Synthesis and biological evaluation of novel pyrazoles and indazoles as activators of the nitric oxide receptor, soluble guanylate cyclase

78

J . Med. Chem. 2001, 44, 78-93

Syn th esis a n d Biologica l Eva lu a tion of Novel P yr a zoles a n d In d a zoles a s

Activa tor s of th e Nitr ic Oxid e Recep tor , Solu ble Gu a n yla te Cycla se

David L. Selwood,*^,†David G. Brummell,^§J oanna Budworth,^§Guillaume E. Burtin,^†Richard O. Campbell,^§

Surinder S. Chana,^†Ian G. Charles,^§Patricia A. Fernandez,^†Robert C. Glen,^‡Maria C. Goggin,^†

Adrian J . Hobbs,^§Marcel R. Kling,^†Qian Liu,^‡David J . Madge,^†Sylvie Meillerais,^§Kenneth L. Powell,^§

Karen Reynolds,^†Graham D. Spacey,^§J eremy N. Stables,^§Mark A. Tatlock,^§Kerry A. Wheeler,^§

Grant Wishart,^†and Chi-Kit Woo^†

Biological & Medicinal Chemistry and Molecular & Cellular Biology, The Wolfson Institute for Biomedical Research,

University College London, The Cruciform Building, Gower Street, London WC1E 6BT, U.K., and Tripos Inc.,

1699 South Hanley Road, St. Louis, Missouri 63144

Received J uly 25, 2000

Database searching and compound screening identified 1-benzyl-3-(3-dimethylaminopropyloxy)-

indazole (benzydamine, 3) as a potent activator of the nitric oxide receptor, soluble guanylate

cyclase. A comprehensive structure-activity relationship study surrounding 3 clearly showed

that the indazole C-3 dimethylaminopropyloxy substituent was critical for enzyme activity.

However replacement of the indazole ring of 3 by appropriately substituted pyrazoles

maintained enzyme activity. Compounds were evaluated for inhibition of platelet aggregation

and showed a general lipophilicity requirement. Aryl-substituted pyrazoles 32, 34, and 43

demonstrated potent activation of soluble guanylate cyclase and potent inhibition of platelet

aggregation. Pharmacokinetic studies in rats showed that compound 32 exhibits modest oral

bioavailability (12%). Furthermore 32 has an excellent selectivity profile notably showing no

significant inhibition of phosphodiesterases or nitric oxide synthases.

In tr od u ction

brain, placenta, spleen, and uterus only.¹⁰Tissue se-

lectivity may derive from the differential desensitiza-

tion¹¹of the enzyme rather than from variation in

isoform expression. sGC has been shown to bind one

molecule of ferrous heme per heterodimer⁴in a five-

coordinate complex with a histidine residue of the â

subunit acting as the proximal ligand.¹²Spectral studies

have demonstrated that NO forms a five-coordinate

nitrosyl heme complex with sGC, breaking the weak

Fe²⁺-His bond and causing conformational changes

leading to enzyme activation.^13,14Carbon monoxide (CO)

also shows limited activation of sGC.¹⁴

Soluble guanylate cyclase (sGC, EC 4.6.1.2) catalyzes

the conversion of guanosine 5′-triphosphate (GTP) to

guanosine 3′,5′-cyclic monophosphate (cGMP) and is the

only known receptor for the signaling molecule nitric

oxide (NO).^1,2NO may also participate in responses that

are not mediated via sGC.³NO is a potent activator of

sGC causing activation up to 600-fold greater than the

basal level.⁴The NO-cGMP signaling pathway is im-

portant in many physiological processes including va-

sodilation, neurotransmission, and platelet aggrega-

tion.^5,6Activators (or inhibitors) of sGC are therefore

very desirable as both pharmacological tools to probe

the NO-cGMP pathway and as therapeutic agents. sGC

activators may be useful in a wide variety of conditions,

and their potential in glaucoma has recently been

highlighted.⁷

sGC exists as a heterodimeric heme protein with a

total molecular mass of about 150 kDa, consisting of R

and â subunits, both of which are required for catalytic

activity.⁸Several isoforms of each subunit are known,

with the R₁â₁heterodimer being the most studied.^2,9The

R and â subunits show considerable sequence homology

with each other, particularly in the C-terminal catalytic

domain, where they also share homology with adenylate

cyclases and membrane-bound guanylate cyclases. sGC

is widely expressed in human tissues with the R₁â₁

isoform being predominant. The R₂isoform shows a

more restricted expression pattern with high levels in

Presently there are few known potent modulators of

sGC.¹⁵ODQ (1, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-

1-one) has been reported as an inhibitor of sGC.¹⁶The

most studied class of sGC modulators are the NO donor

compounds which activate sGC in a manner similar to

endogenous NO by release of NO or an NO-related

species.^6,17The vasodilatory effects of NO donors have

been exploited clinically for many years; however, it is

only recently that their mode of action has been

elucidated. NO donors can cause tolerance upon pro-

longed use, and problems associated with reactions of

the compounds may arise, such as nitration of tyrosine

residues.⁶Therefore there is an obvious need for novel

activators of sGC that circumvent the problems associ-

ated with NO donors. YC-1 (2, 3-(5-hydroxymethyl-2-

furyl)-1-benzylindazole) has been reported as an acti-

vator of sGC.¹⁸This compound does not act as an NO

donor but activates purified sGC at high concentrations

and shows synergistic activation in the presence of NO

or CO.^19,20The mechanism of activation is not yet fully

understood. However YC-1 has recently been shown to

also act as a nonspecific phosphodiesterase inhibitor²¹

* To whom correspondence should be addressed. Tel: +44 (0)20 7679

6716. Fax: +44 (0)20 7679 6799. E-mail: d.selwood@ucl.ac.uk.

^†Biological & Medicinal Chemistry, University College London.

^§Molecular & Cellular Biology, University College London.

^‡Tripos Inc.

Published on Web 11/22/2000

Novel Pyrazoles/ Indazoles as Activators of sGC

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 1 79

Sch em e 2^a

Sch em e 1^a

a

Reagents: (a) HO(CH₂)₃NMe₂, ADDP, PBu₃, toluene, 80 °C;

(b) R-Cl or R-Br, t-BuOK, THF; (c) R-Cl, CH₂Cl₂.

a

Reagents: (a) R-Br or R-Cl, NaH, DMF, 100 °C; (b) with R

) (CH₂)₃NHBoc, (CH₂)₅NHBoc: 50% TFA/CH₂Cl₂, with R )

(CH₂)₄NHBoc: 1 M HCl, Et₂O; (c) HCHO, NaCNBH₃, AcOH,

MeOH.

lowed by sodium hydrosulfite reduction and subsequent

cyclization as described previously.²³Analogues 5 and

6 were synthesized from 4 via alkylation with the

appropriate chloro-substituted alkyldimethylamino com-

pound in the presence of sodium hydride. The first step

in synthesizing analogues 10-12 was again alkylation

of 4 using the appropriate BOC-protected bromoalky-

lamine. Removal of the BOC group of 8 was achieved

using 50% TFA in dichloromethane to give the primary

amine analogue 12. Similarly deprotection of 7 and 9

followed by reductive amination of formaldehyde af-

forded analogues 10 and 11, respectively. In this series

of analogues where the hydroxyindazole 4 was alky-

lated, both N- and O-alkylations were possible. However

IR spectroscopy proved that O-alkylation was the major

product as there was no detection of a carbonyl stretch

in the IR spectrum.

making interpretation of cell and tissue-based experi-

ments difficult.

We have used the structure of compound 2 to generate

2-D substructural queries and performed 2-D substruc-

tural and 2-D similarity searches²²of commercially

available compound databases. From the subsequent

compound screening it was found that benzydamine (3,

1-benzyl-3-(3-dimethylaminopropyloxy)indazole) is a more

potent activator of sGC than YC-1 upon partially

purified sGC. Benzydamine is a known antiinflamma-

tory and analgesic; however, its mechanism of action is

unknown.²³We herein describe the synthesis and

structure-activity relationships (SARs) of novel inda-

zole and pyrazole activators of sGC based upon the

benzydamine structure.

Analogues synthesized to investigate the SAR at the

N-1 position of the indazole are shown in Table 2. All

the analogues (with the exception of 22) were synthe-

sized from the intermediate 13 which was synthesized

from 3-indazolinone and 3-dimethylamino-1-propanol

via a Mitsunobu reaction using 1,1′-(azodicarbonyl)-

dipiperidine (ADDP) and tributylphosphine as the redox

system.²⁴Compounds 14-16 were prepared from 13 by

alkylation with the corresponding halide compound

using potassium tert-butoxide as the base as shown in

Scheme 2. Acylation of 13 afforded 17-20. Analogue 22

was synthesized by alkylation of 21 with 3-dimethyl-

aminopropyl chloride hydrochloride in anhydrous DMF

using sodium hydride as base as shown in Scheme 3.

Intermediate 21 was prepared by treatment of diphe-

nylcarbamoyl chloride with sodium azide using standard

procedures and subsequent conversion of the diphenyl-

carbamoyl azide to 21.²⁵

Ch em istr y

The synthesis of the benzydamine analogues which

have been modified at the indazole C-3 substituent

(Table 1) are depicted in Scheme 1. The starting

material 1-benzyl-3-hydroxy-1H-indazole (4) was pre-

pared by diazotization of N-benzylanthranilic acid fol-

A series of novel pyrazole analogues were also syn-

thesized. Table 3 outlines a wide variety of analogues

80 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 1

Selwood et al.

Sch em e 3^a

Sch em e 5^a

a

Reagents: (a)NaN₃,acetone/H₂O;(b)xylene,4;(c)Cl(CH₂)₃NMe₂‚

HCl, NaH, DMF.

Sch em e 4^a

a

Reagents: (a) with R ) Ph: HO(CH₂)₃NMe₂, ADDP, PBu₃,

toluene, with R ) CH₂Ph: Cl(CH₂)₃NMe₂‚HCl, NaH, DMF.

all of which contain the 3-dimethylaminopropyloxy

substituent at the pyrazole C-3 position but which vary

at the N-1, C-4, and C-5 positions. Compound 23 was

prepared via alkylation of 1-benzyl-3-hydroxy-1H-pyra-

zole²⁶with the hydrochloride salt of 3-dimethylamino-

propyl chloride using sodium hydride in DMF (Scheme

4). Analogue 24 was formed from the reaction of

3-hydroxy-1-phenyl-1H-pyrazole²⁷with 3-dimethylamino-

1-propanol under Mitsunobu conditions as described

previously. The precursor to 26, intermediate 25, was

prepared from 1-benzyl-3-hydroxy-5-methoxycarbonyl-

1H-pyrazole²⁶and 3-dimethylamino-1-propanol using

the same Mitsunobu conditions as for compound 13. The

carboxylic acid 26 was subsequently prepared by sa-

ponification of 25 in alkaline solution and purification

achieved using Dowex AG1 X8 (OH^-form) ion-exchange

resin. Reaction of 26 in anhydrous DMF with the acti-

vating coupling reagent O-(1H-benzotriazol-1-yl)-N,N,-

N′,N′-tetramethylammonium tetrafluoroborate (TBTU)

and appropriate amine gave the amide derivatives 27-

31 as shown in Scheme 5. For compound 32 O-(7-aza-

benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluo-

rophosphate (HATU) was used as the coupling reagent

to combat the decreased nucleophilic character of the

amine. Pyrazole analogue 33 was prepared from 3-hy-

droxy-5-phenyl-1H-pyrazole²⁸via reaction with 3-di-

methylamino-1-propanol in the presence of the Mit-

sunobu reagent ADDP (Scheme 6). The tautomeric 33

gave access to the isomeric N-substituted analogues 34

a

Reagents: (a) HO(CH₂)₃NMe₂, ADDP, PBu₃, toluene; (b) 2.5

M NaOH, MeOH; (c) RR₁NH, TBTU, DIPEA, except for 32:

4-methoxyaniline, O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethy-

luronium hexafluorophosphate, DIPEA.

and 35 in a ratio of 1:1.6, respectively, by reaction of

33 under Mitsunobu conditions with benzyl alcohol. The

more reactive Mitsunobu reagent 1,1-azobisdimethyl-

formamide was used in this case. The regiochemistry

of 34 and 35 was determined on the basis of NOE

experiments. For compound 34 an enhancement was

observed between the benzylic protons and the ortho

protons of the pyrazole C-5 substituted phenyl moiety.

This enhancement was absent for 35. The remaining

pyrazole analogues were prepared by several different

methods. In each case the 3-hydroxypyrazole was pre-

pared initially and subsequently the C-3 substituent

was introduced via Mitsunobu reaction with 3-dimeth-

ylamino-1-propanol using ADDP. The synthesis of pyra-

zoles can be achieved by reaction of alkynoates with a

hydrazine.²⁹The precursor to 43, 1,5-diphenyl-3-hy-

droxy-1H-pyrazole (36), was prepared from phenylhy-

drazine and ethyl phenylpropiolate. Similarly the pre-

cursor to 44, 3-hydroxy-5-trifluoromethyl-1H-pyrazole

(37) was prepared from the reaction of ethyl 4,4,4-

Novel Pyrazoles/ Indazoles as Activators of sGC

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 1 81

Sch em e 7^a

Sch em e 6^a

a

Reagents: (a) ROH, ADDP, PBu₃, toluene; (b) HO(CH₂)₃NMe₂,

ADDP, PBu₃, toluene; (c) PhCH₂OH, 1,1-azobisdimethylforma-

mide, PBu₃, toluene.

trifluoro-2-butynoate with hydrazine hydrate in ethanol

(Scheme 7). Subsequent Mitsunobu reaction afforded

the desired analogues 43 and 44. Compounds 45-47

were all synthesized by a similar method as shown in

Scheme 7. In each case the appropriate diketo ester 38-

40 was reacted with hydrazine hydrate in ethanol to

form the 3-hydroxypyrazole and converted to the desired

product via Mitsunobu reaction. For compound 48 it was

necessary to synthesize the diketo ester intermediate.

This was accomplished as shown in Scheme 8 by

reaction of phenylacetyl chloride with 2,2-dimethyl-1,3-

dioxolane-4,6-dione (Meldrum’s acid) in the presence of

base followed by refluxing in ethanol to give ethyl 3-oxo-

4-phenylbutyrate (41). Intermediate 41 was converted

to the 3-hydroxypyrazole 42, then to 48 as in previous

examples. To form the C-4 substituted pyrazole 51,

methyl 3-dimethylamino-2-phenylacrylate (49) was pre-

pared from the reaction of tert-butoxybis(dimethylami-

no)methane with methyl phenylacetate (Scheme 9).

