Mol Divers (2012) 16:839–846 DOI 10.1007/s11030-012-9400-3
SHORT COMMUNICATION
Synthesis of 3-aryl-1,2,4-benzotriazines via intramolecular cyclization of solid-supported o-hydrazidoanilines Edwar Cortés · Luciana Méndez · Ernesto G. Mata · Rodrigo Abonia · Jairo Quiroga · Braulio Insuasty
Received: 7 May 2012 / Accepted: 24 September 2012 / Published online: 10 October 2012 © Springer Science+Business Media Dordrecht 2012
Abstract An efficient solid-phase protocol for the rapid generation of libraries of biologically promising 1,2,4-benzotriazines, including amino acid-derived components, is described. Keywords Solid-phase organic synthesis · Aromatic nucleophilic substitution (SN Ar) · Hydrazidoanilines · 1,2,4-Benzotriazines
Introduction The synthesis and reactivity of 1,2,4-triazines and related compounds have been widely studied. This family of heterocycles exhibits promising biological activities, such as anticancer, antinociceptive, antibacterial and fungicidal properties [1–7]. For instance, compound 1 is a potent, ligand efficient, selective, and orally efficacious antagonist of the adenosine A2A receptor [8] (Fig. 1). The 1,2,4-benzotriazines, in particular, constitute an important group of heterocyclic compounds due to the broad biological activity spectrum displayed by them [9,10]. For example, tirapazamine 2 is a promising antitumor agent that Electronic supplementary material The online version of this article (doi:10.1007/s11030-012-9400-3) contains supplementary material, which is available to authorized users. E. Cortés · R. Abonia · J. Quiroga · B. Insuasty Grupo de Investigación de Compuestos Heterocíclicos, Departamento de Química, Universidad del Valle, Cali A.A. 25360 Colombia L. Méndez · E. G. Mata (B) Instituto de Química Rosario (IQUIR), Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario-CONICET, Suipacha 531, S2002LRK Rosario, Argentina e-mail:
[email protected]
selectively causes DNA damage in hypoxic tumor cells [11], while their analogs 3 (a: R1 = Cl, R2 = cyclohexyl; b R1 = Cl, R2 = Ph) have even more potent cytotoxic activity and hypoxic selectivity than tirapazamine [12]. On the other hand, the spirobenzotriazine 4 displays high selectivity as a ligand for the σ1 receptor subtype in CNS and peripheral tissues [13], and fluorinated compound 5 has been tested for antiviral and cytotoxic activity on Vero cell cultures showing activity against severe diseases caused by smallpox and some other pathogenic viruses [14]. In addition, antimicrobial [15], herbicidal [16], antimalarial [17], human blood rheology regulant [18], antifungal [19], and antiinflammatory [20] activities have also been reported for these kind of compounds. Combinatorial chemistry-related techniques have been widely applied to the discovery of novel lead molecules and the optimization of compound libraries [21,22]. Particularly, solid-phase organic synthesis (SPOS) has experienced a spectacular growth, since the arrival of the small-molecule combinatorial chemistry to the field of drug discovery [23–26]. For library production, SPOS simplifies the purification step, avoiding tiresome isolation processes and allowing an excess of reagents to be used in order to drive reactions to completion. Also, the fact of having a component linked to a polymeric solid support facilitates the use of high-boiling solvents and, thanks to the “pseudo-dilution effect” [27], the formation of undesired homocoupling products is disfavored [28,29]. Among the organic structural realm, heterocycles are of particular interest in combinatorial synthesis [30]; however, to the best of our knowledge, there have been no reports on the synthesis of the 1,2,4-benzotriazinic framework using SPOS [31,32]. As part of our current interest in the application of solid-phase chemistry to biologically promising structures [33–40], herein, we wish to report a strategy developed for the generation of novel 1,2,4-benzotriazine derivatives via
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840
Mol Divers (2012) 16:839–846 Me O N+ N
N N
F3C
N
N
N + NH2 O
NH2
2
1
1
R
N
O N+ N N+ N O H
N H
R2
N N
Bn
4
3a,b F
N
F
N
N
5 Fig. 1 Bioactive 1,2,4-triazine-containing compounds
the intramolecular cyclization of solid-supported o-hydrazidoanilines.
