Structure-Activity Relationships for Itraconazole-Based Triazolone Analogues as Hedgehog Pathway Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.8b01283

The Food and Drug Administration-approved antifungal agent, itraconazole (ITZ), has been increasingly studied for its novel biological properties. In particular, ITZ inhibits the hedgehog (Hh) signaling pathway and has the potential to serve as an anticancer chemotherapeutic against several Hh-dependent malignancies. We have extended our studies on ITZ analogues as Hh pathway inhibitors through the design, synthesis, and evaluation of novel des-triazole ITZ analogues that incorporate modifications to the triazolone/side chain region of the scaffold. Our overall results suggest that the triazolone/side chain region can be replaced with various functionalities (hydrazine carboxamides and meta-substituted amides) resulting in improved potency when compared to ITZ. Our studies also indicate that the stereochemical orientation of the dioxolane ring is important for both potent Hh pathway inhibition and compound stability. Finally, our studies suggest that the ITZ scaffold can be successfully modified in terms of functionality and stereochemistry to further improve its anti-Hh potency and physicochemical properties.

Full text of DOI:10.1021/acs.jmedchem.8b01283

Subscriber access provided by Universiteit Leiden / LUMC
Article
Structure-Activity Relationships for Itraconazole-Based
Triazolone Analogues as Hedgehog Pathway Inhibitors
Jennifer Pace, Kelly A. Teske, Lianne Chau, Radha Charan Dash,
Angela M. Zaino, Robert Wechsler-Reya, and M. Kyle Hadden
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01283 • Publication Date (Web): 21 Mar 2019
Downloaded from http://pubs.acs.org on March 21, 2019
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Structure-Activity Relationships for Itraconazole-Based
Triazolone Analogues as Hedgehog Pathway Inhibitors
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1†
1‡
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Jennifer R. Pace , Kelly A. Teske , Lianne Q. Chau , Radha Charan Dash , Angela M. Zaino ,
2
1
Robert J. Wechsler-Reya , and M. Kyle Hadden
1
Department of Pharmaceutical Sciences, University of Connecticut, 69 North Eagleville Road,
Unit 3092, Storrs, CT 06269, United States
2
Tumor Initiation and Maintenance Program, NCI-Designated Cancer Center, Sanford Burnham
Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, La Jolla CA 92037, United
States
*To whom correspondence should be addressed. kyle.hadden@uconn.edu Phone: 1-860-486-
446. Fax: 1-860-486-6857.
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ABSTRACT
The FDA-approved antifungal agent, itraconazole (ITZ), has been increasingly studied for its
novel biological properties. In particular, ITZ inhibits the hedgehog (Hh) signaling pathway and
has the potential to serve as an anti-cancer chemotherapeutic against several Hh-dependent
malignancies. We have extended our studies on ITZ analogues as Hh pathway inhibitors through
the design, synthesis, and evaluate of novel des-triazole ITZ analogues that incorporate
modifications to the triazolone/side chain region of the scaffold. Our overall results suggest that
the triazolone/side chain region can be replaced with various functionality (hydrazine
carboxamides and meta-substituted amides) resulting in improved potency when compared to ITZ.
Our studies also indicate that the stereochemical orientation of the dioxolane ring is important for
both potent Hh-pathway inhibition and compound stability. Finally, our studies suggest that the
ITZ scaffold can be successfully modified in terms of functionality and stereochemistry to further
improve its anti-Hh potency and physicochemical properties.
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INTRODUCTION
Itraconazole (ITZ) is an FDA-approved antifungal agent that has several additional
biological activities, including anti-cancer and anti-viral properties.1–3 With respect to its anti-
cancer properties, ITZ inhibits the hedgehog (Hh) pathway, an embryonic cell-signaling cascade
1
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1
responsible for cell proliferation, differentiation, and tissue growth. Aberrant Hh pathway
activation has been identified in a variety of cancers; most notably, basal cell carcinoma (BCC)
4
and medulloblastoma (MB). Previous studies suggest that ITZ inhibits Hh signaling through direct
binding interactions with Smoothened (Smo), a key regulatory protein within the Hh pathway.
These studies also suggest that ITZ binds Smo in a distinct manner from other Hh pathway
inhibitors that function through Smo antagonism.1,5,6,7 A common pitfall for Smo antagonists has
been the emergence of resistance due to mutations in the binding site on Smo;8,9 however, ITZ
maintains potent Hh pathway inhibition in the presence of both wild type and mutant forms of
Smo, presumably through its distinct binding interactions.
N
Linker Region
N
O
2
O
N
4
N
N
2'
O
O
N
N
N
Cl
Cl
1
, ITZ
IC50 = 140 nM
Triazolone/
Side Chain Region
Dioxolane Region
O
O
O
N
O
N
N
N
N
Cl
Cl
2a, cis,des-triazole ITZ
IC50 = 24 nM
2b, trans,des-triazole ITZ
IC50 = 22 nM
Chart 1. Initial SAR for the ITZ scaffold.
ITZ consists of a diverse scaffold that is amenable to modification. Previously, we
performed structure activity relationship (SAR) studies for ITZ, which indicated the optimal
1
0
functionality and stereochemistry for potent Hh pathway inhibition (Chart 1). These SAR studies
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resulted in a novel class of Hh pathway inhibitors: des-triazole ITZ analogues. In addition to
removing the triazole moiety, we identified that the stereochemistry around the dioxolane (4R) is
important for potent Hh pathway inhibition. Our studies also demonstrated that the absolute
stereochemistry of the side chain is less important; however, the methyl of the sec-butyl region is
crucial for maintaining potent Hh pathway inhibition. In addition, several analogues that truncated
or completely removed the triazolone and side chain retained potent Hh pathway inhibition.9
Herein, we present our continued SAR for the des-triazole ITZ scaffold. These studies focus on
the synthesis and evaluation of analogues containing a wide range of structural modifications to
the triazolone/side chain region of lead ITZ analogues 2a and 2b. Our goals with these analogues
were multi-fold. In addition to exploring SAR for this region of the scaffold, we sought to reduce
the number of synthetic steps necessary to generate potent analogues and by extension reduced the
molecular weight of the scaffold. We also sought to evaluate key early stage pharmacokinetic (PK)
parameters for our analogues to ensure that future generations of des-triazole ITZ analogues were
developed with both potency and drug-like properties in mind.
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RESULTS
Chemistry
The preparation of des-triazole ITZ analogues containing a modified triazolone region
starts with direct coupling of the desired dioxolane (3 or 4) with the unprotected linker region (5)
to provide key intermediates 6 or 7.11-13 The dioxolane-containing nitro was reduced to the aniline
in the presence of 10% Pd/C and hydrazine monohydrate (Scheme 1).12,13 These key aniline
intermediates (8 and 9) can be utilized to provide the most efficient synthetic route towards a
diverse set of ITZ analogues that contain modifications to the triazolone/side chain region.
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The majority of des-triazole analogues synthesized and evaluated in this study simplified
the ITZ scaffold by replacing the triazolone and side chain with a substituted aromatic moiety
directly appended to the aniline of 8 or 9. Synthesis towards these amide analogues (10-19) utilized
standard amidation conditions: aniline (8 or 9), activating agent 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride (EDCI·HCl), 4-dimethylaminopyridine
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9
0
(DMAP), and various carboxylic acids stirred in DCM at room temperature for 12 hours (Scheme
2
).14 While the yields obtained with this procedure were less than ideal (< 50%) further
optimization was not undertaken.
Scheme 1. Synthesis of Aniline Intermediatesa
O
+
HO
N
N
NO2
a
O
OTs
Cl
Cl
Cl
3
, trans
4, cis
5
O
O
O
O
b
O
N
N
NO2
O
N
N
NH2
Cl
Cl
Cl
6
, trans
7, cis
8, trans
9, cis
a
Reagents and conditions: (a) Cs CO (10 eq), DMSO, 90 °C, 12 h, 34-85%; (b) 10% Pd/C, hydrazine
2
3
monohydrate, EtOH, reflux, 3.5 h, 42%-94%.
a
Scheme 2. Initial Synthesis of des-Triazole Amide Analogues
O
O
O
a
O
8
or 9
+
O
N
N
N
R
HO
R
H
Cl
Cl
1
0-19
see Table 1 for structrues
aReagents and conditions: (a) EDCI, DMAP, DCM, RT, 12 h, < 50%.
The synthetic route described in Scheme 2 worked well to generate all the amide-
containing analogues with the exception of those incorporating a phenol-substituted phenyl ring.
