Potent and selective bicyclic lactam inhibitors of thrombin. Part 4: Transition state inhibitors

Article abstract of DOI:10.1016/S0960-894X(00)00636-3

Bicyclic piperazinone based thrombin inhibitors of general structure 2 were prepared and evaluated in vitro and in vivo. These inhibitors, having in common an electrophilic basic trans-cyclohexylamine P₁ residue, displayed high thrombin affinity, high selectivity against trypsin and good in vivo efficacy in the rat arterial thrombosis model.

Full text of DOI:10.1016/S0960-894X(00)00636-3

Bioorganic & Medicinal Chemistry Letters 11 (2001) 287±290
Potent and Selective Bicyclic Lactam Inhibitors of Thrombin.
Part 4: Transition State Inhibitors
Benoit Bachand,a,* Micheline Tarazi,a Yves St-Denis,a Jeremy J. Edmunds,b
Peter D. Winocour,a Lorraine Leblonda and M. Arshad Siddiquia
a
Â
BioChem Pharma Inc., 275 Armand-Frappier Blvd., Laval, Quebec, Canada, H7V 4A7
bParke-Davis Pharmaceutical Research, Division of Warner-Lambert Co., Ann Arbor, MI 48105, USA
Received 11 May 2000; accepted 6 November 2000
AbstractÐBicyclic piperazinone based thrombin inhibitors of general structure 2 were prepared and evaluated in vitro and in vivo.
These inhibitors, having in common an electrophilic basic trans-cyclohexylamine P1 residue, displayed high thrombin anity, high
selectivity against trypsin and good in vivo ecacy in the rat arterial thrombosis model. # 2001 Elsevier Science Ltd. All rights
reserved.
Bicyclic piperazinone thrombin inhibitors of general
structure 1 have recently been shown by us to be highly
potent and active in vivo.1 Although these compounds
have potency in the low nanomolar range, none of them
had acceptable bioavailability or a high level of selec-
tivity against other serine proteases, such as trypsin. In
order to increase bioavailability as well as enzyme
selectivity, we needed to replace the arginine moiety by
a much more lipophilic and less basic group, such as a
cyclohexylamine unit, to provide thrombin inhibitors of
general structure 2. This trans-cyclohexylamine moiety
linked to a (d)-Phe-Pro dipeptide has been reported by
others to a€ord selective thrombin inhibitors (Fig. 1).2
carboxylic acid4 3 followed by oxidation using TPAP
a€orded the ketone 4 in 57% overall yield. Reductive
amination of the cyclohexanone 4 using sodium cyano-
borohydride with an excess of ammonium acetate fol-
lowed by protection with Mtr-Cl a€orded, after
puri®cation to remove the minor cis isomer, the trans-
cyclohexylamine 5 in overall yield of 38%. Addition of
2-lithiothiazole, 2-lithiobenzothiazole, or 2-lithio-3-
vinyl-4-methyl thiazole to the Weinreb amide followed
by acidic deprotection provided the cyclohexylamine 6a,
6b and 6c in high yield. Coupling of these amines with
the bicyclic template 71 and deprotection yielded the
thrombin inhibitors 2a and 2b in 20±40% yield. Cou-
pling of the amine 6c followed by oxidation a€orded
analogues 2c bearing a carboxylic side chain on the
thiazole ring.
In this paper, we describe the preparation of cyclohex-
ylamine based thrombin inhibitors of general structure 2
be0aring a variety of di€erent activated carbonyls at the
P1 site, as well as their in vitro (potency, selectivity) and
in vivo ecacy in the rat arterial thrombosis model.
Chemistry
Since the known methods2,3 for the preparation of the
trans-cyclohexylamine moiety su€ered from some lim-
itations, we opted for a novel approach that is depicted
in Scheme 1. Weinreb amide formation of the
*Corresponding author. Tel.: +1-450-687-4910; fax: +1-450-978-
7777; e-mail: bachandb@biochempharma.com
Figure 1.
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00636-3

