Potent Synergy between Spirocyclic Pyrrolidinoindolinones and Fluconazole against Candida albicans

Article abstract of DOI:10.1002/cmdc.201500271

A spiroindolinone, (1S,3R,3aR,6aS)-1-benzyl-6′-chloro-5-(4-fluorophenyl)-7′-methylspiro[1,2,3a,6a-tetrahydropyrrolo[3,4-c]pyrrole-3,3′-1H-indole]-2′,4,6-trione, was previously reported to enhance the antifungal effect of fluconazole against Candida albicans. A diastereomer of this compound was synthesized, along with various analogues. Many of the compounds were shown to enhance the antifungal effect of fluconazole against C. albicans, some with exquisite potency. One spirocyclic piperazine derivative, which we have named synazo-1, was found to enhance the effect of fluconazole with an EC₅₀ value of 300 pM against a susceptible strain of C. albicans and going as low as 2 nM against some resistant strains. Synazo-1 exhibits true synergy with fluconazole, with an FIC index below 0.5 in the strains tested. Synazo-1 exhibited low toxicity in mammalian cells relative to the concentrations required for antifungal synergy. Synergy from stereochemical complexity: An attempt to synthesize analogues of a known spiroindolinone led to a series of diastereomers. One spiroindolinone, termed synazo-1, was shown to exhibit potent activity (300 pM) against C. albicans in the presence of fluconazole. Synazo-1 is a true synergizer and was also highly active against some drug-resistant C. albicans strains.

Full text of DOI:10.1002/cmdc.201500271

DOI: 10.1002/cmdc.201500271
Full Papers
Potent Synergy between Spirocyclic
Pyrrolidinoindolinones and Fluconazole against Candida
albicans
Ilandari Dewage Udara Anulal Premachandra,[a] Kevin A. Scott,[a] Chengtian Shen,[a]
Fuqiang Wang,[b] Shelley Lane,[b] Haoping Liu,[b] and David L. Van Vranken*[a]
A spiroindolinone, (1S,3R,3aR,6aS)-1-benzyl-6’-chloro-5-(4-fluo-
rophenyl)-7’-methylspiro[1,2,3a,6a-tetrahydropyrrolo[3,4-c]pyr-
role-3,3’-1H-indole]-2’,4,6-trione, was previously reported to en-
hance the antifungal effect of fluconazole against Candida albi-
cans. A diastereomer of this compound was synthesized, along
with various analogues. Many of the compounds were shown
to enhance the antifungal effect of fluconazole against C. albi-
cans, some with exquisite potency. One spirocyclic piperazine
derivative, which we have named synazo-1, was found to en-
hance the effect of fluconazole with an EC50 value of 300 pm
against a susceptible strain of C. albicans and going as low as
2 nm against some resistant strains. Synazo-1 exhibits true syn-
ergy with fluconazole, with an FIC index below 0.5 in the
strains tested. Synazo-1 exhibited low toxicity in mammalian
cells relative to the concentrations required for antifungal syn-
ergy.
Introduction
Candida spp. account for 80% of major systemic fungal infec-
tions.[1] The mortality rate for invasive candidiasis has been es-
timated at around 40%.[2] Fluconazole has high bioavailability
and is well tolerated;[3–7] it is the first-line antifungal treatment
for candidiasis and is now available in generic form. However,
there are reports of side effects, including hepatitis, leukope-
nia, thrombocytopenia, gastrointestinal distress, headache,
anaphylaxis, and rash.[8–13] There is a need for drugs that can
potently enhance the efficacy of azoles. Moreover, resistance
to fluconazole and other azole drugs remains a major prob-
lem,[14] particularly among immunocompromised patients.[15]
Compounds that synergize with fluconazole at low concen-
trations (e.g., MIC90 <0.1 mgmLÀ1) are unusual. The antifungal
agents flucytosine[16–19] and fenpropimorph[20] have been
shown to potently synergize with fluconazole against various
strains of C. albicans. Micafungin[21–23] and caspofungin[20] are
highly potent, but not synergistic with fluconazole, with FIC in-
dices above 1.0. A number of drugs commonly used against
non-fungal human diseases have also been shown to synergize
with azoles against C. albicans. The calcineurin-inhibiting drug
tacrolimus[24,25] potently enhances the activity of fluconazole
against C. albicans. Quite a few other compounds have been
reported to inhibit the growth of Candida in synergy with flu-
conazole, with MIC90 values below 1 mgmLÀ1, but not below
0.1 mgmLÀ1: e.g., T. broussonetii extract,[26] terbinafine,[27] amlo-
darone,[28] catechin, quercetin, epigallocatechin,[29] simvasta-
tin,[30] tunicamycin,[20] cationic peptides IJ3, IJ4[31] and VS3,[32]
ketorolac,[33] cyclosporin A,[24,34] nystatin,[35] sanguinarine,[36] alli-
cin,[37] declofenac,[33] leaf extracts of Lippia alba,[38] diphenyldi-
selenide,[39] balcalein,[40] geldanamycin,[36] pseudolaric acid B,[41]
and doxycycline.[42] Hundreds of other compounds have been
reported to exhibit antifungal activity in concert with azoles
but not below 1 mm. Chemical synthesis can be used to im-
prove the potency of lead molecules; in a recent study, several
analogues of the azole synergizer berberine (MIC80:
1.0 mgmLÀ1) were identified with up to eightfold greater po-
tency.[43,44]
Several groups have screened large libraries of compounds
in search for small molecules that synergize with azoles. In
2011, Spitzer et al. screened the Prestwick library of off-patent
drugs[45] and demonstrated that sertraline and thiethylperazine
synergize with fluconazole. The MIC for sertraline was as low
as 0.5 mgmLÀ1 in the presence of fluconazole. Also in 2011, La-
Fleur and co-workers screened a library of 120000 compounds
in search of molecules that can act in synergy with clotrima-
zole against C. albicans biofilms, but none were active below
1 mm.[46] Starting in 2010 Lindquist, Schreiber, and others at the
Broad Institute reported a massive screening campaign to
identify small molecules capable of inhibiting the growth of
C. albicans in synergy with fluconazole with a particular inter-
est in Hsp90 and calcineurin pathways.[47,48] After an initial
screen of over 300000 compounds and a subsequent rescreen-
ing, 296 compounds were found to have fluconazole-depen-
dent potency against a partially resistant clinical strain CaCi-8
as well as a lack of cytotoxicity against mammalian fibroblasts.
Three of those compounds[48–50] were selected for further opti-
[a] I. D. U. A. Premachandra, K. A. Scott, C. Shen, Prof. Dr. D. L. Van Vranken
Department of Chemistry, University of California, Irvine
1102 Natural Sciences 2, Irvine, CA 92697-2025 (USA)
E-mail: david.vv@uci.edu
[b] Dr. F. Wang, S. Lane, Prof. Dr. H. Liu
Department of Biological Chemistry, University of California, Irvine
825 Health Sciences Road, Medical Sciences I, Irvine, CA 92697-1700 (USA)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201500271: synthesis and spectroscopic
characterization of all compounds.
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mization, but none of the resulting compounds (ML189,
ML212, and ML229) were active below 0.7 mm against CaCi-8.
We speculated that some of the other 296 compounds identi-
fied in the Broad screen might merit further study as potentia-
tors of fluconazole activity in C. albicans. We set out to identify
promising candidates from the Broad assay and to design ana-
logues capable of potently inhibiting the growth of C. albicans
in the presence of fluconazole. We were intrigued by the struc-
turally interesting spirocyclic compound (1S,3R,3aR,6aS)-1-
benzyl-6’-chloro-5-(4-fluorophenyl)-7’-methylspiro[1,2,3a,6a-tet-
rahydropyrrolo[3,4-c]pyrrole-3,3’-1H-indole]-2’,4,6-trione (Pub-
Chem CID 6584729) and the potential activity of synthetically
accessible diastereomer 1 and related analogues (Figure 1).
Scheme 2. Synthesis of substituted isatins. Reagents and conditions: a) chlo-
ral hydrate (1.1 equiv), NH2OH·HCl (3.0 equiv), Na2SO4 (9.0 equiv), HCl
(1.1 equiv), H2O, 708C, 1 h; b) H2SO4, 238C, 5 min; c) BnBr (1.1 equiv), NaH
(1.1 equiv), DMF, 238C, 2 h.
Figure 1. Fluconazole synergizer CID 6584729 and diastereomer 1.
Results and Discussion
Chemistry
As shown in Scheme 1, N-phenylmaleimides 4a and 4b were
synthesized through a two-step condensation of substituted
anilines with maleic anhydride.[51] Anilines 2a and 2b were
condensed with maleic anhydride to form the corresponding
N-phenylmaleamic acids 3a and 3b, which were cyclized with
acetic anhydride in the presence of sodium acetate to afford
the corresponding maleimides.
Scheme 3. Synthesis of compound 1 and nOes used in the assignment of
relative configuration. Reagents and conditions: a) MeOH/H2O (3:1), 658C,
16 h.
Various substituted isatins were prepared from the corre-
sponding anilines using the two-step Sandmeyer synthesis
(Scheme 2).[52] Anilines were reacted with the oxime of chloral,
generated in situ, to afford isonitrosoacetanilides, which were
pure, as determined by TLC. The isonitrosoacetanilides were
cyclized, without purification, through an intramolecular Frie-
del–Crafts reaction to afford the corresponding isatins 6a–6d
in good yield. N-Benzylisatins 7d and 7e were prepared
by alkylation with benzyl bromide using sodium hydride as
a base.[53]
Spiroindolinones are readily accessible through one-pot
three-component coupling reactions of isatins, amino acids,
and maleimides.[54–56] The reaction of isatin 6d, l-phenylala-
nine, and maleimide 4a generated compound 1 as a single
diastereomer in 74% yield (Scheme 3). The optically pure
amino acid undergoes decarboxylation during the reaction;
unless otherwise stated, all spiroindolinones were isolated and
tested as racemates. The relative stereochemistry of compound
1 was secured through a NOESY experiment (Scheme 3) and
shown to match that of related spiroindolinones prepared
from isatins and maleimides under the same reaction condi-
tions.[56] In particular, the strong nuclear Overhauser effect
(nOe) between protons on C3’ and C6a’ of the pyrrolidine ring
indicate that they are on the same face, and conversely that
the benzyl group and succinimide ring are both on the oppos-
ing face. Furthermore, the strong nOe between the fluoro-
phenyl proton and the proton on C4 of the indolone ring is
consistent with the stereochemistry of compound 1. Interest-
Scheme 1. Synthesis of substituted N-phenylmaleimides. Reagents and condi-
tions: a) maleic anhydride (1.0 equiv), Et2O, 238C, 15 min; b) NaOAc
(0.7 equiv), (CH3CO)2O, 708C, 30 min.
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products consistent with syn–
anti azomethine ylides (Figure 2,
configuration A).[57–60] However,
in the corresponding reactions
with phenylglyoxylate, the reac-
tive configuration of the azome-
thine ylide seems to depend on
the type of amino acid: proline
gives products consistent with
syn–anti
azomethine
ylides
(Figure 2,
configuration A),[61]
whereas acyclic amino acids give
products consistent with anti–
anti azomethine ylides (Figure 2,
configuration B).[62–64]
The 1,3-dipolar cycloaddition
can proceed through either an
endo or exo transition state. The
products derived from all known
reactions of amino acids, acyclic
enones, and either isatins or
phenylglyoxalates can be ration-
alized to arise through endo
transition
states
(Scheme 5,
path A).[58–62,65] In contrast, reac-
tions of amino acids, maleimides,
and either isatins or phenyl-
Scheme 4. Synthesis of spirocyclic pyrrolidines via three-component, one-pot [1,3]-dipolar cycloaddition with
amino acids. Reagents and conditions: a) MeOH/H2O (3:1), 658C, 4–16 h.
1
ingly, the H and 13C NMR spectra and nOes for the compound
sold as CID 6584729 (by Vitas-M) were indistinguishable from
those of compound 1. Thus, commercial STK 580951 is, in fact,
the same as our synthetic compound 1 and does not match
PubChem CID 6584729.
Other analogues of spirocycle
1
were synthesized
(Scheme 4) using phenylalanine, tryptophan, and Ne-Boc-lysine.
In all cases, the limiting reagent, isatin 6 or 7, was completely
consumed, and the reaction gave the desired cycloadduct as
a single diastereomer. The reac-
Figure 2. Stereochemical configurations of azomethine ylides.
tions of these amino acids were
highly stereoselective, affording
products with a relative configu-
ration analogous to spiroindoli-
none 1. We did not observe or
isolate other diastereomers of
the spiroindolinones 8–13.
The formation of diastereomer
1, and 8–13 was anticipated
based on the work of Pavlov-
skaya et al.,[55] but is best ex-
plained by examining a larger
body of work involving reactions
of amino acids, enones, and
either isatins or phenylglyoxalate
derivatives, which are essentially
acyclic analogues of isatins. All
known reactions of amino acids,
isatins, and enones react to give Scheme 5. Endo/exo selectivity in dipolar cycloadditions of azomethine ylides.
