Discovery of a potent and orally active hedgehog pathway antagonist (IPI-926)

4400 J. Med. Chem. 2009, 52, 4400–4418

DOI: 10.1021/jm900305z

Discovery of a Potent and Orally Active Hedgehog Pathway Antagonist (IPI-926)

ꢀ

Martin R. Tremblay,* Andre Lescarbeau, Michael J. Grogan, Eddy Tan, Grace Lin, Brian C. Austad, Lin-Chen Yu,

Mark L. Behnke, Somarajan J. Nair, Margit Hagel, Kerry White, James Conley, Joseph D. Manna, Teresa M. Alvarez-Diez,

Jennifer Hoyt, Caroline N. Woodward, Jens R. Sydor, Melissa Pink, John MacDougall, Matthew J. Campbell, Jill Cushing,

Jeanne Ferguson, Michael S. Curtis, Karen McGovern, Margaret A. Read, Vito J. Palombella, Julian Adams, and

Alfredo C. Castro

Infinity Pharmaceuticals, Inc., 780 Memorial Drive, Cambridge, Massachusetts 02139

Received March 10, 2009

Recent evidence suggests that blocking aberrant hedgehog pathway signaling may be a promising

therapeutic strategy for the treatment of several types of cancer. Cyclopamine, a plant Veratrum

alkaloid, is a natural product antagonist of the hedgehog pathway. In a previous report, a seven-

membered D-ring semisynthetic analogue of cyclopamine, IPI-269609 (2), was shown to have greater

acid stability and better aqueous solubility compared to cyclopamine. Further modifications of the

A-ring system generated three series of analogues with improved potency and/or solubility. Lead

compounds from each series were characterized in vitro and evaluated in vivo for biological activity and

pharmacokinetic properties. These studies led to the discovery of IPI-926 (compound 28), a novel

semisynthetic cyclopamine analogue with substantially improved pharmaceutical properties and

potency and a favorable pharmacokinetic profile relative to cyclopamine and compound 2. As a result,

complete tumor regression was observed in a Hh-dependent medulloblastoma allograft model after

daily oral administration of 40 mg/kg of compound 28.

Introduction

mutations in PTCH.^7,8Subsequent analysis of various tu-

mor samples demonstrated aberrant Hh signaling in breast,⁹

esophagus,¹⁰gastric,¹⁰medulloblastoma,¹¹pancreatic adeno-

carcinoma,¹²prostate,¹³and small cell lung cancers.¹⁴Accord-

ingly, targeting the Hh pathway is an emerging therapeutic

strategy in cancer.

The hedgehog (Hh^a) signaling pathway regulates cell

growth and differentiation during embryonic tissue patterning

and plays a pivotal role in tissue homeostasis.¹Hh-signal

transduction is initiated by the binding of Hh protein ligand

to its cellular membrane receptor Patched (PTCH), which

relieves PTCH-mediated repression of the seven-transmem-

brane protein Smoothened (SMO). Activated SMO trans-

duces the signal to the GLI family of transcription factors,

which translocate to the nucleus to regulate numerous gene

products involved in tissue patterning and cell differentiation.

In adulthood, the Hh signaling pathway in normal cells is

much less active (Figure 1A). However, an increasing number

of reports over the past decade have implicated the Hh path-

way in human diseases, especially cancer (Figure 1B).^2-5The

first link between the Hh pathway and cancer came from the

discovery that Gorlin’s syndrome, an inherited condition,

results from an autosomal loss of the gene for PTCH.

Children with this condition have multiple physical defects,

including a predisposition for cancers such as medulloblasto-

ma and basal cell carcinoma (BCC).⁶Analyses of tumor tissue

from sporadic BCC and medulloblastoma have also demon-

strated a high incidence of Hh pathway hyperactivation, as

indicated by elevated Gli1 protein expression and inactivating

Several agents acting on various components of the Hh

signaling pathway have been described preclinically, and their

relevance as novel cancer treatments has been recently re-

viewed.¹⁵While most Hh pathway small molecule antagonists

target SMO,^16-21components upstream and downstream of

SMO are also being investigated as potential pharmaceutical

targets.^22-25Four Hh pathwayantagonists are being explored

clinically.^26-29Herein we report preclinical data that sup-

ported the selection of IPI-926 (compound 28) as a develop-

ment candidate that has recently entered clinical phase 1.²⁹

Cyclopamine (1, Figure 2), a natural product isolated from

Veratrum californicum,^30-32has been a significant pharmaco-

logical tool to validate the Hh pathway in cancer. Cyclopa-

mine directly acts on SMO³³and inhibits tumor growth in

several murine models of pancreatic,¹²medulloblastoma,^34,35

prostate,^13,36,37small cell lung,¹⁴and digestive tract¹⁰cancers.

However, the clinical development of cyclopamine as a ther-

apeutic in cancer is hampered by cyclopamine’s poor solu-

bility, acid sensitivity, and weak potency relative to other

reported small-molecule Hh antagonists. Compound 3¹⁷

(Figure 2) is 10- to 20-fold more potent than cyclopamine

but has essentially the same limitations as cyclopamine from

solubility and chemical stability standpoints. Chemical mod-

ifications have improved the pharmaceutical profile of cyclo-

pamine. In one study, the addition of carbohydrate moieties

on the piperidine nitrogen resulted in the identification of

*To whom correspondence should be addressed. Phone: 617-453-

1268. Fax: 617-682-1418. E-mail: Martin.Tremblay@infi.com.

^aAbbreviations: BCC, basal cell carcinoma; BODIPY, 4,4-difluoro-

4-bora-3a,4a-diaza-s-indacene; HPBCD, 2-hydroxypropyl-β-cyclodex-

trin; Hh, Hedgehog; Hic1, hypermethylated in cancer 1; MTD, max-

imum tolerated dose; Ptch, Patched; Smo, Smoothened; SAR,

structure-activity relationship; SuFu, Suppressor of Fused.

r

pubs.acs.org/jmc

Published on Web 06/12/2009

2009 American Chemical Society

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4401

Figure 1. Schematic of the Hedgehog signaling pathway: (A) in the absence of ligand, which represents the situation on most normal cells

during adulthood; (B) in several types of cancer cells, for which the pathway is activated by the presence of ligand and/or loss-of-function of

PTCH and/or gain-of-function of SMO.

compound 2 is readily metabolized to the corresponding

saturated alcohol and glucuronide conjugate after oral ad-

ministration in cynomolgus monkeys.⁴³We chose to explore

the importance of the half-chair conformation of the A-ring

system of compound 2 on the potency. Stereoselective reduc-

tion of the enone of 2 with Li/NH₃provided trans-decalone 4

(Scheme 1),⁴⁴whereas hydrogenation catalyzed with Pd/C in

presence of pyridine⁴⁵yielded the cis-decalone as the major

product (ratio of ∼9:1). Protection of the piperidine nitrogen

of cis-decalone as a benzyl carbamate provided stereochemi-

cally pure material (5) after silica gel chromatography, and

removal of protecting group provided 6. Hedgehog inhibitory

activity of these new compounds was measured by inhibition

of oxysterol-dependent differentiation of C3H10T1/2 cells.⁴⁶

In this assay, cis-decalone 6 was found to be distinctly

more potent (30-fold) than trans-decalone 4 and, more im-

portantly, enone 2 (Table 1). Utilizing the 3-ketone as a

synthetic handle, three series of cis-decalone analogues were

pursued: 3-substituted analogues, fused heterocycles, and

Figure 2. Structures of cyclopamine (1), IPI-269609 (2), and 3.

A-ring lactams.

equipotent analogues of cyclopamine with fair-to-good solu-

The scalable intermediate 5 served as a useful starting point

bility in aqueous media.³⁸Another approach conjugated

for additional elaboration of the A-ring. Schemes 2 and 3

cyclopamine to prostate specific, antigen activated peptides

show the synthesis of compounds that have been modified at

to produce prostate cancer targeted prodrugs.³⁹We have also

the C3⁰-position (Table 1, compounds 6-29). The A-ring

previously reported the development of a prototype analogue

ketone was reductively removed from 6 by a Wolff-Kishner

2 (Figure 2) that exhibited improved chemical stability and

reaction to give cyclohexane analogue 7. Oxime 10 and oxime

aqueous solubility relative to cyclopamine.⁴⁰Compound

methyl ether 11 were prepared by standard procedures from

2 demonstrated in vivo efficacy in several mouse xenograft

compound 5. Third, the ketone of compound 5 was reduced in

models.^41,42However, development of compound 2 as a drug

the presence of bulky potassium tri-sec-butyl borohydride to

candidate was limited by potency and metabolic stability.

the C3⁰(S)-alcohol 12, whereas Luche reduction (CeCl₃,

We now report the further optimization of compound 2,

NaBH₄) gave the C3⁰(R)-alcohol 13. Hydrogenation of these

which has led to the discovery of the more potent cis-decalone

protected derivatives provided the desired analogues 14

analogue 6 from which three series of A-ring modified analo-

and 15. Conversion of the protected alcohols 12 and 13 into

gues of reduced ^D-homocyclopamine were identified. Among

methyl ethers was performed using standard procedures to

them, compound 28 emerged as the most promising clinical

give 16 and 17, after removal of the benzylcarbamate. As

development candidate with improved potency, pharmaco-

described in Scheme 3, epimeric alcohols 12 and 13 were

kinetic, and pharmaceutical properties relative to cyclopa-

readily transformed to the corresponding azides 18 and 19.

mine and compound 2.

These azides were key intermediates in the formation of

triazoles (20, 21), amines (24, 25), acetamides (26, 27), and

sulfonamides (28, 29).

Chemistry

The R/β-unsaturated ketone system found incompound2 is

a very common and structurally important functionality in

endogenous steroid hormones. However, this functionality in

The synthetic approaches to fused-ring analogues (C2⁰/C3⁰

and C3⁰/C4⁰fused ring systems) and their derivatives are

depicted in Schemes 4-6 (Table 1, compounds 32-50). As

4402 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14

Tremblay et al.

described in Scheme 4, formylation of compound 5 under

thermodynamic conditions⁴⁷afforded β-ketoaldehyde 30.

Condensation of the latter with hydrazine followed by ring

closure provided, after benzylcarbamate removal, pyrazole

32. The other regioisomer of 32 was prepared by formylation

of the kinetic TES enol ether 33 to give the rather unstable

β-hydroxyketone 34. Subsequent oxidation with Dess-

Martin periodinane, followed by condensation with hydra-

zine, and deprotection gave pyrazole 36. Evaluation of the

relative potency between the pyrazole regioisomers revealed

that the C2⁰/C3⁰fused ring system (32) was clearly superior

to the C3⁰/C4⁰analogue 36 (Table 1). Additional C2⁰/C3⁰

fused heterocycles were synthesized from intermediate 30

(Schemes 5). Thiazole 38 was prepared by the condensation

of thioacetamide with C2⁰-bromoketone 37.⁴⁸The β-keto-

aldehyde 30 was transformed to β-ketooxime 39,⁴⁹which was

then converted to oxadiazole 40 using conditions adapted

from Ohta and co-workers.⁵⁰On the other hand, β-ketoalde-

hyde 30 was converted in a regioselective manner to [3,2-c]

isoxazole (41) under basic conditions.⁵¹Substitutions around

the C2⁰/C3⁰fused pyrazole were also explored, and the details

of their synthesis are described in Scheme 6. The C3⁰⁰position

of the pyrazole was substituted with trifluoromethyl (42),

which was prepared by Claisen condensation from ketone 5

and ethyl trifluoroacetate, followed by ring-closing condensa-

tion to give, after deprotection, trifluoromethylpyrazole 44.

Pyrazolone 47 was prepared by condensation of hydrazine on

unsaturated β-ketoester 46, which was synthesized from key

intermediate 30. Condensation of β-ketoaldehyde 30 with

N-methylhydrazine gave methylpyrazole 48 as the major

regioisomer. Pyrazole 31 was reacted with the appropriate

sulfonyl chloride to give sulfamylpyrazoles 49 and 50 as the

major regioisomers.

Table 1. Cellular Hh Pathway Inhibition and in Vitro Metabolic Stability

in Human Liver Microsomes of A-Ring Analogues of ^D-Homocyclopamine

A-ring motif

compd

EC₅₀, μM (N)

t_1/2, min

enone

trans-decalone

2

4

0.30 ( 0.05 (9)

0.4 ( 0.2 (2)

75

cis-decalone

cyclohexyl

6

0.013 ( 0.004 (6)

0.76 ( 0.11 (2)

0.015 ( 0.007 (2)

0.16 ( 0.04 (2)

0.023 ( 0.016 (2)

0.009 ( 0.001 (2)

0.49 ( 0.04 (2)

0.18 ( 0.05 (2)

0.024 ( 0.009 (2)

0.13 ( 0.03 (2)

>1 (2)

70

7

>200

45

55

3⁰-oxime

10

11

14

15

16

17

20

21

24

25

26

27

28

29

32

36

38

40

41

44

47

48

49

50

55

56

3⁰-methyloxime

3⁰-(S)-hydroxy

20

40

3⁰-(R)-hydroxy

3⁰-(S)-methoxy

>200

20

40

3⁰-(R)-methoxy

3⁰-(R)-triazole

3⁰-(S)-triazole

70

3⁰-(R)-amino

3⁰-(S)-amino

>1 (2)

3⁰-(R)-acetamido

3⁰-(S)-acetamido

3⁰-(R)-sulfonamido

3⁰-(S)-sulfonamido

2⁰,3⁰-fused pyrazole

3⁰,4’-fused pyrazole

2⁰,3⁰-fused thiazole

2⁰,3⁰-fused oxadiazole

2⁰,3⁰-fused isoxazole

2⁰,3⁰-fused (CF₃)-pyrazole

2⁰,3⁰-fused (OH)-pyrazole

2⁰,3⁰-fused N-Me-pyrazole

2⁰,3⁰-fused N-(Ms)-pyrazole

2⁰,3⁰-fused N-(Ts)-pyrazole

lactam

0.017 ( 0.005 (3)

0.30 ( 0.08 (3)

0.007 ( 0.002 (3)

0.09 ( 0.03 (3)

0.013 ( 0.008 (6)

0.22

65

60

85

30

120

27

A synthetic sequence for the conversion of the cyclohex-

anone A-ring of compound 6 to lactams 55 and 56 is shown in

Scheme 7. The use of nonreducible azasteroids has been very

successful in the development of 5R-reductase inhibitors.^52,53

On the basis of literature precedents,^54,55it was envisioned

that a regioselective Beckmann rearrangement could be

achieved using unsaturated oxime 52. The requisite enone

51 was prepared from key intermediate β-ketoaldehyde 45 via

deformylation with Wilkinson’s catalyst. Ring enlargement of

the oxime 52 through Beckmann rearrangement occurred re-

0.27 ( 0.08 (2)

0.23 ( 0.05 (2)

0.062 ( 0.023 (3)

0.17 ( 0.02 (3)

0.17 ( 0.01 (2)

0.054 ( 0.007 (4)

0.14 ( 0.01 (2)

0.9 ( 0.3 (2)

100

8

200

100

35

180

65

0.025 ( 0.005 (3)

0.040 ( 0.003 (2)

65

46

0

gioselectively to afford Δ^{1 -2}-en-lactam 53. Standard alkylation

N-Me-lactam

Scheme 1^a

^aReagents and conditions: (a) Li, NH₃, THF, t-BuOH, -78 °C; (b) H₂, Pd/C, pyridine, 25 °C; (c) CbzCl, Et₃N, THF, 25 °C; (d) H₂, Pd/C, EtOAc,

toluene, 25 °C.

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4403

Scheme 2^a

^aReagents and conditions: (a) N₂H₄-H₂O, K₂CO₃, triethylene glycol, 150 °C; (b) HONH₂-HCl, NaOAc, wet EtOH, 25 °C; (c) CH₃ONH₂-HCl,

NaOAc, wet EtOH, 25 °C; (d) H₂, Pd/C, 25 °C; (e) K-Selectride, THF, -78 °C; (f) CeCl₃7H₂O, NaBH₄, -78 °C; (g) Me₂SO₄, aq KOH,

3

benzyltriethylammonium chloride, CH₂Cl₂, 25 °C.

gave the N-methyllactam 54. Hydrogenolysis removes the benzyl

carbamate and reduces the olefin of Δ^{1 -2}-en-lactams 53 and 54

to give saturated the desired saturated analogues 55 and 56.

the sp²hybridization at the C3⁰position is not critical for

biological activityasdemonstrated by the potency of hydroxyl

and methoxy analogues (14-17). The R configuration and

again a hydrogen bond donor at the C3⁰position were clearly

preferred for improved biological activity (cf. 15 vs 14, and

17 vs 16). In vitro and in vivo metabolism studies with

compound 2 revealed the formation of metabolite alcohol

15, which is cleared from the plasma along with its glucur-

onide conjugate in cynomolgus monkeys.⁴³In light of this

observation we searched for analogues devoid of this meta-

bolic liability while also maintaining a hydrogen bond donor

at the C3⁰position. Consequently, additional functional

groups were introduced at the C3⁰position to further probe

the SAR. While the primary amine analogues 24 and 25 were

found to be very weak inhibitors regardless of the stereochem-

istry at C3⁰position, the amide and sulfonamide analogues

proved more interesting. As was the case with the C3⁰alcohol

analogues, the R-analogues(26and 28) were morepotent than

the corresponding S-analogues (27 and 29). The sulfonamides

were also 2-fold superior to the amides, possibly because

of a lower pK_aof the N-H, coupled with a larger volume

occupied by the sulfonamide group. Importantly, the C3⁰-

(R)-sulfonamide 28 was found to be similar in potency to the

C3⁰-(R)-alcohol 15 (EC₅₀=7 nM vs 9 nM) and yet had the

advantage of being more metabolically stable in the human

liver microsomes assay (t_1/2=85 min vs 40 min).

