Potent inhibitors of the Hedgehog signaling pathway

Article abstract of DOI:10.1021/jm070694n

A small family of phenyl quinazolinone ureas is reported as potent modulators of Hedgehog protein function. Preliminary SAR studies of the urea substituent led to a nanomolar Hedgehog antagonist.

Full text of DOI:10.1021/jm070694n

1108
J. Med. Chem. 2008, 51, 1108–1110
Letters
Chart 1. Initial Hh Antagonist Screening Hit
Potent Inhibitors of the Hedgehog Signaling
Pathway
Shirley A. Brunton,* John H. A. Stibbard, Lee L. Rubin,†
Lawrence I. Kruse,† Oivin M. Guicherit,† Edward A. Boyd,
and Steven Price
EVotec, 114 Milton Park, Abingdon, Oxfordshire, OX14 4RX,
United Kingdom, and Curis Inc., 61 Moulton Street,
Cambridge, Massachusetts 02138-1118
ReceiVed June 14, 2007
propionic anhydride. This was opened to the bisamide by heating
with 4-fluoroaniline in chloroform then cyclized to the quinazoli-
none by heating in ethylene glycol at 130–140 °C to give 1.
The ethyl side chain was brominated with bromine in acetic
acid buffered with sodium acetate and converted to amine (2)
using methylamine in ethanol. The final urea formation to give
4 was achieved using 3-trifluoromethylphenyl isocyanate in
chloroform. Testing of racemic 4 in the Hh assay13 confirmed
the IC50 as 1.4 µM. Intermediates 1 and 2 were also tested for
activity but were considered to be much less active (IC50 >10
µM).
The initial aims were to identify a compound with improved
potency (<100 nM) over 4. There were three obvious regions
of 4 that could be investigated to reach this goal, as indicated
in Chart 1: the urea group, the 4-fluorophenyl, and the
quinazolinone ring. Here, we describe some preliminary SAR
work that led to tractable, nanomolar potent Hh pathway
antagonists.
Initially, we investigated the importance of the two methyl
groups in 4 for activity and prepared the bis-desmethyl
analogue (7a) starting from methyl anthranilate (Scheme 1,
Route B). A coupling reaction with N-Cbz glycine using CDIa
in THF gave an amide, which was saponified with lithium
hydroxide in aqueous dioxane. The resulting acid was coupled
with 4-fluoroaniline then cyclized to the quinazolinone using
CDI in THF. The Cbz protecting group was removed by
hydrogenation, and the resulting amine was treated with
3-trifluoromethylphenyl isocyanate to give 7a. Testing of this
compound established that both methyl groups were not
required for activity because a 2-fold increase in activity was
obtained over 4 (Table 1).
A monomethyl derivative 5 was also prepared (Scheme 1,
Route A) following the same route as described for 4 except
that ammonium chloride was used as a source of ammonia to
form the primary amine, followed by treatment with 3-trifluoro-
methyl isocyanate to give the final product. Interestingly, 5
proved to be ∼3-fold less active than the parent compound (4)
and ∼7-fold less active than the bis-desmethyl compound (7a).
With that result in mind, three further bis-desmethyl ureas
(7b-d) were prepared (Scheme 1, Route B) to explore SAR
around that region. The final compounds were prepared starting
from methyl anthranilate as described for 7a. Compound 7d
Abstract: A small family of phenyl quinazolinone ureas is reported
as potent modulators of Hedgehog protein function. Preliminary SAR
studies of the urea substituent led to a nanomolar Hedgehog antagonist.
The Hedgehog (Hh) proteins are essential 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 three Hedgehog ligands, SHh, IHh, and DHh, are
morphogens that have different roles in embryogenesis. Thus,
IHh affects cartilage and bone development, SHh influences
neuronal development in the CNS, and DHh regulates peripheral
neuronal development.
Inappropriate activation of the Hedgehog pathway occurs in
several cancers such as basal cell carcinoma (BCC) and
medulloblastoma.4–7
The complex natural product cyclopamine, one of the jervine
family of compounds isolated from the lily Veratrum
californicum, acts upon the Hh pathway as an antagonist.8
Cyclopamine is a teratogenic compound that produces cyclopia,
among other effects, in offspring of sheep that graze upon the
Veratrum lily. Recently, modern cellular pharmacology experi-
ments with cyclopamine have shown it to modify Hedgehog
pathway activation in cell cultures.9 It has been reported that
the proliferation of human tumor cell lines, including medullo-
blastomas10 and basal cell carcinomas (BCCs),11 is inhibited
in culture by cyclopamine. However, cyclopamine presents a
poor lead structure for modification because it is a very complex,
hydrophobic azasteroid with 10 chiral centers.
We therefore became interested in finding an alternative
small molecule inhibitor of the Hh pathway that contained drug-
like characteristics and would lend itself well to chemical
manipulations.
As part of our screening work, we found a hit with weak
antagonist activity, IC50 ∼1.4 µM, whose structure was con-
firmed as racemic quinazolinone 4 (Chart 1) by resynthesis. A
similar route was used to one that has been previously
described12 (Scheme 1, Route A). The first step involved the
formation of an oxazinone by heating anthranilic acid with
a Abbreviations: CDI, 1,1′-carbonyl diimidazole; Cbz, benzyl oxycar-
bonyl; Boc, tert-butyloxycarbonyl; TFA, trifluoroacetic acid; DCM,
dichloromethane.
* To whom correspondence should be addressed. Telephone: +44 1235
441228. Fax: +44 1235 838931. E-mail: shirley.brunton@evotec.com.
†
Curis Inc.
10.1021/jm070694n CCC: $40.75
2008 American Chemical Society
Published on Web 02/15/2008

