Editor-in-Chief Hatice Kübra Elçioğlu Vice Editors Levent Kabasakal Esra Tatar Online ISSN 2630-6344 Publisher Marmara University Frequency Bimonthly (Six issues / year) Abbreviation J.Res.Pharm. Former Name Marmara Pharmaceutical Journal
Journal of Research in Pharmacy 2010 , Vol 14 , Num 2
Synthesis of some novel hydrazone derivatives and evaluation of their antituberculosis activity
Ahmet Özdemir1, Zafer Asım Kaplancıklı1, Gülhan Turan-Zitouni1, Gilbert Revial2
1Anadolu Üniversitesi Eczacılık Fakültesi, Farmasötik Kimya, Eskişehir, Türkiye
2UMR-CNRS 7084, Cnam, 2 rue Conte', 75003 , Laboratoire de Transformations Chimiques et Pharmaceutiques, Paris, France
DOI : 10.12991/201014453

Summary

Heterosiklik hidrazon yapısı antitüberküloz aktiviteleri nedeniyle medisinal kimyacıların dikkatini çeken, cazip biyolojik olarak aktif önemli bir ilaç sınıfıdır. Bu amaçla, yeni hidrazon türevleri sentezlendi ve antitüberküloz etkinlikleri değerlendirilmiştir. (5,6,7,8-Tetrahidronaftalen-1-il) asetik asit hidraziti ile çeşitli benzaldehitlerin reaksiyonu, 5,6,7,8-tetrahidronaftalen asetik asit benziliden hidrazit türevlerini verdi. Bileşiklerin kimyasal yapıları 1H-NMR, EI-MS spectral verileri ve elemental analiz metodları ile aydınlatıldı. BACTEC 460 radyometrik sistem ve BACTEC 12B ortamından yararlanılarak Mycobacterium tuberculosis H37Rv (ATCC 27294)'e karşı bileşiklerin antitüberküloz aktiviteleri değerlendirilmiştir. Bileşik A10 yüksek antitüberküloz etkinlik (IC50: 3.072 μg/mL ve IC90: 3.358 μg/mL) ve düşük sitotoksisite (CC50: >40 μg/mL) gösterdi.

Introduction

In spite of a 5000 year history, tuberculosis (TB) remains the leading single-agent infectious disease killer in the world. Approximately one third of the world's population is infected with TB bacilli, and each year almost 8 million people develop active TB and 2 million die as a result of TB. The major challenges for tuberculosis control are the development of multidrug-resistant tuberculosis (MDRTB) strains and the increasing numbers of immunocompromised individuals with HIV infections who are highly susceptible to the disease. As a result, there is a pressing need for new antitubercular agents acting with greater potency and efficacy than the current existing drugs[1].

To pursue this goal, our research efforts are directed to find new chemical classes of antimycobacterially active agents. The methods of investigation of structure-activity relationships (SARs) enabled us to find some new pharmacophores of the above-mentioned activity. Many studies were carried out on heterocyclic systems bearing a hydrazone structure as a pharmacophore[2-13]. In this study, we planned to synthesize new molecules bearing hydr azone moieties for their potential antituberculosis activity.

Chemistry
The synthetic route of the compounds is outlined in Scheme 1. For the synthesis of the title compounds, 5,6,7,8-tetrahydronaphthalene acetic acid hydrazide required as starting material was prepared by the reaction of 5,6,7,8-tetrahydronaphthalene acetic acid ethyl ester with hydrazine hydrate[14]. The reaction of equimolar quantities of hydrazide with appropriate benzaldehydes in the presence of isopropyl alcohol resulted in the formation of the title compounds (A1-15) (Table 1).


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SCHEME 1: Synthetic protocol of the title compounds

TABLE 1: Some characteristics of the compound

Pharmacology
Antituberculosis activity and Cytotoxicity
The initial screen is conducted against Mycobacterium tuberculosis H37Rv (ATCC 27294) in BACTEC 12B medium using the Microplate Alamar Blue Assay (MABA)[15]. One of the compounds showed significant antituberculosis activity as can be inferred from Table 2.

TABLE 2: Antituberculosis activity and cytotoxicity of the compounds

The VERO cell cytotoxicity assay[16] is done in parallel with the TB Dose Response assay. Viability is assessed using Promega's Cell Titer-Glo Luminescent Cell Viability Assay[17].

EXPERIMENTAL
Chemistry
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 recorded on the following instruments: 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; EI-MS, VG Quattro mass spectrometer.

