Novel bis(indolyl)hydrazide-hydrazones as potent cytotoxic agents

Article abstract of DOI:10.1016/j.bmcl.2011.11.031

A series of bis(indolyl) hydrazide-hydrazones 5a-n were synthesized and evaluated for their cytotoxicity against selected human cancer cell lines. The reaction of indole-3-carboxaldehyde 2 with indole-3-carbohydrazide 4 in presence of catalytic amount of acetic acid afforded 5a-n in good yields. Among the synthesized bis(indolyl)hydrazide-hydrazones, the compound 5b with N-(p-chlorobenzyl) and bromo substituents was found to be the most potent against multiple cancer cell lines (IC₅₀ = 1.0 μM, MDA-MB-231). The compound 5k exhibited selective cytotoxicity against breast cancer cell line MCF7 (IC₅₀ = 3.1 μM).

Full text of DOI:10.1016/j.bmcl.2011.11.031

Bioorganic & Medicinal Chemistry Letters 22 (2012) 212–215
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel bis(indolyl)hydrazide–hydrazones as potent cytotoxic agents
Dalip Kumar a, , N. Maruthi Kumar a, Soumitra Ghosh b, Kavita Shah b,
⇑
⇑
a Department of Chemistry, Birla Institute of Technology and Science, Pilani 333 031, India
b Department of Chemistry and Purdue Cancer Center, Purdue University, 560 Oval Drive, West Lafayette, IN 47907, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of bis(indolyl) hydrazide–hydrazones 5a–n were synthesized and evaluated for their cytotoxicity
against selected human cancer cell lines. The reaction of indole-3-carboxaldehyde 2 with indole-3-carbo-
hydrazide 4 in presence of catalytic amount of acetic acid afforded 5a–n in good yields. Among the syn-
thesized bis(indolyl)hydrazide–hydrazones, the compound 5b with N-(p-chlorobenzyl) and bromo
Received 3 August 2011
Revised 21 October 2011
Accepted 9 November 2011
Available online 16 November 2011
substituents was found to be the most potent against multiple cancer cell lines (IC50 = 1.0
MB-231). The compound 5k exhibited selective cytotoxicity against breast cancer cell line MCF7
(IC50 = 3.1 M).
lM, MDA-
Keywords:
Bisindoles
Hydrazide–hydrazone
Anticancer agents
Bis(indole) alkaloids
l
Ó 2011 Elsevier Ltd. All rights reserved.
Indole ring system is one of the most ubiquitous heterocycles in
nature,1,2 which has become important structural components in
many pharmaceutical agents with diverse biologically activities.3
Of these active scaffolds, bis(indole) alkaloids with a broad spec-
trum of biological activities are being discovered from marine
invertebrates such as sponges, bryozoans coelenterates and tuni-
cates.4–8 For example, Nortopsentine A–C isolated from a marine
sponge Spongosorites ruetzleri exhibit in vitro cytotoxicity against
Many of the indole-based small molecules are identified as
potent anticancer agents, for example indolyl glyoxalyl amide
(D-24851) destabilizes microtubules and blocks cell cycle transi-
tion specifically at G2/M phase.21 Similarly the functional group
such as hydrazide–hydrazone (–CO–NH–N@CH–) have shown
significant role as pharmaceutical agents,22 possessing antiinflam-
matory,23 antimalarial,24 antimicrobial,25 antileishmanial,26 anti-
convulsant,27 antitubercular28 and antitumor29–31 activities.
