Synthesis, molecular docking and cytotoxicity evaluation of novel 2-(4-amino-benzosulfonyl)-5H-benzo[b]carbazole-6,11-dione derivatives as histone deacetylase (HDAC8) inhibitors

Article abstract of DOI:10.1016/j.bioorg.2014.01.002

A new series of 2-(4-aminobenzosulfonyl)-5H-benzo[b]carbazole-6,11-dione derivatives, which has not been reported yet, has been synthesized from 1,4-naphthoquinone and 4-aminophenylsulfone involving an Michael addition, benzoylation and Pd catalyzed coupl

Full text of DOI:10.1016/j.bioorg.2014.01.002

Bioorganic Chemistry 53 (2014) 24–36
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis, molecular docking and cytotoxicity evaluation of novel
2-(4-amino-benzosulfonyl)-5H-benzo[b]carbazole-6,11-dione
derivatives as histone deacetylase (HDAC8) inhibitors
P. Ravichandiran a, A. Jegan b, D. Premnath e, V.S. Periasamy c, S. Muthusubramanian d, S. Vasanthkumar a,
⇑
a Department of Chemistry, School of Science & Humanities, Karunya University, Coimbatore 641 114, India
b Department of Nanosciences & Technology, School of Nanosciences & Technology, Karunya University, Coimbatore 641 114, India
c Department of Animal Science, Bharathidasan University, Tiruchirappalli 620 024, India
d Department of Organic Chemistry, School of Chemistry, Madurai Kamaraj University, Madurai 625 021, India
e Department of Bioinformatics, School of Biotechnology and Health Sciences, Karunya University, Coimbatore 641 114, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 September 2013
Available online 3 February 2014
A new series of 2-(4-aminobenzosulfonyl)-5H-benzo[b]carbazole-6,11-dione derivatives, which has not
been reported yet, has been synthesized from 1,4-naphthoquinone and 4-aminophenylsulfone involving
an Michael addition, benzoylation and Pd catalyzed coupling. This set of compounds has been evaluated
for in vitro cytotoxicity specifically against human cervical cancer cell line (SiHa) and most of the synthe-
sized compounds exhibited good cytotoxic activity. Molecular docking of all the synthesized compounds
was studied; among fourteen molecules docked compound 3 was the one with the best glide and E model
score of ꢀ9.06 and ꢀ73.41, respectively which is close to the glide score of SAHA (standard). In all docked
molecules, the compound 7a exhibits least glide and E model score of ꢀ2.97 and ꢀ71.02 respectively.
Ó 2014 Elsevier Inc. All rights reserved.
Keywords:
1,4-Naphthoquinone
Cervical cancer
Acid chlorides
Carbazole
Sulfone
Molecular docking
1. Introduction
phosphorylation [4]. Heterocyclic quinones are biologically active
[5] and aminoquinones create high interest due to their enhanced
HDAC activity is invariably increased in cancer cells and there is
a need to synthesize novel class of HDAC inhibitors. SAHA (Sube-
royl Anilide Hydroxamic Acid, Fig. 1) is the drug has been recently
validated clinically and approved by FDA for the treatment of cuta-
neous T cell lymphoma [1]. Methyl-Gene’s isotype specific HDAC
inhibitor MGCD0103 (Fig. 1) is currently clinically investigating
for in solid tumors and hematological malignancies [2]. Most of
HDAC inhibitors have three common features such as metal bind-
ing moiety, a carbon linker and a capping group (Fig. 1). In HDAC
inhibitors, the capping group is solvent-exposed and interacts with
amino acids near the entrance of the active site, metal binding moi-
ety binds in the protein interior and complexes the metal ion in-
volved in catalysis. The linker helps for high-affinity interactions
with proteins [3].
anticancer activities. The heterocyclic aminoquiniones have wide
range of biological applications including anticancer [6], antibacte-
rial [7,8], fungicidic [8,9], luciferase inhibition [10], antiprolifera-
tive [11] and tuberculostatic effects [12]. In addition, the
heterocyclic naphthoquinone derivatives exhibit potent properties
like electrochemical capacitance [13], electrochemical redox [14],
electron mediator [15] and electron transfer [16]. They are also
capable of forming complexes with metals [17].
According to Moore [18] and Pindur [19], a molecule with
DNA-intercalating property should have three to four coplanar
rings with a length of 3–4 Å and a width of 6–8 Å. The molecule
must also possess
a para-conjugated quinone ring having
nitrogen atoms, which will help it to make hydrogen bonding
with DNA. The structure–activity relationship of heterocyclic
quinones containing nitrogen atoms shows that the number
and position of nitrogen atoms are very important for the
cytotoxic activity [20]. If the number of nitrogen atoms increases,
the cytotoxicity also increases [21]. The structure of carbazole-
6,11-dione (1a–7a) targeted in the present investigation has a
similar kind of ring arrangement helping it to form a hydrogen
bonding with DNA and hence this investigation assumes
importance.
Quinones are effectively involved in a wide range of biochemi-
cal processes including electron transport and oxidative
⇑
Corresponding author. Address: Department of Chemistry, School of Science &
Humanities, Karunya University, Coimbatore 641114, Tamil Nadu, India. Fax: +91
422 2615615.
E-mail addresses: ravichandru55@gmail.com (P. Ravichandiran), kumar2359@
yahoo.com (S. Vasanthkumar).
http://dx.doi.org/10.1016/j.bioorg.2014.01.002
0045-2068/Ó 2014 Elsevier Inc. All rights reserved.

