Synthesis of Some New Folic Acid-Based Heterocycles of Anticipated Biological Activity

To date, no fused heterocycles have been formed on folic acid molecules; for this reason, and others, our target is to synthesize new derivatives of folic acid as isolated or fused systems. Folic acid 1 reacted with ethyl pyruvate, triethyl orthoformate, ethyl chloroformate, thioformic acid hydrazide, and aldehydes to give new derivatives of folic acid 2–6a,b. Moreover, It reacted with benzylidene malononitrile, acetylacetone, ninhydrin, ethyl acetoacetate, ethyl cyanoacetate, and ethyl chloroacetate to give the pteridine fused systems 10–15, respectively. Ethoxycarbonylamino derivate 5 reacted with some nucleophiles containing the NH2 group, such as aminoguanidinium hydrocarbonate, hydrazine hydrate, glycine, thioformic acid hydrazide, and sulfa drugs in different conditions to give the urea derivatives 16–20a,b. Compound 4 reacted with the same nucleophiles to give the methylidene amino derivatives 21–24a,b. The fused compound 10 reacted with thioglycolic acid carbon disulfide, malononitrile, and formamide to give the four cyclic fused systems 25–30, respectively. The biological activity of some synthesized showed moderate effect against bacteria, but no effect shown towards fungi.


Introduction
Folic acid 1 is essential to the human metabolic process. It is a key factor in the synthesis of nucleic acid. Folic acid (vitamin B 9 ) helps with growth [1] and healthy red blood cells (RBCs) [2]. It is important for cell division. It is essential for growth of the fetus [3]. Folic acid deficiency can lead to human megaloblastic anemia and neural tube defects in fetuses [4], as well as heart diseases, and cancer [5]. To avoid all of these risks, folic acid intake from fortified food has been increasing rapidly [6].
Several novel 2,4-diamino-5-deaza- 6,7,8,9-tetrahydropyrido [3,4-g]pteridine derivatives with different substitution at the N 7 position were designed and synthesized, as classical and non-classical, partially restricted, linear tricyclic 5-deaza antifolates. The purpose was to investigate the effect of conformational restriction of the C 6 −C 9 (τ1) and C 9 −N 10 (τ2) bonds via an ethyl bridge from the N 10 to the C 7 position of 5-deaza methotrexate (MTX) on the inhibitory potency against dihydrofolate reductase (DHFR) from different sources and on antitumor activity [7]. Moreover, some efforts were carried out to synthesize the 7-substituted folic acid derivatives the less toxic, more effective, and selective agents for cancer chemotherapy based upon inhibition of dihydrofolate reductase and thymidylate synthetase [8]. The 10-Alkyl-5,10-dideaza analogs of methotrexate and tetrahydrofolic acid were synthesized and used as potent inhibitors of glycineamide ribonucleotide (GAR) formyltransferase [9]. Moreover, numerous fused cyclopenta[d]pyrimidine antifolate were synthesized and examined for their effects as highly potent as DHFR and cell growth inhibitors, and most of them are more potent than methotrexate (MTX) and 10ethyl-10-deazapterin (10-EDAM) in inhibiting tumor cell growth (P388 MTX-sensitive and

Results and Discussion
The importance of synthesizing a new heterocyclic compound is strongly bonded with the finding of a new drug, capable of destroying any of the diseases that spread around the world. Herein is a new trial, in continuation of our previous work , to synthesize a new compound from folic acid in the hope of getting a new promoting drug.
