Received 7 October 2015; accepted 9 November 2015; published 13 November 2015

ABSTRACT

We herein disclose a series of novel pyrrole derivatives as well as fused pyrrolopyridines 6a,b and 7a,b, pyrrolopyrazoles 8a, b, pyrrolo[2,3-d]pyrimidine derivatives 10a-d, 12a,b, 14a,b, 18a,b, 20a,b, 21a,b, 22a,b, 23a,b, 24a,b, 31a,b, 36a,b, 40a,b, pyrrolo[1,2,6]thiadiazine derivatives 19a,b, pyrrolotriazolopyrimidines 25a,b, 26a,b, 27a,b and 28a,b, pyrrolo[2,3-d][1,2,3]triazine derivatives 32a,b and pyrrolo[2,3-d][1,3]oxazine derivatives 39a,b as novel compounds. All compounds were evaluated for their anti-inflammatory, analgesic (compared to the reference drug Indomethacin) and antimicrobial activities (compared to the reference drug Ampicillin and Fluconazole). Compounds 4d, 5b-d, 6a,b, 9c,d, 10d, 12ab, 13b, 19a,b, 21b, 23b, 31a,b, 38b and 40a were found to be the most active anti-inflammatory drugs exhibiting potency ranging from 1 - 1.01 compared to the reference drug indomethacin. In addition to docking study of these highly active twenty compounds against the active site of cyclooxygenase-2 enzyme (COX-2), among the tested compounds, compounds 5d, 9d, 11b, 12a, 13b and 32a showed multiple activities; anti-inflammatory, analgesic and anti-bacterial activities.

Keywords:

Anti-Inflammatory Activity, Pyrrole, Pyrrolo[2,3-d]Pyrimidine, Molecular Modeling

1. Introduction

Nitrogen heterocycles are of special interest as they constitute an important class of natural and non-natural products as they occupy a key position in the area of drugs and pharmaceuticals [1] [2] . Pyrroles have drawn considerable attention due to their synthetic importance and useful biological activities that are extensively used in drug discovery [3] . Pyrrole derivatives exhibited a vital role in many pharmacological activities including anti-inflammatory [4] - [13] , anti-microbial [14] - [19] , anti-fungal [20] - [22] , antiviral [23] - [25] and anti-cancer [26] - [28] activities.

It is well known that the anti-inflammatory activity is due to the ability to inhibit the cyclooxygenase (COX) activity of prostaglandin H synthase, an enzyme which mediates the production of prostanoids (including prostaglandins, prostacyclins and thromboxanes) from arachidonic acid. Prostaglandins act as mediators in the process of inflammation, thus the discovery of COX-2 specific inhibitors (Coxibs), which their pharmacological properties are correlated to their ability to decrease the COX-2 dependent prostanoid biosynthesis, providing a rational for the development of drug devoid of GIT disorders while retaining clinical efficacy as anti-inflam- matory agent [29] . The recent market withdrawal of some coxibs such as rofecoxibs (Vioxx^®) and valdecoxib (Bextra^®) due to their adverse cardiovascular side effects [30] clearly delineates the need to explore and evaluate alternative templates with COX-2 inhibitory activity.

Therefore, our aim was to design derivatives of existing clinically used NSAIDs, such as Tolmetin and Ketorolac [31] [32] which are well known pyrrole derivatives acting as anti-inflammatory drugs. In the light of these facts, this paper deals with the synthesis of novel pyrrole and condensed pyrrole derivatives and evaluates them for their anti-inflammatory activity. Furthermore, the extent of the pharmacological effects of pyrrole derivatives is reported to depend on the active groups which are attached to it, as several scientists have elucidated that in pyrrole system positions 2 and 3, it can be suitably modified by the introduction of groups [28] , aromatic [19] [25] or heterocyclic moieties to show excellent pharmacological results [12] .

2. Results and Discussion

2.1. Chemistry

Reaction of 4-chlorophenacyl bromide 1 either with 3-trifluoromethyl aniline 2a in refluxing ethanol or by stirring with 3-amino pyridine 2b in diethyl ether afforded 2-(substituted amino)-1-(4-chlorophenyl)ethanones 3a,b; respectively which upon stirring with the active methylene bearing carbonitriles namely; malononitrile and ethyl cyanoacetate in sodium ethoxide afforded 3-substituted-2-amino-4-(4-chlorophenyl)-1-(substitutedaryl)-1H-py- rroles 4a-d which were utilized as building units for novel substituted pyrrole compounds (Scheme 1).

Scheme 1. Synthetic pathways for compounds 3a,b and 4a-d.

Furthermore, the o-aminonitrile derivatives 4a,b were condensed with the appropriate aromatic aldehyde namely; benzaldehyde and 2,4-dichlorobenzaldehyde in absolute ethanolto afford the corresponding benzylidenimino derivatives 5a-d (Scheme 2). Moreover, reaction of compounds 4a,b either with ethyl cyanoacetate or malononitrile in refluxing ethanol yielded 6-oxopyrrolo[2,3-b]pyridines 6a,b and 4,6-diamino-pyrrolo[2,3- b]pyridine derivatives 7a,b; respectively. Additionally, cyclization of compounds 4a,b with hydroxylamine hydrochloride in boiling sodium ethoxide afforded the aminopyrazole derivatives 8a,b.

Furthermore, compounds 4a,b were reacted with urea/thiourea in sodium ethoxide to give the target compounds 9a-d (Scheme 3), which were cyclized upon refluxing in pyridine to afford the pyrrolopyrimidine derivatives 10a-d which can also be obtained through refluxing of the o-aminonitrile derivatives 4a,b with urea/ thiourea in a mixture of glacial acetic acid and concentrated hydrochloric acid (3:1). Furthermore, the target phenylthioureaderivatives 11a,b was obtained via refluxing of compounds 4a,b with phenyl isothiocyanate in absolute ethanol. Also, refluxing of compounds 11a,b in pyridine afforded the cyclized compounds 4-imino- pyrrolopyrimidine-2-thiones 12a,b which were also prepared directly via refluxing of compounds 4a,b with phenyl isothiocyanate in dry pyridine.

Moreover, our goal was directed to synthesize various substituted pyrrole-3-carbonitrile derivatives bearing different substituted imino side chains in position two of pyrrole (Scheme 4). Therefore, refluxing of compounds 4a,b in a mixture of triethyl orthoformate and acetic anhydride furnished compounds 13a,b with a replaceable ethoxymethyleneimino function which were cyclized upon reaction with ammonia in refluxing methanol via elimination of an ethanol moiety to yield 4-aminopyrrolopyrimidine derivatives 14a,b which were also obtained directly through refluxing of compounds 4a,b with formamide. On the other hand, stirring of compounds 13a,b with dimethylamine in absolute ethanol afforded N,N-dimethyl formimidamide derivatives 15a,b which was also synthesized directly via refluxing compounds 4a,b with dimethylformamide/dimethylacetal in xylene. Moreover, acylation of compounds 4a,b was accomplished through refluxing of compounds 4a,b with p-toluene sulphonyl chloride in toluene to afford 4-methylbenzenesulfonamides 16a,b via elimination of a hydrochloride molecule.

Scheme 2. Synthetic pathways for compounds 5a-d, 6a,b, 7a,b and 8a,b.

Scheme 3. Synthetic pathways for compounds 9a-d, 10a-d, 11a,b and 12a,b.

Scheme 4. Synthetic pathways for compounds 13a,b, 14a,b, 15a,b and 16a,b.

Also, partial hydrolysis of compounds 4a,b was accomplishedby stirring at room temperature with concentrated sulfuric acid to afford substituted-2-aminopyrrol-3-carboxamides 17a,b (Scheme 5), which undergo cyclization into pyrrolo[2,3-d]pyrimidin-4-ones 18a,b, pyrrolo[3,2-d]1,2,6-thiadiazine-2,4-diones 19a,b and 2-me- thyl-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-ones 20a,b upon their refluxing with formamide, thionyl chloride and excess acetic anhydride; respectively. However, the target compounds 18a,b was also obtained directly by refluxing the o-aminonitrile derivatives 4a,b with formic acid.

Moreover, chlorination of pyrrolopyrimidin-4-one derivatives 18a,b with excess phosphorus oxychloride followed by alkalinization to pH 10 using sodium bicarbonate furnished 4-chloropyrrolo[2,3-d]pyrimidines 21a,b (Scheme 6). However, the chlorinated compounds 21a,b were reacted with morpholine in ethanol under reflux in presence of a catalytic amount of triethylamine to furnish the 4-morpholino-pyrrolo[2,3-d]pyrimidines 22a,b. Furthermore, hydrazinolysis of chloro derivatives 21a,b was carried out by refluxing with hydrazine hydrate 98% in ethanol in presence of triethylamine as a catalyst to afford 4-hydrazinyl-7H-pyrrolo[2,3-d]pyrimidines 23a,b.

Scheme 5. Synthetic pathways for compounds 17a,b, 18a,b, 19a,b and 20a,b.

Scheme 6. Synthetic pathways for compounds 21a,b, 22a,b and 23a,b.

Furthermore, stirring of compounds 13a,b with hydrazine hydrate 98% in absolute ethanol afforded 4-imino- 4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidines 24a,b (Scheme 7) which were used as good starting materials for preparation of several pyrrolo[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine derivatives. Thus, compounds 24a,b were reacted with ethyl cyanoacetate in absolute ethanol containing few drops of glacial acetic acid to yield the cyanomethyl triazolopyrimidine derivatives 25a,b. While, refluxing of compounds 24a,b with carbon disulphide in absolute ethanol containing potassium hydroxide yielded the corresponding triazolopyrimidine-2-thione derivatives 26a,b. Moreover, the reaction of the precursors 24a,b with triethyl orthoformate in absolute ethanol gave the unsubstituted triazolopyrimidine compounds 27a,b. While, upon refluxing of 24a,b with phenyl isothiocyanate in absolute ethanol in presence of a catalytic amount of triethylamine afforded the phenyl amine substituted triazolopyrimidine derivatives 28a,b.

Heating of the o-aminonitrile derivatives 4a,b with sodium azide in presence of ammonium chloride in dimethylformamide as a solvent to furnish 2-aminopyrrol-3-tetrazole derivatives 29a,b (Scheme 8). However, heating compounds 4a,b with ethylene diamine in the presence of carbon disulphide afforded the target compounds 2-aminopyrrol-3-imidazole derivatives 30a,b. Also, the o-aminonitrile derivatives 4a,b were refluxed with carbon disulphide in dry pyridine to yield the pyrrolopyrimidine-2,4-dithiones 31a,b. Besides, pyrrolo[2,3- d][1,2,3]triazin-4-ones 32a,b were obtained via stirring of compounds 4a,b with sodium nitrite solution in a mixture of glacial acetic acid and concentrated hydrochloric acid at 0˚C - 5˚C. The reaction mechanism is suggested to proceed first through partial hydrolysis of the cyano function to afford the corresponding o-aminocar- boxamide derivatives that undergo subsequent diazotization then coupling with amino function of the carboxamide to yield the target 4-oxotriazine derivatives 32a & 32b.

This work was extended to shed more light on the activity and synthetic potential of the amino and carboxylate groups in compounds 4c,d (Scheme 9). Thus, compounds 4c,d reacted with hydrazine hydrate in absolute ethanol to afford the corresponding acid hydrazide derivatives 33a,b. However, acetylation of amino group in compounds 4c,d with acetic anhydride afforded the target acetamido derivatives 34a,b. However, the synthesis of different hydrazone derivatives through the condensation of hydrazine compounds with monosaccharide is well documented in the literature [33] - [35] . Therefore, this information prompted us to explore the effect of glucose hydrazone derivatives 35a,b whichwere synthesized through the condensation of the hydrazide derivatives 33a,b with glucose in absolute ethanol. Additionally, the reaction of o-acetamidocarboxylate derivatives 34a,b were refluxed with excess hydrazine hydrate in ethanol to afford the target substituted pyrrolopyrimidinone derivatives 36a,b. The reaction mechanism is suggested to proceed first through formation of the hydrazone derivatives followed by tautomerism then intramolecular cyclization via elimination of an ethanol molecule.

Scheme 7. Synthetic pathways for compounds 24a,b, 25a,b, 26a,b, 27a,b and 28a,b.

Scheme 8. Synthetic pathways for compounds 29a,b, 30a,b, 31a,b and 32a,b.

Scheme 9. Synthetic pathways for compounds 33a,b, 34a,b, 35a,b and 36a,b.

Scheme 10. Synthetic pathways for compounds 37a,b, 38a,b, 39a,b and 40a,b.

Finally, the title carboxylic acid derivatives 37a,b was obtained via refluxing of the o-aminoester compounds 4c,d with ethanolic sodium hydroxide (Scheme 10). Also, our aim was extended to develop novel ethyl 1- (substitutedaryl)-4-(4-chlorophenyl)-2-(3-phenylthioureido)-1H-pyrrol-3-carboxylates 38a,b which were obtained via refluxing of 2-aminopyrrole derivatives 4c,d with phenylisothiocyanate in absolute ethanol. Furthermore, fused 2-methyloxazine-4-one rings are reported to be prepared from the reaction of different o-amino- carboxylic acid derivatives with excess acetic anhydride. Thus, the target 2-methyloxazine-4-one derivatives 39a,b were synthesized via refluxing of the o-aminocarboxylic acid compounds 37a,b in excess acetic anhydride. Finally, the thiourea derivatives 38a,b were heated with sodium ethoxide under reflux to furnish the target 2-thioxopyrimidin-4-one derivatives 40a,b.

2.2. Biology

All the newly synthesized compounds 4a-40b were preliminarily evaluated for their anti-inflammatory and analgesic activities (using rat paw edema method and writhing test; respectively) as well as their gastric ulcerative effect (ulcerogenicity) an in-vitro antibacterial activity against Staphylococcus aureus (ATCC 25923) as a representative of Gram-positive bacteria; Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 8739) as representatives of Gram-negative bacteria. The compounds were also evaluated for their in-vitro antifungal activity against Candida albicans (ATCC 10231) (using the cup diffusion technique).

2.2.1. Anti-Inflammatory and Analgesic Screening

For the tested compounds 4a-40b, the percent of edema inhibition after 1-6 h and the percent inhibition of the writhing movements are presented in Tables 1-6.

^a,b: Significantly different from control value and reference value at P < 0.05. ^●S.D. = Standard deviation.

It was revealed from the results that, compounds 4d, 5b, 5c, 5d, 6a, 6b, 9c, 9d, 10d, 12a, 12b, 13b, 19a, 19b, 21b, 23a, 23b, 31a, 31b, 38b and 40a exerted highly potent anti-inflammatory effect, comparable to that of indomethacin (Indocin^®) at 6 h interval post carrageenan showing inhibition potency ranging from 1.01% - 1.05%. While, compounds 4b, 4c, 5a, 7a, 7b, 8b, 9a, 9b, 9c, 10b, 10c, 11a, 11b, 13a, 13b, 14b, 15b, 16a, 16b, 18a, 18b, 20b, 21a, 24a, 25b, 26a, 26b, 28b, 32a, 32b, 34a, 34b, 38a and 40b exerted moderate anti-inflammatory activity at 6 h interval post carrageenan, comparable with that of indomethacin (Indocin^®) showing inhibition potency ranging from 0.68% - 1%.

In addition to, compounds 4a, 8a, 10a, 14a, 15a, 22a, 22b, 25a, 27a, 27b, 28a, 29a, 29b, 30a, 30b, 33a, 33b, 35a, 35b, 36a, 36b, 37a, 37b, 39a and 39b which showed weak anti-inflammatory activity at 6 h interval less than indomethacin showing inhibition potency ranging from 0.36% - 0.67%. The activity profiles of all the previous compounds were the same as indomethacin (response increasing by time).

It is worth mentioning that, the highly potent compounds were those comprising 3-cyanopyrrole rings attached to different side chains in the 2 position, among these chains are the aryl imino function as in compounds 5b-d, thiourea group as in compounds 9c,d and ethoxymethyleneimino chain in compound 13b. Also, compounds containing pyrrole-3-carboxylate with the 2 position either unsubstituted as in compound 4d or substituted with thiourea side chain as compound 38b were highly potent.

Furthermore, among the highly potent compounds were thefused pyrrolopyrimidine compounds bearing 2-thioxo function with different substituents in the 4 position. Among these groups in the 4 position was the amino group as compound 10d, imino function in compounds 12a,b, 4-thioxo function as in compounds 31a,b and 4-oxo group as in compound 40a. Also, the 4-chloropyrrolopyrimidine derivative 21b as well as the 4-hydrazinopyrrolopyrimidine analogue 23b exhibited potent activity comparable to the reference drug Indomethacin (Indocin^®).

Moreover, other fused pyrrole compounds such as the pyrrolopyridine-2-one derivatives 6a,b and the pyrrolo[1,2,6]thiadiazine-2,4-dione analogues 19a,b exerted excellent activity.

As revealed from the results presented in Tables 1-3 that, compounds 4d, 5c, 5d, 6b, 10c, 10d, 17b, 31a and 31b exhibited the most potent analgesic activity with potency ranging from 1 - 1.10 to the reference drug Indomethacin. It is to be noted that some functions are assumed to be responsible for the highly potent analgesic activity of these compounds. Among these functions are the ester function in 3-position as in compound 4d, the 2,4-dichlorobenzylidine imino function in pyrrole-2-position as in compounds 5c and 5d and pyrrolopyridine moiety in compound 6b. Also, the pyrrolopyrimidine thione function as in compounds 10c and 10d.

Furthermore, the carboxamide function as in compound 17b led to high analgesic activity. Additionally, pyrrolopyrimidine dithione function as in compounds 31a and 31b exerted the most potent analgesic activity.

2.2.2. Ulcerogenicity

Five compounds that exhibited the most potent anti-inflammatory activity; 12b, 23b, 31a, 31b and 38b were evaluated for their ulcerative effect on rats as revealed in Table 7.

In general, all the tested compounds showed better results than the reference drug Indomethacin. Especially, compounds 12b and 31b which were devoid of any ulcerative effect compared to 94% of that of Indomethacin as illustrated in the previous table.

2.2.3. Anti-Microbial Screening

For the tested compounds 4a-40b, the resulting inhibition zones were measured in mm diameter, Tables 8-10.

Among the tested compounds, compounds 5d, 9d, 11b, 12a, 13b, 15a, 15b, 17a, 22a, 27a, 32a, 33a, 39a and 39b were found to be the most active.

Among the different functions attached to the free pyrrole ring which exerted potent antimicrobial activity against Gram negative bacteria are the 2,4-dichlorobenzylidineimino, thiourea, phenylurea, ethoxymethyleneimino as well as dimethylaminomethyleneimino groups as in compounds 5d, 9d, 11b, 13b and 15a, 15b; respectively, in addition to, the carboxamide and the acid hydrazide side chains attached to the pyrrole ring in compounds 17a and 33a; respectively.

Furthermore, functions surmounted on the pyrrolopyrimidine skeleton as 4-imino-3-phenyl-2-thione and 4- morpholino groups as in compounds 12a and 22a; respectively, also displayed significant antimicrobial activity. Besides to, the triazolopyrrolopyrimidine derivative 27a, pyrrolotriazine analogue 32a as well as the pyrroloxazine compounds 39a, 39b which exerted high antimicrobial activities against Gram negative bacteria. It is worth mentioning that, all compounds exerted weak activity against Gram positive bacteria except for the 3- imidazolidine substituted pyrrole derivative 30b which exerted moderate antimicrobial activity. However, only the pyrrolopyrimidinone derivative 18a and the 1,2,6-thiadiazine-2,4-dione analogue 19a exerted moderate antifungal activity against C. albicans.

2.3. Computer Aided Docking

The most active twenty compounds as anti-inflammatory agents 4d, 5b, 5c, 5d, 6a, 6b, 9c, 9d, 10d, 12a, 12b, 13b, 19a, 19b, 21b, 23b, 31a, 31b, 38b and 40a were subjected to docking using Molecular Operating Environment (MOE) program [36] on the 3D structure of the cyclooxygenase-2 enzyme (COX-2) in a trial to predict their mode of action as anti-inflammatory drugs.

2.3.1. Docking on the Active Site of Cyclooxygenase-2 Enzyme (COX-2)

1) Diclofenac interactions with the active site of COX-2:

Diclofenac interacted as hydrogen bond acceptor via four hydrogen bonds via both the oxygen atoms of carboxyl group with the amino acid residues Tyr 385 (2.73 A˚) and Ser 530 (2.65 A˚, 2.91 A˚ and 3.04 A˚) as shown in Figure 1.

