HETEROCYCLES

Paper | Regular issue | Vol. 81, No. 1, 2010, pp. 91-115
Received, 24th September, 2009, Accepted, 2nd November, 2009, Published online, 6th November, 2009.
DOI: 10.3987/COM-09-11839

■ The Synthesis of Novel 2,4,6-Trisubstituted 1,3,5-Triazines: A Search for Potential MurF Enzyme Inhibitors

Izidor Sosič, Bogdan Štefane,* Andreja Kovač, Samo Turk, Didier Blanot, and Stanislav Gobec*

Faculty of Chemistry and Chemical Technology, University of Ljubljana, Askerceva 5, 1000 Ljubljana, Slovenia

Abstract

A series of new 2,4,6-trisubstituted 1,3,5-triazines, possessing a variety of substituents (–OH, –SH, –OMe, –Cl, –HNR, –SR and amino acid moieties), were synthesized and evaluated for the inhibition of the bacterial peptidoglycan biosynthesis enzyme MurF. Ethoxycarbonyl isothiocyanate successfully reacted with a variety of amidines, enabling an approach to 6-substituted-4-thioxo-1,3,5-triazin-2-ones. Also, a representative set of 2-thio-, 2-amino-, and 2-oxo-substituted 1,3,5-triazines was synthesized by the SNAr reaction, employing 2,4,6-trichloro-1,3,5-triazine and 2-chloro-4,6-dimethoxy-1,3,5-triazine as the starting materials. One compound displayed notable inhibitory activity against MurF from Escherichia coli.

INTRODUCTION
1,3,5-Triazines (or s-triazines) constitute a class of heterocyclic compounds that have been well known for a long time, and still represent the object of considerable interest, mainly due to their applications in different fields, such as the production of polymeric photostabilisers1 and herbicides.2 Triazine herbicides act as inhibitors of photosynthesis in plants by interrupting the light-driven flow of electrons from water to NADP+.3 Some 1,3,5-triazines also display other important biological properties, of which it is worth mentioning antineoplastic activity4 and antimalarial activity. 5,6 1,3,5-Triazines have also been recognized as antibacterial compounds.7 Recently, Maeda et al. described a series of novel 4,2-di(substituted)amino-1,2-dihydro-1,3,5-triazine derivatives that were evaluated for their antiseptic properties by MIC and MBC tests against Gram-positive and Gram-negative bacteria.8
Peptidoglycan is an essential bacterial cell-wall polymer and its biosynthesis provides a unique and selective target for antibacterial chemotherapy.9 Peptidoglycan biosynthesis consists of 10 biosynthetic transformations, each of them requiring a specific enzyme.10 These enzymes include MurA, MurB, MurC, MurD, MurE, MurF, MraY, MurG, and the transglycosylase and transpeptidase families of enzymes. As a part of our efforts to identify new, small-molecule inhibitors of the intracellular steps of peptidoglycan biosynthesis, the virtual high-throughput screening (VHTS) of the National Cancer Institute “Diversity Set” bank of compounds was performed. We identified the triazine derivative NSC 209931 (Figure 1) as a promising inhibitor of MurF (IC50 = 63 µM).11

MurF belongs to the family of ATP-dependent Mur ligases and catalyzes the formation of the peptide bond between UDP-MurNAc-tripeptide and D-Ala-D-Ala, to form the final soluble peptidoglycan biosynthesis precursor UDP-MurNAc-pentapeptide.10a There are only a few known inhibitors of MurF. The pseudo-tripeptide and pseudo-tetrapeptide aminoalkylphosphinic acids were developed as simplified transition-state analogues.12 A series of small-molecule cyanothiophenes were developed by Abbott Laboratories 13,14 and thiazolylaminopyrimidines were discovered by Johnson & Johnson.15 However, none of these showed significant antibacterial activity. In order to find new inhibitors of MurF as potential antibacterial lead compounds, we initiated the synthesis of a series of 4-thioxo-3,4-dihydro-1,3,5-triazin-2(1H)-one derivatives related to our VHTS hit compound NSC 209931.

RESULTS AND DISCUSSION
The 4-thioxo-1,3,5-triazin-2(1H)-one derivatives 1–8 were prepared from appropriate amidines and ethoxycarbonyl isothiocyanate.16 Independent of the nature of the substituent of the amidine, the reactions of ethoxycarbonyl isothiocyanate with 1 equiv of amidines in the presence of 2 M NaOH proceeded smoothly in toluene at room temperature to give the desired 6-substituted-4-thioxo-3,4-dihydro-1,3,5-triazin-2(1H)-ones 1–5 (Scheme 1).

Furthermore, product 4 (Scheme 1) was readily benzylated in a basic EtOH solution at room temperature, providing 4-thiobenzyl derivatives 6–8 in good yields. However, compounds 1–8 did not show any significant inhibitory activity against MurF from Escherichia coli.
In a second approach, we focused our efforts on the nucleophilic substitution of the chloro substituents in the 1,3,5-triazine derivatives 9a and 9b (Scheme 2). We reasoned that the use of trichloro-1,3,5-triazine (9a) and dimethoxychloro-1,3,5-triazine (9b) as a core scaffold might give us access to multifunctional architectures of the triazine derivatives. Moreover, in the light of the known sequential reactivity of the three chlorine atoms in the trichloro-triazine backbone, such a scaffold is well known to provide a high synthetic variability, useful for combinatorial synthesis.17 With these objectives in mind, series of 2,4,6-trichloro-1,3,5-triazine (9a) and 2-chloro-4,6-dimethoxy-1,3,5-triazine (9b) derivatives were prepared, as outlined in Scheme 2.
Reacting the 2,4,6-trichloro-1,3,5-triazine (9a) at 0 °C with 2-mercaptobenzoic acid resulted in the displacement of one chlorine, yielding the corresponding 1,3,5-triazinethiobenzoic acid 10 in a good yield. The substitution of the chlorine by the 2-substituted primary anilines and benzyl amines was achieved at –15 °C in MeCN, giving products 11−17, with high purity and in reasonable yields. As the replacement of the chloro substituent in 9a appeared straightforward, we decided to investigate its SNAr displacement with aminobenzoic acids. The products of the nucleophilic substitution, (4,6-dichloro- 1,3,5-triazin-2-yl)aminobenzoic acids, could serve us as valuable substrates for the secondary modifications on the carboxylic functionality. Unfortunately, we were unsuccessful in all our attempts to prepare such (4,6-dichloro-1,3,5-triazin-2-yl)aminobenzoic acids in a one-step process.

Nevertheless, we overcame this problem by applying the reverse synthetic strategy. As outlined in Scheme 3, 4-nitrobenzoyl chloride was reacted with isopropyl L-alaninate, yielding 26a, which was subsequently reduced to the amino product 27a in an excellent yield.

Alternatively, amide-bond formation was achieved between the N-benzyl-protected 2-aminobenzoic acid 25b and the D-alanine benzyl ester p-toluenesulfonate salt, using EDC and HOBt as the coupling reagents. The hydrogenolysis (H2, Pd-C) of 26b resulted in the formation of the desired aniline derivative, N-(2-aminobenzoyl)-D-alanine (27b) (Scheme 4).

Finally, target compounds 18 and 19 were obtained in low yields, 20% and 10%, respectively, by the treatment of 2,4,6-trichloro-1,3,5-triazine with 27a and 27b (Scheme 2).
We attempted to hydrolyse the chloro substituents in products 10–19 under basic or acidic conditions. Different reaction conditions (a variation of the reaction temperature and the concentration of the acid or base) were investigated without any success. In most cases, complex reaction mixtures were formed, from which it was not possible to isolate any identifiable products. We proceeded with the synthesis of the dimethoxy analogues 20−24, which were derived from 2-chloro-4,6-dimethoxy-1,3,5-triazine (9b). In this case, 9b was successfully reacted with 2-aminobenzoic acid and 2-mercaptobenzoic acid, giving the corresponding 4,6-dimethoxy-1,3,5-triazine derivatives 20 and 21, respectively, in good yields. The same procedure (MeCN, r.t.) was also used for the synthesis of the methyl ester analogue 22. Furthermore, the treatment of 9b with C-protected D-Glu provided the amino acid derivative 23, which was converted by catalytic hydrogenation (H2, 10% Pd-C) to the target compound 24 (Scheme 2) in an excellent yield.
The 1,3,5-triazin-2-yl amino- and thiobenzoic acid derivatives 20 and 21 were further modified, as shown in Scheme 5. In the course of these SAR studies, we postulated that the introduction of an amino acid side-chain in 20 and 21 might have an impact on the inhibitory activity of such derivatives. Thus, the dimethoxy-1,3,5-triazinethiobenzoic acid derivative 21 was coupled to the D-alanine benzyl ester p-toluenesulfonate salt (28a), the D-glutamic acid dibenzyl ester p-toluenesulfonate salt (29a), and the N6-carbobenzyloxy-L-lysine benzyl ester hydrochloride (30a) to produce the amido derivatives 28, 29 and 30.

The attempted hydrogenolysis of the benzyl groups was unsuccessful. Furthermore, the controlled basic hydrolysis (1 M LiOH, 25 °C) turned out to be problematic as well, resulting in the formation of the complex reaction mixtures. When the amino analogue 20 was reacted with the D-alanine benzyl ester p-toluenesulfonate salt (28a), two products were isolated in low yields (Scheme 5, products 31 and 32). Product 31 resulted from the SNAr substitution of one of the methoxy groups in the dimethoxy-1,3,5-triazine 20 with the D-alanine benzyl ester p-toluenesulfonate salt and the simultaneous cyclization of the EDC-activated carboxylate with the 1,3,5-triazine ring. The structure of 31 was confirmed by HMQC and HMBC NMR spectra. The isolated product 32 indicates that unlike the thio derivatives 21, the 2-amino substituted 4,6-dimethoxy-1,3,5-triazines 20 are subject to the SNAr substitution reaction of the methoxy group under the applied reaction condition for the amide-bond formation.
Target compounds 2−8, 10–24, and 28−32 were tested for the inhibitory activity of the MurF enzyme from E. coli using the Malachite green assay, which detects the free phosphate liberated during the reaction.18 To avoid any possible non-specific (promiscuous) inhibition, all the compounds were tested in the presence of the detergent (Triton X-114, 0.005%).19 The results were obtained as the residual activities (RAs) of the enzyme in the presence of 500 μM concentrations of each compound. Based on the results we can conclude that all of the tested compounds are poor inhibitors of MurF, with the RA values being between 60 and 90%. The only exception is compound 15, with an RA value of 41%, and for which the IC50 was determined to be 450 μM.
On the basis of the results presented above, we used compound 15 as a starting point for further structural modifications. Several analogues of 15 were prepared via the SNAr displacement of chlorine in the 1,3,5-triazine ring with nitrogen nucleophiles such as aniline, morpholine, D-glutamic acid dibenzyl ester p-toluenesulfonate salt, and 2-(methylamino)ethanol (Scheme 6).

