Design, synthesis and evaluation of aspirin analogues having an additional carboxylate substituent for antithrombotic activity

Article abstract of DOI:10.1016/j.bmcl.2009.05.120

Acetylsalicylic acid (aspirin) is an effective long-term prophylaxis of thrombotic events such as heart attacks and strokes. It covalently inhibits prostaglandin-H-synthase by interacting with Arg120 or Tyr385 at the active site allowing delivery of its a

Full text of DOI:10.1016/j.bmcl.2009.05.120

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4213–4216
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and evaluation of aspirin analogues having an additional
carboxylate substituent for antithrombotic activity
*
Ahmed Alagha, Edelmiro Moman, Mauro F. A. Adamo, Kevin B. Nolan, Anthony J. Chubb
Centre for Synthesis and Chemical Biology, Department of Pharmaceutical and Medicinal Chemistry, Royal College of Surgeons in Ireland, 123 St Stephen’s Green, Dublin 2, Ireland
a r t i c l e i n f o
a b s t r a c t
Article history:
Acetylsalicylic acid (aspirin) is an effective long-term prophylaxis of thrombotic events such as heart
attacks and strokes. It covalently inhibits prostaglandin-H-synthase by interacting with Arg120 or
Tyr385 at the active site allowing delivery of its acetyl group to Ser530. However the structure has not
been optimized to fit the active site. We have designed acetylsalicylate analogues with an additional car-
boxylate substituent which allows simultaneous interaction with Arg120 and Tyr385 whilst positioning
the acetyl group in close proximity to Ser530. One of these, an ester derivative which unlike acetylsali-
cylic acid is non-acidic, may act as useful lead compound for further exploitation of this approach.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 18 March 2009
Revised 22 May 2009
Accepted 27 May 2009
Available online 13 June 2009
Keywords:
Aspirin
COX
Prostaglandin H2 synthase
Inhibitor
Arg120
Ser530
Acetylation
Tyr385
Platelet aggregation
Thrombosis
Recognition of vessel rupture induces outside-in signalling in
platelets, which leads to activation of phospholipases that release
arachidonate from the lipid bilayer. Arachidonic acid is then con-
verted to prostaglandin G2 (PGG2) in the cyclooxygenase (COX) site
of the enzyme prostaglandin H2 synthase (PGHS). Following this,
PGG2 is converted to prostaglandin H2 at the peroxidase site
(POX) of PGHS, and this in turn is processed to thromboxane A2
(TxA2) by synthases. TxA2 is a potent stimulator of platelet recruit-
ment and aggregation, resulting in clot formation and haemostasis.
However, thrombus formation in at-risk individuals leads to vessel
occlusion, disruption in blood flow, oxygen starvation of down-
stream tissues and ischaemic heart attack or stroke.
Acetylsalicylic acid (aspirin, 1) shows antithrombotic, antipy-
retic, analgesic and antiproliferative effects due to the irreversible
inhibition of PGHS, the rate limiting enzyme in the production of
prostaglandins and thromboxane. Aspirin acetylates Ser530 found
between the positively charged arginine at the mouth of the COX
channel (Arg120), and a deeply buried tyrosine (Tyr385) that initi-
ates the cyclo oxygenation of arachidonate.2,3 This irreversible
acetylation permanently blocks entry of arachidonate to the active
site. Vascular endothelial synthesis of the antithrombotic prosta-
glandin, prostacyclin, is also blocked. Unlike endothelial cells,
platelets are anucleate and are unable to replace the inactivated
PGHS enzymes, thus giving aspirin a therapeutic advantage against
thrombosis.4 Long-term, low daily doses of aspirin remains an
effective inhibitor of platelet function in cardiovascular patients.
The crystal structure of ovine PGHS-1 shows that the PGHS sub-
strate arachidonic acid binds Arg120 by a salt linkage.1 Most aro-
matic carboxylic acid containing non-steroidal anti-inflammatory
drugs (NSAIDs) interact similarly, and orientate the aromatic func-
tionality of the drug towards the cyclooxygenase site at Tyr385.5,6
Interestingly, Tyr385 is critical for aspirin acetylation of Ser530, as
confirmed by site-directed mutation of Tyr385 to phenylalanine
which reduces aspirin action by over 90%.7 The role of Tyr385 is
to stabilize the negatively charged tetrahedral intermediate
formed during acetylation. It follows that a more effective inhibitor
might result by simultaneous interaction with both Arg120 and
Tyr385, while orientating an acetyl group in close proximity to
the hydroxyl oxygen of Ser530.
