Synthesis, characterization and in vitro biological evaluation of some novel 1,3,5-triazine-Schiff base conjugates as potential antimycobacterial agents

Article abstract of DOI:10.1016/j.bmcl.2013.08.063

A series of some novel 1,3,5-triazine-Schiff base conjugates (1-32) have been synthesized and evaluated for their in vitro antimycobacterial activity against Mycobacterium tuberculosis H37Rv using Alamar Blue assay and the activity expressed as the minimum inhibitory concentration (MIC) in μg/mL. Compounds 4 (4-Methoxy-6-methyl-N-(3,4,5-trimethoxybenzylidene)-1,3,5-triazin-2- amine), 11 (4-Methoxy-6-methyl-N-(2-hydroxy-3-bromo-5-chloro-benzylidene)-1,3,5- triazin-2-amine) and 24 (4-Methoxy-6-methyl-N-(1-(2,5-dihydroxyphenyl) ethylidene)-1,3,5-triazin-2-amine) exhibited a significant activity at 3.125, 6.25 and 6.25 μg/mL, respectively, when compared with the antitubercular drugs such as ethambutol (3.125 μg/mL), pyrazinamide (6.25 μg/mL) and streptomycin (6.25 μg/mL) and it could be a potential starting point to develop new lead compounds in the fight against Mycobacterium tuberculosis H37Rv.

Full text of DOI:10.1016/j.bmcl.2013.08.063

Bioorganic & Medicinal Chemistry Letters 23 (2013) 5968–5970
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, characterization and in vitro biological evaluation of some
novel 1,3,5-triazine–Schiff base conjugates as potential
antimycobacterial agents
a
a
b
Vasudeva Rao Avupati a, , Rajendra Prasad Yejella , Venkateswara Rao Parala , Kishore Naidu Killari ,
Venkata Madhava Reddy Papasani b, Prasad Cheepurupalli a, Venkateswara Rao Gavalapu a,
Bhavani Boddeda c
⇑
a Pharmaceutical Chemistry Division, AU College of Pharmaceutical Sciences, Andhra University, Visakhapatnam 530003, Andhra Pradesh, India
b Pharmacology Division, AU College of Pharmaceutical Sciences, Andhra University, Visakhapatnam 530003, Andhra Pradesh, India
c Pharmaceutical Technology Division, AU College of Pharmaceutical Sciences, Andhra University, Visakhapatnam 530003, Andhra Pradesh, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 28 February 2013
Revised 28 July 2013
Accepted 14 August 2013
Available online 22 August 2013
A series of some novel 1,3,5-triazine–Schiff base conjugates (1–32) have been synthesized and evaluated for
their in vitro antimycobacterial activity against Mycobacterium tuberculosis H37Rv using Alamar
Blue assay and the activity expressed as the minimum inhibitory concentration (MIC) in
lg/mL.
Compounds (4-Methoxy-6-methyl-N-(3,4,5-trimethoxybenzylidene)-1,3,5-triazin-2-amine), 11
4
(4-Methoxy-6-methyl-N-(2-hydroxy-3-bromo-5-chloro-benzylidene)-1,3,5-triazin-2-amine) and 24
(4-Methoxy-6-methyl-N-(1-(2,5-dihydroxyphenyl)ethylidene)-1,3,5-triazin-2-amine) exhibited a signifi-
Keywords:
cant activity at 3.125, 6.25 and 6.25
lg/mL, respectively, when compared with the antitubercular drugs
1,3,5-Triazine–Schiff base conjugate
Antimycobacterial activity
Mycobacterium tuberculosis H37Rv
Alamar Blue assay
such as ethambutol (3.125 g/mL), pyrazinamide (6.25
l
l
g/mL) and streptomycin (6.25 g/mL) and it could
l
be a potential starting point to develop new lead compounds in the fight against Mycobacterium
tuberculosis H37Rv.
Ó 2013 Elsevier Ltd. All rights reserved.
