MK-5204: An orally active β-1,3-glucan synthesis inhibitor

Article abstract of DOI:10.1016/j.bmcl.2020.127357

Our previously reported efforts to produce an orally active β-1,3-glucan synthesis inhibitor through the semi-synthetic modification of enfumafungin focused on replacing the C2 acetoxy moiety with an aminotetrazole and the C3 glycoside with a N,N-dimethyl

Full text of DOI:10.1016/j.bmcl.2020.127357

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MK-5204: 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)30468-6
DOI:
Reference:
https://doi.org/10.1016/j.bmcl.2020.127357
BMCL 127357
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
7 May 2020
11 June 2020
14 June 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., Jo
Hsu, M., 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., MK-5204: an orally active β-1,3-glucan synthesis
inhibitor, Bioorganic & Medicinal Chemistry Letters (2020), doi: https://doi.org/10.1016/j.bmcl.2020.127357
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Bioorganic & Medicinal Chemistry Letters
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MK-5204: 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
bSynexis Inc., 1 Evertrust Plaza, Jersey City, NJ 07302, USA
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Our previously reported efforts to produce an orally active β-1,3-glucan synthesis inhibitor
through the semi-synthetic modification of enfumafungin focused on replacing the C2 acetoxy
moiety with an aminotetrazole and the C3 glycoside with a N,N-dimethylaminoether moiety. This
work details further optimization of the C2 heterocyclic substituent, which identified 3-
carboxamide-1,2,4-triazole as a replacement for the aminotetrazole with comparable antifungal
activity. Alkylation of either the carboxamidetriazole at C2 or the aminoether at C3 failed to
significantly improve oral efficacy. However, replacement of the isopropyl alpha amino
substituent with a t-butyl, improved oral exposure while maintaining antifungal activity. These
two structural modifications produced MK-5204, which demonstrated broad spectrum activity
against Candida species and robust oral efficacy in a murine model of disseminated Candidiasis
without the N-dealkylation liability observed for the previous lead.
Keywords:
MK-5204
β-1,3-Glucan synthesis inhibitor
Enfumafungin
Antifungal
Candidiasis
2009 Elsevier Ltd. All rights reserved.
———
 Corresponding author. Tel.: +1-908-740-5122; fax: +1-908-740-6136; e-mail: james_apgar@merck.com

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Disseminated invasive Candidiasis and Aspergillosis, the most
common serious fungal infections, continue to produce high
mortality rates despite the development of new treatment options
(azoles, echinocandins) over the past few decades.1-6 Once
confined mainly to immunocompromised patients, the incidence
of fungal infections in intensive care units has increased
substantially with Candida species becoming one of the leading
causes of nosocomial bloodstream infections.1-3 Only three main
classes of antifungal agents (polyenes, azoles, and echinocandins)
are available to treat invasive fungal infections. Even though the
rate of antifungal resistant infection is low, increasing reports of
azole and echinocandin resistant Candida and Aspergillus isolates
are of particular concern as affected patients have limited
remaining treatment options and typically result in poor clinical
outcomes.5-10 Consideration of these factors clearly indicates the
need for the development of novel antifungal therapies that
demonstrate benefit over existing options.
(fluconazole, voriconazole, posaconazole), which indirectly affect
the function of the fungal cell membrane through inhibiting a
fungal P450 enzyme involved in ergosterol biosynthesis, provided
a safer treatment option, but their use is complicated by extensive
drug-drug interactions that result from inhibition of host P450
enzymes.3,5,11
Conversely, the most recently developed class of
antifungals,
echinocandins
(caspofungin,
micafungin,
anidulafungin), target the fungal cell wall instead of the cell
membrane through the inhibition of β-1,3-glucan synthase (GS),
which is responsible for the production of a key cell wall
component (β-1,3 glucan). The structurally compromised cell wall
leaves the fungal cell vulnerable to lysis under osmotic stress.5,12
The echinocandins exhibit fewer mechanism based toxic side
effects than the polyenes and fewer drug-drug interactions than the
azoles because of their target specificity to fungal cells.3,5,11 The
improved safety profile and broad spectrum of activity has led to
the adoption of echinocandins as the first line therapy of choice for
Amphotericin B (polyene), which disrupts fungal cell
membranes by binding to ergosterol, was the gold standard in
antifungal therapy for many years because of its broad spectrum
of activity.3,5,11 However, therapy with Amphotericin B is often
limited due to nephrotoxicity, resulting from the binding of
mammalian sterols.3,5,11 The development of azole antifungals
invasive Candida infections in recent years.1,3-4,10,13
The
echinocandins are restricted to parenteral administration as a result
of poor oral bioavailability.11-14 An orally active GS inhibitor,
retaining the safety profile of the echinocandins, would be an
important addition to antifungal therapy options, particularly for
N-dealkylation observed for 2.18 Our original screen of C2
heterocyclic substituents with the naturally occurring glycoside at
C3 indicated that 1,2,4-triazole warranted further optimization as
its antifungal activity was within 2-fold of the tetrazole analog.18
The effect of C2 substitution with 1,2,4-triazoles in combination
with the optimal C3 aminoether from the tetrazole series was
probed by preparing analogs as described in Scheme 1.
O
OH
OH
O
18
25
H
O
O
O
OH
2
O
HO
HO
3
H
OH
Reduction of the bridging hemiacetal of enfumafungin
(1) with triethylsilane afforded C25-deoxyenfumafungin (3) in
good yield.18-21 Treatment of 3 with sulfuric acid in methanol
hydrolyzed the C3 glycoside to the alcohol and displaced the C2
acetoxy group to give the methyl ether.18 4 was attained in good
yield through subsequent protection of the C18 carboxylic acid as
a benzyl ester. 5 was prepared via alkylation of the C3 alcohol of
4 with tosyl protected aziridine 16. Deprotection of the tosyl
protected aminoether at C3 and the benzyl ester at C18 was
achieved through a dissolving metal reduction to give 6.
Borontrifluoride diethyletherate mediated displacement of the C2
methoxy with substituted 1,2,4-triazoles proceeded with retention
of stereochemistry at C2 as previously described.18 The
distribution of regioisomeric products from the displacement
reaction was substrate specific with the 1-[1,2,4-triazole] 7
typically being the major product, though the distribution was
slightly tilted in favor of the 2-[1,2,4-triazole] 8 in some cases. In
general, the 4-[1,2,4-triazole] 9 was only obtained as a minor
product out of the displacement reaction. The regioiosomers were
enfumafungin (1)
O
OH
H2N
N
N
N
18
25
H
O
N
2
N
O
3
H
2
Figure 1. C2 aminotetrazole, C3 aminoether lead
patients with azole resistant infections or needing step-down
therapy from a parenteral antifungal.
Synthetic derivatization of enfumafungin15 (1),
a
naturally occurring GS inhibitor,16-17 previously reported by our
group culminated in the discovery of 2, which had an
aminotetrazole at C2 in place of the acetoxy group, an aminoether
at C3 in place of the glycoside, and a bridging ether at C25 in place
of the hemiacetal (Figure 1).18 2 exhibited robust oral efficacy in
a 7 day murine model of disseminated Candidiasis with an ED99 of
9 mpk (estimated dose required to reduce fungal burden by 99%).18
Herein we present additional optimization of the C2 and C3
substituents aimed at improving oral efficacy and mitigating the
readily separable by reverse phase HPLC.
Mono-methyl
alkylation of the aminoether was achieved by treating the tosyl
protected amine of 5 with iodomethane prior to global deprotection
and C2 displacement. Dimethyl, ethyl, and propyl alkylations of
the aminoether were installed via reductive amination of 6
followed by C2 displacement.20-21
A previously reported method was employed to measure

