Truncated Itraconazole Analogues Exhibiting Potent Anti-Hedgehog Activity and Improved Drug-like Properties

Article abstract of DOI:10.1021/acsmedchemlett.9b00188

We conducted a structure-activity relationship study to explore simplified analogues of the itraconazole (ITZ) scaffold for their ability to inhibit the hedgehog (Hh) signaling pathway. These analogues were based on exploring the effects of chemical modifications to the linker and triazolone/side chain region of ITZ. Analogue 11 was identified as the most potent compound in our first generation, with an IC₅₀ value of 81 nM in a murine Hh-dependent basal cell carcinoma. Metabolic identification studies led us to identify truncated piperazine (26) as the major metabolite in human liver microsomes (HLMs) and an improved Hh pathway inhibitor (IC₅₀ = 22 nM). This work verifies that continued truncation of the ITZ scaffold is a practical method to maintain potent anti-Hh activity while also reducing the molecular weight for the ITZ scaffold and achieving improved pharmacokinetic properties.

Full text of DOI:10.1021/acsmedchemlett.9b00188

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Letter
Truncated Itraconazole Analogues Exhibiting Potent Anti-
Hedgehog Activity and Improved Drug-Like Properties
Jiachen Wen, Divya Chennamadhavuni, Shana R. Morel, and M. Kyle Hadden
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00188 • Publication Date (Web): 23 Aug 2019
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Truncated Itraconazole Analogues Exhibiting Potent Anti-
Hedgehog Activity and Improved Drug-Like Properties
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Jiachen Wen, Divya Chennamadhavuni, Shana R. Morel, M. Kyle Hadden*
Department of Pharmaceutical Sciences, University of Connecticut, 69 North Eagleville Rd, Unit 3092, Storrs, CT 06029-
3092, United States
KEYWORDS. Hedgehog pathway, Itraconazole, Structure–activity relationship, Metabolite, Druggability.
ABSTRACT: We conducted a structure–activity relationship study to explore
simplified analogues of the itraconazole (ITZ) scaffold for their ability to inhibit the
hedgehog (Hh) signaling pathway. These analogues were based on exploring the
effects of chemical modifications to the linker and triazolone/side chain region of
ITZ. Analogue 11 was identified as the most potent compound in our first generation,
with an IC50 value of 81 nM in a murine Hh-dependent basal cell carcinoma.
Metabolic identification studies led us to identify the truncated piperazine (26) as the
major metabolite in human liver microsomes (HLMs) and an improved Hh pathway
inhibitor (IC50 = 22 nM). This work verifies that continued truncation of the ITZ
scaffold is a practical method to maintain potent anti-Hh activity, while also reducing the molecular weight for the ITZ scaffold and
achieving improved pharmacokinetic properties.
The Hedgehog (Hh) pathway is a signaling cascade critical
for cell development and tissue patterning.1 Inappropriate
modulation of Hh signaling has been implicated in
developmental disorders and cancers including, basal cell
significantly more active than the comparable 4S analogues.14
We also
Dioxolane Region Diphenylpiperazine Tether Triazolone/Side Chain
N
2
−5
carcinoma (BCC), medulloblastoma (MB), and leukemia.
The approval of vismodegib and sonidegib as treatments for
advanced BCC confirmed the clinical efficacy of small
molecule pathway inhibitors for the treatment of Hh-dependent
N
O
O
N
2
4
O
N
N
O
N
N
N
6
-9
cancers. Of continued concern in the development of Hh
pathway inhibitors is the identification of multiple mutations in
Smoothened (Smo), the molecular target of vismodegib and
sonidegib, which reduce binding affinity for the drugs and
Cl
Cl
1
, ITZ, IC50 = 140 nM, M.W. = 705.6
O
1
0−12
H3C
decrease overall efficacy.
2S
O
4
R
O
OH
Recent years have seen the identification and exploration of
O
N
N
N
H
itraconazole (1, ITZ, Figure 1), an FDA approved antifungal
Cl
Cl
1
3−15
agent, as an Hh pathway inhibitor.
