Synthesis of analogs of the Gwt1 inhibitor manogepix (APX001A) and in vitro evaluation against Cryptococcus spp

Article abstract of DOI:10.1016/j.bmcl.2019.126713

Fosmanogepix (APX001) is a first-in-class prodrug molecule that is currently in Phase 2 clinical trials for invasive fungal infections. The active moiety manogepix (APX001A) inhibits the novel fungal protein Gwt1. Gwt1 catalyzes an early step in the GPI anchor biosynthesis pathway. Here we describe the synthesis and evaluation of 292 new and 24 previously described analogs that were synthesized using a series of advanced intermediates to allow for rapid analoging. Several compounds demonstrated significantly (8- to 32-fold) improved antifungal activity against both Cryptococcus neoformans and C. gattii as compared to manogepix. Further in vitro characterization identified three analogs with a similar preliminary safety and in vitro profile to manogepix and superior activity against Cryptococcus spp.

Full text of DOI:10.1016/j.bmcl.2019.126713

Journal Pre-proofs
Synthesis of analogs of the Gwt1 inhibitor Manogepix (APX001A) and in vi-
tro evaluation against Cryptococcus spp
Michael Trzoss, Jonathan A. Covel, Mili Kapoor, Molly K. Moloney, Quinlyn
A. Soltow, Peter J. Webb, Karen Joy Shaw
PII:
S0960-894X(19)30671-7
DOI:
https://doi.org/10.1016/j.bmcl.2019.126713
BMCL 126713
Reference:
Bioorganic & Medicinal Chemistry Letters
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19 September 2019
Please cite this article as: Trzoss, M., Covel, J.A., Kapoor, M., Moloney, M.K., Soltow, Q.A., Webb, P.J., Joy Shaw,
K., Synthesis of analogs of the Gwt1 inhibitor Manogepix (APX001A) and in vitro evaluation against
Cryptococcus spp, Bioorganic & Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.
2019.126713
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Synthesis of analogs of the Gwt1 inhibitor Manogepix (APX001A) and in vitro evaluation
against Cryptococcus spp
Michael Trzoss*, Jonathan A. Covel, Mili Kapoor, Molly K. Moloney, Quinlyn A. Soltow, Peter
J. Webb, Karen Joy Shaw*
Amplyx Pharmaceuticals, 12730 High Bluff Dr #160, San Diego, CA 92130
*Corresponding authors
Michael Trzoss, Ph.D.
Email: trzossmichael@gmail.com
Karen Joy Shaw, Ph.D.
Email: kshaw@amplyx.com
Abstract: Fosmanogepix (APX001) is a first-in-class prodrug molecule that is currently in
Phase 2 clinical trials for invasive fungal infections. The active moiety manogepix (APX001A)
inhibits the novel fungal protein Gwt1. Gwt1 catalyzes an early step in the GPI anchor
biosynthesis pathway. Here we describe the synthesis and evaluation of 292 new and 24
previously described analogs that were synthesized using a series of advanced intermediates to
allow for rapid analoging. Several compounds demonstrated significantly (8- to 32-fold)
improved antifungal activity against both Cryptococcus neoformans and C. gattii as compared to
manogepix. Further in vitro characterization identified three analogs with a similar preliminary
safety and in vitro profile to manogepix and superior activity against Cryptococcus spp.
Keywords: APX001, APX001A, Gwt1, GPI anchor biosynthesis, Cryptococcus, antifungal,
fosmanogepix, manogepix, MGX
Cryptococcus neoformans is a major cause of illness in people living with HIV/AIDS and it is
uniformly fatal unless treated. Widespread availability of Antiretroviral Therapy (ART) reduced
the estimated cases of cryptococcal meningitis (CM) to 200,000-300,000 cases per year, but
mortality is still >50% when inferior therapeutic regimens are used.1 A recent report from
Botswana illustrated that the incidence of CM has continued to be high and stable for the last 5
years despite ART use.2 Even in resource-available regions such as North America and Europe,
CM still occurs with a mortality rate in some risk groups of up to 30%.3, 4 The morbidity and
long-term health care costs (5 years post-infection) remain substantial.5 Thus there is an urgent
need for new treatment options for CM.
In August 2019, the FDA added CM to its priority review voucher program to encourage the
development of new treatment options in this area of unmet medical need, and a few companies
have entered this development space. Viamet Pharmaceuticals received Fast Track designation

