Design, synthesis and SAR of a series of 1,3,5-trisubstituted benzenes as thrombin inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.12.105

A novel 1,3,5-trisubstituted benzamide thrombin inhibitor template was designed via hybridization of a known aminopyridinoneacetamide and a known 1,3,5-trisubstituted phenyl ether. Optimization of this lead afforded a novel potent series of biaryl 1,3,5-trisubstituted benzenes with excellent functional anticoagulant potency.

Full text of DOI:10.1016/j.bmcl.2010.12.105

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1536–1540
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and SAR of a series of 1,3,5-trisubstituted benzenes
as thrombin inhibitors
Richard C. A. Isaacs a, , Christina L. Newton a, Kellie J. Cutrona a, Swati P. Mercer a, Linda S. Payne a,
⇑
Kenneth J. Stauffer a, Peter D. Williams a, Jacquelynn J. Cook c, Julie A. Krueger b, S. Dale Lewis b,
Bobby J. Lucas b, Elizabeth A. Lyle c, Joseph J. Lynch c, Daniel R. McMasters d, Adel M. Naylor-Olsen d,
Maria T. Michener c, Audrey A. Wallace c
a Department of Medicinal Chemistry, Merck Research Laboratories, West Point, PA 19486, USA
b Department of Biological Chemistry, Merck Research Laboratories, West Point, PA 19486, USA
c Department of Pharmacology, Merck Research Laboratories, West Point, PA 19486, USA
d Department of Molecular Systems, Merck Research Laboratories, West Point, PA 19486, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel 1,3,5-trisubstituted benzamide thrombin inhibitor template was designed via hybridization of a
known aminopyridinoneacetamide and a known 1,3,5-trisubstituted phenyl ether. Optimization of this
lead afforded a novel potent series of biaryl 1,3,5-trisubstituted benzenes with excellent functional anti-
coagulant potency.
Received 14 August 2010
Revised 18 December 2010
Accepted 21 December 2010
Available online 28 December 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Thrombin
Inhibitor
Anticoagulant
As part of our efforts to design and develop novel small
molecule thrombin inhibitors for the treatment and prevention
of coagulation disorders we routinely seek to exploit new
structure-activity relationships which evolve from our own work
as well as others in the field. We have established a research
interest in 3-aminopyridinone and related thrombin inhibitors
exemplified by 1 (Fig. 1) which is a potent inhibitor (Ki = 0.5 nM).1
The aminopyridine moiety binds to the specificity pocket S1 of the
thrombin active site. The methyl group on the pyridinone ring
binds to the S2 pocket and the benzyl group of the sulfonamide
chain binds to the S3 pocket of the enzyme.1 The related analog
2 was found to be similarly potent (Ki = 2.2 nM). While the binding
mode of 1 in the thrombin active site was established by X-ray
crystallography and has been disclosed,1a an X-ray crystal
structure of 2 bound to thrombin was never obtained. Compound
2 was originally targeted based on molecular modeling, with one
of the isobutyl groups having been designed to interact with the
S3 pocket of thrombin, mimicking the role of the phenyl group in
1. While this work was in progress, a series of patent applications
disclosed,3 we were struck by the obvious structural similarity to
2, namely the similarity of the 1,3 relative disposition of the methyl
and sulfonate groups in 3 when compared to the 1,3 relative
disposition of the methyl and carboxamide groups in 2. This
suggested to us that these two compounds likely had similar
binding modes across the S3S2 span of the enzyme. The ether
linked guanylpiperidine moiety of 3 was presumed to be capable
of binding directly to Asp189 in the S1 pocket in a manner similar
to the aminopyridine in compounds 1 and 2.
We decided to prepare and evaluate hybrids of these two types
of compounds. In this paper we will describe our work to prepare,
evaluate and optimize the inhibitory potency and anticoagulant
properties of said hybrids.
Although we did not have any actual experience handling
compounds such as 3, we did intuitively have reservations about
the hydrolytic susceptibility of the sulfonate linkage and reasoned
that replacement by an amide would likely confer improved
stability. In evaluating our options for surrogates for the
guanylpiperidine moiety at P1 we decided to focus primarily on
weakly basic heterocycles and the noncharged 2,5-disubstituted
phenyls previously pioneered in these labs.4 Based on our
experience, these P1 ligands demonstrated an increased propensity
to confer physicochemical properties conducive to good oral
bioavailability, while minimizing the undesirable side effects
which have been observed with more basic groups.5 Molecular
detailing 1,3,5-trisubstituted benzenes exemplified by
3 was
published.2 Although at the time, no binding data for 3 was
⇑
Corresponding author.
