ELSEVIER
`
`Available online at www.sciencedirect.com
`
`0 ScienceDirect
`
`Bioorganic & Medicinal Chemistry Letters 16 (2006) 517(»5182
`
`Bioorganic &
`Medicinal
`
`Chemistry
`Letters
`
`Preparation of 1—(3—aminobenzo[d]isoxazol-5-yl)—1H-
`pyrazolo[4,3—d]pyrimidin—7(6H)—ones as potent, selective,
`and eflicacious inhibitors of coagulation factor Xa
`
`Yun-Long Li,*’l John M. Fevig,* Joseph Cacciola} Joseph Buriak, Jr.,§
`Karen A. Rossi, Janan Jona,'” Robert M. Knabb, Joseph M. Luettgen,
`Pancras C. Wong, Stephen A. Bai," Ruth R. Wexler and Patrick Y. S. Lam
`
`Bristol-Myers Squibb Pharmaceutical Research Institute, PO Box 5400, Princeton, NJ 08543-5400, USA
`
`Received 6 June 2006; revised 26 June 2006; accepted 5 July 2006
`Available online 25 July 2006
`
`Abstract—Previously, potent factor Xa inhibitors were described based on the lH-pyrazolo[4,3-d]pyrimidin-7(6H)-one bicyclic core
`and a 4-methoxyphenyl Pl moiety. This manuscript describes 1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one and related bicyclic cores
`with the 3-aminobenzisoxazole Pl moiety. Many of these compounds are potent, selective, and eflicacious inhibitors of coagulation
`factor Xa.
`© 2006 Elsevier Ltd. All rights reserved.
`
`Thromboembolic diseases remain the leading cause of
`death and disability in developed countries. This reality,
`combined with the limitations of current therapies, has
`led to extensive efforts to develop novel antithrombotic
`agents.‘ Factor Xa has become a major focus of phar-
`maceutical intervention in the past decade because of
`its central role in the blood coagulation cascade? Exten-
`sive preclinical and clinical evidence has demonstrated
`that inhibition of factor Xa is efficacious in both venous
`and arterial thrombosis.“
`
`Previously, it was demonstrated that a series of non—ben—
`zamidine N-arylpyrazole carboxamides, represented by
`the 3—aminobenzisoxazole Pl analog razaxaban (1),5a
`were highly potent, selective, and orally bioavailable
`small molecule fXa inhibitors (Fig. 1). Razaxaban has
`been shown to be efficacious in phase II deep vein
`thrombosis (DVT) clinical trials.5b Furthermore, it was
`
`Keywords: Factor Xa inhibitors; Anticoagulants; Antithrombotic
`agents; Pyrazole; Bicyclic core; Pyrazolo[4,3-d]pyrimidin-7(6H)-ones.
`*Corresponding authors. Tel.: +1 609 818 5285; fax: +1 609 818
`3450; e-mail: john.fevig@bms.com
`T Present address: Incyte Corporation, Wilmington, DE, USA.
`it Present address: AstraZeneca, Wilmington, DE, USA.
`§ Present address: DuPont Ag Products, Newark, DE, USA.
`l' Present address: Amgen, Thousand Oaks, CA, USA.
`” Present address: Centocor, Malvern, PA, USA.
`
`0960-894X/$ - see front matter © 2006 Elsevier Ltd. All rights reserved.
`doi: l0. l0l6/j.bmcl.2006.07.002
`
`F3C
`
`F3C
`
`N/
`
`l
`
`H
`
`F
`
`.
