Inhibitory Effects of New Mercapto Xanthine Derivatives in Human mcf7 and k562 Cancer Cell Lines

Article abstract of DOI:10.1002/jhet.2602

A series of new 2-methyl-2-[(1,3-Diethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)thio]-N- substituted arylacetamides were synthesized. The antitumor activity of these purine based compounds were evaluated on breast cancer (MCF7) and leukemic cancer (K562) cell lines via cell viability assay utilizing the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). These results were substantiated using computer docking experiments (LigandFit docking engine and PMF scoring function) which predict that the antitumor activity of these new compounds may be attributable to their abilities to effectively bind and block oncogenic tyrosine kinases, particularly bcr/abl.

Full text of DOI:10.1002/jhet.2602

Month 2016 Inhibitory Effects of New Mercapto Xanthine Derivatives in Human mcf7
and k562 Cancer Cell Lines
a
a
b
a
a
*
Haider N. Sultani, Rasha A. Ghazal, Alaa M. Hayallah, Loay K. Abdulrahman, Khaled Abu-Hammour,
Shatha AbuHammad,d Mutasem O. Taha,c and Malek A. Zihlifd
*
aFaculty of Pharmacy, Al-Isra University, Amman, Jordan
bDepartment of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt
cDrug Discovery Unit, Faculty of Pharmacy, The University of Jordan, Amman 11942, Jordan
dDepartment of Biochemistry, Faculty of Medicine, The University of Jordan, Amman 11942, Jordan
*
E-mail: terralunna@yahoo.com; M.zihlif@ju.edu.jo
Received July 2, 2015
DOI 10.1002/jhet.2602
Published online 00 Month 2016 in Wiley Online Library (wileyonlinelibrary.com).
A series of new 2-methyl-2-[(1,3-Diethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)thio]-N- substituted
arylacetamides were synthesized. The antitumor activity of these purine based compounds were evaluated on
breast cancer (MCF7) and leukemic cancer (K562) cell lines via cell viability assay utilizing the tetrazolium
dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). These results were substantiated
using computer docking experiments (LigandFit docking engine and PMF scoring function) which predict
that the antitumor activity of these new compounds may be attributable to their abilities to effectively bind
and block oncogenic tyrosine kinases, particularly bcr/abl.
J. Heterocyclic Chem., 00, 00 (2016).
INTRODUCTION
their cytotoxic effect against two human malignant cancer cell
lines: T-cell leukemia derived SKW-3 and breast cancer de-
rived MDA-MB-231. Some compounds showed interesting
anti-tumor activity [15]. These promising anti-cancer effects
of xanthine derivatives led us to numerous chemical works fo-
cusing on the synthesis of new xanthine derivatives with more
potent anti-cancer activity. Accordingly, we envisaged to pre-
pare a set of xanthine derivatives incorporating N-substituted
arylacetamide moiety and evaluate their antitumor activity
against MCF7 breast cancer and K562 leukemia cell lines.
Purine nucleus is reported to be an important class of
biologically active structures. Xanthine is a purine base that
is found in most human body tissues and fluids [1]. They have
been widely investigated for their biological activities.
Nitrogen-containing compounds from terrestrial and marine
organisms have been intensively investigated over the last
few years for their antitumor activity, which include 1,3,7-
trimethylxanthine or caffeine, a natural methylxanthine present
in coffee and tea, and its various analogues, referred to as 1,3,7-
trialkylxanthines or xanthenes [2–7]. Previous studies revealed
that caffeine inhibits the development of tumors induced by
various carcinogens in numerous organs including skin, lung,
stomach, and liver [8–10]. Oral administration of caffeine
found to be inhibit UVB-induced carcinogenesis in SKH-1
mice [11]. A recent study revealed that caffeine inhibited
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung
tumor [9]. Caffeine was shown to suppress the proliferation
of various cancer and transformed cell lines including human
neuroblastoma, human pancreatic adenocarcinoma, human
A549 lung adenocarcinoma, and mouse epidermal JB6
promotion-sensitive (P) cells, respectively [12–14].
