Novel multidrug resistance reversal agents

Article abstract of DOI:10.1021/jm9804477

A series of 59 α-aryl-α-thioether-alkyl, -alkanenitrile, and - alkanecarboxylic acid methyl ester tetrahydroisoquinoline and isoindoline derivatives (15a-48) were synthesized and evaluated as multidrug resistance (MDR) reversal agents. The compounds were

Full text of DOI:10.1021/jm9804477

J . Med. Chem. 1999, 42, 2145-2161
2145
Novel Mu ltid r u g Resista n ce Rever sa l Agen ts
Dan Berger,* Ron Citarella, Minu Dutia, Lee Greenberger, William Hallett, Rolf Paul, and Dennis Powell
Oncology/ Immunology/ Inflammatory Diseases and Chemical Sciences Sections, Wyeth-Ayerst Research,
Pearl River, New York 10965
Received J uly 31, 1998
A series of 59 R-aryl-R-thioether-alkyl, -alkanenitrile, and -alkanecarboxylic acid methyl ester
tetrahydroisoquinoline and isoindoline derivatives (15a -48) were synthesized and evaluated
as multidrug resistance (MDR) reversal agents. The compounds were tested on S1-B1-20 human
colon carcinoma cells selected for resistance to bisantrene. Both the cytotoxicity of the reversal
agents and their ability to resensitize the cells to bisantrene were determined. All but two of
these compounds (15q, 40) were more effective MDR reversal agents in vitro than verapamil
(VRP), a calcium channel antagonist which also has been shown to possess MDR modulating
activity. Several showed good activity in this assay (IC50’s < 0.5 µM), the most potent being
isoindolines 44 (IC50 0.26 µM) and 46 (IC50 0.26 µM) and tetrahydroisoquinolines 47 (IC50 0.29
µM) and 15m (IC50 0.30 µM). A number of compounds were evaluated in vivo against vincristine
(VCR)-resistant murine P388 leukemia, as well as against human epidermoid carcinoma KB/
8.5 implanted sc in athymic mice. The reversal agents which consistently showed the highest
activity, together with low toxicity, were R-aryl-R-thiotolylalkanenitrile tetrahydroisoquinoline
derivatives with electron-rich alkoxy substituents on the aromatic rings. Of the tested
compounds, the most effective reversal agents for both tumor lines were 15h (33% increased
life span at 12.5 mg/kg, 0.2 mg/kg VCR versus VCR alone in the VCR-resistant P388 leukemia
model and 59% relative tumor growth at 50 mg/kg, 8 mg/kg doxorubicin versus doxorubicin
alone in the KB/8.5 model) and 39a (48% increased life span at 50 mg/kg, 0.2 mg/kg VCR
versus VCR alone in the VCR-resistant P388 leukemia model and 46% relative tumor growth
at 25 mg/kg, 8 mg/kg doxorubicin versus doxorubicin alone in the KB/8.5 model). The mechanism
of action of these compounds is believed to involve blocking the drug efflux pump, P-glycoprotein.
Ch a r t 1
In tr od u ction
Drug resistance is believed to be the single most
important reason for the failure of cancer chemo-
therapy.1 Certain tumors are unresponsive to chemo-
therapy from the outset, through inherent resistance.
However, in many cases this occurs after a period of
responsiveness to chemotherapy, through the process
of acquired resistance. In tissue culture, several cellular
mechanisms are responsible for mediating acquired
resistance.2 Tumor cells selected for resistance to one
drug generally show cross-resistance to a number of
functionally and structurally diverse hydrophobic drugs,
including taxol, vinblastine, adriamycin, mitoxantrone,
etoposide, and bisantrene. This is known as multidrug
resistance (MDR).3 Tumor cells which possess the MDR
phenotype overexpress an energy-dependent drug efflux
pump, P-glycoprotein (P-gp),4 which lowers the intrac-
ellular concentration of cytotoxic agents by pumping
them from the cell. While cells in culture can display
drug resistance up to 1000-fold greater than a sensitive
phenotype, drug-resistant tumors isolated from patients
generally exhibit only a 2-10-fold increase in drug
resistance. Thus, relatively low levels of P-gp in tumors
are believed to contribute to the refractory behavior of
tumors in clinical oncology.5
cium channel antagonists such as verapamil (VRP),6
dihydropyridines,7,8 pyridine analogues,9 vinca alkaloid
analogues,10 antiarrhythmic agents (quinidine),11 im-
munosuppressants (cyclosporin A),12 phenoxazines,13
calmodulin antagonists (trifluoroperazine),14 triazines
(S-9788),15 acridone carboxamides (GF-120918),16 and
pipecolinates (VX-710).17 Several groups have synthe-
sized analogues of dihydropyridines,18 verapamil,19,20
and cyclosporin A21 to optimize their MDR reversal
activity. Clinical trials with several of these compounds
(most of which have been carried out with verapamil,
cyclosporin A, and structurally related analogues) in
conjunction with chemotherapeutic agents have dem-
onstrated some benefit, although the effects have been
modest and thus not universally accepted as useful.22
We used verapamil (Chart 1) as the structural basis
for the program. Our intent was to identify compounds
Studies have demonstrated that a variety of com-
pounds are able to reverse MDR in vitro, thus restoring
the levels of the anticancer agents within the cell to
cytotoxic concentrations. These structurally diverse
compounds are generally hydrophobic and include cal-
10.1021/jm9804477 CCC: $18.00 © 1999 American Chemical Society
Published on Web 05/27/1999