P. Ravichandiran et al. / Bioorganic Chemistry 53 (2014) 24–36
25
Fig. 1. Structure of SAHA, MGCD0103 and pharmacophoric features of HDAC inhibitors in the title compounds.
The second most common cancer in females worldwide has
been identified to be cervical carcinoma. In a year it is calculated
that 12,200 new females suffer from cervical cancer and about
4210 deaths are attributed to cervical cancer in the United States
alone [25,26]. Therefore, in our effort to identify new and effective
therapeutic agents for cancer treatment, selective HDAC inhibitors
have to design and synthesis has become one of our major goals. In
this concern we synthesized a new series of 2-(4-amino-ben-
zosulfonyl)-5H-benzo[b]carbazole-6,11-dione derivatives and
studied their cytotoxicity against human cervical cancer cell line
(SiHa) and the molecular docking studies of all the synthesized
compounds were studied and reported.
docking have not been tested on these systems so far. The syn-
thetic methodology of title compounds are given in the Scheme 1.
2.2. Cytotoxic properties of quinone derivatives
The in vitro cytotoxic activities of the synthesized compounds
(1–7, 1a–7a) are evaluated by cell viability assay method against
a human cervical cancer cell line (SiHa). Though the synthesized
compounds show potent cytotoxic activity (2a, 3a, 4a, 5a, 7a) than
their precursors. Compounds 1a and 6a exhibited less cyctotoxicity
than its precursors. Thus the ring closed carbazole-6,11-dione
derivatives (2a, 3a, 4a, 5a, 7a) that consist of four coplanar
annulated heterocyclic rings show higher antitumor activity than
2-[4-(4-amino-benzene sulfonyl)-phenyl amino]-[1,4] naphtho-
quinone derivatives (2, 3, 4, 5, 7).
2. Results and discussion
Among 1–7, it is noticed that 2-[4-(4-aminobenzenesulfonyl)-
phenylamino]-[1,4] naphthoquinone (1) and N-{4-[4-(1,4-dioxo-
1,4-dihydronaphthalene-2-ylamino)benzene sulfonyl]phenyl}-4-
nitrobenzamide (6) have more toxicity than the other compounds
with the latter having more effect than the former. All the IC50 val-
ues are listed in Fig. 2.
2.1. Chemistry
1,4-Naphthoquinone reacts with 4-aminophenyl sulfone to gen-
erate 2-[4-(4-amino-benzenesulfonyl)-phenylamino]-[1,4]naph-
thoquinone (1). Though this conversion has already been effected
in glacial acetic acid under reflux [12], it is now found that the
reaction take place smoothly in water medium without the aid of
the acid. It is found that the yield in this method is only marginal
(41%) (Method B) [27], but when the reaction is conducted in a
mixture of ethanol and acetic acid, the reaction led to a very good
yield (91%) (Method A). However performing the reaction in etha-
nol alone is not at all successful, the yield being very poor (35%).
Compound 1 is then reacted with several aromatic acid chlorides
to give N-{4-[4-(1,4-dioxo-1,4-dihydro naphthalene-2-yl amino)-
benzene sulfonyl]-phenyl}-aryl benzamide derivatives (2–7) by
conventional method in acetone and yield between 98–99%. Finally
carbazole-6,11-dione derivatives (1a–7a) are synthesized via a typ-
ical intramolecular cyclization with palladium (II) acetate in acetic
acid (Method C).
N-[4-(6,11-dioxo-6,11-dihydro-5H-benzo[b]carbazole-2-sulfo-
nyl)-phenyl]-3,5-dinitro-benzamide (7a), 2-(4-amino-benzosulfo-
nyl)-5H-benzo[b]carbazole-6,11-dione
(1a)
and
N-[4-(6,
11-dioxo-6,11-dihydro-5H-benzo[b]carbazole-2-sulfonyl)-phenyl]-
4-methylbenzamide (4a) exhibited good cytotoxic activity in the
carbazole system than the other compounds of the series (2a, 3a,
5a, 6a). In carbazole system the compound 6a exhibited less
activity than compound 6.
2.3. Molecular docking studies of quinone derivatives
Aromatic carbonyl functional group of all the molecules (3a–h)
were found to be close to Zn2+ atom in the active site, and estab-
lished the hydrogen bond with GLY 151 which is the major inter-
actions of the ligands with HDAC8 (Figs. 3–16). Among the ten
molecules docked the compound 3 was the one with the best glide
and E model score of ꢀ9.06 and ꢀ73.41, respectively which is close
to the glide score of SAHA (standard). The compound 3 exhibited
two hydrogen bonds with GLY 151 (C–O–H) and LYS 33 (N–H–O)
with the bond distance of 1.855 ÅA and 2.070 ÅA respectively
(Fig. 7). All the docking results are presented in Table 1. Compound
3a comes next with a glide and E model score of ꢀ8.96 and ꢀ72.72,
This the first report on the synthesis of N-{4-[4-(1,4-dioxo-1,4-
dihydronaphthalen-2-yl
amino)-benzenesulfonyl]-phenyl}-aryl
benzamide derivatives (2–7) and 2-(4-amino-benzosulfonyl)-5H-
benzo[b]carbazole-6,11-dione derivatives (1a–7a) to the best of
our knowledge. Only few reports are available in literature wherein
compounds with carbazoloquinone nuclei on naphthoquinone and
N-dansyl carbazoloquinone have been found to exhibit antituber-
culosis [12] apart from chemical and electrochemical fluorescent
activities [22]. However, the cytotoxicity and the molecular

