Design, synthesis, and structure-activity relationship studies of novel fused heterocycles-linked triazoles with good activity and water solubility

Article abstract of DOI:10.1021/jm4016284

Triazoles with fused-heterocycle nuclei were designed and evaluated for their in vitro activity on the basis of the binding mode of albaconazole using molecular docking, along with SAR of antifungal triazoles. Tetrahydro-[1,2,4] triazolo[1,5-a]pyrazine and tetrahydro-thiazolo[5,4-c]pyridine nuclei were preferable to the other four fused-heterocycle nuclei investigated. Potent in vitro activity, broad spectrum and better water solubility were attained when triazoles containing nitrogen aromatic heterocycles were attached to these two nuclei. The most potent compounds 27aa and 45x, with low hERG inhibition and hepatocyte toxicity, both exhibited excellent activity against Candida, Cryptococcus, and Aspergillus spp., as well as selected fluconazole-resistant strains. A high water-soluble compound 58 (the disulfate salt of 45x) displayed unsatisfactory in vivo activity because of its poor PK profiles. Mice infected with C.alb. SC5314 and C.alb. 103 (fluconazole-resistant strain) and administered with 27aa displayed significantly improved survival rates. 27aa also showed favorable pharmacokinetic (PK) profiles.

Full text of DOI:10.1021/jm4016284

Article
pubs.acs.org/jmc
Design, Synthesis, and Structure−Activity Relationship Studies of
Novel Fused Heterocycles-Linked Triazoles with Good Activity and
Water Solubility
Xufeng Cao,†,∥ Zhaoshuan Sun,‡,∥ Yongbing Cao,§ Ruilian Wang,§ Tongkai Cai,§ Wenjing Chu,†
Wenhao Hu,*,‡ and Yushe Yang*,†
†State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institute for Biological Sciences, Chinese
Academy of Sciences, Shanghai 201203, China
‡Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development East China Normal University
Shanghai 200062, China
§School of Pharmacy, Second Military Medical University, Shanghai 200433, China
S
* Supporting Information
ABSTRACT: Triazoles with fused-heterocycle nuclei were
designed and evaluated for their in vitro activity on the basis of
the binding mode of albaconazole using molecular docking,
along with SAR of antifungal triazoles. Tetrahydro-[1,2,4]-
triazolo[1,5-a]pyrazine and tetrahydro-thiazolo[5,4-c]pyridine
nuclei were preferable to the other four fused-heterocycle
nuclei investigated. Potent in vitro activity, broad spectrum
and better water solubility were attained when triazoles
containing nitrogen aromatic heterocycles were attached to
these two nuclei. The most potent compounds 27aa and 45x,
with low hERG inhibition and hepatocyte toxicity, both
exhibited excellent activity against Candida, Cryptococcus, and
Aspergillus spp., as well as selected fluconazole-resistant strains. A high water-soluble compound 58 (the disulfate salt of 45x)
displayed unsatisfactory in vivo activity because of its poor PK profiles. Mice infected with C.alb. SC5314 and C.alb. 103
(fluconazole-resistant strain) and administered with 27aa displayed significantly improved survival rates. 27aa also showed
favorable pharmacokinetic (PK) profiles.
The first-generation azoles, fluconazole (1) and itraconazole
(2) (Figure 1), have been widely applied clinically. However,
several factors have limited their practical applications. For
example, Aspergillus infections have become so common in
recent years that many experts believe that Aspergillus rather
than Candida is the major problem.11 Unfortunately, this
organism is intrinsically resistant to fluconazole. In addition, the
number of fluconazole-resistant strains has increased signifi-
cantly with the increased use of fluconazole.12 The use of
another triazole antifungal compound, itraconazole, which
demonstrates stronger activity against Aspergillus than does
fluconazole, is hampered by its poor aqueous solubility and oral
bioavailability.13 During the past decade, many novel azoles
have been developed to overcome these drawbacks. The
second-generation azoles, voriconazole (3), posaconazole (4),
ravuconazole (5), isavuconazole (6), and albaconazole (7)
(Figure 1), which exhibit favorable antifungal spectrum,
improved pharmacokinetic (PK) properties, and acceptable
toxicity profiles, have been properly investigated in clinical
INTRODUCTION
■
During the past two decades, a tremendous growth in the
incidence of systemic fungal infections has become a significant
cause of morbidity and mortality in immunocompromised
patients.1−3 These infections resulted from many factors,
including the growing number of oncology patients undergoing
chemotherapy, the development of transplantation medicine,
the acute immunodeficiency syndrome epidemic,4 and the
more widespread use of potent pharmacologic immunomodu-
lators and broad-spectrum antibiotics. The most severe fungal
infections are associated with three major pathogens from the
Candida spp., Cryptococcus neoformans (C.neo.), and Aspergillus
spp.5 The use of antifungal agents to treat these infections has
increased during last 20 years. These antifungals can be divided
into four different classes based on their mode of action: the
polyenes (such as amphotericin B and nystatin),6 azoles,7
echinocandins (such as caspofungin and micafungin),8 and
antimetabolites (such as 5-fluorocytosine).9 Among these
agents, azoles are the most commonly used antifungal agents
because of their broader antifungal spectrum, better efficacy,
and acceptable safety profile.10
Received: October 21, 2013
Published: February 24, 2014
© 2014 American Chemical Society
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Figure 1. Azole antifungals (names refer to single enantiomers, except itraconazole, which is racemic).
Figure 2. The chemical structures of the prodrugs in clinical trials.
trials.14 Some types of fungal infections such as pulmonary
infections with Aspergillus can cause swallowing difficulties in
severely ill individuals, so there is a pressing medical need for
injectable antifungal agents with a broad spectrum for treating
patients with dysphagia.15 However, most of the second-
generation azoles cannot be administered parenterally because
of their poor water solubility.16 To overcome this problem,
prodrug approaches are employed to improve their aqueous
solubility. For example, isavuconazium chloride hydrochloride
(BAL-8557) (8) and bis-(L-lysine) salt form BMS-379224 (9)
are the prodrugs of isavuconazole and ravuconazole, which have
also been assayed in clinical trials respectively (Figure 2).17−19
However, these prodrugs liberate an equivalent amount of toxic
formaldehyde or acetaldehyde in vivo when cleaved.20 In
addition, the discovery and development of these types of
prodrugs have been proved to be time-consuming and costly.21
Therefore, there is still an urgent need for novel antifungal
agents with broad spectrum, high aqueous solubility, and low
toxicity.
antifungal agent with high potency against pathogenic yeasts
and filamentous fungi, favorable PK, and acceptable toxicity
profile.22 It also showed excellent in vivo activities against
Candida, Aspergillus, and Cryptococcus species; however, its low
water solubility may hinder treatment in acute cases. This has
prompted us to undertake a structure−activity relationship
(SAR) investigation of albaconazole to maintain it potent
activity while significantly increasing its aqueous solubility. Like
the other triazole antifungal agents, albaconazole exerts
antifungal activity via inhibition of the lanosterol 14α-
demethylase (P45014DM, CYP51), which catalyzes the oxidative
removal of the 14α-methyl group of lanosterol to give Δ14,15
-
desaturated intermediates in the ergosterol biosynthetic
pathway.22,23 In yeasts and filamentous fungi (e.g., Candida
spp. and Aspergillus spp.), CYP51 is the key enzyme in sterol
biosynthesis and selective inhibition of CYP51 would cause the
growth inhibition of fungal cells.7 Because of its importance in
antifungal drug studies, CYP51 was studied as a target of
albaconazole for computational molecular docking which has
been widely applied in the rational design of antifungal
agents.5,24−27 Thus, albaconazole was docked into the active
site of Candida albicans (C.alb.) CYP51 (CACYP51) using the
well validated docking software GOLD. Sheng’s modeling
study5 revealed that the active site of CACYP51 can be divided
In our continuing effort to find antifungal triazoles with more
desirable properties, we focused on identifying antifungal agents
with potent activity, broad antifungal spectrum, and high
aqueous solubility through structural modification of the
original compounds. Albaconazole (7) is a novel orally active
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Figure 3. The binding mode of albaconazole (A) and compound 27aa (B) in the active site of CACYP51.
Figure 4. Previous selected compounds and new structures.
into four pockets (S1−S4). As shown in Figure 3A, the triazole
ring and difluorophenyl group of albaconazole formed a
coordination bond and hydrophobic interactions with S2
heme group (Fe atom) and S3 pocket (Phe126 and Tyr132),
respectively. Although the C2 hydroxyl group of albacoanole
did not form direct interaction with CACYP51, it might form
hydrogen-bonding interaction with His310 in the S1 pocket
mediated by crystal waters.5,28 The C3 side chain of albacoanole
was located in the S4 pocket, which represents a hydrophobic
hydrogen bond binding site (facing FG loop). The
quinazolinone ring formed hydrophobic interactions with
surrounding residues such as Tyr118, Leu121, Met508, and
Phe380.
On the basis of the binding mode of albaconazole, along with
the structural features of triazoles reported in the patent and
scientific literature,7,29−31 we designed and synthesized six
series of novel azoles (depicted by general formulas I and II)
with a five-membered heterocyclic ring (B ring) fused to the
piperazine or piperidine (A ring) as side chains (Figure 4).
