5-Amidinoindoles as dual inhibitors of coagulation factors IXa and Xa

Article abstract of DOI:10.1016/j.bmcl.2004.08.037

Structural features of a 5-amidinoindole inhibitor of factor Xa, which displayed modest inhibition of factor IXa were varied to increase potency and improve selectivity for factor IXa. Structural features of a 5-amidinoindole inhibitor of factor Xa, which displayed modest inhibition of factor IXa were varied to increase potency and improve selectivity for factor IXa.

Full text of DOI:10.1016/j.bmcl.2004.08.037

Bioorganic & Medicinal Chemistry Letters 14 (2004) 5269–5273
5-Amidinoindoles as dual inhibitors of coagulation factors
IXa and Xa
Douglas G. Batt,* Jennifer X. Qiao, Dilip P. Modi, Gregory C. Houghton,
Deborah A. Pierson, Karen A. Rossi, Joseph M. Luettgen, Robert M. Knabb,
P. K. Jadhav and Ruth R. Wexler
Bristol-Myers Squibb Pharmaceutical Research Institute, PO Box 4000, Princeton, NJ 08543-4000, USA
Received 2 July 2004; revised 17 August 2004; accepted 17 August 2004
Available online 15 September 2004
Abstract—Structural features of a 5-amidinoindole inhibitor of factor Xa, which displayed modest inhibition of factor IXa were
varied to increase potency and improve selectivity for factor IXa.
Ó 2004 Elsevier Ltd. All rights reserved.
Table 1. Screening leads
Thrombotic diseases, such as myocardial infarction,
stroke, deep vein thrombosis and pulmonary embolism,
are leading causes of mortality. Currently these diseases
are most often treated with heparin or warfarin, both of
which carry significant risks for bleeding complications.
Enormous effort has been expended in the search for
safer agents, mostly aimed at inhibition of thrombin
or coagulation factor Xa (fXa).1 Factor Xa, which con-
verts prothrombin to thrombin in the penultimate step
in the coagulation cascade, can be generated from the
zymogen (factor X) through proteolysis by either factor
VIIa via the extrinsic pathway2 or by factor IXa (fIXa)
via the intrinsic pathway.3 Selective blockade of only the
intrinsic pathway by inhibiting fIXa could provide a
drug with less liability for problem bleeding. The viabil-
ity of fIXa inhibition as an approach to selective anti-
thrombosis is supported by studies with anti-IXa
monoclonal antibodies4,5 and with active-site covalently
inhibited fIXa.6
HN
R
SO2NH2
NH
O
H2N
N
H
Compound
R
H
fIXa Ki, nM fXa Ki, nM fIXa/fXa
1
2
6700
660
3.2
0.63
2100
1050
CH2Ph
benzyl substituent of 2 improved activity 10-fold. Com-
pound 2 was selected for structure–activity studies in an
attempt to increase potency for fIXa inhibition, and to
seek selectivity with respect to inhibition of fXa.
The synthetic approach to compounds 13–24, 26, and
29–32 is shown in Scheme 1. The ketoester 39 was re-
duced to the indoleacetate ester 4 with triethylsilane.
Over-reduction always produced about 30% of the cor-
responding indoline, which was difficult to remove chro-
matographically; it was more expedient to subject the
mixture to re-oxidation using a published procedure10
followed by a much easier chromatographic purification
to provide 4 in 76% overall yield. After methylation or
protection of the ring nitrogen, benzylic alkylation of
the derived anion generally provided 5 in good yields.
Anilides were prepared directly from the esters by treat-
ment with the corresponding aniline and trimethylalum-
inum; benzylamides were prepared from the derived
carboxylic acid using coupling reagents. Pinner reaction
Since the active sites of fIXa and fXa are very similar,7
screening of our proprietary fXa inhibitors in an assay
for fIXa inhibition8 was undertaken to seek an initial
lead. Among the compounds revealed by this procedure
were the 3-substituted 5-amidinoindoles9 1 and 2 (Table
1). Although 1 was only a weak inhibitor of fIXa, and
was 2000-fold selective for fXa, incorporation of the
Keywords: Factor IXa; Anticoagulant; Indole.
*
Corresponding author. Tel.: +1 609 252 4034; fax: +1 609 252
6601; e-mail: douglas.batt@bms.com
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.08.037

