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. 2020 Sep 22;6(9):e04956. doi: 10.1016/j.heliyon.2020.e04956

Synthesis of arylidene hydrazinylpyrido[2,3-d]pyrimidin-4-ones as potent anti-microbial agents

Reda M Abdelhameed a, Osama M Darwesh b, Mahmoud El-Shahat c,∗
PMCID: PMC7511821  PMID: 32995633

Abstract

Combination of arylidene hydrazinyl moiety with pyrido[2,3-d]pyrimidin-4-one skeleton in compounds 7‒26 results in the output of unprecedented anti-microbial agents. Arylidene hydrazinyl based on Pyrido[2,3-d]pyrimidin-4-one analoges 7‒26 prepared by the treatment of [2,3-d]pyrimidin-4-ones 6a,b with various aromatic aldehydes. The antimicrobial action for recently synthesized compounds was considered towards gram positive bacterial species (Staphylococcus aurous ATCC- 47077; Bacillus cereus ATCC-12228), gram negative bacterial species (Escherichia coli ATCC-25922; Salmonella typhi ATCC-15566) and Candida albicans ATCC-10231 as fungal strains. The antimicrobial action expanded by expanding the electron donating group in position 2 and 5 for Pyrido[2,3-d]pyrimidin-4-one core. Derivatives 13, 14, 15, 16 and 12; individually appeared hopeful anti-microbial action towards all strains utilized with inhibition zone higher than that of standard reference drug with lowest MIC.

Keywords: Organic chemistry; Pharmaceutical chemistry; Pyrido[2,3-d]pyrimidin-4-one; Hydrazinylpyrido[2,3-d]pyrimidin-4-one; Anti-bacterial activity; Anti-fungal activity

Organic chemistry, Pharmaceutical chemistry, Pyrido[2,3-d]pyrimidin-4-one; Hydrazinylpyrido[2,3-d]pyrimidin-4-one; Anti-bacterial activity; Anti-fungal activity.

1. Introduction

Chemistry for hydrazinyl derivatives are growing interest [1, 2, 3, 4]. Hydrazinyls are extensively considered as promising reactions intermediates which can be readily undergo various reactions through ring closure [5, 6]. Hydrazinyl moiety possessing heterocyclic compounds displaying a wide spectrum of pharmacological activities which depend on the nature of incorporated substituent. Hydrazinyl derivatives display biological interesting as antioxidant [4, 7], anti-inflammatory [8], anticonvulsant [9], analgesic [10], antimicrobial [11, 12], anticancer [13], antiprotozoal [14], antiparasitic [15, 16], cardioprotective [17, 18], antitubercular [19] and anti-HIV [20, 21]. Hydrazinyls derivative are used as common drug diseases for malaria [22], tuberculosis [23] and mental disorders.

Heterocyclic derivatives builded on Pyrido[2,3-d]pyrimidine backbone have received much attention of synthetic chemist in the last few decades and still study is going on [24, 25, 26]. Pyrido[2,3-d]pyrimidine cores have been cited as a great nucleus with enormous area for pharmaceutical action value such as antimicrobial [27, 28, 29], anti-inflammatory [30, 31], antitumor [32, 33, 34] and antiviral [35, 36]. Moreover, some of pyrido[2,3-d]pyrimidines displayed phosphodiesterase inhibitory [37].

One of the most public health problems is considered in the raise resistance of known antibiotic and it can interpret why remediation for bacterial and fungal infections is disappointing to save countless millions of human lives [38]. So, development of unprecedented drugs with antibacterial and antifungal potency is a significant solution to beat drug-resistance troubles [39]. Researchers and scientists around the world work hard to develop new materials or derivatives for facing the pathogenic microbe's distribution in the environment [40, 41, 42]. These materials may be produced by microbial [43]or plant sources [44]as well as chemical synthesis [45, 46, 47].

According to which we mentioned high; we attempts to combine hydrazine function group and pyrido[2,3-d]pyrimidin-4(3H)-one together with a view to obtain more potent antimicrobial analogues. As a part of our program aims to develop synthesis of new biologically active heterocycles [45, 48, 49, 50, 51], In this study we are targeted to synthesize certain new pyrido[2,3-d]pyrimidin-4(3H)-ones with thiophene nucleus at position 7 and various substituted at position 5 bearing arylidene hydrazinyl groups and study their antimicrobial influence.

2. Results and discussion

2.1. Chemistry

Pyrido[2,3-d]pyrimidines analogue preparation starting from α,β-unsaturated ketones is proceeded through an electrophilic attack on the position 5 of the pyrimidine derivatives like 2,4-diamino-pyrimidin-6(1H)-one, 6-amino-1-ethyl-1H-pyrimidine-2,4-dione, 6-aminouracil, 6-amino-2-thiouracil and 6-amino-1,3-dimethyluracil [52, 53]. 3-(Aryl)-1-(thiophen-2-yl)prop-2-en-1-ones 3a, b heated with 6-amino-2,3-dihydro-2-thioxopyrimidin-4(1H)-one (4) in presence of dimethylformamide as a solvent to give 5-(aryl)-2,3-dihydro-7-(thiophen-2-yl)-2-thioxopyrido[2,3-d]pyrimidin-4(1H)-ones (5a,b), respectively (Figure 1). The constitute of thioxopyrido[2,3-d]pyrimidin-4(1H)-ones 5 were supported by the principle of its spectroscopic data; IR and 1H-NMR spectra elucidated characteristic peaks attributed to the pyrimidine NH groups and absences of amino group significant peak. Additionally, NMR spectra display signals for both of Pyridine proton and carbons; respectively (c.f. experimental, Figure 1).

Figure 1.

Figure 1

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Pathway for preparation of 2-hydrazinylpyrido[2,3-d]pyrimidin-4(3H)-ones 6a,b.

2-Thioxopyridopyrimidines 5 undergoes reacting with hydrazine hydrate afforded the corresponding 2-hydrazinylpyrido[2,3-d]pyrimidin-4(3H)-ones 6 in excellent yield (Scheme 1). The IR spectrum of latter derivatives presented new peaks corresponding to hydrazinyl group with absence for one peak belong to pyrimidine ring NH peaks. 1H NMR data of compound 6a,b exhibited new two exchangeable signals refer to hydrazinyl protons with disappearance for one NH pyrimidine ring signal. Furthermore; 13C NMR data of compound 6a,b showed absence for characteristic significant signal for C=S group(c.f. experimental, Figure 1).

2-Hydrazinylpyrido[2,3-d]pyrimidin-4(3H)-ones 6 is regards as vital starting material for preparation of many diverse pyrido[2,3-d]pyrimidines derivative. The existence of a hydrazinyl group in compounds 6 has been assured by condensation with diverse aromatic aldehydes. So, A series of 2-(2-arylidenehydrazinyl)-5-(aryl)-7-(thiophen-2-yl)pyrido[2,3-d]pyrimidin-4(3H)-ones 7-26 derivatives prepared by reaction of compounds 6 with various aromatic aldehydes in presence of a acid produced the corresponding arylidene hydrazinyl derivatives 7-26 with high yield values. The structure of arylidene hydrazinyl derivatives 7-26 were elucidated via all possible spectral analysis. Both of IR and 1H-NMR spectra of compounds 7-26 presented the absence of the amino group signals for 2-hydrazinylpyrido[2,3-d]pyrimidin-4(3H)-ones analoges besides existence of signals assigned to the benzylic proton and new arylidene SP2 protons in 1H-NMR spectra. 13C-NMR spectra for 7-26 derivatives showed signals characteristic for new arylidene SP2 carbon atoms (c.f. experimental, Figure 2; Figures S1–S40).

