A four component coupling strategy for the synthesis of D-phenylglycinamide-derived non-covalent factor Xa inhibitors

Article abstract of DOI:10.1016/S0960-894X(03)00462-1

A novel isonitrile derivative was synthesized and used in an Ugi four component coupling reaction to explore aryl group substitution effects on inhibition of the coagulation cascade serine protease factor Xa.

Full text of DOI:10.1016/S0960-894X(03)00462-1

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2255–2259
A Four Component Coupling Strategy for the Synthesis of
D-Phenylglycinamide-derived Non-Covalent Factor Xa Inhibitors
Scott M. Sheehan,a,* John J. Masters,a Michael R. Wiley,a Stephen C. Young,b
John W. Liebeschuetz,b Stuart D. Jones,b Christopher W. Murray,b
Jeffrey B. Franciskovich,a David B. Engel,a Wayne W. Weber, II,a
Jothirajah Marimuthu,a Jeffrey A. Kyle,a Jeffrey K. Smallwood,a Mark W. Farmena
and Gerald F. Smitha
aLilly Research Laboratories, A Division of Eli Lilly and Company, Indianapolis, IN 46285, USA
bTularik Ltd., Beechfield House, Lyme Green Business Park, Macclesfield, Cheshire SK11 0JL, UK
Received 25 February 2003; revised 5 May 2003; accepted 5 May 2003
Abstract—A novel isonitrile derivative was synthesized and used in an Ugi four component coupling reaction to explore aryl group
substitution effects on inhibition of the coagulation cascade serine protease factor Xa.
# 2003 Elsevier Science Ltd. All rights reserved.
Discovery of new agents for the treatment and modera-
tion of thrombotic disorders is an area of intensive
research within the pharmaceutical and biotechnology
communities.1 Presently, thrombosis related compli-
cations are the leading cause of death in the industrial
world.2 Current therapies suffer from an unfavorable
margin of safety which requires the blood of patients to
be frequently monitored to prevent excessive pharma-
cology, namely life-threatening bleeding.3 It is well
understood that thrombus formation is dependant upon
activation of the coagulation cascade which ends in the
thrombin mediated conversion of soluble fibrinogen to
insoluble fibrin. Once formed, fibrin then combines with
activated platelets to form the thrombus. The serine
protease enzyme factor Xa is central to both the intrin-
sic and extrinsic coagulation pathways and represents a
promising target for the regulation of thrombin generation
and thus control of clot formation.4
require the individual synthesis of each newly desired
central amino acid. The Ugi four component coupling
(4CC) reaction represented an intriguing approach to
our scaffold and offered the potential advantage of
enabling us to draw upon the commercial availability of
a wide variety of aryl aldehydes as the requisite starting
materials (Scheme 1).
Our team recently discovered a series of non-covalent
inhibitors of factor Xa derived from d-phenyl glycin-
amide and we wished to explore the effect of substitu-
tion on the central phenyl ring. In order to accomplish
this quickly, we desired a synthetic route that would not
Scheme 1. General scheme for the 4CC approach to phenyl glycin-
amide derived factor Xa inhibitors.
*Corresponding author. Tel.: +1-317-277-8633; fax: +1-317-433-
0715; e-mail: sheehan_scott@lilly.com
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00462-1

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S. M. Sheehan et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2255–2259
Table 1. Effect of ortho substitution on binding to factor Xa
Our initial efforts focused on two interconvertible iso-
nitriles that are reported in the literature.5 After some
experimentation, we found that development of a new
isonitrile that already contained much of our desired S4
functionality, offered us the most flexibility with respect to
tolerance of the starting aryl aldehyde component. Iso-
nitrile formation from N-tert-butoxycarbonyl-4-amino-
methyl-piperidene 2 was straightforward and could be
performed on large scale.6,7 Isonitrile 3 was isolated as a
white solid which could be stored for prolonged periods in
the freezer without significant decomposition (Scheme 2).8
Compd
R
Kass
(106 L/mol)a
% Yield of
4CC Reaction
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
7q
7r
H
CH3
CF3
F2.0
Cl
Br
0.71
2.5
3.1
—
42
32
72
5.1
4.6
4.1
1.3
1.9
2.8
2.6
4.1
1.3
4.2
0.75
1.1
2.9
1.7
61
50
65
51b
60
66
51
56
69
56
I
OH
Scheme 2. Preparation of isonitrile 3.