Strong evidence exists
2, IC50 = 160 nM, M.W. = 634.6
to suggest that ITZ binds and directly inhibits Smo, a key
1
3
component of the Hh pathway; however,ITZ retains potent Hh
inhibitory activity in vitro and in vivo in the presence of
H3C
O
1
6,17
O
vismodegib-resistant Smo mutants.
With this in mind, the
R
O
ITZ scaffold represents a promising anti-cancer strategy that
has the possibility to address the acquired resistance after
treatment associated with the clinically approved Smo
antagonists.
O
N
N
X
Cl
Cl
3-20
Figure 1. Structure and regions of ITZ, lead analogue 2, and
analogues made in current work.
As shown in Figure 1, the structure of ITZ can be separated
into three general regions, the dioxolane region, the central
diphenylpiperazine core, and the triazolone/side chain region.
Our research group has been exploring structure-activity
relationships (SARs) for the ITZ scaffold with respect to its
demonstrated that the triazole ring is not required for ITZ-
14
mediated Hh inhibition. Our most recent SAR suggests that
modification in the triazolone/side chain area is well-tolerated.
Potent compounds have been discovered through side chain
1
4,15
anti-Hh activity.
In these previous studies, we demonstrated
14,18
15
group modification,
(hetero)cycle replacement, and even
that the stereochemistry around the dioxolane is critical for
potent Hh inhibition. Analogues with the 4R orientation are
14
direct truncation of the triazolone ring. In the current study,
the simplified triazolone analogue 2, which retained the potent
1
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anti-Hh activity and a decreased molecular weight compared to
Page 2 of 8
corresponding aniline 9 by palladium on charcoal. For
analogues bearing an allyl group at the terminal phenol, free
phenols were protected as allyloxy before coupling with 24. By
treating with sodium borohydride and iodine, the allyl group
was removed under a translocation mechanism, to give phenols
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
ITZ, was used as our lead structure. We sought to begin probing
SAR for the tether region adjacent to the triazolone by directly
appending the aromatic functional group to the central
piperazine through an amide bond. These modifications would
also result in analogues with reduced molecular weight.
1
9
3, 4, 10−14, and 17. Annotated structures for the final
analogues are listed in Table 1.
Following our previously reported procedures, 1-(4-
hydroxyphenyl)piperazine 21 and the stereochemically defined
dioxolane regions 22 (2S,4R-cis) and its enantiomer (2R,4R-
For the analogues bearing a hydrazide, intermediate 21 was
first coupled with phenyl chloroformate under alkaline
conditions. The resulting amide 23 was coupled with dioxolane
22, followed by refluxing with hydrazine hydrate in 1,4-dioxane
to afford hydrazide 24. The hydrazide terminal was later
coupled with appropriate benzaldehydes to give final phenols
18 and 19. When 23 coupled with dioxolane in 2R,4R-trans
configuration, followed by the same hydrazinolysis and
condensation procedures, hydrazide 20 was synthesized
(Structure in Table 1, complete syntheses are shown in the
Supporting Information).
1
4
trans) were synthesized. As shown in Scheme 1, intermediate
1 was coupled with an appropriate carboxylic acid in the
2
0
1
2
3
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5
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7
8
9
0
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0
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5
6
7
8
9
0
presence
of
the
condensing
agent
(EDCI)
1-ethyl-3-(3-
and 4-
dimethylaminopropyl)carbodiimide
dimethylaminopyridine (DMAP) at room temperature.
Selective addition to the piperazine secondary amine was
observed for the majority of our analogues. Amide
intermediates were then coupled with dioxolane 22 (2S,4R-cis)
under alkaline conditions to afford analogues 5−8, 15 and 16.
