C-7 analogues of progesterone as potent inhibitors of the P-glycoprotein efflux pump

Article abstract of DOI:10.1021/jm010126m

The P-glycoprotein product (Pgp) of the MDR1 gene has been implicated in the multiple drug resistance phenotype expressed by many cancers. Functioning as an efflux pump, P-glycoprotein prevents the accumulation of high intracellular concentrations of subs

Full text of DOI:10.1021/jm010126m

390
J . Med. Chem. 2002, 45, 390-398
C-7 An a logu es of P r ogester on e a s P oten t In h ibitor s of th e P -Glycop r otein Efflu x
P u m p
Fabio Leonessa,† J i-Hyun Kim,† Alem Ghiorghis,‡ Robert J . Kulawiec,‡ Charles Hammer,‡
Abdelhossein Talebian,‡,§ and Robert Clarke*,†
Departments of Oncology, Physiology and Biophysics, and Lombardi Cancer Center, Georgetown University School of Medicine,
3970 Reservoir Road Northwest, Washington, DC 20007, and Department of Chemistry, Georgetown University,
37th and O Streets Northwest, Washington, DC
Received March 20, 2001
The P-glycoprotein product (Pgp) of the MDR1 gene has been implicated in the multiple drug
resistance phenotype expressed by many cancers. Functioning as an efflux pump, P-glycoprotein
prevents the accumulation of high intracellular concentrations of substrates. We have taken a
rational approach to designing inhibitors of P-glycoprotein function, selecting a natural substrate
(progesterone) as our lead compound. We hypothesized that progesterone, substituted at C-7
with an aromatic moiety(s), would exhibit reduced Pgp affinity, significantly increased antiPgp
activity, and reduced affinity for progesterone receptors (PGR). We synthesized 7R-[4′-
(aminophenyl)thio]pregna-4-ene-3,20-dione (2), which comprises a C-7R thiol bridge linking
an aminophenyl moiety to progesterone, from pregna-4,6-diene-3,20-dione (1). The subsequent
addition reaction of 2 with the appropriate isocyanate produced an initial series of compounds
(3-6). Compounds 3-5 (respectively, -CH2CH2Cl; -CH2CH3; and -CH(CH3)C6H5) exhibit a
significantly increased ability to inhibit P-glycoprotein. Potency for restoring doxorubicin
accumulation in MDR1-transduced human breast cancer cells is increased up to 60-fold as
compared with progesterone. Compound 5 has greater potency than verapamil and is equipotent
with cyclosporin A, for inhibiting P-glycoprotein function. Furthermore, 5 does not bind to PGR,
implying a potential reduction in in vivo toxicity. These data identify C-7-substituted
progesterone analogues and 5, in particular, as rationally designed antiPgp compounds worthy
of further evaluation/development.
In tr od u ction
expelled from the cell.7 We have shown by meta analysis
that Pgp expression is detected in g50% of breast
cancers and that this expression is associated with prior
chemotherapy, a worse than partial response to chemo-
therapy, and in vitro resistance to Pgp substrates.8
These data suggest that where Pgp expression is
detected, it likely contributes to multiple drug resistance
in some breast cancers. Nonetheless, the likely role of
Pgp in conferring drug resistance remains controversial.
The poor activity of current antiPgp agents in patients
has been attributed to the presence of resistance factors
in addition to Pgp, inappropriate design of clinical trials,
toxicity, and/or lack of specificity of antiPgp reagents.
The ability of reversing agents to alter the pharmaco-
kinetics of the coadministered cytotoxic drugs, and an
inability to achieve adequate levels of some reversing
agents, also are problematic.9 The absence of a series
of nontoxic drugs, specifically designed to reverse Pgp,
limits the design of clinical trials to reverse this form
of multidrug resistance.
One aspect of the controversy regarding the role of
Pgp comes from the relatively poor activity of those few
Pgp reversing agents evaluated in clinical trials. Most
attention has focused on the Pgp reversing agents
verapamil, cyclosporin A, and its nonimmunosuppres-
sant analogue PSC833 (valspodar), but the activity of
other drugs also has been studied in patients. Few of
these compounds were designed as Pgp inhibitors. Thus,
severe side effects, often related to either the “normal”
function of these agents and/or their ability to influence
While many cancers are initially responsive to cyto-
toxic chemotherapy, most acquire a resistant phenotype.
This phenotype is often characterized by crossresistance
to structurally unrelated drugs to which the tumor has
not been exposed. The precise genes that confer this
multidrug resistance phenotype are unknown, but there
are several strong single-gene candidates. These include
several ABC transporters including the P-glycoprotein
product of the MDR1 gene (Pgp; gp170),1 the lung
resistance protein,2 the breast cancer resistance pro-
tein,3 and several members of the multidrug resistance
associated protein family.4,5 The precise contribution of
each potential multidrug resistance mechanism is un-
clear. Indeed, more than one mechanism may operate,
either within the same tumor cell subpopulation and/
or within different subpopulations of the same tumor.
We have chosen to study Pgp-mediated multiple drug
resistance. Pgp confers resistance to drugs by prevent-
ing their accumulation within the cell. Pgp’s efflux
capabilities appear to reflect its ability to bind sub-
strates within the inner leaflet of the plasma mem-
brane.6 Subsequently, and in a potentially adenosine 5′-
triphosphate (ATP)-dependent manner, substrates are
* To whom correspondence should be addressed. Tel: (202)687-3755.
Fax: (202)687-7505. E-mail: clarker@georgetown.edu.
† Departments of Oncology, Physiology and Biophysics, and Lom-
bardi Cancer Center, Georgetown University School of Medicine.
‡ Department of Chemistry, Georgetown University.
§ Deceased.
