Synthesis and pharmacological evaluation of pyrazolopyrimidopyrimidine derivatives: Anti-inflammatory agents with gastroprotective effect in rats

Article abstract of DOI:10.1007/s00044-013-0742-x

We report the synthesis of new anti-inflammatory 1,7- dihydropyrazolo[3′,4′:4,5]pyrimido[1,6-a]pyrimidine 5 from aminocyanopyrazole. All compounds were characterized by physical, chemical and spectral studies. Preliminary pharmacological evaluation of the resulting products showed that compounds 5a, b, f (50-100 mg/kg, i.p) are active anti-inflammatory agents in carrageenan-induced rat paw oedema assay, and their effects are comparable to that of acetylsalicylic-lysine (300 mg/kg, i.p.), used as a reference drug. The nature of substituent (Y, R₃) had a pronounced effect on the anti-inflammatory activity. Studies of structure-activity relationships have led to selection of compound ethyl-3,5-dimethyl-7-imino-N ¹-phenyl-1,7-dihydropyrazolo[3′, 4′:4,5]pyrimido[1,6-a]pyrimidine-6-carboxylate, 5f which exhibited the most potent anti-inflammatory activity. In addition, the compounds 5a, b, f showed a significant gastroprotective effect against HCl/EtOH-induced gastric ulcer.

Full text of DOI:10.1007/s00044-013-0742-x

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2014) 23:1591–1598
DOI 10.1007/s00044-013-0742-x
ORIGINAL RESEARCH
Synthesis and pharmacological evaluation
of pyrazolopyrimidopyrimidine derivatives: anti-inflammatory
agents with gastroprotective effect in rats
•
•
•
Amine Karoui Fatma Allouche Monia Deghrigue Asma Agrebi
•
•
Abderrahman Bouraoui Fakher Chabchoub
Received: 17 April 2013 / Accepted: 17 August 2013 / Published online: 4 September 2013
Ó The Author(s) 2013. This article is published with open access at Springerlink.com
Abstract We report the synthesis of new anti-inflamma-
tory 1,7-dihydropyrazolo[30,40:4,5]pyrimido[1,6-a]pyrimi-
dine 5 from aminocyanopyrazole. All compounds were
characterized by physical, chemical and spectral studies.
Preliminary pharmacological evaluation of the resulting
products showed that compounds 5a, b, f (50–100 mg/kg,
i.p) are active anti-inflammatory agents in carrageenan-
induced rat paw oedema assay, and their effects are compa-
rable to that of acetylsalicylic–lysine (300 mg/kg, i.p.), used
as a reference drug. The nature of substituent (Y, R3) had a
pronounced effect on the anti-inflammatory activity. Studies
of structure–activity relationships have led to selection of
compound ethyl-3,5-dimethyl-7-imino-N1-phenyl-1,7-di-
hydropyrazolo[30,40:4,5]pyrimido[1,6-a]pyrimidine-6-car-
boxylate, 5f which exhibited the most potent anti-
inflammatory activity. In addition, the compounds 5a, b,
f showed a significant gastroprotective effect against HCl/
EtOH-induced gastric ulcer.
Introduction
Nonsteroidal anti-inflammatory drugs (NSAIDs) are most
widely used to treat variety of acute and chronic inflam-
matory diseases. Such drugs are being increasingly used for
the treatment of postoperative pain (Moote, 1992) with or
without supplemental opioid agents. The pharmacological
action of these agents was assigned to inhibit two enzymes,
known as cyclooxygenase-1 (COX-1) and cyclooxygenase-
2 (COX-2) (Vane et al., 1998). The constitutive isoform
COX-1 is present in most tissues and is involved in the
synthesis of prostaglandins vital to normal cell function. In
contrast, the inducible isoform COX-2 appears to be pro-
duced primarily in response to growth factors or inflam-
matory mediators, such as cytokines (Vane and Botting,
1996). Many of the currently available NSAIDs, including
indomethacin and piroxicam, are more potent inhibitors of
COX-1 than that of COX-2 (Vane and Botting, 1995). This
preferential inhibition of COX-1 may be responsible for
many of the adverse effects associated with NSAIDs. It has
been postulated that NSAIDs which preferentially inhibit
COX-2, such as meloxicam (Lipscomb et al., 1998),
celecoxib (Simon et al., 1998) and several experimental
drugs including NS 398, L-745,337 and DFP, should pro-
duce the same or better anti inflammatory effects with less
gastrointestinal, haematological and renal toxicities than
classical NSAIDs (Winter et al., 1962). Pyrazolopyrimi-
dines are a class of sedative and anxiolytic drugs such as
Zaleplon known by its hypnotic effect (Weitzel et al.,
2000). However, pyrazolopyrimidine derivatives become a
new chemical resource for searching of novel bioactive
compounds in drug development.