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glyoxalates can proceed through endo or exo transition states,
depending on the structure of the amino acid. Acyclic amino
acids give products consistent with endo transition states
(Scheme 5, paths B and C).[55,58,62–65] The only known reaction of
a cyclic six-membered ring amino acid, pipecolic acid, with
isatin and an acyclic dipolarophile also gives products consis-
tent with an endo transition state (Scheme 5, path B);[59] how-
ever, the stereochemical outcome in the reaction of azome-
thine ylides with acyclic dipolarophiles cannot be extrapolated
to reactions with maleimides.[56,60a] In contrast, the cyclic five-
membered ring amino acid proline gives products consistent
with an exo transition state (Scheme 5, path D).[56]
chromatography after benzoylation of the hydroxy groups to
afford esters 25a and 25b. The relative stereochemistry was
assigned on the basis of diagnostic nOes. In particular, in both
spiroindolinones 25a and 25b, there is an nOe between the
bridgehead proton Hd and the arene proton Hg on the indo-
lone ring. In spiroindolinone 25a, protons Hf’, Ha, and He’ on
the b-face of the proline ring exhibit vicinal nOes between
each other. Protons He’ and Hf’ on the b-face of the proline ring
exhibit long-range nOes with protons Hc and Hd on the succini-
mide ring, respectively; proton Hf’ exhibits highly diagnostic
long-range nOes to the indolone aryl proton Hg. In spiroindoli-
none 25b, protons Hf’, Ha, He’, and Hb on the b-face of the pro-
line ring exhibit vicinal nOes between each other. Protons He
and Hf on the a-face of the proline ring exhibit long-range
nOes with protons Hc and Hd on the succinimide ring, respec-
tively; proton Hf exhibits a highly diagnostic long-range nOe
to the indolone aryl proton Hg. Thus, the [3+2] cycloaddition
of trans-hydroxyproline proceeds via exo addition of maleimide
Similar ylides can be accessed from three-component reac-
tions with amines instead amino acids, but there are cases in
which the trends in ylide configuration[66] and endo/exo selec-
tivity[67] no longer hold. Notably, Ardill et al. showed that 1,3-di-
poles derived from N-methylpiperazine aminals and related
compounds favor exo adducts over endo adducts—sometimes
exclusively exo—in toluene at reflux.[67]
to an azomethine ylide with
(Scheme 5, path D).
a syn–anti configuration
The assumption that isatins would react through path C
(Scheme 5) may have led to the mis-assignment of the com-
pound CID 6584729 by the commercial supplier along with
over 100 spiroindolinones in the PubChem database. We
cannot be sure of the stereochemistry of the compound
CID 6584729 that was tested by Lindquist and co-workers, as
the experimental data for compounds in PubChem assay IDs
1979, 2467, and 2423 were never reported.[68] One cannot rule
out the possibility that the compound CID 6584729 tested in
those assays was correctly assigned and generated through
a more lengthy synthetic route than the one-pot reaction used
in this and related work.
The reaction of the six-membered ring amino acid Ne-Boc-pi-
perazine-2-carboxylic acid proceeds in manner analogous with
pipecolic acid (Scheme 5, path B) to afford spirocyclic indoli-
none 14 as a single diastereomer. The relative stereochemistry
of spirocyclic piperazine 14 was secured with nOes after re-
moval of the Boc group and shown to match that of com-
pound 1. Related spiroindolinones 15–23 were also prepared
stereoselctively from Ne-Boc-piperazine-2-carboxylic acid
(Scheme 4).
The Boc group was removed from the spirocyclic piperazine
14 using trifluoroacetic acid to give piperazine 26 in 90% yield
(Scheme 7). Piperazine 26 served as the precursor for various
N-acyl derivatives 27–31 in the following reactions (Scheme 7).
Compound 27 was synthesized by acylating 26 with methyl
10-chloro-10-oxodecanoate in the presence of sodium carbon-
ate. Carbodiimide-mediated coupling of (S)-2-methoxy-2-phe-
nylacetic acid with the racemic piperazine 26 in the presence
of triethylamine resulted in a mixture of diastereomers; only
one diastereomer 28 was readily purified, but the relative ste-
reochemistry was not assigned. Acylation of 26 with hydrocin-
namoyl chloride in the presence of triethylamine afforded
amide 29. The reaction of piperazine 26 with benzyl isocyanate
generated the urea 30. Carbamate 31 was synthesized by acy-
lation of piperazine 26 with benzyl chloroformate.
To provide access to diastereomeric spiroindolinones with
defined absolute stereochemistries, we carried out a three-
component coupling with isatin 6d, N-phenylmaleimide, and
(2S,4R)-4-hydroxyproline (Scheme 6). After 16 h the reaction
generated an inseparable mixture of two spiroindolinones 24a
and 24b in 30% yield along with unreacted isatin. The two op-
tically pure stereoisomers were readily separated by silica gel
Structure–activity relationships
CID 6584729 was reported to enhance the effect of fluconazole
against the partially resistant clinical isolate of C. albicans CaCi-
8 at an EC50 value of 0.12 mm.[68] We determined the antifungal
potency of the new spiroindolinones in combination with flu-
conazole against a susceptible strain (HLY4123) derived from
a commonly used laboratory strain of C. albicans. The activity
of compound 1 was promising, with an EC50 value of 0.011 mm.
We then compared the activity of compound 1, derived from
phenylalanine, with the activity of spiroindolinones derived
from other amino acids (Table 1; compounds 1, 8, 9, and 10).
Scheme 6. Stereoselectivity in the three-component, one-pot [1,3]-dipolar
cycloaddition with (2S,4R)-4-hydroxyproline. Stereochemistry was established
by nOes (grey lines). Reagents and conditions: a) MeOH/H2O (3:1), 908C,
16 h; b) BzCl (1.1 equiv), Et3N (1.2 equiv), DMF, 238C, 20 h.
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Compound 13, derived from 6-
chloro-7-methylisatin, but lack-
ing the 4-fluoro substituent, was
exceedingly potent with an EC50
value of 1 nm.
When we used the non-natu-
ral amino acid Ne-Boc-piperazine-
2-carboxylic acid, the resulting
spirocyclic piperazine 14 was still
highly active with an EC50 value
of 5.6 nm. N-Benzylsuccinimide
derivatives 18, 19, and 21 were
not highly active. The substitu-
ents on the indolone ring were
still important, even with the
pentacyclic
piperazine
core
(compounds 17, 20, 22, and 23).
We removed the Boc group
from the piperazine ring of com-
pound 14, leading to a loss of
potency (compound 26). Surpris-
ingly, the carboxyl oxygen atom
of the carbamate moiety ap-
pears to be essential for high
potency, because carbamates 14
and 31 were more potent than
Scheme 7. Synthesis of pentacyclic pyrrolidines by further substitutions to compound 26. Reagents and conditions:
a) TFA/CH2Cl2 (1:1), 238C, 15 min, 90%; b) methyl 10-chloro-10-oxodecanoate (1.04 equiv), Na2CO3 (1.0 equiv),
CH2Cl2, 238C, 30 min, 53%; c) (S)-2-methoxy-2-phenylacetic acid (1.3 equiv), EDC (2.1 equiv), Et3N (3.5 equiv),
CH2Cl2, 238C, 20 min, 45%; d) 3-phenylpropanoyl chloride (1.05 equiv), Et3N (1.1 equiv), CH2Cl2, 238C, 3 h, 64%;
e) (isocyanatomethyl)benzene (1.1 equiv), CH2Cl2, 238C, 4 h, 75%; f) benzyl chloroformate (1.1 equiv), DIPEA
(2.1 equiv), CH2Cl2, 0–238C, 2.5 h, 54%.
Neither tryptophan nor Ne-Boc-lysine derivatives were better
than the parent compound 1 derived from phenylalanine. Re-
gardless of the maleimide substituent, N-benzylisatin deriva-
tives exhibited relatively low activity (compounds 11 and 12).
amides 27 and 28. Even more revealing, the isosteric amide 29
and urea 30 were two and three orders of magnitude less
active, respectively, than benzyloxycarbamate 31. Ultimately,
benzyloxycarbamate 31 proved to be exquisitely active in im-
proving the efficacy of flucona-
zole, with an EC50 value of
300 pm. The two hydroxyproline
Table 1. Structure–activity relationships for polycyclic pyrrolidines against the susceptible strain HLY4123 of
C. albicans in the presence of fluconazole (0.25 mgmLÀ1).
adducts 25a and 25b exhibited
almost no activity under the
assay conditions; these results
are not surprising, given that the
relative stereochemistry of those
compounds is different from all
the other compounds that were
tested.
Compd[a]
R5’
R2’
R3’
R5
R6
R7 R1
EC50 [mm][b]
1
8
9
4-fluorophenyl
H
H
H
H
H
H
H
Ph
H
H
H
H
H
H
H
H
H
H
H
H
H
H
MeO
H
H
H
Cl CH3
H
H
H
H
Bn
0.011Æ0.004
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Ph
3-indolyl
(CH2)3N(Boc)
Ph
H
H
H
H
H
H
H
H
H
H
~10Æ1.3
~10Æ1.8
>100
>10
10
11
12
13
14
15
16
17
18
19
20
21
22
23
26
27
28
29
30
31
Ph
Ph
Bn >100
Cl CH3
Cl CH3
Cl CH3
Cl CH3
H
H
H
H
0.001Æ0.0005
In general, substitution of
small hydrophobic groups at the
6- and 7-positions of the indo-
lone ring and phenyl sub-
stitution at the 3’-position of
the central pyrrolidine improves
antifungal activity. Moreover,
a phenyl moiety—not benzyl—
in the succinamide ring enhan-
ces antifungal activity. When the
piperazine is present in the poly-
cyclic pyrrolidine, a carbamoyl
moiety, but not the acyl groups,
on the piperazine ring signifi-
cantly improves antifungal activi-
ty. On the other hand, fluorinat-
CH2CH2N(Boc)
CH2CH2N(Boc)
CH2CH2N(Boc)
CH2CH2N(Boc)
CH2CH2N(Boc)
CH2CH2N(Boc)
CH2CH2N(Boc)
CH2CH2N(Boc)
CH2CH2N(Boc)
0.0056Æ0.003
0.037Æ0.001
0.0237Æ0.01
4-fluorophenyl
3,5-bis(F3C)phenyl
Ph
Bn
Bn
Ph
Bn
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Cl CH3 Bn >100
H
H
H
H
H
H
H
H
H
CH3
Bn
H
0.0318Æ0.4
>100
Bn >100
H
H
H
H
H
H
H
H
H
230Æ5.7
0.213Æ0.08
CH2CH2N(Boc)
CH3 CH3
Cl CH3
Cl CH3
Cl CH3
Cl CH3
Cl CH3
Cl CH3
0.0057Æ0.006
CH2CH2NH
~10Æ1.5
CH2CH2N[CO(CH2)8CO2Me]
CH2CH2N[COCH(OCH3)C6H5]
CH2CH2N[CO(CH2)2Ph]
CH2CH2N[CONHCH2Ph]
CH2CH2N(Cbz)
H
H
H
H
0.0379Æ0.009
0.035Æ0.007
0.0181Æ0.004
0.256Æ0.1
H
0.0003Æ0.00001
[a] See Schemes 4 and 7 for structures. [b] Each value is the arithmetic mean ÆSD of three independent experi-
ments.
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ed substituents at the para and meta positions of the phenyl
group in the succinamide ring as well as N-substitution of the
indolone moiety diminish the activity of the spiroindolinones
against C. albicans.
Activity against resistant cell lines
A variety of resistant clinical isolates of C. albicans were
screened with 64 mgmLÀ1 fluconazole and compound 31 at
a single dose (3 mm) in a broth microdilution assay (Table 2).[69]
Figure 3. Structure of compound 31, renamed synazo-1.
fluconazole dramatically enhances the activity of compound
31 against C. albicans. Conversely, compound 31 makes fluco-
nazole more potent against those same strains, but the effect
is less dramatic. We have named compound 31 ’synazo-1’
(Figure 3).
Table 2. Effect of compound 31 on the growth of resistant clinical strains
in the presence of fluconazole.
[a]
[b]
Isolate[a]
MICflc
Growth (OD600
)
Potency
+Compd 31
EC50 [nm][c,e]
+Fluc[c]
+Compd 31[d]
+Fluc[c]
Greater than 90% inhibition of C. albicans sterol a-demethy-
lase (a.k.a. Erg11 or CYP51) would be expected at ten times the
Ki value for fluconazole, which has been determined to be
0.03 mm.[71,72] When synazo-1 is present at 300 nm in the sus-
ceptible strain, the MIC90 for fluconazole is decreased from 0.5
to 0.125 mm, consistent with the theoretical limit for flucona-
zole potency of ~0.3 mm.
