Synthesis, structural characterization and antitumor activity of novel 2,4-diamino-1,3,5-triazine derivatives

Article abstract of DOI:10.1016/S0223-5234(00)01194-6

The syntheses, structural elucidation based on NMR spectroscopy and X-ray analysis of 8 as well as antitumor activities of novel 2,4-diamino-1,3,5-triazine derivatives 5 and 7-22 are described. Screenings performed at NCI showed that most derivatives possessed a moderate to strong growth inhibition activity on various tumor panel cell lines between 0.148 and 56.2 μM concentrations. 2-Amino-6-bromomethyl-4-(3,5,5-trimethyl-2-pyrazoline)-l,3,5-triazine 11 showed the most potent antitumor activity with the mean midpoint values of log₁₀ GI50, log₁₀ TGI50 and log₁₀ LC50 of all tests equal to -5.26, -4.81 and -4.37, respectively and therefore, it can be considered as a lead structure for further development of anticancer agents.

Full text of DOI:10.1016/S0223-5234(00)01194-6

Eur. J. Med. Chem. 35 (2000) 1053–1064
´
© 2000 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved
PII: S0223-5234(00)01194-6/FLA
Original article
Synthesis, structural characterization and antitumor activity of novel
2,4-diamino-1,3,5-triazine derivatives
Z. Brzozowskia, F. Sa˛czewskia*, M. Gdaniecb
aDepartment of Chemical Technology of Drugs, Medical University of Gdan´sk, Al. Gen. Hallera 107, P-80-416 Gdan´sk, Poland
bFaculty of Chemistry, A. Mickiewicz University, ul. Grunwaldzka 6, P-60-780 Poznan´, Poland
Received 11 February 2000; revised 30 May 2000; accepted 6 June 2000
Abstract – The syntheses, structural elucidation based on NMR spectroscopy and X-ray analysis of 8 as well as antitumor activities
of novel 2,4-diamino-1,3,5-triazine derivatives 5 and 7–22 are described. Screenings performed at NCI showed that most derivatives
possessed a moderate to strong growth inhibition activity on various tumor panel cell lines between 0.148 and 56.2 mM
concentrations. 2-Amino-6-bromomethyl-4-(3,5,5-trimethyl-2-pyrazoline)-1,3,5-triazine 11 showed the most potent antitumor activ-
ity with the mean midpoint values of log10 GI50, log10 TGI50 and log10 LC50 of all tests equal to −5.26, −4.81 and −4.37,
´
respectively and therefore, it can be considered as a lead structure for further development of anticancer agents. © 2000 Editions
scientifiques et me´dicales Elsevier SAS
2,4-diamino-1,3,5-triazine derivatives / X-ray structure analysis / antitumor effect
1. Introduction
the target compounds 5–18 and 19–22 are described in
figures 1 and 2, respectively.
Numerous 2,4-diamino-1,3,5-triazines possess vari-
ous biological activity, but many of their derivatives
are still with unexplored pharmacological properties.
However, several reports describe their potential as
cardiotonic [1], neuroleptic [2] nootropic [3], antihis-
taminergic [4], tuberculostatic [5], anti-HIV [6, 7],
antiviral [8] and anticancer [9–11] agents. In this
context and in connection with a research program on
2,4-diamino-1,3,5-triazine chemistry and biological
activities undertaken in our laboratories years ago [7],
we have considered that novel, suitably substituted,
2,4-diamino-1,3,5-triazines may act as potential anti-
cancer agents.
Synthesis of triazine derivatives 5–12 were performed
according to a well known procedure [12] consisting of
the reactions of either biguanide hydrochlorides 1–4
(Method A) or their free bases 1a–4a (Method B) with
suitably substituted carboxylic acid esters.
The 6-chloromethyl- or 6-bromomethyl-triazines 8
and 11 were subsequently subjected to the reactions with
selected N, O or S nucleophiles to afford products 13
and 14–16. Among them 7,8-dihydroimidazo[1,2-a]-
1,3,5-triazine-2,4-dithione [13], considered to be 5-aza-7-
deaza-purine [14], was used.
Hydrolysis of the chloromethyl derivative 8 carried
out in aqueous alkaline medium at ambient temperature
led to the formation of a mixture of the alcohol 17 and
ether 18 in 35 and 46% yields, respectively.
