A preliminary investigation of Mesoionic Xanthine analogues as inhibitors of platelet aggregation

Article abstract of DOI:10.1016/S0968-0896(00)00123-1

A series of mesoionic xanthines (e.g. mesoionic thiazolopyrimidines, 3, and thiadiazolopyrimidines, 5) and related analogues were examined as inhibitors of human platelet aggregation. Appropriately substituted compounds were found to fully inhibit platelet aggregation, and anhydro-(6-ethyl-8-isopentyl-7-oxo-5-hydroxy-1,3,4-thiadiazolo[3,2-a]pyrimidinium hydroxide) (5b) was 40 times more potent than the structurally related xanthine theophylline (1). Gel filtration studies suggest that compound 5b irreversibly inhibits aggregation and this might be due to its ability to act as a latent acylation agent. Copyright (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(00)00123-1

Bioorganic & Medicinal Chemistry 8 (2000) 1917±1923
A Preliminary Investigation of Mesoionic Xanthine Analogues
as Inhibitors of Platelet Aggregation
Mark Hellberg,* James F. Stubbins and Richard A. Glennon
Department of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth University,
Richmond, VA 23298-0540, USA
Received 15 February 2000; accepted 13 April 2000
AbstractÐA series of mesoionic xanthines (e.g. mesoionic thiazolopyrimidines, 3, and thiadiazolopyrimidines, 5) and related analogues
were examined as inhibitors of human platelet aggregation. Appropriately substituted compounds were found to fully inhibit platelet
aggregation, and anhydro-(6-ethyl-8-isopentyl-7-oxo-5-hydroxy-1,3,4-thiadiazolo[3,2-a]pyrimidinium hydroxide) (5b) was 40 times
more potent than the structurally related xanthine theophylline (1). Gel ®ltration studies suggest that compound 5b irreversibly inhibits
aggregation and this might be due to its ability to act as a latent acylation agent. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
The serious cardiovascular disorders of myocardial infarc-
tion and unstable angina are associated with the activation
and aggregation of platelets. Ischemic stroke resulting from
the transient or permanent reduction in cerebral blood is
in most cases caused by the occlusion of a cerebral artery
by either embolism or local thrombosis. The utility of
anti-platelet agents in the treatment of cardiovascular
disorders and stroke has recently been reviewed.1
As predicted by molecular orbital calculations, mesoionic
thiazolo[3,2-a]pyrimidines and 1,3,4-thiadiazolo[3,2-a]
pyrimidines undergo nucleophilic attack by amines at the
5-positions.9 12 This is shown in Figure 1. The possibility
exists, then, that these compounds might behave as latent
acylating agents. However the use of these potential acy-
lating agents to thereby irreversibly inhibit a biological
response has not yet been demonstrated. Gel ®ltration
of platelet rich plasma provides a method that results in
the rapid and complete separation of human platelets
from plasma. This procedure is useful in determining the
reversibility of drug e€ects by removing drug from the
platelet-containing suspending medium. The reversibility
of the e€ects of beta lactam antibiotics13 and an N-(2-
chloroethyl)localanesthetic14 on platelets has been assessed
by this method. The possibility that the mesoionic xanthine
derivatives might act by an irreversible mechanism as
inhibitors of platelet aggregation was evaluated using
the gel ®ltration technique.
A large number of agents such as papaverine2 and the
methyl xanthine derivatives theophylline (1) and 1-iso-
butyl-3-methylxanthine (IBMX; 2) inhibit platelet
aggregation through their activity as phosphodiesterase
inhibitors.3,4 In a number of reports Glennon and
co-workers have shown that a series of mesoionic
derivatives have activity as inhibitors of phospho-
diesterase.5 9 The possibility hat these compounds
might also, then, inhibit platelet aggregation led to their
evaluation as platelet aggregation inhibitors. Several
previously published mesoionic compounds were
evaluated, and a few novel mesoionic compounds were
prepared to further develop the structure±activity
relationships associated with the activity of these
compounds as inhibitors of platelet aggregation.
Chemistry
*Corresponding author at current address; Alcon Laboratories, 6201
S Freeway, Fort Worth, TX 76134, USA. Tel.: +1-817-551-4359; fax:
+1-817-568-7640; e-mail: mark.hellberg@alconlabs.com
The mesoionic xanthine analogues were prepared by
the methods of Coburn et al.10 12,16 and Glennon and
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00123-1

1918
M. Hellberg et al. / Bioorg. Med. Chem. 8 (2000) 1917±1923
The inhibition of platelet aggregation by the mesoionic
inhibitors was time dependent. A twenty minute incu-
bation was selected since this incubation time gave a
maximum inhibitory e€ect while allowing for a reasonable
number of evaluations within the two-hour period in
which the platelets were viable.
Figure 1. Attack of a mesoionic xanthine analogue by a nucleophile to
a€ord a ring-opened compound.9
The gel ®ltration studies were carried out by a modi®cation
of Tangen's procedure using a Sepharose 2B column.14,26
Platelets passed rapidly through the column and were
eluted in what was essentially the void volume of the
column. Proteins and presumably other small molecular
species have substantially longer retention times which
allowed for the ecient separation of the platelets from
the other constituents of the platelet rich plasma. Results
are shown in Figure 2; a second run gave similar results.
co-workers.5 9,15,17 Brie¯y, the target compounds were
prepared by the thermal condensation of an alkylamino-
heterocycle and a bis(2,4,6-trichlorophenyl)-2-alkylmalo-
nate ester under a continuous stream of nitrogen.7,15,18,19
See Scheme 1 for the general synthesis of the mesoionic
derivatives.