Subsequent reaction of 49 with hydrazine hydrate in

refluxing ethanol gave 3-hydroxy-4-phenyl-1H-pyrazole

(50). Treatment of 50 as previously described with

3-dimethylamino-1-propanol gave compound 51.

Table 4 depicts a series of pyrazole analogues that

were synthesized to further investigate the pyrazole C-3

substituent. Compounds 54-58 were all synthesized

from 3-hydroxy-5-phenyl-1H-pyrazole²⁸under Mitsuno-

bu conditions with the respective hydroxy compound

using ADDP as shown in Scheme 6. The hydroxy

precursors for 54 and 55 were available commercially.

3-Pyrrolidin-1-yl-1-propanol which was used to synthe-

size 57 was prepared from the alkylation of pyrrolidine

with 3-chloro-1-propanol according to published proce-

dures.³⁰The precursor to 56, 2-amino-3-pyridinemetha-

a

Reagents: (a) with R ) H: NH₂NH₂, EtOH, with R ) Ph:

PhNHNH₂, t-BuOK, THF; (b) HO(CH₂)₃NMe₂, ADDP, PBu₃,

toluene.

Sch em e 8^a

a

Reagents: (a) Py, EtOH.

nol,³¹was prepared in two steps from 2-aminonicotinic

acid. 2-Amino-4-pyridinemethanol (53), the reagent used

in the synthesis of 58, was synthesized in two steps from

2-acetylaminopyridine-4-carboxylic acid³²as depicted in

Scheme 10. Cleavage of the acetyl group and esterifi-

cation of the carboxylate was achieved in one step using

concentrated sulfuric acid in ethanol to give 2-amino-

4-ethoxycarbonylpyridine (52). The final step involved

the reduction of the ester 52 using lithium aluminum

hydride to give 53. The synthesis of 60 is shown in

Scheme 11. First N1,N1-dimethyl-3-[(5-phenyl-1H-3-

pyrazolyl)amino]propanamide (59) was prepared from

82 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 1

Selwood et al.

Sch em e 9^a

Sch em e 12^a

a

Reagents: (a) THF, rt; (b) NH₂NH₂, EtOH; (c) HO(CH₂)₃NMe₂,

ADDP, PBu₃, toluene.

Sch em e 10^a

a

Reagents: (a) concd H₂SO₄, EtOH; (b) LiAlH₄, THF.

Sch em e 11^a

a

Reagents: (a) NH₂NH₂, EtOH; (b) (i) SOCl₂, 60 °C, (ii)

dimethylamine, Et₂O; (c) LiAlH₄, dioxane.

Resu lts a n d Discu ssion

Activa tion of Solu ble Gu a n yla te Cycla se. All

target compounds were assayed for sGC stimulation

using baculovirus expressed and partially purified

bovine sGC. cGMP levels were measured using a com-

mercially available ELISA assay. sGC was submaxi-

mally stimulated with the NO donor compound DEA/

NO (2-(N,N-diethylamino)diazenolate 2-oxide, 300 nM),

and cGMP accumulation was evaluated for each com-

pound at a single concentration of 1 µM. The cGMP

produced by each compound was expressed first as a

percentage of the DEA/NO response. Relative enzyme

activities were calculated as the ratio between the

percentage of the DEA/NO response for the compound

and that for benzydamine (3), the lead compound.

Dose-response curves in the presence of PAPA/NO ((Z)-

1-[N-(3-ammoniopropyl)-N-(n-propyl)amino]diazen-1-

ium-1,2-diolate, 30 nM) were produced for selected

compounds and EC₅₀values calculated.

The first stage of exploring the SAR of benzydamine

analogues was to modify the indazole C-3 substituent

(Table 1) which afforded key information. Complete

replacement of the dimethylamino functionality by

nonbasic groups capable of hydrogen bonding such as

hydroxy, methoxy, and carboxylate completely abolished

enzyme activity (data not shown). Removal of a meth-

ylene unit (5) from the substituent decreased activity,

but 10 with four methylene units only showed a slight

decrease in REA. Indazole 11 with a further methylene

unit gave a further drop in enzyme activation. The

reduced activity of the methyl-substituted racemate 6

a

Reagents: (a) N,N-dimethylacrylamide; (b) LiAlH₄1.0 M in

THF, dioxane.

the Michael addition of commercially available 3-amino-

5-phenyl-1H-pyrazole and N,N-dimethylacrylamide us-

ing previously described methodology.³³Lithium alu-

minum hydride reduction of 59 in dioxane yielded 60.

The synthesis of 63 is depicted in Scheme 12. The

diketone starting material, 7-phenyl-5,7-diketoheptanoic

acid,³⁴was prepared by known methods from acetophe-

none and glutaric anhydride. This was converted to the

intermediate 4-(5-phenyl-1H-3-pyrazolyl)butanoic acid

(61) by reaction with hydrazine hydrate in refluxing

ethanol. Conversion of 61 to the acid chloride followed

by quenching with dimethylamine gave N1,N1-di-

methyl-4-(5-phenyl-1H-3-pyrazolyl)butanamide (62). The

final step involved reduction of 62 with lithium alumi-

num hydride in dioxane to give 63.

Novel Pyrazoles/ Indazoles as Activators of sGC

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 1 83

Ta ble 1. Activity Data for C-3 Substituted

1-Benzyl-1H-indazoles

Ta ble 2. Activity Data for N-1 Substituted

3-(3-Dimethylaminopropyloxy)-1H-indazoles

a

Relative enzyme activity, enzyme activity relative to compound

b

3. Concentration required to inhibit collagen-induced platelet

aggregation by 50%.

further indicates that the SAR surrounding the C-3

substituent is tight and that any bulk is not well-

tolerated. Surprisingly primary amine 12 showed very

little activity. Therefore the more lipophilic dimeth-

ylamino group is critical for enzyme activation.

To investigate the function of the indazole N-1 sub-

stituent (Table 2) a number of suitable substituents

were selected as representatives of the groups most

frequently attached to heteroaryl nitrogen atoms in the

World Drug Index.³⁵The phenyl-substituted compound

22 and benzoyl analogue 18 show comparable activity

to the parent compound. However the phenylethyl-

substituted 15 shows a decreased REA suggesting that

the size increase is detrimental to activity. Conforma-

tional properties of these derivatives appear to be less

important as both benzydamine and 15 have flexible

substituents whereas the N-1 substituents of 18 and 22

will be conformationally restricted. The isopropyl and

alkylamino derivatives 14 and 16 show decreased activ-

ity as do the acetyl- and dimethylamide-substituted

compounds 17 and 20. Surprisingly methylsulfonyl

substitution (19) retained the activity of the parent, and

unsubstituted 13 exhibited almost a 2-fold enhancement

in relative enzyme activity over benzydamine. Therefore

the N-1 unsubstituted indazole is favored for increased

enzyme activity and substitutents containing an aryl

ring also show good activation, but there does appear

to be a steric requirement within the N-1 substituent

binding site.

In an attempt to move into a novel series of com-

pounds, replacement of the indazole ring by pyrazole

was investigated (Table 3). Pyrazoles 23 and 24, sub-

stituted at the N-1 position only, result in decreased

enzyme activity. However the activity is regained upon

the introduction of a phenyl ring substituted at the

pyrazole C-4 or C-5 position. Indeed 34 and 43 show

improved REA over benzydamine. The reduced activity

of 35 suggests the C-3 exocyclic oxygen may play a

crucial role as an electron-donating group via the +M

a

Relative enzyme activity, enzyme activity relative to compound

b

3. Concentration required to inhibit collagen-induced platelet

aggregation by 50%. ^cInhibition at highest concentration tested.

effect. This renders the sp²hybridized nitrogen atom of

the pyrazole ring more susceptible to donation of its lone

pair of electrons in an activator-enzyme interaction.

The N-1 unsubstituted, C-5 substituted pyrazoles 33

and 48 showed enhanced activity over benzydamine.

When the aryl substituent is alternatively at the pyra-

zole C-4 position as in 51, enzyme activity is also

maintained. Replacement of the phenyl ring of 33 with

3-furyl (45) or 4-pyridyl (46) suggests that the electron-

deficient pyridyl substituent was less favored. Tri-

methoxyphenyl analogue 47 also exhibited good enzyme

activity. Further substitutions at the pyrazole C-5

position were also fruitful. Compounds 26, 27, and 44

all showed similar enzyme activation. Due to the ease

of synthesis of 27 from 26 and the potential application

to library synthesis, several amides were synthesized

and evaluated. The amides were all synthesized as N-1

benzyl-substituted pyrazoles to increase lipophilicity

and cell membrane penetration. Compounds 28-32 all

showed enzyme activity similar to benzydamine.

Reduction of the pK_aof the dimethylamino function

of the pyrazole series was studied. Aminopyridines have

been proven to act as bioisosteres of guanidino groups

for thrombin inhibitors.³⁶The aminopyridyl pyrazoles

56 and 58 exhibited reduced enzyme activity in the case

of sGC (Table 4), suggesting either the lowered pK_aor

84 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 1

Selwood et al.

Ta ble 3. Activity Data for Substituted

Ta ble 4. Activity Data for Substituted 5-Phenyl-1H-pyrazoles

3-(3-Dimethylaminopropyloxy)-1H-pyrazoles

a

Platelet aggregation IC₅₀10.33 ( 2.44 µM for compound

b

57. Relative enzyme activity, enzyme activity relative to com-

pound 3.

Dose-response curves were carried out for selected

compounds identified in the screening assay (Table 5).

In general, evaluated EC₅₀’s mirror the trends observed

in the screening assay. Comparison of the EC₅₀’s of YC-1

(2) with benzydamine and compounds 33, 34, 43, and

48 clearly indicates the increase in potency that was

achieved within the series. Furthermore the most active

compound of the series 48 is almost 1 order of magni-

tude more potent against the enzyme than benzy-

damine. However compound 32 showed reduced enzyme

potency upon comparison with benzydamine.

In summary, from our lead compound benzydamine,

the dimethylamino function was proven critical for

enzyme activity. The optimal length of the indazole C-3

substituent was identified as three methylene units, and

substitution of this group is not tolerated. Phenyl,

benzyl, and benzoyl are favored substituents at the

indazole N-1 position, but larger groups appear to be

subject to steric constraints. The unsubstituted analogue

is particularly favored. The indazole moiety may be

replaced by a pyrazole ring provided the pyrazole C-4

or C-5 positions are substituted. Groups with significant

electron delocalization are favored at the C-5 position,

in particular phenyl and benzyl. The pyrazole C-3

exocyclic oxygen is essential for activity and substitution

of the dimethylaminopropyloxy group is unfavorable.

Similarities are drawn between the indazole and pyra-

zole series, and it is speculated that the indazoles and

a

b

For the structure of compound 35, see Scheme 6. Relative

enzyme activity, enzyme activity relative to compound 3. ^cCon-

centration required to inhibit collagen-induced platelet aggregation

by 50%. Inhibition at highest concentration tested.

d

the additional steric bulk is unfavorable. The decrease

in activity also observed for 54 and 55 indicates that

steric bulk in the region of the pyrazole C-3 substituent

is clearly not tolerated and is consistent with the SAR

of the indazole series. When the dimethylamino group

is constrained within a cyclic system as in 57, a modest

drop in activity occurs. Finally the role of the pyrazole

exocyclic oxygen atom was probed. Replacement by

amino (60) or methylene (63) resulted in decreased

activity: this highlights the electronic and conforma-

tional importance of the oxygen atom providing a

conjugated system with the pyrazole ring.

Novel Pyrazoles/ Indazoles as Activators of sGC

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 1 85

Ta ble 5. Activation of sGC: EC₅₀Data for Selected Indazoles

and Pyrazoles in the Presence of 30 nM PAPA/NO

F igu r e 1. Plot of inhibition of collagen-induced platelet

aggregation -log[IC₅₀] versus clogP and REA.

Ta ble 6. Pharmacokinetic Parameters for sGC Activators

compd

C_max(ng/mL)

AUC (ng/h/mL)

t_max(h)

F (%)

22^a

32^b

34^a

43^b

3.2

6.4

3.1

1.4

7.9

61.1

15.5

3.3

0.5

2.7

1.7

0.5

2

12

2

<2

a

b

Cassette dosing at 2 mg/kg po. Dosed at 5 mg/kg po.

3.0 is generally required for potent inhibition of platelet

aggregation. This observation can have important im-

plications in library design of novel analogues. The clogP

constraints from the plot can be used as a filter to

ensure that synthesized compounds would have cell-

based activity.

pyrazoles are binding in a similar manner to the same

site. This assumption allows a pharmacophore to be

proposed consisting of the dimethylaminopropyl group

and a hydrogen bond acceptor atom which is equivalent

to the pyrazole or indazole N-2 nitrogen. However the

enzyme-bound conformation of the flexible dimethyl-

aminopropyl group cannot be predicted with any great

certainty. The hydrogen bond acceptor is proposed on

the basis of the reduced activity for compound 35. This

suggests that the conjugated system of the exocyclic

oxygen and the pyrazole sp²hybridized nitrogen that

is absent for 35 are important for enzyme activity and

may participate in an activator-enzyme interaction.

Therefore it is proposed that the electron-rich pyrazole

or indazole N-2 nitrogen will accept a hydrogen bond

in the activator-enzyme complex.

In h ib it ion of P la t elet Aggr ega t ion . Compounds

exhibiting significant potency in the enzyme assay were

evaluated in a functional assay measuring the com-

pounds ability to inhibit collagen-stimulated platelet

aggregation (Tables 1-4). The most potent analogue 34

(IC₅₀) 2.37 µM) is almost 3-fold more potent than

benzydamine. However compounds 33 and 48, which

both show good activity in the enzyme assay, show a

decreased potency in the platelet assay upon comparison

with benzydamine and 34. This is attributed to poorer

cell penetration, which is supported by a decrease in

lipophilicity due to the lack of a hydrophobic substituent

at the pyrazole N-1 position. A similar case is observed

for the N-1 unsubstituted indazole 13. Optimal inhibi-

tion of platelet aggregation appears to be achieved with

a hydrophobic aryl group at the indazole and pyrazole

N-1 position. The plot of -log[IC₅₀] versus calculated

log P³⁷and REA (Figure 1) clearly indicates there is a

lipophilicity requirement for platelet activity. Com-

pounds with an IC₅₀g 100 µM were capped with an

assigned IC₅₀of 100 µM for the purposes of the plot only.