The mercaptylation of 8 with thiourea gave the de-
sired product 20. The method consists in initial forma-
tion of the isothiouronium salt 19, followed by alkaline
hydrolysis in the presence of hydrazine. Bearing in mind
that the previously described analogous mercaptylation
2. Results
2.1. Synthesis
The synthetic routes utilized for the preparation of
* Correspondence and reprints.
of
4-amino-6-piperidino-2-chloromethyl-1,3,5-triazine

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Z. Brzozowski et al. / European Journal of Medicinal Chemistry 35 (2000) 1053–1064
led to the formation of corresponding disulfide rather
than the anticipated thiol [11], hydrazine was used to
prevent possible oxidation of the mercaptomethyl
derivative 20.
Further reaction of the thiol 20 with ethyl chloroac-
etate in the presence of EtONa gave ester 21, which
upon treatment with biguanide 1a afforded the sulfide
22.
atom at −294 ppm (1J(15N.1H)=94 Hz). Such type of
coupling constants is known to be strongly dependent
on the hybridization of the nitrogen atom. Approximate
percentage of s character in a hybrid orbital was calcu-
lated using the following equation [15]:
1
%s=0.43 · ꢀ J(N.H)ꢀ−6
All of the final compounds were characterized by IR,
NMR and elemental analysis and all were in accordance
with the proposed structures.
and amounted to 33%, thereby suggesting sp2 hybridiza-
tion of the exocyclic nitrogen atom.
Therefore, we have also performed quantum chemical
calculations of energy as functions of C6ꢀN7 enterring
bond rotation using AM1 semiempirical method [16].
The rotational energy profile showed a global minimum
at dihedral (s=N1ꢀC6ꢀN7ꢀN8) angle= −8.9° and a
second minimum ca. −166.6°. The energy difference
An intriguing feature of the 1H-NMR spectrum of the
compound 8 run in CDCl3 at 22°C was pairing of some
signals from protons in the vicinity of the triazine
moiety. Thus, methylene protons were found as two
broadened signals at 4.26 and 4.36 ppm, respectively,
while the NH2 occurred as a broad singlet at 6.0 ppm.
between the two conformers was ca. 1.6 kcal mol−1
,
1
When the temperature of the H- and 13C-NMR experi-
with s= −8.9° the preferred dihedral angle. The energy
barrier between these conformations was ca. 5 kcal
mol−1. These findings confirm that in solution, com-
pound 8 may adopt two major rotamer populations A
and B (figure 3) while in the solid state, rotamer A is
present exclusively due to intermolecular packing forces
(vide infra).
All the above evidence points to a conclusion that
doubling of the signals in NMR spectra resulted from
hindered rotation of pyrazoline ring with respect to
1,3,5-triazine ring system. It is worth noting that a
ments was decreased to −30°C, both spectra exhibited
doubled signals arising from all protons and carbons.
For example, protons of the NH2 appeared as four
resonances at 5.25, 6.33, 6.83 and 7.51 ppm. Moreover,
two distinct signals were found in 15N-NMR spectrum
for exocyclic nitrogen atom at −294 and −296 ppm.
From the N,H heterocorrelated HSQC spectrum it fol-
lowed that signals at 5.25 and 7.51 ppm correlated to
the nitrogen atom at −296 ppm (1J(15N.1H)=90 Hz)
and these at 6.33 and 6.83 correlated to the nitrogen
Figure 1.

Z. Brzozowski et al. / European Journal of Medicinal Chemistry 35 (2000) 1053–1064
1055
Figure 2.
similar effect was not observed in NMR spectra run at
22° for other 1,3,5-triazine derivatives obtained, which
might be explained by either lower energy barrier to the
rotation or, which is more probable, overlapping of the
signals attributed to CH2 bridges for corresponding
rotamers.
within this ring show large discrepancies, ranging from
,
1.321 to 1.364 A, however the pattern of bond lengths
within the ring compares well with that observed in the
other diamino-s-triazine derivative, where similar influ-
ence of the substituents on the ring geometry could be
expected [17]. The N12 amino group lies in the plane of
,
the s-triazine ring and C2ꢀN12 bond length of 1.314 A
indicates significant conjugation of the N12 lone elec-
tron pair with the p-system of the aromatic ring. All
endocyclic torsion angles of the 3,5,5-trimethyl-2-pyra-
zoline fragment are smaller than 3°. Average deviation
of atoms from the least-squares plane calculated
2.2. Crystal structure of 8
The 1,3,5-triazine ring is slightly non-planar with C2,
,
N5 located in the mean plane, N1, C4 0.03 A below and
,
N3, C6 0.03 A above this plane. The CꢀN bond lengths

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Z. Brzozowski et al. / European Journal of Medicinal Chemistry 35 (2000) 1053–1064
Figure 3.
through the five atoms constituting the pyrazoline ring is
Data of the most active compounds 5–8, 11, 13, and
16 are recorded in table I.
