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. 2014 Jun 26;2014:249780. doi: 10.1155/2014/249780

Synthesis and Evaluation of Novel Pyrroles and Pyrrolopyrimidines as Anti-Hyperglycemic Agents

M S Mohamed 1, S A Ali 2, D H A Abdelaziz 2, Samar S Fathallah 1,*
PMCID: PMC4099110  PMID: 25054134

Abstract

A series of pyrrole and pyrrolopyrimidine derivatives were examined for their in vivo antihyperglycemic activity. Compounds Ia–c,e, and IVg showed promising antihyperglycemic activity equivalent to a well-known standard antihyperglycemic drug, Glimepiride (Amaryl, 4 mg/kg). In this paper, we examine and discuss the structure-activity relationships and antihyperglycemic activity of these compounds.

1. Introduction

For several decades, interest in pyrrole derivatives increases due to their pharmaceutical importance [1–3], such as antimicrobial [4–8], antiviral [9, 10], anti-inflammatory [11–13], analgesic [14], antitumor [15, 16], antihyperlipidemic [17], anticonvulsant [18], and antihyperglycemic agents [19, 20], as shown in Figures 1 and 2(a).

Figure 1.

Figure 1

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Pyrrole as valuable leads in the drug discovery field.

Figure 2.

Figure 2

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(a) Pyrroles as antihyperglycemic agents [19, 20], (b) Amaryl, standard antihyperglycemic drug [21, 22], and approved DPP-IV inhibitors [23–25] as type 2 diabetes medications containing a pyrrole moiety. (c) Thieno and Pyrrolo-pyrimidines as DPP-IV inhibitors.

Likewise, the key roles played by purines and pyrimidines in cellular processes have made them valuable lead for drug discovery; among these, pyrrolo[3,2-d]pyrimidines, a class of 7-deazapurine analogs, exhibit interesting biological activity in part due to their resemblance to pyrimidines and purines. These huge therapeutic applications have motivated new efforts in the search for novel derivatives with improved biological activity and diverse applications in the pharmaceutical industry [1–4, 19, 20].

Diabetes mellitus (DM) is a severe metabolic disorder that has a significant impact on the health and quality of patients' life. Treatment of diabetic patients has been focused on dietary management and oral antidiabetics, among these: sulfonylureas, metformin, acarbose, and others. However, some of the currently used antihyperglycemic have several adverse side effects like hepatotoxicity, weight gain, and hypoglycemia.

This situation emphasized the need to develop novel antihyperglycemic agents [21]. Glimepiride (Amaryl) is a sulfonylurea containing a pyrrole group, acting as antihyperglycemic drug [22]. It is sometimes classified either as the first third-generation sulfonylurea or as second-generation. Glimepiride is indicated to treat type 2 diabetes mellitus; its mode of action is to increase insulin production by the pancreas, as shown in Figure 2(b).

Recently, dipeptidyl peptidase IV (DPP-IV) inhibitors [23–25] have been shown to be effective and safe compounds that control blood glucose. Improvement of the inhibitory activity and chemical stability of a series of substituted piperidinyl glycine 2-cyano-4,5-methano pyrroline (DPP-IV) inhibitors was, respectively, achieved by the introduction of pyrroline moiety at the 4 position and 1 position of the piperidinyl glycine, leading to a series of potent and stable DPP-IV inhibitors [25]. Two important DPP-IV inhibitors, having a pyrrole and fused pyrrole, vildaglipin, and saxagliptin [24, 25], are on the market in many countries, as shown in Figure 2(b).

A highly potent DPP-IV inhibitor thienopyrimidine was also reported [24]. While trying to maintain consistency of in vitro and in vivo biological activity, a simple scaffold replacement of thienopyrimidine with pyrrolopyrimidine lead to significantly improved metabolic stability [22–24], as shown in Figure 2(c).

Motivated by the importance of this system and in continuation of our research efforts [26–30], we try to highlight aspects reported on the chemistry of some newly synthesized pyrrole and pyrrolopyrimidine derivatives and evaluate them for the antihyperglycemic activities. The synthetic pathways adopted for the synthesis of these compounds are registered in Schemes 1–3.

Scheme 1.

Scheme 1

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Synthetic pathways for compounds Ia–o: reagents and conditions: (1) pyridine/benzene; (2) CH2(CN)2; (3) NaHCO3/EtOH; (4) CH2(CN)2/NaOEt; (5) TEOF; or (6) Ac2O.

Scheme 3.

Scheme 3

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Synthetic pathways for compounds VII–VIII: reagents and conditions: (1) NaNO2/HCl/Stirring (75 min); (2) NCCH2Y/CH3CO2NH4/EtOH; and (3) N2H4·H2O.

2. Materials and Methods

2.1. Chemistry

All melting points were uncorrected and measured using Electrothermal IA 9100 apparatus (Shimadzu, Japan). IR spectra were recorded as potassium bromide pellets on a Perkin-Elmer 1650 spectrophotometer (USA), Faculty of Science, Cairo University, Cairo, Egypt. 1H NMR and 13CNMR spectra were performed on JOEL NMR FXQ-300 MHz and JOELNMR FXQ-500 MHz spand chemical shifts were expressed as ppm against TMS as internal reference (Faculty of Science, Cairo University, Cairo, Egypt). Mass spectra were recorded at 70 eV EI Ms-QP 1000 EX (Shimadzu, Japan), Faculty of Science, Cairo University, Cairo, Egypt. Microanalyses were operated using Vario, Elementar apparatus (Shimadzu, Japan), Organic Microanalysis Unit, Faculty of Science, Cairo University, Cairo, Egypt. Column chromatography was performed on (Merck) silica gel 60 (particle size 0.06–0.20 mm). Compounds Ia–c, f–h, k–m, IIa–c, f–h, IIIa–c, f–h, IVa–c, Va–c, VIa–c, VIIa–c,and VIIIa–c were prepared as reported in the literature [26–31]. All new compounds yielded spectral data consistent with the proposed structure and microanalysis within ± 0.4% of the theoretical values.

2.1.1. 2-Amino-1-(3,4-dichlorophenyl)-4,5-diphenyl-1H-pyrrole-3-carbonitrile Id (Scheme 1)

A mixture of benzoin (2 g, 0.01 mol), 3,4-dichlorophenyl amine (1.6 g, 0.01 mol) in dry benzene (50 mL), was kept at 80°C for 9 h. The reaction mixture was cooled; then malononitrile (0.66 mg, 0.01 mol) was added, followed by catalytic amount of pyridine (2 mL) portion wise and left to reflex till solid formed. The solvent was evaporated under reduced pressure and the residue was recrystallized from methanol to give Id. Yield: 45%; M.P. 118–122°C; 1H NMR (DMSO-d 6, 300 MHz) δ (ppm): 5.21 (br.s, 2H, NH2, D2O exchangeable), 7.0–7.8 (m, 13H, Ar-H); 13C NMR (DMSO-d 6): δ 114.33, 118.24, 119.37, 125.8, 126.18, 127.80, 128.45, 129.84, 130.29, 132.16, 132.70, 133.62, 134.96, 142.05 ppm; IR (KBr) υ (cm−1): 3410, 3370 (NH2), 2220 (C≡N); MS (EI) m/z: 403 (M+, 14%), 405 (M++ 2, 8.75%), 407 (M++ 4, 1.1%); Anal. Calcd for C23H15Cl2N3 (403.06): C, 68.33; H, 3.74; Cl, 17.54; N, 10.39%. Found: C, 68.55; H, 3.92; Cl, 17.23; N, 10.72%.

