Synthesis and Biological Evaluation of Novel Carbazole Hybrids as Promising Antimicrobial Agents

Article abstract of DOI:10.1002/cbdv.201900550

Two series of carbazole analogs of 8-methoxy-N-substituted-9H-carbazole-3-carboxamides (series 1) and carbazolyl substituted rhodanines (series 2) were synthesized through facile synthetic routes. All the final compounds from these two series were evaluated for their preliminary in vitro antifungal and antibacterial activity against four fungal (Candida albicans, Cryptococcus neoformans, Cryptococcus tropicalis and Aspergillus niger) and four bacterial (Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa) strains, respectively. Among the tested compounds, three compounds of series 1 displayed promising antifungal and antibacterial activity, especially against C. neoformans and S. aureus. In addition, one compound of series 1 displayed notable antimicrobial activity (MIC: 6.25 μg/mL) against clinical isolates of C. albicans and C. neoformans (MIC: 12.5 μg/mL). From the second series, four compounds exhibited significant antifungal and antibacterial activity, especially against C. neoformans and S. aureus. The most active compound of series 2 displayed a prominent antimicrobial activity against C. neoformans (MIC: 3.125 μg/mL) and S. aureus (MIC: 1.56 μg/mL), respectively.

Full text of DOI:10.1002/cbdv.201900550

Title: Synthesis and biological evaluation of novel carbazole hybrids as
promising antimicrobial agents
Authors: Mahamadhanif S. Shaikh, Balakumar Chandrasekaran,
Mahesh B. Palkar, Ashish M. Kanhed, Afsana Kajee, Koleka
P. Mlisana, Parvesh Singh, Meenu Ghai, Mavela Cleopus
Mahlalela, and Rajshekhar V Karpoormath
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of the final Version of Record (VoR). This work is currently citable by
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the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Biodiversity 10.1002/cbdv.201900550
Link to VoR: http://dx.doi.org/10.1002/cbdv.201900550

10.1002/cbdv.201900550
Chemistry & Biodiversity
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Synthesis and biological evaluation of novel carbazole hybrids as
promising antimicrobial agents
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Mahamadhanif S. Shaikha, Balakumar Chandrasekarana,e, Mahesh B. Palkara, Ashish M.
Kanheda, Afsana Kajeea,b, Koleka P. Mlisanab, Parvesh Singhc, Meenu Ghaid, Mavela Cleopus
Mahlalelaa, and Rajshekhar Karpoormath*a
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aDepartment of Pharmaceutical Chemistry, Discipline of Pharmaceutical Sciences, College of Health Sciences,
University of KwaZulu-Natal (Westville), Durban 4000, South Africa.
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bDepartment of Microbiology, School of Laboratory Medicine & Medical Sciences, National Health Laboratory
Services (NHLS), Inkosi Albert Luthuli Central Hospital Academic Complex, University of KwaZulu-Natal,
Durban 4091, South Africa.
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cDepartment of Chemistry, School of Chemistry and Physics, College of Agriculture, Science and Engineering,
University of KwaZulu-Natal (Westville), Durban 4000, South Africa.
dDiscipline of Genetics, School of Life Sciences, College of Agriculture, Science and Engineering, University
of KwaZulu-Natal (Westville), Durban 4000, South Africa.
eFaculty of Pharmacy, Philadelphia University-Jordan, P. O. Box: 1, Philadelphia University-19392, Jordan.
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Prof. Dr. Rajshekhar Karpoormath
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Department of Pharmaceutical Chemistry,
Discipline of Pharmaceutical Sciences,
College of Health Sciences,
University of KwaZulu-Natal (Westville),
Durban 4000, South Africa
Tel: +27-312607179, +27-721107207
Fax: +27-312607792
E-mail: karpoormath@ukzn.ac.za and rvk2006@gmail.com
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10.1002/cbdv.201900550
Chemistry & Biodiversity
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Abstract
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Two series of carbazole analogs of 8-methoxy-N-substituted-9H-carbazole-3-carboxamides
(series-1) and carbazolyl substituted rhodanines (series-2) were synthesized through facile
synthetic routes. All the final compounds from these two series were evaluated for their
preliminary in vitro antifungal and antibacterial activity against four fungal (Candida
albicans, Cryptococcus neoformans, Cryptococcus tropicalis and Aspergillus niger) and four
bacterial (Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas
aeruginosa) strains, respectively. Among the tested compounds, 6d, 6e and 6f (series-1)
displayed promising antifungal and antibacterial activity, especially against C. neoformans
and S. aureus. In addition, compound 6f displayed notable antimicrobial activity (MIC: 6.25
µg/mL) against clinical isolates of C. albicans and C. neoformans (MIC: 12.5 µg/mL). From
the second series, 15f, 15g, 15i and 16f exhibited significant antifungal and antibacterial
activity, especially against C. neoformans and S. aureus. The most active compound 15i
displayed a prominent antimicrobial activity against C. neoformans (MIC: 3.125 µg/mL) and
S. aureus (MIC: 1.56 µg/mL), respectively.
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Keywords: Carbazole, Antifungal, Antibacterial, Clinical isolate of S. aureus.
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Introduction
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It is well documented that the pathogenic microorganisms (bacteria, parasites, viruses and
fungi) are rapidly developing resistance, and their associated infections can range in severity
from asymptomatic to life-threatening diseases. Antimicrobial resistance threatens the
effective prevention and treatment of an ever-increasing range of infections, thus posing a
grave concern to human kind and challenges to the medical community worldwide. In recent
years, there has been rapid rise in the frequency and spectrum of antimicrobial-resistant
infections particularly in hospitals settings and the community at large. This has resulted in
higher morbidity and mortality, with an overall increase in healthcare costs.[1] Across the
globe, there has been a constant rise in common infections such as urinary tract infections,
pneumonia, and blood stream infections due to the antibiotic resistance. A large percentage of
hospital-acquired infections are caused by extremely resistant bacterial strains such as
methicillin-resistant Staphylococcus aureus (MRSA), S. epidermis and vancomycin-resistant
Enterococcus (VRE), or multidrug-resistant Gram-negative bacteria, which are responsible
for an alarming crisis of ever-increasing significance.[2–4] In recent years, the occurrence of
antifungal resistance has grown to be a major health concern in clinical practice as the
morbidity and mortality linked to invasive fungal diseases remains very high despite of the
availability of newer antifungal and therapeutic strategies. In the past two decades,
complications due to fungal infections have been recognized as a major cause of morbidity
and mortality, especially in immune compromised patients including those affected by
tuberculosis, HIV-1, organ-transplants, diabetes and cancer.[5–7] In addition, an increase in the
incidence of fungal infections often follows the frequent use of antibacterial and cytotoxic
drugs. The three most common human pathogens are C. albicans (mortality rate: 20-40%), C.
neoformans (mortality rate: 20-70%) and A. fumigatus (mortality rate: 50-90%).[8,9] This
situation has stimulated an urgent need to develop more effective and novel antimicrobial
agents to overcome the drug resistance and improve the antimicrobial potency.
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The chemistry of heterocyclics has contributed immensly to the drug discovery and
more than 95% of the drugs in the market are heterocyclic compounds. Carbazole is one such
versatile heterocyclic scaffold, obtained either from natural or by synthesis.[10] The synthesis
of novel carbazole derivatives and the investigation of their chemical and biological
behaviour have gained importance in recent decades for their biological, medicinal and
photoelectrical applications.[11,12] Although carbazole is a rigid moiety, but can easily be
functionalised to develop novel bioactive molecules. This has resulted in potential
applications of carbazole-based derivatives as industrial and pharmaceutical products. Many
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recent studies have reported that carbazole derivatives exhibit a variety of biological activities
such as antimicrobial,[13] anticancer,[14] anti-inflammatory,[15] antimalarial,[16] antiviral,[17]
antitubercular,[18] antipsychotic and anticonvulsant[19] (Figure 1).
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Figure 1. Potential carbazole analogs with their reported pharmacological activity.