OMe
OEt
OBn
OCF3
OPh
NO2
NH2
NMe2
SMe
S-tBu
With multigram quantites of isonitrile 3 in hand, the
4CC route proved to be adequate for the investigation
of a variety of aryl substituents. Although not indivi-
dually optimized, purified yields for the Ugi reaction
with aryl aldehydes, isonitrile 3, p-methoxybenzoic acid,
and 2,4-dimethoxybenzyl amine ranged from 15–78%
providing compounds of type 4. Subsequent removal of
the Boc and 2,4-DMB protecting groups with tri-
fluoroacetic acid provided, after basic workup, products 5
which were generally taken on as crude material through a
final reductive amination with acetone to afford the final
products 6 in acceptable yields (Scheme 3).9,10
c
—
—
74
78
d
aKass is the apparent association constant reported in units of 106 L/
mol and is approximately equal to 1/Ki. Kass values were obtained by
the method of Smith et al.14 and are the result of a single experiment
(performed in triplicate at each of four to eight inhibitor concentra-
tions).15
bTwo step yield obtained from deprotection (H2, Pd/C, quantitative
yield) of 2-benzyloxybenzaldehyde 4CC adduct 4k.
cCompound 7o was obtained in quantitative yield from 7n (H2, Pd/C).
dCompound 7p was obtained in 31% yield from 7o ((CH2O)n,
NaCNBH3, HOAc).
The effects of ortho-substitution on the central phenyl
ring of 1 are shown in Table 1.11 A variety of ortho-
substituted aryl aldehydes served as diversity elements
for the Ugi reaction and the 4CC products were carried
on to the corresponding inhibitors of type 7 (Table 1).
Many of the surveyed ortho-substituents provide an
increase in binding affinity for factor Xa.12 Electron-
withdrawing groups such as Cl, and Br provide appar-
ent association constants (Kass) of 5.1 and 4.6 million
L/mol, respectively. Interestingly, electron-donating
groups such as methyl and methoxy boost binding affi-
nity as well. The results in Table 1 also illuminate factor
Scheme 3. The Ugi 4CC route to glycinamide factor Xa inhibitors.

S. M. Sheehan et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2255–2259
Table 3. 3-Chloroindolyl S1 factor Xa inhibitors
2257
Figure 1. A factor Xa inhibitor with a 3-chloro-6-indolyl S1 unit.
Compd
R
Kass
(106 L/mol)
2XPT
(mM)17
11a
11b
11c
11d
1-Naphthalene
2-Naphthalene
3-Quinoline
250
79
0.2
380
3.8
4.1
—
Table 2. Effect of heteroaromatic substitution on binding to factor
Xa
2-Thiazole
0.79
benzylamine, and isonitrile 3 provided 10a in 53%
isolated yield (Scheme 4).18
Compounds 10a–d were then converted in straightfor-
ward fashion under the same conditions shown in
Scheme 3, replacing acetone with cyclopentanone to
afford compounds 11a–d (Table 3). It should be noted
that despite the moderate yield (24%) of the 4CC reac-
tion to form 10d, we found the Ugi route to be superior
to alternate approaches involving the synthesis of
2-thiazolyl glycine for multigram preparation of 11d.19
Compd
R
Kass
,
%Yield of 4CC
Reaction
(106 L/mol)
7a
8a
8b
8c
8d
8e
8f
C6H5
2-Furan
3-Furan
2-Thiophene
3-Thiophene
2-Imidazole
4-Quinoline
3-Quinoline
0.71
0.36
0.20
1.1
0.88
0.21
1.3
—
17
15
15
32
30
21
25
In conclusion, a previously unknown isonitrile 3 was
synthesized and used to develop an Ugi four component
coupling reaction for the synthesis of reversible factor
Xa inhibitors. It was discovered that ortho-substitution
on the central phenyl ring with either electron with-
drawing or electron donating groups provided a sig-
nificant increase in binding affinity. The Ugi 4CC
reaction allowed rapid access to a variety of sub-
stitutuents and heterocyclic replacements for the central
aryl unit and enabled us to readily scale-up compounds
for further evaluation.
