The design and synthesis of thrombin inhibitors: Analogues of MD805 containing non-polar surrogates for arginine at the P1 position

Article abstract of DOI:10.1016/S0960-894X(00)00282-1

A series of monocyclic and bicyclic amino acids have been synthesised and incorporated into thrombin inhibitors based on CGH728, an analogue of the Mitsubishi compound MD805. Benzthiazolylalanine (Bta) was found to be a good non-polar substitute for argin

Full text of DOI:10.1016/S0960-894X(00)00282-1

Bioorganic & Medicinal Chemistry Letters 10 (2000) 1563±1566
The Design and Synthesis of Thrombin Inhibitors: Analogues
of MD805 Containing Non-Polar Surrogates for Arginine
at the P1 Position
Urs Baettig, Lyndon Brown, Derek Brundish, Colin Dell, Alex Furzer, Sheila Garman,
Diana Janus, Peter D. Kane,* Garrick Smith, Clive V. Walker, Xiaoling Cockcroft,
John Ambler, Andrew Mitchelson, Mark D. Talbot, Morris Tweed and Nicholas Wills
Novartis Horsham Research Centre, Wimblehurst Road, Horsham, West Sussex RH12 4AB, UK
Received 21 October 1999; accepted 9 May 2000
AbstractÐA series of monocyclic and bicyclic amino acids have been synthesised and incorporated into thrombin inhibitors based
on CGH728, an analogue of the Mitsubishi compound MD805. Benzthiazolylalanine (Bta) was found to be a good non-polar
substitute for arginine at the P1 position, yielding compounds with low nanomolar potency and good selectivity for thrombin.
# 2000 Elsevier Science Ltd. All rights reserved.
The serine protease enzyme thrombin plays a central role
in the blood coagulation pathway,1 directly triggering
the production of insoluble ®brin while at the same time
providing stimulus for platelet aggregation. The key
position of thrombin has made it a popular target for
antithrombotic therapy; initial research leading to the
discovery of parenterally administered thrombin inhibitors
such as hirudin.2 However, the main e€ort has been
centred on achieving the more dicult goal of an orally
active compound.3
Despite the strong monodentate salt bridge interaction
between the arginine guanidine group and Asp-189 of the
enzyme, the S1 site of serine proteases tends to be largely
lipophilic. This property is accentuated in thrombin over
certain other serine proteases by the presence of an alanine
residue at position 190 (e.g., serine in trypsin and chymo-
trypsin). The prospect of making fuller use of this lipo-
philicity, that is apparently not optimally exploited by
arginine, represented a possible opportunity to obtain
good binding and selectivity while removing the guani-
dine. We were therefore interested to explore the P1 posi-
tion with amino acids totally devoid of amidine or
guanidine, but containing a variety of lipophilic side
chains. A large number of acyclic,9 monocyclic and
bicylic containing structures were investigated, some of
which are shown in Tables 1 and 2.
Previous work in our laboratories, based on the Mitsu-
bishi lead MD805,4 had led to compounds with reduced
stereogenicity and increased potency, exempli®ed by
CGH728 that has a Ki of 6 nM for thrombin (Fig. 1).8
Although such arginine based inhibitors are potent and
selective they tend to have a short duration of action and
poor oral bioavailability, properties that are considered
largely due to the presence of the very basic guanidine
group (pKaꢀ14). Manipulations to reduce the basicity of
the P1 amino acid have been demonstrated to increase cell
permeability5 and with this in mind we sought to ®nd a P1
arginine substitute for MD805 type inhibitors that would
eventually lead to an ecacious oral drug.
*Corresponding author. at current address: Tripos Receptor Research,
Bude, Cornwall, EX23 8LY, UK. Tel.: +44-288-359-359; fax: +44-
288-359-222; e-mail: pkane@tripos.com
Figure 1.
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00282-1