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In our present work, we report thirty four novel compounds consisting of 8-methoxy-
9H-carbazole-3-carboxamides (4a-g and 6a-i), carbazolyl-rhodanine hybrids (15a-l and 16a-
f) along with their antifungal and antibacterial activities.
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11 Results and Discussion
12 Chemistry
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The synthesis of a novel series of 8-methoxy-9H-carbazole-3-carboxamide derivatives (4a-g
and 6a-i) and carbazole tethered rhodanine derivatives (15a-l and 16a-f) was achieved
through versatile synthetic routes (Scheme 1 and 2).
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Scheme 1 Synthetic outline of a novel series of 8-methoxy-N-substituted-9H-carbazole-3-carboxamide
derivatives (4a-g) and (6a-i). Reagents and Conditions: (a) MeOH, Conc. H2SO4, Stir, 60 C, 12 h; (b) 1.1
Equiv of 2-iodoanisole, 7 mol % Pd(OAc)4, 8 mol % rac-BINAP, 1.4 Equiv of Cs2CO3, dry toluene, stir, 110 C,
36 h; (c) 10 mol % Pd(OAc)4, 2.5 Equiv of Cu(OAc)2, HOAc, stir, 117 C, 48 h; (d) 1.2 Equiv of R-NH2, 2M
Trimethylaluminium solution in toluene, reflux, 1-5 h; (e) 2.5 Equiv of Aq. NaOH, MeOH, stir, 50C, 12 h; (f)
primary and/or secondary amines, 1.5 Equiv of HATU, 1.5 Eq. of TEA, stir, RT, 24 h.
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It is clear from the synthetic scheme 1 that two unique final steps were performed in
obtaining the target compounds which have structural variations at the C(3) atom of the
carboxamide bond on the carbazole scaffold. The starting material 1 (methyl ester of 4-amino
benzoic acid) was prepared by an esterification reaction of 4-amino benzoic acid in the
presence of absolute methanol and a few drops of Conc. H2SO4 under reflux for 12 h. This
was subjected to Buchwald-Hartwig coupling reaction with 2-iodo anisole under the
influence of palladium acetate and racemic BINAP catalyst to afford methyl 4-(2-methoxy-
phenylamino) benzoate (2). The compound 2 was then treated with a trace amount of
palladium acetate and copper acetate as catalysts in the presence of glacial acetic acid to yield
methyl 8-methoxy-9H-carbazole-3-carboxylate 3 through a carbon-carbon bond formation.
Compound 3 was further reacted with appropriately substituted aryl and alkyl amines in the
presence of 2 M trimethylaluminium solution in toluene, which gave the desired 8-methoxy-
N-substituted-9H-carbazole-3-carboxamides 4a-g. In addition, alkaline hydrolysis of 3 in the
presence of sodium hydroxide and methanol resulted 8-methoxy-9H-carbazole-3-carboxylic
acid 5. 8-Methoxy-N-substituted-9H-carbazole-3-carboxamides 6a-i were synthesized by
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10.1002/cbdv.201900550
Chemistry & Biodiversity
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reacting compound 5 with appropriately substituted primary and secondary amines in the
presence of coupling reagent like HATU (1-[Bis(dimethylamino)methylene]-1H-1,2,3-
triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate) in THF/DMF mixture. The progress of
the reaction and the purity of the products were established by TLC and other
chromatographic methods.
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The structures of key intermediate 3 and its corresponding carboxamide derivatives
(4a-g and 6a-i) were established on the basis of their physicochemical and spectral data (FT-
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IR, H-NMR, 13C-NMR and HRMS). All the newly synthesized compounds showed
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acceptable results of their anticipated structures, which are summarized in the experimental
section. In general, the IR spectrum of compound 3 presented typical absorption bands
around 3265 cm-1 for N-H and 1696 cm-1 for C=O groups. This was further substantiated by
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the H-NMR spectrum of 3, which revealed the presence of a singlet at  8.80 ppm (4th
proton),  8.48 ppm (N-H proton),  4.02 ppm (acetate protons), and  3.97 ppm for
methoxyl protons confirming the carbon-carbon bond formation. IR spectra of the
compounds 4a-g and 6a-i showed moderately strong bands around 3398-3164 cm-1 and 1699-
1611 cm-1, which are characteristic of the N-H and amide C=O groups, respectively. The H-
1
NMR spectra (400 MHz) recorded in DMSO-d6 displayed some charecteristic singlet signals
at around δ 13.59-11.55 ppm for N-H proton of the carbazole ring, δ 11.89-8.36 ppm for N-H
proton of amide group at the 3rd position of the carbazole, and δ 9.10-8.12 ppm for an
aromatic proton at the 4th position of the carbazole nucleus. The distinctive singlet at around δ
4.02-3.87 ppm specified the presence of the methoxyl group on the C(8) position of the
carbazole ring. These findings were further substantiated from the 13C-NMR spectra of the
compounds with the characteristic signals appearing at around δ 170.36-170.07 ppm for the
carbonyl carbon of the amide and δ 55.50-52.10 ppm for the methoxy group attached to C(3)
and C(8) positions of the carbazole, respectively. The prominent signals appeared around δ
120.9-120.45 ppm indicated the presence of the CF3 group in 4a and 6i, while various
aromatic carbons resonated around δ 149.7-110.5 ppm and the heterocyclic aliphatic carbons
resonated around δ 48.2-21.3 ppm. In addition, the formation of novel titled compounds was
also confirmed based on the high resolution mass spectra (HRMS) which were in agreement
with their expected molecular weights.
Carbazole tethered rhodanine derivatives (15a-l and 16a-f) were prepared (Scheme 2)
according to our earlier reported method[20]
.
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Chemistry & Biodiversity
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Scheme 2 Synthetic outline of carbazolyl-rhodanines (15a-l and 16a-f)[20]. Reagents and conditions: (a)
bis(carboxymethyl) trithiocarbonate, substituted aromatic/heteroaromatic amines (8a-l and 10a-f), H2O, reflux,
100 C, 12-19 h; (b) DMF, NaH, CH3I, stir, RT, 5 h; (c) POCl3, DMF, 0 °C, stir, 90 °C, 6 h, Na2CO3; (d)
Compound 14, Rhodanine derivatives (9a-l and 11a-f), piperidine, ethanol, microwave irradiation, 30 mins, 80
ºC, 150 psi.
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31 Biological evaluation
32 Antifungal activity
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All the synthesized compounds from the two series namely; 8-methoxy-N-substituted-
9H-carbazole-3-carboxamide analogues (4a-g and 6a-i) and carbazolyl substituted rhodanines
(15a-l and 16a-f) were evaluated for their in vitro antifungal activity against a panel of fungi:
Candida albicans (ATCC90028), Cryptococcus neoformans (ATCC6603) and its clinical
isolate, Cryptococcus tropicalis (ATCC66029) and Aspergillus niger (ATCC16404).
Amphotericin B was used as the reference drug and the screening results (MIC values) are
summarized in Table 1. A careful analysis of the screening data in Table 1 revealed that the
compounds from both the series were predominantly more active against C. neoformans
(MIC = 3.125 to 50 µg/mL) and its clinical isolate (MIC = 6.25 to 50 µg/mL). The best active
compounds against C. neoformans (CN) were 15f and 15i (MIC = 3.125 µg/mL), followed by
6f, 15g, and 16f with MIC values at 6.25 µg/mL each and subsequently 6d and 6e displaying
MIC at 12.5 µg/mL, respectively. All the remaining compounds from these two series
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10.1002/cbdv.201900550
Chemistry & Biodiversity
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displayed moderate to low activity with MIC’s 25.0 to 50.0 µg/mL. In addition the most
active compounds from the above-mentioned series were further evaluated against the
clinical isolates of C. neoformans and interestingly four carbazolyl substituted rhodanine
hybrids 15f, 15g, 15i, and 16f displayed significant activity at MIC = 6.25 µg/mL, while from
the 9H-carbazole-3-carboxamide analogues, only 6f indicated a moderate activity (MIC=
12.5 µg/mL). From Table 1, It was also observed that few of the carbazolyl substituted
rhodanine hybrids presented good activity against Candida albicans (CA), with 15f
displaying the best activity at MIC = 6.25 µg/mL, followed by 15g, 15i, and 16f at MIC =
12.5 µg/mL. However, against Cryptococcus tropicalis (CT) and Aspergillus niger (AN),
poor activity (MIC = 25.0 to 50.0 µg/mL) was displayed by both the series. Over all from the
activity data, it was concluded that the best antifungal compound was 15f followed by 15i.