10.1021/jm010126m CCC: $22.00 © 2002 American Chemical Society
Published on Web 12/14/2001

C-7 Analogues of Progesterone as Potent Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2 391
additional targets, may be induced at concentrations
required to affect Pgp function. Many antiPgp drugs
affect the pharmacokinetics of the substrate, signifi-
cantly increasing cytotoxic drug-induced toxicity.10 Other
antiPgp agents cannot readily be delivered at doses that
produce adequate serum levels. For example, the serum
levels of verapamil required to produce in vitro reversal
of Pgp resistance are rarely achieved in patients, despite
administering sufficient doses of verapamil to induce
significant cardiotoxicity.9,11,12 Adequate serum trifluo-
perazine levels are not reached in patients at doses that
induce dose-limiting toxicities.9,13 Peak plasma levels of
the stereoisomer of cis-flupenthixol (trans-flupenthixol)
are 1000-fold less than that necessary to achieve full
chemosensitization in vitro.14,15 Several clinical studies
have used patient populations where tumor Pgp expres-
sion is unknown, complicating a clear determination of
its contribution to multiple drug resistance.
Previously, we have established cellular breast cancer
models in which to study Pgp-mediated efflux and
evaluate inhibitors of this activity. These models have
been generated by inducing a constitutive expression
of Pgp, following transduction with retroviral gene
expression vectors.16,17 The major advantage of these
models is that unlike cells selected for resistance in
vitro, Pgp expression is the only mechanism present to
produce the multiple drug resistance phenotype. For
example, the widely used MCF7ADR cells, which were
selected in vitro for resistance to doxorubicin (DOX) and
recently redesignated NCI/ADR-RES,18 exhibit in-
creased glutathione transferase and topoisomerase II
activities.19,20 Differences in the potency of isomers of
fluphenthixol identified in MCF7ADR cells could not be
confirmed in MDR1-transfected NIH 3T3 cells.15
The major beneficial properties we wished to confer
included but were not restricted to (i) improved potency
for Pgp reversal, (ii) either no change or a reduction in
affinity for PGR, and (iii) no agonist (mitogenic) activi-
ties. Concurrently, we wished to avoid either a substan-
tial increase in PGR binding or a loss of Pgp reversing
potency.
Unfortunately, the precise structure-function char-
acteristics of Pgp reversing agents are unknown. This
is not surprising, given the remarkable structural
diversity of Pgp substrates.30 Nonetheless, several
characteristics are apparent, providing generic guide-
lines for the design of Pgp reversing agents. Lipophi-
licity appears central, with increased lipophilicity strongly
associated with increased antiPgp activity.31-37 Planar
aromatic rings are commonly found in substrates, and
these may contribute to lipophilicity.37 Amphipathicity
also is common, as is the presence of a basic amine,
where primary amines appear most effective.31,33,35,36,38
Size, for example, as determined by calculated molar
refractivity, appears an important factor in several
classes of compounds.32,35,39 C21-aminosteroids have a
structural similarity to progesterone, and in these
compounds, the steroid moiety, lipophilicity, and amphi-
pathicity are considered important attributes.40 For
compounds composed of two structures joined by a
molecular spacer, the length of the spacer seems im-
portant.15,31,34,41,42 This suggests that some part of the
molecule may be oriented into a “pocket” in Pgp.31 This
pocket may have specific requirements for lipophilicity,
size, and charge.
A C-7 addition to the steroid 17â-estradiol, as occurs
in the antiestrogens ICI 182,780 and ICI 164,384,
produces compounds with low toxicity and potentially
appropriate pharmacokinetics.43,44 Limited evidence
suggests that ICI 164,384 can reverse Pgp-mediated
resistance,45 despite the apparent inability of 17â-
estradiol to do so.23 Thus, a bulky C-7 substitution on a
steroid nucleus might increase antiPgp activity. C-7-
substituted progesterone analogues were synthesized 20
years ago, but several exhibit antiprogestational activ-
ity.46 Data from these studies suggest that bulky addi-
tions at C-7, when these include an aromatic ring,
reduce PGR affinity by approximately 10-1000-fold.46
Consequently, it may be possible to reduce the endog-
enous toxicity of progesterone by reducing/eliminating
its ability to bind PGR.
Using our cellular models, we now describe an initial
series of progesterone analogues that exhibit signifi-
cantly increased antiPgp activity as compared with
progesterone and verapamil and comparable to that
seen with cyclosporin A. Importantly, the most potent
of these analogues has lost its ability to activate
progesterone receptors (PGR) and is predicted to exhibit
relatively low intrinsic toxicity in vivo.
Ch em istr y
Con cep tu a liza tion a n d Design . We wished to take
a rational, structure-function-based approach to design
inhibitors of Pgp function. Initially, we hypothesized
that a natural substrate for the pump could provide an
ideal candidate for rational drug design, since it is likely
that Pgp evolved specifically to efflux such molecules.
Evidence shows that several molecules with a steroid
nucleus are Pgp substrates.21-23 Pgp is expressed in the
uterus23,24 and the placenta,25 suggesting a natural role
for protecting secretory cells from the toxic effects of
high local concentrations of steroids. Progesterone is the
most potent of the steroids, including progesterone’s
metabolites, for reversing the effects of Pgp expres-
sion.23,26,27 Progestins have intrinsically lower toxicity
than other reversing agents and are orally active. In
addition, progesterone is readily available and cheap,
and the chemistry for generating several structural
modifications is relatively straightforward.28,29 Thus, we
chose progesterone as our lead compound.