E-mail address: richard_isaacs@verizon.net (Richard C. A. Isaacs).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.12.105

Richard C. A. Isaacs et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1536–1540
1537
P1
P3
P2
N
O
O
N
N
O
O2
S
H
N
O
N
H
N
H
N
O
P1
O
N
NH2
N
1
Ki = 0.5 nM
4
5
O
First target
Targeted hybrids
N
O
Figure 3.
N
H
N
O
2
NH2
O
O
O
Ki = 2.2 nM
a,b
HO
O
O2
S
NH
NH2
OH
OBn
Cl
N
6
7
O
c,d
3
3-D Pharmaceuticals
Ki = 4.6 nM
O
N
O
N
e
Figure 1.
modeling suggested that the optimum tether length for connecting
these P1 groups to the phenolic oxygen of the hybrid template was
two carbons (Fig. 2).
OH
OBn
9
8
Based on this analysis we targeted hybrid compounds
generally and specifically for synthesis to validate our
structure-driven design hypothesis (Fig. 3).
4
5
HO
OHC
N
N
N
f,g
N
Tr
NC
Tr
Compound 5 was synthesized as outlined in Scheme 1. The
methyl ester of the known carboxylic acid 66 was protected as a
benzyl ether. Compound 7 was hydrolyzed and the resulting acid
converted to the diisobutylamide 8. Hydrogenolysis of the benzyl
ether gave phenol 9. Commercially available imidazole aldehyde
10 was protected as the trityl derivative (a mixture of regioisomers)
and the aldehyde group homologated to give nitrile 11 using
potassium t-butoxide and tosylmethylisocyanide (Tosmic). During
hydrolysis of nitrile 11 with 6 M HCl, the trityl protecting group
was compromised and was therefore reinstalled. The resulting acid
12 was esterified and then reduced with LAH to give alcohol 13.
Alcohol 13 was converted to mesylate 14 which was then used to
alkylate phenol 9. Removal of the trityl group with TFA and
triethylsilane furnished compound 5.
h,i,j,k
N
H
10
N
11
13
l
MsO
9,m,n
N
N
Tr
5
14
Scheme 1. Synthesis of hybrid 5. Reagents and conditions: (a) TMSCHN2, CH2Cl2/
MeOH, 0 °C; (b) NaH, BnBr, THF, 0 °C; (c) LiOH, THF, MeOH; (d) iBu2NH, EDC, HOBt,
Et3N, DMF; (e) H2, Pd/C, ethanol; (f) TrCl, TEA, CH2Cl2; (g) tBuOK, Tosmic; (h) 6 M
HCl, dioxane, 80 °C; (i) TMSCHN2, CH2Cl2/MeOH, 0 °C; (j) TrCl, TEA; (k) LiAlH4, THF;
(l) Ms2O, TEA, CH2Cl2; (m) Cs2CO3, DMF, 80 °C, (n) Et3SiH, TFA, CH2Cl2.
Compound 5 was found to be a potent (Ki = 3.5 nM) low
molecular weight (372) thrombin inhibitor which displayed
modest selectivity for thrombin versus trypsin (Ki = 2.6 lM).
The in vitro anticoagulant potency of 5 as measured by the
concentration required to double the activated partial
thromboplastin time in human plasma (2 Â APTT = 4.11
lM) was
poor.7
Based on these results, compound 5 was deemed to be a
reasonable new lead structure for further optimization. We started
by screening a variety of different weakly basic and neutral groups
at P1. Representative results are shown in Table 1.
These data demonstrate that with respect to thrombin binding
affinity, the diisobutylamide template is compatible with the same
variety of weakly basic and neutral P1 groups as the pyridinone
and pyrazinone template from which it was derived in part. The
level of binding potency however did not translate into functional
anticoagulant potency in human plasma (generally, 2 Â APTT
>3 lM). This is not surprising given the lipophilic nature of these
analogs. Compound 21 is a particular standout in that it contains
the most basic group (i.e., piperidine) of all the analogs shown
but is among the least active analogs in the 2 Â APTT assay.8
Figure 2. X-ray crystal structure of inhibitor 1 (in blue) bound to the active site of
human a-thrombin overlayed by a modeled hybrid 5 (in orange).

