Synthesis and biological evaluation of antifungal derivatives of enfumafungin as orally bioavailable inhibitors of β-1,3-glucan synthase

Article abstract of DOI:10.1016/j.bmcl.2012.05.031

Orally bioavailable inhibitors of β-(1,3)-d-glucan synthase have been pursued as new, broad-spectrum fungicidal therapies suitable for treatment in immunocompromised patients. Toward this end, a collaborative medicinal chemistry program was established based on semisynthetic derivatization of the triterpenoid glycoside natural product enfumafungin in order to optimize in vivo antifungal activity and oral absorption properties. In the course of these studies, it was hypothesized that the pharmacokinetic properties of the semisynthetic enfumafungin analog 3 could be improved by tethering the alkyl groups proximal to the basic nitrogen of the C3-aminoether side chain into an azacyclic system, so as to preclude oxidative N-demethylation. The results of this research effort are disclosed herein.

Full text of DOI:10.1016/j.bmcl.2012.05.031

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6811–6816
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of antifungal derivatives of enfumafungin
as orally bioavailable inhibitors of b-1,3-glucan synthase
Brian H. Heasley a, , Gregory J. Pacofsky a, Ahmed Mamai a, Hao Liu a, Kingsley Nelson a, Ghjuvanni Coti a,
⇑
Michael R. Peel a, James M. Balkovec b, Mark L. Greenlee b, Paul Liberator b, Dongfang Meng b,
Dann L. Parker b, Robert R. Wilkening b, James M. Apgar b, F. Racine b, Ming Jo Hsu b, Robert A. Giacobbe b,
Jennifer Nielsen Kahn b
a Scynexis, Inc., PO Box 12878, Research Triangle Park, NC 27709-2878, USA
b Merck Research Laboratories, Rahway, NJ 07065, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 17 May 2012
Orally bioavailable inhibitors of b-(1,3)-D-glucan synthase have been pursued as new, broad-spectrum
fungicidal therapies suitable for treatment in immunocompromised patients. Toward this end, a collab-
orative medicinal chemistry program was established based on semisynthetic derivatization of the triter-
penoid glycoside natural product enfumafungin in order to optimize in vivo antifungal activity and oral
absorption properties. In the course of these studies, it was hypothesized that the pharmacokinetic prop-
erties of the semisynthetic enfumafungin analog 3 could be improved by tethering the alkyl groups prox-
imal to the basic nitrogen of the C3-aminoether side chain into an azacyclic system, so as to preclude
oxidative N-demethylation. The results of this research effort are disclosed herein.
In dedication to Professor Timothy
Macdonald for his many contributions to
the field of Organic and Medicinal
Chemistry
Keywords:
Enfumafungin
Ó 2012 Elsevier Ltd. All rights reserved.
b-1,3-Glucan synthase
Triterpenoid glycoside
Antifungal agents
Medicinal chemistry
Oral bioavailability
The widespread medical use of immunosuppressive drug ther-
apies and broad-spectrum antibacterial agents, in conjunction
with the rise of viral infections that debilitate the human immune
system such as AIDS, have contributed to an increasing annual
death rate due to fungal infections (mycoses) over the last
30 years. During this same time period, frequent and indiscrimi-
nate use of antibacterial1 and antifungal agents has led to in-
creased antimicrobial resistance.2 This alarming scenario has
prompted a search for new, safe and effective human antifungal
drugs.3
The semisynthetic antifungal drug CANCIDAS (caspofungin
acetate)4 was first approved by the US FDA in 2001 for invasive
aspergillosis in patients who are intolerant of standard therapy.
The drug is generally effective against systemic fungal infections
in immunocompromised patients and is the primary treatment
for a variety of Candida infections. CANCIDAS is derived from the
echinocandin macrocyclic lipopeptide natural product, pneumo-
candin B0.5 The potent and broad-spectrum antifungal activity of
the echinocandin family is attributed to inhibition of fungal cell
wall synthesis via blockade of the biosynthesis of b-(1,3)- -glucan,
an essential cell wall component of pathogenic fungi.2a Given that
the molecular target of CANCIDAS, b-1,3- -glucan synthase, is
unique to lower eukaryotes, antifungal agents that share this
fungal-specific mode of action tend to exhibit low toxicity. Unfor-
tunately, echinocandins and derivatives thereof are poorly
absorbed when administered orally6 and, therefore, are used only
for parenteral administration. For this reason, orally bioavailable
D
D
inhibitors of b-1,3-D-glucan synthase have, in recent years, been
pursued3b,c as new, broad-spectrum fungicidal therapies suitable
for treatment in immunocompromised patients.
