NEW CONDENSED BIOACTIVE PYRIMIDINES . SYNTHETIC STRATEGIES , STRUCTURAL CHARACTERIZATION AND BIORATIONAL STUDIES

Abdelmonim El-Torgoman 1 , Sherif M. Sherif 2 , Mamdouh I. Nassar 3 , Hatem M. Gaber 4 and Doaa A. Zaki 1 . 1. Department of Chemistry, Faculty of Science, Menoufia University, Shibin Al kawm , Egypt. 2. Department of Chemistry, Faculty of Science, Cairo University, Giza 12613, Egypt. 3. Department of Entomology, Faculty of Science, Cairo University, Giza 12613, Egypt. 4. National Organization for Drug Control and Research (NODCAR), P.O. Box 29, Cairo, Egypt. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

Versatile 3-aminopyrrolopyridine-2-carboxylate and -2-carboxylic acid derivatives 2a,bwere obtained based on an orthofunctionalized pyrrole derivative 1. Initially cyclization of ester 2a followed by formylation to obtain a tricyclic chloroaldehyde derivative 4, which on further treatment with hydroxylamine hydrochloride yielded the corresponding oxime5a. The latter product was subsequently dehydrated forming the tricyclic 2-cyanopyrimidine derivative 5b. Other different 2-functionalized tricyclic derivatives of N-3substituted pyrimidine were synthesized, for biorational assessment, starting from the key precursors 2a,b through convenient methods. All condensed tricyclic pyrimidine derivatives with the common 7,9diphenyl-8-(4-methoxyphenylazo) substitution patternwere evaluated for their toxicity on the 4 th larval instar of the mosquitoes, Culexpipiens. The compounds under investigation displayed different levels of biorational effects. In general, chloro compounds 4, 5a and 5b that have a chlorine atom as a substituent on the C-4 atom of the pyrimidine ring, showed high insecticidal activity, with compound 5b showing essentially the highest biorational effect. The lethal concentration at LC 50 of the latter compound 5b is very significant (0.22%). Our results may provide some guidance for development of some novel chloropyrimidine-based insecticidal lead structures. The detailed synthesis and biological screening data were reported.

Introduction:-
There has been an increasing demand for new bioactive agents, as the fast development of pests resistance to conventional chemicals is one of the major difficulties in the treatment. Researchers have been used new novel chemicals as bio-agents effects because they attended that the conventional chemicals have been adverse action on the non-target organisms.
In the course of reviewing various structures which may be of use in the design of novel bioactive and biorational agents, condensed pyrimidines have attracted much of our attention because of their synthetic and biological INTERNATIONAL JOURNAL OF ADVANCED RESEARCH (IJAR) ISSN: 2320-5407 Int. J. Adv. Res. 4 (10), 265-279 267 to give a solid product, in 55% yield, identical in every respect (mp, mixed mp and IR data) to that obtained above from method A.

Method B:
This compound was synthesized from N-acetyl derivative 10a (0.002 mol) and hydrazine hydrate (0.02 mol, 1 ml) in a manner similar to that described above in method A (reaction time: 8 h). The material obtained after recrystallization from ethanol and chloroform mixture proved to be 9a (59% yield).

Method B:
To a solution of compound 9b (0.003 mol) in dimethylformamide (20 ml), anhydrous potassium carbonate (0.006 mol) was added and the mixture was stirred at room temperature for 15 min, followed by the addition of ethyl chloroacetate (0.0033 mol) in dimethylformamide (10 ml). Stirring was continued at room temperature overnight, according to thin layer chromatographic (TLC) analysis. And then ice/water mixture was added to the reaction mixture to form a precipitate, which was isolated by filtration, washed with water and dried. The residue was recrystallized from ethanol to give a solid product, in 55% yield, identical in every respect (mp, mixed mp and IR data) to that obtained above from method A.

