Synthesis and antituberculosis activity of some N-pyridyl-N′-thiazolylhydrazine derivatives

Article abstract of DOI:10.1016/j.ejmech.2010.01.017

In this study, new N-(1-arylethylidene)-N′-(4-arylthiazol-2-yl)hydrazine derivatives were synthesized and evaluated for their antituberculosis activity. The chemical structures of the compounds were elucidated by IR, NMR and FAB⁺-MS spectral data and Elemental Analyses. The initial screen was conducted against Mycobacterium tuberculosis H37Rv (ATCC 27294) in BACTEC 12B medium using the Microplate Alamar Blue Assay (MABA). The VERO cell cytotoxicity assay was done in parallel with the TB Dose Response assay. Viability was assessed using Promega's Cell Titer-Glo Luminescent Cell Viability Assay. Cytotoxicity was determined from the dose-response curve as the CC50 using a curve-fitting program. One of the compounds showed high activity with low toxicity.

Full text of DOI:10.1016/j.ejmech.2010.01.017

European Journal of Medicinal Chemistry 45 (2010) 2085–2088
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Preliminary communication
Synthesis and antituberculosis activity of some
N-pyridyl-N0-thiazolylhydrazine derivatives
*
¨
Gu¨lhan Turan-Zitouni, Zafer Asım Kaplancıklı , Ahmet Ozdemir
Anadolu University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 26470 Eskis¸ehir, Turkey
a r t i c l e i n f o
a b s t r a c t
In this study, new N-(1-arylethylidene)-N0-(4-arylthiazol-2-yl)hydrazine derivatives were synthesized
and evaluated for their antituberculosis activity. The chemical structures of the compounds were
elucidated by IR, NMR and FABþ-MS spectral data and Elemental Analyses. The initial screen was con-
ducted against Mycobacterium tuberculosis H37Rv (ATCC 27294) in BACTEC 12B medium using the
Microplate Alamar Blue Assay (MABA). The VERO cell cytotoxicity assay was done in parallel with the TB
Dose Response assay. Viability was assessed using Promega’s Cell Titer-Glo Luminescent Cell Viability
Assay. Cytotoxicity was determined from the dose–response curve as the CC50 using a curve-fitting
program. One of the compounds showed high activity with low toxicity.
Article history:
Received 23 September 2009
Received in revised form
22 December 2009
Accepted 9 January 2010
Available online 20 January 2010
Keywords:
Antituberculosis activity
Hydrazine
Ó 2010 Elsevier Masson SAS. All rights reserved.
Thiazole
Pyridine
Mycobacterium tuberculosis
1. Introduction
tuberculosis, the standard treatment is six to nine months of INH
alone. Non-compliance has contributed to the appearance of XDR-
Tuberculosis (TB) is the leading infectious disease among adults
and youth, with one-third of the world’s population infected with
Mycobacterium tuberculosis. According to the World Health Orga-
nization (WHO), in 2006 there were 9.2 million new cases and 1.7
million deaths from TB around the world. The incidence of TB
infection has steadily risen in the last decade [1]. The reemergence
of TB infection has been further complicated. Two developments
make the resurgence in TB especially alarming. The first is patho-
genic synergy with HIV. The overall incidence of TB in HIV-positive
patients is 50 times that of the rate for HIV-negative individuals [2].
The second is the emergence of drug-resistant and multi-drug-
resistant TB (MDR-TB). In the WHO report, Anti-Tuberculosis Drug
Resistance in the World, is based on information collected between
2002 and 2006 on 90 000 TB patients in 81 countries. It is also
found that extensively drug-resistant tuberculosis (XDR-TB),
a virtually untreatable form of the respiratory disease, has been
recorded in 45 countries [1,3].
TB strains. MDR-TB is resistant to, at least, INH and rifampicin, often
taking a further two years to treat with second-line drugs. XDR-TB
also exhibits resistance to second-line drugs including fluo-
roquinolones and one of capreomycin, kanamycin or amikacin [4,5].
