Novel adamantylated pyrimidines and their preliminary biological evaluations

Article abstract of DOI:10.1016/j.farmac.2004.07.010

The synthesis of adamantylated pyrimidines was based on the reaction of 3-(adamantan-1-yl)-3-oxopropionic acid ethyl ester with urea, thiourea, guanidine as well as acetamidine, respectively. Then the compounds obtained were converted into respective brom

Full text of DOI:10.1016/j.farmac.2004.07.010

IL FARMACO 59 (2004) 929–937
http://france.elsevier.com/direct/FARMAC/
Novel adamantylated pyrimidines and their preliminary
biological evaluations
Barbara Orzeszko a, Zygmunt Kazimierczuk b,c, Jan Krzysztof Maurin d,e
,
Agnieszka Ewa Laudy f, Bohdan Jerzy Staros´ciak f, Juhani Vilpo g, Leena Vilpo g,
Jan Balzarini h, Andrzej Orzeszko b,i,
*
a Faculty of Chemistry, Warsaw University of Technology, Koszykowa 75, 00-662 Warsaw, Poland
b Agricultural University, Institute of Chemistry, Nowoursynowska 159c, 02-787 Warsaw, Poland
c Laboratory of Experimental Pharmacology, Polish Academy of Science Medical Research Center, Pawin´skiego 5, 02-106 Warsaw, Poland
d
´
Institute of Atomic Energy, 05-400 Otwock-Swierk, Poland
e National Institute of Public Health, Chełmska 30/34, 00-725 Warsaw, Poland
f Medical University, Department of Pharmaceutical Microbiology, Oczki 3, 02-007 Warsaw, Poland
g Department of Clinical Chemistry, Tampere University, Tampere University Hospital (Laboratory Center)
and Helsinki University Central Hospital (HUSLAB), Finland
h Rega Institute for Medical Research, Katholieke Universiteit Leuven, Belgium
i Military University of Technology, Kaliskiego, 00-908 Warsaw, Poland
Received 15 May 2004; accepted 22 July 2004
Available online 16 September 2004
Abstract
The synthesis of adamantylated pyrimidines was based on the reaction of 3-(adamantan-1-yl)-3-oxopropionic acid ethyl ester with urea,
thiourea, guanidine as well as acetamidine, respectively. Then the compounds obtained were converted into respective bromo-, thio- and
S-alkyl derivatives. The molecular structures for some compounds were studied by X-ray methods. The significant anticancer and antimicro-
bial properties of [2-(6-adamantan-1-yl-2-methylpyrimidin-4-ylthio)ethyl]dimethylamine were found.
© 2004 Elsevier SAS. All rights reserved.
Keywords: 6-(Adamantan-1-yl)pyrimidines; Anti-HIV; Anticancer and antimicrobial properties
1. Introduction
Some aminoadamantane derivatives show activity against
HIV-1 and HIV-2 [3].Amantadine is used also in treatment of
Parkinson disease [4] and memantine is considered as a
promising drug for the treatment of certain dementia, parti-
cularly Alzheimer’s disease [5]. N-(Adamantan-1-yl)-
maleimide exhibited selected inhibitory activity against dif-
ferent cancer cell lines [6,7]. We have reported previously
that certain novel N-(adamantan-1-yl)phthalimides as well as
adamantylamino-pyrimidines and –pyridines, enhance
TNF-a [7–9]. The combination of an adamantyl moiety with
imides, also gives compounds of significant antimicrobial
properties [10].
The adamantyl moiety present in numerous agents and
drugs improves their lipophilicity due to aliphatic cage-like
structure. Such compounds might be much better taken up by
cells, and have enhanced blood–brain barrier penetration and
increased accumulation in lipids. Therefore adamantane de-
rivatives have received considerable attention because of
their diverse biological activity. Many years ago, 1-amino-
adamantane (amantadine) was shown to be an effective pro-
phylactic agent against type influenza A [1]. Even today
amantadine is tested in combination with other antiviral
agents for treatment hepatitis C [2].
The exocyclic aminoadamantylated pyrimidines were ob-
tained by the attack of an adamantyl cation generated from
1-adamantanol in boiling trifluoroacetic acid on the respec-
tive aminopyrimidine. In the same conditions barbituric acid
* Corresponding author. Agricultural University, Institute of Chemistry,
Nowoursynowska 159c, 02-787 Warsaw, Poland.
E-mail address: orzeszkoa@delta.sggw.waw.pl (A. Orzeszko).
0014-827X/$ - see front matter © 2004 Elsevier SAS. All rights reserved.
doi:10.1016/j.farmac.2004.07.010

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B. Orzeszko et al. / IL FARMACO 59 (2004) 929–937
and 2-thiobarbituric acid gave C-5 adamantylated products
[11,12]. Also 1,3-dimethyluracil and uridine were transfor-
med into their 5-substituted adamantyl derivatives under
such conditions. A ring closure procedure was used for the
synthesis of 5-(1-adamantyl)uracil and its 2-thio congener,
which were further transformed to respective a or b anomers
of 2′-deoxyuridines [13]. In contrast to 5-adamantylated py-
rimidines, their 6(4)-substituted analogues are practically
unknown. Till now only one 6-adamantylated pyrimidine,
namely 6-(adamantan-1-yl)-3-allyl-2-thiouracil was repor-
ted [14].