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B. Bachand et al. / Bioorg. Med. Chem. Lett. 11 (2001) 287±290
Scheme 2 summarizes the preparation of the 1,2-dicar-
bonyl analogues 2d±i. Transformation of the Weinreb
amide 5 to the ketoamide 8 was done using a known
procedure.2b Using standard conditions, coupling with
the bicyclic template 7 and removal of the Mtr protective
group a€orded the inhibitor 2d in 44% yield. Prepara-
tions of the analogues 2e±i were done slightly di€erently.
The ketoester 9 was used as the reagent for coupling
with the template that, upon further manipulations,
a€orded compounds 2e±i.
Preparations of analogues 2j±k having a tetrazole unit
are depicted in Scheme 3. Addition of lithioderivatives
of N-methyl or N-allyl tetrazole to the aldehyde 11
.
Scheme 1. (a) CH3NH(OCH3) HCl, DIEA, BOP, DMF (75%); (b) TPAP, NMO, MS 4 A, DCM (73%); (c) (i) ammonium acetate (10 equiv),
NaCNBH3, isopropanol, MS 4 A; (ii) MTr-Cl, DMAP, DIEA, DCM (38%, 2 steps); for 6a: (d) (i) thiazole, n-BuLi, THF, 40 ꢀC (75%); (ii) 4.0 M
HCl, dioxane (100%); for 6b: (d) (i) benzothiazole, n-BuLi, THF, 40 ꢀC (%); (ii) 4.0 M HCl, dioxane (100%); for 6c: (d) (i) 3-vinyl-4-methyl
thiazole, n-BuLi, THF (95%); (ii) 4.0 M HCl, dioxane (100%); for 2a and 2b: (e) (i) HATU, NMM, DMF, bicyclic template 7 (78%); (ii) TFA,
thioanisole, methanesulfonic acid (20-40%); for 2c: (e) (i) HATU, NMM, DMF, bicyclic template 7 (72%); (ii) O3, CH2Cl2, (100%); (iii) AgNO3,
NaOH, THF/H2O (85%); (iv) TFA, thioanisole, methanesulfonic acid (26%).
Scheme 2. (A) (i) HATU, NMM, DMF, bicyclic template 7 (69%); (ii) TFA, thioanisole, methanesulfonic acid (58%); (B) for 2e: (i) TFA, MSA,
.
thioanisole, HPLC puri®cation (24%); for 2f: (i) LiOH H2O, THF/H2O (94%); (ii) TFA, MSA, thioanisole, HPLC puri®cation (24%); for 2g: (i) n-
BuOH, EEDQ (44%); (ii) TFA, MSA, thioanisole, HPLC puri®cation (69%); for 2h: (i) EtSH, EDC, DMAP (23%); (ii) TFA, MSA, thioanisole,
HPLC puri®cation (59%) for 2i: (i) ClH3NCH2CO2Bn, HATU, 2,4,6-collidine (63%); (ii) H2, Pd/C 5%, methanol (83%); (iii) TFA, MSA, thio-
anisole, HPLC puri®cation (26%).
Scheme 3. (a) (i) N-Alkyltetrazole, n-BuLi, THF, 40 ꢀC (63% for N-methyl for 12a, 55% for N-allyltetrazole 12b); (b) (i) Dess±Martin, CH2Cl2
(79% for 13a, 55% for 13b); (ii) 4.0 M HCl, dioxane (100%). For 2j: (d) (i) HATU, NMM, DMF, bicyclic template 7 (74%); (ii) TFA, thioanisole,
methanesulfonic acid (73%). For 2k: (d) (i) HATU, NMM, DMF, bicyclic template 7 (71%); (ii) O3, (96%); (iii) AgNO3, TFA, thioanisole,
methanesulfonic acid (60%).

