Discovery and preliminary SAR of 5-arylidene-2,2-dimethyl-1,3-dioxane-4,6- diones as platelet aggregation inhibitors

Article abstract of DOI:10.2174/138620712801619122

We herein document the discovery of 5-arylidene-2,2-dimethyl-1,3-dioxane-4, 6-diones as a novel family of platelet aggregation inhibitors. The preliminary optimization study enabled us to establish the most salient features of the structure-activity relat

Full text of DOI:10.2174/138620712801619122

Combinatorial Chemistry & High Throughput Screening, 2012, 15, 551-554
551
Discovery and Preliminary SAR of 5-Arylidene-2,2-Dimethyl-1,3-Dioxane-
4,6-Diones as Platelet Aggregation Inhibitors
Abdelaziz El Maatougui1, JhonnyAzuaje1, Alberto Coelho1,2, Ernesto Cano3, Matilde Yañez3,
Carmen López2, Vicente Yaziji1, Carlos Carbajales1 and Eddy Sotelo*,1,2
1Center for Research in Chemical Biology and Molecular Materials, University of Santiago de Compostela, Santiago de
Compostela, 15782, Spain
2Department of Organic Chemistry, University of Santiago de Compostela, Santiago de Compostela, 15782, Spain
3Department of Pharmacology, Faculty of Pharmacy, University of Santiago de Compostela, Santiago de Compostela,
15782, Spain
Abstract: We herein document the discovery of 5-arylidene-2,2-dimethyl-1,3-dioxane-4,6-diones as a novel family of
platelet aggregation inhibitors. The preliminary optimization study enabled us to establish the most salient features of the
structure-activity relationships in this series as well as to identify novel derivatives that are upto 60 times more potent than
the hit structure 1 and slightly superior to the reference drug Milrinone.
Keywords: Antiplatelet agents, Meldrum´s acid, platelet aggregation inhibitors.
INTRODUCTION
ideal antiplatelet agent [8]. As a result, the discovery of
novel chemotypes that exhibit antiplatelet activity is a goal
of great interest, not only for their possible evolution into
clinical candidates but also because such compounds could
be employed as valuable pharmacological tools to provide
comprehensive information about platelet function.
The hemostatic system is designed to maintain fluid
blood flow under physiological conditions and to react
rapidly to form a clot at sites of vascular damage [1]. In this
exquisitely regulated system platelets play key roles as
sentinels of vascular integrity [2]. The interaction of platelets
Meldrum´s acid (2,2-dimethyl-1,3-dioxane-4,6-dione)
was first synthesized and misassigned by A. N. Meldrum in
1908 [9] and correctly elucidated in 1948 by Davidson and
Bernhard [10]. This compound exhibits unique structural
features that make it a tremendously attractive molecule
from practical and theoretical points of view [11, 12]. While
Meldrum´s acid and its derivatives have been extensively
employed in the preparation of heterocyclic libraries that
show manifold biological activities [13], the pharmaco-
logical properties associated with the 1,3-dioxane-4,6-dione
core remain practically unexplored. Some recent reports on
remarkable biological activities have highlighted the as yet
untapped potential of such a chemotype in medicinal
chemistry [14, 15]. Within the framework of a chemical
genomics project aimed at the discovery of novel
chemotypes that exhibit antiplatelet activity through original
mechanisms of action, we recently started a high-throughput
screening program of our in-house library (ca. 4000
compounds) in the search for new chemotypes with
antiplatelet effect. Herein we document the discovery and
some preliminary features of the structure-activity relation-
ship of 5-arylidene-2,2-dimethyl-1,3-dioxane-4,6-diones as a
novel family of structurally simple and potent antiplatelet
agents.
with collagen and/or thrombin initiates
a
complex
biochemical sequence of signal transduction resulting in a
functional cell response [3]. Although this mechanism is
vital when tissue is damaged, inappropriate activation of
platelets is an important pathogenic factor in widespread
cardiovascular diseases, such as myocardial and cerebral
circulatory disorders, venous thrombosis and arteriosclerosis.
However, failure of platelets to become activated can lead to
excessive bleeding.
Small molecules that are able to modulate platelet
function represent a well-established approach to prevent the
diseases mentioned above [4]. Inhibitors of platelet
aggregation currently employed in therapy (Fig. 1) are
represented by three main drug categories, which include
non-steroidal
anti-inflammatory
drugs
(Aspirin)[5],
adenosine diphosphate receptor antagonists (Clopidogrel or
Ticlopidine) [6], and glycoprotein IIb/IIIa (GPIIb/IIIa)
antagonists (Tirofiban) [7]. Despite the documented utility of
these drugs, their efficacy and selectivity are not sufficiently
high and there are reasons to believe that substantial
improvements in antiplatelet therapy can be made. This issue
is of primary importance since none of the currently
marketed antithrombotic agents, or indeed most of the
compounds in advanced clinical trials, are free of significant
bleeding risk or meet the basic requirements expected for the
Two hit compounds (Fig. 2) were identified during a
screening campaign performed on a library containing
diverse 5-substituted Meldrum´s acid derivatives (e.g. 5-
acyl, 5-alkyl, 5-alkylidene and 5-arylidene). While most of
the 1,3-dioxane-4,6-dione congeners were inactive, 5-
benzylidene derivative 1 and its azaanalog 2 (Fig. 2)
emerged as encouraging hit structures due to their structural
*Address correspondence to this author at the Center for Research in
Chemical Biology and Molecular Materials, University of Santiago de
Compostela, Santiago de Compostela, 15782, Spain; Tel: ++34-881815732,
E-mail: e.sotelo@usc.es
1875-631X/12 $58.00+.00
© 2012 Bentham Science Publishers

