[CANCER RESEARCH 50. 3055-3061, May 15, 1990]

Two Pyridine Analogues with More Effective Ability to Reverse Multidrug

Resistance and with Lower Calcium Channel Blocking Activity ThanTheir Dihydropyridine Counterparts1

Norimasa Shudo, Tetsuro Mizoguchi, Tatsuto Kiyosue, Makoto Arita, Akihiko Yoshimura, Kiyotomo Seto,Ryozo Sakoda, and Shin-ichi Akiyama2Department of Cancer Chemotherapy, Institute of Cancer Research, Faculty of Medicine, Kagoshima L'nirersity, Kagoshima 890 fN. S., T, M., A. Y., S. A.]; Departmentof Physiology, Oita Medical School, Oita 879-56 [T. A'., A/. A.]; and Central Research Institute, Mssan Chemical Ind. Co., Ltd., Chiba [K. S., R. S.]. Japan

ABSTRACT

Four pyridine analogues and their dihydropyridine counterparts »ereexamined for their ability to reverse drug resistance in a multidrug-resistant human carcinoma cell line, KB-C2. Two pyridine analogueswere more able to reverse drug resistance than their dihydropyridinecounterparts. The other two pyridine analogues had an effect on drugresistance similar to their dihydropyridine counterparts. The calciumchannel-blocking activity of all the pyridine analogues was considerablylower than that of the dihydropyridine analogues.

Of the pyridine analogues, 2-|4-(diphenylmethyl)-l-piperazinyl|ethyl5-(fra/js-4,6-dimethyl-l,3,2-dioxaphosphorinan-2-yI)-2,6-dimethyl-4-(3-nitrophenyl)-3-pyridinecarboxylate P-oxide (PAK-104P) was the mosteffective in reversing multidrug resistance. PAK-104P (1 and 5 MM)completely reversed the drug resistance in KB-8-5 and KB-C2 cells,respectively. The reversing effect of PAK-I04P was greater than that ofother multidrug resistance-reversing agents, cepharanthine, verapamil,nimodipine, and nicardipine. PAK-104P at 1 u\i increased about 10-foldthe accumulation of vinblastine in KB-C2 cells, whereas verapamil at thesame concentration increased the accumulation about 2-fold.

The inhibition of j-'Hjazidopine photolabeling of P-glycoprotein by

the pyridine and dihydropyridine analogues except 2-|methyl(phenyl-methyl)amino|ethyl 4-(2-chlorophenyl)-5-(4-methyl-l,3,2-dioxaphos-phorinan-2-yl)-l,4-dihydro-2,6-dimethyl-3-pyridinecarboxylate P-oxidecorrelated with the reversing of drug resistance by the analogues. Somenewly synthesized pyridine analogues seemed to have lower calciumchannel-blocking activity and more potent resistance-reversing abilitythan verapamil and other calcium channel blockers.

INTRODUCTION

Neoplastic cells resistant to multiple drugs including ADR,'

VCR, VBL, actinomycin D, etoposide, and teniposide often

Received 8/31/89; revised 1/24/90.The costs of publication of this article were defrayed in pan by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This study was supported by a Grant-in-Aid for Cancer Research from the

Ministry of Education. Science and Culture. Japan.J To whom requests for reprints should be addressed.' The abbreviations used are: ADR. doxorubicin; MDR. multidrug resistance:

SDB-ethylenediamine. A'-solanesyl-A',A"-bis(3,4-dimethoxjbenzyl)ethylenedia-mine: MTT. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PBS.phosphate-buffered saline; DAU. daunorubiein; VCR. vincristine; VBL, vinblastine. NMR, nuclear magnetic resonance; s, singlet; d, doublet; t. triplet; m.multiplet; MS. mass spectrum; PAK-101. 2-[methyl(phenylmethyl)amino]ethyl5-(diethoxyphosphinyl)-1.4-dihydro-2,6-dimethyI-4-[3-(trifluoromethyl)phenyl]-3-pyridine-carboxylate; PAK-101 P. 2-(methyl(phenylmethyl)amino]ethyl 5-(die-thoxyphosphinyl)-2.6-dimethyl-4-[3-(trifluoromethyl)phenyl]-3-p>ridinecarboxy-late; PAK-102. 2-[methyl(phenylmethyl)amino]ethyl 4-(2-chlorophenyl)-5-<4-methyl-1.3.2-dioxaphosphorinan-2-yl)-1.4-dihydro-2.6-dimethyl-3-pyridinecar-boxylale P-oxide; PAK-102P. 2-[methyl(phenylmethyl)amino]ethyl 4-(2-chloro-phenyl-5-(4-methyl-1.3,2-dioxaphosphorinan-2-yl)-2,6-dimethyl-3-pyridinecar-boxylate P-oxide: PAK-103. 2-(phenylamino)ethyl 5-(5.5-dimethyl-l,3.2-dioxa-phosphorinan-2-yl)-1.4-dihydro-2.6-dimethyl-4-(3-nitrophenyl)-3-pyridi-necarboxylate P-oxide: PAK-103P. 2-(phenylamino)ethyl 5-(5.5-dimethyl-1.3.2-dioxaphosphorinan-2-yl)-2.6-dimethyl-4-(3-nitrophenyl)-3-pyridinecarbox-ylate P-oxide; PAK-104. 2-[4-(diphenylmethyl)-l-piperazinyl]ethyl 5-(/ranj-4.6-dimelhyl-l,3.2-dioxaphosphorinan-2-yl)-l,4-dihydro-2.6-dimethyl-4-(3-nitro-phenyl)-3-pyridinecarboxylate P-oxide; PAK-I04P. 2-[4-(diphenyÃŒmethyl)-l-pi-perazinyl]ethyl 5-(fron.s-4.6-dimethyl-1.3.2-dioxaphosphorinan-2-yl)-2,6-di-methyl-4-(3-nitrophenyl)-3-pyridinecarboxylate P-oxide.

arise after exposure to antineoplastic agents. This problem ofacquired multidrug resistance presents a major obstacle in themanagement of cancer.

