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

Article abstract of DOI:10.1016/j.bmcl.2011.06.023

The Hedgehog (Hh-) signaling pathway is a key developmental pathway which gets reactivated in many human tumors, and smoothened (Smo) antagonists are emerging as novel agents for the treatment of malignancies dependent on the Hh-pathway, with the most advanced compounds demonstrating encouraging results in initial clinical trials. A novel series of potent bicyclic hydantoin Smo antagonists was reported in the preceding article, these have been resolved, and optimized to identify potent homochiral derivatives with clean off-target profiles and good pharmacokinetic properties in preclinical species. While showing in vivo efficacy in mouse allograft models, unsubstituted bicyclic tetrahydroimidazo[1,5-a]pyrazine-1,3(2H,5H)-diones were shown to epimerize in plasma. Alkylation of the C-8 position blocks this epimerization, resulting in the identification of MK-5710 (47) which was selected for further development.

Full text of DOI:10.1016/j.bmcl.2011.06.023

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4429–4435
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-phenylcyclo-
propyl]-N-(1-phenyl-1H-pyrazol-5-yl)hexahydro-imidazo[1,5-a]pyrazine-7
(1H)-carboxamide), a potent smoothened antagonist for use in Hedgehog
pathway dependent malignancies, Part 2
Olaf Kinzel, Anna Alfieri, Sergio Altamura, Mirko Brunetti, Simone Bufali, Fabrizio Colaceci,
Federica Ferrigno, Gessica Filocamo, Massimiliano Fonsi, Paola Gallinari, Savina Malancona,
Jose Ignacio Martin Hernando, Edith Monteagudo, Maria Vittoria Orsale, Maria Cecilia Palumbi,
⇑
Vincenzo Pucci, Michael Rowley, Romina Sasso, 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 gets reactivated in many
human tumors, and smoothened (Smo) antagonists are emerging as novel agents for the treatment of
malignancies dependent on the Hh-pathway, with the most advanced compounds demonstrating encour-
aging results in initial clinical trials. A novel series of potent bicyclic hydantoin Smo antagonists was
reported in the preceding article, these have been resolved, and optimized to identify potent homochiral
derivatives with clean off-target profiles and good pharmacokinetic properties in preclinical species.
While showing in vivo efficacy in mouse allograft models, unsubstituted bicyclic tetrahydroimi-
dazo[1,5-a]pyrazine-1,3(2H,5H)-diones were shown to epimerize in plasma. Alkylation of the C-8 posi-
tion blocks this epimerization, resulting in the identification of MK-5710 (47) which was selected for
further development.
Keywords:
Hedgehog pathway
Smoothened antagonist
Hedgehog inhibitor
Ó 2011 Elsevier Ltd. All rights reserved.
The hedgehog (Hh-) signaling pathway is a one of several key
developmental pathways which regulate patterning, growth and
cell migration in most tissues and organs, and while it plays an
important role during embryonic development its role in adult-
hood is limited to a tissue maintenance and repair.1–4 Hh-signal
transduction is initiated by binding of the Hh-protein to its cellular
membrane receptor Patched (Ptch), thereafter binding relieves
Ptch-mediated repression of the GPCR-like receptor smoothened
(Smo). Activation of Smo then initiates an intracellular signalling
cascade, culminating in the translocation of the Gli-transcription
factors, Gli-1, -2, into the nucleus where they regulate genes in-
volved in differentiation, proliferation and survival. Evidence has
emerged showing that the pathway is aberrantly activated in a
wide number of cancers, through mutations in Ptch and Smo, as
well as downstream factors SUFU and Gli.1,2 Several agents have
been demonstrated to show preclinical antitumor activity in ani-
mal models,3,5 including the natural product cyclopamine, and var-
ious synthetic Smo antagonists. Recently, the most advanced of
these, vismodegib (GDC-0449),6 has demonstrated encouraging re-
sults in phase I studies in advanced basal cell carcinoma.7 These re-
sults suggest blockage of the Hh-pathway is an attractive approach
to target defined tumors dependent on this pathway.