,
0.013 A, with C14 lying in the plane of the ring and C6
,
deviating 0.179(2) A from the plane of the ring. Atoms
Among the 2-chloromethyl series 5–8, the 3,5,5-
trimethylpyrazoline derivative 8 showed significant ac-
tivity against most of the cell lines with the exception of
prostate cancer, while its analogues bearing dimethyl-
amine (5), morpholine (6) or 3-methylpyrazoline (7)
were less active. It was concluded from these results that
a significant antineoplastic activity was associated with
3,5,5-trimethylpyrazoline moiety. Therefore, further
structural modifications conserved this ring system and
a highly active 8 served as a point of departure. Thus, a
set of compounds 9–22 varied on 6-methylene group
was produced. The introduction of any of the sub-
stituents F, OH, MeSO2, OMe and SH, however, re-
N5, N1, N7, C6 are coplanar and atoms N8 and C11
are displaced from their plane by −0.035(4) and
,
0.309(5) A which results in significant piramidalization
of N7. This deformation is also reflected in deviation of
,
N7 (0.091(3) A) from the plane through its three sub-
stituents and in the values of the N5C6N7C11 and
N1C6N7N8 torsion angles which are −15.5(4)° and
−1.3(3)°, respectively.
The molecules in the crystal join via NꢀH···N hydro-
gen bonds forming tapes as shown in figures 4 and 5.
Both hydrogen atoms of the N12 amino group are
involved in this interaction and N1 and N3 atoms of the
1,3,5-triazine ring act as acceptors.
2.3. Biology
Evaluation of anticancer activity was performed on
all compounds described herein at the National Cancer
Institute (NCI) of Bethesda, following the known in
vivo disease oriented antitumor screening program
against a panel of 62 tumor cell lines derived from nine
cancer types (leukemia, lung, colon, brain, melanoma,
ovarian, prostate, renal and breast) according to stan-
dard protocol [18]. In each test, dose–response curves
for each cell line were measured with five different drug
concentrations and the concentration causing 50% cell
growth inhibition (GI50), total cell growth inhibition
(TGI), 0% growth) and 50% cell death (LC50, −50%
growth) compared with the control, was calculated.
Figure 4. Atom labeling of 8.

Z. Brzozowski et al. / European Journal of Medicinal Chemistry 35 (2000) 1053–1064
1057
H522 and HOP-92) as well as melanoma cell line
LOX IMVI.
In conclusion, the high degree of activity of com-
pounds 8 and 11 raises the potentiality of their further
derivatization in the hope of finding even more active
and selective cytotoxic agents.
3. Experimental protocols
3.1. Synthesis
Melting points are uncorrected and were recorded
on a Buchi apparatus. IR spectra were recorded on a
Perkin–Elmer FT spectrometer. 1D 1H- and 13C-
NMR spectra were recorded on a Varian Gemini XL-
200 instrument at 200 and 50 MHz, respectively.
Two-dimensional NMR spectra were recorded on a
Varian Unity Plus 500 instrument. The results of ele-
mental analyses for C, H and N were within 90.4%
of the theoretical values.
3.2. 2-Amino-4-R2-6-haloalkyl-1,3,5-triazines 5–11
3.2.1. Method A
To a solution of sodium methoxide in methanol (2.3
g Na, 90 mL methanol) corresponding biguanide hy-
drochloride 1, 2 or 3 (0.101 mol) [21] was added and
the reaction mixture was stirred at r.t. for 3 h. Then,
a suitably substituted acetic acid methyl ester (figure
1) was added dropwise. The reaction mixture was
stirred for 24 h and subsequently heated under reflux
for 6 h. After cooling to r.t., the product that precipi-
tated was collected by filtration, washed with
methanol (2×5 mL) and water and purified by crys-
tallization from suitable solvent (table II).
