Synthesis and biological evaluation of aminothiazoles against Histoplasma capsulatum and Cryptococcus neoformans

Article abstract of DOI:10.1016/j.bmc.2018.01.024

The design and synthesis of a library of forty novel 2-aminoazole analogues as well as their evaluation as antifungal compounds against Histoplasma capsulatum and Cryptococcus neoformans is described. These structures were derived from N-[5-(1-naphthaleny

Full text of DOI:10.1016/j.bmc.2018.01.024

Accepted Manuscript
Synthesis and biological evaluation of aminothiazoles against Histoplasma cap-
sulatum and Cryptococcus neoformans
Keisuke Ishita, Stavros Stefanopoulos, Ahmed Khalil, Xiaolin Cheng, Werner
Tjarks, Chad Rappleye
PII:
S0968-0896(17)32473-2
https://doi.org/10.1016/j.bmc.2018.01.024
BMC 14178
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
22 December 2017
15 January 2018
26 January 2018
Please cite this article as: Ishita, K., Stefanopoulos, S., Khalil, A., Cheng, X., Tjarks, W., Rappleye, C., Synthesis
and biological evaluation of aminothiazoles against Histoplasma capsulatum and Cryptococcus neoformans,
Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.01.024
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Synthesis and biological evaluation of aminothiazoles against Histoplasma
capsulatum and Cryptococcus neoformans
Keisuke Ishita1, Stavros Stefanopoulos2, Ahmed Khalil1,#, Xiaolin Cheng1, Werner Tjarks1, Chad
Rappleye2,*
1Division of Medicinal Chemistry & Pharmacognosy, The Ohio State University, Columbus, OH, 43210.
2Departments of Microbiology and Microbial Infection and Immunity, The Ohio State University.
*Corresponding Author: Chad Rappleye: 484 West 12th Avenue, Columbus, OH 43210, USA; Tel.: 614-
247-2718; Fax: 614-292-8120; E-mail address: rappleye.1@osu.edu
#Permanent address: Chemistry Department, Faculty of Science, Zagazig University, Zagazig, Egypt;
Current address: Chemistry Department, College of Science, King Faisal University, Al-Hofuf 31982, Al-
Ahssa, KSA.
Keywords
Aminothiazoles; Antifungal activity; Structure-Activity-Relationship; Histoplasma capsulatum;
Cryptococcus neoformans
1

Abstract
The design and synthesis of a library of forty novel 2-aminoazole analogues as well as their evaluation as
antifungal compounds against Histoplasma capsulatum and Cryptococcus neoformans is described. These
structures were derived from N-[5-(1-naphthalenylmethyl)-2-thiazolyl]cyclohexanecarboxamide (41F5), a
fungistatic agent previously identified through phenotypic screening (Antimicrob Agents Chemother.
2013; 57:4349). Modifications to improve potency and water-solubility of 41F5 focused primarily on the
5-naphthalenyl group, the thiazole core, and the methylene linker between these two structural elements.
In general, compounds with lipophilic [5+6] bicyclics ring systems, such as the 7-benzothiophenyl- and 4-
indanyl groups, at the 5-position were 2-3 times more active against both fungal species as compard to
41F5. Also, introduction of a carbonyl group at the methylene linker of 41F5 resulted in a 2-3 fold
increase in potency. These highly active compounds also showed generally low toxicities against murine
P388D1 macrophages resulting in selectivity indices ranging from 63 to >200. Compounds that were
highly active against fluconazole-sensitive C. neoformans strains had almost identical activity against
fluconazole-resistant variants of this fungus indicating that 14α-demethylase is not their molecular target.
Highly active compounds also retained activity against H. capsulatum phagocytosed into P388D1
macrophages.
2

1. Introduction
Histoplasma capsulatum, the fungal pathogen causing histoplasmosis, is endemic to the Ohio- and
Mississippi River valleys of North America as well as parts of Latin American and Africa, where up to
90% of the residents display signs of prior infection.1 Up to 50 million people have had contracted H.
capsulatum and an estimated 500,000 cases of new infections occur annually in the US.2,3 Fortunately,
most infections are subclinical but over 3,000 hospitalizations occur per year.4 Histoplasmosis is the most
common cause for hospitalization among endemic fungal disease.4 The cost for each hospitalization is
estimated to be $20,300 for children and $17,000 for adults.4 Cryptococcus neoformans, the culprit of
cryptococcosis, affects primarily the immunocompromised population in sub-Saharan Africa, where the
human immunodeficiency virus (HIV) burden is the highest in the world. In sub-Saharan Africa, the
approximate number of cases of cryptococcal meningitis is 720,000 and the 90-day case fatality is 70%.5-8
Major classes of current antifungal drugs include allylamines, azoles, pyrimidine analogues,
polyenes, echinocandins, and oxaboroles.9,10 These agents have different molecular targets found in the
fungal cell membrane or in fungal biochemical processes.11 All clinically utilized antifungals suffer from
side effects and limited activity spectra.9,10,12-20 In particular, echinocandins, which have the least host
toxicity potential, are ineffective against H. capsulatum and C. neoformans.12-14 Therefore, it is important
to explore novel structural classes for the development of antifungal agents.
Edwards et al. performed a phenotypic screening of a purinome-focused library of 3600
commercially available compounds against H. capsulatum to find novel antifungal candidates and
identified 41F5, which has a thiazole core structure, as the most active compound of this library (Table
1).21 This agent displayed fungistatic activity with an IC50 value of 0.87 µM against H. capsulatum and a
selectivity index of ~63 over macrophages. It was also found that 41F5 is active against C. neoformans
with an IC50 of 1.25 µM but 41F5 was inactive against the fungi Candida albicans, Aspergillus fumigatus
and Blastomyces dermatitidis.21 41F5 did not enhance sensitivity of H. capsulatum to fluconazole
3