In the existing scenario of mycobacterial infectious diseases,
Tuberculosis (TB) is a serious global public health problem and a
leading cause of death in most of the developing regions of the
world.1–10 World Health Organization (WHO) declared TB ‘a global
health emergency’ since 1993. It is estimated by WHO that approx-
imately one-third of the world’s population (two billion people) is
infected with Mycobacterium tuberculosis (MTB) and two million
people die every year. The emergence of single drug-resistant TB
(SDR-TB), multi-drug resistant TB (MDR-TB) and extensively
drug-resistant TB (XDR-TB) increases the rate of treatment failure
and the costs of TB control. TB in co-occurrence with the spread
of human immunodeficiency virus (HIV) infection is an increasing
problem in industrialized countries.11–18 Although, a number of
new chemical entities have been discovered recently as potential
antimycobacterial agents, so far no new drug has entered into
the market since 1960s. To overcome this rapid development of
drug resistance, there is an urgent need to discover and develop
new drugs, acting through a novel mechanism of action for the effi-
cient chemotherapy of TB infection.19,20
1,3,5-Triazine (s-triazine) is an important class of heterocyclic
compounds found in many synthetic and natural products with a
wide range of biological activities, such as adenosine receptor
antagonist,21antiamoebic,22 anticancer,23antileishmanial,24 anti-
malarial,25 antimicrobial,26 antiviral,27 antitubercular,28 carbonic
anhydrase inhibitor29 and cathepsin B inhibitor.30 Similarly, Schiff
bases have also gained great importance as these compounds have
been found to exhibit antimicrobial,31–34 antiviral,35,36 antioxi-
dant37 and antitumor.38,39 These activities are largely attributed
due to the azomethine (C@N) moiety. Having such varied pharma-
cological activities, these pharmacophores have attracted research-
ers and consequently a number of strategies have been originated
to synthesize them. In the present study 1,3,5-triaizine-azome-
thine conjugates 1–32 have been synthesized and were studied
for their antimycobacterial activity. The rationale behind the selec-
tion of these moieties is that the compounds having either 1,3,5-
triazine or azomethine moieties were earlier reported to possess
significant inhibitory activity against M. tuberculosis H37Rv28 and
thus the synthesized compounds now are expected to possess
additive effect, as both the features form part of these molecules.
The reaction sequence employed in the synthesis of title com-
pounds (1–32) is shown in Scheme 1, and their physical properties
are depicted in Table 1. Schiff base conjugates of 1,3,5-triazine
were prepared by a condensation reaction of the key starting
⇑
Corresponding author.
E-mail addresses: vasudevaraoavupati@rediffmail.com, vasudevaraoavupati@
gmail.com (V.R. Avupati).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.08.063

V. R. Avupati et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5968–5970
5969
Plates were covered and sealed with parafilm and incubated at
37 °C for five days. After this time, 25 L of freshly prepared 1:1
l
mixture of Almar Blue reagent and 10% tween 80 was added to
the plate and incubated for 24 h. A blue color in the well was inter-
preted as no bacterial growth, and pink color was scored as growth.
The MIC was defined as the lowest drug concentration which pre-
vented the color change from blue to pink. Correspondingly, these
compounds were tested for cytotoxicity (IC50) in VERO cells at con-
Scheme 1. Reagents and conditions: (i) Ethanol, substituted aromatic/heteroaro-
matic aldehydes and ketones, stirring, 2–5 h.
centrations (50, 100 and 200 lg/mL) greater than the MIC for
material 4-methoxy-6-methyl-1,3,5-triazin-2-amine with variety
of aromatic/heteroaromatic aldehydes and ketones in ethanol
which afforded a series of 1,3,5-triazine–Schiff base conjugates
(1–32) in good yield. All the synthesized compounds as mentioned
in Table 1 were characterized by spectroscopic methods such as
FTIR, 1H NMR, 13C NMR and LC mass spectral analysis. Eventually
all the spectra of the new products are in keeping with the
expected structures.
The antimycobacterial activity of compounds were assessed
against Mycobacterium tuberculosis (H37Rv) strain using micro
plate Alamar Blue assay (MABA).40 This methodology is non-toxic,
uses a thermally stable reagent and shows good correlation with
Mycobacterium tuberculosis (H37Rv). After 72 h exposure, viability
was assessed on the basis of cellular conversion of MTT into a for-
mazan product using the Promega Cell Titre 96 non-radioactive
cell proliferation assay.41 As a general rule, the MIC data given in
Table 1 was expressed as the average of three independent trials
(n = 3).
