Grinding and Microwave-assisted Synthesis of Heterocyclic Molecules in High Yields and Their Biological Evaluation

Article abstract of DOI:10.1002/jhet.2481

Cis-cyclohexane1,2-dicarboxylic acid (1a), phthalic acid (1b), and pyrazine 2,3- dicarboxylic acid (1c) on grinding with hydrazine hydrate (2a) gave 2-aminohexahydro-1H-isoindole-1,3(2H)-dione (3a), 2-amino-1H-isoindole-1,3(2H)-dione (3b), and 6-amino-5H-pyrrolo[3,4-b]pyrazine-5,7(6H)-dione (3c), respectively. Condensation of (3a, 3b, 3c) with aldehydes (4x, 4y, 4z) and 2-cyanopyridine, 4-cyanopyridine, 2-cyanopyrazine (5x, 5y, 5z) under microwave irradiation gave corresponding azomethine (6ax, 6ay, 6az, 6bx, 6by, 6bz, 6cx, 6cy, 6cz) and amidine (7ax, 7ay, 7az, 7bx, 7by, 7bz, 7cx, 7cy, 7cz) derivatives, respectively. Fully characterized azomethine (6ax, 6ay, 6az, 6bx, 6by, 6bz, 6cx, 6cy, 6cz) and amidine (7ax, 7ay, 7az, 7bx, 7by, 7bz, 7cx, 7cy, 7cz) derivatives were screened for anti-inflammatory and anticancer activity against five human cancer cell lines. Compound 7cx exhibited 35% anti-inflammatory activity at a dose of 50 mg/kg p.o. whereas standard drug ibuprofen showed 39% activity at a dose of 50 mg/kg p.o. Compounds 6bz, 7cx, 7cz (breast T47D), 6bz, 6cy (lung NCI H-522), 6bx, 7bz (colon HCT-15), 6bz (ovary PA-1) and 6bx, and 6cz (liver HepG-2) exhibited good (35–41% inhibition at 10 μM c) anticancer activity. IC₅₀values of 6bx, 6bz, 6cy, 6cz, 7bz, 7cx, and 7cz against various cancer cell lines and normal cell (COS-1) are also reported.

Full text of DOI:10.1002/jhet.2481

Month 2015 Grinding and Microwave-assisted Synthesis of Heterocyclic Molecules in
High Yields and Their Biological Evaluation
a
a
b
b
a
*
Sandeep Kumar, Anuj Kumar, Nikhil Kumar, Partha Roy, and Sham M. Sondhi
aDepartment of Chemistry, Indian Institute of Technology-Roorkee, Roorkee 247667 Uttarakhand, India
bDepartment of Biotechnology, Indian Institute of Technology-Roorkee, Roorkee 247667 Uttarakhand, India
*
E-mail: sondifcy@iitr.ernet.in
Received March 16, 2015
DOI 10.1002/jhet.2481
Published online 00 Month 2015 in Wiley Online Library (wileyonlinelibrary.com).
Cis-cyclohexane1,2-dicarboxylic acid (1a), phthalic acid (1b), and pyrazine 2,3- dicarboxylic acid (1c) on
grinding with hydrazine hydrate (2a) gave 2-aminohexahydro-1H-isoindole-1,3(2H)-dione (3a), 2-amino-
1H-isoindole-1,3(2H)-dione (3b), and 6-amino-5H-pyrrolo[3,4-b]pyrazine-5,7(6H)-dione (3c), respectively.
Condensation of (3a-c) with aldehydes (4x-z) and 2-cyanopyridine, 4-cyanopyridine, 2-cyanopyrazine
(5x-z) under microwave irradiation gave corresponding azomethine (6ax-cz) and amidine (7ax-cz) deriv-
atives, respectively. Fully characterized azomethine (6ax-cz) and amidine (7ax-cz) derivatives were
screened for anti-inflammatory and anticancer activity against five human cancer cell lines. Compound
7cx exhibited 35% anti-inflammatory activity at a dose of 50 mg/kg p.o. whereas standard drug ibuprofen
showed 39% activity at a dose of 50 mg/kg p.o. Compounds 6bz, 7cx, 7cz (breast T47D), 6bz, 6cy (lung
NCI H-522), 6bx, 7bz (colon HCT-15), 6bz (ovary PA-1) and 6bx, and 6cz (liver HepG-2) exhibited
good (35–41% inhibition at 10 μM c) anticancer activity. IC50 values of 6bx, 6bz, 6cy, 6cz, 7bz, 7cx,
and 7cz against various cancer cell lines and normal cell (COS-1) are also reported.
J. Heterocyclic Chem., 00, 00 (2015).
INTRODUCTION
pyrrolopyrazine have been synthesized in high yields using
grinding at room temperature and microwave irradiation
technique. All these compounds were screened for anti-
inflammatory and anticancer activities; the results of these
studies we wish to report in this paper.
Heterocyclic compounds which can be easily synthe-
sized continue to be an active area of research for medici-
nal chemists. Inflammatory diseases and cancer are some
of the major health problems faced by human beings. A
number of anti-inflammatory and a few anticancer drugs
are available in the market, but these drugs also have
serious side effects [1] associated with them. In light of
the preceding discussion, there is an urgent need to
search new biologically active molecules which can be
developed as drugs. Isoindole derivatives exhibiting anti-
fungal [2], antimalarial [3], anticonvulsant [4], anti-
asthamatic [5], antiplasmodial [6], and antimicrobial [7]
and pyrrolopyrazine derivatives exhibiting anti-arrhythmic
activity [8] are reported in literature. Isoindole and
pyrrolopyrazine derivatives possessing anti-inflammatory
[9–13], anticancer [14–19], and antibacterial [20–22] activ-
ities are well-documented in literature. These compounds
are also reported to act as HIV-1 integrase inhibitors
[23–25]. The aforementioned literature reports suggest that
isoindole and pyrrolopyrazine derivatives possess a broad
range of biological activities. In continuation of efforts in
search of biological active molecules [26–31], it was
considered worthwhile to synthesize new isoindole and
pyrrolopyrazine derivatives and screen them for biological
activities. Several new derivatives of isoindole and
RESULTS AND DISCUSSION
Chemistry. Reported methods for the synthesis of
2-aminohexahydro-1H-isoindole-1,3(2H)-dione (3a) [32,33]
and 2-Amino-1H-isoindole-1,3(2H)-dione (3b) [33] involve
two-step synthesis and refluxing in a solvent. We have
synthesized 2-aminohexahydro-1H-isoindole-1,3(2H)-dione
(3a; Scheme 1), 2-Amino-1H-isoindole-1,3(2H)-dione (3b;
Scheme 1), and 6-amino-5H-pyrrolo[3,4-b]pyrazine-5,7
(6H)-dione (3c; Scheme 1) by simply grinding a mixture
of corresponding dicarboxylic acids (1a-c; Scheme 1) and
hydrazine hydrate (1:1.2 mol ratio) for 30min at room
temperature. Crude products so obtained were purified by
crystallization from methanol to give pure products 3a-c
(Scheme 1) in good yields. Spectral data and elemental
analysis of 3a-c reported in this paper fully support the
structures assigned to them.
