Potent agonists of the Hedgehog signaling pathway

Article abstract of DOI:10.1016/j.bmcl.2009.05.096

A family of biaryl substituted 1,4-diaminocyclohexanamides of 3-chlorobenzothiophene-2-carboxylic acid is reported as picomolar modulators of Hedgehog protein function. SAR for the 1,4-diaminocyclohexane group is shown to be exquisitely sensitive to subst

Full text of DOI:10.1016/j.bmcl.2009.05.096

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4308–4311
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Potent agonists of the Hedgehog signaling pathway
a
b
b
b
Shirley A. Brunton a, , John H. A. Stibbard , Lee L. Rubin , Oivin M. Guicherit , Lawrence I. Kruse ,
Stephen Price a, Raffaella di Lucrezia a, Colin H. MacKinnon a, Alaric Avery a, Yvonne Park a,
Danielle Buxton a, Edward A Boyd a
*
a Evotec, 114 Milton Park, Abingdon, Oxfordshire OX14 4SA, UK
b Curis Inc, 61 Moulton St, Boston, MA 02138-1118, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A family of biaryl substituted 1,4-diaminocyclohexanamides of 3-chlorobenzothiophene-2-carboxylic
acid is reported as picomolar modulators of Hedgehog protein function. SAR for the 1,4-diaminocyclohex-
ane group is shown to be exquisitely sensitive to substitution on the 4-amino group, and SAR for the 3-
chlorobenzothiophene group is highly specific. Preliminary SAR studies of the biaryl substituent led to a
picomolar compound with in vivo activity.
Received 23 April 2009
Revised 18 May 2009
Accepted 20 May 2009
Available online 29 May 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Hedgehog signaling pathway
Agonist
SAR study
Small molecule
The Hedgehog (Hh) family of proteins plays an essential role in
embryonic development.1–3 The protein ligands for the receptor,
Sonic (SHh), Indian (IHh), and Desert (DHh), as well as the other
protein components of the Hedgehog cascade, show very high
interspecies homology, from species as diverse as Drosophila and
human. The Hh protein receptor, Patched-1 (Ptch-1), is a trans-
membrane protein with a structure reminiscent of that found in
ion channels. Ptch-1 inhibits Smoothened (Smo), a receptor struc-
turally similar to G protein coupled receptors. Upon binding of an
Hh protein ligand to Ptch-1, the normally inhibited Smo is disin-
hibited and it activates the nuclear transcription factor Gli-1 via
a signaling cascade.
The three Hedgehog ligands, SHh, IHh, and DHh, are morphogens
which have different roles in embryogenesis. Thus, IHh affects carti-
lage and bone development, SHh influences neuronal development
in the CNS, and DHh regulates peripheral neuronal development.
The Hh pathway is essential for the formation of normal nerves
in the central and peripheral nervous system. It has been shown
that treatment with a Hh protein accelerates the restoration of
nerve function in models of trauma and disease.4 This indicates
that the Hh pathway may have a potential therapeutic effect in
treating certain neurological disorders (e.g., stroke, Parkinson’s dis-
ease, spinal cord injury and diabetic neuropathy).
known.6 We previously disclosed the structures of three com-
pounds from a family of biaryls7 (which are also described in this
Letter; 8, 11 and 21i) which were shown to activate the Hh path-
way by binding to Smo and we now wish to report an SAR study
which led to an improved compound (21k) with picomolar po-
tency and in vivo activity.
Biaryl 8 was an HTS hit8 whose structure was confirmed by
resynthesis. This compound has three obvious regions for struc-
tural manipulation as indicated in Figure 1: the 3-chlorobenzothi-
ophene, the 1,4-diaminocyclohexane, and the biaryl group. Here
we report initial results which demonstrate that both the 1,4-
diaminocyclohexane and the 3-chlorobenzo thiophene regions
are exquisitely sensitive to structural variation. Further, prelimin-
ary SAR at the biaryl group led to compounds with picomolar po-
tency and in vivo activity.
Initially, we investigated the importance of the central diamine
functionality for activity. A series of compounds was made with
NC
O
Cl
N
S
MeO
While several small molecule Hh pathway inhibitors have been
reported,5 small molecule activators of this pathway are less well
NH2
8
* Corresponding author. Tel.: +44 1235 441228; fax: +44 1235 838931.
E-mail address: shirley.brunton@evotec.com (S.A. Brunton).
Figure 1. Initial Hh agonist screening hit.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.05.096

