SAR Studies on Aromatic Acylhydrazone-Based Inhibitors of Fungal Sphingolipid Synthesis as Next-Generation Antifungal Agents

Article abstract of DOI:10.1021/acs.jmedchem.9b01004

Recently, the fungal sphingolipid glucosylceramide (GlcCer) synthesis has emerged as a highly promising new target for drug discovery of next-generation antifungal agents, and we found two aromatic acylhydrazones as effective inhibitors of GlcCer synthesis based on HTP screening. In the present work, we have designed libraries of new aromatic acylhydrazones, evaluated their antifungal activities (MIC₈₀ and time-kill profile) against C. neoformans, and performed an extensive SAR study, which led to the identification of five promising lead compounds, exhibiting excellent fungicidal activities with very large selectivity index. Moreover, two compounds demonstrated broad spectrum antifungal activity against six other clinically relevant fungal strains. These five lead compounds were examined for their synergism/cooperativity with five clinical drugs against seven fungal strains, and very encouraging results were obtained; e.g., the combination of all five lead compounds with voriconazole exhibited either synergistic or additive effect to all seven fungal strains.

Full text of DOI:10.1021/acs.jmedchem.9b01004

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Article
SAR Studies on Aromatic Acylhydrazone-Based Inhibitors of Fungal
Sphingolipid Synthesis as Next-Generation Antifungal Agents
Krupanandan Haranahalli, Cristina Lazzarini, Yi Sun, Julia Zambito, Senuri Pathiranage,
John Brian McCarthy, John P. Mallamo, Maurizio Del Poeta, and Iwao Ojima
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01004 • Publication Date (Web): 01 Aug 2019
Downloaded from pubs.acs.org on August 2, 2019
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SAR Studies on Aromatic Acylhydrazone-Based Inhibitors of Fungal
Sphingolipid Synthesis as Next-Generation Antifungal Agents
Krupanandan Haranahallia,d,†, Cristina Lazzarinib,c,†, Yi Sun , Julia Zambito , Senuri
d
d
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Pathiranage , J. Brian McCarthy , John Mallamo , Maurizio Del Poetaa,b,c,f, and Iwao Ojimaa,d,*
d
e
e
aInstitute of Chemical Biology and Drug Discovery, Stony Brook University, Stony Brook, New
York 11794-3400, USA;
bDepartment of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook,
New York 11794-5222, USA;
cVeterans Administration Medical Center, Northport, New York , USA;
dDepartment of Chemistry, Stony Brook University, Stony Brook, New York 11794-3400, USA;
eMicroRid Technologies Inc., 86 Deer Park Road, Dix Hills, New York 11746, USA;
fDivision of Infectious Diseases, School of Medicine, Stony Brook University, New York 11794-
8
434, USA;
†
Contributed equally
Corresponding Author
*
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Abstract
Recently, the fungal sphingolipid glucosylceramide (GlcCer) synthesis has emerged as a highly
promising new target for drug discovery of next-generation antifungal agents, and we found two aromatic
acylhydrazones as effective inhibitors of GlcCer synthesis based on HTP screening. In the present work,
we have designed libraries of new aromatic acylhydrazones, evaluated their antifungal activities (MIC80
and time-kill profile) against C. neoformans and performed an extensive SAR study, which led to the
identification of five promising lead compounds, exhibiting excellent fungicidal activities with very
large selectivity index. Moreover, two compounds demonstrated broad spectrum antifungal
activity against six other clinically relevant fungal strains. These five lead compounds were
examined for their synergism/cooperativity with five clinical drugs against seven fungal strains
and very encouraging results were obtained, e.g., the combination of all five lead compounds with
voriconazole exhibited either synergistic or additive effect to all seven fungal strains.
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.
INTRODUCTION
Invasive fungal infections (IFIs), such as cryptococcosis, aspergillosis and candidiasis, are a serious
threat to human health, as IFIs are associated with a large number of deaths which is similar in number
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to that of tuberculosis or malaria. Recent statistics suggest that more than 150 million people suffer from
serious fungal infections and it is estimated that annually around 1.5-2 million deaths occur as a result of
2, 3
these invasive fungal infections. IFIs are highly prevalent among individuals with low immunity such
as HIV positive patients, organ transplant and cancer patients receiving immunosuppressants, as well as
pediatric and geriatric patients.4-8 Cryptococcus neoformans (C. neoformans) that causes
meningoencephalitis is responsible for 600,000 deaths per year, accounting for 15% AIDS-related deaths
globally.9, 10 Candida species can cause invasive candidiasis that includes blood-derived and deep-tissue
11
infections in hospitalized individuals who are treated for various conditions. Candida species are also
a major concern for immunocompromised patients.12, 13 Candidemia caused by Candida species is
14
associated with poor prognosis and contributes to ~30-60% mortality rate. Aspergillus species was
recently estimated to cause ca, 250,000 cases of invasive aspergillosis.2
Current treatment options for IFIs consist of three major classes of drugs which include azoles
15
(e.g. fluconazole), polyenes (e.g. amphotericin B) and echinocandins (e.g. caspofungin). These
drugs are associated with serious side effects such as nephrotoxicity, narrow spectrum of activity
and drug resistance.16-19 Amphotericin B, the last resort anti-fungal agent, is associated with
adverse drug-drug interactions with anticancer agents and azoles.20, 21 In spite of all those
drawbacks, the same three classes of drugs have been used to date, because no newer and more
efficacious anti-fungal drugs have been approved by FDA for some time. Hence, there is a dire
need for new, efficacious antifungal drugs that can overcome drug resistance with novel
mechanism of action.
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In this context, the fungal sphingolipid glucosylceramide (GlcCer) synthesis has emerged as a
highly promising new target for the development of next-generation antifungal agents.22-24 GlcCer is
essential for the cell division of pathogenic fungi such as C. neoformans, Candida albicans (C. albicans)
and Aspergillus fumigatus (A. funigatus), and responsible for their virulence.23-27 It has been shown that
fungal cells lacking GlcCer cannot replicate in neutral or alkaline environments.23, 24, 27 This finding
clearly indicates the importance of GlcCer for virulence in alveolar spaces, cerebrospinal fluid or
bloodstream of the host wherein the pH is neutral or alkaline, and thus makes GlcCer a promising target
for drug discovery.23, 24 As shown in Figure 1A, aromatic acylhydrazone 1, bearing a salicylaldehyde-
hydrazone moiety, selectively inhibited the synthesis of fungal GlcCer in a dose-dependent manner
without affecting the synthesis of mammalian GlcCer.23, 24 When the efficacy of 1 and 2 was evaluated
in vivo, using mice infected with C. neoformans, both compounds substantially improved the survival of
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mice, compared to the untreated ones (Figure 1B). Moreover, 2 exhibited better antifungal activity than
fluconazole which is a clinically used drug to treat fungal infections.
