Appraisal of GABA and PABA as linker: Design and synthesis of novel benzamide based histone deacetylase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2012.03.058

Histone deacetylase inhibitors have been actively explored as a new generation of chemotherapeutics for cancers, generally known as epigenetic therapeutics. Two novel series of N-(2-amino-phenyl)-4-{[(2/3/4-substituted- phenylcarbamoyl)-methyl]-amino}-but

Full text of DOI:10.1016/j.ejmech.2012.03.058

European Journal of Medicinal Chemistry 53 (2012) 390e397
Contents lists available at SciVerse ScienceDirect
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journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Appraisal of GABA and PABA as linker: Design and synthesis of novel benzamide
based histone deacetylase inhibitors
,
a
a
a
Harish Rajak a , Pramod Kumar , Poonam Parmar , Bhupendra Singh Thakur ,
Ravichandran Veerasamy b, Prabodh Chander Sharma c, Ajay Kumar Sharma d, Arun Kumar Gupta e,
Jawahar Singh Dangi a
*
a Medicinal Chemistry Research Laboratory, SLT Institute of Pharmaceutical Sciences, Guru Ghasidas University, Bilaspur 495 009, CG, India
b Faculty of Pharmacy, AIMST University, Semeling, 08100 Bedong, Kedah Darul Aman, Malaysia
c Institute of Pharmaceutical Sciences, Kurukshetra University, Kurukshetra 136 119, Haryana, India
d Department of Pharmacy, G.S.V.M., Medical College, Kanpur 208002, UP, India
e B.P.S. Mahila Vishwavidyalaya, Khapur-Kalan, Sonepat 131 001, Haryana, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Histone deacetylase inhibitors have been actively explored as a new generation of chemotherapeutics for
cancers, generally known as epigenetic therapeutics. Two novel series of N-(2-amino-phenyl)-4-{[(2/3/4-
Received 15 December 2011
Received in revised form
5 March 2012
Accepted 31 March 2012
Available online 11 April 2012
substituted-phenylcarbamoyl)-methyl]-amino}-butyramide
and
N-(2-amino-phenyl)-4-{[(2/3/4-
substituted-phenylcarbamoyl)-methyl]-amino}benzamide were designed and synthesized as novel
histone deacetylase inhibitors. The anticancer potential of the compounds were determined in-vitro
using MTT assay against HCT-116 and U251 (glioma) cell lines and histone deacetylase inhibitory assay.
The synthesized compounds were investigated for anti-tumor activity against Ehrlich ascites carcinoma
(EAC) cells in Swiss albino mice. The efforts were also made to ascertain structureeactivity relationships
Keywords:
GABA
PABA
among test compounds. The results of the present studying represents appraisal of g-aminobutyric acid
(GABA) and para-aminobenzoic acid (PABA) as linker moiety for development of newer benzamide based
histone deacetylase inhibitor.
Anticancer activity
Histone deacetylase inhibitors
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
class II HDAC4, HDAC6 and HDAC10 with IC50 of 10.7
and 50.1 M, respectively. The IC50 value of MS-275 toward Class
HDACIII SIRT1 was found more than 100 M [7]. The mechanism
mM, >100 mM
m
The epigenetic changes play a vital role in tumorigenesis. The
acetylation and deacetylation of histones have been found to
support to a substantial degree to epigenetic regulation of gene
expression [1,2]. Histone deacetylase inhibitors (HDACi) are a novel
class of chemotherapeutic agent that control gene expression by
increasing the acetylation of histones, leading to induction of
chromatin relaxation and alteration of gene expression [3,4].
Recent findings indicate that HDACi selectively induce growth
arrest, differentiation and apoptosis in tumor cells via transcrip-
tional activation of a small set of genes that control cell prolifera-
tion and cell cycle development like p21 [5,6].
m
inducing cell death in cell lines appears to involve generation of
Reactive Oxygen Species (ROS). MS-275 has demonstrated anti-
proliferative action in an array of in-vitro human cancer cell lines
including breast, ovary, colon, myeloma, pancrease and leukemia. It
has also exhibited in vivo anti-tumor activity in a quite of adult and
pediatric orthotopic tumor xenograft models following oral
administration. Various reports of clinical trials in patients with
refractory solid tumors or refractory hematologic malignancies
have indicated that MS-275 is well tolerated and exhibited poten-
tial anti-tumor activity. MGCD-0103 and CS-055 are other benza-
mide based HDACi, which are in phase II clinical trial.
WO2004035525-Ii is an example of patented benzamide based
HDACi with potent biological activity [8e11]. (Fig. 1).
Entinostat (MS-275, SNDX-275) is a moderately potent benza-
mide based HDACi, which is in phase II clinical trial. MS-275 was
found to be an inhibitor of class I HDACs with a high affinity for
HDAC1 (IC50 ¼ 0.368
m
M) and HDAC3 (IC50 ¼ 0.501
m
M) but rela-
HDACi generally conform to a broadly accepted pharmacophore.
The crystallographic studies for the design of valuable HDACi have
pointed out three structural requirements: a cap group (A) that
provides interaction with the pocket entrance, a terminal group (B)
that can bind to the zinc ion at the bottom of the active site, also
tively weak inhibition of HDAC8 (IC50 ¼ 63.4
m
M). MS-275 inhibits
* Corresponding author. Tel.: þ91 9827911824; fax: þ91 7752260140.
E-mail address: harishdops@yahoo.co.in (H. Rajak).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.03.058

H. Rajak et al. / European Journal of Medicinal Chemistry 53 (2012) 390e397
391
O
N
O
N
H
NH2
N
N
H
NH2
H
N
H
N
N
N
B
O
O
B
A
A
C
C
MS-275
MGCD0103
O
F
O
N
NH
NH
H
N
NH2
NH2
O
F
O
B
B
C
C
A
A
Chidamide (CS-055)
WO2004035525-li
O
NH2
H
H
NH
N
N
H
N
N
H
NH2
R
N
H
O
O
R
O
B
C
B
C
A
01- 11
12-22
A
Fig. 1. Structural features present in clinically established benzamides and synthesized analogs 01e22. A ¼ surface recognition moiety, B ¼ Linker; C ¼ zinc binding group.
known as zinc binding group (ZBG), and between both, a hydro-
phobic linker (C) fitting the tube-like portion of the binding pocket
[12,13] (Fig. 1) The optimum chain length between cap and zinc
binding group for HDACi activity has been found 7e8 atoms [14].
GABA/PABA has resulted in the formation of linker in the title
compounds.