Docking using the MOE-Dock programme allowing receptor
flexibility8,9, accurately predicted the crystallographic placement
of arachidonate in the crystal structure of ovine PGHS-11,10
,
PDB:1DIY (Fig. 1A), with a root-mean-squared deviation (RMSD)
of 1.11 Å and a binding energy of À14.3 kcal/mol. Similar docking
of acetylsalicylate showed carboxylate binding to either Arg120
or Tyr385, but not both simultaneously (Fig. 1B). The optimal bind-
ing energy was significantly higher at À8.8 kcal/mol, although this
* Corresponding author. Tel.: +353 1 4028621; fax: +353 1 4022168.
E-mail address: achubb@rcsi.ie (A.J. Chubb).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.05.120

4214
A. Alagha et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4213–4216
Figure 1. Docked conformations of arachidonate and aspirin in the PGHS-1 cyclooxygenase site. MOE-Dock was used to place arachidonate (A) and aspirin (B) in the active
site of the ovine PGHS-1 COX site crystal structure 1DIY.1 The PGHS-COX site isoelectric surface is shown semi-transparent, where negative regions are red, positive regions
blue, and neutral regions white. Receptor carbons are green, the crystallographic arachidonate carbons are grey, and the binding pose found using MOE-Dock has yellow
carbons. Nitrogens are blue, oxygens are red. Non-polar hydrogens (white) were removed for clarity. Ser530 is shown in space fill, Arg120, Tyr355 and Tyr385 are shown in
ball-and-stick, and other Cox site residues are shown in line notation. The isolated backbone alpha helix is shown in red ribbon, while the loop regions are depicted with cyan.
Half the site has been cropped for visualization. Light green lines indicate distance measures mentioned in the text. In (B), aspirin bound to Arg120 is shown with yellow
carbons.
is compensated for by irreversible inhibition. If bound to Arg120,
the distance between the acetyl group and Ser530 is 5.88 Å, which
is too far for acetyl group transfer. This is corroborated by the crys-
tallographic position of salicylate in PDB:1PTH11 which is bound to
Arg120 and is separated from Ser530 by 5.54 Å (Fig. 2).
To allow the acetylsalicylate moiety to advance further down the
COX active site channel, while retaining a salt bridge with Arg120,
we introduced an n-pentanoate chain at the meta position, giving
2 as a target molecule. In 2 the n-pentanoate group is capable of
interacting with the active site Arg120, while the salicylate carbox-
ylate interacts with Tyr385, and the acetyl group is in close proxim-
ity to Ser530. MOE-docking showed that 2 could indeed form a salt
bridge with Arg120, and a hydrogen bond to Tyr385, while allowing
the acetyl group to interact favourably with Ser530 (Fig. 3A). The
binding energy of À13.7 kcal/mol is similar to that of the substrate,
and significantly lower than that for aspirin.
Compound 2 was obtained in a 6-step process from salicylic
acid 6, in overall 13% yield as shown in Scheme 1. Esterification
of 6 gave 7 (99% yield), which was converted by Fries rearrange-
ment with glutaric anhydride to 8 (29% yield).12 Reduction of the
ketone 8 with triethylsilane/trifluoroacetic acid at room tempera-
ture13 afforded 9 (79% yield), hydrolysis of which gave 10 (93%
yield). Acetylation of the phenolic group in 10 with acetic anhy-
dride and base in dry DMF afforded the target compound 2 in
60% yield.14
Following this result which may have been due to suboptimal
polarity in 2 or in the length of the alkycarboxylate chain it was
decided to synthesize the more polar 3, the less polar 5 and the
shorter chain alkyl carboxylate containing 4. Compound 3 was ob-
tained by hydrolysis of 8 to give 11 (95% yield) followed by acety-
lation (81% yield), Scheme 1.16 Compound 5 was obtained by
esterification of 2 (55% yield).17 Synthesis of target compound 4
was carried out as illustrated in Scheme 2. Reaction of 7 with
maleic anhydride using 2 equiv of AlCl3 in tetrachloroethane gave
12 (44% yield) which upon catalytic hydrogenation gave 13 (97%
yield), followed by hydrolysis gave 14 (90% yield) and finally acet-
ylation gave target compound 4 (40% yield).18
Compounds 3 and 4 were found to be inactive in platelet aggre-
gation assays at the highest concentration tested (1000 lM). The
platelet aggregation assay measures the increased light transmis-
sion through platelet rich plasma (PRP), freshly isolated from
healthy volunteers, in response to addition of 1.5 mM arachidonic
acid as agonist. Various concentrations of each compound (0.1–
1000 lM; final DMSO concentration <0.4%) were added to stirred
PRP for 5 min prior to arachidonate addition.19 However 5, was
found to inhibit platelet aggregation, with a 50% maximal inhibi-
tion concentration (IC50) of 720 85
lM which is 15-fold less po-
tent than aspirin (47 27 M). Molecular modelling shows that 5
l
is capable of interacting simultaneously with Arg120 and Tyr385
by hydrogen bonding while correctly positioning the acetyl group
near Ser530 for acetyl transfer to occur (Fig. 3B). Furthermore,
the binding energy of À13.3 kcal/mol is similar to that of the dicar-
boxylate form 2.
