Synthesis, structure and anticancer activity of novel 2,4-diamino-1,3,5- triazine derivatives

Article abstract of DOI:10.1016/j.ejmech.2005.10.013

A series of 2-(4,6-diamino-1,3,5-triazin-2-yl)-2-{[4-(dimethylamino)- phenyl]imino}acetonitriles 19-27 have been synthesized by the reaction of 2-(4-amino-6-alkylamino-1,3,5-triazin-2-yl)acetonitriles 10-15 with p-nitrosodimethylaniline. Unexpectedly, a s

Full text of DOI:10.1016/j.ejmech.2005.10.013

European Journal of Medicinal Chemistry 41 (2006) 219–225
http://france.elsevier.com/direct/ejmech
Synthesis, structure and anticancer activity
of novel 2,4-diamino-1,3,5-triazine derivatives
F. Sączewski a,*, A. Bułakowska b, P. Bednarski c, R. Grunert c
a
Department of Chemical Technology of Drugs, Medical University of Gdańsk, 80-416 Gdańsk, Poland
b
Department of Organic Chemistry, Medical University of Gdańsk, 80-416 Gdańsk, Poland
c
Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, University of Greifswald, D-17487 Greifswald, Germany
Received 21 July 2005; received in revised form 17 October 2005; accepted 26 October 2005
Available online 27 December 2005
Abstract
A series of 2-(4,6-diamino-1,3,5-triazin-2-yl)-2-{[4-(dimethylamino)-phenyl]imino}acetonitriles 19–27 have been synthesized by the reaction
of 2-(4-amino-6-alkylamino-1,3,5-triazin-2-yl)acetonitriles 10–15 with p-nitrosodimethylaniline. Unexpectedly, a similar reaction of acetonitriles
10, 14, 15, 17 and 18 with nitrosobenzene led to the formation of 4,6-diamino-N-phenyl-1,3,5-triazine-2-carboxamides 28–32. The in vitro
antitumor activity of the compounds obtained has been tested and 2-[4-Amino-6-(4-phenylpiperazin-1-yl)-1,3,5-triazin-2-yl]-2{[4-(dimethyla-
mino)phenyl]imino}acetonitrile (19) having remarkable activity against melanoma MALME-3 M cell line (GI50 = 3.3 × 10–8 M, TGI = 1.1 ×
10–6 M) is a leading candidate for further development.
© 2005 Elsevier SAS. All rights reserved.
Keywords: 2,4-Diamino-1,3,5-triazine derivatives; Synthesis; Antitumor effect
1. Introduction
phostins [12,13] and “stilbenic” tyrosine kinase inhibitors
[14–17].
1,3,5-triazine derivatives containing various amino groups
at position 2, 4 or 6, such as tretamine, furazil and dioxadet,
have been known as anticancer drugs [1]. Structural modifica-
tions consisting in the replacement of ethyleneimino moiety
with either dialkylamino, alkoxy, alkylarylo or hydroxy groups
led to discovery of novel chemotherapeutic agents [2–7].
Moreover, an anti-gastric ulcer agent that is commonly used
in Japan, irsogladine [2,4-diamino-6-(2,5-dichlorophenyl)-
1,3,5-triazine], was shown to possess antiangiogenic properties
which result in anticancer effect of the drug [8,9].
Encouraged by this result and on the basis of a commonly
held view that the CH=N group is bioisosterically equivalent to
a CH=CH group, we synthesized novel compounds of general
formula C (Fig. 1) and investigated their biological activity in
four cancer cell lines.
2. Results and discussion
2.1. Chemistry
As a part of our research program aimed at search for new
pharmacophores as antitumor agents, we have previously de-
scribed syntheses and pronounced anticancer activity of 2,4-
diamino-1,3,5-triazine derivatives of type A and B [10,11]
(Fig. 1). The structure of compounds in the former series con-
taining cyanovinyl group bears resemblance to those of tyr-
As outlined in Scheme 1, the reaction of biguanide hydro-
chlorides 1–6 or free bases 7–9 with ethyl cyanoacetate carried
out in ethanol at room temperature for 1–2 h afforded corre-
sponding acetonitrile derivatives 10–18 in 16–76% yields.
The IR spectra of all the acetonitriles 10–18 showed C≡N
1
group vibrations in the range of 2255–2259 cm–1, and in H-
NMR spectra the signals corresponding to CH2 group appear at
3.63–3.93 ppm.
* Corresponding author.
E-mail address: saczew@amg.gda.pl (F. Sączewski).
Condensation of nitrosobenzenes with compounds contain-
ing active methylene group was described previously and, de-
0223-5234/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2005.10.013

220
F. Sączewski et al. / European Journal of Medicinal Chemistry 41 (2006) 219–225
Fig. 1.
Scheme 2.
posed mechanism for the reaction sequence is presented in
Scheme 3. First, the adduct D is formed which subsequently
loses a water molecule to give iminoacetonitrile E. In case of
19–27 the presence of strong electron-donating dimethylamino
group decreases the electrophilic character of Cα carbon atom
which results in a stable iminoacetonitrile E. On the other
hand, the unsubstituted E should be more reactive, and there-
fore, may undergo addition reaction with water to give the in-
termediate F, in which the leaving group (C≡N) is attached to
the carbon atom. Then, the Na2CO3-promoted elimination of
Scheme 1.
pending on substituents, may lead to the formation of either the
imines or nitrones [18–20]. We found that treatment of the
acetonitrile derivatives 10–18 with p-nitrosodimethylaniline in
anhydrous ethanol in the presence of KOH yielded the desired
iminoacetonitriles 19–27 in 36–89% yields. Apparently, the
adduct of type D formed in the first stage of the reaction se-
quence underwent a facile imine-forming elimination at room
temperature (Scheme 2).
