Identification of 1-(3-(6,7-dimethoxyquinazolin-4-yloxy)phenyl)-3-(5-(1,1, 1-trifluoro-2-methylpropan-2-yl)isoxazol-3-yl)urea hydrochloride (CEP-32496), a highly potent and orally efficacious inhibitor of V-RAF murine sarcoma viral oncogene homologue B1 (BRAF) V600E

Article

pubs.acs.org/jmc

Identification of 1-(3-(6,7-Dimethoxyquinazolin-4-yloxy)phenyl)-3-

5-(1,1,1-trifluoro-2-methylpropan-2-yl)isoxazol-3-yl)urea

(

Hydrochloride (CEP-32496), a Highly Potent and Orally Efficacious

Inhibitor of V-RAF Murine Sarcoma Viral Oncogene Homologue B1

(

BRAF) V600E

,†

†

Martin W. Rowbottom,* Raffaella Faraoni, Qi Chao, Brian T. Campbell, Andiliy G. Lai,

Eduardo Setti, Maiko Ezawa, Kelly G. Sprankle, Sunny Abraham, Lan Tran, Brian Struss,

Michael Gibney, Robert C. Armstrong, Ruwanthi N. Gunawardane, Ronald R. Nepomuceno,

Ianina Valenta, Helen Hua, Michael F. Gardner, Merryl D. Cramer, Dana Gitnick,

Darren E. Insko, Julius L. Apuy, Susan Jones-Bolin, Arup K. Ghose, Torsten Herbertz,

Mark A. Ator, Bruce D. Dorsey, Bruce Ruggeri, Michael Williams, Shripad Bhagwat,

Joyce James, and Mark W. Holladay

†

‡

†

^†Ambit Biosciences, 4215 Sorrento Valley Boulevard, San Diego, California 92121, United States

^‡Cephalon, Inc., 145 Brandywine Parkway, West Chester, Pennsylvania 19380, United States

*

S Supporting Information

ABSTRACT: The Ras/RAF/MEK/ERK mitogen-activated protein kinase (MAPK)

signaling pathway plays a central role in the regulation of cell growth, differentiation,

V600E

and survival. Expression of mutant BRAF

results in constitutive activation of the

MAPK pathway, which can lead to uncontrolled cellular growth. Herein, we describe

V600E

an SAR optimization campaign around a series of quinazoline derived BRAF

inhibitors. In particular, the bioisosteric replacement of a metabolically sensitive tert-

butyl group with fluorinated alkyl moieties is described. This effort led directly to the

identification of a clinical candidate, compound 40 (CEP-32496). Compound 40

V600E

exhibits high potency against several BRAF

mutant BRAF

-dependent cell lines and selective cytotoxicity for tumor cell lines expressing

V600E

versus those containing wild-type BRAF. Compound 40 also exhibits an excellent PK profile across multiple

preclinical species. In addition, significant oral efficacy was observed in a 14-day BRAF

xenograft mouse model, upon dosing at 30 and 100 mg/kg BID.

V600E

-dependent human Colo-205 tumor

rationale that inhibition of BRAF^V⁶⁰⁰^Eshould result in clinica₄l

benefit for patients with carcinomas expressing this enzyme.

As such, numerous reports describing small-molecule inhibitors

INTRODUCTION

The Ras/RAF/MEK/ERK mitogen-activated protein kinase

MAPK) signaling pathway plays a central role in the trans-

duction of signals from cell surface receptors to the nucleus,

regulating both cell proliferation and survival. The RAF

members of this pathway consist of the serine/threonine

kinases ARAF, BRAF, and CRAF (also known as RAF1), which

phosphorylate and activate downstream MEK1/2. A number of

activating mutations of the BRAF gene have been identified,

with a single point mutation (Val600 → Glu) accounting for

■

(

V600E

5

of BRAF

have emerged, with several investigational drugs,

1

including RAF265 (Chiron/Novartis, 1, Figure 1) and vemurafenib

(

PLX-4032, Daiichi Sankyo/Roche, 2, Figure 1) having advanced

6−9

into human clinical trials.

Some time ago, we initiated our own efforts to identify novel

V600E

inhibitors of BRAF

. To identify appropriate chemical start-

ing points, we employed the KINOMEscan technology plat-

form to screen the Ambit compound collection. The utiliza-

tion of KINOMEscan allows for the rapid screening of large

compound collections across several hundred individual human

more than 90% of those described. Constitutively activated

V600E

BRAF

is 500 times more active than wild-type BRAF and is

found to be present in approximately 7% of all cancers, being

most frequently associated with melanoma (50−70%), papillary

thyroid (∼50%), colorectal (∼15%), and ovarian (∼30%)

10

kinase targets (including mutants of known therapeutic value).

This technology represents an extremely powerful tool for aiding

in the identification of hit series for any given kinase target and

2

V600E

carcinomas. The presence of BRAF

in certain tumors is

correlated with increased malignancy and decreased response to

3

V600E

chemotherapy. It is therefore not surprising that BRAF

has emerged as a target of considerable interest, based on the

Received: July 25, 2011

Published: December 14, 2011

©

2011 American Chemical Society

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Article

Figure 1. Structures of known RAF inhibitors.

for gaining a greater understanding of the kinome selectivity

Scheme 1. Formation of Aminoisoxazoles from Ketonitriles

11

profile of such hits. Analysis of the data set obtained from

screening of the Ambit internal compound collection across the

KINOMEscan panel led to the identification of several hit series

exhibiting high affinity for BRAF^V⁶⁰⁰^E. One such series is

represented by the diaryl amide derivative 3 (Figure 2), and an

indicated a general preference for formation of 5-amino-

isoxazoles 6 (or their corresponding hydrolysis products) resulting

from initial attack of the hydroxylamine nitrogen on the keto

group of the ketonitrile. Nevertheless, these workers found

specific conditions (initial reaction at pH ∼ 8 at 60−100 °C,

followed by ring closure under acidic conditions at 70−100 °C)

1

under which, when R = tert-butyl, attack of the hydroxylamine

nitrogen on to the nitrile is favored, leading to formation of the

3

-aminoisoxazole product. However, under these conditions

1

the 5-amino product still prevailed for R = Me, isopropyl, and

Ph, suggesting a considerable steric contribution in the case of

R = tert-butyl. Consistent with the expectations from these

1

14

previously reported results, treatment of several ketonitriles

1

(

R = cyclopentyl, 5b; C(CF )Me , 5f; C(CH F) Me, 5j; and

3 2 2 2

tert-butyl) with hydroxylamine in the presence of an extra

equivalent of hydroxide resulted in predominant formation of

5-aminoisoxazoles (Scheme 2). In our hands, 5-aminoisoxazoles

Figure 2. Evolution of the 4-quinazolinyloxy-diaryl urea series of

V600E

b-Raf

inhibitors.

were also the predominant products when the starting pH was

1

initial hit to lead the optimization campaign described

elsewhere led to the identification of 4-quinazolinyloxy-diaryl

adjusted to ∼7.5 for R = isopropyl (5a), CFMe

2

(5c), CF

(5i). Some

2

Me

(5d), C(CN)Me

(5g), and cyclobutyl-1-CF

2

3

12

urea derivative 4. Herein we describe further optimization

of the 4-quinazolinyloxy-diaryl urea series, in particular SAR

around the left-hand aryl urea moiety. This effort ultimately led

to the discovery of the clinical candidate compound 40 (CEP-

combination of lower steric demand, compared with tert-butyl,

together with increased electrophilicity of the ketonitrile car-

bonyl when electron-withdrawing groups (F, CN) are present,

presumably contributes to disfavoring formation of the 3-amino

isomer under these conditions. This is despite the similarity to

the initial conditions in which the 3-amino isomer was ulti-

1

3

2496), a highly potent and orally efficacious inhibitor of

BRAF^V⁶⁰⁰^E.

1

14

mately formed when R = tert-butyl. For a couple of the above

1

R groups (C(CF )Me , 5f, and C(CH F) Me, 5j), initial

3

2

reaction in the presence of excess NaHCO in aqueous MeOH

3

followed by cyclization under acidic conditions resulted in good

yields of the corresponding 3-aminoisoxazoles. Interestingly,

these conditions also provided a good yield of the 3-amino

1

regioisomer when R = cyclopropyl-1-CF . Comparing this

3

1

result to those described above for R = cyclobutyl-1-CF , the

3

latter bicarbonate conditions seem to be particularly suitable

for favoring initial events (presumably reaction of the hydroxyl-

amine nitrogen with the nitrile group of the ketonitrile) that

lead ultimately to the 3-aminoisoxazole regiochemistry. 3-

CHEMISTRY

■

All compounds described herein were prepared as illustrated

in Schemes 2−5. Final derivatives incorporating substituted

isoxazole urea moieties were obtained via the corresponding inter-

mediate isoxazole carbamates. These isoxazole carbamates were

obtained from the corresponding aminoisoxazoles, which in

turn were prepared from substituted ketonitriles 5 (Schemes 1

and 2). Reaction of substituted ketonitriles with hydroxylamine

can result in the formation of regioisomeric 3- or 5-amino-

1

Aminoisoxazole derivatives 8h (R = isopropyl), 8i (R =

1

cyclopentyl), and 8j (R = CFMe ), inaccessible via the

2

aforementioned procedures, were prepared via alternate routes

involving protection of the ketonitrile carbonyl moiety, as

described in Scheme 3. Protection of the carbonyl of 5a and 5b

via conversion to the 1,3-dioxolane moiety gave intermediates

10a and 10b, respectively. Subsequent reaction of 10a and 10b

with hydroxylamine yielded exclusively the required amidoxime

14

isoxazoles (Scheme 1). Results summarized by Takase et al.

1

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a

Scheme 2. Synthesis of Isoxazol-5-yl-carbamates 7a−k and Isoxazol-3-yl-carbamates 9a−g

a

Reagents and conditions: (a) NaH, MeCN, THF, 75 °C, 15 h, 29−93%; (b) hydroxylamine hydrochloride, NaOH, water, 100 °C, 2.5 h, or

hydroxylamine sulfate, NaOH, water/EtOH, 80 °C, 15 h; or (for 6e) (i) TMSCHN , Et O, rt, 15 h, (ii) hydroxylamine hydrochloride, NaOMe,

2

MeOH, 60 °C, 15 h, (iii) HCl, aq EtOH, 60 °C, 2 h; 11−100%; (c) PhOC(O)Cl or 4-Cl-PhOC(O)Cl, K CO , THF, rt, 33−100%;

2

3

(

2

d) (i) hydroxylamine sulfate, NaHCO , water/MeOH, 65 °C, or hydroxylamine hydrochloride, NaOH, water/EtOH, 65 °C, (ii) conc HCl, reflux,

6−71%.

3

intermediates that upon treatment with concentrated HCl

underwent carbonyl deprotection followed by cyclization to

give the required 3-aminoisoxazole derivatives 8h and 8i, re-

spectively. All attempts to convert ketonitrile 5c to the corre-

sponding 1,3-dioxolane derivative failed. As a result, 5c was

converted to the corresponding methyl enol ether 11 via

treatment with (trimethylsilyl)diazomethane. Subsequent re-

action of 11 with hydroxylamine followed by heating in the

presence of HCl gave the desired 3-aminoisoxazole derivative

(trimethylsilyl)diazomethane. However, subsequent treatment

with hydroxylamine followed by heating in the presence of HCl

failed to yield any of the desired 5-(trifluoromethyl)isoxazole-3-

amine. Rather, for reasons that are unclear, only the corre-

sponding 3-(trifluoromethyl)isoxazole-5-amine (6e) was obtained

albeit in low yield (11%). All 5-aminoisoxazoles (6) and

3-aminoisoxazoles (8) were subsequently converted to the

corresponding isoxazole carbamates 7 and 9, respectively, via

reaction with either phenyl chloroformate or 4-chlorophenyl

chloroformate. The pyrazole carbamates required for the pre-

paration of the pyrazole ring containing urea derivatives were

prepared as described in Scheme 4. The reaction of substituted

ketonitriles with a variety of hydrazines in refluxing EtOH gave

5-aminopyrazoles 12 in moderate to good yields. Subsequent

treatment of 5-aminopyrazoles 12 with phenyl chloroformate

8

j, albeit in low yield (13%). The reason for the lower than

expected yield was not clearly apparent; however, enough 8j

was prepared to be utilized in the next step. In a similar manner,

an attempt was made to gain access to 5-(trifluoromethyl)-

isoxazole-3-amine. Thus, ketonitrile 5e was converted first to

4,4,4-trifluoro-3-methoxybut-2-enenitrile via treatment with

1

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a

Scheme 3. Synthesis of Isoxazol-3-yl-carbamates 9h−j

a

Reagents and conditions: (a) ethylene glycol, TMSCl, DCM, reflux, 15 h, 57−78%; (b) (i) hydroxylamine hydrochloride, NH /MeOH,

3

8

-hydroxyquinoline, 70 °C, 15 h, (ii) conc HCl, EtOH, reflux, 68−69% (over two steps); (c) PhOC(O)Cl, K CO , THF, rt, 77−89%;

2

3

(d) TMSCHN , Et O, rt, 15 h, 90%; (e) (i) hydroxylamine hydrochloride, NaOMe, MeOH, 70 °C, 16 h, (ii) conc HCl, 80 °C, 30 min, 13%;

2 2

(f) 4-Cl-PhOC(O)Cl, K CO , THF, rt, 100%.

2

3

a

Scheme 4. Synthesis of 1H-Pyrazol-5-yl-carbamates 13a−g

a

2

Reagents and conditions: (a) R NHNH , EtOH, reflux, 36−51%; (b) PhOC(O)Cl, K CO , DCM or THF, 0 °C or rt, 30−100%.

2

3

a

Scheme 5. Synthesis of Quinazoline Derivatives 18−51 and 54

a

Reagents and conditions: (a) X = O, (i) Cs CO , THF, rt, 30 min, (ii) 4-chloro-6,7-dimethoxyquinazoline, 50 °C, 24 h, 100%; X = S, (i) NaH,

2

3

THF, rt, 30 min, (ii) 4-chloro-6,7-dimethoxyquinazoline, rt, 15 h, 85%; (b) ArNCO, DMF, 50 °C; or 7a−k or 9a−j or 13a−g, DIEA, cat. DMAP,

THF, rt or 50 °C, 5−75%; (c) PhOC(O)Cl, K CO , THF, rt, 15 h, 89%; (d) 3-aminophenol, THF, 120 °C, 2 h, 20%; (e) (i) Cs CO , THF, rt, 1 h,

2

3

2

3

(

ii) 4-chloro-6,7-dimethoxyquinazoline, 55 °C, 15 h, 43%.

and potassium carbonate in THF or CH Cl gave the required

pyrazole carbamates 13. The desired urea compounds 18−51

and 54 were prepared as described in Scheme 5. 3-Aminophenol

(14) was reacted with cesium carbonate in THF to generate the

2

1

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Table 1. SAR of Quinazoline-Derived BRAF^V⁶⁰⁰^EInhibitors: Variation of the Aryl Urea Moiety

a

Results are presented as mean values from experiments performed in duplicate. Variability between individual values is less than 2-fold.

b

c

Competition binding assay performed at rt for 1 h. K values were determined using eleven serial 3-fold dilutions. Phosphorylation assay in A375 cells

d

V600E

d

V600E

(

expressing BRAF

). Cells were incubated with test compound for 2 h. Cell Titer Blue proliferation assay in A375 cells (expressing BRAF

).

e

Cells were incubated with test compound for 72 h. Compounds were screened at 10 μM concentration against the KINOMEscan panel consisting

of 290 individual wild-type kinases (mutants being excluded from this analysis). Selectivity S(10) scores represent the fraction of kinase targets

exhibiting >90% competition (measured as <10% of control experiment).

corresponding cesium phenoxide, which was coupled in situ

with 4-chloro-6,7-dimethoxyquinazoline to give 16 in quanti-

tative yield. Similarly, 3-aminobenzenethiol (15) was treated

first with sodium hydride in THF, followed by 4-chloro-6,7-

dimethoxyquinazoline to give 17 in high yield. Direct coupling

of 16 with 4-tert-butylphenyl isocyanate gave the 4-oxyquinazo-

line derivative 20, whereas 4-(dimethylamino)pyridine-catalyzed

coupling of 16 with phenyl 3-tert-butylphenylcarbamate gave

compound 21. 4-(Dimethylamino)pyridine-catalyzed coupling

of 16 with isoxazole carbamates 7a−k and 9a−j gave the corre-

sponding 4-oxyquinazoline derivatives 18 and 22−41. Over-

all, yields for the final coupling step proved reasonable;

however, for the case of the trifluoromethyl substituted

derivative 31, the recovery was low (5%) for reasons that are

unclear. The 4-(dimethylamino)pyridine-catalyzed coupling

of 17 with isoxazole carbamates 7c and 9a−c gave the cor-

responding 4-thioquinazoline derivatives 42−45. In an analo-

gous fashion, coupling of 16 with pyrazole carbamates 13a−g

gave the corresponding pyrazole containing 4-oxyquinazoline

derivatives 46−51. Pyrazole containing 4-oxyquinazoline

derivative 54 was prepared in three steps from 1-tert-butyl-

1H-pyrazol-4-amine 52.

RESULTS AND DISCUSSION

■

As part of our SAR campaign, all compounds described (Tables 1

and 2) were profiled in vitro for activity against BRAF

V600E

, both

in a biochemical binding assay and for their ability to inhibit

V600E

BRAF

-mediated phosphorylation of MEK (pMEK for-

mation) in the A375 cell line. In addition, each compound was

further profiled for its ability to inhibit the proliferation of A375

cells over a 72 h period. As part of this profiling process,

particular emphasis was placed on the potencies observed in the

A375 cellular assays. The A375 cell line is dependent on

V600E

signaling through BRAF

, and the observation of activity in

these assays, coupled with suitable ADME properties, would be

considered a reasonable predictive measure of activity in appro-

V600E

priate in vivo models of BRAF

-driven disease. We were

also interested in obtaining a preliminary understanding of the

1

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Table 2. SAR of Quinazoline-Derived BRAF^V⁶⁰⁰^EInhibitors 22−51

1

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Table 2. continued

a

b

Results are presented as mean values from experiments performed in duplicate. Variability between individual values is less than 2-fold. Competition

c

binding assay performed at rt for 1 h. K values were determined using eleven serial 3-fold dilutions. Phosphorylation assay in A375 cells (expressing

BRAF

incubated with test compound for 72 h. Compounds were screened at 10 μM concentration against the KINOMEscan panel consisting of 290 individual

wild-type kinases (mutants being excluded from this analysis). Selectivity S(10) scores represent the fraction of kinase targets exhibiting >90% com-

petition (measured as <10% of control experiment). Not determined due to biphasic dose−response curves. Complete inhibition was observed at 5000 nM.

d

V600E

d

V600E

). Cells were incubated with test compound for 2 h. Cell Titer Blue proliferation assay in A375 cells (expressing BRAF

). Cells were

e

f

selectivity profile across the human kinome. This was facilitated

and proliferation assays; however, potency in the proliferation

assay was still 10-fold lower than that observed with the lead

compound 4. Incorporation of either a 4-(tert-butyl)phenyl

urea (20) or a 3-(tert-butyl)phenyl urea moiety (21) led to a

10

by use of the KINOMEscan technology platform. Compounds

prepared were screened at 10 μM concentration across a diverse

panel of 290 individual wild-type kinases. Selectivity for each com-

pound is represented by a selectivity S(10) score, which equals the

fraction of kinase targets assayed exhibiting >90% competition

modest improvement in kinome selectivity. However, although

V600E

potent in both the BRAF

binding and A375 pMEK cellular

(

measured as <10% of control experiment), with lower S(10)

assays, these compounds again proved 5- to 10-fold less active

than lead compound 4 in the A375 proliferation assay. Due to

the promising cellular activity observed with the isomeric (tert-

butyl)isoxazole-containing derivatives 4 and 18, we decided to

investigate the effect of varying the tert-butyl moiety to other

functional groups. A range of different substituents were in-

10a

scores indicating greater selectivity.

Structure−Activity Relationships. The previously identi-

fied tert-butyl-isoxazole urea-containing quinazoline derivative 4

V600E

(

Table 1) exhibited high binding affinity for BRAF

(K =

d

7

3 nM), and potent inhibition of both BRAF^V⁶⁰⁰^E-mediated

pMEK activity (IC = 121 nM) and proliferation (IC = 328 nM)

corporated as summarized in Table 2 (compounds 22−41).

50

1

2

V600E

in A375 cells. Compound 4 also proved moderately selective

across the panel of 290 kinases, exhibiting an S(10) score of

Overall, it was observed that potency for BRAF

was

governed by both the size and lipophilicity of this substituent.

0.286. An initial scan of alternate aryl urea moieties (Table 1)

For example, substitution of the tert-butyl (4) for the smaller

indicated that the isoxazole regioisomer of 4, the 3-(tert-

butyl)isoxazole-5-urea derivative 18, was equipotent in both

binding and cellular assays and had a very similar S(10) score.

