Synthesis of new piperazinyl-pyrrolo[1,2-: A] quinoxaline derivatives as inhibitors of Candida albicans multidrug transporters by a Buchwald-Hartwig cross-coupling reaction

Article abstract of DOI:10.1039/c9ra09348f

Two series of piperazinyl-pyrrolo[1,2-a]quinoxaline derivatives were prepared via a Buchwald-Hartwig cross-coupling reaction and then evaluated for their ability to inhibit the drug efflux activity of CaCdr1p and CaMdr1p transporters of Candida albicans overexpressed in a Saccharomyces cerevisiae strain. In the initial screening of twenty-nine piperazinyl-pyrrolo[1,2-a]quinoxaline derivatives, twenty-three compounds behaved as dual inhibitors of CaCdr1p and CaMdr1p. Only four compounds showed exclusive inhibition of CaCdr1p or CaMdr1p. Further biological investigations were developed and for example, their antifungal potential was evaluated by measuring the growth of control yeast cells (AD1-8u^-) and efflux pump-overexpressing cells (AD-CDR1 and AD-MDR1) after exposition to variable concentrations of the tested compounds. The MIC₈₀ values of nineteen compounds ranging from 100 to 901 μM for AD-CDR1 demonstrated that relative resistance index (RI) values were between 8 and 274. In comparison, only seven compounds had RI values superior to 4 in cells overexpressing Mdr1p. These results indicated substrate behavior for nineteen compounds for CaCdr1p and seven compounds for CaMdr1p, as these compounds were transported via MDR transporter overexpressing cells and not by the AD1-8u^- cells. Finally, in a combination assay with fluconazole, two compounds (1d and 1f) have shown a synergistic effect (fractional inhibitory concentration index (FICI) values ≤ 0.5) at micromolar concentrations in the AD-MDR1 yeast strain overexpressing CaMdr1p-protein, indicating an excellent potency toward chemosensitization.

Full text of DOI:10.1039/c9ra09348f

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Synthesis of new piperazinyl-pyrrolo[1,2-a]
quinoxaline derivatives as inhibitors of Candida
albicans multidrug transporters by a Buchwald–
Hartwig cross-coupling reaction†
Cite this: RSC Adv., 2020, 10, 2915
Jean Guillon,‡a Shweta Nim,‡b Stephane Moreau,a Luisa Ronga,a
´
Solene Savrimoutou,a Elisabeth Thivet,a Mathieu Marchivie,c Attilio Di Pietro,d
`
f
Rajendra Prasade and Marc Le Borgne
*
Two series of piperazinyl-pyrrolo[1,2-a]quinoxaline derivatives were prepared via a Buchwald–Hartwig
cross-coupling reaction and then evaluated for their ability to inhibit the drug efflux activity of CaCdr1p
and CaMdr1p transporters of Candida albicans overexpressed in a Saccharomyces cerevisiae strain. In
the initial screening of twenty-nine piperazinyl-pyrrolo[1,2-a]quinoxaline derivatives, twenty-three
compounds behaved as dual inhibitors of CaCdr1p and CaMdr1p. Only four compounds showed
exclusive inhibition of CaCdr1p or CaMdr1p. Further biological investigations were developed and for
example, their antifungal potential was evaluated by measuring the growth of control yeast cells (AD1-
8uꢀ) and efflux pump-overexpressing cells (AD-CDR1 and AD-MDR1) after exposition to variable
concentrations of the tested compounds. The MIC80 values of nineteen compounds ranging from 100 to
901 mM for AD-CDR1 demonstrated that relative resistance index (RI) values were between 8 and 274. In
comparison, only seven compounds had RI values superior to 4 in cells overexpressing Mdr1p. These
results indicated substrate behavior for nineteen compounds for CaCdr1p and seven compounds for
CaMdr1p, as these compounds were transported via MDR transporter overexpressing cells and not by
the AD1-8uꢀ cells. Finally, in a combination assay with fluconazole, two compounds (1d and 1f) have
shown a synergistic effect (fractional inhibitory concentration index (FICI) values # 0.5) at micromolar
concentrations in the AD-MDR1 yeast strain overexpressing CaMdr1p-protein, indicating an excellent
potency toward chemosensitization.
Received 10th November 2019
Accepted 30th December 2019
DOI: 10.1039/c9ra09348f
rsc.li/rsc-advances
invasive fungal infections include Candida, Aspergillus, and
Cryptococcus,2 with opportunistic strains of Candida albicans
Introduction
accounting for approximately 50–60% causes of candidiasis,
particularly in immunocompromised patients. The treatment of
these Candida infections relies heavily on azole antifungal
agents,3 for which widespread and prolonged use has led to the
rapid emergence of multidrug resistant (MDR) isolates of C.
albicans as well as of non-albicans species.4 Various mecha-
nisms potentially contributing to the development of MDR have
been identied, and the induction of genes encoding drug-
efflux pumps, like the primary ATP binding cassette (ABC)
transporter genes CaCDR1 and CaCDR2 and the secondary
major-facilitator superfamily (MFS) transporter gene CaMDR1,
has been shown to play a prominent role in the development of
resistance to antifungal drugs.5–7 Overexpression of these pump
proteins may lead to an increased efflux of drug substrates in
MDR clinical isolates.4,8,9
Transplantation surgery, cancer chemotherapy, and HIV infec-
tions have led to a worldwide rise of the immunocompromised
population, and hence also of bacterial and fungal opportu-
nistic infections.1 The fungal genera most oen associated with
aUniv. Bordeaux, INSERM U1212 – UMR CNRS 5320, ARNA Laboratory, UFR des
Sciences Pharmaceutiques, F-33076 Bordeaux Cedex, France
bSchool of Life Sciences, Jawaharlal Nehru University, 110067 New Delhi, India
cCNRS, Univ. Bordeaux, Bordeaux INP, ICMCB, UMR 5026, F-33608 Pessac Cedex,
France
dDRMP Group, IBCP, UMR 5086 (MMSB), CNRS/Lyon I University, 69367 Lyon, France
eAmity Institute of Integrative Sciences and Health, Amity University, Education Valley,
Gurgaon 122413, India
f
´
´
´
Universite de Lyon, Universite Claude Bernard Lyon 1, Faculte de Pharmacie – ISPB,
´
EA 4446 Bioactive Molecules and Medicinal Chemistry, SFR Sante Lyon-Est CNRS
UMS3453 – INSERM US7, Lyon, France. E-mail: marc.le-borgne@univ-lyon1.fr
† Electronic supplementary information (ESI) available. CCDC 891808 and
891809. For ESI and crystallographic data in CIF or other electronic format see
DOI: 10.1039/c9ra09348f
The potent modulators of multidrug transporter CaCdr1p
such as the immunosuppressants cyclosporin, FK520 and
FK506, the natural polyphenol curcumin, the quorum-sensing
‡ These authors contributed equally to this work.
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molecule farnesol, the antabuse drug disulram, the antibiotic cesium carbonate as the base, and THF–H2O as the solvent
milbemycin, some synthetic-D-octapeptides, the anti- system.23 Reaction of 6 and one equivalent of N-bromosuccini-
inammatory drug ibuprofen and the antibacterial unnarmi- mide (NBS) afforded the 1-bromo-4-phenylpyrrolo[1,2-a]qui-
cins have been displayed to prevent drug extrusion and restore noxaline 7 as the sole reaction product.28 The Buchwald–
fungicidal synergism with the azoles and other drugs.10–14 Hartwig Pd-catalyzed amination of the 1-bromo-4-phenylpyrrolo
Unlike CaCdr1p, there is only a handful number of chemo- [1,2-a]quinoxaline 7 using Pd2(dba)3 as catalyst with BINAP as
sensitizers in case of CaMdr1p such as verapamil and enniantin the ligand was then investigated. Under these conditions,
B.15,16 Recently, a further screening from a library of synthetic various substituted piperazines were successfully coupled with
aromatic compounds sharing a cyclobutene-dione moiety was 7 to give the desired piperazinyl-pyrrolo[1,2-a]quinoxaline
investigated for the discovery of new inhibitors of MFS and ABC derivatives 1a–i by using t-BuONa as a base and toluene as the
transporters of C. albicans. A few specic inhibitors of MFS solvent at 100 C.39–41
ꢁ
transporter CaMdr1p were then identied.17 Therefore, the
The 3D structural determinations of 1a and 1h were estab-
search for novel inhibitors able to block the drug extrusion lished by X-ray crystallography (Fig. 2 and 3), and conrmed the
mediated by these efflux proteins represents an attractive structures in the solid state as anticipated on the basis of NMR
approach to reverse MDR.
data.
By using the same cross-coupling catalyzed methodology,
The pyrrolo[1,2-a]quinoxaline heterocyclic framework
constitutes the basis of an important class of compounds pos- the Pd-catalyzed coupling of substituted piperazines with the 4-
sessing interesting biological activities. These compounds have chloropyrrolo[1,2-a]quinoxalines 5a–e30 led to the new 4-(4-
been reported as key intermediates for the assembly of several substituted-piperazinyl)-4-phenylpyrrolo[1,2-a]quinoxaline 1j–w
heterocycles including antipsychotic agent,18 anti-HIV agent,19 (Scheme 2). These compounds 1a–w were then converted into
adenosine A3 receptor modulator,20 antiparasitic agents,21–25 their hydrochloride or oxalate salts (Table 1).