1538
Richard C. A. Isaacs et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1536–1540
Table 1
O
O
Thrombin and trypsin inhibition constants and in vitro anticoagulant potency for
compounds 5 and 15–21
improve
stability
N
N
O
N
O
N
H
P1
O
P1
4
22
OR
1st generation hybrid
2nd generation hybrid
Compound OR
Thrombin Trypsin 2 Â APTT
Figure 4.
Ki (nM)
Ki (
lM)
(lM)
O
N
5
3.5
2.6
4.1
ND
O
O
N
N
H
EDC/HOBt
N
F
F
O
N
H2N
P1
CO2H
15
37
>100
29
N
H
O
P1
23
22 2nd generation hybrid
NH2
O
Scheme 2.
16
2.9
3.6
ND
OMe
replacements with potential for improved chemical and metabolic
stability. Molecular modeling indicated that the ether could be
replaced by an amide group (second generation hybrid 22, Fig. 4)
and still allow for productive binding to all three pockets (S3, S2,
O
17
34
>100
Cl
N
N
N
N
Table 2
N
18
1.7
37
4.4
7.1
Thrombin and trypsin inhibition constants and in vitro anticoagulant potency for
compounds 24–27
O
O
N
N
N
O
19
0.93
28
O
NHR
Cl
O
Compound NHR
Thrombin Ki
(nM)
Trypsin Ki
M)
2 Â APTT
M)
(
l
(l
CO2Me
O
H
N
20
0.73
>100
>8.2
>8.2
H
24
0.68
111
1.6
5.3
HN
Cl
O
H
N
Cl
N
O
O
N
N
N
N
H
21
1.6
78
N
Cl
25
7
>100
HN
Cl
N
N
N
After working with these compounds for some time we
developed concerns about the chemical and metabolic viability of
the ether linkage. All of the analogs shown in Table 1 were
synthesized using an etherification procedure as previously
described for compound 5 using different means of activating the
requisite P1 containing alcohol (e.g., trifilate, bromide, Mitsunobu).
These reactions typically gave only modest yields of the ether.
Competitive elimination to give styrene-like P1 containing
by-products was a recurring issue. In vitro metabolic profiling of
selected analogs in dog, rat and human liver microsomes served
to also highlight the metabolic vulnerability of the ether linkage
in these molecules. For these reasons we sought ether
26
0.27
29
1.2
ND
HN
Cl
N
N
N
27
50
>100
HN

Richard C. A. Isaacs et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1536–1540
1539
Table 3
S1) of the enzyme. We reasoned that the amide could potentially
be a stable replacement for the ether linkage.
Thrombin and trypsin inhibition constants and in vitro anticoagulant potency for
compounds 26 –37
The second generation bis-amide hybrids 22, were prepared in a
straightforward manner (Scheme 2). EDC mediated coupling of
5-methylisophthalic acid with diisobutylamine gave carboxylic
acid 23 which was then coupled with the appropriate aminomethyl
P1 fragments.
R
N
N
N
N
O
N
H
We were pleased to find that this modification did in fact
deliver a potent series of thrombin inhibitors. Table 2 summarizes
thrombin and trypsin binding data as well as functional potency
(2 Â APTT) data for four bisamide analogs (24–27). The
2,5-disubstituted benzylamine P1 groups used in these analogs
delivered the best combination of intrinsic thrombin binding
potency and functional anticoagulant potency (2 Â APTT).
In the context of the 2,5-disubstituted benzylamine P1 ligands,
the bisamide template was capable of yielding sub-nanomolar
thrombin inhibitors (compounds 24 and 26, Ki = 0.68 nM and
0.27 nM respectively) with excellent selectivity versus trypsin
(>100,000 fold). In spite of this level of intrinsic binding potency,
functional potency as measured in the 2 Â APTT assay was
Cl
Compound
R
Thrombin Ki
(nM)
Trypsin Ki
M)
2 Â APTT
M)
(
l
(l
O
N
26
0.27
29
1.2
N
N
28
29
30
67
302
ND
ND
1.5
marginal (1.6 and 1.2 lM respectively). We reasoned based on
N
our previous experience, that functional potency in this series
was being compromised by high plasma protein binding.8 Of the
two amide groups present in the current template, we reasoned
that the di-isobutyl amide was the greater contributor to
lipophilicity and sought polar replacements to address the issue.