Figure 5. H-bonded tape of molecules 8.
sulted in considerable decreasing of the antitumor ac-
tivity. Also, ether (18) or thioether (22) groups were
associated with very poor activity.
The only substituents which gave indisputable activ-
ity were Br (11), morpholine (13) and 7,8-dihydroimi-
dazo[1,2-a]triazine-2,4-dithione (16), with the former
being most active (log10 GI50 ranges from −4.25 to
−6.83).
For the compound 11, the mean logarithmic value
of GI50 in all cell lines is −5.42; this value is the
midpoint of the bar graph. Compound 11 was found
to exhibit moderate selectivity towards renal cell lines
CAKI-1 at the TGI level with a selectivity ratio of 39.
This selectivity was retained at the LC50 level with a
ratio of 49. From the pattern of the mean graph it is
clear that 11 also demonstrates a greater than average
activity towards some cell lines of lung cancer (NCI-
3.2.2. Method B
To a solution of biguanide 4a (10.0 g, 0.059 mol) in
ethanol (100 mL) suitably substituted acetic acid ethyl
ester (figure 1) was added dropwise over 1 h. The
reaction mixture was stirred at ambient temperature
for 12 h and the product that precipitated was sepa-
rated by suction, washed with ethanol (10 mL) and
purified by crystallization.
2-Amino-6-(methylsulfonyl)-4-(3,5,5-trimethyl-2-pyra-
zolino-1,3,5-triazine 12 was obtained according to
method B, with the exception that the reaction mix-
ture was heated under reflux for 8 h.

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Z. Brzozowski et al. / European Journal of Medicinal Chemistry 35 (2000) 1053–1064
1059

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Z. Brzozowski et al. / European Journal of Medicinal Chemistry 35 (2000) 1053–1064
3.3. 2-Amino-6-(morpholinomethyl)-4-(3,5,5-trimethyl-
2-pyrazolino)-1,3,5-triazine 13
3.4.2. Method B
To a suspension of the compound 8 (3.82 g, 0.015
mol) in methanol (30 mL), 7% aqueous sodium hydrox-
ide (10 mL) was added. The reaction mixture was heated
under reflux for 6 h. Then, the solution was concen-
trated under reduced pressure to a volume of 25 mL,
treated with water (25 mL) and the resulting solution
was allowed to stand at r.t. for 12 h. The solid that
precipitated was collected by filtration and purified by
crystallization from isopropanol.
To a solution of morpholine (3.5 g, 0.34 mol) in
benzene (60 mL) chloromethyl-triazine 8 was added.
The reaction mixture was refluxed for 5 h with stirring.
After cooling to r.t., morpholine hydrochloride that
precipitated was separated by filtration and the filtrate
was evaporated to dryness under reduced pressure. The
residue was washed with water (20 mL), collected by
filtration and purified by crystallization from methanol–
water.
3.5. 2-Amino-6-(4-aminophenoxymethyl)-4-(3,5,5-
trimethyl-2-pyrazolino)-1,3,5-triazine 15
3.4. 2-Amino-6-methoxymethyl-4-(3,5,5-trimethyl-
2-pyrazolino)-1,3,5-triazine 14
To a solution of 4-aminophenol (3.3 g, 0.03 mol) in
anhydrous dioxane (50 mL) sodium (0.69 g, 0.03 mol)
was added and the reaction mixture was heated under
reflux for 6 h. Then, triazine 8 (6.37 g, 0.025 mol) was
added and the resulting solution was refluxed for 14 h.
Then, charcoal (0.2 g) was added and the solution was
allowed to stand at r.t. overnight. The charcoal and side
products that precipitated were separated by suction
and the filtrate was concentrated under reduced pressure
to a volume of 20 mL. The solution thus obtained was
treated with water (80 mL) and the crude product 15
3.4.1. Method A
A solution of sodium methoxide (0.46 g Na, 40 mL
methanol) was treated with chloromethyl-triazine 8 (3.82
g, 0.015 mol) and the resulting mixture was heated
under reflux for 8 h. After cooling to r.t. water (60 mL)
was added dropwise and the solution was left overnight.
The solid that deposited was separated by filtration and
purified by recrystallization from isopropanol.
Table II. Physico–chemical properties of the triazine derivatives 5–22
Compd.