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P. Ravichandiran et al. / Bioorganic Chemistry 53 (2014) 24–36
Scheme 1. The synthesis of 2-[4-(4-amino-benzenesulfonyl)-phenylamino]-[1,4]naphthoquinone (1), N-{4-[4-(1,4-dioxo-1,4-dihydro-naphthalene-2-ylamino)-benzenesul-
fonyl]-phenyl}-aryl benzamides (2–7) and carbazole-6,11-dione derivatives (1a–7a).
respectively and it exhibited one hydrogen bond with GLY 151
with the distance of 1.825 ÅA (C–O–H) (Fig. 8). All docked mole-
cules, the compound 7a exhibits least glide and E model score of
ꢀ2.97 and ꢀ71.02 respectively (Fig. 16).
Among fourteen molecules docked the compound 3 exhibited
good glide and E model score and it reveals that the substitution
at the zinc binding group plays an important role to bind with
HDAC8. At the same time the absence of methyl group in carbonyl

P. Ravichandiran et al. / Bioorganic Chemistry 53 (2014) 24–36
3. Experimental
27
3.1. Materials and methods
Melting points (°C, uncorrected) of the synthesized compounds
were checked in open an capillary tubes by using digital auto melt-
ing point apparatus (Labtronics 110, India) and found uncorrected.
All the chemicals and solvents were purchased from Sigma–Aldrich
and Merck, India. All reactions were carried out under atmospheric
air and the products were checked by thin layer chromatography on
TLC silica gel 60 F254 using eluting solvents such as ethyl acetate
and hexane (1:1). The synthesized compounds were purified by col-
umn chromatography using column silica gel 100–200 mesh (ethyl
acetate:hexane 1:2). All the compounds were characterized by UV–
Vis spectrophotometer (UV-1800, Shimadzu, Japan) using acetone
as solvent, FT-IR spectrometer (IR 8400, Shimadzu, Japan) using
KBr pellets, 1H NMR spectroscopy in DMSO (400 MHz, Bruker),
13C NMR spectroscopy in DMSO (100 MHz, Bruker) using tetra-
methylsilane (TMS) as internal standard. Coupling constants (J val-
ues) are reported in Hz. Mass spectra were measured by Electron
Impact (EI) method (Jeol GC-Mate 2). In vitro cytotoxicity of all
the compounds was studied by cell viability assay method. Molec-
ular docking studies of all the synthesized compounds were studies
by GLIDE program (version 8.5, Schrodinger, LLC, New York, 2010).
Fig. 2. Comparison of cytotoxicity (IC50) of derived 2-[4-(4-amino-benzenesulfo-
nyl)-phenyl amino]-[1,4]naphthoquinone (1–7) with 2-substituted 2-(4-amino-
benzosulfonyl)-5H-benzo [b]carbazole-6,11-dione derivatives (1a–7a).
group orient the cap group differently in the pocket. These factors
together has kept not favoring hydrogen bonding interaction with
HDAC8 for other molecules (1, 1a, 7, 7a), a favorable interaction
shown by many HDAC Inhibitors (3, 3a, 6, 2, 2a). This may led to
the reduced potency of 7 and 7a compared with other docked
molecules.
3.2. General procedures for synthesis of 2-[4-(4-amino-benzene
The use of glide and E model scores are questionable for
the type of rank ordering of different derivatives within a series.
The molecular docking and in vitro cytotoxocity study results
suggested that, glide scores and IC50 values of the synthesized
compounds did not correlated because of the glide scores mainly
used to separate the active and inactive compounds. In addition,
glide is primarily concerned with generating an accurate pose for
each ligand and enrichment (the separation of actives from
inactives) [28,29].
sulfonyl)-phenyl amino]-[1,4] naphthoquinone (1)
3.2.1. Method A: [12]
A solution of 1,4-naphthoquinone (1.581 g, 10 mmol) in 95% of
ethyl alcohol (40 mL) was gradually added over a period of 30 min,
to a solution of 4-aminophenyl sulfone (2.048 g, 10 mmol) in gla-
cial acetic acid (10–30 mL) and stirred for 30 min. Then the mix-
ture was refluxed for 1 h. The reaction mixture was cooled and
left overnight at room temperature. The black precipitate formed
Fig. 3. Docking model structures of compound 1 into the HDAC8 binding pocket.