Their design rationale was based on the following consid-
erations: (1) Because of the importance of the 1,2,4-triazole
moieties, 2,4-difluorophenyl, C2 hydroxyl group, and C3-
methyl, these groups were retained and optimization studies
were mainly focused on the C3-side chains. (2) A five-
membered heterocyclic ring (B ring) fused to the piperazine or
piperidine (A ring) was designed to mimick the hydrophobic
interactions of quinazolinone ring of albaconazole. Moreover,
new triazoles with these nitrogen-containing heterocycles were
expected to have better water solubility than albaconazole and
therefore improve the pharmacodynamics and pharmacoki-
netics profiles.32 (3) The linkers can restrict the side chains to
their proper conformation and thereby strongly impact the
activity of compounds.5 (4) Attaching an aryl group to the
linker of triazole derivatives may lead to the improvement of
the antifungal activity, as suggested by a number of recent
studies.24,26,33,34 (5) Aromatic groups were introduced on the
fused heterocyclic ring to enhance the possible interactions
with S4 pocket of CACYP51. Various attaching positions (e.g.,
compounds 12a−l and 19a−l) were also considered to
investigate their effect on the conformation of the side chains
and thereby the influence on the antifungal activity. Moreover,
incorporation of basic N-containing aromatic group into the B
ring may enhance their water solubility or lead to a higher
chance of salt formation of target molecules, which is the most
common and effective route to increasing the solubility. We
therefore focused on SAR studies on R1 and R2-substituted
fused heterocyclic derivatives I and II. In addition, we
performed a detailed, systematic study of salt formation of
the most potent compounds.
During the present study, two more potent classes of
compounds containing triazolopiperazine and thiazolopiper-
idine units have been of interest. These compounds exhibit
strong in vitro antifungal activity against Candida spp.,
Aspergillus spp., and Cryptococcus ssp. In addition, the most
potent compounds 27aa and 45x also exhibited high activity
against fluconazole-resistant strains with relatively low hERG
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a
Scheme 1. Synthesis of R-Substituted [1,2,3]-Triazolo[1,5-a]piperdine Derivatives As Final Products 12a−l
a
Reagents and conditions: (a) LiClO4, CH3CN, 80 °C, 12−24 h; (b) 10% Pd−C, H2, room temp, overnight; (c) R3B(pin) or R3B(OH)2, Cs2CO3,
Pd(PPh3)4, 1,4-dioxane/H2O, 80 °C, 12 h.
a
Scheme 2. Synthesis of [1,2,3]-Triazolo[1,5-a]piperdine Intermediates As Side Chains 11a−g
a
Reagents and conditions: (a) K2CO3, RCCCH2Cl, CH3CN, reflux, 6 h; (b) (i) SOCl2, pyridine, CH2Cl2, 0 °C 2 h; (ii) NaN3, DMF, 80 °C, 8 h;
(c) Pd(OH)2, 50 °C, 12 h; (d) (Boc)2O, triethylamine, room temp, 4 h; (e) NBS, 40 °C overnight; (f) NCS, 40 °C overnight; (g) LiOH·H2O,
MeOH/H2O, room temp, 2 h; (h) 4N HCl/dioxane, room temp, 6 h; (i) SOCl2, Pyridine, CH2Cl2, 0 °C then room temp 6 h; (j) H2, Pd/C, room
temp, overnight; (k) I2, K2CO3, CF3CH2OH, 50 °C, 4 h; (l) K2CO3, dimethylamine hydrochloride, CH3CN, 60 °C 8 h.
inhibition and hepatocyte toxicity. Most importantly, the
disulfate salt of 45x (compound 58) exhibits high aqueous
solubility (>100 mg/mL) at near-neutral pH, which offers
moderate protection in murine models of systemic infection
with C.alb. (strain SC5314). Another potent compound 27aa
conferred significantly improved survival rates for a murine
model infected with C.alb. SC5314 and C.alb. 103.
(fluconazole-resistant strain). It also showed a favorable PK
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a
Scheme 3. Synthesis of R-Substituted 5,6,7,8-Tetrahydro-imidazo[1,2-a]pyrazine Derivatives As Final Products 19a−l
a
Reagents and conditions: (a) LiClO4, CH3CN, 80 °C, 12−24 h.
a
Scheme 4. Synthesis of R-Substituted 5,6,7,8-Tetrahydro-imidazo[1,2-a]pyrazine Intermediates As Side Chains 18a−l
a
Reagents and conditions: (a) reflux in ethanol 12 h; (b) Pd/C(10%), MeOH, room temp, 12 h; (c) React with ammonia in a sealed tube, 100 °C,
48 h; (d) DMF, 35−40 °C, 12 h.
profile. This paper describes the synthesis and SARs of the
novel antifungal agents and the selection of 27aa for further
development.
or boric acid. The R3-substituted [1,2,3]-triazolo[1,5-a]-
piperidine intermediates (16a, 16b, and 11c−g) were prepared
according to published procedures,37 as summarized in Scheme
2. Nucleophilic substitution between N-benzylethanolamine 13
and RCCCH2Cl (R = −H or −CH2OAc) yielded
compounds 14a and 14b, which were converted to benzyl-
substituted [1,2,3]-triazolo[1,5-a]piperidine intermediates (15a
and 15b) through chlorination and intramolecular cyclo-
addition.38 Compounds 15a and 15b were treated with 10%
Pd(OH)2 under a H2 atmosphere to afford intermediates 16a
and 16b. The reaction of compound 16a and 16b with di-tert-
butyl dicarbonate and triethylamine afforded compounds 17a
and 17b. Treatment of 17a with N-bromosuccinimide (NBS)
or N-chlorosuccinimide (NCS) in CH3CN afforded halogen-
ated compounds 17c and 17d, respectively. During hydrolysis
of 17b, compound 17e was obtained in high yield.
Subsequently, these N-Boc-protected compounds were reacted
with hydrochloric acid in 1,4-dioxane to yield the key
CHEMISTRY
■
As shown in Scheme 1, the products 12c−d and 12f−g were
synthesized via ring-opening reaction between R1-substituted
[1,2,3]-triazolo[1,5-a]piperidine side chains (11c−d and 11f−
g) and (2R,3S)-2-(2,4-difluorophenyl)-3-methyl-2-(1H-1,2,4-
triazole-1-yl) methyl oxirane 1035 in the presence of LiClO4
by refluxing in acetonitrile (Scheme 1).36 However, the R2-
substituted final products 12a−b and 12e could not be
obtained in the same manner for unknown reasons. Compound
12a was generated in high yield from bromide-substituted
compound 12c by catalytic hydrogenation. Synthesis of [1,2,3]-
triazolo[1,5-a]piperidine derivatives bearing aryl groups is also
outlined in Scheme 1. Compounds 12h−l were obtained by
Suzuki coupling of compound 12c with various boric acid esters
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Scheme 5. Synthesis of R-Substituted 5,6,7,8-Tetrahydro-[1,2,4]triazolo[1,5-a]pyrazine Derivatives As Final Products 27a−ag
a
and 29
a
Reagents and conditions: (a) LiClO4, CH3CN, 80 °C, 12−24 h; (b) LiClO4, CH3CN, microwave 125 °C P (80 W), 4 h; (c) R2B(OH)2 or
R2B(pin), Cs2CO3, Pd(PPh3)4, 1,4-dioxane/H2O, 80 °C, 10 h; (d) R3B(OH)2 or R3B(pin), Cs2CO3, Pd(PPh3)4, 1,4-dioxane/H2O, 80 °C, 8 h.
Scheme 6. Synthesis of R-Substituted 5,6,7,8-Tetrahydro-[1,2,4]triazolo[1,5-a]pyrazine Intermediates As Side Chains 26a−
a
26b
a
Reagents and conditions: (a) (CF3CO)2O, Et3N, CH2Cl2, 0 °C then room temp, 2 h; (b) PCl5, dichloroethane, reflux, 4 h; (c) 50% aq NH2OH,
THF, rt, 2 h; (d) polyphosphoric acid, 100 °C, 2 h; (e) H2, 10% Pd/C, EtOH, rt, 8 h; (f) N,N-dimethylformamide dimethyl acetal, toluene, reflux, 3
h; (g) hydroxylamine hydrochloride, sodium acetate, MeOH, 0 °C overnight.
intermediates 16a and 16b and 11c−e. Chlorination of 17e
with thionyl chloride and pyridine followed by reductive
dehalogenation and deprotection afforded intermediate 11f.
Meanwhile, 17e was treated with iodine in 2,2,2-trifluoroetha-
nol to form 17h, which was converted to 17i in CH3CN.
Finally, the key intermediate 11g was produced from 17i by
further deprotection.
19a−l were synthesized via ring-opening reactions between
oxirane 10 and intermediates 18a−l, as shown in Scheme 3.