3. Pharmacology
Present studies report the evaluation of
g-aminobutyric acid
(GABA) and para-aminobenzoic acid (PABA) as linker in benzamide
based HDACi.
All the synthesized compounds were tested for their ability to
inhibit HDAC-1 activity and antiproliferative activity by MTT assays
[16]. The test compounds were evaluated in-vivo for their anti-
cancer activity against EAC cells in Swiss albino mice as per
reported procedure [17].
2. Chemistry
The substituted anilines I (iexi) were reacted with chlor-
oacetylchloride resulting in the formation of N-(4-substituted-
phenyl)-2-chloro-acetamide II (iexi). 4-{[(4-substituted-phenyl-
carbamoyl)-methyl]-amino}-butyric acid III (iexi) were prepared
by condensation of II (iexi) with GABA in ethanol in the presence of
triethylamine [15]. The reaction of III (iexi) with 1,2-pheny
lenediamine in the presence of CDI and THF resulted in the
formation of final benzamide analogs i.e., N-(2-amino-phenyl)-4-
{[(4-substituted-phenylcarbamoyl)-methyl]-amino}-butyramides
(01e11) (Scheme 1). Another almost similar reaction scheme was
also performed in which PABA was employed in place of GABA
resulted in the preparation of N-(2-amino-phenyl)-4-{[(4-
substituted-phenylcarbamoyl)-methyl]-amino}-benzamides (12e22)
(Scheme 1). On critical observation, it is clear that the use of
4. Result and discussion
The structures of the title compounds were elucidated on the
basis of elemental analysis, IR, 1H NMR and mass spectral data.
Elemental analyses for C, H and N were found within ꢀ0.4% of the
theoretical value. The spectroscopic data (IR, 1H NMR and mass
spectroscopy) of title compounds were in conformity with the
assigned structure.
All the synthesized compounds were tested for their ability to
inhibit HDAC-1 activity, antiproliferative activity by MTT assays and
in-vivo anticancer activity against EAC cells. The HDAC-1 was
chosen since it is widely implicated in both transcriptional
repression and chromatin remodeling. Test compounds exhibited

392
H. Rajak et al. / European Journal of Medicinal Chemistry 53 (2012) 390e397
O
H
Cl
NH2
N
a
R
R
I (i- xi)
II (i-xi)
b
d
O
OH
H
H
N
N
OH
N
H
N
H
R
R
O
O
O
IV (i-xi)
III (i-xi)
e
c
O
NH2
H
H
NH
H
N
N
N
NH2
N
N
H
R
H
R
O
O
O
01- 11
12-22
Compound Code
I (i)
I (ii)
I (iii)
I (iv)
I (v)
I (vi)
I (vii)
I (viii)
I (ix)
R
H
Compound Code
1
2
3
4
5
6
7
8
9
10
11
R
H
Compound Code
R
H
12
13
14
15
16
17
18
19
20
21
22
4-Br
2-Cl
3-Cl
4-Cl
2- NO2
3- NO2
4- NO2
4-OCH3
4-CH3
4-Br
2-Cl
3-Cl
4-Cl
2- NO2
3- NO2
4- NO2
4-OCH3
4-CH3
4-Br
2-Cl
3-Cl
4-Cl
2- NO2
3- NO2
4- NO2
4-OCH3
4-CH3
I (x)
I (xi)
2,5-Cl
2,5-Cl
2,5-Cl
Scheme 1. Synthesis of GABA and PABA-based benzamides. Reaction conditions: (a) ClCH2COCl, CH2Cl2, 4e6 h stirring, 59e82%; (b) C2H5OH, (C2H5)3N, GABA, 6e7 h reflux, 53e76%;
(c) CDI, THF, 2 h stirring at 60e65 ꢁC, 1,2-phenylenediamine, TFA, 9e10 h stirring, 58e71%; (d) C2H5OH, (C2H5)3N, PABA, 8e9 h reflux, 54e66%; (e) CDI, THF, 2 h stirring at 60e65 ꢁC,
1,2-phenylenediamine, TFA, 11e14 h stirring 56e68%.
significant activity in HDAC-1 inhibitory assay indicating that
inhibition of histone deacetylase-1 might be one of the basis for
anticancer activity. Inhibition of HDAC-1 causes apoptosis leading
to anticancer activity [18]. The antiproliferative activity of test
compounds was evaluated by MTT assays using HCT-116, U251
(glioma) cells [16]. HCT-116 cells were chosen for MTT assays
because they have a high level of HDAC expression [19,20]. The test
compounds were evaluated in-vivo for their anticancer activity
against EAC cells in Swiss albino mice as per reported procedure
[17]. The Mitomycin-C was chosen as standard drug in in-vivo
anticancer studies as its mechasnism of action involves apoptosis
[21]. The tumor weight inhibition (TWI) and tumor cell inhibition
(TCI) were measured as parameters for anticancer evaluation.
The results of in-vitro anticancer studies shows that among
GABA based benzamide analogs (1e11), compound 8 exhibited
GI50 ¼ 0.6
m
M and GI50 ¼ 8.2
mM against HCT-116 and U251 (glioma)
cell lines, respectively (Table 1). In terms of potency of synthesized
benzamide analogs, almost similar results were observed during in-
vivo anticancer studies against EAC cells in Swiss albino mice. The
compound 8 (%TWI ¼ 78.5; %TCI ¼ 76.5) and 19 (%TWI ¼ 83.3; %
TCI ¼ 77.5) were found most potent in GABA and PABA series of
compounds, respectively (Table 2).
The title compounds were designed and synthesized with two
points of diversity i.e., variation of substituents on the surface
recognition moiety (phenyl ring) and variation of linker. It is the
surface recognition moiety along with linker that is responsible for
variation in anticancer activity among different test compounds.
On going through structureeactivity relationship, it has been
observed that compounds bearing the groups like nitro, chloro and
bromo on phenyl ring possess high potency in in-vitro and in-vivo
anticancer evaluation. On the other hand, replacement of these
groups with methyl groups resulted in compounds with lesser
antiproliferative activity. On comparison of results, it has been
found that anticancer activity of test compounds changes on
varying para-substituted group on aryl moiety as follows: nitro
> chloro > bromo > methoxy > methyl > no substitution. It is to be
maximum HDAC inhibitory activity with an IC50 ¼ 0.9
m
M in HDAC-
1 assay. The compound 8 displayed GI50 ¼ 0.6
m
M and GI50 ¼ 9.4
mM
against HCT-116 and U251 (glioma) cell lines, respectively. On the
other hand, compound 19 was found to be most active among PABA
based benzamide analogs (12e22), with HDAC inhibitory activity
IC50 ¼ 0.7
mM in HDAC-1 assay. The compound 19 showed

H. Rajak et al. / European Journal of Medicinal Chemistry 53 (2012) 390e397
393
Table 1
The optimum length of linker is one of the prime requirements
for a benzamide based HDACi to exhibit anti-tumor activity. The
presence of anticancer activity in title compounds may be attrib-
uted to the optimum length of linker i.e., 7e8 atoms incorporated in
the benzamide analogs from GABA and PABA. An overview of the
results of anticancer evaluation shows that PABA based benzamide
analogs were found to possess more activity than corresponding
GABA based benzamide analogs. Thus it is clear that PABA provides
better linker than that contributed by GABA in the design of HDACi.
