Recent patents for Hedgehog pathway inhibitors for the treatment of malignancy

Review

10.1517/13543770903008551 © 2009 Informa UK Ltd ISSN 1354-3776 1039All rights reserved: reproduction in whole or in part not permitted

RecentpatentsforHedgehogpathwayinhibitorsforthetreatmentofmalignancyMartin R Tremblay†, Michael Nesler, Robin Weatherhead & Alfredo C CastroInfinity Pharmaceuticals, Inc., 780 Memorial Drive, Cambridge, MA 02139, USA

Background: There is increasing evidence suggesting that blocking aberrant Hedgehog (Hh) signaling can be a novel therapeutic avenue for the treatment of cancer. During the past decade, efforts from academic and industrial groups have led to the discovery of a variety of Hh pathway inhibitors. Objective: This review covers the patent literature related to Hh pathway inhibitors for the treatment of proliferative diseases, regardless of their modes of action. Methods: A comprehensive survey of the patent literature since 1999 is presented. Results/conclusion: Most reported Hh pathway inhibitors act on the key signaling transducer Smoothened (SMO). Screening of compound libraries using reporter and binding assays have identified a broad diversity of chemical structures that interact with SMO. These screen-ing approaches, followed by conventional medicinal chemistry, have delivered important clinical drug candidates, such as GDC-0449 and XL-139. In addition, modification of the naturally occurring Veratrum alkaloid cyclopamine has resulted in various active analogues, including clinical drug candidate IPI-926. Although there are recent scientific literature reports of small molecules acting downstream of SMO, there is limited patent literature on this mode of Hh pathway inhibition.

Keywords: anticancer, basal cell carcinoma, cyclopamine, GDC-0449, GLI, hedgehog, IPI-926, medulloblastoma, natural product, Patched, Smoothened, XL-139

Expert Opin. Ther. Patents (2009) 19(8):1039-1056

1. Introduction

The Hedgehog (Hh) signaling pathway is highly conserved from Drosophila to human and regulates processes essential to the proper development of embryos. Reviewed more extensively elsewhere [1-5], a simplified schematic of the mammalian Hh pathway is depicted in Figure 1. There are three homologues of the Hh ligand proteins: Sonic (Shh), Indian (Ihh) and Desert (Dhh). Hh ligands bind to the Patched (PTCH) receptor, a 12-transmembrane receptor that exists as two isoforms in humans (PTCH-1 and 2). In the absence of Hh ligand, PTCH inhibits Smoothened (SMO) activity, thus blocking Hh signal transduction. Hh binding to PTCH relieves inhibition of SMO. Subsequently, SMO is further activated by phosphorylation, likely by multiple kinases including G-protein coupled receptor kinase (GRK), thus leading to β-arrestin recruitment [6]. It has been proposed that SMO translocation to the primary cilium is required for proper function [7-9].

Hh pathway activation ultimately leads to the modulation of Gli Zn-finger transcription factors, permitting transactivation of Hh-responsive genes whose products are crucial for tissue patterning, growth and differentiation and tissue homeostasis. When the Hh pathway is not active, Gli2/Gli3 are maintained in a repressor form (GliR), whereas the active form of Gli1 (GliA) is inhibited by the negative regulator Suppressor of Fused (SuFu) (Figure 1). Activation of the Hh pathway results in phosphorylation and nuclear localization of GliA.

1. Introduction

2. Heterocycles and other synthetic

derivatives

3. Natural products and derivatives

4. Biomolecules and analogues

5. Expert opinion

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RecentpatentsforHedgehogpathwayinhibitorsforthetreatmentofmalignancy

1040 ExpertOpin.Ther.Patents(2009) 19(8)

The precise mechanisms by which SMO regulates Gli activity are not fully understood. Cytoplasmic members of the Hh pathway have been identified that act between SMO and Gli. These include both negative regulators (SuFu, Rab23 and Ren) and positive regulators (IFT proteins, Iguana, Tectonic and MIM/BEG4) [3]. Further experimentation is needed to characterize this complex pathway.

Over the past decade, it has become increasingly evident that aberrant expression of Hh pathway members contrib-utes to cancer formation and maintenance [1,10]. Misregula-tion of the Hh pathway can occur by several mechanisms. First, somatic mutations in genes encoding Hh pathway members can disrupt proper Hh signaling. For instance, PTCH loss-of-function mutation is the inherited trait in Gorlin’s syndrome [11], and these mutations have been reported in both basal cell carcinoma [11] and medulloblas-toma [12]. Gain-of-function mutations in SMO have been found in 10 – 20% of basal cell carcinoma [13,14]. Loss- of-function mutations in SuFu have been associated with medulloblastoma [15,16].