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P. Ravichandiran et al. / Bioorganic Chemistry 53 (2014) 24–36
Fig. 4. Docking model structures of compound 1a into the HDAC8 binding pocket.
Fig. 5. Docking model structures of compound 2 into the HDAC8 binding pocket.
was separated by filtration. Water was added to the filtrate, the
brownish material formed was filtered, washed with hot water
(200 mL), dried at 80 °C, and crystallized from 95% ethyl alcohol
to give 1 (3.692 g, 91%) as orange crystals.
(100 mL) and refluxed for 4 h. The reaction mixture was cooled
at room temperature and the brownish precipitate was filtered
and washed with hot water (200 mL). The precipitate was dried
at 80 °C and crystallized from 95% ethyl alcohol to give
1
(1.657 g, 41%) as orange crystals; mp > 300 °C; UV–Vis
(acetone): 451.45 nm; IR (KBr): 1294, 1633, 3381, 3475 cmꢀ1
;
3.2.2. Method B: [27]
4-Aminophenyl sulfone (2.048 g, 10 mmol) was added to a
solution of 1,4-naphthoquinone (1.581 g, 10 mmol) in water
1H NMR (400 MHz, DMSO-d6) d: 6.14 (s, 2H), 6.34 (s, 1H),
6.61 (d, 2H, J = 8.8 Hz), 7.53 (d, 2H, J = 8.8 Hz), 7.56 (d, 2H,

P. Ravichandiran et al. / Bioorganic Chemistry 53 (2014) 24–36
29
Fig. 6. Docking model structures of compound 2a into the HDAC8 binding pocket.
Fig. 7. Docking model structures of compound 3 into the HDAC8 binding pocket.
J = 8.8 Hz), 7.70–7.80 (m, 4H), 7.95 (d, 1H, J = 7.2 Hz), 8.06 (d,
3.3. General procedure for synthesis of N-{4-[4-(1,4-dioxo-1,4-
dihydro-naphthalene-2-ylamino) benzenesulfonyl]-phenyl}-aryl
benzamides (2–7)
1H, J = 7.3 Hz), 9.41 (s, 1H); 13C NMR (100 MHz, DMSO-d6) d:
104.4, 112.9, 122.6, 125.2, 125.5, 126.1, 127.8, 129.2, 130.3,
132.2, 132.8, 134.8, 138.2, 142.3, 144.8, 153.5, 181.2, 183.0;
MS (EI): m/z 403.49 (M+1, 8%), 257 (80), 180.80 (75), 157.78
(45), 142.78 (60), 122.83 (100).
Substituted benzoyl chloride (1 mmol) was added to a solution
of 1 (0.404 g, 1 mmol) in acetone (100 mL). After refluxing for

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P. Ravichandiran et al. / Bioorganic Chemistry 53 (2014) 24–36
Fig. 8. Docking model structures of compound 3a into the HDAC8 binding pocket.
Fig. 9. Docking model structures of compound 4 into the HDAC8 binding pocket.
30 min, the reaction mixture was filtered and concentrated in va-
cuo to give pure samples of 2–7 which required no further
purification.
134.8, 135.2, 136.2, 143.1, 143.7, 144.6, 166.0, 181.1, 183.0; MS
(EI): m/z 508.028 9 (M+, 12%), 444.63 (55), 300.89 (60), 224.76
(100), 123.07 (60).
3.3.1. N-{4-[4-(1,4-dioxo-1,4-dihydro-naphthalene-2-ylamino)-
benzenesulfonyl]-phenyl} benzamide (2)
3.3.2. N-{4-[4-(1,4-dioxo-1,4-dihydro-naphthalene-2-ylamino)-
benzenesulfonyl]-phenyl}-3-methyl-benzamide (3)
Orange solid; Reaction time 25 min (0.501 g, 99%); mp > 300 °C;
UV–Vis (acetone): 447.36 nm; IR (KBr): 1296, 1631, 1676,
Red-brown solid; Reaction time 25 min (0.515 g, 99%);
mp > 300 °C; UV–Vis (acetone): 445.23 nm; IR (KBr): 1298, 1616,
3400 cmꢀ1
;
1H NMR (400 MHz, DMSO-d6) d: 6.39 (s, 1H), 7.54 (d,
1680, 2922, 3309 cmꢀ1 1H NMR (400 MHz, DMSO-d6) d: 2.08 (s,
;
2H, J = 6.8 Hz), 7.61–8.07 (m, 15H), 9.45 (s, 1H), 10.62 (s, 1H); 13C
NMR (100 MHz, DMSO-d6) d: 104.9, 120.2, 122.6, 125.2, 126.2,
127.7, 128.3, 128.4, 128.5, 130.3, 131.9, 132.1, 132.9, 134.2,
3H), 6.40 (s, 1H), 7.36–8.08 (m, 16H), 9.49 (s, 1H), 10.61 (s, 1H);
13C NMR (100 MHz, DMSO-d6) d: 21.3, 105.4, 120.7, 123.1, 125.4,
125.7, 126.7, 126.9, 128.7, 128.8, 128.9, 130.1, 130.8, 131.2,