All the corresponding compounds were isolated in reasonable yields and with high purity. Unfortunately, when tested on MurF they did not show any significant increase in potency against MurF, having RA values >60% in all cases.
To locate the possible binding orientation of inhibitor 15 within the active site of MurF, a docking experiment was performed using the FlexX software.20 The predicted binding pose of compound 15 is presented in Figure 2.

In this docking pose, the hydroxy group of the inhibitor forms hydrogen bonds with Asn326 and Asn328, while the phenyl ring forms hydrophobic interactions with Phe31, Leu45 and Ile139. Phe31 also forms π-stacking interactions with the inhibitor’s phenyl and 1,3,5-triazine rings. It should also be noted that compound 15 is well aligned with the co-crystallized cyanothiophene inhibitor.14 The hydroxy group of compound 15 is aligned with the sulfonylamide group of the co-crystallized inhibitor, forming similar interactions. In addition, the phenyl ring of compound 15 and that of the co-crystallized inhibitor sit in the hydrophobic pocket formed by Phe31, Leu45 and Ile139.
Since it is known that tautomerism occurs in 1,3,5-triazines when they are substituted by hydroxy, sulfanyl, or amino functionality,21 it should be noted that unlike the 4-amino-substituted 1,3,5-triazines, the 2,4-diamino substituted derivatives 33−38 exist in tautomeric equilibria, as is evident from the 1H and 13C NMR spectra recorded in DMSO and CDCl3 solutions at 25 °C. For this reason, some of the NMR spectra were recorded at elevated temperatures for reasons of clarity. For most of the compounds possessing the symmetric 2,4-dichloro-substituted 1,3,5-triazine ring, we have observed in the 13C NMR spectra three distinct resonances (between 150−170 ppm) for the triazine carbons. On the other hand, the 2,4-dimethoxy derivatives 20−22 and 28−30, display the symmetric pattern in the 13C NMR spectra, having only two resonances in the region of 168−172 ppms.

CONCLUSION
In summary, based on encouraging results with the VHTS hit NSC209931, a series of new 1,3,5-triazines were designed and synthesized as putative MurF inhibitors. Ethoxycarbonyl isothiocyanate successfully reacted with a variety of amidines, enabling an approach to the 6(4)-substituted-4-thioxo- 1,3,5-triazin-2-ones. Also, a representative set of 2-thio-, 2-amino-, and 2-oxo-substituted 1,3,5-triazines was synthesised by the SNAr reaction, employing 2,4,6-trichloro-1,3,5-triazine (9a) and 2-chloro-4,6-dimethoxy-1,3,5-triazine (9b) as the starting materials. All the synthesised compounds were tested for their inhibitory activity of the MurF enzyme from E. coli; however, only compound 15 turned out to display significant inhibitory activity, with an IC50 value of 450 μM.

EXPERIMENTAL
General methods. Solvents and starting compounds were obtained from commercial sources (Fluka, Sigma and Aldrich). Light petroleum refers to the fraction with the boiling point 40–60 °C. TLC was carried out on Fluka silica-gel TLC-cards. All mps were determined on a hot-stage apparatus and are uncorrected. IR spectra were recorded on a BioRad FTS 3000MX instrument. NMR spectra were recorded on a Bruker Avance 300 DPX spectrometer at 302 K. Chemical shifts are reported in δ ppm, referenced to an internal TMS standard for 1H NMR, chloroform–d (δ 77.0), DMSO–d6 (δ 39.5) for 13C NMR. Microanalyses were performed on a Perkin-Elmer 2400 series II CHNS/O analyser. Mass spectra and high-resolution mass measurements were performed on a VG-Analytical Autospec EQ instrument. Hardware and software. The docking experiment was made on a computer workstation with four dual-core Opteron processors, 16GB of RAM and 1.2TB of hard-drive space running the Fedora 7 operating system. FlexX 3.1.2 from BioSolveIT GmbH20 was used for the active-site preparation, the docking of the compound 15 and the scoring. PyMol from DeLano Scientific was used for preparation of the graphical representation of the docking results.

General procedure for the preparation of 6-substituted-4-thioxo-3,4-dihydro- 1,3,5-triazin-2(1H)-ones 1-5. To a solution of corresponding amidine (1.0 mmol) in H2O (3 mL), toluene (5 mL) was added and the mixture stirred vigorously. After 5 min, ethoxycarbonyl isothiocyanate (1.4 mmol) in toluene (2 mL) and NaOH (2 M, 4 mL) were simultaneously added over a period of 10 minutes. Additional NaOH (2 M, 2 mL) was added after 15 min and then the reaction mixture was stirred for 1 h at room temperature. The phases were then separated and the organic layer washed with NaOH (2 M, 6 mL). The combined alkaline phases were acidified to pH 1−2 with H2SO4, and in this way the precipitate was formed, which was then filtered off. Pure products were obtained by crystallization from the corresponding solvent.
6-(4-Aminophenyl)-4-thioxo-3,4-dihydro-1,3,5-triazin-2(1H)-one (1): Crystallization from MeCN gave 170 mg (77%) of yellow crystals. mp 283.0–287.0 °C. Rf 0.35 (MeOH-CH2Cl2 = 1/5). IR (KBr): 3442, 3338, 3020, 2891, 1720, 1633, 1601, 1577, 1519, 1490, 1322, 1184, 969, 830, 764, 601 cm–1. 1H NMR (300 MHz, DMSO–d6): δ 6.34 (br s, 2H, PhNH2), 6.62 (d, J = 9.0 Hz, 2H, -Ph), 7.95 (d, J = 9.0 Hz, 2H, -Ph), 12.38 (br s, 1H, -NH-), 12.59 (br s, 1H, -NH-). 13C NMR (75.5 MHz, DMSO–d6): δ 113.0, 114.7, 131.3, 150.3, 154.7, 158.9, 183.8. MS (EI) m/z (%): 220 (M+, 100), 161 (30), 119 (100), 69 (22), 57 (30). HRMS calcd for C9H8N4OS: 220.0419. Found: 220.0234. Anal. Calcd for C9H8N4OS: C, 49.08; H, 3.66; N, 25.44. Found: C, 48.79; H, 3.68; N, 25.69.
6-(3-Nitrophenyl)-4-thioxo-3,4-dihydro-1,3,5-triazin-2(1H)-one (2)22: Crystallization from MeCN gave 461 mg (92%) of orange crystals. mp 99.0–105.0 °C. mp 74 °C.22 Rf 0.18 (MeOH-CH2Cl2 = 1/5). IR (KBr): 3414, 3210, 3107, 1678, 1617, 1528, 1449, 1388, 1350, 1278, 1224, 1150, 918, 702, 578 cm–1. 1H NMR (300 MHz, DMSO–d6): δ 7.86 (t, J = 8.0 Hz, 1H, -Ph), 8.45–8.54 (m, 2H, -Ph), 8.92 (t, J = 2.0 Hz, 1H, -Ph), 12.89 (br s, 1H, -NH-), 13.47 (br s, 1H, -NH-). MS (ESI) m/z: 249 (M-H)–. HRMS calcd for C9H5N4O3S: 249.0082. Found: 249.0078.
6-(Benzylthio)-4-thioxo-3,4-dihydro-1,3,5-triazin-2(1H)-one (3)16: Crystallization from MeCN gave 1.87 g (75%) of pale white crystals. mp 226.0–228.0 °C. mp 230.0–234.0 °C.16 Rf 0.44 (MeOH-CH2Cl2 = 1/5). IR (KBr): 3415, 3140, 3031, 2912, 1655, 1528, 1391, 1239, 1207, 1145, 1031, 882, 701, 614, 577 cm–1. 1H NMR (300 MHz, DMSO–d6): δ 4.38 (s, 2H, -CH2S-), 7.24–7.46 (m, 5H, -Ph), 12.52 (br s, 1H, -NH), 13.58 (br s, 1H, -NH-). MS (ESI) m/z: 274 (M+Na)+. HRMS calcd for C10H9N3ONaS2: 274.0085. Found: 274.0088.
6-Phenyl-4-thioxo-3,4-dihydro-1,3,5-triazin-2(1H)-one (4)16: Crystallization from EtOH gave 1.76 g (86%) of yellow crystals. mp 244.0–248.0 °C. mp 246.0–248.0 °C.16 Rf 0.17 (MeOH-CH2Cl2 = 1/20). IR (KBr): 3421, 3062, 2936, 1678, 1601, 1551, 1385, 1320, 1231, 1164, 941, 816, 766, 696 cm–1. 1H NMR (300 MHz, DMSO–d6): δ 7.52–7.61 (m, 2H, -Ph), 7.64–7.72 (m, 1H, -Ph), 8.07–8.14 (m, 2H, -Ph), 12.74 (br s, 1H, -NH-), 13.18 (br s, 1H, -NH-). MS (ESI) m/z: 206 (M+H)+. HRMS calcd for C9H8N3OS: 206.0388. Found: 206.0380.
6-p-Tolyl-4-thioxo-3,4-dihydro-1,3,5-triazin-2(1H)-one (5)16: Crystallization from EtOH gave 135 mg (62%) of white crystals. mp 245.0–247.5 °C. mp 246.0–247.0 °C.16 Rf 0.63 (MeOH-CH2Cl2 = 1/5). IR (KBr): 3414, 3124, 3051, 2936, 1645, 1615, 1589, 1549, 1397, 1328, 1233, 1169, 952, 832, 783, 732, 626, 564 cm–1. 1H NMR (300 MHz, DMSO–d6): δ 2.40 (s, 3H, -CH3), 7.37 (d, J = 8.0 Hz, 2H, -Ph), 8.04 (d, J = 8.0 Hz, 2H, -Ph), 12.67 (br s, 1H, -NH-), 13.09 (br s, -NH-). MS (EI) m/z (%): 219 (M+, 68), 160 (30), 144 (43), 118 (100), 91 (24). HRMS calcd for C10H9N3OS: 219.0466. Found: 219.0471.

