Synthesis, Antimycobacterial Evaluation and Docking Studies of Some 7-Methyl-5,6,7,8-tetrahydropyrido[4′,3′:4,5]thieno[2,3-d]pyrimidin-4(3H)-ones

Abstract

Two series of 3-substituted-7-methyl-5,6,7,8-tetrahydropyrido[4′,3′:4,5] thieno[2,3-d]pyrimidin-4(3H)-one (6a–k) and 3-substituted-7,2-dimethyl-5,6,7,8-tetrahydropyrido[4′,3′:4,5]thieno[2,3-d]pyrimidin-4(3H)-one (7a–k) derivatives were synthesized and characterized using spectral data i.e., IR, ¹H-, ¹³C-NMR, Mass and CHN elemental analyses. The synthesized compounds were evaluated for antibacterial activity against each of two strains of Gram-positive (Bacillus subtilis and Staphylococcus aureus) and Gram-negative (Escherichia coli and Klebsiella pneumoniae) bacteria and antimycobacterial activity screened against two strains i.e., Mycobacterium tuberculosis (MTB) H37Rv and an isoniazid-resistant clinical sample. Further to validate potentiality of our design was analyzed using molecular docking studies by taking crystal structure of MTB pantothenate synthetase (MTB-PS) (PDB: 3IVX). In this study, some compounds 6k (Minimum Inhibitory Concentration (MIC): MIC-22 µM), 7d (MTB: MIC-22 µM) and 7k (MTB: MIC-11 µM) showed potential antibacterial and antimycobacterial activities.

Tuberculosis (TB) is considered as a serious public health threat worldwide, as current therapy is limited due to the emergence of multi-drug resistance (MDR) and extensively drug resistance (XRD), regimen therapy, and severe side effects of existing drugs and long duration of therapy.^1,2) Moreover, TB is more widespread due to its coincidence with AIDS and Human Immunodeficiency Virus (HIV) co-infection.³⁾ Development of drug resistance (MDR and XDR) to widely used first line drugs is an obstacle in the treatment and control programs of this disease. Resistance to isoniazid (INH) is more predominant in MDR TB as about 30% of clinical isolates found to be resistant to it, worldwide. Several decades of extensive research resulted in identification and development of only few drugs such as bedaquiline and delamanide, which were approved as drugs of choice in drug resistant (MDR and XDR) TB. Several other novel drug candidates for drug resistant TB such as oxazolidinones (linezolid), nitroimidazoles (PA-824) and ethylenediamines (SQ-109) etc., are currently under clinical development.⁴⁾ There is an urgent need of shorter and simple regimens, effective, safe and well tolerated drugs for drug-resistant and drug-susceptible TB.

In view of development of hetero-fused thieno[2,3-d]pyrimidine-4(3H)-one analogues various recent reports prompted us to continue our studies by retaining 4,5,6,7-tetrahydrothieno[2,3-c]pyridine as basic nucleus.⁵⁾ It was a common feature in some reported Mycobacterium tuberculosis Pantothenate synthetase (MTB-PS) inhibitors such as 6-benzyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide (A, SID 92104095; IC₅₀=3.3 µg/mL),⁶⁾ 6-acetyl-4,5,6,7-tetrahydro thieno[2,3-c]pyridine-3-carboxamide (B, SID 92097880; IC₅₀=0.5 µg/mL))⁶⁾ derivatives, as described earlier.⁵⁾ In addition, Rashmi et al. reported potentiality of some thieno[2,3-d]pyrimidines (C) on Mycobacterium.⁷⁾ Narayana et al. reported the antibacterial activity of some 4,5,6,7-tetrahydro[1]benzothieno[2,3-d]pyridimidine-4(3H)-ones (D).⁸⁾ These studies proved the essentiality of 4,5,6,7-tetrahydrothieno[2,3-c]pyridine (A, B) nucleus and arylidineamino side chain at 3rd or 4th position in C and D in their antibacterial and antimycobacterial activity (Fig. 1). In the design of molecules, both the structural features were adopted. The 3-amino-7-methyl-5,6,7,8-tetrahydropyrido[4′,3′:4,5]thieno[2,3-d]pyrimidin-4(3H)-one was considered as a key structure for the synthesis. The bulky (benzyl) group present in the previous series⁵⁾ was substituted with a small (methyl) group at 7th position of 5,6,7,8-tetra-hydropyrido[4′,3′:4,5]thieno[2,3-d]pyrimidine nucleus. Aurelio et al. also reported the allosteric modulation effects of 2-amino-4,5,6,7-tetrahydrothieno[2,3-c]pyridines on A₁ adenosine receptor.⁹⁾ Modifications at 7th position with small and medium pharmacophores such as N-methyl, N-benzyl and N-ethoxycarbonyl resulted differences in their biological activity. As per their studies, it was due to increase in basicity with N-methyl substitution. Although, the structure activity relationship was described for A₁ adenosine receptor modulation, the significance of 7th substitution was considered in this study and attempted with these modifications perhaps it could show a better antimycobacterial activity. In this direction our studies are continued on the development of novel hetero-fused thieno[2,3-d]pyrimidine analogues against drug-resistant TB (INH-resistant TB).⁵⁾ Two series of molecules 7-methyl-3-(substituted-aryledineamino)-5,6,7,8-tetrahydropyrido[4′,3′:4,5]thieno[2,3-d]pyrimidin-4(3H)-ones (6a–k) and 2,7-dimethyl-3-(substituted-aryledineamino)-2-methyl-5,6,7,8-tetrahydropyrido [4′,3′:4,5]thieno[2,3-d]pyrimidin-4(3H)-ones (7a–k) have been synthesized and evaluated for their antitubercular activity. Initial antibacterial screening was performed against each of two Gram-positive (Bacillus subtilis and Staphylococcus aureus) and Gram-negative (Escherichia coli and Klebsiella pneumonia) bacteria. The antimycobacterial activity was conducted for selected compounds based on results obtained in initial antibacterial screening. Mycobacterium tuberculosis (MTB) H37Rv and an INH-resistant clinical sample were used as standard and test organisms.

Fig. 1. General Structures of Some Potent Antibacterial Agents (A, B: MTB-PS Inhibitors)

Further, in silico binding interactions of these analogues were studied using target protein MTB-PS (PDB: 3IVX) with the help of Molegro Virtual Docker (MVD) software. These investigations indicated that some of the compounds expressed promising in vitro antibacterial and antimycobacterial activities against the test strains.

Results and Discussion

Chemistry

The synthesis of target compounds 7-methyl-3-(substituted-aryledineamino)-5,6,7,8-tetrahydropyrido[4′,3′:4,5]thieno[2,3-d]pyrimidin-4(3H)-ones (6a–k) and 2,7-dimethyl-3-(substituted-aryledineamino)-5,6,7,8-tetrahydropyrido[4′,3′:4,5]thieno[2,3-d]pyrimidin-4(3H)-ones (7a–k) was achieved as shown in Chart 1.

Chart 1. General Chart for Synthesis of Compounds 6a–k and 7a–k

i) S₈, morpholine, ethanol, reflux, 1–2 h; ii) Triethylorthoformate/triethylorthoacetate, reflux, 2–4 h; iii) NH₂NH₂H₂O, ethanol, stir, 1–2 h; iv) R¹CHO, glacial CH₃COOH, ethanol, reflux, 12 h.