P. Ravichandiran et al. / Bioorganic Chemistry 53 (2014) 24–36
31
Fig. 10. Docking model structures of compound 4a into the HDAC8 binding pocket.
Fig. 11. Docking model structures of compound 5 into the HDAC8 binding pocket.
132.7, 133.0, 133.4, 134.7, 135.7, 138.3, 143.6, 144.3, 167.8, 181.7,
183.6; MS (EI): m/z 521.60 (Mꢀ1, 15%), 499.17 (25), 457.80 (10),
274.85 (50), 257.85 (25), 175.06 (97), 114.09 (100), 100.07 (76).
144.6, 165.8, 181.1, 183.0; MS (EI): m/z 521.54 (Mꢀ1, 45%),
456.86 (15), 250.75 (80), 184.42 (100).
3.3.4. N-{4-[4-(1,4-dioxo-1,4-dihydro-naphthalene-2-ylamino)-
benzene sulfonyl]-phenyl}-3-nitro-benzamide (5)
3.3.3. N-{4-[4-(1,4-dioxo-1,4-dihydro-naphthalene-2-ylamino)-
benzenesulfonyl]-phenyl}-4-methyl-benzamide (4)
Red-brown solid; Reaction time 25 min (0.545 g, 99%);
mp > 300 °C; UV–Vis (acetone): 447.36 nm; IR (KBr): 1274, 1348,
Crimson red solid; Reaction time 30 min (0.512 g, 98%);
mp > 300 °C; UV–Vis (acetone): 448.38 nm; IR (KBr): 1294, 1616,
1529, 1616, 1687, 3412 cmꢀ1 1H NMR (400 MHz, DMSO-d6) d:
;
1680, 2945, 3307, 3360 cmꢀ1
;
1H NMR (400 MHz, DMSO-d6) d:
6.40 (s, 1H), 7.63 (d, 2H, J = 7.2 Hz), 7.82 (d, 2H, J = 7.2 Hz), 7.85–
8.06 (m, 7H), 8.34 (d, 1H, J = 7.3 Hz), 8.40 (d, 1H, J = 7.3 Hz), 8.47
(d, 1H, J = 7.8 Hz), 8.62 (s, 1H), 9.50 (s, 1H), 10.96 (s, 1H), 13.71
(s, 1H); 13C NMR (100 MHz, DMSO-d6) d: 105.4, 121.0, 123.1,
123.2, 124.1, 125.7, 126.7, 127.7, 128.9, 129.1, 130.7, 130.8,
131.2, 132.6, 132.9, 133.4, 135.3, 135.8, 136.1, 143.8, 145.1,
2.30 (s, 3H), 6.38 (s, 1H), 7.27 (d, 2H, J = 8.0 Hz), 7.32 (d, 2H,
J = 8.0 Hz), 7.61 (d, 2H, J = 8.8 Hz), 7.77–8.06 (m, 10H), 9.45 (s,
1H), 10.53 (s, 1H); 13C NMR (100 MHz, DMSO-d6) d: 20.9, 104.8,
120.1, 122.6, 125.2, 126.1, 127.8, 128.3, 128.9, 129.2, 130.3,
131.3, 132.1, 132.9, 134.8, 135.1, 136.3, 142.1, 143.1, 143.8,

32
P. Ravichandiran et al. / Bioorganic Chemistry 53 (2014) 24–36
Fig. 12. Docking model structures of compound 5a into the HDAC8 binding pocket.
Fig. 13. Docking model structures of compound 6 into the HDAC8 binding pocket.
148.2, 164.4, 181.6, 183.6; MS (EI): m/z 553.12 (M+, 13%), 507.98
(15), 440.31 (38), 366.39 (100), 293.45 (41), 232.44 (40).
7.86 (t, 1H, J = 7.8 Hz), 7.95 (d, 2H, J = 8.0 Hz), 7.98 (d, 2H,
J = 8.0 Hz), 8.02 (d, 2H, J = 8.0 Hz), 8.06 (d, 1H, J = 7.8 Hz), 8.14
(d, 1H, J = 7.8 Hz), 8.18 (d, 2H, J = 8.0 Hz) 8.37 (d, 2H, J = 8.0 Hz),
9.46 (s, 1H), 10.92 (s, 1H); 13C NMR (100 MHz, DMSO-d6) d:
104.9, 120.4, 122.6, 123.5, 125.2, 126.2, 126.2, 128.4, 128.6,
130.3, 132.1, 132.9, 134.3, 135.3, 135.6, 135.8, 136.1, 143.2,
144.6, 149.3, 164.4, 181.1, 183.0; MS (EI): m/z 551.85 (Mꢀ2,
10%), 528.20 (30), 510.51 (25), 268.46 (75), 252.45 (98), 191.36
(100), 177.40 (52).
3.3.5. N-{4-[4-(1,4-dioxo-1,4-dihydro-naphthalene-2-yl amino)-
benzene sulfonyl]-phenyl}-4-nitro-benzamide (6)
Orange solid; Reaction time 25 min (0.543 g, 98%);
mp > 300 °C; UV–Vis (acetone): 453.49 nm; IR (KBr): 1273, 1350,
1529, 1614, 1680, 3248, 3315 cmꢀ1 1H NMR (400 MHz, DMSO-
;
d6) d: 6.39 (s, 1H), 7.62 (d, 2H, J = 8.0 Hz), 8.79 (t, 1H, J = 7.8 Hz),

P. Ravichandiran et al. / Bioorganic Chemistry 53 (2014) 24–36
33
Fig. 14. Docking model structures of compound 6a into the HDAC8 binding pocket.
Fig. 15. Docking model structures of compound 7 into the HDAC8 binding pocket.