On the basis of the various structure-function ob-
servations noted above, we hypothesized that proges-
terone, substituted at C-7 with an aromatic moiety(s),
would exhibit both reduced Pgp affinity and signifi-
cantly increased antiPgp activity. Thus, we designed an
initial compound from which we could derive an ap-
propriate series of progesterone analogues for evalua-
tion. This compound, 7R-[4′-(aminophenyl)thio]pregna-
4-ene-3,20-dione (2), has a C-7 thiol bridge linking an
aminophenyl moiety to progesterone. Subsequent ad-
ditions to the amine with the appropriate isocyanate
generated the corresponding Pgp analogues. For our
initial series of compounds, we selected isocyanates that
would provide analogues with predicted differences in
the size, lipophilicity, and charge of their C-7 additions.
Syn th esis. Compound 1 (Scheme 1) was prepared
from progesterone by a modified Turner and Ringold’s

392 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2
Sch em e 1. Synthesis of C-7 Progesterone Analogues
Leonessa et al.
Ta ble 1. Structure and Physical Properties of C-7
analogues were purified by flash column chromatogra-
phy to yield the corresponding ureas as a white solid
(40-83%). The physical properties of these analogues
are provided in Table 1 as mp and Rf on silica gels (2:3
hexanes-ethyl acetate).
Progesterone Analoguesa
b
compd
R
mp (deg)
Rf
2
3
4
5
6
N/A
-CH2CH2Cl
-CH2CH3
-CH(CH3)C6H5
-SO2C6H4CH3
228-230
137-141
130-135
146-149
128-132
0.23
0.47
0.36
0.46
0.29
Resu lts a n d Discu ssion
Su bstr a te Accu m u la tion Stu d ies. Because Pgp is
an efflux pump, we measured the ability of our com-
pounds to influence the intracellular accumulation of
the cytotoxic drugs vinblastine (VBL) and DOX. Both
drugs are widely used clinically and are efficiently
effluxed by Pgp.50 Activity was evaluated in MDR1-
transduced human breast cancer cells (MDA435/
LCC6MDR1), using the parental cells (MDA435/LCC6) as
the Pgp-negative control. Potency of the compounds was
compared with that of progesterone and the established
Pgp inhibitors verapamil and cyclosporin A. VBL con-
tent in Pgp-positive cells, exposed to media containing
5 nM [3H] VBL, was approximately 6-fold lower than
in parental Pgp-negative cells. Cellular content of DOX,
in cells exposed to 4 µM DOX, was about 8-fold lower
in the presence than in the absence of Pgp.
Results of VBL and DOX accumulation studies, sum-
marized in terms of EC50, are presented in Table 2.
Because these data were estimated from dose response
curves, representative curves are shown in Figure 1.
Treatment with progesterone analogues 3-6 reverses
the difference in VBL and DOX content between Pgp-
positive and Pgp-negative cells. Analogues 3-5 exhibit
significantly increased antiPgp potency as compared
a
b
See Scheme 1 for the structures of 2-6. Rf in hexane-ethyl
acetate (2:3).
method,47-49 using 2,3-dichloro-5,6-dicyano-1,4-benzo-
quinone (DDQ) as an oxidizing agent and p-toluene-
sulfonic acid (p-TsOH) in refluxing benzene via Dean-
Stark distillation. Purification of the crude 1 on silica
gel gives 6-dehydroprogesterone (1) as a yellow solid
(35%, Rf ) 0.44, 2:3 hexanes-ethyl acetate, mp ) 143-
145 °C). Reaction of compound 1 with 4-aminothio-
phenol and NaOH pellets, in degassed dioxane as
solvent for 6 days at 74 °C, provided 7R-[(4′-amino-
phenyl)thio]pregna-4-ene-3,20-dione (2) as an ivory
solid. Crude crystals of 2 were precipitated from a
mixture of hexanes-ethyl acetate and purified by flash
column chromatography to yield 790 mg of white solid
(61%, Rf ) 0.23, 2:3 hexanes-ethyl acetate, mp ) 228-
230 °C).
The additional C-7 progesterone analogues (3-6) were
obtained by reacting compound 2 with the appropriate
isocyanate (Table 1). The general reaction was per-
formed under a N2 atmosphere for 12 h, until no 2 was
detected by thin-layer chromatography (TLC), and the
solvent was removed under reduced pressure. All crude

C-7 Analogues of Progesterone as Potent Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2 393
Ta ble 2. Potency of C-7 Progesterone Analogues, Progesterone, Verapamil, and Cyclosporin A in Reversing the Difference in VBL
and DOX Accumulation between Pgp-Negative and Pgp-Positive Cells
reversal of [3H] VBL accumulation
reversal of DOX accumulation
EC50 µMa
Pgp-specific EC50c µM
(relative potency)
EC50 µM
(relative potency)
Pgp-specific EC50 µM
(relative potency)
compd
(relative potency)b
progesterone
3
4
5
18.7 ( 3.7d (1)
0.8 ( 0.2 (22.5)
1.3 ( 0.1 (14.2)
0.8 ( 0.2 (24.5)
34.8 ( 8.6 (0.5)
1.2 ( 0.2 (16.1)
0.6 ( 0.06 (32.5)
21.0 ( 4.2 (1)
22.3 ( 2.0 (1)
42.2 ( 7.2 (1)
0.9 ( 0.2 (23.5)
1.5 ( 0.2 (14.0)
0.7 ( 0.2 (28.2)
33.4 ( 5.2 (0.6)
3.1 ( 0.9 (6.8)
0.6 ( 0.06 (32.5)
0.6 ( 0.1 (40.5)
0.7 ( 0.07 (31.3)
0.6 ( 0.07 (37.2)
14.7 ( 3.2 (1.5)
2.4 ( 0.3 (9.2)
0.5 ( 0.1 (41.9)
0.7 ( 0.2 (60.2)
1.0 ( 0.06 (42.7)
0.9 ( 0.09 (44.8)
37.0 ( 6.5 (1.1)
4.1 ( 0.5 (10.2)
0.7 ( 0.2 (60.6)
6
verapamil
cyclosporin A
a
EC50 ) drug concentration that reduces the difference in drug accumulation between MDA435/LCC6 and MDA435/LCC6MDR1 cells
by 50%; obtained by interpolation on dose response curves. Representative curves are shown in Figures 1 ([3H] VBL) and 2 (DOX). Values
b
in parentheses represent the potency of each compound relative to the lead compound progesterone. c Pgp-specific EC50 ) data corrected
d
for any effect of test compound on drug accumulation in MDA435/LCC6 (Pgp-negative) cells. Values represent the mean ( SE obtained
from at least three independent experiments.
nates the gain in activity conferred by the C-7 moiety.
