Non-peptidic phenyl-based thrombin inhibitors: Exploring structural requirements of the S1 specificity pocket with amidines

Article abstract of DOI:10.1016/S0960-894X(99)00616-2

We expand the structural requirements and structure-activity relationship of a novel class of non-peptidic aryl-based thrombin inhibitors through exploration of the S1 specificity pocket of thrombin using flexible and constrained amidines. The most active compound of this class is 11 with K(i) = 69 nM, which is ca. 15-fold less potent than constrained guanidine 5.

Full text of DOI:10.1016/S0960-894X(99)00616-2

Bioorganic & Medicinal Chemistry Letters 10 (2000) 83±85
Non-Peptidic Phenyl-Based Thrombin Inhibitors:
Exploring Structural Requirements of the S1 Speci®city
Pocket with Amidines
Tianbao Lu, Bruce Tomczuk, Roger Bone, Larry Murphy, F. Raymond Salemme
and Richard M. Soll*
3-Dimensional Pharmaceuticals, Eagleview Corporate Center, Exton, PA 19341, USA
Received 11 October 1999; accepted 2 November 1999
AbstractÐWe expand the structural requirements and structure±activity relationship of a novel class of non-peptidic aryl-based
thrombin inhibitors through exploration of the S1 speci®city pocket of thrombin using ¯exible and constrained amidines. The most
active compound of this class is 11 with Ki=69 nM, which is ca. 15-fold less potent than constrained guanidine 5. # 1999 Published
by Elsevier Science Ltd. All rights reserved.
A growing body of achiral, non-peptidic thrombin
inhibitors (e.g. 1±6) has emerged recently in which an
aromatic sca€old has replaced the polyamide- or pep-
tide-like backbone of typical thrombin inhibitors.1±10 In
contrast to sca€olds such as those found in UK 156406
(7), LB30057 (8), and L-375378 (9) many of these peptide
replacements, such as 1, 5, and 6 lack hydrogen bonding
interactions with the enzyme, aside from basic group
interactions with Asp 189 of the S1 speci®city pocket.
Given their structural simplicity, drug-like features, low
molecular weight, potency, and selectivity, inhibitors
based on aryl sca€olds may ultimately o€er advantages
in antithrombotic therapy in comparison to aminoacid-
based or polyamide systems.
*Corresponding author. Tel.: +1-610-458-6056; fax: +1-610-458-
8249; e-mail: soll@3dp.com
0960-894X/00/$ - see front matter # 1999 Published by Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(99)00616-2

84
T. Lu et al. / Bioorg. Med. Chem. Lett. 10 (2000) 83±85
We recently reported analogues of 6, which developed
the SAR of allowable aryl groups projecting into the
distal aromatic binding pocket.6 We now extend these
structure±activity studies to explore requirements of
amidino-based moieties in the S1 speci®city pocket of
the enzyme. In appreciation of factors which may ulti-
mately govern overall in vivo performance,11 this study
was undertaken to explore guanidino group replace-
ment with a group of lower pKa but still capable of
maintaining key hydrogen bonding interactions with
Asp189 of the S1 pocket.
Compounds 10±15 were assayed for thrombin inhibition
as well as for selectivity against a panel of other related
serine proteases3 as summarized in Table 1. Overall the
amidine were weaker thrombin inhibitors in comparison
to their guanidino analogues. Compound 11, the most
potent of the amidines, was about 15-fold less potent
than the constrained guanidine (5) and about 5-fold
less potent than unconstained 6. Surprisingly, restricting
the P1 chain into rigid, hydrophobic backbone isosteric
with 5 failed to increase potency in contrast to the guani-
dino analogues. Thus, benzamidine derivative 13 was
slightly less active than 11 while trans-cyclohexyl (12) was
30-fold less potent than 11, 160-fold less potent than 6,
and more than 450-fold less potent than the constrained
guanidine 5. The benzamidine regioisomers 13 and 14
were nearly equipotent with respect to thrombin inhibition.
Replacing the terminal amino group of 5 with a methyl
group was detrimental to activity, as expected based on
crystallographic analysis of 5.3 With respect to selectivity,
no trypsin activity was detected as screening doses with
amidine 11, whereas the selectivity for thrombin versus
trypsin decreased for benzamidines 13 and 14.
Compounds 10±14 were prepared in 2 steps from 16:3
etheri®cation of 16 with corresponding alcohols 17±22
using Mitsunobu conditions (76±93% yield) followed by
amidination ((a) 37% HCl in EtOH, 0 ꢀC, 2 days; (b)
(NH4)2CO3, 0 ꢀC, 2 days; 50±75% overall yield). Com-
pound 15 was similarly synthesized by coupling 16 with
alcohol 23 using Mitsunobu conditions12 (90%), de-
protection (4 N HCl in dioxane, 95%), and conversion
to the amidine (ethyl acetimidate hydrochloride, TEA,
DMF, 86%).
Table 1. Inhibition of proteases: expressed as Ki (mM) or % inhibition at (mM)
Compound
Thrombin
FXa
>15
Chymotrypsin
>15
Trypsin
>15
53
0.0046‹0.001
63
0.013‹0.0016
110‹56.5
>49
42‹0.78
10
11
12
0.46‹0.045
0.069‹0.0032
2.1‹0.33
0% (7.5 mM)
0% (1.5 mM)
0% (1.6 mM)
0% (15 mM)
2.4‹0.37
0% (15 mM)
0% (15 mM)
0% (1.6 mM)
2.9‹0.37
13
14
0.11‹0.0056
0.099‹0.0068
17‹3.8
0% (1.7 mM)
0% (1.3 mM)
2.7‹0.4
2.7‹0.163
1.5‹0.10
15
1.3‹0.066
0% at 1.0 mM
0% at 1.0 mM
0% at 1.0 mM

T. Lu et al. / Bioorg. Med. Chem. Lett. 10 (2000) 83±85
85
In summary, we have expanded further the SAR of a
non-amide-based series of thrombin inhibitors with
respect to S1 speci®city pocket of thrombin. Despite
potential advantages of lower pKa, the amidino based
analogues in this series were less e€ective inhibitors of
thrombin than isosteric guanidino counterpart. Various
modes of rigidi®cation of the ¯exible amidines in the
hydrophobic S1 pocket failed to maintain thrombin
inhibition potency. Nevertheless, given the low molecular
weight and the prospects for achieving low nM thrombin
inhibition, further studies of this series of achiral, aryl-
based systems are in progress and will be reported in
due course.
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