Synthesis and Evaluation of Dihydropyrroloquinolines That Selectively Antagonize P-Glycoprotein

Article abstract of DOI:10.1021/jm0303204

In a search for improved multiple drug resistance (MDR) modulators, we identified a novel series of substituted pyrroloquinolines that selectively inhibits the function of P-glycoprotein (Pgp) without modulating multidrug resistance-related protein 1 (MRP

Full text of DOI:10.1021/jm0303204

J . Med. Chem. 2004, 47, 1413-1422
1413
Syn th esis a n d Eva lu a tion of Dih yd r op yr r oloqu in olin es Th a t Selectively
An ta gon ize P -Glycop r otein
Brian D. Lee, Zhanjiang Li, Kevin J . French, Yan Zhuang, Zuping Xia, and Charles D. Smith*
Department of Pharmacology, The Pennsylvania State University, College of Medicine, Hershey, Pennsylvania 17033
Received J uly 3, 2003
In a search for improved multiple drug resistance (MDR) modulators, we identified a novel
series of substituted pyrroloquinolines that selectively inhibits the function of P-glycoprotein
(Pgp) without modulating multidrug resistance-related protein 1 (MRP1). These compounds
were evaluated for their toxicity toward drug-sensitive tumor cells (i.e. MCF-7, T24) and for
their ability to antagonize Pgp-mediated drug-resistant cells (i.e. NCI/ADR) and MRP1-mediated
resistant cells (i.e. MCF-7/VP). Cytotoxicity and drug accumulation assays demonstrated that
the dihydropyrroloquinolines inhibit Pgp to varying degrees, without any significant inhibition
of MRP1. The compound termed PGP-4008 was the most effective at inhibiting Pgp in vitro
and was further evaluated in vivo. PGP-4008 inhibited tumor growth in a murine syngeneic
Pgp-mediated MDR solid tumor model when given in combination with doxorubicin. PGP-
4008 was rapidly absorbed after intraperitoneal administration, with its plasma concentrations
exceeding the in vitro effective dose for more than 2 h. PGP-4008 did not alter the plasma
distribution of concomitantly administered anticancer drugs and did not cause systemic toxicity
as was observed for cyclosporin A. Because of their enhanced selectivity toward Pgp, these
substituted dihydropyrroloquinolines may be effective MDR modulators in a clinical setting.
In tr od u ction
malignancies. For example, increased expression of Pgp
in neuroblastoma and childhood sarcoma is associated
with poor response to chemotherapy and decreased
survival,14 and breast cancer patients with Pgp-express-
ing tumors are three times more likely to fail chemo-
therapy than those patients with Pgp-negative tumors.15
In contrast, although MRP1 is expressed in a high
percentage of leukemias and solid tumors,16 its over-
expression is not consistently found in tumors. For
example, MRP1 levels detected in normal and malig-
nant hematopoietic cells were equivalent,17,18 and the
MRP1 levels in lung tumors were found to be lower than
those in normal lung tissue.19 The MRP1 mRNA levels
in malignant melanoma,20 acute lymphocytic leuke-
mia,21 or chronic lymphocytic leukemia22 were not
altered by chemotherapy, but did increase moderately
in acute myelogenous leukemia.18,21 Therefore, it seems
that overexpression of Pgp activity is clinically more
significant than elevation of MRP1 levels.
Cancer chemotherapy often fails because of the de-
velopment of multiple drug resistance (MDR) by tumor
cells. A major mechanism of MDR is through the
overexpression of energy-dependent, unidirectional trans-
membrane efflux pumps. The drug transporter, P-
glycoprotein (Pgp), is a 170 kDa protein that belongs to
the ATP-binding cassette superfamily of transporters.1
Its biochemistry and pharmacology have been intensely
studied for the past 25 years.2-4 A series of homologous
proteins termed multidrug resistance-related proteins
(MRPs) have been discovered more recently, and these
proteins share many pharmacological properties with
Pgp.5,6 The first described protein of this series, MRP1,
is a 190 kDa protein that was identified in 1992 in a
drug-resistant lung cancer cell line that does not express
Pgp.7 These transporters function by binding to drugs
within the cell and releasing them to the extracellular
space using energy from the hydrolysis of ATP.8 Tumor
cells that are exposed to cytotoxic compounds often
overexpress these efflux pumps, which allows these cells
to survive even in the presence of anticancer drugs.9
MDR affects patients with a variety of cancers,
including leukemias and solid tumors such as breast,
lung, and brain cancers. Overexpression of Pgp has been
documented in a number of tumor types including acute
leukemia and small-cell lung carcinoma, especially after
the patient has received chemotherapy, indicating that
this mechanism of MDR is clinically important.3,10-13
Additionally, several studies have shown that Pgp
expression may be a prognostic indicator in certain
Because of the importance of Pgp in clinical oncology,
an intensive search has developed for antagonists of
these transport proteins.23 These antagonists, often
termed MDR modulators, function by blocking the
transporter-mediated drug efflux so that a concomit-
antly administered anticancer drug can cause tumor cell
death. Initial attempts to develop MDR modulators
focused on verapamil and cyclosporin A.4,24 These com-
pounds demonstrate excellent in vitro reversal of MDR
but failed to achieve clinical success due to their in-
trinsic toxicity and/or their alteration of the pharmaco-
kinetics of the coadministered anticancer drugs.2,25,26
These clinical results may reflect differences in tissue
distribution between the transport proteins, Pgp and
MRP1. Previous studies have shown that Pgp is ex-
pressed by certain types of secretory cells, including the
* To whom correspondence should be addressed: Penn State College
of Medicine, Department of Pharmacology, H078, 500 University Drive,
Hershey, PA 17033. E-mail: cdsmith@psu.edu. Phone: (717) 531-1672.
Fax: (717) 531-5013.
10.1021/jm0303204 CCC: $27.50 © 2004 American Chemical Society
Published on Web 02/10/2004

1414 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6
Lee et al.