From the plot it can be seen that a clogP greater than

Selectivity a n d P h a r m a cok in etics. Compounds

22, 32, and 34 were evaluated for their selectivity

against a number of enzymes including phosphodi-

esterases and adenylate cyclase at a single concentra-

tion of 10 µM (see Supporting Information). Compound

22 shows minimal inhibition of PDE II (23%), and

compound 34 exhibits no significant activity at any of

the enzymes studied. It is particularly noteworthy that

32 and 34 do not activate adenylate cyclase and unlike

YC-1 (2) show no significant inhibition of any of the

phosphodiesterases, particularly PDE V. Compound 32

was additionally assayed against the NOS isoforms, and

minimal inhibition was observed with iNOS (25%) and

nNOS (17%). Furthermore 32 shows no significant

activity at any of the other enzymes studied.

The oral bioavailability of benzydamine in humans

is claimed to be high,³⁸though it is generally only used

as a topical agent. Four compounds were examined for

oral pharmacokinetics in rats (Table 6). In general the

compounds were not particularly bioavailable though

32 did demonstrate 12% bioavailability. Further opti-

mization of the compounds for oral bioavailability is

likely to be required in order for them to be utilized as

oral agents.

Con clu sion

A series of novel analogues of benzydamine have been

identified as potent activators of the NO receptor sGC.

In our evaluation of sGC activators we chose an isolated

enzyme system as our initial screen and inhibition of

platelet aggregation as a well-characterized cellular

system responsive to cGMP. We considered that an

86 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 1

Selwood et al.

1-Ben zyl-3-(3-d im eth yla m in o-2-m eth ylp r op yloxy)-1H-

in d a zole (6). To 1-benzyl-3-hydroxy-1H-indazole (4) (1.0 g,

4.46 mmol) in anhydrous DMF (40 mL) was added sodium

hydride (60% dispersion in mineral oil) (0.21 g, 5.35 mmol)

and the mixture was stirred under nitrogen at room temper-

ature for 30 min. Simultaneously 3-dimethylamino-2-methyl-

propyl chloride hydrochloride (0.85 g, 4.49 mmol) was stirred

with sodium hydride (60% dispersion in mineral oil) (0.18 g,

4.46 mmol) in anhydrous DMF (10 mL) for 30 min. This

mixture was then added to the flask containing the indazole

and the reaction mixture was heated to 100 °C for 12 h then

cooled to room temperature. Water (20 mL) was added and

the mixture was extracted with diethyl ether (3 × 50 mL). The

combined organic extracts were dried (MgSO₄), filtered and

concentrated under reduced pressure. The crude product was

purified by flash chromatography using 0.88 ammonia/MeOH/

CHCl₃(1:5:94) to give 6 as an oil (650 mg, 45%): ¹H NMR

(300 MHz, CDCl₃) δ 7.60 (d, 1H, J ) 8.1 Hz), 7.23-7.17 (m,

4H), 7.10-7.07 (m, 3H), 6.98-6.95 (m, 1H), 5.31 (s, 2H), 4.37-

4.27 (m, 1H), 4.19-4.10 (m, 1H), 2.42-2.33 (m, 1H), 2.20 (s,

6H), 1.04 (d, 3H, J ) 6.3 Hz); MS (APCI⁺) m/z 324 [MH]⁺.

Anal. (C₂₀H₂₅N₃O) C, H, N.

intact cellular system would provide valuable confirma-

tion of the activity as the isolated enzyme differs in some

important aspects from the sGC present in intact cells.

Thus the potency of NO is at least 10-fold greater in

intact cells than against isolated enzyme. Further,

inactivation of sGC in cells is far faster (seconds) than

for purified enzyme.¹¹

A comprehensive SAR study has been accomplished

which demonstrates the severely restricted allowable

modifications of the C-3 dimethylaminopropyloxy sub-

stituent. However substituted pyrazoles can replace the

indazole ring of benzydamine maintaining the enzyme

potency. More specifically aryl-substituted pyrazole

analogues such as 33, 43, and 48 show enhanced

potency over benzydamine, and a pharmacophore hy-

pothesis has been proposed. Evaluation of compounds

in the cell-based inhibition of platelet aggregation

unveiled a general lipophilicity requirement for cell

membrane penetration. Compounds 22, 32, 34, and 43

were subsequently identified as sGC activators and

potent inhibitors of platelet aggregation. Pharmacoki-

netic and selectivity studies have established that com-

pound 32 (CFM1571) shows modest oral bioavailability

(12%) and is selective over a number of related enzymes.

In conclusion CFM1571 (32) has been identified as the

first potent activator of sGC which is selective over

phosphodiesterases and does not act as an NO donor.

Further optimization of both pharmacokinetic param-

eters and potency is currently under investigation.

1-Ben zyl-3-(4-ter t-bu toxyca r bon yla m in obu tyloxy)-1H-

in d a zole (7). To indazole 4 (0.5 g, 2.23 mmol) in anhydrous

DMF (20 mL) was added sodium hydride (60% dispersion in

mineral oil) (110 mg, 2.68 mmol) and the mixture was stirred

under nitrogen at room temperature for 15 min. The reaction

mixture was then heated to 100 °C while 4-bromobutylamine

tert-butylcarbamate (620 mg, 2.45 mmol) in anhydrous DMF

(10 mL) was added to the reaction mixture by syringe over a

30 min period. Once the reaction mixture reached 100 °C it

was kept at this temperature for 2 h. The reaction mixture

was then cooled to room temperature and stirred overnight.

Water (20 mL) was added and the mixture was extracted with

diethyl ether (3 × 30 mL). The combined organic extracts were

dried (MgSO₄), filtered and concentrated under reduced pres-

sure. The crude product was purified by flash chromatography

using cyclohexane/EtOAc (70:30) to give 7 as a white solid

Exp er im en ta l Section

Gen er a l. All starting materials were either commercially

available or reported previously in the literature unless noted.

Solvents and reagents were used without further purification

except THF which was dried over sodium. Reactions were

monitored by TLC on precoated silica gel plates (Kieselgel 60

1

(481 mg, 55%): mp 53-54 °C; H NMR (300 MHz, CDCl₃) δ

7.67 (d, 1H, J ) 8.1 Hz), 7.34-7.24 (m, 5H), 7.17 (d, 2H, J )

8.5 Hz), 7.04 (t, 1H, J ) 7.5 Hz), 5.40 (s, 2H), 4.68 (br s, 1H),

4.41 (t, 2H, J ) 6.3 Hz), 3.23 (m, 2H), 1.90 (m, 2H), 1.71 (m,

2H), 1.46 (s, 9H); MS (EI) m/z 395 [M]⁺. Anal. (C₂₃H₂₉N₃O₃)

C, H, N.

F

₂₅₄, Merck). Purification was performed by flash chromatog-

raphy using silica gel (particle size 40-63 µM, Merck). ¹H and

¹³C NMR spectra were recorded on a Bruker AMX-300 or

Bruker AMX-400 spectrometer. Chemical shifts are reported

as ppm (δ) relative to TMS as internal standard. Mass spectra

were recorded on either a VG ZAB SE spectrometer (electron

impact and FAB) or a Micromass Quattro electrospray LC-

mass spectrometer. IR spectra were recorded on a Perkin-

Elmer 1600 series FT-IR spectrophotometer. Melting points

were determined on a Gallenkamp melting point apparatus

and are uncorrected. Microanalysis was carried out by the

Analytical Services Section, Department of Chemistry, Uni-

versity College London.

1-Ben zyl-3-(3-ter t-b u t oxyca r b on yla m in op r op yloxy)-

1H-in d a zole (8). From 4 (0.5 g, 2.23 mmol) and 3-bromopro-

pylamine tert-butylcarbamate (0.58 g, 2.45 mmol) using the

same process as for compound 7. The crude product was

purified by flash chromatography using cyclohexane/EtOAc

(70:30) to give 8 as a white solid (0.61 g, 72%) which was

recrystallized from diethyl ether: mp 92-93 °C; ¹H NMR (300

MHz, CDCl₃) δ 7.67 (d, 1H, J ) 8.1 Hz), 7.36-7.22 (m, 7H),

7.05 (dt, 1H, J ) 0.73, 7.4 Hz), 5.40 (s, 2H), 5.06 (br s, 1H),

4.48 (t, 2H, J ) 6.1 Hz), 3.35 (q, 2H, J ) 6.1 Hz), 2.05 (q, 2H,

1-Ben zyl-3-(2-dim eth ylam in oeth yloxy)-1H-in dazole (5).

To 1-benzyl-3-hydroxy-1H-indazole (4) (0.5 g, 2.23 mmol) in

anhydrous DMF (20 mL) was added sodium hydride (60%

dispersion in mineral oil) (0.11 g, 2.68 mmol) and the mixture

was stirred under nitrogen at room temperature for 30 min.

Simultaneously 2-dimethylaminoethyl chloride hydrochloride

(0.35 g, 2.45 mmol) was stirred with sodium hydride (60%

dispersion in mineral oil) (0.09 g, 2.23 mmol) in anhydrous

DMF (10 mL) for 30 min. This mixture was then added to the

flask containing the indazole and the reaction mixture was

heated to 100 °C for 3 h. The reaction mixture was cooled to

room temperature. Water (20 mL) was added and the mixture

was extracted with diethyl ether (3 × 50 mL). The combined

organic extracts were dried (MgSO₄), filtered and concentrated

under reduced pressure. The crude product was purified by

flash chromatography using 0.88 ammonia/EtOH/EtOAc (1:

5:94) to give 5 as a pale yellow oil (436 mg, 66%): ¹H NMR

(300 MHz, CDCl₃) δ 7.61 (d, 1H, J ) 7.7 Hz), 7.22-7.15 (m,

4H), 7.09-7.06 (m, 3H), 6.97-6.91 (m, 1H), 5.31 (s, 2H), 4.43

(t, 2H, J ) 5.6 Hz), 2.74 (t, 2H, J ) 5.6 Hz), 2.29 (s, 6H); MS

(EI) m/z 296 [MH]⁺. Anal. (C₁₈H₂₁N₃O) C, H, N.

J

) 6.3 Hz), 1.46 (s, 9H); MS (EI) m/z 381 [M]⁺. Anal.

(C₂₂H₂₇N₃O₃) C, H, N.

1-Ben zyl-3-(5-ter t-b u t oxyca r b on yla m in op en t yloxy)-

1H-in d a zole (9). From 4 (0.5 g, 2.23 mmol) and 5-bromopen-

tylamine tert-butylcarbamate (0.65 g, 2.45 mmol) using the

same process as for compound 7. The crude product was

purified by flash chromatography using cyclohexane/EtOAc

(70:30) to give 9 as a clear oil (0.68 g, 76%): ¹H NMR (300

MHz, CDCl₃) δ 7.68 (d, 1H, J ) 8.1 Hz), 7.34-7.21 (m, 4H),

7.17 (br d, 3H, J ) 8.1 Hz), 7.04 (m, 1H), 5.40 (s, 2H), 4.57 (br

s, 1H), 4.39 (t, 2H, J ) 6.6 Hz), 3.16 (m, 2H), 1.89 (t, 2H, J )

6.8 Hz), 1.56-1.55 (m, 4H), 1.46 (s, 9H); MS (EI) m/z 409 [M]⁺.

Anal. (C₂₄H₃₁N₃O₃‚0.5H₂O) C, H, N.

1-Ben zyl-3-(4-dim eth ylam in obu tyloxy)-1H-in dazole (10).

To 7 (200 mg, 0.51 mmol) in diethyl ether (10 mL) was added

1 M HCl in diethyl ether (1.0 mL, 1.0 mmol) and the reaction

was stirred overnight at room temperature. Two further

equivalents of 1 M HCl in diethyl ether (1.0 mL, 1.0 mmol)

were added and the reaction mixture stirred for a further 48

h. A white solid precipitated out of solution which was filtered

Novel Pyrazoles/ Indazoles as Activators of sGC

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 1 87

and washed with diethyl ether. To the crude amine (72 mg,

0.22 mmol) in MeOH (5 mL) were added NaCNBH₃, 1 M in

THF (0.32 mL, 0.32 mmol) and glacial acetic acid (0.2 mL).

Formaldehyde (20 mg, 0.66 mmol) in MeOH (2 mL) was then

added to the reaction and the mixture was stirred overnight

at room temperature. The MeOH was removed under reduced

pressure. Water (20 mL) was added to the residue which was

subsequently washed with EtOAc (2 × 10 mL), then saturated

with sodium carbonate and extracted again with EtOAc (3 ×

20 mL). The combined organic layers were dried (MgSO₄) and

concentrated under reduced pressure to give 10 as a pale

yellow oil (67 mg, 41%): ¹H NMR (300 MHz, CDCl₃) δ 7.66 (d,

1H, J ) 8.1 Hz), 7.35-7.26 (m, 4H), 7.20-7.16 (m, 3H), 7.05

(m, 1H), 5.39 (s, 2H), 4.42 (t, 2H, J ) 5.5 Hz), 2.85 (t, 2H, J )

7.5 Hz), 2.58 (s, 6H), 1.90 (m, 4H); MS (EI) m/z 322 [M - H]⁺.

Anal. (C₂₀H₂₅N₃O‚2HCl‚0.5H₂O) C, H, N.

1-Ben zyl-3-(5-d im et h yla m in op en t yloxy)-1H -in d a zole

(11). From 9 (123 mg, 0.30 mmol) using the same process as

for compound 10 except 50% TFA in CH₂Cl₂at room temper-

ature for 1 h was used in place of 1 M HCl in diethyl ether, to

give 11 as a yellow oil (12 mg, 12%): ¹H NMR (300 MHz, CDCl₃

) δ 7.66 (d, 1H, J ) 8.1 Hz), 7.34-7.24 (m, 4H), 7.19-7.16 (m,

3H), 7.04 (t, 1H, J ) 7.4 Hz), 5.40 (s, 2H), 4.39 (t, 2H, J ) 6.3

Hz), 2.88-2.82 (m, 2H), 2.64 (s, 6H), 1.91-1.83 (m, 2H), 1.81-

1.73 (m, 2H), 1.62-1.51 (m, 2H); MS (FAB) m/z 338 [MH]⁺.