17
23
26
33
36
45
32
32
32
64
64
3.96
6.77
6.44
7.38
5.08
9.17
0.76
0.60
0.83
0.88
1.07
1.03
5Æ0.011
53Æ0.005
2Æ0.002
11 Æ0.008
5Æ0.251
16Æ0.002
128
[a] From ref. [69]. [b] Growth measured after 16 h in SC at 308C. [c] [fluco-
nazole]=64 mgmLÀ1
[d] [compound 31]=3 mm. [e] Each value is the
.
arithmetic meanÆSD of three independent experiments.
Cytotoxicity of synazo-1 against mammalian cells
We compared the cytotoxicity of spiroindoline 1 and synazo-
1 against NIH 3T3 cells at higher concentrations, up to 1.5 mm
(Figure 4). Both compounds exhibited only weak cytotoxicity.
Synazo-1 was slightly more cytotoxic, but not at the concentra-
tions required for antifungal synergy. According to PubChem,
CID 6584729 was previously tested for cytotoxicity against
NIH 3T3 cells (PubChem AID 2387), but the EC50 value was
ꢀ160 mm, the limit of the assay. It is unclear how the biological
activities reported for CID 6584729 should relate to that of the
diastereomer 1, or whether they were even distinct com-
pounds.
The strains grow at dramatically different rates. The published
fluconazole minimum inhibitory concentrations (MICs) for
these isolates convey the level of resistance. Strains for which
growth in the presence of compound 31 and fluconazole was
less than 25% of growth in the presence of fluconazole alone
(isolates 17, 23, 26, 33, 36, and 45) were selected for determi-
nation of EC50 values. Compound 31 was particularly active
against clinical isolates 17, 26, and 36 and exhibited good ac-
tivity against the highly resistant isolate 45.
A checkerboard assay was used to determine the fractional
inhibitory concentrations (FICs) for compound 31 and flucona-
zole against the fluconazole-susceptible strain (HLY4123) and
two fluconazole-resistant clinical isolates 26 and 45 (Table 3).[69]
In all of the strains tested, the FIC index was below 0.5, fitting
the classical definition of synergy.[70] Compound 31 alone did
not have measurable toxicity against any of the strains at the
solubility limit (between 30 and 300 mm). In the strains tested,
Table 3. FIC indices for compound 31 and fluconazole in different strains
of C. albicans.[a]
Strain
MICcpd
MICcpd(+Flc)
MICflc
MICflc(+Cpd)
FICI
HLY4123
26
45
>300
>30
>30
0.03
0.3
0.03
0.5
836
836
0.125
105
105
0.25
0.14
0.13
[a] MIC90 measured in mm; MIC data are derived from single replicates of
two independent experiments, and values were consistent between ex-
periments.
Figure 4. Cytotoxicity of synazo-1 and compounds 1 against NIH 3T3 cells;
data are the arithmetic meansÆSD of three independent experiments.
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Drug-like parameters for synazo-1
Experimental Section
Chemistry
A wide range of readily calculated properties are often used as
indicators of oral bioavailability. The calculated physicochemi-
cal properties of synazo-1 were compared with typical ranges
for lead-like molecules.[73–75] Synazo-1 flags just one of the
common warnings for drug lead-like properties (Table 4): mo-
lecular weight. For comparison, the orally available azole poso-
conazole is outside the range on four of the parameters. It is
widely recognized that the average molecular weight and
complexity of newly approved oral drugs has been increasing
with each year.[76,77] Both the carbamate and imide moieties
are potential liabilities for metabolism, yet when the stability
of synazo-1 was tested in 10% FBS/phosphate-buffered saline
at 378C, no decomposition was observed over 16 h.
General procedures: NMR spectral data were recorded at room
temperature using a Bruker 500 or 600 MHz spectrometer, unless
stated otherwise. The NMR data are reported as follows: chemical
shifts in ppm from an internal tetramethylsilane standard on the d
scale, multiplicity (br=broad, app=apparent, s=singlet, d=dou-
blet, t=triplet, q=quartet, and m=multiplet), coupling constants
(Hz), and integration. Analytical thin-layer chromatography (TLC)
was performed using EMD Reagents 0.25 mm silica gel 60-F plates.
“Flash” chromatography on silica gel was performed with Silicycle
silica gel (40-63 mm). All reactions were carried out under an at-
mosphere of nitrogen in glassware that was evacuated and back-
filled with nitrogen three times. Reactions were carried out at
room temperature unless otherwise indicated. Unless otherwise
noted, all reagents were commercially obtained and, where appro-
priate, purified prior to use. THF, Et2O, DMF, and CH2Cl2 were dried
by filtration through alumina according to the procedure of
Grubbs and co-workers.[78] For final compounds the purity was de-
termined by HPLC (Agilent Technologies series 1200). The HPLC in-
strument consisted of an Agilent Technologies series 1200 auto-
sampler, series 1200 UV/Vis detector, series 1100 pump, using
ChemStation software (Agilent Technologies, Santa Clara, CA, USA).
The analytical column was a reversed-phase Waters Nova-pak C18
150 mm”3.9 mm column. A gradient elution was used (flow rate:
0.2 mLminÀ1), starting with 80% H2O and progressing to 100%
CH3CN over a period of 1 h, with both solvents containing 0.1%
formic acid. All compounds have purity ꢀ95% (l=254 nm) by
HPLC.
Table 4. Calculated physicochemical properties of synazo-1.[a]
Property
Range
Synazo-1
logP
À4.0 to 4.2
ꢁ120
ꢁ460
ꢀ1
4.173
99.3
571
9
1
4
1
0.20
5
TPSA [Š2]
Mr [Da]
N+O
H-bond donors
Rotatable bonds
Halogens
Fraction sp3
H-bond acceptors
ꢁ5
ꢁ10
ꢁ7
0.15–0.80
ꢁ9
[a] Calculated with the Molinspiration property calculation service:
www.molinspiration.com/cgi-bin/properties.
Synthesis of pyrrolidines by three-component dipolar cycloaddi-
tion (1, 8–23, 24a, and 24b):[54] A 100 mL round-bottom flask was
charged with substituted isatin (1.0 equiv), N-substituted malei-
mide (1.1 equiv), the amino acid (1.1 equiv), and a stir bar. A 3:1 (v/
v) mixture of H2O and MeOH was added to the reaction flask such
that the concentration of isatin was 0.25m. The reaction was
heated at reflux by immersing the reaction flask in a hot oil bath
at 908C up to the level of the flask’s contents. Initially a clear solu-
tion was obtained, and CO2 evolution was observed. However,
after a few hours the reaction mixture became cloudy. The reaction
was monitored for consumption of the substituted isatin by TLC
(EtOAc/hexanes). Upon consumption of the substituted isatin, the
reaction was cooled to room temperature. Next, the reaction mix-
ture was quenched by pouring it into a mixture of ice and saturat-
ed aqueous NaHCO3. The resulting solid was washed thoroughly
with H2O in Büchner funnel to afford a grey solid. The solid was
then dissolved in a minimum amount of CH2Cl2 and purified by
flash chromatography with EtOAc/hexanes (1:1) to afford the race-
mic substituted pyrrolidine.
Conclusions
In summary, we have designed, synthesized, and studied spi-
roindolinones inspired by CID 6584729, which was previously
reported to exhibit activity against C. albicans in combination
with fluconazole. The relative stereochemistry of compound
1 and analogues was secured through 2D NMR experiments.
The three-component, one-pot [3+2] dipolar cycloaddition of
isatins, amino acids, and maleimides was found to proceed
through endo addition of maleimides to a syn–anti azomethine
ylide in all cases except for a proline derivative. A number of
the new spiroindolinones were substantially more potent
against C. albicans than the original lead compound 1 when
used in combination with fluconazole. In particular, synazo-
1 was exquisitely potent, with an EC50 value of 300 pm against
a susceptible strain. Synazo-1 also exhibited low nanomolar ac-
tivity against a number of resistant isolates of Candida. When
tested in both susceptible and resistant strains of C. albicans,
synazo-1 was a true synergizer with an FIC index below 0.5.
Synazo-1 has many of the calculable parameters associated
with orally available drug molecules and represents a promising
candidate for development as an antifungal synergizer.
(Æ)-(3R,3’R,3a’R,6a’S)-3’-Benzyl-6-chloro-5’-(4-fluorophenyl)-7-
methyl-2’,3’,3a’,6a’-tetrahydro-4’H-spiro[indoline-3,1’-pyrro-
lo[3,4-c]pyrrole]-2,4’,6’(5’H)-trione (1): A 100 mL round-bottom
flask was charged with 1-(4-fluorophenyl)-1H-pyrrole-2,5-dione
(0.50 g, 2.55 mmol, 1.0 equiv), p-fluoro-N-phenylmaleimide (0.53 g,
2.8 mmol, 1.1 equiv), l-phenylalanine (0.46 g, 2.8 mmol, 1.1 equiv),
and a stir bar. A 3:1 mixture of H2O and MeOH (11 mL) was added
to the reaction flask. The content of the reaction flask was heated
at reflux by immersing the reaction flask in a hot oil bath up to the
level of the flask’s contents. Initially a clear solution was obtained,
and CO2 was expelled. After few hours a cloudy solution was ob-
served. Upon consumption of the substituted isatin (16 h), the re-
action was allowed to cool to room temperature. Next, the reac-
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tion mixture was quenched by pouring it into a mixture of ice and
saturated aqueous NaHCO3. The resulting solid was washed thor-
oughly with H2O in Büchner funnel to afford a grey solid. The solid
was then dissolved in a minimum amount of CH2Cl2 and purified
by flash chromatography with various combinations of EtOAc/hex-
anes to afford pyrrolidine 1 as a white solid (0.93 mg, 1.9 mmol,
for the synthesis of pyrrolidines outlined above, indoline-2,3-dione
(0.37 g, 2.5 mmol, 1.0 equiv) was used, and the product was puri-
fied by flash chromatography with EtOAc/hexanes to afford pyrroli-
dine 10 as a white solid (0.42 mg, 0.82 mmol, 33%): Rf =0.35 (4:6
EtOAc/hexanes); mp: 109–111 8C; 1H NMR (600 MHz, CDCl3): d=
7.45–7.44 (m, 2H), 7.38–7.33 (m, 3H), 7.29 (brs, 1H), 7.20–7.17 (m,
1H), 6.79–6.76 (m, 2H), 6.36 (d, J=7.4 Hz, 1H), 4.78 (d, J=14.0 Hz,
1H), 4.70–4.63 (m, 1H), 4.61 (d, J=14.0 Hz, 1H), 4.34–4.32 (m, 1H),
3.50 (t, J=7.7 Hz, 1H), 3.42 (d, J=7.7 Hz, 1H), 3.18–3.08 (m, 2H),
2.06–1.91 (m, 2H), 1.57–1.53 (m, 2H), 1.43 ppm (s, 9H); 13C NMR
(125 MHz, CDCl3, 313 K): d=180.15, 175.8, 174.5, 140.3, 135.8,
129.8, 129.2, 128.8, 128.2, 126.5, 126.4, 122.7, 109.8, 68.1, 58.3, 51.7,
48.1, 42.7, 40.1, 31.2, 30.0, 28.5, 24.6, 14.3 ppm; IR (thin film): n˜ =
3707, 2971, 2843, 1345, 1054, 1032 cmÀ1; HRMS (ESI): m/z calculat-
ed for C29H34N4O5Na [M+Na]+ 541.2427, found 541.2411; HPLC
purity: 97.81%, tR =20.74 min.
1
74%): Rf =0.35 (1:1 EtOAc/hexanes); mp: 212–2148C. The H NMR
chemical shifts were concentration-dependent in CDCl3, particularly
1
within the range 0.5–2 mm. H NMR (600 MHz, CDCl3): d=8.00 (s,
1H), 7.41–7.38 (m, 2H), 7.27–7.16 (m, 6H), 7.02 (d, J=8.0 Hz, 2H),
6.81 (d, J=8.5 Hz, 1H), 4.72–4.71 (m, 1H), 3.75–3.69 (m, 1H), 3.45
(dd, J=14.0, 4.0 Hz, 1H), 2.73 (dd, J=13.8, 10.5 Hz, 1H), 2.16 (s,
1H), 1.99 ppm (s, 3H); 13C NMR (125 MHz, CDCl3): d=180.4, 175.1,
174.4, 163.4, 161.4, 140.5, 139.1, 136.2, 128.9, 128.8, 128.3, 127.6,
126.7, 124.6, 124.5, 123.5, 118.2, 116.6, 116.4, 68.2, 58.9, 51.6, 47.6,
37.9, 13.5 ppm; IR (thin film): n˜ =3201, 3065, 1710, 1696, 1623,
1601, 1510 cmÀ1; HRMS (ESI): m/z calculated for C28H29ClN4O5Na
[M+Na]+ 559.1724, found 559.1743; HPLC purity: 95.76%, tR =
21.73 min.
