Identification of MK-5710 ((8aS)-8a-methyl-1,3-dioxo-2-[(1S,2R)-2- phenylcyclopropyl]-N-(1-phenyl-1H-pyrazol-5-yl)hexahydroimid azo[1,5-a]pyrazine-7(1H)-carboxamide), a potent smoothened antagonist for use in Hedgehog pathway dependent malignancies, Part 1

Article abstract of DOI:10.1016/j.bmcl.2011.06.024

The Hedgehog (Hh-) signaling pathway is a key developmental pathway which controls patterning, growth and cell migration in most tissues, but evidence has accumulated showing that many human tumors aberrantly reactivate this pathway. Smoothened antagonists offer opportunities for the treatment of malignancies dependent on the Hh pathway, and the most advanced clinical candidates are demonstrating encourage initial results. A novel series of [6,5]-bicyclic tetrahydroimidazo[1,5-a]pyrazine-1,3(2H,5H)-dione smoothened antagonists has been identified, and the series has been extensively explored to ascertain the key detriments for activity, demonstrating that the trans-2-phenylcyclopropyl and hydantoin ring systems are critical for potency, while a variety of urea substituents can be tolerated. The combination of these optimal groups gives smoothened antagonists with activity in the low nanomolar range.

Full text of DOI:10.1016/j.bmcl.2011.06.024

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4422–4428
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of MK-5710 ((8aS)-8a-methyl-1,3-dioxo-2-[(1S,2R)-2-phenylcy-
clopropyl]-N-(1-phenyl-1H-pyrazol-5-yl)hexahydroimid azo[1,5-a]pyrazine-
7(1H)-carboxamide), a potent smoothened antagonist for use in Hedgehog
pathway dependent malignancies, Part 1
⇑
Savina Malancona , Sergio Altamura, Gessica Filocamo, Olaf Kinzel, Jose Ignacio Martin Hernando,
⇑
Michael Rowley, Rita Scarpelli, Christian Steinkühler, Philip Jones
IRBM, Merck Research Laboratories Rome, Via Pontina km 30,600, Pomezia, 00040 Rome, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 16 June 2011
The Hedgehog (Hh-) signaling pathway is a key developmental pathway which controls patterning,
growth and cell migration in most tissues, but evidence has accumulated showing that many human
tumors aberrantly reactivate this pathway. Smoothened antagonists offer opportunities for the treatment
of malignancies dependent on the Hh pathway, and the most advanced clinical candidates are demon-
strating encourage initial results. A novel series of [6,5]-bicyclic tetrahydroimidazo[1,5-a]pyrazine-
1,3(2H,5H)-dione smoothened antagonists has been identified, and the series has been extensively
explored to ascertain the key detriments for activity, demonstrating that the trans-2-phenylcyclopropyl
and hydantoin ring systems are critical for potency, while a variety of urea substituents can be tolerated.
The combination of these optimal groups gives smoothened antagonists with activity in the low nanomo-
lar range.
Keywords:
Hedgehog pathway
Smoothened antagonist
Hedgehog inhibitor
Ó 2011 Elsevier Ltd. All rights reserved.
The Hedgehog (Hh-) signaling is essential for development, but
in recent years, evidence has accumulated showing that many hu-
man tumors aberrantly reactivate this developmental pathway and
that interfering with it may provide a new strategy for the develop-
ment of novel anti-cancer therapeutics.1–4
number of proteins important for differentiation, proliferation
and survival.
In line with its primary role as a developmental morphogen, Hh
signaling ceases on completion of development, but remains acti-
vatable in very few tissues after birth (such as CNS, hair follicles,
bone and testes). In adult tissues the Hh pathway may be reacti-
vated during processes such as wound healing and tissue repair.
In fact, in all highly regenerative tissues such as the hematopoietic
system, intestine or skin, a subpopulation of adult stem cells usu-
ally residing in a stem cell niche is maintained to control homeo-
stasis and repair tissues after possible injuries. Given the need of
proliferation, stem cells reactivate development pathways, includ-
ing the Hh signaling cascade.