Preparation of the side chains 18a−l is outlined in Scheme 4.39
Cyclization of 2-aminopyrazine 20 with R1-substituted α-
bromoacetone 21a−c, followed by catalytic hydrogenation,
gave key intermediates 18a−c. However, this similar procedure
was less applicable to the synthesis of side chains bearing the
aryl group. The reaction between 2-aminopyrazine 20 and R2-
substituted α-bromoacetone 21d−l yielded no bicyclic product
even after longer reaction time. Similar to what was described
in detail in a previous study,40 alkylation by α-bromoacetone
21d−l did not take place at the amino group. Instead, it mainly
occurred at the more nucleophilic nitrogen not adjacent to the
amino group, thereby preventing synthesis of the desired side
chains. According to this study, placing a chlorine atom in the
ring adjacent to the amino group can reduce the nucleophilicity
of the nonadjacent nitrogen. Thus, 2,3-dichloropyrazine 23 was
the starting material subjected to amination, cyclization, and
hydrogenation to obtain the desired bicyclic products 18d−l,
which are key intermediates in the preparation of the target
aryl-substituted molecules shown in Scheme 3.41
Synthesis of the title compounds 27a−ag was carried out
according to Scheme 5. Among these compounds, 27a−w were
prepared by ring-opening reaction of epoxide 10 with side
chains 26a−w in the presence of a base at 80 °C. However, the
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a
Scheme 7. Synthesis of R-Substituted 5,6,7,8-Tetrahydro-[1,2,4]triazolo[1,5-a]pyrazine Intermediates As Side Chains 26c−ag
a
Reagents and conditions: (a) 1,4-dioxane, room temp, 12 h; (b) triethylamine, hydroxylamine hydrochloride, MeOH/EtOH, room temp, 2 h, then
reflux 4 h; (c) NaNO2, CuBr, HBr, acetic acid/H2O, 0 °C 2 h then reflux overnight; (d) LiBH4, EtOH, 50 °C, 6 h; (e) (Boc)2O, triethylamine, room
temperature, 2 h; (f) R2,3B(OH)2 or R2, 3B(pin), Cs2CO3, Pd(PPh3)4, 1,4-dioxane/H2O, 80 °C, 10 h; (g) HCl/dioxane, room temp, 6 h.
title compounds (27x−ag), bearing aromatic nitrogen hetero-
cycles, could not be obtained in the same manner described
above. Extremely low yield was obtained when ring-opening
reactions were performed by using the conventional method or
microwave technology. This reaction might not have proceeded
as expected because of the nucleophilic N atom of aromatic
heterocycles, which could also attack the carbon atom of the
epoxide 10, leading to ring-opening.42,43 Thus, bromine-
substituted compound 29 was considered for use as a pivotal
precursor in Suzuki coupling to efficiently prepare R3-
substituted title compounds 27x−ag. R2-substituted title
compounds 27c−w were also more conveniently obtained via
similar procedures.
Scheme 8. Synthesis of 4,5,6,7-Tetra-hydro-thiazolo[5,4-
c]pyridine Intermediates as Side Chain 43
a
As shown in Scheme 6, side chains 26a and 26b were
obtained by slight modifications of published procedures.44−46
Aminopyrazine 20 was acylated with trifluoroacetic anhydride
to afford the acetamide 30, which was treated with phosphorus
pentachloride and then with hydroxylamine to give 31.
Cyclization of hydroxyimideamide 31 with polyphosphoric
acid followed by catalytic hydrogenation gave 26a. The
unsubstituted triazolopiperazine side chain 26b was prepared
from aminopyrazine 20 by using procedures similar to those
described above.
a
Reagents and conditions: (a) (i) pyrrolidine, p-toluenesulfonic acid
monohydrate, cyclohexane, reflux, 2 h, (ii) elemental sulfur,
cyanamide, MeOH, 0 °C then room temp 6 h; (b) tBuONO,
CuBr2, DMF, 50 °C, 4 h. (c) HCl/dioxane, room temp, 8 h.
generated by microwave-assisted ring-opening reaction between
oxirane 10 and bromine-substituted compound 43. Afterward,
the target compounds 45a−ab were prepared conveniently via
palladium-catalyzed Suzuki coupling (Scheme 9).
As outlined in Scheme 7, aryl-substituted triazolopiperazine
derivatives 26c−ag were synthesized via seven steps.
Compound 20 was treated with isothiocyanate 34 to give a
bicyclic product 35,47 which was then brominated and
hydrogenated to afford bromide-substituted compound 28.
The amino group of 28 was protected with (Boc)2O. R2,3-
Substituted compounds 38c−af were assembled by Suzuki
coupling of bromide-substituted compound 37 with a series of
R2,3-substituted boronic acids or boric acid esters. Subsequently,
cleavage of the Boc protecting group of compounds 38c−af
with hydrochloric acid gave R2,3-substituted 5,6,7,8-tetrahydro-
imidazo[1,2-a]pyrazine derivatives 26c−ag as side chains.
Synthesis of tetrahydro-thiazolo[5,4-c]pyridine intermediates
was carried out according to Scheme 8. A known method48 was
applied to convert 40 to 43. Enamine formation of 40 with
pyrrolidine in the presence of p-toluenesulfonic acid mono-
hydrate, followed by reaction with elemental sulfur and addition
of cyanamide, afforded bicyclic product 41. Bromidation with
tBuONO and CuBr2 gave bromine-substituted compound 42,
which was subsequently deprotected by hydrochloric acid to
give tetrahydro-thiazolo[5,4-c]pyridine intermediate 43 as side
chain. The novel fragment 44, a pivotal precursor, was
Triazoles 50a−o with tetrahydro-thieno[3,2-c]pyridine side
chains were synthesized through conventional methods, as
depicted in Scheme 10.49 The amino group of compound 46
was first protected by using di-tert-butyl dicarbonate and then
brominated to give hydrobromide 48, which was subsequently
treated with oxirane 10 to afford key intermediate 49. Finally, a
well-known coupling reaction between 49 and R-substituted
boronic acids or boric acid esters yielded target compounds
50a−o.
Triazoles 57a−d were synthesized from compound 51 in six
steps as shown in Scheme 11.50,51 The reduction of the nitrile
group using borane dimethyl sulfide complex, followed by the
formation of Boc-protected amines and cyclization, constructed
the core bicyclic ring system to give compound 54. Bromine-
substituted intermediate 56 was obtained from compound 55
via ring-opening reaction. In the same way, triazoles 57a−d
having R-substituted tetrahydro-thieno[2,3-c]pyridine were
prepared through Suzuki coupling reaction in high yield.
RESULTS AND DISCUSSION
In Vitro Activity. The target compounds were evaluated for
their antifungal activity against a panel of pathogenic fungi
■
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a
Scheme 9. Synthesis of R-Substituted 4,5,6,7-Tetrahydro-thiazolo[5,4-c]pyridine Intermediates As Final Products 45a−ab
a
Reagents and conditions: (a) LiClO4, CH3CN, microwave 80 °C P (80 W), 10 h; (b) RB(OH)2 or RB(pin), Cs2CO3, Pd(PPh3)4, 1,4-dioxane/
H2O, 80 °C, 10 h.
a
Scheme 10. Synthesis of R-Substituted 4,5,6,7-Tetrahydro-thieno[3,2-c]pyridine Intermediates As Final Products 50a−o
a
Reagents and conditions: (a) (Boc)2O, triethylamine, room temp, 6 h; (b) Br2, CHCl3, 0 °C then room temp, 12 h; (c) LiClO4, CH3CN, 80 °C,
12−24 h; (d) RB(OH)2 or RB(pin), Cs2CO3, Pd(PPh3)4, 1,4-dioxane/H2O, 80 °C, 10 h.
a
Scheme 11. Synthesis of 4,5,6,7-Tetrahydro-thieno[2,3-c]pyridine Intermediates As Final Products 57a−d
a
Reagents and conditions: (a) THF, BH3 Me2S, 0 °C then reflux, 12 h; (b) K2CO3, (Boc)2O, THF/H2O, room temp 6 h; (c) paraformaldehyde, p-
toluenesulfonic acid monohydrate, toluene, reflux, 2 h; (d) CHCl3, Br2, from 0 °C to room temp 12 h; (e) LiClO4, CH3CN, 80 °C, 12−24 h; (f)
RB(OH)2 or RB(pin), Cs2CO3, Pd(PPh3)4, 1,4-dioxane/H2O, 80 °C, 10 h.
obtained from the American Type Culture Collection or
clinical isolates, including fluconazole-resistant strains of C.alb.
The results of in vitro antifungal activities are summarized in
Tables 1−6.
Base on the considerations mentioned above for structural
optimization of albaconazole, the [1,2,3]-triazolopiperidine
nucleus was first chosen to mimick the hydrophobic
interactions of quinazolinone ring of albaconazole. Thus, a
series of R-substituted [1,2,3]-triazolopiperidine derivatives
were synthesized and evaluated for in vitro antifungal activity.
Of these, analogues with halogens (12c, R = Br; 12d, R = Cl)
exhibited the most potent activity against C.alb., Candida
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a
Table 1. In Vitro Antifungal Activity of [1,2,3]-Triazolo[1,5-a]piperidine Derivatives (MIC, μg/mL)
C.alb.
C.par.
C.neo.
C.gla.
