Ibrexafungerp: An orally active β-1,3-glucan synthesis inhibitor

Article abstract of DOI:10.1016/j.bmcl.2020.127661

We previously reported medicinal chemistry efforts that identified MK-5204, an orally efficacious β-1,3-glucan synthesis inhibitor derived from the natural product enfumafungin. Further extensive optimization of the C2 triazole substituent identified 4-pyridyl as the preferred replacement for the carboxamide of MK-5204, leading to improvements in antifungal activity in the presence of serum, and increased oral exposure. Reoptimizing the aminoether at C3 in the presence of this newly discovered C2 substituent, confirmed that the (R) t-butyl, methyl aminoether of MK-5204 provided the best balance of these two key parameters, culminating in the discovery of ibrexafungerp, which is currently in phase III clinical trials. Ibrexafungerp displayed significantly improved oral efficacy in murine infection models, making it a superior candidate for clinical development as an oral treatment for Candida and Aspergillus infections.

Full text of DOI:10.1016/j.bmcl.2020.127661

Journal Pre-proofs
Ibrexafungerp: an orally active β-1,3-glucan synthesis inhibitor
James M. Apgar, Robert R. Wilkening, Dann L. Parker Jr., Dongfang Meng,
Kenneth J. Wildonger, Donald Sperbeck, Mark L. Greenlee, James M.
Balkovec, Amy M. Flattery, George K. Abruzzo, Andrew M. Galgoci, Robert
A. Giacobbe, Charles J. Gill, Ming-Jo Hsu, Paul Liberator, Andrew S. Misura,
Mary Motyl, Jennifer Nielsen Kahn, Maryann Powles, Fred Racine, Jasminka
Dragovic, Weiming Fan, Robin Kirwan, Shu Lee, Hao Liu, Ahmed Mamai,
Kingsley Nelson, Michael Peel
PII:
S0960-894X(20)30772-1
DOI:
Reference:
https://doi.org/10.1016/j.bmcl.2020.127661
BMCL 127661
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
31 August 2020
26 October 2020
28 October 2020
Please cite this article as: Apgar, J.M., Wilkening, R.R., Parker, D.L. Jr., Meng, D., Wildonger, K.J., Sperbeck,
D., Greenlee, M.L., Balkovec, J.M., Flattery, A.M., Abruzzo, G.K., Galgoci, A.M., Giacobbe, R.A., Gill, C.J.,
Hsu, M-J., Liberator, P., Misura, A.S., Motyl, M., Nielsen Kahn, J., Powles, M., Racine, F., Dragovic, J., Fan,
W., Kirwan, R., Lee, S., Liu, H., Mamai, A., Nelson, K., Peel, M., Ibrexafungerp: an orally active β-1,3-glucan
synthesis inhibitor, Bioorganic & Medicinal Chemistry Letters (2020), doi: https://doi.org/10.1016/j.bmcl.
2020.127661
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Ibrexafungerp: an orally active β-1,3-glucan synthesis inhibitor
James M. Apgar a,, Robert R. Wilkening a, Dann L. Parker, Jr. a, Dongfang Meng a, Kenneth J. Wildonger
a, Donald Sperbeck a, Mark L. Greenlee a, James M. Balkovec a, Amy M. Flattery a, George K. Abruzzo a,
Andrew M. Galgoci a, Robert A. Giacobbe a, Charles J. Gill a, Ming-Jo Hsu a, Paul Liberator a, Andrew S.
Misura a, Mary Motyl a, Jennifer Nielsen Kahn a, Maryann Powles a, Fred Racine a, Jasminka Dragovic a,
Weiming Fan b, Robin Kirwan b, Shu Lee b, Hao Liu b, Ahmed Mamai b, Kingsley Nelson b, Michael Peel b
aMerck & Co. Inc., 2000 Galloping Hill Road, Kenilworth, NJ 07033, USA
bScynexis Inc., 1 Evertrust Plaza, Jersey City, NJ 07302, USA
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
We previously reported medicinal chemistry efforts that identified MK-5204, an orally efficacious
β-1,3-glucan synthesis inhibitor derived from the natural product enfumafungin. Further
extensive optimization of the C2 triazole substituent identified 4-pyridyl as the preferred
replacement for the carboxamide of MK-5204, leading to improvements in antifungal activity in
the presence of serum, and increased oral exposure. Reoptimizing the aminoether at C3 in the
presence of this newly discovered C2 substituent, confirmed that the (R) t-butyl, methyl
aminoether of MK-5204 provided the best balance of these two key parameters, culminating in
the discovery of ibrexafungerp, which is currently in phase III clinical trials. Ibrexafungerp
displayed significantly improved oral efficacy in murine infection models, making it a superior
candidate for clinical development as an oral treatment for Candida and Aspergillus infections.
Keywords:
Ibrexafungerp
SCY-078
MK-3118
β-1,3-glucan synthesis inhibitor
Enfumafungin
2009 Elsevier Ltd. All rights reserved.
———
 Corresponding author. Tel.: +1-908-740-5122; fax: +1-908-740-6136; e-mail: james_apgar@merck.com

Even with current antifungal therapy (polyenes, azoles,
echinocandins), high mortality rates are observed for invasive
candidiasis (IC) and aspergillosis, which comprise the majority of
posaconazole), the next class of antifungals to be developed,
disrupt fungal cell membranes by inhibiting the cytochrome P450
enzyme (Cyp51p) responsible for a key step in the biosynthesis of
ergosterol. They are better tolerated than the polyenes and have
the advantage of being orally bioavailable; however, inhibition of
host P450 enzymes result in a plethora of drug-drug interactions
(DDIs) that present challenges for patients taking multiple
systemic,
opportunistic
fungal
infections.1-6
Immunocompromised and intensive care unit (ICU) patients are at
highest risk for developing IC, with candidemia being cited as one
of the most common bloodstream infections in ICUs, particularly
among patients with central venous catheters.1-5 The emergence
of multidrug resistant organisms, including Candida auris with
some isolates showing resistance to all three antifungal classes,
highlights the urgent need for the development of novel antifungal
treatments.4-5,7-11 Reports of azole resistant isolates of Aspergillus
fumigatus arising from the widespread agricultural use of azole
antifungals is also concerning.9-10
medications.3,6,12
Echinocandins (caspofungin, micafungin,
anidulafungin), the most recent class of antifungals, compromise
the integrity of the fungal cell wall, eventually leading to cell lysis
under osmotic stress, through the inhibition of β-1,3-glucan
synthase (GS), which produces β-1,3-glucan, a critical component
of the cell wall.6,13 Due to the specificity of their target for fungal
cells, the echinocandins have demonstrated improved tolerability
and fewer DDIs compared to the polyenes and azoles.3,6,12 The
echinocandins are now recommended as first line therapy for IC,
as a result of these advantages and because of their broad spectrum
of activity for Candida species.1,3-4,8,14 Restriction to parenteral
administration due to poor oral bioavailability constitutes a
significant limitation for echinocandin therapy.12-15 A novel class
of GS inhibitor, combining oral bioavailability with the improved
safety profile of the echinocandins, would have the potential to
The three classes of antifungals available to treat
systemic fungal infections have distinct advantages and
limitations. Polyenes (Amphotericin B), the oldest class of
antifungals, disturb fungal cell membranes by binding to
ergosterol, resulting in the leakage of cell contents. They have
very broad spectrum antifungal activity, but their clinical use is
often limited by nephrotoxicity caused by non-specific binding to
mammalian sterols.3,6,12
Azoles (fluconazole, voriconazole,
O
OH
O
OH
O
OH
O
OH
H2N
N
O
NH2
25
18
N
N
N
18
18
25
H
25
H
H
O
O
O
O
OH
N
N
N
2
2
2
O
HO
HO
N
H2N
O
3
O
O
3
3
H
H
H
OH
2
MK-5204 (3)
enfumafungin (1)
Figure 1. Progression from enfumafungin to MK-5204
provide benefit over existing treatment options for patients with
resistant infections or requiring step-down treatment from a
parenteral antifungal.
The most recent report from our group concerning
synthetic modification of enfumafungin,16 a naturally occurring
GS inhibitor,17-18 focused on replacing the C2 acetoxy group with
a carboxamidetriazole moiety, the C3 glycoside with an
aminoether, and the C25 bridging hemiacetal with an ether
(Figure 1).19 These efforts eventually led to the identification of
MK-5204 (3), which demonstrated improved oral efficacy in a 7-
day murine model of disseminated candidiasis (6.4 mpk ED99 –
effective dose to reduce fungal burden by 99%) compared to an
earlier C2 aminotetrazole lead20 (2) (9 mpk ED99) while also
removing concerns about N-dealkylation metabolism from the C3
aminoether substituent.19 Herein we present efforts to further
optimize the C2 and C3 substituents to produce additional
improvement in oral efficacy. Initially, we focused on expanding
our scan of C2 triazole substituents from previously reported polar
and/or electron withdrawing groups to alkyl and aryl moieties in
conjunction with an (R)-isopropyl, methyl amino ether at C3 using

Route A
O
OH
O
OH
OH
O
O
18
25
H
H
O
O
O
O
O
O
OH
OH
a
2
O
O
HO
HO
HO
HO
3
H
H
OH
OH
enfumafungin (1)
C25-deoxyenfumafungin (4)
Ts
N
R1
R2
O
OBn
O
OBn
6
H
O
H
MeO
O
d or d, e
b, c
MeO
HO
Ts
N
R3
O
H
R1 R2
H
5
7
R4
NH
O
OH
O
OH
N
R4
N
N
H
H
O
O
9
MeO
O
N
O
N
f
g
H
H
N
N
R3
R3
R1 R2
R1 R2
H
H
10
8
R4 BF3K or B(OR)2
Route B
R4
11
Br
N
N
N
MeO
h
i
X =
O
OH
N
N
N
8
10
10a
H
O
O
X
N
H
N
R4
N
13
Route C
R3
O
R4
R1 R2
H
H
N
N
N
MeO
j
k
H2N
X =
N
12
8
10
Reagents and conditions: (a) Et3SiH, TFA, DCM, rt; (b) H2SO4, MeOH, 65 oC; (c) BnBr, NaHCO3, DMF, 65 oC, 76% (3 steps); (d) 1) K t-pentylate,
18-crown-6, DMA, 0 oC, or 2) KH, 18-crown-6, DME, 0 oC, 17-100%; (e) NaH, MeI, DMF, 50 oC, 69-98%; (f) Na, NH3, THF, -35 oC, 70-100%; (g) BF3 OEt2,
DCE, 50 oC, 4-60%; (h) 3-Br-1,2,4-triazole, BF3 OEt2, DCE, 50 oC, 30-49%; (i) 1) Pd(OAc)2 or PdCl2(PPh3)2, XPHOS, Cs2CO3, EtOH, H2O, 80 oC, or
Pd(OAc)2. XPHOS, Cs2CO3, DMA or DME, 100 oC, 6-35%; (j) hydrazine or benzyl carbazate, BF3 OEt2, DCE, 50 oC, 38-59%; (k) AcOH, 90 oC, 42-70%.
Scheme 1. Synthesis of C2 triazole, C3 aminoether derivatives.
R4
NH
the general synthetic route shown in Scheme 1.
O
O
O
C25-Deoxyenfumafungin (4) was obtained via reduction
of the bridging hemiacetal of enfumafungin (1) with
triethylsilane.20-23 Sulfuric acid in methanol was employed to
hydrolyze the C3 glycoside of 4 to the alcohol with concurrent
displacement of the C2 acetoxy moiety to furnish the methyl
ether.20 Benzyl ester protection of the C18 carboxylic acid
afforded 5 in 76% overall yield from 1. Alkylation of the C3
alcohol with tosyl protected aziridine 6 provided access to 7, which
was treated with iodomethane to achieve mono-methyl alkylation
of the protected amine.19,22-23 Intermediate 8 was obtained via
Birch reduction, which simultaneously removed the benzyl ester
from C18 and the tosyl protecting group from the C3 aminoether.
