Synthesis and biology of a 7-nitro-2,1,3-benzoxadiazol-4-yl derivative of 2-phenylindole-3-acetamide: A fluorescent probe for the peripheral-type benzodiazepine receptor

Full text of DOI:10.1021/jm970220w

© Copyright 1997 by the American Chemical Society
Volume 40, Number 16
August 1, 1997
Communications to the Editor
testis, ovary, placenta, and brain tissues.10 In these
Syn th esis a n d Biology of a
7-Nitr o-2,1,3-ben zoxa d ia zol-4-yl
Der iva tive of
2-P h en ylin d ole-3-a ceta m id e:
A F lu or escen t P r obe for th e
P er ip h er a l-Typ e Ben zod ia zep in e
Recep tor
cells, the PBR was found to be localized primarily in
the mitochondria and more specifically in the outer
mitochondrial membrane.11 Nonmitochondrial locations
of PBR have also been described in various tissues.12
An 18 kDa isoquinoline-binding protein was identified
as PBR, cloned, and expressed.13
On the basis of the pharmacological effects of PBR
ligands and the tissue-specific cellular and subcellular
localization of the 18 kDa isoquinoline binding protein,
the PBR has been shown to be involved in numerous
functions including steroid biosynthesis, mitochondrial
respiration, heme biosynthesis, calcium channel modu-
lation, cell proliferation and differentiation, and immu-
nomodulation.10,12 From these functions the role of PBR
in steroidogenesis is fairly well established. Using
adrenal,14 testis Leydig,15 ovarian granulosa,16 pla-
centa,17 and brain glia18 cells, it has been demonstrated
that the PBR is a functional component of the ste-
roidogenic machinery mediating cholesterol delivery
from the outer to the inner mitochondrial membrane.19
Further studies demonstrated that pharmacologically
induced reduction of adrenal PBR levels in vivo resulted
in decreased circulating glucocorticoid levels.20 In ad-
dition, targeted disruption of the PBR gene in ste-
roidogenic cells resulted in inhibition of cholesterol
transport to the inner mitochondrial membrane and
arrest of steroid biosynthesis.21 Thus, the PBR provides
an attractive target molecule for the development of
compounds which may be used for the regulation of
steroid synthesis in the periphery and the central
nervous system and for the regulation of the above-
mentioned PBR-dependent functions.
Alan P. Kozikowski,*,† Maria Kotoula,‡,§ Dawei Ma,|
Noureddine Boujrad,‡ Werner Tu¨ckmantel,† and
Vassilios Papadopoulos*,‡
Drug Discovery Laboratory, Institute of Cognitive and
Computational Sciences, and Department of Cell Biology,
Georgetown University Medical Center,
3970 and 3900 Reservoir Road, NW,
Washington, D.C. 20007
Received March 31, 1997
Benzodiazepines are among the most highly pre-
scribed drugs due to their pharmacological action of
relieving anxiety mediated through modulating the
activity of γ-aminobutyric acid receptors in the central
nervous system.1 The peripheral-type benzodiazepine
receptor (PBR) is another class of binding sites for
benzodiazepines distinct from the aforementioned neu-
rotransmitter receptors. The PBR was originally dis-
covered because it binds the benzodiazepine diazepam
with relatively high affinity.2 Further studies demon-
strated that in addition to the benzodiazepines, PBR
binds with high affinity other classes of organic com-
pounds, such as isoquinolines,3 imidazopyridines,4 in-
dole derivatives,5 and pyrrolobenzoxazepines.6 In ad-
dition to these drug ligands, the polypeptide diazepam
binding inhibitor,7 porphyrins,8 and benzodiazepine-like
molecules9 have been identified as endogenous PBR
ligands. The PBR, although present in all tissues
examined, was found to occur in particularly high
density in steroid-producing tissues, such as adrenal,
The studies described above on the localization and
characterization of the structure and function of the
PBR in fixed cells or isolated subcellular fractions3-20
were performed using high-affinity ligands, antisera
developed against the entire molecule or fragments of
the 18 kDa PBR protein, and cDNA probes. Consider-
ing the problems associated with antiserum specificity
and sensitivity as well as with subcellular fractionation,
one could argue that the results obtained may not
represent the actual situation in a living cell. We report
herein the synthesis, characterization, and biologic
* Authors for correspondence.
† Drug Discovery Laboratory.
‡ Cell Biology.