The requisite key starting material 3 was synthesized by Gewald Reaction (GR) using N-methyl-4-piperidone (1), elemental sulphur and ethylcyanoacetate (2) by following a reported procedure^9,10) which gave the product in 85% of yield. The compound 3 was converted to iminoester derivatives by heating under reflux in triethylorthoformate and triethylorthoacetate, which gave thick brown oily products 4a and 4b, respectively. Compounds 4a and 4b were treated with hydrazine hydrate^5,11–13) to get their cyclized products 5a and 5b. In IR spectrum compound 5b showed two characteristic signals at 3450 cm⁻¹ and 3300 cm⁻¹ assignable to N–H stretching for 3-NH₂ group and another signal at 1670 cm⁻¹ assignable to C=O stretching of carbonyl group of pyrimidine ring. The ¹H-NMR spectrum of compound 5b showed a singlet at δ=2.20 ppm which could be assignable to methyl (2-CH₃) group of pyrimidinone ring. The presence of another broad singlet at the δ=5.13 ppm could be assignable to primary amino functionality.

In the last step different arylidineamino analogues 6a–k and 7a–k were prepared by condensation of 5a and 5b with differently substituted aromatic aldehydes following some reported protocols.^5,7,8,14) The IR spectra of compound 6a–k and 7a–k showed characteristic signals of C=O stretching and C=N stretching (-imine). The loss of strong N–H stretching signals of 3-NH₂ group was noticeable which revealed the formation of imine. The ¹H-NMR spectra for compounds 6a–k showed characteristic singlet at δ=9.4–10.2 ppm could be assignable for single proton of imine (–N=CH) and another sharp singlet at δ=8.2–8.3 ppm could be assignable to single proton (2-H) of pyrimidinone ring. Compounds 7a–k showed singlet at δ=9.2–10.2 ppm could be assignable for single proton of imine (–N=CH) and another singlet at δ=2.59–2.63 ppm could be assignable for methyl (2-CH₃) protons of pyrimidinone ring. The mass spectra (Electron Spray Ionization (ESI) and Electron Impact (EI) mode) showed the molecular ion (M⁺, M⁺+1, M⁺+2) peaks for all the compounds. The halogenated compounds showed the isotopic peaks (M⁺+1 and M⁺+2), which further confirms the presence of chlorine (Cl) as a substituent, in compounds 6b, 6k, 7b and 7k. The physical data of all the final compounds was shown in Table 1.

Table 1. Physical Data of Synthesized Compounds 6a–k and 7a–k

Comp. Code	R	R1	Mol. Wt. (M)	Melting range (°C)	Yield (%)
6a	H	-C6H5	324	164–166	87
6b	H	4-ClC6H4	358	196–198	78
6c	H	4-FC6H4	343	180–182	64
6d	H	4-CNC6H4	350	236–238	67
6e	H	4-OCH3C6H4	354	168–170	80
6f	H	4-OHC6H4	340	220–222	65
6g	H	3,4,5-(OCH3)3C6H2	414	192–194	67
6h	H	2-NO2C6H4	369	159–161	72
6i	H	Thiophene-2-yl	330	120–122	66
6j	H	Furan-2-yl	314	156–158	80
6k	H	2,6-Cl2C6H3	392	190–192	82
7a	CH3	-C6H5	338	185–187	85
7b	CH3	4-ClC6H4	372	153–155	71
7c	CH3	4-FC6H4	355	230–232	65
7d	CH3	4-CNC6H4	363	200–202	68
7e	CH3	4-OCH3C6H4	368	173–175	82
7f	CH3	4-OHC6H4	354	218–220	76
7g	CH3	3,4,5-(OCH3)3C6H2	428	198–200	70
7h	CH3	2-NO2C6H4	383	154–156	78
7i	CH3	Thiophene-2-yl	312	178–180	70
7j	CH3	Furan-2-yl	328	172–174	79
7k	CH3	2,6-Cl2C6H3	406	181–183	80

Antibacterial Activity

All the newly synthesized compounds 6a–k and 7a–k were assessed for their in vitro antibacterial activity against S. aureus (MTCC-96), B. subtilis (MTCC-441), E. coli (MTCC-443) and K. pneumoniae (MTCC-109) using streptomycin as a reference. Broth micro dilution method^15,16) was employed for determination of minimum inhibitory concentration (MIC) for all test compounds. The MIC (µM) data of all the compounds were tabulated in Table 2.

Table 2. Antibacterial Activity (MIC) Data of Test Compounds 6a–k and 7a–k

Comp. Code	R	R1	MIC (µM)
			S. aureus MTCC 96	B. subtilis MTCC 441	E. coli MTCC 443	K. pneumoniae MTCC 109
6a	H	-C6H5	NA	NA	NA	NA
6b	H	4-ClC6H4	43.64	43.64	21.82	87.29
6c	H	4-FC6H4	46	46	23	46
6d	H	4-CNC6H4	48	48	24	48
6e	H	4-OCH3C6H4	44.1	88.2	44.1	NA
6f	H	4-OHC6H4	NA	NA	NA	NA
6g	H	3,4,5-(OCH3)3C6H2	NA	NA	NA	NA
6h	H	2-NO2C6H4	84	84	84	84
6i	H	Thiophene-2-yl	48	48	24	48
6j	H	Furan-2-yl	98	98	98	<98
6k	H	2,6-Cl2C6H3	22	22	11	22
7a	CH3	-C6H5	NA	NA	NA	NA
7b	CH3	4-ClC6H4	44	44	22	44
7c	CH3	4-FC6H4	46	46	23	46
7d	CH3	4-CNC6H4	24	24	12	24
7e	CH3	4-OCH3C6H4	44	44	22	44
7f	CH3	4-OHC6H4	NA	NA	NA	NA
7g	CH3	3,4,5-(OCH3)3C6H2	NA	NA	NA	NA
7h	CH3	2-NO2C6H4	162	162	162	162
7i	CH3	Thiophene-2-yl	28	28	28	28
7j	CH3	Furan-2-yl	50	50	50	50
7k	CH3	2,6-Cl2C6H3	21	21	10	21
Std	—	—	<1	<1	<1	<1
Ctrl	—	—	NA	NA	NA	NA

Std.=Streptomycin; Ctrl=N,N-dimethylformamide (DMF) in water (25% v/v); NA=Not active.

Among all the synthesized compounds, 6b, 6c, 6d, 6i, 6k, 7b, 7c, 7d and 7k exhibited good activity with MIC in the range of 11–24 µg/mL against E. coli and rest of the compounds showed moderate to less activity. The structure activity relationship reveals that presence of thieno(2,3-d)pyrimidine-4-one ring with 3-arylidineamino side chain skeleton is crucial for biological activity. In general, the arylidineamino side chain bearing substituted phenyl and heterocyclic groups showed more activity over unsubstituted analogues. Phenyl ring substituted with electron withdrawing groups such as chloro (4-Cl: 6b, 7b; 2,6-dichloro: 6k, 7k), flouro (4-F: 6c, 7c) (excepting 4-methoxy: 6e, 7e) and also with thiophene (6i, 7i) system exhibited better activity when compared to electron donating groups such as hydroxyl (4-OH: 6f, 7f), 3, 4, 6-trimethoxyl (6g, 7g). However nitro (2-NO₂) containing compounds (6h, 7h) showed less activity. Compounds possessing 2,6-dichloro (6k, 7k) substitution on phenyl ring exhibited potent activity on all the test organisms (Gram-positive and Gram-negative bacteria) used. Variations in the phenyl substitution might have altered the lipophilic character of molecules which is an important feature for permeability across the bacterial cell membrane. The 4-hydroxyl (activating group) substituted compounds (6f and 7f) were found to be less potent might be due to their poor lipophilicity. On overall assessment most of the compounds showed activity against E. coli (Gram-negative bacteria). Methyl substitution at 2nd position of pyrimidine ring relatively enhanced the biological activity.

