Synthesis and evaluation of novel spiro derivatives for pyrrolopyrimidines as anti-hyperglycemia promising compounds

Full text of DOI:10.1080/14756366.2018.1461854

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Synthesis and evaluation of novel spiro derivatives
for pyrrolopyrimidines as anti-hyperglycemia
promising compounds
Samar Said Fatahala, Shahenda Mahgub, Heba Taha & Rania Helmy Abd-El
Hameed
To cite this article: Samar Said Fatahala, Shahenda Mahgub, Heba Taha & Rania Helmy Abd-El
Hameed (2018) Synthesis and evaluation of novel spiro derivatives for pyrrolopyrimidines as anti-
hyperglycemia promising compounds, Journal of Enzyme Inhibition and Medicinal Chemistry, 33:1,
809-817, DOI: 10.1080/14756366.2018.1461854
To link to this article: https://doi.org/10.1080/14756366.2018.1461854
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2018, VOL. 33, NO. 1, 809–817
https://doi.org/10.1080/14756366.2018.1461854
RESEARCH PAPER
Synthesis and evaluation of novel spiro derivatives for pyrrolopyrimidines as
anti-hyperglycemia promising compounds
Samar Said Fatahalaa, Shahenda Mahgubb, Heba Tahab and Rania Helmy Abd-El Hameeda
b
aPharmaceutical Organic Chemistry Department, Helwan University, Helwan, Cairo, Egypt; Biochemistry Department, Faculty of Pharmacy,
Helwan University, Helwan, Cairo, Egypt
ABSTRACT
ARTICLE HISTORY
Received 8 November 2017
Revised 7 March 2018
Accepted 3 April 2018
Pyrrolopyrimidin-4-ylidene-malononitriles IIa–d were prepared as important intermediates for preparation
of a new series of spiro-pyrrolopyrimidines. These intermediates undergo cyclisation via reaction with ace-
tylacetone, guanidine hydrochloride or hydrazine hydrate. Elemental and spectroscopic evidences for the
structures of these compounds are presented. The final compounds have been monitored for in vivo anti-
hyperglycemic activity, compared with Amaryl as standard drug. Among 12 tested compounds, both spiro
(pyrano IIIb and pyrazlo Va) derivatives exhibit promising anti-hyperglycemic activity.
KEYWORDS
Spiropyrimidines; pyrrolo-
pyrimidines; anti-
hyperglycemic assay
Introduction
DPP-IV inhibitors, has been reported in advanced clinical trials. A
highly potent DPP-IV inhibitor with pyrrolopyrimidine was also
reported17,18 (Figure 1).
Diabetes mellitus (DM) is a severe metabolic complaint that has a
significant influence on the health and feature of patients’ life1. In
2013, 382 million adults were diagnosed with diabetes worldwide.
This number is expected to grow to 592 million in 2035, of which
90% will have type 2 diabetes (non-insulin-dependent diabetes
mellitus; T2D)2. Patients with T2D are 2–4 times more likely to have
fatal or non-fatal coronary events or a stroke. Almost 70–80% of
patients die from one of these two conditions. The International
Diabetes Federation (IDF) listed Egypt among the world top 10
countries in the number of patients with diabetes3–5. In Egypt, the
predominance of diabetes is around 15.56% among adults (age: 20
and 79 years), with an annual death of 86,478 related to diabetes2.
Treatment of diabetic patients has been concentrated on diet-
ary controlling and well-known anti-hyperglycemic like sulfonylur-
In 2004, pyrazolopyrimidine APD668 was discovered by Arena
pharmaceutics, was found to exhibition high in vivo activity com-
pared to a known DPP-IV inhibitor. APD668 was found to be
more potent on delaying the onset of hyperglycemia (Figure 2).
Researchers at GlaxoSmithKline replacement of pyrazolopyrimidine
ring system in APD668 with a dihydropyrrolopyrimidine scaffold,
which were described as having therapeutic value for diabetes
and associated conditions, obesity, glucose intolerance, insulin
resistance and atherosclerosis19 (Figure 2).
Spiro-based heterocyclic systems20
, containing one carbon
atom common to two rings, were found to be very motivating21.
The asymmetric nature of these compounds, due to the spiro car-
bon, found to be one of the important criteria of the biological
activities22–26. Several patents described spiroazetidine and spiroa-
zetidinone derivatives as GPR119 receptor agonists for the treat-
ment of diabetes19 (Figure 3).
R
eas, metformin and acarbose. Glimepiride (AmarylV, Sanofi-Aventis,
Gentilly, France), a sulfonylurea containing a pyrrole group, acting
as anti-hyperglycemic drug6. It indicated to treat type 2 diabetes
through increase insulin production by the pancreas (Figure 1).
Recently, urgent requisite to develop novel anti-hyperglycemic
Encouraged by the prominence of spiro containing compounds,
and in maintenance of our research efforts27–31, in this research,
we are going to spot an aspect on the chemistry of some newly
synthesised spiro-pyrrolopyrimidine derivatives and estimate them
for the anti-diabetic activities. The synthetic pathways approved
for the synthesis of these compounds are revealed in Scheme 1.
agents was observed7,8
.
Numerous adverse effects present anti-hyperglycemic were
indicated such as hepatotoxicity, weight gain and hypoglycemia9.