Letters
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1109
Scheme 1. Synthesis of Quinazolinones (1-7)a
a Reagents and conditions: (i) (R2CH2CO)2O, reflux, 4 h, 90%; (ii) R1NH2, CHCl3, reflux, 6 h; (iii) NaOH, ethylene glycol, 130-140 °C, 5 h, quantitative;
(iv) bromine, AcOH/NaOAc, 40 °C, 5 h, 96%; (v) R3NH2, EtOH, 40 °C, 1 h, 62% quantitative; (vi) R4NCO, CHCl3, rt, 1 h, 11% quantitative; (vii)
N-Cbz-amino acid, CDI, THF, rt, 22 h, 14%; (viii) LiOH, dioxane, water, rt, 16 h, 96%; (ix) R1NH2, CDI, THF, reflux, 20 h, 48%; (x) H2, 10% Pd/C, EtOH,
rt, 2 h, 95%.
Scheme 2. Synthesis of pyrimidinone analogue (9)a
a Reagents and conditions: (i) (Boc)2O, dioxan, 1 N NaOH; rt, 6 h, 85%; (ii) i-BuOCOCl, Et3N, THF, -20 °C, 20 min; (iii) 4-fluoroaniline, THF, <-7
°C, 5 h then rt, 18 h, quantitative; (iv) TFA, CH2Cl2, rt, 4 h, 64%; (v) triethyl orthopropionate, 100 °C, 7 h; (vi) toluene, reflux, 26 h, quantitative; (vii)
bromine, AcOH/NaOAc, 40 °C, 2 h, 96%; (viii) MeNH2, EtOH, rt, 3 h, 76%; (ix) R4NCO, CHCl3, rt, 1 h, 66%.
proved to be the most potent derivative from this series, with
in a 3-fold decrease in activity compared to 4, suggesting that
an IC50 of 70 nM, providing a 20-fold increase in activity over
the phenyl ring may be required for activity.
4.
Finally, the requirements of the quinazolinone ring for activity
were explored by preparing the truncated pyrimidinone deriva-
tive 9 (Scheme 2). Aminonorbornene carboxylic acid was
protected with Boc anhydride in dioxane with 1 N NaOH. The
acid was coupled with 4-fluoroaniline via the mixed anhydride
from iso-butyl chloroformate. The Boc protecting group was
removed with TFA in DCM and the annulated pyrimidinone
formed by heating with triethyl orthopropionate. A retro
Diels–Alder reaction was then performed in refluxing toluene
overnight to leave the pyrimidinone. This was brominated,
reacted with methylamine and finally treated with 3-trifluoro-
The requirement of the 4-fluorophenyl group for activity was
next investigated and the NH analog 3 was prepared starting
from the commercially available quinazolinone 8 (Route C).
Treatment of 8 with methylamine followed by 3-trifluoro-
methylphenyl isocyanate gave the target compound 3, which
proved to be much less active (>10 µM). We next explored
alkyl replacement of the 4-fluorophenyl and prepared the
N-isopropyl analogue 6 from methyl anthranilate (Scheme 1,
Route B), as described for 7a, except that isopropyl amine was
used in the quinazolinone forming step. This compound resulted