(Æ)-(3R,3’R,3a’R,6a’S)-1,3’,5’-Tribenzyl-2’,3’,3a’,6a’-tetrahydro-4’H-
spiro[indoline-3,1’-pyrrolo[3,4-c]pyrrole]-2,4’,6’(5’H)-trione (11):
Using the general procedure for the synthesis of pyrrolidines out-
lined above, 1-benzylindoline-2,3-dione (0.07 g, 0.32 mmol,
1.0 equiv) was used, and the product was purified by flash chroma-
tography with EtOAc/hexanes to afford pyrrolidine 11 as a white
solid (0.08 mg, 0.15 mmol, 46%): Rf =0.60 (1:1 EtOAc/hexanes);
mp: 173–1758C; 1H NMR (600 MHz, CDCl3): d=7.50–7.49 (m, 2H),
7.39–7.12 (m, 14H), 6.82–6.79 (m, 1H), 6.64 (d, J=9.3 Hz, 1H), 6.54
(d, J=8.7 Hz, 1H), 4.88–4.81 (m, 2H), 4.80–4.72 (m, 1H), 4.71–4.65
(m, 2H), 3.64–3.60 (m, 1H), 3.44–3.39 (m, 2H), 2.61 (dd, J=16.6,
12.3 Hz, 1H), 2.01 ppm (brs, 1H); 13C NMR (125 MHz, CDCl3, 313 K):
d=178.7, 175.9, 174.4, 142.6, 139.3, 135.8, 135.4, 129.8, 129.0,
128.9, 128.9, 128.8, 128.6, 128.1, 127.8, 127.3, 126.5, 126.4, 125.8,
122.6, 109.1, 67.5, 58.5, 51.8, 47.7, 43.6, 42.7, 38.2 ppm; IR (thin
film): n˜ =3850, 3708, 2971, 2864, 1453, 1054, 1032 cmÀ1; HRMS
(ESI): m/z calculated for C34H29N3O3Na [M+Na]+ 550.2106, found
550.2108; HPLC purity: 98.45%, tR =23.19 min.
(Æ)-(3R,3’R,3a’R,6a’S)-3’,5’-Dibenzyl-2’,3’,3a’,6a’-tetrahydro-4’H-
spiro[indoline-3,1’-pyrrolo[3,4-c]pyrrole]-2,4’,6’(5’H)-trione
(8):
Using the general procedure for the synthesis of pyrrolidines out-
lined above, indoline-2,3-dione (0.37 g, 2.5 mmol, 1.0 equiv) was
used, and the product was purified by flash chromatography with
EtOAc/hexanes to afford pyrrolidine 8 as a white solid (0.65 mg,
1.5 mmol, 60%): Rf =0.50 (1:1 EtOAc/hexanes); mp: 138–1408C;
1H NMR (600 MHz, CDCl3): d=7.50–7.49 (m, 2H), 7.40–7.38 (m, 3H),
7.29 (brs, 1H), 7.26–7.25 (m, 1H), 7.24–7.22 (m, 3H), 7.18–7.16 (m,
2H), 6.80 (t, J=7.5 Hz, 1H), 6.69 (d, J=7.8 Hz, 1H), 6.50 (d, J=
7.5 Hz, 1H), 4.83 (d, J=14.0 Hz, 1H), 4.69 (d, J=14.0 Hz, 1H), 4.68–
4.67 (m, 1H), 3.58–3.56 (m, 1H), 3.42 (d, J=7.6 Hz, 2H), 2.60 (dd,
J=13.8, 10.4 Hz, 1H), 2.01 ppm (brs, 1H); 13C NMR (125 MHz,
CDCl3, 313 K): d=180.1, 175.8, 174.5, 140.3, 139.3, 135.8, 129.8,
129.0, 128.9, 128.8, 128.7, 128.1, 126.9, 126.5, 126.4, 122.6, 109.7,
67.6, 58.5, 51.5, 47.7, 42.7, 38.2 ppm; IR (thin film): n˜ =3850, 3646,
2971, 2843, 1697, 1619, 1054, 1032 cmÀ1; HRMS (ESI): m/z calculat-
ed for C27H23N3O3Na [M+Na]+ 460.1637, found 460.1620; HPLC
purity: 100%, tR =20.04 min.
(Æ)-(3R,3’R,3a’R,6a’S)-1,3’-Dibenzyl-5’-phenyl-2’,3’,3a’,6a’-tetrahy-
dro-4’H-spiro[indoline-3,1’-pyrrolo[3,4-c]pyrrole]-2,4’,6’(5’H)-
trione (12): Using the general procedure for the synthesis of pyrro-
lidines outlined above, 1-benzylindoline-2,3-dione (0.30 g,
1.3 mmol, 1.0 equiv) was used, and the product was purified by
flash chromatography with EtOAc/hexanes to afford pyrrolidine 12
as a yellow solid (0.38 mg, 0.74 mmol, 57%): Rf =0.60 (1:1 EtOAc/
(Æ)-(3R,3’R,3a’R,6a’S)-3’-((1H-Indol-3-yl)methyl)-5’-benzyl-
2’,3’,3a’,6a’-tetrahydro-4’H-spiro[indoline-3,1’-pyrrolo[3,4-c]pyr-
role]-2,4’,6’(5’H)-trione (9): Using the general procedure for the
synthesis of pyrrolidines outlined above, indoline-2,3-dione (0.37 g,
2.5 mmol, 1.0 equiv) was used, and the product was purified by
flash chromatography with EtOAc/hexanes to afford pyrrolidine 9
as a yellow solid (0.83 mg, 1.7 mmol, 70%): Rf =0.35 (1:1 EtOAc/
1
hexanes); mp: 183–1858C; H NMR (600 MHz, [D6]DMSO): d=7.57–
7.52 (m, 2H), 7.46–7.44 (m, 1H), 7.40–7.37 (m, 4H), 7.34–7.33 (m,
3H), 7.31–7.25 (m, 3H), 7.21–7.17 (m, 2H), 7.14 (dd, J=7.4, 1.5 Hz,
1H), 6.97 (t, J=7.5 Hz, 1H), 6.84 (d, J=7.8 Hz, 1H), 4.90 (d, J=
15.6 Hz, 1H), 4.76 (d, J=15.6 Hz, 1H), 4.52–4.50 (m, 1H), 3.73 (dd,
J=7.5, 1.8 Hz, 1H), 3.63 (dd, J=7.8, 2.7 Hz, 1H), 2.61 (dd, J=16.6,
12.3 Hz, 1H), 3.32 (brs, 1H), 2.82–2.78 ppm (m, 1H); 13C NMR
(125 MHz, CDCl3, 313 K): d=178.7, 175.9, 174.4, 142.6, 139.3, 135.8,
135.4, 129.8, 129.0, 128.9, 128.9, 128.8, 128.6, 128.1, 127.8, 127.3,
126.5, 126.4, 125.8, 122.6, 109.1, 67.5, 58.5, 51.8, 47.7, 43.6, 42.7,
38.2 ppm; IR (thin film): n˜ =3680, 2966, 2865, 1706, 1054,
1032 cmÀ1; HRMS (ESI): m/z calculated for C34H29N3O3Na [M+Na]+
550.2106, found 550.2108; HPLC purity: 95.07%, tR =23.09 min.
1
hexanes); mp: 130–1328C; H NMR (600 MHz, CDCl3): d=7.96 (brs,
1H), 7.60 (d, J=7.9 Hz, 1H), 7.52 (d, J=7.3 Hz, 2H), 7.42–7.39 (m,
2H), 7.36–7.33 (m, 1H), 7.30–7.29 (m, 1H), 7.17–7.11 (m, 2H), 7.09–
7.06 (m, 2H), 6.80–6.77 (m, 1H), 6.62 (d, J=7.7 Hz, 1H), 6.49 (d, J=
7.5 Hz, 1H), 4.86 (d, J=14.0 Hz, 1H), 4.80–4.76 (m, 1H), 4.70 (d, J=
14.0 Hz, 1H), 3.60 (t, J=7.7 Hz, 1H), 3.49 (dd, J=14.6, 3.7 Hz, 1H),
3.41 (d, J=7.7 Hz, 1H), 2.82 (dd, J=14.6, 10.3 Hz, 1H), 2.13 ppm
(brs, 1H); 13C NMR (125 MHz, CDCl3, 313 K): d=180.3, 176.0, 174.8,
140.4, 136.2, 135.9, 129.6, 129.0, 128.8, 128.2, 127.5, 126.8, 126.5,
122.5, 122.4, 122.1, 119.5, 119.1, 113.6, 111.1, 109.7, 67.7, 58.1, 51.6,
47.7, 42.6, 27.7 ppm; IR (thin film): n˜ =3679, 2971, 2864, 2843,
1695, 1619, 1054, 1032 cmÀ1; HRMS (ESI): m/z calculated for
C29H24N4O3Na [M+Na]+ 499.1746, found 499.1739; HPLC purity:
98.81%, tR =21.98 min.
(Æ)-(3R,3’R,3a’R,6a’S)-3’-Benzyl-6-chloro-7-methyl-5’-phenyl-
2’,3’,3a’,6a’-tetrahydro-4’H-spiro[indoline-3,1’-pyrrolo[3,4-c]pyr-
role]-2,4’,6’(5’H)-trione (13): Using the general procedure for the
synthesis of pyrrolidines outlined above, 6-chloro-7-methylindo-
line-2,3-dione (0.30 g, 1.7 mmol, 1.0 equiv) was used, and the prod-
uct was purified by flash chromatography with EtOAc/hexanes to
afford pyrrolidine 13 as a white solid (0.21 g, 0.44 mmol, 26%): Rf =
0.60 (1:1 EtOAc/hexanes); mp: 168–1708C; 1H NMR (600 MHz,
(Æ)-tert-Butyl
(4-((3R,3’R,3a’R,6a’S)-5’-benzyl-2,4’,6’-trioxo-
3’,3a’,4’,5’,6’,6a’-hexahydro-2’H-spiro[indoline-3,1’-pyrrolo[3,4-
c]pyrrol]-3’-yl)butyl)carbamate (10): Using the general procedure
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[D6]DMSO): d=10.12 (s, 1H), 7.54–7.51 (m, 2H), 7.45–7.43 (m, 1H),
7.39–7.35 (m, 4H), 7.29–7.27 (m, 2H), 7.19–7.17 (m, 1H), 6.97 (d,
J=8.0 Hz, 1H), 6.90 (d, J=8.0 Hz, 1H), 4.55 (brs, 1H), 3.72 (t, J=
7.5 Hz, 1H), 3.53 (d, J=7.8 Hz, 1H), 3.38–3.35 (m, 2H), 2.27 ppm (s,
3H); 13C NMR (125 MHz, [D6]DMSO): d=142.8, 140.6, 135.1, 134.5,
132.9, 129.4, 129.3, 128.7, 127.6, 127.3, 126.4, 125.0, 121.9,
117.3 ppm; IR (thin film): n˜ =3850, 3626, 2971, 2864, 1710, 1693,
1014, 1032 cmÀ1; HRMS (ESI): m/z calculated for C27H22ClN3O3Na
[M+Na]+ 494.1247, found 494.1255; HPLC purity: 95.00%, tR =
23.41 min.
13C NMR (125 MHz, [D6]DMSO): d=177.6, 174.2, 173.0, 154.4, 141.2,
136.8, 133.0, 132.7, 126.2, 124.5, 123.8, 123.7, 122.5, 118.4, 81.0,
72.9, 62.3, 58.3, 57.6, 50.5, 46.17, 45.6, 28.4, 13.8 ppm; IR (thin film):
n˜ =3187, 1724, 1706, 1680, 1599, 1276, 1132 cmÀ1; HRMS (ESI): m/z
calculated for C30H27ClF6N4O5Na [M+Na]+ 695.1472, found
695.1450; HPLC purity: 98.46%, tR =24.08 min.