Administration of dipeptidyl peptidase IV (DPP-IV)10–13 inhibitors
to diabetic patients results in higher concentrations of endogen-
ous glucagon-like peptide (GLP-1) lead to decrease in plasma glu-
cose. Long-term treatment with a DPP-IV inhibitor reduced HbA1c
(glycosylated haemoglobin), offered prospective improvement in
insulin producing function of the pancreas.
Materials and methods
Synthesis of lead compounds
DPP-IV inhibitors14 were validated to be active and safe com-
pounds that control blood glucose. Vildagliptin, saxagliptin, DPP-IV
All commercial chemicals used as starting materials and reagents
in this study were purchased from Merck (Darmstadt, Germany)
inhibitors, (having pyrrole and fused pyrrole ring15,16, are on the and were of reagent grade. All melting points were uncorrected
market in many countries. Gosogliptin, di-pyrrole containing and measured using Electro-thermal IA 9100 apparatus (Shimadzu,
CONTACT Samar Said Fatahala (Fathallah)
samarradwan1@yahoo.com
Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Helwan University,
Ain-Helwan, Helwan, Cairo 1179, Egypt
Supplemental data for this article can be accessed here.
ß 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

810
S. S. FATAHALA ET AL.
CN
O
H
N
HN
O
N
HO
HN
S
O
vildagliptin
EMEA approved
O
O
CN
O
HN
N
O
N
HO
Glimepiride
NH2
saxagliptin
EMEA/FDA approved
Amaryl®, standard anti-hyperglycemic drug a
sulfonylurea containing
Approved DPP-IV inhibitors as type
2 diabetes medications containing
a pyrrole moiety.
a pyrrole group
N
F
F
N
N
N
O
N
Gosogliptin ; Dipyrrole containg DPP-IV
reported in advanced clinical trials
NH
Figure 1. Pyrroles and pyrrolopyrimidines as anti-diabetic agents.
O
O
O
O
N
N
O
N
CN
R3
R1=H,CH3
R2,R3=H,CH3,Br
N
O
N
O
N
R2
NH2
N
N
N
N
N
R1
N
N
F
F
A highly potent DPP-IV inhibitor with
pyrrolopyrimidine significantly improved
metabolic stability
SO2Me
SO2Me
APD668;
pyrazolopyrimidine
Dihydropyrrolopyrimidine
potent anti-hyperglycemic
Figure 2. Pyrrolopyrimidines as anti-diabetic agents.
F
F
Ar
N
O
N
S
NH
NH2
NH
N
N
O
O
NH
N
N
spiropyran;
DNA-binding
spiro-thiazolidine;
Anticancer
F
O
N
spiroazetidinone;
Anti-diabetic
Cl
Figure 3. Spiro compounds as biological active scaffolds.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
811
CN
CN
S
Ph
Ph
NH
N
NH
i
X
iv
NH2
N
X
N
N
Ar
N
I a-d
Ar
II a-d
ii
CN
NH
HN
Ph
iii
v
COCH3
NH2
N
X
NH2
N
CH3
Ph
N
NH2
SMe
Ar
V a-d
CN
NH
Ph
O
N
CN
NH
HN
Ph
Cpd
X
Ph
Ph
H
Ar
Benzyl
Antipyrine
Antipyrine
a
b
c
N
X
N
X
N
N
N
X
N
Ar
d
H
3,4-dichloro-phenyl
Ar
IV a-d
Ar
VI a-d
III a-d
Scheme 1. Synthetic pathway for preparation of II-V [reagents; i ¼ NC-CH2-CN, ii ¼ (CH3CO)2CH2, iii ¼ (NH2)2C ¼ NH, iv ¼ NH2NH2, v ¼ MeI].
Japan); IR spectra were recorded as potassium bromide pellets on 2-(7-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-5-
a Perkin-Elmer 1650 spectrophotometer (Waltham, MA, USA),
Faculty of Science, Cairo University, Cairo, Egypt. 1H NMR spectra
were determined on a Varian Mercury (300 MHz) spectrometer
(Varian, UK) and chemical shifts were expressed as ppm against
TMS as internal reference (Faculty of Science, Cairo University,
phenyl-7H-pyrrolo[2,3-d]pyrimidin-4-ylidene)-malononitrile
(IIc).
Yield: 54%; m.p.: 173–175 ꢀC; IR (KBr) t (cmꢁ1): 3295 (N–H), 2211
(CꢂN), 1691 (C¼O), 1588 (C¼N); MS (EI) m/z: 445 (Mþ, 73.4%), 1H
NMR (DMSO-d6, 300 MHz) d (ppm): 2.2 (s, 3H, CH3), 3.42 (s, 3H,
NCH3), 6.6–7.8 (m, 11H, Ar–H ), 8.5 (s, 1H, C2–H), 8.9 (s, 1H, NH,
Cairo, Egypt). Mass spectra were recorded on 70 eV (EI Ms-QP 1000 D2O exchangeable); Anal. Calcd for C26H19N7O (445.48): C, 70.11;
EX, Shimadzu, Japan), Faculty of Science, Cairo University, Cairo, H, 4.27; N, 22.02%. Found: C, 70.38; H, 4.61; N, 22.28%.