Antimycobacterial Activity

Few compounds randomly selected for antimycobacterial studies by considering the preliminary antibacterial activity data. The MTB H37Rv and an INH-resistant clinical sample were taken as reference and test organisms, respectively. The MIC was determined on Middlebrook 7H9 medium supplemented with OADC using broth micro dilution method with the help of four well known drugs (INH, Streptomycin, Rifampicin and Ethambutol) as reference.¹⁷ The test samples were prepared by serial dilution method in different strengths in DMF in water (25% v/v) as diluent. Among all the tested compounds 7k, bearing 2, 6-dichloro substitution on arylidineamino side chain showed greater activity with MIC-11 µM against MTB H37Rv and also clinical sample (INH-resistant). Other compounds bearing 2,6-dichloro (6k), 4-Cl (7b), 4-F (7c) and 4-CN (7d) showed activity in the MIC range of 22–25 µM on both the strains. The additional chloro (Cl) group at 2nd position of phenyl ring in 7k, 6k instead monochloro (4-Cl) substitution in 6b, 7b enhanced the biological activity. Compounds, 6c, 6d, 7e and 7i exhibited poor in potency and their MIC range was observed at ≥46 µM. The antimycobacterial activity (MIC in µM) data is tabulated in Table 3.

Table 3. Antimycobacterial Activity (in Vitro) of Test Compounds

Comp. Code	R	R1	MIC (µM)
			MTB H37Rv		Clinical sample of MTB
			14 d	21 d	14 d	21 d
6b	H	4-ClC6H4	46	46	46	46
6c	H	4-FC6H4	88	88	88	88
6d	H	4-CNC6H4	180	180	180	180
6k	H	2,6-Cl2C6H3	22	22	22	22
7b	CH3	4-ClC6H4	24	24	24	24
7c	CH3	4-FC6H4	25	25	25	25
7d	CH3	4-CNC6H4	22	22	22	22
7e	CH3	4-OCH3C6H4	84	84	84	84
7i	CH3	Thiophene-2-yl	98	98	98	98
7k	CH3	2,6-Cl2C6H3	11	11	11	11
Std1	—	—	0.1	0.1	4	4
Std2	—	—	2	2	2	2
Std3	—	—	4	4	4	4
Std4	—	—	0.09	0.09	2	2
Ctrl	—	—	NA	NA	NA	NA

Std¹=INH; Std²=Streptomycin; Std³=Ethambutol; Std⁴=Rifampicin; Ctrl=DMF in water (25% v/v); NA=Not active.

Molecular Modeling

The molecular docking studies were performed on target protein MTB-PS, which has been proven to be valid drug target for rational design of new analogues for TB by several reports.^18,19) The crystal structure of MTB-PS was retrieved from protein data bank ((PDB: 3IVX) (http://www.rcsb.org/pdb/explore.do?structure Id=3ivx). Ligands possessing 4-flouro (6c), 4-hydroxy (6f), 2-nitro (6h), 4-methoxy (7e) and 2,4-dichloro (6k, 7k) substitution on phenyl ring of arylidineamino side chain showed best docking scores, i.e., −118.43, −118.42, −122.87, −121.03, −120.68 and −115.63, respectively.

The carbonyl (C=O, H-bond acceptor) group at 4th position in pyrimidine ring showed strong hydrogen bond interaction with Gly 158 (H-bond donor) amino acid residue in all (6a–k and 7a–k) the ligands. Another H-bond interaction was observed at nitrogen (7-N) atom of the 5,6,7,8-tetrahydrothieno[2,3-d]pyridine ring with Val 187 amino acid residue (Table 4).

Table 4. H-Bond Interactions of Biologically Active Ligands 6k, 7d and 7k

Comp code	Residue	Atom ID	PDB atom name	Ligand name	Distance
6k	Gly 158	1163	N (d)	O (a)	2.93
	Val 187	1397	N (d)	N (a)	2.70
	Thr 186	1386	O (d+a)	N (a+d)	3.14
7d	Gly 158	1163	N (d)	O (a)	1.65
	Val 187	1397	N (d)	N (a)	3.09
7k	Gly 158	1163	N (d)	O (a)	2.60
	Val 187	1397	N (d)	N (a)	3.11

Additionally, the steric interactions observed for all the molecules with amino acid residues such as Leu 50, Val 184, Pro 185, Gln164 and Pro 38. All these molecules 6a–k and 7a–k lack additional π–π stacking and van der Waals interactions due to presence of N-methyl group at 7th position, hence they showed less potency compared to N-benzyl analogues.⁵⁾ The binding mode and orientation pattern at active site were analyzed for all molecules. The docking poses of the experimentally more potent molecules 7b and 7k were showed in Fig. 2.

Fig. 2. Snapshots of the Typical Binding Interactions of Ligands 7b (A) and 7k (B) with Active Site of MTB-PS, Hydrogen Bond Interactions of C=O Group with Residue of Gly 158 and Nitrogen (7-N) with Val 187

Conclusion

In conclusion this study deals with the design and synthesis of novel analogues of 7-methyl-5,6,7,8-tetrahydropyrido[4′,3′:4,5]thieno[2,3-d]pyrimidin-4(3H)-ones. Among all the compounds 6a, 6b, 6c, 6d, 6k, 7b, 7c, 7d, 7e and 7k showed promising antibacterial activity against E. coli in the MIC range 10–24 µM and compounds 6k, 7b, 7c, 7d and 7k further showed antimycobacterial activity in the MIC range 10–25 µM against MTB H37Rv and an INH-resistant clinical sample. Among these compound 7k was found to be more potent (MIC-11 µM). Further the molecular docking studies helped in knowing the binding modes of core structure with active sites and the docking scores obtained were correlated with the MIC values (µM) of most of the compounds. Further investigations are in progress on enzyme inhibitory activity, cytotoxicity and pharmacokinetic properties to validate these results.

Experimental

Chemistry

Melting points of all the compounds were determined in open capillaries on melting point apparatus (Biotechniques India-BTI-34), and are uncorrected. Purity and homogeneity was verified by pre-coated TLC plates (E. Merck silica gel 60 F₂₅₄). IR spectra were recorded using KBr pellet method on Bruker spectrophotometer and Perkin-Elmer FTIR 240-C (υ_max in cm⁻¹). The ¹H-NMR and ¹³C-NMR was recorded on a Bruker spectrometer 500 MHz and 400 MHz respectively using tetramethylsilane (TMS) as internal standard, Central Instrumentation Laboratory, Indian Institute of Chemical Technology, Hyderabad, India. The chemical shifts (δ) are reported in part per million (ppm) relative to TMS using CDCl₃ as solvent. Signal multiplicities are represented by s (singlet), d (doublet), t (triplet), q (quartet), br s (broad singlet), dd (double doublet) and m (multiplet). MS were recorded on ESI and EI mode using QSTARXL hybrid MS/MS system (Applied Bio-systems, U.S.A.), Central Instrumentation Laboratory, Indian Institute of Chemical Technology, Hyderabad, India. Elemental analysis was carried out using vario MICRO CUBE Elementar (2mgChem80s method), Kakatiya University, Warangal. All the chemicals used in the synthesis were procured from Aldrich Company Ltd. (Bengaluru, India) and Himedia Chemicals (Mumbai, India). All the solvents used were purchased from SD Fine Chemicals Ltd. (Mumbai, India) and were employed without further purification.

Ethyl-2-amino-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylate (3)

N-Methyl-4-piperidone (1) (5 g, 26.42 mmol), ethyl cyanoacetate (2) (3.09 mL, 29.06 mmol) and sulphur (1.02 g, 31.70 mmol) were suspended in ethanol (55 mL). Morpholine (4.62 mL, 52.84 mmol) was added and the mixture was heated under reflux gently with stirring for 2 h. The cooled solution was diluted with water and extracted with dichloromethane (3×50 mL). The combined organic layers were washed with water, then brine, dried (MgSO₄), filtered and concentrated. The resultant residue was triturated with hot methanol (20 mL) and washed with ice cold methanol to afford the desired product as an off white powder.

