Repurposing the Clinically Efficacious Antifungal Agent Itraconazole as an Anticancer Chemotherapeutic

Article abstract of DOI:10.1021/acs.jmedchem.5b01718

Itraconazole (ITZ) is an FDA-approved member of the triazole class of antifungal agents. Two recent drug repurposing screens identified ITZ as a promising anticancer chemotherapeutic that inhibits both the angiogenesis and hedgehog (Hh) signaling pathways. We have synthesized and evaluated first- and second-generation ITZ analogues for their anti-Hh and antiangiogenic activities to probe more fully the structural requirements for these anticancer properties. Our overall results suggest that the triazole functionality is required for ITZ-mediated inhibition of angiogenesis but that it is not essential for inhibition of Hh signaling. The synthesis and evaluation of stereochemically defined des-triazole ITZ analogues also provides key information as to the optimal configuration around the dioxolane ring of the ITZ scaffold. Finally, the results from our studies suggest that two distinct cellular mechanisms of action govern the anticancer properties of the ITZ scaffold.

Full text of DOI:10.1021/acs.jmedchem.5b01718

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Article
Repurposing the Clinically Efficacious Anti-Fungal
Agent Itraconazole as an Anti-Cancer Chemotherapeutic
Jennifer R Pace, Albert M DeBerardinis, Vibhavari Sail, Silvia K Tacheva-Grigorova, Kelly
A Chan, Raymond Tran, Daniel S Raccuia, Robert J. Wechsler-Reya, and M. Kyle Hadden
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01718 • Publication Date (Web): 25 Mar 2016
Downloaded from http://pubs.acs.org on March 31, 2016
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Repurposing the Clinically Efficacious Antiꢀ
Fungal Agent Itraconazole as an AntiꢀCancer
Chemotherapeutic
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‡
,§
‡,§
§
¥
Jennifer R. Pace , Albert M. DeBerardinis , Vibhavari Sail , Silvia K. TachevaꢀGrigorova ,
§
§
§
¥
Kelly A. Chan , Raymond Tran , Daniel S. Raccuia , Robert J. WechslerꢀReya , M. Kyle
§,
Hadden *
§
Department of Pharmaceutical Sciences, University of Connecticut, 69 N Eagleville Rd, Unit
3092, Storrs, CT 06269ꢀ3092
¥
Tumor Initiation and Maintenance Program, NCIꢀDesignated Cancer Center, Sanford Burnham
Prebys Medical Discovery Institute, 2880 Torrey Pines Scenic Drive, La Jolla, CA 92037
‡
These authors contributed equally to this work.
*
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To whom correspondence should be addressed. kyle.hadden@uconn.edu Phone: 1ꢀ860ꢀ486ꢀ
446. Fax: 1ꢀ860ꢀ486ꢀ6857.
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Abstract
Itraconazole (ITZ) is an FDAꢀapproved member of the triazole class of antiꢀfungal
agents. Two recent drug repurposing screens identified ITZ as a promising antiꢀcancer
chemotherapeutic that inhibits both angiogenesis and the hedgehog (Hh) signaling pathway. We
have synthesized and evaluated first and second generation ITZ analogues for their antiꢀHh and
antiꢀangiogenic activities to more fully probe the structural requirements for these antiꢀcancer
properties. Our overall results suggest that the triazole functionality is required for ITZꢀmediated
inhibition of angiogenesis, but that it is not essential for inhibition of Hh signaling. The synthesis
and evaluation of stereochemically defined desꢀtriazole ITZ analogues also provides key
information as to the optimal configuration around the dioxolane ring of the ITZ scaffold.
Finally, the results from our studies suggest that two distinct cellular mechanisms of action
govern the antiꢀcancer properties of the ITZ scaffold.
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INTRODUCTION
Identifying novel biological activities of FDAꢀapproved drugs has emerged as a viable
strategy to expedite the drug discovery process. The pharmacokinetic and toxicological profiles
of these compounds are wellꢀunderstood and they are inherently “drugꢀlike.” To this end, drug
development researchers have made a concerted effort to incorporate small molecule libraries
containing approved drugs in their highꢀthroughput screens. Recently, two such screens designed
to repurpose FDAꢀapproved compounds as antiꢀcancer chemotherapeutics identified itraconazole
(ITZ) as both an inhibitor of the hedgehog (Hh) signaling pathway (IC50 = 690 nM) and
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angiogenesis (IC = 160 nM). ITZ is a clinically efficacious member of the 1,2,4ꢀtriazole class
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of antiꢀfungal agents that exerts its antimycotic effects through potent inhibition of lanosterol
14αꢀdemethylase (14LDM, also known as CYP51), a cytochrome P450 enzyme that catalyzes
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the oxidative conversion of lanosterol to ergosterol.
The Hh pathway is a signaling cascade responsible for cell proliferation, differentiation,
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ꢀ5
and tissue growth in embryogenesis. In healthy adults, the Hh pathway is considered less
active and its primary role is maintaining stem cells in the skin and neural systems.
Dysregulation of the Hh pathway, through a variety of different mechanisms, can lead to its
constitutive activation, uncontrolled cellular proliferation, and tumor growth. Aberrant Hh
pathway activation has been identified in a variety of cancers; however, the two most commonly
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ꢀ8
identified as Hhꢀdependent are basal cell carcinoma (BCC) and medulloblastoma (MB).
Because a significant portion of both BCCs (70%) and MBs (25%) rely on Hh signaling for
tumorigenesis, it has drawn considerable interest for the development of targeted antiꢀcancer
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ꢀ10
agents.
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The importance of angiogenesis in tumor formation, growth, and metastasis is wellꢀ
documented and numerous small molecules and biologics that inhibit angiogenesis are clinically
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useful antiꢀcancer agents.
These angiogenesis inhibitors are primarily utilized to indirectly
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arrest tumor growth by inhibiting the growth of blood vessels and depriving tumor cells of
oxygen and nutrients. In certain cases, combination therapy including a traditional firstꢀline
chemotherapy and an antiꢀangiogenic agent has demonstrated improved efficacy when compared
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to the chemotherapy alone. In addition, while most chemotherapeutic agents are toxic and
confer resistance, antiꢀangiogenic agents have only shown mild side effects and there are no
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reported incidents of resistance.
