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

Article abstract of DOI:10.1021/jm200944b

Itraconazole is an antifungal drug that was recently found to possess potent antiangiogenic activity and anti-hedgehog (Hh) pathway activity. To search for analogues of itraconazole with greater potency and to understand the structure-activity relationship in both antiangiogenic and Hh targeting activity, 25 itraconazole side chain analogues were synthesized and assayed for inhibition of endothelial cell proliferation and Gli1 transcription in a medulloblastoma (MB) culture. Through this analysis, we have identified analogues with increased potency for inhibiting endothelial cell proliferation and the Hh pathway, as well as VEGFR2 glycosylation that was recently found to be inhibited by itraconazole. An SAR analysis of these activities revealed that potent activity of the analogues against VEGFR2 glycosylation was generally driven by side chains of at least four carbons in composition with branching at the α or β position. SAR trends for targeting the Hh pathway were divergent from those related to HUVEC proliferation or VEGFR2 glycosylation. These results also suggest that modification of the sec-butyl side chain can lead to enhancement of the biological activity of itraconazole.

Full text of DOI:10.1021/jm200944b

ARTICLE
pubs.acs.org/jmc
Itraconazole Side Chain Analogues: StructureꢀActivity Relationship
Studies for Inhibition of Endothelial Cell Proliferation, Vascular
Endothelial Growth Factor Receptor 2 (VEGFR2) Glycosylation, and
Hedgehog Signaling
Wei Shi,† Benjamin A. Nacev,†,§,|| Blake T. Aftab,†,‡,|| Sarah Head,† Charles M. Rudin,‡ and Jun O. Liu*,†,‡
†Department of Pharmacology and Molecular Sciences, ‡Department of Oncology, §Medical Scientist Training Program,
Johns Hopkins School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205, United States
S Supporting Information
b
ABSTRACT: Itraconazole is an antifungal drug that was recently
found to possess potent antiangiogenic activity and anti-hedgehog
(Hh) pathway activity. To search for analogues of itraconazole
with greater potency and to understand the structureꢀactivity rela-
tionship in both antiangiogenic and Hh targeting activity, 25 itra-
conazole side chain analogues were synthesized and assayed for
inhibition of endothelial cell proliferation and Gli1 transcription in a medulloblastoma (MB) culture. Through this analysis, we have
identified analogues with increased potency for inhibiting endothelial cell proliferation and the Hh pathway, as well as VEGFR2 glyco-
sylation that was recently found to be inhibited by itraconazole. An SAR analysis of these activities revealed that potent activity of the
analogues against VEGFR2 glycosylation was generally driven by side chains of at least four carbons in composition with branching at the α
or β position. SAR trends for targeting the Hh pathway were divergent from those related to HUVEC proliferation or VEGFR2
glycosylation. These results also suggest that modification of the sec-butyl side chain can lead to enhancement of the biological activity of
itraconazole.
’ INTRODUCTION
growth factor receptor 2 (VEGFR2) and blocks VEGF-activated
phosphorylation of the receptor,2 thereby blocking downstream
signaling (Nacev et al., submitted for publication). Despite these
new mechanistic insights, however, the direct target(s) of itraco-
nazole remains unknown.
Itraconazole is used as a clinical agent for the treatment of
a broad spectrum of fungal infections. We previously found that
itraconazole possesses potent antiangiogenic activity both in vitro and
in vivo.1,2 In addition, it has been recently found that itraconazole
inhibits Hh signaling and the growth of MB allografts with deregu-
lated Hh activity.3 These reports form the basis for the expansion of
the potential therapeutic application of itraconazole and have sparked
evaluation of this agent in four ongoing cancer clinical trials.
To better understand the structural parameters that influ-
ence antiangiogenic activity, we have recently synthesized all eight
stereoisomers of itraconazole (1aꢀ1h, Figure 1).4 For the first
time, these individual stereoisomers were evaluated for both their
in vitro antiangiogenic and 14α-demethylase inhibition (14DM)5
dependent antifungal activities.4 The discordance between these
activities in one pair of trans-stereoisomers 1eꢀ1f and the others
further supported our previous hypothesis that the molecular
mechanism of itraconazole in antiangiogenesis is distinct from that
for antifungal activity.1
Itraconazole shares a large degree of structure similarity with
the other azole antifungals, such as terconazole and ketoconazole
(Figure 2), which, despite their equipotent antifungal activity in
comparison to itraconazole, do not inhibit HUVEC proliferation1
and fail to induce the VEGFR2 glycosylation defect (data not
shown). The structural differences are particularly evident with
respect to the triazolone ring and its sec-butyl side chain. This
suggested that perhaps these moieties are responsible for the unique
activities of itraconazole. Thus, we have synthesized a library of 25
itraconazole analogues in which the sec-butyl side chain was replaced
by a group of substituents with diverse structural variations. These
analogues were evaluated in several assays, including HUVEC
proliferation, VEGFR2 glycosylation, medulloblastoma (MB) pro-
liferation, and Hh signaling as measured by Gli1 transcript levels as a
pharmacodynamic marker of pathway activation.
Our attempts to deconvolute the molecular basis of the anti-
angiogenic activity of itraconazole have revealed several cellular
and biochemical effects in human umbilical vein endothelial cells
(HUVEC). Itraconazole inhibits cholesterol trafficking in late end-
somes/lysosomes and blocks mTORC1 and mTORC2 signaling.6
More recently, we observed that itraconazole can also impede the
maturation of N-linked sugars appended to vascular endothelial
’ CHEMISTRY
The syntheses of itraconazole analogues 7aꢀ7n (Table 1)
with linear, branched, or cyclic hydrocarbon side chains followed
Received: July 15, 2011
Published: September 21, 2011
r
2011 American Chemical Society
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the previously reported synthetic route (Scheme 1).4 Because
the 2S,4R-cis-stereochemistry on the 1,3-dioxolane ring is most
favored for antiangiogenic activity, one single diastereomer 6a4
was used as the starting material to make the analogues unless
otherwise stated.
Alkylated compounds 4aꢀ4n (Table 1) were prepared by reac-
tion of the free triazolone precursor 2 with commercially available
alkyl bromides or alkyl tosylates synthesized from commercially
available alcohols (see the Supporting Information). For alkyl bro-
mides or tosylates with a low boiling point, or potentially low stability
or high reactivity at high temperatures, the reactions were carried out
at room temperature with potassium carbonate and 18-crown-6.
Demethylation of 4aꢀ4n with concentrated aqueous hydrobromic
acid at 110 °C afforded the corresponding phenols 5aꢀ5n (Table 1)
in excellent yields. Final coupling of 5aꢀ5n with 1,3-dioxolane
tosylate 6a gave the desired side chain analogues 7aꢀ7n.
three steps. Next, the N-alkylation of 13 with the corresponding
alkylating reagents 3oꢀ3q (see the Supporting Information) under
basic conditions afforded the intermediates 14aꢀ14c (Table 2),
which were in turn treated with 50% trifluoroacetic acid in dichlor-
omethane at room temperature to remove the MOM group. The
acquired phenols 5oꢀ5q were subjected to the aforementioned
coupling conditions to give the final products 7oꢀ7q.
Although the synthetic route described in Scheme 2 can
accommodate a broad range of functional groups, two column
purifications were usually required to remove DMSO and obtain
the final products in good quality. To avoid the use of DMSO as
the reaction solvent, we adopted a previously reported linear synthetic
methodology (Scheme 3).8 The reaction sequence was first exam-
ined by using 2R,4R-cis-tosylate 6b as the starting material due to its
similar biological activity according to the reported stereoisomers.4
N-(4-hydroxyphenyl)-N0-(4-nitrophenyl)-piperazine 10 was purified
by column chromatography before it was coupled with 6b. Forma-
tion of the triazolone moiety in 17b from the nitro group in 15b
was achieved by following the similar reaction conditions described
in Scheme 2. Using potassium carbonate in combination with 18-
crown-6 as the deprotonation reagents,9 the final N-alkylation of 17b
with 4-pentynyl-1-tosylate 3s was performed in acetonitrile at 40 °C
to give the final product 7r in 63% yield. Similarly, analogues 7sꢀ7x
(Table 3) were obtained from 2S,4S-cis-tosylate 6a and the corre-
sponding alkyl bromides or tosylates 3sꢀ3x.
The syntheses of analogues 7oꢀ7q (Table 2) are shown in
Scheme 2. To incorporate the functional groups (azido, internal
alkyne, and benzophenone) into the side chain, the methyl protect-
ing group of the phenolic hydroxyl group was replaced with the
methoxymethyl (MOM) group to avoid the harsh demethylation
conditions (HBr, 110 °C). To start with, N-(4-hydroxyphenyl)-
N0-(4-nitrophenyl)-piperazine 10 was prepared by heating the
mixture of commercially available N-(4-hydroxylphenyl)-piper-
azine 8 and 1-chloro-4-nitrobenzene 9 in N-methylpyrrolidone
(NMP) overnight.7 The crude product isolated from 2-propanol
precipitation was directly used to react with methoxymethyl chloride
to afford the MOM-protected intermediate 11, which was purified
by column chromatography. Subsequently, the nitro group in 11
was reduced to the amino group by refluxing with hydrazine
monohydrate in the presence of 10% palladium on charcoal. In a
three-step sequence, the aniline intermediate 12 was then reacted
with phenyl chloroformate, hydrazine monohydrate, and forma-
midine acetate to construct the triazolone ring in 51% yield over
’ RESULTS AND DISCUSSION
Inhibition of HUVEC Proliferation. We analyzed the pre-
viously determined activity of the analogues against HUVEC
proliferation, which was used as an in vitro proxy for angiogenesis
Table 1. Side Chain Structures of Compounds 4aꢀ4n,
5aꢀ5n, and 7aꢀ7n
Figure 1. Structures of the eight itraconazole diastereomers arising
from three stereogenic centers numbered 2, 4, and 20. The cis- designa-
tion denotes that the two substituents in the boxes are on the same side
of the 1,3-dioxolane ring, while trans- denotes the opposite orientation.3
a Racemic on the side chain.
Figure 2. Structures of terconazole and ketoconazole.
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Scheme 1a
a Reagents and conditions: (a) K2CO3, 18-crown-8, DMSO, rt, or K2CO3, KI, DMSO, 80 °C; (b) 48% aqueous HBr, 110 °C; (c) NaH, DMSO,
50 °C f 85 °C.
inhibition (Table 3). From Table 3, it is clear that modification
of the side chain in most analogues did not dramatically affect
the activity. Those analogues with extremely short side chains
(7a, and 7b) or lacking any side chain (17a) had lower potency
for inhibition of HUVEC proliferation than itraconazole. A few
analogues, however, possess considerably higher potency, in-
cluding 7r and 7u.