RESULT AND DISCUSSION
Chemistry. 1,3-Diethyl-5,6-diaminouracil 3 were synthe-
sized according to previously described methods [16–20].
Thus, 1,3-Diethyl urea was condensed with cyanoacetic acid
to give the 6 aminouracil 1. Standard nitrosation of
compound 1 with sodium nitrite in acetic acid leading to
compound 2 was followed by reduction with sodium
dithionite to give diaminouracil 3.
Finaly, the desired 8-mercapto xanthine derivative 4 was
obtained by reacting the diaminouracil with carbon disulfide
in ethanol solution in the presence of potassium hydroxide
[21]. The N-substituted aryl-2-methyl-2-cloroacetamides
1,3,8-trisubstituted xanthines and 1,3,6-trisubstituted
thiazolo[2,3-f]purine-2,4-diones were assessed in vitro for
© 2016 Wiley Periodicals, Inc.

H. N. Sultani, R. A. Ghazal, A. M. Hayallah, L. K. Abdulrahman, K. Abu-Hammour,
S. AbuHammad, M. O. Taha, and M. A. Zihlif
Vol 000
Table 2
5a-l were prepared by reacting the appropriate amine pyri-
dine in chloroform with 2-chloropropanoyl chloride
[22,23]. The 8-mercapto-cloroacetamides derivatives 6a-l
were obtained by reacting the aqueous solution of compound
4 in sodium hydroxide with the appropriate N- substituted
arylacetamide 5a-l solutions in ethanol [24], Scheme 1.
Effects of compounds that have shown potential activity on the screening
assay MCF7 and K562, Doxorubicin is used as a positive control.
Compound
IC50 MCF7 (μM) SD
IC50 K562 (μM) SD
Doxorubicin
0.31 0.01
29.34 2.4
>100
49.22 2.5
56.54 1.6
17.59 2.715290
60.07 9.9
1.41 0.31
6c
6d
6e
6j
6k
6f
33.72 4.6810468
49.01 1.0465180
41.78 7.2831998
47.5 3.8678740
9.57 0.6222539
52.33 2.0293964
Anti-tumor activity.
The anti-tumor activity of the
synthesized compounds were evaluated for their cell
viability assay using tetrazolium dye 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT). Cultures of
the breast cancer cell line MCF-7 and the Leukemic cell
Scheme 1. Reagents and condition: (i) CNCH2COOH, Ac2O, 80°C, 2 h, 70% NaOH; (ii) NaNO2, 50% aq AcOH, 80°C, 45 min; (iii) Na2S2O4, 12.5% aq
NH4OH, 60°C, 1 h; (iv) CS2, KOH, EtOH, reflux, 4 h; (iv) 5 a-l, 1% NaOH, EtoH.
Table 1
Percentage cell survival of MCF7 and K562 following 72 h exposure to 10 and 50 μM of all compounds.