In the IR spectra of these compounds characteristic absorp-
tions in the range of 2201–2204 cm–1 attributable to conju-
gated C=C–C≡N groups are present.
Next, an attempt was made to synthesize the unsubstituted
analogues, by the same method as 19–27, using nitrosoben-
zene. However, no desired iminoacetonitrile was formed and
it should be noted that a number of experiments were done to
see if condensation with nitrosobenzene would succeed under
more vigorous conditions. Higher concentration of KOH and
higher reaction temperatures were tried, but the complex mix-
ture of products always contained varying amounts of un-
reacted starting material and we were unable to separate the
products by chromatography.
Faced with this unexpected setback, we then performed the
reaction of acetonitriles 10, 14, 15, 17 and 18 with nitrosoben-
zene in DMF or EtOH solution in the presence of 20% aqueous
Na2CO3. To our surprise, the amide derivatives 28–32 were
isolated from the reaction mixtures in 20–72% yields. A pro-
Scheme 3.

F. Sączewski et al. / European Journal of Medicinal Chemistry 41 (2006) 219–225
221
hydrogen cyanide from F gives the final amides 28–32 via the
iminol G.
iminoacetonitrile 19 bearing (4-phenylpiperazin-1-yl)- substitu-
ent at position 6 of triazine ring exhibited remarkable activity
and selectivity for melanoma MALME-3 M cell line (GI50
= 3.3 × 10–8 M, TGI = 1.1 × 10–6 M and Δ = 2.93) [24].
It should be noted that the iminoacetonitrile derivative 24
with 6-(3,5,5-trimethyl-4,5-dihydro-1H-pyrazol-1-yl)- substitu-
ent at triazine ring was inactive, while the previously described
acrylonitriles of type B (Fig. 1) required this substituent for
their cytotoxic activity [11]. These results are rather surprising
from the structure–activity relationships point of view, and
may suggest that the mechanisms by which acrylonitriles of
type B and iminoacetonitriles 19 and 20 exert their antineoplas-
tic effects are different.
Structure of the amides 28–32 was confirmed by IR, NMR
and MS spectroscopic data. For example, in the IR spectrum of
compound 30 no absorption at 2000–2200 cm–1 attributable to
C≡N group is observed. Instead, a strong absorption at
1693 cm–1 confirms the presence of C=O group. The 1H-
NMR spectrum reveals a signal corresponding to the amide
NH proton at 10.12 ppm which shows no correlation to any
carbon atom in HSQC (heteronuclear single quantum correla-
tion) spectrum, while in the HMBC (heteronuclear multiple
bond correlation) spectrum a correlation of the NH proton to
the carbon atom of C=O group at 158.1 ppm is observed. The
MS spectrum of 30 contains a peak of M+ at m/z = 325
(77.2%) which is in agreement with the structure proposed.
3. Experimental protocols
2.2. Biology
Melting points are not corrected and were recorded on a
Buchi apparatus. IR spectra, KBr pellets, 400–4000 cm–1, were
The in vitro cytotoxic activity of iminoacetonitriles 19–27
and amides 28–32 was evaluated [21] using human bladder
cancer cell line 5637; human pancreatic cancer cell line
DAN-G, human breast cancer cell line MCF-7 and human
non-small cell lung cancer cell line LCLC-103H. Primary
screening of the new compounds was done to indicate whether
a substance possesses enough activity at the concentration of
20 μM to inhibit cell growth by 50%.
It should be pointed out that all the amide derivatives 28–32
were inactive. On the other hand, the results of primary screen-
ing for iminoacetonitriles indicated that compounds 19, 20, 21,
25 and 27 were active. Thus, a secondary screening to deter-
mine their potency was performed on a panel of four human
cell lines mentioned above. Table 1 lists the IC50 values calcu-
lated from dose–response data.
1
recorded on a Satellite FTIR spectrophotometer (Mattson). H
and 13C NMR spectra were recorded on a Varian Gemini 200
instrument at 200 and 50 MHz, respectively (chemical shifts
are expressed as δ values relative to TMS as internal standard).
Mass spectra were recorded on Finnigan MAT 95 spectrometer
at 70 eV. Analyses of C, H, N were within 0.4% of the the-
oretical values. Biguanides 1 [25], 5 [26,27], 6 [28], 8 [28] and
9 [29], as well as acetonitrile 15 [11] were prepared according
to the described procedures.
3.1. Synthesis
3.1.1. General procedure for the preparation of biguanides
hydrochlorides 1–7
As shown in Table 1, the highest cytotoxic activity was
found for compound 20 with 4-(2-methylphenyl)piperazin-1-
yl substituent at position 6 of triazine ring. Replacement of
the 2-methyl group for either 3-chloro (21) or 4-nitro (25) of
the phenyl ring results in reductions in activity. Similarly, un-
substituted analogue 19 was much less active, suggesting that
the presence of lipophilic weak electron-donating substituent,
such as methyl group, at position 2 of phenyl ring is important
for inhibitory activity against the investigated cell lines.
The iminoacetonitriles 19, 23, 24, 26 and 27 and amides
28–32 were also selected by the National Cancer Institute
(NCI, Bethesda, USA) for testing against a panel of ca. 60
tumor cell lines. Details of this test system have been published
[21–23]. Again, amide derivatives proved to be inactive, while
An equimolar mixture of the appropriate amine hydrochlor-
ide and dicyandiamide (68 mmol) in dry 1-butanol (20 ml) was
slowly heated with stirring (oil-bath). The mixture began to
melt at 90 °C and fused completely at 122–123 °C. Heating
was continued over 8 h. After cooling to r.t., stirring was con-
tinued for 6 h. The precipitate was separated by suction,
washed 1-butanol and isopropyl alcohol, dried and purified
by crystallization from MeOH.
3.1.1.1. N-[imino(4-phenylpiperazin-1-yl)methyl]guanidine hy-
drochloride 1. Yield: 70%, m.p. 215–217 °C (Ref. [1], m.p.