Although ITZ is clinically efficacious, several of its overall features are not inherently
“drugꢀlike”. The molecular weight of ITZ (706 g/mol) is above the threshold typically targeted
for small molecule therapeutics (500 g/mol). Multiple formulations of ITZ have been developed
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and evaluated to improve its oral bioavailability and overall pharmacokinetic profile.
In
addition, the triazole moiety of ITZ has the ability to coordinate with the heme of CYP450s;
most notably, CYP3A4, and ITZꢀmediated inhibition of CYP3A4 is responsible for multiple
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drugꢀdrug interactions and adverse side effects. If ITZ is to be used as a chemotherapeutic,
drugꢀdrug interactions need to be minimized as cancer patients are often taking multiple
medications to both treat the cancer and manage symptoms. Herein we report the synthesis and
evaluation of an extensive series of itraconazole analogues designed to probe the scaffold for the
identification of functionalities required for its antiꢀHh and antiꢀangiogenic properties.
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RESULTS
Analogue Design
As noted above, the triazole moiety of ITZ is both required for its antiꢀfungal properties
and responsible for its main detrimental side effect, inhibition of CYP3A4. The N4 of the ITZ
triazole interacts with the heme group in CYP51 and CYP3A4 to prevent coordination of the
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molecular oxygen required to initiate oxidation.
In addition to the triazole containing
dioxolane region of the ITZ scaffold, ITZ contains a central phenylꢀpiperazineꢀphenyl linker
region and a triazolone/side chain region (Chart 1). Within the context of its antiꢀfungal activity,
the extended linker and triazolone/side chain regions are not essential for binding the active site
of CYP51; however, they do interact with several amino acid residues in the substrate access
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channel. ITZ contains three chiral centers (2, 4, and 2’) and can potentially exist as a mixture of
eight distinct stereoisomers; however, formation of the cisꢀconfigurations around the dioxolane
ring predominates during its synthesis and pharmaceutical preparations of ITZ are typically
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administered as a 1:1:1:1 mixture of the cis diastereomers.
N
N
Linker Region
O
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N
O
4
2'
O
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N
O
N
N
N
Cl
Cl
Triazolone/
Side Chain Region
Dioxolane Region
Chart 1. Structure and regions of itraconazole (ITZ).
To date, the only structural component of ITZ explored for Hh inhibition is the side chain
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region. These side chain derivatives, as well as a series of stereochemically defined ITZ
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4ꢀ25
analogues, have been explored for their antiꢀangiogenic properties;
however, an inꢀdepth
exploration of the structural requirements of the ITZ scaffold for these antiꢀcancer properties has
not been undertaken. We have prepared and evaluated a first generation series of ITZ analogues
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that systematically truncates the scaffold from both the leftꢀ and rightꢀhand side to identify key
structural features required for inhibition of both Hh signaling and angiogenesis (Table 1).
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Table 1. First generation series of ITZ analogues.
Compound
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ꢀCH
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ꢀNH
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Evaluation of this first generation series provided key structureꢀactivity relationship
SAR) data that was subsequently utilized for the design of a second generation of
(
stereochemically defined ITZ derivatives based on analogue 2 (Table 2). The second generation
series was designed to determine whether a specific stereoisomer of key ITZ analogue 2 was
responsible for its antiꢀHh and/or antiꢀangiogenic properties.
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Table 2. Second generation, stereochemically defined analogues of 2.
Compound
R1
R2
Final Stereochemistry
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Transꢀ2,4
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Cisꢀ2,4
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Transꢀ2S,4S,2’S
Cisꢀ2R,4S,2’S
Transꢀ2R,4R,2’S
Cisꢀ2S,4R,2’S
Transꢀ2S,4S,2’R
Cisꢀ2R,4S,2’R
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Transꢀ2R,4R,2’R
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Transꢀ2S,4S
Cisꢀ2R,4S
Chemistry
Analogues 1ꢀ25 were synthesized following slightly modified literature procedures. All
of these analogues contain the phenylꢀpiperazineꢀphenyl linker region of ITZ, which is initially
constructed by a coupling step between commercially available Nꢀ(4ꢀmethoxyphenyl)ꢀpiperazine
2
6 and 1ꢀchloroꢀ4ꢀnitrobenzene 27 to yield Nꢀ(4ꢀhydroxylphenyl)ꢀN’ꢀ(4ꢀnitrophenyl)ꢀpiperazine
28 (Scheme 1). The nitro moiety in 28 is reduced to the aniline 29 in the presence of hydrazine
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monohydrate and 10% palladium on charcoal. The aniline undergoes a series of wellꢀ
characterized transformations to ultimately provide the key triazolone intermediate 32. The
methoxy substituent in starting material 26 serves as a protecting group throughout Scheme 1
and while other literature sources protect this phenol with a methoxymethyl (MOM) group, we
found the synthetic scheme utilizing the MOM protection to be significantly less efficient in
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generating the corresponding key intermediate.
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a
Scheme 1. Synthesis of linker region intermediate.
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a
Reagents and conditions: (a) K CO , reflux, 12 h, 82%; (b) Pd/C, NH NH •H O (10 eq), reflux,
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.5 h, 71%; (c) Pyr (17 eq), ClCOOPh (1.1 eq), 3 h, 90%; (d) NH NH •H O (5.5 eq), reflux, 3 h,
2 2 2
quant; (e) formamidine acetate (4.5 eq), acetic acid, reflux, 3 h, 91%.
Triazolone 32 was alkylated with either commercially available alkyl bromides (37ꢀ38)
or alkyl brosylates (35ꢀ36), which were prepared from the corresponding commercially available
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and stereochemically defined alcohols (33ꢀ34) (Scheme 2). The methoxy group in the
linker/triazolone/side chain intermediates (39ꢀ42) was removed with 48% hydrobromic acid in
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toluene to afford the phenols 43ꢀ46. It is important to note that alkylation of the triazolone
results in an inversion of stereochemistry for the defined side chains; for example, (R)ꢀbrosylate
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a
Scheme 2. Synthesis of linker/triazolone/side chain intermediates.
a
Reagents and conditions: (a) Et N, BsCl (1.3 eq), RT, 3 h; 30ꢀ50%; (b) Cs CO , brosyl/bromo
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alkyl chain, RT, 12 h, 45ꢀ88% (c) 48% HBr, toluene, reflux, 12 h, 55ꢀ80%.