Egypt. Microanalyses were operated using Vario, Elementary
apparatus (Shimadzu, Japan), Organic Microanalysis Unit, Faculty
of Science, Cairo University, Cairo, Egypt. Column Chromatography
was performed on (Merck) Silica gel 60 (particle size
2-(7-(3,4-dichlorophenyl)-5-phenyl-7H-pyrrolo[2,3-d]pyrimidin-4-yli-
dene)-malononitrile (IId). Yield: 56%; m.p.: 191–193 ꢀC; IR (KBr) t
(cmꢁ1): 3347 (N–H), 2212 (CꢂN), 1581 (C¼N); MS (EI) m/z: 404 (Mþ,
13.5%), 406 (Mþþ2, 8.5%), 408 (Mþþ4, 2.7%) 1H NMR (DMSO-d6,
300 MHz) d (ppm): 6.8–7.8 (m, 9H, Ar–H ), 8.09 (s, 1H, C2–H), 8.83 (s,
1H, NH, D2O exchangeable); Anal. Calcd for C21H11Cl2N5 (404.25): C,
62.38; H, 2.72; N, 17.33%. Found: C, 62.05; H, 2.69; N, 17.71%.
0.06–0.20 mm).
Compounds
Ia–d
were
synthesised
as
reported32–34. The rest of compounds prepared in this paper were
new and their structures were confirmed using spectral data.
General procedure for the synthesis of compounds IIa–d
Compounds Ia–d (0.01 mol) and malononitrile (0.66 g, 0.01 mol)
were heated under reflux in dry ethanol (30 ml) for 8 h, cooled,
poured onto ice-water to give precipitate which was filtered off,
dried and recrystallised from methanol to give IIa–d.
General procedure for the synthesis of compounds IIIa–d
A mixture of compounds IIa–d (0.02 mol), acetylacetone (2g,
0.02 mol) and pyridine (6–8 drops) was heated under reflux in dry
ethanol (50 ml) for 8 h, concentrated, cooled and the separated
compound was filtered off and recrystallised from methanol to
give IIIa–d.
2-(7-benzyl-5,6-diphenyl-3H-pyrrolo[2,3-d]pyrimidin-4-ylidene)-
malononitrile (IIa). Yield: 73%; m.p.: 179–181 ꢀC; IR (KBr) t (cmꢁ1):
3318 (N–H), 2219 (CꢂN), 1607 (C¼N); MS (EI) m/z: 425 (Mþ, 67%),
1H NMR (DMSO-d6, 300 MHz) d (ppm): 5.4 (s, 2H, Ph–CH2), 6.8–8.0
(m, 15H, Ar–H ), 8.18 (s, 1H, C2–H), 8.9 (s, 1H, NH, D2O exchange-
able); Anal. Calcd for C28H19N5 (425.16): C, 79.06; H, 4.47; N,
16.47%. Found: C, 79.38; H, 4.66; N, 16.07%.
5-acetyl-4-amino-70-benzyl-6-methyl-50,60-diphenyl-spiro[3H-pyr-
rolo[2,3-d]pyrimidine-40,2-pyran]-3-carbonitrile (IIIa). Yield: 72%;
m.p.: 187–189 ꢀC; IR (KBr) t (cmꢁ1): 3212–3345 (N–H, NH2), 2199
(CꢂN), 1667 (C¼O), 1598 (C¼N), 1260 (C–O); MS (EI) m/z: 525 (Mþ,
41%), 1H NMR (DMSO-d6, 300 MHz) d (ppm): 2.23(s, 3H, C6–CH3),
2.27(s, 3H, COCH3), 5.8(s, 2H, Ph–CH3), 4.7 (brs, 2H, NH2, D2O
exchangeable), 6.9–7.7 (m, 15H, Ar–H), 8.3 (s, 1H, C20–H). 8.8 (s, 1H,
NH, D2O exchangeable); Anal. Calcd for C33H27N5O2 (525.60): C,
75.43; H, 5.14; N, 13.33%. Found: C, 75.80; H, 5.02; N, 13.61%.
2-(7-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-5,6-
diphenyl-7H-pyrrolo[2,3-d]pyrimidin-4-ylidene)-malononitrile (IIb).
Yield: 67%; m.p.: 186–188 ꢀC; IR (KBr) t (cmꢁ1): 3287 (N–H), 2228
(CꢂN), 1693 (C¼O), 1608 (C¼N); MS (EI) m/z: 521 (Mþ, 28%), 1H
NMR (DMSO-d6, 300 MHz) d (ppm): 2.25 (s, 3H, CH3), 3.5 (s, 3H,
NCH3), 6.6–7.8 (m, 15H, Ar–H ), 8.12 (s, 1H, C2–H), 8.9. (s, 1H, NH,
D2O exchangeable); Anal. Calcd for C32H23N7O (521.57): C, 73.70;
H, 4.41; N, 18.81%. Found: C, 74.01; H, 4.35; N, 18.92%.