In this study we synthesized series of novel 6-adaman-
tylpyrimidines. Also the crystallographic parameters and the
preliminary biological evaluations on these novel modified
heterocyclic compounds were shown.
2. Experimental
Scheme 2. Synthesis of compounds 9–14.
2.1. Synthesis
theoretical values. Their purity was over 95% and was
controlled by TLC (SiO2) and 1H-NMR spectroscopy.
The syntheses were performed according to the Schemes
1 and 2. All chemicals used were analytical-grade commer-
cial products (Aldrich) and were used without any further
purification. The starting substrate 3-(adamantan-1-yl)-3-
oxopropionic acid ethyl ester was obtained according to the
procedure described previously [15]. Structures of novel
compounds were confirmed by H-NMR, UV, MS analysis
and in two cases by X-ray crystallographic methods. Ele-
mental analyses of new compounds were within 0.4% of
2.1.1. Condensation procedure
Reactions were conducted by the Traube method and were
similar to that given for 4-phenylisocytosine synthesis [16].
An equimolar amount of sodium (0.58 g, 25 mmol) was
dissolved in anhydrous ethanol (20 ml). Then the solution
was treated with 35 mmol of urea (for 1) or thiourea (for 2).
For the synthesis of compounds 3 and 4, hydrochloride salts
1
Scheme 1. Synthesis of compounds 1–8.

B. Orzeszko et al. / IL FARMACO 59 (2004) 929–937
931
of guanidine or acetamidine were used, respectively. In these
cases, the amount of sodium was 1.16 g (50 mmol). After
cooling, 12 g (12.5 mmol) of 3-(adamantan-1-yl)-3-
oxopropionic acid ethyl ester was added dropwise to the
mixture and refluxed for 12 h (for 1 48 h). Then alcohol was
evaporated and the residue dissolved in the least possible
volume of cold water. The solution was then carefully acidi-
fied with dilute hydrochloric acid in order to precipitate the
respective pyrimidine. The crude products were filtered off
and crystallized from ethanol.
282 (5900), (0,1 M KOH/MeOH, 1:1) 227 (7200), 283
(5100); m/z (EI) 326 (M+ 99), 325 (17), 324 (100), 246 (30),
135 (39), 79 (16%); dH (200 MHz, DMSO-d6) 1.6–2.19 (m,
15H, H-Ada), 9.50 (s, 1H, N(1)-H), 11.57 (s, 1H, N(3)-H).
2.1.2.2. 6-Adamantan-1-yl-2-amino-5-bromo-3H-pyrimidin-
4-one (7). Yield: 40% (0.3 g); m.p. 275 °C (decomp.); TLC,
chloroform–methanol, 6:1, Rf = 0.61; UV (H2O/MeOH, 1:1)
310 (3500), (0,1 M HCl/MeOH, 1:1) 228 (8200), 281 (6700),
(0,1 M KOH/MeOH, 1:1) 235 (6400), 290(5600); m/z (EI)
325 (M+, 71), 324 (19), 323 (73), 268 (18), 245 (45), 244
(100%); dH (200 MHz, DMSO-d6) 1.68–2.18 (m, 15H,
H-Ada), 6.83 (s, 2H, NH2).
2.1.1.1. 6-Adamantan-1-yl-1H-pyrimidine-2,4-dione (1).
Yield 24% (0.73 g); m.p. 325 °C (decomp.); TLC, chloro-
form–methanol, 6:1, Rf = 0.78; UV (H2O/MeOH, 1:1) 263
(6500), (0,1 M KOH/MeOH, 1:1) 222 (7800), 264 (6300);
m/z (EI) 246 (M+, 100%), 189 (11), 135 (17); dH (200 MHz,
DMSO-d6) 1.68–2.00 (m, 15H, H-Ada), 5.21 (s, 1H, H-5),
10.48 (s, 1H, N(3)-H), 10.91 (s, 1H, N(1)-H).
2.1.3. Thionation procedure
Compounds 6 and 8 were prepared similarly to the method
described previously [18,19]. A mixture of 2 or 4 (5 mmol),
Lawesson’s reagent 1.21 g (3 mmol) and toluene (25 ml) was
stirred and refluxed for 2 h. Compound 6 precipitated from
the reaction mixture, while compound 8 remained in the
solution. The crude products were purified by column chro-
matography and crystallization: 6 (SiO2, hexane–EtOAc, 3:2,
crystallized from EtOH) and 8 (chloroform–methanol, 15:1;
crystallized from MeOH).
