Design and synthesis of peptidomimetic factor VIIa inhibitors

Article abstract of DOI:10.1248/cpb.58.38

Selective factor VIIa-tissue factor complex (FVIIa/TF) inhibition is regarded as a promising target for developing new anticoagulant drugs. In previous reports, we described a S3 subsite found in the X-ray crystal structure of compound 2 that bound to FVIIa/soluble tissue factor (sTF). Based on the X-ray crystal structure information and with the aim of improving the inhibition activity for FVIIa/TF and selectivity against other serine proteases, we synthesized derivatives by introducing substituents at position 5 of the indole ring of compound 2. Among them, compound 16 showed high selectivity against other serine proteases. Contrary to our expectations, compound 16 did not occupy the S3-subsite; X-ray structure analysis revealed that compound 16 improved selectivity by forming hydrogen bonds with Gln217, Thr99 and Asn100.

Full text of DOI:10.1248/cpb.58.38

38
Chem. Pharm. Bull. 58(1) 38—44 (2010)
Vol. 58, No. 1
Design and Synthesis of Peptidomimetic Factor VIIa Inhibitors
Takuya SHIRAISHI,* Shojiro KADONO, Masayuki HARAMURA, Hirofumi KODAMA, Yoshiyuki ONO,
Hitoshi IIKURA, Tohru ESAKI, Takaki KOGA, Kunihiro HATTORI, Yoshiaki WATANABE,
Akihisa SAKAMOTO, Kazutaka YOSHIHASHI, Takehisa KITAZAWA, Keiko ESAKI,
Masateru OHTA, Haruhiko SATO, and Toshiro KOZONO
Fuji Gotemba Research Laboratories, Chugai Pharmaceutical Co., Ltd.; 1–135 Komakado, Gotemba, Shizuoka 412–8513,
Japan. Received July 25, 2009; accepted October 10, 2009; published online October 15, 2009
Selective factor VIIa-tissue factor complex (FVIIa/TF) inhibition is regarded as a promising target for de-
veloping new anticoagulant drugs. In previous reports, we described a S3 subsite found in the X-ray crystal
structure of compound 2 that bound to FVIIa/soluble tissue factor (sTF). Based on the X-ray crystal structure
information and with the aim of improving the inhibition activity for FVIIa/TF and selectivity against other ser-
ine proteases, we synthesized derivatives by introducing substituents at position 5 of the indole ring of compound
2. Among them, compound 16 showed high selectivity against other serine proteases. Contrary to our expecta-
tions, compound 16 did not occupy the S3-subsite; X-ray structure analysis revealed that compound 16 improved
selectivity by forming hydrogen bonds with Gln217, Thr99 and Asn100.
Key words factor VIIa; X-ray; 2ꢀactivated partial thromboplastin time/2ꢀprothrombin time; thromboembolic disorder; pep-
tidomimetic inhibitor
Coagulation factor VIIa (FVIIa; EC 3.4.21.21) is a serine site of FVIIa/sTF.18—24) Surprisingly, in the case of com-
protease enzyme which plays an important role in the blood pound 2, there appears to be a hole extendable to the space
coagulation cascade comprising extrinsic and intrinsic coag- near the S3 site (Fig. 2) not observed upon binding to D-Phe-
ulation pathways. FVIIa in complex with tissue factor (TF) Phe-Arg-chloromethyl-ketone.19,21,26)
(FVIIa/TF) initiates the extrinsic coagulation pathway. The
This condition is caused by the change in the Gln217 side
extrinsic pathway is triggered by formation of the complex, chain when the indole ring of compound 2 binds to a specific
which subsequently activates factor IX to IXa and factor X to position in the S3 site. A channel was found near position 5
Xa, which in turn activates factor X to Xa and prothrombin of the indole ring in P3 and, through this channel, it might be
to thrombin, respectively.1) Thrombin cleaves fibrinogen to possible to access the large cavity consisting of Cys168,
fibrin, which forms blood clots with activated platelets. Inap- Ile176, Cys182, His224, Phe225, Gly226, and Val227 (the
propriate thrombus formation in blood vessels causes cardio- S3 subsite). This large cavity exists only in FVIIa/TF be-
vascular diseases (myocardial infarction, stroke, pulmonary cause, among the coagulation serine proteases, only FVIIa
embolism, etc.), which are the most common causes of mor- has a large 170-loop. Therefore, the introduction of the ap-
tality in industrialized countries.2) Recent studies on blood propriate substituent at position 5 of the indole ring of com-
coagulation have suggested that selective inhibition of extrin- pound 2 was expected to lead to new interactions with the S3
sic coagulation provides effective anticoagulation and low subsite through this channel and to improve the binding
risk of bleeding compared with other antithrombotic mecha- affinity and selectivity for FVIIa/TF.
nisms.3—8) Therefore, specific inhibition of FVIIa/TF and
thus extrinsic coagulation is expected to be a promising tar- pounds is shown in Chart 1. Intermediate 9 was prepared by
Chemistry The typical synthetic route for our com-
get in the development of new anticoagulant drugs.9—17)
Recently, we reported a peptidomimetic FVIIa inhibitor
(2) with a Gln moiety as the P2 from optimization of throm-
bin inhibitor 1 at the substituents to the S2 and S3 pocket
(Fig. 1).18—25) The X-ray structure of 2 suggested that the
glutamine side chain forms potent hydrogen bonds with
Asp60, Tyr94, and Thr98 in the hydrophilic pocket of the S2
starting with condensation of 4-cyanobenzylamine (3) and
Fig. 2. Crystal Structure of Compound 2 Bound to FVIIa/sTF (PDB
1WUN)
The figure on the left shows the molecular surface around the S4 site. Near position 5
of the indole moiety at P3 of compound 2, a tunnel through which it is possible to con-
nect an internal large cavity form S4 site was found. The figure on the right shows the
internal cavity in which two fixed water molecules were found.