We have previously reported the identification and initial SAR
exploration of
a novel series of [6,5]-bicyclic tetrahydroimi-
dazo[1,5-a]pyrazine-1,3(2H,5H)-dione smoothened antagonists
with potential for the treatment of Hh-pathway dependent malig-
nancies, exemplified by 1 and 2 (Fig.1).8
This article describes the subsequent optimization of this series
of hydantoins Smo antagonists, culminating in identification of the
O
O
N
Cl
N
H
N
H
N
N
N
N
Cl
N
Ph
Ph
O
O
O
O
⇑
Corresponding author at present address: Belfer Institute for Applied Cancer
Sciences, Dana Farber Cancer Institute, 450 Brookline Avenue, Boston, MA 02115,
USA. Tel.: +1 617 582 9648.
1
2
E-mail address: philip_jones@dfci.harvard.edu (P. Jones).
Figure 1. Lead Smo antagonists.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.06.023

4430
O. Kinzel et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4429–4435
Table 1
bodipy-cyclopamine from HEK-293 cells expressing recombinant
human Smo is measured (Smo Bind).11 The latter was conducted
in 2% fetal calf serum (FCS), and also in 20% normal human serum
(NHS). The individual stereoisomers displayed slight differences in
activity, around a 4-fold difference (Table 1).
Biological activities of lead Smo antagonists, and their individual stereoisomersa
Compd
Light2 IC50 (nM)
Smo Bind 2% FCS IC50 (nM)
1
2
84
94
340
100
42
220
48
21
3 (Single isomer of 1)
4 (8aS, 1S, 2R isomer of 1)
5 (Single isomer of 1)
6 (Single isomer of 1)
7
8
9
10
In parallel, a chiral synthesis of the four individual stereoiso-
mers was conducted to yield 7–10 (Scheme 1). Firstly, trans 2-phe-
nylcyclopropylamine 11 was resolved into its individual tartaric
acid salts 12 and 13 as described by Newman,12 and converted to
the corresponding free amines (14 and 15). In turn, each enantio-
mer was coupled with homochiral 4-[(benzyloxy)carbonyl]-1-
(tert-butoxycarbonyl)piperazine-2-carboxylic acids 16 and 17.
Following removal of the Boc-protecting group, cyclization to the
desired hydantoin using triphosgene at rt was attempted. Instead
of closure to the desired bicyclic scaffold, cyclization occurred on
the amide oxygen atom to give the isomeric 1-imino-3-oxotetrahy-
dro[1,3]oxazolo[3,4-a]pyrazine core 19 in modest yield. This mate-
rial was carried forward, and following removal of the protecting
group and urea formation, the five membered ring was reopened
to homochiral piperazine carboxamide 22 by stirring with 0.2 M
HCl in dioxane for 1 h at rt. Ring closure with two equivalents of
triphosgene in the presence of DBU in DCM at ꢀ30 to ꢀ10 °C suc-
cessfully yielded the required bicyclic hydantoin 7 after 45 min,
with high stereochemical purity (>90% de by chiral SFC). Similar re-
sults were obtained during the preparation of stereoisomers 8–10.
The four stereoisomers displayed a similar four fold shift in
activity from the most potent to the weakest Smo antagonist (Ta-
ble 1). The stereochemistry of the 2-phenylcyclopropylamine
proved to be the key determinant for activity as the pair of com-
pounds bearing the 1S,2R stereochemistry (7 and 9) displayed
the highest affinity. The chiral centre on the bicyclic framework
influenced the activity to a limited extent, with the S-stereochem-
istry (7) being the more active configuration. The R-diastereomer
(9) was around two fold weaker than 7 in both Light2 (IC50 = 270
140
50
95
35
270
880
430
1400
230
670
650
1700
a
Values are means of at least five experiments (SD were within 25% of the IC50
values).
preclinical candidate MK-5710 (47) that displays good pharmaco-
kinetics in preclinical species, demonstrates sustained target
engagement in vivo, and striking anti-tumor efficacy in a mouse
allograft tumor model dependent on the Hh-signalling pathway.