Natural product screening efforts by Onishi and co-workers7
identified four acidic terpenoid b-1,3-D-glucan synthase inhibitors
that offer improved pharmacokinetic properties compared to those
of the echinocandin-type lipopeptides. The in vitro antifungal
activity of one of the screening hits, the triterpenoid glycoside
enfumafungin (1, Fig. 1), was comparable to that of caspofungin
acetate, particularly with regard to potency against the Candida
and Aspergillus pathogenic fungal strains.4,7 Moreover, in whole
cells of Candida albicans, 1 selectively inhibits the incorporation
of [14C]-glucose into insoluble cell wall glucan without affecting
⇑
Corresponding author. Fax: +1 919 5448697.
E-mail address: brian.heasley@scynexis.com (B.H. Heasley).
URL: http://www.scynexis.com (B.H. Heasley).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.05.031

6812
B. H. Heasley et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6811–6816
CO2H
OH
12
18
HO
25
H
O
AcO
Ref. [9]
2
O
3
AcO
GlucoseO
OH
O
H
O
H
HO
O
OH
enfumafungin (1)
HO
HO
N
O
H
CO2H
CO2H
N
Ref. [10]
H
AcO
N
O
O
N
O
H
N
H
N
O
this work:
H
H
tethering
strategy
2
3
Figure 1. The triterpenoid glycoside, enfumafungin (1), and semisynthetic, orally bioavailable antifungal derivatives (2–3).
other components of the fungal cell wall, indicating that 1 is a spe-
cific inhibitor of glucan synthesis with an IC50 value that is similar
in magnitude to the MIC.7 The primary structure and relative ste-
reochemistry of enfumafungin were determined mainly by de-
tailed spectroscopic (two-dimensional NMR) studies and
derivatization techniques.8 The defining structural feature of the
aglycone portion of 1 is the hemiacetal cyclization of C23–25
across the A ring (on the b-face of the molecule), which gives rise
to interconverting diastereomers at the anomeric carbon position,
C25. It was found that this unique bridging hemiacetal system en-
ables a cationic SN1-type substitution reaction to occur at C2 (vida
infra), providing convenient and stereoselective access to A-ring-
functionalized derivatives.
We, in collaboration with Merck Research Laboratories, estab-
lished a medicinal chemistry program based on semisynthetic
derivatization of the terpenoid natural product enfumafungin in
order to modulate and optimize in vivo antifungal activity and oral
absorption properties.9 In the course of our work, chemical modi-
fication of C2, C3, C12, C18 and C25 was achieved and the resulting
analogs were evaluated for in vitro antifungal activity and for oral
efficacy in a mouse model of disseminated candidiasis. It was
found that 12-oxo-25-deoxy derivatives bearing an ethanol-
amine-based side chain appended via an ether linkage to C3 (e.g.,
2) were efficacious in a candidiasis model when delivered orally.9
Preliminary structure–activity relationship (SAR) studies indicated
that oral activity is strongly dependent upon the nature of the ste-
ric environment proximal to the basic amino group of the C3 side
chain. Eventually, by incorporating various heteroaryl systems at
acceptable pharmacokinetic profile as a C3-glucoside. We therefore
elected to replace the b- -glucose moiety with a chemically and met-
abolically stable system that was more likely to confer oral bioavail-
ability, a primary goal of the program. Acidic methanolysis of the
D
glycoside linkage resulted in conversion to the 2a-methoxy-3b-
hydroxy aglycone 5. As alluded to above, the cationic substitution
reaction at C2 is thought to be facilitated by the proximal bridging
ether that simultaneously stabilizes a transient cationic species
and sterically blocks the b-face, resulting in nucleophilic attack from
the concave a-face. This unique reactivity pattern is exploited again
in the subsequent operation wherein exposure of 5 to Lewis acidic
