Benzimidazole-Based fXa inhibitors with improved thrombin and trypsin selectivity

Article abstract of DOI:10.1016/S0960-894X(02)00145-2

Optimization of the benzimidazole-based fXa inhibitors for selectivity versus thrombin and trypsin was achieved by substitution on the benzimidazole ring and replacement of the naphthylamidine group. Substitution of a nitro group at the 4-position on the benzimidazole improves both potency against fXa and selectivity versus thrombin. Alternatively, replacement of the naphthylamidine with either a biphenylamidine or propenylbenzamidine not only improves fXa potency and selectivity versus thrombin, but selectivity versus trypsin as well.

Full text of DOI:10.1016/S0960-894X(02)00145-2

Bioorganic& Medicinal Chemistry Letters 12 (2002) 1311–1314
Benzimidazole-Based fXa Inhibitors with Improved Thrombin
and Trypsin Selectivity
Kenneth J. Shaw, William J. Guilford,* Brian D. Griedel, Steve Sakata, Lan Trinh,
Shung Wu, Wei Xu, Zuchun Zhao and Michael M. Morrissey
Medicinal Chemistry, Berlex Biosciences, 15049 San Pablo Avenue, PO Box 4099, Richmond, CA 94804-0099, USA
Received 13 December 2001; accepted 8 February 2002
Abstract—Optimization of the benzimidazole-based fXa inhibitors for selectivity versus thrombin and trypsin was achieved by
substitution on the benzimidazole ring and replacement of the naphthylamidine group. Substitution of a nitro group at the
4-position on the benzimidazole improves both potency against fXa and selectivity versus thrombin. Alternatively, replacement of
the naphthylamidine with either a biphenylamidine or propenylbenzamidine not only improves fXa potency and selectivity versus
thrombin, but selectivity versus trypsin as well. # 2002 Elsevier Science Ltd. All rights reserved.
As part of our effort to identify potent and selective
anticoagulants, which target factor Xa (fXa),1 we have
designed a novel series of inhibitors based on our ben-
zimidazole scaffold.2 Although the previously-reported
benzimidazole-based fXa inhibitors were potent against
fXa, they are only moderately selective against thrombin
(fIIa) and trypsin (Trp) and have a poor pharmaco-
kinetic(PK) profile. Efforts to inocrporate acrboxylic
acid functionality onto the benzimidazole template
improved the PK profile, as in our pyridine based fXa
inhibitors,3 but gave little improvement in selectivity
against other serine proteases.4 In this communication
we describe our efforts towards improving the selectivity
profile by substitution on the benzimidazole ring and
replacement of the naphthylamidine group.
carbonyl-4-hydroxyl-piperidine afforded 2. In contrast
to alkylations without the 4-nitro group,2 alkylation of
2 with 7-cyano-2-bromomethylnaphthalene (3) afforded
4 as a single regioisomer. The selective alkylation of the
benzimidazole nitrogen meta to the nitro group may be
due to the sterichindrance of the ortho position. Specific
analogues could be prepared by reduction of 4 to the
corresponding amine 5, followed by acylation or alkyl-
ation to give intermediate 6. A two-step process of
arylamidine formation using Pinner conditions yielded 7
and N-alkylation gave 8 as described previously.2,3
A similar procedure was used to prepare benzimidazoles
bearing a biphenyl or propenylphenyl substituent at C-1
instead of the naphthyl group. As shown in Scheme 2A,
the biphenylmethylbromide 12 was prepared by Stille
coupling between phenyl bromides 9 and 10 followed by
bromination of toluene intermediate 11. Allylbromide 19
was prepared by a three step synthesis starting with
aldehyde 17 and Wittig reagent 16 (Scheme 2B).
Reduction of the resulting unsaturated aldehyde 18 and
replacement of the corresponding alcohol with bromine
gave allyl bromide 19 (Scheme 2B). Alkylation of ben-
zimidazole 13 with either 12 or 19 followed by the ami-
dine-forming procedures outlined above afforded a
separable regioisomericmixture of 14a–e/15a–e (Table
2) or 20a–e/21a–e (Table 3), respectively. The phenolic
group in 20e and 21e was uncovered by removal of the
p-methoxybenzyl protecting group during amidine form-
ation. The regiochemistry of the inhibitors in Tables 1–3
was established by NMR NOE studies.
Chemistry
Compounds (8b–h in Table 1) substituted at the 4-posi-
tion of the benzimidazole ring were prepared according
to Scheme 1. Nitration of 5-hydroxyl-benzimidazole (1)
afforded a mixture of the 4- and 6-nitro isomers that
were readily separated by chromatography. Mitsunobu
reaction of the 4-nitrobenzimidazole with N-t-butoxy-
*Corresponding author. Tel.: +1-510-669-4065; fax: +1-510-669-
4310; e-mail: william_guilford@berlex.com
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00145-2

