Structure- and property-based design of factor Xa inhibitors: Pyrrolidin-2-ones with acyclic alanyl amides as P4 motifs

Article abstract of DOI:10.1016/j.bmcl.2006.09.001

Structure-based drug design was exploited in the synthesis of 3-(6-chloronaphth-2-ylsulfonyl)aminopyrrolidin-2-one-based factor Xa (fXa) inhibitors, incorporating an alanylamide P4 group with acyclic tertiary amide termini. Optimized hydrophobic contacts

Full text of DOI:10.1016/j.bmcl.2006.09.001

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5953–5957
Structure- and property-based design of factor Xa inhibitors:
Pyrrolidin-2-ones with acyclic alanyl amides as P4 motifs
Robert J. Young,a,* Matthew Campbell,a Alan D. Borthwick,a David Brown,a
b
a
a
a
´
Cynthia L. Burns-Kurtis, Chuen Chan, Maire A. Convery, Miriam C. Crowe,
Satish Dayal,a Hawa Diallo,a Henry A. Kelly,a N. Paul King,a Savvas Kleanthous,a
Andrew M. Mason,a Jackie E. Mordaunt,a Champa Patel,a Anthony J. Pateman,a
Stefan Senger,a Gita P. Shah,a Paul W. Smith,a Nigel S. Watson,a
Helen E. Westona and Ping Zhoua
aGlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK
bGlaxoSmithKline, 709 Swedeland Road, King of Prussia, PA 19406, USA
Received 31 July 2006; revised 31 August 2006; accepted 1 September 2006
Available online 18 September 2006
Abstract—Structure-based drug design was exploited in the synthesis of 3-(6-chloronaphth-2-ylsulfonyl)aminopyrrolidin-2-one-
based factor Xa (fXa) inhibitors, incorporating an alanylamide P4 group with acyclic tertiary amide termini. Optimized hydropho-
bic contacts of one amide substituent in P4 were complemented by hydrophobicity-modulating features in the second, producing
potent fXa inhibitors including examples with excellent anticoagulant properties.
ꢀ 2006 Elsevier Ltd. All rights reserved.
In the search for anticoagulant therapies with improved
efficacy/safety profiles, coagulation Factor Xa (fXa) has
provided a major focus.1 The pivotal role of fXa in the
blood coagulation cascade makes it an attractive thera-
peutic target and emerging data support this hypothe-
sis.2 The primary specificity (S1) pocket of fXa
recognizes a basic residue, in common with other such
trypsin-like serine proteases. Consequently, many fXa
inhibitors include a basic amine or amidine P1 motif
to facilitate binding to Asp189 at the base of this pocket;
such charged features limit permeation and generally
engender poor oral pharmacokinetics.
inhibitors, which utilize similar interactions in the S1
pocket, have been reported.5
The application of structure- and property-based design
is described here in the evaluation of a series of acyclic
analogues 2 exploiting the alanyl amide P4 ligand.
R3
H
N
N
O
Me
N
Me
N
N
O
O
S
R1
O
S
O
O
O
N
O
Cl
R2
Cl
O
1
2
We recently described a series of non-basic compounds
exploiting an alternative interaction in S1, specifically
that between an aryl chloride and Tyr228.3 Optimisation
of this aminopyrrolidin-2-one-based series led to mole-
cules, for example, 1, which displayed good anticoagu-
lant potency and attractive oral pharmacokinetics.4
Other structural classes of neutral or weakly basic fXa
Impetus for this work arose from inspection of the crys-
tal structure of 1 bound into fXa;3 one edge of the mor-
pholine ring made hydrophobic contacts with aromatic
residues forming S4, but without filling this space. Initial
molecular modelling supported the hypothesis that a
small aliphatic R1 group, as one substituent on a tertiary
acyclic amide, could better occupy this space (Fig. 1). It
was envisaged that the second amide substituent might
be used to introduce hydrophobicity-modulating fea-
tures to enhance pharmacokinetic and anticoagulant
profiles as discussed in our earlier publications.3,4
Keyword: Factor Xa.
