Design and synthesis of novel carbazolo-thiazoles as potential anti-mycobacterial agents using a molecular hybridization approach

Article abstract of DOI:10.1039/c4ra11752b

Various substituted carbazolo-thiazoles (compounds 6a-6o) were synthesized in good yields using a molecular hybridization approach. The synthesized compounds were evaluated for their in vitro anti-tubercular activity against Mycobacterium tuberculosis H₃₇Rv strain at the National Institute of Allergy and Infectious Diseases (Bethesda, MD, USA). Among the tested series, compound 6c (minimum inhibitory concentration 21 μM) showed the most promising anti-mycobacterial activity. Brief structure-activity relationship studies showed that the electron-donating groups (OCH₃ and OH), particularly on the phenyl ring of the thiazole motif, had a positive correlation with the anti-mycobacterial activity. In addition, they displayed low cytotoxicity against a mammalian Vero cell line using the MTT assay, thereby having a high therapeutic index. This study shows the importance of molecular hybridization and the scope for the development of carbazole-thiazole compounds as potential anti-mycobacterial agents.

Full text of DOI:10.1039/c4ra11752b

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KARPOORMATH, M. Shaikh, M. B. PALKAR, H. Patel, R. rane, W. alwan, M. M. Shaikh, I. M. Shaikh and G.
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Page 1 of 31
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DOI: 10.1039/C4RA11752B
Design and Synthesis of Novel Carbazoloꢀ
Thiazoles by Molecular Hybridization Approach
as Potential Antiꢀmycobacterial Agents
Mahamadhanif S. Shaikh, Mahesh B. Palkar, Harun M. Patel, Rajesh A. Rane, Wesam S. Alwan,
Mahidansha M. Shaikh, Iqbal M. Shaikh, Girish A. Hampannavar, Rajshekhar Karpoormath*
Department of Pharmaceutical Chemistry, Discipline of Pharmaceutical Sciences, College of Health
Sciences, University of KwaZulu-Natal, Westville Campus, Durban – 4000, South Africa.
*Corresponding author
Eꢀmail: karpoormath@ukzn.ac.za, rvk2006@gmail.com
Tel no.: +27(0)312607179, +27721107207; Fax No.:+27(0)312607792
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Abstract
Various substituted carbazoloꢀthiazoles 6 (aꢀo) have been synthesized in good yields by
molecular hybridization approach. These synthesized compounds were evaluated for their in
vitro antiꢀtubercular activity against Mycobacterium tuberculosis H37Rv strain at National
Institute of Allergy and Infectious Diseases (NIAID), Bethesda, MD, USA. Among the tested
series, compound 6c (MIC = 21 µM) exhibited the most promising antiꢀmycobacterial activity. A
brief structure activity relationship (SAR) studies revealed that electron donating groups (OCH3
and OH) especially on the phenyl ring of thiazole motif exhibited positive correlation with antiꢀ
mycobacterial activity. In addition, they displayed low cytotoxicity against a mammalian Vero
cell line using MTT assay, thereby providing a high therapeutic index. This study reveals the
significance of molecular hybridization and the scope for the development of carbazole clubbed
thiazole compounds as potential antiꢀmycobacterial agents.
Keywords: Carbazole; Thiazole; Antiꢀmycobacterialactivity; Cytotoxicity; NIAID.
2

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1 Introduction
Tuberculosis is the leading cause of mortality from a single infectious agent and is responsible
for more than three million deaths worldwide every year.1 Despite half a century of antiꢀ
tubercular chemotherapy, there are still 8ꢀ10 million new cases of active tuberculosis each year
and nearly two billion individuals are believed to harbor latent tuberculosis.In coincidence with
the spread of HIV infection,2 tuberculosis is today amongst the universal health threats. India,
China, the Russian Federation and South Africa have almost 60% of the world’s cases of MDRꢀ
TB.3 It is estimated that nearly 5, 00,000 people die from the disease more than 1000 per day.4
The combination of long treatment duration (6–9 months), increased incidence of (multi or
extensive) drug resistance, coꢀmorbidity with HIVꢀAIDS and lack of investment in antiꢀinfective
drug discovery has led to a situation, where the discovery, development and introduction of new
treatments for tuberculosis has become critical.5 Therefore the presentꢀday situation necessitates
the reꢀengineering and repositioning of old drug families for developing new antiꢀmycobacterial
to achieve effective TB control even against the resistant forms of TB.6ꢀ8
Natural products or their direct derivatives, play crucial role in modern day chemotherapy of
tuberculosis.9 The promising natural antiꢀmycobacterials include carbazole compounds (I to III)
and carbazole alkaloids, such as Clausine (IV) and Micromeline (V), isolated independently
from several sources10 (Fig. 1). Synthetic analogues of natural carbazole alkaloid exhibited
significantly improved in vitro as well as in vivo antiꢀmycobacterial activity against M.
tuberculosisH37Rv.11ꢀ15 On the other hand, thiazole conjugated with a wide range of heterocyclic
moieties (VII to IX) were reported to exhibit potent antiꢀmycobacterial activity16, 17(Fig. 1). In
view of these facts and as a part of our enduring studies in the area of antiꢀmycobacterial
agents18ꢀ21 it was envisaged to synthesize biologically active hybrid pharmacophores (carbazole
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and thiazole) by integrating them in one molecular platform for biological evaluation. This
approach was an attempt to investigate the possible synergistic influence of such structural
hybridizations on the anticipated activity, hoping to discover a new lead molecule. In the present
study, we report the synthesis and in vitro antiꢀmycobacterial activity of some novel carbazoloꢀ
thiazoles 6(aꢀo) hybrid molecules against M. tuberculosis H37Rv and their in vitro cytotoxicity
profile.
2 Chemistry
The synthesis of a novel series of thiazolyl substituted carbazole hydrazine analogues 6 (aꢀo) was
achieved through an efficient and versatile synthetic route as illustrated in Scheme 1. It is quite
clear that unique final steps were involved in the synthesis of target compounds, which have
structural variations on the hydrazine bond attached to 3rd position of carbazole ring. The various
starting material i.e. 9ꢀmethylꢀcarbazole (1) was prepared from commercially available carbazole
by Nꢀmethylation process using iodoꢀmethane and sodium hydride. Further, compound (1) was
subjected to formylation reaction (Vilsmayer Hack) in the presence of POCl3 and DMF to afford
9ꢀmethylꢀcarbazoleꢀ3ꢀcarbaldehyde (2),which was further treated with thiosemicarbazide in the
presence of methanol and catalytic glacial acetic acid to yield 1ꢀ((9ꢀmethylꢀcarbazolꢀ6ꢀyl
methylene)thiosemicarbazide (3). In addition, the desired title compounds 6 (aꢀo) were
synthesized by Hantzsch cycloꢀcondensation reaction of compound (3) with various
appropriately substituted αꢀBromoaromatic/heteroaromatic ketones 5 (aꢀo) in the presence of
methanol.22 Structures of key intermediate compound (3) and its corresponding thiazolyl
substituted carbazole hydrazine derivatives 6 (aꢀo) were established on the basis of their
physicochemical and spectral (IR, 1HꢀNMR, 13CꢀNMR and HRMS) data.
