Solid-phase parallel synthesis of azarene pyrrolidinones as factor Xa inhibitors

Article abstract of DOI:10.1016/S0960-894X(00)00151-7

A focused library (4x14) prepared from 4-aminopyridine and 4-, 5-, and 6-azoindole templates was synthesized using 14 polymer supported 4-amido-2,3,5,6-tetrafluorophenyl (TFP) sulfonate esters inputs. Several compounds were identified as factor Xa inhibitors (IC₅₀≤0.1 μM) helping to establish the SAR among these four series of azarene pyrrolidinones. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00151-7

Bioorganic & Medicinal Chemistry Letters 10 (2000) 1033±1036
Solid-Phase Parallel Synthesis of Azarene Pyrrolidinones as
Factor Xa Inhibitors
Yong Gong, a Michael Becker, a Yong Mi Choi-Sledeski, a Roderick S. Davis, a
Joseph M. Salvino, b Valeria Chu, c Karen D. Brown c and Henry W. Pauls a,*
aDepartment of Medicinal Chemistry, Rhone-Poulenc Rorer, 500 Arcola Rd., Collegeville, PA 19426, USA
bDepartment of Lead Discovery, Rhone-Poulenc Rorer, 500 Arcola Rd., Collegeville, PA 19426, USA
cDepartment of Cardiovascular Biology, Rhone-Poulenc Rorer, 500 Arcola Rd., Collegeville, PA 19426, USA
Received 28 January 2000; accepted 1 March 2000
AbstractÐA focused library (4Â14) prepared from 4-aminopyridine and 4-, 5-, and 6-azoindole templates was synthesized using 14
polymer supported 4-amido-2,3,5,6-tetra¯uorophenyl (TFP) sulfonate esters inputs. Several compounds were identi®ed as factor
Xa inhibitors (IC50ꢀ0.1 mM) helping to establish the SAR among these four series of azarene pyrrolidinones. # 2000 Elsevier
Science Ltd. All rights reserved.
The serine proteases of the coagulation cascade, namely
thrombin (fIIa) and factor Xa (fXa), have been identi®ed
as logical targets for the development of antithrombotic
agents.1 The inhibition of fXa, the central enzyme in the
cascade, has emerged as a particularly active area of
research. Numerous small molecule inhibitors of fXa have
been described,2 4 however, most incorporate highly basic
functions which impart poor physicochemical properties
for oral administration. Previous results from this labora-
tory have shown that pyrrolidinone benzamidines 1 are
potent inhibitors of fXa.5 7 The benzamidine group (pKa
11.6) can be replaced by a less basic aminoisoquinoline 2
(pKa 7.6) with improved Caco-2 cell permeability and oral
bioavailability.8
focused library using an optimized set of sulfonamide
groups identi®ed in the pyrrolidinone benzamidines 1.
4-Aminopyridine (pKa=9.2) derivatives have been used
to replace benzamidines and guanidines in an e€ort to
improve the oral activity of thrombin inhibitors.9 11 In
the course of our continuing work on novel benzami-
dine replacements in fXa inhibitors we have prepared 4-
aminopyridine pyrrolidinones 3.12 As a further exten-
sion of this work 4-, 5-, and 6-azaindoles (pKa=6.9, 8.3,
8.0, respectively) were targeted. These weak bases can
be considered as conformationally restricted aminopyr-
idine analogues. Initial results based on this strategy
were quite promising. Selected compounds 3a, 4a, 5a, 6a
(Ar=6-chlorobenzothiophene-2-yl) demonstrated inhi-
bitory activity with anti-fXa Kis of 17, 109, 96, and 43
nM, respectively.13 This result prompted us to design a
Previous work from our laboratories had demonstrated
the ready derivatization of 4-amido-2,3,5,6-tetra¯uoro-
phenoxy (TFP) resins (polystyrene/1% divinylbenzene)
to form activated sulfonate and carboxylate esters.14 The
resin bound sulfonate esters can be used as electrophilic
partners for the formation of sulfonamide linkages
(Scheme 2). A variety of amines can serve as nucleophiles
and the application of this methodology to the construc-
tion of sulfonamido pyrrolidinone libraries is a logical
extension of this work. The preparation, analysis and
assay of a focused library to quickly expand the SAR of
the pyrrolidinone azarenes 3±6 is described.
*Corresponding author. Tel.: +1-610-454-5636; fax: +1-160-454-
3311; e-mail: heinz.pauls@aventis.com
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00151-7