(Æ)-tert-Butyl (3R,3a’R,3b’S,9a’S)-1,2’-dibenzyl-1’,2,3’-trioxo-2’,3’,
3a’,3b’,4’,6’,7’,9a’-octahydrospiro[indoline-3,9’-pyrrolo[3’,4’:3,4]-
pyrrolo[1,2-a]pyrazine]-5’(1’H)-carboxylate (18): Using the general
procedure for the synthesis of pyrrolidines outlined above, 1-ben-
zylindoline-2,3-dione (0.30 g, 1.3 mmol, 1.0 equiv) was used, and
the product was purified by flash chromatography with EtOAc/hex-
anes to afford pyrrolidine 18 as a white solid (0.39 mg, 0.66 mmol,
56%): Rf =0.8 (1:1 EtOAc/hexanes); mp: 191–1928C; 1H NMR
(600 MHz, [D6]DMSO): d=7.38–7.37 (m, 4H), 7.36–7.32 (m, 5H),
7.31–7.27 (m, 1H), 7.22–7.20 (m, 1H), 6.87 (d, J=7.8 Hz, 1H), 6.82–
6.79 (m, 1H), 6.50 (d, J=7.2 Hz, 1H), 4.87 (s, 2H), 4.65 (AB q, J=
14.7 Hz, 2H), 4.36–4.34 (m, 1H), 3.85–3.83 (m, 1H), 3.79–3.76 (m,
1H), 3.72–3.71 (m, 1H), 3.49 (d, J=7.8 Hz, 1H), 2.68–2.64 (m, 1H),
2.51–2.53 (m, 1H), 2.12–2.09 (m, 1H), 1.44 ppm (s, 9H); 13C NMR
(125 MHz, [D6]DMSO, 313 K): d=176.1, 175.8, 174.5, 155.2, 143.7,
136.6, 136.4, 130.1, 129.2, 129.0, 128.2, 128.1, 127.9, 127.7, 126.7,
124.2, 122.7, 109.7, 79.7, 71.7, 58.2, 50.8, 46.3, 45.5, 43.1, 42.2,
28.5 ppm; IR (thin film): n˜ =3850, 3626, 2971, 2862, 1707, 1693,
1678, 1032 cmÀ1; HRMS (ESI): m/z calculated for C35H36N4O5Na [M+
Na]+ 615.2584, found 615.2572; HPLC purity: 100%, tR =24.32 min.
(Æ)-tert-Butyl (3R,3a’R,3b’S,9a’S)-6-chloro-7-methyl-1’,2,3’-trioxo-
2’-phenyl-2’,3’,3a’,3b’,4’,6’,7’,9a’-octahydrospiro[indoline-3,9’-pyr-
rolo[3’,4’:3,4]pyrrolo[1,2-a]pyrazine]-5’(1’H)-carboxylate
(14):
Using the general procedure for the synthesis of pyrrolidines out-
lined above, 6-chloro-7-methylindoline-2,3-dione (0.81 g, 4.1 mmol,
1.0 equiv) was used, and the product was purified by flash chroma-
tography with EtOAc/hexanes to afford pyrrolidine 14 as a light-
pink solid (1.3 g, 2.5 mmol, 60%): Rf =0.35 (4:6 EtOAc/hexanes);
1
mp: 201–2038C; H NMR (600 MHz, [D6]DMSO, 400 K): d=10.90 (s,
1H), 7.54–7.51 (m, 2H), 7.47–7.46 (m, 1H), 7.31 (d, J=7.8 Hz, 2H),
7.06 (d, J=7.8 Hz, 1H), 6.73 (d, J=7.8 Hz, 1H), 4.31 (brs, 1H), 3.85
(t, J=7.5 Hz, 2H), 3.64–3.65 (m, 1H), 3.55 (d, J=7.8 Hz, 1H), 2.74–
2.58 (m, 2H) 2.25 (apps, 4H), 2.13–2.08 (m, 1H), 1.41 ppm (s, 9H);
13C NMR (125 MHz, [D6]DMSO, 313 K): d=177.9, 174.7, 173.4, 153.6,
142.8, 134.6, 132.1, 128.9, 128.5, 126.9, 124.6, 123.2, 122.8, 117.3,
79.1, 71.7, 62.3, 58.7, 57.6, 50.4, 45.9, 44.9, 27.9, 13.7 ppm; IR (thin
film): n˜ =3840, 3708, 3626, 2971, 2843, 1713, 1695, 1032 cmÀ1
;
HRMS (ESI): m/z calculated for C28H29ClN4O5Na [M+Na]+ 559.1724,
found 559.1743; HPLC purity: 96.96%, tR =22.18 min.
(Æ)-tert-Butyl (3R,3a’R,3b’S,9a’S)-2’-benzyl-1’,2,3’-trioxo-2’,3’,3a’,
3b’,4’,6’,7’,9a’-octahydrospiro[indoline-3,9’-pyrrolo[3’,4’:3,4]-
pyrrolo[1,2-a]pyrazine]-5’(1’H)-carboxylate (19): Using the general
procedure for the synthesis of pyrrolidines outlined above, indo-
line-2,3-dione (0.37 g, 2.5 mmol, 1.0 equiv) was used, and the prod-
uct was purified by flash chromatography with EtOAc/hexanes to
afford pyrrolidine 19 as a white solid (0.66 mg, 1.3 mmol, 53%):
Rf =0.45 (1:1 EtOAc/hexanes); mp: 207–2088C; 1H NMR (600 MHz,
CDCl3): d=7.46–7.43 (m, 3H), 7.36–7.35 (m, 3H), 7.23–7.20 (m, 1H),
6.83–6.81 (m, 1H), 6.77 (d, J=7.7 Hz, 1H), 6.32 (d, J=6.8 Hz, 1H),
4.80 (d, J=14.1 Hz, 1H), 4.63 (d, J=14.1 Hz, 1H), 4.22–4.12 (m, 1H),
3.80–3.78 (m, 1H), 3.60–3.58 (m, 1H), 3.41 (d, J=7.9 Hz, 1H), 2.79–
2.45 (m, 2H), 2.27–2.18 (m, 2H), 1.46 ppm (s, 9H); 13C NMR
(125 MHz, [D6]DMSO, 310 K): d=180.4, 175.8, 174.7, 156.1, 140.5,
135.8, 129.7, 129.2, 128.9, 128.8, 128.2, 126.5, 122.6, 109.9, 68.1,
58.3, 51.6, 48.1, 42.6, 40.1, 31.1, 30.0, 28.5, 24.6 ppm; IR (thin film):
n˜ =3679, 2971, 2864, 1706, 1642, 1054, 1032, 1012 cmÀ1; HRMS
(ESI): m/z calculated for C28H30N4O5Na [M+Na]+ 525.2114, found
525.2106; HPLC purity: 100%, tR =22.09 min.
(Æ)-tert-Butyl (3R,3a’R,3b’S,9a’S)-6-chloro-2’-(4-fluorophenyl)-7-
methyl-1’,2,3’-trioxo-2’,3’,3a’,3b’,4’,6’,7’,9a’-octahydrospiro[indo-
line-3,9’-pyrrolo[3’,4’:3,4]pyrrolo[1,2-a]pyrazine]-5’(1’H)-carboxyl-
ate (15): Using the general procedure for the synthesis of pyrroli-
dines outlined above, 6-chloro-7-methylindoline-2,3-dione (0.12 g,
0.63 mmol, 1.0 equiv) was used, and the product was purified by
flash chromatography with EtOAc/hexanes to afford pyrrolidine 15
as a white solid (0.17 mg, 0.31 mmol, 49%): Rf =0.35 (4:6 EtOAc/
hexanes); mp: 246–2488C; 1H NMR (600 MHz, [D6]DMSO, 400 K):
d=10.42 (s, 1H), 7.39–7.35 (m, 2H), 7.32–7.29 (m, 2H), 7.02 (d, J=
7.8 Hz, 2H), 6.75 (d, J=7.8 Hz, 1H), 4.35 (dd, J=12.6, 1.8 Hz, 1H),
3.89–3.83 (m, 2H), 3.74–3.71 (m, 1H), 3.56 (d, J=7.8 Hz, 1H), 2.74–
2.58 (m, 2H) 2.77–2.73 (m, 1H), 2.74–2.54 (m, 1H), 2.50 (s, 3H),
2.29–2.09 (M, 1H), 1.44 ppm (s, 9H); 13C NMR (125 MHz, [D6]DMSO,
313 K): d=178.5, 175.3, 173.9, 161.0, 154.2, 143.3, 135.1, 129.7,
128.8, 125.1, 123.7, 122.6, 117.9, 116.6, 116.4, 79.6, 72.2, 58.1, 51.0,
46.6, 45.6, 45.9, 28.5, 14.3 ppm; IR (thin film): n˜ =3850, 3708, 2965,
2866, 1706, 1689, 1032 cmÀ1; HRMS (ESI): m/z calculated for
C28H28ClFN4O5Na [M+Na]+ 577.1630, found 577.1644; HPLC purity:
100%, tR =22.20 min.
(Æ)-tert-Butyl (3R,3a’R,3b’S,9a’S)-2’-benzyl-1’,2,3’-trioxo-2’,3’,3a’,
3b’,4’,6’,7’,9a’-octahydrospiro[indoline-3,9’-pyrrolo[3’,4’:3,4]-
pyrrolo[1,2-a]pyrazine]-5’(1’H)-carboxylate (20): Using the general
procedure for the synthesis of pyrrolidines outlined above, 1-ben-
zylindoline-2,3-dione (0.30 g, 1.3 mmol, 1.0 equiv) was used, and
the product was purified by flash chromatography with EtOAc/hex-
anes to afford pyrrolidine 20 as a yellow solid (0.43 mg, 0.75 mmol,
71%): Rf =0.75 (1:1 EtOAc/hexanes); mp: 210–2128C; 1H NMR
(600 MHz, [D6]DMSO, 400 K): d=7.54–7.51 (m, 2H), 7.46–7.44 (m,
1H), 7.35–7.34 (m, 6H), 7.29–7.26 (m, 2H), 7.03–7.02 (m, 2H), 6.92
(d, J=8.4 Hz, 1H), 4.91 (s, 2H), 4.40–4.38 (m, 1H), 3.92–3.89 (m,
2H), 3.81–3.77 (m, 1H), 3.61 (d, J=7.8 Hz, 1H), 2.84–2.77 (m, 1H),
2.68–2.63 (m, 1H), 2.24–2.16 (m, 2H), 1.45 ppm (s, 9H); 13C NMR
(125 MHz, [D6]DMSO, 310 K): d=176.7, 175.7, 174.3, 155.2, 144.2,
137.1, 133.1, 130.7, 130.0, 129.7, 129.5, 128.4, 128.1, 127.9, 126.9,
124.8, 123.4, 110.2, 80.1, 72.4, 58.8, 51.7, 47.0, 46.8, 43.6, 28.9 ppm;
IR (thin film): n˜ =3850, 3671, 2972, 2843, 1712, 1690, 1135,
(Æ)-tert-Butyl (3R,3a’R,3b’S,9a’S)-2’-(3,5-bis(trifluoromethyl)phen-
yl)-6-chloro-7-methyl-1’,2,3’-trioxo-2’,3’,3a’,3b’,4’,6’,7’,9a’-octahy-
drospiro[indoline-3,9’-pyrrolo[3’,4’:3,4]pyrrolo[1,2-a]pyrazine]-
5’(1’H)-carboxylate (16): Using the general procedure for the syn-
thesis of pyrrolidines outlined above, 6-chloro-7-methylindoline-
2,3-dione (0.50 g, 2.6 mmol, 1.0 equiv) was used, and the product
was purified by flash chromatography with EtOAc/hexanes to
afford pyrrolidine 16 as a white solid (0.13 mg, 0.30 mmol, 12%):
Rf =0.5 (2:3 EtOAc/hexanes); mp: 199–2018C; 1H NMR (600 MHz,
CDCl3, 320 K): d=8.51 (s, 1H), 7.93–7.92 (m, 3H), 7.12 (d, J=8.1 Hz,
1H), 6.70 (d, J=8.0 Hz, 1H), 4.59 (brs, 1H), 4.11 (brs, 1H), 3.80–3.82
(m, 1H), 3.81–3.78 (m, 1H), 3.74–3.72 (m, 1H), 2.82–2.89 (m, 1H),
2.65–2.71 (m, 1H), 2.33–2.34 (m, 2H), 2.17 (s, 3H), 1.47 ppm (s, 9H);
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1032 cmÀ1; HRMS (ESI): m/z calculated for C34H34N4O5Na [M+Na]+
601.2427, found 601.2415; HPLC purity: 100%, tR =23.46 min.
Synthesis of (+)-(3S,3a’S,7’R,8a’S,8b’R)-6-chloro-7-methyl-1’,2,3’-
trioxo-2’-phenyl-2’,3’,3a’,6’,7’,8’,8a’,8b’-octahydro-1’H-spiro[indo-
line-3,4’-pyrrolo[3,4-a]pyrrolizin]-7’-yl benzoate (25a) and (À)-
(3R,3a’R,7’R,8a’R,8b’S)-6-chloro-7-methyl-1’,2,3’-trioxo-2’-phenyl-
2’,3’,3a’,6’,7’,8’,8a’,8b’-octahydro-1’H-spiro[indoline-3,4’-pyrrolo-
[3,4-a]pyrrolizin]-7’-yl benzoate (25b): Using the general proce-
dure for the synthesis of pyrrolidines outlined above, 6-chloro-7-
methylindoline-2,3-dione (0.50 g, 2.6 mmol, 1.0 equiv) was used.
The resulting crude reaction mixture was dissolved in cold CH2Cl2,
and the products precipitated as a yellow solid. The solid was then
collected by vacuum filtration and washed with cold CH2Cl2 until
the solid became white. The pyrrolidine products were collected as
a mixture of two diastereomers. The resulting products were
highly insoluble in most of the organic solvents, and this made it
very hard to separate the two diastereomers by flash column chro-
matography.