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3 Results and Discussion
3.1 Spectral Studies
All the newly synthesized compounds exhibited acceptable analysis of their anticipated
structures, which are summarized in experimental section. In general, the IR spectrum of
compound (3) revealed typical absorption bands around 3373.23 cmꢀ1 for NꢀH (of NH2 group),
1
1619.92 cmꢀ1 for C=N and1145.80cmꢀ1 for C=S groups. This was further substantiated from H
NMR spectrum of compound (3), which exhibited the presence of singlet signals at around
δ11.38, 8.58, 8.21 and 3.89 ppm for the NꢀH proton of CH= N protons, 4th proton of carbazole
ring and NꢀCH3 protons indicating its formation by a simple carbonꢀ nitrogen bond formation
process in the presence of thiosemicarbazide and acetic acid as catalyst.22 The IR spectrum of the
compounds 6 (aꢀo) showed disappearance of the typical peaks due to NH2 group (NꢀH Str) and
appearance of moderately strong bands around 3319ꢀ3074 cmꢀ1 and 1629.47ꢀ1572.25 cmꢀ1,
which are characteristic of the NꢀH and C=N groups respectively. In the case of compounds 6i
and 6j, the peaks appeared around 3454.28ꢀ3342.53 cmꢀ1due to OH group, while the prominent
bands around 1721.35ꢀ1711.27 cmꢀ1 was attributed to C=O in compounds 6n and 6o.1HꢀNMR
spectrum (400 MHz) of these title compounds 6 (aꢀo) recorded in DMSOꢀd6 displayed some
distinctive singlet signals at around δ 12.59ꢀ8.38 ppm for NꢀH proton, δ 8.45ꢀ8.19 ppm for
CH=N proton at 3rd position of the carbazole and δ 8.36ꢀ8.20 ppm for a aromatic proton at 4th
position of the carbazole nucleus. Further, the most informative singlet signal resonated between
δ 7.92ꢀ7.20 ppm, which was attributed to the aromatic proton (Hꢀ5) of the thiazole ring
indicating its formation through cycloꢀcondensation process. This observation was consistent
with previously reported similar compounds.22 The unique singlet signal resonating around δ
3.91ꢀ3.87 ppm indicated the presence of methyl protons (NꢀCH3) on the 9th position of the
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carbazole ring. Various aromatic/heteroꢀaromatic proton signals were observed between δ 8.31ꢀ
13
6.92 ppm. These findings were further substantiated from their respective CꢀNMR spectra of
the title compounds. The characteristic 13CꢀNMR signals observed at around δ 148.72ꢀ141.12
and 110.21ꢀ101.48 ppm were assigned to carbons of Schiff base (CH=N) and Cꢀ5 of thiazole
ring. A prominent carbon signal observed around δ 29.65ꢀ28.62 ppm, indicated the presence of
NꢀCH3 carbon in the title compounds. In case of compounds 6n and 6o, a characteristic signal
appeared around δ 167.86ꢀ158.75 ppm due to the presence of carbonyl carbon (C=O) of
coumarin ring, while various aromatic/heteroꢀaromatic carbons resonated around δ 134.76ꢀ
111.08 ppm.
3.2 Antimycobacterial Activity
In-vitro evaluation of the antiꢀmycobacterial activity of newly synthesized title compounds [(3,
and 6 (aꢀo) was carried out at Infectious Disease Research Institute (IDRI) within the National
Institute of Allergy and Infectious Diseases (NIAID) screening program, Bethesda, MD, USA.
Minimum Inhibitory Concentration (MIC) was determined against M. tuberculosis strain H37Rv
grown under aerobic conditions by using a dual readꢀout (OD590 and fluorescence) assay
procedure.23ꢀ25 The assay was based on measurement of growth in liquid medium of a fluorescent
reporter strain of H37Rv, where the readout was either optical density (OD) or fluorescence. The
use of two readouts minimizes problems caused by compound precipitation or auto fluorescence.
The purpose of the screening program was to provide a resource whereby new experimental
compounds could be tested for their capacity to inhibit the growth of virulent M. tuberculosis.
The result of antiꢀmycobacterial activity is presented in Table 1. All the synthesized compounds
exhibited an interesting activity profile with MIC ranging from 21 to 200 µM against the tested
mycobacterial strain. We also studied the effects of various aromatic/heteroꢀaromatic substituents
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at 4th position of the thiazole ring, which was in turn attached to 3rd position of 9ꢀmethyl
carbazole through a hydrazine linkage. Amongst them compound 6c (MIC=21 µM) with 3,4ꢀ
dimethoxyl (OCH3) groups on aromatic nucleus exhibited most promising antiꢀmycobacterial
activity with IC50 value of 28 µM. In the case of compound 6b (MIC=32 µM), with an
unsubstituted aromatic ring exhibited good inhibitory activity of IC50 value of 35µM. The
activity was considerably affected by substituents at positionꢀ4 on the thiazole nucleus. In
general, the electron donating groups on aromatic ring have greatly contributed for antiꢀ
mycobacterial activity while the electron withdrawing substituents have caused decrease in
activity. It was observed that compounds 6i (MIC = 31 µM) and 6j (MIC = 37 µM) with
hydroxyl (OH) group at 2nd and 4th position of aromatic nucleus displayed notable activity with
IC50 values 39 and 41 µM respectively. Compounds 6a, 6d, 6e, 6f, 6h, 6k and 6l having either
nitro or halogen groups on aromatic ring found to be least active with MIC value >200 µM.
Replacing substituted phenyl group at 4th position of thiazole ring with heterocyclic groups such
as thiophene (6m) and coumarin (6n and 6o) resulted in no significant change in antiꢀ
mycobacterial activity with MIC value of >200 µM. In three cases we have replaced the aromatic
ring with thiophene (6m) and coumarin (6n and 6o) ring systems, which resulted in activity with
a MIC value of >200 µM.
3.3 Cytotoxic Activity
The synthesized title compounds [(3, and 6 (aꢀo)] were further screened for cytotoxicity (IC50) in
a mammalian Vero cell line. After 72 h of exposure, viability of cell was assessed on the basis of
cellular conversion of MTT into a formazan product using the Promega Cell Titer 96 nonꢀ
radioactive cell proliferation assay and results are summarized in Table 1. The sixteen tested
derivatives showed IC50 values ranging from 202.4 to 440.8 µM. All the synthesized compounds
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displayed no significant activity against mammalian Vero cell line at concentrations <100 µM.
Among the series tested, compounds 6a, 6b, 6e, 6h, 6i, 6n and 6o demonstrated lower toxicity
with IC50 values of >300 µM. These results are important, since compounds with increased
cytoliability are much attractive in the development of new chemical entities for the treatment of
TB. This is primarily due to the fact that the treatment of TB requires a lengthy course of drug
regimen leading to several side effects. Thus, the need for an agent with a high margin of safety
becomes a primary concern.
4 Conclusion
In conclusion, herein we report the synthesis, spectral studies and preliminary in-vitro antiꢀ
mycobacterial activity of a novel series of thiazolyl substituted carbazolehydrazine analogues
6(aꢀo) by molecular hybridization approach. Structure activity relationship study reveal that the
electron donating groups (OCH3 and OH) on the phenyl ring of thiazole moiety resulted in
excellent antiꢀmycobacterial activity, while the electron withdrawing substituents decreased the
activity. Among the synthesized compounds, compound (6c, MIC = 21µM) emerged as a
promising compound endowed with good antiꢀmycobacterial activity. Further, the title
compounds were also assessed for their cytotoxic activity (IC50) against mammalian Vero cell
line using MTT assay. The results indicated that these compounds exhibit antiꢀtubercular activity
at nonꢀcytotoxic concentrations. These results indicate the importance of molecular hybridization
and the development of carbazole clubbed thiazole based lead candidates as potential antiꢀ
mycobacterial agents. The antiꢀmycobacterial activity can further be enhanced by slight
modifications in the ring substituents and/or extensive additional functionalization, which
warrants further investigation. We believe that the observed outcome should contribute to the
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global efforts in the discovery of new lead compounds with improved antiꢀmycobacterial
activity.
5 Experimental
5.1 Chemistry Protocol
All research chemicals were purchased from Sigma–Aldrich and Merck Millipore, South Africa.
Commercially available chemicals 4ꢀbromo phenacyl bromide (5a) and phenacyl bromide (5b)
were procured from Sigma–Aldrich (South Africa). Solvents except laboratory reagent grade
were dried and purified according to the literature when necessary. The progress of reactions and
purity of compounds were monitored by thinꢀlayer chromatography (TLC) on preꢀcoated silica
gel plates procured from E. Merck and Co. (Darmstadt, Germany) using ethyl acetate (10%) in
dichloromethane as mobile phase and iodine vapours as visualizing agent.
Melting points of synthesized compounds were determined in Thermo Fisher Scientific (IA9000,
Essex, Great Britain) digital melting point apparatus and are uncorrected. IR spectra were
recorded on Bruker Alpha FTꢀIR Spectrometer (Billerica, MA, USA) by using ATR technique.
The 1H NMR and 13C NMR were recorded on Bruker AVANCE 400 (Bruker,
Rheinstetten/Karlsruhe, Germany) using CDCl3 and/or DMSOꢀd6. Chemical shifts are reported
in δ ppm units with respect to TMS as internal standard. HRMS were recorded on Autospec
Mass Spectrometer under the electron impact at 70 eV.
