Synthesis and cytotoxic activity of imidazo[1,2-a]-1,3,5-triazine analogues of 6-mercaptopurine

Article abstract of DOI:10.1002/ardp.200700176

A series of 2-substituted imidazo[1,2-a]-1,3,5-triazines with various aliphatic and aromatic amines has been prepared and characterized by IR, ¹H-NMR, and elemental analysis. The initial in-vitro cytotoxicity studies with five human cancer cell lines showed that all but one of the compounds are without cytotoxic activity. The one active compound, 2-(indolin-1-yl)-7,8-dihydroimidazo[1,2-a]-1,3,5-triazine-4(6H)-thione 12, was tested on 12 human cancer cell lines. Of these, two lines, RT-4 and MCF-7, were the most sensitive to the compound, with IC₅₀ values of 6.98 μM and 8.43 μM, respectively. When compared with the reference anticancer drug 6-mercaptopurine, only the RT-4 urinary bladder and KYSE-70 oesophagus cancer cell lines were more sensitive to the new compound. The antimetabolite thioguanine was more cytotoxic than 12 for all common cell lines tested.

Full text of DOI:10.1002/ardp.200700176

Arch. Pharm. Chem. Life Sci. 2008, 341, 121–125
F. Sa˛czewski et al.
121
Full Paper
Synthesis and Cytotoxic Activity of Imidazo[1,2-a]-1,3,5-triazine
Analogues of 6-Mercaptopurine
Franciszek Saczewski1, Magdalena Maruszak1, 2, Patrick J. Bednarski2
1 Department of Chemical Technology of Drugs, Medical University of Gdansk, Gdansk, Poland
2
Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, University of Greifswald,
Greifswald, Germany
A series of 2-substituted imidazo[1,2-a]-1,3,5-triazines with various aliphatic and aromatic
amines has been prepared and characterized by IR, 1H-NMR, and elemental analysis. The initial
in-vitro cytotoxicity studies with five human cancer cell lines showed that all but one of the com-
pounds are without cytotoxic activity. The one active compound, 2-(indolin-1-yl)-7,8-dihydroimi-
dazo[1,2-a]-1,3,5-triazine-4(6H)-thione 12, was tested on 12 human cancer cell lines. Of these, two
lines, RT-4 and MCF-7, were the most sensitive to the compound, with IC50 values of 6.98 lM and
8.43 lM, respectively. When compared with the reference anticancer drug 6-mercaptopurine,
only the RT-4 urinary bladder and KYSE-70 oesophagus cancer cell lines were more sensitive to
the new compound. The antimetabolite thioguanine was more cytotoxic than 12 for all common
cell lines tested.
Keywords: Aliphatic and aromatic amines / Cytotoxic activity / 6-Mercaptopurine / 2-Substituted imidazo[1,2-a]-1,3,5-
triazines / Thioguanine /
Received: August 20, 2007; accepted: September 20, 2007
DOI 10.1002/ardp.200700176
described procedure [10], since this structure bears
resemblance to the well known chemotherapeutic drug
6-mercaptopurine. The structure of the imidazo[1,2-a]-
1,3,5-triazines can be compared to already known anti-
metabolites and analogues of purines, i. e. 6-mercapto-
purine and thioguanine (Fig. 1), which have been used in
the anticancer therapy successfully for years [11]. There-
fore, we investigated their potential cytotoxic activity on
a panel of 12 human cancer cell lines.
Introduction
Our research interests have been focused on the synthesis
and evaluation of the cytotoxic activity of variously sub-
stituted imidazo[1,2-a]-1,3,5-triazines, which can be
regarded as 5-aza-7-deazapurine derivatives. Depending
on the nature of the substituents, representatives of this
group have been found to possess antidepressant [1], her-
bicidal [2], antiviral [3–5] activities as well as to affect the
circulatory system [6–8] and the thrombocyte aggrega-
tion [9]. In attempting to develop a new class of antitu-
mor agents, we used the imidazo[1,2-a]1,3,5-triazine-2,4-
dithione scaffold, prepared according to a previously
Results and discussion
Chemistry
Correspondence:
The substrate 7,8-dihydroimidazo[1,2-a]-1,3,5-triazine-
2,4(3H,6H)-dithione 1, 2-(alkylthio)-7,8-dihydroimidazo
[1,2-a]-1,3,5-triazine-4(6H)-thiones 2–6 and 2-amino(hy-
drazine)7,8-dihydroimidazo[1,2-a]-1,3,5-triazin-4(6H)-thio-
nes 10, 11, 16, and 17 were prepared according to the pro-
cedure previously described [7, 8, 10]. Thus, the reaction
of 2-chloro-4,5-dihydroimidazolium hydrogen sulfate
with ammonium thiocyanate in acetone led to the forma-
1Franciszek Saczewski, Department of Chemical Technology of Drugs,
Medical University of Gdansk, 80-416 Gdansk, Poland.