Fig. 1. Structure of Compounds 1 and 2
© 2010 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed. e-mail: shiraishitky@chugai-pharm.co.jp

January 2010
39
Chart 1. Synthesis of Compound 16
Boc-L-Gln under 1-ethyl-3-(3-dimethylaminopropyl) car- of the indole ring has hydrophobic residues near the gate.
bodiimide (EDC)/1-hydroxybenzotriazole (HOBt) condi- This is supported by the fact that the introduction of an OH
tions. After cleavage of the Boc-protecting group and the moiety to position 5 of the indole ring of compound 2 re-
condensation amine (5) with Boc-D-Trp(5-OH) under sulted in a decrease in the FVIIa/TF binding affinity; how-
EDC/HOBt conditions, compound 6 was reacted with 3-sub- ever, the introduction of an OMe moiety to the same position
stituted benzyl bromide (7). After cleavage of the Boc-pro- resulted in regaining the FVIIa/TF binding affinity to the
tecting group, the reaction of the amine with ethanesulfonyl same level exhibited by compound 2.
chloride led to intermediate 9. The nitrile was converted to
Based on the crystal structure of FVIIa/sTF bound to com-
the amidine and hydrolysis of the ester led to the desired pound 2, which has the propoxy group at position 5 of the in-
product 16. The other compounds were prepared following dole ring in P3, we synthesized compound 14. Compound 14
the same procedure.
showed improved coagulation over other serine proteases. We
tested compound 14 using standard clotting assays including
a prothrombin time (PT) assay and an activated partial
Results and Discussion
Based on the crystal structure, the NH at position 1 of the thromboplastin time (APTT) assay to confirm the improved
indole ring of D-tryptophan is exposed to the solvent and specificity for FVIIa/TF inhibition. Theoretically, specific in-
does not appear to contribute to the binding of FVIIa/TF. hibition of FVIIa/TF, which blocks only extrinsic coagula-
This is supported by the fact that the introduction of a Me tion, should result in prolongation of only PT without affect-
group to the nitrogen of the indole ring of compound 2 re- ing APTT, thus making the concentration ratio for twofold
sults in conservation of the affinity for FVIIa/TF, as shown in prolongations of APTT and PT (2ꢀAPTT/2ꢀPT) infinitely
Table 1. However, thrombin and FXa have hydrophobic S3 large. The measured 2ꢀAPTT/2ꢀPT ratio of compound 14
pockets so the increased hydrophobicity of the indole ring by was 15.9, higher than that of compound 2 (6.1). To confirm
the introduction of a Me group at position 1 of the indole the binding mode of compound 14 to FVIIa/TF, the crystal
ring of compound 2 reduced selectivities against thrombin structure bound to FVIIa/sTF was solved by X-ray crystal-
and factor Xa.
In the crystal structure of FVIIa/soluble tissue factor (sTF) the S3 subsite was not found.
bound to compound 2, the channel observed near position 5 The binding mode of compound 14 is basically the same
lography (Fig. 3). Unfortunately, the expected binding with

40
Vol. 58, No. 1
Table 1. Inhibition of FVIIa/TF and Related Serine Proteases from Compounds 2, 11—16
IC50 (nM)
FIXa
2ꢀAPTT/
2ꢀPT
Compound
R
FVIIa
62
Thrombin
5900
FXa
FXIa
270
FXIIa
79000
aPC
2
57000
ꢁ100000
2900
6.1
11
12
13
69
140
65
1400
13000
8300
3700
ꢁ100000
ꢁ100000
ꢁ100000
NTa)
210
1600
2600
38000
NTa)
2100
3600
3500
2.0
8.6
NTa)
NTa)
14.0
14
15
56
14000
19000
ꢁ100000
ꢁ100000
ꢁ100000
3100
NTa)
ꢁ100000
4000
7400
15.9
NTa)
204
NTa)
NTa)
16
39
18000
ꢁ100000
NTa)
4700
NTa)
4800
28.1
a) Not tested.
Fig. 3. Crystal Structure of Compound 14 Bound to FVIIa/sTF (PDB
1WV7)
Fig. 4. Crystal Structure of Compound 16 Bound to FVIIa/sTF (PDB
2ZZU)
as that of compound 2. However, the 5-propoxy-indole moi- However, a new hydrogen bond between the NH of the indole
ety in P3 of compound 14 is accommodated in the S3 site in ring and Gln217 was observed. Among coagulation serine
a far different binding mode than for the non-substituted in- proteases, only Gln217 of FVIIa has a uniquely bent confor-
dole moiety in P3 of compound 2. Surprisingly, the 5- mation, which makes possible a hydrogen bond with the NH
propoxy-indole moiety flips and the propoxy moiety extends of the indole.
to the solvent region. As a result of this conformation, the
The abovementioned occupation of the S3 subsite by sim-
hydrophobic interactions with Gln217 and 170-loop are lost. ple hydrophobic substituents was not successful. In another

January 2010
41
drochloride (EDC HCl) (1.7 g, 8.9 mmol) were added and stirred at room
temperature under a nitrogen stream. After 12 h, water was added to the re-
action mixture, which was then extracted with ethyl acetate. The ethyl ac-
etate layer was washed sequentially with 10% aqueous citric acid, saturated
aqueous sodium bicarbonate and saturated brine, and then dried over anhy-
drous magnesium sulfate. The magnesium sulfate was filtered off and the fil-
trate was concentrated under reduced pressure to give 4 (2.9 g, quant.) as a
approach to the forming of new interactions with the S3 sub-
site, we focused on water molecules. In the crystal structures
of FVIIa/sTF bound to our compounds, two highly conserved
water molecules were observed in the S3 subsite. One
formed hydrogen bonds with Ser170B and Phe225, and the
other formed hydrogen bonds with Gln217 and Phe225.
Compounds 15 and 16 were designed by replacing these
water molecules.
As shown in Table 1, compound 15 showed less activity
than compound 2. However, compound 16 showed improved
activity over compound 2 with a slight amelioration of selec-
tivities against other serine proteases and an improved
2ꢀAPTT/2ꢀPT ratio (28.1) compared with compound 2. We
concluded that compound 16 achieves higher selectivity for
extrinsic coagulation inhibition.
To confirm the binding mode of compound 16 to
FVIIa/TF, the crystal structure bound to FVIIa/sTF was
solved by X-ray crystallography (Fig. 4). In this structure, the
expected binding with the S3 subsite was not observed. The
binding of compound 16 to FVIIa/sTF was almost the same
as that of compound 14. The NH of the indole in the P3 moi-
ety formed a hydrogen bond with Gln217. Additionally, the
benzyl carboxylate moiety stacks on Pro170I. The carboxy-
late formed hydrogen bonds with the Thr99 side chain OH
and the main chain NH atoms. And this carboxylate formed a
hydrogen bond with the Asn100 main chain NH atom
through a water molecule. Of the other coagulation serine
proteases, Thr99 is not conserved and FVIIa and aPC have
Thr in this sequence position. However, the main chain con-
formation near this residue was completely different between
FVIIa and aPC. Only FVIIa can form these hydrogen bonds
with the carboxylate of the ligand. Hence, these interactions
contribute to the improvement of selectivity to FVIIa.