A
B
C
Figure 1. (A)Analysis of the synthesis of glucosylceramide (GlcCer) in C. neoformans or J774 cells labelled
3
with [ H] palmitate and treated with 1 at the indicated concentrations; (B) Survival studies of mice infected
5
intranasally with 5 x 10 C. neoformans cells and treated via i.p. injection on the day of infection with 1.2
mg/kg/day of fluconazole (Flu), 1 or 2. *Compound 2 versus no drug, P value of 0.0018; (C) Structures of
1
(BHBM) and 2 (D13).
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Furthermore, 2 was effective against A. fumigatus in vivo, and more efficacious than the clinically
used antifungal drug, voriconazole, with very high “selectivity index” (SI = LD50/MIC80) (>500).22
Although aromatic acylhydrazones are known to be pan assay interference compounds (PAINS), our
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initial hits possessed promising antifungal activity not only in vitro, but also in vivo.22-24, 28 Moreover,
the fact that these aromatic acylhydrazones, bearing a salicylaldehyde-hydrazone moiety, were also
highly fungus-selective (vide supra), gave us confidence to further explore this class of compounds as
29
next-generation antifungal agents. We describe here the library synthesis, biological evaluations and
SAR study of new aromatic acylhydrazones, bearing a salicylaldehyde-hydrazone moiety, against C.
neoformans and six other pathogenic fungi. Our study has led to the identification of five highly potent
and selective lead compounds, which have been further examined for their synergy/cooperativity with
five antifungal drugs currently used in clinic against seven pathogenic fungal strains.
.
RESULTS AND DISCUSSION
Library synthesis. For the synthesis of initial library of aromatic acylhydrazones 5.0~5.7,
commercially available benzoyl chlorides or benzoic acids (1.0~1.3) were converted to their
methyl esters (2.0~2.3), which were reacted with excess hydrazine monohydrate under reflux to
give the corresponding hydrazides 3.0~3.3. Hydrazides 3, thus obtained, were condensed with
different salicylaldehydes 4a-g in the presence of an arenesulfonic acid resin as a catalyst in
DMSO. Excess unreacted aldehyde 4 was removed from the reaction mixture by treating with an
aminomethylated resin. The reaction mixture was then filtered to afford the corresponding
aromatic acylhydrazones 5.0~5.4 (Scheme 1, Table 1).
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Scheme 1. Synthesis of the initial library of acylhydrazones 5.0~5.7
MeOH, RT, 2–4 h
or
O
O
R5
H2SO4, MeOH
R5
NH2-NH2.H2O (15-20 eq.),
MeOH, reflux, overnight
X
reflux, overnight
OMe
A
A
R4
R2
R4
R2
quant.
47–91%
R3
R3
0
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1
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1
.0 R = R = R = H, R = Br, X = Cl
2.0 R = R = R = H, R = Br
.1 R = R = F, R4 = R = H, X = Cl
2
3
5
2.1 R = R = F, R4 = R = H
2
3
5
.2 R = R = Br, R3 = R = H, X = OH
2
4
5
2.2 R = R = Br, R3 = R = H
2
4
5
.3 R2 = R5 = Br, R3 = R4 = H, X = OH
2
5
3
4
2.3 R = R = Br, R = R = H
2
3
5
4
2
.4 R = R = R = H, R = Br
2.5 R3 = R = H, R = OH, R = Br
4
2
5
2
.6 R4 = H, R2 = OH, R = R = Br
3
5
O
2.7
O
N
CHO
B
R6’
OH
(1.05 eq.),
R3’
R3’
HO
R4’
R5’
O
O
R5’
R5
NH2
R5
R4
N
B
R4’
N
N
4
a-g
A
H
A
H
R6’
R4
R2
R2
AcOH (cat.), MeOH,
r.t., overnight
R3
5
R3
5.0-5.6
Br
2
4
3
56-99%
3
3
3
3
.0 R = R = R = H, R = Br
HO
N
.1 R = R = F, R4 = R = H
.2 R = R = Br, R3 = R = H
2
3
5
O
a R3’ = R5’ = R6’ = H, R4’ = Br
4
4
4
4
2
4
5
3’
4’
6’
5’
b R = R = R = H, R = Cl
N
Br
2
5
3
4
H
.3 R = R = Br, R = R = H
3’
4’
6’
5’
c R = R = R = H, R = CH3
3.4 R2 = R = R = H, R = Br
3
5
4
d R = R = Cl, R4’ = R = H
3’
5’
6’
N
5.7
4
2
5
3
3
.5 R3 = R = H, R = OH, R = Br
4e R3’ = R4’ = R6’ = H, R5’ = Br
.6 R4 = H, R2 = OH, R = R = Br
3
5
f R3’ = R5’ = Br, R4’ = R6’ = H
4
O
3.7
NH2
N
4g
H
OH
N
CHO
The same protocol was used for the synthesis of other aromatic acylhydrazones. In some
cases (2.4~2.7, Scheme 1; 2.19~2.23, Scheme 3; 2.24~2.26, Scheme 5; 2.27, Scheme 6), the methyl
or ethyl esters of substituted benzoic acids were commercially available. A variety of hydrazides
3
were condensed with different salicylaldehydes 4 to give the corresponding acylhydrazones 5
(
57 compounds) (Schemes 2~5). For the synthesis of 5.21 (Scheme 4), p-fluoromethylbenzoic acid
was condensed with Boc-hydrazine in the presence of EDC.HCl and DMAP to give Boc-protected
hydrazide 3.21. Then, the Boc group was removed by TFA, followed by the condensation with
salicylaldehyde 4 in the same pot to give 5.21.
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Scheme 2.
1
1
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1
1
1
2
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2
2
2
2
2
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2
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Scheme 3.
