Discovery of amide replacements that improve activity and metabolic stability of a bis-amide smoothened antagonist hit

Article abstract of DOI:10.1016/j.bmcl.2011.07.052

A bis-amide antagonist of Smoothened, a seven-transmembrane receptor in the Hedgehog signaling pathway, was discovered via high throughput screening. In vitro and in vivo experiments demonstrated that the bis-amide was susceptible to N-acyl transferase mediated amide scission. Several bioisosteric replacements of the labile amide that maintained in vitro potency were identified and shown to be metabolically stable in vitro and in vivo.

Full text of DOI:10.1016/j.bmcl.2011.07.052

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5206–5209
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of amide replacements that improve activity and metabolic
stability of a bis-amide smoothened antagonist hit
Matthew L. Brown, Wade Aaron, Richard J. Austin, Angela Chong, Tom Huang, Ben Jiang, Jacob A. Kaizerman,
Gary Lee, Brian S. Lucas, Dustin L. McMinn, Jessica Orf, Minqing Rong, Maria M. Toteva, Guifen Xu,
Qiuping Ye, Wendy Zhong, Michael R. DeGraffenreid, Dineli Wickramasinghe, Jay P. Powers,
⇑
Randall Hungate, Michael G. Johnson
Amgen Inc., 1120 Veterans Blvd., South San Francisco, CA 94080, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A bis-amide antagonist of Smoothened, a seven-transmembrane receptor in the Hedgehog signaling
pathway, was discovered via high throughput screening. In vitro and in vivo experiments demonstrated
that the bis-amide was susceptible to N-acyl transferase mediated amide scission. Several bioisosteric
replacements of the labile amide that maintained in vitro potency were identified and shown to be met-
abolically stable in vitro and in vivo.
Received 29 April 2011
Revised 11 July 2011
Accepted 13 July 2011
Available online 23 July 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Smoothened
Hedgehog
Amide replacement
Hedgehog (Hh) signaling is essential for growth and develop-
ment and regulates, via the transmembrane proteins Smoothened
(SMO) and its repressor Patched (PTCH), the downstream activity
of the GLI family transcription factors.1 Aberrant Hh signaling is
associated with basal cell carcinoma (BCC) and medulloblastoma
(MB).2 In BCC and MB loss-of-function mutations in PTCH or
gain-of-function mutations in SMO elevate GLI1 expression com-
pared to the corresponding normal tissues. Recent studies have
shown that antagonism of Hedgehog signaling can inhibit cancer
growth in mouse models of MB and in human MB and BCC pa-
tients, indicating that antagonism of Hh signaling can provide ther-
apeutic benefit in these cancers.3 In other cancer types, co-
overexpression of GLI1 with Hh has been observed suggesting that
a Hh pathway antagonist may have utility in treating additional
cancer types.1 SMO is an attractive target for pharmaceutical inter-
vention as it signals downstream of PTCH and Hh. In addition, we
and others have shown that SMO can be inhibited with small mol-
ecule compounds.4 Here we describe structurally varied com-
pounds inspired by the discovery of a bis-amide hit. These
compounds antagonize SMO signaling in vitro and have pharmaco-
kinetic properties suitable for evaluation in in vivo models of BCC
or MB.
its in vitro antagonist activity against SMO as well as its in vitro
and in vivo metabolic stability. While 1 exhibited promising inhi-
bition of SMO in a cell-based functional assay, its pharmacokinetic
properties in the rat were suboptimal. Identification of primary
metabolites after incubation of 1 with rat hepatocytes indicated
that the unsubstituted benzamide group was susceptible to enzy-
matic cleavage by N-acyl transferase to afford a compound with
no activity against SMO (Fig. 1). We sought to find a replacement
for the labile amide that maintained potent antagonism of SMO
and demonstrated improved in vivo metabolic stability relative
to our initial lead 1.
A variety of amide replacements maintain or improve in vitro
potency and improve both in vitro and in vivo metabolic stability
relative to the high throughput screening hit 1. The 2-atom
replacements 2–4 (Table 1) afford compounds that are comparable
in potency to 1. Potency of the alkene 3 suggests that polar surface
area within the linker is not required, nor is there any critical
hydrogen bond acceptor/donor interaction between the receptor
and the linker region of the molecule.
Syntheses of compounds with two atom linkers between the
central phenyl and unsubstituted benzamide are represented in
Scheme 1. The lead compound 1 was made from commercially
available 4-aminonitrobenzene in two steps. Compounds 2 and 4
were synthesized by Friedel–Crafts acylation of benzene with 4-
nitrophenylacetyl chloride to 4 and subsequent ketone reduction
to 2. Heck coupling afforded 3 from the readily available iodide
and styrene.
After high throughput screening of our compound library, the
bis-amide hit 1 was identified and characterized with respect to
⇑
Corresponding author.
E-mail address: mgjohnso@amgen.com (M.G. Johnson).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.07.052