5270
D. G. Batt et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5269–5273
O
COOMe
Ph
COOMe
COOMe
NC
a, b, c, d
NC
NC
a,b
NC
N
H
N
N
H
N
H
TIPS
9
3
4
10
e
c,d
O
Ph
O
Ph
NHR'
O
f
R
R
O
NH
COOMe
COOEt
NC
NC
NC
e, f, g
O
H2N
N
N
H
N
N
H
R''
R''
11
12
6a (R'' = H)
5a (R'' = TIPS)
5b (R'' = Me)
6b (R'' = Me)
Scheme 3. Reagents and conditions: (a) CH@CHCOOH, HOAc,
Ac2O, reflux, 15%; (b) MeOH, conc. HCl, rt, 81%; (c) NaH, DMF,
0°C; i-Pr3SiCl, to rt (39%); (d) LiN(TMS)2, THF, ꢀ78°C; PhCH2Br,
to rt, 52%; (e) PhCH2CHO, MeldrumÕs acid, L-proline, MeCN, rt,
32%; (f) pyridine, EtOH, Cu powder, reflux, 79%.
Scheme 1. Reagents and conditions: (a) Et3SiH, CF3COOH, rt; (b)
(PhSeO)2O, indole, THF, 60°C, 76% (2 steps); (c) for 5a: KN(TMS)2,
THF, 0°C; i-Pr3SiCl, to rt (75%); for 5b: NaH, DMF, 0°C; MeI, to rt
(99%); (d) LiN(TMS)2, THF, ꢀ78°C; RBr or RI, to rt (30–80%); (e)
Me3Al, R0NH2, CH2Cl2; or LiOH, then DIC, pyridine/DMSO,
R0NH2, 60–80%; (f) EtOH, HCl, rt; (g) NH3, MeOH, rt; or NH4OAc,
MeOH, rt, 50–80% (2 steps).
urement of inhibition of fIXa in particular is difficult
since the enzyme shows poor activity against small
chromogenic substrates,15 requiring the addition of sub-
stances such as ethylene glycol to obtain useful enzy-
matic activity.8 (A more physiologically relevant assay
of fIXa activity, involving activation of factor X fol-
lowed by monitoring the generated fXa activity via
hydrolysis of a chromogenic substrate,16 was not used
because the strong fXa inhibition would make dissection
of the inhibition of fIXa from the overall inhibition
difficult or impossible.) Since the assays for fIXa and
fXa are performed under different conditions, the actual
magnitudes of the two Ki values are not directly compa-
rable, but the ratio of the two Ki values should be useful
for comparing the selectivities of analogs within a series
of compounds. (In results not shown, compounds 13–32
were generally 10–50 fold less potent against thrombin14
than against fIXa.)
Ph
COOMe
H
NC
NC
a,b
N
N
7
8
Scheme 2. Reagents and conditions: (a) NaH, DMF, 0°C;
BrCH2COOEt, to rt (72%); (b) LiN(TMS)2, THF, ꢀ78°C; PhCH2Br,
to rt (30%).
followed by ammonolysis was accompanied by removal
of the TIPS protecting group (if present) to provide the
desired compounds (represented by structures 6a and
6b) in 40–60% overall yield.
The alternative 1,6-disubstituted indole derivative 25
was prepared similarly, from the known 6-cyanoindole
7,11 as shown in Scheme 2. Alkylation of the sodium salt
of 7 with ethyl bromoacetate proceeded in good yield,
but alkylation of the derived anion provided 8 in only
low yield. Amide formation and amidine elaboration
then proceeded as shown in Scheme 1.
Screening results for other proprietary fXa inhibitors
suggested that replacement of the 4-biphenyl moiety
(which is bound in the S4 region of trypsin,17 and pre-
sumably binds similarly in both fXa and fIXa) with 4-
(1-benzimidazolyl)phenyl18 should boost fIXa activity.
Incorporation of this substituent into 2 provided 13a,
which enhanced fIXa potency by an order of magnitude
and, importantly, failed to produce a similar increase in
fXa potency, thus lowering the selectivity ratio to 25
(Table 2). This racemic compound was resolved by
chiral HPLC, and better potency and selectivity were
displayed by the levorotatory isomer (13b). The
carboxylic acid obtained by hydrolysis of 5a
(R = CH2Ph) was also resolved by chiral HPLC, and a
single crystal X-ray structure of the cinchonidine salt
of the (+) isomer proved this to have the (S) configura-
tion. The (ꢀ) isomer of the acid was converted to 13b,
by coupling the acid directly with the appropriate
R0NH2 using DIC followed by amidine elaboration as
in Scheme 1, establishing the configuration of the more
active isomer as (R).
Two different approaches were used to prepare com-
pounds with longer chains (Scheme 3). 5-Cyanoindole
9 was alkylated with acrylic acid12 in low yield, followed
by conversion to the methyl ester, nitrogen protection
and alkylation to provide 10. Several approaches to
inter-mediate 12 were explored with limited success. Fi-
nally, following a literature method,13 9 was treated with
phenylacetaldehyde and MeldrumÕs acid in the presence
of proline to provide, in moderate yield, 11, which
underwent ester exchange and decarboxylation to pro-
vide useable quantities of 12. Intermediates 10 and 12
were elaborated to 27 and 28 (Table 5), respectively, as
described above.
Inhibitory activities against fIXa and fXa were assayed
using published methods, involving enzymatic hydro-
lysis of small peptidic chromogenic substrates.8,14 Meas-
This stereochemical finding matches the results of mode-
ling des-fluoro 13 in the active site of fIXa, derived from