suggesting the target of 41F5 differs from the cytochrome P450 sterol 14ꢀ-demethylase inhibited by
azole-class antifungals.22
The thiazole core structure has been utilized as a pharmacophore in a number of biologically
active agents.23 Examples are the anticancer agents tiazofurin and dasatinib, the anti-HIV agent ritonavir,
the antiparasitic drug nitazoxanide, the anti-inflammatory agents fanetizole, meloxicam, and fentiazac, the
antiulcer agent nizatidine, and the insecticide thiamethoxam.24-26 Abafungin is an antifungal drug that
contains a thiazole ring systems but is otherwise structurally different from 41F5. This compound has
inhibitory activity against sterol-C24-methyltransferase and can directly damage fungal cell membrane.27
Abafungin is fungicidal rather than fungistatic, and it is active against Candida albicans and Aspergillus
fumigatus.24 Therefore, it was suggested that abafungin and 41F5 have different mechanisms of action.21
Khalil et al. synthesized 68 analogues of 41F5 to improve potency against H. capsulatum and C.
neoformans and to develop a structure-activity relationship (SAR) for 41F5-derived antifungal
compounds.22 Unfortunately, compounds with higher potency than 41F5 were not identified. Therefore,
we synthesized and evaluated further analogues of 41F5 with the intention to improve potency against H.
capsulatum and C. neoformans and to enhance water solubility. Another objective was the synthesis of
41F5 analogues that have the potential to be used for molecular target identification by unbiased affinity
chromatography techniques. The results of these studies are presentend in this paper.
2. Results
2.1. Design strategies
Table 1 summarizes the primary objectives of our studies and aligns these with areas of structural
modifications of the 41F5 structure and the numbering system for all target compounds. Analogues of
41F5 were synthesized to (1) improve potency against H. capsulatum and C. neoformans yeasts, (2)
enhance water solubility, and (3) identify the molecular target(s) of 41F5-derived antigfungal compounds,
which is(are) currently unknown. Additional related objectives were the evaluation of compound
4

toxicities
against
fluconazole-
Table 1. Alignment of research objectives with areas of structural modifications
at the 41F5 structure and the numbering system for all target compounds
resistant C. neoformans yeast,
murine macrophages, and H.
capsulatum phagocytosed into
murine macrophages.
Areas of chemical
Objective
Target compounds
modifications
The
“A”
group
was
1 (Increased potency
against H. capsulatum
and C. neoformans)
All the target compounds
(14-33, 36, 38, 40)
A, B, C, and D
extensively modified to achieve all
the three objectives (Table 1). In
order to investigate the effect of the
size on antifungal activity, the
28 (quinoline-substituted);
29, 31, 32 (pyridine-substituted);
40 (imidazole-substituted)
2 (Increased water
solubility)
A and C
3 (Molecular target
identification)
14, 15, 33 (ester-modified at
2- or 5-substituents)
A, B and D
naphthalenyl group at the 5-position was replaced with tricyclic rings (fluorenyl), bicyclic rings
(benzothiophenyl, benzofuranyl, 2,1,3-benzothiadiazolyl, indanyl, quinolinyl and tetralinyl), monocyclic
rings (pyridinyl, cyclopentyl, and ester-modified phenyl) or the methyl group. Bioisosteric considerations
played an important role in modifying this group (objective 1).28-30 To potentially improve water
solubility (objective 2), pyridine- (pKa = 5.2) (29a/b, 31, 32a/b)31 and quinoline substituents (pKa = 4.85)
(28a/b)32 were introduced at the 5-position. The phenyl ring at the 5-position of the thiazole core was
substituted with an ester group either at the 3- or 4-position (objective 3). Using standard chemical
methodology, such an ester function could be converted to an alkyne-containing amide group, which
could be coupled to an azide-containing solid support matrix via click chemistry. A stationary phase
modified in this way could potentially be used in an unbiased affinity chromatography approach for
molecular target identification,33 which may have the potential to facilitate the structure-based design of
41F5-derived antifungal compounds. A crucial milestone in pursuing this methodololgy would be to
initially evaluate the toxicity of ester containing compounds against H. capsulatum and C. neoformans. If
such compounds would have significantly reduced activity as compared to 41F5, the fairly bulky ester
group must interfere with binding to the target protein. In such a case, it would be reasonable to assume
5

that further increased steric hindrance by subsequent chemical modifications would be even more
detrimental to binding, thus, rendering this affinity chromatography approach futile. In the “B” area, the
methylene linker at the 5-position was replaced with a carbonyl group in order to explore the nature of the
linker on potency (objective 1). In the “C” area, other heteroaromatic ring systems, such as oxazole (38a
and 38b) and imidazole (40) were introduced to address objective 1. In addition, imidazole (pKa = 6.95)34
was also chosen as a core replacement to explore objective 2 . Similar to the attachment of an ester group
to a phenyl group at the 5-position (“A” area), modifications of the “D” area encompassed introduction of
ester groups to a cyclohexane ring at the 2-position (33a, 33b and 33c) to potentially explore affinity
chromatography approaches for molecular target identification (objective 3).
2.2. Chemistry
2.2.1. Precursor synthesis
Compound 2 (Scheme 1) was synthesized from
commercially-available compound 1 as a precursor
molecule according to a published procedure.35 This
Scheme 1. Reagents and conditions: (a) NaNO2,
method was then adapted for the synthesis of 4 from
commercially available compound 3. Briefly, the reactions
18% HCl, H2O, -5 °C, 24 h; (b) Acrolein, CuCl2 × 2
H2O, CaO, acetone, 5 °C, 2 h; (c) Thiourea, ethanol,
reflux, 24 h.
of ethyl 4-aminobenzoate (1) and ethyl 3-aminobenzoate (3) with sodium nitrite resulted in the formations
of corresponding diazonium salts, which were subsequently exposed to acrolein in the presence of
copper(II) chloride dihydrate to yield the α-chloro aldehyde intermediates 1i and 3i. Cyclization of these
intermediates with thiourea afforded products 2 and 4 in 21% and 22% over all yield, respectively.
Intermediates 1i and 3i were not isolated and converted in situ to their respective end products 2 and 4.
Commercially available compound 5 was lithiated using lithium diisopropyl amide (LDA)
followed by reaction with aldehydes to afford 6a-o in yields ranging from 9% to 81% (Scheme 2).36
6

