Design, synthesis and structure-activity relationships of benzoxazinone-based factor Xa inhibitors

Article abstract of DOI:10.1016/S0960-894X(02)00927-7

A series of benzoxazinone derivatives was designed and synthesized as factor Xa inhibitors. We demonstrated that the naphthyl moiety in the aniline-based compounds 1 and 2 can be replaced with benzene-fused heterobicycles and biaryls to give factor Xa inh

Full text of DOI:10.1016/S0960-894X(02)00927-7

Bioorganic & Medicinal Chemistry Letters 13 (2003) 561–566
Design, Synthesis and Structure–Activity Relationships of
Benzoxazinone-Based Factor Xa Inhibitors
Wenrong Huang, Penglie Zhang,* Jingmei F. Zuckett, Lingyan Wang, John Woolfrey,
Yonghong Song, Zhaozhong J. Jia, Lane A. Clizbe, Ting Su, Katherine Tran,
Brian Huang, Paul Wong, Uma Sinha, Gary Park, Andrea Reed, John Malinowski,
Stanley J. Hollenbach, Robert M. Scarborough and Bing-Yan Zhu*
Millennium Pharmaceuticals, Inc., 256 E. Grand Avenue, South San Francisco, CA 94080, USA
Received 19 June 2002; accepted 27 September 2002
Abstract—A series of benzoxazinone derivatives was designed and synthesized as factor Xa inhibitors. We demonstrated that the
naphthyl moiety in the aniline-based compounds 1 and 2 can be replaced with benzene-fused heterobicycles and biaryls to give
factor Xa inhibitors with improved trypsin selectivity. The P4 modifications lead to monoamidines which are moderately active.
The benzoxazinones 41–45 are potent against factor Xa, retain the improved trypsin selectivity of the corresponding aniline-based
compounds, and show strong antithrombotic effect dose responsively.
# 2002 Published by Elsevier Science Ltd.
Factor Xa is a trypsin-like serine protease positioned at
the convergence of the intrinsic and extrinsic blood
coagulation pathways.1 The prothrombinase complex
formed by factor Xa on the phospholipid surface with
factor Va and calcium catalyzes the conversion of pro-
thrombin to thrombin(IIa), which is the last enzyme in
the coagulation cascade leading to fibrin formation and
eventually thrombosis. Inhibition of factor Xa has been
actively pursued to develop novel anticoagulants to
replace existing therapies in the treatment or prevention
of thromboembolic disorders.2 Previously reported
small molecule, direct factor Xa inhibitors include a
variety of diamidines as represented by DX-9065a3 and
YM60828 (1, Fig. 1) and its analogue 2.4 DX-9065a is
currently under clinical investigation as an intravenous
agent. Even though the diamidines are highly efficacious
in animal thrombosis models, they are poorly absorbed
following oral administration due to the presence of the
strongly basic amidino groups.2
synthesis and opportunities for chemical diversification. A
stepwise design strategy was employed. First, it was highly
desired to substitute the P1 naphthylamidine group with
bicyclic heteroaromatic and/or biaryl amidines to expand
the chemical diversity as well as to probe the selectivity of a
factor Xa inhibitor over other serine proteases including
thrombin and trypsin. Structure–activity relationship
studies around the P4 moiety were planned because repla-
cement of the P4 amidino group with less basic functional
groups might result in monoamidines which can poten-
tially display improved oral bioavailability. Finally, com-
pound 2 exhibited an extended L-shaped conformation
when docked in the active site of factor Xa. The P1 ami-
dine binds to the S1 specificity site of factor Xa, while the
iminoethylpiperidine moiety occupies the aryl binding
site. This binding conformation is very similar to that of
DA-9065a co-crystallized with factor Xa.5 It was envi-
sioned that substituting the sulfonyl group with a car-
bonyl followed by tethering the methyl group to the
aniline template with an oxygen atom would yield the
benzoxazinones (structure 3 in Fig. 1) as structurally
novel, bicyclic factor Xa inhibitors.6,7 This sequential
introduction of new P1 benzamidines, P4 modifications
and aniline template cyclization is reported here.
Compounds 1 and 2 were quite attractive to us when our
factor Xa inhibitor medicinal chemistry efforts started
several years ago due to their structure simplicity, ease of
Our studies started with the synthesis of compound 2
according to Scheme 1 (Ar=naphthyl). The IC50 values
*Corresponding author. Fax:+1-650-244-9287; e-mail: penglie.
zhang@mpi.com (P. Zhang); e-mail: bing-yan.zhu@mpi.com (B.-Y. Zhu)
0960-894X/03/$ - see front matter # 2002 Published by Elsevier Science Ltd.
PII: S0960-894X(02)00927-7

