Design, synthesis and discovery of 1-(2-(6-chloro-3-methylsulfonyl)- naphthyl)-1H-pyrazole-5-carboxylamides as highly potent factor Xa inhibitors

Article abstract of DOI:10.1248/cpb.57.1004

Based upon the biphenyl 1-(2-naphthyl)-1H-pyrazole-5-carboxylamides reported in our previous communications, we designed and discovered 2-(6-chloro-3-methylsulfonyl)-naphthyl as an optimal factor Xa S1 binding element. Employing a key Diels-Alder reaction of 1,4-dihydro-2,3-benzoxathiin-3- oxide with maleic anhydride and a key Cu(I)-mediated methylsulfonylation, we prepared two biphenyl 1-(2-(6-chloro-3-methylsulfonyl)-naphthyl)-1H-pyrazole-5- carboxylamides as highly potent factor Xa inhibitors with K_i values of 0.065 nM and 0.045 nM respectively, and demonstrated the synergistically enhanced binding interaction in the factor Xa S1 site.

Full text of DOI:10.1248/cpb.57.1004

1004
Notes
Chem. Pharm. Bull. 57(9) 1004—1007 (2009)
Vol. 57, No. 9
Design, Synthesis and Discovery of 1-(2-(6-Chloro-3-methylsulfonyl)-
naphthyl)-1H-pyrazole-5-carboxylamides as Highly Potent Factor Xa
Inhibitors
Zhaozhong Jon JIA, ,a,f Robert Murry SCARBOROUGH,a,f Penglie ZHANG,b,f Sherin HALFON,c,f
*
e,f
Ann Elizabeth ARFSTEN,d,f Uma SINHA,a,f and Bing-Yan ZHU
a Portola Pharmaceuticals, Inc.; 270 East Grand Ave., Suite 22, South San Francisco, CA 94080, U.S.A.; b ChemoCentryx,
Inc.; 850 Maude Ave., Mountain View, CA 94043, U.S.A.: c Relypsa, Inc.; 5301 Patrick Henry Dr., Santa Clara, CA 95054,
U.S.A.: d Genelabs Technologies, Inc.; 505 Penobscot Dr., Redwood City, CA 94063, U.S.A.: e Genentech, Inc.; 1 DNA Way,
South San Francisco, CA 94080, U.S.A.: and f Millennium Pharmaceuticals, Inc.; 256 East Grand Ave., South San
Francisco, CA 94080, U.S.A. Received March 29, 2009; accepted June 9, 2009; published online June 12, 2009
Based upon the biphenyl 1-(2-naphthyl)-1H-pyrazole-5-carboxylamides reported in our previous communi-
cations, we designed and discovered 2-(6-chloro-3-methylsulfonyl)-naphthyl as an optimal factor Xa S1 binding
element. Employing a key Diels–Alder reaction of 1,4-dihydro-2,3-benzoxathiin-3-oxide with maleic anhydride
and a key Cu(I)-mediated methylsulfonylation, we prepared two biphenyl 1-(2-(6-chloro-3-methylsulfonyl)-naph-
thyl)-1H-pyrazole-5-carboxylamides as highly potent factor Xa inhibitors with Ki values of 0.065 nM and 0.045 nM
respectively, and demonstrated the synergistically enhanced binding interaction in the factor Xa S1 site.
Key words factor Xa inhibitor; 1-naphthyl-1H-pyrazole; 6-chloro-3-iodonaphthyl-2-amine; 1,4-dihydro-2,3-benzoxathiin-3-
oxide; sodium methanesulfinate
Serine protease factor Xa (fXa), positioned at the juncture swer this question, we designed compounds 5 and 6 that bear
of the intrinsic and the extrinsic pathways, plays a pivotal a tri-b-substituted 6-chloro-3-methylsulfonyl naphthalene
role in the blood coagulation cascade.1) Selective inhibition moiety as the fXa S1 binding element.