1110 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Table 1. Quinazolinone SAR
Letters
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cmpd
R1
R2
R3
methyl 3-F3C phenyl
4-fluorophenyl methyl methyl 3-F3C phenyl
R4
IC50,d nM
3c
4a
5a
6b
H
H
>10000
1400 ( 300
4700
4300
700
(8) (a) Keeler, R. F.; Binns, W. Teratogenic compounds of Veratrum
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4-fluorophenyl methyl
isopropyl
H
H
H
H
H
H
3-F3C phenyl
3-F3C phenyl
3-F3C phenyl
3-F3C, 5-OMe phenyl 250
3,5-di Cl, 4-pyridyl
3-F3C, 4-Cl phenyl
H
H
H
H
H
7ab 4-fluorophenyl
7bb 4-fluorophenyl
7cb 4-fluorophenyl
7db 4-fluorophenyl
110
70 ( 20
a Route A. b Route B. c Route C. d See ref 13 and Supporting Information
for assay details.
phenyl isocyanate to give the target urea, as described for Route
A (Scheme 1). This compound proved to be much less active
(>10 µM), which suggests that the quinazolinone ring is
important for activity.
In conclusion, we have described the optimization of a
micromolar hit 4 to afford compound 7d, a nanomolar potent
Hh antagonist. As such, 7d and its structural analogs may have
potential as novel cancer therapeutics.
(10) Berman, D. M.; Karhadkar, S. S.; Hallahan, A. R.; Pritchard, J. I.;
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(13) The induction of cells by Hh proteins sets in motion a cascade
involving the activation and inhibition of downstream effectors, the
ultimate consequence of which is a detectable change in the transcrip-
tion or translation of a gene (e.g., Gli). The Hh assay is a reporter
gene assay that measures the end stage of this cascade (i.e.,
transcriptional modulation). A reporter gene construct, which utilizes
Luciferase, is inserted into the reagent cell to generate a detection
signal, and the amount of transcription from the reporter gene can be
measured. The amount of expression from the reporter gene is
compared to the amount of expression from the same cell in the
absence of the test compound. Any decrease in the amount of
transcription indicates that the test compound is a potential Hh
antagonist. This “Gli-Luc” assay can be applied to both HTS and lead
compound discovery platforms, see: Frank-Kamenetsky, M.; Zhang,
X, M.; Bottega, S.; Guicherit, O.; Wichterle, H.; Dudek, H.; Bumcrot,
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1, 10.
Acknowledgment. The authors thank Jim Marsters and the
team at Genentech for their approval of this manuscript. We
also thank Alysia Parkes at Curis for her help in retrieving
analytical data.
Supporting Information Available: Experimental procedures
and data for all final compounds, and experimental details for the
“Gli-Luc” assay. This material is available free of charge via the
Internet at http://pubs.acs.org.
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JM070694N