Anal. (C₂₁H₂₇N₃O‚0.5CF₃COOH‚0.5H₂O) C, H, N.

1-Ben zyl-3-(3-a m in op r op yloxy)-1H-in d a zole (12). Com-

pound 8 (0.083 g, 0.22 mmol) was stirred in 50% TFA in CH₂-

Cl₂(3 mL) at room temperature for 1 h. The solvent was

removed under reduced pressure. The residue was taken up

in EtOAc (20 mL) washed with saturated NaHCO₃(30 mL)

and saturated brine (30 mL) then concentrated under reduced

pressure. The crude product was purified by flash chromatog-

raphy using 0.88 ammonia/MeOH/CHCl₃(1:5:94) to give 12

as a colorless oil (46 mg, 75%): ¹H NMR (300 MHz, CDCl₃) δ

7.56 (d, 1H, J ) 8.3 Hz), 7.27-7.06 (m, 7H), 6.96 (t, 1H, J )

7.5 Hz), 5.27 (s, 2H), 4.83 (br s, 2H), 4.40 (t, 2H, J ) 5.8 Hz),

3.00 (t, 2H, J ) 6.6 Hz), 2.08 (quintet, 2H, J ) 6.2 Hz); MS

(EI) m/z 281 [M]⁺. Anal. (C₁₇H₁₉N₃O) C, H, N.

3-(3-Dim eth yla m in op r op yloxy)-1H-in d a zole (13). To

3-indazolinone (10.0 g, 74.6 mmol), 3-dimethylamino-1-pro-

panol (9.0 mL, 76.1 mmol), and tributylphosphine (18.5 mL,

75.0 mmol) in toluene (600 mL) was added 1,1′-(azodicarbonyl)-

dipiperidine (19.0 g, 75.3 mmol) portionwise. The mixture was

heated at 80 °C for 15 h. The cooled mixture was filtered and

the residue was washed with EtOAc (200 mL). The combined

filtrate was washed with 10% aqueous NaOH (2 × 200 mL)

and water (200 mL). The organic layer was dried (MgSO₄) and

concentrated under reduced pressure. The crude material was

purified by flash chromatography using 0.88 ammonia/MeOH/

CHCl₃(2:20:78) to give 13 as an oil (5.40 g, 33%): ¹H NMR

(300 MHz, CDCl₃) δ 7.69 (dt, 1H, J ) 8.0, 0.9 Hz), 7.36 (ddd,

1H, J ) 8.4, 6.8, 1.1 Hz), 7.30 (dt, 1H, J ) 8.4, 0.9 Hz), 7.08

(ddd, 1H, J ) 7.9, 6.7, 1.2 Hz), 4.47 (t, 2H, J ) 6.4 Hz), 2.56

(br t, 2H, J ) 7.5 Hz), 2.32 (s, 6H), 2.26-2.07 (m, 2H); ¹³C

NMR (67.8 MHz, CDCl₃) δ 156.9, 142.3, 127.1, 119.6, 119.0,

112.1, 109.6, 67.0, 56.1, 45.0 (2C), 27.1; MS (FAB) m/z 220

[MH]⁺. Anal. (C₁₂H₁₇N₃O‚0.3H₂O) C, H, N.

3-(3-Dim eth yla m in op r op yloxy)-1-(2-p h en yleth yl)-1H-

in d a zole (15). From 13 (127 mg, 0.50 mmol) and 1-bromo-2-

phenylethane (0.2 mL, 1.4 mmol). Purified by flash chroma-

tography using MeOH/CHCl₃(3:97) to give 15 as an oil (51

mg, 32%): ¹H NMR (300 MHz, CDCl₃) δ 7.66 (dt, 1H, J ) 8.0,

0.9 Hz), 7.31-7.14 (m, 6H), 7.07-6.99 (m, 2H), 4.47 (t, 2H, J

) 6.5 Hz), 4.40 (br t, 2H, J ) 7.5 Hz), 3.14 (br t, 2H, J ) 7.5

Hz), 2.58 (br t, 2H, J ) 7.6 Hz), 2.34 (s, 6H), 2.15-2.06 (m,

2H); ¹³C NMR (75.5 MHz, CDCl₃) δ 155.9, 141.4, 138.8, 128.8

(2C), 128.5 (2C), 127.1, 126.5 120.0, 118.8, 112.6, 108.5, 67.3,

56.5, 50.0, 45.5 (2C), 36.2, 27.5; MS (APCI⁺) m/z 324 [MH]⁺.

Anal. (C₂₀H₂₅N₃O‚0.5H₂O) C, H, N.

1-(3-Dim et h yla m in op r op yl)-3-(3-d im et h yla m in op r o-

p yloxy)-1H-in d a zole (16). From 13 (106 mg, 0.48 mmol) and

1-chloro-3-dimethylaminopropane hydrochloride (150 mg, 0.73

mmol of free amine). Purified by flash chromatography using

0.88 ammonia/MeOH/CHCl₃(1:10:89) to give 16 as an oil (117

mg, 80%): ¹H NMR (300 MHz, CDCl₃) δ 7.64 (dt, 1H, J ) 7.1,

0.9 Hz), 7.31 (ddd, 1H, J ) 8.5, 6.6, 1.1 Hz), 7.24 (dt, 1H, J )

8.5, 1.0 Hz), 6.98 (ddd, 1H, J ) 7.8, 6.5, 1.1 Hz), 4.41 (t, 2H,

J ) 6.5 Hz), 4.20 (t, 2H, J ) 6.8 Hz), 2.48 (br t, 2H, J ) 7.5

Hz), 2.24 (s, 6H), 2.17 (s, 6H), 2.09-1.92 (m, 4H); ¹³C NMR

(75.5 MHz, CDCl₃) δ 155.5, 141.3, 126.8, 119.8, 118.5, 112.2,

108.5, 67.1, 56.4, 56.3, 45.8, 45.4 (2C), 45.3 (2C), 27.6, 27.5;

MS (FAB) m/z 305 [MH]⁺. Anal. (C₁₇H₂₈N₄O) C, H, N.

1-Acet yl-3-(3-d im et h yla m in op r op yloxy)-1H -in d a zole

(17). To a solution of 13 (298 mg, 1.36 mmol) in dry CH₂Cl₂(6

mL) was added acetic anhydride (0.30 mL, 3.10 mmol) and

the mixture was stirred at room temperature for 48 h. The

reaction mixture was diluted with CH₂Cl₂(10 mL) and washed

successively with 1 M aqueous NaOH (5 mL) and water (5 mL).

The organic layer was dried (MgSO₄) and concentrated under

reduced pressure. The crude material was purified by flash

chromatography using MeOH/CHCl₃(5:95) to give 17 as an

oily solid (164 mg, 54%): ¹H NMR (300 MHz, CDCl₃) δ 8.39

(dt, 1H, J ) 8.3, 1.0 Hz), 7.67 (dt, 1H, J ) 7.9, 1.0 Hz), 7.55

(ddd, 1H, J ) 8.3, 7.2, 1.2 Hz), 7.31 (ddd, 1H, J ) 7.9, 7.3, 0.8

Hz), 4.50 (t, 2H, J ) 6.5 Hz), 2.66 (s, 3H), 2.53 (br t, 2H, J )

7.4 Hz), 2.31 (s, 6H), 2.10-2.03 (m, 2H); ¹³C NMR (75.5

MHz, CDCl₃) δ 203.0, 159.1, 140.3, 130.1, 123.9, 119.6, 117.7,

115.7, 67.6, 56.2, 45.4 (2C), 27.1, 22.8; IR (film) 1706 (CdO)

cm^-1; MS (FAB) m/z 262 [MH]⁺. Anal. (C₁₄H₁₉N₃O₂‚0.1H₂O)

C, H, N.

1-Be n zoyl-3-(3-d im e t h yla m in op r op yloxy)-1H -in d a -

zole (18). To a solution of 13 (182 mg, 0.83 mmol) and pyridine

(0.13 mL, 1.57 mmol) in CH₂Cl₂(3 mL) at -30 °C was added

benzoyl chloride (0.11 mL, 0.92 mmol) and the mixture was

allowed to warm to room temperature over 6 h. The reaction

mixture was diluted with CH₂Cl₂(10 mL) and washed suc-

cessively with 1 M aqueous NaOH (5 mL) and water (5 mL).

The organic layer was dried (MgSO₄) and concentrated under

reduced pressure. The crude material was purified by flash

chromatography using a 0-3% gradient of MeOH/CHCl₃to

give 18 as an oil (112 mg, 42%): ¹H NMR (300 MHz, CDCl₃)

δ 8.53 (br d, 1H, J ) 8.4 Hz), 8.12 (dt, 2H, J ) 7.0, 1.5 Hz),

7.72 (br d, 1H, J ) 7.9 Hz), 7.64-7.46 (m, 4H), 7.37 (br t, 1H,

J ) 7.6 Hz), 4.45 (t, 2H, J ) 6.5 Hz), 2.48 (br t, 2H, J ) 7.1

Hz), 2.26 (s, 6H), 2.08-1.99 (m, 2H); ¹³C NMR (75.5 MHz,

CDCl₃) δ 167.2, 159.5, 141.5, 133.7, 131.5, 130.7, 130.2, 129.3,

127.7, 127.6, 124.3, 119.6, 117.5, 116.2 67.5, 55.7, 44.7 (2C),

26.4; IR (film) 1676 (CdO) cm^-1; MS (FAB) m/z 324 [MH]⁺.

Anal. (C₁₉H₂₁N₃O₂‚0.1H₂O) C, H, N.

3-(3-Dim et h yla m in op r op yloxy)-1-(2-p r op yl)-1H-in d a -

zole (14). To a solution of indazole 13 (100 mg, 0.46 mmol) in

THF (3.5 mL) were added successively t-BuOK (77 mg, 0.69

mmol) and 2-bromopropane (80 µL, 0.92 mmol). The reaction

mixture was refluxed for 4 h, then allowed to cool and

concentrated under reduced pressure. The crude residue was

purified by flash chromatography using 5-10% MeOH/CHCl₃

to give 14 as an oil (72 mg, 60%): ¹H NMR (300 MHz, CDCl₃)

δ 7.65 (dt, 1H, J ) 8.0, 0.9 Hz), 7.34-7.25 (m, 2H), 7.01 (ddd,

1H, J ) 7.9, 6.5, 1.2 Hz), 4.65 (dt, 1H, J ) 13.3, 6.7 Hz), 4.44

(t, 2H, J ) 6.5 Hz), 2.56-2.39 (m, 2H), 2.30 (s, 6H), 2.10-2.01

(m, 2H), 1.49 (d, 6H, J ) 6.7 Hz); ¹³C NMR (75.5 MHz, CDCl₃)

δ 155.4, 140.4, 126.6, 120.0, 118.6, 112.7, 108.6, 67.2, 56.5, 49.7,

45.4 (2C), 27.5, 21.7 (2C); MS (FAB) m/z 278 [MH]⁺. Anal.

(C₁₅H₂₃N₃O‚0.1H₂O) C, H, N.

The following compounds were prepared similarly.

3-(3-Dim eth yla m in op r op yloxy)-1-m eth ylsu lfon yl-1H-

in d a zole (19). From 13 (100 mg, 0.46 mmol), pyridine (0.22

mL, 2.66 mmol), and methanesulfonyl chloride (85 µL, 1.1

mmol) in dry CH₂Cl₂(3 mL) at room temperature for 20 h.

Purified by flash chromatography using 0.88 ammonia/MeOH/

CHCl₃(1:5:94) to give 19 as a white solid (64 mg, 47%): mp

1

35-36 °C; H NMR (300 MHz, CDCl₃) δ 7.99 (d, 1H, J ) 8.5

Hz), 7.72 (d, 1H, J ) 8.0 Hz), 7.56 (t, 1H, J ) 7.6 Hz), 7.36 (t,

1H, J ) 7.6 Hz), 4.56 (t, 2H, J ) 6.5 Hz), 3.07 (s, 3H), 2.53 (t,

2H, J ) 7.3 Hz), 2.31 (s, 6H), 2.13-2.01 (m, 2H); ¹³C NMR

(75.5 MHz, CDCl₃) δ 161.2, 142.6, 130.2, 124.1, 120.3, 117.5,

The following compounds were prepared similarly.

88 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 1

Selwood et al.

113.8, 67.9, 55.9, 45.1 (2C), 38.7, 26.8; MS (APCI⁺) m/z 298

ammonia/MeOH washings were combined and concentrated

under reduced pressure. The crude product was purified by

flash chromatography using 0.88 ammonia/MeOH/CHCl₃(2:

10:88) to give 24 (100 mg, 20%): ¹H NMR (300 MHz, CDCl₃)

δ 7.73 (d, 1H, J ) 3 Hz), 7.61 (m, 2H), 7.41 (m, 3H), 7.20 (m,

1H), 5.90 (d, 1H, J ) 3 Hz), 4.30 (t, 2H, J ) 4.3 Hz), 2.48 (t,

2H, J ) 8 Hz), 2.27 (s, 6H), 2.00 (m, 2H); ¹³C NMR (67.8 MHz,

CDCl₃) δ 167.6, 140.2, 129.3, 127.6, 125.2, 117.8, 93.6, 67.6,

56.4, 45.5, 27.5; MS (EI) m/z 246 [MH]⁺. Anal. (C₁₄H₁₉N₃O‚

0.1H₂O) C, H, N.

[MH]⁺. Anal. (C₁₃H₁₉N₃O₃S) C, H, N.

1-Dim e t h yla m in oca r b on yl-3-(3-d im e t h yla m in op r o-

p yloxy)-1H-in d a zole (20). From 13 (114 mg, 0.52 mmol),

pyridine (72 µL, 0.89 mmol), and dimethylcarbamyl chloride

(62 µL, 0.68 mmol) in dry CH₂Cl₂(2 mL) at reflux for 15 h.