2.1.1.2. 6-Adamantan-1-yl-2-thioxo-2,3-dihydro-1H-pyri-
midin-4-one (2). Yield 60% (1.96 g); m.p. 285 °C (decomp.);
TLC, chloroform–methanol, 6:1, Rf = 0.92; UV (H2O/
MeOH, 1:1) 275 (5200), (0,1 M KOH/MeOH, 1:1) 236
(10500), 259 (10500) 310 (6400). m/z (EI) 262 (M+, 100%);
dH (200 MHz, DMSO-d6) 1.67–2.00 (m, 15H, H-Ada), 5.54
(s, 1H, H-5), 11.75 (s, 1H, N(3)-H), 12.30 (s, 1H, N(1)-H).
2.1.3.1. 6-Adamantan-1-yl-1H-pyrimidine-2,4-dithione (6).
Yield: 41% (0.57 g); m.p. 300 °C (decomp.); TLC, chloro-
form–methanol, 20:1, Rf = 0.60; UV (H2O/MeOH, 1:1) 290
(9900), 348 (8100), (0,1 M KOH/MeOH, 1:1) 221 (9100),
273 (16600), 370 (2600); m/z (EI) 278 (M+, 80%). dH
(200 MHz, DMSO-d6) 1.69–2.02 (m, 15H, H-Ada), 6.29 (s,
1H, H-5), 12.36 (s, 1H, N(1)-H), 13.48 (s, 1H, N(3)-H).
2.1.1.3.
6-Adamantan-1-yl-2-amino-3H-pyrimidin-4-one
(3). Yield 60% (1.85 g); m.p. 340 °C (decomp.); TLC,
chloroform–methanol, 6:1, Rf = 0.52; UV (H2O/MeOH, 1:1)
262 (365), (0,1 M HCl/MeOH, 1:1) 259 (5800), (0,1 M
KOH/MeOH, 1:1) 231 (7100), 274 (6500); m/z (EI) 245 (M+,
100), 188 (17%); dH (200 MHz, DMSO-d6) 1.68–2.00 (m,
15H, H-Ada), 5.35 (s, 1H, H-5), 6.32 (s, 2H, NH2), 10.52 (s,
1H, NH).
2.1.3.2. 6-Adamantan-1-yl-2-methyl-3H-pyrimidine-4-thione
(8). Yield: 68% (0.89 g); m.p. 266 °C; TLC, chloroform–
methanol, 20:1, Rf = 0.64; UV (H2O/MeOH, 1:1) 283 (5100),
332 (6000), (0,1 M HCl/MeOH, 1:1) 320 (14100), (0,1 M
KOH/MeOH, 1:1) 229 (6300), 300 (9400); m/z (EI) 260 (M+,
74), 203 (19%); dH (200 MHz, DMSO-d6) 1.68-2.01 (m,
15H, H-Ada), 2.38 (s, 3H, CH3), 6.85 (s, 1H, H-5), 13.68
(s,1H, NH).
2.1.1.4. 6-Adamantan-1-yl-2-methyl-3H-pyrimidin-4-one
(4). Yield 75% (2.3 g); m.p. 246 °C (decomp.); TLC, chloro-
form–methanol, 6:1, Rf = 0.85; UV (H2O/MeOH, 1:1) 223
(5000), 263 (3900), (0,1 M HCl/MeOH, 1:1) 229 (9500),
(0,1 M KOH/MeOH, 1:1) 230 (8800), 264 (4100); m/z (EI)
244 (M+, 100), 187(19%); dH (200 MHz, DMSO-d6) 1.70 (s,
6H, H-Ada), 1.79 (s, 6H, H-Ada), 2.01 (s, 3H, H-Ada), 2.26
(s, 3H, CH3), 5.89 (s, 1H, H-5), 12.00 (s, 1H, NH).
2.1.4. Alkylation procedure (method I)
Compounds 9 and 10 were obtained from 2, and the
substrate for 12 and 13 was compound 8. An equimolar
mixture of 2 or 8 (1.6 mmol) with proper alkylating agent and
0.11 g (0.8 mmol) K2CO3 in ethanol (10 ml) was stirred at
room temperature for 4 h. After the reaction completion the
reaction mixtures were poured into water and neutralized
with diluted hydrochloric acid. The crude products were
crystallized from ethanol.
2.1.2. Bromination procedure
Compounds 5 and 7 were obtained by bromination of 1
and 3 in acetic acid, respectively [17]. A solution of bromine
0.39 g (2.46 mmol) in acetic acid (1 ml) was added dropwise
to a hot solution of 1 or 3 (2.28 mmol) in acetic acid (13 ml).
The mixture was heated up to 70 °C for 1°h. After cooling
crude products deposited and were filtered off and crystalli-
zed from ethanol.
2.1.4.1. 6-Adamantan-1-yl-2-[(4-nitrobenzyl)thio]-3H-pyri-
midin-4-one (9). Yield: 50% (0.32 g); mp 263 °C; TLC,
chloroform–methanol, 20:1, Rf = 0.51; UV (H2O/MeOH,
1:1) 286 (8500), (0,1 M HCl/MeOH, 1:1) 237 (9100), 284
2.1.2.1.