0

Potency and Metabolic Stability

The major disadvantages with the prototype compound 2 are

its limited potency and metabolic stability. The remarkable

improvement in potency demonstrated by cis-decalone analo-

gue 6 in the differentiation assay is tempered by the likelihood

that it would suffer from the same metabolic fate as compound

2. Efforts to improve the metabolic stability while maintaining

(or improving) the biological activity of the potent cis-decalone

scaffold led to the synthesis of multiple classes of analogues

without the C3⁰-ketone. New analogues were evaluated for their

ability to inhibit the hedgehog pathway using oxysterol-depen-

dent differentiation of C3H10T1/2 cells,⁴⁶as well as for their

metabolic stability in human liver microsomes (Table 1).

Polar functionalities on the C3⁰position provided analo-

gues with the best biological activity on the hedgehog path-

way. For instance, the more hydrophobic deoxygenated

compound 7 is almost 3 orders of magnitude less potent than

ketone 6 in the differentiation assay. Methyloxime analogue

11 was found to be 10-fold less active than oxime 10, suggest-

ing the preference for a hydrogen bond donor. Although

oxime 10 was found to be as active in the differentiation assay

as ketone 6, it was less metabolically stable. It was found that

4404 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14

Tremblay et al.

Scheme 3^a

^aReagents and conditions: (a) MsCl, Et₃N, CH₂Cl₂, 0 °C; (b) NaN₃, DMPU, 60 °C; (c) TMS-acetylene, toluene, reflux; (d) TBAF, THF, 50 °C;

(e) H₂, Pd/C, 25 °C; (f) PPh₃, wet THF, 55 °C; (g) Ac₂O, pyridine, 25 °C; (h) MsCl, i-Pr₂EtN, CH₂Cl₂, 0 °C.

We envisioned that the metabolic liability of the C3⁰ketone

in compound 6 could be reduced by introducing a heterocycle

such as pyrazole and isoxazole fused onto the A-ring. The

position of the pyrazole on the A ring has a dramatic effect on

the potency and metabolic fate, with the C2⁰-C3⁰fused

analogue 32 being superior with respect to both, compared to

the C3⁰-C4⁰fused analogue 36. The nature of the heterocyclic

ring is also very important for biological activity, favoring

pyrazole (32) over thiazole (38), oxadiazole (40), and isoxazole

(41). Notably, ring systems with a heteroatom at the C3⁰⁰

position (38 and 40) were clearly disfavored compared to the

unsubstituted pyrazole ring (32). Likewise, trifluoromethyl 44

and pyrazolone 47 were also significantly less potent than

compound 32. Substitution on the nitrogen of the pyrazole

ring also resulted in a loss of potency as exemplified with

analogues 48-50, with the least pronounced effect coming

from the N2⁰⁰-methyl substitution (∼3-fold). The metabolic

stability was greatly affected by the nature of the fused hetero-

cycle with pyrazole 32 demonstrating superior biological activ-

ity in the differentiation assay (EC₅₀=13 nM) and superior

metabolic stability in human liver microsomes (t_1/2=120 min).

In another approach to modify the A-ring, we prepared

seven-membered ring lactam analogue 55 and N-methyl

lactam analogue 56. Like pyrazole 32 and sulfonamide 28,

lactam 55 displays a hydrogen bond acceptor/donor pair in

the same region of the molecule and was expected to provide

similar potency. Lactam 55 was only slightly less potent than

the pyrazole 32 and sulfonamide 28, despite the expanded

A-ring (EC₅₀=25 nM vs 13 nM and 7 nM, respectively).

N-Methyllactam 56 showed decreased potency compared to

lactam 55, an effect similar to findings with N-methylation of

pyrazole (48 vs 32) and consistent with the notion that a

hydrogen bond donor is preferable. To our surprise, the in

vitro metabolic stability of lactam 55 was almost the same

as that of the 3-ketone cis-decalone 6 (t_1/2=65 min vs 70 min).

After extensive exploration of the SAR around the

A-ring of reduced ^D-homocyclopamine, three lead com-

pounds emerged based on in vitro assay data (C3H10T1/2

differentiation and human liver microsome stability): sulf-

onamide 28, pyrazole 32, and lactam 55. The pharmaco-

kinetic properties of these three distinct chemotypes were

evaluated to help guide selection of a superior develop-

ment candidate.

Pharmacokinetic Properties in Multiple Mammalian Species

The plasma pharmacokinetic properties of the three lead

compounds (28, 32, and 55) were investigated in mice, rats,

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4405

Scheme 4^a

^aReagents and conditions: (a) HCOOEt, t-BuOK, t-BuOH, 25 °C; (b) N₂H₄-H₂O, EtOH, 80 °C; (c) H₂, Pd/C, 25 °C; (d) KHMDS, Et₃SiCl, THF,

-78 °C; (e) HCHO, TBAF, CH₂Cl₂, -20 °C; (f) Dess-Martin periodinane, CH₂Cl₂, 0 °C.

Scheme 5^a

^aReagents and conditions: (a) NBS, HOAc, NaOAc, wet dioxane, 25 °C; (b) CH₃C(S)NH₂, EtOH, reflux; (c) H₂, Pd/C, 25 °C; (d) NaNO₂, aq AcOH,

CH₂Cl₂, 0 °C; (e) HONH₂-HCl, NaOAc, EtOH, 25 °C; (f) KOH, dioxane, ethylene glycol, 120 °C; (g) HONH₂-HCl, pyridine, 120 °C.

dogs, and cynomolgus monkeys and compared to the proto-

type compound 2 (Table 2). Compound 2 exhibited a rela-

tively short elimination half-life (1.7-3.5 h), high plasma

clearance, and variable oral bioavailability across all species.

Of significance, sulfonamide 28 and lactam 55 have much

lower clearance compared to enone 2 and have excellent

oral bioavailability in rodents and nonrodents. Sulfonamide

28 has a volume of distribution (11-30 L/kg) similar to the

4406 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14

Tremblay et al.

Scheme 6^a

^aReagents and conditions: (a) CF₃COOEt, t-BuOK, t-BuOH, 25 °C; (b) N₂H₄-H₂O, EtOH, 80 °C; (c) H₂, Pd/C, 25°C; (d) DDQ, toluene, 25 °C;

(e) PDC, MeOH, DMF, 25 °C; (f) CH₃NHNH₂, EtOH, 80 °C; (g) MsCl, pyridine, 25 °C; (h) TsCl, pyridine, CH₂Cl₂, 25 °C.

enone 2 (13.9-28 L/kg). Overall, the encouraging pharmaco-

kinetic profile of the potent lead compounds 28, 32, and 55

provided impetus to evaluate them further in a murine tumor

model in vivo.

and compound 2, which showed no activity in this model

when orally administered at a dose near the maximum

tolerated dose (MTD). Growth inhibition of murine medullo-

blastoma model (Ptc1^+/-/p53^+/-; Ptc1^+/-/p53^-/-) has been

shown with cyclopamine administered subcutaneously³⁴

(50 mg/kg) or intraperitoneally³⁵(10 mg/kg). Our results

underscore the superior oral efficacy of these novel analogues

relative to cyclopamine. To better ascertain differences among

these three compounds, time to tumor regrowth, during a

21-day post-treatment phase, was measured. Tumors in ani-

mals treated with compound 28 did not recur during the post-

treatment period, whereas 4/10 and 3/10 animals regrew

tumors when treated with compounds 32 and 55, respectively.

These findings suggest thatcompound 28 demonstrates super-

ior antitumor activity relative to compounds 32 and 55,

possibly resulting in the complete elimination of all viable

tumor cells.

Pharmacology

Antitumor activity and pharmacology of the three lead

compounds were explored using a genetic model of medullo-

blastoma derived from mice engineered to be heterozygous

for both the Ptch1 and Hic1 genes. These animals develop

spontaneous, aggressive medulloblastoma with an incidence

of 40%.⁵⁶B837Tx is a serially passaged subcutaneous allo-

graft derived from a medulloblastoma that developed in these

mice. The B837Tx medulloblastoma tumor is dependent on

the Hh pathway for growth and survival.⁵⁶

Multidose tolerability studies performed on tumor-bearing

immunocompromised mice demonstrated maximum toler-

ated oral doses of 40, 80, and 30 mg/kg for compounds 28,

32, and 55, respectively. Daily oral administration of com-

pounds 28, 32, and 55 at these tolerated doses led to complete

tumor regression during the treatment phase, and median

time to unmeasurable tumor volume was 9 days for all three

compounds (Table 3). This is in stark contrast to cyclopamine

Discussion

The Hh signaling pathway is an emerging target for the

treatmentofcancer. The majorityof anticancerdrugspotently

inhibit the growth of cancer cells in culture, which provides

information on potential sensitivity of tumor types for these

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4407

Table 2. Pharmacokinetic Parameters for Enone 2, Sulfonamide 28, Pyrazole 32, and Lactam 55 in Multiple Mammalian Species

^aParameters obtained from the concentration-time profile after iv administration (1 mg/kg). T_last= 24 h in all studies.

Scheme 7^a

^aReagents and conditions: (a) (PPh₃)₃RhCl, toluene, 80 °C; (b) HONH₂-HCl, NaOAc, EtOH, 25 °C; (c) MsCl, pyridine, 5 °C; (d) HCl, MeOH,

toluene, 60 °C; (e) KHMDS, CH₃I, THF, -78 to 25 °C; (f) H₂, Pd/C, alcohol, 25 °C.

drugs. However, there are significant limitations in using

conventional in vitro growth inhibition assays to predict the

in vivo efficacy of Hh pathway antagonists. Cancer cells can

lose their dependency on Hh signaling pathway for prolifera-

tion and growth when grown in culture.^57-59The antitumor

activity of Hh antagonists is thus best assessed in vivo, but this

poses significant challenges for lead identification and the lead

optimization process. The natural product cyclopamine has

shown in vivo efficacy in several pharmacological models and

served as an excellent starting point for our medicinal chem-

istry effort.

Alterations to the cyclopamine core structure have pro-

duced very potent Hh antagonists. Homologation of the

D-ring of cyclopamine and change in the oxidation state of

the A-B ring system provided first generation compounds

such as compound 2. These modifications were shown

to increase chemical stability and solubility relative to the

natural product. Despite favorable pharmaceutical properties,

4408 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14

Tremblay et al.

Table 3. In Vivo Antitumor Activity of Cyclopamine, Enone 2, Sulfonamide 28, Pyrazole 32, and Lactam 55 in the Ptc1^+/-/Hic1^+/-

B837Tx Allograft Mouse Model

^aThe maximum tolerated dose (MTD) is the dose tolerated by all animals (N = 3) after repeat oral dose administration (5 days).

compound 2 was extensively metabolized in multiple species

after oral administration⁴³(Table 2). Consequently, com-

pound 2 has shownmodest efficacy incertaintumor xenograft

models^41,42and demonstrated no efficacy in the B837Tx

medulloblastoma allograft model when administered orally

at doses approaching the MTD.

expressing recombinant human SMO with an IC₅₀value of

1.4 nM compared to 114 nM for cyclopamine (data not

shown).³³Further preclinical investigation corroborated the

decision to pursue compound 28 as a clinical development

candidate. Compound 28 is currently undergoing clinical

evaluation in a phase 1 clinical trial in cancer patients.

In summary, structural modifications on the first genera-

tion antagonist compound2 led to the discovery of compound

28, a novel Hh antagonist with improved pharmaceutical

properties and potency, as well as a superior plasma pharma-

cokinetic profile relative to cyclopamine and compound 2.

Clinical studies are ongoing to fully scope the full potential

of this novel drug candidate.²⁹

The conversion of the half-chair A-ring system of com-

pound 2 to a cis-ring fusion system provided a significant

improvement in potency of these ^D-homocyclopamine deri-

vatives. Furthermore, the presence of a pair of H-bond

acceptor/donor on the A-ring was a common feature of the

most active analogues such as sulfonamide 28, pyrazole 32,

and lactam 55. It is noteworthy that all three compounds

displayed better activity than compound 2 in Hh pathway-

mediated differentiation in vitro and Hh-dependent tumor

growth in vivo. The clear correlation between these two

activities provides strong evidence that these molecules are

acting on-mechanism in vivo. The A-rings of the three lead

compounds (28, 32, and 55) possess functional moieties that

are metabolically more stable than the enone on first genera-

tion compound 2 and ketone of the compound6. Sulfonamide

28, pyrazole 32, and lactam 55 were all significantly more

potent and less susceptible to metabolism than the first

generation compound 2.

One key discriminating factor between the three leads is

their pharmacokinetic profiles. Although all three leads dis-

played good exposure when administered orally to multiple

species, the sulfonamide 28 showed a significant increase

in plasma half-life due to low clearance and high tissue

distribution. These properties translated into greater tumor-

free intervals following treatment and more robust efficacy

than the two other lead compounds when studied in a

Hh-dependent B837Tx tumor model. Compound 28 (IPI-

926) has also shown in vivo efficacy in lung⁶⁰and pancreatic⁶¹

cancer xenograft models. Compound 28 is a very potent SMO

antagonist, as shown by its ability to inhibit the association of

BODIPY-cyclopamine (5 nM) on C3H10T1/2 transiently

Experimental Section

General Methods. Commercial reagents and solvents were

used as received without further purification or drying. All

experiments involving water-sensitive compounds were carried

out under argon and scrupulously dry conditions, using com-

mercially available anhydrous solvents. Thin-layer chromato-

graphy was performed on glass-backed precoated Merck silica

gel (60 F254) plates, and compounds were visualized using

UV light, ceric ammonium molybdate, or 2% aqueous potas-

sium permanganate solution. Silica gel column chromatography

was carried out using Merck silica gel 60. Flash chromatography

was run using silica gel (200-400 mesh) from Sorbent Techno-

logies. The purity of tested compounds was determined by

analytical liquid chromatography (HPLC) performed on an

Agilent 1100 HPLC system equipped with a Waters C18-Sym-

metry reverse-phase column (4.6 mm ꢀ 150 mm, 5 μm). The

mobile phases were acetonitrile (0.1% trifluoroacetic acid) and

water (0.1% trifluoroacetic acid). After injection the gradient

holds at 10% acetonitrile for 2 min followed by a gradient to

60% over 18 min, a 1 min organic flush at 95% acetonitrile, and

a 6 min re-equilibration at a flow rate of 1.5 mL/min, column

temperature at 40 °C, and detection wavelength of 215 nm.

¹H NMR spectra were recorded on a Bruker 400 spectrometer

(400 MHz). Chemical shifts are reported in ppm with the solvent

resonance as the internal standard (CHCl₃: δ 7.26). Data are

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4409

reported as follows: chemical shift, integration, multiplicity (s=

singlet, d = doublet, dd = doublet of doublet, ddd = doublet

of doublet of doublet, t=triplet, q=quartet, br=broad, m=

multiplet), and coupling constants (Hz). ¹³C NMR spectra were

recorded on a Bruker 400 spectrometer (100 MHz) with com-

plete proton decoupling. Chemical shifts are reported in ppm

with the solvent as the internal reference (CDCl₃: δ 77.16).

Electrospray positive ionization mass spectrometry was per-

formed on samples using a reversed phase HPLC-MS system.

The system was equipped with a diode array UV detector and a

single quadrupole mass spectrometer and utilized the above-

mentioned reversed-phase HPLC gradient method.

Preparation of Compound Included in Scheme 1. Compound 4.

Freshly condensed ammonia (300 mL) at -78 °C was treated

with lithium (1.5 g, 215 mmol, 22.7 equiv) and stirred for 10 min.

The mixture was then charged with a solution of enone 2^40,62

(4.01 g, 9.46 mmol, 1 equiv) in anhydrous tetrahydrofuran

(40 mL) and tert-butanol (2.6 mL) over 15 min. After 2 h, the

mixture was treated with saturated aqueous ammonium chlor-

ide. The reaction mixture was warmed to room temperature and

the ammonia evaporated overnight. The residue was then

partitioned between dichloromethane and water. The aqueous

layer was separated and extracted with dichloromethane. The

combined organic layers were washed with saturated aqueous

ammonium chloride. The organic phase was dried over sodium

sulfate and concentrated to white foam. The residue was pur-

ified using silica gel chromatography (10-20% EtOAc/hex-

anes) to give the desired ketone (3.46 g, 8.14 mmol, 86%).

NMR δ_H(400 MHz, CDCl₃) 3.34 (dt, J=10.6, 3.9 Hz, 1H),

3.03 (dd, J=12.6, 4.2 Hz, 1H), 2.66 (dd, J=9.8, 7.3 Hz, 1H),

2.47-0.95 (m, 31H), 0.93 (s, 3H), 0.90 (s, 3H), 0.88 (s, 3H);

NMR δ_C(100 MHz, CDCl₃) 210.01, 140.75, 124.71, 82.70,

64.18, 54.86, 54.75, 49.89, 49.25, 46.57, 46.52, 44.73, 44.51,

40.30, 39.10, 38.21, 37.30, 35.46, 31.69, 30.77, 30.55, 29.69,

27.88, 23.94, 19.03, 10.68, 10.31; m/z=426.26 [M+H]⁺; HPLC

94.5 a/a % @ 215 nm.

Compound 6. A suspension of enone 2 (4.20 g, 9.94 mmol,

1 equiv) and 10% palladium on carbon (0.850 g) in pyridine

(40 mL) was placed under hydrogen atmosphere and stirred for

16 h at room temperature. The reaction mixture was filtered on

Celite and the filtrate concentrated to dryness. The residue was

purified using silica gel chromatography (0-5% DCM/MeOH).

A solution of the isolated product (4.23 g, 9.94 mmol, 1 equiv) in

tetrahydrofuran (60 mL) was treated with triethylamine (5.03 g,

49.7 mmol, 5.0 equiv) and benzyl chloroformate (1.87 g,

10.9 mmol, 1.1 equiv). The reaction mixture was stirred at room

temperature for 2 h and partitioned between saturated aqueous

sodium bicarbonate and ethyl acetate. The organic phase was

separated, dried over sodium sulfate, and concentrated to

dryness. The residue was purified using silica gel chromatogra-

phy (2-14% EtOAc/hexanes) to afford the desired product as

single isomer 5 (4.65 g, 8.3 mmol, 84%). NMR δ_H(400 MHz,

CDCl₃) 7.39-7.27 (m, 5H), 5.15 (d, J=12.6 Hz, 1H), 5.11 (d,

J=12.7, 1H), 3.82 (dd, J=12.7, 4.4 Hz, 1H), 3.63 (dt, J=10.5,

4.1 Hz, 1H), 3.09 (dd, J=10.1, 6.0 Hz, 1H), 2.69-2.55 (m, 3H),

2.50-2.38 (m, 2H), 2.26-2.14 (m, 3H), 2.10-1.10 (m, H), 0.97

(d, J=6.8, 3H), 0.95 (s, 3H), 0.91 (d, J=7.1, 3H); HPLC 96.6

a/a % at 215 nm.