5.1.1 Synthesis of 9ꢀmethylcarbazole (1) 26
To a constantly stirred solution of carbazole (5 g, 0.025 mol, 1 equiv.) in N,Nꢀ
dimethylformamide (20 mL) maintained at 0°C, NaH (0.028 mol, 1.1 eq) was slowly and
carefully added in portions and stirring was continued for 30 min. To this reaction mass, methyl
iodide (4.34 g, 0.0306 mol, 1.2 equiv.) was added and stirred at room temperature for 5 h. the
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progress of reaction was monitored by TLC. Further, the reaction mixture was poured into ice
cold water, the solid precipitated was filtered and washed with cold water to yield white solid of
compound (1). Yield: 93.33%, M.P.; 86ꢀ88 °C; FTIR (ATR,ν
max, cmꢀ1): 3048.18 (ArꢀH Str.),
1
2953.18 (CꢀH Str. of CH3); HꢀNMR (400 MHz, CDCl3, δ ppm): 8.13 (d, J = 7.80 Hz, 2H, Arꢀ
H),7.50 (m, 2H, ArꢀH), 7.43 (d, J = 8.20 Hz, 2H, ArꢀH), 7.25 (m,2H, ArꢀH), 3.87 (s, 3H,NꢀCH3);
13CꢀNMR (100 MHz, DMSOꢀd6, δ ppm): 140.97, 125.63, 122.74, 120.27, 118.79, 108.38,29.02
(NꢀCH3).
5.1.2 Synthesis of 9ꢀmethylꢀcarbazoleꢀ3ꢀcarbaldehyde (2)26
VilsmeierꢀHaack reagent was freshly prepared by the careful addition of POCl3 (2ml, 0.021 mol)
in DMF (8 ml, 0.103 mol) at 0 °C with constant stirring. To this, 9ꢀMethyl carbazole (1, 5.0 g,
0.0238 mol, 1 equiv.) was added to the reagent with continuous stirring maintaining the
temperature at 0 °C for initial 30 min and later stirred at RT for 2 h, and finally at 60 °C for
another 2 h. The reaction mixture was then poured in sodium carbonate solution and stirring was
continued at 90 °C for 2 h. After cooling to RT, the reaction mixture was suspended into water,
extracted with dichloromethane (20 ml X 3 times) and the collective extracts were washed with
water and dried over anhydrous sodium sulphate. The residue obtained after the in-vacuo
removal of dichloromethane was further recrystallized from ethanol to afford the desired
°
compound (2) as off white crystalline solid. Yield: 75 %, M.P.; 74ꢀ76 C; FTIR (ATR,
ν
max, cmꢀ
1
1): 3022 (ArꢀH Str.) 2993 (CꢀH Str. of CH3), 1684.01 (C = O Str.); HꢀNMR (400 MHz,DMSOꢀ
d6, δ ppm): 10.09 (s, 1H, CHO),8.15 (d, 1H, J = 7.76 Hz, ArꢀH), 8.02 (dd, 1H, J = 8.52 Hz,
ArH), 7.53 (t, 1H, ArꢀH), 7.45 (dd, 2H, J = 8.28 Hz, ArꢀH), 7.34 (t, 1H, ArꢀH), 3.88 (s, 3H,Nꢀ
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13
CH3); CꢀNMR (100 MHz, CDCl3, δ ppm): 191.80 (C = O), 144.51, 141.70, 128.54, 127.24,
126.76, 123.83, 122.99, 120.65, 109.11, 29.38 (NꢀCH3).
5.1.3 Synthesis of 1ꢀ((9ꢀmethylꢀcarbazolꢀ6ꢀyl methylene) thiosemicarbazide (3)22
To a constantly stirred solution of compound 2 (4.0 g, 0.0191 mol, 1 equiv.) and
thiosemicarbazide (1.91 g, 0.021 mol, 1.1 equiv.) in anhydrous methanol (40 mL), a catalytic
amount of glacial acetic acid (0.15 equiv.) was added. The reaction mixture was refluxed for 4 h.
After cooling to room temperature, the solid separated was filtered and washed with cold
methanol to afford off white crystalline solid of compound (3). Yield: 81%, M.P.; 225ꢀ226 °C;
FTIR (ATR,ν
max, cmꢀ1): 3373.23, 3261.31 (NꢀH Str. of NH2), 3174.17(NꢀH Str. of NH), 3036.85
(ArꢀH), 1619.92 (C = N Str.), 1145.80(C = S); 1HꢀNMR (400 MHz,DMSOꢀd6, δ ppm): 11.38 (s,
1H, NH), 8.58 (s, 1H, CH = N), 8.23 (d, 1H, J = 7.76, ArꢀH), 8.21 (s, 1H, Hꢀ4 of carbazole),
8.15ꢀ7.98 (s, 2H, NH2), 7.96 (dd, 1H, J = 8.60 Hz, ArꢀH), 7.62 (d, 2H, J = 8.88 Hz, ArꢀH), 7.49
(t, 1H, J = 8.10 Hz, ArꢀH), 7.26 (t, 1H, J = 7.48 Hz, ArꢀH), 3.89 (s, 3H, NꢀCH3); 13CꢀNMR (100
MHz, DMSOꢀd6, δ ppm): 176.92, 143.04 (CH=N),141.06, 140.56, 125.60, 124.60, 121.67,
119.94, 118.76, 108.86, 28.62 (NꢀCH3); HR ESI m/z: calculated for C15H14N4S [M+H]+:
282.0939 found 283.0931
5.1.4 General procedure for the synthesis of 2ꢀbromoꢀ1ꢀsubstituted phenylethanone 5(cꢀh)
27
To a cooled solution of appropriately substituted acetophenones 4(cꢀh) (1 equiv.) in
chloroform/AcOH was added bromine (1.2 equiv.). The solution was stirred at room temperature
for 3ꢀ5 h until TLC showed full consumption of starting materials. The reaction mass was poured
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in ice cold water and extracted with 10 mL of DCM (3 times). Anhydrous Na2SO4 was added to
the combined organic layer, filtered and the excess solvent was removed in-vacuo under reduced
pressure. The resultant solid/liquid obtained was washed with hexane to yield compounds 5(cꢀh).
5.1.4.1 2ꢀbromoꢀ1ꢀ(3,4ꢀdimethoxyphenyl)ethanone (5c)
1
Off white solid; Yield: 93%; HꢀNMR (400 MHz, CDCl3, δ ppm): 7.61ꢀ7.58 (dd, 1H, J = 8.40
Hz, ArꢀH), 7.52 (s, 1H, ArꢀH), 6.90 (d, 1H, J = 8.36 Hz, ArꢀH), 4.39 (s, 2H, CH2Br), 3.94 (s, 3H,
OCH3), 3.92 (s, 3H, OCH3).
5.1.4.2 2ꢀbromoꢀ1ꢀ(4ꢀchlorophenyl)ethanone (5d)
Off white solid; Yield: 80%, 1H NMR (400 MHz, CDCl3, δ ppm): 7.91 (d, 2H, J = 8.52 Hz, Arꢀ
H), 7.48 (d, 2H, J = 8.52 Hz, ArꢀH), 4.65 (s, 2H, CH2Br).
5.1.4.3 2ꢀbromoꢀ1ꢀ(4ꢀnitrophenyl)ethanone (5e)
Yellow solid; Yield: 90 %, 1H NMR (400 MHz, CDCl3,δ ppm): 8.35 (d, 2H, J = 8.84 Hz, ArꢀH),
8.16 (d, 2H, J = 8.88 Hz, ArꢀH), 4.46 (s, 2H, CH2Br)
5.1.4.4 2ꢀbromoꢀ1ꢀ(4ꢀfluorophenyl)ethanone (5f)
1
White solid; Yield: 75 %, H NMR (400 MHz, CDCl3,δ ppm): 8.04 (m, 2H, ArꢀH), 7.14 ꢀ7.20
(m, 2H, ArꢀH), 4.40 (s, 2H, CH2Br)
5.1.4.5 2ꢀbromoꢀ1ꢀ(pꢀtolyl)ethanone (5g)
1
White solid; Yield: 78 %, H NMR (400 MHz, CDCl3,δ ppm): 7.88 (d, 2H, J = 8.28Hz, ArꢀH),
7.29 (d, 2H, J = 8.12 Hz, ArꢀH), 4.42 (s, 2H, CH2Br), 2.42 (s, 3H, CH3)
5.1.4.6 2ꢀbromoꢀ1ꢀ(3ꢀnitrophenyl)ethanone (5h)
Yellow solid; Yield: 65%, 1H NMR (400 MHz, CDCl3, δ ppm): 8.82 (t, 1H, J = 7.86 Hz, ArꢀH),
8.48 (dd, 1H, J = 8.08 Hz, ArꢀH), 8.31ꢀ8.34 (m, 1H, ArꢀH), 7.75ꢀ7.71 (t, 1H, J = 7.98 Hz, ArꢀH),
4.47 (s, 2H, CH2Br).