M. L. Brown et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5206–5209
5207
Table 2
In vitro antagonist activities of Smoothened receptor by compounds with five-
membered heteroaromatic amide replacements
Figure 1. Predominant metabolism of 1 in rat hepatocytes by N-acyl transferase
(NAT). Rat clearance after iv dosing at 0.5 mg/kg.
a
Compound
hSMO IC50 (nM)
5
27
Table 1
In vitro antagonist activities of Smoothened receptor by compounds with two atom
amide replacement
6
7
8
31
40
44
a
Compound
hSMO IC50 (nM)
9
63
89
1
13
10
11
12
2
3
4
14
49
79
93
109
a
See Ref. 7. Values are represented as an average of two experiments; standard
deviation 20%.
a
See Ref. 7. Values are represented as an average of two experiments; standard
deviation 20%.
Compounds bearing five-membered heteroaromatic replace-
ments for the unsubstituted benzamide were synthesized from
commercially available or readily available starting materials as
described in Scheme 2. Cyclodehydration with the appropriate thi-
obenzamide or benzamide and a 2-bromoacetophenone was the
general method applied to synthesis of thiazoles 5 and 12 and oxa-
zole 11. Synthesis of the trifluoromethyl phenyloxazole 6 followed
a procedure described by Kawase.5 Oxazoles 7–9 were assembled
either by classical Robinson–Gabriel oxazole synthesis or by a pro-
cedure for oxazole synthesis developed by Wipf.6 Lastly, cyclode-
hydration afforded oxadiazole 10.
Exceptional in vitro potencies are observed when the amide lin-
ker is replaced with six-membered aryl groups (Table 3). Six-mem-
bered rings with a meta-disubstitution pattern appear to be good
replacements for the amide. As with the five-membered ring
replacements, position of heteroatoms does not have a significant
effect on potency within the pyrimidine and pyridine series and
the potency of compound 13 with the phenyl replacement indi-
cates no polar surface area on the linker is required for in vitro
antagonist activity. These observations reinforce our suggestion
that the linking unit replacing the amide serves as positioning ele-
ment, not as a binding element.
Synthetic sequences to six-membered aryl replacements for the
western amide are shown in Scheme 3. Suzuki coupling affords the
critical bond formation between the central phenyl ring and wes-
tern phenyl ring of 13. Compounds 14, 17 and 20 were synthesized
by coupling of the appropriate chlorophenylpyridine with the de-
scribed boronate ester. Pyridine 16 is prepared by selective
sequential Suzuki couplings as are pyrimidines 15, 18 and 19.
Compounds with five- and six-membered ring amide replace-
ments have improved in vivo metabolic stability in the rat com-
pared with the lead compound 1, for example, Table 4, 6 and 14.
These compounds are especially promising for evaluation in
in vivo efficacy models, exhibiting moderate oral bioavailability
Scheme 1. Reagents and conditions: (a) Benzoyl chloride, Et3N, DCM, 0 °C, 10%; (b)
2-chlorobenzoyl chloride, pyridine, DCM, 0 °C, 16–54%; (c) NaBH4, MeOH, 0 °C, 93%;
(d) styrene, triethylorthotoluene phosphine, Et3N, Pd2(dba)3, DMF, 100 °C, 27%; (e)
benzene, AlCl3, reflux, 57%; (f) Zn, HOAc, rt, 97%.
We also investigated amide replacements by five-membered
rings, many of which demonstrate both in vitro potency compara-
ble to that of 1 (Table 2) as well as in vitro and in vivo metabolic
stabilities superior to 1 (Table 4). Position of the heteroatoms with-
in the ring system is not relevant to measured functional activity
across the series and both 4-substituted (cf. 6 and 8) and 4-unsub-
stituted compounds (cf. 9) are potent antagonists. These results, ta-
ken together with the SAR of the 2-atom linker analogs, suggest
that distribution of the polar surface area at this linking structural
unit is not important for in vitro activity.
These amide replacements are not susceptible to metabolism by
hydrolytic or N-acyl transferase cleavage, and thus the metabolic
pathway observed with 1 is averted. In most cases the five-mem-
bered ring substitutions afforded compounds with significantly re-
duced rat in vivo clearance compared with 1 (Table 4). Compound
6 is especially noteworthy due to its moderate oral availability.