Thus, the increased activity in 5 is not simply due to
the presence of an aromatic F ring (Scheme 1). Further
structural modifications will allow us to test further the
structure-activity relationships of C-7 progesterone
analogues for Pgp reversal.
A major problem with many existing antiPgp com-
pounds is their intrinsic toxicity. We wished to obtain
an in vitro assessment of the toxicity of our compounds
relative to progesterone, verapamil, and cyclosporin A.
We used our breast cancer cell models because they do
not express PGR and would provide a simple model for
assessing PGR-independent cytotoxicity. Furthermore,
any reduction in toxicity seen in the MDA435/LCC6MDR1
cells, as compared with the MDA435/LCC6 cells (rela-
tive resistance of Pgp-positive cells in Table 3), would
suggest that the compounds were Pgp substrates, not
simply Pgp inhibitors. Results are summarized in terms
of IC50 in Table 3; representative dose response curves
are shown in Figure 2.
To estimate relative activity, each drug’s intrinsic
cytotoxicity was expressed relative to its antiPgp activ-
ity (IC50/EC50; Table 3). We did not detect cytotoxicity
for compounds 4 and 5, due to their low solubility,
rendering our ratios underestimates based on the high-
est (noncytotoxic) concentration tested. Nonetheless, 4
produces ∼40% inhibition of proliferation at 20 µM. In
F igu r e 1. Ability of C-7 progesterone analogues to affect [3H]
marked contrast, 20 µM 5 does not inhibit proliferation
VBL accumulation (A) and DOX accumulation (B) in MDA435/
LCC6 and MDA435/LCC6MDR1 cells. Data (mean ( SE) are
from one of three or more representative experiments used to
obtain the ED50 values presented in Table 2. Progesterone )
b, cyclosporin A ) O, verapamil ) 2, 3 ) 9, 4 ) 4, 5 ) 0, and
6 ) 3.
significantly in either untreated cells or MDA435/LCC6
and MDA435/LCC6MDR1 cells.
When adjusted for cytotoxicity, cyclosporin A and
progesterone exhibit approximately equivalent relative
activities. The low estimates for cyclosporin A reflect
its substantial toxicity. Compound 6 is the least active
compound, and 5 is the most active despite the over-
estimation of its cellular toxicity. While VBL and DOX
may have different recognition sites in Pgp,53 5 shows
broadly comparable activity against both drugs, as does
cyclosporin A.
Having established that the C-7 addition significantly
increased antiPgp activity, we wished to evaluate the
PGR activity of our best compound. Overall, 3 and 5
have antiPgp activity comparable to cyclosporin A.
Because 3 exhibits significant cellular toxicity, we chose
to evaluate the relative affinity of 5 for binding to PGR.
We compared the ability of 5, progesterone, and un-
labeled ORG2058, a synthetic progestin, to compete with
[3H] ORG2058 for binding to PGR. The data in Figure
with progesterone, being 14-60-fold more potent. In
marked contrast, 6 is only equipotent with progesterone.
Three compounds (3-5) are significantly more potent
than verapamil, when Pgp-specific EC50s are compared
for both VBL and DOX accumulation. Compounds 3 and
5 were equipotent with cyclosporin A (p > 0.05 for all
comparisons). Recently, we have established a chro-
matographic approach for assessing relative Pgp binding
affinities.51,52 Studies to measure the affinity of these
analogues are in progress.
While 3 and 5 tend to be slightly more potent than 4,
the difference is not statistically significant. This sug-
gests that addition of either a Cl (3) or a second aromatic
ring (5) does not further increase activity. In marked
contrast, the presence of the sulfonyl group in 6 elimi-

394 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2
Leonessa et al.
Ta ble 3. Growth Inhibitory Activity of C-7 Progesterone Analogue, Progesterone, Verapamil, and Cyclosporin A on MDA435/LCC6
(Pgp-Positive) and MDA435/LCC6MDR1 (Pgp-Negative) Human Breast Cancer Cells
IC50 µM
c
IC50/EC50
MDA435/LCC6
MDA435/LCC6MDR1
(relative cytotoxicity)
relative resistance
compd
(relative cytotoxicity)a
of Pgp-positive cellsb
VBL activity
DOX activity
progesterone
3
4
5
27.4 ( 7.9d(1.0)
3.2 ( 0.09 (8.5)
>20.0 (ND)e
36.4 ( 8.3 (1.0)
7.3 ( 2.5 (5.0)
>20.0 (ND)
1.4 ( 0.09
2.2 ( 0.7
ND
1.3
3.6
>13.3
>28.6
0.7
0.6
3.2
>20.0
>22.2
0.6
>20.0 (ND)
>20.0 (ND)
ND
6
22.1 ( 1.6 (1.2)
65.8 ( 0.04 (0.4)
1.0 ( 0.5 (26.9)
38.2 ( 0.2 (1.0)
63.4 ( 1.2 (0.6)
1.1 ( 0.2 (33.6)
1.7 ( 0.1
1.0 ( 0.02
1.3 ( 0.4
verapamil
cyclosporin A
21.2
1.7
16.0
1.4
a
b
Relative cytotoxicity ) ability of compounds to inhibit cell growth relative to progesterone. Relative resistance of Pgp-positive )
ratio of toxicity in resistant and control cells. Relative resistance > 1 suggests that the compound is at least partly effluxed by Pgp. c This
ratio relates the effect of each drug on either VBL or DOX accumulation to its intrinsic cellular toxicity; higher ratios suggest a greater
degree of safety. Values represent the mean ( SE obtained from at least three independent experiments. e ND ) no data; an IC50 was
not reached at the highest concentration tested (limited by solubility). Some values (>) are underestimates based on nontoxic concentrations.