Many MDR sublines of mammalian cells have been used tounderstand the mechanism of resistance during cancer treatment (1-3). Most cell lines with the MDR phenotype showincreased expression of MDR] gene, which encodes a M,170,000 plasma membrane glycoprotein (P-glycoprotein) (4-9). P-glycoprotein is now thought to be an energy-dependentmultidrug efflux pump (10-14). MDR is reversed by a varietyof compounds including verapamil (15), quinidine (16), reser-pine (17), a synthetic isoprenoid, SDB-ethylenediamine (18),and a biscoclaurine alkaloid, cepharanthine (32). The agentsthat reverse MDR apparently did not seem to have commonfeatures. However, it appears that hydrophobicity at physiological pH, cationic charge, and molar refractivity are importantphysical properties for modulators of MDR (19, 20). They aresupposed to inhibit the pump activity of P-glycoprotein (21,22). When the activity of the pump is inhibited, anticanceragents accumulate in MDR cells and this may be why MDR isreversed.

Recently, dihydropyridine analogues have been shown toreverse MDR (23-25). However, most of such analogues reverse MDR in KB-C2 cells at the 10~5 M level and block the

calcium channel. We need to find or synthesize new agents thatreverse MDR at much lower concentrations and that have lowercalcium channel-blocking activity.

Pyridine analogues are known to have lower calcium channel-blocking activity and higher lipid solubility than their dihydropyridine analogue counterparts. Thus we synthesized four pyridine analogues and their dihydropyridine analogue counterparts and investigated their MDR-reversing activity.

MATERIALS AND METHODS

Chemicals. Dihydropyridine and pyridine analogues were newly synthesized by Nissan Chemical Ind. Co., Ltd. (Chiba, Japan). The structures and purities of these analogues were determined using the following procedures. Melting points were determined on a Yanako micro-melting point apparatus. 'H NMR spectra in CDCI.> solution were

recorded on a Joel PMX60S1 spectrometer and chemical shifts weregiven in ppm with tetramethylsilane as an internal standard. Massspectra were obtained on a Joel JMS D-300 instrument. Columnchromatography was carried out on a Merck Kieselgel 60 (70-230 meshASTM).

For PAK-101, 2-[methyl(phenylmethyl)amino]ethyl 3-aminocroton-ate (1.24 g) was added to a solution of diethyl-l-acetyl-2-(3-trifluoro-methylphenyl)ethenylphosphonate (1.75 g) in toluene (30 ml). Thereaction mixture was heated to reflux for 8 h with azeotropic removalof water. The solvent was evaporated in a vacuum and the residue wassubjected to column chromatography (ethyl acetatc:ethanol, 5:1) to give1.20 g (41%) of pure PAK-101 as pale yellow oil: 'H NMR (CDC1.,)0.96 (t. J = 7.0 Hz, 3H). 1.20 (t, J = 7.0 Hz, 3H), 2.18 (s, 3H), 2.26(d, J = 2.2 Hz, 3H). 2.28 (s, 3H), 2.62 (t, J = 6.0 Hz. 2H), 3.47 (s,

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REVERSAL OF DRUG RESISTANCE BY PYRIDINE ANALOGUES

2H), 3.50-4.30 (m, 6H), 4.77 (d, J = 11.0 Hz. IH), 7.23 (s, 5H), 7.10-7.60 (m, 4H); MS (El), ml e 580 (m+, 6), 432 (5), 288 (8), 147 (100).

For PAK-101P, PAK-101 (300 mg) was added to a solution of 60%nitric acid (1 ml) in water (7 ml). The reaction mixture was allowed toreflux for 1 h. After cooling, the reaction mixture was neutralized withsaturated NaHCO, aqueous solution and then extracted with CH2C12(30 ml). The organic extract was dried over Na2SO4 and was evaporatedin a vacuum. The residue was subjected to column chromatography(ethyl acetate:ethanol, 9:1) to give 150 mg (53%) of pure PAK-101P asyellow oil: 'H NMR (CDCI,) 1.09 (t, J = 6.5 Hz, 3H), 1.15 (t. J = 6.5Hz, 3H), 2.10 (s, 3H), 2.30 (t, J = 6.0 Hz, 2H), 2.55 (s, 3H), 2.91 (d,J = 2.2 Hz, 3H), 3.40 (s, 2H), 3.50-4.20 (m, 6H), 7.19 (s, 5H), 7.10-7.70 (m,4H).