Ta ble 1. Cytotoxicity and MDR Antagonism by Substituted Dihydropyrroloquinolines
a
b
Toxicity is calculated as the percentage of MCF-7 cells killed by 10 µg/mL of the indicated compound. The Pgp antagonism score )
percentage of NCI/ADR cells surviving in the absence of vinblastine/percentage of NCI/ADR cells surviving in the presence of vinblastine.
c The MRP1 antagonism score ) percentage of MCF-7/VP cells surviving in the absence of vincristine/percentage of MCF-7/VP cells surviving
in the presence of vincristine.
capillary endothelial cells of the brain and testis, and
by cells within the pancreas, kidney, liver, and gastro-
intestinal tract.22,27 Conversely, mRNA of MRP1 has
been observed in virtually every type of tissue within
the body28 and is expressed in particular high concen-
tration in peripheral blood mononuclear cells.7,17,18,21,29
The clinical significance of Pgp and its limited expres-
sion in normal tissues makes Pgp a target for the

Dihydropyrroloquinolines as P-Glycoprotein Antagonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1415
Ta ble 2. Reversal of Pgp- and MRP1-Mediated MDR by Dihydropyrroloquinolines
development of drugs to reverse MDR. It is likely that
the lack of drug transporter-selectivity in early MDR
modulators played a role in their ultimate failure.
Therefore, we have hypothesized that improved selec-
tivity of Pgp antagonists will provide more clinically
effective chemotherapeutic agents.30-32 Accordingly,
some newer MDR modulators, including XR957633,34
and LY335979,35 have improved Pgp selectivity and
pharmacological properties.30,31,36,37 To expand the port-
folio of Pgp-selective MDR modulators, we screened a
library of synthetic compounds and identified a series
of dihydropyrroloquinolines that reverse Pgp-mediated
MDR without antagonizing MRP1. We now describe the
synthesis of substituted dihydropyrroloquinolines, struc-
ture-activity relationships for their effects on Pgp and
MRP1, and the in vivo pharmacological properties of the
most promising drug candidate, PGP-4008.
treated with a Pgp-substrate drug, vinblastine, or an
MRP1-substrate drug, vincristine, either alone or in the
presence of a test MDR modulator. Verapamil was used
as the positive control and is highly effective at revers-
ing both Pgp- and MRP1-mediated MDR in vitro.
Screening of approximately 11 000 compounds from
a commercially available library was conducted at a
final concentration of 10 µg/mL (15-25 µM) for the test
compounds. This identified several chemotypes, includ-
ing the dihydropyrroloquinoline heterocycle, with excel-
lent promise as versatile scaffolds with which to study
structure-activity relationships for inhibition of Pgp
activity. The library contained 29 dihydropyrroloquino-
lines, whose biological activities are described in Table
1. The intrinsic cytotoxicity of the compounds is ex-
pressed as the percentage of MCF-7 cells killed by 10
µg/mL of each compound. It can be seen that these
compounds have a wide range of toxicity, varying from
0% of cells killed by compound 27 to 99% of cells killed
by compounds 11 and 15. While toxicity toward cultured
cells is a typical and desired property for cancer drugs,
it is desirable that MDR modulators have low intrinsic
toxicity. The ability of the dihydropyrroloquinolines in
the screening library to reverse Pgp-mediated MDR is
also indicated in Table 1. The Pgp antagonism score is
calculated as the percentage survival of NCI/ADR cells
treated with the compound alone divided by the per-
centage survival of NCI/ADR cells treated with the
compound plus 50 nM vinblastine. Therefore, an an-
tagonism score of 1.0 indicates inactivity of the test
Resu lts a n d Discu ssion
Id en tifica tion of MDR-Rever sin g Dih yd r op yr -
r oloqu in olin es. As with our previous studies,30-32,38
we sought new inhibitors of Pgp and MRP1 using cell-
based cytotoxicity assays with the following tumor cell
lines: MCF-7, a drug-sensitive human breast adeno-
carcinoma line; NCI/ADR, a line selected for resistance
to adriamycin that expresses high levels of Pgp without
the overexpression of MRP1;39 and MCF-7/VP, a subline
of MCF-7 selected for resistance to etoposide in the
presence of verapamil that expresses high levels of
MRP1 without the overexpression of Pgp.40 Cells were

1416 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6
Lee et al.
Sch em e 1
Ta ble 3. Reaction Conditions, Yields, and Melting Points of
Compounds 27 and 31-38
reaction conditions
compound
catalyst
time (h) yield (%)
mp (°C)
31
32
33
34
27
(CH3)3COK
(CH3)3COK
(CH3)3COK
(CH3)3COK
NaH
1.5
2
3
3
4
3
2
1.5
2
61
65
80
84
49
52
55
45
53
132-134
170-171
167-169
133-135
220-222
157-159
189-191
195-197
160-162
35 (PGP-4008) NaH
36
37
38
NaH
NaH
NaH
compound, while larger antagonism scores indicate
increasing activity. It is apparent that several of the
dihydropyrroloquinolines inhibit Pgp function, with
compounds 24-29 being particularly effective. The
abilities of the compounds to reverse MRP1-mediated
MDR are also indicated. The MRP1 antagonism score
is calculated as the percentage survival of MCF-7/VP
cells treated with the compound alone divided by the
percentage survival of MCF-7/VP cells treated with the
compound plus 1 nM vincristine, so that chemosensiti-
zation is indicated by a score greater than 1.0. In
general, the dihydropyrroloquinolines had only marginal
abilities to reverse MRP1-mediated MDR, so that
several compounds are effective inhibitors of Pgp with-
out inhibiting the action of MRP1. For example, com-
pound 27 has a Pgp antagonism score of 18 whereas
the MRP1 antagonism score is only 1.1. Compounds
having a benzyl substitution at R1 were more active
toward Pgp than were corresponding compounds with
methyl groups at that site, e.g. compound 27 compared
with compound 7 and compound 29 compared with
compound 11. Additionally, increasing the size and/or
hydrophobicity of the substituent at position R2 en-
hanced activity toward Pgp, e.g. compound 27 > 26 >
25 ) 24. Thus, the initial screening indicated that
bisubstituted dihydropyrroloquinolines provide a new
and versatile chemotype for the development of Pgp-
selective MDR antagonists.
