptis chinensis (Huang lian) and is one of the main components of R. Coptidis [1]. Magnolol is a 4-allyl-2-(5-allyl2-hydroxy-phenyl) phenol, and is present in considerable quantities in the bark of the Houpu magnolia (Magnolia officinalis) [2]. Previous data showed that berberine and magnolol were extensively employed in traditional Chinese medicine for the treatment of diabetes, cancers, inflammations, and hyperlipoidemia [3–8]. Metformin has been widely applied in clinics since the 1950s [9]. Over the past decades, metformin has been recommended as a first-line antidiabetic therapy for the treatment of type 2 diabetes mellitus (T2DM) based on the official guidelines of the American Diabetes Association (ADA), the European Association for the Study of Diabetes (EASD), and the American Association of Clinical Endocrinologists (AACE) [10,11]. Moreover, it takes part in the regulation of reduced insulin resistance and blood glucose concentration [12]. According to a review of recent advances in structural modifications of metformin, a variety of metformin derivatives have been successfully synthesized. In 2005, Viollet synthesized new 3d and 4f complex compounds with metformin that were utilized for the treatment of diabetic patients with non-insulin-dependent diabetes mellitus [13]. Liu synthesized seven derivatives of 2-amino-4-dimethylamino-1,3,5-triazine and obtained pure target compounds from silica gel column chromatography in the same year [14]. Additionally, berberine and magnolol derivatives have Molecules 2017, 22, 1752; doi:10.3390/molecules22101752

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chromatography in the same Additionally, berberine andberberrubine magnolol derivatives have likewise been synthesized. In year 1995,[14]. K. Iwsav firstly synthesized derivatives [15]. been synthesized. In 1995, K. Iwsav firstly synthesized berberrubine derivatives [15]. Inas2010, In likewise 2010, Huang designed, synthesized, and evaluated a new series of berberine derivatives AChE Huang designed, synthesized, a new series of berberine derivatives as AChE inhibitors inhibitors [16]. In 2012, Khaing and Zin evaluated synthesized clovanemagnolol from the reaction of magnolol and [16]. In 2012, Khaing Zin synthesized clovanemagnolol from the reaction of magnolol and of caryophyllene β-oxide, appending macromolecular groups onto the phenolic hydroxyl groups caryophyllene macromolecular groups onto the phenolic hydroxyl groups of magnolol for the β-oxide, first timeappending [17]. magnolol for the first time [17]. However, these reports on berberine, magnolol, and metformin derivatives described individual However, these reports on berberine, magnolol, and metformin derivatives individual structural transformations, while there are no reports on berberine, magnolol, anddescribed metformin combined structural transformations, while there are no reports on berberine, magnolol, and metformin in the formation of a triazine ring catalyzed by sodium methylate. combined in the formation of a triazine ring catalyzed by sodium methylate. Our continuing interest in the chemistry of metformin, berberine, and magnolol and the absence Our continuing interest in the chemistry of metformin, berberine, and magnolol and the absence of any reported synthetic approaches to the structurally distinct frameworks of products 1–3 (Figure 1) of any reported synthetic approaches to the structurally distinct frameworks of products 1–3 (Figure 1) prompted us to begin investigations in this area. We synthesized compounds 1–3 combined in the prompted us to begin investigations in this area. We synthesized compounds 1–3 combined in the formation of aoftriazine ring from berberine, magnolol, and metformin (published patents patents CN 106966997 formation a triazine ring from berberine, magnolol, and metformin (published CN A and CN 106928246 A) [18,19]. On this basis, we optimized the previous reaction conditions and 106966997 A and CN 106928246 A) [18,19]. On this basis, we optimized the previous reaction 1 13 identified their UV, IR, H-NMR, 2D-NMR, HRESIMS. In this study, 1H-NMR, 13C-NMR,and conditions andstructure identifiedby their structure by UV, IR,C-NMR, 2D-NMR, and HRESIMS. In thethis crystal structures of the target product 2 and the intermediate product 7b were never reported study, the crystal structures of the target product 2 and the intermediate product 7b were never in ourreported cognition. The cytotoxicThe activities of activities these derivatives on INS-1 cells and cells RAW264.1 cells were in our cognition. cytotoxic of these derivatives on INS-1 and RAW264.1 established the MTTby method [20]. We also determined that PEG-2 and COX-2 amounts cells wereby established the MTT method [20]. We also determined that PEG-2 andreleased COX-2 released of amounts these compounds through ELISA assays, in order to investigate their potential biological and of these compounds through ELISA assays, in order to investigate their potential biological and pharmacological activities. pharmacological activities.