2.3.2. Docking of Compound 4d into COX-2

Active site revealed that several molecular interactions were considered to be responsible for the observed affinity, as the N of pyridine moiety acted as a hydrogen bond acceptor with the side chain residue; His 90 (2.25 A˚) with a strength of 81.3%. In addition to a hydrogen bond interaction between the hydrogens of the amino group which acted as a hydrogen bond donor with the side chain residue Tyr 355 (2.61 A˚) with a strength of 5.3%. Besides to, hydrophobic interactions involving the pyridine C₆ and p-chlorophenyl C₃ carbon as well the CH₃ group of ester function and the following amino acid residues: His 90, Met 113, Val 116, Leu 117, Arg 120, Val 349, Leu 352, Ser 353, Tyr 355, Leu 359, Leu 384, Tyr 385, Trp 387, Phe 518, Met 522, Val 523, Gly 526, Ala 527, Ser 530 and Leu 531 as shown in Figure 2.

2.3.3. Docking of Compound 5b into COX-2

Active site revealed the presence of arene cation interaction between the pyrrole ring and the amino acid residue Arg 120. In addition to, hydrophobic interactions involving pyridine C₄ and C₆ carbons and the following amino acid residues: His 90, Met 113, Val 116, Leu 117, Arg 120, Val 349, Leu 352, Ser 353, Tyr 355, Leu 359, Trp 387, Arg 513, Phe 518, Met 522, Val 523, Ala 527, Ser 530 and Leu 531 as shown in Figure 3.

2.3.4. Docking of Compound 5c into COX-2

Active site illustrated the presence of several interactions of the cyano group with different amino acid residues as it acted as a hydrogen bond acceptor with the side chain residues; His 90, Tyr 355 and Arg 513 (3.35 A˚, 2.43 A˚ and 3.16 A˚; respectively) at a strength of 2.1%, 90.6% and 13.4%; respectively. This beside hydrophobic interactions among the cyano function and the following amino acid residues: His 90, Val 116, Leu 117, Arg 120, Gln 192, Val 349, Leu 352, Ser 353, Tyr 355, Leu 359, Tyr 385, Trp 387, Arg 513, Ala 516, Ile 517, Phe 518, Val 523, Gly 526, Ala 527, Ser 530 and Leu 531 as shown in Figure 4.

2.3.5. Docking of Compound 5d into COX-2

Active site revealed the presence of hydrogen bond interaction between the cyano group, as it acted as a hydrogen bond acceptor with the amino acid residue Ser 530 residue (3.32 A˚) with a strength of 1.8%. In addition to, arene cation interactions involving the p-chlorophenyl ring with the amino acid residue Phe 518 and the 2,4-dichlorophenyl ring with the amino acid residue Arg 120. There are also hydrophobic interactions involving the pyridine C₂, C₅, C₆ as well as the pyridine nitrogen atom with the following amino acid residues: His 90, Val 116, Arg 120, Val 349, Leu 352, Ser 353, Tyr 355, Leu 359, Leu 384, Phe 518, Met 522, Gly 526, Ala 527, Ser 530 and Leu 531 as shown in Figure 5.

2.3.6. Docking of Compound 6a into COX-2

Active site revealed the presence of four hydrogen bonds in which the cyano nitrogen acted as a hydrogen bond acceptor for three hydrogen bonds with the amino acid residues His 90, Arg 513 and Tyr 355 (2.82 A˚, 3.44 A˚ and 2.91 A˚; respectively) with a strength of 20.4%, 7% and 7.3%; respectively. While, the amino group acted as a hydrogen bond acceptor with the amino acid residue Tyr 355 residue (3.24 A˚) with a strength 5.5%. In addition to, hydrophobic interactions involving the carbonyl oxygen with the following amino acid residues: His 90, Met 113, Val 116, Leu 117, Arg 120, Ser 530, Val 349, Leu 352, Ser 353, Tyr 355, Leu 384, Try 385, Ala 527 and Leu 531 as shown in Figure 6.

2.3.7. Docking of Compound 6b into COX-2

Active site revealed the presence of four hydrogen bonds and two arenes cation interactions. In which the amino group acted as a hydrogen acceptor via three hydrogen bonds with the amino acid residues His 90, Tyr 355 and Arg 513 (2.25 A˚, 3.32 A˚ and 3.43 A˚; respectively) with a strength of 3.5%, 9.1% and 43.2%; respectively. While the cyano nitrogen atom acted as a hydrogen bond acceptor with the amino acid residue His 90 (3.41 A˚) with strength of 2.2%. In addition to, two arene cation interactions among the p-chlorophenyl moiety and the amino acid residue Arg 120 and Arg 513. Besides to hydrophobic interactions involving the pyridine ring, pyrrole C₂ carbons as well as the chlorine atom and the p-chlorophenyl C₃ carbon with the following amino acid residues: Pro 86, Val 89, His 90, Arg 120, Val 349, Leu 352, Tyr 355, Arg 513, Ala 516, Phe 518, Val 523, Glu 524, Gly 526, Ala 527 and Ser 530 as shown in Figure 7.

2.3.8. Docking of Compound 9c into COX-2

Active site revealed the presence of two hydrogen bond interactions between the cyano nitrogen, as it acted as a hydrogen bond acceptor with the amino acid residue side Arg 120 and Tyr 355 (3.21 A˚ and 1.60 A˚; respectively) with a strength of 12.3% and 95.9%; respectively. In addition to, hydrophobic interactions involving the cyano nitrogen and chlorine atom with many amino acid residues: His 90, Val 349, Leu 352, Ser 353, Tyr 355, Leu 359, Phe 381, Tyr 385, Trp 387, Arg 513, Ala 516, Val 523, Gly 526, Ala 527, Ser 530 and Leu 531 as shown in Figure 8.

2.3.9. Docking of Compound 9d into COX-2

Active site revealed the presence of one hydrogen bond between the pyridyl nitrogen atom as it acted as a hydrogen bond acceptor with the amino acid residue His 90 (2.82 A˚) with a strength of 4.8%. In addition to, hydrophobic interactions concerning 4-chlorophenyl C₂ carbon, pyridine C₂ and C₄ carbons, the thiourea amino group and sulphur atom with the following amino acid residues: His 90, Arg 120, Val 349, Leu 352, Ser 353, Tyr 355, Phe 381, Leu 384, Tyr 385, Trp 387, Met 522, Val 523, Gly 526 and Ala 527 as shown in Figure 9.

2.3.10. Docking of Compound 10d into COX-2

Active site revealed hydrogen bond interaction between the N atom of pyridine moiety as it acted as a hydrogen bond acceptor with the side chain residues His 90 and Arg 513 (2.74 A˚ and 3.54 A˚; respectively) with a strength of 21.7% and 1.2%; respectively. Besides to, arene cation interaction between the p-chlorophenyl ring and the amino acid residue Arg 120. In addition to, hydrophobic interactions among the p-chloro phenylC₂ carbon, chlorine atom as well as the pyrimidine thioxo function, N₃ atom and C₄ carbon with the following amino acid residues: His 90, Val 116, Arg 120, Val 349, Leu 352, Ser 353, Leu 359, Trp 387, Arg 513, Ala 516, Ile 517, Phe 518, Met 522, Val 523 and Ala 527 as shown in Figure 10.

2.3.11. Docking of Compound 12a into COX-2

Active site revealed the presence of only hydrophobic interactions involving the phenyl C₂ carbon and the p-chlorophenyl C₃ and C₆ carbons as well as the chlorine atom with the following amino acid residues: His 90, Met 113, Val 116, Arg 120, Ile 345, Val 349, Leu 352, Ser 353, Tyr 355, Leu 359, Tyr 385, Arg 513, Ala 516, Phe 518, Met 522, Val 523, Gly 526, Ala 527, Ser 530 and Leu 531 as shown in Figure 11.

2.3.12. Docking of Compound 12b into COX-2

Active site showed the presence of a hydrogen bond interaction between imine function, as it acted as a hydrogen bond acceptor with the side chain Tyr 355 residue (3.15 A˚) with a strength of 6.3%. In addition to hydrophobic interactions involving the chlorine atom and the pyridyl C₆ carbon with many amino acid residues: His 90, Val 116, Arg 120, Val 349, Leu 352, Ser 353, Leu 384, Tyr 385, Trp 387, Phe 518, Met 522, Val 523, Gly 526, Ala 527 and Leu 531 as shown in Figure 12.

2.3.13. Docking of Compound 13b into COX-2

Active site showed hydrophobic interactions concerning the chlorine atom, cyano nitrogen and the methylene imino side chain with many amino acid residues: His 90, Met 113, Val 116, Arg 120, Leu 331, Ile 345, Val 349, Leu 352, Ser 353, Tyr 355, Leu 359, Val 523, Ala 527, Ser 530 and Leu 534 as shown in Figure 13.

2.3.14. Docking of Compound 19a into COX-2

Active site revealed the presence of two hydrogen bond interactions between the oxygen atom of pyrimidine C₄ oxo function as it acted as a hydrogen bond acceptor with the side chain residues Arg 120 and Tyr 355 (2.63 A˚ and 3.04 A˚; respectively) with a strength of 19.6% and 12.5%; respectively. In addition to hydrophobic interactions involving the pyrimidine N₃ and 2-oxo functions as well as pyrrole C₅ and p-chlorophenyl C₂ and C₆ carbons with many amino acid residues: Pro 86, Val 89, His 90, Arg 120, Tyr 348, Val 349, Leu 352, Tyr 355, Tyr 385, Trp 387, Arg 513, Met 522, Val 523, Glu 524, Gly 526, Ala 527 and Leu 531 as shown in Figure 14.

2.3.15. Docking of Compound 19b into COX-2

Active site revealed the presence of hydrogen bond interactions between the oxygen atoms of pyrimidine C₂ and C₄ oxo functions as they acted as hydrogen bond acceptor with the side chain residue Arg 513 (3.27 A˚, 2.3%) and Arg 120 (2.28 A˚, 9.5%); respectively. In addition to, another hydrogen bond between the pyridyl nitrogen atom as it acted as hydrogen bond acceptor with the amino acid residue Arg 513 (3.45 A˚, 1.7%). In addition to, hydrophobic interactions concerning the carbon atoms of p-chlorophenyl moiety and the following amino acid residues: Pro 86, His 90, Arg 120, Gln 192, Val 349, Leu 352, Ser 353, Tyr 355, Arg 513, Ala 516, Val 523, Glu 524, Ala 527, Ser 530 and Leu 531 as shown in Figure 15.

2.3.16. Docking of Compound 21b into COX-2

Active site revealed the presence of hydrogen bond interaction between the N₁ of the pyrimidine moiety, as it acted as a hydrogen bond acceptor with the side chain residue Arg 120 (2.35 A˚) with strength of 64.2%. In addition to, arene cation interaction between the pyridine moiety and the amino acid residue Arg 120. Besides to, hydrophobic interactions involving the pyrimidine C₂ carbon, pyrrole C₅ carbon and pyridine C₆ carbon with many amino acid residues: Arg 120, Val 349, Leu 352, Tyr 355, Tyr 385, Trp 387, Arg 513, Val 523, Glu 524, Gly 526, Ala 527 and Leu 531 as shown in Figure 16.

2.3.17. Docking of Compound 23b into COX-2

Active site revealed the presence of three hydrogen bond interactions in which the hydrazine NH₂ proton acted as a hydrogen bond donor with the amino acid residue Val 349 (3.21 A˚, 1.5%), while the nitrogen atom acted as a hydrogen bond acceptor with the amino acid residue Ser 353 (2.62 A˚, 2.5%). Also, the hydrazine NH proton acted as a hydrogen bond donor to the amino acid residue Val 349 (1.38 A˚, 93.7%). Besides to, hydrophobic interactions among the p-chlorophenyl moiety and the pyrimidine C₄ carbon with the following amino acid residues: His 90, Tyr 348, Val 349, Leu 352, Ser 353, Tyr 355, Phe 381, Leu 384, Tyr 385, Phe 518, Met 522, Val 523, Gly 526, Ala 527 and Ser 530 as shown in Figure 17.

2.3.18. Docking of Compound 31a into COX-2

Active site showed arene cation interaction between the 3-triflouromethylphenyl ring and the amino acid residue Arg 513. In addition to, hydrophobic interactions involving the 3-trifluoromethylphenyl C₆ and fluorine atom as well as the two sulphur atoms of the two thioxo functions of pyrimidine ring with the following amino acid residues: His 90, Gln 192, Val 349, Leu 352, Ser 353, Tyr 355, Leu 384, Tyr 385, Trp 387, Arg 513, Ala 516, Ile 517, Phe 518, Met 522, Val 523, Gly 526, Ala 527 and Ser 530 as shown in Figure 18.

2.3.19. Docking of Compound 31b into COX-2

Active site revealed the presence of hydrophobic interactions involving the pyridine C₆ carbon, chlorine atom, the sulphur atom of the 2-thioxo function of pyrimidine ring as well as the pyrimidine N₃ NH moiety with the following amino acid residues: His 90, Val 349, Leu 352, Ser 353, Tyr 355, Leu 384, Tyr 385, Trp 387, Arg 513, Phe 518, Met 522, Val 523, Gly 526, Ala 527 and Leu 531 as shown in Figure 19.

2.3.20. Docking of Compound 38b into COX-2

Active site showed one hydrogen bond between NH-phenyl proton which acted as a hydrogen bond donor with the side chain residue Tyr 355 (2.03 A˚, 0.8%). In addition to, two arene cation interactions between both pyridine and phenyl rings and the amino acid residue Arg 120. Besides to, hydrophobic interactions involving the phenyl C₄ carbon and the sulphur atom with many amino acid residues: Pro 86, Val 116, Arg 120, Tyr 348, Val 349, Leu 352, Ser 353, Tyr 355, Leu 359, Tyr 385, Trp 387, Arg 513, Val 523, Glu 524, Gly 526, Ala 527, Ser 530 and Leu 531 as shown in Figure 20.

2.3.21. Docking of Compound 40b into COX-2

Active site revealed the presence of hydrophobic interactions involving the p-chlorophenyl C₂ carbon and pyrrole C₂ carbon with many amino acid residues: His 90, Met 113, Val 116, Leu 117, Arg 120, Ile 345, Val 349, Leu 352, Ser 353, Tyr 355, Leu 359, Tyr 385, Trp 387, Arg 513, Ala 516, Val 523, Gly 526, Ala 527, Ser 530 and Leu 531 as shown in Figure 21.

3. Experimental

3.1. Chemistry

All melting points were measured on Electro thermal LA 9000 SERIS, Digital Melting point Apparatus and are uncorrected. IR spectra (KBr) were recorded on FT-IR 200 spectrophotometer (ύ∙cm⁻¹), pharmaceutical analytical unit, Faculty of Pharmacy, Al-Azhar University. ¹H-NMR spectra were recorded in (DMSO-d₆) at 300 MHz on a Varian Gemini NMR spectrometer (δ ppm) using TMS as an internal standard, ResearchService Unit, Faculty of Science, Cairo University. Mass spectra were recorded on GC MS-QP 5050A mass spectrometer at 70 eV andmicroanalytical data were performed on Elementar Vario EI III CHN analyzer at the microanalytical unit, in Regional center for Mycology and Biotechnology, Al-Azhar University. Thin layer chromatography was performed on precoated (0.25 mm) silica gel GF254 plates (E. Merck, Germany). Compounds were detected with UV lamp at λ 254 nm.

3.1.1. 1-(4-Chlorophenyl)-2-(3-Trifluoromethylphenylamino)Ethanone; 3a

Equimolar amounts of 4-chlorophenacyl bromide (0.38 g, 2 mmol.) and 3-trifloromethylaniline (0.32 g, 0.25 mL, 2 mmol.) were refluxed for 4 h in absolute ethanol (25 mL) in presence of drops of TEA, then left to cool. The solid product was collected by filtration after cooling and recrystallized from ethanol.

Fluffy golden yellow needle crystals; yield (90%); m.p.:138˚C. IR [KBr, cm⁻¹]: 3370 (NH); 3079 (CH aromatic); 2912 (CH aliphatic); 1691 (C=O); 1522 (C=C); 1108 (p-Cl-phenyl). ¹H NMR(DMSO-d₆, δ ppm): 4.76 (s, 2 H, CH₂CO); 6.39 (s, 1 H, NH, D₂O exchangeable); 6.82 (s, 1 H, 3-CF₃-C₆H₄-C₂-H); 6.94 (d, 1 H, J = 7.2 Hz, 3-CF₃-C₆H₄-C₆-H); 6.97-7.00 (m, 1 H, 3-CF₃-C₆H₄-C₄-H); 7.25-7.30 (m, 1 H, 3-CF₃-C₆H₄-C₅-H); 7.64 (d, 2 H, J = 8.1 Hz, 4-Cl-C₆H₄-C_2,6-H); 8.11 (d, 2 H, J = 8.1 Hz, 4-Cl-C₆H₄-C_3,5-H). Anal. Calc. (%) for C₁₅H₁₁ClF₃NO (313.7): C, 57.43; H, 3.53; N, 4.46. Found (%): C, 57.52; H, 3.55; N, 4.48.

3.1.2. 1-(4-Chlorophenyl)-2-(Pyridin-3-Ylamino)Ethanone; 3b

An equimolar mixture of 4-chlorophenacyl bromide (0.38 g, 2 mmol.) and 3-aminopyridine (0.19 g, 2 mmol.) were stirred for 2 h. at room temperature in (20 mL) diethyl ether. The solid product was collected by filtration and recrystallized from the ethanol.

Buff needle crystals; yield (95%); m.p.: 205 ˚C. IR [KBr, cm⁻¹]: 3332, 3220 (NH); 3060 (CH aromatic); 2929 (CH aliphatic); 1693 (C=O); 1636 (C=N); 1585 (C=C); 1084 (p-Cl-phenyl); MS m/z (relative intensity %): 248 (M^+•+2, 3); 246 (M^+•, 11); 139 (100). Anal. Calc. (%) for C₁₃H₁₁ClN₂O (246.7): C, 63.29; H, 4.49; N, 11.36. Found (%): C, 63.32; H, 4.57; N, 11.33.

3.1.3. General Procedure for Synthesis of Compounds 4a-d

The selected 2-(substitutedamino)-1-(4-chlorophenyl)ethanones 3a,b (0.01 mol.) was stirred at room temperature with the appropriate nitrile (0.01 mol.) namely; malononitrile and ethylcyanoacetate in (40 mL) sodium ethoxide [prepared by dissolving sodium metal (0.23 g, 0.01 mol.) in absolute ethanol (40 mL)] overnight. The obtained product was filtered, washed with water then recrystallized from glacial acetic acid.

1) 2-Amino-4-(4-chlorophenyl)-1-(3-trifluoromethylphenyl)-1H-pyrrole-3-carbonitrile; 4a.

Yellow needle crystals; yield (63%); m.p.: >300˚C. IR [KBr, cm⁻¹]: 3427, 3300 (NH₂); 2900 (CH aliphatic); 2224 (C≡N); 1561 (C=C); 1023 (p-Cl-phenyl). ¹H NMR(DMSO-d₆, δ ppm): 7.07 (s, 1 H, CH-pyrrole); 7.22 (d, 2 H, J = 8.1 Hz, 4-Cl-C₆H₄-C_2,6-H); 7.31-7.38 (m, 3 H, 3-CF₃-C₆H₄-C_4,5,6-H); 7.64 (s, 1 H, 3-CF₃-C₆H₄-C₂-H); 7.86 (d, 2 H, J = 8.1 Hz, 4-Cl-C₆H₄-C_3,5-H); 8.45 (s, 2 H, NH₂, D₂O exchangeable). ¹³C NMR (DMSO-d₆, δ ppm): 108 (pyrrole-C₃); 112 (3-CF₃-C₆H₄-C₂); 115 (C≡N); 118 (pyrrole-C₅); 120 (pyrrole-C₂); 122 (3-CF₃- C₆H₄-C₄); 125.7 (CF₃); 127.5 (3-CF₃-C₆H₄-C₆); 127.6 (pyrrole-C₄); 127.9 (4-Cl-C₆H₄-C_2,6); 128.1 (4-Cl-C₆H₄- C_3,5); 128.5 (3-CF₃-C₆H₄-C₅); 131.4 (3-CF₃-C₆H₄-C₃); 132.2 (4-Cl-C₆H₄-C₄); 132.5 (4-Cl-C₆H₄-C₁); 142 (3-CF₃-C₆H₄-C₁); MS m/z (relative intensity %): 363 (M^+•+2) (0.3); 361 (M^+•) (0.2); 76 (100). Anal. Calc. (%) for C₁₈H₁₁ClF₃N₃ (361.7): C, 59.76; H, 3.06; N, 11.62. Found (%): C, 59.80; H, 3.14; N, 11.68.