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13 Antibacterial activity
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It is well documented from literature reports that natural alkaloid carbazole and its
synthetic analogues have displayed notable antibacterial activity.[21] Hence, we decided to
evaluate these compounds against a panel of Gram positive (S. aureus, B. subtilis) and Gram
negative (E. coli, P. aeruginosa) bacterial strains. The antibacterial activity was carried out in
a MH medium (Mueller-Hinton medium: casein hydrolysate 17.5 g, soluble starch 1.5 g, beef
extract 1000 mL) with amoxicillin as a reference standard.[22] The in vitro antibacterial
screening of the compounds are summarized in Table 1. From both the series, it was
witnessed that 6d, 6f, 15a, 15c, 15d, 15f, 15g, 15i and 16f displayed notable antibacterial
activity specifically against S. aureus (MIC = 1.56 to 12.5 µg/mL). Interestingly, compound
15i presented the highest activity at MIC = 1.56 µg/mL followed by 15c, 15f, and 16f (MIC =
3.125 µg/mL). In addition, compounds 6f, 15a, 15d, and 15g presented good activity at MIC
of 6.25 µg/mL and 15d displayed a worthwhile activity at MIC of 12.5 µg/mL. The
remaining compounds from both the series presented moderate activity with MIC’s 25 to 50
µg/mL. Further, the most active compounds against S. aureus were also evaluated against its
clinical isolates. These compounds displayed significant antibacterial activity with 15i
indicating the best activity at MIC = 3.125 µg/mL, followed by 6f, 15c, 15f, 15g, and 16f at
MIC = 6.25 µg/mL and 15d at MIC = 12.5 µg/mL. Against B. subtilis strain, seven
compounds (15a, 5c, 15d, 15f, 15g, 15i, and 16f) demonstrated moderate inhibition (MIC =
12.5 µg/mL). In the case of E. coli, three compounds (15g, 15i, and 16f) exhibited good
antibacterial activity (MIC = 6.25 µg/mL), while four compounds (15a, 15c, 15d, and 15f)
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displayed moderate activity (MIC = 12.5 µg/mL). Similarly against P. aeruginosa, 15d and
15i exhibited moderate activity at MIC = 12.5 µg/mL, while rest of the compounds presented
poor activity. Overall, it was quite evident from Table 1 that 15i was the most active
compound exhibiting a broad spectrum of antifungal and antibacterial activity.
Table 1. Antifungal and antimicrobial activity[a] of final compounds (4a-4g, 6a-6i, 15a-15l
and 16a-16f).
CN
SA
Comp CA
CN
CT
AN
SA
BS
EC
PA
(Clinical
Isolate)[b]
(Clinical
Isolate)[b]
50
50
50
25
50
50
50
50
50
50
50
50
25
50
50
50
25
100
25
25
25
6.25
25
25
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
25
50
50
25
50
50
50
50
50
50
25
50
50
50
50
50
50
25
100
100
50
50
25
50
100
50
25
50
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
50
100
100
100
100
100
100
50
100
100
25
50
50
50
100
50
50
50
100
50
50
50
50
50
50
50
50
25
50
25
12.5
25
25
4a
4b
4c
25
50
25
100
50
25
50
4d
4e
25
25
100
100
25
25
50
25
4f
25
50
25
4g
25
50
25
100
100
100
100
100
50
100
100
100
100
100
50
6a
25
100
100
50
25
6b
6c
25
25
6d
6e
12.5
12.5
6.25
25
12.5
25
50
50
50
50
6f
12.5
ND
ND
ND
ND
ND
ND
ND
ND
6.25
6.25
25
6.25
ND
ND
ND
12.5
ND
6.25
12.5
ND
6.25
100
50
100
50
100
25
6g
25
25
6h
6i
50
50
25
50
50
25
25
15a
15b
15c
15d
15e
15f
6.25
50
12.5
100
12.5
12.5
25
12.5
50
25
100
25
50
3.125
6.25
25
12.5
12.5
25
25
100
50
25
25
3.125
3.125
12.5
12.5
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Chemistry & Biodiversity
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50
25
50
50
50
50
25
50
50
50
25
100
100
25
50
50
15g
15h
15i
12.5
100
12.5
50
6.25
50
6.25
ND
6.25
ND
ND
ND
ND
ND
ND
ND
ND
6.25
6.25
25
6.25
ND
3.125
ND
ND
ND
ND
ND
ND
ND
ND
6.25
12.5
100
12.5
100
50
6.25
100
6.25
100
50
3.125
25
1.56
25
12.5
50
50
15j
15k
15l
25
25
50
25
50
50
25
50
25
100
100
100
100
50
100
100
100
100
25
100
50
50
25
100
100
50
25
16a
16b
16c
16d
16e
16f
50
25
25
50
50
100
50
100
25
50
50
50
50
50
25
50
25
100
12.5
100
6.25
50
25
25
12.5
6.25
3.125
Amph
oterici
n
1.2
-
25
-
1.2
-
1.95
-
-
-
-
-
-
-
-
-
Amoxi
cillin
<0.39
<0.39
<0.39
<0.39
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3
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[a]Values in MIC (µg/mL); [b]Clinical isolates are well-stored and characterized clinical isolates obtained from
Department of Microbiology, Inkosi Albert Luthuli Hospital, Durban, South Africa. CA: C. albicans; CN: C.
neoformans; CT: C. tropicalis; AN: A. niger; SA: S. aureus; BS: B. subtilis; EC: E. coli; PA: P. aeruginosa;
ND: Not determined.
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Structure-activity relationship (SAR) analysis
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In general, a close inspection of the biological screening results revealed that the
antimicrobial activity of carbazole hybrids was considerably affected by the nature of
substitutions on the carbazole nucleus. Of the various substituents, nitrogen containing
heterocyclic systems such as piperidinyl and pyrimidinyl on the carbazole nucleus exerted a
significant influence on the biological activity in the first series of compounds. Specifically,
2-methylpiperidinyl carboxamide derivative 6f (Figure 2) demonstrate better antifungal and
antibacterial activity than its isomeric (3-methyl or 4-methyl) piperidinyl carboxamides (6d
and 6e) thereby confirming the crucial role of steric bulkiness for the antimicrobial action.
Increasing the nucleophilicity of the substituents by incorporating additional nitrogen atoms
in the ring system (compounds 6g-i) detrimental on the antifungal or antibacterial activity.
In the second series of compounds bearing various substitutions at position-3 on the
rhodanine, which is conjugated to 9-methylcarbazole through an arylidine linkage, we
envisaged to study the impact of some aromatic/hetero-aromatic groups towards the
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antimicrobial activity. Specifically, we have investigated the effect of bioisosteres such as 2-
quinolone (15f) and coumarin (15i) systems towards antifungal or antibacterial activity and
obtained potent inhibitory profiles against the respective microbial strains including clinical
isolates. Captivatingly, coumarin ring substituted rhodanine-tethered carbazole derivative 15i
showed highly potent inhibition of S. aureus and its clinical isolates leading to the discovery
novel therapeutic application for the rhodanine-hybridized carbazoles. Replacement of
aromatic groups on rhodanine moiety with thiadiazole-conjugated aromatic systems (16a-f)
exerted no additional improvement to the activity profile, with an exception of compound 16f
bearing para-methoxy group. It was also observed that the compounds with electron donating
groups such as methoxy (OCH3) and hydroxy (OH) on the phenyl ring greatly contributed to
the antifungal and antibacterial activity for four compounds (15a, 15c, 15d and 15g).