Keywords Aminocyanopyrazole ꢀ Anti-inflammatory ꢀ
Gastroprotective ꢀ Pyrazolo[3,4-d]pyrimidine ꢀ
Dihydropyrazolo[30,40:4,5]pyrimido[1,6-a]pyrimidine
A. Karoui ꢀ F. Allouche (&) ꢀ A. Agrebi ꢀ F. Chabchoub
´
Laboratoire de Chimie Appliquee, Heterocycles, Corps Gras
et Polymeres, Faculte des Sciences de Sfax, Universite de Sfax,
´ ´
`
´
´
3018 Sfax, Tunisia
e-mail: fatmaallouch@yahoo.fr
M. Deghrigue ꢀ A. Bouraoui
On this basis, we directed our attention to the synthe-
sis of novel 1,7-dihydropyrazolo[30,40:4,5]pyrimido[1,6-a]
pyrimidines 5a–i related to aminocyanopyrazole with the
´
Unite de Recherche des Substances Actives Marines (URSAM),
Laboratoire de Pharmacologie, Faculte de Pharmacie, Universite
´
´
de Monastir, 5000 Monastir, Tunisia
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Med Chem Res (2014) 23:1591–1598
a) 4-Amino-N1-phenyl-1H-pyrazolo[3,4-d]pyrimidine 4a
Yield 83 %; mp 228 °C; IR (cm-1); mNH2 3100, 3283;
aim of improving their anti-inflammatory activity and
reducing their ulcerogenic properties as it appeared to be
plausible that variation of the active compound structures
could exert a pronounced influence on activity, as the case
with 5b, f.
1
mC=N 1480, 1500, 1590 cm-1; RMN H (d ppm,
DMSO): 4.69 (2H, s, NH2), 7.36 (1H, t, J = 7.3 Hz,
ArH4), 7.48 (2H, t, J = 7.3 Hz, ArH3 and ArH5), 7.52
(2H, d, J = 7.3 Hz, ArH2 and ArH6), 7.60 (1H, s, H3),
7.72 (1H, s, H6), 13C RMN (d ppm, DMSO): 114.14
(C-3a), 124.27 (C-20 and C-60), 129.00 (C-40), 129.58
(C-30 and C-50), 130.04 (C-3), 136.94 (C-10), 141.36
(C-7a), 149.83 (C-6), 156.84 (C-4); HRMS Calcd. for
C11H9N5: 211.0858, found: 211.0859.
Materials and methods
Chemistry
b) 4-Amino-3-methyl-N1-phenyl-1H-pyrazolo[3,4-d]
Phenyl hydrazine, malononitrile, triethylorthoester and
ammoniac were purchased from Sigma Chemical (Berlin,
Germany). Analytical grade solvents (ethanol, HCl, ethyl
acetate, chloroform) were obtained from Merck.
pyrimidine 4b Yield 68 %; mp 192 °C; IR (cm-1);
1
m
NH2 3083, 3317; mC=N 1626, 1647, 1665; RMN H
(d ppm, DMSO): 2.76 (3H, s, CH3), 5.97 (2H, s,
NH2), 7.33 (1H, t, J = 7.1 Hz, ArH4), 7.57 (2H, t,
J = 7.1 Hz, ArH3 and ArH5), 8.16 (2H, d,
J = 7.1 Hz, ArH2 and ArH6), 8.46 (1H, s, H3);
RMN13C (d ppm, DMSO):14.89 (CH3),101.23 (C-
3a), 121.49 (C-20 and C-60), 126.37 (C-40), 129.19
(C-30 and C-50), 138.81 (C-3), 141.83 (C-10),
154.41 (C-7a), 156.48 (C-4), 158.40 (C-6); HRMS
Calcd. for C12H11N5: 225.1014, found: 225.1018.
c) 4-Amino-6-methyl-N1-phenyl-1H-pyrazolo[3,4-d]
Melting points (mp) were determined on a Buchi cap-
illary apparatus and were uncorrected. Nuclear magnetic
resonance (NMR) spectra were recorded on a Bruker 300
spectrometer (1H at 300 MHz and 13C at 75 MHz) with
deuterio-dimethylsulphoxide (d-DMSO) as solvent and
tetramethylsilane as internal standard reference. Infra-red
(IR) spectra were recorded on a Bio-rad FTS-6000 spec-
trometer. Solvents used in reactions were dried and dis-
tilled before use. The purity of all synthesized compounds
was controlled by thin layer chromatography (TLC; Merck
silica gel plates 60F-254). High resolution masses were
recorded on a spectrometer JEOL JMS-Gcmate II is com-
posed of a GC/MS system from compounds dissolved in
dichloromethane.
pyrimidine 4c Yield 70 %; mp 160 °C; IR (cm-1);
1
m
NH2 3090, 3320; mC=N 1597, 1638, 1663; RMN H
(d ppm, DMSO): 2.65 (3H, s, CH3), 4.28 (2H, s,
NH2), 7.28 (1H, t, J = 7.3 Hz, ArH4), 7.56 (2H, t,
J = 7.3 Hz, ArH3 and ArH5), 8.19 (2H, d,
J = 7.3 Hz, ArH2 and ArH6), 8.29 (1H, s, H6);
RMN13C (d ppm, DMSO): 14.44 (CH3), 100.24 (C-
3a), Carom 120.24 (C-20 and C-60), 124.67 (C-40),
129.16 (C-30 and C-50), 138.8 (C-3), 142.79 (C-10);
C3 154.14 (C-7a), 156.51 (C-4),158.58 (C-6);
HRMS Calcd. for C12H11N5 : 225.1014, found:
225.1016.