2) 2-Amino-4-(4-chlorophenyl)-1-(pyridin-3-yl)-1H-pyrrole-3-carbonitrile; 4b.

Dark grey crystals; yield (77%); m.p.: >300˚C. IR [KBr, cm⁻¹]: 3445, 3200 (NH₂); 3080 (CH aromatic); 2880 (CH aliphatic); 2191 (C≡N); 1638 (C=N); 1598 (C=C); 1091 (p-Cl-phenyl); MS m/z (relative intensity %): 294 (M^+•, 3); 112 (100). Anal. Calc. (%) for C₁₆H₁₁ClN₄ (294.7): C, 65.20; H, 3.76; N, 19.01. Found (%): C, 65.83; H, 4.01; N, 19.54.

3) Ethyl 2-amino-4-(4-chlorophenyl)-1-(3-trifluoromethylphenyl)-1H-pyrrole-3-carboxylate; 4c

Orange needle crystals; yield (57%); m.p.: 250˚C - 252˚C C. IR [KBr, cm⁻¹]: 3442, 3200 (NH₂); 3060 (CH aromatic); 2960 (CH aliphatic); 1728 (C=O); 1280, 1060 (C-O-C); 1121 (p-Cl-phenyl). MS m/z (relative intensity %): 410 (M^+•+2, 2); 409 (M^+•+1, 0.4); 161 (100). Anal. Calc. (%) for C₂₀H₁₆ClF₃N₂O₂ (408.8): C, 58.76; H, 3.94; N, 6.85. Found (%): C, 58.80; H, 4.01; N, 6.82.

4) Ethyl 2-amino-4-(4-chlorophenyl)-1-(pyridin-3-yl)-1H-pyrrole-3-carboxylate; 4d

Brown crystals; yield (69%); m.p.: >300˚C. IR [KBr, cm⁻¹]: 3360, 3250 (NH₂); 3002 (CH aromatic); 2819 (CH aliphatic); 1720 (C=O); 1641 (C=N); 1530 (C=C); 1259, 1089 (C-O-C); 1147 (p-Cl-phenyl). ¹H NMR (DMSO-d₆, δ ppm): 1.25 - 1.30 (m, 3 H, CH₂CH₃); 3.50 - 3.59 (m, 2 H, CH₂CH₃); 6.72 (s, 2 H, NH₂, D₂O exchangeable); 7.30 (d, 2 H, J = 8.3 Hz, 4-Cl-C₆H₄-C_2,6-H); 7.55 - 7.58 (m, 1 H, pyridyl-C₅-H); 7.64 - 7.72 (m, 1 H, pyridyl-C₆-H); 7.85 (d, 2 H, J = 8.3 Hz, 4-Cl-C₆H₄-C_3,5-H); 7.92 (s, 1 H, CH-pyrrole); 8.03 - 8.06 (m, 1 H, pyridyl-C₄-H); 8.51 (s, 1 H, pyridyl-C₂-H). Anal. Calc. (%) for C₁₈H₁₆ClN₃O₂ (341.8): C, 63.25; H, 4.72; N, 12.29. Found (%): C, 63.75; H, 4.98; N, 12.78.

3.1.4. General Procedure for Synthesis of Compounds 5a-d

An equimolar mixture of the appropriate 1-(substitutedaryl)-2-amino-4-(4-chlorophenyl)-1H-pyrrole-3-carboni- trile 4a,b (2 mmol.) and the selected aromatic aldehyde namely; benzaldehyde and 2,4-dichlorobenzaldehyde (2 mmol.) was heated under reflux for 16 h in absolute ethanol (30 mL) containing glacial acetic acid (2 - 3 drops). The reaction mixture was allowed to cool and the product was filtered off, washed with ethanol and recrystallized from dioxane.

1) 2-(Benzylidenimino)-4-(4-chlorophenyl)-1-(3-trifluoromethylphenyl)-1H-pyrrole-3-carbonitrile; 5a

Faint yellow needle crystals; yield (55%); m.p.: 280˚C - 282˚C. IR [KBr, cm⁻¹]: 2927 (CH aromatic); 2863 (CH aliphatic); 2223 (C≡N); 1634 (C=N); 1560 (C=C); 1023 (p-Cl-phenyl). ¹H NMR(DMSO-d₆, δ ppm): 6.77 - 6.88 (m, 3 H, C₆H₅-C_3,4,5-H); 7.03 (d, 2 H, J = 8.1Hz, 4-Cl-C₆H₄-C_2,6-H); 7.25 (s, 1 H, CH-pyrrole); 7.30 (d, 2 H, J = 8.1 Hz, 4-Cl-C₆H₄-C_3,5-H); 7.34 - 7.37 (m, 1 H, 3-CF₃-C₆H₄-C₅-H); 7.45 - 7.50 (m, 2 H, 3-CF₃-C₆H₄-C_4,6-H); 7.85 (d, 2 H, J = 8.1 Hz,C₆H₅, C_2,6-H); 8.36 (s, 1 H, CH-benzylidinimine); 8.49 (s, 1 H, 3-CF₃-C₆H₄-C₂-H); MS m/z (relative intensity %): 450 (M^+•+1) (0.4); 449 (M^+•, 0.5); 448 (M^+•-1, 0.5); 83 (100). Anal. Calc. (%) for C₂₅H₁₅ClF₃N₃ (449.9): C, 66.75; H, 3.36; N, 9.34. Found (%): C, 66.82; H, 3.58; N, 9.74.

2) 2-(Benzylidenimino)-4-(4-chlorophenyl)-1-(pyridin-3-yl)-1H-pyrrole-3-carbonitrile; 5b

Dark brown powder; yield (67%); m.p.: >300˚C. IR [KBr, cm⁻¹]: 2926 (CH aromatic); 2864 (CH aliphatic); 2207 (C≡N); 1585 (C=N); 1562 (C=C); 1105 (p-Cl-phenyl); MS m/z (relative intensity %): 384 (M^+•+2, 0.5); 383 (M^+•+1, 0.1); 382 (M^+•, 0.1); 67 (100). Anal. Calc. (%) for C₂₃H₁₅ClN₄ (382.8): C, 72.16; H, 3.95; N, 14.63. Found (%): C, 72.52; H, 4.01; N, 14.60.

3) 2-(2,4-Dichlorobenzylidenimino)-4-(4-chlorophenyl)-1-(3-trifluoromethylphenyl)-1H-pyrrole-3-carbonitrile; 5c

Yellow needle crystals; yield (49%); m.p.: 276˚C - 278˚C. IR [KBr, cm⁻¹]: 2933 (CH aromatic); 2850 (CH aliphatic); 2220 (C≡N); 1561 (C=N); 1024 (p-Cl-phenyl). ¹H NMR(DMSO-d₆, δ ppm): 7.30 (s, 1 H, CH-pyr- role); 7.33 - 7.36 (m, 1 H, 3-CF₃-C₆H₄-C₅-H); 7.42 - 7.51 (m, 3 H, 2,4-(Cl)₂-C₆H₃-C_3,5,6-H); 7.54 - 7.59 (m, 2 H, 3-CF₃-C₆H₄-C_4,6-H); 7.57 (d, 2 H, J = 8.4 Hz, 4-Cl-C₆H₄-C_2,6-H); 7.81 (d, 2 H, J = 8.4 Hz, 4-Cl-C₆H₄-C_3,5-H); 7.86 (s, 1 H, 3-CF₃-C₆H₄-C₂-H); 8.36 (s, 1 H, CH-benzylidinimine). Anal. Calc. (%) for C₂₅H₁₃Cl₃F₃N₃ (518.7): C, 57.88; H, 2.53; N, 8.10. Found (%): C, 57.65; H, 2.87; N, 8.59.

4) 2-(2,4-Dichlorobenzylidenimino)-4-(4-chlorophenyl)-1-(pyridin-3-yl)-1H-pyrrole-3-carbonitrile; 5d

Dark brown needle crystals; yield (85%); m.p.:240˚C - 242˚C. IR [KBr, cm⁻¹]: 3030 (CH aromatic); 2900 (CH aliphatic); 2199 (C≡N); 1620 (C=N); 1591 (C=C); 1094 (p-Cl-phenyl); MS m/z (relative intensity %): 452 (M^+•+2, 1); 95 (100). Anal. Calc. (%) for C₂₃H₁₃Cl₃N₄ (451.7): C, 61.15; H, 2.90; N, 12.40. Found (%): C, 61.45; H, 3.02; N, 12.56.

3.1.5. General Procedure for Synthesis of Compounds 6a,b

An equimolar mixture of the selected 1-(substitutedaryl)-2-amino-4-(4-chlorophenyl)-1H-pyrrole-3-carbonitrile 4a,b (2 mmol.) and ethyl cyanoacetate (0.23 g, 0.1 mL, 2 mmol.) was heated under reflux in absolute ethanol (30 mL) for 8 h. The reaction mixture was allowed to cool and the product was collected and recrystallized from glacial acetic acid.

1) 4-Amino-3-(4-chlorophenyl)-6-oxo-1-(3-trifluoromethylphenyl)-6,7-dihydro-1H-pyrrolo[2,3-b]pyridine-5- carbonitrile; 6a

Pale yellow needle crystals; yield (77%); m.p.: >300˚C. IR [KBr, cm⁻¹]: 3490, 3461 (br. OH tautomer); 3454, 3366, 3316 (NH₂, NH); 2960 (CH aromatic); 2200 (C≡N); 1660 (C=O); 1566 (C=C); 1022 (p-Cl-phenyl). ¹H NMR(DMSO-d₆, δ ppm): 4.93 (s, 2 H, NH₂, D₂O exchangeable); 7.08 (s, 1 H, CH-pyrrole); 7.34 - 7.38 (m, 1 H, 3-CF₃-C₆H₄-C₅-H); 7.48-7.62 (m, 2 H, 3-CF₃-C₆H₄-C_4,6-H); 7.64 (d, 2 H, J = 8.4 Hz, 4-Cl-C₆H₄-C_2,6-H); 7.86 (d, 2 H, J = 8.1 Hz, 4-Cl-C₆H₄-C_3,5-H); 7.90 (s, 1/2 H, NH, D₂O exchangeable); 8.45 (s, 1 H, 3-CF₃-C₆H₄-C₂-H); 10.28 (s, ½ H, OH tautomer, D₂O exchangeable). Anal. Calc. (%) for C₂₁H₁₂ClF₃N₄O (428.8): C, 58.82; H, 2.82; N, 13.07. Found (%): C, 58.93; H, 2.88; N, 13.18.

2) 4-Amino-3-(4-chlorophenyl)-6-oxo-1-(pyridin-3-yl)-6,7-dihydro-1H-pyrrolo[2,3-b]pyridine-5-carbonitrile; 6b

Pale brown powder; yield (40%); m.p.: >300˚C. IR [KBr, cm⁻¹]: 3462, 3412 (br. OH tautomer); 3380, 3330, 3292, 3225 (NH₂, NH); 2920 (CH aromatic); 2206 (C≡N); 1680 (C=O); 1634 (C=N); 1525 (C=C); 1092 (p- Cl-phenyl); MS m/z (relative intensity %): 361 (M^+•, 2); 360 (M^+•-1, 3); 57 (100). Anal. Calc. (%) for C₁₉H₁₂ClN₅O (361.8): C, 63.08; H, 3.34; N, 19.36. Found (%): C, 63.17; H, 3.76; N, 19.51.

3.1.6. General Procedure for Synthesis of Compounds 7a,b

Equimolar amounts of the appropriate 1-(substitutedaryl)-2-amino-4-(4-chlorophenyl)-1H-pyrrole-3-carbonitrile 4a,b (2 mmol.) and malononitrile (0.13 g, 0.1 mL, 2 mmol.) were heated under reflux in absolute ethanol (20 mL) for 14 h. The reaction mixture was allowed to cool and the product was collected, washed with ethanol and recrystallized from dioxane.

1) 4,6-Diamino-3-(4-chlorophenyl)-1-(3-trifluoromethylphenyl)-1H-pyrrolo[2,3-b]pyridine-5-carbonitrile; 7a

Golden yellow needle crystals; yield (88%); m.p.: >300˚C. IR [KBr, cm⁻¹]: 3375, 3325 (NH₂); 2928 (CH aromatic); 2200 (C≡N); 1646 (C=N); 1560 (C=C); 1024 (p-Cl-phenyl).¹H NMR(DMSO-d₆, δ ppm): 4.18 (s, 2 H, pyridine-C₄-NH₂, D₂O exchangeable); 5.19 (s, 2 H, pyridine-C₂-NH₂, D₂O exchangeable); 6.83 (s, 1 H, CH- pyrrole); 7.10 - 7.13 (m, 1 H, 3-CF₃-C₆H₄-C₅-H); 7.31 (d, 2 H, J = 8.1 Hz, 4-Cl-C₆H₄-C_2,6-H); 7.34 - 7.47 (m, 2 H, 3-CF₃-C₆H₄-C_4,6-H); 7.82 (d, 2 H, J = 8.1 Hz, 4-Cl-C₆H₄-C_3,5-H); 8.38 (s, 1 H, 3-CF₃-C₆H₄-C₂-H). Anal. Calc. (%) for C₂₁H₁₅ClF₃N₅ (429.8): C, 58.96; H, 3.06; N, 16.37. Found (%): C, 58.74; H, 3.21; N, 16.34.

2) 4,6-Diamino-3-(4-chlorophenyl)-1-(pyridin-3-yl)-1H-pyrrolo-[2,3-b]pyridine-5-carbonitrile; 7b

Buff powder; yield (58%); m.p.: >300˚C. IR [KBr, cm⁻¹]: 3428, 3364 (NH₂); 3066 (CH aromatic); 2198 (C≡N); 1638 (C=N); 1550 (C=C); 1092 (p-Cl-phenyl); MS m/z (relative intensity %): 363 (M^+•+3, 2); 58 (100). Anal. Calc. (%) for C₁₉H₁₅ClN₆ (362.8): C, 63.25; H, 3.63; N, 23.29. Found (%): C, 63.92; H, 3.48; N, 23.20.

3.1.7. General Procedure for Synthesis of Compounds 8a,b

A mixture of the selected 1-(substitutedaryl)-2-amino-4-(4-chlorophenyl)-1H-pyrrole-3-carbonitrile 4a,b (2 mmol.) and hydroxylamine hydrochloride (0.14 g, 2 mmol.) in (30 mL) sodium ethoxide [prepared by dissolving sodium metal (0.06 g, 2 mmol.) in absolute ethanol (30 mL)] was refluxed for 12 h. Then the reaction was concentrated under reduced pressure and the residue was collected by filtration, washed thoroughly with water and recrystallized from ethanol/benzene mixture.

1) 4-(4-Chlorophenyl)-6-(3-trifluoromethylphenyl)-1,6-dihydropyrrolo[2,3-c]pyrazol-3-amine; 8a

Yellow powder; yield (75%); m.p.: 293˚C - 295˚C. IR [KBr, cm⁻¹]: 3418, 3290, 3240 (NH₂, NH); 3000, 2925 (CH aromatic); 1650 (C=N); 1560 (C=C); 1090 (p-Cl-phenyl); MS m/z (relative intensity %): 378 (M^+•+2, 1); 377(M^+•+1, 1); 376 (M^+•, 1); 58 (100). Anal. Calc. (%) for C₁₈H₁₂ClF₃N₄ (376.8): C, 57.38; H, 3.21; N, 14.87. Found (%): C, 57.45; H, 3.36; N, 14.92.

2) 4-(4-Chlorophenyl)-6-(pyridin-3-yl)-1,6-dihydropyrrolo[2,3-c]pyrazol-3-amine; 8b

Pale brown powder; yield (64%); m.p.: > 300˚C. IR [KBr, cm⁻¹]: 3428, 3332, 3270, 3214, 3132 (NH₂, NH), 3040 (CH aromatic); 1636 (C=N); 1550 (C=C); 1094 (p-Cl-phenyl). ¹H NMR(DMSO-d₆, δ ppm): 5.27 (s, 2 H, NH₂, D₂O exchangeable); 6.77 (s, 1 H, CH-pyrrole); 7.33 (d, 2 H, J = 7.8 Hz, 4-Cl-C₆H₄-C_2,6-H); 7.56 - 7.70 (m, 3 H, pyridyl-C_4,5,6-H); 7.87 (d, 2 H, J = 7.8 Hz, 4-Cl-C₆H₄-C_3,5-H); 7.92 (s, 1 H, pyridyl-C₂-H); 8.06 (s, 1 H, NH, D₂O exchangeable). Anal. Calc. (%) for C₁₆H₁₂ClN₅ (309.8): C, 62.04; H, 3.90; N, 22.61 Found (%): C, 62.10; H, 3.93; N, 22.69.

3.1.8. General Procedure for Synthesis of Compounds 9a-d

The appropriate compound 4a,b (2 mmol.) was refluxed with equimolar amount of urea or thiourea (2 mmol.) in absolute ethanol (20 mL) containing sodium ethoxide [prepared by dissolving sodium metal (0.03 g, 2 mmol.) in absolute ethanol (20 mL)] for 10 h. The reaction was allowed to cool and the solid product was filtered and washed with ethanol.

1) 1-[4-(4-Chlorophenyl)-3-cyano-1-(3-trifluoromethylphenyl)-1H-pyrrol-2-yl]urea; 9a

Pale yellow crystals; gl. acetic; yield (80%); m.p.: >300˚C. IR [KBr, cm⁻¹]: 3324, 3248, 3164 (NH₂, NH); 3092 (CH aromatic); 2225 (C≡N); 1700 (C=O); 1562 (C=C); 1024 (p-Cl-phenyl);); MS m/z (relative intensity %): 405 (M^+•+1, 0.2); 404 (M^+•, 0.2); 57 (100). Anal. Calc. (%) for C₁₉H₁₂ClF₃N₄O (404.8): C, 56.38; H, 2.99; N, 13.84. Found (%): C, 56.44; H, 3.05; N, 13.96.

2) 1-[4-(4-Chlorophenyl)-3-cyano-1-(pyridin-3-yl)-1H-pyrrol-2-yl]urea; 9b

Buff powder; gl. acetic; yield (67%); m.p.: 324˚C - 326˚C. IR [KBr, cm⁻¹]: 3375, 3310, 3244, 3200 (NH₂, NH); 3025 (CH aromatic); 2225 (C≡N); 1680 (C=O); 1636 (C=N);1456 (C=C); 1100 (p-Cl phenyl). ¹H NMR(DMSO-d₆, δ ppm): 5.46 (s, 2 H, NH₂, D₂O exchangeable); 6.70 (s, 1 H, CH-pyrrole); 7.30 (d, 2 H, J = 8.1 Hz, 4-Cl-C₆H₄-C_2,6-H); 7.57 - 7.65 (m, 3 H, pyridyl-C_4,5,6-H); 7.84 (d, 2 H, J = 8.1 Hz, 4-Cl-C₆H₄-C_3,5-H); 8.04 (s, 1 H, NH, D₂O exchangeable); 8.48 (s, 1 H, pyridyl-C₂-H). ¹³C NMR (DMSO-d₆, δ ppm): 106.2 (pyrrole-C₃); 114 (C≡N); 117 (pyrrole-C₅); 119 (pyrrole-C₂); 124 (pyrrole-C₄); 124.4 (pyridyl-C₅); 126.7 (pyridyl- C₆); 127.1 (4-Cl-C₆H₄-C_2,6); 127.6 (4-Cl-C₆H₄-C_3,5); 130.8 (4-Cl-C₆H₄-C₄); 131.6 (4-Cl-C₆H₄-C₁); 133.5 (pyridyl-C₁); 138.9 (pyridyl-C₂); 148.4 (pyridyl-C₄); 159.8 (C=O) of amide. Anal. Calc. (%) for C₁₇H₁₂ClN₅O (337.8): C, 60.45; H, 3.58; N, 20.73. Found (%): C, 60.71; H, 3.63; N, 20.78.

