Facile Synthesis and Anticancer Activity of Novel 4-Aminothieno[2,3-d]pyrimidines and Triazolothienopyrimidines

Full text of DOI:10.1080/00304948.2019.1666635

Organic Preparations and Procedures International
The New Journal for Organic Synthesis
ISSN: 0030-4948 (Print) 1945-5453 (Online) Journal homepage: https://www.tandfonline.com/loi/uopp20
Facile Synthesis and Anticancer Activity of
Novel 4-Aminothieno[2,3-d]pyrimidines and
Triazolothienopyrimidines
Fatma S. M. Abu El-Azm, Amira T. Ali & Mohamed H. Hekal
To cite this article: Fatma S. M. Abu El-Azm, Amira T. Ali & Mohamed H. Hekal (2019):
Facile Synthesis and Anticancer Activity of Novel 4-Aminothieno[2,3-d]pyrimidines and
Triazolothienopyrimidines, Organic Preparations and Procedures International, DOI:
10.1080/00304948.2019.1666635
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Organic Preparations and Procedures International, 0:1–14, 2019
# 2019 Taylor & Francis Group, LLC
ISSN: 0030-4948 print / 1945-5453 online
DOI: 10.1080/00304948.2019.1666635
Facile Synthesis and Anticancer Activity of Novel
4-Aminothieno[2,3-d]pyrimidines and
Triazolothienopyrimidines
Fatma S. M. Abu El-Azm, Amira T. Ali, and
Mohamed H. Hekal
Chemistry Department, Faculty of Science, Ain Shams University, Abbassia
11566, Cairo, Egypt
Thienopyrimidines play important roles in numerous biological activities.1 They are
known to display multiple therapeutic properties, such as anti-inflammatory,2 antimicro-
bial,3,4 antiproliferative,5 antidiabetic6 and analgesic activities.7 They are structural ana-
logs of biogenic purines and can be considered as potential nucleic acid
antimetabolites.8 Consequently, preparation of thienopyrimidine derivatives with inter-
esting biological activities has attracted particular attention and a variety of methods for
the synthesis of thienopyrimidines have been developed.9–11
The most common method for synthesis of 4-aminothieno[2,3-d]pyrimidines
involves the thermal ring closure of 2-(benzoylthioureido)thiophene-3-carbonitriles
effected by sodium hydroxide in an ethanol–water mixture, followed by the addition of
alkyl halides.12–14 In continuation of our interest in synthesizing biologically active het-
erocyclic compounds,15–17 we therefore designed a simple and facile synthesis of key
reactant 2.
Tetrahydrobenzo[b]thiophene derivative A was reacted with chloroacetyl chloride,
furnishing the corresponding N-chloroacetylamino derivative 1 by a modified procedure
of Sauter et al.18 (Scheme 1). Compound 1 was treated with ammonium thiocyanate in
refluxing ethanol, affording the compound 2 rather than the 2-imino-4-oxothiazolidine
derivative 3 (Scheme 1). In the infrared spectrum of the product, the appearance of
strong absorption bands for the NH2 group at 3296 and 3134 cmꢀ1 and the carbonyl
ester group at 1735 cmꢀ1 along with the absence of any absorption band for the nitrile
group is in agreement with the structure 2 and ruled out the 2-imino-4-oxothiazolidine
1
derivative 3. Moreover, in the H-NMR spectrum of compound 2, the appearance of a
broad signal attributable to the NH2 protons at d 6.8 ppm and a quartet at d 4.12 ppm
and a triplet at d 1.19 ppm confirmed the outcome of the cyclization reaction.
Hydrazinolysis of compound 2 using hydrazine hydrate (1:1) in refluxing ethanol
afforded colorless crystals which were identified as acetohydrazide 4 (Scheme 2). A
number of novel 4-aminothieno[2,3-d]pyrimidines were then synthesized using the acid
hydrazide 4 and treatment with different C-electrophiles. Thus, refluxing compound 4
Received November 24, 2018; in final form June 14, 2019.
Address correspondence to Fatma S. M. Abu El-Azm, Chemistry Department, Faculty of
Science, Ain Shams University, Abbassia 11566, Cairo, Egypt. E-mail: ftmsaber@yahoo.com
1

2
Abu El-Azm et al.
Scheme 1
Scheme 2

4-Aminothieno[2,3-d]pyrimidines and Triazolothienopyrimidines
3
Scheme 3
with 3,4-dimethoxybenzaldehyde in ethanol gave the corresponding hydrazone 5
(Scheme 2). Moreover, when the hydrazide 4 was allowed to react with isatin in boiling
ethanol, it produced the condensation product 6 (Scheme 2).
It has been reported that the reaction of hydrazides with phthalic anhydride can pro-
vide either the N-phthalimide or phthalazine derivatives.19 We now report the reaction of
the hydrazide derivative 4 with 3,4,5,6-tetrachlorophthalic anhydride in refluxing ethanol
which gave 1,3-dioxoisoindoline 7. Also the reaction of 4 with maleic anhydride under the
same conditions afforded 2,5-dioxopyrrole derivative 8 (Scheme 2). Supportive evidence
for the structures 7 and 8 came from the IR spectra which showed the appearance of cou-
pling bands of C ¼ O groups at 1795, 1738 and 1715 cmꢀ1, respectively.