Scheme 2. Reagents and conditions: (a) LDA, THF, -78 °C, 30 min; (b) R1CHO, r.t., 16 h; (c) Et3SiH, TFA, DCM,
r.t., 16 h; (d) 57% aq. HI, acetic acid, 100 °C, 4 h; *compound was purchased.
Compounds 6a-l and 6o were converted to 7a-l and 7o by reduction of secondary alcohols and
deprotection of t-butoxycarbonyl (Boc) with triethylsilane and trifluoroacetic acid (TFA), respectively, in
yields ranging from 14% to 88%.22 Compounds 7m and 7n were prepared by simultaneous reduction of
the hydroxyl group and removal of the Boc protective group of 6m and 6n using hydriodic acid in 10%
and 43% yields, respectively.37 Boc-deprotection and deoxygenation of 6i afforded not only 7i but also 7i’
in a molar ratio of 84:16 as determined by 1H NMR
and HR-ESI-MS (Scheme 2). It was not possible to
separate this mixture by column chromatography.
The deoxygenation of thiophene derivatives with
triethylsilane in the presence of strong acids has
been reported previously.38,39
Scheme 3. Reagents and conditions: (a) Dess-Martin
Aldehyde
9
was synthesized from
periodinane, 4Å molecular sieves, DCM, r.t., 16 h; (b) t-
BuBr, DMSO, acetonitrile, 65 °C, 16 h; (c) Thiourea,
ethanol, reflux, 16 h.
7

commercially available compound 8 according to a published procedure (Scheme 3),40 which was adapted
for the synthesis of 1241 from starting material 11. Briefly, 3- (8) and 4-pyridinepropanol (11) were
oxidized with Dess-Martin periodinane to afford 9 and 12 in 43% and 44% yield, respectively.
Compounds 9 and 12 were then α-brominated with t-butyl bromide and dimethyl sulfoxide (DMSO)
followed by cyclization with thiourea to afford 1040 and 13 in 15% and 13% yield, respectively.
Scheme 4. Reagents and conditions: (a) R2COCl, Et3N, THF, r.t., 15 min; (b) R2COCl, THF, r.t., 15 min.
2.2.1. Target compound synthesis
Reactions of the precursor molecules 2, 4, 7a-n, 7p, 10 and 13 containing free amino groups with
acid chlorides in the presence/absence of triethylamine (Scheme 4) yielded target compounds 14-32. As 7i
was contaminated with 7i’ (vide supra), two target compounds 24 and 24’ were produced in molar ratio of
1
93:7 as determined by H NMR (SM-S.6.17.1) and HR-ESI-MS (SM-S.6.17.3). As in the case of the
precursor molecules, it was not possible to separate this mixture by column chromatography. Precursor
molecule 7o was coupled with commercially-available ester-substituted cyclohexane-1-carboxylic acids
8

in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI)22 to afford 33a, 33b and 33c
in 10%, 16% and 9% yield, respectively (Scheme 5). Carboxylic acids were used in these experiments
because the corresponding acid chlorides were not commercially available.
Scheme 6. Reagents and conditions: (a) Dess-Martin
periodinane, DCM, r.t., 16 h; (b) TFA, DCM, r.t., 16
h; (c) 3-Cyclohexylpropanoyl chloride or
cyclohexanecarbonyl chloride, Et3N, THF, r.t., 15 min.
Scheme 5. Reagents and conditions: (a) (1R/S,4R/S)-4-
(methoxycarbonyl)cyclohexane-1-carboxylic acid or
(1R,4R)-4-(methoxycarbonyl)cyclohexane-1-
carboxylic acid or (1R,2S)-2-
(methoxycarbonyl)cyclohexane-1-carboxylic acid,
EDCI, DMAP, Et3N, DCM/DMF (3:1, v/v), r.t., 16 h.
The secondary alcohol of compound 6o was
oxidized with Dess-Martin periodinane to a
carbonyl group to afford 34 in 54% yield (Scheme
6)42 followed by the removal of the Boc protective
Scheme 7. Reagents and conditions: (a) 3-
Cyclohexylpropanoyl chloride or cyclohexanecarbonyl
group with trifluoroacetic acid (TFA) to yield 35 in
chloride, Et3N, THF, r.t., 15 min.
82% yield. Compounds 36a and 36b were
synthesized from 35 in 53% and 76% yields, respectively, using the method described for the synthesis of
14-27, and 30a/b.
The oxazole derivatives 38a and 38b (Scheme 7) were prepared from a reported compound 3743 in
8% and 27% yields, respectively, using the method described for synthesis of 14-27, 30a/b, and 36a/b.
Imidazole derivative 40 was synthesized in 65% yield from commercially available
9

