Design, synthesis, and structure-activity relationships of substituted piperazinone-based transition state factor Xa inhibitors

Article abstract of DOI:10.1016/S0960-894X(02)01038-7

The structure-activity relationship of a novel series of substituted piperazinone-based factor Xa inhibitors is described. The most potent compound 34 displays IC₅₀ of 0.9 nM.

Full text of DOI:10.1016/S0960-894X(02)01038-7

Bioorganic & Medicinal Chemistry Letters 13 (2003) 729–732
Design, Synthesis, and Structure–Activity Relationships of
Substituted Piperazinone-Based Transition State Factor
Xa Inhibitors
Ting Su,a,* Hua Yang,a Deborah Volkots,a John Woolfrey,a Suiko Dam,b
a
Paul Wong,b Uma Sinha,b Robert M.Scarborough and Bing-Yan Zhua,*
aDepartment of Medicinal Chemistry, Millennium Pharmaceuticals Inc., 256 East Grand Avenue, South San Francisco, CA 94080, USA
bDepartment of Biology, Millennium Pharmaceuticals Inc., 256 East Grand Avenue, South San Francisco, CA 94080, USA
Received 12 February 2002; accepted 1 November 2002
Abstract—The structure–activity relationship of a novel series of substituted piperazinone-based factor Xa inhibitors is described.
The most potent compound 34 displays IC50 of 0.9 nM.
# 2003 Elsevier Science Ltd.All rights reserved.
Factor Xa is a serine protease, which plays a pivotal
role in the blood coagulation cascade.Early animal
model studies of selective and potent factor Xa inhibi-
tors suggest they are effective in preventing venous and
arterial thrombosis formation and may have a much
wider therapeutic index (efficacy/bleeding) than throm-
bin inhibitors.Therefore, factor Xa has emerged as an
attractive biological target for the development of either
parenteral or oral anticoagulants.1
side chains at the 3-position of the piperazinone ring
system and hydrophobic sulfonamide functionality at
the 4-position might result in chemical entities with a
better pharmacological profile.In this report, the
design, synthesis and in vitro structure–activity rela-
tionships of a series of substituted piperazinone-based
transition state factor Xa inhibitors with high potency
and specificity will be discussed.
The synthesis of our key intermediate Arg(Tos)-thiazole
6 is outlined in Scheme 1.Coupling of Boc-Arg(Tos)-
OH 4 and N,O-dimethylhydroxylamine hydrochloride
generated Weinreb amide 5.Treatment of 5 with excess
2-lithiothiazole at À78 ꢀC6 in THF, followed by Boc
deprotection with 50%TFA in DCM yielded the Arg
(Tos)-thiazole 6.The preparation of compounds 11, 12,
and 15–34 is outlined in Scheme 2.Treatment of
2,2-dimethoxyethylamine with tert-butyl chloroacetate
and potassium carbonate, followed by coupling with
various amino acids yielded intermediate 7.The key
cyclic intermediate 8 was obtained when dimethyl acetal
7 was treated with 70% aqueous trifluoroacetic acid or
heated in toluene containing catalytic amount of
p-TsOH.7,8 Hydrogenation of the double bond of 8,
esterification of the acid, methyl or benzyl sulfonylation
at the 4 position, hydrolysis of the ester, and coupling to
Arg(Tos)-thiazole 6 followed by cleavage of the Tos
group afforded compounds 15–28 and 31–34.The
synthesis of compound 13 is shown in Scheme 3.Cou-
pling of Boc-Arg(Tos)-OH with diethyl iminodiacetate,
We previously presented the discovery of a class of tri-
peptide ketoheterocyclic transition state factor Xa
inhibitors represented by BnSO2-d-Arg-Gly-Arg-thia-
zole 12 and BnSO2-d-Phe-Gly-Arg-thiazole 23 (Fig.1 ).
Compound 1 inhibits factor Xa with an IC50 of 0.65
nM.More importantly, it shows strong in vivo antith-
rombotic efficacy (in vivo EC50=30–50 nM) without
significant bleeding complications in our rabbit deep
vein thrombosis model.4 To potentially improve the
pharmacokinetic properties of tri-peptide based factor
Xa inhibitors, we incorporated the conformationally
restricted piperazinone template into these inhibitors.In
the preceding publication,5 the initial unsubstituted
piperazinone compound 3 prepared displayed potent
fXa inhibitory activity (IC50 of 4 nM).To explore this
further, we envisioned that incorporation of various
*Corresponding author.Fax:+1-650-244-9387; e-mail: bingyanz.hu@
mpi.com
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd.All rights reserved.
doi:10.1016/S0960-894X(02)01038-7