However, electron withdrawing substituents like halogens (F, Cl, Br and CF3) decreased the
antibacterial activity profile. Hence, the rational SAR analysis of these carbazole hybrids
unveiled the significance of bulky groups and electron-donating groups in imparting the
antifungal and antibacterial activity, respectively (Figure 2).
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Figure 2. Structure-activity relationship (SAR) analysis of the most active compounds.
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19 Conclusions
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In summary, we have synthesized novel carbazole hybrids and evaluated for preliminary in
vitro antimicrobial activities. Interestingly, three compounds 6d, 6e and 6f from the first
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series displayed promising antifungal (C. neoformans) and antibacterial (S. aureus) activities.
From the second series, 15f, 15g, 15i and 16f exhibited significant activities against the same
microbial strains. Amongst all the screened compounds, 15i was found to be a potent
antimicrobial against the clinical isolates of C. neoformans and S. aureus and can be
considered as a lead compound. These exciting results will assist the scientific community to
develop safer yet potential carbazole-based antimicrobial agents.
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Experimental
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Materials and Instrumentation
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All reagents and fine chemicals were purchased from Sigma–Aldrich and Merck Millipore,
South Africa. Solvents except the laboratory grade reagent were dried and purified according
to the literature, when necessary. The progress of the reactions was monitored by thin-layer
chromatography (TLC) on pre-coated silica gel plates procured from E. Merck and Co.
(Darmstadt, Germany) using ethyl acetate (10%) in dichloromethane as mobile phase and
iodine vapors as visualizing agent. Thermo Fisher Scientific (IA9000, Essex, Great Britain)
digital melting point apparatus was employed for the melting point determination of the
synthesized compounds and were uncorrected. Bruker Alpha FT-IR Spectrometer (Billerica,
1
13
MA, USA) was used for the FT-IR analysis employing ATR technique. H-NMR and C-
NMR spectra of the synthesized compounds were recorded on Bruker AVANCE 400
(Bruker, Rheinstetten/Karlsruhe, Germany) using deuteriated solvents such as CDCl3 and/or
DMSO-d6. Chemical shift values were reported in  ppm units with respect to TMS as an
internal standard. Autospec mass spectrometer under the electron impact at 70 eV was used
for HRMS analysis.
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25 Chemistry
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Synthesis of methyl 4-amino benzoate (1)
To a constantly stirred solution of 4-amino benzoic acid (5 g, 1 Equiv) in 50 ml of MeOH, 3-

4 drops of conc. sulphuric acid were added. The stirring was further continued at 60 C for 12
h. After completing the reaction (monitored by TLC), the excess of MeOH was removed
under reduced vacuum. The reaction mixture was poured onto crushed ice and extracted
successively with 50 ml of DCM (3 times). The combined organic layer was washed with
saturated solution of sodium bicarbonate and dried over anhydrous Na2SO4, filtered, and the
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organic layer was concentrated in vacuo to yield pale brown crystalline solid. Yield: 94% (5.2
1
g), M.P.; 108-112 ºC; H-NMR (400 MHz, CDCl3,  ppm): 7.85 (d, 2H, J = 8.60 Hz, ArH),
3
6.63 (d, 2H, J = 8.52 Hz, ArH), 4.03 (s, 2H, NH2), 3.85 (s, 3H, OCH3).
4
5
Synthesis of methyl-4-(2-methoxyphenylamino)benzoate (2)[23]
6
A mixture 7 mol% Pd (OAc)4, 8 mol% rac-BINAP and Cs2CO3 (1.4 Equiv) were cautiously
added to a constantly stirred solution of the compound 1 (5 g, 1 Equiv) and 2-iodo anisole
(1.1 Equiv) dissolved in 25 ml of dry toluene. The reaction mixture was further stirred at 110
C for 36 h. After completing the reaction (monitored by TLC), the reaction mass was
allowed to attain room temperature, poured onto ice-cold water and extracted successively
with 50 ml of ethyl acetate (3 times). The combined ethyl acetate fraction was dried over
anhydrous Na2SO4 and concentrated in vacuo to obtain crude compound which was further
purified by column chromatography (silica gel) using 5% ethyl acetate in hexane as the
mobile phase to afford the compound 2 as a colorless liquid. Yield: 97% (5.31 g). IR (ATR,
νmax, cm-1): 3398, (N-H), 3064 (Ar-H), 2946 (C-H), 1699 (C=O), 1519 (C=C), 1238 (Ar-O-
C); 1H-NMR (400 MHz, CDCl3,  ppm): 7.93 (d, 2H, J = 8.7 Hz, Ar-H), 7.38 (t, 2H, J = 7.7
Hz, ArH), 7.08 (d, 2H, J = 8.72 Hz, ArH), 6.95-7.02 (m, 2H, Ar-H), 6.37 (s, 1H, N-H), 3.88
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(s, 3H, COOCH3), 3.87 (s, 3H, OCH3); C-NMR (100 MHz, CDCl3,  ppm): 167.2 (C=O),
149.7, 147.7, 131.5, 130.7, 122.4, 120.9, 117.9, 115.3, 111.0, 55.8 (COOCH3), 51.9 (OCH3).
Synthesis of methyl-8-methoxy-9H-carbazole-3-carboxylate (3)[23]
A mixture of 10 mol% Pd (OAc)4 and Cu(OAc)2 (2.5 Equiv) was added to a constantly stirred
solution of compound 2 (5 g, 1 Equiv) dissolved in 25 ml glacial acetic acid. The reaction

mass was further stirred for 48 h at 117 C and was monitored by TLC. The reaction mixture
was added in ice cold water and basified with saturated solution of sodium bicarbonate
(alkaline to litmus). The inorganic solid collected was filtered over celite bed and the filtrate
was extracted successively with 50 ml of DCM (3 times) and dried over anhydrous Na2SO4.
The combined organic layer was concentrated in vacuo and purified by column
chromatography using 6% ethyl acetate in hexane as the mobile phase to obtain compound 3
as a white crystalline solid. Yield: 52% (2.61 g), M.P.; 136-138 ºC; IR (ATR, νmax, cm-1):
3265 (N-H), 3066 (Ar-H), 2930, 2857 (C-H), 1696 (C=O), 1508 (C=C), 1238 (Ar-O-C); 1H-
NMR (400 MHz, CDCl3,  ppm): 8.80 (s, 1H, H-4 of carbazole), 8.48 (s, 1H, N-H), 8.11 (dd,
1H, J = 8.56 Hz, ArH), 7.73 (d, 1H, J = 7.9 Hz, ArH), 7.46 (d, 1H, J = 8.56 Hz, ArH), 7.19
(t, 1H, J = 7.9 Hz, ArH), 6.95 (d, 1H, J = 7.8 Hz, ArH), 4.02 (s, 3H, COOCH3), 3.97 (s, 3H,
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OCH3); 13C-NMR (100 MHz, CDCl3,  ppm): 168.07 (C=O), 145.9, 142.0, 130.4, 127.5,
124.5, 123.6, 121.6, 120.9, 113.2, 110.6, 106.8, 55.8 (COOCH3), 52.1 (OCH3).
3
4
General procedure for the synthesis of 8-methoxy-N-substituted-9H-carbazole-3-
5
carboxamide (4a-g)[24]
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7
Compound 3 (1 Equiv) and the amines (1.2 Equiv) were dissolved in dry toluene, to which
2M solution of trimethylaluminium in toluene (1 ml) was slowly added in a drop-wise
manner. The resulting solution was stirred at 80 C for 1-5 h. After completing the reaction
(monitored by TLC), the mass was cooled and dumped in cold water, extracted successively
with 10 ml of ethyl acetate (3 times) and dried over anhydrous Na2SO4. The combined
organic layer was concentrated in vacuo and purified by trituration with diethyl ether and n-
pentane to obtain the desired compounds 4a-g. The physicochemical and spectroscopic data
of newly synthesized compounds are provided below.