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5
5
5
6
Under these standard conditions, the amide couplings with the phenol carboxylic acids did not
proceed, presumably because the free phenol becomes activated allowing for multiple side
reactions to occur. Synthesis of these analogues was achieved by protecting the free phenols of the
corresponding aromatic esters with a benzyl ether moiety (Scheme 3a). Ideally, the benzyl (Bn)
0
1
2
3
4
5
6
7
8
9
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2
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8
9
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group could be removed under mild conditions (Pd/C and H ) that would not affect the intact
2
1
4
dioxolane. This synthetic approach provided the desired phenol-substituted analogues; however,
these compounds, except for compound 30, were obtained in very low yields after the final
hydrogenation reaction (< 20%).
Scheme 3. Couplings Procedures for Phenols and Second Generation ITZ Amidesa
(A)
O
O
O
+
a
8
or 9
OH
O
O
N
N
N
H
BnO
OBn
Cl
Cl
20-25
O
O
O
b
O
N
N
N
H
OH
Cl
Cl
26-31
See Table 1 for Structures
(B)
O
O
O
c
8
or 9
+
O
O
N
N
N
R
HO
R
H
Cl
Cl
2
6-36
see Table 1 for structures
a
Reagents and conditions: (a) EDCI, DMAP, DCM, RT, 12 h, < 50%; (b) 10% Pd/C, THF:EtOH, H , <
2
2
0%; (c) HATU, NMM, DMF, RT, 12 h, 8-31%.
In an attempt to obtain higher yields for the analogues incorporating a phenol, we utilized
N-methylmorpholine
(NMM)
and
1-[bis(dimethylamino)methylene]-1H-1,2,3-
triazolo[4,5,6]pyridinium3-oxidhexafluorophosphate (HATU) in dimethyl formamide (Scheme
b).15,16 While obtaining higher yields was unsuccessful, these conditions did allow for an efficient
3
direct coupling of the carboxylic acid of the free phenol to the aniline intermediates (8 or 9) to
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yield our final analogues (26-31). With the increased efficiency of these conditions
(
NMM/HATU), they were also utilized for several subsequent second generation des-triazole ITZ
amide analogues (34-36) in combination with our original amide coupling conditions
EDCI/DMAP).
Table 1. First Generation des-Triazole ITZ Amide Analogues.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
R1
O
N
N
R2
Cmpd
6
R1
R2
Cmpd
16
R1
R2
O
2
O
2
O
4
4
4
O
O
O
O
O
N
H
Cl
Cl
Cl
Cl
Cl
O
2
O
2
O
4
N
7
8
O
17
18
19
26
27
28
29
30
31
O
N
H
O
Cl
Cl
Cl
O
2
O
2
O
O
O
4
4
4
4
4
4
4
4
O
O
N
N
N
O
O
O
O
O
O
O
O
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
O
2
O
2
4
4
4
4
4
9
O
O
N
H
O
O
O
Cl
Cl
O
2
O
OH
O
2
10
O
N
H
O
N
H
Cl
Cl
O
2
O
O
O
2
OH
1
1
1
2
O
N
O
N
H
H
Cl
Cl
O
O
2
O
O
O
O
O
2
N
O
O
N
H
N
H
O
Cl
Cl
Cl
OH
OH
OH
O
2
O
2
O
OH
13
O
N
H
N
N
O
N
H
O
Cl
Cl
Cl
O
2
O
O
2
4
4
1
1
4
5
O
N
H
O
N
H
O
Cl
Cl
Cl
O
2
O
O
2
4
O
N
H
O
N
H
O
O
Cl
Cl
Cl
Cl
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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5
5
5
5
6
Des-triazole ITZ analogues containing carbamate, hydrazine carboxamide, or an
unsubstituted triazolone were synthesized in a similar manner as previously described for the
synthesis of ITZ and other analogues (Scheme 4).11,12 The carbamates 37 and 38 were generated
through the addition of phenyl chloroformate to anilines 8 and 9, respectively. Addition of
hydrazine monohydrate to the carbamates provided the hydrazine carboxamides 39 and 40.
Finally, the intact triazolones were generated following the addition of formamidine acetate to
yield 41 and 42.11,12
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 2. Second Generation des-ITZ Amide Analogues.
R1
O
N
N
R2
Compound
R
R2
Compound
35
R1
R2
1
O
2
O
4
4
4
2
4
4
3
2
O
O
Cl
Cl
Cl
Cl
O
2
Cl
Cl
Cl
O
2
33
O
36
O
Cl
O
2
Cl
3
4
O
Cl
Scheme 4. Synthesis of des-Triazole Triazolone Precursorsa
O
O
a
O
b
8 or 9
O
N
N
N
H
O
Cl
N
Cl
3
3
7, trans
8, cis
O
O
O
O
O
c
O
NH
N
O
N
N
NHNH2
O
N
N
N
H
Cl
Cl
Cl
Cl
3
9, trans
41, trans
42, cis
40, cis
a
Reagents and conditions: (a) Pyr (17 eq), ClCOOPh (1.1 eq), 3 h, 78-81%; (b) NH NH -H O (5.5 eq),
2
2
2
reflux, 3 h, 88-89%; (c) formamidine acetate (4.5 eq), acetic acid, reflux, 3 h, 20-46%.
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Biological Evaluation
Hh inhibition studies. All des-triazole ITZ analogues were initially evaluated for Hh pathway
inhibition by monitoring endogenous Gli1 mRNA levels in the murine BCC cell line ASZ-001.
Our previous SAR studies indicated that the ASZ cells represent a more appropriate cellular model
for measuring des-triazole ITZ analogue-mediated Hh pathway inhibition compared to the
C3H10T1/2 and NIH-3T3 mouse embryonic fibroblasts (MEFs) commonly used for evaluating
inhibition of Hh signaling. More specifically, the ability of lead analogues 2a and 2b to down-
regulate Gli1 mRNA expression in the ASZ cell line correlated more closely with our results in
the primary Hh-dependent murine MB cells isolated from conditional patched knockout (Math1-
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
fl/fl
17-19
Cre;Ptc , hereafter termed Ptch-CKO) mice.
As shown above for lead analogue 2, our initial SAR studies suggested that the 4R-trans
9
orientation around the dioxolane was optimal for potent Hh pathway inhibition. In addition, our
compounds suggested that truncation of the triazolone region of the scaffold was tolerated. With
this in mind, our initial plan for this generation of ITZ analogues was to synthesize and evaluate a
series of compounds in which the triazolone moiety was replaced with substituted phenyl groups
directly appended to the linker region through an amide bond. Substituted amides were chosen
based on their general stability (chemical and biological), enhanced solubility, and ease of
synthesis.
Our first series of des-triazole ITZ analogues focused on analogues that contained the
1
0
2R,4R-trans-dioxolane for the reasons noted above (6, 8, 10-14, 26-28, Table 1). Both truncated
analogues (6 and 8) exhibited moderate Hh inhibition (IC = 0.31 µM and 0.9 µM, respectively);
5
0
however, the majority of compounds containing an amide in place of the triazolone were inactive
at the concentrations tested (Table 3). Despite reduced potency within this trans series of des-
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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
triazole ITZ amides, the SAR data did indicate that substitutions in the meta-position of the phenyl
ring were most favorable as 13 (meta-pyridyl) was moderately active (IC = 4.3 µM) and 27
5
0
(meta-phenol) demonstrated potent Hh inhibition (IC = 0.19 µM) compared to the ortho- and
5
0
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
para-substituted analogues. These results suggest that functional groups capable of hydrogen
bonding at the meta-position may be advantageous to binding with Smo, the hypothesized target
of ITZ and des-triazole ITZ analogues.
Table 3. In Vitro Activity of First Generation des-ITZ Amide Analogues
a, b
a, b
Compound
IC (µM)
Ptch-CKO
Compound IC (µM)
Ptch-CKO
5
0
50
a,b
a,b
GI (µM)
GI (µM)
50
50
6
7
8
9
0.31 ± 0.04
0.26 ± 0.04
0.92 ± 0.04
0.39 ± 0.09
>10
>10
>10
4.3 ± 0.2
>10
---
---
---
---
---
---
---
>10
---
---
16
17
18
19
26
27
28
29
30
31
>10
---
---
1.1 ± 0.6
---
---
2.8 ± 1.5
---
3.9 ± 0.3
0.19 ± 0.09
>10
>10
0.19 ± 0.01
>10
2.2 ± 0.3
0.16 ± 0.03
3.0 ± 0.1
1
1
0
1
12
1
1
1
3
4
5
---
0.35 ± 0.06
>10
---
aIC50 values represent the Mean ± SEM of at least two separate experiments performed in triplicate. All analogues
b
evaluated following 48 h incubation.