Folic acid 1 reacted with ethyl pyruvate in different ratios in DMF as a solvent. Refluxing mixture of folic acid and ethyl pyruvate 1:1 ratio for 8 hr, gave imidazo[2,1-b]pteridine derivative 2. While the reaction of folic acid with ethyl pyruvate, in a 1:2 ratio for 2 h, led to the formation of the N,N-disubstituted folic acid (Scheme 1). The structures of the two compounds 2 and 3 were confirmed from their IR and NMR spectra, where in the IR spectra, the band due to the NH 2 group disappeared with appearance of new bands due to the inserted carbonyl groups in the two compounds. While, 1 H-NMR of compound 2 showed singlet signal at δ = 1.30 ppm due to the CH 3 attached to the imidazole ring. The 1 H-NMR of compound 3 also showed more deshielded singlet signal at δ = 2.21 ppm due to the 2CH 3 attached to the carbonyl group. The 13 C-NMR supported the structure proposed where it showed signals due to the CH 3 in the two compounds at δ = 24.51 and 26.79 ppm, respectively.
Formation of ethoxymethyleneaminopetridine derivative 4 and ethoxycarbonylaminop etridine derivative 5 were formed by the reaction of folic acid with excess triethyl orthoformate and/or ethyl chloroformate in presence of TMA as a base (Scheme 1). The elucidation of the structures of the two compounds were recognized from their spectra and elemental analysis where, the two compounds showed the appearance of new signals in the 1 H-NMR, triplets at δ = 1.15 and 1.14 quartets at 3.39 and 4.02 ppm due to the CH 3 and CH 2 , respectively. The condensation of the NH 2 of folic acid with aldehyde to form Schiff base was carried using formaldehyde and benzaldehyde in acetic acid glacial using drops of HCl as an acidic catalyst. The Schiff bases formed 6a,b showed the disappearance of the NH 2 group in folic acid and appearance for the olefinic proton due to the formation of N=CH bond at δ = 7.99 and 8.03 ppm, respectively. 13 C-NMR helped in the structures conforma-tion, where, it showed signals at δ = 139.75 and 164.21 ppm due to the methylidene and benzylidene carbons respectively. The reaction of thioformic acid hydrazide with folic acid in DMF/EtOH mixture yielded the thiosemicarbazide derivative 7 which cyclized with ethyl chloroformate in presence of TMA to give the 1,3,4-triazole thione derivative 9 (Scheme 1). The structures of compounds 7 and 9 were established from IR and 1 HNMR, where the IR showed extra bands due to the NH groups in compound 7 and two bands due to C=S at 1338 and 1347 cm −1 for compounds 7 and 9, respectively. Moreover, 1 H-NMR showed three singlet signals for the thiosemicarbazide group at 5.69 (NH 2 ), 11.31 and 12.89 ppm for (NHCSNH) protons respectively. Folic acid, also, reacted with acid chloride derivative in basic medium to give the N-substituted derivative. Reaction of folic acid with sebacoyl chloride in presence of TMA yielded the bis compound 8 (Scheme 1). The 1 H-NMR confirmed the structure of 8 where it showed characteristic signals for the 8 CH 2 due to the sebacoyl moiety at δ = 1.03, 1.26, 2.23, and 3.34 ppm, respectively.  Benzylidene malononitrile was the first compound was used to form the nucleus compound for synthesizing the more fused rings. The well-known benzylidene malononitrile was synthesized and reacted with folic acid in boiling ethanol to form the three fused rings compound pyrimido[2,1-b]pteridine derivative 10 (Scheme 2) The appearance of the CN band in the IR of compound 10 was the first guide for the structure assertion. The 1 H-NMR was the second guide, where it showed multiplets at δ = 7.62-7.96 ppm for the 7-phenyl group. Reaction of folic acid with acetylacetone, ethyl acetoacetate, ethyl cyanoacetate, and/or ethyl chloroacetate in DMF gave fused pyrimidine, fused primidone and fused imidazolidinone to the pteridine ring 11, 13, 14, and 15, respectively (Scheme 2). While the reaction of folic acid with ninhydrin in boiling ethanol yielded the five fused rings derivative indeno [2 ,1 :4,5]imidazo[2,1-b]pteridine 12. The structures of the resulted compounds stablished from their 1 H-NMR and 13 C-NMR. The 1 H-NMR of compound 11 showed two singlets at δ = 2.26 and 2.40 ppm for the two CH 3 -in pyrimidine ring, Moreover, two signals for the same groups appeared at 24.30 and 26.45 ppm in the 13 C-NMR. Compound 13 showed two singlets one for the CH 3 at 2.25 and the other at 7.21 due to pyrimidone olefinic proton. The structure of compound 14 was confirmed by the singlet appeared in its 1 H-NMR at δ = 2.89 ppm due to pyrimidone CH 2 , which also appeared in its 13 C-NMR at δ = 43.05 ppm. The disappearance of the CN band for compound 14 in its IR proved the cyclic structure of the compound. The 1 H-NMR of compound 15 was the main guide for its structure proven, where the 1 H-NMR of compound 15 showed singlet at δ = 3.51 ppm due to imidazolidinone CH 2 along with the presence of NH singlet signal of imidazolidinone at δ = 10.87 ppm. On the other hand, the structure of compound 12 was certain, by the presence of the singlet due to the imidazoloindene OH group at δ = 5.36 ppm.