One of the initial tasks of the lead optimization program was to
determine which of the stereoisomers of 1 and 2 was responsible
for the biological activity, as the initial characterization of these
compounds was conducted on mixtures of four stereoisomers
resulting from the hydantoin chiral centre and racemic trans 2-
phenylcyclopropylamine. Consequently, the mixture of biphenyl
urea 1 was deconvoluted to the stereoisomers 3–6 by chiral super-
critical fluid chromatography (SFC).9 The individual stereoisomers
were profiled in both a reporter gene assay in an engineered mouse
NIH 3T3 cell line stably incorporating a Gli-dependent firefly lucif-
erase reporter gene where the ability of the compounds to inhibit
Hh-signalling following activation with the agonist Purmorph-
amine is measured (Light2),10 and in a Smo binding assay where
the ability of the antagonists to displace a fluorescently labeled
OH
OH
-
-
CO2
CO2
HO2C
HO2C
iii
i
ii
iii
OH
OH
(R)
(S)
(S)
(R)
H2N
Ph
H3+N
Ph
H2N
Ph
v
H3+N
Ph
H2N
Ph
(+/-)
14
12
11
13
15
O
O
vi
iv
N
N
NH
NBoc
CO2H
O
O
H
CbzN
HN
CbzN
N
CbzN
N
N
O
16
18
19
20
Ph
ix
Ph
Ph
iv-ix
vii
O
O
O
viii
Cl
N
Cl
N
Cl
NH
Cl
N
H
H
H
N
H
N
N
HN
O
O
(S)
(R)
(R)
(S)
Cl
N
N
Cl
N
N
Cl
N
Cl
N
N
(S)
(S)
Ph
Ph
Ph
O
O
N
O
O
O
O
8
7
22
21
Ph
O
O
iv-ix
iv-ix
NBoc
Cl
N
Cl
N
H
H
N
N
(R)
CbzN
(R)
(S)
(S)
Cl
N
N
Cl
N
N
CO2H
(R)
(R)
Ph
Ph
O
O
O
O
9
17
10
Scheme 1. Initial synthetic procedure towards single stereoisomeric Smo antagonists. Reagents and conditions: (i)
D-tartaric acid, EtOH, D, then three recrystallizations,
EtOH/H2O (4:1) (31%). (ii) -tartaric acid, EtOH, , then three recrystallizations, EtOH/H2O (4:1) (32%). (iii) 2 N NaOH/Et2O (95%). (iv) HATU, DIPEA, DMF; then TFA/DCM (85–
L
D
99%). (v) 0.33 equiv triphosgene, 10 equiv DIPEA, DCM, rt (48–55%) (vi) H2, Pd/C, MeOH (quant.). (vii) Ar-NCO, DIPEA, DCE (50–76%). (viii) 0.2 M HCl, dioxane (quant.). (ix)
2 equiv triphosgene, 5 equiv DBU, DCM, -30 to -10 °C (10–55%).

O. Kinzel et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4429–4435
4431
and 430 nM) and Smo binding assays (230 and 640 nM respec-
tively). Subsequent synthesis revealed that the most active biphe-
On the other hand, the 2,3-dichloroanilide 7 although displayed
slightly inferior affinity, with Smo binding IC50 = 230/690 nM (2%
FBS/20% NHS), it proved to have more benign pharmacokinetics
profile. In liver microsomes, 7 displayed modest turnovers with
nyl urea
4 also displayed the 8aS, 1S, 2R stereochemistry
(Scheme 2). Subsequently, further SAR was conducted using this
stereoisomer (Table 2).
Clint = 170, 105, and 73 lL/min/mgP in rat, dog and human micro-
A more robust synthetic pathway to the desired homochiral
framework was established (Scheme 2), whereby the homochiral
1S,2R 2-phenylcyclopropylamine 14 was converted to the corre-
sponding isocyanate 23 with triphosgene. Reaction with 1-tert-bu-
tyl 3-methyl (3S)-piperazine-1,3-dicarboxylate 24 gave first the
corresponding urea 25, which could then be cyclized with 4 M
HCl in dioxane to give the hydantoin scaffold 26 in excellent yield
and high stereochemical purity. Coupling with the desired isocya-
nate formed the required Smo antagonists in good yield, for exam-
ple reaction with [1,10-biphenyl]-2-yl-isocyanate gave 4 in 67%
yield, 92% de.
Given the interest in 4 and 7 they were extensively profiled.
Both compounds were able to inhibit the proliferation of irradiated
Ptch +/ꢀ medulloblastoma cells with CC50 = 2 and 12 nM respec-
tively.13 The biphenyl derivatives typically proved to be the more
potent analogues, and 4 displayed IC50 = 21 nM in the binding as-
say in the presence of 2% FBS, with around a 3-fold serum shift
in the presence of 20% NHS, IC50 = 70 nM, reflecting the relatively
high plasma protein binding (PPB), fu = 4.4%. This derivative dis-
somes respectively. In vivo in both rats and dogs low-moderate
clearances were measured, 46 and 4 mL/min/kg respectively.