E-mail: saczew@amg.gda.pl
Fax: +48 58 3493-257
2Patrick J. Bednarski, Department of Pharmaceutical and Medicinal
Chemistry, Institute of Pharmacy, University of Greifswald, 17487 Greifs-
wald, Germany.
E-mail: bednarsk@uni-greifswald.de
Fax: +49 3834 864-874
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F. Sa˛czewski et al.
Arch. Pharm. Chem. Life Sci. 2008, 341, 121–125
Figure 1. Structure of 6-mercaptopurine and thioguanine and
their 5-aza-7-deaza analogues.
Scheme 1. General procedure for preparation of 7,8-dihydro-2-
alkylthio-imidazo[1,2-a]-1,3,5-triazine-4(6H)-thiones 2-9.
Scheme 2. Synthesis of 2-amino(hydrazino)-imidazo[1,2-a]-
1,3,5-triazines 10–18.
tion of the desired dithione 1. Then, the substrate 1 was S- protons are found as a multiplet at 7.2–7.6 ppm and the
alkylated regioselectively with corresponding iodide or proton of the thioureido NH group is found at 10.6 ppm.
bromide under alkaline conditions, as shown in
Scheme 1.
Compound 2 served as a substrate for further transfor-
mations by means of the nucleophilic displacement of
The identity of the newly obtained compounds 7 and 8 methylthio-leaving group with aliphatic and aromatic
was confirmed by elemental analysis and IR as well as 1H- amines as well as hydrazines (Scheme 2). According to
NMR spectroscopic data. For example, the IR spectrum of this procedure, a series of known 10, 11, 16, 17 and novel
8 exhibited absorption at 3210, 2248 and 1661 cm– 1, cor- compounds 12–15 were prepared.
responding to amide NH, nitrile C:N, and imine C=N
Hydrazine derivative 16 obtained in this manner was
groups, respectively. The 1H-NMR spectrum run in DMSO- then reacted with 3-nitrobenzaldehyde in boiling etha-
d6 revealed the presence of two multiplets at 3.6-3.8 and nol in the presence of catalytic amount of piperidine to
4.1–4.3 ppm attributable to the 1,2-disubstituted imida- give the corresponding hydrazone 18 (Scheme 2). All the
zoline ring, a singlet integrating the two protons of the compounds obtained gave satisfactory results of elemen-
CH2-C:N group, and a singlet at 9.6 ppm, corresponding tal analyses, and their structure was further confirmed
to the exchangeable proton of the NH group.
Then, we turned our attention to the reaction of imida-
by IR and 1H-NMR spectral data (Experimental, Section 4).
It is noteworthy, that, for the first time, we succeeded
zotriazines with isocyanates. Previously, it was found in reacting compound 2 with an aromatic amine. Upon
that compounds of type 2 react with aryl isocyanates at heating 2 with excess aniline at reflux for 1 h, the
the N8-H group to give the corresponding N-arylcarba- expected
2-(phenylamino)-7,8-dihydroimidazo[1,2-a]-
moyl derivatives [7]. As shown in Scheme 1, the reaction 1,3,5-triazine-4(6H)-thione 15 was obtained in 58% yield
of 3 with benzoyl isothiocyanate, carried out in acetone (Scheme 2). Interestingly, the 1H-NMR spectra of 15 run at
at room temperature, gave the N8-benzoylthiocarbamoyl, 200 and 500 MHz exhibited broadened signals attribut-
compound 9.
able to protons 2,6-H of the aromatic ring in the range of
In the 1H-NMR spectrum of 9, the S-C2H5 group appears 7.6–7.9 ppm and four broad signals of NH protons in the
as a triplet of the CH3 group at 1.4 ppm and quartet of the range of 8.6–10.2 ppm. Such a pattern indicates that a
CH2 group at 3.3 ppm; the imidazoline CH2-CH2 moiety dynamic process takes place and two different tauto-
gives a multiplet in the range of 4.0–4.1 ppm; aromatic meric forms exist in DMSO solution.