In the past decade several studies have demonstrated that aber-
rant reactivation of Hh signaling occurs in a variety of human tu-
mors,1–3 the best characterized being basal cell carcinoma (BCC),8
but there is strong evidence of alterations in the pathway in
medulloblastoma, as well as small cell lung, gastric, colorectal,
prostate and pancreatic cancers.1–3
The Hh signaling pathway controls patterning, growth and cell
migration in most tissues and organs throughout development.5
Hedgehog was first identified in the early 1980s in a screen for
genes required for embryonic patterning in Drosophila and re-
ceived its name from the peculiar phenotype arising in deficient
embryos.6 In vertebrates three related orthologs named Sonic
(Shh), Desert (Dhh) and Indian (Ihh) Hedgehog have been identi-
fied. Hedgehog initiates its signaling cascade by interacting with
the 12-pass transmembrane protein patched (Ptch). In the absence
of ligand, this protein exerts an inhibitory action, by a poorly
understood mechanism, on the G-protein-coupled receptor
(GPCR)-like, 7-span transmembrane protein smoothened
(Smo).1,3,7 Binding of Hh to Ptch relieves this inhibition, and trig-
gers a series of intracellular events culminating in the nuclear
translocation and activation of Gli 1–3 transcription factors with
the initiation of the Hh-transcriptional program, upregulating a
Smoothened is a G-protein coupled receptor-like protein that is
essentially involved in Hedgehog signal transduction, and small
molecule Smo antagonists have started to show antitumor activity
in preclinical models, and are being developed clinically. The plant
steroidal alkaloid cyclopamine (1) was first identified as an
inhibitor of Hh signaling due to its teratogenicity in sheep.9,10
⇑
Corresponding authors. Tel.: +1 617 582 9648.
E-mail addresses: savinamal@libero.it (S. Malancona), philip_jones@dfci.harvard.
edu (P. Jones).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.06.024

S. Malancona et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4422–4428
4423
Cl
HN
H
HN
H
N
O
H
H
O
H
HN
O
H
H
O
S
H
Cl
H
N
H
H
H
O
HO
SO2Me
Cyclopamine (1)
Vismodegib (2)
IPI-926 (3)
MeO
F3CO
N
N
O
F
O
N
N
F3C
N
N
N
HN
O
N
O
N N
NVP-LDE225 (4)
5
6
Figure 1. Known Smo antagonists.
Table 1
SAR study of the left hand side chaina
O
N
N
N
R
Ph
O
a
a
Compds
R
Light2 IC50 (nM)
Smo binding IC50 (nM)
Compds
R
Light2 IC50 (nM)
1900
Smo binding IC50 (nM)
Bu2N
MeO
7
NDb
ND
>1.5
>1.5
l
l
M
M
16
17
920
O
O
Me
Cl
H
N
Cl
Cl
8
9
340
780
220
280
O
O
c-Hex
H
N
O
ND
>1.5
l
M
18
O
Cl
t-Bu
Cl
H
N
O
10
11
4500
ND
4000
19
20
140
280
O
Cl
Cl
c-Hex
Me2N
H
N
>2.5
l
M
1100
1100
O
F
H
N
O
12
13
14
15
ND
>2.5
>2.5
250
380
l
l
M
M
21
22
23
24
2700
84
700
94
O
O
O
Ph
H
N
H
N
O
ND
O
O
H
N
Ph
Me
N
c-Hex
600
1500
1600
2000
1000
890
O
O
O
H
N
H
N
n-Hex
O
Ph
a
Values are means of at least two experiments with standard deviations <30% of the mean value.
ND = Not determined.
b

4424
S. Malancona et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4422–4428
Table 2
two diastereomeric racemates, 4 compounds in total, and that the
SAR study of the right hand side chaina
stereochemical aspects are discussed in the accompanying
article.19
O
Initial SAR was determined from interrogation and follow up of
related compound present in the sample collection. Maintaining
the key [6,5]-bicyclic hydantoin scaffold and the trans 2-phenylcy-
clopropyl fragment, the left hand side of these molecules was ini-
tially investigated (Table 1). The simple methyl carbamate (7)
N
H
N
N R
N
O
O
a
a
Compds
R
Light2 IC50 (nM)
Smo binding IC50 (nM)
displayed activity greater than 1.5 lM, as were simple amides such
as acetamide (8) and cyclohexylamide (9). Only when additional
lipophilic groups were added, like the t-butyl group in 10, was
weak micromolar activity observed. Alkyl amines, such as cyclo-
hexylmethylamine (11) displayed only weak activity. In contrast,
the urea fragment was deemed beneficial for activity, as seen with
derivatives 12–24. While the simple dimethyl urea (12) displayed
22
84
94
Ph
25
>2.5
lM
>2.5
l
M
Ph
Ph
26
27
5000
NDb
2000
Ph
only marginal activity at 2
proved Smo affinity, and demonstrated encouraging levels of Hh
pathway inhibition in the absence of cytotoxicity at 2.5 M. The
lM, more elaborate fragments im-
>2.5
>2.5
lM
l
28
ND
lM
introduction of cyclohexyl urea generated 14 with sub-micromolar
activity in Smo binding and Light2 assays (IC50 = 600 and 250 nM
respectively). The corresponding acyclic derivatives (15 and 16)
were slightly less active, also highlighting the importance of the
N–H in the urea for activity.