A.fum.
compd
R
SC5314
Y0109
22019
32609
537
7544
12a
12c
12d
12f
12g
12h
12i
H−
Br−
Cl−
CH3−
(CH3)2NCO−
phenyl−
3-pyridyl−
0.0625
0.125
0.25
0.125
0.125
0.5
1
0.25
0.5
16
8
≤0.03125
≤0.03125
0.25
0.0625
≤0.0625
≤0.0625
4
0.25
0.0625
0.25
8
0.5
0.5
16
>16
>16
8
0.5
1
16
8
1
2
4
8
8
2
4
8
4
8
12j
4-pyridyl−
5-pyrimidinyl−
2-CN-5-pyridyl−
1
4
4
16
8
>16
>16
>16
>64
2
12k
12l
1
2
8
8
8
2
4
4
16
4
fluconazole
0.5
1
1
8
0.25
≤0.125
≤0.125
≤0.03125
0.5
voriconazole
ravuconazole
albaconazole
amphotericin B
≤0.0625
≤0.0625
≤0.03125
1
0.0625
0.0625
≤0.03125
4
0.125
0.125
0.0625
0.5
0.0625
≤0.0625
≤0.03125
4
1
0.5
4
a
Abbreviations: C.alb., Candida albicans; C.par., Candida parapsilosis; C.neo., Cryptococcus neoformans; C.gla., Candida glabrata; A.fum., Aspergillus
fumigatus.
a
Table 2. In Vitro Antifungal Activity of 5,6,7,8-Tetrahydro-imidazo[1,2-a]pyrazine Derivatives (MIC, μg/mL)
C.alb.
C. par.
C.neo.
C.gla.
A.fum.
compd
R
SC5314
Y0109
22019
32609
537
7544
19a
19b
19c
19d
19e
19f
19g
19h
19i
CF3−
EtOC(O)−
(CH3)3C−
4-CH30-phenyl−
4-F-phenyl−
4-CF3-phenyl−
2,4-diF-phenyl−
4-CH3-phenyl−
3-CH30-phenyl−
3-F-phenyl−
0.25
0.25
0.25
1
0.25
>64
>64
>64
32
32
32
32
32
32
64
64
16
>64
4
0.5
0.5
0.5
2
1
0.25
0.125
0.0625
0.125
0.25
0.25
0.25
0.25
0.0625
0.125
0.125
0.125
0.0625
0.0625
0.125
0.125
0.0625
0.5
0.125
0.125
0.25
0.125
0.125
0.125
0.125
0.125
0.0625
0.25
0.125
0.125
0.25
0.125
0.0625
0.0625
0.25
0.125
0.125
0.125
0.125
0.125
0.0625
2
0.125
0.125
0.125
0.25
19j
19k
19l
3-CF3-phenyl−
phenyl−
0.5
0.25
0.25
0.0625
0.5
0.03125
0.5
0.03125
0.5
fluconazole
voriconazole
ravuconazole
albaconazole
amphotericin B
0.0625
0.0625
0.0156
1
0.0625
0.0625
0.0156
2
0.25
0.0625
0.0625
0.0156
2
0.0625
0.0625
0.0156
2
0.125
0.0156
2
2
0.5
4
a
Abbreviations: C.alb., Candida albicans; C.par., Candida parapsilosis; C.neo., Cryptococcus neoformans; C.gla., Candida glabrata; A.fum., Aspergillus
fumigatus.
parapsilosis (C.par.), C.neo., and Candida glabrata (C.gla.),
which are comparable to those of the reference drugs
voriconazole and ravuconazole; however, the activity is still
inferior to that of albaconazole. Catalytic hydrogenation of
compound 12c afforded compound 12a, which showed
decreased potency against all of the tested strains. Methyl
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Article
a
Table 3. In Vitro Antifungal Activity of 5,6,7,8-Tetrahydro-[1,2,4]triazolo[1,5-a]pyrazine Derivatives (MIC, μg/mL)
C.alb.
C. par.
C.neo.
C.gla.
A.fum.
compd
R
SC5314
Y0109
22019
32609
537
7544
27a
27b
27c
27d
27e
27f
27g
27h
27i
H−
CF3−
phenyl−
4-CN-phenyl−
3-CH3-phenyl−
4-Br-phenyl−
0.25
0.25
0.5
0.25
0.5
0.5
0.5
16
32
32
64
32
16
32
16
64
16
16
16
>64
16
32
32
16
16
32
16
8
0.25
0.5
1
0.0625
0.125
0.0625
0.25
0.125
0.125
0.125
0.125
0.125
0.125
0.125
0.125
2
0.125
0.5
0.125
0.125
0.125
0.25
0.125
0.125
2
0.125
0.125
0.125
0.5
0.25
0.5
4-F-phenyl−
0.25
0.125
0.5
0.125
0.5
4-CI-phenyl−
3-CI-phenyl−
3-Br-phenyl−
3-CF3-phenyl−
4-CF3-phenyl−
2,4-diF-phenyl−
3-isopropyl-phenyl−
4-CH3-phenyl−
4-CH30-phenyl−
3-F-phenyl−
3-CN-phenyl−
3-CH30-phenyl−
4-isopropyl-phenyl−
2-thienyl−
2-furyl−
3-thienyl−
0.25
0.25
1
1
27j
0.25
0.5
1
1
27k
27l
0.5
0.5
2
0.25
0.125
0.5
0.25
0.5
0.5
1
27m
0.125
0.25
0.125
4
0.25
4
0.5
27n
8
4
27o
0.0625
0.0625
0.125
0.125
0.0625
0.5
0.125
0.125
0.125
0.125
0.125
0.5
0.125
0.25
0.5
0.125
0.125
0.5
0.125
0.125
0.25
0.5
27p
27q
27r
0.125
0.25
1
0.5
27s
0.25
2
0.125
1
27t
27u
0.125
0.125
0.125
0.125
0.0625
0.03125
0.03125
0.0156
0.0156
0.125
0.125
0.0625
0.0625
0.0625
0.5
0.125
0.125
0.125
0.125
0.0625
0.03125
0.125
0.0156
0.0625
0.125
0.125
0.125
0.125
0.125
1
0.125
0.25
0.125
0.25
0.03125
0.03125
0.0625
0.0156
0.0625
0.125
0.25
0.125
0.125
0.25
1
0.125
0.25
0.125
0.25
0.125
0.03125
0.0625
0.0078
0.03125
0.125
0.125
0.25
0.125
0.5
0.25
0.25
0.125
0.5
27v
16
16
16
16
32
32
1
27w
29
Br−
4-pyridyl−
3-pyridyl−
27x
0.125
0.03125
0.0625
0.0078
0.03125
1
27y
27z
5-pyrimidinyl−
2-CN-5-pyridyl−
2-F-5-pyridyl−
3-F-5-pyridyl−
3-CN-5-pyridyl−
2-methoxy-5-pyridyl−
2-methyl-5-pyrimidinyl−
2-methoxy-5-pyrimidinyl−
27aa
27ab
2
27ac
32
64
32
32
32
64
4
27ad
1
27ae
0.5
27af
0.25
0.25
1
27ag
fluconazole
voriconazole
ravuconazole
albaconazole
amphotericin B
2
0.0625
0.0625
0.0078
1
0.0625
0.0625
≤0.0156
1
0.25
0.125
0.0156
2
0.0625
0.0625
0.0078
1
0.0625
0.0625
0.0156
4
2
0.5
4
a
Abbreviations: C.alb., Candida albicans; C.par., Candida parapsilosis; C.neo., Cryptococcus neoformans; C.gla., Candida glabrata; A.fum., Aspergillus
fumigatus.
substitution of 12f (R = CH3) and introduction of the amide
group 12g (R = CO2NMe2) all resulted in poorer compounds.
To further investigate the effect of the substituent group (R
group) of triazolopiperidine derivatives on the antifungal
activity, aryl-substituted analogues 12h−l were prepared.
Unfortunately, the activity against tested fungi were lost in
these compounds.
Apparently, all of the target compounds discussed above
(Table 1) were ineffective against Aspergillus fumigatus; we
processed to survey other cycles. Considering the significant
impact of various attaching positions (R group) on the
conformation of the side chains and the activity of antifungal
triazoles,29 R-substituted tetrahydro-imidazo[1,2-a]pyrazine
nucleus was introduced to the molecule as a linker. As shown
in Table 2, the title compounds 19a−l were active against six
fungi to some extent. Generally, the aryl-substituted analogues
19d−l were more potent than compounds 19a−c, each of
which bearing a relatively small substituent. In particular, the
unsubstituted phenyl compound 19l displayed significant
activity against C.par., C. C.neo., and C.gla. which was 2−4-
fold greater than that of voriconazole and ravuconazole.
However, introduction of an electron-donating group such as
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Article
a
Table 4. In Vitro Antifungal Activity of 4,5,6,7-Tetrahydro-thiazolo[5,4-c]pyridine Derivatives (MIC, μg/mL)
C.alb.
C. par.
C.neo.
C.gla.
