De novo design of non-coordinating indolones as potential inhibitors for lanosterol 14-α-demethylase (CYP51)

Article abstract of DOI:10.1248/cpb.c13-00003

The development of antifungal drugs that inhibit lanosterol 14-α-demethylase (CYP51) via non-covalent ligand interactions is a strategy that is gaining importance. A series of novel tetraindol-4-one derivatives with 1- and 2-(2,4-substituted phenyl) side chains were designed and synthesized based on the structure of CYP51 and fluconazole. The antifungal activities of these derivatives against eight human pathogenic filamentous fungi and yeast strains were evaluated in vitro by measuring the minimal inhibitory concentrations. Nearly all tested compounds 8a-g displayed activity against Candida tropicalis, Candida guilliermondii and Candida parapsilosis with a minimum inhibitory concentration (MIC) value until 8 μg mL^-1, on the other hand compounds 7a-g showed activity against Aspergillus fumigatus with a MIC value of 31.25 μg mL^-1. A molecular modeling study of the binding interactions between compounds 6, 7d, 8g and the active site of MtCYP51 was conducted based on the computational docking results.

Full text of DOI:10.1248/cpb.c13-00003

16
Regular Article
Chem. Pharm. Bull. 62(1) 16–24 (2014)
Vol. 62, No. 1
De Novo Design of Non-coordinating Indolones as Potential Inhibitors
for Lanosterol 14-α-Demethylase (CYP51)
Rodolfo González-Chávez,a,b Roberto Martínez,c María Eugenia Torre-Bouscoulet,b
,b
Marco Gallo,b and Marco Martín González-Chávez*
a Posgrado en Ciencias en Bioprocesos, Universidad Autónoma de San Luis Potosí; b Facultad de Ciencias Químicas,
Universidad Autónoma de San Luis Potosí; Av. Dr. Manuel Nava No. 6. Zona Universitaria, San Luis Potosí, S.L.P.
c
78210, México: and Instituto de Química, Universidad Nacional Autónoma de México; Circuito Exterior de Ciudad
Universitaria 04510, México D.F.
Received December 28, 2012; accepted September 26, 2013
The development of antifungal drugs that inhibit lanosterol 14-α-demethylase (CYP51) via non-covalent
ligand interactions is a strategy that is gaining importance. A series of novel tetraindol-4-one derivatives
with 1- and 2-(2,4-substituted phenyl) side chains were designed and synthesized based on the structure of
CYP51 and fluconazole. The antifungal activities of these derivatives against eight human pathogenic fila-
mentous fungi and yeast strains were evaluated in vitro by measuring the minimal inhibitory concentrations.
Nearly all tested compounds 8a–g displayed activity against Candida tropicalis, Candida guilliermondii and
Candida parapsilosis with a minimum inhibitory concentration (MIC) value until 8µgmLꢀ1, on the other
hand compounds 7a–g showed activity against Aspergillus fumigatus with a MIC value of 31.25µgmLꢀ1. A
molecular modeling study of the binding interactions between compounds 6, 7d, 8g and the active site of
MtCYP51 was conducted based on the computational docking results.
Key words antifungal agent; azole; Candida spp.; lanosterol 14-α-demethylase (CYP51) inhibitor; tetrahy-
droindol-4-one
Opportunistic mycosis caused by Candida spp. in immuno-
compromised patients,1) pharmaco-resistance due to the modi-
fiedꢀ geneticꢀ expressionꢀ ofꢀ blankꢀ proteinsꢀ inꢀ C. albicans and
C. krusei,2,3) an increase in the pathogenicity of C. glabrata
strains,4) and the limited number of pharmacological therapy
options today5) have together prompted the de novo design of
antifungal drugs with new mechanisms of inhibition.
The 2001 report by Podust et al. of the crystal structure of
lanosterol 14-α-demethylase (CYP51) laid the foundations for
a new strategic approach to the design of inhibitors against
this enzyme.6–8) The new strategy, which used the same
approach employed by azole drugs, relied on imidazole and
Fig.ꢀ 1.ꢀ Non-coordinatingꢀInhibitorsꢀofꢀCYP51
triazole groups to function as electron-donating pairs and
form a coordinating bond with the iron atom of the heme hibitors of CYP5113–15)ꢀandꢀpresentꢀaꢀhighꢀbioisostericꢀaffinityꢀ
group.9) Azole drugs tend to suffer from a low selectivity to- to hydrophobic cavities, forming strong non-covalent interac-
ward the various isoforms of the ubiquitous family of CYPs.10) tions including van der Waals, π–πꢀ stacking,ꢀ andꢀ hydrogenꢀ
Withꢀaꢀhighꢀaffinityꢀtowardꢀtheꢀhemeꢀgroup,ꢀazoleꢀdrugsꢀformꢀ bonds,ꢀseeꢀFig.ꢀ1.
covalentꢀirreversibleꢀbondsꢀthatꢀtranslateꢀintoꢀaꢀhighꢀtoxicity.11)
Here, we have designed several indolone-type compounds
An alternative and promising strategy replaces covalent with thatꢀ doꢀ notꢀ coordinateꢀ theꢀ hemeꢀ Feꢀ atom.ꢀ Theꢀ designꢀ prin-
electrostatic binding by introducing hydrophobic and bioiso- ciples were guided by the isosterism observed in molecular
steric groups that interact strongly with hydrophobic residues fragments of lanosterol 4 and the azoles 5,16,17) as directed
in the cavities of the CYP51 active site. The challenge, then, is towardꢀtheꢀaffinityꢀofꢀtheꢀhydrophobicꢀresiduesꢀ(Tyr76,ꢀPhe78,ꢀ
to identify ancillary groups that enhance binding and inhibi- Met79 Phe83, Arg96, Phe255, Ala256, His259, Leu321, Ile322,
tion without promoting covalent bond formation.
Ile323, Met433 and Val434) in the active site of CYP51.
One of the main disadvantages of azole drugs is their
Inꢀ thisꢀ work,ꢀ weꢀ presentꢀ theꢀ synthesis,ꢀ in vitro antifungal
strongꢀ affinityꢀ toꢀ theꢀ Feꢀ moietyꢀ ofꢀ theꢀ hemoꢀ groupꢀ presentꢀ evaluation, and molecular modeling of derivatives of tetrahy-
inꢀ CYPs,ꢀ causingꢀ lowꢀ selectivityꢀ toꢀ specificꢀ isoformsꢀ ofꢀ thisꢀ droindol-4-ones 6, 7 and 8 (Chart 1).
groupꢀofꢀenzymes,ꢀwhichꢀinꢀturn,ꢀcouldꢀcauseꢀtoxicꢀeffectsꢀonꢀ
Relativelyꢀ fewꢀ studiesꢀ haveꢀ examinedꢀ theꢀ biologicalꢀ ac-
the organism; therefore it is very important to design of non- tivities of indol-4-ones. Such studies have described the cyto-
coordinating compounds, such as tetrahydroindol-4-ones.12) toxicꢀ activitiesꢀ obtainedꢀ uponꢀ introductionꢀ ofꢀ aminoꢀ acids,18)
Recentꢀ studiesꢀ indicateꢀ thatꢀ theꢀ affinitiesꢀ ofꢀ pharmacophoresꢀ sugars,19) sulfonamides,20) or other groups with Lewis base
to CYP51 did not depend on the coordination of a heterocycle characteristics.21,22) Martinez et al.ꢀ preparedꢀ non-cytotoxicꢀ
toꢀtheꢀhemeꢀiron.ꢀForꢀexample,ꢀcompoundsꢀ1–3 are potent in- derivatives of the structures 6, 7 and 8.18) All previous studies
reported the same observation that the introduction of hydro-
phobicꢀgroupsꢀdidꢀnotꢀyieldꢀcytotoxicity,ꢀmakingꢀtheseꢀderiva-
Theꢀauthorsꢀdeclareꢀnoꢀconflictꢀofꢀinterest.
© 2014 The Pharmaceutical Society of Japan
*ꢀToꢀwhomꢀcorrespondenceꢀshouldꢀbeꢀaddressed.ꢀ e-mail:ꢀgcmm@uaslp.mx

January 2014
17
Chart 1. Design of Indol-4-ones from Lanosterol 4 and the Azole Core 5
Reaction conditions: i, 1eq NBS/1.5eq TsOH·H2O/ACN/reflux;ꢀii,ꢀ1ꢀeqꢀClCH2COCH3/1eq EtONa/EtOH/atm N2; iii, X-aniline/90°C IR 4h; iv, 1.8eq K2CO3/CH2Cl2/12h
atm N2; v, NH4OAc/90°C IR 4h.