*
Corresponding author. Tel.: +44 1438 768372; fax: +44 1438
763620; e-mail: Rob.J.Young@gsk.com
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.09.001

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R. J. Young et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5953–5957
Furthermore, distance calculations supported the idea
of R2 substituents forming additional H-bonds with
backbone residues.
and deprotection of the tert-butyl ester provided the ter-
tiary sulfonamide intermediate 6, introducing a particular
modification that significantly enhanced activity in the
cyclic series.3
Available structural information indicated a clear prefer-
ence for molecules with the S,S stereochemistry, so
synthesis of the orthogonally protected intermediate 3
(Scheme 1) exploited the established route from CBZ-
Met-OH and H-Ala-OtBu.3 Hydrogenolysis and sulfony-
lation afforded ester 4, acid deprotection of which gave
chiral acid 5. Alkylation of 4 with 2-bromoacetamide
The targeted acyclic compounds 2 were produced by
amide coupling of primary or secondary amine sub-
strates6 with 5 or 6 (Scheme 2). Terminal amines 8 and
their homologues were produced via N-Boc-protected
intermediates 7, followed by acid deprotection. Sulfona-
mides 9 and ureas 10 were readily derived from these
amines.
A first matrix of compounds investigated the SAR of
lower alkyl combinations and showed a clear preference
for tertiary amides with enhanced activity when one sub-
stituent was isopropyl (Table 1). This was ascribed to
good hydrophobic contacts of the isopropyl group with
the aromatic S4 residues, consistent with modelling
predictions.
R3
O
S
R3
O
S
N
N
O
O
Cl
Cl
O
O
N
N
a
b
O
O
OH
N
R1
R2
5 R3= H
Figure 1. X-ray crystal structure of 1 bound into fXa,3 highlighting
potential S4 interactions for an acyclic structure: (i) better filling of the
S4 ‘aromatic box’ (arrowed) and (ii) accessible backbone residues
2 or
7 R2 = (CH2)2NHBoc
6 R3 = CH2CONH2
R3
R3
O
O
around Lys96 (as starred ).
*
N
S
N
S
O
O
Cl
Cl
O
N
O
N
H
N
c or d
H
H
N
I
S+
O
CBZ
O
S
N
CBZ
CBZ
8
N
R4
N
R1
NH2
R1
N
H
O
O
N
O
O
O
HN
HO
a, b
c
d,e
+
O
9
R4 = SO2Me
NH2
O
10 R4 = CONH2
O
3
O
Scheme 2. Reagents and conditions: (a) R1R2NH, EDC, HOBT,
Et3N, DMF, rt; (b) TFA, DCM; (c)MeSO2Cl, pyridine DCM, rt; (d)
H2NCO2Ph, NMM, THF, D.
O
S
O
H
H
N
N
S
O
O
Cl
O
O
Cl
N
N
O
O
f
Table 1. fXa inhibitory activities7a for compounds 11–16
OH
O
4
5
O
H
N
Me
N
O
O
S
O
H2N
O
S
N
R1
O
O
N
Cl
Cl
g, f
R2
O
N
O
Compound
R1
R2
fXa Ki/nM
6
OH
11
12
13
14
15
16
Me
Et
Me
Me
Et
346
56
Scheme 1. Reagents and conditions: (a) EDC, 1-hydroxybenzotriazole
(HOBT), Et3N, DMF, rt; (b) MeI, acetone, rt; (c) DOWEX [OH]À,
MeCN; (d) H2, Pd–C, EtOH, rt; (e) 6-chloronaphth-2-ylSO2Cl,
pyridine, DCM, rt; (f) TFA, DCM; (g) BrCH2CONH2, K2CO3,
DMF, rt.