A suspension of compound 5 (1.35 g, 2.41 mmol, 1 equiv) and

10% palladium on carbon (300 mg) in ethyl acetate (30 mL) was

placed under hydrogen atmosphere and stirred for 3 h at room

temperature. The reaction mixture was filtered on Celite and the

filtrate concentrated to dryness. The oil was purified by silica

gel chromatography (5% MeOH/DCM) to give compound 6

(950 mg, 2.23 mmol, 93%). NMR δ_H(400 MHz, CDCl₃) 3.37

(dt, J=9.2, 2.9 Hz, 1H), 3.07 (dd, J=12.3, 2.8 Hz, 1H), 2.73-

2.60 (m, 2H), 2.51-2.40 (m, 2H), 2.37-2.16 (m, 5H), 2.11-1.03

(m, 23H), 0.96 (s, 3H), 0.95-0.90 (m, 6H); NMR δ_C(100 MHz,

CDCl₃) 213.45, 140.18, 124.94, 82.64, 64.15, 54.81, 49.89, 49.65,

46.69, 44.72, 43.95, 42.51, 42.12, 40.27, 38.29, 37.56, 37.29,

34.86, 31.66, 30.44, 30.40, 27.98, 27.90, 25.50, 24.00, 21.66,

19.01, 10.29; m/z = 426.19 [M + H]⁺; HPLC 95.4 a/a % at

215 nm.

Preparation of Compound Included in Scheme 2. Compound 7.

A solution of ketone 6 (85 mg, 0.199 mmol, 1 equiv) in

triethylene glycol (2 mL) was treated with hydrazine monohy-

drate (500 mg, 10 mmol, 50 equiv) and potassium carbonate

(138 mg, 1 mmol, 5 equiv). The reaction mixture was sealed in a

tube and heated to 150 °C for 16 h. The reaction mixture was

cooled to room temperature and partitioned between water and

chloroform. The organic layer was washed with water, dried

over sodium sulfate, and concentrated to dryness. The residue

was purified using silica gel chromatography (4% MeOH/

DCM). The purified fractions were pooled and concentrated

to dryness. The resulting oil was dissolved in methyl tert-butyl

ether, washed with water, 2 N aqueous sodium hydroxide, and

saturated aqueous sodium chloride. The combined organic

layers were dried over sodium sulfate, filtered, and concentrated

to afford the desired compound 7 as a white foam (64 mg,

0.155 mmol, 75%). NMR δ_H(400 MHz, CDCl₃) 3.35 (dt, J=

10.1, 3.6 Hz, 1H), 3.21 (s, 1H), 3.05 (dd, J=12.8, 3.8 Hz, 1H),

2.69 (dd, J=9.5, 7.6 Hz, 1H), 2.45 (d, J=14.0 Hz, 1H), 2.31

(t, J=11.6 Hz, 1H), 2.22 (br d, J=11.4 Hz, 1H), 2.12 (dd, J=

14.4, 6.0 Hz, 1H), 2.05-1.00 (m, 28H), 0.92 (d, J=7.3 Hz, 3H),

0.91 (d, J = 6.6 Hz, 3H), 0.86 (s, 3H). NMR δ_C(100 MHz,

CDCl₃) 141.43, 124.02, 82.84, 64.19, 54.87, 49.93, 49.82, 47.12,

44.69, 42.96, 42.04, 40.32, 38.67, 37.33, 35.25, 31.74, 31.36,

30.40, 28.72, 27.90, 27.36, 27.20, 26.30, 23.95, 23.34, 21.76,

19.03, 10.31; m/z = 412.29 [M + H]⁺; HPLC 98.2 a/a %

at 215 nm.

Compound 10. A solution of ketone 5 (185 mg, 0.3 mmol,

1 equiv) in ethanol (3 mL) and water (0.3 mL) was treated with

hydroxylamine hydrochloride (140 mg, 2 mmol, 6 equiv) and

sodium acetate (160 mg, 2 mmol, 6 equiv). The reaction mixture

was stirred for 1 h at room temperature and partitioned between

ethyl acetate and water. The organic layer was washed with

saturated aqueous sodium chloride, dried over sodium sulfate,

and concentrated to a white residue. The crude product was

purified by silica gel chromatography (2:3 to 1:1 Et₂O/hexanes)

to give the oxime 8 (193 mg, 0.3 mmol, 100%). A solution of

compound 8 (65 mg, 0.113 mmol) and 10% palladium on carbon

(20 mg) in ethyl acetate (7 mL) was placed under hydrogen

atmosphere and stirred for 3 h at room temperature. The

reaction mixture was filtered on Celite and the filtrate concen-

trated to dryness. The residue was purified using silica gel

chromatography (0.5:2:97.5 to 0.5:6:93.5 NH₄OH(aq)/MeOH/

DCM) to give compound 10 as a white powder (15 mg,

0.113 mmol, 36%, 1/1 cis and trans oxime isomers). NMR δ_H

(400 MHz, CDCl₃) 3.36 (dt, J=10.8, 4.5 Hz, 1H), 3.16-3.10

(m, 1H), 3.06 (dd, J=12.6, 4.4 Hz, 0.5H), 2.94 (dd, J=15.2, 5.0

Hz, 0.5H), 2.70 (dd, J=9.4, 7.6 Hz, 1H), 2.50-2.40 (m, 2H), 2.31

(t, J=12 Hz, 1H), 2.27-1.14 (m, 28H), 0.95-088 (m, 9H); NMR

δ_C(100 MHz, CDCl₃) 162.08, 161.88, 140.86, 124.89, 83.02,

77.56, 64.33, 56.98, 50.14, 49.94, 44.88, 43.32, 42.25, 42.20,

42.05, 40.49, 38.52, 37.56, 37.53, 35.66, 32.79, 31.89, 31.57,

30.68, 28.23, 28.18, 27.63, 25.98, 25.94, 25.27, 24.24, 22.43,

22.34, 20.30, 10.33; m/z=441.50 [M+H]⁺.

Compound 11. 11 was prepared as described for compound

10. The ketone 5 (100 mg, 0.179 mmol) and methoxyamine

hydrochloride with all other reagents scaled accordingly were

used to generate the desired methyloxime 11 (45 mg, 0.99 mmol,

55% for 2 steps). NMR δ_H(400 MHz, CDCl₃) 4.78 (br s, 3H),

3.33 (dt, J=10.2, 3.8 Hz, 1H), 3.03 (dd, J=12.3, 3.8 Hz, 1H),

2.82 (dd, J=14.9, 5.1 Hz, 1H), 2.66 (dd, J=9.8, 7.2 Hz, 1H),

2.48-2.38 (m, 1H), 2.29 (t, J = 11.5 Hz, 1H), 1.25-1.00 (m,

28H), 0.92-0.86 (m, 9H); NMR δ_C(100 MHz, CDCl₃) 161.27,

161.08, 140.59, 140.57, 124.67, 82.74, 64.18, 61.09, 61.07, 54.85,

49.92, 49.69, 46.86, 46.80, 44.72, 43.73, 42.11, 41.99, 41.83,

40.29, 38.36, 37.49, 37.30, 35.41, 35.31, 32.58, 31.70, 31.36,

30.43, 27.97, 27.91, 27.38, 25.86, 25.76, 25.70, 24.02, 24.01,

4410 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14

Tremblay et al.

22.20, 22.09, 20.89, 19.03, 10.13; m/z=455.30 [M+H]⁺; HPLC

96.5 a/a % at 215 nm.

2.27 (t, J = 12.1 Hz, 1H), 2.21 (br td, J =12.1, 3.5 Hz, 1H),

2.12 (dd, J=15.0, 5.9 Hz, 1H), 2.01-1.00 (m, 28H), 0.91 (d, J=

8.0 Hz, 3H), 0.89 (d, J=6.4 Hz, 3H), 0.85 (s, 3H); NMR δ_C

(100 MHz, CDCl₃) 140.87, 124.26, 82.74, 71.64, 63.90, 54.55,

49.99, 49.87, 47.09, 44.63, 42.05, 41.63, 40.22, 37.41, 36.97,

36.76, 34.53, 31.77, 31.32, 31.27, 30.39, 28.52, 27.87, 26.21,

24.03, 22.50, 18.96, 10.21; m/z=428.30 [M+H]⁺; HPLC 98.9

a/a % at 215 nm.

Compound 14. A solution ketone 5 (300 mg, 0.536 mmol,

1 equiv) in anhydrous tetrahydrofuran (10 mL) was cooled

to -78 °C and treated with 1.0 M potassium tri-sec-butyl

borohydride in tetrahydrofuran (0.536 mL, 0.536 mmol,

1 equiv). The reaction mixture was stirred for 4 h and treated

with methanol. The mixture was warmed to -30 °C and treated

with 3 N aqueous sodium hydroxide and 15% aqueous sodium

peroxide, keeping the temperature below 5 °C. The mixture was

warmed to room temperature and stirred for 20 h. The mixture

was partitioned between ethyl acetate and 1 N aqueous hydro-

chloric acid. The organic phase was separated, washed with

saturated aqueous sodium chloride, dried over sodium sulfate,

and concentrated. The residue was purified using silica gel

chromatography (25-40% EtOAc/hexanes) to give the desired

alcohol 12 (250 mg, 0.446 mmol, 90%). NMR δ_H(400 MHz,

CDCl₃) 7.40-7.29 (m, 5H), 5.17 (d, J=12.6 Hz, 1H), 5.13 (d,

J=12.7, 1H), 4.08 (br s, 1H), 3.82 (dd, J=12.9, 5.3 Hz, 1H), 3.63

(dt, J=10.6, 4.7 Hz, 1H), 3.08 (dd, J=10.3, 7.3 Hz, 1H), 2.68-

2.55 (m, 3H), 2.45 (br d, J=14.1 Hz, 1H), 2.20 (td, J=11.2, 4.1

Hz, 1H), 2.13 (dd, J=14.7, 5.9 Hz, 1H), 2.01-1.00 (m, 25H),

0.97 (d, J=7.1, 3H) 0.94-0.89 (m, 6H).

A suspension of compound 12 (170 mg, 0.303 mmol, 1 equiv)

and 10% palladium on carbon (37 mg) in ethyl acetate (5 mL)

was placed under hydrogen atmosphere, and the mixture was

stirred for 3 h at room temperature. The reaction mixture was

filtered on Celite and the filtrate concentrated to dryness. The oil

was purified by silica gel flash chromatography (0.1:10:89.9

NH₄OH(aq)/MeOH/DCM) to give compound 14 as a white

powder (72 mg, 0.168 mmol, 56%). NMR δ_H(400 MHz,

CDCl₃) 4.05 (br s, 1H), 3.34 (dt, J = 10.3, 3.8 Hz, 1H), 3.04

(dd, J=12.9, 4.1 Hz, 1H), 2.67 (dd, J=10.3, 7.3 Hz, 1H), 2.43 (br

d, J=14.1 Hz 1H), 2.29 (t, J=12 Hz, 1H), 2.21 (br td, J=11.8, 3.9

Hz 1H), 2.11 (dd, J=14.7, 6.4 Hz 1H), 2.01-1.00 (m, 28H),

0.92-0.87 (m, 9H); NMR δ_C(100 MHz, CDCl₃) 141.11, 124.22,

82.80, 67.05, 64.08, 54.72, 49.87, 49.78, 46.84, 44.62, 41.28,

40.24, 37.30, 35.79, 34.96, 33.65, 31.69, 31.32, 31.16, 30.51,

28.06, 27.90, 27.87, 26.02, 23.94, 22.90, 18.99, 10.27;

m/z=428.29 [M+H]⁺; HPLC 99.2 a/a % at 215 nm.

Compound 15. A solution of cerium trichloride heptahydrate

(260 mg, 0.697 mmol, 1.3 equiv) in methanol (10 mL) was

cooled to 0 °C and treated with sodium borohydride (24 mg,

0.643 mmol, 1.2 equiv). After 10 min, the mixture was cooled

to -78 °C and treated with a solution of ketone 5 (300 mg,

0.536 mmol, 1 equiv) in anhydrous tetrahydrofuran (5 mL). The

mixture was stirred for 1 h, treated with water, ethyl acetate, and

warmed to room temperature. The mixture was partitioned

between water and 1 N aqueous hydrochloric acid. The organic

phase was separated, washed with saturated aqueous sodium

chloride, dried over sodium sulfate, and concentrated. The

residue was purified using silica gel chromatography (30-

50% Et₂O/hexanes) to give the desired alcohol 13 (235 mg,

0.418 mmol, 78%). NMR δ_H(400 MHz, CDCl₃) 7.38-7.29 (m,

5H), 5.15 (d, J=12.6 Hz, 1H), 5.11 (d, J=12.7, 1H), 3.81 (dd,

J = 13.7, 3.7 Hz 1H), 3.67-3.54 (m, 1H), 3.08 (dd, J =10.0,

6.7 Hz, 1H), 2.63 (dd, J=12.9, 10.0 Hz, 1H), 2.59 (br t, J=

6.7 Hz, 1H), 2.45 (br d, J=14.1 Hz, 1H), 2.19 (br td, J=12.1, 3.5

Hz, 1H), 2.13 (dd, J=15.0, 5.9 Hz, 1H), 2.06-1.10 (m, H), 0.97

(d, J=6.7 Hz, 3H), 0.90 (d, J=7.0 Hz, 3H), 0.86 (s, 3H).

A suspension of compound 13 (235 mg, 0.418 mmol, 1 equiv)

and 10% palladium on carbon (51 mg) in ethyl acetate (5 mL)

was placed under hydrogen atmosphere and stirred for 3 h at

room temperature. The reaction mixture was filtered on Celite

and the filtrate concentrated to dryness. The oil was purified

by silica gel chromatography (0.1:6:93.9 NH₄OH(aq)/MeOH/

DCM) to give compound 15 as a white foam (130 mg,

0.304 mmol, 73%). NMR δ_H(400 MHz, CDCl₃) 3.52 (br s,

1H), 3.33 (dt, J=10.3, 3.5 Hz, 1H), 3.02 (dd, J=13.0, 4.1 Hz,

1H), 2.66 (dd, J=9.7, 7.1 Hz, 1H), 2.40 (br d, J=13.8 Hz, 1H),

Compound 16. A solution of alcohol 12 (110 mg, 0.196 mmol,

1 equiv) in dichloromethane (4 mL) was treated with benzyl-

triethylammonium chloride (9 mg, 0.039 mmol, 0.2 equiv),

dimethyl sulfate (99 mg, 0.783 mmol, 4 equiv), and 50% (w/w)

aqueous potassium hydroxide (0.300 mL). The mixture was

stirred for 18 h at room temperature and partitioned between

ethyl acetate and water. The organic phase was separated,

washed with aqueous sodium chloride, dried over sodium

sulfate, and concentrated to dryness. The residue was purified

using silica gel chromatography (10% Et₂O/hexanes). A suspen-

sion of isolated product and 10% palladium on carbon (6 mg) in

ethyl acetate (3 mL) was placed under hydrogen atmosphere and

stirred for 3 h at room temperature. The reaction mixture was

filtered on Celite and the filtrate concentrated to dryness. The oil

was purified by silica gel chromatography (0.1:2:97.9 to 0.1/12/

87.9 NH₄OH(aq)/MeOH/DCM) to give compound 16 as a

white foam (9 mg, 0.020 mmol, 43%). NMR δ_H(400 MHz,

CDCl₃) 3.47 (br s, 1H), 3.37 (t, J=10.3 Hz, 1H), 3.28 (s, 3H),

3.07 (br d, J=12.2 Hz, 1H), 2.70 (t, J=8.1 Hz, 1H), 2.45 (br d, J=

14.1 Hz 1H), 2.32 (t, J=12 Hz, 1H), 2.24 (br d, J=11.8 Hz 1H),

2.14 (dd, J=14.7, 6.4 Hz 1H), 2.01-1.00 (m, 27H), 0.95-0.86

(m, 9H); NMR δ_C(100 MHz, CDCl₃) 141.28, 124.23, 82.85,

76.12, 64.20, 55.67, 54.85, 49.96, 49.88, 46.95, 44.71, 41.59,

40.32, 37.37, 36.24, 34.86, 31.70, 31.57, 31.37, 30.65, 30.60,

28.08, 27.94, 26.11, 24.49, 24.00, 22.87, 19.05, 10.36; m/z =

442.30 [M+H]⁺; HPLC 98.0 a/a % at 215 nm.

Compound 17. 13 was prepared as described for compound

16. The alcohol 13 (100 mg, 0.178 mmol) with all other reagents

scaled accordingly were used to generate the desired compound

17 (22 mg, 0.050 mmol, 32% for 2 steps). NMR δ_H(400 MHz,

CDCl₃) 3.40-3.32 (m, 1H), 3.33 (s, 3H), 3.18-3.09 (m, 1H), 3.06

(dd, J=13.6, 4.1 Hz, 1H), 2.69 (dd, J=10.2, 7.3 Hz, 1H), 2.45 (br

d, J=14.1 Hz 1H), 2.31 (t, J=11.8 Hz, 1H), 2.26-2.18 (m, 1H),

2.12 (dd, J=14.7, 6.4 Hz 1H), 2.01-1.00 (m, 28H), 0.92-0.87

(m, 9H); NMR δ_C(100 MHz, CDCl₃) 140.96, 124.21, 82.96,

80.43, 64.21, 55.62, 54.86, 49.94, 49.74, 47.06, 44.67, 41.85,

41.51, 40.33, 37.27, 36.59, 34.87, 33.08, 31.70, 31.37, 30.36,

28.62, 27.88, 27.15, 26.12, 23.99, 22.54, 19.05, 10.36; m/z =

442.31 [M+H]⁺; HPLC 99.7 a/a % at 215 nm.