Substitution of the isoxazole moiety for an N-1-(tert-butyl)-

methyl group (22) led to a loss in both binding affinity (K =

d

678 nM) and cellular potency (IC₅₀> 15000 nM in both

assays). Compound 22 also exhibited reduced potencies across

the broader panel of kinase targets, as indicated by an S(10)

score of 0.062. Incorporation of the larger phenyl moiety,

pyrazole-4-urea motif gave compound 54, which also exhibited

V600E

good binding affinity for BRAF

and a somewhat improved

exemplified by compounds 25 and 28, also led to much

V600E

kinome selectivity profile. However, compound 54 proved to

be essentially inactive in the A375 cell proliferation assay. Com-

pared to 54, the related N-1-methyl-3-(tert-butyl)pyrazole-5-

urea derivative 19 proved more potent in both the A375 pMEK

reduced BRAF

binding affinity and cellular potencies

compared to the corresponding tert-butyl containing analogues

4 and 18, respectively. Again both compounds 25 and 28

exhibited much reduced affinity across the broader panel of

1

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kinases, with the S(10) score being 0.052 for both. Overall, it

was found that incorporation of substituents of similar size and

lipophilicity to the tert-butyl group gave analogues with a more

optimal potency profile. For instance, compared to 4, the

the isoxazole substituent is important for optimal activity. On

the basis of earlier observations, wherein differences in activity

were observed between corresponding isoxazole isomeric pairs,

we also investigated replacement of the tert-butyl group of

isoxazol-3-yl derivative 4 with select fluorine-containing alkyl

motifs. Again, key differences between isoxazole regioisomers

were observed. For instance, compared to 33, the correspond-

ing 5-(2-fluoropropan-2-yl)isoxazol-3-yl derivative 38 proved

approximately 2- and 4-fold less potent in the A375 pMEK and

proliferation assays, respectively. In contrast, both isoxazole-3-yl

derivatives 39 and 40 proved significantly more potent in cells

than the corresponding isoxazol-5-yl isomers 34 and 35, respec-

tively. In fact, the 5-(1,1,1-trifluoro-2-methylpropan-2-yl)-

isoxazol-3-yl derivative 40 proved one of the most potent com-

5-isopropylisoxazol-3-yl derivative (23) and the 5-cyclopenty-

lisoxazol-3-yl derivative (24) exhibited very similar potencies

V600E

toward BRAF

in both binding and cellular assays. How-

ever, incorporation of the more polar hydroxyl-bearing tert-

V600E

butyl group (29), although potent in the BRAF

binding

assay (K = 49 nM), surprisingly led to much reduced cellular

d

potencies. Other more polar substituents such as alkylamine-

bearing tert-butyl groups were also incorporated; however,

incorporation of such groups resulted in significantly lower

V600E

affinity for BRAF

(data not shown). Also worth noting is

V600E

the observation that the initial (tert-butyl)isoxazolyl isomers

and 18 exhibited a very similar potency profile, but such

pounds in this series, exhibiting potent binding (BRAF

4

K = 14 nM) and cellular activity (pMEK IC = 82 nM and

d

50

comparable activity of isoxazole regioisomers did not prove

to be universal. Activity differences between corresponding

isoxazole isomeric pairs depended upon the exact nature of the

isoxazole substituent. For instance, compared with the correspond-

ing 5-substituted isoxazol-3-yl isomers, the 3-isopropylisoxazol-5-yl

A375 proliferation IC₅₀= 78 nM), with activity in the prolif-

eration assay being approximately 4-fold greater than that

observed with the tert-butyl analogue 4.

The analogues described thus far contain an oxygen atom

linking the quinazoline ring with the central phenyl moiety. We

had previously observed that replacement of the oxygen linker

of 4 with the slightly larger and more lipophilic sulfur atom not

only yielded a derivative that proved equipotent in both the

binding and pMEK cellular assays but also exhibited an im-

(

26) and the 3-cyclopentylisoxazol-5-yl derivatives (27) both

exhibited reduced potencies in cells, particularly with respect to

the A375 proliferation assay.

As described, the (tert-butyl)isoxazole-containing derivatives

V600E

12

4

and 18 exhibited good potency against BRAF

. However,

proved S(10) score (0.18 vs 0.29). However, for some of the

in vitro metabolite profiling studies on compound 4 indicated

that, in both rat and human liver microsomes, hydroxylation of

the tert-butyl moiety and subsequent oxidation to the car-

boxylate occurred to a significant degree (data not shown). As

we had already demonstrated that the corresponding hydroxy-

lated tert-butyl-containing derivative (29) exhibits much reduced

cellular activity compared to the nonhydroxylated analogue 4, we

more potent derivatives described herein, replacement of oxygen

with sulfur did not result in greatly improved selectivity profiles,

and in certain cases, decreased activity was observed in the A375

proliferation assay (compare 43 with 40, and 45 with 33). The

complete loss of proliferation activity for 43 could not be readily

explained. However, a similar drop-off in proliferation activity

was observed for certain other closely related sulfur-linked

analogues (data not shown).

15

were interested in finding replacements for the tert-butyl group

V600E

that would both be well tolerated by BRAF

yet be less prone

In addition to the described isoxazole urea-containing

derivatives, we had earlier identified a related 3-tert-butyl-1H-

to such oxidative metabolic processes. It was clear from earlier

SAR that the incorporation of polar groups was not an attractive

option. However, we expected that the selective incorporation

of fluorine would lead to more stable derivatives with similar

potency profiles. A carbon−fluorine bond is often used as a

bioisosteric replacement for a corresponding carbon−hydrogen

bond, due to its similar size and lipophilicity. More importantly,

due to the high electronegativity of fluorine, carbon−fluorine

bonds are much stronger and exhibit a differential dipole-

moment compared to the corresponding carbon−hydrogen

bond. As such, the strategic introduction of a carbon−fluorine

bond into a bioactive molecule can often have positive effects

on overall metabolic stability, while maintaining the desired

pyrazol-5-yl urea derivative 19 that displayed modest potency

V600E

toward BRAF

(K = 87 nM, pMEK IC = 336 nM).

d 50

A number of additional pyrazole-containing analogues were

prepared, represented by compounds 46−51. Overall, SAR

around the pyrazole N-1 substituent indicated that incorpo-

ration of larger alkyl moieties such as isopropyl (46), isobutyl

(47), or cyclohexyl (48), although increasingly more selective

as represented by decreasing S(10) scores, proved less potent in

both the pMEK and proliferation cell assays. Only upon incor-

poration of a phenyl group (49) did we observe robust pMEK

cell activity (IC = 21 nM), although less than optimal activity

50

was observed in the A375 proliferation assay. In addition, 49

exhibited decreased selectivity (S(10) = 0.40). Although signifi-

cant effort was applied toward the optimization of the pyrazole

series, a suitable balance between potency, selectivity, and PK

profile ultimately proved elusive.

16

potency profile at the target of interest. With this in mind, we

prepared a number of fluoroalkyl substituted isoxazole deriv-

atives (compounds 31−36 and 38−45). Direct replacement of

the tert-butyl of 18 with the likely more metabolically stable

trifluoromethyl group (31) resulted in loss of cellular potency,

even though binding affinity remained comparable to that

observed with 18. Replacement of one (32) or two (33) fluoro

groups (of 31) with methyl, thereby increasing both the size

and lipophilicity of this motif, resulted in the restoration of

cellular activity, with the 2-fluoropropyl-containing derivative

The rat PK profile for selected analogues is illustrated in

Table 3. Male Sprague−Dawley rats were dosed with com-

pound either via intravenous (iv) or oral (po) administration

(n = 2 and 3 animals for the iv and po arms, respectively), and

PK parameters were calculated, including clearance (CL), volume

of distribution (V ), terminal half-life (t ), AUC (iv and po), and

d

1/2

(

33) proving equipotent to 18. The 2-(trifluoromethyl)propyl

absolute oral bioavailability (%F). As predicted, replacement of

the isoxazole tert-butyl substituent with an appropriate fluoro-

containing alkyl motif led to improved metabolic stability, as

reflected by decreased in vivo clearance rates. This trend was

further supported by rat liver microsome stability data. For

derivative (35) also proved potent in both binding and cellular

assays. However, incorporation of the slightly larger (trifluoro-

methyl)cyclobutyl group (36) led to a reduction in activity in

the A375 proliferation assay, again suggesting that the size of

1

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a

b

Table 3. Rat Pharmacokinetic and Rat Liver Microsomal Stability Data for Select Compounds

c

d

compd CL [(mL/min)/kg] V_d(L/kg) t₁_/₂(h) AUC₀₋_∞(po) (μM·h) C_m_a_x(po) (μM) t_m_a_x(po) (h)

F (%)

RLM % remaining (at 60 min)

4

1

3

4

(7.3, 17.6)

(9.8, 11.9)

(4.0, 4.1)

(0.5, 2.5)

(1.0, 1.5)

(0.6, 0.6)

19.2

8.2

17.0 ± 5.8

1.9 ± 1.4

11.0 ± 2.0

0.7 ± 0.5

7.9 ± 3.5

4.7 ± 4.0

21.1 ± 4.0

6.5 ± 1.5

6.7 ± 4.6

0.8 ± 0.3

2.0 ± 0

1.7 ± 0.6

2.0 ± 0

49 ± 17

6 ± 4

46

51

82

8

3

5

8

0

9

4.7

42.0 ± 19.2

40.7 ± 12.2

127.0 ± 23.3

74.4 ± 9.7

25.9 ± 5.4

48 ± 22

e

f

ND

3.5

7.8

4.2

ND

(2.9, 3.1)

(3.4, 5.0)

(3.1, 5.2)

(0.5, 0.5)

(1.6, 2.0)

(0.3, 0.3)

100 ± 20

94 ± 12

33 ± 7

92

81

g

2.0 ± 0

1.3 ± 0.6

f

ND

a

Dosed to Sprague−Dawley rats. The parameters were calculated using plasma concentration−time data for individual animals, n = 2 animals

(

iv arm) and 3 animals (po arm). Dosed iv at 1 mg/kg as a solution in 3:1 PEG400/water. Dosed po at 10 mg/kg as a solution in Pharmatek #6

Hot Rod Chemistry vehicle series; Pharmatek Incorporated, San Diego, CA). Results are presented as mean values from experiments performed in

b

c

duplicate. Compound concentration used was 1 μM. Compounds were incubated at 37 °C. Data reported for both individual animals (n = 2).

d

e

f

g

Mean values and standard deviation reported (n = 3). Dosed po (10 mg/kg) only. ND = not determined. Dosed as a free base.

Figure 3. KINOMEscan tree spot graphic illustrating the selectivity profile for compound 40 versus a panel of 290 wild-type kinase targets (plotted

as percent of control at 10 μ M concentration, where 0 and 35% of control equals 100 and 65% competition, respectively). Images courtesy of

KINOMEscan (http://www.kinomescan.com) and used with permission from KINOMEscan, a division of DiscoveRx Corporation.

example, in a rat liver microsome stability assay, the fluorinated

derivatives 33, 38, and 40 exhibit approximately 2-fold impro-

vement in metabolic stability when compared with 4, as reflected

by measured percent remaining of parent at 60 min (Table 3).

A second observation from the data in Table 3 is that while

their clearance rates and volumes of distribution remain

equal, the isoxazol-3-yl-containing urea isomers typically

exhibited significantly improved absorption profiles com-

pared to the corresponding isoxazol-5-yl-containing urea

isomers. For instance, the 5-tert-butyl-isoxazol-3-yl isomer

both binding and cellular assays coupled with excellent oral

exposure in rat, 40 was chosen for further profiling. Compound

40 proved modestly selective when screened at 10 μM concen-

tration across a KINOMEscan panel of 290 diverse wild-type

kinases, as indicated by the selectivity S(10) score of 0.245

(Figure 3). Corresponding K determinations versus select

d

V600E

kinase targets are presented in Table 4. In addition to BRAF

,

40 exhibits high binding affinity for both wild-type BRAF and

related CRAF, as well as certain receptor tyrosine kinases of

known therapeutic utility, such as Abl-1, c-Kit, Ret, PDGFR-β,

and VEGFR-2. However, 40 proved selective for the RAF mem-

bers of the MAPK signal transduction pathway, as no significant

affinity was observed for other key kinases of the MAPK

pathway, including MEK-1, MEK-2, ERK-1, and ERK-2. This

4

exhibited significantly improved oral exposure (AUC₀_‑_α

=

1

7 μM·h; %F = 49) compared to the 3-tert-butyl-isoxazol-5-

yl isomer 18 (AUC₀_‑_α= 1.9 μM·h; %F = 6). A similar trend

was observed between the 2-fluoropropyl-isoxazole isomeric

pairs 33 and 38 and the 2-(trifluoromethyl)propyl-isoxazole

isomeric pairs 35 and 40, with the isoxazol-3-yl isomer

exhibiting the greatest exposure after oral dosing in each

case.

suggests that the observed cellular activity was driven primarily

V600E

through inhibition of BRAF

, which is further supported by

the observation that 40 exhibited selective cytotoxicity for tumor

cell lines expressing mutant BRAF versus those expressing wild-

Selectivity and Further Pharmacokinetic Profiling of

type BRAF (Table 5). When screened across a panel of tumor

V600E

4

0. Due to the combination of high potency for BRAF

in

cell lines expressing BRAF

(A375, SK-MEL-28, Colo-205,

1

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Table 4. Select Kinase K Determinations for Compound 40

human liver microsomes indicates that both mono O-demethyla-

tion (of the quinazoline methoxy groups) and oxidation of the

d

a

kinase

K_d(nM)

kinase

JAK-1

JAK-2

JAK-3

c-Kit

K (nM)

d

(

1,1,1-trifluoro-2-methylpropan-2-yl)isoxazol-3-yl moiety occur

BRAF^V⁶⁰⁰^E

wt BRAF

CRAF

14

36

39

3

>10000

4700

>10000

2

to some extent (data not shown). However, as indicated above,

the measured in vitro clearance rates of such oxidative processes

remain low across all species tested. In line with these obser-

vations, 40 exhibits an exceptional PK profile in mouse, dog, and

cynomolgus monkey, as illustrated in Table 6. For example,

administration of 40 to beagle dogs (single dose of 1 mg/kg

iv and 10 mg/kg po) resulted in low clearance (CL = 5.0

Abl-1

ALK

>10000

9

22

14

LCK

2

Aurora A

Aurora B

CSF-1R

EGFR

EPHA2

ERK-1

MEK-1

MEK-2

c-Met

PDGFR-β

PIK3-CA

PIK3-CB

PLK-1

Ret

7100

8300

513

(

mL/min)/kg) and excellent bioavailability (%F = 100).

2

>10000

2

Similarly, in cynomolgus monkey, the administration of 40

(single dose of 1 mg/kg iv and 10 mg/kg po) led to high oral

exposure due to low clearance (CL = 6.7 mL/min/kg) and

excellent bioavailability (%F = 100). Compound 40 also exhibits a

favorable CYP450 inhibition profile, with measured IC₅₀values

greater than 10 μM versus the CYP1A2, 2C9, 2D6, and 3A4

isoforms and an IC = 3.4 μM versus 2C19 (data not shown). In

>10000

ERK-2

IGFR-1

INSR

VEGFR-2

8

a

Competition binding assays performed at rt for 1 h. K values were

d

determined using eleven serial 3-fold dilutions. Results are presented

as mean values from experiments performed in duplicate. Variability

between individual values is less than 2-fold.

50

addition, as a preliminary measure of potential cardiotoxicity, 40

was assessed for activity against the hERG potassium ion channel

18

in a patch-clamp assay. Compound 40 does not significantly

inhibit the hERG channel, exhibiting <10% inhibition at 10 μM

concentration (data not shown).

Table 5. Cell Titer Blue Viability of Compound 40 across

V600E

Human Tumor Cell Lines Expressing BRAF

Wild-Type BRAF

or

a

In Vivo Efficacy and Pharmacodynamic Profiling of

4

0. Compound 40 was further evaluated for antitumor efficacy

EC₅₀

cell line (BRAF^V⁶⁰⁰^E

A375 (homozygous)

)

(nM)

cell line (wt BRAF)

(nM)

V600E

and tolerability in several BRAF

xenograft mouse models, including both Colo-205 and A375.

-driven human carcinoma

19

78

36

HCT116

Hs578T

LNCaP

DU145

PC-3

669

2736

6631

2911

6257

Colo-205 (heterozygous)

Colo-679 (homozygous)

SK-MEL-28 (homozygous)

HT-144 (homozygous)

Data from the Colo-205 xenograft study is presented here

(Figure 4). Athymic nude mice bearing established Colo-205

tumor xenografts were dosed orally with 40 at 10, 30, and

100 mg/kg twice daily (BID) over a 14-day period. All doses were

well tolerated, with no mortality or significant body weight loss

(<5% relative to vehicle matched controls) observed during

treatment or up to 1 week post dosing. Dosing at 10 mg/kg

BID had little effect on tumor growth compared to the control

group. However, increasing the dose to either 30 or 100 mg/kg

BID resulted in significant efficacy. The 30 mg/kg dose group

exhibited tumor stasis and a 40% incidence of partial tumor

regressions (PRs) (p < 0.008), whereas the 100 mg/kg BID

dose group exhibited both tumor stasis and an 80% incidence

of PRs (p < 0.0001). The observed efficacy is correlated with an

in vivo pharmacodynamic−pharmacokinetic end point as

illustrated in Figure 5. Oral administration of 40 to Colo-205

tumor xenograft-bearing mice resulted in significant inhibition

of pMEK in tumor cell lysates. For instance, a single 30 mg/kg

(po) dose of 40 led to a 50 and 75% inhibition of normalized

pMEK in tumor lysates at the 2 and 6 h postdose time point,

respectively (p < 0.03), while a 55 mg/kg (po) dose resulted

in a 75% to 57% (p < 0.03) inhibition of pMEK at 2 through

10 h post administration, with normalization to baseline by

211

454

228

a

Results are presented as mean values from experiments performed in

duplicate. Cells were cultured in 0.5% serum overnight prior to

incubation with test compound for 72 h. This was followed by addition

of Cell Titer Blue (Promega, Madison, WI) to detect viable cells after

3

h incubation with reagent.

Colo-679, and HT-144) versus a panel expressing only wild-type

BRAF (HCT116, Hs578T, LNCaP, DU145, and PC-3), those

cell lines harboring mutant BRAF^V⁶⁰⁰^Eproved significantly more

sensitive to treatment with 40. The only wild-type cell line in

which 40 had appreciable activity was the HCT116 cell line, a

colonic epithelial line with a known RAS mutation and wild-type

p53, a combination previously shown to confer sensitivity to

inhibition of the MAPK pathway. Compound 40 also exhibited

good stability in mouse, dog, monkey, and human liver micro-

somal preparations (with measured intrinsic clearance values of

17

<

23 (μL/min)/mg and t₁_/₂> 60 min in all assays; data not

shown), thus predicting low phase I hepatic clearance in these

species. Metabolite profiling of 40 in rat, dog, monkey, and

a

b

c

Table 6. Pharmacokinetics Profile of Compound 40 in Mouse, Dog, and Monkey Following Intravenous (iv) or Oral Dosing

d

(

po)

species

CL (mL/min/kg)

V_d(L/kg)

t₁_/₂(h)

AUC₀₋_∞(po) (μM·h)

394 ± 131

C_m_a_x(po) (μM)

t_m_a_x(po) (h)

F (%)

e

mouse

dog

ND

35.6 ± 6.7

5.2 ± 0.6

4.3 ± 0.5

1.3 ± 0.6

2.0 ± 1.0

4.0 ± 2.3

ND

f

5.0 ± 1.0

6.7 ± 3.3

4.5 ± 0.5

2.5 ± 0.6

12 ± 2.8

4.9 ± 0.9

55.1 ± 6.4

92.2 ± 15.4

100 ± 14

100 ± 43

monkey

a

Athymic nude mice (n = 3 animals), dosed at 100 mg/kg po only in Pharmatek #6 (Hot Rod Chemistry vehicle series; Pharmatek Incorporated,

b

San Diego, CA). Beagle dogs (n = 3 animals), dosed iv at 1 mg/kg as a solution in 22% HPβCD, and po at 10 mg/kg as a solution in 22% HPβCD.

c

Male Cynomolgus monkeys (n = 3 animals), dosed iv at 1 mg/kg as a solution in 22% HPβCD, and po at 10 mg/kg as a solution in 22% HPβCD.

d

The parameters were calculated using plasma concentration−time data for individual animals, and mean values are reported with standard

e

f

deviations. ND = not determined. Reported as AUC

.