Further improvements include the synthesis of 3-phenyl-5-sulfon-
amidoindole-2-carboxylic acid hydrazides as anti-depressant
agents at 100 mg/kg.32 This active pharmacophore also reported
for anticancer activity, some pyrazole and benzo[d]isothiazole-3-
carboxylic acid hydrazides are found to be potent antineoplastic
agents. Song and co-workers, reported 3,4,5-trimethoxybenzohyd-
razides for their anticancer activity against PC3 cancer cell line.33
The 5-chloro-3-methyl-indole-2-carboxylic acid benzylidene
hydrazides are identified as potent apoptosis inducers by inhibit-
ing tubulin polymerization in G2/M phase against breast cancer cell
P388 cells (IC50 = 4.5–20.7
further improved P388 activity (IC50, 0.8–2.1
l
M) and their N-methylated derivatives
l
M).9–11 Isolated
from Aspergillus fungi another bis(indole) heterocycle, Asterriqui-
none with a six-membered ring as a spacer is effective in inhibiting
the growth of several transplantable animal tumors in vivo.12 Due
to immense biological activities associated with bis(indole) natural
products, in recent time researchers have paid great attention to
synthetic analogues of bis(indole)alkaloids with variety of hetero-
cyclic spacers such as thiazoles, furans, thiophenes, isoxazoles and
pyridines were reported for their cytotoxicities against several can-
cer cell lines.8,13–17 Apart from heterocyclic spacers other long
chain linkers such as bis(indole)enamides, Coscinamide A–C
(Fig. 1) are isolated from a marine sponge Coscinoderma sp. that
showed HIV-inhibitory activity in the NCI assay.18,19
lines T47D (IC50 = 0.2 l
M).34 Most recently reported aroyl hydra-
zones of 2-phenylindole-3-carboxaldehydes exhibited excellent
anticancer results by blockage of cell cycle in G2/M phase and drive
the tumor cells into apoptosis as demonstrated by the strong in-
crease of caspase-3 activity against breast cancer cells (IC50 = 20–
30 nM).35 In due course hydrazides of oxindoles were reported as
Indole ring substituted with variety of functional groups and
linked to heterocycles via C-3 position has led to a fascinating array
of bioactive natural products and pharmaceutical agents.3,20
potent inhibitors of tubulin polymerization IC50 = 0.19–0.97 l
M).36
Our previous reports include studies of various indolylazoles
linking indole ring with a five-membered heterocycle such as
5-(3-indolyl)-1,3,4-oxadiazoles,37 4-(3-indolyl)oxazoles,38 5-(3-
⇑
indolyl)-1,3,4-thiadiazoles,39
indolyl-1,2,4-triazoles40
and
Corresponding authors. Tel.: +91 1596 245073 229 (D.K.); +1 765 496 9470
(K.S.).
bis(indolyl)-1,2,4-thiadiazoles41 as potential anticancer agents. In
a way to find potent anticancer agents and important biological
E-mail addresses: dalipk@bits-pilani.ac.in (D. Kumar), shah23@purdue.edu (K.
Shah).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.11.031

D. Kumar et al. / Bioorg. Med. Chem. Lett. 22 (2012) 212–215
213
N
O
N
O
O
H
N
R
HO
OH
N
H
N
R
R
R
O
N
H
R
N
H
N
H
N
H
N
H
N
H
N
H
N
H
R
Coscinamide A-C
Bis(indolyl)pyrazines
Hyrtisin B
Nortopsentin A-D
O
O
H
N
NH
O
O
N
R2
R3
4
N
R
N
3
5
6
N
H
O
N
1
2
N
R4
N
H
R'
R
N
H
N
7
R1
Cl
Rhopaladins
Indibulin
Bis(indolyl)hydrazide-hydrazones (5a-n)
Figure 1. Rational approach to bis(indolyl)hydrazide–hydrazones.
features of bisindoles and hydrazide–hydrazones, we have re-
placed central heterocyclic spacer in bis(indole)alkaloids with an
active pharmacophore (–CO–NH–N@CH–) to prepare a novel series
of bis(indolyl)hydrazide–hydrazones 5a–n.
