Sterol 14α-Demethylase Structure-Based Design of VNI ((R)- N-(1-(2,4-Dichlorophenyl)-2-(1 H-imidazol-1-yl)ethyl)-4-(5-phenyl-1,3,4-oxadiazol-2-yl)benzamide)) Derivatives to Target Fungal Infections: Synthesis, Biological Evaluation, and Crystallographic Analysis

Article abstract of DOI:10.1021/acs.jmedchem.8b00641

Because of the increase in the number of immunocompromised patients, the incidence of invasive fungal infections is growing, but the treatment efficiency remains unacceptably low. The most potent clinical systemic antifungals (azoles) are the derivatives of two scaffolds: ketoconazole and fluconazole. Being the safest antifungal drugs, they still have shortcomings, mainly because of pharmacokinetics and resistance. Here, we report the successful use of the target fungal enzyme, sterol 14α-demethylase (CYP51), for structure-based design of novel antifungal drug candidates by minor modifications of VNI [(R)-N-(1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-phenyl-1,3,4-oxadiazol-2-yl)benzamide)], an inhibitor of protozoan CYP51 that cures Chagas disease. The synthesis of fungi-oriented VNI derivatives, analysis of their potencies to inhibit CYP51s from two major fungal pathogens (Aspergillus fumigatus and Candida albicans), microsomal stability, effects in fungal cells, and structural characterization of A. fumigatus CYP51 in complexes with the most potent compound are described, offering a new antifungal drug scaffold and outlining directions for its further optimization.

Full text of DOI:10.1021/acs.jmedchem.8b00641

Subscriber access provided by Hong Kong University of Science and Technology Library
Article
Sterol 14#-Demethylase Structure-Based Design of VNI ((R)-N-(1-
(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-phenyl-1,3,4-
oxadiazol-2-yl)benzamide)) Derivatives to Target Fungal Infections:
Synthesis, Biological Evaluation, and Crystallographic Analysis
Laura Friggeri, Tatiana Y Hargrove, Zdzislaw Wawrzak, Anna L. Blobaum, Girish Rachakonda, Craig W
Lindsley, Fernando Villalta, W. David Nes, Maurizio Botta, F. Peter Guengerich, and Galina I Lepesheva
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00641 • Publication Date (Web): 12 Jun 2018
Downloaded from http://pubs.acs.org on June 12, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 37
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Sterol 14α-Demethylase Structure-Based Design of VNI ((R)-N-(1-(2,4-dichlorophenyl)-2-(1H-
imidazol-1-yl)ethyl)-4-(5-phenyl-1,3,4-oxadiazol-2-yl)benzamide)) Derivatives to Target Fungal
Infections: Synthesis, Biological Evaluation, and Crystallographic Analysis
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
†
†
§
¥
ŧ
Laura Friggeri, Tatiana Y. Hargrove, Zdzislaw Wawrzak, Anna L. Blobaum, Girish Rachakonda, Craig W.
¥
ŧ
ǂ
#
†
Lindsley, Fernando Villalta, W. David Nes, Maurizio Botta, F. Peter Guengerich , and Galina I.
†, ‡
Lepesheva*
†
§
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA
Synchrotron Research Center, Life Science Collaborative Access Team, Northwestern University, Argonne,
Illinois 60439, USA
¥
Vanderbilt Center for Neuroscience Drug Discovery, Franklin, Tennessee 37067, USA
ŧ
Department of Microbiology and Immunology, Meharry Medical College, Nashville, Tennessee 37208, USA
ǂ
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas 79409, USA
#
Department of Biotechnology, Chemistry and Pharmacy, University of Siena, 53100 Siena, Italy
‡
Center for Structural Biology, Vanderbilt University, Nashville, Tennessee 37232, USA
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 37
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Abstract
Due to the increase in the number of immunocompromised patients, the incidence of invasive fungal
infections is growing but the treatment efficiency remains unacceptably low. The most potent clinical
systemic antifungals (azoles) are the derivatives of two scaffolds: ketoconazole and fluconazole. Being the
safest antifungal drugs, they still have shortcomings, mainly because of pharmacokinetics and resistance.
Here we report the successful use of the target fungal enzyme, sterol 14α-demethylase (CYP51), for
structure-based design of novel antifungal drug candidates by minor modifications of VNI [(R)-N-(1-(2,4-
dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-phenyl-1,3,4-oxadiazol-2-yl)benzamide)], an inhibitor of
protozoan CYP51 that cures Chagas disease. Synthesis of fungi-oriented VNI derivatives, analysis of their
potencies to inhibit CYP51s from two major fungal pathogens (Aspergillus fumigatus and Candida albicans),
microsomal stability, effects in fungal cells, and structural characterization of A. fumigatus CYP51 in
complexes with the most potent compound are described, offering a new antifungal drug scaffold and
outlining directions for its further optimization.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
ACS Paragon Plus Environment

Page 3 of 37
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Introduction
In the last two decades, the mortality and morbidity of invasive fungal infections (IFIs) has greatly
increased, and it has been estimated that over 1.6 million infected people die each year. The lack of access
to timely diagnosis, co-infections with other diseases (HIV/tuberculosis), use of immunosuppressive
therapies (chemotherapy/transplants), some medical devices (central venous catheters), and growing
resistance to commercial antifungal drugs significantly contribute to the higher incidence of IFIs. In
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
addition, the socio-economic environment in different countries has a great impact on antifungal treatment
1
(i.e., expensive drugs and commercial availability). Of the different fungal species, Candida and Aspergillus
genera are responsible for the vast majority of life-threatening IFIs. Candida albicans remains the most
prevalent human pathogen, yet non-albicans Candida species represent a growing global emergency.
Highly virulent strains less susceptible to antifungal medicines, e.g. C. auris, a multidrug-resistant deadly
2
pathogen, are often present in healthcare facilities. Aspergillus is a ubiquitous fungus, transmitted to
humans by inhalation of conidia. Approximately 200,000 cases of invasive aspergillosis, mostly in
immunocompromised patients, are caused each year by Aspergillus fumigatus, which is also the major
3
species responsible for chronic pulmonary aspergillosis and asthma.
The therapeutic antifungal drugs available for systemic clinical use act on three different targets: 1)
sterol biosynthesis (six azoles and terbinafine); 2) cell wall integrity (amphotericin B and caspofungin); and
3
) DNA synthesis (flucytosine) (Supporting information, Figure S1A,C). All of these drugs have clinical
4
3, 5
limitations, and therefore even upon treatment the mortality rates from IFIs often exceed 50%. More
efficient new medicines are needed.
Several attempts to discover new fungal drug targets have been undertaken, and a few compounds with
6
different modes of action are in preclinical studies. Whether they will progress into clinical trials remain
uncertain. On the other hand, further development of antifungal azoles, inhibitors of fungal sterol 14α-
demethylase (CYP51, EC 1.14.13.70), represents a successful area for investigation because azoles,
compared to other classes of current systemic antifungals, clearly display higher potency, better safety
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 37
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
6
-7
profile, and broader spectrum of activity. Indeed, in 2015 a new azole, the triazole-based isavuconazole,
8
was approved for treatment of IFIs, while another triazole (albaconazole) and a tetrazole (VT-1161) are in
clinical trials (Supporting information, Figure S1B) [https://clinicaltrials.gov].
9
10
Determination of the crystal structures of A. fumigatus and C. albicans CYP51 in complexes with
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
1
azole drugs (voriconazole and posaconazole) and drug candidates (VNI, VT-1161, and VT-1598 ) opened
new opportunities for rational structure-based design of more efficient compounds. The objective of this
work was to use the knowledge that we acquired from our structure/function studies to modify our
,
1
2-13
experimental inhibitor of protozoan CYP51, VNI,
in such a way that its inhibitory effect on fungal CYP51
is enhanced. We selected VNI because it is active in vivo against Chagas disease and visceral leishmaniasis,
non-toxic, non-mutagenic, with excellent cellular permeability, metabolic stability, and pharmacokinetics
(
including oral bioavailability and tissue distribution that are substantially more favorable than those of
7
systemic clinical antifungal azoles ). Moreover, although VNI is a relatively weaker inhibitor of fungal CYP51
than the protozoan orthologs, we found that it is more effective in killing A. fumigatus cells than
voriconazole, a first line drug against aspergillosis, with the minimum inhibitory concentration (MIC) values
9
being 0.5 vs. 0.7 µg/mL, respectively, for VNI and voriconazole. Very importantly, the VNI scaffold is easy
to synthesize and modify.
The rationale for this study was based on the observation that fungal and protozoan CYP51 structures,
though displaying high 3-dimensional similarity, differ noticeably in the features of substrate entry, which in
the fungal CYP51 structures is open much wider (due to the phylum-specific differences between the
1
0
protozoan and fungal amino acid sequences). The hypothesis to test was whether antifungal potency of
the VNI scaffold can be enhanced through the formation of additional contacts between the inhibitor
molecule and the substrate entrance residues of the fungal CYP51 enzyme.
Figure 1. Sites of modifications in the VNI molecule
4
ACS Paragon Plus Environment