Preparation of Compound Included in Scheme 3. Compound

20. A solution of alcohol 12 (7.60 g, 13.53 mmol, 1 equiv) in

dichloromethane(115mL) was treated with triethylamine(8.21 g,

81 mmol, 6.0 equiv), cooled to 0 °C, and treated with methane-

sulfonyl chloride (1.86 g, 16.2 mmol, 1.2 equiv). After 30 min,

the reaction mixture was partitioned between saturated aqueous

sodium bicarbonate and ethyl acetate. The organic layer was

separated, dried over sodium sulfate, and concentrated to

dryness. The residue was purified using silica gel chromatogra-

phy (10-25% EtOAc/hexanes). A solution of the isolated

product in DMPU (50 mL) was treated with sodium azide

(4.17 g, 64.3 mmol, 5.0 equiv), heated to 60 °C, and stirred for

17 h. The reaction mixture was cooled to room temperature and

partitioned between methyl tert-butyl ether and 5% aqueous

sodium chloride. The organic layer was separated, washed with

5% aqueous sodium chloride, dried over sodium sulfate, and

concentrated to dryness. The residue was purified using silica gel

chromatography (5-15% EtOAc/hexanes) to give the desired

azide 18 (7.16 g, 12.2 mmol, 90% for two steps). NMR δ_H(400

MHz, CDCl₃) 7.40-7.30 (m, 5H), 5.17 (d, J=12.6 Hz, 1H), 5.13

(d, J = 12.7, 1H), 3.83 (dd, J = 12.8, 3.9, 1H), 3.64 (dt,

J=10.9, 3.8 Hz, 1H), 3.32-3.21 (m, 1H), 3.10 (dd, J=13.1,

6.3 Hz, 1H), 2.70-2.56 (m, 2H), 2.47 (br d, J=14.4 Hz, 1H),

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4411

2.33 (br td, J=11.6, 4.3 Hz, 1H), 2.14 (dd, J=14.8, 5.9 Hz, 1H),

2.06-1.12 (m, 25H), 0.98 (d, J=6.6 Hz, 3H), 0.92 (d, J=7.4 Hz,

3H), 0.89 (s, 3H).

2.13 (dd, J=14.8, 5.9 Hz, 1H), 2.06-1.07 (m, 25H), 0.98 (d,

J=6.3 Hz, 3H), 0.91 (d, J=6.8 Hz, 3H), 0.87 (s, 3H).

A solution of compound 22 (206 mg, 0.368 mmol, 1 equiv)

and 10% palladium on carbon (42 mg) in 2-propanol (5 mL) was

placed under hydrogen atmosphere and stirred for 18 h at room

temperature. The reaction mixture was filtered on Celite and the

filtrate concentrated to dryness. The residue was purified using

silica gel chromatography (0-10% MeOH/DCM) to give com-

pound 24 (110 mg, 0.258 mmol, 70%). NMR δ_H(400 MHz,

CDCl₃) 3.35 (dt, J=10.0, 3.2 Hz, 1H), 3.05 (dd, J=12.6, 3.5 Hz,

1H), 2.74-2.60 (m, 2H), 2.44 (br d, J=13.8 Hz, 1H), 2.30 (t, J=

11.9 Hz, 1H), 2.22 (br d, J=11.2 Hz, 1H), 2.11 (dd, J=14.7,

5.6 Hz 1H), 2.01-1.00 (m, 29H), 0.95-0.88 (m, 6H), 0.86

(s, 3H); NMR δ_C(100 MHz, CDCl₃) 140.93, 124.14, 82.78,

76.77, 64.11, 54.76, 51.41, 49.86, 49.68, 46.98, 44.61, 41.87,

40.24, 37.49, 37.28, 37.23, 34.46, 31.64, 31.59, 30.27, 28.42,

27.82, 26.11, 25.47, 23.90, 22.59, 18.97, 10.27; m/z = 427.30

[M+H]⁺; HPLC 95.1 a/a % at 215 nm.

Compound 25. 23 was prepared as described for compound

22. The azide 19 (126 mg, 0.215 mmol) and all other reagents

scaled accordingly were used to generate the desired amine 23

(107 mg, 0.191 mmol, 85%). NMR δ_H(400 MHz, CDCl₃) 7.37-

7.29 (m, 5H), 5.15 (d, J=12.2 Hz, 1H), 5.11 (d, J=12.3 Hz, 1H),

3.81 (dd, J=13.2, 3.8 Hz, 1H), 3.62 (dt, J=10.6, 4.0 Hz, 1H),

3.21 (br s, 1H), 3.08 (dd, J=9.8, 5.4 Hz, 1H), 2.67-2.55 (m, 2H),

2.44 (br d, J=14.0 Hz, 1H), 2.19 (br td, J=11.5, 4.2 Hz, 1H), 2.12

(dd, J=14.2, 5.9 Hz, 1H), 2.04-1.11 (m, 25H), 0.96 (d, J=6.3

Hz, 3H), 0.90 (d, J=6.8 Hz, 3H), 0.89 (s, 3H).

25 was prepared as described for compound 24. The amine 23

(107 mg, 0.191 mmol) and all other reagents scaled accordingly

were used to generate the desired amine 21 (73 mg, 0.171 mmol,

90%). NMR δ_H(400 MHz, CDCl₃) 3.36 (dt, J=10.5, 3.3 Hz,

1H), 3.23 (br s, 1H), 3.06 (dd, J=11.9, 3.3 Hz, 1H), 2.69 (t, J=

8.1 Hz, 1H), 2.46 (br d, J=13.4 Hz, 1H), 2.32 (t, J=11.9 Hz, 1H),

2.24 (br d, J=11.4 Hz, 1H), 2.14 (dd, J=14.4, 6.2 Hz, 1H), 2.04-

1.03 (m, 29H), 0.95-0.89 (m, 9H); NMR δ_C(100 MHz, CDCl₃)

141.16, 124.26, 82.84, 64.20, 54.88, 49.95, 49.85, 46.91, 46.44,

44.73, 41.31, 40.34, 37.37, 35.75, 35.23, 33.99, 31.77, 31.37,

31.18, 30.52, 28.39, 28.18, 27.93, 26.14, 23.99, 23.15, 19.05,

10.34; m/z=427.31 [M+H]⁺; HPLC 92.7 a/a % at 215 nm.

Compound 26. A solution of amine 22 (510 mg, 0.933 mmol,

1 equiv) in pyridine (5 mL) was treated with acetic anhydride

(95 mg, 0.933 mmol, 1 equiv) and stirred for 1 h. The reaction

mixture was partitioned between saturated aqueous sodium

bicarbonate and ethyl acetate. The organic phase was separated,

washed with saturated sodium chloride, dried over sodium

sulfate, and concentrated to an oil. A solution of the residue

(245 mg, 0.406 mmol, 1 equiv) and 10% palladium on carbon

(46 mg) in 2-propanol (5 mL) was placed under hydrogen

atmosphere and stirred for 16 h at room temperature. The

reaction mixture was filtered on Celite and the filtrate concen-

trated to dryness. The residue was purified using silica gel

chromatography (0-10% MeOH/DCM) to give the acetamide

26 (185 mg, 0.406 mmol, 100% for two steps). NMR δ_H(400

MHz, CDCl₃) 5.44 (br d, J=7.8 Hz, 1H), 3.72 (br s, 1H), 3.33 (dt,

J=10.4, 3.5 Hz, 1H), 3.03 (dd, J=12.6, 3.9 Hz, 1H), 2.66 (t,

J=7.3 Hz, 1H), 2.41 (br d, J=13.9 Hz, 1H), 2.29 (t, J=11.7 Hz,

1H), 2.21 (br d, J =11.7 Hz, 1H), 2.10 (dd, J=14.7, 6.0 Hz, 1H),

1.91 (s, 3H), 1.99-1.03 (m, 27H), 0.90 (d, J=3.0 Hz, 3H), 0.88 (d,

J=2.2Hz, 3H), 0.85(s, 3H); NMR δ_C(100MHz, CDCl₃) 169.27,

140.87, 124.41, 82.73, 64.12, 54.77, 49.89, 49.80, 49.40, 46.97,

44.66, 42.02, 41.63, 40.27, 37.33, 37.05, 34.47, 33.92, 31.72, 31.33,

30.31, 28.27, 28.23, 27.88, 26.16, 23.97, 23.67, 22.55, 19.00, 10.30;

m/z=469.32 [M+H]⁺; HPLC 95.8 a/a % at 215 nm.

A solution of azide 18 (49 mg, 0.084 mmol, 1 equiv) in toluene

(4 mL) was treated with trimethylsilylacetylene (164 mg,

1.67 mmol, 20 equiv). The reaction mixture was sealed and

heated to 115 °C for 16 h. The reaction mixture was cooled

to room temperature and the solvent removed under vacuum.

The residue was purified using silica gel chromatography

(2-20% EtOAc/hexanes). A solution of isolated product in

anhydrous tetrahydrofuran (3 mL) was treated with 1.0 M

tetrabutylammonium fluoride in tetrahydrofuran (0.131 mL,

0.131 mmol, 2 equiv) and heated to 55 °C. The mixture was

stirred for 4 h, cooled to room temperature, and partitioned

between saturated aqueous bicarbonate and methyl tert-butyl

ether. The organic phase was separated, dried over sodium

sulfate, and concentrated. The residue was purified by silica

gel chromatography (EtOAc/hexanes). A suspension of isolated

product and 10% palladium on carbon (10 mg) in ethanol

(5 mL) was placed under hydrogen atmosphere and stirred for

1.5 h at room temperature. The reaction mixture was filtered

on Celite and the filtrate concentrated to dryness. The residue

was purified using silica gel chromatography (0-10% MeOH/

DCM) to give compound 20 (8 mg, 0.017 mmol, 26% for

three steps). NMR δ_H(400 MHz, CDCl₃) 7.69 (s, 1H), 7.58

(s, 1H), 4.50 (m, 1H), 3.36 (dt, J=10.2, 4.2 Hz, 1H), 3.06 (dd,

J = 12.7, 5.1 Hz, 1H), 2.70 (dd, J = 9.3, 6.7 Hz, 1H), 2.47

(d, J=13.5 Hz, 1H), 2.32 (t, J=11.9 Hz, 1H), 2.24 (br d, J=

12.7 Hz, 1H), 2.19 (dd, J=15.2, 6.8 Hz, 2H), 2.10-1.00 (m, 26H),

0.96 (s, 3H), 0.93 (d, J=7.6 Hz, 3H), 0.91 (d, J=6 Hz, 3H); NMR

δ_C(100 MHz, CDCl₃) 140.52, 133.47, 124.80, 121.30, 82.75,

64.22, 60.89, 54.90, 49.85, 49.70, 46.99, 44.78, 42.09, 42.07,

40.35, 37.40, 37.14, 34.75, 34.44, 31.75, 31.18 30.33, 28.46,

28.25, 27.95, 26.10, 24.02, 22.52, 19.07, 10.35; m/z = 479.31

[M+H]⁺; HPLC 95.5 a/a % at 215 nm.

Compound 21. 19 was prepared as described for compound

18. The alcohol 13 (174 mg, 0.310 mmol) and all other reagents

scaled accordingly were used to generate the desired azide 19

(174 mg, 0.298 mmol, 96% for two steps). NMR δ_H(400 MHz,

CDCl₃) 7.38-7.30 (m, 5H), 5.16 (d, J=12.3 Hz, 1H), 5.12 (d,

J=12.5 Hz, 1H), 3.93 (br s, 1H), 3.82 (dd, J=12.3, 3.6, 1H), 3.63

(dt, J=10.7, 4.0 Hz, 1H), 3.09 (dd, J=13.3, 6.0 Hz, 1H), 2.68-

2.56 (m, 2H), 2.44 (br d, J=14.0 Hz, 1H), 2.20 (br td, J=11.6, 4.3 Hz,

1H), 2.13 (dd, J=14.8, 5.9 Hz, 1H), 2.06-1.07 (m, 25H), 0.98 (d,

J=6.3 Hz, 3H), 0.91 (d, J=7.1 Hz, 3H), 0.90 (s, 3H).

21 was prepared as described for compound 20. The azide 19

(60 mg, 0.102 mmol) and all other reagents scaled accordingly

were used to generate the desired triazole 21 (8 mg, 0.018 mmol,

12% for three steps). NMR δ_H(400 MHz, CDCl₃) 7.69 (br d, J=

13.1 Hz, 2H), 4.69 (br s, 1H), 3.37 (dt, J=10.2, 3.4 Hz, 1H), 3.07

(dd, J=12.5, 4.2 Hz, 1H), 2.70 (dd, J=10.7, 7.6 Hz, 1H), 2.46

(br d, J=13.6 Hz, 1H), 2.39-1.13 (m, 28H), 1.07 (t, J=11.4 Hz,

2H) 0.98-0.87 (m, 6H), 0.85 (s, 3H); NMR δ_C(100 MHz,

CDCl₃) 140.71, 133.55, 124.68, 122.61, 82.77, 64.18, 56.62,

54.82, 49.96, 49.83, 46.85, 44.73, 42.04, 40.30, 37.38, 36.54,

34.72, 31.87, 31.67, 31.38, 30.48, 30.20, 27.92, 27.29, 25.98,

25.33, 24.04, 22.87, 19.04, 10.36; m/z=479.30 [M+H]⁺; HPLC

98.0 a/a % at 215 nm.

Compound 24. A solution of azide 18 (7.16 g, 12.2 mmol,

1 equiv) in tetrahydrofuran (120 mL) was treated with triphe-

nylphosphine (9.60 g, 36.6 mmol, 3.0 equiv) and water (2.19 g,

122.3 mmol, 10 equiv). The mixture was heated to 55 °C, stirred

for 20 h, cooled to room temperature, and concentrated to

dryness. The residue was purified using silica gel chromatogra-

phy (10-20% MeOH/DCM) to afford the desired amine 22

(5.83 g, 10.4 mmol, 85%). NMR δ_H(400 MHz, CDCl₃) 8.38-

8.34 (m, 1H), 7.39-7.30 (m, 5H), 5.16 (d, J=12.2 Hz, 1H), 5.12

(d, J=12.3 Hz, 1H), 3.83 (dd, J=12.0, 3.9 Hz, 1H), 3.63 (dt, J=

10.8, 4.0 Hz, 1H), 3.10 (dd, J=13.3, 6.0 Hz, 1H), 2.70-2.57 (m,

4H), 2.46 (br d, J=14.0 Hz, 1H), 2.20 (br td, J=11.2, 4.3 Hz, 1H),

Compound 27. 27 was prepared as described for compound

26. The amine 23 (50 mg, 0.089 mmol) and all other reagents

scaled accordingly were used to generate the desired acetamide

27 (34 mg, 0.070 mmol, 78% for two steps). NMR δ_H(400 MHz,

CDCl₃) 5.69 (br d, J = 7.1 Hz, 1H), 4.18 (br s, 1H), 3.38

(dt, J = 10.5, 3.9 Hz, 1H), 3.08 (dd, J = 13.1, 4.5 Hz, 1H),

4412 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14

Tremblay et al.

2.71(dd, J=9.7, 7.9 Hz, 1H), 2.46 (br d, J=14.3 Hz, 1H), 2.33 (t,

J=11.8 Hz, 1H), 2.24 (br d, J=11.7 Hz, 1H), 2.14 (dd, J=15.0,

5.8 Hz, 1H), 2.00 (s, 3H), 1.99-1.03 (m, 27H), 0.96-0.90 (m,

9H); NMR δ_C(100 MHz, CDCl₃) 169.40, 140.79, 124.49, 82.86,

64.10, 54.79, 49.92, 49.70, 46.86, 45.34, 44.64, 41.30, 40.23,

37.30, 37.25, 34.97, 32.49, 31.50, 31.36, 30.76, 30.47, 27.99,

27.90, 25.93, 25.09, 23.99, 23.85, 23.18, 19.00, 10.36; m/z =

469.32 [M+H]⁺; HPLC 98.8 a/a % at 215 nm.

20.42 mmol, 2 equiv), heated to 80 °C, stirred for 30 min, and

concentrated to dryness. The residue was purified by silica gel

chromatography (20-55% EtOAc/hexanes) to give the desired

pyrazole 31 (4.5 g, 7.71 mmol, 76%). NMR δ_H(400 MHz,

CDCl₃) 7.37-7.30 (m, 5H), 7.29 (s, 1H), 5.14 (d, J=12.5 Hz,

1H), 5.10 (d, J=12.4, 1H), 3.81 (dd, J=12.7, 4.1 Hz, 1H), 3.62

(dt, J=10.6, 3.8 Hz, 1H), 3.06 (dd, J=9.5, 6.3 Hz, 1H), 2.80 (dd,

J=17.4, 10.8 Hz, 1H), 2.68-2.52 (m, 4H), 2.37 (br d, J=13.8

Hz, 1H), 2.28 (br d, J=16.1 Hz, 1H), 2.19 (dt, J=11.8, 4.5 Hz,

1H), 2.10-1.18 (m, 22H), 1.05 (s, 3H), 0.97 (d, J=6.8 Hz, 3H),

0.89 (d, J=7.1 Hz, 3H).

Compound 28. A solution of amine 22 (5.10 g, 9.09 mmol,

1 equiv) in dichloromethane (60 mL) was treated with diisopro-

pylethylamine (5.88 g, 45.5 mmol, 5.0 equiv), cooled to 0 °C, and

treated with methanesulfonyl chloride (2.08 g, 18.2 mmol,

2.0 equiv). The reaction mixture was stirred for 30 min and

partitioned between saturated aqueous sodium bicarbonate and

ethyl acetate. The organic layer was separated, dried over

sodium sulfate, and concentrated to dryness. The residue was

purified using silica gel chromatography (10-30% EtOAc/

hexanes). A suspension of the isolated product and 10% palla-

dium on carbon (1.0 g) in 2-propanol (50 mL) was placed under

hydrogen atmosphere and stirred for 4 h at room temperature.