cytotoxicity. The bromo derivative 5b showed good activity against
all tested cancer cell lines, particularly selective cytotoxicity
against MDA-MB-231 cancer cell line (IC50 = 1.0 lM). Substitution
of C-5 bromo with a methoxy group reduces the activity (com-
Synthesis of hydrazide–hydrazones 5a–n was achieved as out-
lined in Scheme 1. The reaction of indole 1 with phosphorus oxy-
chloride in presence of dimethylformamide followed by
alkylation resulted in the formation of indole-3-carboxaldehydes
2 in good yields. On the other hand, indole-3-carbohydrazides 4
were prepared from corresponding indole-3-carboxylic acids 3 by
initial preparation of its ethyl ester followed by reaction with
hydrazine hydrate. Finally, the reaction of indole-3-carbohydra-
zides 4 with indole-3-carboxaldehydes 2 in presence of a catalytic
amount of acetic acid in ethanol led to bis(indolyl) hydrazide–
hydrazones 5a–n in good yields (Scheme 1).42 All the synthesized
compounds were well characterized by IR, 1H NMR and Mass spec-
tral analysis.43
pound 5c) but improve the selectivity against MCF7 cancer cell line
(IC50 = 8.7 lM). Introduction of fluoro substituent at C-6 position of
indole ring is also beneficial for the activity (compound 5d) as well
as selectivity against both the breast cancer cell lines (IC50 = 5.3–
6.0
dole ring in 5c led to compound 5e with 16-folds improved selec-
tivity against MDA-MB-231 (IC50 = 5.0 M). The analogue 5f with
lM). Introduction of bromo substituent at C-5 position of in-
l
additional bromine atom at C-5 position of indole ring improved
the activity against all the tested cancer cell lines with selective
cytotoxicity against breast cancer cell lines MCF7 (IC50 = 8.3
and MDA-MB-231 (IC50 = 5.1 M). Introduction of a methoxy sub-
stituent in compound 5g led to analogue 5h with increased activity
against MCF7 cancer cell line (IC50 = 9.8 M). Analogues of 5g,
lM)
l
l
Synthesized hydrazide–hydrazones 5a–n were screened for
their in vitro cytotoxicity against six human cancer cell lines: pros-
tate (PC3, DU145 and LnCaP), breast (MCF7 and MDA-MB-231) and
pancreatic (PaCa2). Cytotoxicity of bis(indolyl)hydrazide–hydra-
zones 5a–n against cancer cell lines is expressed in terms of IC50
values as mentioned in Table 1. Most of the compounds have
shown significant to moderate cancer cell growth inhibition. The
effect of various substituents on indole ring was examined. The
structure–activity relationship (SAR) study revealed that the sub-
stituents like N1-(4-chlorobenzyl), bromo and fluoro on indole ring
are crucial for inducing cytotoxicity as well as selectivity against
particular cancer cell lines. The bis(indolyl) hydrazide–hydrazone
5a was found to be modest selective cytotoxic against PC3 cancer
compounds 5i and 5j with C-6 fluoro and/or C-5 bromo substitu-
ents decreased the cytotoxicity when compared to parent com-
pound 5g. Introduction of bromo substituent in compound 5h
induces high selectivity (compound 5k) towards MCF7 cancer cell
line (IC50 = 3.1
with improved cytotoxicity, particularly towards LnCaP cancer
cells (IC50 = 9.6 M). However, N-methylation of compounds 5c
lM). The N-methylation of 5i led to compound 5l
l
and 5d resulted in poor activity (compounds 5n and 5m). Doxoru-
bicin was used as a positive control, which showed higher cytotox-
icity against LnCaP and MDA-MB-231 cancer cell lines.
Introduction of hydrazide–hydrazone moiety as
a spacer in
bis(indolyl)hydrazide–hydrazones 5a–n found to be beneficial for
the anticancer activity when compared with a five/six-membered
heterocyclic ring spacer (imidazole, pyrazine, diketone and ketoa-
mide) in bis(indole) alkaloids . Generally, the reported bis(indolyl)
alkaloids8,13–17 showed anticancer activity with IC50 values ranging
cells (IC50 = 16.2 lM). By replacing 4-chlorobenzyl with 4-
methoxybenzyl group at N-1 position led to compound 5g with
modest improvement in cytotoxicity, particularly against breast
cancer cell lines (IC50 = 12–15
l
M). Incorporation of bromo or
from 1.7
lM to 100 lM against broad spectrum of cancer cell lines,
methoxy group at C-5 position of indole ring also improved the
whereas the most cytotoxic bis(indolyl) hydrazide–hydrazone 5b
CHO
R2
R3
R2
R3
a
O
R
N
N
H
1
N
R4
R2
R3
N
H
2
c
CONHNH2
N
N
COOH
R
R1
R4
R
5a-n
b
N
N
R1
R1
4
3
Scheme 1. Reagents: (a) (i) DMF-POCl3; (ii) R4X, 50% NaOH, toluene; (b) (i) dil H2SO4, ethanol, reflux; (ii) NH2NH2ÁH2O, ethanol; (c) AcOH, ethanol, reflux.