537
A.fum.
compd
R
SC5314
Y0109
22019
32609
7544
44
Br−
phenyl−
4-CI-phenyl−
4-CF3-phenyl−
4-CH30-phenyl−
4-CH3-phenyl−
4-F-phenyl−
4-Br-phenyl−
4-isopropyl-phenyl−
3-CI-phenyl−
3-F-phenyl−
3-CH3-phenyl−
3-Br-phenyl−
3-CH30-phenyl−
4-CN-phenyl−
3-CN-phenyl−
2-thienyl−
2-furyl−
3-thienyl−
4-pyridyl−
3-pyridyl−
1
1
1
0.5
1
32
16
32
32
16
16
16
16
16
32
32
32
32
16
16
16
8
45a
45b
45c
45d
45e
45f
45g
45h
45i
45j
45k
45l
0.5
0.5
1
0.5
2
0.125
1
0.5
0.5
1
1
2
2
2
1
1
1
0.5
0.5
2
1
1
0.5
1
0.5
1
1
1
1
4
1
1
4
1
2
0.5
1
0.5
2
1
1
4
4
2
4
2
2
2
2
8
2
2
1
4
4
2
2
2
4
8
45m
1
1
1
2
1
45n
0.0625
0.125
0.25
0.25
0.25
0.03125
0.03125
0.125
0.125
0.0625
0.0156
0.0625
0.25
0.0625
0.0625
0.5
0.125
0.125
0.25
1
0.125
2
0.125
1
0.5
2
45o
45p
1
0.5
0.5
0.5
0.25
0.125
0.125
0.125
0.5
0.5
0.0625
0.125
0.5
0.25
0.25
1
45q
1
0.5
8
45r
0.25
0.03125
0.03125
0.125
0.25
0.0625
0.0156
0.125
0.25
0.0625
0.125
0.5
0.25
0.125
0.125
0.25
0.25
0.25
0.0625
0.25
0.5
0.25
0.25
2
0.25
0.125
0.0625
0.125
0.25
0.25
0.0625
0.125
0.25
0.25
0.5
8
45s
4
45t
2
45u
3-F-5-pyridyl−
3-CN-5-pyridyl−
2-CN-5-pyridyl−
5-pyrimidinyl−
2-F-5-pyridyl−
2-methoxy-5-pyridyl−
2-methyl-5-pyrimidinyl−
2-methoxy-5-pyrimidinyl--
8
45v
8
45w
8
45x
1
45y
8
45z
16
16
8
45aa
45ab
fluconazole
voriconazole
ravuconazole
albaconazole
amphotericin B
1
>64
4
0.0625
0.0625
0.0078
1
0.0625
0.0625
≤0.0156
1
0.25
0.125
0.0156
2
0.0625
0.0625
0.0156
2
0.0625
0.0625
0.03125
4
2
0.5
4
a
Abbreviations: C.alb., Candida albicans; C.par., Candida parapsilosis; C.neo., Cryptococcus neoformans; C.gla., Candida glabrata; A.fum., Aspergillus
fumigatus.
methyl (19f) and methoxy groups (19d and 19i), or an
electron-withdrawing group such as a fluorine atom (19e, 19g,
and 19j) and trifluoromethyl group (19f and 19k), resulted in a
slight decrease in activity in vitro.
Compounds with an electron-withdrawing group (−F, −Cl,
−Br, −CF3) on the phenyl ring showed only moderate activity,
except for CN derivatives 27d and 27r, which had slightly lower
activity. Analogues with a phenyl ring substituted with electron-
donating groups (−CH3, −CH3O) exhibited activity similar to
those observed in unsubstituted phenyl compound 27c;
however, increasing the alkyl size (27n and 27t) significantly
reduced the activity against the six tested strains. To enhance
the possible interactions with S4 pocket of CACYP51, as well
as the water solubility, various aromatic groups were introduced
on the fused heterocyclic ring. A series of aromatic heterocycles
(thiophene ring, furan ring, pyridine ring, and pyrimidine ring)
were introduced at position 2 of the triazole ring of
[1,2,4]triazolo[1,5-a]pyrazine derivatives to afford compounds
27u−ag. The activity of thienyl analogues 27u, 27w, and furyl
analogue 27v were lower than those of the phenyl analogues.
Given the relatively high in vitro activity of compound 19a−
l, their tetrahydro-imidazo[1,2-a]pyrazine nucleus were re-
placed with tetrahydro-[1,2,4]triazolo[1,5-a]pyrazine nucleus
for further optimization. Thus, we first prepared many
tetrahydro-[1,2,4]triazolo[1,5-a]pyrazine derivatives featuring
a relatively small substituent or phenyl ring (Table 3). Similar
to compounds 19a−l, analogues having a hydrogen (27a), alkyl
group (27b), or bromine atom (29) were less active than
phenyl-substituted compounds 27c−t. Substitution within this
phenyl ring was investigated. We found that the in vitro
antifungal activity of para- and meta-substituted compounds
(27d and 27r, 27e and 27o, 27k and 27l) were similar.
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Journal of Medicinal Chemistry
Article
Table 5. In Vitro Antifungal Activity of 4,5,6,7-Tetrahydro-thieno[3,2-c]pyridine Derivatives and 4,5,6,7-Tetrahydro-thieno[2,3-
a
c]pyridine Intermediates (MIC, μg/mL)
C.alb.
C. par.
C.neo.
C.gla.
A.fum.
compd
R
SC5314
Y0109
22019
32609
537
7544
49
Br−
phenyl−
2
4
8
2
4
64
32
64
32
32
8
50a
50b
50c
50d
50e
50f
50g
50h
50i
50j
50k
50l
1
8
8
2
4
4
4-F-phenyl−
4-CF3-phenyl−
4-CI-phenyl−
4-CH3-phenyl−
4-CN-phenyl−
3-CH3-phenyl−
3-F-phenyl−
3-CN-phenyl−
3-CI-phenyl−
3-CF3-phenyl−
5-pyrimidinyl−
2-CN-5-pyridyl−
4-pyridyl−
3-pyridyl−
Br−
4-pyridyl−
3-pyridyl−
5-pyrimidinyl−
2-CN-5-pyridyl−
2
2
8
2
4
8
16
8
16
16
4
4
8
16
8
1
2
4
4
2
8
8
4
8
64
16
8
1
2
2
4
4
1
2
4
1
1
2
4
4
2
8
64
>64
32
>32
32
>32
32
32
32
8
2
2
4
4
8
1
0.5
2
0.5
1
2
4
4
4
8
50m
1
4
2
1
2
50n
1
2
4
4
8
50o
1
8
4
4
2
56
1
0.5
2
0.5
1
57a
1
4
2
1
2
57b
0.5
0.5
1
1
0.5
1
57c
0.25
0.5
4
0.5
1
16
32
>64
2
57d
2
4
4
4
fluconazole
voriconazole
ravuconazole
albaconazole
amphotericin B
0.5
1
1
8
0.25
≤0.0625
≤0.0625
≤0.03125
1
0.0625
0.0625
≤0.03125
4
0.125
0.125
0.0625
0.5
0.0625
≤0.0625
≤0.03125
4
≤0.125
≤0.125
≤0.03125
0.5
1
0.5
4
a
Abbreviations: C.alb., Candida albicans; C.par., Candida parapsilosis; C.neo., Cryptococcus neoformans; C.gla., Candida glabrata; A.fum., Aspergillus
fumigatus.
Most of the pyridine and pyrimidine ring analogues (27x−ag)
were tested and proved to be superior or comparable to those
of the phenyl analogues, except for 27ac and 27ad, which
featured an electron-withdrawing group (−F, −CN) at the
meta position of the m-pyridyl ring. Compared with the
pyridine ring analogue 27ae (−CH3O) and pyrimidine ring
analogues 27af−ag (−CH3 and −CH3O), compounds 27aa
(−CN) and 27ab (−F) with ortho substitution on the meta-
pyridyl ring and unsubstituted pyrimidine ring analogue 27z
exhibited stronger activity against Candida spp. and Crypto-
coccus spp. Most importantly, 27aa and 27ab also showed
remarkable activity against A.fum., which was superior or
comparable to those of the reference drug and the most potent
antifungal agent, albaconazole.
To further investigate the potential mechanism and the SAR
of tetrahydro-[1,2,4]triazolo[1,5-a]pyrazine derivatives 27a−ag,
a representative compound, 27aa, was docked into the active
site. As depicted in Figure 3B, the triazolopyrazine group
formed hydrophobic interaction with Tyr118, Leu121, Met508,
and Phe380, similar to the quinazolinone ring in albaconazole.
Moreover, the pyridinyl group of compound 27aa formed
additional hydrophobic interactions with Tyr505, Tyr64, and
Phe233. Most importantly, two nitrogen atoms in the cyano
and pyridinyl group formed two hydrogen bonds with Ser378,
which was a important residue for the affinity and specificity of
the CYP51 inhibitors.5,52 These results of docking may provide
a good explanation for the excellent in vitro activity of
compound 27aa.
Because of its significant activity and broad antifungal
spectrum, 27aa was subjected to PK studies in rats (Table
10). Compound 27aa exhibited favorable PK profile; however,
the water solubility of this compound was very poor and salt
formation of the weakly basic compound 27aa with various
acids was very difficult.
Accordingly, we surveyed other cycles by an approach similar
to that described above. To further investigate the effect of
different types of fused heterocycles (linker) on their antifungal
activity, many triazoles featuring tetrahydro-thiazolo[5,4-c]-
pyridine side chains were synthesized and tested for antifungal
activity. As shown in Table 4, the antifungal activity of R-
substituted phenyl compounds was very poor compared with
those of the control drug, except for CN-substituted
compounds 45n and 45o, which displayed moderate activity
against C.alb. Thus, we focused our efforts on an approach
similar to that described above. In particular, the benzene ring
on the side chain of compound 45a was replaced with a series
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Journal of Medicinal Chemistry
Article
Table 6. In Vitro Antifungal Activity of Selected Fused Heterocycles against Fluconazole-Resistant Strains of C.alb. (MIC, μg/
a
mL)
C.alb.
b
b
b
b
b
compd
12c
strain 100
strain 103
strain J18
strain J28
strain J38
>32
>32
>32
8
>32
>32
>32
>16
8
>32
>32
2
0.25
19l
0.5
0.125
0.0625
27c
>32
0.5
27aa
>16
0.0625
0.0625
>4
≤0.03125
≤0.03125
>4
45x
>16
512
>16
8
>16
fluconazole
voriconazole
albaconazole
>1024
>16
4
>1024
>16
0.0625
0.0625
>16
≤0.03125
≤0.03125
a
Abbreviations: C.alb., Candida albicans; strain 100, fluconazole-resistant strains of Candida albicans; strain 103, fluconazole-resistant strains of
Candida albicans; strain J18, fluconazole-resistant strains of Candida albicans; strain J28, fluconazole-resistant strains of Candida albicans; strain J38,
b
fluconazole-resistant strains of Candida albicans. Candida albicans (strain 100), Candida albicans (strain 103), Candida albicans (strain J18), Candida
albicans (strain J28), and Candida albicans (strain J38) were provided by Shanghai Changhai Hospital.
of aromatic heterocycle rings to develop compounds 45p−ab.
