Amino(methyl) pyrrolidines as novel scaffolds for factor Xa inhibitors

Article abstract of DOI:10.1016/j.bmcl.2007.07.063

The design and synthesis of a novel class of amino(methyl) pyrrolidine-based sulfonamides as potent and selective FXa inhibitors is reported. The amino(methyl) pyrrolidine scaffolds were designed based on the proposed bioisosterism to the piperazine core in known FXa inhibitors. The SAR study led to compound 15 as the most potent FXa inhibitor in this series, with an IC₅₀ of 5.5 nM and PT EC_2x of 1.7 μM. The proposed binding models show that the pyrrolidine cores are in van der Waals contact with the enzyme surface, and the flexibility of amino(methyl) pyrrolidines allows the two nitrogen atoms to anchor both the P1 and P4 groups to fit similarly in the S1 and S4 pockets.

Full text of DOI:10.1016/j.bmcl.2007.07.063

Bioorganic & Medicinal Chemistry Letters 17 (2007) 5952–5958
Amino(methyl) pyrrolidines as novel scaffolds for factor Xa inhibitors
Yan Shi,* Doree Sitkoff, Jing Zhang, Wei Han, Zilun Hu, Philip D. Stein, Ying Wang,
Lawrence J. Kennedy, Stephen P. O’Connor, Saleem Ahmad, Eddie C.-K. Liu,
Steve M. Seiler, Patrick Y. S. Lam, Jeffrey A. Robl, John E. Macor,
Karnail S. Atwal and Robert Zahler
Bristol-Myers Squibb Pharmaceutical Research Institute, PO Box 5400, Princeton, NJ 08543-5400, USA
Received 15 June 2007; revised 19 July 2007; accepted 19 July 2007
Available online 21 August 2007
Dedicated to the memory of Karnail S. Atwal.
Abstract—The design and synthesis of a novel class of amino(methyl) pyrrolidine-based sulfonamides as potent and selective FXa
inhibitors is reported. The amino(methyl) pyrrolidine scaffolds were designed based on the proposed bioisosterism to the piperazine
core in known FXa inhibitors. The SAR study led to compound 15 as the most potent FXa inhibitor in this series, with an IC50 of
5.5 nM and PT EC2x of 1.7 lM. The proposed binding models show that the pyrrolidine cores are in van der Waals contact with the
enzyme surface, and the flexibility of amino(methyl) pyrrolidines allows the two nitrogen atoms to anchor both the P1 and P4
groups to fit similarly in the S1 and S4 pockets.
Ó 2007 Elsevier Ltd. All rights reserved.
Factor Xa is one of the targets for the design of new
antithrombotics.1 Selective factor Xa inhibitors may
effectively block coagulation, since factor Xa is posi-
tioned at the start of the common pathway of the
extrinsic and intrinsic coagulation systems.2 It has
been hypothesized that, because the amount of serine
protease is amplified at each step of the coagulation
cascade, anticoagulants which target coagulation fac-
tors located higher up in the cascade, such as factor
Xa, might be more effective than those directly tar-
geting thrombin. Moreover, by not inhibiting throm-
bin directly, residual thrombin may still facilitate
hemostasis and reduce unwanted bleeding risk.2c Clin-
ical studies with the parenteral, indirect factor Xa
inhibitor fondaparinux indicate that selective inhibi-
tion of factor Xa is a highly effective approach to
the prevention and treatment of venous thromboem-
bolism.3 Recent clinical trial disclosures on oral, di-
rect factor Xa inhibitors such as razaxaban,4a–d
apixaban,4e rivaroxaban4f,g, and LY7177174h,i have
provided clinical evidence of preclinical findings.4
Factor Xa contains a deep S1 and a box-like S4 recog-
nition site at the enzyme’s active site. Potent FXa
inhibitors generally require both an S1 and an S4 bind-
ing element which are connected through L-shaped or
other ‘bent’ scaffolds.1 Several series of potent FXa
inhibitors containing the 1,4-substituted piperazine or
2-oxo-piperazine cores have been reported.5 Among
them, compound 1 (Fig. 1) was first disclosed in a Zen-
eca patent application in 1996,5a and was subsequently
reported to have an IC50 of 210 nM against human
FXa by researchers from Mochida.5b Compound 2
(Fig. 1) was described to inhibit FXa with an IC50 of
19 nM in a Takeda patent application in 1998.5c More
recently, several X-ray crystal structures of structurally
related molecules bound to FXa have been dis-
closed.5d–f They show that the two nitrogen atoms of
the piperazine or 2-oxopiperazine core are positioned
near the entry points of the S1 and S4 pockets, and
the ‘bent’ conformation of the molecule is created by
appropriate substitutions at these two nitrogen atoms.
We envisioned that the 3-aminomethyl-pyrrolidine
could be a potential bioisostere for the piperazine core
due to the relatively planar conformation of the
pyrrolidine ring in combination with the flexible
Keywords: Aminomethyl pyrrolidines; Factor Xa inhibitor; Scaffold;
Bioisosterism; Piperazine.
*
Corresponding author. Tel.: +1 609 818 4124; fax: +1 609 818
3450; e-mail: yan.shi@bms.com
Present address: Hoffman-La Roche Inc., 340 Kingsland Street,
Nutley, NJ 07110, USA.
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.07.063

