Design, synthesis and evaluation of carbazole derivatives as potential antimicrobial agents

Article abstract of DOI:10.1080/14756366.2020.1850713

Five series of novel carbazole derivatives containing an aminoguanidine, dihydrotriazine, thiosemicarbazide, semicarbazide or isonicotinic moiety were designed, synthesised and evaluated for their antimicrobial activities. Most of the compounds exhibited potent inhibitory activities towards different bacterial strains (including one multidrug-resistant clinical isolate) and one fungal strain with minimum inhibitory concentrations (MICs) between 0.5 and 16 μg/ml. Compounds 8f and 9d showed the most potent inhibitory activities (MICs of 0.5–2 μg/ml). Furthermore, compounds 8b, 8d, 8f, 8k, 9b and 9e with antimicrobial activities were not cytotoxic to human gastric cancer cell lines (SGC-7901 and AGS) or a normal human liver cell line (L-02). Structure–activity relationship analyses and docking studies implicated the dihydrotriazine group in increasing the antimicrobial potency and reducing the toxicity of the carbazole compounds. In vitro enzyme activity assays suggested that compound 8f binding to dihydrofolate reductase might account for the antimicrobial effect.

Full text of DOI:10.1080/14756366.2020.1850713

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Design, synthesis and evaluation of carbazole
derivatives as potential antimicrobial agents
Yi-Jie Xue, Ming-Yue Li, Xue-Jun Jin, Chang-Ji Zheng & Hu-Ri Piao
To cite this article: Yi-Jie Xue, Ming-Yue Li, Xue-Jun Jin, Chang-Ji Zheng & Hu-Ri Piao
(2021) Design, synthesis and evaluation of carbazole derivatives as potential antimicrobial
agents, Journal of Enzyme Inhibition and Medicinal Chemistry, 36:1, 295-306, DOI:
10.1080/14756366.2020.1850713
To link to this article: https://doi.org/10.1080/14756366.2020.1850713
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2021, VOL. 36, NO. 1, 295–306
https://doi.org/10.1080/14756366.2020.1850713
RESEARCH PAPER
Design, synthesis and evaluation of carbazole derivatives as potential
antimicrobial agents
Yi-Jie Xuea,b, Ming-Yue Lia, Xue-Jun Jina, Chang-Ji Zhenga and Hu-Ri Piaoa
b
aKey Laboratory of Natural Medicines of the Changbai Mountain, Ministry of Education, Yanbian University, Yanji, China; School of Pharmacy,
Fudan University, Shanghai, China
ABSTRACT
ARTICLE HISTORY
Received 28 November 2018
Revised 2 November 2020
Accepted 9 November 2020
Five series of novel carbazole derivatives containing an aminoguanidine, dihydrotriazine, thiosemicarba-
zide, semicarbazide or isonicotinic moiety were designed, synthesised and evaluated for their antimicrobial
activities. Most of the compounds exhibited potent inhibitory activities towards different bacterial strains
(including one multidrug-resistant clinical isolate) and one fungal strain with minimum inhibitory concen-
trations (MICs) between 0.5 and 16 mg/ml. Compounds 8f and 9d showed the most potent inhibitory
activities (MICs of 0.5–2 mg/ml). Furthermore, compounds 8b, 8d, 8f, 8k, 9b and 9e with antimicrobial
activities were not cytotoxic to human gastric cancer cell lines (SGC-7901 and AGS) or a normal human
liver cell line (L-02). Structure–activity relationship analyses and docking studies implicated the dihydrotria-
zine group in increasing the antimicrobial potency and reducing the toxicity of the carbazole compounds.
In vitro enzyme activity assays suggested that compound 8f binding to dihydrofolate reductase might
account for the antimicrobial effect.
KEYWORDS
Carbazole; antibacterial
activities; antifungal
activities; structure–activity
relationship; cytotoxicity
1. Introduction
ubiquitous enzyme that catalyses the conversion of 7,8-dihydrofo-
late to 5,6,7,8-tetrahydrofolate, which is involved in metabolic
reactions such as purine and thymidine nucleotide biosyn-
thesis19,20. DHFR is required by all organisms to grow and multi-
ply; however, selective inhibitors of microbial enzymes have been
The steady increase of microbial pathogens that do not respond
to conventional treatments presents a significant threat to global
public health. Fungal and bacterial infections are becoming pro-
gressively more resistant towards currently available antimicrobial
medicines, such as antibiotics. Therefore, there is an urgent
requirement for new drugs that target these pathogens. Without
effective antimicrobials for prevention and treatment of infections,
medical procedures such as organ transplantation, chemotherapy,
diabetes management and routine surgery (e.g. caesarean sections
or hip replacements) have a significantly higher risk of morbidity
and mortality1. The scale of the threat of antimicrobial resistance
has led to the development of numerous strategies to conserve
and make more effective use of existing antibiotics and to pro-
mote the development of novel antimicrobials2. In 2010, the
Infectious Diseases Society of America (IDSA) launched the 10 ꢀ 20
initiative that called for the development of 10 novel, safe and
effective systemic antibiotics by 20203.
utilised as therapeutic agents.
Here, we report the design and synthesis of five series of novel
carbazole derivatives (Figure 1), totaling 30 compounds and the
subsequent in vitro evaluations of their antibacterial and antifun-
gal activities. Several different substituents were systematically
introduced onto the carbazole ring and their effects on the overall
antimicrobial activity were investigated.
2. Experimental
2.1. Chemistry
Carbazole alkaloids have attracted considerable attention in
medicinal chemistry because they exhibit a broad spectrum of
biological and pharmacological activities, including antibacterial,
antituberculous, antitumour, antioxidant and anti-inflammatory
properties4–13. In our previous studies, guanidines exhibited sig-
nificant antibacterial activity and triazine ring-containing com-
pounds displayed hypoglycaemic, antitumour and antibacterial
activities14,15. Dihydrotriazine compounds have a higher hydro-
phobicity compared with guanidine compounds and several mole-
cules containing this scaffold have been reported as antibacterial
agents16–18. Recent studies have identified dihydrotriazine-contain-
All reagents were obtained commercially and were used without
further purification. Solvents were dried according to standard
procedures. Reactions were monitored by thin-layer chromatog-
raphy (TLC) on silica gel plates. Melting points were determined in
1
open capillary tubes and were uncorrected. H NMR and 13C NMR
spectra were measured on an AV-300 (Bruker, Switzerland), and all
chemical shifts were given in ppm relative to TMS. Mass spectra
were measured on a HP1100LC (Agilent Technologies). High-reso-
lution mass spectra were measured on an MALDI-TOF/TOF mass
ing compounds as inhibitors of dihydrofolate reductase (DHFR), a spectrometer (Bruker Daltonik, Germany).
CONTACT Hu-Ri Piao
piaohr@ybu.edu.cn
Key Laboratory of Natural Medicines of the Changbai Mountain, Ministry of Education, Yanbian University College of
Pharmacy, Yanji 133002, China
ß 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

296
Y.-J. XUE ET AL.
Figure 1. The structures of the target compounds.
2.1.1. Procedure to synthesise compound 2a
2.1.5.2.
6-(9-Ethyl-9H-carbazol-3-yl)-N2,N2-dimethyl-3,6-dihydro-
To a solution of carbazole (1.03 g, 6.16 mmol) in DMC (10 ml),
DABCO (0.069 g, 0.62 mmol) was added and the resulting solution
was heated to 90–95 ꢁC for 24 h to give compound 2a21.
1,3,5-triazine-2,4-diamine (8 b). White powder; yield 61.6%; m.p.
256.9–259.8 ꢁC. IR (KBr) cmꢂ1: 3356, 3132 (NH2, NH), 1598 (C ¼ N).
1H NMR (300 MHz, DMSO-d6, ppm) d 8.80 (br s, 2H, NH2),
8.25–8.12 (m, 2H, Ar-H), 7.68 (dd, 2H, J ¼ 15.0, 8.4 Hz, Ar-H), 7.51
(ddd, 2H, J ¼ 16.2, 8.3, 1.5 Hz, Ar-H), 7.24 (t, 1H, J ¼ 7.4 Hz, Ar-H),
5.98 (s, 1H, CH), 5.76 (s, 1H, NH), 4.48 (q, 2H, J ¼ 7.1 Hz, CH2), 3.08
(s, 6H, CH3), 1.31 (t, 3H, J ¼ 7.0 Hz, CH3). 13 C NMR (75 MHz, DMSO-
d6, ppm) d 157.66, 155.83, 139.95, 139.66, 131.06, 126.02, 123.98,
121.95, 121.82, 120.27, 118.98, 118.21, 109.34 (2 C), 62.76, 37.06,
36.94 (2 C), 13.67. HRMS (MALDI) calcd for C19H22N6 [M þ H]þ:
335.1979, found: 335.1967.
2.1.2. Procedure to synthesise compound 2 b
A solution of potassium hydroxide (1.21 g, 21.56 mmol) and bro-
moethane (1.01 g, 9.24 mmol) in acetone (20 ml) was added to car-
bazole (1.03 g, 6.16 mmol) and the mixture was stirred at room
temperature for 2 h to give compound 2b22.