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8-Methoxy-N-(3-(trifluoromethyl) phenyl)-9H-carbazole-3-carboxamide (4a) Yield: 80%,
M.P.; 274-276 C. IR (ATR, νmax, cm-1): 3370 (N-H), 3203, 3008 (Ar-H), 2937, 2908, 2835
1
(C-H), 1645 (C=O), 1596, 1578, 1507 (C=C), 1440, 1400, 1324, 1256, 1225 (Ar-O-C); H
NMR (400 MHz, DMSO-d6,  ppm): 11.72 (s, 1H, NH), 10.52 (s, 1H, CO-NH), 8.80 (s, 1H,
H-4 of carbazole), 8.30 (s, 1H, ArH), 8.12 (d, 1H, J = 8.32 Hz, ArH), 8.04 (dd, 1H, J = 8.57
Hz, ArH), 7.79 (d, 1H, J = 7.76 Hz, ArH), 7.63-7.59 (m, 2H, J = 6.76 Hz, ArH), 7.44 (d, 1H,
J = 7.72 Hz, ArH), 7.17 (t, 1H, J = 7.80 Hz, ArH), 7.06 (d, 1H, J = 7.80 Hz, ArH), 4.01 (s,
13
3H, OCH3); C-NMR (100 MHz, DMSO-d6,  ppm): 166.93 (C=O), 145.2, 141.07, 139.9,
129.72, 129.23, 125.03, 124.86, 124.15, 123.07, 121.61, 120.10, 119.4, 118.92, 115.67,
112.15, 110.45, 106.34, 78.73, 54.90 (OCH3); HRMS (ESI+) m/z: calculated for
C21H15F3N2O2 [M+H]+: 385.1223 found 385.1226.
N-cyclohexyl-8-methoxy-9H-carbazole-3-carboxamide (4b) Yield: 63%, M.P.; 218-220 C;
IR (ATR, νmax, cm-1): 3405 (NH), 3252, 3072, 3011 (Ar-H), 2928, 2853 (C-H), 1621 (C=O),
1579, 1547, 1504 (C=C), 1457, 1395, 1325, 1237 (Ar-O-C); 1H-NMR (400 MHz, DMSO-d6,
 ppm): 11.55 (s, 1H, NH), 8.62 (s, 1H, CO-NH), 8.11 (d, 1H, J = 7.92 Hz, H-4 of carbazole),
7.89 (dd, 1H, J = 8.56 Hz, ArH), 7.72 (d, 1H, J = 7.76 Hz, ArH), 7.47 (d, 1H, J = 8.52 Hz,
ArH), 7.12 (t, 1H, J = 7.82 Hz, ArH), 7.02 (d, 1H, J = 7.80 Hz, ArH), 3.99 (s, 3H, OCH3),
3.81 (q, 1H), 1.87 (d, 2H), 1.76 (t, 2H), 1.63 (d, 1H), 1.34 (q, 4H), 1.15 (q, 1H); 13C-NMR
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(100 MHz, DMSO-d6,  ppm): 165.89 (C=O), 145.74, 141.07, 130.14, 125.41, 123.70, 122.0,
119.83, 12.61, 110.54, 106.61,55.41 (OCH3), 48.27, 32.63, 25.34; HRMS (ESI+) m/z:
calculated for C20H22N2O2[M+H]+: 323.1819 found 323.1819.
3
4
5
N-cyclopropyl-8-methoxy-9H-carbazole-3-carboxamide (4c) Yield: 73%, M.P.; 246-248 C;
IR (ATR, νmax, cm-1): 3164 (NH2), 3071, 3032 (Ar-H), 2933, 2838 (C-H), 1611 (C=O), 1597,
6
1
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1579, 1506 (C=C), 1450, 1408, 1362, 1322, 1277, 1259, 1236 (Ar-O-C); H-NMR (400
8
MHz, DMSO-d6,  ppm): 11.57 (s, 1H, NH), 8.58 (s, 1H, H-4 of carbazole), 8.36 (d, 1H, J =
4Hz, CO-NH), 7.84 (dd, 1H, J = 8.52 Hz, ArH), 7.71 (d, 1H, J = 7.76 Hz, ArH), 7.46 (d, 1H,
J = 8.52 Hz, ArH), 7.12 (t, 1H, J = 7.82 Hz, ArH), 7.02 (d, 1H, J = 7.80 Hz, ArH), 3.99 (s,
3H, OCH3), 2.91-2.85 (m, 1H), 0.73-0.68 (m, 2H), 0.62-0.58 (m, 2H); 13C-NMR (100 MHz,
DMSO-d6,  ppm): 168.11(C=O), 145.75, 141.14, 130.16, 125.03, 123.68, 122.03, 119.80,
112.58, 110.62, 106.66, 55.42 (OCH3), 23.11; HRMS (ESI+) m/z: calculated for C17H16N2O2
[M+H]+: 281.1692 found 281.1694.
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N-(2-chloro-4-fluorophenyl)-8-methoxy-9H-carbazole-3-carboxamide (4d) Yield: 41%, M.P.;
238-240 C. IR (ATR, νmax, cm-1): 3437, 3372 (NH), 3009 (Ar-H), 2932, 2910, 2831 (C-H),
1
1662 (C=O), 1578, 1528 (C=C), 1486, 1459, 1399, 1302, 1290, 1237 (Ar-O-C); H-NMR
(400 MHz, DMSO-d6,  ppm): 11.70 (s, 1H, NH), 10.02 (s, 1H, CO-NH), 8.81 (s, 1H, H-4 of
carbazole), 8.02 (dd, 1H, J = 8.57 Hz, ArH), 7.76 (d, 1H, J = 7.72 Hz, ArH), 7.61 (d, 2H, J =
8.88 Hz, ArH), 7.57-7.54 (m, 1H, ArH), 7.26 (d, 1H, J = 8.65 Hz, ArH), 7.15 (t, 1H, J = 7.8
Hz, ArH), 7.06 (d, 1H, J = 7.80 Hz, ArH), 4.01 (s, 3H, OCH3); 13C-NMR (100 MHz, DMSO-
d6,  ppm): 165.88 (C=O), 158.14, 145.61, 141.42, 132.13, 130.47, 129.79, 125.14, 123.94,
122.02, 120.45, 116.56, 114.42, 112.50, 110.76, 106.68, 70.57, 55.27 (OCH3); HRMS (ESI+)
m/z: calculated for C20H14ClFN2O2 [M+H]+: 369.0871 found 369.0871.
N-(heptan-2-yl)-8-methoxy-9H-carbazole-3-carboxamide (4e) Yield: 68%, M.P.; 140-142 C.
IR (ATR, νmax, cm-1) : 3392, 3235 (NH), 3065 (Ar-H), 2948, 2929, 2852 (C-H), 1624 (C=O),
1
1579, 1545, 1506 (C=C), 1458, 1396, 1321, 1256, 1241 (Ar-O-C); H-NMR (400 MHz,
DMSO-d6,  ppm) : 11.55 (s, 1H, NH), 8.61 (s, 1H, CO-NH), 8.06 (d, 1H, J = 8.16 Hz, H-4
of carbazole), 7.90 (d, 1H, J = 8.40 Hz, ArH), 7.71 (d, 1H, J = 7.72 Hz, ArH), 7.46 (d, 1H, J
= 8.48 Hz, ArH), 7.14 (t, 1H, J = 7.76 Hz, ArH), 7.02 (d, 1H, J = 7.76 Hz, ArH), 4.05 (t,
1H), 3.99 (s, 3H, OCH3), 1.56-1.49 (m, 2H), 1.27 (s, 8H), 1.15 (t, 4H); 13C-NMR (100 MHz,
DMSO-d6,  ppm): 166.08 (C=O), 145.73, 141.05, 130.14, 125.46, 123.68, 122.01, 119.76,
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119.70, 112.58, 110.54, 106.61, 55.40 (OCH3), 44.71, 36.08, 31.22, 25.58, 22.05, 20.93,
13.91; HRMS (ESI+) m/z: calculated for C21H26N2O2 [M+H]+: 339.2146 found 339.2146.
