The synthesis of novel 2,4,6-trisubstituted 1,3,5-triazines: A search for potential murf enzyme inhibitors

Article abstract of DOI:10.3987/COM-09-11839

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.

Full text of DOI:10.3987/COM-09-11839

HETEROCYCLES, Vol. 81, No. 1, 2010
91
HETEROCYCLES, Vol. 81, No. 1, 2010, pp. 91 - 115. © The Japan Institute of Heterocyclic Chemistry
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,a Bogdan Štefane,b* Andreja Kovač,a Samo Turk,a Didier
Blanot,c and Stanislav Gobeca*
a
Faculty of Pharmacy, University of Ljubljana, Aškerčeva 7, 1000 Ljubljana,
Slovenia. Tel.: +386
1
4258031; fax: +386
1
4258031; e-mail:
gobecs@ffa.uni-lj.si
b
Faculty of Chemistry and Chemical Technology, University of Ljubljana,
Aškerčeva 5, 1000 Ljubljana, Slovenia. Tel.: +386 1 2419264; fax: +386 1
2419220; e-mail: bogdan.stefane@fkkt.uni-lj.si
c
Enveloppes Bactériennes et Antibiotiques, IBBMC, UMR 8619 CNRS, Univ
Paris-Sud, 91405 Orsay, France
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 photostabilisers 1 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

92
HETEROCYCLES, Vol. 81, No. 1, 2010
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
Figure 1. VHTS hit NSC 209931; IC50 (MurF, E. coli) = 63 µM.
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
14
Laboratories13,
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 series of
a
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).

HETEROCYCLES, Vol. 81, No. 1, 2010
93
HN
NH3Cl
1: R = p-NH2-C6H4, 77%
2: R = m-NO2-C6H4, 92%
3: R = C6H5-CH2S, 75%
4: R = Ph, 86%
H
N
O
S
EtO
NCS
R
HN
N
a
O
R
5: R = p-Me-C6H4, 62%
1-5
X
H
N
H
N
R2
O
S
N
O
S
R
6: R = C6H5-CH2, 76%
HN
N
N
7: R = o-MeO-C6H4-CH2, 95%
8: R = p-CN-C6H4-CH2, 58%
b
X = Cl, R2 = H
Ph
Ph
X = Cl, R2 = o-MeO
X = Br, R2 = p-CN
4
6-8
Scheme 1. Synthesis of potential inhibitors of MurF based on
4-thioxo-3,4-dihydro-1,3,5-triazin-2(1H)-one core. Reagents and conditions: (a) 2 M NaOH, toluene, H2O,
25 °C, 1h; (b) 2 M NaOH, EtOH, 25 °C, 1 h.
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.