Page 5 of 37
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
A small in-house library of analogues carrying various substituents in the ortho, meta, and para positions of
the distal (5-phenyl) ring of the VNI long arm (Figure 1) has been synthesized and analyzed, with the results
serving as the initial proof of concept. Potencies of most of the new compounds to inhibit C. albicans and A.
fumigatus CYP51 were increased, some of them approaching the potency of posaconazole, which is
presently one of the most powerful inhibitors of fungal CYP51 (example shown in Table of Content
Graphic).
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
Results
Synthesis. The new in-house library of VNI derivatives was prepared using the general procedures outlined
in Scheme 1. The synthesis started by bromination of 1-(2,4-dichlorophenyl)-ethan-1-one, followed by
condensation with the imidazole ring. Subsequently, asymmetric transfer hydrogenation with RuCl(p-
cymene)[(S,S)-Ts-DPEN] catalyst was performed in order to obtain (S)-1-(2,4-dichlorophenyl)-2-(1H-
1
4
imidazol-1-yl)ethan-1-ol, as described previously. The direct conversion of (S)-1-(2,4-dichlorophenyl)-2-
1H-imidazol-1-yl)ethan-1-ol to (R)-1-(2-azido-2-(2,4-dichlorophenyl)ethyl)-1H-imidazole was carried out by
dissolving the alcohol in N,N-dimethylformamide (DMF) and adding diphenyl phosphorazidate (DPPA) and
,5-diazabiciclo[5.4.0]undec-7-ene (DBU) at 0 °C. (R)-1-(2,4-Dichlorophenyl)-2-(1H-imidazol-1-yl)ethan-1-
(
1
4
amine was then prepared by reduction of the azide into an amine using LiAlH .
Scheme 1. Synthesis of new VNI derivatives.
N
N
N
Cl
O
Cl
O
Cl
O
Cl
OH
CuBr
2
N
H
RuCl(p-cymene)[(S,S)-Ts-DPEN]
Br
N
N
CH
3
CHCl
reflux 2h
3
/EtOAc
DMF
2h
T= 0°C
DCM, TEA; HCOOH
Cl
Cl
Cl
Quantitative yield
Cl
Quantitative yield
26h
T = 40°C
60% yield
DPPA
DBU
DMF
T = 0°C to r.t.
overnight
Ar
R1
Cl
Cl
N
N
N
Cl
N
3
N
Cl
NH
2
H
1c-14c
4
LiAlH , THF
R2
R3
N
N
N
O
overnight
r.t.
O
EDC/DMAP
DCM
r.t.
overnight
N
Cl
Cl
R4
2
N
70% yield
Quantitative yield
1
-14
0-60% yield; e.e. 98-99%
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 37
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
The final compounds 1-14 were synthesized by coupling between (R)-1-(2,4-dichlorophenyl)-2-(1H-
imidazol-1-yl)ethan-1-amine and 4-(5-phenyl-1,3,4-oxadiazol-2-yl)benzoic acids containing various
substituents in the 5-phenyl ring (intermediate c, Scheme 2). The synthesis of intermediate c was
performed using three reaction steps. The acylation of the hydrazide with methyl 4-
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
3
(chlorocarbonyl)benzoate was followed by cyclization of the 1,3,4-oxadiazole in POCl , and the hydrolysis of
ester into carboxylic acid by LiOH·H O.
2
Scheme 2. Synthesis of intermediate c.
However, different approaches were used for preparing variously substituted benzohydrazines
(intermediate b). Thus, 1b and 6b were available commercially, the intermediates b for compounds 2, 5, 7,
8
2 4 2
, and 14 were synthesized by the addition of the hydrazine monohydrate (N H ·H O) to the commercial
available carboxylic acid or ester, as shown in Scheme 3. The synthesis of intermediates b for 3, 4, and 9-13
included preparation of intermediates a. Intermediates a for compounds 3 and 4 were made by addition of
the electrophile 2,2,2-trifluoroethyl trifluoromethanesulfonate to the corresponding carboxylic acid.
Intermediates
a
for
9-13
were
synthesized
using
[1,1′-
bis(diphenylphosphino)ferrocene]dichloropalladium(II) (Pd(dppf)Cl
2
) as a catalyst for Suzuki coupling, and it
was performed in a microwave reactor to increase the efficiency (Scheme 3).
6
ACS Paragon Plus Environment

Page 7 of 37
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Scheme 3. Synthesis of benzohydrazines.
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
a
H SO was not used for hydrazinolysis of 9a-13a.
2
4
Evaluation of new compounds as inhibitors of fungal CYP51 activity. Even minor modifications in the 5-
phenyl ring composition produced remarkably strong effects on the potencies of the new compounds to
inhibit enzymatic activity of C. albicans and A. fumigatus CYP51 (Table 1). Thus, quite encouragingly,
addition of only two fluorine atoms, in the meta and para positions relative to 1,3,4-oxadiazole (Figure 1)
(compound 1, 5-(3,4-difluorophenyl)-1,3,4-oxadiazole), enhanced inhibition of C. albicans CYP51 activity
from 20 to 90% and inhibition of A. fumigatus CYP51 activity from 24 to 60% (under the same conditions
the inhibitory effects of posaconazole were 98 and 93%, respectively). When chlorines were substituted for
fluorines (atomic radius 0.99 Å vs. 0.71 Å) in the same meta and para positions (compound 5), the
inhibitory effect on C. albicans increased slightly (92%) but inhibition of A. fumigatus CYP51 became weaker
(55%). Because the larger size of a chlorine atom did not appear to be crucial for inhibition, and
introduction of a fluorine has a lower impact on drug lipophilicity (e.g., the logP values of 1 and 5 are 4.88
and 5.68, respectively, Table 1), we mainly used fluorine instead of chlorine substituents in other (non-
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 37
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
aromatic) analogues, except for compound 14 (logP 3.83), whose potency towards both fungal enzymes
turned out to be about 2-fold lower than that of 5. On the other hand, substitution of a para fluorine with a
trifluoromethyl group in 2 (logP 5.65), increased the ability of the compound to inhibit A. fumigatus (68%)
without influencing its inhibition of C. albicans CYP51 (89%). Our strongest inhibitor, compound 3, has one
fluorine atom in the ortho position and a trifluoroethoxy group in the para position (94% and 82% inhibition
of C. albicans and A. fumigatus CYP51 activity, respectively). However, when the same substituents are in
two meta positions, compound 4, the potency drops, although the inhibitory effect is still more than twice
that of the parent compound VNI. Since all of the modifications above produced inhibitors with higher
potencies, thus supporting our hypothesis, a number of bulkier substituents were introduced to decorate
the 5-phenyl ring of the VNI arm. A single bromine atom (atomic radius 1.14 Å) in the meta position
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
(compound 6) enhanced the inhibition to 84% (C. albicans) and to 41% (A. fumigatus). When the meta
position was substituted with a morpholine ring however (compound 7), the potency for inhibition of A.
fumigatus CYP51 decreased (15% inhibition), and insertion of a morpholine ring into the para position
produced an even weaker inhibitor (compound 8, 29% and 3% inhibition towards C. albicans and A.
fumigatus CYP51, respectively). While it is possible that the morpholine substitutions are unfavorable as a
result of the ring hydrophilicity, because the CYP51 substrate entry, being immersed into the endoplasmic
reticulum membrane in vivo, is composed mainly of hydrophobic amino acid residues, a general trend of
the meta position being more preferable than the para position for a heterocyclic substituent is apparent
(compounds 9, 10, 11, 12, 13, and 15 in Table 1). Among these six analogues, 11 and 15 were most potent
towards both fungal enzymes (87/80% and 92/57%, C. albicans/A. fumigatus CYP51, respectively).
a
Table 1. New VNI analogues: 5-phenyl ring composition and inhibition of fungal CYP51 activity .
CYP51 inhibition, %
CYP51 inhibition, %
Cmp
Cmp
C. albicans
A. fumigatus
C. albicans
A. fumigatus
8
ACS Paragon Plus Environment

Page 9 of 37
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
VNI
1
20 ± 3
90 ± 3
24 ± 6
58 ± 3
8
9
29 ± 3
77 ± 3
3 ± 1
34 ± 4
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
89 ± 2
94 ± 1
65 ± 4
65 ± 13
82 ± 4
51 ± 5
10
66 ± 6
87 ± 3
61 ± 4
23 ± 1
80 ± 5
26 ± 4
3
4
11
12
5
92 ± 3
55 ± 4
13
83 ± 1
24 ± 5
logP 5.4
6
7
84 ± 4
58 ± 2
41 ± 4
15 ± 1
14
15
53 ± 1
92 ± 1
32 ± 3
57 ± 1
Br
a
A 1-hour reaction was used to distinguish the strongest inhibitors, with the potential propensity to act as functionally
irreversible (see Experimental Section, Discussion). The P450 concentration was 0.5 M and the molar excess of each
inhibitor over enzyme was 1.5-fold (0.75 M)
ꢀ
ꢀ
.
Evaluation of selected compounds as CYP51 binding ligands. Five the strongest inhibitors of C. albicans
and A. fumigatus CYP51 activity were also assayed as binding ligands of the enzymes (Table 2). VNI,
posaconazole, and voriconazole were used as controls. Previously we observed a clear lack of correlation
between the apparent spectral binding affinities of some ligands and their potencies to inhibit CYP51
14-16
enzymatic activity, particularly in long reactions.
Accordingly we compared spectral titrations with close
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 37
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
structural analogues (e.g., as observed in ref. 17). Overall, the data support a relationship between binding
and inhibitory potency, particularly for compounds
to VNI (see Table 1 and Supplemental Fig S2).
1, 2, and 3, which have the highest structural similarities
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. Dissociation constants determined by spectral titration of C. albicans and A. fumigatus CYP51 with
VNI, compounds 1, 2, 3, 11, and 15, posaconazole and voriconazole.
Cmpds
C. albicans
, nM
Fold enhancement
A. fumigatus
, nM
Fold enhancement
a
K
d
(K
d
VNI/K
d
n )
K
d
(K
d
d
VNI/K n)
VNI
1
290 ± 27
13 ± 1
-
203 ± 10
57± 6
-
22
22
29
12
3
4
2
13 ± 1
55 ± 3
4
3
10 ± 6
20 ± 4
10
3
1
1
1
5
25 ± 5
61 ± 3
95 ± 11
81 ± 16
165 ± 9
132 ± 2
131 ± 11
56 ±4
1.5
1.5
3.6
Posaconazole
Voriconazole
3.5
1.8
a
n – compound number
Estimated intrinsic and hepatic clearance in human, rat, and mouse microsomes. Since high metabolic
1
8
stability is one of the many advantages of VNI as a potential drug, we examined the parameters for the
new compounds. Moreover, simultaneous analysis in human and animal microsomes is highly informative
in allowing for a prediction of the likelihood of acceptable pharmacokinetics in clinical trials. VNI and
thirteen new compounds with the increased potencies to inhibit both fungal CYP51 enzymes (all except for
the morpholine-containing
7 and 8) were tested in human, rat, and mouse liver microsomes (Table 3). The
data show that overall, none of the modifications drastically affected the stability of the new compounds,
with all but one (11, human microsomes) displaying low to moderate hepatic clearance. The stability of
1
0
ACS Paragon Plus Environment