−24h

0

1

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Figure 4. Efficacy results upon oral dosing of compound 40 at 10, 30, and 100 mg/kg BID (dosed in 22% HPβCD) for 14 days to athymic nu/nu

V600E

nude mice bearing established Colo-205 human colon tumor subcutaneous xenografts with the BRAF

genotype (10 animals per treatment

group). A dose response relationship (left panel) for complete Colo-205 tumor growth inhibition and incidence of tumor regressions (right panel)

was observed. Mice were monitored for signs of morbidity (behavior and body weight loss), and tumors were measured 3 times per week. Statistical

significance (p < 0.05) for antitumor efficacy, based upon tumor growth inhibition relative to vehicle-treated controls, for both the 30 and 100 mg/kg

groups was reached by day 5 of administration and extended to the termination of the study, with a final significance for tumor growth inhibition of

p < 0.008 (30 mg/kg group) and p < 0.0001 (100 mg/kg group).

2

4 h (Figure 5a). In general, the magnitude and duration of

compounds were docked into the BRAF^V⁶⁰⁰^Eprotein, including

22, which contains an isoxazole bearing the much smaller methyl

group at the 5-position of the isoxazol-3-yl ring. Compound 22

scored lower in these docking experiments, presumably due to

less efficient hydrophobic interactions within the hydrophobic

normalized pMEK inhibition by 40 was consistent with plasma

and tumor levels over the dose range and time course of the

pharmacodynamic study (Figure 5b).

Molecular Modeling Studies of Compound 40 with

V600E

BRAF

. In order to gain a better understanding of how the

pocket (data not shown), which may explain the observed reduc-

V600E

described quinazoline-derived inhibitors might bind to BRAF

we carried out docking experiments into the ligand binding site

of the previously reported cocrystal structures of sorafenib

,

tion in the binding affinity of 22 to BRAF

. Compounds such

as 30, which also exhibits reduced binding affinity compared to 40,

has a methoxy group in place of the CF . Compound 30 docked

3

V600E

with this methoxy group pointing away from Thr508 and is

therefore is unable to interact with this residue (Figure 6c).

Figure 6b allows comparison between the binding mode of 40 and

sorafenib. As one might expect, overall, these ligands occupy

similar space within the binding site, with similar positioning of the

urea moieties and interaction of both quinazoline N-1 of 40 and

the pyridyl nitrogen of sorafenib with the same residue (the NH of

Cys532) located on the hinge of the protein.

(

Figure 6d) bound to a DFG-out form of BRAF

(PDB ID:

2

0

1uwj) and a DFG-out form of wt-BRAF (PDB ID: 1uwh).

Select compounds, including 22, 30, and 40 docked to the

DFG-out mode in a consistent manner, and all docking trials

scored compound 40 the best. The predicted binding mode

_o_f_t_h_e_s_e_c_o_m_p_o_u_n_d_s_t_o_D_F_G_-_o_u_t_B_R_A_FV600E is illustrated with

compound 40 (Figure 6a). In this binding mode the

quinazoline N-1 nitrogen participates in a hydrogen bonding

interaction with the NH of Cys532 of the hinge domain. In

addition, the quinazoline core is in close proximity to three

aromatic residues, namely Trp531, Phe583, and Phe595 (of the

DFG motif), which could participate in appropriate π−π or

π−σ aromatic binding interactions with the quinazoline ring

system. In a similar manner, Phe595 could also interact with the

central phenyl ring of 40. The urea moiety of 40 participates in

two key hydrogen bonding interactions with the protein: one

between the urea oxygen and the NH of Asp594 (of the DFG

motif); the other between one or both of the urea NH’s and the

carboxylate of Glu501 (of the α-C Helix). Finally, the 5-(1,1,1-

trifluoro-2-methylpropan-2-yl)isoxazol moiety sits in a hydrophobic

pocket formed between the α-C and α-E helices and the

N-terminal regions of the DFG motif and the catalytic loop, with

the 1,1,1-trifluoro-2-methylpropan-2-yl group in close proximity

to the residues Val504, Leu505, Thr508, Ile513, and Leu567.

The model predicts that along with the expected hydrophobic

CONCLUSION

■

An optimization campaign around the initial lead 5-tert-

butyl-isoxazol-3-yl urea-containing quinazoline 4 was described.

Targeted replacement of the metabolically sensitive tert-butyl

moiety with a variety of alkyl fluoride motifs led to the iden-

tification of 5-(1,1,1-trifluoro-2-methylpropan-2-yl)isoxazol-3-yl

urea-containing quinazoline derivative 40. Compared to 4, com-

pound 40 exhibited improved cellular potency against the

V600E

BRAF

-dependent A375 cell line and much improved oral

exposure in rat by virtue of reduced in vivo clearance. Com-

pound 40 also exhibited excellent oral exposure in other pre-

clinical species. In addition, 40 proved selective for the RAF

kinase members of the MAPK signaling cascade and exhibited

selective cytotoxicity for tumor cell lines expressing mutant

V600E

BRAF

versus those containing wild-type BRAF. Com-

pound 40 also demonstrated significant oral efficacy in a 14-day

V600E

interactions with the residues described, the CF group can

BRAF

-dependent human Colo-205 tumor xenograft mouse

3

interact electrostatically with Thr508 via a hydrogen bonding

interaction between a fluorine atom and the OH of Thr508 (F−H

distance ∼2.97 Å). This interaction with Thr508 may con-

tribute to the enhanced binding affinity observed with 40 relative

to 4. In an attempt to rationalize certain SAR observations, other

model, when dosed 30 and 100 mg/kg BID. These observations,

coupled with excellent druglike properties and a favorable toler-

ability profile, led to the selection of 40 as a clinical candidate,

which represents a new opportunity for the treatment of mutant

V600E

BRAF

-driven human carcinomas.

1

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Figure 5. (a) In vivo inhibition of normalized MEK phosphorylation in Colo-205 tumor xenograft-bearing mice over a 24 h period following a single

oral dose of either vehicle (22% HPβCD) or compound 40 (10, 30, and 55 mg/kg, dosed in 22% HPβCD), and (b) corresponding compound levels

in both plasma and tumor samples. At appropriate time points over a 24 h period, animals were sacrificed and plasma and tumor samples were

collected and lysates prepared. Amounts of total MEK and pMEK in tumor lysates were measured by immunoblotting and ELISA methods.

Compound levels in plasma and tumor tissue samples were analyzed and quantitated by LC-MS/MS. Values shown are mean + SEM from two

studies; n = 3 tumor-bearing mice per time point per study.

HPLC systems with either a Varian Pursuit XRs 10 μm Diphenyl

EXPERIMENTAL SECTION

■

2

50 mm × 50.0 mm column (eluent flow rate 95 mL/min) or a

Phenomenex Luna 10 μm C18(2) 100 Å 250 × 21.20 mm column

eluent flow rate 30 mL/min), eluting with acetonitrile/water, con-

Chemistry. General Methods. All commercially available

solvents and reagents were used as received. Where appropriate,

reactions were carried out under an argon atmosphere. Flash column

chromatography was conducted under medium pressure either on

silica (Merck silica gel 40−63 μM) or on prepacked silica gel cartridges

Biotage), and eluents were monitored by UV light (λ = 254 nm). H

NMR spectra were recorded using a Bruker Avance 300 MHz NMR

spectrometer unless stated otherwise. Chemical shifts (δ) are reported

as parts per million (ppm) relative to tetramethylsilane (internal standard),

and coupling constants (J) are reported in Hz. The following abbreviations

are used for multiplicities: s = singlet; br s = broad singlet; d = doublet;

t = triplet; q = quartet; m = multiplet. Low resolution mass spectra

(

taining 0.05% acetic acid. The purity of all final compounds was

determined to be ≥95% by reverse-phase LC-MS, which was per-

formed on a Shimadzu LC-MS 2010 EV (ESI probe) using a Pheno-

menex Luna 5 μm C18(2) 100 Å 250 mm × 4.60 mm column, eluting

with a gradient of 10−95% acetonitrile in water (containing 0.05%

acetic acid) over 30 min (at rt), with a flow rate of 1 mL/min and

visualized using a wavelength of 254 nm.

1

(

1

-(5-tert-Butylisoxazol-3-yl)-3-(3-(6,7-dimethoxyquinazolin-4-

yloxy)phenyl)urea Hydrochloride (4). A stirred mixture of 3-

aminophenol (4.36 g, 40 mmol) and 5-tert-butyl-3-isocyanatoisoxazole

(6.64 g, 40 mmol) in degassed THF (300 mL) was heated at 50 °C for

15 h. After cooling to rt, the reaction was concentrated under reduced

pressure and the resulting foam purified by silica gel flash chromato-

graphy (eluting with 25% to 75% EtOAc in hexanes) to afford

(

MS) were obtained as electrospray ionization (ESI) mass spectra,

which were recorded on a Shimadzu HPLC/MS instrument using

reverse-phase conditions (acetonitrile/water, containing 0.05% acetic

acid). Preparative reverse-phase HPLC was performed using Varian

1

093

dx.doi.org/10.1021/jm2009925 | J. Med. Chem. 2012, 55, 1082−1105

Journal of Medicinal Chemistry

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Figure 6. (a) Binding mode of the isoxazol-3-yl urea compounds, based on docking experiments with DFG-out BRAF^V⁶⁰⁰^E, illustrated with

compound 40 (carbon-magenta). The urea forms two hydrogen bonding interactions: one between the urea-O and the NH of Asp594 (DFG motif);

the other between the urea NH’s and the carboxylate of Glu501 (α-C helix). A hydrogen bond (electrostatic interaction) between one of the

fluorines of the CF and the Thr508 OH may contribute a key binding interaction. (b) Comparison of the binding mode of sorafenib (carbon-light

3

green) from the X-ray structure and compound 40 (carbon-magenta) from the docking studies. (c) Subtle changes in the binding mode of

compound 30 (carbon-magenta). (d) Chemical structure of sorafenib.

1

8

6

-(5-tert-butylisoxazol-3-yl)-3-(3-hydroxyphenyl)urea as a solid (8.81 g,

(2.56 mL, 48.75 mmol) dropwise over the course of 45 min. The

resulting pale yellow suspension was heated at 70 °C for a further 15 h.

After cooling to rt, the reaction mixture was poured into water

(150 mL) and the resulting solution was extracted with diethyl ether

(2 × 100 mL). The aqueous layer was separated, acidified to pH 2 with

aqueous 2 M HCl, and extracted with diethyl ether (2 × 100 mL). The

1

0%). H NMR (DMSO-d , 300 MHz): δ 9.39 (s, 1H), 9.37 (s, 1H),

6

.69 (s, 1H), 7.01−7.06 (m, 2H), 6.78 (d, J = 6.0 Hz, 1H), 6.49 (s, 1H),

+

.41 (d, J = 6.0 Hz, 1H), 1.29 (s, 9H). LC-MS (ESI) m/z 275 [M + H] .

To a slurry of potassium tert-butoxide (6.73 g, 60 mmol) in THF

300 mL) was added 1-(5-tert-butylisoxazol-3-yl)-3-(3-hydroxyphenyl)-

(

urea (8.25 g, 30 mmol), and the solution was stirred at rt for 1 h, at which

point 4-chloro-6,7-dimethoxyquinazoline (6.74 g, 30 mmol) was added,

followed by potassium carbonate (4.1 g, 30 mmol). After stirring at rt for

combined ether layers were dried over MgSO and then concentrated

4

under reduced pressure to afford the title compound as a yellow oil,

1

which did not require further purification (2.71 g, 78%). H NMR

72 h, the reaction was concentrated under reduced pressure and the

(CDCl , 300 MHz): δ 3.53 (s, 2H), 2.81 (septet, J = 6 Hz, 1H), 1.21

3

resulting solid was partitioned between EtOAc and water. The organic

(d, J = 6 Hz, 6H).

layer was separated, dried over MgSO , filtered, and then concentrated

under reduced pressure. The residue was purified by silica gel flash

chromatography (eluting with 15% to 100% EtOAc in hexanes) to give 1-

3-Cyclopentyl-3-oxopropanenitrile (5b). Prepared from methyl

cyclopentanecarboxylate (4.0 g, 31.25 mmol) according to General

4

Procedure A to afford the title compound as a yellow oil (3.97 g, 93%).

1

(

5-tert-butylisoxazol-3-yl)-3-(3-(2-chloro-6,7-dimethoxyquinazolin-4-

yloxy)phenyl)urea as a white solid. The solid was dissolved in EtOAc

50 mL), and 4 M HCl in 1,4-dioxane (5 mL, 20 mmol) was added and

H NMR (CDCl , 300 MHz): δ 3.5 (s, 2H), 3.02−3.13 (m, 1H),

3

1.62−1.95 (m, 8H).

4-Fluoro-4-methyl-3-oxopentanenitrile (5c). Prepared from ethyl

2-fluoroisobutyrate (10 g, 74.62 mmol) according to General Proce-

(

stirred at rt for 30 min. The mixture was concentrated under reduced

1

pressure to afford the title compound as a solid (6.29 g, 42%). H NMR

dure A to afford the title compound as a yellow oil (8 g, 83%). H

(

7

9

DMSO-d , 300 MHz): δ 9.72 (s, 1H), 9.44 (s, 1H), 8.73 (s, 1H), 7.60−

NMR (CDCl , 300 MHz): δ 3.82 (s, 2H), 1.54 (d, J = 21 Hz, 6H).

3

6

.65 (m, 2H), 7.38−7.45 (m, 2H), 7.29 (d, J = 9.0 Hz, 1H), 6.98 (d, J =

4,4-Difluoro-3-oxopentanenitrile (5d). Prepared from ethyl

2,2-difluoropropionate (3 g, 22 mmol) according to General Procedure

A. The crude product was purified by silica gel flash chromatography

.0 Hz, 1H), 6.48 (s, 1H), 4.02 (s, 3H), 4.00 (s, 3H), 1.28 (s, 9H).

+

LC-MS (ESI) m/z 464 [M + H] .

Preparation of Substituted 3-Oxopropanenitriles. General

Procedure A. 4-Methyl-3-oxopentanenitrile (5a). A stirred suspen-

sion of sodium hydride (1.95 g, 60% dispersion in mineral oil,

(eluting with 10%−40% EtOAc in hexanes) to afford the title compound

1

as a yellow oil (1 g, 31%). H NMR (CDCl , 300 MHz): δ 3.95 (s, 2H),

3

1.86 (t, J = 19 Hz, 3H).

4

8.75 mmol) in THF (25 mL) was heated to 75 °C. To this was added

4,4,4-Trifluoro-3-oxobutanenitrile (5e). Prepared from methyl

2,2,2-trifluoroacetate (10.24 g, 80 mmol) according to General Procedure

a mixture of methyl isobutyrate (3.19 g, 31.25 mmol) and acetonitrile

1

094

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1

A to afford the title compound as an oil (9.08 g, 83%). H NMR

CDCl , 300 MHz): δ 2.93 (s, 2H).

5

dropwise trifluoroacetic anhydride (50.0 g, 177 mmol). The reaction

mixture was stirred for 2 h at −78 °C, after which the reaction was

allowed to warm slowly to rt and was stirred for an additional 2 h. The

mixture was partitioned between CH Cl (200 mL) and 3% aqueous

(

3

,5,5-Trifluoro-4,4-dimethyl-3-oxopentanenitrile (5f). The reac-

tion was carried out in two separate batches, employing 5 g (32 mmol)

of 3,3,3-trifluoro-2,2-dimethylpropionic acid in each batch. To a stirred

solution of 3,3,3-trifluoro-2,2-dimethylpropionic acid (5 g, 32 mmol)

in anhydrous CH Cl (20 mL) at 0 °C was added dropwise a solution

of (trimethylsilyl)diazomethane (18 mL of a 2 M solution in diethyl

ether, 35 mmol) (gas evolution observed). The resulting yellow solu-

tion was allowed to warm to rt and stirred for a further 48 h. Addi-

tional (trimethylsilyl)diazomethane (5 mL of a 2 M solution in diethyl

ether, 10 mmol) was added, and stirring was continued for a further

2

HCl solution (200 mL). The organic layer was separated, washed with

3% aqueous HCl solution (200 mL), dried over MgSO , and then

4

concentrated under reduced pressure to give an oil. The oil was

dissolved in THF (50 mL) and cooled to 0 °C. Tetrabutylammonum

fluoride (200 mL of a 1 M solution in THF, 200 mmol) was added

dropwise, and the reaction mixture was stirred at rt for 15 h. The mix-

ture was concentrated under reduced pressure and the residue dis-

solved in CH Cl (400 mL), which was washed with brine (2 × 200 mL),

2

5

h, whereupon a further amount of (trimethylsilyl)diazomethane

dried over MgSO , and then concentrated under reduced pres-

4

(

6 mL of a 2 M solution in diethyl ether, 12 mmol) was added. After

sure. The residue was purified by vacuum distillation to afford methyl

stirring for a further 15 h, the reaction mixture was concentrated under

reduced pressure (keeping bath temperature below 30 °C). The result-

ing oil was redissolved in diethyl ether (200 mL), washed with satu-

3-fluoro-2-(fluoromethyl)-2-methylpropanoate as an oil (2.89 g, 22%).

1

H NMR (CDCl , 300 MHz): δ 4.33−4.66 (m, 4H), 3.67 (s, 3H),

3

1.14 (s, 3H). Methyl 3-fluoro-2-(fluoromethyl)-2-methylpropanoate

(5.21 g, 34.2 mmol) was treated according to General Procedure

rated aqueous NaHCO solution (100 mL), separated, and dried over

3

1

MgSO . Filtration followed by concentration under reduced pressure

A to afford the title compound as an oil (4.41 g, 80%). H NMR

4

(

3

keeping bath temperature below 30 °C) afforded methyl 3,3,

(CDCl , 300 MHz): δ 4.67−4.72 (m, 2H), 4.45−4.52 (m, 2H), 3.80

3

-trifluoro-2,2-dimethylpropanoate as an oil. Product from both batches

(s, 2H), 1.27 (s, 3H).

was combined to afford methyl 3,3,3-trifluoro-2,2-dimethylpropanoate

5-Hydroxy-4,4-dimethyl-3-oxopentanenitrile (5k). A solution of

methyl 3-hydroxy-2,2-dimethylpropanoate (5.00 g, 38 mmol), N,N-

diisopropylethylamine (7.30 g, 57 mmol), and tert-butyldimethyl-

chlorosilane (6.80 g, 45 mmol) in DMF (70 mL) was stirred at rt for

12 h. The reaction solution was partitioned between water and diethyl

ether. The combined organic layers were washed with water and then

as a yellow oil (7.69 g, 71%), which was used in the next step with-

1

out further purification. H NMR (CDCl , 300 MHz): δ 3.86 (s, 3H),

3

1.40 (s, 6H). Methyl 3,3,3-trifluoro-2,2-dimethylpropanoate (7.69 g,

4

5 mmol) was treated according to General Procedure A to afford

1

the title compound as a yellow oil (4.27 g, 37%). H NMR (CDCl ,

3

00 MHz): δ 3.77 (s, 2H), 1.43 (s, 6H).

,2-Dimethyl-3-oxopentanedinitrile (5g). Prepared from ethyl

-cyano-2-methylpropanoate (3.0 g, 21.25 mmol) according to General

brine and then dried over MgSO . Concentration under reduced

4

2

pressure afforded methyl 3-(tert-butyldimethylsilyloxy)-2,2-dimethyl-

2

propanoate as a colorless oil, which did not require further purifica-

1

Procedure A to afford the title compound as a yellow oil (1.40 g, 48%).

tion (9.34 g, 100%). H NMR (CDCl , 300 MHz): δ 3.64 (s, 3H),

3

1

H NMR (CDCl , 300 MHz): δ 3.98 (s, 2H), 1.60 (s, 6H).

3.55 (s, 2H), 1.13 (s, 6H), 0.85 (s, 9H), 0.00 (s, 6H). Methyl 3-(tert-

butyldimethylsilyloxy)-2,2-dimethylpropanoate (6.0 g, 24.39 mmol)

was treated according to General Procedure A and then purified by

silica gel flash chromatography (eluting with 33% EtOAc in petroleum

3

-Oxo-3-(1-(trifluoromethyl)cyclopropyl)propanenitrile (5h). Pre-

pared from methyl 1-(trifluoromethyl)cyclopropane carboxylate (2.0 g,

1.9 mmol) according to General Procedure A. The crude product was

1

purified by silica gel flash chromatography (eluting with 5%−40%

EtOAc in hexanes) to afford the title compound as an oil (1.03 g,

4

ether) to afford the title compound as a yellow oil (1 g, 29%). H

NMR (CDCl , 300 MHz,): δ 3.76 (s, 2H), 3.61 (s, 2H), 1.19 (s, 6H).

3

+

9%). LC-MS (ESI) m/z 178 [M + H] .

