Synthesis and antimicrobial activity of N1-benzyl or N 1-benzyloxy-1,6-dihydro-1,3,5-triazine-2,4-diamines

Article abstract of DOI:10.1016/j.bmcl.2011.06.125

The emergence and spread of multidrug-resistant strains of Staphylococcus aureus and Mycobacterium tuberculosis are generating a threat to public health worldwide. In the current study, a series of N¹-benzyl and N ¹-benzyloxy-1,6-dihydro-1,3,5-triazine-2,4-diamine derivatives were synthesized and investigated for their antimicrobial activity against S. aureus, and Mycobacterium smegmatis which is taxonomically related to M. tuberculosis. Most of the compounds exhibited good activity against M. smegmatis as determined by comparison of diameters of the zone of inhibition of test compounds and standard antibiotics. Compound 7o showed potent antimycobacterial activity against M. smegmatis without mammalian DHFR inhibition liability. The results from this study indicate that 1-benzyl derivatives of 1,6-dihydro-1,3,5- triazine-2,4-diamines may be used as lead compounds for the discovery of antimycobacterial agents.

Full text of DOI:10.1016/j.bmcl.2011.06.125

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5428–5431
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antimicrobial activity of N1-benzyl or
N1-benzyloxy-1,6-dihydro-1,3,5-triazine-2,4-diamines
Xiang Ma a,b, , Soo-Tong Tan b, Chai-Ling Khoo b, Hong-May Sim b,c, Lai-Wah Chan b, Wai-Keung Chui b,c,
⇑
⇑
a School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan 430030, Hubei, China
b Department of Pharmacy, Faculty of Science, 18 Science Drive 4, National University of Singapore, Singapore 117543, Singapore
c Medicinal Chemistry Programme, Office of Life Science, 18 Science Drive 4, National University of Singapore, Singapore 117543, Singapore
a r t i c l e i n f o
a b s t r a c t
Article history:
The emergence and spread of multidrug-resistant strains of Staphylococcus aureus and Mycobacterium
tuberculosis are generating a threat to public health worldwide. In the current study, a series of N1-benzyl
and N1-benzyloxy-1,6-dihydro-1,3,5-triazine-2,4-diamine derivatives were synthesized and investigated
for their antimicrobial activity against S. aureus, and Mycobacterium smegmatis which is taxonomically
related to M. tuberculosis. Most of the compounds exhibited good activity against M. smegmatis as deter-
mined by comparison of diameters of the zone of inhibition of test compounds and standard antibiotics.
Compound 7o showed potent antimycobacterial activity against M. smegmatis without mammalian DHFR
inhibition liability. The results from this study indicate that 1-benzyl derivatives of 1,6-dihydro-1,3,5-tri-
azine-2,4-diamines may be used as lead compounds for the discovery of antimycobacterial agents.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 20 March 2011
Revised 28 June 2011
Accepted 30 June 2011
Available online 2 July 2011
Keywords:
Antibacterial agents
Dihydro-1,3,5-triazines
Mycobacterium tuberculosis
Staphylococcus aureus
The 2,4-diamino-1,3-diaza (1, Fig. 1) is a common pharmaco-
phoric feature of many antifolates that exhibit antibacterial activ-
ity against pathogens such as gram-positive bacteria,1
Mycobacterium tuberculosis,2,3 and Mycobacterium avium which
are known to cause opportunistic infections in patients with com-
promised immune systems.4–7 For instance, the structural motif is
embedded in the structure of diaminopyrimidines like trimetho-
prim and iclaprim. Trimethoprim (TMP, 2, Fig. 1), is an antibiotic
that has been widely used for over four decades in medical prac-
tice,8 and, most recently, iclaprim (3, Fig. 1), has completed a phase
III clinical trial against complicated skin and skin structure infec-
tion with promising results.9 Studies have also shown that the
biguanide PS-15 (4) and its metabolite, WR99210 (5), which pos-
sesses the same 2,4-diamino-1,3-diaza pharmacophore, have been
found to exhibit reasonable in vitro inhibitory activity against M.
tuberculosis and other mycobacteria.3,6,7 A ternary complex crystal
structure of M. tuberculosis dihydrofolate reductase (DHFR) with
NADP+ and Br-WR99210 (6) was determined in a separate study.10
In the study, structural comparison of the ternary complex with
human DHFR has identified a striking difference in the binding site
region near the N1 and two methyl groups of Br-WR99210 (Fig. 1),
where a glycerol molecule binds in a pocket of the complex and
this pocket is essentially filled with hydrophobic side-chains in hu-
man DHFR.10 The differences provided opportunities for designing
new selective antimycobacterial agents.
In the light of these reports, a series of dihydro-1,3,5-triazines
embedded with the 2,4-diamino-1,3-diaza pharmacophore was
designed for synthesis. The library of compounds contains deriva-
tives of dihydro-1,3,5-triazine-2,4-diamine that carry various
bulky alkyl substitution at the above mentioned C-2 position,
and in addition, some of the compounds have either a benzyl or
benzyloxy bridge that is connected to the triazine ring at its N-1
position. Thus, compounds 7a–7w were synthesized to structurally
mimic the conventional 2,4-diaminopyridine of TMP or iclaprim
due to a similar benzyl substitution. Whereas compounds 8a–8g
were designed with a N-benzyloxy substitution at N-1 position
in an attempt to probe steric and electronic tolerance of the side
chain. The designed compounds were synthesized and evaluated
for their antimicrobial activity against a panel of bacteria including
Staphylococcus aureus, and Mycobacterium smegmatis which is taxo-
nomically related to M. tuberculosis. At the same time, DHFR inhib-
itory assay was conducted to determine the antifolate activity
against mammalian DHFR. This study was conducted to evaluate
whether the compounds would interfere with mammalian DHFR
which could lead to potential side effect liability if inhibition
against bacterial and mammalian DHFR was not selective.11
The synthesis of 7a–7e was reported in our previous publica-
tion,12 and the synthetic route to 7a–7e was applied for the prep-
aration of 7f–7w as described in Scheme 1. Reaction of the
substituted benzylamine hydrochloride and cynoguanidine at
170–180 °C for 30 min provided biguanide hydrochlorides, which
⇑
Corresponding authors. Tel.: +65 6516 2657; fax: +65 6779 1554 (X.M.); tel.:
+65 6516 2933; fax: +65 6779 1554 (W.K.C.).
E-mail addresses: maxwellcn@yahoo.com (X. Ma), phacwk@nus.edu.sg (W.-K.
Chui).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.06.125

