Discovery of N-[(1-aryl-1H-indazol-5-yl)methyl]amides derivatives as smoothened antagonists for inhibition of the hedgehog pathway

Article abstract of DOI:10.1016/j.bmcl.2009.05.112

We report the synthesis and biological evaluation of N-[(1-aryl-1H-indazol-5-yl)methyl]amide derivatives as Smoothened antagonists and inhibitors of the Hedgehog pathway. Identification of the lead structure 1 by HTS, followed by SAR study on the amide and aryl portions led to the discovery of antagonists with nanomolar activity.

Full text of DOI:10.1016/j.bmcl.2009.05.112

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4191–4195
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of N-[(1-aryl-1H-indazol-5-yl)methyl]amides derivatives as
smoothened antagonists for inhibition of the hedgehog pathway
*
Gabriella Dessole , Danila Branca, Federica Ferrigno, Olaf Kinzel, Ester Muraglia, Maria Cecilia Palumbi,
Michael Rowley, Sergio Serafini, 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:
We report the synthesis and biological evaluation of N-[(1-aryl-1H-indazol-5-yl)methyl]amide deriva-
tives as Smoothened antagonists and inhibitors of the Hedgehog pathway. Identification of the lead struc-
ture 1 by HTS, followed by SAR study on the amide and aryl portions led to the discovery of antagonists
with nanomolar activity.
Received 4 May 2009
Revised 26 May 2009
Accepted 27 May 2009
Available online 2 June 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Hedgehog pathway
Smoothened antagonists
N-[(1-aryl-1H-indazol-5-yl)methyl]amide
derivatives
The Hedgehog (Hh) signaling pathway is essential for cell differ-
entiation and organ formation in embryogenesis and is involved in
growth and survival of various cells and tissues.1,2 The Hh signaling
cascade is initiated by Hh binding to Patched (Ptch) protein on the
cell surface. In the absence of the ligand, Ptch represses the activity
of Smoothened (Smo), a G-protein coupled receptor (GPCR)-like
protein. Binding of Hh to Ptch relieves the inhibition of Smo and
triggers a cytoplasmatic signal transduction cascade leading to
the activation of Gli transcription factors that regulate Hh target
gene expression.3,4
While the loss of Hh signaling at the embryonic stage has been
demonstrated to cause developmental defects,4 aberrant activation
of this pathway in adults has been documented to be involved in
the formation of basal cell carcinoma (BCC), medulloblastoma
and rhabdomyosarcoma. Aberrant pathway activation was also re-
ported for pancreatic adenocarcinoma, lung cancer, prostate,
breast, and digestive tract tumors.5 Thus the Hh pathway is an
important pharmacological target for the development of new
treatments for cancer.2
In our company a research program was initiated with the aim
of identifying agents useful for the treatment of Hh dependent
malignancies. An HTS campaign was conducted on Shh-light 2 cells
(NIH 3T3 cells stably incorporating a Gli-dependent firefly lucifer-
ase).10 Indazole 1 was shown to be an inhibitor of the pathway
with IC50 = 150 nM and displayed no cytotoxicity at 5 lM. The
compound was confirmed to bind to Smoothened in a whole cell
Smo binding assay,11 where 1 could compete with the binding of
bodipy-cyclopamine, displaying IC50 = 130 nM. In a phenotypic as-
say 1 was able to inhibit the growth of irradiated Ptch +/ꢀ medul-
loblastoma cells with a CC50 = 3 nM12 (see Fig. 1).
O
N
X
H
N
Recent studies have demonstrated that the natural product
cyclopamine inhibits Hh pathway activation by binding directly
to Smo6 and is able to inhibit tumor cell proliferation in mouse
models,7 thus validating Smo as a therapeutic target in the treat-
ment of Hh related diseases. Recently several small molecule
Smo antagonists have been developed8 in fact clinical proof of con-
cept has been demonstrated for use of a Smo antagonist in BCC.9
F
1 (X= N)
IC 50 = 0.13 µM
IC50 0.15 µM
2 (X=C)
2.0 µM
2.8 µM
Smo Binding
Light 2
=
Medulloblastoma
Proliferation
CC 50= 0.003 µM
Figure 1.
* Corresponding author. Tel.: +39 06 91093726; fax: +39 06 91093654.
