Synthesis and evaluation of (2-(4-methoxyphenyl)-4-quinolinyl)(2- piperidinyl)methanol (NSC23925) isomers to reverse multidrug resistance in cancer

Article abstract of DOI:10.1021/jm300117u

Development of multidrug resistance (MDR) during chemotherapy is a fundamental obstacle associated with cancer care. Prior studies have identified (2-(4-methoxyphenyl)-4-quinolinyl)(2-piperidinyl)methanol (5) (NSC23925) to be a small molecule agent that r

Full text of DOI:10.1021/jm300117u

Article
pubs.acs.org/jmc
Synthesis and Evaluation of (2-(4-Methoxyphenyl)-4-quinolinyl)
(2-piperidinyl)methanol (NSC23925) Isomers To Reverse Multidrug
Resistance in Cancer
Zhenfeng Duan,*,# Xin Li,† Haoxi Huang,† Wei Yuan,† Shao-Liang Zheng,‡ Xianzhe Liu,# Zhan Zhang,#,§
Edwin Choy,# David Harmon,# Henry Mankin,# and Francis Hornicek#
#Sarcoma Biology Laboratory, Center for Sarcoma and Connective Tissue Oncology, Massachusetts General Hospital, Boston,
Massachusetts 02114, United States
†Chengdu ChemPartner Co., Ltd., Floor 3, Building 3, Tianfu Life Science Park, No. 88, Keyuan South Road, Hi-Tech Zone,
Chengdu, 610041, People's Republic of China
‡Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, United States
§The Third Affiliated Hospital of Zhengzhou University, People's Republic of China
S
* Supporting Information
ABSTRACT: Development of multidrug resistance (MDR) during
chemotherapy is a fundamental obstacle associated with cancer care.
Prior studies have identified (2-(4-methoxyphenyl)-4-quinolinyl)
(2-piperidinyl)methanol (5) (NSC23925) to be a small molecule
agent that reverses MDR in cancer cells. We synthesized all four
isomers of 5 and analyzed them by liquid chromatography−mass
spectrometry (LCMS). Structure−activity relationships for revers-
ing MDR were evaluated. Isomer 11 demonstrated the most po-
tent activity. 11 reversed MDR in several drug-resistant cell lines
expressing Pgp, including ovarian, breast, colon, uterine, and sarcoma
cancer. 11 resensitized these cell lines to paclitaxel, doxorubicin,
mitoxantrone, vincristine, and trabectedin with no effect on cell
sensitivity to cisplatin, topotecan, and methotrexate. 11 significantly
enhanced in vivo antitumor activity of paclitaxel in MDR xenograft models, without increasing the level of paclitaxel toxicity. In
conclusion, 11 and derivatives of this compound may hold therapeutic value in the treatment of MDR-dependent cancers.
on MDR-reversing agents such as verapamil, cyclosporine A,
valspodar (PSC833, Amdray),8 and biricodar (VX710,
INCEL).9 Unfortunately, these compounds, initially developed
for pharmacological uses other than MDR reversal, are rela-
tively nonspecific and weak in potency.8,9 For most of these
compounds, clinical toxicities associated with their use at
concentrations required to inhibit Pgp have precluded their
widespread use. For example, the serum concentration of the
calcium channel blocker verapamil that is required to produce
in vitro reversal of MDR cannot be achieved in patients because
of significant cardiotoxicities. Valspodar and biricodar have shown
significant pharmacokinetic interaction with paclitaxel.1,3,10,11 The
addition of biricodar (biricodar blood steady-state concentra-
tions of 10 μM were sustained at a dosage of 120 mg/m2 per
hour) to doxorubicin and vincristine therapy did not signifi-
cantly increase antitumor activity or survival.12
INTRODUCTION
■
A major obstacle to cancer therapy is multidrug resistance
(MDR), which can arise from either intrinsic or acquired mech-
anisms of resistance.1,2 One of the molecular mechanisms for
MDR is overexpression of plasma membrane glycoprotein
(Pgp).1−3 Pgp is the gene product of MDR1(ABCB1), a
member of the ABC (ATP binding cassette) superfamily of a
170 kDa transporter protein, and it acts as an energy-dependent
(ATP) cancer drug efflux by pumping drugs out of the cells.4
Overexpression of Pgp in tumor cells prevents adequate intra-
cellular accumulation of a large number of cytotoxic drugs
including anthracyclines (doxorubicin, daunorubicin), anthra-
cenedione (mitoxantrone), vinca alkaloids (vinblastine, vincris-
tine), taxanes (paclitaxel, docetaxel), and many others.3−5
series of homologous ABC proteins such as multidrug-resis-
tance proteins (MRPs) and breast cancer resistance protein
(BCRP) have also been discovered.6,7
Pgp is a major protein associated with MDR and has a low
level expression in normal tissues. A series of compounds that
interact with Pgp and block drug efflux have been reported to
reverse the MDR phenotype.1,8 Previous studies have focused
A
Previously, in an attempt to identify novel small molecules
that are more potent and selective as MDR inhibitors, we
Received: November 14, 2011
Published: March 8, 2012
© 2012 American Chemical Society
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a
Scheme 1. Synthesis of 5 (NSC23925) Isomers
a
Reagents and conditions: (a) 1-(4-methoxyphenyl)ethanone, KOH, EtOH, 85 °C, 24 h, 50%; (b) concd H2SO4, MeOH, reflux overnight, 70%;
(c) 2-bromopyridine, n-BuLi, Et2O−THF, −78 °C, 2 h, 91%; (d) NaBH4, EtOH, 0 °C, 1 h, 60%; (e) H2, PtO2, concd HCl, MeOH, 20 °C, 2 h;
(f) (Boc)2O, Et3N, THF, 0 to rt,overnight; (g) chiral HPLC; (h) HCl/EtOH, 20 °C.