243–244 °C). Anal. (C12H19ClN6) C, H, N.
3.1.1.2. N-{imino[4-(2-methylphenyl)piperazin-1-yl]methyl}gua-
nidine hydrochloride 2. Yield: 78%, m.p. 230–232 °C; IR
(KBr): 3316, 3204, 2917, 1613, 1548, 1496, 999, 762,
725 cm–1. Anal. (C13H21ClN6) C, H, N.
Table 1
a
IC50 values [μM] in four human cancer cell lines
Cell line
Com-
pound
19
20
21
5631 DAN-G MCF-7 LCLC-103H
4.65 1.082
1.51 0.292
6.55 0.483
5.43 1.331
12.9 0.883
8.93 0.806
2.17 0.370
4.68 0.953
12.6 3.041
> 20
> 20
11.3 2.492
2.14 0.603
7.84 1.235
9.59 1.118
> 20
3.1.1.3. N-{[4-(3-chlorophenyl)piperazin-1-yl]-(imino)methyl}
guanidine hydrochloride 3. Yield: 61%, m.p. 221–222 °C; IR
(KBr): 3308, 3186, 2839, 1649, 1539, 1496, 1239, 1002,
778 cm–1. Anal. (C12H18Cl2N6) C, H, N.
5.97 1.497
6.86 0.824
11.2 2.871
> 20
25
27

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F. Sączewski et al. / European Journal of Medicinal Chemistry 41 (2006) 219–225
3.1.1.4. N-{[4-(4-fluorophenyl)piperazin-1-yl]-(imino)methyl}
guanidine hydrochloride 4. Yield: 55%, m.p. 189–192 °C; IR
(KBr): 3322, 3189, 2833, 1644, 1505, 1441, 1237, 1003,
828 cm–1. Anal. (C12H18ClFN6) C, H, N.
3.86 (m, 4H, CH2), 3.9 (s, 2H, CH2), 6.83 (d, 1H, CH,
J = 7.6 Hz), 6.98 (d, 2H, CH, J = 7.6 Hz), 7.2 (br. s, 2H,
NH2), 7.25 (t, 2H, CH, J = 8.5 Hz) ppm. Anal. (C15H17N7)
C, H, N.
3.1.1.5. N-[imino(pyrrolidin-1-yl)methyl]guanidine hydrochlo-
ride 5. Yield: 47%, m.p. 223–225 °C (Refs. [2,3], m.p. 225–
226 °C). Anal. (C6H14ClN5) C, H, N.
3.1.3.2. 2-{4-Amino-6-[4-(2-methylphenyl)piperazin-1-yl]-1,3,5-
triazin-2-yl}acetonitrile 11. Yield: 34%, m.p. 173–176 °C
(MeOH); IR (KBr): 3350, 3325, 2255, 1650, 1632, 1584,
1
1370 cm–1; H-NMR (DMSO-d6) δ: 2.32 (s, 3H, CH3), 2.84–
2.89 (m, 4H, CH2), 3.35–3.89 (m, 4H, CH2), 3.92 (s, 2H,
CH2), 6.99–7.22 (m, 4H, CH + 2H, NH2) ppm. Anal.
(C16H19N7) C, H, N.
3.1.1.6. N-[imino(3,5,5-trimethyl-4,5-dihydro-pyrazol-1-yl)
methyl]guanidine hydrochloride 6. Yield: 85%, m.p. 228–
232 °C (Ref. [4], m.p. 238–239 °C). Anal. (C8H17ClN6) C, H,
N.
3.1.3.3. 2-{4-Amino-6-[4-(3-chlorophenyl)piperazin-1-yl]-1,3,5-
triazin-2-yl}acetonitrile 12. Yield: 46%, m.p. 160–162 °C
(MeOH); IR (KBr): 3436, 3322, 3155, 2259, 1650, 1553,
3.1.2. General procedure for the preparation of biguanides
7–9
1
1336 cm–1; H-NMR (DMSO-d6) δ: 3.24 (br. s, 4H, CH2),
An equimolar mixture of the appropriate amine hydrochlor-
ide and dicyandiamide (46.8 mmol) in dry 1-butanol (30 ml)
was stirred. The reaction mixture was heated at 90 °C for
40 min and then subsequently heated at 121–123 °C for 8 h.
The solid hydrochloride that precipitated was separated by suc-
tion, washed with 1-butanol (2 × 5 ml), isopropyl alcohol
(4 × 5 ml) and dried. The crude product was dissolved in water
(20 ml), treated with active carbon, was heated at 75 °C and
filtered. To the filtrate 40% sodium hydroxide was added
(4.5 ml). After cooling, the product was separated and was re-
crystallized from MeOH.
3.86 (br. s, 4H, CH2), 3.92 (s, 2H, CH2), 6.83 (d, 1H,
J = 7.8 Hz), 6.96 (d, 1H, CH, J = 8.3 Hz), 7.01 (s, 1H, CH),
7.13 (br. s, 2H, NH2), 7.27 (t, 1H, CH, J = 8.1 Hz, J = 8 Hz)
ppm. Anal. (C15H16ClN7) C, H, N.
3.1.3.4. 2-{4-Amino-6-[4-(4-fluorophenyl)piperazin-1-yl]-1,3,5-
triazin-2-yl}acetonitrile 13. Yield: 73%, m.p. 180–182 °C
(MeOH); IR (KBr): 3416, 3326, 3153, 2262, 1656, 1548,
1
1324 cm–1; H-NMR (DMSO-d6) δ: 3.07–3.18 (m, 4H, CH2),
3.85 (br. s, 4H, CH2), 3.9 (s, 2H, CH2), 6.95–7.12 (m, 4H,
CH + 2H, NH2) ppm. Anal. (C15H16FN7) C, H, N.