E-mail address: gabriella_dessole@merck.com (G. Dessole).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.05.112

4192
G. Dessole et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4191–4195
Table 1
Encouraged by this result a SAR study based on the lead struc-
SAR study around the amide portion
ture was initiated to identify compounds with improved activity
over 1. The modification of structure 1 to the corresponding indole
2 resulted in a more than 10-fold drop in activity in both Smo bind-
ing and light 2 assays, thus the study was focused on the indazole
scaffold. Initial SAR from the HTS campaign suggested that the 2-
propylpentanamide was important for activity so initial explora-
tion was focused on the modifications of the alkyl-amide portion
(Table 1).
O
N
R
R1
N
N
R= H, Me
F
R1
Smo binding 2%
FCS IC50
Light 2 IC50
(lM)
c
Compds
R
a,b
Shortening of the hydrocarbon chain of 1 proved to be detri-
mental for activity, as demonstrated by compound 3, which was
(l
M)
6-fold less active in the Smo binding assay (IC50 = 0.72 lM), and
1
H
0.13
0.16
0.46
N.D.d
6.6
by the isopropyl derivative 4, that displayed an even more marked
drop in potency, showing micromolar binding affinity. Constrain-
ing of the alkyl chains of 3 into a cyclohexyl ring (5) produced more
than a 10-fold drop in activity. The same loss of activity was also
observed by introduction of bulkier tert-butyl (6) group. Activity
could be partially regained by introducing a long linear chain as
in 7, although 7 proved to be 10-fold less potent than the lead 1.
3
4
H
0.72
10.0
10.0
10.0
1.2
The presence of an
a-methyl as in 8, or an a-gem-dimethyl as in
H
9, was beneficial for activity, affording submicromolar compounds
(IC50 = 0.36 and 0.33 lM, respectively), albeit both 3-fold less ac-
tive than 1.
The aliphatic side chain was crucial for activity since substantial
loss of activity was observed for phenyl and benzyl analogues 10
and 11. The importance of the amide hydrogen for activity was ver-
ified as its replacement with a methyl group resulted in a 30-fold
5
H
6
H
3.5
drop in activity with respect to 1, with 12 showing IC50 = 3.0 lM.
The replacement of the amide with a urea functionality proved
to be well tolerated, thus urea derivative 13 (Table 1) showed only
a slight drop in activity with respect to amide 1. The N-methylated
analogue 14 was characterized by 4-fold drop in activity with re-
spect to 13, confirming the importance of the N–H moiety.
The most active compounds in Table 1 (3, 8, 9 and 13) main-
tained the antagonism as seen with 1 in the Light 2 assay, confirm-
ing the inhibitory activity on the pathway, and they were devoid of
7
H
0.84
0.23
0.15
N.D.
N.D.
1.7
8
H
0.36
0.33
10.0
>10.0
3.0
toxicity at 5 lM.
We then turned our efforts to the exploration of the phenyl por-
tion of structure 1 (Table 2). Replacement of the fluorine in 1 with a
chlorine (compound 15) proved detrimental and led to a 10-fold
drop in binding affinity (IC50 = 1.2 lM). The isomeric 2-fluoro and
3-fluoro derivatives 16 and 17 were weaker binders than the 4-flu-
oro compound 1. The unsubstituted phenyl derivative 18 displayed
comparable binding affinity to 1 (IC50 = 0.2 lM). The effect of a
methyl group was also evaluated in all the positions of the phenyl
ring. While the 4-methyl derivative 19 showed a decrease in activ-
ity with respect to 1, 2- and 3-methyl substitution produced antag-
onists 20 and 21, displaying similar levels of activity to 1, although
the 2-methyl analogue 20 was very active in the functional assay.
Combination of the two methyl groups was tolerated, although no
additional improvement in affinity was observed as 2,3-dimethyl
analogue 22 and tetrahydronaphthyl derivative 24 displayed
9
H
10
11
12
13
H
H
Me
H
IC50 = 0.12 and 0.15 lM respectively in the Smo binding assay. This
N
N
is in contrast to the 2,6-dimethyl analogue 23 which lost around
7-fold activity. Interestingly, introduction of the methoxy group
into all the positions was well tolerated (25–27). In particular the
ortho and para isomers 25 and 27 displayed both high affinity with
IC50 = 5 and 40 nM and in the functional assay they inhibited the
pathway with IC50 = 5 and 10 nM, respectively. Introduction of
electron withdrawing groups, such as ketone in 28 and ester in
29, in the 4-position was characterized by a significant loss in
potency.