Synthesis of 4-oxo-3,7-diphenyl-2-phenylazo-4,5-dihydropyrrolo[1,2-a]thieno[3,2-e]pyrimidine-6-carbonitrile (12):-
To a solution of compound 3 (0.005 mol) in 30 ml of dimethylformamide, phenyl isothiocyanate (0.0056 mol) was added dropwise. The reaction content was heated at reflux for 24 h. After cooling to room temperature, the solvent was removed in vacuoand the crude solid product was filtered off, dried and purified by recrystallization from ethanol to give light brown crystals of the title compound 12 (   273 Biorationalactivity:-Rearing mosquitoes:-Fourth-instar larvae of Culexpipienswere obtained from a laboratory colony at Entomology Department, Faculty of Science, Cairo University. Larvae were placed in Pyrex storage jars containing 200 ml of tap water. The colonies of mosquitoes were maintained at 25 ± 2°C and 60 ± 5 % relative humidity, and under 14: 10 h light: dark photoperiod cycle. Larvae were daily fed with fresh food consists in a mixture of Biscuit Petit regal-dried yeast (75:25 by weight) and water was replaced every four days. Colony was continuously as previously described (14) with some modification.

Treatment of Larvae:-
Twenty five fourth instars larvae of C. pipiens, were exposed to three concentrations (0.25; 0.50; and 0.75%) of different pyridine compounds. The treatment has been done under laboratory conditions, according to standard World Health Organization (WHO) methodology (15). Larvae were placed in glass beakers containing 200 ml water. Serial dilution of pyridine compounds (from 1 to 11compounds) were prepared in distillate water and added to the treatment beakers, then added a food source to larvae. Control larvae were reared in jars containing only water. The effects of pyridine compounds were assessed by biorational effect on larvae after 24 hours in parallel with control experiment.

Statistical Analysis:-
The larval mortality was corrected by (16) formula if control mortality was between. Toxicity data were studies by probit analysis (17). The probits regression program was used to determine the LC 50 .