cyclohexanecarboxylic acid 39 by reaction with
hydroxybenzotriazole (HOBt) in the presence of EDCI
hydrochloride followed by reaction with commercially
available 5-benzyl-1H-imidazol-2-amine hydrochloride
(Scheme 8). Acylation occurred exclusively at the
amino group under the applied reaction
Scheme 8. Reagents and conditions: (a) HOBt, Et3N,
EDCI hydrochloride, acetonitrile, r.t., 10 min; (b) 5-
Benzyl-1H-imidazol-2-amine HCl, 80 °C, 16 h.
Table 2. Highly active thiazole antigfungal compounds
synthesized and evaluated by Khalil et al.22
C.
neoformans
H.
capsulatum
Cmpd
(H99 strain)
conditions.
R1
R2
IC50 (µM)
IC50 (µM)
41F5
0.4
0.4
0.4
2.3. Biology
> 10
A
Compounds A-C and 41F5, previously
1.6
0.7
1.3
B
C
reported by Khalil et. al,22 were used in our
biological studies as general reference
> 10
compounds because they showed significant antifungal activity and are structurally similar to the newly
synthesized compounds (Table 2). The cytoxicities of target compounds (14-33, 36, 38 and 40) were
evaluated in vitro against H. capsulatum and C. neoformans yeasts as well as in murine P388D1
macrophages and the obtained data are summarized in the Tables 3-5.
Table 3 shows data of target compounds having a bicyclic ring system at the 5-position. Amongst
the group of compounds with a cyclohexylamide substituent at the 2-position, compounds 17b, 22b and
26b, having a 7-benzothiophenyl-, 4-benzothiophenyl-, and indanyl group, respectively, at the 5-position,
were ~2 times more active than 41F5 both against H. capsulatum and C. neoformans. Compound 36b,
substituted at the 2-position with cyclohexylamide and having a carbonyl linked naphthalenyl group at the
5-position, was also ~ 2 times more active than 41F5 against H. capsulatum but appears to be slightly less
active than 41F5 against C. neoformans. The activities of benzofuran-substituted 21a/b (Table 3) against
H. capsulatum and C. neoformans were similar to that of 41F5 and compound A (Table 2).
10

11

Amongst the group of compounds with a cyclohexylethylamide substituent at the 2-position,
compounds 22a and 23a with a 4- and 3-benzothiophenyl group, respectively, were ~ 2 times more active
against H. capsulatum than 41F5. Compounds 21a and 26a, substituted with a 7-benzofuranyl- and an
indanyl group, respectively, had similar activity against H. capsulatum as 41F5 whereas compound 36a,
with a carboranyl linked naphthalenyl group, was slightly less active against this fungal species. A limited
set of compounds with relatively small alicyclic (25a/b) and aliphatic (30a/b) substituents at the 5-
position was evaluated in assays with H. capsulatum and C. neoformans (Table 4). Compound 25b, with a
cyclohexylamide subsituents at the 2-position and a cyclopentylmethyl group at the 5-position, was active
against H. capsulatum in the same range as reference compounds B and C.
Table 4. Activity of 41F5-derived antifungals with monocyclic ring systems or an acyclic ethyl group at the 5-position
C. neoformans
(H99 strain)
H. capsulatum
P388D1
SI
(Hc)
SI
(Cn)
MIC
MIC
IC50 (µM)
IC50 (µM)
IC50 (µM)
Comp
C
R1
R2
(µM)
(µM)
[ SD]
0.80
[ 0.04]
1.06
[ SD]
[ SD]
1.25
2.50
> 40
1.25
20.00
40.00
> 40
> 40
> 20
> 40
2.50
> 40
> 40
> 40
20.00
> 40
> 40
> 40
> 40
nd
> 50
> 38
nd
nd
> 29
nd
1.36
[ 0.10]
B
[ 0.06]
> 20
> 20
> 20
> 20
25a
25b
0.76
[ 0.05]
> 40
> 40
> 40
nd
> 53
> 5
> 2
nd
nd
8.09
[ 0.70]
nd
29a
29b
30a
30b
18.30
[ 1.06]
7.17
[ 0.84]
> 6
nd
C2H5
C2H5
> 20
> 20
> 20
> 20
nd
nd
nd
> 20
> 40
20.00
> 40
> 20
> 20
> 20
> 40
> 40
> 40
nd
> 40
nd
nd
> 5
nd
nd
nd
nd
31
7.67
[ 0.25]
32a
32b
> 20
SD: Standard deviation; SI: Selectivity index, nd: not determined
Khalil et. al.22 reported that 41F5-derived thiazole antifungal compounds with a
cyclohexylethylamide group at the 2-position (e.g. A and C, Table 2) were highly active only against H.
capsulatum whereas those having a cyclohexylamide group at the 2-position (e.g. 41F5 and B, Table 2)
12