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P. Ravichandiran et al. / Bioorganic Chemistry 53 (2014) 24–36
Fig. 16. Docking model structures of compound 7a into the HDAC8 binding pocket.
3.3.6. N-{4-[4-(1,4-dioxo-1,4-dihydro-naphthalene-2-yl amino)-
benzene sulfonyl]-phenyl}-3, 5-dinitro-benzamide (7)
into ice cold water. The precipitate was filtered, dried at 60 °C and
crystallized from acetone to give 1a–7a.
Red-brown solid; Reaction time 30 min (0.589 g, 98%);
mp > 300 °C; UV–Vis (acetone): 446.12 nm; IR (KBr): 1271, 1346,
3.4.2. 2-(4-amino-benzosulfonyl)-5H-benzo [b]carbazole-6, 11-dione
(1a)
1541,1624, 1691, 3334, 3400 cmꢀ1 1H NMR (400 MHz, DMSO-
;
d6) d: 6.39 (s, 1H), 7.62 (d, 2H, J = 8.8 Hz), 7.78 (t, 1H, J = 6.8 Hz),
7.82 (t, 1H, J = 6.8 Hz) 7.93 (d, 2H, J = 7.2 Hz), 7.93–8.05 (m, 8H),
9.17 (s, 1H), 9.45 (s, 1H), 11.16 (s, 1H); 13C NMR (100 MHz,
DMSO-d6) d: 104.9, 120.7, 121.4, 122.6, 125.2, 126.1, 128.1,
128.6, 130.3, 132.1, 132.9, 134.8, 134.8, 136.2, 136.8, 142.8,
143.2, 144.6, 148.0, 148.2, 161.9, 181.1, 183.0; MS (EI): m/z
596.51 (Mꢀ2, 55%), 566.45 (20), 537.60 (25), 473.23 (15), 399.43
(100), 382.51 (46), 218.53 (45).
Yellow solid; Reaction time 2 h (0.150 g, 75%); mp > 300 °C;
UV–Vis (acetone): 278.71 nm; IR (KBr): 1288, 1629, 1651, 3384,
3478 cmꢀ1 1H NMR (400 MHz, DMSO-d6) d: 6.13 (s, 2H), 6.60 (d,
;
2H, J = 8.8 Hz),, 7.55 (d, 2H, J = 8.8 Hz), 7.70 (d, 1H, J = 8.8 Hz),
7.80–7.90 (m, 3H), 8.09 (d, 1H, J = 5.2 Hz), 8.10 (d, 1H, J = 5.2 Hz),
8.60 (s, 1H), 13.4 (s, 1H); 13C NMR (100 MHz, DMSO-d6) d: 113.5,
118.4, 121.9, 123.6, 125.0, 126.2, 126.7, 129.8, 130.3, 133.0,
134.0, 134.3, 135.0, 139.1, 139.8, 140.0, 151.0, 154.0, 177.9,
180.7; MS (EI): m/z 402.40 (M+, 5%), 342.75 (8), 250.87 (50),
205.06 (98), 162.06 (100), 117.10 (60), 75.10 (45).
3.4. General procedure for synthesis of 2-(4-amino-benzosulfonyl)-
5H-benzo [b]carbazole-6,11-diones (1a–7a)
3.4.3. N-[4-(6,11-dioxo-6,11-dihydro-5H-benzo[b]carbazole-2-
sulfonyl)-phenyl]-benzamide (2a)
3.4.1. Method C: [22,23]
Light yellow solid; Reaction time 2 h (0.195 g, 77%);
mp > 300 °C; UV–Vis (acetone): 278.79 nm; IR (KBr): 1244, 1589,
Mixture of 1–7 (0.5 mmol) in glacial acetic acid (60 mL) and pal-
ladium (II) acetate (0.112 g, 0.5 mmol) were refluxed for 2 h and
the reaction mixture was cooled at room temperature and poured
1668, 3375 cmꢀ1 1H NMR (400 MHz, DMSO-d6) d: 7.58 (t, 2H,
;
J = 8.0 Hz), 7.60 (t, 1H, J = 8.0 Hz), 7.76 (d, 1H, J = 8.8 Hz),