`
`©WN\©\ £NMe2 HCI
`
`N \N
`\=/
`1, razaxaban
`fXa K; = 0.19 nM
`
`0
`
`0- / NH2
`N
`
`R3
`
`Q! OAW,
`
`/
`
`24
`
`NH
`
`N/
`
`l
`
`H
`
`F
`
`gm © some
`
`0
`
`Q
`
`MeO
`
`2
`fXa K: = 3-6 W
`
`HZNOC
`
`Q 30
`
`Meo
`
`fXaKi=1.1nM
`
`demonstrated that the 4—methoxyphenyl residue could
`be an effective P1 group when combined with an opti-
`mized pyrazole—P4 subunit, as in compound 2.6 Recent-
`ly, bicyclic core variants of 2,
`represented by the
`1H—pyrazolo[4,3—d]pyrimidin—7(6H)—one 3,7 have been
`shown to retain potent binding affinity for fXa. These
`bicyclic variants were also expected to be less susceptible
`to in vivo amide hydrolysis, which in the case of N-aryl-
`pyrazole carboxamides such as 1 and 2 would liberate
`a biarylaniline fragment. However, compound 3 was
`BMS 2004
`V. BMS
`CFAD
`|PR2015-01723
`
`

`
`Y.-L. Li et al. / Bioorg. Med. Chem. Lett. 16 (2006) 51 76—5I82
`
`5177
`
`found to have only modest efficacy in a rabbit arterio-
`venous (A—V) shunt thrombosis models relative to razax—
`aban. This was rationalized on the basis of 3 being
`5-fold less potent than razaxaban.7 This manuscript de-
`scribes bicyclic variants in the 3—aminobenzisoxazole P1
`series,9 represented by 4, which were prepared with the
`expectation that this P1 series would display greater
`potency and in vivo efficacy relative to analogs with
`the 4—methoxyphenyl P1, such as 3.10
`
`bicyclic core by refluxing in 95% formic acid. This treat-
`ment also resulted in loss of the TBS group and subse-
`quent conversion of the alcohol to a formate ester, to
`give 10. Treatment of 10 with acetohydroxamic acid
`and KZCO3 to form the 3—aminobenzisoxazole P1 moie-
`ty” was followed by hydrolysis of the formate ester, giv-
`ing 11. Bromination of the alcohol and displacement
`with pyrrolidine afforded the 1H—pyrazolo[4,3—d]pyrimi—
`din—7(6H)—one example 12.
`
`The initial bicyclic core example in the 3—aminobenzis-
`oxazole P1
`series,
`the
`1H—pyrazolo[4,3—d]pyrimidin—
`7(6H)—one 12, was prepared as described in Scheme 1,
`using the synthesis route used for preparing compounds
`such as 3.7 Catalytic hydrogenation of the nitro func-
`tionality of 5 afforded an aniline, which upon diazotiza—
`tion and treatment with ethyl cyanoacetate gave the
`expected hydrazone intermediate. Treatment of this
`hydrazone with methyl bromoacetate and KZCO3 at
`90 °C gave the 4—aminopyrazole diester 6 by N—alkyl—
`ation and in situ ring closure. However, this sequence
`proceeded in very low and irreproducible yield due to
`the poor nucleophilicity of the nitrogen in the N—alkyl—
`ation step. Nevertheless, aminopyrazole 6 was diazo-
`tized and treated with sodium azide to form a
`
`4—azidopyrazole. Selective hydrolysis of the methyl ester
`was accomplished with lithium hydroxide to give 7.
`Conversion of 7 to an acid chloride and subsequent
`treatment with readily available biphenylaniline 8“
`afforded amide 9. Reduction of the azide afforded a
`
`cleanly
`4-aminopyrazole—5—carboxamide, which was
`cyclized to the 1H—pyrazolo[4,3—d]pyrimidin—7(6H)—one
`
`Due to the poor yield of 6 discussed above, additional
`examples in this bicyclic series were prepared via an
`alternate route, described in Scheme 2, wherein the pyr-
`azole R3 substituent was initially a methyl group.%
`Readily available pyrazole ester 139*‘ was efficiently
`and selectively nitrated at the pyrazole 4—position using
`excess ammonium nitrate and trifluoroacetic anhydride
`in TFA.” Subsequent ester hydrolysis afforded acid
`14, which was coupled with 4—bromoaniline to give 15.
`Nitro reduction was unexpectedly diflicult, with several
`reagents (SnCl2, Zn/HCl, and Fe/HCl) resulting in sig-
`nificant decomposition. However, potassium borohy—
`dride and CuCl in ethanol afforded in moderate yield
`the clean 4—aminopyrazole, which was efficiently cyclized
`to the bicyclic core 16 by refluxing in either N,N—DMF
`dimethyl acetal
`(R5 = H) or N,N—dimethylacetamide
`dimethyl acetal (R5 = Me). Suzuki coupling with boron-
`ic acid 17 readily gave the biphenyl aldehyde 18. The
`completion of the synthesis of compounds 19a—h was
`best accomplished by a four—step sequence. Thus, sodi-
`um borohydride reduction of the aldehyde to the
`alcohol was
`followed by the introduction of the
`
`
`
`
`
`F
`
`F
`
`N
`
`3
`
`N
`
`H
`N O OTBS
`
`9,
`
`EtO2C
`N’I
`N
`Q 0
`
`F
`
`CN 9
`
`/
`EtO2C
`MN I
`
`N
`\
`11
`
` O
`
`O\N/ NH2 12
`
`B020
`N’
`I R
`N
`N
`OCHO
`Q 0 0 O
`
`F
`
`CN 10
`I-»
`
`1
`/
`EtO2C
`kl
`NO <— N-N
`
`N
`
`\
`11
`
`l
`
`OH
`
`Q70 3 O
`
`o\N/ NH2
`
`11
`
`;K
`
`F
`
`CN
`13
`Me
`
`::
`
`F
`
`CN
`14
`
`Ci
`
`F
`
`0
`
`CN
`15
`
`Me
`
`
`de
`
`N
`
`R5
`
`HO 3 CH0
`N\ R5
`N\ R5
`91
`N?