were highly active against both H. capsulatum and C. neoformans. Surprisingly, this SAR pattern was not
observed for cyclohexylethylamide substituted compounds 20a and 28a, which were inactive against both
H. capsulatum and C. neoformans, whereas their cyclohexylamide counterparts 20b and 28b were
moderately active against both fungal species in accordance with the previously observed SAR pattern
(Table 3). In contrast, the cyclohexylamide-substituted compounds 23b and 27b were inactive against
both H. capsulatum and C. neoformans whereas their cyclohexylethylamide counterparts 23a and 27a
adhered to the previously established SAR trend and were active/moderately active against H. capsulatum
and inactive against C. neoformans (Table 3). Yet another SAR profile was observed for the 25a/b pair
(Table 4). In this case, activity was observed only for cyclohexylamide-substituted compound 25b in H.
capsulatum. Studies with compounds 20a/b, 23a/b, 25a/b, 27a/b, and 28a/b were repeated several times
to exclude the possibility of experimental error. The obtained set of data indicates that differences in the
SAR pattern between both fungal species are more complex than originally proposed by Khalil et al.22
Compounds 28a/b, 29a/b, 31 and 32a/b were moderately active or inactive against H. capsulatum
and C. neoformans indicating that the presence of a nitrogen in the ring system at the 5-position
negatively affects activity (Tables 3 and 4). No significant differences were observed when the activities
of 20a/b, 28a/b, 29a/b, 31 and 32a/b against both fungal species were evaluated comparatively at pH 5
and and pH 7 (Supplementary Material [SM]-S.2). On the other hand, compounds with oxygen in the ring
system at the 5-position (21a/b) had approximately similar activities as 41F5. These results are consistent
with those reported by Khalil et al.,22 which indicates that polar atoms within the ring systems at the 2-
and 5-positions generally have no positive effect on the activities of 41F5-derived antifungals. In addition,
calculations with ACD/Percepta software (Version 14.1.0, Advanced Chemistry Development, Inc.,
Toronto, ON, Canada) indicated increased aqueous solubilities of compounds 28b, 29a/b, 31, 32a/b by
factors ranging from 15 to 298 at pH 5 and from 10 to 88 at pH 7 as compared to that of 41F5 (SM-S.3),
which suggests that improved water solubility may also have no marked effect on the activities of 41F5-
derived antifungals.
13

The compounds shown in Table 5, having oxazole (38a/b) or imidazole (40) core ring structures,
were evaluated in assays with H. capsulatum and C. neoformans to explore the effect of modifications to
the thiazole core structure on activity. Imidazole 40 was weakly active against H. capsulatum and inactive
against C. neoformans whereas 38a and 38b were inactive against both fungal species. These results are
consistent with those previously reported by Khalil et al.,22 which strongly suggests that the thiazole core
structure is essential for activity.
Table 5. Activity of antifungals with oxazole- and imidazole core structures
C. neoformans
(H99 strain)
H. capsulatum
P388D1
SI
SI
(Hc) (Cn)
MIC IC50
(µM) (µM)
MIC
(µM)
IC50
(µM)
IC50 (µM)
[ SD]
Cmpd
X
Y
R1
R2
CH
CH
O
O
> 20
> 40
> 40
> 20
> 20
> 40
> 40
nd
nd
nd
nd
nd
nd
38a
38b
> 20
8.11
[ 0.33]
CH NH
20.00
> 20
> 40
> 40
> 5
> 2
40
SD: Standard deviation; SI: Selectivity index, nd: not determined
Linking tricyclic fluorene rings via methylene spacer either through the 2- (16a/b), 1- (18), or 4-
position (19) (Scheme 4) to the 5-position of the thiazole core structure resulted in loss of activity both in
H. capsulatum and C. neoformans (IC50s = >20 ꢁM/MIC = >40 ꢁM) indicating that substituents bulkier
than bicyclic naphthalene are not tolerated at this position. Compounds 14, 15, and 33a/b/c (Schemes 4
and 5) were designed and synthesized with the intention of molecular target identification (vide supra).
Unfortunatley, these compounds were inactive (IC50s = >20 ꢁM/MIC = >40 ꢁM) both against H.
capsulatum and C. neoformans, which indicates that the identification of the molecular target of 41F5-
derived antifungals via unbiased affinity chromatography may be difficult to accomplish.
A small libray of highly active compounds, composed of 17b, 21b, 22b, 26b, and 36b, was
evaluated in a comparative assay against C. neoformans using both fluconazole-sensitive (H99 and
B3501) and -resistant (TES9 and MRL862) strains (Table 6).44 Fluconazole and 41F5 were used as
14

Table 6. Activity of 41F5-derived antifungals against fluconazole-resistant Cryptococcus isolates
H99
B3501
TES9a
MRL862a
(serotype A-FluS)
(serotype D-FluS)
(serotype A-FluR)
(serotype A-FluR)
Cmpd
MIC
(µM)
MIC
(µM)
MIC
(µM)
MIC
(µM)
IC50 (µM)
[ SD]b
0.67
[ 0.06]
0.95
[ 0.63]
0.34
[ 0.04]
0.62
[ 0.12]
0.21
[ 0.09]
0.41
[ 0.07]
0.89
[ 0.32]
IC50 (µM)
[ SD]
0.18
[ 0.17]
0.13
[ 0.22]
0.27
[ 0.02]
0.32
[ 0.02]
0.16
[ 0.03]
0.42
[ 0.06]
0.38
[ 0.06]
IC50 (µM)
[ SD]
0.20
[ 0.03]
27.38
[ 2.17]
0.23
[ 0.01]
0.41
[ 0.02]
0.19
[ 0.01]
0.45
[ 0.07]
0.47
[ 0.05]
IC50 (µM)
[ SD]
0.23
[ 0.04]
14.61
[ 0.65]
0.20
[ 0.02]
0.15
[ 0.09]
0.13
[ 0.01]
0.37
[ 0.58]
0.41
[ 0.05]
41F5
Fluconazole
17b
1.25
0.63
0.63
0.63
4.08
0.63
2.50
0.63
1.25
2.50
2.04
0.63
0.63
0.63
1.25
1.25
65.36
0.63
0.63
0.31
1.25
1.25
32.68
0.63
0.63
0.31
1.25
1.25
21b
22b
26b
36b
aTES9 and MRL862 are fluconazole-resistant (FluR) strains due to mutation of the azole target Erg11.
bSD: Standard deviation
reference compounds in this assay. The results indicate that 17b, 21b, 22b, 26b, 36b, and 41F5 were
active to approximately the same extent against both fluconazole-sensitive and -resistant strains. In
contrast, fluconazole was notably less active in the resistant strains as compared to the sensitive strains.
These observations suggest that 14α-demethylase is not the molecular target of 17b, 21b, 22b, 26b, 36b,
and 41F5. It should be noted that clinically-used fluconazole, which has fungistatic or funcicidal dose-
dependent activity in Cryptococcus,45 has approximately the same activity levels against the fluconazole-
sensitive H99- and B3501 C. neoformans strains as the experimental compounds 17b, 21b, 22b, 26b, 36b,
and 41F5.
The activities of compounds 41F5, 17b, 22b and 36b were also evaluated against H. capsulatum
yeasts phagocytozed into murine P388D1 macrophages. Compound 36b displayed slightly improved
IC50/MIC values as compared to those of 41F5 (0.71 0.15 ꢁM/1.25 ꢁM v.s. 1.26 0.17 ꢁM/2.5 ꢁM)
whereas 17b (1.38 0.10 ꢁM/2.5 ꢁM) and 22b (1.21 0.0.7 ꢁM/2.5 ꢁM) had similar inhibitory activities.
As already discussed (vide supra), compounds 17b, 22b and 36b showed ~2-fold increase in activity
against H. capsulatum yeast in vitro as compared to 41F5. However, the same trend was only observed in
the case of 36b for intramacrophage H. capsulatum. In addition, 17b, 22b, 36b, and 41F5 showed
15