94
HETEROCYCLES, Vol. 81, No. 1, 2010
10: R = o-CO2H-C6H4-S, 77%
R1
Cl
Cl
N
11: R = o-Br-C6H4-NH, 53%
R2
N
N
N
N
12: R = o-Et-C6H4-NH, 42%
13: R = o-t-Bu-C6H4-NH, 58%
14: R = o-MeO2C-C6H4-NH, 56%
15: R = o-HO(CH2)2-C6H4-NH, 58%
Cl
N
Cl
Cl
R
a or b
9a
10-19
a, R1 = SH, R2 = o-CO2H
b, R1 = NH2, R2 = o-Br
b, R1 = NH2, R2 = o-Et
b, R1 = NH2, R2 = o-t-Bu
16: R = p-MeO-C6H4-CH2-NH, 96%
17: R = 3,4-MeO-C6H4-(CH2)2-NH, 72%
Me
18: R =
, 29%
CO2i-Pr
CONH
b, R1 = NH2, R2 = o-CO2Me
b, R1 = NH2, R2 = o-(CH2)2OH
b, R1 = CH2NH2, R2 = p-OMe
HN
HO2C
Me
NH
b, R1 = (CH2)2NH2, R2 = 3,4-OMe
CONH
19: R =
, 10%
Me
b, R1 = NH2, R2 =
p-CONH
o-CONH
CO2i-Pr
Me
CO2H
b, R1 = NH2, R2 =
R1
OMe
OMe
N
R2
c or d
20: R = o-CO2H-C6H4-NH, 37%
21: R = o-CO2H-C6H4-S, 61%
22: R = o-MeO2C-C6H4-O, 78%
N
N
N
MeO
N
R
MeO
N
Cl
20-22
9b
c, R1 = NH2, R2 = o-CO2H
c, R1 = SH, R2 = o-CO2H
d, R1 = OH, R2 = o-CO2Me
OMe
OMe
OMe
N
(CH2)2CO2Bn
(CH2)2CO2H
N CO2H
N
N
(CH2)2CO2Bn
N CO2Bn
TsOH3N
CO2Bn
N
N
N
MeO
N
e
MeO
N
f
MeO
N
Cl
H
H
9b
23, 65%
24, 90%
Scheme 2. Synthesis of potential inhibitors of MurF based on triazine core. Reagents and conditions: (a)
H2O, MeCN, 0 °C, 1 h; (b) Et3N, MeCN, –15 °C, 1 h; (c) MeCN, Et3N, 25 °C, 2 h; (d) tBuOK, MeCN,
25 °C, 2 h; (e) K2CO3, MeCN, 2 days; (f) H2, 60 psi, Pd-C, MeOH.
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.
Me
Me
CO2i-Pr
CONH
COCl
Me
CO2i-Pr
NH2
CO2i-Pr
CONH
b
a
NO2
NO2
NH2
27a, 95%
25a
26a, 88%
Scheme 3. Preparation of isopropyl N-(4-aminobenzoyl)- L-alaninate 27a. Reagents and conditions: (a)
Et3N, MeCN, 0 °C, then 25 °C, 14 h; (b) MeOH, H2, 1atm, Pd-C.

HETEROCYCLES, Vol. 81, No. 1, 2010
95
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).
CO2Bn
BnO2C
NHBn
CONH
HO2C
NHBn
CONH
Me
Me
NHBn
Me
HO2C
NH3OTs
a
b
25b
26b, 79%
27b, 57%
Scheme 4. Preparation of N-(2-aminobenzoyl)- D-alanine 27b. Reagents and conditions: (a) EDC·HCl,
HOBt, Et3N, DMF, 0 °C, 24 h; (b) H2, 60 psi, Pd-C.
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.

96
HETEROCYCLES, Vol. 81, No. 1, 2010
CO2Bn
NH
OMe
OMe
N
Me
28: R =
29: R =
, 43%
, 48%
N
N
N
28a or 29a or 30a
(CH2)2CO2Bn
CO2Bn
MeO
N
S
MeO
N
S
a
COR
CO2Bn
Me
CO2H
HN
HN
28a =
NH3OTs
(CH2)4NHCO2Bn
CO2Bn
28-30
(CH2)2CO2Bn
CO2Bn
20
30: R =
, 49%
29a =
30a =
TsOH3N
(CH2)4NHCO2Bn
CO2Bn
ClH3N
Me
Me
CO2Bn
NH
OMe
HN
N
CO2Bn
MeO
O
N
N
N
N
N
N
MeO
N
NH
Me
CO2Bn
MeO
N
NH
28a
a
CO2H
N
CONH
21
31, 15%
32, 18%
Scheme 5. Synthesis of potential inhibitors of MurF based on triazine core. Reagents and conditions: (a)
EDC·HCl, HOBt, Et3N, DMF, 0 °C, 24 h.
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 M 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 M.