Page 11 of 37
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
compounds
1
and
2
in human and mouse systems was even higher than that of VNI. Replacement of the
two fluorine atoms with chlorine (
microsomes. Although the pyridine ring can be metabolically vulnerable, compounds
showed rather good stability, not inferior to compound 15 (5-fluoro-6 pyrimidinyl analogue of 11).
5), however, had slightly negative effects in human/mouse but not in rat
1
9
9
and 12-13 also
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
Table 3. Microsomal stability: Half-lives and estimated clearance of VNI and new derivatives in human,
rat, and mouse microsomes
.
Cmpds
Human
Rat
Mouse
CLHEP
a
b
CLINT
CLHEP t ½ (min)
CLINT
CLHEP
t ½ (min)
CLINT
t ½ (min)
VNI
1
8.31
7.53
6.06
16.5
11.3
11.4
9.47
13.1
10.8
96.6
15
5.95
5.54
4.07
9.24
7.33
7.39
6.53
8.06
7.15
17.3
8.75
6.85
6.45
150
166
206
76
26.4
19.2
21
106
87
44
34
62
31
36
91
77
44
56
40
106
41
30
78
54
57
27
55
60
98
92
62
65
51
43
36
57
99
149
23
2
24
36
3
64
34
88
44
4
111
110
132
95
50.4
70
29
84.7
107
126
152
95
43.6
49
5
35
6
27
19
55
9
68
35
56
10
115
13
94
40
46
1
1
1
2
36.0
51.6
49.4
104
24.0
29.7
29.0
42
55.0
36.5
238
56
34
83
26.0
65
13
10.2
9.31
123
134
1
4
34.5
97
1
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 37
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
15
14.9
8.37
84
27.0
19.5
104
104
48.3
52
a
b
Intrinsic (CLINT
)
and hepatic clearance (CLHEP
)
expressed in mL/min/kg body weight; in vitro half-life values in
minutes.
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
verapamil
1
5
3
2
1
VNI
0
50
100
150
200
Half-life time, min
2
Figure 2. Half-life time of VNI, selected analogues, and a drug verapamil (positive control) in human microsomes.
R
values were all >0.7.
Cellular effects in A. fumigatus strains. Four compounds (1, 2, 3, and 15) that revealed both the highest
potencies to inhibit A. fumigatus CYP51 activity and favorable stability in human microsomes (Figure 2)
were further tested in cellular experiments. VNI was used as a control (Table 4). All the derivatives
displayed stronger inhibition of fungal cell growth, and their fungicidal potencies correlated well with the
effects on the target enzyme. The lowest MIC values, 2.4- and 2.5-fold lower than the MICs of VNI on
average, were observed for compounds
2 and 3, respectively.
Table 4. Antifungal activity of VNI and derivatives (MIC ±SEM, µg/mL)
A. fumigatus
strain
VNI
1
p-value
2
p-value
3
p-value
15
p-value
1
022
0.48 + 0.05
0.08 + 0.03
0.08 + 0.03
0.29(+0.03)
0.08(+0.03)
0.78(+0.03)
0.0001*
0.0009*
0.0035*
0.19(+0.03) 0.0003* 0.19(+0.03) 0.0002*
0.09(+0.02) 0.0027* 0.06(+0.01) 0.0004*
0.39(+0.05) 0.0001* 0.39(+0.05) 00001*
0.48(+0.05)
1
3
2820
0.19(+0.03) 0.21
MYA-3626
0.78(+0.04) 0.0035*
*
Statistical significance of the differences (p values) was analyzed by one-way ANOVA
1
2
ACS Paragon Plus Environment

Page 13 of 37
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
X-ray crystallography. The compound (3) that displayed the strongest inhibition of both fungal CYP51
enzymes (Table 1) and the most expressed enhancement in the apparent binding affinity (29 and 10-fold in
comparison to VNI, C. albicans and A. fumigatus CYP51, respectively, Table 2) was selected for co-
crystallization. In addition to the goal of observing inhibitor-specific interactions with the enzyme (i.e.,
structural basis for the increased inhibitory potency), we examined whether the modifications alter the
ligand orientation in the fungal CYP51 substrate binding cavity. While the co-crystals of C. albicans CYP51
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
with
3 did not diffract well, the A. fumigatus complexes produced crystals that diffracted to ~2.4 Å. Several
datasets were collected, the structures were determined, and each of them indicated that upon binding to
the enzyme the inhibitor can adopt two different conformations, both of them different from the
conformations of VNI in A. fumigatus or in T. brucei CYP51 (Figure 3A). The structure of the best diffracting
crystal has been refined and deposited in the Protein Data Bank (PDB 6CR2). Supplementary Table S4
summarizes the diffraction and refinement statistics. The 2F
o
-F
c
electron density maps of the inhibitor
molecules in both conformations contoured at 1.2
of Content Graphic.
σ and are shown as gray mesh can be seen in the Table
Discussion and Conclusions
It has been known for years that antifungal azoles, both clinical drugs and agricultural fungicides, kill
20
fungal cells by inhibiting their sterol biosynthesis at the stage of 14α-demethylation of sterol precursors.
This is a three-step reaction catalyzed by a single cytochrome P450 monooxygenase, named sterol 14α-
21
demethylase (CYP51). This target enzyme, being a highly hydrophobic membrane-bound protein, has
7
never actually been directly utilized in the process of drug discovery or development.
6
In part because of this, the process of antifungal drug discovery has been slow, costly, and low-efficient.
The six clinical systemic azoles, which represent the major portion of the current arsenal of clinical
antifungal drugs, are derivatives of two inhibitory scaffolds: ketoconazole (itraconazole and posaconazole)
and fluconazole (voriconazole and isavuconazole) (Supplementary Figure S1A). The third, a tetrazole–based
scaffold from Viamet (Supplementary Figure S1B), is currently in trials. All these drugs were discovered
1
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 37
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
rather empirically by monitoring effects of hundreds of compounds on fungal cell growth. Our study shows
that direct analysis of CYP51 enzyme inhibition can be utilized to shift the traditional drug discovery
paradigm, making it potentially much more effective.
9
All of the 15 compounds synthesized and analyzed in this work, including the parent compound VNI,
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
inhibit the initial rate of fungal CYP51 reactions when added at an equimolar ratio to the enzyme, thus
producing IC values of ~0.25 µM, corresponding to one-half of the enzyme concentration in the reaction
5
0
mixture (data not shown). However, as seen in Table 1, with time some of the inhibitors are replaced by the
substrate in the fungal CYP51 active site more easily than the others, although most of the (5-phenyl) ring
substituents (presumably positioned in close proximity to the CYP51 substrate entry) increase potencies of
the inhibitors to hold the enzyme in the catalytically inactive state.
Azoles are generally known as competitive reversible heme coordinating inhibitors of cytochromes
2
2
23
P450. However, as it was first observed in 1987 for CYP51 from S. cerevisiae and later rediscovered by
1
6
our research group for protozoan CYP51, some azole-based inhibitors can act in a functionally irreversible
manner due to their high affinity. Added at a concentration equimolar to the enzyme, they entirely inhibit
CYP51 catalysis and cannot be replaced by the substrate for an extended period of time, even though no
7
covalent bonds with the protein are formed. VNI is an example of such an inhibitor for trypanosomal
CYP51, while VT-1161, VF-1598, and posaconazole can serve as examples of such inhibitors for fungal
CYP51.
The very strong affinity of some of the azoles appears to be somehow connected with the strict
functional phylogenetic conservation of CYP51 and the three-step stereoselective oxidation reaction
2
4
sequence, which together require high structural rigidity of the enzyme active site. Indeed, none of more
than 30 crystal structures of microbial CYP51s bound to various inhibitory chemotypes that we have
determined have displayed any large-scale rearrangements in the active site backbone upon inhibitor
binding. Fortunately for antifungal drug design, the active site of human CYP51 appears to be much more
25-27
flexible,
rendering the human enzyme naturally resistant to inhibition, at least with all ever tested
1
4
ACS Paragon Plus Environment