K562% survival SD
at 10 μM
MCF7% survival SD
at 10 μM
Compound
at 50 μM
at 50 μM
6a
6b
6c
6d
6e
6f
6g
6h
6i
60 12.10
76 3.14
62 5.41
60 12.44
59 4.68
70 3.67
74 13.51
84 23.88
70 17.66
68 2.54
50 7.35
67 10.30
49 16.92
63 11.10
35 5.15
49 2.60
146 7.42
51 3.74
59 3.01
74 4.95
68 1.80
48 9.16
33 0.20
53 5.35
81 4.49
83 5.13
75 43.63
81 5.51
82 7.31
79 3.59
75 9.23
80 5.38
80 2.69
84 13.85
78 0.89
75 7.31
88 3.46
93 13.08
4l2 3.20
64 4.74
48 7.44
81 9.75
85 3.72
85 5.26
87 10.13
75 15.78
77 11.29
71 3.59
6j
6k
6l
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2016
Effects of Mercapto Xanthine Derivatives on Cancer Cell Lines
Figure 1. (A) X-ray crystallographic structure of imitanib co-crystallized within c-abl kinase domain (PDB code: 1IEP, resolution 2.1 Å), (B) The most
potent xanthine analogue docked within the same binding pocket, (C) Superposition of the co-crystalized structure of imitanib over the docked structure of
the most potent xanthine analogue. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

H. N. Sultani, R. A. Ghazal, A. M. Hayallah, L. K. Abdulrahman, K. Abu-Hammour,
S. AbuHammad, M. O. Taha, and M. A. Zihlif
Vol 000
line K562 were treated initially at a concentration of
50 μM, and the results are shown in Table 1. In the
corridor comprised of the -CH3S- of Met290 and the
-(CH2)4- of Lys271 (Fig. 1a) this model can be used to ex-
plain the fitting the nitro aromatic ring of the most potent
analogue within the same corridor (Fig. 1b). Finally, the
apparent electrostatic attraction connecting the piperazine
ring of imitianib with the carboxylate side chain of
Asp381 (Fig. 1a) compares with the hydrogen-bonding in-
teraction connecting the carbonyl of the xanthine terminal
of the most potent analogue with the guanidino NH2 group
of Arg362 in the binding pocket.
However, on the other hand, positioning the pyridinyl-
pyrimidine fragment of imatinib within the aromatic hy-
drophobic pocket of the side chains of Phe382, Tyr253,
and Phe317 (Fig. 1a) is not represented in the binding in-
teractions of the most potent analogue with the binding
pocket, which probably explains the inferior bioactivity
of these compounds compared with imitanib.
MCF-7 screening test, five compounds showed
a
potential anti-MCF7activity. Those compounds were able
to reduce the viability after 72 h to less than 50%. In the
case of K562 cells, only four compounds showed a
potential anti-K562 activity. At 10 μM concentration,
only compound 6k was able to reduce the K262 cell
viability to less than 50%, and none of them has shown
in the case of MCF7. Further, we determined the IC50
values of the potential compounds against the MCF7 and
K562 (Table 2). Unsurprisingly, compound 6k showed
the highest potency against K562 and MCF7cells; it
scored IC50 values of 9.56 and 17.59μM. On the other
hand, the IC50 of the remaining derivatives ranged
between 33 and 59 μM; these values reflected that they
were not potent.
From structure activity relationship point of view, the
type of substitution on phenyl side chain seems to play a
key role in the anti-cancer activity of the targeted com-
pounds. The relatively low IC50 values obtained for com-
pound 6 k reveals the importance of the substitution on the
phenyl side chain. The enhanced anti-cancer activity may
result from the high-electron withdrawing capability of
the NO2 group on the phenyl ring, which in turn has im-
proved the reactivity of compound 6 k toward its cellular
target. To understand inhibitory effects of our new synthe-
sized compounds against K562 and MCF7 cancer cell lines
that over express tyrosine Kinases, namely, bcr/abl tyro-
sine kinase and EGFR tyrosine kinase [1,2], we used the
anti-cancer agent imitanib in the docking experiment,
which is a specific inhibitor of a number of tyrosine kinase
enzymes, combined with the apparent pharmacophoric
commonalities between imitanib, and our new synthesized
compounds prompted us to anticipate that their observed
anti-cancer properties are attributable to their abilities to ef-
fectively bind and block oncogenic tyrosine kinases, par-
ticularly bcr/abl. Figure 1 compares how imitanib binds
within the ATP binding pocket of bcr/abl (PDB code:
1IEP, resolution 2.1 Å) with the way the most active ana-
logue (Table 1) docks into the binding pocket of the same
protein (the docking experiment was performed using
LigandFit docking engine and PMF scoring function).