HETEROCYCLES, Vol. 81, No. 1, 2010
97
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).
Cl
N
R
N
33: R = HO(CH2)2-NMe, 78%
34: R = C6H5-CH2NH, 41%
N
N
N
N
35: R = p-MeO-C6H4-CH2NH, 64%
36: R = morpholino, 38%
33a-38a
a or b
Cl
NH
Cl
NH
(CH2)2OH
(CH2)2OH
37: R = o-HO(CH2)2-C6H4-NH, 30%
(CH2)2CO2Bn
a, 33a = MeNH(CH2)2OH
a, 34a = BnNH2
38: R =
, 42%
33-38
HN
CO2Bn
15
a, 35a = p-MeO-C6H4-CH2NH2
a, 36a = morpholine
b, 37a = o-HO(CH2)2-C6H4-NH2
(CH2)2CO2Bn
b, 38a =
TsOH3N
CO2Bn
Scheme 6. Derivatization of 15. Reagents and conditions: (a) Et3N, MeCN, rt, 3 h; (b) K2CO3, acetone,
50 °C, 24 h.
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.
Figure 2. Docking of compound 15 (magenta) into the active site of MurF (green). The co-crystallized
cyanothiophene inhibitor is also shown (yellow). Amino acid residues that form interactions with
compound 15 are labelled (polar Asn326 and Asn328; hydrophobic Phe31, Leu45 and Ile139, which are
also presented as a yellow surface).

98
HETEROCYCLES, Vol. 81, No. 1, 2010
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
13
possessing the symmetric 2,4-dichloro-substituted 1,3,5-triazine ring, we have observed in the C 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  ppm,
1
13
referenced to an internal TMS standard for H NMR, chloroform–d ( 77.0), DMSO–d6 ( 39.5) for C

HETEROCYCLES, Vol. 81, No. 1, 2010
99
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):
1
3442, 3338, 3020, 2891, 1720, 1633, 1601, 1577, 1519, 1490, 1322, 1184, 969, 830, 764, 601 cm–1. H
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

100
HETEROCYCLES, Vol. 81, No. 1, 2010
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
1
(KBr): 3421, 3062, 2936, 1678, 1601, 1551, 1385, 1320, 1231, 1164, 941, 816, 766, 696 cm–1. H 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,
1
2944, 1672, 1550, 1494, 1420, 1315, 1274, 1252, 1168, 1097, 1026, 850, 751, 710, 624 cm–1. H 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,

HETEROCYCLES, Vol. 81, No. 1, 2010
101
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,
13
-Ph), 7.77–7.84 (m, 2H, -Ph), 8.18 (d, J = 7.0 Hz, 2H, -Ph), 12.99 (br s, 1H, -NHCO-). C 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),
13
11.17 (br s, 1H, -CO2H). C 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.
24
N-(2-Bromophenyl)-2-amino-4,6-dichloro-1,3,5-triazine
(11)
:
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-).

102
HETEROCYCLES, Vol. 81, No. 1, 2010
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,
1
1169, 1018, 835, 797, 615 cm–1. H 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):Purificationon 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
1
cm–1. H 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.
25
Methyl 2-[(4,6-dichloro-1,3,5-triazin-2-yl)amino]benzoate (14) : 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,
13
-NH-). C 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

HETEROCYCLES, Vol. 81, No. 1, 2010
103
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
20
(EtOAc-hexane = 3/5). [α]D +39.2 (c 0.39, DMSO). IR (KBr): 3418, 3273, 1732, 1651, 1608, 1565,
1
1499, 1381, 1216, 824, 794, 624 cm–1. H 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,
13
1H, -NH-). C 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-),
13
11.51 (br s, 1H, -NH-), 12.58 (br s, 1H, -CO2H). C 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

104
HETEROCYCLES, Vol. 81, No. 1, 2010
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.
26
Procedure for the preparation of methyl 2-[(4,6-dimethoxy-1,3,5-triazin-2-yl)oxy]benzoate 22.
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,
1
2953, 2613, 1722, 1584, 1561, 1472, 1363, 1271, 1214, 1123, 1097, 1082, 814, 738, 664, 449 cm–1. H
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,

HETEROCYCLES, Vol. 81, No. 1, 2010
105
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
20
oil. Rf 0.32 (EtOAc-petroleum ether =1/1). [α]D –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)
20
yielding the pure product, 230 mg (90%) as a white solid. mp 151.0–154.0 °C. Rf 0.59 (MeOH). [α]D
+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.