Figure compounds1–3. 1–3. Figure1.1.The The structures structures of compounds

2. Results and Discussion 2. Results and Discussion Chemistry 2.1.2.1. Chemistry The synthetic strategyfor forpreparing preparingcompounds compounds 11 and and thethe strategy The synthetic strategy and 22isisoutlined outlinedininScheme Scheme1,1, and strategy for compound 3 is outlined in Scheme 2. Briefly, it is based on the alkylation of phenolic OH groups for compound 3 is outlined in Scheme 2. Briefly, it is based on the alkylation of phenolic OH groups of magnolol berberrubine (9) with methyl bromoacetate a subsequent ring closure of magnolol (5) (5) andand berberrubine (9) with methyl bromoacetate and aand subsequent ring closure reaction reaction with metformin (a biguanide) in alkaline medium to afford the final 1,3,5-triazine derivatives with metformin (a biguanide) in alkaline medium to afford the final 1,3,5-triazine derivatives (1, 2 and 3). (1, 2 and 3). During the development of compounds 1 and 2, we started by alkylation; reacting During the development of compounds 1 and 2, we started by alkylation; reacting magnolol (5) with magnolol (5) with methyl bromoacetate (6). The reaction proceeded smoothly under mild conditions. methyl bromoacetate (6). The reaction proceeded smoothly under mild conditions. Two phenolic OH Two phenolic OH groups of magnolol (5) engaged in alkylation with the bromine group of methyl groups of magnolol (5) engaged in alkylation with the bromine group of methyl bromoacetate catalyzed

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catalyzed by 3. Because the generated HBr was neutralized by Na2CO3, the bybromoacetate Na2 CO3 . Because the generated HBr was neutralized by NaHBr , theneutralized alkylation reacting was more bromoacetate catalyzed by Na Na22CO CO 3. Because the generated by Na2CO 3, the 2 CO3was alkylation reacting was more rapid compound was to in the alkylation reactingand wascompound more rapid7and and thorough, andpresent compound was found found to be be present inobtain the rapid and thorough, wasthorough, found to and be in the77 form of esters. Inpresent order to form In to dissociated and sodium form of of esters. esters. In order order to obtain obtain dissociated metformin, metformin, metformin hydrochloride and sodiumfor dissociated metformin, metformin hydrochloride and sodiummetformin methoxidehydrochloride were mixed in a solvent methoxide were mixed for at temperature. In process of the were mixed in in aa solvent solvent for 30 30 min min at room roomthe temperature. In the the process of mixing, mixing, the 30 methoxide min at room temperature. In the process of mixing, displacement reaction occurred between displacement reaction occurred between metformin hydrochloride and sodium methoxide. Because displacement reaction occurred between metformin hydrochloride and Because in metformin hydrochloride and sodium methoxide. Because the solution of sodium productmethoxide. 7 could participate the solution product 77 could in the reaction, 7 was dropwise solution of of productproduct could participate indropwise the subsequent subsequent reaction, product product was added added dropwise thethe subsequent reaction, 7participate was added to a cyclization reaction7with metformin. Before to a cyclization reaction with metformin. Before the cyclization reaction, the ester groups of product to a cyclization reaction with metformin. Before the cyclization reaction, the ester groups of product the cyclization reaction, the ester groups of product 7 were hydrolyzed by sodium methoxide and the 77 were hydrolyzed by methoxide and the groups on product 77 engaged in aa ring weregroups hydrolyzed by sodium sodium methoxide andclosure the carbonyl carbonyl groups ontwo product engaged in ring carbonyl on product 7 engaged in a ring reaction with the imino groups of metformin closure closure reaction reaction with with the the two two imino imino groups groups of of metformin metformin (4) (4) in in the the presence presence of of sodium sodium methylate methylate to to (4)afford in the presence of sodium methylate to afford the final 1,3,5-triazine derivatives (1 and 2). afford the the final final 1,3,5-triazine 1,3,5-triazine derivatives derivatives (1 (1 and and 2). 2). OH OH

O O

OH OH

+ +

5 5

Br Br

O O

O O

Na2CO3 Na2CO3 MeOH, 65°C MeOH, 6h 65°C 6h

R1 R1

O O

O O

O O

6 6

R2 R2

yield: 75% yield: 75%

a: R1=OCH3, R2=OH a: RR11=OCH =OCH33,, RR22=OCH =OH 3 b: b: R1=OCH3, R2=OCH3 H + NH N NH2 + N N NH2 NH NH NH NH

4 4

7 7

H2N H2N

N N

N N

N N

Sodium methylate Sodium solution methylate (5mol/L) solution (5mol/L) MeOH, 65°C MeOH, 12h65°C 12h

N N

O O

O O

OH OH

O O

yield: 17% yield: 1 17% H2N H2N

N N

N N

1

N N O O

N N N N

N N

N N

N N

NH2 NH2

O O

yield: 21% yield: 2 21%

2

Scheme Cyclization Scheme of compounds and2. Scheme1.1. 1.Cyclization Cyclization of of compounds compounds 111and and 2.2.