Synthesis and spectral data of compounds 2–5
5-Amino-4-cyano-N1-phenyl pyrazoles (2) 5-Amino-4-
cyano-1-N1-phenyl pyrazoles prepared via a standard
addition of hydrazine derivatives to ketene ethoxymethyl-
ene compounds following the reported procedure. Recrys-
tallization from ethanol afforded pure 2 in good yields.
7-Imino-N1-phenyl-1,7-dihydropyrazolo[30,40:4,5]pyrimido
[1,6-a]pyrimidine 5a–e A mixture of compound 4 (1.0
mmol), ketene ethoxymethylene compounds 1 or ethyl-2-
cyano-3-ethoxyalkyl-2-enoate (1.0 mmol) and a catalytic
amount of acetic acid was refluxed for 2 h in 10 ml etha-
nol. The formed precipitate was filtered, washed by diethyl
ether, dried and recrystallized from ethanol to give com-
pound 5 in good yield.
4-Cyano-N1-phenyl pyrazolo-5-imidates (3) The required
5-amino-4-cyano-N1-phenyl pyrazole (1.0 mmol) was
treated with triethylorthoester 6.0 mmol) and a catalytic
amount of acetic acid and the mixture was refluxed for
24 h. After cooling, the reaction mixture was evaporated.
The product was filtered, washed with diethyl ether then
purified by recrystallisation (ethanol) (Gupta et al., 2008;
Allouche et al., 2013).
a) 6-Cyano-7-imino-3-methyl-N1-phenyl-1,7-dihydropy-
razolo[30,40:4,5]pyrimido[1,6-a]pyrimidine 5a Yield
68 %; mp 290 °C; IR (cm-1); mNH 3356; mC:N 2212;
4-Amino-N1-phenyl pyrazolo[3,4-d]pyrimidine (4)
A
solution of imidate 3 (1.0 mmol) in dry ethanol (5 ml) was
treated with ammoniac (2.0 mmol) and a catalytic amount
of acetic acid. The reaction mixture was refluxed for 6 h,
and the formed solid was collected by filtration, dried and
recrystallized from ethanol to give compound 4.
1
mC=N 1534, 1554, 1587; RMN H (d ppm, DMSO):
2.51 (3H, s, CH3); 7.38 (1H, t, J = 7.3 Hz, ArH4);
7.53 (2H, t, J = 7.3 Hz, ArH3 and ArH5); 7.71 (2H,
d, J = 7.3 Hz, ArH2 and ArH6); 8.02 (1H, s, H5);
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Med Chem Res (2014) 23:1591–1598
1593
8.38 (1H, s, H9); 8.66 (1H, s, NH); RMN13C (d ppm,
DMSO): 14.64 (CH3); 91.81 (C-6); 105.88 (C-3a);
116.24 (CN); Carom 120.46 (C-20 and C-60), 124.17
(C-40), 129.27 (C-30 and C-50), 137.89 (C-10),143.42
(C-10a), 149.71 (C-3),159.61 (C-5),161.88 (C-9),
162.15 (C-4a); 163.43 (C-7); HRMS Calcd. for
C16H11N7 :301.1076, found: 301.1051.
ArH4); 7.52 (2H, t, J = 7.3 Hz, ArH3 and ArH5); 8.04
(2H, d, J = 7.3 Hz, ArH2 and ArH6); 8.18 (1H, s, H5);
8.52 (1H, s, H9); 11.16 (1H, s, NH); RMN13C (d ppm,
DMSO): 9.01 (CH3): 29.31 (CH2); 92.54 (C-6); 106.31
(C-3a); 114.07 (CN); Carom 121.28 (C-20 and C-60),
124.73 (C-40), 126.56 (C-30 and C-50), 141.13 (C-
10),145.82 (C-10a),152.63 (C-3),155.28 (C-9),161.23
(C-4a), 162.07 (C-7); 165.49 (C-5); HRMS Calcd. for
C17H13N7: 315.1232, found: 315.1352.
