Scientific paper
The Reaction of 2-Aminocyclohexeno[b]thiophene Derivatives with Ethoxycarbonyl isothiocyanate: Synthesis of Fused Thiophene Derivatives with Antibacterial and Antifungal Activities
Wagnat W. Wardakhana*, Nadia A. Loucab and Mona M. Kamelc
* Corresponding author: a National organization for Drug Control & Research, P.O. 29, Cairo, A. R. Egypt,
E-mail: wagnatward@hotmail.com
b Hormones Department, National Research Center, Dokki, Giza, A. R. Egypt
c Department of organic Chemistry, Faculty of Pharmacy, Cairo University, Cairo, A. R. Egypt.
Received: 11-05-2006
Abstract
The reaction of 2-amino-tetrahydrobenzo[b]thiophene derivatives 1a-d with ethoxycarbonyl isothiocyanate (2) gave the tetrahydrobenzo[b]thiophen-2-thiourea derivatives 3a-d. The latter products underwent ready cyclizations when heated in sodium ethoxide solution to give annulated derivatives 4a-d. Compounds 3a-d also underwent hetero-cyclizations to give fused thiophene derivatives with antibacterial and antifungal activities.
Keywords: Thiophene, pyrazole, pyrimidine, fused derivatives
1. Introduction
Alkoxycarbonyl, acyl and aroyl isothiocyanates have recently found extensive utility in heterocyclic synthesis.1-6 Their high reactivity is owed to the presence of the multiple bond system which is responsible for either 1,3-dipolar cycloaddition or Michael type addition followed by cyclization through the alkoxycarbonyl, acyl or aroyl moieties, which provides the double bond requisites to a heteroaromatic system. Recently we were investigating a series of reactions involving the uses of isothio-cyanates together with the uses of benzo[ft]thiophene derivatives in heterocyclic synthesis.7-11 The results showed, independently, the formation of fused thiophene derivatives with pharmaceutical interest among which are their antioxidant effects on lipid peroxidation,12 anti-inflammatory, antifungal, antimycotic and antibacterial activi-ties,13-17 some of which have antiproliferative and an-tinoceptive properties.18,19 others are used as dual inhibitors,20 P1 surrogates of inhibitors of blood coagulation factor XA21 and inhibitors for the platelet aggregation.22 In the present work we would like to present a study of the reaction of alkoxycarbonyl isothiocyanate with tetrahy-drobenzo[ft]thiophene derivatives in the aim of connecting the gap between the two annotated series of reactions.
2. Results and Discussion
The reaction of the tetrahydrobenzo[b]thiophene derivatives 1a-d23,24 with ethoxycarbonylisothiocyanate (2) in 1,4-dioxan at room temperature gave the N-ethoxycar-bonylthiourea derivatives 3a-d, respectively. The structures of the latter products were based on analytical and spectral data. Thus, the 13C NMR data of 3a showed 5 14.88 (ester CH3), 20.0, 23.3, 23.9, 24.7 (4CH2); 60.45 (ester CH2); 118.8 (CN); 122.3, 136.7, 135.6, 140.8 (thio-phene-C); 154.7 (amide C=O) and 178.8 (thioamide). The reactions of isothiocyanates with NH2 compounds were reported earlier.25 Compounds 3a-d underwent ready cy-clization when heated in sodium ethoxide solution in a boiling water bath to give the tetrahydro[b]thieno[5,4-d]pyrimidine derivatives 4a-d, respectively.
The reaction of either 3c or 3d with anthranilic acid (5) gave the benzo[d]pyrimidine derivatives 7a,b. The reactions took place through the intermediate formation of the anilide derivatives 6a,b followed by water elimination (scheme 1). Structures of compounds 7a,b were based on analytical and spectral data (see experimental section). The latter compounds underwent ready cyclization when heated in sodium ethoxide to afford the cyclohexeno[b]thieno[5,4-rf]thiazine derivatives 8a and 8b, respectively.
4	r	y	x
A	h	ch2	nh
b	h	ch3	0
c	ch3	c=o	nh
(1	ch3	co	o
3c,d
^¿i^COOH
dmf --)
heat
The reaction of either 4c or 4d with anthranilic acid (5) gave the benzo[d]-1,3-oxazine derivatives 10a and 10b, respectively, formation of these product took place via the intermediacy of 9a and 9b.
The reaction of compound 3c, d with diethyl-malonate (11) gave the pyrimidine derivatives 12a and 12b, respectively. The structures of 12a,b were based on the analytical and spectral data (see experimental section).
Scheme 1
7a, X = CN b,X = COOC2h5
Scheme 2
/ A /COOC2H5
VN N ■S J^c
X
^OH
NHiNHR
1,4-dioxan
heat
12a, X = CN b, X = COOEt
15a, R = H b, R = Ph
NNHR
rv?
OH
x J
I N

16	X	R
a	CN	H
b	CN	Ph
c	COOEt	H
d	COOEt	Ph
COOC2H5
4a, X = NH b, X = O
15a,h
dmf -*
heat
COOC2H5
y^NNHR" NH
17a-d
Scheme 3
19a, X = NH
b, X = O
Further confirmations for these structures were obtained through studying their reactivity with some chemical reagents. Thus, the reaction of either 12a or 12b with ben-zenediazonium chloride (13) gave the phenylazo derivatives 14a and 14b, respectively (scheme 2). on the other hand, the reaction of 12a,b with either hydrazine hydrate 15a or phenylhydrazine 15b gave the 1,2,4-triazolo[4,5-cjpyrimidine derivatives 16a-d (scheme 3). The reaction is explained in terms of reaction with two fold of the hydrazine with one of either 16a or 16b through two reactive sites, the thioxo and the hydroxyl groups followed by ethanol liberation. It should be noted that the ring closure
R = Ph
R = H
18a, X= NH
b, X = O
leading to the formation of the triazolo[4,5-c]pyrimidine enhances the attack of the hydrazine to the thioxo group rather than the a,P-unsaturated carbonyl moiety. The analytical and spectral data are in agreement with the proposed structures.
Scheme 4
The reaction of either 4a and 4b with either hydrazine hydrate (15a) or Phenylhydrazine (15b) gave the corresponding 4,5,6,7-tetrahydrothieno[5,4:4,5]pyrimidi-no[2,1:3,4]-1,2,4-triazole derivatives 18a,b and 19a,b, respectively. Formation of the latter products were based on the formation of the intermediates 17a-d followed by cy-clization (scheme 3). on the other hand the reaction of either 3a or 3b with either hydrazine hydrate or phenylhy-drazine gave the 1,2,4-triazole derivatives 20a-d. The structures of compounds 20a-d were based on analytical
and spectral data (see experimental section). Compounds 20a,c and 20b,d underwent ready cyclization to give the same products 21a and 21b, respectively (m.p., mixed m.p. and finger print IR). Formation of the same compound 21a from either 20a or 20c is explained in terms of the initial addition of NH group to CN group, in the case of 20a, and hydrolysis of the C=NH group,26,27 however, in the case of 20c, loss of ethanol took place. The confirmation that compound 21b with the 3-phenyl group attached to the triazole ring are different than that with the
2-phenyl group in 19b was obtained through carrying the reaction of 4b with phenylhydrazine in 1,4-dioxan at room temperature where the N-phenylhydrazide derivative 22 was separated (scheme 4). Compound 22 underwent ready cyclization when heated in dimethylfor-mamide solution to give the same product 21b (m.p. and mixed m.p.). Therefore, the reaction of 4a,b with phenyl-hydrazine took place through the initial attack at the thione group followed by ethanol liberation, if the reaction is heated under reflux. on the other hand, carrying the reaction at room temperature enhances the first attack of the ester group to give the hydrazide followed by the loss of hydrogen sulphide. In the first case, the 2-phenyl derivative 19b is formed while in the second case the 3-phenyl derivative is formed.