MeOH, r.t., 2-4 h
or
R6
A
O
R6
A
O
NH NH H O,
2
2. 2
R5
H2SO4, MeOH,
OH reflux, overnight
R5
MeOH,
OMe reflux, overnight
R4
R2
quant.
R4
R2
77-89%
R3
R3
2
.11 R2 = R3 = R5 = R = H, R = CN
.14 R2 = R4 = R5 = R6 = H, R = OCHF2
.15 R2 = R3 = R5 = R6 = H, R = OCHF2
6
4
3
5
6
4
1
1
1
1
2.11 R = R = R = R = H, R = CN
3
3
4
5
6
2
2.12 R = R = R = R = H, R = CF3
4
2
3
5
6
4
2.13 R = R = R = R = H, R = CF3
.16 R2 = R3 = R = R6 = H, R = OCF3
5
4
2
4
5
6
3
2.14 R = R = R = R = H, R = OCHF2
1.17 R2 = F, R = OCF3, R = R5 = R6 = H 2.15 R = R3 = R = R6 = H, R = OCHF2
4
3
2
5
4
.18 R2 = F, R = CF , R = R5 = R6 = H
4
3
2.16 R = R3 = R = R6 = H, R = OCF3
2
5
4
1
1
1
3
.19 R2 = R4 = R5 = R6 = H, R3 = F
2.17 R = F, R = OCF3, R = R5 = R6 = H
2
4
3
.20 R2 = R3 = R5 = R6 = H, R4 = F
2.18 R = F, R = CF3, R = R5 = R6 = H
2
4
3
2
4
5
6
3
2
.19 R = R = R = R = H, R = F
2
3
5
6
4
2.20 R = R = R = R = H, R = F
2
2
.22 R2 = R4 = R5 = R6 = H, R3 = OCF3
.23 R2 = R4 = R5 = R6 = H, R3 = CF3
R3’
R6
O
4
a,e,f,h,i
R4’
R5
R4
NH2
HO
R6
O
N
AcoH (cat.)
A
H
R5
B
N
R2
MeOH, r.t., overnight
47-99%
N
5
R ’
A
H
R6’
R3
R4
R2
R3
.11 R2 = R3 = R = R = H, R = CN
5
6
4
5.11-5.23c
3
3
3
3
.12 R3 = R4 = R = R = H, R = CF3
5
6
2
.13 R2 = R3 = R5 = R6 = H, R4 = CF3
.14 R2 = R4 = R5 = R6 = H, R3 = OCHF2
3.15 R2 = R3 = R5 = R6 = H, R4 = OCHF2
.16 R2 = R3 = R = R6 = H, R = OCF3
5
4
3
3
3
3
3
.17 R2 = F, R = OCF3, R = R5 = R = H
4
3
6
.18 R2 = F, R = CF3, R = R5 = R = H
4
3
6
.19 R2 = R4 = R = R = H, R = F
5
6
3
.20 R2 = R = R = R = H, R = F
3
5
6
4
3.22 R2 = R4 = R5 = R6 = H, R3 = OCF3
.23 R2 = R4 = R5 = R6 = H, R3 = CF3
3
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Scheme 4.
1
) EDC.HCl
DMAP, DCM,
r.t., overnight
O
O
H
N
OH
N
H
Boc
F
F
2
) Boc-NH NH2
2
DCM, r.t., overnight
3.21
1
.21
8
6%
0
1
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0
1
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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
Br
HO
1
2
) TFA (5 eq.), DCM,
r.t., overnight
O
N
N
Br
) 4f (0.95 eq.)
H
F
DCM, r.t., 1 h
5
.21
4
6%
Scheme 5.
O
O
NH -NH H O (15 eq.),
2
2. 2
X
NH2
MeOH, reflux, 12 h
O
N
A
A
R1
R1
H
66-80%
N
N
R2
R2
1
2
1
2
2
.24 R = R = H, X = CH CH
3.24 R = R = H,
2
3
1
2
1
2
2.25 R = R = CH , X = CH
3.25 R = R = CH
3
3
3
2
1
2
1
2
.26 R = COCH , R = H, X = CH CH
3.26 R = COCH , R = H
3
2
3
3
R3’
4
a,e,f,g (1.05 eq.),
R4’
R5’
HO
O
AcOH (cat.),
B
N
N
MeOH, r.t., overnight
5-99%
A
H
R1
R6’
N
R2
8
5.24-5.26
Scheme 6.
R6
O
O
1
) H2SO4, MeOH,
reflux, overnight
R5
R4
NH2
OMe
N
A
H
2
) NH2-NH2.H2O (15 eq.),
MeOH, reflux, 12 h
R2
R3
2
.27
99%
.27 R = CH3, R = R4 = R5 = R6 = H
2
3
3
as well as
3
.0, 3.2, 3.3 and 3.4 (see Scheme 1)
R3’
4
j,k,l,m (1.05 eq.),
AcOH (cat.),
MeOH, r.t., overnight
R6
HO
R4’
O
R5
R4
N
B
R5’
N
86-99%
A
H
R6’
R2
j R3’ = R4’ = R6’ = H, R5’ = CF3
4
4
4
4
R3
k R3’ = R4’ = R6’ = H, R5’ = F
5.0a-5.27a
l R3’ = R4’ = R6’ = H, R5’ = CN
m R3’ = R4’ = R6’ = H, R5’ = OCF3
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An oxadiazole mimic of acylhydrazone, 6a, was synthesized by treating 1 with hypervalent
iodine (PhI(OCOCF
3
2
) (Scheme 7a). Another diazole mimic of acylhydrazone, 6b, was
synthesized through diazole formation from 3,5-dibromo-1-benzyloxycinnamaldehyde (4f.2) and
tosylhydrazine, followed by deprotection, 4-bromobenzoylation and debenzylation (Scheme 7b).
Cinnamaldehyde 4f.2 was prepared from 3,5-dibromosalicylaldehyde (4f) in two steps (Scheme
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
7
b). N-Methylacylhydrazone 5.28 was synthesized through the condensation of 3,5-
dibromosalicylaldehyde (4f) with N-methylhydrazide 3.29, which prepared by reacting 4-
bromobenzoyl chloride (1.29) with N-methylhydrazine at -78 oC (Scheme 7c). C-
Methylacylhydrazones 5.2i~5.3h were synthesized by condensing hydrazides 3.2 and 3.3 with 2-
acetylphenols 4.4a and 4.4b (Scheme 7d). Bis-acylhydrazones 5.29 and 5.30 were synthesized
through the condensation of salicylaldehydes with isophthaloyl bishydrazide (3.28), which was
prepared from dimethyl isophthalate (2.28) with hydrazine monohydrate (Scheme 7e).