Compound 2 was found to be inactive in platelet aggregation
assays at the highest concentration tested (1000 lM). In this assay,
arachidonate is used to stimulate human platelets to form aggre-
gates, as would occur at sites of vascular injury, atherosclerotic pla-
que rupture and thrombosis formation. As PGHS-1 is the main form
of prostaglandin H2 synthase in anucleate platelets, it is a conve-
nient and physiologically relevant model for PGHS-1 activity.15
Importantly, 5 had no effect on platelet aggregation stimulated
by 2
centration of inhibitor used (1000
is not due to non-specific destruction of human cells, and that its
lM of the thromboxane analogue U46619, at the highest con-
l
M). This implies that inhibition
CH
O
3
O
O
O
O
OH
O
O
OH
CH
3
CH
3
O
O
CH
O
3
O
O
O
OH
O
OH
OH
O
CH
3
CH3
CH3
O
O
O
5
3
O
O
O
1
OH
OH
2
4
O
Figure 2. Compounds synthesized and tested for anti-platelet activity.

A. Alagha et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4213–4216
4215
Figure 3. Docked conformations of 2 and 5 in the PGHS-1 cyclooxygenase site. MOE-Dock was used to place 2 (A) and 5 (B) in the active site of the ovine PGHS-1 COX site
crystal structure 1DIY.1 Key as per Fig. 1. In (A), the interatomic distances between 2 and Arg120, Ser530 and Tyr385 are 2.34 Å, 2.53 Å and 3.60 Å, respectively. In (B), the
interatomic distances between 5 and Arg120, Ser530 and Tyr385 are 2.51 Å, 3.21 Å, and 2.53 Å, respectively. Arg120 forms hydrogen bonds with either the n-pentanoate or
ester groups, while the salicylate carboxylate or ester groups interact with Tyr385. The acetyl group is in close proximity to Ser530 in both 2 and 5.
COOH
OH
COOCH3
OH
COOCH3
OH
COOCH3
OH
COOH
OH
COOH
OCOCH3
g
a
b
f
e
OH
OH
OH
OH
99%
29%
79%
60%
93%
6
7
8
O
O
9
O
10
O
2
O
95%
55%
c
h
COOH
OH
COOH
COOCH3
OCOCH3
OCOCH3
d
OH
OH
OCH3
81%
11
O
O
3
O
O
5
O
Scheme 1. Reagents and conditions: (a) CH3COCl, MeOH, reflux 12 h; (b) glutaric anhydride 1.0 equiv AlCl3, 2.1 equiv tetrachloroethane (TCE), 1 h rt then 130 °C 4 h; (c)
NaOH 2.0 equiv H2O, reflux 2 h; (d) Ac2O 10 equiv pyridine 100 equiv DMF dry, 24 h rt; (e) (C2H5)3SiH 3.0 equiv TFA, 48 h. rt; (f) NaOH 4.0 equiv H2O, reflux 2 h; (g) Ac2O
10 equiv pyridine 100 equiv DMF dry, 3 h rt; (h) K2CO3 3.0 equiv MeI 10 equiv DMF dry, 12 h rt.