Table 2. Side Chain Structures of Compounds 14aꢀ14c,
5oꢀ5q, and 7oꢀ7q
The similar or even improved potency for compounds 7k, 7l,
and 7v, all of which have bulky side chains, implies a putative
binding site that is not sterically hindered. The relatively rigid
conformation in 7o, 7s, and 7t further supports this idea and
extends it to suggest that the binding pocket in the putative target
may be quite deep. However, the loss of activity in 7w confounds
this hypothesis. One possible explanation is that the loss of
lipophilicity resulting from the incorporation of two nitrogens
may have compromised activity. This is supported by the data for
7x in which an increase in lipophilicity appears to have compen-
sated for the presence of the diazirine. The near total loss of
activity in 7q indicated that there is a limit to the size of the side
chain with respect to inhibition of HUVEC proliferation.
Perhaps most notably, the potency of itraconazole against
HUVEC proliferation was significantly increased by the incor-
poration of relatively small functional groups, such as the azido
group in 7p, the terminal alkyne in 7r, and the cyano group in 7u.
This implies that there are some interactions in the binding site
of the side chain that are not utilized by the sec-butyl group in
itraconazole. These results also suggested that substituents in
place of the sec-butyl group of itraconazole can be further explored
to optimize the potency of itraconazole.
(p < 0.05) greater potencies against proliferation than the group
in which the hypoglycosylation was absent, suggesting that this
phenotype makes an overallcontribution to inhibition of HUVEC
proliferation (Table 4). We also observed that side chains com-
prised of four carbons or greater with branching at the α or β posi-
tion strongly favored robust glycosylation inhibition compared to all
other structural isomers (Figure 3). Compound 7j produced an
intermediate glycosylation defect and was an exception to this rule.
Compared to 7k, 7l, and 7v, the other analogues which contained
bulky side chains and retained glycosylation inhibition, 7j had the
greatest number of possible rotational isomers, suggesting that only
certain conformations may be suitable for glycosylation inhibition.
Inhibition of Ptchꢀ/+ Medulloblastoma Proliferation.
Itraconazole has recently been reported to inhibit Hh pathway
signaling in a 3T3 cell-based reporter system and in medullo-
blastoma allografts exhibiting Hh pathway-dependent growth.3
This reported activity against Hh signaling also seems to be
unrelated to inhibition of 14DM, as other azole antifungal agents
demonstrate decreased potency against this pathway, regardless
of their activity on human 14DM. To explore the relation of the
antiangiogenic and anti-Hh signaling capacity of itraconazole, we
assessed the antiproliferative potency of the side chain analogues
Inhibition of VEGFR2 Glycosylation. Having recently uncov-
ered that itraconazole blocks the normal maturation of N-glycans
on VEGFR2, we analyzed the influence of the side chain structure
on this activity as well (Nacev et al., submitted for publication)
(Table 3). VEGFR2 hypoglycosylation was scored either robust
(++), intermediate (+), or absent (ꢀ) (Supporting Information
Figure S1). Robust inhibitors of glycosylation had significantly
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Scheme 2a
a Reagents and conditions: (a) DIPEA, NMP, 125 °C; (b) MOMCl, DIPEA, rt, 79% over two steps; (c) 10% Pd/C, NH2NH2 H2O, EtOH, reflux, 99%;
3
(d) (i) phenyl chloroformate, pyridine, CH3CN, (ii) NH2NH2 H2O, 1,4-dioxane, reflux, (iii) formamidine acetate, 1-propanol, 110 °C, 51% over 3
3
steps; (e) K2CO3, KI, DMSO, 80 °C; (f) TFA, 0 °C f rt; (g) NaH, DMSO, 50 °C f 85 °C.
in MB cultures. These cultures were derived from Ptchꢀ/+;p53ꢀ/ꢀ
mice that spontaneously develop medulloblastoma associated
with ligand independent Hh pathway activation.10 IC50 values for
proliferation were determined for analogues and are represented
as relative potencies compared to that of itraconazole (Table 3).
Overall, there was poor correlation between analogue potency
ratios for MB and HUVEC proliferation (Spearman r = 0.327;
p = 0.063) (Figure 4). Notably, compounds 7q and 7w, which
lost >69.9- and >13.8-fold of activity against HUVEC, respec-
tively, had respective relative activities of 0.8 and 1.32 against
MB proliferation when compared to itraconazole. Furthermore, the
lack of data points in the lower right quadrant of Figure 4 illustrates
that, without exception, all analogues demonstrating equivalent or
increased potency against HUVEC proliferation with respect to
itraconazole also demonstrate equivalent or increased potency against
MB proliferation. These findings are consistent with the possibility
that the target of itraconazole in HUVEC may also contribute to the
potency of itraconazole against MB proliferation
to the itraconazole analogues at the IC90 for MB proliferation and
quantified transcript levels of Gli1, a transcriptional gene target of
the Hh pathway. These data were then scored based on transcript
levels associated with exposure of MB to synthetic inhibitors
of the signaling protein Smoothened, HhAntag11 and vismodegib
(GDC-0449),12 at the IC90 for proliferation. The results are
listed in Table 3.
Overall, there was a statistically significant association between
inhibition of MB proliferation and inhibition of Hh pathway
activity (p = 0.05; Supporting Information Figure S2). However,
some analogues with relatively weak inhibition of Gli1 expression
did demonstrate potent inhibition of MB proliferation, suggest-
ing that in these instances, antiproliferative activity was predo-
minantly Hh pathway independent.
There was no evident association between inhibition of VEGFR2
glycosylation and targeting of the Hh pathway (Supporting Informa-
tion Figure S3). Nine of 10 analogues demonstrating strong
inhibition of the Hh pathway did not inhibit VEGFR2 glycosyla-
tion. Furthermore, of the six strongest inhibitors of VEGFR2
glycosylation, two were not associated with Hh-dependent in-
hibition, three were intermediate inhibitors, and only onestrongly
inhibited Hh signaling. Notably, with the exception of compound
7h, all analogues demonstrating strong inhibition of VEGFR2
glycosylation also demonstrated increased potency against MB
Inhibition of Hh Pathway Signaling. Inhibitors of the Hh
pathway are known to inhibit MB cell proliferation. However,
MB cell proliferation is also affected by drugs with targets other
than Hh signaling. Therefore, to determine the extent to which
the demonstrated potencies on MB proliferation are associated
with Hh pathway inhibition, we exposed established MB colonies
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Scheme 3a
a Reagents and conditions: (a) NaH, DMF, 50 °C, 88%; (b) 10% Pd/C, NH2NH2 H2O, EtOH, reflux, 99%; (c) (i) Phenyl chloroformate, pyridine,
3
CH3CN, (ii) NH2NH2 H2O, 1,4-dioxane, reflux, (iii) formamidine acetate, 1-propanol, 110 °C, 48% over 3 steps; (d) K2CO3, 18-crown-6, CH3CN,
3
40 °C.
proliferation. These findings reveal a high level of discordance
between these pharmacodynamic markers.
Comparison of Structural Determinants for Inhibition of
Hh Signaling, HUVEC Proliferation, and VEGFR2 Glycosyla-
tion. Taken together, the SAR trends and defined side chain sets
derived from these analyses provide criteria for examining the
relationship between activities against the Hh pathway and those
targeting glycosylation and HUVEC proliferation. Overall, diver-
gence between the activity of 7g and 7m demonstrate MB
selectivity for saturated alkane chains greater than four carbons
in length. Functional groups associated with the distal ends of 7n,
7p, and 7u further differentiate these activities and identify a
preference for small, polar cyano, and azido functional groups in
the pharmacophore associated with effects on HUVEC prolif-
eration and VEGFR2 glycosylation, whereas a more bulky,
hydrophobic phenyl group is preferred for activity against Hh
signaling. Compounds 7k and 7l identify large groups proximal
to the core triazolone ring as having improved activity in HUVEC
proliferation and favoring inhibition of VEGFR2 glycosylation,
whereas these structures were disfavored for inhibition of the Hh
pathway. Interestingly, the sec-pentyl modification associated
with compound 7h represents the only side chain composition
in the analogue set that simultaneously inhibits glycosylation and
the Hh pathway. Most notably, this compound was also the only
analogue demonstrating increased potency across all parameters
as compared with itraconazole.
A study of side chain modifications associated with strong
Hh pathway inhibition reveals several SAR trends. Analogues
with side chains of three carbons or more in length without polar
groups or branching at the β carbon demonstrated strong inhibition
of Hh pathway activity (Figure 5). Compound 7e isan exception, as
it was not associated with strong Hh pathway inhibition, which is
somewhat surprising as structurally closely related analogues 7c
and 7g exhibited strong Hh inhibition.
Examination of the side chains included in this defined set
reveals that the criteria for potent inhibition of the Hh pathway
is permissive to extension of the side chain beyond three carbons
in length, as elongation up to n-octane (compound 7m) gen-
erally resulted in increased potency for Hh pathway inhibition
and MB proliferation. Retention of potency for Hh pathway
inhibition with compound 7n and loss of pathway specific
activity with compound 7q suggests that there is some
allowance for bulk at the distal end of these extensions but
not to the extent necessary to accommodate a benzophenone
group. This was similar to what we observed for HUVEC pro-
liferation. Pathway inhibition was also permissive to branching
on the α or γ carbon as demonstrated by Itra, 7h, 7i, and 7j.
Compound 7k is an exception to this rule. This exception, in
the context of the activity demonstrated by 7j, suggests that
branching from the α position was not permissive to moieties
with either large displacement volume or restricted rotational
degrees of freedom. Pathway inhibition was also favored in
compound 7r, containing a terminal alkyne. Alkene and alkyne
linkages between γ and δ carbons were also active against the
pathway.