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The dioxolane regions were obtained via two different mechanisms due to their structural
differences. Dioxolane regions that contained the triazole moiety could not be directly formed
under standard ketalization procedures due to the basicity of the triazole functionality. For these
intermediates, the triazole moiety (50) was added to various halogenated ketones (47ꢀ49) to form
the triazole containing ketones (51ꢀ53). In parallel, tosylated glycerol 56 was formed through
standard tosylation of the commercially available dioxolane 54 and subsequent acidꢀmediated
hydrolysis (Scheme 3). Final dioxolane region intermediates containing the triazole moiety (57ꢀ
59) were prepared through ketalization of 51ꢀ53 and 56 with triflic acid.
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The desꢀtriazole dioxolane regions (61aꢀc and 64ꢀ67) were synthesized under standard
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ketalization reaction conditions utilizing a DeanꢀStark apparatus (Scheme 4). Stereochemically
defined tosylated glycerols 62 and 63 were prepared via the method described above for the
racemic mixture and each of the protected glycerols was used to ketalize 2,4ꢀ
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dichloroacetophenone. For the stereochemically defined dioxolanes, the cisꢀisomer was
predominantly formed (~3:1 cis:trans) and the isomers were easily separable via column
chromatography.
a
Scheme 3. Synthesis of triazoleꢀcontaining dioxolane region intermediates.
a
Reagents and conditions: (a) NaHCO , toluene, reflux, 3h, 25ꢀ65%; (b) Pyr, TsCl, 0°Cꢀ RT, 12
h, 71%; (c) MeOH, 0.5N HCl, reflux, 5 h, 89%; (d) Triflic acid (3ꢀ4 eq), toluene, RT, 60 h 10ꢀ
3
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a
Scheme 4. Synthesis of desꢀtriazole dioxolane region intermediates.
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a
Reagents and conditions: (a) pꢀTsOH (cat.), toluene, reflux (Dean Stark), 48 h, 70ꢀ90%.
Tosylated dioxolane regions were coupled with the linker/triazolone/side chain region
phenols in anhydrous dimethyl sulfoxide with cesium carbonate to yield the final analogues 1ꢀ8
and 12ꢀ25 (Scheme 5A). The truncated triazolone analogues (10ꢀ11) were synthesized in reverse
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order. The unprotected linker region 68 was prepared in the same manner as 28 in Scheme 1
(
Scheme 5B). This linker region intermediate was then coupled with the tosylated dioxolane
region 59 under similar conditions described above to afford truncated analogue 10, which was
reduced to the aniline with palladium on charcoal (10%) in the presence of hydrazine
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monohydrate to afford ITZ analogue 11. Overall purification methods varied for each ITZ
analogue, particularly amongst the first generation series that contained the triazole moiety.
Purification methods for all compounds are listed in the experimental or supplemental
information.
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Scheme 5. Coupling reactions for final ITZ analogues.
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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
a
Reagents and conditions: (a) Cs CO (10 eq), DMSO, 90ºC, 12 h, 45ꢀ88%; (b) Pd/C,
2
3
NH NH •H O (10 eq), reflux, 3.5 h, 60ꢀ70%. The functional groups represented by X, Y, and Z
2
2
2
above can be found in Schemes 2ꢀ4.
Biological Evaluation
The initial evaluation of first generation ITZ analogues (1ꢀ11) as Hh pathway inhibitors
was performed by monitoring endogenous Gli1 mRNA levels in C3H10T1/2 cells, an Hhꢀ
dependent mouse embryonic fibroblast (MEF) at a single concentration (1 ꢁM). In this wellꢀ
studied model system for evaluating small molecule inhibition of Hh signaling, addition of an
exogenous Hh agonist (recombinant Hh ligand or small molecule) results in a characteristic
28
increase in Gli1 mRNA expression. Concomitant incubation with a pathway inhibitor reduces
Gli1 expression. Analogues that reduced Gli1 mRNA levels below 20% in this assay were
subsequently evaluated for their ability to reduce Gli1 mRNA expression levels in a
concentrationꢀdependent fashion in these cells and the Hhꢀdependent murine BCC cell line
3
0ꢀ31
ASZ.
In addition, several compounds were evaluated for their antiꢀproliferative effects in
primary Hhꢀdependent medulloblastoma cells isolated from conditional patched knockout
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1
2
3
4
5
6
7
8
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fl/fl
32ꢀ34
(
Math1ꢀCreꢀER;Ptc , MERP) mice.
Based on the primary nature of the MERP cells, only a
st
nd
small subset of 1 and 2 generation ITZ analogues were chosen for evaluation in these cells.
Finally, each of these analogues was evaluated for its ability to inhibit proliferation in human
umbilical vein epithelial cells (HUVECs). In vivo angiogenesis is dependent on endothelial cell
proliferation; therefore, the inhibition of HUVEC proliferation is commonly utilized as an early
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
4ꢀ25
stage in vitro model of antiꢀangiogenic activity.
In addition, the identification of ITZ as an
2
antiꢀangiogenic compound was established via this assay. The results for these assays are given
in Table 3.
Evaluation of the first generation of ITZ analogues indicated several structural features
that appear necessary for Hh pathway inhibition. While the absolute stereochemistry at the 2’ꢀ
position of the secꢀbutyl side chain does not appear important, 6 and 7 are equipotent in the MEF
and ASZ cell lines, removing the methyl moiety (8) significantly reduces the overall activity of
the scaffold. Interestingly, removing the side chain (9) or the triazolone/side chain (10ꢀ11) did
not affect the Hh inhibitory activity of the scaffold as each of these analogues also demonstrated
potent inhibition of pathway signaling in each cell line with IC levels comparable to ITZ. In
5
0
regards to modifications to the dioxolane region, removal of the chlorine atoms on the phenyl
ring had minimal effects (3), while complete truncation of the phenyl ring (4) resulted in a
significant loss of antiꢀHh activity. Removal of the triazole moiety (2) had no effect on the
ability of the scaffold to inhibit Hh signaling. Not surprisingly, removal of both the triazole and
phenyl ring (5) or complete truncation to the phenol (45), significantly reduced Hh inhibitory
activity. Our synthesized ITZ (1) demonstrated comparable activity to the ITZ purchased
commercially in each of the cell lines evaluated. Finally, while each of the analogues evaluated
was equipotent in its ability to downꢀregulate Gli1 mRNA expression in both the C3H10T1/2
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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
and ASZ cells (Table 3), the ability of ITZ, 1, and 11 to inhibit the proliferation of Hhꢀdependent
murine MB cells was slightly reduced.