For PAK-102. 2-[methyl(phenylmethyl)amino]ethyl 3-aminocroton-ate (1.0 g) was added to a solution of 3-(4-methyl-l,3,2-dioxaphos-phorinan-2-yl)-4-(2-chlorophenyl)-3-buten-2-one P-oxide (1.05 g) intoluene (20 ml). The reaction mixture was allowed to reflux for 17 h.After concentration of the reaction mixture in a vacuum, the residuewas subjected to column chromatography (ethyl acetate:ethanol. 9:1)to give 1.50 g (83%) of PAK-102 as yellow oil: 'H NMR (CDC1.,)0.90-2.00 (m, 5H), 2.18-2.41 (m, 9H), 2.65 (t, J = 6.5 Hz, 2H), 3.48 (s,2H), 4.18 (t, J = 6.5 Hz, 2H), 4.00-4.90 (m, 3H), 5.05-5.31 (m, 1H),7.24 (s, 5H), 6.80-7.60 (m, 4H).

For PAK-102P, chromium trioxide(IOO mg) was added to a solutionof PAK-102 (550 mg) in acetic acid (2 ml) and the solution was allowedto reflux for 3 h. Then saturated NaHCO, (20 ml) aqueous solutionwas added to the reaction mixture and the product was extracted withethyl acetate (50 ml). The organic extract was dried over Na2SO4 andwas evaporated in a vacuum. The residue was subjected to columnchromatography (ethyl acetate:ethanol. 9:1) to give 400 mg (73%) ofpure PAK-102P as pale yellow oil: 'H NMR (CDCI.,) 0.95-1.80 (m,5H), 2.15 (s, 3H), 2.35 (t. J = 6.5 Hz, 2H), 2.59 (s, 3H), 3.00 (d, J =2.2 Hz, 3H), 3.44 (s, 2H), 3.96 (t, J = 6.5 Hz, 2H), 4.00-4.90 (m, 3H),7.25 (s, 5H), 7.13-7.45 (m, 4H).

For PAK-103, 2-(formylphenylamino)ethyl 3-aminocrotonate (5.0 g)was added to a solution of 3-(5,5-dimethyl-1,3,2-dioxaphosphorinan-2-yl)-4-(3-nitrophenyl)-3-buten-2-one P-oxide (7.0 g) in toluene (50 ml)and heated for 5 h. After cooling, the yellow precipitate was collectedby filtration to give 9.0 g (78%) of 2-(formylphenylamino)ethyl 5-(5,5-dimethyl-l,3,2-dioxaphosphorinan-2-yl)-2,6-dimethyl-4-(3-nitro-phenyl)-l,4-dihydro-3-pyridinecarboxylate P-oxide as yellow crystals.They (1.0 g) were dissolved in methanol (4 ml)-ethanol (4 ml) solutionwith p-toluenesulfonic acid (500 mg) and the solution was heated toreflux for 6.5 h. The reaction mixture was concentrated in a vacuum,and the residue from ethyl acetate was dissolved in ethyl acetate (40ml) and washed with saturated NaHCO.i aqueous solution (40 ml). Theorganic extract was dried over Na2SO4 and evaporated in a vacuum.Recrystallization of the residue gave 600 mg (63%) of pure PAK-103as yellow crystals (m.p. 193-194°C): 'H NMR (CDCI,) 0.90 (s, 3H),

1.01 (s, 3H), 2.28 (s. 3H). 2.30 (d, J = 2.5 Hz, 3H), 3.20-4.40 (m, 8H),4.89 (d, J = 11.0 Hz, 1H). 6.40-8.10 (m, 9H).

For PAK-103P, a solution of 2-(formylphenylamino)ethyl-5-(5,5-dimethyl-l,3,2-dioxaphosphorinan-2-yl)-2,6-dimethyl-4-(3-nitro-phenyl)-l,4-dihydro-3-pyridinecarboxylate P-oxide (5.6 g) and chromium trioxide (2.0 g) in acetic acid (20 ml) was heated to reflux for 30min. Saturated NaHCO.i aqueous solution (100 ml) was added to thereaction mixture and extracted with ethyl acetate (50 ml in 2 additions).The organic extract was evaporated in a vacuum and the residue wasdissolved in methanol (10 ml)-ethanol (10 ml) solution with/Moluene-sulfonic acid (2.0 g). The solution was heated to reflux for 7 h. Aftercooling, ethyl acetate (50 ml) was added to the reaction mixture andthe mixture was washed with saturated NaHCO, aqueous solution (50ml). The organic extract was dried over Na2SO4 and evaporated in avacuum. Recrystallization of the residue from ethyl acetate/diethylether gave 3.0 g (57%) of pure PAK-103P as yellow crystals (m.p. 105-106°C):'H NMR (CDCI,) 0.76 (s,3H), 1.09 (s,3H), 2.60 (s, 3H), 2.87(d, J = 2.5 Hz, 3H), 3.00-4.30 (m, 8H), 6.30-8.30 (m, 9H).

For PAK-104, 2-[4-(diphenylmethyl)-l-piperazinyl]ethyl 3-aminocrotonate (5.6 g) was added to the solution of 3-(r/-a/w-4,6-dimethyl-l,3,2-dioxaphosphorinan-2-yl)-4-(3-nitrophenyl)-3-buten-2-one P-ox

ide (5.0 g) in toluene (100 ml). After heating to reflux for 4 h, thesolvent was removed in a vacuum. The residue was subjected to columnchromatography (ethyl acetate:ethanol, 9:1) to give 6.8 g (66%) of purePAK-104 as yellow oil: 'H NMR (CDCI,) 0.94-1.54 (m, 6H), 1.74 (t,J = 5.4 Hz, 2H), 2.26 (s, 3H), 2.28 (d, J = 2.5 Hz, 3H), 2.10-2.75 (m,8H). 2.61 (t, J = 6.0 Hz, 2H), 4.16 (t, J = 6.0 Hz, 2H), 4.19 (s, 1H),4.80 (d, J = 11.0 Hz, IH), 4.00-4.95 (m, 2H), 7.00-8.10 (m, 14H);MS(EI), mje 700 (M*, 5), 683 (41), 167 (100), 68 (93).