Scheme Cyclization 3. Scheme2.2. 2.Cyclization Cyclization of of compound compound Scheme of compound3.3.

Compared Compared with with compounds compounds 11 and and 2, 2, the the ring ring closure closure reaction reaction of of compound compound 33 underwent underwent an an Compared with According compounds 1a and 2, the ring closure reaction of compound 3 underwent an analogous process. to review of Cao’s and Nechepurenko‘s studies [21,22], analogous process. According to a review of Cao’s and Nechepurenko‘s studies [21,22], the the initial initial analogous process. According to a reviewhydrochloride of Cao’s and Nechepurenko‘s studies [21,22], the initial step step step was was aa pintsch pintsch reaction reaction in in berberine berberine hydrochloride (8), (8), which which led led to to the the product product berberrubine berberrubine (9). (9). was a pintsch reactionradical in berberine hydrochloride (8), which led to the product berberrubine (9). Then, Then, the hydroxyl of berberrubine (9) engaged in the nucleophile substitution reaction Then, the hydroxyl radical of berberrubine (9) engaged in the nucleophile substitution reaction with with thethe hydroxyl (9) engaged in the substitution with bromine group of methyl (6) gave the product (10). the the bromineradical group of of berberrubine methyl bromoacetate bromoacetate (6) and and gavenucleophile the intermediate intermediate productreaction (10). With With thethe similar cyclization process of and 2, product was in the bromine of methyl bromoacetate and gave the intermediate (10). the similar similargroup cyclization process of products products 11(6) and 2, intermediate intermediate product 10 10product was stirred stirred in With the metformin metformin solution, which consisted of metformin hydrochloride and sodium methoxide that had been cyclization process of products 1 and 2, intermediate product 10 was stirred in the metformin solution, solution, which consisted of metformin hydrochloride and sodium methoxide that had been mixed mixed in a solvent for 30 min at room temperature and led to product 3. which consisted of metformin hydrochloride and sodium methoxide that had been mixed in a solvent in a solvent for 30 min at room temperature and led to product 3. for 30 min at room temperature and led to product 3.