The results of in vitro antimycobacterial activity of the synthe-
sized compounds 1–32 against Mycobacterium tuberculosis (H37Rv)
is illustrated in (Table 1). The mycobacterial activity screening data
revealed that the compound 4 demonstrated comparatively the
most potent inhibitory activity, with MIC value 3.125
interesting to note that the compounds 11 and 24 also showed
appreciable inhibitory activity with MIC value 6.25 g/mL. Com-
pounds 7–9, 18, 19 and 28 were also showed satisfactory inhibi-
tory activity with MIC value 12.5 g/mL. The other compounds
such as 2, 3, 5, 10, 12, 13, 21, 22, 23, 27 and 29 showed moderate
level of activity with MIC 25 g/mL. The compounds 1, 6, 14,
15–17, 20, 25, 26, 30, 31 and 32 exhibited comparatively less
lg/mL. It is
BACTEC radiometric method. 200
lL of sterile deionized water
l
was added to all outer perimeter wells of sterile 96 wells plate to
minimize evaporation of medium in the test wells during incuba-
l
tion. The 96 wells plate received 100
lL of the Middlebrook 7H9
broth and serial dilution of compounds were made directly on
plate. The final drug concentrations tested were 100–0.2 lg/mL.
l
Table 1
Physical characterization, antimycobacterial activity and cytotoxicity data of 1,3,5-triazine–Schiff base conjugates 1–32 produced via Scheme 1
Compound
R
R1
Molecular
formula
Molecular
weight
Yield Mp (°C)
MTBa (H37Rv) MICb
mL)
(l
g/
Cytotoxicity (lg/mL) VERO cells
(IC50
)
1
2
3
4
5
6
7
8
H
H
H
H
H
H
H
H
H
H
H
C6H5
4-MeC6H4
4-NMe2C6H4
C12H12N4O
C13H14N4O
C14H17N5O
228
242
271
318
288
273
262
296
306
306
355
64
62
53
71
69
73
72
75
63
61
57
209–214 50
239–244 25
245–250 25
ND
>50
>50
>50
>50
ND
>50
>50
>50
>50
>50
3,4, 5-triOMeC6H2 C15H18N4O4
238–243
3.125
3-OEt,4-OHC6H3
3-NO2C6H4
4-ClC6H4
2,4-diClC6H3
3-BrC6H4
4-BrC6H4
2-OH,3-Br,5-
ClC6H2
C14H16N4O3
C12H11N5O3
227–232 25
234–239 50
204–209 12.5
244–249 12.5
142–147 12.5
234–239 25
C12H11ClN4O
C12H10Cl2N4O
C12H11BrN4O
C12H11BrN4O
C12H10BrClN4O2
9
10
11
219–224
6.25
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
H
H
H
H
H
4-Allyl-OC6H4
Pyrrol-2-yl
Pyridin-3-yl
Indol-3-yl
C15H16N4O2
284
217
229
267
328
242
256
272
272
258
258
274
274
272
272
287
287
248
243
292
330
—
69
69
71
63
69
77
72
82
81
73
81
77
62
63
78
67
65
72
71
73
72
—
225–229 25
231–236 25
232–237 50
184–189 50
102–107 50
220–225 50
256–261 12.5
244–249 12.5
243–248 50
240–245 25
232–237 25
210–215 25
>50
>50
ND
ND
ND
C
C
C
C
10H11N5O
11H11N5O
14H13N5O
20H16N4O
Anthracen-9-yl
CH3 C6H5
CH3 4-MeC6H4
CH3 3-OMeC6H4
CH3 4-OMeC6H4
CH3 2-OHC6H4
C13H14N4O
C14H16N4O
C14H16N4O2
C14H16N4O2
C13H14N4O2
C13H14N4O2
C13H14N4O3
C13H14N4O3
C14H16N4O2
C14H16N4O2
C13H13N5O3
C13H13N5O3
C11H12N4OS
C12H13N5O
C17H16N4O
C20H18N4O
—
ND
>50
>50
ND
>50
>50
>50
>50
ND
CH3 4-OHC6H4
CH3 2,4-diOHC6H3
CH3 2,5-diOHC6H3
CH3 2-OH,5-MeC6H3
CH3 6-OH,5-MeC6H3
CH3 3-NO2C6H4
CH3 4-NO2C6H4
CH3 Thiophen-2-yl
CH3 Pyridin-3-yl
CH3 Naphthalen-2-yl
CH3 Fluoren-2-yl
211–216
6.25
251–256 50
252–257 50
220–225 25
222–227 12.5
245–250 25
216–221 50
195–200 50
128–133 50
ND
>50
>50
>50
ND
ND
ND
>50
>50
>50
28
29
30
31
32
Ethambutol
Pyrazinamide
Streptomycin
—
—
—
—
—
—
—
—
—
3.125
3.125
6.25
—
—
—
—
—
—
ND: Not done.