b) 6-Cyano-7-imino-3,5-dimethyl-N1-phenyl-1, 7-dihyd-
ropyrazolo[30, 40:4, 5]pyrimido[1, 6-a]pyrimidine 5b
Yield 54 %; mp 182 °C; IR (cm-1): mNH 3324; mC:N
f) Ethyl-3,5-dimethyl-7-imino-N1-phenyl-1,7-dihydropy-
razolo[30,40:4,5]pyrimido[1,6-a]pyrimidine-6-carbox-
ylate 5f Yield 71 %; mp 170 °C; IR (cm-1); mNH
1
2230; mC=N 1509, 1562, 1586; RMN H (d ppm,
DMSO): 2.50 (3H, s, CH3), 2.64 (3H, s, CH3); 7.26
(1H, t, J = 7.3 Hz, ArH4); 7.51 (2H, t, J = 7.3 Hz,
ArH3 and ArH5); 7.54 (2H, d, J = 7.3 Hz, ArH2 and
ArH6); 8.19 (1H, s, H9); 8.27 (1H, s, NH); RMN13C (d
ppm, DMSO): 14.42 (CH3); 21.00 (CH3); 87.23 (C-
6); 100.25 (C-3a); 109.00 (CN); 120.22 (C-20 and
C-60), 125.51 (C-40), 128.98 (C-30 and C-50), 138.89
(C-10); 142.79 (C-10a); 154.17 (C-3), 156.49 (C-5),
164.59 (C-9), 165.71 (C-4a), 167.94 (C-7); HRMS
Calcd. for C17H13N7 : 315.1232, found: 315.1214.
c) 6-Cyano-7-imino-9-methyl-N1-phenyl-1,7-dihydropy-
razolo[30,40:4,5]pyrimido[1,6-a]pyrimidine 5c Yield
71 %; mp 166 °C; IR (cm-1); mNH 3321.86; mC:N
1
3081; mCO 1747; mC=N 1510, 1565, 1590; RMN H (d
ppm, DMSO) 1.21 (3H, t, J = 7.2 Hz, CH3); 1.91
(3H, s, CH3); 2.62 (3H, s, CH3); 4.15 (2H, q,
J = 7.2 Hz, CH2); 7.28 (1H, t, J = 7.3 Hz, ArH4);
7.51 (2H, t, J = 7.3 Hz, ArH3 and ArH5); 8.17 (2H, d,
J = 7.3 Hz, ArH2 and ArH6); 8.26 (1H, s, H9); 11.97
(1H, s, NH). RMN13C (d ppm, DMSO) 13.01 (CH3);
14.00 (CH3); 24.45 (CH3); 66.03 (CH2); 105.28 (C-
6); 115.10 (C-3a); 121.07 (C-20 and C-60), 125.50 (C-
40), 129.12 (C-30 and C-50), 138.88 (C-10),142.79 (C-
10a),146.88 (C-3), 148.30 (C-5), 154.14 (C-9), 156.21
(C-4a), 156.48 (C-7), 164.27 (CO); HRMS Calcd. for
C19H18N6O2: 362.1491, found: 362.1478.
1
2223, 1536, 1561, 1599; RMN H (d ppm, DMSO):
2.62 (3H, s, CH3); 7.40 (1H, t, J = 7.3 Hz, ArH4);
7.49 (2H, t, J = 7.3 Hz, ArH3 and ArH5); 7.68 (2H, d,
J = 7.3 Hz, ArH2 and ArH6); 8.19 (1H, s, H5); 8.41
(1H, s, H9); 8.73 (1H, s, NH); RMN13C (d ppm,
DMSO): 14.32 (CH3); 89.64 (C-6); 103.64 (C-3a);
111.83 (CN); Carom 120.38 (C-20 and C-60), 126.65 (C-
40), 138.42 (C-30 and C-50), 140.12 (C-10),143.42 (C-
10a),141.69 (C-3),148.47 (C-5),160.28 (C-9), 161.92
(C-4a); 162.00 (C-7). C16H11N7, 301.1051; HRMS
Calcd. for C16H11N7: 301.1076, found: 301.1087.
d) 6-Cyano-7-imino-N1-phenyl-1,7-dihydropyrazolo[30,40:
4,5]pyrimido[1,6-a]pyrimidine 5d Yield 77 %; mp
248 °C; IR (cm-1); mNH 3189; mC:N 2250; mC=N 1532,
g) Ethyl-5-ethyl-7-imino-3-methyl-N1-phenyl-1,7-dihyd-
ropyrazolo[30,40:4,5]pyrimido[1,6-a]pyrimidine-6-
carboxylate 5g Yield 69 %; mp 181 °C; IR (cm-1);
1
m
NH 3081; mCO 1706; mC=N 1434, 1493, 1589; RMN H
(d ppm, DMSO) 1.06 (3H, t, J = 7.1 Hz, CH3); 1.34
(3H, t, J = 7.0 Hz, CH3); 1.97 (2H, q, J = 7.1 Hz,
CH2); 2.63 (3H, s, CH3); 4.03 (2H, q, J = 7.0 Hz,
CH2); 7.49 (1H, t, J = 7.3 Hz, ArH4); 7.63 (2H, t,
J = 7.3 Hz, ArH3 and ArH5); 8.03 (2H, d, J = 7.3 Hz,
ArH2 and ArH6); 9.57 (1H, s, H9); 11.96 (1H, s, NH).