§ On leave of absence from the Department of Pharmacology,
Aristotelion University, School of Medicine, Thessaloniki, Greece.
| Current address: Shangai Institute of Organic Chemistry, Chinese
Academy of Science, 354 Fengling Lu, Shangai 200032, China.
S0022-2623(97)00220-3 CCC: $14.00 © 1997 American Chemical Society

2436 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16
Communications to the Editor
F igu r e 1. NBD displaces radiolabeled PK 11195. Specific binding of [3H]PK 11195 to MA-10 mouse tumor Leydig (left) and
C6-2B rat glioma (right) cells was measured in the presence of 1 nM [3H]PK 11195 with or without the indicated concentrations
of 4, PK 11195, FGIN-1-27, Ro5-4864, and clonazepam.
Sch em e 1. Synthesis of Fluorescent Probe 4
activity of the fluorescent high-affinity PBR ligand (4)
which provides a tool allowing the direct imaging by
fluorescence microscopy of the 18 kDa PBR protein in
living cells. Moreover, this probe may be used to access
and visualize the PBR in tissues under conditions where
antisera cannot penetrate.
to their PBR expression, localization, and ability to
synthesize steroids.15,18
Ra d ioliga n d Bin d in g Assa ys. Binding assays were
performed as we previously described using [3H]PK
11195 as the radiolabeled ligand.15,18 The IC50 for the
compounds tested was determined using the LIGAND
program.25 Figure 1 shows that 4 displaced [3H]PK
11195 with the same efficacy as the isoquinoline PK
11195, the benzodiazepine Ro5-4864, and the 2-aryl-3-
indoleacetamide prototype FGIN-1-27. The IC50 for all
these compounds was approximately 10 nM for both cell
types. It should be noted that, at nanomolar and low
micromolar concentrations, clonazepam, a benzodiaz-
epine with high affinity for the GABAA receptor, did not
displace [3H]PK 11195. These results demonstrate that
the addition of the fluorigenic 7-nitro-2,1,3-benzoxadia-
zol-4-yl group did not affect the affinity of the indoleac-
etamide to bind to the PBR. In addition, several other
derivatives of 2-phenylindole-3-acetamide were tested
for their ability to displace radiolabeled ligands from
the GABAA, GABAB, dopamine, 5-HT1, 5-HT2, glutamate,
opiate, σ, cholecystokinin, â-adrenergic, and cannabinoid
receptors.5 None of the derivatives tested showed such
activity. Thus, it is unlikely that the addition of the
Ch em istr y. The fluorescent derivative 4 was ob-
tained in two simple steps from the previously de-
scribed22 ethyl ester 1 (Scheme 1). First, the spacer
group was attached by reaction of 1 in boiling toluene
with the aluminum amide formed from hexamethylene-
diamine.23 A small amount of the 2:1 reaction product
3 is inevitably formed despite the use of an excess of
the diamine, but this material is readily separable from
the desired product 2 by chromatography on silica gel,
and 2 is isolated in 46% yield. In the second step, the
remaining amine nitrogen of 2 is alkylated with 4-chloro-
7-nitrobenzofurazan (NBD chloride)24 in DMF to pro-
duce the target compound 4 in 52% yield as an orange-
colored foam. Using CH2Cl2 as the solvent, 4 is also
obtained, but only in 24% yield.
Biology. Cells. The MA-10 mouse tumor Leydig
and the rat C6-2B rat glioma cell lines were used. We
have previously characterized both cell types in regard

Communications to the Editor
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16 2437
F igu r e 2. Effect of 4 on mitochondrial steroidogenesis. 4 and the indicated compounds were tested for their ability to stimulate
MA-10 Leydig (left) and C6-2B (right) glioma mitochondrial ability to synthesize pregnenolone. Mitochondria were isolated and
incubated with the indicated concentrations of different PBR ligands as previously described.7,15 At the end of the incubation,
pregnenolone was extracted and measured by radioimmunoassay.15 The results shown are the means (n ) 4) from two independent
experiments. Standard deviations are not given for the simplicity of the figure and were <15% of the mean value.
F igu r e 3. Fluorescent labeling studies of the PBR in MA-10 Leydig cells. MA-10 cells were cultured on coverslips and incubated
o
with 4 (1 µM) for 45 min at 37 C in the absence (a, b, c) or presence of 100 µM of PK 11195 (d), FGIN-27 (e), Ro5-4864 (f), or
clonazepam (g). At the end of the incubation time, the cells were washed, and PBR was localized by fluorescence microscopy.