Most of the alkylaminoheterocyles were prepared by the
acylation of the aminoheterocycle with the appropriate
acid chloride or anhydride followed by the reduction of
the resulting amide using lithium aluminum hydride or
sodium bis(methoxyethoxy) aluminum hydride. The 2-
aminopyrimidine derivatives were synthesized by the direct
alkylation of 2-aminopyrimidine (ethyl, n-pentyl deriva-
tives) or by the two-step procedure of Kemal and Reese
(benzyl derivative).20 The 3-pyridazine and 4-aminopyr-
imidine derivatives were prepared by amination of and
hydrogenolysis of either 3,6-dichloropyridazine or 4,6-
dichloropyrimidine.21,22 The substituted 4-(methyla-
mino)-2-aminothiazole derivatives were prepared by the
method of Silberg.23
Results and Discussion
The results of the platelet aggregation studies are shown
in Tables 1 and 2. Members of the thiazolopyrimidine
series (i.e., 3) were examined ®rst. The diethyl analogue
3a (MIC100=4mM) was as potent as theophylline (1) and
80-fold less potent than adenosine as an inhibitor of pla-
telet aggregation. Replacement of the N8-ethyl substituent
Pharmacology
The mesoionic compounds were evaluated by the method
of Born24 and Puri25 for their ability to inhibit ADP-
induced platelet aggregation. ADP at a ®nal concentration
of 2.5 mM was used to stimulate platelet aggregation. At
this concentration ADP caused a monophasic tracing
denoting platelet aggregation. In this preliminary study
designed to provide an initial characterization of the
platelet aggregation inhibitory activity of the mesoionic
xanthine analogues, a 100% blockade of ADP-induced
aggregation (minimum concentration necessary to cause
100% inhibition of aggregation, MIC100) was used as a
reference point for two reasons. First, the responsiveness
of the platelets to aggregating agents changes with time.
Using less than a 100% blockade would necessitate
running a control sample between each drug sample.
Secondarily, it allows for a dependable measurement of
the inhibition of primary aggregation since the response
to ADP can be comprised of both primary and secondary
aggregation.
Figure 2. Platelet aggregation as measured by % change in light
transmission before (A) and after (B) gel ®ltration. Platelet rich plasma
was prepared and then incubated either with vehicle, adenosine
(0.05 mM), or 5b (0.8 mM) for 30 min and aggregation was stimulated
by the addition of 2.5 mM ADP (Panel A). Platelet rich plasma was
prepared and then incubated either with vehicle, adenosine (0.05 mM),
or 5b (0.8 mM) for 30 min then ®ltered through a Sepharose 2B col-
umn. Platelet poor plasma was added and aggregation was stimulated
by the addition of 2.5 mM ADP (Panel B).
Scheme 1.

M. Hellberg et al. / Bioorg. Med. Chem. 8 (2000) 1917±1923
1919
Table 1. Minimum 100% inhibitory concentrations (MIC100 values) for platelet aggregation by compounds 3±5
R6
R8
R3
MIC100 (mM)a
Referenceb
3a
3b
Et
Et
Et
Et
H
Me
4
1
7
8
3c
3d
Et
Et
Et
n-Pentyl
Phenyl
H
8
2
3e
3f
3g
3h
3i
3j
4
5a
5b
5c
Et
Et
Et
Et
Benzyl
Benzyl
Et
Me
Me
Et
Phenyl
Benzyl
CH2CH2N(Me)2
Benzyl
Et
CH2CH2N(Me)2
H
H
0.4
0.4
8
6
2
6
6
H
CH2N(Me)2
H
H
7
6
Et
Et
n-Pentyl
Et
Et
Ð
Ð
Ð
Ð
Ð
0.4
0.2
0.1
0.2
0.2
0.05
4
17
7
6
7
6
5d
Adenosine
Theophylline (1)
isoBu
aSee Experimental for explanation of methods.
bLiterature reference for preparation.
Table 2. Minimum 100% inhibitory concentrations (MIC100 values)
for platelet aggregation by compounds 6±10
the alkyl groups of some of the derivatives (i.e. 3g, 3h, 3j);
this only served to reduce potency. Reduction of the C2±
C3 double bond of 3a provided 4 (MIC100=0.4 mM) and
resulted in a 10-fold increase in potency (relative to 3a).
This feature was not further explored, however, because
of the ®ndings with 5c.
Compound 5c represents the 1,3,4-thiadiazole counterpart
of the thiazolopyrimidine 3a, and compound 5c (MIC100=
0.2 mM) was found to be 20 times more potent than 3a.
Shortening the C6-ethyl group to a methyl group (i.e. 5a)
and lengthening it to an isobutyl group (i.e. 5d) had no
e€ect on potency. The C6-methyl-N8-n-pentyl derivative
5b (MIC100=0.1 mM), however, was found to be the
most potent analogue in the series with a potency 40
times that of theophylline and half that of adenosine.