Condensation of 2-aminohexahydro-1H-isoindole-1,3
(2H)-dione (3a) with 4-hydroxy-3-methoxybenzaldehyde
(4x) was carried out by dissolving both of them in 1:1 M
© 2015 HeteroCorporation

S. Kumar, A. Kumar, N. Kumar, P. Roy, and S. M. Sondhi
Vol 000
Scheme 1. Synthesis of azomethine and amidine derivatives of isoindole and pyrrolopyrazine 3a-c, 6ax-cz, and 7ax-cz.
ratio in methanol and then adding silica gel to this reaction
solution and removing the solvent under vacuum to give
both of the reactants adsorbed on silica gel. This silica
gel was subjected to focused microwave irradiation at
120°C for 6 minutes. TLC showed that the reaction is com-
plete. Crude product was obtained by shaking irradiated
silica gel with methanol for 10 min and removing the sol-
vent under vacuum. Crude product so obtained was puri-
fied by crystallization from methanol to give 81% yield
of pure product 6ax. Similarly, condensation of 3b-c with
4-hydroxy-3-methoxybenzaldehyde (4x) and 3a-c with
2,5-dimethoxybenzaldehyde (4y), and 4-(dimethylamino)
benzaldehyde (4z) gave corresponding azomethine deriva-
tives 6ay-cz in good yield. Spectral data and elemental
analysis of 6ax-cz reported in this paper are in full agree-
ment with the structures assigned to them.
Condensation of 2-cyanopyridine (5x), 4-cyanopyridine
(5y), and 2-cyanopyrazine (5z) with 3a-c to give amidine
derivatives 7ax-cz (Scheme 1) was carried out in a similar
way as mentioned earlier. Silica gel adsorbed with both the
reactants in 1:1M ratio was subjected to focused micro-
wave irradiation at 135°C for 6 min. TLC showed that the
reaction is complete. Microwave irradiated silica gel was
shaken with methanol for 10 min and then filtered.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2015
Grinding and Microwave-assisted Synthesis
Removal of the solvent from the filtrate gave crude
amidine derivative, which was purified by crystallization
from methanol. Spectral data and elemental analysis of
7ax-cz reported in this paper fully support the structures
assigned to 7ax-cz.
Table 1. Compounds 6bz, 7cx, 7cz (breast T47D), 6bz,
6cy (lung NCI H-522), 6bx, 7bz (colon HCT-15), 6bz
(ovary PA-1) and 6bx, and 6cz (liver HepG-2) exhibited
good anticancer activity against various cancer cell lines
mentioned earlier. Compounds 6bx, 6bz, 6cy, 6cz, 7bz,
7cx, and 7cz which exhibited good anticancer activity were
further studied, and their IC50 values for various cancer cell
lines and normal cell (COS-1) were determined and re-
ported in Table 2.
Biological results.
Over-expression of COX-2 and
prostaglandin cascade is involved in carcinogenesis [34–36].
Nonsteroidal anti-inflammatory drugs (selective or non
selective COX-2 inhibitors) exhibit strong potential [37–39]
for chemoprevention of cancer. In view of the earlier
information from literature, it was considered worthwhile to
evaluate newly synthesized compounds (6ax-cz and 7ax-cz;
Scheme 1) for anti-inflammatory and anticancer activities.
Compounds 6ax-cz and 7ax-cz (Scheme 1) were screened
for anti-inflammatory activity [40] using carrageenan-
induced paw edema assay and results are summarized in
Table 1. Compound 7cx exhibited good anti-inflammatory
activity, that is, 35% at 50mg/kg p.o. as compared with
ibuprofen which showed 39% activity at 50mg/kg p.o.
Compounds 6ax-cz and 7ax-cz were screened in vitro
for anticancer activity [41] against five human cancer cell
lines, that is, breast (T47D), lung (NCl H-522), colon
(HCT-15), ovary (PA-1), and liver (HepG-2) at a concen-
tration of 1 × 10ꢀ5 M, and results are summarized in
Structure-activity relationship.
Lipophilicity (clogP)
values for compounds 6ax-cz and 7ax-cz were estimated by
using ChemDraw ultra and were found to be 1.779, 2.605,
2.762, 1.403, 2.229, 2.386, ꢀ1.051, ꢀ0.225, ꢀ0.068, 0.916,
0.916, ꢀ0.041, 0.540, 0.540, ꢀ0.417, ꢀ1.914, ꢀ1.914, and
ꢀ2.871, respectively. Compound 7cx showed good (35%)
anti-inflammatory activity, whereas other compounds showed
moderate (25–32%) to low (<25%) anti-inflammatory
activity; it may be due to the fact that compound 7cx can
meet lipophilicity, stereochemical, and electronic
requirements of the target in a better way as compared
with other molecules.
Compounds derived from 3a, that is, 6ax-az and 7ax-az
did not show good anticancer activity, whereas compounds
derived from 3b (6bx, 6bz, and 7bz) and 3c (6cy, 6cz, 7cx,
Table 1
Anti-inflammatory and in vitro anticancer activity of azomethine and amidine derivatives of isoindole and pyrrolopyrazine 6ax-cz and 7ax-cz.
Anti-
*Anticancer activity (% growth inhibition) at a concentration of 1 × 10ꢀ5 M
inflammatory
activity (%) at
50 mg/kg p.o
Compound No.