Preparation of 5,6,7,8-tetrahydronaphthalene acetic acid hydrazide
In a flask equipped with a reflux condenser, a mixture of 5,6,7,8-tetrahydronaphthalene acetic acid ethyl ester (100 mmol) and the hydrazine hydrate (100 mmol) is reacted in ethanol (200 mL). The mixture is then refluxed for 1 h and the obtained solid is filtered and used without further purification[14].

Preparation of 5,6,7,8-tetrahydronaphtalen acetic acid benzylidene hydrazide A1-15
The reaction of equimolar quantities of hydrazide (5 mmol) with appropriate benzaldehyde (5 mmol) in the presence of isopropyl alcohol resulted in the formation of the title compounds. Some characteristics of the synthesized compounds are shown in Table 1.

A1: 1H-NMR: δ 1.60-1.80 (4H, m), 2.60-2.75 (4H, m), 3.74 (2H, s), 6.90-7.10 (3H, m), 7.49 (2H, d, J= 8.5 Hz), 7.70 (2H, dd, J= 8.5, 2.1 Hz), 8.12 (1H, s), 11.51 (1H, s). EIMS (m/z): 326 (M+, 9.5 %), 291 (1), 188 (27), 181 (8), 171 (25), 154 (17), 145 (100), 140 (58), 129 (31). For C19H19ClN2O calculated: 69.83 % C, 5.86 % H, 8.57 % N; found: 69.85 % C, 5.89 % H, 8.56 % N.

A2: 1H-NMR: δ 1.65-1.80 (4H, m), 2.33 (3H, s), 2.65-2.75 (4H, m), 3.71 (2H, s), 6.90-7.10 (3H, m), 7.24 (2H, d, J= 8.0 Hz), 7.57 (2H, dd, J= 8.0, 2.0 Hz), 8.08 (1H, s), 11.37 (1H, s). EIMS (m/z): 306 (M+, 31 %), 288 (9), 262 (1), 248 (1), 188 (30), 171 (25), 161 (31), 145 (100), 134 (37), 120 (40). For C20H22N2O calculated: 78.40 % C, 7.24 % H, 9.14 % N; found: 78.44 % C, 7.25 % H, 9.15 % N.

A3: 1H-NMR: δ 1.65-1.80 (4H, m), 2.65-2.75 (4H, m), 3.72 (2H, s), 3.80 (3H, s), 6.90-7.05 (5H, m), 7.62 (2H, d, J= 6.0 Hz), 8.06 (1H, s), 11.30 (1H, s). EIMS (m/z): 322 (M+, 38 %), 188 (21), 177 (22), 171 (15), 150 (56), 145 (100), 135 (26), 131 (26). For C20H- 22N2O2 calculated: 74.51 % C, 6.88 % H, 8.69 % N; found: 74.52 % C, 6.90 % H, 8.71 % N.

A4: 1H-NMR: δ 1.65-1.80 (4H, m), 2.60-2.75 (4H, m), 3.79 (2H, s), 6.90-7.07 (3H, m), 7.94 (2H, d, J= 8.8 Hz), 8.27 (2H, d, J= 8.8 Hz), 8.23 (1H, s), 11.73 (1H, s). EIMS (m/z): 337 (M+, 2 %), 320 (1), 307 (11), 291 (1), 188 (12), 186 (20), 171 (12), 159 (23), 151 (64), 145 (100), 129 (26). For C19H19N3O3 calculated: 67.64 % C, 5.68 % H, 12.45 % N; found: 67.64 % C, 5.69 % H, 12.47 % N.

A5: 1H-NMR: δ 1.65-1.75 (4H, m), 2.60-2.75 (4H, m), 3.77 (2H, s), 6.90-7.05 (3H, m), 7.82-7.90 (4H, m), 8.17 (1H, s), 11.77 (1H, s). EIMS (m/z): 317 (M+, 3 %), 188 (13), 187 (18), 186 (14), 171 (12), 159 (18), 145 (100), 131 (78). For C20H19N3O calculated: 75.69 % C, 6.03 % H, 13.24 % N; found: 75.71 % C, 6.03 % H, 13.27 % N.

A6: 1H-NMR: δ 1.21 (6H, d, J= 6.9 Hz), 1.65-1.80 (4H, m), 2.60- 2.75 (4H, m), 2.91 (1H, dt, J= 6.9 Hz), 3.73 (2H, s), 6.90-7.10 (3H, m), 7.31 (2H, d, J= 8.2 Hz), 7.60 (2H, dd, J= 8.2, 3.8 Hz), 8.09 (1H, s), 11.42 (1H, s). EIMS (m/z): 334 (M+, 28 %), 316 (1), 290 (1), 276 (1), 189 (38), 188 (34), 171 (22), 161 (31), 148 (39), 147 (27), 145 (100), 131 (41). For C22H26N2O calculated: 79.01 % C, 7.84 % H, 8.38 % N; found: 79.05 % C, 7.87 % H, 8.40 % N.