F igu r e 1. MDR reversal by dihydropyrroloquinolines. The
reversal of Pgp- and MRP1-mediated MDR was assayed as
described in the Experimental Section for the following
compounds: verapamil (9); 18 (2); 27 (3); 31 (1); 32 ([); 33
(b); 34 (0); 35 (]); 36 (O); 37 (×); 38 (4). (A) The Pgp
Antagonism Score is calculated as the percentage of NCI/ADR
cells surviving in the presence of 50 nM vinblastine/percentage
of NCI/ADR cells surviving in the presence of vinblastine plus
the indicated concentration of modulator. (B) The MRP1
Antagonism Score is calculated as the percentage of MCF-7/
VP cells surviving in the presence of 1 nM vincristine/
percentage of MCF-7/VP cells surviving in the presence of
vincristine plus the indicated concentration of modulator.
Syn t h esis a n d In Vit r o E va lu a t ion of Novel
Dih yd r op yr r oloq u in olin es. A series of novel sub-
stituted dihydropyrroloquinolines, shown in Table 2,
was synthesized as indicated in Scheme
1 and
Table 3. 2-Aminobenzonitrile reacted with 1-benzyl-
2-pyrrolidinone in the presence of phosphorus oxy-
chloride and tin(IV) chloride to yield 2-(1-benzylpyrroli-
din-2-ylidenemethyl)benzonitrile (30). Following the
addition of lithium diisopropylamine-tetrahydrofuran
complex, 1-benzyl-2,3-dihydro-1H-pyrrolo[2,3-b]quino-
lin-4-ylamine (18) was produced. Through alkylation
and acetylation reactions, compounds 31-34, 27, and

Dihydropyrroloquinolines as P-Glycoprotein Antagonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1417
Ta ble 4. Cell Line- and Drug-Specificity of Verapamil and PGP-4008
EtOH
verapamil
PGP-4008
IC50
cell line
drug
IC50
IC50
RIa
2.7
RI
3.8
T24
vinblastine (nM)
taxol (nM)
vincristine (nM)
cisplatin (µM)
5-Fluorouracil (µM)
vinblastine (nM)
taxol (nM)
vincristine (nM)
cisplatin (µM)
5-Fluorouracil (µM)
vinblastine (nM)
taxol (nM)
vincristine (nM)
cisplatin (µM)
5-fluorouracil (µM)
vinblastine (nM)
taxol (nM)
1.3 ( 0.3
4.7 ( 0.5
4.8 ( 0.2
6.7 ( 1.1
77 ( 20
0.5 ( 0.1
3.0 ( 0.4
1.3 ( 0.2
7.0 ( 0.8
48 ( 6
0.3 ( 0.1
1.4 ( 0.6
0.1 ( 0.1
14 ( 4
53 ( 20
0.2 ( 0
1.6 ( 0.4
0.5 ( 0.2
10.5 ( 3.2
12 ( 2
0.6 ( 0.1
19 ( 5
0.4 ( 0.1
4.0 ( 0.6
2.2 ( 0.6
5.8 ( 1.2
29 ( 8
1.6
3.7
1.0
1.6
1.2
1.1
4.6
1.1
0.9
2.4
1.1
15.0
0.8
0.9
1.2
1.5
1.1
2.6
1.8
0.9
0.9
0.8
1.6
1.3
0.8
1.0
0.8
0.7
MCF-7
0.4 ( 0.1
1.6 ( 0.3
0.6 ( 0.1
16 ( 5
0.2 ( 0
1.8 ( 0.1
0.4 ( 0.1
19 ( 5
49 ( 31
30 ( 15
0.4 ( 0.1
2.1 ( 0.4
7.3 ( 0.9
11.3 ( 0.5
16 ( 3
MCF7/VP (MRP1)
NCI/ADR (Pgp)
0.6 ( 0.1
1.8 ( 0.4
7.5 ( 1.1
8.5 ( 2.1
11 ( 1
110 ( 17
2020 ( 810
173
106
3.1 ( 2.0
29 ( 5
35
70
doxorubicin (µM)
vincristine (nM)
cisplatin (µM)
5-fluorouracil (µM)
vinblastine (nM)
taxol (nM)
183 ( 14
7.0 ( 0.9
181 ( 84
28 ( 5
1650 ( 470
26.7 ( 10.6
150 ( 24
2.1 ( 0.8
0.22 ( 0.04
3.7 ( 0.8
6.3 ( 0.8
210 ( 99
0.5 ( 0.1
5.0 ( 1.4
0.03 ( 0.01
1.3 ( 0.5
1.1 ( 0.3
0.30 ( 0.08
50
10.5 ( 1.1
6.0 ( 0.5
175 ( 74
0.5 ( 0.1
8.2 ( 2.9
0.13 ( 0.09
5.0 ( 2.9
1.1 ( 0.4
0.32 ( 0.08
18
1.1
0.9
61
330
1000
112
1.2
1.0
63
202
200
30
P388/ADR (Pgp)
doxorubicin (µM)
vincristine (nM)
cisplatin (µM)
2.0
0.7
1.9
0.7
5-fluorouracil (µM)
a
The reversal index (RI) is calculated as the ratio of the IC50 in the absence of modulator to the IC50 in the presence of modulator, so
that larger values indicate increasing activity.
35-38 were obtained. For in vivo studies described
below, compound 35 or PGP-4008 was reacted with 2
equiv of hydrogen chloride in ether to obtain the
corresponding HCl salt.
As detailed in Table 2 and Figure 1, the newly
synthesized dihydropyrroloquinolines were found to
exhibit a broad range of activity against Pgp, with
PGP-4008 having a maximal activity equivalent to
verapamil at a significantly lower concentration (Figure
1A). More importantly, none of the dihydropyrrolo-
quinolines caused any significant antagonism of MRP1
(Figure 1B).