Yield, 7.1 g (85%). mp 110–111°C. IR (KBr) cm⁻¹: 3344, 3254 (N–H, str), 2986, 2878 (C–H, str), 1685 (C=O, str). ¹H-NMR (CDCl₃) δ (ppm): 6.03 (2H, br s, 2-NH₂), 4.27 (2H, q, J=7.2 Hz, CH₂ of carboxylate), 3.39 (2H, s, 7-CH₂), 2.85 (2H, t, J=5.7 Hz, 5-CH₂), 2.68 (2H, t, J=5.8 Hz, 4-CH₂), 2.45 (3H, s, 7-NCH₃), 1.34 (3H, t, J=7.2 Hz, –CH₃ of carboxylate). ESI-MS (m/z; %): 241 (M⁺+1; 100).

3-Amino-7-methyl-5,6,7,8-tetrahydropyrido[4′,3′:4,5]thieno[2,3-d]pyrimidin-4(3H)-one (5a)

A solution of 3 (1⋅10 g, 57 mmol) in triethylorthoformate (5 mL) was heated under reflux for 2–4 h. Excess triethylorthoformate was removed in vacuo. The residue was treated with ethyl acetate in hexane (20% v/v) and dried to obtain light brown oily product (4a). It was used directly in next step without purification. A mixture of 4a and hydrazine hydrate (1 mL) in 10 mL of absolute ethanol was stirred at room temperature for 1–2 h. The separated fine solid was filtered, washed with ethanol and purified by recrystallization from ethanol to afford analytically pure pale yellow granules.

Yield, 0.76 g (71%). mp 194–196°C. IR (KBr) cm⁻¹: 3450, 3300 (N–H, str), 2910 (C–H, str), 1670 (C=O, str). ¹H-NMR (CDCl₃) δ (ppm): 2.50 (3H, s, 7-NCH₃), 2.80 (2H, t, J=6 Hz, 5-CH₂), 3.20 (2H, t, J=6 Hz, 6-CH₂), 3.67 (2H, s, 8-CH₂), 5.13 (2H, br s, 3-NH₂), 8.20 (1H, s, 2-H pyrimidinone). ESI-MS (m/z; %): 236 (M⁺+1; 100).

3-Amino-2,7-dimethyl-5,6,7,8-tetrahydropyrido[4′,3′:4,5]thieno[2,3-d]pyrimidin-4(3H)-one (5b)

A solution of 3 (1⋅10 g, 57 mmol) in triethylorthoacetate (5 mL) was heated under reflux for 2–4 h. Excess triethyorthoacetate was removed in vacuo. The residue was treated with ethyl acetate in hexane (20% v/v) and dried to obtain light brown oily product (4b). It was used directly in next step without purification. A mixture of 4b and hydrazine hydrate (1 mL) in 10 mL of absolute ethanol was stirred at room temperature for 1–2 h. The separated fine solid was filtered, washed with ethanol and purified by recrystallization from ethanol to afford analytically pure pale yellow granules.

Yield, 0.84 g (74%). mp 200–202°C. IR (KBr) cm⁻¹: 3450, 3300 (N–H, str), 2910 (C–H, str), 1670 (C=O, str). ¹H-NMR (CDCl₃) δ (ppm): 2.20 (3H, s, 2-CH₃ of pyrimidinone), 2.50 (3H, s, 7-NCH₃), 2.80 (2H, t, J=5.82 Hz, 5-CH₂), 3.20 (2H, t, J=5.73 Hz, 6-CH₂), 3.67 (2H, s, 8-CH₂), 5.13 (2H, br s, 3-NH₂). ESI-MS (m/z; %): 250 (M⁺+1; 85).

7-Methyl-3-(substituted-aryledineamino)-5,6,7,8-tetrahydropyrido[4′,3′:4,5]thieno[2,3-d]pyrimidin-4(3H)-one (6a–k)

General Method

A mixture of 5a (0.001 M) and appropriate aldehyde (0.001 M) in catalytic amount of glacial acetic acid in 10 mL of absolute ethanol was heated under reflux for 2–6 h. On cooling, the separated solid was filtered, washed with cold ethanol and recrystallized from acetic acid: ethanol to afford desired product.

3-(Benzylideneamino)-7-methyl-5,6,7,8-tetrahydropyrido[4′,3′:4,5]thieno[2,3-d]pyrimidin-4 (3H)-one (6a)

[El-Kashef et al.].¹¹⁾

3-((4-Chlorobenzylidene)amino)-7-methyl-5,6,7,8-tetrahydropyrido[4′,3′:4,5]thieno[2,3-d]pyrimidin-4(3H)-one (6b)

[El-Kashef et al.].¹¹⁾

3-((4-Fluorobenzylidene)amino)-7-methyl-5,6,7,8-tetrahydropyrido[4′,3′:4,5]thieno[2,3-d]pyrimidin-4(3H)-one (6c)

Yield, 0.22 g (64%). mp 180–182°C. IR (KBr) cm⁻¹: 3069, 3031 (aromatic C–H, str), 2919 (aliphatic C–H, str), 1672 (C=O, str), 1601 (C=N, str). ¹H-NMR (CDCl₃) δ (ppm): 2.59 (3H, s, 7-CH₃), 2.79 (2H, t, J=5.88 Hz, 5-CH₂), 3.18 (2H, t, J=5.94 Hz, 6-CH₂), 3.67 (2H, s, 8-CH₂), 7.18 (2H, t, J=8.62 Hz, 3′,5′-Ar-H), 7.85 (2H, dd, J=5.4, 5.4 Hz, 2′,6′-Ar-H), 8.24 (1H, s, 2-H of pyrimidinone), 9.53 (1H, s, N=CH). ¹³C-NMR (CDCl₃) δ (ppm): 26.12, 45.48, 51.89, 53.76, 116.34–116.34 (d), 122.92, 129.31–129.34 (d), 130.13, 130.71–130.80 (d), 132.07, 145.57, 156.37, 160.86, 162.43, 164.01, 166.53. ESI-MS (m/z; %): 343 (M⁺+1; 100). Anal. Calcd for C₁₇H₁₅FN₄OS: C, 59.63; H, 4.42; N, 16.36. Found: C, 58.92; H, 4.46; N, 16.88.

4-(((7-Methyl-4-oxo-5,6,7,8-tetrahydropyrido[4′,3′:4,5]thieno[2,3-d]pyrimidin-3(4H)-yl)imi no)methyl)benzonitrile (6d)

Yield, 0.23 g (67%). mp 236–238°C. IR (KBr) cm⁻¹: 3099 (aromatic C–H, str), 2955 (aliphatic C–H, str), 2229 (CN, str), 1674 (C=O, str), 1545 (C=N, str). ¹H-NMR (CDCl₃) δ (ppm): 2.56 (3H, s, 7-NCH₃), 2.79 (2H, t, J=5.12 Hz, 5-CH₂), 3.18 (2H, t, J=5.22 Hz, 6-CH₂), 3.66 (2H, s, 8-CH₂), 7.78 (2H, d, J=8.86 Hz, 3′,5′-Ar-H), 7.94 (2H, d, J=8.84 Hz, 2′,6′-Ar-H), 8.28 (1H, s, 2-H of pyrimidinone), 9.92 (1H, s, N=CH). ¹³C-NMR (CDCl₃) δ (ppm): 26.12, 45.47, 51.84, 53.73, 115.24, 118.08, 122.87, 128.76, 130.23, 132.51, 132.61, 137.44, 146.02, 156.76, 159.70, 160.69. ESI-MS (m/z; %): 350 (M⁺+1; 100). Anal. Calcd for C₁₈H₁₅N₅OS: C, 61.87; H, 4.33; N, 20.04. Found: C, 61.58; H, 4.76; N, 19.82.