6-Adamantan-1-yl-5-bromo-1H-pyrimidine-2,4-
dione (5). Yield: 66% (0.49 g). m.p. 298 °C (decomp.); TLC,
chloroform–methanol, 6:1, Rf = 0.84; UV (H2O/MeOH, 1:1)

932
B. Orzeszko et al. / IL FARMACO 59 (2004) 929–937
(13500), (0,1 M KOH/MeOH, 1:1) 279 (12100); m/z (EI) 397
(M+, 100), 262 (25), 230 (25%); dH (200 MHz, DMSO-d6)
1.66–1.98 (m, 15H, H-Ada), 4.53 (s, 2H, CH2), 5.89 (s, 1H,
H-5), 7.65–7.76 (m, 2H, ar.), 8.12–8.24 (m, 2H, ar.).
MeOH, 1:1) 278 (6900), (0,1 M HCl/MeOH, 1:1) 230
(6900), 297 (14700); m/z (EI) 331 (M+, 0,5), 260 (10), 71
(100), 58 (47%); dH (200 MHz, DMSO-d6) 1.67–2.05 (m,
15H, H-Ada), 2.20 (s, 9H, (CH3), 2.51 (m, 2H, CH2), 3.32
(m, 2H, CH2), 7.03 (s, 1H, H-5).
2.1.4.2. 6-Adamantan-1-yl-2-[(2-nitrobenzyl)thio]-3H-pyri-
midin-4-one (10). Yield: 72% (0.45 g); mp 268 °C; TLC,
chloroform–methanol, 20:1, Rf = 0.49; UV (H2O/MeOH,
1:1) 282 (7000), (0,1 M HCl/MeOH, 1:1) 280 (11300),
(0,1 M KOH/MeOH, 1:1) 226 (11800), 273 (9100); m/z (EI)
397 (M+, 1), 263 (48), 262 (100%); dH (200 MHz, DMSO-d6)
1.68–2.00 (m, 15H, H-Ada), 4.71 (s, 2H, CH2), 5.90 (s, 1H,
H-5), 7.48–7.63 (m, 1H, ar.), 7.64–7.76 (m, 1H, ar.), 7.77–
7.86 (m, 1H, ar.), 8.00–8.12 (m, 1H, ar.), 12.32 (s, 1H, NH).
2.2. X-ray measurements
Compounds 2 and 4 (10 mg) were dissolved in absolute
ethanol (2 ml). Vials with respective solutions were placed in
hermetic vessels with hexane. After 5–6 days, single crystals
deposited on glass. Then they were carefully filtered-off and
dried. Crystals of 2 and 4 having dimensions of 0.65 × 0.5 ×
0.08 mm and 0.7 × 0.5 × 0.3 mm, respectively, were mounted
consecutively on Kuma KM4 j-axis diffractometer. After
crystals centring the unit cells parameters were determined
based on the least squares procedure performed for 31 strong
reflections from a 2h range of 11.7–21.9° and 12.0–24.0°,
respectively, collected by using MoKa radiation
(k = 0.71073 Å). 4611 and 7400 reflections were collected
for 2 and 4, respectively. Data were corrected for Lorenz-
polarization effects but not for absorption or extinction.
Structures were solved using direct methods from
SHELXS97 program and refined using SHELXL97 software
[20,21].
2.1.4.3. 4-Adamantan-1-yl-2-methyl-6-[(4-nitrobenzyl)thio]-
pyrimidine (12). Yield: 67% (0.42 g); m.p. 157 °C; TLC,
chloroform–methanol, 20:1, Rf = 0.71; UV (H2O/MeOH,
1:1) 280 (8900), (0,1 M HCl/MeOH, 1:1) 305 (17000); m/z
(EI) 395 (M+, 100), 362 (13), 260 (25), 71 (18%); dH
(200 MHz, DMSO-d6) 1.70–2.02 (m, 15H, H-Ada), 2.55 (s,
3H, CH3), 4.58 (s, 2H, CH2), 7.09 (s, 1H, H-5), 7.67–7.77 (m,
2H, ar.), 8.12–8.23 (m, 2H, ar.).
2.1.4.4. 4-Adamantan-1-yl-2-methyl-6-[(2-nitrobenzyl)thio]-
pyrimidine (13). Yield: 60% (0.38 g); m.p. 132 °C; TLC,
chloroform–methanol, 20:1, Rf = 0.72; UV (H2O/MeOH,
1:1) 276 (9700), (0,1 M HCl/MeOH, 1:1) 303 (15300); m/z
(EI) 395 (M+, 4), 260 (100%); dH (200 MHz, DMSO-d6)
1.70–2.00 (m, 15H, H-Ada), 2.55 (s, 3H, CH3), 4.75 (s, 2H,
CH2), 7.07 (s, 1H, H-5), 7.47–7.60 (m, 1H, ar.), 7.65–7.75
(m, 1H, ar.), 7.77–7.86 (m, 1H, ar.), 7.97–8.08 (m, 1H, ar.).
2.2.1. Crystal and experimental data
(2): C14H18N2OS·1/2C6H14, Mr = 305.45; monoclinic
space group C2/c,
a =26.260(17), b = 10.561(4);
c = 12.878(12) Å, b = 112.84(7)°; V = 3291(4) Å3, Z = 8,
q = 1.233 g cm–3, F(000) = 1320, µ(MoKa) = 0.198 mm–1.