conditions in the presence of N-aminophthalimide followed by treat-
ment of the adduct with hydrazine secures the 2a-hydrazino inter-
mediate 7.10 Heating of 7 with the isonicotinamide–formamidine
then fashions the C2 triazole heteroaryl system and the resultant
carboxylic acid intermediate (8) was protected as the corresponding
C18 para-methoxybenzyl ester (9). It is notable in this sequence that
the stereoselective hydrazine installation and heterocycle formation
are conducted efficiently in the presence of unprotected hydroxyl
and carboxylic acid functionality.10
The tethering approach encapsulated in Figure 1 suggested to us
that a cyclic pyrrolidine system would be a beneficial motif for
incorporation into the enfumafungin C3 side chain. Simple pyrrol-
idine systems were accessed by conversion of the enantiomeric
forms of N-Boc-prolinol to their corresponding tosylates in prepa-
ration for a subsequent C3 O-alkylation condensation step with
intermediate 9. The requisite prolinol intermediates bearing a qua-
ternary stereocenter adjacent to nitrogen were obtained as single
stereoisomers by implementation of the ‘memory of chirality’
method of Kawabata.11 Readily available (S)-alanine ethyl ester
was converted into the N-Boc-N-(3-bromopropyl)-alanine deriva-
tive 10 (Scheme 2) in three straight-forward operations.11a The
enantioselective cyclization of 10 proceeded in 96% yield on mul-
C2 on the
a-face of the terpenoid framework, a series emerged
with broad-spectrum fungicidal activity and markedly improved
in vitro potency,10 even in the absence of the 12-oxo moiety. In
light of the previously observed sensitivity of oral efficacy and bio-
availability to the steric nature of the C3-aminoether side chain, we
hypothesized that the pharmacokinetic properties of the lead com-
pound 3 could be improved by tethering the alkyl groups (desig-
nated in Fig. 1 with arrows) proximal to the basic nitrogen into
an azacyclic system, so as to preclude oxidative N-demethylation.
The results of this research effort are dislosed herein.
As depicted in Scheme 1, the epimeric C25 lactol of enfumafungin
was removed by ionic reduction.9,10 Although it is known that the
cardenolide glycoside digoxin is therapeutically effective through
an oral route of administration, enfumafungin does not exhibit an
ti-gram scale to afford N-Boc-a-methyl proline ethyl ester (11)
with exquisite enantioselectivity as determined by a Mosher ester
analysis (cf. 11?13). The cyclization is thought to proceed through
the intermediacy of an axially chiral nonracemic enolate that
undergoes intramolecular alkylation with a total retention of con-
figuration.11 Two additional steps generated the neopentyl tosylate
electrophilic building block 12 in enantiomerically pure form.
Completion of the partial synthesis of final compounds 15e and
15f (from 1) is outlined in Scheme 3. Antifungal derivatives 15a–d

B. H. Heasley et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6811–6816
6813
CO2H
CO2H
H2SO4,
R
O
H
H
MeOH,
65 o
AcO
O
MeO
HO
C
O
86%
(2 steps)
H
H
HO
O
R = OH (1)
Et3SiH,
TFA, tol
OH
5
R = H (4)
HO
HO
N
O
OR
CO2H
O
O
N
NH2
N
NMe2
N
N
H
H
N
O
H
N
R
O
BF3-OEt2,
HOAc, 90 oC,
68%
N
O
DCE, 50 oC,
66%
HO
HO
H
H
R = phthalimide (
R = NH2 (
6)
R = H (
8)
N2H4-H2O,
EtOH, 100%
PMB-Br, K2CO3,
DMF, 80%
R = PMB (
9
)
7
)
Scheme 1. Semisynthetic preparation of the 2a-heteroaryl terpenoid derivative 9 from enfumafungin (1).
KHMDS, DMF,
1. LiBH4,
THF, 97%
Boc
Boc
H3C
CO2Et
Boc
-60 oC, 30 min
CO2Et
CH3
N
N
OTs
Br
N
96%
(2.6 g scale)
CH3
2. pTsCl, DMAP,
pyr, DCM, 79%
10
11
12
1. LiBH4, THF
O
Boc
N
2. RCOCl, pyr,
DMAP, DCM
d.r. >95:5
O
CH3
OMe
F3C
13
a-methyl prolinol building block (12) for eventual incorporation into the C3-aminoether side chain of semisynthetic
Scheme 2. Enantiospecific construction of an
enfumafungin derivatives.