Clearly from the figure, hydrogen-bonding interactions
connecting the amidic linker of imitanib with the carbox-
ylic acid side chain of Glu286 and the peptidic NH of
Asp381 correlate well with hydrogen-bonding interactions
connecting the amidic linker of the most potent analogue
with the same amino-acid residues. Similar analogy can
be noticed between hydrogen-bonding interactions
connecting the hydroxyl of Thr315 with the aromatic NH
of imitanib (Fig. 1a) and the nitro oxygen of the most po-
tent analogue (Fig. 1b). Furthermore, hydrophobic stacking
of the methylbenzene linker of imitanib within a narrow
CONCLUSION
In summary, we have synthesized a series of new
2-methyl-2-[(1,3-Diethyl-2,6-dioxo-2,3,6,7-tetrahydro-
1H-purin-8-yl)thio]-N- substituted arylacetamides through
the reaction of 8-merapto-xanthine with appropriate
Anilide. The prepared compounds were tested in vitro for
their antitumor activity against breast (MCF7) and Leuke-
mic (K562) cancer cell lines. The results revealed that
compound 6 k exerted significant antiproliferative activity
with the aforementioned cancer cell lines, with IC50 value
of 17.59 μM, against MCF7 and IC50 value of 9.57 μM,
against K562 cell line. Compound 6c displayed good activ-
ity, with IC50 value of 29.34μM, against MCF7 cell line
and IC50 value of 33.72 μM, against K562 cell line. The
rest of the compounds showed moderate to weak activities.
The findings would encourage us to do further studies and
testing that prove the usefulness of the prepared com-
pounds as candidate anti-cancer agents.
EXPERIMENTAL
Materials and equipments. Reagents used for the synthesis
were purchased from Sigma-Aldrich (Gillingham– Dorset,
UK), MERCK (Schuchardt, Germany) and Acros organics
(NJ, USA). All solvents were obtained from commercial
suppliers and used without further purification.
Melting points (mp) were determined on an electrother-
mal Stuart Scientific melting point apparatus (uk) and were
uncorrected.
Thin-layer chromatography (TLC) was carried out using
TLC aluminum sheets kieselgel 60F254 (MERCK) and
dichloromethane/methanol (9.5:0.5) as a mobile phase,
and visualization was effected with ultraviolet lamp at
short wavelength (λ = 254nm).
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2016
Effects of Mercapto Xanthine Derivatives on Cancer Cell Lines
All chemical yields are unoptimized and generally repre-
sent the result of a single experiment.
401.1522; found 400.1448. Anal. Calcd for C19H23N5O3S
(401.49g/mol): C, 56.84, H, 5.77, N, 17.44, found: C,
56.78, H, 5.66, N, 17.21.
NMR spectra were recorded on a Bruker Avance III 500,
500MHz spectrometer at Jordan University, college of
chemistry, Amman, Jordan, and we used also Agilent
DD2 400-MHz spectrometer at Lebniz institute for
plant biochemistry NWC department, Halle, Germany;
DMSO-d6 was used as a solvent, unless otherwise speci-
fied, and the chemical shifts are given in δ (ppm), coupling
constants (J) are in Hertz (Hz).
High resolution Mass spectrometer was recorded on
Bruker Daltonics APEX IV, at Jordan University, college
of chemistry, Amman, Jordan.
The microanalyses for C, H, N was performed on Euro
EA elemental analyzer at the college of pharmacy, Jordan
University, Amman, Jordan.