Results and discussion To begin with our strategy, the commercially available 4-fluoro-3-nitrobenzoic acid 6 was chosen as our starting material as shown in Scheme 1 (recently, we have efficiently used acid 6 for the synthesis of novel benzimidazole derivatives. Please see [41]). Initially, compound 6 was linked to Wang resin employing N ,N -diisopropylcarbodiimide (DIC)/ 4-dimethylaminopyridine (4-DMAP) in N ,N −dimethylformamide (DMF), to afford the immobilized ester 7 (step 1). This coupling was confirmed by the presence of the ester carbonyl band at 1,722 cm−1 , the NO2 bands at 1,541 and 1,348 cm−1 in the FT-IR spectrum (KBr disk), and the appearance of a signal at −110.5 ppm in the gel-phase 19 F NMR spectrum. Afterwards, the solid-supported ester 7 was subjected to an aromatic nucleophilic substitution (SN Ar), where the fluorine atom was replaced by a benzoylhydrazide, via an ipso-displacement. In an exploratory sequence, 4-chlorobenzoylhydrazide 8a (Ar = 4-ClPh) was primarily used, yielding the immobilized nitro derivative 9a (step 2). The presence of two broad NH bands (at 3,341 and 3,344 cm−1 ) and two broad C=O bands (at 1,718 and 1,620 cm−1 ) in its FT-IR spectrum and the absence of the 19 F signal at −110.5 ppm in the gel-phase19 F NMR spectrum provided evidence of the convenience of this process. Fluorine displacement was also monitored by gel-phase 19 F NMR [42–44]. Figure 2 clearly shows the disappearance of the 19 F signal after 12 h of reaction in the gel-phase 19 F NMR spectrum (Fig. 2c), indicating that the fluorine replacement was completed.
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Continuing with the sequence described in Scheme 1, the nitro group in 9a was reduced by treatment with SnCl2 ·2H2 O in refluxing DMF (step 3). The absence of the NO2 bands around 1,500 and 1,300 cm−1 in the FT-IR spectrum, indicated the formation of the resin-bound diamine derivative 10a. Subsequently, the solid-supported 1,2,4-benzotriazine 11a was obtained from 10a via an intramolecular cyclization mediated by hot acetic acid (step 4). The cyclization and oxidation processes during the formation of the desired immobilized product 11a were evident from the disappearance of all NH and the C=O amidic bands in its FT-IR spectrum. Finally, to release the heterocycle from the polymer, 11a was treated with 10 % TFA in DCM and was subsequently esterified with diazomethane (step 5), to afford the expected 1,2,4-benzotriazine 12a in a 36 % overall isolated yield (based on the initial loading level of Wang resin) (Scheme 1). Once established the protocol and the correct structure for the 1,2,4-benzotriazine 12a, this procedure was extended to other hydrazides 8b–i in order to evaluate the scope of this approach. In all cases, the sequence proved to be robust, affording the expected 1,2,4-benzotriazine derivatives 12b–i in acceptable overall yields (Table 1). These results encouraged us to investigate a variation of the protocol described in Scheme 1. Our goal was to increase the versatility of the sequence by the attachment of an amino acid moiety to the benzotriazine nucleus. Thus, a new series of amino acid-derived 