of fXa without affecting the existing thrombin levels may
Ethyl 1-(6-chloro-3-(methylsulfonyl)naphthalen-2-yl)-3-
cause less impairment of primary hemostasis and thus should methyl-1H-pyrazole-5-carboxylate 7 was the key building
be a safer anticoagulant therapy than direct inhibition of block needed for the synthesis of compounds 5 and 6, and
thrombin. Clinical findings have confirmed the potential of the tri-b-substituted naphthalene 8 would be the precursor for
fXa inhibition for producing excellent antithrombotic effi- its preparation, as analyzed in Chart 1. The amino group
cacy with minimal bleeding risk when compared to direct would lead to the pyrazole via condensation of the corre-
thrombin inhibitors.2—6)
sponding hydrazine, and the iodo group would serve as a
Our previous communications have reported a series of 1- handle to install the methylsulfonyl substituent through a
(2-naphthyl)-1H-pyrazole-5-carboxylamides as potent and copper-mediated C–S cross coupling with sodium methane-
selective fXa inhibitors with good oral bioavailability and sulfinate.10) 6-Chloro-3-iodo-2-naphthylamine 8 could be
half-life.7—9) This class of fXa inhibitors, as represented by synthesized from methyl 3-amino-2-naphthoate 9 via iodo-
compounds 1—4, possesses a substituent either at the 6-posi- de-diazoniation followed by Curtius rearrangement. Based
tion (chloro) or at the 3-position (methylsulfonyl, aminosul- upon Levy’s precedent work,11) compounds 9—11 could be
fonyl, aminocarbonyl, fluoro or cyano) on the P1 naphthalene prepared from the Diels–Alder product 12 of 4-chloro-o-
moiety. The individual fXa affinity enhancing effect of the 6- quinodimethane (13) and maleic anhydride. To produce the
chloro and the 3-methylsulfonyl substituents prompted us to o-quinodimethane in situ, Levy forced the cheletropic elimi-
wonder if the two substituents could be synergetic to each nation of sulfur dioxide from 1,3-dihydrobenzo[c]thiophene-
other for further fXa binding potency improvement. To an- S,S-dioxide using strong heat at 230 °C. In direct contrast, the
1,4-dihydro-2,3-benzoxathiin-3-oxide (also known as sulfi-
nate or sultine) undergoes the sulfur dioxide cheletropic
elimination smoothly around 80 °C.12—17) Hoey and Dit-
tmer13) have developed a convenient one-step synthesis of
1,4-dihydro-2,3-benzoxathiin-3-oxide in high yield from
a,aꢀ-dihalo-o-xylene and sodium hydroxymethanesulfinate
(rongalite). Townsend and co-workers12) have elegantly ap-
plied this methodology in the preparation of tetra-b-substi-
tuted naphthalenes. We thus chose sulfinate 15 over sulfone
14 as the precursor for 4-chloro-o-quinodimethane 13.
The synthesis of ethyl 1-(6-chloro-3-(methylsulfonyl)-
naphthalen-2-yl)-3-methyl-1H-pyrazole-5-carboxylate 7 and
its corresponding 2-(7-chloro-3-methylsulfonyl)-naphthyl
isomer 7B is illustrated in Chart 2. Diol 18 was readily pre-
pared from commercially available 4-chlorophthalic anhy-
dride 16 using lithium aluminum hydride (LAH) (ꢁ80%
© 2009 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed. e-mail: zjia@portola.com

September 2009
1005
Chart 1. Retrosynthesis of Key Pyrazole Intermediate 7
(a) LAH (1 M at THF, 2.5 eq), THF, room temperature (RT), 2 h, ꢁ80%; (b) BH3·THF (1 M in THF, 10 eq), dioxane, 0 °C to RT, 1 h, ꢁ70%; (c) 48% HBr, reflux, 4 h, 95%; (d)
sodium hydroxymethanesulfinate dihydrate (2 eq), Bu4NBr (0.2 eq), DMF, 0 °C to RT, overnight, 60%; (e) maleic anhydride (1 eq), PhH, reflux, overnight; (f) NBS (2 eq), AIBN
(cat.), Ac2O, 120 °C, 4 h; (g) (1) NaOMe (50 eq), MeOH, reflux, 3 h; (2) conc. HCl; (h) DPPA (1.1 eq), Et3N (1.1 eq), tBuOH, reflux, overnight; flash column purification with 10%
EtOAc in hexane; (i) HCl (4 N in dioxane), 2 h; (j) (1) NaNO2 (1 eq), conc. HCl, 0 °C, 40 min; (2) NaI (4 eq in water), overnight, 0 °C to RT; (k) (1) LiOH·H2O (2 eq), MeOH, THF,
water, RT, 90 min; (2) conc. HCl; (l) (1) NaNO2 (1 eq), conc. HCl, 0 °C, 30 min; (2) SnCl2·2H2O (3 eq), conc. HCl, 0 °C, 30 min; cold filtration to isolate solid product; (m) 25 (1
eq), THF/HOAc (2 : 1), reflux, 3 h; silica flash column purification with 5% EtOAc in hexane to separate 26A and 26B; (n) MeSO2Na (4 eq), (CuOTf)2·PhH (0.5 eq),
MeNHCH2CH2NHMe (0.5 eq), DMSO, 115 °C, 3 h; flash column purification with 10—20% EtOAc in hexane; 50%.