3
4
N-(4-hydroxycyclohexyl)-8-methoxy-9H-carbazole-3-carboxamide (4f) Yield: 68%, M.P.;
252-254 C. IR (ATR, νmax,cm-1): 3432 (OH), 3266 (NH), 2955, 2929, 2856 (C-H), 1621
5
1
6
(C=O), 1580, 1540, 1505 (C=C), 1472, 1456, 1396, 1352, 1330, 1241 (Ar-O-C); H NMR
7
(400 MHz, DMSO-d6,  ppm): 11.55 (s, 1H, NH), 8.61 (s, 1H, CO-NH), 8.09 (d, 1H, J = 7.76
Hz, H-4 of carbazole), 7.89 (d, 1H, J = 8.48 Hz, ArH), 7.71 (d, 1H, J = 7.76 Hz, ArH), 7.46
(d, 1H, J = 8.52 Hz, ArH), 7.14 (t, 1H, J = 7.82 Hz, ArH), 7.02 (d, 1H, J = 7.80 Hz, ArH),
4.55 (d, 1H), 3.99 (s, 3H, OCH3), 3.79-3.73 (m, 1H), 3.44 (s, 1H, OH), 1.86 (t, 1H), 1.41 (q,
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4H), 1.27 (q, 4H); C-NMR (100 MHz, DMSO-d6,  ppm); 166.09 (C=O), 145.73, 141.06,
130.13, 125.32, 123.68, 121.99, 119.81, 112.58, 110.55, 106.61, 68.42 (C-OH), 55.40
(OCH3), 47.84, 34.32, 30.49; HRMS (ESI+) m/z: calculated for C20H22N2O3[M+H]+:
338.1630 found 338.1644.
8-Methoxy-N-morpholino-9H-carbazole-3-carboxamide (4g) Yield: 39%, M.P.; 270-272 C.
IR (ATR, νmax, cm-1): 3270 (NH), 3194, 3039 (Ar-H), 2975, 2955, 2914, 2889, 2859, 2841
(C-H), 1622 (C=O), 1581, 1538, 1507 (C=C), 1465, 1428, 1408, 1366, 1326, 1313, 1296,
1
1240 (Ar-O-C); H -NMR (400 MHz, DMSO-d6,  ppm): 11.59 (s, 1H, NH), 9.44 (s, 1H,
CO-NH), 8.56 (s, 1H, H-4 of carbazole), 7.82 (dd, 1H, J = 8.52 Hz, ArH), 7.73 (d, 1H, J =
7.80 Hz, ArH), 7.48 (d, 1H, J = 8.52 Hz, ArH), 7.15 (t, 1H, J = 7.84 Hz, ArH), 7.03 (d, 1H, J
= 7.80 Hz, ArH), 3.99 (s, 3H, OCH3), 3.69 (s, 4H), 2.94 (t, 4H); 13C-NMR (400 MHz,
DMSO-d6,  ppm): 164.92 (C=O), 145.73, 141.19, 130.14, 124.96, 123.59, 122.0, 119.90,
119.80, 112.62, 110.71, 106.69, 66.09 (C-O), 55.4 (C-N), 54.54 (OCH3); HRMS (ESI+) m/z:
calculated for C18H19N3O3Na [M+H]+: 348.1324 found 348.1330.
Synthesis of 8-methoxy-9H-carbazole-3-carboxylic acid (5)
An aqueous solution of NaOH (2.5 Equiv) was slowly added to a constantly stirred solution
°
of 3 (1 g, 1 Equiv) in MeOH. The reaction mixture was stirred at 50 C for 12 h. After
completion of reaction (monitored by TLC), the mixture was cooled and acidified with dil.
HCl, thus separated solid was filtered and purified by recrystallization to yield compound 4
as a white solid. Yield: 95% (0.900 g). 1H-NMR (400 MHz, DMSO-d6,  ppm): 12.48 (s, 1H,
COOH), 11.72 (s, 1H, NH), 8.72 (s, 1H, H-4 of carbazole), 7.96 (dd, 2H, J = 8.52 Hz, ArH),
7.80 (d, 1H, J = 7.72 Hz, ArH), 7.51 (d, 1H, J = 8.52 Hz, ArH), 7.16 (t, 1H, J = 7.80 Hz,
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ArH), 7.04 (d, 1H, J = 7.80 Hz, ArH), 4.00 (s, 3H, OCH3); 13C-NMR (100 MHz, DMSO-d6, 
ppm): 168.07 (C=O), 145.73, 142.22, 130.20, 126.71, 123.61, 122.63, 121.02, 120.08,
121.91, 110.96, 106.93, 55.47 (OCH3).
3
4
5
General procedure for the synthesis of 8-methoxy-N-substituted-9H-carbazole-3-
carboxamide (6a-i)[25]
6
7
A mixture of compound 4 (1 Equiv), primary/secondary amines (1.5 Equiv), HATU (1.5
Equiv) and TEA (1.5 Equiv) were dissolved in DMF: THF (1:5). The resultant reaction mass
was stirred at room temperature for 24 h. After completing the reaction (monitored by TLC),
it was cooled and dumped in cold water, extracted with ethyl acetate and dried over
anhydrous Na2SO4. The organic layer was concentrated in vacuo and purified through silica
gel column using a mobile phase of 5% MeOH in DCM to obtain the desired target
compounds (6a-i). The physicochemical and spectroscopic data of these compounds are
provided below.
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tert-Butyl-4-(8-methoxy-9H-carbazole-3-carbonyl)piperazine-1-carboxylate (6a) Yield: 51%,
M.P.; 203-205 C. IR (ATR, νmax, cm-1): 3286 (NH), 3208 (Ar-H), 2980, 2914, 2862, 2840
(C=C), 1671 (C=O), 1627, 1580, 1540, 1508 (C-H), 1454, 1427, 1404, 1364, 1323, 1284,
1240 (Ar-O); 1H-NMR (400 MHz, DMSO-d6,  ppm): 11.54 (s, 1H, NH), 8.19 (s, 1H, H-4 of
carbazole), 7.76 (d, 1H, J = 7.72 Hz, ArH), 7.50 (d, 1H, J = 8.28 Hz, ArH), 7.44 (d, 1H, J =
8.36 Hz, ArH), 7.10 (t, 1H, J = 7.80 Hz, ArH), 7.02 (d, 1H, J = 7.80 Hz, ArH), 3.99 (s, 3H,
13
OCH3), 3.54 (s, 4H), 3.41 (s, 4H), 1.41 (s, 9H); C-NMR (100 MHz, DMSO-d6,  ppm):
169.93 (C=O), 153.39, 145.2, 139.7, 129.64, 125.23, 124.43, 122.97, 121.6, 119.59, 112.49,
110.5, 106.21, 78.59, 54.95 (OCH3), 27.57; HRMS (ESI+) m/z: calculated for
C23H27N3O4[M+Na]+: 432.1899 found 432.1895.