Scheme 7.
(
a) Synthesis of oxadiazole mimicking acylhydrazone (6a)
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
b) Synthesis of acyldiazole mimicking acylhydrazone (6b)
Br
CHO
(
1.1 eq.)
CHO
OH
Br
O
Ph
K2CO3 (1.5 eq.), DMF,
r.t., 48 h
Br
Br
Br
4f
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
8
7 %
4f.1
CHO
OHC
PPh3
(
1.1 eq.)
O
Ph
THF, reflux, overnight
86 %
Br
Br
Br
Br
4
f.2
1
) NH2NHTs (1.1 eq.),
CH3CN, r.t, 1 h
O
Br
2) NaOH (1.6 eq.), CH3CN,
reflux, overnight
N
N
3)
COCl
O
Ph
(1.5 + 0.3 eq.),
Br
Br
CH3CN, r.t, 3 +1 h,
NaOH (1.5 eq.)
4
f.3
O
BBr3 (1 M in DCM) (1 eq.)
DCM, 0 oC, 30 min
Br
N
N
50 %
OH
Br
6b
(
c) Synthesis of N-methylacylhydrazone (5.28)
(d) Synthesis of C-methylacylhydrazones (5.2i-5.3h)
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(
e) Synthesis of bis-acylhydrazones (5.29, 5.30)
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
Evaluation of antifungal activities against C. neoformans. All aromatic acylhydrazones
5 and their mimics 6 thus synthesized were evaluated for their antifungal activity against C.
neoformans. The antifungal activities of the compounds are indicated by MIC80 values.
Compounds that displayed MIC80 1 µg/mL were further examined for their fungicidal activities
by a time-kill assay in vitro, indicated by K100 values. Results are summarized in Tables 1~6.
Activities of diarylacylhydrazones 5.0~5.7 in the initial library. Nineteen out of the 20
compounds shown in Table 1 are found to be fungicidal in the time-kill assay in vitro, except for
compound 5.7, which is fungistatic. It is worthy of note that compounds 5.2d, 5.2e and 5.6a are
highly potent and selective to fungus with a selectivity index (SI) (vide supra) of ≥500 based on
their LD50 values for HepG2 and A549.
Table 1. Antifungal activity, time-kill activity (K100) and cytotoxicity of acylhydrazones 5.0~5.7 in
the initial 20 compounds library (µg/mL)
A549c
HepG2c
SI d
A549
SI d
HepG2
Compound
.0
5.1
.1a
Ring A
Ring B
MIC80a
0.5
K100b
(
LD50
)
(LD50
)
5
0.25 (48 h)
64
8
128
72
16
32
32
1
0.5 (48)
72
16
32
16
5
0.5
0.25 (48 h)
32
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1
1
1
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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
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4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
5
.2
5.2a
.2b
.2c
5.2d
.2e
.3
1
2 (24 h)
1 (12 h)
32
64
16
64
32
16
0.25
256
256
5
0.12
1
0.5 (48 h)
0.5 (6 h)
16
64
64
133.3
128
533.3
64
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
128
0.06
0.06 (72 h)
64
128
1066.6
2133.3
5
0.06
1
0.06 (72 h)
0.5 (48 h)
64
32
59
32
32
64
32
32
16
32
1066.6
32
1066.6
32
5
5
.3a
.3b
0.25
0.25
0.5
0.25 (24 h)
0.25 (24 h)
0.25 (24 h)
236
128
64
128
64
5
5.3c
.3d
.3e
64
5
0.12
0.25
0.06
0.25 (6 h)
1 (48 h)
32
16
16
16
32
32
266.6
64
133.3
128
5
5
.4
.5
0.25 (96 h)
266.6
533.3
5
0.25
0.25
0.5 (24 h)
16
4
16
8
64
16
64
32
5.6
.6a
0.25 (24 h)
5
0.06
0.25
0.25 (24 h)
Fungistatic
32
32
32
32
533.3
128
533.3
128
5
.7
a
b
c
d
Minimum concentration of the compound required to inhibit growth of 80% of C. neoformans cells.
Minimum concentration of the compound required to show 100% killing of C. neoformans.
Concentration of the compound required to kill 50% of mammalian cells.
Ratio of LD50 value against mammalian cell line and MIC80 value against C. neoformans, constituting the “selectivity
index”.
Modifications of ring A: Introduction of 2,3-, 3,4- and 3,5-diboromophenyl groups. Since
the majority of compounds with high potency from the initial library was bearing either a 2,4- or
2
,5-dibromophenyl group as the ring A, other dibromobenzoylhydrazones bearing a 2,3-, 3,4- or
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3
,5-diboromophenyl group as ring A were synthesized (Scheme 2) and examined for their
activities against C. neoformans (Table 2). Compounds 5.8 and 5.8a were not only highly potent
(MIC80 0.03-0.06 g/mL), but also showed very low toxicity in mammalian cell lines (SI >1,000).
Most of acylhydazones bearing 2,3-dibromophenyl group as ring A (5.10, 5.10b and 5.10c) were
not potent (MIC80 >16 g/mL) against C. neoformans except 5.10a (Table 2)
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 2. Acylhydrazones 5.8~5.10 bearing dibromophenyl groups in ring A and their biological
activities (µg/mL)
A549c
HepG2c
SI
SI
Compound
Ring A
Ring B
MIC80a
0.06
K100b
A549d
HepG2d
(
LD50
)
(LD50
)
5
.8
0.12 (24 h)
64
64
1066.6 1066.6
1066.6 1066.6
5
.8a
0.03
0.06
0.12
0.25
0.12
1 (6 h)
1 (48 h)
32
16
4
32
32
16
8
5.8b
266.6
33.3
32
533.3
133.3
32
5
.8c
Fungistatic
1 (72 h)
5
.8d
8
5
.8e
0.5 (72 h)
16
16
133.3
133.3
5
.8f
0.12
0.06
0.5 (72 h)
4
8
33.3
66.6
5
.9
Fungistatic
16
16
266.6
266.6
5
.9a
.9b
0.25
0.5
0.5 (24 h)
0.5 (24 h)
2
4
8
16
5
64
64
128
128
5
.9c
1
1 (48 h)
4
16
4
16
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1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
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3
3
3
3
4
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4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
5.9d
5.9e
5.9f
0.12
>16
0.5
1 (12 h)
-
16
-
16
-
133.3
133.3
-
-
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 (24 h)
32
16
64
32
5
.10
>16
-
-
-
-
-
5
.10a
0.5
>16
>16
1
2 (48 h)
32
-
16
-
64
-
32
-
5
.10b
-
5
.10c
-
-
-
-
-
5
.10d
1 (48 h)
64
16
64
16
a-d
See the captions in Table 1.