’ CONCLUSION
Through N-alkylation of the triazolone moiety in three
different synthetic routes, a focused library of 25 side chain analogues
of itraconazole was synthesized. These analogues were screened for
effectsonHUVECproliferation, VEGFR2 glycosylation, inhibition
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Table 3. Inhibition of HUVEC Proliferation and VEGFR2 Glycosylation, MB Proliferation, and Gli1 Transcription by
Itraconazole Stereoisomers 1aꢀ1h and Side Chain Analogues 17a and 7aꢀ7xa
a Stereochemistry on the 1,3-dioxolane ring is 2S,4R for 17a, 7aꢀ7q,and 7sꢀ7x and 2R,4S for 7r. b Proliferation data from ref 3. c Purity is 94.0%, tR =
6.92 min; d Already reported in ref 7. e Mixture of the four cis-diastereomers, from Sigma-Aldrich. f Mixture of stereoisomers on the side chain. g EC50/
IC50 Itra. This comparison was made for analogues with doseꢀresponse curves that did not reach zero proliferation even at the highest doses. * Indicates
a lack of overlap between the 95% confidence interval for the un-normalized itraconazole reference IC50 and the 95% CI for the analogue, which implies a
statistically significant difference in the IC50 values. # p < 0.05, F-test for differences in un-normalized log(IC50 or EC50) compared to the itraconazole
reference value.
of Gli1 transcription in MB cells, and inhibition of MB prolifera-
tion. Analogues that were robust inhibitors of glycosylation were
significantly associated with greater potency against HUVEC
proliferation, suggesting that glycosylation inhibition contributes
to the overall antiangiogenic activity of itraconazole. The SAR
study on antiangiogenic activity suggests that the binding site of
the side chain may be mainly hydrophobic and relatively deep and
flexible. It was also possible to incorporate additional functional
groups, such as terminal alkyne, azido, and cyano, which led to
enhancement of antiangiogenic activity. However, to achieve
potent inhibition against VEGFR2 glycosylation, there were
more stringent structural and functional requirements for the
side chain, mainly that side chains of at least four carbons with
branching at the α or β position were generally required for high
potency. Surprisingly, we found that some compounds with
relatively potent inhibitory activity against MB proliferation were
not similarly potent inhibitors of Gli1 transcription. Of the deriva-
tives tested, 12 were found to exert strong Hh pathway inhibition
associated with their antiproliferative effects in MB, as indicated by
inhibition Gli1 transcript levels. These compounds were generally
Table 4. Correlation between HUVEC Proliferation Inhibi-
tion and Glycosylation Phenotype
glycosylation inhibition
ꢀ
+
++
n
10
6.3
7
16
a
mean IC50
3.4
1.0
ꢀ0.32
0.04
a
mean Log2 (IC50
p-valueb,c
)
1.0
0.9
N/A
0.83
a Relative to Itraconazole (IC50 analogue/IC50 itraconazole). b p-Value
calculated by a two-tailed Student’s t test from log transformed data.
c Compared to the “ꢀ” group.
defined as having side chains with extensions of at least three
carbons in length and lacking branching from the β position. The
distinct trends demonstrated by the SAR of these two molecular
activities, together with the lack of correlation between the potency
of the analogues in HUVEC and MB proliferation, suggest that
itraconazole’s effect on the Hh pathway is largely unrelated to the
activity of itraconazole in HUVEC. It is possible that the effects of
itraconazole on HUVEC and Hh signaling pathway are mediated
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by distinct molecular targets. Together, the results presented herein
have deepened our understanding of the role of the itraconazole side
chain in the antiangiogenic and anti-Hh pathway activities of this
drug and will likely facilitate the design of future analogues with
increased potency for specific activities. Analogues with selectivity for
one or more pathways may be useful in animal studies to clarify the
role for each activity in in vivo tumor suppression.
CHCl3 (δ = 77.0 for 13C) as an internal reference. Chemical shifts are
reported in ppm (δ). Data are presented in the form: chemical shift
(multiplicity, coupling constants, and integration). 1H data are reported
as though they were first-order. The errors between the coupling constants
for two coupled protons were less than 0.5 Hz, and the average number was
reported. Low-resolution mass spectra were obtained on a API 150EX
single quadrupole LC/ESI-MS system in the Department of Pharmacology
and Molecular Sciences or on a Voyager DE-STR, MALDI-TOF instru-
ment at the AB Mass Spectrometry/Proteomics Facility at the Johns
Hopkins University. The MALDI samples were prepared by mixing
droplets of the sample solutions in chloroform or methanol and 2,
5-dihydroxybenzoic acid solution in acetone, where the latter served as
the matrix. The reported purity values were obtained with a JASCO PU-
2089S Plus quaternary pump system, using an MD-2010 Plus PDA
detector at the wavelength of 256 nm and a Varian Microsorb-MV
100ꢀ5 C18 column. The eluant consisted of acetonitrile and 0.125%
diethylamine in water, the ratio and flow rate of which depends on the
compound. The purity of all final compounds synthesized is close to or
above 95%. When the purity derived from HPLC analysis is greater than
99%, it is reported as >99%.
’ EXPERIMENTAL SECTION
General. Reactions were carried out in oven-dried glassware. All
reagents were purchased from commercial sources and were used with-
out further purification unless noted. Unless stated otherwise, all reac-
tions were carried out under a positive pressure of argon monitored by
Merck precoated silica gel 60F-254 plates and visualized using 254 nm
UV light. Column chromatography was performed on silica gel
(200ꢀ400 mesh, Merck). The ratio between silica gel and crude product
ranged from 100 to 50:1 (w/w). NMR data were collected on a Varian
1
Unity-400 (400 MHz H, 100 MHz 13C) machine in the Department
of Pharmacology and Molecular Sciences, the Johns Hopkins University.
1H NMR spectra were obtained in deuteriochloroform (CDCl3) with
either tetramethylsilane (TMS, δ = 0.00 for 1H) or chloroform (CHCl3,
δ = 7.27 for 1H) as an internal reference. 13C NMR spectra were proton
decoupled and were in CDCl3 with either TMS (δ = 0.0 for 13C) or
Typical Procedure A for the Preparation of 7aꢀ7q. To a
solution of the phenol 5aꢀ5q (1 equiv) in DMSO was added NaH (a 60%
dispersion in mineral oil, 4ꢀ5equiv). After the mixture was stirred at 50 °C
Figure 3. The number of carbons and branching of the itraconazole side
chain influences the degree of glycosylation inhibition.
Figure 5. Length, branching, and polarity of the side chain influences
Hh pathway inhibition.
Figure 4. Lack of correlation between potency in MB and HUVEC proliferation.
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under argon for 1 h, a solution of 6a (1.05ꢀ1.1 equiv) in DMSO was
added dropwise. After the addition, the temperature was increased to
90 °C and the solution was stirred under argon for another 3 h. The reaction
was then quenched by the addition of a 50% aqueous NaCl solution, and the
resulting mixture was extracted with CH2Cl2. The organic fractions were
dried (Na2SO4), filtered, and concentrated under vacuum to yield the crude
product, which was purified by column chromatography (1:1 hexanesꢀ
EtOAc fneat EtOAc) to afford the desired products, which could be further
purified by a second column (neat CH2Cl2 f 50:1 CH2Cl2ꢀCH3OH) if
necessary.
Typical Procedure B for the Preparation of 7rꢀ7x. To a
slurry of 17a or 17b (1 equiv) in acetonitrile was added 3rꢀ3x (1.3ꢀ1.6
equiv), K2CO3 (2 equiv), and 18-crown-6 (1 equiv). After the mixture
was stirred at 40ꢀ50 °C under argon for 6ꢀ14 h, the solvent was
removed under vacuum to yield the crude product, which was purified by
column chromatography (neat CH2Cl2 f 50:1 CH2Cl2ꢀCH3OH).
cis-(2S,4R)-4-[4-[4-[4-[[2-(2,4-Dichlorophenyl)-2-(1H-1,2,4-
triazol-l-ylmethyl)-1,3-dioxolan-4-yl]methoxy]phenyl]-
lpiperazinyl]phenyl]-2,4-dihydro-2-methyl-3H-1,2,4-triazol-
3-one (7a). This compound was synthesized as a colorless oil from 5a
(20.1 mg, 0.057 mmol), 6a (29.1 mg, 0.060 mmol), and NaH (10.7 mg
of a 60% dispersion in mineral oil, 0.27 mmol) in 41% yield by following
typical procedure A. 1H NMR (400 MHz, CDCl3, δH) 8.20 (br s, 1H),
7.89 (br s, 1H), 7.63ꢀ7.55 (m, 2H), 7.49ꢀ7.36 (m, 3H), 7.25 (dd, J =
8.7, 2.4 Hz, 1H), 7.05ꢀ6.90 (m, 4H), 6.82ꢀ6.76 (m, 2H), 4.79 (q, J =
14.7 Hz, 2H), 4.40ꢀ4.32 (m, 1H), 3.94ꢀ3.88 (m, 1H), 3.85ꢀ3.75
(m, 2H), 3.52 (s, 3H), 3.46 (dd, J = 9.7, 6.4 Hz, 1H), 3.38ꢀ3.33 (m, 4H),
3.25ꢀ3.19 (m, 4H). 13C NMR (100 MHz, CDCl3, δC) 152.82, 152.60,
151.61, 150.89, 146.20, 145.20, 136.28, 134.26, 133.34, 131.61, 129.84,
127.48, 125.92, 123.91, 118.69, 116.88, 115.47, 107.83, 74.91, 67.83,
67.65, 53.80, 50.80, 49.38, 32.90. MALDI-MS: 663.2 (M + H+), 685.2
(M + Na+). Purity: 98.5%, tR = 6.56 min.
cis-(2S,4R)-4-[4-[4-[4-[[2-(2,4-Dichlorophenyl)-2-(1H-1,2,4-
triazol-l-ylmethyl)-1,3-dioxolan-4-yl]methoxy]phenyl]-
lpiperazinyl]phenyl]-2,4-dihydro-2-isopropyl-3H-1,2,4-triazol-
3-one (7d). This compound was synthesized as a colorless oil from 5d
(26.9 mg, 0.066 mmol), 6a (35.0 mg, 0.072 mmol), and NaH (10.9 mg
of a 60% dispersion in mineral oil, 0.27 mmol) in 50% yield by following
typical procedure A. 1H NMR (400 MHz, CDCl3, δH) 8.23 (s, 1H), 7.90
(s, 1H), 7.67ꢀ7.52 (m, 2H), 7.44ꢀ7.37 (m, 3H), 7.25 (dd, J = 8.3,
2.2 Hz, 1H), 7.07ꢀ6.89 (m, 4H), 6.84ꢀ6.75 (m, 2H), 4.81 (q, J = 14.6
Hz, 2H), 4.60ꢀ4.50 (m, 1H), 4.42ꢀ4.30 (m, 1H), 3.96ꢀ3.72 (m, 3H),
3.56ꢀ3.43 (m, 1H), 3.43ꢀ3.29 (m, 4H), 3.28ꢀ3.17 (m, 4H), 1.41 (d,
J = 6.7 Hz, 6H). 13C NMR (100 MHz, CDCl3, δC) 152.82, 152.09,
150.80, 146.23, 136.29, 134.27, 134.12, 133.37, 131.66, 129.84, 127.48,
126.06, 123.89, 118.69, 116.89, 115.49, 107.84, 74.92, 67.87, 67.68,
53.92, 50.82, 49.44, 47.17, 21.39. ESI-MS: 690.2 (M + H+), 712.2 (M +
Na+). Purity: 94.7%, tR = 8.63 min.
cis-(2S,4R)-4-[4-[4-[4-[[2-(2,4-Dichlorophenyl)-2-(1H-1,2,4-
triazol-l-ylmethyl)-1,3-dioxolan-4-yl]methoxy]phenyl]-
lpiperazinyl]phenyl]-2,4-dihydro-2-butyl-3H-1,2,4-triazol-
3-one (7e). This compound was synthesized as a yellowish oil from 5e
(38.8 mg, 0.099 mmol), 6a (52.6 mg, 0.11 mmol), and NaH (17.8 mg of
a 60% dispersion in mineral oil, 0.45 mmol) in 52% yield by following
typical procedure A. 1H NMR (400 MHz, CDCl3, δH) 8.21 (s, 1H), 7.89
(s, 1H), 7.64ꢀ7.52 (m, 2H), 7.46ꢀ7.38 (m, 3H), 7.24 (dd, J = 8.6,
2.2 Hz, 1H), 7.08ꢀ6.89 (m, 4H), 6.83ꢀ6.77 (m, 2H), 4.79 (q, J =
14.7 Hz, 2H), 4.42ꢀ4.28 (m, 1H), 3.98ꢀ3.71 (m, 5H), 3.48 (dd, J = 9.6,
6.3 Hz, 1H), 3.55ꢀ3.41 (m, 4H), 3.40ꢀ3.27 (m, 4H), 1.87ꢀ1.70
(m, 2H), 1.40 (dq, J = 14.7, 7.4 Hz, 2H), 0.96 (t, J = 7.4 Hz, 3H). 13C
NMR (100 MHz, CDCl3, δC) 152.83, 152.34, 151.55, 150.79, 146.22,
136.26, 134.28, 134.12, 133.34, 131.64, 129.83, 127.46, 126.07, 123.77,
118.67, 116.85, 115.51, 107.83, 74.93, 67.89, 67.66, 53.84, 50.79, 49.41,
45.55, 30.90, 20.00, 13.87. ESI-MS: 705.2 (M + H+), 727.2 (M + Na+).
