Macrocyclic hedgehog pathway inhibitors: Optimization of cellular activity and mode of action studies

Article abstract of DOI:10.1021/ml300172p

Macrocyclic Hedgehog (Hh) pathway inhibitors have been discovered with improved potency and maximal inhibition relative to the previously reported macrocycle robotnikinin. Analogues were prepared using a modular and efficient build-couple-pair (BCP) approach, with a ring-closing metathesis step to form the macrocyclic ring. Varying the position of the macrocycle nitrogen and oxygen atoms provided inhibitors with improved activity in cellular assays; the most potent analogue was 29 (BRD-6851), with an IC₅₀ of 0.4 μM against C3H10T1/2 cells undergoing Hh-induced activation, as measured by Gli1 transcription and alkaline phosphatase induction. Studies with Patched knockout (Ptch^-/-) cells and competition studies with the Smoothened (Smo) agonists SAG and purmorphamine demonstrate that in contrast to robotnikinin, select analogues are Smo antagonists.

Full text of DOI:10.1021/ml300172p

Letter
pubs.acs.org/acsmedchemlett
Macrocyclic Hedgehog Pathway Inhibitors: Optimization of Cellular
Activity and Mode of Action Studies
Chris Dockendorff,*,†,§ Marek M. Nagiec,*,†,§ Michel Weïwer,†,§ Sara Buhrlage,†,§ Amal Ting,†
Partha P. Nag,† Andrew Germain,† Han-Je Kim,† Willmen Youngsaye,† Christina Scherer,†
Melissa Bennion,† Linlong Xue,† Benjamin Z. Stanton,† Timothy A. Lewis,† Lawrence MacPherson,†
Michelle Palmer,† Michael A. Foley,† Jose R. Perez,† and Stuart L. Schreiber†,‡
́
†Chemical Biology Platform and Probe Development Center and ‡Howard Hughes Medical Institute, Broad Institute of Harvard and
MIT, 7 Cambridge Center, Cambridge, Massachusetts 02142, United States
S
* Supporting Information
ABSTRACT: Macrocyclic Hedgehog (Hh) pathway inhibitors have
been discovered with improved potency and maximal inhibition
relative to the previously reported macrocycle robotnikinin. Analogues
were prepared using a modular and efficient build-couple-pair (BCP)
approach, with a ring-closing metathesis step to form the macrocyclic
ring. Varying the position of the macrocycle nitrogen and oxygen
atoms provided inhibitors with improved activity in cellular assays; the
most potent analogue was 29 (BRD-6851), with an IC50 of 0.4 μM
against C3H10T1/2 cells undergoing Hh-induced activation, as
measured by Gli1 transcription and alkaline phosphatase induction.
Studies with Patched knockout (Ptch−/−) cells and competition
studies with the Smoothened (Smo) agonists SAG and purmorphamine demonstrate that in contrast to robotnikinin, select
analogues are Smo antagonists.
KEYWORDS: macrocycle, diversity-oriented synthesis (DOS), Sonic Hedgehog pathway, Smoothened antagonist, C3H10T1/2
differentiation of these cells results in the expression of alkaline
phosphatase, which is readily quantified with fluorescent
substrates; Gli1 expression levels (mRNA) provide another
readout of Hh pathway activation. Our primary objective was
thus to find analogues with improved potency and maximal
inhibition in these assays.18
The preparation of macrocyclic analogues of robotnikinin
used a build/couple/pair19 strategy related to previous
reports15,16,20 that affords rapid access to diverse analogues.
Our general strategy is illustrated in Scheme 1, with only one of
numerous accessible stereoisomers depicted. Amino alcohols
and diamines were coupled with successive alkenoic acid
building blocks, and the resulting dienes were paired in a ring-
closing metathesis (RCM) step. Many compounds underwent
further elaborations at the functional handles included with the
carboxylic acid building blocks.
he Hedgehog (Hh) signaling pathway regulates cell
Tgrowth and migration during embryonic development. It
is normally dormant in adult cells, but elevated activity of this
pathway is associated with cancers such as medulloblastoma
and basal cell carcinoma.1 In recent years, antagonism of
different members of this pathway has been investigated as a
novel mode of cancer chemotherapy, as described in several
reviews.2−6 Inhibition of Smoothened (Smo), a seven-pass
transmembrane receptor with similar topology to G-protein-
coupled receptors (GPCRs), shows promise for the treatment
of cancers driven by activating mutations to the Hh pathway
and also for a subset of epithelial cancers that may require Hh
for growth via a paracrine mechanism.7−10 Several Smo
antagonists have proceeded to advanced clinical trials,11−13
including GDC-0449 (vismodegib),14 which was recently
approved by the Food and Drug Administration (FDA) for
treatment of advanced basal cell carcinoma.