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Arch. Pharm. Chem. Life Sci. 2008, 341, 121–125
Cytotoxic 2-Substituted Imidazo[1,2-a]-1,3,5-triazines
123
Table 2. The relative percent of cell growth (%) compared to
untreated control at a concentration of 20 lM. (N = 1–2 determi-
nations)
Cell lines
Com-
MCF-7a) RT-4a)
LCLC-103H A-427
5637
pound
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
91.56
108.43
112.70
100.03
113.56
112.03
86.76
102.69
105.34
97.36
22.00
113.30
68.99
97.97
94.06
99.86
107.86
105.13
103.24
104.05
106.44
82.37
106.50
107.69
110.12
28.56
106.76
67.37
103.51
93.20
104.70
68.82
56.85a)
91.48
92.79
76.08a)
78.29a)
92.17
102.28
98.37
82.06
64.47a)
97.39
86.58
102.03
97.66
49.77
99.53
70.36
105.46
73.48
98.28
94.70
108.15
110.78
101.58
95.14
93.93
76.07
66.70a)
86.72
66.00a)
126.02
91.42
94.65
112.81
118.33
66.37
105.87
84.52
112.28
81.32
103.02
106.94
106.37
102.08
65.15
105.33
79.45
103.64
80.49
107.28
92.07
Figure 2. Theoretical structures of possible tautomers of imi-
dazo[1,2-a]-1,3,5-triazine 15, optimized by semi-empirical AM1
calculations.
106.60
112.17
Table 1. Heat of formation (DHf), DE, and dipole moments (l) of
possible tautomers 15A–H.
a)
Average of two determinations
Heat of formation DE
Dipole moment
(Debye)
(kcal/mol)
(kcal/mol)
assay based on the staining of cells with crystal violet was
used to determine the antiproliferative activity of the
compounds [12]. The compounds were added to all can-
cer cell lines at the same concentration of 20 lM.
A
B
C
D
E
F
G
H
130.92
131.46
139.46
136.37
134.08
138.34
134.23
137.35
0
6.71
5.49
6.54
6.25
3.11
3.17
3.68
4.66
0.54
8.54
5.45
3.16
7.42
3.31
6.43
Primary screening indicates whether a substance is
active enough to inhibit the cell growth by 50% at 20 lM,
which is a concentration that can typically be attained in
patients. Substances that would inhibit the growth of
these five lines by more than 50% at the concentration of
20 lM would be considered active. As shown in Table 2,
only the indoline derivative 12 was active in this screen.
Thus, compound 12 was examined further for potency of
action in the secondary screening on twelve human can-
cer cell lines.
Compound 12 was tested at five successive concentra-
tions on the cell lines: LCLC-103H, A-427, 5637, KYSE-510,
KYSE-520, YAPC, RT-4, RT-112, DAN-G, KYSE-70, SISO, MCF-
7. The concentration ranges were adjusted depending on
the activity of the tested compound. The results of the
secondary screening tests are shown in Table 3 as the
average IC50 values with standard deviation, calculated
from the results of three independent experiments.
The relatively high values of standard deviations may
be the result of poor solubility of compound 12 in the cul-
ture medium. As a result, the compound may have been
tested in some cases as a suspension in the medium. In
fact, the low water solubility of all tested compounds
might have influenced their activity.
To shed light on this phenomenon, we have calculated
the heats of formation (DHf) of eight possible tautomeric
forms 15A-H (Fig. 2) using quantum chemical calcula-
tions optimized by the semi-empirical AM1 method. The
comparison of the heats of formation (DHf) and dipole
moments (l) for tautomers 15A-H shows that the most
stable structure appears to be tautomer
DHf = 30.92 kcal/mol and l = 6.71 Debye followed by tau-
tomer B (DHf = 131.46 kcal/mol, l = 5.49 Debye).