(Æ)-tert-Butyl (3R,3a’R,3b’S,9a’S)-2’-benzyl-5-methoxy-1’,2,3’-tri-
oxo-2’,3’,3a’,3b’,4’,6’,7’,9a’-octahydrospiro[indoline-3,9’-pyrrolo-
[3’,4’:3,4]pyrrolo[1,2-a]pyrazine]-5’(1’H)-carboxylate (21): Using
the general procedure for the synthesis of pyrrolidines outlined
above, indoline-2,3-dione (0.45 g, 2.5 mmol, 1.0 equiv) was used,
and the product was purified by flash chromatography with
EtOAc/hexanes to afford pyrrolidine 21 as a white solid (0.66 mg,
1.3 mmol, 53%): Rf =0.35 (1:1 EtOAc/hexanes); mp: 218–2208C;
1H NMR (600 MHz, [D6]DMSO): d=10.43 (s, 1H), 7.38–7.36 (m, 2H),
7.33–7.32 (m, 2H), 7.29–7.27 (m, 1H), 6.77 (dd, J=8.7, 2.1 Hz, 1H),
6.73–6.71 (m, 1H), 6.07 (s, 1H), 4.64 (AB q, J=15 Hz, 2H), 4.31 (brs,
1H), 3.87–3.83 (m, 1H), 3.75–3.73 (m, 1H), 3.68–3.64 (m, 1H), 3.45
(d, J=7.8 Hz, 1H), 2.61–2.59 (m, 2H), 2.18–2.16 (m, 1H), 2.04 (td,
J=11.2, 3.0 Hz, 1H), 1.40 ppm (s, 9H); 13C NMR (125 MHz,
[D6]DMSO, 310 K): d=177.7, 176.1, 174.7, 154.9, 153.8, 136.4, 129.1,
127.9, 127.5, 126.0, 115.4, 113.4, 110.5, 79.7, 72.3, 61.2, 57.9, 55.5,
50.6, 46.3, 45.2, 42.0, 28.5 ppm; IR (thin film): n˜ =3850, 3648, 2967,
1710, 1641, 1130, 1032 cmÀ1; HRMS (ESI): m/z calculated for
C28H30N4O5Na [M+Na]+ 555.2222, found 555.2239; HPLC purity:
96.22%, tR =23.24 min.
The mixture of two stereoisomers (0.030 g, 0.07 mmol, 1 equiv)
from the above procedure was added to a 5 mL oven-dried round-
bottom flask. The flask was fitted with a septum and purged with
nitrogen. Next, 1.4 mL of DMF was added to the reaction flask
such that the concentration of the mixture of two diastereomers
was 0.05m. Next, benzoyl chloride (9.0 mL, 0.075 mmol, 1.1 equiv)
and Et3N (0.01 mL, 0.08 mmol, 1.2 equiv) were added to the reac-
tion flask, and the reaction was allowed to stir at room tempera-
ture until the consumption of the alcohols was confirmed by TLC
(9:1 CH2Cl2/MeOH). Upon consumption of the alcohols, the reac-
tion mixture was concentrated under vacuum to give a yellow
solid. The solid was then purified by flash chromatography with
CH2Cl2/Et2O (20:1) to afford esters 25a and 25b.
(Æ)-tert-Butyl (3R,3a’R,3b’S,9a’S)-7-methyl-1’,2,3’-trioxo-2’-phe-
nyl-2’,3’,3a’,3b’,4’,6’,7’,9a’-octahydrospiro[indoline-3,9’-pyrrolo-
[3’,4’:3,4]pyrrolo[1,2-a]pyrazine]-5’(1’H)-carboxylate (22): Using
the general procedure for the synthesis of pyrrolidines outlined
above, 7-methylindoline-2,3-dione (1.0 g, 6.2 mmol, 1.0 equiv) was
used, and the product was purified by flash chromatography with
EtOAc/hexanes to afford pyrrolidine 22 as a white solid (2.0 g,
4.0 mmol, 60%): Rf =0.3 (1:1 EtOAc/hexanes); mp: 212–2148C;
1H NMR (600 MHz, [D6]DMSO, 398 K): d=10.1 (s, 1H), 7.53–7.51 (m,
2H), 7.45–7.42 (m, 1H), 7.34-7.33 (m, 2H), 7.05 (d, J=7.8 Hz, 1H),
6.88–6.86 (m, 1H), 6.76 (d, J=7.2 Hz, 1H), 4.37–4.35 (m, 1H), 3.87–
3.82 (m, 2H), 3.75–3.73 (m, 1H), 3.55–3.54 (m, 1H), 2.79–2.75 (m,
1H), 2.65–2.61 (m, 1H), 2.31–2.22 (m, 4H), 2.21–2.19 (m, 1H),
1.44 ppm (s, 9H); 13C NMR (125 MHz, [D6]DMSO): d=178.6, 175.5,
174.1, 154.5, 141.8, 132.7, 131.5, 129.6, 129.0, 127.5, 124.8, 123.9,
122.2, 119.5, 79.6, 72.3, 58.1, 50.9, 49.1, 46.7, 28.5, 16.7, 14.6 ppm;
The first stereoisomer 25a (0.012 g, 0.02 mmol, 65%) was isolated
as a white solid. Rf =0.55 (20:1 CH2Cl2/Et2O); mp: 236–2388C;
½aŠ20 =54.5 (c=0.13 in MeOH); H NMR (600 MHz, [D6]acetone): d=
1
D
9.67 (s, 1H), 7.97 (dd, J=8.4, 1.2 Hz, 2H), 7.61–7.58 (m, 1H), 7.51
(d, J=7.8 Hz, 1H), 7.48–7.37 (m, 5H), 7.28 (d, J=7.8 Hz, 2H), 7.16
(d, J=7.8 Hz, 1H), 5.63–5.61 (m, 1H), 4.71–4.69 (m, 1H), 4.35 (d, J=
9.6 Hz, 1H), 3.82 (dd, J=10.2, 6.6 Hz, 1H), 3.52 (dd, J=11.1, 5.1 Hz,
1H), 3.01 (d, J=10.8 Hz, 1H), 2.76–2.72 (m, 1H), 2.56–2.52 (m, 1H),
2.33 ppm (s, 3H); 13C NMR (125 MHz, [D6]acetone): d=177.4, 176.6,
174.9, 165.6, 135.6, 133.0, 130.2, 129.3, 128.6, 128.4, 128.1, 127.2,
125.6, 124.4, 122.4, 118.1, 74.7, 65.1, 55.3, 53.4, 53.1, 37.7,
13.3 ppm; IR (thin film): n˜ =3685, 2978, 2851, 1721, 1054, 1032,
1012 cmÀ1; HRMS (ESI): m/z calculated for C30H24ClN3O5Na [M+
Na]+ 564.1302, found 564.1302; HPLC purity: 96.63%, tR =
21.95 min.
IR (thin film): n˜ =3229, 2973, 2360, 2340, 1714, 1696, 1391 cmÀ1
;
HRMS (ESI): m/z calculated for C28H30N4O5Na [M+Na]+ 525.2114,
found 525.2108; HPLC purity: 99.18%, tR =20.85 min.
(Æ)-tert-Butyl
(3R,3a’R,3b’S,9a’S)-6,7-dimethyl-1’,2,3’-trioxo-2’-
phenyl-2’,3’,3a’,3b’,4’,6’,7’,9a’-octahydrospiro[indoline-3,9’-pyrro-
lo[3’,4’:3,4]pyrrolo[1,2-a]pyrazine]-5’(1’H)-carboxylate (23): Using
the general procedure for the synthesis of pyrrolidines outlined
above, 6,7-dimethylindoline-2,3-dione (0.38 g, 2.2 mmol, 1.0 equiv)
was used, and the product was purified by flash chromatography
with EtOAc/hexanes to afford pyrrolidine 23 as a white solid
(0.60 g, 1.20 mmol, 61%): Rf =0.3 (1:1 EtOAc/hexanes); mp: 215–
2178C; 1H NMR (500 MHz, [D6]DMSO): d=10.6 (s, 1H), 7.53–7.52
(m, 2H), 7.50–7.44 (m, 1H), 7.29–7.28 (m, 2H), 6.77 (d, J=7.5 Hz,
1H), 6.60 (d, J=7.5 Hz, 1H), 4.29 (brs, 1H), 3.82–3.79 (m, 2H), 3.64–
3.65 (m, 1H), 3.47–3.46 (m, 1H), 2.62 (brs, 2H), 2.19–2.17 (m, 4H),
2.09–2.07 (m, 4H), 1.39 ppm (s, 9H); 13C NMR (125 MHz, [D6]DMSO,
320 K): d=178.8, 175.5, 174.1, 141.8, 138.7, 132.7, 129.6, 129.0,
127.4, 123.6, 123.5, 122.4, 118.2, 79.6, 72.4, 58.1, 50.9, 46.7, 28.5,
20.1, 13.5 ppm; IR (thin film): n˜ =3739, 3244, 2976, 1712, 1696,
1417, 1390 cmÀ1; HRMS (ESI): m/z calculated for C29H32N4O5Na [M+
Na]+ 539.2271, found 539.2268; HPLC purity: 98.24%, tR =
21.47 min.
The second stereoisomer 25b (0.015 g, 0.03 mmol, 81%) was also
isolated as a white solid. Rf =0.48 (20:1 CH2Cl2/Et2O); mp: 221–
2238C; ½aŠ20 =À80.2 (c=0.23 in MeOH); 1H NMR (600 MHz,
D
[D6]acetone): d=9.69 (s, 1H), 8.09 (dd, J=8.4, 1.2 Hz, 2H), 7.67 (t,
J=7.5 Hz, 1H), 7.54 (t, J=7.8 Hz, 1H), 7.48–7.37 (m, 3H), 7.28 (d,
J=7.8 Hz, 2H), 7.13 (d, J=8.4 Hz, 1H), 5.44–5.42 (m, 1H), 4.55–4.52
(m, 1H), 4.39 (d, J=10.2 Hz, 1H), 4.00 (dd, J=9.9, 6.9 Hz, 1H), 3.38
(dd, J=10.2, 6.0 Hz, 1H), 3.28 (dd, J=9.9, 6.3 Hz, 1H), 2.83–2.78 (m,
1H), 2.39–2.36 (m, 1H), 2.35 ppm (s, 3H); 13C NMR (125 MHz,
[D6]acetone): d=177.4, 176.5, 174.8, 165.6, 135.6, 133.3, 133.0,
130.1, 129.5, 128.6, 128.2, 127.3, 125.5, 125.2, 124.6, 122.4, 74.7,
64.9, 54.4, 53.5, 53.3, 36.6, 13.3 ppm; IR (thin film): n˜ =3686, 2980,
2481, 1720, 1050, 1030, 1014 cmÀ1; HRMS (ESI): m/z calculated for
C30H24ClN3O5Na [M+Na]+ 564.1302, found 564.1305; HPLC purity:
95.36%, tR =21.68 min.
Synthesis of (Æ)-tert-butyl (3R,3a’R,3b’S,9a’S)-1-benzyl-6-chloro-
7-methyl-1’,2,3’-trioxo-2’-phenyl-2’,3’,3a’,3b’,4’,6’,7’,9a’-octahy-
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drospiro[indoline-3,9’-pyrrolo[3’,4’:3,4]pyrrolo[1,2-a]pyrazine]-
5’(1’H)-carboxylate (17):[53] A 5 mL oven-dried round-bottom flask
was charged with NaH (60 wt% in mineral oil, 0.01 g, 0.37 mmol,
1.1 equiv) and the mineral oil dispersed in NaH was removed by
washing with hexanes (3”2 mL). The resulting white solid of NaH
was suspended in 0.2 mL of DMF. The suspension was cooled to
08C in an ice bath, after which pyrrolidine 14 (0.18 g, 0.34 mmol,
1.0 equiv) was added as a solid over the course of 15 min. After
the addition was complete the ice bath was removed, and the so-
lution was stirred for 1 h at room temperature. Next, benzyl bro-
mide (48 mL, 0.40 mmol, 1.2 equiv) was added to the reaction flask,
and the reaction was allowed to stir at room temperature until the
consumption of 15 was confirmed by TLC (2:3 EtOAc/hexanes).