Yield (%)
M.P. (°C)
Analysis for
5
6
60
51
74
69
66
49
58
60
175–176 (MeOH)
C6H10ClN5
C8H12ClN5O
C8H11CLN6
C10H15ClN6
C11H17ClN6
C10H15FN6
C10H15BrN6
C11H18N6O2S
C14H23N7O
C11H18N6O
179–180 (i-propanol)a
214–215 (MeOH)
7
8
9
10
11
12
13
14
225–226 (H2O)
196–198 (ethanol)
250–253 (dec.) (EtOH)
275–278 (dec.) (EtOH)
230–232 (MeOH)
169–171 (dec.) (MeOH/H2O)
226–227 (i-propanol))
70
80 (Method A)
82 (Method B)
15
16
17
18
19
20
21
22
53
48
35
46
85
88
78
72
225–227 (toluene)
217–221 (dec.) (FMF/H2O)
230–232 (EtOH)
326–327 (dec.) (DMF)
226–227 (dec.) (MeOH)
224–226 (H2O)
C16H21N7O
C15H20N10S2
C10H16N6O
C20H30N12O
C11H19ClN8S
C10H16N6S
155–157 (EtOH)
223–225 (MeOH/H2O)
C14H22N6O2S
C16H25N11S
a Ref. [11] m.p. 178°C.

Z. Brzozowski et al. / European Journal of Medicinal Chemistry 35 (2000) 1053–1064
Table III. Spectroscopic data for the triazine derivatives 5–22
1061
Comp. IR (KBr)
1H-NMR (CDCl3)
13C-NMR (CDCl3)
5a
6a
7
3385, 3320, 3175, 1645, 1590,1560
2.49 (s, 6H, CH3), 4.27 (s, 2H, CH2),
6.95 (s, 2H, NH2)
3.55–3.65 (m, HH), 3.65–3.75 (m, 4H), 43.1, 46.5, 65.9, 164.6, 166.9, 172.3
4.3 (s, 2H, CH2) 7.05 (s, 2H, NH2)
2.51 (s, 3H,CH3), 3.26, (t, 2H), 4.37
(t, 2H), 4.69 (s, 2H), 6.29 (s, 2H, NH2) 161.9, 166.9
35.6, 46.7, 165.3, 168.8, 171.9
3330. 3152, 2855, 1648, 1520, 1440
3490, 3275,3140, 1640, 1620, 1570,
1550, 1520
16.3, 35.8, 45.2, 46.0, 111.8, 158.6,
8
3325, 3210, 1635, 1665, 1555, 1535
1.61 (s, 6H, CH3), 2.04 (s, 3H, CH3)
16.3, 26.5, 46.1, 53.6, 64.3, 155.5,
162.4, 172.1
9a
3372, 3319, 3230, 2965, 1657, 1534,
1469, 1378, 1336,
1.55 (s, 6H, CH3), 1.7 (d, 3H, CH3),
2.05 (s, 3H, CH3), 2.85 (s, 2H, CH2),
4.45 (s, 2H, CH2), 4.68 (q, 1H, CH),
7.0 (d, 2H, NH2)
10
3354, 3321, 3218, 2974, 1656, 1569,
1534, 1469, 1451
1.65 (s, 6H, CH3), 2.1 (s, 3H, CH2),
2.76 (s, 2H, CH2), 5.1 (d, 2H, CH2,
J
HF=48 Hz), 6.2 (s, 2H, NH2)
11
12
13
14
15
3323, 3216, 2963, 1657, 1535, 1467,
1448, 1379, 1332
1.58 (s, 6H, CH3), 2.0 (s, 3H, CH2),
2.81 (s, 2H, CH2), 4.15 (s, 2H, CH2),
5.3 (s, 2H, NH2)
3425, 3335, 3115, 1665, 1625, 1570,
1515, 1375, 1302, 1165, 1122
1.65 (s, 6H, CH3), 2.11 (s, 3H, CH3),
2.83 (s, 2H, CH2), 3.23 (s, 3H, CH3),
4.27 (s, 2H, CH2), 5.77 (s, 2H, NH2),
1.66 (s, 6H, CH3), 2.11 (s, 3H, CH3),
2.59 (t, 4H, CH2), 3.41 (s, 2H, CH2),
3.77 (t, 4H, CH2), 5.39 (s, 2H, NH2)
1.65 (s, 6H, CH3), 2.1 (s, 3H, CH3),
2.81 (s, 2H, CH), 3.48 (s, 3H, CH3),
5.59 (s, 2H, NH2)
3325, 3210, 1660, 1640, 1625, 1555,
1530, 1114
3360, 3315, 3205, 1650, 1615, 1560,
1530, 1190, 1070
16.4, 2.3, 53.7, 58.9, 64.1, 74.3,
154.9, 162.2, 166.2, 173.8
3460, 3400, 3300, 3200, 1640, 1625,