5-Methoxy-4,4-dimethyl-3-oxopentanenitrile (5l). Prepared from

2

1

3

-Oxo-3-(1-(trifluoromethyl)cyclobutyl)propanenitrile (5i). To a

stirred solution of 1-trifluoromethylcyclobutane-1-carboxylic acid (10 g,

9.5 mmol) in MeOH (50 mL) was added concentrated sulfuric acid

3 mL), and the resulting mixture was heated at 75 °C for 15 h. After

methyl 3-methoxy-2,2-dimethylpropanoate (8 g, 54.7 mmol)

according to General Procedure A to afford the title compound as a

1

5

yellow oil (2.5 g, 29%). H NMR (CDCl , 300 MHz): δ 3.72 (s, 2H),

3

(

3.32−3.33 (m, 5H), 1.18 (s, 6H).

cooling to rt, the solvent was removed under reduced pressure and the

residue was partitioned between EtOAc and water. The organic phase

was separated and the aqueous solution was further extracted with

Preparation of Substituted Aminoisoxazoles. General Procedure

B. 3-Isopropylisoxazol-5-amine (6a). To a stirred solution of sodium

hydroxide (238 mg, 5.95 mmol) and compound 5a (600 mg, 5.41 mmol)

in a mixture of water (5 mL) and EtOH (5 mL) was added hydroxyl-

amine sulfate (977 mg, 5.95 mmol). The reaction mixture was adjusted

to pH 7.5 with aqueous 1 M sodium hydroxide solution and then

heated at 80 °C for 15 h. After cooling to rt, the solvent was removed

under reduced pressure. The resulting solid was partitioned between

CH Cl (50 mL) and water (50 mL), and the organic layer was

EtOAc. The combined organic layers were dried over MgSO and then

4

concentrated under reduced pressure (water bath at 20 °C) to afford

methyl 1-(trifluoromethyl)cyclobutanecarboxylate as an oil, which did

1

not require further purification (4.5 g, 42%). H NMR (DMSO-d ,

6

3

00 MHz): δ 3.76 (s, 3H), 2.50−2.60 (m, 2H), 2.35−2.45 (m, 2H),

1

.91−2.04 (m, 2H). Methyl 1-(trifluoromethyl)cyclobutanecarboxylate

2

(

2 g, 11 mmol) was treated according to General Procedure A to

separated, washed with brine (50 mL), dried over MgSO , and then

4

1

afford the title compound as a yellow oil (1.68 g, 80%). H NMR

concentrated under reduced pressure to afford the title compound as a

(

CDCl , 300 MHz): δ 3.76 (s, 2H), 2.39−2.65 (m, 4H), 1.95−2.15

cream solid, which did not require further purification (530 mg, 78%).

3

1

m, 2H).

H NMR (CDCl , 300 MHz): δ 5.00 (s, 1H), 4.39 (br s, 2H), 2.89

3

5

-Fluoro-4-(fluoromethyl)-4-methyl-3-oxopentanenitrile (5j). To

(septet, J = 6 Hz, 1H), 1.23 (d, J = 6 Hz, 6H). LC-MS (ESI) m/z 127

[M + H] .

+

a stirred suspension of 3-hydroxy-2-(hydroxymethyl)-2-methylpropanoic

acid (10.06 g, 75 mmol) in MeOH at rt was added dropwise

trimethylsilyl)diazomethane (50 mL of a 2 M solution in diethyl

ether, 100 mmol). The reaction mixture was stirred at rt for 15 h. The

mixture was concentrated under reduced pressure, and the residue was

partitioned between CH Cl and saturated aqueous NaHCO solution.

Preparation of Substituted Aminoisoxazoles. General Procedure

C. 3-Cyclopentylisoxazol-5-amine (6b). To a stirred solution of

compound 5b (645 mg, 4.7 mmol) and sodium hydroxide (386 mg,

9.65 mmol) in water (4.7 mL) was added hydroxylamine hydro-

chloride (343 mg, 4.94 mmol), and the resulting mixture was heated at

100 °C for 2.5 h. After cooling to rt, the mixture was diluted with

(

2

3

The organic layer was separated, dried over MgSO , and then

concentrated under reduced pressure to afford methyl 3-hydroxy-2-

4

CHCl and the organic layer was separated. The aqueous layer was

3

(

hydroxymethyl)-2-methylpropanoate as an oil, which did not require

further extracted with CHCl , and the combined organic layers were

3

1

further purification (3.79 g, 34%). H NMR (CDCl , 300 MHz):

dried over MgSO and then concentrated under reduced pressure to

3

4

δ 3.81 (d, J = 13.8 Hz, 2H), 3.67 (s, 3H), 3.60 (d, J = 13.8 Hz, 2H),

afford the title compound as a colorless solid, which did not require

1

2

.89 (br s, 2H), 0.96 (s, 3H). To a solution of methyl 3-hydroxy-2-

hydroxymethyl)-2-methylpropanoate (13.0 g, 88 mmol) and 2,6-

lutidine (26.79 g, 250 mmol) in CH Cl₂at −78 °C was added

further purification (680 mg, 95%). H NMR (CDCl , 300 MHz):

3

(

δ 5.09 (br s, 2H), 4.93 (s, 1H), 2.94−3.02 (m, 1H), 1.73−2.00 (m, 2H),

+

1.62−1.73 (m, 6H). LC-MS (ESI) m/z 153 [M + H] .

2

1

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1

3

-(2-Fluoropropan-2-yl)isoxazol-5-amine (6c). Prepared from

title compound as a colorless solid (1.07 g, 97%). H NMR (CDCl ,

3

compound 5c (6.0 g, 47 mmol) according to General Procedure B

300 MHz): δ 5.00 (s, 1H), 4.79 (br s, 2H), 1.33 (s, 9H). LC-MS (ESI)

m/z 141 [M + H] .

3-Phenylisoxazole-5-amine (6k). Prepared from benzoylaceto-

nitrile (707 mg, 4.9 mmol) according to General Procedure C. The

crude product was purified by silica gel flash chromatography (eluting

+

to afford the title compound as a light yellow solid (4.83 g, 71%).

1

H NMR (CDCl , 300 MHz): δ 5.19 (s, 1H), 4.48 (br s, 2H), 1.68 (d,

3

+

J = 21 Hz, 6H). LC-MS (ESI) m/z 145 [M + H] .

3

-(1,1-Difluoroethyl)isoxazol-5-ylamine (6d). Prepared from com-

pound 5d (100 mg, 0.75 mmol) according to General Procedure B to

with 20−40% EtOAc in hexanes) to afford the title compound as

1

afford the title compound as a colorless solid (100 mg, 90%). H NMR

a solid (456 mg, 58%). H NMR (CDCl , 300 MHz): δ 7.70−7.76

3

(

3

CDCl , 300 MHz): δ 5.30 (s, 1H), 4.70 (s, 2H), 2.00 (t, J = 19 Hz,

H). LC-MS (ESI) m/z 149 [M + H] .

(m, 2H), 7.40−7.47 (m, 3H), 5.49 (s, 1H), 4.52 (s, 2H). LC-MS (ESI) m/z

3

+

1

61 [M + H] .

3

-(Trifluoromethyl)isoxazol-5-amine (6e). (Trimethylsilyl)-

Preparation of Substituted Isoxazole Carbamates. General

Procedure D. Phenyl 3-Isopropylisoxazol-5-ylcarbamate (7a). To

a stirred mixture of compound 6a (250 mg, 1.98 mmol) and potassium

carbonate (634 mg, 4.59 mmol) in THF (6 mL) was added phenyl

chloroformate (341 mg, 2.18 mmol). The reaction mixture was stirred

at rt for 3.5 h, and then additional phenyl chloroformate (341 mg,

diazomethane (21 mL of a 2 M solution in diethyl ether, 42 mmol)

was added dropwise to a stirred solution of compound 5e (3.79 g,

2

6 mmol) in diethyl ether (25 mL) at 0 °C. The resulting mixture was

allowed to slowly warm to rt and was stirred for 15 h. The mixture was

concentrated under reduced pressure to afford 4,4,4-trifluoro-3-

methoxybut-2-enenitrile as an oil, which did not require further

2

.18 mmol) was added and stirring was continued for a further 15 h.

1

purification (3.93 g, 100%). H NMR (CDCl , 300 MHz): δ 5.00 (s,

3

The resulting mixture was partitioned between CH Cl (50 mL) and

2

1H), 4.16 (s, 3H). To a stirred solution of hydroxylamine hydrochloride

water (50 mL), and the organic layer was separated, washed with brine

50 mL), dried over MgSO , and then concentrated under reduced

(2.88 g, 41.5 mmol) in MeOH (20 mL) at 0 °C was added solid

(

4

sodium methoxide (2.24 g, 41.5 mmol), and the resulting suspension

was stirred at rt for 15 min. The suspension was cooled to 0 °C, and

then 4,4,4-trifluoro-3-methoxybut-2-enenitrile (3.93 g, 26 mmol) was

added dropwise and the mixture was allowed to slowly warm to rt. The

mixture was then heated to 60 °C for 15 h. The colorless precipitate

was removed by filtration, and the filtrate was concentrated under

reduced pressure to afford a solid. The solid was redissolved in EtOH

pressure. The residue was purified by silica gel flash chromatography

(

eluting with 4−40% EtOAc in hexanes) to afford the title compound

1

as a colorless solid (330 mg, 68%). H NMR (CDCl , 300 MHz):

3

δ 7.76 (br s, 1H), 7.40−7.45 (m, 2H), 7.18−7.31 (m, 3H), 6.07 (s, 1H),

3

.02 (septet, J = 6 Hz, 1H), 1.28 (d, J = 6 Hz, 6H). LC-MS (ESI) m/z

+

247 [M + H] .

Phenyl 3-Cyclopentylisoxazol-5-ylcarbamate (7b). Prepared from

(

25 mL) and the solution acidified to pH = 1 with 37% aqueous HCl

compound 6b (675 mg, 4.44 mmol) according to General Procedure

and then stirred at 60 °C for 2 h. After cooling to rt, the mixture was

concentrated under reduced pressure and the residue partitioned

between CH Cl and saturated aqueous NaHCO solution. The organic

1

D to afford the title compound as a colorless solid (528 mg, 50%). H

NMR (CDCl , 300 MHz): δ 8.21 (br s, 1H), 7.39−7.44 (m, 2H),

3

2

3

7

1

.26−7.30 (m, 1H), 7.21 (d, J = 9 Hz, 2H), 6.06 (s, 1H), 3.12 (m,1H),

layer was separated, and the aqueous layer was further extracted with

.99−2.06 (m, 2H), 1.63−1.76 (m, 6H). LC-MS (ESI) m/z 273

CH Cl . The combined organic layers were dried over MgSO and then

2

4

+

[

M + H] .

concentrated under reduced pressure. The residue was purified by silica

gel flash chromatography (eluting with 5% EtOAc in CH Cl ) to

Phenyl 3-(2-Fluoropropan-2-yl)isoxazol-5-ylcarbamate (7c). Pre-

pared from compound 6c (4.83 g, 33.54 mmol) according to General

2

1

afford the title compound as a solid (446 mg, 11%). H NMR (CDCl ,

3

Procedure D to afford the title compound as a colorless solid (6.04 g,

3

00 MHz): δ 5.31 (s, 1H), 5.03 (br s, 2H). LC-MS (ESI) m/z 153

1

+

68%). H NMR (CDCl

2

3

, 300 MHz): δ 7.80 (br s, 1H), 7.39−7.45 (m,

[

M + H] .

H), 7.18−7.32 (m, 3H), 6.27 (s, 1H), 1.74 (d, J = 21 Hz, 6H). LC-

MS (ESI) m/z 265 [M + H] .

3

-(1,1,1-Trifluoro-2-methylpropan-2-yl)isoxazol-5-amine (6f).

+

Prepared from compound 5f (524 mg, 2.9 mmol) according to

Phenyl 3-(1,1-Difluoroethyl)isoxazol-5-ylcarbamate (7d). Pre-

pared from compound 6d (100 mg, 0.68 mmol) according to General

General Procedure C to afford the title compound as a solid (150 mg,

1

2

1

7%). H NMR (CDCl , 300 MHz): δ 5.19 (s, 1H), 4.50 (br s, 2H),

3

1

+

Procedure D to afford the title compound as an oil (141 mg, 77%). H

.54 (s, 6H). LC-MS (ESI) m/z 195 [M + H] .

NMR (CDCl , 300 MHz): δ 8.02 (s, 1H), 7.40−7.45 (m, 2H), 7.18−

2

-(5-Aminoisoxazol-3-yl)-2-methylpropanenitrile (6g). Prepared

3

7

2

.32 (m, 3H), 6.38 (s, 1H), 2.03 (t, J = 19 Hz, 3H). LC-MS (ESI) m/z

69 [M + H] .

from compound 5g (500 mg, 3.68 mmol) according to General Procedure B

+

to afford the title compound as a colorless solid (130 mg, 23%).

1

Phenyl 3-(Trifluoromethyl)isoxazol-5-ylcarbamate (7e). To a

H NMR (CDCl , 300 MHz): δ 5.22 (s, 1H), 4.58 (br s, 2H), 1.72

s, 6H). LC-MS (ESI) m/z 152 [M + H] .

3

+

stirred solution of compound 6e (446 mg, 2.93 mmol) in THF

6 mL) at rt were added triethylamine (1.1 mL, 8.2 mmol), phenyl

chloroformate (0.88 mL, 7.03 mmol), and 4-(dimethylamino)pyridine

357 mg, 2.93 mmol). The reaction mixture was stirred at rt for 3 h.

(

3

-(1-(Trifluoromethyl)cyclobutyl)isoxazol-5-amine (6h). Prepared

from compound 5i (500 mg, 2.6 mmol) according to General Proce-

(

dure B to afford the title compound as a colorless solid (210 mg, 39%).

1

The mixture was filtered through a pad of Celite, and then the filtrate

was concentrated under reduced pressure. The residue was redissolved

H NMR (CDCl , 300 MHz): δ 5.04 (s, 1H), 4.55 (br s, 2H), 2.40−

3

+

2

.59 (m, 4H), 1.90−2.09 (m, 2H). LC-MS (ESI) m/z 207 [M + H] .

in CH Cl , washed with brine, dried over MgSO , and then concen-

3

-(1,3-Difluoro-2-methylpropan-2-yl)isoxazol-5-amine (6i). To a

2

4

trated under reduced pressure. The residue was purified by silica gel

flash chromatography (eluting with 20% EtOAc in hexanes) to afford

stirred solution of compound 5j (1.00 g, 6.2 mmol) and sodium

hydroxide (0.27 g, 6.8 mmol) in EtOH (5 mL) and water (5 mL) at rt

was added a solution of hydroxylamine sulfate (1.12 g, 6.8 mmol) in

water (5 mL). To the mixture was added additional sodium hydroxide

until the pH was 8−9, and the mixture was heated at 100 °C for 2 h.

After cooling to rt, the mixture was partitioned between CH Cl

1

the title compound as a colorless solid (269 mg, 33%). H NMR

(CDCl , 300 MHz): δ 7.99 (br s, 1H), 7.39−7.42 (m, 2H), 7.02−7.35

3

+

(m, 3H), 6.70 (s, 1H). LC-MS (ESI) m/z 273 [M + H] .

4-Chlorophenyl 3-(1,1,1-Trifluoro-2-methylpropan-2-yl)isoxazol-

-ylcarbamate (7f). Prepared from compound 6f (150 mg, 0.77 mmol)

2

5

and water. The organic layer was separated and the aqueous layer re-

extracted with additional CH Cl . The combined organic layers were

and 4-chlorophenyl choloroformate (412 mg, 2.15 mmol) according to

2

washed with water, separated, dried over MgSO , and then concen-

General Procedure D to afford the title compound as a colorless solid

4

1

trated under reduced pressure. The residue was purified by silica gel

(210 mg, 78%). H NMR (CDCl

, 300 MHz): δ 7.72 (br s, 1H), 7.39

3

flash chromatography (eluting with 100% hexanes to 50% EtOAc in

(d, J = 12 Hz, 2H), 7.16 (d, J = 12 Hz, 2H), 6.27 (s, 1H), 1.57 (s, 6H).

1

+

hexanes) to afford the title compound as a solid (191 mg, 17%). H

LC-MS (ESI) m/z 349 [M + H] .

NMR (CDCl , 300 MHz): δ 5.16 (s, 1H), 4.54−4.68 (m, 2H), 4.32−

Phenyl 3-(2-Cyanopropan-2-yl)isoxazol-5-ylcarbamate (7g). Pre-

pared from compound 6g (130 mg, 0.861 mmol) according to General

3

+

4

.50 (m, 4H), 1.37 (s, 3H). LC-MS (ESI) m/z 177 [M + H] .

3

-tert-Butylisoxazol-5-amine (6j). Prepared from pivaloylacetoni-

Procedure D to afford the title compound as a colorless solid (93 mg,

1

trile (1 g, 7.90 mmol) according to General Procedure C to afford the

40%). H NMR (CDCl , 300 MHz): δ 7.82 (br s, 1H), 7.41−7.46

3

1

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(

m, 2H), 7.32 (m, 1H), 7.18−7.21 (m, 2H), 6.29 (s, 1H), 1.83 (s, 6H).

solution, and the organic layer was separated, washed with water, and

+

LC-MS (ESI) m/z 272 [M + H] .

then dried over Na SO₄and then concentrated under reduced

2

Phenyl 3-(1-(Trifluoromethyl)cyclobutyl)isoxazol-5-ylcarbamate

pressure. The residue was purified by recrystallization from diethyl

(

7h). Prepared from compound 6h (210 mg, 1 mmol) according to

ether to afford the title compound as a colorless solid (600 mg, 49%).

1

General Procedure D to afford the title compound as a colorless solid

H NMR (CDCl , 300 MHz): δ 5.64 (s, 1H), 3.65 (s, 2H), 2.30 (br s,

3

1

+

(

7

(

320 mg, 98%). H NMR (DMSO-d , 300 MHz): δ 12.17 (br s, 1H),

6

2H), 1.31 (s, 6H). LC-MS (ESI) m/z 157 [M + H] .

.10−7.54 (m, 5H), 6.08 (s, 1H), 2.50−2.70 (m, 4H), 1.90−2.10

5

-(1-Methoxy-2-methylpropan-2-yl)isoxazol-3-amine (8e). Pre-

+

m, 2H). LC-MS (ESI) m/z 327 [M + H] .

pared from compound 5l (1 g, 6.5 mmol) according to the procedure

described for 8d. Purified by silica gel flash chromatography (eluting

Phenyl 3-(1,3-Difluoro-2-methylpropan-2-yl)isoxazol-5-ylcarba-

mate (7i). Prepared from compound 6i (190 mg, 1.08 mmol)

with a mixture of petroleum ether and EtOAc) to afford the title com-

according to General Procedure D to afford the title compound as a

1

pound as a colorless solid (350 mg, 32%). H NMR (CDCl , 300 MHz):

3

1

solid (0.319 g, 100%). H NMR (CDCl , 300 MHz): δ 7.83 (s, 1H),

3

δ 5.60 (s, 1H), 3.39 (s, 2H), 3.32 (s, 3H), 2.94 (br s, 2H), 1.28 (s, 6H).

7

(

.17−7.45 (m, 5H), 6.23 (s, 1H), 4.68−4.74 (m, 2H), 4.49−4.58

+

LC-MS (ESI) m/z 171 [M + H] .

+

m, 2H), 1.40 (s, 3H). LC-MS (ESI) m/z 297 [M + H] .

5

-Phenylisoxazol-3-amine (8f). Prepared from benzoylacetonitrile

Phenyl 3-tert-Butylisoxazol-5-ylcarbamate (7j). Prepared from

(

1.45 g, 10 mmol) according to the procedure described for 8d.

compound 6j (620 mg, 4.43 mmol) according to General Procedure D

Purified by silica gel flash chromatography (eluting with 20−40%

1

to afford the title compound as a colorless solid (1.02 g, 89%). H

EtOAc in hexanes) to afford the title compound as a colorless solid

NMR (CDCl , 300 MHz): δ 11.81 (br s, 1H), 7.42−7.47 (m, 2H),

1

3

(420 mg, 26%). H NMR (CDCl , 300 MHz): δ 7.72 (d, J = 9 Hz,

3

7

[

.23−7.32 (m, 3H), 6.05 (s, 1H), 1.27 (s, 9H). LC-MS (ESI) m/z 261

2

H), 7.39−7.69 (m, 3H), 6.20 (s, 1H), 4.01 (br s, 2H). LC-MS (ESI)

+

M + H] .

m/z 161 [M + H] .

-Isopropylisoxazol-3-amine (8h). To a stirred solution of hyd-

Phenyl 3-Phenylisoxazol-5-ylcarbamate (7k). Prepared from

5

compound 6k (456 mg, 2.85 mmol) according to General Procedure

roxylamine hydrochloride (6.3 g, 91.7 mmol) in MeOH (2.5 mL) was

added ammonia (15.7 mL of a 7 M solution in MeOH, 110 mmol),

and the resulting suspension was stirred at rt for 30 min. 8-Hydroxy-

quinoline (290 mg, 2 mmol) was added, followed by a solution of

compound 10a (3.38 g, 22 mmol) in MeOH (2.5 mL). The mixture

was heated at 70 °C for 15 h. After cooling to rt, the mixture was

filtered and the filtrate concentrated under reduced pressure and

reconcentrated three times from toluene to afford N′-hydroxy-2-(2-

isopropyl-1,3-dioxolan-2-yl)acetimidamide as a yellow solid, which did

not require further purification (3.9 g, 94%). ¹H NMR (CDCl₃,

1

D to afford the title compound as a colorless solid (675 mg, 84%). H

NMR (CDCl , 300 MHz): δ 7.77−7.81 (m, 3H), 7.40−7.47 (m, 5H),

3

+

7

.19−7.32 (m, 3H), 6.54 (s, 1H). LC-MS (ESI) m/z 281 [M + H] .