Magnification ×217 (reproduced at 75% of original size).
fluorigenic group will confer to compound 4 novel
binding properties for a receptor other than PBR.
F u n ction a l Stu d ies. One of the functions of PBR
ligands is the activation of cholesterol transport to
mitochondria and subsequent steroid formation.14-20
Most of the PBR ligands have been characterized for
their steroidogenesis-inducing activity. Figure 2 shows
that compound 4 stimulated mitochondrial pregnenolo-
ne formation by both testis Leydig and brain glial cells
with the same potency and efficacy as the isoquinoline
PK 11195, the benzodiazepine Ro5-4864, and the 2-aryl-
3-indoleacetamide prototype FGIN-1-27. As previously
shown,15,18 clonazepam did not affect mitochondrial
steroidogenesis.
F lu or escen ce Micr oscop y. Glass coverslips were
incubated for at least 2 h with fetal bovine serum in
order to allow serum components, such as fibronectin,
to coat the surface, thus allowing better attachment of
the cells. Cells were plated on the coverslips and
cultured in media supplemented with serum as previ-
ously described for each cell line.15,18 When cells
reached the required confluency, they were washed
twice with sterile phosphate-buffered saline (PBS) and
incubated for 45 min with 1 µM compound 4 with or
without the indicated competing PBR ligands at 100 µM
concentrations. At the end of the incubation period, the
coverslips were washed with PBS and examined by
fluorescence microscopy using an Olympus BH-2 fluo-
rescence microscope. The incubation time of 45 min was
established based on preliminary experiments where we
observed that intracellular fluorescence was visible after
20 min incubation time and reached the saturation point
at around 40 min. The concentration of 4 used was also
determined based on preliminary dose-response stud-
ies. These experiments showed that intracellular fluo-
rescent labeling could be seen using 10 and 100 nM 4
but that a concentration of 1 µM 4 was required to
obtain a consistent and reproducible labeling. The value
of 1 µM is about 30 times higher than the IC50 obtained
from data in Figure 1, suggesting that compound 4
might be labeling a site other than PBR. However,
higher amounts of compound 4 may be needed because
of the experimental protocol used. It is important to
note that the binding studies were performed in cells
collected from the dishes, centrifuged, distributed to
assay tubes, and incubated at 4 °C whereas fluorescent
studies were performed directly on the living cells in
culture. Figure 3 shows fluorescent labeling of MA-10
Leydig cell PBR. The labeling was notably cytoplasmic,
and when using a low concentration of 4 we could see
fluorescent cytoplasmic spots. These data are in agree-
ment with the observations that in MA-10 Leydig cells,

2438 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16
Communications to the Editor
Su p p or tin g In for m a tion Ava ila ble: Preparation and
spectroscopic and analytical data for compound 4 (2 pages).
Ordering information is given on any current masthead page.
Refer en ces
(1) (a) Costa, E.; Guidotti, A. Molecular Mechanism in the Receptor
Actions of Benzodiazepines. Annu. Rev. Pharmacol. Toxicol.
1979, 19, 531-545. (b) Pritchett, D. B.; Sontheimer, H.; Shivers,
B. D.; Ymer, S.; Kettenmann, H.; Schofield, P. R.; Seeburg, P.
H. Importance of a Novel GABAA Receptor Subunit for Benzo-
diazepine Pharmacology. Nature 1989, 338, 582-585.
(2) Braestrup, C.; Squires, R. F. Specific Benzodiazepine Receptors
in Rat Brain Characterized by High-affinity [3H]diazepam
Binding. Proc. Natl. Acad. Sci. U.S.A. 1977, 74, 3805-3809.
(3) Benavides, J .; Quarteronet, D.; Imbault, F.; Malgouris, C.; Uzan,
A.; Renault, C.; Dubroeucq, M. C.; Gueremy, C.; Le Fur, G.
Labeling of Peripheral-type Benzodiazepine Binding Sites in the
Rat Brain by Using [3H]PK 11195, an Isoquinoline Carboxamide
Derivative: Kinetic Studies and Autoradiographic Localization.
J . Neurochem. 1983, 41, 1744-1750.
F igu r e 4. Fluorescent labeling studies of the PBR in C6 rat
glioma cell line. C6 cells were cultured on coverslips and
incubated with 4 (1 µM) for 45 min at 37 °C (a, b). At the end
of the incubation time, the cells were washed, and the PBR
was localized by fluorescence microscopy. Magnification ×217
(reproduced at 70% of original size).