PGP-4008 was further characterized in a variety of
cell lines with different cytotoxic drugs (Table 4). The
reversal index (RI) was calculated as the ratio of the
IC50 in the absence of modulator to the IC50 in the
presence of modulator, so that larger values indicate
increasing activity. PGP-4008 potentiated the cytotox-
icity of Pgp substrate drugs (vinblastine, vincristine, and
paclitaxel) toward cell lines that overexpress Pgp (NCI/
ADR and P388/ADR), as indicated by the large reversal
index. In contrast, PGP-4008 did not strongly affect the
toxicities of these drugs toward non-Pgp-overexpressing
cell lines (T24 and MCF-7), nor did they affect the
toxicities of non-Pgp substrate drugs (cisplatin and
5-fluorouracil) toward any of the cell lines. PGP-4008
was not cytotoxic to any of the cell lines at doses up to
250 µM, thereby providing a large therapeutic index.
In contrast with verapamil, the selectivity of PGP-4008
toward Pgp is again illustrated by the marked ability
of verapamil to enhance the toxicity of vincristine to
MRP1-overexpressing MCF7/VP cells, whereas PGP-
4008 did not.
mulation of these drugs by MCF-7 or MCF-7/VP cells
(Figure 2). As with the cytotoxicity studies, the optimal
effect of PGP-4008 was reached with doses below 2 µg/
mL (5 µM). The modulatory effects of PGP-4008 ob-
served in the cytotoxicity and drug accumulation assays
are all consistent with selective antagonism of Pgp.
In Vivo Eva lu a tion of P GP -4008. Initial in vivo
experiments were hindered by the low solubility of PGP-
4008; however, conversion to the HCl salt markedly
improved its solubility. The therapeutic effects of PGP-
4008‚HCl were evaluated using a syngeneic solid tumor
model consisting of J C murine mammary adenocarci-
noma cells growing in Balb/c mice.41 These cells are
resistant to a variety of anticancer drugs because of
their high level of expression of Pgp, and PGP-4008
effectively reverses this MDR phenotype. In these
xenograft studies, tumors were allowed to grow to
volumes of approximately 400 mm3 before the animals
were treated with saline (control), 5 mg/kg doxorubicin
(a Pgp substrate), 100 mg/kg PGP-4008, or a combina-
tion of doxorubicin and PGP-4008. As shown in Table
5, tumor volumes in the control group increased 500%
by day 15, while tumor volumes in animals treated with
PGP-4008 alone or doxorubicin alone increased 500%
and 300%, respectively. In contrast, tumors in animals
treated with the combination of doxorubicin and PGP-
4008 increased only 80% by day 15 (p < 0.05). There
were no significant decreases in the weights of mice
receiving the combination of PGP-4008 plus doxorubicin.
This contrasts with the marked weight loss in animals
treated with cyclosporin A, a nonselective MDR modu-
lator.41-43
Pharmacokinetic studies of PGP-4008 were performed
at the same dose as those used in the tumor studies.
The plasma concentration profile of intraperitoneally
administered PGP-4008 is shown in Figure 3, and the
Additional studies have shown that PGP-4008 in-
creases the accumulation of [3H]paclitaxell and [3H]vin-
blastine by NCI/ADR cells, without affecting the accu-

1418 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6
Ta ble 5. In Vivo Tumor Growth Inhibition by PGP-4008a
Lee et al.
day 1
day 5
day 9
day 12
day 15
control
doxorubicin
PGP-4008
PGP-4008 + doxorubicin
380 ( 180
430 ( 180
380 ( 190
390 ( 140
480 ( 130
520 ( 200
470 ( 220
470 ( 140
1220 ( 500
860 ( 280
1030 ( 320
*520 ( 110
1570 ( 560
1210 ( 530
1280 ( 450
*560 ( 160
2120 ( 800
1680 ( 720
2000 ( 550
*670 ( 170
a
Tumor volumes (mm3) are the average ( SD for 4-5 animals per group. *p < 0.05 compared to control.
F igu r e 3. Pharmacokinetic profile of PGP-4008 in blood. PGP-
4008 (100 mg/kg) was administered intraperitoneally to mice
and blood samples were analyzed at the indicated times as
described in the Experimental Section. Values represent the
mean ( SD for triplicate samples in a representative experi-
ment.
Ta ble 6. Pharmacokinetic Parameters for PGP-4008
AUC
AUMC
A
B
kR
kâ
t1/2â
kel
Vdss
V1
V2
17.6 ( 1.4 µg × h/mL
9.4 ( 5.2 µg × h2/mL
440 ( 6 µg/mL
2.25 ( 0.36 µg/mL
49 ( 0.9 h-1
F igu r e 2. Effects of PGP-4008 on intracellular [3H]drug
accumulation. Intracellular accumulation of (A) [3H]vinblastine
and (B) [3H]paclitaxel were performed using MCF-7 (9), NCI/
ADR (2), and MCF-7/VP (1) cells and the indicated concentra-
tions of PGP-4008 as described in the Experimental Section.
Values represent the mean ( SD of triplicate samples in a
representative experiment.