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
Table 3. In vitro activity of first generation ITZ analogues.
c
%
Gli
c
c
GI (µM)
50
IC (ꢁM)
GI (ꢁM)
HUVEC
5
0
50
Compound
expression (1
a
d
MERP
a
C3H10T1/2
ASZ
d
ꢁM)
MB
ITZ
1
2
3
4
5
6
7
8
ꢀꢀꢀ
1.7 ± 0.3
0.074 ± 0.02
0.14 ± 0.02
0.17 ± 0.01
0.17 ± 0.04
0.45 ± 0.06
ND
0.40 ± 0.03
0.49 ± 0.09
23.8 ± 6.7
8.3 ± 0.7
18.4 ± 4.5
>100
2.5 ± 0.3
1.7 ± 0.4
4.7 ± 0.3
5.8 ± 0.8
8.4 ± 0.7
42.7 ± 4.4
>100
0.44 ± 0.08
0.6 ± 0.1
ND
b
0.063 ± 0.003
0.14 ± 0.04
0.42 ± 0.2
ND
17.8 ± 0.5
13.1 ± 1.2
61.4 ± 3.5
71.4 ± 5.5
6.9 ± 3.1
3.0 ± 0.9
58.5 ± 6.4
1.1 ± 0.2
1.5 ± 0.05
6.1 ± 1.6
45.7 ± 3.0
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
0.16 ± 0.04
0.14 ± 0.04
ND
0.043 ± 0.02
0.13 ± 0.03
0.16 ± 0.06
ND
0.14 ± 0.008
0.16 ± 0.07
ND
0.12 ± 0.03
0.09 ± 0.01
0.12 ± 0.05
ND
9
1
1
4
0
1
5
0.9 ± 0.7
ND
a
All analogues evaluated following 24 hr incubation.
Values represent %Gli1 expression relative to recombinant Hh ligand control (set as 100%).
b
c
IC and GI values represent the Mean ± SEM of at least two separate experiments performed in triplicate.
50
50
d
All analogues evaluated following 48 hr incubation.
In addition to exploring Hh pathway inhibition, this first generation of ITZ analogues was
evaluated for their ability to inhibit VEGFꢀinduced proliferation in HUVECs, an initial step
towards determining their ability to inhibit angiogenesis. The ITZ synthesized in the lab (1)
demonstrated antiꢀproliferative activity comparable to the commercially purchased ITZ (GI50
values = 0.49 and 0.40 µM, respectively). The remaining ITZ analogues were significantly less
active than ITZ in this assay. Several analogues were moderately active (GI values, 1.7ꢀ8.4
5
0
µM), but none were comparable to ITZ. Finally, we compared ITZ and 2 for their ability to
inhibit CYP3A4. Not surprisingly, removal of the triazole moiety completely abolished the
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ability of 2 to inhibit CYP3A4 (IC values = 50.4 nM and >10 µM, respectively, Supplementary
5
0
Figure 1).
As noted above, the synthetic route primarily utilized to access ITZ results in a 1:1:1:1
ratio of four stereoisomers that share the cis configuration for the triazole and ether linker around
the dioxolane ring. As a means to optimize our time and efforts related to the synthesis and
evaluation of our first generation series of ITZ analogues, we did not fully characterize the ratio
of stereoisomers present in each of the analogues. Instead they were evaluated as the
stereoisomeric mixtures produced via the synthetic route(s) described. In order to more fully
probe the absolute structural requirements of the scaffold for potent inhibition of Hh signaling
and angiogenesis, we synthesized and evaluated a second generation series of stereochemically
defined ITZ analogues based on the desꢀtriazole ITZ analogue 2. It is important to note that the
nomenclature of the stereochemistry regarding the dioxolane region differs between ITZ and desꢀ
triazole ITZ analogues. The triazole moiety receives priority within the fully intact ITZ
dioxolane region; therefore, the cisꢀorientation is in reference to the triazole and ether linkage
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(
Chart 2). By contrast, removal of the triazole shifts priority to the phenyl ring and a cisꢀ desꢀ
triazole analogue has the opposite absolute configuration between the phenyl ring and ether
linker as ITZ. In addition, coupling of the dioxolane region intermediates to the linker/side chain
phenol results in the opposite nomenclature assignment for the final ITZ analogue, for example,
final analogues that have the 4R stereochemistry were prepared from the (S) tosylate 62.
Chart 2. Cisꢀ and transꢀ nomenclature of dioxolane region: (A) representative cisꢀITZ isomer,
(B) cisꢀ and (C) transꢀ desꢀtriazole analogues.
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1
2
3
4
5
6
7
8
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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
Evaluation of our stereochemically defined analogues for their ability to inhibit Hh
signaling in the C3H10T1/2 and ASZ cell lines provided both interesting and confounding
results. Overall, the majority of the compounds evaluated (12ꢀ25, Table 4) were more active in
the ASZ cells, with numerous analogues exhibiting 100ꢀ to 1000ꢀfold improvement in activity in
these cells when compared to the MEFs. Several analogues that were inactive in the C3H10T1/2
MEFs at concentrations up to 10 µM (15, 24ꢀ25) induced potent downꢀregulation of Gli1 (IC50
values = 0.55, 0.2, and 0.54 µM, respectively). In addition, several other compounds with modest
inhibitory effects in the MEF cell line (13, 17, and 21) exhibited low nanomolar IC values in
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
5
0
the ASZ cells.
Even with the conflicting results between cell lines, several interesting SAR
developments with respect to optimal configuration were identified for our second generation
ITZ analogues. First, these results reiterated that the orientation of the methyl moiety in the secꢀ
butyl side chain is less important, as long as it is present (Table 4). Compounds that included a
propyl moiety were significantly less active than analogues maintaining the same orientation
around the dioxolane ring (i.e. 17 and 23; 18 and 24) and incorporating the secꢀbutyl moiety. ITZ
analogues with the trans- orientation around the dioxolane generally demonstrated enhanced
downꢀregulation of Gli1 mRNA expression in both cell lines when compared to the
corresponding cis analogues. Finally, compounds containing the 4R configuration were generally
more active than corresponding analogues with the 4S configuration and desꢀtriazole analogues
with the trans 2R,4R configuration (16, 20, and 22) demonstrated the most comparable activity
between the two cell lines.