`E 1:]
`(
`I 1:
`N?
`I ][
`O R
`” O L "
`0
`Q °
`3'
`f Q °
`F
`ON 16 R5=H, Me
`F CN1sR5=|—|,|v|e
`Me
`19a R5=H; X:-NMe2
`N/
`19b R5=H; x= N-pyrrolidine
`‘N
`X 19c R5 = H; x =3-(S)-OH-N-pyrrolidine
`19d R5 = H; x = -NHCH2-cyc-Pr
`
`l
`
`\\r
`N
`o 0
`
`Q ‘\‘ 1% R5:H; X:-NH-i-Pr
`
`O , NH,
`‘N
`
`19a-h
`
`19f R5=Me; X=-NMe2
`199 R5 = Me; X = 3-(R)-OH-N-pyrrolidine
`19h R5 = Me; x = -NMe-i-Pr
`
`Scheme 1. Reagents and conditions: (a) H2 (1 atm), 10% Pd/C, EtOH
`(95%); (b) NaNO2, HC1/H20, 0 °C; then ethyl cyanoacetate, NaOAc,
`MeOH/H20, 0 °C (55%); (c) BrCH2CO2Me, KZCO3, DMF, 90 °C (ML
`20%); (d) NaNO2, TFA, 0 °C; then NaN3 (80%); (e) LiOH, THF/H20
`(90%);
`(f)
`(COCl)2, CH2C12;
`then 8, DMAP, CH2C12 (70%);
`(g)
`SnC12-H20, MeOH, 65 °C; (h) 95% HCOZH,
`reflux (70% for two
`steps); (i) AcNHOH, KZCO3, DMF; (j) K2CO3, EtOH, 65 °C (45% for
`two steps); (k) PBr3, CH2C12; (1) pyrrolidine, CH3CN, (50% for two
`steps).
`
`Scheme 2. Reagents and conditions: (a) NH4NO3 (2 equiv), TFAA
`(7 equiv), TFA (90%); (b) LiOH, MeOHH-I20 (80%);
`(c) (COCl)2,
`CH2C12;
`then 4—bromoaniline, DMAP, CH2C12 (80%); (d) KBH4,
`CuCl, EtOH, (50%); (e) N,N—DMF dimethyl acetal, reflux (R= H,
`85%), or N,N—dimethylacetamide dimethyl acetal, reflux (R=Me,
`75%); (f) 17, Pd(PPh3)4, Na2CO3, benzene, H20, 80 °C (70—80%); (g)
`NaBH4, MeOH (5(L65%); (h) HO-NHAc, KZCO3, DMF (60—70%); (i)
`MnO2, CH2C12 (75-85%); (j) HNRR’, NaBH(OAc)3, HOAc, THF,
`(50—70%).
`
`

`
`5178
`
`Y.-L. Li et al. /Bioorg. Med. Chem. Lett. 16 (2006) 5I76—5I82
`
`F3C
`,
`N.
`N
`
`l
`
`F
`
`I
`
`CO2H
`‘cu
`
`20
`
`a
`—> 20
`
`+
`
`F3C
`,
`N.
`N
`
`F
`
`NO
`
`I
`
`2
`co2H
`‘cN
`
`~1 : 2
`
`21
`
`R5
`
`N
`| Y
`N
`
`x
`
`
`b,c
`
`FaC
`N/
`N
`
`[
`
`F
`
`l
`
`NH2
`H
`N
`0 \®\
`C“
`
`Br
`
`22
`
`F30
`N_/
`N
`
`H
`N
`
`I
`
`N
`
`X
`
`F C
`3 /
`N‘
`N
`
`|
`
`F C
`aib 3 /
`N02
`:» N‘
`N
`CO2}-1
`
`I
`
`F C
`3 /
`C
`NH2
`H —> N‘
`N
`
`I
`
`H
`
`Scheme
`2
`O
`if —> 26b-260
`
`z
`
`F
`
`CN
`
`21
`
`[
`
`F
`
`0
`CN
`
`22
`
`Br
`
`Z
`
`F
`
`0
`CN
`
`25
`
`id
`N02
`
`NH2
`
`Br
`
`n
`
`F30
`e,f
`—> N/I
`(27a)
`N
`
`Br Q 0
`
`F
`
`CN
`28
`
`CN
`273 (X=CO2Me)
`27b (X=CN)
`
`F30]
`—g» N.