1570, 1540, 1505, 1225
1.55 (s, 6H, CH3), 2.08 (s, 3H, CH3)
2.78 (s, 2H, CH2), 3.12 (s, 2H, NH2)
4.8 (s, 2H, CH ), 5.74 (s, 2H, NH2),
2
6.51–6.81 (m, 4H)
16
17
3424, 3338, 3159, 2964, 1638, 1526,
1457, 1410, 1379, 1263
1.55 (s, 6H, CH3), 2.1 (s, 3H, CH3),
2.8 (s, 2H, CH2), 3.6 (m, 2H, CH2),
4.0 (s, 2H, CH2), 4.15 (m, 4H, CH2),
6.9 (s, 2H, NH2), 9.2 (s, 1H, NH)
1.66 (s, 6H, CH3), 2.12 (s, 3H, CH3),
2.84 (s, 2H, CH2), 3.01 (t, 1H, OH),
4.49 (d, 2H, CH2), 5.43 (s, 2H, NH2)
3450, 3325, 3215, 166o, 1640, 1570,
1560, 1535, 1085
16.3, 26.6, 53.7, 63.5, 64.1, 155.4,
161.7, 163.3, 175.7
18
3375, 3300, 3215, 1640, 1625, 1580,
1549, 1138
3320, 3205, 3155, 3050, 2970, 2770,
2730, 1655, 1590, 1575, 1540
19a
1.57 (s, 6H, CH3), 2.01 (s, 3H, CH),
2.84 (s, 2H, CH2), 3.01 (t, 1H, OH),
4.49 (d, 2H, CH2), 5.43 (s, 2H, NH2)
1.66 (s, 6H, CH3), 1.98 (t, 1H, SH),
2.09 (s, 3H, CH3), 2.81 (s, 3H, CH2)
3.51 (d, 2H, CH2), 5.53 (s, 2H, NH2)
1.27 (t, 3H, CH3), 165 (s, 6H, CH3),
2.1 (s, 3H, CH3), 2.81 (s, 2H, CH2),
3.36 (s, 2H, CH2), 3.64, (s, 2H, CH2),
4.18 (q, 2H, CH2), 5.44 (s, 2H, NH2)
1.58 (s, 6H, CH3), 2.07 (s, 3H, CH3),
2.99 (s, 2H, CH2), 3.11 (s, 3H,
20
21
3325, 3210, 2540, 1655, 1635, 1555,
1535
3385, 3305, 3205, 1720, 1640, 1630,
1570, 1530,
22
3320, 3160, 1655, 1630, 1560, 1525
NꢀCH3) 3.17 (s, 3H, NꢀCH3), 3.83 (s,
4H, CH2ꢀSꢀCH2), 8.58 (s, 2H, NH2),
8.63 (s, 2H, NH2)
a NMR spectrum run in DMSO-d6.

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Z. Brzozowski et al. / European Journal of Medicinal Chemistry 35 (2000) 1053–1064
,
Table V. Selected bond lengths (A) and angles (°) for 8
that precipitated was collected by filtration and purified
by crystallization from toluene.
Bond lengths
Cl(1)ꢀC(13)
N(1)ꢀC(6)
N(1)ꢀC(2)
C(2)ꢀN(12)
C(2)ꢀN(3)
N(3)ꢀC(4)
C(4)ꢀN(5)
C(4)ꢀC(13)
N(5)ꢀC(6)
1.786(3) C(6)ꢀN(7)
1.327(3) N(7)ꢀN(8)
1.345(3) N(7)ꢀC(11)
1.314(3) N(8)ꢀC(9)
1.364(3) C(9)ꢀC(10)
1.321(3) C(9)ꢀC(14)
1.327(3) C(10)ꢀC(11)
1.503(3) C(11)ꢀC(15)
1.358(3) C(11)ꢀC(16)
1.356(3)
1.401(3)
1.506(3)
1.276(3)
1.480(4)
1.486(4)
1.534(4)
1.507(4)
1.514(4)
3.6. [2-Amino-4-(3,5,5-trimethyl-2-pyrazolino)-1,3,5-
triazin-6-yl-methylthio]-7,8-dihydroimidazo[1,2-a]-
1,3,5-triazine-4H(6H)-thione 16
To a solution of 7,8-dihydroimidazo[1,2-a]1,3,5-tria-
zine-2,4-dithione [13] (1.86 g, 0.01 mol) in methanolic
potassium hydroxide (2.1 g KOH, 12 mL methanol)
bromomethyl-triazine 11 (3.06 g, 0.01 mol) was added
and the reaction mixture was stirred at r.t. for 0.5 h. The
potassium salt of 16 that precipitated was separated by
suction, washed with methanol (5 mL) and dried at r.t.