3. Bioassay
3.1. Materials and Methods
Test organisms. The fungi selected for this study were Fusarium oxysporum f. sp. Lycopersici (SACC.) SNYDER et HANSEN and Helminthosporium oryzae (Cochli-obolus miyabeanus) (ITO and KURIBAYASHI) DPECH-SLER ex DASTUR. The former organism, an important plant pathogen causing tomato wilt in Egypt, was isolated from infected tomato plants. The latter organism was isolated from infected rice plants.
The newly synthesized products were dissolved in aqueous ethanol to give a logarithmic series of concentrations from 2 to 256 mg/L upon tenfold dilution with the growth medium and spore suspension of the test fungi. The toxicity of compounds was determined by sporeling bioassay described by Spendley and Ride28 which is based on the technique of Skipp and Bailey,29 a suspension of fungal spores was prepared in water and pipetted into the wells of multi-well slides, followed with 25 pL of the culture medium. The inoculated slides were then incubated at 25 °C until short germ tubes appeared, approximately 50 pm in length (at 0 h) was calculated. Five pL volumes of the prepared compound test solutions were added to the inoculated wells, one control well on each slide being treated with solvent only. The slides were then returned to the incubator until germ tubes 400±50 pm long were visible in the control wells. Further growth was arrested by the addition of lactophenol aniline blue to each of the wells. Based on these assays, the percent inhibition of germ-tube growth (with respect to the controls) was plotted against the logarithm of concentration of each compound. From this, the concentrations producing 50% inhibition (ED50) and 100% inhibition (MLD) were directly obtained. When the ED50 or MLD values exceeded the maximum concentrations of compound used, extrapolation was performed when the last point was within 5% of the ED50 or MLD line, otherwise the result was expressed as > 256 mg/L.
Growth. Since some compounds are lethal at relatively high doses and others at lower doses, comparison of the effect of compound on the growth, sporulation and nucleic acid synthesis of the test fungi was undertaken at a concentration of 64 mg/L.
A series of conical flasks (250 mL capacity) containing 50 mL Czapek-Dox liquid medium were used for each fungus. Each of three flasks was supplemented with 64 mg/L of each compound. The flasks were inoculated with a 5-mm diameter agar disc cut from the margin of actively growing colonies. The flasks were incubated at 28 °C for 7 d after which the produced mycelial felts were collected, washed several times with distilled water and oven-dried at 80 °C to constant mass.
Sporulation. Plates of Czapek-Dox agar supplemented with 64 mg/L of each compound were inoculated with a 5-mm diameter agar disc of the used fungus. The plates were then incubated for 7 d at 28 °C. A 1 cm2 section was cut from the margin of the colony and transferred to a vial containing 10 mL sterile distilled water. The suspension was spontaneously shaken for 5 min and the concentration of spores per mL was counted in a hemocy-tometer. Three plates were used for each treatment.
Nucleic acids. The nucleic acids (RNA and DNA) of each fungus were estimated in the Mycelia harvested from liquid Czapek-Dox medium amended with 64 mg/L of each thiophene derivative after 7 d of incubation at 28 °C. The method used for quantitative determination of RNA is that of Ashwell.18 It depends on a colorimetric analysis of ribose, using the oreintol reaction. The quantitative estimation of DNA depends on measuring the colour developed after treating the extracted DNA with diphenylamine reagent.
Table 1: Measured concentrations (mg/L) of 18 compounds producing 50% inhibition and 100% inhibition (MLD) of Fusarium oxysporum f. sp. Lycopersici and Helminthosporium oryzae
Compound F. oxysporum f. sp. Lycopersici H. oryzae No.	ED50 MLD	ED50 MLD
3a	10	88	68	50
3b	12	70	15	78
3c	11	80	12	70
3d	12	66	30	66
4a	80	250	196	>256
4b	88	230	190	210
4c	60	180	166	80
4d	78	158	180	60
18a	90	236	244	78
18b	>256	> 256	> 256	>256
19a	12	78	29	82
19b	31	80	36	118
20a	80	199	250	220
20b	20	88	30	112
20c	120	230	110	205
20d	12	60	36	63
21a	11	72	24	68
21b	80	206	73	201
Most of the tested compounds showed significant toxicity which is dependent on their chemical structure. The toxicity pattern of the compounds toward the two fungi is similar although the levels of compounds that were required to produce ED50 and MLD for Helminthospo-rium oryzae were higher than those required for Fusarium oxyporum f. sp. Lycopersici. It is clear from table I that among the 18 tested compounds, the annulated derivative (with the 5-oxo group) 18b showed the highest activity towards Fusarium oxysporum f. sp. Lycopersici and H. oryzae, although 18a with the same structure with 9-imino group showed less activity. Comparing the series of compounds 3a-d, it is obvious that 3a showed the least activity towards F. oxysporum but the highest towards Lycopersici. on the other hand, comparing the triazolyl derivatives 20a-d, one can notice that compound 20c with the substituted ester and N-H groups showed highest activities towards F oxysporum f. sp. Lycopersici but lowed for the ED50 of H. oryzae.
It is clear from table I that among the 18 tested compounds, the annulated derivative (with the 4-oxo group) 18b showed the highest activity towards F. oxysporum f. sp. Lycopersici (ED50 >256) and H. oryzae, although 18a with the same structure with 4-imino group showed less activity (ED50 = 90). Comparing the series of compounds 3a-d, it is obvious that 3a showed the least activity towards F. oxysporum but the highest towards Lycopersici. on the other hand, comparing the 1,2,4-triazolyl derivatives 20a-d, one can notice that compound 20c with the substituted ester and N-H groups showed highest activi-
Table 2: Effect of 64 mg/L of 18 compound on mycelial dry mass, sporulation and nucleic acid synthesis of Fusarium oxysporum f. sp. Lycopersici
Com-	Mycelial	Sporulation	Nucleic acid	
pound	dry mass	spores,	mg/g dry	mass
No.	Mg/50 mL	X 10-5/mL		
		of culture	DNA	RNA
3a	136	30.2	16.0	0.32
3b	104	28.5	10.0	0.23
3c	120	26.6	8.2	0.33
3d	110	22.2	6.1	0.45
4a	228	24.0	11.6	0.44
4b	100	10.2	6.3	0.32
4c	240	12.6	8.4	0.26
4d	120	24.6	10.2	0.22
18a	106	20.2	6.2	0.30
18b	102	23.2	10.3	0.16
19a	214	20.3	10.7	0.34
19b	258	23.8	10.4	0.22
20a	330	20.8	18.7	0.33
20b	225	8.9	6.3	0.84
20c	230	8.5	7.3	0.23
20d	226	36.6	12.5	0.33
21a	130	18.9	5.8	0.26
21b	198	33.5	18.6	0.23
ties towards F. oxysporum f. sp. Lycopersici but lower for the ED50 of H. oryzae.
The effect of all tested compounds on growth, sporulation and nucleic acid synthesis was tested at a concentration of 64 mg/L. Compound 20c allowed good mycelial growth, sporulation and nucleic acid synthesis by the two fungi. This indicates that the two fungi can use the N-containing heterocyclic ring as a nitrogen source.
Table 3: Effect of 64 mg/L each of 18 compound on mycelial dry mass sporulation and nucleic acid synthesis of Helminthosporium oryzae
Compound F. oxysporum f. sp. Lycopersici H. oryzae
No.	ED50		MLD		ED50	MLD
3a	220		28.2		23.0	0.36
3b	240		50.8		22.0	0.26
3c	180		46.6		20.2	0.24
3d	210		48.6		18.2	0.26
4a	140		24.0		18.6	0.32
4b	266		16.2		16.4	0.24
4c	240		18.8		16.8	0.42
4d	233		14.8		14.4	0.26
18a	136		22.2		12.8	0.26
18b	108		14.2		9.7	0.24
19a	245		21.8		9.7	0.26
19b	266		26.8		10.4	0.32
20a	385		26.6		10.2	0.33
20b	205		16.9		8.3	0.14
20c	203		23.9		6.8	0.22
20d	140		22.6		16.5	0.37
21a	270		22.5		10.4	0.22
21b	233		28.5		8.6	0.13
LSDa at	1%	37.2		5.1	3.6	0.05
	5%	21.0		3.5	1.9	0.03
From tables II and III it is clear that the 1,2,4-tria-zolyl compound 20a showed the highest activities towards Mycelial dry. on the other hand comparing 21a (annulated derivative with the NH group) and 21b (annulated derivative with the N-Ph group), it is obvious that 21b showed higher activity towards Mycelial dry. Comparing the iso-meric compounds 19b and 21b, the first showed higher activity towards Mycelial (Mg/50 mg 298) dry and Sporulation and nucleic acid synthesis by the two fungi.
