Synthesis and antiproliferative activity of 2,6-diamino-9-benzyl-9- deazapurine and related compounds

Article abstract of DOI:10.1016/j.bmc.2004.04.003

Treatment of 6-bromomethyl- or 6-dibromomethyl-5-nitropyrimidine-2,4- diamine with KCN gave the same product - (2,6-diamino-5-nitropyrimidinyl) acetonitrile. Benzylation of the nitrile took place on the α-carbon to the cyano group preferentially affording

Full text of DOI:10.1016/j.bmc.2004.04.003

Bioorganic & Medicinal Chemistry 12 (2004) 3187–3195
Synthesis and antiproliferative activity of 2,6-diamino-9-benzyl-9-
deazapurine and related compounds
*
ꢀ
Miroslav Otmar, Milena Masojıdkova, Ivan Votruba and Antonın Holy
ꢀ
ꢀ
ꢀ
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo nꢀam. 2,
166 10 Prague 6, Czech Republic
Received 21 November 2003; accepted 6 April 2004
Available online 10 May 2004
Abstract—Treatment of 6-bromomethyl- or 6-dibromomethyl-5-nitropyrimidine-2,4-diamine with KCN gave the same product––
(2,6-diamino-5-nitropyrimidinyl)acetonitrile. Benzylation of the nitrile took place on the a-carbon to the cyano group preferentially
affording the corresponding mono- and dibenzyl derivative, whose reductive cyclization resulted in 7-benzyl-5H-pyrrolo[3,2-
d]pyrimidine-2,4-diamine and 7,7-dibenzyl-7H-pyrrolo[3,2-d]pyrimidine-2,4,6-triamine, respectively. Suitability of the protection of
N2 and N4 atoms with benzyl, acetyl, or benzoyl groups was also investigated. The in vitro evaluation of cell growth inhibition on
CCRF-CEM, HL-60, HeLa S3, and L1210 cell lines showed significant activity in 8 new compounds. The most potent compounds
were the above mentioned 6-dibromomethyl derivative (IC50 ¼ 0.54, 1.7, 5.0, and 1.9 mol Lꢀ1) and 7,N2,N4-tribenzyl-5H-pyrrolo[3,2-
d]pyrimidine-2,4-diamine (IC50 ¼ 1.9, 2.7, 7.3, and 1.0 mol Lꢀ1).
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
suitably substituted pyrroles introduced by Klein and
co-workers.12
In 1964, Imai reported preparation of 2,6-diamino-9-
deazapurine and its activity against bacteria and pro-
tozoa.1 Since that time, only a few of its derivatives with
alkyl or aryl substituents on the amino groups2;3 and, in
a recent paper,4 with C-phenyl group in the position 8
were described. Quite recently, we published the syn-
thesis of 9-deaza analogue of cytokinin olomoucine,
which bears C-methyl in position 9 (Ref. 5). Surprisingly
enough, no 2,6-diamino-9-deazapurine counterpart to 9-
arylmethyl-9-deazaguanines,6;7 which are extremely po-
tent inhibitors of purine nucleoside phosphorylase, have
been synthesized so far. Therefore, our aim was to assess
the synthetic availability of 2,6-diamino-9-arylmethyl-9-
deazapurines as well as the potential of their biological
activity.8 To simplify our task we selected benzyl as the
arylmethyl substituent, which could be introduced by
alkylation of the appropriate 5-nitro-4(6)-(cyano-
methyl)pyrimidines followed by reductive cycliza-
tion,9–11 which is less laborious than the traditional
approach based on pyrimidine ring construction on
2. Results and discussion
Bromination of the starting 6-methyl-5-nitropyrimidine-
2,4-diamine13;14 (1) with 1 equiv of bromine in acetic acid
according to the described procedure,15;16 which pro-
vides a satisfactory monobromination of 2,4-dimethoxy-
6-methyl-5-nitropyrimidine, results in this case in the
6-dibromomethyl derivative 2. The desired monobromo
derivative 3 was obtained in 7% yield only. This fact is
due to much higher reactivity of the compound 3 in the
bromination reaction compared to the starting com-
pound 1. With excess bromine, the dibromomethyl
derivative 2 was obtained as the only product in high
yield (Scheme 1). Reaction of compound 3 with potas-
sium cyanide afforded the cyanomethyl derivative 4.
Surprisingly, dibromo derivative 2 gave with excess
potassium cyanide also the nitrile 4 in a comparable
yield. The mechanism of the reaction is unknown, nev-
ertheless, TLC analysis showed in situ formation of the
monobromo derivative 3. The nitrile 4 is insoluble in
organic solvents and water; therefore, its benzylation
was carried out without isolation. The reaction afforded
the C-benzyl derivative 5 as the main product.
Keywords: Pyrrolo[3,2-d]pyrimidines; Cytostatics; Aci–nitro Tauto-
merism; C-Alkylation; Reductive cyclization.
* Corresponding author. Tel.: +420-220-183-375; fax: +420-220-183-
560; e-mail: otmar@uochb.cas.cz
0968-0896/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.04.003

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M. Otmar et al. / Bioorg. Med. Chem. 12 (2004) 3187–3195
methyl-5-nitropyrimidine-2,4-diamine (10) was prepared
NH2
NH2
NH2
NO2
CH3
NO2
Br
NO2
Br
(i)
by treatment of 2,4-dichloro-6-methyl-5-nitropyrim-
idine17 (11) with benzylamine. Similarly as in the previ-
ous case, the attempted monobromination gave the
monobromo derivative 12 in 9% yield only, whereas the
dibromomethyl derivative 13 prevailed. The nitrile 14
was prepared from the easily available dibromo deriv-
ative 13 on treatment with potassium cyanide. Alkyl-
ation of the nitrile 14 with benzyl bromide in the
presence of tri-n-butylamine gave the C-benzyl deriva-
tive 15. In 1H as well as 13C NMR spectra of compounds
10 and 12–15, each signal of the mentioned compounds
is accompanied by a minor one (ratio 4–5: 1), whose
chemical shift is only slightly different, however, distinct
enough to be distinguished (see Experimental). Follow-
ing sodium deuteroxide addition this phenomenon dis-
appeared. We assume that this doubling of the chemical
shifts reflects the existence of aci–nitro tautomerism with
minor isomer as the aci-form (tentative structure I and
II, Scheme 3). Contrary to the benzyl derivative 5, in
compound 15 the reactivity of the remaining hydrogen
atom of the acid carbon and the NH-groups toward
alkylation is comparable. Further benzylation of 15 with
1 equiv of benzyl bromide led to a mixture of products
in which the perbenzyl derivative 16 prevailed. Catalytic
hydrogenation of compounds 14 and 15 afforded the
5H-pyrrolo[3,2-d]pyrimidines 17 and 18, respectively,
whereas the perbenzyl derivative 16 gave the 6-amino-
7H-pyrrolo[3,2-d]pyrimidine 19. No cleavage of N-benz-
yl groups by hydrogenation or by other debenzylation
procedures occurred in both cases.18
(i)
(ii)
N
N
N
N
H2N
N
H2N
(ii)
(iii)
N
H2N
N
2
Br
NH2
3
1
NH2
NH2
N
NO2
NO2
NO2
(iv)
N
N
CN
CN
CN
H2N
N
H2N
H2N
N
Bn
Bn
Bn
6
5
4
(v)
(v)
(v)
NH2
NH2
NH2
H
N
NH2
CN
N
(v)
N
N
N
NH2
Bn
H2N
N
H2N
N
H2N
N
Bn
Bn
Bn
8
7
9
Scheme 1. (i) Br2, NaOAc, AcOH; (ii) KCN, DMSO; (iii) BnBr, Bu3N,
DMSO; (iv) BnBr, KOH, dioxane–water 3:1, (v) H2/Pd.
For further benzylation, compound 5 was treated with
excess benzyl bromide in the presence of potassium
hydroxide in a dioxane–water (3:1) mixture. This reac-
tion afforded selectively the C,C-dibenzyl derivative 6.
The monobenzyl derivative 5 was reductively cyclized by
catalytic hydrogenation to the 5H-pyrrolo[3,2-d]pyrim-
idine 7 (the nitrogen atom of the cyano group is liber-
ated as ammonia). A small portion of the uncyclized
intermediate 8, which is unstable and spontaneously
cyclizes to compound 7, was also isolated. To the con-
trary, in the dibenzyl derivative 6 the nitrogen atom of
the cyano group is transformed by catalytic hydroge-
nation to the amino group in position 6, which resulted
in the formation of the 6-amino-7H-pyrrolo[3,2-
d]pyrimidine 9.
