INTRODUCTION:

Heterocyclic chemistry is a branch which is inseparable from mankind because human are totally dependent on the drugs derives from heterocyclic rings. Much attention has paid to the synthesis of nitrogen containing heterocyclic compounds like benzodiazepine mainly due to their broad spectrum of biological and pharmacological activities.

1, 5-Benzodiazepines are bi cyclic compounds with two nitrogen atoms at 1 and 5 positions in a seven membered ring fused to a benzene ring. Benzo di azepines have attracted greater attention as an important class of heterocyclic compounds in the field of drugs and pharmaceuticals. This nucleus is a pharmacophoric scaffold and represent a class of heterocycles with a wide range of biological applications[1].Many of them are widely used as anticonvulsant, antianxiety, sedative, anti depressive, hypnotic and neuroleptic agents[2-5]. Some heterocycles containing benzodiazepines moiety were reported to possess anti inflammatory [6], anti-viral [7], anti-HIV-1[8], antimicrobial[9] and antitumor[10] activities. Other than their biological importance, benzodiazepines are valuable synthons for the preparation of fused ring compounds, such as triazolo[11], thiazolo[12], imidazo[13] and pyrimido-benzodiazepines[14]. It has been noticed that introduction of an additional ring to the benzodiazepine core tends to exert profound influence in conferring novel biological activities in these molecules[11-15]. Although many methods for synthesizing benzodiazepine ring systems have been reported, they continue to receive a great deal attention [16-20].

The core structure of benzodiazepines. "R" labels denote common locations of side chains, which give different benzodiazepines their unique properties [21-22]. A benzodiazepine (sometimes called "benzo"; often abbreviated "BZD") is a psychoactive drug whose core chemical structure is the fusion of a benzene ring and a di azepine ring. The first benzodiazepine, chlordiazepoxide (Librium), was discovered accidentally by Leo Sternbach in 1955, and made available in 1960 by Hoffmann– La Roche, which has also marketed diazepam (Valium) since 1963[23]. Benzodiazepines enhance the effect of the neurotransmitter gamma-amino butyric acid (GABA-A), resulting in sedative, hypnotic (sleep-inducing), anxiolytic (anti anxiety), anticonvulsant, and muscle relaxant properties; also seen in the applied pharmacology of high doses of many shorter-acting benzodiazepines are amnesic-dissociative actions. These properties make benzodiazepines useful in treating anxiety, insomnia, agitation, seizures, muscle spasms, alcohol withdrawal and as a premedication for medical or dental procedures.

1, 5-Benzodiazepines are bicyclic compounds with two nitrogen atoms at 1 and 5 positions in a seven membered ring fused to a benzene ring [Fig 1]. Basically 1, 5- benzodiazepines are the 2, 3-benzo annelated derivatives of 1, 4-diazepines [24].

Fig. 1,5 benzodiazepine

Benzodiazepines are numbered as shown in figure 2

Fig 2

Biological Importance of 1, 5-Benzodiazepines:

The ascendancy of the 1, 5-benzodiazepines in chemical literature is doubtless a consequence of their easy accessibility and the activity they show against a variety of targets including peptide hormones (such as CCK), interleukin converting enzymes (ICE), and potassium blockers (IK). The enormous amount of research work that has been conducted in the pharmaceutical laboratories on these compounds during last several decades derives its inspiration from the discovery of the remarkable tranquilizing, CNS depressants [25], anti-inflammatory [26], antispasmodic, ant depressive [27], antifungal, antibacterial, antifeedant, analgesic, anticonvulsant, muscle relaxant, hypnotic and sedative activity etc. observed with certain members of this heterocyclic system. of particular importance are the substituted and tricyclic derivatives which are not only used as anxiolytic or antineoplastic agents but their novel application is continuously emerging. The recent demonstration that some of their derivatives can serve as potential agents in the control and treatment of AIDS has stimulated further interest in these compounds from yet another perspective.

Combinatorial strategy is used in the design and synthesis of 1, 5-benzodiazepine derivatives [74-75]. Benzodiazepine derivatives belong to an important class of heterocyclic compound and have been the subject of extensive study in recent years [76-79].

In spite of their importance from a pharmaceutical, industrial, synthetic point of view comparatively few methods for their preparation are reported in the literature, a great number of which have appeared only very recently. These include condensation reactions of o-phenylene di amines with α, β unsaturated carbonyl compounds [28], β halo ketones [29] or ketones in the presence of BF₃-Etherate [30],NaBH₄ [31],Poly phosphoric acid [32], SiO₂[33], MgO & POCl₃ [34], Yb(OTf)₃ [35], Al₂O₃/P₂O₅[36].

Thieno Pyrimidine is a bi cyclic heterocyclic compound consists of a five membered thiophene ring is fused to a six membered hetero cylic ring with two nitrogen atoms. The fusion may occur in three different orientations that results in three important types of thienopyrimidines namely; Thieno[2,3-d]Pyrimidine (a), thieno[3,2-d]Pyrimidine (b) and thieno[3,4-d]Pyrimidine (c).Most of the isomeric thienopyrimidines occur as colored amorphous form, some exists as crystalline form.

Synthetic approaches for the construction of a number of thieno Pyrimidines are well established. There exists three possible types of fusion of thiophene to Pyrimidine ring results in corresponding isomeric thienopyrimidines namely; [Fig.3] thieno[2,3-d]pyrimidines (a), thieno[3,4-d]pyrimidines (b) and thieno[3,2-d]pyrimidines (c).

Thieno[2,3-d]pyrimidine (a)

Thieno[3,4-d]pyrimidine (b)

Thieno[3,2-d]pyrimidine (c)

Fig: 3 Structures of different isomers of Thieno Pyrimidine

As a logical consequence of Thiophene – phenyl isosterism, similarly thienopyrimidines can be considered as bioisosteres of Quinazolines, which are extensively described in scientific and patent literature as displaying a plethora of biological activities. The synthesis of thienopyrimidine derivatives as potential surrogates for the quinazoline core structure has therefore, become a routine strategy in modern drug design and development. Thienopyrimidines as isosteres of quinazolines are shown here. figure 4.