So there are three reasons usually given for needing new
tuberculosis drugs: (i) to improve current treatment by shortening
the total duration of treatment and/or by providing for more widely
spaced intermittent treatment, (ii) to improve the treatment of
MDR-TB, and (3) to provide for more effective treatment of latent
tuberculosis infection (LTBI) [6].
There are two basic approaches to develop a new drug for TB: (i)
synthesis of analogues, modifications or derivatives of existing
compounds for shortening and improving TB treatment and, (ii)
searching novel structures which the TB organism has never
encountered with before, for the treatment MDR-TB [7].
To pursue this goal, our research efforts are directed to the
modification of INH, which is a well known antituberculosis agent
bearing pyridine and hydrazine moieties. Modifying either of these
molecules has been a challenge taken up by several research groups
[8–12].
The standard ‘‘short’’ course treatment for tuberculosis (TB), is
isoniazid (INH), rifampicin, pyrazinamide, and ethambutol for two
months, then INH and rifampicin alone for a further four months.
The patient is considered cured at six months. For latent
The literature survey showed an important relationship between
serum levels of INH and the emergence of INH-resistant cultures in
patients with tuberculosis [13]. Serum concentrations are influ-
enced by a number of factors, but among them the most important
factor is the enzymic acetylation of INH by N-acetyltransferase
* Corresponding author. Tel.: þ90 222 335 05 80/3779; fax: þ90 222 335 07 50.
E-mail address: zakaplan@anadolu.edu.tr (Z.A. Kaplancıklı).
0223-5234/$ – see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.01.017

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G. Turan-Zitouni et al. / European Journal of Medicinal Chemistry 45 (2010) 2085–2088
(NAT). This represents a major metabolic pathway for INH in human
beings. NAT is also present in mycobacteria. It was found that
recombinant NAT from M. tuberculosis acetylates INH in vitro and it
was proposed that NAT in mycobacteria could have a role in acety-
lating and hence inactivating INH [14]. It is assumed that resistance
toINH in mycobacteria can thus be related to increased expression of
NAT and it is proposed that the chemical modification of the
hydrazine unit of INH with a functional group blocks acetylation,
maintaining strong activity and improving clinical outcomes [11,12].
This hypothesis was verified by many studies with their significant
results [8–12,15–17].
molecular formula. The IR spectra of the compounds showed NH
bands at 3335–3199 cmꢀ1 and C]C, C]N bands at 1600–
1435 cmꢀ1 regions respectively.
In the 400 MHz 1H NMR spectra of the compounds, the CH3
protons were observed as singlet at 2.30–2.45 ppm. The NH proton
appeared at 10.50–10.90 ppm. The OCH3 and OH protons of 2d–f
were observed at 3.70–3.80 ppm and 11.50–11.90 ppm respectively.
All the other aromatic protons were observed at expected regions.
The results of antituberculosis and cytotoxicity screening of
newly prepared compounds 2a–f are expressed in Table 2. The very
important result was observed at antituberculosis activity
screening for one of the compounds. The compound 2d showed
In this study, we planned to synthesize new molecules bearing
modified hydrazine moieties on different positions of pyridine ring.
We chose the thiazole ring for modification because of the impor-
tance of thiazole derivatives as antituberculosis agents [18–21].
high antituberculosis activity (IC50: 6.22
mg/mL and IC90: 6.78 mg/
mL) and low cytotoxicity (CC50: >40 g/mL). Because of SI value of
m
the compound 2d ꢁ10, further tests are in progress.
2. Chemistry
5. Conclusion
series of novel N-(1-arylethylidene)-N0-(4-arylthiazol-2-
The synthetic route of the compounds is outlined in Scheme 1.