P. Ravichandiran et al. / Bioorganic Chemistry 53 (2014) 24–36
35
Table 1
Molecular docking data of compounds 1–7, 1a–7a.
Compounds
Molecular docking
Glide score Glide energy E model score (kcal/mol) No. of hydrogen bonds
Bond length (ÅA)
XP H bond
(kcal/mol)
1
2
3
4
5
6
7
ꢀ4.38
ꢀ8.52
ꢀ9.06
ꢀ8.25
ꢀ8.17
ꢀ8.59
ꢀ3.42
ꢀ4.35
ꢀ8.35
ꢀ8.96
ꢀ3.74
ꢀ8.03
ꢀ6.55
ꢀ2.97
ꢀ9.42
ꢀ38.55
ꢀ67.78
ꢀ64.78
ꢀ64.04
ꢀ73.60
ꢀ64.52
ꢀ43.17
ꢀ36.57
ꢀ65.44
ꢀ63.87
ꢀ36.27
ꢀ71.89
ꢀ59.68
ꢀ50.69
ꢀ63.82
ꢀ50.17
ꢀ70.06
ꢀ73.41
ꢀ45.50
ꢀ68.17
ꢀ65.50
ꢀ65.34
ꢀ46.54
ꢀ58.16
ꢀ72.72
ꢀ53.67
ꢀ84.18
ꢀ65.27
ꢀ71.02
ꢀ84.04
Hydrophophic interaction
2 (GLY 151, LYS 33)
2 (GLY 151, LYS 33)
1 (GLY 151)
ꢀ
ꢀ0.00 e+
ꢀ1.04
1.915 (C–O–H), 2.152 (N–H–O)
1.855 (C–O–H), 2.070 (N–H–O)
1.829 (C–H–O)
ꢀ1.27
ꢀ0.55
3 (GLY 151, LYS 33, TRP 141) 1.857 (C–H–O), 2.237 (N–H–O), 2.463 (N–H–O) ꢀ0.94
3 (GLY151, LYS 33, HIS 180)
2 (ASP 101, GLY 206)
1 (GLY 151)
1.821 (C–O–H), 2.029 (N–H–O), 1.917 (N–H–O) ꢀ1.32
1.962 (C–O–H), 2.019 (N–H–O)
2.060 (C–H–O)
1.763 (C–O–H)
1.852 (C–O–H)
–
ꢀ0.99
ꢀ0.90
ꢀ0.95
ꢀ0.91
ꢀ0.00 e+
ꢀ0.93
0.97
1a
2a
3a
4a
5a
6a
7a
SAHA
1 (GLY 151)
1 (GLY 151)
Hydrophophic interaction
1 (GLY 151)
2 (HIS 180, GLY 151)
Hydrophophic interaction
PHE 208
1.880 (C–H–O)
1.814 (C–H–O)
–
0.00 e+
ꢀ1.43
1.862 (N–H–O), 1.880 (N–H–O)
7.81–7.92 (m, 5H), 7.90 (d, 2H, J = 8.2 Hz), 8.03 (d, 2H, J = 8.2 Hz),
8.10–8.16 (m, 2H), 8.76 (d, 1H, J = 1.2 Hz), 10.61 (s, 1H), 13.47 (s,
1H); 13C NMR (100 MHz, DMSO-d6) d: 115.3, 117.9, 120.3, 122.2,
123.2, 123.3, 124.8, 126.2, 127.7, 128.4, 131.9, 132.4, 132.5,
133.5, 133.7, 134.2, 134.5, 135.3, 136.8, 139.4, 139.8, 143.7,
166.0, 177.4, 180.2; MS (EI): m/z 505.84 (M+, 5%), 491.09 (35),
474.60 (10), 450.66 (15), 259.18 (20), 218.31 (60), 198.36 (45),
125.41 (35), 81.36 (100).
(10), 408.63 (15), 351.84 (38), 289.86 (95), 233.83 (100),
222.08 (18).
3.4.7. N-[4-(6,11-dioxo-6,11-dihydro-5H-benzo[b]carbazole-2-
sulfonyl)-phenyl]-4-nitro-benzamide (6a)
Yellow solid; Reaction time 2 h (0.215 g, 78%); mp > 300 °C;
UV–Vis (acetone): 258.18 nm; IR (KBr): 1319, 1348, 1529, 1591,
1678, 3437 cmꢀ1 1H NMR (400 MHz, DMSO-d6) d: 7.64–8.39 (m,
;
15H), 9.81 (s, 1H), 10.80 (s, 1H); 13C NMR (100 MHz, DMSO-d6)
d: 117.1, 119.0, 122.0, 122.1, 123.5, 124.6, 126.1, 126.3, 126.8,
127.8, 128.4, 129.0, 131.0, 132.1, 132.7, 132.8, 133.4, 135.1,
135.2, 136.4, 138.0, 140.0, 163.1, 180.7, 182.4; MS (EI): m/z
548.56 (Mꢀ3, 5%), 527.29 (20), 509.61 (20), 251.56 (80), 190.52
(100), 164.77 (18).
3.4.4. N-[4-(6,11-dioxo-6,11-dihydro-5H-benzo[b]carbazole-2-
sulfonyl)-phenyl]-3-methyl-benzamide (3a)
Light yellow solid; Reaction time 2 h (0.199 g, 77%);
mp > 300 °C; UV–Vis (acetone): 339.39 nm; IR (KBr): 1244, 1589,
1668, 2922, 3255 cmꢀ1 1H NMR (400 MHz, DMSO-d6) d: 2.38 (s,
;
3H), 7.40 (m, 2H, J = 7.8 Hz), 7.70–8.20 (m, 12H), 8.77 (s, 1H),
10.59 (s, 1H), 13.54 (s, 1H); 13C NMR (100 MHz, DMSO-d6) d:
21.3, 115.8, 120.7, 122.7, 125.4, 126.7, 128.7, 128.8, 128.9, 132.4,
133.0, 133.5, 134.0, 134.2, 134.7, 135.0, 135.8, 137.3, 138.2,
139.9, 140.3, 142.6, 143.2, 166.6, 178.1, 181.0; MS (EI): m/z
518.45 (Mꢀ2, 15%), 496.07 (15), 477.41 (20), 300.78 (12), 226.92
(35), 171.95 (100), 110.98 (75), 96.96 (52).
3.4.8. N-[4-(6,11-dioxo-6,11-dihydro-5H-benzo[b]carbazole-2-
sulfonyl)-phenyl]-3,5-dinitro-benzamide (7a)
Yellow solid; Reaction time 2 h (0.228 g, 77%); mp > 300 °C;
UV–Vis (acetone): 269.09 nm; IR (KBr): 1244, 1344, 1535, 1629,
1660, 3300 cmꢀ1 1H NMR (400 MHz, DMSO-d6) d: 7.75–8.15 (m,
;
10H), 8.76 (d, 1H, J = 1.2 Hz), 8.98 (t, 1H, J = 2.0 Hz), 9.10 (d, 2H,
3.4.5. N-[4-(6,11-dioxo-6,11-dihydro-5H-benzo[b]carbazole-2-
sulfonyl)-phenyl]-4-methyl-benzamide (4a)
Brick red color solid; Reaction time 2 h (0.201 g, 78%);
mp > 300 °C; UV–Vis (acetone): 346.67 nm; IR (KBr): 1242, 1651,
1666, 2922, 3352 cmꢀ1 1H NMR (400 MHz, DMSO-d6) d: 2.36 (s,
;
3H), 7.31 (d, 2H, J = 8.0 Hz), 7.75–8.16 (m, 12H), 8.75 (s, 1H),
10.51 (s, 1H), 13.50 (s, 1H); 13C NMR (100 MHz, DMSO-d6) d:
20.9, 115.3, 117.9, 120.2, 122.2, 123.1, 124.8, 126.2, 127.8, 128.3,
128.9, 131.3, 131.5, 132.4, 133.5, 133.7, 134.5, 135.2, 136.8,
139.4, 139.8, 142.1, 143.8, 165.8, 177.4, 180.2; MS (EI): m/z
521.85 (M+1, 25%), 471(75), 250.37 (30), 184 (100), 81.79 (65).
3.4.6. N-[4-(6,11-dioxo-6,11-dihydro-5H-benzo[b]carbazole-2-
sulfonyl)-phenyl]-3-nitro-benzamide (5a)
Pale yellow solid; Reaction time 2 h (0.212 g, 77%); mp > 300 °C;
UV–Vis (acetone): 277.58 nm; IR (KBr): 1251, 1350, 1525, 1591,
1670, 3257 cmꢀ1 1H NMR (400 MHz, DMSO-d6) d: 7.70–8.20 (m,
;
15H) 10.94 (s, 1H), 13.55 (s, 1H); 13C NMR (100 MHz, DMSO-d6)
d: 106.9, 119.1, 122.9, 123.2, 124.5, 125.6, 125.6, 125.3, 125.1,
126.4, 127.1, 129.1, 131.2, 132.4, 132.5, 132.9, 133.2, 134.1,
134.4, 134.9, 136.0, 137.1, 139.2, 146.1, 163.4, 180.2, 181.0; MS
(EI): m/z 550.65 (Mꢀ1, 15%), 536.40 (30), 507.28 (10), 437.65
Fig. 17. Docking model structures of compound SAHA into the HDAC8 binding
pocket.