2.1.5.3. 6-(9-Benzyl-9H-carbazol-3-yl)-N2,N2-dimethyl-3,6-dihydro-
1,3,5-triazine-2,4-diamine (8c). White powder; yield 46.1%; m.p.
237.1–238.8 ꢁC. IR (KBr) cmꢂ1: 3440, 3240 (NH2, NH), 1606 (C ¼ N).
1H NMR (300 MHz, DMSO-d6, ppm) d 8.83 (br s, 2H, NH2),
8.27–8.16 (m, 2H, Ar-H), 7.70 (dd, 2H, J ¼ 11.5, 8.4 Hz, Ar-H),
7.55–7.44 (m, 2H, Ar-H), 7.25 (dtd, 4H, J ¼ 8.1, 6.5, 4.4 Hz, Ar-H),
7.16 (dd, 2H, J ¼ 8.0, 1.7 Hz, Ar-H), 5.97 (s, 1H, CH), 5.76 (s, 1H, NH),
5.70 (s, 2H, CH2), 3.07 (s, 6H, CH3). 13 C NMR (75 MHz, DMSO-d6,
ppm) d 157.62, 155.85, 140.60, 140.29, 137.65, 131.37, 128.59 (2 C),
127.28 (2 C), 126.71, 126.20, 124.18, 122.01, 121.92, 120.30, 119.35,
118.29, 109.84 (2 C), 62.76, 45.67, 36.96, 33.9. HRMS (MALDI) calcd
for C24H24N6 [M þ H]þ: 397.2135, found: 397.2134.
2.1.3. General procedure to synthesise compounds 2c–k
To a solution of carbazole (1.67 g, 9.00 mmol) in DMF (20 ml) fol-
lowed by addition of NaH (400 mg, 16.50 mmol) in small portions
at 0 ꢁC, over 5 min, benzyl halide (9.90 mmol) was added drop
wise at 0 ꢁC, over 5 min, the mixture was stirred at room tempera-
ture for 8–18 h to give compounds 2c–k23.
2.1.4. General procedure to synthesise compounds 4a–l
In
a
three-necked round-bottomed flask, POCl3 (700 mg,
4.56 mmol) was added dropwise to DMF (400 mg, 5.42 mmol)
while stirring at 0 ꢁC. The mixture was then stirred at room tem-
perature for 1 h. A solution of 9-substituted carbazole (1.93 mmol)
was slowly added to this mixture over 5 min at 45 ꢁC, and then
the temperature was raised to 95 ꢁC for 8–18 h to yield com-
pounds 4a–l23.
2.5.1.4.
N2,N2-Dimethyl-6-(9-(4-methylbenzyl)-9H-carbazol-3-yl)-
3,6-dihydro-1,3,5-triazine-2,4-diamine (8d). White powder; yield
58.8%; m.p. 237.2–239.8 ꢁC. IR (KBr) cmꢂ1: 3350, 3037 (NH2, NH),
1609 (C ¼ N). 1H NMR (300 MHz, DMSO-d6, ppm) d 8.88 (s, 1H,
NH2), 8.82 (s, 1H, NH2), 8.32–8.14 (m, 2H, Ar-H), 7.69 (dd, 2H,
J ¼ 12.8, 8.2 Hz, Ar-H), 7.49 (dd, 2H, J ¼ 19.7, 8.4 Hz, Ar-H), 7.25 (t,
1H, J ¼ 7.4 Hz, Ar-H), 7.07 (d, 4H, J ¼ 4.7 Hz, Ar-H), 5.97 (s, 1H, CH),
5.77 (s, 1H, NH), 5.65 (s, 2H, CH2), 3.07 (s, 6H, CH3), 2.22 (s, 3H,
CH3). 13 C NMR (75 MHz, DMSO-d6, ppm) d 158.06, 156.35, 141.06,
140.77, 136.92, 135.05, 131.69, 129.58 (2 C), 127.20 (2 C), 126.63,
124.63, 122.47, 122.40, 120.75, 119.77, 118.79, 110.33 (2 C), 63.34,
55.44, 45.95, 37.41, 21.09. HRMS (MALDI) calcd for C25H26N6
[M þ H]þ: 411.2292, found: 411.2299.
2.1.5. General procedure to synthesise compounds 8a–l
A mixture of the required compound 4 (2 mmol) and metformin
hydrochloride (2 mmol) in glacial acetic acid (20 ml) was heated
under reflux at 120 ꢁC for 4–8 h (completion of the reaction was
monitored by TLC). The solvent was then removed under reduced
pressure. The crude products were purified by silica gel column
chromatography using dichloromethane:methanol (20:1) as
the eluent.
2.5.1.5. 6-(9-(2-Fluorobenzyl)-9H-carbazol-3-yl)-N2,N2-dimethyl-3,6-
dihydro-1,3,5-triazine-2,4-diamine (8e). White powder; yield 40.1%;
m.p. 201.2–205.0 ꢁC. IR (KBr) cmꢂ1: 3423, 3144 (NH2, NH), 1603
(C ¼ N). 1H NMR (300 MHz, DMSO-d6, ppm) d 8.90 (s, 1H, NH2),
8.84 (s, 1H, NH2), 8.30–8.16 (m, 2H, Ar-H), 7.68 (dd, 2H, J ¼ 13.2,
8.3 Hz, Ar-H), 7.56–7.43 (m, 2H, Ar-H), 7.33–7.22 (m, 3H, Ar-H), 7.03
(td, 1H, J ¼ 7.4, 1.6 Hz, Ar-H), 6.88–6.78 (m, 1H, Ar-H), 5.97 (s, 1H,
CH), 5.77 (s, 3H, NH and CH2), 3.07 (s, 6H, CH3). 13 C NMR (75 MHz,
DMSO-d6, ppm) d 161.69, 158.44, 157.61, 155.96, 140.57, 140.28,
131.43, 129.50 (d, 1 C, J ¼ 8.1 Hz), 128.71 (d, 1 C, J ¼ 4.2 Hz), 126.23,
124.57 (d, 1 C, J ¼ 3.4 Hz), 124.22, 124.04, 122.08 (d, 1 C, J ¼ 2.3 Hz),
2.1.5.1. N2,N2-Dimethyl-6-(9-methyl-9H-carbazol-3-yl)-3,6-dihydro-
1,3,5-triazine-2,4-diamine (8a). White powder; yield 52.3%; m.p.
267.3–268.9 ꢁC. IR (KBr) cmꢂ1: 3300, 3134 (NH2, NH), 1606 (C ¼ N).
1H NMR (300 MHz, DMSO-d6, ppm) d 8.88 (br s, 2H, NH2),
8.26–8.11 (m, 2H, Ar-H and NH), 7.76–7.46 (m, 5H, Ar-H), 7.25 (t,
1H, J ¼ 7.6 Hz, Ar-H), 5.99 (s, 1H, CH), 3.90 (s, 3H, CH3), 3.08 (s, 6H,
CH3). 13 C NMR (75 MHz, DMSO-d6, ppm) d 157.59, 155.85, 141.05,
140.75, 131.06, 126.01, 123.96, 121.75, 121.63, 120.13, 119.01,
118.02, 109.39 (2 C), 62.77, 36.93 (2 C), 29.12. HRMS (MALDI) calcd
for C18H20N6 [M þ H]þ: 321.1822, found: 321.1806.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
297
120.28, 119.50, 118.34, 115.65, 115.37, 109.75, 62.92, 62.01, 56.01, NH2), 8.99 (s, 1H, NH2), 8.34–8.13 (m, 2H, Ar-H), 7.68 (dd, 2H,
36.95. HRMS (MALDI) calcd for C24H23FN6 [M þ H]þ: 415.2041, J ¼ 15.2, 8.3 Hz, Ar-H), 7.63–7.41 (m, 5H, Ar-H), 7.25 (t, 1H,
found: 415.2037.
J ¼ 7.5 Hz, Ar-H), 7.17–7.06 (m, 2H, Ar-H and NH), 5.99 (s, 1H, CH),
5.69 (s, 2H, CH2), 3.07 (s, 6H, CH3). 13 C NMR (75 MHz, DMSO-d6,
2.5.1.6. 6-(9-(2,4-Dichlorobenzyl)-9H-carbazol-3-yl)-N2,N2-dimethyl-
ppm) d 157.53, 155.87, 140.47, 140.20, 137.10, 131.49(4 C), 128.91
(2 C), 126.28, 124.27, 122.04, 121.98, 120.37, 119.49, 118.39, 109.79
3,6-dihydro-1,3,5-triazine-2,4-diamine (8f). White powder; yield
40.1%; m.p. 255.2–257.1 ꢁC. IR (KBr) cmꢂ1: 3240, 3060 (NH2, NH),
(2 C), 62.86, 45.04, 36.94 (2 C). HRMS (MALDI) calcd for C24H23BrN6
[M þ H]þ: 475.1240, found: 475.1230.