Ligand-dependent activation is another mechanism leading to Hh pathway dysregulation and has been reported in several human malignant tissues, such as lung [17,18], pancreatic [19], prostate [20], digestive tract [21], bone/cartilage [22], colorectal [23] and ovarian tissues [23]. In addition, similar observations were made in some cancer cell lines, particularly in lung [17], colon [24], and pancreatic [19,24] cells. Because of the prevalent involvement of the Hh pathway in cancer, several approaches to block this pathway are under development.

A variety of biological assays have been used to identify and evaluate Hh pathway inhibitors. The most prevalent assay is the cell-based Gli-Luciferase assay [25], in which a Gli-dependent firefly luciferase vector is transfected into competent cells (e.g., NIH 3T3, TM3Hh12, C3H10T1/2). Transfected cells are stimulated with Shh ligand or agonists, and the reduction of luminescence in the presence of increasing concentrations of compounds is measured [26-28]. It should be

mentioned that the vector constructs typically require several copies of the Gli consensus sequence for this assay to give reasonable dynamic range.

Another cell-based assay uses an Hh-dependent pheno-typic readout in C3H10T1/2 cells and is not dependent on transfection of a reporter construct [28,29]. In the presence of Hh ligand or other Smo agonists, murine C3H10T1/2 pro-genitor cells differentiate into osteoblasts through the Hh signaling pathway. Hh-dependent differentiation is accom-panied by production of alkaline phosphatase [29], which can be readily detected and influenced by increasing concentrations of Hh pathway inhibitors.

The biological activity data for compounds included in this review are derived from one of these two cell-based assays. It is worth noting that accumulating evidence indi-cates that cell lines grown in culture lose their dependence on the Hh signaling pathway for growth [30,31]. As a result, a correlation between Hh pathway inhibition and in vitro cancer cell growth inhibition has been controversial.

This review covers the patent literature after 1999 that relates to Hh pathway inhibitors for the treatment of prolif-erative diseases. The various Hh pathway antagonists are categorized by structural class rather than by their modes of action, which are often not reported because of the nature of the primary assays. When appropriate, the current devel-opment status of representative molecules from these patents is included. A list of small molecule Hh antagonists dis-cussed in this review is provided in Table 1 along with their biological data and current development status.

2. Heterocyclesandothersyntheticderivatives

Pyrrolidine (Figure 2). Nearly a decade ago, Curis developed synthetic methodologies to produce libraries of 4-aminopro-line analogues [32]. These libraries were screened for Hh antagonistic activity using C3H10T1/2 cells stably transfected

PtchSmo

SufuGli

Target genes

PtchSmo

SufuGli

Target genes

Hh

Gli

?

Inactive in absence of ligand Active in presence of ligand

Figure1.SchematicofHhpathwaysignalingandtargetsforantagonists.

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Tremblay,Nesler,Weatherhead&Castro

ExpertOpin.Ther.Patents(2009) 19(8) 1041

Tab

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elo

pm

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ph

ase

1[3

2]W

O20

0126

644

Cur

is/G

enen

tech

Smo

100

– 20

0 nM

*Ph

ase

I (ha

lted)

2[3

5]W

O20

0119

800

Cur

is/E

vote

cSm

o70

nM

*Pr

eclin

ical

4[3

7]W

O20

0301

1219

Cur

is/G

enen

tech

Smo

< 1

nM

*Pr

eclin

ical

5[3

7]W

O20

0301

1219

Cur

is/G

enen

tech

Smo

40 n

M*

Prec

linic

al

6[4

0]W

O20

0308

8970

JHU

Smo

30 n

M*

Prec

linic

al

7[4

1]W

O20

0503

3288

JHU

n.d.

–Pr

eclin

ical

8[4

2]W

O20

0602

8958

Gen

ente

chSm

o22

nM

*Ph

ase

II

9[4

5]W

O20

0607

8283

Gen

ente

chSm

o<

1,0

00 n

M*

Prec

linic

al

10[4

6]W

O20

0705

9157

Gen

ente

chSm

o–

Prec

linic

al

11[4

7]W

O20

0801

4291

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LC

Cn.

d.–

Prec

linic

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12[4

8]W

O20

0902

7746

Ast

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n.d.