A with
Biology
Cytotoxic activity
The cytotoxic activities of imidazo[1,2-a]-1,3,5-triazine
derivatives were examined in the primary testing on five
human cancer cell lines: the breast cancer cell line MCF-
7, two urinary bladder cancer cell lines RT-4 and 5637
and two non-small cell lung cancer cell lines – LCLC-
103H and A-427 (Table 2). A well established microtiter
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F. Sa˛czewski et al.
Arch. Pharm. Chem. Life Sci. 2008, 341, 121–125
Table 3. IC50 values (lM) l S.D. in twelve human cancer cell lines (N = 3 determinations)
Cell lines
5637
A-427
Kyse-520
SISO
RT-4
YAPC
LCLC-103H Kyse-510
RT-112
MCF-7
DAN-G
Kyse-70
Compd. 12
22.43 l 1.6 15.1 l 5.1 21.7 l 1.2 26.9 l 5.8
6.98 l1.3
37.57 l 2.4 21.2 l 5.6
26.67 l 2.8
1.78 l 0.88
21.8 l 0.9 8.43 l 2.0 28.8 l 1.6 23.4 l 2.8
6-Mercaptopurine Nda)
0.37 l 0.13 Nda)
2.82 l 0.64 15.8 l 3.5 Nda)
1.50 l 0.23 3.98 l 0.50 Nda)
2.18 l 2.24
1.06 l 0.74 0.016 l 0.015
Nda)
Nda)
2.79 l 0.69 15.2 l 7.4 33.3 l 9.5
2.64 l 1.86 0.72 l 0.15 8.69 l 1.43
6-Thioguanine
3.53 l 1.40 0.31 l 0.28 Nda)
a)
Nd, not determined
were recorded on a Perkin-Elmer 1600 FTIR spectrometer as KBr
pellets (Perkin-Elmer, Norwalk, CT, USA). The 1H-NMR spectra
were measured with the Varian Gemini Plus spectrometer (Var-
ian Inc., Palo Alto, CA, USA) in DMSO-d6 solution at 200 Hz or 500
MHz. The chemical shift values d are expressed in ppm units
towards tetramethylsilane used as internal standard. Molecular
modeling studies were performed using Spartan programs v. 5.0
distributed by Wavefunction Inc. (Irvine, CA, USA) and installed
on a Silicon Graphics O2 workstation.
Although the MCF-7 and RT-4 cell lines are the most
sensitive to compound 12, the results show no great selec-
tivity towards any one specific cancer cell line (Table 3).
For example, this compound 12 is only 5.4 fold less active
on the YAPC cell line than on the RT-4 cell line and 4.5
fold less active than on the MCF-7 cell line. Compound 12
shows relatively low selectivity over all cell lines (relative
standard deviation of mean IC50 is only 39%).
The IC50 values of known anticancer drugs like 6-mer-
captopurine and 6-thioguanine are in general much
lower than for 12, with the exception of the RT-4 and
KYSE-70 cell lines (Table 3). Compound 12 is twice as
active as 6-mercaptopurine on the RT-4 cell line. How-
ever, 12 is only half as active in comparison to 6-thiogua-
nine in the same cell line. Imidazotriazine 12 also shows
somewhat better cytotoxic activity on the KYSE-70 cancer
cell line than 6-mercaptopurine. On the other hand, thio-
guanine was more active than 12 in all the common cell
lines tested.