Given the lipophilic nature of the amino acid residues which line
the S3 pocket of thrombin, we designed amide replacements which
contained both a hydrophobic terminus (for interacting with the S3
pocket) and a polar terminus to be compatible with solvent
exposure. We also evaluated replacing the central phenyl ring with
a pyridine ring; however, that modification led to a significant loss
in thrombin potency. Likewise, attempts to reduce polarity by
replacing the P2 methyl group by polar surrogates (cyano,
aminomethyl), led to loss of thrombin activity. Table 3 summarizes
data for a number of polar di-isobutylamide replacements which
were tolerated by the enzyme.
85
>100
>100
O
N
0.27
O
N
31
32
0.77
1.8
483
278
0.38
1.42
H
N
Replacement of the di-isobutylamide in compound 26 with a
4-isopropylpyridine gave compound 30 which was equipotent
with respect to both thrombin activity and potency in the 2 Â APTT
assay. Activity in the 2 Â APTT assay could be significantly
improved by increasing polarity via oxidation to the pyridine
33
34
9.6
4.1
>100
>100
>8.2
1.67
N-oxide (compound 31, 2 Â APTT = 0.38
lM). Aminocycloalkanes
HN
were also found to be potent replacements for the diisobutylamide
(compounds 33–37) with conversion of the amine to the
methanesulfonamide providing sufficient increase in polarity to
improve activity in the 2 Â APTT assay (compounds 35 and 37).
Summary: Structure based design techniques were used to
exploit the putative similarity in binding mode of an
N
O
S
aminopyridinone thrombin inhibitor
2 and a trisubstituted
benzene inhibitor 3 to generate a new lead inhibitor 5. SAR work
at both the amide and ether carbons of the central benzene ring
in conjunction with optimization of the P1 ligand led to the
development of a new series of potent inhibitors exemplified by
compound 31. Several of these inhibitors displayed good in vitro
functional potency as judged by the concentration of compound
required to double the activated partial thromboplastin time
(2 Â APTT) in human plasma. None displayed animal
pharmacokinetics comparable to compound 1.
35
0.93
>100
0.87
N
O
O
N
36
37
1.6
75
1.48
0.75
H
O
N
H
Acknowledgements
1.25
>100
The primary author wishes to thank Wayne Thompson,
Donnette Staas, and Thomas Tucker for helpful discussions during
the preparation of this Letter.
N
O
S

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Richard C. A. Isaacs et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1536–1540
(b) Young, M. B.; Barrow, J. C.; Glass, K. L.; Lundell, G. F.; Newton, C. L.; Pellicore,
References and notes
J. M.; Rittle, K. E.; Selnick, H. G.; Stauffer, K. J.; Vacca, J. P.; Williams, P. D.; Bohn,
D.; Clayton, F. C.; Cook, J. J.; Krueger, J. A.; Kuo, L. C.; Lewis, S. D.; Lucas, B. J.;
McMasters, D. R.; Miller-Stein, C.; Pietrak, B. L.; Wallace, A. A.; White, R. B.;
Wong, B.; Yan, Y.; Nantermet, P. G. J. Med. Chem. 2004, 47, 2995; (c) Tucker, T. J.;
Brady, S. F.; Lumma, W. C.; Lewis, S. D.; Gardell, S. J.; Naylor-Olsen, A. M.; Yan, Y.;
Sisko, J. T.; Stauffer, K. J.; Lucas, B. J.; Lynch, J. J.; Cook, J. J.; Stranieri, M. T.;
Holahan, M. A.; Lyle, E. A.; Baskin, E. P.; Chen, I.-W.; Dancheck, K. B.; Krueger, J.
A.; Cooper, C. M.; Vacca, J. P. J. Med. Chem. 1998, 41, 3210; (d) Lumma, W. C.;
Witherup, K. M.; Tucker, T. J.; Brady, S. F.; Sisko, J. T.; Naylor-Olsen, A. M.; Lewis,