Quinazoline

Thieno Pyrimidines

Fig: 4 Structures of thiophene – pheyl isoster

Thienopyrimidines can also be considered as structural analogues of five-membered heterocycles such as purines and thiazolopyrimidines. As interesting anti-HIV activity was discovered within the thiazolo [5, 4-d] pyrimidine series, whereas the thiazolo[4,5d]pyrimidines lack antiretroviral activity. The structures of purines and thiazolopyrimidines are shown in the following figure 5.

Purine

thiazolo[5,4-d] pyrimidine

thiazolo[4,5-d] pyrimidine

thieno[2,3-d] pyrimidine

Figure 5: Structures of purines and thiazolo pyrimidine

Thiophene containing compounds are well known to exhibit various biological effects. Heterocycles containing the thienopyrimidine moiety are of interest because of their interesting pharmacological and biological activities [37– 39]. They bear structural analogy and isoelectronic relation to purine and several substituted thieno [2,3-d]Pyrimidine derivatives shown to exhibit prominent and versatile biological activities [40,41]. Over the last two decades, many thieno-pyrimidines have been found to exhibit a variety of pronounced activities. Many of their derivatives have been synthesized as potential anticancer [42], analgesic [43], antimicrobial [44, 45] and antiviral agents [46].

Some reviews on Pyrimidine thiones [47] and condensed pyrimidines, namely pyrazolo-pyrimidines [48] and furo-pyrimidines [49].Thieno-pyrimidines are interesting heterocyclic compounds and a number of derivatives of these compounds display therapeutic activity as antimicrobial [50-53], anti-viral [54-55], anti-inflammatory [56-57], anti-diabetic [58], anti-oxidant [59], anti-tumour [60-64] and anti-cancer agents [65-66], anti-depressant [67], anti-platelet [68], anti-hypertensive [69], herbicidal [70] and plant growth regulatory properties [71].

“Chalcones; either natural or synthetic are well known to exhibit promising biological activities such as antibacterial, antitumor, anti-inflammatory analgesic, anti-malarial, and antituberculosis antipyretic[72]. Chalcones are used new for the synthesis of various classes of heterocyclic compounds such as thiazines, pyrazolines, isoxazolines and to benzodiazepines [73] etc.

The chalcones are unsaturated ketones containing the reactive keto ethylene group

Fig. 6 General structure of chalcones

Encouraged by the significance of benzodiazepines cited in literature and the movement of our work in the bio-organic field, we have studied its anti-microbial activity. In this current investigation, we report the synthesis, biological evaluation and preliminary structure activity relationship (SAR) of Benzodiazepine derivatives.

The synthesis of the compounds as per the following Scheme I given below.

The synthetic route was depicted in scheme I.

The structures of all synthesized compounds were assigned on the basis of IR, Mass, ¹H & ¹³C NMR spectral data analysis. Further these compounds were subjected for antifungal and antibacterial activity.

MATERIALS AND METHODS:

In this Investigation chemicals were purchased from local dealer with S.D fine & Avra labs make was used. Chemicals were 99 % pure; purity has been checked by thin layer chromatography and melting point. Conventional method has been used for synthesis of thieno [2, 3-d] Pyrimidine derivatives. Stirring and reflux method were used for synthesis of Thieno [2, 3-d] Pyrimidine derivatives 6 (a-j) respectively.

The synthetic route was depicted in scheme I.

The title compounds 6(a-j) were synthesized in five sequential steps using different reagents and reaction conditions, the 6(a-j) were obtained in moderate yields. The structure were established by spectral (IR, ¹H-NMR, ¹³C-NMR and mass) data.

EXPERIMENTAL SECTION:

All reactions were carried out under argon in oven-dried glassware with magnetic stirring. Unless otherwise noted, all materials were obtained from commercial suppliers and were used without further purification. All solvents were reagent grade. N, N Di Methyl Formamide (DMF) was distilled from CaH₂ and degassed thoroughly with dry argon directly before use. Unless otherwise noted, organic extracts were dried with anhydrous Na₂SO₄, filtered through a fitted glass funnel, and concentrated with a rotary evaporator (20–30 Torr). Flash chromatography was performed with silica gel (200–300 mesh) by using the mobile phase indicated. The NMR spectra were measured with a 400 MHz Bruker Avance spectrometer at 400.1 and 100.6 MHz, for ¹H for ¹³C, respectively, in CDCl₃ solution with tetra methyl silane as internal standard. Chemical shifts are given in ppm (δ) and are referenced to the residual proton resonances of the solvents. Proton and carbon magnetic resonance spectra (¹H NMR and ¹³C NMR) were recorded using tetra methyl silane (TMS) in the solvent of CDCl₃-d₁ or DMSO-d₆ as the internal standard (¹H NMR: TMS at 0.00 ppm, CDCl₃ at 7.26 ppm ,DMSO at 2.50 ppm; ¹³C NMR: CDCl₃ at 77.16 ppm, DMSO at 40.00 ppm).

The antimicrobial tests were carried out at the Bio-Technology Department, Faculty of Sciences, Sri Krishnadevaraya University. ChemDrawUltra-12.0 has been used for the nomenclature of the prepared compounds.

SYNTHESIS:

General procedure for synthesis of thieno [2, 3-d] pyrimidine-6-carbaldehyde [Compound 2]:

thieno[2,3-d] Pyrimidine (1) (10 g, 0.0735 mol) was dissolved in dry DMF(100 mL),under anhydrous condition, it was cooled to 0^OC, POCl₃ (15 mL) was added drop wise for 30 min. and stirring continued for 4 h at 80^oC After completion of reaction (TLC), The reaction mass was poured over crushed ice, basified with NaOH , Extracted with chloroform and dried over anhydrous Na₂SO₄. Organic layer was concentrated under reduced pressure and purified through silica gel column (Neutral Alumina) using Chloroform as eluting solvent to yield product (2) [yield 60%, 7.2g ].

