Unexpected enhancement of thrombin inhibitor potency with o-aminoalkylbenzylamides in the P1 position

Article abstract of DOI:10.1016/S0960-894X(03)00732-7

Thrombin inhibitors incorporating o-aminoalkylbenzylamides in the P1 position were designed, synthesized and found to have enhanced potency and selectivity in several different structural classes. X-ray crystallographic analysis of compound 24 bound in the α-thrombin-hirugen complex provides an explanation for these unanticipated results.

Full text of DOI:10.1016/S0960-894X(03)00732-7

Bioorganic & Medicinal Chemistry Letters 13 (2003) 3477–3482
Unexpected Enhancement of Thrombin Inhibitor Potency with
o-Aminoalkylbenzylamides in the P1 Position
Kenneth E. Rittle,a,* James C. Barrow,a Kellie J. Cutrona,a Kristen L. Glass,a
Julie A. Krueger,b Lawrence C. Kuo,c S. Dale Lewis,b Bobby J. Lucas,b
Daniel R. McMasters,d Matthew M. Morrissette,a Philippe G. Nantermet,a
Christina L. Newton,a William M. Sanders,a Youwei Yan,c Joseph P. Vaccaa
and Harold G. Selnicka
aDepartment of Medicinal Chemistry, Merck Research Laboratories, West Point, PA 19486, USA
bDepartment of Biological Chemistry, Merck Research Laboratories, West Point, PA 19486, USA
cDepartment of Structural Biology, Merck Research Laboratories, West Point, PA 19486, USA
dDepartment of Molecular Systems, Merck Research Laboratories, West Point, PA 19486, USA
Received 21 March 2003; revised 14 July 2003; accepted 14 July 2003
Abstract—Thrombin inhibitors incorporating o-aminoalkylbenzylamides in the P1 position were designed, synthesized and found
to have enhanced potency and selectivity in several different structural classes. X-ray crystallographic analysis of compound 24
bound in the a-thrombin–hirugen complex provides an explanation for these unanticipated results.
# 2003 Elsevier Ltd. All rights reserved.
Thrombin, a serine protease, plays a critical, multi-
functional role in the mechanism of clot formation
establishing this trypsin-like enzyme as a popular drug
target. Such an entity would have potential for the
treatment and prevention of cardiovascular disorders
such as deep vein thrombosis, pulmonary embolism,
myocardial infarction and stroke.1,2 Existing therapies,
such as orally administered warfarin and subcutaneous
injection with low molecular weight heparin (LMWH),
are burdened by drug and dietary restrictions, bleeding
risk and required monitoring of blood parameters.3,4
Because of these limitations, there is a clear need to
develop orally administered thrombin inhibitors which
are safe, effective anti-coagulants to obtain maximum
patient compliance. In this communication, a series of
o-aminoalkylbenzylamides with unexpected potency
enhancing properties will be discussed as P1 groups in
the search for a clinically useful antithrombotic drug.
basic P1 group such as guanidine or amidine is important
for potency5 (for example, 1a,6 Fig. 1). However, these
moieties have been associated with poor oral
bioavailability.7À9 Alternatively, trans-cylclohexylamine
It is recognized that formation of a salt bridge to
Asp189 in the bottom of the thrombin S1 pocket with a
*Corresponding author. Tel.:+1-215-652-7342; fax:+1-215-652-3971;
e-mail: ken_rittle@merck.com
Figure 1. Potent and selective basic P1 groups.
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00732-7