Outcomes of HDAC-1 inhibition and antiproliferative assays.
Compound HDAC
inhibitory
activitya
HCT-116a
GI50 TGI
(
mM)
U251 (Glioma)a
(m
M)
LC50
LC50
GI50
TGI
(IC50 (mM))
1
2
3
4
5
6
7
8
9.2
1.7
2.9
1.8
1.4
3.1
2.3
0.9
3.6
7.5
8.9
7.4
1.6
2.9
2.5
1.0
2.0
1.4
0.7
3.1
4.5
8.6
0.5
70.4 >100
>100 >100
55.5 >100 10.4
28.6 >100
19.5 >100
11.4
32.0 >100
>100
80.0 >100
>100
7.7
21.8 >100
>100
>100
>100
11.6
5.5
2.8
0.9
0.6
8.6
75.0 >100
45.0 >100
32.0 >100
22.5 >100
8.5 >100
44.0 >100
75.0 >100
>100
5. Conclusion
15.0
9.4
93.5 >100
66.0 >100
9
34.0 >100
57.0 >100
44.0 >100
65.0 >100
>100
>100
>100
>100
The structure of the title compounds fulfilled all the three
pharmacophoric structural requirements for HDAC inhibitors i.e.,
benzamide group as zinc binding group, propylamino-acetamide
moiety (in GABA based analogs)/tolyl-amino-acetamide (in PABA
based analogs) as linker and phenyl moiety as surface recognition
cap group. All the title compounds exhibited varied degree of
anticancer activity and some compounds showed anticancer
activity comparable to standard drug employed in respective
studies. The presence of different substitutions in phenyl ring might
have facilitated stronger cap group interactions with the amino acid
side chains at the entrance of the HDAC active site. The optimum
length of linker might have made possible the binding of benza-
mide group with zinc ion at the bottom of the active site.
Inconclusion, we have designedand synthesizedtwo novel series
of benzamide analogs possessing linker derived from GABA or PABA
for development of newer benzamide based HDACi. Present studies
indicate that PABA provides better linker than that contributed by
GABA in the design of HDACi. These results represent a remarkable
step regarding the discovery of novel benzamide based HDACi.
10
11
12
13
14
15
16
17
18
19
20
21
22
MS-275
17.9 >100
31 78.0 >100
45.3 >100 >100
6.8 47.5 >100
9.5 >100
11.5 >100
>100
10.8
24.2 >100
45.0 >100
>100
>100
>100
15.4
9.4
18.0
13.5
8.2
88.0 >100
62.5 >100
83.0 >100
52.0 >100
42.5 >100
4.2
3.5
1.1
0.6
7.5
62.5 >100
25.0 >100
9.0 >100
6.5 >100
38.0 >100
25.5 >100
84.0 >100
38.6 >100
>100
>100
>100
>100
12.6 >100
7.9 66.0 >100
0.6 >100 >100
>100
6.5
40
Abbreviations: GI50 ¼ Growth Inhibition at 50%; TGI ¼ Tumor Growth Inhibition;
LC50 ¼ Lethal Concentration at 50%.
a
Values are means of observation recorded in two experiments.
noted that para-nitro analogs i.e., 8 and 19 showed greater potency
than ortho-nitro substituted analogs i.e., 6 and 17 or meta-nitro
substituted compound i.e., 7 and 18 in anticancer evaluation
studies. The position of the substitution had dramatic effects on the
HDAC activity. In a similar pattern analogs with ortho- and meta-
chloro substitution were found to be lesser active than para-chloro
analogs. This might be attributed to unfavorable steric effect of
meta- and ortho-substitution or to exceeding the optimum lipo-
philic value of the substituents.
6. Experimental protocols
6.1. Chemistry
All the chemicals and solvents employed in this study were
procured from E-Merck (Germany), Aldrich (Germany), Himedia
Table 2
Anticancer activities of GABA and PABA based benzamide analogs in Swiss albino mice.
Compound
Average wt of
Ascitic fluid (control)
Average wt. of
% inhibition of
Ascitic fluid (TWI)
Average no of
Average no. of
% inhibition of
ascitic cell (TCI)
Ascitic fluid (Test)a
cells/ml in controla ꢂ 105
cells/ml Testa ꢂ 105
1
2
3
4
5
6
7
8
2.8 (ꢀ0.3)
4.2 (ꢀ0.2)
2.8 (ꢀ0.7)
2.8 (ꢀ0.2)
4.4 (ꢀ0.5)
2.9 (ꢀ0.2)
3.2 (ꢀ0.3)
4.2 (ꢀ0.5)
3.6 (ꢀ0.1)
2.8 (ꢀ0.8)
3.4 (ꢀ0.4)
2.7 (ꢀ0.5)
3.8 (ꢀ0.6)
3.0 (ꢀ0.2)
3.8 (ꢀ0.8)
3.9 (ꢀ0.6)
3.1 (ꢀ0.2)
3.4 (ꢀ0.5)
4.2 (ꢀ0.3)
3.3 (ꢀ0.2)
3.0 (ꢀ0.4)
3.6 (ꢀ0.6)
3.9 (ꢀ0.2)
2.1 (ꢀ0.4)
1.1 (ꢀ0.2)
2.2 (ꢀ0.3)
1.9 (ꢀ0.5)
1.2 (ꢀ0.6)
1.7 (ꢀ0.8)
1.9 (ꢀ0.2)
0.9 (ꢀ0.4)
1.3 (ꢀ0.5)
1.6 ꢀ 0.64)
1.7 (ꢀ0.2)
2.2 (ꢀ0.3)
0.9 (ꢀ0.7)
1.7 (ꢀ0.5)
1.2 (ꢀ0.2)
0.8 (ꢀ0.7)
2.7 (ꢀ0.4)
1.1 (ꢀ0.6)
0.7 (ꢀ0.2)
1.1 (ꢀ0.)