Page 15 of 37
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
2
6
antimicrobial azoles, both commercial and experimental, a favorable feature that enables pathogen-
selectivity of CYP51-targeting antimicrobial agents.
1
2
18
In the protozoan CYP51 structures, VNI and its derivatives VFV and VNT all form hydrogen bonds
with the amino acid residues in the central, catalytic area of the CYP51 active site. The VT tetrazoles form
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
H-bonds with the fungal-specific histidine (His-374 in A. fumigatus and His-377 in C. albicans) that lines the
10-11
CYP51 substrate access channel.
CYP51 complexes with posaconazole do not involve any H-bond
10
formation but display multiple enzyme-inhibitor contacts around the substrate entry.
Binding of VNI to A. fumigatus CYP51 does not produce any H-bonds, and its three ring arm is not long
enough to reach the substrate entry site. Nevertheless, in addition to the strong (2.0 Å) coordination bond
between its imidazole (N3) nitrogen and the enzyme heme iron, VNI forms van der Waals contacts with 19
9
amino acid residues, including the sandwich
π
-
π
stacking interactions with Phe-234. This bonding provides
a structural background for the strong inhibitory effect of VNI on the initial fungal CYP51 reaction and the
reversibility of inhibition in the presence of the substrate. The results support our hypothesis that
elongation of the VNI arm is likely to increase its potency as fungal CYP51 inhibitor.
The crystal structure of A. fumigatus CYP51 complexed with compound 3 (ligand PDB ID LFV) shows that
the stronger enzyme inhibition is indeed due to the new interactions around the substrate entry (Figure 3B)
and also suggests that larger substituents here might be even more efficient. In both conformations LFV
forms contacts with an additional seven amino acid residues of the enzyme (Figure 3C). In molecule A
(
CYP51-common portion of the substrate entry), Thr-65, Tyr-68, Gly-69 (helix A´), Leu-91, Leu-92 (β1
hairpin), and Phe-234 (helix F´´) interact with the trifluoroethoxy moiety, and Val-121 (helix B´) interacts
with the fluorine atom inserted into the ortho position relative to the oxadiazole ring. In molecule B (fungal
CYP51-specific portion of the substrate entry), Asn-233, Phe-234, Met-235, Leu-236, Pro-237, and Trp-238
(helix F´´) interact with the trifluoroethoxy moiety, while Val-121 and Phe-124 (helix B´) interact with the
additional ortho fluorine. Interestingly, in both complexes Phe-234 “follows” the inhibitor orientation,
preserving the
π-π stacking interactions with its 5-phenyl aromatic ring (see also Table of Content Graphic,
right panel). It remains to be clarified if one of the two LFV conformations warrants a higher inhibitory
1
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 37
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
potency, but it appears that such a probability for complex B may be higher. In this complex, a fluorine
atom of the LFV trifluoroethoxy moiety displays a propensity to form H-bond like interactions with the main
chain oxygen of Met-235 (2.3 Å) and with the indole nitrogen of Trp-238 (3.2 Å), while the ethoxy oxygen is
positioned only 3.5 Å from the main chain oxygen of Phe-234 (Supplementary Figure S3). All these three
residues are in helix F´´, the CYP51 area that is most likely to serve as part of the “lid” for the substrate
entry. Assuming that binding of the substrate requires opening of the FG-lid, these contacts with the ligand
are likely to restrict the required movement. No such gatekeeping interactions are seen in complex A.
Further efforts directed towards strengthening fungal CYP51-VNI scaffold interactions are in progress,
with our ultimate goal being to design very high affinity compounds that will bind to the fungal CYP51 most
efficiently, keeping the enzyme in its catalytically inactive state and preventing a conformational switch
required for the interaction with the electron donor partner NADPH-cytochrome P450 reductase.
Overall, the results of this study prove that structure-based design is useful for the target fungal CYP51,
meaning that novel potent inhibitory scaffolds can be rationally built. Minor modifications may be sufficient
to optimize pharmacokinetics and thus to improve the IFI treatment efficiency and retard drug resistance.
The availability of various alternative CYP51 inhibitory scaffolds should be extremely useful in overcoming
situations in which patients succumb to fungal sepsis simply because they must be temporarily
immunosuppressed and there are no efficient drugs available.
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
6
ACS Paragon Plus Environment

Page 17 of 37
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
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 3. LFV (compound 3) adopts two alternative conformations in the active site of A. fumigatus
CYP51B. A. Substrate entry area. The carbon atoms in the superimposed structures of complexes A and B
are tan and blue, respectively. The corresponding orientation of VNI in the structures of A. fumigatus
CYP51B (PDB ID 4uyl, gray lines) and Trypanosoma brucei CYP51 (PDB ID 3gw9, purple lines) are shown for
comparison. The A. fumigatus CYP51 substrate entry-defining secondary structural elements are presented
as gray ribbon and labeled. The semitransparent ribbon of the (longer) F/G loop area in the protozoan
CYP51 (3gw9) is purple, and the F´´ helix and the protozoan-specific G´ helix are labeled.
B. Overall view in
semitransparent surface representation. . New ligand/enzyme interactions formed as a result of the VNI
C
modification. Atoms added to the VNI structure are presented as spheres. The amino acid residues of A.
fumigatus CYP51B involved in the additional interactions are shown as stick models and labelled, and
selected distances are displayed.
1
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 37
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Experimental Section
Chemical Synthesis. All reagents and solvents were commercial grade and were purchased from Fisher
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
Scientific, Sigma-Aldrich, Santa Cruz, or Alpha Aesar. Microwave reactions were performed with a Biotage
1
Initiator Microwave System. H NMR spectra were acquired on a Bruker AVANCE-400 MHz spectrometer in
CDCl and DMSO-d6 at 25 °C. Chemical shift values are given in
δ (ppm) relatively to TMS as internal
3
standard. Coupling constants are given in Hz. The following abbreviations are used to set multiplicities: s =
singlet, d = doublet, dd= double of doublets, t = triplet, q= quartet, m = multiplet, br = broad. Mass
spectrometry was done with a LTQ Orbitrap XL Hybrid Ion Trap-Orbitrap Mass Spectrometer. The molecular
+
-
peaks (m/z) was observed as [M+H] or [M-H] ions. Compounds were purified by flash chromatography
using silica gel high purity grade, pore size 60 Å/230–400 mesh/particle size 40-63
µm (Sigma-Aldrich). The
final compounds were filtered through HF Bond Elut-SCX cartridges (500 mg, 6 mL, Agilent Technologies),
using a 2.0 M ammonia solution in methanol (Sigma-Aldrich). LC-MS (UV and ESI-MS) and reverse-phase
HPLC were used for analyzing the purity of the compounds (> 95%). Analytical LC-MS was performed on an
Agilent 1200 series with UV detection at 214 and 254 nm, along with ELSD detection. General LC-MS
parameters were as follow: Phenomenex-C18 Kinetex column, 50 mm × 2.1 mm, 2 min gradient, 5% (0.1%
TFA/CH
3 2 3
CN)/95% (0.1% TFA/H O) to 100% (0.1% TFA/ CH CN) (all v/v). The reversed-phase HPLC system
(Waters) was equipped with a dual-wavelength UV 2489 detector set at 250 and 291 nm (VNI-specific
-
1
-1
13
absorption maximum, ε291 = 36 mM cm ) and a Symmetry C18 (3.5
μ
m) 4.6 mm × 75 mm column. The
mobile phase was 55% 0.015 M ammonium acetate solution (pH 7.2) and 45% CH
3
CN (v/v) with an isocratic
flow rate of 1.0 mL/min. The enantiomeric excess (ee) of the final compounds was evaluated by analytical
chiral chromatography on a Chiralpak IB-3 column, 250 mm × 4.6 mm i.d. (Daicel Corporation, Japan) using
a Waters HPLC system with the detector set at 250 and 291 nm. As eluent, a solution of hexane, ethanol,
diethylamine, and ethylenediamine, (ratio 70:30:0.1:0.1, v/v) was used, and the flow rate was 0.7 mL/min.
High resolution mass spectra were obtained on an Agilent 6540 UHD Q-TOF with ESI source. MS parameters
were as follows: fragmentor: 150, capillary voltage: 3500 V, nebulizer pressure: 60 psig, drying gas flow: 13
1
8
ACS Paragon Plus Environment