2-methyl-2-[(1,3-Diethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-
8-yl)thio]-N-(4-methoxyphenyl)acetamide 6b. Yield: 85%; mp:
262–264°C. 1H NMR (500 MHZ, DMSO): δ 13.6(br s, 1H,
N7-H), 10.2(s, 1H, amide-H), 7.49 (d, J= 8.7 Hz, 2H, 2′,6′
Ar-H), 6.88(d, J=8.7 Hz, 2H, 3′,5′Ar-H), 4.53 (q, 1H,
SCH-), 4.09(q, 2H, N3-CH2), 3.91(q, 2H, N1-CH2), 3.72(s,
3H,-OCH3), 1.59(d,3H,SCHCH3), 1.18 (t, 3H,N3-CH2CH3),
1.12(t,3H, N1-CH2CH3). HRMS (ESI) MS m/z: calcd for
+
C19H23N5O4S[M–H] (417.1471); found: 416.1398. Anal.
Calcd for (C19H23N5O4Sg/mol): C, 54.66, H, 5.55, N,
16.77, found: C, 54.54, H, 5.39, N, 15.608.
N-(4-Chlorophenyl)-2-methyl-2-[(1,3-Diethyl-2,6-dioxo-2,3,6,7-
tetrahydro-1H-purin-8-yl)thio]acetamide 6c. Yield: 74%; mp:
220°C. 1H NMR (500 MHZ, DMSO): δ 13.6(br s, 1H,
N7-H),), 11.27(s, 1H, amide- H), 7.67(d,J= 8.9 Hz, 2H, 2′,6′
Ar-H), 7.36(d,J = 8.9 Hz, 2H, 3′,5′Ar-H), 4.41 (q, 1H, SCH-),
3.97(q, 2H, N3-CH2), 3.91(q, 2H, N1-CH2), 1.53(d,3H,
SCHCH3), 1.16 (t, 3H,N1-CH2CH3), 1.11(t,3H, N1-CH2CH3).
HRMS (ESI) MS m/z: calcd for C18H20ClN5O3S[M–H]+
(421.0975); found: 420.0901. Anal. Calcd for (C18H20ClN5O3S
g/mol): C, 51.24, H, 4.78, N, 16.6, found: C, 51.1, H, 4.7,
N, 16.5.
N-(2-clorophenyl)-2-methyl-2-[(1,3-Diethyl-2,6-dioxo-2,3,6,7-
tetrahydro-1H-purin-8-yl)thio]acetamide 6d. Yield: 88.8%;
mp: 243°C. 1H NMR (500 MHZ, DMSO): δ 13.7(br s, 1H,
N7-H), 9.9(s, 1H, amide-H), 7.21–7.73 (m, 4H, Ar-H),
4.72 (q, 1H, SCH2), 4. 1(q, 2H, N3-CH2), 3.91(q, 2H,
N1-CH2), 1.6(d,3H,SCHCH3), 1.18 (t, 3H,N1-CH2CH3),
The anti-cancer evaluation of our target compounds
were healed in The Molecular Biology Laboratory, college
of medicine, University of Jordan.
General procedure for preparation of 1,3-Diethyl-8-thioxo-
3,7,8,9-tetrahydro-1H-purine-2,6-dione (4).
To a stirred
solution of 5,6-diamino-1,3-dimethyluracil 3 (3.28g,
16.50 mmol) in ethanol (25 mL), carbon disulfide
(1.50 mL, 26.4mmol) was added. The reaction mixture
was refluxed for 4 h, and then cooled. Cold water (25 mL)
was added to the reaction mixture with stirring, the
precipitate formed was filtered, washed successively with
cold water, and then with methanol. The product was
dried and crystallized from water as colorless crystals.
1,3-Diethyl-8-thioxo-3,7,8,9-tetrahydro-1H-purine-2,6-dione (4).
Yield: 61%, mp: 280–283°C. 1H NMR (500 MHZ, DMSO): δ
13.4 (br s, 1H, N7-H), 11.97 (br s, 1H, N9-H), 3.92 (q, 2H,
N3-CH2CH3), 3.86 (q, 2H, N1-CH2CH3), 1.17 (t, 3H,
N3-CH2CH3), 1.12 (t, 3H, N1-CH2CH3). Anal. Calcd for
C9H12N4O2S (240.29): C, 44.99, H, 5.03, N, 23.32, found C,
44.36, H, 4.96, N, 23.10.