3) 1-[4-(4-Chlorophenyl)-3-cyano-1-(3-trifluoromethylphenyl)-1H-pyrrol-2-yl]thiourea; 9c

Yellow powder; petroleum ether 60/80; yield (54%); m.p.: >300˚C. IR [KBr, cm⁻¹]: 3413, 3250, 3183 (NH₂, NH); 2933 (CH aromatic); 2259 (C≡N); 1562 (C=C); 1562, 1416, 1100, 922 (I, II, III, IV bands N-C=S); 1025 (p-Cl-phenyl). ¹H NMR(DMSO-d₆, δ ppm): 5.47 (s, 2 H, NH₂, D₂O exchangeable); 7.05 (s, 1 H, CH-pyrrole); 7.12-7.15 (m, 1 H, 3-CF₃-C₆H₄-C₅-H); 7.30 (d, 2 H, J = 8.1 Hz, 4-Cl-C₆H₄-C_2,6-H); 7.34-7.47 (m, 2 H, 3-CF₃- C₆H₄-C_4,6-H); 7.82 (d, 2 H, J = 8.1 Hz, 4-Cl-C₆H₄-C_3,5-H); 8.40 (s, 1 H, 3-CF₃-C₆H₄-C₂-H); 10.62 (s, 1 H, NH, D₂O exchangeable). Anal. Calc. (%) for C₁₉H₁₂ClF₃N₄S (420.8): C, 54.23; H, 2.87; N, 13.31. Found (%): C, 54.27; H, 2.93; N, 13.56.

4) 1-[4-(4-Chlorophenyl)-3-cyano-1-(pyridin-3-yl)-1H-pyrrol-2-yl]thiourea; 9d

Pale browncrystals; dioxane; yield (89%); m.p.: 334˚C - 336˚C. IR [KBr, cm⁻¹]: 3424, 3380, 3330 (NH₂, NH); 3020 (CH aromatic); 2196 (C≡N); 1628 (C=N); 1552 (C=C); 1500, 1280, 1098, 1006 (I, II, III, IV bands N-C=S); 1110 (p-Cl-phenyl); MS m/z (relative intensity %): 355 (M^+•+2, 0.5); 353 (M^+•, 1); 352 (M^+•−1, 1); 57 (100). Anal. Calc. (%) for C₁₇H₁₂ClN₅S (353.8): C, 57.71; H, 3.42; N, 19.79. Found (%): C, 57.80; H, 3.45; N, 19.88.

3.1.9. General Procedure for Synthesis of Compounds 10a-d

Method 1:

A mixture of the selected 1-(substitutedaryl)-2-amino-4-(4-chlorophenyl)-1H-pyrrole-3-carbonitrile 4a,b (2 mmol.) was refluxed and urea (0.12 g, 2 mmol.) and/or thiourea (0.15 g, 2 mmol.) was refluxed for 12 h. in a mixture of glacial acetic acid and HCl (20 mL) (3:1). The reaction was allowed to cool, filtered and washed with ethanol to yield the target compounds 10a-d in an average yield of 82%.

Method 2:

The selected urea or thiourea derivative 9a-d (2 mmol.) was refluxed in pyridine (10 mL) for 16 h. The solvent was evaporated under reduced pressure and the solid obtained was collected to yield the target compounds 10a-d in an average yield of 63%.

1) 4-Amino-5-(4-chlorophenyl)-7-(3-trifluoromethylphenyl)-1H-pyrrolo[2,3-d]pyrimidine-2(7H)-one; 10a

Light brown crystals; Ethanol/Benzene; yield (73%); m.p.: >300˚C. IR [KBr, cm⁻¹]: 3450 (br. OH tautomer); 3356, 3211 (NH₂, NH); 3080 (CH aromatic); 1654 (C=O); 1458 (C=C); 1100 (p-Cl-phenyl); MS m/z (relative intensity %): 406 (M^+•+2, 1); 405 (M^+•+1, 1); 404 (M^+•, 1); 61 (100). Anal. Calc. (%) for C₁₉H₁₂ClF₃N₄O (404.8): C, 56.38; H, 2.99; N, 13.84. Found (%): C, 56.43; H, 3.07; N, 13.93.

2) 4-Amino-5-(4-chlorophenyl)-7-(pyridin-3-yl)-1H-pyrrolo[2,3-d]pyrimidine-2(7H)-one; 10b

Pale brownpowder; dioxane; yield (40%); m.p.: 286˚C - 288˚C. IR [KBr, cm⁻¹]: 3480 (br. OH tautomer); 3420, 3223 (NH₂, NH); 3100 (CH aromatic); 1690 (C=O); 1095 (p-Cl-phenyl). ¹H NMR (DMSO-d₆, δ ppm): 4.15 (s, 2 H, NH₂, D₂O exchangeable); 4.94 (s, 1/2 H, NH, D₂O exchangeable); 7.39 - 7.41 (m, 3 H, pyridyl- C_4,5,6-H); 7.42 (s, 1 H, CH-pyrrole); 7.50 (d, 2 H, J = 8.4 Hz, 4-Cl-C₆H₄-C_2,6-H); 7.83 (s, 1/2 H, OH, tautomer, D₂O exchangeable); 8.05 (s, 1 H, pyridyl-C₂-H); 8.17 (d, 2 H, J = 8.4 Hz, 4-Cl-C₆H₄-C_3,5-H). Anal. Calc. (%) for C₁₇H₁₂ClN₅O (337.8): C, 60.45; H, 3.58; N, 20.73. Found (%): C, 60.62; H, 3.60; N, 20.81.

3) 4-Amino-5-(4-chlorophenyl)-7-(3-trifluoromethylphenyl)-1H-pyrrolo[2,3-d]pyrimidine-2(7H)-thione; 10c

Pale yellowpowder; Ethanol; yield (65%); m.p.: >360˚C. IR [KBr, cm⁻¹]: 3367, 3265 (NH₂, NH); 3164 (CH aromatic); 1480, 1328, 1120, 1097 (I, II, III, IV bands N-C=S); 1080 (p-Cl-phenyl). ¹H NMR(DMSO-d₆, δ ppm): 4.76 (s, 1 H, NH, D₂O exchangeable); 6.37 (s, 2 H, NH₂, D₂O exchangeable); 6.85 (d, 1 H, J = 7.2 Hz, 3- CF₃-C₆H₄-C₆-H); 6.95 (d, 2 H, J = 8.3 Hz, 4-Cl-C₆H₄-C_2,6-H); 7.00 (s, 1 H, CH-pyrrole); 7.25 - 7.30 (m, 1 H, 3-CF₃-C₆H₄-C₅-H); 7.64 (d, 2 H, J = 8.3 Hz, 4-Cl-C₆H₄-C_3,5-H); 8.08 - 8.12 (m, 2 H, 3-CF₃-C₆H₄-C_2,4-H). ¹³C NMR(DMSO-d₆, δ ppm): 107.8 (pyrrolopyrimidine C_4a); 113 (3-CF₃-C₆H₄-C₂); 116.6 (pyrrolopyrimidine C₆); 120 (pyrrolopyrimidine C_7a); 121.9 (3-CF₃-C₆H₄-C₄); 124.1 (CF₃); 124.8 (3-CF₃-C₆H₄-C₆); 126 (pyrrolopyrimidine C₅); 128.9 (4-Cl-C₆H₄-C_2,6); 129.8 (4-Cl-C₆H₄-C_3,5-H); 130.9 (3-CF₃-C₆H₄-C₅); 131.04 (3-CF₃-C₆H₄-C₃); 131.08 (4-Cl-C₆H₄-C₄); 135.8 (4-Cl-C₆H₄-C₁); 145 (3-CF₃-C₆H₄-C₁); 157 (pyrrolopyrimidine C₄); 185 (C=S). Anal. Calc. (%) for C₁₉H₁₂ClF₃N₄S (420.8): C, 54.23; H, 2.87; N, 13.31. Found (%): C, 54.27; H, 2.91; N, 13.46.

4) 4-Amino-5-(4-chlorophenyl)-7-(pyridin-3-yl)-1H-pyrrolo[2,3-d]pyrimidine-2(7H)-thione; 10d

Pale brown powder; Ethanol; yield (83%); m.p.: >300˚C. IR [KBr, cm⁻¹]: 3428, 3350, 3220 (NH₂, NH); 3080 (CH aromatic); 1624 (C=N); 1404, 1316, 1150, 1000 (I, II, III, IV bands N-C=S); 1100 (p-Cl-phenyl); MS m/z (relative intensity %): 355 (M^+•+2, 0.6); 354 (M^+•+1, 1); 353 (M^+•, 2); 58 (100). Anal. Calc. (%) for C₁₇H₁₂ClN₅S (353.8): C, 57.71; H, 3.42; N, 19.79. Found (%): C, 57.80; H, 3.51; N, 19.84.

3.1.10. General Procedure for Synthesis of Compounds 11a,b

A mixture of the appropriate 1-(substitutedaryl)-2-amino-4-(4-chlorophenyl)-1H-pyrrole-3-carbonitrile 4a,b (2 mmol.) and phenyl isothiocyanate (0.27 g, 0.24 mL, 2 mmol.) was refluxed for 7 h in absolute ethanol (30 mL) and the solid product was filtered and washed with ethanol.

1) 1-[4-(4-Chlorophenyl)-3-cyano-1-(3-trifluoromethylphenyl)-1H-pyrrol-2-yl]-3-phenylthiourea; 11a

Yellow needle crystals; gl.acetic; yield (57%); m.p.: >360˚C. IR [KBr, cm⁻¹]: 3372, 3286 (NH); 3050 (CH aromatic); 2200 (C≡N); 1562 (C=C); 1418, 1282, 1170, 1020 (I, II, III, IV bands N-C=S); 1098 (p-Cl-phenyl). ¹H NMR (DMSO-d₆, δ ppm): 6.90 (s, 1 H, CH-pyrrole); 7.10 - 7.13 (m, 3 H, C₆H₅-C_3,4,5-H); 7.30 (d, 2 H, J = 8.4 Hz, 4-Cl-C₆H₄-C_2,6-H); 7.34-7.39 (m, 3 H, 3-CF₃-C₆H₄-C_4,5,6-H); 7.47 (d, 2 H, J = 8.4 Hz, 4-Cl-C₆H₄-C_3,5-H); 7.80 (d, 2 H, J = 7.2 Hz, C₆H₅-C_2,6-H); 7.83 (s, 1 H, 3-CF₃-C₆H₄-C₂-H); 8.70 (s, 1 H, pyrrole-C₂-NH, D₂O exchangeable); 9.59 (s, 1 H, NH, D₂O exchangeable). Anal. Calc. (%) for C₂₅H₁₆ClF₃N₄S (496.9): C, 60.42; H, 3.25; N, 11.27. Found (%): C, 60.44; H, 3.35; N, 11.31.

2) 1-[4-(4-Chlorophenyl)-3-cyano-1-(pyridin-3-yl)-1H-pyrrol-2-yl]-3-phenylthiourea; 11b

Buff crystals; DMF; yield (70%); m.p.: 288˚C - 290˚C. IR [KBr, cm⁻¹]: 3374, 3326, 3260, 3200 (NH); 3090 (CH aromatic); 2206 (C≡N); 1640 (C=N); 1566 (C=C); 1420, 1396, 1250, 1110 (I, II, III, IV bands N-C=S); 1098 (p-Cl-phenyl); MS m/z (relative intensity %): 429 (M^+•, 2); 83 (100). Anal. Calc. (%) for C₂₃H₁₆ClN₅S (429.9): C, 64.25; H, 3.75; N, 16.29. Found (%): C, 64.29; H, 3.77; N, 16.32.

3.1.11. General Procedure for Synthesis of Compounds 12a,b

Method 1:

A mixture of the appropriate 1-(substitutedaryl)-2-amino-4-(4-chlorophenyl)-1H-pyrrole-3-carbonitrile 4a,b (2 mmol.) and phenyl isothiocyanate (0.27 g, 0.24 mL, 2 mmol.) was refluxed in pyridine (15 mL) for 10 h and the solid product was filtered, washed with ethanol to yield the target compounds 12a,b in an average yield of 53%.

Method 2:

The selected phenyl thiourea derivatives 11a,b was refluxed in pyridine (10 mL) for 18 h. The solvent was evaporated under reduced pressure to yield the target compounds 12a,b in an average yield of 48%.

1) 5-(4-Chlorophenyl)-4-imino-3-phenyl-7-(3-trifluoromethylphenyl)-3,4-dihydro-1H-pyrrolo[2,3-d]pyrimi dine- 2(7H)-thione; 12a

Light brown crystals; dioxane; yield (68%); m.p.: >300˚C. IR [KBr, cm⁻¹]: 3190 (NH); 3050 (CH aromatic); 1546 (C=C); 1434, 1332, 1180, 1020 (I, II, III, IV bands N-C=S); 1100 (p-Cl-phenyl); MS m/z (relative intensity %): 496 (M^+•, 1); 140 (100). Anal. Calc. (%) for C₂₅H₁₆ClF₃N₄S (496.9): C, 60.42; H, 3.25; N, 11.27. Found (%): C, 60.51; H, 3.28; N, 11.29.

2) 5-(4-Chlorophenyl)-4-imino-3-phenyl-7-(pyridin-3-yl)-3,4-dihydro-1H-pyrrolo[2,3-d]pyrimidine-2(7H)-thione; 12b

Dark grey powder; dioxane; yield (59%); m.p.: > 300˚C. IR [KBr, cm⁻¹]: 3317, 3191 (NH); 3020 (CH aromatic); 1625 (C=N); 1520 (C=C); 1406, 1250, 1134, 1026 (I, II, III, IV bands N-C=S); 1080 (p-Cl-phenyl). ¹H NMR (DMSO-d₆, δ ppm): 5.30 (s, 1 H, NH, D₂O exchangeable); 6.63 (s, 1 H, CH-pyrrole); 6.85 - 7.05 (m, 3 H, C₆H₅-C_3,4,5-H); 7.30 - 7.50 (m, 2 H, C₆H₅-C_2,6-H); 7.56 (d, 2 H, J = 8.4 Hz, 4-Cl-C₆H₄-C_2,6-H); 7.62 - 7.78 (m, 2 H, pyridyl-C_5,6-H); 7.94 (d, 2 H, J = 8.4 Hz, 4-Cl-C₆H₄-C_3,5-H); 7.99 - 8.03 (m, 2 H, pyridyl-C_2,4-H); 10.58 (s, 1 H, imine NH, D₂O exchangeable). Anal. Calc. (%) for C₂₃H₁₆ClN₅S (429.9): C, 64.25; H, 3.75; N, 16.29. Found (%): C, 64.29; H, 3.80; N, 16.36.

3.1.12. General Procedure for Synthesis of Compounds 13a,b

A mixture of the appropriate 1-(substitutedaryl)-2-amino-4-(4-chlorophenyl)-1H-pyrrole-3-carbonitrile 4a,b (2 mmol.) and triethyl orthoformate (0.29 g, 0.33 mL, 2 mmol.) in acetic anhydride (25 mL) was heated under reflux for 6 h. The reaction mixture was concentrated to the minimum and left to cool. The obtained product was collected washed with ethanol and recrystallized from dimethylformamide.

1) Ethyl N-4-(4-chlorophenyl)-3-cyano-1-(3-trifluoromethylphenyl)-1H-pyrrol-2-ylformimidate; 13a

Pale yellow crystals; yield (67%); m.p.: 298˚C - 300˚C. IR [KBr, cm⁻¹]: 3100 (CH aromatic); 2900 (CH-aliphatic); 2190 (C≡N); 1620 (C=N); 1560 (C=C); 1270, 1025 (C-O-C); 1090 (p-Cl-phenyl). ¹H NMR (DMSO-d₆, δ ppm): 1.17 (t, 3 H, J = 6.8 Hz, CH₂CH₃); 4.05 - 4.10 (m, 2 H, CH₂CH₃); 6.80 (s, 1 H, CH- pyrrole); 7.15 - 7.18 (m, 1 H, 3-CF₃-C₆H₄-C₅-H); 7.30 - 7.33 (m, 2 H, 3-CF₃-C₆H₄-C_4,6-H); 7.35 (s, 1 H, N=CH); 7.51(d, 2 H, J = 8.4 Hz, 4-Cl-C₆H₄-C_2,6-H); 7.81 (d, 2 H, J = 8.4 Hz, 4-Cl-C₆H₄-C_3,5-H); 8.36 (s, 1 H, 3-CF₃-C₆H₄-C₂-H). Anal. Calc. (%) for C₂₁H₁₅ClF₃N₃O (417.8): C, 60.37; H, 3.62; N, 10.06. Found (%): C, 60.41; H, 3.65; N, 10.12.

2) Ethyl N-4-(4-chlorophenyl)-3-cyano-1-(pyridin-3-yl)-1H-pyrrol-2-ylformimidate; 13b

Dark brown needle crystals; yield (49%); m.p.:< 300˚C. IR [KBr, cm⁻¹]: 3178 (CH aromatic); 2923, 2860 (CH-aliphatic); 2198 (C≡N); 1630 (C=N); 1557 (C=C); 1280, 1023 (C-O-C); 1023 (p-Cl-phenyl); MS m/z (relative intensity %): 352 (M^+•+2, 0.4); 351 (M^+•+1, 0.5); 350 (M^+•, 0.4); 349 (M^+•−1, 0.4); 58 (100). Anal. Calc. (%) for C₁₉H₁₅ClN₄O (350.8): C, 65.05; H, 4.31; N, 15.97. Found (%): C, 65.10; H, 4.37; N, 15.99.

3.1.13. General Procedure for Synthesis of Compounds 14a,b

Method 1:

The appropriate 1-(substitutedaryl)-2-amino-4-(4-chlorophenyl)-1H-pyrrole-3-carbonitrile 4a,b (2 mmol.) was refluxed in excess formamide (30 mL) for 10 h. The reaction mixture was concentrated to the minimum and left to cool then triturated with glacial acetic acid to yield the target compounds 14a,b in an average yield of 66%.

Method 2:

The selected ethyl N-1-(substitutedaryl)-4-(4-chlorophenyl)-3-cyano-1H-pyrrol-2-ylformimidate 13a,b (2 mmol.) was stirred in (100 mL) of methanolic ammonia solution [methanol: ammonia (2:1)] at room temperature for 7 h. The separated solid was then collected to yield the target compounds 14a,b in an average yield of 41%.

1) 5-(4-Chlorophenyl)-7-(3-trifluoromethylphenyl)-7H-pyrrolo[2,3-d]pyrimi-din-4-amine; 14a

Pale yellow powder; gl. acetic; yield (51%); m.p.:< 300˚C. IR [KBr, cm⁻¹]: 3408, 3350, 3240 (NH₂); 3020 (CH aromatic); 1525 (C=C); 1098 (p-Cl-phenyl); MS m/z (relative intensity %): 390 (M^+•+2, 2); 389 (M^+•+1, 1); 388 (M^+•, 1); 387 (M^+•−1, 2); 57 (100). Anal. Calc. (%) for C₁₉H₁₂ClF₃N₄ (388.8): C, 58.70; H, 3.11; N, 14.41. Found (%): C, 58.73; H, 3.18; N, 14.49.

2) 5-(4-Chlorophenyl)-7-(pyridin-3-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine; 14b

Dark brown powder; gl. acetic; yield (67%); m.p.: 320˚C - 322˚C. IR [KBr, cm⁻¹]: 3436, 3382 (NH₂); 3092 (CH aromatic); 1550 (C=N); 1450 (C=C); 1100 (p-Cl phenyl). ¹H NMR (DMSO-d₆, δ ppm): 7.38 (s, 2 H, NH₂, D₂O exchangeable); 7.41 - 7.50 (m, 2 H, pyridyl-C_4,5-H); 7.69 (s, 1 H, CH-pyrrole); 7.78 (d, 2 H, J = 8.4 Hz, 4-Cl-C₆H₄-C_2,6-H); 7.91-8.10 (m, 3 H, pyridyl-C₆-H & 4-Cl-C₆H₄-C_3,5-H); 8.87 (s, 1 H, pyridyl-C₂-H); 9.06 (s, 1 H, pyrimidine-C₂-H). Anal. Calc. (%) for C₁₇H₁₂ClN₅ (321.8): C, 63.46; H, 3.76; N, 21.77. Found (%): C, 63.53; H, 3.80; N, 21.80.

3.1.14. General Procedure for Synthesis of Compounds 15a,b

Method 1:

Equimolar amounts of the selected of 1-(substituted aryl)-2-amino-4-(4-chlorophenyl)-1H-pyrrole-3-carboni- trile 4a,b (2 mmol.) and N,N-dimethylformamide-dimethylacetal ( 0.24 g, 0.27 mL, 2 mmol.) in xylene (20 mL) were refluxed for 6 h. The reaction mixture was concentrated under reduced pressure and the obtained products were filtered, washed with ethanol to yield the target compounds 15a,b in an average yield of 45%.

Method 2:

The appropriate ethyl 1-(substitutedaryl)-N-4-(4-chlorophenyl)-3-cyano-1H-pyrrol-2-ylformimidate 13a,b (2 mmol.) and dimethylamine (0.09 g, 0.1 mL, 2 mmol.) were stirred for 2 h in absolute ethanol (50 mL). The obtained product was filtered off, washed with ethanol to yield the target products 15a,b in an average yield of 57%.