screened 2000 small molecule compounds from the NCI Diver-
sity Set library to look for compounds that can reverse MDR in
drug-resistant cell lines. We identified 2 out 2000 compounds,
tetrandrine (NSC77037)13 and (2-(4-methoxyphenyl)-4-
quinolinyl)(2-piperidinyl)methanol (5) (NSC23925), as com-
pounds with the highest activities as MDR reversal agents.5,13
5 is the most potent MDR reversing compound that we
observed in the NCI compound set.5,14 In comparison with
tetrandrine, 5 is a unique compound with essentially very little
chemical or biologic characterization since its synthesis as an
antimalarial agent in the 1940s.14 Analysis of the structure of
5 indicates that it has two chiral centers which lead to four
different stereoisomers 8, 9, 10, and 11. In theory, each of the
compound 5 isomers may have distinct bioactivities.
30 mol % PtO2 in a mixture of methanol and concd HCl (40:1)
afforded (2-(4-methoxyphenyl)quinolin-4-yl)(piperidin-2-yl)-
methanol (5).16 Because attempts to separate isomers of 5 by
chiral HPLC failed to get optically pure products, derivation of
5 was carried out, and Boc-protected derivatives 6 and 7 were
prepared and separated away from each other on silica gel
chromatography. The structure of 6 and 7 was confirmed to be
threo isomer and erythro isomer, respectively, according to the
common trend in which the coupling constant of CH(NH)-
CH(OH) of the erythro isomer (3J-erythro) is smaller than
that of threo isomer (3J-threo).17 Then threo isomer 6 was
separated by chiral HPLC to give optically pure Boc-8 and Boc-
9, and erythro isomer 7 gave optically pure Boc-10 and Boc-11.
Finally deprotection of these Boc-derivatives of 8 to 11 by
HCl−EtOH solution afforded four isomers 8, 9, 10, and 11.
We cultivated the single crystal from these synthesized
compounds and determined the absolute configuration for all
four isomers (Supporting Information Figure 1) which
confirmed the judgment on the erythro/threo isomers. Liquid
chromatography−mass spectrometry (LCMS, chiral HPLC)
analysis demonstrated that the four isomers 8, 9, 10, and 11
have the desired mass with significant purity (Supporting
Information Figure 2).
In this study, we synthesized and separated all four isomers
of 5 and biologically characterized each of them in several
multidrug-resistant cell lines. The most potent 5 isomer, 11,
was selected for further evaluation in vivo in a multidrug-
resistant ovarian cancer xenograft mouse model.
CHEMISTRY
■
By using the route as indicated in Scheme 1, the synthesis of all
four isomers of 5 was accomplished with Pfitzinger’s method.15
2-(4-Methoxyphenyl)quinoline-4-carboxylic acid (1) was pre-
pared in 50% yield from indoline-2,3-dione and 1-(4-methoxy-
phenyl)ethanone, and then it was esterified to afford methyl
2-(4-methoxyphenyl)quinoline-4-carboxylate (2) in 70% yield.
Using n-BuLi as a base, 2-bromopyridine was reacted with 2 to
give (2-(4-methoxyphenyl)quinolin-4-yl)(pyridin-2-yl)-
methanone (3) in 91% yield. Subsequent reduction of 3 by
NaBH4 afforded (2-(4-methoxyphenyl)quinolin-4-yl)(pyridin-
2-yl)methanol (4) in 60% yield. Hydrogenation of 4 with
BIOLOGICAL EVALUATION AND DISCUSSION
■
Identification of 11 as the Most Potent Isomer That
Reverses MDR in Human Cancer Cell Lines. Structure−
activity relationships of four 5 isomers for reversing MDR were
evaluated in human ovarian cancer MDR cell line SKOV-3TR by
MTT assay. We observed that among the 5 isomers, 11 is the most
potent one in reversing drug resistance (Figure 1). 8 and 9 showed
modest but lesser activity, while 10 showed the least potency.
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Figure 1. Effect of 5 isomers on reverse drug resistance in SKOV-3TR cell line. Cells were treated with the paclitaxel and isomers (8, 9, 10, and 11) in
RPMI1640 complete media at the indicated concentrations. The relative sensitivity of each line to paclitaxel was determined by MTT analysis 6 days
posttreatment.