S. D.; Freidinger, R. M. J. Med. Chem. 1998, 41, 1011.
1. (a) Sanderson, P. E. J.; Cutrona, K. J.; Dorsey, B. D.; Dyer, D. L.; McDonough, C. M.;
Naylor-Olsen, A. M.; Chen, I.-W.; Chen, Z.; Cook, J. J.; Gardell, S. J.; Krueger, J. A.;
Lewis, S. D.; Lin, J. H.; Lucas, B. J., Jr.; Lyle, E. A.; Lynch, J. J., Jr.; Stranieri, M. T.;
Vastag, K.; Shafer, J. A.; Vacca, J. P. Bioorg. Med. Chem. Lett. 1998, 8, 817; (b)
Sanderson, P. E. J.; Lyle, T. A.; Cutrona, K. J.; Dyer, D. L.; Dorsey, B. D.;
McDonough, C. M.; Naylor-Olsen, A. W.; Chen, I.-W.; Chen, Z.; Cook, J. J.; Cooper,
C. M.; Gardell, S. J.; Hare, T. R.; Krueger, J. A.; Lewis, S. D.; Lin, J. H.; Lucas, B. J.;
Lyle, E. A.; Lynch, J. J.; Stranieri, M. T.; Vastag, K.; Yan, Y.; Shafer, J. A.; Vacca, J. P.
J. Med. Chem. 1998, 41, 4466.
2. (a) von der Saal, W.; Heck, R.; Kucznierz, R.; Leinert, H.; Stegmeier, K. Ger. Offen.
DE 4430757 A1, 1996.; (b) Lu, T.; Illig, C. R.; Tomczuk, B. E.; Soll, R. M.;
Subasinghe, N. L.; Bone, R. F. PCT Int. Appl. WO 9711693 A1 1997.; (c) Lu, T.;
Tomczuk, B. E.; Illig, C.; Soll, R. M. PCT Int. Appl. WO 9724135 A1 1997.
3. Our involvement in this series was initiated based solely on the structures
disclosed in the original patent applications in ref 2 above. After the work
commenced, more information about compound 3 specifically (including its
thrombin potency) and this series in general, was published. See: (a) Lu, T.;
Tomczuk, B.; Illig, C. R.; Bone, R. F.; Murphy, L.; Spurlino, J.; Salemme, F. R.; Soll,
R. M. Bioorg. Med. Chem. Lett. 1998, 8, 1595; (b) Lu, T.; Tomczuk, B.; Bone, R.;
Murphy, L.; Salemme, F. R.; Soll, R. M. Bioorg. Med. Chem. Lett. 2000, 10, 83; (c)
Lu, T.; Soll, R. M.; Illig, C. R.; Bone, R.; Murphy, L.; Spurlino, J.; Salemme, F. R.;
Tomczuk, B. E. Bioorg. Med. Chem. Lett. 2000, 10, 79; (d) Tomczuk, B.; Lu, T.; Soll,
R. M.; Fedde, C.; Wang, A.; Murphy, L.; Crysler, C.; Dasgupta, M.; Eisennagel, S.;
Spurlino, J.; Bone, R. Bioorg. Med. Chem. Lett. 2003, 13, 1495.
5. (a) Kikumoto, R.; Tamao, Y.; Ohkubo, K.; Tezuka, T.; Tonomura, S.; Okamoto, S.;
Hijikata, A. J. Med. Chem. 1980, 23, 1293; (b) Kaiser, B.; Hauptmann, J.; Weiss, A.;
Markwardt, F. Biomed. Biochim. Acta 1985, 44, 1204; (c) Reers, M.; Koschinsky,
R.; Dickneite, G.; Hoffmann, D.; Czech, J.; Stuber, W. J. Enzyme Inhib. 1995, 9, 61.
6. Turner, F. A.; Gearien, J. E. J. Org. Chem. 1959, 24, 1952.
7. We consider human plasma 2 Â APTT values of 1
predictive of good in vivo efficacy with lower concentrations being predictive of
greater efficacy. We use this 1 M mark routinely as a gate for determining
lM or less to be generally
l
whether to evaluate a compound further in vivo in the rat ferric chloride efficacy
assay (see Ref. 4). In practice, in optimizing compounds, we strive to achieve
2 Â APTT values of <0.5
lM.
8. For a discussion of the role of lipophilicity in modulating the functional potency
of thrombin inhibitors see: Tucker, T. J.; Lumma, W. C.; Mulichak, A. M.; Chen, Z.;
Naylor-Olsen, A. M.; Lewis, S. D.; Lucas, R.; Freidinger, R. M.; Kuo, L. C. J. Med.
Chem. 1997, 40, 830.
4. (a) Isaacs, R. C. A.; Solinsky, M. G.; Cutrona, K.; Newton, C. L.; Naylor-Olsen, A.
M.; Krueger, J. A.; Lewis, S. D.; Lucas, B. J. Bioorg. Med. Chem. Lett. 2006, 16, 338;