8g
0.05
Xa’s ability to accommodate increasing steric bulk at
this position on inhibitors of type 7.13
Heterocyclic and bicyclic replacements for the central
aryl ring are shown in Table 2. Heterocycles such as
2-thiophene and 2-furan are tolerated, but provide no
additional boost in binding affinity toward factor Xa.
The scope of the Ugi methodology was readily applic-
able to the preparation of derivatives containing the
more potent 3-chloroindole S1 binding element found in
compound 9 (Fig. 1).16,17
Acknowledgements
S.M.S. thanks John M. Schaus for valuable discussions
regarding the research and manuscript. The authors
also thank Tommy Smith for assistance with sample
management.
For example, the 4CC reaction of 3-chloroindole-6-car-
boxylic acid with 1-naphthaldehyde, 2,4-dimethoxy-
Scheme 4. Isolated yields for the Ugi 4CC reaction with 3-chloroindole-6-carboxylic acid.

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S. M. Sheehan et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2255–2259
D. B.; Weber, W. W.; Marimuthu, J.; Kyle, J. A.; Smallwood,
References and Notes
J. K.; Foster, R. S.; Froelich, L. L.; Gifford-Moore, D. S.;
Towner, R. D.; Snyder, D. W.; Chouinard, M. L.; Chastain,
M. K.; Johnson, L. M.; Sipes, P. R.; Tluczek, J. P.; Craft, T. J.;
Smith, G. F. Abstracts of Papers, Part MEDI-230, 225th
National Meeting of the American Chemical Society, New
Orleans, LA, March 23–27, 2003; American Chemical Society:
Washington, DC, 2003.
13. While steric bulk is not problematic when located on one
side of the phenyl ring, 2,6-substitution presents a substantial
challenge to the enzyme. The 2,6-dichloro analogue of 7, pre-
pared from the Ugi reaction with 2,6-dichlorobenzaldehyde
had an apparent Kass of 0.11 million L/mol.
14. (a) Herron, D. K.; Goodson, T., Jr.; Wiley, M. R.; Weir,
L. C; Kyle, J. A.; Yee, Y. K.; Tebbe, A. L.; Tinsley, J. M.;
Mendel, D.; Masters, J. J.; Franciskovich, J. B.; Sawyer, J. S.;
Beight, D. W.; Ratz, A. M.; Milot, G.; Hall, S. E.; Klim-
kowski, V. J.; Wikel, J. H.; Eastwood, B. J.; Towner, R. D.;
Gifford-Moore, D. S.; Craft, T. J.; Smith, G. F. J. Med. Chem.
2000, 43, 859. (b) Smith, G. F.; Gifford-Moore, D. S.; Craft,
T. J.; Chirgadze, N. K.; Ruterbories, K. J.; Lindstrom, T. D.;
Satterwhite, J. H. in New Anticoagulants for the Cardiovascular
Patient; Piffare, R., Ed.; Hanley & Belfus Publishers: Phila-
delphia, 1997; pp 265–300.
1. (a) Agnelli, G.; Sonaglia, F. Haematologica 2002, 87, 757.
(b) Zhu, B.-Y.; Scarborough, R. M. Ann. Rep. Med. Chem.
2000, 38, 83. (c) Turpie, A. G. Am. J. Cardiol. 1998, 82, 11 L.
(d) Wiley, M. R.; Fisher, M. J. Exp. Opin. Ther. Pat. 1997, 7,
1265.
2. Mortality statistics from 1997 can be obtained from the
World Health Organization: Avenue Appia 20, 1211 Geneva
27, Switzerland. Website: http://www.who.int/en/
3. (a) Williams, E. C.; Suttie, J. W. In Cardiovascular Throm-
bosis: Thrombocardiology and Thromboneurology; Verstaete,
M., Fuster, V., Topol, E. J., Eds.; Lippincott-Raven: Phila-
delphia, 1998; pp 285–300. (b) Levine, M. N.; Hirsh, J.;
Salzman, E. W. In Hemostasis and Thrombosis: Basic Principles
and Clinical Paractice; Colman, R. W., Hirsh, J., Marder, V.