Displacement of the C2 methoxy, which occurred with retention
of stereochemistry, by substituted 1,2,4-triazoles was promoted by
borontrifluoride diethyletherate as previously described (Route A
– Scheme 1).20 The triazoles (9a-b) used in the synthesis of 10n
and 10v were prepared according to Scheme 2. Condensation of
commercial aryl-amides (15) with N,N-dimethylformamide
diethyl acetal provided acyl-amidines (13), which were cyclized
with hydrazine hydrate to give 3-aryl-1,2,4-triazoles (9).24
N
N
a
b
c
R4
N
R4 OR
R4 NH2
N
13
14
15
9
Reagents and conditions: (a) NH4OH, 77%-quant.; (b) DMF diethyl acetal,
120 oC, 81%-quant.; (c) hydrazine hydrate, AcOH, 90 oC, 27-65%.
Scheme 2. Synthesis of acyl-amidines and 3-aryl-1,2,4-triazoles.
The identity of the 3-substitutent on the triazole determined the
distribution of regioisomeric products (1-[1,2,4-triazole], the
desired 2-[1,2,4-triazole] (10), 4-[1,2,4-triazole]) from the
displacement reaction in Scheme 1.19 Reverse phase HPLC was
employed to separate the three regioisomers.19 In the displacement
reaction, 3-alkyl-1,2,4-triazoles exemplified by 3-methyl-1,2,4-
triazole, failed to produce enough of the desired 2-[1,2,4-triazole]
isomer to isolate.
3-aryl-1,2,4-triazoles only performed
marginally better with yields of 5% or less for the desired isomer
(10g, 10n, 10v). These limitations forced us to develop alternative
synthetic strategies to access desired chemical space and enable
scale-up of analogs of interest. Fortunately, the displacement
reaction with 3-bromo-1,2,4-triazole, which was synthesized

according to literature procedures,25 gave 10a as the major product
with yields ranging from 30-49% (Route B – Scheme 1). Suzuki
cross-coupling with 10a afforded aryl substituted analogs (10g-
10q) in low to moderate yield (6-35%). This route was also
employed to deliver the ethyl and propyl substituted triazole
analogs (10d-10e) via hydrogenation of olefin Suzuki products.
To further expand access to chemical space, we
chose to construct the C2 triazole substituent stepwise on the
modified enfumafungin scaffold (8). Performing the displacement
reaction with anhydrous hydrazine or benzyl carbazate
(deprotection occurred in situ under the reaction conditions)
provided the C2 hydrazine (12) in yields ranging from 38-59%
(Route C – Scheme 1). Cyclization of 12 with acyl-amidines (13)
proceeded smoothly with exclusive formation of the desired 2-
[1,2,4-triazole] isomer to provide 3-alkyl and aryl triazole analogs
(10c, 10f, 10q-10u, 10w-10y) in 42-70% yield.24 Acyl-amidines
(13) were generated via condensation of N,N-dimethylformamide
diethyl acetal with amides24 (15) that were obtained commercially
or from treatment of commercial esters (14) with ammonium
hydroxide as shown in Scheme 2, except for the amide used in the
synthesis of 10r, which was prepared from the carboxylic acid
using literature procedures.26 This third and final route to install
the C2 triazole
Scheme 3. Synthesis of aziridines.
provided the broadest substrate scope and was employed to scale-
up key analogs.
The tosyl-aziridines (6) used to synthesize the analogs
displayed in Tables 1-2 were prepared via the three methods
shown in Scheme 3. The racemic/achiral tosyl-aziridines (6)
employed in the preparation of 10q, 10z-10ad, and 10ag were
obtained either by treatment of olefins (16) with chloramine-T
(Method A - Scheme 3) or cyclization of amino-alcohols with
tosyl chloride (Method B – Scheme 3).19,22-23 C3 alkylation with
the racemic isopropyl, methyl and t-butyl, methyl aziridines
prepared according to Method A, necessitated a difficult resolution
of diastereomeric products via normal phase chromatography.
Desiring broad, straightforward access to (R)-alpha-disubstituted
aziridines, we successfully developed an enantioselective method
(Method C – Scheme 3). Condensation of ketones (18) with (R)-
p-toluenesulfinamide afforded 19, which was cyclized via
treatment with trimethylsulfoxonium chloride and n-butyllithium
to give chiral toluenesulfinyl aziridines (20).27-28 Oxidation of 20
with mCPBA furnished the tosyl protected (R)-alpha-disubstituted
aziridines (6) used in the synthesis of 10ac-10af and 10ah. Greater
stereochemical control was achieved starting from acyclic ketone
substrates with cyclization producing high diastereomeric ratios
(dr > 10:1) for (R) t-butyl, methyl and methylcyclopropyl, methyl
toluenesulfinyl aziridines (20). In contrast, the cyclic ketone
substrate used to prepare the spirotetrahydropyran aziridine
employed in the synthesis of 10ah only attained a 2:1
diastereomeric ratio upon cyclization.
Method A
Ts
N
R1
R2
R1
a
R2
16
6
Synthesized compounds were evaluated for GS
inhibition (IC50, Table 1) in Candida albicans (MY1055)
microsomal membrane fragments using a previously reported
method.17 Onishi et al established CLSI protocols in RPMI
medium that were employed to measure in vitro antifungal
Method B
Ts
H2N
N
OH
R1
R1 R2
b
R2
17
6
Method C
O
O
S
N
R2
R1
18
c
d
R2
R1
19
Ts
N
O
S
N
R1
R2
e
R1
R2
6
20
Reagents and conditions: (a) PTAB, Chloramine-T, ACN, rt, 5-54%; (b)
Ts-Cl, DMAP, TEA, DCM, rt, 14-60%; (c) (R)-p-toluenesulfinamide,
Ti(OEt)4, DCE, 70 oC, 7-82%; (d) trimethylsulfoxonium chloride, n-
BuLi, THF, 0 oC-rt, 46-100%; (e) mCPBA, 1 M NaHCO3, EtOAc /
hexanes or DCM, 0 oC, 50-99%.