552 Combinatorial Chemistry & High Throughput Screening, 2012, Vol. 15, No. 7
El Maatougui et al.
HN
O
Cl
Cl
MeOOC
N
H
COOH
COOH
N
HN
OCOMe
SO2
S
S
Tirofiban
Aspirin
Ticlopidine
Clopidogrel
Fig. (1). Structures of representative antiplatelet agents in clinical use.
O
O
O
N
O
O
O
O
O
1
2
5-benzylidene-2,2-dimethyl-1,3-dioxane-4,6-dione
2,2-dimethyl-5-(pyridin-2-ylmethylene)-1,3-dioxane-4,6-dione
IC50 = 121.42 μM
IC50 = 82.12 μM
Fig. (2). Structure and antiplatelet activity of the Hit Compounds 1 and 2.
simplicity and relatively potent platelet inhibitory activity
(IC50 = 121.42 μM and 82.12 μM, respectively). As a
preliminary step in assessing the SAR in this series we
decided to evaluate the platelet inhibitory activity of three
structural analogs of hit compound 1 [Fig. 3, 2-benzylidene-
5,5-dimethylcyclohexane-1,3-dione (3), 2-benzylidenemalo-
nic acid (4), and 5-benzyl-2,2-dimethyl-1,3-dioxane-4,6-
dione (5)]. All compounds proved to be inactive, a result that
highlights the contribution of the heterocyclic scaffold and
the exocyclic double bond to the documented biological
activity.
graphy). The analytical and spectral characterization enabled
unequivocal assignments and the results are consistent with
data reported previously.
The platelet aggregation inhibitory activity of the
obtained library (8a-t) and two reference compounds (Sul-
finpyrazone and Milrinone) was examined on washed human
platelets by the turbidimetric method of Born employing
thrombin as agonist [17]. Compounds were tested in DMSO
solutions and reported IC50 values are the mean of at least
three experiments (in duplicate) in which human blood from
different individuals was used. Active compounds inhibited
platelet aggregation in a dose-dependent manner. The results
of these experiments are summarized in Table 1.
In an effort to complete the SAR studies a focused library
of 2,2-dimethyl-1,3-dioxane-4,6-diones (8) incorporating
different arylidene, heteroarylidene, and alkylidene residues
at position 5 of the heterocyclic scaffold was synthesized
(Scheme 1) by following a recently reported environmentally
friendly procedure [16]. Target compounds were isolated by
precipitation and purified by recrystallization (or chromato-
Inspection of the biological data (Table 1) shows that
structural manipulation of hit structure 1 enabled the
identification of novel derivatives that are generally more
potent (2- to 60-fold) than 1 and the reference compound
Sulfinpyrazone. Several of the derivatives described here
O
O
O
O
HO
O
O
O
HO
O
O
O
O
O
1
3
4
5
IC50 = 121.42 μM
Inactive
Inactive
Inactive
Fig. (3). Comparison of the antiplatelet activity of 1 and three analogs (3-5).
O
O
R
R
CTMAB
Water
+
O
O
O
H
O
O
O
O
6
7
8a-t
Scheme 1. Synthesis of the 5-alkylidene-2,2-dimethyl-1-3-dioxane-4,6-dione library.