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K. E. Rittle et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3477–3482
1b6 and the moderately basic 2-amino-6-methyl-pyri-
dine 1c10 have been used successfully as potent and
selective replacements. In a search to improve on the
pharmacokinetic and pharmacodynamic properties
associated with these P1 groups, it was noted that
p-aminomethylaryl groups (Fig. 2, 2a–c11,12) had been
incorporated into several P3P2 scaffolds with good
potency. The interaction of the meta and ortho isomers
within the S1 pocket, however, were unreported and
merited further examination.
Chemistry
Compounds 1a–c were chosen for comparison because
of their reported biological profiles and the availability
of acid 313 which could be coupled and deprotected
(Scheme 1) to give a series of aminoalkylbenzylamides
4. The benzylamine targets for this study were prepared
as in Schemes 2–5. Azide displacement of 4-cyano-
benzylbromide (Scheme 2) gave 5. Hydrogenolysis of
the azide proceeded smoothly, and treatment with di-
tert-butyl dicarbonate gave the protected amine 6. The
nitrile was reduced and the benzylamine 7a was isolated
as its hemisulfate salt with sodium hydrogen sulfate
from ethyl acetate. All three positional benzylamine
isomers 7a–c14 were prepared in this manner.
An eleven step sequence (Scheme 3) was used to prepare
the 2-Boc-aminoethyl analogue of 7c. Commercially
available o-tolylacetic acid was reacted with NBS fol-
lowed by esterification, displacement with azide and
reduction to give benzylamine 8. Treatment of 8 with
benzylchloroformate followed by reduction of the ester
to the alcohol, mesylation and displacement with
sodium azide gave 9. Reduction of azide 9 with triphe-
nylphosphine, protection of the amine as its t-butylcar-
bamate and removal of the Cbz group gave the
homologous benzylamine 10.
Synthesis of the 5-chloro substituted derivative of 7c
(Scheme 4) was accomplished by esterification of 2-
bromo-5-chlorobenzoic acid followed by palladium(0)
mediated cyanation to give nitrile 11. Reduction of both
the nitrile and ester was achieved using LiAlH4 and
treatment of the amine with di-tert-butyl dicarbonate
gave carbamate 12. Conversion of the alcohol to azide
and reduction using triphenylphosphine provided 5-
chlorobenzylamine 13.
Figure 2. P3P2 scaffolds with p-aminomethylbenzyl in P1.
The homologue of 13 was prepared by reacting commer-
cially available 4-chlorophenethylamine with phosgene
Scheme 1. (a) EDC, HOAT, Et3N, DMF, rt, 2–18 h; (b) HCl, EtOAc,
0 ꢀC.
Scheme 3. (a) NBS, AIBN, CCl4, 77 ꢀC, 4 h; (b) Concd H2SO4, iso-
butylene, dioxane, rt, 4 h; (c) NaN3, DMF, 65 ꢀC, 3 h; (d) 5% Pd/C,
H2, 45 psi, THF, rt, 2 h; (e) PhCH2OCOCl, DMAP, CH2Cl2, 0 ꢀC; (f)
2.0 MLiBH 4, THF, 0 ꢀC to rt, 18 h; (g) CH3SO2Cl, TEA, CH2Cl2,
0 ꢀC to rt, 18 h; (h) NaN3, DMF, 40 ꢀC, 4 h; (i) Ph3P, THF, H2O, rt,
18 h; (j) Boc2O, CH2Cl2, 0 ꢀC to rt, 2 h; (k) H2, 10% Pd/C, 1 atm,
EtOH, rt, 2 h.
Scheme 2. (a) NaN3, THF, H2O, rt, 18 h; (b) H2, 5% Pd/C, 45 psi,
THF, rt, 2 h; (c) Boc2O, THF, rt, 2 h; (d) CoCl2, NaBH4, THF, H2O,
35 ꢀC, 24 h.

K. E. Rittle et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3477–3482
3479
Scheme 5. (a) Phosgene, ClPh, reflux; (b) AlCl3, ClPh, 70 ꢀC, 1 h; (c)
Boc2O, DIEA, DMAP, DMF, rt, 2 h; (d) 2.0 M LiBH4, THF, 0 ꢀC, 1.5
h; (e) DPPA, DBU, THF, 0 ꢀC to rt, 3 h; (f) PPh3, THF, H2O, rt, 18 h.
Scheme 4. (a) HCl, MeOH, rt, 18 h; (b) Zn(CN)2, Pd(Ph3P)4, DMF,
90 ꢀC, 8 h; (c) 1.0 MLiAlH 4, THF, 0 ꢀC, 0.5 h; (d) Boc2O, CH2Cl2,
0 ꢀC to rt, 2 h; (e) DPPA, DBU, THF, rt, 18 h; (f) PPh3, THF, H2O,
65 ꢀC, 18 h.
(Scheme 5) followed by treatment in situ of the resulting
isocyanate with aluminum chloride to give a mixture of
regioisomers from which the desired dihydro-
isoquinolinone 14 was obtained by fractional crystal-
lization with toluene/heptane. Protection with Boc
anhydride and reductive cleavage resulted in alcohol 15.
Azide displacement with DPPA followed by reduction
gave the homologous 5-chlorobenzylamine 16.
thrombin potency when compared to the basic P1
groups of 1a–c, the result (49 nM) proved worthy of
further evaluation. Moving the aminomethyl group to
the 3-position (18) produced a 10-fold further loss in
binding affinity (490 vs 49 nM) most likely due to a
reduced interaction between the basic amine and
Asp189. However, introduction of aminomethyl at the
2-position gave an unanticipated result in that com-
pound 19 was nearly an order of magnitude more
potent than its para-isomer 17 (5.2 vs 49 nM) and sig-
nificantly more selective (12,885 vs 26.5-fold). In addi-
tion, it had good activity in the 2Â APTT assay (0.44
mM) and had twice the thrombin selectivity (12,885 vs
6400-fold) of 1c. This discovery was especially remark-
able given that the amino group in the 2-postion was
predicted to have little or no interaction with Asp189.
This exciting result led to the modification of the alkyl
sidechain to determine the optimal length for activity.
Excision of the methylene spacer as in aniline 20 was 90-
fold less potent and had decreased selectivity compared
to 19. However, the phenethylamine homologue 21 was
equipotent with 19 (5.2 vs 4.1 nM) and its selectivity was
improved 8-fold.
Results and Discussion
The results of a study to examine the effect of the ami-
nomethyl position on the phenyl ring are shown in
Table 1. Thrombin (Factor IIa) and trypsin (Tryp)
inhibition constants (Ki) were determined for each
compound in the new series. The concentration needed
to double the activated partial thromboplastin time (2Â
APTT) in human plasma15 was also determined for the
more potent compounds.
Although substitution of aminomethyl in the 4-postion
of benzylamide (17) showed a 10- to 100-fold loss in
Table 1. Benzylsulfonamidopyridinone P1 analogues
Compd
R
Y
IIa
Ki (nM)
Tryp
Ki (nM)
Selectivity ratio
Trp Ki
IIa Ki
2Â APTT
(mM)
1c
17
18
19
20
21
22
23
24
25
—
4-CH2NH2
3-CH2NH2
2-CH2NH2
2-NH2
—
H
H
H
H
H
H
Cl
Cl
Cl
0.5
49
490
5.2
470
4.1
130
7.1
3200
6400
26.5
46.9
0.22
—
—
0.44
—
0.53
—
2.8
0.16
0.18
1300
23,000
67,000
12,885
>2128
106,585
3585
2786
40,217
740,000
>1,000,000
43,7000
46,6000
20,000
2-CH2CH2NH2
H
H
2-CH2NH2
2-CH2CH2NH2
0.046
0.05
1850
37,000