Further studies showed that 11 is able to reverse paclitaxel,
doxorubicin, and mitoxantrone resistance in human breast
cancer MDR cell line MCF-7adr and human colon cancer
MDR cell line SW480TR (Figure 2). In addition, 11 is much
more potent (10 to 60 fold) than that of known drug resistance
reversing agents verapamil or CsA (data not shown). In
summary, 11 is able to reverse MDR in several MDR cell lines,
including ovarian, breast, uterine, colon, and sarcoma MDR
cancer cell lines to paclitaxel, docetaxel, doxorubicin, vincristine,
daunorubicin, gemcitabine, trabectedin (ET-743, Yondelis),5
and mitoxantrone. Table 1 shows representative findings of 11
on reversing drug resistance in ovarian, breast, and colon MDR
cell lines while similar results have been found in uterine
sarcoma, osteosarcoma, and Ewing sarcoma cell lines (data not
shown). In the profile of reverse drug resistance to chemo-
therapy drugs, the results showed that 11 could reverse drug
resistance to paclitaxel, doxorubicin, mitoxantrone, vincristine,
and ET-743 but has no significant effects on drug sensitivities
of cisplatin, carboplatin, topotecan, and methotrexate. Overex-
pression of MDR1 is a well-known mechanism of drug resis-
tance for paclitaxel, doxorubicin, mitoxantrone, and vincristine.
In contrast, the mechanism of drug resistance for cisplatin,
carboplatin, and topotecan are thought to be due to increased
DNA repair mechanisms. Interestingly, our previous study
showed reversal of MDR by 5 was through selective and potent
inhibition of MDR1 (Pgp) function. Furthermore, we also eval-
uated the effects of 11 on MDR1 (Pgp)-independent MRP1 or
BCRP-mediated drug resistance in H-69AR and MCF-7/MX
cell lines. The results showed that 11 was unable to reverse
drug resistance in these non-Pgp-expressing drug-resistant cell
lines, suggesting that 11 activity is specific for Pgp.
11 Modulates Pgp-Mediated Uptake and Efflux of
Calcein AM. Reversal of MDR is usually displayed as an in-
creased intracellular accumulation of chemotherapeutic drugs.
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Figure 2. Reverse multidrug resistance isomer 11 in MCF-7adr and SW480TR cell lines. Cells were treated with paclitaxel, doxorubicin, or
mitoxantrone and isomer 11 in RPMI1640 complete media at the indicated concentrations. The relative sensitivity of each line to the indicated drugs
was determined by MTT analysis 6 days posttreatment.
a
Table 1. Effect of Isomer 11 on Reverse Drug Resistance in MDR Cell Lines
SKOV-3TR IC50 (μM)
MCF-7adr IC50 (μM)
SW480TR IC50(μM)
pactlitaxel
with 11 0.1 μM
0.57 0.03
0.62 0.12
0.12 0.08
0.33 0.04 (2)
0.08 0.06 (7)
0.02 0.03 (28)
38.5 2.6
0.46 0.1 (1.3)
0.16 0.03 (4)
0.05 0.02 (12)
3.8 0.12
0.09 0.01 (1.3)
0.016 0.006 (8)
0.009 0.002 (13)
0.84 0.16
with 11 0.5 μM
with 11 1 μM
doxorubicin
with 11 0.1 μM
with 11 0.5 μM
with 11 1 μM
24.6 1.2 (2)
4.2 0.5 (9)
2.8 0.4 (1.4)
0.96 0.22 (4)
0.25 0.06 (15)
0.32 0.06
0.5 0.1 (1.7)
0.28 0.08 (3)
0.11 0.04 (8)
0.27 0.03
1.2 0.3 (32)
0.46 0.06
mitoxantrone
with 11 0.1 μM
with 11 0.5 μM
with 11 1 μM
0.37 0.05 (1.2)
0.18 0.02 (2.6)
0.06 0.01 (8)
0.3 0.001 (1.1)
0.12 0.02 (3)
0.05 0.01(6)
0.23 0.03 (1.2)
0.09 0.03 (3)
0.02 0.01 (14)
a
Cell survival was determined by MTT assay as described in Materials and Methods. IC50 is the concentration of drug (μM) that produced 50%
inhibition of cell growth. Results were calculated from one experiment with triplicate wells. The numbers in the parentheses represent fold-reversal of
drug resistance (IC50 of drugs divided by IC50 of drugs with 11).
We examined the effects of 11 on the uptake and efflux of cal-
cein AM in MCF-7adr and SW480TR. Fluorescence microscopic
analysis showed 11’s ability to increase intracellular accumulation
of calcein AM in these cell lines in a dose-dependent manner
(Figure 3). 11 has a prominent effect on the accumulation of
calcein AM in these cells starting at a concentration as low as
10 nM. Half-maximal reversal of accumulation deficit was observed
at 100 nM and near maximal at 500 nM.
Combination of 11 and Doxorubicin Induces Apop-
tosis in MDR Cancer Cells. The effect of 11 on the
doxorubicin induction of apoptosis was assessed in MCF-7adr
or SW480TR cell lines. The cells were treated with either doxo-
rubicin (0.5 μM) alone, 11 (1 μM) alone, or with a com-
bination of doxorubicin and 11 for 72 h. Apoptosis was
scored using the M30-Apoptosense ELISA assay. The
combination of doxorubicin and 11 resulted in significantly
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Figure 3. Calcein AM efflux from isomer 11-treated MCF-7adr and SW480TR cell line cells. The calcein AM assay was optimized and performed
using the Vybrant Multidrug Resistance kit. Cells were seeded at 50 000 cells/well (100 μL of culture medium) in a 96-well plate and incubated for
24 h. MCF-7adr or SW480TR cells in triplicate were treated with 11 for 1 h and then incubated in calcein AM for 30 min. The cell fluorescence
images were acquired by a fluorescence microscope.