Chart 2
tivesꢀ excellentꢀ candidatesꢀ forꢀ theꢀ developmentꢀ ofꢀ antifungalꢀ in the presence of the bromoacetophenones 10a–g. We then
drugs that inhibit CYP51 in a non-coordinative manner.
obtained compounds 8a–g via the Paal–Knorr reaction using
NH4OAc (Chart 2).
Chemistry
The synthesis of compounds 6 and 7a–g began with the
Results and Discussion
preparation of the tricarbonyl compound 12 from the dim-
The in vitro activities of compounds 6, 7a–g, and 8a–g
edone 11 via nucleophilic substitution by chloroacetone in were evaluated in a growth assay involving several oppor-
theꢀ presenceꢀ ofꢀ theꢀ baseꢀ sodiumꢀ ethoxideꢀ underꢀ aꢀ nitrogenꢀ tunistic pathogenic yeast (C. albicans, C. glabrata, C. guil-
atmosphere. The formation of the pyrrole ring of compound liermondii, C. krusei, and C. tropicalis) and fungi (A. niger
12 was achieved through the Paal–Knorr reaction in acetic and A. fumigatus), as summarized in Table 1. The minimum
acid in the presence of NH4OAc to form compound 6 or in the inhibitory concentration (MIC) values were compared with
presence of X-anilines to form compounds 7a–g.
fluconazoleꢀ (Flu),ꢀ itraconazoleꢀ (Itr)ꢀ andꢀ amphotericinꢀ (Amp)ꢀ
Forꢀ theꢀ synthesisꢀ ofꢀ compoundsꢀ 8a–g, the enols of com- as references.
pounds 10a–g were synthesized from the X-acetophenones
Analysis of the MIC of compounds 6, 7a–g, and 8a–g
9a–g using p-toluenesulfonic acid and N-bromosuccinimide revealed that inhibitory activity was present in the lead
(NBS). Nucleophilic substitution of the tricarbonyl compounds compound 6 across all Candidaꢀ speciesꢀ (conꢀ excepciónꢀ deꢀ
13a–g using K2CO3 as a base yielded the dimedone enolate C. parapsilosis) evaluated over a concentration range of
11, which was then submitted to nucleophilic substitution 250–500µgmL−1 after 24h incubation. The structural ad-

18
Vol. 62, No. 1

January 2014
19
Tableꢀ 2.ꢀ Comparisonꢀ ofꢀ theꢀ ExperimentallyꢀDeterminedꢀ Activitiesꢀ inꢀ C.
tropicalis and C. guilliermondii, and the Glide XP Ecoul and Glide XP
Energies,ꢀReportedꢀinꢀkcalꢀmol−1
dition of a phenyl group to position 1 of the indolones led
to an increase in the fungal activity, corresponding to MIC
values of 62.5–250µgmL−1 for the indolones 7a–c in the C.
albicans assay and an MIC value of 62.5µgmL−1 for the in-
dolones 7d–g in the C. tropicalis assay after 24h incubation.
The change in position of the phenyl group from position 1
to position 2 in the indolones increased the antifungal activ-
ity across all Candida species tested. The indolones 8c–g, in
particular, presented higher activities against C. tropicalis and
C. guilliermondii, with an MIC of 31.25µgmL−1 after 24h
incubation. The type of halogen and its position on the phenyl
ring did not appreciably affect the antifungal activities against
the majority of Candida species; nevertheless, the greater
antifungal activity change was in the indolones 7b, 7c, and
7d, because indolone 7dꢀincludedꢀtwoꢀfluorineꢀatomsꢀandꢀwasꢀ
eight times more active in C. tropicalis after 48h incubation.
C. parapsilosis showed the highest sensitivity to the indolone-
compoundsꢀ usedꢀ inꢀ thisꢀ work,ꢀ compoundsꢀ 6 and 7a showed
high activity at MIC of 8µgmL−1 after 24h incubation. In
the case of compounds 7b–g and 8a–g a reduction of the
antifungal activity was showed using MICs of 16 and 31.25
respectively, which could be due to the presence of a substi-
tuted phenyl group in these compounds. The majority of the
indolones yielded MIC values after 48h incubation that were
twice the values after 24h incubation for all Candida strains.
Glide XP
ecoulb)
Glide XP
energyb)
No.
C. tropicalisa) C. guilliermondiia)
6
1.4105
0.4934
0.4607
0.4607
0.2160
0.2172
0.2172
0.1940
0.2612
0.2429
0.1215
0.1135
0.1142
0.1142
0.1014
0.0008
1.4105
0.4934
0.4607
0.4607
0.4320
0.4344
0.4344
0.3879
0.2612
0.2429
0.1215
0.1135
0.1142
0.1142
0.1014
0.0016
−1.145
0.229
−24.013
−23.492
−23.33
−30.404
−18.952
−28.279
−14.524
−16.79
7a
7b
7c
7d
7e
7f
0.955
0.847
1.911
0.544
1.093
7g
8a
8b
8c
8d
8e
8f
0.376
−2.178
−2.839
−1.634
−2.298
−3.738
−1.98
−3.233
−12.380
−30.45
−34.058
−32.911
−34.388
−33.143
−35.923
−37.858
−49.401
8g
Flu
a)ꢀ MICꢀ (mmol)ꢀ valuesꢀ wereꢀ determinedꢀ asꢀ describedꢀ inꢀ Experimental.ꢀ b) Data
(kcalꢀmol−1) generated using Glide 5.6.
levelꢀ ofꢀ theory.ꢀ Theꢀ optimizationꢀ wasꢀ verifiedꢀ byꢀ calculatingꢀ
TheꢀMICꢀvaluesꢀforꢀtheꢀfilamentousꢀfungiꢀusedꢀinꢀthisꢀwork,ꢀ the vibrational frequencies. The optimized molecules were
showed that these species are more sensitive to compounds thenꢀdockedꢀinꢀtheꢀactiveꢀsiteꢀofꢀMtCYP51ꢀusingꢀGlideꢀXP.
6,7a–f, contrary to the activity showed for the majority of the
Theꢀ energiesꢀ obtainedꢀ fromꢀ theꢀ Glideꢀ XPꢀ dockingꢀ studyꢀ
Candida strains with a MIC between 31.25–250µgmL−1. It are displayed in Table 2. Shown are the sum of the van der
was observed that the halogen and its position in the phenyl Waals and Coulombic energies, from the Glide XP study,
ringꢀ doesꢀ notꢀ influenceꢀ inꢀ toꢀ antifungalꢀ activityꢀ againstꢀ A. and the Coulombic energy, from the Glide XP ecoul study.
niger.ꢀ Howeverꢀ itꢀ wasꢀ observedꢀ thatꢀ aꢀ chlorineꢀ orꢀ fluorineꢀ Compounds 8a–g gave the minimum Glide XP energies and
moeity on the C2 of the phenyl ring improves the activity Glide XP ecoul energies. These compounds also yielded the
against A. Fumigatus with a MIC of 31.5µgmL−1.
lowest MIC values against two species of Candida. These
Molecular Modelingꢀ ꢀMolecularꢀ dockingꢀ studiesꢀ wereꢀ results suggested that shifting the position of the halophenylic
performed using the structure of lanosterol 14-α-demethylase, group from N-1 to C-2 resulted in an increase in the antifun-
obtained from Mycobacterium tuberculosis (MtCYP51), in galꢀactivity.ꢀAꢀdetailedꢀcomparativeꢀanalysisꢀofꢀtheꢀdockingꢀofꢀ
complexꢀ withꢀ fluconazoleꢀ (Proteinꢀ Dataꢀ Bankꢀ (PDB)ꢀ ID.ꢀ complexesꢀ 8g–MtCYP51 and 7g–MtCYP51ꢀ (Fig.ꢀ 2)ꢀ revealedꢀ
1EA1) as a template. This structure has been used previ- differentꢀ interactionsꢀ thatꢀ playedꢀ keyꢀ roleꢀ inꢀ theꢀ bindingꢀ ofꢀ
ously to study and predict ligand–receptor interactions and to 8a–gꢀtoꢀtheꢀactiveꢀsite.ꢀTheꢀmostꢀsignificantꢀinteractionꢀofꢀ8g
rationallyꢀdesignꢀmoreꢀselectiveꢀandꢀefficientꢀantifungalꢀcom- involvedꢀ aꢀ hydrogenꢀ bondꢀ betweenꢀ theꢀ oxygenꢀ inꢀ theꢀ ketoneꢀ
pounds.23) The use of MtCYP51 has been validated in design group and the Arg96 residue, with a distance of 2.038Å. One
studies of new azole compounds,24) mutational maps,25) and ofꢀ theꢀ methyleneꢀ groupsꢀ ofꢀ theꢀ cyclohexanoneꢀ ringꢀ wasꢀ po-
homology models26) that demonstrated a high degree of simi- sitioned perpendicular to the porphyrin plane, 4.661Å from
larity between the hydrophobic cavities of the catalytic site of theꢀFeꢀatomꢀinꢀtheꢀhemeꢀgroup,ꢀwhereasꢀtheꢀotherꢀmethyleneꢀ
CYP51 from Candida spp. and that of MtCYP51.