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K. E. Rittle et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3477–3482
potency of the 3-chloro analogue 2316 over the unsub-
stituted benzylamine 22 encouraged us to incorporate
this feature into the 5-position of 19 and 21. This resul-
ted in the synthesis of the most potent compounds (24
and 25, respectively) in this series. Binding affinities
were improved 82 to 113-fold over the des-chloro var-
iants and more interestingly, they were 1 to 2 orders of
magnitude more potent then any of the basic P1 groups
present in the lead structure 1. Of particular note was
the large improvement of 24 and 25 over 1c in trypsin
selectivity and their comparable efficacy in the 2Â
APTT assay (0.16/0.18 vs 0.22 mM).
Solving the crystal structure of the thrombin-24 com-
plex17 (Fig. 3) led to a better understanding of these
results. As was observed for 1c, the benzylsulfonamide
group of 24 fills the distal hydrophobic pocket, while
the 6-methyl substituent on the P2 pyridinone ring
occupies the insertion loop of the proximal hydrophobic
pocket. However, in contrast to the interactions of the
aminopyridine of 1c with Asp189, the P1 methylamine of
24 is observed to form a direct salt bridge with Glu192
(3.0 A O–N distance), and is within hydrogen-bonding
Figure 3. X-ray crystal structure of 24.
Substitution at the 3-position of aryl P1 groups with
lipophilic moieties such as chlorine can lead to large
improvements in thrombin inhibitory potency because
this substituent can access a lipophilic recess in the S1
pocket of the enzyme.11 An 18-fold improvement in
Table 2. P1 analogues with d-PhePro as P3P2 scaffold
Compd
R
Y
IIa Ki
(nM)
Tryp Ki
(nM)
Selectivity ratio
Trp Ki
IIa Ki
2Â APTT
(mM)
26
27
28
29
30
H
H
H
Cl
H
Cl
Cl
4600
250
313
3.3
>1,000,000
89,000
>217
356
339
1908
4065
—
—
—
0.34
—
CH2NH2
CH2NH2
CH2CH2NH2
10,6000
6200
252,000
62
Table 3. P1 analogues with phenethylaminopyrazinone as P3P2 scaffold
Compd
P1
IIa
Ki (nM)
Tryp
Ki (nM)
Selectivity ratio
Trp Ki
IIa Ki
2Â APTT
(mM)
31
0.13
1600
1800
12,307
0.41
0.41
32
0.8
1908

K. E. Rittle et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3477–3482
3481
Acknowledgements
We are very grateful to Peter Williams and Phil San-
derson for their helpful scientific discussions and Laurie
Rittle for her assistance in the preparation of this
manuscript.
Figure 4. Pyridine N-oxide 6-chloropyrazinone P3P2 scaffold.
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distance of four additional H-bond acceptors: the
Gly216 backbone carbonyl oxygen (2.9 A O–N), the
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(26 vs 27) in the peptide series d-PhePro18À20 (Table 2).
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(29) improved potency by nearly two orders of magni-
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14. 7a (cat. # 53944-9) and 7b (cat. # 53528-1) are now avail-
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P3P2 scaffold 3321 resulting in benzylamide (33a), one
of the most potent thrombin inhibitors to be prepared
in our laboratories (Fig. 4).
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an R factor of 0.22.
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The discovery of o-aminoalkylbenzylamides as a new
class of thrombin inhibitor P1 groups is described. The
interaction of 24 with the thrombin active site provides
insights into their unanticipated potency and selectivity
enhancing features, the generality of which extended
over several different P3P2 scaffolds. The pharmacody-
namic and pharmacokinetic properties of this new P1
class will be discussed in subsequent publications.
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