25.0
73.8
21.4
32.1
72.7
41.3
40.6
78.5
63.8
42.8
50.0
18.5
76.3
43.3
68.4
79.4
12.9
67.6
83.3
66.6
43.3
58.3
100
310.6 (ꢀ5.8)
474.1 (ꢀ6.5)
353.7 (ꢀ5.8)
382.5 (ꢀ8.6)
511.1 (ꢀ3.4)
347.3 (ꢀ7.2)
454.7 (ꢀ6.5)
522.1 (ꢀ5.8)
470.4 (ꢀ3.4)
366.9 (ꢀ8.6)
442.6 (ꢀ6.5)
292.4 (ꢀ5.8)
516.5 (ꢀ3.4)
439.6 (ꢀ5.3)
465.3 (ꢀ7.2)
490.2 (ꢀ5.8)
322.6 (ꢀ5.3)
444.3 (ꢀ6.5)
508.8 (ꢀ8.6)
459.4 (ꢀ5.8)
361.7 (ꢀ7.2)
443.9 (ꢀ8.6)
463.2 (ꢀ8.4)
251.2 (ꢀ4.7)
150.8 (ꢀ5.8)
308.5 (ꢀ4.2)
296.2 (ꢀ3.3)
127.4 (ꢀ4.7)
310.8 (ꢀ2.5)
290.6 (ꢀ5.8)
122.6 (ꢀ4.2)
172.5 (ꢀ6.5)
297.2 (ꢀ2.5)
217.3 (ꢀ3.3)
203.1 (ꢀ2.5)
136.5 (ꢀ4.2)
301.5 (ꢀ4.7)
224.7 (ꢀ6.5)
130.8 (ꢀ2.5)
259.0 (ꢀ5.8)
264.7 (ꢀ6.5)
114.4 (ꢀ3.3)
172.9 (ꢀ2.5)
270.2 (ꢀ4.2)
194.1 (ꢀ6.5)
0.0 (ꢀ0.0)
19.1
68.7
12.7
22.5
75.0
10.5
36.0
76.5
63.3
18.9
50.9
30.5
73.5
31.4
51.7
73.3
19.7
40.4
77.5
62.3
25.2
56.2
100.0
9
10
11
12
13
14
15
16
17
18
19
20
21
22
MMCb
1.7(ꢀ0.5)
1.5 (ꢀ0.5)
0.0 (ꢀ0.0)
Abbreviations: GABA ¼ -aminobutyric acid; PABA ¼ para-aminobenzoic acid; TWI ¼ Tumor Weight Inhibtion; TCI ¼ Tumor Cell Inhibtion; MMC ¼ Mitomycin-C.
g
a
Observation shown are mean of 6 readings and data in parenthesis indicates standard error of mean.
Mitomycin-C at a dose level of 1 mg/kg body weight was employed as standard, which showed 100% inhibition.
b

394
H. Rajak et al. / European Journal of Medicinal Chemistry 53 (2012) 390e397
(India) and Spectrochem Pvt Ltd (India). Melting points were
determined by open capillary method and are uncorrected. The IR
spectra were recorded on a Perkin Elmer IR spectrophotometer
(KBr disc) (Perkin Elmer, Beaconsfield, UK), NMR spectra on
a Bruker DRX-300 NMR spectrometer (DMSO-d6, TMS) (Bruker
Bioscience, Billerica, MA, USA) and the electrospray mass spectra on
a Micromass Quattro II triple-quadrupole mass spectrometer
(Methanol) (Micromass, Manchester, UK). The title compounds
were prepared using the synthetic strategy described in Scheme 1.
The N-(4-substituted-phenyl)-2-chloro-acetamide II (iexi) and
4-{[(4-substituted-phenylcarbamoyl)-methyl]-amino}-butyric acid
III (iexi) were prepared according to the procedure reported [15].
1670.68 (C]O str of amide), 3462.59 (NeH str of amide), 1715.27
(CeO str of COOH), 2703.94 (OeH str of COOH); 1H NMR (300 MHz,
DMSO-d6, TMS,
d ppm): 6.67e7.84 (m, 9H, ArH), 8.14 (s, 1H, NH),
3.76 (d, 2H, COCH2NH), 4.30 (t, 1H, COCH2NH), 10.85 (s, 2H, COOH);
ESI-MS (Methanol) m/z 271.12 ([M þ H]þ).
Compounds: IV(ii) yield 56% m.p. 139e141 ꢁC; IV(iii) 62%,
111e113 ꢁC; IV(iv) 58%, 110e112 ꢁC; IV(v) 63%, 111e114 ꢁC; IV(vi)
60%, 127e129 ꢁC; IV(vii) 57%, 127e128 ꢁC IV(viii) 64%, 126e128 ꢁC;
IV(ix) 66%, 138e140 ꢁC; IV(x) 54%, 120e122 ꢁC; IV(xi) 59%,
145e146 ꢁC.
6.1.4. General procedure for synthesis of N-(2-amino-phenyl)-4-
{[(4-substituted-phenylcarbamoyl)-methyl]-amino}-butyramides
(01e11)
6.1.1. General procedure for synthesis of N-(4-substituted-phenyl)-
2-chloro-acetamide II (iexi)
Compound III (iexi) (0.01 M) was added to 100 ml THF followed
by proportion wise addition of CDI (0.01 M) at room temperature.
The reaction mixture was stirred for 2 h at 60e65 ꢁC. 1,2-
phenylenediamine (0.01 M) and trifluoroacetic acid (0.01 M) were
added to reaction mixture followed by stirring for 9e10 h. The
solvent was evaporated under vacuum and crude product was
stirred in a mixture of hexane and water (2:5 v/v) for 1 h and
filtered and dried. The crude product was recrystallized using
dichloromethane.
Substituted aniline (0.01 mol) I (iexi) and Dichloromethane
(20 ml) were mixed in a conical flask and Chloroacetylchloride
(1.02 ml; 0.01 M) in a separating funnel was added drop by drop to
the conical flask. The reaction mixture was stirred for 4e6 h
resulting in the formation of white precipitate. The product
obtained was filtered, and washed with a little cold water and
recrystallized from water or from methanol or ethanol either alone
or dilute with water.