-(1,1,1-Trifluoro-2-methylpropan-2-yl)isoxazol-3-amine (8a).

Compound 5f (3 g, 16.76 mmol), hydroxylamine sulfate (3.30 g,

0.11 mmol), and NaHCO (3.52 g, 41.90 mmol) in a mixture of 10%

5

2

3

MeOH in water (60 mL) were heated at 65 °C for 15 h. After cooling

to rt, an additional 30 mL of 10% MeOH in water was added and the

mixture was divided into 9 × 10 mL batches. Each batch was adjusted

to pH 1 with concentrated HCl and placed into a 20 mL volume

microwave vial fitted with a stirrer bar. After sealing, each batch was

placed in a Biotage Microwave Synthesizer and heated (with stirring)

at 140 °C for 5 min (maximum internal pressure attained was 7 bar).

Each batch was cooled and neutralized with saturated aqueous

3

00 MHz): δ 5.01 (br s, 2H), 3.94−4.05 (m, 4H), 2.44 (s, 2H), 1.99

+

(m, 1H), 0.95 (d, J = 12 Hz, 6H). LC-MS (ESI) m/z 189 [M + H] .

N′-Hydroxy-2-(2-isopropyl-1,3-dioxolan-2-yl)acetimidamide (1.8 g,

9.57 mmol) was dissolved in EtOH (12 mL) and acidified to pH 1

with 37% aqueous HCl. The mixture was placed into a 20 mL micro-

wave vial fitted with a stirrer bar. After sealing, the reaction mixture

was placed in a Biotage Microwave Synthesizer and heated (with

stirring) at 120 °C for 30 min. After cooling to rt, the mixture was

concentrated under reduced pressure and the residue diluted with

CH Cl . Saturated aqueous NaHCO solution was added until the

NaHCO solution. All processed batches were combined and then

3

concentrated under reduced pressure, and the aqueous solution was

extracted with 10% isopropanol in CHCl (3 × 150 mL). The com-

3

bined organic layers were washed with brine (200 mL), dried over

MgSO , and then concentrated under reduced pressure to afford the

4

2

3

title compound as a pale yellow solid, which did not require further

mixture became basic (pH = 11) and the organic layer separated. The

aqueous layer was further extracted with CH Cl , and the organic

1

purification (2.34 g, 71%). H NMR (CDCl , 300 MHz): δ 5.80

3

2

+

(

s, 1H), 3.98 (br s, 2H), 1.53 (s, 6H). LC-MS (ESI) m/z 195 [M + H] .

layers were combined, dried over MgSO , and then concentrated

4

5

-(1,3-Difluoro-2-methylpropan-2-yl)isoxazol-3-amine (8b). A

under reduced pressure. The residue was purified by silica gel flash

stirred mixture of compound 5j (483 mg, 3 mmol), hydroxylamine

chromatography (eluting with 20−100% EtOAc in hexanes) to afford

1

sulfate (590 mg, 3.6 mmol), and NaHCO (700 mg, 8.3 mmol) in

the title compound as a solid (819 mg, 68%). H NMR (CDCl , 300 MHz):

3

MeOH (1 mL) and water (10 mL) was heated at 60 °C for 8 h. The

reaction was adjusted to pH =1 with 10% aqueous HCl, and the

resulting mixture was heated at 60 °C for a further 3 h. After cooling to

rt, the mixture was partitioned between CH Cl and saturated aqueous

δ 5.52 (s, 1H), 3.89 (br s, 2H), 2.94 (m, 1H), 1.27 (d, J = 12 Hz, 6H).

LC-MS (ESI) m/z 127 [M + H] .

5-Cyclopentylisoxazol-3-amine (8i). Prepared from compound

10b (1.5 g, 8.3 mmol) according to the procedure described for

+

2

NaHCO solution. The organic layer was separated, and the aqueous

compound 8h, to afford the title compound as a colorless solid (875 mg,

3

1

layer was further extracted with CH Cl . The combined organic layers

69%). H NMR (CDCl , 300 MHz): δ 5.52 (s, 1H), 3.86 (br s, 2H),

2

3

were dried over MgSO and then concentrated under reduced pressure

3.06 (m, 1H), 2.04 (m, 2H), 1.62−1.75 (m, 6H). LC-MS (ESI) m/z

4

+

to afford the title compound as a solid, which did not require further

153 [M + H] .

1

purification (289 mg, 55%). H NMR (CDCl , 300 MHz): δ 5.76

5-(2-Fluoropropan-2-yl)isoxazol-3-amine (8j). To MeOH (10 mL)

at rt was added portionwise sodium metal (145 mg, 6.30 mmol). After

all of the metal had dissolved, the reaction mixture was cooled to 0 °C

and hydroxylamine hydrochloride (438 mg, 6.30 mmol) was added in

one portion. The reaction mixture was stirred for 15 min. A solution of

compound 11 (500 mg, 3.50 mmol) in MeOH (3 mL) was added, and

the mixture was heated at 70 °C for 16 h. Concentrated HCl (0.8 mL,

9.6 mmol) was added and the reaction mixture stirred at 80 °C for

30 min. After cooling to rt, the mixture was concentrated under reduced

pressure to give an orange foam which was dissolved in water (50 mL)

and the solution adjusted to pH 10 using aqueous 1 M sodium

hydroxide solution. The aqueous layer was extracted with CH Cl

3

(

s, 1H), 4.58−4.60 (m, 2H), 4.48−4.51 (m, 4H), 1.37 (s, 3H). LC-MS

+

ESI) m/z 177 [M + H] .

5

-(1-(Trifluoromethyl)cyclopropyl)isoxazol-3-amine (8c). Pre-

pared from compound 5h (1 g, 5.65 mmol) according to the pro-

cedure described for 8a to afford the title compound as a pale yellow

+

solid (687 mg, 64%). LC-MS (ESI) m/z 193 [M + H] .

2

-(3-Aminoisoxazol-5-yl)-2-methylpropan-1-ol (8d). A stirred

solution of compound 5k (1 g, 7.90 mmol), 96% sodium hydroxide

374 mg, 9.35 mmol), and hydroxylamine hydrochloride (650 mg,

.35 mmol) in a mixture of EtOH (100 mL) and water (100 mL) was

(

9

heated at 60 °C for 22 h. Concentrated HCl (3 mL) was added, and

the mixture was heated at 80 °C for 1 h. The reaction mixture was

cooled to rt and then concentrated under reduced pressure. The residue

2

(3 × 50 mL), and the combined organic layers were washed with brine

(50 mL), dried over MgSO , and then concentrated under reduced

4

was partitioned between CHCl and 7 M aqueous sodium hydroxide

pressure. The residue was purified by silica gel flash chromatography

3

1

097

dx.doi.org/10.1021/jm2009925 | J. Med. Chem. 2012, 55, 1082−1105

Journal of Medicinal Chemistry

Article

(

eluting with 12−100% EtOAc in hexanes) to afford the title compound

2-(2-Isopropyl-1,3-dioxolan-2-yl)acetonitrile (10a). A stirred mix-

ture of compound 5a (3.12 g, 28 mmol), ethylene glycol (4.7 mL,

84 mmol), and chlorotrimethylsilane (10.6 mL, 84 mmol) in CH Cl

1

as a cream solid (64 mg, 13%). H NMR (CDCl , 300 MHz): δ 5.82

3

(s, 1H), 4.08 (br s, 2H), 1.71 (d, J = 21 Hz, 6H). LC-MS (ESI) m/z 145

2

+

[M + H] .

(50 mL) was heated at 40 °C for 15 h. After cooling to rt, aqueous 5%

4

-Chlorophenyl 5-(1,1,1-Trifluoro-2-methylpropan-2-yl)isoxazol-

NaHCO (50 mL) was added. The organic layer was separated and the

aqueous phase further extracted with diethyl ether. The combined

3

-ylcarbamate (9a). Prepared from compound 8a (123 mg, 0.63 mmol)

and 4-chlorophenyl chloroformate (180 mg, 0.95 mmol) according to

organic layers were dried over MgSO and then concentrated under

4

General Procedure D to afford the title compound as a colorless solid

reduced pressure. The residue was purified by silica gel flash chro-

1

(

85 mg, 39%). H NMR (CDCl , 300 MHz): δ 7.83 (br s, 1H), 7.38

matography (eluting with 20−40% EtOAc in hexanes) to afford the

3

1

d, J = 9 Hz, 2H), 7.15 (d, J = 9 Hz, 2H), 6.82 (s, 1H), 1.59 (s, 6H).

title compound as a colorless oil (3.38 g, 78%). H NMR (CDCl ,

3

+

LC-MS (ESI) m/z 349 [M + H] .

300 MHz): δ 4.16−4.21 (m, 2H), 3.99−4.07 (m, 2H), 2.69 (s, 2H),

2.07 (m, 1H), 0.96 (d, J = 12 Hz, 6H).

2-(2-Cyclopentyl-1,3-dioxolan-2-yl)acetonitrile (10b). Prepared

from compound 5b (2 g, 14.6 mmol) according to the procedure

Phenyl 5-(1,3-Difluoro-2-methylpropan-2-yl)isoxazol-3-ylcarba-

mate (9b). Prepared from compound 8b (287 mg, 1.6 mmol)

according to General Procedure D to afford the title compound as a

1

solid (358 mg, 76%). H NMR (CDCl , 300 MHz): δ 8.17 (br s, 1H),

described for compound 10a to afford the title compound as an oil

3

1

7

4

.39−7.44 (m, 2H), 7.26 (m, 1H), 7.17−7.20 (m, 2H), 6.81 (s, 1H),

(1.5 g, 57%). H NMR (CDCl , 300 MHz): δ 4.15−4.23 (m, 2H),

3

.63−4.71 (m, 2H), 4.47−4.55 (m, 2H), 1.42 (s, 3H). LC-MS (ESI)

4.01−4.12 (m, 2H), 2.72 (s, 2H), 2.36 (m, 1H), 1.45−1.81 (m, 8H).

4-Fluoro-3-methoxy-4-methylpent-2-enenitrile (11). To a stirred

solution of compound 5c (1 g, 7.75 mmol) in diethyl ether (150 mL)

at 0 °C was added dropwise (trimethylsilyl)diazomethane (4.65 mL of

a 2.0 M solution in diethyl ether, 9.30 mmol). After warming to rt, the

reaction mixture was stirred for a further 15 h. The reaction mixture

was concentrated under reduced pressure to afford the title compound

+

m/z 297 [M + H] .

Phenyl 5-(1-(Trifluoromethyl)cyclopropyl)isoxazol-3-ylcarbamate

9c). Prepared from compound 8c (687 mg, 3.58 mmol) according to

(

General Procedure D to afford the title compound as a colorless solid

1

(

727 mg, 65%). H NMR (DMSO-d , 300 MHz): δ 11.34 (br s, 1H),

6

7

.40−7.47 (m, 2H), 7.20−7.31 (m, 3H), 6.80 (s, 1H), 1.45−1.56

+

(

m, 4H). LC-MS (ESI) m/z 313 [M + H] .

as a yellow oil, which did not require further purification (1 g, 90%).

Phenyl 5-(1-Hydroxy-2-methylpropan-2-yl)isoxazol-3-ylcarba-

mate (9d). Prepared from compound 8d (100 mg, 0.64 mmol)

1

H NMR (CDCl , 300 MHz): δ 4.89 (s, 1H), 4.23 (s, 3H), 1.47 (d, J =

3

2

1 Hz, 6H).

according to General Procedure D to afford the title compound as a

Preparation of Substituted Aminopyrazoles. General Procedure

E. 3-tert-Butyl-1-isopropyl-1H-pyrazol-5-amine Hydrochloride

1

colorless solid (120 mg, 68%). H NMR (CDCl , 300 MHz): δ 8.30

3

(

6

br s, 1H), 7.42−7.43 (m, 2H), 7.26 (m, 1H), 7.18−7.21 (m, 2H),

(

4

12a). A stirred solution of isopropylhydrazine hydrochloride (500 mg,

.54 mmol) and 4,4-dimethyl-3-oxopentanenitrile (679 mg, 5.44 mmol)

.65 (s, 1H), 3.67 (s, 2H), 1.98 (br s, 1H), 1.32 (s, 6H). LC-MS (ESI)

+

m/z 277 [M + H] .

in EtOH (5 mL) was heated under reflux for 15 h. After cooling to rt, the

reaction mixture was concentrated under reduced pressure and the solid

residue was recrystallized from a mixture of diethyl ether and petroleum

Phenyl 5-(1-Methoxy-2-methylpropan-2-yl)isoxazol-3-ylcarba-

mate (9e). Prepared from compound 8e (30 mg, 0.176 mmol)

according to General Procedure D to afford the title compound as an

1

ether to afford the title compound as a colorless solid (500 mg, 51%). H

1

oil (50 mg, 98%). H NMR (CDCl , 300 MHz): δ 8.04 (br s, 1H),

3

NMR (CDCl , 400 MHz): δ 5.55 (s, 1H), 5.05 (br s, 2H), 3.62 (m, 1H),

3

7

3

.42−7.43 (m, 2H), 7.31 (m, 1H), 7.18−7.21 (m, 2H), 6.63 (s, 1H),

+

1

.68 (d, J = 6.4 Hz, 6H), 1.43 (s, 9H). LC-MS (ESI) m/z 182 [M + H] .

.45 (s, 2H), 3.33 (s, 3H), 1.35 (s, 6H). LC-MS (ESI) m/z 291

3

-tert-Butyl-1-isobutyl-1H-pyrazol-5-amine Hydrochloride (12b).

+

[

M + H] .

Prepared from isobutylhydrazine hydrochloride (1 g, 8 mmol) accord-

Phenyl 5-Phenylisoxazol-3-ylcarbamate (9f). Prepared from

ing to General Procedure E to afford the title compound as a colorless

compound 8f (428 mg, 2.67 mmol) according to General Procedure

1

solid (800 mg, 43%). H NMR (DMSO-d , 400 MHz): δ 14.06 (br s,

6

1

D to afford the title compound as a colorless solid (599 mg, 80%). H

1

H), 6.93 (br s, 2H), 5.52 (s, 1H), 3.92 (m, 2H), 2.16 (m, 1H), 1.26

NMR (CDCl , 300 MHz): δ 7.92 (br s, 1H), 7.77−7.80 (m, 2H),

+

3

(s, 9H), 0.83 (m, 6H). LC-MS (ESI) m/z 196 [M + H] .

7

.40−7.51 (m, 6H), 7.20−7.32 (m, 2H), 7.12 (s, 1H). LC-MS (ESI)

3

-tert-Butyl-1-cyclohexyl-1H-pyrazol-5-amine Hydrochloride

+

m/z 281 [M + H] .

(12c). Prepared from cyclohexylhydrazine hydrochloride (1.5 g, 9.96

Phenyl 5-Methylisoxazol-3-ylcarbamate (9g). Prepared from

mmol) according to General Procedure E to afford the title compound

1

5

-methylisoxazol-3-amine 8g (490 mg, 5.0 mmol) according to General

as a colorless solid (1.0 g, 39%). H NMR (DMSO-d , 400 MHz):

6

1

Procedure D to afford the title compound as a solid (425 mg, 39%). H

δ 13.24 (br s, 1H), 7.02 (br s, 2H), 5.52 (s, 1H), 4.30 (m, 1H), 1.63−

+

NMR (DMSO-d , 300 MHz): δ 11.14 (s, 1H), 7.41−7.46 (m, 2H), 7.29

1.98 (m, 7H), 1.10−1.40 (m, 12H). LC-MS (ESI) m/z 222 [M + H] .

6

(

d, J = 6 Hz, 1H), 7.21 (m, 2H), 6.47 (s, 1H), 2.38 (s, 3H). LC-MS (ESI)

1-Benzyl-3-tert-butyl-1H-pyrazol-5-amine (12d). Prepared from

benzylhydrazine (977 mg, 8.0 mmol) according to General Procedure E.

The crude product was purified by silica gel flash chromatography

+

m/z 219 [M + H] .

Phenyl 5-Isopropylisoxazol-3-ylcarbamate (9h). Prepared from

compound 8h (816 mg, 6.5 mmol) according to General Procedure D

(eluting with 5% to 25% EtOAc in hexanes) to afford the title

1

to afford the title compound as a solid (1.24 g, 77%). H NMR

compound as a solid (666 mg, 36%). H NMR (DMSO-d , 300 MHz):

6

(

CDCl , 300 MHz): δ 8.05 (br s, 1H), 7.38−7.45 (m, 2H), 7.18−7.30

δ 7.21−7.32 (m, 3H), 7.09 (d, J = 9 Hz, 2H), 5.17 (s, 1H), 5.07 (s, 2H),

3

+

m, 3H), 6.55 (s, 1H), 3.06 (m, 1H), 1.30 (d, J = 12 Hz, 6H). LC-MS

5.04 (s, 2H), 1.15 (s, 9H). LC-MS (ESI) m/z 230 [M + H] .

+

ESI) m/z 247 [M + H] .

1-Phenyl-3-(1,1,1-trifluoro-2-methylpropan-2-yl)-1H-pyrazol-5-

amine (12e). To a stirred solution of compound 5f (1 g, 5.58 mmol)

in 50% aqueous EtOH (25 mL) was added phenylhydrazine sulfate

(1.15 g, 5.58 mmol), and the reaction mixture was heated at 80 °C for

15 h. After cooling to rt, the reaction mixture was concentrated under

reduced pressure and the residue was partitioned between EtOAc and

aqueous 1 M sodium hydroxide. The organic layer was separated and

the aqueous layer was further extracted multiple times with EtOAc.

Phenyl 5-Cyclopentylisoxazol-3-ylcarbamate (9i). Prepared from

compound 8i (875 mg, 5.75 mmol) according to General Procedure D

to afford the title compound as a colorless solid (1.4 g, 89%). H NMR

1

(

CDCl , 300 MHz): δ 7.97 (br s, 1H), 7.39−7.44 (m, 2H), 7.18−7.29

3

m, 3H), 6.54 (s, 1H), 3.16 (m, 1H), 2.04−2.10 (m, 2H), 1.58−1.78

+

m, 6H). LC-MS (ESI) m/z 273 [M + H] .

4

-Chlorophenyl 5-(2-Fluoropropan-2-yl)isoxazol-3-ylcarbamate

(9j). Prepared from compound 8j (40 mg, 0.278 mmol) and 4-

The combined organic layers were dried over MgSO and then con-

4

chlorophenyl chloroformate (54 mg, 0.28 mmol) according to General

centrated under reduced pressure. The residue was purified by silica

Procedure D to afford the title compound as a colorless solid (83 mg,

gel flash chromatography (eluting with 7−60% EtOAc in hexanes) to

1

00%). H NMR (CDCl , 300 MHz): δ 8.11 (br s, 1H), 7.36−7.40

afford the title compound as a yellow solid (590 mg, 40%). H NMR

3

(

m, 2H), 7.12−7.17 (m, 2H), 6.83 (s, 1H), 1.76 (d, J = 21 Hz, 6H).

(CDCl , 300 MHz): δ 7.30−7.59 (m, 5H), 5.53 (s, 1H), 5.37 (br s, 2 H),

3

+

LC-MS (ESI) m/z 299 [M + H] .

1.45 (s, 6 H). LC-MS (ESI) m/z 270 [M + H] .

1

098

dx.doi.org/10.1021/jm2009925 | J. Med. Chem. 2012, 55, 1082−1105

Journal of Medicinal Chemistry

Article

Preparation of Substituted Pyrazole Carbamates. General

Procedure F. Phenyl 3-tert-Butyl-1-isopropyl-1H-pyrazol-5-ylcar-

bamate (13a). To a stirred mixture of phenyl chloroformate (170 mg,

EtOAc and water, and the organic layer was separated, washed with

brine, dried over MgSO , and then concentrated under reduced

4

pressure. The residue was purified by trituration with diethyl ether to

1

2

^.¹^m^m^o^l⁾^aⁿ^d^p^o^t^a^s^sⁱ^u^m^c^a^r^b^oⁿ^a^t^e⁽²¹⁰^m^g^,¹^.⁵^m^m^o^l⁾ⁱⁿ^C^H^C^l2

afford the title compound as a solid (21.3 g, 85%). H NMR (DMSO-

(3 mL) at 0 °C was added dropwise a solution of compound 12a

d₆, 300 MHz): δ 8.70 (s, 1H), 7.33 (s, 1H), 7.31 (s, 1H), 7.13

(

220 mg, 1 mmol) and N,N-diisopropylethylamine (130 mg, 1 mmol)

(

t, J = 7.6 Hz, 1H), 6.81 (s, 1H), 6.67−6.74 (m, 2H), 5.35 (br s, 2H),

+

in CH Cl (2 mL), and the reaction mixture was stirred at 0 °C for 3 h.