for VT-1129 for the treatment of CM. This orally available inhibitor of fungal CYP51
additionally received orphan drug designation for the treatment of CM and has been designated a
Qualified Infectious Disease Product (QIDP) by the FDA. Similarly, Amplyx Pharmaceuticals
has received Orphan drug and QIDP designations for fosmanogepix (APX001), a first-in class
broad spectrum antifungal agent. The active moiety of fosmanogepix inhibits the fungal enzyme
Gwt1.
The Gwt1 enzyme catalyzes an early step in the glycosylphosphatidylinositol (GPI)-anchor
biosynthesis pathway and was explored by Eisai as an antifungal target6. It has been shown that
inhibition of Gwt1 prevents the appropriate localization of fungal cell wall mannoproteins, thus
compromising cell wall integrity, germ tube formation, biofilm formation and fungal growth.7, 8
Multiple series of Gwt1 inhibitors have been optimized through extensive medicinal chemistry
efforts at Eisai and other groups.9-12 E1210 [now manogepix (MGX), APX001A] is one of the
most potent inhibitors and the phosphonooxymethyl prodrug (fosmanogepix, APX001,
previously E1211) is currently in clinical development for the treatment of invasive fungal
infections. While optimization efforts at Eisai focused on improving the activity against Candida
albicans and Aspergillus fumigatus, little was known about the SAR of these compounds against
Cryptococcus spp.13
In this study we synthesized a number of analogs of manogepix and evaluated their activity against
two species of Cryptococcus. Compounds with minimum inhibitory concentration (MIC) values less
than or equal to 0.016 µg/mL against both C. neoformans and C. gattii were assessed for stability in
human liver microsomes (HLM) and cytotoxicity using a HepG2 assay. The best performing
compounds were then evaluated in a Gwt1 overexpression assay to confirm the mechanism of action
(MoA). The final selection criterion was lack of activity against PigW7, 14 (the closest human
homolog of Gwt1) to decrease the risk of mechanism-based toxicity in humans.
Data on Gwt1 inhibitors published by Eisai suggest that compounds with potent antifungal
activity against Aspergillus and Candida are composed of four structural elements (A to D),
which are interconnected as shown in Figure 1.11
2-atom
linker
L
D
D
C
C
B
isoxazole
isoxazole
N
O
A
hetero
hetero
X
O
N
O
N
hetero
hetero
L
pyridine
L
Y
aryl
aryl
aryl
aryl
phenyl
ring
R
= H,NH2
N
N
316 compounds
24 known (Eisai)
292 new analogs
NH2
NH2
R
N
NH2
W,X,Y,Z
= CH,O,N
aminopyridine
aminopyridine
Eisai patent US7691882.
Analogs synthesized and activity against
Cryptococcus evaluated.
APX001A
Figure 1. Structural elements of Gwt1 inhibitors and rationale for designing novel analogs.

Within the described compounds, most potent analogs have an aminopyridine or
diaminopyridine head group A directly connected to a 5-membered aromatic heterocycle B,
preferentially an isoxazole. This two-ring system is connected via a -CH2-group to a 6-membered
(hetero-)aryl ring C, preferably a phenyl ring, which is substituted at the meta- or para-position
with group D. Group D consists of a one or two atom linker attached to a 5- or 6-membered
(hetero-)aryl ring. A large variety of different linkers and aryl groups are tolerated at position D
while maintaining potent antifungal activity against Aspergillus and Candida.
In order to establish SAR for manogepix based inhibitors of Gwt1 against Cryptococcus, we
synthesized a collection of analogs of manogepix and evaluated their activity against C.
neoformans and C. gattii. Based on the analysis above, we opted to hold constant the
aminopyridine as head group A and the isoxazole as heterocycle B and focused on modifying
groups C and D. Eisai has published several patents outlining the syntheses of manogepix related
compounds.11, 15 While the synthetic routes were highly convergent, they did not allow for rapid
exploration of group D analogs for various A-B-C-ring scaffolds. Therefore, we adopted Eisai’s
procedures for the syntheses of the advanced intermediates I to V. From these intermediates,
final compounds were accessible in only one synthetic step. The synthesis of the intermediates is
outlined in Scheme 1. Eisai introduced the Boc-protected chloromethylisoxazole 115, which was
used in a Suzuki coupling reaction in the synthesis of manogepix (previously known as E1210),
compound 3. We observed that the hydroxypyridyl group in compound 3 (manogepix) would
substitute quantitatively with chloride when refluxed in dioxane in presence of concentrated HCl.
Thus, we used this method for the synthesis of the advanced intermediate I after following
Eisai’s procedure for the synthesis of compound 3. The Boc-protected chloromethylisoxazole 1
also reacted smoothly with a variety of other boronic acids and boronic esters. Direct Boc-
deprotection of the coupling products 4, 6 and 7 in formic acid gave access to the intermediates
II, IV and V. The global deprotection of coupling product 5 was achieved by using 4 M aq. HCl
in dioxane to form advanced intermediate III.