The impossibility to cleave the N-benzyl groups attached
to pyrrolo[3,2-d]pyrimidine nucleus led us to examine,
whether the acyl substituents in N2 and N4 positions
could be used instead of benzyl groups to make the
starting compound 1 more lipophilic (Scheme 4). The
amino groups in compound 1 are not sufficiently
nucleophilic due to the presence of the nitro group,
however, refluxing of compound 1 with acetic anhydride
To avoid the difficult isolation of polar and insoluble
pyrimidine-2,4-diamines, we used their N2,N4-dibenzyl
derivatives instead (Scheme 2). N2,N4-Dibenzyl-6-
Cl
NHBn
NHBn
NO2
CH3
NO2
NO2
(ii)
BnHN
(i)
(iii)
N
N
N
Br
Cl
N
BnHN
(iv)
N
CH3
N
R
11
10
12 R = H
13 R = Br
NHBn
NO2
NHBn
NBn2
NO2
NO2
CN
Bn
(v)
N
N
N
CN
CN
BnHN
N
BnHN
N
Bn2N
N
16
Bn
Bn
(vi)
15
14
(vi)
(vi)
NHBn
NHBn
NBn2
H
H
N
N
N
N
N
N
NH2
Bn
Bn
BnHN
N
BnHN
N
Bn2N
N
Bn
19
17
18
Scheme 2. (i) BnNH2, K2CO3; (ii) Br2, AcOH, NaOAc, 60 °C; (iii) KCN, DMSO; (iv) BnBr, Bu3N, DMSO; (v) BnBr, NaH, DMSO; (vi) H2/Pd.

M. Otmar et al. / Bioorg. Med. Chem. 12 (2004) 3187–3195
3189
3. Conclusion
Bn
N
Bn
N
Bn
NH
O
N
NH
N
NH
O
N
NO2
R
O
N
O
The assay showed a potent antiproliferative activity of
the 6-dibromomethyl-5-nitropyrimidines 2, 13, and 23,
which is slightly lower than the activity of 6-(dibro-
momethyl)-2-methoxy-4-morpholino-5-nitropyrimidine
(L1210, IC50 ¼ 0.32 lM; H.Ep.2, IC50 ¼ 1.6 lM) and
related compounds described by a Townsend’s group.16
However, the IC50 values of the 5-nitropyrimidines 10
and 24 show that the activity is not necessarily associ-
ated with the presence of dibromomethyl group. With
regard to the in vivo toxicity of 6-dibromomethyl-5-ni-
tropyrimidines,16 the activity of the pyrrolo[3,2-d]pyr-
imidines 7, 17, and 18 seems to be more interesting. The
mechanism of their cytostatic activity is so far unknown.
The analysis of cell-cycle profiles (CCRF-CEM cells,
G1/G0, S+G2/M) at various concentrations of tested
compounds (IC25, IC50, IC75) by flow cytometry did not
show any differencies between control and treated
samples (data not shown). These results reveal a certain
potential of substituted 2,6-diamino-9-deazapurines for
the design of antineoplastic compounds.
Bn
Bn
Bn
N
N
R
N
N
H
N
R
H
H
I
II
10, 12-15
Scheme 3.
NH2
NHR
NO2
NHR
N
NO2
CH3
NO2
(i)
(ii)
N
N
N
H2N
N
RHN
N
CH3
RHN
CHBr2
1
20 R = Ac
21 R = Bz
22 R = Ac
23 R = Bz
(iii)
NHBz
NHBz
NO2
NO2
(iv)
N
N
CN
Bn
CN
BzHN
N
BzHN
N
Bn
24
25
Scheme 4. (i) Ac2O, reflux or Bz2O, 150 °C; (ii) Br2, AcOH, NaOAc,
60 °C; (iii) KCN, DMSO; (iv) BnBr, NaH.
4. Experimental
4.1. General
Table 1. Cytostatic activity of selected compounds
Melting points were determined on a Kofler block and
are uncorrected. Analytical TLC was performed on sil-
ica gel pre-coated aluminum plates with fluorescent
indicator (Merck 5554, 60 F254). Spots were visualized
with UV light (254 nm) or by spraying with ninhydrin
(1% solution in ethanol) followed by a short heating to
300–400 °C. Column chromatography was carried out
on silica gel (Sigma S-0507, 40–63 lm). Mass spectra
were measured on a ZAB-EQ (Micromass, Manchester,
UK) spectrometer using the EI (electron energy 70 eV)
or FAB (ionization with Xe, accelerating voltage 8 kV,
thioglycerol–glycerol 3:1 mixture or bis(2-hydroxyethyl)
disulfide were used as matrix). 1H and 13C NMR spectra
were recorded at 500 and 125.7 MHz on a Varian Unity
500 instrument in DMSO-d6 (referenced to the solvent
signal d ¼ 2:50 and 39.70 ppm, respectively). Chemical
shifts are in ppm and coupling constants (J) in Hz. UV
spectra were taken on a Beckman DU-65 spectropho-
tometer in methanol solution. IR spectra were obtained
on an FT IR Bruker Equinox IFS 55 spectrometer in
chloroform or KBr pellets. Elemental analyses were
carried out on a Perkin Elmer CHN Analyser 2400,
Series II Sys (Perkin Elmer, Norwolk, CT USA).
IC50 (lmol Lꢀ1
)
CCRF-CEM
HL-60
HeLa S3
L1210
2
0.54
3.6
8.0
1.5
4.0
1.9
4.2
3.5
1.7
2.7
18.0
1.0
4.9
2.7
2.2
3.1
5.0
––
1.9
2.0
––
7
10
13
17
18
23
24
––
23.5
16.0
7.3
5.0
4.5
1.0
4.2
5.2
––
25.0
afforded the diacetate 20. Similarly, melting compound 1
with benzoic anhydride at 150 °C gave the dibenzoate
21. Treatment with excess bromine smoothly gave the 6-
dibromomethyl derivatives 22 and 23, respectively.
However, their reaction with potassium cyanide gave a
complex mixture of products. From the reaction mixture
containing the dibromo derivative 23 and excess potas-
sium cyanide in dimethyl sulfoxide, to which an excess
of benzyl bromide was given, the dibenzyl derivative 24
was isolated in 9% yield only, which confirmed the in
situ formation of the nitrile 25.
In vitro cell growth inhibition by compounds 2, 5–7, 9,
10, 13–15, 17, 18, and 20–24 was evaluated in the fol-
lowing cell lines:19;20 human T-lymphoblastoids CCRF-
CEM line (ATCC CCL 119), human promyelocytic
leukemia HL-60 cells (ATCC CCL 240), human cervix
carcinoma HeLa S3 cells (ATCC CCL 2.2), and mouse
lymphocytic leukemia L1210 cells (ATCC CCL 219).
For compounds, whose inhibition of the cell growth at
c ¼ 10 lM was higher than 30%, IC50 values were
determined (Table 1).
4.2. Cytostatic activity assays
Inhibition of the cell growth was estimated in CCRF-
CEM T-lymphoblastoid cells (human acute lympho-
blasic leukemia, ATCC CCL 119), human promyelocytic
leukemia HL-60 cells (ATCC CCL 240), human cervix
carcinoma HeLa S3 cells (ATCC CCL 2.2), and mouse
lymphocytic leukemia L1210 cells (ATCC CCL 219)
(Ref. 19). CCRF-CEM, HL-60, and L1210 cells were
cultivated in RPMI 1640 medium supplemented with

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M. Otmar et al. / Bioorg. Med. Chem. 12 (2004) 3187–3195
10% calf fetal serum using 24-well tissue culture plates.
The endpoint of the cell growth was 72 h following the
drug addition. HeLa S3 cells were seeded to 24-well
dishes in RPMI 1640 HEPES modification with fetal
calf serum. After 48 h following the drug addition the
cultivation was stopped and the cell growth was evalu-
ated. In parallel, the cell viability was examined using
MTT standard spectrophotometric assay.20 The inhibi-
tory potency of the tested compounds was expressed as
IC50 values.
within 15 min and the reaction mixture was left at room
temperature for additional 20 min. Benzyl bromide
(6.0 g, 35 mmol) was added and the reaction mixture was
stirred overnight. The reaction mixture was taken into
ethyl acetate and washed with brine. Chromatography
on a silica gel column (chloroform–methanol 98:2) fol-
lowed by crystallization (petroleum ether–ethyl acetate
1:1) afforded compound 5 (580 mg, 41%) as yellowish
crystals, mp 240–241 °C. MS (EI) m=z (rel. intensity):
284 (4, M), 267 (6), 178 (100, M)Bn–NH), 152 (11), 123
1
(18), 91 (87), 43 (28). H NMR (500 MHz, DMSO-d6):
8.04 (s, 1H, NH), 8.02 (s, 1H, NH), 7.66 (s, 1H, NH),
7.46 (s, 1H, NH), 7.35–7.30 (m, 5H, Ph), 5.05 (dd, 1H,
J ¼ 10:5, 4.5, CH–CN), 3.32 (dd, 1H, J ¼ 13:3, 4.5,
CH2 Ph), 3.01 (dd, 1H, J ¼ 13:3, 10.5, CH2 Ph). 13C
NMR (125.7 MHz, DMSO-d6): 163.19 (C-4), 161.04 (C-
6), 158.83 (C-2), 137.28 (Ph), 129.19 (2C, Ph), 128.74
(2C, Ph), 127.33 (Ph), 119.83 (C-5), 119.32 (CN), 40.43
(CH–CN), 37.91 (CH2–Ph). IR (KBr): 3469, 3419, 3353,
3199, 2258, 1662, 1602, 1560, 1547, 1473, 1429, 1271,
4.2.1. 6-Dibromomethyl-5-nitropyrimidine-2,4-diamine (2).