A7: 1H-NMR: δ 1.65-1.80 (4H, m), 2.60-2.75 (4H, m), 3.72 (2H, s), 5.15 (2H, s), 6.90-7.10 (5H, m), 7.30-7.50 (5H, m), 7.62 (2H, d, J= 8.8 Hz), 8.09 (1H, s), 11.35 (1H, s). EIMS (m/z): 398 (M+, 15 %), 307 (1), 253 (16), 225 (9), 212 (3), 188 (9), 171 (5), 145 (45), 91 (100). For C26H26N2O2 calculated: 78.36 % C, 6.58 % H, 7.03 % N; found: 78.38 % C, 6.59 % H, 7.00 % N.

A8: 1H-NMR: δ 1.60-1.75 (4H, m), 2.60-2.75 (4H, m), 3.75 (2H, s), 6.90-7.10 (3H, m), 7.48 (1H, d, J= 8.5 Hz), 7.68 (1H, s), 7.90- 7.97 (1H, m), 8.44 (1H, s), 11.74 (1H, s). EIMS (m/z): 360 (M+, 5 %), 325 (1), 215 (5), 189 (12), 188 (33), 187 (14), 176 (42), 174 (67), 171 (26), 159 (9), 145 (100), 129 (29). For C19H18Cl2N2O calculated: 63.17 % C, 5.02 % H, 7.75 % N; found: 63.19 % C, 5.04 % H, 7.71 % N.

A9: 1H-NMR: δ 1.65-1.80 (4H, m), 2.65-2.80 (4H, m), 3.72 (2H, s), 3.81 (3H, s), 6.82 (1H, d, J= 8.1 Hz), 6.90-7.07 (4H, m), 7.26 (1H, s), 7.99 (1H, s), 9.49 (1H, s), 11.30 (1H, s). EIMS (m/z): 338 (M+, 49 %), 320 (12), 294 (1), 193 (36), 188 (25), 171 (17), 166 (47), 165 (40), 145 (100), 129 (28). For C20H22N2O3 calculated: 70.99 % C, 6.55 % H, 8.28 % N; found: 70.96 % C, 6.59 % H, 8.31 % N.

A10: 1H-NMR: δ 1.65-1.80 (4H, m), 2.60-2.75 (4H, m), 3.74(2H, s), 6.90-7.05 (3H, m), 7.19 (1H, dd, J= 8.7, 2.1 Hz), 7.88 (1H, dt, J= 8.7, 2.1 Hz), 7.97 (1H, s), 8.13-8.20 (2H, m), 11.51 (1H, s). EIMS (m/z): 353 (M+, 6 %), 335 (1), 188 (26), 171 (18), 167 (72), 159 (10), 145 (100), 129 (25). For C19H19N3O4 calculated: 64.58 % C, 5.42 % H, 11.89 % N; found: 64.61 % C, 5.45 % H, 11.92 % N.

A11: 1H-NMR: δ 1.65-1.80 (4H, m), 2.60-2.75 (4H, m), 3.72 (2H, s), 6.08 (2H, s), 6.90-7.05 (4H, m), 7.12 (1H, dd, J= 8.2, 1.6 Hz), 7.25 (1H, d, J= 1.6 Hz), 8.02 (1H, s), 11.37 (1H, s). EIMS (m/z): 336 (M+, 51 %), 318 (1), 292 (1), 278 (1), 191 (26), 188 (31), 171 (14), 164 (52), 163 (42), 149 (39), 145 (100), 129 (27). For C20H- 20N2O3 calculated: 71.41 % C, 5.99 % H, 8.33 % N; found: 71.44 % C, 5.95 % H, 8.37 % N.

A12: 1H-NMR: δ 1.65-1.80 (4H, m), 2.60-2.75 (4H, m), 3.74 (2H, s), 3.78 (6H, s), 6.55 (1H, s), 6.93 (2H, s), 6.90-7.05 (3H, m), 8.05 (1H, s), 11.46 (1H, s). EIMS (m/z): 352 (M+, 43 %), 334 (1), 308 (1), 294 (2), 188 (29), 179 (29), 171 (16), 165 (35), 145 (100), 129 (29). For C21H24N2O3 calculated: 71.57 % C, 6.86 % H, 7.95 % N; found: 71.60 % C, 6.89 % H, 7.99 % N.