`N
`
`|
`
`|
`
`Br Q 0
`
`F
`
`CN 29
`
`B’
`
`_
`
`\'
`F30
`N_’
`N
`
`“N
`.
`
`I
`
`Q 0
`
`F
`
`C”
`
`Scheme
`2
`
`26d
`
`Scheme
`2
`
`Br
`
`26f
`
`31
`
`Scheme
`—2» 26e
`
`Br
`
`F3C
`N,’
`N
`
`I
`
`X
`
`Q 0
`
`1
`
`h
`
`(27b)
`
`H
`
`|
`
`I
`
`NH2
`
`Fae
`N_/
`N
`
`Q 0
`
`F
`
`C“
`
`30
`
`F3C
`L, N/
`N
`then see
`Scheme 2
`steps f-j
`
`O
`
`O
`
`0
`ow’ NH2 23a.d
`
`0
`
`0
`
`O~N’ NH2 24a-b
`
`Scheme 3. Reagents and conditions: (a) NH4NO3 (2 equiv), TFAA
`(7 equiv), TFA (60%); (b) (COC1)2, CH2C12;
`then 4-bromoaniline,
`DMAP, CH2C12 (90%); (c) KBH4, CuCl, EtOH, (70%); (d) N,N-DMF
`dimethyl acetal, (R = H, 90%), or N,N-dimethylacetamide dimethyl
`acetal, reflux; then HOAc, reflux (R = Me, 75%).
`
`3—aminobenzisoxazole P1 moiety using acetohydroxa—
`mic acid. The alcohol was then re—oxidized to the alde-
`
`hyde with MnO2 and treated under standard reductive
`amination conditions with an appropriate cyclic or acy-
`clic amine, affording compounds 19a—h. Alternatively,
`the alcohol could be brominated with PBr3 followed
`by displacement with the appropriate amine as previous-
`ly described.
`
`Slight modification of this sequence, as shown in Scheme
`3, afforded compounds 23a—d, where the pyrazole R3
`group is trifluoromethyl.9b In this case,
`the pyrazole
`nitration was best performed on the carboxylic acid
`20.53 However, even after using an excess of reagents
`and prolonged reaction times, it generally afforded only
`an approximately 2:1 mixture of nitropyrazole 21 along
`with recovered starting material 20. These materials
`could be efficiently separated by prolonged stirring of
`the solid in water, wherein the desired nitro compound
`21 slowly went almost completely into solution. Filtra-
`tion and extraction afforded clean product in about
`60% yield. The starting material 20 could then be recy-
`cled. Coupling with 4—bromoaniline and nitro reduction
`as before gave 22. Where R5 = H, formation of the bicy-
`clic core was accomplished as in Scheme 2 by refluxing
`in N,N-DMF dimethyl
`acetal. However, where
`R5 = Me,
`the cyclization was best accomplished by a
`two—step sequence, wherein 22 was first
`refluxed in
`N,N—dimethylacetamide dimethyl acetal and the result-
`ing acetamidine intermediate was refluxed in glacial ace-
`tic acid to effect the final cyclization.7 Once the CF3
`bicyclic core was established, the synthetic sequence fol-
`lowed that described in Scheme 2, to afford compounds
`23a—d. The 5,6—dihydro-1H—pyrazolo[4,3-d]pyrimidin-
`7(4H)—one examples 24a,b were prepared from a core
`reduction side product isolated from the sodium boro-
`hydride reduction of the corresponding biphenyl alde-
`hyde, as described in Scheme 2.
`
`Additional bicyclic core analogs were prepared as de-
`scribed in Scheme 4. Aminopyrazole 22 was cyclized
`by heating with phosgene in a sealed tube to afford the
`1H—pyrazolo[4,3—d]pyrimidin—5,7(4H,6H)—dione core 25.
`
`then
`(COC1)2, CHZCIZ;
`(a)
`Scheme 4. Reagents and conditions:
`reflux
`4-bromoaniline, DMAP, CH2C12 (90%);
`(b) SnC12, MeOH,
`(70%); (c) C1COC1/PhMe, sealed tube, 110°C (85%); (d) (COC1)2,
`DMF, CHZCIZ,
`then methyl p-bromophenylacetate, LDA, THF,
`—78 °C (X=CO2Me, 60%), or (COC1)2, DMF, CH2C12,
`then p-
`bromophenylacetonitrile, LDA, THF, —78 °C (X=CN, 75%);
`(e)
`1.5 N HC1, dioxane, reflux; (0 Fe, 1N HC1, EtOH, reflux (65% for two
`steps); (g) HC(OEt)3, 120 °C (85%); (h) SnC12, EtOH, reflux (50%); (i)
`NaNO2,H2SO4, (80%).