Y. Shi et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5952–5958
5953
O
S
O
O
S
O
O
O
N
N
N
N
S
N
N
N
Zeneca
1 IC50 = 210 nM5a,b
Takeda
Cl
2 IC50 = 19 nM5c
Cl
N
Figure 1. Substituted piperazine-based inhibitors of human factor Xa 1 and 2.
O
O
O
S
O
S
O
S
H
H
O
H
N
N
O
N
O
N
N
O
S
N
S
N
N
N
N
N
Cl
N
Cl
Cl
3 IC50 = 7800 nM
4 IC50 = 1100 nM
5 IC50 = 247 nM
Figure 2. 3-Aminomethyl-pyrrolidine-based inhibitors of human factor Xa 3–5.
3-aminomethyl group. To test this hypothesis, we syn-
thesized three racemic compounds 3, 4, and 5 (Fig. 2),
which combine the new 3-aminomethyl-pyrrolidine
core with known sulfonamide and amide moieties in
1 and 2. While both 3 and 4 showed weak activity
against human FXa, a clear preference for the 3-aryl-
sulfonylaminomethyl-1-acylpyrrolidine 4 was observed.
The exchange of the thiazole-pyridine moiety in 4 with
a 1-(4-pyridyl)piperidine group provided the more po-
tent analog 5, which has an IC50 of 247 nM6 against
human FXa. Based on this lead, we initiated SAR
studies to further improve the potency. This communi-
cation describes the in vitro SAR of this novel series of
aminomethylpyrrolidine-based FXa inhibitors.7
Replacement of the 4-chlorophenyl group in 11 with
a 5-bromo-2-yl-thiophene moiety affords the more po-
tent analog 13 (IC50 = 86 nM). Its (R)-enantiomer,
compound 14, appears to be the more active stereo-
isomer with an IC50 of 22 nM. The (E)-2-(5-chloro-
thiophen-2-yl)ethene derivatives (compounds 15 and
16) further demonstrate the preference for the (R)-
enantiomer 15. The (R)-enantiomer 15 has an IC50
of 5.5 nM (PT EC2x = 1.7 lM),6 and is about 25-fold
more
potent
than
its
(S)-enantiomer
16
(IC50 = 150 nM). The 50-chloro-2,20-bithiophene moi-
ety also provides potent compounds, for example,
17 (IC50 = 18 nM).
To explore the SAR of the amide moiety appended to
the pyrrolidine ring nitrogen, additional compounds
based on 50-chloro-2,20-bithiophene-5-yl- and (E)-2-
(5-chlorothiophen-2-yl)ethane-sulfonamide were syn-
thesized according to Scheme 2. Selected analogs are
shown in Table 2 to illustrate the SAR. These com-
pounds were prepared by formation of sulfonamide
19 from optically pure tert-butyl 3-(aminomethyl)pyr-
rolidine-1-carboxylate 18 and appropriate sulfonyl
chlorides; subsequent Boc deprotection of 19 and
amide formation reaction with a diverse set of carbox-
ylic acids affords 20–28.
Early efforts in this study focused on analogs of 5 where
the arylsulfonyl group was varied. Selected analogs are
shown in Table 1 to illustrate the SAR. These com-
pounds were prepared according to Scheme 1. Acylation
of the pyrrolidine nitrogen in 6 with 1-(pyridin-4-
yl)piperidine-4-carboxylic acid 7 affords 8. Subsequent
deprotection of the Boc group and reaction with appro-
priate arylsulfonyl chlorides provides 5 (65% yield) and
9–17 (15–73% yield).
As shown in Table 1, the unsubstituted 2-naphthyl com-
pound 9 is about 5-fold less potent than the 6-chloro-
naphthyl analog 5, which indicates the importance of
the chlorine atom. Replacement of the 6-chloro-2-naph-
thyl group in 5 with a (E)-3-chlorostyryl group provides
10, which is about 20-fold less active. However, the reg-
ioisomeric (E)-4-chlorostyryl compound 11 (racemate) is
only about 2-fold less potent than 5, and is about 2-fold
less potent than the piperazine derivative 1 (210 nM,
Fig. 1) with the same pharmacophores at both ends.
This indicates a trans olefin-aryl group may be a viable
pharmacophore in this region. The 6-chloro-2-yl-
benzo[b]thiophene compound 12 has similar FXa activ-
ity to 5.
As shown in Table 2, compounds with a biaryl amide
substrate are generally disfavored in both R and S ser-
ies (20 and 21). The 2-methylpyrimidine compounds
are less potent than their pyridine analogs (compare
compounds 24–26 with 15–17). Interestingly, some
substituted oxalic pyrrolidines in the S series exhibit
potent FXa inhibition activities (compounds 23, 27,
and 28), with compound 28 having an IC50 = 40 nM
and PT EC2x = 13 lM.
The proposed binding models of compounds 17
and 28 with FXa are shown in Figures 3 and 4,