moderately reduced activity against intramacrophage H. capsulatum as compared to H. capsulatum yeast
in vitro (Tables), which might be due to the fact that the compounds have to permeate into the phagosome
to inhibit the intracellular yeasts.
3. Summary and Conclusions
The highest activities against both C. neoformans and H. capsulatum were found for compounds
containing [5,6]- and [6,5]-bicyclic ring systems, such as benzothiophene or indane groups, at the 5-
position (17b, 22a, 22b, 23a, 26a, 26b) and a [6,6]-bicyclic ring system linked via carbonyl spacer (36b)
to the same position of the thiazole core structure. These compounds also showed low toxicities against
murine P388D1 macrophages resulting in excellent selectivity indices (SIs). Highly active compounds,
such as 17b, 21b, 22b, 26b, and 36b, remained active against fluconazole-resistent C. neoformans TES9
and MRL862 strains and H. capsulatum yeasts phagocytozed into murine P388D1 macrophages.
Compounds with reduced activity had ester functions in
cyclic systems either at the 2- or the 5-position (14, 15,
33), a tricyclic ring system at the 5-position (16, 18, 19),
an ionizable nitrogen atom in aromatic systems at the 5-
position (28, 29, 31, 32), a small ethyl substituent at the
5-position (30), or a modified core structure (38, 40).
Several other modifications did also not lead to
improved activity (17a, 20, 21, 23b, 24, 25, 27, 36a).
Based on the obtained results we were able to extend our
understaning of the H. capsulatum specific SAR for
Figure 1. Updated Histoplasma SAR for 41F5-
derived antifungal compounds. Areas circled with
41F5-derived antifungals, which is illustrated in Figure 1.
dotted lines indicate findings described in this paper.
All other findings were reported previously by
Khalil et al.
16

As discussed (vide supra), the C. neoformans specific SAR deviates from this pattern to some degree.
The results obtained for the 36a/b tandem (carbonyl linker at the 5-position) indicate that the
exploration of e.g. ether-, thioether-, thiocarbonyl-, thionyl- and sulfonamide linkers at the 2- and 5
position have the potential to further improve activity of 41F5-derived antifungals. Overall, however, the
chemical space specific for this compound class has been explored fairly exhaustively by our group in the
present and a previous study.22 Although compounds such as 17b, 21b, 22b, 26b, and 36b appeared to
have a ~2 fold increased activity both against H. capsulatum and C. neoformans as compared to 41F5,
compounds with markedly improved potency could not be indentified. Therefore, it is doubtful if
additional SAR studies using a phenotypic screening approach with a further extended compound library
can produce any dramatic advancement.
As discussed (vide supra), it is conceivable that structural information for the molecular target(s)
of 41F5-derived antifungal compounds could potentially facilitate a more rational structure-based drug
design approach. However, low antifungal activities obtained for ester-modified compounds make it
questionable if an unbiased affinity chromatography approach for molecular target indentification is a
viable strategy.
Acknowledgements
We thank June Kwon-Chung at the NIH for providing fluconazole-resistant Cryptococcus strains for
testing. The project described was supported by Award Number Grant 8UL1TR000090-05 from the
National Center For Advancing Translational Sciences and, in part, NIH (NIAID) grant R21AI109437.
The content is solely the responsibility of the authors and does not necessarily represent the official views
of the National Center For Advancing Translational Sciences or the National Institutes of Health. This
work was initiated with funds provided by the OSU Public Health Preparedness for Infectious Diseases
(PHPID) program (phpid.osu.edu) and by the OSU College of Pharmacy.
17