Page 19 of 37
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
L/min, drying gas temperature: 275° C. Samples were introduced via an Agilent 1290 UHPLC comprised of a
G4220A binary pump, G4226A ALS, G1316C TCC, and G4212A DAD with ULD flow cell. UV absorption was
observed at 215 nm and 254 nm with a 4 nm bandwidth. Column: Waters Acquity BEH C18, 1.0 x 50 mm,
3 2 3
1.7 um. Gradient conditions: 5% to 95% CH CN in H O (0.1% Formic Acid) over 1.25 min, hold at 95% CH CN
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
for 0.25 min, 0.3 mL/min, 40° C.
Preparation of (R)-1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethan-1-amine.
Synthesis of 2-bromo-1-(2,4-dichlorophenyl)ethan-1-one (step 1). CuBr (11.8 g, 53 mmol) was ground
2
with a mortar and pestle to ensure a large surface area for the reaction and placed in a round bottom flask.
EtOAc (10 mL) was added and the solution was heated until reflux. Subsequently, 2’,4′-
dichloroacetophenone (5 g, 26.5 mmol) was dissolved in CHCl
dropwise to the CuBr solution. The resulting mixture was stirred for 2 hours under reflux. The reaction was
allowed to cool at room temperature, filtrated, diluted with EtOAc, and washed with aqueous NH Cl. The
organic layer was then washed with brine, dried over anhydrous Na SO , filtered, and the solvent removed
3
(10 mL), stirred at 40 °C, and added
2
4
2
4
in vacuo. The crude product was purified by flash column chromatography (hexane/EtOAc, 8:2, v/v)
1
affording the title product as a yellow oil in quantitative yield. H NMR (CDCl ):
δ
ppm 7.56 (d,
J= 8.4 Hz,
3
1
H), 7.48 (d,
J
= 1.9 Hz, 1H), 7.36 (dd,
267.90.
J
= 8.4, 1.9 Hz, 1H), 4.50 (s, 2H,). ESI MS, mass calcd for C
8
H
5
BrCl
2
O
+
[M+H] 267.89, found
m
/z
Synthesis of 1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethan-1-one (step 2). To a solution of imidazole (9
g, 133 mmol) in DMF (10 mL) was added (dropwise) 2-bromo-1-(2,4-dichlorophenyl)ethan-1-one (7 g, 27
mmol). The reaction was stirred for 2 hours at 0 °C. The reaction mixture was diluted with H O, extracted
2
2
into EtOAc, washed with LiCl (5%, w/v) and brine, and dried over anhydrous Na SO4, filtered, and the
solvent was removed in vacuo. The crude product was purified by flash column chromatography (CH Cl /
2
2
1
CH
3
OH, 9:1, v/v) affording the title product as a white solid, isolated in 60% yield. H NMR (CDCl
3
): δ ppm
= 8.4, 1.9 Hz, 1H), 7.11 (s, 1H), 6.93 (s, 1H), 5.32 (s,
255.3.
7
2
.56 (d,
J= 8.9 Hz, 1H), 7.49 (d,
J= 1.9 Hz, 2H), 7.36 (dd, J
+
H). ESI MS, mass calcd for C11
H
8
2
Cl N
2
O [M+H] 255.10, found
m/z
1
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 37
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Synthesis of (S)-1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethan-1-ol (step 3). 1-(2,4-Dichlorophenyl)-2-
(1
imidazol-1-yl)ethan-1-one. The title compound was isolated as white solid in quantitative yield. H NMR
(CDCl ): ppm 7.59 (d, = 8.4 Hz, 1H), 7.39 (d, = 2.0 Hz, 1H), 7.37 (s, 1H), 7.30 (dd, = 8.4, 2.0 Hz, 1H), 6.89
s, 1H), 6.82 (s, 1H), 5.21 (dd, = 14.0, 2.2 Hz, 1H), 3.84 (dd, = 14.0, 8.3 Hz, 1H).
256.9.
H-imidazol-1-yl)ethan-1-ol was prepared as previously described using of 1-(2,4-dichlorophenyl)-2-(1H-
2
8
1
3
δ
J
J
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
(
J= 8.3, 2.0 H, 1H), 4.19 (dd,
J
J
+
ESI MS, mass calcd for C H Cl N O [M+H] 257.02, found
m/z
1
1
10
2
2
Synthesis of (R)-1-(2-azido-2-(2,4-dichlorophenyl)ethyl)-1H-imidazole (step 4). To a cold solution of (
S
)-1-
-imidazol-1-yl)ethan-1-ol (230 mg, 0.9 mmol) and diphenylphosphoryl azide,
DPPA) (194 µL, 0.9 mmol) in anhydrous DMF (1.8 mL), was added dropwise 1,5-diazabiciclo[5.4.0]undec-5-
L, 1.26 mmol). The solution was stirred at 0 °C for 2 hours and then at room temperature
overnight. Then the reaction mixture was diluted with water, extracted into EtOAc, washed with LiCl (5%,
w/v) and brine, and dried over anhydrous Na SO4, filtered, and the solvent removed in vacuo. The crude
product was purified by flash column chromatography (CH Cl /CH OH, 9:1, v/v) affording the title product
(2,4-dichlorophenyl)-2-(1H
(
ene (DBU) (188
µ
2
2
2
3
1
as a white solid, isolated in 70% yield. H NMR (CDCl
s, 1H), 6.90 (s, 1H), 5.24 (dd, = 7.6, 3.2 Hz, 1H), 4.21 (dd,
ESI MS, mass calcd for C11 Cl
Synthesis of (R)-1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethan-1-amine (step 5). To a suspension of
LiAlH (87.5 mg, 2.30 mmol) in dry THF (3 mL), ( )-1-(2-azido-2-(2,4-dichlorophenyl)ethyl)-1 -imidazole
542 mg, 1.92 mmol) (dissolved 2 mL of dry THF), was added dropwise at 0 °C. The suspension was warmed
3
):
δ
ppm 7.43 (d,
= 14.4, 3.2 Hz, 1H), 4.01 (dd,
2 5
N [M+H] 282.02, found m/z 282.0.
J
= 2.0 Hz, 1H), 7.36-7.29 (m, 3H), 7.05
(
J
J
J= 14.4, 7.6 Hz, 1H).
+
H
9
4
R
H
(
to room temperature and stirred overnight. The reaction mixture was quenched by Rochelle’s salt and
filtered through Celite. The crude product was extracted into EtOAc, washed with brine, dried on
anhydrous Na SO filtered, and the solvent removed in vacuo. The crude product was purified by flash
2
4,
1
chromatography (CH
2
Cl
2
/CH
3
OH, 9:1, v/v) to give a white solid in quantitative yield. H NMR (CDCl
3
):
δ ppm
7
1
2
.44-7.40 (m, 3H), 7.27 (d,
J
= 2.0 Hz, 1H), 7.06 (s, 1H), 6.90 (s, 1H), 4.72 (dd,
J
= 8.0, 3.7 Hz, 1H), 4.19 (dd, J=
+
4.0, 3.7 Hz, 1H), 3.94 (dd,
56.0.
J= 14.0, 8.0 Hz, 1H). ESI MS, mass calcd for C11
2 3
H11Cl N [M+H] 256.03, found m/z
2
0
ACS Paragon Plus Environment

Page 21 of 37
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Preparation of the side chains. Intermediates 1b, 2a, 5a
,
6b
,
7a, 8a, and 14a were commercially available.
CO (5
General synthesis of intermediates (3a and 4a). To a mixture of selected acid (1 equiv.) and K
2
3
equiv.) in DMF (0.5 M) was added 2,2,2-trifluoroethyl trifluoromethanesulfonate (1.5 equiv.), and the
solution was stirred at 80 °C for 2 hours. The reaction mixture was cooled to room temperature, poured
into water (100 mL), and the aqueous layer was extracted by EtOAc, washed with brine, dried with
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
anhydrous Na SO4, and filtered, and the solvent removed in vacuo. The crude product was purified by flash
chromatography (CH Cl /CH OH, 9:1) to give a white solid.
2
2
3
1
2
-Fluoro-4-(2,2,2-trifluoroethoxy)benzoic acid (3a). Yield 78%. H NMR (DMSO-
d
6
)
δ
11.0 (br, 1H), 7.77 (t,
J= 8.8 Hz, 1H), 6.73 (dd,
J= 8.7, 2.3 Hz, 1H), 6.65 (dd,
J= 13.1, 2.2 Hz, 1H), 4.90 (q,
J
= 8.8 Hz, 2H). ESI MS,
-
mass calcd for C
9
H
6
F
4
O
3
[M-H] 237.03, found
m/z
236.9.
1
3-Fluoro-5-(2,2,2-trifluoroethoxy)benzoic acid (4a). Yield 50%. H NMR (DMSO-
d
6
)
δ
11.0 (br, 1H), 7.20 (s,
1H), 7.07 (d,
J
= 8.8 Hz, 1H), 6.90 (d,
237.0.
J= 10.6 Hz, 1H), 4.92 (q, J= 9.0 Hz, 2H). ESI MS, mass calcd for C H F O
9 6 4 3
-
[
M-H] 237.03, found
m/
z
General synthesis of intermediates (9a-13a). The selected boronic acid (1 equiv.), 2-bromopyridine (1
equiv.), [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (Pd(dppf)Cl ) (0.6 mol%), dioxane, and
O (ratio 4:1, v/v) were stirred under argon. The reaction was performed in the microwave reactor for 30
min at 120 °C. The solvent was evaporated, and the crude residue was extracted with EtOAc, washed with
brine, and dried over anhydrous Na SO4, filtered, and the solvent removed in vacuo. The crude product was
2
H
2
2
purified by flash chromatography (hexanes/EtOAc, 4:1, v/v) to give a white solid in 50-70% yield.
1
Methyl 3-(pyridin-2-yl)benzoate (9a). Yield 60%. H NMR (CDCl
3
):
= 7.8 Hz, 1H), 7.30-7.27 (m, 1H), 3.96 (s,
214.10.
): ppm 8.74-8.72 (m, 1H), 8.15 (d, J= 8.6
δ ppm 8.74-8.72 (m, 1H), 8.65-8.64 (m,
1H), 8.27-8.24 (m, 1H), 8.11-8.09 (m, 1H), 7.80 (m, 2H), 7.57 (t,
J
+
3H). ESI MS, mass calcd for C13
H
11NO
2
[M+H] 214.08, found
m
/
z
1
Methyl 4-(pyridin-2-yl)benzoate (10a). Yield 50%. H NMR (CDCl
3
δ
Hz, 2H), 8.08 (d, J= 8.6 Hz, 2H), 7.80-7.79 (m, 2H), 7.31-7.28 (m, 1H), 3.95 (s, 3H). ESI MS, mass calcd for
+
C
13
H
11NO
2
[M+H] 214.08, found
m/
z
214.05.
2
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 37
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
Methyl 3-fluoro-5-(pyridin-2-yl)benzoate (11a). Yield 70%. H NMR (CDCl ):
δ ppm 8.73-8.72 (m, 1H), 8.45-
3
8.44 (m, 1H), 8.03-7.99 (m, 1H), 7.82-7.75 (m, 3H), 7.33-7.30 (m, 1H), 3.97 (s, 3H). ESI MS, mass calcd for
+
C
13
H
10FNO
2
[M+H] 232.07, found
m/z
232.1.
1
Methyl 2-fluoro-4-(pyridin-2-yl)benzoate (12a). Yield 50 %. H NMR (CDCl
3
): δ ppm 8.73-8.71 (m, 1H), 8.05-
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
7
.95 (m, 1H), 7.86 (s, 1H), 7.84-7.76 (m, 3H), 7.34-7.31 (m, 1H), 3.96 (s, 3H). ESI MS, mass calcd for
+
C H FNO [M+H] 232.07, found
m/z
232.1.
1
3
10
2
1
Methyl 2-fluoro-5-(pyridin-2-yl)benzoate (13a). Yield 60 %. H NMR (CDCl
3
):
δ
ppm 8.69-8.67 (m, 1H), 8.54
= 1.5 Hz, 1H), 3.96
(dd,
J
= 9.4, 2.5 Hz, 1H), 8.23-8.19 (m, 1H), 7.79-7.72 (m, 2H), 7.27-7.26 (m, 1H), 7.24 (d, J
+
(s, 3H). ESI MS, mass calcd for C13
H10FNO
2
[M+H] 232.07, found
m/z
232.09.
1
Methyl 3-fluoro-5-(5-fluoropyrimidin-4-yl)benzoate (15a). Yield 92 %. H NMR (CDCl
3
): δ ppm 9.13 (d,
J
=
3
.0 Hz), 8.72 (s, 1H), 8.65 (s, 1H), 8.12-8.09 (m, 1H), 7.91-7.88 (m, 1H), 3.4 (s, 3H). ESI MS, mass calcd for
+
C
12
8 2 2 2
H F N O [M+H] 251.06, found m/z 251.10.
General synthesis of intermediates b and c. To a solution of substituted benzoic acid (1 equiv.) in EtOH,
hydrazine monohydrate (2 equiv.) and few drops of concentrated H SO were added. The reaction mixture
2
4
was heated under reflux for 6 hours and then, after cooling, the white solid was filtered to give desired
product (intermediate b) in quantitative yield, which was used for the next step. (The same procedure was
2 4
performed with substituted methyl benzoate without adding concentrated H SO ).
Subsequently, methyl 4-(chlorocarbonyl)benzoate, dissolved in anhydrous THF, was added
dropwise to a dried round bottom flask containing the appropriate intermediate b, under an argon flow.
The mixture was stirred at room temperature for 6 hours. Then the solvent was removed under reduced
pressure. The corresponding
N´-benzoyl-(hydrazinocarbonyl)esters, differently substituted, were used
without further purification in the next step, when POCl was added dropwise. The mixture was heated at
3
reflux overnight (85 °C), then cooled to room temperature and poured into ice and solid NaHCO
resulting solution was extracted with EtOAc, washed with H O, and dried with Na SO . Differently
substituted methyl 4-(5-phenyl-1,3,4-oxadiazol-2-yl)benzoates were purified by flash chromatography
hexane/EtOAc, 3:1, v/v). The hydrolysis of the benzoate ester to benzoic acid was performed by adding, to
3
. The
2
2
4
(
2
2
ACS Paragon Plus Environment