Purified by flash chromatography using a 3-10% gradient of

MeOH/CHCl₃to give 20 as an oil (65 mg, 43%): ¹H NMR (300

MHz, CDCl₃) δ 8.02 (dt, 1H, J ) 8.6, 0.9 Hz), 7.63 (dt, 1H, J

) 8.0, 1.0 Hz), 7.46 (ddd, 1H, J ) 8.4, 7.1, 1.2 Hz), 7.18 (ddd,

1H, J ) 8.0, 7.1, 0.8 Hz), 4.45 (t, 2H, J ) 6.5 Hz), 3.21 (s, 6H),

2.49 (br t, 2H, J ) 7.1 Hz), 2.26 (s, 6H), 2.09-2.00 (m, 2H);

¹³C NMR (75.5 MHz, CDCl₃) δ 157.5, 154.3, 142.5, 129.2, 122.3,

119.5, 115.7, 114.9, 67.5, 56.3, 45.5 (2C), 38.8 (2C), 27.3; IR

(film) 1680 (CdO) cm^-1; MS (APCI⁺) m/z 291 [MH]⁺. Anal.

(C₁₅H₂₂N₄O₂) C, H, N.

1-Ben zyl-3-(3-d im et h yla m in op r op yloxy)-5-m et h oxy-

ca r bon yl-1H-p yr a zole (25). To 3-dimethylamino-1-propanol

(5.61 mL, 47.4 mmol) in toluene (250 mL) were added 1-benzyl-

3-hydroxy-5-methoxycarbonyl-1H-pyrazole²⁶(11.0 g, 47.4 mmol)

and tributylphosphine (9.6 g, 11.7 mL, 47.4 mmol) followed

by 1,1′-(azodicarbonyl)dipiperidine (13.1 g, 52.1 mmol) por-

tionwise. The mixture was heated to 100 °C for 2 h then cooled

to room temperature and the reduced ADDP product was

filtered off and washed with EtOAc (100 mL). The filtrate was

extracted with 1 M HCl (2 × 100 mL) and the combined acidic

extracts were washed with EtOAc (3 × 100 mL) and then

basified with 0.88 ammonia solution (40 mL). This was

extracted with CH₂Cl₂(3 × 100 mL) and the combined organic

phase was dried (MgSO₄) and concentrated under reduced

pressure to give 16.2 g of a semisolid which still contained

some reduced ADDP. The crude product was dissolved in THF

(300 mL) and 20 equivalents of Dowex 50W X8 resin (200 g, 1

mol) was added. The mixture was stirred gently for 1 h then

filtered and washed with methanol. The resin was then stirred

in piperidine/methanol (5:95) for 3 h, filtered and the process

repeated twice. The piperidine/methanol combined washes

were concentrated under reduced pressure and the resulting

residue was filtered through silica using MeOH/CHCl₃(5:95)

3-Hyd r oxy-1-p h en yl-1H-in d a zole (21). A solution of di-

phenylcarbamoyl chloride (10 g, 43.16 mmol) in acetone (20

mL) was added dropwise to a solution of NaN₃(4.2 g, 64.61

mmol) in H₂O (14 mL) at 0 °C. The mixture was allowed to

stir overnight at room temperature. The layers were then

separated and the aqueous layer was extracted with EtOAc

(2 × 100 mL). The combined organic material was dried

(MgSO₄) and concentrated under reduced pressure to give the

crude diphenylcarbamoyl azide as a solid (8.84 g, 86%). The

material was used without further purification. A solution of

diphenylcarbamoyl azide (4.0 g, 16.79 mmol) in xylenes (40

mL) was heated to reflux for 5 h. The solution was then cooled

to 0 °C. The precipitate that formed was collected to give 21

as a white solid (1.21 g, 34%): mp 206-207 °C (lit.²⁵mp 206-

1

207 °C); H NMR (300 MHz, DMSO-d₆) δ 7.16 (br t, 1H, J )

7.5 Hz), 7.26 (br t, 1H, J ) 7.4 Hz), 7.49 (m, 3H), 7.69 (br d,

2H, J ) 7.4 Hz), 7.77 (m, 2H), 11.25 (br s, 1H); ¹³C NMR (67.8

MHz, CDCl₃) δ 156.64, 140.58, 139.55, 129.85 (2C), 128.73,

125.13, 120.97 (2C), 120.87, 120.67, 115.10, 110.68; MS (EI)

m/z 210 [M]⁺. Anal. (C₁₃H₁₀N₂O) C, H, N.

1

to give 25 as a solid (1.24 g, 39%): mp 141-142 °C; H NMR

(300 MHz, CDCl₃) δ 7.27 (m, 5H), 6.22 (s, 1H), 5.59 (s, 2H),

4.25 (t, 2H, J ) 6 Hz), 3.84 (s, 3H), 3.15 (m, 2H), 2.76 (s, 6H),

2.34 (m, 2H); ¹³C NMR (67.8 MHz, CDCl₃) δ 137.1, 132.7, 128.5,

127.7, 127.5, 95.4, 66.0, 55.6, 54.4, 52.0, 43.0, 24.4; MS (EI)

m/z 318 [MH]⁺. Anal. (C₁₇H₂₃N₃O₃) C, H, N.

3-(3-Dim e t h yla m in op r op yloxy)-1-p h e n yl-1H -in d a -

zole (22). Indazole 21 (0.50 g, 2.38 mmol) was added portion-

wise as a solid to a solution of NaH (60% dispersion in mineral

oil, 0.10 g, 2.5 mmol) in DMF (20 mL) at room temperature.

The mixture was stirred for 30 min, then a further portion of

NaH (0.10 g, 2.5 mmol) was added and stirring was continued

for another 15 min. 3-Dimethylaminopropyl chloride hydro-

chloride (0.41 g, 2.59 mmol) was then added and the mixture

was heated to 100 °C for 2 h. The mixture was cooled to

room temperature and the solvent was removed under re-

duced pressure. The crude residue was recrystallized (EtOH/

H₂O) to give 22 as a solid (0.44 g, 63%): mp 63.5-64.5 °C;

¹H NMR (300 MHz, CDCl₃) δ 7.71 (m, 4H), 7.46 (m, 3H),

7.26 (m, 1H), 7.15 (m 1H), 4.54 (t, 2H, J ) 6.5 Hz), 2.53 (t,

2H, J ) 7.6 Hz), 2.30 (s, 6H), 2.10 (m, 2H); ¹³C NMR (67.8

MHz, CDCl₃) δ 158.57, 141.57, 140.99, 130.22 (2C), 129.03,

126.03, 122.45 (2C), 121.23, 121.14, 115.75, 110.99, 68.38,

57.37, 46.48 (2C), 28.47; MS (EI) m/z 295 [M]⁺. Anal.

(C₁₈H₂₁N₃O) C, H, N.

1-Ben zyl-3-(3-d im eth yla m in op r op yloxy)-1H-p yr a zole

(23). From 3-dimethylaminopropyl chloride hydrochloride (158

mg, 1.0 mmol) and 1-benzyl-3-hydroxy-1H-pyrazole²⁶(174 mg,

1.0 mmol) using the same process as for compound 7 to give

23 (19 mg, 7%): ¹H NMR (300 MHz, CDCl₃) δ 7.27 (m, 5H),

7.15 (d, 1H, J ) 2 Hz), 5.67 (d, 2H, J ) 2 Hz), 5.13 (s, 2H),

4.17 (t, 2H, J ) 6 Hz), 2.46 (t, 2H, J ) 8 Hz), 2.26 (s, 6H), 1.95

(m, 2H); MS (EI) m/z 260 [MH]⁺. Anal. (C₁₅H₂₁N₃O‚0.3H₂O)

C, H, N.

1-Ben zyl-3-(3-d im eth yla m in op r op yloxy)-1H-p yr a zole-

5-ca r boxylic Acid (26). To 25 (2.43 g, 7.66 mmol) in MeOH

(24 mL) was added 2.5 M aqueous NaOH solution (4 mL, 10

mmol) and the reaction mixture stirred at room temperature

overnight. The MeOH was removed under reduced pressure

and water (10 mL) was added. The solution was added to an

AG1 X8 (OH^-) form ion-exchange column and eluted with

water (100 mL) and 0.1 M aqueous HCl. Fractions containing

the product were concentrated under reduced pressure to give

26 as a solid (1.60 g, 69%): mp 169-174 °C; ¹H NMR (300

MHz, D₂O) δ 7.29 (m, 3H), 7.12 (m, 2H), 6.32 (s, 1H), 5.56 (s,

2H), 4.14 (t, 2H, J ) 6 Hz), 3.14 (m, 2H), 2.72 (s, 6H), 2.11 (m,

2H); ¹³C NMR (67.8 MHz, D₂O) δ 168.5, 163.6, 143.9, 140.3,

131.2, 130.1, 129.3, 101.1, 95.5, 69.4, 57.7, 55.5, 45.2, 26.2; MS

(FAB) m/z 304 [MH]⁺. Anal. (C₁₆H₂₁N₃O₃‚0.12HCl) C, H, N.

1-Ben zyl-3-(3-d im eth yla m in op r op yloxy)-5-m eth yla m i-

n oca r bon yl-1H-p yr a zole (27). To 26 (1 M solution in DMF,

0.5 mL, 0.5 mmol), 40% aqueous methylamine (0.5 mL) and

DIPEA (2 M solution in DMF, 2 mL, 2 mmol) was added TBTU

(1 M solution in DMF, 0.7 mL, 0.7 mmol) and the reaction

mixture stirred at room temperature overnight. The DMF was

removed under reduced pressure (∼1 mmHg) and the residue

taken up in saturated brine (20 mL), made basic with 0.88

ammonia and extracted with THF (3 × 20 mL). The THF

extracts were dried (Na₂SO₄) and purified by flash chroma-

tography using 0.88 ammonia/EtOH/CHCl₃(2:14:84) to give

27 (26 mg, 16%): ¹H NMR (300 MHz, CDCl₃) δ 7.28 (m, 5H),

5.93 (br s, 1H), 5.87 (s, 1H), 5.61 (s, 2H), 4.17 (t, 2H, J ) 6

Hz), 2.92 (d, 3H, J ) 5 Hz), 2.43 (m, 2H), 2.24 (s, 6H), 1.93

(m, 2H); MS (EI) m/z 317 [MH]⁺. Anal. (C₁₇H₂₄N₄O₂‚0.3CHCl₃)

C, H, N.

3-(3-Dim e t h yla m in op r op yloxy)-1-p h e n yl-1H -p yr a -

zole (24). From 3-hydroxy-1-phenyl-1H-pyrazole²⁷(320 mg,

2.0 mmol) and 3-dimethylamino-1-propanol (206 mg, 2.0 mmol)

using the same process as for compound 13. The workup

employed involved the addition of MeOH (20 mL) and Dowex

50W X8 (4 g) to the mixture, which was then swirled at room

temperature for 1 h. The mixture was filtered and the resin

washed with MeOH (50 mL). The resin was suspended in 0.88

ammonia/MeOH (15:85) (20 mL) and swirled for 30 min. Then

filtered and washed with the same mixture (20 mL). The

The following compounds were prepared similarly.

1-Ben zyl-3-(3-dim eth ylam in opr opyloxy)-5-dipr opylam i-

n oca r bon yl-1H-p yr a zole (28). From 26 (102 mg, 0.33 mmol)

and dipropylamine (33 mg, 0.33 mmol). The reaction mixture

Novel Pyrazoles/ Indazoles as Activators of sGC

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 1 89

1

was heated to 110 °C and the following workup was employed.

The crude reaction mixture was cooled and water (1 mL) was

added followed by MeOH (5 mL) and Dowex 50W X8 ion-

exchange resin (3 g). The mixture was stirred for 30 min,

filtered and washed with MeOH/H₂O (90:10) (10 mL). The

product was eluted from the resin with MeOH/0.88 ammonia

(70:30) (10 mL). Fractions containing product were concen-

trated under reduced pressure and purified by flash chroma-

tography using 0.88 ammonia/MeOH/CHCl₃(2:10:88) to give

28 as an oil (39.5 mg, 31%): ¹H NMR (300 MHz, CDCl₃) δ

7.26 (m, 5H), 5.71 (s, 1H), 5.28 (s, 2H), 4.19 (t, 2H, J ) 6 Hz),

3.31 (t, 2H, J ) 7 Hz), 3.03 (t, 2H, J ) 8 Hz), 2.45 (t, 2H, J )

8 Hz), 2.26 (s, 6H), 1.95 (m, 2H), 1.52 (m, 2H), 1.34 (m, 2H),

0.89 (t, 3H, J ) 7 Hz), 0.72 (t, 3H, J ) 7 Hz); ¹³C NMR (67.8

MHz, CDCl₃) δ 162.1, 161.6, 137.4, 137.1, 128.4, 127.7, 127.6,

91.0, 67.7, 56.3, 53.8, 50.3, 46.7, 45.5, 27.5, 21.8, 20.4, 11.4,

10.9; MS (EI) m/z 387 [MH]⁺. Anal. (C₂₂H₃₄N₄O₂‚0.2H₂O) C,

H, N.

1-Be n zyl-3-(3-d im e t h yla m in op r op yloxy)-5-(d im e t h -

yla m in oca r bon yl)-1H-p yr a zole (29). From 26 (306 mg, 1.0

mmol) and dimethylamine (45 mg, 1 mmol) using NMP as

solvent at 100 °C. Purification by flash chromatography using

0.88 ammonia/MeOH/CHCl₃(1:5:94) to give 29 as an oil (125

mg, 38%): ¹H NMR (300 MHz, CDCl₃) δ 7.25 (m, 5H), 5.74 (s,

1H), 5.29 (s, 2H), 4.18 (t, 2H, J ) 6.4 Hz), 2.96 (s, 3H), 2.76 (s,

3H), 2.45 (t, 2H, J ) 8 Hz), 2.25 (s, 6H), 1.94 (m, 2H); ¹³C NMR

(67.8 MHz, CDCl₃) δ 161.5, 137.4, 136.5, 128.4, 127.8, 127.6,

91.8, 67.6, 56.3, 53.9, 45.5, 38.7, 35.0, 27.5; MS (EI) m/z 331

[MH]⁺. Anal. (C₁₈H₂₆N₄O₂‚0.2H₂O) C, H, N.

49%): mp 70-72 °C; H NMR (300 MHz, CDCl₃) δ 7.57 (m,

2H), 7.42-7.26 (m, 3H), 5.92 (s, 1H), 4.13 (t, 2H, J ) 6 Hz),

2.41 (t, 2H, J ) 7 Hz), 2.21 (s, 6H), 1.90 (m, 2H); ¹³C NMR

(67.8 MHz, CDCl₃) δ 130.2, 128.9, 128.4, 125.4, 87.3, 67.8, 56.3,

45.4, 27.5; MS (FAB) m/z 246 [MH]⁺. Anal. (C₁₄H₁₉N₃O) C, H,

N.