`
`Methods described in Scheme 2 allowed for the prepara-
`tion of compounds 26b,c. Compound 26a was prepared
`in a similar fashion, except that the fully elaborated P4
`aniline was used in coupling with the carboxylic acid
`21. For bicyclic cores where the amide nitrogen is re-
`placed by carbon, conversion of 21 to the acid chloride
`followed by treatment at -78 °C with the lithium anion
`of methyl p—bromophenylacetate and p—bromophenyl—
`acetonitrile gave good yields of oc—ketoester 27a and
`oucyanoketone 27b, respectively. Acid—catalyzed decar—
`boxylation of 27a followed by nitro reduction afforded
`28. Heating 28 in neat triethyl orthoformate induced
`ring closure to the 1H—pyrazolo[4,3—b]pyridin—7(4H)—
`one core 29. The oc—cyanoketone 27b was treated with
`SnCl2 in refluxing ethanol to effect nitro reduction and
`subsequent ring closure to give the 5—amino substituted
`1H—pyrazolo[4,3—b]pyridin—7(4H)—one bicyclic core 30.
`Diazotization of 28 was followed by in situ trapping
`by the adjacent enol
`to afford the 1H—pyrazolo[4,3-
`c]pyridazin—7(4H)—one bicyclic core 31. Compounds
`29—31 were converted by the methods described previ-
`ously into 26d—f, respectively.
`
`The SAR for the 1—(3—aminobenzo[a1isoxazol—5—yl)—1H-
`pyrazolo[4,3—d]pyrimidin—7(6H)—one core examples are
`shown in Table 1. The initial bicyclic example, 12, was
`an order of magnitude less potent than razaxaban 1 in
`the in vitro fXa Ki assay, but was 4-fold more potent
`and equipotent to 1 in the in vitro aPTT and PT clotting
`
`

`
`Table 1. 1H-Pyrazolo[4,3-d]pyrimidin-7(6H)-one core
`
`Y.-L. Li et al. / Bioorg. Med. Chem. Lett. 16 (2006) 51 76—5I82
`
`5179
`
`R3NF{5
`N.’I‘Y
`N N X
`0
`
`0w’ NH2 12, 19a-h, 23a—d
`
`R3|-|
`N/IN
`
`O
`/ NH
`°‘N
`
`2
`
`O
`24a-b
`
`Compound“
`1
`
`12
`192
`19b
`19c
`19d
`19e
`19f
`19g
`19h
`23a
`23b
`23c
`23d
`24a
`24b
`
`R3
`_
`
`CO2Et
`Me
`Me
`Me
`Me
`Me
`Me
`Me
`Me
`CF3
`CF3
`CF3
`CF3
`CF3
`CF3
`
`R5
`_
`
`H
`H
`H
`H
`H
`H
`Me
`Me
`Me
`H
`Me
`Me
`Me
`—
`—
`
`X
`_
`
`N-Pyrrolidine
`NMe2
`N-Pyrrolidine
`3-(S)-OH-N-Pyrrolidine
`—NHCH2-c-Pr
`—NH-i-Pr
`NMe2
`3-(R)-OH-N-Pyrrolidine
`—NMe-i-Pr
`NMe2
`NMe2
`—NHCH2-c-Pr
`3-(R)-OH-N-Pyrrolidine
`NMe2
`3-(R)-OH-N-Pyrrolidine
`
`fXa K,” (nM)
`0.19
`1.6
`0.53
`1.3
`0.50
`1.7
`1.5
`0.27
`0.37
`0.35
`0.35
`0.17
`0.50
`0.18
`0.25
`0.21
`
`aP’IT IC2x° (uM)
`6.1
`1.6
`2.1
`1.6
`4.3
`8.1
`3.0
`1.7
`5.8
`2.0
`3.7
`9.2
`18.6
`28
`1.7
`2.9
`
`PT IC2x° (uM)
`2.1
`2.3
`1.3
`1.1
`2.1
`3.1
`1.9
`1.4
`2.5
`4.0
`2.6
`4.1
`10.9
`6.6
`1.4
`1.6
`
`“All final compounds were purified by prep HPLC and gave satisfactory spectral data.
`"K; values were obtained from purified human enzyme and were averaged from multiple determinations (n = 2), as described in Ref. 5a.
`° The aPTT (activated partial thromboplastin time) and PT (prothrombin time) in vitro clotting assays were performed in human plasma as described
`in Ref. 5a.