As expected, heterocycle-substituted analogues (45p−ab),
especially unsubstituted pyridine-ring analogues 45s and 45t
and unsubstituted pyrimidine analogue 45x, showed excellent
activity against Candida spp., Cryptococcus spp., and A.fum.
However, compounds 45u (−F) and 45v (−CN), which
featured meta substitution on the m-pyridyl ring, as well as 45y
(−F), 45w (−CN), and 45z (−CH3O), which feature ortho
substitution on the m-pyridyl ring, exhibited reduced activity.
The most potent compound, pyrimidinyl-substituted com-
pound 45x, exhibited 2−4-fold lower MICs than those of the
control drugs voriconazole and ravuconazole. However,
introduction of methyl and methoxy group in position 2 of
the pyrimidine ring resulted in poorer compounds 45aa and
45ab, respectively.
Because of the relatively high in vitro activity of tetrahydro-
thiazolo[5,4-c]pyridine derivatives 45a−ab, a series of tetrahy-
dro-thieno[3,2-c]pyridine derivatives (49 and 50a−o) and
tetrahydro-thieno[2,3-c]pyridine derivatives (56 and 57a−d)
were prepared through the bioisosteric approach. Results of in
vitro activity are summarized in Table 5. The tetrahydro-
thieno[3,2-c]pyridine analogue 49 (R = Br) and phenyl-
substituted analogues 50a−k showed very weak activity in vitro.
The in vitro antifungal activity of these analogues 50a−k was
independent of the electronic nature and position of the
substituent in the phenyl ring. In addition, similar poor activity
of analogues 50l−o substituted with aromatic heterocycle was
observed.
Exchanging the tetrahydro-thieno[3,2-c]pyridine side chains
of more potent target compounds 49 and 50l−o for tetrahydro-
thieno[2,3-c]pyridine intermediates produced novel fused
heterocycles 56 and 57a−d, respectively. However, none of
these target compounds exhibited interesting activity levels
compared with the reference drug.
Tables 1−5 suggest that both potent in vitro antifungal
activity and broad spectrum were attained when triazole-
containing nitrogen aromatic heterocycles were attached to the
2-triazole ring of tetrahydro-[1,2,4]triazolo[1,5-a]pyrazine
nucleus or 2-thiazole ring of the tetrahydro-thiazolo[5,4-
c]pyridine nucleus.
The widespread use of fluconazole has greatly increased the
number of C.alb. strains resistant to fluconazole, which has
been a major clinical problem for therapy for systemic
candidiasis.53 Therefore, there is an urgent need for novel
antifungal agents with improved activity against fluconazole-
resistant strains of C. alb. Thus, the in vitro antifungal activity of
more potent compounds against fluconazole-resistant strains of
C.alb. was further evaluated. As shown in Table 6, all of the
selected fused heterocycles displayed 16−32-fold more potency
against fluconazole-resistant strains of C.alb. (strains 100, 103,
and J18) than fluconazole. Among these compounds, 27aa and
45x showed remarkable activity against all fluconazole-resistant
strains tested, which was superior or comparable to those of
voriconazole and albaconazole.
hERG K+ Channel Inhibition. During the 10-year period, a
number of noncardiovascular drugs that block human ether-a-
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go-go related gene (hERG) K+ channels and delay cardiac
repolarization have been withdrawn because of sudden cardiac
deaths associated with drug-induced acquired length of time
between the start of the Q wave and end of the T wave on an
electrocardiogram (QT) interval prolongation.54,55 To reduce
risks of drugs as early as possible during development, QT
prolongation risks are predicted through the in vitro hERG
channel inhibition activity (IC50) in mammalian cell lines.56
Thus, the most potent compounds 27aa and 58 were selected
to evaluate their hERG K+ channel inhibition using Qpatch 16X
assay.57−62 As shown in Table 7, all tested compounds showed
mild inhibitory effects on the hERG channel compared with the
control drugs.
water solubility of the more potent compounds 27c, 27y, 27aa,
45a, 45t, and 45x at pH 1.2 and 7.4. As shown in Table 8,
Table 8. Solubility Data for Selected Compounds (Mean
SD, n = 3)
aqueous solubility
a
b
compd
27c
pH 1.2
pH 7.4
0.316 0.0600
5.900 0.0600
0.044 0.0060
1.250 0.0960
10.073 0.2580
14.673 0.7090
0.001 0.0006
0.003 0.0006
1.263 0.1060
0.097 0.0150
1.816 0.0930
27y
c
27aa
BDL
45a
0.130 0.0260
2.360 0.2460
3.093 0.2100
45t
45x
c
Table 7. hERG K+ Channel Inhibition of Selected
albaconazole
ravuconazole
voriconazole
BDL
c
a
BDL
Compounds (IC50, μM)
0.6820 0.0270
compd
27aa
hERG K+ channel inhibition
a
In mg/mL, determined in pH =1.2 hydrochloric acid solution, at 30
b
>40
°C. In mg/mL, determined in pH 7.4 phosphate buffer, at 30 °C.
c
58
>40
BDL, below the detection limit.
albaconazole
voriconazole
itraconazole
ravuconazole
cisapride
34.86
>40
pyridyl- or pyrimidinyl-substituted compounds 27y, 45t, and
45x were more soluble than were phenyl-substituted
compounds and the reference drugs albaconazole and
ravuconazole, indicating that the incorporation of nitrogen
aromatic heterocycles might have sufficiently improved their
water solubility, especially under acidic conditions. However,
these compounds still exhibited moderate solubility in neutral
water, which was still not suitable for injection. Additionally, we
attempted to prepare a suitable salt form of 45x to significantly
increase its water solubility because of the basic nature of the
pyrimidinyl-substituted compound 45x at physiological pH.
As shown in Table 9, various acid salts (e.g., hydrochloric
acid, methanesulfonic acid, p-toluene sulfonic acid, L(−)-malic
acid, sulfuric acid, oxalic acid, hydrobromic acid, phosphoric
acid, phosphoric acid, and maleic acid) were examined for their
ability to enhance the aqueous solubility of pyrimidinyl-
containing compound 45x. Finally, disulfate salt 58 was
found to be most preferable, as it had higher solubility and
better property (Scheme 12).
>40
10.37
0.08
a
Measured in hERG-expressing CHO cells using Qpatch 16X assay.
In Vitro Cytotoxic Activity. It is well-known that most of
triazole antifungal agent are inhibitors of both fungal
cytochrome P450 and, to a certain extent, mammalian P450,
which may play arole in the metabolism of azole antifungals in
the liver.7,63,64 It is possible to indirectly assess the hepatotoxic
potential of compounds using in vitro method of rat
hepatocytes. Thus, the most potent compounds 27aa and 58
were selected to evaluate their cytotoxicity in rat primary
hepatocytes by sulforhodamine B (SRB) assay according to the
protocol described previously.65,66 As shown in Figure 5, the
survival rates of rat hepatocytes were not reduced significantly
in all doses of compounds 27aa and 58 after 48 h incubation
compared with DMSO-treated cells. Thus, compounds 27aa
and 58 displayed low cytotoxicity to rat primary hepatocytes.
Aqueous Solubility of Selected Compounds and Salt
Formation of Most Potent Compounds. We evaluated the
In Vivo Activity. In vivo efficacy of the selected analogues
27aa and 58, which showed significant in vitro activity as well
as broad antifungal spectrum, were evaluated in a systemic
Institute of Cancer Research (ICR) mice model infected with
Figure 5. Cytotoxic effects of compounds 27aa, 58, voriconazole, and albaconazole on rat primary hepatocytes. Rat primary hepatocytes were
inoculated in 96-well plates. All molecules dissolved in DMSO were administered to the cells with four different concentrations: 40, 20, 10, and 5 μM
in parallel with DMSO-only treated cells as negative controls. After 48 h of incubation, cell viability was determined using the SRB cytotoxicity assay.
The results are expressed as mean SD (n = 3). Differences between the groups were analyzed by Student’s t-test. P values were calculated using
paired t-tests. P < 0.05 was considered to be statistically significant. &P < 0.05 versus DMSO-treated cells.
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that this result may be caused by poor metabolic stability of
compound 58 in rat liver microsomes.