It was reported in the literature that the reaction of an acid hydrazide with ethoxy-
methylenemalononitrile yielded the corresponding 5-amino-4-substituted pyrazole
derivative.20 In this study, treatment of the hydrazide 4 with ethoxymethylenemalononi-
trile21 in boiling ethanol furnished pale yellow crystals with molecular formula
C24H26N8O2S4 which was analyzed and assigned as the bis-hydrazide 9 (Scheme 2).
The reactivity of the hydrazide 4 towards phenyl isothiocyanate, chloroacetyl chlor-
ide, acetic anhydride and carbon disulfide was also studied. It is well known that the
reaction of hydrazides with phenyl isothiocyanate can produce thioxo triazole

4
Abu El-Azm et al.
Scheme 4
derivatives.22 Here, we found that the reaction of hydrazide 4 with phenyl isothiocyan-
ate in aqueous sodium hydroxide (10%), followed by acidification gave the acetic acid
derivative 10 (Scheme 3). The formation of compound 10 was deduced from its IR
spectrum which confirmed the presence of an OH group of a carboxylic acid as a broad
band at 3498 cmꢀ1 and C ¼ O group at 1695 cmꢀ1. The presence of a broad singlet in
1
the H-NMR spectrum at d 12.66 ppm argued against the formation of the putative tri-
azole derivative 11. Furthermore, acylation of the hydrazide 4 using chloroacetyl chlor-
ide in DMF in the cold afforded the chloroacetyl derivative 12. Also, stirring the
hydrazide 4 with acetic anhydride at room temperature yielded the N-acylated product
13. Thiol 14 was obtained upon refluxing the hydrazide 4 with carbon disulfide in alco-
holic potassium hydroxide (Scheme 3).
a-Aminocarbonitriles23,24 have recently been utilized as general precursors for the
synthesis of biologically active thienopyrimidines and triazolothienopyrimidines.
Tetrahydrobenzo[b]thiophene derivative A25 has attracted considerable attention over the
years. We found26 that its reaction with triethylorthoformate in the presence of freshly dis-
tilled acetic anhydride gave ethyl formimidate 15, which we then exploited for the synthe-
ses of novel thienopyrimidines and triazolothienopyrimidines. Thus, subjecting the ethyl
formimidate 15 to react with trimethoprim in dry pyridine afforded the uncyclized forma-
midine derivative 16 (Scheme 4). The existence of mCꢁN at 2210cmꢀ1 in addition to
mNH NH2
at 3327, 3184 and 3143 cmꢀ1 supported the suggested structure. On the other
,
hand, refluxing compound 15 with p-toluenesulphonohydrazide yielded sulfonamide
derivative 17 (Scheme 4).

4-Aminothieno[2,3-d]pyrimidines and Triazolothienopyrimidines
5
Table 1
Cytotoxicity (IC50) of the Tested Compounds on Different Cell Lines
IC50 (mg/ml)a
Compd. no.
HePG2
MCF-7
1
2
4
5
6
7
8
33.27 2.9
>100
19.28 1.5
87.25 5.3
7.49 0.7
58.55 3.9
63.86 4.5
12.67 1.2
5.38 0.4
10.81 0.9
53.42 3.8
80.51 4.9
40.03 3.0
61.14 4.2
49.11 3.6
28.47 1.9
32.70 2.4
45.39 3.3
>100
6.83 0.5
54.36 3.8
84.38 4.8
17.04 1.5
9.71 0.8
13.45 1.2
47.65 3.6
91.16 5.1
65.02 3.9
74.48 4.3
38.40 3.3
25.82 2.1
20.34 1.8
42.91 3.5
>100
10
12
13
14
16
17
18a
18b
18c
18d
18e
18f
DOX
69.32 4.0
31.20 2.4
4.50 0.3
75.92 4.7
37.50 2.7
4.17 0.2
aIC50 (lg/mL): 1–10 (very strong), 11–20 (strong), 21–50 (moderate), 51–100 (weak), above
100 (non-cytotoxic).
DOX: Doxorubicin (standard anticancer drug for comparison).
Treatment of ethyl formimidate 15 with acid hydrazides has been reported to afford
two ring annulations leading to construction of fused triazolopyrimidine systems.26
Thus, triazolothienopyrimidines 18a-f were obtained via reaction of ethyl formimidate
15 with different acid hydrazides (Scheme 4). The structures of compounds 18a-f were
educed by studying their spectroscopic data. Thus, the IR spectra showed absence of
the absorption band of the CꢁN group in addition to the presence of C ¼ O groups at
1664, 1675 cmꢀ1 for compounds 18d,e and bands for NH2 groups at 3365, 3318, 3345,
3223 cmꢀ1 for compounds 18a,f. In addition there was a band for the OH group at
3424 cmꢀ1 for compound 18b.