12
(4 eq)
N
O
OPMB
N
O
OPMB
Boc
N
OTs
CH3
N
N
H
H
N
O
N
N
N
Boc
O
NaH, DMF,
50 oC,
23% (HPLC)
O
N
HO
CH3
H
H
9
14
O
N
O
OH
N
OH
formalin,
NaCNBH3,
HOAc,
MeOH
TFA,
anisole,
DCM
N
N
N
H
H
N
N
O
N
47%
H3C
90%
(HPLC)
O
O
H
(HPLC)
N
N
O
CH3
H
CH3
H
15e
15f
Scheme 3. Completion of antifungal synthetic targets 15e–f.
were obtained in a similar fashion.10 The intermolecular C3 O-
alkylation reaction between terpenoid derivative 9 and tosylate
12 was conducted using sodium hydride as a base under thermally
forcing conditions. The modest isolated yield12 is attributed to the
relatively unreactive nature of the neopentyl tosylate electrophile
due to steric hindrance. Utilization of N-Boc- -methyl prolinol io-
dide or triflate species in the O-alkylation reaction returned only
a
unreacted 9, presumably due to decomposition of the electrophile

Table 1
In vitro inhibition of (1,3)-glucan synthase and antifungal activity against C. albicans
H
N
Me
N
H
N
Me
N
H
N
Me
N
H
N
N
COOH
O
O
O
O
O
O
O
H
H
H
H
N
3
15a
H
15b
15c
15d
15e
15f
N
N
O
RO
H
a
GS IC50 (ng/mL)
18
1000
61
1000
22
500
19
500
4.0
125
6.4
125
2.0
30
MIC (ng/mL) C. albicans
a
See Ref. 13 for assay conditions.
Table 2
Oral efficacy (TOKA) data and pharmacokinetic profiling (murine).
H
N
Me
N
H
Me
H
Me
H
N
N
N
N
N
N
COOH
O
O
O
O
O
O
O
H
H
H
H
N
3
15b
15c
15d
15e
15f
H
15a
N
N
O
RO
H
TOKAa
log CFU:
D
at 12.5 mpk/IP
at 25 mpk/PO
at 12.5 mpk/PO
at 6.25 mpk/po
À2.18
À1.32
À0.26
À0.40
N/D
N/D
À1.69
À2.09
À1.81
À0.27
À1.84
À1.14
À0.90
À1.22
À2.83
À2.16
À1.80
À2.00
À1.40
À0.90
À4.37
À4.25
À3.88
À2.72
Mouse PK
Clearance
N/D
16.3
0.03
1.7
13.1
0.06
23
19.2
0.06
4.7
N/D
16.0
0.62
42
po nAUC (
F (%)
lM  hr  kg/mg)
N/D = not determined.
a
See Ref. 14 for assay conditions.

B. H. Heasley et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6811–6816
6815
under the vigorous reaction conditions. Alkylations of 9 with the
enantiomeric forms of N-Boc- -H-prolinol tosylate provided ca.
efficacy. Derivatives bearing the (R)-prolinol stereochemical con-
figuration (15c–f) were more orally efficacious than the represen-
tative (S)-prolinol analog 15a, reducing kidney fungal burden by ca.