For PAK-104P, 36% nitric acid (10 ml) was added to a solution ofPAK-104 (6.8 g) in acetic acid (20 ml). After stirring for 30 min atroom temperature, saturated NaHCO, aqueous solution (100 ml) wasadded to the reaction mixture, followed by extraction with ethyl acetate(100 ml in 2 additions). The organic extract was dried over Na2SO4and evaporated in a vacuum. The residue was subjected to columnchromatography (ethyl acetate:ethanol, 19:1) to give 6.0 g (88%) ofpure PAK-104P as yellow oil: 'H NMR (CDCI,) 0.97-1.36 (m, 6H),1.40-1.80 (m, 2H), 2.00-2.60 (m, 10H), 2.59 (s, 3H), 2.95 (s, 3H),3.90-4.10 (m, 3H), 4.19 (s, IH), 4.60-4.80 (m, 1H), 7.10-8.30 (m,14H); MS(FD), m/e 699 (M+ + 1, 10), 485 (4), 354 (20), 157 (100).

['H]VBL (17 Ci/mmol) and ('H)azidopine (40 Ci/mmol) were pur

chased from Amersham Corp., Arlington. Heights, IL. VCR, VBL,ADR, DAU, MTT, and verapamil were obtained from Sigma ChemicalCo., St. Louis. MO. Cepharanthine was kindly donated to us by KakenShoyaku Co., Ltd.. Osaka, Japan.

Cell Culture and Cell Lines. Human epidermal KB carcinoma cellswere obtained from Dr. Gottesman (National Cancer Institute, Be-thesda, MD). The multidrug-resistant mutants KB-8-5 and KB-C2 wereselected from KB cells with increasing concentrations of colchicine andmaintained as described previously (3).

Cell Survival by MTT Assay. MTT colorimetrie assay performed ina 96-well plate was used for an in vitro chemosensitivity test (26). The

assay is dependent on the reduction of MTT by the mitochondrialdehydrogenase of viable cells to a blue formazan product which can bemeasured spectrophotometrically. Equal numbers of cells (2000 for KB.3000 for KB-8-5, and 5000 for KB-C2) were inoculated into each wellwith 0.18 ml of culture medium. After overnight incubation (37°C,5%

CO2), 20 fi\ of vincristine solution and 0.5 ^1 of sample solution wereadded and incubated for 4 days. Then 50 ¿Jof MTT (1.1 mg/ml PBS)were added to each well and incubated for a further 4 h. The resultingformazan was dissolved with 100 /jl of dimethyl sulfoxide after aspiration of the culture medium. Plates were placed on a plate shaker for 5min and read immediately at 570 nm.

Determination of Calcium Antagonistic Activities. The calcium-antagonistic activity of each compound was determined by the ability of thecompound to inhibit the contraction of the taenia caecum from a guineapig in the presence of calcium (25).

A taenia caecum was excised from a male Hartley guinea pig (300-400 g) and suspended in an organ bath containing physiological saltsolution (135 m,\t NaCl, 5 mM KC1, 2 m\i CaCl2, 1 mM MgCI2 15 mMNaHCO,, and 5.5 mM glucose) aerated with 95% O2 and 5% CO2 at37°C.The taenia caecum was then stretched to an initial tension of 1

g and isometric contraction was measured with a force displacementtransducer. After 30 min equilibration, the bathing solution was replaced by a Ca2*-free, high K* (100 mM KCI, 40 mM NaCl) solution.

The taenia caecum relaxed completely within 30 min. Then CaCI2 (10mM) was added. After further equilibration (30 min), the test compoundsolution (10~'°-10~5M) was added cumulatively. The result was ex

pressed as a minus logarithm of the dose required for 50% of themaximum relaxation produced by 10~4M papaverine.

Calcium Current Measurement in Guinea Pig Ventricular Myocytes.Single ventricular myocytes were prepared as described by Taniguchiet al. (27). Cells were superfused with modified Tyrode's solution with

the following composition (mM): NaCl 137, KCI 5.4, CaCI2 1.8, MgCl20.5, NaHCO, 3, NaH,PO4 0.16, glucose 5.5, and yV-2-hydroxyethylpi-perazine-fY'-2-ethanesulfonic acid 5 (pH was adjusted to 7.4 by adding

NaOH). Glass electrodes were filled with the solution which contained(mM): KCI 140. MgCl2 2, CaCl2 1, ethyleneglycol bis (fi-aminoethylether)-Ar,A',Ar,A''-tetraaceticacid 11. A'-2-hydroxyethylpiperazine-A''-

2-ethanesulfonic acid 5, creatine phosphate 5, ATP-Na2 2 (pH wasadjusted to 7.2 by adding KOH). Whole cell voltage clamp studies were

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REVERSAL OF DRUG RESISTANCE BY PYR1DINE ANALOGUES

conducted as described by Marty and Neher (28). Compounds werefirst dissolved in dimethyl sulfoxide at a concentration of 10"' M andthen diluted in Tyrode's solution to make a desired final concentration.Experiments were done at 33°C.