Figure 4. Isomer 11 in combination with doxorubicin significantly induces cell death and apoptosis. A: Cell death induced by isomer 11 in
combination with doxorubicin on MCF-7adr and SW480TR cells. B: Cell apoptosis induced by 11 in combination with doxorubicin on MCF-7adr
and SW480TR cells. Cells were seeded at a density of 8000 cells per well in a 96-well plate for 24 h. The cells were then treated with 11, doxorubicin,
or a combination of the two drugs for an additional 48 h. The cells were lysed with 10% NP-40, and the M30-Apoptosense ELISA assay was
performed as described in Materials and Methods.
greater cell death as compared with doxorubicin or 11 alone
(Figure 4). Additionally, MTT (3-(4,5-dimethylthiazol-2-yl)-
2,5- diphenyltetrazolium bromide) cytotoxicity assay also
showed that 11 has a synergistic effect on doxorubicin-induced
cell death.
Effect of Combination Therapy with 11 on Tumor
Growth in Vivo. To further evaluate the effects of 11 and
paclitaxel combination therapy on the tumor growth of resistant
cells, the growth of xenograft tumors was examined. A well-
characterized ovarian cancer MDR cell line, SKOV-3TR, was
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Figure 5. Effect of combination treatment with 11 and paclitaxel on tumor growth in vivo. A: Tumors were measured twice a week with a digital
caliper. Tumor volume (mm3) was calculated as (W2 × L)/2, where W is width and L is length. B: Representative images of dissected tumor tissues
from treated nude mice.
chosen for in vivo studies. The results showed that 11 or paclitaxel
alone had no significant effects on the paclitaxel-resistant tumor cell
growth in xenograft model. In contrast, the combination of
paclitaxel and 11 produced an inhibitory effect on the resistant
tumor growth as compared with animals treated with paclitaxel
alone (20 mg/kg, Figure 5). Intraperitoneal (ip) injection of 11
(50 mg/kg) was found to almost completely reverse resistance to
paclitaxel in this model. In addition, on the basis of animal weight
and mortality, no significant toxicity was observed and the animals
appeared to have tolerated it well when 11 was administered alone.
potent activity after ip administration in mice bearing MDR hu-
man xenografts, restoring antitumor activity of paclitaxel with-
out an apparent increase in toxicity. All of the efficacious
combination schedules with 11 appeared to be well tolerated, as
indicated by the lack of significant changes in body weights of
various treatment groups. These results demonstrate that the
5 isomer 11 is an extremely potent, selective, and effective inhibi-
tor of Pgp with a long duration of action. It exhibits potent
activity without apparently enhancing the toxicity of coadmin-
istrated drugs.
In conclusion, 11 is a potent in vitro and in vivo modulator
of MDR1 (Pgp)-mediated MDR. The properties of 11 indicate
that it will be useful in the treatment of Pgp-mediated chemo-
resistant cancers.
CONCLUSIONS
■
5 is a newly identified small molecular compound found to
efficiently reverse MDR in a wide variety of MDR cell lines.5
The present studies were undertaken to synthesize 5 isomers 8,
9, 10, and 11 based on its molecular chirality and to charac-
terize these novel isomers in vitro and in vivo. We observe that
11 is a selective and very potent inhibitor of Pgp-mediated
MDR, and the data provide valuable clues for the further
optimization of this structure. The in vitro potency was evalua-
ted by several assays (including cytotoxic drug activity by MTT
assay, enhancement of drug uptake by calcein AM assay, and
increased drug-induced apoptosis by M-13 apoptosense assay)
using a panel of human MDR cell lines with different expressions
of Pgp, MRP1, and BCRP. Not surprisingly, we observe that 11
recapitulates the activity of the diasteromeric mixture of 5 isomers;
11 reverses MDR in (Pgp) overexpressing cell lines for several
chemotherapy drugs, is a highly potent, specific, nontoxic Pgp
inhibitor, and does not inhibit MRP1 or BCRP1 at therapeutically
relevant doses. 11 appears to possess all of the desired properties
for treating Pgp-mediated MDR.
Direct comparison with 8, 9, 10, and 11 shows that 11 is the
most potent modulator (Figure 1). Previous and current studies
have clearly indicated that reversal of MDR by 5 and its iso-
mer 11 was through selective and potent inhibition of Pgp
function.5 For example, in contrast to reversing MDR in Pgp-
overexpressed cell lines, 11 had no effect on drug sensitivities
in MRP1- or BCRP-overexpressed MDR cell lines, nor did
it affect the sensitivities to cisplatin or carboplatin (data not shown).
In this study, we show new in vivo data confirming in mouse
xenografts the expected activities seen in vitro. 11 exhibited
EXPERIMENTAL SECTION
■
General Procedures for the Synthesis of 5 Isomers. All
evaporations were carried out in vacuo with a rotary evaporator.