1
colorless solid. H-NMR (400 MHz, CD3OD) d: 1.45 (9H, s), 1.83—2.10
(2H, m), 2.31 (2H, d, Jꢂ7.6 Hz), 4.06 (1H, dd, Jꢂ4.9, 8.8 Hz), 4.46 (2H, dd,
Jꢂ15.6, 20.0 Hz), 7.47 (2H, d, Jꢂ8.3 Hz), 7.67 (2H, d, Jꢂ8.3 Hz); ESIꢃ
383 [MꢃNa]ꢃ.
(S)-2-Amino-pentanedioic Acid 5-Amide 1-(4-Cyano-benzylamide) (5)
To 4 (2.9 g, 8.1 mmol), a solution of 4 N hydrogen chloride/ethyl acetate
(20 ml) was added and stirred at room temperature under a nitrogen stream.
After 1 h, the solvent was distilled off under reduced pressure and the
residue was applied to column chromatography (Fuji Silysia NH-DM-1020;
mobile phase: dichloromethane : methanolꢂ1 : 1) to give 5 (2.1 g, quant.) as
a colorless solid. 1H-NMR (400 MHz, CD3OD) d: 1.78—2.03 (2H, m), 2.29
(2H, t, Jꢂ7.8 Hz), 3.38 (1H, dd, Jꢂ5.9, 7.8 Hz), 4.47 (2H, s), 7.48 (2H, d,
Jꢂ8.3 Hz), 7.69 (2H, d, Jꢂ8.3 Hz); ESIꢄ 259 (Mꢃꢄ1).
[(R)-1-[(S)-3-Carbamoyl-1-(4-cyano-benzylcarbamoyl)-propylcar-
bamoyl]-2-(5-hydroxy-1H-indol-3-yl)-ethyl]-carbamic Acid tert-Butyl
Ester (6) To a stirred suspension of 5 (510 mg, 1.96 mmol) and tert-bu-
toxycarbonyl-D-(5-hydroxy)tryptophan (624 mg, 1.96 mmol) in THF (50 ml)
was added HOBt (345 mg, 2.55 mmol), EDC HCl (488 mg, 2.55 mmol) and
triethylamine (480 mg, 4.71 mmol) and stirred at room temperature under a
nitrogen stream. After 15 h, water was added to the reaction mixture, which
was then extracted with ethyl acetate. The ethyl acetate layer was washed se-
quentially with saturated aqueous ammonium chloride, saturated aqueous
sodium bicarbonate and saturated brine, and then dried over sodium sulfate.
The sodium sulfate was filtered off and the filtrate was concentrated under
reduced pressure. The residue was applied to flash column chromatography
(Merck silica gel 60; mobile phase: dichloromethane : methanolꢂ10 : 0 to
9 : 1) to give 6 (882 mg, 80%). 1H-NMR (300 MHz, DMSO-d6) d: 1.25 (9H,
s), 1.60—2.03 (4H, m), 2.70—3.02 (2H, m), 4.08—4.45 (4H, m), 6.58 (1H,
dd, Jꢂ1.8, 8.3 Hz), 6.74 (1H, br s), 6.88 (1H, d, Jꢂ5.3 Hz), 6.98 (1H, d,
Jꢂ5.9 Hz), 7.06 (1H, d, Jꢂ1.8 Hz), 7.10 (1H, d, Jꢂ8.3 Hz), 7.42 (2H, d,
Jꢂ7.3 Hz), 7.76 (2H, d, Jꢂ7.3 Hz); ESIꢃ 563 (Mꢃꢃ1).
3-(3-{(R)-2-tert-Butoxycarbonylamino-2-[(S)-3-carbamoyl-1-(4-cyano-
benzylcarbamoyl)-propylcarbamoyl]-ethyl}-1H-indol-5-yloxymethyl)-
benzoic Acid Methyl Ester (8) To a solution of 6 (327 mg, 0.58 mmol) in
acetone (4 ml), 3-(methoxycarbonyl)benzylbromide (7) (267 mg, 1.2 mmol)
and cesium carbonate (378 mg, 1.2 mmol) were added and stirred at reflux
under a nitrogen stream. After 4 h, the reaction mixture was filtered and the
filtrate was concentrated under reduced pressure. The residue was applied
to flash column chromatography (Merck silica gel 60; mobile phase:
dichloromethane : methanolꢂ10 : 1) to give 8 (347 mg, 84%). 1H-NMR
(270 MHz, CD3OD) d: 1.28 (9H, s), 1.50—2.05 (4H, m), 3.03—3.19 (2H,
m), 3.91 (3H, s), 4.01—4.24 (2H, m), 4.43 (2H, s), 5.19 (2H, s), 6.87 (1H,
dd, Jꢂ2.2, 8.8 Hz), 7.11 (1H, s), 7.18 (1H, d, Jꢂ2.3 Hz), 7.28 (1H, d,
Jꢂ8.9 Hz), 7.41 (2H, d, Jꢂ8.3 Hz), 7.50 (1H, t, Jꢂ7.8 Hz), 7.65 (2H, d,
Jꢂ8.3 Hz), 7.75 (1H, d, Jꢂ7.5 Hz), 7.96 (1H, d, Jꢂ7.5 Hz), 8.16 (1H, s);
ESIꢃ 711 (Mꢃꢃ1).
Conclusion
We have designed and synthesized peptidomimetic FVIIa
inhibitors based on the X-ray crystallographic structure of
compound 2. Among these compounds, compound 16
showed the highest selectivity and a high 2ꢀAPTT/2ꢀPT
ratio (28.1). X-Ray structure analysis revealed that com-
pound 16 exhibited improved selectivity by forming un-
expected hydrogen bonds with Gln217, Thr99 and Asn100.
This structural information will be of benefit to the develop-
ment of FVIIa-specific inhibitors.