1
1603 (C ¼ N). H NMR (300 MHz, DMSO-d6, ppm) d 8.93–8.72 (br s,
2H, NH2), 8.35–8.18 (m, 2H, Ar-H), 7.75 (d, 1H, J ¼ 2.1 Hz, Ar-H),
2.1.5.11. 4-((3-(4-Amino-6-(dimethylamino)-2,5-dihydro-1,3,5-tria-
7.68–7.17 (m, 7H, Ar-H), 6.33 (d, 1H, J ¼ 8.4 Hz, CH), 5.97 (s, 1H,
NH), 5.76 (s, 2H, CH2), 3.08 (s, 6H, CH3). 13C NMR (75 MHz, DMSO-
zin-2-yl)-9H-carbazol-9-yl)methyl)benzonitrile (8k). White powder;
yield 52.9%; m.p. 234.1–235.4 ꢁC. IR (KBr) cmꢂ1: 3306, 3061 (NH2,
d6, ppm) d 157.55, 155.97, 140.52, 140.25, 133.76, 132.77, 132.69,
131.74, 129.10, 128.50, 127.69, 126.43, 124.41, 122.17, 120.43 (2 C),
119.76, 118.48, 109.63 (2 C), 62.92, 45.32, 43.47, 36.95. HRMS
(MALDI) calcd for C24H22Cl2N6 [M þ H]þ: 465.1356, found:
465.1359. Purity: 97.41% by HPLC (A: 0.1% FA in H2O; B: 0.1% FA
in CH3CN, graded: 20–100%), tR 12.273 min, k: 250 nm.
1
NH), 1605 (C ¼ N). H NMR (300 MHz, DMSO-d6, ppm) d 9.06 (s, 1H,
NH2), 9.01 (s, 1H, NH2), 8.35–8.15 (m, 2H, Ar-H), 7.78–7.62 (m, 4H,
Ar-H), 7.56 (d, 1H, J ¼ 8.4 Hz, Ar-H), 7.51–7.43 (m, 1H, Ar-H),
7.36–7.23 (m, 3H, Ar-H), 6.01 (s, 1H, CH), 5.83 (s, 2H, CH2), 5.77 (s,
1H, NH), 3.09 (s, 6H, CH3). 13 C NMR (75 MHz, DMSO-d6, ppm) d
158.06, 156.33, 143.92, 140.95, 140.66, 133.04 (2 C), 132.15, 127.98
(2 C), 126.82, 124.80, 122.58, 122.54, 120.87, 120.10, 119.11, 118.88,
110.59, 110.18 (2 C), 63.28, 55.41, 49.03, 37.42. HRMS (MALDI) calcd
for C25H23N7 [M þ H]þ: 422.2088, found: 422.2077.
2.1.5.7. 6-(9-(2-Chlorobenzyl)-9H-carbazol-3-yl)-N2,N2-dimethyl-3,6-
dihydro-1,3,5-triazine-2,4-diamine (8 g). White powder; yield
37.6%; m.p. 240.7–243.2 ꢁC. IR (KBr) cmꢂ1: 3396, 3211 (NH2, NH),
1603 (C ¼ N). 1H NMR (300 MHz, DMSO-d6, ppm) d 8.86 (s, 1H,
NH2), 8.81 (s, 1H, NH2), 8.38–8.20 (m, 2H, Ar-H), 7.70–7.39 (m, 6H,
Ar-H), 7.37–7.23 (m, 2H, Ar-H), 7.11 (dd, 1H, J ¼ 8.8, 6.3 Hz, Ar-H),
6.36 (d, 1H, J ¼ 8.0 Hz, CH), 5.98 (s, 1H, NH), 5.84–5.75 (m, 2H, CH2),
3.09 (s, 6H, CH3). 13 C NMR (75 MHz, DMSO-d6, ppm) d 157.99,
156.37, 141.07, 140.80, 134.95, 132.24, 132.07, 130.10, 129.52,
127.97, 127.59, 126.86, 124.86, 122.57, 122.54, 120.90, 120.13,
118.95, 110.13 (2 C), 63.36, 55.43, 44.22, 37.42. HRMS (MALDI) calcd
for C24H23ClN6 [M þ H]þ: 431.1745, found: 431.1733.
2.1.5.12.
6-(6-Bromo-9-phenyl-9H-carbazol-3-yl)-N2,N2-dimethyl-
3,6-dihydro-1,3,5-triazine-2,4-diamine (8 l). White powder; yield
41.4%; m.p. 221.5–224.8 ꢁC. IR (KBr) cmꢂ1: 3383, 3070 (NH2, NH),
1600 (C ¼ N). 1H NMR (300 MHz, DMSO-d6, ppm) d 9.19 (s, 2H,
NH2), 8.44 (d, 2H, J ¼ 19.4 Hz, Ar-H), 7.64 (dd, 4H, J ¼ 15.7, 8.1 Hz,
Ar-H), 7.55 (d, 3H, J ¼ 7.6 Hz, Ar-H), 7.39 (d, 1H, J ¼ 8.4 Hz, Ar-H),
7.29 (t, 1H, J ¼ 6.9 Hz, Ar-H), 6.05 (s, 1H, CH), 5.76 (s, 1H, NH), 3.08
(s, 6H, CH3). 13 C NMR (75 MHz, DMSO-d6, ppm) d 158.01, 156.25,
141.08, 139.79, 136.62, 133.51, 130.71 (2 C), 129.41, 128.52, 127.02
(2 C), 125.85, 124.88, 123.47, 121.88, 119.35, 112.82, 112.18, 110.60,
63.04, 55.41, 49.03. HRMS (MALDI) calcd for C23H21BrN6 [M þ H]þ:
461.1084, found: 461.1066. Purity: 98.69% by HPLC (A: 0.1% FA in
H2O; B: 0.1% FA in CH3CN, graded: 20–100%), tR 11.660 min,
k: 250 nm.
2.1.5.8. 6-(9-(3-Chlorobenzyl)-9H-carbazol-3-yl)-N2,N2-dimethyl-3,6-
dihydro-1,3,5-triazine-2,4-diamine (8 h). White powder; yield
54.9%; m.p. 207.0–209.2 ꢁC. IR (KBr) cmꢂ1: 3313, 3029 (NH2, NH),
1600 (C ¼ N). 1H NMR (300 MHz, DMSO-d6, ppm) d 8.87 (s, 1H,
NH2), 8.81 (s, 1H, NH2), 8.34–8.16 (m, 2H, Ar-H), 7.71 (dd, 2H,
J ¼ 13.6, 8.3 Hz, Ar-H), 7.59–7.45 (m, 2H, Ar-H), 7.39–7.20 (m, 4H,
Ar-H), 7.06 (dd, 1H, J ¼ 6.3, 2.9 Hz, Ar-H), 5.97 (s, 1H, CH), 5.77 (s,
1H, NH), 5.74 (s, 2H, CH2), 3.12 (s, 6H, CH3). 13 C NMR (75 MHz,
DMSO-d6, ppm) d 158.06, 156.36, 140.96, 140.74, 140.69, 133.67,
131.94, 131.03, 127.78, 127.03, 126.80, 125.77, 124.82, 122.52,
122.46, 120.85, 120.02, 118.91, 110.23 (2 C), 63.34, 55.44, 45.55,
37.42. HRMS (MALDI) calcd for C24H23ClN6 [M þ H]þ: 431.1745,
found: 431.1749.
2.1.6. General procedure to synthesise compounds 9a–k
A mixture of the required compound 4 (2.3 mmol) and aminogua-
nidine hydrochloride (2 mmol) in ethanol (20 ml) in the presence
of 5 drops of concentrated hydrochloric acid was stirred at
50–60 ꢁC for 8–12 h. The solvent was then removed under reduced
pressure. The crude products were purified by silica gel column
chromatography using dichloromethane:methanol (10:1) as
the eluent.
2.1.5.9. 6-(9-(4-Chlorobenzyl)-9H-carbazol-3-yl)-N2,N2-dimethyl-3,6-
dihydro-1,3,5-triazine-2,4-diamine (8i). White powder; yield 36.7%;
m.p. 262.4–264.1 ꢁC. IR (KBr) cmꢂ1: 3373, 3059 (NH2, NH), 1603
(C ¼ N). 1H NMR (300 MHz, DMSO-d6, ppm) d 9.00 (s, 1H, NH2),
8.94 (s, 1H, NH2), 8.36–8.14 (m, 2H, Ar-H), 7.69 (dd, 2H, J ¼ 14.6,
8.2 Hz, Ar-H), 7.60–7.43 (m, 3H, Ar-H), 7.35 (dd, 2H, J ¼ 8.6, 2.1 Hz,
Ar-H), 7.29–7.13 (m, 3H, Ar-H and NH), 5.98 (s, 1H, CH), 5.72 (s, 2H,
CH2), 3.07 (s, 6H, CH3). 13 C NMR (75 MHz, DMSO-d6, ppm) d
158.02, 156.35, 140.95, 140.67, 137.15, 132.36, 131.92, 129.05 (4 C),
126.75, 124.73, 122.52, 122.46, 120.83, 119.96, 118.85, 110.26 (2 C),
63.32, 49.04, 45.47, 37.41. HRMS (MALDI) calcd for C24H23ClN6
[M þ H]þ: 431.1745, found: 431.1761.