29
2500
ND
>2.5
lM
Ph
Further investigation of these urea derivatives focused in the
phenyl series (17–24). Several compounds from this subseries
demonstrated submicromolar Smo antagonism, notably 17–19
and 22. Disubstitution with two lipophilic groups appeared to
be beneficial on activity, with the 2,3- and the 2,6-dichloro-
phenyl fragments (17 and 19) displaying activity in the low
nanomolar range in Smo binding assay (IC50 = 220 and 280 nM
respectively) and inhibiting the Hh pathway in Light2 cells
(IC50 = 340 and 140 nM respectively). Another interesting deriva-
tive was the 2-biphenylurea 22, which displays double digit
nanomolar activity in both Smo binding (IC50 = 94 nM) and
Light2 (IC50 = 84 nM) assays. As previously mentioned the substi-
tution at NH of urea appeared detrimental for the activity, and
similarly alkylation of 22 to give the methylated analog 23 re-
sulted in an 11-fold drop in potency. The position of the phenyl
group was critical for activity, as the isomeric 4-biphenyl 24 was
10-fold less active.
Having demonstrated the striking Smo antagonism of 22, atten-
tion focused on exploration of the right hand side of these
compounds, and investigation of the SAR around the trans 2-phe-
nylcyclopropyl fragment (Table 2). Strikingly it was demonstrated
that the regiochemistry of cyclopropyl ring was critical for activity,
and while trans-isomer 22 displayed activity in the low nanomolar
range, the cis-isomer 25 showed only modest micromolar Smo
30
31
>2.5
>2.5
l
M
M
>2.5 lM
l
32
33
Ph
130
320
440
880
<30% of the mean value.
a
Values are means of at least 2 experiments with standard deviations.
ND = Not determined.
b
Cyclopamine was later shown to directly bind and inhibit Smo, and
inhibit the proliferation of medulloblastomas and BCC. Subsequent
research has identified a number of small molecule Smo antago-
nists which are advancing in clinical trials (Fig. 1).3,4 The most ad-
vanced of these is Vismodegib (GDC-0449) (2),11 which has
demonstrated encouraging results in phase I studies in advanced
BCC,12 and is currently in phase II trials in BCC, colorectal, ovarian
and pancreatic cancer. Other clinical candidates include a semi-
synthetic cyclopamine analogue IPI-926 (3),13 and NVP-LDE225
(4),14 as well as the structurally undisclosed BMS-833923 (XL-
139), PF-04449913, TAK-441 and LEQ506.15 Recently, a number
of publications have appeared describing other classes of Smo
antagonists, such as pyrazole (5) and phthalazine (6).16,17
Our program began with an uHTS campaign to identify small
molecules useful for the treatment of Hh dependent malignancies.
Compounds were screened for their ability to inhibit the Hh path-
way in Shh-Light2 cells expressing a Gli-dependent reporter gene,
simultaneously monitoring for cytotoxicity.18 Hits were then pro-
filed in a whole cell Smo binding assay, measuring the ability of
compounds to displace a Bodipy-labeled cyclopamine derivative.10
Screening identified a series of bicyclic hydantoins as Smo antago-
nists, and inhibitors of the Hh-pathway. The characterization of
these hits and exploratory SAR work is the subject of this article.