Et
24
i-Pr
i-Pr
i-Pr
H
2720
22
Me
Et
11

R. J. Young et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5953–5957
5955
Table 2. fXa inhibitory activities7a for compounds 17–23
A subsequent subset of compounds, derived from avail-
able monomers incorporating a common cyanoethyl
group, provided further empirical evidence to support
the hypothesis concerning the required hydrophobic
contacts (Table 2). The high potency of isopropyl and
cyclopropylmethyl analogues corroborated the likely
optimum size and fit of the R1 substituent.
H
N
Me
R1
N
O
S
O
O
O
N
Cl
CN
Compound
R1
fXa Ki/nM
The finding that good intrinsic activity could be main-
tained in analogues containing a hydrogen bond accept-
ing nitrogen was also demonstrated in a series of
alkyl(pyridinylalkyl)amines (Table 3). As expected, this
gave compounds with enhanced potency with the iso-
propyl R1 group; no clear preference for the pyridine
orientation was apparent. Members of these series
showed only moderate anticoagulant activities, as as-
sessed in the prothrombin time (PT) assay;8 for example,
17
18
19
20
21
22
23
i-Pr
i-Bu
5
136
57
3
c-Pr
CH2c-Pr
c-Bu
15
11
73
c-Pentyl
c-Hexyl
9
17 Ki = 5 nM, 1.5 · PT 9.4 lM, clogD7.4 2.3; 27
Ki = 9 nM, 1.5 · PT 18.7 lM, clogD7.4 3.0; 28,
Ki = 6 nM, 1.5 · PT 8.8 lM, clogD7.4 3.0.
Table 3. fXa inhibitory activities7a for compounds 24–29
H
N
At this stage, the incorporation of more hydrophilic R2
substituents was investigated, to capitalise on previous
findings that reducing hydrophobicity9 promoted a bet-
ter translation of intrinsic potency into plasma-based
activity.3,4 Gratifyingly, both intrinsic potency and anti-
coagulant activity were enhanced (Table 4), with amino
substituted analogues having particularly attractive pro-
files (e.g., 33, Ki = 4 nM, 1.5 · PT 1.3 lM, clogD7.4 0.6;
42 Ki = 2 nM, 1.5 · PT 1.6 lM, clogD7.4 1.3). Results in
this matrix also supported isopropyl as the R1 group of
choice; related cyclopropylmethyl derivatives gave
broadly similar fXa activities that did not translate as
Me
N
O
O
S
R1
O
O
N
X
Cl
R2
Compound
R1
Et
X
R2
fXa Ki/nM
24
25
26
27
28
29
CH2
CH2
CH2
CH2
CH2
4-Pyridinyl
Ph
27
24
11
9
i-Pr
i-Pr
i-Pr
i-Pr
i-Pr
2-Pyridinyl
3-Pyridinyl
4-Pyridinyl
2-Pyridinyl
6
13
CH2CH2
Table 4. fXa inhibitory activities,7a,b anticoagulant potency8 and hydrophobicity calculations9 for compounds 30–48
H
N
Me
R1
N
O
S
O
O
O
N
R2
Cl
9
Compound
R1
R2
clogD7.4
fXa Ki/nM
1.5 · PT/lM
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
1
i-Pr
OH
OH
2.3
2.3
—
6
9.7
16.7
—
CH2c-Pr
H
i-Pr
7
NH2
NH2
NH2
CONH2
4100a
0.6
0.6
1.5
1.5
1.5
—
4
15
1.3
2.8
8.0
8.1
16
CH2c-Pr
i-Pr
i-Pr
11
2
NHCONH2
NHCONH2
NHSO2Me
NHSO2Me
NHSO2Me
SO2NH2
CH2c-Pr
H
i-Pr
7
39,000a
—
1.8
1.8
1.4
1.3
2.4
2.2
3
1
2
3
2
2
4
2
2
5
3
6
2.5
10.5
4.8
1.6
1.7
5.8
1.6
2.2
6.7
9.5
5.0
CH2c-Pr
i-Pr
i-Pr