1312
K. J. Shaw et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1311–1314
Table 1. In vitro inhibitory activities of 1,4,5-substituted benzimidazoles
increase in potency and selectivity was independent of
the substituent at C-2 as seen in the 2-methyl (8g) and 2-
isopropyl analogues (8h). Thus, substitution of a nitro
group at the 4-position on the 1,5-benzimidazole tem-
plate affords potent fXa inhibitors with excellent
thrombin selectivity (8f, 8g, and 8h) and improved
trypsin selectivity.
fKi (nM)a
To evaluate the importance of the naphthylamidine
group to the biological activity of the benzimidazole
template, we replaced the naphthylamidine with differ-
ent substituted biphenylamidines. Table 2 shows the
disappointing in vitro profile for the four meta and para
substituted isomers in both the 1,5-disubstituted and
1,6-disubstituted regioisomers. Little difference was seen
in the activity between the 1,5- (15a–b) and 1,6-dis-
ubstituted (14a–b) regioisomers in the 3,40 and 4,40 series
of analogues. The 3,30 (14d–15d) and 4,30 (14c–15c) ser-
ies of analogues were more potent and selective than the
other biphenyl analogues, with 15d affording a sub-
nanomolar fXa inhibitor with over 1000-fold thrombin
and 200-fold trypsin selectivity.
No.
R1
R2
fXa
fIIa
Trp
8a
8b
8c
8d
8e
8f
Et
Et
Et
Et
Et
Et
Me
i-Pr
H
NH2
NMe2
4
8
5
50
50
0.1
0.3
0.2
2000
3500
>5000
>5000
>5000
3000
5
24
10
25
15
5
NHCOMe
N(CH2CO2H)2
NO2
8g
8h
NO2
NO2
>5000
>5000
3
3
aKi values for these competitive inhibitors are averaged from multiple
determinations (nꢀ2) and the standard deviations are <30% of the
mean.
Results and Discussion
Simplification of the naphthylamidine group was
accomplished by removal of carbon atoms from the
naphthalene ring to yield a propenylbenzene group,
which we hoped would have a similar binding mode to
trypsin as the 1,5-substituted analogue of 8a.5 The pro-
penylbenzene series of analogues (20b–c and 21b–c)
showed dramatically improved potency and selectivity
(Table 3) over the corresponding unsubstituted naph-
thalene analogue. In particular, propenylbenzene ana-
logue 21b shows a 10-, 4-, and 64-fold increase in fXa
potency, thrombin selectivity, and trypsin selectivity,
respectively, over the corresponding naphthalene ana-
logue 8a. Methyl substitution on the propenyl group in
the 1,6-series as in 20c decreased the potency 40-fold
The effect of varying the substitution at the 4-position
on the benzimidazole ring on potency against fXa and
selectivity versus fIIa and trypsin is shown in Table 1.
Substitution of an amino group at C-4 (8b, 8c) increased
trypsin and thrombin selectivity up to a 2-fold when
compared with unsubstituted analogue 8a with minimal
impact on the inhibition of fXa. Substitution with acet-
amide (8d) or iminodiacetic acid (8e) decreased the fXa
inhibitory activity more than 10-fold versus 8a. How-
ever, substitution with a nitro group enhanced fXa
potency about 40-fold (8f vs 8a) and enhanced selectiv-
ity against thrombin 60- and 40-fold versus trypsin. The
Scheme 1. Reagents and conditions: (i) (a) HNO3, TFA, separate isomers; (b) N-t-butoxycarbonyl-4-hydroxyl-piperidine, DEAD, PPh3; (ii) NaH,
DMF; (iii) SnCl2, pyr; (iv) MeOCOCl, Et3N, CH2Cl2 or RBr, K2CO3, DMF; (v) (a) HCl, EtOH; (b) NH3, EtOH; (vi) (a) LiOH or aq HCl;
.
(b) MeCNHOEt HCl, Et3N, MeOH.