RMN13C (dppm, DMSO) 11.26 (CH3); 14.03 (CH3);
14.07 (CH3); 30.19 (CH2); 67.92 (CH2); 105.58
(C-6); 114.96 (C-3a); 120.64 (C-20 and C-60), 125.99
(C-40), 129.69 (C-30 and C-50), 139.45 (C-10),143.25
(C-10a),154.76 (C-3), 156.97 (C-5), 159.15 (C-9),
162.04 (C-4a), 162.50 (C-7), 164.09 (CO); HRMS
Calcd. for C20H20N6O2: 376.1648, found 376.1621.
h) Ethyl-7-imino-N1-phenyl-1,7-dihydropyrazolo[30,40:
4,5]pyrimido[1,6-a]pyrimidine carboxylate 5h Yield
89 %; mp 184 °C; IR (cm-1); mNH 3227; mCO 1710; mC=N
1
1559, 1562; RMN H (d ppm, DMSO): 7.33 (1H, t,
J = 7.3 Hz, ArH4), 7.55 (2H, t, J = 7.3 Hz, ArH3 and
ArH5), 8.03 (1H, s, H5), 8.21 (2H, d, J = 7.3 Hz, ArH2
and ArH6), 8.31 (1H, s, H9), 8.36 (1H, s, H3), 8.37 (1H, s,
NH); RMN13C (d ppm, DMSO): 89.87 (C-6); 101.37 (C-
3a); 120.45 (CN); Carom 126.00 (C-20 and C-60), 129.10
(C-40), 13015 (C-30 and C-50), 134.04 (C-10); 138.94 (C-
10a); 139.11 (C-3); 142.14 (C-5);153.19 (C-9); 156.68
(C-4a); 158.26 (C-7); HRMS Calcd. for C15H9N7:
287.0976, found: 287.0919.
1
1539, 1552, 1574.17; RMN H (d ppm, DMSO) 1.29
(3H, t, J = 7.0 Hz, CH3); 4.24 (2H, q, J = 7.0 Hz,
CH2); 7.37 (1H, t, J = 7.3 Hz, ArH4); 7.55 (2H, t,
J = 7.3 Hz, ArH3 and ArH5); 8.14 (2H, d, J = 7.3 Hz,
ArH2 and ArH6); 8.75 (1H, s, H5); 8.83 (1H, s, H9); 9.18
(1H, s, H3); 12.11 (1H, s, NH). RMN13C (d ppm, DMSO)
14.11 (CH3); 61.36 (CH2); 103.83 (C-6); 114.46 (C-
3a); 120.62 (C-20 and C-60), 126.73 (C-40), 129.20 (C-30
e) 6-Cyano-7-imino-5-ethyl-N1-phenyl-1,7-dihydropyraz-
olo[30,40:4,5]pyrimido[1,6-a]pyrimidine 5e Yield
70 %; mp 168 °C; IR (cm-1); mNH 3332; mC:N 2218;
1
m
(3H, t, CH3); 2.30 (2H, q, CH2); 7.30 (1H, t, J = 7.3 Hz,
C=N 1568, 1589, 1620; RMN H (d ppm, DMSO): 1.23
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and C-50), 134.35 (C-10),138.10 (C-10a),148.14 (C-3),
151.37 (C-5), 153.53(C-9), 154.00 (C-4a), 155.18 (C-7),
163.36 (CO). 120.62-126.73-129.20-134.35, C17H14
N6O2, 334.1171; HRMS Calcd. for: C17H14N6O2:
334.1178, found: 334.1171.
30 min, 0.05 ml of 1 % carrageenan suspension was
injected into the left hind paw. The paw volume up to the
tibiotarsal articulation was measured using a plethys-
mometer (model 7150, UgoBasile, Italy) at 0 h (V0) (before
carrageenan injection) and 1, 3 and 5 h later (VT) (after
carrageenan injection). Paw swelling was determined for
each rat and the difference between VT and V0 was taken as
the oedma value. The percent inhibition was calculated
according to the following formula:
i) Ethyl-5-methyl-7-imino-N1-phenyl-1,7-dihydropyrazol-
o[30,40:4,5]pyrimido[1,6-a] pyrimidine-6-carboxylate
5i Yield 78 %; mp 166 °C; IR (cm-1); mNH 3059; mCO
1
1718; mC=N 1579, 1591, 1612; RMN H (d ppm, DMSO)
Â
Ã
1.34 (3H, t, J = 7.0 Hz, CH3); 1.92 (3H, s, J = 7.1 Hz,
CH3); 4.02 (2H, q, J = 7.0 Hz, CH2); 7.30 (1H, t,
J = 7.3 Hz, ArH4); 7.61 (2H, t, J = 7.3 Hz, ArH3 and
ArH5); 8.10 (2H, d, J = 7.3 Hz, ArH2 and ArH6); 9.29
(1H, s, H3); 9.49 (1H, s, H9); 11.95 (1H, s, NH). RMN13C
(d ppm, DMSO); 15.06 (CH3); 23.14 (CH3); 69.54
(CH2); 102.85 (C-3a); 117.05 (C-6); 121.637 (C-20 and
C-60), 126.41 (C-40), 128.65 (C-30 and C-50), 139.24 (C-
10),143.92 (C-10a),144.17 (C-3), 159.62 (C-5), 161.45
(C-9), 167.12 (C-4a), 167.83 (C-7), 168.28 (CO); HRMS
Calcd. for C18H16N6O2: 348,1335, found 348,1274.