a
Mycobacterium tuberculosis H37Rv.
Average of three independent trials (n = 3).
b

5970
V. R. Avupati et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5968–5970
inhibitory activity with MIC value 50
the standard drugs (Ethambutol, MIC: 3.125
l
g/mL in comparison with
g/mL; Pyrazinamide,
g/mL and Streptomycin, MIC: 6.25 g/mL).
Supplementary data
l
MIC: 3.125
l
l
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
08.063.
An examination of the SAR (Structure–Activity Relationship) of
these compounds clearly exhibited the intrinsic phenomenon of
Mycobacterium tuberculosis (H37Rv) inhibitory activity associated
with the basic skeleton consisting of 1,3,5-triazine and azomethine
References and notes
moieties with MIC values ranging from 50–3.125 lg/mL. The ob-
1. http://www.who.int/tb/en/.
2. Janin, Y. L. Bioorg. Med. Chem. 2007, 15, 2479.
3. Goldman, R. C.; Plumley, K. V.; Laughon, B. E. Infect. Disord. Drug Targets 2007, 7,
73.
4. Ballell, L.; Field, R. A.; Duncan, K.; Young, R. J. Antimicrob. Agents Chemother.
2005, 49, 2153.
5. Iseman, M. D. Eur. Respir. J. Suppl. 2002, 36, 87.
6. Migliori, G. B.; Ambrosetti, M.; Fattorini, L.; Penati, V.; Vaccarino, P.; Besozzi, G.;
Ortona, L.; Saltini, C.; Orefici, G.; Moro, M. L.; Lona, E.; Cassone, A. Int. J. Tuberc.
Lung Dis. 2000, 4, 940.
7. Migliori, G. B.; Ambrosetti, M.; Fattorini, L.; Penati, V.; Vaccarino, P.; Besozzi, G.;
Ortona, L.; Saltini, C.; Orefici, G.; Moro, M. L.; Lona, E.; Cassone, A. Int. J. Tuberc.
Lung Dis. 2000, 4, 940.
8. Harries, A. D. M. D. TB/HIV, A Clinical Manual; World Health Organization:
Geneve, 1996.
9. Nunn, P.; Williams, B.; Floyd, K.; Dye, C.; Elzinga, G.; Raviglione, M. Nat. Rev.
Immunol. 2005, 5, 819.
10. Murray, C. J. L.; Styblo, K.; Rouillon, A. In Disease Control Priorities in Developing
Countries; Jamison, D. T., Mosely, W. H., Eds.; Oxford University Press: New
York, 1993; p 233.
11. Styblo, K. Bull: Int. Union Tuberc. 1990, 65, 24.
12. Baker, R. W.; Mitscher, L. A.; Arain, T. M.; Shawar, R.; Stover, C. K. Annu. Rep.
Med. Chem. 1996, 31, 161.