5954
Y. Shi et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5952–5958
Table 1. SAR of the aromatic binding element
O
O
O
O
S
O
S
O
H
S
H
H
R
S
N
N
N
O
N
O
N
O
N
Ar
Ar
Ar
N
N
N
5, 9-13
14, 15, 17
Chirality
( )
16
N
N
N
a
b
Compound
Ar
IC50 (nM)
247
PT EC2x (lM)
5
40
—
—
32
45
8.7
5
Cl
9
10
11
12
13
14
15
16
17
( )
( )
( )
( )
( )
R
1100
4300
493
255
86
Cl
Cl
S
Cl
Br
S
S
S
S
Br
Cl
Cl
22
R
5.5
1.7
13
6.8
S
150
18
S
Cl
R
S
a IC50 values are measured against human factor Xa utilizing the cleavage of a synthetic substrate S-2222.
b Concentration of inhibitor required to double the prothrombin-based clotting time in human plasma; data are the average of two independent
determinations.
O
O
O
S
H
H
*
*
N
N
N
O
N
a
b, c
BOC
H
*
NH
Ar
N
BOC
N
N
6
5, 9-17
8
N
N
Scheme 1. The synthesis of 5, 9–17. Reagents: (a) 1-(pyridin-4-yl)piperidine-4-carboxylic acid (7), EDCI, Et3N, CH2Cl2; (b) TFA, CH2Cl2;
(c) arylsulfonyl chloride, EtOAc/NaHCO3 (satd), 15–73% yield for three steps.
respectively.8 In both models, the aminomethyl pyrrol-
idine cores lie along the surface of the protein. The
two nitrogen atoms of the aminomethyl pyrrolidine
group provide the anchor points for the P1 and P4
moieties. In the P1 direction, the arylsulfonyl group
creates the needed L-shaped turn seen in many FXa
inhibitors, allowing the chlorothiophene group to fit
deep into the S1 pocket where the chlorine atom fills
a key hydrophobic interaction site above Tyr228. In
the S4 pocket, the protonated 4-(piperidin-1-yl)pyri-
dine group of 17 participates in a hydrogen bond with
a conserved water molecule, and forms a p–cation