5-acetyl-4-amino-70-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-
pyrazol-4-yl)-6-methyl-50,60-diphenyl-spiro[3H-pyrrolo[2,3-d]pyrimi-
dine-40,2-pyran]-3-carbonitrile (IIIb). Yield: 55%; m.p.: 195–197 ꢀC;
IR (KBr) t (cmꢁ1): 3250–3387 (N–H, NH2), 2207 (CꢂN), 1678, 1693

812
S. S. FATAHALA ET AL.
(C¼O), 1612 (C¼N), 1270 (C–O); MS (EI) m/z: 621 (Mþ, 26%), 1H (ppm): 2.2 (s, 3H, CH3), 3.5 (s, 3H, NCH3), 4.05–4.4 (brs, 4H, 2NH2,
NMR (DMSO-d6, 300 MHz) d (ppm): 2.2 (s, 3H, C6–CH3), 2.3(s, 3H, D2O exchangeable), 6.8–7.8 (m, 12H, Ar–H þ NH ), 8. 2 (s, 1H,
COCH3), 2.37 (s, 3H, CH3), 3.52 (s, 3H, NCH3), 5.2 (brs, 2H, NH2, D2O C20–H), 8.8 (s, 1H, NH, D2O exchangeable); Anal. Calcd for
exchangeable), 6.9–7.9 (m, 15H, Ar–H), 8.1(s, 1H, C20–H). 8.9 (s, 1H, C27H24N10O (504.25): C, 64.29; H, 4.76; N, 27.78%. Found: C, 63.93;
NH, D2O exchangeable); Anal. Calcd for C37H31N7O3 (621.12): C, H, 4.65; N, 27.40%.
71.50; H, 4.99; N, 15.78%. Found: C, 71.39; H, 5.18; N, 15.66%.
2,4-diamino-70-(3,4-dichlorophenyl)-50-phenyl-spiro[1H-pyrimidine-
5-acetyl-4-amino-70-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-
pyrazol-4-yl)-6-methyl-50-phenyl-spiro[3H-pyrrolo[2,3-d]pyrimidine-
40,2-pyran]-3-carbonitrile (IIIc). Yield: 65%; m.p.: 188–190 ꢀC; IR
(KBr) t (cmꢁ1): 3145–3348 (N–H, NH2), 2213 (CꢂN), 1661, 1688
(C¼O), 1602 (C¼N), 1305 (C–O); MS (EI) m/z: 545 (Mþ, 31.4%), 1H
NMR (DMSO-d6, 300 MHz) d (ppm): 2.2 (s, 3H, C6–CH3), 2.34 (s, 3H,
COCH3), 2.39 (s, 3H, CH3), 3.48 (s, 3H, NCH3), 4.82 (brs, 2H, NH2,
D2O exchangeable), 7.0–7.9 (m, 11H, Ar–H), 8.3(s, 1H, C20–H). 8.9 (s,
1H, NH, D2O exchangeable); Anal. Calcd for C31H27N7O3 (545.32): C,
68.26; H, 4.95; N, 17.98%. Found: C, 68.03; H, 5.20; N, 18.22%.
6,40-3H-pyrrolo[2,3-d]pyrimidine]-5-carbonitrile (IVd). Yield: 35%;
m.p.: 212–214 ꢀC; IR (KBr) t (cmꢁ1): 3209–3345 (N–H, NH2), 2218
(CꢂN), 1626 (C¼N), MS (EI) m/z: 462 (Mþ, 58%), 464 (Mþþ2,
18.3%), 466 (Mþþ4, 5.7%), 1H NMR (DMSO-d6, 300 MHz) d (ppm):
4.1– 4.4 (brs, 4H, 2NH2, D2O exchangeable), 6.9–8.0 (m, 10H,
Ar–H þ NH), 8.23 (s, 1H, C20–H), 9.1 (s, 1H, NH, D2O exchangeable);
Anal. Calcd for C22H16N8Cl2 (462.34): C, 57.14; H, 3.46; N, 24.24%.
Found: C, 57.08; H, 3.62; N, 24.53%.
General procedure for the synthesis of compounds Va–d
A mixture of compound IIa–d (0.02 mol), hydrazine hydrate (0.64g,
0.02 mol) and pyridine (6–8 drops) was heated under reflux in dry
ethanol (50 ml) for 8 h, concentrated, cooled, and the separated
compound was filtered off and recrystallised from methanol to
give Va–d.
5-acetyl-4-amino-70-(3,4-dichlorophenyl)-6-methyl-50-phenyl-
spiro[3H-pyrrolo[2,3-d]pyrimidine-40,2-pyran]-3-carbonitrile
(IIId).
Yield: 33%; m.p.: 203–205 ꢀC; IR (KBr) t (cmꢁ1): 3225–3419 (N–H,
NH2), 2222 (CꢂN), 1709 (C¼O), 1614 (C¼N), 1312 (C–O); MS (EI) m/
z: 503 (Mþ, 60%), 505 (Mþþ2, 20.3%), 507 (Mþþ4, 8.3%) 1H NMR
(DMSO-d6, 300 MHz) d (ppm): 2.3 (s, 3H, C6–CH3), 2.41(s, 3H,
COCH3), 4.8 (brs, 2H, NH2, D2O exchangeable), 6.9–7.8 (m, 9H, Ar-
H), 8.4 (s, 1H, C20–H). 8.9 (s,1H, NH, D2O exchangeable); Anal.
Calcd for C26H19Cl2N5O2 (503.3): C, 62.03; H, 3.78; N, 13.92%.
Found: C, 62.34; H, 3.52; N, 14.12%.