A key aspect of this hit cluster from the initial screen was the
presence of the [6,5]-bicyclic tetrahydroimidazo-[1,5-a]pyrazine-
1,3(2H,5H)-dione scaffold (Tables 1 and 2), and the majority of
the active hits conserved the trans 2-phenylcyclopropyl fragment
in the right hand region (Table 1). The preliminary result presented
in this article focus in the investigation of a library of racemic bicy-
clic hydantoins, it should be highlighted that these are mixtures of
binding affinity (IC50 >2.5 lM). Replacement of the trans 2-phenyl-
cyclopropyl with a 2-phenethyl group as in 26 resulted in only
micromolar activity. Other more radical changes such as replace-
ment of the trans 2-phenylcyclopropyl group with benzyl, naph-
thyl, meta-biphenyl and butyl derivatives (27–30) were not
tolerated. Similarly, spiro fused analogue 31 displayed only
micromolar activity on the Hh-pathway. Two structural changes
which were tolerated, although resulting in several fold loss in
activity, were the replacement with 3-phenylcyclobutane (32)
and indane (33). These two derivatives displayed Smo binding
affinities of 440 and 880 nM respectively, compared to 94 nM
for 22.
Compounds 17 and 22 were selected for further characteriza-
tion, both compounds were able to inhibit the growth of irradiated
Ptch +/- medulloblastoma cells with CC50 = 3.3 and 12 nM respec-
tively.20 Profiling in liver microsomes revealed that whilst the
dichlorophenyl urea 17 display medium turnover in rat micro-
somes (Clint = 180
logue 22 displayed inferior stability (Clint >300
l
L/min/mgP), the more potent 2-biphenyl ana-
L/min/mgP).
l

S. Malancona et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4422–4428
4425
Table 3
SAR monophenyl seriesa
O
N
H
N
N
N
R
Ph
O
O
a
a
Compds
R
Light2 IC50 (nM)
340
Smo binding IC50 (nM)
Compds
R
Light2 IC50 (nM)
140
Smo binding IC50 (nM)
Cl
Cl
Cl
17
220
39
130
F3CO
Cl
Cl
Cl
MeO2C
34
2200
1100
40
330
280
F3C
Cl
Me
Cl
Cl
35
36
37
38
370
530
510
220
460
770
480
310
41
42
43
53
77
62
84
MeO2S
Cl
Cl
Cl
120
150
Me
Cl
NC
NC
Cl
Cl
Cl
F
Me
Cl
F
Cl
a
Values are means of at least two experiments with standard deviations <30% of the mean value.
Table 4
SAR biphenyl seriesa
O
N
H
N
N
N
R
Ph
O
O
a
a
Compds
R
Light2 IC50 (nM)
Smo binding IC50 (nM)
Compds
R
Light2 IC50 (nM)
Smo binding IC50 (nM)
Ph
Cl
22
84
94
52
890
680
N
i-Pr
Cl
44
45
46
180
210
890
53
54
55
18
19
N
CF3
CN
Cl
1100
2400
210
120
140
N
F3C
>2.5
l
M
160
N
(continued on next page)

4426
S. Malancona et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4422–4428
Table 4 (continued)
a
a
Compds
R
Light2 IC50 (nM)
Smo binding IC50 (nM)
Compds
R
Light2 IC50 (nM)
Smo binding IC50 (nM)
OMe
MeO
47
2400
1700
56
82
47
88
N
S
S
48
49
210
220
57
240
N
N
MeN N
O
2000
1800
58
59
1200
16
620
13
N
Ph
N
50
51
28
89
12
88
N
N
Ph
N
a
Values are means of at least two experiments with standard deviations <30% of the mean value.
Encouraged by these results a more extensive SAR study was
introduction of heteroatoms in the internal phenyl ring was toler-
ated, and the pyridine analog 50 demonstrated very high activity in
both assays (Smo binding IC50 = 12 nM, Light2 IC50 = 28 nM).
Although less active, the regioisomer 51 presents comparable
activity to 22, displaying IC50 <100 nM. Given the superior activity
of 50 additional substituents were introduced on the distill phenyl
ring (52–54), and it was discovered that meta-substitution gave the
most active compound (IC50 <20 nM). While the ortho- and para-
substituents were poorly tolerated losing 10- and 30-fold activity
respectively. Further meta-substitutions were examined, like tri-
fluoromethyl (55) and methoxy (56), which both showed good
activity, although less active than 53. Replacement of the terminal
aromatic ring with more polar heterocycles like in 58 was detri-
mental for the activity. Finally, the biphenyl fragment and 2-aryl-
pyrid-3-yl motif was exchanged for the N-phenylpyrazole moiety
in 59 giving one of the most potent compounds identified in this
series, in fact 59 displays Smo binding IC50 = 13 nM.