Purity: >99%, tR = 9.89 min.
cis-(2S,4R)-4-[4-[4-[4-[[2-(2,4-Dichlorophenyl)-2-(1H-1,2,4-
triazol-l-ylmethyl)-1,3-dioxolan-4-yl]methoxy]phenyl]-
lpiperazinyl]phenyl]-2,4-dihydro-2-ethyl-3H-1,2,4-triazol-3-
one (7b). This compound was synthesized as a colorless oil from 5b
(18.6 mg, 0.051 mmol), 6a (27.7 mg, 0.057 mmol), and NaH (10.0 mg
of a 60% dispersion in mineral oil, 0.25 mmol) in 58% yield by following
typical procedure A. 1H NMR (400 MHz, CDCl3, δH) 8.21 (s, 1H), 7.90
(s, 1H), 7.67ꢀ7.53 (m, 2H), 7.49ꢀ7.37 (m, 3H), 7.25 (dd, J = 8.7, 2.3
Hz, 1H), 7.09ꢀ6.87 (m, 4H), 6.83ꢀ6.77 (m, 2H), 4.80 (q, J = 14.7 Hz,
2H), 4.46ꢀ4.27 (m, 1H), 3.99ꢀ3.73 (m, 5H), 3.46 (dt, J = 23.5, 11.8
Hz, 1H), 3.43ꢀ3.32 (m, 4H), 3.32ꢀ3.16 (m, 4H), 1.46ꢀ1.32 (t, J = 7.3
Hz, 3H). 13C NMR (100 MHz, CDCl3, δC) 152.86, 152.11, 151.68,
150.84, 146.20, 136.29, 134.24, 133.35, 131.66, 129.84, 127.48, 126.02,
123.90, 118.71, 116.89, 115.49, 107.85, 74.92, 67.86, 67.67, 53.83, 50.82,
49.40, 40.79, 14.10. MALDI -MS: 677.2 (M + H+), 699.2 (M + Na+).
Purity: >99%, tR = 7.04 min.
cis-(2S,4R)-4-[4-[4-[4-[[2-(2,4-Dichlorophenyl)-2-(1H-1,2,4-
triazol-l-ylmethyl)-1,3-dioxolan-4-yl]methoxy]phenyl]-
lpiperazinyl]phenyl]-2,4-dihydro-2-propyl-3H-1,2,4-triazol-
3-one (7c). This compound was synthesized as a colorless oil from
5c (22.9 mg, 0.060 mmol), 6a (32.0 mg, 0.066 mmol), and NaH (10.0
mg of a 60% dispersion in mineral oil, 0.25 mmol) in 53% yield by
following typical procedure A. 1H NMR (400 MHz, CDCl3, δH) 8.24
(s, 1H), 7.90 (s, 1H), 7.65ꢀ7.54 (m, 2H), 7.47ꢀ7.38 (m, 3H), 7.25
(dd, J = 8.4, 2.1 Hz, 1H), 7.13ꢀ6.95 (m, 4H), 6.85ꢀ6.76 (m, 2H), 4.80
(q, J = 14.7 Hz, 2H), 4.45ꢀ4.28 (m, 1H), 3.97ꢀ3.73 (m, 5H),
3.60ꢀ3.10 (m, 9H), 1.94ꢀ1.68 (m, 2H), 0.97 (t, J = 7.4 Hz, 3H).;
13C NMR (100 MHz, CDCl3, δC) 152.38, 151.69, 150.59, 140.03,
136.29, 134.25, 134.12, 133.34, 131.66, 129.83, 127.49, 126.09, 123.80,
119.06, 117.02, 115.54, 107.85, 74.87, 67.81, 67.62, 53.81, 51.09, 49.23,
47.41, 22.26, 11.34. ESI-MS: 690.2 (M + H+), 712.2 (M + Na+). Purity:
97.4%, tR = 7.45 min.
cis-(2S,4R)-4-[4-[4-[4-[[2-(2,4-Dichlorophenyl)-2-(1H-1,2,4-
triazol-l-ylmethyl)-1,3-dioxolan-4-yl]methoxy]phenyl]-
lpiperazinyl]phenyl]-2,4-dihydro-2-isobutyl-3H-1,2,4-triazol-
3-one (7f). This compound was synthesized as a yellowish oil from 5f
(20.0 mg, 0.051 mmol), 6a (27.1 mg, 0.058 mmol), and NaH (10.2 mg
of a 60% dispersion in mineral oil, 0.26 mmol) in 63% yield by following
typical procedure A. 1H NMR (400 MHz, CDCl3, δH) 8.22 (s, 1H), 7.89
(s, 1H), 7.66ꢀ7.51 (m, 2H), 7.48ꢀ7.37 (m, 3H), 7.25 (dd, J = 8.6,
2.2 Hz, 1H), 7.05ꢀ6.88 (m, 4H), 6.83ꢀ6.76 (m, 2H), 4.80 (q, J =
14.7 Hz, 2H), 4.41ꢀ4.29 (m, 1H), 3.91 (dd, J = 8.3, 6.7 Hz, 1H),
3.86ꢀ3.72 (m, 2H), 3.65 (d, J = 7.3 Hz, 2H), 3.47 (dd, J = 9.6, 6.4 Hz,
1H), 3.38ꢀ3.32 (m, 4H), 3.25ꢀ3.19 (m, 4H), 2.19 (dt, J = 13.7, 6.9 Hz,
1H), 0.97 (d, J = 6.7 Hz, 6H). 13C NMR (100 MHz, CDCl3, δC) 152.82,
152.64, 150.79, 146.22, 136.28, 134.28, 134.06, 133.34, 131.65, 129.84,
127.47, 126.11, 123.72, 118.68, 116.88, 115.48, 107.83, 74.92, 67.86,
67.66, 53.87, 53.04, 50.80, 49.43, 28.46, 20.12. ESI-MS: 705.2 (M + H+),
727.2 (M + Na+). Purity: >99%, tR = 9.63 min.
cis-(2S,4R)-4-[4-[4-[4-[[2-(2,4-Dichlorophenyl)-2-(1H-1,2,4-
triazol-l-ylmethyl)-1,3-dioxolan-4-yl]methoxy]phenyl]-
lpiperazinyl]phenyl]-2,4-dihydro-2-pentyl-3H-1,2,4-triazol-
3-one (7g). This compound was synthesized as a yellowish oil from 5g
(23.4 mg, 0.057 mmol), 6a (30.0 mg, 0.062 mmol), and NaH (11.4 mg
of a 60% dispersion in mineral oil, 0.28 mmol) in 57% yield by following
typical procedure A. 1H NMR (400 MHz, CDCl3, δH) 8.24 (s, 1H), 7.90
(s, 1H), 7.62ꢀ7.55 (m, 2H), 7.48ꢀ7.38 (m, 3H), 7.25 (dd, J = 8.3,
2.1 Hz, 1H), 7.15ꢀ6.95 (m, 4H), 6.85ꢀ6.77 (m, 2H), 4.80 (q, J = 14.7
Hz, 2H), 4.42ꢀ4.30 (m, 1H), 3.99ꢀ3.71 (m, 5H), 3.58ꢀ3.19 (m, 9H),
1.88ꢀ1.70 (m, 2H), 1.35 (dd, J = 7.2, 3.7 Hz, 4H), 0.90 (dd, J = 8.8,
5.0 Hz, 3H). 13C NMR (100 MHz, CDCl3, δC) 152.32, 150.59, 136.31,
134.22, 134.12, 133.34, 131.67, 129.83, 127.50, 123.83, 117.07, 116.68,
115.57, 107.85, 74.86, 67.80, 67.60, 53.82, 51.42, 49.13, 45.85, 28.92,
7370
dx.doi.org/10.1021/jm200944b |J. Med. Chem. 2011, 54, 7363–7374

Journal of Medicinal Chemistry
ARTICLE
28.58, 22.51, 14.21. ESI-MS: 719.3 (M + H+), 741.3 (M + Na+). Purity: >99%,
tR = 13.19 min.
50.80, 49.41, 31.69, 25.65, 25.45. ESI-MS: 731.3 (M + H+), 753.3 (M +
Na+). Purity: >99%, tR = 7.77 min.
cis-(2S,4R)-4-[4-[4-[4-[[2-(2,4-Dichlorophenyl)-2-(1H-1,2,4-
triazol-l-ylmethyl)-1,3-dioxolan-4-yl]methoxy]phenyl]-
lpiperazinyl]phenyl]-2,4-dihydro-2-(1-methylbutyl)-3H-1,2,4-
triazol-3-one (7h). This compound was synthesized as a yellowish oil
from 5h (23.4 mg, 0.057 mmol), 6a (29.8 mg, 0.062 mmol), and NaH
(10.3 mg of a 60% dispersion in mineral oil, 0.26 mmol) in 66% yield by
cis-(2S,4R)-4-[4-[4-[4-[[2-(2,4-Dichlorophenyl)-2-(1H-1,2,4-
triazol-l-ylmethyl)-1,3-dioxolan-4-yl]methoxy]phenyl]-
lpiperazinyl]phenyl]-2,4-dihydro-2-(1-cyclopentylmethyl)-3H-
1,2,4-triazol-3-one (7l). This compound was synthesized as a yellow-
ish oil from 5l (20.0 mg, 0.048 mmol), 6a (25.4 mg, 0.052 mmol), and
NaH (10.2 mg of a 60% dispersion in mineral oil, 0.26 mmol) in 68%
yield by following typical procedure A. 1H NMR (400 MHz, CDCl3, δH)
8.21 (s, 1H), 7.89 (s, 1H), 7.66ꢀ7.53 (m, 2H), 7.51ꢀ7.37 (m, 3H), 7.25
(dd, J = 8.1, 1.7 Hz, 1H), 7.14ꢀ6.89 (m, 4H), 6.82ꢀ6.72 (m, 2H), 4.80
(q, J = 14.7 Hz, 2H), 4.40ꢀ4.32 (m, 1H), 4.01ꢀ3.71 (m, 5H), 3.54ꢀ3.15
(m, 9H), 2.43 (dt, J = 15.3, 7.6 Hz, 1H), 1.95ꢀ1.47 (m, 6H), 1.40ꢀ1.25
(m, 2H). 13C NMR (100 MHz, CDCl3, δC) 152.83, 152.52, 151.61,
150.78, 146.21, 136.28, 134.27, 134.00, 133.34, 131.65, 129.84, 127.48,
126.12, 123.74, 118.70, 116.89, 115.48, 107.84, 74.92, 67.86, 67.66, 53.83,
50.81, 50.53, 49.44, 39.53, 30.36, 25.30. ESI-MS: 731.3 (M + H+), 753.3
(M + Na+). Purity: >99%, tR = 7.74 min.