Chart 2
yield) and also from commercially available 4-chlorophthalic Compounds 20A/B were purified by flash column and their
acid 17 using borane-tetrahydrofuran (THF) (ꢁ70% yield). overall yield from 15A/B was 60%. The BOC protecting
a,aꢀ-Dibromo-o-xylene 19 was produced from diol 18 in group was then cleaved by HCl, and the resulted naphthy-
refluxed 48% HBr quantitatively. Under Dittmer’s condi- lamines 9A/B (1 : 1 by HPLC, yet separable by challenging
tion,12,13) compound 19 reacted smoothly with sodium hy- flash column work) were converted to iodonaphthalenes
droxymethanesulfinate to afford a mixture of 1,4-dihydro- 21A/B (inseparable) through iodo-de-diazoniation.18) Methyl
2,3-benzoxathiin-3-oxides 15A/B in 1 : 1 ratio in 60% yield. esters 21A/B were hydrolyzed into naphthyl carboxylic acids
In refluxed benzene, mixture 15A/B decomposed to 4- 22A/B, which were converted to the corresponding BOC-
chloro-o-quinodimethane 13, which then underwent Diels– protected naphthylamines 23A/B via another Curtius re-
Alder reaction with maleic anhydride to cleanly generate arrangement. Compounds 23A/B (inseparable) were purified
6-chloro-1,2,3,4-tetrahydro-2,3-naphthalic anhydride 12. Its by flash column, and their overall yield from 20A/B was
aromatization to 6-chloro-2,3-naphthalic anhydride 11 was 37%. Treatment of 23A/B with 4 N HCl in dioxane offered
accomplished using NBS (N-bromosuccinimide) in boiling the HCl salt of 6-chloro-3-iodonaphthyl-2-amine 8A and 7-
acetic anhydride.11)
chloro-3-iodonaphthyl-2-amine 8B.
Naphthalic anhydride 11 was converted to its half esters
Naphthylamines 8A/B were converted to hydrazines
10A/B (inseparable) with sodium methoxide and next to 24A/B using the same procedure we had reported earlier.7) It
the corresponding tert-butoxycarbonyl (BOC)-protected was then condensed with mono-protected diketone 25 to af-
naphthylamine derivatives 20A/B (inseparable) via Curtius ford naphthyl pyrazoles 26A/B (1 : 1 by HPLC; overall 12%
rearrangement using diphenylphosporyl azide (DPPA).11,12) yield from 23A/B). 6-Chloro-3-iodo-2-naphthyl pyrazole

1006
Vol. 57, No. 9
Table 1. Effects of Naphthalene Substituents on fXa Potency
fXa IC50 fXa Ki 2ꢂTG
Compound
R
Z3
Z6
Z7
(nM)
(nM)
(mM)
1
2
3
4
5
6
29
30
NH2
Me
H
H
Cl
Cl
H
H
H
H
H
H
H
Cl
Cl
3
6
5
3.4
1.8
1.1
ꢁ5
ꢁ5
4.5
1.0
2.5
4.6
nd
NH2 SO2Me
Me SO2Me
NH2 SO2Me
Me SO2Me
NH2 SO2Me
Me SO2Me
H
9
1.7
Cl
Cl
H
0.5
0.6
56
65
0.065
0.045
19
H
22
nd
(a) Me3Al (2 M in hexane, 5 eq), DCM, RT, 1 d; Rochelle’s salt (aq.) quench; 40—
60%; (b) TFA, 50 °C, 1 h, 90%.