COOCH3
OH
COOCH3
OH
COOCH3
OH
COOH
OH
COOH
OCOCH3
a
b
c
d
40%
44%
97%
90%
7
12
O
13
14
4
O
OH
O
OH
O
OH
O
OH
Scheme 2. Reagents and conditions: (a) maleic anhydride 1.0 equiv AlCl3 2.1 equiv tetrachloroethane (TCE), 1 h rt then 130 °C 2 h; (b) Pd/C 0.1 equiv, MeOH, 12 h rt; (c) NaOH
2.0 equiv H2O, reflux 12 h; (d) Ac2O 4.0 equiv pyridine 3.3 equiv DMF dry, 24 h rt.
mechanism of action is specific to the cyclooxygenase pathway,
downstream of arachidonate and upstream of thromboxane.
Despite the poor correlation between docking studies and data,
compound 5 is sufficiently diverse from aspirin to constitute a new
starting point for investigation. In particular, compound 5 contains
an extra aliphatic chain and two ester groups, offering the possibil-
ity of further derivatization at each of these sites. An added benefit
of this type of derivative is that it is non-acidic and would not
cause topical irritation as aspirin does due to the presence in its
structure of an acidic carboxylic acid group. Finally, compound 5
has been shown by us to specifically inhibit the COX pathway of
platelet activation.
Acknowledgements
We thank the Irish Government under its Programme for Re-
search in Third Level Institutions and the Health Research Board
for financial support.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.05.120.
We had predicted that the introduction of an n-pentanoate
group meta to the carboxylate group of acetylsalicylic acid would
form a salt bridge with Arg120 at the active site of PGHS, whilst
the salicylate carboxylate group would interact with Tyr385 and
allow delivery of the acetyl group to Ser530. Our results show that
an esterified n-pentanoate group forms a sufficiently strong inter-
action with Arg120 for this to occur and we have produced a novel
compound that inhibits PGHS and can be further developed to give
a new type of non-steroidal anti-inflammatory drug.
References and notes
1. Malkowski, M. G.; Ginell, S. L.; Smith, W. L.; Garavito, R. M. Science 2000, 289,
1933.
2. Numbering. The traditional ovine COX-1 numbering system is used in this
report.
3. Mancini, J. A.; Riendeau, D.; Falgueyret, J. P.; Vickers, P. J.; O’Neill, G. P. J. Biol.
Chem. 1995, 270, 29372.
4. Cheng, Y.; Austin, S. C.; Rocca, B.; Koller, B. H.; Coffman, T. M.; Grosser, T.;
Lawson, J. A.; FitzGerald, G. A. Science 2002, 296, 539.
5. Picot, D.; Loll, P. J.; Garavito, R. M. Nature 1994, 367, 243.

4216
A. Alagha et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4213–4216
6. Kurumbail, R. G.; Stevens, A. M.; Gierse, J. K.; McDonald, J. J.; Stegeman, R. A.;
Pak, J. Y.; Gildehaus, D.; Miyashiro, J. M.; Penning, T. D.; Seibert, K.; Isakson, P.
C.; Stallings, W. C. Nature 1996, 384, 644.
7. Hochgesang, G. P.; Rowlinson, S. W.; Marnett, L. J. J. Am. Chem. Soc. 2000, 122,
6514.
8. Docking MOE-Dock 2008.10 function was used for docking. Ligand placement
was performed using alpha sphere triangle matching, with London dG scoring.
The top 30 hits were retained and refined using MMFF94Â forcefield energy
minimization with Generalized Born solvation model, allowing the receptor
residues within 6 Å to relax around the mobile ligand. The receptor side chains
were tethered with a force constant of 1.0 kcal/(mol Å2). Energy minimization
was stopped when the root-mean-squared gradient cutoff of 0.01 kcal/(mol Å)
was reached. Final poses were ranked using the London dG method to calculate
the free energy of binding.
(KBr disc) mmax 3435 br, 2919, 1754, 1695, 1434, 1304 cmÀ1. Elemental Anal.
Calcd for C14H16O6: C, 59.99; H, 5.75. Found: C, 60.54; H, 5.69. HRMS found:
279.0882 [MÀH+] C14H15O6 requires 279.0869.