As noted, we initially focused our synthesis and evaluation of ITZ amides on the trans-
dioxolane because of the activity of this stereochemical orientation in our first generation series;10
however, several cis-dioxolane ITZ analogues also demonstrated potent Hh inhibition. With this
in mind, we prepared cis-dioxolane amides with the same substituents to determine what effect
this modification would have on Hh inhibitory activity of the scaffold (7, 9, 15-19, 29-31, Table
1
). Interestingly, each of the cis-dioxolane analogues was more potent than its corresponding trans-
dioxolane suggesting that this orientation was preferred when the triazolone was substituted with
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the simplified aromatic moiety. In addition, analogues containing the meta-pyridyl (18, IC = 0.19
5
0
µM) or meta-phenol (30, IC = 0.16 µM) were the most potent for the cis-dioxolane.
5
0
Based on the SAR generated from our first series of amide-containing ITZ analogues, we
synthesized and evaluated several more compounds that incorporated the cis-dioxolane and a
meta-substituted aromatic moiety (Table 3). These analogues were designed to further probe the
sterics, electronics, and hydrogen bonding of the substituents in the meta-position. Analogues
containing a furan (35) or thiophene (36) were inactive, suggesting 5-membered heterocycles are
potentially less active (Table 4). Masking the phenol of 30 as the methoxy (33) significantly
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
decreased potency, supporting a role for the free phenol in the meta-position (IC values = 0.16
50
µM and 5.7 µM, respectively). The most potent analogue in this second series, and the most potent
amide analogue we prepared, was 34 (IC = 0.089 µM), which contains the meta-acetyl phenyl
5
0
ring. Based on our results, potent inhibition of the Hh pathway can be correlated to the ability to
hydrogen bond and/or increased polarity at the meta-position of the phenyl ring for cis-dioxolane
analogues that incorporate an amide moiety in place of the triazolone/side chain.
Table 4. In Vitro Evaluation of Second Generation des-ITZ Amide Analogues
Ptch-CKO
a, b
Compound
IC (µM)
50
a,b
GI (µM)
5
0
3
3
3
35
36
2
3
4
>10
---
---
1.0 ± 0.3
5.7 ± 1.9
0.089 ± 0.01
>10
---
---
>10
aIC50 values represent the Mean ± SEM of at least two separate experiments performed in triplicate. All analogues
b
evaluated following 48 h incubation.
In parallel with our studies of ITZ analogues that contain amides as isosteres of the
triazolone ring, we synthesized and evaluated the stereochemically defined unsubstituted
triazolones (41 and 42). Our earlier SAR demonstrated that the side chain was not required for Hh
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2
2
2
2
2
2
2
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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
10
inhibitory activity, but the defined cis- and trans-dioxolane des-triazole analogues were not
evaluated (40 and 41). We also explored the Hh inhibitory activity of several synthetic precursors
to the triazolone (37-40). In addition to a shorter synthetic route, the functional groups on these
precursors may aid in improving the overall drug-like properties of this scaffold. Carbamates have
been widely studied within the field of medicinal chemistry and are key structural motifs in many
FDA-approved compounds and prodrugs because they offer chemical stability and enhanced cell
permeability.10 Hydrazine carboxamides are derivatives of urea and have thus been studied
extensively within the field of drug development and may aid in hydrogen-bonding and
solubility.20 Not surprisingly, both of the intact triazolones (41 and 42) were potent inhibitors of
Hh signaling (IC values = 0.14 and 0.21 µM, respectively), similar to our previous results for the
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
9
undefined triazolone analogue (IC = 0.12 µM ) (Table 5). The most potent analogues for the
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triazolones and precursors were the two hydrazine carboxamides (39 and 40) and similar to the
ITZ amides, the cis-hydrazine was more potent than the corresponding trans-analogue. In fact, the
most potent analogue generated through these studies was the cis-carboxamide (40, IC = 0.009
5
0
µM). Based on our results from the ITZ amide analogues, the enhanced inhibitory activity for the
carboxamides was not surprising given their increased polarity and hydrogen bond
donating/accepting properties. In addition, the carboxamide is an acyclic functionality, providing
strong evidence that anti-Hh activity can still be achieved without the cyclic functionality of the
triazolone and aromatic isosteres. The carbamate analogues were inactive, which may be attributed
to the presence of the bulky non-substituted hydrophobic ring.
Based on their activity in ASZ cells, several compounds were evaluated for their anti-
proliferative activity in Hh-dependent MB Ptch-CKO cells. Overall, the ability of our ITZ
analogues to reduce the growth of the Ptch-CKO cells was greatly reduced compared to their anti-
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Hh activity in the ASZ cell line. The one compound that did not follow this trend was 30, which
contains the meta-phenol (GI = 0.35 µM). Similar to the ASZ cells, the cis-amide analogues 18
5
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and 30 were more potent than the corresponding trans-amides, 13 and 27. Interestingly, this
activity trend did not hold for the carboxamides 39 and 40; for these analogues, the trans-dioxolane
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
0 was more potent in the Ptch-CKO cells. This difference in anti-Hh activity could be attributed
to differing permeability across the various cellular models or because down-regulation of Gli1
does not directly result in anti-proliferative activity in either of these cell models.
Table 5. In Vitro Evaluation of des-ITZ Intermediate Analogues
a, b
Compound
IC (µM)
Ptch-CKO
5
0
a,b
GI (µM)
5
0
3
3
3
4
7
8
9
0
>10
>10
0.11 ± 0.01
0.009 ± 0.0002
0.14 ± 0.03
---
---
0.5 ± 0.4
2.8 ± 0.9
---
41
4
2
0.21 ± 0.04
---
aIC50 values represent the Mean ± SEM of at least two separate experiments performed in triplicate. All analogues
b
evaluated following 48 h incubation.
Preliminary In Vitro Pharmacokinetic Assays. Several representative compounds were chosen for
evaluation in a series of preliminary pharmacokinetic (PK) assays (Table 6). The amide
substituents did not enhance solubility as all these analogues were significantly less soluble than
ITZ and our initial analogues (2a and 2b). Interestingly, the trans isomers (13, 27, and 39)
demonstrated enhanced metabolic stability compared to the cis analogues (18, 30, 34, and 40),
suggesting that the stereochemistry around the dioxolane moiety plays a role in compound
metabolism and clearance. We previously verified that removal of the triazole abolishes inhibition
of CYP3A4 for the ITZ scaffold and this also translates to the des-triazole ITZ analogues prepared
in our current series. Neither ITZ nor any of the analogues evaluated demonstrated the ability to
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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
passively diffuse through a hexadecane membrane in a parallel artificial membrane permeability
assay (PAMPA). Finally, as these compounds are being developed to target MB, we evaluated
whether ITZ and several analogues are substrates of the two primary efflux pumps in the blood
brain barrier, Breast Cancer Resistance Protein (BCRP) and P-glycoprotein (P-gp). ITZ was not a
substrate for either efflux pump; however, none of the analogues evaluated were soluble enough
under the assay conditions to determine efflux parameters. Interestingly, our results demonstrating
that ITZ is not a P-gp substrate are different than those previously reported, in which P-gp efflux
was identified as a mechanism through which ITZ is actively removed from mouse brain.20
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 6. Pharmacokinetic Evaluation of des-Triazole ITZ Analogues.a
T
1/2
hERG
Efflux Pump
Substrates
Solubility
Clearance
(mL/min/mg)
CYP3A4
(µM)
PAMPA
-log P
e
a,b
(HLM,
min)
Inhibition
a
a
a
a
(µM)
a
a
(%)
BCRP
P-gp
No
ND
ND
ND
ND
---
ITZ
2a
2b
13
18
27
0.8
0.8
1.56
0.0042
0.0069
0.0014
0.0016
0.0014
0.0093
0.0014
27.0 ± 6.2
---
---
---
---
0.04
>20
1.02
>30
>20
>30
11.4
>30
>30
>30
< 1
ND
ND
ND
< 1
1.2
< 1
< 1
2.2
ND
8.8 ± 0.2
7.7 ± 0.2
9.0 ± 0.1
7.9 ± 0.03
8.0 ± 0.2
---
No
b
ND
---
ND
ND
ND
---
59.9 ± 2.0
8.9 ± 0.03
>231
19.9 ± 0.6
11.4 ± 0.1
20.1 ± 0.6
11.5 ± 0.1
11.6 ± 0.4
78.3 ± 0.2
< 3
34.7 ± 1.0
61.0 ± 0.3
34.5 ± 0.9
60.4 ± 0.7
3
3
3
4
0
4
9
0
7.6 ± 0.2
---
ND
---
ND
---
---
---
---
---
---
---
a
b
Additional experimental information for all assays provided in the supplementary information. ND = Not determined
based on poor compound solubility under the assay conditions.