Asꢀtheꢀfirstꢀstep,ꢀweꢀvalidatedꢀourꢀdockingꢀprocedureꢀusingꢀ
group was stabilized by hydrophobic interactions with Ala256.
The 2,4-dichlorophenyl ring was stabilized by face-to-
Glideꢀ extraꢀ precisionꢀ (XP)27,28) by identifying a binding con- face π–πꢀ stackingꢀ withꢀ Tyr76ꢀ andꢀ edge-to-faceꢀ π–πꢀ stackingꢀ
formation that resembled, as predicted by the Glide XP score, with residue Phe78, located in the upper portion of the ring.
theꢀ experimentalꢀ positionꢀ fromꢀ theꢀ X-rayꢀ crystallographyꢀ The lower portion of the ring was stabilized on one side by
study.
Leu321, His259, Met433, and Val435 and on the other side by
Toꢀ thisꢀ end,ꢀ theꢀ fluconazoleꢀ moleculeꢀ wasꢀ removedꢀ fromꢀ Ile323 and Leu324.
the active site of the PDB structure, and the molecule was
Aꢀ dockingꢀ analysisꢀ ofꢀ complexꢀ 7g–MtCYP51 revealed an
re-dockedꢀinꢀtheꢀactiveꢀsite.ꢀOurꢀdockingꢀprocedureꢀproducedꢀ adjustment in the orientation of the indolone group to avoid
aꢀ structureꢀ thatꢀ agreedꢀ wellꢀ withꢀ theꢀ experimentallyꢀ deter- formingꢀaꢀhydrogenꢀbondꢀbetweenꢀtheꢀindoloneꢀoxygenꢀgroupꢀ
minedꢀdockingꢀconformationꢀofꢀfluconazoleꢀinꢀtheꢀactiveꢀsite,ꢀ and the Arg96 residue. One of the methylene groups in the
with a root mean square deviation (RMSD) of 1.34Å. This cyclohexanoneꢀ ringꢀ wasꢀ positionedꢀ perpendicularꢀ toꢀ theꢀ por-
RMSDꢀvalueꢀprovidedꢀsupportꢀforꢀtheꢀvalidityꢀofꢀourꢀdockingꢀ phyrinꢀ planeꢀ 3.044ꢀÅꢀ fromꢀ theꢀ Feꢀ atomꢀ inꢀ theꢀ hemeꢀ group,ꢀ
procedure.29)ꢀTheꢀscoresꢀandꢀdockingꢀconformationsꢀofꢀtheꢀin- whereas the other methylene was stabilized by hydrophobic
dolones were obtained by optimizing the molecular structures
interactions with the Thr260 residue. The 2,4-dichlorophenyl
ring occupied the same hydrophobic cavity as compound
using the Gaussian 03 software30)ꢀatꢀtheꢀDFT/B3LYP/6-311G**ꢀ

20
Vol. 62, No. 1
Fig.ꢀ 2.ꢀ PredictedꢀBindingꢀModesꢀandꢀDockingꢀConformationsꢀofꢀtheꢀRepresentativeꢀCompoundsꢀa)ꢀ7g and b) 8g in the Active Site of MtCYP51
Atomꢀcolors:ꢀwhiteꢀ(H),ꢀgrayꢀ(C),ꢀblueꢀ(N),ꢀredꢀ(O),ꢀdarkꢀgreenꢀ(Cl),ꢀturquoiseꢀ(Fe)ꢀandꢀpinkꢀ(heme).ꢀAminoꢀacidꢀcolors:ꢀpurpleꢀ(positiveꢀpolar)ꢀandꢀgreenꢀ(hydrophobic).ꢀ
Hydrogenꢀbondsꢀareꢀindicatedꢀbyꢀdashedꢀlinesꢀinꢀred.ꢀHydrogenꢀatomsꢀareꢀomittedꢀforꢀtheꢀsakeꢀofꢀclarity,ꢀexceptꢀforꢀthoseꢀinvolvedꢀinꢀbondingꢀinteractions.ꢀ(Colorꢀimagesꢀ
were converted into gray scale.)
8g, stabilized by π–πꢀ face-to-faceꢀ stackingꢀ interactionsꢀ withꢀ aꢀmoderateꢀbutꢀsignificantꢀinhibitoryꢀeffectꢀoverꢀ C. tropicalis
residue Tyr76, and the lower portion was stabilized by residue and C. guilliermondii. These results indicated that a change
Leu321.
in the position of the halophenyl regioisomers from N-1 to
The differences between the interactions present for com- C-2ꢀ increasedꢀ theꢀ antifungalꢀ activity.ꢀ Theꢀ molecularꢀ dockingꢀ
pounds 8 and 7ꢀareꢀreflectedꢀbyꢀaꢀdecreaseꢀinꢀtheꢀXPꢀGlideꢀen- studyꢀprovidedꢀaꢀbetterꢀunderstandingꢀofꢀtheꢀkeyꢀinteractionsꢀ
ergy and an increase in the antifungal activity of compounds between the drugs and the active site of MtCYP51. C. para-
8a–g.ꢀ Replacementꢀ ofꢀ theꢀ fluorineꢀ groupꢀ toꢀ aꢀ chlorineꢀ groupꢀ psilosis was the most sensitive yeast strain to the indolone-
increased contact with the hydrophobic residues Met433 and compoundsꢀ usedꢀ inꢀ thisꢀ work,ꢀ showingꢀ compoundsꢀ 6 and 7a
Val435. This suggested that a change in the antifungal activi- theꢀ highestꢀ activity.ꢀ Inꢀ theꢀ caseꢀ ofꢀ filamentousꢀ fungiꢀ strains,ꢀ
ties via regioisomeric changes in the tetrahydroindol-4-ones A. fumigatus showed the highest sensitivity to the compounds
was related not only to the position of the indolone group, but 7b and 7e. Recent advances in SAR studies of fragments of
also to the position of the halogen in the halophenyl ring in tetrahydroindol-4-ones are helpful toward elucidating new
the active site of MtCYP51.
non-coordinating antifungal compounds with broad activ-
The minimum Glide XP energies and Glide XP ecoul ener- ityꢀ spectraꢀ andꢀ highꢀ potency.ꢀ Furtherꢀ structuralꢀ optimizationꢀ
gies of indolones and MIC to Aspergillus spp. not correlate.
studies, such as estereoelectronic effects due to electron-
releasing and electron-withdrawing groups and bioisosteric
effects in indolones are in progress.
Conclusion
A structure–activity relationship (SAR) analysis was per-
formedꢀtoꢀexamineꢀtheꢀbindingꢀmodesꢀofꢀmolecularꢀfragmentsꢀ
of the principal active substrates to the active site of CYP51.