Figure 2.

Figure 2

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Synthetic pathways for preparation of arylidene hydrazinyl pyrido [2,3-d] pyrimidin-5-ones 7-26.

2.2. Anti-microbial activity

One of the main health attention globally is the microbial infections that occurred by multi-drug resistant microbial species. In particular, these infections are occured via the gram positive bacteria, Staphylococcus aureus and species of the genus Enterococcus in hospitals and communities. The semi-synthetic streptogramins, quinupristin/dalfopristin and daptomycin as some of most applicable antibacterial agents are disadvantageous with serious side effects. These side effects have challenged to the researchers to improve a new modern potent biocidal agents [54].

In this study, series of certain pyrido[2,3-d]pyrimidin-4-one 5a,b; 6a,b and hydrazinyl pyrido[2,3-d]pyrimidin-4-ones 7-26 analogue have been prepared to evaluate their biocidal activities against gram positive bacteria Staphylococcus aurous ATCC- 47077 (St.), Bacillus cereus ATCC-12228 (B.C.), and gram negative bacteria species Escherichia coli ATCC-25922 (E.C.), Salmonella typhi ATCC-15566 (Salm.), in addition to fungi strain namly; Candida albicans ATCC-10231 (C. Alb.). Antimicrobial activity results were illustrated in Table 1 and Figure 3. Antibacterial activity varied between all tested compounds.

Table 1.

Anti-microbial activity of new synthesized Pyrido[2,3-d]pyrimidin-4-one 5a,b; 6a,b and hydrazinyl Pyrido[2,3-d]pyrimidin-4-one 7-26 derivatives (mm)∗.

Compounds No. Gram-positive bacteria
 Gram-negative bacteria
 Fungi
St. B.C. E.C. Salm. C. Alb.
5a 12 11 12 13 12
6a 17 16 13 17 18
6b - - 12 13 16
9 21 19 23 21 28
12 26 20 25 27 31
13 35 33 40 35 35
14 35 32 35 30 35
15 28 23 27 30 38
16 25 25 30 21 25
23 21 19 23 26 30
24 18 19 22 25 28
25 18 20 19 20 21
26 18 17 20 20 28
Ampicillin 25 20 16 19 9
Vancomycin 14 15 15 17 15
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∗

The diameter of the well (6 mm) is included.

Figure 3.

Figure 3

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Anti-microbial activity of new synthesized Pyrido[2,3-d]pyrimidin-4-one 5a,b; 6a,b and hydrazinyl pyrido[2,3-d]pyrimidin-4-one 7-26 derivatives.

The anti-bacterial and antifungal efficiency of the recently prepared pyrido[2,3-d]pyrimidin-4-ones and hydrazinylpyrido[2,3-d]pyrimidin-4-ones are illustrated in Table (1). From the data, it is observed that, compound 6a which contain 4-methoxyphenyl group in position 5 revealed a higher antimicrobial activity than compound 6b which contain 4- fluorophenyl group in position 5; compound 5a show higher activity than 6b due to the existence of electron donating group in position 5 and less activity than 6a which contain the same electron donating group in the same position due to presence of hydrazinyl group in position 2 in compound 6a rather than 5a; the compounds 5a; 6a,b display moderate antibacterial and antifungal activity when compared to reference drugs. The hydrazinyl pyrido[2,3-d]pyrimidin-4-one analogues 12-16 revealed excellent antimicrobial activity action against all strain used when compared with common reference drugs within higher inhibition zones. All of hydrazinylpyrido[2,3-d]pyrimidin-4-one analogues 12-16 contain the same electron donating group in position 5 (R = OCH3) also contain various electron donating group at phenyl ring in position 2; Compound 12 has no substitution on phenyl ring (X = H); Compound 13 has substitution in para position of phenyl ring (X = 4-OCH3); Compound 14 has substitution in meta position of phenyl ring (X = 3-OCH3); Compound 15 has substitution in para position of phenyl ring (X = 4-CH3); Compound 16 has substitution at the meta position of phenyl ring (X = 3-CH3). Presence of substituent in phenyl ring in position 2 for hydrazinylpyrido[2,3-d]pyrimidin-4-one analogues play vital role to increase both of anti bacterial and antifungal action; The activities against all the used microbial strains increased by increasing electron donating in position 2 so compound activity order is 13 > 14> 15 > 16 >12 > reference common drugs.

The hydrazinylpyrido[2,3-d]pyrimidin-4-one analogues 9 and 23-26 revealed good antimicrobial activity action against all strain used when compared with common reference drugs with inhibition zone equal or slightly less than reference drugs. hydrazinylpyrido[2,3-d]pyrimidin-4-one derivative 9 has electron donating group in position 5 (R = OCH3) and electron withdrawing substitution in para position of phenyl ring (X = 4-F); while hydrazinyl pyrido[2,3-d]pyrimidin-4-one derivatives 23-26 have electron withdrawing group in position 5 (R = F) with various electron donating group in phenyl ring at position 2; Compound 23 has substitution in para position of phenyl ring (X = 4-OCH3); Compound 24 has substitution in meta position of phenyl ring (X = 3-OCH3); Compound 25 has substitution in para position of phenyl ring (X = 4-CH3); Compound 26 has substitution in meta position of phenyl ring (X = 3-CH3). Existence of electron donating group in position 5 raise the anti microbial action so the activity of compound 9 > 23-26; while the activity of compounds 23-26 which contain electron withdrawing group improved by increased electron donating substitute in position 7 i.e activity order 23 > 24> 25 > 66 >12.

Minimal Inhibitory Concentration (MIC) of the examined novel Pyrido[2,3-d]pyrimidin-4-one and hydrazinylpyrido[2,3-d]pyrimidin-4-one nucleus was studied after utilizing various concentrations of each new derivatives and studied about the least concentration gave inhibition of the studied pathogenic microbes growth. The results of this part are illustrated in Table 2. These results indicated that some derivatives need concentrations over 200 ppm to effect on microbial growth. Furthermore, derivatives 13; 14 and 15 required 8–10 ppm of concentration to kill the studied microbes. For all indications, the MIC of the studied novel derivatives is depended on both of the class of derivative and the type of microbes. The MIC diversified among all new synthetic derivatives and between pathogenic microbes into the same derivative.

Table 2.

Minimum Inhibition Concentration (ppm) of new synthesized Pyrido[2,3-d]pyrimidin-4-one 5a,b; 6a,b and hydrazinyl Pyrido[2,3-d]pyrimidin-4-one 7-26 derivatives.