3-amino-70-benzyl-50,60-diphenyl-spiro[1,4-dihydropyrazole-5,40-3H-
pyrrolo[2,3-d]pyrimidine]-4-carbonitrile (Va). Yield: 72%; m.p.:
192–194 ꢀC; IR (KBr) t (cmꢁ1): 3197–3342 (N–H, NH2), 2226 (CꢂN),
1633 (C¼N); MS (EI) m/z: 457 (Mþ, 29%), 1H NMR (DMSO-d6,
300 MHz) d (ppm): 4.1(s, 1H, C–4H), 4.9 (s, 2H, Ph–CH2), 5.82 (s, 2H,
NH2,D2O exchangeable), 6.8–7.9 (m, 16H, Ar–H þ NH), 8.32 (s, 1H,
C–20H), 8.7(s,1H, NH, D2O exchangeable); Anal. Calcd for C28H23N7
(457.34): C, 73.52; H, 5.03; N, 21.44%. Found: C, 73.58; H, 4.80;
N, 21.71%.
General procedure for the synthesis of compounds IVa–d
A
mixture of compounds IIa–d (0.02 mol), guanidine (1.18g,
0.02 mol) and pyridine (6–8 drops) was heated under reflux in dry
ethanol (50 ml) for 8 h, concentrated, cooled, and the separated
compound was filtered off and recrystallised from methanol to
give IVa–d.
3-amino-70-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-
yl)-50,60-diphenyl-spiro[1,4-dihydropyrazole-5,40-3H-pyrrolo[2,3-
d]pyrimidine]-4-carbonitrile (Vb). Yield: 65%; m.p.: 201–203 ꢀC; IR
(KBr) t (cmꢁ1): 3139–3442 (N–H, NH2), 2216 (CꢂN), 1688 (C¼O),
1622 (C¼N); MS (EI) m/z: 553 (Mþ, 91%), 1H NMR (DMSO-d6,
300 MHz) d (ppm): 2.27 (s, 3H, CH3), 3.4 (s, 3H, NCH3), 4.3 (s, 1H,
C–4H), 5.1 (s, 2H, NH2,D2O exchangeable), 7.0–8.0 (m, 16H,
Ar–H þ NH), 8.32 (s, 1H, C–20H), 9.0 (s,1H, NH, D2O exchangeable);
Anal. Calcd for C32H27N9O (553.39): C, 69.44; H, 4.88; N, 22.78%.
Found: C, 69.80; H, 4.62; N, 22.45%.
2,4-diamino-70-benzyl-50,60-diphenyl-spiro[1H-pyrimidine-6,40-3H-
pyrrolo[2,3-d]pyrimidine]-5-carbonitrile (IVa). Yield: 68%; m.p.:
195–197 ꢀC; IR (KBr) t (cmꢁ1): 3126–3419 (N–H, NH2), 2212 (CꢂN),
1618 (C¼N); MS (EI) m/z: 484 (Mþ, 61%), 1H NMR (DMSO-d6,
300 MHz) d (ppm): 5.26 (s, 2H, Ph–CH2), 4.2–4.6 (brs, 4H, 2NH2, D2O
exchangeable), 6.9–8.1 (m, 16H, Ar–H þ NH), 8.33 (s, 1H, C20–H), 8.9
(s, 1H, NH, D2O exchangeable); Anal. Calcd for C29H24N8 (484.4): C,
71.90; H, 4.96; N, 23.14%. Found: C, 71.75; H, 4.98; N, 23.42%.
3-amino-70-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-
yl)-50-phenyl-spiro[1,4-dihydropyrazole-5,40-3H-pyrrolo[2,3-d]pyr-
imidine]-4-carbonitrile (Vc). Yield: 53%; m.p.: 182–184 ꢀC; IR (KBr) t
(cmꢁ1): 3231–3448 (N–H, NH2), 2207 (CꢂN), 1682 (C¼O), 1633
2,4-diamino-70-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyra-
zol-4-yl)-50,60-diphenyl-spiro[1H-pyrimidine-6,40-3H-pyrrolo[2,3-
d]pyrimidine]-5-carbonitrile (IVb). Yield: 53%; m.p.: 197–199 ꢀC; IR
(KBr) t (cmꢁ1): 3153–3320 (N–H, NH2), 2227 (CꢂN), 1698 (C¼O),
1622 (C¼N); MS (EI) m/z: 580 (Mþ, 23.9%), 1H NMR (DMSO-d6,
300 MHz) d (ppm): 2.3 (s, 3H, CH3), 3.41 (s, 3H, NCH3), 4.2–4.5 (brs,
4H, 2NH2, D2O exchangeable), 7.0–7.7 (m, 16H, Ar–H þ NH), 8.09 (s,
1H, C20–H), 8.94 (s, 1H, NH, D2O exchangeable); Anal. Calcd for
C33H28N10O (580.04): C, 68.28; H, 4.83; N, 24.14%. Found: C, 68.39;
H, 5.09; N, 24.45%.
1
(C¼N); MS (EI) m/z: 477 (Mþ, 19.4%), H NMR (DMSO-d6, 300 MHz)
d (ppm): 2.31 (s, 3H, CH3), 3.38 (s, 3H, NCH3), 3.42 (s, 1H, C–4H), 4.4
(s, 2H, NH2,D2O exchangeable), 6.8–7.9 (m, 12H, Ar–H þ NH), 8.35
(s, 1H, C–20H), 8.9 (s,1H, NH, D2O exchangeable); Anal. Calcd for
C26H23N9O (477.52): C, 65.41; H, 4.82; N, 26.42%. Found: C, 65.62;
H, 4.71; N, 26.79%.