Some exploratory SAR work into alternatives core scaffolds was
also conducted (Table 5), which demonstrated the critical impor-
tance of the bicyclic hydantoin system. The corresponding phthal-
imide 60 resulted in a 34-fold loss in potency compared to 22.
Similarly, removal of one carbonyl groups of the hydantoin as in
61 was also detrimental for the activity, resulting in a 15-fold loss
of activity. Manipulations of the hydantoin ring by ring expansion
to 6-membered rings (62 and 63) resulted in an even more sub-
stantial loss in affinity.
conducted, aiming to further improve Smo activity and increase
metabolic stability. We decided to optimize the activity in the race-
mic series, preparing mixtures of four isomers with achiral trans 2-
phenylcyclopropyl, and racemic [6,5]-bicyclic hydantoin, relying
on robust chemistry to quickly prepare analogues.
In the optimization work around 17 attempts were made to
modulate potency by variation of the substituents on the phenyl
group (Table 3). When one of the chlorine atom in the 2,3-disubsti-
tution pattern is replaced with methyl ester (34) or methyl (35)
groups a drop in potency was observed. Maintaining the dichloro
substitution pattern attempts were made to modulate the com-
pound properties introducing a further substituent on the phenyl
ring. The introduction of a fluorine in the para-position of the
2,3-dichloro (37) and the 2,6-dichloro systems (38) resulted in a
small drop in binding affinity compared to the corresponding
des-fluorinated analogues. However, the addition of stronger elec-
tron withdrawing groups in the para-position of the 2,6-dichloro-
phenyl motif gave some interesting derivatives (39–42). In
particular, the 4-methylsulfonyl (41) and 4-cyano (42) were very
active compounds. The former was one of the most potent com-
pounds displaying IC50 = 53 nM in the Light2 assay, and
IC50 = 77 nM in the Smo binding assay. These two derivatives illus-
trate that it is possible to derive potent Smo antagonists without
the need to resort to the bulky biphenyl urea.
Meanwhile, the results for the optimization of 2-biphenyl urea
22 are reported in Table 4. Replacement of the ortho-phenyl ring
with small substituents (44–47) was initially attempted, due to
suspicions that this group could be the cause for the high intrinsic
clearance, however, trifluoromethyl, cyano and methoxy groups
were detrimental for affinity. The larger isopropyl substituent
(44) retained some activity, Smo binding IC50 = 210 nM. Com-
pounds where the phenyl had been replaced by five-membered
heterocycles were also prepared, and while thiophene (48) main-
tained activity, the polar 1,3-oxazol-5-yl derivative (49) was poorly
tolerated, resulting in a 20-fold loss of activity. In contrast, the
Table 5
Core structure modificationsa
a
Compds
Light2 IC50 (nM)
Smo binding IC50 (nM)
60
61
62
63
4600
3000
ND
3200
1400
7800
5100
ND
a
Values are means of at least two experiments with standard deviations <30% of
the mean value. ND = not determined.

S. Malancona et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4422–4428
4427
O
O
NH
CO2Me
N
a
b, c or d
Ph
N
H
N
N
BocN
N
N
HN
R
Ph
60 %
O
O
O
64
65
66
Scheme 1. General hydantoin synthesis. Reagents and conditions: (a) (i) trans 2-Phenylcyclopropyl isocyanate, toluene, rt; (ii) DIPEA, reflux; (iii) TFA, DCM; (b) R-NCO, DIPEA,
DCM; (c) RNH2, CDI, TEA, MeCN; (d) RNHC(O)OPh, DIPEA, DCE.
O
O
O
a
b
Ph
Ph
H
N
N
O
N
HO2C
HO2C
Ph
Ph
Ph
Ph
O
O
O
O
67
65
68
60
O
O
d
N
c
N
H
N
N
N
N
HN
O
69
61
O
NBn
NH
f
e
Ph
N
N
N
Ph
H
N
BnN
BocN
N
O
O
N
CO2Et
70
CO2Et
71
O
62
H
N
Ph
N
N
h
g
N
H
N
N
N
64
BocN
O
N
CO2Me
Ph
O
O
Ph
72
63
Scheme 2. Core structure modifications. Reagents and conditions: (a) trans 2-Phenylcyclopropyl amine, DIPEA,
lW (150 °C, 1 h); (b) Biphenyl-amine, HATU, DIPEA, DMF; (c)
BH3.THF, THF, ; (d) RNHC(O)OPh, DCM, TEA; (e) H2, Pd/C, HCl, MeOH then K2CO3, BOC-ON; (f) (i) trans 2-Phenylcyclopropyl isocyanate, DCM; (ii) 4 N HCl in dioxane; (iii)
D
RNHC(O)OPh, DCM, TEA; (g) (i) BrCH2CO2Bn, K2CO3, acetone; (ii) H2, Pd/C, MeOH; (iii) trans 2-Phenylcyclopropylamine, HATU, DIPEA; (g) (i) K2CO3, toluene; (ii) TFA, DCM; (iii)
RNHC(O)OPh, DCM, TEA.