X. Ma et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5428–5431
5429
NH2
X
NH2
NH2
O
N
N
O
O
N
Y
H2N
N
H2N
N
O
H2N
N
O
O
1
TMP (2)
iclaprim (3)
NH2
N
Cl
Cl
NH2
Cl
O
O
O
O
N
N
N
H
NH
H2N
N
Cl
H2N
Cl
Cl
WR 99210 (5)
PS-15 (4)
NH2
N
NH2
NH2
R
1
O
O
O
N
N
N
2
R
N
N
R1
R2
R1
R2
H2N
N
H2N
N
Br
H2N
N
7
8
Br-WR99210 (6)
Figure 1. Structures of some antibacterial or antimycobacterial agents with 2,4-diamino-1,3-diaza pharmacophore.
NH2
N
NH NH
.
HCl
NH2
c
a, b
NH2
N
N
H
N
H
R
R1
R
R
.
H2N
N
R2
HCl
7f-7w
Scheme 1. Synthesis of 7f–7w. Reagents and conditions: (a) HCl, ether, rt; (b) cynoguanidine, 170–180 °C; (c) ketones, 2,2-dimethoxypropane, concd. HCl, ethanol, reflux.
were subjected to HCl-catalyzed cyclocondensation with various
ketones to afford the target compounds 7f–7w. The progress of
the reaction was followed by the observation of shifts in ultraviolet
absorption spectrum of the reaction mixture, or by the measure-
ment of biguanide disappearance which was accomplished by
means of the colored copper complex formed characteristically
by biguanide with cuprammonium ion.13 The synthesis of 8a–8d
and 8g was accomplished using a reported procedure14 for prepa-
ration of 8e and 8f as described in Scheme 2. Under room temper-
ature, a three component synthesis was carried out to give the
targeted compounds with a spectrum of reaction time using differ-
ent ketone or aldehyde reagents. The relative reaction speed of ke-
tone or aldehyde reagents employed was observed in the following
trend: acetone (24 h) = methyl ethyl ketone (24 h) > cyclohepta-
none (48 h) > c-propyl formaldehyde (120 h) > cyclopentanone
(7 d) = cyclohexanone (7 d) > cyclobutanone (8 d). The details of
synthetic procedures and structural characterizations are provided
in Supplementary data.
NH2
O
O
Ph
N
N
N
R1
R2
R1
.
HCl
NH
NH2
H2N
N
R2
HCl
or
.
NC
+
PhCH2ONH2 HCl
8a-8c
+
N
H
EtOH, rt, 24h-7d
or
O
NH2
N
O
Ph
n
.
HCl
H2N
N
n
8d and 8g
Scheme 2. Synthesis of 1-benzyloxy-1,3,5-triazines.