The compound displayed acceptable bioavailability (F = 30%) and
T1/2 = 2.2 h in rats; and excellent bioavailability (F = 97%) and long
terminal half life in dogs, T1/2 = 10 h. In ancillary screening both
compounds displayed micromolar activity on IKr, IKs and Cav1.2
cardiac ion channels, as well binding to the adenosine transporter
and histamine H1.
In the interests of optimizing this series further (Table 2), SAR
was conducted with the aims to improve the pharmacokinetics
properties in the bi(hetero)aryl ureas, and improving the potency
whilst maintaining the good PK profile in the monoaryl ureas. In
both cases given the relatively high lipophilicity of these com-
pounds with high PPB and micromolar ion channel activity and
CYP inhibition, efforts focused on the introduction of polar groups
and heteroatoms, building on previous knowledge from the race-
mic series.8
In the monoaryl series derivatives 34–37 were prepared intro-
ducing the electron deficient substituents in the para position of
a 2,6-dichloroanilide. All four derivatives displayed higher affinity
than 7, most notably the dimethylcarboxamide 36, and the meth-
ylsulfone 37, both of which displayed double digit affinity in the
Smo binding assay (IC50 = 77 and 42 nM respectively), with the lat-
ter displaying minimal serum shift. The introduction of these
played low microsomal stability, with Clint >300 and 120 lL/min/
mgP in rat and human microsomes respectively, and relatively
high clearance in rats, Cl = 56 mL/min/kg (Table 3). Furthermore,
4 was also a weak inhibitor of CYP450s 2C9 and 3A4, 50% inhibition
at 10 lM.
O
O
O
(R)
N
(S)
i
ii
iiii
iv
N
N
H
Ph
N
H
N
(S)
(R)
N
(R)
(S)
(S)
N
(S)
N
N
H2+N
Cl-
(R)
(S)
(S)
BocN
(S)
(S)
OCN
Ph
H2N
Ph
(S)
CO2Me
Ph
O
Ph
O
O
O
NH
14
23
25
26
4
(S)
BocN
v
CO2Me
vi
O
24
O
N
H
N
N
N
(R)
N
(R)
(S)
(S)
BocN
Ar
(S)
27
Ph
O
Ph
O
34-44
vii, viii
O
O
O
O
O
N
N
v
ix
N
(S)
N
N
(S)
N
(R)
(R)
BocN
H
N
(R)
N
N
(S)
(R)
(R)
BocN
(R)
(R)
(S)
(S)
HN
N
Ph
Ph
O
Ph
Ph
O
O
O
30
Peak 2: 29
Peak 1: 28
ix
NH2
N
45
N
O
x
N
32
N
(S)
(R)
Ph
HN
(S)
H
N
OPh
N
O
N
31
v
O
N
xi
33
O
O
N
O
N
H
H
N
(S)
(S)
(R)
(R)
(S)
(S)
N
N
N
N
N
N
Ph
Ph
O
O
O
46
47
Scheme 2. Modified synthetic procedure for the preparation of single stereoisomeric Smo antagonists. Reagents and conditions: (i) triphosgene, NaHCO3 (aq)/DCM (98%). (ii)
DCM, 1 h. (iii) 4 M HCl/dioxane, 2 h (95%, 92%de). (iv) [1,10-biphenyl]-2-yl-isocyanate, DIPEA, DCM, 1 h (67%, 92%de). (v) [1,10-biphenyl]-2-yl-isocyanate, DIPEA, DCM, 1 h. (vi)
Boc2O, DIPEA, DCM (quant.). (vii) LiHMDS, THF, ꢀ78 °C, 30 min then MeI ꢀ78 °C to rt (77%) (viii) Chiral SFC purification. (ix) TFA, DCM. (x) PhOCOCl, DIPEA, DCE, 1 h (94%). (xi)
33, DCM, Et3N (90%).