5-Arylidene-2,2-Dimethyl-1,3-Dioxane-4,6-Diones
Combinatorial Chemistry & High Throughput Screening, 2012, Vol. 15, No. 7 553
have IC50 values in the low micromolar range (2-25 μM). In
particular, compounds 8j and 8m (IC50 = 2.10 μM and 3.40
μM, respectively) exhibit antiplatelet activity that is slightly
superior to that determined for Milrinone. Analysis of the
data in Table 1 reveals the substantial contribution of the
group(R) on the exocyclic double bond to the documented
platelet inhibitory activity. As it can be observed, active
compounds incorporate aryl (8a-n) or heteroaryl (8o-q)
fragments, whereas derivatives containing alkyl residues (8r-
t) were inactive. This result confirms the relevance of the
(het)aryl group, which together with the 1,3-dioxane-4,6-
dione and the exocyclic double bond can be considered part
of the pharmacophore.
The screening of compounds that incorporate substituents
at diverse positions of the phenyl residue (Table 1,
compounds 8b-n) showed that such a modification provides
derivatives with improved platelet aggregation inhibitory
activity in comparison to the reference compound (8a, R =
Ph IC50 = 121.42μM). Although favorable, the change in
biological profile was clearly dependent on the nature of the
introduced group and, more importantly, on the substitution
pattern at the phenyl ring. A common structural feature of all
compounds that showed potent antiplatelet activity (IC50< 20
μM) was the presence of a substituent at position 2, a factor
that is particularly important for derivatives that have a 2,4-
or 2,6-substitution pattern (e.g., 8g, 8h, 8i, 8m, 8n). The
exceptions to this trend are those derivatives that contain
methoxy groups (8f, 8i, 8k and 8l). Although the limited
number of compounds incorporating heterocyclic moieties
precludes a detailed analysis of the structure-activity
relationship, the superior antiplatelet effect observed for the
2-thiophenyl and 2-furyl derivatives suggests that a similar
SAR trend could be operating in these series. It is important
to highlight that compounds 8j and 8m exhibited outstanding
antiplatelet activity (IC50 = 2.10 μM and 3.40 μM,
respectively) -i.e. 2-times higher than that determined for the
Table 1. Antiplatelet Activity of 5-Arylidene-2,2-dimethyl-1-
3-dioxane-4,6-diones (8)
O
O
R
O
O
Antiplatelet Activity
Compound
R
Yield (%)
IC50 (μM)ꢀ
reference compound Milrinone under the same experimental
conditions.
8a
8b
8c
8d
8e
8f
Ph
82
72
45
64
58
68
90
74
76
71
77
59
56
63
71
65
76
37
43
48
121.42
43.21
47.60
21.58
33.94
57.83
19.45
16.33
39.27
2.10
In summary, we have documented the discovery and
structural optimization of a new family of potent antiplatelet
agents. The preliminary exploration around the hit structure
1 generated new potent derivatives and also highlighted the
most salient features of the SAR in this series. Further
studies are in progress in our laboratories to identify novel