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5.1.5 General Procedure for the αꢀBromination of aromatic/heteroaromatic ketones 5(iꢀo) 28
To a hot solution of copper(II)bromide (1.07 g, 0.007 mol, 2 equiv.) in ethyl acetate (10 mL) was
added a solution of appropriately substituted aromatic/heteroaromatic ketones 4 (iꢀo) (0.003 mol,
1 equiv.) in chloroform (10 mL) drop wise over 30 min. The reaction mixture was then refluxed
for 12 h and cooled to room temperature, filtered through Celite bed. The filtrate was washed
with saturated NaHCO3/brine solution and dried over anhydrous Na2SO4. The resultant solution
was concentrated under reduced pressure to afford the desired compounds 5 (iꢀo).
5.1.5.1 2ꢀbromoꢀ1ꢀ(2ꢀhydroxyphenyl)ethanone (5i)
1
Yelow oil; Yield: 65%, H NMR (400 MHz, CDCl3, δ ppm): 12.25 (s, 1H, OH), 7.75 (m, 2H,
ArꢀH) 7.54 (m, 2H, ArꢀH), 4.44 (s, 2H, CH2Br).
5.1.5.2 2ꢀbromoꢀ1ꢀ(4ꢀhydroxyphenyl)ethanone (5j)
1
Yellow oil; Yield:75 % , HNMR (400 MHz , CDCl3, δ ppm): 11.72 (s, 1H, OH), 7.94 – 7.90
(dd, 2H, J = 8 .72ꢀ 8.53 Hz, ArꢀH), 6.95 – 6.91 (t, 2H, J = 8.78 Hz, ArꢀH ), 4.41 (s, 2H, CH2Br).
5.1.5.3 2ꢀbromoꢀ1ꢀ(3ꢀchlorophenyl)ethanone (5k)
1
Off white solid; Yield: 67 % , H NMR (400 MHz , CDCl3, δ ppm ): 7.95 (s, 1H, ArꢀH), 7.86 –
7.82 (d, 1H, J = 7.76 Hz, ArꢀH), 7.58ꢀ7.55 (dd, 1H, J = 8.00 Hz, ArꢀH) , 7.45 – 7.38 (m, 1H,
ArꢀH), 4.41 (s, 2H, CH2Br).
5.1.5.4 2ꢀbromoꢀ1ꢀ(3ꢀfluorophenyl)ethanone (5l)
White solid; Yield: 70%, 1H NMR (400 MHz, CDCl3, δ ppm): 7.86ꢀ7.84 (d, 1H, J = 5.88 Hz, Arꢀ
H), 7.76ꢀ7.75 (d, 2H, J = 6.28 Hz, ArꢀH), 7.49ꢀ7.47 (d, 1H, J = 5.40 Hz, ArꢀH), 4.42 (S, 2H,
CH2Br).
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5.1.5.5 2ꢀbromoꢀ1ꢀ(thiophenꢀ2ꢀyl)ethanone (5m)
1
Yellow oil; Yield: 80%, H NMR (400 MHz, CDCl3, δ ppm): 7.81ꢀ 7.80 (dd, 1H, J = 4.00 Hz,
ArꢀH), 7.72ꢀ 7.71 (dd, 1H, J = 5.00 Hz, ArꢀH), 7.18 ꢀ7.16 (dd, 1H, J = 7.76 Hz, ArꢀH), 4.36 (s,
2H, CH2Br)
5.1.5.6 3ꢀ(2ꢀbromoacetyl)ꢀ2Hꢀchromenꢀ2ꢀone (5n)
Yellow solid; Yield: 90%, 1H NMR (400 MHz, CDCl3, δ ppm): 8.63 (s, 1H, ArꢀH), 7.72ꢀ7.70 (d,
1H, J = 7.48 Hz, ArꢀH), 7.41 (m, 3H, ArH), 4.75 (s, 2H, CH2Br)
5.1.5.7 6ꢀbromoꢀ3ꢀ(2ꢀbromoacetyl)ꢀ2Hꢀchromenꢀ2ꢀone (5o)
Yellow solid; Yield, 90% , 1H NMR (400 MHz , CDCl3 , δ ppm): 8.52 (s, 1H, ArꢀH), 7.82ꢀ7.80
(d, 1H, J = 7.24 Hz, ArꢀH), 7.78ꢀ7.77 (d, 1H, J = 7.36 Hz, ArꢀH), 7.30ꢀ7.28 (d, 1H, J = 8.84 Hz,
ArꢀH), 4.71 (s, 2H, CH2Br).
5.1.6 General procedure for the synthesis of thiazolyl substituted carbazole hydrazine
derivatives 6 (aꢀo) 22
A solution of carbazole thiosemicarbazide compound (3, 150 mg, 0.531 mol, 1 equiv.) and αꢀ
bromoaromatic/heteroaromatic ketones 5 (aꢀo), (0.638 mol, 1.2 equiv.) in anhydrous methanol
(10 mL) was refluxed for 3ꢀ5 h until TLC showed full consumption of starting materials. The
residue was collected by filtration and was stirred in saturated NaHCO3 solution for 30 min. The
resultant solid was filtered, dried and purified by recrystallization with ethanol to afford the title
compounds 6 (aꢀo).
5.1.6.1 4ꢀ(4ꢀbromophenyl)ꢀ2ꢀ(2ꢀ((9ꢀmethylꢀ9Hꢀcarbazolꢀ3yl)methylene)hydrazinyl)thiazole (6a)
Brown solid, Yield: 62%, M.P.; 262ꢀ264ºC; FTIR (ATR,
ν
max, cmꢀ1): 3319.96 (NꢀH Str.),
1
3043.75 (ArꢀH Str.), 2965.52 (CꢀH Str. of CH3), 1618.20 (C = N Str.); HꢀNMR (400
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MHz,DMSOꢀd6, δppm): 12.39 (s, 1H, NH), 8.38 (s, 1H, CH = N), 8.22 (s, 1H, Hꢀ4 of carbazole),
8.20 (s, 1H, ArꢀH), 7.88ꢀ7.87 (dd, 1H, J = 8.56 ꢀ 8.60 Hz, ArꢀH), 7.85ꢀ 7.81 (d, 2H, J = 8.48 Hz,
ArꢀH), 7.67ꢀ7.59 (m, 4H, ArꢀH), 7.52ꢀ7.50 (t, 1H, J = 7.60 Hz, ArꢀH), 7.40 (s, 1H, Hꢀ5 of
thiazole), 7.25ꢀ7.23 (t, 1H, J = 7.54 Hz, ArꢀH), 3.90 (s, 3H, NꢀCH3). 13CꢀNMR (100 MHz,
DMSOꢀd6, δ ppm): 168.05, 148.72(CH = N), 142.60, 140.79, 140.58, 133.44, 131.05, 127.07,
125.67, 124.89, 123.08, 121.64, 119.99, 119.06, 118.83, 109.27, 103.69 (Cꢀ5 of thiazole), 28.69
(NꢀCH3). HR ESI m/z: calculated for C23H17BrN4S [M+H]+: 460.0357 found 461.0359.
5.1.6.2 2ꢀ(2ꢀ((9ꢀmethylꢀ9Hꢀcarbazolꢀ3ꢀyl) methylene)hydrazinyl)ꢀ4ꢀphenylthiazole (6b)
brown solid, Yield: 58%, M.P.;246ꢀ248ºC; FTIR (ATR,
ν
max, cmꢀ1): 3295.05 (NꢀH Str.), 3049.53
1
(ArꢀH Str.), 2915.12 (CꢀH Str. of CH3), 1620.14 (C = N Str.); HꢀNMR (400 MHz,DMSOꢀd6, δ
ppm): 12.00 (s,1H, NH), 8.37 (s, 1H,CH = N), 8.24 (s, 1H, Hꢀ4 of carbazole), 8.21 (s, 1H, ArꢀH),
7.88ꢀ7.86 (d, 2H, J = 8.44 Hz, ArꢀH), 7.67ꢀ7.60 (m, 2H, ArꢀH), 7.52ꢀ7.48 (t, 2H, J = 7.72 Hz,
ArꢀH), 7.41ꢀ7.39 (t, 2H, J = 7.62 Hz, ArꢀH), 7.32 (s,1H, Hꢀ5 of thiazole), 7.25ꢀ7.23 (m, 2H, Arꢀ
H), 3.90 (S,3H,N-CH3). 13CꢀNMR (100 MHz, DMSOꢀd6, δ ppm): 168.37, 142.74 (CH = N),
141.09, 141.02, 134.76, 128.56, 127.44, 126.10, 125.47, 123.49, 122.09, 121.88, 120.43, 119.44,
109.71, 109.45, 103.18 (Cꢀ5 of thiazole), 55.99, 29.19 (NꢀCH3), 18.51. HR ESI m/z: calculated
for C23H18N4S [M+H]+: 382.1252 found 383.1253.