Page 23 of 37
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
a suspension of the benzoate in THF, LiOH·H O and CH OH (THF/CH OH /H O, 3:1:1, v/v/v). The reaction
2
3
3
2
mixture was stirred at room temperature for 3 hours, and then the solvent was removed under reduced
pressure, and the residue was extracted with an aqueous HCl solution and EtOAc. The solvent was
evaporated and a white pure solid was obtained in quantitative yield (intermediate c).
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
General Procedure for amide coupling (step 6). (
R)-1-(2,4-Dichlorophenyl)-2-(1H-imidazol-1-yl)ethan-1-
amine (1.2 equiv.) was dissolved in CH Cl (4 mL) and the corresponding acid (intermediate c, 1.0 equiv.)
2
2
was added to the round bottom flask, which was cooled to 0 °C before adding N-(3-dimethylaminopropyl)-
N′-ethylcarbodiimide hydrochloride (EDC/HCl) (1.0 equiv.) and 4-dimethylaminopyridine (DMAP). The
reaction mixture was warmed to room temperature and stirred overnight. The mixture was diluted with
CH
2
Cl
2
, washed with NaHCO
3
, and brine dried on Na
2
SO
4
. The solvent was evaporated and the final
OH, 9:1, v/v). The yields were calculated as
compound was purified by flash chromatography (CH
pure compounds, obtained as a white solid.
2
Cl
2
/CH
3
(R)-N-(1-(2,4-Dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-(3,4-difluorophenyl)-1,3,4-oxadiazol-2-
1
yl)benzamide (compound 1). Yield 50 %. H NMR (CDCl
3
):
δ
ppm 9.97 (s, 1H), 9.55 (d,
J
= 8.8 Hz, 1H), 8.04 (d,
J= 8.4 Hz, 2H), 7.98-7.89 (m, 2H), 7.79 (dd,
J= 8.3, 5.2 Hz, 3H), 7.47-7.45 (m, 2H), 7.40-7.35 (m, 3H), 6.08-
+
6
.04 (m, 1H), 5.27 (dd,
J= 13.8, 12.0 Hz, 1H), 4.41 (dd,
J= 14.0, 3.2 Hz, 1H). LC-MS, found [M+H]
m/z 539.8;
+
ESI MS, mass calcd for C26
H17Cl
2
F
2
N
5
O
2
[M] 539.0727, found
M
obs 539.0733. HPLC,
t
R
12.6 min, purity 98 %.
Enantiomeric excess > 97 %.
(
R)-N-(1-(2,4-Dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-(3-fluoro-4-(trifluoromethyl)phenyl)-1,3,4-
1
oxadiazol-2-yl)benzamide (compound 2). Yield 50 %. H NMR (CDCl ):
δ
ppm 9.92 (s, 1H), 9.49 (d,
J= 8.5 Hz,
3
1
H), 8.08 (d,
J= 8.4 Hz, 2H), 8.04 (d,
J= 8.2 Hz, 1H), 7.97 (d,
J= 10.2 Hz, 1H), 7.82-7.77 (m, 4H), 7.47-7.45 (m,
2H), 7.40 (s, 1H), 7.35 (dd,
J
= 8.4, 2.0 Hz, 1H), 6.09-6.04 (m, 1H), 5.26 (dd,
J= 14.0, 12.0 Hz, 1H), 4.42 (dd, J=
+
+
14.0, 3.3 Hz, 1H). LC-MS found, [M+H]
found obs 598.0696. HPLC,
m/z
590.18; ESI MS, mass calcd for C27
H
17Cl
2
F
N
4 5
O
2
[M] 598.0695,
M
t
R
11.4 min, purity 99 %. Enantiomeric excess > 97 %.
(
R)-N-(1-(2,4-Dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-(2-fluoro-4-(2,2,2-trifluoroethoxy)phenyl)-
1
1
,3,4-oxadiazol-2-yl)benzamide (compound 3).Yield 40 %. H NMR (CDCl
3
): δ ppm 9.79 (s, 1H), 9.47 (d,
2
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 37
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
J
=8.4 Hz, 1H), 8.13 (t,
J= 8.4 Hz, 1H), 8.02 (d, J= 8.3 Hz, 2H), 7.80 (dd, J= 8.3, 3.4 Hz, 3H), 7.45 (d, J= 2.0 Hz,
1H), 7.44 (s, 1H), 7.38 (s, 1H), 7.34 (dd,
J
= 8.4, 2.0 Hz, 1H), 6.94-6.86 (m, 2H), 6.07-6.02 (m, 1H), 5.23 (dd, J=
+
13.6, 12.4 Hz, 1H), 4.49-4.39 (m, 3H). LC-MS, found [M+H]
m/z 618.10; ESI MS, mass calcd for
+
C
28
H
19Cl
2
F
4
N
5
O
3
[M] 619.0801, found
M
obs 619.0805. HPLC,
t
R
17.6 min, purity 99 %. Enantiomeric excess >
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
9
7 %.
(
R)-N-(1-(2,4-Dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-(3-fluoro-5-(2,2,2-trifluoroethoxy)phenyl)-
1
1
,3,4-oxadiazol-2-yl)benzamide (compound 4) . Yield 30 %. H NMR (CDCl
3
):
δ
ppm 10.0 (s, 1H), 9.55 (d,
J
=
9
.0 Hz, 1H), 8.04 (d, J= 8.3 Hz, 2H), 7.78 (dd, J= 11.4, 8.4 Hz, 3H), 7.55-7.53 (m, 2H), 7.46-7.45 (m, 2H), 7.42
(s, 1H), 7.36 (dd,
J= 8.4, 2.0 Hz, 1H), 6.93-6.90 (m, 1H), 6.08-6.04 (m, 1H), 5.30 (dd, J= 14.0, 11.8 Hz, 1H),
+
4
.49-4.45 (m, 2H), 4.42-4.38 (m, 1H). LC-MS, found [M+H]
m/z
620.09; ESI MS, mass calcd for
+
C
28
H
19Cl
2
F
4
N
5
O
3
[M] 619.0801, found
M
obs 619.0802. HPLC,
t
R
13.4 min, purity 98 %. Enantiomeric excess >
97 %.
(R)-4-(5-(3,4-Dichlorophenyl)-1,3,4-oxadiazol-2-yl)-N-(1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-
1
yl)ethyl)benzamide (compound 5). Yield 20%. H NMR (CDCl
3
):
δ
ppm 8.21 (br, 1H), 8.16 (d,
J= 2.0 Hz, 1H),
8
2
4
.04 (d,
J
= 8.4 Hz, 2H), 7.96 (d,
J= 8.4 Hz, 2H), 7.93 (dd,
J= 8.4, 2.0 Hz, 1H), 7.61 (d,
J= 8.4 Hz, 1H), 7.45 (d, J=
.0 Hz, 1H), 7.42 (d,
J= 8.4 Hz, 1H), 7.23 (dd,
J
= 8.4, 2.0 Hz, 1H), 7.09 (s, 1H), 7.05 (s, 1H), 5.92-5.87 (m, 1H),
+
.76 (dd,
J
= 14.0, 8.9 Hz, 1H), 4.26 (dd,
J
= 14.0, 5.1 Hz, 1H). LC-MS, found [M+H]
obs 571.1046. HPLC,
m/z 573.91; ESI MS, mass
+
calcd for C26
H
17Cl
4
N
5
O
2
[M] 571.1036, found
M
t
R
13.4 min, purity 98 %. Enantiomeric
excess > 97 %.
(
R)-4-(5-(3-Bromophenyl)-1,3,4-oxadiazol-2-yl)-N-(1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-
1
yl)ethyl)benzamide (compound 6). Yield 50%. H NMR (CDCl
3
):
= 7.8 Hz, 1H), 7.51 (s, 1H), 7.45 (m, 1H), 7.35 (s, 1H),
= 7.8 Hz, 1H), 7.06 (s, 1H), 6.95-6.93 (m, 1H), 6.86 (s, 1H), 5.79 (m, 1H), 4.56 (d, = 6.48 Hz, 2H). LC-
[M] 581.0021, found obs 581.0026.
δ ppm 8.30 (s, 1H), 8.24 (d, J= 7.4 Hz, 2H),
8.11 (d,
7.12 (d,
J= 7.5 Hz, 1H), 7.91 (d, J= 7.5 Hz, 2H), 7.72 (d, J
J
J
+
+
MS, found [M+H]
m/
z
581.9; ESI MS, mass calcd for C26
H
18BrCl
2
N
5
O
2
M
HPLC, 17.0 min, purity 98 %. Enantiomeric excess > 97 %.
t
R
2
4
ACS Paragon Plus Environment