Experimental
General General methods: All reagents and solvents were
Theꢀ SARꢀ results,ꢀ inꢀ combinationꢀ withꢀ aꢀ molecularꢀ dockingꢀ reagent-grade and were used as received from Sigma-Aldrich
study of select antifungal agents, guided the design, synthesis, Co.ꢀ (St.ꢀ Louis,ꢀ MO,ꢀ U.S.A.).ꢀ Flashꢀ chromatographyꢀ wasꢀ
and in vitro evaluation of a series of derivatives of the substi- performedꢀ usingꢀ E.ꢀ Merckꢀ Kieselgelꢀ 60ꢀ silicaꢀ gelꢀ (230–400ꢀ
tuted tetrahydroindol-4-ones. Some of the newly synthesized mesh).ꢀ Reportedꢀ meltingꢀ pointsꢀ areꢀ uncorrected.ꢀ Theꢀ FT-IRꢀ
compounds were found to be effective against Candida spp. wereꢀ recordedꢀ onꢀ aꢀ Thermoꢀ Nicoletꢀ Nexusꢀ 470ꢀ FT-IRꢀ asꢀ
Compounds with hydrophobic halophenyl groups at the C-2 thinꢀ filmsꢀ onꢀ aꢀ KBrꢀ diskꢀ (forꢀ solids)ꢀ andꢀ asꢀ aꢀ germaniumꢀ
1
position (8a–g)ꢀ demonstratedꢀ excellentꢀ antifungalꢀ activitiesꢀ ATR crystal (for liquids). H- and 13C-NMR were obtained
against all yeast strains evaluated. Compounds 8e–g presented on an Eclipse Jeol (operated at 300, 75MHz, respectively)

January 2014
21
and a Varian–Gemini (operating at 200MHz and 50MHz, 1,3-dione (13b): The title compound was obtained as a white
respectively).ꢀChemicalꢀshiftsꢀareꢀexpressedꢀasꢀδ values (ppm) powder (0.9g, 29%): Rf=0.42ꢀ (hexane–AcOEt,ꢀ 70ꢀ:ꢀ30);ꢀ mpꢀ
1
relative to tetramethylsilane (TMS) as an internal standard (s= 156°C. H-NMR (200MHz, DMSO-d6) δ: 0.97 (s, 6H), 2.1 (s,
singlet, d=doublet, t=triplet, q=ꢀquadruplet,ꢀ sext=ꢀsextuplet,ꢀ 2H), 2.18 (s, 2H), 3.70 (d, 2H, J=2.2Hz), 7.24–7.35 (m, 2H),
m=multiplet and br=broad). Coupling constants (J) are given 7.54–7.65 (m, 1H), 7.71 (ddd, 1H, J=8.8, 8.5, 0.8Hz) 10.67 (s,
in Hertz (Hz). All mass spectra (MS) were recorded on a 1H). 13C-NMR (50MHz, DMSO-d6) δ: 27.8, 31.7, 35.6, 36.4,
Jeol AX505HA mass spectrometer. Elemental analyses were 38.2, 102.4, 107.8, 116.6, 124.5, 126.0, 130.0, 134.2, 158.0,
performedꢀ onꢀ aꢀ CE-440ꢀ Exeterꢀ Analyticalꢀ Inc.ꢀ Theꢀ purityꢀ ofꢀ 163.0, 196.0. MS (EI): m/z (%): 276 (M+, 56), 153 (30), 123
compounds 6, 7a–g and 8a–g (>97%) was analyzed using (100), 97 (25), 83 (21), 55 (12). IR (KBr): ṽ: 3127, 2958, 2892,
an Agilent Technology GC 6890N coupled with a MS 5973N 1683, 1608, 1247, 1195, 765cm−1.
mass spectrometer operated at 70eV and equipped with a DB-
2-[2-(4-Fluorophenyl)-2-oxoethyl]-5,5-dimethylcycloꢀhexane-
5HT capillary column (15m, 0.25 i.d.) with a (5%-phenyl)- 1,3-dione (13c): The title compound was obtained as a yellow-
methylpolysiloxaneꢀ (0.10ꢀµmꢀ film).ꢀ Heliumꢀ wasꢀ usedꢀ asꢀ theꢀ orange powder (1.5g, 68%): Rf=0.47ꢀ(hexane–AcOEt,ꢀ70ꢀ:ꢀ30);ꢀ
1
carrierꢀ gasꢀ atꢀ aꢀ flowꢀ rateꢀ ofꢀ 1ꢀmL/min.ꢀ Theꢀ injectorꢀ andꢀ MSꢀ mp 96°C. H-NMR (200MHz, DMSO-d6) δ: 0.97 (s, 6H), 2.1
transfer line temperatures were set at 300°C. Diluted samples (s, 2H), 2.38 (s, 2H), 3.77 (s, 2H), 7.31 (dd, 2H, J=8.8, 9Hz),
(1:10 v/v, in acetone) 2.0µL in volume were manually inject- 8.01 (dd, 2H, J=9Hz), 10.99 (s, 1H). MS (EI): m/z (%): 276
ed. After sample injection, the initial temperature in the oven (M+, 26), 162 (14), 123 (100), 95 (14), 83 (29), 55 (13). IR
(100°C) was held constant for 2min, then increased to 300°C (KBr) ṽ: 3081, 2960, 2871, 2628, 2578, 1691, 1602, 1515, 1469,
(at a rate of 20°C/min). This temperature was then maintained 1349, 1307, 1228, 1147, 1039, 998, 831cm−1.
for 3min. After a delay of 1.8min to permit passage of the
2-[2-(2,4-Difluorophenyl)-2-oxoethyl]-5,5-dimethylcyclo-
solvent, the mass spectra were scanned from 15 to 800 m/z.
hexane-1,3-dioneꢀ(13d): The title compound was obtained as a
5,5-Dimethyl-2-(2-oxopropyl)cyclohexane-1,3-dione (12) yellow powder (2.1g, 73%): Rf=0.47ꢀ(hexane–AcOEt,ꢀ70ꢀ:ꢀ30);ꢀ
1
Aꢀ mixtureꢀ ofꢀ 14ꢀmmolꢀ sodiumꢀ ethoxide,ꢀ 2ꢀgꢀ (14ꢀmmol)ꢀ mp 138°C. H-NMR (200MHz, DMSO-d6) δ: 0.97 (s, 6H),
5,5-dimethylcyclohexane-1,3-dioneꢀ andꢀ 1.33ꢀmLꢀ (14ꢀmmol)ꢀ 2.22 (s, 4H), 3.68 (d, 2H, J=2.4Hz), 7.18 (ddd, 1H, J=7.2, 9.6,
chloroacetoneꢀ inꢀ 20ꢀmLꢀ ethanolꢀ wasꢀ heatedꢀ underꢀ refluxꢀ forꢀ 0.8Hz), 7.37 (ddd, 1H, J=11.45, 9.3, 2.6Hz), 7.81 (ddd, 1H,
30min and then cooled. The sodium chloride that formed J=16, 7.6, 1.8Hz), 10.61 (s, 1H). 13C-NMR (50MHz, DMSO-
wasꢀremovedꢀbyꢀfiltration,ꢀandꢀtheꢀfiltrateꢀwasꢀconcentratedꢀin d6) δ: 27.8, 31.8, 36.3, 44.6, 104.9, 107.7, 112.1, 122.7, 132.2,
vacuo.ꢀTheꢀresidualꢀsyrupꢀwasꢀdissolvedꢀinꢀaꢀmixtureꢀofꢀchlo- 158.8, 1162.0, 163.9, 167.03, 172.9, 194.7. MS (EI): m/z (%):
roformꢀ(20ꢀmL)ꢀandꢀ10%ꢀsodiumꢀhydroxideꢀ(20ꢀmL).ꢀTheꢀaque- 294 (M+, 46), 153 (30), 141 (100), 97 (24), 83 (14), 55 (8). IR
ousꢀ phaseꢀ wasꢀ separatedꢀ andꢀ re-extractedꢀ withꢀ chloroformꢀ (KBr) ṽ: 3077, 2964, 2892, 2657, 1695, 1608, 1577, 1243, 1199,
(20mL). The aqueous phase was cooled in an ice bath, made 1147, 1099, 1039cm−1.
acidicꢀ withꢀ theꢀ additionꢀ ofꢀ hydrochloricꢀ acid,ꢀ andꢀ extractedꢀ
2-[2-(2-Chlorophenyl)-2-oxoethyl]-5,5-dimethylcyclo-
with chloroform (3×20ꢀmL).ꢀ Theꢀ combinedꢀ organicꢀ extractsꢀ hexane-1,3-dioneꢀ(13e): The title compound was obtained as a
wereꢀ driedꢀ withꢀ anhydrousꢀ sodiumꢀ sulfateꢀ andꢀ filtered,ꢀ andꢀ white powder (0.7g, 20%): Rf=ꢀ0.32ꢀ (hexane–AcOEt,ꢀ 70ꢀ:ꢀ30);ꢀ
1H-NMR (200MHz, DMSO-d6) δ: 0.97 (s, 6H),
theꢀ filtrateꢀ wasꢀ evaporatedꢀ in vacuo to dryness. Recrystal- mp 142°C.
lization of the crude product from acetone gave compound 12
2.1 (s, 4H), 5.70 (s, 2H), 7.6 (m, 4H) 10.98 (s, 1H). 13C-NMR
(50MHz, DMSO-d6) δ: 27.9, 31.1, 32.1, 53.2, 63.3, 102.4, 126.7,
as colorless crystals (2.2g, 80%): Rf=0.22ꢀ (hexane–AcOEt,ꢀ
70:30); mp 133–135°C.
1H-NMR (200MHz, DMSO-d6) δ: 130.0, 131.4, 138.5, 200.1, 204.7. MS (EI): m/z (%): 292 (M+,
0.99 (s, 6H), 1.98 (s, 3H), 2.22 (s, 4H), 3.16 (s, 1H), 3.33 (s, 1), 140 (57), 112 (23), 83 (100), 56 (49) 55 (35). IR (KBr) ṽ:
1H), 10.62 (s, 1H). MS (electron ionization (EI)): m/z (%): 196 2960, 2869, 2680, 2576, 2530, 1907, 1617, 1581, 1519, 1471,
(M+, 51), 154 (89), 125 (24), 98 (100), 55 (45). IR (KBr) ṽ: 1349, 1305, 1228, 1145cm−1.