cis-(2S,4R)-4-[4-[4-[4-[[2-(2,4-Dichlorophenyl)-2-(1H-1,2,4-
triazol-l-ylmethyl)-1,3-dioxolan-4-yl]methoxy]phenyl]-
lpiperazinyl]phenyl]-2,4-dihydro-2-(n-octyl)-3H-1,2,4-triazol-
3-one (7m). This compound wassynthesized as a yellowishoilfrom 5m
(27.4 mg, 0.055 mmol), 6a (29.3 mg, 0.060 mmol), and NaH (10.9 mg of
a 60% dispersion in mineral oil, 0.27 mmol) in 72% yield by following
typical procedure A. 1H NMR (400 MHz, CDCl3, δH) 8.21 (s, 1H), 7.90
(s, 1H), 7.70ꢀ7.51 (m, 2H), 7.50ꢀ7.36 (m, 3H), 7.25 (dd, J = 8.4,
1.9 Hz, 1H), 7.13ꢀ6.90 (m, 4H), 6.84ꢀ6.73 (m, 2H), 4.80 (q, J =
14.7 Hz, 2H), 4.42ꢀ4.29 (m, 1H), 3.99ꢀ3.71 (m, 5H), 3.57ꢀ3.12
(m, 9H), 1.85ꢀ1.72 (dt, J = 14.6, 7.4 Hz, 2H), 1.48ꢀ1.16 (m, 10H), 0.87
(t, J = 6.8 Hz, 3H). 13C NMR (100 MHz, CDCl3, δC) 152.83, 152.33,
151.62, 150.81, 146.22, 136.29, 134.27, 134.11, 133.35, 131.66, 129.83,
127.47, 126.08, 123.80, 118.70, 116.89, 115.49, 107.85, 74.92, 67.86, 67.67,
53.84, 50.82, 49.43, 45.88, 32.00, 29.38, 28.88, 26.80, 22.86, 14.33. ESI-MS:
761.3 (M + H+), 783.3 (M + Na+). Purity: >99%, tR = 14.67 min.
cis-(2S,4R)-4-[4-[4-[4-[[2-(2,4-Dichlorophenyl)-2-(1H-1,2,4-
triazol-l-ylmethyl)-1,3-dioxolan-4-yl]methoxy]phenyl]-
lpiperazinyl]phenyl]-2,4-dihydro-2-(6-phenylhexyl)-3H-1,2,4-
triazol-3-one (7n). This compound was synthesized as a yellowish oil
from 5n (25.3 mg, 0.051 mmol), 6a (25.9 mg, 0.054 mmol), and NaH
(10.9 mg of a 60% dispersion in mineral oil, 0.27 mmol) in 70% yield
by following typical procedure A. 1H NMR (400 MHz, CDCl3, δH) 8.21
(s, 1H), 7.90 (s, 1H), 7.69ꢀ7.53 (m, 2H), 7.52ꢀ7.35 (m, 3H), 7.33ꢀ7.22
(m, 3H), 7.22ꢀ7.12 (m, 3H), 7.08ꢀ6.99 (m, 2H), 6.97ꢀ6.89 (m, 2H),
6.83ꢀ6.76 (m, 2H), 4.80 (q, J = 14.7 Hz, 2H), 4.39ꢀ4.32 (m, 1H),
4.01ꢀ3.72 (m, 5H), 3.47 (dd, J = 9.7, 6.4 Hz, 1H), 3.41ꢀ3.32 (m, 4H),
3.28ꢀ3.18 (m, 4H), 2.67ꢀ2.52 (d, J = 7.5 Hz, 2H), 1.83ꢀ1.72 (m, 2H),
1.70ꢀ1.56 (m, 2H), 1.46ꢀ1.33 (m, 4H). 13C NMR (100 MHz, CDCl3,
δC) 152.82, 152.33, 151.62, 150.81, 146.20, 142.88, 136.28, 134.27, 134.17,
133.34, 131.66, 129.84, 128.62, 128.46, 127.49, 126.04, 125.83, 123.81,
118.70, 116.89, 115.47, 107.84, 74.92, 67.83, 67.66, 53.81, 50.81, 49.42,
45.81, 36.07, 31.53, 29.07, 28.83, 26.66. ESI-MS: 808.3 (M + H+), 830.3
(M + Na+). Purity: >99%, tR = 12.78 min.
1
following typical procedure A. H NMR (400 MHz, CDCl3, δH) 8.26
(s, 1H), 7.91 (s, 1H), 7.72ꢀ7.53 (m, 2H), 7.48ꢀ7.37 (m, 3H), 7.25 (dd,
J = 8.3, 2.1 Hz, 1H), 7.13ꢀ6.96 (m, 4H), 6.86ꢀ6.79 (m, 2H), 4.81 (q, J =
14.7 Hz, 2H), 4.51ꢀ4.29 (m, 2H), 3.98ꢀ3.86 (m, 1H), 3.86ꢀ3.71
(m, 2H), 3.58ꢀ3.10 (m, 8H), 1.93ꢀ1.77 (m, 1H), 1.62 (ddt, J = 13.6,
9.9, 5.9 Hz, 1H), 1.47ꢀ1.18 (m, 6H), 0.92 (t, J = 7.4 Hz, 3H). 13C NMR
(100 MHz, CDCl3, δC) 152.10, 150.56, 136.29, 134.23, 134.11, 133.34,
131.66, 129.83, 127.49, 123.75, 119.06, 117.05, 115.55, 107.84, 74.86,
67.80, 67.61, 53.84, 51.07, 49.24, 37.69, 29.93, 19.88, 19.70, 14.00. ESI-
MS: 719.3 (M + H+), 741.3 (M + Na+). Purity: >99%, tR = 9.85 min.
cis-(2S,4R)-4-[4-[4-[4-[[2-(2,4-Dichlorophenyl)-2-(1H-1,2,4-
triazol-l-ylmethyl)-1,3-dioxolan-4-yl]methoxy]phenyl]-
lpiperazinyl]phenyl]-2,4-dihydro-2-(3-methylbutyl)-3H-1,2,4-
triazol-3-one (7i). This compound was synthesized as a yellowish oil
from 5i (23.4 mg, 0.057 mmol), 6a (30.3 mg, 0.063 mmol), and NaH
(11.1 mg of a 60% dispersion in mineral oil, 0.28 mmol) in 63% yield by
1
following typical procedure A. H NMR (400 MHz, CDCl3, δH) 8.23
(s, 1H), 7.90 (s, 1H), 7.67ꢀ7.51 (m, 2H), 7.51ꢀ7.34 (m, 3H), 7.25 (dd,
J = 8.4, 2.0 Hz, 1H), 7.15ꢀ6.97 (m, 4H), 6.88ꢀ6.80 (m, 2H), 4.80 (q, J =
14.7 Hz, 2H), 4.43ꢀ4.27 (m, 1H), 3.98ꢀ3.70 (m, 5H), 3.56ꢀ3.08
(m, 9H), 1.73ꢀ1.60 (m, 3H), 0.96 (d, J = 6.3 Hz, 6H). 13C NMR (100
MHz, CDCl3, δC) 152.23, 150.60, 136.29, 134.24, 134.08, 133.34, 131.66,
129.83, 127.48, 123.76, 119.07, 117.03, 115.55, 107.84, 74.86, 67.81, 67.61,
53.83, 51.18, 49.21, 44.19, 37.56, 25.81, 22.61. ESI-MS: 719.3 (M + H+),
741.3 (M + Na+). Purity: >99%, tR = 10.10 min.
cis-(2S,4R)-4-[4-[4-[4-[[2-(2,4-Dichlorophenyl)-2-(1H-1,2,4-
triazol-l-ylmethyl)-1,3-dioxolan-4-yl]methoxy]phenyl]-
lpiperazinyl]phenyl]-2,4-dihydro-2-(1-ethylpropyl)-3H-1,2,4-
triazol-3-one (7j). This compound was synthesized as a yellowish oil
from 5j (23.3 mg, 0.057 mmol), 6a (30.1 mg, 0.062 mmol), and NaH
(10.4 mg of a 60% dispersion in mineral oil, 0.28 mmol) in 63% yield by
1
following typical procedure A. H NMR (400 MHz, CDCl3, δH) 8.26
(s, 1H), 7.91 (s, 1H), 7.69ꢀ7.53 (m, 2H), 7.51ꢀ7.37 (m, 3H), 7.25 (dd,
J = 8.4, 2.1 Hz, 1H), 7.10ꢀ6.93 (m, 4H), 6.85ꢀ6.77 (m, 2H), 4.81 (q, J =
14.7 Hz, 2H), 4.47ꢀ4.29 (m, 1H), 4.12ꢀ3.99 (m, 1H), 3.99ꢀ3.86
(m, 1H), 3.87ꢀ3.71 (m, 2H), 3.57ꢀ3.13 (m, 9H), 1.94ꢀ1.62 (m, 4H),
0.88 (t, J = 7.4 Hz, 6H). 13C NMR (100 MHz, CDCl3, δC) 153.05,
150.53, 136.31, 134.22, 133.34, 131.67, 129.84, 127.50, 123.74, 119.15,
117.06, 115.57, 107.84, 74.87, 67.81, 67.61, 59.06, 53.86, 51.34, 49.22,
27.00, 10.97. ESI-MS: 719.3 (M + H+), 741.3 (M + Na+). Purity: 95.1%,
tR = 9.17 min.