1) N'-[4-(4-Chlorophenyl)-3-cyano-1-(3-trifluoromethylphenyl)-1H-pyrrol-2-yl]-N,N-dimethylformimidamide; 15a

Faint yellow crystals; Ethanol/Benzene.; yield (67%); m.p.: <300˚C. IR [KBr, cm⁻¹]: 3040 (CH aromatic); 2900, 2850 (CH-aliphatic); 2220 (C≡N); 1648 (C=N); 1560 (C=C); 1024 (p-Cl-phenyl); MS m/z (relative intensity %): 418 (M^+•+2, 0.6); 415 (M^+•−1, 0.4); 284 (100). Anal. Calc. (%) for C₂₁H₁₆ClF₃N₄ (416.8): C, 60.51; H, 3.87; N, 13.44. Found (%): C, 60.55; H, 3.93; N, 13.51.

2) N'-[4-(4-Chlorophenyl)-3-cyano-1-(pyridin-3-yl)-1H-pyrrol-2-yl]-N,N-dimethylformimidamide; 15b

Buff crystals; Ethanol/Benzene.; yield (59%); m.p.: 288˚C - 290˚C. IR [KBr, cm⁻¹]: 3030 (CH aromatic); 2930, 2840 (CH-aliphatic); 2202 (C≡N); 1648 (C=N); 1564 (C=C); 1090 (p-Cl-phenyl). ¹H NMR (DMSO-d₆, δ ppm): 3.80 (s, 6 H, two CH₃); 7.30 (d, 2 H, J = 8.4 Hz, 4-Cl-C₆H₄-C_2,6-H); 7.45-7.65 (m, 3 H, CH-pyrrole, pyridyl-C_5,6-H); 7.82 (d, 2 H, J = 8.4 Hz, 4-Cl-C₆H₄-C_3,5-H); 7.98 (s, 1 H, N=CH); 8.21 (d, 1 H, J = 6.7 Hz, pyridyl-C₄-H); 8.41 (s, 1 H, pyridyl-C₂-H). Anal. Calc. (%) for C₁₉H₁₆ClN₅ (349.8): C, 65.24; H, 4.61; N, 20.02. Found (%): C, 65.28; H, 4.72; N, 20.10.

3.1.15. General Procedure for Synthesis of Compounds 16a,b

A mixture of the selected 1-(substitutedaryl)-2-amino-4-(4-chlorophenyl)-1H-pyrrole-3-carbonitrile 4a,b (2 mmol.) and p-toluene sulphonyl chloride (0.38 g, 2 mmol.) was refluxed for 7 h. in toluene (15 mL) containing drops of TEA. The solid obtained was filtered off, washed with ethanol and recrystallized from ethanol/benzene mixture.

1) N-[4-(4-Chlorophenyl)-3-cyano-1-(3-trifluoromethylphenyl)-1H-pyrrol-2-yl)-4-methylbenzenesulfonamide; 16a

Orange yellow crystals; yield (59%); m.p.: >300˚C. IR [KBr, cm⁻¹]: 3230, 3198 (NH); 3000 (CH aromatic); 2921, 2858 (CH-aliphatic); 2260 (C≡N); 1522 (C=C); 1452, 1350, 1120 (SO₂); 1035 (p-Cl-phenyl); MS m/z (relative intensity %): 516 (M^+•+1, 2); 57 (100). Anal. Calc. (%) for C₂₅H₁₇ClF₃N₃O₂S (515.9): C, 58.20; H, 3.32; N, 8.14. Found (%): C, 58.26; H, 3.37; N, 8.17.

2) N-[4-(4-Chlorophenyl)-3-cyano-1-(pyridin-3-yl)-1H-pyrrol-2-yl]-4-methylbenzenesulfonamide; 16b

Buff powder; yield (63%); m.p.: >360˚C. IR [KBr, cm⁻¹]: 3310, 3180 (NH); 3050 (CH aromatic); 2923, 2858 (CH-aliphatic); 2209 (C≡N); 1543 (C=C); 1420, 1300, 1177 (SO₂); 1026 (p-Cl phenyl). ¹H NMR (DMSO- d₆, δ ppm): 2.29 (s, 3 H, CH₃); 7.11 (d, 2 H, J = 8.1 Hz, 4-CH₃-C₆H₄-C_3,5-H); 7.18 (s, 1 H, CH-pyrrole); 7.38 - 7.42 (m, 4 H, 4-Cl-C₆H₄-C_2,6-H & 4-CH₃-C₆H₄-C_2,6-H); 7.48 (d, 2 H, J = 8.4 Hz, 4-Cl-C₆H₄-C_3,5-H); 7.60 - 7.85 (m, 3 H, pyridyl-C₄,_5,6-H); 8.07 (s, 1 H, pyridyl-C₂-H); 9.40 (s, 1 H, NH, D₂O exchangeable). ¹³C (DMSO-d₆): 20.7 (CH₃); 109 (pyrrole-C₃); 114 (C≡N); 117 (pyrrole-C₅); 119 (pyrrole-C₂); 124 (pyrrole-C₄); 124.4 (pyridyl- C₅); 125.4 (pyridyl-C₆); 128 (4-CH₃-C₆H₄-C_2,6); 128.9 (4-Cl-C₆H₄-C_2,6); 131 (4-Cl-C₆H₄-C_3,5& 4-CH₃-C₆H₄- C_3,5); 133 (4-Cl-C₆H₄-C₆); 134 (4-Cl-C₆H₄-C₁); 135 (pyridyl-C₁); 137.8 (4-CH₃-C₆H₄-C₁); 139 (4-CH₃-C₆H₄-C₄); 143.9 (pyridyl-C₂); 145.2 (pyridyl-C₄). Anal. Calc. (%) for C₂₃H₁₇ClN₄O₂S (448.9): C, 61.54; H, 3.82; N, 12.48. Found (%): C, 61.58; H, 3.88; N, 12.51.

3.1.16. General Procedure for Synthesis of Compounds 17a,b

The selected compound 4a,b (2 mmol.) was stirred at room temperature for 3 h. inconc. sulfuric acid (15 mL) then poured drop by drop on to crushed ice. The reaction mixture was neutralized with ammonium hydroxide and the obtained product was filtered, washed thoroughly with water, left to dry and recrystallized from ethanol.

1) 2-Amino-4-(4-chlorophenyl)-1-(3-trifluoromethylphenyl)-1H-pyrrole-3-carboxamide; 17a

Brown needle crystals; yield (43%); m.p.: >300˚C. IR [KBr, cm⁻¹]: 3390, 3167 (NH₂); 3020 (CH aromatic); 1683 (C=O); 1110 (p-Cl-phenyl). ¹H NMR (DMSO-d₆, δ ppm): 6.37 (s, 2 H, NH₂, D₂O exchangeable); 6.94 (d, 1 H, J = 7.2 Hz, 3-CF₃-C₆H₄-C₆-H); 7.00 (s, 1 H, CH-pyrrole); 7.25 - 7.30 (m, 2 H, 3-CF₃-C₆H₄-C_4,5-H); 7.34 (d, 2 H, J = 8.1 Hz, 4-Cl-C₆H₄-C_2,6-H); 7.63 (d, 2 H, J = 8.1 Hz, 4-Cl-C₆H₄-C_3,5-H); 8.11 (s, 1 H, 3-CF₃-C₆H₄-C₂-H); 8.60 (s, 2 H, CONH₂, D₂O exchangeable). Anal. Calc. (%) for C₁₈H₁₃ClF₃N₃O (379.8): C, 56.93; H, 3.45; N, 11.06. Found (%): C, 56.97; H, 3.52; N, 11.13.

2) 2-Amino-4-(4-chlorophenyl)-1-(pyridin-3-yl)-1H-pyrrol-3-carboxamide; 17b

Dark brown powder; yield (65%); m.p.: 305˚C - 307˚C. IR [KBr, cm⁻¹]: 3430, 3292, 3182 (NH₂); 3015 (CH aromatic); 1672 (C=O); 1620 (C=N); 1512 (C=C); 1069 (p-Cl-phenyl); MS m/z (relative intensity %): 314 (M^+•+2, 1); 313 (M^+•+1, 2); 312 (M^+•, 0.3); 197 (100). Anal. Calc. (%) for C₁₆H₁₃ClN₄O (312.8): C, 61.44; H, 4.19; N, 17.91. Found (%): C, 61.47; H, 4.23; N, 17.96.

3.1.17. General Procedure for Synthesis of Compounds 18a,b

Method 1:

The selected 1-(substitutedaryl)-2-amino-4-(4-chlorophenyl)-1H-pyrrole-3-carbonitrile 4a,b (2 mmol.) was refluxed for 9 h. in excess formic acid 80% (30 mL). The reaction mixture was then concentrated and the obtained product was filtered, washed with ethanol to yield the target compounds 18a,b in an average yield of 52%.

Method 2:

The selected 1-(substitutedaryl)-2-amino-4-(4-chlorophenyl)-1H-pyrrole-3-carboxamide 17a,b (2 mmol.) was refluxed in excess formamide (30 mL) for 8 h. The reaction mixture was concentrated to yield the target compounds 18a,b in an average yield of 67%.

1) 5-(4-Chlorophenyl)-7-(3-trifluoromethylphenyl)-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one; 18a

Faint yellow powder; Ethanol; yield (48%); m.p.: >300˚C. IR [KBr, cm⁻¹]: 3386 (br. OH, tautomer); 3280 (NH); 3000 (CH aromatic); 1680 (C=O); 1470 (C=C); 1090 (p-Cl-phenyl). ¹H NMR(DMSO-d₆, δ ppm): 5.27 (s, 1/2 H, NH, D₂O exchangeable); 6.76 (s, 1 H, CH-pyrrole); 6.80 - 6.90 (m, 1 H, 3-CF₃-C₆H₄-C₅-H); 6.98 (d, 1 H, J = 7.2 Hz, 3-CF₃-C₆H₄-C₆-H); 7.33 (d, 2 H, J = 8.4 Hz, 4-Cl-C₆H₄-C_2,6-H); 7.54 (d, 1 H, J = 6.9 Hz, 3-CF₃-C₆H₄-C₄-H); 7.65 (d, 2 H, J = 8.4 Hz, 4-Cl-C₆H₄-C_3,5-H);7.88 (s, 1 H, CH-pyrimidine); 7.91 (s, 1 H, 3-CF₃-C₆H₄-C₂-H); 8.06 (s, 1/2 H, OH tautomer, D₂O exchangeable). Anal. Calc. (%) for C₁₉H₁₁ClF₃N₃O (389.8): C, 58.55; H, 2.84; N, 10.78. Found (%): C, 58.58; H, 2.89; N, 10.81.

2) 5-(4-Chlorophenyl)-7-(pyridin-3-yl)-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one; 18b

Grey powder; Ethanol; yield (54%); m.p.: >300˚C. IR [KBr, cm⁻¹]: 3429 (br. OH tautomer); 3200 (NH); 3048 (CH aromatic); 1670 (C=O); 1598 (C=C); 1098 (p-Cl-phenyl); MS m/z (relative intensity %): 324 (M^+•+2, 0.1); 323 (M^+•+1, 0.1); 78 (100). Anal. Calc. (%) for C₁₇H₁₁ClN₄O (322.7): C, 63.26; H, 3.44; N, 17.36. Found (%): C, 63.31; H, 3.54; N, 17.41.

3.1.18. General Procedure for Synthesis of Compounds 19a,b

The selected 1-(substitutedaryl)-2-amino-4-(4-chlorophenyl)-1H-pyrrol-3-carboxamide 17a,b (2 mmol.) was refluxed in excess thionyl chloride (15 mL) for 6 h. The reaction mixture was evaporated under reduced pressure. The residue was triturated with ethanol, filtered then recrystallized from glacial acetic acid.

1) 7-(4-Chlorophenyl)-5-(3-trifluoromethylphenyl)-1H-pyrrolo[3,2-d]1,2,6-thiadiazine-2,4(1H,3H)-dione; 19a

Faint orange powder; yield (48%); m.p.: 294˚C - 296˚C. IR [KBr, cm⁻¹]: 3181 (NH); 3070 (CH aromatic); 1706 (C=O); 1520 (C=C); 1139 (S=O); 1050 (p-Cl-phenyl).¹H NMR(DMSO-d₆, δ ppm): 4.20 (s, 1 H, NH, D₂O exchangeable); 7.03 (s, 1 H, CH-pyrrole); 7.10 - 7.20 (m, 3 H, 3-CF₃-C₆H₄-C_4,5,6-H); 7.37 (d, 2 H, J = 8.1 Hz, 4-Cl-C₆H₄-C_2,6-H); 7.68 (d, 2 H, J = 8.1 Hz, 4-Cl-C₆H₄-C_3,5-H); 7.92 (s, 1 H, 3-CF₃-C₆H₄-C₂-H); 8.06 (s, 1 H, NH, D₂O exchangeable); MS m/z (relative intensity %): 427 (M^+•+2, 0.1); 426 (M^+•+1, 0.1); 425 (M^+•, 0.2); 424 (M^+•−1, 52); 229 (100). Anal. Calc. (%) for C₁₈H₁₁ClF₃N₃O₂S (425.8): C, 50.77; H, 2.60; N, 9.87. Found (%): C, 50.81; H, 2.68; N, 9.91.

2) 7-(4-Chlorophenyl)-5-(pyridin-3-yl)-1H-pyrrolo[3,2-d]1,2,6-thiadiazin-2,4(1H,3H)-dione; 19b

Dark brown powder; yield (53%); m.p.: 310˚C - 312˚C. IR [KBr, cm⁻¹]: 3354 (NH); 3090 (CH aromatic); 1690 (C=O); 1650 (C=N); 1524 (C=C); 1112 (S=O); 1046 (p-Cl-phenyl); MS m/z (relative intensity %): 360 (M^+•+2, 2); 358 (M^+•, 2); 71 (100). Anal. Calc. (%) forC₁₆H₁₁ClN₄O₂S(358.8): C, 53.56; H, 3.09; N, 15.61. Found (%): C, 53.58; H, 3.12; N, 15.67.

3.1.19. General Procedure for Synthesis of Compounds 20a,b

The selected 1-(substitutedaryl)-2-amino-4-(4-chlorophenyl)-1H-pyrrole-3-carboxamide 17a,b (2 mmol.) was refluxed in excess acetic anhydride (15 mL) for 10 h. The reaction mixture was concentrated to the minimum. The solid product was collected, washed with ethanol then recrystallized from dioxane.

1) 5-(4-Chlorophenyl)-2-methyl-7-(3-trifluoromethylphenyl)-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one; 20a

Faint brown needle crystals; yield (51%); m.p.: >300˚C. IR [KBr, cm⁻¹]: 3418 (br. OH tautomer); 3150 (NH); 3060 (CH aromatic); 1616 (C=N); 1539 (C=C); 1095 (p-Cl-phenyl). ¹H NMR (DMSO-d₆, δ ppm): 2.73 (s, 3 H, CH₃); 7.04 (s, 1 H, CH-pyrrole); 7.31 (d, 2 H, J = 8.1 Hz, 4-Cl-C₆H₄-C_2,6-H); 7.42-7.54 (m, 1 H, 3-CF₃-C₆H₄- C₅-H); 7.60 (d, 1 H, J = 7.2 Hz, 3-CF₃-C₆H₄-C₆-H); 7.69 (d, 1 H, J = 6.9 Hz, 3-CF₃-C₆H₄-C₄-H); 7.81 (d, 2 H, J = 8.1 Hz, 4-Cl-C₆H₄-C_3,5-H); 7.96 (s, 1 H, 3-CF₃-C₆H₄-C₂-H); 9.63 (s, 1/2 H, NH, D₂O exchangeable); 11.50 (s, 1/2 H, OH tautomer, D₂O exchangeable). Anal. Calc. (%) for C₂₀H₁₃ClF₃N₃O (403.8): C, 59.49; H, 3.25; N, 10.41. Found (%): C, 59.53; H, 3.31; N, 10.48.

2) 5-(4-Chlorophenyl)-2-methyl-7-(pyridin-3-yl)-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one; 20b

Dark brown needle crystals; yield (47%); m.p.: 272˚C - 274˚C. IR [KBr, cm⁻¹]: 3140 (NH); 3030 (CH aromatic); 2890 (CH aliphatic); 1652 (C=O); 1610 (C=N); 1107 (p-Cl-phenyl). ¹H NMR (DMSO-d₆, δ ppm): 2.07 (s, 3 H, CH₃); 7.30 - 7.40 (m, 2 H, pyridyl-C_5,6-H); 7.58 (d, 2 H, J = 8.4 Hz, 4-Cl-C₆H₄-C_2,6-H); 7.64 (s, 1 H, CH-pyrrole); 7.95 (d, 2 H, J = 8.4 Hz, 4-Cl-C₆H₄-C_3,5-H); 7.99 - 8.16 (m, 2 H, pyridyl-C_2,4-H); 9.29 (s, 1/2 H, NH, D₂O exchangeable); 10.99 (s, 1/2 H, OH tautomer, D₂O exchangeable). Anal. Calc. (%) for C₁₈H₁₃ClN₄O (336.8): C, 64.19; H, 3.89; N, 16.64. Found (%): C, 64.22; H, 3.91; N, 16.65.

3.1.20. General Procedure for Synthesis of Compounds 21a,b

The selected 7-(substitutedaryl)-5-(4-chlorophenyl)-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one 18a,b (2 mmol.) was refluxed in excess POCl₃ (15 mL) for 18 h. The reaction mixture was allowed to cool then poured on to crushed ice then alkalinized with sodium bicarbonate till pH 10. The obtained product was filtered, washed with water then recrystallized from glacial acetic acid.

1) 4-Chloro-5-(4-chlorophenyl)-7-(3-trifluoromethylphenyl)-7H-pyrrolo[2,3-d]pyrimidine; 21a

Yellow crystals;yield (71%); m.p.: >300˚C. IR [KBr, cm⁻¹]: 2924 (CH aromatic); 1620 (C=N); 1542 (C=C); 1100 (p-Cl-phenyl); MS m/z (relative intensity %): 408 (M^+•+1, 0.2); 407 (M^+•, 0.2); 406 (M^+•−1, 0.2); 405 (M^+•−2) (0.2); 58 (100). Anal. Calc. (%) for C₁₉H₁₀Cl₂F₃N₃ (408.2): C, 55.90; H, 2.47; N, 10.29. Found (%): C, 55.93; H, 2.51; N, 10.32.

2) 4-Chloro-5-(4-chlorophenyl)-7-(pyridin-3-yl)-7H-pyrrolo[2,3-d]pyrimidine; 21b

Brown crystals; yield (83%); m.p.: >300˚C. IR [KBr, cm⁻¹]: 3080 (CH aromatic); 1654 (C=N); 1524 (C=C); 1059 (p-Cl-phenyl). ¹H NMR (DMSO-d₆, δ ppm): 7.29 (d, 1 H, J = 7.8 Hz, pyridyl-C₆-H); 7.59 (s, 1 H, CH- pyrrole); 7.84 (d, 2 H, J = 8.4 Hz, 4-Cl-C₆H₄-C_2,6-H); 8.00 - 8.10 (m, 3 H, 4-Cl-C₆H₄-C_3,5-H & pyridyl-C₅-H); 8.22 (d, 1 H, J = 7.8 Hz, pyridyl-C₄-H); 8.52 (s, 1 H, pyridyl-C₂-H); 10.23 (s, 1 H, pyrimidine-C₂-H). Anal. Calc. (%) for C₁₇H₁₀Cl₂N₄ (341.2): C, 59.84; H, 2.95; N, 16.42. Found (%): C, 59.92; H, 2.97; N, 16.51.

3.1.21. General Procedure for Synthesis of Compounds 22a,b

An equimolar mixture of the appropriate 7-(substitutedaryl)-4-chloro-5-(4-chlorophenyl)-7H-pyrrolo[2,3-]py- rimidine 21a,b (2 mmol.) and morpholine (0.17 g, 0.2 mL, 2 mmol.) was refluxed in absolute ethanol for 9 h (30 mL) in presence of a catalytic amount of TEA (3 - 5 drops). The reaction mixture was allowed to cool then treated with 10% acetic acid. The obtained product was filtered, washed with ethanol then recrystallized from ethanol.