d
F igu r e 3. Competitive binding of progesterone, ORG2058,
and 5 to PGR. [3H] ORG2058 was used as the radiolabeled
ligand. Progesterone ) b, ORG2058 ) O, and 5 ) 0.
vations can guide future studies. The molecules are
clearly amphipathic, with lipophilicity greatest around
the “E” and/or “F” rings and the polarity greatest around
C-17-C-21. These observations suggest that effective
substrates may concurrently interact with both hydro-
philic and hydrophobic regions. However, it is not clear
whether these are both in Pgp as previously suggested31
or whether they represent pockets at the plasma
membrane/Pgp interface. The possibility that drugs are
removed from within the plasma membrane6 may favor
the model that invokes a plasma membrane component
F igu r e 2. Cytotoxicity of progesterone analogues in MDA435/
to the binding interaction.
LCC6 (A) and MDA435/LCC6MDR1 cells (B). Data (mean ( SE)
Compounds 3-5 are significantly more potent than
are from one of three or more representative experiments used
progesterone at specifically increasing Pgp substrate
to obtain the IC50 values presented in Table 3. Progesterone
accumulation. These observations are consistent with
) b, cyclosporin A ) O, verapamil ) 2, 3 ) 9, 4 ) 4, 5 ) 0,
and 6 ) 3.
our initial hypothesis that aromatic C-7 substitutions
of progesterone will increase activity and with the
known contribution of aromatic moieties in other modu-
lating agents.37,42 Compound 6 also contains a C-7
aromatic addition but is essentially equipotent with
progesterone. Perhaps the simplest explanation is that
this compound is the least lipophilic of the analogues,
since lipophilicity appears to be a major factor in the
activity of other Pgp substrates.31-35
The significant increase in potency observed with
compounds 3-5 supports our initial structure-function-
based hypothesis, based on previous published observa-
tions. The activity of our existing compounds already
compares well with that of cyclosporin A. C-7 progest-
3 show that 5, at concentrations up to its EC50, does
not significantly compete with ORG2058. This repre-
sents a reduction of >100-fold in its PGR affinity as
compared with ORG2058. Thus, 5 has antiPgp activity
comparable to cyclosporin A, exhibits potentially low
intrinsic cellular toxicity, and does not bind to its
predicted cellular target (PGR) at its EC50 for inhibition
of Pgp activity.
Con clu sion s
While we cannot draw definitive structure-activity
conclusions, some potentially useful preliminary obser-

C-7 Analogues of Progesterone as Potent Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2 395
1H NMR spectroscopy. Elemental analysis was performed by
Atlantic Microlab (Norcross, GA).
erone analogues have the potential to provide more
potent, selective, and safe inhibitors of Pgp function
than others that have currently completed clinical trials.
We believe that the observations reported here, com-
bined with the lack of receptor binding activity, identify
5 as the next logical lead compound for further develop-
ment and provide valuable clues for the further opti-
mization of this structure. We are currently synthesiz-
ing a larger series of compounds to further optimize the
MDR1 reversing potency and effectively define the
structure-activity relationships of these compounds.
Our ability to increase the potency of progesterone
up to 60-fold (3; Pgp-specific EC50 for DOX accumula-
tion) supports the use of relatively limited structure-
function data in the design of effective antiPgp com-
pounds. Furthermore, by including structure-activity
information on the binding characteristics of the lead’s
natural intracellular target (PGR) in our conceptual-
ization, we reduced affinity of 5 for a target that could
produce toxicity in normal cells. We are now poised to
evaluate our compounds in vivo, to pursue further
modifications that may increase antiPgp activity, and
to explore the structure-activity relationship for C-7
progesterone analogues in detail. Overall, the data in
this study identify C-7-substituted progesterone ana-
logues and 5, in particular, as rationally designed
antiPgp compounds worthy of further evaluation/
development.
P r egn a -4,6-d ien e-3,20-d ion e (1). Compound 1 was pre-
pared by the method of Tuner and Ringold.47,49 Thus, p-TsOH
monohydrate (11.0 g, 63.9 mmol) was dehydrated in freshly
distilled benzene (320 mL) by azeotropic refluxing using a
Dean-Stark trap. After 1 h, the solution was cooled for 0.5 h,
and progesterone (5.0 g, 15.9 mmol) and DDQ (4.6 g, 20.3
mmol) were added. The olive mixture was refluxed for 3 h and
then filtered through a pad of Celite. The filtrate was washed
with saturated NaCl (5 × 20 mL) followed by 1% NaOH
solution until it gave clear solution and dried over anhydrous
MgSO4. Solvent was removed under reduced pressure, and the
filtrate was purified by chromatography; 1.69 g of product
(35%, Rf ) 0.44, 2:3 hexanes-ethyl acetate); yellow solid (mp
1
) 143-145 °C). H NMR: δ 6.12 (s, 1H), 5.69 (s, 1H), 2.84-
1.12 (complex, 12H), 2.17 (s, 3H), 2.14 (s, 3H), 1.12 (s, 3H),
1.10 (s, 1H), 1.00 (s, 1H), 0.72 (s, 3H). IR: 3855, 3745, 3678,
2953, 1700, 1663, 1457, 1361, 1223, 875, 754.