Compound 1 (3.4 g, 20 mmol) and sodium acetate (3.4 g,
41 mmol) were added to a solution of bromine (6.6 g,
41 mmol) in acetic acid (200 mL) and the reaction mix-
ture was heated to 65 °C for 20 min. The solvent was
evaporated to a small volume, taken into ethyl acetate
and washed with water and brine. Chromatography on a
silica gel column (chloroform–methanol 97:3) followed
by crystallization (ethyl acetate) afforded compound 2 as
yellow crystals (5.1, 78%) g, mp 185–187 °C. MS (EI)
m=z (rel. intensity): 329 (29), 327 (59, M), 325 (31), 248
(35, M)Br), 246 (35, M)Br), 220 (28, M)Br–NO), 218
1226, 793, 699, 501 cmꢀ1
. UV/vis (MeOH): 348
(e ¼ 11400), 260 (e ¼ 6800). Anal. Calcd for
C20H18N6O2: C, 64.16; H, 4.85; N, 22.45. Found: C,
64.03; H, 4.84; N, 22.32. Anal. Calcd for C13H12N6O2:
C, 54.93; H, 4.25; N, 29.56. Found: C, 54.69; H, 3.97; N,
29.33.
1
(31, M)Br–NO), 167 (58, M)Br2), 57 (41), 43 (100). H
NMR (500 MHz, DMSO-d6): 8.09 (s, 1H, NH), 7.98
(s, 1H, NH), 7.74 (s, 1H, NH), 7.47 (s, 1H, NH), 7.41
(s, 1H, CHBr2). 13C NMR (127.5 MHz, DMSO-d6):
163.32 (C-6), 161.58 (C-4), 158.52 (C-2), 116.59 (C-5),
40.94 (CHBr2). IR (KBr): 3501, 3383, 1649, 1608, 1545,
1421, 1336, 1267, 1199, 1149, 737, 516 cmꢀ1. UV/vis
(MeOH): 360 (e ¼ 9700), 276 (e ¼ 6400). Anal. Calcd
for C5H5Br2N5O2:C, 18.37; H, 1.54; Br, 48.88; N, 21.42.
Found: C, 18.19; H, 1.75; Br, 48.60; N, 21.19.
4.2.4. 2-Benzyl-2-(2,6-diamino-5-nitropyrimidin-4-yl)-3-
phenylpropionitrile (6). Benzyl bromide (480 lL,
4 mmol) was added to a solution of compound 5
(280 mg, 1 mmol) and potassium hydroxide (340 mg,
6 mmol) in a dioxane–water mixture (3:1, 10 mL) and
the reaction mixture was stirred overnight. Methanol
(5 mL) was added, the reaction mixture was taken into
ethyl acetate, washed with brine and evaporated.
Chromatography on a silica gel column (chloroform)
followed by crystallization (petroleum ether–ethyl ace-
tate, 5:1) afforded compound 6 (240 mg, 64%) as yellow
crystals, mp 237–238 °C. MS (EI) m=z (rel. intensity):
374 (4, M), 357 (7, M)NH3), 344 (7, M)NO), 283 (10,
M)Bn), 251 (67, M)Bn–O2), 236 (9, M)Bn–NO2+H),
177 (25, M)2Bn–O+H), 91 (100, Bn). 1H NMR
(500 MHz, DMSO-d6): 7.59 (s, 2H, NH), 7.48 (s, 1H,
NH), 7.39 (s, 1H, NH), 7.32–7.23 (m, 10H, Ph), 3.90 (d,
2H, J ¼ 13:4, CH2–Ph), 3.00 (d, 2H, J ¼ 13:4, CH2–Ph).
13C NMR (500 MHz, DMSO-d6): 161.25 (C-4), 160.45
(C-6), 158.26 (C-2), 135.51 (2C, Ph), 130.81 (4C, Ph),
128.25 (4C, Ph), 127.46 (2C, Ph), 122.21 (C-5), 118.95
(CN), 51.03 (C–CN), 42.53 (2C, CH2–Ph). IR (CHCl3):
3495, 3425, 2235, 1648, 1615, 1594, 1548, 1426, 1333,
4.2.2. 6-Bromomethyl-5-nitropyrimidine-2,4-diamine (3)
and 6-Dibromomethyl-5-nitropyrimidine-2,4-diamine (2).
Compound 1 (3.4 g, 20 mmol) and sodium acetate (1.6 g,
20 mmol) were added to a solution of bromine (3.2 g,
20 mmol) in acetic acid (200 mL) and the reaction mix-
ture was heated to 65 °C for 20 min. The solvent was
evaporated to a small volume, taken into ethyl acetate
and washed with water and brine. Chromatography on a
silica gel column (chloroform–methanol 97:3) followed
by crystallization (ethyl acetate) afforded, besides the
unreacted starting compound 1 (1.2 g, 35%) and the
dibromo derivative 2 (2.5 g, 38%), compound 3 (350 mg,
7%) as yellowish crystals, mp 142–143 °C. MS (EI) m=z
(rel. intensity): 249 (8, M), 247 (8, M), 232 (5, M)NH3),
230 (5, M)NH3), 168 (100, M)Br). 1H NMR (500 MHz,
DMSO-d6): 8.01 (s, 2H, NH), 7.56 (s, 1H, NH), 7.38 (s,
1H, NH), 4.60 (s, 2H, CH2 Br). 13C NMR (127.5 MHz,
DMSO-d6): 164.52 (C-6), 161.21 (C-4), 159.13 (C-2),
119.17 (C-5), 33.64 (CH2 Br). Anal. Calcd for
C5H6BrN5O2: C, 24.21; H, 2.44; Br, 32.21; N, 28.24.
Found: C, 24.38; H, 2.63; Br, 31.98; N, 28.01.
1279 cmꢀ1
.
UV/vis (MeOH): 364 (e ¼ 6000), 265
(e ¼ 7200). Anal. Calcd for C20H18N6O2: C, 64.16; H,
4.85; N, 22.45. Found: C, 64.03; H, 4.84; N, 22.32.
4.2.5. 7-Benzyl-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine
(7) Hydrochloride. 3-Phenyl-2-(2,5,6-triaminopyrimidin-
4-yl)propionitrile (8). Compound 5 (570 mg, 2 mmol) in
methanol (100 mL) acidified with methanolic hydrogen
chloride (1 M, 5 mL) was hydrogenated under slight
overpressure in the presence of Pd/C catalyst (10 wt %,
40 mg) overnight. The catalyst was filtered off through a
4.2.3. 2-(2,6-Diamino-5-nitropyrimidin-4-yl)-3-phenylprop-
ionitrile (5). To a suspension of potassium cyanide (1.6 g,
25 mmol) in dimethyl sulfoxide (125 mL) cooled to 0 °C
compound 2 (1.6 g, 5 mmol) was added portionwise

M. Otmar et al. / Bioorg. Med. Chem. 12 (2004) 3187–3195
3191
Celite pad and the filtrate evaporated. Chromatography
on a silica gel column (chloroform–methanol, 96:4)
followed by crystallization (ethyl acetate–methanol, 6:1)
afforded hydrochloride of compound 7 (200 mg, 42%) as
white crystals, mp 244–246 °C. MS (EI) m=z (rel. inten-
sity): 240 (26, M+1), 239 (100, M), 238 (61, M)H), 223
(22, M)NH2), 194 (19, M)NO2+H), 162 (40, M)Ph).
1H NMR (500 MHz, DMSO-d6): 10.67 (s, 1H, NH),
7.25 (m, 4H, Ph), 7.15 (m, 1H, Ph), 7.05 (s, H-6), 6.80 (s,
2H, NH), 5.72 (s, 2H, NH), 3.05 (s, 2H, CH2–Ph). 13C
NMR (500 MHz, DMSO-d6): 157.33 (s, C-4), 151.73 (s,
C-2), 144.05 (C-7a), 141.99 (Ph), 128.50 (2C, Ph), 128.32
(2 C, Ph), 125.98 (C-6), 125.74 (Ph), 111.50 (C-7),
109.425 (C-4a), 29.60 (CH2–Ph). IR (KBr): 3326, 3186,
1636, 1560, 1543, 1472, 1396, 960 cmꢀ1. UV (MeOH):
287 (e ¼ 11900), 231 (e ¼ 21400). Anal. Calcd for
C13H14ClN5: C, 56.63; H, 5.12; Cl, 12.86; N, 25.40.
Found: C, 56.91; H, 5.26; Cl, 12.59; N, 25.23.
4.2.7.