4. Experimental
Melting points are uncorrected and were determined in open capillary tubes on a digital Gallen Kamp MFB-595. IR spectra were taken on a Perkin-Elmer FT-IR 1650 spectrophotometer (v, cm-1), using samples in KBr disks, 1H NMR spectra were recorded on a Bruker AC 200 (200 Mz) spectrometer (5 ppm) using DMSO-d6 as solvent and TMS as internal standard.
4.1. Ethyl [(3-cyano-4,5,6,7-tetrahydro-1-benzo-thien-2-yl)amino]carbothioylcarbamate (3a), ethyl (3-(ethoxycarbonyl)-4,5,6,7-tetrahydro-benzo[b ]thiophen-2-ylamino)carbothioyl carbamate (3b), ethyl [(3-cyano-5,5-dimet-hyl-7-oxo-4,5,6,7-tetrahydro-1-benzothien-2-yl)amino]carbothioylcarbamate (3c) and ethyl (3-(ethoxycarbonyl)-4,5,6,7-tetrahydro-5,5-dimethyl-7-oxobenzo[b]thiophen-lami-no)carbothioylcarbamate (3d)
General procedure: Equimolar amounts of either 1a (1.78 g, 0.01 mol), 1b (2.25 g, 0.01 mol), 1c (1.72 g, 0.01 mol) or 1d (2.19 g, 0.01 mol) in 1,4-dioxan (30 mL), ethoxycarbonyl isothiocyanate (1.31 g, 0.01 mol) [prepared by adding ammonium isothiocyanate (0.01 mol) to a solution of ethyl chloroformate (0.01 mol) in 1,4-dioxan (20 mL) and heat for 1/2 h followed by isolation of the byproduct, ammonium chloride] was added. The whole reaction mixture, in each case, was stirred at room temperature overnight and the solid product formed upon pouring onto ice/water was collected by filtration.
Compound 3a: Yellow crystals from acetic acid, yield 70% (2.16 g), m.p. 188-190 °C. Anal. Calculated for C13H15N3O2S2 (309.41): C, 50.46; H, 4.89; N, 13.58; S, 20.73. Found: C, 50.07; H, 5.42; N, 13.88; S, 20.57. IR (v/cm-1): 3460-3324 (2NH), 2980, 2888 (CH3, CH2), 2225 (CN), 1687 (CO), 1638 (C=C), 1205-119(5 (C=S). 1H NMR: 5 1.61 (t, 3H, J = 7.02 Hz, CH3), 2.14-2.16 (m, 4H, 2CH2), 2.23-2.26 (m, 4H, 2CH2), 43.11 (s, 1H, NH), 4.24 (q, 2H, J = 7.02 Hz, CH2), 8.32 (s, 1H, NH). 13C NMR: 5 14.88 (ester CH3), 20.0, 23.3, 23.9, 24.7 (4CH2), 60.45 (ester CH2), 118.8 (CN), 122.3, 136.7, 135.6, 140.8 (thiophene-C), 154.7 (amide C=O), 178.8 (C=S).
Compound 3b: Pale yellow crystals from acetic acid, yield 66% (2.34 g), m.p. 105 °C. Anal. Calculated for C15H20N2O4S2 (356.46): C, 50.54; H, 5.66; N, 7.86; S, 17.99. Found: C, 50.87; H, 5.24; N, 8.31; S, 18.44. IR (v/cm-1): 3456-3339 (2NH), 2986, 2893 (CH3, CH2), 1690, 1685 (2 CO), 1636 (C=C), 1205-1196 (C=S). 1H NMR: 5 1.62, 1.65 (2t, 6H, J = 6.22, 7.04 Hz, 2 CH3), 2.16-2.19 (m, 4H, 2CH2), 2.25-2.29 (m, 4H, 2CH2), 4.30 (s, 1H, NH), 4.22, 4.25 (2q, 4H, J = 6.22, 7.04 Hz, 2 CH2), 8.30 (s, 1H, NH).
Compound 3c: Yellow crystals from acetic acid, yield 66% (2.31 g), m.p. 203-206 °C. Anal. Calculated for C15H17N3O3S2 (351.44): C, 51.26; H, 4.88; N, 11.96; S, 18.25. Found: C, 51.66; H, 5.21; N, 12.08; S, 18.88. IR (v/cm-1): 3465-3323 (2 NH), 2988, 2875 (CH3, CH2), 2225 (CN), 1693, 1687 (2 C=O), 1638 (C=C), 12(35-1198 (C=S). 1H NMR: 5 1.09, 1.10 (2s, 6H, 2CH3), 1.30 (t, 3H, J = 5.66 Hz, CH3), 2.58, 2.86 (2m, 4H, 2CH2), 4.20 (q, 2H, J = 5.66 Hz, CH2), 4.89, 8.30 (2s, 2H, 2NH).
Compound 3d: Pale yellow crystals from acetic acid, yield 62% (2.47 g), m.p. 170 °C. Anal. Calculated for C17H22N2O5S2 (398.50): C, 51.24; H, 5.56; N, 7.03; S, 16.09. Found: C, 50.87; H, 5.99; N, 7.21; S, 15.92. IR
(v/cm-1): 3550-3312 (2 NH), 2991, 2882 (CH3, CH2), 1686, 1682 (2 C=O), 1641 (C=C), 1203-1195 (C=S). 1H NMR: 5 1.11, 1.13 (2s, 6H, 2CH3), 1.32, 1.35 (2t, 6H, J = 6.72, 7.11 Hz, 2CH3), 2.56, 2.83 (2m, 4H, 2CH2), 4.21, 4.23 (2q, 4H, J = 6.72, 711 Hz, 2CH2), 4.92, 8.34 (2s, 2H, 2NH).
4.2. 2-Aminothioxo-(2-hydroxy-8-oxobenzo[d] pyrimidino-1-yl)-4,5,6,7-tetrahydro-5,5-di-methyl-7-oxobenzo[6]thiophen-3-carbonitri-le (7a), ethyl 2-aminothioxo-(2-hydroxy-8-oxobenzo[d]pyrimidino-1-yl)-4,5,6,7-tetrahy-dro-5,5-dimethyl-7-oxobenzo[6]thiophen-3-carboxylate (7b) 6,6-dimethyl-4-imino-3-(8-oxo-benzo[d]1,3-oxazino-2-yl)-8-oxo-1[#]-2-thioxo-5,6,7,8-tetrahydrobenzo[6]thieno[5,4 :4,5]pyrimidine (10a) and 6,6-dimethyl-3-((8-oxo-benzo[d ]1,3-oxazino-2-yl)-4,8-dioxo-1[#]-2-thioxo-5,6,7,8-tetrahydrobenzo[6]thi-eno-[5,4:4,5]-pyrimidin-3-carboxylate (10b)
General procedure: To a solution of either 3c (3.03 g, 0.01 mol), 3d (3.50 g, 0.01 mol), 4c (4.94 g, 0.01 mol) or 4d (5.41 g, 0.01 mol) in dimethylformamide (40 mL), anthranilic acid (1.37 g, 0.01 mol) was added. The reaction mixture was heated under reflux for 10 h then poured onto ice/water. The formed solid product was collected by filtration.