The resulting solid was suspended in water (10 mL) and
neutralized with 10% hydrochloric acid to give crude
product 16 that was purified by crystallization from
dimethylformamide–water.
Bond angles
C(6)ꢀN(1)ꢀC(2)
N(12)ꢀC(2)ꢀN(1)
N(12)ꢀC(2)ꢀN(3)
N(1)ꢀC(2)ꢀN(3)
C(4)ꢀN(3)ꢀC(2)
N(3)ꢀC(4)ꢀN(5)
N(3)ꢀC(4)ꢀC(13)
N(5)ꢀC(4)ꢀC(13)
C(4)ꢀN(5)ꢀC(6)
N(1)ꢀC(6)ꢀN(7)
N(1)ꢀC(6)ꢀN(5)
N(7)ꢀC(6)ꢀN(5)
C(6)ꢀN(7)ꢀN(8)
C(6)ꢀN(7)ꢀC(11)
115.1(2) N(8)ꢀN(7)ꢀC(11)
117.9(2) C(9)ꢀN(8)ꢀN(7)
117.9(2) N(8)ꢀC(9)ꢀC(10)
124.3(2) N(8)ꢀC(9)ꢀC(14)
113.0(2)
108.3(2)
114.0(2)
121.0(3)
113.6(2) C(10)ꢀC(9)ꢀC(14) 125.0(3)
128.1(2) C(9)ꢀC(10)ꢀC(11) 105.3(2)
116.2(2) N(7)ꢀC(11)ꢀC(15) 110.2(2)
115.7(2) N(7)ꢀC(11)ꢀC(16) 111.4(2)
112.9(2) C(15)ꢀC(11)ꢀC(16) 110.8(3)
117.8(2) N(7)ꢀC(11)ꢀC(10)
99.3(2)
3.7. [2-Amino-4-(3,5,5-trimethyl-2-pyrazolino)-1,3,5-
triazin-6-yl]methanol 17 and bis [2-amino-4-(3,5,5-
trimethyl-2-pyrazolino)-1,3,5-triazin-6-ylmethyl] ether
18
125.6(2) C(15)ꢀC(11)ꢀC(10) 111.3(3)
116.6(2) C(16)ꢀC(11)ꢀC(10) 113.3(3)
117.6(2) C(4)ꢀC(13)ꢀCl(1) 109.0(2)
128.2(2)
To a suspension of the triazine 8 (12.7 g, 0.05 mol) in
water (80 mL) was added 16% aqueous sodium hydrox-
ide and the reaction mixture was stirred at ambient
temperature for 12 h followed by refluxing for 6 h. After
cooling to r.t., product 18 that precipitated was collected
by filtration, washed with water (6×5 mL) (the aqueous
filtrate which contained product 17 was used for further
workup), dimethylformamide (4 mL) and ethanol (2×3
mL) and purified by crystallization from ethanol.
Aqueous filtrate from the experiment described above
was neutralized with 15% hydrochloric acid, filtered
with charcoal and evaporated under reduced pressure to
dryness. The dry residue thus obtained was extracted
with chloroform (100 mL). The organic layer was con-
centrated to a small volume and the crude product 17
that precipitated was collected by filtration and purified
by crystallization from dimethylformamide.