Compounds No. Gram-positive bacteria
 Gram-negative bacteria
 Fungi
St. B.C. E.C. Salm. C. Alb.
5a 100 100 100 100 100
6a 66.7 66.7 100 66.7 40
6b 200 200 100 100 66.7
9 25 25 18.2 25 16.7
12 16.7 25 16.7 16.7 13.3
13 10 13.3 8 10 10
14 10 13.3 10 13.3 10
15 16.7 20 16.7 13.3 8
16 20 20 13.3 25 20
23 25 40 16.7 20 13.3
24 40 40 20 20 16.7
25 40 25 25 25 25
26 40 66.7 25 25 16.7
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2.3. Stracture activity relationship (SAR)

The synthesis of novel high bioactive pyridopyrimidine heterocyclic derivatives is important because pyrimidine derivatives are commercially important and they have unique application in medicinal drugs. However, it is still very challenging to develop suitable pyridopyrimidine derivatives capable of producing high biological activity. This work gives some details about how we can improve the antibacterial and antifungal activity for pyrido[2,3-d]pyrimidin-4-one. We introduce arylidene hydrazinyl moiety to pyrido[2,3-d]pyrimidin-4-one framework and study the effect of substituted electron donating/withdrawing group in positions number 2 and 5. compound 6a which contain (4-OMe.C6H4) group in position 5 (R = OMe) revealed a higher antimicrobial activity than compound 6b which contain (4-F.C6H4) group in position 5 (R = F); compound 5a (R = OMe; R' = SH) show higher activity than 6b (R = F; R' = NHNH2) due to existence of electron donating group in position 5 and less activity than 6a (R = OMe; R' = NHNH2) which contain the same electron donating group in the same position due to presence of (-NHNH2) group in position 2 in compound 6a rather than 5a; the compounds 5a; 6a,b show moderate both of antibacterial and antifungal action when compared to reference drugs (Ampicillin and Vancomycin) Figure (4a). The hydrazinylpyrido[2,3-d]pyrimidin-4-one analogues 12-16 revealed excellent antimicrobial activity action against all strain when compared with common reference drugs within higher inhibition zones. All of hydrazinylpyrido[2,3-d]pyrimidin-4-one analogues 12-16 contain the same electron donating group in position 5 (R = OMe) but contain electron donating substituted group at phenyl ring in position 2; Compound 12 has no substitution on phenyl ring (X = H); Compound 13 has substitution at para position of phenyl ring (X = 4-OMe); Compound 14 has substitution at meta position of phenyl ring (X = 3-OMe); Compound 15 has substitution at para position of phenyl ring (X = 4-Me); Compound 16 has substitution at meta position of phenyl ring (X = 3-Me). The substituent at phenyl ring in position 2 for hydrazinyl Pyrido[2,3-d]pyrimidin-4-one analogues play vital role to enhancement both of anti bacterial and antifungal action; The activities against all the used microbial strains increased by increasing electron donating in position 2; so compound activity order is 13 > 14> 15 > 16 >12 > reference common drugs Figure (4b).

Figure 4.

Figure 4

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Summary of SAR of (a) pyrido[2,3-d]pyrimidin-4(3H)-ones derivatives; (b) arylidene hydrazinyl pyrido [2,3-d] pyrimidin-5-ones derivatives.

3. Conclusion

This study represents a simple and proper route for the preparation of certain Pyrido[2,3-d]pyrimidin-4-one 5a,b; 6a,b and hydrazinylpyrido[2,3-d]pyrimidin-4-one 7-26 derivatives. The antimicrobial potency was estimated against four bacterial species and one fungal strain. Compounds 13, 14, 15, 16 and 12; respectively showed promising anti-microbial potency which showed the highest activities when compared with the standard reference drug. Incorporation of electron donating group into pyrido[2,3-d]pyrimidin-4-one in both of position 5 and phenyl ring in position 2 resulted in the production of superior anti-candidal laborers.

4. Experimental

4.1. Chemistry

4.1.1. General

Melting points are determined by utilizing digital melting point apparatus (Electro-Thermal IA 9100; Büchi, Flawil, Switzerland). Infrared spectra are listed on a Perkin-Elmer 1600 FTIR (Perkin-Elmer, Waltham, MA, USA) discs. NMR spectra are resolved on a Jeol-Ex-300 NMR spectrometer (JEOL, Tokyo, Japan) and chemical shift was represented as part per million; (δ values, ppm) against TMS as internal reference. Chemical shifts for both of 1H and 13C are pointed to the solvent signal (DMSO) at 2.50 and 39.52 ppm, respectively. Data are presented as follows: chemical shift, integration, multiplicity (br = broad, s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet) and coupling constant in Hertz (Hz). Mass spectra are hold at 70 eV with a Finnigan SSQ 7000 spectrometer (Thermo Electron Corporation, Madison, WI, USA) utilizing EI and the values of m/z are expressed by Dalton. Elemental analyses are done on a Perkin-Elmer 2400 analyzer (Perkin-Elmer) with agreeable range (±0.30) of the calculated values. Monitoring of the reaction and purity of new compounds was achieved by using thin layer chromatography on silica gel pre-coated aluminum sheets (type 60 F254, Merck, Darmstadt, Germany). All chemical reagents and solvents were purchased from Aldrich (Munich, Germany). Compounds 2 [54]; 3a,b [55] and 5a,b [11] were synthesized according to their previous method.

4.1.2. General procedure for preparation of compounds 6a,b

Under solvent free reaction condition; heated an equal molar ratio of hydrazine hydrate (99%) and 2-thioxopyridopyrimidine 5a,b for 2h. Then stand the reaction at room temperature to cool. Collect the formed precipitate after cooling and re-crystallized from EtOH: DMF mixture (v: v; 2:1).

4.1.2.1. 2-hydrazinyl-5-(4-methoxyphenyl)-7-(thiophen-2-yl)pyrido[2,3-d]pyrimidin-4(3H)-one (6a)

Yield 92%, mp 266–268 °C; IR (KBr, υ, cm−1): 3344(NH2); 32895(NH); 3222(NH), 1677 (C=O). 1H NMR (DMSO-d6, 300 MHz): δ (ppm) 3.78 (s, 3H, OCH3), 5.40 (br s, 2H, NH2; D2O exchangeable), 6.14 (br s, 1H, NH; D2O exchangeable), 6.95–6.99 (m, 2H, thiophene-H+ pyrimidine NH; D2O exchangeable), 7.17–7.34 (m, 5H, 4 Ar-H + pyridine-H), 7.46 (d, J = 3.8 Hz, 1H, thiophene -H), 7.50 (d, J = 3.5 Hz, 1H, thiophene-H). 13C NMR (75 MHz, DMSO) δ = 160.15, 153.09, 150.00, 141.23, 132.11, 129.00, 128.66, 127.80, 127.80, 126.37, 124.55, 123.76, 115.31, 114.17, 101.14, 55.33. MS, m/z (%): 365 (M+, 88); Anal. Calcd. for C18H15N5O2S (365.09): C, 59.17; H, 4.15; N, 19.18; S, 8.77. Found: C, 58.90; H, 3.89; N, 18.94; S, 8.50.

4.1.2.2. 5-(4-fluorophenyl)-2-hydrazinyl-7-(thiophen-2-yl)pyrido[2,3-d]pyrimidin-4(3H)-one (6b)

Yield 87%, mp 277–279 °C; IR (KBr, υ, cm−1): 3340(NH2); 3295(NH); 3220(NH), 1689 (C=O). 1H NMR (DMSO-d6, 300 MHz): δ (ppm) 5.47 (br s, 2H, NH2; D2O exchangeable), 6.19 (br s, 1H, NH; D2O exchangeable), 6.97–7.00 (m, 2H, thiophene-H+ pyrimidine NH; D2O exchangeable), 7.19–7.37 (m, 5H, 4 Ar-H + pyridine-H), 7.49 (d, J = 3.8 Hz, 1H, thiophene -H), 7.52 (d, J = 3.5 Hz, 1H, thiophene-H). 13C NMR (75 MHz, DMSO) δ = 159.97, 153.49, 150.10, 141.90, 132.53, 129.08, 128.94, 127.88, 126.97, 124.75, 123.97, 115.67, 114.34, 101.27. MS, m/z (%): 353 (M+, 69); Anal. Calcd. for C17H12FN5OS (353.07): C, 57.78; H, 3.42; N, 19.83; S, 9.07. Found: C, 57.55; H, 3.16; N, 19.58; S, 8.80.

