Discovery of octahydroindenes as PAR1 antagonists

Article abstract of DOI:10.1021/ml400235c

Octahydroindene was identified as a novel scaffold for protease activated receptor 1 (PAR1) antagonists. Herein, the 2-position (C2) was explored for structure-activity relationship (SAR) studies. Compounds 14, 19, and 23b showed IC₅₀ values of 1.3, 8.6, and 2.7 nM in a PAR1 radioligand binding assay, respectively, and their inhibitory activities on platelet activation were comparable to that of vorapaxar in a platelet rich plasma (PRP) aggregation assay. This series of compounds showed high potency and no significant cytotoxicity; however, the compounds were metabolically unstable in both human and rat liver microsomes. Current research efforts are focused on optimizing the compounds to improve metabolic stability and physicochemical properties as well as potency.

Full text of DOI:10.1021/ml400235c

Letter
pubs.acs.org/acsmedchemlett
Discovery of Octahydroindenes as PAR1 Antagonists
Sunkyung Lee,*,† Jong-Hwan Song,† Chul Min Park,† Jin-Seok Kim,† Ji-Hye Jeong,† Woo-Young Cho,‡
and Dong-Chul Lim‡
†Division of Drug Discovery Research, Korea Research Institute of Technology, 141 Gajeongno, Yuseong, Deajeon 305-600, Korea
‡R&D Park, LG Life Sciences, 104-1 Munji-Dong, Yuseong, Deajeon 305-380, Korea
S
* Supporting Information
ABSTRACT: Octahydroindene was identified as a novel scaffold
for protease activated receptor 1 (PAR1) antagonists. Herein, the 2-
position (C2) was explored for structure−activity relationship
(SAR) studies. Compounds 14, 19, and 23b showed IC50 values
of 1.3, 8.6, and 2.7 nM in a PAR1 radioligand binding assay,
respectively, and their inhibitory activities on platelet activation were
comparable to that of vorapaxar in a platelet rich plasma (PRP)
aggregation assay. This series of compounds showed high potency
and no significant cytotoxicity; however, the compounds were
metabolically unstable in both human and rat liver microsomes.
Current research efforts are focused on optimizing the compounds
to improve metabolic stability and physicochemical properties as
well as potency.
KEYWORDS: Octahydroindene, PAR1 antagonist, PRP aggregation, antiplatelet, bleeding
he protease activated receptor 1 (PAR1) plays a critical
Trole in thrombin mediated platelet aggregation but not in
fibrin generation. It is thought to be a promising antithrombosis
target with potentially less severe bleeding side effects.1,2 PAR1
is activated through proteolytic cleavage of its extracellular loop
by thrombin, and the resulting new amino acid terminus
(SFLLRN) intramolecularly binds to the proximally located
portion of the receptor, acting as a tethered ligand to cause
transmembrane signaling.3−6 Consequently, a strong inter-
action between PAR1 and its antagonist is required for the
efficient blockage of the intramolecular binding of a tethered
ligand with a very high local concentration.7−13 Vorapaxar is a
very potent and virtually irreversible PAR1 antagonist with very
slow receptor association and dissociation rates.14−17 Regard-
less the observation of an increased risk of bleeding in a phase
III trial of vorapaxar on patients who had previously suffered a
stroke,18 there is still the potential to dissociate antiplatelet
activity from bleeding risk.
We have designed the compounds described herein using the
structure of vorapaxar as a starting point to identify new PAR1
antagonists. We kept its (3-fluorophenyl)pyridine-2-vinyl
moiety fixed and attempted to modify the tricyclic ring of
vorapaxar. We found that a 6/5 bicycle,19 octahydroindene
could serve as a core scaffold (Figure 1). The middle 6-
membered ring of vorapaxar was replaced with a 5-membered
ring, and its lactone ring was opened. The 2-position (C2) of
the octahydroindene was further studied to determine
structure−activity relationships (SARs). In this letter, we report
the SAR of this series of compounds as PAR1 antagonists and
Figure 1. Design of octahydroindene core.
we further describe the biological and physicochemical profiles
of some of the active compounds.