B. Bachand et al. / Bioorg. Med. Chem. Lett. 11 (2001) 287±290
289
provided the corresponding alcohol 12a±b in good yield.
Dess±Martin oxidation of the alcohol followed by acidic
deprotection furnished ketones 13a±b, which after cou-
pling with bicyclic template 7, oxidation of the vinyl
group with ozone followed by silver oxide a€orded after
usual deprotection analogues 2j±k.
ranging from 0.09 (2f) to 5 nM (2j) except for com-
pound 2k, which has a Ki of 2.6 mM. The use of ben-
zylsulfonamide at the P3 site was necessary to obtain a
high level of potency. For example, compound 2a has a
Ki of 3 nM, which represents a 10-fold increase in
potency compared to the previously reported analogue
having a phenylpropanoyl group at the P3 site.6 As can
be seen, the presence of a cyclohexylamine moiety sub-
stantially increased the selectivity over trypsin. In fact,
the analogue of compound 2a, which had a regular
arginine at the P1 instead of a trans-cyclohexylamine,
did not show any selectivity.7 From Table 1, all com-
pounds produced good selectivity ratios ranging from
150 for compound 2k to a maximum value of 24,400
displayed by compound 2f. In some cases, the presence
of a carboxylic function seemed to increase the selectiv-
ity ratio, for example if one compares compound 2g
(Tryp/Thr: 5500) with compound 2f (Tryp/Thr: 24,400)
or compound 2d (Tryp/Thr: 7340) with compound 2i
(Tryp/Thr: 12,960). But in other cases the presence of
the same functionality decreased the selectivity ratio if
one compares compounds 2a and 2b (Tryp/Thr: 2500
Biology
Inhibition of the proteolytic activity of thrombin and
trypsin (Ki), in vivo coagulation parameters such as the
mean occlusion time (MOT), the activated partial
thromboplastin time (aPTT), the thrombin time (TT) and
bioavailability were measured according to published
procedures.5
Results and Discussion
All compounds prepared and listed in the Table 1 dis-
played high potency towards thrombin with Ki's
Table 1. In vitro activity against human a-thrombin and trypsin, selectivity, in vivo activity and bioavailability of inhibitors 2a±ka in the rat arterial
thrombosis model
Rat arterial thrombosis modelb
Entry
R
Ki (nM) (Thrombin)
Ki (nM) (Trypsin)
Tryp/Thr
2500
MOTe (min)
>60
APTTf (s)
TTg (s)
% Fh
1.8
2a
3.2(S)c
8000
66 Æ 6
829 Æ 208
2b
2c
2d
0.72(S)
80
1400
65,000
2400
1930
812
>60
16 Æ 1
20 Æ 4
25 Æ 5
20 Æ 1
47 Æ 6
339 Æ 55
99 Æ 9
999
0.6
0.4
ndi
0.33(S)
7340
2e
2f
0.13(S)
0.09(S)
nd
nd
>60
>60
46 Æ 4
54 Æ 2
999
1.1
0.4
2200(S)
24,400
938 Æ 75
2g
2h
2i
(0.1)d(S)
0.26(S)
2.14(S)
5.0
(550)d
150
5500
580
47 Æ 17
49 Æ 14
nd
53 Æ 10
33 Æ 1
nd
883 Æ 143
999
0.7
0.3
nd
nd
nd
29,000
35,000
>400,000
12,960
7000
150
nd
2j
46 Æ 12
nd
46 Æ 2
nd
322 Æ 38
nd
2k
2600
aAll new targets were characterized by 1H NMR, reverse HPLC and mass spectroscopy.
bDose: intravenous bolus dose (0.75 mg/kg) followed by an infusion (50 mg/min).
c(S)=slow binder.
dIC50
.
eMean occlusion time (control: 17±19 min).
fActivated partial thromboplastin time (20±22 s).
gThrombin time (control: 40±45 s).
hBioavailability, based on oral administration (30 mg/kg).
iNot determined.