3-(3-{(R)-2-[(S)-3-Carbamoyl-1-(4-cyano-benzylcarbamoyl)-propyl-
carbamoyl]-2-ethanesulfonylamino-ethyl}-1H-indol-5-yloxymethyl)-ben-
Experimental
Chemistry In general, reagents and solvents were used as purchased zoic Acid Methyl Ester (9) To a solution of 8 (347 mg, 0.5 mmol) in
without further purification. Column chromatography was carried out on
dichloromethane (10 ml), trifluoroacetic acid (10 ml) was added and stirred
Merck silica gel 60 (230—400 mesh) if not otherwise specified. H-NMR at room temperature under a nitrogen stream. After 1 h, the solvent was dis-
spectra were recorded on JEOL EX-270, JEOL ECP-400 or a Mercury
tilled off under reduced pressure. The residue was applied to column chro-
1
300 MHz instrument in CDCl3, DMSO-d6 or CD3OD solutions. Low resolu- matography (Fuji Silysia NH-DM-1020; mobile phase: dichloromethane :
tion mass spectra were determined on an LC/photodiode array (PDA)/MS methanolꢂ1 : 1) to give 3-(3-{(R)-2-amino-2-[(S)-3-carbamoyl-1-(4-cyano-
(HP 1100/TSP UV 6000/LCQ Classic, LCMS) system using Cadenza CD-
benzylcarbamoyl)-propylcarbamoyl]-ethyl}-1H-indol-5-yloxymethyl)-ben-
C18 column (3.0 mm i.d.ꢀ20 mm) at 35 °C. The accurate mass of the target zoic acid methyl ester (277 mg, 93%).
compound was measured using an Agilent 1100 series (Agilent Technolo-
To a solution of 3-(3-{(R)-2-amino-2-[(S)-3-carbamoyl-1-(4-cyano-ben-
gies, Inc. Santa Clara, CA, U.S.A.) and QSTAR XL system (Applied zylcarbamoyl)-propylcarbamoyl]-ethyl}-1H-indol-5-yloxymethyl)-benzoic
Biosystems/MDS Analytical Technologies, Toronto, Canada) in the electro- acid methyl ester (0.45 mmol) in DMF (10 ml), triethylamine (137 mg,
spray ionization (ESI) positive ion mode. The sample solution was sepa- 1.4 mmol) and ethanesulfonyl chloride (174 mg, 1.4 mmol) were added
rated using an Inertsil octadecyl silica (ODS)-3.3 mm column (4.0 mm and stirred at room temperature under a nitrogen stream. After 2 h, the
i.d.ꢀ33 mm; GL Science) at 30 °C.
solvent was distilled off under reduced pressure and the residue was applied
[(S)-3-Carbamoyl-1-(4-cyano-benzylcarbamoyl)-propyl]-carbamic to flash column chromatography (Merck silica gel 60; mobile phase:
1
Acid tert-Butyl Ester (4) To a solution of 4-aminomethyl-benzonitrile dichloromethane : methanolꢂ8 : 1) to give 9 (158 mg, 50%). H-NMR (270
(1.6 g, 12.2 mmol) in N,N-dimethylformamide (DMF) (20 ml), tert-butoxy-
carbonyl-L-glutamine (2.0 g, 8.1 mmol), 1-hydroxybenzotriazole (HOBt)
MHz, CD3OD) d: 0.95 (3H, t, Jꢂ7.3 Hz), 1.60—2.05 (4H, m), 2.59—2.84
(2H, m), 2.97—3.27 (2H, m), 3.90 (3H, s), 4.09—4.20 (2H, m), 4.40 (2H, d,
(1.4 g, 8.9 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hy- Jꢂ2.5 Hz), 5.18 (2H, s), 6.88 (1H, dd, Jꢂ2.3, 8.7 Hz), 7.14 (1H, s), 7.22

42
Vol. 58, No. 1
(S)-2-[(R)-2-Ethanesulfonylamino-3-(5-methoxy-1H-indol-3-yl)-propi-
onylamino]-pentanedioic Acid 5-Amide 1-(4-Carbamimidoyl-benzyl-
amide) Trifluoroacetate (13) Compound 13 was synthesized from (S)-2-
[(R)-2-ethanesulfonylamino-3-(5-methoxy-1H-indol-3-yl)-propionylamino]-
pentanedioic acid 5-amide 1-(4-cyano-benzylamide) according the proce-
(1H, d, Jꢂ2.3 Hz), 7.26 (1H, d, Jꢂ8.7 Hz), 7.43 (2H, d, Jꢂ8.7 Hz), 7.48
(1H, t, Jꢂ7.9 Hz), 7.64 (2H, d, Jꢂ8.6 Hz), 7.73 (1H, d, Jꢂ8.1 Hz), 7.95 (1H,
d, Jꢂ7.9 Hz), 8.15 (1H, s); ESIꢃ 703 (Mꢃꢃ1).
3-(3-{(R)-2-[(S)-1-(4-Carbamimidoyl-benzylcarbamoyl)-3-carbamoyl-
propylcarbamoyl]-2-ethanesulfonylamino-ethyl}-1H-indol-5-
yloxymethyl)-benzoic Acid Methyl Ester (10) A solution of 9 (158 mg,
0.22 mmol) in pyridine (10 ml) and triethylamine (2 ml) was bubbled with
hydrogen sulfide gas. After bubbling for 30 min, the solution was allowed to
stand. After 12 h, water/ethyl acetate was added to the reaction mixture and
the aqueous layer was adjusted to pH 4 with 2 N aqueous hydrogen chloride,
followed by extraction. The organic layer was washed with saturated brine
and then dried over anhydrous magnesium sulfate. The magnesium sulfate
was filtered off and the filtrate was concentrated under reduced pressure.
The residue was dissolved in acetone (10 ml), to which methyl iodide
(312 mg, 2.2 mmol) was then added and stirred at 50 °C under a nitrogen
stream. After 1 h, the reaction mixture was concentrated under reduced pres-
sure.
1
dure described for 11 (65%, 68 mg). H-NMR (270 MHz, CD3OD) d: 1.04
(3H, t, Jꢂ7.3 Hz), 1.60—2.08 (4H, m), 2.72—2.92 (2H, m), 3.03—3.31
(2H, m), 3.87 (3H, s), 4.12—4.22 (2H, m), 4.38—4.55 (2H, m), 6.80 (1H,
dd, Jꢂ2.4, 8.8 Hz), 7.13 (1H, d, Jꢂ2.4 Hz), 7.17 (1H, s), 7.26 (1H, d,
Jꢂ8.9 Hz), 7.53 (2H, d, Jꢂ8.6 Hz), 7.76 (2H, d, Jꢂ8.6 Hz); HR-MS (ESIꢃ)
Calcd for C27H36N7O6S: 586.2442 (Mꢃꢃ1). Found: 586.2419.