5.1.6.3
4ꢀ(3,4ꢀdimethoxyphenyl)ꢀ2ꢀ(2ꢀ((9ꢀmethylꢀ9Hꢀcarbazolꢀ3ꢀyl)methylene)hydrazinyl)thiazole
(6c)
brown solid, Yield: 50%, M.P.; 242ꢀ244ºC; FTIR (ATR, max, cmꢀ1): 3074.36 (NꢀH), 3008.28 (Arꢀ
ν
1
H), 2930.41 (CꢀH Str. of CH3), 1616.36 ( C = N ); HꢀNMR (400 MHz,DMSOꢀd6, δ ppm): 8.38
(s, 1H, NH), 8.31 (s, 1H,CH = N), 8.23 (s, 1H, Hꢀ4 of carbazole), 8.21ꢀ8.20 (d, 1H, J = 7.88 Hz,
ArꢀH), 7.89ꢀ7.87 (dd, 1H, J = 8.68 ꢀ8.60 Hz, ArꢀH), 7.67ꢀ7.63 (m, 2H, ArꢀH), 7.52ꢀ7.48 (m, 1H,
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ArꢀH), 7.43ꢀ7.41 (m, 2H, ArꢀH), 7.27ꢀ7.25 (t, 1H, J = 7.52 Hz, ArꢀH), 7.20 (s, 1H, Hꢀ5 thiazole),
7.00ꢀ6.98 (d, 1H, J = 8.36 Hz, ArꢀH), 3.91 (s, 3H, NꢀCH3), 3.82 (s, 3H, OCH3), 3.78 (s,
3H,OCH3). 13CꢀNMR (100 MHz, DMSOꢀd6, δ ppm): 168.16, 148.68(CH=N), 141.26, 126.12,
125.31, 123.56, 122.10, 121.89, 120.44, 119.57, 118.03, 111.79, 109.72, 101.48 (Cꢀ5 of
thiazole), 55.51, 29.14 (NꢀCH3). HR ESI m/z: calculated for C25H22N4O2S [M+H]+: 442.1463
found 443.1464.
5.1.6.4 4ꢀ(4ꢀchlorophenyl)ꢀ2ꢀ(2ꢀ((9ꢀmethylꢀ9Hꢀcarbazolꢀ3ꢀyl)methylene)hydrazinyl)thiazole (6d)
brown solid, Yield: 48%, M.P.; 236ꢀ238ºC; FTIR (ATR, max, cmꢀ1): 3181.95 (NꢀH ), 3054.74
ν
1
(ArꢀH ), 2926.22 (CꢀH Str. of CH3), 1592.71 (C = N); H NMR (400 MHz,DMSOꢀd6, δ ppm):
12.08 (s, 1H,NH ), 8.36 (s, 1H,CH = N), 8.22 (s, 1H , Hꢀ4 of carbazole), 8.20 (s, 1H, ArꢀH),
7.89ꢀ7.85 (m, 3H, ArꢀH), 7.67ꢀ7.64 (d, 1H, J = 8.60 Hz, ArꢀH), 7.62ꢀ7.60 (d, 1H, J = 8.16 Hz,
ArꢀH), 7.51ꢀ7.49 (m, 3H, ArꢀH), 7.37 (s, 1H, Hꢀ5 of thiazole), 7.26ꢀ7.22 (t, 1H, J = 7.36 Hz),
3.90(s, 3H, NꢀCH3), 13CꢀNMR(100MHz, DMSOꢀd6, δppm): 168.51, 149.14, 143.04 (CH=N),
141.23, 141.02, 133.54, 131.84, 128.58, 127.19, 126.12, 125.34, 123.53, 122.09, 121.87, 120.42,
119.49, 119.28, 109.71, 109.46, 104.03 (Cꢀ5 of thiazole), 56.00, 29.11 (NꢀCH3), 18.49; HR ESI
m/z: calculated for C23H17ClN4S [M+H]+: 416.0862 found 417.0865
5.1.6.5 2ꢀ(2ꢀ((9ꢀmethylꢀ9Hꢀcarbazolꢀ3ꢀyl)methylene)hydrazinyl)ꢀ4ꢀ(4ꢀnitrophenyl)thiazole (6e)
brown solid, Yield: 55%, M.P.; 236 – 238ºC; FTIR (ATR, max, cmꢀ1): 3312.86 (NꢀH ), 3046.57
ν
1
(ArꢀH), 2928.85 (CꢀH Str. of CH3), 1621.16 (C = N); H NMR (400 MHz,DMSOꢀd6, δ ppm):
12.16 (s, 1H, NꢀH) , 8.38 (s, 1H, CH = N), 8.29 ꢀ 8.26 (d, 2H, J = 8.92 Hz, ARꢀH), 8.24 (s, 1H,
Hꢀ4 of carbazole) , 8.22ꢀ8.21 (d, 1H, J = 7.76 Hz, ArꢀH), 8.13ꢀ8.11 (d, 2H, J = 8.9 Hz, ARꢀH),
7.88ꢀ7.86 (dd, 1H, J = 8.62 Hz, ArꢀH), 7.70 (s, 1H, Hꢀ5thiazole), 7.67ꢀ7.65 (d, 1H, J = 8.64, Arꢀ
H), 7.63ꢀ7.61(d, 1H, J = 8.16 Hz, ArꢀH ), 7.50ꢀ7.48 (t, 1H, J = 8.16 Hz, ArꢀH), 7.27ꢀ7.25 (t,
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1H, J = 7.50 Hz, ArꢀH), 3.90 (s, 3 H, NꢀCH3). 13CꢀNMR (400 MHz, DMSOꢀd6, δ ppm): 168.78,
148.52, 146.14, 143.30 (CH = N), 141.27, 141.03, 140.75, 126.29, 126.13, 125.26, 124.08,
123.55, 122.10, 121.88, 119.58, 119.29, 109.72, 109.47, 108.17 (Cꢀ5 of thiazole), 29.13 (Nꢀ
CH3); HR ESI m/z: calculated for C23H17N5O2S [M+H]+: 427.1103 found 428.1107
5.1.6.6 4ꢀ(4ꢀfluorophenyl)ꢀ2ꢀ(2ꢀ((9ꢀmethylꢀ9Hꢀcarbazolꢀ3ꢀyl)methylene)hydrazinyl)thiazole (6f)
dark brown solid Yield: 60 %, M.P ; 228ꢀ229 ºC; FTIR (ATR, max, cmꢀ1): 3180 ( NH ),3047.51
ν
1
(ArꢀH), 2926.67 (CꢀH Str. of CH3), 1624.30 (C = N); H NMR (400 MHz,DMSOꢀd6, δ ppm):
11.69 (s, 1H ,NꢀH), 8.37 (s, 1H, CH = N), 8.23 (s, 1H, Hꢀ4 of carbazole), 8.22ꢀ8.20 (d, 1H, J =
7.76 Hz, ArꢀH), 7.91ꢀ7.85 (m, 3H, ArꢀH), 7.67ꢀ7.64 (d, 1H, J = 8.59 Hz, ArꢀH ), 7.62ꢀ7.60 (d,
1H, J = 8.28 Hz, ArꢀH), 7.51ꢀ7.47 (t, 1H J = 7.72 Hz, ArꢀH), 7.28 (s, 1H,Hꢀ5 of thiazole), 7.26ꢀ
7.24 (m, 3 H, ArꢀH), 3.90 (s,3H, NꢀCH3), 13CꢀNMR (100 MHz, DMSOꢀd6, δ ppm): 168.47,
160.35, 149.20, 143.06 (CH=N), 141.23, 141.01, 131.24, 127.52, 127.44, 126.13, 125.33,
123.53, 122.08, 121.87, 120.41, 119.48, 119.28, 115.51, 115.29, 109.70, 109.45, 102.98 (Cꢀ5 of
thiazole), 29.12 (NꢀCH3); HR ESI m/z: calculated for C23H17FN4S [M+H]+: 400.1158 found
401.1156
5.1.6.7 2ꢀ(2ꢀ((9ꢀmethylꢀ9Hꢀcarbazolꢀ3ꢀyl)methylene)hydrazinyl)ꢀ4ꢀ(pꢀtolyl)thiazole (6g)
off white solid, Yield: 60%, M.P; 252–254ºC; FTIR (ATR, max, cmꢀ1): 3109.40 (NꢀH), 3048.24
ν
1
(ArꢀH), 2917.32 (CꢀH Str. of CH3), 1613.95 (C = N); H NMR (400 MHz,DMSOꢀd6, δppm):
12.30 (s, 1H, NꢀH ), 8.38 (s, 1H, CH = N), 8.24 (s, 1H, Hꢀ4 of carbazole) , 8.22ꢀ8.21 (d, 1H, J =
7.68 Hz, ArꢀH), 7.89ꢀ7.88 (dd, 1H, J = 8.56 Hz, ArꢀH), 7.86ꢀ7.85 (d, 2H, J = 8.08 Hz, ArꢀH),
7.76ꢀ7.74 (d, 1H, J = 8.60 Hz, ArꢀH ), 7.67ꢀ7.65 (d, 1H, J = 8.24 Hz, ArꢀH), 7.63ꢀ7.61 (t, 1H, J =
7.52 Hz, ArꢀH), 7.26ꢀ7.24 (m, 3H, ArꢀH), 7.21 (s, 1H, Hꢀ5 of thiazole) 3.90 (s, 3H, NꢀCH3), 2.32
(s,CH3), 13CꢀNMR (100 MHz, DMSOꢀd6, δ ppm): 168.28, 141.26 (CH=N), 141.02, 136.89,