Primarily, ketal analogues at C2 were prepared to confirm
that the substitution of an octahydroindene scaffold could
retain the compound’s ability to inhibit PAR1. The synthesis
followed the conventional procedures outlined in Scheme 1
(see the Supporting Information for details). Reductive ring-
opening of both cis- and trans-fused hexahydroisobenzofuran-
1,3-diones using LiAlH4 generated diols (1a and 1b) and O-
mesylation of alcohols were subsequently performed. An SN2
reaction of mesylates (2a and 2b) with cyanides (3a and 3b)
and a Dieckmann cyclization of dinitriles yielded three
octahydroindenes (4a, 4b, and 4c).20−22 The trans-fused
Received: June 20, 2013
Accepted: September 10, 2013
Published: September 10, 2013
© 2013 American Chemical Society
1054
dx.doi.org/10.1021/ml400235c | ACS Med. Chem. Lett. 2013, 4, 1054−1058

ACS Medicinal Chemistry Letters
Letter
a
Scheme 1. Syntheses of cis- and trans-Octahydroindenes
Table 2. SAR at C2 of Octahydroindene Derivatives
Table 1. Binding Data of cis- and trans-6/5 Bicyclic
Dioxolanes
b
cmpd
vorapaxar
0.0011
8a
8b
8c
a
IC50 (μM)
0.47
0.026
2.1
a
PAR1 binding assay ligand [3H]haTRAP, 10 nM. The value is an
b
average of three measurements. Vorapaxar was synthesized and
evaluated by our laboratory as a reference standard.
Scheme 2. Syntheses of 2-Oxy and 2-Amine Derivatives
a
See Table 1 footnotes.
formation, reduction of nitriles to aldehydes (6a, 6b, and 6c)
using DIBAL-H and a Horner−Wadsworth−Emmons (HWE)
reaction with phosphonate 7 were performed to produce the
corresponding dioxolanes (8a, 8b, and 8c).10 Racemic mixtures
were used for the biological assay without separation. A PAR1
binding assay was carried out to evaluate the activity of
compounds to inhibit PAR1 using human platelet membrane as
the receptor source and [3H]-labeled high affinity thrombin
receptor activation peptide ([3H]−haTRAP, alanine-p-fluoro-
compound 3a yielded only one epimer (4a), which might be
thermodynamically stable; however, the cis-fused compound 3b
gave a mixture of epimers of 4b and 4c as a 3:1 ratio. After ketal
1055
dx.doi.org/10.1021/ml400235c | ACS Med. Chem. Lett. 2013, 4, 1054−1058

ACS Medicinal Chemistry Letters
Letter
The addition of another H-bond acceptor or a bulky
substituent at C2 might be beneficial for activity.
Scheme 3. Syntheses of 2-Quaternary and Spiro Derivatives
Amine 12 was prepared by nucleophilic substitution of
mesylate 11d to an azide and a subsequent reduction using
PPh3. The compound 12 was further derivatized to carbamate,
amide, and methanesulfonamide compounds. The potencies of
amine 12 and cyclopropylamide 13f were similar to that of
alcohol 10, but carbamates 13a−13c showed a marked
reduction in affinity, especially when the size was increased.
The incorporation of a large substituent at C2 was not
tolerated. Methanesulfonamide 13d and acetamide 13e showed
IC50 values of 0.050 and 0.053 μM, respectively, and these
values were similar to those of methoxy compound 11a and
acetate 11c. N-Methylation of 13d and 13e led to a 10−25-fold
improvement in activity with IC50 values of 1.3 and 5.4 nM,
respectively. The potency of N-methylmethanesulfonamide
(14) was comparable to that of vorapaxar. The bulky
substituent, which is close to C2, seemed to be favorable for
PAR1 binding, but a large substituent far from C2 was
deleterious, as observed with carbamates 13a−13c. For
vorapaxar, it has been reported previously that the methyl at
the lactone ring gives optimal activity.11
Because the compounds with a short and bulky substituent at
C2 exhibited improved PAR1 inhibition, quaternary and spiro
compounds were additionally synthesized (Scheme 3). Initially,
we attempted to form the spiro ring after introducing a
pyridine-2-vinyl moiety to improve the synthetic throughput.