Results and Discussion:-
Chemical compounds:-Heterocyclization of ethylthio derivative 1 [14] with ethyl glycinate was carried out to afford a bicyclic product for which the pyrrolopyridine2a with o-aminoesterstructurewas established on the basis of spectroscopic studies. The IR spectrum of the latter product showed no nitrile absorption, but a carbonyl absorption was observed at ν1664 cm -1 corresponding to a chelated conjugated ester. Similarly, the 1 H NMR spectrum was also informative in establishing the structure of aminoester2a. The spectrum was well characterized by the presence of two D 2 O-exchangeable signals arising from the exocyclic NH 2 and endocyclic NH protons at δ H 6.30 and 11.17 ppm, respectively, besides signals for the expected protons of an ethyl ester and aryl moieties. Alkaline hydrolysis of ethyl ester 2a followed by acidification gave the corresponding carboxylic acid 2b. It is worth mentioning that the same product 2b could also be obtained directly by reacting compound 1 with glycine. Compound 2b prepared by the latter route was found to be identical to that isolated by the former method as confirmed by TLC analysis, m.p., IR data and undepressed mixed melting point with the previously isolated material. Treating aminoester2a with formamide led to closure of the pyrimidine ring and accordingly led to the formation of the tricyclic pyridinopyrrolopyrimidine derivative 3 (Scheme 1). Compound 3 might be exist in one or more of three tautomeric structures A-C (Fig. 1). The most stable tautomeric form for compound 3 was determined through the analysis of IR and 1 H NMR spectral data. The possibility of the tautomerism involving lactim (enol) form 3Cwas excluded by the evident absence of typical signals for a hydroxylic group in the IR and 1 H NMR spectra. In the 1 H NMR spectrum, the most diagnostic signal is due to the NH proton (exchangeable with D 2 O). The spectrum showed this proton, appearing in the downfield region (δ H 12.42 ppm) typical for an 3-unsubstituted lactam (keto) form 3A, where the NH proton was deshielded by the adjacent carbonyl group. This shift is characteristic for lactam species, for which a lactam NH group has been reported [15-18a,b] to experience a further downfield shift to around δ H ~ 12.00 ppm due to the additional deshielding effect of the adjacent carbonyl moiety. On the contrary, the alternative tautomeric 1H-form 3B, if isolated, should lead to an upfield shift at N-1-unsubstituted resonance position due to the lesser deshielding effect. As a result, the NH proton of 1H-form 3B would be expected to appear at a lower chemical shift value (δ H < 12.00 ppm) than the corresponding one for tautomeric 3H-form 3A that have a lactam proton with a greater deshielding effect. The prevalence of the lactam (keto) form in compound 3 was further verified based on its IR spectrum. The spectrum was well characterized by the presence of a strong band for the lactam carbonyl group at a typical low frequency (ν1659 cm -1 ) together with an absorption band for the cyclic secondary amide at ν3175 cm -1 . Similar stretching frequencies have been reported on closely related heterocyclic amido compounds [21a,c -22].The prevalence of the 3H-rather than the 1H-form in a number of 3-unsubstituted pyrimidin-4-ones is well documented [18, 19, 20 and 26]. These findings affirm that the isolated product exists mainly in one tautomeric form, namely lactam (keto) form 3A. Vilsmeier-Haackformylation of compound 3, using two molar equivalent amount of the 274 Vilsmeier-Haack reagent, occurred not only with the formation of an aldehyde group in the 2-position of the pyrimidine ring but also with the chlorination of the amidic carbonyl function, leading eventually to the tricyclic chloroaldehyde analogue 4 as evidenced by elemental and spectroscopic investigations. In its IR spectrum, an aldehydic functional group was clearly observed with a carbonyl absorption frequency at ν1691 cm -1 . Moreover, the 1 H NMR spectrum of the isolated product could give unequivocal proof for its structure by the observation of a new diagnostic signal, due to the aldehyde proton, appearing in a typical downfield region (δ H 10.06 ppm). These assignments are in line with prior observations of spectra of analogous heterocyclic aldehydes [27,28]. The absence of signals belonging to the protons of the pyrimidine methine and amidic NH, present in the spectrum of the parent lactam 3, also lent additional support to the structural assignments. The structural elucidation of the reaction product 4 revealed that the electrophilic attack of the Vilsmeier reagent took place on the C-2 methine atom of compound 3, generating an enamine salt 3', which after hydrolysis yielded the corresponding chloroaldehyde compound 4 (Fig.  2). This assumption is consistent with the prior findings of related reports on the well establishedVilsmeier -Haackformylations [27][28][29][30].
The constitution of aldehyde 4 was also well supported by its conversion, on treatment with hydroxylamine hydrochloride, to the corresponding oxime5a, which in turn was dehydrated by the action of acetic anhydride to form the required nitrile 5b (Scheme 1). The IR spectrum of the latter product 5b lacked an absorption band due to the hydroxylic group, but contained stretching frequency at ν2240 cm -1 characteristic for a CN frequency. Also, the resonances of the hydroxy and methine protons, present in the 1 H NMR spectrum of the original oxime5a, were not detectable for the isolated product 5b, providing confirmatory evidence in support of nitrile formation through dehydration. This observation is also supported by previous literature reports on a similar transformation of an aldehyde moiety to a nitrile group in closely related systems via an oximation/dehydration sequence [31,32].
The bicyclic o-aminoester2a proved to be a useful precursor for the synthesis of other tricyclic pyrimidinones (Scheme 2). Thus, a new N-aminolactam7a could also be synthesized via initial treatment of compound 2a with phenyl isothiocyanate, followed by subsequent cyclization of the isolated thiourea product 6a with hydrazine hydrate via loss of hydrogen sulfide. Nevertheless, the configuration of the previously isolated 2phenylaminopyrimidine derivative 7a with N-3-aminolactam structurewas further supported chemically by its successful deamination, where treatment of compound 7a with sodium nitrite and acetic acid led to the corresponding 3-protodeamino analogue 7b. 1 H NMR spectroscopy could firmly confirm the structure of theseproducts. The resonance of amino protons allowed us to identify the latter products. In the amino derivative 7a, the N-3-amino protons appeared as a D 2 O-exchangeable singlet resonating at a typical shift (δ H 5.82 ppm) characteristic for an N-NH 2 resonance, whereas the corresponding deamino analogue 7b showed no N-NH 2 resonance, which is generally found at around δ H ~ 5.75 ppm by analogy with related N-3-aminoquinazolin-4-one analogues [13,33], thus indicating the disappearance of that N-3-amino group in the spectrum of 7b. The remaining resonances were also observed at the expected frequencies (see Experimental section). In addition, the mass spectrum of compound 7b showed a molecular ion peak at m/z 563 corresponding to its molecular weight, which is indicative of an decrease of 15 mass units from the parent 7a, confirming the assigned structure. This observation is also supported by prior literature reports on the well established nitrous acid deamination of the exocyclic amino group in closely related cyclic N-aminoamides [34][35][36][37].
The bicyclic o-aminocarboxylic acid 2b has also been employed as a synthetic foundation for the target condensed pyrimidine system with the common 7,9-diphenyl-8-(4-methoxyphenylazo) substitution pattern. Thus for example, heterocyclization of compound 2b with phenyl isothiocyanate was carried out to afford a tricyclic product with an annelated pyrimidine ring for which the thione structure 8 could be formulated (Scheme 2). It is remarkable to report here that an unexpected reaction took place on reacting compound 8 with hydrazine hydrate in an attempt to obtain the hydrazino compound 9. To our surprise, this reaction did not give the anticipated product 9 and instead the phenylamino derivative 7a was again isolated, the structure of which was unambiguously confirmed by comparison of TLC analysis, m.p., mixed m.p. and IR data with that of the previously obtained sample 7a. This result can be explained, as illustrated in Figure 3, by assuming that the reaction occurs firstly with the anticipated formation of the hydrazino compound 9, that underwent a Dimroth-type rearrangement [12, 37 and 38] in which the initially formed product 9 was subsequently hydrolyzed to the final rearranged phenylamino derivative 7a via N-phenyllactam ring opening, followed by dehydrativerecyclization of the acyclic acid intermediate 9'.
On the other hand, heterocyclization of carboxylic acid 2b with one-carbon building blocks such as triethylorthoformate and benzoyl chloride afforded lactones 10a,b, whereas acetylation of compound 2b with acetic 275 anhydride readily occurred with the formation of the corresponding 2-methyl analogue 10c, which could be transformed to the desired tricyclic pyrimidinones upon treatment with different nitrogen nucleophiles (Scheme 3). On reaction with hydrazine hydrate, the lactone 10c gave a product which might be assigned the structure of an N-3aminolactam 12a. The pathway of the studied reaction is two-step processes and probably involves initial nucleophilic attack by the amino group of hydrazine hydrate on the electrophilic carbonyl carbon center (C-4 carbon) with subsequent cleavage of the C-O bond and as a result the oxazinone ring opening leads to the intermediate formation of acyclic bisamide adduct 11a. Subsequent recyclization could then take place via nucleophilic attack by the carbohydrazide NH moiety on the amidic carbonyl group. This would be accompanied by cyclodehydration, leading eventually to the pyridopyrrolopyrimidine12a with N-3-aminolactam structure. The results of several investigations reported recently support this hypothesis [39][40][41][42]. It is worth mentioning that Naminolactam structure 12a was confirmed by an alternative synthetic route involving initial acetylation of the ester 2a with acetic anhydride and subsequent cyclization of the formed N-acetyl derivative 6b with hydrazine hydrate. Obviously, this reaction could also proceed, in this case, through the intermediacy of the carbohydrazide11a followed by intramolecular cyclocondensation under the applied reaction conditions. Compound 12a prepared by the latter route was found to be identical to that obtained by the former method.
As discussed above, tricyclic pyrimidinone analogues 12b-d could also be obtained in a similar manner by reacting compound 10c with each of ammonium acetate, aniline and ethyl glycinate, respectively. By analogy with hydrazine, intermediacy of open chain bisamides11b-d, respectively, are most likely. This would also be followed by dehydrativerecyclization, giving the target condensed pyrimidinones12b-d, respectively. Alternative synthesis of N-deamino derivative 12b was achieved by its deamination through treating N-amino compound 12a with sodium nitrite and acetic acid. Furthermore, another synthesis of N-alkylated compound 12d involved the alkylation of 3unsubstituted pyrimidinone derivative 12b with ethyl chloroacetate in the presence of potassium carbonate (Scheme 3). Better yield of the required product was obtained in this case. Elucidation of structure for the latter products was established on the basis of elemental and spectroscopic analyses in each case.