2-(4-thioxo-4,6,7,8-tetrahydroimidazo[1,2-a]-1,3,5-triazin-
2-ylthio)acetamide 7
Compound 1 was treated with 1.5 molar excess of chloroaceta-
mide in methanol in the presence of KOH and the reaction mix-
ture was left at room temperature for 1 h. The solid that precipi-
tated was filtered off, dried, and dissolved in water. After acidifi-
cation with aqueous solution of HCl the precipitated product 7
was collected by suction and recrystallized from DMF. Yield
0.27 g (26%), mp. 218–2258C. IR (KBr) m (cm– 1): 3420, 3170 (NH),
1
1654 (C=O), 1517, 1446, 1423, 1347, 1290, 1268, 1227. H-NMR
(DMSO) d = 3.6–4.0 (m, 4H, CH2), 4.1–4.3 (t, 2H, CH2), 7.2 (s, 2H,
NH2), 9.3 (s, 1H, NH). Anal. calcd. for C7H9N5OS2 (243.309): N,
28.78. Found N, 28.52.
Conclusions
2-(4-thioxo-4,6,7,8-tetrahydroimidazo[1,2-a]-1,3,5-triazin-
2-ylthio)acetonitrile 8
Here, we have synthesized a series of imidazo[1,2-a]-1,3,5-
triazines by substituting an aliphatic or aromatic amine
at the ring position 2. Five novel compounds were
obtained, among them a derivative with an aromatic sub-
stituent was synthesized for the first time. This group of
compounds possessed a general lack of cytotoxic activity
on human cancer cells in culture. Only 2-(indolin-1-yl)-
7,8-dihydroimidazo[1,2-a]-1,3,5-triazine-4(6H)-thione 12
was active enough to enter the secondary screening and
was tested on 12 cancer cell lines. The lines MCF-7 and RT-
4 were the ones most sensitive to this compound. In com-
parison to the established cytotoxic activity of 6-mercap-
topurine, 12 is more active only on the RT-4 and KYSE-70
cancer cell lines.
Prepared as described for compound 7.
Mp. 246–2538C. IR (KBr) v (cm– 1): 3210 (NH), 2248 (CN), 1661,
1634, 1510, 1434, 1340, 1269, 1225. 1H-NMR (DMSO) d = 3.6–3.8
(m, 2H, CH2 imidazoline), 4.2 (s, 2H, CH2-CN), 4.1–4.3 (m, 2H, CH2
imidazoline), 9.6 (s, 1H, NH). Anal. calcd. for C7H7N5S2 (225.294):
C, 37.32, H, 3.13, N, 31.09. Found C, 37.57, H, 3.17, N, 31.99.
{[2-(ethylthio)-4-thioxo-6,7-dihydroimidazo[1,2-a][1,3,5]-
triazin-8-(4H)-yl]carbonthioyl}benzamide 9
To a suspension of compound 3 in anhydrous acetone was added
an equimolar amount of benzoyl isothiocyanate. The reaction
mixture was stirred at room temperature for 12 h and the pre-
cipitating yellowish solid 9 was collected by filtration. Yield
79%, mp. 214–2168C. IR (KBr) m (cm– 1): 3335, 3128 (NH), 1674
(C=O), 1517, 1438, 1427, 1376, 1245, 1234, 1216. 1H-NMR (DMSO)
d = 1.4 (t, 3H, CH3), 3.3 (q, 2H, CH2), 4.0–4.1 (m, 4H, CH2CH2),
7.2–7.6 (m, 5H, aromat), 10.6 (s, 1H, NH). Anal. calcd. for
C15H15N5OS3 (377.507): N, 18.55. Found N, 18.54.
The authors have declared no conflict of interest.
Experimental
2-(alkyl(aryl)amino)-7,8-dihydroimidazo[1,2-a]-1,3,5-
Chemistry
triazine-4(6H)-thiones 12–15
Compound 2 was dissolved in an excess of corresponding amine
and heated at reflux for 1 h. After cooling to room temperature,
Melting points were defined on a Boetius 545 apparatus (Franz
Kiistner, Dresden, Germany) and are uncorrected. The IR spectra
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Arch. Pharm. Chem. Life Sci. 2008, 341, 121–125
Cytotoxic 2-Substituted Imidazo[1,2-a]-1,3,5-triazines
125
the precipitate was separated by suction and purified by recrys-
tallization from a suitable solvent. According to this procedure,
the following novel compounds were obtained.
The cytotoxicity testing was carried out in 96-well microtiter
plates, whereby 100 lL of cell suspensions per each well were
seeded at a density of 1000 cells per well (with an exception for
the LCLC-103H line with only 500 cells).
For the secondary screening, cells were treated with substance
at five successive concentrations; i. e. the 1000-fold concentrated
stock solution in DMF was serially diluted by 50% in DMF to give
feed solutions, which were diluted 500-fold into the culture
medium. Controls wells received just 100 lL of culture medium
containing 0.1% DMF. One plate was untreated for each cell line
and fixed immediately with glutaraldehyde. Later, this plate
served as the ,C,0' control.