1-Be n zyl-3-(3-d im e t h yla m in op r op yloxy)-5-p h e n yl-

1H-p yr a zole (34) a n d 1-Ben zyl-5-(3-d im eth yla m in op r o-

p yloxy)-3-p h en yl-1H-p yr a zole (35). Benzyl alcohol (132 mg,

0.126 mL, 1.22 mmol), tributylphosphine (246 mg, 0.3 mL, 1.22

mmol) and 33 (300 mg, 1.22 mmol) were dissolved in toluene

(10 mL) and 1,1-azobisdimethylformamide (211 mg, 1.22

mmol) added and the reaction mixture was stirred overnight.

Further equivalents of benzyl alcohol, tributylphosphine and

1,1-azobisdimethylformamide were added and the reaction

mixture stirred for 4 h, then MeOH (20 mL) and Dowex 50W

X8 (4 g) were added and the mixture swirled at room

temperature for 1 h. The mixture was filtered and the resin

washed with MeOH (50 mL). The resin was suspended in 0.88

ammonia/MeOH (15:85) (20 mL) and swirled for 30 min then

filtered and washed with the same mixture (20 mL). The

ammonia/MeOH washings were combined and concentrated

under reduced pressure. The products were purified by flash

chromatography using MeOH/CHCl₃(8:92) to give 34 as a solid

(90 mg, 22%): mp 60-61 °C; ¹H NMR (300 MHz, CDCl₃) δ

7.42-7.20 (m, 8H), 7.08 (m, 2H), 5.80 (s, 1H), 5.18 (s, 2H), 4.22

(t, 2H, J ) 7 Hz), 2.48 (t, 2H, J ) 8 Hz), 2.26 (s, 6H), 1.97 (m,

2H); MS (EI) m/z 336 [MH]⁺. Anal. (C₂₁H₂₅N₃O) C, H, N. Also

obtained was 35 as an oil (146 mg, 36%): ¹H NMR (300 MHz,

CDCl₃) δ 7.78 (d, 2H, J ) 7 Hz), 7.43-7.21 (m, 8H), 5.88 (s,

1H), 5.22 (s, 2H), 4.14 (t, 2H, J ) 6 Hz), 2.34 (t, 2H, J ) 6 Hz),

2.22 (s, 6H), 1.93 (m, 2H); ¹³C NMR (67.8 MHz, CDCl₃) δ 155.1,

149.6, 137.3, 134.0, 128.5, 128.5, 127.5, 127.4, 127.3, 125.3,

82.5, 70.0, 55.9, 50.9, 45.4, 27.2; MS (EI) m/z 336 [MH]⁺. Anal.

(C₂₁H₂₅N₃O‚0.5H₂O) C, H; N: calcd, 12.20; found, 12.75.

1,5-Dip h en yl-3-h yd r oxy-1H-p yr a zole (36). A solution of

t-BuOK (1.0 M in THF, 41 mL, 41 mmol) was added to a

mixture of phenylhydrazine (2.0 mL, 20.33 mmol) in THF (350

mL) at room temperature. To this was added ethyl phenyl-

propiolate (3.40 mL, 20.59 mmol) and the mixture stirred at

room temperature overnight, then heated to reflux for 4 h. The

solution was allowed to cool to room temperature and the

solvent was removed under reduced pressure. The residue was

dissolved in water and extracted with CH₂Cl₂(2 × 100 mL).

The aqueous layer was acidified with concentrated HCl and

then the solid was collected by filtration. The crude solid was

recrystallized (cyclohexane/CH₂Cl₂) to give 36 as a white solid

1-Ben zyl-3-(3-d im eth yla m in op r op yloxy)-5-ben zyla m i-

n oca r bon yl-1H-p yr a zole (30). From 26 (306 mg, 1 mmol)

and benzylamine (107 mg, 1 mmol) using NMP as solvent at

100 °C. Purification by flash chromatography using 0.88

ammonia/MeOH/CHCl₃(1:5:94) to give 30 as a solid (125 mg,

1

32%): mp 78-80 °C; H NMR (300 MHz, CDCl₃) δ 7.28 (m,

10H), 6.19 (m, 1H), 5.90 (s, 1H), 5.63 (s, 2H), 4.54 (d, 2H, J )

6 Hz), 4.17 (t, 2H, J ) 6 Hz), 2.42 (t, 2H, J ) 7 Hz), 2.23 (s,

6H), 1.92 (m, 2H); ¹³C NMR (67.8 MHz, CDCl₃) δ 137.7, 137.6,

128.8, 128.4, 127.7, 127.5, 91.1, 67.7, 56.3, 54.1, 45.4, 43.5, 27.4;

MS (EI) m/z 393 [MH]⁺. Anal. (C₂₃H₂₈N₄O₂) C, H, N.

1-Ben zyl-3-(3-dim eth ylam in opr opyloxy)-5-(N-m or ph oli-

n oca r bon yl)-1H-p yr a zole (31). From 26 (102 mg, 0.33 mmol)

and morpholine (29 mg, 0.33 mmol). The workup followed that

described for 28 to give 31 as a solid (53 mg, 43%): mp 75-76

1

°C; H NMR (300 MHz, CDCl₃) δ 7.32-7.19 (m, 5H), 5.70 (s,

1H), 5.32 (s, 2H), 4.21 (t, 2H, J ) 6 Hz), 3.59 (m, 4H), 3.27 (m,

2H), 3.17 (m, 2H), 2.46 (t, 2H, J ) 8 Hz), 2.27 (s, 6H), 1.96 (m,

2H); ¹³C NMR (67.8 MHz, CDCl₃) δ 160.9, 137.5, 128.6, 127.9,

127.8, 91.9, 67.7, 66.4, 56.3, 53.9, 45.5, 27.5; MS (EI) m/z 373

[MH]⁺. Anal. (C₂₀H₂₈N₄O₃) C, H, N.

1

(1.75 g, 36%): mp 251-253 °C; H NMR (DMSO-d₆) δ 7.18-

6.96 (m, 10H), 5.75 (s, 1H); MS (EI) m/z 236 [M]⁺.

3-Hyd r oxy-5-tr iflu or om eth yl-1H-p yr a zole (37). A solu-

tion of ethyl 4,4,4-trifluoro-2-butynoate (0.50 g, 3.01 mmol) and

hydrazine hydrate (0.15 mL, 3.09 mmol) in EtOH (5 mL) was

stirred at room temperature for 15 h. The solvent was removed

under reduced pressure and the residue was purified by flash

chromatography using MeOH/CHCl₃(15:85) to give 37 as a

solid (263 mg, 57%): mp 205-208 °C; ¹H NMR (300 MHz,

DMSO-d₆) δ 12.84 (br, 1H), 11.25 (br, 1H), 5.67 (s, 1H); MS

(EI) m/z 152 [M⁺].

The following compounds were prepared similarly.

3-Hyd r oxy-5-(3-fu r yl)-1H-p yr a zole (38). From ethyl 3-(3-

furyl)-3-oxopropanoate (0.50 g, 2.75 mmol). Precipitation of the

reaction mixture afforded 38 as a white solid (0.21 g, 50%):

1-Be n zyl-3-(3-d im e t h yla m in op r op yloxy)-5-(4-m e t h -

oxyp h en yla m in oca r bon yl)-1H-p yr a zole (32). To 26 (1.51

g, 5.0 mmol), 4-methoxyaniline (0.61 g, 5.0 mmol) and DIPEA

(1.29 g, 1.74 mL, 10 mmol) in THF (20 mL) was added O-(7-

azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluoro-

phosphate (1.90 g, 5 mmol) and the reaction was heated to

100 °C for 1 h. The reaction mixture was cooled, poured onto

aqueous 1 M NaOH (100 mL) and extracted with EtOAc (3 ×

50 mL). The extracts were dried (Na₂SO₄) and the product

purified by flash chromatography using 0.88 ammonia/MeOH/

CHCl₃(2:10:88) to give 32 (1.4 g, 69%): ¹H NMR (300 MHz,

DMSO-d₆) δ 10.69 (br s, 1H), 7.75 (d, 2H, J ) 9.0 Hz), 7.43

(m, 4H), 7.31 (d, 1H, J ) 6.8 Hz), 7.05 (d, 2H, J ) 9.0 Hz),

6.80 (s, 1H), 5.73 (s, 2H), 4.30 (t, 2H, J ) 6.0 Hz), 3.88 (s, 3H),

3.32 (m, 2H), 2.90 (s, 3H), 2.88 (s, 3H), 2.27 (m, 2H); ¹³C NMR

(67.8 MHz, CDCl₃) δ 160.9, 157.7, 156.2, 138.4, 136.7, 131.6,

128.8, 127.7, 127.5, 122.6, 114.1, 93.1, 66.5, 66.5, 55.6, 54.1,

53.6, 42.3, 24.2; MS (FAB) m/z 409 [MH]⁺. Anal. (C₁₀H₁₇N₃O₃‚

0.1H₂O) C, H, N.

1

mp >200 °C dec; H NMR (DMSO-d₆) δ 11.80 (br, 1H), 9.49

(br, 1H), 7.99 (s, 1H), 7.70 (s, 1H), 6.79 (s, 1H), 5.67 (s, 1H);

MS (FAB) m/z 151[MH]⁺.

3-Hyd r oxy-5-(4-p yr id yl)-1H-p yr a zole (39). From ethyl

isonicotinoylacetate (1.0 g, 5.18 mmol). Precipitation of the

reaction mixture and recrystallization (EtOH/H₂O) afforded

1

39 (0.73 g, 88%): mp >250 °C; H NMR (DMSO-d₆) δ 12.44

(br, 1H), 9.81 (br, 1H), 8.58 (br s, 2H), 7.64 (d, 2H, J ) 5.5

3-(3-Dim e t h yla m in op r op yloxy)-5-p h e n yl-1H -p yr a -

zole (33). From 3-hydroxy-5-phenyl-1H-pyrazole²⁸(1.6 g, 10

mmol) as the starting material using the same process as for

compound 13. Purification by flash chromatography using 0.88

ammonia/EtOH/EtOAc (2:10:88) to give 33 as a solid (1.2 g,

Hz), 6.14 (br s, 1H); MS (EI) m/z 161 [M]⁺.

3-Hydr oxy-5-(3,4,5-tr im eth oxyph en yl)-1H-pyr azole (40).

From ethyl 3,4,5-trimethoxybenzoylacetate (2.0 g, 7.09 mmol).

Precipitation of the reaction mixture afforded 40 as a white

90 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 1

Selwood et al.

solid (1.47 mg, 78%): mp 230-232 °C; ¹H NMR (300 MHz,

DMSO-d₆) δ 7.02 (s, 2H), 5.96 (s, 1H), 3.86 (s, 6H), 3.71 (s,

3H); MS (EI) m/z 250 [M]⁺.

2H, J ) 6.9 Hz); MS (EI) m/z 335 [M]⁺. Anal. (C₁₇H₂₅N₃O₄‚

1.1H₂O) C, H, N.

3-(3-Dim e t h yla m in op r op yloxy)-5-b e n zyl-1H -p yr a -

zole (48). From 42 (0.50 g, 2.87 mmol) using the same process

as for compound 13. Purified by flash chromatography using

0.88 ammonia/MeOH/CHCl₃(1:10:89) followed by recrystalli-

zation (hexane) to give 48 as a solid (393 mg, 53%): mp 52-

Eth yl 3-Oxo-4-p h en ylbu tyr a te (41). Phenylacetyl chlo-

ride (5.0 mL, 37.81 mmol) was added dropwise to a solution

of 2,2-dimethyl-1,3-dioxolane-4,6-dione (Meldrum’s acid; 5.45

g, 37.81 mmol) and pyridine (6.2 mL) in CH₂Cl₂(70 mL) at 0

°C. The solution was stirred for a further 30 min at 0 °C, then

allowed to warm slowly to room temperature overnight. The

reaction mixture was then washed with 10% aqueous HCl (2

× 50 mL) and H₂O (50 mL). The organic layer was dried

(MgSO₄), filtered and concentrated under reduced pressure.

The crude residue was dissolved in EtOH (100 mL) and heated

to reflux for 4 h. The mixture was allowed to cool to room

temperature and then concentrated under reduced pressure.

The dark, oily residue was purified by flash chromatography

using EtOAc/cyclohexane (20:80) followed by Kugelrohr distil-

lation (oven temperature 130 °C/ca. 1 mmHg) to give 41 as a

pale yellow oil (5.68 g, 73%): ¹H NMR (300 MHz, CDCl₃) δ

7.30 (m, 5H), 4.18 (q, 2H, J ) 7.0 Hz), 3.84 (s, 2H), 3.46 (s,

2H), 1.27 (t, 3H, J ) 7.0 Hz); MS (EI) m/z [M]⁺206.

1

54 °C; H NMR (300 MHz, CDCl₃) δ 7.51-7.36 (m, 5H), 5.68

(s, 1H), 4.29 (t, 2H, J ) 6.4 Hz), 4.08 (s, 2H), 2.59 (t, 2H, J )

7.5 Hz), 2.40 (s, 6H), 2.08 (quintet, 2H, J ) 7.0 Hz); MS (EI)

m/z 259 [M]⁺. Anal. (C₁₅H₂₁N₃O‚0.65H₂O) C, H; N: calcd,

15.50; found, 15.03.

Meth yl 3-Dim eth yla m in o-2-p h en yla cr yla te (49). To a

solution of methyl phenylacetate (2.0 mL, 13.9 mmol) in THF

(30 mL) was added tert-butoxybis(dimethylamino)methane (3.2

mL, 15.5 mmol). The resultant solution was stirred at room

temperature for 15 h and then the solvent was removed under

reduced pressure. The residual oil was purified by flash

chromatography EtOAc/cyclohexane (20:80) followed by Kugel-

rohr distillation to give 49 as an oil (1.70 g 59%): bp 150 °C/

1

∼1 mmHg (oven); H NMR (300 MHz, CDCl₃) δ 7.58 (s, 1H),

7.36-7.20 (m, 5H), 3.64 (s, 3H), 2.68 (s, 6H); MS (FAB) m/z

206 [MH]⁺205.