The reaction mixture was then filtered on Celite and the filtrate

concentrated to dryness. The residue was then purified using

silica gel chromatography (0-5% DCM/MeOH) to give the

desired compound 28 (4.06 g, 8.05 mmol, 95% for two steps).

NMR δ_H(400 MHz, CDCl₃) 6.90 (br s, 1H), 3.31 (dt, J =

10.6, 3.8 Hz, 1H), 3.20 (br s, 1H), 3.10 (dd, J=13.7, 4.5 Hz, 1H),

2.91 (s, 3H), 2.62 (dd, J=9.9, 7.6 Hz, 1H), 2.33 (br d, J=14.5 Hz,

1H), 2.27-2.15 (m, 1H), 2.10 (dd, J=14.5, 6.9 Hz, 1H), 1.99-

1.17 (m, 28H), 1.05 (q, J=11.6 Hz, 1H), 0.93 (d, J=7.4 Hz, 3H),

0.88 (d, J = 6.6 Hz, 3H), 0.86 (s, 3H); NMR δ_C(100 MHz,

CDCl₃) 140.47, 124.53, 82.48, 76.97, 63.73, 54.08, 53.87, 50.12,

49.98, 47.19, 44.73, 42.27, 42.10, 40.24, 37.55, 37.44, 36.04,

34.44, 31.87, 31.33, 30.46, 29.79, 28.37, 27.94, 26.26, 24.19,

22.70, 18.92, 10.19; m/z=505.29 [M+H]⁺; HPLC 99.1 a/a %

at 215 nm.

Compound 29. 29 was prepared as described for compound

28. The amine 23 (50 mg, 0.089 mmol) and all other reagents

scaled accordingly were used to generate the desired compound

29 (35 mg, 0.072 mmol, 90% for two steps). NMR δ_H(400 MHz,

CDCl₃) 4.47 (d, J=7.5 Hz, 1H), 3.81-3.74 (m, 1H), 3.36 (dt, J=

10.0, 3.5 Hz, 1H), 3.07 (dd, J=12.7, 3.6 Hz, 1H), 2.96 (s, 3H),

2.69 (dd, J=9.6, 7.7 Hz, 1H), 2.44 (d, J=13.6 Hz, 1H), 2.32 (t, d,

J=13.4 Hz, 1H), 2.26-2.19 (m, 1H) 2.13(dd, J=14.3, 6.1 Hz,

1H), 2.08-1.02 (m, 28H) 0.96-0.88 (m, 9H); NMR δ_C

(100 MHz, CDCl₃) 140.66, 124.61, 82.77, 64.11, 54.73, 50.14,

49.91, 49.70, 46.80, 44.66, 41.66, 41.48, 40.24, 37.31, 36.59,

34.87, 32.21, 31.71, 31.58, 31.36, 30.47, 27.95, 27.88, 26.53,

25.98, 24.01, 23.01, 19.01, 10.35; m/z=505.38 [M+H]⁺; HPLC

98.6 a/a % at 215 nm.

A suspension of pyrazole 31 (4.5 g, 7.71 mmol, 1 equiv) and

10% palladium on carbon (350 mg) in ethanol (50 mL) and ethyl

acetate (50 mL) was placed under hydrogen atmosphere and

stirred for 1.5 h at room temperature. The reaction mixture was

filtered on Celite and the filtrate concentrated to dryness. The

residue was purified using silica gel chromatography (0.5/2/97.5

to 0.5/6/93.5% NH₄OH/DCM/MeOH) to afford the desired

product 32 (3.0 g, 6.67 mmol, 87%). NMR δ_H(400 MHz,

CDCl₃) 3.32 (dt, J = 10.2, 3.5 Hz, 1H), 3.02 (dd, J = 12.5,

4.1 Hz, 1H), 2.75 (dd, J=17.1, 11.5 Hz, 1H), 2.63 (t, J=8.6 Hz,

1H), 2.58-2.49 (m, 2H), 2.37-2.15 (m, 4H), 2.07-1.18 (m,

24H), 1.00 (s, 3H), 0.9-0.85 (m, 6H); NMR δ_C(100 MHz,

CDCl₃) 140.64, 124.38, 113.97, 82.86, 64.14, 54.76, 49.99, 49.89,

46.55, 44.68, 42.30, 40.27, 39.07, 37.29, 35.62, 32.73, 31.65,

31.37, 30.85, 27.84, 27.20, 25.42, 23.97, 23.03, 22.23, 19.03,

10.35; m/z=450.19 [M+H]⁺; HPLC 99.8 a/a % at 215 nm.

Compound 36. A solution of potassium hexamethyldisilazane

(310 mg, 1.55 mmol, 1.5 equiv) in anhydrous THF (10 mL)

was cooled to -78 °C and treated with a solution of ketone 5

(580 mg, 1.036 mmol, 1 equiv) in anhydrous tetrahydrofuran

(2 mL) followed by triethylsilyl chloride (234 mg, 1.55 mmol,

1.5 equiv). The reaction mixture was stirred for 15 min, parti-

tioned between water and ethyl acetate, and warmed to room

temperature. The organic phase was separated, dried over

sodium sulfate, and concentrated to a colorless oil. The residue

was purified by silica gel chromatography (2-6% EtOAc/

hexanes) to give the desired compound 33 (546 mg, 0.810 mmol,

78%). NMR δ_H(400 MHz, CDCl₃) 7.39-7.31 (m, 5H), 5.14 (d,

J=12.5 Hz, 1H), 5.10 (d, J=12.4, 1H), 4.73 (br d, 1H minor C2

isomer), 4.50 (s, 1H major C4 isomer), 3.82 (dd, J=13.1, 4.3 Hz,

1H), 3.63 (dt, J=10.7, 4.9 Hz, 1H), 3.19 (dd, J=10.7, 6.3 Hz,

1H), 2.66 (dd, J=13.1, 10.1 Hz, 1H), 2.59 (t, J=6.3 Hz, 1H), 2.43

(br d, J=14.1 Hz, 1H), 2.24-1.03 (m, 25H), 1.00-0.95 (m, 12H),

0.93-0.87 (s, 6H), 0.69-0.61 (m 6H).

A

37% aqueous formaldehyde solution (0.302 mL,

4.05 mmol, 5 equiv) was diluted in tetrahydrofuran (10 mL),

stirred for 10 min, and cooled to -20 °C. The reaction mixture

was treated with a solution of the compound 33 (546 mg, 0.810

mmol, 1 equiv) and 1.0 M of tetrabutylammonium fluoride in

tetrahydrofuran (4.05 mL, 4.05 mmol, 5 equiv), keeping the

internal temperature below -20 °C. The mixture was stirred for

30 min and quenched with saturated aqueous sodium chloride.

The mixture was warmed to room temperature and extracted

with ethyl acetate. The combined extracts were dried over

sodium sulfate and concentrated to dryness. The residue was

purified by silica gel chromatography (10-20% EtOAc/hex-

anes) to give the desired alcohol 34 (330 mg, 0.560 mmol, 69%).

NMR δ_H(400 MHz, CDCl₃) 7.37-7.30 (m, 5H), 5.14 (d, J=

12.5 Hz, 1H), 5.10 (d, J = 12.4 Hz, 1H), 3.90-3.78 (m, 2H),

3.70-3.58 (m, 2H), 3.08 (dd, J=13.1, 4.3 Hz, 1H), 2.77-2.41

(m, 6H), 2.26-2.16 (m, 3H), 2.08-1.11 (m, 23H), 0.99-0.88

(m, 9H).

A solution of alcohol 34 (100 mg, 0.170 mmol, 1 equiv) in

dichloromethane (5 mL) was cooled to 5 °C and treated with

Dess-Martin periodinane (144 mg, 0.339 mmol, 2 equiv). The

reaction mixture was stirred for 30 min and partitioned between

saturated aqueous bicarbonate and dichloromethane. The or-

ganic phase was separated, dried over sodium sulfate, and

concentrated to dryness. The residue was purified by silica gel

Preparation of Compound Included in Scheme 4. Compound

32. A solution of ketone 5 (6.3 g, 11.25 mmol, 1 equiv) in tert-

butanol (65 mL) was treated with potassium tert-butoxide

(8.84 g, 79 mmol, 7 equiv) and stirred for 10 min. The mixture

was treated with ethyl formate (5.00 g, 67.5 mmol, 6 equiv) and

stirred for 48 h at room temperature. The mixture was parti-

tioned between diethyl ether and 1 N aqueous sodium hydro-

xide. The aqueous layer was separated and acidified to pH 1-2

with concentrated aqueous hydrochloric acid and extracted with

dichloromethane. The combined organic extracts were dried

with sodium sulfate and concentrated to give the desired com-

pound 30 (6.0 g, 10.21 mmol, 91%, 9/1 C2/C4 epimer). NMR

δ

_H(400 MHz, CDCl₃) 8.75 (d, J=4 Hz, 1H C4 epimer), 8.45 (d,

J=3.4 Hz, 1H C2 epimer), 7.39-7.29 (m, 5H), 5.16 (d, J=12.6

Hz, 1H), 5.12 (d, J=12.7, 1H), 3.82 (dd, J=12.6, 4.0 Hz, 1H),

3.63 (dt, J=10.8, 3.8 Hz, 1H), 3.08 (dd, J=9.7, 6.2 Hz, 1H), 2.64

(dd, J=13.1, 9.4 Hz, 1H), 2.59 (t, J=6.2 Hz, 1H), 2.53-2.39 (m,

2H), 2.33-1.15 (m, 27H), 1.00 (s, 3H), 0.97 (d, J=6.2 Hz, 3H),

0.90 (d, J=6.8 Hz, 3H).

A solution of ketone 30 (6.0 g, 10.21 mmol, 1 equiv) in etha-

nol (100 mL) was treated with hydrazine hydrate (1.022 g,

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4413

chromatography (5-10% EtOAc/hexanes). A solution of the

isolated product in ethanol (5 mL) was treated with hydrazine

(27 mg, 0.85 mmol, 10 equiv) and heated to 80 °C. The reaction

mixture was stirred for 15 min and concentrated to dryness. The

residue was purified by silica gel chromatography (50% EtOAc/

hexanes) to give the desired pyrazole 35 (43 mg, 0.073 mmol,

65% for two steps). NMR δ_H(400 MHz, CDCl₃) 7.38-7.31

(m, 5H), 7.30 (br s, 1H), 5.15 (d, J=12.5 Hz, 1H), 5.11 (d, J=

12.7 Hz, 1H), 3.81 (dd, J=13.1, 4.3 Hz, 1H), 3.62 (dt, J=10.7,

4.9 Hz, 1H), 3.08 (dd, J=10.1, 6.2 Hz, 1H), 2.70-2.53 (m, 5H),

2.37 (br d, J=13.9 Hz, 1H), 2.24-2.13 (m, 2H), 2.10-1.03 (m,

22H), 1.02 (s, 3H), 0.98 (d, J=6.9 Hz, 3H), 0.89 (d, J=7 Hz, 3H).

A suspension of pyrazole 35 (43 mg, 0.073 mmol, 1 equiv) and

10% palladium on carbon (8 mg) in ethyl acetate (3 mL) was

placed under hydrogen atmosphere and stirred for 16 h at room

temperature. The reaction mixture was filtered on Celite and

the filtrate concentrated to dryness. The residue was purified

using silica gel chromatography (5% DCM/MeOH) to afford

the desired product 36 (30 mg, 0.067 mmol, 91%). NMR δ_H

(400 MHz, CDCl₃) 7.30 (br s, 1H), 3.35 (dt, J=10.6, 3.8 Hz, 1H),

3.06 (dd, J=12.6, 4.2 Hz, 1H), 2.90-2.61 (m, 4H), 2.39-2.27 (m,

2H), 2.23 (td, J=11.8, 3.4, Hz, 1H), 2.16 (dd, J=14.8, 5.4 Hz,

1H), 2.10-1.03 (m, 23H), 1.00 (s, 3H), 0.93-0.86 (s, 6H); NMR

δ_C(100 MHz, CDCl₃) 140.77, 124.17, 119.01, 82.88, 76.62,

64.02, 54.55, 49.89, 49.43, 46.63, 44.57, 42.23, 40.22, 39.29,

37.29, 35.39, 35.35, 31.50, 31.37, 30.84, 27.90, 26.78, 26.35,

24.05, 21.22, 19.00, 18.82, 10.33; m/z=450.28 [M+H]⁺; HPLC

97.3 a/a % at 215 nm.

Preparation of Compound Included in Scheme 5. Compound

38. A solution of hydroxymethylene 30 (300 mg, 0.510 mmol,

1 equiv) in dioxane (5 mL) and water (0.5 mL) was treated with a

solution of sodium acetate (42 mg, 0.510 mmol, 1 equiv) in acetic

acid (31 mg, 0.510 mmol, 1 equiv) and N-bromosuccinimide

(95 mg, 0.536 mmol, 1.05 equiv). The mixture was stirred at

room temperature for 16 h and partitioned between ethyl acetate

and aqueous sodium sulfite. The organic phase was separated,

washed with water, washed with saturated aqueous sodium

chloride, dried over sodium sulfate, and concentrated to dry-

ness. A solution of the crude residue in ethanol (5 mL) and

sodium methoxide (0.05 mL) was stirred for 40 min and parti-

tioned between ethyl acetate and water. The organic phase was

separated, washed with water, washed with saturated aqueous

sodium chloride, dried over sodium sulfate, and concentrated to

dryness. The residue was purified by silica gel chromatography

(5-30% EtOAc/hexanes) to give the desired bromoketone 37

(159 mg, 0.249 mmol, 49%). NMR δ_H(400 MHz, CDCl₃) 7.39-

7.30 (m, 5H), 5.15 (d, J=12.6 Hz, 1H), 5.11 (d, J=12.7, 1H), 4.82

(dd, J=13.8, 6.4 Hz, 1H), 3.82 (dd, J=12.8, 4.0 Hz, 1H), 3.63 (dt,

J=11.1, 3.7 Hz, 1H), 3.10 (dd, J=10.2, 6.4 Hz, 1H), 2.81 (t, J=

13.8, 1H), 2.69-2.16 (m, 7H), 2.14-1.08 (m, 20H), 1.00-0.82

(m, 9H).

1H), 2.12-1.00 (m, 24H), 1.04 (s, 3H), 0.91-0.89 (m, 6H); NMR

δ_C(100 MHz, CDCl₃) 162.66, 148.34, 140.53, 126.43, 124.59,

82.86, 64.09, 54.68, 49.95, 49.88, 46.65, 44.60, 42.52, 40.24,

39.01, 37.24, 36.19, 35.20, 31.48, 30.95, 29.85, 28.16, 27.83,

26.96, 25.48, 24.01, 21.94, 19.26, 19.02, 10.39; m/z = 481.23

[M+H]⁺; HPLC 97.7 a/a % at 215 nm.

Compound 40. A solution of ketone 30 (500 mg, 0.851 mmol,

1 equiv) in dichloromethane (5 mL), acetic acid (20 mL), and

water (2.5 mL) was cooled to 0 °C. The mixture was treated with

sodium nitrite (59 mg, 0.851 mmol, 1 equiv) and stirred for

30 min. The reaction mixture was concentrated to dryness and

purified by silica gel chromatography (50% EtOAc/hexanes) to

give the desired oxime 39 (120 mg, 0.204 mmol, 24%).

A solution of compound 39 (120 mg, 0.204 mmol, 1 equiv) in

ethanol (5 mL) and water (0.25 mL) was treated with sodium

acetate (50 mg, 0.611 mmol, 3 equiv) and hydroxylamine

hydrochloride (43 mg, 0.611 mmol, 3 equiv). The mixture was

stirred for 3 h at room temperature and partitioned between

ethyl acetate and water. The organic layer was separated, dried

over sodium sulfate, and concentrated to dryness. The crude

product was purified by silica gel chromatography (20-50%

EtOAc/hexanes). A solution of the isolated product in dioxane

(2 mL) and ethylene glycol (3 mL) was placed in a sealed tube

and treated with potassium hydroxide (1 pellet). The mixture

was heated to 120 °C for 90 min and partitioned between ethyl

acetate and water. The organic layer was separated, dried over

sodium sulfate, and concentrated to dryness. The crude product

was purified by silica gel chromatography (0.5/1/98.5 to 0.5/10/

89.5 NH₄OH/DCM/MeOH). A suspension of the isolated

product and 10% palladium on carbon (30 mg) in ethanol

(2.5 mL) and ethyl acetate (2.5 mL) was placed under hydrogen

atmosphere and stirred for 2 h at room temperature. The

reaction mixture was filtered on Celite and washed with ethanol

and the filtrate concentrated to dryness. The residue was

purified by silica gel chromatography (0.5-6% MeOH/DCM)

to give the desired compound 40 (11 mg, 0.024 mmol, 12% for

three steps). NMR δ_H(400 MHz, CDCl₃) 3.36 (dt, J =10.1,

4.0 Hz, 1H), 3.11-2.89 (m, 3H), 2.68 (dd, J=9.8, 7.4 Hz, 1H),

2.45 (d, J=17.2 Hz, 1H), 2.38 (br d, J=14.0 Hz, 1H), 2.32 (t, J=

11.7 Hz, 1H), 2.22 (td, J=11.3, 3.2 Hz, 1H), 2.12 (dd, J=14.5,

6.2 Hz, 1H), 2.07-1.16 (m, 20H), 1.14 (s, 3H), 1.11-0.97 (m,

2H), 0.94-0.89 (m, 6H); NMR δ_C(100 MHz, CDCl₃) 51.36,

151.30, 139.31, 125.42, 82.66, 64.14, 54.82, 49.87, 49.66, 46.55,

44.74, 43.02, 40.27, 38.24, 37.27, 36.02, 31.66, 31.63, 31.39,

30.52, 27.86, 26.76, 24.82, 24.01, 22.00, 20.68, 19.04, 10.35;

m/z=452.29 [M+H]⁺; HPLC 100.0 a/a % at 215 nm.

Compound 41. A solution of hydroxymethylene 30 (200 mg,

0.340 mmol, 1 equiv) in pyridine (4.5 mL) was heated to 110 °C.