We assessed the cytotoxic activity of the compounds against two human tumor cell
lines (Table 1). The cell lines were hepatocellular carcinoma HePG2 and mammary
gland breast cancer MCF-7. In general, the cytotoxic activity of the tested compounds
ranged from very strong to weak activity. The optimal results were observed for com-
pounds 4 and 8 with IC50 22 1.6 lM and 24.9 2 lM for the HePG-2 cell line,
respectively.
In summary we have explored the ready synthesis of some novel 4-amino-
thieno[2,3-d]pyrimidines and triazolothienopyrimidines and explored their anticancer
activity. We have prepared and fully characterized twenty new compounds and tested
their properties against HePG2 and MCF-7 cancer cells lines. Some of the compounds

6
Abu El-Azm et al.
were highly active. We hope that our experiments will encourage more research into
these potentially useful anticancer materials and that the selective toxicity of the more
active compounds will be the subject of future work.
Experimental Section
Commercially available solvents and reagents were purified according to standard pro-
cedures. All melting points were taken on a Griffin and Geory melting point apparatus
and are uncorrected. IR spectra were recorded on a Pye Unicam SP1200 spectropho-
1
tometer using the KBr wafer technique. H-NMR and 13C NMR spectra (ppm d) were
determined on a Varian Gemini 300 MHz instrument using tetramethylsilane as an
internal standard. In compounds 5 and 16, we observed a single one-hydrogen singlet
for the azomethine proton, and we attributed this to the E-isomer. By analogy, we have
shown the E-stereochemistry for compound 6. Elemental analyses were carried out at
the Microanalytical Unit, Faculty of Science, Ain Shams University, using a Perkin-
Elmer 2400 CHN elemental analyzer, and satisfactory analytical data ( 0.4) were
obtained for all compounds. The homogeneity of the synthesized compounds was con-
trolled by thin layer chromatography (TLC) using silica gel F254 (Merck). The pharma-
cological activity assays were carried out at the Pharmacology Department, Faculty of
Pharmacy, El-Mansoura University, El-Mansoura, Egypt.
2-Chloro-N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)acetamide (1)
A
mixture of 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbonitrile (1.78 g,
10 mmol) and chloroacetyl chloride (1.11 g, 10 mmol) in dioxane (10 mL) was stirred at
0 ꢂC for 30 min. The separated solid was collected by filtration, then washed by ethanol
and recrystallized from toluene to give 1 as white crystals (80%), mp 188-190 ꢂC. IR(m/
cmꢀ1): 3225 (NH), 2221 (CꢁN), 1708 (C ¼ O). 1H NMR (300 MHz, DMSO-d6) d
(ppm): 1.75 (m, 4H, 2CH2), 2.49-2.59 (m, 4H, 2CH2), 4.43 (s, 2H, COCH2Cl), 11.80
(br.s, 1H, NH, exchangeable with D2O). 13C NMR (DMSO-d6) d (ppm): 21.7, 22.6,
23.4, 42.2, 93.7, 113.9, 127.9, 131.1, 145.8, 164.5.
Anal. Calcd. for C11H11ClN2OS: C, 51.87; H, 4.35; Cl, 13.92; N, 6.28; S, 12.59.
Found: C, 51.81; H, 4.30; Cl, 13.99; N, 6.32; S, 12.63.
Ethyl 2-((4-amino-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-2-yl)
thio)acetate (2)
A mixture of compound 1 (1.27 g, 5 mmol) and ammonium thiocyanate (0.38 g, 5 mmol)
was heated under reflux in absolute ethanol (30 mL) for 4h. The deposited precipitate
was filtered off, dried and recrystallized from dioxane to give 2 as pale yellow crystals
(72%), mp 138-140 ꢂC. IR(m/cmꢀ1): 3296, 3134 (NH2), 1735 (C ¼ Oester), 1635
1
(C ¼ N). H NMR (300 MHz, DMSO-d6) d (ppm): 1.19 (t, 3H, CH2-CH3, J ¼ 8.4 Hz),
1.78 (m, 4H, 2CH2), 2.68-2.84 (m, 4H, 2CH2), 3.92 (s, 2H, SCH2CO), 4.12 (q, 2H,
CH2-CH3, J ¼ 6.9 Hz), 6.81 (brs, 2H, NH2, exchangeable with D2O). 13C NMR
(DMSO–d6) d (ppm): 13.9, 21.8, 22.1, 24.6, 25.2, 32.5, 60.6, 112.2, 126.6, 129.3,
157.5, 162.8, 165.9, 169.

4-Aminothieno[2,3-d]pyrimidines and Triazolothienopyrimidines
7
Anal. Calcd. for C14H17N3O2S2: C, 51.99; H, 5.30; N, 12.99; S, 19.83. Found: C,
52.09; H, 5.28; N, 12.86; S, 19.79.