Compound 7a: Yellow crystals from 1,4-dioxan, yield 80% (3.39 g), m.p. 258-262 °C. Anal. Calculated for C20H16N4O3S2 (424.50): C, 56.59; H, 3.80; N, 13.20; S, 1511. Found: C, 56.31; H, 4.09; N, 13.62; S, 14.93. IR (v/cm-1): 3540-3338 (OH, NH), 2984, 2883 (CH3, CH2), 2223 (CN), 1692, 1688 (2 C=O), 1665 (C=N), 1636 (C=C), 1206-1195 (C=S). 1H NMR: 5 1.09, 1.13 (2s, 6H, 2CH3), 2.55, 2.87 (2m, 4H, 2CH2), 7.33-7.39 (m, 4H, C6H4), 8.32 (s, 1H, NH), 10.22 (s, 1H, OH).
Compound 7b: Yellow crystals from 1,4-dioxan, yield 63% (2.96 g), m.p. 164 °C. Anal. Calculated for C22H21N3O5S2 (471.55): C, 56.04; H, 4.49; N, 8.91; S, 133.60. Found: C, 55.88; H, 4.69; N, 9.28; S, 13.44. IR (v/cm-1): 3560-3321 (OH, NH), 2980, 2881 (CH3, CH2), 1690-1684 (3 C=O), 1660 (C=N), 1639 (C=C), 1202-1198 (C=S). 1H NMR: 5 1.05, 1.10 (2s, 6H, 2CH3), 1.16 (t, 3H, J = 6.29 Hz, CH3), 2.56, 2.84 (2m, 4H, 2CH2), 4.22 (q, 2H, J = 6.29 Hz, CH2), 8.30 (s, 1H, NH), 7.307.39 (m, 4H, C6H4), 10.42 (s, 1H, OH).
Compound 10a: Pale orange crystals from 1,4-diox-an, yield 68% (2.88 g), m.p. 120 °C. Anal. Calculated for C20H16N4O3S2 (424.50): C, 56.59; H, 3.80; N, 13.20; S, 1511. Found: C, 56.08; H, 4.32; N, 13.29; S, 14.88. IR (v/cm-1): 3438-3320 (2 NH), 2980, 2869 (CH3, CH2), 1694, 1684 (2 C=O), 1668 (C=N), 1630 (C=C). 1H NMR: 5 1.05, 1.10 (2s, 6H, 2CH3), 2.50, 2.87 (2m, 4H, 2CH2), 7.32-7.39 (m, 4H, C6H4), 8.32, 8.80 (2s, 2H, 2NH).
Compound 10b: orange crystals from 1,4-dioxan, yield 69% (2.93 g), m.p. 189-94 °C. Anal. Calculated for
C20H15N3O4S2 (425.48): C, 56.46; H, 3.55; N, 9.88; S, 152.07. Found: C, 56.23; H, 3.52; N, 10.09; S, 15.28. IR (v/cm-1): 3427-3332 (NH), 2984, 2874 (CH3, CH2), 1693-1682 (3 C=O), 1656 (C=N), 1632 (C=C). 1H NMR: 5 1.08, 1.13 (2s, 6H, 2CH3), 2.52, 2.85 (2m, 4H, 2CH2), 7.33-7.41 (m, 4H, C6H4), 8.30 (s, 1H, NH).
4.3. Ethyl 1-imino-3-thioxo-4#-6,7,8,9-tetrahy-dro[1]benzothieno[2,3-d]-pyrimidin-3-car-boxylate (4a), ethyl 1-oxo-3-thioxo-4#-6,7,8, 9-tetra-hydro[1]benzothieno[2,3-d]-pyrim-idin-3-carboxylate (4b), ethyl 4-imino-6,6-di-methyl-8-oxo-1.ff-2-thioxo-5,6,7,8-tetrahy-drobenzo[6]thieno-[5,4:4,5]-pyrimidin-3-car-boxylate (4c), ethyl 6,6-dimethyl-4,8-dioxo-1[ff]-2-thioxo-5,6,7,8-tetrahydrobenzo[# ] thieno[5,4:4,5]-pyrimidin-3-carboxylate (4d), 4-imino-6,6-dimet-hyl-2-(2-hydroxy-8-oxobe-nzo[d]-pyrimidin-1-yl)-8-oxo-2-thioxo-5,6,7, 8-tetrahydrobenzo[6]thieno[5,4-d]-1,3-thi-azine (8a) and 4-oxo -6,6-dimethyl-2-(2-hy-droxy-8-oxobenzo[d]-pyrimidin-1-yl)-8-oxo-2-thioxo-5,6, 7,8-tetrahydrobenzo-[6]thieno [5,4-d]-1,3-thiazine (8b)
General procedure: A suspension of either 3a (3.09 g, 0.01 mol), 3b (3.56 g, 0.01 mol), 3c (3.03 g, 0.01 mol), 3d (3.50 g, 0.01 mol), 7a (3.76 g, 0.01 mol) or 7b (4.23 g, 0.01 mol) in sodium ethoxide (0.01 mol) [prepared by dissolving sodium metal (0.23 g, 0.01 mol) in absolute ethanol (40 mL)] was heated in a boiling water bath for 6 h then left to cool. The solid product formed upon pouring onto ice/water containing hydrochloric acid (till pH 6) was collected by filtration.
Compound 4a: Colourless crystals from 1,4-dioxan, yield 62% (1.91 g), m.p. 233-235 °C. Anal. Calculated for C13H15N3O2S2 (309.41): C, 50.46; H, 4.89; N, 13.58; S, 20.73. Found: C, 50.22; H, 5.31; N, 13.88; S, 21.12. IR (v/cm-1): 3442-3326 (2NH), 2982, 2887 (CH3, CH2), 1688 (CO), 1639 (C=C), 1207-1193 (C=S). 1H NMR: 5 1.36 (t, 3H, J = 7.66 Hz, CH3), 1.69-1.72 (m, 4H, 2CH2), 2.20-2.23 (m, 4H, 2CH2), 4.13 (s, 1H, NH), 4.20 (q, 2H J = 7.66 Hz, CH2), 8.26 (s, 1H, NH).
Compound 4b: Pale yellow crystals from ethanol, yield 55% (1.91 g), m.p. 233-235 °C. Anal. Calculated for C13H14N2O3S2 (310.04): C, 50.30; H, 4.55; N, 9.03; S, 2(X66. Found: C, 50.07; H, 4.88; N, 8.88; S, 20.38. IR (v/cm-1): 3456-3336 (NH), 2980, 2890 (CH3, CH2), 1693, 1685 (2 CO), 1636 (C=C), 1204-1190 (C=S). 1H NMR: 5 1.38 (t, 3H, J = 7.21 Hz, CH3), 1.66-1.70 (m, 4H, 2CH2), 2.22-2.26 (m, 4H, 2CH2), 4.22 (s, 1H, NH), 4.24 (q, 2H J = 7.21 Hz, CH2).
Compound 4c: Yellow crystals from acetic acid, yield 70% (2.12 g), m.p. 205-208 °C. Anal. Calculated for C15H17N3O3S2 (351.44): C, 51.26; H, 4.88; N, 11.96; S, 18.25. Found: C, 51.52; H, 4.94; N, 11.36; S, 18.46. IR
(v/cm-1): 3465-3347 (2 NH), 2982, 2877 (CH3, CH2), 1693, 1685 (2 C=O), 1666 (C=N), 1636 (C=C). 1H NMR: 5 1.07, 1.10 (2s, 6H, 2CH3), 1.16 (t, 3H, J = 5.99 Hz, CH3), 2.53, 2.80 (2m, 4H, 2CH2), 4.23 (q, 2H, J = 5.99 Hz, CH2), 8.33, 10.24 (2s, 2H, 2NH).
Compound 4d: Buff crystals from acetic acid, yield 60% (1.82 g), m.p. 180-183 °C. Anal. Calculated for C15H16N2O4S2 (352.36): C, 51.12; H, 4.58; N, 7.95, S, 181.20. Found: C, 51.08; H, 4.89; N, 7.89; S, 18.42. IR (v/cm-1): 3475-3312 (NH), 2976, 2867 (CH3, CH2), 1689-1683 (2 C=O), 1660 (C=N), 1637 (C=C). 1H NMR: 5 1.06, 1.12 (2s, 6H, 2CH3), 1.15 (t, 3H, J = 6.81 Hz, CH3), 2.53, 2.82 (2m, 4H, 2CH2), 4.23 (q, 2H, J = 6.81 Hz, CH2), 8.30 (s, 1H, NH).