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2
3
4
5
6
7
8
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Table 4. In vitro activity of second generation ITZ analogues.
a
a
a
GI50 (µM)
MERP
IC (ꢁM)
GI (ꢁM)
HUVEC
5
0
50
Compound
b
c
C3H10T1/2
ASZ
c
MB
12
13
14
0.091 ± 0.02
0.85 ± 0.1
1.1 ± 0.17
>10
0.47 ± 0.001
1.85 ± 0.09
0.54 ± 0.24
6.6 ± 0.3
0.19 ± 0.04
2.4 ± 0.6
4.1 ± 1.5
>10
0.077 ± 0.01
0.071 ± 0.02
2.5 ± 0.7
0.55 ± 0.07
0.38 ± 0.07
0.15 ± 0.038
0.13 ± 0.04
2.8 ± 0.9
0.022 ± 0.01
0.024 ± 0.02
1.3 ± 0.65
1.4 ± 0.5
0.2 ± 0.08
0.54 ± 0.06
0.54 ± 0.05
3.32 ± 0.55
3.69 ± 1.1
7.7 ± 4.4
78.3 ± 17.3
18.3 ± 11.8
2.5 ± 0.7
12.1 ± 4.7
65.0 ± 11.5
53.2 ± 25.2
12.8 ± 2.9
26.5 ± 3.5
49.9 ± 15.9
84.1 ± 33.5
24.9 ± 2.2
1.6 ± 0.02
2.0 ± 1.3
2.9 ± 1.9
ND
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
15
16
17
18
19
20
21
22
23
ND
ND
0.39 ± 0.2
ND
ND
0.6 ± 0.2
1.0 ± 0.5
ND
ND
22.4 ± 12
ND
24
25
PSZ
>10
>10
0.14 ± 0.02
1.5 ± 0.3
a
b
c
IC and GI values represent the Mean ± SEM of at least two separate experiments performed in triplicate.
All analogues evaluated following 24 hr incubation.
All analogues evaluated following 48 hr incubation.
50
50
Based on the antiꢀHh activity of the stereochemically defined compounds in the
C3H10T1/2 and ASZ cells, we evaluated several of these ITZ analogues in the primary MB cell
line. The specific analogues selected for the antiꢀproliferative studies were chosen either because
they were potent in both cell lines (12 and 20) or because they were significantly less active in
the MEFs (13, 17, 21, 24). Measuring the antiꢀproliferative effects of these analogues in the Hhꢀ
dependent primary cell culture would not only provide additional in vitro data for the most active
trans analogues, but might also serve to clarify the discrepancies in antiꢀHh activity between the
two immortalized cell lines. Not surprisingly, the mixtures of trans- (12) and cis- (13)
stereoisomers were less active than the single stereochemically defined analogues. The most
active analogue in this assay was 17 (GI = 0.39 µM), which has the cis 2R,4R configuration
5
0
around the dioxolane moiety. Finally, the reduced antiꢀproliferative activity demonstrated for
analogue 24 further highlights the importance of the methyl group on the side chain for Hh
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8
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
inhibition. Based on the antiꢀproliferative results in the MB cells, we chose to evaluate ITZ and
three analogues (17, 20, and 21) for their ability to downꢀregulate endogenous Gli1 mRNA
expression in the MERP MB cell line. Along with ITZ, each of these compounds exhibited
potent downꢀregulation of mRNA expression in this assay further demonstrating their ability to
inhibit Hh signaling (Table 5). It is important to note that the antiꢀHh activity of the ITZ
analogues in the MERP MB cells more closely correlated with the data obtained in the ASZ
cells, suggesting that the immortalized BCC cell line may be a more appropriate early stage in
vitro cellular model of Hh signaling for evaluating analogues based on the ITZ scaffold.
Table 5. Downꢀregulation of Gli1 mRNA in MERP MB cells.
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
a
b
Compound
IC (µM)
50
ITZ
0.39 ± 0.06
0.26 ± 0.12
0.19 ± 0.07
0.29 ± 0.08
1
2
7
0
21
a
All analogues evaluated following 48 hr incubation.
IC values represent the Mean ± SEM of two separate experiments.
b
50
Chart 3. Structure of posaconazole (PSZ).
Following the initial identification of ITZ as an Hh pathway inhibitor, several additional
1
azole antiꢀfungals were evaluated for their antiꢀHh properties. Interestingly, the closely related,
stereochemically defined triazole antiꢀfungal posaconazole (PSZ, Chart 2) was not explored in
these early studies. Based on our findings that ITZ analogue 20 was the most consistent in its
ability to inhibit pathway activity across multiple Hhꢀdependent cell lines, we also evaluated
PSZ, which shares the same orientation around its tetrahydrofuran (THF) ring, in each of these
assays. PSZ demonstrated potent downꢀregulation of Gli1 expression in the MEF and ASZ cell
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5
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lines; however, it was significantly less active in the MERP cells. Taken together, these data
strongly suggesting that the dioxolane and hydrophobic side chain of ITZ are more active than
the corresponding THF and hydroxylated side chains of PSZ.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The second generation of ITZ analogues was also evaluated in the HUVECs for their
antiꢀangiogenic activity. In a fashion similar to our initial series, each analogue evaluated in this
assay was significantly less active than the commercially purchased ITZ. Interestingly, the transꢀ
(12) and cisꢀ (13) dioxolane mixtures were both more potent than the complete mixture (2). Our
results did not yield a clear SAR pattern that distinguished one stereoꢀdefined dioxolane region
to be more potent against HUVEC antiꢀproliferation. The stereoisomer that had the lowest IC ,
5
0
1
7, contains the (R)ꢀcisꢀdioxolane region and the (S)ꢀsecꢀbutyl group (IC = 2.5 ± 0.7 ꢁM).
50
When only the stereochemistry of the secꢀbutyl moiety is inverted (21), activity is attenuated
IC = 12.8 ± 2.9 ꢁM). Overall, the (S)ꢀsecꢀbutyl analogues have lower IC values than the
(
5
0
50
corresponding (R)ꢀsecꢀbutyl analogues. The same trend held true for the (R)ꢀtransꢀdioxolane
analogues (16 and 20); this was the most potent orientation determined from the Hh pathway
evaluation. These results differ from the Hh pathway inhibitory activity of the scaffold in that the
stereochemistry of the side chain region did not play a significant role in achieving potent
inhibition. Taken together, there is not one dioxolane region that can be readily determined as the
most potent across the second generation of ITZ analogues for inhibiting proliferation of
HUVEC cells.