Membrane Vesicle Preparation. Membrane vesicles from KB-C2 cellswere prepared as described (29) from cells grown in 24- x 24-cm dishes(GIBCO) under standard growth conditions (3). Protein concentrationswere determined by the method of Bradford (30).

Photoaffinity Labeling. Membrane vesicles were incubated with 0.75MM[3H]azidopine for 15 min at room temperature in the presence and

absence of various drugs. After continuous irradiation at 366 nm for20 min at 25°C,samples were solubilized in a sodium dodecyl sulfate

sample buffer as described by Debenham et al. (31).Sodium Dodecyl Sulfate Gel Electrophoresis. Samples labeled with

['Hjazidopine were analyzed by electrophoresis using a modification of

the system described by Debenham et al. (31) on a 5% polyacrylamide/4.5 M urea gel. pH 7.6, without a stacking gel as described previously(25). Proteins were stained with 0.25% Coomassie blue in 50% (w/v)trichloroacetic acid.

Drug Accumulation and Efflux. For the study of drug accumulation,confluent monolayers of KB and KB-C2 cells in a 24-well plate wereincubated with 1 ^M ['H]VBL in the absence and presence of indicatedconcentrations of agents for l h at 37°C.After washing with ice-cold

PBS three times, the cells were solubilized in 0.2% Triton X-100 in 10miviphosphate buffer (pH 7.4), harvested, and then counted.

For efflux study, the confluent cells were incubated with 1 /JM 1'H]VBL in the presence and absence of 10 ^M agents for l h at 37°C.After

two washings with ice-cold PBS, the cells were further incubated in themedium with and without 10 ^M agents. At the indicated times thecells were harvested and counted as described above.

PAK -101 PAK-101 P

CHjCHzO

CH3CH20

SCF3

cooc

ru.

COOCH2CH2N<,CH3

H3C f¡ CH3

PAK-102

CH2-O

•HCl

PAK-103

>P^JyCOOCHzCHzNH-Q

1 MCI

PAK-104

/N02

CH3CH2O^VCOOCH m N<CH3CH3CH2°rX ' CH2~OH3C N CH3 .2HCI

PAK-102 P

:OOCH2CH2N' „CH2-O

2HCI

COOCH2CH2NH

H3C 2HCI

PAK-104 P

yN02

COOCH2CH2N N-CHJOX)

H3C H CH3 2HC| H3C CH3 .3HC|

Fig. 1. Chemical structures of 1,4-dihydropyridine and pyridine analogues.

RESULTS

Reversing of Drug Resistance in KB-C2 Cells by Pyridine andDihydropyridine Analogues. We synthesized 4 pyridine analogues and their dihydropyridine analogue counterparts andexamined their ability to reverse drug resistance in KB-C2 cells.The chemical structures of these newly synthesized compoundsare shown in Fig. 1. PAK-101, PAK-102, PAK-103, and PAK-104 are dihydropyridine analogues, and PAK-101P, PAK-102P, PAK-103P. and PAK-104P are their pyridine analoguecounterparts.

We examined the cytotoxic effect of the analogues on KB-C2 cells in the presence and absence of 22 n\i VCR by theMTT method. PAK-101, PAK-101P, PAK-102, PAK-Õ02P.PAK-104, and PAK-104P at 20 MMand PAK-103 and PAK-103P at 10 MMhad no cytotoxic effect on KB-C2 cells (Fig. 2).

VCR at 22 nivi had no cytotoxic effect on KB-C2 cells (datanot shown) but increased the sensitivity of KB-C2 to PAK-101,PAK-101P, PAK-102, PAK-102P, PAK-103P, PAK-104, andPAK-104P about 4.1, 4.8, 2.3, 2.1, <1.7, 5.3, 5.0, and 20 times,respectively. VCR at the concentration did not increase thecytotoxic effect of PAK-103 on KB-C2. PAK-104P reversedthe resistance to 22 nivi VCR in KB-C2 cells at about 1/20 ofthe concentration required to kill the cells (50% growth inhibition concentration of PAK-104P without VCR/50% growthinhibition concentration of PAK-104P with VCR = 20.0).

Next, the dose-response curves of various anticancer agentswith and without the compounds were assayed by the MTTmethod. Table 1 summarizes the data from the curves. PAK-101 at 5 MMmoderately reversed the resistance to VCR, VBL,ADR, and DAU in KB-C2 cells. PAK-102 at 10 //M moderatelyreversed the resistance to VCR and VBL and completely reversed the resistance to ADR and DAU. PAK-102 at 5 MMhadlittle effect on drug resistance (data not shown). The pyridineanalogue counterparts of these two dihydropyridine analogueshad effects similar to the dihydropyridine analogues.

E 100

; so

2 5 10 20 40 1 2 5 10 20 40

Concentration I ^jM )

Fig. 2. Sensitivity of KB-C2 cells to the dihydropyridine and pyridine analogues in the presence and absence of 22 nM VCR. A. effect of PAK-101 (O).PAK-101P (D), PAK-102 (•).and PAK-102P (•)on cell survival of KB-C2 inthe absence ( ) or presence ( ) of 22 nM VCR. B. effect of PAK-103 (O).PAK-103P (D). PAK-104 (•).and PAK-104P (•)on cell survival of KB-C2 inthe absence ( ) or presence ( ) of 22 nM VCR. Each point represents themean of 2 experiments.