Page 25 of 37
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
(
R)-N-(1-(2,4-Dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-(3-morpholinophenyl)-1,3,4-oxadiazol-2-
1
yl)benzamide (compound 7). Yield 60%. H NMR (CDCl
3
):
= 7.0 Hz, 1H), 7.45 (s, 1H), 7.41-7.29 (m, 3H), 7.22 (d,
= 7.5 Hz, 1H), 7.0 (s, 1H), 6.92 (s, 1H), 5.88-5.79 (m, 1H), 4.55 (dd, = 13.7, 6.8 Hz, 1H), 4.45
589.6. HPLC, 7.3 min, purity
δ
ppm 8.06 (d,
J
= 7.6 Hz, 2H), 7.91 (d,
J
= 7.6 Hz,
1H), 7.87 (d,
Hz, 1H), 7.08 (d,
dd, = 13.0, 4.4 Hz, 1H), 3.87 (s, 4H), 3.22 (s, 4H). LC-MS, found [M+H]
8 %. Enantiomeric excess > 97 %.
J
= 7.6 Hz, 2H), 7.60 (s, 1H) 7.53 (d,
J
J= 8.2
J
J
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
+
(
J
m
/
z
t
R
9
(
R)-N-(1-(2,4-Dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-(4-morpholinophenyl)-1,3,4-oxadiazol-2-
1
yl)benzamide (compound 8). Yield 40 %. H NMR (CDCl ):
δ
ppm 8.27 (br, 1H), 8.04 (d,
= 8.4, 2.0 Hz, 1H), 7.08 (br, 2H), 6.97 (s, 1H), 6.95
= 13.2, 8.1 Hz, 1H), 4.45 (dd, = 14.0, 4.4 Hz, 1H), 3.87 (m, 4H), 3.22 (m,
6.8 min, purity 98 %. Enantiomeric excess > 97 %.
J= 8.2 Hz, 2H), 7.98-
3
7
.95 (m, 4H), 7.48 (s, 1H), 7.45 (d, J= 2.0 Hz, 2H), 7.24 (dd, J
(s, 1H), 5.95-5.90 (m, 1H), 4.79 (dd,
J
J
+
4
H). LC-MS, found [M+H]
m/z
589.23. HPLC,
t
R
(R)-N-(1-(2,4-Dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-(3-(pyridin-2-yl)phenyl)-1,3,4-oxadiazol-2-
1
yl)benzamide (compound 9). Yield 20 %. H NMR (CDCl ):
δ
ppm 8.75-8.74 (m, 2H), 8.48 (s, 1H), 8.38 (s,
= 8.4 Hz, 2H), 7.84-7.82 (m, 2H), 7.65 (t, = 7.8 Hz,
= 2.0 Hz, 1H), 7.33-7.30 (m, 1H), 7.28 (d, = 2.2 Hz, 1H), 7.20 (s, 1H),
3
1
1
5
H), 8.21-8.18 (m, 2H), 8.04 (d,
H), 7.50 (d, = 8.6 Hz, 1H), 7.44 (d,
.96-5.90 (m, 1H), 4.89 (dd,
J
= 8.4 Hz, 2H), 7.99 (d,
J
J
J
J
J
J
= 14.0, 9.5 Hz, 1H), 4.46 (dd,
J= 14.0, 4.9 Hz, 1H). (One signal was overlapped
+
+
with CDCl
3
). LC-MS, found [M+H]
m/z
581.2; ESI MS, mass calcd for C31
H
2
22Cl N
O
6 2
[M] 580.1181, found
M
obs 580.1192. HPLC,
t
R
11.0 min, purity 99 %. Enantiomeric excess > 97 %.
(
R)-N-(1-(2,4-Dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-(4-(pyridin-2-yl)phenyl)-1,3,4-oxadiazol-2-
1
yl)benzamide (compound 10). Yield 30 %. H NMR (CDCl ):
δ
ppm 8.75 (d,
J
= 4.5 Hz, 1H), 8.67-8.62 (m, 2H),
= 2.0 Hz, 1H), 7.33-7.28
= 14.0, 4.4 Hz, 1H). (One
3
8
.23-8.17 (m, 4H), 8.05 (s, 3H), 7.82-7.81 (m, 2H), 7.58 (d,
J= 8.4 Hz, 1H), 7.46 (d, J
(m, 2H), 7.19 (s, 2H), 6.00-5.95 (m, 1H), 4.95 (dd,
J
= 14.0, 9.6 Hz, 1H), 4.45 (dd, J
+
signal is overlapped with CDCl
3
). LC-MS, found [M+H]
m/z
581.09; ESI MS, mass calcd for C31
H
2
22Cl N
6
O
2
+
[M] 580.1181, found
M
obs 580.1195. HPLC,
t
R
10.9 min, purity 99 %. Enantiomeric excess > 97 %.
(
R)-N-(1-(2,4-Dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-(3-fluoro-5-(pyridin-2-yl)phenyl)-1,3,4-
1
oxadiazol-2-yl)benzamide (compound 11). Yield 20 %. H NMR (CDCl
3
):
δ
ppm 10 (s, 1H), 9.58 (d,
J= 8.7 Hz,
2
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 37
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
H), 8.75 (d,
J
= 4.6 Hz, 1H), 8.59 (s, 1H), 8.04 (d,
J
= 8.4 Hz, 2H), 7.88-7.86 (m, 2H), 7.82-7.75 (m, 4H), 7.69 (s,
1H), 7.46 (s, 1H), 7.42 (d,
J= 2.0 Hz, 1H), 7.38-7.32 (m, 3H), 6.09-6.03 (m, 1H), 5.30 (dd,
J= 13.4, 12.0 Hz, 1H),
+
4.41 (dd,
J= 13.6, 3.0 Hz, 1H). LC-MS, found [M+H]
m/z
599.09; ESI MS, mass calcd for C31
H21Cl
FN O
2 6 2
+
[M+H] 598.1087, found
M
obs 5998.1092. HPLC,
t
R
8.5 min, purity 98 %. Enantiomeric excess > 97 %.
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
(
R)-N-(1-(2,4-Dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-(4-(3-fluoropyridin-2-yl)phenyl)-1,3,4-
1
oxadiazol-2-yl)benzamide (compound 12). Yield 40 %. H NMR (CDCl ):
δ
ppm 10.0 (s, 1H), 9.55 (d,
J= 8.7
3
Hz, 1H), 8.76 (d,
J= 4.6 Hz, 1H), 8.28 (t,
J= 7.8 Hz, 1H), 8.05-8.01 (m, 3H), 7.98 (dd, = 8.2, 1.4 Hz, 1H), 7.86-
J
7
1
C
9
.77 (m, 5H), 7.52 (s, 1H), 7.44 (d,
J
= 2.0 Hz, 2H), 7.38-7.33 (m, 2H), 6.09-6.03 (m, 1H), 5.24 (dd, J= 13.8,
+
2.5 Hz, 1H), 4.43 (dd,
J
= 14.0, 3.2 Hz, 1H). LC-MS, found [M+H]
obs 619.0802. HPLC,
m/z 599.14; ESI MS, mass calcd for
+
28
H
19Cl
2
F
4
N
5
O
3
[M] 619.0801, found
M
t
R
6.2 min, purity 99 %. Enantiomeric excess >
7 %.
(R)-N-(1-(2,4-Dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-(2-fluoro-5-(pyridin-2-yl)phenyl)-1,3,4-
1
oxadiazol-2-yl)benzamide (compound 13). Yield 20 %. H NMR (CDCl ):
δ ppm 10.0 (s, 1H), 9.77 (d, J= 9.6
3
Hz, 1H), 8.96 (d,
J
= 5.6 Hz, 1H), 8.90 (dd,
.4 Hz, 1H), 8.07 (d, = 8.3 Hz, 2H), 8.01 (s, 1H), 7.86 (dd,
.42 (d, = 2.0 Hz, 1H), 7.33 (dd,
J= 6.0, 2.2 Hz, 1H), 8.45-8.41 (m, 1H), 8.35-8.30 (m, 1H), 8.18 (d,
J=
8
7
J
J= 8.4, 4.0 Hz, 3H), 7.53-7.50 (m, 2H), 7.46 (s, 1H),
J
J
= 8.5, 2.0 Hz, 1H), 6.09-6.03 (m, 1H), 5.34 (dd,
J= 13.8, 12.5 Hz, 1H), 4.43
+
+
(dd,
J= 13.2, 2.5 Hz, 1H). LCMS, found [M+H]
2 6 2
m/z 599.18; ESI MS, mass calcd for C31H21Cl FN O [M]
5
98.1087, found obs 598.1090. HPLC,
M
t
R
10.3 min, purity 99 %. Enantiomeric excess > 97 %.
(
R)-4-(5-(3-Chloro-4-(methylsulfonyl)phenyl)-1,3,4-oxadiazol-2-yl)-N-(1-(2,4-dichlorophenyl)-2-(1H-
1
imidazol-1-yl)ethyl)benzamide (compound 14). Yield 50 %. H NMR (CDCl ):
δ
ppm 10.0 (s, 1H), 9.57 (d,
= 8.3 Hz, 2H) 7.78 (dd,
J
=
=
3
8
.6 Hz, 1H), 8.35 (s, 1H), 8.34-8.33 (m, 1H), 8.22 (dd,
J
= 8.2, 1.5 Hz, 1H), 8.07 (d,
J
J
8.2, 5.04 Hz, 3H), 7.50 (s, 1H), 7.47 (d,
J= 2.0 Hz, 1H), 7.44 (s, 1H), 6.10-6.05 (m, 1H), 5.30 (dd, J= 13.8, 12.4
+
Hz, 1H), 4.42 (dd,
J= 13.6, 2.6 Hz, 1H), 3.35 (s, 3H). LC-MS, found [M+H]
m/z
617.7; ESI MS, mass calcd for
+
C
27 3 5 4 R
H20Cl N O S [M] 6175.0302, found Mobs 615.0315. HPLC, t 5.8 min, purity 99 %. Enantiomeric excess >
9
7 %.
2
6
ACS Paragon Plus Environment