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Then, we studied the reaction under various conditions of alkylation reactions (Tables S5 and S6). Our studies were initiated using dichloromethane (DCM) with Na2CO3 for compound 7, methanol Then, studied the10, reaction under various conditions alkylation reactions (Tables (NaOCH S5 and S6). (MeOH) forwecompound and methanol (MeOH) with 8 of mL of a sodium methylate 3) Our studies were initiated using dichloromethane (DCM) with Na CO for compound 7, methanol 2 3 solution (5 mol/L) for compounds 1–3 at room temperature (25 °C). After screening the reaction (MeOH) forusing compound 10, and (MeOH) 8 mL of sodium methylate (NaOCH 3 ) solution conditions, Na2CO 3 as themethanol base catalyst andwith methanol as athe solvent reacted at 65 °C for 6 h was ◦ C). After screening the reaction conditions, (5 mol/L) for compounds 1–3 at room temperature (25 determined to be optimal for the synthesis of product 7 (Table S5, entry 11). DCM as the solvent using Naat2 CO asfor the8base catalyst and methanol as the solvent at 65of◦ C for 6 h 10 wasindetermined reacted 45 3°C h was determined to be optimal for thereacted synthesis product 80% yield to be optimal for9).the synthesis of product 7 (Table S5, entry 11). DCM as the solvent reacted at 45 ◦ C (Table S6, entry for 8Using h was these determined to beconditions optimal for(Table the synthesis product 10 inS6, 80% yield9),(Table S6, entry the 9). optimized S5, entryof11 and Table entry we examined Usingscope these of optimized conditions (Table(Table S5, entry and best Tableconditions S6, entry 9), examined substrate the cyclization reaction S7).11The forwe the reaction the of substrate scope the cyclization reaction (Table S7).asThe conditions for the reactionas of the compounds compounds 1–3ofwere determined to be CH 3ONa thebest base catalyst and methanol solvent, 1–3 wereatdetermined CH3 S7, ONa as the base catalyst and methanol as the solvent, reacted at 65 ◦ C reacted 65 °C for 12toh be (Table entry 12). for 12 h (Table S7, entry 12). 2.2. Characterization of Compounds 2.2. Characterization of Compounds Target compound 1 was confirmed to possess a molecular formula of C26H29N5O4 by highTarget electrospray compound ionization 1 was confirmed to possess a(HRESIMS) molecular (m/z formula of C26 H 5 O4 by = resolution mass spectrometry = 476.2322 M29+,Ncalcd +, high-resolution electrospray ionization mass spectrometry (HRESIMS) (m/z = 476.2322 M 13 476.2298) and C-NMR data, requiring 13 degrees of unsaturation. Compound 2 was confirmed to 13 C-NMR data, requiring 13 degrees of unsaturation. Compound 2 was confirmed calcd = 476.2298) andformula possess a molecular of C30H36N10O2 by HRESIMS (m/z = 569.3101 M+, calcd = 569.3101) and toC-NMR possessdata. a molecular formula of C30 H36infrared N10 O2 by HRESIMS (m/z =of569.3101 M+S7) , calcd = 569.3101) 13 The Fourier transform (FT-IR) spectrum 1 (Figure suggested the 13 and C-NMR data. The Fourier transform infrared (FT-IR) spectrum of 1 (Figure S7) suggested −1 −1 (1680.66 presence of a hydroxyl group (3225.36 cm ), a methyl group (2929.34 cm ), a carbonyl group −1 ), a carbonyl group the−1presence of a hydroxyl groupcm (3225.36 cm−1 ), acm methyl group (2929.34 cmFT-IR −1 and 1630.52 −1). A comparison cm ), and benzene rings (1571.7 of the data of 2 and 1 −1 ), and benzene rings (1571.7 cm−1 and 1630.52 cm−1 ). A comparison of the FT-IR data (1680.66 cm (Figures S15 and S7) suggested that these compounds shared the same methyl group and benzene of 2 and 1 (Figures S7) suggested that these compounds the same methyl group 13C-NMRS15 1H-NMR rings. The andand spectra of 1 clearly showed theshared two methyl groups located on and the 13 C-NMR and 1 H-NMR spectra of 1 clearly showed the two methyl groups located benzene rings. The N atom (Tables S8 and S9). A detailed examination of the 2D-NMR spectra of 1 confirmed the on the N atom (Tables S8 and S9). A detailed examination the of the 2D-NMR spectra 1 confirmed existence of two benzene rings—ring-A and ring-B—and triazine ring-C. Theofheteronuclear the existence of two benzene rings—ring-A and ring-B—and the triazine ring-C. The heteronuclear multiple bond correlation (HMBC) of H-5/C-7, C-9, C-8; H-6/C-4, C-9; H-5′/C-7′, C-9′; and H-8′/C-6′, 0 ; and H-80 /C-60 , multiple correlation (HMBC) H-5/C-7, C-9, C-8; H-6/C-4, C-9;correlations H-50 /C-70 , C-9 1H-1Hof C-5′, C-4′,bond combined with the correlation spectroscopy (COSY) of H-5/H-6, clearly 0 0 1 1 C-5 , C-4 the , combined thebenzene H- H correlation (COSY) correlations of H-5/H-6, showing locationswith of the ring-A andspectroscopy -B. The triazine ring-C was established based clearly on the showingcorrelations the locations the benzeneH-15′ ring-A and -B. The triazine was established based on HMBC ofofC-13′/H-14′, and H-10′/C-11′, C-12′.ring-C The relative configurations of 0 /H-140 , H-150 and H-100 /C-110 , C-120 . The relative configurations of the HMBC correlations of C-13 compounds 1 and 7 were determined by X-ray diffractions using Cu-Kα radiation with a refinement compounds and The 7 were determined diffractions using Cu-Kα a refinement parameter of10.04. crystal structureby ofX-ray compound 1 is deposited in the radiation CCDC as with number 1,547,785 parameter of 0.04. The crystal structure of compound 1 is deposited in the CCDC as number 1,547,785 (Figure 2), while compound 7b is 1,547,784 (Figure 3). Furthermore, the HMBC and 1H-1H COSY 1 1 (Figure 2), while compound 7b is 1,547,784 (Figure 3). Furthermore, the HMBC and HH COSY correlations are shown in Figure 4. correlations are shown in Figure 4.