Table I Effects of dihydropyridine and pyridine analogues on drug resistance ofKB-C2 cells

Each value represents the mean of duplicate determinations. KB 50% growthinhibition concentrations: VCR, 3.3 nM; VBL. 11.0 nM; ADR, 51.7 nM; DAU,93.1 n\i.

ChemicalsRelative resistance to

CelllineKBKBC2(UM)PAK-101PAK-10IPPAK-102PAK-102PPAK-

103PAK-103PPAK-104PAK-104P00551010101055VCR1.0939.449.418.519.115.5272.72.415.2<1.5VBL1.0100.09.45.55.17.427.40.516.20.7ADR1.0122.59.63.11.33.738.02.71.40.7DAU1.0425.13.51.31.04.575.21.172.00.9

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The effect of PAK-103 on drug resistance was nil at 5 MM(data not shown) and weak at 10 MM.PAK-104 at 5 MMalmostcompletely reversed the resistance to ADR and moderatelyreversed the resistance to VCR, VBL, and DAU. The pyridineanalogues, PAK-103Pand PAK-104P, had a more potent effecton drug resistance than their dihydropyridine analogue counterparts. PAK-103Pat 10 MMcompletely reversed the resistanceto VBL and DAU and moderately reversed the resistance toVCR and ADR. PAK-104P completely reversed the resistanceto all agents tested and was the most potent MDR-reversingagent among the tested compounds.

Calcium Antagonism. The calcium-antagonistic activities ofthe 4 dihydropyridine analogues and their pyridine analoguecounterparts were measured (Table 2). The antagonistic activities of all the pyridine analogues were considerably lower thanthose of their dihydropyridine analogue counterparts. PAK-103was the most active agent among them, and PAK-101P was theweakest antagonist. No correlation was observed between thecalcium-antagonistic activity of the analogues and the reversingof drug resistance.

Effect of PAK-103, PAK-103P, PAK-104, and PAK-104P onCalcium Current of Guinea Pig Ventricular Cells. We comparedthe potencies of 4 agents (PAK-103, PAK-103P, PAK-104, andPAK-104P) to block the calcium current of guinea pig ventricular myocytes using a whole-cell voltage clamp technique. Inthe absence of agents (controls in Fig. 3), inward calciumcurrents (transient downward deflections) with amplitudesranging from 0.5 to 1.5 nA were recorded. PAK-103 and PAK-104, at a concentration of 1 MM, completely abolished thecalcium current (the right current traces in Fig. 3, A and C)with remaining time-independent outward current. On the con-

Table2 Calciumantagonisticactivityofdihydropyridineandpyridineanalogues

ChemicalsPAK-101PAK-101PPAK-

102PAK-I02PPAK-103PAK-103PPAK-104PAK-104PCa2*

antagonism

(pIC»)7.495.918.495.978.646.317.536.27

20ms

Fig. 3. Effects of PAK-103. PAK-103P, PAK-104. and PAK-104P at a concentration of 1 JIMon calcium current of guinea pig ventricular myocytes. Stepdepolarizations (500 ms duration) to 0 mV from a holding potential of—40 mVevoked transient inward calcium current (left current trace of each panel). Horizontal line, zero current level; upward deflection, outward current: downwarddeflection, inward current. PAK-103 and PAK-104 (right current traces in A andC, respectively) completely abolished calcium current, while PAK-103P and PAK-104P (right current traces in B and D, respectively) decreased the calcium currentonly slightly.

trary, PAK-103P and PAK-104P at the same concentrationdecreased the calcium current only slightly (the right currenttraces in Fig. 3, B and D).

These results suggest that the potencies of calcium channel-blocking activity of PAK-103P and PAK-104P is much less orvirtually nil, as compared to those of PAK-103 and PAK-104.Similar results were observed for PAK-101P and PAK-102P(data not shown).

Comparison of the Ability of PAK-104P to Reverse MDR withThat of Other Reversing Agents. The pyridine analogue, PAK-104P, was the most effective MDR-reversing agent and had anexceedingly low calcium channel-blocking activity in this study.We compared the ability of this agent to reverse MDR in KB-8-5 cells with that of other reversing agents, verapamil, cephar-anthine, nimodipine, and nicardipine. Since drug resistance intumors in patients is usually at a low level, we used a KB-8-5cell line because of its low resistance to drugs. Fig. 4 showsMTT assays to test the reversing effects of PAK-104P and otheragents on resistance to VCR in KB-8-5 cells. KB-8-5 showedabout 40 times greater resistance to VCR than KB. PAK-104Pat l /¿Mcompletely reversed the resistance in KB-8-5. Thesensitivity of KB to VCR was also increased 3 times by 1 MMPAK-104P. None of the other MDR-reversing agents at 1 MMcould completely reverse the resistance in KB-8-5. Cepharan-thine, verapamil, nimodipine, and nicardipine increased thesensitivity of KB-8-5 to VCR 12, 4.3, 2.3, and 2.7 times,respectively. Cepharanthine and verapamil at 1 MMincreasedthe sensitivity of KB to VCR only 1.5 times. Thus we observedthat PAK-104P had the most potent ability to reverse MDR invitro among MDR-reversing agents tested.

Effect of Agents on Cellular Accumulation of | '111\ mlihisiinc.