1,2,4-benzotriazines 18a–c were synthesized following the procedure described in Scheme 2. Our initial attempts were addressed to the synthesis of Fmoc-protected amino hydrazides, in order to apply them to a direct substitution of the fluorine atom in resin 7, such as in the sequence depicted in Scheme 1. However, premature deprotection of the Fmoc was observed when the corresponding amino acid ester was treated with neat hydrazine, probably due to the basicity of the hydrazine (this drawback has also been observed by Bibian et al. [45]). Then, a new strategy was outlined based on an intermolecular reaction between Fmoc-protected amino acids 14a–c and the solid-supported hydrazine 13 (Scheme 2). To achieve this goal, hydrazine was initially linked to the immobilized 4-fluoro-3-nitrobenzoate 7 using hydrazine monohydrate in DMSO at room temperature for 6 h, to afford the solid-supported hydrazine 13 (step 1). Formation of hydrazine 13 was corroborated by the disappearance of the signal at −110.5 ppm in the gel-phase 19 F NMR spectrum and by the presence of a broad NH band (at 3,300 cm−1 ) in the FT-IR spectrum. For the coupling of the Fmoc-protected amino acids 14a–c with the immobilized hydrazide 13, the best conditions were found when the activation of the amino acids was achieved by the combination of 1-hydroxybenzotriazole (HOBt) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). Thus, the formation of the corresponding immobilized acylhydrazines 15a-c (step
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Fig. 2 Gel-phase 19 F NMR study. a Immobilized ester 7 before reaction. b Mixture of the ester 7 and hydrazide 8a after 6 h of reaction. c Reaction of the ester 7 and hydrazide 8a after 12 h of reaction HO
Wang´s resin
O
O
O
O
OH
F
DIC, DMAP/DMF 12 h
6
step 1
NO2
NO2
DM SO, rt, 12 h
5
7 8
4 N N 1
Ar N
ii. CH2 N2 /DCM
reflux 5 min
O N N
step 5
12a-i
SnCl2.2H2O DMF
O O
O
step 2
7
i. 15% TFA DCM
Ar
9a-i
F
step 3
H3CO2C
NO2 H N N H
O
ArCONHNH2 , 8a-i
Ar N
AcOH 80ºC, 7 h step 4
11a-i
O
NH2 H N N H
10a-i
Ar O
Scheme 1 Solid-phase-based synthesis of 1,2,4-benzotriazines
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Conclusions
Table 1 Synthesis and analytical data of the solid-phase synthesized 1,2,4-benzotriazines 12a–i mp (◦ C)
Yielda (%)
Product
Ar–
12a
4-Chlorophenyl
36
178–180
12b
Benzyl
23
107–109
12c
2-Fluorophenyl
24
125–127
12d
Pyridin-3-yl
28
121–123
12e
2-Chlorophenyl
10
150–152
12f
3-Methoxyphenyl
52
124–126
12g
4-(N ,N -dimethyl)phenyl
54
128–130
12h
4-Fluorophenyl
14
184–185
12i
4-Methoxyphenyl
13
166–168
In summary, the sequence developed in this study broadens the scope of the application of the solid-phase approach to heterocyclic chemistry by adding an efficient protocol for the rapid generation of libraries of biologically promising 1,2,4-benzotriazines. A key feature of this sequence is the acid-mediated intramolecular cyclization/oxidation of immobilized o-hydrazidoanilines to yield the 1,2,4-benzotriazine framework. In addition, the synthesis of 1,2,4-benzotriazines derived from amino acids increases the versatility of the reaction sequence and opens the possibility of preparing large libraries of biologically promising compounds. To the best of our knowledge, this is the first synthesis of 1,2, 4-benzotriazines using a solid-phase methodology (for report dealing with the solid-phase synthesis of 1,2,3-benzotriazin4-ones, see [46,47]).