and antitumor agents.26–31 We also previously demonstrated
The series of piperazinylalcohol pyrrolo[1,2-a]quinoxaline
that the pyrrolo[1,2-a]quinoxaline heterocyclic scaffold could derivatives 2a–f was synthesized as previously described by the
lead to the preparation of bacterial multidrug resistance pump authors starting from 5a or 8 (Schemes 3 and 4).42
inhibitors.32,33
In this context and as part of a programme on the develop-
ment of new efflux pump inhibitors (EPIs), we decided to
Biological assays
broaden the structural diversity and used the pyrrolo[1,2-a] Piperazinyl-pyrrolo[1,2-a]quinoxaline derivatives were evaluated
quinoxaline moiety as a template for the design of new deriva- for their ability to inhibit the drug-efflux activity of CaCdr1p and
tives 1 and 2 in which a piperazine is incorporated in position 1, CaMdr1p transporters of C. albicans overexpressed in a Saccha-
4 or 9 of the heterocyclic core in analogy with the EPI pyrimidine romyces cerevisiae strain. The transport assay was performed by
and quinoline derivatives I–III, quinine and MS-209 used in the monitoring Nile Red (NR) efflux in cells overexpressing the
various multidrug resistance therapies (Fig. 1).34–38
referred efflux pumps. In this assay, compounds were compared
to wild-type control cells, and when the Nile Red efflux was
lower than 60%, the compound was indeed considered to have
inhibitory activity (Fig. 4). The efflux study performed on twenty-
nine compounds revealed the total of twenty-three compounds
Results and discussion
Chemistry
The reported piperazinyl-pyrrolo[1,2-a]quinoxaline derivatives (1a, 1b, 1e, 1g, 1h, 1i, 1j, 1k, 1l, 1m, 1n, 1o, 1p, 1s, 1t, 1u, 1v, 1w,
1a–i were synthesized in ve steps from 2-nitroaniline (Scheme 2a, 2c, 2d, 2e, 2f) that behaved as dual inhibitors of CaCdr1p
1). Preparation of 1-(2-nitrophenyl)pyrrole 2 was performed and CaMdr1p. Compounds 2c > 2d > 1j ¼ 1l ¼ 1o ¼ 1v revealed
according to the Clauson–Kaas reaction run under micro-wave the strongest inhibitory activity of Cdr1p efflux pump, ranging
irradiation starting from 2-nitroaniline and 2,5-dimethoxyte- from 77 to 82%. Regarding S. cerevisiae cells overexpressing
trahydrofuran in acetic acid. This pathway partially involved Mdr1p, the most active compounds were 2f > 2c > 1a ¼ 1b ¼ 1j ¼
synthetic methodologies already described by our group.21–24,28 1k ¼ 1n ¼ 1s ¼ 1t ¼ 2a ¼ 2d > 1l > 1o ranging from 78 to 84%
The resulting 1-(2-nitrophenyl)pyrrole intermediate 2 was efflux inhibition. This initial screening identied compounds
subsequently reduced into the attempted 1-(2-aminophenyl) 1u, 1w and 2e as dual inhibitors with intermediate potency.
pyrrole 3 using a sodium borohydride-copper(II) sulfate treat- Compounds 1e, 1g, 1h, 1i, 1m, 1p nally revealed a weak
ment in ethanol at room temperature. This NaBH4–CuSO4 inhibitory activity in both cell lines overexpressing the two types
system was found to be quite powerful in reducing our aromatic of efflux pumps. Compound 1c and 1f showed exclusive but
nitro group with excellent yield (85%). The reaction of 3 with weak inhibition of CaCdr1p, whereas compounds 1d and 1q
triphosgene in toluene gave the lactam 4, which was subse- demonstrated their exclusive impact on CaMdr1p with efflux
quently chlorodehydroxylated with phosphorous oxychloride, inhibition ranged from 29 to 50%. Compounds 1r and 2b are
leading to the 4-chloropyrrolo[1,2-a]quinoxaline 5a. The 4-phe- not active on both efflux pumps.
nylpyrrolo[1,2-a]quinoxaline 6 was easily prepared by a direct
Suzuki–Miyaura cross-coupling reaction of
The antifungal potential of all piperazinyl-pyrrolo[1,2-a]qui-
4-chlor- noxaline derivatives was also evaluated by measuring the
opyrroloquinoxalines 5a with potassium phenyltriuoroborate growth of control yeast cells (AD1-8uꢀ) and efflux pump-
performed in the presence of PdCl2(dppf)$CH2Cl2 as a catalyst, overexpressing cells (AD-CDR1 and AD-MDR1) when exposed
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Fig. 1 Structures of previously described EPIs I–III, quinine and MS-209, and new synthesized substituted pyrrolo[1,2-a]quinoxaline derivatives 1–2.
to variable concentrations of the tested compounds for 48 h. needed to decrease 80% of cells growth (ESI Table S1†). In case
The yeast growth in the absence of inhibitor was considered as of the control yeast cells (AD1-8uꢀ), compounds did not reveal
100%. The results were expressed as MIC80, the concentration any signicant antifungal activity, as demonstrated by their
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Scheme 1 Synthesis of 1-(4-substituted-piperazinyl)-4-phenylpyrrolo[1,2-a]quinoxalines 1a–i; reagents and conditions: (i) 2,5-diMeOTHF,
AcOH, D; (ii) CuSO4/NaBH4, EtOH, RT; (iii) (Cl3CO)2CO, toluene, D; (iv) POCl3, D; (v) (OHC–C6H4)–BF3K, PdCl2(dppf)$CH2Cl2, Cs2CO3, THF–H2O,
D; (vi) NBS, CH2Cl2, RT; (vii) R-piperazine, Pd2(dba)3, BINAP, t-BuONa, toluene, 100 ꢁC.
relative resistance index (RI) values close to 1. However the compounds (1c, 1e, 1g, 1j, 1k, 1m, 1o) had RI values superior to
MIC80 values of nineteen selected compounds (Table 2) ranging 4 in cells overexpressing Mdr1p. These results indicate the
from 100 to 901 mM for AD-CDR1 demonstrated that RI values substrate behavior for both 19 compounds for CaCdr1p and
were comprised between 8 and 274. In comparison, only seven seven compounds for CaMdr1p, as these compounds are
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Fig. 2 The ORTEP drawing of pyrrolo[1,2-a]quinoxaline 1a with thermal ellipsoids at 30% level.
transported via MDR transporter overexpressing cells and not as the fractional inhibitory concentration index (FICI), are
by the AD1-8uꢀ cells. Interestingly, all the nineteen compounds summarized in ESI Table S2.† FICI values # 0.5 indicate
were observed to inhibit the Nile Red transport from the AD- synergistic interaction between the inhibitor and the
CDR1 cells and simultaneously behaved as substrate of substrate.44 It was observed that the two piperazinyl-pyrrolo[1,2-
CaCdr1p (Table 2). Then it could be suggested that the Nile Red a]quinoxaline derivatives 1d and 1f with FIC ¼ 0.0076 and 0.25,
and these compounds seem to share the same drug binding respectively, displayed strong synergistic effects (FICI ¼ 0.15
pocket of CDR1, undergoing the kinetics of competitive inhi- and 0.4, respectively) when both are combined with uconazole
bition. By contrast, in the case of AD-MDR1 cells, the route of (FIC ¼ 0.15) in the AD-MDR1 yeast strain overexpressing the
efflux transport for Nile Red and these compounds did not MFS CaMdr1p, decreasing 129-fold the MIC80 of the antifungal
overlap as only seven compounds showed substrate behavior. agent. High FICI values ($1) were found for the remaining
Here the results suggest the presence of an allosteric drug compounds (ESI Table S2†). Similarly, high FICI values were
binding pocket for MDR1 and thus following the path of non- also found in the AD-CDR1.
competitive kinetics.
The effect of the compounds 1d and 1f was examined by
The ability of the compounds to sensitize yeast growth to the confocal imaging of GFP-tagged Cdr1p and Mdr1p, and revealed
antifungal agent uconazole was evaluated by the checkerboard the non-effect of these compounds on the intactness of the
method.43 In this assay, the control (AD1-8uꢀ) cells and the overexpressing strains AD-CDR1 and AD-MDR1 (Fig. 5).
CaCdr1p- and CaMdr1p-overexpressing cells were grown in the
Finally, compounds 1d and 1f were further evaluated for
presence of either uconazole alone or a combination therapy their ability to chemosensitize the azole-resistant clinical isolate
(efflux pump inhibitor plus uconazole). The results, expressed (F5) of C. albicans together with the azole-susceptible strain
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Fig. 3 The ORTEP drawing of pyrrolo[1,2-a]quinoxaline 1h (molecules A and B) with thermal ellipsoids at 30% level.
(F2).45,46 As can be observed in Table 3, when combined with derivatives 1a–w. In parallel, six other derivatives containing as
uconazole, compounds 1d and 1f (FICI ¼ 0.6 and 0.78, well a quinoxaline moiety (compounds 2a–f, previously syn-
respectively) were able to reduce the effective concentration of thetized42) were added to this study. Then twenty-nine
uconazole.
compounds have been selected on their potential to inhibit
About the structure–activity relationships on both series of fungal multidrug resistance pumps as pyrrolo[1,2-a]quinoxaline
piperazinyl-pyrrolo[1,2-a]quinoxaline derivatives, the three best template was already used and efficient for the inhibition of
dual inhibitors belonging to the series 2 (2c, 2d, 2f) has bacterial efflux pumps.32,33
a common structural feature, namely the presence of a spacer
Currently, we broadened our horizon to look into the role of
(oxy-propan-2-ol or amino-propan-2-ol) between the tricyclic these compounds to inhibit the CaCdr1p and CaMdr1p trans-
scaffold and the piperazinyl moiety. On the other hand, exclu- porters in pathogenic yeast C. albicans. Our study based on the
sive inhibitors of CaCdr1p (1c, 1f) or CaMdr1p (1d, 1q) have the biological assays corroborated with the piperazinyl-pyrrolo[1,2-
piperazinyl moiety directly linked to the pyrroloquinoxaline. a]quinoxaline derivatives to be the putative and promising
Four of these compounds (2c, 2f, 1d and 2d) have a benzhydryl modulators of efflux pumps in the pathogenic yeast C. albicans.
substituent or related on the piperazine ring. Further pharma- The results of this work have demonstrated that most of the
comodulation works will be carried out to extend and deepen compounds could inhibit the efflux of Nile Red mediated by
our knowledge.
both the ABC transporter CaCdr1p and the MFS pump
CaMdr1p. Some compounds were able to inhibit specically the
efflux of Nile Red without being themselves substrates of the
efflux-pump proteins CaMdr1p and CaMdr1p. This assumption
was corroborated by the relative resistance index values close to
1 obtained from the cytotoxicity assays, showing that the pres-
ence of efflux-pump proteins did not affect the growth and the
viability of yeast cells.