Breast T47D
Lung NCl H-522
Colon HCT-15
Ovary PA-1
Liver HepG-2
6ax
6ay
6az
6bx
6by
6bz
6cx
6cy
6cz
7ax
7ay
7az
7bx
7by
7bz
7cx
09
06
32
22
24
18
26
14
29
17
20
17
17
23
20
35
12
14
39
—
—
—
22
14
15
34
24
35
13
25
11
13
14
15
17
13
27
37
25
41
—
18
16
26
16
12
14
25
05
40
10
37
28
28
25
20
16
11
22
33
10
23
—
24
15
11
19
19
10
38
04
25
05
16
18
23
05
01
29
24
36
05
12
07
—
20
13
12
18
21
09
31
19
36
11
29
16
11
21
25
19
18
14
30
19
26
—
21
30
15
21
23
17
35
14
24
19
25
39
19
17
20
24
32
30
13
27
17
—
18
26
18
7cy
7cz
Ibuprofen
a5-FU
bCYC-PHO
cCYC-HEXI
*Compounds tested in triplicate, data expressed as mean value of three independent experiments.
a5-FU 5-Fluorouracil.
bCYC-PHO Cyclophosphamide.
cCYC-HEXI Cycloheximide.
Bold values represent compounds showing good anti-inflammatory and good anticancer activity.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

S. Kumar, A. Kumar, N. Kumar, P. Roy, and S. M. Sondhi
Vol 000
Table 2
IC50 valuesa,* of in vitro antitumor activity of active compounds.
IC50 (μM)
Compound
No.
Breast T47D
Lung NCI H-522
Colon HCT-15
Ovary PA-1
Liver HepG-2
Normal cell COS-1
6bx
6bz
6cy
6cz
7bz
7cx
7cz
13.55 2.54
15.32 2.78
21.13 4.31
38.92 3.91
20.21 2.08
13.22 1.74
11.23 2.98
51.8 2.34
70.1 2.32
65.13 7.31
22.43 1.84
11.33 3.11
16.11 2.65
19.13 3.14
28.34 2.56
15.16 1.91
25.08 3.41
56.76 3.4
67.9 3.09
60.1 5.34
12.78 3.13
23.32 4.03
34.73 1.93
26.32 2.34
14.07 2.54
67.63 3.75
65.47 2.44
45.01 1.45
74.32 4.98
54.13 4.65
18.02 2.71
15.67 2.35
21.07 4.27
31.26 1.74
37.66 3.31
16.88 4.13
23.68 2.66
39.5 4.32
64.12 5.43
40.6 2.09
14.06 3.39
24.64 2.74
23.78 1.46
12.33 2.07
15.46 4.04
38.39 2.78
28.77 3.33
29.87 1.82
55.3 3.59
123.76 3.98
117.97 2.56
109.77 1.99
132.44 4.11
128.46 2.18
116.51 2.37
110.25 3.25
110 8.98
b5-FU
bCYC-PHO
cCYC-HEXI
125.43 9.24
128.31 7.89
57.12 4.65
a50% growth inhibition as determined by MTT assay (24 h drug exposure).
*Compounds tested in triplicate, data expressed as mean value SD of three independent experiments.
b5-FU 5-Fluorouracil.
bCYC-PHO Cyclophosphamide.
cCYC-HEXI Cycloheximide.
Bold values represent compounds showing good anticancer activity.
and 7cz) exhibited good anticancer activity. From this
observation, it may be concluded that aromatic (3b) or
heteroaromatic (3c) rings in place of cyclohexyl (3a) ring
made the structures of these molecules suitable for interaction
with the cancerous cell. Compounds 6bx, 6bz, 6cy, 6cz,7bz,
7cx, and 7cz exhibited good anticancer activity; it may be due
to the fact that these compounds meet lipophilicity, stereo-
chemical, and electronic requirements of the target in a better
way as compared with other molecules.
elementor. Thin-layer chromatography (TLC) was
performed on silica gel G for TLC (Merck), and spots
were visualized by iodine vapors or by irradiation with
ultraviolet light (254nm). Compounds 6ax-cz and 7ax-cz
were purified by crystallization from methanol.
General procedure for synthesis of isoindole and
pyrrolopyrazine derivatives (3a-c).
aminohexahydro-1H-isoindole-1,3(2H)-dione
Synthesis of 2-
(3a).
Cis-
cyclohexane-1,2-dicarboxylic acid (0.344 g; 2.0mmol) and
hydrazine hydrate (0.12 mL; 2.4 mmol) were grinded
together in a small mortar with a pestle for 30 min. TLC
of the reaction mixture on silica gel using ethyl acetate:
methanol (7:3) as mobile phase exhibited that the
reaction is complete. The crude product so obtained was
purified by crystallization from methanol to give pure
product 2-aminohexahydro-1H-isoindole-1,3(2H)-dione
(3a; Scheme 1). Yield 272 mg (82%). mp: 63–65°C (lit
[32,33] mp 60–62°C). IR (KBr) νmax: 3288 (NH2),
CONCLUSIONS
A number of azomethine and amidine derivatives of
isoindole and pyrrolopyrazine (3a-c, 6ax-cz, 7ax-cz) have
been synthesized by using simple grinding and microwave
irradiation methods. Compounds 6ax-cz and 7ax-cz were
screened for anti-inflammatory and anticancer activities.
Compound 7cx exhibited good anti-inflammatory activity,
whereas 6bx, 6bz, 6cy, 6cz, 7bz, 7cx, and 7cz exhibited
good anticancer activity.
1
1687 (C = O) cmꢀ1. H NMR (500 MHz, DMSO-d6) δ:
1.36–1.44 (m, 4H, 2 × CH2), 1.62–1.67 (m, 2H, CH2),
1.76–1.82 (m, 2H, CH2), 2.66–2.75 (m, 2H, CH+CH),
6.00 (bs, 2H, NH2, exch). 13C NMR (125 MHz, DMSO-d6)
δ: 25.0, 26.7, 38.9, 178.1. GC–MS: m/z 169 (M+• +1, 1%),
168 (M+•, 15%), 152 (M+•-NH2; 100%). Anal. Calcd for
C8H12N2O2: C, 57.14; H, 7.14; N, 16.67. Found: C, 57.29;
H, 7.23; N, 16.78.
