Synthesis and Antibacterial Activities of Different Five-Membered Heterocyclic Rings Incorporated with Pyridothienopyrimidine.

Certain pyridothienopyrimidine derivatives exhibit antiatheroscleorotic, antibacterial, antiviral, antidepressant, antidiabetic, antihypertensive, anticancer, antihistaminic, antiallergic, anti-inflammatory, spasmolytic, analgesic, and neurotropic activities. 4-Hydrazino-7,9-dimethylpyrido[3',2':4,5]thieno[3,2-d]pyrimidine (1) is a reported pyridothienopyrimidine derivative. In the current study, (1) has been reacted with different reagents to obtain 12 new pyridothienopyrimidine derivatives. The newly synthesized five-membered heterocyclic rings incorporated with pyridothienopyrimidines have been screened for their antibacterial activities. The results encourage further studies on other possible biological activities.

Introduction

As an example of a triheterocyclic system, pyridothienopyrimidine has interesting biological activities among its derivatives which have attracted much attention. Pyridothienopyrimidines are a special class of thienopyridine derivatives. There are two regioisomeric structures, namely, 5H-thieno[2,3,4-e,d]pyrido[4,3-d]pyrimidine (Figure A) or pyrido[3′,2′:4,5] thieno[3,2-d]pyrimidine (Figure B). These two structures give all pyridothienopyrimidine derivatives which are prepared by annulation of thieno[2,3-b] pyridines.[1]

Figure 1

Chemical structures of 5H-thieno[2,3,4-e,d]pyrido[4,3-d]pyrmidine (A) and pyrido[3′,2′:4,5] thieno[3,2-d]pyrimidine (B).

Among all fused thienoazines, pyridothienopyrimidine derivatives have been studied the most. These derivatives act as bactericides[2−6] fungicides,[2−6] antiprotozoals,[7] or immunomodulators; exhibit antianaphylactic, antiallergic,[8] anticonvulsive,[9] analgetic, or anti-inflammatory activities;[10,11] and are used as intermediate products in chemical pharmaceutical industry.[1]

In addition to antimicrobial,[2−6] anticonvulsant,[9] and anticancer activities[12−17] of pyridothienopyrimidine derivatives, recent studies showed that they have the ability to specifically activate testicular steroidogenesis without affecting thyroid functions.[18] They also have inhibitory effects on some closely linked factors to human malignancies such as cellular mesenchymal–epithelial transition factor;[19] the Moloney murine leukemia virus-1 (pim-1)[20] proviral integration site; some signaling pathways that play an important role in tumorigenesis;[21] VEGFR-2 receptors to reduce angiogenesis or lymphangiogenesis;[22] and act as alpha-1 blockers[23] that are mainly used as a prescribed drugs for benign prostatic hyperplasia, manage hypertension, and treatment of posttraumatic stress disorder.

Within this respect, the current study has aimed to examine the chemistry of 4-hydrazino-7,9-dimethylpyrido[3′,2′:4,5]thieno[3,2-d]pyrimidine[24] to be used as a starting material for the synthesis of some new pyrazole, pyrazolopyrimidine, and thiazole derivatives and investigate their antibacterial activities.