cis-(2S,4R)-4-[4-[4-[4-[[2-(2,4-Dichlorophenyl)-2-(1H-1,2,4-
triazol-l-ylmethyl)-1,3-dioxolan-4-yl]methoxy]phenyl]-
lpiperazinyl]phenyl]-2,4-dihydro-2-(1-cyclohexyl)-3H-1,2,4-
triazol-3-one (7k). This compound was synthesized as a yellowish oil
from 5k (21.8 mg, 0.052 mmol), 6a (25.4 mg, 0.055 mmol), and NaH
(10.0 mg of a 60% dispersion in mineral oil, 0.25 mmol) in 69% yield by
cis-(2S,4R)-4-[4-[4-[4-[[2-(2,4-Dichlorophenyl)-2-(1H-1,2,4-
triazol-l-ylmethyl)-1,3-dioxolan-4-yl]methoxy]phenyl]-
lpiperazinyl]phenyl]-2,4-dihydro-2-(hex-3-ynyl)-3H-1,2,4-triazol-
3-one (7o). This compound was synthesized as a yellowish oil from 5o
(25.9 mg, 0.062 mmol), 6a (31.6 mg, 0.065 mmol), and NaH (12.8 mg
of a 60% dispersion in mineral oil, 0.32 mmol) in 65% yield by following
typical procedure A. 1H NMR (400 MHz, CDCl3, δH) 8.19 (s, 1H), 7.88
(s, 1H), 7.69ꢀ7.51 (m, 2H), 7.51ꢀ7.33 (m, 3H), 7.24 (dd, J = 8.2,
2.0 Hz, 1H), 7.06ꢀ6.87 (m, 4H), 6.79 (d, J = 9.0 Hz, 2H), 4.78 (q, J =
14.7 Hz, 2H), 4.48ꢀ4.25 (m, 1H), 4.06ꢀ3.72 (m, 5H), 3.54ꢀ3.12 (m,
9H), 2.63 (tt, J = 7.3, 2.3 Hz, 2H), 2.13 (qt, J = 7.5, 2.3 Hz, 2H),
1.17ꢀ0.99 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, CDCl3, δC) 152.81,
1
following typical procedure A. H NMR (400 MHz, CDCl3, δH) 8.20
(s, 1H), 7.88 (s, 1H), 7.71ꢀ7.51 (m, 2H), 7.47ꢀ7.36 (m, 3H), 7.24
(dd, J = 8.7, 2.2 Hz, 1H), 7.05ꢀ6.87 (m, 4H), 6.82ꢀ6.74 (m, 2H), 4.79
(q, J = 14.7 Hz, 2H), 4.40ꢀ4.28 (m, 1H), 4.20ꢀ4.05 (m, 1H), 3.98ꢀ3.71
(m, 3H), 3.46 (dd, J = 9.6, 6.3 Hz, 1H), 4.43ꢀ4.25 (m, 4H), 3.27ꢀ3.12
(m, 4H), 1.95ꢀ1.63 (m, 6H), 1.49ꢀ1.32 (m, 2H), 1.30ꢀ1.13 (m, 2H).
13C NMR (100 MHz, CDCl3, δC) 152.80, 151.73, 151.60, 150.75,
146.19, 136.26, 134.26, 134.04, 133.33, 131.64, 129.83, 127.48, 126.06,
123.89, 118.68, 116.87, 115.45, 107.82, 74.91, 67.82, 67.65, 54.31, 53.79,
7371
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Journal of Medicinal Chemistry
ARTICLE
152.25, 151.60, 150.84, 146.19, 145.15, 136.26, 134.51, 134.27, 133.33,
131.65, 129.84, 127.48, 125.92, 123.83, 118.68, 116.87, 115.46, 107.83,
83.95, 75.41, 74.91, 67.83, 67.65, 53.80, 50.79, 49.38, 45.02, 19.26, 14.34,
12.62. ESI-MS: 729.2 (M + H+), 751.2 (M + Na+). Purity: >99%,
tR = 7.93 min.
from 17a (19.8 mg, 0.031 mmol), 3s (12.3 mg, 0.048 mmol), K2CO3
(8.4 mg, 0.061mmol), and18-crown-6(8.1 mg, 0.031 mmol) in55%yield
by following typical procedure B. 1H NMR (400 MHz, CDCl3, δH) 8.23
(s, 1H), 7.90 (s, 1H), 7.61ꢀ7.55 (m, 2H), 7.51ꢀ7.37 (m, 3H), 7.25 (dd,
J = 8.4, 1.8 Hz, 1H), 7.03 (d, J = 9.0 Hz, 2H), 6.95 (d, J = 8.9 Hz, 2H), 6.81
(d, J = 8.9 Hz, 2H), 5.50 (dtt, J = 15.3, 6.3, 1.3 Hz, 1H), 5.31ꢀ5.15 (m,
1H), 4.84 (q, J = 14.7 Hz, 2H), 4.49ꢀ4.32 (m, 1H), 3.92ꢀ3.73 (m, 6H),
3.52 (dd, J = 9.7, 6.5 Hz, 1H), 3.41ꢀ3.33 (m, 4H), 3.26ꢀ3.17 (m, 4H),
2.43 (q, J = 7.0 Hz, 2H), 2.08ꢀ1.92 (m, 2H), 0.92 (t, J = 7.5 Hz, 3H). 13C
NMR (100 MHz, CDCl3, δC) 152.83, 152.36, 151.66, 150.85, 146.21,
136.28, 134.77, 134.29, 133.34, 131.67, 129.84, 127.49, 126.00, 124.52,
123.81, 118.70, 116.88, 115.48, 107.85, 74.92, 67.83, 67.66, 53.82, 50.82,
49.42, 45.57, 25.90, 21.82, 14.03. ESI-MS: 731.2 (M + H+), 753.2 (M +
Na+). Purity: 95.3%, tR = 9.15 min.
cis-(2S,4R)-4-[4-[4-[4-[[2-(2,4-Dichlorophenyl)-2-(1H-1,2,4-
triazol-l-ylmethyl)-1,3-dioxolan-4-yl]methoxy]phenyl]-
lpiperazinyl]phenyl]-2,4-dihydro-2-((Z)-hex-3-enyl)-3H-1,2,4-
triazol-3-one (7t). This compound was synthesized as a yellowish oil
from 17a (18.7 mg, 0.029 mmol), 3t (11.0 mg, 0.043 mmol), K2CO3
(8.0 mg, 0.058 mmol), and18-crown-6(7.6 mg, 0.029 mmol) in 70% yield
by following typical procedure B. 1H NMR (400 MHz, CDCl3, δH) 8.21
(s, 1H), 7.89 (s, 1H), 7.62ꢀ7.55 (m, 2H), 7.50ꢀ7.36 (m, 3H), 7.25 (dd,
J = 8.5, 1.7 Hz, 1H), 7.02 (d, J = 9.0 Hz, 2H), 6.94 (d, J = 8.9 Hz, 2H), 6.80
(d, J = 8.9 Hz, 2H), 5.50 (dt, J = 10.7, 7.2 Hz, 1H), 5.44ꢀ5.31 (m, 1H),
4.85 (q, J = 14.7 Hz, 2H), 4.48ꢀ4.30 (m, 1H), 3.95ꢀ3.75 (m, 6H), 3.49
(dd, J = 9.9, 6.3 Hz, 1H), 3.42ꢀ3.34(m, 4H), 3.27ꢀ3.19 (m, 4H), 2.54 (q,
J = 7.0 Hz, 2H), 2.12ꢀ1.97 (m, 2H), 0.93 (t, J = 7.5 Hz, 3H). 13C NMR
(100 MHz, CDCl3, δC) 152.83, 152.34, 151.64, 150.84, 146.19, 136.29,
134.93, 134.21, 133.34, 131.66, 129.84, 127.49, 126.01, 124.33, 123.83,
118.71, 116.89, 115.47, 107.84, 74.92, 67.83, 67.66, 53.82, 50.82, 49.42,
45.60, 26.89, 20.80, 14.47. ESI-MS: 731.2 (M + H+), 753.2 (M + Na+).
Purity: 95.0%, tR = 8.91 min.
cis-(2S,4R)-4-[4-[4-[4-[[2-(2,4-Dichlorophenyl)-2-(1H-1,2,4-
triazol-l-ylmethyl)-1,3-dioxolan-4-yl]methoxy]phenyl]-
lpiperazinyl]phenyl]-2,4-dihydro-2-(5-cyanopentyl)-3H-1,2,4-
triazol-3-one (7u). This compound was synthesized as a yellowish oil
from 17a (40.0 mg, 0.062 mmol), 3u (16.3 mg, 0.092 mmol), K2CO3
(17.1 mg, 0.124 mmol), and 18-crown-6 (16.3 mg, 0.062 mmol) in 41%
yield by following typical procedure B. 1H NMR (400 MHz, CDCl3, δH)
8.25 (s, 1H), 7.90 (s, 1H), 7.65ꢀ7.55 (m, 2H), 7.48 (d, J = 1.7 Hz, 1H),
7.41 (d, J = 8.8 Hz, 2H), 7.25 (dd, J = 8.4, 2.0 Hz, 1H), 7.03 (d, J = 8.9 Hz,
2H), 6.94 (d, J = 8.7 Hz, 2H), 6.81 (d, J = 8.7 Hz, 2H), 4.81 (q, J = 14.7
Hz, 2H), 4.50ꢀ4.25 (m, 1H), 4.05ꢀ3.71 (m, 5H), 3.49 (t, J = 7.8 Hz,
1H), 3.43ꢀ3.33 (m, 4H), 3.29ꢀ3.20 (m, 4H), 2.37 (t, J = 7.1 Hz, 2H),
1.93ꢀ1.81 (m, 2H), 1.80ꢀ1.69 (m, 2H), 1.60ꢀ1.49 (m, 2H). 13C NMR
(100 MHz, CDCl3, δC) 152.85, 152.44, 150.91, 146.24, 136.30, 134.42,
134.28, 133.35, 131.66, 129.84, 127.48, 125.87, 123.87, 118.70, 116.87,
115.51, 107.83, 74.93, 67.89, 67.67, 53.95, 50.80, 49.39, 45.19, 28.01,
25.80, 25.15, 17.31. ESI-MS: 744.2 (M + H+), 766.2 (M + Na+). Purity:
94.9%, tR = 5.36 min.
cis-(2S,4R)-4-[4-[4-[4-[[2-(2,4-Dichlorophenyl)-2-(1H-1,2,4-
triazol-l-ylmethyl)-1,3-dioxolan-4-yl]methoxy]phenyl]-
lpiperazinyl]phenyl]-2,4-dihydro-2-(6-azidohexyl)-3H-1,2,4-
triazol-3-one (7p). This compound was synthesized as a yellowish oil
from 5p (33.1 mg, 0.072 mmol), 6a (36.4 mg, 0.075 mmol), and NaH
(14.4 mg of a 60% dispersion in mineral oil, 0.36 mmol) in 44% yield by
following typical procedure A. 1H NMR (400 MHz, CDCl3, δH) 8.19
(s, 1H), 7.87 (s, 1H), 7.60 (s, 1H), 7.55 (d, J = 8.4 Hz, 1H), 7.44 (d, J =
2.1 Hz, 1H), 7.39 (t, J = 6.0 Hz, 2H), 7.23 (dd, J = 8.4, 2.1 Hz, 1H), 7.00
(d, J = 9.0 Hz, 2H), 6.93 (d, J = 8.8 Hz, 2H), 6.78 (d, J = 9.0 Hz, 2H), 4.77
(q, J = 14.7 Hz, 2H), 4.41ꢀ4.25 (m, 1H), 3.94ꢀ3.70 (m, 5H), 3.44 (dd,
J = 9.7, 5.9 Hz, 1H), 3.41ꢀ3.30 (m, 4H), 3.28ꢀ3.16 (m, 6H), 1.88ꢀ1.71
(m, 2H), 1.65ꢀ1.52 (m, 2H), 1.45ꢀ1.33 (m, 4H). 13C NMR (100 MHz,
CDCl3, δC) 152.93, 152.32, 151.59, 150.76, 136.24, 134.25, 133.32,
131.63, 129.84, 127.47, 126.00, 123.77, 118.75, 116.87, 115.47, 107.82,
74.90, 67.81, 67.62, 53.78, 51.53, 50.87, 49.33, 45.60, 28.91, 28.72, 26.50,
26.31. ESI-MS: 774.2 (M + H+), 796.2 (M + Na+). Purity: >99%,
tR = 7.26 min.