Conclusions
The chemical approach by Buchwald–Hartwig Pd-catalyzed
amination of the 1-bromo-4-phenylpyrrolo[1,2-a]quinoxaline 7
or the 4-chloropyrrolo[1,2-a]quinoxalines 5a–e was successfully
used to access to new piperazinyl-pyrrolo[1,2-a]quinoxaline
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Scheme 2 Synthesis of 4-(4-substituted-piperazinyl)-4-phenylpyrrolo[1,2-a]quinoxalines 1j–w; reagents and conditions: (i) R-piperazine,
Pd2(dba)3, BINAP, t-BuONa, toluene, 100 ꢁC.
In S. cerevisiae cells expressing CaCdr1p and CaMdr1p, the not observed with compound 1f. In this context, it is important
greater inhibitory effect on Nile Red efflux was obtained with to mention that each drug/compound may interact differently
compounds 2c–f (5 < MIC80 (mM) < 14) on CaMdr1p. For with different amino acid residues within the binding pocket of
compounds 2c–e, the best three compounds (MIC80 ¼ 5–6 mM), CaMdr1p, which could explain the different behavior of Nile
the main structural feature is the presence of a bulky group R Red and uconazole with these compounds. As no synergy has
(e.g. compound 2c with a diphenylmethyl moiety).
been found in the clinical isolate F5 overexpressing CaMdr1p,
In the combination assay with uconazole, the two a signicant decreasing of the effective concentration of the
compounds 1d and 1f have shown a synergistic effect (FICI antifungal agent was also observed, corroborating the results
values # 0.5) at micromolar concentrations in the AD-MDR1 obtained in the AD-MDR1 strain. It is also interesting to note
yeast strain overexpressing CaMdr1p-protein, indicating an that compound 1d is the unique active compound bearing
excellent potency toward chemosensitization. Interestingly, a benzhydryl moiety in the sub-series 1.
compound 1d showed exclusive and maximum Nile Red efflux
Finally, this study has shown that piperazinyl-pyrrolo[1,2-a]
inhibition on AD-MDR1 strain and showed excellent chemo- quinoxaline derivatives are able to reverse antifungal resistance,
sensitization in the presence of uconazole, whereas this was mediated by efflux pumps belonging to both ABC and MFS
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Table 1 Physical properties of the final amines 1a–w
superfamilies of transporters of the pathogenic yeast C. albi-
cans. Therefore, at non-inhibitory concentrations, these
compounds stand as wise candidates chosen to be potential
modulators in MDR reversal. For example, compound 1d could
offer a new treatment strategy known as combo-therapy in the
use of new azole antifungals recently designed.47,48 Nevertheless
further chemical modications will be carried out to synthetize
a second generation of piperazinyl-pyrrolo[1,2-a]quinoxaline
derivatives designed specically as EPIs of the pathogenic yeast
C. albicans. Once again, compound 1d will be investigated for
assessing the structural importance of its benzhydryl moiety
(e.g. nature and position of additional substituents). By simi-
larity, a pharmacomodulation study around compound 2c will
be also managed, using rational drug design tools such as 3D
structural characteristics of efflux pumps49 and recent chemical
features50 to design new piperazinyl-pyrrolo[1,2-a]quinoxaline
derivatives as specic EPIs of C. albicans.
Compound
a,b,cSalt
dmp (ꢁC)
eYield (%)
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1n
1o
1p
1q
1r
3HCl
3HCl
114
88
71
68
58
61
52
59
53
64
59
66
61
72
74
67
82
59
63
67
64
71
60
75
62
60
(COOH)2, H2O
(COOH)2, H2O
(COOH)2, H2O
(COOH)2, H2O
(COOH)2, H2O
(COOH)2, H2O
(COOH)2, H2O
2(COOH)2
2(COOH)2
2(COOH)2
2(COOH)2
2(COOH)2
2(COOH)2
2(COOH)2
2(COOH)2
2(COOH)2
2(COOH)2
2(COOH)2
2(COOH)2
2(COOH)2
2(COOH)2
2(COOH)2
172
109
175
156
241
238
93
203
144
189
236
228
215
194
190
189
212
225
190
180
251
253
Experimental section
Chemistry
1s
1t
1u
1v
1w
General information. Commercially reagents were used as
received without additional purication. Melting points were
determined with an SM-LUX-POL Leitz hot-stage microscope
and are uncorrected. NMR spectra were recorded with tetra-
methylsilane as an internal standard using a Bruker Avance 300
spectrometer. Splitting patterns have been designated as
follows: s ¼ singlet; bs ¼ broad singlet; d ¼ doublet; t ¼ triplet;
q ¼ quartet; dd ¼ double doublet; ddd ¼ double double
doublet; dt ¼ double triplet; m ¼ multiplet. For all compounds
1a–w, all NMR spectra are available in the ESI (Fig. S1–S45†).
Analytical TLC were carried out on 0.25 precoated silica gel
plates (POLYGRAM SIL G/UV254) and visualization of
compounds aer UV light irradiation. Silica gel 60 (70–230
a
The amines 1a, b were dissolved in 20 mL of anhydrous diethyl ether,
and treated with HCl gas. Aer crystallization, the ammonium chlorides
were collected by ltration and were washed with Et2O, then dried under
b
reduced pressure. The amines 1c–w were dissolved in 30 mL of 2-
propanol, heated to boiling, and treated with oxalic acid (4 or 5
equiv., based on the amount of the starting material). The oxalate
salts crystallized upon cooling were collected by ltration, and were
c
washed with 2-propanol and Et2O.
The stoichiometry and
composition of the salts were determined by elemental analyses
d
(within ꢂ 0.4% of the theoretical values). Crystallization solvent: 2-
e
PrOH–H2O. The yields only included the conversions into the
ammonium chlorides or oxalates.
Scheme 3 Synthesis of piperazinylalcohol-pyrrolo[1,2-a]quinoxalines 2a–e; reagents and conditions: (i) K2CO3, DMF, 120 ꢁC.
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Scheme 4 Synthesis of piperazinylalcohol-pyrrolo[1,2-a]quinoxaline 2f; reagents and conditions: (i) (C6H5)2CH-piperazine, isopropanol, D.
mesh) was used for column chromatography. Elemental anal- pip), 3.18–3.09 (m, 2H, CH2-pip). 13C NMR (CDCl3) d 156.1 (C-4),
yses were found within ꢂ0.4% of the theoretical values.
152.6 (C-100), 145.1 (C-3a), 139.4 (C-5a), 138.4 (C-10), 131.2 (C-7),
General procedure: synthesis of 1-piperazinyl-4-phenyl- 130.7 (C-8, C-300 and C-500), 130.2 (C-30 and C-50), 130.1 (C-9a),
pyrrolo[1,2-a]quinoxalines 1a–w. To a solution of 2.5 mmol of 1- 130.0 (C-20 and C-60), 127.7 (C-9), 126.5 (C-6), 123.5 (C-1), 121.8
bromo-4-phenylpyrrolo[1,2-a]quinoxaline 7 or 4-chloropyrrolo (C-40), 117.9 (C-400), 117.8 (C-200 and C-600), 109.9 (C-2), 104.7 (C-
[1,2-a]quinoxaline 5a–e in 18 mL of anhydrous toluene were 3), 53.8 (CH2-pip), 50.6 (CH2-pip). Anal. calcd for C27H24N4: C,
added 3 mmol (1.2 equiv.) of substituted piperazine, 0.333 g 80.17; H, 5.98; N, 13.85. Found: C, 81.04; H, 6.02; N, 13.94.
(3.5 mmol, 1.4 equiv.) of t-BuONa, 0.046 g (0.05 mmol, 0.02
1-[4-(2-Methoxyphenyl)piperazinyl]-4-phenylpyrrolo[1,2-a]qui-
equiv.) of tris(dibenzylideneacetone)dipalladium (0) [Pd2(dba)3] noxaline (1b). Yellow crystals (21%) mp 38 ꢁC. 1H NMR (CDCl3)
and 0.062 g (0.1 mmol, 0.04 equiv.) of 2,20-bis(diphenylphos- d 9.23 (dd, 1H, J ¼ 7.85 and 2.40 Hz, H-9), 8.01 (dd, 1H, J ¼ 7.85
phino)-1,10-binaphthalene [(ꢂ)-BINAP]. The reaction mixture and 2.40 Hz, H-6), 7.96–7.94 (m, 2H, H-20 and H-60), 7.56–7.53
was then heated at 100 ꢁC during 15 h. Aer cooling, the (m, 3H, H-30, H-40 and H-50), 7.52–7.42 (m, 2H, H-7 and H-8),
mixture was diluted with methylene chloride. The reaction 7.13–7.06 (m, 2H, H-400 and H-500), 7.05–7.01 (m, 1H, H-600),
mixture was then ltered on Celite and then diluted with water. 6.96 (dd, 1H, J ¼ 8.50 and 2.15 Hz, H-300), 6.92 (d, 1H, J ¼
The organic layer was separated and the aqueous layer was 4.30 Hz, H-2), 6.61 (d, 1H, J ¼ 4.30 Hz, H-3), 3.93 (s, 3H, CH3O),
extracted with methylene chloride (20 mL). The organic layers 3.65–3.61 (m, 2H, CH2-pip), 3.49–3.43 (m, 2H, CH2-pip), 3.31–
were collected, dried over magnesium sulfate, ltered and 3.12 (m, 4H, CH2-pip). 13C NMR (CDCl3) d 156.2 (C-4), 153.7 (C-
evaporated to dryness. Column chromatography of the residue 200), 145.2 (C-100), 142.4 (C-3a), 139.7 (C-5a), 138.7 (C-10), 131.0 (C-
on silica gel using ethyl acetate–methanol (8/2) as eluent gave 7), 130.8 (C-8), 130.2 (C-9a), 130.1 (C-30 and C-50), 129.9 (C-20 and
the nal product 1.