Table 9. Solubility Data for Salts of Compound 45x (Mean
SD, n = 3)
Metabolic Stability of Selected Antifungal Agents. To
further investigate the pharmacokinetic profile of these two
compounds and validate our hypothesis, compound 27aa and
compound 58 were evaluated for their metabolic stability using
rat liver microsomes (Table 11).67−69 With a long half-life (t1/2
= 430.2 min) and low in vitro rat liver microsomal clearance
(Clint = 5.0 μL/min/mg), compound 27aa is metabolically
sufficiently stable. In contrast, compoud 58 possessed lower
metabolic stability (t1/2 = 32.4 min Clint = 65.0 μL/min/mg),
which may be one of the main causes for its low oral
bioavailability.
a
b
salt form
aqueous solubility
appearance
hydrochloric acid
methanesulfonic acid
p-toluene sulfonic acid
L(−)-malic acid
sulfuric acid
14.800 0.9165
11.967 0.4726
5.800 0.6245
pale-yellow solid
viscous solid
pale-yellow solid
absorb moisture easily
pale-yellow solid
absorb moisture easily
viscous solid
107.633 8.1990
oxalic acid
hydrobromic acid
phosphoric acid
maleic acid
12.067 0.5033
8.333 0.2512
viscous solid
absorbs moisture easily
a
b
In mg/mL, determined in pH 7.4 phosphate buffer, at 30 °C. Visual
observation of products after filtration at room temp (within 12 h).
CONCLUSIONS
■
We identified a series of novel azoles containing different types
of fused-heterocycle nucleus. Summarizing the results of Tables
1−6, we can conclude that triazoles featuring tetrahydro-
[1,2,4]triazolo[1,5-a]pyrazine and tetrahydro-thiazolo[5,4-c]-
pyridine nuclei are more suited for improving activity against
Candida spp. and Cryptococcus ssp. In these phenyl-substituted
analogues (R = phenyl or substituted phenyl), antifungal
activity was independent of the electronic nature and position
of the substituent in the phenyl ring. In addition, replacement
of the phenyl ring containing nitrogen-substituted aromatic
heterocycles resulted in development of a more potent
compound with broader antifungal spectrum. Among these
compounds, analogues 27aa and 45x were selected for further
development based on their significant in vitro activity against a
variety of clinical pathogens, including Candida spp.,
Cryptococcus spp., Aspergillus spp., and fluconazole-resistant
C.alb. strains, as well as their relatively low hERG inhibition and
hepatocyte. Additionally, the disulfate salt of 45x (compound
58) was highly water-soluble and thus suitable for intravenous
administration. Compared with the vehicle group, the murine
infection model orally administrated with 27aa also showed
excellent response against C.alb. SC5314 and C.alb. 103
(fluconazole-resistant strain). Further developments of com-
pound 27aa are ongoing in our laboratory.
C.alb. (SC5314) and Fluconazole-resistant C.alb. (strain 103)
(Figure 6−8).
As shown in Figure 6, the efficacy (mean survival days) of
water-soluble compound 58 at a dose of 0.32−10.0 mg/kg less
than that of orally administered fluconazole at a dose of 0.5
mg/kg in the C.alb. SC5314 infection model following
intravenous administration. In addition, compound 58 at a
high dose (10.0 mg/kg) still did not show significantly
improved survival rates of the infected mice. The lack of
activity of compound 58 could be related to its poor PK profiles
(see Table 10).
Compared with less active compound 58, compound 27aa at
dose 1.0−10.0 mg/kg exhibited high and significantly improved
survival rates of the infected mice (Figure 7). The efficacy of
orally administered 27aa at a dose of 1.0−10.0 mg/kg in the
C.alb. SC5314 infection model were comparable or slightly
superior to the efficacy of fluconazole at a dose of 0.5 mg/kg.
Apart from that, it also showed that compound 27aa protected
mice infected with C.alb. SC5314 in a dose-dependent fashion.
On the basis of the above findings (moderate in vitro activity
against resistant C.alb. (strains 100 and 103)) and its good PK
profile (Table 10), compound 27aa was also tested in systemic
infection of an ICR mouse model with fluconazole-resistant
C.alb. (strain 103) (Figure 8). Compared with the vehicle
group and fluconazole treated group (at a dose of 0.5 mg/kg,
qd, po), compound 27aa (at a dose of 3.2−10.0 mg/kg, qd, po)
provided stronger protection for ICR mice infected with
fluconazole-resistant C.alb. (strain 103).
PK Profiles of Selected Antifungal Agents. The PK
properties of compounds 58 and 27aa in rats after oral
administration and intravenous injection were measured. As
summarized in Table 10, compound 27aa exhibited favorable
PK profiles with excellent half-lives, areas under the
concentration−time curve (AUCs), as well as high oral
bioavailability. In contrast, the water-soluble compound 58
displayed an unacceptably short half-life and poor oral
absorption, as well as higher clearance. Therefore, we speculate
EXPERIMENTAL SECTION
■
Minimum Inhibitory Concentration Testing. The in vitro
minimum inhibitory concentration (MIC) of all title compounds were
determined by the method recommended by the National Committee
for Clinical Laboratory Standards (NCCLS) using the serial dilution
method in 96-well microtest plates.70,71 The tested fungi species,
including eight pathogenic fungi, C.alb., C.par. 22019, and C. neo.
32609, were purchased from ATCC, and other strains were provided
by Shanghai Changhai Hospital. C.par. 22019 was used as the quality-
controlled strain and tested in each assay. Fluconazole, voriconazole,
ravuconazole albaconazole, and amphotericin B purchased from
respective manufacturers were served as the positive control drugs.
All of test compounds were dissolved in DMSO serially diluted in
a
Scheme 12. Synthesis of Disulfate Salt of 45x (58)
a
Reagents and conditions: (a) 0.67N sulfuric acid in ethyl acetate, room temp 1 h then 0 °C 2 h.
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Figure 6. In vivo efficacy (mean survival days SD) of compound 58 in a systemic infection of an ICR mouse model (n = 10) with C.alb. SC5314;
FCZ, fluconazole. aIn vivo efficacy of 58 was assessed by mean survival days upon systemic C.alb. (SC5314) infection (ICR mouse model, n = 10), iv,
b
qd, 5 days. p < 0.05 versus vehicle control.
a
Table 10. Pharmacokinetic Parameters of Compounds 27aa and 58
b
c
d
compd
route
dose (mg/kg)
Cmax (ng/mL)
Tmax (h)
t1/2 (h)
AUC0−∞(ng·h/mL)
clearance (L/h/kg)
F (%)
27aa
po
iv
10
3
1420
na
1.75
na
3.63
1.20
0.46
0.42
7913
2245
141
nc
1.36
nc
105.4
58
po
iv
10
5
164
na
0.25
na
5.8
1189
4.35
a
Cmax, peak plasma concentration of the drug after administration; Tmax, time to reach Cmax; t1/2, elimination half-life; AUC, area under the
b
concentration−time curve; F, bioavailability. Doses of 10 and 5 mg/kg of 58 were equivalent to 7.1 and 3.5 mg/kg of 45x, respectively. All values
were measured based on the amount of the active antifungal agent 45x detected. na: not applicable. nc: not calculated.
c
d
Figure 7. In vivo efficacy (mean survival days SD) of compound 27aa in a systemic C.alb. SC5314 infection model in ICR mice (n = 10); FCZ,
fluconazole. aIn vivo efficacy of 27aa was assessed by mean survival days upon systemic C.alb. SC5314 infection (ICR mouse model, n = 10), po, qd,
b
c
5 days. p < 0.05 versus vehicle control. p < 0.05 versus fluconazole treated group.
Figure 8. In vivo efficacy (mean survival days SD) of compound 27aa in a systemic infection of an ICR mouse model with fluconazole-resistant
C.alb. (strain 103) (n = 10). aIn vivo efficacy of 27aa was assessed by mean survival days upon a systemic infection with fluconazole-resistant C.alb.
b
c
(strain 103) (ICR mice, n = 10), po, qd, 5 days. p < 0.05 versus vehicle control. p < 0.05 versus fluconazole treated group.
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200 μL) in 96-well plates. The compounds dissolved in DMSO were
applied in concentrations between 5 and 40 μM in parallel with
DMSO-only treated cells as negative controls. SRB cytotoxicity assay
in rat primary hepatocytes were treated with compounds at indicated
concentrations for 48 h, followed by SRB assay as previously
described.66
Table 11. Half-Lives and Intrinsic Clearances of Compounds
27aa and 58 in Rat Liver Microsomes
a
b
c
compd
microsomal t1/2 (min)
Clint (μL/min/mg protein)
27aa
58
430.2
32.4
5.0
65.0
Determination of Aqueous Solubility in Hydrochloric Acid
Solution (pH 1.2) and Phosphate Buffer. (pH 7.4). Aqueous solubility
of compounds were determined by high performance liquid
chromatography (HPLC) method (column: Dikma C 18, 250 mm
× 4.6 mm, 5 μm particle size; isocratic elution with a mobile phase of
25% CH3CN/75% H2O; flow rate, 1.0 mL/min; UV detection at 210
nm; column temperature, 30.0 °C; injection volume, 20 μL). A
standard solution was prepared by dissolving a precisely weighted
amount (generally 1 mg) of sample in 10 mL of CH3OH. The
standard solution was scanned by UV to determine the wavelength of
maximum absorbance, diluting as necessary. A saturated solution was
prepared by stirring magnetically a small volume of pH 1.2
hydrochloric acid solution or pH 7.4 phosphate buffer solution in
the presence of excess sample for 3 h. To remove solid compound, the
saturated solution was filtered to remove excess compound through a
Millipore 0.45 μm filter. Then, the saturated solution was scanned by
UV at the wavelength of the absorption maximum. Total solubility was
then determined by the relationship C′ = A′C/A, where C =
concentration of standard solution in mg/mL, A = absorbance of the
standard solution (correcting for any dilutions), A′ = absorbance of the
saturated solution (correcting for any dilutions), and C′ =
concentration of saturated solution in mg/mL.