Modifications of ring A: Introduction of bioisosteres of bromine and fluorine-containing
groups. Since the vast majority of active compounds contained one or more bromine atoms on
ring A, bromine appears to have a favorable size and polarity as substituent(s) in ring A. However,
it is rather rare to have many bromines in a pharmaceutical drug candidate. Thus, we examined if
bromine could be replaced with its bioisosters such as CN and CF
that indeed 4-cyanobenzoyl- and 4-trifluoromethylbenzoylhydrazones (5.11 and 5.13) exhibited
good MIC80 values (Table 3). Accordingly, other acylhydrazones containing F, OCF or OCHF
group, as well as 2-F-4-OCF or 2-F-4-CF groups in ring A, were synthesized (Schemes 3 and 4)
3
groups (Scheme 3), and found
3
2
3
3
and their biological activities examined. As Table 3 shows, all compounds, except 5.22 and 5.23c,
in this series exhibited ≤1 µg/mL MIC80 values against C. neoformans. Compounds 5.11, 5.14a,
5
.15, 5.15b, 5.17, 5.18a and 5.20 possess good selectivity index (SI >100). Except for compound
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.19, all compounds with ≤1µg/mL MIC80 values are fungicidal.
Table 3. Acylhydrazones bearing bromine-bioisosteres or fluorine-contaning groups in ring A and
their biological activities (µg/mL)
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
A549c HepG2c
(LD50
SId
SId
Compound
Ring A
Ring B
MIC80a
0.06
K100b
)
(LD50
)
A549 HepG2
5
5
5
5
.11
.12
.13
.14
1 (72 h)
64
16
1066.6 266.6
1
1 (72 h)
1 (12 h)
1 (24 h)
16
4
16
8
16
16
0.12
0.12
33.3
66.6
33.3
533.3
8
64
5
.14a
0.12
0.12
0.25 (96 h)
1 (24 h)
16
32
16
32
133.3
266.6
133.3
266.6
5
.15
5
.15a
0.25
0.12
0.06
0.5
2 (72 h)
1 (6 h)
32
16
4
16
16
16
16
16
128
133.3
66.6
128
64
5
.15b
133.3
266.6
32
5
.16
1 (12 h)
4 (72 h)
0.5 (48 h)
5
.16a
64
16
5
.17
0.06
266.6
266.6
5.17a
0.5
0.5
0.5 (72 h)
1 (48 h)
64
16
64
8
128
32
16
32
5
.18
16
16
5
.18a
0.12
0.5 (48 h)
533.3
133.3
5
.19
0.5
Fungistatic
32
32
64
64
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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
5
.19a
1
0.12
0.5
4
2 (12 h)
0.25 (24 h)
0.5 (24 h)
-
16
32
16
-
16
32
16
-
16
266.6
32
16
266.6
32
5.20
5
5
5
.21
.22
.23
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
2 (96 h)
2 (48 h)
32
16
32
16
32
16
32
16
5
.23a
5
.23b
0.5
2
1 (48 h)
-
64
-
16
-
128
-
32
-
5
.23c
a-d
See the captions in Table 1.
Modifications of ring A: Introduction of 4-aminobenzoyl groups. Several
benzoylhydrazones bearing an amino-, N,N-dimethylamino or N-acetamido group were
synthesized and examined for their biological activities (Scheme 5, Table 4). As Table 4 shows,
the introduction of a 4-aminobenzoyl group as ring A reduced the potency (5.24, MIC80 4 µg/mL)
and that of a 4-acetamidobenzoyl group was detrimental to the potency (5.26, MIC80 16 µg/mL).
Compounds with a 4-N,N-dimethylaminobenzoyl group exhibited mixed results, i.e., 5.25 did not
show appreciable potency, while 5.25a, 5.25b and 5.25c exhibited good potency (MIC80 0.25-0.5)
with modest SI values. However, 5.25a and 5.25b were fungistatic. Compound 5.25c, bearing a 2-
hydroxynaphthyl group as ring B, was found to be fungicidal with a good time-kill profile.
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8
9
Table 4. 4-Aminobenzoylhydrazones 5.24~5.26 and their biological activities (µg/mL)
A549c
HepG2c
SI
SI
Compound
Ring A
Ring B
MIC80a
4
K100b
-
(LD50
)
(LD50
)
A549d HepG2d
5
.24
-
-
-
-
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
5.25
>16
0.5
-
-
-
-
-
5
.25a
Fungistatic
16
16
32
32
5
.25b
0.25
0.5
Fungistatic
2 (24 h)
-
16
32
-
16
16
-
64
64
-
64
64
-
5
.25c
5
.26
>16
a-d
See the captions in Table 1.
Modifications of ring B. Introduction of bioisosteres of bromine and fluorine-containing
groups. In a manner similar to that performed on ring A, a bromine substituent on ring B was
replaced with a CN, CF , F or OCF group and examined for their activity against C. neoformans
Scheme 6, Table 5). As Table 5 shows, 5.2f, 5.2h, 5.4a and 5.4b exhibited good potency (MIC80
3
3
(
≤1 µg/mL) and were fungicidal in the time-kill assay. Compound 5.2g and 5.27a were weakly
active (MIC80 8 and 4 µg/mL, respectively), while compound 5.3f did not show appreciable
activity (MIC80 >16 µg/mL).