Based on the lack of clear SAR in the HUVEC cells, we sought to further explore the
antiꢀangiogenic properties of ITZ and our analogues by evaluating their ability to inhibit tube
formation in HUVECs grown on Matrigel. Under normal conditions, HUVECs plated and grown
on Matrigel migrate towards each other, align, and form tubes that resemble in vivo capillary
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0
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
beds. This assay is generally considered a more robust model of angiogenesis as it requires
2
9
several aspects of proper vessel formation, including adhesion, migration, and tube formation.
As tube formation assays are inherently lowerꢀthroughput, we chose to evaluate only a few select
compounds, including, ITZ, 2, 18, 21, and the wellꢀcharacterized angiogenesis inhibitor suramin
(positive control). These analogues were chosen for evaluation based on both their activity in the
antiꢀproliferation assays and their overall structure in an attempt to provide the most relevant
preliminary information with respect to the ability of this scaffold to inhibit angiogenesis.
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
Figure 1. Tube formation assays for ITZ and its analogues. (A) DMSO, (B) Suramin, (C) ITZ,
D) 2, (E) 18, (F) 21. All compounds were evaluated at 10 µM and each image is a representative
(
visual field from a single assay. Each assay was repeated at least three distinct times.
Inhibition of tube formation (as measured by overall tube length and total tube junctions)
by ITZ and 2 were comparable to that of the positive control suramin at 10 µM; however, neither
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5
6
7
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of the stereochemically defined analogues (18 and 21) were active at this concentration (Figures
and 2). ITZ also demonstrated significant inhibition of tube formation at 1 µM. A decrease in
1
activity was observed at 1 µM for analogue 2, highlighting the importance of the triazole moiety
for optimal antiꢀangiogenic activity of the scaffold. Interestingly, there was minimal correlation
between the ability of a compound to inhibit antiꢀproliferation and tube formation in the HUVEC
cells. ITZ was most active in both assay systems; however, analogues 18 and 21 were twoꢀfold
more active in the antiꢀproliferation assay than 2, yet they were essentially inactive in the tube
formation assay. Taken together, these data suggest that the triazole of ITZ is important for the
antiꢀangiogenic activity of the scaffold and further SAR with respect to this region and its antiꢀ
cancer properties is warranted.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
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2
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
(
A)
1
1
50
00
50
0
1
1
0 uM
uM
0
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(B)
1
1
50
00
50
0
Figure 2. Comparison of total tube length (A) and total tube junctions (B) for ITZ, 2, 18, and 21.
DMSO (negative control) was set as 100% tube formation for analysis purposes. Suramin (10
µM) was used as the positive control for each experiment and its ability to inhibit tube length
51.9 ± 7.8%) and tube junctions (57.6 ± 6.1%) was consistent. Data represent the Ave ± SEM of
at least 3 separate experiments in which ≥ 5 fields of vision were quantified using ImageJ
software.
(
DISCUSSION AND CONCLUSIONS
The identification of two distinct antiꢀcancer activities for the FDA approved drug ITZ
prompted our exploration of the structural characteristics of this scaffold that govern these
2
4
activities. Based on previously published SAR for the side chain region of the ITZ scaffold, it
was not surprising that our preliminary data suggests no correlation between the antiꢀHh and
antiꢀangiogenic activity of these scaffolds. Significant modifications to multiple regions of the
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scaffold resulted in compounds that retained the ability to inhibit Hh signaling in Hhꢀdependent
MEFs, murine BCC cells, and murine MB cells. By contrast, every analogue evaluated was
significantly less active than ITZ in the antiꢀangiogenic assays performed in HUVECs.
Preliminary data provides strong evidence that ITZ inhibits the Hh pathway through Smoothened
(Smo), a key regulator of this signaling cascade. As neither Smo nor Hh signaling are known to
play a role in angiogenesis, it is reasonable to hypothesize that the antiꢀangiogenic properties of
ITZ are mediated through a distinct, as yet unidentified, cellular target not associated with this
developmental pathway; however, further studies are needed to definitively characterize
Smo/ITZ binding interactions and identify the cellular protein(s) responsible for its ability to
inhibit cellular angiogenesis.
10
11
12
13
14
15
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24
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46
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49
50
51
52
53
54
55
56
57
58
59
60
A key goal for our initial ITZ analogue series was to determine whether the triazole
moiety was essential for the antiꢀcancer properties of the scaffold. Removal of the triazole from
the dioxolane region (2) had no effect on the ability to inhibit Hh signaling in either cell line;
however, it did significantly affect the antiꢀangiogenic properties of the scaffold. This also
provides further evidence that the antiꢀcancer activities of ITZ are mediated through distinct
cellular mechanisms and highlights the importance of the triazole for the ability of ITZ to inhibit
angiogenesis. Not surprisingly, removal of the triazole completely abolished inhibition of
CYP3A4, the main detrimental side effect of ITZ, providing an improved lead scaffold for
further development as an inhibitor of Hh signaling.
After establishing that the triazole functionality is not required for Hh inhibition, we
prepared and evaluated two additional series of analogues to (1) determine whether truncation at
other locations affected the antiꢀHh properties of the scaffold and (2) identify the optimal
stereochemical orientation of our new lead 2. The truncated analogues were not only designed to
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1
1
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1
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2
2
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identify the pharmacophore for ITZ inhibition of Hh signaling, but also to reduce the overall size
of ITZ (molecular weight = 705.6 g/mol) to improve both its drugꢀlike properties and the overall
synthetic efficiency of preparing these compounds. Overall, the dioxolane region was not
amenable to further truncation; however, removing the triazolone/side chain region did not affect
the ability of the scaffold to inhibit Hh signaling in any of the Hhꢀdependent cell lines (analogue
0
1
2
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8
9
0
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1
1). For the stereochemically defined analogues of 2, the trans orientation was preferred for the
dioxolane region and more specifically the trans 2R,4R analogues were consistently more active
than other analogues with their respective side chain orientation in the immortalized cell lines. In
the primary murine MB cells, a cis analogue (17) with the 4R orientation also demonstrated
potent antiꢀproliferative effects, suggesting that the (R)ꢀconfiguration at the 4ꢀposition of the
dioxolane ring might be more important for Hh inhibition than the overall cis- or trans-
orientation. This is further highlighted by the significant reduction in antiꢀproliferative activity
for analogue 24 (trans-2S,4S).