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W. Huang et al. / Bioorg. Med. Chem. Lett. 13 (2003) 561–566
Figure 1. Design of benzoxazinone derivatives as factor Xa inhibitors.
Scheme 2. (a) Tf2O, Py; (b) KCN, DPPF, (dba)3Pd2(CHCl3), ace-
tone; (c) BBr3, CH2Cl2; (d) SnMe4, LiCl, PdCl2(PPh3)2, DMF; (e)
NBS, (PhCO2)2, CCl4; (f) LDA, DMF, THF; (g) HSCH2CO2Me,
TEA, DMSO; (h) NaBH4, CaI2, NaHCO3, THF; (i) TsCl, Py,
CH2Cl2; (j) NaOEt, (CO2Et)2, EtOH; (k) Zn, HOAc; (l) MeI or EtI,
i
Cs2CO3, DMF; (m) bromotoluene, Pd(PPh3)4, NaOH, H2O, PrOH;
(n) NH2OH/HCl, TEA, EtOH; (o) NCS, DMF; (p) propargyl alco-
hol, TEA; (q) ethyl 3-methylpyrazole-5-carboxylate, Cu(OAc)2, Py,
CH2Cl2; (r) LiAlH4, THF.
Scheme 1. (a) 1-Fluoro-4-nitrobenzene, NaH, THF; (b) Pd/C (10%),
H2 (1 atm), MeOH; (c) MsCl, Py; (d) XCH2–Ar–CN, Cs2CO3, DMF
(X=Br, Cl); (e) (1) NH2OH, EtOH; (2) Ac2O, AcOH; (3) Pd/C (10%),
H2 (1 atm), MeOH; (f) (1) TFA, CH2Cl2; (2) ethyl acetimidate/HCl,
Et3N, EtOH.
hand, the P1 moieties have a marked effect on trypsin
selectivity (trypsin/Xa in Table 1). For example, com-
pounds 8, 9, and 16 are less selective than the lead
compound 2. Interestingly, moving the amidino group
from the 5-position of the indole ring as in compound 9
to the 6-position as in compound 10 improves potency
as well as trypsin selectivity. Increased trypsin selectivity
was also observed for compounds 11, 14, and especially
15, which has a trypsin/Xa >700.
of compound 2 for factor Xa, thrombin and trypsin
were determined as 6 nM, >10 mM and 95 nM, respec-
tively, in our in-house assay. It is noted that compound 2
has a rather low trypsin selectivity (IC50, trypsin/IC50, Xa
=15).
To synthesize the analogues of compound 2 with P1 aryl
variations, a variety of the halide intermediates were
prepared according to Scheme 2. The halides were uti-
lized in the alkylation step of Scheme 1 to obtain com-
pounds 8–17.
The benzothiophene compound 8 and biphenyl com-
pound 14 were selected for the attachment of a carboxyl
group to evaluate its effect on in vitro potency and
enzyme selectivity. Scheme 3 describes the syntheses of
compounds 19–23 as well as YM60828 (1). The alde-
hydes used in the amination reactions were prepared
according to Scheme 4.
Table 1 shows that replacing the naphthyl moiety with a
benzothiophene unit affords the equipotent compound
8. While the 5-amidinoindole compound 9 is less active
than compound 2, a 40-fold improvement in potency
was observed with the 6-isomer (10, IC50=0.8 nM).
Table 2 shows that the acids 22 and 23 are more potent
than the parent compounds 8 and 14, as well as the
ethyl esters 20 and 21. This trend is similar to that
observed with the naphthyl compounds 1, 2, and 19.
The esters (19, 20, and 21) have trypsin selectivity com-
parable to that of the methylsulfonyl compounds 2, 8,
and 14. A slight increase in trypsin selectivity was
observed with the carboxyl containing compounds 1,
22, and 23 (the in vitro IC50 assays were repeated at
least three times). Interestingly, the improved trypsin
selectivity of 14 over 2 is retained by the ester 21 and
acid 23. All the compounds have thrombin IC50s>10
mM.
The replacement of the naphthyl moiety with a biaryl
substructure was then investigated. Compound 11,
which contains a para–meta substituted biphenyl group,
has an IC50=25 nM. The survey of the regio-isomers of
compound 11 demonstrates the importance of the meta–
meta substitution (14).8 Further studies on the P1 moiety
involve replacement of the distal phenyl ring in com-
pound 14 with heteroaromatic structures. Compound 15,
which bears an isoxazole moiety,9 shows a minor (2-fold)
decrease in potency. Compounds 16 and 17 incorporat-
ing a pyrazole ring10 are substantially less active.
The replacement of the P4 amidino group in the diami-
dines with less basic functional groups would result in
monoamidine compounds which may improve the poor
Table 1 also shows that compounds 8–17 are all highly
selective for factor Xa over thrombin(IIa). On the other