< 3

nM

‡Pr

eclin

ical

13[4

9]W

O20

0712

0827

Nov

artis

Smo

17 n

M*

Prec

linic

al

14[5

1]W

O20

0713

1201

IRM

LC

Cn.

d.–

Prec

linic

al

15[5

3]W

O20

0811

2913

Exel

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Smo

3.4

nM*

Prec

linic

al

16[5

3]W

O20

0811

2913

Exel

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Smo

2.6

nM*

Prec

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17[5

3]W

O20

0811

2913

Exel

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Smo

3.6

nM*

Prec

linic

al

18[5

5]W

O20

0807

5196

Pfize

rSm

o<

2,0

00 n

M*

Prec

linic

al

19[5

5]W

O20

0807

5196

Pfize

rSm

o<

2,0

00 n

M*

Prec

linic

al

20[5

5]W

O20

0807

5196

Pfize

rSm

o<

2,0

00 n

M*

Prec

linic

al

21[4

1]

[57]

WO

2005

0332

88W

O20

0605

0351

JHU

GIN

RFA

lcoh

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nase

/m

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tubu

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30 n

M‡

Prec

linic

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22[6

0]W

O20

0705

4623

Lice

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unkn

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> 1

00 n

M*

Prec

linic

al

23[6

0]W

O20

0705

4623

Lice

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unkn

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> 1

00 n

M*

Prec

linic

al

24[6

1]W

O20

0811

0611

Nov

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Smo

< 1

00 n

M*

Prec

linic

al

25[6

2]W

O20

0900

2469

Am

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Smo

< 1

,000

nM

*Pr

eclin

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26[6

2]W

O20

0900

2469

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Smo

< 1

,000

nM

*Pr

eclin

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27[6

3]W

O20

0713

9492

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BG

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000

nM*

Prec

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28[6

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O20

0713

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29[6

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0,00

0 nM

*Pr

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ical

30[6

5]W

O20

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UC

SFG

li-3

< 2

0,00

0 nM

*Pr

eclin

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31[6

6]W

O20

0813

0552

Mer

ckSm

o<

25,

000

nM*

Prec

linic

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32[6

6]W

O20

0813

0552

Mer

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o<

25,

000

nM*

Prec

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33[7

2]W

O20

0127

135

JHU

/Cur

isSm

o50

0 nM

*Pr

eclin

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34[7

2]W

O20

0127

135

JHU

/Cur

isSm

o30

0 nM

*Pr

eclin

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35[7

2]W

O20

0127

135

JHU

/Cur

isSm

o–

Prec

linic

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36[7

2]W

O20

0127

135

JHU

/Cur

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o20

nM

*Pr

eclin

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37[9

4]U

S740

7967

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ity P

harm

aceu

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sSm

o>

2,5

00 n

M‡

Prec

linic

al

38[9

5]U

S 7,

230,

004

Infin

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harm

aceu

tical

sSm

o20

0 nM

‡Pr

eclin

ical

39[9

7]W

O20

0808

3248

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ity P

harm

aceu

tical

sSm

o7

– 10

nM

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ase

I

40[9

8]W

O20

0810

9184

Infin

ity P

harm

aceu

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sSm

o10

– 2

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‡Pr

eclin

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41[9

9]W

O20

0810

9829

Infin

ity P

harm

aceu

tical

sSm

o30

– 4

0 nM

‡Pr

eclin

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42[1

00]

WO

2008

1313

54U

Mis

sour

in.

d.30

,000

nM

*Pr

eclin

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43[1

00]

WO

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Mis

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d.20

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*Pr

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44[1

01]

WO

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0675

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Tab

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(C

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).

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1 (Cur61414; G-024856)Cell IC50 = 100 – 200 nM

WO200126644

N

N

O

N

NH

OCH3

O

O

O

Figure2.PyrrolidineHhantagonist1.

N

N

NH

NH

O

OF

CF3

Cl 2 (Compound Z)Cell IC50 = 70 nMWO200119800

Figure3.QuinazolinoneHhantagonist2.

with Gli-luciferase (s12 cells) as a reporter. This screen iden-tified SMO antagonist 1 (Cur61414) [33], which emerged as a development candidate and was evaluated as a topical Hh antagonist in clinical studies. The clinical development of Cur61414 was halted because it failed to produce clinical changes and pharmacodynamic responses when applied topically to basal cell carcinoma lesions [34].