N2,N4-Dibenzyl-6-methyl-5-nitropyrimidine-2,4-
diamine (10). 6-Methyl-5-nitrouracil (8.6 g, 50 mmol)
was heated in aqueous sodium hydroxide (1 M, 50 mL)
until a clear yellow solution arised. Water was evapo-
rated under reduced pressure and the resulting sodium
salt of 6-methyl-5-nitrouracil was co-evaporated with
ethanol and toluene and dried in vacuo. The salt was
added portionwise to phosphoryl chloride (50 mL,
540 mmol) at 0 °C and the reaction mixture was refluxed
for 4 h. The resulting 2,4-dichloro-6-methyl-5-nitroura-
cil (11) was extracted from the reaction mixture with 3
portions (100 mL, each) of petroleum ether. Collected
petroleum ether extracts were evaporated to a volume of
about 60 mL, mixed with neutral aluminum oxide (10 g)
and filtered through an aluminum oxide pad (10 g).
Benzyl amine (approx. 15 mL) was added portionwise to
the filtrate until it became basic. The alkalified filtrate
was refluxed for 10 min, taken into ethyl acetate, washed
with aqueous sodium hydrogen carbonate and brine.
Chromatography on a silica gel column (chloroform)
followed by crystallization (petroleum ether–ethyl ace-
tate, 4:1) afforded compound 10 (9.4 g, 54%, yellow
crystals, mp 117–118 °C [at 70–73 °C change of modifi-
cation]) as a mixture of tautomers (5:1). MS (EI) m=z
(rel. intensity): 349 (41, M), 332 (63, M)OH), 319 (9,
M)NO), 301 (21), 243 (30, M)BnNH), 228 (7), 106 (17,
The aforementioned chromatography afforded also a
less polar uncyclized byproduct––compound 8 (30 mg,
6%), which was obtained by crystallization from ethyl
acetate as white crystals with mp 182–184 °C. After
several days storage it spontaneously cyclized to 7. Data
of compound 8: MS (EI) m=z (rel. intensity): 254 (100,
M), 238 (7, M)NH2), 214 (18), 177 (20, M)Ph), 163 (61,
1
1
M)Bn), 91 (97, Bn). H NMR (500 MHz, DMSO-d6):
BnNH), 91 (100, Bn). H NMR (500 MHz, DMSO-d6
7.35–7.25 (m, 5H, Ph), 6.24 (br s, 2H, NH2), 5.43 (br s,
2H, NH2), 4.56 (dd, J ¼ 9:8, 6.2, CH–CN), 3.75 (br s,
2H, NH2), 3.09 (dd, J ¼ 13:6, 6.2, CH2–Ph), 3.04
(dd, J ¼ 13:6, 9.8, CH2–Ph). 13C NMR (125.7 MHz,
DMSO-d6): 157.845 (2C, C-2, C-6), 157.24 (C-4), 137.86
(Ph), 129.28 (2C, Ph), 128.84 (2C, Ph), 126.99 (Ph),
120.45 (CN), 114.16 (C-5). Anal. Calcd for C13H14N6: C,
61.40; H, 5.55; N, 33.05. Found: C, 61.59; H, 5.76; N,
32.91.
minor tautomer in italic): 9.28 (t, 1H, J ¼ 6:1, NH), 9.03
(t, 1H, J ¼ 6:0, NH), 8.58 (t, 1H, J ¼ 6:5, NH), 8.33 (t,
1H, J ¼ 6:4, NH) 7.40–7.10 (m, 10H, Ph), 7.40–7.10 (m,
10H, Ph), 4.70 (d, 2H, J ¼ 6:0, CH2–Ph), 4.65 (d, 2H,
J ¼ 6:1, CH2–Ph), 4.56 (d, 2H, J ¼ 6:5, CH2–Ph), 4.43
(d, 2H, J ¼ 6:5, CH24–Ph), 2.56 (s, 3H, CH3) 2.55 (s,
3H, CH3). 13C NMR (125.7 MHz, DMSO-d6, minor
tautomer in italic): 167.74 (C-6), 167.53 (C-6), 159.32
(C-4), 159.24 (C-4), 156.70 (C-2), 156.48 (C-2), 139.82
(Ph), 139.50 (Ph), 139.31 (Ph), 139.24 (Ph), 128.46
(2C, Ph), 128.42 (2C, Ph), 128.42 (2 C, Ph), 128.40 (2C,
Ph), 127.75 (2C, Ph), 127.48 (2C, Ph), 127.59 (2C, Ph),
127.44 (2C, Ph), 127.03 (2C, Ph), 126.90 (2C, Ph),
121.76 (C-5), 120.78 (C-5), 44.38 (CH2–Ph), 44.25
(CH2–Ph), 44.14 (CH2–Ph), 43.84 (CH2–Ph), 26.95
(CH3), 26.53 (CH3). IR (CHCl3): 3436, 3366, 3019, 1591,
1568, 1546, 1510, 1435, 1346, 1266 cmꢀ1. UV/vis
(MeOH): 362 (e ¼ 16000), 265 (e ¼ 8100), 224
(e ¼ 19300). Anal. Calcd for C19H19N5O2: C, 65.32; H,
5.48; N, 20.04. Found: C, 65.14; H, 5.40; N, 19.95.
4.2.6. 7,7-Dibenzyl-7H-pyrrolo[3,2-d]pyrimidine-2,4,6-tri-
amine (9) dihydrochloride. Compound
6 (750 mg,
2 mmol) in methanol (100 mL) acidified with methanolic
hydrogen chloride (1 M, 5 mL) was hydrogenated under
slight overpressure in the presence of Pd/C catalyst
(10 wt %, 40 mg) overnight. The catalyst was filtered off
through a Celite pad and the filtrate evaporated.
Chromatography on a silica gel column (chloroform–
methanol, 97:3) followed by crystallization (ethyl ace-
tate–methanol, 6:1) afforded dihydrochloride of com-
pound 7 (530 mg, 58%) as white crystals, mp 199–
201 °C. MS (EI) m=z (rel. intensity): 344 (26, M), 253
(48, M)Bn), 36 (100, HCl). 1H NMR (500 MHz,
DMSO-d6): 11.40 (br s, 1H, NH), 10.88 (br s, 1H, NH),
8.40 (br s, 2H, NH2), 7.53 (br s, 2H, NH2), 7.23 (m, 6H,
Ph), 7.07 (m, 4H, Ph), 3.81 (d, 2H, J ¼ 13:7, CH2–Ph),
3.75 (d, 2 H, J ¼ 13:7, CH2–Ph). 13C NMR (500 MHz,
DMSO-d6): 169.31 (C-6), 153.92 (C-4), 151.34 (C-2),
146.90 (C-7a), 133.94 (2C, Ph), 129.06 (4C, Ph), 128.74
(4C, Ph), 127.89 (2C, Ph), 112.205 (C-4a), 61.35 (C-7),
40.935 (C2–Ph). IR (KBr): 3303, 3110, 1658, 1550, 1516,
4.2.8. N2,N4-Dibenzyl-6-bromomethyl-5-nitropyrimidine-
2,4-diamine (12). Compound 10 (700 mg, 2 mmol) and
sodium acetate (160 g, 2 mmol) were added to a solution
of bromine (320 mg, 2 mmol) in acetic acid (20 mL) and
the reaction mixture was heated to 65 °C for 20 min. The
solvent was evaporated to a small volume, taken into
ethyl acetate and washed with water and brine. Chro-
matography on a silica gel column (petroleum ether–
ethyl acetate 9:1) followed by crystallization (petroleum
ether–ethyl acetate 19:1) afforded, besides the unreacted
starting compound 10 (210 mg, 30%) and the dibromo
derivative 13 (360 mg, 36%), compound 12 (80 mg, 9%,
yellow crystals, mp 118–120 °C) as a mixture of 2 tau-
tomers (5:1). MS (EI) m=z (rel. intensity): 429 (15, M),
1497, 1457, 750, 703 cmꢀ1
.
UV (MeOH): 278
(e ¼ 12600). Anal. Calcd for C20H26Cl2N6O2: C, 52.98;
H, 5.78; Cl, 15.64, N, 18.54. Found: C, 52.88; H, 5.80;
Cl, 15.47, N, 18.11.