13. Dunkan, K. Expert Opin. Ther. Pat. 1997, 7, 129.
14. Niccolai, D.; Tarsi, L.; Thomas, R. J. Chem. Commun. 1997, 33.
15. Horsburgh, C. R., Jr. N. Engl. J. Med. 1991, 324, 1332.
16. Heifets, L. Antimicrob. Agents Chemother. 1996, 40, 1759.
17. Harries, A. D.; Mahler, D. TB/HIV, A Clinical Manual; World Healt Organization:
Geneve, 1996.
served inhibitory property of 1,3,5-triazine-azomethine conjugates
(1–32) against Mycobacterium tuberculosis (H37Rv) revealed the
importance of the nature of substituted aromatic/heteroaromatic
aldehyde or ketone from which the corresponding Schiff bases
(1–32) were obtained, which in some cases was enhanced by the
influence of some substituents and decreased by some other sub-
stituents. The aromatic/heteroaromatic aldehyde or ketone derived
azomethine analogs of 1,3,5-triazine, as seen in the case of com-
pounds 14–17 and 30–32 containing pyridine-3-yl, indol-3-yl,
anthracen-9-yl, naphthalen-2-yl and fluoren-2-yl ring structure
showed moderate MIC value 50
rived from pyrrole-2-carboxaldehyde) and 29 (from 2-acetylthio-
phene) displayed better inhibitory potency with MIC 25 g/mL
lg/mL. The compounds 13 (de-
l
indicating the significance of five membered heterocycle ring to-
wards its observed antimycobacterial activity. The compounds
having halogen substituents either at meta or para positions on
the phenyl ring significantly enhanced the activity with MIC value
25–6.25 lg/mL as seen in case of compounds 7–10. This observa-
tion indicates the usefulness of phenyl ring than the other ring sys-
tems to improve inhibitory activity against Mycobacterium
tuberculosis (H37Rv). The compounds substituted with electron
releasing groups was found to be biologically relevant and the
18. Hamilton, C. D. Curr. Infect. Dis. Rep. 1999, 1, 80.
19. Zhang, Y.; Post-Martens, K.; Denkin, S. Drug Discovery Today 2006, 11, 21.
20. Brown, E. D.; Wright, G. D. Chem. Rev. 2005, 105, 759.
21. Pastorin, G.; Federico, S.; Paoletta, S.; Corradino, M.; Cateni, F.; Cacciari, B.;
Klotz, K.; Gao, Z.; Jacobson, K. A.; Spalluto, G.; Moro, S. Bioorg. Med. Chem. 2010,
18, 2524.
activity order was (4 (3,4,5-triOCH3, MIC: 3.125
CH3, MIC: 12.5 g/mL), 19 (3-OCH3, MIC: 12.5 g/mL) >2 (4-CH3,
25 g/mL), 3 (N(CH3), MIC: 25 g/mL)), respectively. Considerable
activity was observed when the hydroxyl groups are substituted
at different positions on the phenyl ring as seen in the case of com-
pounds 11, 24, 5 and 21–23 and the order of activity was 11 (2-
lg/mL) >18 (4-
l
l
l
l
22. Wani, M. Y.; Bhat, A. R.; Azam, A.; Choi, I.; Athar, F. Eur. J. Med. Chem. 2012, 48,
313.
OH,3-Br,5-Cl, MIC: 6.25
>5 (3-OC2H5,4-OH, MIC: 25
(4-OH, MIC: 25 g/mL), 23 (2,4-diOH, MIC: 25
The compounds 25 (MIC: 50 g/mL) and 26 (MIC: 50
ing the methyl group substitution on the phenyl ring at position
five along with the hydroxyl group substitution at six (26) and
two (25) positions, respectively showed lower level of inhibitory
activity when compared with that of the compounds (21–24) pos-
sessing only hydroxyl group substitution. With respect to the cyto-
toxicity (IC50) in VERO cells at different concentrations, most of the
l
g/mL), 24 (2,5-diOH, MIC: 6.25
g/mL), 21 (2-OH, MIC: 25 g/mL), 22
g/mL) respectively.
g/mL) hav-
l
g/mL)
23. Popowycz, F.; Schneider, C.; DeBonis, S.; Skoufias, D. A.; Kozielski, F.; Galmarini,
C. M.; Joseph, B. Bioorg. Med. Chem. 2009, 17, 3471.
24. Sunduru, N.; Agarwal, A.; Katiyar, S. B.; Nishi, N.; Goyal, N.; Guptab, S.;
Chauhana, P. M. S. Bioorg. Med. Chem. 2006, 14, 7706.