C-60), 127.5 (C-9), 126.3 (C-6), 124.8 (C-500), 123.5 (C-1), 122.4 (C-
1-(4-Phenylpiperazinyl)-4-phenylpyrrolo[1,2-a]quinoxaline (1a). 40), 119.7 (C-400), 117.9 (C-600), 112.7 (C-300), 109.5 (C-2), 104.7 (C-
1
ꢁ
Yellow crystals (48%) mp 203 C. H NMR (CDCl3) d 9.21 (dd, 3), 56.8 (CH3O), 54.0 (CH2-pip), 52.0 (CH2-pip). Anal. calcd for
1H, J ¼ 8.10 and 2.80 Hz, H-9), 8.02 (dd, 1H, J ¼ 8.10 and
2.80 Hz, H-6), 7.96–7.93 (m, 2H, H-20 and H-60), 7.57–7.52 (m, 5.94; N, 13.04.
3H, H-30, H-40 and H-50), 7.47–7.43 (m, 2H, H-7 and H-8), 7.35 (t,
1-(4-Benzylpiperazinyl)-4-phenylpyrrolo[1,2-a]quinoxaline (1c).
C28H26N4O: C, 77.39; H, 6.03; N, 12.89. Found: C, 77.41; H,
1
2H, J ¼ 7.70 Hz, H-300 and H-500), 7.07 (d, 2H, J ¼ 7.70 Hz, H-200 Yellow oil (41%). H NMR (CDCl3) d 9.15 (dd, 1H, J ¼ 8.00 and
and H-600), 6.96 (t, 1H, J ¼ 7.70 Hz, H-400), 6.92 (d, 1H, J ¼ 1.70 Hz, H-9), 8.00 (dd, 1H, J ¼ 8.00 and 1.70 Hz, H-6), 7.96–7.92
4.30 Hz, H-2), 6.59 (d, 1H, J ¼ 4.30 Hz, H-3), 3.78–3.74 (m, 2H, (m, 2H, H-20 and H-60), 7.54–7.51 (m, 3H, H-30, H-40 and H-50),
CH2-pip), 3.51–3.47 (m, 2H, CH2-pip), 3.31–3.26 (m, 2H, CH2- 7.48–7.45 (m, 2H, H-7 and H-8), 7.43–7.41 (m, 2H, H-200 and H-
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Fig. 4 Effects of piperazinyl-pyrrolo[1,2-a]quinoxaline derivatives 1a–w and 2a–f on Nile Red (NR) efflux in S. cerevisiae cells overexpressing (A)
the CaCdr1p ABC-transporter (AD-CDR1) and (B) the CaMdr1p MFS-transporter (AD-MDR1). Inhibition of rhodamine 6G (R6G) efflux in the
presence of curcumin (70 mM) (CUR) was taken as positive control for CaCdr1p. Inhibition of [3H] fluconazole ([3H] Flu) efflux in the presence of
verapamil (90 mM) was taken as a positive control for CaMdr1p. Values are the means ꢂ standard deviations (error bars) for three independent
experiments. Nile Red was used as the transport substrate at 7 mM, and its efflux was measured by fluorescence. Each inhibitor was used
individually at a 10-fold excess over substrate (70 mM). The first column of each graph shows the Nile Red efflux in absence of any inhibitor.
600), 7.41–7.28 (m, 3H, H-300, H-400 and H-500), 6.88 (d, 1H, J ¼
C34H30N4: C, 82.56; H, 6.11; N, 11.33. Found: C, 83.27; H,
4.30 Hz, H-2), 6.54 (d, 1H, J ¼ 4.30 Hz, H-3), 3.68 (sl, 2H, CH2), 5.82; N, 10.91.
3.33–3.29 (m, 2H, CH2-pip), 3.06–2.97 (m, 4H, CH2-pip), 2.60–
1-(4-Phenethylpiperazinyl)-4-phenylpyrrolo[1,2-a]quinoxaline (1e).
2.55 (m, 2H, CH2-pip). 13C NMR (CDCl3) d 156.2 (C-4), 145.2 (C- Yellow oil (56%). H NMR (CDCl3) d 9.15 (dd, 1H, J ¼ 7.90 and
3a), 139.8 (C-5a), 139.3 (C-100), 138.8 (C-10), 130.9 (C-7), 130.8 (C- 2.35 Hz, H-9), 7.99 (dd, 1H, J ¼ 7.90 and 2.35 Hz, H-6), 7.96–7.92
8), 130.6 (C-30 and C-50), 130.2 (C-9a), 130.1 (C-200 and C-600), (m, 2H, H-20 and H-60), 7.56–7.50 (m, 3H, H-30, H-40 and H-50),
129.9 (C-300 and C-500), 129.8 (C-20 and C-60), 128.7 (C-9), 127.5 (C- 7.48–7.45 (m, 2H, H-7 and H-8), 7.37–7.32 (t, 2H, J ¼ 7.20 Hz, H-
6), 126.2 (C-40), 123.4 (C-1), 118.0 (C-400), 109.3 (C-2), 104.4 (C-3), 200 and H600), 7.33–7.25 (m, 3H, H-300, H-400 and H-500), 6.89 (d, 1H,
64.6 (NCH2), 54.4 (CH2-pip), 53.7 (CH2-pip). Anal. calcd for J ¼ 4.30 Hz, H-2), 6.55 (d, 1H, J ¼ 4.30 Hz, H-3), 3.38–3.34 (m,
1
C
28H26N4: C, 80.35; H, 6.26; N, 13.39. Found: C, 80.09; H, 2H, CH2-pip), 3.14–3.01 (m, 4H, CH2-pip), 2.95–2.90 (ddd, 2H, J
6.34; N, 13.57.
¼ 12.20, 8.55 and 7.40 Hz, CH2), 2.82–2.76 (ddd, 2H, J ¼ 12.20,
1-[4-(Diphenylmethyl)piperazinyl]-4-phenylpyrrolo[1,2-a]quinoxa- 8.55 and 7.40 Hz, CH2), 2.65–2.58 (m, 2H, CH2-pip). 13C NMR
line (1d). Yellow crystals (68%) mp 69 ꢁC. 1H NMR (CDCl3) d 9.12 (CDCl3) d 156.1 (C-4), 145.2 (C-3a), 141.3 (C-5a), 139.8 (C-100),
(dd, 1H, J ¼ 7.40 and 2.20 Hz, H-9), 8.00–7.92 (m, 3H, H-6, H-20 138.8 (C-10), 131.0 (C-7), 130.9 (C-8), 130.2 (C-30 and C-50), 130.1
and H-60), 7.55–7.52 (m, 7H, H-30, H-40, H-50, H-200 and H-600), (C-9a), 130.0 (C-200 and C-600), 129.9 (C-300, C-500, C-20 and C-60),
7.45–7.41 (m, 2H, H-7 and H-8), 7.37–7.31 (m, 4H, H-300 and H- 127.6 (C-9), 127.5 (C-6), 126.2 (C-40), 123.5 (C-1), 117.9 (C-400),
500), 7.28–7.21 (m, 2H, H-400), 6.90 (d, 1H, J ¼ 4.30 Hz, H-2), 6.55 109.4 (C-2), 104.5 (C-3), 61.8 (NCH2), 54.4 (CH2-pip), 53.6 (CH2-
(d, 1H, J ¼ 4.30 Hz, H-3), 4.41 (s, 1H, CH), 3.09–3.00 (m, 4H, pip), 35.0 (CH2). Anal. calcd for C29H28N4: C, 80.52; H, 6.52; N,
CH2-pip), 2.48–2.41 (m, 2H, CH2-pip). 13C NMR (CDCl3) d 156.1 12.95. Found: C, 79.96; H, 6.12; N, 12.92.
(C-4), 145.3 (C-3a), 144.0 (2 C-100), 139.8 (C-5a), 138.7 (C-10), 130.9
1-{4-[(2-Triuoromethyl)phenethyl]piperazinyl}-4-phenylpyrrolo
(C-7), 130.7 (C-8), 130.2 (C-9a), 130.1 (2 C-300, 2 C-500, C-30 and C- [1,2-a]quinoxaline (1f). Yellow crystals (63%); mp 34 ꢁC. 1H NMR
50), 129.9 (C-200 and C-600), 129.2 (C-20 and C-60), 128.7 (C-9), 127.5 (CDCl3) d 9.16 (dd, 1H, J ¼ 8.20 and 2.00 Hz, H-9), 8.01 (dd, 1H, J
(C-6), 126.2 (C-40), 123.4 (C-1), 118.0 (2 C-400), 109.4 (C-2), 104.3 ¼ 8.20 and 2.00 Hz, H-6), 7.97–7.93 (m, 2H, H-20 and H-60), 7.68
(C-3), 77.9 (NCH), 54.0 (CH2-pip), 53.3 (CH2-pip). Anal. calcd for (d, 1H, J ¼ 7.80 Hz, H-300), 7.57–7.48 (m, 5H, H-40, H-30, H-50, H-7
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Table 2 Effects of compounds 1 and 2 on Nile Red (NR) efflux by Table 2 (Contd.)
CaCdr1p and CaMdr1p in S. cerevisiae cells either overexpressing the
Compounds
Yeast strains
aMIC80 (mM)
bRI
Cdr1p ABC-transporter (AD-CDR1) or the Mdr1p MFS-transporter (AD-
MDR1)
presence of a range of concentrations of the different compounds. Yeast
growth in the absence of inhibitor was considered as 100%, and the
concentration where the growth was decreased to 80% was taken as
MIC80. The values are the means ꢂ standard deviations of three
Compounds
Yeast strains
aMIC80 (mM)
bRI
1c
AD1-8uꢀ
CDR1
MDR1
AD1-8uꢀ
CDR1
MDR1
AD1-8uꢀ
CDR1
MDR1
AD1-8uꢀ
CDR1
MDR1
AD1-8uꢀ
CDR1
MDR1
AD1-8uꢀ
CDR1
MDR1
AD1-8uꢀ
CDR1
MDR1
AD1-8uꢀ
CDR1
MDR1
AD1-8uꢀ
CDR1
MDR1
AD1-8uꢀ
CDR1
MDR1
AD1-8uꢀ
CDR1
MDR1
AD1-8uꢀ
CDR1
MDR1
AD1-8uꢀ
CDR1
MDR1
AD1-8uꢀ
CDR1
MDR1
AD1-8uꢀ
CDR1
MDR1
AD1-8uꢀ
CDR1
99 ꢂ 10
817 ꢂ 87
400 ꢂ 55
12 ꢂ 1.3
810 ꢂ 86
789 ꢂ 85
50 ꢂ 6
1
b
independent experiments. The resistance index (RI) was calculated
8.25
4.04
1
67.5
65.75
1
16
4.2
1
33.3
8.33
1
133.3
4
as the ratio between the MIC80 value determined for the strain
overexpressing the transporter relatively to that of the control strain
(AD1-8uꢀ).