Contrary to our expectations, the reaction was very restricted,
presumably due to the presence of the pyridine-2-vinyl group,
which can act as a Michael acceptor. In a subsequent synthesis
attempt, the spiro oxazolidinone ring was incorporated before
performing the HWE reaction according to the established
procedures.25,26 Oxidation of alcohol 18 to aldehyde using 2-
iodobenzoic acid (IBX), followed by an immediate HWE
reaction produced compound 19.27 2-Methyl-2-alcohol 20 was
prepared by the same procedure as 19 and was subsequently
derivatized to methyl ether 23a and carbamate 23b.28 As
anticipated, the introduction of an additional 2-methyl group
and spiro ring made the compounds 5 to 10 times more potent.
Spiro oxazolidinone 19 and its open structure 23b were highly
active with IC50 values of 8.6 and 2.7 nM, respectively.
phenylalanine-arginine-cyclohexylalanine-homoarginine-[3H]-
tyrosine-NH2) as the ligand (Table 1).23,24 In this assay,
vorapaxar, which was synthesized in our laboratory for use as a
reference standard, showed an IC50 value of 0.0011 μM. One of
the cis-fused dioxolanes (8b, IC50 = 0.026 μM) exhibited a
much higher activity level than the trans compound (8a, IC50
=
0.47 μM) and the other cis compound (8c, IC50 = 2.1 μM).
We also synthesized the 2-oxy and 2-amine derivatives shown
in Scheme 2 (see the Supporting Information for details) and
evaluated their PAR1 inhibitory activity to study the SAR at C2
of cis-octahydroindene (Table 2). The ketal 8b was hydrolyzed
to ketone 9, and then reduced to alcohol 10, which resulted in
the formation of two diastereoisomers that were separated and
evaluated. However, the diastereoisomers showed no marked
differences in activity. We then decided to prepare the mixture
for further manipulation until we found a good activity profile
for the compound. The yield of the ketone to alcohol reduction
reaction was unusually low (20%) because a compound in
which the double bond had migrated was substantially formed
along with several unidentified side products. Alkylation and
acylation of alcohol 10 were performed under standard
conditions. The ketone 9 exhibited significantly less activity
(IC50 = 1.1 μM), while alcohol 10, benzyloxy derivative 11b,
phenylcarbamate 11e, and dimethylcarbamothioate 11f were
moderately active (IC50 = 0.12−0.36 μM). The methyl ether
11a and acetate 11c showed a slightly improved potency over
alcohol 10 but were weaker than ketal 8b. The mesylate 11d
showed the highest increase in activity (IC50 = 0.0064 μM).
We also examined the inhibitory effects of several of the
active compounds on platelet activation via a human platelet
aggregation assay induced with haTRAP (Table 3).29,30 While
the inhibitory activity on washed platelet aggregation (WPA)
was well correlated with PAR1 binding affinity, only 3
Table 3. Functional Assays and Properties
b
metabolic stability (R50, min)
a
a
c
cmpd
WPA IC50 (μM)
PRP IC50 (μM)
human
rat
cytotox. at 30 μM (% ATP)
d
vorapaxar
8b
0.0015
0.41
0.12
4.1
83.2
4.5
3.5
273
2.5
5.6
4.4
4.3
32.4
3.4
6.5
105
2.2
5.2
6.7
5.8
102%
97%
94%
N.A
11d
13d
14
0.006
0.14
N.A.
3.9
0.0022
0.21
0.097
8.9
95%
98%
101%
99%
15
19
0.0072
0.0029
0.21
0.10
23b
a
b
The value is an average of three measurements. Aggregation inducer, haTRAP; preincubation 10 min. Time when 50% of the compound remains
c
upon incubation with human and rat liver microsomes. In HepG2 cell line, ATP contents were determined by a % compared to those of vehicle-
treated control at 24 h after addition of compound (30 μM). See the Table 1 footnotes.
d
1056
dx.doi.org/10.1021/ml400235c | ACS Med. Chem. Lett. 2013, 4, 1054−1058

ACS Medicinal Chemistry Letters
Letter
(4) Vu, T. K.; Wheaton, V. I.; Hung, D. T.; Charo, I.; Coughlin, S. R.