`
`assays, respectively. The 3—methylpyrazole series 19a—h
`afforded compounds approaching the binding potency
`of razaxaban, especially when R was also methyl, as
`in compounds 19f—h. The 3—methyl series also exhibited
`generally favorable potency in the clotting assays rela-
`tive to 1. The 3—trifluoromethylpyrazole series 23a—d
`provided even more potent compounds, but generally
`at the expense of lower potency in the in vitro clotting
`assays, presumably due to the higher protein binding
`imparted by the CF3 group. For example, compounds
`23b and 23d are essentially equipotent with razaxaban
`
`in binding potency, but are less potent in the clotting as-
`says. The reduced analogs of this core, the 5,6—dihydro—
`1H—pyrazolo[4,3-d]pyrimidin—7(4H)—ones 24a,b,
`retain
`excellent potency and now have very favorable clotting
`assay activity relative to compounds 23a—d and to
`razaxaban.
`
`The SAR for additional bicyclic core analogs are shown
`in Table 2. Compounds 26a—c contain the 1H—pyrazolo—
`[4,3—a]pyrimidin—5,7(4H,6H)—dione bicyclic core.7 These
`examples are characterized by excellent in vitro binding
`
`Table 2. Examples of other bicyclic cores
`
`F3C
`
`H
`
`N‘/
`
`|
`
`“ T1 1‘
`N \N\=/
`
`F
`
`O
`
`o\N“
`
`“H2 26a
`
`ow’
`
`“H2 26b-f
`
`Compound“
`1
`2621
`26b
`26c
`26d
`26e
`26f
`
`Y
`_
`C=O
`C=O
`C=O
`CH
`C—NH2
`N
`
`Z
`_
`N
`N
`N
`C
`C
`C
`
`X
`_
`NMe2
`3-(R)-OH-N-Pyrrolidine
`4-OH-N-Piperidine
`NMe2
`4-OH-N-Piperidine
`NMe2
`
`fXa K9’ (nM)
`0.19
`0.35
`0.11
`0.22
`3.2
`0.77
`5.5
`
`apt IC2x° (uM)
`6.1
`37.8
`74.5
`352
`ND
`638
`ND
`
`PT IC2x° (uM)
`2.1
`14.9
`24.1
`92.6
`ND
`230
`ND
`
`“All final compounds were purified by prep HPLC and gave satisfactory spectral data.
`'’K; values were obtained from purified human enzyme and were averaged from multiple determinations (n = 2), as described in Ref. 5a.
`° The aPTT (activated partial thromboplastin time) and PT (prothrombin time) in vitro clotting assays were performed in human plasma as described
`in Ref. 5a.
`
`

`
`5180
`
`Y.-L. Li et al. /Bioorg. Med. Chem. Lett. 16 (2006) 5I76—5I82
`
`potency, especially 26b, but also by uniformly poor
`activity in the in vitro clotting assays, suggesting that
`this core contributes to high protein binding. Examples
`26d—f were prepared to determine if it was possible to re-
`place the carboxamide nitrogen with carbon, thus negat-
`ing the potential for generating an aniline degradant
`in vivo. Only the 5-amino-1H-pyrazolo[4,3—b]pyridin—
`7(4H)—one example 26e retains subnanomolar fXa bind-
`ing potency. Unfortunately,
`this compound has poor
`activity in the clotting assays, again suggesting that it
`is highly protein bound.
`
`Compounds with the 3—aminobenzisoxazole P1 residue
`have previously been reported to show high selectivity
`for fXa versus thrombin, trypsin and related serine pro-
`teases.5‘“’°’” Additional selectivity data are shown for
`selected compounds in Table 3. Compounds 19f, 19g,
`23b, and 24b all showed >1000—fold selectivity relative
`to trypsin, aPC, factor IXa, factor VIIa, plasmin, tPA,
`plasma kallikrein, urokinase, and chymotrypsin. They
`are less selective relative to thrombin, but still show
`
`Table 3. Human enzyme selectivity profile
`
`1
`24b
`23b
`19g
`Enzyme Ki (nM) 19f
`0.19
`0.21
`0.17
`0.37
`fXa
`0.27
`540
`130
`130
`245
`Thrombin
`305
`> 10,000
`>4200
`>4200
`>4200
`Trypsin
`>4200
`>76,000 >37,000 >20,000 19,700
`aPC
`19,000
`fIXa
`>41,000 >41,000 37,000
`>41,000 9000
`Plasmin
`>15,000 >15,000 >25,000 >15,000 >15,000
`tPA
`23,000
`>33,000 >40,000 >33,000 >33,000
`Urokinase
`>13,000 >13,000 >40,000 >13,000 >13,000
`Chymotrypsin
`960
`580
`>1 1,000 3240
`8500
`
`All Kfs were obtained from purified human enzymes and are averaged
`from multiple determinations (n = 2). See Ref. 5a for more details.