106
HETEROCYCLES, Vol. 81, No. 1, 2010
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
20
(EtOAc-petroleum ether = 3/5). [α]D +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
13
(AA’BB’, J = 8.5 Hz, 2H, -Ph), 9.14 (br d, J = 6.5 Hz, 1H, -NH-). C 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
20
ether = 3/5). [α]D +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,
13
-Ph) 8.21 (br d, J = 6.5 Hz, 1H, -NHCH-). C 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

HETEROCYCLES, Vol. 81, No. 1, 2010
107
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%)
20
as a yellowish oil. Rf 0.34 (EtOAc-petroleum ether = 1/3). [α]D –19.1 (c 1.94, CH2Cl2). IR
1
(NaCl-plates): 3354, 3030, 2937, 1740, 1638, 1580, 1518, 1452, 1200, 1169, 747, 697 cm–1. H 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
20
mg (57%) as a white solid. mp 129.5–132.0 °C. Rf 0.54 (CH2Cl2-MeOH-AcOH = 30/10/1). [α]D –30.0
(c 0.51, DMSO). IR (KBr): 3466, 3395, 3363, 3096, 2988, 1717, 1651, 1613, 1583, 1522, 1526, 1212,
1
1182, 845, 756, 528 cm–1. H 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

108
HETEROCYCLES, Vol. 81, No. 1, 2010
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 =
20
1/1). [α]D +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
20
ether = 1/1). [α]D –4.5 (c 1.45, CH2Cl2). IR (NaCl-plates): 3323, 2947, 1738, 1665, 1542, 1504, 1456,
1
1353, 1296, 1165, 1106, 1046, 815, 750, 698 cm–1. H 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
20
(EtOAc-petroleum ether = 1/1). [α]D +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,
13
-Ph). C 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.

HETEROCYCLES, Vol. 81, No. 1, 2010
109
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
20
mg (18%) of a yellowish oil. Rf 0.66 (EtOAc-petroleum ether = 1/1). [α]D + 31.6 (c 0.50, CH2Cl2). IR
1
(NaCl-plates): 3064, 3034, 1986, 1742, 1669, 1633, 1589, 1545, 1450, 1306, 1157, 750, 697 cm–1. H
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
13
(br d, J = 6.0 Hz, 1H, -NH-). C 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,

110
HETEROCYCLES, Vol. 81, No. 1, 2010
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,
13
-Ph), 7.52 (d, J = 7.5 Hz, 1H, -Ph), 9.20 (br s, 1H, -NH-). C 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,
1
1354, 1246, 1103, 1047, 990, 800, 756, 697, 610 cm–1. H 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.

HETEROCYCLES, Vol. 81, No. 1, 2010
111
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,
1
1605, 1574, 1536, 1446, 1406, 1285, 1243, 1117, 798, 757, 537 cm–1. H 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
1
(KBr): 3313, 3245, 2889, 1617, 1596, 1528, 1454, 1390, 1231, 1073, 995, 801, 748, 610, 586 cm–1. H
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
20
(EtOAc-petroleum ether = 3/5). [α]D –0.8 (c 1.46, CH2Cl2). IR (NaCl-plates): 3324, 3034, 2950, 2881,
1
1737, 1572, 1522, 1455, 1385, 1245, 1170, 989, 805, 752, 734, 698 cm–1. H 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,

112
HETEROCYCLES, Vol. 81, No. 1, 2010
13
-Ph), 7.53–7.71 (m, 1H, -Ph), 8.63 in 8.79 (br s, 1H, -NH-). C 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--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--D-Glu-meso-A2pm, 600 µM D-Ala-D-Ala, 500 μM ATP,
27
purified MurF from E.coli
(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)13 was 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).
Figure 3. Validation of FlexX docking. Conformation of co-crystallized inhibitor (yellow) and the same
compound re-docked with FlexX (orange). The structure of the enzyme is not shown for reasons of
clarity.