1) 7-(3-Trifluoromethylphenyl)-5-(4-chlorophenyl)-4-morpholino-7H-pyrrolo[2,3-d]pyrimidine; 22a

Yellow cubes; yield (76%); m.p.: >300˚C. IR [KBr, cm⁻¹]: 3090 (CH aromatic); 2924, 2862 (CH aliphatic); 1660 (C=N); 1580 (C=C); 1250, 1046 (C-O-C); 1110 (p-Cl-phenyl). ¹H NMR (DMSO-d₆, δ ppm): 3.43 (t, 4 H, J = 4.5 Hz, morpholine-C_2,6-H); 3.93 (t, 4 H, J = 4.5 Hz, morpholine-C_3,5-H); 7.33 (d, 2 H, J = 8.1 Hz, 4-Cl-C₆H₄-C_2,6-H); 7.46 - 7.51 (m, 3 H, 3-CF₃-C₆H₄-C_5,6-H & CH-pyrrole); 7.60 - 7.64 (m, 2 H, 3-CF₃-C₆H₄- C_2,4-H); 7.79 (d, 2 H, J = 8.1 Hz, 4-Cl-C₆H₄-C_3,5-H); 8.30 (s, 1 H, pyrimidine-C₂-H). Anal. Calc. (%) for C₂₃H₁₈ClF₃N₄O (458.9): C, 60.20; H, 3.95; N, 12.21. Found (%): C, 60.23; H, 3.97; N, 12.28.

2) 7-(Pyridine-3-yl)-5-(4-chlorophenyl)-4-morpholino-7H-pyrrolo[2,3-d]pyrimidine; 22b

Dark brown powder; yield (83%); m.p.: > 300˚C. IR [KBr, cm⁻¹]: 3000 (CH aromatic); 2924, 2861 (CH aliphatic); 1620 (C=N); 1550 (C=C); 1270, 1040 (C-O-C); 1110 (p-Cl-phenyl). ¹H NMR(DMSO-d₆, δ ppm): 3.40 - 3.43 (m, 4 H, morpholine-C_2,6-H); 4.13 (t, 4 H, J = 4.8 Hz, morpholine-C_3,5-H); 7.04 (s, 1 H, CH-pyrrole); 7.32 (d, 2 H, J = 8.4 Hz, 4-Cl-C₆H₄-C_2,6-H); 7.54 - 7.70 (m, 3 H, pyridyl-C_4,5,6-H); 7.81 (d, 2 H, J = 8.4 Hz, 4-Cl- C₆H₄-C_3,5-H);7.88 (s, 1 H, pyridyl-C₂-H); 8.00 (s, 1 H, pyrimidine-C₂-H). Anal. Calc. (%) for C₂₁H₁₈ClN₅O (391.9): C, 64.37; H, 4.63; N, 17.87. Found (%): C, 64.41; H, 4.67; N, 17.92.

3.1.22. General Procedure for Synthesis of Compounds 23a,b

A mixture of compounds 21a,b (2 mmol.) and hydrazine hydrate 98% (0.1 g, 0.1 mL, 2 mmol.) was refluxed in absolute ethanol (30 mL) in presence of TEA as a catalyst for 10 h. The reaction mixture was allowed to cool and poured on to crushed ice. The obtained solid was filtered, washed with water and recrystallized from dioxane to yield the target compounds 23a,b; respectively.

1) 5-(4-Chlorophenyl)-4-hydrazinyl-7-(3-trifluoromethylphenyl)-7H-pyrrolo[2,3-d]pyrimidine; 23a

Faint brown needle crystals; yield (45%); m.p.: > 300˚C. IR [KBr, cm⁻¹]: 3400, 3200, 3110 (NH₂, NH); 2923 (CH aromatic); 1590 (C=N); 1573 (C=C); 1031 (p-Cl-phenyl). ¹H NMR (DMSO-d₆, δ ppm): 4.06 (s, 2 H, NH₂, D₂O exchangeable); 7.01 (s, 1 H, CH-pyrrole); 7.15 - 7.22 (m, 3 H, 3-CF₃-C₆H₄-C_4,5,6-H); 7.36 (d, 2 H, J = 8.1 Hz, 4-Cl-C₆H₄-C_2,6-H); 7.67 (d, 2 H, J=8.1 Hz, 4-Cl-C₆H₄-C_3,5-H); 8.10 (s, 1 H, 3-CF₃-C₆H₄-C₂-H); 9.20 (s, 1 H, pyrimidine-C₂-H); 10.45 (s, 1 H, NH, D₂O exchangeable). Anal. Calc. (%) for C₁₉H₁₃ClF₃N₅ (403.8): C, 56.52; H, 3.25; N, 17.34. Found (%): C, 56.54; H, 3.29; N, 17.37.

2) 5-(4-Chlorophenyl)-4-hydrazinyl-7-(pyridin-3-yl)-7H-pyrrolo[2,3-d]pyrimidine; 23b

Dark brown powder; yield (56%); m.p.: >300˚C. IR [KBr, cm⁻¹]: 3382, 3230, 3120 (NH₂, NH); 2924 (CH aromatic); 1651 (C=N); 1527 (C=C); 1044 (p-Cl-phenyl). ¹H NMR (DMSO-d₆, δ ppm): 4.52 (s, 2 H, NH₂, D₂O exchangeable); 7.00 (s, 1 H, CH-pyrrole); 7.32 (d, 2 H, J = 8.4 Hz, 4-Cl-C₆H₄-C_2,6-H); 7.47-7.52 (m, 1 H, pyridyl-C₅-H); 7.56 (d, 1 H, J = 6.4 Hz, pyridyl-C₆-H); 7.76 (d, 2 H, J = 8.4 Hz, 4-Cl-C₆H₄-C_3,5-H); 7.83 - 7.86 (m, 2 H, pyridyl C_2,4-H); 7.88 (s, 1 H, pyrimidine-C₂-H); 8.00 (s, 1 H, NH, D₂O exchangeable). Anal. Calc. (%) for C₁₇H₁₃ClN₆ (336.8): C, 60.63; H, 3.89; N, 24.95. Found (%): C, 60.66; H, 3.91; N, 24.97.

3.1.23. General Procedure for Synthesis of Compounds 24a,b

To a stirred solution of the selected ethyl N-1-(substitutedaryl)-4-(4-chlorophenyl)-3-cyano-1H-pyrrol-2-ylfor- mimidate 13a,b (2 mmol.) in absolute ethanol (50 mL), hydrazine hydrate 98% (0.1 g, 0.1 mL, 2 mmol.) was added and stirring was continued for 5 h at room temperature. The solid product was filtered, washed with ethanol and recrystallized from ethanol/benzene.

1) 3-Amino-5-(4-chlorophenyl)-4-imino-7-(3-trifluoromethylphenyl)-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidines; 24a

Faint yellow powder; yield (42%); m.p.: >300˚C. IR [KBr, cm⁻¹]: 3447, 3292, 3183 (NH₂, NH); 2928 (CH aromatic); 1643 (C=N); 1556 (C=C); 1025 (p-Cl-phenyl); MS m/z (relative intensity %): 403 (M^+•, 1); 85 (100). Anal. Calc. (%) for C₁₉H₁₃ClF₃N₅ (403.8): C, 56.52; H, 3.25; N, 17.34. Found (%): C, 56.57; H, 3.31; N, 17.42.

2) 3-Amino-5-(4-chlorophenyl)-4-imino-7-(pyridin-3-yl)-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidines; 24b

Dark grey powder; yield (57%); m.p.: > 300˚C. IR [KBr, cm⁻¹]: 3310, 3192 (NH_2, NH); 2921 (CH aromatic); 1650 (C=N); 1558 (C=C); 1091 (p-Cl-phenyl). ¹H NMR (DMSO-d₆, δ ppm): 3.48 (s, 2 H, NH₂, D₂O exchangeable); 6.70 (s, 1 H, CH-pyrrole); 7.24 - 7.31 (m, 2 H, 4-Cl-C₆H₄-C_2,6-H); 7.42 - 7.51 (m, 3 H, pyridyl-C_4,5,6-H); 7.82 (d, 2 H, J = 8.4 Hz, 4-Cl-C₆H₄-C_3,5-H); 8.28 (s, 1 H, pyridyl-C₂-H); 9.40 (s, 1 H, pyrimidine-C₂-H); 10.60 (s, 1 H, imino NH, D₂O exchangeable). Anal. Calc. (%) for C₁₇H₁₃ClN₆ (336.8): C, 60.63; H, 3.89; N, 24.95. Found (%): C, 60.68; H, 3.92; N, 24.97.

3.1.24. General Procedure for Synthesis of Compounds 25a,b

An equimolar mixture of the selected 7-(substitutedaryl)-5-(4-chlorophenyl)-4-imino-4,7-dihydro-3H-pyrro- lo[2,3-d]pyrimidin-3-amine 24a,b (2 mmol.) and ethyl cyanoacetate (0.23 g, 0.1 mL, 2 mmol.) was refluxed in absolute ethanol (30 mL) containing few drops of glacial acetic acid (3 - 5 drops) for 18 h. The reaction mixture was concentrated, then left to cool and the solid product was filtered, washed with ethanol then recrystallized from ethanol.

1) 2-[9-(4-Chlorophenyl)-7-(3-trifluoromethylphenyl)-7H-pyrrolo[3,2-e][1,2,4]triazolo[1,5-c]pyrimidin-2-yl]- acetonetrile; 25a

Faint brown powder; yield (47%); m.p.: 280˚C - 282˚C. IR [KBr, cm⁻¹]: 2934 (CH aromatic); 2850 (CH aliphatic); 2263 (C≡N); 1632 (C=N); 1569 (C=C); 1091 (p-Cl-phenyl); MS m/z (relative intensity %):454 (M^+•+2, 0.3); 452 (M^+•, 1); 58 (100). Anal. Calc. (%) for C₂₂H₁₂ClF₃N₆ (452.8): C, 58.35; H, 2.67; N, 18.56. Found (%): C, 58.41; H, 2.69; N, 18.62.

2) 2-[9-(4-Chlorophenyl)-7-(pyridin-3-yl)-7H-pyrrolo[3,2-e][1,2,4]triazolo[1,5-c]pyrimidin-2-yl]acetonitrile; 25b

Dark brown powder; yield (57%); m.p.: >300˚C. IR [KBr, cm⁻¹]: 3000 (CH aromatic); 2924, 2868 (CH aliphatic); 2200 (C≡N); 1641 (C=N); 1533 (C=C); 1026 (p-Cl-phenyl). ¹H NMR (DMSO-d₆, δ ppm): 4.16 (s, 2 H, CH₂); 7.10 (s, 1 H, CH-pyrrole); 7.32 - 7.34 (m, 2 H, 4-Cl-C₆H₄-C_2,6-H); 7.42 - 7.62 (m, 3 H, pyridyl-C_4,5,6-H); 7.87 (d, 2 H, J = 8.4 Hz, 4-Cl-C₆H₄-C_3,5-H); 8.09 (s, 1 H, pyridyl-C₂-H); 9.70 (s, 1 H, pyrimidine-C₂-H). Anal. Calc. (%) for C₂₀H₁₂ClN₇ (385.8): C, 62.26; H, 3.14; N, 25.41. Found (%): C, 62.32; H, 3.18; N, 25.47.

3.1.25. General Procedure for Synthesis of Compounds 26a,b

An equimolar mixture of the appropriate 7-(substitutedaryl)-3-amino-5-(4-chlorophenyl)-4-imino-4,7-dihydro- 3H-pyrrolo[2,3-d]pyrimidines 24a,b (2 mmol.), carbon disulfide (0.15 g, 2 mmol.) and KOH (0.11 g, 2 mmol.) was refluxed for 9 h in absolute ethanol (30 mL). The reaction mixture was allowed to cool then poured on to crushed ice and neutralized with few drops of c.HCl. The obtained solid product was filtered off, washed thoroughly with water then recrystallized from dioxane.

1) 9-(4-Chlorophenyl)-7-(3-trifluoromethylphenyl)-3,7-dihydro-2H-pyrrolo[3,2-e][1,2,4]triazolo[1,5- c]pyrimidin- 2-thione; 26a

Yellow crystals; yield (57%); m.p.: 274˚C - 276˚C. IR [KBr, cm⁻¹]: 3230, 3140 (NH); 2922 (CH aromatic); 1650 (C=N); 1522 (C=C); 1464, 1320, 1170, 1002 (I, II, III, IV bands N-C=S); 1108 (p-Cl-phenyl); MS m/z (relative intensity %): 447 (M^+•+2, 6); 446 (M^+•+1, 7); 56 (100). Anal. Calc. (%) for C₂₀H₁₁ClF₃N₅S (445.8): C, 53.88; H, 2.49; N, 15.71. Found (%): C, 53.91; H, 2.52; N, 15.76.

2) 9-(4-Chlorophenyl)-7-(pyridin-3-yl)-3,7-dihydro-2H-pyrrolo[3,2-e][1,2,4]triazolo[1,5-c]pyrimidin-2-thione; 26b

Brown needle crystals; yield (75%); m.p.: 310˚C - 312˚C. IR [KBr, cm⁻¹]: 3275, 3125 (NH); 3005 (CH aromatic); 1605 (C=N); 1542 (C=C); 1460, 1398, 1260, 1108 (I, II, III, IV bands N-C=S); 1020 (p-Cl-phenyl). ¹H NMR (DMSO-d₆, δ ppm): 3.40 (s, 1 H, NH, D₂O exchangeable); 7.00 (s, 1 H, CH-pyrrole); 7.34 (d, 2 H, J = 8.4 Hz, 4-Cl-C₆H₄-C_2,6-H); 7.48 - 7.51 (m, 1 H, pyridyl-C₅-H); 7.58 (d, 1 H, J = 6.4 Hz, pyridyl-C₆-H); 7.79 (d, 2 H, J = 8.4 Hz, 4-Cl-C₆H₄-C_3,5-H); 7.86 (d, 1 H, J = 6.4 Hz, pyridyl-C₄-H); 8.30 (s, 1 H, pyridyl-C₂-H); 8.47 (s, 1 H, pyrimidine-C₂-H). Anal. Calc. (%) for C₁₈H₁₁ClN₆S (378.8): C, 57.07; H, 2.93; N, 22.18. Found (%): C, 57.12; H, 2.96; N, 22.21.

3.1.26. General Procedure for Synthesis of Compounds 27a,b

The appropriate 7-(substitutedaryl)-3-amino-5-(4-chlorophenyl)-4-imino-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimi- dine 24a,b (2 mmol.) was refluxed in excess triethyl orthoformate (10 mL) for 12 h. The reaction mixture was left to cool and the solid product was filtered, washed with ethanol then recrystallized from glacial acetic acid.

1) 9-(4-Chlorophenyl)-7-(3-trifluoromethylphenyl)-7H-pyrrolo[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine; 27a

Faint yellow crystals; yield (47%); m.p.: >300˚C. IR [KBr, cm⁻¹]: 3000 (CH aromatic); 1620 (C=N); 1561 (C=C); 1025 (p-Cl-phenyl). ¹H NMR (DMSO-d₆, δ ppm): 6.40 (s, 1 H, CH-pyrrole); 6.80 - 6.85 (m, 3 H, 3-CF₃-C₆H₄-C_4,5,6-H); 6.92 - 6.97 (m, 2 H, 4-Cl-C₆H₄-C_2,6-H); 7.28 - 7.35 (m, 2 H, 4-Cl-C₆H₄-C_3,5-H); 7.45 (s, 1 H, 3-CF₃-C₆H₄-C₂-H); 7.48 (s, 1 H, triazole-C₃-H); 8.58 (s, 1 H, pyrimidine-C₂-H). Anal. Calc. (%) for C₂₀H₁₁ClF₃N₅ (413.8): C, 58.05; H, 2.68; N, 16.93. Found (%): C, 58.10; H, 2.71; N, 16.96.

2) 9-(4-Chlorophenyl)-7-(pyridin-3-yl)-7H-pyrrolo[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine; 27b

Dark brown powder, yield (56%); m.p.: 278˚C - 280˚C. IR [KBr, cm⁻¹]: 2924 (CH aromatic); 1647 (C=N); 1562 (C=C); 1027 (p-Cl-phenyl). ¹H NMR (DMSO-d₆, δ ppm): 6.92 - 6.97 (m, 1 H, pyridyl-C₅-H); 7.26 (s, 1 H, CH-pyrrole); 7.30 (d, 1 H, J = 7.8 Hz, pyridyl-C₆-H); 7.46 (d, 2 H, J = 8.4 Hz, 4-Cl-C₆H₄-C_2,6-H); 7.78 (d, 2 H, J = 8.4 Hz, 4-Cl-C₆H₄-C_3,5-H); 8.18 - 8.22 (m, 1 H, pyridyl-C₄-H); 8.50 (s, 1 H, pyridyl-C₂-H); 8.63 (s, 1 H, triazole-C₃-H); 9.00 (s, 1 H, pyrimidine-C₂-H). Anal. Calc. (%) for C₁₈H₁₁ClN₆ (346.8): C, 62.34; H, 3.20; N, 24.23. Found (%): C, 62.38; H, 3.27; N, 24.28.

3.1.27. General Procedure for Synthesis of Compounds 28a,b

An equimolar mixture of the selected compound 24a,b (2 mmol.) and phenyl isothiocyanate (0.27 g, 0.24 mL, 2 mmol.) was refluxed for 7 h in absolute ethanol (30 mL) containing few drops TEA (2 - 4 drops). The reaction mixture was left to cool and the obtained product was filtered off, washed with ethanol then recrystallized from ethanol.

1) 2-Amino-9-(4-chlorophenyl)-N-phenyl-7-(3-trifluoromethylphenyl)-7H-pyrrolo[3,2-e][1,2,4]triazolo[1,5-c] pyrimidine; 28a

Pale yellowish needle crystals; yield (42%); m.p.: >300˚C. IR [KBr, cm⁻¹]: 3220 (NH); 2924 (CH aromatic); 1627 (C=N); 1554 (C=C); 1091 (p-Cl-phenyl). ¹H NMR (DMSO-d₆, δ ppm): 7.00 (s, 1 H, CH-pyrrole); 7.12 - 7.15 (m, 3 H, C₆H₅-C_3,4,5-H); 7.32 (d, 2 H, J = 8.4 Hz, 4-Cl-C₆H₄-C_2,6-H); 7.36-7.40 (m, 3 H, 3-CF₃-C₆H₄-C_4,5,6- H); 7.47 (d, 2 H, J = 8.4 Hz, 4-Cl-C₆H₄-C_3,5-H); 7.80 (d, 2 H, J = 7.2 Hz, C₆H₅-C_2,6-H); 7.83 (s, 1 H, 3-CF₃- C₆H₄-C₂-H); 9.70 (s, 1 H, pyrimidine-C₂-H); 10.40 (s,1 H, NH, D₂O exchangeable). Anal. Calc. (%) for C₂₆H₁₆ClF₃N₆ (504.9): C, 61.85; H, 3.19; N, 16.65. Found (%): C, 61.88; H, 3.23; N, 16.71.

2) 2-Amino-9-(4-chlorophenyl)-N-phenyl-7-(pyridin-3-yl)-7H-pyrrolo[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine; 28b

Grey crystals; yield (56%); m.p.: >300˚C. IR [KBr, cm⁻¹]: 3189 (NH); 3002, 2924 (CH aromatic); 1610 (C=N); 1550 (C=C); 1087 (p-Cl phenyl). ¹H NMR(DMSO-d₆, δ ppm): 6.93 (t, 1 H, J = 7.5 Hz, C₆H₅-C₄-H); 7.27 (s, 1 H, CH-pyrrole); 7.31 (d, 2 H, J = 7.5 Hz, C₆H₅-C_3,5-H); 7.38 - 7.48 (m, 2 H, pyridyl-C_5,6-H); 7.55 (d, 2 H, J = 8.4 Hz, 4-Cl-C₆H₄-C_2,6-H); 7.65 (d, 2 H, J = 7.5 Hz, C₆H₅-C_2,6-H); 7.87 (d, 2 H, J = 8.4 Hz, 4-Cl- C₆H₄-C_3,5-H); 8.22 - 8.32 (m, 1 H, pyridyl-C₄-H); 8.67 (s, 1 H, pyridyl-C₂-H); 9.87 (s, 1 H, pyrimidine-C₂-H); 10.20 (s, 1 H, NH, D₂O exchangeable). ¹³C (DMSO-d₆): 107.9 (pyrrolotriazolopyrimidine-C_9a); 116.5 (pyrrolotriazolopyrimidine-C₈); 116.8 (N-C₆H₅-C_2,6); 120.9 (N-C₆H₅-C₄); 123.6 (4-Cl-C₆H₄-C₁); 124 (pyridyl-C₅); 126 (pyrrolotriazolopyrimidine-C₉); 128 (pyridyl-C₆); 128.9 (4-Cl-C₆H₄-C_2,6); 129.7 (4-Cl-C₆H₄-C_3,5); 129.9 (N- C₆H₅-C_3,5); 131.5 (4-Cl-C₆H₄-C₆); 133.9 (pyridyl-C₁); 136 (N-C₆H₅-C₁); 139.4 (pyrrolotriazolopyrimidine-C₅); 141.2 (pyridyl-C₂); 144.6 (pyridyl-C₄); 146 (pyrrolotriazolopyrimidine-C_9b); 155.6 (pyrrolotriazolopyrimidine- C_6a); 157.9 (pyrrolotriazolopyrimidine-C₂). Anal. Calc. (%) for C₂₄H₁₆ClN₇ (437.9): C, 65.83; H, 3.68; N, 22.39. Found (%): C, 65.87; H, 3.72; N, 22.43.