Y. Shi et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5952–5958
5955
O
R
O
O
S
H
H
a
b, c
*
*
N
N
BOC
O
N
O
N
*
S
N
BOC
H2N
Ar
Ar
18
20-28
19
Scheme 2. The synthesis of 20–28. Reagents: (a) arylsulfonyl chloride, EtOAc/NaHCO3 (satd); (b) TFA, CH2Cl2; (c) RCOOH, EDCI, Et3N,
CH2Cl2, 10–71% yield over three steps.
Table 2. SAR of the amide pharmacophore
O
O
S
H
*
N
O
N
R
Ar
20-28
a
IC50 (nM)
b
Compound
Ar
R
Chirality
PT EC2x (lM)
H2NO2S
S
S
Cl
Cl
20
R
1900
1300
—
—
N
21
22
S
N
N
O
O
S
Cl
R
436
—
N
N
S
S
S
Cl
Cl
Cl
23
24
25
26
27
S
R
S
R
S
70
8
N
N
N
N
230
718
61
28
—
50
130
N
N
N
N
N
N
S
S
Cl
Cl
S
S
O
O
130
N
N
N
S
Cl
28
S
40
13
S
a IC50 values are measured against human factor Xa utilizing the cleavage of a synthetic substrate S-2222.
b Concentration of inhibitor required to double the prothrombin-based clotting time in human plasma; data are the average of two independent
determinations.
interaction with the aromatic residues lining the pock-
et. For compound 28, the 2-pyrrolidinomethylpyrroli-
dine fills the S4 pocket with the distal pyrrolidine
forming an intramolecular hydrogen bond with the
amide carbonyl seen in many j Opioid agonists9; the
presence of this additional pyrrolidine enhances the
interaction between the acyl pyrrolidine and S4 pocket
(compare compounds 27 and 28). In addition, the po-
tential hydrogen bonding interaction between Gln192
side chain and one of the sulfonamide oxygens in both
models may also contribute to the binding activity.
Docking studies of 15 versus 16 suggest the activity
difference (25-fold) may be due to the energetics
of the different conformations of the aminomethyl

5956
Y. Shi et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5952–5958
role played by the cores seen in crystal structures of
Gln192
piperazine-based ligands in FXa.5e,f The flexibility of
the aminomethyl pyrrolidine group allows both enantio-
meric cores to project the P1 and P4 pharmacophores
into the S1 and S4 pockets. It could be proposed that
2-aminomethyl pyrrolidine and 3-aminopyrrolidine with
a two-carbon spacer between two nitrogens may also be
good scaffolds for FXa inhibitors. Indeed, compounds
such as racemate 29 and 30 (Fig. 5), with the same P1
and P4 pharmacophores as in 17, are also potent FXa
inhibitors with FXa IC50 of 5.5 nM and 7.9 nM, respec-
tively. Figure 6 shows the overlay of models of 17, 29
(S enantiomer), and 30 (S enantiomer) bound to FXa,
which demonstrates that the three central core variants
enable the P1 and P4 groups to fit similarly in the S1
and S4 pockets.
His57
Tyr99
Cys191
Thr98
Trp215
Phe174
Tyr228
Asp189
Ile175
Figure 3. Model of 17 bound to FXa.
Asp97
Lys96
Although the preferred absolute stereochemistry for 29
and 30 has not been determined, similar SAR in the
P1 binding elements have been observed for both the
3-aminomethyl pyrrolidine and 3-aminopyrrolidine
series when the P4 pharmacophore is a 1-(4-pyridyl)-
piperidine group (data not shown).
Gln192
Tyr99
Thr98
Cys191
While the compounds described above are potent and
selective FXa inhibitors relative to related trypsin-
like serine proteases,10 compounds containing
a
Cys220
1-(4-pyridyl)piperidine group as P4 pharmacophore are
generally moderate inhibitors of cytochrome P450 and
have limited Caco-2 cell permeability.11 Further work
would be necessary to address these issues before this ser-
ies can furnish acceptable antithrombotic drug candidates.
Trp215
Ile175
Asp189
Tyr228
Figure 4. Model of 28 bound to FXa.
In summary, we have discovered a novel series of FXa
inhibitors. These compounds exhibit potent and highly
selective anti-FXa activity. The SAR demonstrates that
both 2- and 3-aminomethyl pyrrolidines and 3-amino-
pyrrolidine are excellent bioisosteres for the piperazine
core in FXa inhibitors with similar P1 and P4 pharma-
cophores. The SAR is consistent with binding
models which show that the pyrrolidine cores are in
van der Waals contact with the enzyme surface, and
the flexibility of amino(methyl) pyrrolidines allows the
two nitrogen atoms to anchor both the P1 and P4
groups to fit similarly in the S1 and S4 pockets.
substituent relative to the pyrrolidine core—the R con-
formation is equatorial, whereas the S conformation is
axial and must fold back on the pyrrolidine to dock
well.
From these two models, it is clear that the center pyrrol-
idine cores function as scaffolds, where the primary
interaction with the enzyme is through van der Waals
contacts on the bottom face of the core, similar to the
O
O
O
S
O
S
O
N
H
H
O
N
O
N
O
N
S
N
N
R
H
S
S
S
O
N
N
N
N
S
S
S
N
N
Cl
Cl
Cl
17
29
30
IC50 = 18 nM
IC50 = 5.5 nM
IC50 = 7.9 nM
EC2 x PT = 6.8 μM
EC2 x PT = 3.3 μM
EC2 x PT = 3.0 μM
Figure 5. The SAR of amino(methyl)pyrrolidines.