To study how PAK-104P reverses MDR, we examined its effecton the accumulation of VBL in KB and KB-C2 cells andcompared this with the effect of other MDR-reversing agents(Fig. 5). The intracellular level of VBL in KB-C2 cells was one-tenth ofthat in KB cells. The addition of PAK-104P at 2.5 MMenhanced the accumulation of VBL in KB-C2 cells 12-fold tothe level ofthat in KB cells without the pyridine. PAK-104 hadlittle effect at 2.5 MMon the accumulation of VBL in KB-C2

cells.

100

¿50D2

0.1 1Vincristine

100

Fig. 4. Effect of PAK-104P and other MDR-reversing agents on resistance tovincristine in KB-8-5 cells. The effect of verapamil (• •).cepharanthine(A A), nimodipine (D D), nicardipine (A A), and PAK-104P(• •)at 1 /¿Mon the resistance to vincristine in KB-8-5 cells (x x) wasexamined by MTT assay. The effect of some agents at I JIM on sensitivity tovincristine of KB cells (x x) was also examined; verapamil (• •),cepharanthine (A A). PAK-104P (• •).Points represent the mean of duplicatedeterminations.

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REVERSAL OF DRUG RESISTANCE BY PYRIDINE ANALOGUES

20 0 S 10 20

Concentration ( ^jM)

Fig. 5. Effect of agents on the accumulation vinblastine in KB and KB-C2cells. Effect of agents on accumulation of vinblastine in KB cells (A) and KB-C2cells (B) at the indicated concentrations; PAK-101 (O). PAK-lOlP(fí), PAK-104(D), PAK-104P (•),verapamil (A), cepharanthine (A). Points represent the meanof duplicate determinations.

At 2.5 MM,verapamil, cepharanthine, PAK-101, and PAK-101P enhanced the cellular accumulation of VBL in KB-C2cells by factors of 5.1, 4.0, 2.6, and 2.0. respectively. Accumulation of VBL in KB-C2 cells plateaued at higher concentrationsthan 2.5 MMfor PAK-104P and 10 MMfor cepharanthine andverapamil.

The effect of PAK-104P on the accumulation of VBL in KB-C2 cells was greater than the effect of cepharanthine andverapamil at any concentrations tested. The accumulation ofVBL in KB-C2 cells increased with increasing concentrationsof PAK-101 P, PAK-101, and PAK-104 added to the mediumuntil 20 MM, and above 20 MMthese 3 agents enhanced theaccumulation of VBL more effectively than PAK-104P. In thesensitive KB cells, the accumulation of VBL was enhanced upto 1.4 times by PAK-104P, 1.8 times by PAK-101 and PAK101P, and 1.3 times by verapamil. Cepharanthine had no enhancing effect.

Effect of PAK-104P on Efflux of VBL. We examined whetherincreased accumulation of anticancer agents in KB-C2 cells byPAK-104P was due to the inhibition of drug efflux. Afterincubation of the cells for 3 min in the absence of PAK-104P,about 60% of VBL was lost from KB-C2 cells, whereas about70% of VBL was retained in KB cells. Addition of 10 MMPAK-104P to the culture medium completely terminated this effluxof VBL from KB-C2 cells at 1 min and thereafter. PAK-104Pdid not affect the efflux of VBL from KB cells (Fig. 6).

Effect of Agents on Photoaffinity Labeling of P-glycoproteinby [3H]Azidopine. The radioactive photoactive dihydropyridinecalcium channel blocker, [3H]azidopine, photolabeled P-glycoprotein in membrane vesicles from KB-C2 cells (25). Thisphotolabeling was almost completely inhibited by dihydropyridine analogues that reversed drug resistance but was not significantly inhibited by analogues that do not reverse resistance(25).

Since the reversing of the drug resistance by dihydropyridineanalogues seems to be correlated with the inhibition of the [3H]-azidopine photolabeling of P-glycoprotein by dihydropyridineanalogues, we studied the effect of newly synthesized dihydropyridine analogues and their pyridine analogue counterparts onthe labeling. The effect of verapamil and cepharanthine on thelabeling was also investigated and compared with that of newlysynthesized agents (Fig. 7).

Two pyridine analogues, PAK-103P and PAK-104P, that

15 5 10 15

Time ( min )

Fig. 6. Effect of P/K-104P on the extracellular transport of vinblastine.Release of vinblastine in the absence (O) and presence (A) of PAK-104P from KB(A) and KB-C2 (B) is shown. Points represent the mean of duplicate determinations.

10 11

KOa

200 -

116 -

97 -

66 -

Density 100 36.7 23.3 56.7 36.7 40.0 6.7 10.0 3.3 43.3 23.31% of control)

Fig. 7. Inhibition of |'H]azidopine labeling of P-glycoprotein in membranevesicles from KB-C2 cells. KB-C2 vesicles were incubated with [3H]azidopine inthe absence and presence of agents at 5 ^M; Lane I, no agent; Lane 2, PAK-101;Lane 3. PAK- 101P; Lane 4. PAK-102: Lane 5. PAK-102P; Lane 6, PAK-103;Lane 7. PAK-103P; Lane 8. PAK-104; Lane 9, PAK-104P; Lane 10. verapamil;Lane II, cepharanthine. The density of the bands on the autoradiogram wasdetermined and the data are expressed as a percentage of the density of thephotolabeling of P-glycoprotein in the absence of the compounds under each lane.kDA. molecular weight in thousands.

had a greater ability to reverse drug resistance than their dihydropyridine analogue counterparts were more potent inhibitorsof the photolabeling than their counterpart dihydropyridineanalogues, PAK-103 and PAK-104. PAK-104P, the most potent MDR-reversing agent among the tested compounds, had

the greatest ability to inhibit photolabeling.The MDR-reversing ability of the compounds seems to be

correlated with their binding activity to P-glycoprotein exceptPAK-102. PAK-102 was the weakest inhibitor of the photo-labeling among the used compounds, but it had greater abilityto reverse drug resistance than PAK-103.