D. G. Batt et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5269–5273
5271
˚
Table 2. Effect of absolute configuration
bonding distance (3.1A) of the backbone nitrogen of
Glu 219 at the edge of the S1 pocket. The benzimidazol-
ylphenyl group lies in the S4 region, interacting with Tyr
99, Phe 174, and Trp 215. The (R) benzyl substituent
could engage in a hydrophobic interaction with the
Cys191-Cys220 disulfide bond near the edge of the S1
pocket, whereas such interactions would not be possible
for the (S) enantiomer; in fact, the (S) benzyl would
clash with the edge of the S1 pocket if the rest of the
molecule bound as shown in Figure 1.
F
N
N
HN
NH
O
H2N
N
H
Compound Config. fIXa Ki, nM fXa Ki, nM fIXa/fXa
13a
13b
13c
(RS)
(R)-(ꢀ)
(S)-(+)
63
19
2.5
1.3
25
15
32
820
26
Variation of the benzyl substituent showed no strong
structure–activity trends, as the examples in Table 3
demonstrate. (The presence or absence of the 2-fluoro
substituent on the aniline ring had a minimal effect on
either potency or selectivity.)18 The presence of at least
a 2-carbon chain seemed required for better selectivity
(compare 14 and 15 with 16), although longer groups
did not help significantly (18 vs 13a). Larger lipophilic
groups (19, 24) seemed in general to provide slightly bet-
ter selectivity, due to their negative impact on fXa activ-
ity. The naphthyl derivative 19 showed the weakest fXa
activity of the substituents explored. This could suggest
unfavorable steric interactions with fXa, although no
such interactions were apparent from computer mode-
ling. The cyclopropylmethyl compound 17 was anoma-
lously weak against fIXa. Since a major difference
Table 4. Effect of indole core changes
F
N
N
HN
Figure 1. Model of Compound 13b in the active site of IXa. The
inhibitor, shown in blue, has the amidine positioned near Asp 189 in
the back of the S1 pocket, with the S4 pocket to the right.
NH
O
Y
X
H2N
Compound
X
Y
fIXa Ki, nM fXa Ki, nM fIXa/fXa
a published X-ray structure, as shown in Figure 1.19 In
this model, the indole lies in the S1 pocket, with the ami-
dine interacting with Asp 189 at the bottom of this
pocket. The amide carbonyl of 13 lies within hydrogen
13a
25
NH
CH
C
N
C
63
300
360
2.5
5.6
25
54
2
26
NMe
160
Table 3. Effect of chain substitution
Y
N
N
HN
NH
X
O
H2N
N
H
Compound
X
Y
fIXa Ki, nM
fXa Ki, nM
fIXa/fXa
13a
14
15
16
17
18
19
20
21
22
23
24
CH(CH2Ph)
CH(Me)
F
63
480
750
160
3800
240
98
2.5
3
25
160
340
57
H
H
H
H
F
C(Me)2
CH(Et)
2.2
2.8
CH(CH2-cyclopropyl)
CH(CH2CH2Ph)
CH(CH2-2-naphthyl)
CH(CH2-Ph-3-COOH)
CH(CH2-Ph-4-NH2)
CH(CH2-3-pyridyl)
CH(CH2-Ph-4-NHCOPh)
CH(CH2-Ph-4-NHSO2Ph)
14
6.1
28
270
39
F
4
33
F
59
75
1.8
4
H
H
H
H
19
1200
270
130
7
170
15
18
19
7