R
W
X
Y
Z
MIC100, mM
6a
6b
6c
6d
7
8
9
10a
10b
10c
Et
Et
n-Pentyl
Benzyl
Et
Et
Et
Et
n-Pentyl
Benzyl
CH
CH-CH3
CH
CH
N
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
2
4
4
2
>8
2
4
6
1
4
Because the introduction of a limited amount of bulk at
the 3-position (compare 3b with 3a), and incorporation of
a ring nitrogen atom (compare 5c with 3a) was bene®cial
for activity, we prepared a series of ring-expanded analo-
gues (i.e. 6±10). Although nearly all of these compounds
retained activity (with the exception of 7 which failed to
produce 100% inhibition at the highest concentration
evaluated), none was more potent than any members of
the thiadiazolopyrimidine series 5.
N
N
with larger or bulkier phenyl or benzyl substituents
resulted in enhanced potency; N8-substituent (followed
by MIC100 values): phenyl (3e; 0.4 mM), benzyl (3f;
0.4 mM). Compounds 3e and 3f were ten times more
potent than theophylline (1) and one-tenth as potent as
adenosine. The N8-pentyl substituent caused little change
in potency (3d, 2 mM). Replacement of the C6-ethyl sub-
stituent with the larger benzyl group (3i; MIC100=2mM)
resulted in little change in potency. Introduction at the
3-position of a methyl group (3b; MIC100=1 mM)
enhanced potency by 4-fold whereas incorporation of a
phenyl substituent (i.e. 3c) decreased potency. Because
many of the compounds display only limited aqueous
solubility, an N,N-dimethylamino function was added to
Gel ®ltration studies were conducted to provide evidence
that the mesoionic xanthine derivatives might be irre-
versible inhibitors of platelet aggregation. Gel ®ltration
of platelet rich plasma separates platelets from proteins
and small molecular weight compounds. This procedure
removes most reversibly acting platelet aggregation
inhibitors. The mesoionic thiadiazole derivative 5b was
selected for these studies in part because it was the most

1920
M. Hellberg et al. / Bioorg. Med. Chem. 8 (2000) 1917±1923
potent inhibitor of platelet aggregation, and in part
because it has been demonstrated that the mesoionic
thiadiazolo[3,2-a]pyrimidines are fairly reactive to
nucleophilic attack.9 12
0.4% of theoretical values. Melting points were deter-
mined using a Thomas-Hoover or Mel-Temp melting
point apparatus. All melting points are uncorrected.
Solvents were puri®ed using the methods reported by
Gordon and Ford.27
After a 30 min incubation at a concentration of 0.8 mM
5b completely inhibited platelet aggregation stimulated by
2.5 mM ADP. Following gel ®ltration, platelet aggregation
was still completely inhibited when the platelet-poor
plasma was added to the ®ltered platelets and the platelets
were stimulated with 2.5 mM ADP. A similar study using
0.05 mM of adenosine showed the expected complete
inhibition of platelet aggregation prior to ®ltration and
a complete reversal of platelet aggregation inhibition
following gel ®ltration, the addition of platelet poor
plasma and stimulation with 2.5 mM ADP. Concurrent
control studies with untreated platelets showed that they
retained their response to ADP following gel ®ltration
and addition of platelet poor plasma.
Anhydro-(6,8-diethyl-3-phenyl-7-oxo-5-hydroxythiazole
[3,2-a]pyrimidinium hydroxide) (3c). A mixture of 4-
phenyl-2-(ethylamino)thiazole (0.7 g, 3.6 mmol) and bis
(2,4,6-trichlorophenyl) ethylmalonate (1.8 g, 3.6 mmol)
was warmed at 160 ꢀC under a stream of nitrogen for
4 min. The cooled melt was triturated with anhydrous
ether to produce a tan solid. Recrystallization from 2-
propanol yielded 0.6 g (56%) of a white solid, mp 212±
214 ꢀC. IR (KBr) 3050, 2960 (C H), 1690, 1620 (CˆO),
1590cm 1 (CˆC, CˆN). 1H NMR (CDCl3) d 7.40 (s, 5H,
Ph), 6.80 (s,1, thiazole H), 4.20 (q, 2, CH2-N), 2.50 (q, 2,
CH2-C), 1.45 (t, 3, CH3-CH2-N), 1.05 (t, 3, CH3-CH2-
CH2-C). Anal. (C16H16N2O2S) C, H, N.
The gel ®ltration studies clearly demonstrate that platelet
aggregation by the mesoionic thiadiazolo[3,2-a]pyr-
imidine 5b is irreversible. It is interesting to speculate
that the irreversible inhibition of platelet aggregation
may be the result of acylation of the receptor or active
site by the mesoionic compound. This represents the
®rst reported irreversible inhibition of a biological
response by a mesoionic compound.
Anhydro-(6-ethyl-7-oxo-8-pentyl-5-hydroxythiazolo[3,2-a]
pyridinium hydroxide) (3d). The procedure described for
3c was used with 2-(pentylamino)thiazole (0.6g, 3.6 mmol)
and bis(2,4,6-trichlorophenyl) ethylmalonate (1.8 g, 3.6
mmol) to give 3d. Recrystallization from 2-propanol
yielded 0.6 g (63%) as a white solid, mp 132±134 ꢀC. IR
(KBr) 2950 (C H), 1680, 1640 (CˆO), 1580cm 1 (CˆC,
1
CˆN). H NMR (CDCl3) d 8.25 (d, 1, thiazole H), 7.45
(d, 1, thiazole H), 4.15 (t, 2, CH2-N), 2.55 (q, 2, CH3-
CH2-C), 2.00±0.80 (m, 12, (CH2)3CH3, C-CH2-CH3).