Table 1
C2 2-[1,2,4-triazole] Derivatives
O
OH
R2
N
H
O
N
O
N
2
H
N
R1
3
H
All concentrations in μg/mL
Mouse PK and PO Efficacy

TOKA
Δlog CFUse
nAUCoral
± S.D.c
A. fumigatus μM·hr·kg/mg
IC50
GS
MICa (+ ser)
C. albicans
MECb
± S.D.c
Compound
Route
R1
R2
Forald (%)
(dose mpk)
-4.2 ± 0.7 (0.5)f,g,***
-4.2 ± 0.7 (0.125)f,h,***
-2.3 ± 1.1 (0.03)f,***
-
Caspofungin
-
-
-
0.001
0.06 (0.25)
<0.03
-
-
10b
10c
10d
A
C
B
H
H
H
H
Me
Et
0.007
0.005
0.010
0.25 (4)
0.25 (4)
0.25 (8)
<0.03
0.03
-
-
-
-
-
-
-
-
-
<0.03
10e
10f
B
C
H
H
Pr
0.003
0.006
8 (16)
<0.03
0.008
-
-
-
-
cPr
0.125 (16)
-
-2.4 ± 0.7 (25)***
-1.7 ± 1.2 (12.5)**
-0.2 ± 1.1 (6.25)
-
10g
A and B
H
Phenyl
0.003
0.25 (4)
<0.03
0.50 ± 0.13
28
10h
B
H
2-F Phenyl
-
0.5 (8)
<0.03
-
-
10i
10j
B
B
H
H
3-F Phenyl
4-F Phenyl
0.011
0.011
0.25 (8)
0.25 (8)
<0.03
<0.03
-
-
-
-
-
-
-
10k
B
H
3-OMe Phenyl
0.011
0.25 (32)
<0.03
-
-
10l
B
B
H
4-OMe Phenyl
2-Pyridyl
0.015
0.003
0.5 (8)
<0.03
<0.03
-
-
-
-
-
-
10m
Me
0.25 (16)
-3.0 ± 0.4 (25)***
-2.4 ± 0.9 (12.5)*
-1.1 ± 0.5 (6.25)*
-3.7 ± 0.7 (25)***
-3.1 ± 0.4 (12.5)***
-0.9 ± 0.6 (6.25)*
-3.1 ± 0.3 (25)***
-2.9 ± 0.5 (12.5)***
-1.4 ± 0.8 (6.25)*
-4.5 ± 0.6 (25)i,***
-2.9 ± 0.3 (12.5)***
-2.4 ± 1.0 (6.25)*
10n
10o
10p
10q
A and B
Me
Me
H
3-Pyridyl
4-Pyridyl
0.005
0.001
0.004
0.002
0.25 (1)
0.06 (0.5)
0.06 (1)
<0.03
<0.03
<0.03
<0.03
0.59 ± 0.13
0.83 ± 0.34
0.17 ± 0.03
0.42 ± 0.14
21
29
11
31
B
B
3-Pyridyl
B and C
H
4-Pyridyl
<0.03 (0.5)
-4.2 ± 0.3 (25)i,***
-2.5 ± 0.6 (12.5)***
-2.6 ± 0.5 (6.25)***
10r
10s
C
C
H
H
2-F, 4-Pyridyl
0.001
0.008
0.015 (1)
0.25 (4)
0.008
<0.03
0.34 ± 0.10
-
26
-
3-OMe, 4-
Pyridyl
-
10t
10u
10v
10w
C
C
A
C
H
H
H
H
2,3-Pyridazine
2,4-Pyrimidine
2,5-Pyrazine
0.005
0.005
0.004
0.004
<0.03 (2)
<0.03 (4)
0.5 (4)
<0.03
<0.03
<0.03
<0.03
-
-
43
-
-
-
-
-
2.38 ± 1.20
-
-
2,6-Pyrimidine
0.25 (8)
-
-2.6 ± 0.8 (25)**
-1.2 ± 0.4 (12.5)**
-1.2 ± 2.2 (6.25)
-3.0 ± 0.5 (25)***
-3.0 ± 1.1 (12.5)**
-2.1 ± 0.3 (6.25)j,***
10x
C
H
3,4-Pyridazine
3,5-Pyrimidine
0.002
0.006
0.125 (1)
0.06 (1)
<0.03
<0.03
0.23 ± 0.06
0.33 ± 0.10
29
26
10y
C
H
aMinimum inhibitory concentration.
bMinimum effective concentration.
cStandard deviation.
dOral bioavailability = oral area under the concentration time curve normalized to intravenous (IV) dose / IV area under the concentration time curve.
eChange in colony forming units/g kidney relative to sham treated control animals.
fDosed intraperitoneally (IP).
gKidneys from 100% of animals were below the limit of detection (100% clearance).
hKidneys from 82% of animals were below the limit of detection (82% clearance).
iKidneys from 75% of animals were below the limit of detection (75% clearance).
j25% mortality (1 of 4 animals).
*p < 0.05 for CFUs/g kidney versus sham treated control animals (two tail t test).
**p < 0.01 for CFUs/g kidney versus sham treated control animals (two tail t test).
***p < 0.001 for CFUs/g kidney versus sham treated control animals (two tail t test).
susceptibilities of clinically relevant fungal species (minimum
inhibitory concentration for Candida albicans (MY1055) and
minimum effective concentration for Aspergillus fumigatus
(MF5668), Table 1).17 Mouse pharmacokinetics (PK) and oral
antifungal efficacy in a murine model of disseminated candidiasis
(Target Organ Kidney Assay – TOKA) using C. albicans
(MY1055) as the pathogen were utilized to further profile analogs
of interest.29-30 Caspofungin, an echinocandin GS inhibitor, was
employed as the positive control for in vivo efficacy studies.