2-((4-Amino-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-2-
yl)thio)acetohydrazide (4)
A mixture of compound 2 (1.61 g, 5 mmol) and hydrazine hydrate (60%) (0.25 g,
5 mmol) in absolute ethanol (30 mL) was refluxed for 3 h. The separated solid was fil-
tered off, dried and then recrystallized from ethanol to give 4 as white crystals (62%),
mp 258-260 ꢂC. IR (m/cmꢀ1): 3477 (NH), 3316,3248, 3194, 3118 (2NH2), 1661
1
(C ¼ O), 1625 (C ¼ N). H NMR (300 MHz, DMSO-d6) d (ppm): 1.79 (m, 4H, 2CH2),
2.71-2.86 (m, 4H, 2CH2), 3.75 (s, 2H, SCH2CO), 4.28 (br.s, 2H, NH2
,
hydrazide
exchangeable with D2O), 6.88 (br.s, 2H, NH2 pyrimidine, exchangeable with D2O), 9.10
(br.s, 1H, NH, exchangeable with D2O). 13C NMR (DMSO–d6) d (ppm): 21.8,
22.1,24.6, 25.2, 32.4, 112.2, 120.6, 126.7, 129.2, 157.5, 163.3, 166.1, 167.3.
Anal. Calcd. for C12H15N5OS2: C, 46.58; H, 4.89; N, 22.64; S, 20.72. Found: C,
46.53; H, 4.91; N, 22.70; S, 20.68.
(E)-2-((4-Amino-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-2-yl)thio)-N'-
(3,4-dimethoxy benzylidene)acetohydrazide (5)
A mixture of compound 4 (1.55 g, 5 mmol) and 3,4-dimethoxylbenzaldehyde (0.97 g,
5 mmol) in ethanol (20 mL) was heated under reflux for 4 h. The solid that separated
while heating was collected by filtration and recrystallized from dioxane to give 5 as
white crystals (90%), mp 218-220 ꢂC. IR (m/cmꢀ1): 3296, 3165 (NH2), 1679 (C ¼ O),
1660 (C ¼ N). 1H NMR (300 MHz, DMSO-d6) d (ppm): 1.78 (m, 4H, 2CH2), 2.69
-2.88 (m, 4H, 2CH2), 3.76 (s, 3H, OCH3), 3.78 (s, 3H, OCH3), 3.90 (s, 2H, SCH2CO),
6.85 (br.s, 2H, NH2, exchangeable with D2O), 6.94-7.30 (m, 3H, ArH), 8.13 (s, 1H,
CH ¼ N), 11.42 (br.s, 1H, NH, exchangeable with D2O). 13C NMR (DMSO–d6) d
(ppm): 21.9, 22.2, 24.7, 25.3, 32.1, 33.4, 55.5, 108.6, 111.5, 112.3, 126.8, 129.3, 143.1,
146.9, 148.9, 150.7, 163.3, 164.3, 166.1, 169.6.
Anal. Calcd. for C20H21N5O3S2: C, 55.12; H, 5.07; N, 15.31; S, 14.01. Found: C,
55.23; H, 4.99; N, 15.48; S, 14.11.
(E)-2-((4-Amino-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-2-yl)thio)-N'-(2-
oxoindolin-3-ylidene)acetohydrazide (6)
A mixture of compound 4 (1.55 g, 5 mmol) and isatin (0.74 g, 5 mmol) in dioxane
(20 mL) was refluxed for 6 h. The solid that formed while heating was collected and
recrystallized from dioxane to give 6 as yellow crystals (80%), mp 282-284 ꢂC. IR
(m/cmꢀ1): 3348 (NH), 3200, 3148 (NH2), 1709, 1685 (2 C ¼ O), 1619 (C ¼ N). 1H
NMR (300 MHz, DMSO-d6) d (ppm): 1.77 (m, 4H, 2CH2), 2.68-2.84 (m, 4H, 2CH2),
4.02 (s, 2H, SCH2CO), 6.90 (br.s, 2H, NH2, exchangeable with D2O), 6.92 (d, 1H,
ArH, J ¼ 7.5 Hz), 7.09 (t, 1H, ArH, J ¼ 7.2 Hz), 7.39 (t, 1H, ArH, J ¼ 7.8 Hz), 7.54 (d,
1H, ArH, J ¼ 7.5 Hz), 11.21 (br.s, 1H, NH, exchangeable with D2O), 13.41 (br.s, 1H,
NH, exchangeable with D2O).

8
Abu El-Azm et al.
Anal. Calcd. for C20H18N6O2S2: C, 54.78; H, 4.14; N, 19.16; S, 14.62. Found: C,
54.71; H, 4.16; N, 19.27; S, 14.73.
2-((4-Amino-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-2-yl)thio)-N-(4,5,6,7-
tetrachloro-1,3-dioxoisoindolin-2-yl)acetamide (7)
A mixture of compound 4 (1.55 g, 5 mmol) and tetrachlorophthalic anhydride (1.34 g,
5 mmol) in ethanol (30 mL) was heating under reflux for 3h. The precipitated solid
which formed while heating was filtered off and recrystallized from DMF to give 7 as
yellow crystals (73%), mp> 300 ꢂC. IR (m/cmꢀ1): 3478 (NH), 3365, 3318 (NH2), 1795,
1
1738 (C ¼ O vibrational coupling), 1709 (C ¼ O amide). H NMR (300 MHz, DMSO-d6) d
(ppm): 1.80 (m, 4H, 2CH2), 2.72-2.86 (m, 4H, 2CH2), 4.07 (s, 2H, SCH2CO), 6.90
(br.s, 2H, NH2 exchangeable with D2O), 11.01 (br.s, 1H, NH, exchangeable with D2O).