3.1.2.1. N-{imino{4-(4-nitrophenyl)piperazin-1-yl]methyl}gua-
nidine 7. Yield: 90%, m.p. 216–220 °C; IR (KBr): 3311,
3157, 2728, 1637, 1555, 1330, 1244, 1115, 999 cm–1. Anal.
(C10H14ClN3O2) C, H, N.
3.1.3.5. 2-(4-Amino-6-pyrrolidin-1-yl-1,3,5-triazin-2-yl)aceto-
nitrile 14. Yield: 76%, m.p. 174–176 °C (MeOH); IR (KBr):
1
3459, 3339, 3200, 2255, 1660, 1516, 1340 cm–1; H-NMR
(CDCl3) δ: 1.95–1.96 (m, 4H, CH2), 3.5–3.52 (m, 2H, CH2),
3.58–3.6 (m, 2H, CH2), 3.63 (s, 2H, CH2), 5.3 (s, 2H, NH2)
ppm. Anal. (C9H12N6) C, H, N.
3.1.2.2. N-[(5-ethyl-4-methyl-4,5-dihydro-pyrazol-1-yl)-(imino)
methyl]guanidine 8. Yield: 75%, m.p. 157–158 °C (Ref. [4],
m.p. 189–191 °C). Anal. (C8H17ClN6) C, H, N.
3.1.3.6. 2-[4-Amino-6-(3,5,5-trimethyl-4,5-dihydro-1H-pyrazol-
1-yl)-1,3,5-triazin-2-yl]acetonitrile 15. Yield: 50%, m.p. 236–
237 °C (1-butanol), (Ref. [6] m.p. 246–247 °C); IR (KBr):
3.1.2.3. N-[imino(morpholin-4-yl)methyl]guanidine 9. Yield:
40%, m.p. 173–176 °C (Ref. [5], m.p. 195–202 °C). Anal.
(C6H13N5O) C, H, N.
1
3350, 3315, 3200, 2255, 1650, 1539, 1335 cm–1; H-NMR
(CDCl3) δ: 1.68 (s, 6H, CH3), 2.13 (s, 3H, CH3), 2.85 (s, 2H,
CH2), 3.67 (s, 2H, CH2), 7.28 (s, 2H, NH2) ppm. Anal.
(C11H15N7) C, H, N.
3.1.3. General procedure for the preparation of 2-(4-amino-6-
alkylamino-1,3,5-triazin-2-yl)acetonitriles 10–15
To a solution of sodium ethoxide in ethanol (0.58 g Na,
36 ml ethanol) corresponding biguanide hydrochloride 1–6
(25.4 mmol) was added and the reaction mixture was stirred
at r.t. for 3 h. Then ethyl cyanoethanoate (2.71 ml, 25.4 mmol)
was added drop-wise and stirring was continued at r.t. for 1 h.
The precipitate was separated by suction, washed with ethanol
(2 × 5 ml) and purified by crystallization from a suitable sol-
vent.
3.1.4. General procedure for the preparation of 2-(4-amino-6-
alkylamino-1,3,5-triazin-2-yl)acetonitriles 16–18
To a solution of biguanide 7–9 (30.6 mmol) in dry methanol
(48 ml) ethyl cyanoethanoate (3.3 ml, 30.6 mmol) was added
drop-wise over 1 h. The reaction mixture was stirred at ambient
temperature for 2 h and the product that precipitated was sepa-
rated by suction, washed with methanol (5 ml) and purified by
crystallization.
3.1.3.1. 2-[4-Amino-6-(4-phenylpiperazin-1-yl)-1,3,5-triazin-2-
yl]acetonitrile 10. Yield: 40%, m.p. 205–208 °C (DMF–
water); IR (KBr): 3433, 3323, 3156, 2259, 1653, 1523,
3.1.4.1. 2-{4-Amino-6-[4-(4-nitrophenyl)piperazin-1-yl]-1,3,5-
triazin-2-yl}acetonitrile 16. Yield: 59%, m.p. 263–265 °C
(DMF-water); IR (KBr): 3455, 3332, 3196, 2259, 1630, 1534,
1
1332 cm–1; H-NMR (DMSO-d6) δ: 3.14–3.19 (m, 4H, CH2),

F. Sączewski et al. / European Journal of Medicinal Chemistry 41 (2006) 219–225
223
1
1322 cm–1; H-NMR (DMSO-d6) δ: 3.56–3.61 (m, 4H, CH2),
4H, CH), 7.22–7.3 (m, 2H, CH), 7.62 (d, 1H, CH, J = 9.1 Hz)
3.88 (br. s, 4H, CH2), 3.93 (s, 2H, CH2), 7.06 (d, 2H, CH,
J = 9.5 Hz), 7.17 (br. s, 2H, NH2), 8.1 (d, 2H, CH,
J = 9.4 Hz) ppm. Anal. (C15H16N8O2) C, H, N.
ppm. Anal. (C23H24ClN9) C, H, N.
3.1.5.4. 2-{4-Amino-6-[4-(4-fluorophenyl)piperazin-1-yl]-1,3,5-
triazin-2-yl}-2-{[4-(dimethylamino)phenyl]imino}acetonitrile 22
.
3.1.4.2. 2-[4-Amino-6-(5-ethyl-4-methyl-4,5-dihydro-1H-pyrazol-
1-yl)-1,3,5-triazin-2-yl] acetonitrile 17. Yield: 63%, m.p. 278–
280 °C (MeOH); IR (KBr): 3503, 3120, 2977, 2257, 1642,
Yield: 50%, 236–240 °C (DMF-water); IR (KBr): 3318, 3176,
2906, 2202, 1644, 1557 cm–1; 1H-NMR (DMSO-d6) δ: 3.08 (s,
6H, CH3), 3.17 (br. s, 4H, CH2), 3.94 (br. s, 4H, CH2), 6.87 (d,
2H, CH, J = 9.3 Hz), 6.98–7.14 (m, 4H, CH), 7.16 (br. s, 2H,
NH2), 7.62 (d, 2H, CH, J = 9.1 Hz) ppm. Anal. (C23H24FN9)
C, H, N.