4.1.3. Common method for synthesis of compounds 7–26

In 15 ml glacial acetic acid; heated an equal molar ratio of compounds 6 and diversified aldehydes under reflux condition for 4h. Then stand the reaction at room temperature to cool. Collect the formed precipitate after cooling and re-crystallized from EtOH: DMF mixture (v: v; 1:2).

4.1.3.1. 5-(4-methoxyphenyl)-2-(2-(4-nitrobenzylidene)hydrazinyl)-7-(thiophen-2-yl)pyrido [2,3-d]pyrimidin-4(3H)-one(7)

Yield 65%; m.p. over 310 °C; IR (KBr, υ, cm−1): 3382 and 3371(2NH), 1685(C=O). 1H NMR (DMSO-d6, 300 MHz): δ (ppm) 3.82 (s, 3H, OCH3), 6.96 (d, J = 8.8 Hz, 2H, Ar-H), 7.21 (td, J = 5.3, 3.7 Hz, 1H, Thiophene-H), 7.37–7.45 (m, 3H, 2Ar-H + Pyridine-H), 7.53–7.61 (m, 2H, Ar-H), 7.78 (dd, J = 12.7, 4.5 Hz, 2H, Ar-H), 8.01 (d, J = 3.5 Hz, 1H, Thiophene-H), 8.06 (d, J = 3.8 Hz, 1H, Thiophene-H), 8.37 (s, 1H, N=CH), 11.51 (s, 1H, NH; D2O exchangeable), 11.91 (brs, 1H, NH; D2O exchangeable). 13C NMR (75 MHz, DMSO) δ = 160.55, 159.25, 153.10, 151.17, 148.46, 143.77, 143.08, 137.84, 136.97, 136.13, 132.09, 131.43, 130.59, 130.19, 129.16, 128.77, 127.17, 122.30, 116.02, 112.85, 108.02, 55.18. MS, m/z (%): 498 (M+, 47); Analysis calc. for C25H18N6O4S (498.52): C, 60.23; H, 3.64; N, 16.87; S, 6.43. Found: C, 59.96; H, 3.39; N, 16.64; S, 6.18.

4.1.3.2. 2-(2-(4-chlorobenzylidene)hydrazinyl)-5-(4-methoxyphenyl)-7-(thiophen-2-yl) pyrido[2,3-d]pyrimidin-4(3H)-one(8)

Yield 67%; m.p.over 310 °C; IR (KBr, υ, cm−1): 3379 and 3368(2NH), 1677(C=O).1H NMR (DMSO-d6, 300 MHz): δ (ppm) 3.79 (s, 3H, OCH3), 6.96 (d, J = 8.7 Hz, 2H,Ar-H), 7.18–7.27 (m, 3H, 2Ar-H + Thiophene-H), 7.45–7.50 (m, 3H, 2Ar-H + Pyridine-H), 7.76 (d, J = 4.9 Hz, 1H, Thiophene-H), 7.90 (d, J = 8.7 Hz, 2H, Ar-H), 8.01 (d, J = 3.2 Hz, 1H, Thiophene-H), 8.06 (s, 1H,N=CH), 11.27 (s, 1H,NH; D2O exchangeable), 11.70 (s, 1H,NH; D2O exchangeable). 13C NMR (75 MHz, DMSO) δ = 164.65, 160.87, 160.60, 160.17, 155.38, 152.15, 151.16, 143.92, 135.92, 130.74, 130.63, 129.21, 128.75, 127.92, 127.01, 115.61, 114.35, 114.01, 55.28. MS, m/z (%): 487 (M+, 35), 489(M++2, 11); Analysis calc. for C25H18ClN5O2S (487.96): C, 61.54; H, 3.72; Cl, 7.26; N, 14.35; S, 6.57. Found: C, 61.32; H, 3.47; Cl, 7.06; N, 14.11; S, 6.37.

4.1.3.3. 2-(2-(4-fluorobenzylidene)hydrazinyl)-5-(4-methoxyphenyl)-7-(thiophen-2-yl) pyrido[2,3-d]pyrimidin-4(3H)-one (9)

Yield 68%; m.p.over 310 °C; IR (KBr, υ, cm−1): 3380 and 3374(2NH), 1682(C=O). 1H NMR (DMSO-d6, 300 MHz): δ (ppm) 3.81 (s, 3H, OCH3), 6.96 (d, J = 8.2 Hz, 2H, Ar-H), 7.18–7.27 (m, 3H, 2Ar-H + Thiophene-H), 7.42–7.51 (m, 4H, Ar-H), 7.62 (s, 1H,Pyridine-H), 7.77 (d, J = 4.9 Hz, 1H, Thiophene-H), 8.03 (d, J = 3.8 Hz, 1H, Thiophene-H), 8.11 (s, 1H, N=CH), 11.39 (s, 1H, NH; D2O exchangeable), 11.83 (s, 1H, NH; D2O exchangeable). 13C NMR (75 MHz, DMSO) δ = 163.64, 160.93, 160.41, 159.54, 155.29, 152.18, 151.21, 143.83, 135.78, 130.78, 130.67, 129.58, 128.77, 128.05, 120.63, 115.73, 114.36, 114.08, 111.91, 107.99, 55.31. MS, m/z (%): 471 (M+, 56); Analysis calc. for C25H18FN5O2S (471.12): C, 63.68; H, 3.85; N, 14.85; S, 6.80. Found: C, 63.41; H, 3.55; N, 14.61; S, 6.55.

4.1.3.4. 2-(2-(4-bromobenzylidene)hydrazinyl)-5-(4-methoxyphenyl)-7-(thiophen-2-yl) pyrido[2,3-d]pyrimidin-4(3H)-one (10)

Yield 62%; m.p. over 310 °C; IR (KBr, υ, cm−1): 3379 and 3366(2NH), 1680(C=O). 1H NMR (DMSO-d6, 300 MHz): δ (ppm) 3.80 (s, 3H, OCH3), 6.97 (d, J = 8.7 Hz, 2H,Ar-H), 7.18–7.28 (m, 3H, 2Ar-H + Thiophene-H), 7.45–7.51 (m, 3H, 2Ar-H + Pyridine-H), 7.77 (d, J = 4.9 Hz, 1H, Thiophene-H), 7.90 (d, J = 8.7 Hz, 2H, Ar-H), 8.02 (d, J = 3.2 Hz, 1H, Thiophene-H), 8.06 (s, 1H, N=CH), 11.28 (s, 1H, NH; D2O exchangeable), 11.70 (brs, 1H, NH; D2O exchangeable). 13C NMR (75 MHz, DMSO) δ = 163.51, 160.73, 160.46, 155.25, 152.02, 151.03, 143.78, 135.78, 130.60, 130.60, 130.50, 129.07, 128.61, 127.78, 126.87, 115.47, 114.21, 114.21, 113.92, 113.87, 55.15. MS, m/z (%): 531 (M+, 39), 533 (M+ +2, 35); Analysis calc. for C25H18BrN5O2S (531.04): C, 56.40; H, 3.41; N, 13.15; S, 6.02. Found: C, 56.14; H, 3.15; N, 12.88; S, 5.77.