Compound 8a: Yellow crystals from DMF, yield 56% (2.37 g), m.p. 184-187 °C. Anal. Calculated for C20H16N4O3S2 (424.50): C, 56.59; H, 3.80; N, 13.20; S, 15i.11. Found: C, 56.77; H, 3.88; N, 13.48; S, 14.79. IR (v/cm-1): 3570-3322 (OH, NH), 2975, 2880 (CH3, CH2), 2223 (CN), 1690, 1688 (2 C=O), 1660 (C=N), 1636 (C=C). 1H NMR: 5 1.06, 1.12 (2s, 6H, 2CH3), 2.51, 2.82 (2m, 4H, 2CH2), 7.31-7.36 (m, 4H, C6H4), 8.35 (s, 1H, NH), 10.28 (s, 1H, OH).
Compound 8b: yellow crystals from acetic acid, yield 48% (2.04 g), m.p. 145 °C. Anal. Calculated for C20H15N3O4S2 (425.48): C, 56.46; H, 3.55; N, 9.88; S, 152.07. Found: C, 56.27; H, 3.73; N, 9.40; S, 15.29. IR (v/cm-1): 3566-3320 (OH, NH), 2988, 2872 (CH3, CH2), 1693, 1684 (2 C=O), 1661 (C=N), 1636 (C=C). 1H NMR: 5 1.02, 1.16 (2s, 6H, 2CH3), 2.55, 2.84 (2m, 4H, 2CH2),
7.30-7.38 (m, 4H, C6H4), 8.35 (s, 1H, NH), 10.40 (s, 1H, OH). 6 4
4.4. Ethyl 3-(3-cyano-4,5,6,7-tetrahydro-5-dimet-hyl-7-oxobenzo[ö]thiophen-2-yl)-3,4-dihy-dro-6-hydroxy-4-oxo-2-thioxopyrimidine-1 (2H)-carboxylate (12a) and ethyl 3-(3-ethoxy-carbonyl)-4,5,6,7-tetrahydro-5-dimethyl-7-oxobenzo[ö]thiophen-2-yl)-3,4-dihydro-6-hy-droxy-4-oxo-2-thioxopyrimidine-1(2.ff)-car-boxylate (12b)
General procedure: To a solution of either 4c (3.03 g, 0.01 mol) or 4d (3.50 g, 0.01 mol) in 1,4-diox-an (40 mL) containing piperidine (0.5 mL), diethyl-malonate (1.60 g, 0.01 mol) was added. The reaction mixture was heated under reflux for 14 h then evaporated under vacuum. The resitue was triturated with carbontetrachloride and the solidified product was collected by filtration.
Compound 12a: orange crystals from acetic acid, yield 50% (2.09 g), m.p. > 300 °C. Anal. Calculated for C18H17N3O5S2 (419.47): C, 51.54; H, 4.08; N, 10.02; S, 15.29. Found: C, 51.88; H, 4.29; N, 10.52; S, 15.44. IR (v/cm-1): 3566-3342 (OH), 3052 (CH aromatic), 2990, 2880 (CH3, CH2), 2223 (CN), 1690-1687 (3 C=O), 1642
(C=C). *H NMR: 5 1.04, 1.13 (2s, 6H, 2CH3), 1.16 (t, 3H, 7.33 Hz, CH3), 2.57, 2.78 (2m, 4H, 2CH2), 4.24 (q, 2H, 7.33 Hz, CH2), 6.95 (s, 1H, pyrimidine H-52), 10.33 (s, 1H, OH).
Compound 12b: orange crystals from acetic acid, yield 59% (2.74 g), m.p. 288-293 °C. Anal. Calculated for C20H22N2O7S2 (466.53): C, 51.49; H, 4.75; N, 6.00; S, 13.75. Found: C, 51.82; H, 4.93; N, 6.31; S, 13.85. IR (v/cm-1): 3550-3326 (OH), 3055 (CH aromatic), 2986, 2880 (CH3, CH2), 1690-1683 (4 C=O), 1662 (C=N), 1642 (C=C). 1H NMR: 5 1.04, 1.13 (2s, 6H, 2CH3), 1.15, 1.17 (2t, J = 6.40, 7.11 Hz, 6H, 2CH3), 2.53, 2.82 (2m, 4H, 2CH2), 4.21, 4.24 (2d, J = 6.40, 7.11 Hz, 4H, 2CH2), 6.87 (s, 1H, pyrimidine H-5), 10.41 (s, 1H, OH).
4.6. Ethyl 5-(2-phenyldiazenyl)-3-(3-cyano-4,5,6,7 -tetrahydro-5,5-dimethyl-7-oxobenzo[6]thio-phen-2-yl)-3,4-dihydro-6-hydroxy-4-oxo-2-thioxopyrimidine-1(2.ff)-carboxylate (14a) and ethyl 5-(2-phenyldiazenyl)-3-(3-(ethoxy-carbonyl)-4,5,6,7-tetrahydro-5,5-dimethyl-7-oxobenzo[6]thiophen-2-yl)-3,4-dihydro-6-hy-droxy-4-oxo-2-thioxopyrimidine-1(2.ff)-car-boxylate (14b)
General procedure: To a cold solution (0-5 °C) of either 12a (3.71 g, 0.01 mol) or 12b (4.18 g, 0.01 mol) in ethanol (80 mL) containing sodium hydroxide (10 mL, 10%), benzenediazonium chloride [prepared by the addition of sodium nitrite solution (0.7 g, 0.01 mol) to a cold solution (0-5 °C) of aniline (0.94 g, 0.01 mol) dissolved in the appropriate amount of hydrochloric acid with continuous stirring] was added with continuous stirring for 3 h. The formed solid product was collected by filtration.
Compound 14a: Redish brown crystals from acetic acid, yield 72% (3.76 g), m.p. 205-207 °C. Anal. Calculated for C24H21N5O5S2 (523.58): C, 55.05; H, 4.05; N, 13.38; S, 12.25. Found: C, 55.47; H, 4.39; N, 13.88; S, 12.28. IR (v/cm-1): 3549-3322 (OH), 3062 (CH aromatic), 2987, 2878 (CH3, CH2), 2227 (CN), 1692-1684 (3 C=O), 1635 (C=C), 1204-1198 (C=S). 1H NMR: 5 1.06, 1.14 (2s, 6H, 2CH3), 1.15 (t, J = 6.21 Hz, 3H, CH3), 2.59, 2.74 (2m, 4H, 2C3H2), 4.23 (q, 2H, J = 6.21 Hz3, CH2), 7.31-7.37 (m, 5H, C6H5), 10.32 (s, 1H, OH).