Anal. (C13H18N2O2S) C, H, N.
Conclusion
In summary, a series of mesoionic xanthine analogues
demonstrated activity as inhibitors of platelet aggregation.
In these preliminary studies, the thiadiazolo[3,2-a]pyr-
imidine (i.e. 5) series was clearly more potent than the
corresponding thiazolo[3,2-a]pyrimidines 3, pyrido[1,2-a]
pyrimidines 6, pyrimido[1,2-a]pyrimidine 8, pyrimido[1,6-
a] pyrimidine 9, and pyrazine[1,2-a]pyrimidines 10. The
pyridazo[1,2-b]pyrimidine 7 was only a partial inhibitor
at 8 mM, the highest concentration tested. Further studies
are needed to completely characterize the potency of
these compounds as inhibitors of platelet aggregation.
Anhydro-(6-ethyl-7-oxo-8-benzyl-5-hydroxythiazole[3,2-a]
pyrimidinium hydroxide (3f). The procedure described
for 3c was used with 2-(benzylamino)thiazole (0.68 g,
3.6 mmol) and bis(2,4,6-trichlorophenyl) ethylmalonate
(1.8 g, 3.6 mmol) to give 3f. Recrystallization from tolu-
ene yielded 0.58 g, (56%) of 3f as an o€-white solid, mp
144±145 ꢀC. IR (KBr) 2990 (C H), 1690, 1660 (CˆO),
1570 cm 1 (CˆC, CˆN). 1H NMR (CDCl3) d 8.10 (d, 1,
thiazole H), 7.40 (s, 5, Ph), 7.10 (d, 1, thiazole H), 5.40
(s, 2, CH2-Ph), 2.65 (q, 2, CH2-CH3), 1.20 (t, 3, CH3).
Anal. (C16H14N2O2S) C, H, N.
The most potent compound anhydro-(6-methyl-7-oxo-8-
pentyl-5-hydroxythiadiazolo[3,2-a]pyrimidinium hydrox-
ide) (5b) was demonstrated to be an irreversible inhibitor
of platelet aggregation as demonstrated by gel ®ltration
studies. This represents the ®rst reported irreversible inhi-
bition of a biological response by this series of mesoionic
xanthines. The potential utility of the mesoionic com-
pounds as irreversible inhibitors in other biological systems
remains to be explored.
Anhydro-(6-ethyl-7-oxo-8-(2-dimethylaminoethyl)-5-hy-
droxythiazolo[3,2 - a]pyrimidinium hydroxide) HCl (3g).
The procedure described for 3c was used with 2-[2-dim-
ethylamino)ethylamino]thiazole (0.62 g, 3.6 mmol) and
bis(2,4,6-trichlorophenyl) ethylmalonate (1.8 g, 3.6 mmol)
to give a white solid following column chromatography
(chloroform:methanol, 9:1). Recrystallization from tet-
rahydrofuran/hexane yielded 0.53 g of a white solid, mp
123±124 ꢀC. To form the hydrochloride salt, the free base
was dissolved in anhydrous tetrahydrofuran and HCl gas
was bubbled through the solution. The white solid that
formed was collected by ®ltration and recrystallized
from methanol yielding 0.57 g (62%) of 3g as a white
solid, mp 250±251 ꢀC. IR (free base, KBr) 2950 (C H),
2950 (C H), 1665, 1620 (CˆO) 1565 cm 1 (CˆC, CˆN).
1H NMR (free base, CDCl3) d 8.15 (d, 1, thiazole H),
7.15 (d, 1 thiazole), 4.15 (t, 2, CH2-N-(CH3)2), 2.60±2.30
(m, 8, CH3-CH2-, (CH3)2N-), 1.15 (t, 3, CH2-CH3).
Anal. (C12H17N3O2S HCl) C, H, N.
Experimental
1H NMR spectra were recorded on a Perkin±Elmer R-24
high resolution spectrophotometer and chemical shifts
were reported relative to tetramethylsilane. IR spectra
were obtained on a Beckman Acculab 8 grating spec-
trophotometer. Elemental analysis were performed by
Atlantic Microlab Inc., Norcross GA and are within

M. Hellberg et al. / Bioorg. Med. Chem. 8 (2000) 1917±1923
1921
Anhydro-[3-dimethylaminomethyl)-6-ethyl-7-oxo-8-benzyl-
5 - hydrothiazolo[3,2 - a]pyridinium hydroxide) HCl] (3h).