C.alb. SC5314 Infection Model. Compounds 27aa, 58, and
fluconazole were studied in a systemic C.alb. SC5314 infection
model in mice. ICR mice (female, SLAC Lab Animal Ltd.) weighing
18−22 g were used in the study, with 10 mice in each group. Mice
were inoculated iv with 0.2 mL of a suspension containing 5 × 106
CFU/mL of C.alb. SC5314. In this model, compound 27aa (as a
suspension in 0.5% carboxymethylcellulose in distilled water), 58 (as a
solution in distilled water), and fluconazole (as a solution in distilled
water) were administered orally or intravenously 2 h after infection (5
days, qd). Reference compound, fluconazole, was administered at daily
dose of 0.5 mg/kg, and compounds 27aa and 58 were administered at
daily doses of 0.32 , 1.0, 3.2, and 10.0 mg/kg, respectively. The vehicle
control group received only the vehicle (5 days, qd). In vivo efficacy of
test compounds was determined by mean of survival days for 30 days
after infections.
Fluconazole-Resistant C.alb. 103 Infection Model. Compounds
27aa and fluconazole were also studied in a mouse systemic C.alb. 103
(fluconazole-resistant strain) infection model. Female ICR mice
(SLAC Lab Animal Ltd.), weighing between 18 and 22 g, were
rendered neutropenic by treatment with 80 mg/kg of cyclo-
phosphamide prior to infection (3 days, qd, iv). Systemic C.alb. 103
infection was given to the mice by the injection of 0.2 mL of an
inoculum of C.alb. 103 1 × 106 CFU/mL via intravenous injection. In
this model, compound 27aa (as a suspension in 0.5% carboxyme-
thylcellulose in distilled water) and fluconazole (as a solution in
distilled water) were administered orally or intravenously 2 h post
infection (5 days, qd). Reference compound, fluconazole, was
administered at daily dose of 0.5 mg/kg, and compounds 27aa was
administered at daily doses of 0.32, 1.0, 3.2, and 10.0 mg/kg. The
vehicle control group received only the vehicle (5 days, qd). In vivo
efficacy of test compounds was determined by mean of survival days
for 30 days after infections.
Pharmacokinetic Studies. Compounds 27aa and 58 were tested in
pharmacokinetic studies on Sprague−Dawley rats weighing 200−250
g, with seven mice (male) in each group. Compound 27aa (5%
DMSO + 5% Tween 80 in 90% saline) was administered orally at a
dose of 10 mg/kg or was administered intravenously at a dose of 3
mg/kg. Compound 58 (5% DMSO + 5% Tween 80 in 90% saline)
was taken orally at a dose of 10 mg/kg or was taken intravenously at a
dose of 5 mg/kg. Serial blood samples (0.3 mL) were collected via the
retrobulbar vein, and quantification was performed by LC-MS.
a
0.33 mg/mL microsomal protein, NADP+-regenerating system,
[inhibitor], 0.1 μM, incubation at 37 °C, samples taken at 0, 7, 17,
b
30, and 60 min, determination of parent compound by MS. t1/2
elimination half-life in rat liver microsomes. Clint: intrinsic body
clearance.
:
c
growth medium. The strains were incubated at 35 °C. The MIC values
was determined at 24 h for Candida spp. at 48 h for A.fum. and 72 h
for C. neo. 32609.
Molecular Docking. Homology model of CACYP51 were
obtained from previous studies.72 GOLD73 was used for molecular
docking and the docking parameters were defined the same with
previous reports.74
Biology. Cell Culture and Transfection. A Chinese hamster ovary
(CHO) cell line stably expressing hERG potassium channels were
used. Cells were grown in Ham F12 (catalogue no. 11765-054, Life
Technologies) supplemented with 10% (v/v) heat inactivated fetal
bovine serum (FBS), 100 μg/mL hygromycin B, and 100 μg/mL
geneticin (catalogue no. 10687−010, Life Technologies). Cell density
should be ranged within 3−8 × 106 cells/mL in the final suspension
before applying to the QPatch stir chamber. After leaving CO2
incubator, cells are maintained in serum-free medium buffered
HEPES. Cells in such condition can be used for recording only for
4 h after harvesting.
Solutions and Drugs. Cells were automatically prepared for
application to the chips (centrifuged and washed twice, then
resuspended in extracellular solution) as described previously.57
Composition of the intracellular solution was: (in mM): 145 NaCl,
4 KCl, 2 CaCl2, 1 MgCl2, 10 Gluose, 10 HEPES, adjusted to pH 7.4
with NaOH, osmolarity ∼305 mOsm. Composition of the internal
solution was (in mM): 120 KCl, 5.374 CaCl2, 1.75 MgCl2, 10 HEPES,
5 EGTA, 4 Na-ATP, adjusted to pH 7.25 with NaOH, osmolarity
∼280 mOsm. Drugs were obtained from from respective manufac-
turers, or synthesized in our laboratory.
Electrophysiology. Whole-cell recordings were performed using
automated QPatch (Sophion). The cells were voltage clamped at a
holding potential of −80 mV. The hERG current was activated by
depolarizing at +20 mV for 5 s, after which the current was taken back
to −50 mV for 5 s to remove the inactivation and observe the
deactivating tail current. The maximum amount of tail current was
used to determine hERG current amplitude. Compound powder will
be made into 10 or 30 mM DMSO stock right before the experiments.
All compounds will be diluted freshly in the external solution to the
desired concentrations from compound stock in DMSO. After
achieving break-in (whole-cell) configuration, the cells were recorded
for 120 s to assess current stability. The voltage protocol described
above was then applied to the cells every 20 s throughout the whole
procedure. Only stable cells with recording parameters above
threshold were allowed to enter the drug addition procedure. External
solution containing 0.1% DMSO (vehicle) was applied to the cells to
establish the baseline. After allowing the current to stabilize for 3 min,
compound was applied. Compound solution was added. and the cells
were kept in the test solution until the compound’s effect reached a
steady state or for a maximum of 3 min. Washout with external
solution might be performed until the recovery of the current reached
a steady state. Positive control cisapride is used in the experiments to
ensure the normal response and quality of the cells. Data were
analyzed using Assay Software provided by Sophion, XLFit, or
Graphpad Prism 6.0.
Sulforhodamine B Assay for Cytotoxicity Screening. Rat primary
hepatocytes were isolated using Selgen’s two-step perfusion method65
and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM).
Rat primary hepatocytes were inoculated (2000−10000 cells/well in
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Pharmacokinetic parameters were calculated from the mean plasma
concentration by noncompartmental analysis.
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ASSOCIATED CONTENT
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S
* Supporting Information
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AUTHOR INFORMATION
■
Corresponding Author
*Phone: 86-21-5080 6786. Fax: 86-21-5080 6786. E-mail:
ysyang@mail.shcnc.ac.cn.
̂
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Author Contributions
∥These authors contributed equally to this work.
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drugs: review of the Phase II and III trials. Clin. Invest. 2011, 1, 1577−
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Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
The authors thank Prof. Chunquan Sheng and Dr. Shengzheng
Wang from School of Pharmacy, the Second Military Medical
University, for molecular docking experiments; We also thank
Prof. Chunquan Sheng for writing the script for the molecular
docking analysis presented in the paper. We thank Prof. Jingya
Li and Dr. Jin Zhang from the National Center for Drug
Screening, Shanghai Institute of Materia Medica for cytotoxicity
assay. We thank Prof. Zhaobing Gao and Jinfeng Yue from
Shanghai Institute of Materia Medica for performing the hERG
assay. We also thank Dr. Jia Liu for performing the metabolic
stability assay. Critical comments of Prof. Chunquan Sheng,
Prof. Xiaoyan Chen and Prof. Zhaobing Gao are greatly
appreciated. We are grateful to the National Science and
Technology Major Project for the support of this research. The
project described was supported by Key New Drug Creation
and Manufacturing Program, China (No. 2009ZX09301-001).
ABBREVIATIONS USED
■
C.alb., Candida albicans; C.par., Candida parapsilosis; C.neo.,
Cryptococcus neoformans; C.gla., Candida glabrata; A.fum.,
Aspergillus fumigatus; MIC, minimum inhibitory concentration;
SAR, structure−activity relationship; NBS, N-bromosuccini-
mide; NCS, N-chlorosuccinimide; ICR, Institute of Cancer
Research; hERG, human ether-a-go-go related gene; QT, the
length of time between the start of the Q wave and end of the T
wave on an electrocardiogram; SRB, sulforhodamine B; CHO,
Chinese hamster ovary; DMEM, Dulbecco’s Modified Eagle’s
Medium; CFU, colony-forming units
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NOTE ADDED AFTER ASAP PUBLICATION
■
This paper was published ASAP on March 7, 2014. A text
change was made in the Chemistry section, and the revised
version was reposted on April 17, 2014.
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