3-Hyd r oxy-5-ben zyl-1H-p yr a zole (42). From ethyl 3-oxo-

4-phenylbutyrate (41) (2.0 g, 9.70 mmol) using the same

process as for compound 37. Precipitation of the reaction

mixture afforded 42 as a white solid (1.26 g, 75%): mp 196-

197 °C; ¹H NMR (DMSO-d₆) δ 11.39 (br s, 1H), 9.34 (br s, 1H),

7.32-7.17 (m, 5H), 5.21 (s, 1H), 3.78 (s, 2H); MS (EI) m/z 174

[M]⁺.

3-Hyd r oxy-4-p h en yl-1H-p yr a zole (50). A solution of 49

(1.0 g, 4.87 mmol) and hydrazine hydrate (0.26 mL, 5.36 mmol)

in EtOH (20 mL) was heated to reflux for 5 h. The cooled

solution was concentrated under reduced pressure and the

solid was recrystallized (MeOH) to give 50 (0.67 g, 86%): mp

221.5-223 °C; ¹H NMR (300 MHz, DMSO-d₆) δ 11.76 (br, 1H),

10.12 (br, 1H), 7.90 (s, 1H), 7.67 (d, 2H, J ) 7.4 Hz), 7.29 (t,

2H, J ) 7.7 Hz), 7.09 (t, 1H, J ) 7.4 Hz); MS (EI) m/z 160

[M]⁺.

1,5-Dip h en yl-3-(3-d im eth yla m in op r op yloxy)-1H-p yr a -

zole (43). From 36 (0.20 g, 0.85 mmol) using the same process

as for compound 13. Purified by flash chromatography using

0.88 ammonia/EtOH/EtOAc (1:5:94) to give 43 as an oil (0.39

g, 64%): ¹H NMR (300 MHz, CDCl₃) δ 7.2-7.1 (m, 10H), 5.9

(s, 1H), 4.2 (t, 2H, J ) 6.4 Hz), 2.4 (t, 2H, J ) 7.5 Hz), 2.15 (s,

6H), 1.85-1.81 (m, 2H); MS (EI) m/z 321 [M]⁺. Anal.

(C₂₀H₂₃N₃O) C, H, N.

3-(3-Dim e t h yla m in op r op yloxy)-4-p h e n yl-1H -p yr a -

zole (51). From 50 (0.40 g, 2.50 mmol) using the same process

as for 13. Purified by flash chromatography using 0.88

ammonia/MeOH/CHCl₃(1:10:89) to give 51 as a solid (0.48 g,

78%) which was recrystallized (hexane/CH₂Cl₂): mp 98.5-100

3-(3-Dim eth yla m in op r op yloxy)-5-tr iflu or om eth yl-1H-

p yr a zole (44). From 37 using the same process as for

compound 13. Purified by flash chromatography using 0.88

ammonia/MeOH/CHCl₃(1:5:94) followed by recrystallization

(hexane/CH₂Cl₂) to give 44 as a solid (0.16 g, 51%): mp 85.5-

86.5 °C; ¹H NMR (300 MHz, CDCl₃) δ 5.86 (s, 1H), 4.24 (t, 2H,

J ) 5.9 Hz), 2.66 (t, 2H, J ) 6.8 Hz), 2.44 (s, 6H), 2.06 (m,

2H); MS (EI) m/z 237 [M]⁺. Anal. (C₉H₁₄N₃OF₃) C, H, N.

1

°C; H NMR (300 MHz, CDCl₃) δ 7.68-7.65 (m, 3H), 7.36 (t,

2H, J ) 7.9 Hz), 7.21 (t, 1H, J ) 7.4 Hz), 4.36 (t, 2H, J ) 6.3

Hz), 2.52 (t, 2H, J ) 7.5 Hz), 2.29 (s, 6H), 2.04 (m, 2H); ¹³C

NMR (67.8 MHz, CDCl₃) δ 160.75, 132.61, 128.91, 127.89,

126.22, 126.05, 106.93, 67.72, 56.80, 45.80 (2C), 27.91; MS (EI)

m/z 245 [M]⁺. Anal. (C₁₄H₁₉N₃O) C, H, N.

2-Am in o-4-eth oxyca r bon ylp yr id in e (52). 2-Acetylami-

nopyridine-4-carboxylic acid³²(2.0 g, 11.1 mmol) and concen-

trated H₂SO₄(3 mL) in dry EtOH (20 mL) was refluxed for 6

h. The cooled mixture was basified with aqueous ammonia

solution and extracted with EtOAc (3 × 100 mL). The organic

layer was dried (MgSO₄) and concentrated under reduced

pressure. The crude material was purified by flash chroma-

tography using a 0-2% gradient of MeOH/CHCl₃to give 52

3-(3-Dim et h yla m in op r op yloxy)-5-(3-fu r yl)-1H -p yr a -

zole (45). From 38 (0.18 g, 1.20 mmol) using the same process

as for compound 13. Purified by flash chromatography using

0.88 ammonia/MeOH/CHCl₃(1:10:89) to give 45 as a solid (0.11

g, 39%) which was recrystallized (hexane/CH₂Cl₂): mp 100-

101 °C; ¹H NMR (300 MHz, CDCl₃) δ 7.70 (s, 1H), 7.49 (s, 1H),

6.59 (s, 1H), 5.86 (s, 1H), 4.22 (t, 2H, J ) 6.3 Hz), 2.47 (t, 2H,

J ) 7.4 Hz), 2.27 (s, 6H), 1.97 (m, 2H); ¹³C NMR (67.8 MHz,

CDCl₃) δ 143.95, 139.74, 117.06, 108.99, 87.57, 68.33, 56.64,

45.81 (2C), 27.84; MS (EI) m/z 235 [M]⁺. Anal. (C₁₂H₁₇N₃O₂)

C, H, N.

1

as a white solid (1.40 g, 75%): mp 85-86 °C; H NMR (300

MHz, CD₃OD) δ 8.05 (d, 1H, J ) 5.3 Hz), 7.16 (br d, 1H, J )

1.5 Hz), 7.09 (dd, 1H, J ) 5.3, 1.5 Hz), 4.40 (q, 2H, J ) 7.1

Hz), 1.42 (t, 3H, J ) 7.1 Hz); ¹³C NMR (75.5 MHz, CD₃OD) δ

166.7, 161.6, 148.9, 140.9, 112.4, 110.0, 62.6, 14.4; MS FAB

m/z 167 [MH]⁺.

3-(3-Dim eth yla m in op r op yloxy)-5-(4-p yr id yl)-1H-p yr a -

zole (46). From 39 (0.40 g, 2.48 mmol) using the same process

as for compound 13. Purified by flash chromatography using

0.88 ammonia/MeOH/CHCl₃(1:10:89) to give 46 as a solid (0.39

g, 64%) which was recrystallized (hexane/CH₂Cl₂): mp 103-

105 °C; ¹H NMR (300 MHz, CDCl₃) δ 8.76 (d, 2H, J ) 6.1 Hz),

7.66 (d, 2H, J ) 6.1 Hz), 6.18 (s, 1H), 4.36 (t, 2H, J ) 6.3 Hz),

2.65 (t, 2H, J ) 7.2 Hz), 2.44 (s, 6H), 2.13 (t, 2H, J ) 6.8 Hz);

13C NMR (67.8 MHz, CDCl₃) δ 164.00, 163.20, 151.07 (2C),

139.53, 120.48 (2C), 87.98, 69.33, 56.87, 46.20 (2C), 28.19; MS

(EI) m/z 246 [M]⁺. Anal. (C₁₃H₁₈N₄O) C, H, N.

2-Am in o-4-p yr id in em et h a n ol (53). To a solution of 52

(1.1 g, 6.62 mmol) in dry THF (20 mL) at 0 °C was added a 1

M solution of LiAlH₄in THF (8.6 mL, 8.6 mmol). The mixture

was stirred at room temperature for 2 h and an excess of

MeOH was added. The mixture was concentrated under

reduced pressure and the crude material purified by flash

chromatography using a 5-10% gradient of MeOH/CHCl₃to

give 53 as a white solid (721 mg, 90%): mp 43-44 °C; ¹H NMR

(300 MHz, CD₃OD) δ 7.84 (d, 1H, J ) 5.5 Hz), 6.63 (s, 1H),

6.60 (d, 1H, J ) 5.5 Hz), 4.54 (s, 2H); ¹³C NMR (75.5 MHz,

CD₃OD) δ 160.6, 154.4, 147.4, 111.9, 107.1, 63.4; MS (APCI⁺)

m/z 125 [MH]⁺. Anal. (C₆H₈N₂O) C, H, N.

3-(3-Dieth ylam in opr opyloxy)-5-ph en yl-1H-pyr azole (54).

From 3-hydroxy-5-phenyl-1H-pyrazole²⁸(0.25 g, 1.56 mmol)

and 3-diethylamino-1-propanol (0.205 g, 1.56 mmol) using the

same process as for compound 13 except the reaction was

carried out in THF at 70 ^ïC for 3.5 h and a Dowex resin workup

3-(3-Dim et h yla m in op r op yloxy)-5-(3,4,5-t r im et h oxy-

p h en yl)-1H-p yr a zole (47). From 40 (500 mg, 1.90 mmol)

using the same process as for 13. Purified by flash chroma-

tography using 0.88 ammonia/EtOH/EtOAc (1:10:89) to give

47 as an oil (507 mg, 79%): ¹H NMR (300 MHz, CDCl₃) δ 6.71

(s, 2H), 5.83 (s, 1H), 4.15 (t, 2H, J ) 6.3 Hz), 3.83 (s, 6H), 3.80

(s, 3H), 2.46 (t, 2H, J ) 7.4 Hz), 2.25 (s, 6H), 1.92 (quintet,

Novel Pyrazoles/ Indazoles as Activators of sGC

J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 1 91

was employed. Purified by flash chromatography using 0.88

ammonia/MeOH/CHCl₃(1:5:94) to give 54 (0.12 g, 30%): ¹H

NMR (300 MHz, CDCl₃) δ 7.50-7.47 (m, 2H), 7.40-7.27 (m,

3H), 5.91 (s, 1H), 4.17 (t, 2H, J ) 6.3 Hz), 2.61-2.49 (m, 6H),

1.95-1.86 (m, 2H), 0.10 (t, 6H, J ) 7.2 Hz); MS (EI) m/z 273

[M]⁺. Anal. (C₁₆H₂₃N₃O‚1H₂O) C, H, N.

(1:95:5) to give 60 as a clear oil (80 mg, 57%) which was

converted to its hydrochloride salt with HCl (1.0 M in diethyl

1

ether): mp 225 °C (HCl salt); H NMR (300 MHz, CDCl₃) δ

7.75 (d, 2H, J ) 8 Hz), 7.40-7.20 (m, 3H), 5.75 (s, 1H), 4.80

(br s, 2H), 4.10 (m, 2H), 2.20 (s, 6H), 2.15 (m, 2H), 2.05 (m,

2H); MS (APCI⁺) m/z 245 [MH]⁺. Anal. (C₁₄H₂₀N₄‚2HCl‚1H₂O)

C, H, N.

3-(1-Meth yl-3-piper idin yl)m eth yloxy-5-ph en yl-1H-pyr a-

zole (55). From 3-hydroxy-5-phenyl-1H-pyrazole²⁸(250 mg,

1.56 mmol) and 1-methyl-3-hydroxymethylpiperidine (202 mg,

1.56 mmol) using the same process as for compound 13 except

the reaction was carried out in THF at 70 °C for 3.5 h and a

Dowex resin workup was employed. The crude compound was

purified by flash chromatography using 0.88 ammonia/MeOH/

CHCl₃(1:5:94) followed by recrystallization (cyclohexane/

4-(5-P h en yl-1H-3-p yr a zolyl)bu ta n oic Acid (61). 7-Phen-

yl-5,7-diketoheptanoic acid³⁴(2 g, 8.5 mmol) and hydrazine

hydrate (2 mL) were refluxed in EtOH (20 mL). After 2 h the

cooled solution was concentrated under reduced pressure to

give 61 as a crude oil (1.8 g, 92%) which solidified on standing

and was used without further purification or characteriza-

tion: MS (APCI^-) m/z 229 [M - H]⁺.

1

EtOAc) to give 55 (138 mg, 33%): mp 110-111 °C; H NMR

N1,N1-Dim et h yl-4-(5-p h en yl-1H -3-p yr a zolyl)b u t a n a -

m id e (62). Crude compound 61 (1.5 g, 6.5 mmol) was treated

with thionyl chloride (5 mL) at 60 °C for 3 h then cooled and

concentrated under reduced pressure. The resulting dark oil

was suspended in diethyl ether and dimethylamine (2.0 M in

THF, 5 mL) and the mixture was stirred overnight. The

solution was concentrated under reduced pressure and purified

by flash chromatography using CH₂Cl₂/MeOH (95:5), to give

(300 MHz, CDCl₃) δ 7.55 (d, 2H, J ) 7.0 Hz), 7.45-7.33 (m,

3H), 5.96 (s, 1H), 4.07 (dd, 1H, J ) 5.9, 9.9 Hz), 4.00 (dd, 1H,

J ) 7.4, 9.6 Hz), 2.98 (d, 1H, J ) 11.0 Hz), 2.79 (d, 1H, J )

11.0 Hz), 2.28 (s, 3H), 2.14 (m, 1H), 1.94 (td, 1H, J ) 11.0, 2.9

Hz), 1.84-1.58 (m, 4H), 1.07 (m, 1H); MS (EI) m/z 273 [M +

2H]⁺. Anal. (C₁₆H₂₁N₃O) C, H, N.

5-P h en yl-3-[(2-a m in op yr id in -3-yl)m eth yloxy]-1H-p yr a -

zole (56). From 3-hydroxy-5-phenyl-1H-pyrazole²⁸(550 mg,

3.44 mmol) and 2-amino-3-pyridinemethanol³¹(640 mg, 5.2

mmol) using the same process as for 13 except the reaction

was carried out in dry THF at 80 °C for 15 h. The crude

material was purified by flash chromatography using a 0-5%

gradient of MeOH/CHCl₃followed by recrystallization (CH₂-

Cl₂/cyclohexane) to give 56 as a solid (64 mg, 7%): mp 151-

1

62 as a solid (450 mg, 20%): mp 148 °C; H NMR (300 MHz,

CDCl₃) δ 7.75 (d, 2H, J ) 8 Hz), 7.45-7.25 (m, 3H), 6.40 (s,

1H), 2.95 (s, 6H), 2.80 (t, 3H, J ) 6 Hz), 2.45 (m, 2H), 2.05 (m,

2H); MS (APCI⁺) m/z 258 [MH]⁺.