The kallikrein IC50 values for compounds 5 and 6 are 1.6 mM
and 1.9 mM, respectively. Unfortunately, fXa inhibitors 5
(2ꢂmaximum thrombin generation (TG) 2.5 mM) and 6
Chart 3
26A and its 7-chloro-3-iodo-2-naphthyl regioisomer 26B (2ꢂTG 4.6 mM) have not displayed strong in vitro anticoagu-
were separated from each other by silica flash column using lant activity in our human plasma thrombin generation
5% EtOAc in hexane. Compound 26B has a slightly higher assay,27) probably due to their poor hydrophilicity and the re-
Rf value than its regioisomer 26A. Ethyl 1-(6-chloro-3- sulted high plasma protein binding. However, as we have re-
(methylsulfonyl)naphthalen-2-yl)-3-methyl-1H-pyrazole-5- ported previously, the fXa inhibitors’ hydrophilicity and in
carboxylate 7 was then successfully prepared from com- vitro anticoagulant potency can be significantly improved by
pound 26A in about 50% yield by the Cu(I)-promoted C–S changing and optimizing the P4 moieties, without com-
cross coupling reaction.10,19) So was ethyl 1-(7-chloro-3- promising the potent fXa binding affinity.8,9) Hopefully, the
(methylsulfonyl)naphthalen-2-yl)-3-methyl-1H-pyrazole-5- optimal P4 motifs we have discovered can lead us to
carboxylate 7B prepared from compound 26B. The struc- potent 1-(2-(6-chloro-3-methylsulfonyl)-naphthyl)-1H-pyra-
tures of 7 and 7B were determined by proton NMR NOE zole-5-carboxylamide-based fXa inhibitors with improved
study.20)
Finally, the synthesis of 6-chloro-3-methylsulfonyl fXa in- ties.
hibitors 5 and 6, along with their corresponding 7-chloro-3-
anticoagulant activity and desired pharmacokinetic proper-
In conclusion, we have designed and synthesized biphenyl
methylsulfonyl regioisomers 29 and 30, was completed by 1-(2-(6-chloro-3-methylsulfonyl)-naphthyl)-1H-pyrazole-5-
the route shown in Chart 3.21,22) Weinreb reactions23) were carboxylamides 5 and 6 as highly potent fXa inhibitors.
used to couple the biphenylamines (27, 28)24) with ethyl es- We have discovered that the 2-(6-chloro-3-methylsulfonyl)-
ters 7 and 7B in 40—60% yield. The amino-protecting t- naphthyl is a more potent fXa S1 binding element than the
butyl groups were cleaved using warm trifluoroacetic acid 2-(6-chloro)-naphthyl and the 2-(3-methylsulfonyl)-naphthyl
(TFA) to liberate the sulfonamide functionality in com- in the class of 1-(2-naphthyl)-1H-pyrazole-5-carboxylamide-
pounds 5 and 29.
based fXa inhibitors, due to the synergetic effect of the 6-
The biological activity data for the biphenyl 1-(2-naph- chloro and the 3-methylsulfonyl groups to the S1 binding in-
thyl)-1H-pyrazole-5-carboxylamides are summarized in teraction.