a Overall
isolated yield after flash column chromatography (based on initial loading of Wang resin, five reaction steps)
2), was confirmed by treating an aliquot with 10 % trifluoroacetic acid in CH2 Cl2 for 1 h at ambient temperature. The analysis of the reaction crudes by 1 H and 13 C NMR revealed the presence of the corresponding soluble acylhydrazines. The desired amino acid-derived 1,2,4-benzotriazines 18a–c were obtained after reduction of 15a–c with SnCl2 · 2H2 O and cyclization with AcOH, followed by resin cleavage and methylation (steps 3–5) (Table 2). R (3eq)
O
General Chemical reagents were purchased from commercial sources and were used without further purification. Solvents were analytical grade or were purified by standard procedures prior
HOBt (3eq),
FmocHN CO2H 14a-c O
Experimental section
EDC (3eq) R O
O
DMSO, rt, 6h
F
O N
O
N N
I
NH2 NH2 .H2 O (5eq) NO2
O
FmocHN
O
NO2 HN
step 1
7
DMF:DCM (2:1), 6h step 2
NH2
13
NO2 O
HN
R
O O
OCH3
CH2N2
N
N
N
N R
17a-c
Scheme 2 Solid-phase synthesis of amino acid-derived 1,2,4-benzotriazines
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Fmoc NH
N
R
18a-c
O
AcOH, 80ºC
10% TFA step 5
step 3
O
DCM Fmoc NH
N
Fmoc
15a-c SnCl 2.H2O (8eq) 4h, 80 ºC
O
H N
N H
12h step 4
NH2 O
HN
H N
N H R
16a-c
Fmoc
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Table 2 Synthesis and analytical data of the new amino acid-derived 1,2,4-benzotriazines 18a–c Yield (%)a
Mp (◦ C)
Product
R
18a
H
10
67–68
18b
CH2 Ph
15
84–85
18c
CH3
14
76–77
a Overall
isolated yield after flash column chromatography (based on initial loading of Wang resin, six-step synthesis)
to use. Melting points were determined on a Büchi melting point apparatus and are uncorrected. IR spectra were recorded on a Shimadzu Prestige 21 spectrophotometer and only partial spectral data are listed. 1 H NMR spectra were recorded on a Bruker Avance 300 spectrophotometer operating at 300 MHz, using CDCl3 as solvent and tetramethylsilane as internal standard. Conventional and gel-phase 13 C and 19 F NMR spectra were recorded on the same apparatus using CDCl3 as solvent, at 75.5 and 282.4 MHz, respectively. For gel-phase 13 C and19 F NMR spectra, 50–80 mg of resin was placed in a conventional NMR tube and about 0.5 mL of CDCl3 was added to obtain a gel that was finally homogenized by sonication. Mass spectra were performed on a LC-MS Bruker micrOTOF-Q II spectrophotometer. Microanalyses were performed on an Agilent elemental analyzer and the values are within ±0.4 % of the theoretical values. Silica gel aluminum plates (Merck 60 F254) were used for analytical TLC. Flash column chromatography was performed using Merck silica gel 60 (230–400 mesh), according to the procedure reported by Still et al. [48]. Representative procedure for the solid-phase synthesis of methyl 3-arylbenzo[e][1,2,4]triazine-6-carboxylates 12: methyl3-(4-chlorophenyl)benzo[e][1,2,4]triazine-6carboxylate 12a To a suspension