J., Salzman, E. W., Eds.; J.B. Lippincott Co.: Philadelphia,
1994; pp 936–955. (c) Hirsh, J. N. Engl. J. Med. 1991, 324,
1865. (d) Amerena, J.; Mashford, M. L.; Wallace, S. Adverse
Drug React. Acute Poisoning Rev. 1990, 9, 1.
4. Leadley, R. J., Jr. Curr. Top. Med. Chem. 2001, 1, 151.
5. (a) Lindhorst, T.; Bock, H.; Ugi, I. Tetrahedron 1999, 55,
7411. (b) Keating, T. A.; Armstrong, R. W. J. Am. Chem. Soc.
1995, 117, 7842.
15. (a) Utilizing the statistical method of Bland and Altman,
apparent Kass data generated in this fashion from a single
experiment has been determined to have a minimum sig-
nificant ratio of 2.13
(b) Bland, J. M.: Altman, D. G. Lancet 1986, 1, 307. (c) For a
description of statistical analysis for precision of a single Kass
determination, see ref 11a.
6. Weber, W. P.; Gokel, G. W. Tetrahedron Lett. 1972, 13, 1637.
7. Preparation of isonitrile 3: To a solution of 2 (77 g, 360 mmol)
in 110 mL of CH2Cl2 at room temperature was added chloro-
form (40 mL, 500 mmol) and benzyl triethylammonium chloride
(1.6 g, 7.2 mmol). 110 mL of a 50% (w/v) sodium hydroxide
solution was added and the flask was fitted with a reflux con-
denser. The reaction spontaneously reached and maintained a
gentle reflux for 90 min and the reaction was allowed to stir
overnight at room temperature. The reaction was diluted with
water and the product was extracted into CH2Cl2. The organic
layer was dried over K2CO3, filtered, and concentrated under
reduced pressure. The resultant oil was filtered through a SiO2
plug (2:1 EtOAc:Hexane) to afford, after concentration 28 g
(35%, 83% based on recovered starting material) of 3 as a
white amorphous solid. The SiO2 plug was then washed with
3:1 EtOAc:Isopropyl amine to provide 45g of recovered 2.
3: Mp 58–59 ꢀC; IR (neat) 2147, 1691, 1171 cmÀ1. 1H NMR
(400 MHz, CDCl3) 1.16–1.26 (m, 2H), 1.44 (s, 9H), 1.72–1.84
(m, 3H), 2.67 (t, br, 2H, J=12.4 Hz), 3.28 (d, 2H, J=6.0), 4.13
(s, br, 2H); 13C NMR (100 MHz, CDCl3) 28.8, 29.5, 36.3, 47.4,
47.5, 79.9, 154.7, and 157.1.
16. For a discussion of this and other bicyclic non-amidine S1
moieties, see: Masters, J. J.; Weber, W. W.; Franciskovich,
J. B.; Wiley, M. R.; Halligan, N. G.; Camp, N. P.; Jones, S. D.;
Richards, S.; Lyons, A.; Morgan, P.; Liebeschuetz, J. W.;
Murray, C. W.; Young, S. C.; Chirgadze, N. Y.; Briggs, S. L.;
Craft, T. J.; Towner, R. D.; Smallwood, J. K.; Smith, G. F.
Abstracts of Papers, Part MEDI-284, 224th National Meeting
of the American Chemical Society, Boston, MA, March 23–
27, 2002; American Chemical Society: Washington, DC, 2003.
17. (a) 2XPT is defined as the concentration of test compound
required to double the time to clot formation in reconstituted
human plasma, using the prothrombin time assay as described
in: Wiley, M. R.; Weir, L. C.; Briggs, S.; Bryan, N. A.; Buben,
J.; Campbell, C.; Chirgadze, N. Y.; Conrad, R. C.; Craft, T. J.;
Ficorilli, J. V.; Franciskovich, J. B.; Froelich, L. L.; Gifford-
Moore, S. S.; Goodson, T., Jr.; Herron, D. K.; Klimkowski,
V. J.; Kurz, K. D.; Kyle, J. A.; Masters, J. J.; Ratz, A. M.;
Milot, G.; Shuman, R. T.; Smith, T.; Smith, G. F.; Tebbe,
A. L.; Tinsley, J. M.; Towner, R. D.; Wilson, A.; Yee, Y. K. J.
Med. Chem. 2000, 43, 883.