2

3.98 (s, 3H), 3.97 (s, 3H). LC-MS (ESI) m/z 314 [M + H] .

Preparation of Quinazolin-4-yloxy Phenylurea Derivatives.

General Procedure H. 1-(3-tert-Butylisoxazol-5-yl)-3-(3-(6,7-di-

methoxyquinazolin-4-yloxy)phenyl)urea (18). To a stirred solution of

compound 16 (90 mg, 0.3 mmol), N,N-diisopropylethylamine (78 μL,

0.45 mmol), and 4-(dimethylamino)pyridine (1.8 mg, 0.015 mmol) in

THF (1.5 mL) was added compound 7j (118 mg, 0.45 mmol). The

reaction mixture was heated at 50 °C for 2.5 h. After cooling to rt, the

The mixture was filtered, and the filtrate was washed with water and

brine and then dried over MgSO . Concentration under reduced

4

pressure gave a solid, which was recrystallized from diethyl ether to

afford the title compound as a colorless solid (300 mg, 100%). LC-MS

+

(

ESI) m/z 302 [M + H] .

Phenyl 3-tert-Butyl-1-isobutyl-1H-pyrazol-5-ylcarbamate (13b).

Prepared from compound 12b according to General Procedure F to

+

afford the title compound as a solid. LC-MS (ESI) m/z 316 [M + H] .

mixture was partitioned between CH Cl₂and saturated aqueous

2

Phenyl 3-tert-Butyl-1-cyclohexyl-1H-pyrazol-5-ylcarbamate

NaHCO . The organic layer was separated and the aqueous layer

3

(

13c). Prepared from compound 12c (260 mg, 1 mmol) according

was further extracted with CH Cl . The combined organic layers

2

to General Procedure F to afford the title compound as a solid (300 mg,

were dried over MgSO and then concentrated under reduced pres-

4

+

8

8%). LC-MS (ESI) m/z 342 [M + H] .

sure. The residue was purified by preparative reverse-phase HPLC, and

Preparation of Substituted Pyrazole Carbamates. General

the obtained solid was triturated with diethyl ether to afford the title

1

Procedure G. Phenyl 3-tert-Butyl-1-benzyl-1H-pyrazol-5-ylcarba-

compound as a colorless solid (41 mg, 29%). H NMR (DMSO-d ,

6

mate (13d). To a stirred mixture of compound 12d (666 mg,

3

2

6

00 MHz): δ 10.19 (s, 1H), 9.04 (s, 1H), 8.57 (s, 1H), 7.56−7.59 (m,

2

(

.91 mmol) and potassium carbonate (522 mg, 3.78 mmol) in THF

10 mL) at rt was added dropwise a solution of phenyl chloroformate

499 mg, 3.2 mmol) in THF (5 mL). The reaction mixture was stirred

H), 7.39−7.44 (m, 2H), 7.30 (d, J = 9 Hz, 1H), 6.98 (d, J = 9 Hz, 1H),

.04 (s, 1H), 3.99 (s, 6H), 1.25 (s, 9H). LC-MS (ESI) m/z 464

+

[

M + H] .

2

^a^t^r^t^f^o^r¹⁵^h^.^T^h^e^mⁱ^x^t^u^r^e^w^a^s^p^a^r^tⁱ^tⁱ^oⁿ^e^d^b^e^t^w^e^eⁿ^w^a^t^e^r^aⁿ^d^C^H^C^l2

1-(3-tert-Butyl-1-methyl-1H-pyrazol-5-yl)-3-(3-(6,7-dimethoxy-

quinazolin-4-yloxy)phenyl)urea (19). Prepared from compound 13f

and the organic layer was separated, dried over MgSO , and then

4

concentrated under reduced pressure. The residue was purified by

silica gel flash chromatography (eluting with 100% hexanes to 50%

(123 mg, 0.45 mmol) and compound 16 (89 mg, 0.30 mmol) accord-

ing to General Procedure H. The crude product was purified by silica

gel flash chromatography (eluting with 100% hexanes to 100% EtOAc

EtOAc in hexanes) to afford the title compound as a solid (565 mg,

1

5

(

3

6%). H NMR (DMSO-d , 300 MHz): δ 10.20 (br s, 1H), 7.10−7.43

6

in hexanes) to afford the title compound as a solid (102 mg, 71%). H

m, 10H), 6.14 (s, 1H), 5.29 (s, 2H), 1.22 (s, 9H). LC-MS (ESI) m/z

50 [M + H] .

NMR (DMSO-d , 300 MHz): δ 9.20 (s, 1H), 8.56−8.58 (m, 2H), 7.53

6

+

(

m, 2H), 7.50 (m, 2H), 7.30 (m, 1H), 6.95 (m, 1H), 6.08 (s, 1H), 3.99

s, 6H), 3.54 (s, 3H), 1.25 (s, 9H). LC-MS (ESI) m/z 477 [M + H] .

+

Phenyl 1-Phenyl-3-(1,1,1-trifluoro-2-methylpropan-2-yl)-1H-pyrazol-

-ylcarbamate (13e). Prepared from compound 12e (590 mg,

5

2

1

-(4-tert-Butylphenyl)-3-(3-(6,7-dimethoxyquinazolin-4-yloxy)-

.2 mmol) according to General Procedure G to afford the title

phenyl)urea (20). To a stirred solution of compound 16 (89 mg,

0.3 mmol) in DMF (3 mL) at rt was added 4-tert-butylphenyl iso-

cyanate (54 μL, 0.3 mmol), and the mixture was heated at 50 °C for

1

compound as a solid (748 mg, 87%). H NMR (CDCl , 300 MHz):

3

δ 7.02−7.55 (m, 11H), 6.65 (br s, 1H), 1.60 (s, 6H). LC-MS (ESI)

+

m/z 390 [M + H] .

4

h. After cooling to rt, the mixture was partitioned between EtOAc

Phenyl 3-tert-Butyl-1-methyl-1H-pyrazol-5-ylcarbamate (13f).

Prepared from 3-tert-butyl-1-methyl-1H-pyrazol-5-amine 12f (1.0 g,

and water. The organic layer was separated and further washed with

brine. The organic layer was separated, dried over MgSO , and then

4

6

.5 mmol) according to General Procedure G to afford the title

concentrated under reduced pressure. The residue was purified by

silica gel flash chromatography (eluting with 25% to 100% EtOAc in

1

compound as a solid (530 mg, 30%). H NMR (DMSO-d , 300

6

1

MHz): δ 10.20 (s, 1H), 7.40−7.49 (m, 2H), 721−7.30 (m, 3H), 6.06

hexanes) to afford the title compound as a solid (54 mg, 38%). H

+

(

s, 1H), 3.66 (s, 3H), 1.25 (s, 9H). LC-MS (ESI) m/z 274 [M + H] .

Phenyl 3-tert-Butyl-1-phenyl-1H-pyrazol-5-ylcarbamate (13g).

NMR (DMSO-d , 300 MHz): δ 8.83 (s, 1H), 8.65 (s, 1H), 8.57 (s,

6

1

H), 7.60 (s, 1H), 7.56 (s, 1H), 7.21−7.40 (m, 7H), 6.91 (d, J = 6 Hz,

+

Prepared from 3-tert-butyl-1-phenyl-1H-pyrazol-5-amine 12g (2.00 g,

.3 mmol) according to General Procedure G to afford the title

H), 3.99 (s, 6H), 1.25 (s, 9H). LC-MS (ESI) m/z 473 [M + H] .

9

1

-(3-tert-Butylphenyl)-3-(3-(6,7-dimethoxyquinazolin-4-yloxy)-

1

compound as a solid (1.3 g, 42%). H NMR (DMSO-d , 300 MHz): δ

phenyl)urea (21). A mixture of 3-tert-butylaniline (447 mg, 3 mmol),

potassium carbonate (828 mg, 6 mmol), phenyl chloroformate (1.13 mL,

9 mmol), and 4-(dimethylamino)pyridine (36 mg, 0.30 mmol) in

THF (15 mL) was stirred at rt for 15 h. The reaction mixture was

diluted with EtOAc, filtered, and then concentrated under reduced

pressure. The residue was purified by silica gel flash chromatography

6

1

3

0.00 (s, 1H), 7.42−7.57 (m, 4H), 7.33−7.44 (m, 3H), 7.08−7.23 (m,

+

H), 6.37 (s, 1H), 1.30 (s, 9H). LC-MS (ESI) m/z 336 [M + H] .

3

-(6,7-Dimethoxyquinazolin-4-yloxy)aniline (16). To a stirred

slurry of cesium carbonate (28.20 g, 86 mmol) in THF (300 mL) at

rt was added 3-aminophenol (4.83 g, 43 mmol). After stirring for

0 min at rt, 4-chloro-6,7-dimethoxyquinazoline (10 g, 43 mmol) was

added and the reaction mixture was heated at 50 °C for 24 h. After

cooling to rt, the mixture was partitioned between EtOAc and 1 M

aqueous sodium hydroxide. The organic layer was separated, washed

3

(eluting with 5−15% EtOAc in hexanes) to afford phenyl 3-tert-

1

butylphenylcarbamate as a solid (458 mg, 57%). H NMR (DMSO-d ,

6

300 MHz): δ 10.14 (s, 1H), 7.59 (s, 1H), 7.10−7.49 (m, 7H), 7.08 (d,

+

J = 9 Hz, 1H), 1.25 (s, 9H). LC-MS (ESI) m/z 270 [M + H] . Phenyl

with brine, dried over MgSO , and then concentrated under reduced

3-tert-butylphenylcarbamate (114 mg, 0.42 mmol) and compound 16

(90 mg, 0.3 mmol) were allowed to react according to General Proce-

dure H. The crude product was purified by silica gel flash chromato-

4

pressure. The residue was purified by silica gel flash chromatography

(

eluting with 1% MeOH in CH Cl ) to afford the title compound as a

2 2

1

solid (12.7 g, 100%). H NMR (DMSO-d , 300 MHz): δ 8.55 (s, 1H),

graphy (eluting with 25−100% EtOAc in hexanes) to afford the title

6

1

7

(

.51 (s, 1H), 7.37 (s, 1H), 7.09 (m, 1H), 6.38−6.49 (m, 3H), 5.30

compound as a solid (83 mg, 59%). H NMR (DMSO-d , 300 MHz):

6

+

br s, 2H), 3.99 (s, 3H), 3.97 (s, 3H). LC-MS (ESI) m/z 298 [M + H] .

-(6,7-Dimethoxyquinazolin-4-ylthio)aniline (17). To a stirred

slurry of sodium hydride (3.52 g of a 60% dispersion in mineral oil,

8 mmol) in THF (500 mL) at rt was added portionwise 3-amino-

benzenethiol (10 g, 80 mmol). After stirring for 30 min at rt, 4-chloro-

,7-dimethoxyquinazoline (18 g, 80 mmol) was added and the reaction

mixture was stirred at rt for 15 h. The mixture was partitioned between

δ 8.83 (s, 1H), 8.70 (s, 1H), 8.57 (s, 1H), 7.61 (s, 1H), 7.57 (s, 1H),

7.36−7.50 (m, 3H), 7.14−7.31 (m, 3H), 6.86−7.05 (m, 2H), 4.00

3

+

(s, 6H), 1.28 (s, 9H). LC-MS (ESI) m/z 473 [M + H] .

8

1-(3-(6,7-Dimethoxyquinazolin-4-yloxy)phenyl)-3-(5-methylisox-

azol-3-yl)urea (22). Prepared from compound 9g (98 mg, 0.42 mmol)

and compound 16 (89 mg, 0.3 mmol) according to General Procedure

6

1

H to afford the title compound as a solid (31 mg, 25%). H NMR

1

099

dx.doi.org/10.1021/jm2009925 | J. Med. Chem. 2012, 55, 1082−1105

Journal of Medicinal Chemistry

Article

(

DMSO-d , 300 MHz): δ 9.53 (s, 1H), 9.01 (s, 1H), 8.56 (s, 1H), 7.57

(m, 1H), 6.50 (s, 1H), 3.99 (s, 6H), 3.38 (s, 2H), 3.23 (s, 3H), 1.24

(s, 6H). LC-MS (ESI) m/z 494 [M + H] .

1-(3-(6,7-Dimethoxyquinazolin-4-yloxy)phenyl)-3-(3-

(trifluoromethyl)isoxazol-5-yl)urea (31). Prepared from compound 7e

6

+

s, 1H), 7.55 (s, 1H), 7.35−7.45 (m, 2H), 7.26 (d, J = 9 Hz, 1H), 6.97

d, J = 6 Hz, 1H), 6.51 (s, 1H), 3.99 (s, 6H), 2.35 (s, 3H). LC-MS

+

ESI) m/z 422 [M + H] .

(124 mg, 0.45 mmol) and compound 16 (104 mg, 0.35 mmol) accord-

1

-(3-(6,7-Dimethoxyquinazolin-4-yloxy)phenyl)-3-(5-isopropyl-

isoxazol-3-yl)urea (23). Prepared from compound 9h (110 mg,

.45 mmol) and compound 16 (90 mg, 0.3 mmol) according to General

Procedure H to afford the title compound as a colorless solid (79 mg,

ing to General Procedure H to afford the title compound as a colorless

1

0

solid (9 mg, 5%). H NMR (DMSO-d , 300 MHz): δ 10.90 (br s, 1H),

6

9.30 (br s, 1H), 8.59 (br s, 1H), 7.20−7.80 (m, 5H), 7.06 (br s, 1H),

1

+

5

8

6

9%). H NMR (DMSO-d , 300 MHz): δ 9.57 (s, 1H), 9.01 (s, 1H),

6.50 (s, 1H), 4.09 (s, 6H). LC-MS (ESI) m/z 476 [M + H] .

6

.56 (s, 1H), 7.57 (s, 2H), 7.38−7.43 (m, 2H), 7.27 (d, J = 9 Hz, 1H),

1-(3-(1,1-Difluoroethyl)isoxazol-5-yl)-3-(3-(6,7-dimethoxyquina-

zolin-4-yloxy)phenyl)urea (32). Prepared from compound 7d

(141 mg, 0.52 mmol) and compound 16 (156 mg, 0.52 mmol) accord-

.99 (d, J = 9 Hz, 1H), 6.49 (s, 1H), 4.00 (s, 6H), 3.00 (m, 1H), 1.22

+

(d, J = 12 Hz, 6H). LC-MS (ESI) m/z 450 [M + H] .

ing to General Procedure H to afford the title compound as a colorless

1

-(5-Cyclopentylisoxazol-3-yl)-3-(3-(6,7-dimethoxyquinazolin-4-

yloxy)phenyl)urea (24). Prepared from compound 9i (130 mg,

.48 mmol) and compound 16 (95 mg, 0.32 mmol) according to

General Procedure H to afford the title compound as a colorless solid

1

solid (66 mg, 27%). H NMR (DMSO-d , 300 MHz): δ 10.70 (s, 1H),

6

9

.17 (s, 1H), 8.56 (s, 1H), 7.56−7.57 (m, 2H), 7.39−7.45 (m, 2H),

0

7

.33 (m, 1H), 7.00 (m, 1H), 6.27 (s, 1H), 3.99 (s, 3H), 3.98 (s, 3H),

+

1

2.00 (t, J = 19 Hz, 3H). LC-MS (ESI) m/z 472 [M + H] .

(

81 mg, 53%). H NMR (DMSO-d , 300 MHz): δ 9.58 (s, 1H), 9.03

6

1

-(3-(6,7-Dimethoxyquinazolin-4-yloxy)phenyl)-3-(3-(2-fluoro-

propan-2-yl)isoxazol-5-yl)urea (33). Prepared from compound 7c

95 mg, 0.36 mmol) and compound 16 (89 mg, 0.3 mmol) according

s, 1H), 8.56 (s, 1H), 7.57 (s, 2H), 7.38−7.43 (m, 2H), 7.26 (d, J = 9

Hz, 1H), 6.97 (d, J = 9 Hz, 1H), 6.50 (s, 1H), 4.00 (s, 6H), 3.11

(

m, 1H), 1.64−1.66 (m, 2H), 1.18−1.20 (m, 6H). LC-MS (ESI) m/z

+

to General Procedure H, to afford the title compound as a colorless

4

76 [M + H] .

-(3-(6,7-Dimethoxyquinazolin-4-yloxy)phenyl)-3-(5-phenyl-

isoxazol-3-yl)urea (25). Prepared from compound 9f (126 mg,

.45 mmol) and compound 16 (90 mg, 0.3 mmol) according to General

Procedure H to afford the title compound as a colorless solid (47 mg,

1

solid (63 mg, 45%). H NMR (DMSO-d , 300 MHz): δ 10.50 (br s,

6

1

2

3

H), 9.15 (s, 1H), 8.56 (s, 1H), 7.57−7.58 (m, 2H), 7.40−7.45 (m,

H), 7.32 (m, 1H), 7.00 (m, 1H), 6.14 (s, 1H), 4.00 (s, 3H), 3.99 (s,

0

+

H), 1.67 (d, J = 21 Hz, 6H). LC-MS (ESI) m/z 468 [M + H] .

1

1-[3-(1,3-Difluoro-2-methylpropan-2-yl)isoxazol-5-yl]-3-[3-(6,7-

3

8

2%). H NMR (DMSO-d , 300 MHz): δ 9.75 (s, 1H), 9.08 (s, 1H),

6

dimethoxyquinazolin-4-yloxy)phenyl]urea (34). Prepared from

compound 7i (158 mg, 0.5 mmol) and compound 16 (119 mg,

.58 (s, 1H), 7.86 (d, J = 6 Hz, 2H), 7.51−7.87 (m, 4H), 7.40−7.51

(

m, 2H), 7.21−7.31 (m, 3H), 7.00 (d, J = 6 Hz, 1H), 4.00 (s, 6H). LC-MS

0

.4 mmol) according to General Procedure H. The crude product was

+

(

ESI) m/z 484 [M + H] .

-(3-(6,7-Dimethoxyquinazolin-4-yloxy)phenyl)-3-(3-isopropyl-

isoxazol-5-yl)urea (26). Prepared from compound 7a (89 mg,

.36 mmol) and compound 16 (89 mg, 0.30 mmol) according to General

Procedure H to afford the title compound as a colorless solid (25 mg,

purified by silica gel flash chromatography (eluting with 40−50%

EtOAc in hexanes) to afford the title compound as a solid (115 mg,

1

5

8

8%). H NMR (DMSO-d , 300 MHz): δ 10.33 (s, 1H), 9.00 (s, 1H),

6

0

.55 (s, 1H), 7.55−7.58 (m, 3H), 7.39 (s, 1H), 7.26 (d, J = 6 Hz, 2H),

1

6.24 (s, 1H), 4.72−4.76 (m, 2H), 4.56−4.57 (m, 2H), 3.98 (s, 6H),

1

6

1

9%). H NMR (DMSO-d , 300 MHz): δ 10.30 (br s, 1H), 9.14 (s,

6

+

.30 (s, 3H). LC-MS (ESI) m/z 500 [M + H] .

-(3-(6,7-Dimethoxyquinazolin-4-yloxy)phenyl)-3-(3-(1,1,1-tri-

H), 8.56 (s, 1H), 7.57 (s, 2H), 7.39−7.44 (m, 2H), 7.31 (m, 1H),

1

.99 (m, 1H), 5.99 (s, 1H), 4.00 (s, 6H), 2.90 (septet, J = 6 Hz, 1H),

fluoro-2-methylpropan-2-yl)isoxazol-5-yl)urea (35). Prepared from

compound 7f (104 mg, 0.3 mmol) and compound 16 (89 mg, 0.3 mmol)

according to General Procedure H. The crude product was purified by

silica gel flash chromatography (eluting with 100% CH Cl to 50% EtOAc

+

.19 (d, J = 6 Hz, 6H). LC-MS (ESI) m/z 450 [M + H] .

1

-(3-Cyclopentylisoxazol-5-yl)-3-(3-(6,7-dimethoxyquinazolin-4-

yloxy)phenyl)urea (27). Prepared from compound 7b (124 mg, 0.45

mmol) and compound 16 (104 mg, 0.35 mmol) according to General

Procedure H to afford the title compound as a colorless solid (75 mg,

4

8

6

1

2

in CH Cl ) to afford the title compound as a colorless solid (80 mg,

2

1

5

2%). H NMR (DMSO-d , 300 MHz): δ 10.44 (s, 1H), 9.11 (s, 1H),

1

6

5%). H NMR (DMSO-d , 300 MHz): δ 10.18 (s, 1H), 9.05 (s, 1H),

6

8

.56 (s, 1H), 7.56−7.58 (m, 2H), 7.40−7.45 (m, 2H), 7.31 (m, 1H), 7.01

.56 (s, 1H) 7.57 (s, 2H), 7.39−7.56 (m, 2H), 7.29 (d, J = 9 Hz, 1H),

(m, 1H), 6.18 (s, 1H), 4.00 (s, 6H), 1.24 (s, 6H). LC-MS (ESI) m/z 518

.98 (d, J = 9 Hz, 1H), 5.95 (s, 1H), 4.00 (s, 6H), 3.03 (m, 1H), 1.93−

+

[

M + H] .