Final R and wR were 0.0463 and 0.1378, respectively, for
2124 observed data [with I > 2r(I)].
2.1.5. Alkylation procedure (method II)
(4): C15H20N2O, Mr = 244.33; triclinic space group P–1,
a = 6.532(5), b =13.296(11), c = 15.372(11) Å, ␣ = 87.90(7),
b = 83.41(6), c = 84.82(7)°, V = 1320.3(18) Å3, Z = 4,
q = 1.229 g cm–3; F(000) = 528, µ(MoKa) = 0.078 mm–1.
Final R and wR were 0.0580 and 0.1807, respectively, for
3452 observed data [with I > 2r(I)].
Crystallographic data (excluding structure factors) for the
structures 2 and 4 in this paper have been deposited with the
Cambridge Crystallographic Data Centre as supplementary
publication numbers CCDC 229643 and 229644, respecti-
vely.
Compounds 11 and 14 were obtained from 2 and 8, res-
pectively. Equimolar mixtures of 2 or 8 (2.5 mmol) with
0.36 g (2.5 mmol) of 2-chloro–N,N-dimethyletylamine hy-
drochloride and 0.76 g DBU (5 mmol) in acetonitrile (20 ml)
were stirred for 48 h at room temperature. The solvent was
evaporated and the crude products were purified by column
chromatography (SiO2) using as an eluent chloroform–
methanol mixture (for 11 5:1 and for 14 9:1).
2.1.5.1. 6-Adamantan-1-yl-2-[(2-dimethylaminoethyl)thio]-
3-H-pyrimidin-4-one (11). Yield: 55% (0.46 g); m.p. 169 °C;
TLC, chloroform–methanol, 6:1, Rf = 0.48; UV (H2O/
MeOH, 1:1) 279 (6000), (0,1 M HCl/MeOH, 1:1) 231
(11200), 276 (7000), (0,1 M KOH/MeOH, 1:1) 244 (4800),
277 (4000); m/z (EI) 333 (M+, 1), 120 (28), 71 (100), 58 (49),
44 (31), 40 (56%); dH (200 MHz, DMSO-d6) 1.68–2.00 (m,
15H, H-Ada), 2.21 (s, 6H, CH3), 2.53 (t, 2H, J = 7.2 Hz,
CH2), 3.22 (t, 2H, J = 7.2 Hz, CH2), 5.83 (s, 1H, H-5), 11.48
(s, 1H, NH).
2.3. Biological evaluations
2.3.1. Antimicrobial studies
The following microorganisms were used: Gram-positive
bacteria: Staphylococcus aureus ATCC 6538P, Staphylococ-
cus aureus NCTC 4163, Enterococcus faecalis ATCC
29212, Bacillus subtilis ATCC 6633, Bacillus stearothermo-
philusATCC 7953; Gram-negative bacteria: Escherichia coli
ATCC 25922, Escherichia coli NCTC 8196, Proteus vulga-
ris NCTC 4635, Klebsiella pneumoniae ATCC 13883, Pseu-
domonas aeruginosa ATCC 27853, Psudomonas aeruginosa
2.1.5.2.
[2-(6-Adamantan-1-yl-2-methylpyrimidin-4-
yl)thioethyl]dimethylamine (14). Yield: 88% (0.73 g); m.p.
65 °C. TLC, chloroform–methanol, 6:1, Rf = 0.74; UV (H2O/

B. Orzeszko et al. / IL FARMACO 59 (2004) 929–937
933
NCTC 6749, Stenotrophomonas maltophilia ATCC 13637,
Burkholderia cepacia ATCC 25416, Acinetobacter bauma-
nii ATCC 18606, Bordetella bronchiseptica ATCC 4617;
fungi: Candida albicans ATCC 10231, Candida albicans
ATCC 90028, Candida parapsilosis ATCC 22019. The mi-
croorganisms came from the State Institute of Hygiene (War-
saw, Poland), the Children Memorial Health Institute (War-
saw, Poland), and from the own collection of the Department
of Pharmaceutical Microbiology, Medical University of War-
saw (Warsaw, Poland).
Antibacterial activity was examined by the disc-diffusion
method and the MIC method under standard conditions using
Mueller–Hinton II agar medium (Becton–Dickinson) accor-
ding to guidelines established by the NCCLS [22,23]. Solu-
tions of tested agents, depending on their structures, were
prepared in different mixtures of EtOH, CHCl3 and 2.5 M
NaOH. For disc-diffusion assays, sterile filter paper discs
(9 mm diameter, Whatman No. 3 chromatographic paper)
were dripped with tested compound solutions to load 400 µg
of a given compound per disc. For MIC determinations,
concentrations of tested compounds in solid medium ranged
from 0.78 to 400 µg/ml, and agar plates were inoculated
using 2 µl aliquots. The final inoculum of all studied orga-
nisms were 104 CFU/ml (colony forming units per ml),
except the final inoculum of E. faecalis ATCC 29212 which
was 105 CFU/mL. Results of antimicrobial activity were read
after 18 h incubation at 35 °C.
lowed to proliferate for 96 h at 37 °C in a humidified CO2-
controlled atmosphere. At the end of the incubation period,
the cells were counted in a Coulter counter (model ZB;
Coulter Electronics Ltd., Harpenden, Hertfordshire, En-
gland). The 50% cytostatic concentration (CC50) was defined
as the concentration of compound that inhibited CEM cell
proliferation by 50%.