Domains specifying thrombin-receptor interaction. Nature 1991, 353,
674−677.
compounds (14, 19, and 23b) represented significant efficacy
(IC50 = 0.097, 0.21, and 0.10 μM, each) in the human platelet
rich plasma (PRP) aggregation assay. These results are
comparable to that obtained for vorapaxar (IC50 = 0.12 μM).
In addition to the affinity of the compound for PAR1, the PRP
assay reflects the compound’s physicochemical properties,
including plasma stability. None of the compounds exhibited
cytotoxicity as determined by ATP contents in the HepG2 cell
line; however, the compounds were not stable when incubated
in human and rat liver microsomes. The compounds must be
optimized to improve their metabolic stability.
(5) Chen, J.; Ishii, M.; Wang, L.; Ishii, K.; Coughkin, S. R. J. Biol.
Chem. 1994, 269, 16041−16045.
(6) Zhang, C.; Srinivasan, Y.; Arlow, D. H.; Fung, J. J.; Palmer, D.;
Zheng, Y.; Green, H. F.; Pandey, A.; Pror, R. O.; Shaw, D. E.; Weis, W.
I.; Coughlin, S. R.; Kobilka, B. K. High-resolution crystal structure of
human protease-activated receptor 1. Nature 2012, 492, 387−392.
(7) Chackalamannil, S.; Xia, Y. Thrombin receptor (PAR1)
antagonists as novel antithrombotic agents. Expert Opin. Ther. Pat.
2006, 16, 493−505.
In conclusion, we have identified a novel 6/5 bicycle core,
octahydroindene scaffold that functions as a PAR1 antagonist.
From the SAR studies at the C2 position, it was found that
short and bulky substitution at C2 led to an improvement in
activity. The compounds 14, 19, and 23b were comparably
potent to vorapaxar in PAR1 binding and PRP aggregation
assays. While this series of compounds was generally not
cytotoxic, the compounds were not metabolically stable in
human and mouse liver microsomes. We are currently trying to
optimize the compounds described herein to improve
metabolic stability as well as potency.
(8) Ishii, K.; Hein, L.; Kobilka, B.; Coughlin, S. R. Kinetics of
thrombin receptor cleavage on intact cells. Relations to signaling. J.
Biol. Chem. 1993, 268, 9780−9786.
(9) Trejo, J.; Hammes, S. R.; Coughlin, S. R. Termination of
signaling by protease-activated receptor-1 is linked to lysosomal
dorting. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 13698−13702.
(10) Hein, L.; Ishii, K.; Coughlin, S. R.; Kobilka, B. K. Intracellular
targeting and trafficking of thrombin receptors. A novel mechanism for
resensitization of a G protein-coupled receptor. J. Biol. Chem. 1994,
269, 27719−27726.
(11) Clasby, M. C.; Chackalamannil, S.; Czarniecki, M.; Doller, D.;
Eagen, K.; Greenlee, W.; Kao, G.; Lin, Y.; Tsai, H.; Xia, Y.; Ahan, H.-
S.; Agans-Fantuzzi, J.; Boykow, G.; Chintala, M.; Foster, C.; Smith-
Torhan, A.; Alton, K.; Bryant, M.; Hsieh, Y.; Lau, J.; Palamanda, J.
Metabolism-based indentification of a potent thrombin receptor
antagonist. J. Med. Chem. 2007, 50, 129−138.
(12) Dockendorff, C.; Aisiku, O.; VerPlanl, L.; Dilks, J. R.; Smith, D.
A.; Gunnik, S. F.; Dowal, L.; Negri, J.; Palmer, M.; MacPherson, L.;
Schreiber, S. L.; Flaumenhaft, R. Discovery of 1,3-diaminobenzenes as
selective inhibitors of platelet activation at the PAR1 receptor. ACS
Med. Chem. Lett. 2012, 3, 232−237.