3-((4-Methoxybenzylidene)amino)-7-methyl-5,6,7,8-tetrahydropyrido[4′,3′:4,5]thieno[2,3-d]pyrimidin-4(3H)-one (6e)

[El-Kashef et al.].¹¹⁾

3-((4-Hydroxybenzylidene)amino)-7-methyl-5,6,7,8-tetrahydropyrido[4′,3′:4,5]thieno[2,3-d]pyrimidin-4(3H)-one (6f)

[El-Kashef et al.].¹¹⁾

7-Methyl-3-((3,4,5-trimethoxybenzylidene)amino)-5,6,7,8-tetrahydropyrido[4′,3′:4,5]thieno[2,3-d]pyrimidin-4(3H)-one (6g)

Yield, 0.28 g (67%). mp 192–194°C. IR (KBr) cm⁻¹: 3059 (aromatic C–H, str), 2937, 2776 (aliphatic C–H, str), 1672 (C=O, str), 1576 (C=N, str). ¹H-NMR (CDCl₃) δ (ppm): 2.52 (3H, s, 7-NCH₃), 2.81 (2H, t, J=5.86 Hz, 5-CH₂), 3.22 (2H, t, J=5.89 Hz, 6-CH₂), 3.69 (2H, s, 8-CH₂), 3.94 (6H, s, 3′,5′-Ar-OCH₃), 3.96 (3H, s, 4′-Ar-OCH₃), 7.12 (2H, s, 2′,6′-Ar-H), 8.26 (1H, s, 2-H of pyrimidinone), 9.44 (1H, s, N=CH). ESI-MS (m/z; %): 415 (M⁺+1; 100). Anal. Calcd for C₂₀H₂₂N₄O₄S: C, 57.96; H, 5.35; N, 13.52. Found: C, 57.92; H, 5.46; N, 13.68.

7-Methyl-3-((2-nitrobenzylidene)amino)-5,6,7,8-tetrahydropyrido[4′,3′:4,5]thieno[2,3-d]pyrimidin-4(3H)-one (6h)

Yield, 0.27 g (72%). mp 159–161°C. IR (KBr) cm⁻¹: 3019 (aromatic C–H, str), 2932, 2774 (aliphatic C–H, str), 1686 (C=O, str), 1524 (C=N, str). ¹H-NMR (CDCl₃) δ (ppm): 2.51 (3H, s, 7-NCH₃), 2.79 (2H, t, J=5.88 Hz, 5-CH₂), 3.18 (2H, t, J=5.88 Hz, 6-CH₂), 3.67 (2H, s, 8-CH₂), 7.72 (1H, dt, J=8.02 Hz, 4′-Ar-H), 7.78 (1H, dt, J=8.02 Hz, 5′-Ar-H), 8.18 (2H, dd, J=8.12, 1.52 Hz, 3′,6′-Ar-H), 8.28 (1H, s, 2-H of pyrimidinone),10.18 (1H, s, N=CH). ¹³C-NMR (CDCl₃) δ (ppm): 26.04, 45.43, 51.84, 53.72, 122.94, 124.86, 128.49, 129.42, 130.36, 131.97, 132.28, 133.73, 145.57, 148.86, 156.20, 159.57, 160.75. ESI-MS (m/z; %): 370 (M⁺+1; 100). Anal. Calcd for C₁₇H₁₅N₅O₃S: C, 55.27; H, 4.09; N, 18.96. Found: C, 55.42; H, 4.46; N, 18.88.

7-Methyl-3-((thiophen-2-ylmethylene)amino)-5,6,7,8-tetrahydropyrido[4′,3′:4,5]thieno[2,3-d]pyrimidin-4(3H)-one (6i)

Yield, 0.22 g (66%). mp 120–122°C. IR (KBr) cm⁻¹: 3051 (aromatic C–H, str), 2938, 2785 (aliphatic C–H, str), 1683 (C=O, str), 1581 (C=N, str). ¹H-NMR (CDCl₃) δ (ppm): 2.51 (3H, s, 7-NCH₃), 2.81 (2H, t, J=5.88 Hz, 5-CH₂), 3.19 (2H, t, J=5.79 Hz, 6-CH₂), 3.68 (2H, s, 8-CH₂), 7.26 (1H, dt, J=5, 1.12 Hz, 4′-Ar-H of thiophene), 7.54 (1H, dd, J=3.88, 1.53 Hz, 3′-Ar-H of thiophene), 7.58 (1H, dd, J=5.01, 3.12 Hz, 5′-Ar-H of thiophene), 8.25 (1H, s, 2-H of pyrimidinone), 9.78 (1H, s, N=CH). ¹³C-NMR (CDCl₃) δ (ppm): 25.80, 49.87, 51.64, 62.01, 112.54, 118.74, 127.36, 128.44, 129.14, 130.28, 137.96, 146.90, 153.58, 155.08, 161.19. ESI-MS (m/z; %): 331 (M⁺+1; 100). Anal. Calcd for C₁₅H₁₄N₄OS₂: C, 54.52; H, 4.27; N, 16.96. Found: C, 54.82; H, 4.46; N, 16.88.

3-((Furan-2-ylmethylene)amino)-7-methyl-5,6,7,8-tetrahydropyrido[4′,3′:4,5]thieno[2,3-d]pyrimidin-4(3H)-one (6j)

Yield, 0.25 g (80%). mp 156–158°C. IR (KBr) cm⁻¹: 3104 (aromatic C–H, str), 2920, 2841 (aliphatic C–H, str), 1662 (C=O, str), 1539 (C=N, str). ¹H-NMR (CDCl₃) δ (ppm): 2.54 (3H, s, 7-NCH₃), 2.82 (2H, t, J=5.82 Hz, 5-CH₂), 3.20 (2H, t, J=5.83 Hz, 6-CH₂), 3.68 (2H, s, 8-CH₂), 6.58 (1H, dd, J=3.48, 1.76 Hz, 4′-Ar-H of furan), 7.02 (1H, d, J=3.22 Hz, 5′-Ar-H of furan), 7.69 (1H, d, J=1.55 Hz, 3′-Ar-H of furan), 8.29 (1H, s, 2-H of pyrimidinone), 9.54 (1H, s, N=CH). EI-MS (m/z; %): 314 (M⁺; 62), 315 (M⁺+1; 12). Anal. Calcd for C₁₅H₁₄N₄O₂S: C, 57.31; H, 4.49; N, 17.82. Found: C, 57.72; H, 4.46; N, 16.18.

3-((2,4-Dichlorobenzylidene)amino)-7-methyl-5,6,7,8-tetrahydropyrido[4′,3′:4,5]thieno[2,3-d]pyrimidin-4(3H)-one (6k)

Yield, 0.32 g (82%). mp 190–192°C. IR (KBr) cm⁻¹: 3083 (aromatic C–H, str), 2938, 2783 (aliphatic C–H, str), 1681(C=O, str), 1582 (C=N, str). ¹H-NMR (CDCl₃) δ (ppm): 2.50 (3H, s, 7-NCH₃), 2.79 (2H, t, J=5.98 Hz, 5-CH₂), 3.19 (2H, t, J=5.56 Hz, 6-CH₂), 3.71 (2H, s, 8-CH₂), 7.34 (1H, dd, J=1.6, 2.5 Hz, 5′-Ar-H), 7.47 (1H, d, J=2 Hz, 3′-Ar-H), 8.13 (1H, d, J=8.2 Hz, 6′-Ar-H), 8.23 (1H, s, 2-H of pyrimidinone), 10.09 (1H, s, N=CH). ¹³C-NMR (CDCl₃) δ (ppm): 26.14, 45.44, 51.84, 53.73, 122.93, 127.74, 128.45, 129.60, 129.98, 130.30, 132.17, 136.80, 138.56, 145.80, 156.40, 158.37, 160.67. ESI-MS (m/z; %): 394 (M⁺+2; 35), 392 (M⁺; 100). Anal. Calcd for C₁₇H₁₄Cl2N₄OS: C, 51.92; H, 3.59; N, 14.25. Found: C, 51.72; H, 3.46; N, 14.48.