2962, 2931, 1720, 1645, 1155, 1064cm−1.
2-[2-(4-Chlorophenyl)-2-oxoethyl]-5,5-dimethylcyclo-
General Procedure for the Synthesis of 2-(2-(X-Phenyl)- hexane-1,3-dioneꢀ (13f): The title compound was obtained as
2-oxoethyl)-5,5-dimethylcyclohexane-1,3-dione, 13a–g a white powder (0.9g, 47%): Rf=0.5ꢀ(hexane–AcOEt,ꢀ70ꢀ:ꢀ30);ꢀ
A
1
slurry of dimedone 11ꢀ (1ꢀeq),ꢀ bromoketoneꢀ 10a-g (1eq), and mp 164°C. H-NMR (200MHz, DMSO-d6) δ: 0,99 (s, 6H),
anhydrous K2CO3(1.8eq) in chloroform was stirred at room 2,23 (s, 4H), 3,77 (s, 2H), 7,55 (dd, 2H, J=8.6, 1.6Hz), 7,94
temperatureꢀ forꢀ 12ꢀh.ꢀ Theꢀ mixtureꢀ wasꢀ thenꢀ filtered.ꢀ Theꢀ in- (dd, 2H, J=8.6, 1.6Hz) and 10,65 (s, 1H). MS (EI): m/z (%):
solubleꢀsaltsꢀwereꢀdissolvedꢀinꢀwater,ꢀandꢀtheꢀfilteredꢀsolutionꢀ 292 (M+, 51), 153 (14), 139 (100), 111 (16), 97 (16), 55 (5). IR
was made acidic by the addition of concentrated HCl. The (KBr) ṽ: 3162, 2956, 2917, 2871, 1685, 1600, 1411, 1378, 1321,
precipitateꢀ wasꢀ filteredꢀ off,ꢀ washedꢀ withꢀ water,ꢀ andꢀ crystal- 1251, 1193, 1145, 1039, 993, 813, 777cm−1.
lized from acetone.
2-[2-(2,4-Chlorophenyl)-2-oxoethyl]-5,5-dimethylcyclo-
5,5-Dimethyl-2-(2-oxo-2-phenylethyl)ꢀcyclohexane-1,3-dioneꢀ hexane-1,3-dioneꢀ (13g): The title compound was obtained as
(13a): The title compound was obtained as a white pow- a white powder (0.4g, 10%): Rf=0.27ꢀ(hexane–AcOEt,ꢀ70ꢀ:ꢀ30);ꢀ
1
der (1.3g, 39.5%): Rf=0.47ꢀ (hexane–AcOEt,ꢀ 70ꢀ:ꢀ30);ꢀ mpꢀ mp 140°C. H-NMR (200MHz, DMSO-d6) δ: 0.97 (s, 6H),
1
136–138°C. H-NMR (200MHz, DMSO-d6) δ: 0.97 (s, 6H), 2.10 (s, 4H), 5.17 (s, 2H), 7.48 (dd, 1H, J=8.4, 1.8Hz), 7.58 (d,
2.10 (s, 4H), 3.79 (s, 2H), 7.44–7.63 (m, 3H), 7.90–7.95 (m, 1H, J=8.4Hz), 7.66 (d, 1H, J=1.8Hz), 10.96 (s, 1H). 13C-NMR
2H), 10.96 (s, 1H). MS (EI): m/z (%): 258 (M+, 11), 140 (30), (50MHz, DMSO-d6) δ: 27.9, 32.1, 46.1, 102.4. MS (EI): m/z
105 (34), 83 (100), 56 (55). IR (KBr) ṽ: 2960, 2869, 2680, (%): 326 (M+, 9), 173 (42), 140 (79), 112 (32), 83 (100) 56 (74),
2630, 2576, 2532, 1617, 1577, 1519, 1471, 1349, 1305, 1226, 55(63). IR (KBr) ṽ: 2960, 2871, 2630, 2576, 2532, 1700, 1617,
1145 cm−1.
1577, 1521, 1471, 1349, 1305, 1251, 1226, 1145cm−1.
2-[2-(2-Fluorophenyl)-2-oxoethyl]-5,5-dimethylcycloꢀhexane-
General Procedure for the Synthesis of 1-(X-Phenyl)-2,6,6-

22
Vol. 62, No. 1
trimethyl-6,7-dihydro-1H-indol-4(5H)-one (6, 7a–g, 8a–g) J=1, 0.4Hz), 7.28–7.35 (m, 1H), 7.54–7.66 (m, 2H). 13C-NMR
To a vigorously stirred suspension of tricarbonyl compounds (50MHz, DMSO-d6) δ: 11.9, 27.8, 35.2, 51.5, 103.0, 105.4,
13a–g (3.88mmol) in AcOH (5mL) R-aniline was added to 112.6, 118.7, 120.5, 131.3, 131.6, 143.5, 154.9, 159.8, 164.6,
7a–g or NH4OAc to yield 6 and 8a–g (3.88mmol). The result- 192.0. MS (EI): m/z (%): 289 (M+, 89), 233 (94), 205 (100),
ing slurry was heated to 90°C for 4h using an IR lamp. The 154 (54), 113 (9). IR (KBr) ṽ: 3073, 2956, 2931, 2869, 1648,
reactionꢀ mixtureꢀ wasꢀ thenꢀ allowedꢀ toꢀ coolꢀ toꢀ roomꢀ tempera- 1608, 1517, 1465, 1436, 1272, 1201, 1145, 1105, 962, 860cm−1.
tureꢀandꢀpouredꢀintoꢀiceꢀwaterꢀ(10ꢀmL).ꢀTheꢀsolidꢀwasꢀfilteredꢀ Anal. Calcd for C17H17F2NO:ꢀC,ꢀ70.57;ꢀH,ꢀ5.92;ꢀN,ꢀ4.84.ꢀFound:ꢀ
andꢀ washedꢀ withꢀ cooledꢀ water.ꢀ Theꢀ crudeꢀ mixtureꢀ wasꢀ puri- C, 70.69; H, 6.09; N, 4.98. GC-MS purity=99.2%; tR=7.0 min.
fiedꢀbyꢀcolumnꢀchromatographyꢀonꢀsilicaꢀgelꢀusingꢀaꢀgradientꢀ
1-(2-Chlorophenyl)-6,7-dihidro-2,6,6-trimetil-1H-indol-
ofꢀ 30–50%ꢀ AcOEtꢀ inꢀ hexaneꢀ asꢀ theꢀ eluent.ꢀ Evaporationꢀ ofꢀ 4(5H)-one (7e): The title compound was obtained as a color-
the collected fractions gave indolones 6, 7a–g and 8a–g. The less crystals (0.8g, 57%): Rf=0.63ꢀ (hexane–AcOEt,ꢀ 70ꢀ:ꢀ30);ꢀ
1
spectral data for the tetrahydroindol-4-ones 6, 7a, 7c, 7f, 8a, mp 121°C. H-NMR (200MHz, DMSO-d6) δ: 0.97 (d, 6H),
8c, and 8f were in agreement with the reported data.31) Spec- 1.88 (d, 3H), 2.24 (q, 4H), 6.21 (d, 1H, J=1.2Hz), 7.47–7.63
tralꢀ dataꢀ forꢀ theꢀ unknownꢀ tetrahydroindol-4-onesꢀ 7b, 7d, 7e, (m, 3H), 7.69–7.78 (m, 1H). 13C-NMR (50MHz, DMSO-d6) δ:
7g, 8b, 8d, 8e and 8g are described below.
11.9, 27.8, 35.2, 51.5, 103.0, 105.4, 112.6, 118.7, 120.5, 131.3,
2,6,6-Trimethyl-6,7-dihydro-1H-indol-4(5H)-one (6): The 131.6, 143.5, 154.9, 159.8, 164.6, 192.0. MS (EI): m/z (%): 287
title compound was obtained as a yellow powder (1.2g, (M+, 89), 233 (94), 205 (100), 154 (54), 113 (9). IR (KBr) ṽ:
72%): Rf=0.38ꢀ (hexane–AcOEt,ꢀ 70ꢀ:ꢀ30);ꢀ mpꢀ 184°C;ꢀ 1H-NMR 3073, 2956, 2931, 2869, 1648, 1608, 1517, 1465, 1436, 1272,
(200MHz, DMSO-d6) δ: 0.99 (s, 6H), 2.11 (s, 3H), 2.13 (s, 1201, 1145, 1105, 962, 860cm−1. Anal. Calcd for C17H18ClNO:
2H), 2.55 (s, 2H), 5.88 (q, 1H, J=2.1, 1Hz), 11.02 (s, 1H). C,ꢀ70.95;ꢀH,ꢀ6.30;ꢀN,ꢀ4.87.ꢀFound:ꢀC,ꢀ70.83;ꢀH,ꢀ6.15;ꢀN,ꢀ4.70.ꢀ
MS (EI): m/z (%): 177 (M+, 65), 121 (92), 93 (100), 66 (7), 42 GC-MS purity=99.9%; tR=7.9 min.