214
D. Kumar et al. / Bioorg. Med. Chem. Lett. 22 (2012) 212–215
Table 1
Anticancer activity of bis(indolyl)hydrazide–hydrazones 5a–n against selected human cancer cell lines IC50 (lM)
Compound
R
R1
R2
R3
R4
LnCaP
MCF7
MDA-MB-231
DU145
PaCa2
PC3
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
H
H
H
H
Br
Br
H
H
H
Br
Br
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
CH3
CH3
H
Br
OCH3
H
OCH3
H
H
OCH3
H
H
OCH3
H
H
H
H
F
H
F
H
H
F
F
H
F
F
H
4-ClC6H4CH2
4-ClC6H4CH2
4-ClC6H4CH2
4-ClC6H4CH2
4-ClC6H4CH2
4-ClC6H4CH2
4-OCH3C6H4CH2
4-OCH3C6H4CH2
4-OCH3C6H4CH2
4-OCH3C6H4CH2
4-OCH3C6H4CH2
4-OCH3C6H4CH2
4-ClC6H4CH2
>100
3.7
96.7
3.2
8.7
6.0
29.1
8.3
12.4
15.0
13.1
31.7
3.1
22.6
90.6
99.1
11.67
66.4
1.0
82.7
5.3
5.0
5.1
52.1
3.6
44.3
8.7
16.2
12.4
30.3
19.5
15.9
12.4
14.6
13.6
18.4
21.8
28.0
12.8
22.1
21.2
62.26
>100
55.2
>100
24.8
31.9
>100
>100
>100
>100
9.6
50.0
18.6
57.0
12.5
43.9
19.1
16.2
67.5
84.4
15.2
84.2
93.0
15.65
26.7
18.0
26.3
10.6
41.6
30.5
43.3
45.5
44.5
55.8
50.4
56.4
18.23
15.1
9.8
71.2
99.8
61.2
17.7
>100
>100
3.07
5m
5n
Doxorubicin
H
OCH3
>100
>100
5.23
4-ClC6H4CH2
Bold values show IC50 of <10 lM.
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exhibited cytotoxicity with an IC50 value of 1.0
venient to synthesize a diverse library of bis(indolyl)hydrazide–
hydrazones when compared with bis(indole) alkaloids.
In conclusion, we have reported synthesis and anticancer activ-
ity of novel bis(indolyl) hydrazide–hydrazones 5a–n. Most of the
compounds showed significant cytotoxicity against multiple can-
l
M. Also it is con-
cer cell lines (IC50 < 10
tive cytotoxicity among the tested cancer cell lines, compound 5k
was most selective against MCF7 (IC50 = 3.1 M). Compound 5b
was found to be most potent in the series with IC50 values 1.0,
3.2, 3.6 and 3.7 M against MDA-MB-231, MCF7, DU145 and LnCaP
lM). A few compounds also exhibited selec-
l
l
cancer cell lines, respectively. Further we found that N1, (4-
methoxybenzyl) and substituents on indole ring are important
for the anticancer activity of hydrazide–hydrazones 5. Present
study revealed that replacement of central ring of bis(indolyl)het-
erocycles with hydrazide–hydrazone is beneficial for the cytotoxic-
ity. Further studies to improve cytotoxicity and identify cellular
targets of bis(indolyl) hydrazide–hydrazones 5 are in progress
and will be reported in due course.
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Acknowledgments
37. Kumar, D.; Sundaree, S.; Johnson, E. O.; Shah, K. Bioorg. Med. Chem. Lett. 2009,
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Authors acknowledge financial support from the Council of Sci-
entific and Industrial Research (CSIR), New Delhi. N.M.K. thanks to
CSIR for Senior Research Fellowship.
40. Kumar, D.; Narayanam, M. K.; Chang, K.-H.; Shah, K. Chem. Biol. Drug Des. 2011,
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References and notes
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carboxaldehyde
2 (1 mmol) and indole-3-carbohydrazide 4 (1 mmol) in
ethanol(10 mL) was added catalytic amount of acetic acid (0.1 mL). The
reaction mixture was refluxed for 3–4 h. Progress of the reaction was
monitored by thin layer chromatography. Upon completion, the reaction
mixture was cooled, and solid mass so obtained was filtered and recrystallized
to afford pure bis(indolyl)hydrazide–hydrazones 5a–n in good yields.