7r-[4′-(Am in op h en yl)th io]p r egn a -4-en e-3,20-d ion e (2).
We obtained 2 using the method of Brueggemeier et al.56
Briefly, 1 (1.65 g, 5.28 mmol), 4-aminothiophenol (1.32 g, 10.56
mmol), and NaOH (pellet, 116 mg, 2.9 mmol) were placed in
a Schlenk tube, which was purged with a constant flow of
N2(g). Deoxygenated anhydrous dioxane (25 mL) was added
and heated at 74 °C for 6 days. The mixture was concentrated
under reduced pressure and purified by chromatography; 790
mg white solid (61%, Rf ) 0.23, 2:3 hexanes-ethyl acetate);
mp ) 228-230 °C). 1H NMR: δ 7.26-7.21 (q, J ) 8.5 Hz, 2H),
6.64-6.61 (q, J ) 8.5 Hz, 2H), 5.73 (s, 1H), 3.77 (s, 2H), 3.24
(s, 1H), 2.14 (s, 3H), 2.63-1.10 (complex, 11H), 1.19 (s, 3H),
0.69 (s, 3H). IR: 3420, 3360, 3250, 2930, 1700. 13C NMR: δ
209.3, 199.0, 167.6, 147.1, 136.6, 127.3, 121.2, 115.7, 63.4, 17.7,
13.1.
Exp er im en ta l Section
Gen er a l P r oced u r e for th e P r ep a r a tion of P r ogester -
on e An a logu es. A suspension of 2 in degassed chloroform was
treated with the appropriate isocyanates under N2. The
mixture was stirred for 12 h and then chromatographed
directly on silica gel to afford the corresponding ureas as oil.
The resulting oil was stirred in anhydrous ether until white
powder came out.
Ch em istr y. Gen er a l P r oced u r es. All reactions were
carried out under an atmosphere of nitrogen using standard
Schlenk techniques.54 Benzene and chloroform were distilled
from CaH2, stored over 3D molecular sieves, and deaerated
by purging with nitrogen immediately before use. TLC was
performed using Merck glass plates precoated with F254 silica
gel 60; compounds were visualized by UV and/or with p-
anisaldehyde stain solution. Flash chromatography was per-
formed using EM Science silica gel 60, following the procedure
of Still,55 with the solvent mixtures indicated. Melting points
were measured on a Thomas-Hoover capillary melting point
apparatus and are uncorrected (Table 1). The broad melting
points for compounds 3-6 suggest the presence of minor
impurities. All reagents were purchased from commercial
suppliers and used as received unless indicated otherwise.
Dioxane was purchased from Aldrich in Sure-Seal bottles.
Nuclear magnetic resonance (NMR) spectra were measured
on Nicolet NT 270 and Varian Mercury 300 MHz instruments
at the Georgetown NMR Facility. Chemical shifts are reported
in units of parts per million relative to Me4Si. All spectra are
recorded in CDCl3. Significant 1H NMR data are tabulated in
the following order: multiplicity (s, singlet; d, doublet; t,
triplet; q, quartet; m, multiplet), coupling constants in Hertz,
and number of protons. 13C NMR spectra were recorded at
frequencies of 67.9 and 75.6 MHz. Infrared (IR) spectra were
measured on a MIDAC Corp. or a Mattson Galaxy 2020 Series
7r-[4′-(N-Ch lor oet h yla m in oa cyla m in op h en yl)t h io]-
p r egn a -4-en e-3,20-d ion e (3). Reaction of 2 (0.10 g, 0.23
mmol) with 2-chloroethylisocyanate (38 µL, 0.46 mmol) in
CHCl3 (3.0 mL) for 12 h gave 50 mg of product (40%, mp )
1
137-141 °C, Rf ) 0.47, 2:3 hexanes-ethyl acetate). H NMR:
δ 7.34-7.25 (m, 4H), 5.69 (s, 1H), 5.18 (s, 1H), 3.68-3.62 (m,
4H), 3.38 (s, 1H), 2.64-0.84 (complex, 18H), 2.14 (s, 3H), 1.19
(s, 3H), 0.69 (s, 3H). IR: 3312, 2964, 1700, 1630, 1587, 1517,
1488, 1449, 1394, 1238, 1013, 831, 734. 13C NMR: δ 231.5,
210.3, 196.2, 193.9, 181.9, 156.5, 149.3, 146.4, 141.4, 132.9,
125.1, 119.5, 118.5, 103.2, 94.2, 75.9, 75.8, 71.9, 69.3, 49.0, 35.8,
24.2, 14.4. MS: m/e ) 543 (24, M+ + 1), 507 (10), 313 (27),
230 (23), 185 (50), 149 (69), 125 (57), 119 (23), 107 (38), 105
(48), 91 (50), 81 (50), 57 (73), 55 (100). HRMS: calcd for
C
30H39N2O3SCl [M + H]+, 543.24481; found, 543.24248. Anal.
Calcd for (C30H40O3N2SCl): C, 66.22; H, 7.41; N, 8.82; S, 6.52.
Found: C, 66.38; H, 7.27; N, 8.78; S, 6.28.