0.58 ( 0.19 h-1
1.20 ( 0.39 h
35 ( 2.7 h-1
43 ( 15 mL
2.3 ( 0.3 mL
40.5 ( 7.4 mL
80.4 ( 6.4 mL/h
pharmacokinetic parameters are summarized in Table
6. Analysis of the concentration-time profile revealed
that it best fits a two-compartment model, with rapid
alpha-phase clearance and a beta-phase terminal half-
life of 1.2 h. The highest concentration of PGP-4008 was
observed at the earliest time point (1 min), indicating
that PGP-4008 is rapidly absorbed into the systemic
circulation from the intraperitoneal cavity. However,
less than 1% of the peak concentration of PGP-4008
remained in the plasma after 4 h. The area under the
concentration-time curve (AUC) for PGP-4008 was 17.6
µg‚h/mL, with a clearance rate from the plasma (Clp) of
80.4 mL/h, corresponding to a large apparent volume
of distribution (Vdss ) 43 mL). The second compartment
constitutes the majority of the volume of distribution
of PGP-4008 (V2 ) 41 mL). Most importantly, however,
is the fact that the maximum concentration in plasma
Clp
(Cmax, 197 µg/mL) was well above the in vitro effective
dose of approximately 0.8 µg/mL. Furthermore, this
effective concentration is maintained for at least 2 h,
indicating that PGP-4008 is rapidly absorbed into the
blood stream at therapeutically significant levels. Com-
parative studies in which PGP-4008 was administered
intravenously indicated a bioavailability of 60% after
intraperitoneal administration. Plasma distribution
studies of vinblastine coadministered with PGP-4008
were also performed. As indicated in Figure 4, the
clearance of [3H]vinblastine was not altered by the
presence of PGP-4008. This contrasts with the altered
pharmacokinetic properties commonly seen with non-
specific MDR modulators.2

Dihydropyrroloquinolines as P-Glycoprotein Antagonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1419
mL) containing 1-benzyl-2-pyrrolidinone (5.16 g, 29.4 mmol)
were added phosphorus oxychloride (5.2 mL) and tin(IV)
chloride (1.0 mL). The mixture was stirred at room tempera-
ture for 1.5 h, before anthranilonitrile (3.3 g, 27.9 mmol) was
added in portions and the mixture was stirred at 50 °C for 5
h. Ice-cold water (15 mL) was then added, and a 30% aqueous
sodium hydroxide solution was further added to make a weak
alkaline mixture. The organic solvent was removed under
reduced pressure, and the residue was extracted with chloro-
form. The extract was dried over anhydrous sodium sulfate
and condensed under vacuum. The crude product was purified
by flash chromatography (chloroform:methanol, 100:1) to yield
30 (7.0 g, 91%) as slightly yellow needles, mp 59-61 °C; IR
(KBr): 2217, 1628, 1439, 1279 cm-1 1H NMR (DMSO-d6):
;
7.06-7.69 (m, 9H), 4.83 (s, 2H), 3.46 (t, 2H), 2.60 (t, 2H), 2.10
(m, 2H); 13C NMR (CDCl3): 162.8, 156.3, 137.6, 133.7, 132.9,
128.7(2C), 128.3(2C), 127.4, 122.8, 121.7, 118.8, 105.9, 48.3,
47.3, 27.7, 19.6. Anal. Calcd for C18H17N3 (275.35): C, 78.52;
H, 6.22; N, 15.26. Found: C, 78.37; H, 6.20; N, 15.17.
1-Be n zyl-2,3-d ih yd r o-1H -p yr r olo[2,3-b]q u in olin -4-
yla m in e (18). A solution of tetrahydrofuran (350 mL) contain-
ing 30 (37.6 g, 0.14 mol) under argon was cooled to -35 °C,
and 1.5 M lithium diisopropylamine-tetrahydrofuran complex
(233 mL, 0.35 mol) in cyclohexane was added dropwise. After
the addition was complete, the temperature of the mixture was
gradually raised to -10 °C, and ice-cold water (30 mL) was
added dropwise. After the organic solvent was evaporated
under reduced pressure, the residue was extracted with
chloroform. The combined organic extracts were dried over
anhydrous sodium sulfate and condensed under vacuum. The
residue was dissolved in ethanol (15 mL), and crystals were
precipitated, filtered, washed with cold ethanol, and dried
under vacuum yielding 14.6 g (39%) of 18 as needles, mp 174-
F igu r e 4. Effect of PGP-4008 on blood concentrations of
[3H]vinblastine. DMSO (9) or 100 mg/kg of PGP-4008 (2) was
administered intraperitoneally to mice followed by [3H]vin-
blastine as described in the Experimental Section, and samples
were analyzed at the indicated times for blood levels of
[3H]vinblastine as described in the Experimental Section.
Values represent the mean ( SD for triplicate samples in a
representative experiment.
176 °C; IR (KBr): 3411, 3116, 1654, 1502, 1350, 756 cm-1
;
1H NMR (DMSO-d6): 7.01-7.93 (m, 9H), 6.05 (s, 2H), 4.60 (s,
2H), 3.37 (t, 2H), 2.87 (t, 2H); 13C NMR (CDCl3): 162.7, 149.3,
144.8, 138.8, 128.7(2C), 128.2(2C), 128.1, 127.2, 126.2, 121.8,
119.9, 117.6, 100.7, 48.4(2C), 23.3. Anal. Calcd for C18H17N3
(275.35): C, 78.52; H, 6.22; N, 15.26. Found: C, 78.07; H, 6.07;
N, 15.08.
Gen er a l P r oced u r e for th e Syn th esis of Com p ou n d s
31-34. To a solution of 18 (1 mmol) in tetrahydrofuran (15
mL) was added potassium tert-butoxide at 0 °C under nitrogen.
After being stirred at room temperature for 1.5 h, the reaction
mixture at -5 °C was added dropwise to a solution of
corresponding alkyl chloride (1 mmol) in tetrahydrofuran (10
mL) using a syringe. The mixture was warmed to room
temperature, stirred for 1.5-3 h, and then poured into water
(50 mL) to precipitate a solid that was collected by filteration
and dried under vacuum. The crude products were purified
by flash chromatography (chloroform:methanol, 100:1) on silica
gel to give the corresponding product (Table 3).
(1-Be n zyl-2,3-d ih yd r o-1H -p yr r olo[2,3-b]q u in olin -4-
yla m in o)a cetic Acid Eth yl Ester (31). Yield 61%; mp 132-
134 °C; IR (KBr): 3410, 1745, 1620, 1502, 1215 cm-1; 1H NMR
(CDCl3): 7.16-7.72 (m, 9H), 4.77 (s, 2H), 4.25 (q, 2H), 3.51 (t,
2H), 3.49 (s, 2H), 3.11 (t, 2H), 1.34 (t, 3H); 13C NMR (CDCl3):
171.1, 157.4, 139.6, 137.1, 135.5, 132.1, 128.8(2C), 128.5(2C),
128.3, 127.3, 126.5, 124.5, 121.7, 120.5, 61.9, 48.7, 48.0, 26.1,
24.9, 14.6. Anal. Calcd for C22H23N3O2 (361.18): C, 73.11; H,
6.41; N, 11.63. Found: C, 73.04; H, 6.08; N, 11.90.