The procedure described for 3c was used with 2-(benzyl-
amino)-4-(dimethylaminomethyl)thiazole(0.8 g, 3.6 mmol)
and bis(2,4,6-trichlorophenyl) benzylmalonate (1.8 g, 3.6
mmol) to give a clear oil following column chromato-
graphy (chloroform) which solidi®ed on standing. The
solid was dissolved in anhydrous tetrahydrofuran and HCl
gas was bubbled through the solution. The white solid that
formed was collected by ®ltration and recrystallized from
absolute ethanol yielding 0.62 g (52%) of a white solid,
mp 251±253 ꢀC. IR (free base, KBr) 3100, 2940 (C H),
2950 (C H), 1685, 1640 (CˆO), 1600 cm (C H). H
NMR (free base, CDCl3) d 7.40 (s, 5, Ph), 7.10 (s, 1
thiazole H), 5.30 (s, 2, CH2-Ph), 4.20 (s, CH2-N(CH3)2),
2.80±2.30 (m, 8, CH3-CH2-, (CH3)2-N-), 1.20 (t, 3, CH3-
CH2). Anal. (C18H28N3O2S.HCl) C, H, N.
Anhydro-(1,3-diethyl-4-oxo-7-methyl-2-hydroxypyrido
[1,2-a]pyridinium hydroxide) (6b). The procedure descri-
bed for 3c was used with 2-(ethylamino)-5-methylpyridine
(0.80 g, 5.9 mmol) and bis(2,4,6-trichlorophenyl) ethyl-
malonate (2.9 g, 4.9 mmol) to give 6b. Recrystallization
from 2-propanol yielded 0.85 g (62%) of a green±yellow
solid, mp 233±235 ꢀC. IR (KBr) 2960 (C H), 1690, 1620
(CˆO), 1570 cm (CˆC, CˆN). H NMR (CDCl3) d
9.20 (s, 1, pyridine H), 8.00 (d, 1, pyridine H), 7.45 (d, 1,
pyridine), 4.40 (q, 2, CH2-N), 2.80 (q, 2, CH2-C), 1.40
(t, 3, CH3-CH2-N), 1.20 (t, 3, CH3-CH2-C). Anal.
(C13H16N2O2) C, H, N.
1
1
1
1
Anhydro-(1-pentyl-3-ethyl-4-oxo-2-hydroypyrido[1,2-a]
pyrimidinium hydroxide) (6c). The procedure described
for 3c was used with 2-(pentylamino)pyridine (0.60 g,
3.6 mmol) and bis(2,4,6-trichlorophenyl) ethylmalonate
(1.80g, 3.6mmol) to give a yellow oil following column
chromatography (chloroform:methanol, 95:5). Trituration
with anhydrous ether produced a yellow solid, which
was recrystallized from 2-propanol to yield 0.45 g (48%)
Anhydro-(6-benzyl-7-oxo-8-ethyl-5-hydrothiazolo[3,2-a]
pyrimidinium hydroxide) (3i). The procedure described
for 3c was used with 2-(ethylamino)thiazole (0.45 g, 3.6
mmol) and bis(2,4,6-trichlorophenyl) benzylmalonate
(2.0 g, 3.6 mmol) to give 3i after column chromatography
(chloroform:methanol, 9:1). Recrystallization from tol-
uene yielded 0.39 g (38%) of 3i mp 167±168.5 ꢀC. IR
(KBr) 3100 (C H), 1685, 1620 (CˆO), 1600 cm 1 (CˆC,
CˆN). 1H NMR (CDCl3) d 7.95 (d, 1, thiazole H), 7.50±
7.10 (m, 5, Ph), 6.95 (d, 1, thiazole-H), 4.10±3.80 (m, 4,
N-CH2, Ph-CH2), 1.25 (t, 3, CH3). Anal. (C15H14N2O2S)
C, H, N.
of 6c as a yellow solid, mp 85±87 ꢀC. IR (free base, KBr)
1
2970 (C H), 1690, 1650 (CˆO), 1555 cm
(CˆC,
1
CˆN). H NMR (free base, CDCl3) d 9.50 (d, 1, pyri-
dine H), 7.80±7.30 (m, 2, pyridine H), 4.35 (t, 2, CH2-
N), 2.60 (q, 2, CH2-C), 1.80±0.85 (m, 12, CH3-(CH2)3-,
CH3-CH2-C). Anal. (C15H20N2O2) C, H, N.
Anhydro-(1-benzyl-3-ethyl-4-oxo-2-hydroxypyrido[1,2-a]
pyrimidinium hydroxide) (6d). The procedure described
for 3c was used with 2-(benzylamino)pyridine (0.70 g,
3.8 mmol) and bis(2,4,6-trichlorophenyl) ethylmalonate
(1.90 g, 3.8 mmol) to give 6d. Recrystallization from
absolute ethanol yielded 0.70 g (70%) of a white solid,
mp 181±183 ꢀC. IR (free base, KBr) 3150, 2970 (C H),
Anhdro-[6-benzyl-7-oxo-8-(2-dimethylaminoethyl)-5-hy-
droxythiazolo[3,2 - a]pyrimidinium hydroxide] HCl (3j).
The procedure described for 3c was used with 2-[2-dime-
thylamino)ethylamino]thiazole (1.2g, 7.2 mmol) and bis
(2,4,6-trichlorophenyl) benzylmalonate (1.2 g, 7.2 mmol)
to give a clear oil following column chromatography
(ethyl acetate:hexane, 20:1), which solidi®ed upon tri-
turation with anhydrous ether. Recrystallization from 2-
propanol yielded 1.3 g of a white solid, mp 123±124 ꢀC.