For the synthesis of the title compounds, 1-(1-arylethylidene)-
thiosemicarbazide (1a–c) required as starting material was
prepared by the reaction of acetylpyridines with thiosemicarbazide
[22]. The reaction of equimolar quantities of thiosemicarbazide
(1a–c) with 1-(2-thiophenyl)-2-bromoethanone or 1-(2-hydroxy-
5-methoxyphenyl)-2-bromoethanone in the presence of isopropyl
alcohol resulted in the formation of the title compounds (2a–f)
(Table 1).
A
yl)hydrazine derivatives 2 were synthesized and their antituber-
culosis activities have been evaluated. Among these series,
compound 2d including 2-pyridyl and 2-hydroxy-5-methox-
yphenyl showed significant antituberculosis activity. On the other
hand compound 2a, which also includes 2-pyridyl moiety, showed
notable antituberculosis activity. As a result, we can say that the 2-
pyridyl derivatives are more active than other pyridyl derivatives
when their antituberculosis activity is compared.
3. Pharmacology
6. Experimental
3.1. Antituberculosis activity and cytotoxicity
6.1. Chemistry
The initial screen is conducted against M. tuberculosis H37Rv
(ATCC 27294) in BACTEC 12B medium using the Microplate Alamar
Blue Assay (MABA) [23]. One of the compounds showed significant
antituberculosis activity as can be inferred from Table 2.
The VERO cell cytotoxicity assay [24] is done in parallel with the
TB Dose Response assay. Viability is assessed using Promega’s Cell
Titer-Glo Luminescent Cell Viability Assay [25].
All melting points (m.p.) were determined in open capillaries on
a Gallenkamp apparatus and are uncorrected. The purity of the
compounds was routinely checked by thin layer chromatography
(TLC) using silica gel 60G (Merck). Spectroscopic data were recor-
ded on the following instruments: IR, Shimadzu 435 IR spectro-
photometer; 1H NMR, Bruker 400 MHz NMR spectrometer in
DMSO-d6 using TMS as an internal standard; elemental analyses
were performed on a Perkin Elmer EAL 240 elemental analyser;
MS-FABþ, VG Quattro mass spectrometer.
4. Results and discussion
The structures of compounds 2a–f were confirmed by elemental
analyses, MS-FAB, IR and 1H NMR spectral data. All compounds
gave satisfactory elemental analysis. The mass spectra (MS (FAB)) of
the compounds showed M þ 1 peaks, in agreement with their
6.1.1. Preparation of 1-(1-arylethylidene)thiosemicarbazide (1a–c)
In a flask equipped with a reflux condenser, a mixture of thio-
semicarbazide (50 mmol) and the appropriate acetylpyridine
(50 mmol) is reacted in n-butanol (100 ml) in the presence of
H
O
H
N
N
NH2
+
NH2
N
H2N
Ar1
Ar1
S
S
1a-c
Ar2
S
Ar1
Br
+
N
1a-c
N
H
N
Ar2
O
2a-f
Ar1: 2-pyridyl, 3-pyridyl, 4-pyridyl
Ar2: 2-thiophenyl, 2-hydroxy-5-metoxyphenyl
Scheme 1. Synthetic protocol of the title compounds.

G. Turan-Zitouni et al. / European Journal of Medicinal Chemistry 45 (2010) 2085–2088
2087
Table 1
Some characteristics of the compounds.
Comp.
Ar1
Ar2
m.p. (ꢂC)
244–246
Yield (%)
75
Mol. formula
C14H12N4S2
M.W.
2a
300
S
N
2b
2c
247–249
227–228
72
78
C14H12N4S2
300
300
S
S
N
N
C14H12N4S2
OH
OH
OH
2d
2e
2f
249–251
219–221
275 dec.
85
88
82
C17H16N4O2S
C17H16N4O2S
C17H16N4O2S
340
340
340
N
H3CO
H3CO
H3CO
N
N
a catalytic amount of acetic acid. The mixture is then refluxed for
3 h and the obtained solid is filtered and used without further
purification [22].
Compounds 2a–f: IR (KBr) ymax (cmꢀ1): 3335–3199 (NH), 1600–
1435 (C]C and C]N).