3.1.28. General Procedure for Synthesis of Compounds 29a,b

A mixture of the appropriate 1-(substitutedaryl)-2-amino-4-(4-chlorophenyl)-1H-pyrrole-3-carbonitrile 4a,b (2 mmol.) and sodium azide (0.13 g, 2 mmol.) and ammonium chloride (0.22 g, 2 mmol.) was heated in water bath for 10 h. in excess DMF (15 mL). The reaction mixture was allowed to cool, then triturated with water. The solid product was filtered off, washed with ethanol then recrystallized from glacial acetic acid.

1) 2-Amino-4-(4-chlorophenyl)-1-(3-trifluoromethylphenyl)-3-(1H-tetrazol-5-yl)-1H-pyrrole; 29a

Pale orange crystals; yield (73%); m.p.: >300˚C IR [KBr, cm⁻¹]: 3280, 3180 (NH₂, NH); 2923 (CH aromatic); 1559 (C=C); 1383 (N=N); 1081 (p-Cl-phenyl); MS m/z (relative intensity %): 405 (M^+•+1, 1); 58 (100). Anal. Calc. (%) for C₁₈H₁₂ClF₃N₆ (404.8): C, 53.41; H, 2.99; N, 20.76. Found (%): C, 53.48; H, 2.97; N, 20.88.

2) 2-Amino-4-(4-chlorophenyl)-1-(pyridin-3-yl)-3-(1H-tetrazol-5-yl)-1H-pyrrole; 29b

Dark brown needle crystals; yield (92%); m.p.: >300˚C. IR [KBr, cm⁻¹]: 3345, 3200 (NH₂, NH); 2926 (CH aromatic); 1520 (C=C); 1419 (N=N); 1096 (p-Cl-phenyl). ¹H NMR (DMSO-d₆, δ ppm): 5.20 (s, 2 H, NH₂, D₂O exchangeable); 6.80 (s, 1 H, CH-pyrrole); 7.23 - 7.28 (m, 1 H, pyridyl-C₅-H); 7.45 (d, 1 H, J = 6.5 Hz, pyridyl-C₆-H); 7.53 (d, 2 H, J = 8.4 Hz, 4-Cl-C₆H₄-C_2,6-H); 7.61 (d, 1 H, J = 6.5 Hz, pyridyl-C₄-H); 7.92 (d, 2 H, J = 8.4 Hz, 4-Cl-C₆H₄-C_3,5-H); 8.20 (s, 1 H, pyridyl-C₂-H); 9.92 (s, 1 H, NH, D₂O exchangeable). Anal. Calc. (%) for C₁₆H₁₂ClN₇ (337.8): C, 56.89; H, 3.58; N, 29.03. Found (%): C, 56.91; H, 3.54; N, 29.08.

3.1.29. General Procedure for Synthesis of Compounds 30a,b

A mixture of the appropriate 1-(substitutedaryl)-2-amino-4-(4-chlorophenyl)-1H-pyrrole-3-carbonitrile 4a,b (2 mmol.), CS₂ (0.15 g, 0.13 mL, 2 mmol.) and excess ethylene diamine (5 mL) was refluxed in a water bath for 7 h. The reaction mixture was allowed to cool then triturated with ethanol. The obtained product was filtered off, washed with ethanol then recrystallized from dioxane.

1) 2-Amino-4-(4-chlorophenyl)-3-(4,5-dihydro-1H-imidazol-2-yl)-1-(3-trifluoromethylphenyl)-1H-pyrrole; 30a

Yellow powder; yield (67%); m.p.: >300˚C. IR [KBr, cm⁻¹]: 3250, 3120 (NH₂, NH); 3000 (CH aromatic); 2950, 2850 (CH aliphatic); 1514 (C=C); 1100 (p-Cl-phenyl); MS m/z (relative intensity %): 406 (M^+•+2, 0.1); 405 (M^+•+1, 0.1); 404 (M^+•, 0.1); 56 (100). Anal. Calc. (%) for C₂₀H₁₆ClF₃N₄ (404.8): C, 59.34; H, 3.98; N, 13.84. Found (%): C, 59.39; H, 3.92; N, 13.87.

2) 2-Amino-4-(4-chlorophenyl)-3-(4,5-dihydro-1H-imidazol-2-yl)-1-(pyridin-3-yl)-1H-pyrrole; 30b

Grey crystals;yield (83%); m.p.: >300˚C. IR [KBr, cm⁻¹]: 3360; 3145 (NH₂, NH); 3050 (CH aromatic); 2890 (CH aliphatic); 1654 (C=N); 1520 (C=C); 1090 (p-Cl-phenyl). ¹H NMR (DMSO-d₆, δ ppm): 4.75 (s, 1 H, NH, D₂O exchangeable); 5.64 - 5.67 (m, 4 H, imidazolidine-C_4,5-H); 7.37 (s, 1 H, CH-pyrrole); 7.40 (s, 2 H, NH₂, D₂O exchangeable); 7.51 - 7.54 (m, 2 H, 4-Cl-C₆H₄-C_2,6-H); 7.57 - 7.62 (m, 1 H, pyridyl-C₅-H); 7.65 - 7.66 (m, 1 H, pyridyl-C₆-H); 8.01 - 8.03 (m, 2 H, 4-Cl-C₆H₄-C_3,5-H); 8.04 - 8.05 (m, 1 H, pyridyl-C₄-H); 8.06 (s, 1 H, pyridyl-C₂-H). Anal. Calc. (%) for C₁₈H₁₆ClN₅ (337.8): C, 64.00; H, 4.77; N, 20.73. Found (%): C, 64.16; H, 4.81; N, 20.79.

3.1.30. General Procedure for Synthesis of Compounds 31a,b

A mixture of the appropriate 1-(substitutedaryl)-2-amino-4-(4-chlorophenyl)-1H-pyrrole-3-carbonitrile 4a,b (2 mmol.) and CS₂ (0.13 mL, 0.15 g, 2 mmol.) was heated in excess pyridine (10 mL) in a water bath for 10 h, then left to cool. The solid product collected, washed with ethanol then recrystallized from ethanol.

1) 5-(4-Chlorophenyl)-7-(3-trifluoromethylphenyl)-1H-pyrrolo[2,3-d]pyrimidin-2,4(3H,7H)-dithione; 31a

Faint yellow needle crystals;yield (62%); m.p.: >300˚C. IR [KBr, cm⁻¹]: 3236 (NH); 3050 (CH aromatic); 1622 (C=N); 1560 (C=C); 1424, 1328, 1129, 1005 (I, II, III, IV bands N-C=S); 1129 (p-Cl-phenyl). ¹H NMR (DMSO-d₆, δ ppm): 8.10 (s, 1 H, pyrimidine-N₃-H, D₂O exchangeable); 7.35 - 7.41 (m, 1H, 3-CF₃-C₆H₄-C₅-H); 7.50 (s, 1H, CH-pyrrole); 7.56 (d, 2 H, J = 8.4 Hz, 4-Cl-C₆H₄-C_2,6-H); 7.61 - 7.63 (m, 1 H, 3-CF₃-C₆H₄-C₆-H); 7.78 - 7.82 (m, 1 H, 3-CF₃-C₆H₄-C₄-H); 7.93 (d, 2 H, J = 8.4 Hz, 4-Cl-C₆H₄-C_3,5-H); 8.58 (_S, 1 H, 3-CF₃- C₆H₄-C₂-H); 8.76 (s, 1 H, pyrimidine-N₁-H, D₂O exchangeable). Anal. Calc. (%) for C₁₉H₁₁ClF₃N₃S₂ (437.9): C, 52.11; H, 2.53; N, 9.60. Found (%): C, 52.17; H, 2.63; N, 9.71.

2) 5-(4-Chlorophenyl)-7-(pyridin-3-yl)-1H-pyrrolo[2,3-d]pyrimidin-2,4(3H,7H)-dithione; 31b

Brown needle crystals; yield (78%); m.p.: > 300˚C. IR [KBr, cm⁻¹]: 3306, 3207 (NH); 2923 (CH aromatic); 1641 (C=N); 1534 (C=C); 1480, 1390, 1128, 1000 (I, II, III, IV bands N-C=S); 1128 (p-Cl-phenyl); MS m/z (relative intensity %): 372 (M^+•+2, 1); 371 (M^+•+1, 1); 58 (100). Anal. Calc. (%) for C₁₇H₁₁ClN₄S₂ (370.9): C, 55.05; H, 2.99; N, 15.11. Found (%): C, 55.01; H, 2.86; N, 15.16.

3.1.31. General Procedure for Synthesis of Compounds 32a,b

The selected 1-(substitutedaryl)-2-amino-4-(4-chlorophenyl)-1H-pyrrole-3-carbonitrile 4a,b (2 mmol.) was dissolved in a mixture of glacial acetic acid/c.HCl (10:7) (20 mL). To such solution, a solution of sodium nitrite (0.14 g, 2 mmol.) in (1 mL) water was added drop wise while stirring at 0˚C - 5˚C then stirring was continued for 3 h while maintaining the reaction temperature at 5˚C. The solid product was then collected, washed with ethanol then recrystallized from glacial acetic acid.

1) 5-(4-Chlorophenyl)-7-(3-trifluoromethylphenyl)-3H-pyrrolo[2,3-d][1,2,3]triazine-4(7H)one; 32a

Yellow powder; yield (62%); m.p.:287˚C - 289˚C. IR [KBr, cm⁻¹]: 3100 (NH); 3050 (CH aromatic); 1691 (C=O); 1528 (C=C); 1411 (N=N); 1112 (p-Cl-phenyl).¹H NMR (DMSO-d₆, δ ppm): 7.25 (s, 1 H, CH-pyrrole); 7.41 (s, 1 H, NH, D₂O exchangeable); 7.54 (d, 2 H, J = 8.4 Hz, 4-Cl-C₆H₄-C_2,6-H); 7.62 - 7.86 (m, 4 H, 3-CF₃- C₆H₄-C_2,4,5,6-H); 7.92 (d, 2 H, J = 8.4 Hz, 4-Cl-C₆H₄-C_3,5-H). Anal. Calc. (%) for C₁₈H₁₀ClF₃N₄O (390.7): C, 55.33; H, 2.58; N, 14.34. Found (%): C, 55.33; H, 2.58; N, 14.34.

2) 5-(4-Chlorophenyl)-7-(pyridin-3-yl)-3H-pyrrolo[2,3-d][1,2,3]triazin-4(7H)-one; 32b

Dark grey crystals; yield (78%); m.p.: 267˚C - 269˚C. IR [KBr, cm⁻¹]: 3114 (NH); 3010 (CH aromatic); 1690 (C=O); 1531 (C=C); 1407 (N=N); 1088 (p-Cl-phenyl). ¹H NMR(DMSO-d₆, δ ppm): 7.28 (s, 1 H, CH-pyrrole); 7.45 (s, 1 H, NH, D₂O exchangeable); 7.52 (d, 2 H, J = 8.1 Hz, 4-Cl-C₆H₄-C_2,6-H); 7.55 - 7.62 (m, 4 H, pyridyl-C_2,4,5,6-H); 7.91 (d, 2 H, J = 8.1 Hz, 4-Cl-C₆H₄-C_3,5-H). Anal. Calc. (%) for C₁₆H₁₀ClN₅O (323.7): C, 59.36; H, 3.11; N, 21.63. Found (%): C, 59.36; H, 3.11; N, 21.63.

3.1.32. General Procedure for Synthesis of Compounds 33a,b

To a stirred solution of the selected ethyl 1-(substitutedaryl)-2-amino-4-(4-chlorophenyl)-1H-pyrrol-3-carbox- ylate 4c, d (2 mmol.) in absolute ethanol (50 mL), hydrazine hydrate 98% (0.1 g, 0.12 mL, 2 mmol.) was added and the reaction was refluxed for 6 h. The solid product was isolated by filtration, washed with ethanol and recrystallized from ethanol.

1) 2-Amino-4-(4-chlorophenyl)-1-(3-trifluoromethylphenyl)-1H-pyrrol-3-carbohydrazide; 33a

Pale yellow crystals; yield (67%); m.p.: >300˚C. IR [KBr, cm⁻¹]: 3450, 3401, 3200 (NH₂, NH); 2934 (CH aromatic); 1690 (C=O); 1520 (C=C); 1021 (p-Cl-phenyl).¹H NMR (DMSO-d₆, δ ppm): 5.30 (s, 2 H, NH-NH₂, D₂O exchangeable); 6.74 (s, 1 H, CH-pyrrole); 7.31 (d, 2 H, J = 8.1 Hz, 4-Cl-C₆H₄-C_2,6-H); 7.38 - 7.45 (m, 1 H, 3-CF₃-C₆H₄-C₅-H); 7.50 - 7.60 (m, 2 H, 3-CF₃-C₆H₄-C_4,6-H); 7.87 (d, 2 H, J = 8.1 Hz, 4-Cl-C₆H₄-C_3,5-H), 8.35 (s, 1 H, 3-CF₃-C₆H₄-C₂-H); 8.50 (s, 2 H, pyrrole-C₂-NH₂, D₂O exchangeable); 8.59 (s, 1 H, NH, D₂O exchangeable). Anal. Calc. (%) for C₁₈H₁₄ClF₃N₄O (394.8): C, 54.76; H, 3.57; N, 14.19. Found (%): C, 54.65; H, 3.43; N, 14.23.

2) 2-Amino-4-(4-chlorophenyl)-1-(pyridin-3-yl)-1H-pyrrole-3-carbohydrazide; 33b

Faint brown crystals; yield (72%); m.p.: >300˚C. IR [KBr, cm⁻¹]: 3390, 3213 (NH₂, NH); 2980 (CH aromatic); 1680 (C=O), 1522 (C=C); 1031 (p-Cl-phenyl); MS m/z (relative intensity %): 329 (M^+•+2, 1); 327 (M^+•, 9); 140 (100). Anal. Calc. (%) for C₁₆H₁₄ClN₅O (327.8): C, 58.63; H, 4.31; N, 21.37. Found (%): C, 58.71; H, 4.42; N, 21.43.

3.1.33. General Procedure for Synthesis of Compounds 34a,b

The selected ethyl 1-(substitutedaryl)-2-amino-4-(4-chlorophenyl)-1H-pyrrol-3-carboxylate 4c, d (2 mmol.) was refluxed for 7 h in excess acetic anhydride (30 mL). The reaction mixture was concentrated under reduced pressure and the residue was collected by filtration, washed with ethanol and recrystallized from ethanol.

1) Ethyl 2-acetamido-4-(4-chlorophenyl)-1-(3-trifluoromethylphenyl)-1H-pyrrol-3-carboxylate; 34a

Faint orange powder; yield (59%); m.p.: > 300˚C. IR [KBr, cm⁻¹]: 3293, 3180 (NH); 2927 (CH aromatic); 2860 (CH-aliphatic); 1689, 1650 (C=O); 1421 (C=C); 1250, 1026 (C-O-C); 1120 (p-Cl-phenyl). ¹H NMR (DMSO-d₆, δ ppm): 1.17 - 1.23 (m, 3 H, CH₂CH₃); 2.05 (s, 3 H, COCH₃); 3.91 - 4.01 (m, 2 H, CH₂CH₃); 7.30 (d, 1 H, J = 7.8 Hz, 3-CF₃-C₆H₄-C₆-H); 7.37 (d, 1 H, J = 7.8 Hz, 3-CF₃-C₆H₄-C₄-H); 7.50 - 7.55 ( m, 3 H, 4-Cl- C₆H₄-C_2,6-H & 3-CF₃-C₆H₄-C₅-H); 7.75 (s, 1 H, CH-pyrrole); 7.84 (d, 2 H, J = 8.4 Hz, 4-Cl-C₆H₄-C_3,5-H); 8.08 (s, 1 H, 3-CF₃-C₆H₄-C₂-H); 10.45 (s, 1 H, NH, D₂O exchangeable). Anal. Calc. (%) for C₂₂H₁₈ClF₃N₂O₃ (450.8): C, 58.61; H, 4.02; N, 6.21. Found (%): C, 58.64; H, 4.10; N, 6.28.

2) Ethyl 2-acetamido-4-(4-chlorophenyl)-1-(pyridin-3-yl)-1H-pyrrole-3-carboxylate; 34b

Dark brown needle crystals; yield (71%); m.p.: > 300˚C. IR [KBr, cm⁻¹]: 3220 (NH); 3096 (CH aromatic); 2921, 2858 (CH-aliphatic); 1693 (C=O); 1544 (C=C); 1245, 1024 (C-O-C); 1110 (p-Cl-phenyl). MS m/z (relative intensity %): 385 (M^+•+2, 0.6); 384 (M^+•+1, 0.2); 383 (M^+•, 0.2); 58 (100). Anal. Calc. (%) for C₂₀H₁₈ClN₃O₃ (383.8): C, 62.58; H, 4.73; N, 10.95. Found (%): C, 62.55; H, 4.81; N, 10.97.

3.1.34. General Procedure for Synthesis of Compounds 35a,b

An equimolar mixture of the selected 1-(substitutedaryl)-2-amino-4-(4-chlorophenyl)-1H-pyrrole-3-carbohy- drazide 33a,b (2 mmol.) and glucose (0.22 g, 2 mmol.) was refluxed in absolute ethanol (30 mL) for 12 h, then left to cool. The obtained product was filtered off, washed with ethanol then recrystallized from ethanol.

1) 2-Amino-4-(4-chlorophenyl)-N'-2,3,4,5,6-pentahydroxy-1-hexylidene)-1-(3-trifluoromethylphenyl)-1H-pyr-rol- 3-carbohydrazide; 35a

Yellow needle crystals; yield (89%); m.p.: 296˚C - 298˚C. IR [KBr, cm⁻¹]: multiple absorption bands at 3496, 3438 (OH); 3360, 3310, 3250 (NH₂, NH); 3000 (CH aromatic); 2924, 2860 (CH-aliphatic); 1710 (C=O); 1644 (C=N); 1542 (C=C); 1036 (p-Cl-phenyl). MS m/z (relative intensity %): 558 (M^+•+2, 0.1); 57 (100). Anal. Calc. (%) for C₂₄H₂₄ClF₃N₄O₆ (556.9): C, 51.76; H, 4.34; N, 10.06. Found (%): C, 51.81; H, 4.31; N, 10.15.

2) 2-Amino-4-(4-chlorophenyl)-N'-2,3,4,5,6-pentahydroxy-1-hexylidene)-1-(pyridin-3-yl)-1H-pyrrol-3-car-bohy- drazide; 35b

Buff needle crystals; yield (91%); m.p.: 326˚C - 328˚C. IR [KBr, cm⁻¹]: 3400, 3386 (OH); 3300, 3210(NH_2, NH); 3000 (CH aromatic); 2920 (CH-aliphatic); 1700 (C=O), 1640 (C=N); 1470 (C=C); 1090 (p-Cl-phenyl). MS m/z (relative intensity %): 490 (M^+•+1, 0.2); 57 (100). Anal. Calc. (%) for C₂₂H₂₄ClN₅O₆ (489.9): C, 53.94; H, 4.94; N, 14.30. Found (%): C, 53.97; H, 4.91; N, 14.38.

3.1.35. General Procedure for Synthesis of Compounds 36a,b

The selected ethyl 1-(substitutedaryl)-2-acetamido-4-(4-chlorophenyl)-1H-pyrrol-3-carboxylates 34a,b (2 mmol.) was refluxed with hydrazine hydrate 98% (0.1 g, 0.12 mL, 2 mmol.) in absolute ethanol (30 mL) for 6 h. The reaction mixture was allowed to cool and the solid product was filtered, washed with ethanol then recrystallized from dioxane.