Quinazolinones (Figure 3). In 2001, Curis in collaboration with Evotec reported a series of quinazolinones [35] exempli-fied by compound Z (2). The structure–activity relation-ships (SARs) that led to the identification of 2 was subsequently reported (IC50 of 70 nM in a Gli-Luciferase assay) [36].

Bisarylcarboxamides (Figures 4 – 6). This class of Hh antagonists has been broadly explored by different groups, and several analogues with the common bisarylcarboxamide structural motif 3 have been disclosed (Figure 4).

As the first example around that core structure, Curis described a series of benzimidazoles [37,38] that includes the very potent compound 4 and the related, more broadly pub-lished compound 5 (also referred to as HhAntag and HhAn-tag691; Figure 5). The latter has shown efficacy in a variety of pharmacological models [23,39]. Concurrently, Johns Hop-kins University filed a patent on closely related compounds, such as compound 6 (SANT-2; Figure 5) [40]. During high-throughput screening for Hh pathway antagonists, Johns Hopkins University identified several small heterocyclic molecules, such as 2-phenylpyridyl derivative 7 [41], but no biological data were reported.

Curis, in collaboration with Genentech, also discovered a series of 2-arylpyridines [42], which ultimately led to the development of SMO antagonist GDC-0449 (8; Figure 5). Orally administered GDC-0449 is being investigated in Phase II clinical trials at present for the treatment of solid tumors, including basal cell carcinoma, colorectal and ovar-ian tumors and medulloblastoma [43]. This molecule was selected from optimization studies [44] of the benzimidazole series, exemplified by compounds 4 and 5, as well as the quinaxoline series, represented by compound 9 [45]. Interest-ingly, another patent application from Genentech and Curis described a series of bisamide analogues, exemplified by compound 10 [46].

In 2008, a group at the Genomics Institute for the Novartis Research Foundation (GINRF) disclosed a series of diarylimida-zoles, exemplified by compound 11 (Figure 5) [47]. More recently, AstraZeneca also disclosed a group of 2-phenylimidazoles, including compound 12 (Figure 5). This compound inhibits the differentiation of C3H10T1/2 with IC50 value < 3 nM, although it only reached 58% inhibition at 3 μM [48].

A group at Novartis identified a series of ortho-biphenyl carboxamide antagonists of SMO, represented by compound 13 (Figure 6) [49]. During the exploration of SAR on this class of compounds, it was found that S-configuration of the aminoindane moiety was preferred for the Hh antagonistic activity [50]. In the same year, a group at GINRF filed two

HN

OR

1

R2

3

Figure4.Commonbisarylcarboxamidestructuralmotif3.

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Cl

NH

O

S

N

O

O

Cl

NH

O

Cl

N

Cl

8 (GDC-0449)Cell IC50 = 22 nMWO2006028958

7 WO2005033288

Cl

NH

O

S

N

N

O

O

N

9 WO2006078283

NH

N

Cl

NH

O

N

O

O

NH

N

N

Cl

NH

OOMe

OMe

NH

N

Cl

NH

OOEt

OEt

OEt

4 Cell IC50 < 1 nMWO2003011219WO2006050506

5 (HhAntag; HhAntag691)Cell IC50 = 40 nMWO2003011219WO2006050506

6 (SANT-2)Cell IC50 = 30 nMWO2003088970

Figure5.Hhantagonistsaroundthebisarylcarboxamidemotif. Benzimidazoles (4–6), 2-arylpyridine (7,8), quinaxoline (9).

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patent applications related to ortho-biphenyl carboxamides, such as compound 14 [51,52].

In 2008, a series of quinazolines and pyridopyrimidines were disclosed by Exelixis [53]. Representative compounds 15, 16 and 17 (Figure 6) demonstrated single digit nM IC50 values in a Gli-Luciferase assay. Exelixis in collaboration with Bristol-Myers Squibb is pursuing XL-139 as a systemic Hh antagonist for the treatment of solid tumors [54], but at present it is unknown whether XL-139 belongs to this class of compounds.

Benzimidazoles (Figure 7). Pfizer reported a series of benz-imidazoles [55] containing a 4-aminopiperidine moiety that is reminiscent of the 4-aminoproline 1 (Figure 2) and quinazolinone 2 (Figure 3) derivatives discussed previously. Representative compounds 18 – 20 are benzimidazoles that have the same configuration on the piperidine moiety as well as an electron-withdrawing group at the 4-position on the phenyl urea.