Scheme 1. Synthesis of advanced intermediates I to V. Reagents and conditions: (i) 2 M sodium
carbonate in water, palladium(0)tetrakis(triphenylphosphine), dimethoxyethane, 90 °C; (ii)
formic acid, 21-25 °C; (iii) 12 M aq. HCl, dioxane, reflux; (iv) 4 M aq. HCl, dioxane, 50 °C.
Scheme 2. Representative examples for the synthesis of analogs from intermediate I.
Reagents and conditions: (i) 2 M sodium carbonate in water,
palladium(0)tetrakis(triphenylphosphine), dimethoxyethane, 90 °C, analogous procedures
for 8b-8e; (ii) sodium hydride (60% in mineral oil), NMP, 21-25 °C then 50 °C, analogous
procedure for 9b; (iii) DMF, 90 °C, analogous procedure for 10b.

Scheme 3. Representative examples for the synthesis of analogs from intermediates II to V.
Reagents and conditions: (i) N-ethyl-N-isopropylpropan-2-amine, DMSO, 120 °C,
analogous procedure for 11b; (ii) potassium tert-butoxide (1M in THF), DMF, 21-25 °C
then 90 °C, analogous procedures for 12b-12d; (iii) potassium tert-butoxide (1 M in THF),
analogous procedures for 13b-13n and 14b.
Table 1. Antifungal activity of compounds targeting Gwt1 against Cryptococcus spp.
MIC (µg/mL)b
MIC (µg/mL)b
C.
C.
Compda
C. gattii Compda
C. gattii
neoformans
neoformans
MGX
8a
0.25
0.031
0.5
0.125
0.008
0.5
13a
0.016
0.008
0.016
0.016
0.016
0.016
13b
13c
8b
8c
8d
8e
9a
0.031
0.016
0.016
0.25
0.016
0.016
0.016
0.063
13d
13e
13f
13g
0.016
0.031
0.25
0.031
0.031
0.5
0.031
0.031

9b
0.125
0.125
> 4
0.063
0.063
> 4
13h
13i
13j
0.031
0.016
0.016
0.016
0.008
0.031
10a
10b
11a
11b
0.25
> 4
0.25
> 4
13k
13l
2
2
0.016
0.016
12a
0.008
0.016
0.004
0.008
13m
13n
0.008
0.008
12b
0.063
0.063
12c
12d
0.031
0.031
14a
14b
0.031
0.5
0.031
1
0.016
0.008
a Italicized compound numbers indicate a compound that has been previously synthesized by Eisai.
b MIC values were determined according to the methods under a) Antifungal susceptibility testing. Values in bold
fulfill the selection criteria.
c Abbreviation: MGX, manogepix
With the advanced intermediates I to V in hand, we synthesized 24 previously described Gwt1
inhibitors and 292 novel compounds. Intermediate I was most versatile and underwent Suzuki
coupling reactions to form either 1- or 2-carbon atom linked analogs (8a and 8b), as well as
substitution reactions with a variety of nucleophiles, such as pyrazoles, phenols, anilines or
amines. In that manner we were able to synthesize -CH2-linked pyrazole 9a, -CH2O-linked
aryloxy analog 9b and the -CH2NH-linked derivatives 10a and 10b (Scheme 2). The free amino
group in intermediate II allowed for nucleophilic aromatic substitution reactions with a variety
of activated 5- and 6-membered aromatic heterocycles forming the -CH2NH-linked analogs 11a
and 11b. In a similar fashion we reacted the free phenol in intermediate III to furnish the aryl
ethers 12a and 12b. Phenol III also reacted smoothly with several benzyl or alkyl halides
delivering -OCH2-linked compounds 12c and 12d. The fluoropyridine intermediates IV and V
were equally reactive and underwent nucleophilic aromatic substitutions with a large variety of
alcohols, phenols, amines and anilines to give analogs 13a-n and 14a-b, respectively (Scheme
3). Schemes 2 and 3 exemplify representative reactions for each of the intermediates, while
additional analogs were synthesized in the same manner. Table 1 summarizes the activities for
selected compounds against C. neoformans and C. gattii. MIC values for manogepix were of