Computational Modeling
We recently developed a homology model of Smo and used it to perform docking and molecular
dynamics studies for the structurally related triazole anti-fungal posaconazole (PSZ) and several
7
PSZ analogues. These studies provided strong evidence that the triazolone/side chain region of
the scaffold penetrates deeply into the Smo binding site on the seven-transmembrane domain (7-
TM) while the dioxolane/triazole region extends out of the binding pocket and is solvent exposed.
This binding conformation also supports the in vitro SAR that determined for both of these
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scaffolds with respect to Hh inhibition. To explore whether our amide-based ITZ analogues adopt
the same binding conformation in the Smo 7-TM we performed docking studies for 30 and 34 with
our homology model (Figure 1).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 1. Predicted binding mode of 30 and 34 in complex with Smo. Compared to 30, compound
3
4 penetrates more deeply inside the binding pocket (A). Key intermolecular interactions between
Smo:34 (B) and Smo:30 (C) include a network of π –π (orange mesh), cation–π (pink mesh) and
hydrogen bonds (yellow dotted line). The pdb file of our homology model of Smo is included in
the Supplementary Information.
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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
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5
5
5
5
6
Compounds 30 and 34 adopt similar orientations and form comparable binding interactions
with Smo inside the previously determined binding pocket. The dichlorophenyl ring is oriented
towards the extracellular surface with the linker/amide region penetrating deeply into the Smo
binding pocket. Interestingly, the dioxolane region of 30 is significantly more exposed to the solvent
accessible surface of the binding pocket when compared to compound 34, which penetrates more
deeply into the binding grove. Our docking studies predict that the shallower penetration of
compound 30 is due to a hydrogen bond between the meta-phenol and the side chain of E518; an
intermolecular interaction not present in the Smo:34 structure. The dioxolane and dicholorophenyl
rings of both compounds are primarily stabilized by a network of cation–π interactions with the
positively charged basic side chain residues (K395 and R296) present at the opening of the binding
site. The biaryl piperazine groups of 30 and 34 exhibit favorable non-polar interactions with
several hydrophobic side chains in the binding pocket (I215, M301, F222, V386 and L515). The
deeper penetration of analogue 34 allows for a cation–π interaction between the protonated
nitrogen of the piperazine and the side chain of F484, an additional intermolecular interaction that
may underlie its enhanced activity. The amide nitrogen of both compounds forms a hydrogen bond
with D384, a linker and phenyl moiety of both the compounds demonstrate a network of π-π
stacking and hydrogen bonds with Y394 and D384, respectively.
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
DISCUSSION AND CONCLUSIONS
We have extended our studies on des-triazole ITZ analogues as Hh pathway inhibitors by
designing, synthesizing, and evaluating a new series of compounds that incorporate extensive
modifications to the triazolone/side chain region of the scaffold. Our primary goal for these
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analogues was three-fold. First, we sought to further probe the sterics, electronics, and hydrogen
bonding requirements in this region of the ITZ scaffold for optimal anti-Hh potency. Second, we
wanted to identify a simplified triazolone mimic that could be prepared more rapidly than the
triazolone/side chain region of the parent scaffold. Finally, we evaluated preliminary PK properties
of our most potent analogues to determine how our modifications affect drug-like properties of the
scaffold and to determine what potential PK liabilities should be addressed in subsequent
compounds. Interestingly, des-triazole analogues that simplified the triazolone/side chain to an
aromatic amide demonstrated a preference for the 2S,4R-cis-orientation around the dioxolane ring.
The most potent des-triazole ITZ amide analogues contained the 4R-cis-dioxolane region and
meta-substitutions on the phenyl ring. In addition, the anti-Hh activity of the scaffold increases
with increasing polarity at the meta-position. The most potent amide analogues incorporate either
a meta-phenol (30) or meta-acetyl (34) functional group. In addition to the amide-containing
compounds, several analogues that truncate the triazolone of ITZ were potent inhibitors of the Hh
pathway, verifying that this region of the scaffold is amenable to a wide range of structural
modifications. The most potent analogue in the ASZ cell line was the cis-carboxamide 40. Finally,
these compounds were significantly more potent in the immortalized murine ASZ BCC cell line
compared to the primary murine Ptch-CKO cells. We previously noted a similar difference in
cellular activity for the ITZ scaffold across several Hh-dependent cell lines, highlighting the
importance of comparing activity across several model systems.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
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
As many of our analogues exhibit potent inhibition of Hh activity, a key factor for
delineating between their potential as therapeutic agents relies on which compounds exhibit
improved drug-like properties. This is especially important for this scaffold, considering ITZ is a
‘large’ small molecule (MW = 705 g/mol) and is poorly soluble. Unfortunately, the majority of
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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
our compounds were less soluble than ITZ and the most potent (30, 34, 40) demonstrated
comparable or reduced metabolic stability compared to ITZ. Not surprisingly, each analogue was
also poorly permeable and solubility issues prevented us from determining whether the analogues
were substrates of the two primary efflux pumps in the blood brain barrier. Taken together, these
results highlight the potential of the ITZ scaffold to treat Hh-dependent cancers while also
demonstrating the need for continued improvements in the drug-like properties and formulation of
the ITZ scaffold.
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
EXPERIMENTAL SECTION
Chemical Synthesis.
General Information. All chemicals were purchased from either Sigma-Aldrich or Fisher
Scientific. ACS grade methanol, ethyl acetate, toluene, anhydrous DMF, DCM, and DMSO were
purchased from Fisher Scientific or Sigma-Aldrich. All reactions were performed under an argon
atmosphere. NMR data were collected on a Bruker AVANCE 500 MHz spectrometer, and
analysis was carried out using MestReNova. HRMS data was obtained at the Mass Spectrometry
Facility at the University of Connecticut. FT-IR analysis was carried out on a Bruker Alpha
Platinum ATR instrument using OPUS software (v7.2). The preparation of previously
characterized ITZ intermediates (3-5) followed known procedures with minor
modifications.11,12,21,22 All ITZ analogues evaluated in the biological assays were greater than 95%
pure based on the HPLC methods described below.
Purity Analysis of Final Analogues. Purity analysis of all final des-triazole ITZ analogues was
determined via one of the methods described below.
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Method A. ITZ analogues were dissolved in HPLC-grade MeCN and injected (25 μL of a 1 mM
solution) into an Agilent Manual FL-Injection 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
1
1
1
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1
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0
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9
0
Photodiode Array Detector. The mobile phase consisted of 70% MeCN/30% H O. All analogues
2
were run at a flow rate of 1.0 mL/min for 20 min and purity was assessed at 254 nm.
Method B. ITZ analogues were dissolved in HPLC-grade MeCN and injected (25 μL of a 1mM
solution) 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 was a gradient of 25% - 100% MeCN (0.1 % formic acid) in
H O over 75 min. All analogues were run at a flow rate of 0.7 mL/min for 30 min and purity was
2
assessed at 254 nm.