IR (KBr, cm^-1): 3110 cm^-1 (ArC-H stret), 2720 (C-H Stretch), 1725 cm^-1 (C=O Stretch),1550 cm^-1 (C=C Stret), Wave numbers respectively.

¹H NMR (400 MHz; CDCl₃): δH 8.2 (S, 1H, Ar-H), 8.8 (S, 1H, Ar-H), 9.4 (S, 1H, Ar-H), 10.04 (S,-H-C=O).

¹³C NMR (100 MHz; CDCl₃): δC 130, 135, 145, 149, 158, 190.

MS (70 eV): m/z = 165(M+H)⁺.

General procedure for synthesis of (E)-1-phenyl-3-(thieno [2, 3-d]pyrimidin-6-yl)prop-2-en-1-one (4a), (E)-3-(thieno[2,3-d]pyrimidin-6-yl)-1-p-tolylprop-2-en-1-one(4b), (E)-1-(4-methoxyphenyl)-3-(thieno[2,3-d]pyrimidin-6-yl)prop-2-en-1-one (4c), (E)-3-(thieno[2,3-d]pyrimidin-6-yl)-1-(4-(trifluoromethoxy)phenyl)prop-2-en-1-one (4d), (E)-3-(thieno[2,3-d]pyrimidin-6-yl)-1-(4-(trifluoromethyl)phenyl)prop-2-en-1-one (4e), (E)-1-(4-nitrophenyl)-3-(thieno[2,3-d]pyrimidin-6-yl)prop-2-en-1-one (4f), (E)-1-(4-chlorophenyl)-3-(thieno[2,3-d]pyrimidin-6-yl)prop-2-en-1-one (4g), (E)-1-(4-bromophenyl)-3-(thieno[2,3-d]pyrimidin-6-yl)prop-2-en-1-one(4h),(E)-3-(thieno[2,3-d]pyrimidin-6-yl)-1-(thiophen-2-yl)prop-2-en-1-one(4i), (E)-1-(pyrazin-2-yl)-3-(thieno[2,3-d]pyrimidin-6-yl)prop-2-en-1-one (4j):

Various acetyl derivatives (3 a-j) (10 m.mol) were dissolved in ethanol, 5 mL 20% NaOH Solution was added to it. And stirred for 10 min at RT. Then thieno[2,3-d]pyrimidine-6-carbaldehyde (2) was added and stirring continued for 24h at Room temperature, after completion of reaction (TLC), reaction mixture was poured over crushed ice and stirred. The precipitate obtained was filtered and recrystallised by using Ethanol to obtain the chalcone derivatives (4a-j).

(E)-1-phenyl-3-(thieno [2, 3-d] pyrimidin-6-yl) prop-2-en-1-one (4a):

Yield: 85% (yellow colour solid);

IR (KBr, cm^-1): 3140(-Ar CH), 1652 (C=O Stretching), 1620(C=C Stretching), 675(C-S-C).

¹H NMR (400 MHz; CDCl₃): δH 9.4 (S, 1H, Ar-H), 8.8 (S, 1H, - Ar-H), 8.3(S,1H), 7.56 (d, 1H, CO-CH=), 7.95 (d, 1H, β C-H), 7.6-7.9(5H,m).

¹³C NMR (100 MHz; CDCl₃): δC 128.92, 124.03, 128.11, 151.67, 154.75, 159.62, 195.

MS (70 eV): m/z = 266(M+H)⁺.

(E)-3-(thieno [2, 3-d] pyrimidin-6-yl)-1-p-tolylprop-2-en-1-one (4b):

Yield: 86% (light yellow colour solid);

IR (KBr, cm^-1): 3120(-Ar CH), 2970(SP³ CH), 1675 (C=O Stretching), 1630(C=C Stretching), 668(C-S-C).

¹H NMR (400 MHz; CDCl₃): δH 9.4 (S, 1H, Ar-H), 8.8 (S, 1H, - Ar-H), 8.23(S,1H), 7.56 (d, 1H, CO-CH=), 7.95 (d, 1H, β C-H), 7.98(2H,d), 7.4(2H,d), 2.3(3H,S).

¹³C NMR (100 MHz; CDCl₃): δC 23, 125, 128.92, 124.03,135, 151.67, 154.75, 159.62, 190.

MS (70 eV): m/z = 281(M+H)⁺.

(E)-1-(4-methoxyphenyl)-3-(thieno [2, 3-d] pyrimidin-6-yl) prop-2-en-1-one (4c):

Yield: 90% (yellow colour solid);

IR (KBr, cm^-1):

3120(-Ar CH), 2970(SP³ CH), 1655 (C=O Stretching), 1630(C=C Stretching), 1160(C-O-C Stretching), 668(C-S-C).

¹H NMR (400 MHz; CDCl₃):

δH 9.4 (S, 1H, Ar-H), 8.8 (S, 1H, - Ar-H), 8.23(S,1H), 7.56 (d, 1H, CO-CH=), 7.95 (d, 1H, β C-H), 8.2(2H,d), 7.2(2H,d), 3.9(3H,S).

¹³C NMR (100 MHz; CDCl₃):

δC 56, 120,125, 128.92, 124.03,135, 149,151.67, 154.75, 159.62, 185.

MS (70 eV): m/z = 297(M+H)⁺.