0.31
1.3
0.49
>5.0
14
Me
a
The affinity of compounds for Smo receptor is measured as reported in Ref. 11.
Values are means of at least two experiments. SD are 30% of the mean value.
The Light 2 activities were measured as reported in Ref. 10.
Not determined.
b
c
We then evaluated replacement of the phenyl moiety with a
heteroaromatic ring (Table 3). While the 2-pyridine isomer 30
d

G. Dessole et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4191–4195
4193
Table 2
Table 2 (continued)
SAR study around the aryl portion
c
Compds
R
Smo binding 2%
Light 2 IC50
(lM)
O
a,b
FCS IC50
(lM)
N
H
N
N
R
27
0.04
0.01
1.5
c
Compds
R
Smo binding 2%
Light 2 IC50
M)
a,b
OCH3
FCS IC50
(l
M)
(l
28
1.4
1.8
1
0.13
0.16
O
F
29
5.9
15
16
17
18
1.2
0.9
0.98
0.15
1.4
OC(CH3)3
O
Cl
a–c
See footnotes in Table 1.
F
F
was devoid of activity, both the 3- and 4-isomers 31 and 32 main-
tained micromolar activity. The activity of 31 could be enhanced,
however, by the introduction of a methoxy-group in positions 2
0.68
0.2
or 4 (33 and 34, Smo binding IC50 = 0.34 and 0.13 lM, respectively),
as already observed for phenyl derivatives 18 and 27 (Table 2).
Both compounds retained antagonistic activity in the light 2 cells.
In contrast introduction of a pyridazine (35) and pyrimidine (36)
proved to be detrimental for activity.
0.12
Table 3
Heterocyclic replacement
19
20
21
0.9
3.0
Compds
R
SMO binding
Light 2
IC50 (lM)
a,b
c
IC50
(l
M) 2% FCS
0.2
0.04
0.17
30
>10
N.D.d
N
N
0.07
31
32
33
1.83
4.5
0.87
2.6
22
23
0.12
0.95
0.14
0.41
N
OCH3
0.34
0.39
N
N
34
35
0.13
>25
6.3
0.32
N.D.
N.D.
24
25
26
0.15
0.005
0.10
0.11
0.005
0.11
OCH3
OCH3
OCH3
N
N
N
N
36
a–d
See footnotes in Table 1.

4194
G. Dessole et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4191–4195
OHC
F
CN
around the aryl portion has revealed that a range of aryl groups are
tolerated and has resulted in the identification of 25 and 27 as
nanomolar Smo antagonists.
37
Route A
Route B
a
e
Acknowledgments
The authors acknowledge Danielle McMaster for the first syn-
thesis of 1.
CN
CN
N
N
N
N
H
R1
38
References and notes
40
1. Jiang, J.; Hui, C. Dev. Cell 2008, 15, 801.
b
b
2. Rubin, L. L.; de Sauvage, F. J. Nat. Rev. Drug Disc. 2006, 5, 1026.
3. Di Magliano, M. P.; Hebrok, M. Nat. Rev. Cancer 2003, 3, 903.
4. Chiang, C.; Litingtung, Y.; Lee, E.; Young, K. E.; Corden, J. L.; Westphal, H.;
Beachy, P. A. Nature 1996, 383, 407; Roessler, E.; Belloni, E.; Gaudenz, K.; Jay, P.;
Berta, P.; Scherer, S. W.; Tsui, L. C.; Muenke, M. Nat. Genet. 1996, 14, 357;
Belloni, E.; Muenke, M.; Roessler, E.; Traverso, G.; Siegel-Bartelt, J.; Frumkin, A.;
Mitchell, H. F.; Donis-Keller, H.; Helms, C. Nat. Genet. 1996, 14, 353.
5. Evangelista, M.; Tian, H.; de Sauvage, F. J. Clin. Cancer Res. 2006, 12, 5924.
6. Chen, J. K.; Taipale, J.; Cooper, M. K.; Beachy, P. A. Genes Dev. 2002, 16, 2743.
7. 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, 1159.
NH2
NH2
N
N
N
R1
N
H
39
41
c, d (R3= Me)
f
or
d (R3= H)
8. Mahindroo, N.; Chandanamali, P.; Fujii, N. J. Med. Chem. 2009. doi:10.1021/
jm801420y; Gallinari, P.; Filocamo, G.; Jones, P.; Pazzaglia, S.; Steinkühler, C.