(13) Chackalamannil, S. Thrombin receptor (protease activated
receptor 1) antagonist as potent antithrombotic agents with strong
anti-platelet effects. J. Med. Chem. 2006, 49, 5389−5403.
(14) Chackalamannil, S.; Wang, Y.; Greenlee, W. J.; Hu, J.; Ahn, H.-
S.; Boykow, G.; Hsieh, Y.; Palamanda, J.; Agans-Fantuzzi, J.; Kurowski,
S.; Graziano, M.; Chintala, M. Discovery of a novel, orally active
himbacine-based thrombin receptor antagonist (SCH 530348) with
potent antiplatelet activity. J. Med. Chem. 2008, 51, 3061−3064.
(15) Chackalamannil, S.; Xia, Y.; Greenlee, W. J.; Clasby, M.; Dollar,
D.; Tsai, H.; Asberom, T.; Czarniecki, M.; Ahn, H.-S.; Boykow, G.;
Foster, C.; Agans-Fantuzzi, J.; Bryant, M.; Lau, J.; Chintala, M.
Discovery of potent orally active thrombin receptor (protease
activated recptor 1) antagonists as novel antithrombotic agents. J.
Med. Chem. 2005, 48, 5884−5887.
(16) Xia, Y.; Chackalamannil, S.; Greenlee, W. J.; Wang, Y.; Hu, Z.;
Root, Y.; Wong, J.; Kong, J.; Ahn, H.-S.; Boykow, G.; Hsieh, Y.;
Kurowski, S.; Chintala, M. Discovery of a vorapaxar analog with
increased aquwous solubility. Bioorg. Med. Chem. Lett. 2010, 20, 6676−
36679.
(17) Kosoglou, T.; Reyderman, L.; Tiessen, R. G.; van Vilet, A. A.;
Fales, R. R.; Keller, R.; Yang, V.; Culter, D. L. Pharmacodynamics and
pharmacokinetics of the novel PAR-1 antagonist vorapaxar (formerly
SCH 530348) in healthy subjects. Eur. J. Clin. Pharmacol. 2012, 68,
249−258.
ASSOCIATED CONTENT
* Supporting Information
■
S
Synthetic procedures and characterization data; assay protocols.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*(S.L.) Tel: +82-42-860-7148. E-mail: leesk@krict.re.kr.
■
Funding
This work was supported by the Global Frontier Project grant
NRF-2011-0032185 and the center for Biological Modulator of
the 21st century Frontier R&D program by the Ministry of
Science, ICT, and Future Planning of Korea.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
[3H]−haTRAP (Ala-Phe(p-F)-Arg-ChA-Har-[3H]Tyr-NH2):
Cold peptides were prepared by Peptron (Korea, www.
peptron.co.kr), and the radiolabeling of ligands was done by
Moravek, USA.
ABBREVIATIONS
■
PAR1, protease-activated receptor 1; SAR, structure−activity
relationship; HWE, Horner−Wadsworth−Emmons; DIBAL-H,
diisobutylaluminum hydride; haTRAP, high affinity thrombin
receptor activation peptide; IBX, 2-iodobenzoic acid; WPA,
washed platelet aggregation; PRP, platelet rich plasma
(18) Morrow, D. A.; Braunwald, E.; Bonaca, M. P.; Ameriso, S. F.;
Dalby, A. J.; Fish, M. O.; Fox, K. A. A.; Lipka, L. J.; Liu, X.; Nicolau, J.
C.; Theroux, P.; Wiviott, S. D.; Strony, J.; Murphy, S. A. Vorapaxar in
the secondary prevention of atherothrombotic events. N. Engl. J. Med.
2012, 366, 1404−1413.
(19) Chelliah, M. V.; Chackalamannil, S.; Xia, Y.; Eagen, K.;
Greenlee, W. J.; Ahn, H.-S.; Agans-Fantuzzi, J.; Boykow, G.; Hsieh, Y.;
Bryant, M.; Chan, T.-M.; Chintala, M. Discovery of nor-seco
himbacine analogs as thrombin receptor antagonists. Bioorg. Med.
Chem. Lett. 2012, 22, 2544−2549.