Analytical samples were dried in vacuo (1−5 mmHg) at room
temperature. Thin layer chromatography (TLC) was performed on
silica gel plates with fluorescent indicator. Spots were visualized by UV
light (214 and 254 nm). Purification by column and flash chroma-
tography was carried out using silica gel (300−400 mesh). Solvent
systems are reported as volume percent mixture. All NMR spectra
1
were recorded on a Bruker WH-300 (300 MHz) spectrometer. H
chemical shifts are reported in δ values in ppm with the deuterated
solvent as the internal standard. Data are reported as follows: chemical
shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, br =
broad, m = multiplet), coupling constant (Hz), integration. LCMS
spectra were obtained on an Agilent 1200 series 6110 or 6120 mass
spectrometer with electrospray ionization. LCMS: Method 1 (5-100a
and 5-100b) = (Waters X Bridge C18, 50 mm × 4.6 mm × 3.5 μm,
2.3 min gradient, 5% [0.05% TFA/CH3CN]: 95% [0.05% TFA/H2O]
to 100% [0.05% TFA/CH3CN] for 1.6 min and hold 100% [0.05%
TFA/CH3CN] for 0.7 min; method 2 (5-100a (test) and 5-100b
(test)) = (Waters X Bridge C18, 50 mm × 4.6 mm × 3.5 μm, 3.5 min
gradient, 5% [0.05% TFA/CH3CN]: 95% [0.05% TFA/H2O] to 100%
[0.05% TFA/CH3CN] for 1.6 min and hold 100% [0.05% TFA/
CH3CN] for 1.9 min; method 3 (5−100A 5) = (Waters X Bridge C18,
50 mm × 4.6 mm × 3.5 μm, 5.8 min gradient, 5% [0.05% TFA/
CH3CN]: 95% [0.05% TFA/H2O] to 100% [0.05% TFA/CH3CN]
for 5.0 min and hold 100% [0.05% TFA/CH3CN] for 0.8 min.
Preparative chiral HPLC purification was performed on a Shimadzu
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LC 20 with UV detector SPD-20A at 35 °C using CHIRALPAK IA
(0.46 cm I.D. × 25 cm length) and using hexane/ispropanol/DEA =
70/30/0.1 (V/V/V) as the mobile phase. Flow rate was 1.0 mL/min
(all solvents were HPLC grade). The HPLC system was monitored at
254 nm. Analytical chiral HPLC spectra were run on a Shimadzu
LC 20 with UV detector SPD-20A at 35 °C using Chiralpak AD-H
(0.46 × 25 cm) and using hexane/ispropanol/DEA = 70/30/0.1
(V/V/V) during 20 min as the mobile phase. Flow rate was
1.0 mL/min (all solvents were HPLC grade). The HPLC system
was monitored at 254 nm. Optical rotation was run on a Polarimeter
Perkin-Elmer Model 341 at 20 °C.
2-(4-Methoxyphenyl)quinoline-4-carboxylic Acid (1). Indo-
line-2,3-dione (30.6 g, 203.9 mmol), 1-(4-methoxyphenyl)ethanone
(25.0 g, 169.9 mmol), and KOH (28.6 g, 509.7 mmol) were dissolved
in EtOH (200 mL). The mixture was stirred at 80 °C for 24 h.
Evaporation of the solvent afforded a residue which was diluted in
water, and then the solution was extracted by ether. The aqueous
phase was acidified at 0 °C to pH = 1 with concd HCl, and then the
precipitate was collected by suction filtration, washed with water and
CH2Cl2, and dried in vacuo to afford 28.4 g (50% yield) of analyti-
cally pure 1 (yellow solid) confirmed by LCMS. Analytical LCMS
(method 1): single peak (214, 254 nm), tR = 1.65 min, MS (ESI+) m/z
279.09 (M + H)+.
in THF (15 mL) dropwise. The mixture was stirred at r.t. overnight,
and then water (50 mL) was added. The mixture was extracted with
AcOEt (3 × 100 mL), and the combined organic layers were washed
with brine, dried over anhydrous Na2SO4, and filtered. The filtrate was
evaporated in vacuo, and the crude residue was flash chromatographed
on silica gel and eluted with 1:10−1:6 EA/PE to afford 1.0 g of 6
(threo isomer) (analytical LCMS (method 1): single peak (214,
254 nm), tR = 1.62 min, MS (ESI+) 449.0 (M + H)+) and 1.3 g of 7
(erythro isomer) (analytical LCMS (method 1): single peak (214,
254 nm), tR = 1.68 min, MS (ESI+) 449.3 (M + H)+).
(2-(4-Methoxyphenyl)quinolin-4-yl)(piperidin-2-yl)methanol
Hydrochloride (8−11). After purification of 7 (1.3 g) by chiral
HPLC, two Boc-protected chiral compounds were separated from
each other. Then each compound was deprotected with a solution of
HCl in EtOH to give the corresponding HCl salt. Thus, 10 (0.6 g) and
11 (0.6 g) were obtained, respectively. Using the same methods, 8
(0.37 g) and 9 (0.26 g) were produced, respectively, from 1 g of 6.