(13). IR (KBr) ṽ: 3243, 3170, 2954, 2921, 1629, 1482, 1128,
1-(4-Chlorophenyl)-2,6,6-trimethyl-6,7-dihydro-1H-indol-
4(5H)-one (7f): The title compound was obtained as a color-
808cm−1. GC-MS purity=99.8%; tR=4.0min.
2,6,6-Trimethyl-1-phenyl-6,7-dihydro-1H-indol-4(5H)-one less crystals (0.9g, 53,5%): Rf=0.65ꢀ (hexane–AcOEt,ꢀ 70ꢀ:ꢀ30),ꢀ
(7a): The title compound was obtained as a colorless crystals mp 174°C. 1H-NMR (200MHz, DMSO-d6) δ: 0.96 (s, 6H),
(0.6g, 67%): Rf=0.68ꢀ (hexane–AcOEt,ꢀ 70ꢀ:ꢀ30);ꢀ mpꢀ 141°C.ꢀ 2.00 (d, 3H), 2.21 (s, 2H), 2.39 (s, 2H), 6.20 (d, 1H, J=1Hz),
1H-NMR (200MHz, DMSO-d6) δ: 0.96 (s, 6H), 1.98 (s, 3H), 7.40 (dd, 2H, J=8.8Hz), 7.61 (dd, 2H, J=8.8Hz). MS (EI):
2.16 (s, 2H), 2.38 (s, 2H), 6.19 (q, 1H, J=1.2, 0.4Hz), 7.32–7.37 m/z (%): 287 (M+, 86), 231 (100), 203 (47), 168 (93), 152 (23),
(m, 2H), 7.45–7.60 (m, 3H). MS (EI): m/z (%): 253 (M+, 79), 111 (7). IR (KBr) ṽ: 3060, 2956, 2933, 2869, 1648, 1496,
197 (94), 169 (100), 168 (52), 118 (31), 77 (35). IR (KBr) ṽ: 1465, 1436, 1407, 1272, 1228, 1203, 1087, 863, 790, 619cm−1.
3048, 2954, 2925, 2867, 1652, 1529, 1463, 1436, 1403, 1274, GC-MS purity=99.9%; tR=8.2min.
1203, 1120, 1076, 788, 767, 701cm−1. GC-MS purity=99.9%;
tR=7.4 min.
1-(2,4-Dichlorophenyl)-2,6,6-trimethyl-6,7-dihydro-1H-
indol-4(5H)-one (7g): The title compound was obtained as a
1-(2-Fluorophenyl)-2,6,6-trimethyl-6,7-dihydro-1H-indol- yellow crystals (0.6g, 30%): Rf=0.68ꢀ(hexane–AcOEt,ꢀ70ꢀ:ꢀ30);ꢀ
1
4(5H)-one (7b): The title compound was obtained as a yellow- mp 176°C. H-NMR (200MHz, DMSO-d6) δ: 0.97 (d, 6H),
orange crystals (0.4g, 55%): Rf=0.68ꢀ(hexane–AcOEt,ꢀ70ꢀ:ꢀ30);ꢀ 1.89 (d, 3H), 2.49 (q, 4H), 6.21 (d, 1H, J=1Hz), 7.57 (dd,
1
mp 137°C. H-NMR (200MHz, DMSO-d6) δ: 0.97 (d, 6H), 1H, J=8.4, 0.4Hz), 7.65 (dd, 1H, J=8.5, 2.2Hz), 7.96 (d, 1H,
1.95 (s, 3H), 2.23 (s, 2H), 2.32 (q, 2H), 6.22 (d, 1H, J=1Hz), J=2Hz). 13C-NMR (50MHz, DMSO-d6) δ: 11.7, 27.4, 28.5,
7.37 (dd, 1H, J=7.9, 1.8Hz), 7.41 (dd, 1H, J=6.9, 1.6Hz), 7.52 35.2, 51.6, 102.9, 118.6, 128.8, 129.9, 130.8, 131.8, 133.1, 134.9,
(dd, 1H, J=9.9, 1.8Hz), 7.57–7.65 (m, 1H). 13C-NMR (50MHz, 143.1, 191.9. MS (EI): m/z (%): 321 (M+, 81), 265 (100), 237
DMSO-d6) δ: 11.7, 27.9, 35.2, 51.5, 103.0, 116.8, 118.6, 123.9, (46), 202 (71), 145 (8), 109 (5). IR (KBr) ṽ: 3073, 3035, 2954,
125.5, 130.2, 131.1, 131.3, 143.3, 154.6, 159.5, 192.0. MS (EI): 2919, 2867, 1648, 1531, 1490, 1463, 1407, 1280, 1201, 1101,
m/z (%): 271 (M+, 82), 215 (75), 187 (100), 167 (4), 136 (52), 1072, 1000, 863, 802cm−1. Anal. Calcd for C17H17Cl2NO: C,
95 (11), 75 (6), 41 (4). IR (KBr) ṽ: 3046, 2954, 2904, 2869, 63.37;ꢀ H,ꢀ 5.32;ꢀ N,ꢀ 4.35.ꢀ Found:ꢀ C,ꢀ 63.05;ꢀ H,ꢀ 5.34;ꢀ N,ꢀ 4.82.ꢀ
1654, 1508, 1463cm−1. Anal. Calcd for C17H18FNO:ꢀ C,ꢀ 75.25;ꢀ GC-MS purity=99.0%; tR=8.5min.
H,ꢀ 6.69;ꢀ N,ꢀ 5.16.ꢀ Found:ꢀ C,ꢀ 75.48;ꢀ H,ꢀ 6.31;ꢀ N,ꢀ 5.05.ꢀ GC-MSꢀ
purity=99.9%; tR=7.3 min.
6,6-Dimethyl-2-phenyl-6,7-dihydro-1H-indol-4(5H)-one
(8a): The title compound was obtained as a white powder
1-(4-Fluorophenyl)-2,6,6-trimethyl-6,7-dihydro-1H-indol- (0.2g, 88%): Rf=0.51ꢀ (hexane–AcOEt,ꢀ 70ꢀ:ꢀ30),ꢀ mpꢀ 227°C.ꢀ
4(5H)-one (7c): The title compound was obtained as a color- 1H-NMR (200MHz, DMSO-d6) δ: 1.04(s, 6H), 2.23 (s, 2H),
less crystals (0.8g, 59%): Rf=0.68ꢀ (hexane–AcOEt,ꢀ 70ꢀ:ꢀ30);ꢀ 2.70 (s, 2H), 6.70 (d, 1H, J=2.4Hz), 7.19 (dd, 1H, J=7.3,
mp 155–158°C. 1H-NMR (200MHz, DMSO-d6) δ: 0.96 (s, 7.2Hz), 7.36 (dd, 2H, J=7.5, 7.2Hz), 7.64 (dd, 2H, J=7.9,
6H), 1.95 (d, 3H), 2.21 (s, 2H), 2.38 (s, 2H), 6.19 (d, 1H, 1.4Hz), 11.71 (s, 1H). MS (EI): m/z (%): 239 (M+, 239), 183
J=1Hz), 7.33–7.47 (m, 4H). MS (EI): m/z (%): 271 (M+, 90), (78), 155 (100), 128 (9), 104 (24), 77 (8). IR (KBr) ṽ: 3513,
215 (100), 187 (95), 172 (9), 136 (31), 95 (24). IR (KBr) ṽ: 3338, 3224, 2964, 2929, 1608, 1488, 1228, 1143, 823, 757, 692,
3066, 2956, 2904, 2871, 1648, 1513, 1465, 1413, 1224, 1120, 626cm−1. GC-MS purity=99.7%; tR=8.5min.
1045, 863, 794cm−1. GC-MS purity=99.9%; tR=7.3 min.