2-(Indolin-1-yl)-7,8-dihydroimidazo[1,2-a]-1,3,5-triazine-
4(6H)-thione 12
Yield 59%, mp. 336–3388C (EtOH). IR (KBr) m (cm– 1): 3221 (NH),
1636 (C=C), 1522, 1465, 1280. 1H-NMR (DMSO) d = 3.2–3.3 (t, 2H,
CH2), 3.6-3.7 (t, 2H, CH2), 4.1–4.3 (m, 4H, CH2CH2), 7.0–7.1 (m,
2H, Ar-H), 7.2–7.35 (m, 2H, Ar-H), 9.0 (s, 1H, NH). Anal. calcd. for
C13H13N5S (271.341): N, 25.81. Found: N, 26.06.
After 96-hours of exposure to the drug, the medium was
removed and 1% glutaraldehyde buffer solution (GBS) was added
to fix the cells for 20 min. Cells were stored at 48C under PBS.
Staining with crystal violet was done as previously described
[11]. The optical density (OD) was measured at k = 570 nm with
an Anthos 2010 microplate reader (Salzburg, Austria). To calcu-
late the T/C values, the equation was used as explained below:
2-(Furan-2-ylmethylamino)-7,8-dihydroimidazo[1,2-a]-
1,3,5-triazine-4(6H)-thione 13
Yield 69%, mp. 253–2598C (DMF). IR, (KBr) m (cm– 1): 3420, 3360,
3202 (NH), 3096, 2917, 1660, 1602, 1508, 1341. Anal. calcd. for
C10H11N5OS (249.292): N, 28.09. Found: N, 28.17.
T/C corr. = (ODT – ODC,0)/(ODC – ODC,0) N 100
2-((Tetrahydrofuran-2-yl)methylamino)-7,8-
dihydroimidazo[1,2-a]-1,3,5-triazine-4(6H)-thione 14
Yield 76%, mp. 259–2638C (EtOH). IR (KBr) m (cm– 1): 3366, 3214,
3098, 1658, 1644, 1577, 1514, 1428, 1372, 1114. Anal. calcd. for
C10H15N5OS (253.324): N, 27.65. Found: N, 27.34.
where ODT is mean OD of the treated cells; ODC the mean
OD of the controls, and ODC,0 the mean OD at the time the
drug was added. IC50 values were obtained by linear
regression of the T/C values that ranged between 90 and
10%. The reported IC50 values are the average of three
independent experiments.
2-(Phenylamino)-7,8-dihydroimidazo[1,2-a]-1,3,5-
triazine-4(6H)-thione 15
Yield 58%, mp. 223–2278C (DMF / H2O). IR (KBr) m (cm– 1): 3403,
3252, 1654, 1599, 1532, 1490, 1446, 1337, 1277. 1H-NMR (DMSO)
d = 3.6–3.75 (t, 2H, CH2), 4.2–4.35 (t, 2H, CH2) 7.0–7.15 (t, 1H, aro-
mat), 7.25–7.4 (t, 2H, aromat), 7.6–7.8 (br s, 2H, aromat), 9.0 (br
s1H, NH) 10.1 (br s, 1H, NH). Anal. calcd. for C11H11N5S (245.304):
N, 28.55. Found: N, 28.89.
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2-(2-(3-nitrobenzylideno)hydrazinyl)-7,8-
dihydroimidazo[1,2-a]-1,3,5-triazine-4(6H)-thione 18
To a solution of the hydrazine compound 16 (0.5 g, 3 mmol) and
m-nitrobenzaldehyde (0.45 g, 3 mmol) in ethanol (10 mL) was
added five drops of piperidine. The reaction mixture was heated
at reflux for 10 h. Upon cooling to room temperature, a precipi-
tate formed; it was separated by filtration and purified by crys-
tallization from DMF. Yield 0.6 g (69.8%) of 18 as a yellow pow-
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(C=C), 1578 (NO2), 1348 (NO2), 1120. 1H-NMR (DMSO) d = 3.6–3.8
(t, 2H, CH2), 4.1–4.3 (t, 2H, CH2), 7.7–7.84 (m, 4H, aromat.), 8.0–
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pyruvate (1 mM), MEM salts and amino acid. The cells were incu-
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