(4) Langer, S. Z.; Arbilla, S. Imidazopyridines as a Tool for Char-
acterization of Benzodiazepine Receptors: A Proposal for
a
Pharmacological Classification as Omega Receptor Subtypes.
Pharmacol. Biochem. Behav. 1988, 29, 763-766.
(5) Romeo, E.; Auta, J .; Kozikowski, A. P.; Ma, A.; Papadopoulos,
V.; Puia, G.; Costa, E.; Guidotti, A. 2-Aryl-3-Indoleacetamides
(FGIN-1): A New Class of Potent and Specific Ligands for the
Mitochondrial DBI Receptor. J . Pharmacol. Exp. Ther. 1992,
262, 971-978.
(6) Campiani, G.; Nacci, V.; Fiorini, I.; De Filippis, M. P.; Garofalo,
A.; Ciani, S. M.; Greco, G.; Novellino, E.; Williams, D. C.;
Zisterer, D. M.; Woods, M. J .; Mihai, C.; Manzoni, C.; Mennini,
T. Synthesis, Biological Activity, and SARs of Pyrrolobenzox-
azepine Derivatives, a New Class of Specific “Peripheral-Type”
Benzodiazepine Receptor Ligands. J . Med. Chem. 1996, 39,
3435-3450.
which have high numbers of mitochondria, 90% of the
PBR is on the mitochondria as shown in our previous
studies using anti-PBR antisera and confocal micros-
copy.26 The fluorescent staining was completely dis-
placed by a 100-fold excess of the isoquinoline PK 11195,
the benzodiazepine Ro5-4864, and the 2-aryl-3-indole-
acetamide prototype FGIN-1-27, further demonstrating
the specificity of the interaction 4 with the PBR.
(7) (a) Papadopoulos, V.; Berkovich, A.; Krueger, K. E.; Costa, E.;
Guidotti, A. Diazepam Binding Inhibitor (DBI) and its Process-
ing Products Stimulate Mitochondrial Steroid Biosynthesis via
an Interaction with Mitochondrial Benzodiazepine Receptors.
Endocrinology 1991, 129, 1481-1488. (b) Garnier, M.; Boujrad,
N.; Ogwuegbu, S. O.; Hudson, J . R., J r.; Papadopoulos, V. The
Polypeptide Diazepam Binding Inhibitor and a Higher Affinity
Peripheral-type Benzodiazepine Receptor Sustain Constitutive
Steroidogenesis in the R2C Leydig Tumor Cell Line. J . Biol.
Chem. 1994, 269, 22105-22112.
(8) Snyder, S. H.; Verma, A.; Trifiletti, R. R. The Peripheral-type
Benzodiazepine Receptor: a Protein of Mitochondrial Outer
Membranes Utilizing Porphyrins as Endogenous Ligands. FASEB
J 1987, 1, 282-288.
(9) Rothstein, J . D.; Garland, W.; Puia, G.; Guidotti, A.; Weber, R.
J .; Costa, E. 1992 Purification and Characterization of Naturally
Occuring Benzodiazepine Receptor Ligands in Rat and Human
Brain. J . Neurochem. 1992, 58, 2102-2115.
(10) (a) Papadopoulos, V. Peripheral-type Benzodiazepine/diazepam
Binding Inhibitor Receptor: Biological Role in Steroidogenic Cell
Function. Endocrine Rev. 1993, 14, 222-240. (b) Gavish, M.;
Katz, Y.; Bar-Ami, S.; Weizman, R. Biochemical, Physiological,
and Pathological Aspects of the Peripheral Benzodiazepine
Receptor. J . Neurochem. 1992, 58, 1589-1601.
The possibility that some of the fluorescent labeling
seen may be due to nonspecific trapping of the com-
pound in the lysosomal fraction of the cells should be
also considered. Taking into account the subcellular
fractionation studies performed with radiolabeled
ligands,10-12 the colocalization of PBR with mitochodrial
markers and enzymes,10-12 the high number of mito-
chondria in these cells, and the observation that both
the fluorescent and the radioactive labeling were dis-
placed by unlabeled ligands, a nonspecific lysosomal
labeling is highly unlikely to occur.27
Figure 4 shows fluorescent labeling of the PBR of the
C6-2B glioma cell line. This cell line was also previously
characterized for its PBR content.18 The fluorescent
cytoplasmic spots suggest a mitochondrial localization
of the receptor. Because these cells contain smaller
amounts of the PBR (25 pmol/mg of protein)18 than the
MA-10 cells (56 pmol/mg of protein),15 the spots are
easier to distinguish.