2.1.6.1. (E)-2-((9-Methyl-9H-carbazol-3-yl)methylene)hydrazine-1-
carboximidamide (9a). White powder; yield 52.3%; m.p. 199.1-
200.8 ꢁC. IR (KBr) cmꢂ1: 3330, 3147 (NH2, NH), 1629 (C ¼ N). 1H
NMR (300 MHz, DMSO-d6, ppm) d 11.89 (br s, 1H, NH), 8.63 (s, 1H,
CH ¼ N), 8.33 (s, 1H, Ar-H), 8.18 (d, 1H, J ¼ 8.0 Hz, Ar-H), 8.05 (d,
1H, J ¼ 8.4 Hz, Ar-H), 7.91–7.46 (m, 6H, Ar-H and NH), 7.29 (d, 1H,
J ¼ 7.5 Hz, Ar-H), 3.94 (s, 3H, CH3). 13 C NMR (75 MHz, DMSO-d6,
ppm) d 155.41, 147.77, 141.81, 141.07, 126.20, 125.14, 124.36,
122.08, 121.96, 120.59, 120.36, 119.41, 109.53, 109.40, 29.16. HRMS
(MALDI) calcd for C15H15N5 [M þ H]þ: 266.1400, found: 266.1405.
6-(9-(4-Bromobenzyl)-9H-carbazol-3-yl)-N2,N2-dimethyl-
2.1.6.2. (E)-2-((9-Ethyl-9H-carbazol-3-yl)methylene)hydrazine-1-car-
2.1.5.10.
boximidamide (9b). White powder; yield 59.2%; m.p.
3,6-dihydro-1,3,5-triazine-2,4-diamine (8j). White powder; yield
35.1%; m.p. 248.7–252.1 ꢁC. IR (KBr) cmꢂ1: 3311, 3054 (NH2, NH), 199.4–200.5 ꢁC. IR (KBr) cmꢂ1: 3354, 3120 (NH2, NH), 1628 (C ¼ N).
1603 (C ¼ N). 1H NMR (300 MHz, DMSO-d6, ppm) d 9.06 (s, 1H, 1H NMR (300 MHz, DMSO-d6, ppm) d 11.85 (br s, 1H, NH), 8.63 (s,

298
Y.-J. XUE ET AL.
1H, CH ¼ N), 8.33 (d, 1H, J ¼ 1.9 Hz, Ar-H), 8.18 (d, 1H, J ¼ 7.7 Hz, 109.73, 43.59. HRMS (MALDI) calcd for C21H17Cl2N5 [M þ H]þ:
Ar-H), 8.03 (d, 1H, J ¼ 8.6 Hz, Ar-H), 7.77–7.46 (m, 6H, Ar-H and 410.0934, found: 410.0932.
NH), 7.27 (t, 1H, J ¼ 7.3 Hz, Ar-H), 4.49 (q, 2H, J ¼ 6.9 Hz, CH2), 1.33
(t, 3H, J ¼ 7.1 Hz, CH3). 13 C NMR (75 MHz, DMSO-d6, ppm) d 155.42,
2.1.6.7. (E)-2-((9-(2-Chlorobenzyl)-9H-carbazol-3-yl)methylene)hy-
147.76, 140.83, 140.04, 131.54, 128.68, 126.26, 125.23, 124.44,
drazine-1-carboximidamide (9 g). White powder; yield 45.4%; m.p.
249.7–250.4 ꢁC. IR (KBr) cmꢂ1: 3386, 3159 (NH2, NH), 1629 (C ¼ N).
122.31, 120.72, 120.53, 119.42, 109.56, 109.40, 37.18, 18.68. HRMS
(MALDI) calcd for C16H17N5 [M þ H]þ: 280.1557, found: 280.1548.
1H NMR (300 MHz, DMSO-d6, ppm) d 11.88 (br s, 1H, NH), 8.70 (s,
1H, CH ¼ N), 8.33 (s, 1H, Ar-H), 8.24 (d, 1H, J ¼ 7.6 Hz, Ar-H), 8.00
2.1.6.3.
(E)-2-((9-Benzyl-9H-carbazol-3-yl)methylene)hydrazine-1-
(dd, J ¼ 8.6, 1H, 1.7 Hz, Ar-H), 7.96–7.43 (m, 7H, Ar-H and NH),
7.37–7.24 (m, 2H, Ar-H), 7.18–7.06 (m, 1H, Ar-H), 6.51–6.40 (m, 1H,
Ar-H), 5.77 (s, 2H, CH2). 13 C NMR (75 MHz, DMSO-d6, ppm) d
155.88, 147.98, 141.96, 141.05, 134.82, 132.26, 130.10, 129.55,
127.95, 127.71, 126.99, 125.94, 125.55, 122.97, 122.83, 121.20,
121.10, 120.45, 110.29, 110.18, 44.30. HRMS (MALDI) calcd for
C21H18ClN5 [M þ H]þ: 376.1326, found: 376.1322.
carboximidamide (9c). White powder; yield 63.1%; m.p.
271.0–272.4 ꢁC. IR (KBr) cmꢂ1: 3319, 3154 (NH2, NH), 1600 (C ¼ N).
1H NMR (300 MHz, DMSO-d6, ppm) d 11.91 (br s, 1H, NH), 8.67 (s,
1H, CH ¼ N), 8.34 (s, 1H, Ar-H), 8.21 (d, 1H, J ¼ 7.7 Hz, Ar-H), 8.01
(d, 1H, J ¼ 8.5 Hz, Ar-H), 7.84–7.65 (m, 5H, Ar-H and NH), 7.47 (t,
1H, J ¼ 7.7 Hz, Ar-H), 7.24 (dt, 6H, J ¼ 18.8, 7.5 Hz, Ar-H), 5.71 (s, 2H,
CH2). 13 C NMR (75 MHz, DMSO-d6, ppm) d 155.82, 148.14, 141.94,
141.09, 137.95, 132.14, 131.97, 129.05, 127.80, 127.21, 126.85,
125.85, 125.26, 122.87, 122.73, 121.10, 120.99, 120.21, 110.50,
110.33, 46.20. HRMS (MALDI) calcd for C21H19N5 [M þ H]þ:
342.1713, found: 342.1701.
2.1.6.8. (E)-2-((9-(3-Chlorobenzyl)-9H-carbazol-3-yl)methylene)hy-
drazine-1-carboximidamide (9 h). White powder; yield 41.5%; m.p.
256.1–257.5 ꢁC. IR (KBr) cmꢂ1: 3389, 3137 (NH2, NH), 1634 (C ¼ N).
1H NMR (300 MHz, DMSO-d6, ppm) d 11.93 (s, 1H, NH), 8.68 (s, 1H,
CH ¼ N), 8.33 (s, 1H, Ar-H), 8.22 (d, 1H, J ¼ 7.7 Hz, Ar-H), 8.09–7.58
(m, 6H, Ar-H and NH), 7.49 (t, 1H, J ¼ 7.7 Hz, Ar-H), 7.29 (t, 4H,
J ¼ 6.0 Hz, Ar-H), 7.08 (dd, 1H, J ¼ 6.6, 3.1 Hz, Ar-H), 5.75 (s, 2H,
CH2). 13 C NMR (75 MHz, DMSO-d6, ppm) d 155.89, 148.00, 141.82,
140.97, 140.57, 133.66, 131.02, 127.81, 127.07, 126.96, 125.94,
125.83, 125.46, 122.91, 122.77, 121.19, 121.07, 120.38, 110.43,
110.28, 45.59. HRMS (MALDI) calcd for C21H18ClN5 [M þ H]þ:
376.1326, found: 376.1319.
2.1.6.4. (E)-2-((9-(4-Methylbenzyl)-9H-carbazol-3-yl)methylene)hy-
drazine-1-carboximidamide (9d). White powder; yield 45.0%; m.p.
295.5–298.0 ꢁC. IR (KBr) cmꢂ1: 3382, 3134 (NH2, NH), 1629 (C ¼ N).
1H NMR (300 MHz, DMSO-d6, ppm) d 11.82 (br s, 1H, NH), 8.59 (s,
1H, CH ¼ N), 8.25 (s, 1H, Ar-H), 8.13 (d, 1H, J ¼ 7.7 Hz, Ar-H), 7.94
(dd, 1H, J ¼ 8.7, 1.6 Hz, Ar-H), 7.75–7.58 (m, 4H, Ar-H and NH), 7.40
(t, 2H, J ¼ 7.7 Hz, Ar-H), 7.20 (t, 1H, J ¼ 7.4 Hz, Ar-H), 7.01 (s, 4H, Ar-
H), 5.59 (s, 2H, CH2), 2.14 (s, 3H, CH3). 13 C NMR (75 MHz, DMSO-d6,
ppm) d 155.46, 147.63, 141.47, 140.63, 136.53, 134.46, 129.14 (2 C),
126.81 (2 C), 126.37, 125.39, 124.78, 122.43, 122.30, 120.65, 120.54,
119.71, 110.09, 109.91, 45.56, 20.63. HRMS (MALDI) calcd for
C22H21N5 [M þ H]þ: 356.1869, found: 356.1854. Purity: 96.74% by
HPLC (A: 0.1% FA in H2O; B: 0.1% FA in CH3CN, graded: 20–100%),
tR 11.533 min, k: 250 nm.