1
1530, 1378 cm–1; H-NMR (DMSO-d6) δ: 1.21–1.28 (m, 6H,
CH3), 2.38–2.43 (m, 1H, CH), 2.59–2.67 (m, 1H, CH), 3.25–
3.3 (m, 1H, CH), 3.69 (m, 3H, CH + CH2), 4.22 (s, 1H, CH),
5.41 (br. s, 2H, NH2) ppm. Anal. (C11H15N7) C, H, N.
3.1.5.5. 2-(4-Amino-6-pyrrolidin-1-yl-1,3,5-triazin-2-yl)-2-{[4-
(dimethylamino)phenyl]imino} acetonitrile 23. Yield: 65%, m.
p. 295–299 °C (DMF); IR (KBr): 3407, 3327, 3210, 2201,
3.1.4.3. 2-(4-Amino-6-morpholin-4-yl-1,3,5-triazin-2-yl)acetoni-
trile 18. Yield: 16%, m.p. 178–180 °C (MeOH); IR (KBr):
1
3369, 3223, 2924, 2259, 1646, 1558, 1384 cm–1; H-NMR
1
1661, 1613 cm −1; H-NMR (CDCl3) δ: 1.98 (s, 4H, CH2),
(DMSO-d6) δ: 3.64 (s, 2H, CH2), 3.73 (m, 4H, CH2), 3.8–
3.87 (m, 4H, CH2), 5.2 (s, 2H, NH2) ppm. Anal. (C9H12N6O)
C, H, N.
3.1 (s, 6H, CH3), 3.55 (s, 2H, CH2), 3.75 (s, 2H, CH2), 5.4
(br. s, 2H, NH2), 6.75 (d, 2H, CH , J = 8.3 Hz), 7.84 (d, 2H,
CH, J = 6.8 Hz) ppm. Anal. (C17H20N8) C, H, N.
3.1.5. General procedure for the preparation of 2-(4,6-
diamino-1,3,5-triazin-2-yl)-2-{[4-(dimethylamino)phenyl]
imino}acetonitriles 19–27
3.1.5.6. 2-[4-Amino-6-(3,5,5-trimethyl-4,5-dihydro-1H-pyrazol-1-
yl)-1,3,5-triazin-2-yl]-2-{[4-(dimethylamino)phenyl]imino}aceto-
nitrile 24. Yield: 89%, m.p. > 350 °C (DMF-water); IR (KBr):
1
3398, 3306, 3225, 2202, 1633, 1615 cm–1, H-NMR (DMSO-
A mixture of appropriate triazine 10–18 (3.2 mmol) and p-
nitrosodimethylaniline (0.48 g, 3.2 mmol) in ethanol (80 ml)
was refluxed for 0.5 h. Then 30% aqueous potassium hydro-
xide (0.1 ml) was added. The reaction mixture was stirred at r.t.
for 12 h. The solid that precipitated was separated by suction
and purified by crystallization from suitable solvent.
d6) δ: 1.64 (s, 6H, CH3), 1.99 (s, 3H, CH3), 2.86 (s, 2H, CH2),
3.1 (s, 6H, CH3), 6.85 (d, 2H, CH, J = 8.8 Hz), 7.3 (br. s, 2H,
NH2), 7.6 (d, 2H, CH, J = 8.3 Hz) ppm. Anal. (C19H23N9) C,
H, N.
3.1.5.7. 2-{4-Amino-6-[4-(4-nitrophenyl)piperazin-1-yl]-1,3,5-tria-
zin-2-yl}-2-{[4-(dimethylamino)phenyl]imino}acetonitrile
25.
3.1.5.1. 2-[4-Amino-6-(4-phenylpiperazin-1-yl)-1,3,5-triazin-2-
yl]-2{[4-(dimethylamino)phenyl] imino}acetonitrile 19. Yield:
68%, m.p. 243–246 °C (DMF-water); IR (KBr): 3317, 3180,
Yield: 60%, m.p. 273–276 °C (DMF-water); IR (KBr): 3348,
1
3135, 2908, 2201, 1633, 1599 cm–1; H-NMR (DMSO-d6) δ:
1
2914, 2204, 1653, 1614 cm–1; H-NMR (CDCl3) δ: 3.11 (s,
3.06 (s, 6H, CH3), 3.61 (br. s, 4H, CH2), 3.92 (br. s, 4H, CH2),
6.84 (d, 2H, CH, J = 9.1 Hz), 7.06 (d, 2H, CH, J = 9.2 Hz), 7.3
(br. s, 2H, NH2), 7.6 (d, 2H, CH, J = 8.9 Hz), 8.08 (d, 2H, CH,
J = 9 Hz) ppm. Anal. (C23H24N10O2) C, H, N.
6H, CH3), 3.25 (s, 4H, CH2), 3.89–3.99 (m, 4H, CH2), 5.62
(br. s, 2H, NH2), 6.75 (d, 2H, CH, J = 9.3 Hz), 6.91 (t, 1H,
CH, J = 7.3 Hz, J = 6.8 Hz), 6.97 (d, 2H, CH, J = 7.8 Hz),
7.28–7.32 (m, 2H, CH), 7.86 (d, 2H, J = 8.8 Hz) ppm. EI-
MS m/z (M+): 427.2. Anal. (C23H25N9) C, H, N.