In 2009, a macrocyclic compound derived from a diversity-
oriented synthesis (DOS) library [robotnikinin (1)] was
described that inhibits the Hh signaling pathway.15,16 Hit
compounds were identified by their interaction with Sonic
Hedgehog (Shh) in a small-molecule microarray screen. We
aimed to build upon this work by developing analogues with
improved activity in cellular assays. We ultimately used an assay
based on the differentiation of murine mesenchymal
C3H10T1/2 cells, induced by the Shh protein.17 The
One focus of our medicinal chemistry studies was the
determination of the optimal linker joining the alkenoic acids.
To this end, a variety of amino alcohols and diamines were
obtained or prepared, and these building blocks were
incorporated into different macrocyclic products. A selection
of these compounds is depicted in Chart 1, along with their
Received: June 27, 2012
Accepted: August 18, 2012
© XXXX American Chemical Society
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Scheme 1. General Strategy for Macrocycle Synthesis
Chart 2. Analogues Probing Key SARs
Chart 1. Analogues with Alternative Amino Alcohol Linkers
Chart 3. Analogues with Alternative Side Chains at the 6-
Position
half-maximal inhibitory concentrations in the Shh-induced21
C3H10T1/2 alkaline phosphatase assay, and their maximal
activity relative to the prototypical Shh pathway inhibitor
cyclopamine.
Table 1. Activity of 2-Substituted Macrocycles in Shh-
Induced C3H10T1/2 Cell Differentiation
Cyclopamine produced an half-maximal inhibitory concen-
tration (IC50) of 0.6 μM and reduced the alkaline phosphatase
activity to levels measured in the cells without Shh treatment.
As previously reported, robotnikinin (1) proved to be only
weakly active in this assay.18 Removal of the 2-phenyl
substituent from the macrocycle of 1 obviated all activity
(compound 2). Norephedrine-based compound 3 and
norpseudoephedrine-based 4 had improved maximal activity
over 1 and slightly improved potency in the C3H10T1/2 assay,
as did the prolinol derivative 5. A significant improvement was
observed with compound 6 (IC50 = 5 μM), where the positions
of the macrocyclic oxygen and nitrogen are reversed. Indane 7
was also prepared, but its potency and maximal activity were
poor.
We systematically explored several other structure−activity
relationships (SARs) (Charts 2 and 3 and Table 1).
Methylation of the macrocyclic nitrogen (compound 8) gave
a slight improvement versus 3, but inversion of stereochemistry
at the 2-position of 6 (compound 9) decreased potency.
Substitution at the 11-position was well-tolerated; methyl (10)
and benzyl-substituted (11) analogues of 6 maintained potency
with good to excellent maximal activity. The 11-isopropylami-
no-substituted analogue 12 was weakly active. Certain
modifications of the olefin were also tolerated. For example,
compound 13, possessing a Z-olefin derived from the minor
product of a metathesis reaction, was more potent than the
analogous E-olefin 10. A number of hydrogenated analogues
showed activity comparable to the parent olefin series; for
example, ephedrine derivative 14 showed moderate potency but
low maximal activity. Noncyclic dienes such as 15 were inactive.
a
b
Relative to cyclopamine (100%). Measured by alkaline phosphatase
c
activity. Measured by Gli1 levels. See the Supporting Information for
details.
B
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Figure 1. Use of Smo agonists and antagonists demonstrates Shh pathway specificity of macrocyclic inhibitors. Shh-induced activation of C3H10T1/
2 cells is inhibited by cyclopamine (A), 25 (B), and 29 (C). Pathway inhibition is rescued by the Smo agonist SAG (20 nM). Gli1 mRNA levels were
measured by qPCR using Actb as an internal control.
We next modified the substituent at position 6 (Chart 3) of
the various scaffold variants. Truncated analogues such as 16
and 17 were inactive in the cell assay or were only partial
inhibitors. Amides such as 18 possessing solubilizing groups
had poor activity, suggesting that a lipophilic chain is necessary
at position 6. Compound 19, possessing a trifluorobutyl group
in place of the 4-chlorobenzylamide, showed a dose−response
in this assay, but with poor maximal inhibition. Compounds 20
(IC50 = 7 μM) and 21 (IC50 = 8 μM) demonstrate that the
amide moiety is not critical for activity. Interestingly, movement
of the aromatic chloride of 1 from the para to the meta position
(22) gave improved potency in this assay relative to 1 (IC50 = 8
μM), although the moderate maximal inhibition was not
improved and reached only 50%. The macrocyclic carbamate
23 was prepared to remove the chiral center at the 6-position
and because it would be expected to have improved plasma
stability. Unfortunately, it showed poor activity and decreased
maximal inhibition relative to 6.