Bioassay of the novel pyridopyrrolopyrimidine on the 4 th larval instars of the Culexpipiens:-
This study was obviously the pyridine chemical compounds, from 1-11 have distinct biological effects on theculexpipens larvae. The lethal effect of these compounds was gradually increased by increasing concentrations of each compounds. Exposure of early fourth instar larvae of C. pipiensto the different concentrations of pyridine compounds caused a significant larvicidal effect (Table 1). At the lower concentration 0.25%, the larval mortality was lower corresponding to 0.75 % . The lethal concentration of pyridine compounds, 1, 2, 3 and 4 produced 67, 58, 85, and 95% of larval mortality at the higher concentration (0.75%) respectively. The population, LC 50 is determined from regression line measured as 0.61, 0.72, 0.43 and 0.22 of the previous compounds. Most of deaths at higher concentrations occurred in larvae produced deformation stages as larval-pupal intermediates.
The Biorational assessment of the chemical compounds in Tables 1. As shown by these results, the new condensed tricyclic pyrimidine derivatives under investigation displayed variable biorational effects. In general, the chemical structure, comprising the nature of the heterocyclic system as well as the substituted function present in the heterocyclic ring, has a pronounced effect on insecticidal activity. In particular, it was found that the attachment of a chloro moiety to the pyridopyrrolopyrimidine core produced the highest effects on mosquitoes (Culexpipiens) as observed for 4-chloro compounds 4, 5a and 5b, with LC 50 values 0.43, 0.61 and 0.22%, respectively ( Table 1).
The lethal effect of those derivatives was gradually increased by increasing concentrations of each compound. Exposure of early fourth instar larvae of C. pipiensto the different concentrations of condensed pyrimidine compounds caused a significant larvicidal effect (Table 1). At the lower concentration 0.25%, the larval mortality was lower corresponding to 0.75%. Most of deaths at higher concentrations occurred in larvae produced deformation stages as larval-pupal intermediates. Based on the biorational evaluation, 4-chloro compound 5b was the highest toxic and hence could be considered as a lead compound in this field. Similar compounds 4 and 5a showed also high toxicity. Other compounds 3, 7a,b, 8 and 12a-d were strikingly less toxic than those 4-chloro compounds. This would be as a result of something in the combination of pyridine with pyrrole and pyrimidine without a chlorine moiety.
From the structure-activity relationship (SAR), we can conclude that the chloro moiety is important for insecticidal activity, especially against the Culexpipiens larvae as observed for compound 5b. Further studies are in progress on 276 that compound to increase its efficacy and understand its QSAR. The overall results of the present study can be considered promising in the perspective of new insecticides discovery.
Pyridine compounds proved to be highly efficient to the C. pipiens (43 and 44). LC 50 values due to effect pyridine derivatives on C. pipienswere 60 and 43% (47). Our results were comparable with findingsfrom other researchers as the Pyridine solution (5 ) with sculpture, was highest their toxic compound to Culexlarvae in comparison with other compounds of pyridine base at the same concentrations. Experiments of pyrrolidine of concentration show the compound (4) was highly toxic may due chlorine attached to pyrimidine. The other chemical compounds of pyridine compounds, are strikingly less toxic than compounds (1, 2, 3 and 4) which results from something in the combination of pyridine with pyrrole and pyrimidine without sulphure and chlorine groups.
In harmony to these results, the idea that nicotine attached to pyridine is more effective against insects than its salts.
(44) reported some experiments in which silkworms (BombyxmoriL.) were sprayed with aqueous solutions of nicotine, nicotine containing alkali, and nicotine containing acid. No differences were noticed in the effects produced, but the acidulated nicotine was less active than the other solutions , while free nicotine is more toxic than nicotine salts.