3192
M. Otmar et al. / Bioorg. Med. Chem. 12 (2004) 3187–3195
427 (15, M), 349 (25, M)Br), 333 (23, M)Br–O), 332
(27, M)Br–OH), 301 (12), 243 (14), 106 (19, BnNH), 91
at room temperature for additional 20 min. Acetic acid
(5 mL) was added at 0 °C, the reaction mixture was ta-
ken into ethyl acetate and washed with brine. Chroma-
tography on a silica gel column (petroleum ether–ethyl
acetate 9:1) followed by crystallization (petroleum
ether–ethyl acetate 4:1) afforded compound 14 (3.2 g,
87%, yellow crystals, mp 172–173 °C) as a mixture of 2
tautomers (3.8:1). MS (EI) m=z (rel. intensity): 374 (34,
M), 357 (51, M)OH), 268 (14, M)BnNH), 256 (19), 91
1
(100, Bn). H NMR (500 MHz, DMSO-d6 minor tauto-
mer in italic): 9.41 (t, 1H, J ¼ 6:1, NH), 9.29 (t, 1H,
J ¼ 6:1, NH), 8.83 (t, 1H, J ¼ 6:5, NH), 8.58 (t, 1H,
J ¼ 6:4, NH), 7.40–7.10 (m, 10H, Ph), 7.40–7.10 (m,
10H, Ph), 4.70 (s, 2 H, CH2Br), 4.69 (d, 2H, J ¼ 6:1,
CH2Ph), 4.67 (d, 2H, J ¼ 6:1, CH2–Ph), 4.64 (s, 2H,
CH2Br), 4.58 (d, 2 H, J ¼ 6:5, CH2Ph), 4.44 (d, 2H,
J ¼ 6:5, CH2–Ph). 13C NMR (125.7 MHz, DMSO-d6
minor tautomer in italic): 164.54 (C-6), 164.50 (C-6)
159.39 (C-4), 159.21 (C-4), 156.78 (C-2), 156.54 (C-2),
139.13 (Ph), 139.04 (Ph), 128.48–126.90 (10C, Ph),
120.10 (C-5), 119.03 (C-5), 44.50 (CH2–Ph), 44.37
(CH2–Ph), 44.16 (CH2–Ph) 44.06 (CH2–Ph), 34.62
1
(100, Bn). H NMR (500 MHz, DMSO-d6, minor tau-
tomer in italic): 9.43 (t, 1H, J ¼ 6:0, NH), 9.21 (t, 1H,
J ¼ 6:1, NH), 8.86 (t, 1H, J ¼ 6:4, NH), 8.66 (t, 1H,
J ¼ 6:5, NH), 7.40–7.10 (m, 10H, Ph), 7.40–7.10 (m,
10H, Ph), 4.70 (d, 2H, J ¼ 6:1, CH2–Ph), 4.68 (d, 2H,
J ¼ 6:0, CH2–Ph), 4.61(d, 2H, J ¼ 6:5, CH2–Ph), 4.51
(s, 2H, CH2CN), 4.43 (d, 2H, J ¼ 6:4, CH2–Ph), 4.42 (s,
2H, CH2CN). 13C NMR (125.7 MHz, DMSO-d6, minor
tautomer in italic): 161.07 (C-4), 160.81 (C-4), 158.90
(C-6), 158.83 (C-6), 156.54 (C-2), 156.43 (C-2), 139.51
(Ph), 139.14 (Ph), 139.04 (Ph), 138.97 (Ph), 128.46
(4C, Ph), 128.44 (2C, Ph), 128.41 (2C, Ph), 128.10 (2C,
Ph), 127.71 (2C, Ph), 127.54 (2C, Ph), 127.41 (2C, Ph),
127.18 (Ph), 127.06 (Ph), 126.99 (Ph), 126.954 (Ph),
119.62 (CN), 118.86 (CN), 116.98 (C-5), 116.92 (C-5),
44.65 (CH2–Ph), 44.52 (CH2–Ph), 44.43 (CH2–Ph),
44.27 (CH2–Ph), 29.20 (CH2–CN), 28.53 (CH2–CN). IR
(CHCl3): 3430, 3020, 2260, 2191, 1594, 1574, 1553, 1514,
1304, 1196 cmꢀ1. UV/vis (MeOH): 363 (e ¼ 16400), 269
(e ¼ 7800). Anal. Calcd for C20H18N6O2: C, 64.16; H,
4.85; N, 22.45. Found: C, 63.96; H, 4.92; N, 22.30.
(CH2Br),
34.05
(CH2Br).
Anal.
Calcd
for
C19H18BrN5O2: C, 53.28; H, 4.24; Br, 18.66; N, 16.35.
Found: C, 53.19; H, 4.41; Br, 18.85; N, 16.12.
4.2.9.
N2,N4-Dibenzyl-6-dibromomethyl-5-nitropyrim-
idine-2,4-diamine (13). Compound 10 (7.0 g, 20 mmol)
and sodium acetate (3.3 g, 40 mmol) were added to a
solution of bromine (6.4 g, 40 mmol) in acetic acid
(200 mL) and the reaction mixture was heated to 65 °C
for 20 min. The solvent was evaporated to a small vol-
ume, taken into ethyl acetate and washed with water and
brine. Chromatography on a silica gel column (petro-
leum ether–ethyl acetate 9:1) followed by crystallization
(petroleum ether–ethyl acetate 19:1) afforded compound
13 (6.6 g, 65%, yellow crystals, mp 130–131 °C) as a
mixture of 2 tautomers (4:1). MS (EI) m=z (rel. inten-
sity): 509 (1.5), 507 (3), 505 (1.5) [M]; 492 (1.5), 490 (3),
488 (1.5) [M)OH]; 229 (1.5), 227 (1.5) [M)Br]; 421 (2),
410 (2) [M)Br–O]; 403 (1), 401 (2), 399 (1) [M)BnNH];
106 (96) [BnNH]; 105 (68) [BnN]; 91 (100) [Bn]. 1H
NMR (500 MHz, DMSO-d6, minor tautomer in italic):
9.35 (t, 1H, J ¼ 6:0, NH), 9.12 (t, 1H, J ¼ 6:0, NH),
9.03 (t, 1H, J ¼ 6:5, NH), 8.71 (t, 1H, J ¼ 6:5, NH),
7.51 (s, 1H, CHBr2), 7.50 (s, 1H, CHBr2), 7.40–7.10 (m,
10H, Ph), 7.40–7.10 (m, 10H, Ph) 4.69 (d, 2H, J ¼ 6:0,
CH2–Ph), 4.68 (d, 2H, J ¼ 6:0, CH2–Ph), 4.62 (d, 2H,
J ¼ 6:5, CH2-Ph), 4.43 (d, 2H, J ¼ 6:5, CH2–Ph). 13C
NMR (125.7 MHz, DMSO-d6, minor tautomer in italic):
163.34 (C-6), 163.01 (C-6), 159.57 (C-4), 159.46 (C-4),
156.19 (C-2), 156.11 (C-2), 139.45 (Ph), 139.01 (Ph),
138.87 (Ph), 138.81 (Ph), 128.43 (4C, Ph), 128.27 (4C,
Ph), 127.75 (4C, Ph), 127.63 (2C, Ph), 127.40 (2C, Ph),
127.19 (Ph), 127.06 (Ph), 127.015 (Ph), 126.945 (Ph),
117.25 (C-5), 116.53 (C-5), 44.58 (2C, CH2–Ph), 44.28
(2C, CH2Ph), 41.91 (CHBr2), 41.17 (CHBr2). IR
(CHCl3): 3430, 3375, 1593, 1584, 1569, 1546, 1508,
1278 cmꢀ1. UV/vis (MeOH): 379 (e ¼ 12800), 288
(e ¼ 7200). Anal. Calcd for C19H17Br2N5O2: C, 45.00;
H, 3.38; Br, 31.51; N, 13.18. Found: C, 45.06; H, 3.52;
Br, 31.65; N, 13.38.
4.2.11. 2-[2,6-Bis(benzylamino)-5-nitropyrimidin-4-yl]-3-
phenylpropionitrile (15). Compound 14 (3.7 g, 10 mmol)
was dissolved in dimethyl sulfoxide (30 mL), tributyl-
amine (5 mL) and then benzyl bromide (2.6 g, 15 mmol)
were added, and the reaction mixture was stirred over-
night. Methanol (5 mL) was added, the reaction mixture
was taken into ethyl acetate and washed with sodium
hydrogencarbonate and brine. Chromatography on a
silica gel column (chloroform) followed by crystalliza-
tion (petroleum ether–ethyl acetate 3:1) afforded com-
pound 15 (3.3 g, 71%, yellow crystals, mp 187–188 °C) as
a mixture of 2 tautomers (4.3:1). MS (EI) m=z (rel.