(S)-2-[(R)-2-Ethanesulfonylamino-3-(5-propoxy-1H-indol-3-yl)-propi-
onylamino]-pentanedioic Acid 5-Amide 1-(4-Carbamimidoyl-benzyl-
amide) Trifluoroacetate (14) Compound 14 was synthesized from (S)-2-
[(R)-2-ethanesulfonylamino-3-(5-propoxy-1H-indol-3-yl)-propionylamino]-
pentanedioic acid 5-amide 1-(4-cyano-benzylamide) according the proce-
1
dure described for 11 (83%, 98 mg). H-NMR (270 MHz, CD3OD) d: 1.04
(3H, t, Jꢂ7.3 Hz), 1.10 (3H, t, Jꢂ7.4 Hz), 1.63—2.09 (6H, m), 2.69—2.92
(2H, m), 3.02—3.31 (2H, m), 4.01 (2H, t, Jꢂ6.6 Hz), 4.14—4.24 (2H, m),
4.39—4.57 (2H, m), 6.82 (1H, dd, Jꢂ2.3, 8.9 Hz), 7.14 (1H, d, Jꢂ2.3 Hz),
7.17 (1H, s), 7.27 (1H, d, Jꢂ8.9 Hz), 7.53 (2H, d, Jꢂ8.6 Hz), 7.76 (2H, d,
Jꢂ8.2 Hz); HR-MS (ESIꢃ) Calcd for C29H40N7O6S: 614.2755 (Mꢃꢃ1).
Found: 614.2762.
5-(3-{(R)-2-[(S)-1-(4-Carbamimidoyl-benzylcarbamoyl)-3-carbamoyl-
propylcarbamoyl]-2-ethanesulfonylamino-ethyl}-1H-indol-5-yloxy)-pen-
tanoic Acid Trifluoroacetate (15) Compound 15 was synthesized from 5-
(3-{(R)-2-[(S)-3-carbamoyl-1-(4-cyano-benzylcarbamoyl)-propylcar-
The residue was dissolved again in methanol (10 ml), followed by the ad-
dition of ammonium acetate (170 mg, 2.2 mmol) and heating to reflux under
a nitrogen stream. After 4 h, the solvent was distilled off under reduced pres-
sure and the residue was applied to column chromatography (Fuji Silysia
NH-DM-1020; mobile phase: dichloromethane : methanolꢂ4 : 1, 2 : 1) to
give 10 (124 mg, 78%). 1H-NMR (270 MHz, CD3OD) d: 0.94 (3H, t,
Jꢂ7.4 Hz), 1.63—2.06 (4H, m), 2.54—2.79 (2H, m), 2.96—3.27 (2H, m),
3.89 (3H, s), 4.08—4.21 (2H, m), 4.40 (2H, s), 5.18 (2H, s), 6.87 (1H, dd,
Jꢂ2.5, 8.7 Hz), 7.15 (1H, s), 7.23 (1H, s), 7.25 (1H, d, Jꢂ11.7 Hz), 7.40
(2H, d, Jꢂ8.4 Hz), 7.48 (1H, t, Jꢂ7.7 Hz), 7.68 (2H, d, Jꢂ8.4 Hz), 7.72 (1H,
d, Jꢂ7.7 Hz), 7.94 (1H, d, Jꢂ7.7 Hz), 8.14 (1H, s); ESIꢃ 720 (Mꢃꢃ1).
3-(3-{(R)-2-[(S)-1-(4-Carbamimidoyl-benzylcarbamoyl)-3-carbamoyl-
propylcarbamoyl]-2-ethanesulfonylamino-ethyl}-1H-indol-5-
yloxymethyl)-benzoic Acid Trifluoroacetate (16) To a solution of 10
(124 mg, 0.17 mmol) in ethanol (3 ml), 1 N aqueous sodium hydroxide (3 ml)
was added and stirred at room temperature. After 2 h, the reaction mixture
was adjusted to pH 6 with 1 N aqueous hydrogen chloride and then concen-
trated under reduced pressure. The residue was applied to preparative HPLC
(YMC-pack ODS: gradient of 95% A/B to 25% A/B over 10 min, Aꢂ0.1%
trifluoroacetic acid (TFA)-H2O, Bꢂ0.1% TFA-CH3CN) to give 16 (85 mg,
bamoyl]-2-ethanesulfonylamino-ethyl}-1H-indol-5-yloxy)-pentanoic
acid
ethyl ester according the procedure described for 11 and 16 (51%, 20 mg).
1H-NMR (300 MHz, CD3OD) d: 1.02 (3H, t, Jꢂ7.4 Hz), 1.58—2.06 (8H,
m), 2.41 (3H, dd, Jꢂ3.6, 6.8 Hz), 2.68—3.41 (4H, m), 4.02—4.10 (2H, m),
4.11—4.21 (2H, m), 4.37—4.54 (2H, m), 6.80 (1H, dd, Jꢂ2.3, 8.8 Hz), 7.13
(1H, d, Jꢂ2.2 Hz), 7.15 (1H, s), 7.24 (1H, d, Jꢂ8.8 Hz), 7.52 (2H, d,
Jꢂ8.4 Hz), 7.74 (1H, d, Jꢂ8.4 Hz), 8.31—8.42 (2H, m); HR-MS (ESIꢃ)
Calcd for C31H42N7O8S: 672.2810 (Mꢃꢃ1). Found: 672.2806.
Crystal Structures. Cloning, Expression, Purification, and Crystal-
lization Purified human FVIIa/sTF and crystals of human FVIIa/sTF in
complex with compounds 2, 14 and 16 were prepared as described previ-
ously.21,24,27)
Data Collection After soaking in a cryoprotectant solution of 10% PEG
5000, 100 mM cacodylate buffer, pH 5.0, 100 mM NaCl, and 5 mM CaCl2,
30% (v/v) glycerol, the crystals were frozen at 100 K in a nitrogen gas
stream. X-Ray diffraction data on the FVIIa/sTF crystals in complex with
compounds 2, 14 and 16 were collected on an R-axis IV (Rigaku) mounted
on a copper rotating-anode X-ray generator ultraX 18 (Rigaku) equipped
with a Confocal Mirror (Osmic). The data were processed using Denzo and
Scalepack software.28)
Structure Determination and Refinement The crystals of FVIIa/sTF
in complex with compound 16 were prepared as described previously.24,27)
X-Ray diffraction data of this crystal was collected on an R-axis IV
(Rigaku) mounted on a copper rotating-anode X-ray generator ultraX 18
(Rigaku) equipped with Confocal Mirror (Osmic) at 100 K. The data was
processed using Denzo and Scalepack.
The model phases of FVIIa/sTF in complex with compounds 16 were im-
proved by rigid body refinement with CNX (Accelrys), using the previously
published structure of FVIIa/sTF in complex with D-Phe-Phe-Arg
chloromethyl ketone (PDB code 1DAN)29) as a starting model. The inhibitor
molecules were identified using the difference Fourier method. A model of
the protein and inhibitor was built with Quanta (Accelrys) and the structure
was refined with CNX (Accelrys).