of Wang resin (1 g, 1.1 mmol, 1.1 mmol/g loading) in DMF (8 mL) was added successively 4-fluoro3-nitrobenzoic acid (6; 815 mg, 4.4 mmol), DIC (0.75 mL, 2.2 mmol), and N ,N -dimethylaminopyridine (4-DMAP, 14 mg, 0.11 mmol). The suspension was stirred for 12 h at ambient temperature, and the yellow solid-supported 4-fluoro-3nitrobenzoate 7 obtained was then filtered and washed with DMF (2× 6 mL), MeOH (2× 6 mL), and CH2 Cl2 (2× 6 mL) and dried under reduced pressure. An aliquot of resin 7 (100 mg, 0.929 mmol) was then treated with 4-chlorophenylhydrazide 8a (79 mg, 5 mmol) in DMSO (2 mL) at ambient temperature for 12 h to afford the resin-bound 2-(4-chlorobenzoyl)hydrazinyl)-3-nitrobenzoate 9a. The green resin was filtered, washed successively with DMF (2× 4 mL), MeOH (2× 4 mL) and CH2 Cl2 (1×4 mL) and dried under reduced pressure. The immobilized 3-nitrobenzoate 9a was treated with a 2.0 M solution of SnCl2 · 2H2 O (1 mL, 2.0
mmol) in DMF (1.5 mL) at reflux for 5 min. The reaction mixture was filtered and the resin was washed successively with DMF (2× 4 mL), 20 % H2 O-THF (at 60 ◦ C, 2× 4 mL), MeOH (2× 4 mL) and CH2 Cl2 (1× 4 mL), affording the solid-supported o-hydrazido aniline 10a. After that the resin was dried under vacuum, suspended in AcOH (1.5 mL) and heated to 80 ◦ C for 7 h. Then, the AcOH excess was removed under reduced pressure affording the solid-supported 1,2,4-benzotriazine 11a. Resin-bound 11a was stirred with a 10 % TFA/CH2 Cl2 (3 mL) for 1 h. After that, the mixture was filtered, the resin washed with CH2 Cl2 , and the filtrate concentrated under reduced pressure. Finally, esterification with diazomethane afforded the crude material that was then purified by column chromatography on silica gel and (hexane/EtOAc, 60:40) as eluent, to provide 17.0 mg of benzotriazine 12a (36 % overall yield based on the initial loading level of the Wang resin). Methyl 3-(4-chlorophenyl)benzo[e][1,2,4]triazine-6-carboxylate (12a): Orange powder; m.p. 178–180 ◦ C; IR (KBr) (νmax , cm−1 ): 2956, 2924, 2852, 1728, 1506, 1327, 1269, 1089. 1 H NMR (300 MHz, CDCl3 ): δ 4.07 (s, 3H, OCH3 ), 7.57 (d, J = 8.4 Hz, 2H, Ar–H ), 8.40 (dd, 1H, J = 8.8 Hz, J = 1.6 Hz, 7-H ), 8.60 (d, 1H, J = 8.8 Hz, 8-H ), 8.71 (d, 2H, J = 8.4 Hz, Ar–H ), 8.78 (d, 1H, J = 1.6 Hz, 5-H ) ppm. 13 C NMR (75 MHz, CDCl3 ): δ 53.1, 129.3, 129.7, 129.9, 130.1, 132.1, 133.6, 136.2, 138.3, 140.3, 146.9, 159.4, 165.2 ppm. HRMS m/z 300.0525 (M+H)+ ; calcd for C15 H11 ClN3 O2 + : 300.0534. Methyl 3-benzylbenzo [e] [1, 2, 4] triazine-6-carboxylate (12b): Orange powder; m.p. 107–109 ◦ C; IR (KBr) (νmax , cm−1 ): 2956, 2933, 2848, 1728, 1512, 1330, 1255, 1089. 1 H NMR (300 MHz, CDCl3 ): δ 4.03 (s, 3H, OCH3 ), 4.76 (s, 2H, CH2 ), 7.23 (t, 1H, J = 7.3 Hz, Ar–H ), 7.31 (t, 2H, J = 7.5 Hz, 7.3 Hz, Ar–H ), 7.49 (d, 2H, J = 7.5H z, Ar–H ), 8.38 (dd, 1H, J = 8.8 Hz, J = 1.6 Hz, 7-H ), 8.57 (d, 1H, J = 8.4 Hz, 8-H ), 8.73 (d, 1H, J = 1.1 Hz, 5-H ) ppm. 13 C NMR (75 MHz, CDCl3 ): δ 44.2, 53.0, 126.9, 128.7, 129.3, 129.5, 129.8, 131.7, 135.9, 137.2, 140.3, 146.7, 165.3, 165.7 ppm. HRMS m/z 302.0893 (M+Na)+ ; calcd for C16 H13 N3 NaO2 + : 302.0900. Methyl 3-(2-fluorophenyl)benzo[e][1,2,4]triazine-6-carboxylate (12c): Orange powder, m.p. 125–127 ◦ C; IR (KBr) ( νmax , cm−1 ): 2960, 2924, 2856, 1732, 1614, 1508, 1325, 1226, 1087. 