2.1.2. 2-Amino-1-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H -pyrazol-4-yl)-4-phenyl-1H -pyrrole-3-carbonitrile Ie (Scheme 1)

1,5-Dimethyl-4-(2-oxo-2-phenylethylamino)-2-phenyl-1H-pyrazol-3(2H)-one [27–30](3.22 g, 0.01 mol) was dissolved in dry ethanol (20 mL); then malononitrile (0.66 g, 0.01 mol) was added, followed by sodium ethoxide (0.01 mol) portion wise, and left to reflux till solid formed. The solvent was evaporated under reduced pressure and the residue was recrystallized from methanol to give Ie.Yield: 66%; M.P. 163–166°C; 1H NMR (DMSO-d 6, 300 MHz) δ (ppm): 2.43 (s, 3H, CH3), 3.12 (s, 3H, N-CH3), 6.13 (br.s, 2H, NH2, D2O exchangeable), 6.8–7.8 (m, 10H, Ar-H and 1H, C6-H); IR (KBr) υ (cm−1): 3410, 3350 (NH2), 2210 (C≡N), 1703 (C=O); MS (EI) m/z: 369 (M+, 23%), 370 (M++ 1, 6.1%); Anal. Calcd for C22H19N5O (369.16): C, 71.53; H, 5.18; N, 18.96; O, 4.33%. Found: C, 71.55; H, 5.26; N, 18.70; O, 3.05%.

2.1.3. 2-Ethoxymethylenamino-1,5-disubstituted-4-phenyl-1H -pyrrole-3-carbonitriles Ii,j (Scheme 1)

Compound I, d, or e (0.01 mol) in triethyl orthoformate (20 mL) was refluxed for 9 h. The solvent was removed under reduced pressure and the residue was recrystallized from methanol/water to give the target compounds Ii, j.

2.1.4. Ethyl N-3-Cyano-1-(3,4-dichlorophenyl)-4,5-diphenyl-1H -pyrrol-2-ylformimidate Ii

Yield: 60%; M.P. 120–122°C; 1H NMR (DMSO-d 6, 300 MHz) δ (ppm): 1.30 (t, 3H, J = 7.1 Hz, CH3), 4.2 (q, 2H, J = 7.1 Hz, OCH2), 6.9–7.8 (m, 14H, Ar-H and N=CH); IR (KBr) υ (cm−1): 3070, 2900(CH), 2310(CN), 1620 (C=C), 1560 (C=N); MS (EI) m/z: 460 (M+, 13.5%), 462 (M++ 2, 8.5%), 464 (M++ 4, 2.71%); Anal. Calcd for C26H19Cl2N3O (460.35): C, 67.83; H, 4.16; Cl, 15.40; N, 9.13; O, 3.48%, Found: C, 68.03; H, 4.11; Cl, 15.63; N, 8.91; O, 3.69%.

2.1.5. Ethyl N-3-Cyano-1-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H -pyrazol-4-yl)-4-phenyl-1H -pyrrol-2-ylacetimidate Ij

Yield: 56%; M.P. 120–122°C; 1H NMR (DMSO-d 6, 300 MHz) δ (ppm): 1.20 (t, 3H, J = 7.2 Hz, CH3), 2.41 (s, 3H, CH3), 3.1 (s, 3H, N-CH3), 4.01 (q, 2H, J = 7.1 Hz, OCH2), 6.9–7.8 (m, 12H, Ar-H, C6-H and N=CH); IR (KBr) υ (cm−1): 3030, 2910(CH), 2240(CN), 1610 (C=C), 1570 (C=N); MS (EI) m/z: 425 (M+, 6.5%), 426 (M++ 1, 1.81%); Anal. Calcd for C26H23N5O2 (425.19): C, 70.57; H, 5.45; N, 16.46; O, 7.52%. Found: C, 70.21; H, 5.17; N, 16.12; O, 7.18%.

2.1.6. N-(4-Phenyl-1,3,5-trisubstituted-1H -pyrrol-2-yl)-acetamides I n,o (Scheme 1)

Compound I, d, or e (0.01 mol) in acetic anhydride (30 mL) was refluxed for 2 h, cooled, poured onto ice water, neutralized with ammonia to give a precipitate which was filtered off, dried, and recrystallized from methanol to give the target compounds In, o.

2.1.7. N-(3-Cyano-1-(3,4-dichlorophenyl)-4,5-diphenyl-1H -pyrrol-2-yl) acetamide In

Yield: 66%; M.P. 135–138°C; 1H NMR (DMSO-d 6, 300 MHz) δ (ppm): 2.23 (s, 3H, CO-CH3), 7.0–7.8 (m, 13H, Ar-H), 9.5 (s, 1H, NH, D2O exchangeable); IR (KBr) υ (cm−1): 3430 (NH), 2330 (C≡N), 1710 (C=O); MS (EI) m/z: 445 (M+, 13.1%), 447 (M+ + 2, 7.9%), 449 (M+ + 4, 0.91%); Anal. Calcd for C25H17Cl2N3O (445.07): C, 67.28; H, 3.84; Cl, 15.89; N, 9.41; O, 3.58%. Found: C, 67.47; H, 4.06; Cl, 16.22; N, 9.57; O, 3.66%.

2.1.8. N-(3-Cyano-1-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H -pyrazol-4-yl)-4-phenyl-1H -pyrrol-2-yl)acetamide Io

Yield: 72%; M.P. 135–138°C; 1H NMR (DMSO-d 6, 300 MHz) δ (ppm): 2.23 (s, 3H, CO-CH3), 2.43 (s, 3H, CH3), 3.12 (s, 3H, N-CH3), 7.0–7.8 (m, 11H, Ar-H and C6-H), 9.4 (s, 1H, NH, D2O exchangeable); IR (KBr) υ (cm−1): 3350 (NH), 2310 (C≡N), 1715, 1705 (C=O); MS (EI) m/z: 411 (M+, 15.4%), 412 (M++ 1, 3.73%); Anal. Calcd for C24H21N5O2 (411.17): C, 70.06; H, 5.14; N, 17.02; O, 7.78%. Found: C, 70.37; H, 5.45; N, 17.34; O, 7.95%.

2.1.9. 7-(2,4-Dimethyl-5-oxo-1-phenyl-2,5-dihydro-1H -pyrazol-3-yl)-5,6-diphenyl -3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one IId,e (Scheme 2)

Scheme 2.

Scheme 2

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Synthetic pathways for compounds I–VI: reagents and conditions: (1) HCO2H; (2) AcOH/HCl; (3) HCONH2; (4) NH2CSNH2; (5) POCl3; (6) N2H4·H2O; and (7) NH2CSNH2.

Compound Id or e (0.01 mol) in formic acid (20 mL, 85%) was refluxed for 12 h. The reaction mixture was cooled, poured onto ice/water to give a precipitate which was filtered, dried, and recrystallized from ethanol to give the target compounds IId, e.