EXPERIMENTAL
General. Microwave reactor Anton Paar (monowave
300) was used for microwave irradiation. Melting points
(mp) were determined on a Jain Scientific Glass Works
(JSGW) apparatus and are uncorrected. IR spectra were
Similarly, compounds 3b and 3c were synthesized.
1
recorded using a Perkin Elmer 1600 FT spectrometer. H
2-Amino-1H-isoindole-1,3(2H)-dione (3b).
Yield: 77%.
and 13C NMR spectra were recorded on a Bruker WH-
500 spectrometer at a ca 5–15% (w/v) solution in
deuterated solvent (TMS as internal standard). GC–MS
was recorded on Perkin Elmer Clarus 500 gas
chromatograph, where a built-in MS detector was used.
Elemental analysis was carried out on a Vario EL III
mp: 202–205°C (lit [33] mp 200–205°C). IR (KBr) νmax
:
3377 (NH2), 1669 (C = O), 1476 (Ar) cmꢀ1 1H NMR
.
(500 MHz, DMSO-d6) δ: 6.09 (s, 2H, NH2, exch),
7.58–7.61 (m, 2H, Ar), 7.65–7.68 (m, 2H, Ar). 13C
NMR (125MHz, DMSO-d6) δ: 127.2, 132.7, 160.1,
167.6. GC–MS: m/z 163 (M+• + 1, 1%), 162 (M+•, 38%),
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2015
Grinding and Microwave-assisted Synthesis
146 (M+•-NH2; 100%), 76 (
, 14%). Anal. Calcd for
(
(
, 100%), 150 (
, 15%), 122
C8H6N2O2: C, 59.26; H, 3.70; N, 17.28. Found: C, 59.41;
H, 3.81; N, 17.42.
6-Amino-5H-pyrrolo[3,4-b]pyrazine-5,7(6H)-dione (3c). Yield:
77%. mp: 218°C. IR (KBr) νmax: 3315 (NH2), 1679 (C =O),
, 15%). Anal. Calcd for C16H18N2O4: C,
1
1536, 1507 (Ar) cmꢀ1. H NMR (500MHz, DMSO-d6) δ:
63.57; H, 5.96; N, 9.27. Found: C, 63.66; H, 6.05; N, 9.39.
Compounds 6ay-cz were similarly synthesized (Scheme 1).
Physical constants, spectral data, and elemental analysis of
6ay-cz are summarized subsequently.
6.04 (s, 2H, NH2, exch), 8.86 (s, 2H, Ar). 13C NMR
(125 MHz, DMSO-d6) δ: 143.4, 145.9, 168.0. GC–MS:
m/z 165 (M+• + 1, 1%), 164 (M+•, 46%), 148 (M+•-NH2,
100%). Anal. Calcd for C6H4N4O2: C, 43.90; H, 2.44;
N, 34.14. Found: C, 43.90; H, 2.44; N, 34.14.
2-[(2,5-Dimethoxybenzylidene)amino]hexahydro-1H-isoindole-
1,3(2H)-dione (6ay).
ν
Yield: 77%. mp: 174°C. IR (KBr)
max: 1688 (C =O), 1635 (C = N), 1594, 1497 (Ar) cmꢀ1. 1H
General procedure for synthesis of azomethine derivatives
NMR (500MHz, DMSO-d6) δ: 1.35–1.44 (m, 4H, 2×CH2),
1.62–1.68 (m, 2H, CH2), 1.76–1.83 (m, 2H, CH2), 2.64–
2.75 (m, 2H, CH+CH), 3.74 (s, 3H, OCH3), 3.87 (s, 3H,
OCH3), 7.16–7.19 (m, 2H, Ar), 7.24–7.27 (m, 1H, Ar), 8.13
(s, 1H, Ar). 13C NMR (125 MHz, DMSO-d6) δ: 25.1, 26.7,
39.0, 55.0, 55.9, 114.0, 115.1, 117.1, 117.7, 143.3, 152.1,
153.8, 178.1. GC–MS: m/z 317 (M+•+1, 1%), 316 (M+•,
(6ax-cz).
Synthesis of 2-[(4-hydroxy-3-methoxybenzylidene)
amino]hexahydro-1H-isoindole-1,3(2H)-dione (6ax). 2-
Aminohexahydro-1H-isoindole-1,3(2H)-dione (0.168g, 1mmol)
(3a; Scheme 1) and 4-hydroxy-3-methoxybenzaldehyde
(0.152 g; 1 mmol) were dissolved in methanol (5 mL).
To this solution, silica gel (5 g, 60–120 mesh) was
added, and then the solvent was removed under vacuum
to give dry silica gel adsorbed with the aforementioned
reactants. This silica gel was subjected to focused
microwave irradiation at 120°C for 6 min. A small
portion of the aforementioned silica gel was shaken
with methanol (5 mL) and filtered. Filtrate was reduced
to one milliliter (1 mL) and was checked for progress of
the reaction. TLC of this reaction solution over silica gel
using ethyl acetate:methanol (4:1) as mobile phase
showed absence of starting materials and, hence,
completion of reaction. Total amount of microwave-
irradiated silica gel was shaken with methanol (20 mL)
for 10 min and then filtered. Silica gel on filter paper was
further washed with methanol (2 × 10 mL). Solvent from
the combined filtrate was removed under vacuum to give
crude product 6ax. This crude product was purified by
crystallization from methanol to give pure 2-[(4-hydroxy-3-
methoxybenzylidene)amino]hexahydro-1H-isoindole-1,3(2H)-
dione (6ax) Yield 0.244g (81%).
26%), 180 (
164 (
, 4%), 179 (
, 20%),
, 13%), 153 (
, 3%), 152
(
, 100%), 136 (
, 25%), 82 (
,
30%). Anal. Calcd for C17H20N2O4: C, 64.55; H, 6.32; N, 8.86.
Found: C, 64.63; H, 6.41; N, 8.97.
2-{[4-(Dimethylamino)benzylidene]amino}hexahydro-1H-
isoindole-1,3(2H)-dione (6az) [42]. Yield: 83%. mp: 146°C.