(E)-3-(thieno [2, 3-d] pyrimidin-6-yl)-1-(4-(tri fluoro methoxy) phenyl)prop-2-en-1-one (4d):

Yield: 90% (yellow colour solid);

IR (KBr, cm^-1):

3110(-Ar CH), 1640 (C=O Stretching), 1625(C=C Stretching), 1340(C-F), 1160(C-O-C), 675(C-S-C).

¹H NMR (400 MHz; CDCl₃):

δH 9.4 (S, 1H, Ar-H), 8.8 (S, 1H, - Ar-H), 8.23(S,1H), 7.56 (d, 1H, CO-CH=), 7.95 (d, 1H, β C-H), 8.2(2H,d), 7.2(2H,d), 3.9(3H,S).

¹³C NMR (100 MHz; CDCl₃):

δC 56, 120,125, 128.92, 124.03,135, 149,151.67, 154.75, 159.62, 185.

MS (70 eV): m/z = 351(M+H)⁺.

(E)-3-(thieno [2, 3-d] pyrimidin-6-yl)-1-(4-(trifluoromethyl) phenyl) prop-2-en-1-one (4e):

Yield: 90% (yellow colour solid);

IR (KBr, cm^-1):

3130(Ar CH), 1665 (C=O Stretching), 1640(C=C Stretching), 1360(C-F), 1160(C-O-C), 685(C-S-C).

¹H NMR (400 MHz; CDCl₃):

δH 9.4 (S, 1H, Ar-H), 8.8 (S, 1H, - Ar-H), 8.23(S,1H), 7.55 (d, 1H, CO-CH=), 7.90 (d, 1H, β C-H), 8.1(2H,d), 7.8(2H,d).

¹³C NMR (100 MHz; CDCl₃):

δC 120,125, 128.92, 124.03,135, 149,151.67, 154.75, 159.62, 189.

MS (70 eV): m/z = 335(M+H)⁺.

(E)-1-(4-nitrophenyl)-3-(thieno [2, 3-d] pyrimidin-6-yl) prop-2-en-1-one (4f):

Yield: 80% (yellow colour solid);

IR (KBr, cm^-1):

3110(Ar CH), 1655 (C=O Stretching), 1646(C=C Stretching), 1336 & 1550 (N-O Symmetric & Asymmetric stretching in Nitro Group), 680(C-S-C).

¹H NMR (400 MHz; CDCl₃):

δH 9.4 (S, 1H, Ar-H), 8.8 (S, 1H, - Ar-H), 8.23(S,1H), 7.55 (d, 1H, CO-CH=), 7.95 (d, 1H, β C-H), 8.2(2H,d), 8.5(2H,d).

¹³C NMR (100 MHz; CDCl₃):

δC 125, 128.92, 124.03,135, 149,151.67, 154.75, 159.62, 189.

MS (70 eV): m/z = 310(M-H)⁺.

(E)-1-(4-chlorophenyl)-3-(thieno [2, 3-d]pyrimidin-6-yl)prop-2-en-1-one (4g):

Yield: 85% (yellow colour solid);

IR (KBr, cm^-1):

3110(Ar CH), 1655 (C=O Stretching), 1646(C=C Stretching), 1140 (C-O-C stretching in Furan ring), 685(C-S-C).

¹H NMR (400 MHz; CDCl₃):

δH 9.4 (S, 1H, Ar-H), 8.8 (S, 1H, - Ar-H), 8. 3(S,1H), 6.75 (d, 1H, CO-CH=), 7.6 (d, 1H, β C-H), 8(1H,d), 7.9(1H,t), 8.7(1H,d).

¹³C NMR (100 MHz; CDCl₃):

δC 115,125, 128.92, 124.03,135, 149,151.67, 154.75, 159.62, 195.

MS (70 eV): m/z = 257(M-H)⁺.

(E)-1-(4-bromophenyl)-3-(thieno [2, 3-d]pyrimidin-6-yl)prop-2-en-1-one(4h):

Yield: 85% (pale yellow colour solid);

IR (KBr, cm^-1):

3120(Ar CH), 1665 (C=O Stretching), 1650(C=C Stretching), 680(C-S-C).

¹H NMR (400 MHz; CDCl₃):

δH 9.4 (S, 1H, Ar-H), 8.8 (S, 1H, - Ar-H), 8. 3(S, 1H), 6.75 (d, 1H, CO-CH=), 7.6 (d, 1H, β C-H), 8(1H, d), 7.4(1H,t), 8.2(1H,d).

¹³C NMR (100 MHz; CDCl₃):

δC 125, 128.92, 124.03,135, 149,151.67, 154.75, 159.62, 195.

MS (70 eV): m/z = 273(M-H)⁺.

(E)-1-(pyrazin-2-yl)-3-(thieno [2, 3-d] pyrimidin-6-yl) prop-2-en-1-one (4i):

Yield: 80% (yellow colour solid);

IR (KBr, cm^-1):

3100(Ar CH), 1670 (C=O Stretching), 1655(C=C Stretching), 1470(C=N), 685(C-S-C).

¹H NMR (400 MHz; CDCl₃):

δH 9.3 (S, 1H, Ar-H), 8.9 (S, 1H, - Ar-H), 8. 3(S, 1H), 6.75 (d, 1H, CO-CH=), 7.65 (d, 1H, β C-H), 8.8(1H, d), 8.6(1H,d), 9.4(1H,S).

¹³C NMR (100 MHz; CDCl₃):

δC 120,125, 128.92, 124.03,135, 149,151.67, 154.75, 159.62, 195.

MS (70 eV): m/z = 269(M+H)⁺.

(E)-1-(pyridin-2-yl)-3-(thieno [2, 3-d] pyrimidin-6-yl) prop-2-en-1-one (4j):

Yield: 82% (yellow colour solid);

IR (KBr, cm^-1): 3100(Ar CH), 1670 (C=O Stretching), 1655(C=C Stretching), 1460(C=N), 685(C-S-C).