2,7-Dimethyl-3-(substituted-aryledineamino)-5,6,7,8-tetrahydropyrido[4′,3′:4,5]thieno[2,3-d]pyrimidin-4(3H)-one (7a–k)

General Method

A mixture of 5b (0.001 M) and appropriate aldehyde (0.001 M)) in catalytic amount of glacial acetic acid in 10 mL of absolute ethanol was heated under reflux for about 2–6 h. On cooling, the separated solid was filtered, washed with cold ethanol and recrystallized from acetic acid: ethanol to afford desired product.

3-(Benzylideneamino)-2,7-dimethyl-5,6,7,8-tetrahydropyrido[4′,3′:4,5]thieno[2,3-d]pyrimidin-4(3H)-one (7a)

Yield, 0.29 g (85%). mp 185–187°C. IR (KBr) cm⁻¹: 3066 (aromatic C–H, str), 2957 (aliphatic C–H, str), 1640 (C=O, str), 1547 (C=N, str). ¹H-NMR (CDCl₃) δ (ppm): 2.53 (3H, s, 7-NCH₃), 2.62 (3H, s, 2-CH₃ of pyrimidinone), 2.80 (2H, t, J=5.83 Hz, 5-CH₂), 3.16 (2H, t, J=5.81 Hz, 6-CH₂), 3.65 (2H, s, 8-CH₂), 7.51 (2H, t, J=7.38 Hz, 3′,5′-Ar-H), 7.56 (1H, dt, J=2.67, 1.85 Hz, 4′-Ar-H), 7.90 (2H, d, J=7.07 Hz, 2′,6′-Ar-H), 8.94 (1H, s, N=CH). EI-MS (m/z; %): 338 (M⁺; 31), 339 (M⁺+1; 6). Anal. Calcd for C₁₇H₁₆N₄OS: C, 62.94; H, 4.97; N, 17.27. Found: C, 63.06; H, 4.66; N, 17.16.

3-((4-Chlorobenzylidene)amino)-2,7-dimethyl-5,6,7,8-tetrahydropyrido[4′,3′:4,5]thieno[2,3-d]pyrimidin-4(3H)-one (7b)

Yield, 0.26 g (71%). mp 153–155°C. IR (KBr) cm⁻¹: 3069 (aromatic C–H, str), 2919 (aliphatic C–H, str), 1672 (C=O, str), 1546 (C=N, str). ¹H-NMR (CDCl₃) δ (ppm): 2.50 (3H, s, 7-NCH₃), 2.61 (3H, s, 2-CH₃ of pyrimidinone), 2.84 (2H, t, J=5.88 Hz, 5-CH₂), 3.18 (2H, t, J=5.91 Hz, 6-CH₂), 3.68 (2H, s, 8-CH₂), 7.39 (2H, d, J=7.89 Hz, 3′,5′-Ar-H), 7.50 (2H, d, J=7.98 Hz, 2′,6′-Ar-H), 10.42 (1H, s, N=CH). ¹³C-NMR (CDCl₃) δ (ppm): 22.52, 26.10, 45.45, 51.87, 53.74, 122.91, 129.29, 129.72, 130.15, 131.62, 132.12, 138.41, 145.68, 156.46, 160.81, 161.98. ESI-MS (m/z; %): 374 (M⁺+2; 33), 372 (M⁺; 100). Anal. Calcd for C₁₇H₁₅ClN₄OS: C, 56.90; H, 4.21; N, 15.61. Found: C, 56.52; H, 4.16; N, 15.16.

3-((4-Fluorobenzylidene)amino)-2,7-dimethyl-5,6,7,8-tetrahydropyrido[4′,3′:4,5]thieno[2,3-d]pyrimidin-4(3H)-one (7c)

Yield, 0.23 g (65%). mp 230–232°C. IR (KBr) cm⁻¹: 3069 (aromatic C–H, str), 2919 (aliphatic C–H, str), 1672 (C=O, str), 1601 (C=N, str). ¹H-NMR (CDCl₃) δ (ppm): 2.50 (3H, s, 7-CH₃), 2.59 (3H, s, 2-CH₃ of pyrimidinone), 2.77 (2H, t, J=6 Hz, 5-CH₂), 3.13 (2H, t, J=6 Hz, 6-CH₂), 3.63 (2H, s, 8-CH₂), 7.18 (2H, t, J=8.8 Hz, 3′,5′-ArH), 7.90 (2H, dd, J=5.6, 5.6 Hz, 2′,6′-Ar-H), 8.91 (1H, s, N=CH). ¹³C-NMR (CDCl₃) δ (ppm): 22.52, 25.97, 45.50, 51.94, 53.73, 116.14–116.36 (d), 120.85, 128.90–128.93 (d), 129.87, 130.24, 131.00–131.09 (d), 153.99, 155.51, 161.19, 164.19, 165.76, 166.72. EI-MS (m/z; %): 356 (M⁺; 38), 234 (M⁺-C₇H₄Cl₂N; 45), 192 (M⁺-C₉H₄Cl₂NO; 100). Anal. Calcd for C₁₇H₁₅FN₄OS: C, 59.63; H, 4.42; N, 16.36. Found: C, 59.22; H, 4.86; N, 16.06.

4-(((2,7-Dimethyl-4-oxo-5,6,7,8-tetrahydropyrido[4′,3′:4,5]thieno[2,3-d]pyrimidin-3(4H)-yl)imino)methyl)benzonitrile (7d)

Yield, 0.25 g (68%). mp 200–202°C. IR (KBr) cm⁻¹: 3050 (aromatic C–H, str), 2981 (aliphatic C–H, str), 2234 (CN, str), 1676 (C=O, str), 1550 (C=N, str). ¹H-NMR (CDCl₃) δ (ppm): 2.52 (3H, s, 7-NCH₃), 2.63 (3H, s, 2-CH₃ of pyrimidinone), 2.79 (2H, t, J=5.22 Hz, 5-CH₂), 3.14 (2H, t, J=5.54 Hz, 6-CH₂), 3.64 (2H, s, 8-CH₂), 7.79 (2H, d, J=8.82 Hz, 3′,5′-Ar-H), 8.01 (2H, d, J=8.89 Hz, 2′,6′-Ar-H), 9.22 (1H, s, N=CH). ¹³C-NMR (CDCl₃) δ (ppm): 22.64, 25.98, 45.48, 51.88, 53.69, 115.56, 118.02, 120.87, 129.02, 129.96, 130.68, 132.63, 136.97, 153.66, 155.65, 161.10, 163.32. ESI-MS (m/z; %): 364 (M⁺+1; 100). Anal. Calcd for C₁₉H₁₇N₅OS: C, 62.79; H, 4.71; N, 19.27. Found: C, 62.42; H, 4.86; N, 19.66.