Compound II (i): Yield 82%, m.p. 86e88 ꢁC. IR (cmꢃ1) (KBr)
3048.4 (Aromatic CeH str), 1600.4 & 1503.7 (Aromatic CeC str),
1670.24 (C]O str of amide), 3463.92 (NeH str of amide), 750.26
Compound 1: Yield 63%, m.p. 109e112 ꢁC. IR (cmꢃ1) (KBr)
3063.11 (aromatic CeH str), 1600.47 & 1501.81 (aromatic CeC str),
1674.68 (C]O str of amide), 3433.19 (NeH str of amide), 3365.38
(NeH str amino group), 2893.62 (aliphatic CeH str), 1442.64
(CeCl); 1H NMR (300 MHz, DMSO-d6, TMS,
d ppm): 6.95e7.68 (m,
5H, ArH), 8.16 (s, 1H, NH), 4.47 (s, 2H, CH2); ESI-MS (Methanol) m/z
(aliphatic CeH def); 1H NMR (300 MHz, DMSO-d6, TMS,
d ppm):
170.05 ([M þ H]þ).
6.65e7.61 (m, 9H, ArH), 9.94 (s, 1H, NH), 3.55 (d, 2H, COCH2NH),
2.58 (q, 2H, NHCH2CH2CH2CO), 1.75 (p, 2H, NHCH2CH2CH2CO), 2.31
(t, 2H, NHCH2CH2CH2CO), 4.97 (s, 2H, NH2); ESI-MS m/z 327.18
([M þ H]þ).
Compounds: II(ii) yield 77% m.p. 110e112 ꢁC; II(iii) 60%,
93e95 ꢁC; II(iv) 62%, 93e94 ꢁC; II(v) 60%, 94e96 ꢁC; II(vi) 59%,
121e122 ꢁC; II(vii) 63%, 122e124 ꢁC II(viii) 66%, 122e125 ꢁC; II(ix)
67%, 136e137 ꢁC; II(x) 64%, 116e118 ꢁC; II(xi) 62%, 131e133 ꢁC.
Compound 2: Yield 69%, m.p. 124e126 ꢁC; IR (cmꢃ1) (KBr)
3048.78 (aromatic CeH str), 1601.22 & 1500.73 (aromatic CeC str),
1677.24 (C]O str of amide), 3429.90 (NeH str of amide), 3357.48
(NeH str amino group), 2898.71 (aliphatic CeH str), 1436.83
(aliphatic CeH def), 824.59 (para-disubstituted benzene CeH def);
6.1.2. General procedure for synthesis of 4-{[(4-substituted-pheny
lcarbamoyl)-methyl]-amino}-butyric acid III (iexi)
4-Amino butanoic acid (GABA) was dissolved in ethyl alcohol
followed by addition of II (iexi) (0.01 M) and triethylamine
(0.01 mol). The reaction mixture was refluxed for 6e7 h. The
reaction mixture was poured on crushed ice. The resulting solid
was separated, dried, and recrystallized from ethanol.
1H NMR (300 MHz, DMSO-d6, TMS,
d ppm): 6.41e7.63 (m, 8H, ArH),
9.86 (s, 1H, NH), 3.51 (d, 2H, COCH2NH), 2.56 (q, 2H,
NHCH2CH2CH2CO), 1.77 (p, 2H, NHCH2CH2CH2CO), 2.28 (t, 2H,
NHCH2CH2CH2CO), 4.94 (s, 2H, NH2); ESI-MS m/z 405.10 ([M þ H]þ).
Compound 3: Yield 59%, m.p. 118e120 ꢁC; IR (cmꢃ1) (KBr)
3036.35 (aromatic CeH str), 1600.48 & 1502.85 (aromatic CeC str),
1679.04 (C]O str of amide), 3432.62 (NeH str of amide), 3353.93
(NeH str amino group), 2893.14 (aliphatic CeH str), 1439.13
(aliphatic CeH def), 750.48 (ortho-disubstituted CeC def), 789.25
Compound III (i): Yield 76%, m.p. 195e197 ꢁC. IR (cmꢃ1) (KBr)
3063.92 (Aromatic CeH str), 1601.85 & 1502.47 (Aromatic CeC str),
1674.77 (C]O str of amide), 3468.82 (NeH str of amide),, 2894.33
(CeH str alkane), 1710.25 (CeO str of COOH), 2728.43 (OeH str of
COOH); 1H NMR (300 MHz, DMSO-d6, TMS,
d ppm): 6.90e7.62 (m,
5H, ArH), 8.11 (s, 1H, NH), 3.53 (d, 2H, COCH2NH), 2.18 (q, 2H,
NHCH2CH2CH2COOH), 1.75 (p, 2H, NHCH2CH2CH2COOH), 2.33 (t,
2H, NHCH2CH2CH2COOH), 10.78 (s, 2H, COOH); ESI-MS (Methanol)
m/z 237.16 ([M þ H]þ).
(CeCl str); 1H NMR (300 MHz, DMSO-d6, TMS,
d ppm): 6.45e7.66
(m, 8H, ArH), 9.83 (s, 1H, NH), 3.54 (d, 2H, COCH2NH), 2.58 (q, 2H,
NHCH2CH2CH2CO), 1.75 (p, 2H, NHCH2CH2CH2CO), 2.29 (t, 2H,
NHCH2CH2CH2CO), 4.92 (s, 2H, NH2); ESI-MS m/z 361.14 ([M þ H]þ).
Compound 4: Yield 62%, m.p. 117e119 ꢁC; IR (cmꢃ1) (KBr)
3040.22 (aromatic CeH str), 1603.13 & 1503.46 (aromatic CeC str),
1672.97 (C]O str of amide), 3425.71 (NeH str of amide), 3350.54
(NeH str amino group), 2898.26 (aliphatic CeH str), 1444.18
(aliphatic CeH def), 700.94, 770.83 (meta-disubstituted CeH def),
Compounds: III(ii) yield 72% m.p. 122e124 ꢁC; III(iii) 55%,
96e98 ꢁC; III(iv) 53%, 95e98 ꢁC; III(v) 56%, 97e99 ꢁC; III(vi) 62%,
104e106 ꢁC; III(vii) 61%, 105e106 ꢁC III(viii) 66%, 104e107 ꢁC;
III(ix) 60%, 123e125 ꢁC; III(x) 59%, 108e110 ꢁC; III(xi) 57%,
116e118 ꢁC.
785.92 (CeCl str); 1H NMR (300 MHz, DMSO-d6, TMS,
d ppm):
6.1.3. General procedure for synthesis of 4-{[(4-substituted-phenyl
carbamoyl)-methyl]-amino}-benzoic acid IV (iexi)
6.47e7.66 (m, 8H, ArH), 9.91 (s, 1H, NH), 3.54 (d, 2H, COCH2NH),
2.59 (q, 2H, NHCH2CH2CH2CO), 1.77 (p, 2H, NHCH2CH2CH2CO), 2.32
(t, 2H, NHCH2CH2CH2CO), 4.88 (s, 2H, NH2); ESI-MS m/z 361.14
([M þ H]þ).