Biology. Human Factor VIIa (FVIIa) Inhibition Activity A 10%
(v/v) DMSO solution of each test compound (20 ml) was mixed with 40 ml
Thromborel S (50 mg/ml; Dade Behring), 20 ml Spectrozyme® FVIIa
(CH3SO2-D-CHA-But-Arg-pNA·AcOH) (5 mM; American Diagnostica
Inc.), 20 ml MgCl2 (10 mM), 60 ml ethylene glycol, 20 ml buffer (500 mM
Tris–HCl, pH 7.5, 1500 mM NaCl, 50 mM CaCl2) and 40 ml distilled water in
a 96-well assay plate. The reaction was initiated by the addition of 20 ml
human FIXa solution (20 nM; Enzyme Research Laboratories) and then the
absorbance at 405 nm was monitored to measure the initial velocity of the
reaction. Percent inhibition at each concentration was calculated from the
experimental and control samples. IC50 values were calculated from the con-
centration–reaction activity curve of each test compound.
1
61%). H-NMR (270 MHz, CD3OD) d: 0.97 (3H, t, Jꢂ7.3 Hz), 1.59—2.06
(4H, m), 2.60—2.86 (2H, m), 2.98—3.28 (2H, m), 4.09—4.20 (2H, m),
4.35—4.54 (2H, m), 5.19 (2H, s), 6.89 (1H, dd, Jꢂ2.3, 8.7 Hz), 7.15 (1H, s),
7.23 (1H, d, Jꢂ2.3 Hz), 7.27 (1H, d, Jꢂ8.9 Hz), 7.50 (3H, d, Jꢂ8.4 Hz),
7.73 (3H, d, Jꢂ8.4 Hz), 7.97 (1H, d, Jꢂ7.9 Hz), 8.16 (1H, s); HR-MS
(ESIꢃ) Calcd for C34H40N7O8S: 706.2653 (Mꢃꢃ1). Found: 706.2664.
Nitrile intermediates were prepared using the same methods described
above for the synthesis of 9.
(S)-2-[(R)-2-Ethanesulfonylamino-3-(1-methyl-1H-indol-3-yl)-propi-
onylamino]-pentanedioic Acid 5-Amide 1-(4-Carbamimidoyl-benzyl-
amide) (11) (S)-2-[(R)-2-Ethanesulfonylamino-3-(1-methyl-1H-indol-3-
yl)-propionylamino]-pentanedioic acid 5-amide 1-(4-cyano-benzylamide)
(77 mg, 0.14 mmol) was dissolved in saturated hydrogen chloride-ethanol
solution (10 ml) and allowed to stand at room temperature for 23 h. After the
solvent was removed under reduced pressure, the residue was dissolved in
ethanol (2 ml) and further dissolved in ammonium acetate (190 mg,
2.4 mmol) and saturated ammonia-ethanol solution (1 ml), followed by heat-
ing at reflux. After 2 h, the solvent was distilled off under reduced pressure
and the residue was applied to column chromatography (Fuji Silysia NH-
DM-1020; mobile phase: dichloromethane : methanolꢂ3 : 1, 1 : 1) to give 11
(23 mg, 29%). 1H-NMR (270 MHz, CD3OD) d: 1.00 (3H, t, Jꢂ7.4 Hz),
1.42—2.03 (4H, m), 2.61—2.92 (2H, m), 2.98—3.28 (2H, m), 3.74 (3H, s),
4.13—4.21 (2H, m), 4.41 (2H, s), 7.05 (1H, d, Jꢂ7.9 Hz), 7.08 (1H, s), 7.17
(1H, t, Jꢂ8.3 Hz), 7.32 (1H, d, Jꢂ7.9 Hz), 7.42 (2H, d, Jꢂ8.3 Hz), 7.61 (1H,
d, Jꢂ7.8 Hz), 7.68 (2H, d, Jꢂ8.3 Hz); HR-MS (ESIꢃ) Calcd for
C27H36N7O5S: 570.2493 (Mꢃꢃ1). Found: 570.2513.
(S)-2-[(R)-2-Ethanesulfonylamino-3-(5-hydroxy-1H-indol-3-yl)-propi-
onylamino]-pentanedioic Acid 5-Amide 1-(4-Carbamimidoyl-benzyl-
amide) Trifluoroacetate (12) Compound 12 was synthesized from (S)-2-
[(R)-2-ethanesulfonylamino-3-(5-benzyloxy-1H-indol-3-yl)-propionyl-
amino]-pentanedioic acid 5-amide 1-(4-cyano-benzylamide) according the
1
procedure described for 11 (49%, 26 mg). H-NMR (270 MHz, CD3OD) d:
1.01 (3H, t, Jꢂ7.3 Hz), 1.59—2.06 (4H, m), 2.67—2.90 (2H, m), 2.95—
3.23 (2H, m), 4.08—4.20 (2H, m), 4.35—4.52 (2H, m), 6.67 (1H, dd,
Jꢂ2.3, 8.7 Hz), 6.97 (1H, d, Jꢂ2.3 Hz), 7.09 (1H, s), 7.17 (1H, t, Jꢂ8.7 Hz),
7.49 (2H, d, Jꢂ8.4 Hz), 7.72 (2H, d, Jꢂ8.4 Hz); HR-MS (ESIꢃ) Calcd for
C26H34N7O6S: 572.2285 (Mꢃꢃ1). Found: 572.2318.
Human Thrombin Inhibition Activity A 10% (v/v) DMSO solution of
test compound (20 ml) was mixed with 40 ml buffer (200 mM Tris–HCl, pH

January 2010
43
cal Co., Ltd. for HR-MS measurements of the compounds and Ms. Frances
Ford of Chugai Pharmaceutical Co., Ltd. for proofreading the manuscript.
8.0), 20 ml NaCl solution (1 M), 20 ml FVR-pNA (2 mM; Sigma), and 80 ml
distilled water in a 96-well assay plate. The reaction was initiated by the ad-
dition of 20 ml human thrombin solution (5 U/ml, Sigma) and then the ab-
sorbance at 405 nm was monitored to measure the initial velocity of the re-
action. Percent inhibition at each concentration was calculated from the
experimental and control sample. IC50 values were calculated from the con-
centration–reaction activity curve of each test compound.