General procedure for the preparation of benzylated derivatives 6–8. To a solution of compound 4 (1.0 mmol) in EtOH (8 mL) and NaOH (2 M, 5 mL), the corresponding benzyl halide (1.1 mmol) was slowly added. The reaction mixture was stirred for 1 h at room temperature. After the reaction was complete, H2O (10 mL) was added, followed by the addition of H2SO4 (2 M, 5 mL). The precipitate formed was filtered off and the pure product obtained by crystallization from the corresponding solvent.
6-Phenyl-4-(benzylthio)-1,3,5-triazin-2(3H)-one (6)23: Crystallization from dioxane gave 224 mg (76%) of white crystals. mp 220.0–222.0 °C. mp 229.0 °C.23 Rf 0.45 (MeOH-CH2Cl2 = 1/20). IR (KBr): 3414, 3078, 3025, 2939, 1668, 1538, 1494, 1423, 1312, 1276, 1236, 1167, 1099, 1008, 848, 778, 711, 696 cm–1. 1H NMR (300 MHz, DMSO–d6): δ 4.45 (s, 2H, -SCH2-), 7.23–7.38 (m, 3H, -Ph), 7.42–7.50 (m, 2H, -Ph), 7.52–7.62 (m, 2H, -Ph), 7.64–7.73 (m, 1H, -Ph), 8.21 (d, J = 6.5 Hz, 2H, -Ph), 12.96 (br s, 1H, -NHCO-). MS (ESI) m/z: 294 (M-H)–. HRMS calcd for C16H12N3OS: 294.0701. Found: 294.0707.
6-Phenyl-4-(2-methoxybenzylthio)-1,3,5-triazin-2(3H)-one (7): Crystallization from MeCN gave 308 mg (95%) of a white foamy solid. mp 224.5–226.0 °C. Rf 0.47 (MeOH-CH2Cl2 = 1/20). IR (KBr): 3414, 2944, 1672, 1550, 1494, 1420, 1315, 1274, 1252, 1168, 1097, 1026, 850, 751, 710, 624 cm–1. 1H NMR (300 MHz, DMSO–d6): δ 3.85 (s, 3H, -OCH3), 4.40 (s, 2H, -SCH2-), 6.90 (dt, J = 7.5, 1.0 Hz, 1H, -Ph), 7.03 (d, J = 8.0 Hz, 1H, -Ph), 7.29 (dt, J = 7.5, 1.5 Hz, 1H, -Ph), 7.43 (dd, J = 7.5, 1.5 Hz, 1H, -Ph), 7.54–7.62 (m, 2H, -Ph), 7.64–7.73 (m, 1H, -Ph), 8.21 (d, J = 7.0 Hz, 2H, -Ph), 12.91 (br s, 1H, -NHCO-). 13C NMR (75.5 MHz, DMSO–d6): δ 29.3, 55.6, 111.0, 120.4, 124.4, 128.6 (2C), 128.9 (2C), 129.1, 130.3, 133.5, 154.7, 157.2, 164.6. MS (ESI) m/z: 326 (M+H)+. HRMS calcd for C17H16N3O2S: 326.0963. Found: 326.0960. Anal. Calcd for C17H15N3O2S: C, 62.75; H, 4.65; N, 12.91. Found: C, 62.53; H, 4.72; N, 12.94.
6-Phenyl-4-(4-cyanobenzylthio)-1,3,5-triazin-2(3H)-one (8): Crystallization from MeCN gave 185 mg (58%) of a white foamy solid. mp 229.5–232.5 °C. Rf 0.26 (MeOH-CH2Cl2 = 1/20). IR (KBr): 3549, 3413, 3080, 2940, 2225, 1671, 1532, 1493, 1423, 1311, 1272, 1168, 1100, 1008, 944, 847, 776, 709, 684, 554 cm–1. 1H NMR (300 MHz, DMSO–d6): δ 4.52 (s, 2H, -SCH2-), 7.53–7.61 (m, 2H, -Ph), 7.64–7.72 (m, 3H, -Ph), 7.77–7.84 (m, 2H, -Ph), 8.18 (d, J = 7.0 Hz, 2H, -Ph), 12.99 (br s, 1H, -NHCO-). 13C NMR (75.5 MHz, DMSO–d6): δ 33.5, 110.0, 118.7, 128.6, 128.9, 129.9 (2C), 132.4 (2C), 133.7, 143.7, 154.6, 164.5. MS (ESI) m/z: 321 (M+H)+. HRMS calcd for C17H13N4OS: 321.0810. Found: 321.0825. Anal. Calcd for C17H12N4OS: C, 63.73; H, 3.78; N, 17.49. Found: C, 63.50; H, 3.85; N, 17.52.

Procedure for the preparation of 2-[(4,6-dichloro-1,3,5-triazin-2-yl)thio]benzoic acid 10. Thiosalicylic acid (308 mg, 2.0 mmol) was added to an ice-cooled solution of 2,4,6-trichloro-1,3,5-triazine (369 mg, 2.0 mmol) in MeCN (20 mL). The reaction mixture was allowed to react for 1 h at 0 °C and then H2O (5 mL) was added. The white solid formed was filtered off and washed with H2O (5 mL) and MeCN (5 mL), yielding a pure product, 465 mg (77%). mp 140.0–146.0 °C. Rf 0.70 (MeOH). IR (KBr): 3067, 1989, 1882, 1664, 1686, 1585, 1524, 1467, 1278, 1260, 846, 745, 560 cm–1. 1H NMR (300 MHz, DMSO–d6): δ 7.55–7.66 (m, 2H, -Ph), 7.70–7.77 (m, 1H, -Ph), 7.88–7.94 (m, 1H, -Ph), 11.17 (br s, 1H, -CO2H). 13C NMR (75.5 MHz, DMSO–d6): δ 125.7, 129.9, 130.4, 131.8, 135.3, 137.2, 151.8, 166.9, 169.1, 169.2. MS (EI) m/z (%): 301 (M+, 8), 256 (100), 195 (35), 136 (45). HRMS calcd for C10H5N3O2SCl2: 300.9479. Found: 300.9480.