1e
1g
1j
803 ꢂ 83
221 ꢂ 31
3 ꢂ 0.4
and H-8), 7.46–7.41 (m, 2H, H-400 and H-600), 7.35 (t, 1H, J ¼
7.60 Hz, H-500), 6.90 (d, 1H, J ¼ 4.25 Hz, H-2), 6.55 (d, 1H, J ¼
4.25 Hz, H-3), 3.38–3.34 (m, 2H, CH2), 3.13–3.00 (m, 6H, CH2-
pip and CH2), 2.81–2.76 (m, 2H, CH2-pip), 2.68–2.61 (m, 2H,
CH2-pip). 13C NMR (CDCl3) d 156.2 (C-4), 145.1 (C-3a), 140.1 (q, J
¼ 1.6 Hz, C-100), 139.8 (C-10), 138.8 (C-5a), 133.2 (C-500), 133.1 (C-
600), 133.0 (C-400), 131.0 (C-7), 130.9 (C-8), 130.3 (q, J ¼ 29.5 Hz, C-
200), 130.2 (C-9a), 130.1 (C-30 and C-50), 129.9 (C-20 and C-60),
127.5 (C-9), 127.4 (q, J ¼ 5.6 Hz, C-300), 126.2 (C-6), 126.0 (q, J ¼
272.2 Hz, CF3), 123.4 (C-1), 117.9 (C-40), 109.3 (C-2), 104.4 (C-3),
61.6 (NCH2), 54.3 (CH2-pip), 53.7 (CH2-pip), 31.7 (CH2). Anal.
calcd for C30H27F3N4: C, 71.98; H, 5.44; N, 11.19. Found: C,
72.23; H, 4.98; N, 11.02.
1-[4-(3-Fluorophenethyl)piperazinyl]-4-phenylpyrrolo[1,2-a]qui-
noxaline (1g). Yellow crystals (49%) mp 56 ꢁC. 1H NMR (CDCl3)
d 9.15 (dd, 1H, J ¼ 7.80 and 2.10 Hz, H-9), 8.01 (dd, 1H, J ¼ 7.80
and 2.10 Hz, H-6), 7.96–7.93 (m, 2H, H-20 and H-60), 7.55–7.52
(m, 2H, H-30 and H-50), 7.48–7.43 (m, 2H, H-7 and H-8), 7.31–
7.21 (m, 2H, H-500 and H-600), 7.08–6.88 (m, 2H, H-200 and H-400),
6.90 (d, 1H, J ¼ 4.20 Hz, H-2), 6.56 (d, 1H, J ¼ 4.20 Hz, H-3),
3.39–3.35 (m, 2H, CH2-pip), 3.14–3.07 (m, 4H, CH2-pip), 2.94–
2.89 (m, 2H, CH2), 2.83–2.80 (m, 2H, CH2-), 2.64–2.61 (m, 2H,
CH2-pip). Anal. calcd for C29H27FN4: C, 77.31; H, 6.04; N, 12.43.
Found: C, 77.46; H, 6.15; N, 12.61.
100 ꢂ 12
25 ꢂ 3
1k
1l
3 ꢂ 0.2
400 ꢂ 49
12 ꢂ 1
6 ꢂ 0.5
1
803 ꢂ 102
12 ꢂ 2
133.8
2
1
34
4.41
1
16
2
1
33.3
4.1
1
129.3
2
1m
1n
1o
1r
12 ꢂ 1.6
408 ꢂ 52
53 ꢂ 6.2
25 ꢂ 3.1
400 ꢂ 53
50 ꢂ 6.2
6 ꢂ 0.71
200 ꢂ 31
25 ꢂ 3.4
6 ꢂ 0.5
776 ꢂ 81
12 ꢂ 1.7
28 ꢂ 1.7
387 ꢂ 47
59 ꢂ 4.7
94 ꢂ 7.3
821 ꢂ 75
102 ꢂ 9.3
104 ꢂ 8.4
901 ꢂ 77
187 ꢂ 16
47 ꢂ 2.9
412 ꢂ 27
91 ꢂ 7.9
94 ꢂ 110
811 ꢂ 119
104 ꢂ 121
3 ꢂ 0.1
1s
1t
1
13.8
2.1
1
8.7
1.08
1
8.6
1.7
1
8
1.9
1
8.6
1.1
1
272.3
2
1w
2a
2b
2c
2d
2e
2f
1-[4-(4-Fluorophenethyl)piperazinyl]-4-phenylpyrrolo[1,2-a]qui-
noxaline (1h). Yellow crystals (55%) mp 150 ꢁC. 1H NMR (CDCl3)
d 9.15 (dd, 1H, J ¼ 7.70 and 2.10 Hz, H-9), 8.00 (dd, 1H, J ¼ 7.70
and 2.10 Hz, H-6), 7.96–7.93 (m, 2H, H-20 and H-60), 7.55–7.51
(m, 2H, H-30 and H-50), 7.48–7.44 (m, 2H, H-7 and H-8), 7.30–
7.21 (m, 3H, H-200, H-600 and H40), 7.05 (t, 2H, J ¼ 5.55 Hz, H-300
and H-500), 6.90 (d, 1H, J ¼ 4.20 Hz, H-2), 6.56 (d, 1H, J ¼ 4.20 Hz,
H-3), 3.39–3.35 (m, 2H, CH2-pip), 3.12–3.07 (m, 4H, CH2-pip),
2.92–2.88 (m, 2H, CH2), 2.81–2.76 (m, 2H, CH2), 2.63–2.60 (m,
817 ꢂ 77
6 ꢂ 0.2
MDR1
AD1-8uꢀ
CDR1
MDR1
AD1-8uꢀ
CDR1
MDR1
AD1-8uꢀ
CDR1
13
2H, CH2-pip). C NMR (CDCl3) d 162.9 (d, J ¼ 242.6 Hz, C-400),
6 ꢂ 0.2
1
156.2 (C-4), 144.8 (C-3a), 139.7 (C-5a), 138.8 (C-10), 136.7 (d, J ¼
3.5 Hz, C-100), 131.5 (d, J ¼ 7.8 Hz, C-200 and C-600), 131.0 (C-7),
130.9 (C-8), 130.1 (C-9a), 130.2 (C-30 and C-50), 130.0 (C-20 and
C-60), 127.5 (C-9), 126.3 (C-6), 123.5 (C-1), 117.8 (C-40), 116.7 (d, J
¼ 20.9 Hz, C-300 and C-500), 109.4 (C-2), 104.5 (C-3), 61.7 (NCH2),
54.3 (CH2-pip), 53.4 (CH2-pip), 34.0 (CH2). Anal. calcd for
831 ꢂ 88
5 ꢂ 0.2
138.5
0.83
1
273.6
2
1
134
2.33
3 ꢂ 0.1
821 ꢂ 86
6 ꢂ 0.2
6 ꢂ 0.1
804 ꢂ 71
14 ꢂ 1.2
MDR1
C
29H27FN4: C, 77.31; H, 6.04; N, 12.43. Found: C, 77.52; H,
a
The MIC80 values of cytotoxicity were determined by measuring the
optical density of cultures of each strain in the absence and the
6.17; N, 12.38.
1-[4-(3,4-Dichlorophenylethyl)piperazinyl]-4-phenylpyrrolo[1,2-
a]quinoxaline (1i). Yellow oil (47%). 1H NMR (CDCl3) d 9.13 (dd,
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Fig. 5 Effect of compounds 1d and 1f on the expression of the GFP-tagged Cdr1p and Mdr1p. Panels (A) AD-CDR1 and (B) AD-MDR1 were
evaluated in the presence or in the absence of compounds 1d and 1f.
1H, J ¼ 8.35 and 2.30 Hz, H-9), 8.00 (dd, 1H, J ¼ 8.35 and H-1), 7.76 (dd, 1H, J ¼ 7.80 and 1.60 Hz, H-9), 7.72 (dd, 1H, J ¼
2.30 Hz, H-6), 7.96–7.93 (m, 2H, H-20 and H-60), 7.56–7.51 (m, 7.80 and 1.60 Hz, H-6), 7.37–7.25 (m, 7H, H-7, H-8, H-20, H-30, H-
3H, H-20 and H-60), 7.56–7.51 (m, 3H, H-30, H-40 and H-50), 7.49– 40, H-50 and H-60), 6.83 (dd, 1H, J ¼ 4.00 and 1.30 Hz, H-3), 6.78
7.43 (m, 2H, H-7 and H-8), 7.42–7.37 (m, 2H, H-300 and H-600), (dd, 1H, J ¼ 4.00 and 2.70 Hz, H-2), 3.92–3.88 (m, 4H, CH2-pip),
7.10 (dd, 1H, J ¼ 8.35 and 2.30 Hz, H-200), 6.89 (d, 1H, J ¼ 2.94–2.88 (m, 2H, CH2), 2.78–2.70 (m, 6H, CH2 and CH2-pip).
4.30 Hz, H-2), 6.54 (d, 1H, J ¼ 4.30 Hz, H-3), 3.37–3.33 (m, 2H, 13C NMR (CDCl3) d 154.0 (C-4), 141.8 (C-10), 137.6 (C-5a), 130.2
CH2), 3.08–2.98 (m, 2H, CH2-pip and CH2), 2.88–2.72 (m, 2H, (C-30 and C-50), 129.9 (C-20 and C-60), 128.9 (C-7), 127.6 (C-9),
CH2-pip), 2.64–2.56 (m, 2H, CH2-pip). 13C NMR (CDCl3) d 156.2 127.2 (C-3a), 126.6 (C-6), 125.4 (C-40), 121.6 (C-9a), 115.8 (C-8),
(C-4), 144.9 (C-3a), 141.8 (C-5a), 139.8 (C-100), 138.8 (C-10), 133.7 114.7 (C-1), 113.9 (C-2), 108.3 (C-3), 62.0 (NCH2), 54.7 (CH2-
(C-300), 132.1 (C-500), 131.7 (C-7), 131.5 (C-400), 131.0 (C-8), 130.9 pip), 49.5 (CH2-pip), 35.1 (CH2). Anal. calcd for C23H24N4: C,
(C-200), 130.1 (C-9a), 130.0 (C-30and C-50), 129.9 (C-20 and C-60), 77.49; H, 6.78; N, 15.72. Found: C, 77.61; H, 6.92; N, 15.94.