4.1.3.5. 2-(2-(3-bromobenzylidene)hydrazinyl)-5-(4-methoxyphenyl)-7-(thiophen-2-yl) pyrido[2,3-d]pyrimidin-4(3H)-one(11)

Yield 54%; m.p. over 310 °C; IR (KBr, υ, cm−1): 3387 and 3370(2NH), 1679(C=O). 1H NMR (DMSO-d6, 300 MHz): δ (ppm) 3.81 (s, 3H, OCH3), 6.93–6.98 (m, 2H, Ar-H), 7.20 (td, J = 5.3, 3.7 Hz, 1H, Thiophene-H), 7.32–7.41 (m, 3H,2 Ar-H + Pyridine-H), 7.54 (d, J = 6.0 Hz, 1H, Ar-H), 7.60 (s, 1H, Ar-H), 7.77 (dd, J = 12.7, 4.5 Hz, 2H, Ar-H), 8.00 (d, J = 3.5 Hz, 1H, Thiophene-H), 8.07 (d, J = 3.5 Hz, 1H, Thiophene-H), 8.37 (s, 1H, N=CH), 11.50 (s, 1H, NH; D2O exchangeable), 11.91 (brs, 1H, NH; D2O exchangeable). 13C NMR (75 MHz, DMSO) δ = 160.91, 159.25, 153.10, 151.17, 148.45, 148.42, 142.69, 132.09, 131.46, 131.42, 130.58, 130.19, 129.16, 128.76, 127.71, 127.17, 122.29, 116.02, 112.84, 108.02, 55.18. MS, m/z (%): 531 (M+, 22), 533(M+, 20); Analysis calc. for C25H18BrN5O2S (531.04): C, 56.40; H, 3.41; N, 13.15; S, 6.02. Found: C, 56.19; H, 3.14; N, 12.92; S, 5.77.

4.1.3.6. 2-(2-benzylidenehydrazinyl)-5-(4-methoxyphenyl)-7-(thiophen-2-yl)pyrido[2,3-d] pyrimidin-4(3H)-one (12)

Yield 41%; m.p. over 310 °C; IR (KBr, υ, cm−1): 3387 and 3369 (2NH), 1677(C=O). 1H NMR (DMSO-d6, 300 MHz): δ (ppm) 3.81 (s, 3H, OCH3), 6.95–6.98 (m, 2H, Ar-H), 7.18–7.20 (m, 3H, Ar-H), 7.29 (t, J = 7.6 Hz, 1H, Thiophene-H), 7.37–7.40 (m, 2H, Ar-H), 7.44 (s, 1H, Pyridine-H), 7.71 (d, J = 7.9 Hz, 1H, Thiophene-H), 7.76 (dd, J = 6.6, 1.5 Hz, 2H, Ar-H), 8.00 (dd, J = 3.8, 1.2 Hz, 1H, Thiophene-H), 8.11 (s, 1H,N=CH), 11.27 (brs, 2H, 2NH; D2O exchangeable). 13C NMR (75 MHz, DMSO) δ = 160.68, 159.24, 153.12, 150.68, 137.62, 134.38, 131.34, 130.70, 130.69, 130.43, 130.32, 130.18, 129.94, 128.76, 128.44, 128.43, 127.37, 125.02, 124.17, 115.74, 112.84, 55.19. MS, m/z (%): 453 (M+, 11); Analysis calc. for C25H19N5O2S (453.13): C, 66.21; H, 4.23; N, 15.43; S, 7.08. Found: C, 65.94; H, 3.98; N, 15.18; S, 6.84.

4.1.3.7. 2-(2-(4-methoxybenzylidene)hydrazinyl)-5-(4-methoxyphenyl)-7-(thiophen-2-yl) pyrido[2,3-d]pyrimidin-4(3H)-one (13)

Yield 55%; m.p.over 310 °C; IR (KBr, υ, cm−1): 3379 and 3368 (2NH), 1677(C=O). 1H NMR (DMSO-d6, 300 MHz): δ (ppm) 3.81 (s, 6H, 2OCH3), 6.96 (d, J = 8.8 Hz, 4H, Ar-H), 7.19–7.22 (m, 2H, Thiophene-H + Pyridine-H), 7.38 (d, J = 8.7 Hz, 4H, Ar-H), 7.57 (s, 1H, N=CH), 7.81 (dd, J = 5.0, 1.2 Hz, 1H, Thiophene-H), 8.06 (dd, J = 3.8, 1.2 Hz, 1H, Thiophene-H), 12.31 (s, 1H, NH; D2O exchangeable), 12.99 (s, 1H, NH; D2O exchangeable). 13C NMR (75 MHz, DMSO) δ = 175.31, 159.56, 158.64, 154.80, 153.34, 152.79, 142.44, 131.70, 130.87, 130.37, 130.26, 129.03, 128.92, 117.69, 112.91, 107.43, 55.24. MS, m/z (%): 483 (M+, 33); Analysis calc. for C26H21N5O3S (483.14): C, 64.59; H, 4.37; N, 14.47; S, 6.62. Found: C, 64.34; H, 4.11; N, 14.25; S, 6.37.

4.1.3.8. (2-(2-(3-methoxybenzylidene)hydrazinyl)-5-(4-methoxyphenyl)-7-(thiophen-2-yl) pyrido[2,3-d]pyrimidin-4(3H)-one (14)

Yield 47%; m.p.over 310 °C; IR (KBr, υ, cm−1): 3384 and 3370(2NH), 1681(C=O). 1H NMR (DMSO-d6, 300 MHz): δ (ppm) 3.82 (s, 6H, 2OCH3), 6.94–6.99 (m, 3H, Ar-H), 7.19 (dd, J = 5.2, 3.7 Hz, 1H, Thiophene-H), 7.36–7.44 (m, 5H, Ar-H), 7.61 (s, 1H, Pyridine-H), 7.76 (d, J = 4.9 Hz, 1H, Thiophene-H), 8.01 (d, J = 2.8 Hz, 1H, Thiophene-H), 8.10 (s, 1H, N=CH), 11.31 (brs, 1H, NH; D2O exchangeable), 11.79 (brs, 1H, NH; D2O exchangeable). 13C NMR (75 MHz, DMSO) δ = 161.35, 159.56, 159.24, 153.11, 130.68, 130.18, 129.60, 128.76, 120.63, 116.03, 115.43, 115.14, 112.84, 55.33, 55.19. MS, m/z (%): 483 (M+, 15); Analysis calc. for C26H21N5O3S (483.14): C, 64.59; H, 4.39; N, 14.47; S, 6.62. Found: C, 64.35; H, 4.10; N, 14.22; S, 6.38.