¹H NMR (400 MHz; CDCl₃):

δH 9.4 (S, 1H, Ar-H), 8.8 (S, 1H, - Ar-H), 8. 25(S, 1H), 6.75 (d, 1H, CO-CH=), 7.55 (d, 1H, β C-H), 8.3(1H, d), 8.1(1H,d), 8(1H,d), 8.9(1H,d).

¹³C NMR (100 MHz; CDCl₃):

δC 120,125, 128.92, 124.03,135, 149,151.67, 154.75, 159.62, 195.

MS (70 eV): m/z = 266(M-H)⁺.

General Procedure for the Preparation of

6-(4-phenyl-2, 3-dihydro-1H-benzo[b][1,4]diazepin-2-yl)thieno[2,3-d]pyrimidine (6a),

6-(4-p-tolyl-2, 3-dihydro-1H-benzo[b][1,4]diazepin-2-yl)thieno[2,3-d]pyrimidine (6b),

6-(4-(4-methoxy phenyl)-2,3-dihydro-1H-benzo[b][1,4]diazepin-2-yl)thieno[2,3-d]pyrimidine (6c),

6-(4-(4-(tri fluoro methoxy) phenyl)-2,3-dihydro-1H-benzo[b][1,4]diazepin-2-yl)thieno[2,3-d]pyrimidine (6d),

6-(4-(4-(tri fluoro methyl) phenyl)-2,3-dihydro-1H-benzo[b][1,4]diazepin-2-yl)thieno[2,3-d]pyrimidine (6e),

6-(4-(4-nitrophenyl)-2,3-dihydro-1H-benzo[b][1,4]diazepin-2-yl)thieno[2,3-d]pyrimidine (6f),

6-(4-(4-chlorophenyl)-2, 3-dihydro-1H-benzo[b][1,4]diazepin-2-yl)thieno[2,3-d]pyrimidine (6g),

6-(4-(4-bromophenyl)-2,3-dihydro-1H-benzo[b][1,4]diazepin-2-yl)thieno[2,3-d]Pyrimidine (6h),

6-(4-(thiophen-2-yl)-2, 3-dihydro-1H-benzo[b][1,4]diazepin-2-yl)thieno[2,3-d]pyrimidine (6i),

6-(4-(pyrazin-2-yl)-2,3-dihydro-1H-benzo[b][1,4]diazepin-2-yl)thieno[2,3-d]pyrimidine (6j):

A reaction mixture of new chalcones 4 a-j (1 m.mol) and o-phenylene di amine(1.5m.mol) in DMF (15 ml) with few drops of piperidine was heated to reflux for 4-6 h. The progress of the reaction was monitored by using TLC. After completion of reaction, the reaction mixture was distilled to remove the excess solvent and poured into crushed ice. The crude solid product obtained was filtered, washed with water and recrystalized from Ethanol to get product 6 a-j in good yields with high purity.

6-(4-phenyl-2, 3-dihydro-1H-benzo[b][1,4]diazepin-2-yl)thieno[2,3-d]pyrimidine (6a):

Fig. 7 Structure of 6-(4-phenyl-2,3-dihydro-1H-benzo[b][1,4]diazepin-2-yl)thieno[2,3-d]pyrimidine

Yield: 75%

mp 142-143℃.

IR (KBr, cm^-1): 3415(N-H Stretching), 3110(Ar CH), 1455 (C=N Stretching), 680(C-S-C).

¹H NMR (400 MHz; DMSO-d₆): δH 3.8(1H,dd, NH-CH),2.1(1H,dd), 1.8(1H,dd),9.4 (S, 1H, Ar-H), 8.8 (S, 1H, - Ar-H), 6.7(S, 1H), 7.3-7.5(5H,m), 7.6-8.1(5H,m).

¹³C NMR (100 MHz; DMSO-d₆): δC 58, 40,125, 128.92, 124.03,135,140, 149,151.67, 154.75, 159,162.

MS (EI): m/z = 357(M-H)⁺.

6-(4-p-tolyl-2, 3-dihydro-1H-benzo[b][1,4]diazepin-2-yl)thieno[2,3-d]Pyrimidine (6b):

Fig. 8. Structure of 6-(4-p-tolyl-2, 3-dihydro-1H-benzo[b][1,4]diazepin-2-yl)thieno[2,3-d]Pyrimidine

Yield: 78%

mp 147-148°C.

IR (KBr, cm^-1): 3420(N-H Stretching), 3140(-Ar CH), 2960(SP³ CH), 1440(C=N Stretching), 665(C-S-C).

¹H NMR (400 MHz; DMSO-d₆): δH 2.3(3H,S), 3.8(1H,dd, NH-CH),2.0(1H,dd), 1.9(1H,dd),9.4 (S, 1H, Ar-H), 8.8 (S, 1H, - Ar-H), 6.7(S,1H), 7.2-7.4(4H,m), 7.8(2H,d), 7.3(2H,d).

¹³C NMR (100 MHz; DMSO-d₆): δC 23, 59, 41,125, 128.92, 124.03,135, 153, 156, 160.

MS (EI): m/z = 369(M-H)⁺.

6-(4-(4-methoxy phenyl)-2,3-dihydro-1H-benzo[b][1,4]diazepin-2-yl)thieno[2,3-d]pyrimidine (6c):

Fig. 9. Structure of 6-(4-(4-methoxy phenyl)-2,3-dihydro-1H-benzo[b][1,4]diazepin-2-yl)thieno[2,3-d]pyrimidine

Yield: 80% ,

mp 139-140°C.

IR (KBr, cm^-1): 3410(N-H Stretching), 3130(-Ar CH), 2990(SP³ CH), 1445(C=N Stretching), 1040(C-O-C Stretching), 685(C-S-C).

¹H NMR (400 MHz; DMSO-d₆): δH 3.9(1H,dd, NH-CH),2.1(1H,dd), 1.9(1H,dd),9.4 (S, 1H, Ar-H), 8.9 (S, 1H, - Ar-H), 6.73(S,1H), 8.1(2H,d), 7.1(2H,d), 3.9(3H,S).