3-((4-Methoxybenzylidene)amino)-2,7-dimethyl-5,6,7,8-tetrahydropyrido[4′,3′:4,5]thieno[2,3-d]pyrimidin-4(3H)-one (7e)

Yield, 0.31 g (82%). mp 173–175°C. IR (KBr) cm⁻¹: 3095 (aromatic C–H, str), 2921 (aliphatic C–H, str), 1684 (C=O, str) 1567 (C=N, str). ¹H-NMR (CDCl₃) δ (ppm): 2.53 (3H, s, 7-NCH₃), 2.60 (3H, s, 2-CH₃ of pyrimidinone), 2.80 (2H, t, J=5.82 Hz, 5-CH₂), 3.16 (2H, t, J=5.79 Hz, 6-CH₂), 3.66 (2H, s, 8-CH₂), 3.91 (3H, s, Ar-OCH₃), 7.01 (2H, d, J=8.81 Hz, 3′,5′-Ar-H), 7.86 (2H, d, J=8.80 Hz, 2′,6′-Ar-H), 8.75 (1H, s, N=CH). ¹³C-NMR (CDCl₃) δ (ppm): 22.66, 26.10, 45.46, 51.91, 53.76, 55.44, 114.43, 122.92, 125.47, 130.07, 130.57, 131.76, 145.28, 156.19, 160.93, 163.12, 164.29. EI-MS (m/z; %): 368 (M⁺ ; 18), 192 (M⁺-C₁₀H₉O₂N; 15), 57 (M⁺-C₁₆H₁₃N₃O₂S; 100). Anal. Calcd for C₁₉H₂₀N₄O₂S: C, 61.94; H, 5.47; N, 15.21. Found: C, 61.58; H, 5.12; N, 15.06.

3-((4-Hydroxybenzylidene)amino)-2,7-dimethyl-5,6,7,8-tetrahydropyrido[4′,3′:4,5]thieno[2,3-d]pyrimidin-4(3H)-one (7f)

Yield, 0.27 g (76%). mp 218–220°C. IR (KBr) cm⁻¹: 3250 (aromatic C–H, str), 2949 (aliphatic C–H, str), 1647 (C=O, str), 1546 (C=N, str). ¹H-NMR (CDCl₃) δ (ppm): 2.41 (3H, s, 7-NCH₃), 2.48 (3H, s, 2-CH₃ of pyrimidinone), 2.71 (2H, t, J=5.86 Hz, 5-CH₂), 2.97 (2H, t, J=5.80 Hz, 6-CH₂), 3.59 (2H, s, 8-CH₂), 6.98 (2H, d, J=5.14 Hz, 3′,5′-Ar-H), 7.84 (2H, d, J=8.32 Hz, 2′,6′-Ar-H), 8.67 (1H, s, N=CH), 10.47 (1H, s, Ar-OH). ¹³C-NMR (CDCl₃) δ (ppm): 22.51, 26.13, 45.48, 51.90, 53.77, 122.91, 129.32, 129.76, 130.18, 131.64, 132.15, 138.45, 145.70, 156.49, 160.85, 162.03. ESI-MS (m/z; %): 355 (M⁺+1; 100). Anal. Calcd for C₁₈H₁₈N₄O₂S: C, 61.00; H, 5.12; N, 15.81. Found: C, 61.38; H, 4.96; N, 16.12.

2,7-Dimethyl-3-((3,4,5-trimethoxybenzylidene)amino)-5,6,7,8-tetrahydropyrido[4′,3′:4,5]thieno[2,3-d]pyrimidin-4(3H)-one (7g)

Yield, 0.3 g (70%). mp 198–200°C. IR (KBr) cm⁻¹: 3059 (aromatic C–H, str), 2937 (aliphatic C–H, str), 1672 (C=O, str), 1576 (C=N, str). ¹H-NMR (CDCl₃) δ (ppm): 2.53 (3H, s, 7-NCH₃), 2.62 (3H, s, 2-CH₃ of pyrimidinone), 2.80 (2H, t, J=5.81 Hz, 5-CH₂), 3.16 (2H, t, J=5.76 Hz, 6-CH₂), 3.66 (2H, s, 8-CH₂), 3.94 (6H, s, 3′,5′-Ar-OCH₃), 3.96 (3H, s, 4′-Ar-OCH₃), 7.16 (2H, s, 2′,6′-Ar-H), 8.77 (1H, s, N=CH). ¹³C-NMR (CDCl₃) δ (ppm): 22.48, 25.97, 45.47, 51.93, 53.71, 56.29, 60.99, 106.28, 120.81, 127.60, 129.85, 130.15, 142.29, 153.36, 153.60, 155.49, 161.21, 167.28. ESI-MS (m/z; %): 429 (M⁺+1; 100). Anal. Calcd for C₂₁H₂₄N₄O₄S: C, 58.86; H, 5.65; N, 13.07. Found: C, 58.58; H, 5.26; N, 13.12.

2,7-Dimethyl-3-((2-nitrobenzylidene)amino)-5,6,7,8-tetrahydropyrido[4′,3′:4,5]thie no[2,3-d]pyrimidin-4(3H)-one (7h)

Yield, 0.3 g (78%). mp 154–156°C. IR (KBr) cm⁻¹: 3019 (aromatic C–H, str), 2932 (aliphatic C–H, str), 1686 (C=O, str), 1524 (C=N, str). ¹H-NMR (CDCl₃) δ (ppm): 2.51 (3H, s, 7-NCH₃), 2.62 (3H, s, 2-CH₃ of pyrimidinone), 2.79 (2H, t, J=6 Hz, 5-CH₂), 3.24 (2H, t, J=6 Hz, 6-CH₂), 3.67 (2H, s, 8-CH₂), 7.66 (1H, dt, J=8.12 Hz, 4′-Ar-H), 7.80 (1H, dt, J=8.12 Hz, 5′-Ar-H), 8.20 (2H, dd, J=8.34, 1.46 Hz, 3′,6′-Ar-H), 9.44 (1H, s, N=CH). ESI-MS (m/z; %): 384 (M⁺+1; 26), 340 (M⁺−C₂H₃O; 100). Anal. Calcd for C₁₈H₁₇N₅O₃S: C, 56.38; H, 4.47; N, 18.27. Found: C, 56.48; H, 4.91; N, 17.98.

2,7-Dimethyl-3-((thiophen-2-ylmethylene)amino)-5,6,7,8-tetrahydropyrido[4′,3′:4,5]thieno[2,3-d]pyrimidin-4(3H)-one (7i)

Yield, 0.24 g (70%). mp 178–180°C. IR (KBr) cm⁻¹: 3051 (aromatic C–H, str), 2938, 2785 (aliphatic C–H, str), 1683 (C=O, str), 1581 (C=N, str). ¹H-NMR (CDCl₃) δ (ppm): 2.53 (3H, s, 7-NCH₃), 2.62 (3H, s, 2-CH₃ of pyrimidinone), 2.80 (2H, t, J=5.82 Hz, 5-CH₂), 3.16 (2H, t, J=5.80 Hz, 6-CH₂), 3.65 (2H, s, 8-CH₂), 7.18 (1H, dd, J=5.00, 3.70 Hz, 4′-Ar-H), 7.58 (1H, dd, J=3.67, 1.01 Hz, 5′-Ar-H), 7.61 (1H, td, J=4.97, 0.90 Hz, 3′-Ar-H), 9.13 (1H, s, N=CH). ¹³C-NMR (CDCl₃) δ (ppm): 22.51, 25.76, 49.83, 51.59, 61.96, 112.49, 118.66, 127.33, 128.40, 129.10, 130.25, 137.92, 146.85, 153.55, 155.02, 155.53, 161.16. ESI-MS (m/z; %): 315 (M⁺−CO; 92), 344.9 (M⁺+1; 45). Anal. Calcd for C₁₆H₁₆N₄OS₂: C, 55.79; H, 4.68; N, 16.27. Found: C, 55.88; H, 4.61; N, 16.32.