The analogue bearing a para-nitro (8) was reduced to the
a
Scheme 1. General Synthesis of Amides and Hydrazides
O
O
O
e
25, b
O
NH2
HO
N
NH
HO
N
N
OPh
O
N
N
N
H
f
Cl
Cl
21
23
24
g
a
O
O
O
O
OH
R
n
N
HO
N
N
O
N
N
N
H
Cl
Cl
Amide intermediates
18, 19
O
O
n = null, -CH2-, -CH2-CH2-, -CH=CH-, -CH2-CH2-CH2-
NO2
OTs b, c or d
Cl
Cl 22
R =
R =
R =
Allyloxy
OH
O
OCH3
O
c
d
NH2
O
R
n
R =
R =
R =
O
N
N
Cl
Cl
3
- 17
aReagents and conditions: (a) Appropriate carboxylic acid, EDCI, DMAP, TEA, DMF, RT, overnight, 31−89%; (b) Cs
2
3
CO , DMSO,
o
60 C, 4 h, 51−93%; (c) Pd/C, hydrazine hydrate, EtOH, reflux, 4 h, 91%; (d) NaBH
4
, I
2
, THF, RT, 2 h, 31−62%; (e) Phenyl chloroformate,
, DCM, 0 C to RT, 4 h, 62%; (f) Hydrazine hydrate, 1,4-diox, reflux, 12 h, 89−91%; (g) Appropriate benzaldehyde, EtOH, reflux, 4
o
K
2
CO
3
h, 62−92%.
As noted above, our previous SAR studies for the ITZ
scaffold demonstrated that the triazolone/side chain region of
ITZ is amenable to modification. In order to continue our SAR
exploration of this region of the scaffold, we sought to
synthesize and evaluate analogues that continue the scaffold
truncation that we previously probed by removing the phenyl
ring on the ‘right side’ of the scaffold and directly appending
the 3-phenolic carboxylic acid of 2 to the piperazine amine to
provide analogue 3. We evaluated this analogue for its ability
to decrease Gli1 mRNA expression in the Hh-dependent murine
To further probe SAR for functionality on the aromatic
moiety, aromatic carboxylic acids with a range of substitutions
4−9 were synthesized and evaluated. Analogue 4 (IC50 = 0.58
μM), with the phenol in the para-position, was less active than
3. Replacing the phenol with pyridines resulted in compounds
5−7 with reduced anti-Hh activity, with the 4-pyridine (7, IC50
= 0.57 μM) representing the best in this small collection.
Replacing the phenol with a nitro group (8, IC50 = 0.16 μM)
demonstrated improved activity compared to 3 and equivalent
activity to the lead 2. Reduction of the nitro to the 4-aniline (9,
IC50 = 0.28 μM) resulted in a modest decrease in activity.
20-22
BCC cell line ASZ001.
Analogue 3 demonstrated
comparable anti-Hh activity compared to 2 (IC50 values = 0.26
µM and 0.16 µM, respectively) verifying that this series of
compounds retains potent Hh inhibition and encouraging us to
synthesize additional analogues that incorporate the 4-
phenylpiperazinyl amide.
Next, we sought to increase the distance between the amide
bond and the aromatic moiety to understand the optimal
distance between these functional groups for potent Hh
inhibition (10−20). The addition of a single methylene unit (10)
resulted in a slight decrease in Hh inhibition compared to 2;
however, the increase to two methylenes (11, IC50 = 0.081 µM)
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aIC50 values represent the Mean ± SEM of at least two separate
experiments performed in triplicate.
significantly enhanced the anti-Hh activity of the scaffold.