To form the hydrochloride salt the free base was dis-
solved in anhydrous tetrahydrofuran and HCl gas was
bubbled through the solution. The white solid that
formed was collected by ®ltration and recrystallized
from methanol yielding 0.95 g (37%) of a white solid, mp
254±256 ꢀC. IR (free base, KBr) 3100, 2940 (C H), 2950
1
1
1700, 1650 (CˆO), 1620, 1580 cm (CˆC, CˆN). H
NMR (free base, CDCl3) d 9.50 (d, 1, pyridine H), 7.95
(t, 1, pyridine H), 7.50±7.10 (m, 6, Ph H, pyridine H),
5.55 (s, 2, CH2-Ph), 2.75 (q, 2, CH2-CH3), 1.30 (t, 2,
CH3). Anal. (C17H16N2O2) C, H, N.
Anhydro-(1,3-diethyl-4-oxo-2-hydroxypyridazino[1,2-b]
pyrimidinium hydroxide) (7). The procedure described
for 3c was used with 2-(ethylamino)pyridazine (0.60 g,
4.9 mmol) and bis(2,4,6-trichlorophenyl) ethylmalonate
(2.4 g, 4.9 mmol) to give 7. Recrystallization from 2-
propanol yielded 0.65 g (61%) of a yellow solid, mp
226±228 ꢀC. IR (KBr) 2960 (C H), 1660, 1630 (CˆO),
1600 cm 1 (CˆC, CˆN). 1H NMR (CDCl3) d 8.90 (d, 1,
pyridazine H), 8.20±8.00 (m, 2, pyridazine H), 4.50 (q,2,
CH2-N), 2.70 (q, 2, CH2-C), 1.40 (t, 3, CH3-CH2-N),
1.15 (t, 3, CH3-CH2-C). Anal. (C11H13N3O2) C, H, N.
1
(C H), 1680, 1640 (CˆO) cm 1. H NMR (free base,
CDCl3) d 8.00 (d, 1, thiazole H), 7.50±7.10 (m, 5, Ph), 6.90
(d, 1 thiazole), 4.10 (t, 2, CH2-CH2-N-(CH3)2), 3.85 (s, 2,
PH-CH2-), 2.70 (t, 2, CH2-CH2-N-(CH3)2), 2.30 (s, 6,
(CH3)2N-). Anal. (C17H19N3O2S.HCl.H2O) C, H, N.
Anhydro-(1,3-diethyl-4-oxo-2-hydroxypyrido[1,2-a]pyri-
midinium hydroxide) (6a). The procedure described for 3c
was used with 2-(ethylamino)pyridine (0.25 g, 2.1 mmol)
and bis(2,4,6-trichlorophenyl) ethylmalonate (1.1 g, 2.1
mmol) to give 6a. Recrystallization from absolute ethanol
yielded 0.24g (52%) of a yellow solid, mp 178.5±179.5 ꢀC.
Anhydro-(1,3-diethyl-4-oxo-2-hydroxypyrimido[1,6-a]
pyrimidinium hydroxide) (8). The procedure described
for 3c was used with 4-(ethylamino)pyridine (0.60 g,
4.9 mmol) and bis(2,4,6-trichlorophenyl) ethylmalonate
(2.4 g, 4.9 mmol) to give 8. Recrystallization from 2-
propanol yielded 0.92 g (85%) of a yellow solid, mp
165±167 ꢀC. IR (KBr) 2980 (C H), 1690, 1640 (CˆO),
1605 cm 1 (CˆC, CˆN). 1H NMR (CDCl3) d 10.0 (s, 1,
pyrimidine H), 8.20 (d, 1, pyrimidine H), 7.40 (d, 1,
pyrimidine H), 4.40 (q, 2, CH2-N), 2.70 (q, 2, CH2-C),
1
IR (KBr) 3080, 2950 (C H), 1685, 1640 (CˆO), 1555 cm
(CˆC, CˆN). 1H NMR (CDCl3) d 9.50 (d, 1, pyridine H),
8.15 (t, 1, pyridine H), 7.70±7.20 (m, 2, pyridine H), 4.40 (t,
2, CH2-N), 2.70 (q, 2, CH2-C), 1.60±1.00 (m, 6, CH3-CH2-
N, CH3-CH2-C). Anal. (C11H13N2O2) C, H, N.

1922
M. Hellberg et al. / Bioorg. Med. Chem. 8 (2000) 1917±1923
1.40 (t, 3, CH3-CH2-N), 1.20 (t, 3, CH3-CH2-C). Anal.
(C11H13N3O2) C, H, N.
Measurement of aggregation
Aggregation was determined by the turbidometric
method of Born24 using a Bio/Data Corporation Platelet
Aggregation Pro®ler PAP/2A. platelet-rich plasma
(400 mL) and barbital bu€ered saline (pH 7.4, 100 mL) or
baribtal bu€ered saline solution of test drug (pH 7.4,
100 mL) were incubated for 20 min at 37 ꢀC. Ten microliters
of a barbital bu€ered saline solution of ADP (the ®nal
concentration of ADP was 2.5 mL) was added to the
stirred sample and the change in optical density was recor-
ded. An initial minimum 100% inhibitory concentration
was determined by ®nding a concentration at which the
inhibitor gave a complete blockade of ADP-induced
aggregation. A series of dilutions was prepared and
evaluated in subsequent tests until the agent no longer
gave 100% blockade (data not shown). The initial
minimum 100% inhibitory concentration was the lowest
concentration of agent that still produced 100% inhibition
of aggregation. The actual value is bounded by the
minimum 100% inhibitory concentration and the next
lowest concentration employed. Values for the initial
minimum 100% inhibitory concentration are within a
factor of two as the next lowest concentration examined
di€ered by a factor of two. To con®rm the ®nal minimum
100% inhibitory concentration (MIC100), tests were
repeated using the minimum 100% inhibitory concentra-
tion and the next lowest concentration. The MIC100 values
reported in Tables 1 and 2 represent the smaller of the two
concentrations used to produce the e€ect.