-(3-(6,7-Dimethoxyquinazolin-4-yloxy)phenyl)-3-(3-(1-

trifluoromethyl)cyclobutyl)isoxazol-5-yl)urea (36). Prepared from

compound 7h (147 mg, 0.45 mmol) and compound 16 (89 mg,

.30 mmol) according to General Procedure H. The crude product was

+

.99 (m, 2H), 1.61−1.69 (m, 6H). LC-MS (ESI) m/z 476 [M + H] .

1

-(3-(6,7-Dimethoxyquinazolin-4-yloxy)phenyl)-3-(3-phenylisox-

(

azol-5-yl)urea (28). Prepared from compound 7k (126 mg, 0.45 mmol)

and compound 16 (90 mg, 0.3 mmol) according to General Procedure H

to afford the title compound as a colorless solid (63 mg, 43%). H NMR

0

1

purified by silica gel flash chromatography (eluting with 100% CH Cl

2

(

7

DMSO-d , 300 MHz): δ 10.40 (s, 1H), 9.14 (s, 1H), 8.50 (s, 1H), 7.74−

6

to 10% MeOH in CH Cl ) to afford the title compound as a colorless

2

.83 (m, 2H), 7.48−7.83 (m, 7H), 7.42 (d, J = 9 Hz, 1H), 7.00 (d, J =

1

solid (81 mg, 51%). H NMR (DMSO-d , 300 MHz): δ 10.50 (br s,

6

+

9

Hz, 1H), 6.56 (s, 1H), 4.00 (s, 6H). LC-MS (ESI) m/z 484 [M + H] .

1

H), 9.11 (s, 1H), 8.56 (s, 1H), 7.56 (d, J = 2.3 Hz, 2H), 7.29−7.44

1

-(3-(6,7-Dimethoxyquinazolin-4-yloxy)phenyl)-3-(5-(1-hydroxy-

(

m, 3H), 7.00 (d, J = 7.9 Hz, 1H), 6.08 (s, 1H), 3.99 (s, 6H), 2.50−2.56

2

-methylpropan-2-yl)isoxazol-3-yl)urea (29). Prepared from com-

+

(

m, 4H), 2.02 (m, 2H). LC-MS (ESI) m/z 530 [M + H] .

pound 9d (41 mg, 0.15 mmol) and compound 16 (30 mg, 0.10 mmol)

according to General Procedure H. The crude product was purified by

preparative silica gel TLC (eluting with 10% MeOH in CH Cl ) to

afford the title compound as a colorless solid (30 mg, 63%). H NMR

1

-(3-(2-Cyanopropan-2-yl)isoxazol-5-yl)-3-(3-(6,7-dimethoxy-

quinazolin-4-yloxy)phenyl)urea (37). Prepared from compound 7g

(90 mg, 0.332 mmol) and compound 16 (89 mg, 0.30 mmol) accord-

ing to General Procedure H. The crude product was purified by silica

gel flash chromatography (eluting with 100% CH Cl to 10% MeOH

2

1

(

DMSO-d , 300 MHz): δ 9.57 (br s, 1H), 8.99 (br s, 1H), 8.56

6

2

s, 1H), 7.56−7.58 (m, 2H), 7.38−7.42 (m, 2H), 7.25 (m, 1H), 6.97

in CH Cl ) to afford the title compound as a colorless solid (55 mg,

2 2

1

m, 1H), 6.49 (s, 1H), 4.95 (br s, 1H), 3.99 (s, 3H), 3.98 (s, 3H), 3.43

39%). H NMR (DMSO-d , 300 MHz): δ 10.51 (br s, 1H), 9.12 (br s,

6

+

s, 2H), 1.20 (s, 6H). LC-MS (ESI) m/z 480 [M + H] .

1H), 8.57 (s, 1H), 7.56−7.57 (m, 2H), 7.31−7.45 (m, 3H), 7.01 (m,

1

-(3-(6,7-Dimethoxyquinazolin-4-yloxy)phenyl)-3-(5-(1-methoxy-

1H), 6.27 (s, 1H), 4.00 (s, 6H), 1.68 (s, 6H). LC-MS (ESI) m/z 475

+

2

-methylpropan-2-yl)isoxazol-3-yl)urea (30). Prepared from com-

[

M + H] .

pound 9e (50 mg, 0.17 mmol) and compound 16 (50 mg, 0.16 mmol)

according to General Procedure H. The crude product was purified by

preparative silica gel TLC (eluting with 10% MeOH in CH Cl ) to

1

-(3-(6,7-Dimethoxyquinazolin-4-yloxy)phenyl)-3-(5-(2-fluoro-

propan-2-yl)isoxazol-3-yl)urea (38). Prepared from compound 9j

(90 mg, 0.302 mmol) and compound 16 (90 mg, 0.302 mmol)

according to General Procedure H. The crude product was purified by

silica gel flash chromatography (eluting with 100% CH Cl to 10%

2

1

afford the title compound as a colorless solid (38 mg, 49%). H NMR

(

DMSO-d , 300 MHz): δ 9.57 (br s, 1H), 9.01 (br s, 1H), 8.56

6

2

(

s, 1H), 7.57−7.58 (m, 2H), 7.38−7.42 (m, 2H), 7.25 (m, 1H), 6.97

MeOH in CH Cl ) to afford the title compound as a colorless solid

2 2

1

100

dx.doi.org/10.1021/jm2009925 | J. Med. Chem. 2012, 55, 1082−1105

Journal of Medicinal Chemistry

Article

1

(

37 mg, 26%). H NMR (DMSO-d , 300 MHz): δ 9.75 (br s, 1H),

compound as a colorless solid (134 mg, 55%). H NMR (DMSO-d ,

6

9

7

2

.04 (br s, 1H), 8.56 (s, 1H), 7.56−7.58 (m, 2H), 7.40−7.41 (m, 2H),

300 MHz): δ 9.77 (s, 1H), 9.04 (s, 1H), 8.70 (s, 1H), 7.86 (s, 1H),

7.28−7.54 (m, 5H), 6.89 (s, 1H), 3.99 (s, 6H), 1.54 (s, 6H). LC-MS

.29 (m, 1H), 7.00 (m, 1H), 6.86 (s, 1H), 4.00 (s, 6H), 1.72 (d, J =

+

1 Hz, 6H). LC-MS (ESI) m/z 468 [M + H] .

(ESI) m/z 534 [M + H] .

1

-[5-(1,3-Difluoro-2-methylpropan-2-yl)isoxazol-3-yl]-3-[3-(6,7-

1-(3-(6,7-Dimethoxyquinazolin-4-ylthio)phenyl)-3-(5-(1-

(trifluoromethyl)cyclopropyl)isoxazol-3-yl)urea (44). Prepared from

compound 9c (112 mg, 0.36 mmol) and compound 17 (95 mg,

dimethoxyquinazolin-4-yloxy)phenyl]urea (39). A mixture of com-

pound 9b (89 mg, 0.3 mmol), compound 16 (89 mg, 0.3 mmol), and

4

-(dimethylamino)pyridine (30 mg, 0.25 mmol) in THF (6 mL) was

0.3 mmol) according to General Procedure I to afford the title compound

1

stirred at rt for 15 h. The reaction mixture was partitioned between

CH Cl and water, and the organic layer was separated, dried over

MgSO , and then concentrated under reduced pressure. The residue

as a solid (108 mg, 68%). H NMR (DMSO-d , 300 MHz): δ 9.73

6

(s, 1H), 9.01 (s, 1H), 8.69 (s, 1H), 7.84 (s, 1H), 7.52 (m, 1H), 7.45

(t, J = 9 Hz, 1H), 7.35 (s, 1H), 7.34 (s, 1H), 7.29 (d, J = 6 Hz, 1H),

6.86 (s, 1H), 3.99 (s, 6H), 1.45−1.56 (m, 4H). LC-MS (ESI) m/z 532

2

4

was purified by silica gel flash chromatography (eluting with 70−95%

+

EtOAc in hexanes) to afford the title compound as a solid (55 mg, 31%).

[M + H] .

1

H NMR (DMSO-d , 300 MHz): δ 9.70 (s, 1H), 9.02 (s, 1H), 8.57

1-(3-(6,7-Dimethoxyquinazolin-4-ylthio)phenyl)-3-(3-(2-fluoro-

propan-2-yl)isoxazol-5-yl)urea (45). Prepared from compound 7c

6

(

6

s, 1H), 7.57−7.58 (m, 2H), 7.38−7.43 (m, 2H), 7.27 (d, J = 9 Hz, 1H),

(253 mg, 0.96 mmol) and compound 17 (200 mg, 0.64 mmol)

.98 (d, J = 9 Hz, 1H), 6.77 (s, 1H), 4.71 (m, 2H), 4.56 (m, 2H), 4.00

+

according to General Procedure I. The crude product was purified by

(s, 3H), 3.98 (s, 3H), 1.28 (s, 3H). LC-MS (ESI) m/z 500 [M + H] .

trituration with MeOH to afford the title compound as a colorless

1

-(3-(6,7-Dimethoxyquinazolin-4-yloxy)phenyl)-3-(5-(1,1,1-tri-

1

fluoro-2-methylpropan-2-yl)isoxazol-3-yl)urea Hydrochloride (40).

To a solution of compound 16 (3.52 g, 11.9 mmol) in THF (140 mL)

were added compound 9a (4.1 g, 11.8 mmol) and 4-dimethylamino-

pyridine (100 mg, 0.8 mmol), and the solution was stirred at rt for

solid (142 mg, 46%). H NMR (DMSO-d

6

, 300 MHz): δ 10.43 (br s,

1H), 9.13 (br s, 1H), 8.70 (s, 1H), 7.84 (s, 1H), 7.57 (m, 1H), 7.46

(m, 1H), 7.30−7.35 (m, 3H), 6.16 (s, 1H), 3.99 (s, 6H), 1.67 (d, J =

+

21 Hz, 6H). LC-MS (ESI) m/z 484 [M + H] .

1

5 h before heating at 45 °C for a further 24 h. After cooling to rt, the

1-(3-tert-Butyl-1-isopropyl-1H-pyrazol-5-yl)-3-(3-(6,7-dimethoxy-

quinazolin-4-yloxy)phenyl)urea (46). A stirred solution of compound

mixture was concentrated under reduced pressure. The residue was

purified by silica gel flash chromatography (eluting with 20−100%

EtOAc in hexanes) to afford 1-(3-(6,7-dimethoxyquinazolin-4-yloxy)-

phenyl)-3-(5-(1,1,1-trifluoro-2-methylpropan-2-yl)isoxazol-3-yl)urea

as a colorless solid (4.55 g, 75%). H NMR (DMSO-d , 300 MHz):

1

3a (150 mg, 0.50 mmol), N,N-diisopropylethylamine (80 mg, 0.62 mmol),

and compound 16 (92 mg, 0.31 mmol) in THF (1 mL) was heated

at 60 °C for 15 h. After cooling to rt, the reaction mixture was

1

^p^a^r^tⁱ^tⁱ^oⁿ^e^d^b^e^t^w^e^eⁿ^C^H^C^l2 and saturated aqueous Na CO . The

2

3

6

organic phase was separated, dried over MgSO , and then concen-

δ 9.74 (s, 1H), 9.01 (s, 1H), 8.56 (s, 1H), 7.56−7.59 (m, 2H), 7.38−

4

trated under reduced pressure. The residue was purified by silica gel

7

1

.41 (m, 2H), 7.26 (m, 1H), 6.98 (m, 1H), 6.87 (s, 1H), 3.99 (s, 6H),

.54 (s, 6H). LC-MS (ESI) m/z 518 [M + H] . To a stirred solution

+

flash chromatography (eluting with 2−4% MeOH in CH Cl ) followed

2

by preparative reverse-phase HPLC to afford the title compound as a

of 1-(3-(6,7-dimethoxyquinazolin-4-yloxy)phenyl)-3-(5-(1,1,1-tri-

fluoro-2-methylpropan-2-yl)isoxazol-3-yl)urea (3.49 g, 6.75 mmol) in

CH Cl (150 mL) at 0 °C was added slowly a 1 M solution of HCl in

diethyl ether (13.50 mL, 13.50 mmol). A colorless precipitate was ob-

served. After stirring for a further 5 min, the mixture was concentrated

under reduced pressure. The resulting solid was triturated with diethyl

ether,then filtered and dried, to afford the title compound as a color-

less solid (3.25 g, 87%). H NMR (DMSO-d , 300 MHz): δ 10.00 (br

s, 1H), 9.73 (br s, 1H), 8.83 (s, 1H), 7.66 (s, 1H), 7.62 (dd, J = 2.1,

2

1

colorless solid (62 mg, 40%). H NMR (DMSO-d , 400 MHz): δ 9.08

6

(

2

s, 1H), 8.57 (s, 1H), 8.43 (s, 1H), 7.55−7.59 (m, 2H), 7.37−7.40 (m,

2

H), 7.24 (m, 1H), 6.93 (m, 1H), 6.00 (s, 1H), 4.35 (m, 1H), 4.00

(

s, 3H), 3.99 (s, 3H), 1.34 (d, J = 6.4 Hz, 6H), 1.24 (s, 9H). LC-MS

ESI) m/z 505 [M + H] .

+

1

-(3-tert-Butyl-1-isobutyl-1H-pyrazol-5-yl)-3-(3-(6,7-dimethoxy-

quinazolin-4-yloxy)phenyl)urea (47). Prepared from compound 13b

150 mg, 0.47 mmol) and compound 16 (92 mg, 0.31 mmol)

1

6

(

according to the procedure described for compound 46 to afford the

.1 Hz, 1H), 7.48 (s, 1H), 7.43 (dd, J = 8.1, 8.1 Hz, 1H), 7.31

1

title compound as a pale yellow solid (60 mg, 38%). H NMR

(

m, 1H), 7.00 (m, 1H), 6.87 (s, 1H), 4.03 (s, 3H), 4.02 (s, 3H), 1.54

s, 6H). LC-MS (ESI) m/z 518 [M + H] .

+

(DMSO-d , 400 MHz): δ 9.09 (s, 1H), 8.57 (s, 1H), 8.49 (s, 1H), 7.61

6

(

1

2

m, 1H), 7.57 (m, 1H), 7.37−7.41 (m, 2H), 7.24 (m, 1H), 6.94 (m,

1

-(3-(6,7-Dimethoxyquinazolin-4-yloxy)phenyl)-3-(5-(1-(trifluoro-

H), 6.01 (s, 1H), 4.00 (s, 3H), 3.99 (s, 3H), 3.71 (d, J = 7.6 Hz, 2H),

methyl)-cyclopropyl)isoxazol-3-yl)urea (41). Prepared from com-

pound 9c (112 mg, 0.36 mmol) and compound 16 (90 mg, 0.3 mmol)

according to General Procedure H to afford the title compound as a

.07 (m, 1H), 1.20 (s, 9H), 0.83 (d, J = 6.4 Hz, 6H). LC-MS (ESI)

+

m/z 519 [M + H] .

1

-(3-tert-Butyl-1-cyclohexyl-1H-pyrazol-5-yl)-3-(3-(6,7-dimethoxy-

quinazolin-4-yloxy)phenyl)urea (48). Prepared from compound 13c

200 mg, 0.58 mmol) and compound 16 (80 mg, 0.26 mmol) accord-

solid (107 mg, 69%). H NMR (DMSO-d , 300 MHz): δ 9.73 (s, 1H),

6

9

8

1

.10 (s, 1H), 8.56 (s, 1H), 7.58 (s, 1H), 7.57 (s, 1H), 7.41 (t, J =

.1 Hz, 1H), 7.39 (s, 1H), 7.26 (d, J = 6 Hz, 1H), 6.98 (d, J = 6 Hz,

H), 6.85 (s, 1H), 3.99 (s, 3H), 3.98 (s, 3H), 1.56−1.41 (m, 4H). LC-

(

ing to the procedure described for compound 46 to afford the title

1

+

compound as a pale yellow solid (55 mg, 39%). H NMR (DMSO-d ,

MS (ESI) m/z 516 [M + H] .

6

4

00 MHz): δ 9.10 (s, 1H), 8.57 (s, 1H), 8.47 (s, 1H), 7.57−7.60 (m,

Preparation of Quinazolin-4-ylthio Phenylurea Derivatives.

General Procedure I. 1-[5-(1,3-Difluoro-2-methylpropan-2-yl)-

isoxazol-3-yl]-3-[3-(6,7-dimethoxyquinazolin-4-ylthio)phenyl]urea

2

H), 7.37−7.41 (m, 2H), 7.24 (m, 1H), 6.93 (m, 1H), 6.01 (s, 1H),

4.00 (s, 3H), 3.99 (s, 3H), 3.94 (m, 1H), 1.62−1.82 (m, 8H), 1.24−

+

(42). A mixture of compound 9b (89 mg, 0.3 mmol), compound 17

1.35 (m, 2H), 1.24 (s, 9H). LC-MS (ESI) m/z 545 [M + H] .

(

94 mg, 0.3 mmol), and 4-(dimethylamino)pyridine (30 mg, 0.25 mmol)

1-(3-tert-Butyl-1-phenyl-1H-pyrazol-5-yl)-3-(3-(6,7-dimethoxy-

quinazolin-4-yloxy)phenyl)urea (49). Prepared from compound 13g

(151 mg, 0.45 mmol) and compound 16 (89 mg, 0.30 mmol) accord-

ing to General Procedure H. The crude product was purified by silica

gel flash chromatography (eluting with 100% hexanes to 85% EtOAc

in THF (6 mL) was stirred at rt for 15 h. The reaction mixture was

partitioned between CH Cl and water, and the organic layer was

separated, dried over MgSO , and then concentrated under reduced

pressure. The residue was purified by silica gel flash chromatography

(

2

4

eluting with 70−95% EtOAc in hexanes) to afford the title compound

in hexanes) to afford the title compound as a solid (68 mg, 42% yield).

1

as a solid (48 mg, 31%). H NMR (DMSO-d , 300 MHz): δ 9.71

H NMR (DMSO-d , 300 MHz): δ 9.13 (s, 1H), 8.55 (s, 1H), 8.47

6

(

7

(

s, 1H), 9.02 (s, 1H), 8.70 (s, 1H), 7.85 (s, 1H), 7.45−7.54 (m, 2H),

(s, 1H), 7.52−7.55 (m, 6H), 7.36−7.40 (m, 3H), 7.15 (s, 1H), 6.92

(d, J = 7.9 Hz, 1H), 6.36 (s, 1H), 4.00 (s, 6H), 1.25 (s, 9H). LC-MS

(ESI) m/z 539 [M + H] .

1-(1-Benzyl-3-tert-butyl-1H-pyrazol-5-yl)-3-(3-(6,7-dimethoxy-

quinazolin-4-yloxy)phenyl)urea (50). Prepared from compound

13d (105 mg, 0.30 mmol) and compound 16 (90 mg, 0.30 mmol)

according to General Procedure H to afford the title compound as a

.28−7.36 (m, 3H), 6.78 (s, 1H), 4.72 (m, 2H), 4.56 (m, 2H), 3.99

+

s, 6H), 1.29 (s, 3H). LC-MS (ESI) m/z 516 [M + H] .

-(3-(6,7-Dimethoxyquinazolin-4-ylthio)phenyl)-3-(5-(1,1,1-tri-

1

fluoro-2-methylpropan-2-yl)isoxazol-3-yl)urea (43). Prepared from

compound 9a (161 mg, 0.46 mmol) and compound 17 (144 mg,

0

.46 mmol) according to General Procedure I to afford the title

1

101

dx.doi.org/10.1021/jm2009925 | J. Med. Chem. 2012, 55, 1082−1105

Journal of Medicinal Chemistry

Article

1

solid (50 mg, 30%). H NMR (DMSO-d , 300 MHz): δ 9.00 (s, 1H),

before being washed and lysed using cell extraction buffer (Invitrogen,

# FNN0011). Phosphor-MEK Elisa assays were carried out per manu-

facture’s specifications (Biosources, KHO0321). Total MEK Elisa

assays from the same lysates were also run in parallel and used for nor-

malization of the phosphor- signal (Biosource, KHO0291). IC₅₀values

were derived using a 9-point curve fitted with Igor Pro (WaveMetrics,

Inc.) and are presented as mean values from experiments performed in

duplicate. Variability between individual values was less than 2-fold.