Results and Discussion

Ethoxymethylene malononitrile and 4-hydrazino-7,9-dimethylpyrido[3′,2′:4,5]thieno[3,2-d]pyrimidine (1) were refluxed in absolute ethanol to give 5-amino-1-(7,9-dimethylpyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4-yl)-1H-pyrazole-4-carbonitrile (2) in 58% yield. Nitrile derivative (2) was refluxed in 80% formic acid and/or acetic acid to afford 1-(7,9-dimethylpyrido[3′,2′:4,5] thieno[3,2-d]pyrimidin-4-yl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (3) and 1-(7,9-dimethylpyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4-yl)-6-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (4), respectively. A mixture of ethoxymethylene cyanoactate and (1) was refluxed in absolute ethanol to give 2-(7,9-dimethylpyrido [3′,2′:4,5]thieno[3,2-d]pyrimidin-4-yl)-3-oxo-2,3-dihydro-1H-pyrazole-4-carbonitrile (5) in 72% yield. Acid hydrolysis occurred for (5) which was refluxed in 10% sulfuric acid to give 2-(7,9-dimethylpyrido [3′,2′:4,5]thieno[3,2-d]pyrimidin-4-yl)-3-oxo-2,3-dihydro-1H-pyrazole-4-carboxylic acid (6) in 41% yield. Diethyl ethoxymethylenemalonate and (1) were refluxed in absolute ethanol to obtain a powder of ethyl 2-(7,9-dimethylpyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4-yl)-3-oxo-2,3-dihydro-1H-pyrazole-4-carboxylate (7) in 95% yield. Hydrazinolysis of (7) gave a powder of 2-(7,9-dimethylpyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4-yl)-3-oxo-2,3-dihydro-1H-pyrazole-4-carbohydrazide (8) in 53% yield. Treatment of (1) with phenyl isothiocyanate afforded a solid product of 2-(7,9-dimethylpyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4-yl)-N-phenylhydrazinecarbothioamide (9) in 58% yield. A mixture of anhydrous sodium acetate in absolute ethanol was prepared to obtain (9), and finally phenacyl bromide and/or ethylbromoacetate were/was added to get a solid product of (2E)-3,4-diphenyl-1,3-thiazol-2(3H)-one (7,9-dimethylpyrido [3′,2′:4,5]thieno[3,2-d]pyrimidin-4-yl)hydrazone (10) in 71% yield and (2E)-3-phenyl-1,3-thiazolidine-2,4-dione 2-[(7,9-dimethylpyrido [3′,2′:4,5]thieno[3,2-d]pyrimidin-4-yl)hydrazone] (11) in 71% yield. A mixture of acetylacetone and/or ethylacetoacetate and (1) was refluxed in absolute ethanol to give 4-(3,5-dimethyl-1H-pyrazol-1-yl)-7,9-dimethylpyrido[3′,2′:4,5]thieno [3,2-d]pyrimidine (12) in 78% yield and 2-(7,9-dimethylpyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4-yl)-5-methyl-2,4-dihydro-3H-pyrazol-3-one (13) in 84% yield. The structures of (2–13) were established by infrared (IR) as well as proton nuclear magnetic resonance (1H NMR) spectra. All reactions are illustrated in Scheme . Physical properties and structural data for the newly synthesized pyridothienopyrimidine derivatives are shown in Table .

Scheme 1

Synthesis of New Pyridothienopyrimidine Derivatives

Table 1

Physical Properties and Structural Data for the Synthesized Compounds

comp. no.	mp (°C)	yield (%)	mol. formula	MW (g/mol)
2	127–129	58	C15H11N7S	321.36
3	213–215	55	C16H11N7SO	349.37
4	228–231	45	C17H13N7SO	363.40
5	220–222	72	C15H10N6SO	322.34
6	271–273	41	C15H11N5SO3	341.34
7	203–205	95	C17H15N5SO3	369.40
8	191–193	53	C15H13N7SO2	355.37
9	181–183	58	C18H16N6S2	380.49
10	207–209	71	C26H20N6S2	480.61
11	181–183	71	C20H16N6S2O	420.51
12	219–221	78	C16H15N5S	309.39
13	237–239	84	C15H12N5SO	310.35

A screening for antibacterial activities of the newly synthesized pyridothienopyrimidine derivatives has been performed against three microorganisms (Table ).

Table 2

MIC (in μg/mL) of the Title Compoundsa

comp.	B. subtilis (Gram +ve)	P. aeruginosa (Gram −ve)	Streptomyces species (actinomycetes)
penicillin	31	46	33
2	100	125	b
3	100	100	250
4	100	250	75
5		75	75
6	75	125	100
7	75	100	75
8	75	100	75
9	125	500	250
10	250		125
11	75		125
12	125	125	500
13	250	125

The negative control DMSO showed no activity.

Totally inactive (MIC > 500 μg/mL).

Materials and Methods

Synthetic Methods, Analytical and Spectral Data

A Büchi melting point apparatus was used to measure the newly synthesized pyridothienopyrimidine derivatives’ melting points, which were uncorrected. A Bruker-Vector22 spectrometer (Bruker, Bremen, Germany) was used to record IR spectra (KBr). A Varian Gemini spectrometer (300 MHz, DMSO-d6) was used to record 1H NMR with tetramethylsilane (TMS) as an internal reference. The chemical shifts are expressed in δ scale (ppm) relative to TMS as a reference, and the coupling constants (J values) are given in hertz (Hz). Analytical thin layer chromatography was performed to monitor the progress of the reactions using aluminum silica gel 60 F245 plates (Merck, Darmstadt, Germany). Antimicrobial activities were screened at the College of Medicine, Dar Al Uloom University, Riyadh, Kingdom of Saudi Arabia.