2.1.6.9. (E)-2-((9-(4-Chlorobenzyl)-9H-carbazol-3-yl)methylene)hy-
drazine-1-carboximidamide (9i). White powder; yield 38.7%; m.p.
239.5–240.8 ꢁC. IR (KBr) cmꢂ1: 3266, 3057 (NH2, NH), 1629 (C ¼ N).
1H NMR (300 MHz, DMSO-d6, ppm) d 12.07 (br s, 1H, NH), 8.68 (s,
1H, CH ¼ N), 8.35 (d, 1H, J ¼ 5.4 Hz, Ar-H), 8.21 (d, 1H, J ¼ 7.7 Hz,
Ar-H), 8.01 (d, 1H, J ¼ 8.3 Hz, Ar-H), 7.68 (m, 5H, Ar-H and NH), 7.47
(t, 1H, J ¼ 7.7 Hz, Ar-H), 7.38–7.24 (m, 3H, Ar-H), 7.18 (d, 2H,
J ¼ 8.7 Hz, Ar-H), 5.72 (s, 2H, CH2). 13 C NMR (75 MHz, DMSO-d6,
ppm) d 155.44, 147.58, 141.38, 140.53, 136.56, 131.96, 128.66 (2 C),
128.62 (2 C), 126.48, 125.47, 124.96, 122.49, 122.35, 120.72, 120.62,
119.88, 110.02, 109.86, 45.08. HRMS (MALDI) calcd for C21H18ClN5
[M þ H]þ: 376.1326, found: 376.1329.
2.1.6.5. (E)-2-((9-(2-Fluorobenzyl)-9H-carbazol-3-yl)methylene)hy-
drazine-1-carboximidamide (9e). White powder; yield 39.3%; m.p.
248.3–249.9 ꢁC. IR (KBr) cmꢂ1: 3356, 3163 (NH2, NH), 1637 (C ¼ N).
1H NMR (300 MHz, DMSO-d6, ppm) d 11.88 (s, 1H, NH2), 8.67 (s,
1H, CH ¼ N), 8.32 (s, 1H, Ar-H), 8.21 (d, 1H, J ¼ 7.8 Hz, Ar-H), 8.02
(dd, 1H, J ¼ 8.7, 1.6 Hz, Ar-H), 7.89–7.41 (m, 6H, Ar-H and NH),
7.37–7.19 (m, 3H, Ar-H), 7.04 (td, 1H, J ¼ 7.4, 1.4 Hz, Ar-H), 6.89 (td,
1H, J ¼ 7.7, 1.7 Hz, Ar-H), 5.77 (d, 2H, J ¼ 2.2 Hz, CH2). 13 C NMR
(75 MHz, DMSO-d6, ppm) d 161.68, 158.43, 155.40, 147.52, 141.38,
140.51, 128.75 (d, 1 C, J ¼ 4.1 Hz), 126.40, 125.36, 124.93, 124.57 (d,
1 C, J ¼ 3.3 Hz), 124.12, 123.92, 122.43, 122.30, 120.58 (d, 1 C,
J ¼ 8.3 Hz), 119.85, 115.67, 115.39, 109.89 (d, 1 C, J ¼ 8.6 Hz), 65.01.
HRMS (MALDI) calcd for C21H18FN5 [M þ H]þ: 360.1619,
found: 360.1611.
2.1.6.10. (E)-2-((9-(4-Bromobenzyl)-9H-carbazol-3-yl)methylene)hy-
drazine-1-carboximidamide (9j). White powder; yield 41.1%; m.p.
239.5–240.7 ꢁC. IR (KBr) cmꢂ1: 3377, 3161 (NH2, NH), 1629 (C ¼ N).
1H NMR (300 MHz, DMSO-d6, ppm) d 11.86 (s, 1H, NH), 8.67 (s, 1H,
CH ¼ N), 8.32 (s, 1H, Ar-H), 8.21 (d, 1H, J ¼ 7.7 Hz, Ar-H), 8.09–7.98
(m, 1H, Ar-H), 7.97–7.56 (m, 5H, Ar-H and NH), 7.48 (dt, 3H, J ¼ 7.0,
3.5 Hz, Ar-H), 7.29 (t, 1H, J ¼ 7.5 Hz, Ar-H), 7.13 (d, 2H, J ¼ 8.5 Hz,
Ar-H), 5.72 (s, 2H, CH2). 13 C NMR (75 MHz, DMSO-d6, ppm) d
155.43, 147.62, 141.39, 140.54, 136.98, 131.54 (2 C), 129.00 (2 C),
126.49, 125.48, 124.96, 122.49, 122.35, 120.71, 120.61, 120.46,
119.89, 110.02, 109.85, 45.16. HRMS (MALDI) calcd for C21H18BrN5
[M þ H]þ: 420.0818, found: 420.0829.
2.1.6.6. (E)-2-((9-(2,4-Dichlorobenzyl)-9H-carbazol-3-yl)methylene)-
hydrazine-1-carboximidamide (9f). White powder; yield 58.2%;
m.p. 240.7–242.8 ꢁC. IR (KBr) cmꢂ1: 3397, 3146 (NH2, NH), 1629
(C ¼ N). 1H NMR (300 MHz, DMSO-d6, ppm) d 12.12 (br s, 1H, NH),
8.71 (s, 1H, CH ¼ N), 8.35 (s, 1H, Ar-H), 8.24 (d, 1H, J ¼ 7.7 Hz, Ar-H),
8.11–7.66 (m, 5H, Ar-H and NH), 7.59 (d, 1H, J ¼ 8.6 Hz, Ar-H),
7.54–7.41 (m, 2H, Ar-H), 7.29 (t, 1H, J ¼ 7.3 Hz, Ar-H), 7.19 (dd, 1H,
J ¼ 8.3, 2.3 Hz, Ar-H), 6.41 (d, 1H, J ¼ 8.4 Hz, Ar-H), 5.75 (s, 2H, CH2).
2.1.6.11. (E)-2-((9-(4-Cyanobenzyl)-9H-carbazol-3-yl)methylene)hy-
drazine-1-carboximidamide (9k). White powder; yield 45.7%; m.p.
240.6–243.9 ꢁC. IR (KBr) cmꢂ1: 3300, 3123 (NH2, NH), 1631 (C ¼ N).
1H NMR (300 MHz, DMSO-d6, ppm) d 11.92 (br s, 1H, NH), 8.68 (s,
13 C NMR (75 MHz, DMSO-d6, ppm) d 155.52, 147.63, 141.45, 1H, CH ¼ N), 8.33 (s, 1H, Ar-H), 8.22 (d, 1H, J ¼ 7.7 Hz, Ar-H),
140.55, 133.66, 132.83, 132.76, 129.13, 128.66, 127.72, 126.61, 8.17–7.55 (m, 8H, Ar-H and NH), 7.48 (t, 1H, J ¼ 7.7 Hz, Ar-H), 7.30
125.56, 125.24, 122.63, 122.46, 120.74, 120.68, 120.12, 109.83, (dd, 3H, J ¼ 7.9, 2.2 Hz, Ar-H), 5.85 (s, 2H, CH2). 13 C NMR (75 MHz,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
299
DMSO-d6, ppm) d 155.31, 147.68, 143.30, 141.37, 140.50, 132.61 2.1.8.1. (E)-N’-((9-Benzyl-9H-carbazol-3-yl)methylene)isonicotinohy-
drazide (12a). Yellow solid; yield 80.0%; m.p. 198.0–199.5 ꢁC. IR
(KBr) cmꢂ1: 3250, 3046 (NH), 1646 (C ¼ O), 1601 (C ¼ N). 1H NMR
(300 MHz, DMSO-d6, ppm) d 12.02 (s, 1H, NH), 8.90–8.73 (m, 2H,
Ar-H), 8.65 (s, 1H, CH ¼ N), 8.55 (s, 1H, Ar-H), 8.31 (dd, 1H, J ¼ 9.6,
6.5 Hz, Ar-H), 7.97–7.63 (m, 5H, Ar-H and pyridine-H), 7.48 (dd, 1H,
J ¼ 10.7, 4.7 Hz, Ar-H), 7.38–7.06 (m, 6H, Ar-H), 5.73 (s, 2H, CH2).
13 C NMR (75 MHz, DMSO-d6, ppm) d 161.44, 150.29 (2 C), 141.50,
140.77, 140.70, 137.43, 128.59 (2 C), 127.32 (2 C), 126.70 (2 C),
126.40, 125.41, 125.04, 122.60, 122.25, 121.54 (2 C), 120.69, 120.29,
119.74, 110.03, 109.92, 45.81. HRMS (MALDI) calcd for C26H20N4O
[M þ H]þ: 405.1710, found: 405.1719.
(2 C), 127.56 (2 C), 126.60, 125.57, 125.09, 122.54, 122.35, 120.71
(2 C), 120.05, 118.65, 110.15, 109.91, 109.79, 45.43. HRMS (MALDI)
calcd for C22H18N6 [M þ H]þ: 367.1666, found: 367.1656.