Upon consumption of 14, the reaction mixture was concentrated
under vacuum to give a yellow solid. The solid was then purified
by flash chromatography with EtOAc/hexanes (4:1) to afford pyrro-
lidine 17 as a white solid (0.17 g, 0.27 mmol, 82%): Rf =0.65 (2:3
EtOAc/hexanes); mp: 145–1468C; 1H NMR (600 MHz, [D6]DMSO,
400 K): d=7.51–7.48 (m, 2H), 7.42–7.41 (m, 1H), 7.34–7.31 (m, 4H),
7.26–7.24 (m, 1H), 7.18–7.17 (m, 2H), 7.13 (d, J=8.1 Hz, 1H), 6.86
(d, J=8.1 Hz, 1H), 5.21–5.16 (m, 2H), 4.37–4.35 (m, 1H), 3.87–3.86
(m, 2H), 3.74–3.72 (m, 1H), 3.60 (d, J=8.1 Hz, 1H), 2.80–2.78 (m,
1H), 2.67–2.65 (m, 1H), 2.34–2.32 (m, 1H), 2.27 (s, 3H), 2.19–2.17
(m, 1H), 1.42 ppm (s, 9H); 13C NMR (125 MHz, [D6]DMSO, 310 K):
d=177.6, 175.2, 173.8, 154.2, 143.4, 137.8, 136.6, 132.6, 129.6,
129.5, 129.0, 127.7, 127.5, 125.8, 125.1, 124.5, 124.0, 118.6, 79.7,
71.0, 63.9, 58.6, 51.9, 46.5, 45.8, 45.1, 30.7, 28.5, 14.7 ppm; IR (thin
film): n˜ =3680, 2971, 2843, 1713, 1054, 1032, 1012 cmÀ1; HRMS
(ESI): m/z calculated for C35H35ClN4O5Na [M+Na]+ 649.2194, found
649.2194; HPLC purity: 100%, tR =23.13 min.
spiro[indoline-3,9’-pyrrolo[3’,4’:3,4]pyrrolo[1,2-a]pyrazin]-5’(1’H)-
yl)-10-oxodecanoate (27):[80] A 15 mL round-bottom flask was
charged with pyrrolidine 26 (0.13 g, 0.30 mmol, 1 equiv), Na2CO3
(0.03 g, 0.30 mmol, 1.0 equiv) and a stir bar. The flask was fitted
with a septum and purged with nitrogen. Then CH2Cl2 (6 mL) was
added to the reaction flask such that the concentration of 26 was
0.05m. Next, methyl 10-chloro-10-oxodecanoate (0.07 mL,
0.31 mmol, 1.04 equiv) was added dropwise to the reaction mix-
ture by syringe. The reaction was allowed to stir at room tempera-
ture until the consumption of 26 was confirmed by TLC (20:80:5
EtOAc/hexanes/Et3N). Upon consumption of 26, the reaction mix-
ture was concentrated under vacuum to give a yellow oil. The oil
was then purified by flash chromatography with EtOAc/hexanes/
Et3N (80:20:5) to afford pyrrolidine 27 as a yellow solid (0.10 g,
0.16 mmol, 53%): Rf =0.75 (20:80:5 EtOAc/hexanes/Et3N); mp: 110–
1
111 8C; H NMR (600 MHz, [D6]DMSO, 400 K): d=10.42 (s, 1H), 7.53–
7.51 (m, 2H), 7.45–7.43 (m, 1H), 7.33 (d, J=7.8 Hz, 2H), 7.03 (d, J=
8.4 Hz, 1H), 6.78 (d, J=7.8 Hz, 1H), 4.58 (brs, 1H), 4.08 (brs, 1H),
3.86 (t, J=7.8 Hz, 1H), 3.73–3.72 (m, 1H), 3.61–3.58 (m, 4H), 2.61–
2.82 (m, 4H), 2.34–2.27 (m, 5H), 2.22–2.18 (m, 1H), 1.59–1.53 (m,
4H), 1.22–1.42 ppm (m, 9H); 13C NMR (125 MHz, CDCl3, 313 K): d=
178.2, 174.6, 174.3, 172.2, 171.7, 141.8, 136.6, 131.7, 129.4, 128.9,
126.3, 124.4, 123.5, 122.3, 118.7, 72.7, 58.7, 51.4, 50.4, 48.7, 46.2,
45.3, 44.5, 41.1, 34.1, 33.4, 29.7, 29.3, 29.2, 25.3, 24.9, 13.6 ppm; IR
(thin film): n˜ =3817, 3671, 2921, 2863, 1710, 1617, 1054, 1032 cmÀ1
;
HRMS (ESI): m/z calculated for C34H39ClN4O6Na [M+Na]+ 657.2456,
found 657.2435; HPLC purity: 97.45%, tR =23.69 min.
Synthesis of (Æ)-(3R,3a’R,3b’S,9a’S)-6-chloro-5’-((S)-2-methoxy-2-
phenylacetyl)-7-methyl-2’-phenyl-3a’,4’,5’,6’,7’,9a’-hexahydrospir-
o[indoline-3,9’-pyrrolo[3’,4’:3,4]pyrrolo[1,2-a]pyrazine]-
1’,2,3’(2’H,3b’H)-trione (28):[81] A 15 mL round-bottom flask was
charged with pyrrolidine 26 (0.20 g, 0.46 mmol, 1.0 equiv), (S)-2-
methoxy-2-phenylacetic acid (0.10 g, 0.60 mmol, 1.3 equiv), N-(3-di-
methylaminopropyl)-N’-ethylcarbodiimide hydrochloride (0.18 g,
0.96 mmol, 2.1 equiv), and a stir bar. The flask was fitted with
a septum and purged with nitrogen. Then CH2Cl2 (6 mL) was
added to the reaction flask such that the concentration of 26 was
0.07m. Next Et3N (0.22 mL, 1.6 mmol, 3.5 equiv) was added to the
reaction mixture, and the reaction was allowed to stir at room tem-
perature until the consumption of 26 was confirmed by TLC
(20:80:5 EtOAc/hexanes/Et3N). Upon consumption of 26, the reac-
tion mixture was quenched with 5 mL of 1n aqueous HCl, and the
aqueous layer was then extracted with CH2Cl2 (3”5 mL). The com-
bined organic layers were then washed with brine and dried over
Na2SO4. The resulting organic solution was concentrated under
vacuum to give a yellow oil. The oil was then purified by flash
chromatography with EtOAc/hexanes/Et3N (80:20:5) to afford pyr-
rolidine 28 as a white solid (0.12 g, 0.21 mmol, 45%): Rf =0.65 (1:1
EtOAc/hexanes); mp: 127–1298C; 1H NMR (600 MHz, [D6]DMSO,
400 K): d=10.42 (s, 1H), 7.51–7.49 (m, 2H), 7.44–7.41 (m, 1H),
7.39–7.35 (m, 4H), 7.32–7.31 (m, 3H), 7.01 (d, J=8.0 Hz, 1H), 6.74
(d, J=8.0 Hz, 1H), 5.17–5.16 (m, 1H), 4.71–4.70 (m, 1H), 4.19–4.17
(m, 1H), 3.79–3.75 (m, 1H), 3.64–3.60 (m, 1H), 3.54 (appdd, J=8.0,
2.9 Hz, 1H), 3.39–3.37 (m, 3H), 2.73–2.64 (m, 2H), 2.27–2.22 (m,
4H), 2.07–2.05 ppm (m, 1H); 13C NMR (125 MHz, [D6]DMSO, 310 K):
(two rotamers observed) d=178.5, 175.3, 173.9, 154.9, 137.4, 135.2,
132.6, 129.5, 129.0, 128.9, 128.6, 127.6, 127.5, 127.3, 125.2, 123.6,
122.6, 117.8, 81.8, 81.0, 72.1, 68.9, 63.9, 58.6, 58.2, 57.3, 56.3, 50.9,
46.5, 45.3, 44.5, 41.7, 32.5, 30.6, 30.1, 21.8, 19.0, 17.1 ppm; IR (thin
film): n˜ =3850, 3708, 2971, 2921, 1712, 1643, 1054, 1032,
1012 cmÀ1; HRMS (ESI): m/z calculated for C32H29ClN4O5Na [M+
Na]+ 607.1724, found 607.1736; HPLC purity: 97.04%, tR =
20.02 min.
Synthesis of (Æ)-(3R,3a’R,3b’S,9a’S)-6-chloro-7-methyl-2’-phenyl-
3a’,4’,5’,6’,7’,9a’-hexahydrospiro[indoline-3,9’-pyrrolo[3’,4’:3,4]-
pyrolo[1,2-a]pyrazine]-1’,2,3’(2’H,3b’H)-trione (26):[79]
A 10 mL
round-bottom flask was charged with pyrrolidine 14 (0.57 g,
1.1 mmol, 1 equiv) and a stir bar. The flask was fitted with
a septum and purged with nitrogen. Next, 3.1 mL CH2Cl2 was
added to the reaction flask such that the concentration of 14 was
0.34m and stirred for 10 min. A pink solution resulted. Then 3.1 mL
trifluoroacetic acid was slowly added to the reaction mixture by sy-
ringe. A dark-brown solution resulted. The deprotection reaction
was stirred until TLC indicated complete consumption of pyrroli-
dine 14 (15 min). Upon consumption of 14, the reaction mixture
was concentrated under vacuum to give a brown oil. Saturated
aqueous NaHCO3 (5 mL) was added to the oil, and the aqueous
layer was then extracted with CH2Cl2 (3”5 mL). The combined or-
ganic layers were then washed with brine and dried over Na2SO4.
The resulting organic solution was concentrated under vacuum to
afford 26 as a pink solid (0.42 g, 0.96 mmol, 87%): Rf =0.81
1
(20:80:5 EtOAc/hexanes/Et3N); mp: 196–1998C; H NMR (600 MHz,
[D6]DMSO): d=10.89 (s, 1H), 7.58–7.53 (m, 2H), 7.47–7.45 (m, 1H),
7.30 (d, J=7.4 Hz, 2H), 7.06 (d, J=8.0 Hz, 1H), 6.71 (d, J=8.0 Hz,
1H), 3.77–3.76 (m, 2H), 3.51–3.50 (m, 1H), 3.42 (brs, 1H), 3.30 (d,
J=11.9 Hz, 1H), 2.79 (appd, J=12.1 Hz, 1H), 2.43–2.41 (m, 1H),
2.25 (s, 3H), 2.24–2.21 ppm (m, 2H); 13C NMR (125 MHz, CDCl3,
310 K): d=178.8, 175.5, 174.2, 143.4, 134.9, 132.7, 129.6, 128.9,
127.4, 125.2, 124.1, 122.5, 117.7, 72.6, 58.7, 50.7, 48.7, 47.1, 46.7,
44.9, 14.3 ppm; IR (thin film): n˜ =3850, 3671, 2971, 2864, 1709,
1054, 1032, 1013 cmÀ1
;
HRMS (ESI): m/z calculated for
C23H21ClN4O3Na [M+Na]+ 459.1200, found 459.1190; HPLC purity:
100%, tR =17.25 min.
Synthesis of (Æ)-methyl 10-((3R,3a’R,3b’S,9a’S)-6-chloro-7-
methyl-1’,2,3’-trioxo-2’-phenyl-2’,3’,3a’,3b’,4’,6’,7’,9a’-octahydro-
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Synthesis of (Æ)-(3R,3a’R,3b’S,9a’S)-6-chloro-7-methyl-2’-phenyl-
5’-(3-phenylpropanoyl)-3a’,4’,5’,6’,7’,9a’-hexahydrospiro[indoline-
3,9’-pyrrolo[3’,4’:3,4]pyrrolo[1,2-a]pyrazine]-1’,2,3’(2’H,3b’H)-
trione (29): A 10 mL round-bottom flask was charged with pyrroli-
dine 26 (0.05 g, 0.11 mmol, 1.0 equiv), Et3N (18.0 mL, 0.13 mmol,
1.1 equiv), and a stir bar. The flask was fitted with a septum and
purged with nitrogen. Then CH2Cl2 (2.0 mL) was added to the
round-bottom flask by syringe. Hydrocinnamoyl chloride (18.0 mL,
0.12 mmol, 1.05 equiv) was then added to the reaction flask with
a syringe. The reaction mixture was allowed to stir for 3 h at room
temperature. The reaction was monitored for consumption of 26
by TLC (100:5 EtOAc/Et3N). Upon consumption of 26, the reaction
mixture was quenched with 5 mL of saturated aqueous NaHCO3,
and the aqueous layer was then extracted with CH2Cl2 (3”5 mL).