6.1.2.1. N-[1-(2-Pyridyl)ethylidene]-N0-[4-(2-thiophenyl)thiazol-2-yl]-
6.1.2. Preparation of N-(1-arylethylidene)-N0-(4-arylthiazol-2-yl)-
hydrazines (2a–f)
hydrazine (2a). 1H NMR (400 MHz, DMSO-d6)
d (ppm): 2.40 (3H, s,
CH3), 7.90–9.40 (8H, m, aromatic protons), 10.90 (1H, s, NH). For
C14H12N4S2 calculated: 55.98% C, 4.03% H,18.65% N; found: 55.99% C,
4.07% H, 18.69% N. MS-FABþ: m/z: 301(100%) [M þ 1].
1-(1-Arylethylidene)thiosemicarbazide (1a–c) (20 mmol) and
1-(2-thiophenyl)-2-bromoethanone or 1-(2-hydroxy-5-methox-
yphenyl)-2-bromoethanone (20 mmol) are stirred in refluxing
isopropyl alcohol (50 ml) for 3 h. The mixture is then allowed to
cool, basified with saturated NaHCO3 water solution, filter and then
wash with cool water, dried, and crystallized from ethanol. Some
characteristics of the synthesized compounds are shown in Table 1.
6.1.2.2. N-[1-(3-Pyridyl)ethylidene]-N0-[4-(2-thiophenyl)thiazol-2-yl]-
hydrazine (2b). 1H NMR (400 MHz, DMSO-d6)
d (ppm): 2.40 (3H, s,
CH3), 7.70–9.30 (8H, m, aromatic protons), 10.50 (1H, s, NH). For
C14H12N4S2 calculated: 55.98% C, 4.03% H, 18.65% N; found: 55.95% C,
4.00% H, 18.66% N. MS-FABþ: m/z: 301(100%) [M þ 1].
Table 2
6.1.2.3. N-[1-(4-Pyridyl)ethylidene]-N0-[4-(2-thiophenyl)thiazol-2-yl]-
Antituberculosis activity and cytotoxicity of the compounds
hydrazine (2c). 1H NMR (400 MHz, DMSO-d6)
d (ppm): 2.30 (3H, s,
Comp.
MABA: H37Rv data
IC50 g/mL) IC90 (mg/mL)
Cell Titer-Glo:
Vero Cell CC50
(mg/mL)
SI (CC50/IC90)
CH3), 7.10–8.90 (8H, m, aromatic protons), 10.60 (1H, s, NH). For
C14H12N4S2 calculated: 55.98% C, 4.03% H, 18.65% N; found: 56.01% C,
4.01% H, 18.61% N. MS-FABþ: m/z: 301(100%) [M þ 1].
(
m
2a
2b
2c
2d
2e
2f
48.39
92.16
>100
6.22
55.23
>100
>100
6.78
–
–
–
>40
–
–
–
6.1.2.4. N-[1-(2-Pyridyl)ethylidene]-N0-[4-(2-hydroxy-5-methoxy-
phenyl)thiazol-2-yl]hydrazine (2d). 1H NMR (400 MHz, DMSO-d6)
>5.89
–
–
>100
>100
>100
>100
d
(ppm): 2.35 (3H, s, CH3), 3.80 (3H, s, OCH3), 6.70–8.60 (8H, m,
–
aromatic protons), 10.80 (1H, s, NH), 11.60 (1H, s, OH). For

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G. Turan-Zitouni et al. / European Journal of Medicinal Chemistry 45 (2010) 2085–2088
C17H16N4O2S calculated: 59.98% C, 4.74% H, 16.46% N; found: 59.95%
Acknowledgements
C, 4.77% H, 16.48% N. MS-FABþ: m/z: 341(100%) [M þ 1].
Authors are thankful to the Tuberculosis Antimicrobial Acqui-
sition and Coordinating Facility (TAACF) in the USA for the in vitro
evaluation of antimycobacterial activity and cytotoxicity.