2.3.3.3. Viruses. HIV-1(IIIB) was kindly provided by R.C.
Gallo and M. Popovic (at that time at the National Cancer
Institute, Bethesda, MD). HIV-2(ROD) was from L. Monta-
gnier (at that time at the Pasteur Institute, Paris, France).
2.3.3.4. Antiviral activity of tested compounds in cell cultu-
res. CEM cells were suspended at ca. 300,000 cells per ml of
culture medium and infected with ca. 100 times the 50%
cell-culture-infective doses of HIV-1(IIIB) or HIV-2(ROD).
Then, 100 µl of the infected cell suspensions were added to
200 µl microtiter plate wells containing 100 µl of appropriate
serial (five-fold) dilutions of the tested compounds. The
inhibitory effect of compounds on HIV-induced syncytium
formation in CEM cells was examined microscopically on
day 4 or 5 post infection. The EC50 was defined as the
compound concentration that inhibits syncytium formation
in the HIV-infected cell cultures by 50%.
3. Results and discussion
2.3.2. Anticancer studies
The compounds tested were added to exponentially
growing cell cultures (105/ml). After three days, the number
of living cells was determined by using their capability to
exclude the trypan blue dye. Following cell lines were used:
HL-60 and KG-1 (acute myelogenous leukemia); CEM
(acute T-cell leukemia); U-937 (acute monocytic leukemia);
Jok-1 (hairy cell leukemia); BALL (acute B-cell leukemia),
and Daudi (Burkitt’s B-cell lymphoma). The cell lines were a
generous gift from Professor Leif Andersson (Department of
Pathology, Haartman Institute, University of Helsinki, Fin-
land). Cell viability at the beginning of the tumor cultures
was always >97%.
The known drug bropirimine i.e. 2-amino-5-bromo-6-
phenyl-3H-pyrimidin-4-one, is an orally active immunomo-
dulator that stimulates production of endogenous TNF-a and
other cytokines and is now under phase II clinical trials for
treatment of bladder carcinoma [21]. It is a low molecular
weight compound acting as a ligand for TLR7 (Toll-like
receptor) [24]. The inspiration for this study was the search
for brompirimine analogues and expanding the list of 6(4)-
adamantylated pyrimidines for pilot studies on their biologi-
cal activity. The formulas of bropirimine and one of the
compounds described below (6-adamantan-1-yl-2-amino-5-
bromo-3H-pyrimidin-4-one) are presented in Fig. 1, for a
comparison.
2.3.3. Antiviral studies
The synthesis of pyrimidines 1–4 similar to bropirimine
was based on the Traube reaction of 3-(adamantan-1-yl)-3-
oxopropionic acid ethyl ester with urea, thiourea, guanidine
and acetamidine, respectively. In this case the adamantyl
moiety was present in the starting reactant. Next,
2.3.3.1. Cells. Human lymphocyte CEM cells were obtained
from American Type Culture Collection and grown in RPMI
1640 medium supplemented with 10% (v/v) inactivated fetal
calf serum (Gibco), 2 mM L-glutamine (Flow Laboratories),
and 0.075% (v/v) NaHCO3. Cells were subcultured every 3 to
4 days.
2.3.3.2. Cytostatic activity of test compounds in cell cul-
ture. All assays were performed in 96-well microtiter plates
(Falcoln 3072; Becton Dickinson, Paramus, NJ). To each
well were added ca. 6 × 104 CEM cells (100 µl) and a given
amount of the test compound (100 µl). The cells were al-
Fig. 1. Structures of bropirimine and a related adamantyl derivative.

934
B. Orzeszko et al. / IL FARMACO 59 (2004) 929–937
tor interaction of such compounds, better knowledge of their
molecular parameters is necessary. We have succeeded to
obtain single crystals of 2 and 4 suitable for X-ray measure-
ments. Structure of 2 unexpectedly contained disordered
molecules of hexane which was used in preparation of mono-
crystals, while this compound obtained according to the
procedure described in the Experimental part, was free from
solvents and other impurities. The incorporation of hexane
into space lattice during growth of crystals is a good illustra-
tion of the amphiphilic character of such molecules. This
property is a very important factor in respect of biological
evaluations. Fig. 2 shows the independent part of the unit
cell, whereas Fig. 3 illustrates crystal packing of 2 shown
along the c-axis.
The structure (2) is composed of molecules connected into
infinite chains (perpendicular to the drawing and hence not
visible) with N(1)–H(1)·O(1) hydrogen bonds between mo-
lecules related by the c-glide plane symmetry. The chains are
interlinked by series of centrosymmetric N(3)–H(3)·S(1) hy-
drogen bonds between neighbouring molecules. The hydro-
phobic channels passing in the c-direction are filled out with
disordered hexane molecules (shown with thin lines in the
drawing).