7-(3,4-Dichlorophenyl)-5,6-diphenyl-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one IId. Yield: 56%; M.P. 172–176°C; 1H NMR (DMSO-d 6, 300 MHz) δ (ppm): 6.9–7.8 (m, 13H, Ar-H), 8.4 (s, 1H, C2-H), 12.40 (s, 1H, NH, D2O exchangeable): 13C NMR (DMSO-d 6): δ118.24, 119.37, 125.8, 126.18, 127.80, 128.45, 129.84, 130.29, 132.16, 132.70, 133.62, 134.96, 138.25, 142.05, 146.2, 163.15 ppm; IR (KBr) υ (cm−1): 3410, 3350 (NH2), 1720, 1705 (C=O), 1550(C=N); MS (EI) m/z: 432 (M+, 21.5%), 434 (M++ 2, 13.9%), 436 (M++ 4, 4.2%); Anal. Calcd for C24H15Cl2N3O (432.30): C, 66.68; H, 3.50; Cl, 16.40; N, 9.72; O, 3.70%. Found: C, 66.32; H, 3.18; Cl, 16.21; N, 9.50; O, 3.46%.

7-(1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-5-phenyl-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one IIe. Yield: 65%; M.P. 205–208°C; 1H NMR (DMSO-d 6, 300 MHz) δ (ppm): 2.41 (s, 3H, CH3), 3.21 (s, 3H, N-CH3), 6.9–7.8 (m, 11H, Ar-H and C6-H), 8.32 (s, 1H, C2-H), 12.32 (s, 1H, NH, D2O exchangeable); IR (KBr) υ (cm−1): 3410, 3320 (NH2), 1720, 1680 (C=O), 1560(C=N) and disappearance of the CN group; MS (EI) m/z: 379 (M+, 23.5%), 380 (M++ 1, 7.1%); Anal. Calcd for C23H19N5O2 (379.43): C, 69.51; H, 4.82; N, 17.62; O, 8.05%. Found: C, 69.89; H, 5.16; N, 17.97; O, 8.41%.

2.1.10. 7-Disubstituted-2-methyl-5-phenyl-7H-pyrrolo[2,3-d]pyrimidin-4(3H)-one IIi, j (Scheme 2)

Compound Id or e (0.01 mol) in acetic acid/HCl (3 : 1) (30 mL) was refluxed for 12 h. The reaction mixture was cooled, poured onto ice/water, neutralized with ammonia to give a precipitate which was filtered, dried, and recrystallized from methanol to give the target compounds II i, j.

7-(3,4-Dichlorophenyl)-2-methyl-5,6-diphenyl-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one II i. Yield: 67%; M.P. 200–202°C; 1H NMR (DMSO-d 6, 300 MHz) δ (ppm): 2.35 (s, 3H, C2-CH3), 6.9–7.8 (m, 13H, Ar-H), 12.40 (s, 1H, NH, D2O exchangeable); IR (KBr) υ (cm−1): 3430, 3330 (NH2), 1710 (C=O), 1570 (C=N); MS (EI) m/z: 445 (M+, 26.2%), 447 (M++ 2, 15.3%), 449 (M++ 4, 1.04%); Anal. Calcd for C25H17Cl2N3O (445.07): C, 67.28; H, 3.84; Cl, 15.89; N, 9.41; O, 3.58%. Found: C, 67.45; H, 4.11; Cl, 16.22; N, 9.74; O, 3.82%.

7-(1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-2-methyl-5-phenyl-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one II j. Yield: 67%; M.P. 225–227°C; 1H NMR (DMSO-d 6, 300 MHz) δ (ppm): 2.32 (s, 3H, C2-CH3), 2.41 (s, 3H, CH3), 3.22 (s, 3H, N-CH3), 7–7.8 (m, 11H, Ar-H and C6-H), 12.36 (s, 1H, NH, D2O exchangeable); IR (KBr) υ (cm−1): 3430, 3330 (NH2), 1730, 1700 (C=O), 1560 (C=N); MS (EI) m/z: 487 (M+, 45%), 488 (M++ 1, 15.2%); Anal. Calcd for C24H21N5O2 (411.46): C, 70.06; H, 5.14; N, 17.02; O, 7.78%. Found: C, 70.24; H, 5.32; N, 17.31; O, 7.96%.

2.1.11. 6,7-Disubstituted-5-phenyl-7H-pyrrolo[2,3-d]pyrimidin-4-ylamines IIId, e (Scheme 2)

A suspension of the appropriate aminopyrrole I, d, or e (0.01 mol) and formamide (30 mL, 0.066 mol) were heated under reflux for 9 h, cooled, poured onto ice/water to give precipitates which were filtered off, dried, and recrystallized from ethanol to give the target compounds III d, e.

7-(3,4-Dichlorophenyl)-5,6-diphenyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine III d. Yield: 63%; M.P. 115–118°C; 1H NMR (DMSO-d 6, 300 MHz) δ (ppm): 5.4 (brs, 2H, NH2, D2O exchangeable), 7.1–7.7 (m, 13H, Ar-H), 8.2 (s, 1H, C2-H); IR (KBr) υ (cm−1): 3440, 3350 (NH2), 1570 (C=N), 1610 (C=C); MS (EI) m/z: 430 (M+, 31.2%), 432 (M++ 2, 17.6%), 434 (M++ 4, 0.98%); Anal. Calcd for C24H16Cl2N4 (430.08): C, 66.83; H, 3.74; Cl, 16.44; N, 12.99%. Found: C, 67.07; H, 5.11; Cl, 16.81; N, 13.36%.

4-(4-Amino-5-phenyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one III e. Yield: 69%; M.P. 152–155°C; 1H NMR (DMSO-d 6, 300 MHz) δ (ppm): 2.41 (s, 3H, CH3), 3.14 (s, 3H, N-CH3), 5.8 (brs, 2H, NH2, D2O exchangeable), 7.1–7.8 (m, 11H, Ar-H, and C6-H), 8.3 (s, 1H, C2-H); IR (KBr) υ (cm−1): 3430, 3330 (NH, NH2), 1705 (C=O), 1600 (C=C), 1540 (C=N); MS (EI) m/z: 396 (M+, 13.7%), 397 (M++ 1, 1.91%); Anal. Calcd for C23H20N6O (396.44): C, 69.68; H, 5.08; N, 21.20; O, 4.04%. Found: C, 69.42; H, 4.92; N, 20.89; O, 4.32%.

2.1.12. 5-(5,6-Diphenyl-4-thioxo-3H-pyrrolo[2,3-d]pyrimidin-7(4H)-yl)-1,4-dimethyl-2-phenyl-1H -pyrazol-3(2H)-one IIIi, j (Scheme 2)

Compound I, d, or e (0.01 mol) and thiourea (1.2 g, 0.02 mol) were refluxed in dry ethanol (20 mL) for 12 h. The reaction mixture was evaporated under reduced pressure and the residues were recrystallized from methanol to give the target compounds III i, j.

4-Amino-7-(3,4-dichlorophenyl)-5,6-diphenyl-1H-pyrrolo[2,3-d] pyrimidine-2(7H)-thione IIIi. Yield: 70%; M.P. 90–95°C; 1H NMR (DMSO-d 6, 300 MHz) δ (ppm): 6.62 (s, 2H, NH2, D2O exchangeable), 7.1–7.9 (m, 13H, Ar-H), 9.22 (s, 1H, NH, D2O exchangeable); IR (KBr) υ (cm−1): 3440, 3340 (NH, NH2), 1610 (NH–C=S); MS (EI) m/z: 463 (M+, 22.36%), 465 (M++ 2, 14.23%), 467 (M+ + 4, 4.54%); Anal. Calcd for C24H16Cl2N4S (463.38): C, 62.21; H, 3.48; Cl, 15.30; N, 12.09; S, 6.92%. Found: C, 62.45; H, 3.62; Cl, 15.49; N, 12.41; S, 7.21%.