(1R,2R)-(2-(4-Methoxyphenyl)quinolin-4-yl)(piperidin-2-yl)-
methanol Hydrochloride (8). 1H NMR (300 MHz, DMSO-d6)
δ 1.35 (m, 2H), 1.72 (m, 4H), 2.81 (m, 1H), 3.24 (m, 1H), 3.49 (m,
1H), 3.89 (s, 3H), 4.5 (brs, 1H), 5.68 (s, 1H), 7.20 (d, J = 8.7 Hz,
2H), 7.74 (m, 1H), 7.93 (m, 1H), 8.26−8.38 (m, 5H), 8.53 (m, 1H),
9.25 (m, 1H); analytical LCMS (method 3): 97% purity (214 nm),
20
tR = 1.77 min, MS (ESI+) m/z 349.0 (M + H)+; [α]D = −15.3 (c =
Methyl 2-(4-Methoxyphenyl)quinoline-4-carboxylate (2). To
a solution of 1 (15 g, 53.8 mmol) in MeOH (50 mL) was added concd
H2SO4 (13 mL). The mixture was stirred under reflux overnight,
diluted by water, and extracted by AcOEt. The organic layer was
separated, washed with brine, dried over anhydrous Na2SO4, and
filtered. The filtrate was evaporated under reduced pressure to afford
11.1 g (70%) of analytically pure 2 (yellow solid) confirmed by LCMS.
0.16, MeOH); chiral HPLC purity: 99.9% ee (254 nm) (tR (major) =
6.52 min, tR (minor) = 8.69 min).
(1S,2S)-(2-(4-Methoxyphenyl)quinolin-4-yl)(piperidin-2-yl)-
1
methanol Hydrochloride (9). H NMR (300 MHz, DMSO-d6) δ
1.35 (m, 2H), 1.72 (m, 4H), 2.81 (m, 1H), 3.24 (m, 1H), 3.49 (m,
1H), 3.89 (s, 3H), 4.5 (brs, 1H), 5.68 (s, 1H), 7.20 (d, J = 8.7 Hz,
2H), 7.74 (m, 1H), 7.93 (m, 1H), 8.26−8.38 (m, 5H), 8.53 (m, 1H),
9.25 (m, 1H); analytical LCMS (method 3): 99% purity (214 nm),
tR = 1.78 min, MS (ESI+) m/z 349.0 (M + H)+; [α]D20 = +15.0 (c = 0.16,
MeOH); chiral HPLC purity: 99.9% ee (254 nm) (tR (major) = 8.70 min,
tR (minor) = 6.53 min).
Analytical LCMS (method 2): single peak (214, 254 nm), tR
=
2.05 min, MS (ESI+) m/z 293.11 (M + H)+.
(2-(4-Methoxyphenyl)quinolin-4-yl)(pyridin-2-yl)methanone
(3). To a solution of n-BuLi (2.5 M, 26.4 mL, 66.1 mmol) in Et2O
(80 mL) cooled to −40 °C under N2 was added a solution of
2-bromopyridine (10.4 g, 66.1 mmol) in Et2O (20 mL). The reaction
was maintained at −78 °C for 20 min, and then a solution of 2 (6.5 g,
22.0 mmol) in THF was added at −70 °C under N2. The reaction
mixture was stirred at −70 °C for 1.5 h, quenched with water, and
extracted by AcOEt. The organic phase was washed with brine, dried
over anhydrous Na2SO4, and filtered. The filtrate was evaporated in
vacuo to give a residue. The residue was triturated with Et2O to afford
7 g (91%) of analytically pure 3 (yellow solid) confirmed by
LCMS. Analytical LCMS (method 1): single peak (214, 254 nm), tR =
1.87 min, MS (ESI+) m/z 341.2 (M + H)+.
(2-(4-Methoxyphenyl)quinolin-4-yl)(pyridin-2-yl)methanol
(4). To a solution of 3 (7.7 g, 22.6 mmol) in EtOH (110 mL) cooled
to 0 °C was added NaBH4 (2.12 g, 45.3 mmol). After the addition was
complete, the mixture was stirred for 0.5 h and then quenched with
water. Then AcOEt was added to the solution, and the organic layer
was separated, washed with brine, dried over anhydrous Na2SO4, and
filtered. The filtrate was evaporated in vacuo to afford 4.6 g (60%
yield) of 4 (white solid) which was used for the next step without
further purification. Analytical LCMS (method 2): single peak (214,
254 nm), tR = 1.53 min, MS (ESI+) 343.2 (M + H)+.
(2-(4-Methoxyphenyl)quinolin-4-yl)(piperidin-2-yl)methanol
(5) (NSC23925). To a solution of 4 (3.72 g, 10.88 mmol) in
MeOH (360 mL) were added concd HCl (9 mL) and PtO2 (741 mg,
3.26 mmol). The mixture was then hydrogenated at 20 °C for about
2 h. The catalyst was filtered off and washed with MeOH (3 × 10 mL),
and the filtrate was concentrated under reduced pressure. The residue
obtained was a dark green solid, which was used for the next step
reaction without further purification. (We tried to directly separate the
four isomers by using chiral prep HPLC but failed.) Analytical LCMS
(method 2): single peak (214, 254 nm), tR = 1.28 min, MS (ESI+)
349.3 (M + H)+.