Human Factor FIXa (FIXa) Inhibition Activity A 10% (v/v) DMSO
solution of test compound (20 ml) was mixed with 20 ml Spectrozyme® FXa
(MeO-CO-D-CHG-Gly-Arg-pNA·AcOH) (5 mM; American Diagnostica
Inc.), 20 ml buffer (500 mM Tris–HCl, pH 7.5, 1500 mM NaCl, 50 mM CaCl2)
and 80 ml distilled water in a 96-well assay plate. The reaction was initiated
by the addition of 20 ml human FVIIa solution (20 nM; Enzyme Research
Laboratories) and then the absorbance at 405 nm was monitored to measure
the initial velocity of the reaction. Percent inhibition at each concentration
was calculated from the experimental and control sample. The IC50 value
was calculated from the concentration–reaction activity curve of each test
compound.
Human Factor Xa (FXa) Inhibition Activity A 10% (v/v) DMSO so-
lution of test compound (20 ml) was mixed with 20 ml buffer (500 mM
Tris–HCl, pH 8.4, 1500 mM NaCl), 20 ml S-2222 (Bz-Ile-Glu(g-OR)-Gly-
Arg-pNA·HCl) (5 mM; Daiichi Pure Chemical), and 120 ml distilled water in
a 96-well assay plate. The reaction was initiated by the addition of 20 ml
human FXa solution (50 mU/ml; Enzyme Research Laboratories) and the
absorbance at 405 nm was then monitored to measure the initial velocity of
the reaction. Percent inhibition at each concentration was determined from
the experimental and control samples. The IC50 value was calculated from
the concentration–reaction activity curve of each test compound.
Human Factor XIa (FXIa) Inhibition Activity A 10% (v/v) DMSO
solution of test compound (20 ml) was mixed with 100 ml buffer (200 mM
Tris–HCl, pH 7.2, 300 mM NaCl), 20 ml S-2366 (pyroGlu-Pro-Arg-
pNA·HCl) (2 mM, Daiichi Pure Chemical), and 40 ml distilled water in a 96-
well assay plate. The reaction was initiated by the addition of 20 ml human
FXIa solution (5 nM, Enzyme Research Laboratories) and the absorbance at
405 nm was then monitored to measure the initial velocity of the reaction.
Percent inhibition at each concentration was determined from the experi-
mental and control samples. The IC50 value was calculated from the concen-
tration–reaction activity curve of each test compound.
Human Factor XIIa (FXIIa) Inhibition Activity A 10% (v/v) DMSO
solution of test compound (20 ml) was mixed with 100 ml buffer (200 mM
Tris–HCl, pH 8.3, 300 mM NaCl), 20 ml S-2302 (1H D-Pro-Phe-Arg-
pNA·2HCl) (2 mM, Daiichi Pure Chemical), and 40 ml distilled water in a
96-well assay plate. The reaction was initiated by the addition of 20 ml
human FXIIa solution (50 nM, Enzyme Research Laboratories) and the ab-
sorbance at 405 nm was then monitored to measure the initial velocity of the
reaction. Percent inhibition at each concentration was determined from the
experimental and control samples. The IC50 value was calculated from the
concentration–reaction activity curve of each test compound.
Human Activated Protein C (APC) Inhibition Activity A 10% (v/v)
DMSO solution of test compound (20 ml) was mixed with 40 ml buffer
(200 mM Tris–HCl, pH 8.0), 40 ml S-2366 (pyroGlu-Pro-Arg-pNA·HCl)
(2 mM, Daiichi Pure Chemical), 20 ml NaCl (1 M), 20 ml CaCl2 (20 mM), and
40 ml distilled water in a 96-well assay plate. The reaction was initiated by
the addition of 20 ml human APC (1 mg/ml, Sigma) and the absorbance at
405 nm was then monitored to measure the initial velocity of the reaction.
Percent inhibition at each concentration was determined from the experi-
mental and control samples. The IC50 value was calculated from the concen-
tration–reaction activity curve of each test compound.
Prothrombin Time (PT) One hundred microliters human plasma (Dade
Behring) containing the test compound was incubated at 37 °C for 3 min and
was then mixed with 100 ml Thromborel S (Dade Behring). The plasma clot-
ting time was then measured using a KC-10A coagulometer (Amelung). The
concentration for the two-fold prolongation of PT (2ꢀPT) was calculated
from the concentration–reaction activity curve of each test compound.
Activated Partial Thromboplastin Time (APTT) Human plasma
(100 ml; Dade Behring) containing the test compound was incubated at
37 °C for 1 min and then mixed with 100 ml APTT reagent (Sigma). After
3 min, the human plasma was mixed with 100 ml CaCl2 (20 mM). The plasma
clotting time was then measured using a KC-10A coagulometer (Amelung).
The concentration for the two-fold prolongation of APTT (2ꢀAPTT) was
calculated from the concentration–reaction activity curve of each test com-
pound.
References
1) Davie E. W., Fujikawa K., Kisiel W., Biochemistry, 30, 10363—10370
(1991).
2) Braunwald E., Califf R. M., Cannon C. P., Fox K. A., Fuster V., Gibler
W. B., Harrington R. A., King S. B. 3rd, Kleiman N. S., Theroux P.,
Topol E. J., Van de Werf F., White H. D., Willerson J. T., Am. J. Med.,
108, 41—53 (2000).
3) Himber J., Refino C. J., Burcklen L., Roux S., Kirchhofer D., Thromb.
Haemostasis, 85, 475—481 (2001).
4) Harker L. A., Hanson S. R., Wilcox J. N., Kelly A. B., Haemostasis,
26 (Suppl. 1), 76—82 (1996).
5) Suleymanov O. D., Szalony J. A., Salyers A. K., LaChance R. M., Par-
low J. J., South M. S., Wood R. S., Nicholson N. S., J. Pharmacol.
Exp. Ther., 306, 1115—1121 (2003).
6) Szalony J. A., Taite B. B., Girard T. J., Nicholson N. S., LaChance R.
M., J. Thromb. Thrombolysis, 14, 113—121 (2002).
7) Zoldhelyi P., McNatt J., Shelat H. S., Yamamoto Y., Chen Z. Q.,
Willerson J. T., Circulation, 101, 289—295 (2000).
8) Kelley R. F., Refino C. J., O’Connell M. P., Modi N., Sehl P., Lowe D.,
Pater C., Bunting S., Blood, 89, 3219—3227 (1997).