While the active first generation ITZ analogues demonstrated comparable Hh inhibition
in both the C3H10T1/2 and ASZ cell lines, the second generation, stereochemically defined
analogues were generally more active in the ASZs. Several possibilities exist to explain the
differential activity identified for the second generation analogues. First, it is possible that
decreased permeability for the stereochemically defined compounds in the C3H10T1/2 cells
prevents their intracellular accumulation at concentrations required for potent activity. Second,
the cellular target that mediates the antiꢀHh activity of the ITZ scaffold (presumably Smo) may
be more responsive to the stereochemically defined analogues in the ASZ cells. In addition, Hh
signaling in the C3H10T1/2 cells must be activated through the addition of an exogenous agonist
(
for these assays, recombinant sonic Hh ligand), while Hh signaling and Gli1 overexpression are
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30ꢀ31
constitutively active in the ASZ cells due to a heterozygous mutation in the PTCH1 allele.
Upꢀregulation of pathway signaling with the Hh ligand in the MEFs may result in a level of Gli1
overexpression that cannot be fully overcome by the ITZ analogue(s). Finally, complete
inhibition of Hh signaling for the ITZ scaffold may rely on the presence of multiple
stereoisomers to fully inhibit its target, which could explain why the isomeric mixtures in the
first generation demonstrated comparable activity across both cell lines. These discrepancies are
currently being addressed in ongoing studies to more fully understand the mechanisms that
govern ITZꢀmediated inhibition of both cell lines.
10
11
12
13
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15
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46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
A consideration for the further development of these and other ITZ analogues as potential
Smo antagonists is that multiple forms of mutant Smo have been identified in both BCC and MB
36ꢀ40
patients receiving a small molecule Smo antagonists.
These mutations in Smo oftentimes
render patients insensitive to further treatment with the Smo antagonists that have been approved
by the FDA. A key rationale for developing ITZ analogues as Hh pathway inhibitors is that ITZ
has previously demonstrated the ability to inhibit several resistant forms of Smo in vitro and in
1,41
vivo; however, any further development of these analogues as Hh inhibitors must demonstrate
that they also prevent pathway signaling in the presence of mutant Smo. Attempts to circumvent
mutant Smo by Developing small molecule Hh pathway inhibitors that function downstream of
Smo at the level of the Gli transcription factors has emerged as a potential strategy to circumvent
mutant Smo; however, all of the Gli inhibitors reported to date demonstrate only modest
inhibition of Hh signaling, suggesting more studies are necessary to determine whether directly
4
2
targeting Gli(s) is a valid therapeutic strategy.
In conclusion, we have synthesized and evaluated two series of ITZ analogues for their
ability to inhibit both Hh signaling and angiogenesis. These studies have provided preliminary
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insight into the structural features required for potent inhibition of both of these cancer related
pathways. Ongoing studies for the ITZ scaffold include further SAR exploration of the side chain
and dioxolane region as well as studies designed to more fully understand the discrepancies in
Hh inhibition demonstrated across the different Hhꢀdependent cell lines.
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EXPERIMENTAL SECTION
Chemical Synthesis
General Information. Starting materials were purchased from SigmaꢀAldrich or Fisher
Scientific. ACS grade methanol, ethyl acetate, toluene, anhydrous DMF, NMP, and DMSO were
purchased from Fisher Scientific or SigmaꢀAldrich. ITZ analogue 9 was purchased from Toronto
Research Chemicals. All reactions were run under an argon atmosphere. NMR data was
collected on a Bruker AVANCE 500 MHz spectrometer and analysis performed using
MestReNova. HRMS data was analyzed at the Mass Spectrometry Facility at the University of
Connecticut by Dr. YouꢀJun Fu. FTꢀIR analysis was performed on a Bruker Alpha Platinum
ATR instrument using OPUS software (v 7.2). The preparation of previously characterized ITZ
2
4ꢀ26,32
intermediates followed known procedures with minor modifications.
Xꢀray crystals were
prepared using vapor diffusion techniques (pentanes:chloroform) and analysis performed by Dr.
Victor Day at the SmallꢀMolecule Xꢀray Crystallography Lab at the University of Kansas on a
Bruker MicroStar microfocus Cu rotating anode generator with two CCD detectors or a Bruker
Apex II CCD detector equipped with Helios multilayer optics instruments. Mercury (v3.0)
software was used to visualize Xꢀray structural analysis. All ITZ analogues evaluated in the
biological assays (1ꢀ25) were greater than 95% pure based on the HPLC methods described
below.
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9
Purity Analysis of Final Analogues. It is important to note that final ITZ analogues 1ꢀ11 were
synthesized and evaluated as stereoisomeric mixtures. For this initial series, we did not separate
individual stereoisomers nor did we determine the ratios of cis:trans dioxolanes produced in the
10
11
12
13
14
15
16
17
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24
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52
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58
59
60
1
13
ketalization reaction. These mixtures are reflected in the H and C NMR characterization data
described below and the spectra provided in the supplementary information. Purity analysis for
all final analogues was determined via one of the methods described below.
Method A. ITZ analogues were dissolved in HPLC grade MeCN and injected (20 µl of a 1 mM
soln) into an Agilent Manual FLꢀInjection Valve (600 bar) on an Agilent 1100/1200 Series
HPLC equipped with an Agilent Eclipse Plus C18 (4.6 X 100 mm) column and Agilent 1100
Series Photodiode Array Detector. The mobile phase consisted of 60% MeCN:40% H O for
2
analogues containing the triazole moiety and 70% MeCN:30% H O for desꢀtriazole analogues.
2
All analogues were run at a flow rate of 1.0 mL/min for 20 mins and purity was assessed at 254
nm.
Method B. ITZ analogues were dissolved in HPLC grade MeCN and injected (20 µl of a 1 mM
soln) into an Agilent HPLC system coupled to an Agilent ESI single quadrupole mass
spectrometer equipped with a Kinetix C18 (150 X 4.6 mm) column and an Agilent G1315 diode
array detector. The mobile phase consisted of 70% MeCN:30% H O. All analogues were run at a
2
flow rate of 0.7 mL/min for 30 mins and purity was assessed at 254 nm.
Previously Characterized ITZ Intermediates. Common ITZ linker region (28ꢀ32 and 68),
dioxolane region (53ꢀ56, 59, 62ꢀ63), side chain (35ꢀ36), and coupled intermediates were
prepared primarily as described previously for the ITZ scaffold with the minor modifications
2
4ꢀ26,35
described in the supplementary info.