25. Vilaivan, T.; Saesaengseerung, N.; Jarprung, D.; Kamchonwongpaisan, S.;
Sirawaraporn, W.; Yuthavong, Y. Bioorg. Med. Chem. 2003, 11, 217.
26. Zhou, C.; Min, J.; Liu, Z.; Young, A.; Deshazer, H.; Gao, T.; Chang, Y. T.;
Kallenbach, N. R. Bioorg. Med. Chem. Lett. 2008, 18, 1308.
27. Krecmerova, M.; Masojidkova, M.; Holy, A. Bioorg. Med. Chem. 2010, 18, 387.
28. Sunduru, N.; Gupta, L.; Chaturvedi, V.; Dwivedi, R.; Sinha, S.; Chauhan, P. M. S.
Eur. J. Med. Chem. 2010, 45, 3335.
29. Garaj, V.; Puccetti, L.; Fasolis, G.; Winum, J.; Montero, J.; Scozzafava, A.; Vullo,
D.; Innocentia, A.; Supurana, C. T. Bioorg. Med. Chem. Lett. 2005, 15, 3102.
30. Sosic, I.; Mirkovi, B.; Turk, S.; Stefane, B.; Kos, J.; Gobec, S. Eur. J. Med. Chem.
2011, 46, 4648.
31. Jarrahpour, A.; Khalili, D.; De Clercq, E.; Salmi, C.; Brunel, J. M. Molecules 2007,
12, 1720.
l
l
l
l
l
l
active compounds were found to be non-toxic (IC50 >50 lg/mL).
In summary, we could synthesis and characterize a novel series
of 1,3,5-triazine–Schiff base conjugates (1–32) which earlier were
not reported. These compounds were screened for antimycobacte-
rial activity and the results revealed the positive contribution of
azomethine conjugation on the 1,3,5-triazine ring towards the ob-
served antimycobacterial activity.
32. Perumal, P.; Bilal, A. R.; Dontireddy, R. S. R.; Natesh, R. K. Eur. J. Med. Chem.
2009, 44, 2328.
33. Kui, C.; Qing, Z.; Yong, Q.; Lei, S.; Jing, Z.; Hai, Z. Bioorg. Med. Chem. 2009, 17,
7861.
34. Panchal, P. K.; Pansuriya, P. B.; Patel, M. N. J. Enz. Inhib. Med. Chem. 2006, 21,
453.
35. Jarrahpour, A.; Khalili, D.; De, C. E.; Salmi, C.; Brunel, J. M. Molecules 2007, 12,
1720.
Acknowledgments
36. Koneni, V. S.; Jammikuntla, N. R.; Gitika, B.; Saxena, J. K. Eur. J. Med. Chem. 2008,
43, 2592.
37. Vanco, J.; Svajlenova, O.; Racanska, E.; Muselik, J.; Valentova, J. J. Trace Elem.
Med. Biol. 2004, 18, 155.
38. Sinha, D.; Tiwari, A. K.; Singh, S.; Shukla, G.; Mishra, P.; Chandra, H.; Mishra, A.
K. Eur. J. Med. Chem. 2008, 43, 160.
39. Gihane, N.; Eddy, P.; Claudiu, T. S.; Jean-Yves, W.; Mihail, B. Bioorg. Med. Chem.
Lett. 2009, 19, 6014.
40. Maria, C. S. L.; Marcus, V. N.; Alessandra, C. P.; Marcelle, L. F.; Goncalves, T. M.;
Nogneira, M. A. P. ARKIVOC 2007, xv, 181.
41. Heifets, L. B.; Flory, M. A.; Lindholm-Levy, P. J. Antimicrob. Agents Chemother.
1989, 33, 1252.
One of the authors (VasudevaRaoAvupati) is thankful to the
Council of Scientific and Industrial Research (CSIR), Government
of India, New Delhi for providing Senior Research Fellowship
(SRF). We also thank Dr. K.G. Bhat, Department of Microbiology,
Maratha Mandal’s NGH Institute of Dental Sciences and Research
Centre for providing facilities to carry out antimycobacterial activ-
ity and to the Principal, Andhra University College of Pharmaceuti-
cal Sciences, Visakhapatnam for providing required help in
carrying out the Biological activity.