NMe2
i-Pr
i-Pr
Piperidine
Morpholine
Azepine
i-Pr
i-Pr
CH2NH2
CH2NHCONH2
CH2NHSO2Me
0
i-Pr
i-Pr
1.7
2
Morpholine
2.2
a Data secured using a fluorogenic assay.7b

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R. J. Young et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5953–5957
Table 5. fXa inhibitory activities7a,7b and anticoagulant potency8 for
tertiary sulfonamides 49–55
An X-ray structure of 39 bound into fXa confirmed bind-
ing in the manner predicted, with the molecule unambig-
uously fitted to the Fo–Fc electron density map.13 In S4,
the isopropyl group clearly fitted well into the aromatic
box formed by the residues Tyr99, Phe174 and Trp215
and, gratifyingly, the sulfonamide NH makes a hydrogen
bond with the carbonyl of Lys96 (Fig. 2).14
NH2
O
N
S
Me
N
N
O
O
O
O
R2
Cl
In summary, the synthesis, activities and structure–
property relationships have been explored within a
rationally designed series of 3-(6-chloronaphth-2-yls-
ulfonyl)aminopyrrolidin-2-one-based fXa inhibitors
incorporating an acyclic alanyl amide P4 motif. Using
structure- and property-based approaches has provided
a novel series with excellent intrinsic and anticoagulant
activities. These studies gave clear insight into optimal
features for incorporation into further series of fXa
inhibitors. Findings from these studies will be reported
in separate publications.15
Compound
R2
fXa Ki/nM
1.5 · PT/lM
49
50
51
53
53
54
55
OH
NH2
1a
<1
1
1.9
0.8
1.5
1.9
0.6
1.7
0.4
NHCONH2
NHSO2Me
Piperidine
Morpholine
Azepine
<1
1
1
<1
a Data secured using a fluorogenic assay.7b
References and notes
1. (a) Quan, M. L.; Smallheer, J. M. Curr. Opin. Drug Disc.
Dev. 2004, 7, 460; (b) Gould, W. R.; Leadley, R. J. Curr.
Pharm. Des. 2003, 9, 2337; (c) Hirsh, J.; O’Donell, M.;
Weitz, J. I. Blood 2005, 105, 453; (d) Golino, P.; Loffredo,
F.; Riegler, L.; Renzullo, E.; Cocchia, R. Curr. Opin.
Invest. Drugs 2005, 6, 298.
2. (a) Rajagopal, V.; Bhatt, D. L. J. Thromb. Haem. 2005, 3,
436; (b) Viles-Gonzalez, J. F.; Gaztanaga, J.; Zafar, U.
M.; Fuster, V.; Badimon, J. J. Am. J. Cardiovasc. Drugs
2004, 4, 379.
3. Watson, N. S.; Brown, D.; Campbell, M.; Chan, C.;
Chaudry, L.; Convery, M. A.; Fenwick, R.; Hamblin, J.
N.; Haslam, C.; Kelly, H. A.; King, N. P.; Kurtis, C. L.;
Leach, A. R.; Manchee, G. R.; Mason, A. M.; Mitchell,
C.; Patel, C.; Patel, V. K.; Senger, S.; Shah, G. P.; Weston,
H. E.; Whitworth, C.; Young, R. J. Bioorg. Med. Chem.
Lett. 2006, 16, 3784.
4. Chan, C.; Borthwick, A. D.; Brown, D.; Campbell, M.;
Chaudry, L.; Chung, C.-W.; Convery, M. A.; Hamblin, J.
N.; Johnstone, L.; Kelly, H. A.; Kleanthous, S.; Kurtis, C.
L.; Patikis, A.; Patel, C.; Pateman, A. J.; Senger, S.; Shah,
G. P.; Toomey, J. R.; Watson, N. S.; Weston, H. E.;
Whitworth C.; Young, R. J.; Zhou, P.; J. Med. Chem.,
submitted for publication.