All of the 3-alkyl and aryl 2-[1,2,4-triazole]

analogs synthesized according to Scheme 1 exhibited potent GS
inhibition (Table 1), but significant trends were identified for
whole cell antifungal activity in the presence and absence of 50%
mouse serum. With the exception of cyclopropyl (10f), alkyl
substituents (10c-10f) did not confer improvement to the inherent
antifungal activity against C. albicans relative to the unsubstituted
triazole (10b). Furthermore, in the presence of serum, a clear trend
was observed whereby increasing lipophilicity of the alkyl
substituent produced a 2-fold loss in potency for each additional
methylene moving from 10c to 10f. In contrast, the increased
lipophilicity of phenyl analog (10g) did not diminish antifungal
activity relative to 10b, even in the presence of serum, and
contributed reasonable oral exposure, translating into moderate
TOKA efficacy at 25 mpk. Installing a fluoro or methoxy
substituent (10h-10l) at various positions on the phenyl produced
little impact as 10h-10l were all within 2-fold of the antifungal
potency of the unsubstituted phenyl (10g), except the 3-methoxy
(10k), which was 8-fold less potent in the presence of serum.
Incorporation of a nitrogen into the aryl ring was explored in
analogs containing a N-methyl aminoether (10m-10o) with
profound effects observed on antifungal potency and efficacy
based on the regioisomeric position of the nitrogen. While the 2
and 3-pyridyl analogs (10m-10n) were equipotent with phenyl
(10g), the 4-pyridyl (10o) showed a 4-fold improvement. The
importance of the nitrogen position was pronounced in the
presence of serum, with a drastic 32-fold shift in potency observed
as the nitrogen moved around the ring from the 2 to the 4 position.
The 3-pyridyl analog (10n) maintained similar oral exposure to the
phenyl (10g), but the 4-pyridyl analog (10o) exhibited a roughly
1.5X improvement over either analog. Compounds 10n and 10o
both demonstrated excellent efficacy in TOKA with over a 3 log
reduction in fungal CFUs at 25 mpk. Analog 10o still provided a
robust 3 log reduction in fungal burden at 12.5 mpk, but both
analogs failed to retain efficacy at 6.25 mpk. Compounds 10p and
10q showed a 2-4 fold improvement in antifungal potency over
their N-methyl aminoether counterparts (10n-10o), though
potency in the presence of serum remained unchanged. Despite a
5X lower oral exposure versus 10n, 10p demonstrated similar
TOKA efficacy at 25 mpk and had a half-log improvement in
fungal CFU reduction at 12.5 mpk. Compound 10q only produced
a 2X loss in oral exposure compared to 10o and maintained similar
exposure to the parent phenyl analog (10g). The reasonable oral
exposure of 10q coupled with outstanding antifungal potency in
the presence of serum resulted in exceptional oral efficacy in
TOKA at 25 mpk with a 4.5 log reduction in fungal CFUs and
clearance of the infection in 75% of the animals. Compound 10q
maintained robust efficacy at 12.5 mpk and demonstrated
reasonable efficacy at 6.25 mpk with over a 99% reduction in
fungal CFUs at 25 mpk, while retaining efficacy through the lower
end of the dose titration. Unlike the 2-fluoro, combining the 3-
methoxy substituent with the 4-pyridyl (10s) only modestly
improved antifungal potency in the presence of serum relative to
the 3-methoxy phenyl (10k), leaving 10s significantly less potent
than the 4-pyridyl analog (10q). Di-aza aryl analogs (10t-10y)
displayed similar trends to those observed for the pyridyl analogs
(10m-10q) with distal positioning of the nitrogen favored over
substitution proximal to the neighboring triazole for maintaining
antifungal activity in the presence of serum. The 3,4-pyridazine
(10x) and 3,5-pyrimidine (10y) analogs, displayed antifungal
activity in the presence of serum that warranted evaluation in
TOKA, but both analogs produced diminished efficacy relative to
10q due to decreased oral exposure.
Having
identified
aryl-triazole
substituents with improved oral efficacy, we turned our attention
to reoptimizing the combination of the C2 triazole and the C3
aminoether. With the exceptional antifungal potency of 10q,
especially in the presence of serum, and its outstanding oral
efficacy in TOKA, the 4-pyridyl substitution was selected for
further exploration over the 2-fluoro-4-pyridyl of 10r, which
exhibited a similar profile. Holding the 4-pyridyl-triazole constant
at C2, various alkyl substitutions alpha to the amine on the C3
aminoether (Table 2) were prepared according to Route C of
Scheme 1 with the aim of increasing oral exposure while
maintaining antifungal activity in the presence of serum to
maximize oral efficacy.
Potent
GS activity was maintained across the series, but differences in
antifungal activity and oral exposure produced profound effects on
TOKA efficacy. The gem-dimethyl analog (10z) retained
equivalent antifungal potency relative to the isopropyl, methyl
(10q) compound. Compound 10z only produced a 1 log reduction
in fungal burden after oral dosing at 12.5 mpk in TOKA, but
produced over a 4 log reduction when dosed intraperitoneally at
12.5 mpk, indicating that the gem-dimethyl substitution failed to
produce sufficient oral exposure to achieve the TOKA activity
observed for 10q. The increased lipophilicity of the gem-diethyl
compound (10aa) resulted in improved oral TOKA efficacy
compared to 10z, but a 2-4 fold loss in antifungal activity
prevented 10aa from equaling the TOKA efficacy of 10q. The t-
butyl analog (10ab) lost 4-8 fold in antifungal activity relative to
10q, resulting in marginal efficacy at 25 and 12.5 mpk.
Conversely, the t-butyl, methyl analog (10ac), maintained
antifungal activity compared to the isopropyl, methyl analog
(10q), while achieving a slight increase in oral exposure.