intensity): 464 (32, M), 447 (11, M)OH), 358 (6,
M)BnNH2), 341 (12, M)BnNH2-OH), 253 (11,
M)2BnNH2+H), 106 (8, BnNH2), 91 (100, Bn). 1H
NMR (500 MHz, DMSO-d6, minor tautomer in italic):
9.41 (t, 1H, J ¼ 6:0, NH), 9.15 (t, 1H, J ¼ 6:0, NH),
8.92 (t, 1H, J ¼ 6:4, NH), 8.68 (t, 1H, J ¼ 6:4, NH),
7.40–7.10 (m, 15H, Ph), 7.40–7.10 (m, 15H, Ph), 5.10
(dd, 1H, J ¼ 10:6, 4.4, CHCN), 5.09 (dd, 1H, J ¼ 10:3,
4.5, CHCN), 4.69 (d, 2H, J ¼ 6:0, N–CH2–Ph), 4.68 (d,
2H, J ¼ 6:0, N–CH2–Ph), 4.56 (d, 2H, J ¼ 6:4, N–CH2–
Ph), 4.45 (d, 2H, J ¼ 6:4, N–CH2–Ph), 3.33 (dd, 1H,
J ¼ 13:4, 4.4, C–CH2–Ph), 3.26 (dd, 1H, J ¼ 13:4, 4.5,
CCH2–Ph), 2.99 (dd, 1H, J ¼ 13:4, 10.6, C–CH2–Ph),
2.95 (dd, 1H, J ¼ 13:4, 10.3, C–CH2–Ph). 13C NMR
(125.7 MHz, DMSO-d6, minor tautomer in italic): 163.37
(C-4), 163.23 (C-4), 159.02 (C-6), 158.96 (C-6), 156.66
(C-2), 156.57 (C-2), 139.88 (Ph), 139.39 (Ph), 139.28
(Ph), 139.21 (Ph), 137.57 (Ph), 137.47 (Ph), 129.49 (2C,
Ph), 128.99 (2C, Ph), 128.73 (2C, Ph), 128.72 (2C, Ph),
4.2.10. [2,6-Bis(benzylamino)-5-nitropyrimidin-4-yl]ace-
tonitrile (14). To a suspension of potassium cyanide
(3.2 g, 50 mmol) in dimethyl sulfoxide (150 mL) cooled
to 0 °C compound 12 (5.1 g, 10 mmol) was added por-
tionwise during 15 min and the reaction mixture was left

M. Otmar et al. / Bioorg. Med. Chem. 12 (2004) 3187–3195
3193
127.88 (2C, Ph), 127.71 (2C, Ph), 119.82 (C-5), 119.79
(C-5), 119.39 (CN), 119.37 (CN), 44.60 (N–CH2–Ph),
44.48 (N–CH2–Ph), 44.43 (N–CH2–Ph), 44.32 (N–CH2–
Ph), 41.16 (C–CN), 40.89 (C–CN), 38.03 (C–CH2–Ph),
37.96 (C–CH2–Ph). IR (CHCl3): 3430, 3370, 3022, 2250,
1593, 1583, 1571, 1550, 1509 cmꢀ1. UV/vis (MeOH): 369
(e ¼ 8700), 268 (e ¼ 4300). Anal. Calcd for C27H24N6O2:
C, 69.81; H, 5.21; N, 18.09. Found: C, 70.04; H, 5.35; N,
17.88.
CH2Ph). 13C NMR (125.7 MHz, DMSO-d6): 151.95 (C-
2), 151.29 (C-4), 139.09 (Ph), 138.41 (Ph), 134.43 (C-7a),
128.99 (C-6), 128.63 (2C, Ph), 128.55 (2C, Ph), 127.89
(2C, Ph), 127.36 (3C, Ph), 127.16, 108.46 (C-4a), 95.66
(C-7), 44.50 (NCH2), 43.66 (NCH2). IR (KBr): 3254,
3110, 1649, 1619, 1570, 1478, 1391, 1352, 1169, 766, 739,
578 cmꢀ1
.
UV (MeOH): 293 (e ¼ 15; 600), 239
(e ¼ 23; 800). Anal. Calcd for C20H20ClN5 (365.9): C,
65.66; H, 5.51; Cl, 9.69; N, 19.14. Found: C, 65.34; H,
5.60; Cl, 9.70; N, 19.19.
4.2.12. 2-Benzyl-2-[2,6-bis(dibenzylamino)-5-nitropyrim-
idin-4-yl]-3-phenylpropionitrile (16). Compound 15
(930 mg, 2 mmol) was dissolved in dimethyl sulfoxide
(6 mL), sodium hydride (60% dispersion in mineral oil,
100 mg, 2.5 mmol) and then benzyl bromide (430 mg,
2.5 mmol) was added and the reaction mixture was
stirred overnight. Methanol (2 mL) was added, the
reaction mixture was taken into ethyl acetate and
washed with sodium hydrogen carbonate and brine.
Chromatography on a silica gel column (petroleum
ether–ethyl acetate 14:1) afforded compound 16 as yel-
low amorphous solid (380 mg, 26%; When 3 equiv of
BnBr and NaH were used the yield amounted to 1.2 g,
82%). MS (EI) m=z (rel. intensity): 734 (1.1, M), 717 (0.5,
M)OH), 704 (1.4, M)NO), 688 (1.1, M)NO2), 643 (8,
M)Bn), 609 (7), 432 (4), 427 (6), 91 (100, Bn). MS
4.2.14. 7,N2,N4-Tribenzyl-5H-pyrrolo[3,2-d]pyrimidine-
2,4-diamine (18) hydrochloride. Compound 15 (930 mg,
2 mmol) in methanol (100 mL) acidified with methanolic
hydrogen chloride (1 M, 5 mL) was hydrogenated under
slight overpressure in the presence of Pd/C catalyst
(10 wt %, 40 mg) overnight. The catalyst was filtered off
through a Celite pad and the filtrate evaporated.
Chromatography on a silica gel column (chloroform–
methanol, 98:2) followed by crystallization (ethyl ace-
tate–methanol, 9:1) afforded hydrochloride of com-
pound 18 (565 mg, 62%), mp 236–237 °C. MS (EI) m=z
(rel. intensity): 419 (100, M), 328 (31, M)Bn), 314 (30,
M)BnNH2+H), 91 (92, Bn). 1H NMR (500 MHz,
DMSO-d6): 12.44 (s, 1H, NH), 11.68 (s, 1H, NH), 9.38
(t, 1H, J ¼ 6:0, NH), 7.83 (t, 1H, J ¼ 6:4), 7.40–7.10 (m,
15H, Ph), 7.27 (s, 1H, H-6), 4.70 (d, 2H, J ¼ 6:0,
CH2–Ph), 4.61 (d, 2H, J ¼ 6:4, CH2–Ph), 3.93 (s, 2H,
CH2–Ph). 13C NMR (125.7 MHz, DMSO-d6): 152.12
(C-4), 151.27 (C-2), 141.99 (Ph), 140.69 (Ph), 139.12
(Ph), 138.40 (Ph), 132.63 (C-7a), 128.60 (2C, Ph), 128.54
(4C, Ph), 128.50 (2C, Ph), 128.43 (2C, Ph), 128.32 (2C,
Ph), 127.85 (2C, Ph), 127.75 (C-6), 127.39 (2C, Ph),
127.33 (Ph), 127.15 (Ph), 126.15 (Ph), 125.74 (Ph),
108.36 (C-4a), 109.01 (C-7), 44.73 (N–C2–Ph), 44.53 (N–
CH2–Ph), 29.07 (C–CH2–Ph). IR (KBr): 3248, 3150,
3106, 3027, 1648, 1616, 1569, 1469, 1454, 1394, 733,
1
(FAB) m=z (rel. intensity): 735 (10, M+H). H NMR
(500 MHz, DMSO-d6): 7.35–7.05 (m, 25H, Ph), 4.59 s
(2H, CH2–Ph), 4.53 (s, 2H, CH2–Ph), 4.48 (s, 4H, CH2–
Ph), 3.61 (d, 2H, J ¼ 13:7, CH2-Ph), 3.23 (d, 2H,
J ¼ 13:7, CH2–Ph). 13C NMR (125.7 MHz, DMSO-d6):
158.84 (C-6), 158.55 (C-4), 156.16 (C-2), 137.92 (Ph),
137.71 (Ph), 136.89 (2C, Ph), 135.51 (2C, Ph), 130.81
(2C, Ph), 128.66 (2C, Ph), 128.62 (4C, Ph), 128.50 (2C,
Ph), 128.25 (4C, Ph), 127.88 (2C, Ph), 127.46 (2C, Ph),
127.40 (2C, Ph), 127.39 (4C, Ph), 127.23 (4C, Ph), 127.10
(2C, Ph), 125.14 (C-5), 119.59 (CN), 53.37 (4C, CH2–
Ph), 49.93 (C–CN), 42.84 (CH2–Ph), 39.53 (CH2–Ph).
IR (CHCl3): 3032, 3020, 2240, 1546, 1523, 1496, 1454,
1328, 1250, 1029, 971 cmꢀ1. UV/vis (MeOH): 302 (9200),
245 (23,800). Anal. Calcd for C48H42N6O2: C, 78.45; H,
7.76; N, 11.44. Found: C, 78.26; H, 5.98; N, 11.23.
696 cmꢀ1
.
UV (MeOH): 296 (e ¼ 19; 000), 242
(e ¼ 23; 700). Anal. Calcd for C27H26ClN5: C, 71.12; H,
5.75; Cl, 7.77; N, 15.36. Found: C, 70.93; H, 5.85; Cl,
8.03; N, 15.19.
4.2.15. 7,7,N2,N2,N4,N4-Hexabenzyl-7H-pyrrolo[3,2-d]-
pyrimidine-2,4,6-triamine (19). Compound 16 (1.5 g,
2 mmol) in methanol (100 mL) acidified with methanolic
hydrogen chloride (1 M, 5 mL) was hydrogenated under
slight overpressure in the presence of Pd/C catalyst
(10 wt %, 40 mg) overnight. The catalyst was filtered off
through a Celite pad and the filtrate evaporated.