9) Miura M., Seki N., Koike T., Ishihara T., Niimi T., Hirayama F., Shige-
naga T., Sakai-Moritani Y., Tagawa A., Kawasaki T., Sakamoto S.,
Okada M., Ohta M., Tsukamoto S., Bioorg. Med. Chem., 15, 160—
173 (2007).
10) Young W. B., Mordenti J., Torkelson S., Shrader W. D., Kolesnikov A.,
Rai R., Liu L., Hu H., Leahy E. M., Green M. J., Sprengeler P. A.,
Katz B. A., Yu C., Janc J. W., Elrod K. C., Marzec U. M., Hanson S.
R., Bioorg. Med. Chem. Lett., 16, 2037—2041 (2006).
11) Hu H., Kolesnikov A., Riggs J. R., Wesson K. E., Stephens R., Leahy
E. M., Shrader W. D., Sprengeler P. A., Green M. J., Sanford E.,
Nguyen M., Gjerstad E., Cabuslay R., Young W. B., Bioorg. Med.
Chem. Lett., 16, 4567—4570 (2006).
12) Zbinden K. G., Obst-Sander U., Hilpert K., Kuhne H., Banner D. W.,
Bohm H. J., Stahl M., Ackermann J., Alig L., Weber L., Wessel H. P.,
Riederer M. A., Tschopp T. B., Lave T., Bioorg. Med. Chem. Lett., 15,
5344—5352 (2005).
13) Schweitzer B. A., Neumann W. L., Rahman H. K., Kusturin C. L.,
Sample K. R., Poda G. I., Kurumbail R. G., Stevens A. M., Stegeman
R. A., Stallings W. C., South M. S., Bioorg. Med. Chem. Lett., 15,
3006—3011 (2005).
14) Sagi K., Fujita K., Sugiki M., Takahashi M., Takehana S., Tashiro K.,
Kayahara T., Yamanashi M., Fukuda Y., Oono S., Okajima A., Iwata
S., Shoji M., Sakurai K., Bioorg. Med. Chem., 13, 1487—1496 (2005).
15) Riggs J. R., Hu H., Kolesnikov A., Leahy E. M., Wesson K. E.,
Shrader W. D., Vijaykumar D., Wahl T. A., Tong Z., Sprengeler P. A.,
Green M. J., Yu C., Katz B. A., Sanford E., Nguyen M., Cabuslay R.,
Young W. B., Bioorg. Med. Chem. Lett., 16, 3197—3200 (2006).
16) Rai R., Kolesnikov A., Sprengeler P. A., Torkelson S., Ton T., Katz B.
A., Yu C., Hendrix J., Shrader W. D., Stephens R., Cabuslay R., San-
ford E., Young W. B., Bioorg. Med. Chem. Lett., 16, 2270—2273
(2006).
17) Groebke Zbinden K., Banner D. W., Hilpert K., Himber J., Lave T.,
Riederer M. A., Stahl M., Tschopp T. B., Obst-Sander U., Bioorg.
Med. Chem., 14, 5357—5369 (2006).
18) Shiraishi T., Kadono S., Haramura M., Kodama H., Ono Y., Iikura H.,
Esaki T., Koga T., Hattori K., Watanabe Y., Sakamoto A., Yoshihashi
K., Kitazawa T., Esaki K., Ohta M., Sato H., Kozono T., Bioorg. Med.
Chem. Lett., 18, 4533—4537 (2008).
19) Shiraishi T., Kadono S., Haramura M., Kodama H., Ono Y., Iikura H.,
Esaki T., Koga T., Hattori K., Watanabe Y., Sakamoto A., Yoshihashi
K., Kitazawa T., Esaki K., Ohta M., Sato H., Kozono T., Lett. Drug
Des. Discov., 6, 86—92 (2009).
20) Kadono S., Sakamoto A., Kikuchi Y., Oh-Eda M., Yabuta N., Yoshi-
hashi K., Kitazawa T., Suzuki T., Koga T., Hattori K., Shiraishi T.,
Haramura M., Kodama H., Ono Y., Esaki T., Sato H., Watanabe Y.,
Itoh S., Ohta M., Kozono T., Biochem. Biophys. Res. Commun., 326,
859—865 (2005).
21) Kadono S., Sakamoto A., Kikuchi Y., Oh-eda M., Yabuta N., Yoshi-
hashi K., Kitazawa T., Suzuki T., Koga T., Hattori K., Shiraishi T.,
Haramura M., Kodama H., Ono Y., Esaki T., Sato H., Watanabe Y.,
Itoh S., Ohta M., Kozono T., Biochem. Biophys. Res. Commun., 327,
Acknowledgments We thank Ms. Hitomi Suda of Chugai Pharmaceuti-

44
Vol. 58, No. 1
589—596 (2005).
22) Kadono S., Sakamoto A., Kikuchi Y., Oh-Eda M., Yabuta N., Koga T., 25) Shiraishi T., Kadono S., Haramura M., Sato H., Kozono T., Koga T.,
Hattori K., Shiraishi T., Haramura M., Kodama H., Ono Y., Esaki T., Sakamoto A., Japan Patent WO02062829A1 (2002).
Sato H., Watanabe Y., Itoh S., Ohta M., Kozono T., Acta Crystallogr. 26) Bode W., Mayr I., Baumann U., Huber R., Stone S. R., Hofsteenge J.,
Sect. F: Struct. Biol. Cryst. Commun., 61, 169—173 (2005). EMBO J., 8, 3467—3475 (1989).
23) Kadono S., Sakamoto A., Kikuchi Y., Oh-eda M., Yabuta N., Koga T., 27) Kirchhofer D., Guha A., Nemerson Y., Konigsberg W. H., Vilbois F.,
Hattori K., Shiraishi T., Haramura M., Kodama H., Esaki T., Sato H., Chene C., Banner D. W., D’Arcy A., Proteins, 22, 419—425 (1995).
Watanabe Y., Itoh S., Ohta M., Kozono T., Lett. Drug Des. Discov., 2, 28) Otwinowski Z., Minor W., Methods Enzymol., 276, 307—326 (1997).
Commun., 324, 1227—1233 (2004).
165—171 (2005).
29) Banner D. W., D’Arcy A., Chene C., Winkler F. K., Guha A., Konigs-
berg W. H., Nemerson Y., Kirchhofer D., Nature (London), 380, 41—
46 (1996).
24) Kadono S., Sakamoto A., Kikuchi Y., Oh-eda M., Yabuta N., Koga T.,
Hattori K., Shiraishi T., Haramura M., Kodama H., Esaki T., Sato H.,
Watanabe Y., Itoh S., Ohta M., Kozono T., Biochem. Biophys. Res.