6.1.2.5. N-[1-(3-Pyridyl)ethylidene]-N0-[4-(2-hydroxy-5-methox-
yphenyl)thiazol-2-yl]hydrazine (2e). 1H NMR (400 MHz, DMSO-d6)
d
(ppm): 2.40 (3H, s, CH3), 3.75 (3H, s, OCH3), 6.80–9.00 (8H, m,
References
aromatic protons), 10.75 (1H, s, NH), 11.50 (1H, s, OH). For
C17H16N4O2S calculated: 59.98% C, 4.74% H, 16.46% N; found: 59.97%
C, 4.78% H, 16.50% N. MS-FABþ: m/z: 341(100%) [M þ 1].
[1] World Health Organization Tuberculosis Programme Available from: http://
www.who.int/tb/en/June (2009).
[2] P.G. Smith, A.R. Moss, Epidemiology of tuberculosis. in: B.R. Bloom (Ed.),
Tuberculosis. ASM Press, Washington D.C., 1994, pp. 47–59.
[3] T.S. Cole, W. Phillip, B. Heym, Curr. Top. Microbiol. 215 (1996) 49–69.
[4] E.C. Rivers, R.L. Mancera, Drug Discov. Today 13 (2008) 1090–1098.
[5] R. Johnson, E.M. Streicher, G.E. Louw, R.M. Warren, P.D. Van Helden, T.C. Victo,
Curr. Issues Mol. Biol. 8 (2006) 97–112.
[6] R.J. O’Brien, P.P. Nunn, Am. J. Respir. Crit. Care Med. 163 (2001) 1055–1058.
[7] C. Crabb, Bull. World Health Organ. 80 (2002) 517.
6.1.2.6. N-[1-(4-Pyridyl)ethylidene]-N0-[4-(2-hydroxy-5-methox-
yphenyl)thiazol-2-yl]hydrazine (2f). 1H NMR (400 MHz, DMSO-d6)
d
(ppm): 2.35 (3H, s, CH3), 3.70 (3H, s, OCH3), 6.70–8.80 (8H, m,
aromatic protons), 10.60 (1H, s, NH), 11.90 (1H, s, OH). For
C17H16N4O2S calculated: 59.98% C, 4.74% H, 16.46% N; found: 60.01%
C, 4.74% H, 16.42% N. MS-FABþ: m/z: 341(100%) [M þ 1].
[8] R. Maccari, R. Ottana`, M.G. Vigorita, Bioorg. Med.Chem. Lett.15 (2005)2509–2513.
[9] D. Sriram, P. Yogeeswari, K. Madhu,Bioorg. Med. Chem. Lett.15 (2005) 4502–4505.
[10] N. Sinha, S. Jain, A. Tilekar, R.S. Upadhayaya, N. Kishore, G.H. Jana, S.K. Arora,
Bioorg. Med. Chem. Lett. 15 (2005) 1573–1576.
6.2. Pharmacological evaluation
[11] M.J. Hearn, M.H. Cynamon, J. Antimicrob, Chemotherapy 53 (2004) 185–191.
[12] M.J. Hearn, M.H. Cynamon, M.F. Chen, R. Coppins, J. Davis, H. Joo-On Kang,
A. Noble, B. Tu-Sekine, M.S. Terrot, D. Trombino, M. Thai, E.R. Webster,
R. Wilson, Eur. J. Med. Chem. 44 (2009) 4169–4178.
[13] J. Selkon, S. Devadatta, K. Kulkarni, Bull. WorldHealth Organ. 31 (1964) 273–294.
[14] M. Payton, R. Auty, R. Delgoda, J. Bacteriol. 181 (1999) 1343–1347.