Thiazoles (Figure 8). Several marketed and investigational drugs in various therapeutic areas are based on the 2-amino-thiazole

motif [56]. The GINRF identifissed several 2-amino-thiazoles as potent Hh antagonists [57]. Interestingly, compound 21 (JK-184) and closely related analogues bearing imidazopyridines at the 4-position have been claimed as Hh antagonists in patent appli-cations from Johns Hopkins University [41] and GINRF [57]. A few modes of action have been proposed for compound 21: the Hh antagonistic activity may be linked to inhibition of class IV alcohol dehydrogenase [58] or destabilization of microtubules [59]. Licentia also discovered a series of 2-aminothiazoles represented by compounds 22 and 23 [60].

Phthalazines (Figure 9). A group at Novartis discovered Hh antagonists based on a phthalazine scaffold, exemplified by the structure of compound 24 [61]. These Hh antagonists and compound 14 share the common structural feature of an N-(2-pyrido)-piperazine group (Figure 6). Other phthala-zine Hh antagonists, such as compounds 25 and 26, were described in a patent application from Amgen published earlier this year [62]. Notably, compound 25 leads to a > 99% reduction in tumor size relative to vehicle when administered for 6 days at 10 mg/kg/day in a mouse medulloblastoma

10 WO2007059157

HN

Cl

NH

O

S

O

O

ON Cl

11 WO2008014291

12 WO2009027746

NH

N

Cl

NH

O

S

O

O

Cl

N

N

NH

O

O

N

Figure5.Hhantagonistsaroundthebisarylcarboxamidemotif(continued). Bisamide (10) and arylimidazoles (11,12).

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allograft model. The development status of these promising Hh antagonists has not been disclosed yet.

Hexahydropyrimidine, tetrahydroimidazole and derivatives (Figure 10). A group from Actar AB found a family of sym-metric and non-symmetric N,N-bisbenzylated hexahydropy-rimidines (e.g., compound 27) and tetrahydroimidazoles (e.g., compound 28) that block Gli-mediated expression in a variety of cellular systems [63]. A representative compound

from this series (27, GANT-61; Figure 10) acts downstream of SuFu in vitro and inhibits tumor growth in a 22Rv1 prostate tumor xenograft mouse model when administered subcutaneously at 50 mg/kg every other day [64].

Dihydropyrazolecarboxamides (Figure 11). A group from Univer-sity of California San Francisco described a rational approach to small molecules mimicking the transcriptional activation domain of Gli-3 polypeptide [65]. Dihydropyrazolecarboxamide compounds

NHO

HN N

N

O NH

N

N NHN

O NH

HN

N

N

N

15 Cell IC50 = 3.4 nMWO2008112913

16 Cell IC50 = 2.6 nMWO2008112913

17 Cell IC50 = 3.6 nMWO2008112913

N N

N

HN

O

CN

HN

O

CF3

NH

N

S

13 Cell IC50 = 17 nMWO2007120827

14 WO2007131201WO2008154259

Figure6.Hhantagonistsaroundthebisarylcarboxamidemotif. Ortho-biphenylcarboxamide (13,14), quinazoline (15,16) and

pyridopyrimidine (17).

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18 US 2009/0005416 19 US 2009/0005416 20 US 2009/0005416

NH

NN

HN

O

HN

CN

NH

NN

HN

O

HN

CN

CF3

NH

NN

HN

O

HN

OCF3

Figure7.BenzimidazoleHhantagonists(18–20).

21 (JK-184)Cell IC50 = 30 nMWO2005033288WO2006050351

23 Cell IC50 > 100 nMWO2007054623

N

S

HN

N

N

S

HN OEt

N

N

22 Cell IC50 > 100 nMWO2007054623

N

S

HN

N

Cl

MeO

Figure8.ThiazolesHhantagonists21–23.

such as 29 and 30 are described as analogues of the α-helical domain comprising the FDAII motif of Gli-3. Compound 30 (FN1-8) has shown cell killing effects in several cancer cell lines at a concentration (50 μM) that did not affect normal renal cells. Notably, compound 30 inhibits Gli-Luciferase activity in mela-noma cells transfected with Gli-expression plasmid. Finally, com-pound 30 suppresses tumor growth in various tumor xenograft mouse models (NSCLC H460, NSCLC A549 and LOX) when administered daily via intraperitoneal injection.