Using 6 as a lead compound, we reexamined the SAR at the
2-position of the scaffold (Table 1). The potency was
maintained when the arene was replaced with a cyclohexyl
(24) or benzyl group (25); however, replacement with a n-
butyl group gave weak, difficult-to-quantify activity (26), and
the isopropyl analogue 27 had significantly weaker activity than
6. Potency and maximal inhibition were unaffected by
introduction of a fluorine to the para position of the arene of
6 (compound 28). A log gain in activity [IC50 = 0.6 μM
(alkaline phosphatase readout); 0.4 μM (Gli1 expression)] was
observed with the introduction of a 4-chloro substituent (29),
and this compound also attained the maximal inhibition of
cyclopamine. The synthesis of 29 is depicted in the Supporting
Information. Heteroarene 30 had lower activity, pointing to the
importance of a hydrophobic aromatic ring at the 2-position of
the scaffold.
respectively (Figure 1B,C). These findings suggest that both
compounds act at or above Smo in the Shh-signaling pathway.
To narrow the range of possible targets of 25 and 29, we
used constitutively active Ptch−/− mouse embryonic fibroblast
(MEF) cells carrying Gli-responsive β-galactosidase (β-gal)
reporter.17 Because Patched functions upstream of Smo and
acts as its repressor, inhibition of the Shh pathway at or
upstream from Patched is prevented in this cell line. Measuring
both β-gal activity and Gli1 transcription levels, we found that
both 25 [IC50 = 8.8 μM (Gli1 readout); 8.1 μM (β-gal
readout)] and 29 [IC50 = 3.5 μM (Gli1 readout); 2.0 μM (β-gal
readout)] behave similar to cyclopamine [IC50 = 1.5 μM (Gli1
readout); 1.9 μM (β-gal readout)] and fully inhibit Shh
pathway activity. This indicates that in contrast to robotnikinin,
part or all of their activities are derived from interactions
downstream from Patched. As in the studies with the wild-type
(Ptch+/+) cell line, these activities are abrogated by the addition
of 20 nM SAG. A representative dose−response curve for 25 is
shown in Figure 2. Additional evidence of interaction with Smo
was provided by BODIPY-cyclopamine competitive binding
assays15,23 (see the Supporting Information).
Figure 2. Dose−response curves of 25 in Ptch−/− MEF cells carrying
β-gal reporter gene. The activity in this assay is evidence for action of
the inhibitor downstream of Patched. Pathway inhibition is rescued by
the Smo agonist SAG (20 nM). β-Gal activity levels in untreated cells
(dashed line) and in cells treated with 100 nM KAAD-cyclopamine
(complete inhibition, dotted line) are indicated.
To confirm specificity of the new macrocyclic inhibitors for
the Shh pathway, a SAG rescue test was performed, in which
inhibition of Shh-induced Gli1 expression in C3H10T1/2 cells
was measured in the presence of the Smo agonist SAG22,23 for
two of the most potent compounds, 25 and 29. We used SAG
at 20 nM concentration as it was the minimal concentration
that produced a nearly maximal effect in this assay (see the
Supporting Information). Gli1 mRNA transcript levels were
measured using real-time polymerase chain reaction (PCR)
(Figure 1). Similar to cyclopamine (Figure 1A), both
macrocycles showed a significant rightward shift of the dose−
response curve in the presence of 20 nM SAG, resulting in 18-
and 32-fold increases in the IC50 values for 25 and 29,
To obtain further mechanistic insight, a Schild type analysis24
was undertaken using double titration experiments with the
Smo agonist SAG (Figure 3). We measured downstream Gli1
expression induced by different concentrations of SAG in
C3H10T1/2 cells in the presence of different concentrations of
the inhibitors 1 (robotnikinin), 25 and 29, as well as the Smo
antagonists cyclopamine and GDC-0449 (vismodegib). Cyclop-
amine (Figure 3A) and GDC-0449 (Figure 3B) both induced
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Figure 3. Double-titration experiment with the Smo agonists SAG and purmorphamine (PUR) in the presence of cyclopamine (A, D), GDC-0449
(vismodegib) (B, E), and 29 (C, F). Gli1 mRNA levels were measured by qPCR using Actb as an internal control.
rightward shifts in the dose−response curves without affecting
the maximal induction by SAG, consistent with their actions as
competitive inhibitors of SAG at Smo. In contrast, robotnikinin
(1) showed only marginal effects on the induction of Gli1
transcription by SAG (see the Supporting Information).
