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

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.

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 Recently, dipeptidyl peptidase IV (DPP-IV) inhibitors [23][24][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).
Motivated by the importance of this system and in continuation of our research efforts [26][27][28][29][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.

5-(5,6-Diphenyl
. 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.

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.

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/H 2 O. The product was filtered off, washed with water, dried, and recrystallized from ethanol to give VIII.

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.

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].

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.

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.

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].

3.7.
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).

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. value less than 0.05 was considered statistically significant. On the other hand, the pyrrole derivatives Ia-e were converted to the corresponding pyrrolo [2,3-d]pyrimidine-4ones IIa-j via condensation with formic acid [39,40] and/or AcOH/HCl [28,41], as revealed in Scheme 2.

Results and Discussion
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.
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.

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][33][34][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.
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.
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.
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.
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).

Conclusion
In the present study, we described a straightforward and efficient synthesis of some pyrroles and pyrrolo [2,3d]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.