4.1.3.9. 5-(4-methoxyphenyl)-2-(2-(4-methylbenzylidene)hydrazinyl)-7-(thiophen-2-yl) pyrido[2,3-d]pyrimidin-4(3H)-one (15)

Yield 54%; m.p.over 310 °C; IR (KBr, υ, cm−1): 3378 and 3365(2NH), 1679(C=O). 1H NMR (DMSO-d6, 300 MHz): δ (ppm) 2.34 (s, 3H, CH3), 3.82 (s, 3H, OCH3), 6.97 (d, J = 7.7 Hz, 2H, Ar-H), 7.22–7.31 (m, 3H, 2Ar-H + Thiophene-H), 7.38–7.44 (m, 3H, 2Ar-H + Pyridine-H), 7.75–7.87 (m, 3H, 2Ar-H + Thiophene-H), 8.01–8.10 (m, 2H, Thiophene-H + N=CH), 11.27 (brs, 2H, 2NH; D2O exchangeable). 13C NMR (75 MHz, DMSO) δ = 159.24, 154.23, 138.31, 134.96, 134.19, 131.37, 131.34, 130.70, 130.43, 130.18, 128.76, 128.44, 127.89, 127.37, 124.17, 112.37, 55.19, 20.98. MS, m/z (%): 467 (M+, 22); Analysis calc. for C26H21N5O2S (467.14): C, 66.79; H, 4.53; N, 14.98; S, 6.86. Found: C, 66.54; H, 4.28; N, 14.70; S, 6.59.

4.1.3.10. 5-(4-methoxyphenyl)-2-(2-(3-methylbenzylidene)hydrazinyl)-7-(thiophen-2-yl) pyrido[2,3-d]pyrimidin-4(3H)-one (16)

Yield 50%; m.p. over 310 °C; IR (KBr, υ, cm−1): 3376 and 3362(2NH), 1674(C=O). 1H NMR (DMSO-d6, 300 MHz): δ (ppm) 2.34 (s, 3H,CH3), 3.82 (s, 3H,O CH3), 6.95–6.98 (m, 2H,Ar-H), 7.20 (dd, J = 5.0, 3.7 Hz, 2H, Ar-H), 7.30 (t, J = 7.6 Hz, 1H, Thiophene-H), 7.38–7.41 (m, 2H, Ar-H), 7.44 (s, 1H, Pyridine-H), 7.71 (d, J = 7.9 Hz, 1H, Thiophene-H), 7.76 (dd, J = 6.6, 1.5 Hz, 2H, Ar-H), 8.01 (dd, J = 3.8, 1.2 Hz, 1H, Thiophene-H), 8.11 (s, 1H, N=CH), 11.28 (brs, 2H, 2NH; D2O exchangeable). 13C NMR (75 MHz, DMSO) δ = 159.24, 154.23, 138.30, 134.95, 134.36, 131.34, 130.69, 130.42, 130.18, 128.76, 128.44, 127.37, 124.16, 112.36, 55.19, 20.98. MS, m/z (%): 467(M+, 13); Analysis calc. for C26H21N5O2S (467.14): C, 66.79; H, 4.53; N, 14.98; S, 6.86. Found: C, 66.55; H, 4.28; N, 14.74; S, 6.60.

4.1.3.11. 5-(4-fluorophenyl)-2-(2-(4-nitrobenzylidene)hydrazinyl)-7-(thiophen-2-yl) pyrido [2,3-d]pyrimidin-4(3H)-one (17)

Yield 72%; m.p.over 310 °C; IR (KBr, υ, cm−1): 3384 and 3368(2NH), 1675(C=O). 1H NMR (DMSO-d6, 300 MHz): δ (ppm) 6.97 (d, J = 8.8 Hz, 2H, Ar-H), 7.20 (dd, J = 5.2, 3.6 Hz, 1H, Thiophene-H), 7.37–7.45 (m, 3H, 2Ar-H + Pyridine-H), 7.56 (d, J = 8.8 Hz, 2H, Ar-H), 7.78 (d, J = 8.6 Hz, 2H, Ar-H), 8.01 (d, J = 3.5 Hz, 1H, Thiophene-H), 8.06 (d, J = 3.8 Hz, 1H, Thiophene-H), 8.37 (s, 1H, N=CH), 11.51 (s, 1H, NH; D2O exchangeable), 11.91 (s, 1H, NH; D2O exchangeable). 13C NMR (75 MHz, DMSO) δ = 164.82, 163.71, 161.51, 153.46, 145.90, 137.78, 130.65, 128.78, 128.63, 128.43, 128.05, 126.37, 125.02, 114.35, 114.07. MS, m/z (%): 486 (M+, 63); Analysis calc. for C24H15FN6O3S (486.09): C, 59.26; H, 3.12; N, 17.27; S, 6.58. Found: C, 58.99; H, 2.88; N, 17.01; S, 6.33.

4.1.3.12. 2-(2-(4-chlorobenzylidene)hydrazinyl)-5-(4-fluorophenyl)-7-(thiophen-2-yl)pyrido [2,3-d]pyrimidin-4(3H)-one (18)

Yield 74%; m.p.over 310300 °C; IR (KBr, υ, cm−1): 3386 and 3370(2NH), 1680(C=O). 1H NMR (DMSO-d6, 300 MHz): δ (ppm) 7.19–7.33 (m, 4H, Ar-H), 7.47–7.51 (m, 4H, 2Ar-H + Pyridine-H + Thiophene-H), 7.72 (d, J = 3.8 Hz, 1H, Thiophene-H), 7.78 (d, J = 6.0 Hz, 2H, Ar-H), 8.03 (d, J = 3.8 Hz, 1H, Thiophene-H), 8.09 (s, 1H, N=CH), 11.31 (brs, 1H, NH; D2O exchangeable), 11.77 (brs, 1H, NH; D2O exchangeable). 13C NMR (75 MHz, DMSO) δ = 165.10, 163.99, 161.79, 152.29, 146.18, 138.06, 131.05, 130.94, 130.52, 129.06, 128.71, 128.33, 126.65, 125.29, 114.63, 114.35. MS, m/z (%): 475 (M+, 40), 477(M++2, 13); Analysis calc. for C24H15ClN5OS (475.07): C, 60.57; H, 3.18; Cl, 7.45; N, 14.72; S, 6.74. Found: C, 60.33; H, 2.93; Cl, 7.19; N, 14.44; S, 6.45.

4.1.3.13. 2-(2-(4-fluorobenzylidene)hydrazinyl)-5-(4-fluorophenyl)-7-(thiophen-2-yl) pyrido[2,3-d]pyrimidin-4(3H)-one (19)

Yield 70%; m.p.over 310 °C; IR (KBr, υ, cm−1): 3380 and 3362(2NH), 1678(C=O). 1H NMR (DMSO-d6, 300 MHz): δ (ppm) 6.96 (d, J = 8.8 Hz, 4H, Ar-H), 7.20 (dd, J = 5.0, 3.8 Hz, 2H, Ar-H), 7.37–7.40 (m, 4H, 2Ar-H + Thiophene-H + Pyridine-H), 7.57 (s, 1H, N=CH), 7.81 (dd, J = 5.0, 1.2 Hz, 1H, Thiophene-H), 8.06 (dd, J = 3.8, 1.2 Hz, 1H, Thiophene-H), 12.31 (s, 1H, NH; D2O exchangeable), 12.99 (s, 1H, NH; D2O exchangeable). 13C NMR (75 MHz, DMSO) δ = 163.92, 161.18, 160.69, 155.67, 152.46, 151.54, 144.05, 137.16, 136.05, 132.39, 131.06, 130.95, 130.86, 129.43, 129.06, 128.33, 127.47, 122.56, 116.21, 114.65, 114.36, 108.23. MS, m/z (%): 459 (M+, 37); Analysis calc. for C24H15F2N5OS (459.10): C, 62.74; H, 3.29; N, 15.24; S, 6.98. Found: C, 62.49; H, 3.02; N, 14.97; S, 6.72.