Anhydro-(1,3-diethyl-4-oxo-2-hydroxypyrazino[1,2-a]pyr-
imidinium hydroxide) (9). The procedure described for 3c
was used with 2-(ethylamino)pyrazine (0.51 g, 4.2 mmol)
and bis(2,4,6-trichlorophenyl) ethylmalonate (2.0 g,
4.2 mmol) to give 9. Recrystallization from 2-propanol
yielded 0.59g (64%) of an orange solid, mp 198±99.5ꢀC.
1
IR (KBr) 2970 (C H), 1676, 1645 (CˆO), 1550cm
1
(CˆC, CˆN). H NMR (free base, CDCl3) d 9.30 (s
imposed on d, 2, pyrazine H), 8.60 (d, 1, pyrazine H), 4.60
(q, 2, CH2-N), 2.70 (q, 2, CH2-C), 1.80 (t, 3, CH3-CH2-N),
1.20 (t, 3, CH3-CH2-C). Anal. (C11H13N3O2) C, H, N.
Anhydro-(1,3-diethyl-4-oxo-2-hydroxypyrimido[1,2-a]
pyrimidinium hydroxide) (10a). The procedure described
for 3c was used with 2-(ethylamino)pyrimidine (0.60 g,
4.9 mmol) and bis(2,4,6-trichlorophenyl) ethylmalonate
(2.2 g, 4.9 mmol) to give 10a. Recrystallization from 2-
propanol yielded 0.74g (69%) of an orange solid, mp 169±
171 ꢀC. IR (KBr) 2950 (C H), 1700, 1690 (CˆO), 1600,
1
1
1580 cm (CˆC, CˆN). H NMR (free base, CDCl3) d
9.70 (d, 1, pyrimidine H), 9.15 (dd, 1, pyrimidine H), 7.55
(dd, 1, pyrimidine H), 4.60 (q, 2, CH2-N), 3.70 (q, 2, CH2-
C), 1.40 (t, 3, CH3-CH2-N), 1.20 (t, 3, CH3-CH2-C). Anal.
(C11H13N3O2) C, H, N.
Anhydro-(3-ethyl-4-oxo-1-pentyl-2-hydroxypyrimido[1,2-a]-
pyrimidinium hydroxide) (10b). The procedure described
for 3c was used with 2-(pentylamino)pyridine (0.69 g,
4.2 mmol) and bis(2,4,6-trichlorophenyl) ethylmalonate
(2.1 g, 4.2 mmol) to give 10b. Recrystallization from ethyl
acetate yielded 0.77 g (62%) of a yellow green solid, mp
114.5±115.5 ꢀC. IR (KBr) 2960 (C H), 1690, 1650
Gel ®ltration-column preparation
A cylinder of a 50 mL plastic syringe (Becton-Dickson)
with a 3.0 cm internal diameter was used as a chromato-
graphy column. Sepharose 2B (Pharmacia Fine Chemicals)
was prepared and stored in calcium-free Tyrodes bu€er
(267). The column was prepared by inserting a porous
plastic sleeve (Fenwal Lab). The gel slurry was added,
allowed to pack to the 50 mL mark on the syringe and
stored at 4 ꢀC between uses. Prior to use the column was
allowed to warm to room temperature and to equilibrate
with modi®ed calcium-free Tyrodes bu€er which was
used as an elution bu€er (267).
1
1
(CˆO), 1600 cm (CˆC, CˆN). H NMR ( CDCl3) d
9.60 (dd, 1, pyrimidine H), 9.05 (dd, 1, pyrimidine H),
7.40 (t, 1, pyrimidine H), 4.50 (q, 2, CH2-N), 2.70 (q, 2,
CH2-C), 2.30±0.85 (m, 12, (CH2)3CH3, CH3-CH2-C).
Anal. (C14H19N3O2) C, H, N.