1 H NMR (300 MHz, CDCl3 ): δ 4.06 (s, 3H, OCH3 ), 7.32 (dd, 1H, J = 8.3 Hz, 1.1 Hz, Ar–H ), 7.40 (dt, 1H, J = 7.5 Hz, 1.1 Hz, Ar–H ), 7.54–7.65 (m, 1H, Ar–H ), 8.38 (t, 1H, J = 7.8 Hz, J = 1.7 Hz, Ar–H ), 8.46 (dd, 1H, J = 8.9 Hz, J = 1.5 Hz, 7-H ), 8.65 (d, 1H, J = 8.8 Hz, 8-H ), 8.86 (d, 1H, J = 1.6 Hz, 5-H ) ppm. 13 C NMR (75 MHz, CDCl ): δ 53.1, 117.2, 124.1, 124.5, 3 129.9, 130.1, 132.1, 132.4, 133.0, 136.3, 140.2, 146.4, 159.7, 161.7, 165.2 ppm. HRMS m/z 306.0654 (M+Na)+ ; calcd for C15 H10 FN3 NaO2 + : 306.0649.
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844
Methyl 3-(pyridin-3-yl)benzo[e][1,2,4]triazine-6-carboxylate (12d): Orange powder, m.p. 121–123 ◦ C; IR (KBr) (νmax , cm−1 ): 2957, 2924, 2852, 1728, 1625, 1504, 1327, 1238, 1084. 1 H NMR (300 MHz, CDCl3 ): δ 4.07 (s, 3H, OCH3 ), 7.55 (dd, 1H, J = 8.0 Hz, J = 5.0 Hz, Ar–H ), 8.45 (dd, 1H, J = 8.7 Hz, J = 1.6 Hz, 7-H ), 8.66 (d, 1H, J = 8.7 Hz, 8-H ), 8.83 (bs, 1H, Ar–H ), 8.85 (d, 1H, J = 1.6 Hz, 5-H ), 9.04 (dt, 1H, J = 8.1 Hz, J = 1.2 Hz, Ar–H ), 9.97 (s, 1H, Ar–H ) ppm. 13 C NMR (75 MHz, CDCl3 ): δ 53.1, 123.8, 130.0, 130.2, 130.9, 132.0, 136.0, 136.5, 140.3, 147.3, 150.4, 152.4, 158.9, 165.2 ppm. HRMS m/z 289.0686 (M+Na)+ ; calcd for C14 H10 N4 NaO2 + : 289.0696. Methyl 3-(2-chlorophenyl)benzo[e][1,2,4]triazine-6-carboxylate (12e): Orange powder, m.p. 150–152 ◦ C; IR (KBr) ( νmax , cm−1 ): 2954, 2920, 2850, 1726, 1502, 1325, 1263, 1095. 1 H NMR (300 MHz, CDCl3 ): δ 4.06 (s, 3H, OCH3 ), 7.49–7.52 (m, 2H, Ar–H ), 7.61–7.64 (m, 1H, Ar–H ), 8.00– 8.04 (m, 1H, Ar–H ), 8.49 (dd, 1H, J = 8.8 Hz, J = 1.7 Hz, 7-H ), 8.68 (d, 1H, J = 8.8 Hz, 8-H ), 8.87 (d, 1H, J = 1.7 Hz, 5-H ) ppm. 13 C NMR(75 MHz, CDCl3 ): δ 53.1, 127.2, 129.6, 129.9, 130.4, 131.5, 132.0, 132.5, 133.7, 135.1, 136.3, 139.9, 146.4, 161.7, 165.2 ppm. HRMS m/z 322.0349 (M+Na)+ ; calcd for C15 H10 ClN3 NaO2 + : 322.0354. Methyl 3-(3-methoxyphenyl)benzo[e] [1, 2, 4] triazine6-carboxylate (12f): Orange powder, m.p. 124–126 ◦ C; IR (KBr) (νmax , cm−1 ): 2956, 2924, 2841, 1718, 1595, 1504, 1267, 1085. 1 H NMR (300 MHz, CDCl3 ): δ 3.98 (s, 3H, OCH3 ), 4.07 (s, 3H, OCH3 ), 7.16 (dd, 1H, J = 8.1 Hz, J = 2.4 Hz, Ar–H ), 7.52 (t, 1H, J = 8.1 Hz, Ar–H ), 8.33 (bd, 1H, J = 2.2 Hz, Ar–H ), 8.39 (bd, 1H, J = 7.8 Hz, Ar–H ), 8.41 (dd, 1H, J = 8.8 Hz, J = 1.6, 7-H ), 8.61 (d, 1H, J = 8.9 Hz, 8-H ), 8.82 (d, 1H, J = 1.2 Hz, 5-H ) ppm. 13 C NMR (75 MHz, CDCl3 ): δ 53.0, 55.5, 113.1, 118.6, 121.5, 129.5, 129.9, 130.1, 132.1, 136.1, 136.5, 140.5, 147.0, 160.1, 160.2, 165.4 ppm. HRMS m/z 318.0844 (M+Na)+ ; calcd for C16 H13 N3 NaO3 + : 318.0849. Methyl3-(4-(dimethylamino)phenyl)benzo[e][1,2,4] triazine-6-carboxylate (12g): Orange powder, m.p. 128–130 ◦ C; IR (KBr) (νmax , cm−1 ): 2949, 2922, 2852, 1724, 1606, 1500, 1286, 1242, 1087. 1 H NMR (300 MHz, CDCl3 ): δ 3.10 (s, 6H, 2× CH3 ), 4.03 (s, 3H, OCH3 ), 6.83 (d, 2H, J = 8.8 Hz, Ar– H ), 8.23 (dd, 1H, J = 8.9 Hz, J = 1.7 Hz, 7-H ), 8.48 (d, 1H, J = 8.8 Hz, 8-H ), 8.62 (d, 2H, J = 8.8 Hz, Ar–H ), 8.65 (d, 1H, J = 1.6 Hz, 5-H ) ppm. 13 C NMR (75 MHz, CDCl3 ): δ 40.1, 52.9, 111.8, 122.3, 127.9, 129.8, 130.4, 131.8, 135.7, 140.8, 146.2, 152.8, 160.9, 165.6 ppm. HRMS m/z 309.1348 (M+H)+ ; calcd for C17 H17 N4 O2 + : 309.1346. Methyl 3-(4-fluorophenyl)benzo[e][1,2,4]triazine-6-carboxylate (12h): Orange powder, m.p.184–185 ◦ C; IR (KBr) (νmax , cm−1 ): 2952, 2924, 2850, 1734, 1600, 1504, 1321, 1230, 1089. 1 H NMR (300 MHz, CDCl3 ): δ 4.05 (s, 3H,