43. Spectral data: Compound 5a: Yield 92%, white solid; mp 240–241 °C; 1H NMR
(400 MHz, DMSO-d6): d 11.40 (s, 1H), 11.07 (s, 1H), 8.57 (s, 1H), 8.38–8.14 (m,
3H), 7.85 (s, 1H), 7.59 (s, 1H), 7.45 (d, J = 7.60 Hz, 1H), 7.38 (d, J = 8.00 Hz, 1H),
7.20–7.15 (m, 5H), 6.84 (d, J = 8.40 Hz, 2H), 5.23 (s, 2H); IR (KBr,
m
cmÀ1): 3390,
3144 (N–H), 2954, 1656 (C@O), 1546, 1513, 1452, 1242, 1139, 846, 804, 750;
MS (ESI) m/z calcd for C25H20ClN4O (M+H)+ 427.13, obsd 427.3. Compound 5b:
Yield 90%, off-white solid, mp 170–172 °C; 1H NMR (400 MHz, DMSO-d6): d
11.34(s, 1H), 11.05(s, 1H), 8.50 (s, 1H), 8.20–8.10 (m, 2H), 7.90 (s, 1H), 7.48 (s,
1H), 7.43 (d, J = 8.00 Hz, 2H), 7.35 (d, J = 8.00 Hz, 1H), 7.22–7.18 (m, 4H), 6.88
(d, J = 8.00 Hz, 2H), 5.28 (s, 2H); IR (KBr,
(C@O), 1614, 1573, 1377, 1238, 1203, 798, 748; MS (ESI) m/z calcd for
m
cmÀ1): 3390, 3145 (N–H), 2893, 1633
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C
25H18BrClN4O (M)+ 504.03, obsd 504.2.. Compound 5c: Yield 85%, white solid,
mp 152–153 °C; 1H NMR (400 MHz, DMSO-d6): d 11.67 (s, 1H), 11.11 (s, 1H),
8.52 (s, 1H), 8.20–8.14 (m, 2H), 7.91 (s, 1H), 7.88 (s, 1H), 7.46 (d, J = 8.0 Hz, 1H),
7.39 (dd, J = 8.4 Hz, 2.4 Hz, 3H), 7.25 (d, J = 8.4 Hz, 2H), 7.19–7.11(m, 2H), 6.84
19. Bokesch, H. R.; Pannell, L. K.; McKee, T. C.; Boyd, M. R. Tetrahedron Lett. 2000,
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(d, J = 8.0 Hz, 1H), 5.41 (s, 2H), 3.79 (s, 3H); IR (KBr,
2970, 1660 (C@O), 1602, 1537, 1484, 1229, 1178, 794, 752; MS (ESI) m/z calcd
m
cmÀ1): 3226 (N–H), 3024,

D. Kumar et al. / Bioorg. Med. Chem. Lett. 22 (2012) 212–215
215
for C26H22ClN4O2 (M+H)+ 457.14, obsd 457.7. Compound 5d: Yield 73%, pale
yellow solid, mp 163–164 °C; 1H NMR (400 MHz, DMSO-d6): d 11.52 (s, 1H),
11.10 (s, 1H), 8.48 (s, 1H), 8.35 (s, 1H), 8.23 (d, J = 8.0 Hz, 2H), 8.09 (s, 1H), 7.75
(s, 1H), 7.42 (d, J = 8.0 Hz, 1H), 7.31 (d, J = 8.0 Hz, 2H), 7.23–7.20 (m, 2H), 7.17–
3H), 7.15–7.09 (m, 2H), 6.92 (t, J = 8.0 Hz, 1H), 6.84 (d, J = 8.0 Hz, 2H), 5.29 (s,
2H), 3.72 (s, 3H); IR (KBr,
m
cmÀ1): 3243, 3038(N–H), 2926, 1654 (C@O), 1603,
1537, 1434, 1246, 845, 819, 754; MS (ESI) m/z calcd for C26H22FN4O2 (M+H)+
440.2, obsd 441.4. Compound 5j: Yield 78%, light brown solid, mp 248–249 °C;
1H NMR (400 MHz, DMSO-d6): d 11.62 (s, 1H), 11.11 (s, 1H), 8.46 (s, 1H), 8.41