N-(3-(1H-imidazol-1-yl)propyl)-8-methoxy-9H-carbazole-3-carboxamide (6b) Yield: 48%,
M.P.; 188-190 C; IR (ATR, νmax, cm-1): 3390 (NH), 3260, 3065 (Ar-H), 2924, 2857 (C=C),
1671 (C=O), 1579, 1542, 1508 (C-H), 1458, 1406, 1365, 1325, 1263, 1240 (Ar-O-C), 1169,
1095, 1072, 1021, 955, 841, 786; 1H-NMR (400 MHz, DMSO-d6,  ppm): 11.60 (s, 1H, NH),
8.63 (s, 1H, H-4 of carbazole), 8.48 (t, 1H, J = 5.26 Hz, CO-NH), 7.90 (dd, 1H, J = 8.53 Hz,
ArH), 7.86 (s, 1H, ArH), 7.74 (d, 1H, J = 7.76 Hz, ArH), 7.50 (d, 1H, J = 8.48 Hz, ArH), 7.32
(s, 1H, ArH), 7.16 (t, 1H, J = 7.80 Hz, ArH), 7.04 (d, 1H, J = 7.80 Hz, ArH), 7.00 (s, 1H,
ArH), 4.09 (t, 2H, J = 6.86 Hz), 4.01 (s, 3H, OCH3), 3.31 (q, 2H J = 6.23 Hz), 2.03 (t, 2H, J =
17
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1
2
6.78 Hz); C-NMR (100 MHz, DMSO-d6,  ppm): 167.06 (C=O), 145.74, 141.16, 137.12,
130.16, 127.43, 125.06, 123.64, 122.06, 119.85, 112.57, 110.66, 106.67, 55.40 (OCH3),
45.68, 44.14, 40.12, 36.48, 30.84; HRMS (ESI+) m/z: calculated for C20H20N4O2 [M+H]+:
348.1586 found 348.1589.
3
4
5
6
(8-Methoxy-9H-carbazol-3-yl)(4-methylpiperazin-1-yl)methanone (6c) Yield 28%, M.P.;
166-167 C. IR (ATR, νmax, cm-1): 3392 (NH), 3248, 3000 (Ar-H), 2924, 2856 (C=C), 1671
(C=O), 1579, 1542 (C-H), 1457, 1427, 1406, 1365, 1322, 1239 (Ar-O-C), 1168, 1134, 1094,
7
8
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9
1072, 1022, 999, 842, 785, 733, 703; H-NMR (400 MHz, DMSO-d6,  ppm): 11.53 (s, 1H,
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NH), 8.16 (s, 1H, H-4 of carbazole), 7.76 (d, 1H, J = 7.76 Hz, ArH), 7.50 (t, 1H, J = 6.66 Hz,
ArH), 7.41 (d, 1H, J = 8.32 Hz, ArH), 7.10 (t, 1H, J = 7.78 Hz, ArH), 7.02 (d, 1H, J = 7.80
Hz, ArH), 3.99 (s, 3H, OCH3), 3.57 (s, 4H), 2.39 (s, 4H), 2.24 (s, 3H); 13C-NMR (100 MHz,
DMSO-d6,  ppm): 170 (C=O), 145.65, 140.04, 130.06, 125.72, 124.72, 123.39, 122.08,
119.86, 119.53, 112.91, 110.90, 106.62, 55.38 (OCH3), 54.46, 40.12; HRMS (ESI+) m/z:
calculated for C19H21N3O2[M+H]+: 324.1779 found 324.1779.
(8-Methoxy-9H-carbazol-3-yl)(4-methylpiperidin-1-yl)methanone (6d) Yield: 37%, M.P.;
181-182 C. IR (ATR, νmax, cm-1): 3393 (NH), 3233, 3066 (Ar-H), 2952, 2923, 2862 (C=C),
1654 (C=O), 1580, 1534 (C-H), 1508, 1458, 1407, 1364, 1320, 1263, 1238 (Ar-O), 1171,
1092, 1071, 1022, 975, 837, 785, 746; 1H-NMR (400 MHz, DMSO-d6,  ppm): 11.50 (s, 1H,
NH), 8.13 (s, 1H, H-4 of carbazole), 7.75 (d, 1H, J = 7.76 Hz, ArH), 7.49 (d, 1H, J = 8.32 Hz,
ArH), 7.37 (dd, 1H, J = 8.2 Hz, ArH), 7.12 (t, 1H, J = 7.82 Hz, ArH), 7.01 (d, 1H, J = 7.76
Hz, ArH), 3.99 (s, 3H, OCH3), 3.09 (q, 1H), 2.92 (s, 1H), 2.86 (d, 3H), 2.84 (s, 1H), 1.66 (t,
13
4H), 1.10 (q, 2H); C-NMR (100 MHz, DMSO-d6 ,  ppm): 170.07 (C=O), 161.94, 145.67,
139.92, 130.06, 126.6, 124.53, 123.42, 122.08, 119.55, 112.91, 110.87, 106.59, 55.39
(OCH3), 48.16, 33.91, 32.19, 30.56, 29.70, 21.65; HRMS (ESI+) m/z: calculated for
C20H22N2O2[M+Na]+: 345.1579 found 345.1570.
(8-Methoxy-9H-carbazol-3-yl)(3-methylpiperidin-1-yl)methanone (6e) Yield: 42%, M.P.;
183-184 C. IR (ATR, νmax,cm-1): 3395 (NH), 3231, 3001 (Ar-H), 2945, 2922, 2861 (C=C),
1654 (C=O), 1591, 1579, 1543, 1508 (C-H), 1459, 1440, 1405, 1365, 1318, 1260, 1238 (Ar-
1
O), 1173, 1119, 1093, 1023, 968, 838, 788, 750, 731; H-NMR (400 MHz, DMSO-d6, 
ppm): 11.51 (s, 1H, NH), 8.12 (s, 1H, H-4 of carbazole), 7.75 (d, 1H, J = 7.72 Hz, ArH), 7.49
(d, 1H, J = 8.32 Hz, ArH), 7.36 (dd, 1H, J = 8.16 Hz, ArH), 7.12 (t, 1H, J = 7.78 Hz, Ar-H),
18
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7.01 (d, 1H, J = 7.76 Hz, ArH), 3.99 (s, 3H, OCH3), 2.92 (s, 1H), 1.80 (d, 1H), 1.62 (s, 1H),
1.46 (d, 1H), 1.12-1.21 (m, 2H), 0.84 (s, 3H); 13C-NMR (100 MHz, DMSO-d6,  ppm):
170.03 (C=O), 145.66, 139.89, 130.04, 126.59, 124.49, 123.41, 122.07, 119.55, 112.88,
110.85, 106.58, 55.39 (OCH3), 32.60, 18.79; HRMS (ESI+) m/z: calculated for
C20H22N2O2[M+Na]+: 345.1579 found 345.1570.
3
4
5
6
7
(8-Methoxy-9H-carbazol-3-yl)(2-methylpiperidin-1-yl)methanone (6f) Yield: 40%, M.P.;
201-203 C. IR (ATR, νmax, cm-1): 3421 (NH), 3251, 3000 (Ar-H), 2949, 2927, 2863 (C=C),
1654 (C=O), 1596, 1579, 1508 (C-H), 1458, 1432, 1365, 1318, 1260, 1239 (Ar-O-C), 1172,
8
9
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1119, 1099, 1022, 1009, 887, 840, 787, 767, 747; H-NMR (400 Hz, DMSO-d6,  ppm):
11.49 (s, 1H, NH), 8.10 (s, 1H, H-4 of carbazole), 7.76 (d, 1H, J = 7.76 Hz, ArH), 7.49 (d,
1H, J = 8.32 Hz, ArH), 7.34 (dd, 1H, J = 8.37 Hz, ArH), 7.11 (t, 1H, J = 7.80 Hz, ArH),
7.01 (d, 1H, J = 7.76 Hz, ArH), 4.47 (s, 1H), 3.99 (s, 3H, OCH3), 3.01 (t, 1H), 1.63 (q, 4H),
1.40-1.53 (m, 2H), 1.21 (d, 3H); 13C-NMR (100 MHz, DMSO-d6,  ppm): 170.36 (C=O),
145.67, 139.82, 130.06, 127.17, 124.09, 123.43, 122.16, 119.49, 112.93, 110.94, 106.58, 55.4
(OCH3), 29.91, 25.64, 18.54, 15.83; HRMS (ESI+) m/z: calculated for C20H22N2O2[M+Na]+:
345.1579 found 345.1570.