HETEROCYCLES, Vol. 81, No. 1, 2010
113
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.
REFERENCES
1. (a) E. Hollink, E. E. Simanek, and D. E. Bergbreiter, Tetrahedron Lett., 2005, 46, 2005, and
references cited therein; (b) A. Herrera, R. P. Martínez-Alvarez, M. Chioua, and R. Chioua,
Synthesis, 2004, 503, and refrences cited therein; (c) B. Barton, S. Gouwns, M. C. Schaefer, and B.
Zeelie, Org. Process Res. Dev., 2003, 7, 1071, and references cited therein; (d) M. E. D. G Azenha,
H. D. Burrows, M. L. Canle, R. Coimbra, M. I. Fernández, M. V. García, A. E. Rodrigues, J. A.
Santaballa, and S. Steenken, Chem. Comm., 2003, 112; (e) W. Draber, K. Tietjen, J. F. Kluth, and A.
Trebst, Angew. Chem., Int. Ed. Engl., 1991, 30, 1621; (f) J. M. E. Quirke, 'Comprehensive
Heterocyclic Chemistry', ed. by A. R. Katritzky, C. W. Rees, Pergamon Press, London, 1984, 3, pp.
457-530.
2. (a) W. Giencke, L. Willms, H. Dietrich, T. Auler, H. Bieringer, and H. Menne. EP 02 92 580. 2002
[Chem. Abstr., 2002, 137, 370115]; (b) H. J. Riebel, A. Yanagi, Y. Watanabe, T. Goto, K. Kather, S.
Lehr, K. Voigt, M. W. Drewes, P. Dahmen, D. Feucht, and R. Pontzen. EP 00 32 580. 1999 [Chem.
Abstr., 2000, 133, 17484]; (c) Y. Watanabe, T. Goto, C. Ueno, M. W. Drewes, and D. Feucht. EP 01
17 977. 2000 [Chem. Abstr., 2001, 134, 237501]; (d) K. Koizumi, N. Kuboyama, K. Tomono, K.
Wakabayashi, and P. Boger, Pestic. Sci., 1999, 55, 642.
3. A. Ohki, N. Kuboyama, K. Koizumi, A. Tanaka, Y. Sato, H. Kohno, P. Boger, and K. Wakabayashi,
J. Agric. Food Chem., 1999, 47, 4398.
4. J. M. Blaney, C. Hansch, C. Silipo, and A. Vittoria, Chem Rev., 1984, 84, 333. For recent examples
see: (a) H.–S. Moon, E. M. Jacobson, S. M. Khersonsky, M. R. Luzung, D. P. Walsh, W. Xiong, J.
W. Lee, P. B. Parikh, J. C. Lam, T.–W. Kang, G. R. Rosania, A. F. Schier, and Y.–T. Chang, J. Am.
Chem. Soc., 2002, 124, 11608; (b) M. Zheng, C. Xu, J. Ma, Y. Sun, F. Du, H. Liu, L. Lin, C. Li, J.
Ding, K. Chen, and H. Jiang, Bioorg. Med. Chem., 2007, 15, 1815; (c) I. Pakuin, S. Raepel, S. Leit, F.
Gaudette, N. Zhou, O. Moradei, O. Saavedra, N. Bernstein, F. Raeppel, G. Bouchain, S. Frechette, S.
H. Woo, A. Vaisburg, M. Fournel, A. Kalita, M.–F. Robert, A. Lu, M.–C. Trachy-Bourget, P. T. Yan,