15. Born, G. V. Nature 1962, 194, 927.
16. 2-Acetoxy-3-(4-carboxy-butyryl)-benzoic acid (3): 1H NMR (400 MHz, DMSO-d6)
d 1.84 (2H, quintet, J = 7.2 Hz), 2.28 (3H, s, CH3), 2.32 (2H, t, J = 7.6 Hz), 3.10
(2H, t, J = 7.2 Hz), 7.37 (1H, d, J = 8.4 Hz, aromatic), 8.17 (1H, dd, J = 8.4, J = 2 Hz,
aromatic), 8.45 (1H, d, J = 2 Hz, aromatic), 12.60 (2H, br, COOH). 13C NMR
(400 MHz, DMSO-d6) d 19.5, 21.3, 33.2, 37.6, 124.9, 125, 131.5, 133.7, 134.8,
153.9, 165.5, 169.4, 174.7, 198.5. IR (KBr disc) mmax 3386–3000 br, 1783, 1707,
1600 cmÀ1. Elemental Anal. Calcd for C14H14O6: C, 58.64; H, 5.30. Found: C,
58.55; H, 5.31. HRMS found: 293.0674 [MÀH+] C14H13O7 requires 293.0661.
17. 2-Acetoxy-3-(4-methoxycarbonyl-butyl)-benzoic acid methyl ester (5): 1H NMR
(400 MHz, DMSO-d6) d 1.59–1.54 (4H, m), 2.27 (3H, s, CH3), 2.34 (2H, t,
J = 6.8 Hz), 2.64 (2H, t, J = 7.2 Hz), 3.58 (3H, s, OCH3), 3.80 (3H, s, OCH3), 7.14
(1H, d, J = 8.4 Hz, aromatic), 7.50 (1H, dd, J = 8.4, J = 2.4 Hz, aromatic), 7.75 (1H,
d, J = 2 Hz). 13C NMR (400 MHz, DMSO-d6) d 20.7, 23.9, 30.1, 33.0, 33.7, 51.2,
122.5, 123.8, 130.7, 134.0, 140.1, 148.0, 164.5, 169.3, 173.3. HRMS found:
309.1341 [M+H+] C16H21O6 requires 309.1338.
9. Colotta, V.; Capelli, F.; Lenzi, O.; Catarzi, D.; Varano, F.; Poli, D.; Vincenzi, F.;
Varani, K.; Borea, P. A.; Dal Ben, D.; Volpini, R.; Cristalli, G.; Filacchioni, G.
Bioorg. Med. Chem. 2009, 17, 401.
10. Structure, The ovine crystal structure 1DIY was used as there are no crystal
structures of human PGHS-1.
11. Loll, P. J.; Picot, D.; Garavito, R. M. Nat. Struct. Biol. 1995, 2, 637.
12. Vijayasarathy, P. R.; Mane, R. B.; Rao, G. S. K. J. Chem. Soc., Perkin Trans. 1 1977,
1, 34.
13. Vorogushin, A. V.; Wulff, W. D.; Hansen, H.-J. J. Am. Chem. Soc. 2002, 124, 6512.
14. 2-Acetoxy-3-(4-carboxy-butyl)-benzoic acid (2): 1H NMR (400 MHz, DMSO-d6) d
1.59–1.51 (4H, m), 2.24 (5H, t, J = 7.2 Hz), 2.63 (2H, t, J = 7.2 Hz), 7.12–7.07 (1H,
m, aromatic), 7.45 (1H, dd, J = 8.4, J = 2.4 Hz, aromatic), 7.73 (1H, d, J = 2.4 Hz,
aromatic), 12.59 (2H, br, COOH). 13C NMR (400 MHz, DMSO-d6) d 20.8, 24.0,
30.2, 33.4, 33.9, 123.5, 123.6, 130.9, 133.5, 139.9, 148.2, 165.7, 169.3, 174.4. IR
18. 2-Acetoxy-3-(3-carboxy-propyl)-benzoic acid (4): 1H NMR (400 MHz, DMSO-d6)
d 1.80 (2H, quintet, J = 7.6 Hz), 2.23 (5H, s), 2.64 (2H, t, J = 7.6 Hz), 7.10 (1H, d,
J = 8 Hz, aromatic), 7.46 (1H, dd, J = 8.4, J = 2 Hz, aromatic), 7.74 (1H, s,
aromatic), 12.60 (2H, br, COOH). 13C NMR (400 MHz, DMSO-d6) d 21.3, 26.6,
33.4, 33.9, 124.2, 124.1, 131.5, 134.1, 140, 148.9, 166.2, 169.8, 174.7. IR (KBr
disc) mmax 3121, 1763, 1686, 1609, 1488, 1449 cmÀ1. HRMS found: 265.0720
[MÀH+] C13H13O6 requires 265.0712.
19. Dooley, C. M.; Devocelle, M.; McLoughlin, B.; Nolan, K. B.; Fitzgerald, D. J.;
Sharkey, C. T. Mol. Pharmacol. 2003, 63, 450.