1
-(4-(((2R,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4-yl)methoxy)phenyl)-4-(4-
nitrophenyl)piperazine (6). To a solution of 5 (0.77 g, 2.61 mmol) in DMSO (30 mL) was added
Cs CO (8.5 g, 26.14 mmol) and 3 (1.2 g, 2.87 mmol). The solution was warmed to 90 °C and
2
3
stirred for 12 h. The mixture was cooled to room temperature and water was added slowly with
vigorous stirring (~20 mL). A yellow precipitate formed, which was filtered and recrystallized in
1
EtOH to yield 6 (1.2 g, 85%). H NMR (500 MHz, Chloroform-d) δ 8.20 (d, J = 9.4 Hz, 2H), 7.66
(d, J = 8.4 Hz, 1H), 7.46 (d, J = 2.1 Hz, 1H), 7.28 (d, J = 8.5 Hz, 1H), 7.03 – 6.87 (m, 6H), 4.37
(h, J = 5.4 Hz, 1H), 4.16 (dd, J = 9.5, 5.1 Hz, 1H), 4.11 – 3.95 (m, 2H), 3.95 – 3.84 (m, 1H), 3.69
1
3
–
3.53 (m, 4H), 3.35 – 3.18 (m, 4H), 1.86 (s, 3H). C NMR (126 MHz, CDCl ) δ 154.79, 153.29,
3
146.68, 138.10, 134.59, 132.83, 131.21, 128.89, 126.79, 125.98 (2), 118.58 (2), 115.56 (2), 112.90
(2), 109.99, 109.12, 73.94, 69.27, 66.99, 50.39 (2), 47.31 (2), 25.73. DART-HRMS: m/z calcd. for
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+
C H Cl N O [MH] , 544.1406; Found: 544.1388. IR (solid) vmax: 3060, 2926, 2880, 2828,
2
7
27
2
3
5
1
556, 1507, 1485, 1446, 1376, 1222, 1190, 1142, 1047, 995, 942, 821, 737. Purity, Method A:
95.2%.
0
1
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3
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
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0
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0
1
-(4-(((2S,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4-yl)methoxy)phenyl)-4-(4-
nitrophenyl)piperazine (7). To a solution of 5 (260 mg, 0.87 mmol) in DMSO (20 mL) was added
Cs CO (3.1 g, 9.58 mmol) and 4 (400 mg, 0.0958 mmol). The solution was warmed to 90 °C and
2
3
stirred for 12 h. The mixture was cooled to room temperature and water was added slowly with
vigorous stirring (~10 mL). A yellow precipitate formed, which was filtered and recrystallized in
1
EtOH to yield 8 (175 mg, 34%). H NMR (500 MHz, Chloroform-d) δ 8.20 (d, J = 9.4 Hz, 2H),
7
.68 (d, J = 8.5 Hz, 1H), 7.42 (d, J = 2.1 Hz, 1H), 7.24 (d, J = 8.5 Hz, 1H), 6.93 (dd, J = 9.1, 5.4
Hz, 4H), 6.78 (d, J = 9.0 Hz, 2H), 4.65 (p, J = 6.1 Hz, 1H), 4.35 (dd, J = 8.6, 6.4 Hz, 1H), 3.99
(dd, J = 9.7, 5.4 Hz, 1H), 3.81 – 3.76 (m, 2H), 3.64 – 3.59 (m, 4H), 3.29 – 3.23 (m, 4H), 1.83 (s,
13
3
1
5
H). C NMR (126 MHz, CDCl ) δ 154.78, 153.09, 145.50, 139.34, 138.76, 134.35, 132.82,
3
30.97, 128.55, 126.70, 125.98 (2), 118.53 (2), 115.31 (2), 112.89 (2), 109.18, 75.05, 68.41, 67.33,
+
0.38 (2), 47.30 (2), 25.90. DART-HRMS: m/z calcd. for C H Cl N O [MH] , 544.1406;
27 27
2
3
5
Found: 544.1409. IR (solid) vmax: 2922, 2850, 1596, 1555, 1509, 1450, 1375, 1321, 1228, 1195,
151, 1036, 944, 827, 753. Purity, Method B: 95.1%
1
4-(4-(4-(((2R,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4-
yl)methoxy)phenyl)piperazin-1-yl)aniline (8). 10% palladium on carbon (24.4 mg, 5% mole
ratio) was added to a dry round bottom flask. Ethanol (100 mL) was added followed by slow
addition of 6 (250 mg, 0.459 mmol). Hydrazine monohydrate (0.14 mL, 4.59 mmol) was added
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dropwise and the mixture was stirred at reflux for 2 h. Upon cooling to RT, the mixture was
filtered through celite. The celite was washed with ethanol (500 mL) to ensure complete elution of
the aniline. The filtrate was concentrated to afford a yellow solid, which was recrystallized in
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
EtOH to afford 8 (100 mg, 42%). H NMR (500 MHz, Chloroform-d) δ 8.16 (d, J = 9.2 Hz, 1H),
7
.61 (d, J = 8.4 Hz, 1H), 7.41 (t, J = 1.9 Hz, 1H), 7.23 (d, J = 8.5 Hz, 1H), 6.94 (dd, J = 9.3, 3.0
Hz, 2H), 6.91 – 6.83 (m, 4H), 6.68 (d, J = 8.6 Hz, 1H), 4.33 (d, J = 6.9 Hz, 1H), 4.12 (dd, J = 9.5,
5
–
1
.1 Hz, 1H), 4.08 – 3.89 (m, 2H), 3.89 – 3.78 (m, 1H), 3.58 (t, J = 5.2 Hz, 2H), 3.45 (s, 1H), 3.31
1
3
3.11 (m, 6H), 1.82 (s, 3H). C NMR (126 MHz, CDCl ) δ 152.77, 146.21, 144.46, 132.83,
3
31.20, 128.91, 126.79, 125.99, 118.85 (2), 118.59, 118.25 (2), 116.23 (2), 115.56, 115.46 (2),
112.90, 73.97, 69.29, 67.04, 51.25 (2), 50.86 (2), 25.74. DART-HRMS: m/z calcd. for
+
C H Cl N O [MH] , 514.1664; Found: 514.1648. IR (solid) vmax: 2919, 2850, 1595, 1560,
2
7
29
2
3
3
1
510, 1464, 1450, 1375, 1228, 1196, 1150, 1037, 877, 825, 754. Purity, Method A: 98.0%.
4
-(4-(4-(((2S,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4-
yl)methoxy)phenyl)piperazin-1-yl)aniline (9). 10% palladium on carbon (10 mg, 5% mole ratio)
was added to a dry round bottom flask. Ethanol (50 mL) was added followed by slow addition of
8
(100 mg, 0.184 mmol). Hydrazine monohydrate (0.06 mL, 1.84 mmol) was added dropwise and
the mixture was stirred at reflux for 2 h. Upon cooling to RT, the mixture was filtered through
celite. The celite was washed with ethanol (300 mL) to ensure complete elution of the aniline. The
filtrate was concentrated to afford a yellow solid, which was recrystallized in EtOH to afford 9 (89
1
mg, 94%). H NMR (500 MHz, Chloroform-d) δ 8.20 (d, J = 8.8 Hz, 1H), 7.68 (d, J = 8.7 Hz,
1
H), 7.43 (s, 1H), 7.24 (d, J = 8.6 Hz, 1H), 6.92 (q, J = 8.5 Hz, 4H), 6.81 – 6.68 (m, 3H), 4.70 –
4.59 (m, 1H), 4.35 (t, J = 7.4 Hz, 1H), 3.99 (s, 1H), 3.78 (d, J = 6.8 Hz, 2H), 3.61 (t, J = 5.2 Hz,
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13
2
1
H), 3.49 (s, 1H), 3.24 (d, J = 8.2 Hz, 6H), 1.83 (s, 3H). C NMR (126 MHz, CDCl ) δ 140.38,
3
39.34, 136.37, 134.35, 130.97, 128.59, 128.35, 126.70, 125.99, 118.85 (2), 118.54, 118.21 (2),
116.24 (2), 115.21(2), 112.90, 75.07, 68.43, 67.40, 51.24 (2), 50.85 (2), 25.90. DART-HRMS: m/z
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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9
0
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8
9
0
1
2
3
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8
9
0
+
calcd. for C H Cl N O [MH] , 514.1664; Found: 514.1666. IR (solid) vmax: 2921, 2823, 1590,
2
7
29
2
3
3
1
510, 1450, 1375, 1321, 1227, 1193, 1149, 1037, 944, 824, 753. Purity, Method B: 95.2%
General Amide Coupling Procedures.
Method A. Aniline 8 or 9 (20 mg, 0.039 mmol), carboxylic acid (0.117 mmol), EDCI (0.117 mmol),
and DMAP (0.117 mmol) were dissolved in anhydrous DCM (10 mL) and stirred under argon for
12 h at room temperature. The crude reaction was immediately purified via column
chromatography (SiO , 0-80% acetone in hexanes) to afford the ITZ amide analogue.
2
Method B. Acid (0.038 mmol) and HATU (0.083 mmol) were added to a round bottom flask.