Fig. 2. Independent part of the unit cell. The non-hydrogen atoms are shown
as 30% probability ellipsoids. Only the half of the solvent molecule is
shown.
6-adamantan-1-yl-2-thioxo-2,3-dihydro-1H-pyrimidin-4-one
(2) and 6-adamantan-1-yl-2-methyl-3H-pyrimidin-4-one (4)
were treated with Lawesson’s reagent to yield the respective
4-thioderivatives 6 and 8. The compounds 1 and 3 were
brominated with bromine to give the 5-substituted uracil 5
and isocytidine 7, respectively. The alkylation of thioderiva-
tives 2 and 8 with nitrobenzylchlorides and dimethylami-
noethylchloride yielded the respective S-alkyl derivatives
9–11. The general synthetic pathway is given in Schemes
1 and 2. Structures and purity of novel compounds were
confirmed using chromatographic, spectroscopic and, in two
cases, X-ray methods. Their full characterization is given in
section 2.
Structure of 4 is triclinic with 2 independent molecules in
an asymmetric part of the unit cell. Fig. 4 shows a molecular
structure of one of them (Fig. 4).
The molecules are connected into centrosymmetic hydro-
gen bonded dimers (see Fig. 5) with N(3) – H(3) { O(1).
The planar fragments of molecules A and B are inclined
by 58.98(8)° to each other. The molecule of 4 has two
possible hydrogen bond acceptor sites: carbonyl oxygen and
pyrimidine N1 nitrogen; however, only the first one is active.
The pyrimidine nitrogen N1 is completely shielded by a
methyl and an adamantyl moiety. The adamantyl fragment is
a symmetric cage-like structure and one can assume that two
As it has been mentioned, adamantyl derivatives are very
interesting from a pharmacological point of view. For the
better understanding of biological activity and agent–recep-
Fig. 3
. The unit cell shown along the c-axis. The hydrogen bonds are shown as dashed lines. The disordered solvent molecules, drawn with thin lines, occupy
hydrophobic channels passing in c direction.

B. Orzeszko et al. / IL FARMACO 59 (2004) 929–937
935
Table 1
Antimicrobial activities of 6-(adamantan-1-yl)pyrimidines 11 and 14
against bacteria and fungi (the rest of compounds were completely inactive)
Bacteria strains
Diameter of the growth inhibition area
[mm] and MICa (in parenthesis)
11
14
Staphylococcus aureus
ATCC 6538P
12 (>400)
18 (50)
Staphylococcus aureus
NCTC 4163
13 (>400)
17 (100)
19 (100)
23 (100)
21 (50)
na
Enterococcus faecalis
ATCC 29212
nab
Bacillus subtilis
ATCC 6633
12 (>400)
Bacillus stearothermophilus
ATCC 7953
15 (400)
na
Proteus vulgaris
NCTC 4635
Escherichia coli
ATCC 25922
na
na
Fig. 4. Conformation of molecule A and numbering scheme.
Escherichia coli
NCTC 819
na
11 (400)
na
Klebsiella pneumoniae
ATCC 13883
na
Pseudomonas aeruginosa
ATCC 27853
na
na
Psudomonas aeruginosa
NCTC 6749
na
na
Stenotrophomonas maltophilia
ATCC 13637
na
16(100)
na
Burkholderia cepacia
ATCC 25416
na
Acinetobacter baumannii
ATCC 18606
na
na
Bordetella bronchiseptica
ATCC 4617
na
na
Candida albicans
ATCC 10231
na
17 (100)
17 (100)
20 (100)
Candida albicans
ATCC 90028
na
Candida parapsilosis
na
Fig. 5. Crystal packing shown along the a-axis. Two different molecules A
ATCC 22019
a Minimal inhibitory concentration , µg/ml.
b No activity.
and B are present in the asymmetric part of the unit cell. Molecules related
by centre of symmetry are bonded in pairs with the N – H { O intermolecular
hydrogen bonds (shown as the dashed lines).
60° rotamers are energetically equivalent. Our ab initio quan-
tum chemical calculations (RHF 6-31G**) showed, howe-
ver, that the conformation present in a crystal is by
2.437 kcal/mol more stable than the alternative one.
Gram-positive bacteria. Moreover, some of the Gram-
negative bacteria, as Bordetella bronchiseptica ATCC
4617 and Stenotrophomonas maltophilia ATCC 13637, and
all tested fungi were sensitive to 14.