S. A. Brunton et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4308–4311
4309
NC
NC
NC
NH2
X
O
O
Cl
Cl
(
iii) or (iv)
(ii)
(i)
NH
X
N
X
N
Y
S
S
O
O
O
1a: x = NHBoc
1b: x = NMeBoc
(ii) or (vi)
(iii)
(
iii) or (v)
NC
NC
O
X =
O
Cl
2: H
Y =
8: trans-4-NH2
R1
N
3: trans-4-OH
4: trans-4-NHBoc
N
Z =
S
O
13: trans-4-NHEt
9: trans-4-NEtBoc
O
10: cis-4-NH2
5: cis-4-NHBoc
14: trans-4-NMe2
15: trans-4-CH2NHMe
6: trans-4-NMeBoc
11: trans-4-NHMe
NH
7: trans-4-CH2NHBoc
12: trans-4-CH2NMeBoc
z
16 a-l
R2
Scheme 1. Synthesis of 1,4-diaminocyclohexane analogs. Reagents and conditions: (i) Na(OAc)3BH, 20a, EtOH; (ii) acid chloride, Et3N, CH2Cl2, rt, 18 h; (iii) concd HCl, EtOH
(1:2), rt, 3 h; (iv) NaH, alkyl halide, THF, rt, 2 h; (v) alkyl aldehyde, Na(OAc)3BH, toluene, EtOH, rt, 18 h; (vi) carboxylic acid, TBTU, CH2Cl2, rt, 18 h.
Table 1
SAR at the 1,4-diaminocyclohexane ring
a
a
a
X
Entry
EC50 (nM)
Y
Entry
EC50 (nM)
Z
Entry
EC50 (nM)
H
2
3
>10,000
5000
trans-4-NH2
cis-4-NH2
trans 4-NHMe
8
10
11
5000
>10,000
40 ( 20), Amax = 80%
trans-4-NHEt
trans-4-NMe2
trans-4-CH2NHMe
13
14
15
>10,000
>10,000
>10,000
trans-4-OH
a
EC50 and Amax values of final product defined according to Ref. 8.
modifications to this part of the molecule (Scheme 1, Table 1). The
general route to the target compounds (Scheme 1) involved the
reductive amination of aldehyde 20a (Scheme 2) with a variety
of Boc protected amines to give the corresponding secondary
amines. Amide formation from these amines with 3-chlorobenzo-
thiophene-2-carbonyl chloride gave final compounds 2 and 3 and
intermediates 4–7. Boc-deprotection of intermediates 4–6 gave
target amines 8, 10–11. Target amines 13 and 15 were prepared
by alkylation of the corresponding Boc-protected amines (4 and
7) using the relevant alkyl halide, followed by Boc-deprotection.
An alternative procedure was used to prepare the tertiary amine
14, by reductive alkylation of 11 with formaldehyde.
hexyl analog 2 which lacks the 4-amino group. The hydroxy analog
3 was also only weakly active. A 100-fold boost in potency over 8
occurred with the trans-4-NH-methyl analog 11, whereas the lar-
ger trans-4-NH-ethyl analog 13 was much less active, as was the
extended trans-4CH2NH-methyl derivative 15. The trans-4-N,N-di-
methyl analog 14 also showed very little activity. An extended ser-
ies of other much less active analogs (Table 1a, Supplementary
data) further underscores the exceptional activity of the N-methyl
analog 11. The very specific and potent activity of 11 may result
from a more basic secondary amine9 with a very small, lipophilic
substituent (methyl) which binds a receptor region that is quite
intolerant of steric bulk.
The SAR data in Table 1 demonstrate the critical dependence of
activity upon the trans-1,4-diaminocyclohexane scaffold. The cis
analog 10 was much less active than 8 (>10 lM), as was the cyclo-
SAR of the 3-chlorobenzothiophene group was next investi-
gated and a series of structurally related amides was prepared. A
few derivatives were based around the original hit 8, but the
O
Cl
R3
(OH)2B
(OH)2B
N
NH
O
2
ii)
i)
1
S
3
R1
R2 R3
(OH)2B
R1
H
R1
19a: 6-OMe
19b: 4-OMe
19c: 4-OMe
H
H
F
H
H
F
R1
R1
4
R2
6
17a: 6-OMe
17b: 6-Me
17c: 6-F
18a: 6-OMe
18b: 6-Me
18c: 6-F
R1
5
NBoc
NBoc
17d: H
18d: H
17e: 4-OMe
18e: 4-OMe
iii)
iii), iv)
O
iii)
Cl
R3
Ar
Ar
NC
i)
ii), iv)
N
NH
O
S
R1
H
R1
21a-k
R1
20a: OMe
20b: Me
R2
R1
NH
N
Boc
Scheme 2. Synthesis of biaryl analogs. Reagents and conditions: (i) trans-N-methyl-N-tert-butoxycarbonyl-1,4-diaminocyclohexane, acetic acid, Na(OAc)3BH, THF, toluene;
(ii) acid chloride, DIPEA, CH2Cl2; (iii) aryl bromide, Pd(PPh3)4, Cs2CO3, toluene, EtOH, MW 180 °C, 20 min; (iv) concd HCl, EtOH, 3 h, rt.