4432
O. Kinzel et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4429–4435
Table 2
Biological activities of homochiral Smo antagonistsa
Compd
Light2 IC50 (nM)a
Smo Bind 2% FCS IC50 (nM)a
Smo Bind 20% NHS IC50 (nM)a
4
42
21
70
7
270
230
690
Cl
34
140
120
120
180
100
77
520
450
120
F
Cl
Cl
35
36
37
NC
Cl
Cl
Me2NOC
MeO2S
Cl
Cl
41
28
42
12
68
12
Cl
38
39
N
Cl
5
5
4
15
22
N
NC
40
16
N
S
41
42
43
89
18
8
21
15
18
42
18
32
N
N
N
Cl
N
N
F
44
6
7
9
N
N
45
46
47
1400
23
1200
19
1600
60
17
13
22
a
Values are means of at least five experiments (SD were within 25% of the IC50 values).
trisubstituted anilines also improved the PK properties (Table 3),
and all four compounds showed improved stability compared to
7 in liver microsomes, notably 35–37 also displayed lower clear-
ance in rats. Indeed 35 showed remarkable low rat clearance,
Cl = 2 mL/min/kg, but this was probably a result of very high PPB
in rats (fu = 0.7%). The overall profile of 37 in rats was encouraging,
with low clearance (Cl = 5 mL/min/kg), excellent oral bioavailabil-
ity (F = 74%) and moderate half life (T1/2 = 3.2 h). However, in dogs

O. Kinzel et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4429–4435
4433
Table 3
Pharmacokinetic properties of individual stereoisomeric Smo antagonists
Microsomes Clint
(
l
L/min/mgP)
Rat PK
T1/2 (h)
Dog PK
T1/2 (h)
Compd
Rat
Dog
Human
Cl (mL/min/kg)
Vdss (L/kg)
F (%)
Cl (mL/min/kg)
4
Vdss (L/kg)
F (%)
4
>300
170
117
109
58
ND
105
ND
83
ND
39
120
73
64
66
49
58
45
90
37
40
31
62
45
36
56
46
2.7
2.2
8
8
ND
30
7
10
2.5
97
34
35
36
37
38
39
40
41
42
43
44
47
2
41
5
54
54
48
58
43
5.4
0.6
3.2
4.9
1.3
3.8
10
0.5
2.1
0.6
6.7
4.1
6
56
ND
74
31
ND
43
41
88
53
81
1.7
1.0
0.3
ND
60
133
43
147
40
91
49
50
39
10
2.9
7.6
5.5
3.1
2.5
1.0
1.0
ND
82
1.3
113
80
ND
39
55
94
22
5.1
2.9
40
22
1.1
2.0
52
despite the low clearance (Cl = 1.7 mL/min/kg) the low volume of
distribution resulted in a short terminal half life (T1/2 = 1.0 h).
Previous work had illustrated that 2-aryl-3-aminopyridines and
N-aryl-5-aminopyrazoles to be suitable replacements to the biphe-
nyl-2-amine,8 and accordingly these groups were explored further.
As expected, the 2-aryl-3-aminopyridines 38–41 proved to be po-
tent Smo antagonists, with 39 and 40 bearing the meta substitu-
ents on the remote phenyl ring being some of the most potent
compounds identified, displaying IC50 = 4 and 16 nM in 2% FCS
respectively, and IC50 = 15 and 22 nM in the high serum conditions.
The introduction of the pyridyl group served both to alleviate the
micromolar ion channel and CYP inhibition activities, and also to
remedy the high microsomal turnover. In rats all four derivatives
(38–41) displayed clearances in the range 48–58 mL/min/kg, but
as a result of larger Vdss they displayed longer half lives than the
corresponding dichloroanilines. Oral bioavailabilities were also
2,500.0
2,000.0
1,500.0
1,000.0
500.0
vehicle BID
40 mg/kg BID
80 mg/kg BID
120 mg/kg BID
160 mg/kg BID
80 mg/kg QD
0.0
0
5
10
15
20
Days
200
180
160
140
120
100
80
60
40
20
0
0
10
Time (hrs)
20
Figure 2. Efficacy of 42 in medulloblastoma bearing mice,13 following oral dosing at 40, 80, 120 and 160 mg/kg bid and 80 mg/kg qd as suspension in 0.24% SDS/0.5%
Methocel. PD of 42 in tumor biopsies from medulloblastoma bearing mice, following a single oral 80 mg/kg dose.