IR (KBr) νmax: 1679 (C = O), 1657 (C = N), 1533, 1476,
1453 (Ar) cmꢀ1. H NMR (500MHz, DMSO-d6) δ: 1.37–
1
1.44 (m, 4H, 2 ×CH2), 1.62–1.68 (m, 2H, CH2), 1.76–1.84
(m, 2H, CH2), 2.64–2.73 (m, 2H, CH+CH), 2.97 (s, 6H,
2×CH3), 6.68 (d, 2H, J=9 Hz, Ar), 7.35 (d, 2H, J=9Hz,
Ar), 8.12 (s, 1H, Ar). 13C NMR (125 MHz, DMSO-d6) δ:
25.1, 26.6, 39.0, 40.9, 114.7, 123.5, 130.2, 143.3, 151.5,
178.0. GC–MS: m/z 300 (M+• +1, 1%), 299 (M+•, 44%), 180
Yield (81%). mp: 161°C. IR (KBr) νmax: 1675 (C = O),
1657 (C = N), 1536, 1506 (Ar) cmꢀ1. 1H NMR (500MHz,
DMSO-d6) δ: 1.37–1.45 (m, 4H, 2 ×CH2), 1.62–1.67 (m,
2H, CH2), 1.76–1.83 (m, 2H, CH2), 2.64–2.73 (m, 2H,
CH + CH), 3.86 (s, 3H, CH3), 6.95 (d, 1H, J = 9Hz, Ar),
7.38–7.43 (m, 2H, Ar), 8.12 (s, 1H, Ar), 10.25 (bs, 1H,
OH, exch). 13C NMR (125 MHz, DMSO-d6) δ: 25.1,
26.9, 39.0, 55.9, 102.1, 108.0, 109.1, 131.4, 143.4,
150.0, 151.9, 178.1. GC–MS: m/z 303 (M+• + 1, 2%),
(
, 8%), 179 (
, 25%), 153
(
, 9%) 152 (
, 100%), 147 (
,
11%), 119 (
, 13%), 82 (
, 17%). Anal.
302 (M+•, 33%), 180
(
,
,
5%), 179
6%), 152
Calcd for C17H21N3O2: C, 68.22; H, 7.02; N, 14.04. Found:
C, 68.29; H, 7.11; N, 14.09.
2-[(4-Hydroxy-3-methoxybenzylidene)amino]-1H-isoindole-1,3
(2H)-dione (6bx). Yield: 88%. mp: 234°C. IR (KBr) νmax
(
, 32%), 153 (
:
3377 (O-H), 1683 (C= O), 1664 (C =N), 1612, 1477 (Ar)
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

S. Kumar, A. Kumar, N. Kumar, P. Roy, and S. M. Sondhi
.
1H NMR (500MHz, DMSO-d6) δ: 3.80 (s, 3H,
Vol 000
cmꢀ1
OCH3), 6.95 (d, 1H, J=8Hz, Ar), 7.38–7.43 (m, 2H, Ar),
7.58–7.61 (m, 2H, Ar), 7.65–7.69 (m, 2H, Ar), 8.12 (s,
1H, Ar), 10.25 (bs, 1H, OH, exch). 13C NMR (125MHz,
DMSO-d6) δ: 55.9, 102.8, 108.1, 109.2, 127.6, 132.0,
132.8, 143.0, 153.0, 154.3, 156.7, 168.7. GC–MS: m/z 297
(
(
(
, 28%), 174 (
, 2%), 173
3%), 146
, 15%).
,
22%), 147
(
,
(M+• + 1, 1%), 296 (M+•, 22%), 174 (
, 2%),
, 9%), 147
, 100%), 119 (
, 6%),76 (
Anal. Calcd for C17H15N3O2: C, 69.;2, H, 5.12;;N, 14.33.
Found: C, 69.73; H, 5.19; N, 14.47.
173 (
, 10%), 150 (
6-[(4-Hydroxy-3-methoxybenzylidene)amino]-5H-pyrrolo[3,4-
b]pyrazine-5,7(6H)-dione (6cx). Yield: 85%. mp: 256°C. IR
(KBr) νmax: 1687 (C=O), 1672 (C=N), 1608, 1593,
(
(
,
13%), 146
, 19%), 76 (
(
,
100%), 122
1
1498 (Ar) cmꢀ1. H NMR (500MHz, DMSO-d6) δ: 3.84
(s, 3H, OCH3), 6.95 (d, 1H, J=8Hz, Ar), 7.38–7.43 (m,
2H, Ar), 8.12 (s, 1H, Ar), 8.86 (s, 2H, Ar), 10.25 (bs, 1H,
OH, exch). 13C NMR (125MHz, DMSO-d6) δ: 55.9,
102.1, 108.3, 109.0, 131.0, 142.4, 143.7, 145.9, 153.0,
155.9, 168.0. GC–MS: m/z 299 (M+• +1, 1%), 298 (M+•,
, 12%). Anal. Calcd for
C16H12N2O4: C, 64.86; H, 4.05; N. 9.46. Found: C. 64.95;
H. 4.12; N, 9.57.
2-[(2,5-Dimethoxybenzylidene)amino]-1H-isoindole-1,3(2H)-
dione (6by). Yield: 82%. mp: 267°C. IR (KBr) νmax: 1682
(C = O), 1665 (C =N), 1610, 1510, 1476 (Ar) cmꢀ1. H
1
53%), 176 (
22%), 150 (
148 (
, 10%), 175 (
, 2%), 149 (
,
, 10%),
NMR (500 MHz, DMSO-d6) δ: 3.74 (s, 3H, OCH3), 3.87
(s, 3H, OCH3), 7.16–7.19 (m, 2H, Ar), 7.24–7.27 (m,
1H, Ar), 7.58–7.61 (m, 2H, Ar), 7.65–7.68 (m, 2H, Ar),
8.13 (s, 1H, Ar). 13C NMR (125 MHz, DMSO-d6) δ:
55.0, 55.8, 114.2, 115.3, 117.1, 117.9, 127.6, 132.0,
132.8, 143.1, 152.5, 153.2, 168.3. GC–MS: m/z 311
, 100%), 122 (
, 13%). Anal.