W. Huang et al. / Bioorg. Med. Chem. Lett. 13 (2003) 561–566
563
Table 1. SARs of aniline-based dibasic factor Xa inhibitors:
P1 modifications
Compd
P1
IC50s (nM)
IIa Trypsin Trypsin/Xa
Scheme 4. (a) Trimethylamine N-oxide, CHCl3; (b) NaBH4, CaI2,
NaHCO3, THF; (c) IBX, THF.
Xa
2
8
6
6
>10,000
>10,000
94
21
15
Table 2. In vitro potency of dibasic factor Xa inhibitors: modifi-
cation of the methylsulfonyl group by introduction of an ester or acid
functionality
<5
9
31 >10,000
0.8 >10,000
25 >10,000
167
25
5
30
Compd
P1
R
IC50s (nM)
10
11
12
13
14
15
16
17
Xa Trypsin Trypsin/Xa
2
19
1
SO2Me
SO2CH2CO2Et
SO2CH2CO2H
6
6
2
94
83
63
15
15
30
769
30
8
20
22
SO2Me
SO2CH2CO2Et
SO2CH2CO2H
6
9
3
21
86
63
<5
10
20
7000 >10,000 >10,000
—
14
21
23
SO2Me
SO2CH2CO2Et
SO2CH2CO2H
7
7
3
360
292
269
50
40
90
>10,000 >10,000 >10,000
—
7
>10,000
360
50
and 27 with an amidinonaphthyl or amidinobiphenyl as
P1 motifs, respectively, are only moderately potent
(Table 3). Further decrease of potency was observed
with pyrazoles 28 and 29.
14 >10,000 >10,000
>700
<5
—
934 >10,000
2110
Compounds 33–35 containing a pyrrolidinylcarbonyl
group12 were synthesized from the commercially avail-
able compound 30 according to Scheme 6. Compound
31 also served as the starting material to afford 36.
6920 >10,000 >10,000
Compound 33 is, unfortunately, a poor factor Xa inhib-
itor (Table 3). Compounds 34 and 35 possess an amide
group on the pyrrolidine ring to mimic the sulfonamide
functionality in 26. However, both compounds have
insignificant potency. The low activity of compound 36
with a reinstalled P4 amidino group clearly indicates the
importance of the ether linkage in the P4 region probably
due to its rotational flexibility which allows optimal inter-
action with factor Xa. Further studies on substituting the
P4 amidine with a neutral group were discontinued due to
the difficulties in achieving good potency.
After mapping out the SAR profile of the P1 and P4
moieties, attention was focused on bicyclic factor Xa
inhibitors. Benzoxazinone is among a variety of the
heterobicyclic templates we have designed.6 The naph-
thyl, indole, biphenyl and phenylisoxazole moieties were
selected as the P1 groups based on the factor Xa
potency and trypsin selectivity in the aniline series.
Scheme 7 describes the syntheses of benzoxazinone-
based factor Xa inhibitors.
Scheme 3. (a) CHO–Ar–CN, NaBH(OAc)3, AcOH, CH2Cl2; (b
ClSO2CH2CO2Et, Py; (c) (1) NH2OH, EtOH; (2) Ac2O, AcOH; (3) Pd/C
(10%), H2 (1 atm), MeOH; (d) (1) TFA, CH2Cl2; (2) ethyl acetimidate/
HCl, Et3N, EtOH; (e) 4 N HCl.
)
oral bioavailability of the diamidines due to the
decrease of overall basicity.2 Scheme 5 shows the
synthesis of compounds 26–29, which incorporate a S4
binding biphenylsulfonamide moiety.11 Compounds 26