5. Choi-Sledeski, Y. M.; Kearney, R.; Poli, G.; Pauls, H.;
Gardner, C.; Gong, Y.; Becker, M.; Davis, R.; Spada, A.;
Liang, G.; Chu, V.; Brown, K.; Collussi, D.; Leadley, R.;
Rebello, S.; Moxey, P.; Morgan, S.; Bentley, R.; Kasiew-
ski, C.; Maignan, S.; Guilloteau, J-P.; Mikol, V. J. Med.
Chem. 2003, 46, 681, and selected citations in Ref 1a.
6. The majority of amine substrates employed were available
from the GSK monomer collection; or prepared as
described in Borthwick, A. D.; Campbell, M,; Chan, C.;
Kelly, H. A.; King, N. P.; Kleanthous, S.; Mason, A. M.;
Senger, S.; Smith, P. W.; Watson, N. S.; Young, R. J. WO
03043981 A1, 2003.
Figure 2. X-ray crystal structure of 39 bound into fXa, showing S4
interactions as described in the text.
well into plasma-based potency. Furthermore the prefer-
ence for tertiary amides was supported by the poor
activity seen with 32 and 38.
Mirroring a trend seen in the cyclic alanyl amide series
for tertiary sulfonamides,3 a series of tertiary glycina-
mides showed enhanced affinity with good levels of anti-
coagulant activity (Table 5) when compared with their
secondary analogues (Table 4).
Pharmacokinetic studies in the rat with selected examples
in this series generally revealed poor profiles characterised
by increased plasma clearance and poorer oral exposure
compared with related cyclic morpholine-based ana-
logues.10 Increased flexibility and high polar surface
areas have both been implicated as factors that impact
negatively on oral bioavailability11 and both parameters
may have contributed to the lower oral exposure of the
more hydrophilic acyclic alanyl amides compared with
that seen with morpholine-based analogues.12
7. (a) FXa inhibitory activities were determined using N-a-Z-
D-Arg-Gly-Arg-p-nitroanilide as chromogenic substrate;
(b) Rhodamine 110, bis-CBZ-glycylglycyl-L-arginine
amide as fluorogenic substrate; details are described in
Ref. 4. For compounds tested in both assays, comparable
levels of activity were observed.
8. Anticoagulant activities were determined in the prothrom-
bin time (PT) assay; see Ref. 4.

R. J. Young et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5953–5957
5957
9. Hydrophobicity predictions, expressed as clogD7.4, were
calculated using Advanced Chemistry Development soft-
ware v8.0. These correlated well with measured Chroma-
tographic Hydrophobicity Index (CHI) logD7.4 vales; see
Valko, K.; Du, C. M.; Bevan, C.; Reynolds, D. P.;
Abraham, M. H. Curr. Med. Chem. 2001, 8, 1137.
10. Compounds were administered either iv as a bolus or po
via gavage to male Han Wistar rats at nominal doses of
1 mg/kg iv and 2.5 mg/kg po. For example, compound 1
has Clp of 13 mL/min/kg, compared with values of 27.5,
42.6 and 46.3 for compounds 30, 36 and 45, respectively.
11. Veber, D. F.; Johnson, S. R.; Cheng, H.-Y.; Smith, B. R.;
Ward, K. W.; Kopple, K. D. J. Med. Chem. 2002, 45, 2615.
12. Representative examples: compound 1 rotatable bonds 3
2
and PSA 96 A , F = 91%; 33, RB 6, PSA 113 A , F = 2%;
2
˚
˚
2
39, RB 7, PSA 133 A , F = 3%.
˚
˚
13. The structure for 39 was refined at 1.8 A (overall Rmerge
is 0.055) in Refmac5 to a final Rfactor of 0.191 and Rfree
of 0.233, using procedures described in Ref. 4. Co-
ordinates are deposited in the protein data bank with
code 2j4i.
14. Preliminary data for the complex structure with 45
revealed that the protonated amine was also likely to be
in position to make an H-bond interaction with the
backbone carbonyl of Lys96.
15. Watson, N. S. et al, manuscripts in preparation.