3-((Furan-2-ylmethylene)amino)-2,7-dimethyl-5,6,7,8-tetrahydropyrido[4′,3′:4,5]thieno[2,3-d]pyrimidin-4(3H)-one (7j)

Yield, 0.26 g (79%). mp 172–174°C. IR (KBr) cm⁻¹: 3123 (aromatic C–H, str), 2936, 2803 (aliphatic C–H, str), 1671 (C=O, str), 1557 (C=N, str). ¹H-NMR (CDCl₃) δ (ppm): 2.51 (3H, s, 7-NCH₃), 2.60 (3H, s, 2-CH₃ of pyrimidinone), 2.77 (2H, t, J=5.98 Hz, 5-CH₂), 3.13 (2H, t, J=5.61 Hz, 6-CH₂), 3.62 (2H, s, 8-CH₂), 6.60 (1H, t, J=1.22 Hz, 4′-Ar-H of furan), 7.26 (1H, d, J=3.22 Hz, 3′-Ar-H of furan), 7.68 (1H, d, J=3.22 Hz, 5′-Ar-H of furan), 8.77 (1H, s, N=CH). ESI-MS (m/z; %): 329 (M⁺+1; 100). Anal. Calcd for C₁₆H₁₆N₄O₂S: C, 58.52; H, 4.91; N, 17.06. Found: C, 58.48; H, 4.61; N, 16.72.

3-((2,4-Dichlorobenzylidene)amino)-2,7-dimethyl-5,6,7,8-tetrahydropyrido[4′,3′:4,5]thieno[2,3-d]pyrimidin-4(3H)-one (7k)

Yield, 0.32 g (80%). mp 181–183°C. IR (KBr) cm⁻¹: 3083 (aromatic C–H, str), 2938 (aliphatic C–H, str), 1681 (C=O, str), 1582 (C=N, str). ¹H-NMR (CDCl₃) δ (ppm): 2.54 (3H, s, 7-NCH₃), 2.63 (3H, s, 2-CH₃ of pyrimidinone), 2.80 (2H, t, J=5.79 Hz, 5-CH₂), 3.17 (2H, t, J=5.82 Hz, 6-CH₂), 3.66 (2H, s, 8-CH₂), 7.39 (1H, dd, J=8.51, 1.50 Hz, 5′-Ar-H), 7.51 (1H, d, J=1.99 Hz, 3′-Ar-H), 8.17 (1H, d, J=8.48 Hz, 6′-Ar-H), 9.42 (1H, s, N=CH). ¹³C-NMR (CDCl₃) δ (ppm): 22.51, 26.14, 45.44, 51.84, 53.73, 122.93, 127.74, 128.45, 129.60, 129.98, 130.30, 132.17, 136.80, 138.56, 145.80, 156.40, 158.37, 166.67. EI-MS (m/z; %): 408 (M⁺+2, 14), 406 (M⁺; 20), 234 (M⁺−C₇H₄Cl₂N; 45), 192 (M⁺−C₉H₄Cl₂NO; 100). Anal. Calcd for C₁₈H₁₆Cl₂N₄OS: C, 53.08; H, 3.96; N, 13.76. Found: C, 52.86; H, 3.67; N, 14.52.

Antibacterial Activity

The synthesized final compounds were screened for their in vitro antibacterial activity against mentioned strains of Gram-positive and Gram-negative bacteria. The MIC values were determined by employing similar techniques of our studies,⁵⁾ and the results were compared with standard (streptomycin). The MIC values were expressed in micro moles (µM) and the results were listed in Table 1.

Antimycobacterial Activity

The MTB H37Rv and an INH-resistant clinical sample of Mycobacteria were used for screening and the MIC values were determined using a broth micro dilution method as described in our previous protocols.⁵⁾ The stock solution of all compounds was prepared in the concentration of 1 mg/mL dissolved in DMF in water (25% v/v). About ten test concentrations ranging from 1000–1 µg/mL were used to assess the efficacy of each compound.

Molecular Docking

The molecular docking studies were performed using Molegro Virtual Docker (MVD) tools24 on targeted protein MTB-PS (PDB: 3IVX).

Acknowledgments

One of the authors (MN) thanks UGC-BSR (No. F.7-106/2007) for providing a merit scholarship. The authors are thankful to the Principal, University College of Pharmaceutical Sciences (UCPSc), Kakatiya University for providing facilities and also Principal and Management, Sri Shivani College of Pharmacy, Warangal for permitting to carry out the biological activity.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

References

• 1) WHO, “Global tuberculosis report.”: ‹http://www.who.int/tb/publications›, 2014.
• 2) Szumowski J. D., Lynch J. B., Drug Des. Devel. Ther., 9, 677–682 (2015).
• 3) Salvador M. K., Baraldi P. G., Romagnoli R., Drug Discov. Today. Technol., 10, 285–296 (2013).
• 4) Lienhardt C., Vernon A., Raviglione M. C., Curr. Opin. Pulm. Med., 16, 186–193 (2010).
• 5) Malothu N., Bhandaru J. S., Kulandaivelu U., Jojula M., Adidala R. R., Umadevi K. R., Dusthackeer A. V. N., Kaki V. R., Akkinepally R. R., Bioorg. Med. Chem. Lett., 26, 836–840 (2015).
• 6) Robert C. R., Subramaniam A., Ellen F., Judith V. H., Cecil D. K., Clinton M., Lynn R., Melinda I. S., Elizabeth T., Lucile W. E., Wei Z., John A. S. III, Tuberculosis, 92, 72–83 (2012).
• 7) Rashmi P., Nargund L. V. G., Hazra K., Chandra J. N. N., S. Arch. Pharm. Chem. Life Sci., 344, 459–465 (2011).
• 8) Narayana B., Ashalatha B. V., Raj K. K. V., Kumari N. S., Indian J. Chem., 45B, 2696–2703 (2006).
• 9) Aurelio L., Valant C., Figler H., Flynn B. L., Linden J., Sexton P. M., Christopoulos A., Scammell P. J., Bioorg. Med. Chem., 17, 7353–7361 (2009).
• 10) Puterova Z., Krutosikova A., Vegh D., ARKIVOC, 1, 209–246 (2010).
• 11) El-Kashef H., Farghaly A. R., Al-Hazmi A., Terme T., Vanelle P., Molecules, 15, 2651–2666 (2010).
• 12) Al-Taisan K. M., Al-Hazimi H. M. A., Al-Shihry S. S., Molecules, 15, 3932–3957 (2010).
• 13) Nitinkumar S. S., Ravi S. L., Imtiyaz A. M. K., J. Chem. Sci., 121, 301–307 (2009).
• 14) Chambhare R. V., Khadse B. G., Bobde A. S., Bahekar R. H., Eur. J. Med. Chem., 38, 89–100 (2003).
• 15) Dewal M. B., Wani A. S., Vidaillac C., Oupický D., Rybak M. J., Firestine S. M., Eur. J. Med. Chem., 51, 145–153 (2012).
• 16) Raghavendra N. M., Thampi P., Gurubasavarajaswamy P. M., Sriram D., Arch. Pharm. Chem. Life Sci, 340, 635–641 (2007).
• 17) Tran T., Saheba E., Arcerio A. V., Chavez V., Li Q., Martinez L. E., Primm T. P., Bioorg. Med. Chem., 12, 4809–4813 (2004).
• 18) Wang S., Eisenberg D., Protein Sci., 12, 1097–1108 (2003).
• 19) Hung A. W., Silvestre H. L., Wen S., Ciulli A., Blundell T. L., Abell C., Angew. Chem. Int. Ed., 48, 8452–8456 (2009).