General procedure for the preparation of 2-arylamino-4,6-dichloro-1,3,5-triazines 11–19. To a stirred, cooled solution (–15 °C) of 2,4,6-trichloro-1,3,5-triazine (1.0 mmol) in dry MeCN (10 mL), the corresponding substituted aniline (1.0 mmol) was added over a period of 5 min, followed by the addition of Et3N (1.3 mmol). The reaction mixture was stirred at –15 °C for 1 h. After the reaction was complete (monitored by TLC), the solvent was removed under reduced pressure, then H2O (10 mL) was added to give solid crude products, which were filtered off and subsequently purified by silica gel radial chromatography.
N-(2-Bromophenyl)-2-amino-4,6-dichloro-1,3,5-triazine (11)24: Purification on SiO2 (EtOAc-petroleum ether = 3/5) gave 170 mg (53%) of a brown solid. mp 155.5–157.0 °C. mp 158.5–159.0 °C.24 Rf 0.61 (EtOAc-petroleum ether = 3/5). IR (KBr): 3366, 1582, 1541, 1519, 1385, 1314, 1251, 1226, 1166, 1025, 870, 793, 760, 606 cm–1. 1H NMR (300 MHz, DMSO–d6): δ 7.26–7.35 (m, 1H, -Ph), 7.42–7.52 (m, 2H, -Ph), 7.74 (dd, J = 8.5, 1.0 Hz, 1H, -Ph), 10.96 (br s, 1H, -NH-).
N-(2-Ethylphenyl)-2-amino-4,6-dichloro-1,3,5-triazine (12)24: Purification on SiO2 (EtOAc-petroleum ether = 3/5) gave 225 mg (42%) of a white solid. mp 109.0–113.0 °C. mp 119.5–120.5 °C.24 Rf 0.57 (EtOAc-petroleum ether = 3/5). IR (KBr): 3237, 3109, 2968, 1596, 1577, 1546, 1512, 1391, 1317, 1225, 1169, 1018, 835, 797, 615 cm–1. 1H NMR (300 MHz, DMSO–d6): δ 1.11 (t, J = 7.5 Hz, 3H, -CH2CH3), 2.56 (q, J = 7.5 Hz, 2H, -CH2CH3), 7.21–7.36 (m, 4H, -Ph), 10.69 (br s, 1H, -NH-).
N-(2-Tert-butylphenyl)-2-amino-4,6-dichloro-1,3,5-triazine (13): Purification on SiO2 (EtOAc-petroleum ether = 3/5) gave 345 mg (58%) of a white solid. mp 154.5–159.5 °C. Rf 0.62 (EtOAc-petroleum ether = 3/5). IR (KBr): 3230, 3107, 2967, 1609, 1591, 1549, 1512, 1390, 1211, 1171, 1019, 845, 801, 756, 664 cm–1. 1H NMR (300 MHz, DMSO–d6): δ 1.29 (s, 9H, -C(CH3)3), 7.17 (dd, J =, 7.5, 2.0 Hz, 1H, -Ph), 7.21–7.34 (m, 2H, -Ph), 7.48 (dd, J = 8.0, 2.0 Hz, 1H, -Ph), 10.65 (br s, 1H, -NH-). 13C NMR (75.5 MHz, DMSO–d6): δ 30.7, 34.8, 126.9, 127.2, 128.0, 130.6, 134.2, 146.3, 165.6, 168.8, 169.4. MS (ESI) m/z: 297 (M+H)+. HRMS calcd for C13H15N4Cl2: 297.0674. Found: 297.0682. Anal. Calcd for C13H14N4Cl2: C, 52.54; H, 4.75; N, 18.85. Found: C, 52.41; H, 4.89; N, 18.93.
Methyl 2-[(4,6-dichloro-1,3,5-triazin-2-yl)amino]benzoate (14)25: Purification on SiO2 (EtOAc- petroleum ether = 3/5) gave 1.35 g (56%) of a white solid. mp 196.0–197.5 °C. mp 192.0–194.0 °C.)25 Rf 0.59 (EtOAc-petroleum ether = 3/5). IR (KBr): 3140, 2958, 2338, 1686, 1619, 1566, 1508, 1458, 1428, 1388, 1316, 1247, 1168, 1090, 820, 795, 759, 699 cm–1. 1H NMR (300 MHz, DMSO–d6): δ 3.78 (s, 3H, -CO2CH3), 7.35–7.45 (m, 1H, -Ph), 7.63–7-75 (m, 2H, -Ph), 7.86–7.93 (m, 1H, -Ph), 11.19 (br s, 1H, -NH-). 13C NMR (75.5 MHz, DMSO–d6): δ 52.8, 125.4, 126.3, 130.8, 133.5, 133.7, 136.2, 154.6, 164.6, 166.8, 167.0.
N-[2-(2-Hydroxyethyl)phenyl]-2-amino-4,6-dichloro-1,3,5-triazine (15): Purification on SiO2 (EtOAc- petroleum ether = 3/5) gave 165 mg (58%) of a white solid. mp 110.5–114.5 °C. Rf 0.39 (EtOAc-petroleum ether = 3/5). IR (KBr): 3266, 2942, 2884, 1616, 1549, 1456, 1394, 1231, 1018, 825, 794, 761, 612 cm–1. 1H NMR (300 MHz, DMSO–d6): δ 2.73 (t, J = 7.0 Hz, 2H, -CH2CH2OH), 3.58 (t, J = 7.0 Hz, 2H, -CH2CH2OH), 4.90 (br s, 1H, -CH2CH2OH), 7.20–7.40 (m, 4H, -Ph), 10.67 (br s, 1H, -NH-). 13C NMR (75.5 MHz, DMSO–d6): δ 34.5, 61.3, 126.5, 126.6, 127.0, 130.3, 134.4, 135.2, 165.0, 168.8, 169.6. MS (ESI) m/z: 285 (M+H)+. HRMS calcd for C11H11N4OCl2: 285.0310. Found: 285.0317. Anal. Calcd for C11H10N4OCl2: C, 46.34; H, 3.54; N, 19.65. Found: C, 46.82; H, 3.80; N, 19.70.
N-(4-Methoxybenzyl)-2-amino-4,6-dichloro-1,3,5-triazine (16): Purification on SiO2 (EtOAc-hexane = 3/5) gave 273 mg (96%) of a white solid. mp 113.0–115.0 °C. Rf 0.52 (EtOAc-hexane = 3/5). IR (KBr): 3551, 3414, 3266, 1640, 1551, 1514, 1404, 1324, 1236, 1170, 1104, 1032, 848, 795 cm–1. 1H NMR (300 MHz, DMSO–d6): δ 3.81 (s, 3H, -OCH3), 4.59 (d, J = 6.0 Hz, 2H, -NHCH2Ph), 6.26 (br s, 1H, -NHCH2-), 6.88 (d, J = 8.5 Hz, 2H, -Ph), 7.23 (d, J = 8.5 Hz, 2H, -Ph). 13C NMR (75.5 MHz, DMSO–d6): δ 43.4, 55.0, 113.7, 128.7, 129.2, 158.4, 165.2, 168.5, 169.4. MS (ESI) m/z: 285 (M+H)+. HRMS calcd for C11H11N4OCl2: 285.0310. Found: 285.0315. Anal. Calcd for C11H10N4OCl2: C, 46.34; H, 3.54; N, 19.65. Found: C, 46.16; H, 3.61; N, 19.73.
N-(3,4-Dimethoxyphenethyl)-2-amino-4,6-dichloro-1,3,5-triazine (17): Purification on SiO2 (EtOAc- hexane = 3/5) gave 1.187 g (72%) of a yellowish solid. mp 121.5–122.0 °C. Rf 0.32 (EtOAc-hexane = 3/5). IR (KBr): 3479, 3414, 3332, 2945, 1611, 1516, 1433, 1324, 1236, 1156, 1140, 1098, 1020, 838, 626 cm–1. 1H NMR (300 MHz, DMSO–d6): δ 2.86 (t, J = 7.0 Hz, 2H, -NHCH2CH2Ph), 3.75 (q, J = 6.5 Hz, 2H, -NHCH2CH2Ph), 3.87 (s, 3H, -OCH3), 3.88 (s, 3H, -OCH3), 5.91 (br s, 1H, -NHCH2CH2Ph), 6.69–6.76 (m, 2H, -Ph), 6.82 (d, J = 8.0 Hz, 1H, -Ph). 13C NMR (75.5 MHz, DMSO–d6): δ 33.6, 42.3, 55.3, 55.4, 111.8, 112.5, 120.5, 131.0, 147.3, 148.5, 165.1, 168.3, 169.3. MS (ESI) m/z: 329 (M+H)+. HRMS calcd for C13H15N4O2Cl2: 329.0572. Found: 329.0577. Anal. Calcd for C13H14N4O2Cl2: C, 47.43; H, 4.29; N, 17.02. Found: C, 47.15; H, 4.41; N, 16.88.
Isopropyl N-{4-[(4,6-dichloro-1,3,5-triazin-2-yl)amino]benzoil}-L-alaninate (18): Purification on SiO2 (EtOAc-hexane = 3/5) gave 67 mg (29%) of a white solid. mp 228.0–234.0 °C. Rf 0.20 (EtOAc-hexane = 3/5). [α]D20 +39.2 (c 0.39, DMSO). IR (KBr): 3418, 3273, 1732, 1651, 1608, 1565, 1499, 1381, 1216, 824, 794, 624 cm–1. 1H NMR (300 MHz, DMSO–d6): δ 1.19 (t, J = 7.0 Hz, 6H, -CH(CH3)2). 1.39 (d, J = 7.0 Hz, 3H, -CH3), 4.39 (m, 1H, -CH(CH3)2), 4.91 (sim m, 1H, -NHCH-), 7.71 (d, J = 8.0 Hz, 2H, -Ph), 7.91 (d, J = 8.5 Hz, 2H, -Ph), 8.67 (br d, J = 6.5 Hz, 1H, -NHCH-), 11.30 (br s, 1H, -NH-). 13C NMR (75.5 MHz, DMSO–d6): δ 16.6, 21.4, 21.4, 48.4, 67.6, 119.7, 120.5, 128.2, 129.8, 163.8, 165.5, 165.5, 172.1, 172.1. MS (ESI) m/z: 398 (M+H)+. HRMS calcd for C16H18N5O3Cl2: 398.0787. Found: 398.0805. Anal. Calcd for C16H17N5O3Cl2: C, 48.25; H, 4.30; N, 17.59. Found: C, 48.32; H, 4.29; N, 17.37.
N-{2-[(4,6-Dichloro-1,3,5-triazin-2-yl)amino]benzoyl}-D-alanine (19): Purification on SiO2 (EtOAc- petroleum ether-AcOH = 10/50/1) gave 35 mg (10%) of a white solid. mp 165.0–167.0 °C. Rf 0.22 (EtOAc-petroleum ether-AcOH = 10/50/1). IR (KBr): 3374, 3072, 2988, 1721, 1649, 1602, 1572, 1528, 1503, 1382, 1318, 1246, 1225, 1173, 873, 195, 612 cm–1. 1H NMR (300 MHz, DMSO–d6): δ 1.38 (d, J = 7.5 Hz, 3H, -CH3), 4.29–4.44 (m, 1H, -NHCH-), 7.34 (t, J = 7.5 Hz, 1H, -Ph), 7.61 (t, J = 7.5 Hz, 1H, -Ph), 7.80 (d, J = 7.5 Hz, 1H, -Ph), 7.97 (d, J = 8.0 Hz, 1H, -Ph), 8.88 (br d, J = 7.0 Hz, 1H, -NHCH-), 11.51 (br s, 1H, -NH-), 12.58 (br s, 1H, -CO2H). 13C NMR (75.5 MHz, DMSO–d6): δ 17.6, 49.1, 124.6, 124.8, 125.0, 129.2, 132.2, 137.7, 164.8, 168.2, 168.3, 174.6, 174.7. MS (ESI) m/z: 354 (M-H)–. HRMS calcd for C13H10N5O3Cl2: 354.0161. Found: 354.0167.

General procedure for the preparation of 2-arylamino-4,6-dimethoxy-1,3,5-triazine and of 2-arylthio-4,6-dimethoxy-1,3,5-triazine 20–21. To a stirred solution of 2-chloro-4,6-dimethoxy- 1,3,5-triazine (3.0 mmol) in dry MeCN (15 mL), 2-aminobenzoic acid (or 2-thiobenzoic acid) (3.0 mmol) and Et3N (4.0 mmol) were added. The reaction mixture was stirred at room temperature for 2 h. After the reaction was complete (monitored by TLC), the solvent was removed under reduced pressure to obtain solid crude products.
2-[(4,6-Dimethoxy-1,3,5-triazin-2-yl)amino]benzoic acid (20): The title compound was obtained after the crude material was suspended in a mixture of H2O (25 mL) and EtOH (25 mL), filtered off and washed with the same mixture yielding an off-white solid, 306 mg (37%). mp 154.5–156.0 °C. Rf 0.67 (MeOH). IR (KBr): 3318, 3022, 2965, 2556, 1674, 1607, 1562, 1494, 1459, 1377, 1350, 1260, 1132, 810, 750 cm–1. 1H NMR (300 MHz, DMSO–d6): δ 3.93 (s, 6H, 2 × -OCH3), 7.15 (ddd, J = 8.0, 7.5, 1.0 Hz, 1H, -Ph), 7.63 (ddd, J = 8.5, 7.5, 1.5 Hz, 1H, -Ph), 8.01 (dd, J = 8.0, 1.5 Hz, 1H, -Ph), 8.62 (dd, J = 8.5, 1.0 Hz, 1H, -Ph), 11.16 (br s, 1H, -NH-), 13.60 (br s, 1H, -CO2H). 13C NMR (75.5 MHz, DMSO–d6): δ 54.6 (2C), 116.9, 120.6, 122.1, 131.1, 133.8, 140.5, 165.7, 169.4 (2C), 171.9. MS (EI) m/z (%): 276 (M+, 15), 257 (9), 231 (100), 159 (16). HRMS calcd for C12H12N4O4: 276.0859. Found: 276.0860. Anal. Calcd for C12H12N4O4: C, 52.17; H, 4.38; N, 20.28. Found: C, 52.29; H, 4.43; N, 19.99.
2-[(4,6-Dimethoxy-1,3,5-triazin-2-yl)thio]benzoic acid (21): The title compound was obtained after the crude material was suspended in HCl (1 M, 15 mL), filtered off and washed with HCl (1 M, 5 mL) yielding a white solid, 533 mg (61%). mp 180.0–182.0 °C. Rf 0.70 (MeOH). IR (KBr): 3016, 2968, 2667, 1679, 1561, 1532, 1347, 1298, 1192, 1011, 1042, 941, 808, 743 cm–1. 1H NMR (300 MHz, DMSO–d6): δ 3.83 (s, 6H, 2 × -OCH3), 7.53–7.65 (m, 2H, -Ph), 7.73–7.80 (m, 1H, -Ph), 7.86–7.93 (m, 1H, -Ph), 13.06 (br s, 1H, -CO2H). 13C NMR (75.5 MHz, DMSO–d6): δ 54.9 (2C), 127.0, 129.6, 130.1, 131.5, 135.6, 136.6, 167.3, 170.6 (2C), 184.3. MS (EI) m/z (%): 293 (M+, 4), 276 (1), 248 (100), 176 (32). HRMS calcd for C12H11N3O4S: 293.0470. Found: 293.0480. Anal. Calcd for C12H11N3O4S: C, 49.14; H, 3.78; N, 14.33. Found: C, 48.83; H, 3.73; N, 14.12.