Chromatography on a silica gel column (chloroform)
afforded compound 19 (780 mg, 55%) as yellowish foam.
MS (EI) m=z (rel. intensity):704 (58, M), 613 (49,
M)Bn), 523 (6, M)2Bn+H), 521 (6, M)2Bn–H), 431
4.2.13. N2,N4-Dibenzyl-5H-pyrrolo[3,2-d]pyrimidine-2,4-
diamine (17) hydrochloride. Compound 14 (750 mg,
2 mmol) in methanol (100 mL) acidified with methanolic
hydrogen chloride (1 M, 5 mL) was hydrogenated under
slight overpressure in the presence of Pd/C catalyst
(10 wt %, 40 mg) overnight. The catalyst was filtered off
through a Celite pad and the filtrate evaporated.
Chromatography on a silica gel column (chloroform–
methanol, 97:3) followed by crystallization (ethyl ace-
tate–methanol, 6:1) afforded hydrochloride of com-
pound 17 (380 mg, 52%) mp 206–208 °C. MS (EI) m=z
(rel. intensity): 329, 238 (M)Bn), 224 (M)BnNH+H),
106 (34, BnNH), 91 (100, Bn). MS (FAB) m=z (rel.
intensity): 330 (100, M+H). 1H NMR (500 MHz,
DMSO-d6): 12.41 (br s, 1H, NH), 12.14 (br s, 1H, NH),
9.66 (br s, 1H, NH), 8.24 (br s, 1H, NH), 7.28 (m, 10H,
Ph), 7.49 (t, 1H, J ¼ 2:8, 3.1, H-6), 6.21 (t, 1H, J ¼ 2:8,
2.1, H-7), 4.70 (d, 2H, J ¼ 5:9, CH2Ph), 4.58 (d, J ¼ 6:1,
1
(100, M)3Bn), 338 (5, M)4Bn)2H), 91 (74, Bn). H
NMR (500 MHz, DMSO-d6): 8.02–7.52 (m, 10H, Ph),
7.35–7.05 (m, 20H, Ph), 6.74 (s, 2H, NH2), 4.81 (s, 4H,
N–CH2–Ph), 4.50 (s, 4H, N–CH2–Ph), 3.20 (s, 4H, C–
CH2–Ph). 13C NMR (125.7 MHz, DMSO-d6): 168.72 (C-
7a), 165.58 (C-6), 157.01 (C-4), 150.84 (C-2), 140.11
(Ph), 139.15 (Ph), 136.82 (4C, Ph), 129.77–127.42
(Ph, 24C), 124.45 (C-4a), 59.53 (C-7), 49.93 (4C,

3194
M. Otmar et al. / Bioorg. Med. Chem. 12 (2004) 3187–3195
N–CH2–Ph), 41.58 (2C, C-CH2–Ph). IR (CHCl3): 3401,
3011, 2916, 1620, 1587, 1566, 1495, 1473, 1452, 1416,
1338, 1264, 972, 700 cmꢀ1. UV/vis (MeOH): 354 (e ¼
5800), 297 (e ¼ 16; 250), 231 (e ¼ 21; 340). Anal. Calcd
for C48H44N6: C, 81.79; H, 6.29; N, 11.92. Found: C,
81.53; H, 6.42; N, 11.76.
acetate 8:1) afforded compound 22 (3.0 g, 73%) as white
crystals, mp 178–180 °C. MS (EI) m=z (rel. intensity):
413 (2), 411 (4), 409 (2, M), 367 (54), 365 (100), 363 (52,
M)NO2), 325 (17), 323 (24), 321 (12, M)NO2–Ac), 287
(14), 285 (15, M)Br), 244 (14), 242 (14, M)Br-Ac), 220
(16), 218 (18), 167 (24), 122 (32), 81 (35), 79 (33), 68 (48).
1H NMR (500 MHz, DMSO-d6): 11.42 (s, 1H, NH),
11.18 (s, 1H, NH), 7.30 (s, 1H, CHBr2), 2.32 (s, 3H, CH3
CO), 2.14 (s, 3H, CH3 CO). 13C NMR (125.7 MHz,
DMSO-d6): 170.27 (CO), 169.97 (CO), 159.76 (C-6),
157.25 (C-4), 152.76 (C-2), 127.31 (C-5), 36.93 (CHBr2),
25.47 (C3 CO), 23.56 (CH3 CO). IR (KBr): 3447, 3291,
3198, 1679, 1592, 1543, 1293, 1281 cmꢀ1. UV/vis
(MeOH): 356 (e ¼ 9780), 264 (e ¼ 9850), 229
(e ¼ 21; 450). Anal. Calcd for C9H9Br2N5O4: C, 26.30;
H, 2.21; Br, 38.88; N, 17.04. Found: C, 26.02; H, 2.43;
Br, 39.12; N, 16.84.
4.2.16. N-(4-Acetylamino-6-methyl-5-nitropyrimidin-2-
yl)acetamide (20). Compound 1 (1.7 g, 10 mmol) in a
mixture of acetic anhydride and acetic acid (1:1, 40 mL)
was heated to reflux for 3 h; then the volatiles were
evaporated. Chromatography on a silica gel column
(chloroform–methanol, 98:2) followed by crystallization
(ethyl acetate) afforded compound 20 (2.0 g, 79%) as
white crystals, mp 160–161 °C. MS (EI) m=z (rel. inten-
sity): 253 (4, M), 211 (22, M)Ac+H), 207 (100,
M)NO2), 169 (29, M)2Ac+2H), 165 (44, M)NO2–
1
Ac+H), 152 (43, M)2Ac–Me), 43 (76, Ac). H NMR
(500 MHz, DMSO-d6): 11.13 (s, 1H, NH), 10.90 (s, 1H,
NH), 2.52 (s, 3H, Me), 2.22 (s, 3H, CH3 CO), 2.11 (s,
3H, CH3CO). 13C NMR (125.7 MHz, DMSO-d6):
169.91 (CO), 169.49 (CO), 163.72 (C-6), 156.40 (C-4),
151.43 (C-2), 132.67 (C-5), 25.08 (CH3CO), 23.51
(CH3CO), 22.41 (Me). IR (CHCl3): 3389, 3260, 3183,
3020, 1731, 1714, 1686, 1564, 1373, 1304 cmꢀ1. UV
(MeOH): 275 (sh, e ¼ 8100), 242 (e ¼ 22; 260). Anal.
Calcd for C9H11N5O4: C, 42.69; H, 4.38; N, 27.66.
Found: C, 42.66; H, 4.55; N, 27.94.
4.2.19. N-(4-Benzoylamino-6-dibromomethyl-5-nitropyr-
imidin-2-yl)benzamide (23). Compound 21 (2.3 g,
6 mmol) and sodium acetate (980 mg, 12 mmol) were
added to a solution of bromine (1.9 g, 12 mmol) in acetic
acid (60 mL) and the reaction mixture was heated to
65 °C for 20 min. The solvent was evaporated to a small
volume, taken into ethyl acetate and washed with water
and brine. Chromatography on a silica gel column
(chloroform) followed by crystallization (diethylether–
ethyl acetate 9:1) afforded compound 23 (2.6 g, 81%) as
white crystals, mp 115–117 °C. MS (EI) m/z (rel. inten-
sity): 491 (52), 489 (92), 487 (61, M)NO2), 411 (42), 409
1
4.2.17. N-(4-Benzoylamino-6-methyl-5-nitropyrimidin-2-
yl)benzamide (21). Compound 1 (1.7 g, 10 mmol) was
melted with benzoic anhydride (9.0 g, 40 mmol) at
100 °C for 3 h. The reaction mixture was taken into ethyl
acetate and washed with sodium hydrogen carbonate
and brine. Chromatography on a silica gel column
(chloroform) followed by crystallization (ethyl acetate)
afforded compound 21 (3.2 g, 85%) as white crystals, mp
131–132 °C.MS (EI) m=z (rel. intensity): 377 (0.2, M),
361 (0.3), 348 (4, M)NO+H), 331 (42, M)NO2), 105
(43, M)NO2–Br), 226 (100, M)NO2–2Br–Bz+2H). H
NMR (500 MHz, DMSO-d6): 12.07 (s, 1H, NH), 11.78
(s, 1H, NH), 8.02–7.52 (m, 10H, Ph), 7.38 (s, 1H,
CHBr2). 13C NMR (125.7 MHz, DMSO-d6): 167.15
(CO), 166.43 (CO), 159.92 (C-6), 158.34 (C-4), 154.06
(C-2), 133.85 (Ph), 133.32 (Ph), 132.73 (Ph), 132.22 (Ph),
128.85 (2C, Ph), 128.75 (2C, Ph), 128.75 (C-5), 128.65
(4C, Ph), 36.84 (CHBr2). IR (CHCl3): 3470, 3417, 1711,
1571, 1545, 1480, 1390, 1349, 1288, 1004, 844, 707,
597 cmꢀ1. UV (MeOH): 260 (e ¼ 25; 000). Anal. Calcd
for C19H13Br2N5O4: C, 42.64; H, 2.45; Br, 29.86; N,
13.09. Found: C, 42.41; H, 2.56; Br, 29.62; N, 12.92.
1
(100, Bz), 77 (47, Ph). H NMR (500 MHz, DMSO-d6):
11.76 (s, 1H, NH), 11.46 (s, 1H, NH), 8.02–7.52 (m,
10H, Ph), 2.63 (s, 3H, Me). 13C NMR (125.7 MHz,
DMSO-d6): 166.84 (CO), 165.94 (CO), 163.84 (C-6),
157.29 (C-4), 152.72 (C-2), 134.13 (C-5), 133.85 (Ph),
133.32 (Ph), 132.73 (Ph), 132.22 (Ph), 128.85 (2C, Ph),
128.75 (2C, Ph), 128.65 (4C, Ph), 22.40 (Me). IR
(CHCl3): 3426, 1713, 1575, 1482, 1432, 1356, 1232, 842,
706 cmꢀ1. UV (MeOH): 260 (e ¼ 37; 000). Anal. Calcd
for C19H15N5O4: C, 60.48; H, 4.01; N, 18.56. Found: C,
60.22; H, 4.09; N, 18.43.