3-amino-70-(3,4-dichlorophenyl)-50-phenyl-spiro[1,4-dihydropyra-
zole-5,40-3H-pyrrolo[2,3-d]pyrimidine]-4-carbonitrile (Vd). Yield:
39%; m.p.: 206–208 ꢀC; IR (KBr) t (cmꢁ1): 3196–3335 (N–H, NH2),
2,4-diamino-70-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyra-
zol-4-yl)-50-phenyl-spiro[1H-pyrimidine-6,40-3H-pyrrolo[2,3-d]pyrimi-
dine]-5-carbonitrile (IVc). Yield: 43%; m.p.: 194–196 ꢀC; IR (KBr) t 2230 (CꢂN), 1590 (C¼N), MS (EI) m/z: 435 (Mþ, 32.7%), 437
(cmꢁ1): 3239–3485 (N–H, NH2), 2205 (CꢂN), 1682 (C¼O), 1598 (Mþþ2, 11.3%), 439 (Mþþ4, 3.9%), 1H NMR (DMSO-d6, 300 MHz) d
(C¼N), MS (EI) m/z: 504 (Mþ, 22%), H NMR (DMSO-d6, 300 MHz) d (ppm): 3.48 (s, 1H, C–4H), 4.2 (s, 2H, NH2, D2O exchangeable),
1

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
813
6.9–8.0 (m, 10H, Ar–H þ NH), 8.48 (s, 1H, C20–H), 8.91 (s, 1H, NH, Dose determination
D2O exchangeable); Anal. Calcd for C21H15N7Cl2 (435.28): C, 57.93;
H, 3.45; N, 22.53%. Found: C, 57.78; H, 3.65; N, 22.16%.
Glimepiride (Amaryl) was used as a standard anti-diabetic (4 mg/
kg) in 1% of gum acacia and administered orally35. Equivalent
doses of all derivatives were calculated according to their molecu-
lar weight (M.wt).
General procedure for the synthesis of compounds VIa–d
A mixture of compounds Ia–d (0.02 mol) and methyl iodide
(0.02 mol) was stirred in 10% NaOH solution at room temperature
Assessment of improvement on oral glucose tolerance and blood
for 8 h, poured onto acidified ice-water to give a precipitate which glucose lowering activity: sucrose loaded normal rats (SLM)
Male albino Wistar rats (200–250 g) were chosen and kept back on
an overnight fasting. Next morning, the blood glucose level
(0 min) of each animal was stated by glucometer using glucostrips.
The animals presenting their fasting blood glucose levels in the
range of 60–80 mg/dL were selected and separated into one con-
trol group and 13 experimental groups with six animals in each.
Each rat of experimental groups was given suspension of the test
compounds made in 1% of gum acacia at a dosage of (4 mg/kg)
for the standard drug Glimepiride and Equivalent doses of all
derivatives.
was filtered off, dried and crystallised from methanol to afford
compounds VIa–d.
7-benzyl-4-methylsulfanyl-5,6-diphenyl-pyrrolo[2,3-d]pyrimidine
(VIa). Yield: 73%; m.p.: 187–189 ꢀC; IR (KBr) t (cmꢁ1): 1583 (C¼N);
MS (EI) m/z: 407 (Mþ, 52.3%), 1H NMR (DMSO-d6, 300 MHz) d
(ppm): 3.12 (s, 3H, S–CH3), 4.97 (s, 2H, Ph–CH2), 6.9–7.8 (m, 15H,
Ar–H), 8.3 (s, 1H, C–2H); Anal. Calcd for C26H21N3S (407.32): C,
76.66; H, 5.16; N, 10.32%. Found: C, 76.51; H, 4.93; N, 10.70%.
The animals of the control group received vehicle (1.0% of
gum acacia) only. Exactly 30 min post-administration of the test
samples/vehicle, an oral sucrose load of 10 g/kg body weight (bw)
was given to each animal and the blood glucose level of each ani-
mal were measured at 30, 60, 90 and 120 min36. The percentages
(%) decreased in blood glucose level were calculated conferring to
the AUC method.
7-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-4-
methylsulfanyl-5,6-diphenyl-pyrrolo[2,3-d]pyrimidine (VIb). Yield:
51%; m.p.: 183–185 ꢀC; IR (KBr) t (cmꢁ1): 1708 (C¼O), 1618 (C¼N);
1
MS (EI) m/z: 503 (Mþ, 73%), H NMR (DMSO-d6, 300 MHz) d (ppm):
2.31 (s, 3H, CH3), 3.27 (s, 3H, S–CH3), 3.37 (s, 3H, N–CH3), 7.0–8.1
(m, 15H, Ar–H), 8.4 (s, 1H, C–2H); Anal. Calcd for C30H25N5OS
(503.32): C, 71.57; H, 4.97; N, 13.92%. Found: C, 71.82; H, 4.66;
N, 13.77%.
Streptozotocin-induced diabetic rats
7-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-4-
methylsulfanyl-5-phenyl-pyrrolo[2,3-d]pyrimidine (VIc). Yield: 49%;
m.p.: 167–169 ꢀC; IR (KBr) t (cmꢁ1): 1685 (C¼O), 1614 (C¼N); MS
(EI) m/z: 427 (Mþ, 20.7%), 1H NMR (DMSO-d6, 300 MHz) d (ppm):
2.27 (s, 3H, CH3), 3.2 (s, 3H, S–CH3), 3.4 (s, 3H, N–CH3), 7.0–7.9
(m, 11H, Ar–H), 8.2 (s, 1H, C–2H); Anal. Calcd for C24H21N5OS
(427.3): C, 67.45; H, 4.92; N, 16.39%. Found: C, 67.69; H, 4.67;
N, 16.55%.