4-(4-Amino-5-phenyl-2-thioxo-1H-pyrrolo[2,3-d]pyrimidin-7(2H)-yl)-1,5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one IIIj. Yield: 74%; M.P. 172–174°C; 1H NMR (DMSO-d 6, 300 MHz) δ (ppm): 2.34 (s, 3H, CH3), 3.11 (s, 3H, N-CH3), 6.51 (brs, 2H, NH2, D2O exchangeable), 7.1–7.8 (m, 11H, Ar-H, and C6-H), 8.3 (s, 1H, C2-H), 8.92 (s, 1H, NH, D2O exchangeable); IR (KBr) υ (cm−1): 3440, 3370 (NH, NH2), 1610 (C=C), 1600 (NH–C=S); MS (EI) m/z: 428 (M+, 14.8%), 429 (M+ + 1, 2.31%); Anal. Calcd for C23H20N6OS (428.51): C, 64.47; H, 4.70; N, 19.61; O, 3.73; S, 7.48%. Found: C, 64.78; H, 4.97; N, 19.92; O, 3.91; S, 7.62%.

2.1.13. General Procedure for the Preparation of 4-Chloropyrrolopyrimidines IVd–j (Scheme 2)

The appropriate compound II (0.01 mol) was refluxed in phosphorus oxychloride (30 mL) for 12 h. The solution was cooled and poured with stirring onto ice/water and the formed precipitated was filtered, washed several times with water, dried, and recrystallized from ethanol to give the target compounds IVd–j.

4-Chloro-7-(3,4-dichlorophenyl)-5,6-diphenyl-7H-pyrrolo[2,3-d]pyrimidine IVd. Yield: 76%; M.P. 124–128°C; 1H NMR (DMSO-d 6, 300 MHz) δ (ppm): 6.9–7.8 (m, 13H, Ar-H), 8.6 (s, 1H, C2-H); 13C NMR (DMSO-d 6): δ117.5, 118.8, 126.18, 127.80, 128.45, 129.84, 130.21, 132.46, 132.64, 133.76, 134.84, 138.27, 141.05, 150.2, 151.24, 153.8 ppm; IR (KBr) υ (cm−1): 3080, 2840(CH), 1612 (C=C), 1580 (C=N); MS (EI) m/z: 449 (M+, 29.98%), 451 (M+ + 2, 23.6%), 453 (M+ + 4, 6.9%), 455 (M+ + 6, 0.66%); Anal. Calcd for C24H14Cl3N3 (449.03): C, 63.95; H, 3.13; Cl, 23.60; N, 9.32%. Found: C, 64.23; H, 3.42; Cl, 23.91; N, 9.49%.

4-(4-Chloro-5-phenyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one IVe. Yield: 76%; M.P. 125–130°C; 1H NMR (DMSO-d 6, 300 MHz) δ (ppm): 2.43 (s, 3H, CH3), 3.12 (s, 3H, N-CH3), 6.9–7.8 (m, 11H, Ar-H and C6-H), 8.5 (s, 1H, C2-H); IR (KBr) υ (cm−1): 3080, 2840 (CH), 1730 (C=O), 1612 (C=C), 1580 (C=N); MS (EI) m/z: 415 (M+, 20%), 417 (M++ 2, 5.5%); Anal. Calcd for C23H18ClN5O (415.87): C, 66.43; H, 4.36; Cl, 8.52; N, 16.84; O, 3.85%. Found: C, 66.67; H, 4.71; Cl, 8.68; N, 16.94; O, 3.99%.

7-Benzyl-4-chloro-2-methyl-5,6-diphenyl-7H-pyrrolo[2,3-d]pyrimidine IVf. Yield: 46%; M.P. 120–124°C; 1H NMR (DMSO-d 6, 300 MHz) δ (ppm): 2.32 (s, 3H, CH3), 5.12 (s, 2H, Ph-CH2), 6.9–7.8 (m, 15H, Ar-H and C6-H); IR (KBr) υ (cm−1): 3080, 2840 (CH), 1610 (C=C), 1560 (C=N); MS (EI) m/z: 409 (M+, 14%), 411 (M+ + 2, 4.5%); Anal. Calcd for C26H20ClN3 (409.91): C, 76.18; H, 4.92; Cl, 8.65; N, 10.25%. Found: C, 76.47; H, 5.23; Cl, 8.96; N, 10.59%.

4-(4-Chloro-2-methyl-5,6-diphenyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one IVg. Yield: 42%; M.P. 158–160°C; 1H NMR (DMSO-d 6, 300 MHz) δ (ppm): 2.30–2.44 (s, 6H,2∗CH3), 3.13 (s, 3H, N-CH3), 6.9–7.8 (m, 15H, Ar-H); IR (KBr) υ (cm−1): 3080, 2840 (CH), 1710 (C=O), 1605 (C=C), 1577 (C=N); MS (EI) m/z: 505 (M+, 13%), 507 (M+ + 2, 2.5%); Anal. Calcd for C30H24ClN5O (505.17): C, 71.21; H, 4.78; Cl, 7.01; N, 13.84; O, 3.16%. Found: C, 71.02; H, 4.97; Cl, 7.23; N, 14.06; O, 3.45%.

4-Chloro-7-(3,4-dichlorophenyl)-2-methyl-5,6-diphenyl-7H-pyrrolo[2,3-d]pyrimidine IVh. Yield: 45%; M.P. 108–110°C; 1H NMR (DMSO-d 6, 300 MHz) δ (ppm): 2.36 (s, 3H, CH3), 7.2–7.8 (m, 13H, Ar-H); IR (KBr) υ (cm−1): 3080, 2840 (CH), 1600 (C=C), 1565 (C=N); MS (EI) m/z: 464 (M+, 11%), 466 (M+ + 2, 6.5%), 468 (M+ + 4, 2.3%), 394 (M+ + 6, 0.88%); Anal. Calcd for C25H16Cl3N3 (464.77): C, 64.61; H, 3.47; Cl, 22.88; N, 9.04%. Found: C, 64.99; H, 3.73; Cl, 23.08; N, 9.29%.

4-(4-Chloro-2-methyl-5-phenyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one IVi. Yield: 42%; M.P. 95-100°C; 1H NMR (DMSO-d 6, 300 MHz) δ (ppm): 2.28–2.46 (s, 6H, 2∗CH3), 3.27 (s, 3H, N-CH3), 7.0–7.8 (m, 11H, Ar-H and C6-H); IR (KBr) υ (cm−1): 3070, 2850 (CH), 1700 (C=O), 1600 (C=C), 1570 (C=N); MS (EI) m/z: 429 (M+, 16%), 431 (M+ + 2, 4.7%); Anal. Calcd for C24H20ClN5O (429.90): C, 67.05; H, 4.69; Cl, 8.25; N, 16.29; O, 3.72%. Found: C, 67.42; H, 5.08; Cl, 8.57; N, 16.65; O, 4.02%.