4.1.3.14. 2-(2-(4-bromobenzylidene)hydrazinyl)-5-(4-fluorophenyl)-7-(thiophen-2-yl) pyrido[2,3-d]pyrimidin-4(3H)-one (20)

Yield 73%; m.p.over 310 °C; IR (KBr, υ, cm−1): 3379 and 3363(2NH), 1675(C=O). 1H NMR (DMSO-d6, 300 MHz): δ (ppm) 7.24 (d, J = 8.0 Hz, 2H, Ar-H), 7.48 (d, J = 7.8 Hz, 2H, Ar-H), 7.58–7.66 (m, 3H, 2 Ar-H + Thiophene-H), 7.84 (d, J = 7.8 Hz, 2H, Ar-H), 7.98 (s, 1H, Pyridine-H), 8.03–8.16 (m, 2H, Thiophene-H), 10.00 (s, 1H, N=CH), 10.67 (s, 1H, NH; D2O exchangeable), 11.50 (s, 1H, NH; D2O exchangeable). 13C NMR (75 MHz, DMSO) δ = 161.56, 160.58, 155.31, 152.17, 143.99, 139.46, 137.34, 133.86, 132.33, 130.75, 130.63, 129.74, 129.60, 129.30, 129.14, 128.75, 128.02, 127.60, 114.34, 114.06. MS, m/z (%): 519 (M+, 11) 521(M++2, 10); Analysis calc. for C24H15BrFN5OS (519.02): C, 55.39; H, 2.91; N, 13.46; S, 6.16. Found: C, 55.14; H, 2.66; N, 13.20; S, 5.90.

4.1.3.15. 2-(2-(3-bromobenzylidene)hydrazinyl)-5-(4-fluorophenyl)-7-(thiophen-2-yl) pyrido[2,3-d]pyrimidin-4(3H)-one (21)

Yield 61%; m.p. over 310 °C; IR (KBr, υ, cm−1): 3377 and 3362(2NH), 1675(C=O). 1H NMR (DMSO-d6, 300 MHz): δ (ppm) 7.18–7.27 (m, 3H, Ar-H), 7.35 (t, J = 7.8 Hz, 1H, Thiophene-H), 7.46–7.56 (m, 4H, 3Ar-H + Pyridine-H), 7.76 (d, J = 5.2 Hz, 1H, Thiophene-H), 7.81 (d, J = 8.0 Hz, 1H, Ar-H), 8.02 (d, J = 3.9 Hz, 1H, Thiophene-H), 8.08 (s, 1H, Ar-H), 8.37 (s, 1H, N=CH), 11.56 (brs, 1H, NH; D2O exchangeable), 11.84 (brs, 1H, NH; D2O exchangeable). 13C NMR (75 MHz, DMSO) δ = 163.92, 161.18, 160.69, 155.67, 152.46, 151.53, 144.05, 137.16, 136.05, 132.39, 131.06, 130.95, 130.86, 129.43, 129.06, 128.33, 127.47, 122.56, 116.21, 114.65, 114.36. MS, m/z (%): 519 (M+, 19), 521(M++2, 17); Analysis calc. for C24H15BrFN5OS (519.02): C, 55.40; H, 2.90; N, 13.46; S, 6.16. Found: C, 55.11; H, 2.66; N, 3.19; S, 5.91.

4.1.3.16. 2-(2-benzylidenehydrazinyl)-5-(4-fluorophenyl)-7-(thiophen-2-yl)pyrido[2,3-d] pyrimidin-4(3H)-one (22)

Yield 72%; m.p. over 310300 °C; IR (KBr, υ, cm−1): 3386 and 3370(2NH), 1680(C=O). 1H NMR (DMSO-d6, 300 MHz): δ (ppm) 7.17–7.31 (m, 6H,5Ar-H + Thiophene-H), 7.45–7.50 (m, 3H, 2Ar-H + Pyridine-H), 7.71 (d, J = 8.2 Hz, 1H, Thiophene-H), 7.76 (d, J = 6.0 Hz, 2H, Ar-H), 8.02 (d, J = 3.8 Hz, 1H, Thiophene-H), 8.08 (s, 1H, N=CH), 11.30 (brs, 1H, NH; D2O exchangeable), 11.76 (brs, 1H, NH; D2O exchangeable). 13C NMR (75 MHz, DMSO) δ = 164.82, 163.71, 161.51, 153.46, 145.90, 137.78, 130.77, 130.66, 130.43, 128.78, 128.43, 128.05, 128.04, 126.37, 125.02, 114.93, 113.61. MS, m/z (%): 441(M+, 30); Analysis calc. for C24H16FN5OS (441.11): C, 65.30; H, 3.65; N, 15.85; S, 7.26. Found: C, 65.05; H, 3.39; N, 15.66; S, 7.00.

4.1.3.17. 5-(4-fluorophenyl)-2-(2-(4-methoxybenzylidene)hydrazinyl)-7-(thiophen-2-yl) pyrido[2,3-d]pyrimidin-4(3H)-one (23)

Yield 65%; m.p.over 310 °C; IR (KBr, υ, cm−1): 3380 and 3370(2NH), 1675(C=O). 1H NMR (DMSO-d6, 300 MHz): δ (ppm) 3.79 (s, 3H, OCH3), 6.96 (d, J = 8.7 Hz, 2H, Ar-H), 7.17–7.26 (m, 3H, 2 Ar-H + Thiophene-H), 7.44–7.50 (m, 3H, 2 Ar-H + Pyridine-H), 7.75 (d, J = 4.9 Hz, 1H, Thiophene-H), 7.89 (d, J = 8.7 Hz, 2H, Ar-H), 8.01 (d, J = 3.2 Hz, 1H, Thiophene-H), 8.05 (s, 1H, N=CH), 11.27 (s, 1H, NH; D2O exchangeable), 11.69 (brs, 1H, NH; D2O exchangeable). 13C NMR (75 MHz, DMSO) δ = 163.49, 160.87, 160.60, 155.38, 152.15, 151.16, 143.92, 135.92, 130.74, 130.63, 129.21, 128.75, 127.92, 127.01, 115.61, 114.35, 114.01, 55.28. MS, m/z (%): 471 (M+, 21); Analysis calc. for C25H18FN5O2S (471.12): C, 63.68; H, 3.85; N, 14.85; S, 6.80. Found: C, 63.40; H, 3.57; N, 14.65; S, 6.53.