1034
Y. Gong et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1033±1036
Experimental
Assays were run in triplicate by a modi®cation of the
published procedure.19 Stock solutions were diluted to a
®nal concentration of 1 mM in inhibitor using bu€er
containing 0.05 M Tris, 0.15 M NaCl and 0.1% of
PEG-8000 (pH 7.5). Inhibitor solutions were treated
with 1 nM fXa, incubated for 30 min at rt, then assayed
with 200 mM of Spectrozyme substrate (American
Diagnostica Inc.). The initial rates were read at 405 nm
for 5 min with a Molecular Devices microtiterplate
reader in kinetic mode. Any compound with ꢁ65%
inhibition at 1 mM in the initial screen was assayed fur-
ther by serial dilution until ꢂ50% inhibition was
observed.
The 4-aminopyridine sca€old 7 was prepared (Scheme
1) by cyanomethylation of pyrrolidinone 11 followed by
reduction to amine 12. Subsequent reaction with 4-
nitrotetrachloropyridine yields the 4-substituted regioi-
somer 13. Hydrogenolysis followed by deprotection
gave sca€old 7. Sca€olds 8, 9, and 10 were prepared as
previously described.13,15
Results and Discussion
The aryl sulfonyl chlorides used in this work are commer-
cially available (14k±n) or have been previously described
(14a±i).6,7,16 Sulfonyl chloride 14j was prepared by litera-
ture methods.17 The tetra¯uorophenol (TFP) derivatized
resin,18 was loaded with 14 di€erent sulfonyl chlorides 14.
In a typical procedure, TFP resin was agitated with excess
14 and DIEA in CH2Cl2 at rt overnight. The loaded resins
were washed repeatedly (aq DMF; THF; CH2Cl2) and
dried in vacuo. Loading was con®rmed by 19F NMR on a
representative sampling. For example 15c,f,h showed: d
141.7/151.4, 141.8/ 151.4 and 142.1/ 151.6, respec-
tively, versus 148/ 166 for the TFP resin.
The results of the library are given in Table 1; desired
product (as determined by MS) was found in every well.
The highest purity (as determined by analytical LC
using ELSD) was observed for the aminopyridine
series 3; excepting 3i, the compounds in this series had
purities greater than 90% (ELSD). The remaining
azaindoles, series 4±6, also had acceptable purity.
Overall, better than 70% of the compounds had purities
>70%, as determined by ELSD. The average purity of
Solid-phase sulfonylation of templates was carried out
in parallel fashion (Scheme 2). Fifty-six reaction vessels
were charged with a combination of four templates (7±
10) and 14 loaded resins 15 (4Â14). In a typical reac-
tion, loaded resin and sca€old were agitated in DMF at
rt for 3 days. The reaction was monitored for dis-
appearance of starting materials by TLC. Resins were
collected by ®ltration and washed with MeOH. For the
reactions with the aminopyridine template 7, con-
centration of ®ltrate gave product 3. In the case of the
azaindole templates 8±10, products 4±6 were obtained
after removal of the Boc group. Residues were made up
to 10 mM DMSO stock solutions, in a microtiter plate
and analyzed by LC±MS and ELSD (evaporative light
scattering detection).
Scheme 1. Preparation of aminopyridine template 7: (i) NaH, TBAI,
BrCH2CN; (ii) PtO2, H2; (iii) 4-nitrotetrachloropyridine, NMM; (iv)
10% Pd/C, NaOMe, H2; (v) TFA/DCM.
Scheme 2. Parallel synthesis of azarene sulfonamides 3, 4, 5, and 6: (vi)
DIEA, CH2Cl2; (vii) 7, DMF; (viii) 8±10, DMF; (ix) 20% TFA/CH2Cl2.