Figure 2. 2. X-ray X-ray ORTEP ORTEP (Oak (Oak Ridge Ridge Thermal Thermal Ellipsoid Ellipsoid Plot) Plot) drawing drawing of of compound compound 1. 1. Figure

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Figure 3. 3. X-ray X-ray ORTEP ORTEP (Oak (Oak Ridge Ridge Thermal Thermal Ellipsoid Ellipsoid Plot) drawing of coampound 7b. Figure Plot)drawing drawingof ofcoampound coampound7b. 7b. Figure 3. X-ray ORTEP (Oak Ridge Thermal Ellipsoid Plot)

Figure 4. Key heteronuclear multiple bond and 111HH COSY correlations correlations of 4. Key Keyheteronuclear heteronuclear multiple bond correlation correlation (HMBC) (HMBC) and and H-111H HCOSY of Figure compounds 1–3. compounds 1–3. 13C-NMR data Compound hasaaacompletely completely symmetricstructure. structure.A Adetailed detailedcomparison comparisonof ofthe the1313 Compound C-NMR Compound222has has completelysymmetric symmetric structure. A detailed comparison of the C-NMRdata data (TableS10) S10)and and2D-NMR 2D-NMRspectra spectraof of222and and111revealed revealedthat thatthe thestructure structureof of222was wasvery verysimilar similarto tothat that (Table (Table S10) and 2D-NMR spectra of and revealed that the structure of was very similar to that of 1. The only difference was the replacement of the carbonyl group in 1 by an additional moiety of of of1.1.The Theonly onlydifference differencewas wasthe thereplacement replacementof ofthe thecarbonyl carbonylgroup groupin in11by byan anadditional additionalmoiety moietyof of444 in as confirmed bythe theHMBC HMBCof ofH-10/C-11, H-10/C-11,H-14/C-13, H-14/C-13, and H-15/C-13. in in2,2, 2,as asconfirmed confirmedby by the HMBC of H-10/C-11, H-14/C-13,and andH-15/C-13. H-15/C-13. The molecular formula of compound 3, C 25H25N6O4++, was by HRESIMS HRESIMS (m/z == + established The 3, C 4N , was by (m/z The molecular molecularformula formulaof ofcompound compound 3,25HC2525NH6O O was established by HRESIMS 25 6 4 , established + 13 calcd and 13C-NMR C-NMR data, requiring 13 degrees13 of unsaturation. unsaturation. The FT-IR FT-IR 473.1936 M+,, calcd 13 and requiring degrees of The 473.1936 M (m/z = 473.1936 M==+473.1937) ,473.1937) calcd = 473.1937) and data, C-NMR data,13 requiring degrees of unsaturation. spectrum of compound 1 (Figure S23) suggested the presence of a primary amine group (1674.87 spectrum compound 1 (Figure S23) suggested the presence a primary group (1674.87 The FT-IRofspectrum of compound 1 (Figure S23) suggested theof presence of aamine primary amine group 1H-NMR data of 3 cm−1−1)) and and benzene benzene rings (1600.63 (1600.63 cm cm−1−1,, 1553.38 1553.38 cm−1−1,, and and−1491.67 1491.67 cm cm−1−1).). The The 1H-NMR − 1 − 1 1 − 1 1 cm rings cm data 3 (1674.87 cm ) and benzene rings (1600.63 cm , 1553.38 cm , and 1491.67 cm ). The H-NMRof data displayed signals for two methyl groups at δH = 2.179 (d) and a methoxyl group at δH = 3.10 (s) displayed signals for two methyl groups at δH = 2.179 (d)(d) and a methoxyl group of 3 displayed signals for two methyl groups at δH = 2.179 and a methoxyl groupatatδH δH==3.10 3.10(s) (s) (Figure S17). S17). The The HMBC HMBC of ofH-3/2-C, H-3/2-C, 17-C, 17-C, 16-C; 16-C; 1-H/2-C; 1-H/2-C; and 17H/15-C indicated the existence of (Figure H-3/2-C, (Figure S17). The HMBC 17-C, 16-C; 1-H/2-C;and and 17H/15-C 17H/15-Cindicated indicatedthe theexistence existenceof of benzene ring-A. Ring-B contained an N atom between C-5 and C-15, as confirmed from the HMBC benzene ring-A. Ring-B contained an N atom between C-5 and C-15, as confirmed from the HMBC benzene ring-A. Ring-B N atom between C-5 and C-15, as confirmed from the HMBC of H-1H COSY COSY correlations correlations of of H-5/H-6. H-5/H-6. of 5-H/4-C, 5-H/4-C, 6-C and and 6-H/5-C, 6-H/5-C, 15-C, 15-C, combined combined with with the 11Hof 5-H/4-C, 6-C6-C and 6-H/5-C, 15-C, combined with the 1 H-1the H COSY1H correlations of H-5/H-6. Meanwhile, Meanwhile, ring-C was deduced by the HMBC of 8H/13C, 7C, 15C, 14C and 14H/8C, 15C, 16C.The The Meanwhile, ring-C was by of the8H/13C, HMBC of 8H/13C, 7C,and 15C,14H/8C, 14C and15C, 14H/8C, 16C. ring-C was deduced bydeduced the HMBC 7C, 15C, 14C 16C. 15C, The HMBC of 1 1 HMBCof of11H/9C, 11H/9C,13C, 13C,12C, 12C,10C 10Cand and12H/13C, 12H/13C,10C, 10C,11C, 11C,combined combinedwith with1the the11HH-1H HCOSY COSYcorrelations correlations HMBC 11H/9C, 13C, 12C, 10C and 12H/13C, 10C, 11C, combined with the H- H COSY correlations of of12H/11H, 12H/11H,elucidated elucidatedthe thestructure structureof ofring-D. ring-D.The TheHMBC HMBCof of20H/21C; 20H/21C;24H/22C, 24H/22C,23C; 23C;and and25H/22C, 25H/22C, of 23C suggested the presence of the triazine ring. 23C suggested the presence of the triazine ring.