4-Amino benzoic acid (PABA) (0.01 mol) was dissolved in ethyl
alcohol followed by addition of III (iexi) (0.01 M) and triethylamine
(0.1 mol). The reaction mixture was refluxed for 7e8 h. The solution
was poured on crushed ice. The resulting solid was separated, dried,
and recrystallized from ethanol.
Compound 5: Yield 64%, m.p. 118e121 ꢁC; IR (cmꢃ1) (KBr)
3034.45 (aromatic CeH str), 1601.73 & 1502.83 (aromatic CeC str),
1674.42 (C]O str of amide), 3427.38 (NeH str of amide), 3356.02
(NeH str amino group), 2890.42 (aliphatic CeH str), 1446.62
(aliphatic CeH def), 828.93 (para-disubstituted benzene CeH def),
Compound IV (i): Yield 62%, m.p. 224e226 ꢁC. IR (cmꢃ1) (KBr)
3082.75 (Aromatic CeH str), 1602.60 & 1501.55 (Aromatic CeC str),

H. Rajak et al. / European Journal of Medicinal Chemistry 53 (2012) 390e397
395
780.84 (CeCl str); 1H NMR (300 MHz, DMSO-d6, TMS,
d
ppm):
6.1.5. General procedure for synthesis of N-(2-amino-phenyl)-4-
{[(4-substituted-phenylcarbamoyl)-methyl]-amino}-benzamides
(12e22)
6.46e7.62 (m, 8H, ArH), 9.86 (s, 1H, NH), 3.52 (d, 2H, COCH2NH),
2.55 (q, 2H, NHCH2CH2CH2CO), 1.74 (p, 2H, NHCH2CH2CH2CO), 2.33
(t, 2H, NHCH2CH2CH2CO), 4.94 (s, 2H, NH2); ESI-MS m/z 361.14
([M þ H]þ).
Compound IV (iexi) (0.01 M) was added to 100 ml THF followed
by proportion wise addition of CDI (0.01 M) at room temperature.
The reaction mixture was stirred for 2 h at 60e65 ꢁC. 1,2-
phenylenediamine (0.01 M) and trifluoroacetic acid (0.01 M) were
added to reaction mixture followed by stirring for 11e14 h. The
solvent was evaporated under vacuum and crude product was
stirred in a mixture of hexane and water (2:5 v/v) for 1 h and
filtered and dried. The crude product was recrystallized using
dichloromethane.
Compound 6: Yield 68%, m.p. 137e139 ꢁC; IR (cmꢃ1) (KBr)
3043.49 (aromatic CeH str), 1600.89 & 1501.54 (aromatic CeC str),
1672.82 (C]O str of amide), 3423.18 (NeH str of amide), 3353.42
(NeH str amino group), 2888.58 (aliphatic CeH str), 1440.39
(aliphatic CeH def), 1521.68 & 1365.82 (N]O str of AreNO2 group),
753.31 (ortho-disubstituted CeC def); 1H NMR (300 MHz, DMSO-
d6, TMS,
d ppm): 6.42e8.06 (m, 8H, ArH), 9.87 (s, 1H, NH), 3.55 (d,
2H, COCH2NH), 2.57 (q, 2H, NHCH2CH2CH2CO), 1.75 (p, 2H,
NHCH2CH2CH2CO), 2.36 (t, 2H, NHCH2CH2CH2CO), 4.89 (s, 2H,
NH2); ESI-MS m/z 372.16 ([M þ H]þ).
Compound 12: Yield 60%, m.p. 124e126 ꢁC. IR (cmꢃ1) (KBr)
3054.29 (aromatic CeH str), 1597.16 & 1502.51 (aromatic CeC str),
1670.83 (C]O str of amide), 3431.62 (NeH str of amide), 3361.42
(NeH str amino group), 2887.70 (aliphatic CeH str), 1438.81
Compound 7: Yield 66%, m.p. 138e139 ꢁC; IR (cmꢃ1) (KBr)
3056.42 (aromatic CeH str), 1601.48 & 1502.11 (aromatic CeC str),
1675.27 (C]O str of amide), 3425.74 (NeH str of amide), 3351.31
(NeH str amino group), 2885.03 (aliphatic CeH str), 1442.72
(aliphatic CeH def), 1524.50 & 1368.64 (N]O str of AreNO2 group),
703.64, 775.28 (meta-disubstituted CeH def); 1H NMR (300 MHz,
(aliphatic CeH def); 1H NMR (300 MHz, DMSO-d6, TMS,
d ppm):
6.52e7.75 (m, 13H, ArH), 9.84 (s, 1H, NH), 3.76 (d, 2H, COCH2NH),
4.18 (d, 2H, COCH2NH), 4.88 (s, 2H, NH2); ESI-MS m/z 361.18
([M þ H]þ).
Compound 13: Yield 58%, m.p. 138e140 ꢁC; IR (cmꢃ1) (KBr)
3062.92 (aromatic CeH str), 1603.41 & 1501.56 (aromatic CeC str),
1679.04 (C]O str of amide), 3435.72 (NeH str of amide), 3359.37
(NeH str amino group), 2885.52 (aliphatic CeH str), 1442.80
(aliphatic CeH def), 828.29 (para-disubstituted benzene CeH def);
DMSO-d6, TMS,
d ppm): 6.45e8.10 (m, 8H, ArH), 9.88 (s, 1H, NH),
3.57 (d, 2H, COCH2NH), 2.54 (q, 2H, NHCH2CH2CH2CO), 1.73 (p, 2H,
NHCH2CH2CH2CO), 2.30 (t, 2H, NHCH2CH2CH2CO), 4.92 (s, 2H,
NH2); ESI-MS m/z 372.16 ([M þ H]þ).
Compound 8: Yield 71%, m.p. 137e140 ꢁC; IR (cmꢃ1) (KBr)
3060.85 (aromatic CeH str), 1600.52 & 1501.28 (aromatic CeC str),
1672.94 (C]O str of amide), 3434.39 (NeH str of amide), 3355.85
(NeH str amino group), 2882.47 (aliphatic CeH str), 1438.61
(aliphatic CeH def), 1519.31 & 1360.36 (N]O str of AreNO2 group),
825.39 (para-disubstituted benzene CeH def); 1H NMR (300 MHz,
1H NMR (300 MHz, DMSO-d6, TMS,
d ppm): 6.48e7.58 (m, 12H,
ArH), 9.88 (s, 1H, NH), 3.79 (d, 2H, COCH2NH), 4.19 (d, 2H,
COCH2NH), 4.93 (s, 2H, NH2); ESI-MS m/z 439.10 ([M þ H]þ).