% Inhibition: ðVT ꢁ V0Þcontrol ꢁ ðVT ꢁ V0Þtreated
ꢂ 100=ðVT ꢁ V0Þcontrol
Gastroprotective activity
The gastroprotective activity of pyrazolopyrimidopyr-
imidines 5a, b, f, g was studied in 150 mM HCl/EtOH-
induced gastric ulcer (Hara and Okabe, 1985). Rats were
fasted for 24 h prior receiving any treatment and were
divided into six groups of six animals each. Group I was
kept as control group and received the vehicle (Tween
80/Absolute ethanol/Saline solution (0.9 %): 1/1/18).
Group II and III received compound 5a (50, 100 mg/kg,
i.p.), respectively, and Group IV and V received compound
5b (50, 100 mg/kg, i.p.), respectively. Group VI and VII
received compound 5f (50, 100 mg/kg, i.p.), respectively,
and group VIII and IX received compound 5g (50, 100
mg/kg, i.p.), respectively. Group X received cimetidine
(100 mg/kg, i.p.) as reference drug. After 30 min, all
groups were orally treated with 1 ml/100 g of 150 mM
HCl/EtOH (40:60, v/v) solution for gastric ulcer induction.
Animals were sacrificed 1 h after the administration of
ulcerogenic agent; their stomachs were excised and opened
along the great curvature, washed and stretched on cork
plates. The surface was examined for the presence of
lesions and the extent of the lesions was measured. The
summative length of the lesions along the stomach was
recorded (mm) as lesion index.
Pharmacology
Carrageenan (BDH Chemicals Ltd., Poole, England),
cimetidine and acetylsalicylic–lysine were purchased from
pharmacie Centrale of Tunisia.
Animals
Adult Male Wistar rats weighing 150–170 g were obtained
from Pasteur Institute (Tunis, Tunisia). They were housed
in polypropylene cages and left for 2 days for acclimati-
zation to animal room maintained under controlled condi-
tions: a 12 h light–dark cycle (at 22 2 °C) on standard
pellet diet and water ad libitum. Rats were fasted overnight
with free access to water before the experiments. Housing
conditions and in vivo experiments were approved,
according to the guidelines established by the European
´
Union on Animal Care (Communautes Economiques Eu-
´
Statistics
´
ropeennes Council [86/609]).
Results are expressed as the mean SEM of six animals
per group. The data were analysed using Student’s t test,
*p \ 0.05, **p \ 0.01 and ***p \ 0.001 was considered
significant.
Carrageenan-induced rat paw oedema
The anti-inflammatory activity of compounds (5a, b, f,
g) on carrageenan-induced rat paw oedema was determined
according to Winter et al. (1962). The animals were divi-
ded into three groups of six rats each. The control group
received intraperitoneally 2.5 ml/kg of vehicle solution
(Tween 80/absolute ethanol/saline solution (0.9 %) in the
ratio 1:1:18). The reference group received acetylsalicylic–
lysine (300 mg/kg i.p.), and the test groups received
compounds 5a, b, f, g (50 and 100 mg/kg, i.p.). After
Results and discussion
Chemistry
The synthetic routes to target compounds 5a–i are outlined
in Scheme 1. The 5-amino-4-cyano-N1-phenylpyrazole 2,
123

Med Chem Res (2014) 23:1591–1598
1595
Scheme 1 Synthetic procedure
of compounds 5a–i. Reagents:
i H2N–NHPh, CH3CO2H,
CH3CO2H; ii R2C(OEt)3,
CH3CO2H; iii NH3; iv
R2
C
Ph
N
Ph
N
NH2
CN
OEt
R1
NC
NC
N
OEt
i
ii
C C
N
N
R3
Y
CN
R1
,
CH3CO2H
R1
C C
CN
EtO
1
2
3
iii
Ph
N
Ph
N
R2
N
N
R1: H, Me,
R2:H, Me
N
R2
N
N
iv
NH
Y
N
R3:H, Me, Et
N
Y: CN , CO2Et
R1
R1
NH2
R3
4
5a-i
used as a starting material, was prepared in two steps fol-
lowing a similar method reported by Petrie et al. (1985),
Anderson et al., (1990), Aggarwal et al., (2011). The first
step involves acid-catalysed condensation of orthoester with
malonate to form ethoxymethylene malononitrile 1. This
later reacts then with substituted hydrazine to give the am-
inocyanopyrazole 2. Treatment of 2 with orthoester in the
presence of catalytic amount of acid furnished the corre-
sponding cyano-pyrazoloimidates 3 which subsequently
were transformed to the corresponding amino pyrazolopyr-
imidines 4 (Booth et al., 1999; Gupta et al., 2008; Oliveira-
Campos et al., 2007; Bakavoli et al., 2010) upon treatment
with ammoniac. Reaction of compound 4 with ketene eth-
oxymethylene compounds 1 in ethanol in presence of cata-
lytic amount of acid furnished the desired 6-cyano-1,7-
dihydropyrazolo[30,40:4,5]pyrimido[1,6-a]pyrimidine 5a–e
in 70 % yield as a yellow solid. The same procedure gave a
crystalline ethyl-1,7-dihydro pyrazolo [30,40:4,5]pyrimido
[1,6-a]pyrimidine-6-carboxylate 5f–i from ethyl-2-cyano-3-
ethoxyalkyl-2-enoate in 80 % yield. Scheme 1 shows the
synthetic strategy to obtain the target compounds by the
four-steps method, yielding the compounds with structure
5a–i listed in Table 1.