REFERENCES
■
(1) Coughlin, S. R. Thrombin signaling and protease-activated
receptors. Nature 2000, 407, 258−264.
(2) Ramachandran, R.; Noorbakhsh, F.; Defea, K.; Hollenberg, M. D.
Targeting protease-activated receptors: therapeutic potential and
challenges. Nat. Rev. Drug Discovery 2012, 11, 69−86.
(3) Coughlin, S. R. Protease-activated receptors in hemostasis,
thrombosis and vascular biology. J. Thromb. Hemost. 2005, 3, 1800−
1814.
1057
dx.doi.org/10.1021/ml400235c | ACS Med. Chem. Lett. 2013, 4, 1054−1058

ACS Medicinal Chemistry Letters
Letter
(20) McDermott, T. S.; Mortlock, A. A.; Heathcock, C. H. Total
synthesis of (−)-papuamine and (−)-haliclonadiamine. J. Org. Chem.
1996, 61, 700−709.
(21) Krawczyk, A. R.; Jones, J. B. Enzymes in organic synthesis. 46.
Regioselective ans stereoselective horse liver alcohol dehydrogenase
catalyzed reduction of cis- and trans-bicyclo[4,3,0]nonanones. J. Org.
Chem. 1989, 54, 1795−1801.
(22) Taber, D. F.; Wang, Y. Total synthesis of (−)-haliclonadiamine.
J. Am. Chem. Soc. 1997, 119, 22−26.
(23) Andrade-Gordon, P.; Maryanoff, B. E.; Derian, C. K.; Zhang, H.-
C.; Addo, M. F.; Darrow, A. L.; Eckardt, A. J.; Hoekstra, W. J.;
McComsey, D. F.; Oksenberg, D.; Reynolds, E. E.; Santulli, R. J.;
Scarborough, R. M.; Smith, C. E.; White, K. B. Design, synthesis, and
biological characterization of a peptide-mimetic antagonist for a
tethered ligand receptor. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 12257−
12262.
(24) Ahn, H.-S.; Foster, C.; Boykow, G.; Stamford, A.; Manna, M.;
Craziano, M. Inhibition of cellular action of thrombin by N3-
cyclopropyl-7{[4-(1-methylethyl)phenyl]methyl}-7H-pyrrolo[3,2-f ]-
quinazoline-1,3-diamine (SCH 79797), a nonpeptide thrombin
receptor antagonist. Biochem. Phamacol. 2000, 60, 1425−1434.
(25) Campbell, M. M.; Abbas, N.; Sainsbury, M. Spiroheterocycles
derived from tetralone. Tetrahedron 1985, 41, 5637−5644.
(26) Sugiyama, S.; Arai, S.; Ishii, K. Gram-scale synthesis of (S)-5,5-
dimethyl-4-phenyloxazolidin-2-one. Heterocycles 2005, 65, 149−159.
(27) Maruoka, K.; Imoto, H.; Yamamoto, H. Chemoselective
functinalization two different ketones with a series of bulky
organoaluminum receptors. Synlett 1994, 441−443.
(28) Kenworthy, M. N.; McAllister, G. D.; Taylor, R. J. K. The
preparation of simolified scyphostatin analogues using a tethered
aminohydroxylation (TA) strategy. Tetrahedron Lett. 2004, 45, 6661−
6664.
(29) Kato, Y.; Hirasawa-Taniyama, Y.; Nishio, M.; Mihara, K.; Ito, K.;
Yamanaka, T.; Seki, J.; Miyata, S.; Mutoh, S. Inhibition of arterial
thrombosis by a protease-activated receptor 1 antagonist, FR171113,
in the guinea pig. Eur. J. Phamacol. 2003, 473, 163−169.
(30) Kato, Y.; Kita, Y.; Nishio, M.; Hirasawa, Y.; Ito, K.; Yamanaka,
T.; Motoyama, Y.; Seki, J. In vitro antiplatelet profile of FR171113, a
novel non-peptide thrombin receptor antagonist. Eur. J. Phamacol.
1999, 384, 197−202.
1058
dx.doi.org/10.1021/ml400235c | ACS Med. Chem. Lett. 2013, 4, 1054−1058