Experimental Section

5-Amino-1-(7,9-dimethylpyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4-yl)-1H-pyrazole-4-carbonitrile (2)

A mixture of ethoxymethylene malononitrile (0.55 g, 4 mmol) and 4-hydrazino-7,9-dimethylpyrido[3′,2′:4,5]thieno[3,2-d]pyrimidine (1) (0.98 g, 4 mmol) was refluxed in absolute ethanol (30 mL) for 10 h. The resulting precipitate was filtered off with suction, washed with ethanol, and recrystallized from ethanol to give a colored powder of (2) (58%), mp 127–129 °C. IR (KBr) ν cm–1: 3350–3280 (NH2), 1940, 1888 (CH aliphatic), 2230 (C≡N) cm–1. 1H NMR (DMSO-d6) δ in ppm: 2.49, 2.60 (2s, 6H, 2CH3), 4.72 (br s, 2H, NH2), 7.32 (s, 1H, Ar-H), 8.15 (s, 1H, Ar-H), 9.11 (s, 1H, Ar-H).

1-(7,9-Dimethylpyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4-yl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (3)

Nitrile derivative (2) (0.31 g, 1 mmol) was refluxed in 80% formic acid (10 mL) for 11 h and then poured on cold water. The solid product of (3) (55%) was collected after filtration with suction and recrystallized from ethanol, mp 213–215 °C. IR (KBr) ν cm–1: 2920, 2890 (CH aliphatic), 1680 (C=O), 3200 (NH). 1H NMR (DMSO-d6) δ in ppm: 2.48, 2.56 (2s, 6H, 2CH3), 7.00 (s, 1H, Ar-H), 7.28 (s, 1H, Ar-H), 8.97 (s, 1H, Ar-H), 9.25 (br s, 1H, NH).

1-(7,9-Dimethylpyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4-yl)-6-methyl-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (4)

Nitrile derivative (2) (0.31 g, 1 mmol) was refluxed in 80% acetic acid (10 mL) and HCl (0.5 mL) for 10 h and then poured on cold water. The solid product of (4) (45%) was collected after filtration with suction and recrystallized from ethanol, mp 228–231 °C. IR (KBr) ν cm–1: 2920, 2890 (CH aliphatic), 1680 (C=O), 3200 (NH). 1H NMR (DMSO-d6) δ in ppm: 2.49, 2.51, 2.86 (3s, 9H, 3CH3), 6.17 (s, 1H, Ar-H), 7.11 (s, 1H, Ar-H), 8.20 (s, 1H, Ar-H), 9.10 (br s, 1H, NH).

2-(7,9-Dimethylpyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4-yl)-3-oxo-2,3-dihydro-1H-pyrazole-4-carbonitrile (5)

A mixture of ethoxymethylene cyanoactate (0.68 g, 4 mmol) and (1) (0.98 g, 4 mmol) was refluxed in absolute ethanol (30 mL) for 8 h. The resulting precipitate was filtered off with suction, washed with ethanol, and recrystallized from ethanol to give (5) (72%), mp 220–222 °C. IR (KBr) ν cm–1: 2980, 2975 (CH aliphatic), 1665 (C=O), 2120 (C≡N), 3320–3250 (NH). 1H NMR (DMSO-d6) δ in ppm: 2.62, 2.97 (2s, 6H, 2CH3), 7.27, 7.81, 8.88 (3s, 3H, Ar-H), 9.14 (br s, 1H, NH).

2-(7,9-Dimethylpyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4-yl)-3-oxo-2,3-dihydro-1H-pyrazole-4-carboxylic Acid (6)

Acid hydrolysis occurred for (5) (0.32 g, 1 mmol) which was refluxed in 10% sulfuric acid (10 mL) for 12 h. The reaction mixture was poured on cold water, filtered off with suction, and dried to obtain a powder of (6) (41%), mp 271–273 °C. IR (KBr) ν cm–1: 3300–3200 (NH, OH), 1670 (C=O).