Expert Opin. Drug Disc. 2009, 4, 525.
9. LoRusso, P. M.; Rudin, C. M.; Borad, M. J.; Vernillet, L.; Darbonne, W. C.; Mackey,
H.; DiMartino, J. F.; de Sauvage, F.; Low, J. A.; Von Hoff, D. D. J. Clin. Oncol.
(Meeting Abstracts) 2008, 26, 3516.
O
O
g
R2
N
H
N
N
N
N
R3
N
H
R1
R1= Aryl, Het
R2= Alk, NAlk2
R3= H, Me
42
10. Shh Light II cells (ATCC catalog No. CRL-2795) were seeded into a 384-well
Matrix white microplate at an initial concentration of 15,000 cells/well in
20
FCS, 0.1 mg/mL Penicillin-Streptomycin, 2 mM
dilutions of antagonists in DMSO/H2O (0.25% DMSO, 2
plate was incubated for 16 h at 37 °C under 10% CO2, and then agonist
Purmorphamine (Calbiochem catalog No. 540220) was added at final
concentration of 3 M. The plate was then incubated for 30 h at 37 °C under
l
L of assay medium (DMEM without Phenol Red complemented with 2%
-Glutamine) and serial
L) were added. The
L
Scheme 1. Synthesis of compounds in Tables 1–3. Reagents and conditions: (a) (1)
ArNHNH2, W (80 °C, 5 min); (2) K2CO3, W (150 °C, 20 min) (Y = 90%); (b) LiAlH4,
THF, reflux, 15 min; (c) (1) 2,2,2-trifluoroethylformate, K2CO3, THF; (2) BH3ꢁDMS,
l
l
l
a
THF; (d) R2COCl or dipropylcarbamoyl chloride, TEA, DMF; (e) NH2NH2ꢁH2O,
lW
l
(80 °C, 25 min); (f) valproyl chloride, TEA, DMF; (g) ArI or ArBr, N,N0-dimethylcy-
clohexane-1,2-diamine, CuI, K3PO4, dioxane, 100–110 °C, 12 h.
10% CO2, and then Gli-dependent firefly luciferase measured with Bright-Glo
Kit and cell viability measured using CellTiter Blue Kit on View Lux. The IC50
and CC50 value was determined based on the residual Gli-dependent
transcriptional activity, and cell viability.
11. Human HEK293 Flag-Smo cells were seeded into a 384-well Matrix black
For the synthesis of the compounds described in Tables 1–3,
two different routes were developed, with the aim to introduce
diversity in the final step. Compounds reported in Table 1 were
prepared following route A (Scheme 1): condensation of 5-cyano-
2-fluoro-benzaldehyde 37 with 4-fluorophenyl hydrazine pro-
duced 38 (R1 = 4-fluorophenyl), that was smoothly reduced with
microplate (Poly-lys) at an initial concentration of 10,000 cells/well in 20
assay medium (DMEM without Phenol Red complemented with 2% FCS,
0.1 mg/mL Penicillin-Streptomycin, 2 mM -Glutamine). The plate was
incubated for 16 h at 37 °C under 5% CO2, and then Smo antagonists in
DMSO/H2O were added with serial dilutions points (0.25% DMSO, 2 L). The
lL of
L
l
plate was incubated for 2 h at 37 °C under 5% CO2, and then BODIPY-
cyclopamine (Toronto Research Chemical Catalog No. B674800) was added at a
final concentration of 15 nM. The plate was then incubated for 3 h at 37 °C
13
LiAlH4 to the corresponding amine 39, then acylated with the
under 5% CO2, and then the cells were fixed 10 min in 20
formaldehyde. After washing five times with PBS, cell nuclei were stained with
0.5 M Propidium Iodide, and the plate was read using an Acumen Explorer
lL/well of 7%
appropriate acyl chlorides to yield the final products of Table 1.
Formylation of 39 followed by formyl reduction and acylation were
performed to prepare methyl amide 12. This route was also fol-
lowed for the preparation of the two urea derivatives 13 and 14.
The aminomethyl indazole 39 was reacted with dipropylcarbamoyl
chloride to obtain 13 while formylation followed by formyl reduc-
tion and urea formation were performed for preparation of 14.