DISCUSSION

The focus of our research has been to find potent MDR-reversing agents which have low calcium channel-blocking activity (18, 25, 32). In this study all the newly synthesizedpyridine analogues showed more than 1 order of magnitudelower calcium-antagonistic activity than their dihydropyridineanalogue counterparts.

Further, the ability of two pyridines, PAK-103P and PAK-

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REVERSAL OF DRUG RESISTANCE BY PYRIDINE ANALOGUES

104P, to reverse MDR was greater than that of their dihydro-pyridine analogue counterparts. PAK-104P had the greatestability to reverse MDR among agents used in this study. Thereversing ability of PAK-104P was about 10 times that ofverapamil, and the calcium-antagonistic activity of the pyridineanalogue was about 50% ofthat of verapamil.

PAK-104P at 5 x 10~6 and 10~6 M completely reversed

resistance to VCR in strongly resistant KB-C2 and weaklyresistant KB-8-5 cells, respectively. KB-C2 is 939 times andKB-8-5 is 40 times as resistant to VCR as drug-sensitive KB.

Since the tumor cells in patients are expected to have a relativelylow degree of resistance, there seems to be a good prospect thatwe will be able to reverse drug resistance in such tumors atlower doses of PAK-104P.

PAK-104P also enhanced the sensitivity of KB cells to vin-cristine by a factor of 3. The pyridine analogue increased theaccumulation of vinblastine in KB cells. Since we could notdetect any expression of P-glycoprotein in KB cells with Western blotting analysis, we assume that molecules other than P-glycoprotein are involved in these phenomena.

It is known that a photoaffinity analogue of vinblastine labelsP-glycoprotein (33, 34) and the photolabeling was inhibited bymost of the agents that reverse MDR (22). Safa et al. (35)reported that a dihydropyridine analogue, azidopine, photola-beled P-glycoprotein and that this labeling was inhibited byvinblastine and actinomycin D. A photoanalogue ofother MDR-reversing agents, verapamil (36) and SDB-ethylenediamine4 (submitted) was also shown to label P-glyco

protein. These results indicate that hydrophobic anticanceragents that are involved in MDR bind to P-glycoprotein andthat the binding site of these agents may be identical with thatof MDR-reversing agents, verapamil, SDB-ethylenediamine,

and azidopine.Recently, we suggested a role for P-glycoprotein in the re

versing of resistance by dihydropyridine analogues by demonstrating the correlation between the reversing of drug resistanceand the inhibition of [JH]azidopine photolabeling of P-glyco

protein by dihydropyridine analogues (25). Now it seems thatpotent MDR-reversing agents have a high affinity for P-glycoprotein. Two pyridine analogues, PAK-103P and PAK-104P,had a greater ability to inhibit ['H]azidopine photolabeling ofP-glycoprotein than their dihydropyridine analogue counterparts. PAK-104P was the most potent inhibitor in this study,suggesting that it has a higher affinity to P-glycoprotein thanthe other agents. Since [3H]azidopine labeling was inhibited byPAK-104P and since PAK-104P inhibited the efflux of VBLfrom KB-C2 cells and increased the accumulation of VBL, weconclude that PAK-104P binds to the same site on P-glycoprotein as that where the hydrophobic anticancer agent vinblastinebinds. Inhibition of photoaffinity labeling correlated well withMDR-reversing activity of each of the compounds used in thisstudy except PAK-102. MDR-reversing activity of PAK-102was similar to that of PAK-102P and higher than that of PAK-103, but the PAK-102 was the weakest inhibitor of the labelingamong them. PAK-102 may need to be metabolized by a cellbefore functioning as a blocker of P-glycoprotein.

Zamora et al. (19) and Ford et al. (20) reported that hydro-phobicity seems to be one of the common features of MDR-reversing agents. The pyridine analogues used in this study aremore hydrophobic than their dihydropyridine counterparts.Increased hydrophobicity of the pyridines may be correlatedwith their increased MDR-reversing activity and their binding

4S-i. Akiyama el al., submitted for publication.

to P-glycoprotein. However, the increased hydrophobicity aloneseems not to be sufficient. Other features of two pyridines,PAK-103P and PAK-104P, must also be concerned with theirincreased MDR-reversing activity, because the reversing activityof PAK-101P and PAK-102P was similar to that of theirdihydropyridine analogue counterparts. More precise studieswith more analogues are needed to formulate a hypothesis aboutstructure-activity relationships of these new compounds.

This is the first report that has shown the high ability of twopyridine analogues to reverse MDR. We may be able to findeven more potent MDR-reversing agents by further screeningpyridine analogues.

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1990;50:3055-3061. Cancer Res   Norimasa Shudo, Tetsuro Mizoguchi, Tatsuto Kiyosue, et al.   Activity Than Their Dihydropyridine Counterparts

BlockingMultidrug Resistance and with Lower Calcium Channel Two Pyridine Analogues with More Effective Ability to Reverse

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