Con clu sion s
We sought to develop MDR modulators that selec-
tively antagonized Pgp to effectively potentiate the
cytotoxicity of chemotherapeutic anticancer drugs to-
ward resistant tumors. The dihydropyrroloquinoline
heterocycle was chosen for its ease of synthesis and the
presence of several substitution sites. Biological evalu-
ation of several dihydropyrroloquinolines demonstrated
their highly selective modulation of Pgp with maximal
activity demonstrated by PGP-4008. In vivo testing of
PGP-4008 demonstrated its efficacy for reversing Pgp-
mediated MDR in a solid tumor model, its rapid
systemic absorption, and its lack of interaction with a
concomitantly administered chemotherapeutic agent.
These results indicate the effectiveness of PGP-4008 in
the reversal of Pgp-mediated MDR and its potential for
clinical utility.
Exp er im en ta l Section
Ma ter ia ls a n d Meth od s. Solvents were either purchased
as “anhydrous” or “ACS grade” and stored over 4 Å molecular
sieves. “Flash chromatography” refers to the method of Still
et al.44 and used Selecto Scientific silica gel (32-63 µm).
Melting points were determined in an open capillary on a
MelTemp II melting point apparatus and are uncorrected.
Infrared spectra were measured on a Nicolet Avatar 360 FT-
1-Ben zyl-2,3-d ih yd r o-1H -p yr r olo[2,3-b]q u in olin -4-yl
3-flu or oben zyla m in e (32). Yield 65%; mp 170-171 °C; IR
1
(KBr): 3400, 3063, 1622, 1504, 1217 cm-1; H NMR (CDCl3):
7.00-8.15 (m, 13H), 4.73 (s, 2H), 4.19 (s, 2H), 3.21 (t, 2H),
2.45 (t, 2H); 13C NMR (CDCl3): 164.7, 162.5, 159.8, 149.8,
148.6, 137.8, 136.4, 133.5, 133.4, 130.4, 130.2, 128.6, 128.4,
128.2, 127.2, 126.6, 123.6, 121.7, 119.1, 115.4, 115.0, 54.5, 48.8,
47.8, 25.4. Anal. Calcd for C25H22FN3 (383.46): C, 78.30; H,
5.78; N, 10.96. Found: C, 78.23; H, 5.32; N, 11.21.
1-Ben zyl-2,3-d ih yd r o-1H -p yr r olo[2,3-b]q u in olin -4-yl
4-flu or oben zyla m in e (33). Yield 80%; mp 167-169 °C; IR
(KBr): 3405, 1620, 1502, 1215 cm-1; 1H NMR (CDCl3): 6.92-
8.10 (m, 13H), 4.72 (s, 2H), 4.21 (s, 2H), 3.21 (t, 2H), 2.48 (t,
1
IR, and values are expressed in wavenumbers (cm-1). H and
13C NMR spectra were obtained with
a Bruker 200AM
spectrometer. Chemical shifts are reported in ppm (δ) using
tetramethylsilane as the reference and coupling constants (J )
are reported in Hz. Mass spectra were obtained from Mass
Consortium (San Diego, CA), and elemental analyses were
performed by Midwest Microlab (Indianapolis, IN).
2-(1-Ben zylpyr r olidin -2-yliden eam in o)ben zon itr ile (30).
To a solution of chloroform (25 mL) and tetrahydrofuran (25

1420 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6
Lee et al.
2H); 13C NMR (CDCl3): 165.5, 162.5, 160.6, 149.9, 148.5, 140.3,
137.8, 129.9, 128.7, 128.5, 128.2, 127.2, 126.7, 124.3, 123.5,
121.8, 119.1, 115.7, 115.3, 114.7, 114.2, 54.9, 48.9, 47.9, 25.5.
Anal. Calcd for C25H22FN3 (383.46): C, 78.30; H, 5.78; N, 10.96.
Found: C, 78.73; H, 5.75; N, 11.08.
N-(1-Ben zyl-2,3-d ih yd r o-1H-p yr r olo[2,3-b]q u in olin -4-
yl)-2-p h en yla ceta m id e h yd r och lor id e Sa lt (P GP -4008‚
HCl). PGP-4008 (400 mg) was suspended in 1M HCl in ether
(8 mL) and the mixture was stirred at room temperature for
12 h before the solvent was removed with nitrogen flushing.
The residue was washed with 10% EtOAc in hexane twice and
dried in vacuo to yield the yellowish solid, PGP-4008‚HCl salt,
470 mg (Yield 99%). 1H NMR (DMSO-d6): 11.03 (s, 1H), 7.20-
8.20 (m, 14H), 5.13 (s, 2H), 3.89 (s, 2H), 3.74 (t, 2H), 2.94 (t,
2H); 13C NMR (DMSO-d6): 168.5, 155.7, 138.4, 135.4, 134.2,
130.7, 129.1, 128.9 (2C), 128.6 (2C), 128.1 (2C), 128.0 (2C),
127.8, 126.4, 124.2, 123.6, 122.5, 119.0, 118.0, 50.0, 49.5, 42.2,
24.8.
1-Ben zyl-2,3-dih ydr o-1H-pyr r olo[2,3-b]qu in olin -4-yl 3,5-
d im eth oxyben zyla m in e (34). Yield 84%; mp 133-135 °C;
IR (KBr): 3420, 1621, 1509, 1218 cm-1 1H NMR (CDCl3):
;
6.57-8.15 (m, 12H), 4.71 (s, 2H), 4.13 (s, 2H), 3.67 (s, 6H),
3.23 (t, 2H), 2.56 (t, 2H); 13C NMR (CDCl3): 162.1, 160.6(2C),
149.5, 149.0, 141.4, 139.2, 1291, 128.4(2C), 128.2(2C), 127.2,
126.5, 123.7, 121.7, 121.5, 119.8, 106.6, 106.3, 99.6, 55.6(2C),
55.2, 48.8, 47.9, 25.5. Anal. Calcd for C27H27N3O2 (425.52): C,
76.21; H, 6.40; N, 9.87. Found: C, 76.23; H, 6.36; N, 9.50.