(1R,2S)-(2-(4-Methoxyphenyl)quinolin-4-yl)(piperidin-2-yl)-
1
methanol Hydrochloride (10). H NMR (300 MHz, DMSO-d6) δ
1.29 (m, 2H), 1.68 (m, 4H), 2.97 (m, 1H), 3.25 (m, 1H), 3.45 (m,
1H), 3.90 (s, 3H), 4.6 (brs, 1H), 6.11 (s, 1H), 7.23 (d, J = 8.7 Hz,
2H), 7.78 (m, 1H), 8.01 (m, 1H), 8.26 (m, 3H), 8.50 (m, 2H), 8.69 (t,
J = 8.7 Hz, 1H), 10.46 (m, 1H); analytical LCMS (method 3): 95%
purity (214 nm), tR = 1.83 min, MS (ESI+) m/z 349.0 (M + H)+;
[α]D20 = −15.8 (c = 0.2, MeOH); chiral HPLC purity: 99.9% ee (254
nm) (tR (major) = 4.96 min, tR (minor) = 12.7 min).
(1S,2R)-(2-(4-Methoxyphenyl)quinolin-4-yl)(piperidin-2-yl)-
1
methanol Hydrochloride (11). H NMR (300 MHz, DMSO-d6)
δ1.29 (m, 2H), 1.68 (m, 4H), 2.97 (m, 1H), 3.25 (m, 1H), 3.45
(m, 1H), 3.90 (s, 3H), 4.6 (brs, 1H), 6.11 (s, 1H), 7.23 (d, J = 8.7 Hz,
2H), 7.78 (m, 1H), 8.01 (m, 1H), 8.26 (m, 3H), 8.50 (m, 2H), 8.69
(t, J = 8.7 Hz, 1H), 10.46 (m, 1H); analytical LCMS (method 3): 98%
purity (214 nm), tR = 1.83 min, MS (ESI+) m/z 349.0 (M + H)+;
20
[α]D = +15.6 (c = 0.2, MeOH); chiral HPLC purity: 97.2% ee
(254 nm) (tR (major) = 12.7 min, tR (minor) = 4.7 min).
X-ray Crystallography of 5 Isomers. The absolute configuration
of the isomer was determined by X-ray crystallography technique at
the Center for Crystallographic Studies at Department of Chemistry
and Chemical Biology, Harvard University. A crystal of a 5 isomer was
mounted on a diffractometer, and data were collected at 100 K. The
intensities of the reflections were collected by means of a Bruker
APEX II CCD diffractometer (MoKα radiation, λ = 0.71073 Å) and
equipped with an Oxford Cryosystems nitrogen flow apparatus. The
collection method involved 0.5° scans in ω at 28° in 2θ. Data inte-
gration down to 0.76 Å resolution was carried out using SAINT V7.46
A with reflection spot size optimization. Absorption corrections were
made with the program SADABS. The structure was solved by the
direct methods procedure and refined by least-squares methods again
F2 using SHELXS-97 and SHELXL-97. Non-hydrogen atoms were
refined anisotropically, and hydrogen atoms were allowed to ride on
the respective atoms. Crystal data, hydrogen bond parameters, and
geometric parameters of the isomer were collected. The Ortep plots
tert-Butyl 2-(Hydroxy(2-(4-methoxyphenyl)quinolin-4-yl)-
methyl)piperidine-1-carboxylate (6, 7). To a solution of 5 (the
residue above) and Et3N (3.48 g, 34.48 mmol) in THF (50 mL)
cooled at 0 °C was added a solution of (Boc)2O (4.0 g, 11.49 mmol)
3119
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Journal of Medicinal Chemistry
Article
produced with the SHELXL-97 program, and the other drawings, were
produced with Accelrys DS Visualizer 2.0.
Effect of Combination 11 and Paclitaxel on Tumor Growth
in Vivo. The Crl:SHO-PrkdcSCIDHrhr nude female mice at approx-
imately 3−4 weeks of age were purchased from The Charles River
Laboratories (Ann Arbor, MI). Animal studies were carried out with
protocols approved by the Massachusetts General Hospital Sub-
committee on Research Animal Care (SRAC). To determine the effect
of 11 on paclitaxel sensitivity in the xenograft model, 5 × 106 SKOV-3
Pharmacology, Cell Lines, Cell Culture, and Drugs. A panel of
a parental cancer cell line and their daughter resistant cell lines,
displaying a MDR phenotype, were used in this study. Human ovarian
cancer cell line SKOV-3, breast cancer cell line MCF-7, colon cancer
cell line SW480, osteosarcoma cell line U-2OS, KHOS, uterine
sarcoma cell line MESSA and its doxorubicin selected drug-resistant
cell line MESSA/Dx5, and nonsmall cell lung cancer cell line H-69 and
it is doxorubicin-selected drug-resistant cell line H-69AR (over-
expresses MRP1) were obtained from the ATCC (Rockville, MD).
The multidrug-resistant SKOV-3TR, MCF-7adr, and SW480TR cell
lines were established as previously reported. Dr. Efstathios Gonos
(Institute of Biological Research & Biotechnology, Athens, Greece)
provided the doxorubicin-resistant U-2OS R2 (referred in the text
below as U-2OSDR), KHOS R2 cell lines. Dr. Erasmus Schneider
(Wadsworth Center, Albany) provided the mitoxantrone-resistant
breast cancer MCF-7/MX (overexpresses BCRP) cell line. All the cell
lines were cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA)
supplemented with 10% fetal bovine serum, 100 units/mL penicillin
and 100 μg/mL streptomycin (Invitrogen). Cells were incubated at
37 °C in 5% CO2−95% air atmosphere and passaged when near-con-
fluent monolayers were achieved using 2% trypsin−EDTA solution.