Table 1. It is clear that the 7-chloro (Z7) substituent in com-
References and Notes
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Quan M. L., Knabb R. M., J. Thromb. Thrombolysis, 24, 43—51
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N., Nagahara T., Morishima Y., Shibano T., J. Thromb. Haemost., 6,
1542—1549 (2008).
pounds 29 and 30 is not tolerated in the fXa S1 pocket, due
to its unfavorable geometrical orientation. We were delighted
to learn that the 6-chloro-3-methylsulfonyl compounds 5
(fXa IC50 0.5 nM; Ki 0.065 nM) and 6 (fXa IC50 0.6 nM; Ki
0.045 nM) are highly potent fXa inhibitors.25,26) Their fXa
binding affinity is about 10-fold better than that of the corre-
sponding 6-chloro analogs (1, 2), and is also about 10-fold
better than that of the corresponding 3-methylsulfonyl
analogs (3, 4). This observation confirms that the Z3 (3-
methylsulfonyl) and Z6 (chloro) substituents are synergetic to
each other for the 2-naphthyl’s binding interaction in the fXa
S1 pocket. Like inhibitors 1—4, compounds 5 and 6 have
displayed excellent enzyme selectivity toward fXa. Their
IC50 values for thrombin, trypsin, tissue plasminogen acti-
vator, activated protein C and plasmin are all above 10 mM.
7) Jia Z. J., Wu Y., Huang W., Goldman E., Zhang P., Woolfrey J., Wong

September 2009
P., Huang B., Sinha U., Park G., Reed A., Scarborough R. M., Zhu
B.-Y., Bioorg. Med. Chem. Lett., 12, 1651—1655 (2002).
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Sinha U., Arfsten A. E., Edwards S. T., Alphonso M., Hutchaleelaha
A., Park G., Scarborough R. M., Zhu B.-Y., Bioorg. Med. Chem. Lett.,
14, 1221—1227 (2004).
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Arfsten A. E., Edwards S. T., Hutchaleelaha A., Hollenbach S. J.,
Lambing J. L., Scarborough R. M., Zhu B.-Y., Bioorg. Med. Chem.
Lett., 14, 1229—1234 (2004).
10) Baskin J. M., Wang Z., Org. Lett., 4, 4423—4425 (2002).
11) Levy L. A., Synth. Commun., 13, 639—648 (1983).
12) Zhu Z., Drach J. C., Townsend L. B., J. Org. Chem., 63, 977—983
(1998).
13) Hoey M. D., Dittmer D. C., J. Org. Chem., 56, 1947—1948 (1991).
14) Jung F., Molinc M., Van Den Elzen R., Durst T., J. Am. Chem. Soc.,
96, 935—936 (1974).
15) Charlton J. L., Durst T., Tetrahedron Lett., 25, 5287—5290 (1984).
16) Durst T., Charlton J. L., Can. J. Chem., 64, 246—249 (1986).
17) Attardo G., Wang W., Kraus J.-L., Belleau B., Tetrahedron Lett., 35,
4743—4746 (1994).
1007
water to quench the reaction, followed by addition of 100 ml DCM.
The mixture was stirred for 1 h. The organic phase was separated and
washed with brine twice. The organic phase was dried and concen-
trated in vacuo. The residue was then treated with 10 ml TFA at 50 °C
for 1 h. The mixture was concentrated in vacuo and directly subjected
to reverse phase preparative HPLC to isolate compound 5 (47 mg, 51%
yield). Compound 29 was prepared using the same procedure. Com-
pounds 6 and 30 were prepared using the same procedure without the
TFA treatment step.