114
HETEROCYCLES, Vol. 81, No. 1, 2010
J. Liu, J. Rahil, A. R. MacLeod, J. M. Besterman, Z. Li, and D. Delorme, Bioorg. Med. Chem. Lett.,
2008, 18, 1067.
5. P. Mamalis and J. Doskocil, 'Handbook of Experimental Pharmacology, Antimalarial drugs II', Vol.
68, ed. by W. Peters, W. H. G. Michaels, Springer-Verlag, Berlin, 1984, pp.387.
6. S. Melato, D. Prosperi, P. Coghi, N. Basilico, and D. Monti, Chem. Med. Chem., 2008, 3, 873.
7. L. N. Yakhontov and G. M. Vakhatova, Khim. Farm. Zh., 1981, 15, 27.
8. S. Maeda, T. Kita, and K. Meguro, J. Med. Chem., 2009, 52, 597.
9. T. D. H. Bugg and C. T. Walsh, Nat. Prod. Rep., 1992, 9, 199.
10. (a) H. Barreteau, A. Kovač, A. Boniface, M. Sova, S. Gobec, and D. Blanot, FEMS Microbiol. Rev.,
2008, 32, 168; (b) A. Bouhss, A. E. Trunkfield, T. D. Bugg, and D. Mengin-Lecreulx, FEMS
Microbiol. Rev., 2008, 32, 208; (c) E. Sauvage, F. Kerff, M. Terrak, J. A. Ayala, and P. Charlier,
FEMS Microbiol. Rev., 2008, 32, 234.
11. S. Turk, A. Kovač, A. Boniface, J. M. Bostock, I. Chopra, D. Blanot, and S. Gobec, Bioorg. Med.
Chem., 2009, 17, 1884.
12. D. J. Miller, S. M. Hammond, D. Anderluzzi, and T. D. H. Bugg, J. Chem. Soc., Perkin Trans. 1,
1998, 131.
13. K. L. Longenecker, G. F. Stamper, P. J. Hajduk, E. H. Fry, C. G. Jakob, J. E. Harlan, R. Edalji, D. M.
Bartley, K. A. Walter, L. R. Solomon, T. F. Holzman, Y. G. Gu, C. G. Lerner, B. A. Beutel, and V.
S. Stoll, Protein Science, 2005, 14, 3039.
14. G. F. Stamper, K. L. Longenecker, E. H. Fry, C. G. Jakob, A. S. Florjancic, Y. G. Gu, D. D.
Anderson, C. S. Cooper, T. Zhang, R. F. Clark, Y. Cia, C. L. Black-Schaefer, J. Owen McCall, C. G.
Lerner, P. J. Hajduk, B. A. Beutel, and V. S. Stoll, Chem. Biol. Drug. Res., 2006, 67, 58.
15. E. Z. Baum, S. M. Crespo-Carbone, D. Abbanat, B. Foleno, A. Maden, R. Goldschmidt, and K. Bush,
Antimicrob. Agents Chemother., 2006, 50, 230.
16. J. Goerdeler and J. Neuffer, Chem. Ber., 1971, 104, 1606.
17. (a) G. R. Gustafson, C. M. Baldino, M.–M. E. O’Donnell, A. Sheldon, R. J. Tarsa, C. J. Verni, and D.
L. Coffen, Tetrahedron, 1998, 54, 4051; (b) M. Uttamchandani, D. P. Walsh, S. M. Kersonsky, S. Q.
Yao, and Y. C. Chang, J. Comb. Chem., 2004, 6, 862.
18. P. A. Lanzetta, L. J. Alvarez, P. S. Reinach, and O. Candia, Anal. Biochem., 1979, 100, 95.
19. S. L. McGovern and B. K. Shoichet, J. Med. Chem., 2003, 43, 1478.
20. M. Rarey, B. Kramer, T. Lengauer, and G. Klebe, J. Mol. Biol., 1996, 261, 470.
21. S. Van Angerer, 'Science of Synthesis', vol. 17, ed. by S. M. Weinreb, Thieme, Stuttgart, 2006, pp.
449–583.

HETEROCYCLES, Vol. 81, No. 1, 2010
115
22. S. N. Pandeya, A. K. Jain, and N. Siddiqui, Acta Pharm. Turc., 1989, 31, 67.
23. M. N. Basyouni and A. M. El-Khamry, Bull. Chem. Soc. Jpn., 1979, 52, 3728.
24. C. N. Wolf, United States, Patent number 2720480, 1955.
25. N. Nohara, S. Sekiguchi, and K. Matsui, J. Heterocycl. Chem., 1970, 7, 519.
26. Y. Nezu, M. Miyazaki, K. Sugiyama, N. Wada, I. Kajiwara, and T. Miyazawa, Pestic. Sci., 1996, 47,
115.
27. S. Dementin, A. Bouhss, G. Auger, C. Parquet, D. Mengin-Lecreulx, O. Dideberg, J. van Heijenoort,
and D. Blanot, Eur. J. Biochem., 2001, 268, 5800.