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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 (3)
Ts
N
R1
R2
16
d or d, e
O
OBn
O
OBn
H
O
H
MeO
O
b, c
MeO
HO
Ts
N
R3
O
R1 R2
H
H
4
5
O
OH
O
OH
H
H
O
O
MeO
O
triazole
O
R4
R4
N
R1 R2
f or f, g
h
N
R3
R3
R1 R2
H
H
7 = 1-[1,2,4-triazole]
8 = 2-[1,2,4-triazole]
9 = 4-[1,2,4-triazole]
6
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; (e) NaH, MeI, DMF, 50 oC; (f) Na, NH3, THF, -35 oC; (g) RCHO, NaBH3CN, AcOH, MeOH, rt;
(h) triazole, BF3 OEt2, DCE, 50 oC.
Scheme 1. Synthesis of C2 triazole, C3 aminoether derivatives.
Table 1
C2 Triazole Regioisomer Derivatives
mouse pharmacokinetics (PK) and oral antifungal efficacy in the
target organ kidney assay (TOKA), a murine model of
disseminated Candidiasis utilizing C. albicans (MY1055) as the
pathogen.22
Table 1 displays the comparison of antifungal activity for
the 1-[1,2,4-triazole] 7, 2-[1,2,4-triazole] 8, and 4-[1,2,4-triazole]
9 isomers with chloro and cyano substitution that were prepared
according to the route shown in Scheme 1. These substituents
were chosen for comparison because the displacement reaction
produced sufficient quantities of all three regioisomers to allow for
R
O
OH
N
N
N
R
N
N
N
H
O
X
2
8
7
N
N
H2N
N
O
3
X =
R
H
9
O
OH
H
All concentrations in μg/mL
O
X
IC50
GS
MICa + (ser)
MECb
2
Compound
R
H2N
C. albicans
A. fumigatus
O
3
H
0.019
0.334
0.003
0.006
>1
8 (>32)
8 (>32)
0.25
4
7a
7b
8a
8b
9a
9b
Cl
CN
Cl
CN
Cl
0.06 (8)
0.125 (8)
32 (>32)
16 (>32)
<0.03
<0.03
16
Br
N
N
N
N
NH2
a
NO2
N
N
N
X =
CN
>1
8
aMinimum inhibitory concentration
bMinimum effective concentration
8g
8f
the inhibition of GS (IC50, Table 1) by synthesized analogs in
Candida albicans (MY1055) microsomal membrane fragements.16
The CLSI protocols in RPMI medium reported by Onishi et al
were used to determine the in vitro antifungal susceptibilities for
clinically relevant fungal species (minimum inhibitory
concentration for Candida albicans (MY1055) and minimum
effective concentration for Aspergillus fumigatus (MF5668,
Table1)).16 Analogs of interest were further profiled by evaluating
N
N
N
N
N
Br
S
S
O
O
N
N
N
b
c
8d
8j
8k
Reagents and conditions: (a) Pd-C, H2, EtOH, rt; (b) NaSEt, DMF, 50 oC;
(c) mCPBA, DCE, rt.
Scheme 2. Post displacement triazole modification.