22) Compound 5: MS found for C28H22ClFN4O5S2 as [MꢃH]ꢃ 613.2 with
pattern of 1 chlorine; HR-MS (ESI) m/z calcd for [MꢃH]ꢃ 613.0782,
found: 613.0782; 1H-NMR (CD3OD) d: 8.59 (1H, s), 8.13 (1H, d,
Jꢄ2.0 Hz), 7.97 (1H, s), 7.96—7.92 (2H, m), 7.61 (1H, dd, Jꢄ8.4,
2.0 Hz), 7.48—7.44 (2H, m), 7.39 (1H, td, Jꢄ7.6, 1.6 Hz), 7.17 (1H,
dd, Jꢄ7.2, 1.2 Hz), 7.10 (1H, dd, Jꢄ11.2, 1.6 Hz), 7.01 (dd, Jꢄ8.8,
1.6 Hz), 6.92 (1H, s), 3.00 (3H, s), 2.32 (3H, s) ppm. Compound 6:
MS found for C29H23ClFN3O5S2 as [MꢃH]ꢃ 612.2 with pattern of 1
chlorine; HR-MS (ESI) m/z calcd for [MꢃH]ꢃ 612.0830, found:
612.0830; 1H-NMR (CD3OD) d: 8.71 (1H, s), 8.24 (1H, d, Jꢄ2.0 Hz),
8.12 (1H, dd, Jꢄ7.6, 1.6 Hz), 8.08 (1H, s), 8.04 (1H, d, Jꢄ8.8 Hz),
7.74—7.67 (2H, m), 7.61 (1H, td, Jꢄ8.0, 1.6 Hz), 7.36 (1H, dd, Jꢄ7.2,
1.6 Hz), 7.25 (1H, dd, Jꢄ11.2, 2.0 Hz), 7.13 (1H, dd, Jꢄ8.0, 2.4 Hz),
7.04 (1H, s), 3.12 (3H, s), 2.72 (3H, s), 2.43 (3H, s) ppm. Compound
29: MS found for C28H22ClFN4O5S2 as [MꢃH]ꢃ 613.3 with pattern of
1 chlorine; HR-MS (ESI) m/z calcd for [MꢃH]ꢃ 613.0782, found:
613.0782; 1H-NMR (CD3OD) d: 8.63 (1H, s), 8.07 (1H, d, Jꢄ8.8 Hz),
7.99 (1H, d, Jꢄ2.4 Hz), 7.95 (1H, dd, Jꢄ8.0, 1.2 Hz), 7.92 (1H, s),
7.59 (1H, dd, Jꢄ9.2, 2.4 Hz), 7.48—7.44 (2H, m), 7.39 (1H, td, Jꢄ7.6,
1.6 Hz), 7.17 (1H, dd, Jꢄ7.2, 1.2 Hz), 7.10 (1H, dd, Jꢄ11.6, 2.0 Hz),
7.01 (dd, Jꢄ8.4, 1.2 Hz), 6.93 (1H, s), 3.00 (3H, s), 2.32 (3H, s) ppm.
Compound 30: MS found for C29H23ClFN3O5S2 as [MꢃH]ꢃ 612.3
with pattern of 1 chlorine; HR-MS (ESI) m/z calcd for [MꢃH]ꢃ
18) Lucas H. J., Kennedy E. R., Org. Syn., Coll. Vol. II, 351—352 (1943).
19) Preparation of compound 7: To the solution of compound 26A
(430 mg, 1.0 mmol) in 10 ml DMSO were added sodium methane-
sulfinate (Aldrich #433063, 400 mg, 4.0 mmol), copper(I) trifluo-
romethanesulfonate benzene complex (Aldrich #407240, 250 mg,
0.5 mmol) and N,Nꢀ-dimethylethylenediamine (55 ml, 0.5 mmol). The
mixture was stirred for 3 h in 115 °C bath. After cooling to RT, the
mixture was treated with 10 ml 1N HCl solution for a few minutes. The
mixture was diluted using 250 ml ethyl acetate and washed with brine
three times. The organic phase was dried, concentrated and subjected
to silica flash column using 20% ethyl acetate in hexane to isolate
compound 7 (192 mg, 49% yield). Compound 7B was prepared using
compound 26B by the same procedure.
1
612.0830, found: 612.0842; H-NMR (CD3OD) d: 8.75 (1H, s), 8.17
(1H, d, Jꢄ8.4 Hz), 8.11 (1H, dd, Jꢄ8.0, 1.6 Hz), 8.08 (1H, d,
Jꢄ2.0 Hz), 8.03 (1H, s), 7.72—7.66 (3H, m), 7.60 (1H, td, Jꢄ8.0,
1.6 Hz), 7.35 (1H, dd, Jꢄ7.6, 1.6 Hz), 7.25 (1H, dd, Jꢄ11.6, 2.0 Hz),
7.13 (1H, dd, Jꢄ8.0, 2.4 Hz), 7.04 (1H, s), 3.11 (3H, s), 2.71 (3H, s),
2.43 (3H, s) ppm. The ESI-HR-MS experiments were performed by
HT Laboratories, Inc., 9823 Pacific Heights Blvd, Suite F, San Diego,
CA 92121, U.S.A.