4.2.20.
N-[6-(1,1-Dibenzyl-1-cyanomethyl)-2-benzoyl-
amino-5-nitropyrimidin-4-yl]benzamide (24). To a sus-
pension of potassium cyanide (1.0 g, 16 mmol) in di-
methyl sulfoxide (40 mL) cooled to 0 °C compound 23
(1.7 g, 3.2 mmol) was added portionwise within 15 min
and the reaction mixture was left at room temperature
for additional 20 min. Benzyl bromide (3.4 g, 20 mmol)
was added and the reaction mixture was stirred over
night. The reaction mixture was taken into ethyl acetate
and washed with brine. Chromatography on a silica gel
column (chloroform) followed by crystallization
(petroleum ether–ethyl acetate 3:1) afforded compound
24 (150 mg, 9%) as white crystals, mp 264–265 °C. MS
(FAB) m=z (rel. intensity): 583 (24, M+H), 355 (100).
MS (EI) m=z (rel. intensity): 446 (21, M)Bn–NO2+H),
the molecular peak does not manifest. 1H NMR
(500 MHz, DMSO-d6): 11.70 (s, 1H, NH), 11.61 (s, 1H,
NH), 8.02–7.52 (m, 10H, Ph), 7.35–7.30 (m, 5H, Ph),
4.2.18. N-(4-Acetylamino-6-dibromomethyl-5-nitropyrim-
idin-2-yl)acetamide (22). Compound 20 (2.5 g, 10 mmol)
and sodium acetate (1.6 g, 20 mmol) were added to a
solution of bromine (3.2 g, 20 mmol) in acetic acid
(100 mL)and the reaction mixture was heated to 65 °C
for 20 min. The solvent was evaporated to a small vol-
ume, taken into ethyl acetate and washed with water and
brine. Chromatography on a silica gel column (chloro-
form) followed by crystallization (diethyl ether–ethyl

M. Otmar et al. / Bioorg. Med. Chem. 12 (2004) 3187–3195
3195
4. Norman, M. H.; Chen, N.; Chen, Z.; Fotsch, C.; Hale, C.;
Han, N.; Hurt, R.; Jenkins, T.; Kincaid, J.; Liu, L.; Lu, Y.;
Moreno, O.; Santora, V. J.; Sonnenberg, J. D.; Karbon,
W. J. Med. Chem. 2000, 43, 4288–4312.
3.92 (d, 2H, J ¼ 13:4, CH2–Ph), 3.23 (d, 2H, J ¼ 13:4,
CH2–Ph). 13C NMR (125.7 MHz, DMSO-d6): 166.87
(CO), 166.44 (CO), 158.40 (C-4), 156.51 (C-6), 154.85
(C-2), 135.51 (2C, Ph), 132.08 (C-5), 133.85 (Ph), 133.32
(Ph), 132.73 (Ph), 132.22 (Ph), 130.81 (4C, Ph), 128.85
(2C, Ph), 128.75 (2C, Ph), 128.65 (4C, Ph), 128.25 (4C,
Ph), 127.46 (2C, Ph), 117.70 (CN), 51.06 (C–CN), 43.22
(CH2–Ph), 39.86 (C2–Ph). IR (KBr): 3385, 3301, 1729,
1685, 1569, 1522, 1455, 1234, 701 cmꢀ1. UV (MeOH):
260 (e ¼ 30400). Anal. Calcd for C34H26N6O4: C, 70.09;
H, 4.50; N, 14.42. Found: C, 69.83; H, 4.20; N, 14.35.
ꢁ
ꢀ
5. Capek, P.; Otmar, M.; Masojıdkova, M.; Votruba, I.;
Holy, A. Collect. Czech. Chem. Commun. 2003, 68, 779–
ꢀ
ꢀ
791.
6. Montgomery, J. A.; Niwas, S.; Rose, J. D.; Secrist, J. A.,
III; Babu, S.; Bugg, C. E.; Erion, M. D.; Guida, W. C.;
Ealick, S. E. J. Med. Chem. 1993, 36, 55–69.
7. Wada, J.; Yagihashi, A.; Terasawa, K.; Miyao, N.; Hirata,
K.; Cicciarelli, J.; Iwaki, Y. Artif. Organs 1996, 20, 849–
852.
8. This work was presented in part: Otmar, M.;
ꢀ
ꢀ
ꢀ
ꢁꢀ
Masojıdkova, M; Votruba, I.; Holy, A. In Tocık, Z.,
Hocek, M., Eds.; Collection Symposium Series, Vol. 5, pp
38–39. Institute of Organic Chemistry and Biochemistry,
Academy of Sciences of the Czech Republic, Prague 2002.
Acknowledgements
ꢀ
ꢀ
ꢁꢁꢀ
9. Otmar, M.; Masojıdkova, M.; Budesınsky, M.; Holy, A.
ꢀ
ꢀ
This study was made as a part of research project
# Z4055905 of the Institute. Financial support was
provided by the COST Programme # D.13.20 of the
Ministry of Education of the Czech Republic. The
authors wish to thank the staff of the Laboratories of
Mass Spectroscopy (Dr. K. Ubik, Head), Elemental
Tetrahedron 1998, 54, 2931–2940.
ꢀ
10. Otmar, M.; Masojıdkova, M.; Holy, A. Tetrahedron 1997,
ꢀ
ꢀ
53, 391–400.
11. Cupps, T. L.; Wise, D. S., Jr.; Townsend, L. B. J. Org.
Chem. 1986, 51, 1058–1064.
12. Lim, M.-I.; Ren, W.-Y.; Otter, B. A.; Klein, R. S. J. Org.
Chem 1983, 48, 780–788.
13. Gabriel, S.; Colman, J. Chem. Ber. 1901, 34, 1234–1257.
14. Robins, R. K.; Dille, K. J.; Willits, C. H.; Christensen,
B. E. J. Am. Chem. Soc. 1953, 75, 263–266.
ꢀ
Analysis (Dr. L. Valkova, Head) and IR Spectroscopy
(Dr. P. Fiedler, Head). An excellent technical assistance
ꢀ
ꢁ
ꢀ
of Ms. Y. Cerna is gratefully acknowledged.
15. Cupps, T. L.; Wise, D. S.; Townsend, L. B. J. Org. Chem.
1983, 48, 1060–1064.
16. Thompson, M. D.; Cupps, T. L.; Wise, D. S.; Wotring,
L. L.; Townsend, L. B. J. Med. Chem. 1997, 40, 766–770.
17. Marshall, J. R.; Walker, J. J. Chem. Soc. 1951, 1004–1017.
18. Evans, G. B.; Furneaux, R. H.; Hutchinson, T. L.; Kezar,
H. S.; Morris, P. E., Jr.; Schramm, V. L.; Tyler, P. C.
J. Org. Chem. 2001, 66, 5723–5730.
References and notes
1. Imai, K. Chem. Pharm. Bull. 1964, 12, 1030–1042.
2. Modnikova, G. A.; Titkova, R. M.; Glushkov, R. G.;
Sokolova, A. S.; Silin, V. A.; Chernov, V. A. Khim.-Farm.
Zh. 1988, 22, 185–191.
ꢀ
ꢁꢀ
19. Hocek, M.; Holy, A.; Votruba, I.; Dvorakova, H. J. Med.
ꢀ
Chem. 2000, 43, 1817–1825.
20. Carmichael, J.; DeGraff, W. G.; Gazdar, A. F.; Minna,
J. D.; Mitchell, J. B. Cancer Res. 1987, 47, 936–942.
3. McGuire, J. J.; Bergoltz, V. V.; Heitzman, K. J.; Haile, W. H.;
Russell, C. A.; Bolanowska, W. E.; Kotake, Y.; Haneda,
T.; Nomura, H. Cancer Res. 1994, 54, 2673–2679.