2.1.7. General procedure to synthesise compounds 10a–b and 11
A mixture of the required compound 4 (2 mmol) and either thio-
semicarbazide hydrochloride or semicarbazide hydrochloride(2 m-
mol) in ethanol (20 ml) was stirred at 50–60 ꢁC for 8–12 h in the
presence of five drops of concentrated hydrochloric acid. The
solution was evaporated to dryness under reduced pressure, and
the residue was purified by silica gel column chromatography
using dichloromethane:methanol (80:1) as the eluent.
2.1.8.2. (E)-N’-((9-(4-Methylbenzyl)-9H-carbazol-3-yl)methylene)iso-
nicotinohydrazide (12 b). Yellow solid; yield 74.7%; m.p.
196.0–197.8 ꢁC. IR (KBr) cmꢂ1: 3320, 3060 (NH), 1647 (C ¼ O), 1595
(C ¼ N). 1H NMR (300 MHz, DMSO-d6, ppm) d 12.02 (s, 1H, NH),
8.80 (d, 2H, J ¼ 5.0 Hz, Ar-H), 8.63 (s, 1H, CH ¼ N), 8.54 (s, 1H,
pyridine–H), 8.29 (d, 1H, J ¼ 7.4 Hz, pyridine–H), 7.95–7.81 (m, 3H,
Ar-H), 7.71 (dd, 2H, J ¼ 23.9, 8.3 Hz, pyridine–H), 7.48 (t, 1H,
J ¼ 7.6 Hz, Ar-H), 7.27 (t, 1H, J ¼ 7.4 Hz, Ar-H), 7.09 (s, 4H, Ar-H),
5.66 (s, 2H, CH2), 2.22 (s, 3H, CH3). 13 C NMR (75 MHz, DMSO-d6,
ppm) d 161.40, 150.29 (2 C), 150.21, 141.43, 140.72, 140.63, 136.52,
134.40, 129.13 (2 C), 126.73 (2 C), 126.37, 125.28, 124.95, 122.52,
122.19, 121.57, 120.69, 120.33, 119.68, 110.08, 109.97, 45.53, 20.58.
HRMS (MALDI) calcd for C27H22N4O [M þ H]þ: 419.1866,
found: 419.1854.
2.1.7.1. (E)-2-((9-Methyl-9H-carbazol-3-yl)methylene)hydrazine-1-
carbothioamide (10a). White solid; yield 60.5%; m.p.
222.4–223.8 ꢁC. IR (KBr) cmꢂ1: 3238, 3026 (NH2, NH), 1630 (C ¼ N),
1120 (C ¼ S). 1H NMR (300 MHz, DMSO-d6, ppm) d 11.42 (s, 1H,
NH), 8.59 (s, 1H, CH ¼ N), 8.21 (d, 3H, J ¼ 7.8 Hz, Ar-H), 8.08–7.89
(m, 2H, NH2), 7.62 (d, 2H, J ¼ 8.6 Hz, Ar-H), 7.50 (t, 1H, J ¼ 7.6 Hz,
Ar-H), 7.25 (t, 1H, J ¼ 7.4 Hz, Ar-H), 3.90 (s, 3H, CH3). 13 C NMR
(75 MHz, DMSO-d6, ppm) d 177.45, 143.65, 141.54, 141.02, 126.11,
125.11, 125.02, 122.19, 122.00, 120.47, 120.19, 119.30, 109.41,
109.36, 29.09. HRMS (MALDI) calcd for C15H14N4S [M þ H]þ:
283.1012, found: 283.1020.
2.1.7.2. (E)-2-((9-Ethyl-9H-carbazol-3-yl)methylene)hydrazine-1-car-
2.1.8.3. (E)-N’-((9-(2-Fluorobenzyl)-9H-carbazol-3-yl)methylene)iso-
nicotinohydrazide (12c). Yellow solid; yield 73.8%; m.p.
220.3–221.5 ꢁC. IR (KBr) cmꢂ1: 3366, 3050 (NH), 1651 (C ¼ O), 1589
(C ¼ N). 1H NMR (300 MHz, DMSO-d6, ppm) d 12.07 (s, 1H, NH),
8.82 (s, 2H, Ar-H), 8.68 (s, 1H, CH ¼ N), 8.57 (s, 1H, pyridine–H),
8.26 (s, 1H, pyridine–H), 8.00–7.83 (m, 3H, Ar-H), 7.66 (dd, 2H,
J ¼ 26.4, 8.4 Hz, pyridine–H), 7.46 (t, 1H, J ¼ 7.6 Hz, Ar-H), 7.35–7.16
(m, 3H, Ar-H), 7.00 (t, 1H, J ¼ 7.2 Hz, Ar-H), 6.89 (t, 1H, J ¼ 7.7 Hz,
Ar-H), 5.74 (s, 2H, CH2). 13 C NMR (75 MHz, DMSO-d6, ppm) d
161.68, 161.43, 150.29 (2 C), 150.14, 141.38, 140.70, 140.55, 129.53
(d, 1 C, J ¼ 8.1 Hz), 128.66 (d, 1 C, J ¼ 4.3 Hz), 126.44, 125.48, 125.05,
124.58 (d, 1 C, J ¼ 3.4 Hz), 122.59, 122.24, 121.56 (2 C), 120.71,
120.28, 119.86, 115.67, 115.39, 110.03, 109.86, 40.23. HRMS
bothioamide (10 b). White solid; yield 64.6%; m.p. 199.8–202.0 ꢁC.
1
IR (KBr) cmꢂ1: 3280, 3043 (NH2, NH), 1594 (C ¼ N), 1200 (C ¼ S). H
NMR (300 MHz, DMSO-d6, ppm) d 11.40 (s, 1H, NH), 8.58 (s, 1H,
CH ¼ N), 8.27–8.13 (m, 3H, Ar-H), 8.04–7.91 (m, 2H, Ar-H), 7.64 (s,
1H, NH2), 7.61 (s, 1H, NH2), 7.53–7.44 (m, 1H, Ar-H), 7.24 (t, 1H,
J ¼ 7.4 Hz, Ar-H), 4.45 (q, 2H, J ¼ 6.9 Hz, CH2), 1.32 (t, 3H, J ¼ 6.8 Hz,
CH3). 13 C NMR (75 MHz, DMSO-d6, ppm) d 177.45, 143.56, 140.52,
139.96, 126.12, 125.13, 125.08, 122.39, 122.21, 120.61, 120.24,
119.25, 109.39, 109.29, 37.10, 18.64. HRMS (MALDI) calcd for
C16H16N4S [M þ H]þ: 297.1168, found: 297.1161.
2.1.7.3. (E)-2-((9-Ethyl-9H-carbazol-3-yl)methylene)hydrazine-1-car-
boxamide (11). White solid; yield 42.3%; m.p. 211.1–212.0 ꢁC. IR
(KBr) cmꢂ1: 3343, 3049 (NH2, NH), 1691 (C ¼ O), 1595 (C ¼ N). 1H
NMR (300 MHz, DMSO-d6, ppm) d 10.17 (s, 1H, NH), 8.51 (s, 1H,
CH ¼ N), 8.20 (d, 1H, J ¼ 7.7 Hz, Ar-H), 8.01 (s, 1H, Ar-H), 7.86 (d,
1H, J ¼ 8.5 Hz, Ar-H), 7.61 (dd, 2H, J ¼ 8.4, 3.7 Hz, Ar-H), 7.47 (t, 1H,
J ¼ 7.7 Hz, Ar-H), 7.23 (t, 1H, J ¼ 7.4 Hz, Ar-H), 6.52 (br s, 2H, NH2),
4.46 (q, 2H, J ¼ 7.0 Hz, CH2), 1.32 (t, 3H, J ¼ 6.9 Hz, CH3). 13 C NMR
(75 MHz, DMSO-d6, ppm) d 157.03, 140.48, 140.05, 139.93, 126.01,
125.89, 124.46, 122.33, 122.22, 120.58, 119.22, 119.08, 109.31,
109.19, 37.06, 13.72. HRMS (MALDI) calcd for C16H16N4O [M þ H]þ:
281.1397, found: 281.1392.
(MALDI)
calcd
for
C26H19FN4O
[M þ H]þ:
423.1616,
found: 423.1612.
2.1.8.4.
(E)-N’-((9-(2,4-Dichlorobenzyl)-9H-carbazol-3-yl)methyle-
ne)isonicotinohydrazide (12d). Yellow solid; yield 64.2%; m.p.
209.1–210.3 ꢁC. IR (KBr) cmꢂ1: 3400, 3137 (NH), 1657 (C ¼ O), 1597
(C ¼ N). 1H NMR (300 MHz, DMSO-d6, ppm) d 12.04 (s, 1H, NH),
8.88–8.73 (m, 2H, Ar-H), 8.64 (s, 1H, CH ¼ N), 8.58 (d, 1H, J ¼ 1.6 Hz,
pyridine–H), 8.33 (d, 1H, J ¼ 7.7 Hz, pyridine–H), 7.96–7.69 (m, 4H,
Ar-H and pyridine–H), 7.68–7.17 (m, 5H, Ar-H), 6.42 (d, 1H,
J ¼ 8.4 Hz, Ar-H), 5.76 (s, 2H, CH2). 13 C NMR (75 MHz, DMSO-d6,
ppm) d 161.38, 150.28 (2 C), 150.01, 141.34, 140.67, 140.50, 133.60,
132.73, 132.67, 129.09, 128.41, 127.68, 126.60, 125.74, 125.18,
122.67, 122.31, 121.54 (2 C), 120.84, 120.34, 120.07, 109.92, 109.73,
54.91. HRMS (MALDI) calcd for C26H18Cl2N4O [M þ H]þ: 473.0930,
found: 473.0941.