cis-(2S,4R)-4-[4-[4-[4-[[2-(2,4-Dichlorophenyl)-2-(1H-1,2,4-
triazol-l-ylmethyl)-1,3-dioxolan-4-yl]methoxy]phenyl]-
lpiperazinyl]phenyl]-2,4-dihydro-2-(6-(4-benzoylphenoxy)-
hexyl)-3H-1,2,4-triazol-3-one (7q). This compound was synthe-
sized as a yellowish oil from 5q (38.8 mg, 0.063 mmol), 6a (34.1 mg, 0.070
mmol), and NaH (12.3 mg of a 60% dispersion in mineral oil, 0.31 mmol) in
74% yield by following typical procedure A. 1H NMR (400 MHz, CDCl3,
δH) 8.22 (s, 1H), 7.89 (s, 1H), 7.84ꢀ7.77 (m, 2H), 7.73 (dd, J =8.3, 1.2 Hz,
2H), 7.61 (s, 1H), 7.58ꢀ7.52 (m, 2H), 7.48ꢀ7.42 (m, 3H), 7.39 (d, J = 8.9
Hz, 2H), 7.24 (dd, J = 8.4, 2.0 Hz, 1H), 7.01 (d, J = 9.0 Hz, 2H), 6.93 (d, J =
8.8 Hz, 4H), 6.79 (d, J = 8.8 Hz, 2H), 4.79 (q, J = 14.7 Hz, 2H), 4.46ꢀ4.25
(m, 1H), 4.02 (t, J = 6.4 Hz, 2H), 3.96ꢀ3.71 (m, 5H), 3.47 (dd, J = 9.5,
6.4 Hz, 1H), 3.42ꢀ3.28 (m, 4H), 3.28ꢀ3.12 (m, 4H), 1.83 (dq, J = 12.9,
6.6 Hz, 4H), 1.64ꢀ1.36 (m, 4H). 13C NMR (100 MHz, CDCl3, δC)
195.78, 163.01, 152.82, 152.36, 151.68, 150.82, 146.21, 138.54, 136.26,
134.24, 133.33, 132.77, 132.05, 131.64, 130.11, 129.93, 129.84, 128.39,
127.47, 125.96, 123.77, 118.67, 116.84, 115.49, 114.23, 107.82, 74.92, 68.26,
67.86, 67.64, 53.86, 50.78, 49.37, 45.66, 29.14, 28.76, 26.46, 25.81. ESI-MS:
929.3 (M + H+), 951.3 (M + Na+). Purity: >99%, tR = 15.68 min.
cis-(2R,4S)-4-[4-[4-[4-[[2-(2,4-Dichlorophenyl)-2-(1H-1,2,4-
triazol-l-ylmethyl)-1,3-dioxolan-4-yl]methoxy]phenyl]-
lpiperazinyl]phenyl]-2,4-dihydro-2-(pent-4-ynyl)-3H-1,2,4-
triazol-3-one (7r). This compound was synthesized as a yellowish oil
from 17b (25.6 mg, 0.039 mmol), 3r (14.1 mg, 0.059 mmol), K2CO3
(10.9 mg, 0.079 mmol), and 18-crown-6 (10.4 mg, 0.039 mmol) in 40%
yield by following typical procedure B. 1H NMR (400 MHz, CDCl3, δH)
8.22 (s, 1H), 7.90 (s, 1H), 7.62 (s, 1H), 7.58 (d, J = 8.4 Hz, 1H), 7.48 (d,
J = 2.1 Hz, 1H), 7.42 (dd, J = 7.1, 5.1 Hz, 2H), 7.25 (dd, J = 8.7, 2.3 Hz, 1H),
7.03 (d, J = 9.1 Hz, 2H), 6.94 (d, J = 9.0 Hz, 2H), 6.80 (d, J = 9.0 Hz, 2H),
4.81 (q, J = 14.7 Hz, 2H), 4.41ꢀ4.33 (m, 1H), 4.04ꢀ3.88 (m, 3H),
3.87ꢀ3.74 (m, 2H), 3.49 (dd, J = 9.7, 6.4 Hz, 1H), 3.42ꢀ3.34 (m, 4H),
3.27ꢀ3.19 (m, 4H), 2.32 (td, J = 7.0, 2.6 Hz, 2H), 2.11ꢀ1.96 (m, 3H). 13C
NMR (100 MHz, CDCl3, δC) 152.84, 152.38, 151.68, 150.86, 146.24,
136.29, 134.35, 133.35, 131.66, 129.83, 127.47, 125.95, 123.79, 118.69,
116.86, 115.51, 107.85, 83.19, 74.93, 69.28, 67.89, 67.67, 53.87, 50.80, 49.40,
44.74, 27.71, 16.16. ESI-MS: 715.2 (M + H+), 737.2 (M + Na+). Purity:
>99%, tR = 7.35 min.
cis-(2S,4R)-4-[4-[4-[4-[[2-(2,4-Dichlorophenyl)-2-(1H-1,2,4-
triazol-l-ylmethyl)-1,3-dioxolan-4-yl]methoxy]phenyl]-
lpiperazinyl]phenyl]-2,4-dihydro-2-((3-ethyloxetan-3-yl)methyl)-
3H-1,2,4-triazol-3-one (7v). This compound was synthesized as a yellow-
ish oil from 17a (30.0 mg, 0.046 mmol), 3v (18.7 mg, 0.069 mmol), K2CO3
(12.8 mg, 0.092 mmol), and 18-crown-6 (12.2 mg, 0.046 mmol) in 40% yield
1
by following typical procedure B. H NMR (400 MHz, CDCl3, δH) 8.21
(s, 1H), 7.90 (s, 1H), 7.72ꢀ7.52 (m, 2H), 7.52ꢀ7.36 (m, 3H), 7.25 (dd, J =
8.5, 2.2 Hz, 1H), 7.13ꢀ6.72 (m, 6H), 4.91ꢀ4.69 (m, 4H), 4.45 (d, J = 6.3 Hz,
2H), 4.41ꢀ4.32 (m, 1H), 4.07 (s, 2H), 3.92 (dd, J = 8.3, 6.7 Hz, 1H),
3.87ꢀ3.73 (m, 2H), 3.54ꢀ3.44 (m, 1H), 3.43ꢀ3.32 (m 4H), 3.30ꢀ3.23 (m,
4H), 1.74 (dd, J = 14.9, 7.4 Hz, 2H), 1.00 (t, J = 7.4 Hz, 3H). 13C NMR (100
MHz, CDCl3, δC) 152.84, 151.60, 150.92, 146.20, 145.18, 136.28, 134.63,
134.27, 133.34, 131.65, 129.84, 127.48, 125.80, 123.79, 118.70, 116.87, 115.49,
cis-(2S,4R)-4-[4-[4-[4-[[2-(2,4-Dichlorophenyl)-2-(1H-1,2,4-
triazol-l-ylmethyl)-1,3-dioxolan-4-yl]methoxy]phenyl]-
lpiperazinyl]phenyl]-2,4-dihydro-2-((E)-hex-3-enyl)-3H-1,2,4-
triazol-3-one (7s). This compound was synthesized as a yellowish oil
7372
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Journal of Medicinal Chemistry
ARTICLE
107.84, 78.88, 74.92, 67.86, 67.66, 53.82, 50.80, 49.38, 49.26, 44.25, 27.57, 8.34.
ESI-MS: 747.3 (M + H+), 769.3 (M + Na+). Purity: 94.6%, tR = 5.85 min.
cis-(2S,4R)-4-[4-[4-[4-[[2-(2,4-Dichlorophenyl)-2-(1H-1,2,4-
triazol-l-ylmethyl)-1,3-dioxolan-4-yl]methoxy]phenyl]-
lpiperazinyl]phenyl]-2,4-dihydro-2-(2-(3-methyl-3H-diazirin-
3-yl)ethyl)-3H-1,2,4-triazol-3-one (7w). This compound was synthe-
sized as a yellowish oil from 17a (20.2 mg, 0.031 mmol), 3w (10.3 mg, 0.040
mmol), K2CO3 (8.6 mg, 0.062 mmol), and 18-crown-6 (8.2 mg, 0.031
mmol) in 55% yield by following typical procedure B. 1H NMR (400 MHz,
CDCl3, δH) 8.24 (s, 1H), 7.90 (s, 1H), 7.64 (s, 1H), 7.58 (d, J = 8.4 Hz,
1H), 7.48 (d, J = 2.1 Hz, 1H), 7.45ꢀ7.39 (m, 2H), 7.26 (dd, J = 8.6, 2.1 Hz,
1H), 7.04 (d, J = 9.0 Hz, 2H), 6.94 (d, J = 8.6 Hz, 2H), 6.81 (d, J = 8.7 Hz,
2H), 4.81 (q, J = 14.7 Hz, 2H), 4.43ꢀ4.31 (m, 1H), 3.98ꢀ3.85 (m, 3H),
3.85ꢀ3.75 (m, 2H), 3.49 (dd, J = 9.4, 6.4 Hz, 1H), 3.40ꢀ3.34 (m, 4H),
3.28ꢀ3.20 (m, 4H), 1.81 (t, J = 7.1 Hz, 2H), 1.10 (s, 3H). 13C NMR (100
MHz, CDCl3, δC) 152.84, 152.36, 150.95, 146.23, 136.29, 134.68, 134.28,
133.35, 131.66, 129.84, 127.47, 125.85, 123.95, 118.70, 116.87, 115.51,
107.84, 74.93, 67.88, 67.67, 53.90, 50.80, 49.39, 41.08, 33.91, 24.16, 19.57.