In Vitr o Cytotoxicity Assa y. MCF-7 and NCI/ADR cells
were obtained from the Division of Cancer Treatment of the
National Cancer Institute.39 MCF-7/VP cells were provided by
Drs. Schneider and Cowan.40 Cells were maintained in RPMI
1640 (Life Technologies, Inc., Rockville, MD) with L-glutamine
containing 10% FBS and 50 µg/mL gentamicin at 37 °C and
5% CO2. Cells were seeded into 96-well tissue culture dishes
at approximately 20% confluency and allowed to recover and
attach for 24 h. Cells were then treated in triplicate with
varying concentrations of test modulators in the presence or
absence of a cytotoxic drug for 48 h. The number of surviving
cells remaining in each well was quantified with the sulfo-
rhodamine B (SRB) colorimetric assay.45 Briefly, cells were
washed with phosphate-buffered saline and fixed to the plate
with 10% trichloroacetic acid. The cells were then washed with
water and stained with 0.4% SRB in 1% acetic acid. Cells were
then rinsed with 1% acetic acid, and 10 mM Tris buffer was
added to dissolve the remaining SRB. The absorbance of each
well was determined with a PerkinElmer HTS 7000 Plus
Gen er a l P r oced u r e for th e Syn th esis of Com p ou n d s
27 a n d 35-38. A solution of 18 (1 mmol) in DMF (10 mL)
was added dropwise to a suspension of sodium hydride (1
mmol) in DMF (20 mL) at 0 °C under nitrogen. After 5 min,
an acyl chloride was slowly added via syringe over a 20 min
period maintaining a temperature of -5 °C. The reaction
mixture was stirred at room temperature for 1-4 h (Table 3)
and then filtered through silica gel. The solvent was removed
under reduced pressure, and the residue was purified by flash
chromatography (chloroform:methanol, 100:1) using silica gel
to give the corresponding product (Table 3).
N-1-Ben zyl-2,3-d ih yd r o-1H-p yr r olo[2,3-b]qu in olin -4-yl-
2-p ip er id in -1-yla ceta m id e (27). Yield 49%; mp 220-222 °C;
IR (KBr): 3410, 1699, 1660, 1505 cm-1; 1H NMR (DMSO-d6):
7.05-7.78 (m, 9H), 4.60 (s, 2H), 4.46 (s, 2H), 3.46 (m, 2H), 3.10
(m, 4H), 2.86 (t, 2H), 1.72 (m, 4H), 1.52 (m, 2H); 13C NMR
(DMSO-d6): 168.6, 163.6, 149.6, 145.9, 139.5, 129.9(2C),
129.7(2C), 129.5, 128.6, 126.6, 122.7, 121.9, 118.4, 102.4, 60.6,
54.5(2C), 49.5(2C), 24.1, 23.9(2C), 22.7. Anal. Calcd for
BioAssay plate reader at
a wavelength of 570 nm. The
percentage of cells killed is calculated as the percentage
decrease in SRB binding as compared with control cultures.
Reversal of Pgp-mediated MDR was indicated if the compound
enhanced the toxicity of vinblastine toward the NCI/ADR cells.
The Pgp Antagonism Score was calculated as the percentage
of surviving NCI/ADR cells in the absence of vinblastine
divided by the percentage of surviving NCI/ADR cells in the
presence of vinblastine. Control cultures included equivalent
amounts of ethanol (1%, as the solvent control), which did not
modulate the growth or drug-sensitivity of these cells. To
assess the toxicity of the compounds toward drug-sensitive
cells, the effects of the test modulators on the growth of drug-
sensitive MCF-7 cells were determined by the same methods.
Reversal of MRP1-mediated MDR was indicated if the com-
pound enhanced the toxicity of vincristine toward MCF-7/VP
cells. The MRP1 Antagonism Score was calculated as the
percentage of surviving MCF-7/VP cells in the absence of
vincristine divided by the percentage of surviving MCF-7/VP
cells in the presence of vincristine.
In Vitr o Dr u g Accu m u la tion Assa y. Cells were seeded
in 24-well tissue culture dishes at approximately 25% conflu-
ency and allowed to recover and grow to near confluency,
typically 3-4 days. Media was aspirated and replaced with
serum-free media, and varying concentrations of PGP-4008
were added to the wells and cultures were incubated for 30
min at 37 °C. Approximately 0.1 µCi of [3H]paclitaxel (75 Ci/
mmol) or [3H]vinblastine (7.3 Ci/mmol) (Moravek Biochemicals,
Brea, CA) was then added per well, and the cultures were
incubated for 60 min at 37 °C. Radioactive media was
aspirated and cells were rapidly washed twice with ice-cold
phosphate-buffered saline. Intracellular [3H]drug was solu-
bilized with 1% sodium dodecyl sulfate in water and quantified
by liquid scintillation counting using UniverSol (ICN, Costa
Mesa, CA) as previously described.38
C
25H28N4O‚HCl (437.02): C, 68.70; H, 6.70; N, 12.81. Found:
C, 68.81; H, 6.43; N, 12.76.
N-1-Ben zyl-2,3-d ih yd r o-1H-p yr r olo[2,3-b]qu in olin -4-yl-
2-p h en yla ceta m id e (35 or P GP -4008). Yield 52%; mp 157-
159 °C; IR (KBr): 3431, 2959, 1690, 1665, 1507, 1310 cm-1
;
1H NMR (DMSO-d6): 7.29-8.37 (m, 14H), 5.17 (s, 2H), 3.93
(s, 2H), 3.77 (t, 2H), 2.97 (t, 2H); 13C NMR (DMSO-d6): 169.5,
156.5, 139.4, 137.2, 136.4, 135.1, 131.7, 129.9, 129.5(2C),
129.1(2C), 129.0(2C), 128.8(2C), 127.4, 125.3, 124.6, 123.6,
119.9, 118.8, 51.0, 50.5, 43.1, 25.8. Anal. Calcd for C26H23N3O‚
H2O (411.50): C, 75.88; H, 6.13; N, 10.21. Found: C, 76.29;
H, 6.41; N, 9.82. The purity of 35 was confirmed to be >95%
by HPLC analyses as described below.