Anal. Calcd. for C20H13Cl4N5O3S2: C, 41.61; H, 2.27; Cl, 24.56; N, 12.13; S,
11.11. Found: C, 41.54; H, 2.21; Cl, 24.49; N, 12.08 S, 11.07.
2-((4-Amino-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-2-yl)thio)-N-(2,5-
dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamide (8)
A mixture of compound 4 (1.55 g, 5 mmol) and maleic anhydride (0.49 g, 5 mmol) in
absolute ethanol was heated under reflux for 5 h. The solid product was filtered off after
cooling the reaction mixture and recrystallized from ethanol to give 8 as white crystals
(66%), mp 162-164 ꢂC. IR (m/cmꢀ1): 3287 (NH), 3194, 3131 (NH2), 1715, 1681 (2
1
C ¼ O), 1635 (C ¼ N). H NMR (300 MHz, DMSO-d6) d (ppm): 1.79 (m, 4H, 2CH2),
2.70-2.85 (m, 4H, 2CH2), 3.89 (s, 2H, SCH2CO), 6.27 (d, 1H olefinic, J ¼ 12.1 Hz), 6.37
(d, 1H olefinic, J ¼ 12 Hz), 6.90 (br.s, 2H, NH2, exchangeable with D2O), 10.39 (br.s,
1H, NH, exchangeable with D2O).
Anal. Calcd. for C16H15N5O3S2: C, 49.35; H, 3.88; N, 17.98; S, 16.46. Found: C,
49.49; H, 3.70: N, 17.82: S, 16.35.
2-((4-Amino-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-2-yl)thio)-N'-(2-((4-
amino-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-2-
yl)thio)acetyl)acetohydrazide (9)
A mixture of compound 4 (1.55 g, 5 mmol) and ethoxymethylenemalononitrile (0.61 g,
5 mmol) in ethanol (30 mL) was subjected to heating under reflux for 1h. The solid that
precipitated after cooling was filtered off and recrystallized from ethanol to give 9 as
pale yellow crystals (72%), mp 296-298 ꢂC. IR (m/cmꢀ1): 3485 (NH), 3322,3217 (NH2),
1
1632 (C ¼ O). H NMR (300 MHz, DMSO-d6) d (ppm): 1.78 (m, 4H, 2CH2), 2.69-2.79
(m, 2H, 2CH2), 3.84 (s, 2H, SCH2CO), 6.85 (br.s, 2H, NH2, exchangeable with D2O),
10.23 (br.s, 1H, NH, exchangeable with D2O).
Anal. Calcd. for C24H26N8O2S4: C, 49.13; H, 4.47; N, 19.10; S, 21.86. Found: C,
49.02; H, 4.31; N, 19.28; S, 21.91.

4-Aminothieno[2,3-d]pyrimidines and Triazolothienopyrimidines
9
2-((4-Amino-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-2-yl)thio)acetic
acid (10)
A mixture of compound 4 (1.55 g, 5 mmol) and phenyl isothiocyanate (0.68 g, 5 mmol)
in aqueous sodium hydroxide 10% (30 mL) was heating under reflux for 4 h. The reac-
tion mixture was poured onto ice cold dilute hydrochloric acid with stirring. The crude
material was filtered off and recrystallized from dioxane to give 10 as buff crystals
(88%), mp 250-252 ꢂC. IR (m/cmꢀ1): 3498 (OH), 3300, 3168 (NH2), 1695 (C ¼ Oacid),
1
1638 (C ¼ N). H NMR (300 MHz, DMSO-d6) d (ppm): 1.78 (m, 4H, 2CH2), 2.68-2.83
(m, 4H, 2CH2), 3.91 (s, 2H, SCH2CO), 6.86 (br.s, 2H, NH2, exchangeable with D2O),
12.66 (br.s, 1H, OH, exchangeable with D2O).
Anal. Calcd. for C12H13N3O2S2: C, 48.80; H, 4.44; N, 14.23; S, 21.71. Found: C,
48.61; H, 4.40; N, 14.46; S, 21.83.
2-((4-Amino-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-2-yl)thio)-N'-(2-
chloroacetyl) acetohydrazide (12)
A mixture of compound 4 (1.55 g, 5 mmol) and chloroacetyl chloride (0.56 g, 5 mmol)
in DMF (20 mL) was stirred at room temperature for 1 h. The reaction mixture was
poured onto ice cold water, the precipitated solid was filtered off, dried and recrystal-
lized from dioxane to give 12 as white crystals (60%), mp 224-226 ꢂC. IR (m/cmꢀ1):
3498 (NH), 3302, 3169 (NH2), 1695 (C ¼ O), 1633 (C ¼ N). 1H NMR (300 MHz,
DMSO-d6) d (ppm): 1.78 (m, 4H, 2CH2), 2.69-2.84 (m, 4H, 2CH2), 3.91 (s, 2H,
SCH2CO), 4.12 (s, 2H, COCH2Cl), 6.87 (br.s, 2H, NH2, exchangeable with D2O),
10.24 (br.s 1H, NH, exchangeable with D2O), 12.59 (br.s, 1H, NH, exchangeable
with D2O).