A375 Cell Proliferation Assay. A375 cells were seeded at 10,000

cells per well in DMEM with 10% fetal calf serum and allowed to

attach. The cells were washed with PBS and switched to DMEM with

0.5% of serum and incubated overnight. The test compounds were

then added at various concentrations with a final DMSO concentration

of 0.5% and incubated for 72 h. At the end of incubation, a Cell Titer

Blue (Promega, Madison, WI) was added per instructions, and incubation

was continued for 3 h. Remaining viable cells were quantified by

measuring the strength of the fluorescence signal using SoftMax Pro

(excitation at 560 nm and emission at 590 nm). IC₅₀values were derived

using a 9-point curve fitted with Igor Pro (WaveMetrics, Inc.) and are

presented as mean values from experiments performed in duplicate.

Variability between individual values was less than 2-fold.

Cellular Cytotoxicity Assays. For a comparison of activity in mutated

versus wild-type BRAF cell lines, A375, Colo-679, SK-MEL-28, HT-144,

Colo-205, HCT 116, Hs578T, LNCaP, DU 145, and PC-3 cell lines

were obtained from and cultured in media recommended by ATCC

(Manassas, VA). All cell lines were MAP (Mouse Antibody Production)

and mycoplasma-tested and deemed contaminant-free prior to in vitro

and in vivo studies. Cells were cultured in 0.5% serum overnight prior

to incubation with test compound for 72 h, followed by addition of

Cell Titer Blue (Promega, Madison, WI) to detect viable cells after

3 h incubation with reagent. Concentration−response curves were gener-

ated from the fluorometric product, and EC50 values were determined.

Each experiment was conducted in duplicate and repeated at least once.

Microsomal Stability Determinations. Pooled liver microsomes for

rat, dog, cynomolgus monkey (In Vitro Technologies, Baltimore,

MD), and human (CellzDirect, Dallas, TX) (0.5 mg/mL), 0.1 M phos-

6

8

3

.59 (br s, 2H), 7.58−6.93 (m, 11H), 6.16 (s, 1H), 5.20 (br s, 2H),

.98 (s, 6H), 1.21 (s, 9H). LC-MS (ESI) m/z 553 [M + H] .

-(3-(6,7-Dimethoxyquinazolin-4-yloxy)phenyl)-3-(1-phenyl-3-

1,1,1-trifluoro-2-methylpropan-2-yl)-1H-pyrazol-5-yl)urea (51).

Prepared from compound 13e (104 mg, 0.27 mmol) and compound

6 (89 mg, 0.3 mmol) according to the procedure described for

+

1

(

1

compound 46 to afford the title compound as a solid (103 mg, 64%).

1

H NMR (DMSO-d , 300 MHz): δ 9.28 (s, 1H), 8.55−8.59 (m, 2H),

6

7

6

.34−7.57 (m, 9H), 7.17 (d, J = 9 Hz, 1H), 6.94 (d, J = 9 Hz, 1H),

.55 (s, 1H), 4.02 (s, 6H), 1.56 (s, 6H). LC-MS (ESI) m/z 593

+

[

M + H] .

1

-(1-tert-Butyl-1H-pyrazol-4-yl)-3-(3-hydroxyphenyl)urea (53).

To a stirred solution of 1-tert-butyl-1H-pyrazol-4-amine (995 mg,

.16 mmol) in THF (20 mL) were added phenyl chloroformate (1.25 g,

.02 mmol) and potassium carbonate (1.32 g, 9.52 mmol), and the

7

8

reaction mixture was stirred at rt for 15 h. The mixture was filtered, and

the filtrate was concentrated under reduced pressure. The residue was

dissolved in CH Cl and washed with brine. The organic layer was

2

separated, dried over MgSO , and then concentrated under reduced

pressure to afford phenyl 1-tert-butyl-1H-pyrazol-4-ylcarbamate as a

solid which did not require further purification (1.65 g, 89%). H

NMR (CDCl , 300 MHz): δ 7.85 (1H, s), 7.37−7.45 (m, 5H), 7.16−

.19 (m, 2H), 1.60 (s, 9H). To a solution of phenyl 1-tert-butyl-1H-

pyrazol-4-ylcarbamate (782 mg, 3.02 mmol) in THF (10 mL) was

added 3-aminophenol (329 mg, 3.02 mmol), and the mixture was

heated in a sealed vial at 120 °C for 2 h. After cooling to rt, the reac-

tion mixture was concentrated under reduced pressure. The residue

was dissolved in EtOAc, and the solution was washed with water and

brine. The organic layer was dried over MgSO and then concentrated

under reduced pressure. The residue was purified by silica gel flash

chromatography (eluting with 2−20% MeOH in CH Cl ) to afford

the title compound as a solid (169 mg, 20%). H NMR (DMSO-d ,

4

1

3

7

4

2

1

6

3

7

00 MHz): δ 9.25 (s, 1H), 8.48 (s, 1H), 8.20 (s, 1H), 7.80 (s, 1H),

.39 (s, 1H), 6.98−7.03 (m, 2H), 6.77 (d, J = 7.9 Hz, 1H), 6.35 (d, J =

.9 Hz, 1H), 1.49 (s, 9H). LC-MS (ESI) m/z 275 [M + H] .

+

1

-(1-tert-Butyl-1H-pyrazol-4-yl)-3-(3-(6,7-dimethoxyquinazolin-

phate buffer pH 7.4, 6.5 mM MgCl , and test compound (1 μM; 0.2%

2

4

0

(

-yloxy)phenyl) Urea (54). A mixture of compound 53 (169 mg,

final DMSO concentration) were premixed prior to the addition of

NADPH (1 mM) to initiate the reaction. A minus NADPH control

and testosterone positive control were included. All incubations were

carried out at 37 °C. All incubations were performed in duplicate.

Aliquots (50 μL) were taken at 0, 5, 15, 30, and 60 min and added to

200 μL acetonitrile containing an internal standard to quench the

reaction. Samples were diluted with 100 μL 50/50 acetonitrile/water,

vortexed, and centrifuged at 36,000 rpm for 10 min at 4 °C to precip-

itate the protein. The sample supernatants were analyzed by LC-MS/MS.

The t = 0 time point normalized peak area was set to 100%, and the

natural log of the percent compound remaining was plotted versus

.62 mmol) and cesium carbonate (403 mg, 1.24 mmol) in THF

8 mL) was stirred at rt for 1 h. 4-Chloro-6,7-dimethoxyquinazoline

138 mg, 0.62 mmol) was added, and the mixture was heated at 55 °C

for 15 h. The mixture was filtered, and the filtrate was concentrated

under reduced pressure. The residue was purified by preparative

reverse-phase HPLC to afford the title compound as a colorless solid

122 mg, 43%). H NMR (DMSO-d , 300 MHz): δ 8.86 (s, 1H), 8.55

s, 1H), 8.40 (s, 1H), 7.80 (s, 1H), 7.59 (s, 1H), 7.54 (s, 1H), 7.35−

.40 (m, 3H), 7.23 (d, J = 9 Hz, 1H), 6.88 (d, J = 6 Hz, 1H), 3.98

1

(

7

(

6

+

s, 6H), 1.50 (s, 9H). LC-MS (ESI) m/z 463 [M + H] .

Biology. Kinase Competition Binding Assays. KINOMEscan

time. The elimination rate constant (k ) was determined by linear

el

competition binding assays (www.kinomescan.com) were performed

regression. Half-life (t₁_/₂) and intrinsic clearance (Cl ) were calcu-

int

10

as described previously. Kinases were produced displayed on T7

phage or by expression in HEK-293 cells and tagged with DNA. Bind-

ing reactions were performed at rt for 1 h, and the fraction of kinase

not bound to test compound was determined by capture with an

immobilized affinity ligand and quantitation by quantitative PCR. Each

lated using the following equations: t₁_/₂= 0.693/k and CL (μL/min/mg

el

int

protein) = 0.693V/t₁_/₂, where V is expressed as μL/mg protein. All

microsomes were stored at −80 °C before use.

CYP450 Inhibition Assays. Pooled human liver microsomes (male

and female) were purchased from CellzDirect, Dallas, TX (lot

HMMC-PL020). The CYP450 inhibition assay was multiplexed, such

that individual CYP substrates at 5-fold stock concentration were used,

including testosterone (375 μM), diclofenac (80 μM), dextromethorphan

(40 μM), mephenytoin (255 μM), and phenacetin (390 μM). Equi-

volume amounts of the five substrates were mixed to prepare the

pooled substrate stock solution. Individual control CYP inhibitors at

5-fold stock concentration were used, including ketoconazole (2.5 μM),

sulfaphenazole (10 μM), quinidine (1 μM), ticlopidine (1 μM), and

furafylline (0.6 μM). Equivolume amounts of the five control inhib-

itors were mixed to prepare the pooled inhibitor stock solution. Eight

test inhibitor concentrations (0.31, 0.62, 1.25, 2.5, 5, 10, 20, and

40 μM in DMSO; 0.2% final DMSO concentration) were incubated

with human liver microsomes (0.25 mg/mL) and NADPH (1 mM) in

the presence of the five pooled CYP probe substrates for 20 min at

37 °C. The five pooled CYP inhibitors were screened as positive

kinase was tested individually against each compound. K values were

d

determined using eleven serial 3-fold dilutions and presented as mean

values from experiments performed in duplicate. Variability between

individual values was less than 2-fold.

A375 Cell Phospho-MEK Inhibition Assay. In order to quantify

V600E

cellular inhibition of BRAF

, the A375 human melanoma cell line

which carries this activating mutation was acquired from the American

Type Culture Collection. The cells were seeded at 50,000 per well in a

9

6-well plate in DMEM (Dulbecco’s Modification of Eagle’s Medium)

(

Mediatech) with 10% of Fetal Calf Serum (Omega Scientific) and

allowed to attach for at least 6 h. The cells were then washed once with

PBS (phosphate buffered saline) and incubated overnight in DMEM

with 0.5% of serum. Compounds of interest were added at various

concentrations to the cells, and the final DMSO concentration was

maintained at 0.5%. The cells were incubated with compounds for 2 h

1

102

dx.doi.org/10.1021/jm2009925 | J. Med. Chem. 2012, 55, 1082−1105

Journal of Medicinal Chemistry

Article

controls in parallel with test compounds. Reactions were terminated

by the addition of 3:1 acetonitrile/water containing 0.05% formic acid

and an internal standard and centrifuged, and the formation of the five

CYP isoform-specific metabolites was monitored by LC-MS/MS.

hERG Inhibition Assay. Assays were performed at Aviva Biosciences

ATCC (Manassas, VA). Six to eight week old athymic nu/nu nude

mice (Charles River; San Diego, CA, 20−25 g) were inoculated

6

subcutaneously with Colo-205 tumor cells (1 × 10 /mouse) in the

3

right flank. Upon reaching an average tumor volume of 150−200 mm

(10−12 days post implantation), animals were randomized into

treatment groups (n = 10 mice/group). Each group was dosed orally

for 14 days with either vehicle only (22% HPβCD) or with compound

40 at 10, 30, or 100 mg/kg twice daily (BID), and each dose of drug

was given in a volume of 0.1 mL per 20 g of body weight, adjusted for

the body weight of the animal. Tumor volumes were measured three

times weekly using vernier calipers, and volumes were calculated using

(

San Diego, CA). Aviva’s CHO cell line, which stably expresses hERG

channels, was employed. Cells were cultured in DMEM/F12

containing 10% FBS (fetal bovine serum), 1% penicillin/streptomycin,

and 500 μg/mL Geneticin. Before testing, cells were harvested using

Accumax (Innovative Cell Technologies). For electrophysiology

experiments, whole cell recordings were performed using PX7000A

3

2

(

Molecular Devices) with Aviva’s SealChip technology. An external

the following formula: tumor volume (mm ) = W (L/2), where W =

width and L = length in mm. The reported incidence of partial tumor

regressions refers to the percent of tumor bearing mice showing a

reduction in tumor volume (from initial tumor volume) in 3 or more

successive tumor measurements, culminating in tumors whose final

volume is smaller than the starting tumor volume but that are still

palpable, measurable tumors. Mice were monitored for signs of morbidity

(behavioral abnormalities and body weight loss). Upon discontinuation of

dosing, animals were evaluated for a period of 7−10 days to assess any

potential residual side effects of treatment.

solution containing 0.1% DMSO (vehicle) was applied to the cells to

establish a baseline. After allowing the current to stabilize for 3−10 min,

the test article was applied. Test article solutions and cells were kept in

test solution until the effect of the test article reached steady state, to a

maximum of 12 min. Next, 1 μM of cisapride (positive control) was

added. Finally, washout with external solution was performed until the

recovery current reached steady state. Data analysis was performed using

DataXpress (Molecular Devices), Clampfit (Molecular Devices), and

Origin (OriginLab) software.

Rodent Pharmacokinetic Studies. Pharmacokinetic (PK) studies

were conducted in accordance with the ILAR Guide for the Care and

Use of Laboratory Animals and the USDA Animal Welfare Act

and Cephalon Inc. IACUC guidelines. In pharmacokinetic assays,

precatheterized (jugular vein) male Sprague−Dawley rats (Charles

River, Hollister, CA, 250−350 g, n = 2 (iv) and 3 (po) animals) or

female athymic nude mice (Charles River; San Diego, CA, 20−25 g,

n = 3 animals) were administered a single dose of compound either iv

or po formulated in either a screening vehicle (Pharmatek #6 of the

Hot Rod Chemistry vehicle series; Pharmatek, Inc., San Diego, CA) or

a safety vehicle, 3:1 PEG400/water or 22−50% HPβCD, at various

doses (calculated as free base equivalents), following a minimum of

Mouse Tumor Pharmacodynamic−Pharmacokinetic Studies.

Studies were conducted in accordance with the ILAR Guide for the

Care and Use of Laboratory Animals and the USDA Animal Welfare

Act and Cephalon Inc. IACUC guidelines. Athymic nude mice bearing

Colo-205 subcutaneous tumors were treated with a single oral dose of

vehicle (22% HPβCD) or compound 40 (10, 30, and 55 mg/kg, dosed

in 22% HPβCD). At appropriate time points over a 24 h period,

animals were sacrificed and plasma and tumor samples were collected

and lysates prepared. Amounts of total MEK and pMEK in tumor

lysates were measured by immunoblotting and ELISA methods. Com-

pound levels in plasma and tumor tissue samples were analyzed and

quantitated by LC-MS/MS. Values given are mean ± SEM from two

studies, n = 3 tumor-bearing mice per time point per study.

2

5

days acclimation to the vivarium. Blood samples were collected at

(iv only), 15, and 30 min and 1, 2, 4, 6, and 24 h into K3EDTA tubes

Computational Chemistry: Ligand Docking Studies. The

and kept on ice until centrifugation. Following centrifugation, plasma

was pipetted into 96 well plates and stored at −20 °C for LC-MS

analysis. Compound levels in plasma were quantitatively analyzed by

LC-MS/MS (API 4000-Qtrap, Applied Biosystems) following protein

precipitation with acetonitrile containing an internal standard, and PK

parameters were calculated from the normalized LC-MS/MS peak

areas using a noncompartmental model with WinNonlin version 5.2,

using the linear trapezoidal estimation method. The mean parameters

were calculated using plasma concentration−time data for individual

animals.

computational work was carried out using the Schro

molecular modeling package (Maestro Version 9.1.107, Schro

LLC, New York, NY). The essential steps in the current docking

experiment were (1) preparation of two DFG-out BRAF structures

from the Protein Data Bank (PDB) (PDB ID: 1uwh, 1uwj), using

the Maestro protein preparation workflow, (2) creation of a grid around

the ligand, (3) preparation of ligands 4, 22, 25, 30, and 40 using the

LigPrep module, (4) use of Glide/XP docking to keep the top 10 bind-

ing poses for each compound, and (5) selection of the binding mode

̈

dinger/Maestro

dinger,

̈

20

22

using our previously described knowledge based approach.

Dog and Monkey Pharmacokinetic Studies. Compound 40 was

administered to nondrug-naive male cynomolgus monkeys weighing

between 2.6 and 3.4 kg and to beagle dogs weighing between 10.3 and

ASSOCIATED CONTENT

■

11.9 kg. Animals received a single 10 mg/kg po dose and a single

1 mg/kg IV dose (n = 3 animals) in a crossover design with a minimum

7 day washout period separating successive phases of dosing. All oral

*

S

Supporting Information

The complete list of the 290 individual wild-type kinase targets

used for the KINOMEscan selectivity profiling of the com-

pounds. This material is available free of charge via the Internet

at http://pubs.acs.org.

doses were free base equivalents formulated in 22% HPβCD admini-

stered via nasogastric gavage at a fixed volume of 3 mL/kg (monkeys)

and 1 mL/kg (dogs). Intravenous doses were prepared in the same

vehicle and administered as bolus injection via saphenous vein at a

fixed volume of 0.5 mL/kg. Animals were fasted overnight prior to

dosing and through approximately 4 h post dose for a total fasting time

of 18−23 h. During all phases of dosing, blood samples for pharmaco-

kinetic profiling were collected via a femoral vein immediately prior to

dosing and at preselected time points through 24 h post dose. Con-

centrations of compound 40 in plasma samples were determined by

LC-MS/MS following acetonitrile precipitation of proteins. PK param-

eters were calculated from the normalized LC-MS/MS peak areas

using a noncompartmental model with WinNonlin version 5.2, using the

linear trapezoidal estimation method. The mean parameters were calcu-

lated using plasma concentration−time data for individual animals.

Mouse Tumor Xenograft Efficacy Studies. All efficacy studies were

conducted in accordance with the ILAR Guide for the Care and Use of

Laboratory Animals and the USDA Animal Welfare Act and Cephalon

Inc. IACUC guidelines. The Colo-205 cell line was obtained from

AUTHOR INFORMATION

■

*

Corresponding Author

Phone: 1-858-334-2115. Fax: 1-858-334-2192. E-mail:

mrowbottom@ambitbio.com.

ACKNOWLEDGMENTS

■

We thank Paul Gallant, Mazen W. Karaman, Antonio Torres,

Gabriel Pallares, and the KINOMEscan team for the generation

of K and kinome selectivity data. We also thank Dan Treiber

d

(

of KINOMEscan), Patrick P. Zarrinkar, Wendell Wierenga,

and John P. Mallamo for their valued insight and support.

KINOMEscan is a division of DiscoveRx Corporation.

1

103

dx.doi.org/10.1021/jm2009925 | J. Med. Chem. 2012, 55, 1082−1105

Journal of Medicinal Chemistry

Article

Zhang, D. M.; Zhang, Q.; Galvin, K. M. Design and optimization of

potent and orally bioavailable tetrahydronaphthalene Raf inhibitors.

J. Med. Chem. 2011, 54, 1836−1846. (e) Whittaker, S.; Menard, D.;

́

Kirk, R.; Ogilvie, L.; Hedley, D.; Zambon, A.; Lopes, F.; Preece, N.;

Manne, H.; Rana, S.; Lambros, M.; Reis-Filho, J. S.; Marais, R.;

ABBREVIATIONS USED

■

ADME, absoption−distribution−metabolism−excretion;

BRAF, V-RAF murine sarcoma viral oncogene homologue

B1; CYP450, cytochrome P450; ERK, extracellular regulated

kinase; hERG, human ether-a-

̀

go-go related gene; HPβCD, 2-

Springer, C. J. A novel, selective, and efficacious nanomolar

V600E

hydroxypropyl-beta-cyclodextrin; iv, intravenous; MAPK,

mitogen-activated protein kinase; MEK, mitogen-activated

protein kinase/extracellular regulated kinase kinase; PK,

pharmacokinetic; po, per os; RAF, rapidly growing fibrosarcoma;

SAR, structure−activity relationship

pyridopyrazinone inhibitor of

BRAF. Cancer Res. 2010, 70,

8036−8044. (f) Zambon, A.; Men

́

ard, D.; Suijkerbuijk, B. M. J. M.;

Niculescu-Duvaz, I.; Whittaker, S.; Niculescu-Duvaz, D.; Nourry, A.;

Davies, L.; Manne, H. A.; Lopes, F.; Preece, N.; Hedley, D.; Ogilvie,

L. M.; Kirk, R.; Marais, R.; Springer, C. J. Novel hinge binder improves

activity and pharmacokinetic properties of BRAF inhibitors. J. Med.

Chem. 2010, 53, 5639−5655 (and references contained therein).

(g) Wang, X.; Berger, D. M.; Salaski, E. J.; Torres, N.; Dutia, M.;

Hanna, C.; Hu, Y.; Levin, J. L.; Powell, D.; Wojciechowicz, D.; Collins,

K.; Frommer, E.; Lucas, J. Indazolylpyrazolopyrimidines as highly

potent B-Raf inhibitors with in vivo activity. J. Med. Chem. 2010, 53,

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dx.doi.org/10.1021/jm2009925 | J. Med. Chem. 2012, 55, 1082−1105

Article Doi

Identification of 1-(3-(6,7-dimethoxyquinazolin-4-yloxy)phenyl)-3-(5-(1,1, 1-trifluoro-2-methylpropan-2-yl)isoxazol-3-yl)urea hydrochloride (CEP-32496), a highly potent and orally efficacious inhibitor of V-RAF murine sarcoma viral oncogene homologue B1 (BRAF) V600E

DOI: 10.1021/jm2009925

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