1.11(t,3H, N1-CH2CH3). HRMS (ESI) MS m/z: calcd for
+
C18H20ClN5O3S[M–H]
(421.0975); found: 420.0901.
Anal. Calcd for (C18H20ClN5O3S g/mol): C, 51.24, H,
4.78, N, 16.6, found: C, 51.2, H, 4.65, N, 16.1.
N-(4-bromophenyl)-2-methyl-2-[(1,3-Diethyl-2,6-dioxo-2,3,6,7-
tetrahydro-1H-purin-8-yl)thio]acetamide 6e. Yield: 85%; mp:
269–272°C. 1H NMR (500 MHZ, DMSO): δ 13.7(br s, 1H,
N7-H), 10.5(s, 1H, amide-H), 7.57(d, 2H, 2′,6′Ar-H),
7.5(d, 2H, 3′,5′Ar-H), 4.58 (q, 1H, SCH2), 3.99(q, 2H,
N3-CH2), 3.91(q, 2H, N1-CH2), 1.16 (t, 3H,N1-CH2CH3),
1.59(d,3H,SCHCH3), 1.11(t,3H, N1-CH2CH3).HRMS (ESI)
2-methyl-2-[(1,3-Diethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-
8-yl)thio]-N-substituted arylacetamide (6 a-l). To a stirred
solution of compound 4 (0.30 g, 1.28mmol) in aqueous
sodium hydroxide 1% w/v (5.0 ml), the prepared Anilide
(1.28 mmol) dissolved in ethanol (3.0 ml) was added
portion wise. The reaction mixture was stirred at the
ambient temperature for 4 h and then cooled in a
refrigerator for 3 h. The product was filtered, washed with
water, then diethyl ether, and dried. All the compounds
6a-l was crystallized from ethanol.
+
MS m/z: calcd for C18H20BrN5O3S[M–H] (466.0470);
found: 465.0301. Anal. Calcd for (C18H20BrN5O3S
g/mol): C,46.36, H, 4.32, N, 15.02, found: C, 45.2, H,
4.2, N, 14.9.
2-methyl-2-[(1,3-Diethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-
2-methyl-2-[(1,3-Diethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-
8-yl)thio]-N-phenylacetamide 6f. Yield: 66%; mp: 243°C. 1H
NMR (500 MHZ, DMSO): δ 13.7(br s, 1H, N7-H), 10.3(s,
1H, amide-H), 7.21–7.6 (m,5H,Ar-H), 4.56 (q, 1H, SCH2),
3.99(q, 2H, N3-CH2), 3.91(q, 2H, N1-CH2), 1.6(d,3H,
SCHCH3), 1.16 (t, 3H,N1-CH2CH3), 1.11(t,3H, N1-CH2CH3).
HRMS (ESI) MS m/z: calcd for C18H21N5O3S[M–H]+
(387.1365); found: 386.1291. Anal. Calcd for (C18H21N5O3S
8-yl)thio]-N-(3-methylphenyl)acetamide 6a.
Yield: 60%;
1
mp: 218–220°C. H NMR (500 MHZ, DMSO): δ 13.7(br
s, 1H, N7-H), 10.3(s, 1H, amide-H), 6.88–7.43(m,4H,Ar-
H), 4.55 (q, 1H, SCH-), 4.1(q, 2H, N3-CH2), 3.91(q, 2H,
N1-CH2), 1.11(t,3H, N1-CH2CH3), 1.18 (t, 3H,N1-
CH2CH3), 1.59 (d,3H,SCHCH3), 2.23(s,3H, 3’-CH3).
HRMS (ESI) MS m/z: calcd for C19H23N5O3S [M–H]+
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

H. N. Sultani, R. A. Ghazal, A. M. Hayallah, L. K. Abdulrahman, K. Abu-Hammour,
S. AbuHammad, M. O. Taha, and M. A. Zihlif
Vol 000
g/mol): C,51.24, H, 4.78, N, 16.6, found: C, 51.01, H, 4.65,
N, 16.42.