Procedure for the preparation of methyl 2-[(4,6-dimethoxy-1,3,5-triazin-2-yl)oxy]benzoate 22. 26 To a stirred solution of methyl 2-hydroxybenzoate (182 mg, 1.2 mmol) in dry MeCN (5 mL), potassium tert-butoxide (123 mg, 1.1 mmol) was added. The reaction mixture was stirred at room temperature for 5 min and then 2-chloro-4,6-dimethoxy-1,3,5-triazine (176 mg, 1.0 mmol) was added. The mixture was stirred for 2 hours at room temperature and then diluted with H2O (20 mL). The aqueous layer was extracted with EtOAc (3 × 20 mL). The combined organic phases were washed with brine (2 × 20 mL) and dried over Na2SO4. The solvent was evaporated and the residue was purified by silica gel radial chromatography (EtOAc-petroleum ether = 1/1) to yield the pure product, 227 mg (78 %) as a white solid. mp 69.0–73.5 °C. mp 64.0–68.0 °C.26 Rf 0.11 (EtOAc-petroleum ether = 1/1). IR (KBr): 3430, 3029, 3012, 2953, 2613, 1722, 1584, 1561, 1472, 1363, 1271, 1214, 1123, 1097, 1082, 814, 738, 664, 449 cm–1. 1H NMR (300 MHz, CDCl3): δ 3.76 (s, 3H, -CO2CH3), 3.98 (s, 6H, 2 × -OCH3), 7.22 (dd, J = 8.0, 1.0 Hz, 1H, -Ph), 7.35 (ddd, J = 7.5, 7.5, 1.0 Hz, 1H, -Ph), 7.59 (ddd, J = 8.0, 7.5, 1.5 Hz, 1H, -Ph), 8.03 (dd, J = 8.0, 1.5 Hz, 1H, -Ph). MS (ESI) m/z: 292 (M+H)+. HRMS calcd for C13H14N3O5: 292.0933. Found: 292.0943.

Procedure for the preparation of dibenzyl N-(4,6-dimethoxy-1,3,5-triazin-2-yl)-D-glutamate 23. To a stirred solution of 2-chloro-4,6-dimethoxy-1,3,5-triazine (176.0 mg, 1.0 mmol) in dry MeCN (10 mL), d-glutamic acid dibenzyl ester p-toluenesulfonate salt (520 mg, 1.0 mmol) and K2CO3 (414 mg, 3.0 mmol) were added. The mixture was allowed to react at ambient temperature for 2 days. After the reaction was complete (monitored by TLC), H2O (10 mL) was added and the aqueous layer extracted with EtOAc (3 × 20 mL). The combined organic phases were washed with brine (2 × 20 mL) and dried over Na2SO4. The solvent was removed under reduced pressure and the residue was purified by silica gel radial chromatography (EtOAc-petroleum ether = 3/5) to yield the pure product, 307 mg (65%) as a yellowish oil. Rf 0.32 (EtOAc-petroleum ether =1/1). [α]D20 –3.6 (c 1.06, CH2Cl2). IR (NaCl-plates): 3354, 3257, 3150, 2955, 1738, 1577, 1550, 1482, 1466, 1378, 1362, 1192, 1165, 819, 698 cm–1. 1H NMR (300 MHz, CDCl3): δ 2.03–2.57 (m, 4H, 2 × -CO2CH2Ph), 3.85 (s, 3H, -OCH3), 3.91 (s, 3H, -OCH3), 4.78–4.88 (sim m, 1H, -NHCH-), 5.07 and 5.11 (AB, JAB = 12.5 Hz, 2H, -CO2CH2Ph-), 5.15 and 5.19 (AB, JAB = 12.0 Hz, 2H, -CO2CH2Ph-), 6.10 (br d, J = 8.0 Hz, 1H, -NHCH-), 7.27–7.41 (m, 10H, 2 × -Ph). 13C NMR (75.5 MHz, CDCl3): δ 27.3, 30.1, 53.2, 54.7 (2C), 66.5, 67.3, 128.2, 128.3, 128.3, 128.5, 128.5, 128.6, 135.1, 135.6, 167.8, 171.5, 172.2, 172.2, 172.4. MS (ESI) m/z: 467 (M+H)+. HRMS calcd for C24H27N4O6: 467.1931. Found: 467.1950. Anal. Calcd for C24H26N4O6: C, 61.79; H, 5.62; N, 12.01. Found: C, 61.78; H, 5.85; N, 11.82.

Procedure for the preparation of N-(4,6-dimethoxy-1,3,5-triazin-2-yl)-D-glutamic acid 24. To a solution of compound 23 (416 mg, 0.9 mmol) in MeOH (20 mL), 10% Pd-C (105 mg) was added, and the mixture was hydrogenated for 7 h at room temperature and 60 psi. The suspension was then filtered through a pad of celite and washed with MeOH (50 mL). The solvent was removed under reduced pressure. The residue was treated with CH2Cl2 (5 mL), filtered off and washed with CH2Cl2 (10 mL) yielding the pure product, 230 mg (90%) as a white solid. mp 151.0–154.0 °C. Rf 0.59 (MeOH). [α]D20 +7.9 (c 0.29, DMSO). IR (KBr): 3246, 3154, 2961, 1561, 1737, 1704, 1589, 1554, 1479, 1385, 1348, 1192, 1108, 813 cm–1. 1H NMR (300 MHz, DMSO–d6): δ 1.82–2.15 (m, 2H, -CH2CH2CO2H), 2.34 (t, J = 7.5 Hz, 2H, -CH2CH2CO2H ), 3.80 (s, 3H, -OCH3), 3.84 (s, 3H, -OCH3), 4.29–4.41 (m, 1H, -NHCH-), 8.08 (br d, J = 7.5 Hz, 1H, -NH-), 12.09 (br s, 1H, -CO2H). 13C NMR (75.5 MHz, DMSO–d6): δ 26.9, 31.2, 54.2, 55.0, 55.1, 168.6, 172.6, 172.6, 174.3, 174.6. MS (ESI) m/z: 285 (M-H)–. HRMS calcd for C10H13N4O6: 285.0835. Found: 285.0840.
Procedure for the preparation of (S)-isopropyl 2-(4-nitrobenzamido)propanoate 26a. To a stirred, cooled solution (–10 °C) of 4-nitrobenzoyl chloride (1.00 g, 5.39 mmol) in dry MeCN (25 mL), (S)-isopropyl 2-aminopropanoate hydrochloride (0.903 g, 5.39 mmol) was added, followed by the slow addition of Et3N (1.361 g, 13.48 mmol). The reaction mixture was stirred at –10 °C for 1 h and then allowed to reach 25 °C. The stirring was continued at this temperature for an additional 14 h. After the reaction was complete (monitored by TLC), the solvent was removed under reduced pressure, H2O (50 mL) was added and the resulting mixture extracted with CH2Cl2 (3 × 25 mL). The combined organic phases were washed with brine (2 × 20 mL) and dried over Na2SO4. The solvent was then removed under reduced pressure to yield the pure product, 1.33 g (88%) as a white solid. mp 154.0–159.0 °C. Rf 0.30 (EtOAc-petroleum ether = 3/5). [α]D20 +36.7 (c 2.00, CH2Cl2) IR (KBr): 3315, 2986, 1746, 1645, 1603, 1543, 1349, 12251179, 1018 cm–1. 1H NMR (300 MHz, DMSO–d6): 1.17 (d, J = 7.5 Hz, 3H, i-Pr), 1.20 (d, J = 7.5 Hz, 3H, i-Pr), 1.37 (d, J = 8.0 Hz, 3H, -(NH)CHCH3), 4.51 (pent, J = 7.0 Hz, 1H, -NHCH-), 4.91 (sept, J = 7.5 Hz, 1H, -CH-iPr), 5.62 (br s, 2H, -NH2), 8.13 (AA’BB’, J = 8.5 Hz, 2H, -Ph), 8.35 (AA’BB’, J = 8.5 Hz, 2H, -Ph), 9.14 (br d, J = 6.5 Hz, 1H, -NH-). 13C NMR (75.5 MHz, DMSO–d6): δ 16.8, 21.6, 53.0, 67.9, 123.5, 128.9, 139.3, 149.1, 164.7, 172.3. MS (ESI) m/z: 281 (M+H)+. HRMS calcd for C13H17N2O5: 281.1137. Found: 281.1140.