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131.67, 129.15, 128.96, 127.37, 126.12, 125.50, 125.31, 123.56, 122.11, 121.88, 120.43, 119.56,
119.28, 109.72, 109.46, 102.38 (Cꢀ5 of thiazole), 29.13 (NꢀCH3), 20.78 (ArꢀCH3); HR ESI m/z:
calculated for C24H20N4S [M+H]+: 396.1409 found 397.1409
5.1.6.8 2ꢀ(2ꢀ((9ꢀmethylꢀ9Hꢀcarbazolꢀ3ꢀyl)methylene)hydrazinyl)ꢀ4ꢀ(3ꢀnitrophenyl)thiazole (6h)
Yellow solid, Yield: 68 %, M.P.; 220– 222ºC; FTIR (ATR, max, cmꢀ1): 3107.31 (NꢀH), 3085.87
ν
1
(ArꢀH) , 2925.61 (CꢀH Str. of CH3), 1572.25 (C = N ); H NMR (400 MHz,DMSOꢀd6 ,δ ppm):
8.68 (s, 1H, NH), 8.36 (s, 1H, CH = N), 8.31ꢀ8.29 (d, 1H, J = 7.84 Hz, ArꢀH), 8.22ꢀ8.21 (s,
1H, Hꢀ4 of carbazole), 8.19 (d, 1H, J = 7.72 Hz, ArꢀH), 8.14ꢀ8.12 (dd, 1H, J = 8.04 Hz, ArꢀH),
7.88ꢀ 7.85 (dd, 1H, J = 8.56 – 8.60 Hz, ArꢀH), 7.71ꢀ7.60 (m, 4H, ArꢀH), 7.57 (s, 1H, Hꢀ5 of
thiazole) 7.51 ꢀ7.47 (t, 1H, J = 7.56 Hz, ArꢀH), 7.26ꢀ7.22 (t, 1H, J = 7.44 Hz, ArꢀH), 3.89 (s, 3
13
H, NꢀCH3), CꢀNMR (100MHz, DMSOꢀd6, δ ppm): 169.14, 148.24, 148.07, 142.99 (CH = N),
141.19, 141.01, 136.33, 131.50, 130.15, 126.10, 125.44, 123.53, 122.09, 121.88, 120.40, 119.86,
119.42, 119.26, 109.68, 109.43, 105.70 (Cꢀ5 of thiazole), 30.64, 29.11 (NꢀCH3); HR ESI m/z:
calculated for C23H17N5O2S [M+H]+: 427.1103 found 428.1106
5.1.6.9 2ꢀ(2ꢀ(2ꢀ((9ꢀmethylꢀ9Hꢀcarbazolꢀ3ꢀyl)methylene)hydrazinyl)thiazolꢀ4ꢀyl)phenol (6i)
Brown solid, Yield: 65%, M.P.; 245 – 247ºC; FTIR (ATR,
ν
max, cmꢀ1): 3342.53 (OH), 3116.66 (
1
NH ), 3057.16 (ArꢀH), 2894.84 (CꢀH Str. of CH3), 1621.58 (C = N); H NMR (400 MHz,
DMSOꢀd6, δ ppm) : 12.59 (s, 1H, NꢀH), 8.41 (s, 1H,CH = N), 8.29 (s, 1H, Hꢀ4 of carbazole),
8.23ꢀ8.21 (d, 1H, J = 7.68 Hz, ArꢀH), 7.91ꢀ7.89 (dd, 1H, J = 8.64 – 8.72 Hz, ArꢀH), 7.80 ꢀ 7.78
(dd, 1H, J = 7.84 ꢀ 7.80 Hz, ArꢀH), 7.69ꢀ7.66 (d, 1H, J = 8.64 Hz, ArꢀH), 7.63ꢀ7.61 (d, 1H, J =
8.20 Hz, ArꢀH), 7.52ꢀ7.47 (t, 1H, J = 7.30 Hz, ArꢀH ), 7.40 (s, 1H, Hꢀ5 of thiazole), 7.27ꢀ7.23 (t,
1H, J =7.48 Hz, ArꢀH), 7.20ꢀ7.18 (t, 1H , J = 8.42 Hz, ArꢀH ), 6.92ꢀ6.88 (m, 2H,ArꢀH), 5.10 (s,
1H, OH), 3.91 (s, 3H, NꢀCH3) , 13CꢀNMR (100 MHz, DMSOꢀd6, δ ppm): 167.67, 156.65,
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155.13, 141.12 (CH=N), 141.04, 129.20, 127.19, 126.16, 124.98, 123.71, 122.09, 121.87,
120.43, 119.91, 119.35, 119.11, 116.70, 109.75, 109.51, 103.70 (Cꢀ5 of thiazole), 30.66, 29.15
(NꢀCH3); HR ESI m/z: calculated for C23H18N4OS [M+H]+: 398.1201 found 399.1203
5.1.6.10 4ꢀ(2ꢀ(2ꢀ((9ꢀmethylꢀ9Hꢀcarbazolꢀ3ꢀyl)methylene)hydrazinyl)thiazolꢀ4ꢀyl)phenol (6j)
Brown solid, Yield: 55%, M.P.; 284 – 285ºC; FTIR (ATR,
ν
max, cmꢀ1): 3454.28 (OH), 3124.46
1
(NꢀH), 3059.35 (ArꢀH), 2940.97 (CꢀH Str. of CH3), 1629.47 (C = N); HNMR (400 MHz,
DMSOꢀd6, δ ppm) : 12.40 (s, 1H, NꢀH ), 8.37 (s, 1H, CH = N), 8.23 (s, 1H, Hꢀ4 of carbazole),
8.21 (s, 1H, ArꢀH ), 7.88ꢀ7.86 (d, 1H, J = 8.56 Hz, ArꢀH), 7.65ꢀ7.59 (m, 4H, ArꢀH), 7.52ꢀ7.50 (
t, 1H, J = 7.76 Hz, ArꢀH), 7.26ꢀ7.24 (t, 1H, J = 7.42 Hz, ArꢀH), 7.05 (s, 1H, Hꢀ5 of thiazole),
6.80ꢀ6.74 ( d, 2H, J = 8.56 Hz, ArꢀH), 4.66 (s, OH), 3.91 (s, 3H, NꢀCH3); 13CꢀNMR (100 MHz,
DMSOꢀd6, δ ppm): 157.68, 141.80 (CH = N), 141.57, 129.41, 127.51, 126.62, 125.86, 124.12,
122.64, 122.43, 120.93, 119.79, 115.81, 110.21, 109.97, 100.80 (Cꢀ5 of thiazole), 29.65 (Nꢀ
CH3); HR ESI m/z: calculated for C23H18N4OS [M+H]+: 398.1201 found 399.1204
5.1.6.11 4ꢀ(3ꢀchlorophenyl)ꢀ2ꢀ(2ꢀ((9ꢀmethylꢀ9Hꢀcarbazolꢀ3ꢀyl)methylene)hydrazinyl)thiazole (6k)
º
Brown solid, Yield: 48%, M.P.; 238 – 240 C; FTIR (ATR,
ν
max, cmꢀ1): 3215.93 (NꢀH), 3052.00
1
(ArꢀH), 2936.70 (CꢀH Str. of CH3), 1593.86 (C = N); HNMR (400 MHz,DMSOꢀd6, δ ppm):
12.29 (s, 1H, NꢀH ), 8.37 (s, 1H, CH = N), 8.31 (s, 1H, Hꢀ4 of carbazole), 8.22ꢀ8.21 (d, 1H, J =
7.56 Hz, ArꢀH ), 7.92 (s, 1H, Hꢀ5 of thiazole), 7.88 ꢀ7.85 (dd, 1H, J = 8.72 Hz, ArꢀH), 7.84ꢀ7.82
(d, 1H, J = 7.76 Hz, ArꢀH), 7.67ꢀ7.65 (d, 1H , J = 8.64 Hz, ArꢀH), 7.63ꢀ7.61 ( d, 1H, J = 8.24 Hz,
ArꢀH), 7.52ꢀ7.44 (m, 3H, ArꢀH ), 7.36ꢀ7.34 (m, 1H, ArꢀH), 7.26ꢀ7.23 (t, 1H, ArꢀH), 3.91 (s, 3H,
NꢀCH3), 13CꢀNMR (100 MHz, DMSOꢀd6, δ ppm): 141.76 (CH = N), 141.57, 133.94, 130.97,
126.61, 125.70, 124.51, 124.06, 122.64, 120.93, 119.78, 110.21, 109.96 (Cꢀ5 of thiazole), 29.65
(NꢀCH3); HR ESI m/z: calculated for C23H17ClN4S [M+H]+: 416.0862 found 417.0864
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5.1.6.12 4ꢀ(3ꢀfluorophenyl)ꢀ2ꢀ(2ꢀ((9ꢀmethylꢀ9Hꢀcarbazolꢀ3ꢀyl)methylene)hydrazinyl)thiazole (6l)
Brown solid, Yield: 45%, M.P.; 235 – 2370C; FTIR (ATR, max, cmꢀ1): 3187.06 (NꢀH), 3046.62 (
ν
1
ArꢀH ), 2929.54 (CꢀH Str. of CH3), 1590.14 (C = N); H NMR (400 MHz,DMSOꢀd6, δ ppm) :
12.24 (s, 1H,NH ), 8.37 (s, 1H,CH = N), 8.22 (s, 1H, Hꢀ4 of carbazole), 8.21 (s, 1H, ArꢀH ),