277
The Biorational assessment of the chemical compounds in Tables 1. As shown by these results, the new condensed tricyclic pyrimidine derivatives under investigation displayed variable biorational effects. In general, the chemical structure, comprising the nature of the heterocyclic system as well as the substituted function present in the heterocyclic ring, has a pronounced effect on insecticidal activity. In particular, it was found that the attachment of a chloro moiety to the pyridopyrrolopyrimidine core produced the highest effects on mosquitoes (Culexpipiens) as observed for 4-chloro compounds 4, 5a and 5b, with LC 50 values 0.43, 0.61 and 0.22%, respectively ( Table 1).
The lethal effect of those derivatives was gradually increased by increasing concentrations of each compound. Exposure of early fourth instar larvae of C. pipiensto the different concentrations of condensed pyrimidine compounds caused a significant larvicidal effect (Table 1). At the lower concentration 0.25%, the larval mortality was lower corresponding to 0.75%. Most of deaths at higher concentrations occurred in larvae produced deformation stages as larval-pupal intermediates. Based on the biorational evaluation, 4-chloro compound 5b was the highest toxic and hence could be considered as a lead compound in this field. Similar compounds 4 and 5a showed also high toxicity. Other compounds 3, 7a,b, 8 and 12a-d were strikingly less toxic than those 4-chloro compounds. This would be as a result of something in the combination of pyridine with pyrrole and pyrimidine without a chlorine moiety.
From the structure-activity relationship (SAR), we can conclude that the chloro moiety is important for insecticidal activity, especially against the Culexpipiens larvae as observed for compound 5b. Further studies are in progress on that compound to increase its efficacy and understand its QSAR. The overall results of the present study can be considered promising in the perspective of new insecticides discovery.