Triazoles (Figure 12). A group at Merck has discovered that some triazoles, also useful as inhibitors of 11β-steroid dehydrogenase, are potent inhibitors of the Hh pathway [66]. One of the claimed compound in the patent application, exemplified by 1,2,4-oxadiazole 31 and 1,3,4-oxadiazole 32 (Figure 12), is an SMO antagonist and has shown tumor

shrinkage in allograft Ptch+/- medulloblastoma model when administered orally twice daily at 80 mg/kg.

3. Naturalproductsandderivatives

Veratrum alkaloids (Figure 13). Jervine (33), cyclopamine (34) and cycloposine (35) are natural steroidal alkaloids iso-lated from Veratrum plants that have played a historical role in the discovery of biological responses related to Hh path-way modulation. Indeed, cyclopamine exerts its teratogenic effect [67,68] through inhibition of the Hh signaling path-way [69,70], specifically by targeting SMO [25,71]. Cyclopamine and more potent analogues, such as compound 36, were claimed in a patent assigned to Johns Hopkins University [72] that was later licensed to Curis. Cyclopamine has proven to

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be a valuable pharmacological tool to validate the Hh path-way as a promising cancer target. Indeed, cyclopamine inhibits tumor growth in several xenograft models, including those of pancreatic [19,73], medulloblastoma [74,75], pros-tate [20,76,77], glioma [78], melanoma [79], small cell lung [17] and digestive tract [21] origin. Moreover, cyclopamine has been shown to affect a sub-population of tumor cells in glioma [78,80], multiple myeloma [81], leukemias [82] and pancreatic cancers [73].

Several methods of using cyclopamine and its analogues for the treatment of malignant diseases have been described in the patent literature [83-93].

D-ring analogues of cyclopamine (Figure 14). Although cyclopamine has shown very promising Hh antagonistic and anticancer activity, it suffers from poor pharmaceutical prop-erties. The development of an oral agent based on the cyclo-pamine scaffold is hindered by its inherent low aqueous solubility and, more importantly, by its chemical instability at low pH. Structural modifications to resolve stability and solubility issues associated with cyclopamine were explored by a group at Infinity Pharmaceuticals, Inc. A synthetic methodology was developed to access 12,13-cyclopropyl cyclopamine (37) [94] and D-ring-expanded analogues (e.g., 38) [95]. Exploration of the SAR of D-ring expanded

N N

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25 WO200900246924 WO2008110611

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Figure9.PhthalazineHhantagonists24–26.

28 WO200713949227 (GANT-61)WO2007139492

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Figure10.Hexahydropyrimidine(27)andtetrahydroimidazole(28)Hhantagonists.

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30 (FN1-8)WO2007067814

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Figure11.DihydropyrazolecarboxamideHhantagonists29and30.

31 WO2008130552 32 WO2008130552

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Figure12.TriazoleHhantagonists31and32.

cyclopamine analogues revealed that the Hh antagonistic activity tracks with cyclopamine analogues. For example, compound 38 was found to be equipotent while also being more stable to low pH and more soluble in aqueous media relative to cyclopamine [96].

More recently, the same group filed several patent applications related to three series of A-ring modified D-homocyclopamine analogues, exemplified by compounds 39 [97], 40 [98] and 41 [99]. Infinity Pharmaceuticals, Inc. is developing a cyclopamine derivative (39, IPI-926) with improved drug-like properties and potency relative to cyclopamine.

Phytoestrogens (Figure 15). A group from University of Missouri has shown that the phytoestrogens genistein (42) and epigallocatechin gallate (43, EGCG) can inhibit Gli-1 expression in a dose-dependent manner in TRAMP-C2 cell lines, which are derived from a mouse model of pros-tate cancer [100]. Approximate IC50 values for in vitro inhibition of the Hh pathway were 30 μM for genistein and 20 μM for EGCG.

Analogues of antiprogestins mifepristone (Figure 16). Mife-pristone (44) is a derivative of the 19-norprogestin norethin-drone, a drug that acts as a competitive receptor antagonist for both progesterone receptors and is available for the ter-mination of pregnancy. In a patent application from the Centre National de la Recherche Scientifique, mifepristone was reported to inhibit differentiation of the mesenchymal cell C3H10T1/2 with an IC50 of 3.7 μM [101].