2-(2-Fluorophenyl)-6,6-dimethyl-6,7-dihydro-1H-indol-
1-(2,4-Difluorophenyl)-2,6,6-trimethyl-6,7-dihydro-1H- 4(5H)-one (8b): The title compound was obtained as a white
indol-4(5H)-one (7d): The title compound was obtained as powder (0.3g, 73%): Rf=0.54ꢀ (hexane–AcOEt,ꢀ 70ꢀ:ꢀ30);ꢀ mpꢀ
1
a colorless crystals (1.1g, 68%): Rf=0.68ꢀ (hexane–AcOEt,ꢀ 229°C. H-NMR (300MHz, DMSO-d6) δ: 1.04 (s, 6H), 2.24
70:30); mp 137°C. 1H-NMR (200MHz, DMSO-d6) δ: 0.97 (s, 2H), 2.72 (s, 2H), 6.69 (t, 1H, J=3Hz), 7.21–7.29 (m, 3H),
(d, 6H), 1.95 (s, 3H), 2.23 (s, 2H), 2.32 (q, 2H), 6.21 (q, 1H, 7.68–7.76 (m, 1H), 11.70 (s, 1H). 13C-NMR (75MHz, DMSO-

January 2014
23
d6) δ: 28.1, 35.5, 51.7, 105.2, 116.2, 119.5, 119.7, 124.7, 126.0, J=8.6, 2Hz), 7.60 (d, 1H, J=8.6Hz), 7.68 (d, 1H, J=2.2Hz),
127.9, 143.9, 156.5, 159.8, 192.1. MS (EI) m/z (%): 257 (M+, 11.74 (s, 1H). 13C-NMR (75MHz, DMSO-d6) δ: 28.1, 35.2,
85), 201 (54), 173 (100), 146 (9), 122 (25), 102 (7), 75 (2), 51 35.8, 51.7, 106.4, 119.2, 127.5, 127.6, 129.4, 129.9, 130.3, 130.8,
(2). IR (KBr) ṽ: 3286, 3166, 2954, 2919, 1629, 1569, 1492, 131.6, 143.8, 192.2. MS (EI) m/z (%) 307 (M+, 100), 251 (86),
1218, 1168, 1139, 819, 755cm−1. Anal. Calcd for C16H16FNO:ꢀ 223 (77), 172 (19), 136 (5), 69 (3), 28 (15). IR (KBr) ṽ: 3251,
C,ꢀ74.69;ꢀH,ꢀ6.27;ꢀN,ꢀ5.44.ꢀFound:ꢀC,ꢀ74.01;ꢀH,ꢀ6.34;ꢀN,ꢀ5.44.ꢀ 3154, 2958, 2929, 2869, 1631, 1490, 1461, 1382, 1220, 1137,
GC-MS purity=97.9%; tR=8.0min.
1105, 1076, 808cm−1. Anal. Calcd for C16H15Cl2NO: C, 60.35;
2-(4-Fluorophenyl)-6,6-dimethyl-6,7-dihydro-1H-indol- H,ꢀ 4.91;ꢀ N,ꢀ 4.54.ꢀ Found:ꢀ C,ꢀ 62.95;ꢀ H,ꢀ 5.14;ꢀ N,ꢀ 4.53.ꢀ GC-MSꢀ
4(5H)-one (8c): The title compound was obtained as a white purity=97.8%; tR=9.5 min.
powder (0.25g, 44%): Rf=0.49ꢀ (hexane–AcOEt,ꢀ 70ꢀ:ꢀ30);ꢀ mpꢀ
Antifungal Activity The title compounds 6, 7a–g, and
1
242°C. H-NMR (200MHz, DMSO-d6) δ: 1.04 (s, 6H), 2.22 (s, 8a–g were evaluated for their in vitro antifungal activity
2H), 2.69 (s, 2H), 6.67 (d, 1H, J=2.6Hz), 7.20 (dd, 2H, J=8.9, inꢀ comparisonꢀ withꢀ theꢀ commercialꢀ antifungalꢀ fluconazole,ꢀ
8.8Hz), 7.64–7.71 (m, 2H), 11.70 (s, 1H). MS (EI) m/z (%): itraconazole and amphotericin. The activities were measured
257 (M+, 88), 201 (68), 173 (100), 122 (28), 91 (15). IR (KBr) in terms of the minimal inhibitory concentrations (MIC)
ṽ: 3253, 3153, 3060, 2958, 2929, 2869, 1625, 1577, 1527, 1488, using the serial dilution method in 96-well microtest plates.
1409, 1230, 1160, 1141, 829, 794cm−1. GC-MS purity=99.3%; Test fungal strains were obtained from the Candida albicans
tR=8.7 min.
ATCC 24433, C. glabrata ATCC 66032, C. guilliermondii
2-(2,4-Difluorophenyl)-6,6-dimethyl-6,7-dihydro-1H-indol- ATCC 6260, C. krusei ATCC 14243, C. tropicalis ATCC
4(5H)-one (8d): The title compound was obtained as a white 750, C. parapsilosis ATCC 22019, Aspergillus niger ATCC
powder (0.4g, 60%): Rf=0.56ꢀ (hexane–AcOEt,ꢀ 70ꢀ:ꢀ30);ꢀ mpꢀ 16404, A. fumigatus ATCC 204305. The MIC determination
1
247°C. H-NMR (300MHz, DMSO-d6) δ: 1.04 (s, 6H), 2.23 (s, was performed according to method M27-A3 to yeast and
2H), 2.71 (s, 2H), 6.64 (t, 1H, J=2.8Hz), 7.16 (ddd, 1H, J=7.5, M38-Aꢀ toꢀ filamentousꢀ fungiꢀ asꢀ outlinedꢀ inꢀ theꢀ Clinicalꢀ andꢀ
9.5, 2.2Hz), 7.33 (ddd, 1H, J=9.2, 11.7, 2.6Hz), 7.74 (dd, 1H, Laboratory Standards Institute (CLSI) recommendations using
J=7.7, 2.6Hz) 11.71 (s, 1H). 13C-NMR (75MHz, DMSO-d6) δ: RPMI 1640 (Sigma) buffered with 0.165M N-(2-hydroxyethyl)-
28.1, 35.5, 51.7, 104.3, 104.7, 104.9, 111.9, 116.6, 119.4, 125.3, piperazine-N′-2-ethanesulfonic acid (HEPES) (Sigma) as the
127.8, 143.9, 156.5, 158.9, 159.8, 162.2, 192.1. MS (EI) m/z (%): testꢀ medium.ꢀ MICꢀ valuesꢀ wereꢀ definedꢀ asꢀ theꢀ concentrationsꢀ
275 (M+, 65), 219 (49), 191 (100), 164 (9), 140 (27), 120 (7). IR that inhibited growth of Candida spp. and Aspergillus spp. by
(KBr) ṽ: 3282, 3170, 2958, 2927, 2871, 1621, 1577, 1525, 1490, 50% relative to the control, as recommended by the M27-A3
1459, 1409, 1288, 1241, 1135, 971, 929, 850, 815cm−1. Anal. and M38-A guidelines.32,33) Test compounds were dissolved
Calcd for C16H15F2NO:ꢀ C,ꢀ 69.81;ꢀ H,ꢀ 5.49;ꢀ N,ꢀ 5.09.ꢀ Found:ꢀ C,ꢀ in EtOH and serially diluted in growth medium. The yeast
69.13; H, 5.56; N, 5.06. GC-MS purity=97.6%; tR=8.1 min.
2-(2-Chlorophenyl)-6,6-dimethyl-6,7-dihydro-1H-indol- 24–48h for Candida spp. and 48–72h for Aspergillus spp.
4(5H)-one (8e): The title compound was obtained as a white Molecular Modelingꢀ ꢀMolecularꢀ dockingꢀ computationsꢀ
cells were incubated at 37°C. Growth MIC was determined at
powder (0.5g, 48%) : Rf=0.60ꢀ (hexane–AcOEt,ꢀ 70ꢀ:ꢀ30);ꢀ mpꢀ wereꢀ carriedꢀ outꢀ onꢀ aꢀ Dellꢀ Precisionꢀ workstationꢀ withꢀ theꢀ
1
225°C. H-NMR (300MHz, DMSO-d6) δ: 1.05 (s, 6H), 2.24 RHEL X.0 operating system using Glide 5.6 (Schrodinger,
(s, 2H), 2.71 (s, 2H), 6.72 (d, 1H, J=2.6Hz), 7.28 (ddd, 1H, L.L.C.,ꢀNewꢀYork,ꢀNY,ꢀU.S.A.).