It is interesting to note that time reaction and yield of
products are directly related to the nature of substituent
(R3 and Y). The yields of compounds 5h and 5d are 89
and 77 %, respectively. Hydrogen substituent R3 gave
superior yields in short time. In all cases, reaction leads
to pyrazolo pyrimido pyrimidine only when R1 or R2 is a
hydrogen atom. However, steric effect decreased yields
of the reaction, as in the case of 5g, and may even
prevent the progress of the reaction when R2 and R3 are
methyl groups. Analysis of the NMR and IR spectra
indicated that compounds 5f–i has ester functional group
in their structures so ethoxymethylene cyanoacetate reacts
with pyrazolopyrimidine and in both cases Y is CN or
CO2Et, nitrogen attacked on the nitrile function as the
first attack.
Table 1 Synthesis of 7-imino-
Compounds
R1
R2
R3
Y
Yields (%)
Reaction time (h)
N1-phenyl-1,7-dihydro
pyrazolo[30,40:4,5]pyrimido
[1,6-a]pyrimidine 5a–i
5a
5b
5c
5d
5e
5f
CH3
CH3
H
H
H
CN
68
54
71
77
70
71
69
89
78
24
71
24
5
H
CH3
H
CN
CH3
H
CN
H
H
CN
H
H
C2H5
CH3
C2H5
H
CN
48
75
84
7
CH3
CH3
H
H
CO2Et
CO2Et
CO2Et
CO2Et
5g
5h
5i
H
H
H
H
CH3
24
123

1596
Med Chem Res (2014) 23:1591–1598
Fig. 1 Anti-inflammatory
effect of the intraperitoneal
administration of 5a, b, f, g and
of the reference drug
(acetylsalicylic–lysine: ASL) in
carrageenan-induced rat paw
oedema. The values represent
the means difference of volume
of paw SEM (n = 6).
*p \ 0.01 and **p \ 0.001
significantly different from the
control group
Fig. 2 Percentage inhibition of
the oedema after the
intraperitoneal administration of
5a, b, f, g and the reference drug
(acetylsalicylic–lysine: ASL) in
carrageenan-induced rat paw
oedema
Biological activity
group, even and for more important anti-inflammatory
activity, the cyano function is replaced by ester function.
Table 2 reveals the results of the intraperitoneal
administration of the compounds 5a, b, f, g in carrageenan-
induced rat paw oedema. The compounds 5a, b, f, g tested
at 50 and 100 mg/kg, i.p. produced a significant reduction
of the oedema throughout the entire period of observation
in a dose-dependent manner. The highest reduction of the
oedema was at 3 h after carrageen injection with a percent
inhibition ranged, from 40.64 to 56.81 % for compound 5a,
from 58.98 to 71.36 % for compound 5b, from 60.02 to
82.83 % for compound 5f and from 28.75 to 42.87 % for
compound 5g, whereas the reference drug (acetylsalicylic–
lysine, 300 mg/kg, i.p.) produced 48.03 % reduction in
paw volume. The influence of the substituent R2 on activity
is remarkable. Compound 5a is less potent than the
5-methyl derivatives 5b, so a methyl group linked to the
pyrimidine cycle increases the activity compared to the
case of a hydrogen atom. At the same dose (100 mg/kg),
compound 5b produced 71.36 % inhibition of oedema
against 56.81 % for 5a. In addition, the compound 5f is
Anti-inflammatory and gastroprotective activities
of compounds 5a, b, f, g
The pyrazolopyrimidine derivatives are a well-known class
of NSAIDs with several products in market (Russo et al.,
1992; El-Kateb et al., 2012) (Figs. 1, 2).
The structure–activity relationships (SAR) for these
compounds have been extensively explored for optimiza-
tion of anti-inflammatory activity last three decades, since
this class was introduced (Lombardino and Wiseman,
´
1972; Farre et al., 1986; Berq et al., 1999; Lee et al., 1999).