Ethoxycarbonylamino derivative 15 was used as starting material for the synthesis of other types of derivatives, which are not attached directly to the pteridine ring. We exploited the chance presence of an ester group in the compound and subjected it to react with some nucleophiles containing NH 2 , such as aminoguanidinium hydrocarbonate, hydrazine hydrate, glycine, thioformic acid hydrazide, and sulfa drugs in different conditions. structures were elucidated form their spectral and analysis data, where all of the 1 H-NMR spectra of all the compounds missing the ethyl group, triplet and quartet signals, with appearance of other signals due to the new functional groups, for example, compound 126, showed in its 1 H-NMR signals at δ = 4.33, 8.69 and 9.61 ppm for the NH 2 , and two NH proton neighboring to the carbonyl group. Compound 18 showed the signals corresponding to the glycine molecule, the CH 2 protons appeared at δ = 4.03 ppm and the acidic proton of the glycine carboxylic group found at 11.60 ppm. The 13 C-NMR of compound 18 also supported the structure elucidation, where it showed a signal for CH 2 carbon at 41.10 ppm and another signal for the glycine carboxylic carbon at 173.17 ppm. Compound 19 was the highest data help in the structure confirmation, where, Its IR showed bands due to thiocarboxylic group at 2627 cm −1 for (SH) and 1351 cm −1 for (C=S) group. Th 1 H-NMR of compound 19 gave more conformational data, it showed signal due to the SH group at δ = 13.98 ppm, all of this data are supported with the elemental analysis for the sulfur. The presence of the SH group in compound 19 rejected the idea that compound 19 may cyclize to give 1,3,4-triazole thione derivative.
The combination between the sulfa drug and folic acid was an idea for increasing the biological activity of folic acid, especially antibacterial activity. Thus, folic acid reacted with sulfadiazine and/or sulfadimidine in DMF to yield the corresponding aminocarbonyl sulfadiazine derivative 20a and aminocarbonyl sulfadimidine derivative 20b, respectively (Scheme 3). The structures of 20a and 20b were confirmed from their spectral data (experimental part]. Ethoxymethylene derivative 14 was used for synthesizing a new folic acid derivative; thus, compound 113 reacted with some nucleophilic amino compounds, such as, semicarbazide hydrochloride, aminoguanidine hydrocarbonate, glycine, and sulfa drugs, to obtain some new Schiff base derivatives. Reaction of 4 with semicarbazide hydrochloride in boiling DMF in presence of drops of TMA as a base yielded "N- H-NMR with appearance of new signals due to NH 2 , and NH groups at the ranges 6.21-6.98 ppm for NH2 group and 10.13-10.99 ppm for NH groups. Compound 23 showed in its 1 H-NMR two signals for the glycine moiety, at singlet at δ = 4.18 ppm for CH 2 and at 11.58 for the acidic proton CH 2 COOH. Compound 14 reacted with sulfa drugs, namely, sulfadiazine and sulfadimidine, to give the folic acid substituted with sulfa drug moiety, 24a,b (Scheme 4). The structures of compounds 24a,b proved with the aid of their 1 H-NMR, where, the 1 H-NMR showed the appearance of the multiplets due to the phenyl group of the sulfa drug, with disappearance of the ethyl group signals.  [5 ,4 :5,6]pyrimido-[2,1-b]pteridine 30 were prepared by the reaction of 10 with malononitrile and/or formamide on hot. Compound 29 showed, in its 1 H-NMR, two signals for the two NH 2 groups at δ = 6.12 and 6.58 ppm, while compound 30 showed only one signal for the NH 2 groups at δ = 6.50 ppm.