36
P. Ravichandiran et al. / Bioorganic Chemistry 53 (2014) 24–36
J = 2.0 Hz) 11.14 (s, 1H), 13.50 (s, 1H); 13C NMR (100 MHz, DMSO-
d6) d: 115.3, 117.9, 120.8, 212.4, 211.3, 123.2, 124.8, 126.1, 128.1,
128.5, 132.4, 133.5, 133.7, 134.4, 136.3, 136.5, 136.8, 139.4,
139.8, 142.8, 148.0, 148.9, 161.9, 177.4, 180.2; MS (EI): m/z
595.42 (Mꢀ1, 35%), 562.58 (15), 543.04 (40), 407.72 (45), 386.74
(60), 328.63 (100), 249.72 (75), 214.68 (32).
pound 3 was the one with the best glide and E model score of
ꢀ9.06 and ꢀ73.41, respectively which is close to the glide score
of SAHA (standard). In all docked molecules, the compound 7a
exhibits least glide and E model score of ꢀ2.97 and ꢀ71.02
respectively.
Acknowledgments
3.5. Measurement of cytotoxicty
The authors thank the Management and the authorities of Karu-
nya University, Coimbatore, for their kind support, constant
encouragement and providing KSJR fellowship to PR. We extend
our thanks to SAIF, IISC, Bangalore, India for NMR spectral analysis
and SAIF, IIT, Madras, India for the Mass spectral analysis.
To evaluate the cytotoxic property of the synthesized quinone
derivatives, the MTT assay was carried out [24]. A stock solution
of 20 mg/mL was prepared in dimethyl sulfoxide (DMSO) (Sigma
Chemical Co., St. Louis, MO, USA). The solution was stored in ali-
quots at ꢀ20 °C. Further dilutions were made in Dulbecco’s Modi-
fied Eagle Medium (DMEM) to required concentrations between 5
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