4434
O. Kinzel et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4429–4435
number of very interesting compounds (42–44). The prototype
120
100
80
60
40
20
0
42 proved to be a very potent Smo antagonist, Light2 IC50 = 18 nM,
with no serum shift in the binding assay (IC50 = 15/18 nM, 2% FCS/
20% NHS) as a result of the large free fraction in human plasma,
fu = 17%. Unlike the pyridine series, the introduction of meta sub-
stituents on the remote phenyl ring had minimal influence on
the affinity. All three derivatives were devoid of ion channel and
CYP liabilities at 10 lM, while in microsomal turnover studies all
three compounds showed substantial improvements compared to
the biphenyl derivative 4. Interestingly, the meta substituted ana-
logues 43 and 44 showed slightly inferior stability compared to
the parent 42. Accordingly 42 was profiled in vivo in both rats
and dogs, where it displayed excellent oral bioavailability in both
species (F = 88 and 82% respectively) with moderate clearance
(Cl = 43 and 5.5 mL/min/kg) and comparable half lives in both spe-
cies (T1/2 = 1.3 and 2.5 h).
0
50
100
Time (Min)
150
200
In view of the encouraging overall profile 42 was profiled to see
if it could inhibit the proliferation of irradiated Ptch +/ꢀ medullo-
blastoma cells which are dependent on the pathway,13 and 42 dis-
played CC50 = 1 nM. Good exposure of 42 could be obtained in mice
upon oral administration (AUC = 6.4[40 mg/kg], 15[80 mg/kg], 24[160 mg/kg]
Figure 3. Epimerization of 42 in rat (squares) and human (diamonds) plasma, and
formation of diastereomer (dashed lines).
acceptable (F = 31–43%). Encouraged by the rat PK where 41 dis-
played a long terminal half life (T1/2 = 10.4 h), 41 was profiled also
in dogs where despite displaying moderate clearance (Cl = 7.6 mL/
min/kg), the T1/2 was a more modest 3.1 h.
Turning attention to the N-aryl-5-aminopyrazoles, incorpora-
tion of this functionality into the bicyclic hydantoin series gave a
lM h), and therefore 42 was evaluated in a primary mouse
medulloblastoma allograft model. When the subcutaneous tumors
reached an average volume of 400 mm3 mice were randomized
and treated orally at 40, 80, 120 and 160 mg/kg bid for 3 weeks,
and also 80 mg/kg qd. All doses were well tolerated, with less than
3000
2500
2000
1500
1000
500
Control
40 mg/kg BID
80 mg/kg BID
120 mg/kg BID
160 mg/kg BID
0
0
5
10
15
20
25
30
Days
120
100
80
60
40
20
0
0
2
4
6
8
10 12 14 16 18 20 22 24
Time
Figure 4. Efficacy of 47 in medulloblastoma bearing mice,13 following oral dosing at 40, 80, and 160 mg/kg bid as suspension in 0.24% SDS/0.5% Methocel. PD of 47 in tumor
biopsies from medulloblastoma bearing mice following a single oral 80 mg/kg dose.

O. Kinzel et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4429–4435
4435
10% body weight loss, and no visible physical signs or mortality.
Tumor growth inhibition was observed in all groups (Fig. 2), with
regression being observed at doses of 80 mg/kg bid and upwards.
It appears that in this preclinical model it is necessary to inhibit
the pathway completely for 24 h in order to see optimal anti-tu-
mor activity, as 80 mg/kg qd gives only modest tumor growth inhi-
bition, and this is inferior to the same quantity of compound
administered twice-a-day (i.e., 40 mg/kg bid). Similar finding have
been seen by other groups.14 Parallel, pharmacodynamic (PD) stud-
ies in tumor bearing mice demonstrated that following a single
oral 80 mg/kg dose strong inhibition of the Hh-pathway is ob-
served in tumor biopsies, causing a >80% reduction of Gli-1 mRNA
between 4 and 12 h.
Despite the encouraging profile, and the demonstration of
in vivo anti-tumor activity concern about possible epimerization
of the hydantoin ring stimulated deeper investigation.15 Indeed
epimerization occurs in both rat and human plasma (Fig. 3), with
around 30% conversion within 3 h to a biologically active epimer
(Smo binding IC50 = 50 nM (2% FCS)).
doses of 47 at 40, 80, and 160 mg/kg bid all gave rise to sustained
tumor regression and no body weight loss or adverse effects
were observed in any group. Efficacious exposures correspond to
AUC0-inf = 10.9, 24.6, and 133
lM h with Cmax = 7.8, 19.6 and
41.7 M. Pharmacodynamic studies in tumor biopsies revealed
l
47 leads to a time-dependent down-regulation of Gli-1 mRNA with
77% down regulation at 4 h, and >85% pathway inhibition at 6 and
8 h following dosing at 80 mg/kg.