The combined organic layers were then washed with brine and
dried over Na2SO4. The resulting organic solution was concentrated
under vacuum to give a yellow oil. The oil was then purified by
flash chromatography with EtOAc/hexanes/Et3N (60:40:5) to afford
pyrrolidine 29 as a white solid (0.04 g, 0.07 mmol, 64%): Rf =0.2
Synthesis of (Æ)-benzyl (3R,3a’R,3b’S,9a’S)-6-chloro-7-methyl-
1’,2,3’-trioxo-2’-phenyl-2’,3’,3a’,3b’,4’,6’,7’,9a’-octahydrospiro[in-
doline-3,9’-pyrrolo[3’,4’:3,4]pyrrolo[1,2-a]pyrazine]-5’(1’H)-car-
boxylate (31):[82] A 5 mL round-bottom flask was charged with pyr-
rolidine 26 (0.20 g, 0.45 mmol, 1.0 equiv), N,N-diisopropylethyla-
mine (0.17 mL, 0.96 mmol, 2.1 equiv), and a stir bar. The flask was
fitted with a septum and purged with nitrogen. Then CH2Cl2
(0.92 mL) was added to the round-bottom flask by syringe. The re-
action mixture was cooled to 08C with an ice bath. Meanwhile an-
other 5 mL pear-shaped flask was charged with benzyl chlorofor-
mate (0.07 mL, 0.50 mmol, 1.1 equiv), and the flask was fitted with
a septum and purged with nitrogen. Then CH2Cl2 (0.34 mL) was
added to the pear-shaped vial containing the benzyl chloroformate
and stirred for 10 min. Next the contents of the 5 mL pear-shaped
vial were slowly added to the 5 mL round-bottom flask containing
26. The ice bath was removed, and the reaction mixture was al-
lowed to stir for 2.5 h at room temperature. The reaction was
monitored for consumption of 26 by TLC (50:50:5 EtOAc/Hex/
Et3N). Upon consumption of 26, the reaction mixture was
quenched with 5 mL of saturated aqueous NaHCO3, and the aque-
ous layer was then extracted with CH2Cl2 (3”5 mL). The combined
organic layers were then washed with brine and dried over Na2SO4.
The resulting organic solution was concentrated under vacuum to
give a yellow oil. The oil was then purified by flash chromatogra-
phy with EtOAc/hexanes/Et3N (60:40:5) to afford pyrrolidine 31 as
a white solid (0.14 g, 0.25 mmol, 54%): Rf =0.81 (1:1 EtOAc/hex-
anes); mp: 209–2118C; 1H NMR (600 MHz, [D6]DMSO, 400 K): d=
10.40 (s, 1H), 7.53–7.51 (m, 2H), 7.45–7.42 (m, 1H), 7.37–7.35 (m,
6H), 7.32–7.31 (m, 1H), 7.03 (d, J=8.0 Hz, 1H), 6.80 (d, J=8.0 Hz,
1H), 5.16 (s, 2H), 4.48 (d, J=12.7 Hz, 1H), 3.99 (d, J=13.1 Hz, 1H),
3.89–3.87 (m, 1H), 3.82–3.79 (m, 1H), 3.61 (d, J=8.0 Hz, 1H), 2.91–
2.86 (m, 2H), 2.78–2.75 (m, 1H), 2.34–2.25 ppm (m, 5H); 13C NMR
(125 MHz, [D6]DMSO): d=178.5, 175.3, 173.9, 154.9, 143.4, 137.3,
135.2, 132.6, 129.5, 129.0, 128.9, 128.3, 128.1, 127.4, 72.3, 66.9, 58.2,
51.0, 47.2, 43.5, 30.9, 21.1, 14.3 ppm; IR (thin film): n˜ =3850, 3678,
3262, 2966, 2864, 1715, 1694, 1032 cmÀ1; HRMS (ESI): m/z calculat-
ed for C31H27ClN4O5Na [M+Na]+ 593.1567, found 593.1584; HPLC
purity: 97.60%, tR =21.96 min.
1
(60:40:5 EtOAc/hexanes/Et3N); mp: 236–2398C; H NMR (600 MHz,
[D6]DMSO, 400 K): d=10.40 (s, 1H), 7.53–7.51 (m, 2H), 7.45–7.42
(m, 1H), 7.34–7.33 (m, 2H), 7.27–7.23 (m, 4H), 7.17–7.15 (m, 1H),
7.03 (d, J=7.8 Hz, 1H), 6.78 (d, J=8.4 Hz, 1H), 4.59 (s, 1H), 7.09–
7.06 (m, 1H), 3.87–3.84 (m, 1H), 3.73–3.69 (m, 1H), 3.59–3.57 (m,
1H), 2.90–2.88 (m, 2H), 2.84–2.82 (m, 2H), 2.78–2.74 (m, 2H), 2.31–
2.28 (m, 4H), 2.19–2.15 ppm (m, 1H); 13C NMR (125 MHz,
[D6]DMSO, 310 K): d=178.6, 178.5, 175.4, 175.3, 174.0, 170.6, 143.4,
141.9, 135.2, 132.7, 129.6, 129.0, 128.8, 128.7, 127.5, 126.3, 125.2,
123.9, 122.6, 117.9, 72.2, 60.2, 58.9, 51.2, 48.5, 45.5, 44.9, 34.5, 31.2,
14.3 ppm; IR (thin film): n˜ =3423, 2917, 1708, 1633, 1620, 1384,
1204 cmÀ1; HRMS (ESI): m/z calculated for C32H29ClN4O4Na [M+
Na]+ 591.1775, found 591.1763; HPLC purity: 98.20%, tR =
21.15 min.
Synthesis of (3R,3a’R,3b’S,9a’S)-N-benzyl-6-chloro-7-methyl-
1’,2,3’-trioxo-2’-phenyl-2’,3’,3a’,3b’,4’,6’,7’,9a’-octahydrospiro[in-
doline-3,9’-pyrrolo[3’,4’:3,4]pyrrolo[1,2-a]pyrazine]-5’(1’H)-car-
boxamide (30): A 10 mL round-bottom flask was charged with pyr-
rolidine 26 (0.06 g, 0.15 mmol, 1.0 equiv), and a stir bar. The flask
was fitted with a septum and purged with nitrogen. Then CH2Cl2
(3.0 mL) was added to the round-bottom flask by syringe. Next,
(isocyanatomethyl)benzene (0.02 mL, 0.16 mmol, 1.1 equiv) was
added to the reaction flask by syringe. Then the reaction mixture
was allowed to stir for 4 h at room temperature. The reaction was
monitored for consumption of 26 by TLC (50:50:5 EtOAc/Hex/
Et3N). Upon consumption of 26, the resulting organic solution was
concentrated under vacuum to give a yellow oil. The oil was then
purified by flash chromatography with EtOAc/hexanes/Et3N
Biological evaluations
Strains, media, and compounds: The C. albicans strain HLY4123 was
used as the susceptible laboratory strain for the antifungal evalua-
tion in this study. HLY4123 carries a GFP reporter for ERG3 expres-
sion and was constructed by plasmid transformation of the com-
monly used laboratory C. albicans strain CAI4. Selected resistant
C. albicans strains with different mechanisms of becoming drug re-
sistant were obtained from Dr. David Rogers (Department of Clini-
cal Pharmacy, University of Tennessee Health Sciences Center,
Memphis, Tennessee, USA). The strains were cultured at 308C
under constant shaking (200 rpm) in synthetic complete (SC)
medium containing 2% glucose. The stock solution of fluconazole
(Sigma–Aldrich, USA) was prepared in sterile H2O (0.1 mgmLÀ1),
whereas the other test compounds were prepared in DMSO. The
commercial sample sold as CID 6584729 was obtained from Vitas-
M (supplier number STK 580951).
(60:40:5) to afford pyrrolidine 30 as
a white solid (0.63 g,
0.11 mmol, 75%): Rf =0.24 (50:50:5 EtOAc/Hex/Et3N); mp: 258–
2598C; 1H NMR (600 MHz, [D6]DMSO, 400 K): d=10.44 (s, 1H),
7.53–7.51 (m, 2H), 7.45–7.44 (m, 1H), 7.33–7.32 (m, 2H), 7.29–7.28
(m, 4H), 7.20 (m, 1H), 7.03 (d, J=8.0 Hz, 1H), 6.76 (d, J=8.0 Hz,
1H), 4.47–4.45 (m, 1H), 4.29–4.27 (m, 2H), 3.95 (d, J=14.1 Hz, 1H),
3.82–3.77 (m, 2H), 3.56 (d, J=7.9 Hz, 1H), 2.75–2.71 (m, 1H), 2.64–
2.58 (m, 1H), 2.28 (s, 3H), 2.25–2.23 ppm (m, 2H); 13C NMR
(125 MHz, [D6]DMSO): d=178.7, 175.4, 174.1, 157.6, 143.4, 141.4,
135.1, 132.7, 129.6, 129.1, 128.6, 127.5, 126.9, 125.1, 123.9, 122.6,
117.9, 72.3, 58.3, 51.1, 47.5, 46.7, 45.7, 44.1, 43.6 ppm; IR (thin film):
n˜ =3618, 2922, 2360, 2340, 1712, 1616, 1536, 1387 cmÀ1; HRMS
(ESI): m/z calculated for C31H28ClN5O4Na [M+Na]+ 592.1727, found
592.1724; HPLC purity: 97.18%, tR =19.94 min.
Dose–response curves for test compounds against C. albicans with
and without fluconazole: C. albicans was grown in SC medium over-
night and then diluted to an effective OD600 of 0.0625. Serial ten-
fold dilutions of the test compounds (0.15–1500 mm) were pre-
pared in DMSO in 1.5 mL Eppendorf tubes. To each well in columns
B–D (triplicate analysis) of a 24-well Palcon plate was added 2.5 mL
of fluconazole solution. To each well in all four columns of the
ChemMedChem 2015, 10, 1672 – 1686
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Full Papers
[1] Candidiasis (Invasive) (Eds.: S. G. Revankar, J. D. Sobel), Merck Manual,
2014; www.merckmanuals.com/professional/infectious-diseases/fungi/
candidiasis-invasive.
[2] A. Ahmadi, S. H. Ardehali, M. T. Beigmohammadi, M. Hajiabdolbaghi,
S. M. R. Hashemian, M. Kouchek, A. Majidpour, M. Mokhtari, O. M. Mog-
haddam, A. Najafi, R. Negat, M. Niakan, A. H. Lotfi, A. Amirsavadkouhi, F.
Shirazian, P. Tabarsi, M. T. Taher, M. Torabi-Nami, JRSM Open 2014, 5,
2042533313517689.
[3] C. A. Kauffman, UpToDate (Eds.: K. A. Marr, A. R. Thorner), Sep 23, 2014:
www.uptodate.com/contents/treatment-of-candidemia-and-invasive-
candidiasis-in-adults.
[4] J. H. Rex, J. E. Bennett, A. M. Sugar, P. G. Pappas, C. M. van der Horst,
J. E. Edwards, R. G. Washburn, W. M. Scheld, A. W. Karchmer, A. P. Dine,
N. Engl. J. Med. 1994, 331, 1325–1330.
plate was added 1 mL of cells in SC medium such that column A
served as a control to assess the EC50 value of the compound in
the absence of fluconazole. Then to each well in rows 2–5 was
added a solution of the compound in DMSO (2 mL each) such that
the final fluconazole concentration in columns 2–4 was
0.25 mgmLÀ1, and the concentration of compound in each row
varied from 0.003 to 30 mm. The plates were incubated in a rotary
shaker/incubator at 308C for 16 h. The contents of each well were
re-suspended with a micropipettor and a 20 mL aliquot was added
to a polystyrene cuvette and diluted with 680 mL deionized water.
The suspension was triturated again immediately before measuring
the absorbance at l=600 nm (OD600) for cell densities. EC50 values
were determined by fitting to a standard curve using the Excel-
based tool ED50PLUS v. 1.0 (Mario H. Vargas).
[5] P. Phillips, S. Shafran, G. Garber, C. Rotstein, F. Smaill, I. Fong, I. Salit, M.
Miller, K. Williams, J. M. Conly, J. Singer, S. Ioannou, Eur. J. Clin. Microbiol.
Infect. Dis. 1997, 16, 337–345.
Determination of MIC90 values by checkerboard assay: Checkerboard
assays were carried out using four 24-well plates. The results on
each plate were normalized by duplication of one row and one
column with a row and column on another plate. C. albicans was
grown in SC medium overnight and then diluted to an effective
OD600 of 0.0625. Serial tenfold dilutions of the test compounds
(150 mm) were prepared in DMSO in 1.5 mL Eppendorf tubes.
Serial twofold dilutions of fluconazole (6.53 mm) were prepared in
sterile H2O in 1.5 mL Eppendorf tubes. To each well was added
2 mL of a stock solution of compound in DMSO and 2.5 mL of
a stock solution of fluconazole in H2O. Concentrations of com-
pound in rows 1–11 varied from 300 mm to 0.0003 nm; the last
row 12 contained no compound. Concentrations of fluconazole in
each column B–H varied from 0.0625 to 2 mm; the first column A
contained no fluconazole. The plates were incubated in a rotary
shaker/incubator at 308C for 16 h. The contents of each well were
re-suspended with a micropipettor, and a 20 mL aliquot was added
to a polystyrene cuvette and diluted with 680 mL deionized H2O.
The suspension was triturated again immediately before measuring
the absorbance at l=600 nm. MIC90 values were determined as
the lowest drug concentrations (alone or in combination) that in-
hibited fungal growth by 90% compared with that of the drug-
free wells.
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Acknowledgements
This work was supported by the US National Institutes of Health
(NIH) R01 AI099190. We thank Nathan J. Oldenhuis (Department
of Chemistry, University of California, Irvine, USA) for carrying out
the NIH 3T3 cell cytotoxicity study. We are grateful to Drs. David
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Health Sciences Center, Memphis, Tennessee, USA) and Ted White
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