Male albino Wister rats (200–250 g) were designated for this study.
Diabetes was prompted in the rats by intraperitoneally (i.p.) inject-
ing freshly prepared solution of Streptozotocin (STZ) (Sigma-
Aldrich, Co., MO; catalogue number: 1001062761) in ice cold 0.1 M
citrate buffer (pH 4.5)37 at a dosage of 50 mg/kg bw38. The blood
glucose of each animal was tested after 48 h and animals display-
ing fasting blood glucose level ꢃ200 mg/dl were elected39. These
diabetic rats were unsystematically scattered into groups consist-
ing of six animals in each.
7-(3,4-dichlorophenyl)-4-methylsulfanyl-5,6-diphenyl-pyrrolo[2,3-
d]pyrimidine (VId). Yield: 41%; m.p.: 183–185 ꢀC; IR (KBr) t (cmꢁ1):
1596 (C¼N), MS (EI) m/z: 385 (Mþ, 22%), 387 (Mþþ2, 7.3%), 389
(Mþþ4, 2.6%), 1H NMR (DMSO-d6, 300 MHz) d (ppm): 3.3 (s, 3H,
S–CH3), 6.8–7.8 (m, 9H, Ar–H), 8.3 (s, 1H, C–2H); Anal. Calcd for
C19H13N3SCl2 (385.32): C, 59.22; H, 3.38; N, 10.91%. Found: C, 59.61;
H, 3.56; N, 11.13%.
Experimental design
Five groups (eight rats each) were used to investigate the anti-
hyperglycemic effect of the derivatives which showed promising
anti-hyperglycemic effect in SLM (compounds IIIa, Va and IIIb).
Group 1: diabetic control and Group 2: diabetic and Glimepiride
(Amaryl) (4 mg/kg) served as a reference anti-diabetic drug. Groups
3–5 were given the various pyrrole derivatives (compounds IIIa,
Va and IIIb). The treated groups administered the standard drug
(Amaryl) and different derivatives orally. For each group, blood
samples were collected by tail nipping and blood glucose level
was estimated at 0, 1, 2, 4 and 6 h after oral administration of the
tested compounds using glucometer (Gluco Dr Super Sensor, All
Medicus Co., Ltd., Anyang, Gyeonggi, Korea).
Biological screening
Animals
The complete progress of the experiment was conducted using
male Wistar albino rats (200–250 g), delivered by the Institutional
Breeding House, Egypt, reared and maintained in the animal
house of the institution. The animals had free access to food and
water ad libitum and maintained in a controlled environment
Statistical analysis
under standard conditions of temperature and humidity with an Data were represented as mean area under curve (AUC) SD.
alternating 12 h light and dark cycle for about a week for acclima- Significant differences between groups was tested using GraphPad
tisation. The protocol of the study was approved by the Animal InStat (Graph software Inc., V 3.05, Ralph Stahlman, Purdue
Ethics Committee of the Faculty of Pharmacy, Helwan University University, Lafayette, IN). Appropriate graphs were plotted using
on November 2016. The study was conducted in accordance with Microsoft Excel 2016. p Value less than .05 was considered statis-
the EC, directive 86/609/EEC for animal experiments.
tically significant.

814
S. S. FATAHALA ET AL.
normoglycemic rats compared to the sucrose-loaded untreated
control, as revealed in Figure 5. From those active derivatives,
treatment of derivatives IIIa, IIIb and Va only to STZ model of dia-
betes caused lowering on the blood glucose profile to the average
Discussion
Chemistry
The synthetic route to compounds Ia–d was reported in our previ-
ous work40–42. Amino-cyano-pyrroles 1 were reacted with HCO2H
to produce pyrrolopyrimidin-4-ones 2, which on react with POCl3,
250.00
4-chloro-pyrrolopyrimidines
3 were obtained in good yield.
214.39
4-Chloro derivatives 3 on react with thiourea adapted to pyrrolo-
pyrimidin-4-thiones I, To date, and to the best of our knowledge,
formation of the 4-thione analogues has been reported numer-
ously in literature29,32,43,44; but not mechanistically explained.
Herein, the proposed mechanism of the reaction was believed to
proceed via initial nucleophilic attack by the thiol group of thio-
urea on C-4 of the pyrimidine ring with proton transfer to N-3 and
the formation of the potentially unstable intermediate [A]. This
intermediate lose carbodiimide and HCl to give the pyrrolopyrimi-
din-4-thione, as revealed (Figure 4).
200.00
150.00
100.00
50.00
0.00
185.94
144.67
100.00
Amaryl
For preparation of spiro-pyrrolopyrimidines III–V; pyrrolopyrimi-
din-4-ylidene-malononitrile IIa–d was accomplished by the reac-
tion of Ia–d with malononitrile in absolute ethanol using same
procedure reported on our previous work45. On treat thione deriv-
atives II with acetylacetone guanidine hydrochloride and/or hydra-
zine hydrate in ethanol, containing catalytic amount of
pyridine, the corresponding spiro-pyrrolopyrimidine derivatives of
pyrazole, pyrimidine or pyran III–V were afforded in good yield, as
revealed in Scheme 1. All novel compounds were confirmed with
IIIa
Va
IIIb
Figure 5. Potency of anti-hyperglycemic derivatives compared to Amaryl.