5208
M. L. Brown et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5206–5209
Scheme 2. Reagents and conditions: (a) 2-Bromoacetophenone, NMP, 130 °C, 39%; (b) 2-chlorobenzoyl chloride, pyridine, DCM, rt, 13–82%; (c) TFAA, pyridine, benzene, 0 °C–
rt, 61%; (d) Zn, HOAc, rt, 57–99%; (e) 2-amino-1-phenylethanol, EDCI, HOBT, Et3N, DMF, rt, 88%; (f) (i) Dess-Martin periodinane, DMF–DCM, RT; (ii) POCl3, pyridine, 70 °C, 5–
19% over two steps; (g) 1-amino-1-phenylpropan-2-ol, EDCI, HOBT, NMM, DMF, rt, 68%; (h) (R)-(À)-2-phenylglycinol, EDCI, HOBT, Et3N, DMF, RT, 97%; (i) (i) Dess-Martin
periodinane, DMF–DCM, rt; (ii) PPh3, I2, Et3N, DMF, rt, 22% over two steps; (j) POCl3, 100 °C; (k) benzamide, NMP, 135 °C, 78%; (l) 4’-nitro-2-bromoacetophenone, NMP, 130 °C,
55%.
Table 3
Table 4
In vitro antagonist activities of Smoothened receptor by compounds with six-
membered heteroaromatic amide replacements
In vitro and in vivo pharmacokinetic profile of selected compounds
Compound
Rat microsomal
clearancea
Rat in vivo
clearanceb
(L/h/kg)
Rat oral
availabilityc
(%F)
(% turnover)
1
6
14
<10
<10
41
1.4
0.23
0.17
12
47
27
a
a
Values represent
a single experiment and are expressed as percent drug
Compound
hSMO IC50 (nM)
metabolized after 30 min incubation with rat liver microsomes.
b
iv dose = 0.5 mpk.
po dose = 5.0 mpk.
c
13
14
15
6.4
7.2
and good antagonist activity against hSMO as well as the murine
SMO receptor (6 IC50 = 17 nM and 14 IC50 = 26 nM).8
27b
We discovered a novel Smoothened antagonist 1. Metabolic
studies using rat hepatocytes identified the unsubstituted benzam-
ide group as the main metabolic site of cleavage by N-acyl transfer-
ase. A number of non-cleavable amide isosteres were synthesized
and evaluated for potency versus SMO and replacement of this
functional group was shown to be highly tolerant. Several of the
amide replacements afford compounds that are more potent than
1 as well as being more stable both in vitro and in vivo. The lacking
correlation between polar surface area distribution in these linkers
and antagonist activity suggests that the structural motif between
the central phenyl ring and western phenyl ring functions as a
positioning element rather than as a binding element to the
receptor.
16
28b
33
17
18
19
37
48
93
20
a
See Ref. 7.
b
Values are represented as a single experiment, otherwise values are repre-
sented as an average of two experiments; standard deviation 20%.
Scheme 3. Reagents and conditions: (a) Biphenyl-3-ylboronic acid, Pd(PPh3)4, Na2CO3, MeCN–water, 90 °C, 70%; (b) concentrated HCl (aq), 120 °C, 46–93%;
(c) 2-chlorobenzoyl chloride, pyridine, DCM, 0 °C, 42–82%; (d) N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)acetamide, Pd(PPh3)4, Na2CO3, MeCN–water,
90 °C, 24–90%; (e) phenylboronic acid, Pd(PPh3)4, Na2CO3, MeCN–water, 90 °C, 59–81%; (f) (i) nBuLi, 2-(dimethylamino)ethanol, THF, À78 °C; (ii) C2Cl6, THF À78 °C–rt, 72%
over two steps; (g) 4-nitrophenylboronic acid, Pd(PPh3)4, Na2CO3, MeCN–water, 90 °C, 84%; (h) Zn, HOAc, rt, 28%; (i) POCl3, 100 °C, 18%.

M. L. Brown et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5206–5209
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7. IC50 assay for human SMO: HEPM cells were used to measure human SMO
activity in vitro using a modified version of the method described in US Patent
6613,798. In 96 well tissue culture plates compounds were incubated with
HEPM cells in the presence of MEM media supplemented with 0.5% charcoal-
dextran treated fetal bovine serum (HyClone) and 50 mM myristoylated mouse
Shh protein (Williams, K. P.; Rayhorn, P.; Chi-Rosso, G.; Garber, E. A.; Strauch, K.
L.; Horan, G. S. B.; Reilly, J. O.; Baker, D. P.; Taylor, F. R.; Koteliansky, V.; Pepinsky,
R. B. J. Cell Sci. 1999, 112, 4405). Twenty-four hours after the addition of
compound and Shh, GLI expression was measured using a Quantigene assay
(Affymetrix, Santa Clara, CA, USA).
8. IC50 assay for mouse SMO: The mouse Smoothened activity was measured in vitro
using a modified version of the method described by Chen et al. (Chen, J. K.;
Taipale, J.; Young, K. E.; Maiti, T.; Beachy, P. A. Proc. Natl. Acad. Sci. U.S.A. 2002,
14071). An oligonucleotide cassette with 5 consensus Gli1 binding sites was
ligated into the luciferase reporter plasmid pGL4.16 (Promega, Madison, WI,
USA). A stable clone of NIH-3T3 cells stably transfected with the Gli1 binding
site plasmid was used for the reporter assay. To measure compound activity,
compounds were incubated with cells for 15 h in the presence of Optimem
medium (Invitrogen, Carlsbad, CA, USA) supplemented with 0.5% charcoal-
dextran treated fetal bovine serum (HyClone) and 10 mM myristolated mouse
Shh protein (Williams et al 1999). Luciferase activity was measured by addition
of Bright-Glo (Promega) and reading on a luminometer. For a single IC50 assay,
compounds were tested in quadruplicate using a three fold dilution series.