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W. Huang et al. / Bioorg. Med. Chem. Lett. 13 (2003) 561–566
The high potency of the compounds shown in Table 4 vali-
dated the design of bicyclic compounds as factor Xa inhi-
bitors even though no significant activity enhancing effect
by the rotation constraint was observed. Interestingly,
replacing the N-methyl in the indole compound 42 with an
ethyl group resulted in the equipotent compound 43.
Scheme 5. (a) MsCl, Py; (b) XCH2–Ar–CN, Cs2CO3, DMF (X=Br,
Cl); (c) (1) HCl(g), MeOH; (2) NH4OAc, MeOH.
While the good thrombin selectivity is retained by com-
pounds 41, 44, and 45, the indoles (42 and 43) have
IC50s against thrombin in the sub-micromolar range.
The trypsin selectivity of the benzoxazinones is similar
to that of the aniline series, with the naphthyl- contain-
ing compound 41 the least selective and compound 45
which has an isoxazole moiety the most selective.
Table 3. SARs of aniline-based factor Xa inhibitors: P4
modifications
The difference in selectivity noted between compounds
41 and 45 is supported by molecular modeling. Docking
studies using Gold13 suggest both compounds bind to
factor Xa (Fig. 2a) in a manner consistent with pub-
lished and in-house crystallography data.5 The P1-ben-
zamidine moiety clearly interacts with the Asp189 side
Compd
P4
P1
IC50 (nM)
153
26
27
28
29
33
34
35
36
191
500
3933
1050
14,900
4060
1570
Scheme 7. (a) Pd/C (10%), H2 (1 atm), MeOH; (b) chloroacetyl chlor-
ide, satd NaHCO3; (c) BBr3, CH2Cl2; (d) N-Boc-4-hydroxypiperidine,
PPh3, DEAD, THF; (e) XCH2–Ar–CN, Cs2CO3, DMF (X=Br, Cl); (f)
(1) NH2OH, EtOH; (2) Ac2O, AcOH; (3) Pd/C (10%), H2 (1 atm),
MeOH; (g) TFA, CH2Cl2; (h) ethyl acetimidate/HCl, Et3N, EtOH.
Table 4. In vitro potency of benzoxazinone-based dibasic factor Xa
inhibitors
Compd
P1
IC50s (nM)
Trypsin Trypsin/Xa
Xa
6
IIa
41
42
>10,000
28
76
5
2
2
4
882
877
40
43
44
45
58
30
35
>10,000
143
Scheme 6. (a) MsCl, Py; (b) XCH2–Ar–CN, Cs2CO3, DMF (X=Br,
Cl); (c) LiOH; (d) BOP, pyrrolidine(s), TEA, DMF; (e) (1) HCl(g),
MeOH; (2) NH4OAc, MeOH; (f) TFA, reflux (for compounds 34 and
35); (g) BOP, N-Boc-piperazine, TEA, DMF; (h) (1) HCl(g), MeOH;
(2) NH4OAc, MeOH; (i) ethyl acetimidate/HCl, Et3N, EtOH.
52 >10,000 250,000
5000

W. Huang et al. / Bioorg. Med. Chem. Lett. 13 (2003) 561–566
565
chain carboxylate. The P4-amidine is held in the S4
binding pocket by pi-cation interaction with Tyr99,
Phe174, and Trp215. However, contrary to the docking
results with factor Xa, the best binding conformation of
compound 45 predicted by Gold displays an inverted
binding mode, with the benzamidino group in the S4 site
of trypsin (Fig. 2b). A less favorable conformer does
indeed show normal binding (not shown here); however,
this conformation of 45 is significantly different from
that of compound 41 in both the S1 and S4 binding
pockets. This observation may explain why compound
45 is a poor trypsin inhibitor.
compound 41 is 0.78 nM. It doubles the thrombin gen-
eration time of rabbit plasma at a concentration of 0.5
mM. The half lives of compound 41 are 0.5 h in rats and
0.9 h in rabbits following intravenous administration at
2 mg/kg. The bioavailability in rats is 4.6% at an oral
dose of 2 mg/kg in 0.5% methylcellulose. This low oral
bioavalibility is characteristic of diamidine compounds.
The antithrombotic effect of compound 41 in our rabbit
deep vein thrombosis model is dose responsive.14 It
shows a 7, 44, and 99% inhibition of thrombosis at the
dose of 25, 50, and 100 mg/kg, respectively. The rabbit
bleeding time is prolonged by 1.7-fold and the plasma
concentration of compound 41 is 200 nM at the 100 mg/
kg dose.
Selected compounds were subjected to more detailed in
vitro and in vivo tests. For example, the Ki value of
In summary, starting with the aniline-based lead com-
pound 2, we have demonstrated that the naphthyl moiety
is tolerant of replacement with a variety of benzene-fused
heterobicycles or biaryls. Compounds 10, 14, and espe-
cially 15 which contain an indole, biphenyl or phenyl-
isoxazole moiety as P1, respectively, display improved
trypsin selectivity. Improvements in potency and trypsin
selectivity were observed when a carboxyl group was
attached to the aniline nitrogen as in the biphenyl and
benzothiophenyl compounds 22 and 23, similar to that
observed for YM-60828. Considerable potency drop
was observed in compounds with a biphenylsulfon-
amide moiety or a pyrrolidinylcarbonyl phenyl group as
the P4 motifs. The benzoxazinone-based compounds
41–45 are active factor Xa inhibitors. The improved
trypsin selectivity profile of the aniline series of com-
pounds is retained by the corresponding benzox-
azinones. Compound 41 shows strong antithrombotic
effect in a dose responsive manner. The compounds
with an amidinobiaryl group as the P1 moiety paved the
way to the convenient introduction of benzamidine
surrogates such as aminoisoquinolines and amino-
benzoisoxazoles, which offer possibility to achieve good
oral bioavailability.15 The details will be reported in due
course.
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