(C18H20N6O5S 432g/mol): C, 50, H, 4.66, N, 19.43, found:
C, 49.1, H, 4.57, N, 19.36.
N-Benzyl-2-[(1,3-Diethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-
8-yl)thio]acetamide 6l. Yield 70%; mp:192°C. 1H NMR (500
MHZ, DMSO): δ 13.7(br s, 1H, N7-H), 10.3(t, J =6 Hz, 1H,
amide-H), 7.21–7.6 (s,5H,Ar-H), 4.56 (q, 1H, SCH2), 4.46
(d, J= 7Hz, 2H, HNCH2), 3.99 (q, 2H, N3-CH2), 3.91(q,
2H, N1-CH2), 1.6(d,3H,SCHCH3), 1.16 (t, 3H,N1-CH2CH3),
2-methyl-2-[(1,3-Diethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-
8-yl)thio]-N-(4-methylphenyl)acetamide 6g. Yield: 92%; mp:
1
235–240°C. H NMR (500 MHZ, DMSO): δ 13.8 (br s,
1H, N7-H), 10.2(s,1H, NH-amide), 7.48 (d, J= 8.7Hz,
2H, 2′,6′Ar-H),7.13 (d, J=8.7 Hz, 2H, 3′,5′Ar-H), 4.55
(q, 1H, SCH2), 3.99 (q, 2H, N3-CH2), 3.9 (q, 2H, N1-
CH2), 1.6 (d,3H,SCHCH3), 1.2 (t, 3H,N1-CH2CH3), 1.1
(t,3H, N1-CH2CH3). HRMS (ESI) MS m/z: calcd for
C19H23N5O3S[M–H] + (401.1522); found: (400.1444). Anal.
Calcd for (C19H23N5O3S 401 g/mol): C,56.84, H, 5.77, N,
17.44, found: C, 56.78, H, 5.66, N, 17.21.
1.11(t,3H, N1-CH2CH3). HRMS (ESI) MS m/z: calcd for
+
C19H23N5O3S [M+H]
(401.1522), found (401.1448).
Anal. Calcd for (C19H23N5O3S 402 g/mol): C, 56.84, H,
55.77, N, 17.44, found: C, 56.72, H, 5.69, N 17.37.
Cell lines and cell culture. The K562 leukemia cell line
was obtained from Dr Mona Hassona (Faculty of Science,
The University of Jordan) and was cultured in RPMI
while the T47D and MCF-7 breast cancer cells were
obtained from American Type culture collections (ATCC)
and were cultured in DMEM/F12. All media were
supplemented with 10% heat-inactivated fetal bovine
serum (FBS) (Gibco Invitrogen), 1% of 2mM L-glutamine
(Lonza), 50IU/mL penicillin (Lonza), and 50 μg/mL
streptomycin (Lonza) and cells were maintained at 37°C,
5% CO2 humidified incubator.
Cell proliferation assay. MCF-7, T47D, and K562 cells
were seeded at a density of 1 × 104, 1 × 104 and 4 × 104 cells
per well in 96-well plates in appropriate medium. For anti-
MCF7 and anti-K562 screening, the cells were treated with
50 μM concentrations of the tested compounds. For the
IC50 determination, the cells were treated with increasing
concentrations of the tested compound (1.56–100μM). In
all assays, the drugs were dissolved in DMSO immediately
before the addition to cell cultures, and equal amounts
of the solvent were added to control cells. Cell viability
was assessed, after 3days of treatment, with tetrazolium
dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT), obtained from Sigma (Dorset, UK). IC50
concentrations were obtained from the dose–response
curves using Graph Pad Prism Software 5 (San Diego
California USA, www.graphpad.com).