`
`Table 4. Dog pharmacokinetic profiles
`
`Compound
`
`19a“
`191*‘
`19g*‘
`2315*‘
`2311*‘
`2415*‘
`Razaxabanb
`
`Cl
`(L/h/kg)
`3.3
`1.7
`1.2
`0.7
`0.6
`0.98
`1.1
`
`Vdss
`(L/kg)
`9.5
`8.9
`6.6
`13
`6.6
`4.2
`5.3
`
`[1/2
`(h)
`2.5
`4.2
`3.8
`14
`8.4
`4.3
`3.4
`
`F
`(%)
`2
`23
`48
`63
`58
`27
`84
`
`Caco-2
`(P,p,,x10‘5cm/s)
`11
`6.1
`4.7
`19
`ND
`3.9
`5.6
`
`“Compounds were dosed as the TFA salts in an N-in-1 format at
`0.5 mg/kg iv and 0.2 mg/kg po (11 = 2).
`1’ Ref. 5a. ND = not determined.
`
`>500—fold selectivity in all cases. The overall selectivity
`profile for these compounds in most cases is comparable
`to that of razaxaban 1.
`
`The pharmacokinetic profiles of several compounds
`were studied in dogs via a cassette dosing format, with
`dosing at 0.5 mg/kg intravenously and 0.2 mg/kg orally
`(Table 4). The 3—methylpyrazole 1921 has high clearance
`and only 2% bioavailability. Given its good Caco-2 per-
`meability, this low bioavailability is more likely to be
`related to high first pass metabolism, rather than poor
`absorption. Addition of the 5—methyl substituent im-
`proves the pharmacokinetic profile of this series, with
`19f and 19g exhibiting lower clearance and higher bio-
`availability relative to 19a and a half—life comparable
`to razaxaban. The PK profile is further improved by
`the 3—trifluoromethylpyrazole substituent, as shown by
`23b and 23d. These compounds have lower clearance
`and longer half—life than razaxaban, together with better
`bioavailability than the 3—methylpyrazole examples.
`Compound 24b has a profile similar to 19f and 19g.
`
`Three of these compounds were also studied in the rab-
`bit arterio—venous
`(A—V) shunt
`thrombosis model.3
`Upon intravenous dosing, compounds 19f,19g, and
`23b inhibited thrombus formation with an ID50 of 2.0,
`2.4, and 1.9 umol/kg/h,
`respectively (Table 5). Com-
`pound 23b had an IC50 in this same assay of 290 nM.
`This activity compares well to that of razaxaban in this
`model.
`
`Several examples from the 1H—pyrazolo[4,3—d]pyrimidin—
`7(6H)—one core series were examined for their stability
`under conditions of varying pH in order to assess their
`potential for liberation of the P4 aniline via hydrolytic
`cleavage of the bicyclic core. Data are shown for com-
`pounds 19a, 19f, and 23a in Table 6. The results indicate
`that the pyrimidinone ring of the bicyclic core decom-
`poses at various rates depending on the pH and on the
`substitution pattern of the bicyclic core. In most cases
`two major degradants were observed, the identities of
`which were determined by LC/MS. Compound II results
`from h drolysis of the C5—N6 bond to afford the N—for—
`myl (R = H) or N—acetyl (R5 = Me) derivative. Further
`hydrolysis affords the free amine III. For compound
`19a, II was the predominant degradant observed at
`pH 4.0, while the free amine III was the predominant
`degradant at pH 1.0. Mechanistically, the N—formyl II
`was thought to be an intermediate in the formation of
`HI. Evidence for this was gathered experimentally by
`taking one half of the pH 4.0 samples and lowering them
`to pH 1.0. After one week, the pH 1.0 samples showed
`
`Table 5. Anticoagulant activity in rabbits
`
`Compound
`
`19f
`
`19g
`23b
`Razaxabana
`
`fXa K;
`(rabbit) (nM)
`0.30
`
`PT IC2x
`(rabbit) (uM)
`1.8
`
`Rabbit A-V shunts
`IC50 (HM)
`ND
`
`0.59
`0.20
`0.19
`
`3.4
`2.7
`1.9
`
`ND
`290
`340
`
`Rabbit A-V shunts ID50
`(umol/kg/h)
`2.0
`2.4
`1.9
`1.6
`
`Protein binding
`(rabbit) (% bound)
`ND
`91
`97
`93
`
`“ Ref. 5a. ND = not determined.