Footnotes
Abbreviations: Boc, tert-butyloxycarbonyl; DMAP, 4-dimethylaminopyridine; DCM, dichloromethane;
DMF, dimethylformamide; DMSO, dimethyl sulfoxide; EDCI, 1-ethyl-3-(-3-dimethylaminopropyl)
carbodiimide; HIV, human immunodeficiency virus; HOBt, hydroxybenzotriazole; HR-ESI, high
resolution–electrospray ionization; LDA lithium diisopropyl amide; MIC, minimal inhibitory
concentration; rxn, reaction; SAR, structure-activity-relationship; SD, standard deviation; SM,
supplementary material; SI, selectivity index; TFA, trifluoroacedic acid; THF, tetrahydrofurane.
A.
Supplementary material
Supplementary material associated with this article can be found, in the online version, at…… This
material includes data on antifungal activities and calculated aqueous solubilities of 41F5-derived
antifungal compounds at pH5 and pH7, calculated ionization energy levels of 41F5-derived antifungal
compounds, a full experimental section, and NMR/MS spectra for target compound
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21

Table 1. Alignment of research objectives with areas of structural modifications
at the 41F5 structure and the numbering system for all target compounds
Areas of chemical
modifications
Objective
Target compounds
1 (Increased potency
against H.
capsulatum and C.
All the target compounds
(14-33, 36, 38, 40)
A, B, C, and D
neoformans)
28 (quinoline-substituted);
29, 31, 32 (pyridine-substituted);
40 (imidazole-substituted)
2 (Increased water
solubility)
A and C
3 (Molecular target
identification)
14, 15, 33 (ester-modified at
2- or 5-substituents)
A, B and D
22

Table 2. Highly active thiazole antigfungal compounds
synthesized and evaluated by Khalil et al.22
C.
N
H
N
H.
capsulatum
R1
R2
Cmpd
neoformans
(H99 strain)
IC50 (ꢁM)
S
O
R1
R2
IC50 (ꢁM)
41F5
0.4
0.4
0.4
> 10
A
1.6
0.7
1.3
B
C
> 10
23

Table 3. Activity of 41F5-derived antifungals with bicyclic ring systems at the 5-position
N
H
C. neoformans
(H99 strain)
R1
N
R2
H. capsulatum
P388D1
S
SI
SI
O
(Hc)
(Cn)
IC50 (ꢁM)
[ SD]
MIC
(ꢁM)
IC50 (ꢁM) MIC
IC50 (ꢁM)
[ SD]
Cmpd
R1
R2
[ SD]
> 20
(ꢁM)
0.60
[ 0.03]
27.59
[ 4.12]
1.25
> 40
46
> 83
21
nd
> 60
nd
A
0.48
[ 0.01]
0.67
[ 0.06]
41F5
17a
17b
20a
1.25
5.00
0.31
> 40
1.25
> 40
0.63
> 40
> 40
0.92
[ 0.38]
18.87
[ 3.05]
S
> 20
0.20
[ 0.01]
0.34
[ 0.04]
S
> 40
nd
> 200 > 118
N
N
> 20
> 20
nd
nd
S
1.54
[ 0.42]
1.52
[ 0.22]
2.50
0.63
1.25
0.31
5.00
> 40
2.50
> 40
> 40
> 26
44
> 26
nd
20b
21a
21b
22a
0.40
[ 0.01]
17.75
[ 5.00]
> 20
0.42
[ 0.01]
0.62
[ 0.12]
> 40
> 40
> 40
> 95
> 200
> 65
nd
0.20
[ 0.01]
> 20
0.20
[ 0.01]
0.37
[ 0.09]
0.31
0.63
1.25
> 40
> 200 > 108
22b
23a
0.27
[ 0.02]
16.90
[ 1.75]
> 20
63
nd
nd
> 20
> 40
> 40
> 20
> 20
> 40
> 40
nd
nd
nd
nd
23b
24
> 20
nd
SD: Standard deviation; SI: Selectivity index, nd: not determined
continued
24

Table 3. continued
C. neoformans
(H99 strain)
H. capsulatum
P388D1
SI
SI
(Hc) (Cn)
IC50 (ꢁM)
[ SD]
0.38
[ 0.02]
MIC
(ꢁM)
IC50 (ꢁM)
[ SD]
MIC
(ꢁM)
IC50 (ꢁM)
[ SD]
Cmpd
26a
R1
R2
0.63
> 20
> 40
> 40
> 40
> 105
nd
0.25
[ 0.02]
0.41
[ 0.07]
0.63
2.50
1.25
> 40
> 160 > 98
26b
0.90
[ 0.29]
> 20
> 20
> 20
24.27
27
nd
27a
> 20
> 20
> 40
> 40
> 40
> 40
nd
nd
nd
nd
5
nd
nd
5
27b
28a
28b
3.05
[ 0.25]
3.24
[ 0.42]
13.92
[ 1.19]
10.00
10.00
0.64
[ 0.03]
1.25
0.31
> 20
> 40
2.50
> 40
> 40
> 63
nd
36a
36b
0.19
[ 0.01]
0.89
[ 0.32]
> 211 > 45
SD: Standard deviation; SI: Selectivity index. Not determined
25

Table 4. Activity of 41F5-derived antifungals with monocyclic ring systems or an acyclic ethyl group at
the 5-position
C. neoformans
(H99 strain)
H. capsulatum
P388D1
SI
SI
(Hc) (Cn)
IC50 (ꢁM)
[ SD]
0.80
MIC
(ꢁM)
IC50 ( M) MIC
IC50 (ꢁM)
[ SD]
Comp
R1
R2
[ SD]
> 20
(ꢁM)
1.25
> 40
> 40
> 50
nd
C
[ 0.04]
1.06
[ 0.06]
1.36
[ 0.10]
2.50
> 40
1.25
2.50
> 40
> 40
> 40
nd
> 38 > 29
B
> 20
> 20
nd
nd
nd
25a
25b
0.76
[ 0.05]
8.09
[ 0.70]
18.30
> 20
> 40
> 53
20.00
> 20
> 40
> 40
> 5
nd
29a
7.17
[ 0.84]
40.00
> 40
> 40
> 40
20.00
> 40
> 40
> 40
> 40
nd
> 2
nd
nd
nd
> 6
nd
nd
nd
29b
30a
30b
31
[ 1.06]
C2H5
C2H5
> 20
> 20
> 20
> 20
> 20
> 20
nd
nd
7.67
[ 0.25]
20.00
> 40
> 20
> 20
> 40
> 40
> 40
nd
> 5
nd
nd
nd
32a
32b
> 20
SD: Standard deviation; SI: Selectivity index, nd: not determined
26