20) Compound 7: MS found for C18H17ClN2O4S as [MꢃH]ꢃ 393.1 with
1
pattern of 1 chlorine; H-NMR (CDCl3) d: 8.58 (1H, s), 7.98 (1H, d,
Jꢄ2.0 Hz), 7.86 (1H, s), 7.80 (1H, d, Jꢄ8.8 Hz), 7.58 (1H, dd, Jꢄ8.8,
2.0 Hz), 6.82 (1H, s), 4.08 (2H, q, Jꢄ7.0 Hz), 2.96 (3H, s), 2.33 (3H,
s), 1.04 (3H, t, Jꢄ7.0 Hz) ppm; NOE: irradiation at 8.58 ppm caused
positive response at 7.98 (d, Jꢄ2.0 Hz) ppm; irradiation at 7.98 ppm
caused positive response at 8.58 (s) ppm. Compound 7B: MS found
23) Basha A., Lipton M., Weinreb S. M., Tetrahedron Lett., 18, 4171—
4172 (1977).
1
for C18H17ClN2O4S as [MꢃH]ꢃ 393.1 with pattern of 1 chlorine; H-
NMR (CDCl3) d: 8.70 (1H, s), 8.01 (1H, d, Jꢄ8.8 Hz), 7.90 (1H, d, 24) Quan M. L., Liauw A. Y., Ellis C. D., Pruitt J. R., Carini D. J., Bostrom
Jꢄ1.6 Hz), 7.84 (1H, s), 7.63 (1H, dd, Jꢄ8.8, 1.6 Hz), 6.87 (1H, s),
3.01 (3H, s), 4.12 (2H, q, Jꢄ7.0 Hz), 2.38 (3H, s), 1.10 (3H, t,
Jꢄ7.0 Hz) ppm; NOE: irradiation at 8.70 ppm caused positive re-
sponse at 8.01 (d, Jꢄ8.8 Hz) ppm.
L. L., Huang P. P., Harrison K., Knabb R. M., Thoolen M. J., Wong P.
C., Wexler R. R., J. Med. Chem., 42, 2752—2759 (1999).
25) The fXa IC50 values were determined by the method described in:
Sinha U., Ku P., Malinowski J., Zhu B. Y., Scarborough R. M., Mar-
lowe C. K., Wong P. W., Lin P. H., Hollenbach S. J., Eur. J.
Pharmacol., 395, 51—59 (2000).
26) The fXa Ki values were determined by the method described in: Betz
A., Wong P. W., Sinha U., Biochemistry, 38, 14582—14591 (1999).
27) For description of our human plasma-based thrombin generation assay,
see: Sinha U., Lin P. H., Edwards S. T., Wong P. W., Zhu B., Scarbor-
ough R. M., Su T., Jia Z. J., Song Y., Zhang P., Clizbe L., Park G.,
Reed A., Hollenbach S. J., Malinowski J., Arfsten A. E., Arterioscler.
Thromb. Vasc. Biol., 23, 1098—1104 (2003). This is an in vitro func-
tional assay run in human plasma to measure the overall anticoagulant
activity of a fXa inhibitor. The assay result (2ꢂTG) is expressed by the
required concentration of this inhibitor to extend the time for reaching
the maximum thrombin generation point by one fold. The targeted
2ꢂTG values for our fXa inhibitors were 1.0 mM or less.
21) Preparation of compound 5: Ethyl ester 7 (60 mg, 0.15 mmol) and ani-
line 27 (100 mg, 0.31 mmol) were dissolved in 10 ml dichloromethane
(DCM). To this solution was added trimethylaluminum (2.0 M solution
in hexane, Aldrich #268569, 0.45 ml, 0.90 mmol), and the mixture was
stirred for 1 d at RT. To it was carefully added 10 ml of a saturated so-
lution of Rochelle’s salt (potassium sodium tartrate tetrahydrate) in