Ethyl 2-(7,9-Dimethylpyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4-yl)-3-oxo-2,3-dihydro-1H-pyrazole-4-carboxylate (7)

A mixture of diethyl ethoxymethylenemalonate (1.3 g, 6 mmol) and (1) (0.98 g, 4 mmol) was refluxed in absolute ethanol (30 mL) for 15 h. The resulting precipitate was filtered off with suction, washed with ethanol, and recrystallized from ethanol to give a powder of (7) (95%), mp 203–205 °C. IR (KBr) ν cm–1: 3270 (NH); 1890, 1870 (CH aliphatic), 1730, 1680 (2C=O), 1590 (C=N). 1H NMR (DMSO-d6) δ in ppm: 1.02 (t, 3H, J = 1.5 Hz, CHCH2) 2.49, 2.58 (2s, 6H, 2CH3), 4.28 (q, 2H, J = 11 Hz, CH3CH), 7.16 (s, 1H, Ar-H), 8.66 (s,1H, Ar-H), 9.87 (s, 1H, Ar-H).

2-(7,9-Dimethylpyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4-yl)-3-oxo-2,3-dihydro-1H-pyrazole-4-carbohydrazide (8)

A mixture of hydrazine hydrate (0.25 g, 5 mmol) and (7) (0.36 g, 1 mmol) was refluxed in absolute ethanol (30 mL) for 6 h. The reaction mixture was cooled, filtered off, and then dried to give a powder of (8) (53%), mp 191–193 °C. IR (KBr) ν cm–1: 3300–3200 (NH, NH2), 1670, 1663 (2C=O).

2-(7,9-Dimethylpyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4-yl)-N-phenylhydrazinecarbothioamide (9)

A mixture of phenyl isothiocyanate (0.54 g, 4 mmol) and (1) (0.98 g, 4 mmol) was refluxed in absolute ethanol for 6 h. The reaction mixture was cooled and then filtered off with suction to get a solid product of (9) (58%), mp 181–183 °C. IR (KBr) ν cm–1: 3310, 3270 (NH), 1610 (C=N), 1507 (C=S). 1H NMR (DMSO-d6) δ in ppm: 2.49, 2.59 (s, 6H, 2CH3), 6.96–7.48 (m, 7H, Ar-H); 9.10 (br s, 1H, NH), 9.90 (br s, 1H, NH), 10.09 (br s, 1H, NH).

(2E)-3,4-Diphenyl-1,3-thiazol-2(3H)-one (7,9-Dimethylpyrido [3′,2′:4,5]thieno[3,2-d]pyrimidin-4-yl)hydrazone (10)

A mixture of anhydrous sodium acetate (0.33 g, 4 mmol) and absolute ethanol (10 mL) was prepared to obtain (9) (0.38 g, 1 mmol), and finally phenacyl bromide (0.22 g, 1 mmol) was added. The whole mixture was refluxed for 9 h. The mixture was cooled and then filtered off with suction to get a solid product of (10) (71%), mp 207–209 °C. IR (KBr) ν cm–1: 3306 (NH), 2950, 2910 (CH aliphatic), 1600, 1580 (C=C). 1H NMR (DMSO-d6) δ in ppm: 2.43, 2.49 (2s, 6H, 2CH3), 6.68 (s, 1H, Ar-H), 6.95–8.00 (m, 12H, Ar-H).

(2E)-3-Phenyl-1,3-thiazolidine-2,4-dione 2-[(7,9-dimethylpyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4-yl)hydrazone] (11)

A mixture of anhydrous sodium acetate (0.33 g, 4 mmol) and absolute ethanol (10 mL) was prepared to obtain (9) (0.38 g, 1 mmol), and finally ethylbromoacetate (0.29 g, 1 mmol) was added. The above mixture was refluxed for 9 h. The mixture was cooled and then filtered off with suction to get a solid product of (11) (71%), mp 181–183 °C. IR (KBr) ν cm–1: 3280 (NH), 2980, 2897 (CH aliphatic), 1650 (C=O), 1590 (C=N). 1H NMR (DMSO-d6) δ in ppm: 2.49, 2.52 (2s, 6H, 2CH3), 4.16 (s, 2H, CH2), 6.96–8.00 (m, 7H, Ar-H), 10.03 (br s, 1H, NH).