After 5 min, the mixture was treated with a solution of hydro-

xylamine hydrochloride (71 mg, 1.02 mmol, 3 equiv) in water

(1.2 mL) and stirred for 4 h, cooled to room temperature, and

partitioned between water and dichloromethane. The organic

phase was separated, washed with 2 N aqueous hydrochloric

acid, washed with saturated aqueous sodium chloride, dried

with sodium sulfate, and concentrated to an oil. The residue was

purified by silica gel chromatography (10-40% Et₂O/hexanes).

A suspension of the isolated product and 10% palladium on

carbon (25 mg) in ethyl acetate (3 mL) was placed under

hydrogen atmosphere and stirred for 2 h at room temperature.

The reaction mixture was then filtered on Celite and the filtrate

concentrated to dryness. The residue was then purified using

silica gel chromatography (0.5/1/98.5 to 0.5/10/89.5 NH₄-

OH/DCM/MeOH) to afford the desired isoxazole 41 (37 mg,

0.082 mmol, 24% for two steps). NMR δ_H(400 MHz, CDCl₃)

8.06 (br s, 1H), 3.34 (dt, J=10.2, 3.5 Hz, 1H), 3.04 (dd, J=12.6,

4.3 Hz, 1H), 2.85 (dd, J=18.2, 11.4 Hz, 1H), 2.90-2.61 (m, 4H),

2.38 (br d, J=14.6 Hz, 1H), 2.30 (t, J=12.2 Hz, 1H), 2.26-2.18

(m, 1H), 2.10-1.00 (m, 25H), 1.05 (s, 3H), 0.97 (d, J=6.6 Hz,

3H), 0.89 (d, J = 7.3 Hz, 3H); NMR δ_C(100 MHz, CDCl₃)

159.95, 153.06, 140.03, 124.81, 113.75, 82.74, 64.13, 54.79, 49.82,

A solution of bromoketone 37 (97 mg, 0.152 mmol, 1 equiv) in

absolute ethanol (1 mL) was treated with thioacetamide (53 mg,

0.70 mmol, 4.6 equiv) and refluxed for 3 h. The reaction mixture

was cooled to room temperature and partitioned between water

and ethyl acetate. The organic phase was separated, washed with

saturated aqueous sodium chloride, dried over sodium sulfate,

and concentrated to dryness. The residue was purified by silica

gel chromatography (5-35% EtOAc/hexanes). A suspension of

the isolated product and 10% palladium on carbon (8 mg) in

ethanol (6 mL) was placed under hydrogen atmosphere and

stirred for 3 h at room temperature. The reaction mixture was

filtered on Celite and the filtrate concentrated to dryness. The

residue was purified using silica gel flash chromatography (5%

DCM/MeOH) to afford the desired product (16 mg, 0.034

mmol, 22% for two steps). NMR δ_H(400 MHz, CDCl₃) 3.34

(dt, J=10.6, 4.0 Hz, 1H), 3.04 (dd, J=12.8, 4.0 Hz, 1H), 2.84 (br

dd, J=17.7, 10.6 Hz, 1H), 2.71 (t, J=16.8, 1H), 2.68-2.62 (m,

1H), 2.61 (s, 3H), 2.48 (br d, J=16.8 Hz, 1H), 2.37 (br d, J=

14.0 Hz, 1H), 2.29 (t, J=11.8 Hz, 1H), 2.22 (td, J=11.5, 3.8 Hz,

4414 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14

Tremblay et al.

49.75, 46.50, 44.67, 42.33, 40.27, 38.40, 37.22, 35.27, 31.64, 31.35,

30.66, 30.57, 27.83, 27.04, 24.96, 23.96, 22.00, 21.65, 19.01, 10.30;

m/z=451.27 [M+H]⁺; HPLC 98.4 a/a % at215 nm.

δ

_H(400 MHz, CDCl₃) 7.43 (s, 1H), 7.38-7.30 (m, 5H), 5.15 (d,

J=12.6 Hz, 1H), 5.11 (d, J=12.7 Hz, 1H), 3.83 (dd, J=12.9,

4.3 Hz, 1H), 3.81 (s, 3H), 3.63 (dt, J=10.8, 3.8 Hz, 1H), 3.09 (dd,

J=9.4, 6.2 Hz, 1H), 2.80 (t, J=15.6 Hz, 1H), 2.64 (dd, J=12.8,

10.3 Hz, 1H), 2.60 (t, J=6.2 Hz, 1H), 2.43 (br d, J=14.2 Hz, 1H),

2.30-2.10 (m, 5H), 2.06-1.22 (m, 17H), 1.18 (s, 3H), 0.98 (d,

J=6.8 Hz, 3H), 0.91 (d, J=6.8 Hz, 3H); m/z=638.41 [M+H]⁺.

A suspension of compound 46 (35 mg, 0.057 mmol, 1 equiv)

and 10% palladium on carbon (7 mg) in ethanol (3 mL) was

placed under hydrogen atmosphere and was stirred for 16 h at

room temperature. The mixture was treated with hydrazine

hydrate (12 mg, 0.223 mmol, 4 equiv), heated to 80 °C, and

stirred for 4 h. The mixture was cooled to room temperature and

concentrated to a yellow oil. The residue was purified by silica

gel chromatography (10% MeOH/DCM) to give the desired

pyrazolone 47 (14 mg, 0.023 mmol, 40%). NMR δ_H(400 MHz,

CDCl₃) 8.15 (br s, 1H), 3.36 (dt, J=10.0, 4.0 Hz, 1H), 3.10-3.00

(m, 2H), 2.73-2.60 (m, 2H), 2.51-1.20 (m, 28H), 0.96 (s, 3H),

0.94-0.89 (m, 6H); m/z=466.30 [M+H]⁺; HPLC 89.2 a/a % at

215 nm.

Compound 48. A solution of compound 30 (100 mg,

0.170 mmol, 1 equiv) in ethanol (2 mL) was treated with methylhy-

drazine (16 mg, 0.340 mmol, 2 equiv), heated to 80 °C, and stirred

for 2 h. The mixture was cooled to room temperature and

concentrated to a yellow oil. The residue was purified by silica

gel chromatography (20-50% EtOAc/hexanes). A suspension of

the isolated product and 10% palladium on carbon (10 mg) in ethyl

acetate (3 mL) was placed under hydrogen atmosphere and stirred

for 2 h at room temperature. The reaction mixture was filtered on

Celite and the filtrate concentrated to dryness. The residue was

purified using silica gel flash chromatography (0.5/1/98.5 to 0.5/14/

85.5 NH₄OH/DCM/MeOH) to afford a mixture of methylpyr-

azole (34 mg, 0.073 mmol, 43% for two steps, 7:3 mixture of

regioisomer). NMR δ_H(400 MHz, CDCl₃) 7.00 (br s, 1H) 3.82 (s,

3H minor isomer), 3.80 (s, 3H major isomer), 3.35 (dt, J=10.2,

3.9 Hz, 1H), 3.05 (dd, J=12.7, 4.1 Hz, 1H), 2.84-2.73 (m, 1H),

2.71-2.61 (m, 1H), 2.61-2.50 (m, 2H), 2.48-2.11 (m, 4H), 2.09-

1.22 (m, 21H) 1.11-0.97 (m, 1H), 1.02 (s, 3H) 0.90 (d, J=11.1 Hz,

6H); NMR δ_C(100 MHz, CDCl₃) 148.17, 140.55, 127.19,

124.21, 114.70, 82.88, 76.72, 64.12, 54.71, 49.95, 49.87, 46.59,

44.62, 42.25, 40.27, 39.21, 38.66, 37.25, 35.42, 32.78, 31.58, 30.83,

27.82, 27.27, 25.32, 23.98, 23.94, 22.24, 19.02, 10.34; m/z=464.27

[M+H]⁺; HPLC 100 a/a % at 215 nm (71.9/28.1 regioisomers

mixture).

Preparation of Compound Included in Scheme 6. Compound

44. A solution of ketone 5 (500 mg, 0.893 mmol, 1 equiv) in

tert-butanol (5 mL) and potassium tert-butoxide (702 mg,

6.25 mmol, 7 equiv) was treated with ethyl trifluoracetate

(761 mg, 5.36 mmol, 6 equiv) dropwise. The mixture was stirred

at room temperature for 48 h and partitioned between diethyl

ether and 1 N aqueous sodium hydroxide. The organic phase

was separated, washed with 1 N aqueous sodium hydroxide,

dried with sodium sulfate, and concentrated to desired com-

pound 42 (550 mg, 0.839 mmol, 94%) as a pale-yellow foam. The

residue was used without further purification.

A solution of crude compound 42 (210 mg, 0.320 mmol,

1 equiv) in ethanol (6 mL) was treated with hydrazine hydrate

(32 mg, 0.640 mmol, 2 equiv) and heated to 80 °C for 3 h. The

mixture was cooled to room temperature and concentrated to a

yellow oil. The residue was purified by silica gel chromatogra-

phy (20-55% EtOAc/hexanes) to give the desired pyrazole 43

(95 mg, 0.146 mmol, 46%). NMR δ_H(400 MHz, CDCl₃) 7.37-

7.29 (m, 5H), 5.15 (d, J=12.6 Hz, 1H), 5.11 (d, J=12.7 Hz, 1H),

3.82 (dd, J=13.2, 4.3 Hz, 1H), 3.62 (dt, J=10.8, 4.6 Hz, 1H),

3.08 (dd, J=10.2, 6.2 Hz, 1H), 2.86-2.54 (m, 4H), 2.38 (br d, J=

13.8 Hz, 1H), 2.32 (br d, J=16.1 Hz, 1H), 2.19 (td, J=11.9,

4.2 Hz, 1H), 2.12-1.13 (m, 22H), 1.08 (s, 3H), 0.97 (d, J=6.9 Hz,

3H), 0.90 (d, J=7.3 Hz, 3H).

A suspension of pyrazole 43 (95 mg, 0.146 mmol, 1 equiv) and

10% palladium on carbon (30 mg) in ethyl acetate (6 mL) was

placed under hydrogen atmosphere and stirred for 2 h at room

temperature. The reaction mixture was then filtered on Celite

and the filtrate concentrated to dryness. The residue was then

purified using silica gel chromatography (0.5/1/98.5 to 0.5/14/

85.5 NH₄OH/DCM/MeOH) to afford the desired trifluoro-

methylpyrazole 44 (42 mg, 0.081 mmol, 56%). NMR δ_H

(400 MHz, CDCl₃) 3.36 (dt, J = 10.2, 3.8 Hz, 1H), 3.05 (dd,

J=12.9, 3.7 Hz, 1H), 2.81-2.63 (m, 3H), 2.55 (dd, J=17.2,

7.1 Hz, 1H), 2.36 (br d, J=14.7 Hz, 1H), 2.30 (t, J=8.9 Hz, 1H),

2.25-2.17 (m, 2H), 2.10-1.00 (m, 23H), 0.91 (s, 3H), 0.89

(s, 3H); NMR δ_C(100 MHz, CDCl₃) 140.35, 140.07, 124.72,

112.87, 82.79, 76.68, 63.70, 54.52, 49.96, 49.92, 46.48, 44.63,

42.43, 40.17, 38.33, 37.26, 35.35, 31.79, 31.53, 31.33, 30.77,

27.79, 26.93, 25.39, 24.00, 22.00, 21.96, 18.97, 10.37; m/z =

518.28 [M+H]⁺; HPLC 97.8 a/a % at215 nm.

Compound 47. A solution of compound 30 (360 mg, 0.612

mmol, 1 equiv) in toluene (10 mL) was treated with DDQ (153

mg, 0.674 mmol, 1.1 equiv) and stirred at room temperature for

5 min. The mixture was concentrated to dryness and purified by

silica gel chromatography (10-20% EtOAc/hexanes) to give the

desired compound 45 (244 mg, 0.417 mmol, 68%). NMR δ_H

(400 MHz, CDCl₃) 10.08 (s, 1H), 7.54 (s, 1H), 7.38-7.29 (m,

5H), 5.15 (d, J=12.6 Hz, 1H), 5.11 (d, J=12.7 Hz, 1H), 3.82 (dd,

J=13.3, 4.4 Hz, 1H), 3.62 (dt, J=10.8, 3.8 Hz, 1H), 3.08 (dd, J=

10.3, 6.8 Hz, 1H), 2.81 (dd, J=16.6, 15.1 Hz, 1H), 2.64 (dd, J=

11.8, 10.2 Hz, 1H), 2.59 (t, J=6.2 Hz, 1H), 2.42 (br d, J=14.2

Hz, 1H), 2.27-2.10 (m, 5H), 2.05-1.22 (m, 17H), 1.21 (s, 3H),

0.98 (d, J=6.8 Hz, 3H), 0.91 (d, J=6.8 Hz, 3H); m/z=558.48

[M+H]⁺.

A solution of compound 45 (123 mg, 0.210 mmol, 1 equiv)

in N,N-dimethylformamide (6 mL) was treated with methanol

(67 mg, 2.10 mmol, 10 equiv) and PDC (474 mg, 1.26 mmol,

6 equiv). The mixture was stirred for 4 days at room temperature

(∼50% conversion). The reaction mixture was partitioned

between water and ethyl acetate, filtered on Celite, and washed

with ethyl acetate. The filtrate was separated and the aqueous

layer back-extracted with ethyl acetate. The combined organics

were washed with 1 N aqueous hydrochloric acid, dried with

sodium sulfate, and concentrated to dryness. The residue was

purified by silica gel chromatography (80% EtOAc/hexanes)

to give the desired ester 46 (36 mg, 0.059 mmol, 28%). NMR

Compound 49. A solution of pyrazole 31 (200 mg, 0.346 mmol,

1 equiv) in pyridine (4 mL) was treated with methanesulfonyl

chloride (117 mg, 1.03 mmol, 3 equiv) and stirred at room

temperature for 30 min. The reaction mixture was partitioned

between water and ethyl acetate. The organic layer was sepa-

rated, washed with saturated aqueous sodium chloride, dried

over sodium sulfate, and concentrated to dryness. The residue

was purified by silica gel chromatography (10-20% EtOAc/

hexanes). A suspension of the isolated product and 10% palla-

dium on carbon (81 mg) in ethyl acetate (15 mL) was placed

under hydrogen atmosphere and stirred for 1.5 h at room

temperature. The reaction mixture was then filtered on Celite

and the filtrate concentrated to dryness. The residue was

purified using silica gel chromatography (0.5/1/98.5 to 0.5/10/

89.5 NH₄OH/DCM/MeOH) to afford the desired methanesul-

fonylpyrazole 49 (78 mg, 0.148 mmol, 51% for two steps). NMR

δ_H(400 MHz, CDCl₃) 7.64 (s, 1H), 3.32 (dt, J=10.0, 3.8 Hz,

1H), 3.25 (s, 3H), 3.02 (dd, J = 12.7, 3.9 Hz, 1H), 2.82 (dd,

J=18.6, 11.0 Hz, 1H), 2.69-2.58 (m, 3H), 2.36 (br d, J=14.2 Hz,

1H), 2.27 (t, J=11.9 Hz, 1H), 2.24-2.15 (m, 2H), 2.10-0.98

(m, 25H), 0.89 (s, 3H), 0.87 (s, 3H); NMR δ_C(100 MHz,

CDCl₃) 156.04, 140.20, 127.24, 124.83, 118.67, 82.73, 64.11,

54.76, 49.82, 49.77, 46.57, 44.64, 42.41, 41.40, 40.25, 38.65,

37.23, 35.27, 32.43, 31.60, 31.34, 30.63, 27.83, 27.06, 25.04,

24.07, 23.98, 22.06, 19.01, 10.30; m/z=528.30 [M+H]⁺; HPLC

100.0 a/a % at 215 nm.

Article

Compound 50. A solution of pyrazole 30 (140 mg, 0.240 mmol,

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4415

hydrochloric acid (1.19 g, 32.7 mmol, 5 equiv). The resulting

mixture was heated to 60 °C and stirred for 16 h. The mixture

was cooled to room temperature and partitioned between

saturated aqueous bicarbonate and toluene. The organic phase

was separated and concentrated to a brown foam. The residue

was crystallized from ethyl acetate to give the desired lactam 53

as a tan solid (14.35 g, 25 mmol, 61%). NMR δ_H(400 MHz,

CDCl₃) 7.39-7.31 (m, 5H), 6.33 (br s, 1H), 6.10 (d, J=13.3 Hz,

1H), 5.70 (d, J=13.6 Hz, 1H), 5.16 (d, J=12.2 Hz, 1H), 5.12 (d,

J=12.3, 1H), 3.83 (dd, J=12.6, 4.2 Hz, 1H), 3.71-3.59 (m, 2H),

3.09 (dd, J=10.6, 6.4 Hz, 1H), 2.85 (dd, J=14.3, 7.7 Hz, 1H),

2.68-2.57 (m, 2H), 2.44 (br d, J = 15.8 Hz, 1H), 2.39 (dd,

J=14.3, 6 Hz, 1H), 2.23-2.10 (m, 2H), 2.07-1.18 (m, 18H),

1.07 (s, 3H), 0.98 (d, J=6.8 Hz, 3H), 0.91 (d, J=7 Hz, 3H);

m/z=573.45 [M+H]⁺.