0.25 µg/mL and 0.125 µg/mL, respectively. More than half of the synthesized compounds had
significantly improved MIC values against both strains, demonstrating the great potential for
optimization of this compound series towards the activity against Cryptococcus. The obtained
MIC values result from interactions of our compounds with whole fungal cells and thus are a
summary of multiple processes, such as cellular penetration (active/passive transport), efflux and
target engagement. Each of these events reacts differently to structural changes within our
compounds. This inherent drawback of phenotypical assays made it difficult to extract sharp
SAR, however, we identified several trends. We did not observe any strong preference for the
type (-CH2-, -O-, -NH-) or the length of the linker (1-, 2- or 3-atoms) between the C- and D-ring
of the compounds with respect to their activity against Cryptococcus, though there was some
interplay between various heteroaryl rings and linker types that affected activity. Meta fluoro-
and chloro-substituents on the D-ring were well tolerated or improved activity, while any ortho-
substituents larger than a fluorine atom decreased potency. The ortho-methyl group in 13k, for
example, showed about 100-fold weaker antifungal activity compared to the unsubstituted analog
13j. Highly polar D-rings (4-amino-2-methylpyrimidine in 11b) or basic amines (10b) led to
inactive compounds. Decreased antifungal activity was also observed when substituents were
introduced to the linker between ring C and group D. For example, the addition of a methyl-
group to the -OCH2-linker reduced the activity by 8- to 16-fold (13e vs. 13f). Compounds
derived from intermediate V (linker to D-ring is meta substituent on the C-ring) were generally
less active than the corresponding analogs derived from intermediate IV. A total of 11
compounds demonstrated MIC values less than or equal to 0.016 µg/mL against both strains and
were advanced to in vitro metabolic stability studies. The HLM half-life (T1/2) for these
compounds was determined according to the methods described under b) HLM half-life. In this
experiment, the half-life of manogepix was about 50 min and we decided to advance compounds
with a half-life of at least 40 min. Compound 8d with an unsubstituted furan as ring D had an
extremely short half-life of about 3 min. To increase metabolic stability, a fluorine atom was
introduced. The half-life of fluoro-analog 8e increased to 14 min and the compound maintained
its antifungal activity. For five compounds with a half-life of 40 min or longer, we assessed the
cytotoxicity in a HepG2 viability assay as described under c) Cytotoxicity assay. Only analog
12b showed significant liability at a concentration of 3 µM and was not further evaluated. The
remaining four compounds were, alongside manogepix, subjected to MoA studies.
Overexpression/resistance assays are a valuable tool for confirming the target-based activity of
inhibitors.16 Overexpression of a target in bacteria or fungi often leads to enhanced resistance to
compounds that inhibit that enzyme target. We evaluated the MIC of compounds against S.
cerevisiae strains overexpressing high and low levels of Gwt1. MIC values of the compounds
were determined for each strain followed by determining the ratio of the MIC values. An
overexpression ratio of ≥8 is consistent with on-target activity. 12a, 13a and 13b showed ratios
of 8-fold or greater against the overexpression strain, which are comparable to manogepix and
are consistent with Gwt1 as the target of these inhibitors.

Table 2. Metabolic stability, Cytotoxicity, and On-target activity of selected compounds.
Gwt1
T1/2
HepG2
PigW
Compda
Overexpression
HLM (min)b
cytotoxicityc
Inhibitione
ratiod
MGX
8d
8e
12a
12b
12d
13a
13b
13c
13i
49.9
3.15
14.4
43.0
61.5
17.3
62.3
93.9
37.1
4.65
68.2
20.3
98
-
-
93
77
-
97
102
-
8
-
-
16
-
-
8
8
-
-
4
-
0
-
-
0
-
-
5
0
-
-
80
-
-
98
-
13l
13m
a Italicized compound numbers indicate a compound that has been previously synthesized by Eisai.
MGX, manogepix. Values were determined according to the methods for b under b) HLM half-life, for
c under c) Cytotoxicity assay (% viability at 3 µM)), for d under d) Overexpression assay and for e
under e) PigW assay (% inhibition at 16 µM). Values in bold fulfill the selection criteria.
Interestingly, compound 13l, which only showed a 4-fold shift in MIC values in the
overexpression assay, was also the only compound that showed significant activity against the
human homolog PigW7, 14 and was deprioritized. Compounds 12a, 13a and 13b are currently
under further investigation.
In summary, we have synthesized over 300 analogs of manogepix and evaluated their activity
against of C. neoformans and C. gattii. We were able to improve the antifungal activity up to 32-
fold against both strains while maintaining a preliminary PK and toxicity profile similar to
manogepix. We have selected three compounds for further in vivo studies.
Conflict of interest statement. The authors are current employees of Amplyx Pharmaceuticals
which has Gwt1 antifungal programs.
Acknowledgements. We thank Dr. Katsura Hata and Eisai Co. Ltd. for providing strains for the
overexpression assays.
Research Support. This project was supported by Amplyx Pharmaceuticals and in part by the
National Institute of Allergy and Infectious Diseases of the National Institutes of Health under
SBIR grant award 5R44AI131864-02.
Methods and Experimental details for the synthesis of key intermediates and compounds are
available in the Supporting Information document.

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