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D. G. Batt et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5269–5273
Table 5. Effect of S4 substituent
W
Y
HN
NH
X
O
Z
H2N
N
H
Compd
X
Y
Za
W
fIXa Ki, nM
fXa Ki, nM
fIXa/fXa
13a
27
28
29
30
31
32
32
CH(CH2Ph)
CH2CH(CH2Ph)
CH(CH2Ph)CH2
CH(CH2Ph)
CH(CH2Ph)
CH(CH2Ph)
CH(CH2Ph)
CH(CH2Ph)
Bond
Bond
Bond
CH2
4-(1-bzim)
4-(1-bzim)
4-(1-bzim)
4-(1-bzim)
F
63
130
250
310
930
1900
270
660
2.5
45
25
3
H
H
H
H
H
H
H
36
72
7
4
CH2
CH2
4-(CONH-2-bzim)
3-(CONH-2-bzim)
810
2600
79
1
0.7
3
Bond
CH2
4-(4-Phenylimidazolyl)
4-(CONH-6-MeSO2-2-bzim)
1100
0.6
a bzim = benzimidazolyl.
between fXa and fIXa is the replacement of Gly 219
with Glu 219,7 the activity of basic and acidic groups,
which might interact with Glu 219 was of special inter-
est.18 However, neither acidic (20) nor basic (21) groups
on the benzyl substituent had much effect, while basic
heterocycles (22, for example) lost significant fIXa
potency. The broad, shallow nature of the SAR may
suggest that a major role for this substituent could be
to provide a conformational bias for the flexible side-
chain, favoring the modeled disposition of the S4-bind-
ing group relative to the amidinoindole, with added
potency arising from nonobvious interactions of lipo-
philic groups with the enzyme.
enhance selectivity through a detrimental effect on fXa
potency. This was found to be the case, as shown in
Table 5. Adding a carbon atom between the indole
and amide group, on either side of the benzyl substituent
(27, 28), caused a 2- to 4-fold decrease in fIXa activity,
but a 10- to 20-fold decrease in fXa activity, leading to
an improved selectivity ratio. Replacement of the aniline
moiety of the amide with longer groups weakened fIXa
activity further, but led to dramatic increases in selectiv-
ity, with two analogs (31, 33) representing the first exam-
ples of compounds, which are equipotent to slightly
more potent against fIXa than fXa.
In summary, a screening lead with half-micromolar fIXa
potency but 1000-fold fXa selectivity was examined for
structural variations, which would favor fIXa inhibition
over that of fXa. An alpha-substituent favors IXa more
than Xa, with SAR suggesting beneficial effects from
lipophilicity or a possible conformational biasing effect.
Steric interference in the region of the catalytic triad is
poorly tolerated by fXa, while fIXa seems to prefer
the indole NH more than does fXa. Larger, more ex-
tended groups expected to bind in the S4 pocket of the
enzymes also seem to differentiate between the two, pos-
sibly due to steric interference with a less flexible region
of fXa. Thus, several analogs of compound 2 with
greatly diminished fXa selectivity were prepared, and
structural features favorable to fIXa potency were iden-
tified, setting the stage for further optimization of this
series.22
Modeling of compound 13b in the active site of fIXa
(Fig. 1) positioned the indole NH near the catalytic ser-
˚
ine 195. Although the N–O distance in this model (4.3A)
is long for a hydrogen bond, the possibility of an inter-
action clearly exists. Two compounds were prepared to
explore the importance of any such interaction (Table
4). Compound 25, lacking the NH but isosteric with
13b, showed a five-fold loss of potency against fIXa,
suggesting the possibility of an interaction with serine
195. A two-fold loss against fXa was seen. The N-methyl
analog 26, interestingly, had about the same potency
against fIXa as 25, yet showed a very dramatic loss of
potency against fXa. This suggests that the increased
size of 26 does not further damage the binding with
fIXa, but that fXa is much more intolerant of increased
size in this region, possibly due to greater rigidity in the
catalytic triad of the enzyme. Examination of published
X-ray structures of the two enzymes7 shows that in fXa,
the Ô60Õs loopÕ lies closer to serine 195 than in fIXa, pos-
sibly providing a buttressing effect, which could limit the
ability of fXa to accommodate a larger inhibitor.
References and notes
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pocket, is significantly altered in fIXa relative to fXa,
due in large part to a two-residue insertion in fIXa.7
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ing binding to cofactors and macromolecular substrates
could be important in affecting the catalytic efficiency of
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14. Factor Xa and thrombin inhibition was assayed using
human enzymes with
a chromogenic substrate, as