Table 5. Acylhydrazones with CN, CF
activities (µg/mL)
3
, F or OCF substitution in ring B and their biological
3
A549c
HepG2c
(LD50)
SI d
SI
d
MIC80a
K100b
Compound
Ring A
Ring B
(
LD50
)
A549 HepG2d
5
a
0.5
Fungistatic
16
16
32
32
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2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
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4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
5
.2f
1
8
2 (96 h)
16
-
16
-
16
-
16
-
5.2g
-
5
.2h
0.25
>16
0.25
1
2 (24 h)
32
-
64
-
128
-
256
-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
.3f
-
5.4a
5.4b
5.27
1 (72 h)
16
8
64
16
-
64
8
256
16
-
4 (96 h)
8
-
-
-
-
5.27a
4
-
-
-
-
a-d
See the captions in Table 1.
Modifications of the central acylhydrazone moiety. In order to explore structural
variations, as well as examine the effects of conformational rigidification on antifungal activity,
heterocyclic mimics of aromatic acylhydrazones, 6a and 6b, were synthesized (Scheme 7a and 7b)
and their activity against C. neoformans was examined. Rather unexpectedly, these two mimics
did not show appreciable activity (MIC80 >16 µg/mL, Table 6). The results indicate that the
acylhydrazone structure appears to be essential for antifungal activity. It is noteworthy that N-
methylated (5.28) and C-methylated (5.2i, 5.2j, 5.3g and 5.3h) acylhydrazones also did not exhibit
appreciable activity (MIC80 >16 µg/mL, Table 6). The results clearly indicate that the NH and CH
groups in the acylhydrazone moiety are essential for their antifungal activity, which is also
consistent with the lack of appreciable activity in the heterocyclic mimics, 6a and 6b, wherein both
NH and CH groups are eliminated by the introduction of oxadiazole and diazole moieties. Bis-
acylhydrazones, 5.29 and 5.30 (Scheme 7e) did not exhibit appreciable activity (MIC80 >16
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µg/mL, Table 6). Thus, the double units of the acylhydrazone moieties did not enhance binding,
but deteriorated the affinity. The result may indicate that the binding site of aromatic
acylhydrazones 5, especially around the ring A, is rather compact.
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 6. Triclyclic, N-methylated and C-methylated acylhydrazones, bis-hydrazones and their MIC80
values (µg/mL)
Compound
Structure
MIC80
>16
6a
6
b
>16
>16
5.28
5
.2i
>16
>16
5.2j
5.3g
>16
5.3h
5.29
5.30
>16
>16
>16
H
N
H
N
Br
Br
Br
Br
N
N
O
O
O
O
OH
HO
H
N
H
N
N
N
OH
HO
Br
Br
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Structure-activity relationship (SAR) analysis. A library of acylhydrazones (192
compounds) was synthesized and screened for activity against C. neoformans H99. Out of the 192
compounds, 42 of them exhibited strong potency (MIC80 1 µg/mL) against C. neoformans and
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
0 of these hit compounds have not been reported in literature (Scheme 1, Table 1). The SAR of
these early hit compounds shown in Table 1 indicated that bromine was well-tolerated on both
rings A and B. 2-Hydroxyl group on ring B was found to be essential for antifungal activity. Other
halogens such as chlorine and iodine were tolerated on ring B, as well. Most of aromatic
acylhydrazones with 2-hydroxyl-5-methylphenyl as ring B showed lower selectivity indices.
Compounds 5.1 and 5.1a with 2,3-difluorophenyl as ring A displayed low SI, as well. Compounds,
bearing 2,4-dibromophenyl as ring A and 2-hydroxy-4-bromophenyl (5.2d) and 2-hydroxy-5-
bromophenyl (5.2e) as ring B were not only highly selective (SI >1,000), but also fungicidal at a
very low concentration (0.06 µg/mL). Compounds 5.2c and 5.3d exhibited an excellent time-kill
profile by completely eradicating C. neoformans in 6 h at very low concentrations, i.e., 0.5 and
0
.25 µg/mL, respectively. Compound 5.4 needed a much longer incubation time (96 h) to show
fungicidal activity. Even though compound 5.7, bearing a quinolin-3-yl group as ring A, displayed
a low MIC80 value (0.25 µg/mL) and fairly good SI (>100), it was found to be fungistatic in the
time-kill assay.
Compounds, bearing 3,4-dibromophenyl as ring A and 2-hydroxy-3,5-dibromophenyl (5.8)
or 2-hydroxy-3,5-dichlorophenyl (5.8a) as ring B exhibited excellent antifungal activity (MIC80
0
2
.06 and 0.03 µg/mL, respectively) as well as very high selectivity to the fungus (SI >1,000) (Table
). However, compounds, bearing 2-hydroxy-5-bromophenyl (5.8d) or 2-hydroxy-4-bromophenyl
(
5.8f, 5.9c, and 5.10) as ring B, were less selective to the fungus. Compound 5.9, bearing 3,5-
dibromophenyl as ring A and 2-hydroxy-5-bromophenyl as ring B displayed good selectivity (SI
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66.6), but was found to be fungistatic. Compounds, bearing 2-hydroxy-4-bromophenyl (5.9c) and
-hydroxy-3,5-dibromophenyl (5.9e) as ring B did not show good selectivity and appreciable
activity, respectively. In contrast, compound, bearing 2-hydroxy-5-chlorophenyl (5.9b) or 2-
hydroxy-3,5-dichlorophenyl (5.9d) as ring B was fungicidal with fairly good selectivity (SI >100).
Accordingly, for compounds with 3,5-dibromophenyl as ring A, chlorine substitutions on ring B
were better tolerated than the bromine counterparts. Having 2,3-dibromophenyl as ring A appears
to impair aromatic acylhydrazone’s antifungal activity, since 3 out of 5 compounds in this series
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
(
5.10, 5.10b and 5.10c) (Table 2) did not exhibit appreciable activity (MIC80 >16) and even the
remaining 2 compounds (5.10a and 5.10d, Table 2) exhibited low selectivity (SI = 32 and 16,
respectively).