Anal. Calcd. for C14H16ClN5O2S2: C, 43.58; H, 4.18; Cl, 9.19; N, 18.15; S, 16.62.
Found: C, 43.68; H, 4.26; Cl, 9.26; N, 18.01; S, 16.78.
N'-Acetyl-2-((4-amino-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-2-
yl)thio)acetohydrazide (13)
A mixture of compound 4 (1.55 g, 5 mmol) and acetic anhydride (15 mL) was stirred at
room temperature for 1 h. The reaction mixture was poured onto ice cold water. The
precipitated solid was collected by filtration, dried and recrystallized from dioxane to
give 13 as white crystals (76%), mp> 300 ꢂC. IR (m/cmꢀ1): 3298 (NH), 3194, 3100
1
(NH2), 1716 (C ¼ O), 1638 (C ¼ N). H NMR (300 MHz, DMSO-d6) d (ppm): 1.79 (m,
4H, 2CH2), 1.85 (s, 3H, CH3CO), 2.71-2.85 (m, 4H, 2CH2), 3.83 (s, 2H, SCH2CO),
6.93 (br.s, 2H, NH2, exchangeable with D2O), 9.88 (s, 1H, NH, exchangeable with
D2O), 9.96 (s, 1H, NH, exchangeable by D2O).
Anal. Calcd. for C14H17N5O2S2: C, 47.85; H, 4.88; N, 19.93; S, 18.24. Found: C,
47.99; H, 4.70: N, 19.79; S, 18.37.

10
Abu El-Azm et al.
5-(((4-Amino-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-2-yl)thio)methyl)-
1,3,4-oxadiazole-2-thiol (14)
A mixture of compound 4 (1.55 g, 5 mmol) and carbon disulfide (Caution! Carbon
disulfide is toxic. All workers must be thoroughly trained on its safe use, to include per-
sonal protective gear and adequate ventilation; 7 mL) in alcoholic potassium hydroxide
10% (20 mL) was subjected to heating under reflux on a water bath for 6 h, then the
reaction mixture was poured onto ice cold hydrochloric acid. The crude product was fil-
tered off, dried and recrystallized from DMF to give 14 as yellow crystals (53%), mp
296-298 ꢂC. IR (m/cmꢀ1): 3485 (SH), 3323, 3217 (NH2), 1632 (C ¼ N). 1H NMR
(300 MHz, DMSO-d6) d (ppm): 1.77 (m, 4H, 2CH2), 2.68-2.79 (m, 4H, 2CH2), 3.83 (s,
2H, SCH2-), 6.85 (br.s, 2H, NH2, exchangeable with D2O), 10.23 (br.s, 1H, SH,
exchangeable with D2O).
Anal. Calcd. for C13H13N5OS3: C, 44.43; H, 3.73; N, 19.93; S, 27.37. Found: C,
44.30; H, 3.93; N, 20.01; S, 27.44.
General Procedure for Synthesis of Compounds 16, 17, 18a-f
A mixture of compound 15 (1.17g, 5 mmol) and trimethoprim (5 mmol) or hydrazide
derivatives (5 mmol) in dry pyridine (10 mL) was heated under reflux for 4-6 h, The
precipitated solid product was filtered, washed with ethanol, dried, and finally recrystal-
lized from the appropriate solvent to give 16, 17, 18a-f.
(E)-N-(4-Amino-5-(3,4,5-trimethoxybenzyl)pyrimidin-2-yl)-N'-(3-cyano-4,5,6,7-
tetrahydrobenzo[b] thiophen-2-yl)formimidamide (16)
Recrystallized from dioxane, yellow crystals (64%), mp> 300 ꢂC. IR (m/cmꢀ1): 3327
1
(NH), 3184, 3143 (NH2), 2210 (CꢁN), 1638 (C ¼ N). H NMR (300 MHz, DMSO-d6)
d (ppm): 1.76 (m, 4H, 2CH2), 2.49-2.59 (m, 4H, 2CH2), 3.57 (s, 3H, OCH3), 3.63 (s,
3H, OCH3), 3.65 (s, 3H, OCH3), 3.73 (s, 2H, CH2-Ar), 6.59 (s, 2H, ArH), 6.78 (br.s,
2H, NH2, exchangeable with D2O), 7.83 (s, 1H pyrimdine), 8.91 (s, 1H, CH¼N-), 11.02
(br.s, 1H, NH, exchangeable with D2O). .13C NMR (DMSO-d6) d (ppm): 21.5, 22.6,
23.8, 24.1, 32.7, 55.8, 59.8, 66.3, 99.1, 106.1, 111.7, 115.1, 126.6, 132.9, 134.5, 136.1,
150.5, 152.7, 154.8, 155.7, 162.1, 162.2.