In summary, a novel series of potent bicyclic hydantoin Smo
antagonists has been developed, with clean off-target profiles
and good pharmacokinetic properties in preclinical species. This
series of compounds demonstrates target engagement in tumor
biopsies, and anti-tumor activity in mouse allograft models depen-
dent on the Hh signalling pathway. On the basis of this data MK-
5710 (47) was selected for further development.
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.
In order to avoid this epimerization, attempts were made to
block this position by the addition of a methyl group (Scheme 2).
The hydantoin scaffold 47 could readily be alkylated in excellent
yield by adding LiHMDS to a thoroughly degassed solution of 27
in THF at ꢀ78 °C, and quenching with MeI. The diastereomers were
separated by chiral SFC chromatography on a Chiralpak AD-H col-
umn eluting with 15% IPA/CO2 to give first 8S-diastereomer 28 and
then 8R-diastereomer 29. Boc-deprotection and urea formation
readily gave 46 and 45, the direct analogues of 4 and 9. Interest-
ingly, unlike the unsubstituted compounds where there was only
a two-fold difference in activity between diastereomers, in the case
of the methylated scaffold there was a striking difference. Whilst
46 displayed comparable activity to 4 in the Smo binding assays,
IC50 = 19 and 21 nM respectively in 2% FCS, and IC50 = 60 and
70 nM in 20% NHS, the other diastereomer 45 proved to be more
3. Gallinari, P.; Filocamo, G.; Jones, P.; Pazzaglia, S.; Steinkuhler, C. Exp. Opin. Drug
Discov. 2009, 4, 525.
4. Ingham, P. W.; McMahon, A. P. Genes Dev. 2001, 15, 3059.
5. Peukert, S.; Miller-Moslin, K. ChemMedChem 2010, 5, 500.
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than 50-fold less active (IC50 = 1.1 lM). Encouraged by this result,
47 the direct analogue of 42 was prepared from key intermediate
31 using the phenyl carbamate 33 generated in situ. This derivative
maintained all the beneficial characteristics of the unsubstituted
compound (activity, selectivity, PK and activity in vivo), whilst
eliminating the potential for epimerization. In fact, 47 proved to
be an extremely potent Smo antagonist displaying IC50 = 17 nM
in the Light2 cells, whilst binding to Smo with IC50 = 22 nM in
20% NHS. Indeed, 47 inhibited the medulloblastoma proliferation
with CC50 = 0.4 nM.16
No unwanted off-target activities were detected at 10 lM on a
panel of over 150 enzyme and receptor binding assays, while pro-
filing on the cardiac ion channels showed only minimal functional
inhibition of hERG (27% inh. at 30
30 M). In an anesthetized, vagotomized CV dog study no signifi-
cant changes to CV or hemodynamic parameters were measured
following iv infusion (Cmax = 16.0 M). Furthermore, no inhibition
of CYP450s 2D6, 2C9 or 3A4 was observed (IC50 >40 M).
lM), and INa (45% block at
l
l
l
The pharmacokinetic properties of 47 were similar to those of
the unsubstituted derivative. In rats, 47 displayed good PK proper-
ties, with low clearance (Cl = 22 mL/min/kg), T1/2 = 5.1 h, and good
oral bioavailability (F = 40%). The dog PK of 47 is acceptable, and
despite the high Clint in microsomes, in vivo the compound dis-
plays Cl = 21 mL/min/kg and good oral bioavailability (F = 46%).
Efficacy in the primary mouse medulloblastoma allograft model
was also demonstrated at well tolerated doses (Fig. 4). Daily oral
16. The stereochemistry of the 8a-methyltetrahydroimidazo[1,5-a]pyrazine-
1,3(2H,5H)-dione scaffold was confirmed by independent synthesis starting
from
a-aminomethylalanine methyl ester. Yi, H.; Hegedus, L. S. J. Org. Chem.
1997, 62, 3586.