7r-[4′-(N-Eth yla m in oa cyla m in op h en yl)th io]p r egn a -4-
en e-3,20-d ion e (4). Reaction of 2 (0.10 g, 0.23 mmol) with
ethylisocyanate (37 µL, 0.46 mmol) in CHCl3 (3.0 mL) for 12
h gave 78 mg of product (67%, mp ) 130-135 °C, Rf ) 0.36,
2:3 hexanes-ethyl acetate). 1H NMR: δ 7.36-7.25 (m, 4H),
6.38 (s, 1H), 5.69 (s, 1H), 4.18-4.03 (m, 2H), 3.38-3.26 (m,
2H), 2.67-0.68 (complex, 17H), 2.14 (s, 3H), 2.05 (s, 2H), 1.20
(s, 3H), 0.69 (s, 3H). IR: 3855, 3745, 3678, 3373, 2953, 2359,
1700, 1663, 1539, 1457, 1223. 13C NMR: δ 228.5, 222.5, 193.9,
171.5, 141.5, 135.0, 128.3, 127.0, 123.4, 118.5, 108.7, 96.2, 84.5,
69.3, 67.7, 66.0, 62.6, 52.2, 48.4, 46.3, 43.9, 39.8, 34.1, 22.9,
21.2, 13.4. MS: m/e ) 509 (62, M+ + 1), 438 (8), 313 (32), 196
(47), 125 (100), 117 (57), 97 (52), 95 (85), 79 (68), 71 (59).
HRMS: calcd for C30H40N2O3S [M + H]+, 509.28378; found,
509.28372. Anal. Calcd for (C30H41O3N2S): C, 70.69; H, 8.11;
N, 5.49; S, 6.29. Found: C, 70.46; H, 8.06; N, 5.52; S, 6.20.
FTIR, as neat films; absorption bands are reported in cm-1
.
Low-resolution mass spectra were measured on a Fisons
Instruments MD 800 quadrupole mass spectrometer, with 70
EV electron ionization and a GC 8000 Series gas chromato-
graph inlet, and using a J & W Scientific DB-5MS column (15
m length, 0.25 mm internal diameter, 0.25 µm film thickness).
Mass spectra data are given as mass-to-charge ratio, with the
relative peak height following in parentheses. All new com-
pounds were characterized by 1H NMR, IR, and13C NMR
spectroscopies. Fast atom bombardment mass spectra (FABMS)
were recorded at the University of Maryland College Park of
Mass Spectrometry Facility. Literature references are given
for all known compounds, except for those that are com-
mercially available; all known compounds were identified by

396 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 2
Leonessa et al.
7r-[4′-(N-r-(+)-Meth ylben zyla m in oa cyla m in op h en yl)-
th io]p r egn a -4-en e-3,20-d ion e (5). Reaction of 2 (0.10 g, 0.23
mmol) with (R)-(+)-R-methylbenzylisocyanate (66 µL, 0.46
mmol) in CHCl3 (3.0 mL) for 12 h gave 56 mg of product (46%,
monolayers were removed by trypsinization and diluted with
10 mL of scintillation fluid (Ultima Gold XR, Packard Bio-
science, Meriden, CT). Drug accumulation was radiometrically
assessed by scintillation spectrometry.
Results of substrate accumulation assays were plotted as
cellular concentration of substrate against the concentration
of the respective test compound. Pgp reversing potency was
expressed as the EC50, defined as the concentration of a test
drug that reduced the difference in substrate accumulation
between Pgp-negative and Pgp-positive cells by 50%. Proges-
terone and the standard Pgp reversing agents verapamil and
cyclosporin A were used as positive controls and as a reference
to establish relative potency.
Cytotoxicity. Twenty-four hours after they were plated in
96 well plates, MDA435/LCC6 and MDA435/LCC6MDR1 cells
were exposed to growth media containing different concentra-
tions of the test agents for 5 days. Cell cultures were then
fixed/stained by incubation in a 0.5% (w/v) crystal violet
solution in 25% methanol (v/v). After plates had dried, the dye
was extracted in 0.1 M sodium citrate in 25% methanol (v/v)
and absorbance was read at 540 nm using a microplate
spectrophotometer. Absorbance directly correlates with cell
number in this assay.50 Cell survival curves were obtained by
plotting absorbance values (as percent of untreated controls)
against drug concentration. The toxicity of each drug was
expressed as an IC50, defined as the concentration inhibiting
cell density by 50% at the end of the treatment period. To
estimate the extent of resistance conferred by Pgp, the ratio
of each drug’s IC50 in MDA435/LCC6MDR1 and MDA435/LCC6
cells (relative resistance of Pgp-positive cells) was used for
those drugs that produced a detectable IC50 value.
Ra d ioliga n d Bin d in g Stu d ies. These were performed as
previously described, using a whole cell competitive binding
assay.57,58 Briefly, MCF-7 cells were grown in 24 well dishes
and incubated at 37 °C with 100 nM hydrocortisone for 30 min,
before determining PGR binding, to eliminate residual binding
to glucocorticoid receptors. Subsequently, cells were incubated
for 60 min at 37 °C with 5 nM [3H] ORG2058 (specific activity
50.6 Ci/mmol) in the absence or presence of increasing
concentrations of unlabeled competitor (0.5 nM-1 µM; proges-
terone, ORG2058, 5). Radioactivity was extracted into ethanol
and measured in a liquid scintillation spectrometer.
1
mp ) 146-149 °C, Rf ) 0.46, 2:3 hexanes-ethyl acetate). H
NMR: δ 7.32-7.25 (m, 5H), 5.79-5.77 (m, 1H), 5.70-5.68 (s,
1H), 4.97-4.92 (m, 1H), 4.13-4.06 (m, 1H), 3.28 (s, 1H), 2.64-
1.49 (complex, 7H), 2.14 (s, 3H), 1.45 (d, J ) 9.3 Hz, 3H), 1.19
(s, 3H), 0.68 (s, 3H). IR: 3353, 3273, 2949, 2854, 2362, 2340,
1700, 1653, 1595, 1539, 1457, 1460, 1376, 1343, 1159, 1089,
916. 13C NMR: δ 209.4, 199.0, 167.6, 147.0, 136.6, 127.2, 121.2,
115.8, 63.4, 17.7, 13.1. MS: m/e ) 585 (11, M+ + 1), 135 (12),
125 (20), 105 (100), 103 (22), 91 (29), 77 (22), 55 (26). HRMS:
calcd for C36H44N2O3S [M + H]+, 585.31506; found, 585.31501.