4-(3,5-Dimethyl-1H-pyrazol-1-yl)-7,9-dimethylpyrido[3′,2′:4,5]thieno [3,2-d]pyrimidine (12)

A mixture of acetylacetone (0.8 g, 8 mmol) and (1) (0.98 g, 4 mmol) was refluxed in absolute ethanol (30 mL) for 7 h. The excess of ethanol was removed under reduced pressure. The resulting precipitate was filtered off with suction, washed with ethanol, and recrystallized from ethanol to give a colored powder of (12) (78%), mp 219–221 °C. IR (KBr) ν cm–1: 2950, 2980 (CH aliphatic), 1590 (C=N). 1H NMR (DMSO-d6) δ in ppm: 2.29, 2.59, 2.85, 2.91 (4s, 12H, 4CH3), 6.28 (s, 1H, Ar-H), 9.04 (s, 1H, Ar-H).

2-(7,9-Dimethylpyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4-yl)-5-methyl-2,4-dihydro-3H-pyrazol-3-one (13)

A mixture of ethylacetoacetate (0.78 g, 6 mmol) and (1) (0.98 g, 4 mmol) was refluxed in absolute ethanol (30 mL) for 9 h. The resulting precipitate was filtered off with suction, washed with ethanol, and recrystallized from ethanol to give a powder of (13) (84%), mp 237–239 °C. IR (KBr) ν cm–1: 1997, 1899 (CH aliphatic), 1670 (C=O), 1650 (C=N). 1H NMR (DMSO-d6) δ in ppm: 2.08, 2.93, 2.58 (3s, 9H, 3CH3), 3.67 (s, 2H, CH2), 7.22 (s, 1H, Ar-H), 8.67 (s, 1H, Ar-H).

Antimicrobial Screening

Preparation of the Sample

Dimethyl sulfoxide (DMSO) (12.5%) solvent was used to dissolve each of the test compounds and standards at 500 μg/mL concentration. Further diluted compounds and standards were prepared at the required quantities in the test medium.

Microorganisms’ Culture

Reference cultures of Bacillus subtilis (ATCC-6633) (Gram-positive), Pseudomonas aeruginosa (ATCC-27853) (Gram-negative), and Streptomyces species (actinomycetes) were supplied from the Microbiology Department, College of Medicine, Dar Al Uloom University, Riyadh, Kingdom of Saudi Arabia as the bacterial strains for determining antibacterial activities of the newly synthesized pyridothienopyrimidine derivatives. A Mueller–Hinton agar (MHA) medium (Oxoid Chemical Co., UK) was used to maintain the bacterial strains for 24 h at 37 °C. A formed layer of about 3–4 mm thickness was obtained by melting the medium on a water bath, inoculating with 0.5 mL of the culture of the specific microorganism and poured into sterile Petri dishes. The layer was allowed to cool and harden. With the aid of a cork-borer, cups of about 10 mm diameter were produced.[25]

Agar Diffusion Technique

Newly synthesized pyridothienopyrimidine derivatives were screened for their antibacterial activities against B. subtilis (Gram-positive), P. aeruginosa (Gram-negative), and Streptomyces species (actinomycetes) using MHA medium (17.5 g casein hydrolysate, 1.5 g soluble starch, and 1000 mL beef extract). A stock solution of each synthesized compound (500 μg/mL) in DMSO was prepared, and graded quantities of the test compounds were incorporated in the specified quantity of sterilized liquid MHA medium. Different concentrations of pyridothienopyrimidine derivatives in dimethylformamide were placed separately in cups in the agar medium. Overnight incubation was applied for all plates at 37 °C. The measurement of inhibition zones was performed after 24 h. The minimum inhibitory concentration (MIC) was defined as the intercept of the grave of logarithm concentrations versus diameter of the inhibition zones.[26,27]

The antibacterial activities of the newly synthesized pyridothienopyrimidine derivatives have been screened against three microorganisms; B. subtilis (ATCC-6633) (Gram-positive), P. aeruginosa (ATCC-27853) (Gram-negative), and Streptomyces species (actinomycetes) (Table ).

The results of the antibacterial activities screening have shown that compounds 6, 7, 8, and 11 have the highest activity against B. subtilis with MIC values of 75 μg/mL followed by compounds 2, 3, and 4. Compound 5 has the highest inhibition power against P. aeruginosa. Among the series of screened derivatives, compounds 4, 5, 7, and 8 are the most active against Streptomyces species with MIC values of 75 μg/mL. In addition, there are compounds that have shown limited biological activities and others that have not had any antibacterial activity. The MIC values of all screened compounds are shown in Table .