¹³C NMR (100 MHz; DMSO-d₆): δC 60, 41, 56, 120,125, 128.92, 124.03,135, 149,151.67, 154.75, 159,162.

MS (70 eV): m/z = 387(M+H)⁺.

6-(4-(4-(tri fluoro methoxy) phenyl)-2,3-dihydro-1H-benzo[b][1,4]diazepin-2-yl)thieno[2,3-d]pyrimidine (6d):

Fig. 10. Structure of 6-(4-(4-(tri fluoro methoxy) phenyl)-2,3-dihydro-1H-benzo[b][1,4]diazepin-2-yl)thieno[2,3-d]pyrimidine

Yield: 65%,

mp 133-134°C

IR (KBr, cm^-1): 3406(N-H Stretching), 3110(-Ar CH), 1440 (C=N Stretching), 1625(C=C Stretching), 1340(C-F), 1140(C-O-C), 685(C-S-C).

¹H NMR (400 MHz; DMSO-d₆): δH 3.9(1H,dd, NH-CH),2.1(1H,dd), 1.9(1H,dd),9.4 (S, 1H, Ar-H), 8.8 (S, 1H, - Ar-H), 6.63(S,1H), 8.1(2H,d), 7.1(2H,d),7.2 (2H,t), 7.4(2H,t).

¹³C NMR (100 MHz; DMSO-d₆): δC 60, 40, 120,125, 128.92, 124.03,135, 149,151.67, 154.75, 160,162.

MS (70 eV): m/z = 439(M-H)⁺, 462[M+Na]

6-(4-(4-(tri fluoro methyl) phenyl)-2,3-dihydro-1H-benzo[b][1,4]diazepin-2-yl)thieno[2,3-d]pyrimidine (6e):

Fig. 11. Structure of 6-(4-(4-(tri fluoro methyl) phenyl)-2,3-dihydro-1H-benzo[b][1,4]diazepin-2-yl)thieno[2,3-d]pyrimidine

Yield: 63%

mp 137-138°C

IR (KBr, cm^-1): 3425(N-H Stretching), 3130(Ar CH), 1470(C=N Stretching), 1370(C-F), 1150(C-O-C), 685(C-S-C).

¹H NMR (400 MHz; DMSO-d₆): δH 3.9(1H,dd,NH-CH),2.1(1H,dd), 1.9(1H,dd),9.4 (S, 1H, Ar-H), 8.7 (S, 1H, - Ar-H), 6.7(S,1H), 7.8(2H,d), 7.7(2H,d),7.2 (2H,t), 7.4(2H,t).

¹³C NMR (100 MHz; DMSO-d₆): δC 60, 40, 120,125, 128.92, 124.03,135, 149,153, 154.75, 160, 164.

MS (70 eV): m/z = 425(M+H)⁺, 443[M+NH₄⁺].

6-(4-(4-nitrophenyl)-2,3-dihydro-1H-benzo[b][1,4]diazepin-2-yl)thieno[2,3-d]pyrimidine (6f):

Fig. 12. Structure of 6-(4-(4-nitrophenyl)-2,3-dihydro-1H-benzo[b][1,4]diazepin-2-yl)thieno[2,3-d]pyrimidine

Yield: 60%

mp 150-151°C

IR (KBr, cm^-1): 3420(N-H Stretching), 3120(Ar CH), 1445 (C=N Stretching), 1360 & 1540 (N-O Symmetric & Asymmetric stretching in Nitro Group), 665(C-S-C).

¹H NMR (400 MHz; DMSO-d₆):

δH 3.9(1H,dd,NH-CH),2.1(1H,dd), 1.9(1H,dd),9.4 (S, 1H, Ar-H), 8.7 (S, 1H, - Ar-H), 6.5(S, 1H), 8.2(2H,d), 8.5(2H,d), 7.2 (2H,t), 7.4(2H,t).

¹³C NMR (100 MHz; DMSO-d₆):

δC 40, 45,125, 128.92, 124.03,135, 149,151.67, 154.75, 159.62, 164.

MS (70 eV): m/z = 400(M-H)⁺.

6-(4-(4-chlorophenyl)-2, 3-dihydro-1H-benzo[b][1,4]diazepin-2-yl)thieno[2,3-d]pyrimidine (6g):

Fig. 13. Structure of 6-(4-(4-chlorophenyl)-2, 3-dihydro-1H-benzo[b][1,4]diazepin-2-yl)thieno[2,3-d]pyrimidine

Yield: 60%

mp 172-174°C

IR (KBr, cm^-1): 3415(N-H Stretching), 3120(Ar CH), 1445 (C=N Stretching), 640 (C-Cl stretching), 665(C-S-C).

¹H NMR (400 MHz; DMSO-d₆): δH 3.9(1H,dd,NH-CH),2.1(1H,dd), 1.9(1H,dd),9.4 (S, 1H, Ar-H), 8.7 (S, 1H, - Ar-H), 6.5(S, 1H), 8.05(2H,d), 7.5(2H,d), 7.2 (2H,t), 7.4(2H,t).

¹³C NMR (100 MHz; DMSO-d₆): δC 40, 45,125, 128.92, 124.03,135, 149,151.67, 154.75, 159.62, 164.

MS (EI): m/z = 391(M⁺,100%)⁺, 393[M+2, 33%] it indicated molecule contains one –Chlorine atom.

6-(4-(4-bromophenyl)-2,3-dihydro-1H-benzo[b][1,4]diazepin-2-yl)thieno[2,3-d]Pyrimidine (6h):

Fig. 14. Structure of 6-(4-(4-bromophenyl)-2,3-dihydro-1H-benzo[b][1,4]diazepin-2-yl)thieno[2,3-d]Pyrimidine

Yield: 60%

mp 172-174°C

IR (KBr, cm^-1): 3405(N-H Stretching), 3105(Ar CH), 1455 (C=N Stretching), 560 (C-Br stretching), 685(C-S-C).