Constraining the linker as a trans-alkene 12 resulted in an
approximately 3-fold loss in Hh inhibitory activity, a trend that
was also present when the phenol was located in the 4-position
1
2
3
4
5
6
7
8
9
Finally, we sought to vary the chemical composition of the
linker by introducing heteroatoms; therefore, a small collection
of hydrazide-containing analogues was synthesized and
evaluated. Analogue 18 (IC50 = 0.12 μM) demonstrated superior
Hh inhibitory activity to 2 and ITZ, once again suggesting this
region of the scaffold is amenable to modification. Not
surprisingly, analogue 19 (IC50 = 0.96 μM), which incorporates
the para-phenol, was less active compared to the analogous
meta-phenol 18. Our previous truncated ITZ analogue series
identified the cis-dioxolane as the ideal orientation for these
compounds; however, we also synthesized and evaluated the
(13 and 14). Deletion of the phenol (15, IC50 = 0.23 μM) or
masking it as the methoxy (16, IC50 = 0.46 μM) decreased
activity, suggesting the free phenol is not essential for anti-Hh
activity, but it is optimal in our scaffold. Due to the commercial
availability of 3-hydroxybenzenebutanoic acid, analogue 17
with a propyl linker, but a para-phenol tail was synthesized and
evaluated. A decrease in activity was observed with the
extended linker in 17 (IC50 = 0.47 µM).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
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5
5
5
5
5
5
5
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0
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2
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0
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0
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0
1
2
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9
0
Table 1. Anti-Hh Activity for ITZ and its Analoguesa
2
R,4R-trans-dioxolane hydrazide to determine whether this
H3C
O
H3C
O
2R
O
2
S
4
R
4R
trend is replicated for the analogues described herein. This
analogue, 20 (IC50 = 0.58 μM), was approximately 5-fold less
active, highlighting the importance of the cis-orientation around
the dioxolane ring for this series of compounds.
O
O
N
N
R
O
N
N
R
cis
Cl
Cl
Cl
Cl trans
3
- 19
20
Cmpd
ITZ
2
R
ASZ001, IC50 (μM)
0.14 ± 0.02
Table 2. In Vitro Pharmacokinetic Dataa
---
---
0.16 ± 0.03
Solubility, PBS (µM)
Microsomal Stability
3
4
5
6
7
8
0.26 ± 0.08
0.58 ± 0.02
1.42 ± 0.30
1.30 ± 0.31
0.57 ± 0.11
0.16 ± 0.02
Cmpd
Clint
pH 4.0
pH 7.4
T
1/2 (min)
(
µl/min/mg)
25.2 ± 3.1
34.7 ± 1.0
43 ± 0.6
33.9 ± 0.6
218 ± 9.7
60.0 ± 1.1
b
ITZ
2
---
---
0.31
0.25
0.13
0.05
0.80
0.02
0.17
0.13
0.03
0.02
27.0 ± 6.2
19.9 ± 0.6
15.9 ± 0.2
20.4 ± 0.4
3.2 ± 0.1
b
3
10
1
1
1
9
11.4 ± 0.2
a
More information about the assay protocols are shown in the
Supporting Information.
Values for ITZ and 2 are taken from reference 15.
b
In addition to determining whether significant truncation to
the triazolone/side chain region results in compounds that retain
potent Hh inhibition, a key reason for the preparation of these
analogues was to evaluate whether truncation also provides
compounds with improved solubility and/or stability. Several
representative compounds (3, 10, 11, and 15) were chosen to
evaluate these two parameters for this series of analogues.
Unfortunately, all tested analogues failed to improve the overall
solubility as each analogue was less soluble than ITZ at both pH
9
0.28 ± 0.11
0.38 ± 0.04
10
11
0.081 ± 0.022
4
.0 and pH 7.4 (Table 2). Overall, our analogues were less
stable than ITZ in human liver microsomes (HLMs) and
demonstrated predicted intrinsic clearance levels significantly
higher than the parent compound. Based on the reduced stability
previously determined for amide-containing analogue 2, it was
not too surprising that these compounds were also less stable.
1
1
2
3
0.22 ± 0.03
0.26 ± 0.03
Disappointingly, our most active analogue 11, was also the
least stable (T1/2 = 3.2 mins). The only structural difference
between 11 and 10, which was approximately 6.5-fold more
stable, is the addition of a single methylene between the amide
and the phenol-substituted benzene ring. In order to more fully
determine how the additional methylene influences stability, we
performed follow-up metabolic identification (MetID) studies
in HLMs for both 10 and 11.