1) 3-Amino-5-(4-chlorophenyl)-2-methyl-7-(3-trifluoromethylphenyl)-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one; 36a

Pale orange crystals; yield (71%); m.p.: >300˚C. IR [KBr, cm⁻¹]: 3280, 3200 (NH₂); 2924 (CH aromatic); 2861 (CH-aliphatic); 1680 (C=O); 1428 (C=C); 1105 (p-Cl-phenyl). ¹H NMR (DMSO-d₆, δ ppm): 2.07 (s, 3 H, CH₃); 4.91 (s, 2 H, NH₂, D₂O exchangeable); 7.30 (d, 2 H, J = 8.4 Hz, 4-Cl-C₆H₄-C_2,6-H); 7.33 (s, 1 H, CH- pyrrole); 7.38 - 7.48 (m, 2 H, 3-CF₃-C₆H₄-C_5,6-H); 7.50 - 7.60 (m, 1 H,3-CF₃-C₆H₄-C₄-H); 7.85 (d, 2 H, J = 8.4 Hz, 4-Cl-C₆H₄-C_3,5-H); 8.09 (s, 1 H, 3-CF₃-C₆H₄-C₂-H). Anal. Calc. (%) for C₂₀H₁₄ClF₃N₄O(418.8): C, 57.36; H, 3.37; N, 13.38. Found (%): C, 57.44; H, 3.31; N, 13.42.

2) 3-Amino-5-(4-chlorophenyl)-2-methyl-7-(pyridin-3-yl)-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one; 36b

Dark brown crystals; yield (87%); m.p.: >300˚C. IR [KBr, cm⁻¹]: 3391, 3185 (NH₂); 2929 (CH aromatic); 2860 (CH-aliphatic); 1690 (C=O), 1543 (C=C); 1033 (p-Cl-phenyl). MS m/z (relative intensity %): 353 (M^+•+2, 0.2); 351 (M^+•, 1); 58 (100). Anal. Calc. (%) for C₁₈H₁₄ClN₅O (351.8): C, 61.46; H, 4.01; N, 19.91. Found (%): C, 61.51; H, 4.07; N, 19.96.

3.1.36. General Procedure for Synthesis of Compounds 37a,b

A solution of the appropriate ethyl 1-(substitutedaryl)-2-amino-4-(4-chlorophenyl)-1H-pyrrol-3-carboxylate 4c, d (2 mmol.) in (30 mL) ethanolic sodium hydroxide solution [prepared by dissolving sodium hydroxide (0.1 g, 2 mmol.) in (30 mL) absolute ethanol] was heated under reflux for 9 h. The reaction mixture was allowed to cool, then acidified with dilute acetic acid (1.3 mL) and extracted with ether. The obtained product from ethereal solution was collected and recrystallized from ethanol.

1) 2-Amino-4-(4-chlorophenyl)-1-(3-trifluoromethylphenyl)-1H-pyrrol-3-carboxylic acid; 37a

Pale yellow powder; yield (57%); m.p.: >300˚C. IR [KBr, cm⁻¹]: 3350 (br. OH); 3119 (NH₂); 2996 (CH aromatic); 1696 (C=O); 1522 (C=C); 1108 (p-Cl-phenyl). MS m/z (relative intensity %): 382 (M^+•+2, 1); 380 (M^+•, 2); 139 (100). Anal. Calc. (%) for C₁₈H₁₂ClF₃N₂O₂ (380.7): C, 56.78; H, 3.18; N, 7.36. Found (%): C, 56.82; H, 3.21; N, 7.47.

2) 2-Amino-4-(4-chlorophenyl)-1-(pyridin-3-yl)-1H-pyrrol-3-carboxylic acid; 37b

Grey powder; yield (67%); m.p.: > 300˚C. IR [KBr, cm⁻¹]: 3391 (br. OH); 3219 (NH₂); 3050 (CH aromatic); 1684 (C=O); 1517 (C=C); 1089 (p-Cl phenyl). ¹H NMR (DMSO-d₆, δ ppm): 5.37 (s, 2 H, NH₂, D₂O exchangeable); 7.38 (d, 2 H, J = 8.4 Hz, 4-Cl-C₆H₄-C_2,6-H); 7.52 - 7.58 (m, 1 H, pyridyl-C₅-H); 7.59 - 7.68 (m, 1 H, pyridyl-C₆-H); 7.89 (s, 1 H, CH-pyrrole); 7.92 - 7.96 (m, 2 H, 4-Cl-C₆H₄-C_3,5-H); 8.01 - 8.06 (m, 1 H, pyridyl- C₄-H); 8.07 (s, 1 H, pyridyl-C₂-H); 9.47 (s, 1 H, OH, D₂O exchangeable). Anal. Calc. (%) for C₁₆H₁₂ClN₃O₂ (313.7): C, 61.25; H, 3.86; N, 13.39. Found (%): C, 61.27; H, 3.89; N, 13.43.

3.1.37. General Procedure for Synthesis of Compounds 38a,b

An equimolar mixture of the selected ethyl 1-(substitutedaryl)-2-amino-4-(4-chlorophenyl)-1H-pyrrol-3-car- boxylate 4c,d (2 mmol.) and phenyl isothiocyanate (0.27 g, 0.24 mL, 2 mmol.) was refluxed in for 12 h in absolute ethanol (20 mL). The reaction mixture was left to cool and the obtained product was filtered, washed with ethanol then recrystallized from glacial acetic acid.

1) Ethyl 4-(4-chlorophenyl)-2-(3-phenylthioureido)-1-(3-trifluoromethylphenyl)-1H-pyrrol-3-carboxylate; 38a

Faint orange needle crystals; yield (62%); m.p.: >300˚C. IR [KBr, cm⁻¹]: 3200 (NH); 2924 (CH aromatic); 2861 (CH-aliphatic); 1720 (C=O); 1560 (C=C); 1457, 1380, 1140, 998 (I, II, III, IV bands N-C=S); 1250, 1031 (C-O-C); 1031 (p-Cl-phenyl). ¹H NMR (DMSO-d₆, δ ppm): 1.12 - 1.20 (m, 3 H, CH₂CH₃); 4.38 (q, 2 H, J = 6 Hz, CH₂CH₃); 6.90 (s, 1 H, CH-pyrrole); 7.10 - 7.17 (m, 3 H, C₆H₅-C_3,4,5-H); 7.20 - 7.28 (m, 1 H, 3-CF₃-C₆H₄- C₅-H); 7.31 - 7.38 (m, 1 H, 3-CF₃-C₆H₄-C₆-H); 7.51 - 7.58 (m, 3 H, 4-Cl-C₆H₄-C_2,6-H & 3-CF₃-C₆H₄-C₄-H); 7.70 - 7.78 (m, 2 H, C₆H₅-C_2,6-H); 7.81 (d, 2 H, J = 8.1 Hz, 4-Cl-C₆H₄-C_3,5-H); 8.01 (s, 1 H, 3-CF₃-C₆H₄-C₂-H); 11.40 (s, 1 H, pyrrole-C₂-NH, D₂O exchangeable); 11.83 (s, 1 H, C₆H₅-NH, D₂O exchangeable). Anal. Calc. (%) for C₂₇H₂₁ClF₃N₃O₂S (544): C, 59.61; H, 3.89; N, 7.72. Found (%): C, 59.67; H, 3.93; N, 7.81.

2) Ethyl 4-(4-chlorophenyl)-2-(3-phenylthioureido)-1-(pyridin-3-yl)-1H-pyrrole-3-carboxylate; 38b

Dark brown needle crystals; yield (76%); m.p.: >300˚C. IR [KBr, cm⁻¹]: 3390, 3213 (NH); 3080 (CH aromatic); 2927, 2820 (CH-aliphatic); 1680 (C=O); 1525 (C=C); 1411, 1360, 1134, 1033 (I, II, III, IV bands N-C=S); 1250, 1090 (C-O-C); 1040 (p-Cl-phenyl). MS m/z (relative intensity %): 478 (M^+•+2, 1); 94 (100). Anal. Calc. (%) for C₂₅H₂₁ClN₄O₂S (477): C, 62.95; H, 4.44; N, 11.75. Found (%): C, 62.98; H, 4.51; N, 11.78.

3.1.38. General Procedure for Synthesis of Compounds 39a,b

A mixture of the appropriate 1-(substitutedaryl)-2-amino-4-(4-chlorophenyl)-1H-pyrrole-3-carboxylic acid 37a,b (2 mmol.) and excess acetic anhydride (20 mL) was heated under reflux for 10 h. The reaction was left to cool and the solid product which was formed, was filtered off, washed with ethanol then recrystallized from glacial acetic acid.

1) 5-(4-Chlorophenyl)-2-methyl-7-(3-trifluoromethylphenyl)pyrrolo[2,3-d][1,3]oxazin-4(7H)-one; 39a

Yellow powder; yield (73%); m.p.: >300˚C. IR [KBr, cm⁻¹]: 3040 (CH aromatic); 2922, 2852 (CH-aliphatic); 1686 (C=O); 1550 (C=C); 1290, 1088 (C-O-C); 1025 (p-Cl-phenyl). MS m/z (relative intensity %):404 (M^+•, 0.2); 57 (100). Anal. Calc. (%) for C₂₀H₁₂ClF₃N₂O₂ (404.8): C, 59.35; H, 2.99; N, 6.92. Found (%): C, 59.41; H, 2.95; N, 6.97.

2) 5-(4-Chlorophenyl)-2-methyl-7-(pyridin-3-yl)pyrrolo[2,3-d][1,3]oxazin-4(7H)-one; 39b

Dark brown crystals; yield (83%); m.p.: >300˚C. IR [KBr, cm⁻¹]: 3060 (CH aromatic); 2925, 2850 (CH-ali- phatic); 1688 (C=O); 1426 (C=C); 1236, 1084 (C-O-C); 1020 (p-Cl-phenyl). MS m/z (relative intensity %): 338 (M^+•+1, 0.2); 337 (M^+•, 0.2); 57 (100). Anal. Calc. (%) for C₁₈H₁₂ClN₃O₂ (337.8): C, 64.01; H, 3.58; N, 12.44. Found (%): C, 64.07; H, 3.62; N, 12.51.

3.1.39. General Procedure for Synthesis of Compounds 40a,b

A solution of the selected ethyl 1-(substituted aryl)-4-(4-chlorophenyl)-2-(3-phenylthioureido)-1H-pyrrol-3- carboxylate 38a,b (2 mmol.) in sodium ethoxide [prepared by dissolving sodium metal (0.1 g, 2 mmol.) in absolute ethanol (30 mL)] was heated under reflux for 8 h. The reaction mixture was acidified with 10% HCl and the obtained product was collected, washed with ethanol and recrystallized from ethanol.

1) 5-(4-Chlorophenyl)-3-phenyl-7-(3-trifluoromethylphenyl)-2-thioxo-2,3-dihydro-1H-pyrrolo[2,3-d]pyrimi- din-4(7H)-one; 40a

Faint orange needle crystals; yield (69%); m.p.: >300˚C. IR [KBr, cm⁻¹]: 3180 (NH); 2922 (CH aromatic); 1700 (C=O); 1550 (C=C); 1460, 1350, 1134, 1036 (I, II, III, IV bands N-C=S); 1036 (p-Cl-phenyl). ¹H NMR (DMSO-d₆, δ ppm): 5.02 (s,1 H, NH, D₂O exchangeable); 6.60 - 6.78 (m, 5 H, C₆H₅); 7.31 (d, 2 H, J = 8.4 Hz, 4-Cl-C₆H₄-C_2,6-H); 7.40 (s, 1 H, CH-pyrrole); 7.48 - 7.61 (m, 2 H, 3-CF₃-C₆H₄-C_5,6-H); 7.85 (d, 2 H, J = 8.4 Hz, 4-Cl-C₆H₄-C_3,5-H); 7.98 - 8.10 (m, 2 H, 3-CF₃-C₆H₄-C_2,4-H). Anal. Calc. (%) for C₂₅H₁₅ClF₃N₃OS(497.9): C, 60.30; H, 3.04; N, 8.44. Found (%): C, 60.38; H, 3.10; N, 8.48.

2) 5-(4-Chlorophenyl)-3-phenyl-7-(pyridin-3-yl)-2-thioxo-2,3-dihydro-1H-pyrrolo[2,3-d]pyrimidin-4(7H)-one; 40 b

Dark brown needle crystals; yield (69%); m.p.: >300˚C. IR [KBr, cm⁻¹]: 3200 (NH); 2924 (CH aromatic); 1690 (C=O); 1541 (C=C); 1455, 1250, 1122, 1037 (I, II, III, IV bands N-C=S); 1037 (p-Cl-phenyl). MS m/z (relative intensity %): 432 (M^+•+2, 0.4); 57 (100). Anal. Calc. (%) for C₂₃H₁₅ClN₄OS (430.9): C, 64.11; H, 3.51; N, 13.00. Found (%): C, 64.17; H, 3.61; N, 13.08.

3.2. Biology

3.2.1. Anti-Inflammatory Screening

1) Animals

The screening for anti-inflammatory activity for all the newly synthesized compounds 4a-40b was carried out by using adult albino rats of both sexes weighing 120 - 150 g which were obtained from animal house laboratory of Nile company, Cairo, Egypt. Rats were divided into eighty four groups; each group consists of five rats per cage in the Department of Pharmacology, Faculty of Medicine, Al-Azhar University.

The rats were kept under constant temperature 30˚C and 12 hours light/dark cycle. All animals were acclimatized in the animal facility for at least two weeks prior the experiments.

The animals were kept fastened for 24 hours prior to the experiment, but they were allowed free access to water [37] . The animal experiments described below comply with the ethical principles and guidelines for the care and use of laboratory animals adopted by the National Egyptian Community.

The equipment used was Dial micrometer model (120 - 1206 Baty, Sussex, England).

2) Anti-inflammatory activity:

Rat paw edema assay was carried out according to Winter et al. [38] . Prepared compounds (equimolar to the referencedrug) were dissolved in DMSO and administrated subcutaneously.

One hour after drug administration acute inflammation was induced by injection of 0.05 mL of 1% of carrageenan sodium (Sigma-Aldrich, St. Louis, USA) subcutaneously into the sub planter region of the right hind paw.

The thickness of the injected paw was measured (from dorsal to ventral surfaces) immediately after carrageenan injection and after (1, 2, 3, 4, 5 and 6 hours) by using a micrometer. The size of edema was expressed as the increase in the thickness in mm after carrageenan injection.

The percentage inhibition of edema thickness at each time interval was calculated from the mean effect in control and treated animals according to the equation [39] [40] .

where, Tc and Tt are the mean increase in thickness of the carrageenan injected paw of the control group and drug treated groups; respectively.

Control group: received the excipients (water mixed with few drops of tween 80) followed by carrageenan after 1 hour. Indomethacin (Indocin^®) (5 mg/kg) was used as the referencedrug [41] . Then the potencies of compounds were calculated after 6 hours of carrageenan injection where the % edema inhibition reached maximum.

3) Statistical analysis

Results are expressed as (mean ± standard deviation) statistically analyzed using two way analysis of variance (ANOVA) followed by Bonferroni test [42] .

3.2.2. Ulcerogenicity

All animals subjected to this experimental test were sacrificed immediately after the last measurement (6 hour), by diethyl ether and stomachs were separated. An opening at the great curvature was made and the stomachs were washed with distilled water and cleaned gently by dipping in normal saline. The mucosal damage was inspected with a 3× magnifying lens for any evidence of hyperemia, hemorrhage or ulcer. For each stomach the mucosal damage was assessed [43] .

The percentage ulceration for each group was calculated as follows:

3.2.3. Analgesic Screening

1) Animals:

The screening for analgesic activity for all synthesized compounds 4a-40b was carried out by using mices of both sexes weighing 25 - 30 g which were obtained from animal house laboratory, Nile company, Cairo, Egypt.

Mices were divided into eighty four groups; each group consists of four mices per cage in the animal facility of Faculty of Medicine, Al-Azhar University. The mices were kept under constant temperature 25˚C and 12 hours light/dark cycle. All animals were acclimatized in the animal facility for at least two weeks prior the experiments.The animals were kept fastened for 24 hours prior to the experiment, but they were allowed free access to water.

2) Assessment of analgesic screening:

The analgesic activity was evaluated according to writhing test reported by Koster et al. [44] . The newly synthesized compounds (equimolar to the reference drug) were dissolved in DMSO and administrated to the groups orally (using intragastric tube) followed by injection of 0.6% acetic acid solution (10 mL/kg) after 1 hour [45] . Indomethacin (Indocin^®) (2.5 mg/kg) was used as the reference drug.

Stretching movements (arching of the back, developments of the tension in the abdominal muscles, elongation of the body and extension of the forelimbs) were counted as a writhing response. The number of writhes was counted for 15 minutes immediately after the acetic acid injection. The percentage of inhibition of writhes number was calculated as follows:

where Nc and Nt are number of writhes in the control group and drug groups; respectively.

3.2.4. Antimicrobial Activity

1) Materials and methods

Antimicrobial activity was examined by the cup-diffusion method [46] . The in-vitro antimicrobialactivity of the synthesized compounds was investigated against several pathogenic representatives; Gram-negative bacteria; Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 8739) and Gram-positive bacteria; Staphylococcus aureus (ATCC 25923) and Candida albicans (ATCC 10231) as a representative for fungi. All microorganisms used were obtained from the culture collection of the Department of Microbiology and Immunology, Faculty of Pharmacy (boys), Al-Azhar University, Cairo, Egypt.

Media for disc sensitivity tests were the nutrient agar and Muller-Hinton agar (MHA) purchased from Difco (USA). Non-sterile powder of the tested compounds was dissolved in dimethylformamide in a concentration of 1 mg/mL. Each 100 mL of sterile molten agar (at 45˚C) received 1 mL of 6 h cultured broth with the microorganism, then the seeded agar was poured into sterile petri dishes. Cups (8 mm in diameter) were cut in the agar. Each cup received 0.1 mL of the 1 mg/mL solution of the test compounds. Plates were then incubated at 37˚C for 24 h. for bacteria and 48 h. for fungi. Ampicillin (Bioanalyses Turkey) and Fluconazole (Sigma-Aldrich, USA) were used as reference substances.

3.3. Computer Aided Docking

3.3.1. Materials

All the molecular studies were carried out on an Intel Pentium 1.6 GHz processor, 512 MB memory with windows XP operating system using Molecular Operating Environment (MOE 10.2008) software provided by chemical computing group, Montreal, Canada.

All the minimizations were performed with MOE until a RMSD gradient of 0.05 Kcal∙mol⁻¹∙A⁰⁻¹ with MMFF94X force field and the partial charges were automatically calculated.

3.3.2. General Methodology

The coordinates of the X-ray crystallographic structure of the COX-2 complex with its co-crystallized ligand (Diclofenac) in the file (PDP ID: 1CX2) was obtained from the protein data bank (PDB). Enzyme structures were checked for missing atoms, bonds and contacts. The ligand molecules were constructed using the builder molecule and were energy minimized. The enzyme was prepared for docking studies where Ligand molecule was removed from the enzyme active site. Also, hydrogen atoms were added to the structure with their standard geometry. However, MOE Alpha site Finder was used for the active sites search in the enzyme structure and dummy atoms were created from the obtained alpha spheres.

4. Conclusions

It can be concluded that, among the newly synthesized compounds, compounds 5d, 9d, 11b, 12a, 13b and 32a showed multiple activities; anti-inflammatory, analgesic and anti-bacterial activities. Furthermore, it is to be noted that, some functions exerted multiple activities among these functions are 2,4-dichlorobenzylidine imino function in 2-position of pyrrole as in compound 5d. Also, the thiourea and phenylurea function as in compounds 9d and 11b, respectively. In addition to, imino function such as iminopyrrolopyrimidine thione and ethoxymethyleneimino functions as in compounds 12a and 13b, respectively, besides to, the pyrrolo[1,2,3]tri- azine derivative 32a.

In addition to, the molecular docking for the twenty most active anti-inflammatory compounds was performed on the active site of COX-2 enzyme in a trial to predict their mode of action as anti-inflammatory drugs, in which the compounds showed several interactions leading to the conclusion that they might exert their action through inhibition of COX-2 enzyme.

Acknowledgements

The biological anti-inflammatory screening was performed in the Histology Department, Faculty of Medicine (Girls), Al-Azhar University. While, the biological anti-microbial screening was performed in the Microbiology Department, Faculty of Pharmacy (Girls), Al-Azhar University. The Molecular docking study was performed in the pharmaceutical Chemistry Department, Faculty of Pharmacy, Alexandria University.

Cite this paper

M. T. Sarg,M. M. Koraa,A. H. Bayoumi,S. M. Abd El Gilil, (2015) Synthesis of Pyrroles and Condensed Pyrroles as Anti-Inflammatory Agents with Multiple Activities and Their Molecular Docking Study. Open Journal of Medicinal Chemistry,05,49-96. doi: 10.4236/ojmc.2015.54005

References

NOTES

^*Corresponding author.