Interestingly, the related macrocycles 25 (see the Supporting
Information) and 29 (Figure 3C) behaved differently by
strongly decreasing maximal activity of SAG and only weakly
(25) or moderately (29) increasing its EC50 value. This is
consistent with a mechanism that involves allosteric inhibition
of SAG at Smo.25
These double-titration studies were repeated with the
alternative Smo agonist purmorphamine.26,27 In contrast to
its effects with SAG, cyclopamine had a very weak effect on
purmorphamine-induced Gli1 expression (Figure 3D), and
GDC-0449 affected Gli1 expression strongly but in a manner
consistent with allosteric inhibition25 (Figure 3E). Robotnikinin
showed little effect on purmorphamine-induced Gli-expression
(see the Supporting Information), consistent with its weak
inhibition in the original C3H10T1/2 screen (Chart 1).
Interestingly, the responses to increasing concentrations of 25
(see the Supporting Information) and in particular to 29
(Figure 3F) were more pronounced. This resulted in the
reduction of purmorphamine EC50 without a strong effect on its
maximal induction, thus supporting a competitive interaction
between 25 and 29 and purmorphamine. It should be
emphasized that although the Gli mRNA levels may be
modulated by binding events at the Smo receptor, it is
impossible to rule out additional interactions with downstream
pathway components, so interpretations should be made
cautiously.
forms of Smo, such as the D473H mutation characterized after
clinical treatment with GDC-0449 (vismodegib).29
In summary, several novel macrocyclic compounds are
reported that appear to block the Shh pathway by inhibiting
the membrane protein Smo. These compounds were assembled
with a modular build/couple/pair synthetic strategy using
different olefin-containing carboxylic acid and amino alcohol
building blocks. SAR studies determined that a lipophilic side
chain at the 6-position is required for activity. Several amino
alcohol linkers provided decent activity; interestingly, improved
potency was observed when the macrocycle nitrogen and
oxygen of 1 were reversed. With this scaffold, maximal activity
was observed with an aromatic moiety at the 2-position with
(R) stereochemistry. The most potent compound was the 4-
chlorophenyl analogue 29 (IC50 = 0.4 μM). Competition
studies with the Smo agonists SAG and purmorphamine, as
well as activity in a Ptch−/− cell line, suggest that 25 (BRD-
0607) and 29 (BRD-6851), in contrast to robotnikinin, act
predominantly as Smo antagonists. It is interesting to note that
the subtle structural changes between robotnikinin (a direct
binder of Shh) and compounds such as 29 (characterized here
as a Smo antagonist) apparently lead to an additional mode of
Shh pathway inhibition. These studies also provided evidence
for a two-site binding model at Smo. Compound 29 is
particularly interesting for its activity as an allosteric inhibitor of
SAG but a competitive inhibitor of purmorphamine-induced
Gli1 expression. Its activity in cell lines carrying Smo mutations
is presently under investigation.
ASSOCIATED CONTENT
■
The studies described in Figure 3 support a two-site binding
model at Smo, whereby SAG and purmorphamine bind to
unique sites on the receptor. Related competition studies with
alternative Smo antagonists that also provide evidence for
allosteric binding modes have been reported by Rominger24
and Tao.28 Such novel allosteric inhibitors of Smo could show
important utility for the treatment of cancers with mutated
S
* Supporting Information
Assay protocols, structures of tool compounds, double-titration
experimental data with 25, BODIPY-cyclopamine competitive
binding assay data, and protocols for the preparation of 25 and
29. This material is available free of charge via the Internet at
http://pubs.acs.org.
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AUTHOR INFORMATION
■
Corresponding Author
*E-mail: christopher.dockendorff@marquette.edu (C.D.) or
marek@broadinstitute.org (M.M.N.).
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Author Contributions
§These authors contributed equally to this work.
Funding
Funding for this work was provided in part by the Broad
Institute Gift (M.M.N., J.R.P., and C.S.) and NIH Genomics
Based Drug Discovery Grants RL1GM084437 and
UL1RR024924, administratively linked to NIH Grants
RL1HG004671 and RL1CA133834. This work was supported
in part by GM38627 (awarded to S.L.S.).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank James Chen (Stanford University) for Light-2,
Ptch−/− cells, and Smo expression constructs; Stephen
Johnston, Chris Johnson, and Mike Lewandowski for analytical
chemistry support; Giannina Schafer and Tom Hasaka for help
with Smo binding assays; Lili Wang, Yan-Ling Zhang, Katie
Doud, LaTese Briggs, and Angela Koehler for additional studies
not described here; and Robert Gould, Lee Peng, and Aly
Shamji for helpful comments.
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ABBREVIATIONS
■
β-gal, β-galactosidase; DOS, diversity-oriented synthesis; EDCI,
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; FDA, Food
and Drug Administration; GPCR, G-protein-coupled receptor;
Hh, Hedgehog; IC50, half-maximal inhibitory concentration;
MEF, mouse embryonic fibroblasts; NPG, nitrogen with
protecting group; PCR, polymerase chain reaction; RCM,
ring-closing metathesis; SAR, structure−activity relationship;
Shh, Sonic Hedgehog; Smo, Smoothened
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