Procedures and characterization for newly reported
key intermediates and all final analogues is provided below.
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First Generation ITZ Intermediates and Final Analogues (1ꢀ11).
1
ꢀ(1Hꢀ1,2,4ꢀtriazolꢀ1ꢀyl)propanꢀ2ꢀone (51). A solution of chloroacetone (47) (1.72 mL, 21.6
mmol), 1Hꢀ1,2,4ꢀtriazole (50) (2.98 g, 43.2 mmol), NaHCO (2.89 g, 34.5 mmol), and toluene
3
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0
(
100 mL) were heated to reflux (110ꢀ120°C) for 3 h. The reaction vessel was cooled to ꢀ20°C for
12 h. The resulting precipitate was filtered, dissolved in H O, and extracted with EtOAc (50 mL
2
x 3). The organic layer was collected, washed with saturated sodium chloride (100 mL), and
dried (Na SO ). The solvent was evaporated and the crude product was purified via column
2
4
1
chromatography (SiO , 0 ꢀ 3% MeOH in DCM) resulting in a yellow oil (600 mg, 22.2%). H
2
13
NMR (500 MHz, CDCl ) δ 8.01 (s, 1H), 7.76 (s, 1H), 4.90 (s, 2H), 2.00 (s, 3H). C NMR (126
3
MHz, CDCl ) δ 199.58, 151.41, 144.15, 57.52, 26.63. DARTꢀHRMS: m/z calcd. for C H N O
3
5
7
3
+
[MH] , 126.0667; Found: 126.0691. IR (solid) vmax: 3122, 2935, 2241, 1728, 1507, 1352, 1273,
1137, 1115, 727, 677, 646.
1
ꢀphenylꢀ2ꢀ(1Hꢀ1,2,4ꢀtriazolꢀ1ꢀyl)ethanone (52). A solution of 2ꢀbromoacetophenone (48) (2.0
g, 10.0 mmol), 1Hꢀ1,2,4ꢀtriazole (50) (1.4 g, 20.1 mmol), NaHCO (1.3 g, 16.1 mmol), and
3
toluene (100 mL) were heated to reflux (110ꢀ120°C) for 3 h. The reaction vessel was cooled to ꢀ
20°C for 12 h. The resulting precipitate was filtered, dissolved in H O, and extracted with EtOAc
2
(
3 X 50 mL). The organic layer was collected, washed with saturated sodium chloride, and dried
over sodium sulfate. The solvent was evaporated and the crude product was purified via column
1
chromatography (SiO , 0 ꢀ 3% MeOH in DCM) resulting in a yellow solid (1.2 g, 64.1%). H
2
13
NMR (500 MHz, CDCl ) δ 8.28 (s, 1H), 8.04 (s, 3H), 7.70 (s, 1H), 7.57 (s, 2H), 5.71 (s, 2H). C
3
NMR (126 MHz, CDCl ) δ 190.84, 152.02, 145.07, 134.69, 129.30, 128.25, 55.19. DARTꢀ
3
+
HRMS: m/z calcd. for C H N O [MH] , 188.0824; Found: 188.0822. IR (solid) vmax: 3113,
1
0
9
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062, 2992, 2953, 1697, 1596, 1504, 1450, 1345, 1225, 1207, 1135, 1021, 888, 752, 687, 675,
56, 634.
6
(2ꢀ((1Hꢀ1,
2,4ꢀtriazolꢀ1ꢀyl)
methyl)ꢀ2ꢀmethylꢀ1,
3ꢀdioxolanꢀ4ꢀyl)
methyl
4ꢀ
10
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60
methylbenzenesulfonate (57). Ketone 51 (1.4 g, 11.9 mmol) and 56 (2.8 g, 11.5 mmol) were
added to a dry round bottom flask. Anhydrous toluene (10 mL) was added and the mixture was
cooled to 0° C at which time TfOH (4.0 mL, 45.9 mmol) was added dropwise with a glass
syringe. The solution was stirred at RT for 60 h. The mixture was diluted with 50 mL of EtOAc
and slowly added to a solution of K CO (5 g) in water (40 mL). The aqueous layer was washed
2
3
with EtOAc (3 X 50 mL) and the organic layers were combined, dried (Na SO ), filtered, and
2
4
concentrated. The crude product was purified via column chromatography (SiO , 0 ꢀ 10% MeOH
2
1
in DCM) (200 mg, <10%). H NMR (500 MHz, CDCl ) δ 8.10 (d, J = 8.3 Hz, 1H), 7.86 (s, 1H),
3
7.81 – 7.75 (m, 2H), 7.37 (m, 2H), 4.39 – 4.24 (m, 3H), 4.06 (m, 1H), 4.03 – 3.92 (m, 1H), 3.86
(m, 1H), 3.83 – 3.74 (m, 1H), 3.69 (m, 1H), 3.53 (m, 1H), 2.46 (d, J = 5.7 Hz, 3H), 1.32 (d, J =
13
3
5
.9 Hz, 3H). C NMR (126 MHz, CDCL ) δ 152.04, 151.89, 145.75, 144.96, 132.89, 130.45,
130.39, 128.40, 74.56, 74.07, 68.83, 68.68, 67.17, 67.03, 55.93, 55.88, 23.77, 22.66, 22.09.
+
DARTꢀHRMS: m/z calcd. for C H N O S [MH] , 354.1124; Found: 354.1124. IR (solid)
15
19
3
5
vmax: 3007, 2989, 2916, 2894, 1594, 1557, 1359, 1172, 1093, 1035, 961, 873, 724, 662, 563.
2ꢀ((1Hꢀ1,2,4ꢀtriazolꢀ1ꢀyl)methyl)ꢀ2ꢀphenylꢀ1,3ꢀdioxolanꢀ4ꢀyl)methyl
(
4ꢀ
methylbenzenesulfonate (58). Crude dioxolane 58 was prepared through a procedure analogous
to that described above for 57. Following extraction with EtOAc, the combined organic layers
were dried (Na SO ), filtered, and concentrated to ~70 mL EtOAc. A solution of TsOH
2
4
monohydrate (2.0 g) in EtOAc (13 mL) was slowly added at RT. The product precipitated as a
salt and was filtered (3.9 g). The salt was dissolved in aqueous saturated K CO (100 mL) and
2
3
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