J=6.6, 8.3, 1.8Hz), 7.38 (ddd, 1H, J=7.5, 8.2, 1.6Hz), 7.52
Ligand Structure Preparation The molecular structures
(dd, 1H, J=7.2, 1.6Hz), 7.58 (dd, 1H, J=7.7, 1.6Hz), 11.6 (s, wereꢀ firstꢀ optimizedꢀ usingꢀ theꢀ Gaussianꢀ 03ꢀ softwareꢀ atꢀ theꢀ
1H). 13C-NMR (75MHz, DMSO-d6) δ: 28.1, 35.2, 35.8, 51.7, DFT/B3LYP/6-311G**ꢀ levelꢀ ofꢀ theory.ꢀ Theꢀ optimizationꢀ wasꢀ
105.9, 119.1, 127.3, 128.2, 128.7, 129.3, 130.1, 130.5, 143.5, verifiedꢀbyꢀshowingꢀthatꢀtheꢀcalculatedꢀvibrationalꢀfrequenciesꢀ
192.2. MS (EI) m/z (%): 273 (M+, 70), 217 (69), 189 (69), 138 were realistic.
(19), 69 (57), 28 (100). IR (KBr) ṽ: 3224, 3147, 3060, 2960,
Protein Structure Preparation The X-ray crystal struc-
2925, 2892, 1641, 1488, 1367, 1220, 1164, 1124, 1079, 939, 752, tureꢀ ofꢀ MtCYP51ꢀ inꢀ complexꢀ withꢀ theꢀ fluconazoleꢀ (Flu)ꢀ
723cm−1. Anal. Calcd for C16H16ClNO: C, 70.20; H, 5.89; N, ligand (PDB ID: 1EA1) was obtained from the RCSB PDB
5.12.ꢀFound:ꢀC,ꢀ69.37;ꢀH,ꢀ5.98;ꢀN,ꢀ5.03.ꢀGC-MSꢀpurity=97.9%; andꢀusedꢀtoꢀmodelꢀtheꢀproteinꢀstructureꢀexaminedꢀinꢀtheꢀpres-
tR=8.8min.
entꢀ study.ꢀ Theꢀ proteinꢀ wasꢀ optimizedꢀ forꢀ dockingꢀ usingꢀ theꢀ
2-(4-Chlorophenyl)-6,6-dimethyl-6,7-dihydro-1H-indol- “ProteinꢀPreparationꢀWizard”ꢀandꢀ“Prime-RefinementꢀUtility”ꢀ
4(5H)-one (8f): The title compound was obtained as a white of Maestro 9.1 (Schrodinger, L.L.C.). The heme cofactor and
powder (0.7g, 48%): Rf=0.53ꢀ (hexane–AcOEt,ꢀ 70ꢀ:ꢀ30);ꢀ mpꢀ the iron charges and connectivity were carefully inspected. A
1
225°C. H-NMR (300MHz, DMSO-d6) δ: 1.04 (s, 6H), 2.22 zero-order bond was present between the heme iron and the
(s, 2H), 2.69 (s, 2H), 6.75 (d, 1H, J=1.1Hz), 7.41 (dd, 2H, fluconazoleꢀ ligand.ꢀ Thisꢀ bondꢀ wasꢀ brokenꢀ priorꢀ toꢀ “picking”ꢀ
J=8.6Hz), 7.67 (dd, 2H, J=8.6Hz), 11.77 (s, 1H). MS (EI) the ligand during the Glide grid generation.
m/z (%): 273 (M+, 30), 217 (24), 189 (30), 139 (100), 111 (13),
Glide Dockingꢀ ꢀTheꢀ XPꢀ Glideꢀ dockingꢀ methodꢀ wasꢀ usedꢀ
97 (12), 69 (8), 28 (11). IR (KBr) ṽ: 3241, 2956, 2927, 2867, toꢀ dockꢀ compoundsꢀ 6, 7a–g, and 8a–g into the MtCYP51
1625, 1486, 1405, 1222, 1139, 1091, 1033, 933, 819, 794cm−1. bindingꢀsite.ꢀTheꢀdockingꢀmethodologyꢀisꢀdescribedꢀIꢀdetailꢀinꢀ
GC-MS purity=99.0%; tR=9.4 min.
theꢀmainꢀtext.ꢀTheꢀXPꢀmodeꢀisꢀaꢀrefinementꢀtoolꢀdesignedꢀforꢀ
2-(2,4-Dichlorophenyl)-6,6-dimethyl-6,7-dihydro-1H-indol- use only on good ligand conformations. The XP mode is less
4(5H)-one (8g): The title compound was obtained as a white forgiving than the regular Glide mode, so it effectively screens
powder (0.2g, 89%): Rf=0.60ꢀ (hexane–AcOEt,ꢀ 70ꢀ:ꢀ30);ꢀ mpꢀ out false positives. The XP mode is designed to identify ac-
1
290°C. H-NMR (300MHz, DMSO-d6) δ: 1.04 (s, 6H), 2.24 tive compounds that bind to a particular conformation of the
(s, 2H), 2.70 (s, 2H), 6.76 (d, 1H, J=4.6Hz), 7.48 (dd, 1H, receptor.28)

24
Vol. 62, No. 1
Yuan T. T., Lee Y. S., Chen P., Chi W. K., Yang J. Y., Huang H. J.,
Liao C. B., Huang J. J., J. Med. Chem., 53, 5929–5941 (2010).
21) Vanotti E., Amici R., Bargiotti A., Berthelsen J., Bosotti R., Cia-
volellaꢀA.,ꢀCirlaꢀA.,ꢀCristianiꢀC.,ꢀD’AlessioꢀR.,ꢀForteꢀB.,ꢀIsacchiꢀA.,ꢀ
Martina K., Menichincheri M., Molinari A., Montagnoli A., Orsini
P.,ꢀPillanꢀA.,ꢀRolettoꢀF.,ꢀScolaroꢀA.,ꢀTibollaꢀM.,ꢀValsasinaꢀB.,ꢀVarasiꢀ
M., Volpi D., Santocanale C., J. Med. Chem., 51, 487–501 (2008).
22)ꢀ Huangꢀ K.ꢀ H.,ꢀ Vealꢀ J.ꢀ M.,ꢀ Faddenꢀ R.ꢀ P.,ꢀ Riceꢀ J.ꢀ W.,ꢀ Eavesꢀ J.,ꢀ Stra-
chanꢀJ.ꢀP.,ꢀBarabaszꢀA.ꢀF.,ꢀFoleyꢀB.ꢀE.,ꢀBartaꢀT.ꢀE.,ꢀMaꢀW.,ꢀSilinskiꢀ
M.ꢀ A.,ꢀ Huꢀ M.,ꢀ Partridgeꢀ J.ꢀ M.,ꢀ Scottꢀ A.,ꢀ DuBoisꢀ L.ꢀ G.,ꢀ Freedꢀ T.,ꢀ
SteedꢀP.ꢀM.,ꢀOmmenꢀA.ꢀJ.,ꢀSmithꢀE.ꢀD.,ꢀHughesꢀP.ꢀF.,ꢀWoodwardꢀA.ꢀ
R.,ꢀHansonꢀG.ꢀJ.,ꢀMcCallꢀW.ꢀS.,ꢀMarkworthꢀC.ꢀJ.,ꢀHinkleyꢀL.,ꢀJenksꢀ
M.,ꢀGengꢀL.,ꢀLewisꢀM.,ꢀOttoꢀJ.,ꢀPronkꢀB.,ꢀVerleysenꢀK.,ꢀHallꢀS.ꢀE.,ꢀ
J. Med. Chem., 52, 4288–4305 (2009).
Acknowledgments Weꢀ thankꢀ M.ꢀ E.ꢀ Núñezꢀ Pastranaꢀ andꢀ
P.ꢀ Hernández-Moralesꢀ fromꢀ Facultadꢀ deꢀ Cienciasꢀ Químicas,ꢀ
CIEP,ꢀ Universidadꢀ Autónomaꢀ deꢀ Sanꢀ Luisꢀ Potosí,ꢀ forꢀ techni-
calꢀ assistance.ꢀ Financialꢀ supportꢀ fromꢀ FAI-UASLPꢀ Grantsꢀ
C10-FAI-05-16.43ꢀ andꢀ C10-FAI-05-14.41ꢀ areꢀ gratefullyꢀ ac-
knowledged.ꢀThanksꢀtoꢀCONACYTꢀforꢀaꢀgraduateꢀfellowshipꢀ
217383ꢀforꢀR.ꢀGonzález-ChávezꢀandꢀweꢀalsoꢀthankꢀR.ꢀO.ꢀTor-
res, E. Garcia, E. Bonilla, H. Rios, and J. Perez for technical
assistanceꢀfromꢀtheꢀInstitutoꢀdeꢀQuímica,ꢀUNAM.
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