4.1.3.18. 5-(4-fluorophenyl)-2-(2-(3-methoxybenzylidene)hydrazinyl)-7-(thiophen-2-yl) pyrido[2,3-d]pyrimidin-4(3H)-one (24)

Yield 52%; m.p.over 310 °C; IR (KBr, υ, cm−1): 3386 and 3370(2NH), 1678(C=O). 1H NMR (DMSO-d6, 300 MHz): δ (ppm) 3.81 (s, 3H, OCH3), 6.96 (d, J = 8.2 Hz, 1H, Ar-H), 7.18–7.27 (m, 3H, 2 Ar-H + Thiophene-H), 7.33 (d, J = 7.9 Hz, 1H, Ar-H), 7.42–7.51 (m, 4H, Ar-H), 7.61 (s, 1H, Pyridine-H), 7.77 (d, J = 4.9 Hz, 1H, Thiophene-H), 8.02 (d, J = 3.8 Hz, 1H, Thiophene-H), 8.10 (s, 1H, N=CH), 11.39 (brs, 1H, NH; D2O exchangeable), 11.82 (brs, 1H, NH; D2O exchangeable). 13C NMR (75 MHz, DMSO) δ = 163.64, 160.92, 160.40, 159.54, 152.18, 151.21, 135.78, 130.78, 130.66, 129.58, 128.77, 128.05, 120.63, 115.73, 114.36, 114.07, 111.91, 55.31. MS, m/z (%): 471 (M+, 13); Analysis calc. for C25H18FN5O2S (471.12): C, 63.68; H, 3.85; N, 14.85; S, 6.80. Found: C, 63.42; H, 3.55; N, 14.62; S, 6.55.

4.1.3.19. 5-(4-fluorophenyl)-2-(2-(4-methylbenzylidene)hydrazinyl)-7-(thiophen-2-yl)pyrido [2,3-d]pyrimidin-4(3H)-one (25)

Yield 49%; m.p.over 310 °C; IR (KBr, υ, cm−1): 3378 and 3365(2NH), 1674(C=O). 1H NMR (DMSO-d6, 300 MHz): δ (ppm) 2.33 (s, 3H, CH3), 7.20–7.31 (m, 4H, Ar-H), 7.39–7.58 (m, 3H, 2Ar-H + Thiophene-H), 7.78–7.86 (m, 4H, 2Ar-H + Pyridine-H + Thiophene-H), 8.02–8.10 (m, 2H, Thiophene-H + N=CH), 11.36 (brs, 2H, 2NH; D2O exchangeable). 13C NMR (75 MHz, DMSO) δ = 161.56, 160.58, 155.31, 152.17, 143.98, 139.46, 137.34, 133.86, 132.33, 130.75, 130.63, 129.74, 129.60, 129.14, 128.75, 128.02, 127.60, 114.34, 114.06, 21.08. MS, m/z (%): 455 (M+, 28); Analysis calc. for C25H18FN5OS (455.12): C, 65.92; H, 3.98; N, 15.38; S, 7.04. Found: C, 65.68; H, 3.72; N, 15.11; S, 6.80.

4.1.3.20. 5-(4-fluorophenyl)-2-(2-(3-methylbenzylidene)hydrazinyl)-7-(thiophen-2-yl)pyrido [2,3-d]pyrimidin-4(3H)-one (26)

Yield 41%; m.p.over 310 °C; IR (KBr, υ, cm−1): 3374 and 3362(2NH), 1671(C=O). 1H NMR (DMSO-d6, 300 MHz): δ (ppm) 2.35 (s, 3H,CH3), 7.19–7.32 (m, 5H,4Ar-H+ pyridine-H), 7.46–7.51 (m, 3H, 2Ar-H+ thiophene-H), 7.72 (d, J = 8.2 Hz, 1H, thiophene-H), 7.77 (d, J = 6.0 Hz, 2H, Ar-H), 8.03 (d, J = 3.8 Hz, 1H, thiophene-H), 8.09 (s, 1H, N=CH), 11.31 (brs, 1H, NH; D2O exchangeable), 11.77 (brs, 1H,NH; D2O exchangeable). 13C NMR (75 MHz, DMSO) δ = 164.65, 159.47, 151.85, 145.90, 137.78, 130.76, 130.65, 130.42, 128.77, 128.42, 128.04, 127.94, 126.37, 125.01, 114.35, 114.07, 20.96. MS, m/z (%): 455 (M+, 10); Analysis calc. for C25H18FN5OS (455.12): C, 65.92; H, 3.98; N, 15.38; S, 7.04. Found: C, 65.67; H, 3.73; N, 15.15; S, 6.77.

4.2. Anti-microbial activity

The antimicrobial action of the tested samples were examined against some targeted pathogenic microorganisms gained from the American kind culture collection (ATCC; Rockville, MD, USA). The tested organisms were Staphylococcus aurous ATCC- 47077 (St.), Bacillus cereus ATCC-12228 (B.C.), Escherichia coli ATCC-25922 (E.C.), Salmonella typhi ATCC-15566 (Salm.) and Candida albicans ATCC-10231 (C. Alb.). The stock cultures of pathogens utilized in this study were keeped on nutrient agar slants at 4 °C. The Agar well diffusion procedure was employed to study the antimicrobial activities of the samples according to the method described [56, 57]. Reference antibacterial drugs ampicillin and vancomycin were estimated for their antibacterial and antifungal potency and compared with the tested samples. Seventy microliters of bacterial and yeast cells (106 CFU/mL) were spread on plates of nutrient agar media. The wells (6 mm diameter) were excavated on the injected agar plates then 100 μl of the samples and its derivatives suspended in DMSO, were added up to the wells. The reference antibiotics disks (10 and 30 μg/disk of ampicillin and vancomycin, respectively) were potted onto surface of agar inoculated plates. The plates were kept at 4 °C for 2h before incubation to permit diffusion to occur. The plates were kept at 37 °C for 24 h except yeast strain that were incubated at 28 °C for 24h then followed by measure of the diameter of the inhibition zone (mm), and this was replicate for five times and the average was taken [56, 58, 59, 60, 61].

4.2.1. Minimum inhibition concentration (MIC)

The MIC was known as the concentration at which the bacteria and yeast do not presented visible growth with regard to the positive control and was done for new synthesized samples with a little modulation for previous reported procedure [62]. In summarized, serial dilutions were prepared for the examined materials dissolved in DMSO. 150 μL of double strength Mueller Hinton broth medium were loaded in each well of the 96 well micro liter plate followed up by 150 μL of the 2-fold appropriate concentration and mixed well to gain the final concentration. After 24h broth cultures of the screened bacterial and yeast strains prepared as an inoculums of 5 % (V/V) (OD = 0.5 McFarland standard) was inoculated into the respective wells. For the positive growth control, the same inoculums size of each test strain was inoculated in wells that didn't including any of the screened materials. DMSO solution was evaluated as negative control. The plates were statically incubated for 24 h at 37 °C. 30 μL of prepared solution (0.18 %) was added to each well to work as an electron acceptor and the inhibition of bacterial growth was easily visible as a dark blue well and the presence of growth was noticed by existence of red, pink or purple color.

4.2.2. Statistical analysis

Statistical analyses were accomplished utilizing GraphPad Prism 5.0 (Graph Pad Software, LaJolla, CA). In One-way model ANOVA, the detected variance is divided into sections due to different explanatory variables. A level of P less than 0.05 was count to be statistically significant.

Declarations

Author contribution statement

Reda M. Abdelhameed: conceived and designed the experiments; analyzed and interpreted the data; contributed reagents, materials, analysis tools or data; wrote the paper.

Osama M. Darwesh: performed the experiments; analyzed and interpreted the data; contributed reagents, materials, analysis tools or data.

Mahmoud El-Shahat: conceived and designed the experiments; performed the experiments; analyzed and interpreted the data; contributed reagents, materials, analysis tools or data; wrote the paper.

Funding statement

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Competing interest statement

The authors declare no conflict of interest.

Additional information

No additional information is available for this paper.

Appendix A. Supplementary data

The following is the supplementary data related to this article:

Supplementary Material Revised_V2

mmc1.doc (6.3MB, doc)

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