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OCH3 ), 7.25–7.30 (m, 2H, Ar–H ), 8.40 (dd, 1H, J = 8.9 Hz, J = 1.6 Hz, 7-H ), 8.58 (d, 1H, J = 8.8 Hz, 8-H ), 8.76–8.81 (m, 3H, Ar–H ) ppm. 13 C NMR (75 MHz, CDCl3 ): δ 53.1, 116.3, 126.2, 129.9, 131.1, 131.2, 132.0, 140.4, 146.8, 159.5, 163.7, 165.3, 167.1 ppm. HRMS m/z 306.0650 (M+Na)+ ; calcd for C15 H10 FN3 NaO2 + : 306.0649. Methyl 3- (4-methoxyphenyl)benzo[e] [1, 2, 4]triazine6-carboxylate (12i): Orange powder, m.p. 166–168 ◦ C; IR (KBr) (ν max , cm−1 ): 2954, 2921, 2853, 1741, 1609, 1506, 1243, 1089. 1 H NMR (300 MHz, CDCl3 ): δ 3.93 (s, 3H, OCH3 ), 4.04 (s, 3H, OCH3 ), 7.10 (d, 2H, J = 8.8 Hz, Ar–H ), 8.33 (dd, 1H, J = 8.7 Hz, J = 1.4 Hz, 7-H ), 8.55 (d, 1H, J = 8.9 Hz, 8-H ), 8.72 (d, 2H, J = 8.8 Hz, Ar–H ), 8.73 (s, 1H, Ar–H ) ppm. 13 C NMR (75 MHz, CDCl3 ): δ 52.9, 55.46, 114.4, 127.7, 128.8, 129.8, 130.7, 131.9, 135.9, 140.5, 146.6, 160.2, 162.8, 165.4 ppm. HRMS m/z 318.0844 (M+Na)+ ; calcd for C16 H13 N3 NaO3 + : 318.0851. Representative procedure for the solid-phase synthesis of the amino acid-derived 1,2,4-benzotriazines 18: synthesis of (9H -fluoren-9-yl)methyl(6-acetylbenzo[e][1,2,4]triazin3-yl)methyl)carbamate 18a Resin 7 (300 mg, 0.33 mmol, 1.1 mmol/g loading) was swollen in dimethylsulfoxide (3 mL), hydrazine monohydrate (80 μL, 1.65 mmol, 5 eq) was added and the mixture was stirred for 6 h at ambient temperature. The resin was filtered, washed with DMF (3× 5 mL), CH2 Cl2 (3× 5 mL), MeOH (3× 5 mL), and CH2 Cl2 (3× 5 mL), and finally dried under reduced pressure to afford the immobilized hydrazine 13. Then, Fmoc-glycine 14a (294.3 mg, 0.99 mmol, 3 eq) was dissolved in a 2:1 mixture of DMF:CH2 Cl2 (3 mL), HOBt (133.8 mg, 0.99 mmol, 3 eq) and EDC (189.8 mg, 0.99 mmol, 3 eq) were added and the solution was stirred at ambient temperature for 15 min. After this time, the solution was added to a suspension of the resin 13 in DMF: CH2 Cl2 (2:1) (3 mL). The mixture was stirred at ambient temperature for 6 h after which the resin was filtered, washed with DMF (3× 5 mL), CH2 Cl2 (3× 5 mL), MeOH (3× 5 mL), and CH2 Cl2 (3× 5 mL), and finally dried under reduced pressure affording the solid-supported hydrazide 15a. Resin 15a was then suspended in DMSO (3 mL), a 2 M solution of SnCl2 · 2H2 O (8 eq) in DMF was added and the mixture was stirred for 4 h at 80 ◦ C. After filtration, the product was washed with DMF (3× 5 mL), CH2 Cl2 (3× 5 mL), MeOH (3× 5 mL), and CH2 Cl2 (3× 5 mL), and finally dried under reduced pressure affording the immobilized triazine 16a. Finally, resin 16a was suspended in acetic acid (3 mL) and stirred overnight at 80 ◦ C. The acetic acid was evaporated under reduced pressure and the resulting resin was treated with 5 mL of 10 % TFA in CH2 Cl2 for 1 h. The mixture was filtered and washed with CH2 Cl2 , and the combined filtrates were evaporated under reduced pressure. Esterification with