Compound 14b: Reddish orange crystals from acetic acid, yield 83% (4.73 g), m.p. 188-192 °C. Anal. Calculated for C26H26N4O7S2 (570.64): C, 54.72; H, 4.59; N, 9.82; S, 11.24. Found: C, 54.45; H, 4.88; N, 10.23; S, 11.45. IR (v/cm-1): 3562-3343 (OH), 3057 (CH aromatic), 2982, 2879 (CH3, CH2), 1688-1680 (4 C=O), 1640 (C=C), 1205-1196 (C=S). 1H NMR: 5 1.02, 1.14 (2s, 6H, 2CH3), 1.16, 1.18 (2t, 6H, J = 5.98, 6.71 Hz, 2CH3), 2.55, 23.80 (2m, 4H, 2CH2), 4.22, 4.26 (2q, 4H, J = 5.98, 6.71 Hz, 2CH2), 7.27-7.34 (m, 5H, C6H5),
10.33 (s, 1H, OH).	2	6 5
4.7. 2-(5-Hydrazono-3-hydroxy-7-oxo-[1,2,4]tria-zolo[4,3-/]pyrimidm-6(1tf,5tf,7tf)-yl)-4,5,6, 7-tetrahydro-5,5-dimethyl-7-oxobenzo[#]thi-ophene-3-carbonitrile (16a), 2-(5-hydrazono-7-oxo-3-phenyl-[1,2,4]triazolo[4,3-/jpyrimi-din-6(1H,5H,7H)-yl)-4,5,6,7-tetrahydro-5,5-dimethyl-7-oxobenzo[#]thiophene-3-carboni-
trile (16b), ethyl 2-(5-hydrazno-7-oxo-3-phe-
nyl-[1,2,4]triazolo[4,3-/]pyrimidin-6(1H, 5H,7H)-yl)-4,5,6,7-tetrahydro-5,5-dimethyl-7-oxobenzo[#]thiophene-3-carboxylate (16c) and ethyl 2-(5-(2-phenylhydrazono)-7-oxo-1,3-diphenyl[1,2,4]triazolo[4,3-/]pyrimidin-6(1H,5H,7H)-yl)-4,5,6,7-tetrahydro-5,5-di-methyl-7-oxobenzo[#]thiophene-3-carboxy-late (16d)
General procedure: To a solution of either 12a (3.71 g, 0.01 mol) or 12b (4.18 g, 0.01 mol) in 1,4-dioxan (40 mL), either hydrazine hydrate (1.0 g, 0.02 mol) or phenyl-hydrazine (2.16 g, 0.01 mol) was added. The reaction mixture was heated under reflux for 2 h then poured onto ice/water containing few drops of hydrochloric acid and the solid product formed was collected by filtration.
Compound 16a: Yellowish white crystals from 1,4-dioxan, yield 70% (2.35 g), m.p. 190-193 °C. Anal. Calculated for C16H15N7O3S (385.40): C, 49.86; H, 3.92; N, 25.44; S, 8.32. Found: C, 49.57; H, 3.66; N, 25.06; S, 8.45. IR (v/cm-1): 3533-3324 (OH), 3050 (CH aromatic), 2983, 2874 (CH3, CH2), 2223 (CN), 1690-1686 (3 C=O), 1630 (C=C). 1H NMR.: 5 1.05, 1.15 (2s, 6H, 2CH3), 2.53, 2.67 (2m, 4H, 2CH2), 4.66 (s, 2H, NH2), 6.91 (s 1H, pyrimidine H-5), 8.21 (s, 1H, NH), 10.30 (s, 1H, OH).
Compound 16b: Yellow crystals from 1,4-dioxan, yield 55% (2.53 g), m.p. 209-212 °C. Anal. Calculated for C28H23N7O3S (537.59): C, 62.56; H, 4.31; N, 18.24; S,
5.2988.2 3Fo7un3d: C, 62.09; H, 4.09; N, 18.78; S, 6.34. IR
(v/cm-1): 3488-3326 (OH), 3053 (CH aromatic), 2980, 2866 (CH3, CH2), 2225 (CN), 1684, 1682 (2 C=O), 1655 (C=N), 1636 (C=C). 1H NMR: 5 1.06, 1.13 (2s, 6H, 2CH3), 2.52, 2.76 (2m, 4H, 2CH2), 6.99 (s, 1H, pyrimidine H-5), 7.27-7.38 (m, 10H, 2C6H5), 8.22 (s, 1H, NH), 10.28 (s, 1H, OH).	6 5
Compound 16c: Yellow crystals from 1,4-dioxan, yield 63% (2.72 g), m.p. 177-182 °C. Anal. Calculated for C18H20N6O5S (432.45): C, 49.99; H, 4.66; N, 19.43; S, 7.481. Found: C, 50.33; H, 4.38; N, 19.06; S, 7.83. IR (v/cm-1): 3529-3320 (OH, NH, NH2), 3050 (CH aromatic), 2984, 2873 (CH3, CH2), 2222 (CN), 1695-1683 (3 C=O), 1636 (C=C). 1H NMR: 5 1.03, 1.15 (2s, 6H, 2CH3), 1.16 (t, 3H, J = 6.99 Hz, CH3), 2.50, 2.68 (2m, 4H, 2CH2), 4.24 (q, 2H, J = 6.99 Hz, CH2), 4.77 (s, 2H, NH2), 6.92 (s, 1H, pyrimidine H-5), 8.02 (s, 1H, NH), 10.28 (s, 1H, OH).
Compound 16d: orange crystals from 1,4-dioxan, yield 48% (2.80 g), m.p. 120 °C. Anal. Calculated for C^H^NgOjS (584.65): C, 61.63; H, 4.83; N, 14.37; S, 5.408. Found: C, 61.44; H, 4.67; N, 14.86; S, 5.92. IR
(v/cm-1): 3534-3343 (OH, NH), 3062 (CH aromatic), 2980, 2879 (CH3, CH2), 1689-1681 (3 C=O), 1637 (C=C). 1H NMR: 5 1.04,1.13 (2s, 6H, 2CH3), 1.16 (t, 3H, J = 7.51 Hz, CH3), 2.52, 2.84 (2m, 4H, 2CH2), 4.24 (q, 2H, J = 7.51 Hz, CH2), 6.92 (s, 1H, pyrimidine H-5),
7.28-7.36 (m, 10H, 2C6H5), 8.09 (s, 1H, NH), 10.26 (s, 1H, OH). 6 5
4.8. 3-Hydroxy-4-imino-10#-5,6,7,8-tetrahydro-benzo[ö]thieno[2,3:4,5]- pyrimidine[1,2:4,5] 1,2,4-triazole (18a), 3-hydroxy-4-oxo-10#-5, 6,7,8-tetrahydrobenzo[#]thieno[2,3:4,5]pyri-midine[1,2:4,5]1,2,4-triazole (18b), 5-imino-11[#]-2-phenyl-6,7,8,9-tetrahydrobenzo[£] thienothieno[2,3:4,5]- pyrimidine[1,2:4,5]1, 2,4-triazole (19a) and 3,4-dioxo-10#-2-phenyl-5,6,7,8-tetrahydrobenzo[ö]thieno[2,3 :4,5]pyrimidine[1,2:4,5]1,2,4-triazole (19b)
General procedure: To a solution of either 4a (3.09 g, 0.01 mol) or 4b (3.10 g, 0.01 mol) in DMF (40 mL) either hydrazine hydrate (0.50 g, 0.01 mol) or Phenylhydrazine (1.08 g, 0.01 mol) was added. The reaction mixture, in each case was heated under reflux for 2 h till evolution of hydrogen sulphide ceased. The reaction mixture, in each case, was left to cool then poured onto ice/water containing few drops of hydrochloric acid (till pH 6) and the formed solid product was collected by filtration.
Compound 18a: White crystals from 1,4-dioxan, yield 55% (1.43 g), m.p. 166-169 °C. Anal. Calculated for C11H11N5OS (261.30): C, 50.56; H, 4.24; N, 26.80; S, 12.27. Found: C, 50.93; H, 4.47; N, 27.31; S, 12.58. IR (v/cm-1): 3555-3312 (OH, 2NH), 1670 (exocyclic C=N), 1643 (C=C). 1H NMR: 5 1.66-1.70 (m, 4H, 2CH2), 2.23-2.27 (m, 4H, 2CH2), 4.42 (s, 1H, NH), 5.88 (s, 1H, OH), 8.33 (s, 1H, NH). 13C NMR: 5 23.5, 23.8, 25.9, 33.4 (4 CH2), 126.2, 128.1, 136.9, 141.2 (thiophene C), 156.1, 159.6 (2 C=N), 166.2 (C=NH).
Compound 18b: Yellowish white crystals from 1,4-dioxan, yield 62% (1.62 g), m.p. 233-236 °C. Anal. Calculated for C11H10N4O2S (262.29): C, 50.37; H, 3.84; N, 21.36; S, 12.23. Found: C, 50.56; H, 4.22; N, 21.67; S, 12.62. IR (v/cm-1): 3465-3334 (NH), 1690 (CO), 1640 (C=C). 1H NMR: 5 1.68-1.74 (m, 4H, 2CH2), 2.20-2.27 (m, 4H, 2CH2), 5.88 (s, 1H, NH), 6.02 (s, 1H, OH).