Drug-resistant cell lines were periodically cultured in the respective
drug to confirm their drug resistance characteristics. The chemo-
therapy drugs were obtained from the pharmacy at the Massachusetts
General Hospital and stored at −20 °C.
Cytotoxicity MTT Assay and Reversals of MDR. To evaluate
for reversal of Pgp-mediated MDR, SKOV-3TR, MCF-7adr, and
SW480TR cell lines were seeded into a 96-well tissue culture plate at
2000 cells per well with varying concentrations of chemotherapy drugs
and 11 for 6 days. Cell proliferation IC50 (concentration required for
50% inhibition) and MDR reversal IC50 were determined from dose−
response curves carried out in triplate in a 96-well plate. The fold of
reverse resistance for individual drugs were defined as the IC50 of
MDR cells without 11 treatment divided by the IC50 of MDR cells
treated with 11. To test for the effect of reversal of MRP1- or BCRP-
mediated MDR, H-69AR or MCF-7/MX cells were placed into
96-well plates and treated with chemotherapy drugs similar to that
described above. Drug cytotoxicity was assessed in vitro using the
MTT assay as previously described.18 Drugs at the concentrations
utilized in the MTT assay were performed in the absence of cells to
verify no change in absorbances. Response curves were fitted with the
use of GraphPad PRISM 4 software (GraphPad Software).
Drug Efflux Fluorescence Microscopy Assay. The Vybrant
multidrug resistance assay kit (Invitrogen/Molecular Probes) was used
to measure drug efflux properties of different resistant cell lines. This
assay utilizes the fluorogenic dye calcein acetoxymethyl ester (calcein
AM) as a substrate for efflux activity of Pgp or other membrane pump
ABC proteins. Calcein AM is taken up by cells and hydrolyzed by
cytoplasmic esterases to fluorescent calcein. Calcein AM is well
retained in the cytosol. However, multidrug-resistant cells expressing
high levels of Pgp rapidly extrude nonfluorescent calcein AM from the
plasma membrane, reducing accumulation of fluorescent calcein in the
cytosol. Studies were carried out as described. In brief, drug-resistant
cells (5000 cells per well) were cultured in a 96-well plate for 24 h.
Triplicated cultures of cells were treated with different concentrations
of 11 for 1 h and then incubated in calcein AM in 100 μL of total
volume. After 30 min, images were acquired by Nikon Eclipse Ti-U
fluorescence microscope (Nikon Corp.) equipped with a SPOT RT
digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI).
Apoptosis Assays. Apoptosis was evaluated using the M30-
Apoptosense ELISA assay kit (Peviva AB, Bromma, Sweden) ac-
cording to the manufacturer’s instructions. For the doxorubicin and 11
treatment, MCF-7adr or SW480TR cells were seeded at 8000 cells/per
well in a 96-well plate for 24 h before treatment. The cells were then
treated with 0.5 μM doxorubicin, 1 μM 11, or a combination of the
two drugs for an additional 72 h. The cells were then lysed by an
additional 10 μL of 10% NP40 per well, following the manufacturer’s
instructions for apoptosis assay.
cells were injected subcutaneously with matrigel (BD Biosciences,
TR
San Jose, CA) on day one. Two weeks after injection, the mice were
divided into four groups to start the treatments. Group one received
intraperitoneal (ip) injection with normal saline, group two with
paclitaxel (20 mg/kg), and group three with 11 (50 mg/kg). The final
group of mice received paclitaxel (20 mg/kg) and 11 (50 mg/kg). The
dose of paclitaxel (20 mg/kg) was based on previous studies.19,20 All
four groups were treated twice a week for two weeks. The health of the
mice and evidence of tumor growth were examined daily. Tumors
were measured twice a week with a digital caliper. Tumor volume
(mm3) was calculated as (W2 × L)/2, where W is width and L is
length. The curve of tumor growth was drawn according to tumor
volume by using of GraphPad PRISM 4 software.
ASSOCIATED CONTENT
* Supporting Information
■
S
Perspective views showing 50% probability displacement ellipsoids
of absolute configuration for all four isomers (Figure 1). Chiral
HPLC spectra of four compounds 8, 9, 10 and 11 (Figure 2).
This material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*Telephone: (617) 724-3144. Fax: (617) 726-3883. E-mail:
duanz@helix.mgh.harvard.edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported in part by grants from the Gattegno
and Wechsler funds. Support has also been provided by the
Kenneth Stanton Fund. Dr. Duan is supported, in part, through
a grant from Sarcoma Foundation of America (SFA), a grant
from National Cancer Institute (NCI)/National Institutes of
Health (NIH), UO1, CA 151452.
ABBREVIATIONS USED
■
MDR, multidrug resistance; Pgp, P-glycoprotein; MRP1,
multidrug-resistant protein 1; BCRP, breast cancer-resistant
protein
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