(s, 1H), 8.34 (s, 1H), 8.02 (s, 1H), 7.65 (s, 1H), 7.35 (d, J = 8.4 Hz, 1H), 7.23 (d,
J = 8.4 Hz, 1H), 7.16 (d, J = 8.4 Hz, 3H), 6.89 (t, J = 8.0 Hz , 1H), 6.82 (d, J = 8.4 Hz,
7.09 (m, 2H), 6.95–690 (m, 1H), 5.38 (s, 2H); IR (KBr,
m
cmÀ1): 3223 (N–H),
3120, 2913, 1661 (C@O), 1614, 1529, 1454, 1225, 862, 813, 752; MS (ESI) m/z
calcd for C25H19ClFN4O (M+H)+ 445.12 obsd 445.4. Compound 5e: Yield 85%,
light brown solid, mp 150–151 °C; 1H NMR (400 MHz, DMSO-d6): d 11.86 (s,
1H), 11.16 (s, 1H), 8.51 (s, 1H), 8.37 (s, 1H), 8.20 (s, 1H), 7.92 (s, 1H), 7.58 (s,
1H), 7.45 (d, J = 8.00 Hz, 1H), 7.39–7.36 (m, 3H), 7.30 (dd, J = 8.40, 2.00 Hz, 1H),
7.24 (d, J = 8.40 Hz, 2H), 6.85 (d, J = 7.60 Hz, 1H), 5.41 (s, 2H), 3.79 (s, 3H); IR
2H), 5.26 (s, 2H), 3.71 (s, 3H); IR (KBr,
(C@O), 1537, 1454, 1249, 825, 794, 751; MS (ESI) m/z calcd for
m
cmÀ1): 3232, 3018 (N–H), 2958, 1664
C
26H21BrFN4O2(M+H)+ 519.18, obsd 519.6. Compound 5k: Yield 85%, pale
yellow solid, mp 260–261 °C; 1H NMR (400 MHz, DMSO-d6): d 11.75 (s, 2H),
11.40 (s, 1H), 8.54 (s, 1H), 8.21 (s, 1H), 8.13–8.07 (m, 2H), 7.86 (s, 1H), 7.47–
7.40 (m, 3H), 7.22 (d, J = 8.00 Hz, 2H), 7.03–6.97 (m, 2H), 5.31 (s, 2H), 3.79 (s,
(KBr,
m
cmÀ1): 3236 (N–H), 3106, 2933, 1664 (C@O), 1612, 1552, 1484, 1257,
889, 796, 752; MS (ESI) m/z calcd for C26H20BrClN4O2 (M)+ 534.04 obsd, 534.1.
Compound 5f: Yield 78%, pale yellow solid, mp 261–262 °C; 1H NMR (400 MHz,
DMSO-d6): d 11.65 (s, 1H), 11.15 (s, 1H), 8.48 (s, 1H), 8.42–8.36 (m, 2H), 8.12 (s,
1H), 8.00 (s, 1H), 7.69 (s, 1H), 7.36 (d, J = 8.0 Hz, 1H), 7.28 (d, J = 8.0 Hz, 2H),
7.23 (d, J = 8.0 Hz, 1H), 7.19–7.13 (m, 2H), 6.93–688 (m, 1H), 5.35 (s, 2H); IR
3H), 3.69 (s, 3H); IR (KBr,
1537, 1444, 1247, 887, 794; MS (ESI) m/z calcd for, C27H24BrN4O3 (M+H)+
530.1, obsd 531.2. Compound 5l:Yield 85%, off-white solid, mp 281–283 °C; 1
m
cmÀ1): 3213, 3076 (N–H), 2934, 1655 (C@O), 1612,
H
NMR (400 MHz, DMSO-d6): d 11. 16 (s, 1H), 8.46 (s, 1H), 8.31 (s, 1H), 8.22–8.13
(m, 2H), 7.96 (s, 1H), 7.52 (d, J = 8.40 Hz, 1H), 7.45–7.36 (m, 3H), 7.29 (d,
J = 8.40 Hz, 2H), 7.25–7.16 (m, 2H), 7.06 (d, J = 8.00 Hz, 1H), 5.43 (s, 2H), 3.87 (s,
(KBr,
m
cmÀ1): 3238, 3045 (N–H), 2913, 1674 (C@O), 1605, 1537, 1454, 1220,
810, 771, 734; MS (ESI) m/z calcd for C25H18BrClFN4O (M+H)+ 523.03 obsd