derivatives exhibiting sub-micromolar activity derived from
the chemotype documented here and to establish the
mechanism of action of these promising derivatives.
Ph-4-OMe
Ph-4-OH
Ph-4-Cl
Ph-4-F
Ph-2-OMe
Ph-2-Cl
8g
8h
8i
Ph-2-NO2
Ph-2,4-OMe
Ph-2,4-Cl
EXPERIMENTAL
The compounds described here were obtained according
to previously reported procedures. The synthesis, isolation
and purification of all compounds were accomplished using
equipment routinely available in laboratories for parallel
synthesis. Commercially available starting materials and
reagents were purchased and used without further purificat-
ion from freshly opened containers. Isolation of precipitated/
triturated products was performed in a 12-channel vacuum
manifold from Aldrich. The NMR spectra were recorded on
a Bruker AM300 MHz (1H) and 75 MHz (13C) and XM500
spectrometers. The spectroscopic and analytical data, which
are consistent with previously reported data, for representa-
tive compounds are presented in the experimental section.
The platelet aggregation inhibitory activity of the obtained
library was determined according to a previously described
procedure.
8j
8k
8l
Ph-2-Cl-5-OMe
Ph-2,6-OMe
Ph-2,6-Cl
73.25
28.03
3.40
8m
8n
8o
8p
8q
8r
Ph-2-Cl-6-F
2-Thiophene
2-Furane
6.84
17.36
29.46
82.12
*
2-Pyridine
CH2CH3
8s
8t
CH(CH3)2
*
CH2 CH2CH3
Sulfinpyrazone
Milrinone
*
509.49
4.70
5-Benzylidene-2,2-dimethyl-1,3-dioxane-4,6-dione (8a).
Yield: 82%, mp 83-85oC (i-PrOH). IR (KBr): ꢀmax= 1765,
1
1730, 1580 cm-1. H NMR (300 MHz, CDCl3, ppm) ꢁ 8.36
ꢀThe antiplatelet activities of the standards and the synthesized compounds were tested
by the turbidimetric method in human washed platelets using thrombin as agonist.
Methods for these assays have been published elsewhere [17]. * Inactive or IC50
>200μM. Results shown are means of at least three experiments (in duplicate). The
highest mean standard error of IC50 values was 10%.
(s, 1H), 7.18 - 7.16 (d, 2H, J = 8.2Hz), 7.05 (t, 2H, J = 8.2
Hz), 6.93 - 6.90 (m, 1H), 1.70 (s, 6H). 13C NMR (75 MHz,
CDCl3, ppm) ꢁ 162.9, 159.4, 157.7, 133.3, 131.5, 128.5,
114.7, 104.3, 27.4.

554 Combinatorial Chemistry & High Throughput Screening, 2012, Vol. 15, No. 7
El Maatougui et al.
5-(4-Methoxybenzylidene)-2,2-dimethyl-1,3-dioxane-4,
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ACKNOWLEDGEMENTS
This work was financially supported by a Consolidation
Group Research Grant from the Consellería de Educación to
E. Sotelo (Xunta de Galicia). A. Coelho is a research fellow
of the Isidro Parga Pondal program (Xunta de Galicia,
Spain). J.A. thanks FUNDAYACUCHO (Venezuela) for a
pre-doctoral grant.
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Sotelo, E. Design, synthesis, and structure-activity relationships of
a novel series of 5-alkylidenepyridazin-3(2H)-ones with a non-
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CONFLICT OF INTEREST
Declared none.
Received: December 22, 2011
Revised: January 18, 2012
Accepted: February 10, 2012