129.6 (C-600), 127.5 (C-9), 126.2 (C-6), 123.5 (C-1), 117.9 (C-40),
4-[4-(3,4-Dichlorophenethyl)piperazinyl]pyrrolo[1,2-a]quinoxa-
1
109.3 (C-2), 104.4 (C-3), 61.2 (NCH2), 54.4 (CH2-pip), 53.7 (CH2- line (1k). Yellow oil (75%). H NMR (CDCl3) d 7.82 (dd, 1H, J ¼
pip), 34.1 (CH2). Anal. calcd for C29H26Cl2N4: C, 69.46; H, 2.70 and 1.40 Hz, H-1), 7.74 (dd, 1H, J ¼ 7.60 and 1.40 Hz, H-9),
5.22; N, 11.17. Found: C, 69.41; H, 5.47; N, 10.95.
7.70 (dd, 1H, J ¼ 7.60 and 1.40 Hz, H-6), 7.38–7.25 (m, 4H, H-7,
4-(4-Phenethylpiperazinyl)pyrrolo[1,2-a]quinoxaline (1j). Yellow H-8, H-20 and H-50), 7.09 (dd, 1H, J ¼ 8.10 and 1.80 Hz, H-60),
oil (81%). 1H NMR (CDCl3) d 7.84 (dd, 1H, J ¼ 2.70 and 1.30 Hz, 6.80 (dd, 1H, J ¼ 4.00 and 1.30 Hz, H-3), 6.77 (dd, 1H, J ¼ 4.00
Table 3 Ability of compounds 1d and 1f to sensitize yeast growth to fluconazole cytotoxicity
Strain
Compound
aFIC of uconazole
bFIC of compound
cFICI
AD1-8uꢀ
AD1-CDR1
AD1-MDR1
F2
1d
1f
1d
1f
1d
1f
1d
1f
1 (1.5/1.5)
1 (1.5/1.5)
1 (781/781)
1 (811/811)
1 (791/791)
1 (791/791)
0.0076 (6.25/812)
0.25 (200/799)
1 (618/618)
1 (400/400)
0.2 (150/720)
0.43 (350/799)
2 (1 + 1)
2 (1 + 1)
0.3 (81/209)
0.1 (40/209)
0.15 (10/65)
0.15 (10/65)
1 (13/13)
0.5 (7.5/13)
0.4 (200/418)
0.35 (150/418)
1.3 (0.3 + 1)
1.1 (0.1 + 1)
0.15 (0.15 + 0.0076)
0.4 (0.15 + 0.25)
2 (1 + 1)
1.5 (0.5 + 1)
0.6 (0.2 + 0.4)
0.78 (0.35 + 0.43)
F5
1d
1f
a
Evaluated by the checkerboard method, and expressed as the fractional inhibitory concentration (FIC) values for the uconazole (¼MIC80 of
b
uconazole in combination/MIC80 of uconazole alone). Each compound (¼MIC80 of compound in combination/MIC80 of compound alone).
c
The values in brackets are expressed in mM. FIC index (FICI) value #0.5 indicates synergistic interaction between the compound and the
uconazole.
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and 2.70 Hz, H-2), 3.87–3.83 (m, 4H, CH2-pip), 2.85–2.80 (m, 2H, 8), 7.21 (dd, 2H, J ¼ 8.70 and 5.40 Hz, H-20 and H-60), 7.04–6.97
CH2), 2.76–2.64 (m, 6H, CH2 and CH2-pip). 13C NMR (CDCl3) (m, 2H, H-30 and H-50), 6.81 (dd, 1H, J ¼ 3.90 and 1.20 Hz, H-3),
d 153.9 (C-4), 142.0 (C-10), 137.5 (C-5a), 133.5 (C-30), 132.0 (C-50), 6.77 (dd, 1H, J ¼ 3.90 and 2.70 Hz, H-2), 3.91–3.86 (m, 4H, CH2-
131.6 (C-20), 131.4 (C-40), 129.6 (C-60), 128.9 (C-7), 127.2 (C-3a), pip), 2.89–2.84 (m, 2H, CH2), 2.77–2.66 (m, 6H, CH2 and CH2-
126.6 (C-9), 125.4 (C-6), 121.5 (C-9a), 115.8 (C-8), 114.7 (C-1), pip). 13C NMR (CDCl3) d 162.8 (d, J ¼ 242.6 Hz, C-40), 153.8 (C-4),
113.9 (C-2), 108.2 (C-3), 61.2 (NCH2), 54.5 (CH2-pip), 49.3 137.4 (C-5a), 137.0 (d, J ¼ 3.2 Hz, C-10), 131.5 (d, J ¼ 7.7 Hz, C-20
(CH2-pip), 34.0 (CH2). Anal. calcd for C23H22Cl2N4: C, 64.94; H, and C-60), 128.9 (C-7), 127.2 (C-3a), 126.6 (C-9), 125.5 (C-6), 121.5
5.21; N, 13.17. Found: C, 65.08; H, 5.36; N, 13.35.
4-[4-(2-Triuoromethylphenethyl)piperazinyl]pyrrolo[1,2-a]qui-
(C-9a), 116.6 (d, J ¼ 21.2 Hz, C-30 and C-50), 115.8 (C-8), 114.7 (C-
1), 113.9 (C-2), 108.1 (C-3), 61.8 (NCH2), 54.5 (CH2-pip), 49.1
noxaline (1l). Yellow oil (79%). 1H NMR (CDCl3) d 7.84 (dd, 1H, J (CH2-pip), 33.9 (CH2). Anal. calcd for C23H23FN4: C, 73.77; H,
¼ 2.70 and 1.30 Hz, H-1), 7.75 (dd, 1H, J ¼ 7.80 and 1.40 Hz, H- 6.19; N, 14.96. Found: C, 73.94; H, 6.05; N, 15.09.
9), 7.71 (dd, 1H, J ¼ 7.80 and 1.40 Hz, H-6), 7.66 (d, 1H, J ¼
4-[4-(3-Chlorophenyl)piperazinyl]pyrrolo[1,2-a]quinoxaline (1p).
7.60 Hz, H-30), 7.50 (t, 1H, J ¼ 7.60 Hz, H-50), 7.42 (d, 1H, J ¼ Pale-yellow oil (73%). 1H NMR (CDCl3) d 7.90–7.88 (m, 1H, H-1),
7.60 Hz, H-60), 7.36–7.26 (m, 3H, H-7, H-8 and H-40), 6.81 (dd, 7.82–7.76 (m, 2H, H-9 and H-6), 7.43–7.28 (m, 2H, H-7 and H-8),
1H, J ¼ 4.00 and 1.30 Hz, H-3), 6.78 (dd, 1H, J ¼ 4.00 and 7.26 (t, 1H, J ¼ 8.10 Hz, H-50), 7.00 (dd, 1H, J ¼ 1.50 and 1.50 Hz,
2.70 Hz, H-2), 3.91–3.87 (m, 4H, CH2-pip), 3.11–3.06 (m, 2H, H-20), 6.93–6.83 (m, 4H, H-40, H-60, H-2 and H-3), 4.01–3.97 (m,
CH2), 2.81–2.70 (m, 6H, CH2 and CH2-pip). 13C NMR (CDCl3) 4H, CH2-pip), 3.47–3.43 (m, 4H, CH2-pip). 13C NMR (CDCl3)
d 153.9 (C-4), 140.2 (q, J ¼ 1.6 Hz, C-10), 137.6 (C-5a), 133.2 (q, J ¼ d 153.9 (C-4), 153.6 (C-10), 137.3 (C-5a), 136.4 (C-30), 131.5 (C-50),
0.8 Hz, C-50), 132.1 (C-60), 130.0 (q, J ¼ 29.4 Hz, C-20), 128.9 (C-7), 128.9 (C-7), 127.2 (C-3a), 126.7 (C-9), 125.7 (C-6), 121.5 (C-9a),
127.6 (C-40), 127.3 (q, J ¼ 5.6 Hz, C-30), 127.2 (C-3a), 126.5 (C-9), 120.8 (C-8), 117.1 (C-40), 116.0 (C-20), 115.2 (C-60), 114.8 (C-1),
126.1 (q, J ¼ 272.1 Hz, CF3), 125.3 (C-6), 121.6 (C-9a), 115.8 (C-8), 114.0 (C-2), 108.2 (C-3), 49.9 (CH2-pip), 49.2 (CH2-pip), 33.9
114.7 (C-1), 113.9 (C-2), 108.2 (C-3), 61.6 (NCH2), 54.5 (CH2-pip), (CH2). Anal. calcd for C21H19ClN4: C, 69.51; H, 5.28; N, 15.44.
49.4 (CH2-pip), 31.4 (CH2). Anal. calcd for C24H23F3N4: C, 67.91; Found: C, 69.67; H, 5.07; N, 15.30.
H, 5.46; N, 13.20. Found: C, 68.05; H, 5.22; N, 13.38.