The bicyclic hydantoins were synthesized according to Scheme
1, whereby the required isocyanate was reacted with known 64 at
RT in toluene.21 After initial urea formation, DIPEA was added and
the reaction heated at reflux to afford the Boc-protected bicyclic
hydantoin, which after deprotection gave 65. The urea side chains
were installed by reaction with isocyanates, prepared when neces-
sary from the corresponding amine by reaction with triphosgene or
via Curtius rearrangement from the carboxylic acid. Alternatively,
65 was reacted with either an intermediate formed in situ from
the corresponding amine and CDI, or with a phenyl carbamate
intermediate derived from the amine and phenyl chloroformate.
The alternative ring systems were prepared according to estab-
lished procedures (Scheme 2), either: by condensation with phtha-
lic anhydride (67); from ethyl (1,4-dibenzylpiperazin-2-yl)acetate
(70) using similar chemistry; or by alkyation of 64 with benzyl
bromoacetate, followed by deprotection, and coupling to phenylcy-
clopropylamine and cyclization.
hydantoin ring systems are critical for potency, while a variety of
urea substituents can be tolerated. The combination of these opti-
mal groups gives smoothened antagonists with activity in the low
nanomolar range. Further optimization of this series is the subject
of the accompanying paper.19
Acknowledgments
P.K. Chakravarty and M. Fisher are recognized for their design of
the library from which the leads were identified.
References and notes
1. Rubin, L. L.; de Sauvage, F. J. Nat. Rev. Drug Disc. 2006, 5, 1026.
2. Xie, J. Curr. Oncol. Rep. 2008, 10, 107.
3. Gallinari, P.; Filocamo, G.; Jones, P.; Pazzaglia, S.; Steinkuhler, C. Exp. Opin. Drug
Discov. 2009, 4, 525.
4. Peukert, S.; Miller-Moslin, K. ChemMedChem 2010, 5, 500.
5. Ingham, P. W.; McMahon, A. P. Genes Dev. 2001, 15, 3059.
6. Nusslein-Volhard, C.; Wieschaus, E. Nature 1980, 287, 795.
7. Rohatgi, R.; Scott, M. P. Nat. Cell Biol. 2007, 9, 1005.
8. Epstein, E. H. Nat. Rev. Cancer 2008, 8, 743.
9. 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.
Science 2002, 297, 1559.
In summary, a novel series of [6,5]-bicyclic tetrahydroimi-
dazo[1,5-a]pyrazine-1,3(2H,5H)-dione smoothened antagonists
has been identified, with potential for the treatment of malignan-
cies dependent on the Hh-pathway. This series of compounds has
been extensively explored to ascertain the key detriments for
activity, demonstrating that the trans-cyclopropyl phenyl and
10. Chen, J. K.; Taipale, J.; Cooper, M. K.; Beachy, P. A. Genes Dev. 2002, 16, 2743.

4428
S. Malancona et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4422–4428
11. Robarge, K. D.; Brunton, S. A.; Castanedo, G. M.; Cui, Y.; Dina, M. S.; Goldsmith,
R.; Gould, S. E.; Guichert, O.; Gunzner, J. L.; Halladay, J.; Jia, W.; Khojasteh, C.;
Koehler, M. F. T.; Kotkow, K.; La, H.; LaLonde, R. L.; Lau, K.; Lee, L.; Marshall, D.;
Marsters, J. C.; Murray, L. J.; Qian, C.; Rubin, L. L.; Salphati, L.; Stanley, M. S.;
Stibbard, J. H. A.; Sutherlin, D. P.; Ubhayaker, S.; Wang, S.; Wong, S.; Xie, M.
Bioorg. Med. Chem. Lett. 2009, 19, 5576.