Compound 19a: Yellow crystals from DMF, yield 56% (1.88 g), m.p. 166-169 °C. Anal. Calculated for C17H15N5OS (337.4): C, 60.52; H, 4.48; N, 20.76; S, 9.50. Found: C, 60.33; H, 4.82; N, 20.68; S, 9.91. IR (v/cm-1): 3555-3312 (NH), 1670 (exocyclic C=N), 1643 (C=C). 1H NMR: 5 1.64-1.72 (m, 4H, 2CH2), 2.25-2.29 (m, 4H, 2CH2), 4.48 (s, 1H, NH), 5.87 (s, 1H, NH), 7.32-7.43 (m, 5H, C6H5).
Compound 19b: Yellow crystals from DMF, yield 70% (2.36 g), m.p. > 300 °C. Anal. Calculated for C17H14N4O2S (338.38): C, 60.34; H, 4.17; N, 16.56; S,
9.48. Found: C, 60.31; H, 4.26; N, 16.84; S, 9.78. IR (v/cm-1): 3555-3312 (OH, NH), 1660 (C=N), 1640 (C=C). 1H NMR: 5 1.68-1.74 (m, 4H, 2CH2), 2.24-2.28 (m, 4H, 2CH2), 4.48 (s, 1H, NH), 7.29-7.35 (m, 5H, C6H5).
4.9. 2-(5-Hydroxy-2#-1,2,4-triazol-3-ylamino)-4, 5,6,7-tetrahydrobenzo[6]thiophene-3-caboni-trile (20a), 2-(5-hydroxy-2-phenyl-2#-1,2,4-triazol-3-ylamino)-4,5,6,7-tetrahydroben-zo[6]thiophene-3-carbonitrile (20b), ethyl 2-(5-hydroxy-2.ff-1,2,4-triazol-3-ylamino)-4,5,6,7-tetrahydrobenzo[6]thiophene-3-car-
boxylate (20c) and ethyl 2-(5-hydroxy-2-
phenyl-2#-1,2,4-triazol-3-ylamino)-4,5,6,7-tetrahydrobenzo[6]thiophene-3-carboxylate (20d)
General procedure: To a solution of either 3a (3.09 g, 0.01 mol) or 3b (3.65 g, 0.01 mol) in 1,4-dioxan (40 mL) either hydrazine hydrate (0.5 g, 0.01 mol) or phenyl-hydrazine (1.08 g, 0.01 mol) was added. The reaction mixture, in each case, was heated under reflux for 6 h then poured onto ice/water containing hydrochloric acid (till pH 6) and the solid product formed was collected by filtration.
Compound 20a: Yellowish white crystals from acetic acid, yield 72% (1.87 g), m.p. 140 °C. Anal. Calculated for C11H11N5OS (261.30): C, 50.56; H, 4.24; N, 26.80; S, 12.21?. Found: C, 50.83; H, 4.66; N, 27.31; S, 11.92. IR (v/cm-1): 3578-3312 (OH, 2 NH), 2220 (CN), 1660 (C=N), 1636 (C=C). 1H NMR: 5 1.70-1.74 (m, 4H, 2CH2), 2.23-2.28 (m, 4H, 2CH2), 4.46, 6.22 (2s, 2H, 2NH), 12.02 (s, 1H, OH).
Compound 20b: Yellow crystals from acetic acid, yield 54% (1.82 g), m.p. 268-272 °C. Anal. Calculated for C17H15N5OS (337.40): C, 60.52; H, 4.48; N, 20.76; s, 9.50. Found: C, 60.32; H, 4.79; N, 20.85; S, 9.72. IR (v/cm-1): 3566-3332 (OH, NH), 3055 (CH aromatic), 2222 (CN), 1662 (C=N), 1633 (C=C). 1H NMR: 5 1.66-1.70 (m, 4H, 2CH2), 2.24-2.27 (m, 4H, 2CH2), 4.39,
5.99 (2s, 2H, 2NH), 7.08-7.38 (m, 5H, C6H5), 12.21 (s, 1H, OH). 6 5
Compound 20c: orange crystals from acetic acid, yield 69% (1.82 g), m.p. 268-272 °C. Anal. Calculated for C13H16N4O3S (308.36): C, 50.64; H, 5.23; N, 18.17; s, 10.40. Found: C, 50.28; H, 4.88; N, 17.79; S, 10.68. IR (v/cm-1): 3569-3322 (OH, 2 NH), 3057 (CH aromatic), 1689 (CO), 1670 (C=N), 1636 (C=C). 1H NMR: 5 1.33 (t, 3H, J = 7.43 Hz, CH3), 1.64-1.72 (m, 4H, 2CH2), 2.26-2.29 (m, 4H, 2CH2), 4.23 (q, 2H, J = 7.43 Hz, CH2), 6.20 (2s, 2H, 2NH), 11.87 (s, 1H, OH).
Compound 20d: Buff crystals from DMF, yield 60% (2.30 g), m.p. 180-183 °C. Anal. Calculated for C19H20N4O3S (384.45): C, 59.36; H, 5.24; N, 14.57; S, 8.334. Found: C, 59.04; H, 4.92; N, 14.79; S, 8.02. IR
(v/cm-1): 3544-3339 (OH, NH), 3051 (CH aromatic), 1687 (CO), 1663 (C=N), 1639 (C=C). 1H NMR: 5 1.36 (t, 3H, J = 7.03 Hz, CH3), 1.68-1.73 (m, 4H, 2CH2), 2.24-2.28 (m, 4H, 2CH2), 4.26 (q, 2H, J = 7.03 Hz, CH2), 4.38 (s, 1H, NH), 7.33-7.42 (m, 5H, C6H5), 11.92 (s, 1H, OH).
4.10.	3-Hydroxy-1#-4-oxo-5,6,7,8-tetrahydroben-zo[#]thieno[2,3:4,5]-pyrimidine[1,2:4,5]1, 2,4-triazole (21a) and 3-hydroxy-1-phenyl-5-oxo- 5,6,7,8-tetrahydrobenzo[#]thieno[2, 3:4,5]pyrimidine[1,2:4,5]1,2,4-triazole (21b)
General Procedure: A solution of either 20a (2.61 g, 0.01 mol), 20b (3.37 g, 0.01 mol), 20c (3.08 g, 0.01 mol) or 20d (3.84 g, 0.01 mol) in ethanol (40 mL) containing sodium hydroxide (0.40 g, 0.01 mol) was heated under reflux for 8 h then left to cool. The solid product formed, in each case, upon pouring onto water containing hydrochloric acid (till pH 6) was collected by filtration and identified as either 11a from 10a,c or 11b from 10b,d.
Compound 21a: Yellow crystals from DMF, yield 73% (1.91 g) from 20a, and 58% (1.52 g) from 20c, m.p. >300 °C. Anal. Calculated for C11H10N4O2S (262.29): C, 50.37; H, 3.84; N, 21.36; S, 12.23. Found: C, 50.08; H, 4.31; N, 21.57; S, 11.92. IR (v/cm-1): 3585-3312 (OH, NH), 3058 (CH aromatic), 1692 (CO), 1660 (C=N), 1636 (C=C). 1H NMR: 5 1.68-1.73 (m, 4H, 2CH2), 2.22-2.26 (m, 4H, 2CH2), 4.44 (s, 1H, NH), 12.24 (s, 1H, OH). MS: m/z 262.05 (M+, 100%).
Compound 21b: Pale brown crystals from DMF, yield 58% (1.96 g) from 20b, and 67 (2.26 g) from 20d, m.p. 222-225 °C. Anal. Calculated for C17H14N4O2S (338.38): C, 60.34; H, 4.17; N, 16.56; S, 9.48. Found: C, 60.07; H, 3.77; N, 16.52; S, 9.79. IR (v/cm-1): 3577-3330 (OH, NH), 3063 (CH aromatic), 1688 (CO), 1663 (C=N), 1633 (C=C). 1H NMR: 5 1.67-1.70 (m, 4H, 2CH2), 2.24-2.37 (m, 4H, 2CH2), 7.27-7.39 (m, 5H, C6H5), 12.18
(s, 1H, OH).	2	6 5
4.11.	4-Oxo-3-phenylhydrazido-2-thioxo-1# -5,6,7,8-tetrahydro[#]-benzothieno[2,3:4,5] pyrimidine (22)
General procedure: To a solution of 4b (3.10 g, 0.01 mol) in 1,4-dioxan (40 mL) phenylhydrazine (1.08 g, 0.01 mol) was added. The reaction mixture was stirred at room temperature for 24 h and the formed crystals were collected by filtration.