4. Biomoleculesandanalogues

Antibodies. In 2000, a group at Genentech provided the basis for the production of neutralizing SMO antibod-ies [102]. Given its subcellular localization and possible inactive/active conformations, SMO may represent a dif-ficult target for a neutralizing antibody. However, anti-bodies to Hh ligands may prove to be more tractable, representing an attractive therapeutic avenue for the inhibition of the Hh pathway.

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34 (cyclopamine)Cell IC50 = 300 nM

WO200127135

33 (jervine)Cell IC50 = 500 nM

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35 (cycloposine)WO200127135

36 (KAAD-cyclopamine)Cell IC50 = 20 nMWO200127135

Figure13.VeratrumalkaloidsHhantagonists33–35andsyntheticanalogue36.

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A monoclonal antibody to rat Shh, 5E1, was discovered and used as a tool to investigate the importance of Hh signaling in motor neuron differentiation [103]. This antibody recognizes a 20-kDa N-terminal fragment of Shh. The use of an Hh antibody, such as 5E1, for the treatment of malignant diseases was later disclosed in a patent application filed by Curis [24]. In mice, administration of 5E1 decreases the tumor size of

Hh-producing colon cancer (HT-29) and pancreatic cancer (SW1990, CF PAC) cell lines.

Peptides and peptidomimetics. A group at Genentech pro-vided evidence for the importance of type 1 cell surface receptor proteins BOC (brother of CDO) and CDO (cell adhesion molecule-related/down-regulated by oncogenes) in the regulation of Hh pathway [104]. BOC protein can sequester

38 (IPI-269609)Cell IC50 = 200 nM

US 7,230,004

39 (IPI-926)Cell IC50 = 7 – 10 nM

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40 Cell IC50 = 10 – 20 nMWO2008109184

41 Cell IC50 = 30 – 40 nMWO2008109829

Figure14.D-ringanaloguesofcyclopamineasHhantagonists37–41.

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42 (genistein)WO2008131354

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Figure15.PhytoestrogensasHhantagonists42and43.

44 (mifepristone,RU-486)Cell IC50 = 3.7 µMWO2004067550

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Figure16.Mifepristone(44)asHhantagonist.

Shh, thereby preventing activation of the Hh signaling path-way. In contrast, CDO can amplify Hh signaling resulting from Shh binding to Ptc. Consequently, this group has filed a patent for a method of inhibiting the Hh pathway by BOC polypeptides or anti-CDO antibodies.

In 2000, functional antagonists of Shh were reported by a group at Biogen [105]. Truncated analogues of Shh were found to bind PTCH with similar affinity as native Shh, but did not result in activation of the Hh pathway. For instance, a Shh analogue missing nine amino acids on the N terminus and three amino acids on the C terminus (ShhN9/C3) retains affinity for PTCH (IC50 = 2.7 nM relative to 3.7 nM for native Shh) yet only reduces C3H10T1/2 alkaline phosphatase activity by ∼ 50% at 63 nM.

A group from the National Cancer Institute designed and synthesized several peptides that resemble the intracellular loops of SMO with the goal of blocking downstream signaling

by SMO [106]. They identified N-palmitoylated peptide sequences that mimic the second and third loop of SMO and showed that these peptides reduce Hh-pathway gene expression in prostate cancer cell lines DU-145 with potency similar to cyclopamine [107]. Optimizing against cytotoxicity in SK-Mel2 melanoma cell lines, this group identified short peptides (10 amino acids) with retro-inverso sequences of an epitope on the third human SMO loop that are very potent cytotoxic (single-digit nanomolar IC50).

SuFu forms a complex with Gli transcription factors, which prevents their translocation into the nucleus for target gene transactivation [108]. A group at Karolinska Innovations revealed that peptides consisting of fragments from Gli-1 or SuFu are able to bind specifically to SuFu and Gli-1, respec-tively. Expression of plasmids encoding the SuFu and Gli-1 fragment peptides inhibit the Hh pathway in 293 cells and in C3H10T1/2 cells transfected with the Gli-reporter [109].

Antisense and RNA interference. A group from Isis Pharma-ceuticals disclosed a series of oligonucleotides targeting the Shh gene [110]. These 20mers were composed of a central gap con-sisting of ten nucleotides flanked by five 2′methoxyethyl nucle-otides on the 5′ and 3′ regions. The internucleoside linkages were phosphorothioate throughout the sequence. Three chime-ric phosphorothioate oligonucleotides were found to reduce Shh mRNA levels in Jurkat cells by > 80% as monitored by RT-PCR. Curis reported an approach to generate five potential siRNA antagonists of the human Shh [111]. One of the oligo-nucleotides completely blocked expression of Shh in HEK-293 cells transfected with a human Shh plasmid. In addition, the same siRNA was shown to be specific for Shh and did not inhibit Dhh or Ihh expression.