(M+• + 1, 1%), 310 (M+•, 35%), 174 (
, 3%),
Calcd for C14H10N4O4: C, 56.37; H, 3.35; N, 18.79.
Found: C, 56.49; H, 3.44; N, 18.73.
6-[(2,5-Dimethoxybenzylidene)amino]-5H-pyrrolo[3,4-b]
pyrazine-5,7(6H)-dione (6cy). Yield: 81%. mp: 273°C. IR
173 (
, 23%), 164 (
, 11%),
(KBr) νmax: 1698 (C=O), 1668 (C=N), 1536, 1506 (Ar)
cmꢀ1 1H NMR (500MHz, DMSO-d6) δ: 3.74 (s, 3H,
.
OCH3), 3.87 (s, 3H, OCH3), 7.26–7.29 (m, 2H, Ar), 7.34–
7.37 (m, 1H, Ar), 8.12 (s, 1H, Ar), 8.86 (s, 2H, Ar). 13C
NMR (125 MHz, DMSO-d6) δ: 55.1, 55.9, 114.3, 115.1,
117.0, 117.8, 143.1, 143.8, 145.7, 152.5, 153.3, 167.8.
GC–MS: m/z 313 (M+• +1, 1%), 312 (M+•, 32%), 280
147 (
(
, 11%), 146 (
, 10%), 76 (
, 100%), 136
, 18%). Anal. Calcd
for C17H14N2O4: C, 65.81; H, 4.52; N, 9.03. Found: C,
65.96; H, 4.58; N, 9.15.
2-{[4-(Dimethylamino)benzylidene]amino}-1H-isoindole-1,3
(2H)-dione (6bz) [43]. Yield: 83%. mp: 224°C. IR (KBr)
(
, 13%) 176 (
, 32%), 164 (
, 5%),
ν
max: 1684 (C = O), 1669 (C = N), 1611, 1576, 1543, 1508,
1478 (Ar) cmꢀ1. 1H NMR (500 MHz, DMSO-d6) δ: 2.97
(s, 6H, 2 × CH3), 6.68 (d, 2H, J = 9 Hz, Ar), 7.35–7.37
(d, 2H, J=9Hz, Ar), 7.58–7.61 (m, 2H, Ar), 7.65–7.68 (m,
2H, Ar), 8.12 (s, 1H, Ar). 13C NMR (125 MHz, DMSO-d6)
δ: 40.9, 114.2, 124.1, 127.3, 130.0, 132.0, 132.9,
143.0, 151.5, 167.8. GC–MS: m/z 294 (M+• +1, 1%),
175 (
, 18%),
149 (
(
, 3%), 148 (
, 100%), 136
, 11%). Anal. Calcd for C15H12N4O4: C,
293 (M+•, 53%), 249 (
, 6%), 248
57.69; H, 3.85; N, 17.95. Found: C, 57.57; H, 3.83; N,
17.89.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2015
Grinding and Microwave-assisted Synthesis
6-{[4-(Dimethylamino)benzylidene]amino}-5H-pyrrolo[3,4-b]
pyrazine-5,7(6H)-dione (6cz).
DMSO-d6) δ: 25.0, 26.9, 39.0, 120.0, 125.1, 136.4,
149.4, 154.0, 155.9, 178.2. GC–MS: m/z 273 (M+• +1,
Yield: 86%. mp: 243°C. IR
(KBr) νmax: 1678 (C =O), 1660 (C= N), 1537, 1506 (Ar)
cmꢀ1 1H NMR (500MHz, DMSO-d6) δ: 2.96 (s, 6H,
.
1%), 272 (M+•, 19%), 256 (
, 72%),
2×CH3), 6.68 (d, 2H, J=9Hz, Ar), 7.36 (d, 2H, J=9Hz,
Ar), 8.12 (s, 1H, Ar), 8.87 (s, 2H, Ar). 13C NMR
(125MHz, DMSO-d6) δ: 40.8, 114.1, 123.3, 130.0, 143.0,
143.9, 145.0, 151.4, 167.9. GC–MS: m/z 296 (M+• +1, 1%),
179 (
, 12%), 152 (
, 22%), 82 ( , 13%), 78 (
, 100%), 104
, 30%), 77
(
(
295 (M+•, 53%), 251 (
, 12%), 250
, 9%),
, 23%). Anal. Calcd for C14H16N4O2: C, 61.76;
H, 5.88; N, 20.59. Found: C, 61.81; H, 5.96; N, 20.72.
Compounds 7ay-cz were similarly synthesized
(Scheme 1). Physical constants, spectral data, and elemen-
(
8%), 176 (
tal analysis of 7ay-cz are summarized subsequently.
N′-(1,3-dioxooctahydro-2H-isoindol-2-yl)pyridine-4-
175 (
(
, 30%), 149 (
, 10%), 148
carboximidamide (7ay).
Yield: 73%. mp: 195°C. IR
(KBr) νmax: 3288 (NH2), 1697 (C = O), 1663 (C = N),
1599, 1545, 1502 (Ar) cmꢀ1 1H NMR (500 MHz,
, 100%), 147(
, 3%). 119 (
,
.