4-[4-(2-Fluorophenethyl)piperazinyl]pyrrolo[1,2-a]quinoxaline
4-[4-(6-Chloro-5-triuoromethylpyridin-2-yl)piperazinyl]pyrrolo
[1,2-a]quinoxaline (1q). White crystals (43%); mp 146 ꢁC. 1H
(1m). Yellow oil (74%). 1H NMR (CDCl3) d 7.85–7.82 (m, 1H, H- NMR (CDCl3) d 7.88–7.86 (m, 1H, H-1), 7.77–7.73 (m, 2H, H-9
1), 7.76 (dd, 1H, J ¼ 7.80 and 1.40 Hz, H-9), 7.70 (dd, 1H, J ¼ 7.80 and H-6), 7.72 (d, 1H, J ¼ 8.70 Hz, H-40), 7.39–7.29 (m, 2H, H-
and 1.40 Hz, H-6), 7.36–7.04 (m, 6H, H-7, H-8, H-30, H-40, H-50 7 and H-8), 6.86–6.80 (m, 2H, H-2 and H-3), 6.51 (d, 1H, J ¼
and H-60), 6.81–6.77 (m, 2H, H-2 and H-3), 3.97–3.94 (m, 4H, 8.70 Hz, H-30), 4.00–3.97 (m, 4H, CH2-pip), 3.89–3.86 (m, 4H,
CH2-pip), 3.01–2.98 (m, 2H, CH2), 2.87–2.83 (m, 6H, CH2 and CH2-pip). 13C NMR (CDCl3) d 160.6 (C-20), 153.7 (C-4), 149.0 (q, J
CH2-pip). 13C NMR (CDCl3) d 162.6 (d, J ¼ 243.3 Hz, C-20), 153.9 ¼ 1.6 Hz, C-60), 139.0 (q, J ¼ 4.5 Hz, C-40), 137.2 (C-5a), 128.9 (C-
(C-4), 137.6 (C-5a), 132.4 (d, J ¼ 5 Hz, C-60), 129.3 (d, J ¼ 8 Hz, C- 7), 127.2 (C-3a), 126.7 (C-9), 125.7 (C-6), 124.7 (q, J ¼ 269.0 Hz,
40), 128.9 (C-7), 128.5 (d, J ¼ 15.8 Hz, C-10), 127.2 (C-3a), 126.5 (C- CF3), 121.3 (C-9a), 116.0 (C-8), 114.7 (C-1), 114.1 (C-2), 113.8 (q, J
9), 125.4 (d, J ¼ 3.2 Hz, C-50), 125.3 (C-6), 121.6 (C-9a), 116.7 (d, J ¼ 32.2 Hz, C-50), 108.2 (C-3), 104.5 (C-30), 48.6 (CH2-pip), 45.7
¼ 22.1 Hz, C-30), 115.8 (C-8), 114.7 (C-1), 113.8 (C-2), 108.2 (C-3), (CH2-pip). Anal. calcd for C21H17ClF3N5: C, 58.40; H, 3.97; N,
60.2 (NCH2), 54.5 (CH2-pip), 49.4 (CH2-pip), 28.2 (CH2). Anal. 16.22. Found: C, 58.28; H, 4.07; N, 16.12.
calcd for C23H23FN4: C, 73.77; H, 6.19; N, 14.96. Found: C, 73.85;
H, 6.26; N, 15.12.
4-[4-(3-Fluorophenethyl)piperazinyl]pyrrolo[1,2-a]quinoxaline
7-Methoxy-4-[4-(2-triuoromethylphenethyl)piperazinyl]pyrrolo
[1,2-a]quinoxaline (1r). Yellow oil (79%). 1H NMR (CDCl3) d 7.78–
7.74 (m, 1H, H-1), 7.65 (d, 1H, J ¼ 7.80 Hz, H-30), 7.64 (d, 1H, J ¼
(1n). Yellow oil (81%). 1H NMR (CDCl3) d 7.83 (dd, 1H, J ¼ 2.70 8.00 Hz, H-9), 7.51 (t, 1H, J ¼ 7.80 Hz, H-50), 7.43 (d, 1H, J ¼
and 1.30 Hz, H-1), 7.73 (dd, 1H, J ¼ 8.00 and 1.50 Hz, H-9), 7.70 7.80 Hz, H-60), 7.33 (t, 1H, J ¼ 7.80 Hz, H-40), 7.20 (d, 1H, J ¼
(dd, 1H, J ¼ 8.00 and 1.50 Hz, H-6), 7.38–7.23 (m, 3H, H-7, H-8 2.60 Hz, H-6), 6.91 (dd, 1H, J ¼ 8.00 and 2.60 Hz, H-8), 6.80–6.78
and H-50), 7.05–6.89 (m, 3H, H-20, H-40 and H-60), 6.81 (dd, 1H, J (m, 1H, H-3), 6.76–6.74 (m, 1H, H-2), 3.91–3.84 (m, 7H, CH3O
¼ 3.90 and 1.30 Hz, H-3), 6.77 (dd, 1H, J ¼ 3.90 and 2.70 Hz, H- and CH2-pip), 3.13–3.07 (m, 2H, CH2), 2.84–2.74 (m, 6H, CH2
2), 3.91–3.86 (m, 4H, CH2-pip), 2.93–2.87 (m, 2H, CH2), 2.78– and CH2-pip). 13C NMR (CDCl3) d 158.7 (C-7), 154.3 (C-4), 140.3
2.69 (m, 6H, CH2 and CH2-pip). 13C NMR (CDCl3) d 164.3 (d, J ¼ (q, J ¼ 3.2 Hz, C-10), 138.7 (C-5a), 133.1 (q, J ¼ 0.8 Hz, C-60), 133.0
244.1 Hz, C-30), 153.7 (C-4), 143.5 (d, J ¼ 7.35 Hz, C-10), 137.3 (C- (C-50), 130.0 (q, J ¼ 29.4 Hz, C-20), 127.6 (C-9), 127.3 (q, J ¼
5a), 131.3 (d, J ¼ 8.25 Hz, C-50), 128.9 (C-7), 127.2 (C-3a), 126.6 5.6 Hz, C-30), 126.0 (q, J ¼ 272.2 Hz, CF3), 121.5 (C-3a), 121.1 (C-
(C-9), 125.8 (d, J ¼ 2.80 Hz, C-60), 125.6 (C-6), 121.4 (C-9a), 117.0 9a), 115.5 (C-40), 115.4 (C-8), 113.9 (C-1), 113.5 (C-2), 110.7 (C-3),
(d, J ¼ 20.85 Hz, C-20), 115.9 (C-8), 114.7 (C-1), 114.5 (d, J ¼ 107.9 (C-6), 61.6 (NCH2), 57.0 (CH3O), 54.5 (CH2-pip), 49.4 (CH2-
21.5 Hz, C-40), 113.9 (C-2), 108.1 (C-3), 61.2 (NCH2), 54.3 (CH2- pip), 31.4 (CH2). Anal. calcd for C25H25F3N4O: C, 66.07; H,
pip), 48.8 (CH2-pip), 34.1 (CH2). Anal. calcd for C23H23FN4: C, 5.54; N, 12.33. Found: C, 65.94; H, 5.67; N, 12.20.
73.77; H, 6.19; N, 14.96. Found: C, 73.71; H, 6.33; N, 15.16.
4-[4-(4-Fluorophenethyl)piperazinyl]pyrrolo[1,2-a]quinoxaline
(1o). Yellow oil (85%). 1H NMR (CDCl3) d 7.83 (dd, 1H, J ¼ 2.70 7.71 (m, 1H, H-1), 7.66 (d, 1H, J ¼ 8.70 Hz, H-6), 7.65 (d, 1H, J ¼
and 1.20 Hz, H-1), 7.75 (dd, 1H, J ¼ 8.00 and 1.50 Hz, H-9), 7.70 7.60 Hz, H-30), 7.48 (t, 1H, J ¼ 7.60 Hz, H-50), 7.40 (d, 1H, J ¼
(dd, 1H, J ¼ 8.00 and 1.50 Hz, H-6), 7.37–7.26 (m, 2H, H-7 and H- 7.60 Hz, H-60), 7.31 (t, 1H, J ¼ 7.60 Hz, H-40), 7.19 (d, 1H, J ¼
8-Methoxy-4-[4-(2-triuoromethylphenethyl)piperazinyl]pyrrolo
[1,2-a]quinoxaline (1s). Yellow oil (64%). 1H NMR (CDCl3) d 7.73–
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2.40 Hz, H-9), 6.95 (dd, 1H, J ¼ 8.70 and 2.40 Hz, H-7), 6.79–6.77 pip), 49.3 (CH2-pip). Anal. calcd for C29H25F3N4: C, 71.59; H,
(m, 2H, H-2 and H-3), 3.90 (s, 3H, CH3O), 3.79–3.78 (m, 4H, CH2- 5.18; N, 11.52. Found: C, 71.75; H, 5.08; N, 11.57.
pip), 3.11–3.06 (m, 2H, CH2), 2.80–2.69 (m, 6H, CH2 and CH2-
2-Phenyl-4-[4-(4-triuoromethylbenzyl)piperazinyl]pyrrolo[1,2-
pip). 13C NMR (CDCl3) d 158.2 (C-8), 152.9 (C-4), 140.2 (q, J ¼ a]quinoxaline (1w). Yellow oil (76%). 1H NMR (CDCl3) d 8.08 (d,
1.65 Hz, C-10), 133.2 (q, J ¼ 0.9 Hz, C-60), 133.1 (C-50), 131.6 (C- 1H, J ¼ 0.90 Hz, H-1), 7.79 (dd, 1H, J ¼ 7.80 and 1.80 Hz, H-9),
5a), 130.1 (q, J ¼ 29.3 Hz, C-20), 130.0 (C-6), 127.8 (C-3a), 127.6 7.71–7.67 (m, 3H, H-6, H-200 and H-600), 7.64–7.55 (m, 2H, H-7
(C-40), 127.3 (q, J ¼ 5.6 Hz, C-30), 126.0 (q, J ¼ 272.1 Hz, CF3), and H-8), 7.48–7.42 (m, 2H, H-300 and H-500), 7.35–7.29 (m, 3H,
121.9 (C-9a), 115.4 (C-7), 114.0 (C-1), 113.6 (C-2), 107.8 (C-3), H-20, H-60 and H-400), 7.02 (d, 1H, J ¼ 0.90 Hz, H-3), 3.88–3.85 (m,
99.3 (C-9), 61.7 (NCH2), 57.1 (CH3O), 54.5 (CH2-pip), 49.7 4H, CH2-pip), 3.68 (s, 2H, NCH2), 2.72–2.69 (m, 4H, CH2-pip).
(CH2-pip), 31.4 (CH2). Anal. calcd for C25H25F3N4O: C, 66.07; H, 13C NMR (CDCl3) d 153.8 (C-4), 143.8 (q, J ¼ 1.0 Hz, C-100), 137.5
5.54; N, 12.33. Found: C, 66.20; H, 5.44; N, 12.46.