K. J. Shaw et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1311–1314
1313
Scheme 2. Reagents and conditions: (i) Sn2(Bu)6, Pd(PPh3)4; (ii) NBS, AIBN, CCl4; (iii) (a) 12, NaH, DMF; (b) HCl, EtOH; (c) NH3, EtOH;
.
(d) MeC (NH)OEt HCl, Et3N, MeOH and HPLC separation; (iv) MeOH, rt; (v) (a) NaBH4; (b) PPh3, Br2; (vi) (a) 18, NaH, DMF; (b) HCl, EtOH;
.
(c) NH3, EtOH; (d) MeC(NH)OEt HCl, Et3N, MeOH and HPLC separation.
Table 2. In vitro inhibitory activities of biphenylamidino benzimidazole regioisomers
Biphenyl
No.
1,6-Regioisomer, Ki (nM)a
No.
1,5-Regioisomer, Ki (nM)
fXa
fIIa
Trp
fXa
260
670
17
0.46
fIIa
Trp
3,40
4,40
4,30
3,30
14a
14b
14c
14d
150
180
2.2
34
3600
22
770
190
430
91
73
190
15a
15b
15c
15d
>5000
1500
210
690
150
140
67
1120
aKi values for these competitive inhibitors are averaged from multiple determinations (n52) and the standard deviations are <30% of the mean.
Table 3. In vitro inhibitory activities of propenylbenzamidino benzimidazole regioisomers
R1
R2
R3
R4
No.
1,6-Regioisomer, Ki (nM)a
No.
1,5-Regioisomer, Ki (nM)a
fXa
fIIa
Trp
fXa
74
0.40
0.31
fIIa
Trp
H
H
Me
H
H
H
H
H
OMe
OH
H
Am
H
H
H
H
20a
20b
20c
20d
20e
64
0.028
1.3
13
0.10
880
200
380
2600
75
320
21
73
130
43
21a
21b
21c
21d
21e
2500
870
>5000
5000
610
350
32
6.9
410
120
Am
Am
Am
Am
58
0.10
aKi values for these competitive inhibitors are averaged from multiple determinations (nꢀ2) and the standard deviations are <30% of the mean.

1314
K. J. Shaw et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1311–1314
and selectivity about 20-fold versus 20b, but had a more
positive effect in the 1,5-regioisomer series (21b vs 21c).
The importance of the substitution pattern on the
benzamidine ring is demonstrated by the over 100-fold
increase in potency and selective difference between the
the propenyl-para-benzamidine analogues (20a and 21a)
and the corresponding propenyl-meta-benzamidine
analogues (20b and 21b). Saturation of the double bond
in the alkyl chain resulted in a 10-fold loss of activity
versus the propenyl analogues, 20b and 21b (data not
shown). Introduction of a methoxy group on the ben-
zamidine ring causes over a 100-fold decrease in the fXa
inhibitory activity in both the 1,5- and 1,6-regioisomer
series (20d and 21d). The para-hydroxyl group on the
benzamidine ring had minimal effect on the potency and
selectivity over the corresponding unsubstituted com-
pound in both the 1,5-series (21e vs 21b) and the 1,6-
regioisomer series (20b vs 20e).
References and Notes
1. Fevig, J. M.; Wexler, R. R. Annu. Rep. Med. Chem. 1999,
34, 81.
2. Zhao, Z.; Arnaiz, D.; Griedel, B.; Sakata, S.; Dallas, J.;
Whitlow, M.; Trinh, L.; Post, J.; Liang, A.; Morrissey, M.;
Shaw, K. Bioorg. Med. Chem. Lett. 2000, 10, 962.
3. (a) Light, D. R.; Guilford, W. J. Curr. Top. Med. Chem.
2001, 1, 121. (b) Phillips, G. B.; Buckman, B. O.; Davey, D. D.;
Eagen, K. E.; Guilford, W. J.; Hinchman, J.; Ho, E.; Koo-
vakkat, S.; Liang, A.; Light, D. R.; Mohan, R.; Ng, H. P.;
Post, J. M.; Shaw, K. J.; Smith, D.; Subramanyam, B.; Sulli-
van, M. E.; Trinh, L.; Vergona, R.; Walters, J.; White, K.;
Whitlow, M.; Wu, S.; Xu, W.; Morrissey, M. M. J. Med.
Chem. 1998, 41, 3557.
4. Arnaiz, D.; Chou, Y. L., Griedel, B.; Sakata, S.; Shaw, K.;
Zhao, Z.; Koovakkat, S.; Whitlow, M.; Liang, A.; Trinh, L.;
Hinchman, J.; Post, J.; Ho, E.; Subramanyam, B.; Vergona,
R.; Walters, J.; White, K.; Sullivan, M.; Morrissey, M.
Abstract of Papers, 215th National Meeting of the American
Chemical Society, Dallas, TX; American Chemical Society:
Washington, DC, 1998; MedChem 129.
In summary, selective, subnanomolar fXa inhibitors in
the benzimidazole series can be prepared by placement
of a nitro group at the 4-position on 1,5-benzimidazole
template or by replacement of the naphthylamidine with
either a biphenylamidine or a propenylbenzamidine
group.
5. Whitlow, M.; Arnaiz, D. O.; Buckman, B. O.; Davey,
D. D.; Griedel, B.; Guilford, W. J.; Koovakkat, S. K.; Liang,
A.; Mohan, R.; Phillips, G. B.; Seto, M.; Shaw, K. J.; Xu, W.;
Zhao, Z. Z.; Light, D. R.; Morrissey, M. M. Acta Cryst. D
1999, 55, 1395.