290
B. Bachand et al. / Bioorg. Med. Chem. Lett. 11 (2001) 287±290
thrombosis model system, doubling and even tripling
the mean occlusion time in some cases.
Acknowledgements
The authors would like to acknowledge Ms. Celine
Locas and Ms. Brigitte Grouix for providing binding
constants, Ms. Annie St-Pierre for performing the in
vivo experiments and Dr. Mirek Cygler of the Bio-
technology Research Institute of Montreal for the X-
ray structure. Finally, we would like to thank Dr.
Alan Cameron for reading the manuscript and Ms.
Lyne Marcil for her help in the preparation of this
manuscript.
Figure 2.
and 1930, respectively) with compounds 2c (Tryp/Thr:
812) or even more dramatically when one compares
analogue 2j with 2k (Tryp/Thr: 7000 and 150, respec-
tively). All these data taken together suggest that the
cyclohexylamine moiety is obviously essential to obtain
a good level of selectivity and moreover that this selec-
tivity could 0be substantially varied by having a polar
group on P1 .
References and Notes
1. St-Denis, Y.; Augelli-Szafran, C. E.; Bachand, B.; Berry-
man, K. A.; DiMaio, J.; Doherty, A. M.; Edmunds, J. J.;
Leblond, L.; Levesque, S.; Narasimhan, L. S.; Penvose-Yi,
J. R.; Rubin, J. R.; Tarazi, M.; Winocour, P. D.; Siddiqui,
M. A. Bioorg. Med. Chem. Lett. 1998, 8, 3193.
2. (a) Brady, S. F.; Lewis, S. D.; Colton, C. D.; Stau€er, K. J.;
Sisko, J. T.; Ng, A. S.; Homnick, C. F.; Bogusky, M. J.; Sha-
fer, J. A.; Veber, D. F.; Nutt, R. F. Pept.: Chem., Struct. Biol.,
Proc. Am. Pept. Symp., 14th 1996, 331. (b) Veber, D. F.;
Lewis, S. D.; Shafer, J. A.; Feng, D.-M.; Nutt, R.-F.; Brady,
S. F. WO9425051, 1994; Chem. Abstr. 1994, 122, 17839.
3. Lyle, T. A.; Chen, Z.; Appleby, S. D.; Freidinger,
R. M. G. J.; Lewis, S. D.; Li, Y.; Lyle, E. A.; Lynch, J. J., Jr.;
Mulichak, A. M.; Ng, A. S.; Naylor-Olsen, A. M.; Sanders,
W. M. Bioorg. Med. Chem. Lett. 1997, 7, 67.
Most of the compounds with the exception of 2c and 2d
displayed a substantial increase (2- to 3-fold) in anti-
thrombotic activity in the rat arterial thrombosis model
as measured by the MOT. Unfortunately, when these
compounds were given orally to rats, low levels of
bioavailability were observed for all the compounds listed
in Table 1.
4. Ban®, A.; Benedini, F.; Sala, A.; Russo, G. Synth. Com-
mun. 1990, 20, 1531.
5. Finkle, C. D.; St-Pierre, A.; Leblond, L.; Deschenes, I.;
DiMaio, J.; Winocour, P. D. Thromb. Haemostasis 1998, 79,
431.
6. Plummer, J. S.; Berryman, K. A.; Cai, C.; Cody, W. L.;
DiMaio, J.; Doherty, A. M.; Edmunds, J. J.; He, J. X.; Hol-
land, D. R.; Levesque, S.; Kent, D.; Narasimhan, L. S.;
Rubin, R. J.; Rapundalo, S. T.; Siddiqui, M. A.; Susser, A. J.;
St-Denis, Y.; Winocour, P. D. Bioorg. Med. Chem. Lett. 1998,
8, 3409.
Figure 2 shows the X-ray structure8 of inhibitor 2g with
thrombin. The mode of interaction is very similar to the
structure already reported by us on a compound having
a regular arginine at P1.1 Brie¯y, the benzylsulfonamide
group penetrates deeply in the S3 pocket, while the
amino group of trans-cyclohexylamine makes a salt
bridge interaction with the Asp 189 at the bottom of the
S1 pocket. Since thrombin has a more lipophilic S1
pocket (Ala 190) than trypsin (Ser 190), this di€erence
might explain why the more lipophilic P1 side chains are
preferred in thrombin compared to trypsin. This sug-
gests why all the analogues prepared in this paper
showed remarkably good selectivity. Finally, the acti-
vated carbonyl group makes a strong covalent bond
with the Ser 195 forming a tetrahedral intermediate
providing additional binding anity.
7. Compound 14, Ki (thrombin) 0.23 nM, Ki (trypsin) 0.23
nM; Siddiqui, M. A., Unpublished results
.
Conclusion
We have demonstrated that incorporation of a cyclo-
hexylamine unit in the P1 site bearing an electrophilic
carbonyl a€ords very potent thrombin inhibitors in the
low nanomolar range. These compounds, moreover,
displayed good antithrombotic activity in the rat arterial
8. The authors have deposited X-ray crystallographic data
with the Brookhaven Protein Data Bank. Deposition code
1G37.