8. Stored under nitrogen at À20 ꢀC in a sealed flask.
9. By the nature of its mechanism, the 4CC reaction as shown
provides products in racemic form. Isolation of material enri-
ched in the desired enantiomer requires subsequent separation
by chiral HPLC. We have found that the majority of Xa
binding affinity results from one enantiomer (unpublished
results), and have tested the 4CC products as racemates for
expedience.
(b) Inhibitor 9 has >1000Â selectivity with respect to the fol-
lowing enzymes: Thrombin, Plasmin, Bovine Trypsin, Uroki-
nase, aPC, Kallikrein, and tPA.
10. All final products were purified by silica gel column chro-
matography and were judged to be of greater than 95% purity
18. Representative Ugi procedure: To a solution of 1-naph-
thaldehyde (0.69 g, 4.4 mmol) in 2 mL of methanol was added
2,4-dimethoxybenzyl amine (0.74 mL, 4.4 mmol) and the
reaction was allowed to stir at room temperature for 30 min.
The reaction was diluted with 2 mL of methanol after which
3-chloroindole-6-carboxylic acid (0.67 g, 4.4 mmol) and iso-
nitrile 3 (1.0 g, 4.4 mmol) were added. The reaction was then
allowed to stir overnight at room temperature. The reaction
was concentrated under reduced pressure and the resultant
crude residue was subjected to flash silica gel chromatography
to provide 1.7 g (53%) of 10a as a white crystalline solid. Mp
1
by H NMR and HPLC analysis.
11. Jones, S. D.; Wiley, M. R.; Masters, J. J.; Young, S. C;
Leibeschuetz, J. W.; Murray, C. W.; Richards, S. J.; Webber
III, W. W.; Marimuthu, J.; Kyle, J. A.; Sheehan, S. M.;
Watson, B. W.; Smallwood, J. K.; Smith G. F. Abstracts of
Papers, Part MEDI-231, 225th National Meeting of the
American Chemical Society, New Orleans, LA, March 23–27,
2003; American Chemical Society: Washington, DC, 2003.
12. For a depiction of the conformational constraint imparted
by ortho-substitution in a computationally derived model of
an ortho-substituted factor Xa inhibitor from this series
docked into the factor Xa active site, see: Sheehan, S. M.;
Masters, J. J.; Wiley, M. R.; Young, S. C.; Liebeschuetz, J.
W.; Jones, S. D.; Murray, C. W.; Franciskovich, J. B.; Engel,
150–151 ꢀC; IR (neat) 1666, 1613, 1423 cmÀ1 1H NMR
;
(400 MHz, CDCl3) 0.90–0.98 (m, 2H), 1.24–1.57 (m, 2H), 1.41
(s, 9H), 2.51 (s, br, 2H), 3.04 (s, br, 2H), 3.41 (s, 3H), 3.60 (s,
3H), 3.96 (s, br, 2H), 4.57 (dd, 2H, J=16 and 45 Hz, 2H), 5.97

S. M. Sheehan et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2255–2259
2259
(s, br, 2H), 6.19 (t, 1H, J=6.0 Hz), 6.41 (s, br, 1H), 6.88 (d,
1H, J=8.8 Hz), 7.13 (d, 1H, J=2.4 Hz), 7.34–7.38 (m, 2H),
7.43–7.48 (m, 2H), 7.54 (d, 1H, J=7.6 Hz), 7.60–7.71 (m, 1H),
7.73–7.80 (m, 4H), 7.90 (s, br, 1H), and 9.78 (s, br, 1H);
13CNMR (100 MHz, CDCl3) 28.9, 30.0, 36.5, 45.5, 55.1, 55.5,
55.6, 79.7, 98.0, 103.7, 106.0, 111.9, 117.3, 118.1, 119.0, 123.2,
123.5, 125.2, 126.0, 126.6, 126.9, 127.8, 129.1, 129.5, 130.5,
132.4, 133.8, 134.6, 154.9, 157.5, 159.9, 170.5, and 174.5;
HRMS(Acc ES+) calcd for C41H45ClN4O6=725.3105;
Found=725.3091.
19. Hatanaka, M.; Ishimaru, T. Bull. Chem. Soc. Jpn. 1973,
46, 3600.