two logarithmic units when dosed orally at 25 mg/kg (mpk). Com-
a
50% yields10,12 of C3 O-alkylated products and these intermediates
were further elaborated to final compounds 15a–d.10 In the penul-
timate step of the synthesis of 15f, global deprotection of the acid-
labile side chain N-Boc group and C18 para-methoxybenzyl ester
was accomplished in a single operation (14?15e). Finally, the
somewhat hindered pyrrolidine 15e could be N-monomethylated
in acceptable yield12 under standard reductive alkylation condi-
tions to complete the partial synthesis of derivative 15f. The semi-
synthetic preparation of 15f from enfumafungin depicted in
Schemes 1–3 is representative of the general methodologies used
to access other final compounds described in this work.10
pound 15e, the a-methyl (R)-prolinol derivative, exhibited notable
oral efficacy in the TOKA mouse model (À2.16 log unit reduction at
25 mpk/po) but was considerably less active in this assay than lead
compound 3. The demonstrated oral efficacy of compounds 3, 15e
and 15f is consistent with the previously asserted9c notion that
incorporation of a quaternary stereocenter adjacent to the basic
amine of the C3 enfumafungin side chain confers improved oral
activity in the TOKA mouse model of disseminated candidiasis.9,10
Based upon the promising oral efficacy observed in the pyrroli-
dine series, three compounds were advanced to murine pharmaco-
kinetic profiling studies (Table 2).10 Unfortunately, following oral
administration of compounds 15c–e in mice, poor plasma exposure
was indicated by relatively low dose-normalized area under curve
(nAUC) values in conjunction with high rates of clearance (13–19
L/h). Area under the curve values for 15c–e were at least 10-fold
less than the nAUC determined for lead compound 3 after identical
po dosing in mice. Compound 3 was also significantly more bio-
available than derivatives from the pyrrolidine series (F = 42% for
3 vs 23% for 15d). In spite of potent in vitro inhibitory properties
against fungal b-(1,3)-glucan synthase, the moderate TOKA activity
and low plasma exposure (for 15c–e) short-circuited further devel-
opment of C3-aminoether pyrrolidine enfumafungin derivatives as
orally bioavailable antifungal agents. Pyrrolidine systems are
known to undergo bioactivation involving hydroxylation at C5
(pyrrolidine numbering), leading to ring-opening of the resultant
N,O-hemiaminal.15 This metabolic process may be more detrimen-
tal to plasma exposure than oxidative N-demethylation in the case
of 3.
Compounds 15a–f were evaluated as direct inhibitors of
b-(1,3)-glucan synthase, the essential fungal enzyme that forms
b-(1,3)-glucan fibrils from UDP-glucose. In brief, the in vitro assay
for inhibition of b-(1,3)-glucan synthase from Candida albicans
quantifies the incorporation of radioactivity from UDP-[3H]-glu-
cose into precipitated glucan.10,13 The IC50 of enfumafungin (1) in
the glucan synthase inhibition assay is approximately 50 ng/mL,
a value comparable to the MIC (200 ng/mL) against Candida albi-
cans.7,8 Semisynthetic enfumafungin derivatives 15a–f are potent
inhibitors of fungal cell wall glucan assembly (Table 1). The most
potent in vitro inhibitors of glucan synthase from this series are
compounds 15e and 15f, each bearing a quaternary stereocenter
adjacent to the basic amine of the pyrrolidine in the C3-aminoether
side chain. The IC50 potencies of 15e and 15f (4.0 and 6.4 ng/mL,
respectively) in this assay are within an order of magnitude of
the IC50 of the lead compound (3). Compounds 15a-d, lacking the
side chain quaternary carbon substitution, lose about one log order
of potency against glucan synthase relative to 3.
Antifungal susceptibility testing was conducted in order to
determine minimum inhibitory concentrations (MIC values) for
test compounds as a measurement of antifungal activity. Deriva-
tives 15a–f exhibited good to moderate MIC values against C. albi-
cans (Table 1) that were generally comparable in potency to their
corresponding IC50 values in the in vitro glucan synthase inhibition
assay. As a point of reference, the MIC of enfumafungin (1) against
C. albicans is 200 ng/mL.7,8 Two of the compounds tested, 15e and
15f, displayed antifungal activity that was similar to that of 1.
However, the MIC values of these two derivatives (125 ng/mL for
each) against C. albicans were less potent than that of the lead com-
pound (3) by about one order of magnitude. In addition, the
remaining analogs from this set (15a–d) were significantly less po-
tent than 3. Given the general loss of in vitro antifungal activity in
the ‘tethered’ pyrrolidine series relative to 3, it is evident that sub-
tle structural modifications of the C3-aminoether side chain of
enfumafungin derivatives can significantly modulate antifungal
potency. In that respect, the C3 side chain of compound 3 is consid-
ered a highly desirable structural feature that contributes to potent
in vitro fungicidal properties in the enfumafungin series.
In vivo anti-Candida activity and oral efficacy of the experimen-
tal enfumafungin derivatives were evaluated using the target or-
gan kidney assay (TOKA) mouse model of disseminated
candidiasis.10,14 In brief, a disseminated Candida infection is in-
duced in mice by intravenous inoculation of C. albicans into the lat-
eral tail vein. Test compounds are then administered (ip or po) bid
for 2 days and the reduction in kidney fungal burden compared to
sham-treated control groups is determined. TOKA-active com-
pounds generally lower the fungal colony forming units (CFU)
per gram of kidney by 1–2 logarithmic units compared to sham-
treated controls.10 Enfumafungin derivatives 15a and 15c–f were
shown to be efficacious in the mouse model of disseminated can-
didiasis when delivered orally (Table 2). In the pyrrolidine series,
the configuration of the stereogenic carbon position in the C3-
aminoether side chain influenced the magnitude of observed oral
References and notes
1. Heasley, B. H. Angew. Chem., Int. Ed. 2011, 50, 8474. and references cited
therein.