2.1.14. General Procedure for the Preparation of 4-Hydrazinopyrrolopyrimidines Vd–i (Scheme 2)

Method A. Compound IV (0.01 mol) and hydrazine hydrate (8 mL, 0.015 mol, 98%) were refluxed in dry ethanol (30 mL) for 12 h. The solvent was removed under reduced pressure and the residues were recrystallized from methanol to give the target compounds V.

Method B. Compounds Ii, j (0.01 mol) in dry toluene (20 mL) and hydrazine hydrate (5 mL, 0.015 mol, 98%) were added with stirring at room temperature for 14 h. The solvent was removed under reduced pressure, and the residue was recrystallized from methanol to give Vd, e; Compounds Vd, e prepared by this method are identical in all respects (physical and spectral data) to that prepared from Method A.

7-(3,4-Dichlorophenyl)-4-hydrazinyl-5,6-diphenyl-7H-pyrrolo[2,3-d]pyrimidine Vd. Yield: (A; 68%, B; 56%); M.P. 122–126°C; 1H NMR (DMSO-d 6, 300 MHz) δ (ppm): 4.9–5.2 (brs, 2H, NH2, D2O exchangeable), 7.1–7.8 (m, 14H, Ar-H and NH, D2O exchangeable), 8.3 (s, 1H, C2-H); 13C NMR (DMSO-d 6): δ117.45, 118.69, 126.21, 127.47, 128.4, 129.74, 130.2, 132.39, 132.64, 133.76, 134.84, 138.27, 141.05, 153.2, 153.8, 168.71 ppm; IR (KBr) υ (cm−1): 3420, 3350 (NH2) 3210 (NH), 1610 (C=C), 1580 (C=N); MS (EI) m/z: 446 (M+, 23%), 448 (M+ + 2, 14%), 450 (M+ + 4, 3.4%); Anal. Calcd for C24H17Cl2N5 (446.33): C, 64.58; H, 3.84; Cl, 15.89; N, 15.69%. Found: C, 64.95; H, 4.20; Cl, 16.23; N, 16.04%.

4-(4-Hydrazinyl-5-phenyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one Ve. Yield: (A) 71%, (B) 60%; M.P. 142–146°C; 1H NMR (DMSO-d 6, 300 MHz) δ (ppm): 2.42 (s, 3H, CH3), 3.13 (s, 3H, N-CH3), 4.8–5.1 (brs, 2H, NH2, D2O exchangeable), 7.1–7.8 (m, 11H, Ar-H, C6-H and NH, D2O exchangeable), 8.2 (s, 1H, C2-H); IR (KBr) υ (cm−1): 3440, 3330 (NH2) 3260 (NH), 1705 (C=O), 1600 (C=C), 1580 (C=N); MS (EI) m/z: 411 (M+, 28%), 412 (M+ + 1, 6.5%); Anal. Calcd for C23H21N7O (411.46): C, 67.14; H, 5.14; N, 23.83; O, 3.89%. Found: C, 67.52; H, 5.48; N, 24.15; O, 4.52%.

7-Benzyl-4-hydrazinyl-2-methyl-5,6-diphenyl-7H-pyrrolo[2,3-d] pyrimidine Vf. Yield: 67%; M.P. 147–152°C; 1H NMR (DMSO-d 6, 300 MHz) δ (ppm): 2.29 (s, 3H, CH3), 4.9–5.2 (brs, 2H, NH2, D2O exchangeable), 5.78 (s, 2H, Ph-CH2), 7.23–7.8 (m, 16H, Ar-H and NH, D2O exchangeable); IR (KBr) υ (cm−1): 3430, 3350 (NH2) 3250 (NH), 1605 (C=C), 1570 (C=N); MS (EI) m/z: 405 (M+, 29%), 406 (M+ + 1, 4.1%); Anal. Calcd for C26H23N5 (405.49): C, 77.01; H, 5.72; N, 17.27%. Found: C, 77.38; H, 6.05; N, 17.62%.

4-(4-Hydrazinyl-2-methyl-5,6-diphenyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one Vg. Yield: 68%; M.P. 185–188°C; 1H NMR (DMSO-d 6, 300 MHz) δ (ppm): 2.22–2.46 (s, 6H, 2∗CH3), 3.11 (s, 3H, N-CH3), 4.34–4.8 (brs, 2H, NH2, D2O exchangeable), 7.2–7.8 (m, 16H, Ar-H and NH, D2O exchangeable): IR (KBr) υ (cm−1): 3420, 3350 (NH2) 3240 (NH), 1700 (C=O), 1605 (C=C), 1560 (C=N); MS (EI) m/z: 501 (M+, 31%), 502 (M++ 1, 5.4%); Anal. Calcd for C30H27N7O (501.58): C, 71.84; H, 5.43; N, 19.55; O, 3.19%. Found: C, 72.20; H, 5.81; N, 19.92; O, 3.55%.

7-(3,4-Dichlorophenyl)-4-hydrazinyl-2-methyl-5,6-diphenyl-7H-pyrrolo[2,3-d]pyrimidine Vh. Yield: 59%; M.P. 142–146 °C; 1H NMR (DMSO-d 6, 300 MHz) δ (ppm): 2.25 (s, 3H, CH3), 4.5–4.9 (brs, 2H, NH2, D2O exchangeable), 7.3–7.8 (m, 14H, Ar-H, NH, D2O exchangeable); IR (KBr) υ (cm−1): 3440, 3360 (NH2) 3250 (NH), 1610 (C=C), 1560 (C=N); MS (EI) m/z: 460 (M+, 31%), 462 (M+ + 2, 18%), 464 (M+ + 4, 4.9%); Anal. Calcd for C25H19Cl2N5 (460.36): C, 65.22; H, 4.16; Cl, 15.40; N, 15.21%. Found: C, 65.57; H, 4.52; Cl, 15.78; N, 15.56%.

4-(4-Hydrazinyl-2-methyl-5-phenyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-1,5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one Vi. Yield: 69%; M.P. 148–150°C; 1H NMR (DMSO-d 6, 300 MHz) δ (ppm): 2.23–2.44 (s, 6H, 2∗CH3), 3.13 (s, 3H, N-CH3), 4.4–4.78 (brs, 2H, NH2, D2O exchangeable), 7.2–7.8 (m, 12H, Ar-H, C6-H and NH, D2O exchangeable); IR (KBr) υ (cm−1): 3430, 3360 (NH2) 3250 (NH), 1705 (C=O), 1600 (C=C), 1580 (C=N); MS (EI) m/z: 501 (M+, 31%), 502 (M+ + 1, 5.4%); Anal. Calcd for C24H23N7O (425.49): C, 67.75; H, 5.45; N, 23.04; O, 3.76%. Found: C, 68.12; H, 5.76; N, 23.37; O, 4.11%.

2.1.15. General Procedure for the Preparation of 4-Thienopyrrolopyrimidine VI d–f (Scheme 2)

Compound III (0.01 mol) and thiourea (1.2 g, 0.02 mol) were refluxed in dry ethanol (20 mL) for 14 h. The reaction mixture was evaporated under reduced pressure and the residues were recrystallized from methanol to give the target compounds VI.