Replacement of 4-bromophenyl with 4-CN-phenyl in ring A was well tolerated (5.11,
2
2
Table 3), showing activity equivalent to that of 2. Replacement of bromine with CF
resulted in acylhydrazones that displayed good MIC80 values and time-kill activities, but with low
to modest selectivity. Replacement of 3- or 4-bromophenyl with 3- or 4-CHF O-phenyl was
relatively well-tolerated. Use of 3-CF O-phenyl as ring A significantly reduced the antifungal
activity (5.22, Table 3). Compounds with 2-F, 4-OCF or 2-F, 4-CF substitutions on the phenyl
3
in ring A
2
3
3
3
group as ring A and 2-hydroxy-3,5-dibromophenyl as ring B possessed better selectivity than that
of the counterparts bearing 2-hydroxy-5-bromophenyl as ring B (Table 3). Compounds with a 3-
fluorophenyl as ring A were either fungistatic or not selective. However, compound bearing a 4-
fluorophenyl as ring A exhibited good antifungal activity, good selectivity and excellent time-kill
activity at a very low concentration (5.20, Table 3). Replacement of 4-fluorophenyl with 4-FCH
phenyl resulted in drastic reduction of selectivity (5.21, Table 3).
2
-
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2
2
2
2
2
2
2
3
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3
3
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3
3
3
4
4
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4
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4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
The use of 4-amino- and 4-acetamidophenyl as ring A was found to be detrimental to
antifungal activity (Table 4). Compounds with 4-dimethylaminophenyl as ring A were either
fungistatic or lacking appreciable activity, except for a compound bearing 2-hydroxynaphthyl as
ring B (5.25c, Table 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
Ring B was found to be more sensitive to the replacement of bromine with its bioisosteres
and fluorine-containing groups, as compared to ring A (Table 5). The replacement of bromine at
the 5-poisiton of the phenyl group in ring B with CN, CF or F drastically affected either the
3
antifungal activity or selectivity, whereas replacing bromine with OCF
tolerated.
3
on ring B was well-
As described above, tricyclic analogs of 1 or 2, bearing either an oxadiazole or diazole
moiety as the third ring, did not show appreciable antifungal activity (Table 6). Also,
acylhydrazone derivatives with N-methylation of the hydrazone’s NH moiety or C-methylation of
the hydrazone’s methylidene CH moiety did not exhibit appreciable activity, either (Table 6).
Furthermore, the bis-acylhydrazone skeleton was detrimental to the antifungal activity (Table 6).
Based on the findings and analysis described above, a summary of the SAR of aromatic
acylhydrazones in this study is illustrated in Figure 2.
Figure 2. SAR of aromatic acylhydrazones
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Five most potent and selective acylhydrazones. Among the 83 new aromatic
acylhydrazones evaluated, 5 of them exhibited excellent potency and time-kill profile against C.
neoformans with very high selectivity indices (SI >500). These 5 lead compounds are summarized
in Table 7.
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 7. Five most potent and selective acylhydrazones and their biological activities (µg/mL)
A549c
HepG2c
SId
A549
SId
HepG2
Compound
.2d
Ring A
Ring B
MIC80a
0.06
K100b
(
LD50
)
(LD50
)
5
0.06 (72 h)
64
128
1066.6
1066.6
533.3
2133.3
1066.6
533.3
5.2e
0.06
0.06
0.06 (72 h)
0.25 (12 h)
64
32
64
32
5
.6a
5.8
0
.06
0.12 (24 h)
1 (6 h)
64
32
64
32
1066.6
1066.6
1066.6
1066.6
5.8a
0.03
a-d
See the captions in Table 1.
Also, the time-kill activities of the 5 compounds are shown in Figure 3. All 5 compounds
were fungicidal at very low concentrations. Compounds 5.2d and 5.2e were fungicidal at
extremely low concentrations (0.06 µg/mL). Compound 5.6a even though displayed low MIC80
and good selectivity, it behaved slightly erratic in the time-kill assay (Figure 3C). Compound 5.8a
showed fungicidal activity at a slightly higher concentration (1 µg/mL), but was able to completely
eradicate the fungi in a short span of 6 h.
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1
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2
2
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2
2
2
2
2
2
3
3
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3
3
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3
3
3
4
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4
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4
4
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5
5
5
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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. In vitro time-kill activities of (A) 5.2d, (B) 5.2e, (C) 5.6a, (D) 5.8 and (E) 5.8a. Time-kill activity was
determined using an in vitro time-kill kinetics assay in which the compounds were co-incubated with C. neoformans
cells at 37°C, 5% CO
2
, pH 7.4. The number of CFU is counted during 96 h of incubation. All of the compounds
displayed antifungal activity in a dose-dependent manner.
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Evaluation of antifungal activities against other pathogenic fungal strains. These 5
lead compounds were evaluated for their antifungal activities against other pathogenic strains of
fungi such as C. albicans, C. auris, C. krusei, C. krusei R (resistant to a majority of current
antifungal agents), C. parapsilosis and A. fumigatus. As Table 8 shows, compounds 5.6a and 5.8a
exhibited broad spectrum antifungal activity. It should be noted that 5.6a and 5.8a were active
against C. auris, an emerging pathogen that is resistant to all currently available antifungal
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
30
agents. Compound 5.2d was only modestly active against C. parapsilosis, but did not show
appreciable activity to other fungi. Compound 5.2e showed modest activity against C. krusei and
C. parapsilosis, while 5.8 exhibited moderate activity against the resistant strain of C. krusei and
weak activity against C. auris and C. parapsilosis . It is worthy of note that each fungal strain
appears to be very sensitive to rather small variations in the substitution patterns in ring A and ring
B.
Table 8. MIC80 (µg/mL) values of lead compounds against different pathogenic strains of fungi
C.
C.
albicans
C.
auris
C.
krusei
C.
krusei R
C.