Procedure for the preparation of (S)-isopropyl 2-(4-aminobenzamido)propanoate 27a. To a solution of compound 26a (1.00 g, 3.57 mmol) in MeOH (20 mL), 10% Pd-C (200 mg) was added, and the mixture was hydrogenated for 24 h at room temperature and 1 bar. The solution was then filtered through a pad of celite and washed with MeOH (50 mL). The solvent was removed under reduced pressure, yielding the pure product, 849 mg (95%) as a white solid. mp 148.0–148.5 °C. Rf 0.15 (EtOAc-petroleum ether = 3/5). [α]D20 +40.6 (c 2.54, CH2Cl2). IR (KBr): 3449, 3352, 2986, 1741, 1634, 1513, 1501, 1299, 1215, 1182, 1107. 1H NMR (300 MHz, DMSO–d6): 1.17 (d, J = 7.5 Hz, 3H, i-Pr), 1.20 (d, J = 7.5 Hz, 3H, i-Pr), 1.37 (d, J = 8.0 Hz, 3H, -(NH)CHCH3), 4.38 (pent, J = 7.0 Hz, 1H, -NHCH-), 4.91 (sept, J = 7.5 Hz, 1H, -CH-iPr), 5.62 (br s, 2H, -NH2), 6.57 (AA’BB’, J = 8.5 Hz, 2H, -Ph), 7.64 (AA’BB’, J = 8.5 Hz, 2H, -Ph) 8.21 (br d, J = 6.5 Hz, 1H, -NHCH-). 13C NMR (75.5 MHz, DMSO–d6): 16.8, 21.47, 21.53, 48.3, 67.6, 112.5, 120.5, 129.1, 151.8, 166.4, 172.7. MS (ESI) m/z: 250 (M+H)+. HRMS calcd for C13H19N2O3: 251.1396. Found: 251.1385. Anal. Calcd for C13H18N2O3: C, 62.38; H, 7.52; N, 11.19. Found: C, 62.38; H, 7.48; N, 11.30.

Procedure for the preparation of benzyl N-[2-(benzylamino)benzoyl]-D-alaninate 26b. To a stirred ice-cooled solution of the 2-(benzylamino)benzoic acid 25b (725 mg, 3.2 mmol) and the D-alanine benzyl ester p-toluenesulfonate salt (1.23 g, 3.5 mmol) in DMF (8 mL), HOBt×H2O (582 mg, 3.8 mmol) and Et3N (960 mg, 9.5 mmol) were slowly added, followed by the addition of EDC (790 mg, 4.1 mmol) after 5 min. The reaction mixture was stirred for 1 h at 0 °C and then at room temperature for 24 h. After the reaction was complete EtOAc (50 mL) was added and then the reaction mixture was washed consecutively with HCl (1 M, 3 × 20 mL), satd aq NaHCO3 (3 × 20 mL), brine (2 × 20 mL) and then dried over Na2SO4. The solvent was removed under reduced pressure and the residue was purified by silica gel radial chromatography (EtOAc-petroleum ether = 1/7) to yield the pure product, 980 mg (79%) as a yellowish oil. Rf 0.34 (EtOAc-petroleum ether = 1/3). [α]D20 –19.1 (c 1.94, CH2Cl2). IR (NaCl-plates): 3354, 3030, 2937, 1740, 1638, 1580, 1518, 1452, 1200, 1169, 747, 697 cm–1. 1H NMR (300 MHz, CDCl3): δ 1.51 (d, J = 7.0 Hz, 3H, -CH(CH3)CO2-), 4.39 (d, J = 4.5 Hz, 2H, -NHCH2Ph), 4.78 (m, 1H, -NHCH-), 5.18 and 5.24 (AB, JAB = 12.0 Hz, 2H, -CO2CH2Ph), 6.53-6.70 (m, 3H, -Ph), 7.18-7.26 (m, 2H, -NH-, and -Ph), 7.27-7.39 (m, 9H, -Ph), 7.41 (dd, J = 8.0, 1.5 Hz, 1H, -Ph), 8.03 (br s, 1H, PhNHCH2Ph). 13C NMR (75.5 MHz, CDCl3): δ 18.6, 47.1, 48.3, 67.2, 112.2, 114.5, 115.0, 127.0, 127.1, 127.5, 128.1, 128.4, 128.6, 128.6, 133.1, 135.4, 139.1, 149.6, 169.2, 173.1. MS (ESI) m/z: 389 (M+H)+. HRMS calcd for C24H25N2O3: 389.1865. Found: 389.1850.

Procedure for the preparation of N-(2-aminobenzoyl)-D-alanine 27b. To a solution of the compound 26b (980 mg, 2.5 mmol) in MeOH (20 mL), 10% Pd-C (204 mg) was added, and the mixture was hydrogenated for 6 h at room temperature and 60 psi. The suspension was then filtered through a pad of celite and washed with MeOH (50 mL). The solvent was removed under reduced pressure. The residue was treated with Et2O (10 mL), filtered off and washed with Et2O (5 mL) yielding the pure product, 237 mg (57%) as a white solid. mp 129.5–132.0 °C. Rf 0.54 (CH2Cl2-MeOH-AcOH = 30/10/1). [α]D20 –30.0 (c 0.51, DMSO). IR (KBr): 3466, 3395, 3363, 3096, 2988, 1717, 1651, 1613, 1583, 1522, 1526, 1212, 1182, 845, 756, 528 cm–1. 1H NMR (300 MHz, DMSO–d6): δ 1.35 (d, J = 7.5 Hz, 3H, -CH3), 4.22–4.36 (m, 1H, -NHCH-), 6.31 (br s, 2H, PhNH2), 6.46–6.55 (m, 1H, -Ph), 6.68 (dd, J = 8.0, 1.0 Hz, 1H, -Ph), 7.08–7.17 (m, 1H, -Ph), 7.53 (dd, J = 8.0, 1.5 Hz, 1H, -Ph), 8.23 (br d, J = 7.0 Hz, 1H, -CONH-). MS (ESI) m/z: 209 (M+H)+. HRMS calcd for C10H13N2O3: 209.0926. Found: 209.0930.