7-(3,4-Dichlorophenyl)-5,6-diphenyl-3H-pyrrolo[2,3-d]pyrimidine-4(7H)-thione VId. Yield: 66%; M.P. 142–166°C; 1H NMR (DMSO-d 6, 300 MHz) δ (ppm): 7.3–7.8 (m, 13H, Ar-H), 9.02 (s, 1H, C2-H), 11.71 (s, 1H, NH, D2O exchangeable); IR (KBr) υ (cm−1): 3250 (NH), 1630 (NH–C=S), 1560 (C=N); MS (EI) m/z: 447 (M+, 28%), 449 (M+ + 2, 18%), 451 (M++ 4, 0.98%); Anal. Calcd for C24H15Cl2N3S (447.04): C, 64.29; H, 3.37; Cl, 15.81; N, 9.37; S, 7.15%. Found: C, 64.64; H, 3.74; Cl, 16.14; N, 9.73; S, 7.52%.

1,5-Dimethyl-2-phenyl-4-(5-phenyl-4-thioxo-3H-pyrrolo[2,3-d]pyrimidin-7(4H)-yl)-1H-pyrazol-3(2H)-one VIe. Yield: 61%; M.P. 142–166°C; 1H NMR (DMSO-d 6, 300 MHz) δ (ppm): 2.44 (s, 3H, CH3), 3.13 (s, 3H, N-CH3), 7.2–7.8 (m, 11H, Ar-H, and C6-H) 9.12 (s, 1H, C2-H), 12.10 (s, 1H, NH, D2O exchangeable); IR (KBr) υ (cm−1): 3230 (NH), 1700 (C=O), 1610 (NH–C=S), 1550 (C=N); MS (EI) m/z: 413 (M+, 30%), 414 (M+ + 1, 8.4%); Anal. Calcd for C23H19N5OS (413.49): C, 66.81; H, 4.63; N, 16.94; O, 3.87; S, 7.75%. Found: C, 67.16; H, 4.98; N, 17.31; O, 4.24; S, 8.12%.

7-Benzyl-2-methyl-5,6-diphenyl-3H-pyrrolo[2,3-d]pyrimidine-4(7H)-thione VIf. Yield: 47%; M.P. 165–167°C; 1H NMR (DMSO-d 6, 300 MHz) δ (ppm): 2.29 (s, 3H, CH3), 5.78 (s, 2H, Ph-CH2), 7.23–7.8 (m, 16H, Ar-H and NH, D2O exchangeable); IR (KBr) υ (cm−1): 3250 (NH), 1605 (NH–C=S), 1570 (C=N); MS (EI) m/z: 407 (M+, 26%), 408 (M++ 1, 3.91%), 409 (M++ 2, 0.81%); Anal. Calcd for C26H21N3S (407.53): C, 76.63; H, 5.19; N, 10.31; S, 7.87%. Found: C, 76.56; H, 5.11; N, 10.26; S, 7.80%.

2.1.16. General Procedure for the Preparation of Substituted Carbonohydrazonoyl Derivatives VII (Scheme 3)

A mixture of I (0.01 mol) in concentrated HCl (10 ml) was cooled with stirring to 0–5°C under ice, and cooled sodium nitrite solution (2.5 g in 10 mL of water) was added to it dropwise during 30 minutes. The reaction mixture was then stirred for 30 minutes. Without separation, an ice-cold mixture of active methylene compounds (malononitrile and/or ethyl cyanoacetate) (0.015 mol) and sodium acetate (4.10 g; 0.05 mole) in ethanol (50 mL) were added dropwise with stirring for 15 min. The stirring was continued for 30 minutes under ice and the reaction mixture was then left for 12 h at room temperature. The precipitate was filtered off and recrystallized from ethanol/H2O to give VII.

(3-Cyano-1-(3,4-dichlorophenyl)-4,5-diphenyl-1H-pyrrol-2-yl)carbon-hydrazonoyl dicyanide VIId. Yield: 56%; M.P. 102–106°C; 1H NMR (DMSO-d 6, 300 MHz) δ (ppm): 6.71 (s, 1H, NH, hydrazone, D2O exchangeable), 7.3–7.8 (m, 13H, Ar-H); IR (KBr) υ (cm−1): 3290 (NH), 2320 (C≡N), 1695 (C=O), 1585 (C=N); MS (EI) m/z: 481 (M+, 19%), 482 (M+ + 2, 12.8%), 483 (M+ + 4, 2.3%); Anal. Calcd for C26H14Cl2N6 (481.33): C, 64.88; H, 2.93; Cl, 14.73; N, 17.46%. Found: C, 64.67; H, 2.78; Cl, 14.51; N, 17.12%.

(3-Cyano-1-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-4-phenyl-1H-pyrrol-2-yl)carbonohydrazonoyl dicyanide VIIe. Yield: 51%; M.P. 110–114°C; 1H NMR (DMSO-d 6, 300 MHz) δ (ppm): 2.42 (s, 3H, CH3), 3.11 (s, 3H, N-CH3), 6.71 (s, 1H, NH, hydrazone, D2O exchangeable), 7.2–7.8 (m, 11H, Ar-H, and C6-H); IR (KBr) υ (cm−1): 3290 (NH), 2320 (C≡N), 1695 (C=O), 1585 (C=N); MS (EI) m/z: 446 (M+, 17.8%), 447 (M+ + 1, 8.21%); Anal. Calcd for C25H18N8O (446.46): C, 67.25; H, 4.06; N, 25.10; O, 3.58%. Found: C, 67.54; H, 4.12; N, 25.23; O, 3.69%.

Ethyl 2-(2-(1-Benzyl-3-cyano-4,5-diphenyl-1H-pyrrol-2-yl)hydrazono)-2-cyano-acetate VIIf. Yield: 48%; M.P. 125–130°C; 1H NMR (DMSO-d 6, 300 MHz) δ (ppm): 1.31 (t, 3H, J = 6.8, CH2-CH3∗), 4.4 (q, 2H, J = 6.8, O-CH2), 5.62 (s, 2H, Ph-CH2), 6.8 (s, 1H, NH, hydrazone, D2O exchangeable), 7.2–7.8 (m, 15H, Ar-H, and C6-H); IR (KBr) υ (cm−1): 3290 (NH), 2320 (C≡N), 1695 (C=O), 1585 (C=N); MS (EI) m/z: 473 (M+, 18%), 474 (M+ + 1, 5.1%); Anal. Calcd for C29H23N5O2 (473.53): C, 73.56; H, 4.90; N, 14.79; O, 6.76%. Found: C, 73.48; H, 4.64; N, 14.63; O, 6.70%.

2.1.17. General Procedure for the Preparation of Pyrazolyl Derivatives VIII (Scheme 3)

A mixture of compound VII (0.01 mol) and hydrazine hydrate (0.64 ml, 0.02 mole) in ethanol (30 mL) were heated under reflux for 8 h controlled by TLC. The solvent was concentrated and the reaction product was allowed to cool then pour on acidified ice/H2O. The product was filtered off, washed with water, dried, and recrystallized from ethanol to give VIII.