Compound 10ac rivaled the exceptional dose-dependent oral
TOKA efficacy observed for 10q, producing more than a 99%
reduction in fungal burden even at 6.25 mpk, with 50% clearance
of the infection at 25 mpk. N-methylation of the aminoether of
10ac (10ad) and cyclizing two methyl groups from the t-butyl of
10ac to a methylcyclopropyl (10ae), both resulted in similar
antifungal potency and oral exposure relative to 10ac. Compounds
10ad-10ae demonstrated excellent TOKA efficacy with over a 4
log reduction in fungal burden at 25 mpk with 75% (10ad) and
100% (10ae) clearance. Analog 10ad maintained superior
fungal burden.
With
10q as the new benchmark, substituted 4-pyridyl and an array of
6-membered dinitrogen heterocycles were evaluated. Combining
2-fluoro substitution with the 4-pyridyl (10r) drastically improved
antifungal activity versus the 2-fluorophenyl analog (10h), and
was within 2-fold of the antifungal potency of the 4-pyridyl analog
(10q) in the presence of serum. Compound 10r had slightly lower
oral exposure than 10q, but displayed a similarly excellent efficacy
profile in TOKA with 75% clearance and over a 4 log reduction in
Table 2
C3 Aminoether Derivatives

N
O
OH
N
H
O
N
O
N
2
H
N
R1
3
R2 R3
H
All concentrations in μg/mL
Mouse PK and PO Efficacy
TOKA
nAUCoral
± S.D.
Δlog CFUsb
± S.D.
(dose mpk)
IC50
GS
MIC (+ ser)
C. albicans
MEC
Compound
Method
R1
R2, R3
A. fumigatus μM·hr·kg/mg Forala (%)
-4.2 ± 0.7 (0.5)c,d,***
-4.2 ± 0.7 (0.125)c,e,***
-2.3 ± 1.1 (0.03)c,***
Caspofungin
-
-
-
0.001
0.06 (0.25)
<0.03
-
-
-4.5 ± 0.6 (25)f,***
-2.9 ± 0.3 (12.5)***
-2.4 ± 1.0 (6.25)*
10q
10z
A
A
A
H
H
H
iPr, Me
Me, Me
Et, Et
0.002
0.001
0.001
<0.03 (0.5)
0.015 (0.5)
0.06 (1)
<0.03
<0.03
<0.03
0.42 ± 0.14
31
-
-4.2 ± 0.5 (12.5)c,***
-
-
-1.1 ± 0.8 (12.5)*
-3.3 ± 0.5 (25)***
-2.3 ± 0.3 (12.5)***
-1.3 ± 1.1 (6.25)
10aa
-
-2.6 ± 0.6 (25)***
-2.4 ± 1.4 (12.5)*
-1.0 ± 0.7 (6.25)*
-4.6 ± 0.4 (25)h,***
-3.2 ± 0.4 (12.5)***
-2.2 ± 0.8 (6.25)**
-4.2 ± 0.5 (25)f,***
-3.2 ± 0.9 (12.5)i,***
-1.7 ± 1.7 (6.25)
-4.3 ± 0.5 (25)d,***
-2.6 ± 1.1 (12.5)**
-1.5 ± 0.8 (6.25)*
-2.6 ± 0.6 (25)***
-1.5 ± 0.9 (12.5)*
-0.5 ± 0.6 (6.25)
-3.9 ± 0.8 (25)***
-3.1 ± 0.4 (12.5)***
-1.4 ± 0.8 (6.25)*
-3.2 ± 1.0 (25)**
-2.2 ± 0.9 (12.5)**
-0.9 ± 0.5 (6.25)*
10ab
10ac
10ad
10ae
10af
10ag
10ah
Bg
H
H
t-Bu, H
0.007
0.001
0.002
0.002
0.006
0.002
0.004
0.25 (2)
0.06 (0.5)
0.03 (1)
<0.03
<0.03
0.008
<0.03
0.125
<0.03
0.004
-
-
A and C
t-Bu, Me
t-Bu, Me
0.59 ± 0.19
0.62 ± 0.03
0.65 ± 0.23
-
34
42
36
-
A and C
Me
H
C
C
B
C
<0.03 (0.5)
0.25 (4)
H
Phenyl, Me
H
<0.03 (0.25)
0.06 (1)
0.18 ± 0.08
0.35 ± 0.16
13
25
O
O
H
aOral bioavailability = oral area under the concentration time curve normalized to intravenous (IV) dose / IV area under the concentration time curve.
bChange in colony forming units/g kidney relative to sham treated control animals.
cDosed intraperitoneally (IP).
dKidneys from 100% of the animals were below the limit of detection (100% clearance).
eKidneys from 82% of animals were below the limit of detection (82% clearance).
fKidneys from 75% of the animals were below the limit of detection (75% clearance).
gChiral amino alcohol starting material used.31
hKidneys from 50% of the animals were below the limit of detection (50% clearance).
iKidneys from 25% of the animals were below the limit of detection (25% clearance).
*p < 0.05 for CFUs/g kidney versus sham treated control animals (two tail t test).
**p < 0.01 for CFUs/g kidney versus sham treated control animals (two tail t test).
***p < 0.001 for CFUs/g kidney versus sham treated control animals (two tail t test).
efficacy at 12.5 mpk relative to 10ae with over a 3 log reduction
in fungal burden and 25% clearance; however, both analogs
diminished in efficacy more rapidly between 12.5 mpk and 6.25
mpk compared to 10ac. Replacing the t-butyl of 10ac with a
phenyl (10af) produced a 4-8 fold loss of antifungal activity,
resulting in only marginal TOKA efficacy at 25 mpk. The
spirotetrahydropyran analog (10ag) possessed exquisite antifungal
activity with a 2-fold potency improvement in the presence of
serum compared to 10ac; however, a 3X drop in oral exposure
accompanied 10ag. As a result of these opposing trends, 10ag
produced robust TOKA efficacy at 25 and 12.5 mpk, but abruptly
declined at 6.25 mpk. Increasing the lipophilicity of the
spirotetrahydropyran through the incorporation of gem-dimethyl
substitution alpha to the quaternary center (10ah), resulted in a 4-
fold loss of antifungal activity in the presence of serum relative to
10ag. Compound 10ah produced a 2X increase in oral exposure
compared to 10ag, but demonstrated diminished TOKA efficacy,
indicating that the modest gains in oral exposure were insufficient
to compensate for the loss of antifungal potency in the presence of
serum.