A suspension of lactam 53 (12.9 g, 22.5 mmol, 1 equiv) and

10% palladium on carbon (2.5 g) in 2-propanol (195 mL) was

placed under hydrogen atmosphere and stirred for 5 h at room

temperature. The reaction mixture was treated with ethylene-

diamine (1.35 g, 22.5 mmol, 1 equiv) and stirred for 20 min. The

resulting mixture was filtered on Celite and the cake washed with

2-propanol. The filtrate was concentrated until solid started to

separate. Ethyl acetate was added and the mixture further

concentrated to 10% of the original reaction volume. The

precipitate was collected by filtration and air-dried to yield the

desired lactam 55 (8.5 g, 19.2 mmol, 86%) as a white solid. NMR

δ_H(400 MHz, CDCl₃) 7.04-6.95 (br s, 1H), 3.66 (dd, J =

11.0, 3.7 Hz, 1H), 3.27 (dt, J=10.7, 3.8 Hz, 1H), 2.97 (dd, J=

12.9, 4.0 Hz, 1H), 2.71-2.51 (m, 3H), 2.37 (br d, J=13.6 Hz,

1H), 2.23 (t, J=13.6 Hz, 1H), 2.18-2.02 (br m, 3H), 1.99-1.81

(m, 5H), 1.80-1.14 (m, 18H), 0.87 (s, 3H), 0.85-0.80 (m, 6H);

NMR δ_C(100 MHz, CDCl₃) 178.89, 139.91, 124.82, 82.46,

64.02, 54.00, 49.75, 49.35, 46.72, 44.73, 44.56, 44.02, 43.03,

40.13, 37.15, 37.11, 34.84, 31.53, 30.22, 30.20, 29.07, 27.69,

26.61, 25.37, 23.90, 22.71, 18.89, 10.16; m/z = 441.29 [M +

H]⁺; HPLC 95.0 a/a % at 215 nm.

Compound 56. A solution of lactam 53 (50 mg, 0.090 mmol,

1 equiv) in anhydrous tetrahydrofuran (5 mL) was cooled

to -78 °C and treated with 0.5 M potassium hexamethyldisila-

zane in toluene (0.215 mL, 0.107 mmol, 1.2 equiv) and methyl

iodide (25.4 mg, 0.179 mmol, 2 equiv). The solution was stirred

for 30 min, warmed to room temperature, and stirred for 18 h.

The reaction mixture was partitioned between ethyl acetate and

water. The organic phase was separated, dried with sodium

sulfate, and concentrated to dryness. The residue was purified

by silica gel chromatography (20-50% EtOAc/hexanes) to give

the desired lactam 54 (50 mg, 0.089 mmol, 99%). NMR δ_H

(400 MHz, CDCl₃) 7.39-7.30 (m, 5H), 6.02 (d, J=13.2, 1H),

5.84 (d, J=12.9, 1H), 5.16 (d, J=12.6 Hz, 1H), 5.12 (d, J=12.7,

1H), 3.92-3.80 (m, 2H), 3.63 (dd, J=10.4, 4.0 Hz, 1H), 3.11-

3.06 (m, 1H), 3.08 (s, 3H), 2.85 (d, J=14.7, 1H), 2.68-2.57 (m,

2H), 2.44 (br d, J=14.7, 1H), 2.39 (dd, J=14.4, 6.1 Hz, 1H),

2.23-1.17 (m, 20H) 1.03 (s, 3H), 0.98 (d, J=6.8 Hz, 3H), 0.91

(d, J=6.9 Hz, 3H); m/z=587.44 [M+H]⁺.

A suspension of lactam 54 (50 mg, 0.89 mmol, 1 equiv) and 10%

palladium on carbon (10 mg) in ethanol (3 mL) was placed under

hydrogen atmosphere and stirred for 4 h at room temperature. The

reaction mixture was filtered on Celite and washed with ethanol

and the filtrate concentrated to dryness. The residue was purified

by silica gel chromatography (8% MeOH/DCM) to give the

desired lactam 56 (25 mg, 0.055 mmol, 65%). NMR δ_H(400

MHz, CDCl₃)4.21(t, J=7.7 Hz, 1H), 4.05(dd, J=7.3, 4.4Hz, 1H),

3.36 (dt, J=9.9, 3.9 Hz, 1H), 3.06 (dd, J=12.0, 4.3 Hz, 1H), 2.94

(s, 3H), 2.74-2.62 (m, 3H), 2.45 (d, J=13.8 Hz, 1H), 2.31 (t, J=

13.8 Hz, 1H), 2.26-2.10 (br m, 3H), 2.07-1.23 (m, 18H), 1.13-

1.00 (m, 1H), 0.99-0.82 (m, 12H); NMR δ_C(100 MHz, CDCl₃)

175.79, 140.09, 125.09, 82.72, 64.18, 54.83, 53.17, 49.95, 49.57,

46.89, 44.73, 43.17, 40.30, 38.86, 37.30, 36.87, 35.67, 35.36, 31.68,

31.38, 31.09, 30.51, 29.95, 27.90, 26.77, 24.09, 23.20, 19.04, 10.35;

m/z=455.30 [M+H]⁺; HPLC 97.0 a/a % at 215 nm.

1 equiv) in dichloromethane (3 mL) was treated with pyridine

(95 mg, 1.2 mmol, 5 equiv) and p-toluenesulfonyl chloride

(55 mg, 0.288 mmol, 1.2 equiv). The mixture was stirred at

room temperature for 2 h and partitioned between water and

ethyl acetate. The organic layer was washed with saturated

aqueous sodium chloride, dried with sodium sulfate, and con-

centrated to dryness. The residue was purified by silica gel

chromatography (5-30% EtOAc/hexanes). A suspension of

the isolated product and 10% palladium on carbon (14 mg) in

ethanol (2 mL) and ethyl acetate (2 mL) was placed under

hydrogen atmosphere and stirred for 1.5 h at room temperature.

The reaction mixture was then filtered on Celite and the filtrate

concentrated to dryness. The residue was then purified using

silica gel chromatography (0.5/1/98.5 to 0.5/14/85.5 NH₄OH/

DCM/MeOH) to afford the desired p-toluenesulfonylpyrazole

50 (64 mg, 0.106 mmol, 45% for two steps, 88/12 regioisomer

ratio). NMR δ_H(400 MHz, CDCl₃) 7.88 (d, J=8.1 Hz, 2H),

7.72 (br s, 1H), 7.31 (d, J=8.4 Hz, 2H), 3.35 (dt, J=10.5, 3.8 Hz,

1H), 3.05 (dd, J=12.6, 3.8 Hz, 1H), 2.77 (dd, J=17.9, 11.3 Hz,

1H), 2.67 (dd, J=9.6, 7.7 Hz, 1H), 2.63-2.53 (m, 2H), 2.41 (br s,

3H), 2.40-2.18 (m, 3H), 2.07-1.03 (m, 23H), 1.01 (s, 3H), 0.93-

0.88 (m, 6H); NMR δ_C(100 MHz, CDCl₃) 156.04, 140.20,

127.24, 124.83, 118.67, 82.73, 64.11, 54.76, 49.82, 49.77, 46.57,

44.64, 42.41, 41.41, 40.25, 38.65, 37.23, 35.27, 32.43, 31.60,

31.34, 30.63, 27.83, 27.06, 25.04, 24.07, 23.98, 22.06, 19.01,

10.30; m/z = 604.43 [M + H]⁺; HPLC 99.4 a/a % at 215 nm

(82/18 epimer ratio).

Preparation of Compound Included in Scheme 7. Compound

55. A solution of compound 45 (500 mg, 0.854 mmol, 1 equiv) in

toluene (25 mL) was treated with Wilkinson’s catalyst (806 mg,

0.871 mmol, 1.02 equiv). The mixture was stirred at 80 °C for

30 min and cooled to room temperature. The solvent was

removed under vacuum and the residue was purified by silica

gel chromatography (10-15% EtOAc/hexanes) to give the

desired enone 51 (306 mg, 0.549 mmol, 64%). NMR δ_H

(400 MHz, CDCl₃) 7.37-7.30 (m, 5H), 6.73 (d, J =10.1 Hz,

1H), 5.88 (d, J=10.1 Hz, 1H), 5.15 (d, J=12.2 Hz, 1H), 5.11 (d,

J=12.3, 1H), 3.82 (dd, J=13.0, 4.1 Hz, 1H), 3.62 (dt, J=10.8,

4.1 Hz, 1H), 3.08 (dd, J=10.4, 6 Hz, 1H), 2.75-2.54 (m, 3H),

2.43 (d, J = 14.1 Hz, 1H), 2.25-1.14 (m, 22H), 1.10 (s, 3H),

0.97 (d, J=6.7 Hz, 3H), 0.90 (d, J=7.1 Hz, 3H); m/z=580.46

[M+Na]⁺.

A solution of enone 51 (42.3 g, 76 mmol, 1 equiv) in ethanol

(420 mL was treated with sodium acetate (9.33 g, 114 mmol,

1.5 equiv) and hydroxylamine hydrochloride (7.91 g, 114 mmol,

1.5 equiv). The mixture was stirred at room temperature for 3 h

and partitioned between ethyl acetate and water. The organic

layer was separated, dried over sodium sulfate, and concen-

trated to dryness. The residue was purified by silica gel chro-

matography (20-50% EtOAc/hexanes) to give the desired

oxime 52 (43 g, 76 mmol, 100%) as a mixture of regioisomers

(2/1 anti/syn). NMR δ_H(400 MHz, CDCl₃) 7.37-7.29 (m, 5H),

1

6.66 (d, J=10.3 Hz, 1/3H minor), 6.12 (d, J=10.6 Hz, /₃H

minor), 6.01 (dd, J=10.6 Hz, ²/₃H major), 5.97 (d, J=10.6 Hz,

²/₃H major), 5.15 (d, J=12.2 Hz, 1H), 5.11 (d, J=12.3, 1H), 3.82

(dd, J=13, 4.1 Hz, 1H), 3.62 (dt, J=10.6, 4.2 Hz, 1H), 3.08 (dd,

J=10.4, 6.3 Hz, 1H), 2.84-2.53 (m, 3H), 2.46-2.36 (m, 2H),

2.23-1.18 (m, 22H), 1.03 (s, 3H), 0.97 (d, J=6.7 Hz, 3H), 0.90

(d, J=7.1 Hz, 3H); m/z=573.56 [M+H]⁺.

A solution of oxime 52 (5.3 g, 9.25 mmol, 1 equiv) in pyridine

(100 mL) was cooled to 5 °C and treated with methanesulfonyl

chloride (2.12 g, 18.5 mmol, 2 equiv). The solution was stirred

for 1 h, partitioned between water and ethyl acetate, and

warmed to room temperature. The organic phase was separated

and concentrated under vacuum. The residue was purified

by silica gel chromatography (15-20% EtOAc/hexanes) to give

the desired intermediate (5.43 g, 8.34 mmol, 90%). A solution

of the intermediate (4.26 g, 6.55 mmol, 1 equiv) in methanol

(125 mL) and toluene (12.5 mL) was treated with concentrated

4416 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14

Tremblay et al.

Hh-Dependent C3H10T1/2 Differentiation Assay. The assay

was performed according to the procedure described by Dwyer

and co-workers.⁴⁶Briefly, mouse embryonic mesoderm fibro-

blasts C3H10T1/2 cells (obtained from ATCC catalogue no.

CCL-226) were cultured in basal MEM medium (Gibco/Invi-

trogen) supplemented with 10% heat inactivated FBS (Hy-

clone), 50 units/mL penicillin, 50 μg/mL streptomycin (Gibco/

Invitrogen), and 2 mM glutamine (Gibco/Invitrogen) at 37 °C

with 5% CO₂in air atmosphere. Cells were dissociated with

0.05% trypsin and 0.02% EDTA in PBS for passage and

plating. C3H10T1/2 cells were plated in 96 wells with a density

of 8 ꢀ 10³cells/well. Cells were grown to confluence (72 h).

Medium containing 5 μM of 20(S)-hydroxycholesterol and

5 μM of 22(S)-hydroxycholesterol and/or compound was added

at the start of the assay and left for 72 h. The medium was

aspirated, and cells were washed once in PBS. Then 100 μL

of Tropix CDP-Star with Emerald II (0.4 mM, catalogue

no. MS100RY) was added per well and the plate was incubated

at room temperature in the dark for 1 h. The plates were

read for fluorometric measurement on an Envision plate reader

at 405 nm. The percent inhibition with respect to com-

pound concentration was plotted using Prism graphing software

on a semilog plot, and EC₅₀values were determined by

nonlinear regression analysis with a four-parameter logistic

equation.

In Vitro Metabolism Assay. Test compounds and controls

(alprenolol and acebutolol) were tested at 500 nM in pooled

human liver microsomes (BD Biosciences, Woburn, MA). An

incubation solution was made in 85 mM phosphate buffer

containing 1 mM EDTA, 3 mM MgCl₂, and 0.5 mg/mL human

liver microsomes. Compounds were added to a clear flat bottom

96-well plate (Costar, Corning, NY) using a Platemate Plus

liquid handler (Matrix). Reaction solution was preincubated at

37 °C for 5 min. Reaction mixture was added to each 96-well

plate. To start the reaction, NADPH was added to each plate at

a final concentration of 1 mg/mL. Reaction plates were incu-

bated at 37 °C. Reaction was stopped at designated time points

(0, 5, 15, 30, 45, and 60 min) by adding 2 volumes of acetonitrile

containing internal standard (jervine, AG Scientific). Samples

were placed in plate centrifuge (Eppendorf) and spun at 1000 rcf

for 10 min. The supernatant was taken and diluted with

1 volume of water in a 96-well autosampler plate with inert

glass inserts (MicroLiter). Samples were analyzed by LC-MS.

A 2.1 mm ꢀ 20 mm, 3.5 μm Waters C18 Synergy column

(Milford, MA) was used, and samples were analyzed using an

Applied Biosystems 4000QTrap mass spectrometer. Half-lives

were determined by the slope of the line for the percent remain-

ing parent compound over time course.

(Foster City, CA) with an API4000 mass spectrometer from

Applied Biosystems (Foster City, CA). Samples were injected

on an analytical column (Symmetry IS, 2.1 mm ꢀ 20 mm,

C18, 3.5 μm from Waters, Milford, MA) and compounds

eluted from the analytical column with a 5 min gradient from

0 to 95% acetonitrile in H₂O, 0.1% (v/v) formic acid. Mass

spectrometric detection of the drug as well as jervine as the

internal standard was performed by MRM in positive mode.

The data were acquired and processed using the software

Analyst 1.4 (Applied Biosystems, Foster City, CA). The phar-

macokinetics of each compound were analyzed by WinNonlin

5.0.1 (Pharsight, Mountain View, CA) via noncompartmental

analysis.

Efficacy Studies in B837Tx-Bearing Nude Mice. The antitu-

mor activity of cyclopamine and the three lead compounds was

evaluated in mice bearing Ptc^+/-Hic1^+/-medulloblastoma

B837Tx allografts.⁵⁶The B837Tx tumor line originated as

a medulloblastoma in this mouse and is propagated as a

mouse-to-mouse allograft. NOD/SCID mice were inoculated

in the right hind flank with 1 ꢀ 106 B837Tx cells in a 100 μL

injection volume. Once the tumors reached an approximate size

of 100-200 mm³, mice (N=10/group) were dosed orally with

vehicle, 30% hydroxypropyl-β-cyclodextrin (HPBCD), or for-

mulated compounds (28, 40 mg/kg; 32, 80 mg/kg; 55, 30 mg/kg)

daily for 21 days. Tumors were measured in millimeters in two

perpendicular dimensions with calipers 3 times per week using

calipers. In all cases, the tumors regressed with compound

treatment. After 21 days of treatment, mice were monitored

for regrowth of tumors.

Acknowledgment. The authors thank the U.S. Forestry

Services, Joseph McPherson, Charles W. Johannes, and

David Mann for their invaluable help in sourcing Veratrum

californicum. The authors thank Dr. Neil Watkins for the kind

gift of the B837Tx tumor brei. The authors are grateful

to all past and current members of the Hedgehog team at

Infinity Pharmaceuticals, Inc., for preliminary results, scien-

tific guidance, and technical assistance that facilitated this

work.

Supporting Information Available: Physical data (¹H and

¹³C NMR spectra, mass spectrometry, HPLC) for all tested

compounds. This material is available free of charge via the

Internet at http://pubs.acs.org.

References

(1) Ruiz i Altaba, A.; Sanchez, M. P.; Dahmane, N. Gli and Hedgehog

in cancer: tumours, embryos and stem cells. Nat. Rev. Cancer 2002,

2, 361–372.

(2) Lum, L.; Beachy, P. A. The Hedgehog response network: sensors,

switches, and routers. Science 2004, 304, 1755–1759.

Dosing Formulation of Cyclopamine and Compounds 28, 32,

and 55. In the appropriate size vessel, the Hh antagonists (free

bases) were dissolved in a vehicle containing 30% (w/w) of

2-hydroxypropyl-β-cyclodextrin (HPBCD) in 0.1 M citrate

phosphate buffer (pH 3) at a nominal concentration of

1.25 mg/mL for pharmacokinetic studies and at concentration

of 5-10 mg/mL for efficacy studies.

Pharmacokinetic Studies. Dosing. Aqueous formulations of

each compound (vide supra) were administered intravenously or

orally via gavage to CD-1 mice, Sprague-Dawley rats, beagle

dogs, and cynomolgus monkeys. At 0.083, 0.25, 0.5, 1, 2, 4, 6,

and 24 h postdose, blood was collected and processed to plasma

by centrifugation and stored at -80 °C until analysis.

Bioanalytical Analysis. Plasma samples were thawed at room

temperature and processed by protein precipitation with

2 volumes of acetonitrile containing 25 ng/mL jervine (AG

Scientific) as an internal standard. After centrifugation, the

supernatants of the precipitated plasma samples were diluted

1:1 with water and analyzed by LC/MS/MS. A standard curve

was prepared in plasma from 0.5 to 250 ng/mL and processed in

the same way with internal standard solution as the samples.

Sample analysis was performed on an Agilent 1100 series

(3) di Magliano, M. P.; Hebrok, M. Hedgehog signalling in cancer

formation and maintenance. Nat. Rev. Cancer 2003, 3, 903–911.

(4) Beachy, P. A.; Karhadkar, S. S.; Berman, D. M. Tissue repair and

stem cell renewal in carcinogenesis. Nature 2004, 432, 324–331.

(5) Ruiz i Altaba, A. Hedgehog-Gli Signaling in Human Disease;

Landes Bioscience/Eurakah.com: Georgetown, TX, 2006; p 226.

(6) Bale, A. E.; Yu, K.-p. The hedgehog pathway and basal cell

carcinomas. Hum. Mol. Genet. 2001, 10, 757–762.

(7) Dahmane, N.; Lee, J.; Robins, P.; Heller, P.; Ruiz i Altaba, A.

Activation of the transcription factor Gli1 and the Sonic hedgehog

signalling pathway in skin tumours. Nature 1997, 389, 876–881.