Table 5. Activity of antifungals with oxazole- and imidazole core structures
C. neoformans
H. capsulatum
P388D1
(H99 strain)
SI
SI
(Hc) (Cn)
IC50 (ꢁM) MIC IC50
MIC
IC50
Cmpd
X
Y
R1
R2
[ SD]
(ꢁM) (ꢁM)
(ꢁM)
(ꢁM)
CH
CH
O
O
> 20
> 40
> 40
> 20
> 20
> 40
> 40
nd
nd
nd
nd
nd
nd
38a
38b
> 20
8.11
[ 0.33]
CH NH
20.00
> 20
> 40
> 40
> 5
> 2
40
SD: Standard deviation; SI: Selectivity index, nd: not determined
27

Table 6. Activity of 41F5-derived antifungals against fluconazole-resistant Cryptococcus isolates
H99
B3501
TES9a
MRL862a
(serotype A-FluS)
(serotype D-FluS)
(serotype A-FluR)
(serotype A-FluR)
Cmpd
IC50 (ꢁM) MIC IC50 (ꢁM)
MIC
(ꢁM)
IC50 (ꢁM) MIC IC50 (ꢁM) MIC
[ SD]b
0.67
(ꢁM)
[ SD]
[ SD]
(ꢁM)
[ SD]
(ꢁM)
0.18
[ 0.17]
0.13
0.20
0.23
41F5
Fluconazole
17b
1.25
0.63
2.04
0.63
0.63
0.63
1.25
1.25
0.63
0.63
[ 0.06]
[ 0.03]
27.38
[ 2.17]
0.23
[ 0.01]
0.41
[ 0.02]
0.19
[ 0.01]
0.45
[ 0.07]
0.47
[ 0.04]
14.61
[ 0.65]
0.20
[ 0.02]
0.15
[ 0.09]
0.13
[ 0.01]
0.37
[ 0.58]
0.41
0.95
[ 0.63]
0.34
[ 0.04]
0.62
[ 0.12]
0.21
[ 0.09]
0.41
[ 0.07]
0.89
4.08
0.63
2.50
0.63
1.25
2.50
65.36
0.63
0.63
0.31
1.25
1.25
32.68
0.63
0.63
0.31
1.25
1.25
[ 0.22]
0.27
[ 0.02]
0.32
[ 0.02]
0.16
[ 0.03]
0.42
[ 0.06]
0.38
[ 0.06]
21b
22b
26b
36b
[ 0.32]
[ 0.05]
[ 0.05]
aTES9 and MRL862 are fluconazole-resistant (FluR) strains due to mutation of the azole target Erg11.
bSD: Standard deviation
28

Captions
Figure 1. Updated Histoplasma SAR for 41F5-derived antifungal compounds. Areas circled with dotted
lines indicate findings described in this paper. All other findings were reported previously by Khalil et al.
Scheme 1. Reagents and conditions: (a) NaNO2, 18% HCl, H2O, -5 °C, 24 h; (b) Acrolein, CuCl2 × 2
H2O, CaO, acetone, 5 °C, 2 h; (c) Thiourea, ethanol, reflux, 24 h.
Scheme 2. Reagents and conditions: (a) LDA, THF, -78 °C, 30 min; (b) R1CHO, r.t., 16 h; (c) Et3SiH,
TFA, DCM, r.t., 16 h; (d) 57% aq. HI, acetic acid, 100 °C, 4 h; *compound was purchased.
Scheme 3. Reagents and conditions: (a) Dess-Martin periodinane, 4Å molecular sieves, DCM, r.t., 16 h;
(b) t-BuBr, DMSO, acetonitrile, 65 °C, 16 h; (c) Thiourea, ethanol, reflux, 16 h.
Scheme 4. Reagents and conditions: (a) R2COCl, Et3N, THF, r.t., 15 min; (b) R2COCl, THF, r.t., 15 min.
Scheme 5. Reagents and conditions: (a) (1R/S,4R/S)-4-(methoxycarbonyl)cyclohexane-1-carboxylic acid
or (1R,4R)-4-(methoxycarbonyl)cyclohexane-1-carboxylic acid or (1R,2S)-2-
(methoxycarbonyl)cyclohexane-1-carboxylic acid, EDCI, DMAP, Et3N, DCM/DMF (3:1, v/v), r.t., 16 h.
Scheme 6. Reagents and conditions: (a) Dess-Martin periodinane, DCM, r.t., 16 h; (b) TFA, DCM, r.t., 16
h; (c) 3-Cyclohexylpropanoyl chloride or cyclohexanecarbonyl chloride, Et3N, THF, r.t., 15 min.
Scheme 7. Reagents and conditions: (a) 3-Cyclohexylpropanoyl chloride or cyclohexanecarbonyl
chloride, Et3N, THF, r.t., 15 min.
Scheme 8. Reagents and conditions: (a) HOBt, Et3N, EDCI hydrochloride, acetonitrile, r.t., 10 min; (b) 5-
Benzyl-1H-imidazol-2-amine HCl, 80 °C, 16 h.
29