The isopropyl, methyl (10q) and the t-butyl, methyl
(10ac) analogs were selected for further evaluation as a result of
displaying exceptional TOKA efficacy with partial clearance
observed at 25 mpk as well as maintenance of efficacy through the
low end of the dose titration at 6.25 mpk.
The
spirotetrahydropyran analog (10ag) was also chosen for additional
Table 3
PK evaluation of 10q, 10ac, and 10ag

Pharmacokinetics across species
10q
Clearance ± S.D. (mL/min/kg)
t1/2 ± S.D. (h)
Mouse
17 ± 1
4.9 ± 0.4
0.42 ± 0.14
30%
Rat
12 ± 1
3.9 ± 1.3
0.56 ± 0.17
28%
Dog
6 ± 2
9.0 ± 1.4
0.90 ± 0.27
Rhesus
14 ± 1
6.1 ± 0.1
0.08 ± 0.06
5%
nAUCoral ± S.D. (μM·hr·kg/mg)
a
Foral
21%
10ac
Mouse
14 ± 2
4.4 ± 1.8
0.59 ± 0.19
34%
Rat
8 ± 2
8.3 ± 3.8
0.69 ± 0.27
23%
Dog
4 ± 1
11.3 ± 1.2
1.07 ± 1.31
Rhesus
11 ± 2
5.7 ± 0.7
0.33 ± 0.05
16%
Clearance ± S.D. (mL/min/kg)
t1/2 ± S.D. (h)
nAUCoral ± S.D. (μM·hr·kg/mg)
a
Foral
18%
10ag
Mouse
Rat
Dog
Rhesus
Clearance ± S.D. (mL/min/kg)
t1/2 ± S.D. (h)
18 ± 6
20 ± 2
-
-
-
-
-
-
-
-
2.1 ± 0.3
0.18 ± 0.08
13%
1.9 ± 0.1
0.34 ± 0.16
30%
nAUCoral ± S.D. (μM·hr·kg/mg)
a
Foral
aOral bioavailability = oral area under the concentration time curve normalized to intravenous (IV) dose / IV area under the concentration time curve.
study to see, if its exquisite antifungal activity in the presence of
serum would translate to improved efficacy upon extended dosing.
Dog displayed the best overall profile in a PK assessment across
preclinical species (Table 3). Clearance dropped while half-life
and oral exposure increased moving from mouse to dog for 10q
and 10ac, but all three parameters eroded moving from dog to
rhesus. The oral bioavailability of both analogs slowly diminished
moving from mouse to rhesus. Oral bioavailability and exposure
increased for 10ag moving from mouse to rat, while clearance and
half-life remained flat. Compounds 10q and 10ac demonstrated
superior PK profiles in rodents compared to 10ag with
improvements in clearance (rat), half-life (mouse/rat), oral
exposure (mouse/rat), and oral bioavailability (mouse). In general,
10q and 10ac produced similar PK profiles across preclinical
species with 10ac holding advantages in rat half-life (2X), rhesus
oral exposure (4X), and rhesus oral bioavailability (3X).
An extended 7-day survival version of TOKA
PO control groups had survival rates of 40% and 80%,
respectively. Impressively, all three analogs produced 100%
clearance of the infection in study animals when dosed at 12.5 mpk
IP. In addition, all compounds reduced fungal burden by over 4.5
logs with 100% (10q and 10ac) or 80% (10ag) clearance at 25 mpk
PO. 10ac maintained 100% clearance at 12.5 mpk PO with 10q
and 10ag dropping to 40% and 20%, respectively. Nevertheless,
10q and 10ag lowered the fungal burden at this dose by over 3.9
logs, indicating that the infection was nearly eradicated. Moving
to the 6.25 mpk PO dose, 10q and 10ac achieved robust 3.5 log
reductions in fungal burden, while 10ag fell below a 3 log
reduction. These results led to a 3-4X improvement in ED99
(effective dose to reduced fungal burden by 99%) for 10q and 10ac
over 10ag. A 21-day murine survival model of disseminated
aspergillosis utilizing Aspergillus fumigatus (MF5668) as the
pathogen was employed to further evaluate the efficacy of 10q,
10ac, and 10ag (Table 4).15,33 The sham treated control group had
a mean survival of 3.9 days with 100% mortality. Compounds 10q
and 10ag produced dose-dependent survival with 60% and 40%
was employed to further evaluate the efficacy of 10q, 10ac, and
10ag versus Candida albicans (Table 4).32 Sham treated IP and
Table 4
Extended efficacy evaluation of 10q, 10ac, and 10ag
Efficacy in 7-day TOKA
Dose
Δ log CFUsa (% clearance)
Caspofungin
10q
10ac
10ag
25 mpk PO
-
-
-
-
-4.57 ± 0.85 (100)***
-4.62 ± 0.85 (100)***
-4.62 ± 0.85 (80)***
12.5 mpk PO
6.25 mpk PO
12.5 mpk IP
0.5 mpk IP
0.125 mpk IP
0.03 mpk IP
-4.37 ± 0.93 (40)***
-4.62 ± 0.85 (100)***
-3.92 ± 0.98 (20)***
-3.50 ± 1.10 (0)***
-3.62 ± 1.03 (0)***
-2.89 ± 1.40 (0)**
-4.02 ± 0.53 (100)***
-3.99 ± 0.53 (100)***
-4.00 ± 0.53 (100)***
-3.96 ± 0.53 (100)***
-4.00 ± 0.53 (100)***
-3.28 ± 0.93 (20)***
-3.62 ± 0.75 (60)***
<0.0001
-
-
-
-
-
-
-
-
-
0.0078 mpk IP
-
-
-
ED99 mpk (95% C.I.)b
1.2 (0.22 – 6.3)
0.84 (0.11 – 6.2)
3.8 (2.1 – 7.0)
Efficacy in Aspergillosis Survival Model
Dose
% Survival (Mean Survival Time)
Caspofungin
10q
10ac
10ag
12.5 mpk IP
-
60 (14.7 days)
90 (19.4 days)
60 (14.6 days)
6.25 mpk IP
-
40 (12.4 days)
90 (19.2 days)
40 (12.5 days)
3.125 mpk IP
2 mpk IP
1.56 mpk IP
-
30 (10.7 days)
20 (12.2 days)
10 (7.3 days)
90 (19.8 days)
-
-
-
-
0 (4.2 days)
0 (5.1 days)
0 (4.1 days)
0.5 mpk IP
90 (19.4 days)
60 (14.7 days)
50 (13.1 days)
0.07 (0.03 – 0.16)
0.83 (0.22 – 3.1)
-
-
-
-
-
-
-
-
-
0.125 mpk IP
0.03 mpk IP
ED50 mpk (95% C.I.)b
ED90 mpk (95% C.I.)b
8.2 (4.9 – 14)
31 (10 – 96)
4.5 (3.4 – 6.0)
8.9 (5.8 – 14)
11 (6.5 – 17)
26 (10 – 66)
aChange in colony forming units/g kidney relative to sham treated control animals.