DMSO-d6) δ: 1.37–1.44 (m, 4H, 2 × CH2), 1.62–1.67
(m, 2H, CH2), 1.76–1.83 (m, 2H, CH2), 2.65–2.76 (m,
2H, CH + CH), 6.75 (bs, 2H, NH2, exch), 7.97 (d, 2H,
J = 9 Hz, Ar), 8.55 (d, 2H, J = 9 Hz, Ar,). 13C NMR
(125 MHz, DMSO-d6) δ: 25.0, 26.9, 39.0, 121.4, 133.6,
149.1, 162.0, 178.1. GC–MS: m/z 273 (M+• + 1, 1%),
6%). Anal. Calcd for C15H13N5O2: C, 61.01; H, 4.41; N,
23.73. Found: C, 61.12; H, 4.49; N, 23.81.
General procedure for synthesis of amidine derivatives
(7ax-cz). Synthesis of N′-(1,3-dioxooctahydro-2H-isoindol-2-
yl)pyridine-2-carboximidamide (7ax). 2-Aminohexahydro-1H-
isoindole-1,3(2H)-dione (3a; Scheme 1) (0.168 g, 1mmol) and
2-cyanopyridine (5a; Scheme 1) (0.104g; 1 mmol) were
dissolved in methanol (5mL). To this solution, silica gel
(5 g, 60–120mesh) was added, and the then solvent was
removed under reduced pressure to give reactant adsorbed
silica gel. This silica gel was subjected to focused
microwave irradiation at 135°C for 6 min. A small portion
of microwave-irradiated silica gel was shaken with
methanol (5mL) for 10 min and then filtered. Solvent of
the reaction mixture was reduced to 1 mL. TLC of this
reaction mixture on silica gel using ethyl acetate:methanol
(3:2) as mobile phase showed absence of starting
materials, and hence, reaction is complete. Total amount
of microwave irradiated silica gel was shaken with
methanol (20 mL) for 10 min and then filtered. Silica gel
on the filter paper was washed with methanol
(2 × 10 mL). Solvent from the total filtrate was removed
under reduced pressure to give crude product 7ax
(Scheme 1). Crude product so obtained was purified by
crystallization from methanol to give pure product N′-(1,3-
dioxooctahydro-2H-isoindol-2-yl)pyridine-2-
272 (M+•, 8%), 256 (
, 31%), 179
(
,
8%), 152
(
,
100%), 104
(
, 14%), 82 (
, 19%), 78 ( , 23%),
77 (
, 9%). Anal. Calcd for C14H16N4O2: C,
61.76; H. 5.88; N. 20.59. Found: C, 61.84; H, 5.97; N,
20.63.
N′-(1,3-dioxooctahydro-2H-isoindol-2-yl)pyrazine-2-
carboximidamide (7az). Yield: 72%. mp: 109°C. IR (KBr)
ν
max: 3264 (NH2), 1684 (C=O), 1644 (C=N), 1513, 1484
(Ar) cmꢀ1. H NMR (500MHz, DMSO-d6) δ: 1.36–1.44
(m, 4H, 2×CH2), 1.62–1.68 (m, 2H, CH2), 1.76–1.83 (m,
2H, CH2), 2.66–2.71 (m, 2H, CH+CH), 6.68 (bs, 2H,
NH2, exch), 8.86 (t, 1H, J=2Hz, Ar,), 8.97 (d, 1H,
J=2Hz, Ar,), 9.22 (d, 1H, J=2Hz, Ar). 13C NMR
(125MHz, DMSO-d6) δ: 25.1, 26.8, 39.0, 141.2, 143.3,
144.4, 145.5, 155.5, 178.1. GC–MS: m/z 274 (M+• +1,
1
carboximidamide (7ax) Yield 0.198g (73%).
1%), 273 (M+•, 13%), 257 (
, 61%),
Yield (73). mp: 104°C. IR (KBr) νmax: 3403 (NH2),
1684 (C = O), 1647 (C = N), 1565, 1476 (Ar) cmꢀ1. H
1
NMR (500 MHz, DMSO-d6) δ: 1.37–1.44 (m, 4H,
2 × CH2), 1.62–1.67 (m, 2H, CH2), 1.76–1.83 (m, 2H,
CH2), 2.66–2.73 (m, 2H, CH +CH), 6.76 (bs, 2H, NH2,
exch), 7.72–7.75 (m, 1H, Ar), 8.01–8.07 (m, 2H, Ar,),
8.74 (dd, 1H, J = 1 and 4.5 Hz, Ar). 13C NMR (125MHz,
179 (
(
, 18%), 152 (
, 12%), 82 ( , 21%), 79 (
, 100%), 105
, 22%), 78
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

S. Kumar, A. Kumar, N. Kumar, P. Roy, and S. M. Sondhi
Vol 000
, 34%).
(
, 14%), 79 (
, 23%), 78 (
(
, 16%). Anal. Calcd for C13H15N5O2: C, 57.14; H,
Anal. Calcd for C13H9N5O2: C, 58.43; H, 3.37; N,
26.21. Found: C, 58.59; H, 3.43; N, 26.35.
N′-(5,7-dioxo-5,7-dihydro-6H-pyrrolo[3,4-b]pyrazin-6-yl)
pyridine-2-carboximidamide (7cx). Yield: 77%. mp: 219°C.
5.49; N, 25.64. Found: C, 57.25; H, 5.58; N, 25.69.
N′-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)pyridine-2-
carboximidamide (7bx). Yield: 78%. mp: 227°C. IR (KBr)
ν
max: 3424 (NH2), 1685 (C= O), 1645 (C= N), 1522, 1492
IR (KBr) νmax: 3267 (NH2), 1684 (C=O), 1647 (C=N),
(Ar) cmꢀ1. 1H NMR (500MHz, DMSO-d6) δ: 6.67 (bs, 2H,
NH2, exch), 7.58–7.61 (m, 2H, Ar), 7.65–7.69 (m, 2H, Ar),
7.72–7.75 (m, 1H, Ar), 8.01–8.07 (m, 2H, Ar), 8.74 (q, 1H,
J=1 and 4.5 Hz, Ar). 13C NMR (125MHz, DMSO-d6) δ:
120.9, 125.7, 127.2, 131.7, 132.0, 136.1, 149.0, 154.2,
155.9, 167.3. GC–MS: m/z 267 (M+• +1, 1%), 266 (M+•,
1
1519, 1449 (Ar) cmꢀ1. H NMR (500MHz, DMSO-d6) δ:
6.66 (bs, 2H, NH2, exch), 7.72–7.75 (m, 1H, Ar), 8.01–
8.07 (m, 2H, Ar,), 8.74 (q, 1H, J=1 and 4.5Hz, Ar,), 8.86
(s, 2H, Ar). 13C NMR (125MHz, DMSO-d6) δ: 120.7,
125.2, 136.7, 143.3, 145.7, 149.4, 154.2, 155.8, 167.6.
GC–MS: m/z 269 (M+• +1, 1%), 268 (M+•, 10%), 252
12%), 250 (
6%), 146 (
, 69%), 173 (
,
(
(
, 55%), 175 (
, 9%), 148
, 100%), 104 (
, 10%), 78
, 100%), 104 (
, 22%), 78 (
,
(
,
16%), 77
(
, 25%). Anal. Calcd for
16%), 77 (
, 12%). Anal. Calcd for C12H8N6O2: C,
C14H10N4O2: C, 63.15; H. 3.76; N. 21.05. Found: C. 63.28;
H. 3.86; N. 21.12.
N′-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)pyridine-4-
carboximidamide (7by). Yield: 80%. mp: 263°C. IR (KBr)
53.73; H, 2.98; N, 31.34. Found: C, 53.64; H, 3.05; N,
31.41.