Anhydro-(1-benzyl-3-ethyl-4-oxo-2-hydroxypyrimido[1,2-
a]pyrimidinium hydroxide) (10c). The process described
for 3c was used with 2-(benzylamino)pyridine (0.40 g,
4.2 mmol) and bis(2,4,6-trichlorophenyl) ethylmalonate
(2.1 g, 4.2 mmol) to give 10c. Recrystallization from
ethyl acetate yielded 0.77 g (62%) of a orange solid, mp
120±22 ꢀC. IR (KBr) 2960 (C H); 1690, 1640 (CˆO);
Gel ®ltration of platelets
Platelet-rich plasma (6.5 mL) was treated with either
barbital bu€ered saline (pH 7.4, 0.5 mL) or drug dissolved
in barbital bu€ered saline (0.5 mL). The treated platelet-
rich plasma was allowed to incubate for 30 min at room
temperature. After the incubation period, duplicate sam-
ples of platelet-rich plasma (400 mL of platelet-rich
plasma, 100 mL of platelet poor plasma) and treated
platelet-rich plasma (400 mL of platelet-rich plasma,
100 mL of platelet poor plasma) were tested for platelet
aggregation stimulated by 2.5 mM ADP. The remaining
treated plasma was gel-®ltered. The gel ®ltered platelets
were collected in 3 mL fractions in plastic tubes. The
appearance of gel-®ltered platelets was detected visually
and platelet counts were done to insure that the platelet
count was within the bounds required by the platelet
aggregation pro®ler. Duplicate samples of gel-®ltered
1
1
1610 cm (CˆC, CˆN). H NMR (CDCl3) d 9.65 (dd,
1, pyrimidine H), 9.10 (dd, 1, pyrimidine H), 7.40 (m, 6,
Ph, pyrimidine H), 6.70 (s,2, CH2-Ph), 2.70 (q, 2, CH2-
C), 1.15 (t, 3, CH3). Anal. (C14H19N3O2) C, H, N.
Preparation of platelet suspensions
Venous blood was obtained from normal male volun-
teers who had not ingested any known platelet inhibitory
drugs for at least 72 h. The blood (9 mL) was added to
3.9% sodium citrate (1 mL) in a plastic tube. The blood
was mixed and centrifuged at 150Âg for 20 min, and the
supernatant was removed and was designated as platelet-
rich plasma. The remainder of the blood was centrifuged
at 2000Âg for 20 min to obtain platelet poor plasma.

M. Hellberg et al. / Bioorg. Med. Chem. 8 (2000) 1917±1923
1923
(400 mL of platelet-rich plasma, 100 mL of platelet poor
plasma) and the corresponding controls (400mL of plate-
let-rich plasma, 100 mL of platelet poor plasma) were
tested for platelet aggregation when stimulated by
2.5 mM ADP. The study was performed in duplicate.
11. Coburn, R. A.; Taylor, M. D.; Wright, W. L. J. Pharm.
Sci. 1981, 70, 1322.
12. Coburn, R. A.; Bhooshan, B. J. Org. Chem. 1975, 38,
3868.
13. Flethcher, C.; Pearson, C.; Choi, S. C.; Duma, R. J.;
Evans, H. J.; Qureshi, G. D. J. Lab. Clin. Med. 1986, 108, 217.
14. Stubbins, J. F.; Pearson, C.; Qureshi, G. D. Thrombosis
Res. 1988, 51, 417.
15. Glennon, R. A.; Rogers, M. E.; El-Said, M. K. J. Het-
erocycl. Chem. 1980, 17, 337.
References and Notes
16. Coburn, R. A.; Glennon, R. A.; Chmielewicz, Z. J. Med.
Chem. 1974, 17, 1025.
17. Glennon, R. A.; Bass, R. G.; Schubert, E. J. Heterocycl.
Chem. 1976, 16, 903.
18. Kappe, T. Monatsch. Chem. 1967, 98, 874.
19. Kappe, T.; Lube, W. Monatsch. Chem. 1971, 102, 781.
20. Kemal, O.; Reese, C. B. J. Chem. Soc. Perkins Trans. 1
1981, 1569.
1. White, H. D. Am. Heart J. 1999, 138, S570.
2. Markwardt, F.; Ho€man, A. Biochem. Pharmacol. 1970,
19, 2519.
3. Varagaftig, B. B.; Chignard, M.; Benveniste, J. Biochem.
Pharmacol. 1981, 30, 263.
4. Holmsen, H. Ser. Haematol 1975, 8, 50.
5. Glennon, R. A.; Rogers, M. E.; Bass, R. G.; Ryan, S. B. J.
Pharm. Sci. 1978, 67, 1762.
21. Steck, E. A.; Fletcher, L. T. J. Heterocycl. Chem. 1974, 11,
1077.
22. Brown, D. J. The Pyrimidines; Interscience: NY, 1962, p
162.
23. Silberg, A.; Frenkel, A. Bull. Chem. Soc. Fr 1967, 6, 2235.
24. Born, G. V. R. Nature 1962, 194, 927.
25. Puri, R. N. Biochem. Pharmacol. 1999, 57, 851.
26. Tangen, O.; Berman, H. J.; Marfay, P. Thrombosis. Dia-
thes. Haemorrh 1971, 21, 268±278.
6. Rogers, M. E.; Glennon, R. A.; Smith, J. D.; Boots, M. R.;
Nanavati, N.; Maconaughey, J. E.; Aud, D.; Mbagwu, G. J.
Med. Chem. 1981, 24, 1284.
7. Glennon, R. A.; Rogers, M. E.; Smith, J. D.; El-Said, M.
K.; Egle, J. L. J. Med. Chem. 1981, 24, 658.
8. Glennon, R. A.; Gaines, J. J.; Rogers, M. E. J. Med. Chem.
1981, 24, 766.
9. Mbagwu, G. O.; Bass, R. G.; Glennon, R. A. J. Heterocycl.
Chem. 1985, 22, 465.
27. Gordon, A. J., Ford, R. A. The Chemist's Companion,
Wiley-Interscience: NY, 1972.
10. Coburn, R. A.; Glennon, R. A. J. Heterocycl. Chem. 1973,
10, 487.