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U. Baettig et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1563±1566
Chemistry
The majority of compounds were synthesised from a sui-
tably protected aspartic acid precursor. Thus Cbz pro-
tected Bta 3 could be prepared in four steps via the acid
chloride of Cbz±Asp±OBn and 2-aminophenyl disulphide,
using a procedure based on that of Nestor.6 Sequential
coupling with piperidine and sulphonyl chloride was
carried out using standard conditions (Scheme 1).
The nitrogen containing analogues of Bta were prepared
by the coupling of the hydroxy-amino pyridine 8 or
pyrimidine 9 using a reversed mode of thiazolyl ring
formation (Scheme 2). The nucleophilicity of benzimida-
zoles can cause problems during syntheses, a late stage
heterocyclic ring formation strategy was therefore
favoured for the synthesis of 24. Cl-DMTHQS±Asp±AEP
(12) (AEP=acetoxyethylpiperidine) was prepared in 4
steps from Boc-Asp(OBzl)-OH which was then con-
verted to the benzimidazole (24).
Scheme 1. Synthesis of benzthiazolylalanine (Bta) containing inhibi-
tors. Reaction conditions: (i) SOCl2 (99%); (ii) i-Pr2EtN, (2-H2N-
C6H4)2S2 (88%); (iii) Zn, AcOH, then TEA, dioxan (81%); (iv) 1M
NaOH (EtOH/H2O) (98%); (v) 4-(R-ethyl)piperidine, DCC, HOBt
(>90%); (vi) HBr, AcOH (97%); (vii) RSO2Cl, NMM, DCM (50±
70%); (viii) NaOMe, MeOH (90%).
The amino acid precursors to the 4-substituted thiazolyl
compounds 27 to 29 were easily obtained from asparagine,
®rst by conversion to the thioamide with Lawsson's
reagent,7 followed by cyclisation onto the appropriate
chloromethyl ketone. The fully assembled 4-chloromethyl-
thiazolyl compound was converted to the azide and then
smoothly reduced to the amine 29 with triphenylphospine
in the presence of water. Other amino acids were obtained
from commercial sources (Scheme 3).
Compounds containing the monomethyl tetrahydro-
quinolyl (MTHQS) P3 were prepared by coupling the
respective amino precursor to the known 3-methyl-
quinoline-8-sulfonyl chloride with subsequent catalytic
Table 1. Monocyclic and bicyclic amino acids at the P1 position
Compd
R2
R4
R1
Ki (mM)
Comp
R2
R4
R1
Ki (mM)
16
17
18
19
20
21
22
23
H
H
HEP
HEP
HEP
HEP
HEP
HEP
HEP
HEP
6.7
47.3
24a
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
AEP
HEP
FEP
HEP
HEP
HEP
FEP
FEP
0.452
0.0798
0.026
2.19
25
CH3
CH3
H
132.7
19.58
1.57
26 (CGH752)
27
28
29
30
31
0.76
H
d-Tryp
59.9
3.28
H
3.1
0.81
H
0.118
4.56
aContains 6-chlorodimethyltetrahydroquinoline at the P3 position.

U. Baettig et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1563±1566
1565
Table 2. Serine protease selectivity pro®le of thrombin inhibitors containing Benzthiazolylalanine (Bta) and Aminopropylcysteine (APC) at P1
Ki (mM)
Compound
P1
R1, R2
R3
Thrombin
Trypsin
Chymotrypsin
Plasmin
Kallikrein
Xa
34
35
32
26
33
APC
APC
BTA
BTA
BTA
R1=R2=CH3
R1=R2=CH3
R1=R2=CH3
R1=R2=CH3
R1=H,R2=CH3
F
OH
H
F
OH
0.126
0.28
0.248
0.026
0.118
1.32
1.78
260
NDb
126
11.95
384
4.91
8.4
4.0
6.2
240
ND
109
È a 87
È87
È92
ND
Èa87
È87
È92
ND
7.55
È45
È45
aÈ=Inactive at stated concentration.
bND=not determined.
Scheme 3. Synthesis of thiazolyl containing inhibitors. Reaction con-
ditions: (i) Lawesson reagent, toluene, 80 ꢁC; (ii) RCOCH2Cl, mole-
cular sieve, THF; (iii) (a) 1M NaOH, MeOH/H2O; (b) 4-acetoxyethyl-
piperidine, DCC, HOBt; (iv) (a) TFA, DCM; (b) DMTHQSO2Cl,
NMM, DCM; (c) 1M NaOH, MeOH/H2O; (v) (a) NaN3, NaI, DMF;
(b) Ph3P, H2O.
Scheme 2. Synthesis of analogues of Bta. Reaction conditions:
(i) SOCl2 (99%); (ii) pyridine, DMAP, 8 or 9 (93% and 21% respec-
Results and Discussion
tively); (iii) Lawesson's reagent, toluene, 80 ꢁC (68%
& 48%);
From Table 1 it can be seen that benzthiazolylalanine
(Bta) compounds (23, 25 and 26) are the most potent,
indeed Bta in combination with DMTHQS and FEP at P3
and P2 respectively9,10 (26, CGH752) gave a Ki of 26nM, a
potency comparable to arginine based inhibitors.
(iv) (a) iBuOCOCl, phenylenediamine, TEA, THF; (b) AcOH;
(v) (a) iBuOCOCl, o-aminophenol (55%); (b) AcOH, re¯ux
(42%).
reduction of the quinoline ring. Dimethyl tetrahydro-
quinolyl sulphonyl chloride (DMTHQSO2Cl) was pre-
pared from p-chloroaniline and dimethyl malonic acid
in 7 steps,8 4- ¯uoroethylpiperidine (FEP) was prepared
from N-Boc-4-hydroxyethylpiperidine (Boc-HEP) by
treatment with DAST.8
Alterations to this ring system always led to a reduction
in potency. The enhanced activity of Bta can mainly be
rationalised in terms of good lipophilic interaction with
the P1 pocket, an explanation born out by modelling11
(Fig. 2) and later con®rmed by the crystal structure of
an inhibitor/thrombin complex. While the sulphur atom