Con clu sion s. We report herein the synthesis of a
fluorescent derivative related to the 2-aryl-3-indoleac-
etamide prototype FGIN-1-27. This derivative (i) re-
tained full ability to displace the radiolabeled isoquin-
oline from the isoquinoline binding site on the 18 kDa
PBR protein, (ii) retained full steroidogenesis-activating
property when added to mitochondria of both testis
Leydig and brain glial cells, and (iii) specifically labeled
the intracellular localization of the PBR in a manner
consistent with the previously reported localization of
the PBR in these cell types.15,18 We believe that this
compound will be a useful tool to probe the localization
and function of the PBR in different tissues.28
(11) Anholt, R. R. H.; Pedersen, P. L.; DeSouza, E. B.; Snyder, S. H.
The Peripheral-type Benzodiazepine Receptor: Localization to
the Mitochondrial Outer Membrane. J . Biol. Chem. 1986, 261,
576-583.
(12) Woods, M. J .; Zisterer, D. M.; Williams, D. C. Multiple Forms
and Locations for the Peripheral-type Benzodiazepine Receptor.
Biochem. Pharmacol. 1996, 51, 1283-1292.
(13) (a) Sprengel, R.; Werner, P.; Seeburg, P. H.; Mukhin, A. G.; Santi,
M. R.; Grayson, D. R.; Guidotti, A.; Krueger, K. E. Molecular
Cloning and Expression of cDNA Encoding a Peripheral-type
Benzodiazepine Receptor. J . Biol. Chem. 1989, 264, 20415-
20421. (b) Riond, J .; Mattei, M. G.; Kaghad, M.; Dumont, X.;
Guillemot, J . C.; Le Fur, G.; Caput, D.; Ferrara, P. Molecular
Cloning and Chromosomal Localization of a Human Peripheral-
type Benzodiazepine Receptor. Eur. J . Biochem. 1991, 195, 305-
311. (c) Chang, Y. J .; McCabe, R. T.; Rennert, H.; Budarf, M. L.;
Sayegh, R.; Emanuel, B. S.; Skolnick, P.; Srauss, J . F. The
Human “Peripheral-type” Benzodiazepine Receptor: Regional
Mapping of the Gene and Characterization of the Receptor
Expressed from cDNA. DNA Cell Biol. 1992, 11, 471-480. (d)
Garnier, M.; Dimchev, A.; Boujrad, N.; Price, M. J .; Musto, N.
A.; Papadopoulos, V. In vitro Reconstitution of a Functional
Peripheral-type Benzodiazepine Receptor. Mol. Pharmacol. 1994,
45, 201-211.
Ack n ow led gm en t. This work was supported by
Grant ES-07747 by the National Institute of Environ-
mental Health Sciences, National Institutes of Health
(to V.P.) and by Alexis Biochemicals (A.P.K.).
(14) (a) Yanagibashi, K.; Ohno, Y.; Nakamichi, N.; Matsui, T.;
Hayashida, K.; Takamura, M.; Yamada, K.; Tou, S.; Kawamura,
M. Peripheral-type Benzodiazepine Receptors are Involved in

Communications to the Editor
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16 2439
the Regulation of Cholesterol Side Chain Cleavage in Adreno-
cortical Mitochondria. J . Biochem. 1989, 106, 1026-1029. (b)
Mukhin, A. G.; Papadopoulos, V.; Costa, E.; Krueger, K. E.
Mitochondrial Benzodiazepine Receptors Regulate Steroid Bio-
synthesis. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 9813-9816.
(c) Thompson, I.; Fraser, R.; Kenyon, C. J . Regulation of
Adrenocortical Steroidogenesis by Benzodiazepines. J . Steroid
Biochem. Mol. Biol. 1995, 53, 75-79.
(21) Papadopoulos, V.; Amri, H.; Boujrad, N.; Cascio, C.; Culty, M.;
Garnier, M.; Hardwick, M.; Li, H.; Vidic, B.; Brown, A. S.;
Reversat, J . L.; Bernassau, J . M.; Drieu, K. Peripheral Benzo-
diazepine Receptor in Cholesterol Transport and Steroidogen-
esis. Steroids 1997, 62, 21-28.