In continual efforts to find potentially safer and more
efficacious parent agents through further exploration of
SAR of this class, we decided to study the pharmacological
profiles of compounds 5a, b, f, g belonging to pyr-
azolopyrimidopyrimidine family. We examined the effect
of modification of the electronic nature of substituents on
various portions of type NSAIDs. For this objective the
hydrogen atom (position 5) is replaced by methyl or ethyl
123

Med Chem Res (2014) 23:1591–1598
1597
Table 2 Anti-inflammatory effect of the intraperitoneal administration of 5a, b, f, g and of the reference drug (acetylsalicylic–lysine: ASL) in
carrageenan-induced rat paw oedema
Sample
Dose
(mg/kg)
Oedema (10-2 ml) (mean SEM)
Oedema inhibition (%)
1 h
3 h
5 h
1 h
3 h
5 h
Vehicle (2,5 ml/kg)
–
35.87 4.48
50.66 3.68
56.04 2.91
–
–
–
Acetylsalicylic–lysine
(reference drug)
300
13.23 2.69**
26.32 2.44**
29.15 2.87**
63.10
48.03
47.98
5a
5b
5f
50
100
50
20.59 2.51*
7.01 3.41**
14.62 3.21*
2.81 2.06***
13.51 3.4**
2.07 2.8***
24.37 2.7*
12.31 3.2**
30.07 3.51*
21.88 1.89**
20.78 2*
33.73 4.16*
23.45 2.5**
23.56 2*
42.59
80.44
59.25
92.17
62.31
94.22
32.04
65.66
40.64
56.81
58.98
71.36
60.02
82.83
28.75
42.87
39.8
58.15
57.95
62.76
59.42
68.85
25.13
40.18
100
50
14.51 2.98***
20.25 2.8**
8.69 2.3***
36.09 2.9*
28.94 2.4*
20.86 2.21***
22.74 3.2**
17.45 2.5***
41.95 2.8
100
50
5g
100
33.52 2.3
The values represent the mean difference of volume of paw SEM (n = 6)
* p \ 0.05, ** p \ 0.01, *** p \ 0.001 significantly different from control group
more potent than the ethyl derivatives 5g, so an ethyl group
linked to the pyrimidine cycle decreases the activity
compared to the methyl group.
acid on the stomach lining leading to mucosal damage
(Fig. 3).
The results of gastroprotective activity of compounds 5a,
b, f, g on gastric ulcer induced by HCI/ethanol solution are
shown in Table 3. Oral administration of the ulcerogenic
agent to the control group clearly showed a mucosal damage
characterized by multiple haemorrhage red bands of differ-
ent sizes along the long axis of the glandular stomach as
described in other studies (Shay et al., 1945; Yassir et al.,
1999). When we compared the gastroprotective activity of
On the other hand, mucosal erosion and ulceration are
produced by most NSAIDs with varying degrees. Inhibition
of synthesis of gastroprotective prostaglandins (PGE2) is
clearly involved (Nezamis et al., 1967) and due to the
inhibition of the constitutive isoform COX-1 (Main and
Whittle, 1973; Cryer and Feldman, 1992). Thus, deficiency
of PGs reduces the mucosal secretions along with hydrogen
carbonate that ultimately aggravates the lethal effects of
compounds 5a, b, f,
g
we observed that
Table 3 Effect of compounds 5a, b, f, g and the reference drug
(cimetidine) on gastric ulcer induced by HCl/ethanol in rats
Treatment
Dose
(mg/kg)
Ulcer index
(mm)
Inhibition
(%)
Vehicle (2.5 ml/kg)
(control)
–
85 2.82
–
Compounds
5a
50
100
50
43.66 2.58
30 3.03*
48.63
64.7
5b
5f
26.83 3.43**
11.83 0.75***
23.34 2.9**
7.29 0.3***
50.81 3.2
68.43
86.08
72.53
91.42
40.22
52.17
74.03
100
50
100
50
5g
100
100
40.65 2.8
´
Cimetidine (reference
drug)
22.07 2.12**
Fig. 3 Effect of compounds 5a, b, f, g and the reference drug
(cimetidine) on gastric ulcer induced by HCl/ethanol in rats. Data
expressed as mean SEM (n = 6). *p \ 0.05, **p \ 0.01,
***p \ 0.001 significantly different from control group
Data expressed as mean SEM (n = 6)
* p \ 0.05, ** p \ 0.01, *** p \ 0.001 significantly different from
control group
123

1598
Med Chem Res (2014) 23:1591–1598
´
Gupta S, Rodrigues LM, Esteves AP, Oliveira-Campos AMF, Jose
pyrazolopyrimidopyrimidine 5b (100 mg/kg) demonstrated
the higher significant inhibition of gastric lesion (91, 42 %).
In conclusion, we have synthesized a new series of 1,7-
dihydropyrazolo [30,40:4,5]pyrimido[1,6-a]pyrimidine 5a–i
derivatives. The yield of the reaction seems to be signifi-
cantly influenced by the nature of substituent. The highest
yield is obtained for more hydrogen atom substituent.
However, test (or experimental) compounds 5a, b,
f showed that the methyl group increases the anti-inflam-
matory activity, contrary to ethyl group which decreases
this activity. The same interpretation is found with gas-
troprotective effect. Indeed, our results on the gastropro-
tective effects of compounds 5a, b, f compared with
cimetidine indicate that replacement of hydrogen by
methyl reduces the gastrointestinal adverse effects.
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