12. Von Hoff, D. D.; LoRusso, P. M.; Rudin, C. M.; Reddy, J. C.; Yauch, R. L.; Tibes, R.;
Weiss, G. J.; Borad, M. J.; Hann, C. L.; Brahmer, J. R.; Mackey, H. M.; Lum, B. L.;
Darbonne, W. C.; Marsters, J. C., Jr.; de Sauvage, F. J.; Low, J. A. N. Engl. J. Med.
2009, 361, 1164.
13. Tremblay, M. R.; Lescarbeau, A.; Grogan, M. J.; Tan, E.; Lin, G.; Austad, B. C.; Yu,
L.-C.; Behnke, M. L.; Nair, S. J.; Hagel, M.; White, K.; Conley, J.; Manna, J. D.;
Alvarez-Diez, T. M.; Hoyt, J.; Woodward, C. N.; Sydor, J. R.; Pink, M.;
MacDougall, J.; Campbell, M. J.; Cushing, J.; Ferguson, J.; Curtis, M. S.;
McGovern, K.; Read, M. A.; Palombella, V. J.; Adams, J.; Castro, A. C. J. Med.
Chem. 2009, 52, 4400.
16. Li, J. J.; Shanmugasundaram, V.; Reddy, S.; Fleischer, L. L.; Wang, Z.; Smith, Y.;
Harter, W. G.; Yue, W.-S.; Swaroop, M.; Li, L.; Ji, C. X.; Dettling, D.; Osak, B.;
Fitzgerald, L. R.; Conradi, R. Bioorg. Med. Chem. Lett. 2010, 20, 4932.
17. Kaizerman, J. A.; Aaron, W.; An, S.; Austin, R.; Brown, M.; Chong, A.; Huang, T.;
Hungate, R.; Jiang, B.; Johnson, M. G.; Lee, G.; Lucas, B. S.; Orf, J.; Rong, M.;
Toteva, M. M.; Wickramasinghe, D.; Xu, G.; Ye, Q.; Zhong, W.; McMinn, D. L.
Bioorg. Med. Chem. Lett. 2010, 20, 4607.
18. Taipale, J.; Chen, J. K.; Cooper, M. K.; Wang, B.; Mann, R. K.; Milenkovic, L.;
Scotts, M. P.; Beachy, P. A. Nature 2000, 406, 1005; 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. J. Biol. 2002, 1, 10.
19. Kinzel, O.; Alfieri, A.; Altamura, S.; Brunetti, M.; Bufali, S.; Colaceci, F.; Ferrigno,
F.; Filocamo, G.; Fonsi, M.; Gallinari, P.; Malancona, S.; Martin Hernando, J. I.;
Monteagudo, E.; Orsale, M. V.; Palumbi, M. C.; Pucci, V.; Rowley, M.; Sasso, R.;
Scarpelli, R.; Steinkühler, C.; Jones, P. Bioorg. Med. Chem. Lett. 2011, 21,
doi:10.1016/j.bmcl.2011.06.023.
14. Pan, S. F.; Wu, X.; Jiang, J. Q.; Gao, W.; Wan, Y.; Cheng, D.; Han, D.; Liu, J.;
Englund, N. P.; Wang, Y.; Peukert, S.; Miller-Moslin, K.; Yuan, J.; Guo, R.;
Matsumoto, M.; Vattay, A.; Jiang, Y.; Tsao, J.; Sun, F.; Pferdekamper, A. C.; Dodd,
S.; Tuntland, T.; Maniara, W.; Kelleher, J. F.; Yao, Y. M.; Warmuth, M.; Williams,
J.; Dorsch, M. A. C. S. Med. Chem. Lett. 2010, 1, 130.
20. Pazzaglia, S.; Tanori, M.; Mancuso, M.; Rebessi, S.; Leonardi, S.; Di Majo, V.;
Covelli, V.; Atkinson, M. J.; Hahn, H.; Saran, A. Oncogene 2006, 25, 1165; Pazzaglia,
S.; Tanori, M.; Mancuso, M.; Gessi, M.; Pasquali, E.; Oliva, M. A.; Rebessi, S.; Di
Majo, V.; Covelli, V.; Giangaspero, F.; Saran, A. Oncogene 2006, 25, 5575.
21. Bigge, C. F.; Hays, S. J.; Novak, P. M.; Drummond, J. T.; Johnson, G.; Bobovski, T.
P. Tetrahedron Lett. 1989, 30, 5193.
15. http://www.clinicaltrials.gov/ct2/results?term=hedgehog.