Downstream of the Hh ligands, Ruiz i Altaba and collabo-rators have developed lentivector-encoded shRNAs to SMO, which have also shown tumor growth inhibitory effect in xenograft model of human melanomas [79] and glioma [78].

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Further down the Hh signaling pathway, the same group reported siRNA sequences of ∼ 20 nucleotides that are com-plementary to a portion of Gli-1, Gli-2 and Gli-3 genes [79,112]. Notably, lipofection of siRNA against Gli-1 results in a reduc-tion of BrdU incorporation in primary melanomas and mela-noma cell lines. Likewise, Lawman and collaborators also report siRNA sequences for both Gli-1 and Shh and demon-strated their effect on primitive neuroectodermal tumors [113]. Finally, Genentech has claimed RNAi against CDO as part of their patent application filed for BOC polypeptides or anti-CDO antibodies [104] (vide supra).

5. Expertopinion

The academic groups of Beachy, Ruiz i Altaba and Roelink pioneered the field of research of Hh pathway antagonists and have used cyclopamine as key tool to validate the Hh pathway as an intriguing target for anticancer therapeutics. Among the first drug discovery groups working on the development of Hh pathway modulators, Curis has identi-fied a series of small heterocyclic molecule antagonists (and agonists) of the Hh pathway [26]. In the past few years, other pharmaceutical companies have joined Curis with potent antagonists possessing drug-like properties and impressive preclinical efficacy data.

There have been various challenges that complicated the development of Hh antagonists as a treatment for cancer. Although successful in several cases, it was particularly dif-ficult to establish a reliable link between Hh pathway inhi-bition and anticancer activity because it was problematic to generate in vitro systems [30,31]. The discovery of Hh path-way antagonists has principally relied on the screening of compound libraries using reporter assays and binding assays that use unnatural ligands. The seven-transmembrane protein SMO is related to GPCR, but the mechanism for signal transduction still remains largely unknown. Among substructures found frequently in GPCR ligands are 2-arylin-doles, biphenyl substructure and, to a lesser extent, benzimi-dazoles [56,114,115]. Consequently, it is not surprising that

many of the SMO antagonists described in this review exploit these structural motifs. Screening approaches, fol-lowed by conventional medicinal chemistry, delivered impor-tant drug candidates such as GDC-0449 and XL-139 in clinical Phase II and Phase I, respectively, at present. In addition, innovative and daring synthetic modifications of the naturally occurring Veratrum alkaloid cyclopamine led to the discovery of clinical drug candidate IPI-926 in Phase I for the treatment of solid tumors at present.

Despite these successful approaches, there are opportunities to develop more powerful and selective Hh antagonists. Even today, very little is known about the three-dimensional struc-ture and intracellular trafficking of SMO and other members of the Hh pathway. There is increasing interest in developing small molecules that act upstream [116] and down-stream [58,64,117] of SMO, but this field is still relatively under-developed. In the first decade of research targeting the Hh pathway, it is clear that SMO has been the most ‘druggable’ target in the pathway. As the clinical relevance of current Hh pathway antagonists continues to evolve and our understand-ing of the Hh pathway signal transduction increases, there will undoubtedly be additional attractive targets in this pathway for therapeutic intervention in the future.

Acknowledgments

The authors thank Karen McGovern and Margaret A. Read for discussions during the preparation of the manuscript. The authors also thank Dianne Barry, consultant in medical communication, for assistance in writing the manuscript. We sincerely apologize for any omissions; the task of sum-marizing the many exciting recent developments in this field was enormous.

Declarationofinterest

The authors are employees and stock holders of Infinity Pharmaceuticals.

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AffiliationMartin R Tremblay†, Michael Nesler, Robin Weatherhead & Alfredo C Castro†Author for correspondenceInfinity Pharmaceuticals, Inc.,780 Memorial Drive,Cambridge, MA 02139, USA Tel: +1 617 453 1268; E-mail: [email protected]

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Documents

Recent patents for Hedgehog pathway inhibitors for the treatment of malignancy