Compound 14: Yield 59%, m.p. 147e148 ꢁC; IR (cmꢃ1) (KBr)
3048.77 (aromatic CeH str), 1603.94 & 1503.41 (aromatic CeC str),
1671.64 (C]O str of amide), 3431.12 (NeH str of amide), 3358.40
(NeH str amino group), 2886.63 (aliphatic CeH str), 1433.25
(aliphatic CeH def), 753.35 (ortho-disubstituted CeC def), 793.69
DMSO-d6, TMS,
d ppm): 6.40e8.02 (m, 8H, ArH), 9.84 (s, 1H, NH),
3.55 (d, 2H, COCH2NH), 2.58 (q, 2H, NHCH2CH2CH2CO), 1.74 (p, 2H,
NHCH2CH2CH2CO), 2.28 (t, 2H, NHCH2CH2CH2CO), 4.91 (s, 2H,
NH2); ESI-MS m/z 372.16 ([M þ H]þ).
(CeCl str); 1H NMR (300 MHz, DMSO-d6, TMS,
d ppm): 6.40e7.62
(m, 12H, ArH), 9.91 (s, 1H, NH), 3.76 (d, 2H, COCH2NH), 4.16 (d,
2H, COCH2NH), 4.95 (s, 2H, NH2); ESI-MS m/z 395.12 ([M þ H]þ).
Compound 15: Yield 57%, m.p. 146e148 ꢁC; IR (cmꢃ1) (KBr)
3053.84 (aromatic CeH str), 1600.38 & 1499.10 (aromatic CeC str),
1670.84 (C]O str of amide), 3428.65 (NeH str of amide), 3348.91
(NeH str amino group), 2892.74 (aliphatic CeH str), 1443.96
(aliphatic CeH def), 705.16, 771.44 (meta-disubstituted CeH def),
Compound 9: Yield 65%, m.p. 145e147 ꢁC; IR (cmꢃ1) (KBr)
3067.39 (aromatic CeH str), 1601.74 & 1500.52 (aromatic CeC str),
1670.97 (C]O str of amide), 3438.11 (NeH str of amide), 3351.24
(NeH str amino group), 2880.63 (aliphatic CeH str), 1432.74
(aliphatic CeH def), 832.19 (para-disubstituted benzene CeH def),
1258.42 (C-O of OCH3 group); 1H NMR (300 MHz, DMSO-d6, TMS,
d
ppm): 6.42e7.53 (m, 8H, ArH), 9.92 (s, 1H, NH), 3.59 (d, 2H,
785.52 (CeCl str); 1H NMR (300 MHz, DMSO-d6, TMS,
d ppm):
COCH2NH), 2.57 (q, 2H, NHCH2CH2CH2CO), 1.72 (p, 2H,
NHCH2CH2CH2CO), 2.32 (t, 2H, NHCH2CH2CH2CO), 4.84 (s, 2H,
NH2), 3.74 (s, 3H, OCH3); ESI-MS m/z 357.23 ([M þ H]þ).
6.45e7.66 (m, 12H, ArH), 9.90 (s, 1H, NH), 3.81 (d, 2H, COCH2NH),
4.14 (d, 2H, COCH2NH), 4.90 (s, 2H, NH2); ESI-MS m/z 395.12
([M þ H]þ).
Compound 10: Yield 60%, m.p. 131e133 ꢁC; IR (cmꢃ1) (KBr)
3053.41 (aromatic CeH str), 1600.58 & 1503.46 (aromatic CeC str),
1673.82 (C]O str of amide), 3432.75 (NeH str of amide), 3355.57
(NeH str amino group), 2886.18 (aliphatic CeH str), 1436.92
(aliphatic CeH def), 835.72 (para-disubstituted benzene CeH def);
Compound 16: Yield 62%, m.p. 147e149 ꢁC; IR (cmꢃ1) (KBr)
3038.39 (aromatic CeH str), 1600.82 & 1501.81 (aromatic CeC str),
1679.21 (C]O str of amide), 3429.39 (NeH str of amide), 3361.72
(NeH str amino group), 2897.31 (aliphatic CeH str), 1451.89
(aliphatic CeH def), 829.11 (para-disubstituted benzene CeH def),
1H NMR (300 MHz, DMSO-d6, TMS,
d
ppm): 6.46e7.58 (m, 8H, ArH),
784.97 (CeCl str); 1H NMR (300 MHz, DMSO-d6, TMS,
d ppm):
9.86 (s, 1H, NH), 3.54 (d, 2H, COCH2NH), 2.58 (q, 2H,
NHCH2CH2CH2CO), 1.75 (p, 2H, NHCH2CH2CH2CO), 2.36 (t, 2H,
NHCH2CH2CH2CO), 4.94 (s, 2H, NH2), 2.42 (s, 3H, CH3); ESI-MS m/z
341.15 ([M þ H]þ).
6.49e7.29 (m, 12H, ArH), 9.85 (s, 1H, NH), 3.84 (d, 2H, COCH2NH),
4.17 (d, 2H, COCH2NH), 4.92 (s, 2H, NH2); ESI-MS m/z 395.12
([M þ H]þ).
Compound 17: Yield 67%, m.p. 156e158 ꢁC; IR (cmꢃ1) (KBr)
3038.35 (aromatic CeH str), 1602.75 & 1502.92 (aromatic CeC str),
1677.52 (C]O str of amide), 3417.96 (NeH str of amide), 3347.84
(NeH str amino group), 2895.42 (aliphatic CeH str), 1446.28
(aliphatic CeH def), 1524.29 & 1366.85 (N]O str of AreNO2 group),
757.96 (ortho-disubstituted CeC def); 1H NMR (300 MHz, DMSO-
Compound 11: Yield 58%, m.p. 115e116 ꢁC; IR (cmꢃ1) (KBr)
3048.73 (aromatic CeH str), 1601.52 & 1502.88 (aromatic CeC str),
1678.28 (C]O str of amide), 3430.52 (NeH str of amide), 3351.39
(NeH str amino group), 2893.38 (aliphatic CeH str), 1434.81
(aliphatic CeH def), 748.59 (ortho-disubstituted CeC def), 708.21,
776.04 (meta-disubstituted CeH def), 805.16 (CeCl str); 1H NMR
d6, TMS,
d ppm): 6.56e8.14 (m, 12H, ArH), 9.87 (s, 1H, NH), 3.83 (d,
(300 MHz, DMSO-d6, TMS,
d
ppm): 6.42e7.55 (m, 7H, ArH), 9.90 (s,
2H, COCH2NH), 4.19 (d, 2H, COCH2NH), 4.90 (s, 2H, NH2); ESI-MS m/
1H, NH), 3.56 (d, 2H, COCH2NH), 2.62 (q, 2H, NHCH2CH2CH2CO),
1.79 (p, 2H, NHCH2CH2CH2CO), 2.38 (t, 2H, NHCH2CH2CH2CO), 4.94
(s, 2H, NH2); ESI-MS m/z 395.10 ([M þ H]þ).
z 406.11 ([M þ H]þ).