5430
X. Ma et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5428–5431
Table 1
Inhibition of S. aureus, M. smegmatis and bovine DHFR by test compounds and TMP
No.
R
R1
R2
Zone ratioa,c (100
l
g)
DHFRc (IC50
)
S. aureusb
M. smegmatisb
d
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
7q
7r
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
C2H5
C2H5
C2H5
c-Pr
–
–
–
–
–
0.5
–
0.8
–
–
–
0.35
0.17
0.53
0.27
0.98
0.45
0.41
0.24
0.67
0.56
–
0.85
0.66
2.5
ND
ND
ND
2.5
2.5
2.0
–
19.512
24.612
58.812
29.912
12.812
21.8
40-CH3
40-OCH3
40-Cl
0.30
–
0.31
0.35
–
–
–
–
0.19
0.3
0.15
0.18
0.25
0.51
0.18
0.15
0.11
0.3
30,40-diCl
40-F
30,40,50-triOMe
40-CH3
40-OCH3
40-Cl
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
0.069
0.155
0.548
0.134
0.17314
1.7914
5.33
CH3
CH3
CH3
40-Cl
40-CH3
40-OCH3
40-Cl
–(CH2)4–
–(CH2)4–
–(CH2)4–
–(CH2)4–
–(CH2)5–
–(CH2)5–
–(CH2)5–
–(CH2)5–
–(CH2)5–
–(CH2)5–
–(CH2)6–
–(CH2)6–
CH3
CH3
H
–(CH2)3–
–(CH2)4–
–(CH2)5–
–(CH2)6–
30,40-diCl
40-CH3
40-OCH3
40-F
7s
7t
40-Cl
30,40-diCl
30,40,50-triOMe
30,40-diCl
40-CH3
H
0.18
–
7u
7v
7w
8a
8b
8c
8d
8e
8f
0.43
0.18
0.51
NDd
ND
CH3
C2H5
c-Pr
H
H
H
H
H
H
ND
0.55
0.74
0.21
1.09
8g
TMP
>100
a
b
c
inhibition zone of test compound
inhibition zone of standard
Zone ratio ¼
.
Standard drugs for the calculation: S. aureus: cephaloridine disk, M. smegmatis: Streptomycin disk.
The results are averages of three separate experiments.
ND = not determined, ‘–’ = not active.
d
Compound 7a–7w and 8a–8g were screened at the loading of
100 g per disc against a panel of bacteria (namely S. aureus ATCC
bacterial agents was limited by the toxicity in the DHFR assay.
Among the benzyl substituted series 7a–7w, compound 7o was
the best with the zone ratio of 0.98, comparable to the positive
control, streptomycin. Moreover, derivatives 7g–7w and 7o were
l
6538P, Pseudomonas aeruginosa ATCC 9027, M. smegmatis ATCC
607, Bacillus subtilis ATCC 6633, Klebsiella aerogenes ATCC 9621,
Escherichia coli ATCC 25922) and two fungus (Candida albicans
ATCC 10231 and Aspergillus niger ATCC 16404) using the paper disc
diffusion method,15 and compared with TMP tested in the same
in vitro model. DHFR inhibition assay was used to determine
whether the mammalian antifolate activity of target compounds
was present, and bovine DHFR was used as the mammalian stan-
dard. The zone ratios which were used to express the comparative
efficiency to the standard drugs and DHFR inhibitory activity are
summarized in Table 1. In general, only S. aureus and M. smegmatis
were sensitive to most of the tested compounds. All these com-
pounds showed relatively weaker activity against Gram positive
bacteria S. aureus than positive control, cephaloridine with the
zone ratio <1, and none of these compounds showed superiority
to TMP which demonstrated about equal potency to the cephalor-
idine (zone ratio = 1.09). These compounds exhibited more potent
activity against M. smegmatis than those against S. aureus. N-Ben-
zyloxy substituted compounds 8a–8f were found to be the most
active compounds against M. smegmatis in the both series with
the zone ratios of 2.5. However, compounds 8a–8f, bearing a N-
benzyloxy side chain, proved to have potent mammalian DHFR
found to be devoid of DHFR inhibitory activity (IC50 >100 lM).
On the basis of these observation, compound 7o, showed a promise
as a lead in searching new antimycobacterial agents, for its good
activity against M. smegmatis and it did not possess side-effect lia-
bility through mammalian DHFR inhibition. The preliminary re-
sults suggested that the bulky spiro cycloalkyl group at C-2
position was not well tolerated in their inhibitory activity against
mammalian DHFR in both series, and it corroborated the study that
the exact position for the drug-enzyme binding was filled with
hydrophobic side-chains in the human DHFR, which provides the
opportunity to design new selective antimycobacterial agent.10
In conclusion, a series of dihydro-1,3,5-triazines bearing 2,4-
diamino-1,3-diaza pharmacophore was synthesized and screened
against microbes and bovine DHFR. Compound 7o apparently have
better inhibitory activity against M. smegmatis compared to TMP or
standard drug without mammalian DHFR inhibition liability. Here-
in, these results suggest that N1-benzyl-1,3,5-triazines, based on
the unique 2,4-diamino-1,3-diaza pharmacophore, may represent
promising lead candidates that are worthy of further development
into potential antimycobacterial agents.
inhibitory activity, with IC50 values ranged from 0.069
lM to
1.79 M. DHFR inhibitory activity decreased as the size of the sub-
stituent at C-2 of triazine increased. Substitution at C-2 by the very
l
Acknowledgment
bulky seven membered spiro ring as in compound 8g led to a still
fairly good DHFR inhibitory activity (IC50 = 5.33 lM). However, the
activity still suggested an unwanted side effect liability as antimi-
crobial agents. Thus, the potential of 8a–8f as leads for antimyco-
The authors would like to thank the Academic Research Funds
of National University of Singapore (R148-000-052-112) for sup-
porting this piece of work.

X. Ma et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5428–5431
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