b95% confidence interval.
*p < 0.05 for CFUs/g kidney versus sham treated control animals (two tail t test).
**p < 0.01 for CFUs/g kidney versus sham treated control animals (two tail t test).
***p < 0.001 for CFUs/g kidney versus sham treated control animals (two tail t test).

survival at 12.5 and 6.25 mpk, respectively. Mean survival time
at these doses was nearly identical for 10q and 10ag, but at 3.125
mpk, 10q achieved 30% survival with a mean survival time of 10.7
days versus 10% and 7.3 days for 10ag. Compound 10ac
demonstrated exceptional efficacy with 90% survival at both 12.5
and 6.25 mpk with mean survival of over 19 days. The efficacy of
10ac fell precipitously with only 20% survival at 3.125 mpk,
though the mean survival time (12.2 days) was longer than 10q or
10ag. At the 1.56 mpk dose, 100% mortality was observed for all
three analogs with only a 1-2 day extension in mean survival time.
The higher survival rates observed for 10ac translated into 2-3X
improvements in ED50 (effective dose for 50% survival) and ED90
(effective dose for 90% survival) relative to 10q and 10ag.
Due to its superior efficacy in the aspergillosis survival
compound (MK-3118, SCY-078, ibrexafungerp). Owing to its
improved efficacy profile in the 7-day TOKA and aspergillosis
survival model, the clinical development of ibrexafungerp was
prioritized over MK-5204. Having progressed to phase III clinical
trials, ibrexafungerp has the potential to become an important
antifungal therapy with benefits over existing options due to its
oral efficacy and broad spectrum antifungal activity, which
includes echinocandin resistant isolates and Candida auris.
Acknowledgements
The authors wish to thank Dr. Ashok Arasappan, Dr. Jason Cox,
and Ms. Helen Chen for their helpful suggestions in the
preparation of this manuscript.
model, antifungal susceptibility testing against additional fungal
species was performed with 10ac to determine its spectrum of
activity (Table 5). Compound 10ac demonstrated antifungal
activity against the clinically relevant yeast and mold species
displayed in Table 5. Higher concentrations of 10ac were required
to achieve complete inhibition of growth (MIC-100) for Candida
krusei and Candida tropicalis, though prominent inhibition (MIC-
50) was observed at 2-8 fold lower concentrations. Testing against
expanded panels of Candida (113 isolates) and Aspergillus (71
isolates) species further established 10ac as a potent, broad
spectrum antifungal.34-35 Additional studies demonstrated that
10ac is active against a majority of echinocandin resistant Candida
and Aspergillus isolates as well as the recently emerging multi-
drug resistant Candida auris.36-38
Appendix A – Supplemental Data
Supplementary data for this article can be found online at…
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Table 5
Extended antifungal activity spectrum of 10ac
Antifungal activity of 10ac verses Candida and Aspergillus species
Species
MIC-100a μg/mL
MIC-50b μg/mL
C. albicans (MY1055)
(+50% mouse serum)
C. glabrata (MY1381)
C. krusei (ATCC6258)
C. tropicalis (MY1012)
C. lusitaniae (MY1396)
C. parapsilosis (ATTC22109)
Species
0.06
0.5
1
4
8
<0.03
0.25
1
2
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2
0.5
1
<0.03
MEC μg/mL
A. fumigatus (MF5668)
(+50% human serum)
<0.03
0.25
aLowest concentration of compound to completely inhibit fungal growth.
bLowest concentration of compound to restrict fungal growth by 50%.
In conclusion, a scan of 3-alkyl and aryl-2-[1,2,4-
triazole] substituents identified 3-(4-pyridyl)-2-[1,2,4-triazole] as
the optimal replacement for the 3-carboxamide-2-[1,2,4-triazole]
substituent of MK-5204 (3).19 The 4-pyridyl substituent of 10q
((R) isopropyl, methyl aminoether) produced
a
4-fold
improvement in antifungal activity in the presence of serum
relative to 3 in conjunction with a 1.5X increase in oral exposure.
Re-optimization of the alkyl substituents alpha to the amine of the
C3 aminoether in the presence of the 3-(4-pyridyl)-2-[1,2,4-
triazole] substituent at C2, established (R) t-butyl, methyl (10ac)
as the preferred C3 aminoether with a 2X increase in oral exposure
over 3, while maintaining the 4-fold improvement in antifungal
activity in the presence of serum observed for 10q. The concurrent
improvements in these two parameters, which had previously
correlated with oral efficacy, resulted in a drastic improvement in
the 7-day TOKA ED99 for 10ac (0.84 mpk) relative to MK-5204
(3) (6.4 mpk).19 Compound 10q demonstrated similar efficacy to
10ac in the 7-day TOKA, but 10ac displayed superior efficacy in
the aspergillosis survival model and exhibited a favorable rhesus
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