4310
S. A. Brunton et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4308–4311
Table 2
Table 3
SAR at the 3-chlorothiophenecarboxamide group
SAR around the biaryl region
R1
R2
H
EC50 (nM)
Entry
Ar
R1
R2
H
R3
H
EC50 (nm)
a
a
Entry
Cl
Cl
NC
11
6-OMe
40
8
5000
S
21a
21b
6-OMe
6-OMe
H
H
H
H
300
11
Me
H
40
S
S
CN
90
16a
16b
16c
16d
16e
16f
16g
>10,000
300
O
O
S
Cl
21c
21d
21e
21f
21g
21h
21i
6-OMe
6-OMe
6-F
H
H
H
H
H
H
H
H
F
H
H
H
H
H
H
H
H
F
15
Me
Me
Me
Me
Me
Me
S
S
F
MeO
NC
100
Cl
Cl
Cl
15
150
S
S
S
F
NC
NC
40
H
200
6-Me
4-OMe
H
2000
>10,000
>10,000
10
OMe
NO2
NC
Cl
25
S
S
N
N
N
30
Cl
21j
4-OMe
4-OMe
2
F
F
Cl
21k
0.3 ( 0.5), Amax = 80%
16h
16i
Me
Me
H
2 ( 3), Amax = 80%
S
S
F
a
EC50 and Amax values determined according to Ref. 8.
Cl
Cl
2000
tion of the secondary amines 1a or 1b with the corresponding acid
chlorides10 whereas amides 16i,k,l were prepared by coupling
reactions of 1a or 1b to the corresponding carboxylic acids11,12
using TBTU.
F
16j
>10,000
The data in Table 2 demonstrate that removal of the chlorine
atom (16a), or the phenyl ring (16j) results in a loss of activity rel-
ative to 8, whereas replacement of chlorine with a methyl group
(16k) retained weak activity similar to 8. Continued SAR work re-
sulted in a few derivatives that showed a significant increase in
activity compared to 11. Substitution of fluorine into the 3-chloro-
benzothiophene in the 4-, 5-, or 7-positions gave the potent ana-
logs 16c,d,g, respectively. However the 6-fluoro analog 16i was
only weakly active. Additional six-substituted analogs (16e,f) were
prepared and these were also much less active. Heterocyclic
replacements (16b,l) of the phenyl ring in the 3-chlorobenzothi-
ophene were prepared, but this resulted in a significant loss of
activity. Combining the 4- and 7-fluorine substituents into the
4,7-difluorothiophene 16h, led to an EC50 of 2 nM.
S
S
16k
H
5000
Cl
16l
Me
>10,000
S
N
a
EC50 and Amax values determined according to Ref. 8.
majority were derived from the more potent N-methyl analog 11.
Amides 16a–h and 16j (Scheme 1, Table 2) were prepared by reac-