Anal. Calcd. for C24H26N6O3S: C, 60.23; H, 5.48; N, 17.56; S, 6.70. Found: C,
60.36; H, 5.55; N, 17.40; S, 6.65.
N-(4-Imino-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-3(4H)-yl)-4-
methylbenzene sulfonamide (17)
Recrystallized from DMF, white crystals (53%), mp 262-264 ꢂC. IR (m/cmꢀ1): 3365,
1
3249 (2NH), 1637 (C ¼ N), 1364 (S ¼ O). H NMR (300 MHz, DMSO-d6) d (ppm):
1.75 (m, 4H, 2CH2), 2.30 (s, 3H, ArCH3), 2.49-2.76 (m, 4H, 2CH2), 7.19 (d, 2H, ArH,
J ¼ 7.8 Hz), 7.51 (d, 2H, ArH, J ¼ 7.8), 8.03 (br.s, 1H, C ¼ NH exchangeable with
D2O), 8.15 (s, 1H pyimidine), 8.56 (br.s, 1H, NHSO, exchangeable with D2O). 13C NMR
(DMSO-d6) d (ppm): 20.5, 21.5, 24.8, 114.9, 123.7, 126.3, 128.1, 129.0, 137.2, 141.1,
147.0, 149.4, 151.1, 160.6.

4-Aminothieno[2,3-d]pyrimidines and Triazolothienopyrimidines
11
Anal. Calcd. for C17H18N4O2S2: C, 54.53; H, 4.85; N, 14.96; S, 17.12. Found: C,
54.66; H, 4.80: N, 14.80; S, 17.21.
8,9,10,11-Tetrahydrobenzo[4,5]thieno[3,2-e][1,2,4]triazolo[1,5-c]pyrimidin-2-
amine (18a)
Recrystallized from dioxane, yellow crystals (44%), mp 298-300 ꢂC. IR (m/cmꢀ1):
1
3365, 3318 (NH2), 1649 (C ¼ N). H NMR (300 MHz, DMSO-d6) d (ppm): 1.81 (m,
4H, 2CH2), 2.73-2.90 (m, 4H, 2CH2), 6.71 (br.s, 2H, NH2, exchangeable with D2O),
8.17 (s, 1H pyrimidine). 13C NMR (DMSO-d6) d (ppm): 21.86, 22.11, 24.72, 25.30,
114.96, 126.75, 130.94, 152.67, 157.97, 165.33.
Anal. Calcd. for C11H11N5S: C, 53.86; H, 4.52; N, 28.55; S, 13.07. Found: C,
53.70; H, 4.41; N, 28.70; S, 13.37.
8,9,10,11-Tetrahydrobenzo[4,5]thieno[3,2-e][1,2,4]triazolo[1,5-c]pyrimidin-2-ol (18b)
Recrystallized from dioxane, white crystals (60%), mp> 300 ꢂC. IR (m/cmꢀ1): 3424
1
(OH), 1617 (C ¼ N). H NMR (300 MHz, DMSO-d6) d (ppm): 1.87 (m, 4H, 2CH2),
2.86-2.97 (m, 4H, 2CH2), 9.27 (s, 1H pyrimidine), 12.38 (br.s, 1H, OH, exchangeable
with D2O).
Anal. Calcd. for C11H10N4OS: C, 53.64; H, 4.09; N, 22.75; S, 13.02. Found: C,
53.48; H, 4.20; N, 22.68; S, 13.31.
2-(Pyridin-4-yl)-8,9,10,11-tetrahydrobenzo[4,5]thieno[3,2-e][1,2,4]triazolo[1,5-
c]pyrimidine (18c)
Recrystallized from dioxane, yellow crystals (77%), mp 264-266 ꢂC. IR (m/cmꢀ1): 3053
(CH aromatic), 2943 (CH aliphatic), 1620 (C ¼ N). 1H NMR (300 MHz, DMSO-d6) d
(ppm): 1.88 (m, 4H, 2CH2), 2.87-3.03 (m, 4H, 2CH2), 8.05 (d, 2H, ArH, J ¼ 5.7 Hz),
8.77 (d, 2H, ArH, J ¼ 6.3 Hz), 9.60 (s, 1H pyrimidine).
Anal. Calcd. for C16H13N5S: C, 62.52; H, 4.26; N, 22.78; S, 10.43. Found: C,
62.41; H, 4.17; N, 22.91; S, 10.35.
4-(8,9,10,11-Tetrahydrobenzo[4,5]thieno[3,2-e][1,2,4]triazolo[1,5-c]pyrimidin-2-
yl)phthalazin-1(2H)-one (18d)
Recrystallized from DMF, white crystals (65%), mp> 300 ꢂC. IR (m/cmꢀ1): 3165 (NH),
1
1664 (C ¼ O), 1618 (C ¼ N). H NMR (300 MHz, DMSO-d6) d (ppm): 1.92 (m, 4H,
2CH2), 2.94-3.12 (m, 4H, 2CH2), 7.95-8.00 (m, 2H, ArH), 8.37 (d, 1H, ArH,
J ¼ 7.8 Hz), 8.78 (d, 1H, ArH, J ¼ 7.8 Hz), 9.71 (s, 1H pyrimidine), 13.14 (br.s, 1H, NH,
exchangeable with D2O).