(22) Kozikowski, A. P.; Ma, D.; Brewer, J .; Sun, S.; Costa, E.; Romeo,
E.; Guidotti, A. Chemistry, Binding Affinities, and Behavioral
Properties of a New Class of “Antineophobic” Mitochondrial DBI
Receptor Complex (mDRC) Ligands. J . Med. Chem. 1993, 36,
2908-2920.
(23) Basha, A.; Lipton, M.; Weinreb, S. M. A Mild, General Method
for Conversion of Esters to Amides. Tetrahedron Lett. 1977,
4171-4174.
(24) (a) Ghosh, P. B.; Whitehouse, M. W. 7-Chloro-4-nitrobenzo-
2-oxa-1,3-diazole: A New Fluorigenic Reagent for Amino Acids
and Other Amines. Biochem. J . 1968, 108, 155-156. (b) Fager,
R. S.; Kutina, C. B.; Abrahamson, E. W. The Use of NBD
Chloride (7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole) in Detecting
Amino Acids and as an N-Terminal Reagent. Anal. Biochem.
1973, 53, 290-294.
(25) Munson, P. J .; Rodbard, D. LIGAND: a Versatile Computerized
Approach for Characterization of Ligand Binding Systems. Anal.
Biochem. 1980, 107, 220-239.
(26) Garnier, M.; Boujrad, N.; Oke, B. O.; Brown, A. S.; Riond, J .;
Ferrara, P.; Shoyab, M.; Suarez-Quian, C. A.; Papadopoulos,
V.Diazepam Binding Inhibitor is a Paracrine/autocrine Regula-
tor of Leydig Cell Proliferation and Steroidogenesis. Action via
Peripheral-type Benzodiazepine Receptor and Independent Mech-
anisms. Endocrinology 1993, 132, 444-458.
(15) (a) Papadopoulos, V.; Mukhin, A. G.; Costa, E.; Krueger, K. E.
The Peripheral-type Benzodiazepine Receptor is Functionally
Linked to Leydig Cell Steroidogenesis. J . Biol. Chem. 1990, 265,
3772-3779. (b) Papadopoulos, V.; Nowzari, F. B.; Krueger, K.
E. Hormone-Stimulated Steroidogenesis is Coupled to Mitochon-
drial Benzodiazepine Receptors. J . Biol. Chem. 1991, 266, 3682-
3687.
(16) Amsterdam, A.; Suh, B. S. An Inducible Functional Peripheral
Benzodiazepine Receptor in Mitochondria of Steroidogenic Gran-
ulosa Cells. Endocrinology 1991, 128, 503-510.
(17) Barnea, E. R.; Fares, F.; Gavish, M. Modulatory Action of
Benzodiazepines on Human Term Placental Steroidogenesis in
vitro. Mol. Cell. Endocr. 1989, 64, 155-159.
(18) (a) Papadopoulos, V.; Guarneri, P.; Krueger, K. E.; Guidotti, A.;
Costa, E. Pregnenolone Biosynthesis in C6 Glioma Cell Mito-
chondria: Regulation by a Diazepam Binding Inhibitor Mito-
chondrial Receptor. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 5113-
5117. (b) McCauley, L. D.; Park, C. H.; Lan, N. C.; Tomich, J .
M.; Shively, J . E.; Gee, K. W. Benzodiazepines and Peptides
Stimulate Pregnenolone Synthesis in Brain Mitochondria. Eur.
J . Pharm. 1995, 276, 145-153.
(19) Krueger, K. E.; Papadopoulos, V. Peripheral-type Benzodiazepine
Receptors Mediate Translocation of Cholesterol from Outer to
Inner Mitochondrial Membranes in Adrenocortical Cells. J . Biol.
Chem. 1990, 265, 15015-15022.
(20) Amri, H.; Ogwuegbu, S. O.; Boujrad, N.; Drieu, K.; Papadopoulos,
V. In vivo Regulation of the Peripheral-type Benzodiazepine
Receptor and Glucocorticoid Synthesis by the Ginkgo biloba
Extract EGb 761 and Isolated Ginkgolides. Endocrinology 1996,
137, 5727-5730.
(27) (a) Allison, A. C.; Young, M. R. Uptake of Dyes and Drugs by
Living Cells in Culture. Life Sci. 1964, 3, 1407-1414. (b) De
Duve, C.; De Barsy, T.; Poole, B.; Trouet, A.; Tulkens, P.; Van
Hoff, F. Lysosomotropic Agents. Biochem. Pharm. 1974, 23,
2495-2531.
(28) Compound 4 can be obtained from Alexis Biochemicals, San
Diego, CA.
J M970220W