2-methyl-2-[(1,3-Diethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-
8-yl)thio]acetylamino-benzoic acid 6h. Yield: 81%; mp: 280°C.
1H NMR (500 MHZ, DMSO): δ 13.8 (br s, 1H, N7-H),
10.7(s,1H, NH-amide), 8.1 (d, J = 8.7 Hz, 2H, 2′,6′Ar-H),
7.8 (d, J = 8.7 Hz, 2H, 3′,5′Ar-H), 4. 55 (q, 1H, SCH2),
3.96(q, 2H, N3-CH2), 3.9 (q, 2H, N1-CH2), 1.6(d,3H,
SCHCH3), 1.12 (t, 3H, N1-CH2CH3), 1.12(t, 3H,
N1-CH2CH3). HRMS (ESI) MS m/z: calcd for C19H21-
+
N5O5S[M–H] (431.1263); found (430.1189).Anal. Calcd
for (C19H21N5O5S 431g/mol): C, 54.53, H, 5.39, N, 16.23,
found: C, 52.89, H, 4.91, N, 15.6.
N-(4- Acetylphenyl)-2-methyl-2-[(1,3-Diethyl-2,6-dioxo-2,3,6,7-
tetrahydro-1H-purin-8-yl)thio]acetamide 6i. Yield: 65%; mp:
1
230–232°C. H NMR (500 MHZ, DMSO): δ 13.8 (br s,
1H, N7-H), 10.7(s,1H, NH-amide), 7.9 (d, J = 8.7 Hz,
2H, 2′,6′ArH), 7.75 (d, J = 8.7 Hz, 2H, 3′,5′Ar-H), 4.6 (q,
1H, SCH2), 3.95(q, 2H, N3-CH2), 3.9 (q, 2H, N1-CH2),
1.6 (d,3H,SCHCH3), 1.1 (t, 3H, N1-CH2CH3), 1.1 (t,
3H, N1-CH2CH3). HRMS (ESI) MS m/z: calcd for
+
C20H23N5O4S[M–H] (429.1471); found (428.1398). Anal.
Calcd for (C20H23N5O4S 429 g/mol): C, 55.93, H, 5.4, N,
16.31, found: C, 55.45, H, 5.32, N, 16.18.
N-(3-chlorophenyl)-2-methyl-2-[(1,3-Diethyl-2,6-dioxo-2,3,6,7-
tetrahydro-1H-purin-8-yl)thio]acetamide 6j.
Yield 74%;
mp:230°C. 1H NMR (500 MHZ, DMSO): δ 13.8 (br s, 1H,
N7-H), 10.6(s,1H, NH-amide), 7.21–7.73 (m,4H,Ar-H),
4.53 (q, 1H, SCH2), 3.98(q, 2H, N3-CH2), 3.9 (q, 2H,
N1-CH2), 1.58(d,3H,SCHCH3), 1.13 (t, 3H, N1-CH2CH3),
1.13 (t, 3H, N1-CH2CH3). HRMS (ESI) MS m/z: calcd for
Acknowledgments. The authors thank Prof. Bernhard Westermann
from Lebniz Institute for Plant Biochemistry Halle (IPB), for his
support.
+
C18H20ClN5O3S [M–H] (421.0975); found (420.0901).
Anal. Calcd for (C18H20ClN5O3S 421 g/mol): C, 51.24, H,
4.78, N, 16.6, found: C, 51.2, H, 4.65, N, 16.1.
2-methyl-2-[(1,3-Diethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-
REFERENCES AND NOTES
8-yl)thio]-N-(4-nitrophenyl)acetamide 6k.
Yield 74%; mp:
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230–235°C. H NMR (500 MHZ, DMSO): δ 13.8(br s, 1H,
N7-H), 11 (s,1H, NH-amide), 8.25 (d, J =8.7Hz, 2H, 2′,6′
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Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

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Journal of Heterocyclic Chemistry
DOI 10.1002/jhet