Anal. Calcd. for C19H14N6OS: C, 60.95; H, 3.77; N, 22.45; S, 8.56. Found: C,
60.81; H, 3.71; N, 22.58; S, 8.72.

12
Abu El-Azm et al.
6-(4-Chloro-3-methylphenyl)-2-((8,9,10,11-tetrahydrobenzo[4,5]thieno[3,2-
e][1,2,4]triazolo [1,5-c]pyrimidin-2-yl)methyl)pyridazin-3(2H)-one (18e)
Recrystallized from dioxane, yellow crystals (53%) mp 228-230 ꢂC. IR (m/cmꢀ1): 3069
(CH aromatic), 2933 (CH aliphatic), 1675 (C ¼ O), 1617 (C ¼ N). 1H NMR (300 MHz,
DMSO-d6) d (ppm): 1.83 (m, 4H, 2CH2), 2.31 (s, 3H, ArCH3), 2.83-2.94 (m, 4H,
2CH2), 5.65 (s, 2H, CH2NCO), 7.14 (d, 1H pyridazinone, J ¼ 9.9 Hz), 7.43 (d,1H
pyridazi-
none, J ¼ 8.4 Hz), 7.68 (d, 1H, ArH, J ¼ 8.4 Hz), 7.81 (s, 1H, ArH), 8.08 (d, 1H, ArH,
J ¼ 9.6 Hz), 9.46 (s, 1H pyrimidine). 13C NMR (DMSO–d6) d (ppm): 19.2, 21.6, 22.4,
24.8, 49.6, 66.6, 119.3, 120.4, 124.9, 125.8, 128.4, 129.2, 129.9, 131.1,132.9, 134.5,
135.9, 136.7, 138.3, 142.9, 148.6, 152.8, 158.7, 163.1.
Anal. Calcd. for C23H19ClN6OS: C, 59.67; H, 4.14; Cl, 7.66; N, 18.15; S, 6.93.
Found: C, 59.50; H, 4.26; Cl, 7.73; N, 18.28; S, 6.81.
4-(8,9,10,11-Tetrahydrobenzo[4,5]thieno[3,2-e][1,2,4]triazolo[1,5-c]pyrimidin-2-
yl)aniline (18f)
Recrystallized from Ethanol, white crystals (47%), mp 230-232 ꢂC. IR (m/cmꢀ1): 3354,
1
3223 (NH2), 3061 (CH aromatic), 2935 (CH aliphatic), 1607 (C ¼ N). H NMR (300 MHz,
DMSO-d6) d (ppm): 1.90 (m, 4H, 2CH2), 2.89-3.01 (m, 4H, 2CH2), 5.64 (br.s, 2H,
NH2, exchangeable with D2O), 6.69 (d, 2H, ArH, J ¼ 7.8 Hz), 7.94 (d, 2H, ArH,
J ¼ 8.7 Hz), 9.48 (s, 1H pyrimidine).
Anal. Calcd. for C17H15N5S: C, 63.53; H, 4.70; N, 21.79; S, 9.98. Found: C, 63.68;
H, 4.72; N, 21.84; S, 10.05.
Cytotoxicity Assay
The cytotoxic activity of thirteen compounds was tested against two human tumor cell
lines namely: hepatocellular carcinoma HePG-2, and mammary gland cancer MCF-7.
The cell lines were obtained from the ATCC via the Holding Company for Biological
Products and Vaccines (VACSERA, Cairo, Egypt). Doxorubicin was used as a standard
anticancer drug for comparison. The reagents used were RPMI-1640 medium, MTT,
DMSO and doxorubicin (Sigma Co., St. Louis, MO, USA), and fetal bovine serum
(GIBCO, Paisley, UK). The different cell lines27,28 mentioned above were used to
determine the inhibitory effects of compounds on cell growth using the MTT assay.
This colorimetric assay is based on the conversion of the yellow tetrazolium bromide
(MTT) to a purple formazan derivative by mitochondrial succinate dehydrogenase in
viable cells. The cells were cultured in RPMI-1640 medium with 10% fetal bovine
serum. Antibiotics added were 100 units/mL penicillin and 100 mg/mL streptomycin at
29
37 ꢂC in a 5% CO2 incubator. The cell lines were seeded in a 96-well plate at a dens-
ity of 1.0 x 104 cells/well at 37 ꢂC for 48 h under 5% CO2. After incubation the cells
were treated with different concentration of compounds and incubated for 24 h. After
24 h of drug treatment, 20 mL of MTT solution at 5 mg/mL was added and incubated
for 4 h. Dimethyl sulfoxide (DMSO) in volume of 100 mL was added into each well to
dissolve the purple formazan formed. The colorimetric assay was measured and
recorded at an absorbance of 570 nm using a plate reader (EXL 800, BioTech,
Winoosky, VT, USA). The relative cell viability in percentage was calculated as (A570
of treated samples/A570 of untreated sample) x 100%.

4-Aminothieno[2,3-d]pyrimidines and Triazolothienopyrimidines
13
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