730
T. Su et al. / Bioorg. Med. Chem. Lett. 13 (2003) 729–732
Scheme 2. Synthesis of compounds 11, 12, and 15–34: (a) K2CO3,
.
ClCH2CO2tBu, DMF; (b) Z-AA-OH HCl, BOP, DIEA, DMF; (c)
70% aq TFA; (d) H2, Pd(OH)2/C, EtOH, cat.Amount concd HCl; (e)
SOCl2, MeOH; (f) RSO2Cl, DIEA, CH2Cl2; (g) LiOH, THF/H2O; (h),
(j) Arg(Tos)-thiazole 6, BOP, DIEA, DMF; (i) , (k) HF.
Figure 1. Design of substituted piperazinone based factor Xa inhibitors.
.
Scheme 1. Synthesis of compound 6: (a) CH3NHOCH3 HCl, BOP,
DIEA, DMF, rt; (b) nBuLi, thiazole, THF; (c) 50% TFA in DCM.
followed by Boc deprotection yielded 9.Piperazinone 10
was formed in EtOH upon neutralization of 9 with
DIEA and stirring at rt for 5 h.Hydrolysis of the ester,
coupling to Arg(Tos)-thiazole 6 and subsequent clea-
vage of Tos group afforded analogue 13.Analogue 14
was synthesized employing a similar procedure as 13
using Boc-d-Arg(Tos)-OH.
Scheme 3. Synthesis of compounds 13: (a) Boc-Arg(Tos)-OH, BOP,
DIEA, DMF; (b) 50% TFA/DCM; (c) EtOH, DIEA; (d) LiOH, THF/
H2O; (e) Arg(Tos)-thiazole 6, BOP, DIEA, DMF; (f) HF.
The biological activity for compounds 11–34 against
fXa and the structurally related serine protease throm-
bin are summarized in Table 1.4
Although diketopiperazine derivatives 13 and 14 were
comparable to 11 in fXa inhibitory activity, they are the
most selective inhibitors (Xa vs thrombin) in this series
of analogues.Introduction of benzylsulfonamide at the
4-position of 11 afforded 15 with 7-fold enhancement in
the potency, suggesting a hydrophobic group is pre-
ferred at this position.The 4-fold difference in potency
between 15 and 16 indicates a fXa preference for
S-configuration at the 3-position in 3-guanidinopropyl-
substituted inhibitors.The potency and selectivity of
compound 15 encouraged us to explore a variety of dif-
ferent substituents at the 3-position of piperazinone
The basic 3-guanidinopropyl side chain was initially
chosen as the substitute at 3-position of the piper-
azinone ring (11–16) based on the excellent fXa
inhibitory activity of its tri-peptide counterpart
BnSO2-d-Arg-Gly-Arg-thiazole 1 (Fig.1 ).Fully satu-
rated piperazinone ring-containing analogue 11 is
15-fold more potent in fXa inhibitory activity than the
unsaturated analogue 12.This suggests the preference
for a relatively flexible ring at the central piperazinone
unit.