6.2.1. Primary screen (dose response)
6.2.1.1. Determination of a 90% inhibitory concentration (IC90). The
initial screen is conducted against M. tuberculosis H37Rv (ATCC
27294) in BACTEC 12B medium using the Microplate Alamar Blue
Assay (MABA) [23]. Compounds are tested in ten 2-fold dilutions,
´
[15] G. Navarrete-Vazquez, G.M. Molina-Salinas, Z.V. Duarte-Fajardo, J. Vargas-
Villarreal, S. Estrada-Soto, F. Gonza´lez-Salazar, Herna´ndez-Nu´ n˜ez, S. Said-
Ferna´ndez, Bioorg. Med. Chem. 15 (2007) 5502–5508.
typically from 100 mg/mL to 0.19 mg/mL. The IC90 is defined as the
concentration effecting a reduction in fluorescence of 90% relative
to controls. This value is determined from the dose–response curve
[16] J.L. Santos, P.R. Yamasaki, C.M. Chin, C.H. Takashi, F.R. Pavan, C.Q.F. Leite, Bio-
org. Med. Chem. 17 (2009) 3795–3799.
ꢀ
ˇ
´
ˇ
´
ˇ
´
[17] A. Imramovsky, S. Polanc, J. Vinsova, M. Kocevar, J. Jampılek, Z. Reckova,
J. Kaustova´, Bioorg. Med. Chem. 15 (2007) 2551–2559.
using a curve-fitting program. Any IC90 value of ꢃ10
mg/mL is
considered ‘‘Active’’ for antitubercular activity. The ‘‘Active’’
compounds are considered for ‘‘Secondary Screening’’.
[18] M. Shiradkar, G.V.S. Kumar, V. Dasari, S. Tatikonda, K.C. Akula, R. Shah, Eur. J.
Med. Chem. 42 (2007) 807–816.
¨
[19] G. Turan-Zitouni, A. Ozdemir, Z.A. Kaplancikli, K. Benkli, P. Chevallet, G. Akalin,
Eur. J. Med. Chem. 43 (2008) 981–985.
6.2.2. Secondary screen
[20] N. Karali, N. Terzioglu, A. Gursoy, Arzneim. Forsch. 48 (1998) 758–763.
[21] M.R. Shiradkar, K.K. Murahari, H.R. Gangadasu, T. Suresh, C.A. Kalyan,
D. Panchal, R. Kaur, P. Burange, J. Ghogare, V. Mokale, M. Raut, Bioorg. Med.
Chem. 15 (2007) 3997–4008.
[22] D.L. Klayman, J.F. Bartosevich, T.S. Griffin, C.J. Mason, J.P. Scovill, J. Med. Chem.
29 (1979) 855–862.
6.2.2.1. Determination of mammalian cell cytotoxicity (CC50). The
VERO cell cytotoxicity assay [24] is done in parallel with the TB
Dose Response assay. After 72 h exposure, viability is assessed
using Promega’s Cell Titer-Glo Luminescent Cell Viability Assay
[25], a homogeneous method of determining the number of viable
cells in culture based on quantitation of the ATP present. Cytotox-
icity is determined from the dose–response curve as the CC50 using
a curve-fitting program. Ultimately, the CC50 is divided by the IC90
to calculate an SI (Selectivity Index) value. SI values of ꢁ10 are
considered for further testing.
[23] L.A. Collins, S.G. Franzblau, Antimicrob. Agents Chemother. 41 (1997)
1004–1009.
´
[24] V.J. Lara-Dı´az, Angel A. Gayta´n-Ramos, A.J. Da´valos-Balderas, J. Santos-Guz-
ma´n, B.D. Mata-Ca´rdenas, J. Vargas-Villarreal, A. Barbosa-Quintana, M. Sanson,
A.G. Lo´pez-Reyes, J.E. Moreno-Cuevas, Basic Clin. Pharmacol. Toxicol. 104
(2009) 81–86.
[25] CellTiter-GloÒ Luminescent Cell Viability Assay. Promega Corporation’s, June
2009, Available from: http://www.promega.com/tbs/tb288/tb288.html.