Compound 18: Yield 65%, m.p. 155e158 ꢁC; IR (cmꢃ1) (KBr)
3062.52 (aromatic CeH str), 1600.31 & 1508.59 (aromatic CeC str),

396
H. Rajak et al. / European Journal of Medicinal Chemistry 53 (2012) 390e397
1678.92 (C]O str of amide), 3422.27 (NeH str of amide), 3356.62
(NeH str amino group), 2874.83 (aliphatic CeH str), 1438.78
(aliphatic CeH def), 1522.95 & 1362.89 (N]O str of AreNO2 group),
704.82, 779.11 (meta-disubstituted CeH def); 1H NMR (300 MHz,
In-vivo anticancer evaluation [17]: EAC cells were maintained
in-vivo in Swiss albino mice (18e20 g; 10 week old), by passage
after every 10 days. EAC cells 9 days old were employed for anti-
cancer evaluation of test compounds. Mitomycin-C at a dose level of
1 mg/kg body weight was used as standard, which showed 100%
inhibition. The compounds were dissolved/suspended in phos-
phate buffered saline with pH 7.2. EAC cells were collected from the
donor mouse and suspended in sterile isotonic saline. The EAC cells
were counted under the microscope and adjusted to 10 ꢂ 106 cells/
mL. On day zero, 0.1 mL of EAC cells per 10-g body weight of the
animal was injected (ip) followed by a day for incubation to permit
proliferation of the cells. Seven doses of compound (0.2 mmol/kg,
0.1 mL per 10-g body weight) were injected ip from the first day up
to the seventh day with 24-h intervals. Control group of animals
received only vehicle. On eighth day, food and water were with-
drawn 6 h before sacrificing the animals. All the animals were
sacrificed; peritoneal fluid was collected for cell count. All the fluid
in the peritoneal cavity was wiped off with absorbent cotton. The
weight of the animals was measured before sacrificing and after
removing the fluid from their peritoneal cavity. The difference in
weight is considered as tumor weight. The percentage inhibition of
cell count was calculated by equation as follows: Percentage inhi-
bition of Ascitic cell (TCI) ¼ [(1 ꢃ T/C) ꢂ 100]; Percentage inhibition
of Ascitic fluid (TWI) ¼ [(1 ꢃ T/C) ꢂ 100].
DMSO-d6, TMS,
d ppm): 6.40e8.42 (m, 12H, ArH), 9.95 (s, 1H, NH),
3.88 (d, 2H, COCH2NH), 4.17 (d, 2H, COCH2NH), 4.92 (s, 2H, NH2);
ESI-MS m/z 406.11 ([M þ H]þ).
Compound 19: Yield 68%, m.p. 156e159 ꢁC; IR (cmꢃ1) (KBr)
3056.93 (aromatic CeH str), 1601.32 & 1504.86 (aromatic CeC str),
1677.35 (C]O str of amide), 3439.27 (NeH str of amide), 3359.36
(NeH str amino group), 2884.25 (aliphatic CeH str), 1439.84
(aliphatic CeH def), 1523.81 & 1366.17 (N]O str of AreNO2 group),
820.63 (para-disubstituted benzene CeH def); 1H NMR (300 MHz,
DMSO-d6, TMS,
d ppm): 6.49e8.17 (m, 12H, ArH), 9.88 (s, 1H, NH),
3.84 (d, 2H, COCH2NH), 4.11 (d, 2H, COCH2NH), 4.86 (s, 2H, NH2);
ESI-MS m/z 406.11 ([M þ H]þ).
Compound 20: Yield 63%, m.p. 168e170 ꢁC; IR (cmꢃ1) (KBr)
3073.47 (aromatic CeH str), 1602.84 & 1501.47 (aromatic CeC str),
1671.34 (C]O str of amide), 3439.31 (NeH str of amide), 3355.07
(NeH str amino group), 2883.73 (aliphatic CeH str), 1434.11
(aliphatic CeH def), 835.61 (para-disubstituted benzene CeH def),
1262.83 (CeO of OCH3 group); 1H NMR (300 MHz, DMSO-d6, TMS,
d
ppm): 6.82e7.58 (m, 12H, ArH), 9.85 (s, 1H, NH), 3.86 (d, 2H,
COCH2NH), 4.14 (d, 2H, COCH2NH), 4.91 (s, 2H, NH2), 3.65 (s, 3H,
OCH3); ESI-MS m/z 391.22 ([M þ H]þ).
Compound 21: Yield 62%, m.p. 140e142 ꢁC; IR (cmꢃ1) (KBr)
3058.81 (aromatic CeH str), 1602.75 & 1505.92 (aromatic CeC str),
1678.52 (C]O str of amide), 3437.51 (NeH str of amide), 3348.33
(NeH str amino group), 2889.86 (aliphatic CeH str), 1438.52
(aliphatic CeH def), 837.82 (para-disubstituted benzene CeH def);
Acknowledgments
Three of the authors, Pramod Kumar, Poonam Parmar and
Bhupendra Singh Thakur, are thankful to AICTE New Delhi, India for
awarding Junior Research Fellowship and financial assistance. The
help rendered by SAIF, CDRI Lucknow for elemental and spectral
analysis is gratefully acknowledged.
1H NMR (300 MHz, DMSO-d6, TMS,
d ppm): 6.52e7.78 (m, 12H,
ArH), 9.80 (s, 1H, NH), 3.83 (d, 2H, COCH2NH), 4.19 (d, 2H,
COCH2NH), 4.95 (s, 2H, NH2), 2.42 (s, 3H, CH3); ESI-MS m/z 375.18
([M þ H]þ).
Compound 22: Yield 56%, m.p. 135e137 ꢁC; IR (cmꢃ1) (KBr)
3052.79 (aromatic CeH str), 1604.80 & 1505.41 (aromatic CeC str),
1657.94 (C]O str of amide), 3435.36 (NeH str of amide), 3355.98
(NeH str amino group), 2890.32 (aliphatic CeH str), 1437.94
(aliphatic CeH def), 750.07 (ortho-disubstituted CeC def), 710.43,
775.61 (meta-disubstituted CeH def), 807.58 (CeCl str); 1H NMR
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