ESI-MS: 731.2 (M + H+), 753.2 (M + Na+). Purity: 93.7%, tR = 9.84 min.
cis-(2S,4R)-4-[4-[4-[4-[[2-(2,4-Dichlorophenyl)-2-(1H-1,2,4-
triazol-l-ylmethyl)-1,3-dioxolan-4-yl]methoxy]phenyl]-
lpiperazinyl]phenyl]-2,4-dihydro-2-(7-(3-methyl-3H-diazirin-
3-yl)heptyl)-3H-1,2,4-triazol-3-one (7x). This compound was syn-
thesized as a yellowish oil from 17a (21.9 mg, 0.034 mmol), 3x (16.4 mg,
0.051 mmol), K2CO3 (9.3 mg, 0.067 mmol), and 18-crown-6 (8.9 mg,
0.034 mmol) in 57% yield by following typical procedure B. 1H NMR
(400 MHz, CDCl3, δH) 8.22 (s, 1H), 7.90 (s, 1H), 7.64ꢀ7.52 (m, 2H),
7.52ꢀ7.33 (m, 3H), 7.25 (dd, J = 8.5, 1.9 Hz, 1H), 7.05ꢀ6.88 (m, 4H),
6.80 (d, J = 8.6 Hz, 2H), 4.80 (q, J = 14.7 Hz, 2H), 4.46ꢀ4.27 (m, 1H),
3.99ꢀ3.71 (m, 5H), 3.57ꢀ3.42 (m, 1H), 3.43ꢀ3.28 (m, 4H), 3.28ꢀ3.15
(m, 4H), 1.78 (dt, J = 14.7, 7.3 Hz, 2H), 1.44ꢀ1.08 (m, 10H), 1.02ꢀ0.92
(m, 3H). 13C NMR (100 MHz, CDCl3, δC) 152.84, 152.33, 150.81,
146.19, 136.28, 134.27, 134.16, 133.34, 131.65, 129.83, 127.47, 126.04,
123.80, 118.70, 116.88, 115.49, 107.84, 74.92, 67.86, 67.66, 53.86, 50.81,
49.41, 45.76, 34.47, 29.21, 29.15, 28.78, 26.59, 24.15, 20.11. ESI-MS:
801.3 (M + H+), 823.3 (M + Na+). Purity: 92.9%, tR = 10.84 min.
HUVEC Culture and Proliferation Assays. Pooled HUVEC
(Lonza) were grown in EGM-2 bullet kit media (Lonza) and used at
passage eight or lower. The proliferation assays were conducted as
previously described.13 GraphPad Prism (v4.00) was used to per-
form statistical comparisons of the IC50 and log(IC50) values for the
itraconazole reference samples and the analogues.
25 min over a ficoll gradient. The viable cell layer formed at the ficoll
boundary was then collected, suspended in PBS, and pelleted by
centrifugation. The resulting pellet was then suspended and cultured
as “neurospheres” in Neurobasal Media-A supplemented with reti-
noic acid deficient B-27 extract (NBMedia). Cells were cultured for
three passages, with neurospheres disaggregated using Accumax
(Innovative Cell Technologies) between passages. Prior to assaying,
CD15 expression was confirmed by flow cytometric analysis. Inhibi-
tion of proliferation and Gli transcription was also confirmed in
response to HhAntag and vismodegib (Supporting Information
Figure S4).
Medulloblastoma Proliferation Assay. Cultured MB neuro-
spheres were disaggregated as described and 1 ꢁ 104 cells were seeded into
wells of a 96-well assay plate in NBMedia and exposed to multiple analogue
concentrations. Relative cell numbers following a 96 h incubation were
quantified by CellTiter 96 AQueous One Solution Cell Proliferation Assay
(Promega) per manufacturer’s recommendations using a SpectraMax M2e
spectrophotometer and SoftMax Pro software (Molecular Devices). Data
was analyzed using Prism5 (GraphPad Software) and IC50 and IC90 values
were determined from doseꢀresponse curves fitted to mean corrected
absorbance normalized to control treatment. GraphPad Prism (v5.0a) was
used to perform statistical comparisons of the IC50 and log(IC50) values for
the itraconazole reference samples and the analogues.
Quantification of Gli1 Transcript by TaqMan Analysis. MB
neurospheres were cultured in NBMedia to confluence in 25 cm3 culture
flasks and exposed for 24 h to analogues at the experimentally determined
IC90 for proliferation. Following drug exposure, cells were washed in PBS
and pelleted by centrifugation at 300g. Cell pellets were lysed in Trizol
reagent (Invitrogen). Total RNA was separated and collected in the
aqueous phase following centrifugation of lysate in the presence of
chloroform. Total RNA was ethanol precipitated and purified over
RNeasy Mini Kit (Qiagen) filter columns per manufacturer’s recom-
mendations. Transcript levels were quantified using gene-specific Taq-
Man primer/probe sets and the StepOne Plus Real-Time PCR system
(Applied Biosystems) on cDNA reverse transcribed using the Quanti-
Tect Reverse Transcription Kit (Qiagen) per manufacturers’ recommen-
dations. Results were quantified using StepOne Plus software v2.1
(Applied Biosystems) and were expressed as fold induction relative to
control samples using the ΔΔCt (2ꢀΔΔCt) method with actin as an
internal control. The primer/probe set (Applied Biosystems) for Taq-
Man PCR for mouse Gli-1 was Mm00494645_m1 and for mouse actin
was Mm00607939_s1. Fold induction values for Gli1 transcript at the
IC90 for MB proliferation were scored on a three-point semiquantitative
scale as follows: ++, e0.15; +, 0.16ꢀ0.44; ꢀ, g0.45.
VEGFR2 Glycosylation. HUVEC were seeded at 5 ꢁ 104 per well
of a 6-well plate in 3 mL of media. After an overnight recovery, the media
was replaced with 2 mL of fresh media and the analogues were added
from 200X stocks in DMSO. Following a 24 h incubation, the media was
aspirated and 2X SDS sample buffer was added to the cells, which were
incubated on ice for 10 min and then boiled for 10 min. The lysate was
then subjected to 6% SDS-PAGE and transferred to PVDF (Bio-Rad)
membranes, which were subsequently blocked in 5% BSA (Sigma) in
TBS-T (10 mM Tris pH 8.0, 150 mM NaCl, 0.05% Tween 20 [Sigma])
and then incubated with 1% anti-VEGFR2 in 1% BSA in TBS-T (Cell
Signaling no. 2749). Following three washes in TBS-T, the membrane
was incubated with antirabbit horseradish peroxidase conjugated IgG
(GE Healthcare) (1:5000ꢀ1:10000 dilution) in 1% BSA in TBS-T. The
membranes were washed three times in TBS-T, incubated for 1ꢀ5 min
with ECL substrate (Immobilon Wester, Milipore), and visualized
(Kodak Image Staion 440 CF).
’ ASSOCIATED CONTENT
S
Supporting Information. Experimental procedures and
b
analytical data for new compounds 3h, 3l, 3nꢀ3t, 3vꢀ3x, 4aꢀ
4n, 5aꢀ5q, 11ꢀ13, and 14aꢀ14c; VEGFR2 glycosylation scor-
ing, characterization of MB culture system, experimentally deter-
mined IC90, and associated ΔΔCt values, and correlations
between Gli1 inhibition and MB proliferation as well as VEGFR2
glycosylation. This material is available free of charge via the
Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Medulloblastoma Culture. MB cultures were derivedfrommouse
Ptchꢀ/+; p53ꢀ/ꢀ MB grown as hind-flank allografts in nude mice
(Harlan). Briefly, tumors were mechanically disrupted and made into
single cellsuspensions bytwo passagesthrougha 70 μm nylon filter. Cells
were pelleted by centrifugation and resuspended in PBS pH 7.4 twice.
The cell suspension was then subjected to centrifugation at 1000g for
Corresponding Author
*Phone: 410-955-4619. Fax: 410-955-4620. E-mail: joliu@jhu.edu.
Author Contributions
These authors contributed equally to the work.
7373
dx.doi.org/10.1021/jm200944b |J. Med. Chem. 2011, 54, 7363–7374

Journal of Medicinal Chemistry
ARTICLE
Notes
The authors report no conflicts of interest.
297, 1559–1561. (c) Ward, R. J.; Lee, L.; Graham, K.; Satkunendran, T.;
Yoshikawa, K.; Ling, E.; Harper, L.; Austin, R.; Nieuwenhuis, E.; Clarke,
I. D.; Hui, C. C.; Dirks, P. B. Multipotent CD15+ cancer stem cells in patched-
1-deficient mouse medulloblastoma. Cancer Res. 2009, 69, 4682–4690.
(11) Romer, J. T.; Kimura, H.; Magdaleno, S.; Sasai, K.; Fuller, C.;
Baines, H.; Connelly, M.; Stewart, C. F.; Gould, S.; Rubin, L. L.; Curran,
T. Suppression of the Shh pathway using a small molecule inhibitor
eliminates medulloblastoma in Ptc1(()p53(ꢀ/ꢀ) mice. Cancer Cell
2004, 6 (3), 229–240.
(12) Von Hoff, D. D.; LoRusso, P. M.; Rudin, C. M.; Reddy, J. C.; Yauch,
R. L.;Tibes, R.;Weiss, G. J.;Borad, M. J.;Hann, C. L.;Brahmer, J. R.;Mackey,
H. M.; Lum, B. L.; Darbonne, W. C.; Marsters, J. C.; de Sauvage, F. J.; Low,
J. A. Inhibition of the Hedgehog Pathway in Advanced Basal-Cell Carcinoma.
New Engl. J. Med. 2009, 361, 1164–1172.
’ ACKNOWLEDGMENT
This work was supported by NCI, FAMRI, the Commonwealth
Foundation, and a SPORE grant under NIH P50 CA058184 (J.O.L.
and C.M.R.) and the NIH Medical Scientist Training Program grant
T32GM07309 (B.A.N.). It was also supported in part by grant no.
UL1 RR 025005 from the National Center for Research Resources
(NCRR), a component of the National Institutes of Health (NIH)
and NIH Roadmap for Medical Research, and its contents are solely
the responsibility of the authors and do not necessarily represent the
official view of NCRR or NIH.
(13) Nacev, B.; Low, W. K.; Huang, Z.; Su, T.; Su, Z.; Alkuraya, H.;
Kasuga, D.; Sun, W.; Trager, M.; Braun, M.; Fischer, G.; Zhang, K.; Liu,
J. O. A calcineurin-independent mechanism of angiogenesis inhibition
by a non-immunosuppressive Cyclosporin A analog. J. Pharmacol. Exp.
Ther. 2011, 338, 466–475.
’ ABBREVIATIONS USED
DIPEA, N,N-diisopropylethylamine; DMSO, dimethyl sulfoxide;
EC50, half maximal effective concentration; HBr, hydrobromic acid; Hh,
hedgehog; HUVEC, human umbilical vein endothelial cells; IC50, half-
maximal inhibitory concentration; IC90, 90% maximal inhibitory con-
centration; Itra, itraconazole (commercial 1:1:1:1 mixture of cis-dia-
stereomers); MB, medulloblastoma; MOMCl, methoxymethyl chlo-
ride; mTORC, mammalian target of rapamycin complex; NMP,
methylpyrrolidone; SAR, structureꢀactivity relationship; VEGFR2,
vascular endothelial growth factor receptor 2; PCR, polymerase chain
reaction
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