Conclusion:-
We have demonstrated that the heterocyclization of 2-aminopyrrolopyridine-3-carboxylate and -2-carboxylic acid derivatives 2 and 3 provided easy and versatile access to a variety of tricyclic pyrimidines, which were of significant interest for biorational investigations. The biorational potential of all new tricyclic pyrimidine derivatives was further investigated by evaluating their insecticidal activity against Culexpipiens larvae. Biorational study of the compounds under investigation indicated that the highest insecticidal activity was observed for compound 5b with a chloro moiety attached to the pyrimidine ring at the 4-position. Its LC 50 value (0.22%) towards Culexpipiens is very significant. Other chloro compounds 4 and 5a showed also an appreciable toxicity. Their LC 50 values (0.43 and 0.61%) are very interesting. On the other hand, deformation of the treated larvae was observed by effect of different compounds due to molting inhibition for the next stages.The treatment of C. pipiens larvae with different compounds of condensed pyrimidine resulted in a significant inhibition of adult emergence of C. pipiens. In light of the results presented in this work and taking into account that this preliminary study does not produce conclusive evidence regarding a structure-activity relationship, we have focused our attention on the most promising compound 5b as an interesting starting point for the development of a new class of chemical insecticides. Further structural modifications might lead to the discovery of more potent insecticides and this work is in progress. We believe that research in this direction should be encouraged in order to broaden the applicability of these new heterocyclic frameworks to serve as leads for designing novel chemical insecticides.