Table 1. Assessment of various treatments on oral glucose tolerance and blood
glucose lowering activity in sucrose load model and diabetic rats.
Reduction in blood
glucose compared
to control (%)
Mean (AUC SEM)
STZ
Tested
1
compounds
SLM
SLM STZ
spectroscopic analysis (MS, IR, H NMR and microanalysis).
***
*
***
**
*
Amaryl
IIa
88.2 5.3
191.6
177 12.2
214.4 5.2
1373.64 49.55
60.97
15.18
21.54
NA
12.32
12.09
NA
NA
**
3
NA
NA
—
**
**
IIb
IId
Biological activities
—
**
*
*
*
**
*
*
*
IIIa
IIIb
IIIc
IIId
IVa
Va
Vb
Vc
VIa
ꢄ
198.2 3.1
193.2 2.6
222.3 8.6
203.8 6.5
223.3 7.7
195.9 2.2
207.4 4.6
220 12.8
204 9.2
ꢄꢄꢄ
1289.16 90.16
1157.5 102.31
17.49
25.92
—
Twelve of synthesised spiro-pyrrolopyrimidines and pyrrolopyrimi-
dine-4-one were evaluated for their anti-hyperglycemic activity
using both sucrose load model and streptozotocin models of dia-
betes7,36,46–49. The synthesised compounds were assessed for their
anti-hyperglycemic activity, which is comparable to Glimepiride
(Amaryl) the standard anti-hyperglycemic drug, by comparing the
mean area under the curve (AUC) for the blood glucose level
between the different studied groups.
14.52
—
—
—
NA
NA
NA
—
—
**
*
**
*
1211.20 89.86
13.3
22.48
—
—
—
—
NA
NA
NA
—
—
ꢄꢄ
p < .01 and
p < .05,
group (active compounds).
p < .001 significant different compared to control
Among the 12 tested compounds; five compounds showed sig-
nificant improvement (12.32%, 13.3%, 14.52%, 15.18% and 21.54%,
respectively) on oral glucose tolerance post-sucrose-loaded
Values were expressed as mean SEM. Using parametric unpaired t-test. NA: not
active; SLM: sucrose-loaded model; STZ: streptozotocin model of diabetes.
S
O
Cl
Ph
Ph
Ph
CN HCO2H
Ph
NH
NH
N
(NH2)2C=S
POCl3
NH2
1
X
N
N
X
X
N
X
N
N
N
N
Ar
I a-d
2
Ar
Ar
3
Ar
Ar; Benzyl , dichlorophenyl, Antipyrine
X; H, Ph
Cl
N
N
S
Ph
N
Cl
NH
Ph
NH
S
-H+
S
Ph
-(HN)2C
-HCl
+
X
+H+
N
N
N
X
H2N
NH2
N
N
X
Ar
N
Ar
Ar
[A]
Figure 4. Synthetic and mechanistic pathway for preparation of Pyrrolopyrimidine-4-thione Ia–d.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
815
COCH3
NH2
NH2
CN
N
H3C
[IIIa,b] showed higestactivity
[Va] Showed significant activity
over Thio but less than Pyrano
{Ar; Benzyl ring}
over all tested compounds
N
O
CN
S
Spiro
Ph
Ar
[IIa,b[
H
Decrease activity over Spiro
rings(pyrazolo or pyrano)
Replace Ph qp by H
cause loss activity
NH
Ph
N
N
Inactive, loss activity over
Antipyrinyl or Benzyl
[IIIa]
benzyl showed significant
activity, {spiro; pyrano}
Cl
Cl
H3C
Most Active Compounds for Both SLM and STZ Test
N
O
H3C
N
[IIIb]
NH2
H3COC
antipyrinyl showed the highest
activity {Spiro; pyrano ring}
Ph
N
NH2
H3C
Ph
Ph
N
CN
O
CN
NH
Ph
NH
Ph
N
N
N
N
IIIb
Benzyl
>>
Va
Antipyrinyl
Figure 6. Active compounds structural analysis and discussion.
of (17.49%, 22.48% and 25.92%, respectively) compared to the dia-
betic control group, as depicted in Table 1.
Disclosure statement
We wish to declare that there are no recognised conflicts of inter-
est connected with this publication and there has been no remark-
able financial funding for this work that could have influenced its
outcome. We authorise that the manuscript has been read and
approved by all named authors, and that there are no other per-
sons who fulfilled the standards for authorship but are not listed.
Comparing the anti-hyperglycemic activity of these compounds
to that of the reference anti-diabetic drug (Amaryl), compounds
IIIa, Va and IIIb showed significant decrease in the blood glucose
level (144.67%, 185.94% and 214.39%, respectively) compared to
the activity of Amaryl, as shown in Figure 6. Studying these anti-
hyperglycemic derivatives IIIa, Va and IIIb showed that the rats
survived and showed no toxicity symptoms, as revealed in
Table 1.
Active compounds were classified into two main sets: first, the
4-malononitrile derivative of pyrrolopyrimidines, namely, IIa,b (Ar
¼ benzyl and anti-pyrine). Also, the spiro derivatives containing
pyrane ring IIIa,b, spiro-containing pyrazole ring Va.
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