Table IV. Atomic coordinates (×104) and equivalent isotropic
displacement parameters (A2×103) for 8a
x
y
z
Ueq
Cl(1)
N(1)
C(2)
N(3)
C(4)
N(5)
C(6)
N(7)
N(8)
C(9)
C(10)
C(11)
N(12)
C(13)
C(14)
C(15)
C(16)
2145(1)
4061(2)
3051(3)
3019(2)
4039(3)
5145(2)
5129(3)
6304(3)
6278(3)
7635(3)
8780(5)
7916(3)
2042(3)
3948(3)
7957(5)
7305(5)
9120(4)
4860(1)
8959(2)
7534(3)
6028(2)
6108(3)
7393(2)
8786(3)
10114(3)
11586(2)
12556(3)
11852(6)
10088(3)
7591(3)
323(1)
66(1)
39(1)
38(1)
41(1)
39(1)
45(1)
40(1)
53(1)
47(1)
52(1)
84(1)
53(1)
53(1)
48(1)
72(1)
71(1)
78(1)
1928(1)
2052(1)
1723(1)
1250(1)
1089(1)
1465(1)
1352(1)
1725(1)
1606(1)
1130(2)
953(1)
3.8. S-[2-amino-4-(3,5,5-trimethyl-2-pyrazolino)-1,3,5-
triazin-6-ylmethyl] isothiourea hydrochloride 19
A suspension of chloromethyl-triazine 8 (1.,7 g, 0.05
mol) and thiourea (3.9 g, 0.051 mol) in methanol (70
mL) was heated under reflux for 5 h. After cooling to
r.t., the solid that precipitated was collected by filtra-
tion, washed with methanol (3×4 mL) and dried in a
desiccator to give pure isothiouronium salt 19, which
was used for further transformations.
2524(1)
843(1)
1922(2)
317(1)
4543(3)
14245(4)
10100(5)
8514(5)
1080(2)
a Ueq is defined as one third of the trace of the orthogonalized
Uij tensor.

Z. Brzozowski et al. / European Journal of Medicinal Chemistry 35 (2000) 1053–1064
1063
3.9. [2-Amino-4-(3,5,5-trimethyl-2-pyrazolino)-1,3,5-
triazin-6-yl]methanethiol 20
2165 measured reflections, 2123 were independent and
used in further calculations. The structure was solved
by direct methods with the program SHELXS-86 [19].
Full-matrix least-squares refinement was carried out
on F2 with SHELXL-93 [20]. Hydrogen atoms have been
located on DF map and their parameters included in
the refinement process. Final R indices for reflections
with I\2|(I) and 215 refined parameters are: R1 =
0.0558, wR2 =0.1462 (R1 =0.0635, wR2=0.1680 for
all data). Final atomic coordinates, bond lengths and
bond angles are listed in table IV and table V, respec-
tively. Atom labeling is shown in figure 4.
To a suspension of isothiouronium salt 19 (9.92 g, 0.03
mol) in water (40 mL), 5% aqueous sodium hydroxide
and 98% hydrazine hydrate (5 mL) were subsequently
added and the reaction mixture was heated under reflux
for 1 h. After cooling to r.t., pH of the solution was
adjusted to 7.5 with 1% hydrochloric acid. The crude
product 20 that precipitated was washed with water and
purified by recrystallization from water.
3.10. Ethyl [2-amino-4-(3,5,5-trimethyl-2-pyrazolino)-
1,3,5-triazin-6-ylmethylthio] acetate 21
Supplementary data
Compound 20 (3.78 g, 0.015 mol) was dissolved in a
solution of sodium ethoxide (0.016 mol) in ethanol (40
mL) and to the resulting solution ethyl chloroacetate (2.0
g, 0.016 mol) was added dropwise. The reaction mixture
was heated under reflux for 35 h. After cooling to r.t.
water was added (50 mL) and heating was continued for
an additional 2 h. The solid that precipitated was sepa-
rated by suction and purified by crystallization from
ethanol.
Further details of the crystal structure investigation
may be obtained from the Director of the Cambridge
Crystallographic Data Centre, 12 Union Road, Cam-
bridge CB2 1EZ, UK, on quoting the full journal
citation.
Acknowledgements
The authors wish to thank the National Cancer Insti-
tute, Bethesda, USA, for carrying out the in vitro antitu-
mor screening.
3.11. 2-Amino-6-[(2-amino-4-dimethylamino-1,3,5-
triazin-6-yl)-methyl thiomethyl]-4-(3,5,5-trimethyl-2-
pyrazolino)-1,3,5-triazine 22
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To a solution of sodium methoxide (0.4 g Na, 60 mL
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resulting suspension was heated under reflux for 9 h,
cooled to r.t. and treated with water (30 mL). After
standing overnight, product 22 that precipitated was
collected by filtration and purified by crystallization from
methanol–water.
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