Y. Gong et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1033±1036
1035
Table 1. Summary of 56-member library and fXa inhibition assay19
results
tors. The 5-azaindole series 5 was the least e€ective and
the 4-azaindole 4 was intermediary in activity. More
speci®cally, chlorobenzothiophene a (included as an
internal standard) is the only sulfonamide group e€ec-
tive in all four series. The relative potencies for library
compounds 3a, 4a, 5a, 6a tracked that seen for pure
inhibitors (vida supra) in that 3a and 6a are particularly
e€ective fXa inhibitors.
Compd
%
ELSDb
Conversiona purity (%) [M+1]
LC±MS Test concn Inhibition of
fXa (%)
(mM)
3a
3b
3c
3d
3e
3f
3g
3h
3i
94
88
94
88
82
99
99
76
40
85
99
94
97
96
81
90
87
89
92
88
82
36
40
67
38
59
80
65
63
80
73
83
84
78
67
23
36
60
26
44
62
49
77
88
85
88
91
88
80
31
56
69
36
52
76
61
99
99
99
99
99
99
99
95
50
95
99
99
99
99
90
98
99
99
99
96
92
49
47
65
37
73
92
76
77
92
90
92
95
86
79
28
27
64
28
54
83
59
86
97
96
97
99
95
88
44
48
75
37
66
91
76
451
452
452
452
452
418
418
483
444
435
444
434
417
465
461
462
462
462
462
428
428
493
454
444
454
444
427
475
461
462
462
462
462
428
428
493
454
444
454
444
427
475
461
462
462
462
462
428
428
493
454
444
454
444
427
475
0.1
0.4
0.1
1
1
0.7
1
0.1
1
1
1
1
57
50
53
39
23
50
23
77
41
29
18
22
14
47
64
48
23
50
45
58
44
42
35
14
8
31
22
40
53
58
51
35
25
61
18
62
21
31
11
17
14
37
50
57
50
52
48
52
43
53
50
21
17
49
57
52
Closer examination of the table reveals that the struc-
ture±activity relationships (SAR) between the series was
quite variable. For example entry h, (i.e., the chloro-
bithienyl) ligand is very e€ective in series 3 and 5, mod-
erately e€ective in 6 and weakly active in 4. In contrast
the thienopyridyl f, a potent ligand in pyrrolidinone
benzamidines 1, is very e€ective in series 6, moderately
e€ective in 3 and 4, and weakly active in 5. This suggests
that the binding modes for the various sca€old/azarene
combinations may di€er appreciably.
3j
3k
3l
3m
3n
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
1
0.5
0.5
0.5
1
0.5
1
0.3
1
1
1
1
1
1
1
1
0.5
1
1
1
1
1
1
0.1
1
1
1
1
Compounds 3c, f, h, 4f, h, 5f, h and 6f, h were resyn-
thesized; the activities of the pure compounds con®rmed
the SAR established by the library. For example, in
keeping with the library trends, resynthesized samples of
3h, 6h and 4h20 showed anti-fXa Kis of 15, 60, and 310
nM, respectively.
1
1
0.1
0.3
1
0.2
0.5
0.1
0.5
0.3
1
1
1
1
1
0.5
The details of this work and the implications for inhi-
bitor binding modes in fXa will be published in due
course. In summary, a focused 56-member library was
prepared using TFP resins. Library purity (70% of wells
with greater than 70% by ELSD) was sucient to
establish SAR patterns. New fXa inhibitors (e.g. 3c, 3h,
5h, 6f) with potencies less than or equal to 0.1 mM were
identi®ed.
6m
6n
Acknowledgements
aAs estimated by analytical LC area% (UV detection).
bDetermined by analytical LC (evaporative light scattering detection).
The authors would like to thank Dr. S.-Y. Tang for
LC±MS analysis and acknowledge Dr. W. He for the
discovery of ligand j.
this 56-member library is almost 80% with a median of
91%. The major impurity as determined by LC±MS is
unconverted starting sca€olds. None of the sca€olds
show anti-fXa activity at 1 mM concentration.
References and Notes
Inhibition data show that the four series 3±6 were not
equally e€ective fXa inhibitors. Aminopyridines 3 and
6-azaindoles 6 were generally the most e€ective inhibi-
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1
(>95A%) and gave satisfactory H NMR and MS spectra.