14
0.36 ± 0.05
0.23 ± 0.16
0.46 ± 0.03
1
1
5
6
Incubation of 11 with HLMs resulted in the identification of
two primary metabolites (Figure 2 and Figure S1−S4). Peak #1
indicated the metabolite (26, 30.09%) had undergone
hydrolysis of the amide bond, an unexpected metabolite
considering amides are generally deemed as a reliable tether and
exist in many marketed drugs. The second major metabolite
(Peak #2, 15.94%) resulted from single oxidation to the 3-
hydroxyphenethyl region. While the exact location of the
hydroxyl/phenol in metabolite #2 could not be conclusively
17
18
19
0.47 ± 0.11
0.12 ± 0.01
0.96 ± 0.15
0.58 ± 0.07
2
0
3
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determined; CYP-mediated oxidation at the para-phenyl Based on the results of the metabolism studies, our follow-up
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2
3,24
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
position is well-documented.
Based on the measured m/z+
experiments were focused in two directions. First, we
synthesized and evaluated the metabolites (26 and 28) of our
most active analogue 11 to determine if they retain potent anti-
Hh activity. In parallel, we sought to enhance the stability of 11
by eliminating the common metabolic soft spot, which led to
the design of saturated analogue 31. The syntheses are
described in Scheme 2. For the analogue truncated at the
piperazine, selective protection of 21 as the secondary Boc
amine afforded phenol 25. After coupling with dioxolane 22,
Boc removal was performed by stirring with TMSOTf under
alkaline conditions to give the free piperazine 26. Starting with
3,4-bis-allyloxybenzenepropanoic acid and using the same
procedures as in Scheme 1, bis-phenol 28 was synthesized. For
the des-amide analogue, intermediate 29 was synthesized after
reduction and tosylation. Selective addition to the piperazine
amine of 21 was observed by heating in the presence of 29 to
give phenol 30. Dioxolane coupling and final deprotection were
performed under the same conditions as described earlier to
give amine 31.
peaks, we presumed that 3,4-bis-phenol 28 is the most likely
structure for metabolite #2.
For analogue 10, our MetID studies suggested this analogue
was involved in more complicated metabolic pathways,
including hydrolysis, hydrogenation, de-oxidation, and then
oxidation (Figure S5−S10). Interestingly, no direct oxidation to
the phenol region was observed in the metabolites of 10. The
difference in metabolic pathways for 10 and 11 may explain the
difference in HLM stability. Of note, 26 was the primary
metabolite for both 10 and 11, indicating the N-piperazine
amide bond was a clear metabolic soft spot on our amide ITZ
analogues.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
O
N
NH
Cl
Cl
Peak #1, 30.09%
Hydrolysis
O
We were excited to determine that both metabolites retain
potent inhibition of Hh signaling (Table 3). Analogue 28 (IC50
O
O
=
0.089 µM), which incorporates the bis-phenol, showed
OH
OH
O
N
N
equivalent activity to the parent 11; whereas the truncated
piperazine 26 (IC50 = 0.022 µM) was more potent than 11.
Metabolite 23 was significantly less active than 11 or the other
two metabolites (IC50 = 0.87 µM). In C3H10T1/2 cells, an Hh-
dependent mouse embryonic fibroblasts (MEFs), 11 and 26
were also more potent than ITZ when using endogenous
oxysterols as the Hh pathway activator (Table S1). Based on the
improved activity of the truncated analogue 26, which contains
approximately half of the intact ITZ scaffold, we also evaluated
Cl
Cl
1
1, 53.97%
Oxidation
O
O
O
O
N
N
O
Cl
Cl
Peak #2, 15.94%
-/-
this analogue in Sufu mouse embryonic fibroblasts (MEFs) to
determine whether Smo is still the most likely target of the
metabolite. The anti-Hh activity of 26 was completely
abolished in this cell line, suggesting 26 (and ITZ) is
functioning upstream of Sufu, most likely at the level of Smo
(Figure S11).
Figure 2. Metabolite identification for 11 in the presence of HLMs.
Percentages indicate the relative peak area under experimental
conditions.