`
`

`
`Y.-L. Li et al. / Bioorg. Med. Chem. Lett. 16 (2006) 51 76—5I82
`
`5181
`
`Table 6. Stability of 1H-pyrazolo[4,3-d]pyrimidin-7(6H)-ones
`
`R3
`N’
`N
`
`I
`
`NHCOR5
`H
`N‘Ar
`
`R3
`N
`as
`
`N’
`I
`\\r
`‘N
`N‘Ar
`
`R3
`N’
`‘N
`
`I
`
`NH2
`H
`N‘Ar
`
`O
`
`pH=4.0
`(0.1M acetate)
`
`O
`
`pH=1_o
`(()_1 N HCD
`
`O
`
`o\N/ NH2
`II
`
`o\N/ NH2
`I
`
`O\N/ NH2
`III
`
`pH=1.0 (O.1N HCI)
`
`
`Compound“ Conditions“
`19a
`pH 1.0
`pH 4.0
`
`I remaining” (%)
`97.2
`90.1
`
`Ilb (%)
`0.6
`8.5
`
`III" (%)
`2.4
`0.12
`
`19f
`
`23a
`
`pH 1.0
`pH 4.0
`
`pH 1.0
`pH 4.0
`
`89.7
`94.7
`
`83.2
`72.3
`
`5.5
`3.4
`
`1.3
`23.4
`
`1.4
`—
`
`5.6
`—
`
`“pH 1.0 = 0.1 N HCl buffer; pH 4.0 = 0.1 M acetate buffer. Stability
`studies were conducted at 40 °C with an initial concentration of 5 pg]
`mL.
`
`"Values of % I—III were determined by HPLC after 2 days. The
`identities of II and III were determined by LC/MS.
`
`complete conversion to the amine III, while samples
`remaining at pH 4.0 still showed a preponderance of
`II. Thus, it appears that at both pH 1.0 and 4.0, initial
`formation of II occurs, but only at pH 1.0 is H further
`hydrolyzed appreciably to III. The degradation of 19f
`is similar to that of 19a, with II (R5 = Me) being the ma-
`jor component at pH 4.0. At pH 1.0 an appreciable
`amount of II remains, while conversion to III is slower.
`This observation presumably reflects the slower acid
`hydrolysis of an N—acetyl group relative to an N—formyl
`group. The 3—trifluoromethylpyrazole analog 23a under-
`goes a more rapid decomposition than the 3—methylpy—
`razole analogs, with 23% conversion to II at pH 4.0
`after 2 days. Apparently, the CF3 group is activating
`the pyrimidinone ring toward the initial hydrolysis,
`thereby accelerating the decomposition. The observed
`formation of II and III in these studies of 19a, 19f,
`and 23a leads to the possibility that the free P4 aniline
`could still be generated in vivo, although this risk is
`slight, since it would require further hydrolysis of an
`amide such as III, which can be viewed as a vinylogous
`urea. The free P4 aniline was not observed in these
`
`stability studies. Still, advancement of this series would
`require use of an Ames negative P4 aniline.
`
`In summary, several examples of pyrazole—fused bicyclic
`core fXa inhibitors in the 3—aminobenzisoxazole P1 ser-
`
`ies have been described. The 1H—pyrazolo[4,3—d]pyrimi—
`din—7(6H)—one core is especially promising as a means
`to maintain potent fXa inhibition while also reducing
`the probability that in vivo amide hydrolysis will liberate
`a biarylaniline fragment. Within this series, analogs 19f,
`19g, and 23b are not only potent fXa inhibitors, but they
`are also highly selective versus relevant serine proteases.
`Compound 23b has a favorable pharmacokinetic profile
`in dogs, with lower clearance and longer half—life relative
`to razaxaban. Furthermore, 19f,19g, and 23b are highly
`efficacious in the rabbit A—V shunt thrombosis model,
`
`with activity comparable to that of the clinical candi-
`date, razaxaban. However, in aqueous solution stability
`studies, these compounds were found to undergo vari-
`able degrees of hydrolytic cleavage of the pyrimidinone
`ring to generate pyrazole—5—carboxamide degradants.
`While this core series is still promising, a major empha-
`sis has been geared toward finding bicyclic cores with
`greater aqueous stability.9b>‘4 Further efforts along these
`lines will be described in due course.
`
`Acknowledgments
`
`The authors thank Bruce Aungst, Frank Barbera, Tracy
`Bozarth, Earl Crain, Andrew Leamy, Dale McCall, and
`Carol Watson for technical assistance.
`
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