7.88ꢀ7.86 (dd, 1H, J = 8.58 Hz ,ArꢀH), 7.73ꢀ7.71 (d, 1H, J = 7.84 Hz, ArꢀH), 7.67ꢀ7.61 (m, 3H,
ArꢀH), 7.50ꢀ7.46 (m, 2H, ArꢀH ), 7.44 (s, 1H, Hꢀ5 of thiazole), 7.26 ꢀ7.23 (t, 1H, J = 7.18 Hz,
ArꢀH), 7.15 ꢀ7.10 (t, 1H, J = 8.45 Hz, ArꢀH), 3.91 (s, 3H, NꢀCH3) ; 13CꢀNMR (100 MHz,
DMSOꢀd6, δ ppm): 168.47, 163.70, 142.93 (CH = N), 141.22, 141.02, 130.59, 130.51, 126.10,
125.38, 123.50, 122.10, 121.89, 121.48, 120.43, 119.49, 119.26, 112.09, 111.86, 109.71, 109.45,
104.69 (Cꢀ5 of thiazole), 29.13 (NꢀCH3); HR ESI m/z: calculated for C23H17FN4S [M+H]+:
400.1158 found 401.1159
5.1.6.13 2ꢀ(2ꢀ((9ꢀmethylꢀ9Hꢀcarbazolꢀ3ꢀyl)methylene)hydrazinyl)ꢀ4ꢀ(thiophenꢀ2ꢀyl)thiazole (6m)
º
Brown solid, Yield; 45%, M.P.; 241– 243 C; FTIR (ATR,
ν
max, cmꢀ1): 3152 (NꢀH ) 3055.87 (Arꢀ
H), 2928.05 (CꢀH Str. of CH3), 1621.29 (C = N); 1H NMR (400 MHz, DMSOꢀd6 , δ ppm): 12.19
(s, 1H, NH), 8.37 (s, 1H, CH = N), 8.22ꢀ8.20 (d, 1H, J = 7.84 Hz, ArꢀH), 8.19 (s, 1H, Hꢀ4 of
carbazole), 7.87ꢀ7.84 (dd, 1H, J = 8.44ꢀ8.48 Hz, ArꢀH), 7.66ꢀ7.64 (d, 1H, J = 8.24 Hz, ArꢀH),
7.62ꢀ7.60 (d, 1H, J = 8.60 Hz, ArꢀH), 7.51ꢀ7.45 (m, 3H, ArꢀH), 7.26ꢀ7.22 (m, 1H, ArꢀH), 7.15 (s,
1H, Hꢀ5 of thiazole), 7.09ꢀ7.07 (m, 1H, ArꢀH), 3.90 (s, 3H, NꢀCH3); 13CꢀNMR (100 MHz,
DMSOꢀd6, δ ppm):168.2, 145.1, 143.0 (CH=N), 141.2, 141.0, 138.9, 127.8, 126.1, 125.3, 123.1,
122.0, 121.8, 120.4, 119.5, 119.2, 109.7, 101.5 (Cꢀ5 of thiazole), 29.13 (NꢀCH3); HR ESI m/z:
calculated for C21H16N4S2 [M+H]+: 388.0816 found 389.0814
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5.1.6.14 3ꢀ(2ꢀ(2ꢀ((9ꢀmethylꢀ9Hꢀcarbazolꢀ3ꢀyl)methylene)hydrazinyl)thiazolꢀ4ꢀyl)ꢀ2Hꢀchromenꢀ2ꢀone
(6n)
º
Yellow solid; Yield: 67%, M.P.; 267– 269 C; FTIR (ATR,
ν
max, cmꢀ1): 3164.52 (NꢀH ), 3015.73
(ArꢀH), 2871.89 (CꢀH Str. of CH3), 1721.35 (C=O), 1604.68 (C = N); 1H NMR (400 MHz,
DMSOꢀd6, δ ppm) : 8.52 (s, 1H, NꢀH), 8.35 (s, 1H, CH = N), 8.23 (s, 1H, Hꢀ4 of carbazole), 8.20
(s, 1H, Hꢀ4 of coumarin), 7.86ꢀ7.84 (dd, 1H, J = 8.60 Hz, ArꢀH) , 7.83ꢀ7.81 (dd, 1H, J = 7.80
Hz, ArꢀH), 7.75 (s, 1H, Hꢀ5 of thiazole), 7.64ꢀ7.58 (m, 3H, ArꢀH), 7.50ꢀ7.48 (m, 4H, ArꢀH),
7.26ꢀ7.22 (t, 1H, J = 7.48 Hz, ArꢀH), 3.91 (s, 3H, NꢀCH3); 13CꢀNMR (100 MHz, DMSOꢀd6, δ
ppm): 158.75 (C=O), 152.22, 150.22, 143.81 (CH = N), 143.32, 142.92, 141.24, 138.09, 132.61,
131.64, 129.13, 128.73, 126.13, 125.25, 124.80, 123.55, 122.07, 120.38, 119.29, 117.77, 116.04,
112.91, 110.21 (Cꢀ5 of thiazole), 29.09 (NꢀCH3), 18.47; HR ESI m/z: calculated for C26H18N4O2S
[M + Na]+: 450.1150 found 473.1153
5.1.6.15 6ꢀbromoꢀ3ꢀ(2ꢀ(2ꢀ((9ꢀmethylꢀ9Hꢀcarbazolꢀ3ꢀyl)methylene)hydrazinyl)thiazolꢀ4ꢀyl)ꢀ
2Hꢀchromenꢀ2ꢀone (6o)
º
Yellow solid; Yield: 67%, M.P.; 267– 269 C; FTIR (ATR,
ν
max, cmꢀ1): 3201.01, 3119.73 (NꢀH),
3042.34 (ArꢀH), 2929.74, 1711.27 (C = O), 1627.14 (C = N ); 1H NMR (400 MHz, DMSOꢀd6, δ
ppm) : 12.11 (s, 1H, NꢀH ), 8.45 (s, 1H, CH = N), 8.36 (s, 1H, Hꢀ4 of carbazole ), 8.24 (s, 1H, Hꢀ
4 of coumarin), 8.21ꢀ8.19 (d, 1H, J =7.64 Hz, ArꢀH ), 8.10ꢀ8.09 (d, 1H, J = 7.31 Hz, ArꢀH),
7.87ꢀ7.84 (dd, 1H, J = 8.64 Hz, ArꢀH), 7.79 (s, 1H, Hꢀ5 of thiazole), 7.75ꢀ7.72 (dd, 1H, J = 8.72ꢀ
8.88 Hz, ArꢀH), 7.65ꢀ7.63 (d, 1H, J = 8.64 Hz, ArꢀH), 7.61ꢀ7.59 (d, 1H, J = 8.32 Hz, ArꢀH),
7.51ꢀ7.47 (t, 1H, J = 7.54 Hz, ArꢀH), 7.40ꢀ7.38 (d, 1H, J = 8.76 Hz, ArꢀH), 7.22ꢀ7.26 (t, 1H, J =
7.52 Hz, ArꢀH), 3.89 (s, 3 H, NꢀCH3); 13CꢀNMR (100 MHz, DMSOꢀd6, δ ppm): 167.86 (C=O),
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158.27, 151.22, 143.88 (CH=N), 141.24, 136.56, 133.57, 130.58, 126.10, 125.27, 123.54,
122.08, 121.86, 120.40, 119.51, 118.06, 116.32, 111.08, 109.69 (Cꢀ5 of thiazole), 29.11 (Nꢀ
CH3); HR ESI m/z: calculated for C26H17BrN4O2S [M+Na]+: 528.0256 found 551.0253
5.2 In-vitro antiꢀmycobacterial evaluation
The in-vitro antiꢀmycobacterial activity of synthesized compounds 6 (aꢀo) was carried out at
Infectious Disease Research Institute (IDRI) within the National Institute of Allergy and
Infectious Diseases (NIAID) screening program, Bethesda, MD, USA. The activity was assessed
against M. tuberculosis H37Rv grown under aerobic conditions by using a dual readꢀout
(OD590and fluorescence) assay procedure23ꢀ25. Test compounds were prepared as 10ꢀpoint twoꢀ
fold serial dilutions in DMSO and diluted into 7H9ꢀTwꢀOADC medium in 96ꢀwell plates with a
final DMSO concentration of 2%. The highest concentration of compound was 200µM and
compounds were soluble in DMSO at 10 µM. For compounds with limited solubility, the highest
concentration was 50X less than the stock concentration e.g. 100 µM for 5 mM DMSO stock, 20
µM for 1 µM DMSO stock. For potent compounds, assays were repeated at lower starting