Scheme 2. Synthesis of Metabolites 26, 28 and Amine 31a
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O
O
O
1
2
3
4
5
6
7
8
9
Allyloxy
Allyloxy
b, e
O
OH
OH
HO
N
d
N
O
N
N
Cl
Cl
2
8
27
O
O
a
b, c
HO
N
NH
HO
N
25
N
Boc
O
N
NH
Cl
Cl
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
1
2
6
Allyloxy
TsO
f
29
O
Allyloxy
b, e
O
OH
HO
N
N
O
N
N
Cl
Cl
3
0
31
a
o
Reagents and conditions: (a) Di-tert-butyl dicarbonate, TEA, DCM, RT, 2 h, 93%; (b) 22, Cs
2
3
CO , DMSO, 60 C, 4 h, 39−98%; (c)
TMSOTf, 2,6-lutidine, DCM, 0 C to RT, overnight, 72%; (d) 3,4-Bis-allyloxybenzenepropanoic acid, EDCI, DMAP, TEA, DMF, RT,
o
o
4 2 2 3
overnight, 81%; (e) NaBH , I , THF, RT, 2 h, 41−71%; (f) 29, K CO , DMF, 40 C, 4 h, 41%.
a
b
Table 3. Anti-Hh activity , and Pharmacokinetic Data for Metabolite-based ITZ Analogues
H3C
O
O
O
N
N
R
Cl
Cl
2
6, 28, and 31
Solubility, PBS (µM)
Microsomal Stability
Clint
Cmpd
R
ASZ001, IC50 (μM)
0.022 ± 0.011
pH 4.0
pH 7.4
T
1/2 (min)
(
µl/min/mg)
2
2
6
8
-H
169.3
11.7
0.84
50.7 ± 1.5
13.7 ± 0.4
0.089 ± 0.06
---
6.6 ± 0.1
104.7 ± 0.1
50.0 ± 0.8
3
1
0.87 ± 0.6
0.13
0.02
14.0 ± 0.2
a
IC50 values represent the Mean ± SEM of at least two separate experiments performed in triplicate.
More information about the assay protocols are shown in the Supporting Information.
b
The activity for both metabolites prompted us to study their
In conclusion, we have developed a series of ITZ analogues
focused on continued truncation of the ‘right-side’ of the
scaffold. Analogue 11 represents the most potent analogue in
the first-round SAR study, with an IC50 value of 81 nM,
approximately two-fold more potent than lead 2 and ITZ. Our
initial SAR suggested a proper length linker with certain
flexibility is critical for potent anti-Hh activity. In addition, a
stereochemically defined dioxolane cap (2S,4R-cis) and a para-
phenol tail are also important for the anti-Hh activity of 11. A
metabolite of compound 11, analogue 26, demonstrated
improved Hh pathway inhibition, increased stability and
enhanced water solubility. Moreover, the molecular weight of
26 (MW = 423.3) is significantly decreased compared to ITZ
(MW = 705.6) establishing analogue 26 as a promising
candidate for further in vitro and in vivo analysis. Overall, these
studies provide clear rationale for continued modification to the
central core of the scaffold as a practical design strategy to
PK profile (Table 3). Not surprisingly, analogue 26
demonstrated improved stability (T1/2 = 50.7 mins) compared to
parent compound 11. The stabilities of compound 28 and 31
(T1/2 = 6.6 mins and 14.0 mins, respectively) were minimally
improved compared to 11. In addition, significant
improvements in solubility at pH 7.4 and pH 4.0 compared to
ITZ were found for analogue 26. Finally, we evaluated the
general toxicity of compounds 11 and 26 by exploring their
anti-proliferative activity in HC-04 cells, a human hepatocyte-
derived cell line that abundantly expresses drug metabolizing
enzymes (Table S2). Both compounds demonstrated moderate
toxicity (26, GI50 = 27.3 µM; 11, GI50 = 13.18 µM) in the HC-
4 cells, indicating their anti-Hh activity is not a result of broad
toxicity and further highlighting their potential as selective anti-
cancer therapeutics.
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retain potent anti-Hh activity while improving the overall drug-
like properties of the scaffold.
oncogenic transformation in high-risk T-cell acute lymphoblastic
leukemia. Leukemia 2018, 32, 2126−2137.