3.1.5.8. 2-[4-Amino-6-(5-ethyl-4-methyl-4,5-dihydro-1H-pyra-
zol-1-yl)-1,3,5-triazin-2-yl]-2-{[4-(dimethylamino)phenyl]imi-
no}acetonitrile 26. Yield: 79%, m.p. 271–274 °C (DMF-
water); IR (KBr): 3313, 3179, 2932, 220, 1652, 1613 cm–1,
1H-NMR (DMSO-d6) δ: 1.1–1.17 (m, 6H, CH3), 2.29–2.43
(m, 2H, CH2), 3.04 (s, 6H, CH3), 3.2–3.23 (m, 1H, CH),
3.49–3.51 (m, 1H, CH), 4.13 (s, 1H, CH), 6.84 (d, 2H, CH,
J = 8.8 Hz), 7.35 (s, 2H, NH2), 7.58 (d, 2H, CH , J = 8.8 Hz)
ppm. Anal. (C19H23N9) C, H, N.
3.1.5.2. 2-{4-Amino-6-[4-(2-methylphenyl)piperazin-1-yl]-1,3,5-
triazin-2-yl}-2-{[4-(dimethylamino)phenyl]imino}acetonitrile 20
.
Yield: 84%, m.p. 294–296 °C (DMF); IR (KBr): 3473, 3278,
3134, 2202, 1629, 1557 cm–1; 1H-NMR (DMSO-d6) δ: 2.33 (s,
3H, CH3), 2.91 (br. s, 4H, CH2), 3.08 (s, 6H, CH3), 3.36 (br. s,
4H, CH2), 6.87 (d, 2H, CH, J = 9.3 Hz), 6.9–7.25 (m, 4H,
CH), 7.4 (br. s, 2H, NH2), 7.61 (d, 2H, CH, J = 9.1) ppm.
Anal. (C24H27N9) C, H, N.
3.1.5.9. 2-(4-Amino-6-morpholin-4-yl-1,3,5-triazin-2-yl)-2-{[4-
(dimethylamino)phenyl]imino} acetonitrile 27. Yield: 79%, m.
p. 239–241 °C (DMF-water); IR (KBr): 3330, 3184, 2858,
2203, 1655, 1614 cm–1; 1H-NMR (CDCl3) δ: 3.11 (s, 6H,
CH3), 3.76 (s, 4H, CH2), 3.82 (s, 2H, CH2), 3.99 (s, 2H,
CH2), 5.5 (br. s, 2H, NH2), 6.75 (d, 2H, CH, J = 9.3 Hz),
7.86 (d, 2H, J = 9.3 Hz) ppm. Anal. (C17H20N8) C, H, N.
3.1.5.3. 2-{4-Amino-6-[4-(3-chlorophenyl)piperazin-1-yl]-1,3,5-
triazin-2-yl}-2-{[4-(dimethylamino)phenyl]imino}acetonitrile 21
.
Yield: 36%, m.p. 250–254 °C (DMF-water); IR (KBr): 3311,
1
3213, 2908, 2203, 1644, 1615 cm–1; H-NMR (DMSO-d6) δ:
3.08 (s, 6H, CH3), 3.29 (br. s, 4H, CH2), 3.92 (br. s, 2H, NH2),
3.93 (br. s, 4H, CH2), 6.58–6.63 (m, 1H, CH), 6.82–7.04 (m,

224
F. Sączewski et al. / European Journal of Medicinal Chemistry 41 (2006) 219–225
3.1.5.10. 4-Amino-N-phenyl-6-(4-phenylpiperazin-1-yl)-1,3,5-
triazine-2-carboxamide 28. To a solution of triazine 10 (0.5 g,
1.69 mmol) in ethanol (5 ml) 20% aqueous sodium carbonate
(0.4 ml) and nitrosobenzene (0.22 g, 2.03 mmol) were added.
The reaction mixture was heated under reflux for 10 min. After
cooling to r.t. and standing overnight, the product 28 that pre-
cipitated was separated by suction and washed with ethanol,
water, dried and purified by crystallization from THF–water:
36% yield, m.p. 237–240 °C; IR (KBr): 3490, 3330, 3139,
1H, CH), 4.15 (s, 1H, CH), 7.12 (t, 1H, CH, J = 7.3 Hz),
7.34–7.36 (m, 4H, CH + NH2), 7.74 (d, 2H, CH, J = 7.8 Hz),
10.28 (s, 1H, NH) ppm. Anal. (C16H19N7O) C, H, N.
3.1.5.15. 4-Amino-6-morpholin-4-yl-N-phenyl-1,3,5-triazine-2-
carboxamide 32. To a solution of triazine 18 (1 g, 4.54 mmol)
in DMF (14 ml) 20% aqueous sodium carbonate (1 ml) and
nitrosobenzene (0.58 g, 5.45 mmol) were added. The reaction
mixture was heated under reflux for 10 min. After cooling to r.
t. and standing for 24 h, the product 32 that precipitated was
separated by suction and washed with DMF, water, dried and
purified by crystallization from DMF-water: 27% yield, m.p. >
350 °C; IR (KBr): 3496, 3334, 2954, 2256, 1702, 1626 cm–1;
1H-NMR (DMSO-d6) δ: 3.63 (m, 4H, CH2), 3.7–3.83 (m, 4H,
CH2), 7.11 (t, 1H, CH, J = 7.32 Hz, J = 7.3 Hz), 7.2 (s, 2H,
NH2), 7.34 (t, 2H, CH, J = 7.8 Hz, J = 7.3 Hz), 7.73 (d, 2H,
CH, J = 8.3 Hz), 10.27 (s, 1H, NH) ppm. EI-MS m/z (M+):
300.1. Anal. (C14H16N6O2) C, H, N.
1
1686, 1636, 1515 cm–1; H-NMR (DMSO-d6) δ: 3.2 (t, 4H,
CH2, J = 5.1 Hz, J = 4.8 Hz), 3.92–3.99 (m, 4H, CH2), 6.81
(t, 1H, CH, J = 7.3 Hz, J = 7 Hz), 6.99 (d, 2H, CH,
J = 7.7 Hz), 7.13 (t, 1H, CH, J = 7.3 Hz, J = 7.7 Hz), 7.22–
7.38 (m, 6H, CH + NH2), 7.76 (d, 2H, CH, J = 7.7 Hz),
10.27 (s, 1H, NH) ppm. Anal. (C20H21N7O) C, H, N.