(C-5a), 135.8 (C-10), 130.7 (q, J ¼ 28.3 Hz, C-400), 130.6 (C-7), 130.3
Ethyl 4-[4-(2-triuoromethylphenethyl)piperazinyl]pyrrolo[1,2- (C-30 and C-50), 129.7 (C-3a), 129.0 (C-9), 128.3 (C-40), 127.4 (C-20
a]quinoxaline-2-carboxylate (1t). Pale-yellow crystals (66%); mp and C-60), 126.7 (C-6), 126.6 (q, J ¼ 6.3 Hz, C-300 and C-500), 126.5
1
118 ꢁC. H NMR (CDCl3) d 8.33 (s, 1H, H-1), 7.75 (d, 1H, J ¼ (q, J ¼ 3.5 Hz, C-200 and C-600), 125.6 (q, J ¼ 267.8 Hz, CF3), 125.5
8.00 Hz, H-9), 7.67 (d, 1H, J ¼ 8.00 Hz, H-6), 7.64 (d, 1H, J ¼ (C-8), 122.4 (C-9a), 114.6 (C-2), 112.4 (C-1), 105.7 (C-3), 63.9
7.50 Hz, H-30), 7.49 (t, 1H, J ¼ 7.50 Hz, H-50), 7.43 (d, 1H, J ¼ (NCH2), 54.5 (CH2-pip), 49.4 (CH2-pip). Anal. calcd for
7.50 Hz, H-60), 7.41–7.26 (m, 3H, H-7, H-8 and H-40), 7.19 (s, 1H,
C29H25F3N4: C, 71.59; H, 5.18; N, 11.52. Found: C, 71.72; H,
H-3), 4.39 (q, 2H, J ¼ 7.20 Hz, OCH2), 3.89–3.87 (m, 4H, CH2- 5.04; N, 11.48.
pip), 3.10–3.05 (m, 2H, CH2), 2.79–2.69 (m, 6H, CH2 and CH2-
pip), 1.42 (t, 2H, J ¼ 7.20 Hz, CH3). 13C NMR (CDCl3) d 165.7
X-ray crystallography studies
(CO), 153.6 (C-4), 140.2 (C-10), 137.9 (C-5a), 133.1 (C-50), 133.0 (C-
7), 130.0 (q, J ¼ 30.0 Hz, C-20), 129.0 (C-9), 127.7 (C-6), 127.6 (C-
60), 127.3 (q, J ¼ 5.6 Hz, C-30), 126.3 (C-3a), 126.0 (q, J ¼ 272.2 Hz,
CF3), 125.6 (C-40), 121.9 (C-9a), 120.5 (C-2), 119.0 (C-8), 114.9 (C-
1), 109.2 (C-3), 61.9 (OCH2), 61.6 (NCH2), 54.4 (CH2-pip), 49.2
(CH2-pip), 31.4 (CH2), 15.9 (CH3). Anal. calcd for C27H27F3N4O2:
C, 65.31; H, 5.48; N, 11.28. Found: C, 65.39; H, 5.29; N, 11.42.
2-Phenyl-4-[4-(2-triuoromethylphenethyl)piperazinyl]pyrrolo
[1,2-a]quinoxaline (1u). Pale-yellow crystals (67%); mp 136 ꢁC. 1H
NMR (CDCl3) d 8.08 (s, 1H, H-1), 7.77 (d, 1H, J ¼ 8.10 Hz, H-9),
7.74–7.68 (m, 3H, H-6, H-200 and H-600), 7.66 (d, 1H, J ¼ 7.50 Hz,
H-30), 7.51 (t, 1H, J ¼ 7.50 Hz, H-50), 7.49–7.28 (m, 7H, H-40, H-60,
H-7, H-8, H-300, H-400 and H-500), 7.06 (s, 1H, H-3), 3.93–3.90 (m,
4H, CH2-pip), 3.13–3.07 (m, 2H, CH2), 2.84–2.72 (m, 6H, CH2
and CH2-pip). 13C NMR (CDCl3) d 153.4 (C-4), 140.2 (C-100), 137.3
(C-5a), 135.7 (C-10), 133.4 (C-500), 133.3 (C-7), 130.3 (C-30 and C-
50), 129.9 (q, J ¼ 29.3 Hz, C-200), 129.1 (C-9), 128.4 (C-40), 128.0 (C-
6), 127.6 (C-3a), 127.5 (C-20 and C-60), 127.4 (C-600), 127.0 (C-2),
126.7 (C-300), 125.9 (C-8), 125.7 (q, J ¼ 272.2 Hz, CF3), 125.6 (C-
400), 122.2 (C-9a), 114.7 (C-2), 112.6 (C-1), 105.7 (C-3), 61.2
(NCH2), 53.9 (CH2-pip), 48.7 (CH2-pip), 30.7 (CH2). Anal. calcd
for C30H27F3N4: C, 71.98; H, 5.44; N, 11.19. Found: C, 72.06; H,
5.53; N, 11.35.
Crystallographic data of compounds 1a and 1h were collected at
293 K with an Enraf-Nonius CAD-4 diffractometer with mono-
˚
chromatic Cu-Ka radiation (l ¼ 1.54178 A). The collected data
were reduced using the NONIUS CAD4 soware and all reec-
tions were used for unit-cell renement. Then the data were
corrected for Lorentz and polarization effects and for empirical
absorption correction.51 The structure was solved by direct
methods Shelx 2013 (ref. 52) and rened using Shelx 201352
suite of programs.
Colorless single crystal of 1a was obtained by slow evapora-
tion from dichloromethane/methanol solution (70/30; v/v):
˚
˚
ꢀ
triclinic, space group P1, a ¼ 9.5233(14) A, b ¼ 10.8011(19) A,
ꢁ
ꢁ
˚
c ¼ 11.8823(16) A, a ¼ 107.688(11) , b ¼ 110.089(10) , g ¼
3
ꢀ3
ꢁ
˚
100.552(11) , V ¼ 1035.6(3) A , Z ¼ 2, d (calcd) ¼ 1.259 Mg m
,
FW ¼ 392.49 for C26H24N4, F(000) ¼ 416. Colorless single crystal
of 1h was obtained by slow evaporation from methanol/
ꢀ
dichloromethane (20/80; v/v): triclinic, space group P1, a ¼
ꢁ
˚
˚
˚
9.471(10) A, b ¼ 13.021(6) A, c ¼ 19.887(6) A, a ¼ 77.83(4) , b ¼
3
ꢁ
ꢁ
˚
81.76(8) , g ¼ 87.25(6) , V ¼ 2372(3) A , Z ¼ 2, d (calcd) ¼ 1.266
Mg mꢀ3, FW ¼ 904.29 for 2C29H27FN4, 0.1H2O, F(000) ¼ 955.
Full crystallographic results have been deposited at the
Cambridge Crystallographic Data Centre (CCDC-891809, CCDC-
891808, respectively), UK, as ESI.†53
2-Phenyl-4-[4-(3-triuoromethylbenzyl)piperazinyl]pyrrolo[1,2-
1
a]quinoxaline (1v). Yellow oil (54%). H NMR (CDCl3) d 8.07 (s,
Biological studies
1H, H-1), 7.76 (d, 1H, J ¼ 7.80 Hz, H-9), 7.73–7.69 (m, 4H, H-6, H-
40, H-200 and H-600), 7.63–7.56 (m, 2H, H-7 and H-8), 7.51–7.43 (m,
Yeast strains and growth media. All the yeast strains were
3H, H-20, H-300, H-500), 7.37–7.28 (m, 3H, H-50, H-60 and H-400), grown in yeast extract peptone-dextrose (YEPD) broth (HiMedia)
7.04 (s, 1H, H-3), 3.89–3.86 (m, 4H, CH2-pip), 3.67 (s, 2H, NCH2), and for agar plates, 2.5% (w/v) Bacto agar (Difco, BD Biosci-
2.71–2.69 (m, 4H, CH2-pip). 13C NMR (CDCl3) d 153.8 (C-4), ences) was added to the medium. The S. cerevisiae strain used as
140.3 (C-100), 137.4 (C-5a), 135.8 (C-10), 133.9 (C-600), 132.0 (q, J a heterologous host for the expression of Cdr1 and Mdr1
¼ 32.0 Hz, C-300), 130.3 (C-30 and C-50), 130.2 (C-7), 129.8 (C-3a), proteins was AD1-8uꢀ, provided by Richard D. Cannon,
128.9 (C-9), 128.3 (C-40), 127.4 (C-20 and C-60), 127.2 (C-500), 127.1 University of Otago, Dunedin, New Zealand. The host AD1-8uꢀ
(q, J ¼ 3.75 Hz, C-200), 126.9 (C-2), 126.7 (C-6), 125.6 (C-8), 125.5 having seven major ABC transporters deleted was suitably
(q, J ¼ 3.9 Hz, C-400), 125.4 (q, J ¼ 271.3 Hz, CF3), 122.3 (C-9a), modied to clone GFP-tagged Cdr1protein and its mutant
114.6 (C-2), 112.4 (C-1), 105.8 (C-3), 63.8 (NCH2), 54.4 (CH2- variants.54 The yeast strains AD1-8uꢀ, AD-CDR1, AD-MDR1 (ref.
55–57) were cultured at 30 ꢁC. No trailing effect of the
2928 | RSC Adv., 2020, 10, 2915–2931
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compound or uconazole was observed and the false negatives the inhibitors. FICI values were calculated as the sum of the
were ruled out as we compare our experimental data with FICs of each agent (uconazole and inhibitors). The FIC of each
negative control that is AD1-8uꢀ strain (empty vector strain). agent was calculated as the MIC80 of the agent in combination
15% glycerol stocks of these strains were maintained in ꢀ80 ꢁC divided by the MIC80 of the agent alone.
storage that were freshly revived on YEPD before use.
Reagents and media. Nile Red (>98%), curcumin (purity $
99.5%), and verapamil (purity $ 99%) were obtained from
Conflicts of interest
Sigma Chemical Co. Fluconazole (>98%) was obtained from
There are no conicts to declare.
Ranbaxy and [3H]-uconazole (20 Ci mmolꢀ1) was provided by
Moravek Biochemicals and Radiochemicals. All routine chem-
icals were obtained from HiMedia and were of analytical grade.
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