A13: 1H-NMR: δ 1.65-1.80 (4H, m), 2.65-2.77 (4H, m), 3.70 (3H, s), 3.75 (2H, s), 3.82 (6H, s), 6.90-7.05 (5H, m), 8.06 (1H, s), 11.43 (1H, s). EIMS (m/z): 382 (M+, 84 %), 364 (1), 352 (11), 338 (10), 324 (19), 237 (41), 210 (30), 209 (33), 195 (87), 193 (100), 178 (30), 163 (10), 145 (74), 129 (24). For C22H26N2O4 calculated: 69.09 % C, 6.85 % H, 7.32 % N; found: 69.12 % C, 6.88 % H, 7.35 % N.

A14: 1H-NMR: δ 1.65-1.80 (4H, m), 2.60-2.75 (4H, m), 3.74 (2H, s), 6.90-7.10 (3H, m), 7.60-7.70 (1H, m), 7.75-7.83 (1H, m), 8.00- 8.10 (2H, m), 8.51 (1H, s), 11.77 (1H, s). EIMS (m/z): 337 (M+, 1 %), 320 (10), 303 (2), 246 (1), 201 (10), 188 (10), 186 (12), 171 (20), 151 (35), 145 (100), 130 (30). For C19H19N3O3 calculated: 67.64 % C, 5.68 % H, 12.45 % N; found: 67.66 % C, 5.71 % H, 12.48 % N.

A15: 1H-NMR: δ 1.65-1.80 (4H, m), 2.62-2.75 (4H, m), 3.78 (2H, s), 6.90-7.10 (3H, m), 7.68-7.75 (1H, m), 8.08-8.15 (2H, m), 8.20-8.25 (1H, m), 8.49 (1H, s), 11.70 (1H, s). EIMS (m/z): 337 (M+, 2 %), 307 (1), 279 (1), 188 (12), 187 (31), 186 (20), 171 (10), 159 (22), 151 (46), 145 (100), 129 (25). For C19H19N3O3 calculated: 67.64 % C, 5.68 % H, 12.45 % N; found: 67.69 % C, 5.70 % H, 12.43 % N.

Pharmacological evaluation
Primary Screen (Dose Response)
(Determination of a 90% Inhibitory Concentration (IC90))
The initial screen is conducted against Mycobacterium tuberculosis H37Rv (ATCC 27294) in BACTEC 12B medium using the Microplate Alamar Blue Assay (MABA)[14]. Compounds are tested in ten 2-fold dilutions, typically from 100 μg/mL to 0.19 μg/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 using a curve-fitting program. Any IC90 value of ≤10μg/mL is considered “Active” for antitubercular activity. The “Active” compounds are considered for “Secondary Screening”.

Secondary Screen
Determination of Mammalian Cell Cytotoxicity (CC50)
The VERO cell cytoto xicity assay[15] is done in parallel with the TB Dose Response assay. After 72 hours exposure, viability is assessed using Promega’s Cell Titer-Glo Luminescent Cell Viability Assay[16], a homogeneous method of determining the number of viable cells in culture based on quantitation of the ATP present. Cytotoxicity 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.

Results

The structures of compounds A1-15 were confirmed by elemental analyses, MS-FAB and 1H- NMR spectral data. All compounds gave satisfactory elemental analysis. The mass spectra (MS (FAB)) of the compounds showed M+ peaks, in agreement with their molecular formula.

In the 400 MHz 1H-NMR spectra of the compounds, the C6 and C7 protons of 5,6,7,8-tetrahydronaphthalene were observed at 1.60-1.80 ppm. The C5 and C8 protons of 5,6,7,8-tetrahydronaphthalene were observed at 2.60-2.80 ppm. The CH2CO protons appeared as singlet at 3.70-3.79 ppm. The N=CH and NH protons were observed at 7.90-8.23 ppm and 11.30-11.77 ppm respectively. All the other aliphatic and aromatic protons were observed at expected regions.

The results of antituberculosis and cytotoxicity screening of newly prepared compounds A1-15 are expressed in Table 2. The very important result was observed at antituberculosis activity screening for one of the compounds. The compound A10 showed high antituberculosis activity (IC50: 3.072 μg/mL and IC90: 3.358 μg/mL) and low cytotoxicity (CC50: >40 μg/mL). Because of SI value of the compound A10 ≥ 10, further tests are in progress.

ACKNOWLEDGEMENTS
Authors are thankful to the Tuberculosis Antimicrobial Acquisition and Coordinating Facility (TAACF) in the USA for the in vitro evaluation of antimycobacterial activity and cytotoxicity.

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