Page 27 of 37
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
(
R)-N-(1-(2,4-Dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-(3-fluoro-5-(5-fluoropyrimidin-4-yl)phenyl)-
1
1,3,4-oxadiazol-2-yl)benzamide (compound 15). Yield 30 %. H NMR (CDCl
3
):
δ
ppm 10.04 (s, 1H), 9.54 (d,
= 8.5
= 8.4,
= 14.0, 3.0 Hz). LC-MS, found
J
= 8.7 Hz, 1H), 9.16 (d,
Hz, 2H), 8.02-7.99 (m, 1H), 7.80 (dd,
.0 Hz, 1H), 6.09-6.03 (m, 1H), 5.30 (dd,
J
= 3.0 Hz, 1H), 8.76 (d,
J
= 3.4 Hz, 1H), 8.73 (s, 1H), 8-11-8.08 (m, 1H), 8.06 (d,
J
J
= 8.4, 3.0 Hz, 3H), 7.55 (s, 1H), 7.45 (d,
J= 2.0 Hz, 2H), 7.35 (dd, J
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
J
= 14.0, 12.0 Hz, 1H), 4.42 (dd,
J
+
+
[M+H]
m/z
617.8; ESI MS, mass calcd for C H Cl F N O [M] 617.0945, found Mobs 617.0949. HPLC, tR
3
0
19
2
2
7
2
1
3.0 min, purity 99 %. Enantiomeric excess > 97 %.
CYP51 activity assay. A. fumigatus and C. albicans CYP51 were expressed in Escherichia coli and purified
9
-10
9
10
as described earlier. The enzymatic reaction mixture contained 0.5
µM
A. fumigatus or C. albicans
CYP51 and 1.0 rat NADPH-cytochrome P450 reductase, 100
µM
µM
L-α-1,2-dilauroyl-sn-
glycerophosphocholine, 0.4 mg/mL isocitrate dehydrogenase, and 25 mM sodium isocitrate in 50 mM
potassium phosphate buffer (pH 7.2) containing 10% glycerol (v/v) and 0.1% Triton X-100 (v/v). After
3
addition of the radiolabeled ([3- H]) sterol substrate (eburicol for A. fumigatus and lanosterol for C.
albicans CYP51), final concentration 25
preincubated for 60 seconds at 37 °C in a shaking water bath, and the reaction was initiated by the addition
of 100 M NADPH and stopped by extraction of the sterols with EtOAc. The extracted sterols were dried,
dissolved in CH OH, and analyzed using a reversed-phase HPLC system (Waters) equipped with a -RAM
detector (INUS Systems, Inc.) using a NovaPak octadecylsilane (C18) column (particle size 4 m, 3.9 mm
The inhibitory potencies of VNI and
µM, and an inhibitor, final concentration 0.75 µM, the mixture was
µ
3
β
µ
×
9
-10
1
50 mm) and a linear gradient of H
2 3 3
O, CH CN, and CH OH.
1
6, 18
derivatives were compared as percentage of inhibition of sterol 14
α
-demethylation in 60-min reactions;
the molar ratio inhibitor/enzyme/substrate in the reaction mixture was 1.5:1:50. Two clinical systemic
antifungal drugs, fluconazole and posaconazole, were used as controls. The experiments were performed in
triplicate, and the results are presented as means ± SD.
2
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 37
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
CYP51 ligand binding assay. Spectral titrations of C. albicans and A. fumigatus CYP51 with selected VNI
derivatives were carried out in 5 cm optical path length cuvettes at 0.5
µ
M P450 concentration as
previously described. The ligands were added from 0.2 mM stock solutions in DMSO, and the titration
range was 0.1-0.8 M. The apparent dissociation constants of the enzyme-inhibitor complex were
calculated as red shifts in the P450 Soret band maximum (from 417 to 424 nm ) using GraphPad Prism 6
1
5
µ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
9
max
software by fitting the data for the ligand-induced absorbance changes in the difference spectra Δ(A -
14
A
min) versus ligand concentration to the quadratic equation 1 (tight-binding ligands),
2
0.5
Δ
A=(Δ
d d
Amax/2E)((L+E+K )-((L+E+K ) -4LE) ) (Eq.1)
where [L] and [E] are the total concentrations of ligand and enzyme used for the titration, respectively.
Microsomal stability assay. Intrinsic clearance in hepatic microsomes (human, rat, mouse). Human
(mixed gender), rat (male Sprague-Dawley), or mouse (male CD-1) pooled liver microsomes (0.5 mg/mL, BD
Biosciences) and 1
µM test compound were incubated in 100 mM potassium phosphate buffer (pH 7.4)
with 3 mM MgCl at 37 °C with constant shaking. After a 5 min preincubation, each reaction was initiated
2
by the addition of 1 mM NADPH. At selected time intervals (0, 3, 7, 15, 25, and 45 min), 50
taken and subsequently placed into a 96-well plate containing 150 L of cold CH CN with internal standard
50 ng/mL carbamazepine). Plates were then centrifuged at 3000 g (4 °C) for 10 min, and the supernatants
were transferred to a separate 96-well plate and diluted 1:1 (v/v) with H O for LC/MS/MS analysis. The
µL aliquots were
µ
3
(
2
experiments were performed in triplicate. The in vitro half-life (T1/2, min, Eq. 1), estimated intrinsic
clearance (CLINT, mL/min/kg, Eq. 2) and subsequent predicted hepatic clearance (CLHEP, mL/min/kg, Eq. 3)
were determined employing the following equations:
(
1)
represents the slope from linear regression analysis of the natural log percent remaining of test
compound as a function of incubation time
where
k
(2)
2
8
ACS Paragon Plus Environment

Page 29 of 37
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
a
scaling factor
(3)
h
(hepatic blood flow) is 21 mL/min/kg for human, 70 mL/min/kg for rat, and 90 mL/min/kg for
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
where Q
mouse.
Antifungal drug susceptibility test. The Minimal Inhibitory Concentration (MIC) of each drug against the
various A. fumigatus strains was investigated using the Clinical and Laboratory Standards Institute broth
3
0
microdilution method, document M38-A2. The tests were performed in triplicate. Various strains of A.
®
30
fumigatus, 1022, 32820, and MYA626 (ATCC MP-12 kit), were grown according to the protocol. Briefly,
o
the strains were initially plated onto potato dextrose agarose plates for 5 to 6 days at 35 C to generate
conidias. The agar plate surfaces were washed with 5 mL of sterile 1x phosphate-buffered saline with 0.05%
Tween-80 (v/v) to obtain the independent conidias, and the number of conidias were counted with a
4
hemocytometer. The conidias were further diluted in RPMI media to obtain 1 x 10 conidias/mL and seeded
into each well of 96-well plates. The testing media was RPMI 1640 (Gibco-BRL, Uxbridge, UK) with L-
glutamine (without sodium bicarbonate, pH 7.0) adjusted with 0.165 M morpholinepropanesulphonic acid
31
(MOPS). Serial base 2x dilutions of various drugs (50 to 0.006 mg/L) were prepared in RPMI-1640 and
added to the wells of microtiter plates in 100-µL aliquots. Media alone (200 µL) was used as positive control
of mycelia growth and 200
µ
L of media alone without conidias was used as negative control. The plates
o
were further incubated for 48 hours at 35 C and observed under light microscopy for the growth of
mycelia. MICs were the lowest drug concentrations that produced complete growth inhibition (100%) at 48
h of incubation.
Protein Crystallization, Data Collection, Structure Refinement, and Analysis. For crystallographic
experiments, A. fumigatus CYP51B was purified in three steps, including anion exchange chromatography
2
9
ACS Paragon Plus Environment