2.1.8. General procedure for the synthesis of compounds 12a–12d
The intermediate 4 (2 mmol) reacted with isonicotinic acid hydra-
zide (2 mmol) in the presence of catalytic amounts of hydrochloric
acid (5 drops) in ethanol (20 ml) at 70 ꢁC for 5 h. The solution was
evaporated to dryness under reduced pressure, and the residue
was purified by silica gel column chromatography with (dichloro-
methane: methanol ¼ 60:1).
2.2. Evaluation of antibacterial activity in vitro
The in vitro antimicrobial and antifungal activities of the synthes-
ised compounds were evaluated to obtain the minimum inhibitory

300
Y.-J. XUE ET AL.
Scheme 1. Synthetic scheme for the synthesis of the target compounds. Reagents and conditions: (a) DMC, DABCO, 95 ꢁC, 24 h (2a); KOH, 25 ꢁC, 2 h (2b); NaH, DMF,
25 ꢁC, 16 h (2c–k) (b) POCl3, DMF, 90 ꢁC, 8–18 h (c) AcOH, 120ꢁC, 4–8 h (d) CH2CH3OH, HCl, 40 ꢁC, 6 h (9a–k); CH3OH, AcOH, 68ꢁC, 4 h (10a–b,11) (e) CH2CH3OH,
AcOH, 70 ꢁC, 5 h.
concentrations (MICs), using a 96-well microtiter plate and a serial grown in complete medium. Before being treated, cells have been
dilution method. Gatifloxacin and moxifloxacin were used as posi- starved in serum-free medium for 24 h for allowing cell cycle syn-
tive controls and DMSO as a negative control. The micro-organ- chronisation. 72 h after treatments, fresh MTT, re-suspended in
isms used in the present study were two Gram-positive strains PBS, was added to each well (final concentration (0.5 mg/mL).
(Staphylococcus aureus 4220, Streptococcus mutans 3289), one clin- After 2 h incubation at 37 ꢁC, cells have been lysed with DMSO,
ical isolate of multidrug-resistant Gram-positive bacterial strain and then optical density was measured at 570 nm using a micro-
(Methicillin-resistant Staphylococcus aureus CCARM 3167), one plate reader. At least six doses of the studied compounds, solubi-
Gram-negative strain (Escherichia coli 1924) and one fungus lised in DMSO (0.1% final concentration), have been evaluated
(Candida albicans 7535). The bacteria were grown to mid-log and each experiment has been performed in triplicate. The
phase in Mueller-Hinton broth and diluted 1000-fold in the same absorbance values have been used to determine the IC50 for each
medium. Stock solutions of the test compounds in dimethyl sulf- cell line using GraphPad Prism 5 Software (GraphPad Inc., San
oxide were prepared and then poured into 96-well plates. The Diego, CA).
final concentration of 0.5–64 mg/mL underwent a twofold serial
dilution14,24. The bacteria were suspended and contained approxi-
mately 105 CFU/mL. These were applied to 96-well plates with a
2.4. Docking study
serial dilution and incubated at 37 ꢁC for 24 h. The bacteria growth
Preliminary docking was performed to evaluate whether binding
was measured from the turbidity at 630 nm using a microplate
reader. All experiments were carried out in triplicate. Their MBC
and MFC values were also determined (only for the compounds
with MIC values of < 256 lg/mL).
to DHFR might account for the bactericidal effect of the com-
pounds. All docking studies were carried out using Discovery
Studio 2017 (Accelrys, San Diego, CA). Docking studies were per-
formed according to our previous report25. The crystal structure
data were obtained from the protein data bank (E. coli DHFR
PDB_ID: 1RX7). Enzyme structures were checked for missing
atoms, bonds and contacts. Hydrogen atoms were added to the
2.3. MTT assay
Cell viability has been determined using the 3-(4,5-dimethylthia- enzyme structure. Water molecules and bound ligands were
zol-2-y1)-2,5-diphenyltetrazolium Bromide (MTT, Sigma–Aldrich, manually deleted. Then the protein was refined with CHARMm.
Milan, Italy) assay. Cells have been seeded on 48 well plates and The structures of compounds were sketched in 2D and converted

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
301
Table 1. Inhibitory activities (MIC/MBC or MFC, lg/mL) of compounds 8a–l, 9a–k, 10a–b, 11 and 12a–d against various bacteria and fungi.
Gram-positive strains
Gram-negative strain
Fungus
R
S. aureus
MRSA CCARM
3167b
S. mutans
E. coli
C. albicans
Compd
Y
R
X
4220a
3289c
1924d
7535e
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
9a
9b
9c
9d
H
H
H
H
H
H
H
H
H
CH3
C2H5
benzyl
—
—
—
—
—
—
—
—
—
—
—
—
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
S
16/16
64/64
4/4
1/1
4/4
0.5/0.5
2/2
4/4
2/2
2/2
16/16
1/1
2/4
4/4
2/2
1/2
2/4
2/4
1/2
1/2
1/4
2/2
2/4
256/256
>256
>256
>256
>256
>256
>256
0.25/0.25
0.25/0.25
16/16
8/8
64/64
32/128
2/4
8/8
8/16
0.5/1
0.5/0.5
0.5/1
0.5/0.5
1/1
1/1
1/1
1/1
8/8
0.5/1
2/2
4/4
1/2
0.5/1
1/1
2/2
1/1
2/2
2/2
2/2
2/2
>256
>256
>256
>256
>256
>256
>256
2/2
128/128
64/128
32/32
4/8
32/32
2/2
1/1
2/2
1/1
2/2
8/8
1/1
4/8
4/8
1/4
1/2
1/4
16/16
2/4
2/8
2/8
4/8
2/4
>256
>256
>256
>256
>256
>256
>256
0.5/0.5
0.5/0.5
0.5/1
0.5/0.5
0.5/0.5
0.5/0.5
0.5/1
0.5/1
0.5/0.5
0.5/1
16/32
0.5/0.5
2/2
8/8
1/2
0.5/0.5
0.5/0.5
1/2
4-CH3C6H4CH2-
2-FC6H4CH2-
2,4-2ClC6H3CH2-
2-ClC6H4CH2-
3-ClC6H4CH2-
4-ClC6H4CH2-
4-BrC6H4CH2-
4-CNC6H4CH2-
C6H5-
2/2
0.5/4
0.5/1
2/4
2/8
2/2
2/2
16/16
1/2
4/4
8/16
1/2
0.5/1
1/1
2/2
1/4
1/2
1/1
2/16
2/4
>256
>256
>256
>256
>256
>256
>256
0.25/n.t.f
0.25/n.t.
H
H
Br
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
CH3
C2H5
benzyl
4-CH3C6H4CH2-
2-FC6H4CH2-
2,4-2ClC6H3CH2-
2-ClC6H4CH2-
3-ClC6H4CH2-
4-ClC6H4CH2-
4-BrC6H4CH2-
4-CNC6H4CH2-
CH3
C2H5
C2H5
benzyl
4-CH3C6H4CH2-
2-FC6H4CH2-
2,4-2ClC6H3CH2-
9e
9f
9g
9h
9i
9j
9k
10a
10b
11
12a
12b
12c
12d
Gatifloxacin
Moxifloxacin
1/1
0.5/0.5
0.5/0.5
0.5/1
2/8
>256
>256
>256
>256
>256
>256
>256
2/2
S
O
—
—
—
—
1/2
2/2
CCARM: Culture Collection Antimicrobial Resistant Microbes; KCTC: Korean Collection for Type Cultures.
aStaphylococcus aureus RN 4220.
bMethicillin-resistant S. aureus CCARM 3167.
cStreptococcus mutans 3289.
dEscherichia coli KCTC 1924.
eCandida albicans 7535.
fn.t.: Not test.