N-(1-Ben zyl-2,3-d ih yd r o-1H-p yr r olo[2,3-b]q u in olin -4-
yl)-2-flu or o-6-tr iflu or om eth ylben za m id e (36). Yield 55%;
mp 189-191 °C; IR (KBr): 3427, 2933, 1691, 1628, 1119 cm-1
;
1H NMR (DMSO-d6): 7.35-8.25 (m, 12H), 5.02 (s, 2H), 3.66
(t, 2H), 2.98 (t, 2H); 13C NMR (DMSO-d6): 168.5, 165.3, 164.8,
161.2, 156.4, 148.4, 138.6, 136.4, 134.5, 131.1, 129.3(2C),
128.7(2C), 128.3, 124.1, 123.3, 122.5, 119.4, 119.1, 118.6, 116.3,
99.7, 50.3, 49.8, 23.8. Anal. Calcd for C26H19F4N3O (465.44):
C, 67.09; H, 4.11; N, 9.03. Found: C, 67.45; H, 4.19; N, 9.15.
N-(1-Ben zyl-2,3-d ih yd r o-1H-p yr r olo[2,3-b]q u in olin -4-
yl)-4-flu or o-3-tr iflu or om eth ylben za m id e (37). Yield 45%;
mp 195-197 °C; IR (KBr): 3435, 1691, 1626, 1375, 1118 cm-1
;
1H NMR (DMSO-d6): 7.25-8.62 (m, 12H), 4.95 (s, 2H), 3.37
(t, 2H), 3.00 (t, 2H); 13C NMR (DMSO-d6): 169.0, 165.0, 164.9,
161.2, 155.4, 147.9, 138.8, 136.0, 134.3, 131.0, 129.1, 128.7(2C),
128.2(2C), 124.1, 123.1, 122.5, 119.4, 119.1, 118.3, 116.7, 99.5,
50.8, 49.5, 23.5. Anal. Calcd for C26H19F4N3O (465.44): C,
67.09; H, 4.11; N, 9.03. Found: C, 67.50; H, 4.05; N, 9.18.
N-(1-Ben zyl-2,3-d ih yd r o-1H-p yr r olo[2,3-b]q u in olin -4-
yl)-2,3,6-tr iflu or oben za m id e (38). Yield 53%; mp 160-162
°C; IR (KBr): 3430, 1689, 1378, 1119 cm-1; 1H NMR (DMSO-
d6): 7.33-8.31 (m, 11H), 5.14 (s, 2H), 3.84 (t, 2H), 3.13 (t, 2H);
13C NMR (DMSO-d6): 168.9, 165.3, 164.8, 161.2, 159.2, 159.1,
159.0, 138.2, 135.6, 132.3, 130.0, 129.4, 129.3, 125.9, 125.6,
124.5, 120.9, 120.5, 120.2, 119.6, 113.9, 113.4, 50.7, 49.8, 23.6.
Anal. Calcd for C25H18F3N3O (433.43): C, 69.28; H, 4.19; N,
9.69. Found: C, 69.28; H, 4.07; N, 9.62.
In Vivo Toxicity Assa y. Animal care and treatments were
in accordance with guidelines and regulations of the Institu-
tional Animal Care and Use Committee of The Penn State
College of Medicine. Female Swiss-Webster mice (Charles
River Laboratories, Wilmington, MA), 6-8 weeks old, were
acclimated to their environment during quarantine for ap-
proximately 10 days before being released into the mouse

Dihydropyrroloquinolines as P-Glycoprotein Antagonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1421
colony. Mice were housed (5 per cage) under 12 h light/dark
cycles with food and water provided ad libitum. To evaluate
the in vivo toxicity of PGP-4008, mice were injected in the
intraperitoneal cavity with 20 mg/mL of PGP-4008 to give a
total body concentration of 100 mg/kg (approximately 2.0 mg).
Doses were administered once daily for 5 consecutive days,
and mice were monitored for 2-3 weeks after the final
injection.
anesthesia. For the analysis of [3H]vinblastine concentrations,
0.1 mL of 100 mM EDTA and 0.3 mL of 30% H2O2 were added
to decolorize the samples, followed by incubation for 1 h at 50
°C, cooling to room temperature, addition of 15 mL of Univer-
Sol (ICN, Costa Mesa, CA), and quantification by liquid
scintillation counting.
Ack n ow led gm en t. This work was supported by
In Vivo Tu m or Gr owth Assay. Balb/c female mice (Charles
River Laboratories, Wilmington, MA), 6-8 weeks old, were
injected in the subcutaneous space of the right hind quarter
with 106 J C cells (murine mammary adenocarcinoma, Ameri-
can Type Culture Collection CRL-2116, Manassas, VA) sus-
pended in phosphate-buffered saline. After palpable tumor
growth, approximately 2-3 weeks after injection, the tumor
volumes were determined (day 1) using calipers measuring the
length (L) and width (W) of the tumor. Tumor volumes were
calculated using the equation: (L × W2)/2, and animals were
randomized into four groups (five per group). Treatment was
then administered on days 1, 5, and 9 and consisted of either
intravenous administration of 50 mg/kg of cyclosporine injec-
tion USP42 (Bedford Laboratories, Bedford, OH), with or
without 5 mg/kg of doxorubicin hydrochloride (Sigma-Aldrich
Co., St. Louis, MO); or intraperitoneal administration of 100
mg/kg of PGP-4008‚HCl with or without doxorubicin hydro-
chloride. Tumor volumes were monitored until day 15 when
the animals were euthanized, and treatment effects were
compared by unpaired t-test with Welch correction using
InStat (GraphPad Software, San Diego, CA).
National Institute of Health Grant CA088243 (to C.D.S.).
Ap p en d ix
Abbreviations used: MDR, multiple drug resistance;
MRP, multidrug resistance-related protein; Pgp, P-
glycoprotein; RI, reversal index; SRB, sulforhodamine
B.
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