General procedure for the preparation of amido derivatives 28–32. To a stirred, ice-cooled solution of the compound 20 (or 21) (1.0 mmol) and a corresponding C-protected amino acid derivative (1.1 mmol) in DMF (3 mL), HOBt×H2O (1.2 mmol) and Et3N (3.2 mmol) were slowly added, followed by the addition of EDC (1.3 mmol) after 5 min. The reaction mixture was stirred for 1 h at 0 °C and then at room temperature for 24 h. After the reaction was complete, EtOAc (20 mL) was added and then the reaction mixture was washed consecutively with HCl (1 M, 3 × 10 mL), satd aq NaHCO3 (3 × 10 mL), brine (2 × 10 mL) and then dried over Na2SO4. The solvent was removed under reduced pressure and the residue was purified by silica gel radial chromatography.
Benzyl N-{2-[(4,6-dimethoxy-1,3,5-triazin-2-yl)thio]benzoyl}-L-alaninate (28): Purification on SiO2 (EtOAc-petroleum ether = 3/5) gave 194 mg (43%) of a colourless oil. Rf 0.31 (EtOAc-petroleum ether = 1/1). [α]D20 +11.7 (c 1.05, CH2Cl2). IR (NaCl-plates): 3585, 3323, 2941, 1740, 1661, 1541, 1456, 1352, 1296, 1045, 815, 752, 698 cm–1. 1H NMR (300 MHz, CDCl3): δ 1.34 (d, J = 7.0 Hz, 3H, -CH3), 3.90 (s, 6H, 2 × -OCH3), 4.73 (dq, J = 7.0, 7.0 Hz, 1H, -NHCH-), 5.09 and 5.13 (AB, JAB = 12.5 Hz, 2H, -CO2CH2Ph), 7.22 (br d, J = 7.0 Hz, 1H, -CONHCH-), 7.27–7.41 (m, 5H, -Ph), 7.42–7.57 (m, 2H, -Ph), 7.63 (dd, J = 7.5, 1.0 Hz, 1H, -Ph), 7.68 (dd, J = 7.5, 1.5 Hz, 1H, -Ph). 13C NMR (75.5 MHz, CDCl3): δ 18.3, 48.6, 55.3 (2C), 67.1, 124.2, 128.1, 128.4, 128.6, 129.2, 130.7, 135.3 (2C), 137.3, 142.0, 167.3, 171.4 (2C), 172.3, 184.8. MS (EI) m/z (%): 454 (M+, 2), 320 (1), 276 (88), 248 (100), 159 (23), 91 (40). HRMS calcd for C22H22N4O5S: 454.1311. Found: 454.1320. Anal. Calcd for C22H22N4O5S: C, 58.14; H, 4.88; N, 12.33. Found: C, 57.87; H, 5.05; N, 12.34.
Dibenzyl N-{2-[(4,6-dimethoxy-1,3,5-triazin-2-yl)thio]benzoyl}-D-glutamate (29): Purification on SiO2 (EtOAc-petroleum ether = 3/5) gave 288 mg (48%) of a colourless oil. Rf 0.36 (EtOAc-petroleum ether = 1/1). [α]D20 –4.5 (c 1.45, CH2Cl2). IR (NaCl-plates): 3323, 2947, 1738, 1665, 1542, 1504, 1456, 1353, 1296, 1165, 1106, 1046, 815, 750, 698 cm–1. 1H NMR (300 MHz, CDCl3): δ 1.88–2.50 (m, 4H, -(CH2)2-), 3.87 (s, 6H, 2 × -OCH3), 4.80 (sim m, 1H, -NHCH-), 5.01–5.13 (m, 4H, -CO2CH2Ph), 7.27–7.38 (m, 11H, -NH-, and 2 × -Ph), 7.42–7.57 (m, 2H, -Ph), 7.59–7.68 (m, 2H, -Ph). 13C NMR (75.5 MHz, CDCl3): δ 27.6, 29.9, 52.0, 55.3 (2C), 66.4, 67.3, 124.1, 128.2, 128.2, 128.3, 128.5, 128.5, 128.6, 129.3, 130.7, 130,8, 135.1, 135.8, 137.5, 142.0, 167.8, 171.2, 171.4 (2C), 172.2, 184.8. MS (ESI) m/z: 603 (M+H)+. HRMS calcd for C31H31N4O7S: 603.1913. Found: 603.1914. Anal. Calcd for C31H30N4O7S: C, 61.78; H, 5.02; N, 9.30. Found: C, 61.96; H, 5.28; N, 9.53.
Benzyl N6-[(benzyloxy)carbonyl]-N2-{2-[(4,6-dimethoxy-1,3,5-triazin-2-yl)thio]benzoyl}-L-lysinate (30): Purification on SiO2 (EtOAc-petroleum ether = 3/5) gave 157 mg (49%) of a colourless oil. Rf 0.23 (EtOAc-petroleum ether = 1/1). [α]D20 +2.2 (c 1.01, CH2Cl2). IR (NaCl-plates): 3320, 2942, 1719, 1660, 1542, 1504, 1547, 1353, 1597, 1106, 1046, 815, 735, 698 cm–1. 1H NMR (300 MHz, CDCl3): δ 1.08–1.51 (m, 4H, -CH2CH2CH2CH2NH-), 1.52–1.92 (m, 2H, -CH2CH2CH2CH2NH-), 2.99–3.19 (m, 2H, -CH2CH2CH2CH2NH-), 3.88 (s, 6H, 2 × -OCH3), 4.74 (sim m, 1H, -CONHCH-), 4.95 (br s, 1H, -NHCO2CH2Ph), 5.03–5.16 (m, 4H, -NHCO2CH2Ph), 7.19 (br d, J = 7.5 Hz, 1H, -NHCO-), 7.27–7.41 (m, 10H, 2 × -Ph), 7.41–7.55 (m, 2H, -Ph), 7.62 (dd, J = 7.5, 2.0 Hz, 1H, -Ph), 7.67 (dd, J = 7.0, 2.0 Hz, 1H, -Ph). 13C NMR (75.5 MHz, CDCl3): δ 22.2, 29.3, 32.2, 40.7, 52.5, 55.4 (2C), 66.5, 67.1, 124.0, 128.0, 128.0, 128.4, 128.5, 128.5, 128.6, 129.4, 130.7, 130.8, 135.2, 136.7, 137.4, 142.1, 156.4, 167.7, 171.4 (2C), 171.7, 184.9. MS (ESI) m/z: 646 (M+H)+. HRMS calcd for C33H36N5O7S: 646.2335. Found: 646.2354. Anal. Calcd for C33H35N5O7S: C, 61.38; H, 5.46; N, 10.85. Found: C, 61.18; H, 5.63; N, 10.83.
Benzyl N-(4-methoxy-6-oxo-6H-[1,3,5]triazino[2,1-b]quinazolin-2-yl)-L-alaninate (31): Purification on SiO2 (EtOAc-petroleum ether = 1/3) gave 60 mg (15%) of a yellowish oil. Rf 0.20 (EtOAc-petroleum ether = 1/1). [α]D20 +85.2 (c 1.26, CH2Cl2). IR (NaCl-plates): 3160, 2982, 2952, 1737, 1691, 1639, 1579, 1548, 1470, 1380, 1326, 1194, 1134, 777, 700, 618 cm–1. 1H NMR (300 MHz, CDCl3): δ 1.62 (d, J = 7.0 Hz, 3H, -CH3), 4.05 (s, 3H, -OCH3), 4.88 (dq, J = 7.0, 7.0 Hz, 1H, -NHCH-), 5.25 (s, 2H, -CO2CH2Ph), 7.30–7.42 (m, 6H, -Ph), 7.64 (d, J = 8.0 Hz, 1H, -Ph), 7.76 (ddd, J = 8.5, 7.0, 1.5 Hz, 1H, -Ph), 8.21 (dd, J = 8.0, 1.0 Hz, 1H, -Ph), 11.36 (br d, J = 6.5 Hz, 1H, -NH-). 13C NMR (75.5 MHz, CDCl3): δ 17.6, 50.8, 55.5, 67.4, 117.5, 125.1, 126.6, 127.5, 128.2, 128.4, 128.6, 135.1, 136.5, 148.4, 148.8, 156.0, 164.6, 164.9, 171.3. MS (ESI) m/z: 406 (M+H)+. HRMS calcd for C21H20N5O4: 406.1515. Found: 406.1520.
Benzyl (2S)-2-({2-[(4-{[(1S)-2-(benzyloxy)-1-metil-2-oxoethyl]amino}-6-methoxy-1,3,5-triazin-2-yl)- amino]benzoyl}amino)propanoate (32): Purification on SiO2 (EtOAc-petroleum ether = 1/3) gave 103 mg (18%) of a yellowish oil. Rf 0.66 (EtOAc-petroleum ether = 1/1). [α]D20 + 31.6 (c 0.50, CH2Cl2). IR (NaCl-plates): 3064, 3034, 1986, 1742, 1669, 1633, 1589, 1545, 1450, 1306, 1157, 750, 697 cm–1. 1H NMR (300 MHz, CDCl3): δ 1.49 (d, J = 7.0 Hz, 3H, -CH3), 1.59 (d, J = 7.0 Hz, 3H, -CH3), 3.63 (s, 3H, -OCH3), 4.39 (dq, J = 7.0, 7.0 Hz, 1H, -NHCH-), 4.65–4.77 (m, 1H, -CONHCH-), 5.20 (s, 2H, -CO2CH2Ph), 5.25 (s, 2H, CO2CH2Ph), 7.27–7.41 (m, 12H, -Ph), 7.58 (ddd, J = 8.5, 7.0, 1.5 Hz, 1H, -Ph), 8.18 (dd, J = 8.0, 1.5 Hz, 1H, -Ph), 8.85 (br s, 1H, -ArNHPh), 11.24 (br d, J = 6.0 Hz, 1H, -NH-), 11.41 (br d, J = 6.0 Hz, 1H, -NH-). 13C NMR (75.5 MHz, CDCl3): δ 18.2, 19.0, 50.2, 51.0, 54.2, 66.7, 67.5, 119.1, 123.5, 125.3, 126.5, 128.2 (2C), 128.3, 128.4, 128.5, 128.6, 134.1, 135.2, 135.7, 149.4, 153.1, 158.7, 160.2, 163.4, 172.6, 173.2. MS (ESI) m/z: 585 (M+H)+. HRMS calcd for C31H33N6O6: 585.2462. Found: 585.2470.

General procedure for the preparation of 2,6-diamino-4-chloro-1,3,5-triazines 33–36. To a stirred solution of the compound 15 (1.0 mmol) in dry MeCN (10 mL), the corresponding amino derivative (1.1 mmol) was added, followed by the addition of Et3N (1.0 mmol) after 5 min. The reaction mixture was stirred at room temperature for 3 h. After the reaction was complete (monitored by TLC), the solvent was removed under reduced pressure to obtain crude products.
N2-[2-(2-Hydroxyethyl)phenyl]-2-amino-4-chloro-6-((2-hydroxyethyl)(methyl)amino)-1,3,5-triazine (33): The title compound was obtained after the crude material was suspended in H2O (10 mL) and extracted with EtOAc (3 × 10 mL). The combined organic phases were washed with brine (2 × 20 mL), dried over Na2SO4 and subsequently the solvent was removed under reduced pressure. The oily residue was treated with a mixture of hexane (8 mL) and EtOAc (3 mL) and left to stand at room temperature for 1 h. The white precipitate formed was filtered off and washed with hexane (5 mL) to yield 180 mg (78%) of a white solid. mp 144.5–145.0 °C. Rf 0.35 (EtOAC). IR (KBr): 3403, 3277, 2901, 1606, 1578, 1528, 1455, 1412, 1392, 1239, 1205, 1049, 988, 795, 763, 615 cm–1. 1H NMR (300 MHz, DMSO–d6, 80 °C): δ 2.78 (t, J = 6.5 Hz,, 2H, -CH2CH2OH), 3.06 (s, 3 H, -N(CH3)-), 3.50–3.62 (m, 4H, -N(CH3)CH2CH2OH and -CH2CH2OH), 3.66 (t, J = 6.5 Hz, 2H, -N(CH3)CH2CH2OH), 4.45 (br s, 1H, -OH), 4.78 (br s, 1H, -OH), 7.12 (dt, J = 7.5, 1.5 Hz, 1H, -Ph), 7.21 (dt, J = 7.5, 1.5 Hz, 1H, -Ph), 7.26 (dd, J = 7.5, 1.5 Hz, 1H, -Ph), 7.52 (d, J = 7.5 Hz, 1H, -Ph), 9.20 (br s, 1H, -NH-). 13C NMR (75.5 MHz, DMSO–d6, 80 °C): δ 34.4, 34.8, 50.3, 58.0, 61.4, 124.6, 125.1, 125.5, 129.4, 133.9, 136.0, 163.9, 164.4, 167.9. MS (ESI) m/z: 324 (M+H)+. HRMS calcd for C14H19N5O2Cl: 324.1227. Found: 324.1233. Anal. Calcd for C14H18N5O2Cl: C, 51.93; H, 5.60; N, 21.63. Found: C, 51.89; H, 5.69; N, 21.34.
N2-[2-(2-Hydroxyethyl)phenyl]-2-amino-4-chloro-6-benzylamino-1,3,5-triazine (34): The title compound was obtained after the crude material was suspended in H2O (10 mL), filtered off and washed with H2O (5 mL) obtaining a white solid, which was subsequently purified by silica gel radial chromatography (EtOAc-petroleum ether = 1/3). A white solid, 145 mg (41%). mp 159.0–160.0 °C. Rf 0.24 (EtOAc-petroleum ether = 3/5). IR (KBr): 3258, 3123, 2937, 2874, 1612, 1577, 1531, 1452, 1389, 1354, 1246, 1103, 1047, 990, 800, 756, 697, 610 cm–1. 1H NMR (300 MHz, CDCl3, mixture of tautomers): δ 2.87 (t, J = 5.5 Hz, 2H, -CH2CH2OH), 3.90–4.00 (m, 2H, -CH2CH2OH), 4.52 and 4.61 (d, J = 5.5 Hz, 2H, -NHCH2Ph), resonance for –OH missing, 5.53 and 5.59 (br s, 1H, -NH-), 7.07–7.38 (m, 8H, 2 × -Ph), 7.56–7.72 (m, 1H, -Ph), 8.46 and 8.63 (br s, 1H, -NH-). 13C NMR (75.5 MHz, CDCl3, mixture of tautomers): δ 29.7, 45.0, 64.5, 125.0 in 125.1 (1C), 127.4, 127.5, 127.7 in 127.8 (1C), 128.6, 128.7, 130.3 in 130.4 (1C), 132.8, 136.3 in 136.4 (1C), 137.9, 164.5, 165.9, 166.0. MS (ESI) m/z: 356 (M+H)+. HRMS calcd for C18H19N5OCl: 356.1278. Found: 356.1292. Anal. Calcd for C18H18N5OCl: C, 60.76; H, 5.10; N, 19.68. Found: C, 60.89; H, 5.00; N, 19.72.
N2-[2-(2-Hydroxyethyl)phenyl]-2-amino-4-chloro-6-(4-methoxybenzylamino)-1,3,5-triazine (35): The title compound was obtained after the crude material was suspended in H2O (10 mL), filtered off and washed with H2O (5 mL), obtaining a white solid, which was then purified by silica gel radial chromatography (EtOAc-hexane = 3/5). A white solid, 249 mg (64%). mp 138.5–139.0 °C. Rf 0.23 (EtOAc-petroleum ether = 3/5). IR (KBr): 3546, 3413, 3260, 2934, 1612, 1577, 1537, 1513, 1456, 1395, 1275, 1248, 1175, 1032, 987, 799, 752, 608, 595 cm–1. 1H NMR (300 MHz, DMSO–d6, 80 °C): δ 2.74 (t, J = 6.5 Hz, 2H, -CH2CH2OH), 3.58­–3.68 (m, 2H, -CH2CH2OH), 3.72 (s, 3H, -OCH3), 4.28 (d, J = 6.5 Hz, 2H, -NHCH2Ph), 4.72 (t, J = 4.5 Hz, 1H, -CH2CH2OH), 6.83 (d, J = 7.5 Hz, 2H, -Ph), 7.02–7.30 (m, 5H, -Ph), 7.41 (d, J = 7.5 Hz, 1H, -Ph), 8.11 (br s, 1H, -NHCH2-), 8.22 (br s, 1H, -NH-). 13C NMR (75.5 MHz, DMSO–d6, 80 °C): δ 34.4, 42.8, 54.7, 61.4, 113.4, 124.9, 125.6, 125.7, 128.1, 128.3, 129.6, 130.6, 134.3, 136.0, 158.0, 164.4, 165.3. MS (ESI) m/z: 408 (M+Na)+. HRMS calcd for C19H20N5O2ClNa: 408.1203. Found: 408.1183. Anal. Calcd for C19H20N5O2Cl: C, 59.14; H, 5.22; N, 18.15. Found: C, 59.30; H, 5.03; N, 17.91.
N-[2-(2-Hydroxyethyl)phenyl]-2-amino-4-chloro-6-morpholino-1,3,5-triazine (36): The title compound was obtained after the crude material was suspended in a mixture of H2O (10 mL) and EtOH (4 mL), filtered off and washed with the same mixture, obtaining an off-white solid, which was subsequently purified by silica gel radial chromatography (EtOAc-petroleum ether = 3/5). A white solid, 126 mg (38%). mp 171.5–176.0 °C. Rf 0.19 (EtOAc-petroleum ether = 3/5). IR (KBr): 3329, 2961, 1895, 1605, 1574, 1536, 1446, 1406, 1285, 1243, 1117, 798, 757, 537 cm–1. 1H NMR (300 MHz, CDCl3): δ 2.89 (t, J = 5.5 Hz, 2H, -CH2CH2OH), 3.62–3.87 (m, 8H, morpholino-CH2), 3.92–4.03 (m, 2H, -CH2CH2OH), resonance for –OH missing, 7.09–7.17 (m, 1H, -Ph), 7.18–7.29 (m, 2H, -Ph), 7.74 (dd, J = 8.0, 1.0 Hz, 1H, -Ph), 8.67 (br s, 1H, -NH-). 13C NMR (75.5 MHz, CDCl3): δ 34.8, 43.9, 64.5, 66.6, 124.6, 125.2, 126.8, 130.4, 132.9, 136.7, 164.2, 164.5, 164.6. MS (ESI) m/z: 336 (M+H)+. HRMS calcd for C15H19N5O2Cl: 336.1227. Found: 336.1235.