Anhydrous DMF was added (5 mL) followed by NMM (0.15 mmol); and this mixture was stirred
at room temperature for 30 mins. Aniline 8 or 9 (20 mg, 0.038 mmol) in anhydrous DMF (4 mL)
was added to the mixture and stirred for 12 h at room temperature. The solution was added to ice
cold water and washed with EtOAc (3 x 20 mL). The organic layers were combined and washed
with H O (2 x 20 mL) and brine (1 x 20 mL). The organic layers were dried over Na SO and
2
2
4
evaporated to dryness. Crude products was purified via preparative thin layer chromatography
Analtech Uniplate 20x20 cm 2000 micron, 3% acetone in hexanes) or column chromatography
(
(SiO , 0-80% acetone in hexanes) to afford the ITZ amide analogue.
2
N-(4-(4-(4-(((2R,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4-
yl)methoxy)phenyl)piperazin-1-yl)phenyl)benzamide (10). Amide coupling method A; Yield =
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3.0 mg, 54%. H NMR (500 MHz, Chloroform-d) δ 7.92 (d, J = 7.3 Hz, 2H), 7.73 (s, 1H), 7.66
d, J = 8.4 Hz, 1H), 7.63 – 7.57 (m, 3H), 7.54 (t, J = 7.5 Hz, 2H), 7.46 (d, J = 2.1 Hz, 1H), 7.28
(dd, J = 8.4, 2.0 Hz, 1H), 7.05 (d, J = 8.6 Hz, 2H), 7.00 (d, J = 9.0 Hz, 2H), 6.94 (d, J = 9.0 Hz,
H), 4.37 (q, J = 5.5 Hz, 1H), 4.17 (dd, J = 9.5, 5.0 Hz, 1H), 4.09 – 3.98 (m, 2H), 3.89 (t, J = 7.7
(
1
1
1
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1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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8
9
0
1
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8
9
0
1
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8
9
0
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6
7
8
9
0
2
1
3
Hz, 1H), 3.41 – 3.33 (m, 4H), 3.33 – 3.22 (m, 4H), 1.86 (s, 3H). C NMR (126 MHz, CDCl ) δ
3
1
1
6
65.48, 152.92, 148.50, 146.09, 138.15, 135.17, 134.56, 132.83, 131.69, 131.19, 130.61, 128.91,
28.78 (2), 126.95, 126.79 (2), 121.64 (2), 118.40 (2), 116.95 (2), 115.48 (2), 109.09, 73.96, 69.29,
+
7.03, 50.74 (2), 49.88 (2), 25.74.DART-HRMS: m/z calcd. for C H Cl N O [MH] , 618.1926;
3
4
33
2
3
4
Found: 618.1919. IR (solid) vmax: 3367, 3333, 2958, 2924, 2853, 1693, 1638, 1547, 1511, 1450,
1375, 1248, 1230, 1194, 1157, 1037, 941, 822, 703. Purity, Method A: 96.2%
N-(4-(4-(4-(((2R,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4-
yl)methoxy)phenyl)piperazin-1-yl)phenyl)-2-naphthamide (11). Amide coupling method A;
1
Yield = 6.0 mg, 23%. H NMR (500 MHz, Chloroform-d) δ 8.39 (d, J = 8.0 Hz, 1H), 7.97 (d, J =
8
2
.3 Hz, 1H), 7.90 (d, J = 6.9 Hz, 1H), 7.75 (d, J = 7.0 Hz, 1H), 7.66 – 7.48 (m, 7H), 7.41 (d, J =
.1 Hz, 1H), 7.24 (dd, J = 8.4, 2.1 Hz, 1H), 7.03 (d, J = 8.8 Hz, 2H), 6.96 (d, J = 9.0 Hz, 2H), 6.90
(
(
(
d, J = 9.1 Hz, 2H), 4.32 (dq, J = 11.4, 6.5, 5.6 Hz, 1H), 4.12 (dd, J = 9.5, 5.1 Hz, 1H), 4.05 – 3.93
1
3
m, 2H), 3.89 – 3.81 (m, 1H), 3.38 – 3.30 (m, 4H), 3.29 – 3.20 (m, 4H), 1.82 (s, 3H). C NMR
126 MHz, CDCl ) δ 167.31, 152.94, 148.59, 146.09, 138.15, 134.75, 134.56, 133.81, 132.84,
3
131.20, 130.94, 130.75, 130.17, 128.91, 128.43, 127.33, 126.79, 126.59, 125.38, 125.02, 124.79,
1
2
21.41 (2), 118.41 (2), 117.01 (2), 115.49 (2), 109.09, 73.96, 69.29, 67.03, 50.74 (2), 49.92 (2),
+
5.74. DART-HRMS: m/z calcd. for C H Cl N O [MH] , 668.2083; Found: 668.2075. IR
3
8
35
2
3
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(
solid) vmax: 3294, 3247, 3042, 2985, 2873, 1639, 1586, 1509, 1450, 1373, 1226, 1192, 1146,
1
035, 942, 821, 733. Purity, Method A: 97.0%.
0
1
2
3
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6
7
8
9
0
1
2
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8
9
0
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N-(4-(4-(4-(((2R,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4-
yl)methoxy)phenyl)piperazin-1-yl)phenyl)picolinamide (12). Amide coupling method A; Yield
1
= 9.0 mg, 37. H NMR (500 MHz, Chloroform-d) δ 9.91 (s, 1H), 8.61 (d, J = 4.7 Hz, 1H), 8.30 (d,
J = 7.8 Hz, 1H), 7.90 (t, J = 7.7 Hz, 1H), 7.71 (d, J = 8.8 Hz, 2H), 7.62 (d, J = 8.4 Hz, 1H), 7.50 –
7
.43 (m, 1H), 7.41 (d, J = 2.1 Hz, 1H), 7.23 (dd, J = 8.4, 2.1 Hz, 1H), 7.01 (d, J = 8.7 Hz, 2H),
6.96 (d, J = 8.8 Hz, 2H), 6.89 (d, J = 8.8 Hz, 2H), 4.33 (p, J = 5.7 Hz, 1H), 4.12 (dd, J = 9.5, 5.1
Hz, 1H), 4.05 – 3.92 (m, 2H), 3.85 (t, J = 7.7 Hz, 1H), 3.38 – 3.29 (m, 4H), 3.29 – 3.21 (m, 4H),
1
3
1
1
1
.82 (s, 3H). C NMR (126 MHz, CDCl ) δ 161.67, 152.90, 150.10, 148.20, 147.94, 146.11,
3
38.16, 137.64, 134.56, 132.84, 131.20, 130.66, 128.91, 126.79, 126.26, 122.32, 120.91 (2),
18.38 (2), 116.97 (2), 115.48 (2), 109.09, 73.97, 69.30, 67.04, 50.75 (2), 49.91 (2), 25.74.DART-
+
HRMS: m/z calcd. for C H Cl N O [MH] , 619.1879; Found: 619.1872. IR (solid) vmax: 3367,
3
3
32
2
4
4
2
923, 2851, 1672, 1585, 1552, 1509, 1454, 1385, 1268, 1227, 1192, 1148, 1020, 940, 912, 814,
747, 620. Purity, Method A: 96.9%.
N-(4-(4-(4-(((2R,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4-
yl)methoxy)phenyl)piperazin-1-yl)phenyl)nicotinamide (13). Amide coupling method A; Yield
1
=
6.2 mg, 25%. H NMR (500 MHz, Chloroform-d) δ 9.14 (s, 1H), 8.83 (s, 1H), 8.26 (d, J = 8.0
Hz, 1H), 7.72 (s, 1H), 7.66 (d, J = 8.5 Hz, 1H), 7.59 (d, J = 8.4 Hz, 2H), 7.49 (d, J = 7.2 Hz, 1H),
7.46 (d, J = 2.2 Hz, 1H), 7.28 (dd, J = 8.4, 2.1 Hz, 1H), 7.05 (d, J = 8.7 Hz, 2H), 7.00 (d, J = 9.0
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Hz, 2H), 6.96 – 6.92 (m, 2H), 4.37 (p, J = 6.0 Hz, 1H), 4.17 (dd, J = 9.5, 5.0 Hz, 1H), 4.08 – 4.00
1
3
(
m, 2H), 3.89 (dd, J = 8.4, 6.9 Hz, 1H), 3.41 – 3.36 (m, 4H), 3.32 – 3.27 (m, 4H), 1.86 (s, 3H). C
NMR (126 MHz, CDCl ) δ 184.50, 170.35, 152.52, 147.78, 146.05, 138.15, 136.05, 135.22,
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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8
9
0
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0
1
34.57, 132.84, 131.20, 129.90, 128.91, 126.80, 123.69, 121.87 (2), 118.43 (2), 116.86 (2), 115.49
(2), 109.10, 73.96, 69.29, 67.03, 50.73 (2), 49.72 (2), 25.74. DART-HRMS: m/z calcd. for
+
C H Cl N O [MH] , 619.1879; Found: 619.1876. IR (solid) vmax: 3639, 3570, 2920, 2824,
33 32
2
4
4
1
644, 1509, 1447, 1374, 1227, 1194, 1150, 1037, 941, 828, 733, 534. Purity, Method A: 96.9%.