523.6. Compound 5g: Yield 80%, white solid, mp 212–213 °C; 1H NMR
(400 MHz, DMSO-d6): d 10.02 (s, 1H), 8.63 (s, 1H), 8.46 (s, 1H), 8.33 (d,
J = 8.0 Hz, 2H), 8.09 (d, J = 8.0 Hz, 1H), 7.76 (s, 1H), 7.47 (d, J = 8.0 Hz, 1H), 7.40
(d, J = 8.0 Hz, 1H), 7.38–7.29 (m, 6H), 7.11 (d, J = 8.0 Hz, 2H), 5.35 (s, 2H), 3.83
3H), 3.70 (s, 3H); IR (KBr,
m
cmÀ1): 3243 (N–H), 3020, 2904, 1649 (C@O), 1601,
1537, 1458, 1242, 836, 800, 736; MS (ESI) m/z calcd for C27H23FN4O2 (M)+
454.2, obsd 454.8. Compound 5m: Yield 88%, off-white solid, mp 235–236 °C;
1H NMR (400 MHz, DMSO-d6): d 11. 00 (s, 1H), 8.49 (s, 1H), 8.39 (s, 1H), 7.83 (s,
1H), 7.54 (s, 1H), 7.41 (d, J = 8.00 Hz, 1H), 7.23 (d, J = 8.4 Hz, 1H), 7.27 (t,
J = 8.00 Hz, 1H), 7.23–7.15 (m, 3H), 7.10 (d, J = 9.20 Hz, 1H), 6.95–6.90 (m, 1H),
(s, 3H); IR (KBr,
m
cmÀ1): 3236 (N–H), 3011, 2924, 1654 (C@O), 1572, 1537,
1436, 1214, 864, 769, 746; MS (ESI) m/z calcd for C26H23N4O2 (M+H)+ 423.2,
obsd 423.3. Compound 5h: Yield 85%, white solid, mp 150–151 °C; 1H NMR
(400 MHz, DMSO-d6): d 11. 97 (s, 1H), 11.15 (s, 1H), 8.51 (s, 1H), 8.35–8.15 (m,
2H), 8.00 (s, 2H), 7.64 (s, 1H), 7.46–7.40 (m, 2H), 7.30 (d, J = 8.40 Hz, 1H), 7.23–
7.21 (m, 2H), 6.86 (d, J = 8.40 Hz, 3H), 5.31 (s, 2H), 3.78 (s, 3H), 3.69 (s, 3H); IR
6.86 (d, , J = 8.80 Hz, 2H), 5.26 (s, 2H), 3.90 (s, 3H); IR (KBr,
H), 2904, 1644 (C@O), 1536, 1424, 1256, 831, 795, 738; MS (ESI) m/z calcd for
m
cmÀ1): 3219 (N–
C
26H20ClFN4O (M)+ 458.2 obsd 458.7. Compound 5n: Yield 90%, white solid, mp
211–212 °C; 1H NMR (400 MHz, DMSO-d6, d): d 11. 00 (s, 1H), 8.54 (s, 1H), 8.29
(s, 1H), 7.96 (s, 1H), 7.65 (s, 1H), 7.43 (d, J = 8.00 Hz, 1H), 7.30 (d, J = 8.40 Hz,
2H), 7.23–7.21 (m, 2H), 7.18–7.13 (m, 4H), 6.85 (d, J = 8.00 Hz, 1H), 5.37 (s, 2H),
(KBr,
m
cmÀ1): 3233, 3012 (N–H), 2964, 1654 (C@O), 1610, 1532, 1484, 1246,
859, 794, 752; MS (ESI) m/z calcd for C27H24N4O3 (M)+ 452.18 obsd 452.7.
Compound 5i: Yield 75%, light yellow solid, mp 193–195 °C; 1H NMR (400 MHz,
DMSO-d6): d 11.53 (s, 1H), 11.07 (s, 1H), 8.42 (s, 1H), 8.33 (s, 1H), 8.23 (d,
J = 7.6 Hz, 1H), 8.11 (s, 1H), 7.72 (s, 1H), 7.41 (d, J = 8.0 Hz, 1H), 7.25–7.16 (m,
3.90 (s, 3H), 3.73 (s, 3H); IR (KBr,
m
cmÀ1): 3217 (N–H), 2936, 1644 (C@O),
1602, 1537, 1469, 1236, 852, 796, 742; MS (ESI) m/z calcd for C27H23ClN4O2
(M)+ 470.15, obsd 470.2.