3-(4-Dim eth yla m in obu tyl)-5-p h en yl-1H-p yr a zole (63).

Compound 62 (150 mg, 0.58 mmol) was added to dioxane (20

mL) and LiAlH₄(1.0 M in THF, 2 mL) was added. The

resulting solution was heated at 80 °C for 6 h then cooled.

Dioxane moistened with water (2 mL) was added, followed by

1 M NaOH (1 mL) and the mixture heated at 80 °C for 45 min

then cooled and filtered. The filtrate was concentrated under

reduced pressure, toluene added and evaporation repeated.

Purification was by flash chromatography using 0.88 ammonia/

CH₂Cl₂/MeOH (1:89:10) to give 63 as a solid (120 mg, 82%):

1

152 °C; H NMR (300 MHz, CD₃OD) δ 7.84 (dd, 1H, J ) 5.2,

1.8 Hz), 7.57-7.52 (m, 3H), 7.36-7.23 (m, 3H), 6.60 (dd, 1H,

J ) 7.3, 5.2 Hz), 6.07 (s, 1H), 5.04 (s, 2H); MS (APCI⁺) m/z

267 [MH]⁺. Anal. (C₁₅H₁₄N₄O) C, H, N.

5-P h en yl-3-[(3-p yr r olid in -1-yl)-p r op yloxy]-1H -p yr a -

zole (57). From 3-hydroxy-5-phenyl-1H-pyrazole²⁸(500 mg,

3.13 mmol) and 3-pyrrolidin-1-yl-propan-1-ol³⁰(400 mg, 3.13

mmol) using the same process as for compound 13. Purified

by flash chromatography using 0.88 ammonia/EtOH/EtOAc (1:

5:94) followed by recrystallization (cyclohexane/EtOAc) to give

1

mp 91-93 °C; H NMR (300 MHz, CDCl₃) δ 7.75 (d, 2H, J )

8 Hz), 7.45-7.25 (m, 3H), 6.35 (s, 1H), 2.70 (t, 2H, J ) 7 Hz),

2.20 (s, 6H), 1.70 (m, 2H), 1.55 (m, 2H); MS (FAB) m/z 244

[MH]⁺. Anal. (C₁₅H₂₁N₃‚0.25H₂O) C, H, N.

1

57 as a solid (440 mg, 52%): mp 109-110 °C; H NMR (300

MHz, CDCl₃) δ 7.56 (d, 2H, J ) 7.2 Hz), 7.46-7.37 (m, 3H),

5.98 (s, 1H), 4.26 (t, 2H, J ) 6.4 Hz), 2.66 (t, 2H, J ) 7.6 Hz),

2.57 (br s, 4H), 2.09-2.00 (m, 2H), 1.81 (t, 4H, J ) 3.6 Hz);

MS (EI) m/z 271 [M]⁺. Anal. (C₁₆H₂₁N₃O) C, H, N.

sGC En zym e. Recombinant bovine sGC was obtained from

Sf21 cells infected with baculovirus carrying sGC cDNA.

Original clones containing bovine sGC R₁and â₁cDNA were

a kind gift from Doris Koesling (Germany). The cDNA for both

subunits was subcloned into p2Bac, a dual promoter baculovi-

rus vector (Invitrogen). Sf21 cells (1 × 10⁸) were seeded on 4-

× 600-cm²plates in 60 mL of complete TC 100 medium. Cells

were allowed to attach overnight and then medium removed.

40 mL of TC 100 medium, 6 mL of high-titer virus (>10⁸

plaque-forming units/mL) and 2.5 µg/mL hemin were added

and the cells were incubated at 27 °C for 48 h. The cells were

collected by centrifugation at 300g for 5 min. The cells were

washed three times in cold PBS and then resuspended in 10

mL of buffer A (25 mM TEA pH 7.5, 100 mM NaCl, 1 mM

EDTA, 0.2 mM benzamidine, 5 mM DTT). After sonication,

the particulate fraction was pelleted by centrifugation at

15000g for 10 min. The soluble fraction was then applied to a

1 mL Mono-Q column (Amersham-Pharmacia) that had been

equilibrated with buffer A. A 40-mL linear gradient of 100 mM

to 1 M NaCl in buffer A was used to elute sGC. The 1-mL

collected fractions were assayed for sGC activity using a cyclic

GMP ³H assay system (Amersham-Pharmacia). The peak

fractions of activity were pooled, made 50% with glycerol and

stored at -80 °C for subsequent use.

5-P h en yl-3-[(2-a m in op yr id in -4-yl)m eth yloxy]-1H-p yr a -

zole (58). From 3-hydroxy-5-phenyl-1H-pyrazole²⁸(300 mg,

1.88 mmol) and 53 (303 mg, 2.44 mmol) using the same process

as for compound 13 except the reaction was carried out in dry

THF at 80 °C for 15 h. Purified by flash chromatography using

a 2-4% gradient of MeOH/CHCl₃to give 58 as a solid (261

mg, 52%): mp 134-135 °C; ¹H NMR (300 MHz, CDCl₃) δ 8.08

(d, 1H, J ) 5.3 Hz), 7.56 (br d, 2H, J ) 6.8 Hz), 7.48-7.39 (m,

3H), 6.72 (br d, 1H, J ) 5.6 Hz), 6.63 (s, 1H), 6.05 (s, 1H), 5.21

(s, 2H); MS (APCI⁺) m/z 267 [MH]⁺. Anal. (C₁₅H₁₄N₄O) C, H,

N.

N1,N1-Dim et h yl-3-[(5-p h en yl-1H-3-p yr a zolyl)a m in o]-

p r op a n a m id e (59). A mixture of 3-amino-5-phenyl-1H-pyra-

zole (159 mg, 1 mmol) and dimethylacrylamide (120 mg, 1.2

mmol) was heated at 80 °C for 2 h then allowed to cool.

Purification was by flash chromatography using CH₂Cl₂/MeOH

(95:5), to give 59 (175 mg, 68%): ¹H NMR (300 MHz, CDCl₃)

δ 7.80-7.70 (m, 2H), 7.50-7.30 (m, 3H), 5.75 (s, 1H), 4.70 (br

s, 2H), 4.18 (m, 2H), 3.00 (s, 3H), 2.95 (s, 3H), 2.95 (m, 2H);

MS (EI) m/z 258 [M]⁺.

3-(3-Dim et h yla m in op r op yla m in o)-5-p h en yl-1H -p yr a -

zole Dih yd r och lor id e (60). Lithium aluminum hydride

powder (40 mg, 1.05 mmol) was added to a solution of 59 (150

mg, 0.58 mmol) in dry dioxane (15 mL) and the mixture heated

at 80 °C for 6 h. After cooling, water (0.5 mL) was added

followed by 1.0 M NaOH solution (0.5 mL). The solution was

refluxed for 1 h then filtered and the precipitated salts washed

with more dioxane. The combined filtrates were concentrated

under reduced pressure and the crude product was purified

by flash chromatography using 0.88 ammonia/CH₂Cl₂/MeOH

sGC En zym e Assa y. Test compounds (10 mM) were

dissolved in DMSO and diluted with Tris buffer (40 mM trizma

hydrochlorate, 5 mM magnesium chloride hexahydrate, pH 7.4,

1 M NaOH). Into each well 10 µL of the test compound at 20×

the final concentration was added with 100 µL of reaction

buffer (120 µg/mL recombinant sGC, 1.1 mg/mL IBMX (Sigma),

2.6 mg/mL GTP (Sigma) in Tris buffer). The reaction was

started with the addition of 90 µL of 667 nM DEA/NO (RBI)

to give a reaction DEA/NO concentration of 300 nM. The plates

were then incubated at room temperature for 10 min. To

92 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 1

Selwood et al.

determine the amount of cGMP produced the Biotrak cGMP

enzyme immunoassay (EIA) system from Amersham was used

according to manufacturer’s instructions. Briefly the assay was

based upon the competition between unlabeled cGMP and a

fixed quantity of peroxidase labeled cGMP for a limited amount

of cGMP specific antibody. The peroxidase ligand that was

bound to the antibody was immobilized on precoated microtiter

wells and the amount of labeled cGMP was determined using

a one pot stabilized substrate. The concentration of unlabeled

cGMP in a sample was subsequently determined by interpola-

tion from a standard curve. The standard curve was prepared

in duplicate and each test compound was evaluated in dupli-

cate with at least n ) 2. The measured cGMP for each

compound was expressed as a percentage of the DEA/NO

response, and relative enzyme activities (REA) were calculated

as a ratio between the percentage DEA/NO response for the

compound and that for benzydamine (3).

Dose-response curves were carried out using an identical

assay except the recombinant sGC was stimulated by the

addition of 30 nM of PAPA/NO (Alexis Biochemicals). EC₅₀’s

were calculated by nonlinear regression and reported as means

( standard error of the mean of at least 3 independent

experiments. Data analysis was performed with Origin soft-

ware (Microcal Software).

P la telet Aggr ega tion Assa y. Platelets were prepared as

described previously.³⁹Briefly, fresh human blood was col-

lected into tubes as a 1:9 solution of sodium citrate solution

(3.15%):blood and centrifuged immediately at 260g for 20 min

to separate the red cells from the platelet-rich plasma (PRP).

The PRP was decanted and PGI₂(0.3 µg/mL, ICN Pharma-

ceuticals) in Tris buffer (0.05 M, pH 9) was added. The PRP

was then centrifuged at 180g for 10 min to sediment the

remaining red and white cells. The resulting PRP was de-

canted into a new tube, PGI₂(0.15 µg/mL) in Tris buffer was

added and centrifuged at 950g for 10 min to sediment the

platelets. The resultant platelet-poor plasma (PPP) was dis-

carded and the platelet pellet was resuspended in an equal

volume of Tyrodes buffer without calcium (140 mM NaCl, 3

mM KCl, 12 mM NaHCO₃, 0.4 mM NaH₂PO₄‚H₂O, 2 mM

MgCl₂‚6H₂O, 0.1% glucose, pH 7.4 with 0.05 M Hepes) by

gently pipetting up and down. The suspension was centrifuged

at 870g for 10 min at 4 °C. The supernatant was discarded

and the platelet pellet was resuspended in an equal volume

of Tyrodes buffer as before. The platelets were counted (using

a Coulter Counter model T540) and normalized to 250 000

cells/µL using Tyrodes buffer. The resultant suspension was

placed on ice for approximately 1 h until use.

Test compounds (10 mM) were dissolved in DMSO and

subsequent dilutions made in Tyrodes buffer. The final assay

concentration of DMSO did not exceed 0.1% (which is without

effect on platelet reactivity). Platelet aggregation was moni-

tored using either a Chrono-Log model 560-CA dual channel

or a 570-4S four-channel aggregometer (Chrono-Log Corp.,

Havertown, PA). Aggregation was analyzed by using 0.5-mL

aliquots of the platelet suspension at 37 °C using percent (%)

light transmittance. For each sample a baseline reading was

established for a 3-min period, followed by addition of test

compound or buffer. An EC₅₀dose of collagen (collagenreagent

Horm, Nycomed Arzneimittel GmbH, Munchen) was added 1

min later and the response measured 3 min after addition of

collagen. The amplitude of each aggregatory response, normal-

ized to the collagen control, was used to plot dose-response

curves. The concentration of drug that inhibited collagen-

induced platelet aggregation by 50% (IC₅₀) was calculated from

the dose-response curves and is presented as means (

standard error of the mean of at least 2 independent experi-

ments. Data analysis was performed with Origin software

(Microcal Software).

compounds and procedures were as follows: COX₁, diclofenac;⁴⁰

COX₂, NS398;⁴⁰PDE I, 8-methoxy-3-isobutyl-1-methylxan-

thine;⁴¹PDE II, erythro-9-(2-hydroxy-3-nonyl)adenine;⁴²PDE

III, milrinone;⁴³PDE IV, rolipram;⁴²PDE V, dipyridamole;⁴³

adenylate cyclase (stimulated), SQ 22,536;⁴⁴adenylate cyclase

(basal), forskolin;⁴⁴iNOS, L-NMMA;⁴⁵eNOS, L-NMMA;⁴⁶

nNOS, L-NMMA.⁴⁷

P h a r m a cok in etics Stu d ies. Pharmacokinetics studies

were carried out by TNO BIBRA (U.K.). Pharmacokinetics

parameters were determined in male Sprague-Dawley rats.

Compounds 32 and 43 were dosed at a level of 5 mg/kg

prepared in a PBS solution. For the animals dosed iv blood

samples were taken before dosing and at 0.25, 0.5, 1, 1.5, 2, 4,

6, and 8 h after dosing. For the animals dosed po blood samples

were taken before dosing and at 0.5, 1, 2, 3, 4, 6, 10 and 24 h

after dosing. Compounds 22 and 34 were dosed at a level of 2

mg/kg prepared in a PBS solution. For the animals dosed iv

blood samples were taken before dosing and at 5, 15 and 30

min and 1, 1.5, 2, 4, 6, and 8 h after dosing. For the animals

dosed po blood samples were taken before dosing and at 15

min and 0.5, 1, 2, 3, 4, 6 and 10 h after dosing.

Serum was separated by centrifugation and stored at -20

°C prior to analysis. For analysis, compounds were extracted

by liquid-liquid extraction with diethyl ether. Chromatogra-

phy was performed on a 3.9- × 150-mm Nova-Pak C18 column

(Waters Chromatography) using a mobile phase of acetonitrile/

methanol/0.25% ammonia solution 50:45:5. Mass spectrometry

was performed on a Micromass Quattro-LC QLC 9025 quad-

rupole instrument in the positive ion electrospray (ES+) mode.

Ack n ow led gm en t. This work was supported by a

generous grant from the BBSRC under the U.K. Gov-

ernment Technology Foresight initiative involving Tri-

pos Inc. (St. Louis, MO) and Automation Partnership

(Melbourn, Herts, U.K.).

Su p p or tin g In for m a tion Ava ila ble: Elemental analysis,

calculated log P values, and activity summary for compounds

22, 32, and 34 against various enzymes. This material is

available free of charge via the Internet at http://pubs.acs.org.

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J M001034K

Article Doi

Synthesis and biological evaluation of novel pyrazoles and indazoles as activators of the nitric oxide receptor, soluble guanylate cyclase

DOI: 10.1021/jm001034k

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Full text of DOI:10.1021/jm001034k

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