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12H/11H, elucidated Molecules 2017, 22, 1752 the structure of ring-D. The HMBC of 20H/21C; 24H/22C, 23C; and 25H/22C, 6 of 11 23C suggested the presence of the triazine ring. 2.3. Biological Evaluation 2.3. Biological Evaluation In order to evaluate the anti-inflammatory activities and antidiabetic activities of the synthesized In order to evaluate the anti-inflammatory and antidiabetic activities of thecells synthesized compounds 1–3, we chose RAW264.1 cells activities (mice inflammatory cells) and INS-1 (mouse compounds 1–3, we chose RAW264.1 cells (mice inflammatory cells) and INS-1 cells (mouse insulinoma insulinoma cells). Cell viability was measured by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl cells). Cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay.byRAW264.7 cell cultures were pretreated with tetrazolium a series of bromide (MTT) assay. RAW264.7 cell cultures were pretreated with a series of compounds 1–3 or compounds 1–3 or vehicle to evaluate the effects of compounds 1–3 on the release of COX-2/PEG-2. vehicle to evaluate the effects of compounds 1–3 oninthe release of COX-2/PEG-2. Two targetRAW264.7 cytokines Two target cytokines were markedly increased LPS (Lipopolysaccharide)-stimulated were markedly increased in LPS (Lipopolysaccharide)-stimulated RAW264.7 cells; the increase cells; the increase was dramatically diminished by compounds 1, 2 at 10 µMol/L and compound 3 at was dramatically diminished by compounds 1, 2 at 10 µMol/L and compound 3 at 25 25 µMol/L concentrations. The level of COX-2 was decreased by 13.06%, 14.24%, andµMol/L 25.41%, concentrations. level of COX-2 was by 13.06%, 14.24%, and respectively, by The compounds 1–3, and thedecreased level of PEG-2 was decreased by 25.41%, 27.05%, respectively, 32.26%, and by compounds 1–3, and the level of PEG-2 was decreased by 27.05%, 32.26%, and 36.41%. Ibuprofen 36.41%. Ibuprofen was used as positive control and the level of COX-2/PEG-2 was decreased by was used as positive control and the level of COX-2/PEG-2 was decreased by 32.82% and 42.31% 32.82% and 42.31% (Figure 5A,B). In general, compounds 1–3 all performed remarkable anti(Figure 5A,B). In general, compounds 1–3 all performed remarkable anti-inflammatory activity. inflammatory activity.

Figure Figure5.5.(A,B) (A,B)The Thelevels levelsof ofCOX-2 COX-2and andPEG-2 PEG-2which whichinfluenced influencedby bycompounds compounds 1–3 1–3 and and ibuprofen. ibuprofen. The blank group was not stimulated by LPS. The model group and compound 1–3 groups The blank group was not stimulated by LPS. The model group and compound 1–3 groups were were stimulated stimulated by byLPS. LPS.**pp

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