2-(2-(3,5-Diamino-4H-pyrazol-4-ylidene)hydrazinyl)-1-(3,4-dichloro phenyl)-4,5-diphenyl-1H-pyrrole-3-carbonitrile VIIId. Yield: 55%; M.P. 120–124°C; 1H NMR (DMSO-d 6, 300 MHz) δ (ppm): 6.5 (s, 4H, 2∗NH2, D2O exchangeable), 7.2–8 (m, 14H, Ar-H and NH, hydrazone, D2O exchangeable); IR (KBr) υ (cm−1): 3350–3280 (broad NH and NH2), 2320 (C≡N), 1695 (C=O), 1585 (C=N); MS (EI) m/z: 513 (M+, 12%), 515 (M+ + 2, 7.8%), 516 (M+ + 4, 2.4%); Anal. Calcd for C26H17Cl2N7O (513.09): C, 60.71; H, 3.33; Cl, 13.79; N, 19.06; O, 3.11%. Found: C, 60.98; H, 3.41; Cl, 13.95; N, 19.43; O, 3.27%.

2-(2-(3,5-Diamino-4H-pyrazol-4-ylidene)hydrazinyl)-1-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-4-phenyl-1H-pyrrole-3-carbonitrile VIIIe. Yield: 61%; M.P. 135–138°C; 1H NMR (DMSO-d 6, 300 MHz) δ (ppm): 2.42 (s, 3H, CH3), 3.11 (s, 3H, N-CH3), 6.48 (s, 4H, 2∗NH2, D2O exchangeable), 6.89 (s, 1H, NH, hydrazone, D2O exchangeable), 7.3–8 (m, 11H, Ar-H, and C6-H); IR (KBr) υ (cm−1): 3340–3290 (broad NH and NH2), 2320 (C≡N), 1695 (C=O), 1585 (C=N); MS (EI) m/z: 478 (M+, 15.2%), 479 (M+ + 1, 4.46%); Anal. Calcd for C25H22N10O (478.51): C, 62.75; H, 4.63; N, 29.27; O, 3.34%. Found: C, 62.64; H, 4.47; N, 29.02; O, 3.09%.

2-(2-(3-Amino-5-hydroxy-4H-pyrazol-4-ylidene)hydrazinyl)-1-benzyl-4,5-diphenyl-1H-pyrrole-3-carbonitrile VIIIf. Yield: 56%; M.P. 97–100°C; 1H NMR (DMSO-d 6, 300 MHz) δ (ppm): 5.61 (s, 2H, CH2), 6.45 (s, 4H, 2∗NH2, D2O exchangeable), 6.8 (s, 1H, NH, hydrazone, D2O exchangeable), 7.2–7.9 (m, 15H, Ar-H).; IR (KBr) υ (cm−1): 3340–3270 (broad NH and NH2), 2310 (C≡N), 1690 (C=O), 1575 (C=N); MS (EI) m/z: 459 (M+, 10%), 460 (M+ + 1, 2.91%); Anal. Calcd for C27H21N7O (459.50): C, 70.57; H, 4.61; N, 21.34; O, 3.48%. Found: C, 70.65; H, 4.69; N, 21.07; O, 3.41%.

3. Biological Screening

3.1. Animals

The complete course of the experiment was conducted using male Wistar albino rats (200–250 g), reared and maintained in the animal house of the institution and provided free access to pelleted food and water ad libitum. The rats were maintained in a controlled environment (12 h light and dark cycle) for about a week for acclimatization. The protocol of the study was approved by the animal ethics committee of the Faculty of Pharmacy, Helwan University (10-01-2012). The study was conducted in accordance with the EC, directive 86/609/EEC for animal experiments.

3.2. Dose Determination

Glimepiride (Amaryl) was used as a standard antidiabetic (4 mg/kg) in 1% gum acacia and administered orally [32]. Equivalent doses of all derivatives were calculated according to their molecular weight [M·wt].

3.3. Sucrose-Loaded Model (SLM)

Male Wistar rats were fasted overnight. Blood was collected initially and then the compounds were given to corresponding groups consisting of six rats each by oral gavage. A sucrose load of (10 gm/kg) body weight was given to each rat after half an hour posttest treatment. Blood was collected in 30, 60, 90, and 120 min after sucrose load [33]. The percentages (%) fallen in blood glucose level were calculated according to the AUC method.

3.4. Toxicity Study

The derivatives, which showed antihyperglycemic activity in this study, were subjected to in vivo acute toxicity study by testing their effect on serum liver and kidney markers.

3.5. Induction of Experimental Diabetes

Diabetes was induced in overnight fasted rats with a single intraperitoneal injection of streptozotocin (STZ) (Sigma-Aldrich, Co., St. Louis, USA. Catalog number: 1001062761) in a dose of 65 mg/kg. STZ was freshly dissolved in ice cold citrate buffer (0.01 M, pH 4.5) prior to injection [34]. After 48 h, rats showing blood glucose level  ≥ 200 mg/dl were included in the experiment [35].

3.6. Experimental Design

Seventy-six rats (fourteen groups of 5-6 rats each) were used to investigate the antihyperglycemic effect of 12 pyrrole and pyrrolo pyrimidine derivatives. Group 1 was diabetic control; Group 2, diabetic + Glimepiride (Amaryl) (4 mg/kg), served as a reference antidiabetic drug. Groups (3–14) were given the various pyrrole derivatives (Ia–e, IVg, VIf, VIIa, b, f, and VIIIf, a, resp.). The treated groups administered the Amaryl and different derivatives orally.

3.7. Methodology

For each group, blood glucose was estimated at zero, one, two, four, and six hours after oral administration of derivatives using glucometer (Gluco Dr Super Sensor, AllMedicus Co., Ltd., Anyang, Gyeonggi, Korea).

Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in serum were measured according to the Reitman-Frankel calorimetric transaminase procedure [36], whereas alkaline phosphatase (ALP) was assayed by the kinetic enzymatic method by measuring the rate of hydrolysis of p-nitrophenyl phosphate by ALP according to Henry [37]; all were measured as indicators of hepatic injury. Serum creatinine levels were assayed as an indicator for renal injury in the samples by a calorimetric method [38], using commercial diagnostic kits (Diamond Diagnostics, Egypt).

3.8. Statistical Analysis

Data were represented as mean area under curve (AUC) ± SD. Significant differences between groups was tested using GraphPad InStat (Graph software Inc., V 3.05, Ralph Stahlman, Purdue University). Appropriate graphs were plotted using Microsoft Excel 2007. P value less than 0.05 was considered statistically significant.

4. Results and Discussion

4.1. Chemistry

The target pyrrole o-amino carbonitriles Ia, b, and d were prepared by the reaction [26–31] of benzoin with appropriate amines and malononitrile in nonpolar solvent. On the other hand, Ic and e were obtained by condensation of α-(arylamino)-acetophenone with malononitrile in sodium ethoxide/ethanol.

Compounds Ia–e were utilized for the preparation of pyrrole derivatives If–o using appropriate reagents and reaction conditions; heating Ia–e with triethyl orthoformate (TEOF) afforded the corresponding 2-ethoxy methylamino derivative If–j, while, on react with acetic anhydride, the corresponding 2-acetylamino Ik–o were afforded, as revealed in Scheme 1.

On the other hand, the pyrrole derivatives Ia–e were converted to the corresponding pyrrolo[2,3-d]pyrimidine-4-ones IIa–j via condensation with formic acid [39, 40] and/or AcOH/HCl [28, 41], as revealed in Scheme 2.

Interaction [41] of Ia–e with formamide afforded the corresponding 4-amino pyrrolo[2,3-d]pyrimidines IIIa–e, which can also be prepared via stirring of the imidate I f–j with ammonium hydroxide at room temperature, as revealed in Scheme 2. The reaction of pyrrole o-amino carbonitriles Ia–e with thiourea in ethanol was reported [42] to afford the corresponding 4-aminopyrimidine-2-thione IIIf–j.