N-(4-(4-(4-(((2R,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4-
yl)methoxy)phenyl)piperazin-1-yl)phenyl)isonicotinamide (14). Amide coupling method A;
1
Yield = 12.0 mg, 49%. H NMR (500 MHz, Chloroform-d) δ 8.85 (d, J = 5.2 Hz, 2H), 7.75 (d, J
=
6.0 Hz, 3H), 7.66 (d, J = 8.4 Hz, 1H), 7.60 (d, J = 8.6 Hz, 1H), 7.46 (d, J = 2.2 Hz, 1H), 7.27 (d,
J = 2.2 Hz, 2H), 7.05 (d, J = 8.8 Hz, 2H), 7.00 (d, J = 9.0 Hz, 2H), 6.94 (d, J = 9.0 Hz, 2H), 4.38
(d, J = 5.8 Hz, 1H), 4.17 (dd, J = 9.7, 5.1 Hz, 1H), 4.08 – 3.99 (m, 2H), 3.92 – 3.87 (m, 1H), 3.42
1
3
–
1
1
4
6
3.36 (m, 4H), 3.32 – 3.27 (m, 4H), 1.86 (s, 3H). C NMR (126 MHz, CDCl ) δ 163.41, 152.98,
3
50.79 (2), 148.93, 146.03, 142.18, 138.14, 134.57, 132.83, 131.19, 129.64, 128.90, 126.79,
21.81 (2), 120.79 (2), 118.43 (2), 116.79 (2), 115.50 (2), 109.09, 73.96, 69.30, 67.02, 50.72 (2),
+
9.66 (2), 25.73. DART-HRMS: m/z calcd. for C H Cl N O [MH] , 619.1879; Found:
3
3
32
2
4
4
19.1871. IR (solid) vmax: 3302, 3290, 2950, 2936, 1662, 1587, 1512, 1458, 1377, 1249, 1225,
1194, 1151, 1035 943, 823, 690. Purity, Method A: 94.1%
N-(4-(4-(4-(((2S,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4-
yl)methoxy)phenyl)piperazin-1-yl)phenyl)benzamide (15). Amide coupling method A; Yield =
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Journal of Medicinal Chemistry
Page 26 of 46
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2
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2
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3
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4
4
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5
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5
5
5
6
1
5
.0 mg, 20.6%. H NMR (500 MHz, Chloroform-d) δ 7.91 (d, J = 7.4 Hz, 2H), 7.73 (s, 1H), 7.69
d, J = 8.5 Hz, 1H), 7.59 (dd, J = 8.0, 5.2 Hz, 3H), 7.54 (t, J = 7.5 Hz, 2H), 7.43 (d, J = 2.2 Hz,
1H), 7.25 (dd, J = 8.5, 2.2 Hz, 1H), 7.04 (d, J = 8.7 Hz, 2H), 6.95 (d, J = 8.7 Hz, 2H), 6.78 (d, J =
(
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
8
3
.9 Hz, 2H), 4.65 (p, J = 6.2 Hz, 1H), 4.35 (dd, J = 8.6, 6.4 Hz, 1H), 4.00 (dd, J = 9.7, 5.3 Hz, 1H),
1
3
.82 – 3.74 (m, 2H), 3.36 (d, J = 5.4 Hz, 4H), 3.28 (d, J = 5.2 Hz, 4H), 1.83 (s, 3H). C NMR
(
126 MHz, CDCl ) δ 165.58, 152.78, 151.10, 148.49, 139.40, 135.17, 134.36, 132.81, 131.68,
3
1
30.97, 130.61, 128.78, 128.58 (2), 126.95, 126.70 (2), 121.64 (2), 118.35 (2), 116.95 (2), 115.24
(
2), 109.16, 75.07, 68.44, 67.39, 50.73 (2), 49.88 (2), 25.90. DART-HRMS: m/z calcd. for
+
C H Cl N O [MH] , 618.1926; Found: 618.1916. IR (solid) vmax: 3305, 2964, 2824, 1637,
34 33
2
3
4
1592, 1511, 1448, 1374, 1226, 1192, 1152, 1034, 943, 818, 728, 703. Purity, Method B: 95.6%.
N-(4-(4-(4-(((2S,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4-
yl)methoxy)phenyl)piperazin-1-yl)phenyl)-2-naphthamide (16). Amide coupling method A;
1
Yield = 3.0 mg, 11.5%. H NMR (500 MHz, Chloroform-d) δ 8.43 (s, 1H), 8.08 – 7.92 (m, 4H),
7
.89 (s, 1H), 7.73 – 7.58 (m, 5H), 7.43 (d, J = 2.1 Hz, 1H), 7.25 (dd, J = 8.5, 2.1 Hz, 1H), 7.06 (d,
J = 8.8 Hz, 2H), 6.96 (d, J = 9.0 Hz, 2H), 6.78 (d, J = 9.1 Hz, 2H), 4.65 (p, J = 6.1 Hz, 1H), 4.35
(
dd, J = 8.6, 6.4 Hz, 1H), 4.00 (dd, J = 9.7, 5.3 Hz, 1H), 3.86 – 3.71 (m, 2H), 3.45 – 3.33 (m, 4H),
1
3
3
1
.33 – 3.21 (m, 4H), 1.83 (s, 3H). C NMR (126 MHz, CDCl ) δ 165.53, 152.75, 148.50, 146.03,
3
39.34, 134.83, 134.36, 132.80, 132.69, 132.36, 130.97, 130.68, 128.96, 128.73, 128.59, 127.83,
127.41, 126.94, 126.71, 123.57, 121.69 (2), 118.36 (2), 116.96 (2), 115.24 (2), 109.16, 75.07,
+
6
6
8.44, 67.40, 50.74 (2), 49.87 (2), 25.91. DART-HRMS: m/z calcd. for C H Cl N O [MH] ,
3
8
35
2
3
4
68.2083; Found: 668.2116. IR (solid) vmax: 3326, 2920, 2851, 1663, 1596, 1558, 1513, 1450,
1373, 1229, 1195, 1151, 1037, 948, 822, 760. Purity, Method A: 96.3%.
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Journal of Medicinal Chemistry
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N-(4-(4-(4-(((2S,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4-
yl)methoxy)phenyl)piperazin-1-yl)phenyl)picolinamide (17). Amide coupling method A; Yield
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
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5
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5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
=
9.0 mg, 37%. H NMR (500 MHz, Chloroform-d) δ 9.96 (s, 1H), 8.66 (dt, J = 3.8, 1.1 Hz, 1H),
8
.34 (dt, J = 7.9, 1.1 Hz, 1H), 7.95 (td, J = 7.7, 1.7 Hz, 1H), 7.76 (d, J = 8.5 Hz, 2H), 7.69 (d, J =
.5 Hz, 1H), 7.57 – 7.48 (m, 1H), 7.43 (d, J = 2.1 Hz, 1H), 7.25 (dd, J = 8.5, 2.1 Hz, 1H), 7.06 (d,
8
J = 8.6 Hz, 2H), 6.96 (d, J = 8.4 Hz, 2H), 6.78 (d, J = 8.6 Hz, 2H), 4.65 (p, J = 6.1 Hz, 1H), 4.40
–
4
4.30 (m, 1H), 4.00 (dd, J = 9.7, 5.3 Hz, 1H), 3.78 (dd, J = 8.6, 7.2 Hz, 2H), 3.39 (d, J = 12.8 Hz,
H), 3.28 (s, 4H), 1.83 (s, 3H).
1
3
C NMR (126 MHz, CDCl ) δ 178.75, 147.93, 146.04, 139.33, 137.64, 134.73, 134.36, 132.79,
3
1
30.97, 130.88, 128.58, 126.71, 126.25, 122.98, 122.31, 120.91 (2), 118.33 (2), 118.16, 116.96
(2), 115.25 (2), 109.16, 75.06, 68.44, 67.39, 50.74 (2), 49.90 (2), 25.91. DART-HRMS: m/z calcd.