3.1.5.11. 4-Amino-N-phenyl-6-pyrrolidin-1-yl-1,3,5-triazine-2-
carboxamide 29. To a solution of triazine 14 (0.3 g,
1.47 mmol) in DMF (4.5 ml) 20% aqueous sodium carbonate
(0.36 ml) and nitrosobenzene (0.19 g, 1.76 mmol) were added.
The reaction mixture was heated under reflux for 15 min. After
cooling to r.t. and standing overnight, the product 29 that pre-
cipitated was separated by suction and washed with DMF,
water, dried and purified by crystallization from DMF-water:
39% yield, m.p. > 350 °C; IR (KBr): 3322, 3162, 2977,
3.2. Cytotoxicity studies
All reagents were purchased from Sigma (Deisenhofen,
Germany) and the cell lines were obtained from the German
Collection of Microorganisms and Cell Cultures (DSMZ,
Brauschweig, Germany). Cell lines used here were: human
bladder cancer 5637; human pancreatic cancer DAN-G, human
breast cancer MCF-7 and human non-small cell lung cancer
LCLC-103H. The culture medium was RPMI-1640 medium
containing 2 g/l HCO3 and 10% FCS. Cells were grown in
75 cm2 plastic culture flasks (Sarstedt, Nümbrecht, Germany)
in a humid atmosphere of 5% CO2 at 37 °C and passaged
shortly before becoming confluent.
For the cytotoxicity studies, 100 μl of a cell suspension
were seeded into 96-well microtiter plates (Sarstedt) at a den-
sity of 1000 cell per well except for the LCLC-103H cell line,
which was plated out at 250 cells per well. One day after plat-
ing, the cells were treated with test substance at five concentra-
tions per compound. The 1000-fold concentrated stock solu-
tions in DMF were serially diluted by 50% in DMF to give
the feed solutions, which were diluted 500-fold into culture
medium. The controls received just DMF. Each concentration
was tested in eight wells, with each well receiving 100 μl of the
medium containing the substance. The concentration ranges
were chosen to bracket the expected IC50 values as best as
possible. Cells were then incubated for 96 h, after which time
the medium was removed and replaced with 1% glutaralde-
hyde/PBS solution for 20 min. Cells were stored at 4 °C under
PBS. Staining with crystal violet was done as previously de-
scribed [30]. O.D. was measured at λ = 570 nm with an Anthos
2010 plate reader (Salzburg, Austria).
1
1655, 1540, 1499 cm–1; H-NMR (DMSO-d6) δ: 1.9 (s, 4H,
CH2), 3.43–3.59 (m, 4H, CH2), 7.12 (t, 1H, CH, J = 7.3 Hz,
J = 7.3 Hz), 7.17 (br. s, 2H, NH2), 7.36 (t, 2H, CH, J = 7.8 Hz,
J = 7.3 Hz), 7.54 (d, 2H, CH, J = 7.8 Hz), 10.24 (s, 1H, NH)
ppm. Anal. (C14H16N6O) C, H, N.
3.1.5.12. 4-Amino-N-phenyl-6-[(3,5,5-trimethyl or 5-ethyl-4-
methyl)-4,5-dihydro-1H-pyrazol-1-yl]-1,3,5-triazine-2-carbox-
amides 30–31. To a solution of triazine 15 or 17 (2.04 mmol)
in ethanol (6 ml) 20% aqueous sodium carbonate (0.45 ml) and
nitrosobenzene (0.26 g, 2.45 mmol) were added. The reaction
mixture was heated under reflux for 24 h. After cooling to r.t.
the product that precipitated was separated by suction and
washed with ethanol, water, dried and purified by crystalliza-
tion.
3.1.5.13. 4-Amino-N-phenyl-6-(3,5,5-trimethyl-4,5-dihydro-1H-
pyrazol-1-yl]-1,3,5-triazine-2-carboxamide 30. Yield: 26%, m.
p. 282–283 °C (DMF-water); IR (KBr): 3335, 3226, 2919,
1
1693, 1637, 1600 cm–1, H-NMR (DMSO-d6) δ: 1.59 (s, 6H,
CH3), 1.98 (s, 3H, CH3), 2.85 (s, 2H, CH2), 7.1 (t, 1H,
J = 7.3 Hz, J = 7.3 Hz), 7.2 (br. s, 1H, NH2), 7.35 (t, 2H,
CH, J = 7.8 Hz, J = 7.8 Hz), 7.45 (br. s, 1H, NH2), 7.71 (d,
2H, CH, J = 7.8 Hz), 10.12 (s, 1H, NH) ppm. EI-MS m/z
(M+): 325.1. Anal. (C16H19N7O) C, H, N.
Corrected T/C values were calculated by the equation:
3.1.5.14. 4-Amino-N-phenyl-6-(5-ethyl-4-methyl-4,5-dihydro-1H-
pyrazol-1-yl)-1,3,5-triazine-2-carboxamide 31. Yield: 72%, m.p.
236–239 °C (DMF-water); IR (KBr): 3504, 3147, 2977, 1691,
ðT=CÞcorrð%Þ ¼ ðO:D:T ꢀ O:D:c;0Þ=ðO:D:c ꢀ O:D:c;0Þ ꢁ 100
where O.D.T is the mean absorbance of the treated cells; O.D.c
the mean absorbance of the controls and O.D.c,0 the mean ab-
sorbance at the time drug was added. The IC50 values were
1
1638, 1559 cm–1; H-NMR (DMSO-d6) δ: 1.1–1.17 (m, 6H,
CH3), 2.3–2.44 (m, 2H, CH2), 3.23 (m, 1H, CH), 3.56 (s,

F. Sączewski et al. / European Journal of Medicinal Chemistry 41 (2006) 219–225
225
[14] J.R. Dimmock, P. Kumar, J.W. Quail, U. Pagazhenthi, J. Yang, M. Chen,
R.S. Reid, T.M. Allen, G.Y. Kao, S.P.C. Cole, G. Batist, J. Balzarini, E.