Pyrrolopyrimidinones IIa–j were converted [41, 43, 44] to its corresponding 4-chloro derivative IVa–j by refluxing with phosphorus oxychloride, as revealed in Scheme 2.

The 4-chloro IVa–j were utilized for the preparation of pyrrolopyrimidine derivatives Va–j and VIa–j using appropriate reagents and reaction conditions [40, 44]: the synthesis of certain 4-hydrazino-7H-pyrrolo[2,3-d]pyrimidines Va–j by hydrazinolysis of the corresponding 4-chloro analogues. Yet, when 4-chloro analogues IVa–j and thiourea were heated [45] in absolute ethanol, the pyrrolopyrimidine-4(3H)-thiones VIa–f were obtained, as revealed in Scheme 2. Diazotisation reaction of amino group in 2-amino-pyrrole, followed by coupling of the diazonium salt with active methylenes (ex: malononitrile) has been reported [46–48].

Diazotization of Ia–e using a mixture of sodium nitrite and HCl (without acetic acid) at 0–5°C, without separation, adding an active methylene compounds, namely, malononitrile and/or ethyl cyanoacetate in ethanol in the presence of sodium acetate afforded the corresponding hydrazono derivatives VIIa–i. This reaction could be explained via formation of the diazonium chlorides at first, which in addition to malononitrile afforded VIIa–i. Cyclization of hydrazono derivatives 2 using hydrazine hydrate in boiling ethanol leads to the formation of the corresponding pyrazolin-5-one derivatives VIIIa–i, as revealed in Scheme 3.

4.2. Biological Activities

Twelve of the synthesized Pyrroles and pyrrolopyrimidines were evaluated for their antihyperglycemic activity using both streptozotocin models of diabetes and sucrose load model [32–35]. The synthesized compounds were assessed for their antihyperglycemic activity, which is comparable to Glimepiride (Amaryl) the standard antihyperglycemic drug, by comparing the mean area under the curve (AUC) for the blood glucose level between the different studied groups. The proved pyrrole derivatives, which showed promising decrease in the serum blood glucose level, were subjected to test their toxicity in vivo on serum liver and kidney markers.

The tested compounds were classified into 2 main groups: first, the open form pyrrole derivatives, namely, Ia–e (pyrrole o-amino carbonitriles), hydrazone derivatives VIIa, b, and f, and pyrazolin-5-one derivatives VIIIa, f;second, the pyrrolopyrimidines, namely, 4-chloro IVg and 4-thio derivatives VIf.

Only the open form pyrrole derivatives, namely, I a, c, and e (pyrrole o-amino carbonitriles), induced a significant decrease in blood glucose level in the sucrose load model (17.4%, 18%, and 16.7%, respectively) compared to the untreated normal control. Moreover, they induced significant decrease in blood glucose level in the STZ model of diabetes (33.3%, 35.3%, and 29.5%, respectively) compared to the diabetic control group, as depicted in Table 1.

Table 1.

Effect of various treatments on the mean area under curve (AUC) of blood glucose levels in rats.

Tested
compound(s)		 % reduction in blood glucose compared to control
SLM STZ
Amaryl
[Standard drug]		 27.7 a 30.4 a
[Ia] 17.4 a 33.3 a
[Ib] 10.9 a NA
[Ic] 18 a 35.3 a
[Id] NA NA
[Ie] 16.7 a 29.5 a
[IVg] 13 a 11.2 a
[VIf] NA NA
[VIIa] NA NA
[VIIb] NA NA
[VIIf] NA NA
[VIIIa] NA NA
[VIIIf] NA NA
Open in a new tab

NA = not active.

aConsidered significant compared to control (P ≤ 0.05).

SLM: Sucrose-Loaded Model; STZ: Streptozotocin model of diabetes.

Comparing the antihyperglycemic activity of the these compounds with that of the reference antidiabetic drug (Amaryl) showed that compounds Ia, Ic, and Ie showed significant decrease in the blood glucose level (109.4%, 116.2%, and 97%, respectively) when compared to the activity of Amaryl, as shown in Figure 3.

Figure 3.

Figure 3

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Potency of antihyperglycemic derivatives compared to Amaryl.

Among the pyrrolopyrimidines, only the 4-chloro IVg (also bearing the antipyrine moiety at N-pyrrole) showed marked but not significant decrease in blood glucose level 11.2% compared to the diabetic control group, as shown in Table 1.

Studying the acute toxicity of the promising antihyperglycemic derivatives Ia, c, and e on the rats showed that the levels of sera ALT, AST, ALP, and creatinine were not significantly changed from that of the control untreated group and, also, the rats did not die or show any toxicity symptoms, as shown in Table 2.

Table 2.

Effect of compounds Ia, Ic, and Ie on ALT, AST, ALP, and creatinine.

Parameter Control Ia Ic Ie
ALT (U/L) 22.6 ± 4.6 17.9 ± 2.7 27.16 ± 6.3 20.5 ± 3.1
AST (U/L) 63.4 ± 14.6 69.4 ± 9.5 72 ± 7.8 62.7 ± 9.7
ALP (U/L) 70.6 ± 15 68.5 ± 12.3 63.8 ± 15.4 73.9 ± 13.2
Creatinine (mg/dL) 0.83 ± 0.13 0.8 ± 0.16 0.63 ± 0.09 0.77 ± 0.14
Open in a new tab

To analyze structure-activity relationships, three structural components were considered: the nature of the heterocycle nucleus, the nature of the side chain of the heterocycle system, and the function of the side chain, as shown in Figure 4.

Figure 4.

Figure 4

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Pyrrole and Pyrrolopyrimidines derivatives; evaluated as antihyperglycemic agents.

First, the influence of the nature of the heterocyclic system was easily observed as pyrrole (I a, c, and e) derivatives have show superior activity over pyrrolopyrimidines IVg and VIf.

Regarding the side chain function, for the pyrroles derivatives, the free amino group in pyrrole o-amino carbonitriles I a, c, and e conferred the greater activity over the hydrazone derivatives VIIa, b, and f which showed a marked activity over the pyrazolin-5-one derivatives VIIIa, f, which have no activity. For the pyrrolopyrimidines, the 4-chloro IVg confers markedly but not significantly higher activity than the 4-thio derivatives VIf.

Finally, the influence of the nature of the side chain on the heterocycle system, among the active compounds the antipyrine bearing N7-pyrrole (I e and IVg) showing a good activity over the benzyl (VIf, VIIa and VIIIa).

5. Conclusion

In the present study, we described a straightforward and efficient synthesis of some pyrroles and pyrrolo[2,3-d]pyrimidine and also,we examined their effects as antihperglycemic agents. The structure-activity relationship (SAR) results indicated that the pyrroles Ia, c, and e containing amino and cyano groups displayed good to moderate antihyperglycemic activity profile compared to control. On diazotization of the amino group in VII and VIII, this did not enhance the activity. The introduction of chloro group to IVg resulted in an enhanced antihyperglycemic activity of the pyrrolopyrimidine analogs over the hydrazine derivatives. These results and others demonstrated that the synthesized pyrrole and pyrrolopyrimidine compounds are promising antihyperglycemic agents.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

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