S. A. Brunton et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4308–4311
4311
Preliminary SAR of the biaryl region was explored last. Target
compounds 21a–k were prepared from the boronic acid benzalde-
hydes13 17a–e in a four step synthesis (Scheme 2). The first three
steps involved a reductive amination with trans-N-methyl-N-tert-
good PK profile. As such, 21k has potential as a novel therapy for
stroke and other neurological disorders.
Acknowledgements
butoxycarbonyl-1,4-diaminocyclohexane,14
a
Suzuki coupling
reaction with the corresponding aryl bromide and an amide forma-
tion but the order of these steps varied between compounds. Tar-
get compound 21g was obtained by a Suzuki coupling reaction on
17b to give the biaryl aldehyde 20b followed by reductive amina-
tion then amide formation followed by Boc-deprotection, while
target compounds 21c,j,k were obtained by reductive aminations
on aldehydes 17a and 17e to give amines 18a and 18e respectively.
The amide formations then gave 19a–c and Suzuki coupling reac-
tions followed by Boc-deprotections gave the desired compounds.
Finally, targets 21a–b, d–f, h–i were obtained by reductive amina-
tions on aldehydes 17a,c–e to give amines 18a–d followed by Su-
zuki coupling reactions then the amide formation followed by Boc-
deprotection.
The proximal aryl ring of the biaryl group was first chosen for
exploration. Deletion of the methoxyl group (21f) (Table 3) re-
sulted in loss of activity, as did replacement with methyl (21g)
or fluoro (21e). The isomeric methoxyl analog (21h), showed
slightly improved activity relative to the parent (11).
Changes to the distal aryl group demonstrated that deletion of
the para-cyano group (21a) or replacement by methoxyl (19d)
both led to some reduction of activity, as did the isomeric cyano
analog (19b). Replacement of the para-cyano group by sulfone
(21c) led to an enhancement of activity. A number of heterocyclic
analogs of the distal ring were prepared, of which the 4-pyridyl
compound (21i) was notable in potency. Combining this preferred
structure with the potent 4,7-difluoro substituent pattern on the 3-
chlorobenzothiophene led to 21k, a compound of exceptional,
picomolar activity.
The authors thank Dane Springer, Warren Hirst and the team at
Wyeth for their helpful discussions and support in publishing this
work.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.05.096.
References and notes
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8. Screening for agonists was done in an isolated stable clone of the C3H10 T1/2 cell
line transfected with a luciferase reporter downstream of multimerized Gli
binding sites and
a minimal promoter as previously reported (US Patent
6,613,798). In this system a 10–20-fold up-regulation of luciferase activity was
observed upon 24 h stimulation by Hh protein. The EC50, the concentration of
half maximal stimulation by small molecule, as well as the Amax, the maximal
response relative to the Hh protein control was determined for selected
compounds. For a more detailed description of the GLi-luc assay see: Brunton,
S. A.; Stibbard, J. H. A.; Rubin, L. L.; Kruse, L. I.; Guicherit, O. M.; Boyd, E. A.;
Price, S. J. Med. Chem. 2008, 51, 1108.
The compounds in Tables 1 and 2 were tested for in vitro Hh
agonist activity in a Gli-luc reporter assay using mouse cell
lines.7,8,15 Compound 21k was also tested using daoy (human) cell
lines and gave an EC50 = 0.4 nM ( 0.8), Amax = 86%. The pharmaco-
kinetic (PK) profile of 21k was then measured in mouse; after an
oral dose of the hydrochloride salt at 5 mg/Kg administered as a
suspension in 0.5% methyl cellulose 21k showed a Cmax of 50 ng/
ml and T1/2 of 4 h in plasma. The compound also showed a good
distribution into the brain, where a Cmax of 81 ng/ml and T1/2 of
4 h was achieved.16
9. Carey, F. A. In Organic Chemistry; McGraw-Hill, 1996; p 901.
10. Reid, W.; Oremek, G.; Ocakcioglu, B. Liebigs Ann. Chem. 1980, 9, 1424.
11. Shirley, D. A.; Danzig, M. J.; Canter, F. C. J. Am. Chem. Soc. 1953, 75, 3278.
12. Schneller, S. W.; Clough, F. W.; Hardee, L. E. J. Heteroat. Chem. 1976, 13, 273.
13. Eicher, T.; Fey, S.; Puhl, W.; Buechel, E.; Speicher, A. Eur. J. Org. Chem. 1998, 5,
877.
14. Prepared by N-methylation of the mono-Boc diamine. Synthesis of that
intermediate is described by: Ducruet, A. P.; Rice, R. L.; Tamura, K.; Yokokawa,
F.; Yokokwara, S.; Wipf, P.; Lazo, J. S. Bioorg. Med. Chem. 2000, 8, 1451.
15. A member of this class of compounds was shown to activate Shh signaling
using a primary astrocyte model. See: Atkinson, P. J.; Dellovade, T.; Albers, D.;
Von Schack, D.; Saraf, K.; Needle, E.; Reinhart, P. H.; Hirst, W. J. Neurochem.
2009, 108, 1539.
In conclusion, we have described the optimization of a micro-
molar hit 8 to afford compound 21k, a potent Hh agonist with a
16. Compound 21K showed a high volume of distribution (Vss = 23.4 L/kg at 3 mg/
kg iv in rats) and the compound was detected in skin (data not shown).