Y. Shi et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5952–5958
5957
Gln192
17, 29, 30
Tyr99
Thr98
Cys191
Cys220
Tyr228
Trp215
Phe174
Asp189
Ile175
Figure 6. The overlay of models of 17, 29, and 30 (S enantiomers) bound to FXa.
Luettgen, J. M.; Wong, P. C.; Knabb, R. M.; Wexler, R.
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Acknowledgments
We thank Dr. P. T. Cheng and Dr. R. R. Wexler for
insightful review of this work.
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Scarborough, R. M.; Zhu, B.-Y. Bioorg. Med. Chem. Lett.
2004, 14, 2073.
9. At physiologic pH (7.2–7.4), the distal pyrrolidine N
(ACD pKa = 9.69) is protonated and forms an intramo-
lecular hydrogen bond with the amide carbonyl. This
preferred conformation of similar moieties which contain
a N-(2-aminoethyl)acetamide had been intensively inves-
tigated in many j Opioid agonists by molecular model-
ing, NMR studies, and X-ray analyses. See: (a)
Froimowitz, M.; Dimeglio, C. M.; Makriyannis, A. J.
J. Med. Chem. 1992, 35, 3087; (b) Subramanian, G.;
Paterlini, M. G.; Larson, D. L.; Portoghese, P. S.;
Ferguson, D. M. J. Med. Chem. 1998, 41, 4777; (c)
Vecchietti, V.; Giordani, A.; Giardina, G.; Colle, R.;
Clarke, G. D. J. Med. Chem. 1991, 34, 397; (d) Doi, M.;
Ishida, T.; Inoue, M. Acta Crystallogr. 1990, C46, 676;
(e) Doi, M.; Ishida, T.; Inoue, M. Chem. Pharm. Bull.
1990, 38, 1815; (f) Chang, A.-C.; Takemori, A. E.; Ojala,
W. H.; Gleason, W. B.; Portoghese, P. S. J. Med. Chem.
1994, 37, 4490.
10. For example, compound 17 is inactive (IC50 > 30 lM)
when tested against thrombin, trypsin, Factor IXa, Factor
VIIa, and plasma kallikrein.
11. For example, compound 17 has Caco-2 cell permeability
(apical to basal) < 15 nm/s at pH 6.5; it inhibits CYP450
isozymes with the following IC50 values: 3A4 (7-benzyl-
oxy-4-trifluoromethylcoumarin), 2.33 lM; 3A4 (7-benzyl-
oxyresorufin), 1.0 lM; 2D6, 7.97 lM; 2C9, 1.47 lM;
2C19, 14.1 lM; 1A2, >20 lM.
6. IC50s are measured against human factor Xa utilizing the
cleavage of a synthetic substrate S-2222. The PT EC2x is
the concentration of inhibitor required to double the
prothrombin-based clotting time in human plasma. See:
Kettner, C. A.; Mersinger, L. J.; Knabb, R. M. J. Biol.
Chem. 1990, 265, 18289.
7. This work was presented in part by: Shi, Y.; Zhang, J.;
Stein, P. D.; Wang, Y.; Kennedy, L.; O’Connor, S. P.;
Ahmad, S.; Atwal, K. S.; Han, W.; Hu, Z.; Sitkoff, D.;
Lam, P. Y. S.; Robl, J. A.; Liu, E. C.-K.; Seiler, S. M.;
Hartl, K. S.; Wexler, R. R.; Zahler, R. Abstr. Pap. Am.
Chem. Soc. 2006, 232, MEDI-395.
8. Compounds were docked into the FXa enzyme structure
from pdb entry 2bmg (Matter, H.; Will, D. W.; Nazare,
M.; Schreuder, H.; Laux, V.; Wehner, V.; Liesum, A.
J. Med. Chem. 2005, 48, 3290) using Glide (v. 4.0,
Schrodinger, LLC, New York, NY, 2005). Prior to
docking, all waters were removed except the water in the
S4 pocket, and hydrogens were assigned using the prot-
assign utility. The enzyme was then run through the Glide
proteinprep and refine utilities. Docking was performed in
sp mode with default parameters except that the grids for
the enzyme active site were calculated using an 0.9 (instead
of 1.0) scaling factor on the vdw radii of protein atoms
with an absolute value of partial atomic charge less than