6) Robarge, K. D.; Brunton, S. A.; Castanedo, G. M.; Cui, Y.; Dina,
M. S.; Goldsmith, R.; Gould S. E.; Guichert, O.; Gunzner, J. L.;
Halladay, J.; Jia, W.; Khojasteh, C.; Koehler, M. F.; Kotkow, K.; La,
H.; Lalonde, R. L.; Lau, K.; Lee, L.; Marshall, D.; Marsters, J. C.;
Murray, L. J.; Qian, C.; Rubin, L. L.; Salphati, L.; Stanley, M. S.;
Stibbard, J. H.; Sutherlin, D. P.; Ubhayaker, S.; Wang, S.; Wong, S.;
Xie, M. GDC-0449-a potent inhibitor of the hedgehog pathway.
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(
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website.
Detailed MetID results, general experimental methods, detailed
(7) Pan, S.; Wu, X.; Jiang, J.; Gao, W.; Wan, Y.; Cheng, D.; Han, D.;
1
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synthetic procedures, and compound characterization ( H,
C
Liu, J.; Englund, N. P.; Wang, Y.; Peukert, S.; Miller-Moslin, K.; Yuan,
J.; Guo, R.; Matsumoto, M.; Vattay, A.; Jiang, Y.; Tsao, J.; Sun, F.;
Pferdekamper, A. C.; Dodd, S.; Tuntland, T.; Maniara, W.; Kelleher, J.
F.; Yao, Y.-M.; Warmuth, M.; Williams, J.; Dorsch, M. Discovery of
NVP-LDE225, a potent and selective smoothened antagonist. ACS
Med. Chem. Lett. 2010, 1, 130-134.
(8) Sekulic, A.; Migden, M. R.; Oro, A. E.; Dirix, L.; Lewis, K. D.;
Hainsworth, J. D.; Solomon, J. A.; Yoo, S.; Arron, S. T.; Friedlander,
P. A; Marmur, E. Efficacy and safety of Vismodegib in advanced basal-
cell carcinoma. N. Engl. J. Med. 2012, 366, 2171−2179.
NMR and MS). (PDF)
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AUTHOR INFORMATION
Corresponding Author
* E-mail: kyle.hadden@uconn.edu. Phone: 1-860-486-8446. Fax:
-860-486-6857.
1
(9) Migden, M. R.; Guminski, A.; Gutzmer, R.; Dirix, L.; Lewis, K. D.;
Combemale, P.; Herd, R. M.; Kudchadkar, R.; Trefzer, U.; Gogov, S.;
Pallaud, C. Treatment with two different doses of Sonidegib in patients
with locally advanced or metastatic basal cell carcinoma (BOLT): a
multicentre, randomised, double-blind phase 2 trial. Lancet Oncol.
ORCID
M. Kyle Hadden: 0000-0001-9482-1712
Author Contributions
2
015, 16, 716−728.
(10) Yauch, R. L.; Dijkgraaf, G. J.; Alicke, B.; Januario, T.; Ahn, C. P.;
Holcomb, T.; Pujara, K.; Stinson, J.; Callahan, C. A.; Tang, T.; Bazan,
J. F. Smoothened mutation confers resistance to a Hedgehog pathway
inhibitor in medulloblastoma. Science 2009, 326, 572−574.
J. W. synthesized and evaluated ITZ analogues and wrote the
manuscript. D. C. synthesized ITZ analogues. S. R. M. evaluated
ITZ analogues. All authors have given approval to the final version
of the manuscript.
(11) Brinkhuizen, T.; Reinders, M. G.; van Geel, M.; Hendriksen, A.
J.; Paulussen, A. D.; Winnepenninckx, V. J.; Keymeulen, K. B.;
Soetekouw, P. M.; van Steensel, M. A.; Mosterd, K. Acquired
resistance to the Hedgehog pathway inhibitor vismodegib due to
smoothened mutations in treatment of locally advanced basal cell
carcinoma. J. Am. Acad. Dermatol. 2014, 71, 1005−1008.