N′-(5,7-dioxo-5,7-dihydro-6H-pyrrolo[3,4-b]pyrazin-6-yl)
pyridine-4-carboximidamide (7cy). Yield: 80%. mp: 249°C.
ν
max: 3283 (NH2), 1686 (C =O), 1656 (C=N), 1519, 1456
IR (KBr) νmax: 3241 (NH2), 1678 (C= O), 1644 (C= N),
(Ar) cmꢀ1. H NMR (500MHz, DMSO-d6) δ: 6.68 (bs,
2H, NH2, exch), 7.58–7.61 (m, 2H, Ar), 7.65–7.69 (m, 2H,
Ar), 7.97 (d, 2H, J=9Hz, Ar,), 8.55 (d, 2H, J=9Hz, Ar,).
13C NMR (125MHz, DMSO-d6) δ: 121.1, 127.6, 131.9,
132.9, 136.7, 149.5, 162.8, 168.0. GC–MS: m/z 267
1
.
1582, 1512, 1474 (Ar) cmꢀ1 1H NMR (500MHz,
DMSO-d6) δ: 6.69 (bs, 2H, NH2, exch), 7.97–7.99 (m, 2H,
J=9Hz, Ar,), 8.55 (d, 2H, J=9Hz, Ar,), 8.88 (s, 2H, Ar).
13C NMR (125MHz, DMSO-d6) δ: 121.2, 136.7, 143.8,
145.6, 149.2, 162.3, 167.9. GC–MS: m/z 269 (M+• +1,
(M+• +1, 1%), 266 (M+•, 12%), 250 (
,
1%), 268 (M+•, 15%), 252 (
, 67%), 175
60%), 173 (
104 (
, 17%), 146 (
, 10%), 78 (
, 100%),
(
(
,
8%), 148
(
,
100%), 104
, 12%).
, 32%), 78 (
, 30%), 77 (
, 15%), 77 (
,
Anal. Calcd for C12H8N6O2: C, 53.73; H, 2.98; N, 31.34.
24%). Anal. Calcd for C14H10N4O2: C, 63.15; H, 3.76; N,
Found: C, 53.81; H, 3.04; N, 31.39.
N′-(5,7-dioxo-5,7-dihydro-6H-pyrrolo[3,4-b]pyrazin-6-yl)
pyrazine-2-carboximidamide (7cz). Yield: 76%. mp: 224°C.
21.05. Found: C, 63.27; H, 3.83; N, 21.16.
N′-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)pyrazine-2-
carboximidamide (7bz). Yield: 76%. mp: 226°C. IR (KBr)
ν
max: 3368 (NH2), 1688 (C =O), 1643 (C =N), 1584, 1554,
IR (KBr) νmax: 3361 (NH2), 1688 (C= O), 1649 (C= N),
1497 (Ar) cmꢀ1. H NMR (500MHz, DMSO-d6) δ: 6.68
(bs, 2H, NH2, exch), 7.58–7.61 (m, 2H, Ar), 7.65–7.69 (m,
2H, Ar), 8.86 (t, 1H, J=2 Hz, Ar,), 8.97 (d, 1H, J=2.5Hz,
Ar), 9.22 (d, 1H, J=1.5Hz, Ar,). 13C NMR (125MHz,
DMSO-d6) δ: 127.2, 132.0, 132.8, 141.2, 143.130,
144.1, 145.2, 155.9, 168.0. GC–MS: m/z 268 (M+• + 1,
1
1
1572, 1512 (Ar) cmꢀ1. H NMR (500 MHz, DMSO-d6) δ:
6.68 (bs, 2H, NH2, exch), 8.86 (t, 3H, J=2 Hz, Ar), 8.97
(d, 1H, J=2Hz, Ar), 9.23 (d, 1H, J=2.5Hz, Ar,). 13C
NMR (125MHz, DMSO-d6) δ: 141.2, 143.0, 143.8, 144.5,
145.7, 146.0, 155.0, 168.0. GC–MS: m/z 270 (M+• +1,
1%), 269 (M+•, 9%), 253 (
, 73%), 175
1%), 267 (M+•, 10%), 251 (
, 69%),
(
,
17%), 148 (
, 100%), 105
173 (
, 15%), 146 (
, 100%), 105
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2015
Grinding and Microwave-assisted Synthesis
The percentage inhibition was calculated as
(
, 37%), 79 (
, 29%), 78 (
, 24%). Anal.
Mean OD of vehicle treated cellsðnegative controlÞ–Mean OD of treated cellsꢂ100
Mean OD of vehicle treated cellsðnegative controlÞ
Calcd for C11H7N7O2: C, 49.07; H, 2.60; N, 36.43%. Found:
C, 49.16; H, 2.66; N, 36.39%.
The IC50 values were calculated using graph pad prism,
version 5.02 software (Graph Pad Software Inc., CA, USA).
Pharmacology.
Anti-inflammatory activity. Paw edema
inhibition test was used on albino rats of Charles Foster
by adopting the method of Winter et al. [40]. Groups of
five animals of both sexes (body weight 120–160g),
excluding pregnant females, were given a dose of test
compound. Thirty minutes later, 0.20mL of 1% freshly
prepared carrageenan suspension in 0.9% NaCl solution
was injected subcutaneously into the planter aponeurosis
of the hind paw, and the volume was measured by a
water plethysmometer apparatus and then measured again
1–3h later. The mean increase of paw volume at each
interval was compared with that of the control group (five
rats treated with carrageenan, but not with test
compound) at the same intervals and percent inhibition
value calculated by the formula given subsequently.
Acknowledgments. We are thankful to the technical staff of the
Chemistry Department, I. I. T. Roorkee, for spectroscopic studies and
elemental analysis. Thanks are also due to Head I.I.C. for providing
the NMR facility. Mr. Sandeep Kumar and Mr. Anuj Kumar are
thankful to MHRD New Delhi for the financial assistance.
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