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diazomethane afforded the crude material that was then purified by column chromatography on silica gel to give 11 mg of (9H -fluoren-9-yl)methyl (6-acetylbenzo[e][1,2,4]triazin3-yl)methyl)carbamate 18a (15 % overall yield, based on the initial loading level of the Wang resin). (9H -Fluoren-9-yl)methyl-((6-acetylbenzo[e][1,2,4] triazin-3-yl)methyl)carbamate (18a): Orange powder, m.p. 67–68 ◦ C; IR (KBr) (νmax , cm−1 ): 1728, 1519, 1251. 1 H NMR (300 MHz, CDCl3 ): δ 4.05 (s, 3H, OCH3 ), 4.29 (t, 1H, J = 7.1 Hz, CH ), 4.48 (d, 2H, J = 7.1 Hz, CH2 ), 5.20 (d, 2H, J = 5.4 Hz, CH2 ), 6.02 (s, 1H, NH ), 7.30–7.43 (m, 4H, Ar–H ), 7.66 (d, 2H, J = 7.2 Hz, Ar–H ), 7.77 (d, 2H, J = 7.2 Hz, Ar–H ), 8.45 (d, 1H, J = 8.9 Hz, 7-H ), 8.63 (d, 1H, J = 8.9 Hz, 8-H ), 8.76 (s, 1H, 5-H ) ppm. 13 C NMR (75 MHz, CDCl3 ): δ 45.8, 47.2, 53.1, 67.2, 119.9, 125.1, 127.1, 127.7, 130.0, 130.1, 131.4, 136.4, 139.9, 141.3, 143.8, 147.4, 162.3, 165.1 ppm. HRMS m/z 463.1377 (M+Na)+ ; calcd for C25 H20 N4 NaO4 + : 463.1377. (S)-(9H -Fluoren-9-yl)methyl-(1-(6-acetylbenzo[e][1,2, 4]triazin-3-yl)-2-phenylethyl)carbamate (18b): Orange powder, m.p. 84–85 ◦ C; IR (KBr) (νmax , cm−1 ): 1726, 1514, 1448, 1323, 1249. 1 H NMR (300 MHz, CDCl3 ): δ 3.42–3.59 (m, 2H, CH2 ), 4.04 (s, 3H, OCH3 ), 4.22–4.25 (m, 1H, CH ), 4.40 (d, 2H, J = 7.1 Hz, CH2 ), 5.84–5.91 (m, 1H, CH ), 5.98 (d, 1H, J = 8.2 Hz, NH ), 6.90–6.93 (m, 2H, Ar–H ), 7.14–7.16 (m, 3H, Ar–H ), 7.30- -7.39 (m, 4H, Ar–H ), 7.76– 7.60 (m, 4H, Ar–H ), 8.46 (dd, 1H, J = 8.8 Hz, J = 1.6 Hz, 7-H ), 8.65 (d, 1H, J = 8.8 Hz, 8-H ), 8.71 (s, 1H, 5-H ) ppm. 13 C NMR (75 MHz, CDCl3 ): δ 41.4, 47.4, 53.1, 56.7, 66.9, 119.9, 125.1, 126.9, 127.0, 127.6, 128.4, 129.5, 129.9, 131.5, 135.8, 136.4, 139.6, 141.3, 143.9, 147.2, 155.7, 164.6, 165.1 ppm. HRMS m/z 531.2027 (M+H)+ ; calcd for C32 H27 N4 O4 + : 531.2027. (S)-(9H -Fluoren-9-yl)methyl(1-(6-acetylbenzo[e][1,2,4] triazin-3-yl)ethyl) carbamate (18c): Brown powder, m.p. 76–77 ◦ C; IR (KBr) (νmax , cm−1 ): 1730, 1714, 1514, 1450, 1251. 1 H NMR (300 MHz, CDCl3 ): δ 1.77 (d, 3H, J = 6.8 Hz, CH3 ), 4.05 (s, 3H, OCH3 ), 4.41 (d, 2H, J = 7.1 Hz, CH2 ), 5.60–5.64 (m, 1H, CH ), 6.09 (d, 1H, J = 7.1 Hz, NH ), 7.29–7.42 (m, 5H, Ar–H ), 7.58–7.66 (m, 3H, Ar–H ), 7.76 (d, 2H, J = 7.2 Hz, Ar–H ), 8.44 (dd, 1H, J = 8.8 Hz, J = 1.4 Hz, 7-H ), 8.64 (d, 1H, J = 8.8 Hz, 8-H ), 8.77 (s, 1H, 5-H ) ppm. 13 C NMR (75 MHz, CDCl3 ): δ 22.2, 47.3, 52.9, 66.9, 119.9, 124.9, 127.0, 127.6, 127.7, 129.9, 130.0, 131.5, 136.4, 139.8, 141.3, 143.8, 147.4, 155.7, 165.1, 166.2 ppm. HRMS m/z 477.1533 (M+Na)+ ; calcd for C26 H22 N4 NaO4 + : 477.1533. Acknowledgments RA, EC, JQ, and BI thank COLCIENCIAS and Universidad del Valle for financial support. EGM and LM are grateful to Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Agencia Nacional de Promoción Científica y Tecnológica and Universidad Nacional de Rosario for their economic support.
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