Most of the compounds reported in Tables 2 and 3 were pre-
pared following route B (Scheme 1). Indazole nitrile intermediate
40, obtained by condensation of 37 with hydrazine, was reduced
to amine 41,13 that was acylated with valproyl chloride to provide
42.14 The indazole intermediate 42 was then functionalized with
aryl or heteroaryl iodides or bromides via copper catalyzed cou-
pling reaction.15,16
l
(488 nm laser kex, kem 535–618 nm). The IC50 value was determined based on
the residual fluorescence normalised for PI values.
12. 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.
13. Sanderson, P.; Lyle, T.; Dorsey, B.; Santon, M.; Nylor-Olsen, A. M. WO 00/26210.
14. 1H-Indazole-5-carbonitrile (40). A solution of 5-cyano-2-fluorobenzaldehyde
(10.0 mmol) and hydrazine hydrate (101 mmol) in DMF (33.5 mL) was heated
under microwave irradiation at 80 °C for 25 min. After aqueous work up, the
residue was purified through a SCX cartridge. (Y = 83%). 1H NMR (300 MHz,
DMSO-d6) d 13.56 (br s, 1H), 8.39 (s, 1H), 8.25 (s, 1H), 7.74–7.60 (m, 2H). MS
(ES) C8H5N3 requires 143, found 144 (M+H)+.
1-(1H-Indazol-5-yl)methanamine (41): Obtained by LiAlH4 reduction of 40, as
described in Ref. 13.
N-(1H-Indazol-5-ylmethyl)-2-propylpentanamide (42): To a solution of 1-(1H-
indazol-5-yl)methanamine 41 (1.02 mmol) in DMF (5 mL) 2-propylpentanoyl
chloride (1.12 mmol) and TEA (1.12 mmol) were added. The reaction was
stirred at room temperature for 2 h. Aqueous work up provided the title
compound (Y = 57%). 1H NMR (300 MHz, DMSO-d6) d 8.29 (m, 1H), 7.99 (s, 1H),
7.57 (s, 1H), 7.47 (d, J = 8.6 Hz, 1H), 7.23 (d, J = 8.6 Hz, 2H), 4.33 (d, J = 5.8 Hz,
2H), 2.19 (m, 1H), 1.54–1.38 (m, 2H), 1.31–1.1 (m, 6H), 0.81 (t, J = 7.0 Hz, 6H).
MS (ES) C16H23N3O requires 273, found 274 (M+H)+.
Unfortunately, the 2-substituted aryl halides did not react un-
der copper catalyzed conditions, thus they were prepared follow-
ing route A, using the appropriate aryl hydrazines in the
condensation step.17
In summary, we have reported the synthesis and biological
evaluation of N-1-aryl-indazole-5-yl-methylamide derivatives as
inhibitors of the Hedgehog pathway. Our work focused on the
improvement of the Smo binding affinity over the lead compound
1, identified by HTS. The alkyl chain of the amide moiety of 1 ap-
peared to be important for activity. On the other hand a SAR study
15. Antilla, J. C.; Baskin, J. M.; Barder, T. E.; Buchwald, S. L. J. Org. Chem. 2004, 69,
5578.
16. As an example, synthesis of N-[1(3-methoxyphenyl)-1H-indazol-5-ylmethyl]-2-
propylpentanamide (26). Indazole 42 was arylated with 1-iodo-3-
methoxybenzene according to ref. 15 (Y = 59%). 1H NMR (300 MHz,

G. Dessole et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4191–4195
4195
DMSO-d6) d 8.37 (m, 1H), 8.32(s, 1H), 7.82 (d, J = 8.7 Hz, 1H), 7.70 (s, 1H), 7.54–
7.21 (m, 4H), 6.96 (dd, J1 = 8.2, J2 = 1.8 Hz, 1H), 4.38 (d, J = 5.8 Hz, 2H), 3.84
(s, 3H), 2.21 (m, 1H), 1.55–1.37 (m, 2H), 1.31–1.1 (m, 6H), 0.81 (t, J = 7.0 Hz,
6H). MS (ES) C23H29N3O2 requires 379, found 380 (M+H)+.
All the compounds prepared as described for 26 were obtained in comparable
yields (>50%).
17. Compounds synthesized acconding to route A were obtained in 25–50% yields
from 37. Compound 25 was obtained in 35% yields.