Compound 22: Pale yellow crystals from ethanol, yield 55% (1.91 g), m.p. 266-269 °C. Anal. Calculated for C17H16N4O2S2 (372.46): C, 54.77; H, 4.79; N, 15.03; S, 17.181; Found; IR (v/cm-1): 3463-3318 (3NH), 2890 (CH2), 1690, 1685 (2 CO), 1633 (C=C), 1202-1193 (C=S). 1H NMR: 5 1.65-1.69 (m, 4H, 2CH2), 2.21-2.26
(m, 4H, 2CH2), 7.29-7.38 (m, 5H, C6H5), 8.30, 8.32, 8.35 (3s, 3H, 3NH).
4.12. Conversion of Compound 22 into 21b
A solution of 12 (3.72 g, 0.01 mol) in dimethylfor-mamide (30 mL) was heated under reflux for 4 h (till all evolution of H2S ceased). The reaction mixture was poured onto ice/water and the formed solid product was filtered off, crystallized from 1,4-dioxan and identified as compound 12 (m.p., mixed m.p.), yield 60% (1.98 g).
5. Conclusions
The work described presents the synthesis of fused thiophene derivatives, most of the newly synthesized products showed high antifungal activities. Among the tested compounds the tetrahydrobenzo[b]thieno[2,3:4,5] pyrimidine-[1,2:4,5]1,2,4-triazolo derivative 18b showed the highest activity towards F. oxysporum f. sp. Lycopersi-ci and H. oryzae, although 18a with the same structure with 9-imino group showed less activities. Different isomers of the 6,7,8,9-tetrahydro[b]thieno [2,3:4,5]-1,2,4-tri-azolo[1,2:3,4]pyrimidine derivatives like 19b (with the 2-phenyl group) and 21b (with 1-phenyl group) were synthesized and different activities were noticed. Thus, 19b showed higher activity (Mg/50 mg 258) towards Mycelial dry and Sporulation and nucleic acid synthesis by the two fungi (Table II) while 21b showed lower activities (Mg/50 mg 198).
6. Acknowledgements
The author thanks Professor S. A. ouf, Professor at Botany Department, Cairo University, Faculty of Science, A. R. Egypt for recording biological tests for the synthesized compounds.
7. References and Notes
1.	S. Reyes, K. Burgess, J. org. Chem. 2006, 71, 2507-2509.
2.	P. Edman, G. Begg, Eur. J. Biochem. 1967, 1, 80-91.
3.	A. LeTiran, J. P. Stables, H. Kohn, Bioorg. Med. Chem. 2001, 9, 2693-2708.
4.	G. Evindar, R. A. Batey, org. Lett. 2003, J, 1201-1204.
5.	R. A. Mekheimer, R. M. Shaker, J. Chem. Research (S) 1999, 76-77, J. Chem. Research (M) 1999, 449-459.
6.	R. Murugan, E. F. V. Scriven, Aldrich Chim. Acta 2003, 36(1), 21-27.
7.	W. W. Wardakhan, H. M. Gaber, S. A. ouf, S. M. Sherif, Phosphorous, Sulfur & Silicon 2005, 180(2), 601-618.
8.	W. W. Wardakhan, Egypt. J. Chem. 2005, 48(3), 325-338.
9. W. W. Wardakhan, S. A. ouf, Egypt. J. Chem. 2005, 48(4), 393-406.
10.	H. F. Zohdi, W. W. Wardakhan, S. H. Doss, R. M. Mohareb, J. Chem. Research (S) 1996, 440-441, J. Chem. Research (M) 1996, 2526-2545.
11.	R. M. Mohareb, M. H. Mohamed, W. W. Wardakhan, Phosphorous, Sulfur and Silicon 2000, 167, 29-39.
12.	M.-J. R. P. Queiroz, I. C. F. R. Ferreira, R. C. Calhelha, L. M. Estevinho, Bioorg. Med. Chem. 2007, 15, 1788-1794.
13.	A. D. Pillai, P. D. Rathod, F. P. Xavier, H. Padh, V. Sudarsa-nam, K. K. Vasu, Bioorg. Med. Chem. 2005, 13, 6685-6692.
14.	A. D. Pillai, S. Rani, P. D. Rathod, F. P. Xavier, K. K. Vasu, H. Padh, V. Sudarsanam, Bioorg. Med. Chem. 2005, 13, 1275-1283.
15.	N. Fokialakis, C. L. Cantrell, S. o. Duke, A L. Skaltsounis, D. E. Wedge, J. Agric. Food Chem. 2006, 54(5), 1651-1655.
16.	S. Tehranchian, T. Akbarzadeh, M. R. Fazeli, H. Jamalifar, A. Shafiee, Bioorg. Med. Chem. Lett. 2005, 15, 1023-1025.
17.	M. Wujec, M. Pitucha, M. Dobosz, U. Kosikowska, A. Malm, Acta Pharm. 2004, 54(3), 251-260.
18.	I. K. Kostakis, R. Tenta, N. Pouli, P. Marakos, A.-L. Skalt-sounis, H. Pratsinis, D. Kletsas, Bioorg. Med. Chem. Lett. 2005, 15, 5057-5060.
19.	C. E. P. Goncales, D. Araldi, R. B. Panatieri, J. B. T. Rocha, G. Zeni, C. W. Nogueira, Life Sci. 2005, 76(19), 2221-2234.
20.	J. X. Qiao, X. Cheng, D. P. Modi, K. A. Rossi, J. M. Luet-tgen, R. M. Knabb, P. K. Jadhav, R. R. Wexler, Bioorg. Med. Chem. Lett. 2005, 15, 29-35.
21.	W. W. K. R. Mederski, B. Cezanne, C. van Amsterdam, K.-U. Bühring, D. Dorsch, J. Gleitz, J. März, C. Tsaklakidis, Bioorg. Med. Chem. Lett. 2004, 14, 5817-5822.
22.	J. Yang, W.-Y. Hua, F.-X. Wang, Z.-Y. Wang, X. Wang, Bioorg. Med. Chem. 2004, 12, 6547-6557.
23.	H. Fuerstenwerth, Ger. offen. DE 1985, 3, 294-344, Chem Abstr. 1985, 103, 215152.
24.	E. Palitis, E. Gudriniece, V. Barkane, Latv. PSR Zinat. Akad. Vestis., Kim. Ser. 1986, (5), 633-642, Chem. Abstr. 1987, 107, 77573.
25.	S. Reyes, K. Burgess, J. org. Chem. 2006, 71, 2507-2509.
26.	R. M. Mohareb, A. Habashi, H. Z. Shams, S. M. Fahmy, Arch. Pharm. 1987, 320, 599-604.
27.	R. M. Mohareb, S. El-Kousy, A. M. El-Torgoman, Collect. Czech. Chem. Commun. 1992, 57, 1747-1754.
28.	P. J. Spendley, J. P. Ride, Mycol. Soc. 1984, 82, 283-292.
29.	R. A. Skipp, J. A. Bailey, Physiol. Plant Pathol. 1976, 9, 253-264.
Povzetek
V prispevku je opisana reakcija derivatov 2-amino-tetrahidrobenzoMofena 1a-d z etoksikarbonilizotiocianatom (2) do derivatov tetrahidrobenzoMofen-2-tiouree 3a-d. Ti produkti hitro ciklizirajo, če jih segrevamo v raztopini natrijevega etoksida, do anulenov 4a-d. V primeru, da spojine 3a-d podvržemo hetero-ciklizaciji, nastanejo kondenzirani derivati tiofena z antimikrobnim in antifungicidnim učinkom.