+
for C H Cl N O [MH] , 619.1879; Found: 619.1862. IR (solid) vmax: 3365, 2955, 2849, 1675,
3
3
32
2
4
4
1
586, 1568, 1510, 1453, 1373, 1268, 1229, 1193, 1151, 1097, 1037, 942, 823, 747. Purity, Method
B: 92.7%.
N-(4-(4-(4-(((2S,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4-
yl)methoxy)phenyl)piperazin-1-yl)phenyl)nicotinamide (18). Amide coupling method A; Yield
1
=
6.0 mg, 24.7%. H NMR (500 MHz, Chloroform-d) δ 9.15 (s, 1H), 8.83 (s, 1H), 8.27 (s, 1H),
7.86 (s, 1H), 7.68 (d, J = 8.5 Hz, 1H), 7.60 (d, J = 8.4 Hz, 2H), 7.47 (d, J = 16.7 Hz, 1H), 7.43 (d,
J = 2.1 Hz, 1H), 7.28 – 7.20 (m, 1H), 7.01 (dd, J = 29.6, 8.5 Hz, 4H), 6.83 – 6.73 (m, 2H), 4.65 (p,
J = 6.1 Hz, 1H), 4.35 (dd, J = 8.6, 6.4 Hz, 1H), 4.00 (dd, J = 9.7, 5.3 Hz, 1H), 3.83 – 3.74 (m, 2H),
1
3
3.43 – 3.36 (m, 4H), 3.29 (t, J = 5.0 Hz, 4H), 1.83 (s, 3H). C NMR (126 MHz, CDCl ) δ 171.38,
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2
2
2
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60.06, 148.66, 142.60, 139.33, 136.75, 135.23, 134.36, 132.80, 130.97, 130.06, 128.58, 127.85,
26.71, 121.92 (2), 118.62, 118.54 (2), 116.92 (2), 115.27 (2), 109.17, 75.05, 68.43, 67.36, 50.88
+
(2), 49.67 (2), 25.90. DART-HRMS: m/z calcd. for C H Cl N O [MH] , 619.1879; Found:
3
3
32
2
4
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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7
8
9
0
1
2
3
4
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7
8
9
0
1
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5
6
7
8
9
0
1
2
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8
9
0
6
1
19.1866. IR (solid) vmax: 3364, 3315, 2960, 2924, 2853, 1673, 1511, 1456, 1372, 1259, 1227,
193, 1097, 1035, 826, 807. Purity, Method A: 96.5%.
N-(4-(4-(4-(((2S,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4-
yl)methoxy)phenyl)piperazin-1-yl)phenyl)isonicotinamide (19). Amide coupling method A;
1
Yield = 8.0 mg, 33%. H NMR (500 MHz, Chloroform-d) δ 8.85 (s, 2H), 7.77 (d, J = 13.1 Hz,
3H), 7.68 (d, J = 8.5 Hz, 1H), 7.59 (d, J = 8.5 Hz, 2H), 7.43 (d, J = 2.1 Hz, 1H), 7.24 (dd, J = 8.5,
2
6
.2 Hz, 1H), 7.04 (d, J = 8.7 Hz, 2H), 6.98 – 6.92 (m, 2H), 6.78 (d, J = 9.0 Hz, 2H), 4.65 (p, J =
.1 Hz, 1H), 4.35 (dd, J = 8.6, 6.4 Hz, 1H), 4.00 (dd, J = 9.7, 5.3 Hz, 1H), 3.82 – 3.75 (m, 2H),
1
3
3.38 (q, J = 4.2, 3.0 Hz, 4H), 3.32 – 3.24 (m, 4H), 1.83 (s, 3H). C NMR (126 MHz, CDCl ) δ
3
1
1
6
63.39, 152.80, 150.80 (2), 148.93, 145.98, 142.18, 139.36, 134.36, 132.81, 130.97, 129.65,
28.58, 126.70, 121.81 (2), 120.80 (2), 118.38 (2), 116.79 (2), 115.27 (2), 109.16, 75.07, 68.46,
+
7.39, 50.71 (2), 49.65 (2), 25.90. DART-HRMS: m/z calcd. for C H Cl N O [MH] , 619.1879;
33 32
2
4
4
Found: 619.1866. IR (solid) vmax: 3256, 2963, 2854, 1660, 1587, 1510, 1449, 1376, 1261, 1226,
193, 1150, 1034, 940, 826, 740. Purity, Method A: 95.1%.
1
N-(4-(4-(4-(((2R,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4-
yl)methoxy)phenyl)piperazin-1-yl)phenyl)-2-hydroxybenzamide (26). Amide coupling
1
method B; Yield = 10.0 mg, 16%. H NMR (500 MHz, Chloroform-d) δ 12.14 (s, 1H), 7.87 (s,
1H), 7.66 (d, J = 8.4 Hz, 1H), 7.58 – 7.44 (m, 5H), 7.28 (dd, J = 8.4, 2.2 Hz, 1H), 7.07 (dd, J =
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Journal of Medicinal Chemistry
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3.2, 8.5 Hz, 3H), 6.99 (t, J = 7.9 Hz, 2H), 6.95 (t, J = 8.0 Hz, 3H), 4.41 – 4.33 (m, 1H), 4.17 (dd,
J = 9.4, 5.1 Hz, 1H), 4.09 – 3.98 (m, 2H), 3.93 – 3.85 (m, 1H), 3.44 – 3.35 (m, 4H), 3.29 (t, J =
1
3
5.0 Hz, 4H), 1.86 (s, 3H). C NMR (126 MHz, CDCl ) δ 206.89, 168.26, 161.91, 152.97, 149.12,
3
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
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5
5
5
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5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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7
8
9
0
1
2
3
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7
8
9
0
1
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0
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0
1
1
46.04, 138.14, 134.52, 132.83, 131.20, 128.91, 128.83, 126.79, 125.25, 122.85 (2), 118.97,
18.85, 118.43 (2), 116.79 (2), 115.49 (2), 114.62, 109.09, 73.96, 69.29, 67.03, 50.72 (2), 49.65
+
(2), 29.72. DART-HRMS: m/z calcd. for C H Cl N O [MH] , 634.1865; Found: 634.1875. IR
34 33
2
3
5
(
solid) vmax: 3310, 2956, 2916, 2848, 1646, 1596, 1511, 1453, 1373, 1250, 1228, 1196, 1152,
036, 942, 821, 755. Purity, Method B: 98.1%.
1
N-(4-(4-(4-(((2R,4R)-2-(2,4-dichlorophenyl)-2-methyl-1,3-dioxolan-4-
yl)methoxy)phenyl)piperazin-1-yl)phenyl)-3-hydroxybenzamide (27). Amide coupling
1
method B; Yield = 6.0 mg, 25%. H NMR (500 MHz, Chloroform-d) δ 7.74 (d, J = 23.9 Hz, 1H),
7.63 – 7.54 (m, 3H), 7.47 – 7.39 (m, 4H), 7.10 (s, 1H), 7.04 (d, J = 8.6 Hz, 3H), 7.00 (d, J = 8.5
Hz, 2H), 6.96 – 6.92 (m, 2H), 5.21 (s, 1H), 4.37 (s, 1H), 4.16 (dd, J = 9.3, 5.3 Hz, 1H), 4.12 – 3.95
(m, 2H), 3.88 (d, J = 8.0 Hz, 1H), 3.37 (d, J = 5.2 Hz, 4H), 3.29 (t, J = 4.9 Hz, 4H), 1.88 (dd, J =
1
3
1
1
1
7.2, 1.6 Hz, 3H). C NMR (126 MHz, CDCl ) δ 165.41, 156.15, 151.12, 147.60, 145.64, 141.66,
3
38.16, 131.21, 130.09, 128.92, 128.26, 127.93, 127.18, 126.80, 125.31, 121.69 (2), 118.75,
18.42 (2), 116.95 (2), 115.50 (2), 114.43, 110.65, 73.97, 69.30, 67.04, 50.75 (2), 49.85 (2), 25.74.
+
DART-HRMS: m/z calcd. for C H Cl N O [MH] , 634.1876; Found: 634.1847. IR (solid)
34 33
2
3
5
vmax: 3311, 2967, 2926, 2877, 1736, 1644, 1586, 1513, 1449, 1375, 1230, 1198, 1038, 945, 825,
46. Purity, Method A: 97.8%.
7
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