concentrations. Each plate included assay controls for background (medium/DMSO only, no
bacterial cells), zero growth (100 µM Rifampicin) and maximum growth (DMSO only), as well
as a rifampicin dose response curve. Plates were inoculated with M. tuberculosis and incubated
for 5 days: growth was measured by OD590and fluorescence (Ex 560/Em 590) using a BioTek™
Synergy4 plate reader. Growth was calculated separately for OD590 and RFU. MIC was
calculated on the basis of 10ꢀpoint dose response curve which was plotted as % growth. The
MIC was defined as the minimum concentration at which growth was completely inhibited and
was calculated from the inflection point of the fitted curve to the lower asymptote (zero growth).
In addition dose response curves were generated using the LevenbergꢀMarquardt algorithm and
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the concentrations that resulted in 50% and 90% inhibition of growth were determined (IC50 and
IC90respectively).
5.3 Cytotoxicity studies: MTT assay
Mammalian VERO cells were cultured in Dulbecco Modified Eagle Medium (DMEM)
containing 2 µM Na2CO3 supplemented with 10% (v/v) fetal bovine serum (FBS). The cells were
incubated at 37 °C under 5% CO2 and 95% air in a humidified atmosphere until confluent and
then diluted with phosphateꢀbuffered saline to 106 cells/mL. Stock solutions were prepared in
dimethyl sulfoxide (DMSO) and further dilutions were made with fresh culture medium. The
concentration of DMSO in the final culture medium was 1%, which had no effect on the cell
viability. In a transparent 96ꢀwell plate, serially diluted stock solutions were placed at 37 °C for
72 h then the medium was removed and monolayer was washed twice with 100 µL of warm
Hanks’ balanced salt solution (HBSS). 100 µL of warm medium and 20 µL of freshly made
MTSꢀPMS
[3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ5ꢀ(3ꢀcarboxymethoxyphenyl)ꢀ2ꢀ(4ꢀsulfophenyl)ꢀ2Hꢀ
tetrazolium and phenyl methasulfazone] (Promega) were added to each well, plates were
incubated for 3 h and absorbance was determined at 490 nm using a plate reader. The same
experimental conditions were provided for all compounds and analysis was repeated three times
for each cell line. 29
Acknowledgment
We are thankful to Department of Pharmaceutical Chemistry, Discipline of Pharmaceutical
Sciences, College of Health Sciences, University of KwaZuluꢀNatal, South Africa for their
constant support, encouragement, and financial assistance. Authors are grateful to National
Institute of Allergy and Infectious Diseases (NIAID), Bethesda, MD, USA for antiꢀ
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mycobacterial screening, Mr. Dilip Jagjivan (UKZN, South Africa) for his assistance with the
NMR experiments.
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13 A. Termentzi, I. Khouri, T. Gaslonde, S. Prado, B. SaintꢀJoanis, F.Bardou, E. P.
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Reddy,S. G .Franzblau,H.J. Knolker, Med. Chem. Res, 2008, 17, 374.
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and Chemotherapy, 2005, 49,1447
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Table 1 Antimycobacterial activity and cytotoxicity data of a novel series of thiazolyl substituted carbazole hydrazine analogues (3, 6aꢀo).
Cytotoxicity
IC50 (µM) d
Compound
Code
Structure
MIC (µM) a
IC50 (µM) b
IC90 (µM) c
S
HN
N
NH2
3
ND
202.4
393.0
1.6
5.9
N
6a
>200
110
160
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DOI: 10.1039/C4RA11752B
6b
321.5
32
35
62
6c
>50
220.8
21
28
Cl
N
HN
N
S
6d
6e
6f
>200
>200
>200
>200
>200
61
>200
>200
170
389.6
421.5
268.5
N
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Table 1 Continue
Cytotoxicity
IC50 (µM) d
Compound
Code
Structure
MIC (µM) a
IC50 (µM) b
IC90 (µM) c
ND
ND
ND
ND
6g
NO2
N
HN
N
S
>200
>200
>200
306.0
6h
N
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DOI: 10.1039/C4RA11752B
>50
440.8
6i
31
39
>200
271.1
6j
37
41
Cl
N
HN
N
S
>200
>200
>200
>200
>200
>50
>200
>200
>50
259.9
248.5
210.2
6k
N
6l
6m
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Table 1 Continue
Cytotoxicity
IC50 (µM) d
Compound
Code
Structure
MIC (µM) a
IC50 (µM) b
IC90 (µM) c
>200
>25
>25
399.2
6n
>200
>200
>200
0.007
401.7
6o
View Article Online
DOI: 10.1039/C4RA11752B
ꢀ
0.0067
0.0037
ꢀꢀ
Rifampicin
a MIC is minimum inhibitory concentration at which M. tuberculosis H37Rv growth was completely inhibited; b IC50 value is the concentration at which growth is inhibited by
50%; c IC90 value is the concentration at which growth is inhibited by 90%; d cytotoxicity activity was determined on mammalian Vero cell line; ND = Not Determined.
29