(12) Atwood, S. X.; Sarin, K. Y.; Li, J. R.; Yao, C.; Urman, N. M.;
Chang, A. L. S.; Tang, J. Y; Oro, A. E. Rolling the genetic dice: neutral
and deleterious Smoothened mutations in drug-resistant basal cell
carcinoma. J. Invest. Dermatol. 2015, 135, 2138−2141.
Funding Sources
We gratefully acknowledge support of this work by the National
Institutes of Health/National Cancer Institute (CA190617).
ACKNOWLEDGMENT
The authors kindly thank Dr. Ervin Epstein (Children’s Hospital
Oakland Research Institute) for providing the ASZ001 cells. HC-
0
4 cells were provided by Dr. José Manautou (University of
(13) Kim, J.; Tang, J. Y.; Gong, R.; Kim, J.; Lee, J. J.; Clemons, K. V.;
Connecticut).
Chong, C. R.; Chang, K. S.; Fereshteh, M.; Gardner, D; Reya, T.
Itraconazole, a commonly used antifungal that inhibits Hedgehog
pathway activity and cancer growth. Cancer cell 2010, 17, 388−399.
ABBREVIATIONS
(14) Pace, J. R.; DeBerardinis, A. M.; Sail, V.; Tacheva-Grigorova, S.
ITZ, Itraconazole; Hh, Hedgehog; BCC, basal cell carcinoma; MB,
medulloblastoma; Gli, glioblastoma associated oncogene; Smo,
Smoothened; SAR, structure–activity relationship; EDCI, 1-ethyl-
K.; Chan, K. A.; Tran, R.; Raccuia, D. S.; Wechsler-Reya, R. J.;
Hadden, M. K. Repurposing the clinically efficacious antifungal agent
itraconazole as an anticancer chemotherapeutic. J. Med. Chem. 2016,
59, 3635−3649.
3
-(3-dimethylaminopropyl)carbodiimide;
DMAP,
4-
dimethylaminopyridine; Boc, tert-butyloxycarboryl; TMSOTf,
trimethylsilyl trifluoromethanesulfonate; HLMs, human liver
microsomes; MEFs, mouse embryonic fibroblasts; MetID,
metabolic identification.
(15) Pace, J. R.; Teske, K. A.; Chau, L. Q.; Dash, R. C.; Zaino, A. M.;
Wechsler-Reya, R. J.; Hadden, M. K. Structure-activity relationships
for itraconazole-based triazolone analogues as hedgehog pathway
inhibitors. J. Med. Chem. 2019, 62, 3873−3885.
(16) Kim, J.; Aftab, B. T.; Tang, J. Y.; Kim, D.; Lee, A. H.; Rezaee,
M.; Kim, J.; Chen, B.; King, E. M.; Borodovsky, A.; Riggins, G. J.
Itraconazole and arsenic trioxide inhibit Hedgehog pathway activation
and tumor growth associated with acquired resistance to smoothened
antagonists. Cancer Cell 2013, 23, 23−34.
(17) Tao, H.; Jin, Q.; Koo, D. I.; Liao, X.; Englund, N. P.; Wang, Y.;
Ramamurthy, A.; Schultz, P. G.; Dorsch, M.; Kelleher, J.; Wu, X.
Small molecule antagonists in distinct binding modes inhibit drug-
resistant mutant of smoothened. Chemistry & Biology 2011, 18,
432−437.
(18) Shi, W.; Nacev, B. A.; Aftab, B. T.; Head, S.; Rudin, C. M.; Liu,
J. O. Itraconazole side chain analogues: structure–activity relationship
studies for inhibition of endothelial cell proliferation, vascular
endothelial growth factor receptor 2 (VEGFR2) glycosylation, and
hedgehog signaling. J. Med. Chem. 2011, 54, 7363−7374.
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Cap
Linker
Tail
H C
3
O
O
O
N
N R
Cl
Cl
1
1, R = 3-Hydroxyphenyl propionyl, IC50 = 81 nM,
Solubility = 0.03 M, HLM T1/2 = 3.2 min;
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