2. (a) Odds, F. C.; Brown, A. J. P.; Gow, N. A. R. Trends Microbiol. 2003, 11, 272; (b)
Ghannoum, M. A.; Rice, L. B. Clin. Microbiol. Rev. 1999, 12, 501.
3. (a) Brown, E. D.; Wright, G. D. Chem. Rev. 2005, 105, 759; (b) Vicente, M. F.;
Basilio, A.; Cabello, A.; Pelaez, F. Clin. Microbiol. Infect. 2003, 9, 15; (c) http://
www.merck.com/licensing/news-and-events/scynexis-press-release.html.
4. (a) Bouffard, F. A.; Zambias, R. A.; Dropinski, J. F.; Balkovec, J. M.; Hammond, M.
L.; Abruzzo, G. K.; Bartizal, K. F.; Marrinan, J. A.; Kurtz, M. B.; McFadden, D. C.;
Nollstadt, K. H.; Powles, M. A.; Schmatz, D. M. J. Med. Chem. 1994, 37, 222; (b)
Balkovec, J. M.; Black, R. M.; Hammond, M. L.; Heck, J. V.; Zambias, R. A.;
Abruzzo, G.; Bartizal, K.; Kropp, H.; Trainor, C.; Schwartz, R. E.; McFadden, D. C.;
Nollstadt, K. H.; Pittaressi, L. A.; Powles, M. A.; Schmatz, D. M. J. Med. Chem.
1992, 35, 194.
5. Leonard, W. R.; Belyk, K. M.; Conlon, D. A.; Bender, D. R.; DiMichele, L. M.; Liu, J.;
Hughes, D. L. J. Org. Chem. 2007, 72, 2335.
6. The %F of caspofungin acetate in mice is approximately 0.3–1%: Abruzzo, G. K.;
Flattery, A. M.; Gill, C. J.; Kong, L.; Smith, J. G.; Pikounis, V. B.; Balkovec, J. M.;
Bouffard, F. A.; Dropinski, J. F.; Rosen, H.; Kropp, H.; Bartizal, K. Antimicrob.
Agents Chemother. 1997, 41, 2333.
7. Onishi, J.; Meinz, M.; Thompson, J.; Curotto, J.; Dreikorn, S.; Rosenbach, M.;
Douglas, C.; Abruzzo, G.; Flattery, A.; Kong, L.; Cabello, A.; Vicente, F.; Pelaez, F.;
Diez, M. T.; Martin, I.; Bills, G.; Giacobbe, R.; Dombrowski, A.; Schwartz, R.;
Morris, S.; Harris, G.; Tsipouras, A.; Wilson, K.; Kurtz, M. B. Antimicrob. Agents
Chemother. 2000, 44, 368.
8. Schwartz, R. E.; Smith, S. K.; Onishi, J. C.; Meinz, M.; Kurtz, M.; Giacobbe, R. A.;
Wilson, K. E.; Liesch, J.; Zink, D.; Horn, W.; Morris, S.; Cabello, A.; Vicente, F. J.
Am. Chem. Soc. 2000, 122, 4882.
9. (a) Balkovec, J. M., Bouffard, F. A., Tse, B., Dropinski, J., Meng, D., Greenlee, M. L.,
Peel, M. R., Fan, W., Mamai, A., Liu, H., Li, K. PCT International Patent
Application, WO 2007127012 A1, 2007.; (b) Balkovec, J. M., Siliphaivanh, P.,
Bouffard, F. A., Peel, M. R., Fan, W., Mamai, A., Dimick Gray, S. M. PCT
International Patent Application, WO 2009045311 A1, 2009.; (c) Peel, M. R.,
Mamai, A., Fan, W., Dimick-Gray, S., Hong, J., Orr, M. D., Balkovec, J. M.,
Greenlee, M. L., Waddell, S., Abruzzo, G. Flattery, A., Giacobbe, R., Gill, C., Hsu,
M.-J., Racine, F., Liberator, P., Nielsen Kahn, J. Poster F1–848 ICAAC 2010.