Anal. Calcd for (C36H45O3N2S): C, 73.81; H, 7.74; N, 4.78; S,
5.47. Found: C, 73.76; H, 7.79; N, 4.81; S, 5.39.
7r-[4′-(N-p -Tolu en esu lfon yla m in oa cyla m in op h en yl)-
th io]p r egn a -4-en e-3,20-d ion e (6). Reaction of 2 (0.10 g, 0.23
mmol) with p-toluenesulfonylisocyanate (59 µL, 0.46 mmol) in
CHCl3 (3.0 mL) for 12 h gave 120 mg of product (83%, mp )
1
128-132 °C, Rf ) 0.29, 2:3 hexanes-ethyl acetate). H NMR:
δ 8.38 (s, 1H), 7.88 (d, J ) 8.4 Hz, 2H), 7.80 (d, J ) 8.3 Hz,
2H), 7.37-7.25 (m, 4H), 5.70 (s, 1H), 3.36 (s, 1H), 2.67-1.13
(complex, 20H), 2.41 (s, 3H), 2.15 (s, 3H), 1.55 (m, 2H), 1.20
(s, 3H), 0.69 (s, 3H). IR: 3855, 3745, 2359, 1700, 1539, 1457,
1160, 1086, 668. 13C NMR: δ 198.6, 148.6, 141.4, 136.6, 134.6,
129.9, 129.7, 129.6, 127.7, 127.2, 126.4, 120.5, 118.5, 92.4, 76.1,
69.3, 63.3, 52.1, 51.1, 47.0, 46.3, 39.8, 39.4, 38.5, 38.1, 35.4,
34.0, 31.6, 23.7, 22.9, 21.8, 21.1, 17.9, 13.4. MS: m/e ) 635
(29, M+ + 1), 313 (39), 155 (33), 135 (36), 125 (65), 119 (64),
91 (100), 85 (92), 77 (47), 59 (50), 47 (45). HRMS: calcd for
C
35H42N2O5S2 [M + H]+, 635.26135; found, 635.26130. Anal.
Calcd for (C35H43O5N2S2): C, 66.11; H, 6.82; N, 4.41; S, 10.09.
Found: C, 66.05; H, 6.79; N, 4.45; S, 10.02.
P h a r m a cology. Cell Lin es. For the studies of antiPgp
activity, we used cells transduced with a retroviral vector
directing the constitutive expression of the Pgp gene (MDA435/
LCC6MDR1) and their parental, Pgp-negative, MDA435/LCC6
human breast cancer cells.17 Both MDA435/LCC6 and MDA435/
LCC6MDR1 cells are estrogen receptor and PGR negative and
grow as monolayer cultures in vitro and as rapidly proliferat-
ing solid tumors and malignant ascites in vivo in nude mice.17
We used MCF-7 human breast cancer cells57 to measure
binding to PGR. These cells were routinely grown in vitro in
Improved Minimal Essential Media (Biofluids) containing 5%
fetal bovine serum in a 5% CO2:95% air atmosphere.
Su bstr a te Accu m u la tion Assa ys. Pgp reversing activity
of all test agents was evaluated by measuring the ability of
the agents to affect accumulation of DOX and VBL in MDA435/
LCC6MDR1 (resistant) and MDA435/LCC6 (control) cells. Cells
were plated at 2.5 × 105 cells/well into 24 well culture dishes,
in routine growth media, and incubated at 37 °C. Forty-eight
hours after they were plated, cells were treated by replacing
growth media with media containing the test compounds at
five different concentrations and either DOX (4 µM) or [3H]
VBL (5 nM). All treatments were carried out in triplicate. After
3 h of incubation, treatments were stopped by washing each
well with 0.5 mL of ice-cold NaCl (0.15 M). Cells from reference
wells in each plate were counted to enable accumulation to be
corrected for cell number.
For the DOX accumulation assays, DOX was extracted from
cell monolayers by adding 1.5 mL of 20% trichloroacetic acid
to each well and incubating overnight at 4 °C in the dark. DOX
concentrations in the extracts were evaluated fluorometrically.
Thus, extracts were transferred into 13mm × 100 mm boro-
silicate glass tubes, placed in the 10 × 10 rack of a Hitachi
A3000 Autosampler and connected to a Hitachi F-4500 fluo-
rescence spectrophotometer. Fluorescence was read at 500 nm
excitation and 580 nm emission wavelengths. Concentrations
of DOX were obtained by interpolation on a DOX standard
curve and normalized on the extraction volume and number
of cells per well. For the VBL accumulation assays, at the end
of treatment, wells were rinsed with phosphate-buffered saline
(0.5 mL/well) and left to dry at room temperature. Cell
Da ta An a lysis. DOX accumulation and cytotoxicity dose
response data were processed and graphed using SigmaPlot
4.0 (SPSS Science, Chicago, IL). EC50 (DOX accumulation
assays) and IC50 values (cytotoxicity assays) were calculated
by interpolation on the respective dose response curves. The
EC50 and IC50 values reported in Tables 2 and 3 represent the
mean and standard error (SE) obtained from at least three
independent experiments. Descriptive statistics were obtained
using SigmaStat 2.0 (SPSS Science).
Ack n ow led gm en t. This work was supported in part
by the Department of Defense, United States Army
Medical Research and Materiel Command, Award
RP950649, and by a grant from the Susan G. Komen
Breast Cancer Foundation, Award PDF 2000 186.
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