¹H NMR (400 MHz; DMSO-d₆): δH 3.9(1H,dd,NH-CH),2.1(1H,dd), 1.9(1H,dd),9.4 (S, 1H, Ar-H), 8.7 (S, 1H, - Ar-H), 6.5(S, 1H), 7.8(2H,d), 7.5(2H,d), 7.2 (2H,t), 7.4(2H,t).

¹³C NMR (100 MHz; DMSO-d₆): δC 41, 43,125, 128.92, 124.03,135, 149,151.67, 154.75, 159.62, 164.

MS (EI): m/z = 435(M⁺,100%)⁺, 437[M+2, 98%] it indicated molecule contains one –Bromine atom.

6-(4-(thiophen-2-yl)-2,3-dihydro-1H-benzo[b][1,4]diazepin-2-yl)thieno[2,3-d]pyrimidine (6i):

Fig. 15. Structure of 6-(4-(thiophen-2-yl)-2,3-dihydro-1H-benzo[b][1,4]diazepin-2-yl)thieno[2,3-d]pyrimidine

Yield: 60%

mp 160-162°C

IR (KBr, cm^-1): 3420(N-H Stretching), 3120(Ar CH), 1460 (C=N Stretching), 1140 (C-O-C stretching in Furan ring), 680(C-S-C).

¹H NMR (400 MHz; DMSO-d₆): δH 3.9(1H,dd,NH-CH),2.1(1H,dd), 1.9(1H,dd),9.4 (S, 1H, Ar-H), 8.8 (S, 1H, - Ar-H), 6.6(S, 1H), 7.7(1H, d), 7.2(1H, t), 7.5(1H, d), 7.5(2H,d), 7.4(2H,d).

¹³C NMR (100 MHz; DMSO-d₆): δC 39, 45,115,125, 128.92, 124.03,135, 149,151, 155, 160, 163.

MS (70 eV): m/z = 363(M+H)⁺.

6-(4-(pyrazin-2-yl)-2,3-dihydro-1H-benzo[b][1,4]diazepin-2-yl)thieno[2,3-d]pyrimidine (6j):

Fig. 16. Structure of 6-(4-(pyrazin-2-yl)-2,3-dihydro-1H-benzo[b][1,4]diazepin-2-yl)thieno[2,3-d]pyrimidine

Yield: 65%

mp 160-162°C

IR (KBr, cm^-1):

3430(N-H Stretching), 3130(Ar CH), 1455(C=N Stretching), 665(C-S-C).

¹H NMR (400 MHz; DMSO-d₆):

δH 3.8(1H,dd,NH-CH),2.2(1H,dd), 1.9(1H,dd),9.3 (S, 1H, Ar-H), 8.8 (S, 1H, - Ar-H), 6.6(S, 1H), 8.5(1H, d), 8.8(1H,d), 9.4(1H,S) 7.6(4H,m).

¹³C NMR (100 MHz; DMSO-d₆): δC 40, 46,120,125, 128.92, 124.03,135, 149,151.67, 154.75, 160, 165.

MS (70 eV): m/z = 359(M+H)⁺.

Antimicrobial Activity:

The cup plate agar diffusion method [80] was employed for determining the antimicrobial activity of the newly synthesized compounds 6 a-j against two gram positive bacteria viz., Bacillus subtilis, Staphylococci aureus and two gram negative bacteria viz., Escherichia coli, Pseudomonas aeruginosa in addition to fungi(Candida albicans). The solutions of different compounds under test at a concentration of 500 and 600μg/mlin 5% DMSO were poured in the cup/well of bacteria seeded agar plates. These plates were incubated at 37℃ for 24 hours for E. coli, whereas plates of other three bacteria were incubated at 27℃ for 24 hr. The standard antibiotics used were ampicillin (all at 500 μg/ml). and standard antifungal used were nystatin at 500 μg/ml. The solution without compound i.e. only 5% DMSO was used as control which did not reveals any inhibition. The zone of inhibition produced by each compound was measured in mm. The results of antimicrobial studies are given in Table 1. From screening results, it was observed that final compounds 6i, 6e Shows significant antimicrobial activity but other compound showed moderate antimicrobial activity. The discussion and comparison of antibacterial activity were given with respect to ampicillin antibiotic and antifungal screenings were compared with Nystatin. The bacterial zone of inhibition values are given in (Table 1). The order of activity was 6i>6e>6j>6d>6f >6g >6h>>6b>6a>6c.

Microbiological testing of the newly synthesized compounds were performing in the Department of Bio-Technology, Faculty of Science, Sri Krishnadevaraya University, Ananthapuramu, INDIA.

Table 1.Antimicrobial activity of novel synthesized 1,5-Benzodiazepines 6a-j:

Comp. NO.	Concentration (μg/ml)	Microorganism (inhibition zone (mm) )
Comp. NO.	Concentration (μg/ml)	*Basillus Subtilis*	*Staphylococcus aureus*	*Escherichia coli*	*Pseudomonas aeruginosa*	*Candida albicans*
6a	500	10	10.2	10.1	9.5	7.0
6a	600	10.3	10.3	10.4	9.7	7.2
6b	500	11	13	11	10.1	8
6b	600	12.4	13.4	11.3	10.5	8.5
6c	500	10	10.2	10.1	10.5	7.2
6c	600	10.3	10.3	10.4	10.9	7.4
6d	500	14	16	16	14	13
6d	600	14.4	16.5	16.5	15	13.9
6e	500	17	17	17	16	14
6e	600	18	17.5	17.5	16.5	14.4
6f	500	13.5	15	14.5	12.6	12
6f	600	14.4	15.3	14.8	12.7	12.6
6g	500	13	12.4	11.6	11.2	9.5
6g	600	13.2	12.7	11.8	11.4	9.7
6h	500	11.6	13.5	11.5	10	9
6h	600	13	14	12	10.5	9.3
6i	500	19	17	18	17	15
6i	600	19.5	17.5	18.5	17.5	15.5
6j	500	15	16	17	15	12
6j	600	15.4	16.6	17.4	15.5	12.5
Ampicillin	500	24	22	25	21	---
Nystatine	500	---	---	----	----	19.8