(8-Methoxy-9H-carbazol-3-yl)(4-(2-methoxyphenyl)piperazin-1-yl)methanone (6g) Yield:
33%, M.P.; 214-216 C. IR (ATR, νmax, cm-1): 3421 (NH), 3251, 3058, 3001 (Ar-H), 2927,
2858, 2832 (C=C), 1636 (C=O), 1596, 1580, 1508 (C-H), 1458, 1406, 1366, 1319, 1294,
1
1263, 1238 (Ar-O), 1172, 1116, 1101, 1014, 946, 893, 843, 816, 786, 748; H-NMR (400
MHz, D MSO-d6,  ppm): 11.54 (s, 1H, NH), 8.21 (s, 1H, H-4 of carbazole), 7.76 (d, 1H, J =
7.73 Hz, ArH), 7.50 ( d, 1H, J = 8.28 Hz, ArH), 7.45 (d, 1H, J = 8.16 Hz, ArH), 7.13 (t, 2H,
J = 7.76 Hz, ArH), 7.01 (d, 1H, J = 7.80 Hz, ArH), 6.95 (d, 1H, J = 8.00 Hz, ArH), 6.91 (d,
1H, J = 7.40 Hz, ArH), 6.88 (d, 1H, J = 7.08 Hz, ArH), 3.99 (s, 3H, OCH3), 3.79 (s, 3H,
13
OCH3), 3.72 (s, 4H), 3.00 (s, 4H); C-NMR (100 MHz, DMSO-d6,  ppm): 170.13 (C=O),
152.04, 145.69, 140.82, 130.11, 125.90, 124.92, 123.46, 122.90, 122.13, 120.82, 120.01,
119.59, 118.40, 112.99, 111.91, 110.97, 106.68, 55.43 (OCH3), 50.41 (OCH3); HRMS (ESI+)
m/z: calculated for C25H25N3O3[M+Na]+: 438.1794 found 438.1794.
N-(1-benzylpiperidin-4-yl)-8-methoxy-9H-carbazole-3-carboxamide (6h) Yield: 33%, M.P.;
232-234 C. IR (ATR, νmax, cm-1): 3388 (NH), 3251 (Ar-H), 2930, 2859, 2842, 2800 (C=C),
1672 (C=O), 1622, 1579, 1539, 1506 (C-H), 1455, 1405, 1365, 1326, 1239 (Ar-O), 1169,
19
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Chemistry & Biodiversity
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1100, 1071, 1022, 839, 785, 732; 1H-NMR (400 MHz, DMSO-d6,  ppm): 11.57 (s, 1H, NH),
8.62 (s, 1H, H-4 of carbazole), 8.20 (s, 1H, CO-NH), 7.89 (d, 1H, J = 8.44 Hz, ArH), 7.71 (d,
1H, J = 7.72 Hz, ArH), 7.46 (d, 1H, J = 8.52 Hz, ArH), 7.36 (s, 5H, ArH), 7.13 (t, 1H, J =
7.78 Hz, ArH), 7.01 (d, 1H, J = 7.80 Hz, ArH), 3.99 (s, 3H, OCH3), 3.87 (s, 1H), 3.56 (s,
2H), 3.08 (d, 1H, J = 6.92 Hz), 2.92 (s, 2H), 1.86 (s, 2H), 1.68 (s, 2H); 13C-NMR (100 MHz,
DMSO-d6,  ppm): 145.73 (C=O), 141.11, 130.14, 128.24, 125.21, 123.66, 122.0, 119.88,
112.59, 110.57, 106.64, 55.4 (OCH3); HRMS (ESI+) m/z: calculated for C26H27N3O2[M+H]+:
414.2103 found 414.2104.
3
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8-Methoxy-N-(5-(trifluoromethyl)-1,3,4-thiadiazol-2-yl)-9H-carbazole-3-carboxamide
(6i)
Yield: 15%, M.P.; 279-281 C. IR (ATR, νmax, cm-1): 3390 (NH), 3262, 3065, 3000 (Ar-H),
2925, 2856 (C=C), 1671 (C=O), 1654, 1624, 1579, 1542, 1508, 1458, 1406, 1365, 1326,
1
1263, 1240 (Ar-O), 1169, 1096, 1072, 1021, 842, 786, 767, 741; H-NMR (400 MHz,
DMSO-d6,  ppm): 13.59 (s, 1H, NH), 11.89 (s, 1H, CO-NH), 9.10 (s, 1H, H-4 of carbazole),
8.19 (dd, 1H, J = 8.72 Hz, ArH), 7.76 (d, 1H J = 7.76 Hz, ArH), 7.59 (d, 1H, J = 8.64 Hz,
ArH), 7.20 (t, 1H, J = 7.84 Hz, ArH), 7.07 (d, 1H, J = 7.84 Hz, ArH), 4.02 (s, 3H, OCH3);
13C-NMR (100 MHz, DMSO-d6,  ppm): 145.8 (C=O), 142.51, 130.31, 126.06, 123.61,
122.43, 120.48, 112.72, 111.36, 107.22, 55.5 (OCH3); HRMS (ESI+) m/z: calculated for
C17H11F3N4O2S [M+H]+: 393.0695 found 393.0698.
Synthesis and spectral characterization of second series of compounds 3-(substitutedphenyl)-
5-((9-methyl-9H-carbazol-6-yl) methylene)-2-thioxothiazolidin-4-ones (15a-l) and 2-(9-
methyl-9H-carbazol-6-yl) methylene)-4-(5-substitutedphenyl-1,3,4-thiadiazol-2-yl)-5-thioxo-
thiazolidin-4-ones (16a-f) were reported in our recently published article [20]
.
25
26 Biological evaluation
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The synthesized final compounds (series-1: 4a-g; 6a-i) and (series-2: 15a-l and 16a-f) were
assessed for their antimicrobial activity against a panel of bacterial and fungal strains by
following MIC assay method using resazurin dye.[26] The protocols corresponding to
microorganisms, preparations of medium, solutions, inoculums, and standard assay by broth
micro-dilution method employed in this work were based on our earlier published paper.[22]
The antifungal and antibacterial activity on the clinical isolates was carried out at the
Department of Microbiology, Inkosi Albert Luthuli Hospital, Durban, South Africa.
20
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10.1002/cbdv.201900550
Chemistry & Biodiversity
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Supporting Information
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Spectral images of FT-IR, H-NMR, 13C-NMR and HRMS are provided in the Supporting
Information Section.
4
5
Acknowledgements
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Authors are grateful to Discipline of Pharmaceutical Sciences, College of Health Sciences,
University of KwaZulu-Natal (UKZN), Durban, South Africa for providing access to
necessary facilities. Author BC gratefully acknowledge National Research Foundation
(NRF), South Africa (SA) for research funding in the form of NRF-Innovation Post-Doctoral
Research Fellowship (Grant No. 99546). RK is also thankful to NRF-SA for funding this
project (Grant No. 103728 and 112079). Authors express heartfelt thanks to Mr. Dilip
Jagjivan and Dr. Caryl Janse Van Rensburg (UKZN, South Africa) for their assistance in
NMR and HRMS experiments.
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15 Author Contribution Statement
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Mahamadhanif S. Shaikh and Mahesh B. Palkar synthesized the compounds. Balakumar
Chandrasekaran, Ashish M. Kanhed and Parvesh Singh contributed to the data analysis and
manuscript drafting. Afsana Kajee, Koleka P. Mlisana, Meenu Ghai, and Mavela Cleopus
Mahlalela contributed to the biological experiments and data analysis. Rajshekhar
Karpoormath designed the experiments and supervised the project.
Conflict of interest: Authors declare that they have no conflict of interest.
24 References
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Graphical Illustration
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Novel carbazole analogs were synthesized and characterized by spectral methods.
Antimicrobial evaluation was conducted for these hybrids against four bacterial, four fungal
and a couple of clinical isolates. The lead compound 15i displayed a prominent antimicrobial
activity against the clinical isolates of C. neoformans (MIC = 6.25 µg/mL) and S. aureus
(MIC = 3.125 µg/mL) which explored the potential of carbazole-based antimicrobial agents.
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