Biological Activity Results and Discussion
The studying of the biological screening of the tested compounds, Table 1, showed folic acid had a moderate inhibitory effect against each of the G + and G − bacteria compared to the standard antibacterial agent (Ampicillin).  Compounds 6a, 8, 22, 26, and 27 showed inhibitory effects slightly more than folic acid against all types of bacteria, and this may be due to the presence a new group exceeded to folic acid structure, such as, terminal C=N in compounds 6a and 22, SH in compounds 26 and 27, and 8 CH 2 in compound 8.
Inden-1-one present in compound 12 may be the cause for more activity than folic acid against G − bacteria E. coli and P. aeruginosa.
Compound 9 as well as compound 20b showed somewhat strange inhibitory effects, where compound 9 showed higher effect against P. aeruginosa than folic acid, and at the same time, it showed null effects against E. coli and S. aureus; this may be due to the presence of triazole thione ring in its structure. Moreover, compound 20b showed null effects against B. subtilis and P. aeruginosa and moderate effect against E. coli and S. aureus, the diamidine nucleus in its structure.
Compounds 2, 4, 10, 14, 15, 23, and 24b showed nearly the same effects similar to folic acid; this means that the new groups added to the structure of folic acid did not have any effect against microorganisms in general.
Compounds 5 and 18 had no effect against all microorganisms; this may be due to the presence of each of glycine unit and/or the ethyl carbamate unit in the structure.
In comparison, among the different effects of all compounds under study, we found: • Compound 22 showed higher effect against B. subtilis than other compounds that showed an average effect. • Compounds 5, 18, 20b did not show the effect against B. subtilis.
The effect of compounds 6a, 8, 22, 27 was slightly higher than 11, 12, 14, 23 against S. aureus while other compounds have a moderate effect against S. aureus and compounds 1, 5, 18 had no effect against them.
The effect of compounds against E. coli was close to the effect of compounds against bacteria, while the effect of compounds against P. aeruginosa was dissimilar. Compound 27 showed a higher effect against P. aeruginosa than other compounds.
On the other hand, folic acid, as well as all of the synthesized compounds, had no effect against Fungi (A. flavus and C. albicans).

Conclusions
Herein, novel derivatives of folic acid were prepared by direct reactions of different reagents, with folic acid, or by reaction of some derivatives prepared with the same (or another) reagent. The study was directed to find new derivatives of folic acid having a promising biological activity, but, from the study carried out and the results obtained, all of the derivatives prepared were inactive against fungi, while some of these derivatives had a moderate antibacterial activity. The conclusion of this study is that the reactions done on the NH 2 group of folic acid, either substituted groups formed or fused systems, are not valuable as antifungal or antibacterial agents. Future work will be directed to the carboxylic groups of folic acid to synthesize a new isolated or fused system from folic acid in hopes of getting a more promising drug.

Experimental Chemistry
All chemicals used were supplied by Sigma (New York, NY, USA). Digital Electro thermal IA 9100 Series used for measuring melting points and they were uncorrected. Infra-red spectra were examined on ATRAlpha FTIR spectrophotometer (Billerica, MA, USA). 1 H-NMR and 13 C-NMR spectra examined on a Bruker AC-850 MHz apparatus (Bruker, Billerica, Massachusetts). Chemical shifts expressed as (ppm) relative to internal standard (TMS), and DMSO-d6 used as the solvent and in 13