10. (a) Greenlee, M. L., Wilkening, R., Apgar, J., Sperbeck, D., Wildonger, K. J., Meng,
D., Parker, D. L., Pacofsky, G. J., Heasley, B. H., Mamai, A., Nelson, K. PCT
International Patent Application, WO 2010019204 A1, 2010.; (b) Wilkening, R.,
Apgar, J., Meng, D., Parker, D. L., Greenlee, M. L., Sperbeck, D., Wildonger, K. J.,

6816
B. H. Heasley et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6811–6816
Abruzzo, G., Flattery, A., Galgoci, A., Giacobbe, R., Gill, C., Hsu, M.-J., Liberator, P.,
1 mL/well each cycle) using a Packard FILTERMATE HARVESTER. 40
scintillation fluid (Packard ULTIMA GOLD TM-XR) was added and the sealed
plates counted in WALLAC BETA counter in top-counting mode at an
lL/well
Misura, A., Motyl, M., Nielsen Kahn, J., Powles, M. A., Racine, F., Dragovic, J.,
Habulihaz, B., Fabre, E., Heasley, B. H., Kirwan, R., Lee, S., Liu, H., Mamai, A.,
Nelson, K., Pacofsky, G. J., Peel, M. R., Balkovec, J. M. Poster F1–846 ICAAC, 2010.
11. (a) Kawabata, T.; Moriyama, K.; Kawakami, S.; Tsubaki, K. J. Am. Chem. Soc.
2008, 130, 4153; (b) Kawabata, T.; Kawakami, S.; Majumdar, S. J. Am. Chem. Soc.
2003, 125, 13012.
12. The reported yield reflects an isolated quantity of HPLC-purified material from
a single experiment and can, therefore, be considered unoptimized.
13. Glucan synthase inhibition: The in vitro evaluation of glucan synthase inhibitory
activity of compounds was measured in a polymerization assay in 96-well
a
efficiency of approximately 40%.
14. In vivo anti-candida activity: A disseminated Candida infection is induced in
DBA/2 mice by the iv inoculation of 0.2 mL of
a yeast cell suspension
containing 3.0 Â 104 CFU of C. albicans MY1055 into the lateral tail vein.
Therapy is initiated within 15–30 min after challenge. Mice are treated with
test compound, either (1) ip, bid for a total of 2 days, or (2) po, bid for a total of
2 days. For each route of administration and diluent, an appropriate sham-
treated control group is included. Kidneys from euthanized mice (4–5/group)
are removed 4 days after challenge using aseptic techniques, weighed and
placed in sterile WHIRL PAK bags containing 5 mL sterile saline. Kidneys are
homogenized in the bags, serially diluted in saline and aliquots are plated on
SD agar plates. Plates are incubated at 35 °C and enumerated after 30–48 h for
C. albicans colony forming units (CFUs). Means from CFU/g of paired kidneys of
treated groups are compared to the means from sham-treated controls.
15. (a) Xu, S.; Zhu, B.; Teffera, Y.; Pan, D. E.; Caldwell, C. G.; Doss, G.; Stearns, R. A.;
Evans, D. C.; Beconi, M. G. Drug Metab. Dispos. 2005, 33, 121; (b) McKennis, H.;
Schwartz, S. L.; Bowman, E. R. J. Biol. Chem. 1964, 239, 3990.
format. Each well contained 100 lL of 3H-UDPG at 0.5 mM (6000–8000 dpm/
nmol), 50 mM HEPES pH 7.5 (Sigma), 10% w/v glycerol (Sigma), 1.5 mg/mL
bovine serum albumin (Sigma A 9647. Lot 44H0190), 25 mM KF (Fisher), 1 mM
EDTA (Gibco ULTRAPURE), 25
incorporation during the 60 min incubation at 22 °C, and test compound added
from wells in threefold serial dilutions in 100% DMSO (1 L/well). The reaction
was stopped by the addition of 100 L of 20% trichloroacetic acid. Plates were
lM GTP-c-S, enzyme sufficient to give 3–6 nmol
l
l
chilled for a minimum of 10 min, and precipitated glucan collected by filtration
on GF/C plates (Packard UNIFILTERÒ-96), washed with 5 cycles of water (about