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U. Baettig et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1563±1566
Conclusion
In summary a series of novel, reversible thrombin
inhibitors have been synthesised in which the cationic
arginine moiety has been substituted by neutral, low
polarity groups. Bta derived inhibitors exhibit excellent
potency and selectivity for thrombin, and represent
a major step forward in the search for orally active
compounds.
References and Notes
1. Fenton, J. W. Semin. Thromb. Haemost. 1988, 14, 234. (b)
Vu, T. K. H.; Hung, D. T.; Wheaton, V. I.; Coughlin, S. R.
Cell. 1991, 64, 1057.
2. (a) Callas, D.; Fareed, J. Thromb. Haemost. 1995, 74, 473.
(b) Weitz, J. Int. J. Clin. Pract. 1997, Issue Suppl. 90, 25. (c)
Verstraete, M. Thromb. Haemost. 1997, 78, 357.
3. (a) Brundish, D. E. Current Opinion in Therapeutic Patents,
Current Drugs Ltd, 1992, pp 1457±1466. (b) Ripka, W. C. Curr.
Opin. Chem. Biol. 1997, 1, 242. (c) Wiley, M. R.; Fisher, M. J.
Expert Opin. Ther. Pat. 1997, 7, 1265. (d) Hauptmann, J; Sturze-
becher, J. Thromb. Res. 1999, 93, 203.
4. Okamoto, S.; Hijikata, A. J. Med. Chem. 1980, 23, 1293.
5. Misra, R. N.; Kelly, Y. F.; Brown, R. B.; Roberts, D. G. M.;
Chong, S.; Seiler, S. M. Bioorg. Med. Chem. Lett. 1994, 4,
2165.
6. Nestor, J. J. Jr.; Horner, B. L.; Ho, T. L.; Jones, G. H.;
McRae, G. I.; Vickery, B. H. J. Med. Chem. 1984, 27, 320.
7. Cava, P.; Levinson, M. I. Tetrahedron 1985, 41, 5061.
8. Brundish, D.; Bull, A.; Donovan, D.; Fullerton, J. D.;
Garman, S.; Hayler, J.; Janus, D.; Kane, P. D.; McDonnell,
M.; Smith, G. P.; Wakeford, R.; Walker, C. V.; Howarth, G.;
Hoyle, W.; Allen, M. C.; Ambler, J.; Butler, K.; Talbot, M. D.
J. Med. Chem. 1999, 42, 4584.
Figure 2.
and benzene ring of the inhibitor make good contact in
a low energy conformation with the S1 pocket of
thrombin, the thiazolyl ring nitrogen hydrogen bonds
with a water molecule associated with serine. This same
water interacts with the piperidine carboxyl group of
MD805 in thrombin. The di€erence in activity observed
between 22 and 23 probably re¯ects a combination of
the superior lipophilic in¯uence of sulphur on the whole
system and the increased bond length facilitating better
®t. It is more dicult to interpret the intermediate
activity of 24, but it is feasible that this compound may
adopt a di€erent binding conformation.
In Table 2 a selectivity comparison is made between Bta
compounds and inhibitors from an acyclic series (34, 35).12
From this it can be seen that Bta is a P1 amino acid
which confers good selectivity for thrombin over a num-
ber of other serine proteases, especially trypsin, a poten-
tially important pre-requisite for oral activity. Although
the Bta compounds prepared had low aqueous solubility
and no appreciable oral bioavailable, they represented a
unique starting point to totally circumvent the problems
inherent to guanidine and amidine based thrombin inhi-
bitors. A detailed description of our work to introduce the
desired properties into Bta based inhibitors is covered in
the following paper.
9. MD805 and Bta containing inhibitors bind to thrombin with
a di€erent backbone orientation to that of ®brinogen, with the
MTHQS ring occupying S8 and S9, termed P3* by Banner,10 and
the piperidine moiety occupying S2. For convenience we term
MTHQS and DMTHQS P3 and the piperidide moiety P2.
10. Banner, D.; Ackermann, J.; Gast, A.; Gubernator, K.;
Hadvary, P.; Hilpert, K.; Labler, L.; Mueller, K.; Schmid, G.
Serine Proteases: 3-D Structures, Mechanisms of Action and
Inhibitors. In Perspectives in Medicinal Chemistry; Testa, B., Ed.;
Verlag Helvetica Chim. Acta: Basel, Switzerland, 1993, 27.
11. Allen, M. C.; Cockcroft, X. L.; Gruetter, M. G.; Priestle,
J. P. J. Comp.-Aided Mol. Des. 1999, 13, 579.
12. Details concerning acyclic P1 inhibitors of this type will be
published elsewhere.