As first biological examinations we performed antibacte-
rial and antifungal studies. The activity in vitro of newly
obtained derivatives of 6-(adamantan-1-yl)pyrimidines was
tested against a wide range of bacteria and fungi including
Gram-positive cocci and Gram-negative rods. The evaluation
of the antimicrobial properties was carried out using the disc
diffusion method. Compounds showing significant activity in
the above test were next examined for their minimal inhibi-
tory concentration (MIC). The results of tests are summari-
zed in Table 1. Of all agents tested only two, 11 and 14,
shown modest antimicrobial activity. In comparison to 11,
compound 14 had from 5 to 8-fold higher activity against
Then, compounds 1–14 were examined as anticancer
agents. The pyrimidines tested were added to cultures of
growing tumor cells. Following cell lines were used: HL-60
and KG-1 (acute myelogenous leukemia); CEM (acute T-cell
leukemia); U-937 (acute monocytic leukemia); Jok-1 (hairy
cell leukemia); BALL (acute B-cell leukemia, and Daudi
(Burkitt’s B-cell lymphoma). In the initial screening we
tested each compound in 25 µg/ml concentration against two
different cell lines (BALL and HL-60). As shown in Table 2,
only compound 14 significantly inhibited the cell growth of
BALL or HL-60 cells at 25 µg/ml. This compound was,
therefore selected for further testing at several doses and

936
B. Orzeszko et al. / IL FARMACO 59 (2004) 929–937
Table 2
Table 4
Inhibition of the growth of two human cancer cell lines in vitro by
6-(adamantan-1-yl)pyrimidines. The test concentration was 25 µg/ml
Anti-HIV-1 and HIV-2 activity and cytotoxic properties of 6-(adamantan-1-
yl)pyrimidines expressed as EC50 and CC50 (in µg/ml; in brackets in µM),
respectively
Compounds
Cell number (% of untreated control cultures)
a
b
Compound
EC50
CC50
BALL
81
HL-60
98
HIV-1
HIV-2
1
1
2
3
4
5
6
>100 [406]
>20 [76]
>100 [382]
>20 [82]
>100 [307]
>4 [12]
>100 [406]
>20 [76]
>100 [382]
>20 [82]
>100 [307]
>4 [12]
>100 [406]
75.2 [287]
100 [382]
41.8 [171]
>100 [307]
4.6 [14]
2
3
4
5
6
7
67
76
96
77
96
67
104
68
85
32
89
83
7
8
9
10
11
12
13
14
>20 [72]
>20 [77]
>4 [10]
>4 [14]
12.1 [43]
49.3 [189]
21.7 [55]
10.2 [27]
44.1 [111]
>100 [253]
18.7 [56]
6.2 [18]
8
9
31
43
>20 [77]
>4 [10]
101
70
78
10
11
12
13
14
50
>4 [10]
>4 [10]
47
41
>20 [51]
>100 [253]
>4 [12]
>20 [51]
>100 [253]
>4 [12]
75
81
29
35
<1
<1
>4 [12]
>4 [12]
a 50% Effective concentration or compound concentration, required to
protect CEM cell cultures against the cytopathogenicity of HIV by 50%.
b 50% Cytotoxic concentration or compound concentration, required to
reduce CEM cell proliferation by 50%.
Table 3
Results of inhibition (ID80 in µg/ml; in brackets in µM) of cell growth by
compound 14
Cancer cell lines
BALL
ID80
17.3 [52.3]
17.5 [52.8]
20.0 [60.4]
16.4 [49.5]
7.0 [21.2]
18.4 [57.1]
4.8 [014.5]
mentioned in the Introduction such molecular architecture is
very promising for medicinal applications. For this reason we
performed several preliminary biological studies. Although
the studied compounds had no antiviral activity, during the
tests we observed pronounced cytotoxicity for same of the
compounds. Therefore we studied activity of these com-
pounds against several neoplastic cell lines. Anticancer eva-
luations indicate that only [2-(6-adamantan-1-yl)-2-
methylpyrimidin-4-ylthioethyl]-dimethylamine (14) has
significant activity against different human cancer cell lines,
particularly against monocytic leukemia (U-937). The same
compound 14 is also the most potent as antimicrobial agent.
This observation indicates a direction for future syntheses
and studies. It appears that the most promising compounds
might be derivatives of 6-adamantan-1-yl-2-methyl-3H-
pyrimidine-4-thione (8) with diverse 4-S- polar substituents
as pharmacophoric groups.
CEM
Daudi
HL-60
Jok-1
KG-1
U-937
against seven different tumor cell lines. Dose–response cur-
ves for each target cell line were calculated (drawn) using
four doses of the tested compound. Compound concentra-
tions inhibiting the cell growth by 80% (ID80) were determi-
ned from dose–response curves. The comparison of the sen-
sitivities of seven different human cancer cell lines to this
compound was performed by calculating the ID80 values.
The ID80 values are shown in Table 3. As can be seen
monocytic leukemia (U-937) and hairy cell leukemia (Jok-1)
are the most, and Burkitt’s lymphoma (Daudi) the least
sensitive to 14.
Next the compounds were evaluated for their inhibitory
activity against HIV-1(IIIB) and HIV-2(ROD)-induced cyto-
pathicity in CEM cell cultures. As shown in Table 4 none of
the compounds were inhibitors against these viruses in CEM
cell cultures at subtoxic concentrations. Compounds 6 and 14
proved most cytotoxic (CC50 < 10 µg/ml).
Acknowledgements
This study was supported in a part by the Foundation for
the Development of Diagnostic and Therapy, Warsaw, Po-
land. This work was supported in a part by a grant from the
European Commission (HPAW-2002-90001) and by
ISEP/FORTIS .
4. Conclusion
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