RESULT AND DISCUSSION:

Chemistry:

The Title Compounds Novel 1, 5-Benzodiazepines Containing Thieno [2, 3-d] Pyrimidine based derivatives 6(a-j) were synthesized in good yields (scheme-I). All these compounds were tested for Anti-microbial activity showed considerable activity when compared to the standard drug.

In the present communication thieno [2, 3-d] pyrimidine-6-carbaldehyde (2) was synthesised from thieno [2, 3-d] pyrimidine (1) According to the reported procedure [81]. Various chalcone derivatives 4(a-j) having Thieno[2,3-d] Pyrimidine core According to the reported procedure [82], These are further reacted with O-Phenyl Di amine (5) to get target Novel 1, 5-Benzodiazepines Containing Thieno [2, 3-d] Pyrimidine based derivatives 6(a-j) According to the reported procedure[83].

Characterization:

The IR spectrum of the title Compounds 6(a-j) has given stretching vibration at 3110cm^-1, due to the stretching vibration corresponding to Ar-H Stretching vibrations. The absorption peak at 2935 cm^-1 is due to The stretching vibration corresponding to the SP³ C-H ( methyl group).The strong Intensity absorption at 1350 & 1530 cm^-1 is due to The stretching vibration of -N-O Stretching in Nitro group, The weak Intensity absorption at 1620 cm^-1 corresponds to a C=N Stretching vibration.1150cm^-1 corresponding to C-O-C Stretching.

It has been observed from chemical structure of compound 6(a-j) that different pair of protons. The protons of Methyl group which is attached to benzene ring appeared as a singlet at δ =2.3 ppm, the protons of Methoxy group appeared as a Singlet at δ =3.8 ppm. The protons attached benzene & Pyrimidine rings appeared between δ =6.8-8.8 ppm respectively.

The chemical shifts of the final compounds carbon chemical shifts are vary from δ = 195 to 23 ppm. The carbon nucleus under the influence of a strong electronegative environment appeared down field, the carbon chemical shift of the methyl group at δ= 23 ppm. The carbon chemical shift of the Methoxy group at δ= 55 ppm. The carbon chemical shift of the aldehyde carbon at δ= 195 ppm.

Readily available starting materials and simple synthesizing procedures make this method very attractive and convenient for the synthesis of 1, 5 Benzo Di azepine derivatives. Formation of products was confirmed by recording their ¹H NMR, ¹³C NMR, FT-IR, mass spectra.

The IR spectra of new Chalcones confirmed by the presence of two stretching bands at 1660-1590, this due to C=O and CH=CH, in addition the ¹HNMR of Chalcone have two doublet signal one at 7.40 ppm which belong CHβ, and one at 6.90 which belong CHα. upon cyclocondensation of Chalcones with o-phenelendiamine it produce new 1,5-benzodiazepine derivatives 6(a-j) respectively, where the spectral data confirm the existence of this cyclocondensation reaction where the ¹HNMR of new 1,5-benzodiazepines contain two signal one at 2.50 ppm due one hydrogen of (C-2) in benzodiazepine ring and another one at 3.80 ppm due two hydrogen of (C-3) in benzodiazepine ring that prove the cyclocondensation well occur. in addition the disappearance of sharp stretching of carbonyl and appearance of starching at 1590 (C=N of diazepine ) and all this fact will prove this cyclocondensation reaction and formation of 1,5-Benzo Diazepine ring with good yield 65-85%. It was observed clearly that carbonyl Acetyl derivatives (5 a-j) carrying electron withdrawing substituents of the phenyl ring afforded low yields of [1, 5]-benzodiazepines. The structures of some the compounds were established from the spectral data of the resulting compounds.

Anti microbial activity screening:

The results of Anti microbial studies of newly synthesized compounds reveal that the compounds possess significant Anti-microbial activities. The results of these studies are given in Table 1. From anti-bacterial and anti-fungal activity screening results, it has been observed that compounds 6i, 6e, 6j and 6d possess good activity.

CONCLUSION:

We have synthesized a series of new 1, 5-benzodiazepines 6a-j containing bioactive hetero aryl pharmacophore such as Thieno [2, 3,-d] Pyrimidine using convenient method. The antimicrobial activity of representative 1, 5-benzothiazepines 6 a-j showed Excellent antimicrobial activity. An accessible approach for the synthesis of 1,5-benzodiazepines was presented. The potential antimicrobial activity of the synthesised compounds validates the significance of this study. Among the synthesised compounds, 6-(4-(thiophen-2-yl)-2, 3-dihydro-1H-benzo[b][1,4]diazepin-2-yl)thieno[2,3-d]pyrimidine (6i) and 6-(4-(4-(tri fluoro methyl) phenyl)-2,3-dihydro-1H-benzo[b][1,4]diazepin-2-yl)thieno[2,3-d]pyrimidine (6e) acts as potential antifungal and antibacterial agents.

ACKNOWLEDGEMENT:

The authors are also thankful to Sapala organics, Hyderabad for providing NMR spectra and LC-MS data and Pragna generics pvt ltd, Hyderabad for providing laboratory facilities.

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Received on 09.12.2016 Modified on 15.12.2016

Asian J. Research Chem. 2017; 10(2):71-84.

DOI: 10.5958/0974-4150.2017.00012.8