T. Su et al. / Bioorg. Med. Chem. Lett. 13 (2003) 729–732
731
Table 1. In vitro activity for compounds 10–33
Table 1 (continued)
Compd
R
Factor Xa
IC50, mM
Thrombin
IC50, mM
23
24
25
26
27
28
29
30
31
32
33
34
0.514
0.037
0.004
0.001
0.004
0.006
0.888
5
60
8
Compd
R
Factor Xa
IC50, mM
Thrombin
IC50, mM
11
0.02
0.307
0.011
0.021
0.003
0.013
0.37
12
140
150
200
12
3
5
12
13
14
15
16
17
18
19
20
21
22
2
3
2
2
22
325
40
1
0.144
0.459
0.059
0.0009
2
0.031
0.085
0.008
0.032
0.007
0.642
2
5
0.507
4
ring.The acid, ester, amide and hydroxymethyl-con-
taining side chain substituted piperazinone analogues
were synthesized next (17–28).These substituents were
chosen based on the reported substrates for Factor Xa
[Bz-Ile-Glu-Gly-Arg-pNA, Bz-Ile-Glu(g-Pip)-Gly-Arg-
pNA]1g and the cleavage site sequences recognized by
factor Xa in the activation of prothrombin (Ile-Glu-Gly-
Arg, Ile-Asp-Gly-Arg).1f The differences in fXa inhibi-
tory activity between S and R analogue in analogues
3

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T. Su et al. / Bioorg. Med. Chem. Lett. 13 (2003) 729–732
In general, enzyme selectivity shows the following trend
with the following side chains: 3-guanidinopropyl >3-
carboxylethyl>3-carboxymethyl>3-benzyl-substituted
inhibitors.This is consistent with the pattern we
observed with d-Phe-Gly-Arg based ketoheterocyclic
inhibitors.3 Ester, amide, acid and hydroxymethyl
groups are all tolerated as side chains at the 3-position.
Based on the unsubstituted piperazinone based factor
Xa inhibitor5 and our previously reported tripeptide
ketoheterocyclic lead, we have designed, synthesized
and evaluated a series of potent and selective substituted
piperazinone analogues.A systematic SAR study
revealed the structural basis for the potency and selec-
tivity.The fact that various acidic, basic and neutral
groups are well tolerated at the 3-position suggests that
changes in this portion of the molecule may allow for
modulation of PK profiles, while retaining potency and
selectivity.Further SAR studies on improving pharma-
cokinetic properties using piperazinone as templates will
be the subject of additional communications from our
laboratories.
Figure 2. Proposed binding conformation of compound 25 to fXa.
The Connelly surface was generated using InsightII (Accelrys, Inc.).
Red, blue and yellow surface colors represent proximal oxygen, nitrogen
and sulfur atoms, respectively.The carbonyl group involved in the cov-
alant transition state interaction with SER195 is hidden by the surface.
17–24 suggests a factor Xa preference for the R-con-
figuration at the 3-position when neutral and acidic side
chains are present.The homologous compounds 25, 26,
and 27 are 7- to 9-fold more potent than compounds 18,
20, and 24.Homologue acid 25 is as selective for fXa as
analogue 18, but homologue ester 26 and amide 27 are
3- to 4-fold more selective than analogues 20 and 24.
Finally, based on the excellent potency of its tri-peptide
counterpart BnSO2-d-Phe-Gly-Arg-thiazole 2, a benzyl
side chain was introduced into the 3-position of the
piperazinone ring (29–34).This resulted in the most
potent inhibitor 34 in this substituted piperazinone ser-
ies.Similar to the trend in analogues 17–24, analogue 34
with R-configuration at the 3-position is more potent
than its S-isomer 33.
References and Notes
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81.(d) Scarborough, R. M. J. Enzyme. Inhibit. 1998, 14, 15.(e)
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Several compounds were also evaluated in a tissue fac-
tor mediated plasma thrombin generation assay.5 The
assay was attempted to mimic thrombin generation in
the presence of components of whole blood and evalu-
ate the effect of the specific fXa inhibitors on the rate of
thrombin generation.The compound 28 was capable of
producing a 2-fold extension of the lag time at a con-
centration of 760 nM.
Molecular modeling of compound 25 (Fig.2 ) in the fXa
active site shows the 3-guanidinopropyl group (Arg side
chain) fitting into the S1 pocket, forming an ionic
interaction with the Asp189 side chain.The benzylsul-
phonyl group at the N4-position of the piperazinone
projects toward the S4 pocket, and is held tightly by
p-stacking interactions with the Tyr99, Trp215, and
Phe174 side chains.The ketone carbonyl forms a rever-
sible covalent bond with the hydroxyl group of Ser195.
6.Nahm, S;.Weinreb, S.M.
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