General procedure for the preparation of 2,6-diamino-4-chloro-1,3,5-triazines 37–38. To a stirred solution of the compound 15 (1.0 mmol) in acetone (8 mL), the corresponding amino derivative (1.1 mmol) and K2CO3 (3.0 mmol) were added. The reaction mixture was refluxed for 24 h at 50 °C. After the reaction was complete (monitored by TLC), H2O was added and the resulting mixture extracted with EtOAc (3 × 20 mL). The combined organic phases were washed with brine (2 × 20 mL) and dried over Na2SO4. The solvent was then removed under reduced pressure to obtain crude products, which were subsequently purified by silica gel radial chromatography.
2,6-Bis-[2-(2-hydroxyethyl)phenylamino]-4-chloro-1,3,5-triazine (37): Purification on SiO2 (EtOAc-hexane = 1/1) gave 117 mg (30%) of a white solid. mp 170.5–172.0 °C. Rf 0.53 (EtOAc). IR (KBr): 3313, 3245, 2889, 1617, 1596, 1528, 1454, 1390, 1231, 1073, 995, 801, 748, 610, 586 cm–1. 1H NMR (300 MHz, DMSO–d6, 80 °C): δ 2.45–2.78 (m, 4H, 2 × -CH2CH2OH), 3.46–3.75 (m, 4H, 2 × -CH2CH2OH), 5.03 (br s, 2H, 2 × -CH2CH2OH), 7.06–7.61 (m, 8H, 2 × -Ph), 9.56 (br s, 2H, 2 × -NH-). 13C NMR (75.5 MHz, DMSO–d6, 80 °C): δ 34.6, 61.6, 125.3, 125.8, 126.0, 129.8, 134.3, 136.0, 164.8, 168.6. MS (ESI) m/z: 386 (M+H)+. HRMS calcd for C19H21N5O2Cl: 386.1384. Found: 386.1401. Anal. Calcd for C19H20N5O2Cl: C, 59.14; H, 5.22; N, 18.15. Found: C, 59.38; H, 5.39; N, 17.99.
Dibenzyl N-(4-chloro-2-{[2-(2-hydroxyethyl)phenyl]amino}-1,3,5-triazin-6-yl)-D-glutamate (38): Purification on SiO2 (EtOAc-petroleum ether = 1/3) gave 243 mg (42%) of a colourless oil. Rf 0.42 (EtOAc-petroleum ether = 3/5). [α]D20 –0.8 (c 1.46, CH2Cl2). IR (NaCl-plates): 3324, 3034, 2950, 2881, 1737, 1572, 1522, 1455, 1385, 1245, 1170, 989, 805, 752, 734, 698 cm–1. 1H NMR (300 MHz, CDCl3, mixture of tautomers): δ 1.97–2.53 (m, 4H, -NHCH(CH2CH2CO2CH2Ph)CO2CH2Ph), 2.84 (t, J = 5.0 Hz, 2H, -CH2CH2OH), 3.86–3.98 (m, 2H, -CH2CH2OH), resonance for –OH missing, 4.58 in 4.85 (m, 1H, -NHCH-), 4.99–5.24 (m, 4H, -CO2CH2Ph), 5.90 in 6.03 (br d, J = 7.0 Hz, 1H, -NH-), 7.01–7.40 (m, 13H, -Ph), 7.53–7.71 (m, 1H, -Ph), 8.63 in 8.79 (br s, 1H, -NH-). 13C NMR (75.5 MHz, CDCl3, mixture of tautomers): δ 27.6, 30.1, 34.8, 52.3, 64.5, 66.6, 67.4, 124.9 and 125.1 (1C), 125.5 and 125.7 (1C), 128.2, 128.3, 128.3, 128.4, 128.5, 128.6, 128.7 (2C), 130.4, 135.1 in 135.2 (1C), 135.6 in 135.7 (1C), 136.2, 164.3, 165.4, 165.8, 171.2, 172.3. MS (ESI) m/z: 576 (M+H)+. HRMS calcd for C30H31N5O5Cl: 576.2014. Found: 576.1993.

Biochemical evaluation. The inhibitory activity of the compounds against MurF from E. coli was tested for their ability to inhibit the addition of D-Ala-D-Ala to UDP-MurNAc-L-Ala-γ-D-Glu-meso-A2pm. The detection of orthophosphate generated during the reaction was based on the colorimetric Malachite green method18 with slight modifications in a mixture (final volume, 50 μl) containing 50 mM Hepes, pH 8.0, 50 mM MgCl2, 100 µM UDP-MurNAc-L-Ala-γ-D-Glu-meso-A2pm, 600 µM D-Ala-D-Ala, 500 μM ATP, purified MurF from E.coli 27 (diluted with 50 mM Hepes, 1 mM dithiothreitol), and 500 µM of the tested compound dissolved in DMSO. The final concentration of DMSO was 5% (v/v). The mixture was incubated at 37 °C for 20 min and then quenched with 100 µl of Biomol® reagent. The absorbance was measured after 5 min at 650 nm. All the experiments were run in duplicates. The residual activity (RA) was calculated with respect to a similar assay without the inhibitor. The IC50 value was determined by measuring the residual activity at seven different inhibitor concentrations and represents the concentration of the inhibitor for which RA is 50%.
Docking. The crystal structure of MurF (PDB entry: 2AM1)13was used for our docking experiment. The active site was defined as the area within a distance of 5Å around the co-crystallized inhibitor (2,4-dichloro-N-(3-cyano-4,5,6,7-tetrahydrobenzothiophen-2yl)-5-(morpholine-4-sulfonyl)-benzamide). The docking and scoring were made with the default parameters of the FlexX program. The docking program was validated by the removal and subsequent re-docking of the co-crystallized inhibitor. FlexX successfully replicated the conformation from the crystal structure (Figure 3).

ACKNOWLEDGEMENTS
The Ministry of Higher Education, Science and Technology of the Republic of Slovenia, the Slovenian Research Agency (P1-0230-0103 and J1-6693-0103), the European Union FP6 Integrated Project EUR-INTAFAR (Project No. LSHM-CT-2004-512138), the Centre National de la Recherche Scientifique (PICS 3729) and the Franco-Slovene Proteus programme (no. 14007PH) are gratefully acknowledged for their financial support. We thank Dr. D. Žigon and Dr. B. Kralj (Center for Mass Spectroscopy, “Jožef Stefan” Institute, Ljubljana, Slovenia) for the mass measurement.

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