Compound
.2d
.2e
.6a
A. fumigatus
neoformans
parapsilosis
5
0.06
0.06
0.06
0.06
0.03
>16
>16
2
>16
>16
0.5
16
>16
8
>16
>16
0.25
4
8
8
>
16
16
5
>
5
0.25
>16
0.25
2
4
>16
8
5
.8
>16
2
16
1
5.8a
0.5
0.25
Drug combination studies. Since the next-generation antifungal agents are likely to be
used in combination with antifungal drugs that are currently available in clinic, it is important to
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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
examine possible drug-drug interactions or synergy/antagonism between our lead compounds and
clinically used antifungal drugs. Accordingly, the 5 lead compounds, 5.2d, 5.2e, 5.6a, 5.8 and 5.8a
(Tables 7 and 8) were examined for their potential synergism/antagonism with 5 clinical drugs,
i.e., fluconazole (Flu), voriconazole (Vori), itraconazole (Itra), caspofungin (Caspo); amphotericin
B (AB) against 7 fungal strains, i.e., C. neoformans, C. albicans, fluconazole-resistant C. krusei
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
(
Ck Flu R), C. krusei 6258 (Ck6258)
in Tables 9~13, in which each Table summarizes the activity of each of 5 lead compounds against
fungal strains when combined with clinically used drugs. The level of cooperativity was
estimated based on the fractional inhibitory concentration index (FICI). The FICI is defined as
,
C. auris, C. parapsilosis and A. fumigatus. Results are shown
7
3
1
(MICcombined/MICdrugA) + (MICcombined/MICdrugB). The level of cooperativity is categorized as
follows: strongly synergistic: FICI <0.5; synergistic: FICI <1; autonomous: FICI = 1; additive:
FICI 1 ~ 4; antagonistic: FICI >4.31, 32
All 5 lead compounds showed synergistic effect (i.e., FICI <1) with these clinical drugs
against C. neoformans, while they were not tested in combination with caspofungin against C.
3
3
neoformans, since caspofungin is known to be inactive against this fungal strain. In all other
fungal strains, drug combinations with amphotericin B were not studied since Amphotericin B is
3
4
the first line treatment only for cryptococcosis clinically. For C. albicans, 5.2d, 5.8 and 5.8a
showed very to fairly strong synergistic effects (FICI = 0.077~0.625) with only exception of
5
.8a/Caspo combination, which was autonomous (FICI = 1). Compound 5.2e exhibited strong
synergistic effects with voriconazole, itraconazole and caspofungin, but the 5.2e/Flu and 5.6/Flu
combinations were additive (FICI = 2). For fluconazole-resistant C. krusei (Ck Flu R), 5.6a
demonstrated exceptionally strong synergism with all 4 clinical drugs (FICI = 0.00016~0.018),
especially with fluconazole, restoring and enhancing the drug sensitivity (Table 11). Compound
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3
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5
6
7
8
9
5
.8 also exhibited strong synergism with all 4 drugs (FICI = 0.121~0.528) and 5.8a showed
synergistic and additive effects. For C. krusei 6258 (Ck6258), 5.8 and 5.8a exhibited strong
synergism (FICI <0.5) in most combinations, except for two combinations that were autonomous
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
(
FICI = 1). Compounds 5.6a showed strong synergism with 2 clinical drugs, but became additive
with fluconazole and caspofungin, depending on fungal strains. Other two compounds, 5.2d and
.2e did not show synergistic effects in all combinations. For C. auris, 5.8a and 5.8 exhibited
5
strongly synergistic and autonomous effects in all drug combinations, while 5.6a showed strong
synergism with 3 drugs, but the combination with caspofungin was additive. For C. parapsilosis,
only 5.8a exhibited strong synergistic and additive effects with two drugs each. All other drug
combinations resulted in mixed results, although the 5.2d/Vori combination showed extremely
strong synergism (FICI = 0.076). Finally, for A. fumigatus, combinations with caspofungin in
addition to amphotericin B were not studied because caspofungin is not active against this fungal
strain. For the combinations with remaining three drugs, only 5.2d/Vori, 5.2d/Itra and 5.6a/Vori
showed synergistic effects, i.e., all other combinations were additive.
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2
3
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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
Table 9. FICI values of 5.2d combined with clinical drugs against 7 fungal strains
Fungal strain
C. neoformans
C. albicans
Ck Flu R
5.2d/Flu*
5.2d/Vori*
0.072
5.2d/Itra*
0.072
0.128
0.252
1.063
0.508
0.124
0.484
5.2d/Caspo*
5.2d/AB*
0.086
ND
0.065
ND
0.625
0.256
0.502
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
0.281
2
2
ND
Ck6258
2
2
1.250
ND
C. auris
1.016
2
ND
C. parapsilosis
A. fumigatus
1.002
2
0.076
2
ND
0.515
ND
ND
*
Flu: fluconazole; Vori: voriconazole; Itra/itraconazole; Caspo: caspofungin; AB: amphotericin B.
Table 10. FICI values of 5.2e combined with clinical drugs against 7 fungal strains
Fungal strain
C. neoformans
C. albicans
Ck Flu R
5.2e/Flu
5.2e/Vori
0.072
0.256
1.063
1.060
1.125
0.135
0.515
5.2e/Itra
0.072
0.128
0.375
2.015
1.016
2.098
0.484
5.2e/Caspo
5.2e/AB
0.086
ND
0.065
ND
0.502
2
2
2
ND
Ck6258
1.002
2
1.002
2
ND
C. auris
ND
C. parapsilosis
A. fumigatus
1.015
2
1.5
ND
ND
ND
For abbreviation of drug names, see the captions in Table 9.
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Table 11. FICI values of 5.6a combined with clinical drugs against 7 fungal strains
Fungal strain
C. neoformans
C. albicans
Ck Flu R
5.6a/Flu
0.240
2
5.6a/Vori
0.240
0.375
0.018
0.129
0.252
0.135
0.188
5.6a/Itra
0.247
0.188
0.023
0.183
0.180
0.508
1.016
5.6a/Caspo
ND
5.6a/AB
0.261
ND
0.508
0.0005
2.002
2
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
0.00016
0.501
0.531
1.015
2
ND
Ck6258
ND
C. auris
ND
C. parapsilosis
A. fumigatus
2
ND
ND
ND
For abbreviation of drug names, see the captions in Table 9.
Table 12. FICI values of 5.8 combined with clinical drugs against 7 fungal strains
Fungal strain
C. neoformans
C. albicans
Ck Flu R
5.8/Flu
0.237
0.563
0.501
1
5.8/Vori
0.247
0.271
0.310
0.254
1.001
1.03
5.8/Itra
0.247
0.370
0.528
0.153
1.12
5.8/Caspo
ND
5.8/AB
0.247
ND
ND
ND
ND
ND
ND
0.500
0.121
0.125
0.501
1.001
ND
Ck6258
C. auris
1.001
2.015
2
C. parapsilosis
A. fumigatus
2.007
0.281
1.031
For abbreviation of drug names, see the captions in Table 9.
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