De Clerq, Eur. J. Med. Chem. 30 (1995) 209–217.
[15] N.W. Haman, D.L. Bassendowski, D.E. Wright, J.R. Dimmock, L.
M. Noble, J. Pharm. Sci. 67 (1978) 1539–1542.
[16] L.M. Wattenberg, J.B. Coccia, A.R. Galhaith, Cancer Lett. 83 (1983)
165–169.
[17] J.R. Dimmock, S.K. Raghaven, B.M. Logan, G.E. Bigam, Eur. J. Med.
Chem. 18 (1983) 248–254.
[18] F. Barrow, F.J. Thorneycroft, J. Chem. Soc. (1934) 722–727.
[19] F. Barrow, F.J. Thorneycroft, J. Chem. Soc. (1939) 769–773.
[20] R.V. Hoffman, R.A. Bartsh, B.R. Cho, Acc. Chem. Rev. 22 (1989) 211–
217.
estimated by a linear least-square regression of the T/Ccorr va-
lues versus the logarithm of the substance concentration; only
concentrations that yielded T/Ccorr values between 10% and
90% were used in the calculation. The reported IC50 values
are the averages of 3–4 independent experiments and these var-
ied less than 20% from the individual values.
References
[1] M. Negwer, Organic-Chemical Drugs and their Synonyms, Wiley-VCH
Verlag GmbH, Weinheim, 2001.
[21] M.R. Boyd, Am. Assoc. Cancer Res. 30 (1989) 652–653.
[22] A.P. Monks, D.A. Scudiero, P. Skehan, R. Shoemaker, K.D. Poull, D.
Vistica, C. Hose, J. Langley, P. Cronise, A. Vaigro-Wolff, J. Natl. Can-
cer Inst. 83 (1991) 757–776.
[2] M. Jarman, H.M. Coleg, I.R. Judson, T.J. Thornton, D.E.V. Wilman, J.
Med. Chem. 36 (1993) 4195–4200.
[3] J. Soudon, M. Berlion, C. Lucas, P. Haddad, F. Calvo, Cancer Che-
mother. Pharmacol. 36 (1995) 195–199.
[23] J.N. Weinstein, T.G. Myers, P.M. O’Connor, S.H. Friend, A.
J. Fornance Jr., K.W. Kohn, J.K. Buolamwini, W.W. van Osdol, A.
P. Monks, D.A. Scudeiro, E.A. Sansville, D.W. Zaharevitz, R.E. Bunow,
K.D. Paull, Science 275 (1997) 343–349.
[4] J.P. Tassin, J. Dubois, M. Hancoq, G. Atassi, Talanta 42 (1995) 747–
752.
[5] M. Ono, N. Kawahara, D. Goto, Y. Wakabayashi, S. Ushiro, Cancer Res.
56 (1996) 1512.
[6] R. Menicagli, S. Samaritani, G. Signore, F. Vaglini, L.D. Via, J. Med.
Chem. 47 (2004) 4649–4652.
[24] GI50 represents the molar concentration that inhibits 50% net cell growth;
TGI represents the molar concentration giving total growth inhibition; Δ
represents the logarithmic difference between the parametric value re-
ferred to the most sensible cell line (MALME-3M) and the same arithme-
tical mean value for all tested cancer cell lines.
[25] P.C. Das, B.B. Patra, A.N. Bose, U.P. Basu, J. Indian, Chem. 6 (12)
(1968) 691–693.
[26] W.K. Detweiler, E.D. Amstutz, J. Am. Chem. Soc. 74 (1952) 1483–
1487.
[7] N. Baindur, N. Chadha, B.M. Brandt, D. Asgari, R.J. Patch, C. Schalk-
HiHi, T.E. Carver, I.P. Petrounia, C.A. Baumann, H. Ott, C. Manthey, B.
A. Springer, M.H. Player, J. Med. Chem. 48 (2005) 1717–1720.
[8] M. Ono, N. Kawahara, D. Goto, Y. Wakabayashi, S. Ushira, S. Yoshida,
H. Izumi, M. Kuwano, Y. Sato, Cancer Res. 56 (1996) 1512.
[9] S. Nozaki, M. Maeda, H. Truda, G.W. Sledze, Breast Cancer Res. Treat.
83 (2004) 195–199.
[10] Z. Brzozowski, F. Sączewski, M. Gdaniec, Eur. J. Med. Chem. 35 (2000)
1053.
[11] Z. Brzozowski, F. Sączewski, Eur. J. Med. Chem. 37 (2002) 709–720.
[12] A. Gazit, N. Osherov, C. Giton, A. Levitzki, J. Med. Chem. 39 (1996)
4905–4911.
[27] Z. Brzozowski, Acta Pol. Pharm. 55 (1) (1998) 49–56.
[28] Z. Brzozowski, Z. Kamiński, I. Kozakiewicz, S. Angielski, J. Rogulski,
Acta Pol. Pharm. 36 (1979) 401–410.
[29] S.L. Shapiro, V.A. Pavrino, L. Freedman, J. Am. Chem. Soc. 81 (1959)
3728–3736.
[13] A. Gazit, H. App, G. McMahon, J. Chen, A. Levitzki, F.F. Bohmer, J.
Med. Chem. 39 (1996) 2170–2177.
[30] G. Bernhardt, H. Reile, H. Birnböck, T. Spruss, H. Schönenberger, J.
Cancer Res. Clin. Oncol. 118 (1992) 35–39.