Table 2. Cytotoxic activity (IC50a, mg/mL) of compounds 8b, 8d, 8f, 8k, 9b and
9e against human cell lines.
concentrations (0, 0.1, 0.3, 1, 3, 10 mmol/L), according to the manu-
facturer’s instructions.
a
In vitro cytotoxicity IC50 (lg/mL)
Compd
SGC-7901b
AGSb
L-02c
8b
8d
8f
8k
9b
9e
>33.4
7.1
10.0
>42.1
1.8
>33.4
9.9
7.8
>42.1
1.2
>33.4
10.1
9.4
>42.1
3.8
3. Results and discussion
3.1. Chemistry
The synthetic routes used for the construction of target com-
pounds 8a–l, 9a–k, 10a–b, 11 and 12a–d are outlined in Scheme
1. A series of intermediates (2) was obtained from carbazole (1)
via N-alkylation or N-arylation. Next, we performed a formylation
reaction (Vilsmayer-Hack) on the intermediates (2) and commer-
cially available compound 3 in the presence of POCl3 and DMF to
obtain 9-substituted carbazole-3-carbaldehyde analogues 4 in
good yields. Compounds 8a–l were prepared by the reaction of
each of the series 4 compounds with metformin hydrochloride in
acetic acid. Compounds 9a–k were synthesised via the reactions
of each of the series 4 compounds with aminoguanidine hydro-
chloride in ethanol with a catalytic amount of concentrated hydro-
chloric acid. Compounds 10a–b and 11 were prepared by
reacting compounds 4 with thiosemicarbazide and semicarbazide,
respectively, in the presence of acetic acid in methanol under
1.5
0.4
1.1
aIC50 is the concentration required to inhibit the cell growth by 50%. Data repre-
sent the average of three independent experiments running in triplicate.
Variation was generally between 5 and 10%.
bHuman gastric cancer cells.
cHuman normal hepatic cells.
into 3D using the DS molecule editor. Automated docking studies
were carried out to investigate the binding mode of compounds
8b and 8f utilizing DS-CDOCKER protocol. The pose with the top
CDOCKER_INTERACTION_ENERGY was chosen for analysing the
binding features of compounds 8b and 8f with DHFR.
2.5. Inhibition of DHFR activities in vitro
The inhibition of DHFR activities of compound 8f was measured reflux. Next, we reacted compounds 4 with isonicotinic in the
using ELISA kits (Mlbio, Shanghai, China) under different presence of acetic acid in alcohol to obtain compounds 12a–d.

302
Y.-J. XUE ET AL.
Figure 2. Binding mode of folate inside the E. coli DHFR active site.
3.2. Evaluation of in vitro antimicrobial activities
Staphylococcus aureus (MRSA CCARM 3167) and Staphylococcus
aureus RN4220) and one Gram-negative strain (Escherichia coli
1924) with MIC values of 0.5 or 1 mg/ml. Furthermore, the antibac-
terial activities of compound 8f against Gram-positive bacterial
strains (S. aureus RN4220 and Streptococcus mutans 3289) were
comparable to the positive control drugs gatifloxacin and moxi-
floxacin. In addition, compound 8f displayed considerable potency
against MRSA CCARM 3167 (MIC of 0.5 lg/ml) that was two- and
four-fold greater compared with moxifloxacin (MIC of 1 lg/ml) and
gatifloxacin (MIC of 2 lg/ml) respectively. In addition, compound
8f demonstrated a strong inhibitory activity (MIC of 0.5 lg/ml)
against E. coli 1924, which was four-fold greater than the activities
The antimicrobial activities of the synthesised compounds were
then tested against a series of pathogenic bacterial and fungal
strains. Several different bacterial strains and one fungal strain
were susceptible to most of the compounds tested with MICs in
the range of 1–64 mg/ml. N-aryl carbazole derivatives containing
an aminoguanidine or 1,4-dihydro-1,3,5-triazine moiety (8c–l and
9c–k) exhibited potent antibacterial activities with MICs ranging
from 0.5 to 16 mg/ml. These compounds also exhibited antifungal
activity against Candida albicans 7535 with MICs ranging from 1
to 32 mg/ml.
Compounds 8f, 8l, 9d and 9e exhibited strong antibacterial
activity against two Gram-positive strains (methicillin-resistant of moxifloxacin (MIC of 2 lg/ml) and gatifloxacin (MIC of 2 lg/ml),

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
303
Figure 3. Binding mode of 8f inside the E. coli DHFR active site.
respectively. In contrast, compound 8f showed a weaker activity the fungal strain over a fixed incubation time. Most of the series 8
against the fungus C. albicans 7535 compared with the posi- compounds displayed MBCs at 1–2 fold higher than their MICs
tive controls.
against the strains tested. Compounds 9i and 9k also had four-
Moreover, we determined the minimum bactericidal concentra- fold higher MIC values compared with their MBCs against S. aur-
tions (MBCs) and minimum fungicidal concentrations (MFCs) for eus RN4220 and MRSA CCARM 3167, respectively. The MBC/MIC
compounds whose MIC value less than 256 mg/ml against strains. ratio of compounds 9 g and 9j were ꢃ4 for S. mutans 3289.
MBCs and MFCs are defined as the lowest bactericidal or fungi- Compounds 9c, 9e, 9h and 9i demonstrated MFC values that
cidal concentration required to kill a particular bacterial strain or were four-fold higher than their MICs against Candida albicans

304
Y.-J. XUE ET AL.
Figure 4. Binding mode of 8b inside the E. coli DHFR active site.
7535. Notably, bacteria appeared to be less tolerant to the series tested in this study, with MICs greater than 256 mg/ml. As
8 compounds compared with series 9 compounds. When we shown in Table 1, most of the compounds exhibited equivalent
altered the phenyl ring substituents of compounds 8a–l, we or higher potency (with MIC values in the range of 0.5 to 16 mg/
observed a significant effect on the potency of their antimicrobial ml) towards MRSA CCARM 3167 compared with the positive
activities. The order of antibacterial activities were as follows: phe- controls gatifloxacin and moxifloxacin (MICs of 2 and 1 mg/ml,
nyl group > 2,4-dichloro-substitutions > 4-CH3(for compounds respectively).
bearing an electron-donating group)
>
halogen substitu-
To summarise, our findings revealed that the compounds in
tions > benzyl group > 4-CN (for compounds bearing an electron- series 8 exhibited significantly higher antimicrobial activities com-
withdrawing group) > alkyl group. Furthermore, bromo- and pared with the compounds in the other four series. This suggests
chloro-substitutions on the phenyl ring in compounds 8a–l were that the presence of a dihydrotriazine moiety is critical to the
observed to improve their antifungal activity against C. albicans potency of these carbazole derivatives. These results therefore
7535. The antibacterial activity order of compounds 9a–k was provide further evidence to suggest that aryl groups and the dihy-
similar to compounds 8a–l. Compounds in series 10a–b, 11 and drotriazine moiety play critical roles in the activity of these carba-
12a–d generally showed weak activity against all the strains zole compounds.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
305
therapeutic potential, has interacted with the critical active-site
residues of E. coli DHFR.
3.5. Inhibition studies of compound 8f with DHFR
To investigate whether compound 8f can bind to and block the
active site of DHFR, we performed in vitro enzyme assays to test
the inhibitory effect of compound 8f (MIC of 0.5 lg/ml) on DHFR
activity (Figure 5). At concentrations of 3 and 10 lmol/L, com-
pound 8f decreased DHFR activity by 71% compared with the
negative control. The results indicated that compound 8f exerts
its antibacterial activity via binding to DHFR, which however
might not be the only mechanism of action.
4. Conclusions
Figure 5. Inhibition of DHFR of compound 8f.
We have designed, synthesised and evaluated the antibacterial
and antifungal activities of five series of novel carbazole deriva-
tives. Compound 8f showed the most potential as a therapeutic
agent, with an MIC of 0.5–2 mg/ml against selected bacterial
strains. Furthermore, compound 8f also was the least cytotoxic to
SGC-7901, AGS and L-02 cells. Compound 9d also exhibited strong
antibacterial activity with the cancer therapeutic potential.
Therefore, the clinical potential of carbazole derivatives 8f and 9d
could be explored further for future applications in antitumour
and antimicrobial therapies.
Docking simulation and in vitro enzyme activity assays sug-
gested that binding to DHFR might account for the antimicrobial
activity of the compounds. Further studies of the mechanisms of
action of these compounds are currently underway in our labora-
tories and will be reported in due course.
3.3 Cell viability
To determine whether the synthesised compounds were select-
ively toxic towards microbes and not human cells, we evaluated
the cytotoxicity of six compounds in two gastric cancer cell lines
(SGC-7901 and AGS) and a normal human liver cell line (L-02)
using a standard technique. As shown in Table 2, the cytotoxicities
of compounds 8b, 8d, 8f and 8k were lower than that of com-
pounds 9b and 9e. Compounds 8b, 8d, 8f and 8k had no effect
on human liver cell viability at their MICs and, in addition, their
IC50 values were significantly higher than their MIC values.
However, the IC50 values of the series 9 compounds were close to
their MIC values, which make it likely that the promising antibac-
terial activity of these compounds is due to their cyto-
toxic properties.
Compounds in series 8a–l, containing a dihydro-1,3,5-triazine
ring moiety, were generally found to have more potent antimicro-
bial effects but less cytotoxicity compared with the corresponding
series 9a–l compounds that comprised an aminoguanidine moiety.
Interestingly, our results also showed that compound 9 b was
selectively more toxic to human cancer cells compared with nor-
mal human cells. The IC50 value is about three times higher in
normal cells than AGS cell lines. Further studies are required to
investigate if this compound has any potential as a new anti-
cancer agent.
Disclosure statement
We declare that we have no conflict of interest with respect to
this study.
Funding
This work was supported by the National Natural Science
Foundation of China [Grant numbers 81260468 and 81360473].
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