(8) Hutchin, M. E.; Kariapper, M. S. T.; Gratchtchouk, M.; Wang, A.;

Wei, L.; Cummings, D.; Liu, J.; Michael, L. R.; Glick, A.; Dlugosz,

A. A. Sustained Hedgehog signaling is required for basal cell

carcinoma proliferation and survival: conditional skin tumorigen-

esis recapitulates the hair growth cycle. Genes Dev. 2004, 19, 214–

224.

(9) Kubo,M.;Nakamura,M.; Tasaki, A.; Yamanaka, N.; Nakashima,

H.; Nomura, M.; Kuroki, S.; Katano, M. Hedgehog signaling

pathway is a new therapeutic target for patients with breast cancer.

Cancer Res. 2004, 64, 6071–6074.

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4417

(10) Berman, D. M.; Karhadkar, S. S.; Maitra, A.; Montes de Oca, R.;

Gerstenblith, M. R.; Briggs, K.; Parker, A. R.; Shimada, Y.;

Eshleman, J. R.; Watkins, D. N.; Beachy, P. A. Widespread

requirement for Hedgehog ligand stimulation in growth of diges-

tive tract tumors. Nature 2003, 425, 846–851.

(11) Goodrich, L. V.; Scott, M. P. Hedgehog and Patched in neural

development and disease. Neuron 1998, 21, 1243–1257.

(12) Thayer, S. P.; diMagliano, M. P.; Heiser, P. W.; Nielsen, C. M.;

Roberts, D. J.; Lauwers, G. Y.; Qi, Y. P.; Gysin, S.; Fernandez-del

Castillo, C.; Yajnik, V.; Antoniu, B.; McMahon, M.; Warshaw, A.

L.; Hebrok, M. Hedgehog is an early and late mediator of

pancreatic cancer tumorigenesis. Nature 2003, 425, 851–856.

(13) Karhadkar, S. S.; Bova, G. S.; Abdallah, N.; Dhara, S.; Gardner,

D.; Maitra, A.; Isaacs, J. T.; Berman, D. M.; Beachy, P. A.

Hedgehog signalling in prostate regeneration, neoplasia and me-

tastasis. Nature 2004, 431, 707–712.

(30) Keeler, R. F. Teratogenic compounds of Veratrum californicum

(Durand)-IV. First isolation of veratramine and alkaloid Q and a

reliable method for isolation of cyclopamine. Phytochemistry 1968,

7, 303–306.

(31) Keeler, R. F. Teratogenic compounds of Veratrum californicum

(Durand)-VI. The structure of cyclopamine. Phytochemistry 1969,

8, 223–225.

(32) Keeler, R. F.; Binns, W. Teratogenic compounds of Veratrum

californicum (Durand)-V. Comparison of cyclopian effects of

steroidal alkaloids from the plant and structurally related com-

pounds from other sources. Teratology 1968, 1, 5–10.

(33) Chen, J. K.; Taipale, J.; Cooper, M. K.; Beachy, P. A. Inhibition of

hedgehog signaling by direct binding of cyclopamine to Smooth-

ened. Genes Dev. 2002, 16, 2743–2748.

(34) Berman, D. M.; Karhadkar, S. S.; Hallahan, A. R.; Pritchard, J. I.;

Eberhart, C. G.; Watkins, D. N.; Chen, J. K.; Cooper, M. K.;

Taipale, J.; Olson, J. M.; Beachy, P. A. Medulloblastoma growth

inhibition by hedgehog pathway blockade. Science 2002, 297,

1559–1561.

(35) Sanchez, P.; Ruiz i Altaba, A. In vivo inhibition of endogenous

brain tumors through systemic interference of Hedgehog signaling

in mice. Mech. Dev. 2005, 122, 223–230.

(14) Watkins, D. N.; Berman, D. M.; Burkholder, S. G.; Wang, B.;

Beachy, P. A.; Baylin, S. B. Hedgehog signalling within airway

epithelial progenitors and in small-cell lung cancer. Nature 2003,

422, 313–317.

(15) Rubin, L. L; de Sauvage, F. J. Target the hedgehog pathway in

cancer. Nat. Rev Cancer 2006, 5, 1026–1033.

(16) Frank-Kamenetsky, M.; Zhang, X. M.; Bottega, S.; Guicherit, O.;

Wichterle, H.; Dudek, H.; Bumcrot, D.; Wang, F. Y.; Jones, S.;

Shulok, J.; Rubin, L. L.; Porter, J. A. Small-molecule modu-

lators of Hedgehog signaling: identification and characteriza-

tion of Smoothened agonists and antagonists. J. Biol. 2002, 1,

10.1–10.19.

(17) Chen, J. K.; Taipale, J.; Young, K. E.; Maiti, T.; Beachy, P. A.

Small molecule modulation of Smoothened activity. Proc. Natl

Acad. Sci. U.S.A. 2002, 99, 14071–14076.

(18) Bijlsma, M. F.; Spek, C. A.; Zivkovic, D.; van de Water, S.; Rezaee,

F.; Peppelenbosch, M. P. Repression of smoothened by patched-

dependent (pro-)vitamin D3 secretion. PLoS Biol. 2006, 4, 1397–

1410.

(19) Remsberg, J. R.; Lou, J.; Tarasov, S. G.; Dean, M.; Tarasova, N. I.

Structural analogues of Smoothened intracellular loops as potent

inhibitors of hedgehog pathway and cancer cell growth. J. Med.

Chem. 2007, 50, 4534–4538.

(20) Brunton, S. A.; Stibbard, J. H. A.; Rubin, L. L.; Kruse, L. I.;

Guicherit, O. M.; Boyd, E. A.; Price, S. Potent inhibitors of the

hedgehog signaling pathway. J. Med. Chem. 2008, 51, 1108–1110.

(21) Peukert, S.; Jain, R.; Geisser, A.; Sun, Y.; Zhang, R.; Bourret, A.;

Carlson, A.; DaSilva, J.; Ramamurthy, A.; Kelleher, J. F. Identi-

fication and structure-activity relationships of ortho-biphenyl

carboxamides as potent Smoothened antagonists inhibiting the

Hedgehog signaling pathway. Bioorg. Med. Chem. Lett. 2009, 19,

328–331.

(22) Lauth, M.; Bergstroem, A.; Shimokawa, T.; Toftgard, R. Inhibi-

tion of GLI-mediated transcription and tumor cell growth by

small-molecule antagonists. Proc. Natl. Acad. Sci. U.S.A. 2007,

104, 8455–8460.

(23) Lee, J.; Wu, X.; diMagliano, M. P.; Peters, E. C.; Wang, Y.; Hong,

J.; Hebrok, M.; Ding, S.; Cho, C. Y.; Schultz, P. G. A Small-

molecule antagonist of the Hedgehog signaling pathway. Chem-

BioChem 2007, 8, 1916–1919.

(24) Arai, M. A.; Tateno, C.; Hosoya, T.; Koyano, T.; Kowithayakorn,

T.; Ishibashi, M. Hedgehog/GLI-mediated transcriptional inhibi-

tors from Zizyphus cambodiana. Bioorg. Med. Chem. 2008, 16,

9420–9424.

(36) Sanchez, P.; Hernandez, A. M.; Stecca, B.; Kahler, A. J.; De-

Gueme, A. M.; Barrett, A.; Beyna, M.; Datta, M. W.; Datta, S.;

Ruiz i Altaba, A. Inhibition of prostate cancer proliferation by

interference with SONIC HEDGEHOG-GLI1 signaling. Proc.

Natl. Acad. Sci. U.S.A. 2004, 101, 12561–12566.

(37) Sheng, T.; Li, C.; Zhang, X.; Chi, S.; He, N.; Chen, K.;

McCormick, F.; Gatalica, Z.; Xie, J. Activation of the hedgehog

pathway in advanced prostate cancer. Mol. Cancer 2004, 3, 29–42.

(38) Zhang, J.; Garrossian, M.; Gardner, D.; Garrossian, A.; Chang,

Y.-T.; Kim, Y. K.; Chang, C. W. T. Synthesis and anticancer

activity studies of cyclopamine derivatives. Bioorg. Med. Chem.

Lett. 2008, 18, 1359–1363.

(39) Kumar, S. K.; Roy, I.; Anchoori, R. K.; Fazli, S.; Maitra, A.;

Beachy, P. A.; Khan, S. R. Targeted inhibition of Hedgehog

signaling by cyclopamine prodrugs for advanced prostate cancer.

Bioorg. Med. Chem. 2008, 16, 2764–2768.

(40) Tremblay, M. R.; Nevalainen, M.; Nair, S. J.; Porter, J. R.;

Castro, A. C.; Behnke, M. L.; Yu, L.-C.; Hagel, M.; White, K.;

Faia, K.; Grenier, L.; Campbell, M. J.; Cushing, J.; Woodward,

C. N.; Hoyt, J.; Foley, M. A.; Read, M. A.; Sydor, J. R.; Tong, J.

K.; Palombella, V. J.; McGovern, K.; Adams, J. Semi-synthetic

cyclopamine analogues as potent and orally bioavailable

Hedgehog pathway antagonists. J. Med. Chem. 2008, 51,

6646–6649.

(41) Feldmann, G.; Fendrich, V.; McGovern, K.; Bedja, D.; Bisht, S.;

Alvarez, H.; Koorstra, J.-B. M.; Habbe, N.; Karikari, C.;

Mullendore, M.; Gabrielson, K. L.; Sharma, R.; Matsui, W.;

Maitra, A. An orally bioavailable small-molecule inhibitor of

Hedgehog signaling inhibits tumor initiation and metastasis in

pancreatic cancer. Mol. Cancer Ther. 2008, 7, 2725–2735.

(42) McGovern, K. J.; Pien, C. S.; Wright, J. L. Dosing Regimen for the

Treatment of Cancer. WO2007123511, November 1, 2007.

(43) Manna, J. D.; Alvarez-Diez, T. M.; Grogan, M. J.; Porter, J. R.;

Tremblay, M. R.; Castro, A. C.; Sydor, J. R. Metabolite Identifica-

tion of IPI-609, a Novel and Potent Inhibitor of the Hedgehog

Pathway, in Different Species. Presented at the ASMS Conference,

Denver, CO, June 1-5, 2008.

(44) Masamune, T.; Sato, N.; Kobayashi, K.; Yamazaki, I.; Mori, Y.

Syntheses and NMR spectra of 22,27-imino-17,23-oxidojervane

derivatives. Tetrahedron 1967, 23, 1591–1612.

(45) Tsuji, N.; Suzuki, J.; Shiota, M.; Takahashi, I.; Nishimura, S.

Highly stereoselective hydrogenation of 3-oxo-4-ene and -1,4-diene

steroids to 5β-compounds with palladium catalyst. J. Org. Chem.

1980, 45, 2729–2731.

(25) Stanton, B. Z.; Peng, L. F.; Maloof, N.; Nakai, K.; Wang, X.;

Duffner, J. L.; Taveras, K. M.; Hyman, J. M.; Lee, S. W.; Koehler,

A. N.; Chen, J. K.; Fox, J. L.; Mandinova, A.; Schreiber, S. L. A

small molecule that binds Hedgehog and blocks its signaling in

human cells. Nat. Chem. Biol. 2009, 5, 154–156.

(26) Von Hoff, D. D.; Rudin, C. M.; LoRusso, P. M.; Borad, M. J.;

Korn, R. L.; Heath, E. I.; Yauch, R.; Darbonne, W. C.; Kadel, E.

E., III; Zerivitz, K. L.; Nelson, L. E.; Mackey, H.; Marsters, J. C.,

Jr.; de Sauvage, F. J.; Low, J. A. Efficacy Data of GDC-0449, a

Systemic Hedgehog Pathway Antagonist, in a First-in-Human,

First-in-Class Phase I Study with Locally Advanced, Multifocal or

Metastatic Basal Cell Carcinoma Patients. Presented at the AACR

Annual Meeting, San Diego, CA, 2008.

(46) Dwyer, J. R.; Sever, N.; Carlson, M.; Nelson, S. F.; Beachy, P. A.;

Parhami, F. Oxysterols are novel activators of the Hedgehog

signaling pathway in pluripotent mesenchymal cells. J. Biol. Chem.

2007, 282, 8959–8968.

(47) Clinton, R. O.; Clarke, R. L.; Stonner, F. W.; Manson, A. J.;

Jennings, K. F.; Phillips, D. K. Steroidal heterocycles. VI. For-

mylation of A/B-cis 3-ketosteroids. Preparation of 5β-steroidal

[3,2-c]pyrazoles. J. Org. Chem. 1962, 27, 2800–2807.

(27) A Phase 1 Study of BMS-833923 (XL139) Is Subjects with Ad-

vanced of Metastatic Cancer. http://clinicaltrial.gov/ct2/show/

NCT00670189.

(48) Holton, P. G.; Necoechea, E. Steroids. CLXXV. Further steroidal

anabolic agents. J. Med. Chem. 1962, 5, 1352–1357.

(28) Dose Finding and Safety of Oral LDE225 in Patients with Ad-

vanced

NCT00880308?term=LDE-225&rank=1.

(29) A Phase 1 Study of IPI-926 in Patients with Advanced and/or

Metastatic Solid Tumor Malignancies http://clinicaltrial.gov/ct2/

show/NCT00761696.

(49) Mrozik, H.; Buchschacher, P.; Hannah, J.; Fried, J. H. Hetero-

cyclic steroids in the antiinflammatory series. J. Med. Chem. 1964,

7, 584–589.

(50) Ohta, G.; Takegoshi, T.; Ueno, K.; Shimizu, M. Investigations on

steroids. XI. Synthesis of steroidal oxazole, imidazole, and triazole.

Chem. Pharm. Bull. 1965, 13, 1445–1459.

Solid

Tumors.

http://clinicaltrials.gov/ct2/show/

4418 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14

Tremblay et al.

(51) Marchetti, E.; Donini, P. The synthesis of some isoxazole deriva-

tives of androstane. Chim. Ital. 1961, 91, 1133–1141.

Hedgehog signaling in small-cell lung cancer: frequent in vivo but a

rare event in vitro. Lung Cancer 2006, 52, 281–290.

(52) Bakshi, R. K.; Patel, G. F.; Rasmusson, G. H.; Baginsky, W. F.;

Cimis, G.; Ellsworth, K.; Chang, B.; Bull, H.; Tolman, R. L.;

Harris, G. S. 4,7β-Dimethyl-4-azacholestan-3-one (MK-386) and

related 4-azasteroids as selective inhibitors of human type 1 5R-

reductase. J. Med. Chem. 1994, 37, 3871–3874.

(59) Yauch, R. L.; Gould, S. E.; Scales, S. J.; Tang, T.; Tian, H.; Ahn,

C. P.; Marshall, D.; Fu, L.; Januario, T.; Kallop, D.; Nannini-Pepe,

M.; Kotkow, K.; Marsters, J. C., Jr.; Rubin, L. L.; DeSauvage, F. J.

A paracrine requirement for hedgehog signaling in cancer. Nature

2008, 455, 406–410.

(53) Rasmusson, G. H.; Reynolds, G. F.; Utne, T.; Jobson, R. B.;

Primka, R. L.; Berman, C.; Brooks, J. R. Azasteroids as inhibitors

of rat prostatic 5R-reductase. J. Med. Chem. 1984, 27, 1690–1701.

(54) Mazur, R. Azasteroids. III. 3-Aza-A-homo androgens. J. Org.

Chem. 1963, 28, 248–250.

(55) Shroff, A. P.; Harper, C. H. 3-Aza-A-homoandrostenes. J. Med.

Chem. 1969, 12, 190–191.

(56) Briggs, K. J.; Corcoran-Schwartz, I. M.; Zhang, W.; Harcke, T.;

Devereux, W. L.; Baylin, S. B.; Eberhart, C. G.; Watkins, D. N.

Cooperation between the Hic1 and Ptc1 tumor suppressors in

medulloblastoma. Gene Dev. 2008, 22, 770–785.

(57) Sasai, K.; Romer, J. T.; Lee, Y.; Finkelstein, D.; Fuller, C.;

McKinnon, P. J.; Curran, T. Shh pathway activity is down-

regulated in cultured medulloblastoma cells: implications for pre-

clinical studies. Cancer Res. 2006, 66, 4215–4222.

(58) Vestergaard, J.; Pedersen Mikkel, W.; Pedersen, N.; Ensinger, C.;

Tumer, Z.; Tommerup, N.; Poulsen Hans, S.; Larsen Lars, A.

(60) Travaglione, V.; Peacock, C. D.; MacDougall, J.; McGovern, K.;

Cushing, J.; Yu, L.-C.; Trudeau, M.; Palombella, V.; Adams, J.;

Hierman, J.; Rhodes, J.; Devereux, W. L.; Watkins, D. N. A Novel

Hh Pathway Inhibitor, IPI-926, Delays Recurrence Post-Che-

motherapy in a Primary Human SCLC Xenograft Model. Pre-

sented at the Annual Meeting of AACR, San Diego, CA, 2008.

(61) Travaglione, V.; Deyneko, I.; Oaks, Z.; Pink, M.; Proctor, J.;

Mandley, E.; Gu, L.; Osswalt, M.; Read, M.; Sydor, J.; McGo-

vern, K.; Palombella, V.; MacDougall, J. Activity of IPI-926, a

Novel Inhibitor of the HH Pathway, in Subcutaneous and

Orthotopically Implanted Xenograft Tumors That Express

SHH Ligand. Presented at the EORTC-NCI-AACR Sympo-

sium on “Molecular Targets and Cancer Therapeutics”, Geneva,

Switzerland, 2008.

(62) Austad, B.; Behnke, M. L.; Castro, A. C.; Charette, A. B.; Grogan,

M. J.; Janardanannair, S.; Lescarbeau, A.; Peluso, S.; Tremblay,

M. Cyclopamine Analogs. US 2009/0012109 A1, January 8, 2009.

Article Doi

Discovery of a potent and orally active hedgehog pathway antagonist (IPI-926)

DOI: 10.1021/jm900305z

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Full text of DOI:10.1021/jm900305z

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