2146 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12
Sch em e 1. Preparation of 4a (Table 1)a
Berger et al.
Sch em e 2. Preparation of Intermediates 6a -d and
9a -e (Table 2)a
a
Reagents and reaction conditions: (a) formaldehyde, ZnI2,
ether, HCl gas addition, 1.5 h, then 1.5 h, 25 °C; (b) C6H5CH2NH2,
(Bu)4NCl, toluene, aq NaOH, 25 °C, 48 h; (c) 35% Pd(OH)2/C, H2
(Parr, 50 lb/in.2), EtOH, 21 h.
with greater potency and specificity for P-gp, but
without the calcium channel-blocking activity of VRP.
A similarity analysis of VRP to an in-house library of
compounds revealed a number of related structures.23
These compounds demonstrated good levels of in vitro
biological activity, initiating a program to further modify
their structures in order to optimize activity.
This paper describes our efforts to optimize the
properties of these new compounds. Structurally, they
are described by the general formula shown in Chart 1.
Ch em istr y
Tetrahydroisoquinolines which were not commercially
available were prepared by literature methods (Table
1). The synthesis of isoindoline 4a was achieved (Scheme
1) via an initial Friedel-Crafts acylation of 1 with
paraformaldehyde, in the presence of HCl to produce
dichloride 2. Reaction of 2 with benzylamine and
subsequent hydrogenation produced 4a .
Scheme 2 outlines the synthesis of thioethers 6a -d
and 9a -e (see Table 2). All were synthesized by reaction
of a benzyl halide with p-thiocresol anion, except 6c,d ,
which were produced by the reaction of the sodium
anions of 7a ,b with 2-pyridyl and p-tolyl disulfide,
respectively. The latter, shorter method subsequently
became the preferred procedure for the large-scale
preparation of 6a .
a
Reagents and reaction conditions: (a) R5SH, K2CO3, CH3CN,
65 °C, 15 h; (b) NaN(TMS)2, THF, 0 °C, 50 min, then R5SSR5, 5
°C, 40 min, 25 °C, 1.5 h; (c) p-thiocresol, (Bu)4NCl, toluene, aq
NaOH, 25 °C, 15 h, or p-thiocresol, NaH, DMF, 25 °C, 40 min; (d)
HBr gas, toluene, 0 °C, 20 min, then p-thiocresol, (Bu)4NHSO4,
toluene, aq NaOH, 25 °C, 15 h; (e) TBDMSCl, pyridine, reflux 3
h.
Following alkylation of 6a with m-carboxybenzyl bro-
mide, the benzoic acid residue was reduced to a benzyl
alcohol, then converted to chloride 23, and reacted with
tetrahydroisoquinoline 14a to provide target compound
24.
Isoindolines 28a -c were constructed as shown in
Scheme 5 (method E). The alkyl chlorides 12e,h were
converted to the corresponding amines 26a ,b by reaction
with potassium phthalimide and subsequent treatment
with hydrazine. Reaction of the amines with o-bisbenzyl
halides 27a 24 and 27b25 produced the target compounds.
An alternative synthetic approach to compounds
possessing various thioether substituents is outlined in
Scheme 6. This reaction sequence was useful in cases
where the required disulfides were not commercially
available. Friedel-Crafts acylation of 29 with 30 fol-
lowed by hydrolysis and coupling to tetrahydroisoquino-
line 14a produced intermediate 32. Selective reduction
of the ketone, followed by zinc(II) iodide-mediated
alkanethiol displacement of benzyl alcohol 33a provided
compounds 34a -d (method F).26 This displacement
reaction was particularly effective, as even the sterically
congested tert-butyl thiol reacted readily. The final
products 35a -d were obtained by reduction of the
amide linkages. Compound 35e was obtained by treat-
ing ketone 32 with ethyl Grignard and then repeating
the thiol displacement and reduction steps.
Schemes 3-7 describe the syntheses of the target
compounds listed in Tables 4 and 5. A significant
number of the target compounds were constructed by
thioether anion alkylation (methods A, A′) followed by
N-alkylation (methods B, B′) as described in Scheme 3.
Deprotonation of the nitrile- and ester-containing thio-
ethers 6a -d was readily achieved with sodium hydride,
while n-butyllithium was required to produce the less
stabilized anions of 9a -f. Following reaction of these
anions with an appropriate dihalide, the resulting
intermediates 12a -k and 13a -h (Table 3) were N-
alkylated by substituted isoindolines 4a ,b or tetrahy-
droisoquinolines 14a -i to produce target compounds
15a -e′ (Table 4).
Compound 19, possessing a more rigid alkynyl linker
chain, was synthesized by a longer reaction sequence
(method C, Scheme 4). Alkylation of 6a with bromide
16 produced 17. Following removal of the silyl protecting
group, alcohol 18 was mesylated and N-alkylated by
tetrahydroisoquinoline 14a to provide the target com-
pound 19. The synthesis of 24 similarly required a
somewhat lengthier reaction sequence (method D).
Scheme 7 outlines the chemistry utilized to attach

Novel MDR Reversal Agents
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12 2147
Sch em e 3. Methods A and A′, Preparation of Intermediates 12a -k and 13a -h (Table 3), and Methods B and B′,
Preparation of Target Compounds 15a -e′ (Tables 4 and 5)a
a
Reagents and reaction conditions (X ) Br or Cl): (a) NaH, DMF (or DMSO), 1.5 h, 25 °C, then Br(CH2)nX, 3 h (method A); (b) K2CO3,
NaI, DMF (or 2-pyrrolidinone), 95 °C, 4-24 h (method B); (c) n-BuLi, THF, -78 °C, 30 min, then Br(CH2)nX, -78 to 25 °C (method A′);
(d) (i-Pr)2NEt, NaI, CH3CN, reflux 24-48 h (method B′).
Sch em e 4. Method C, Preparation of Target Compound 19 (Table 5), and Method D, Preparation of Target Compound
24 (Table 5)a
a
Reagents and reaction conditions: (a) NaH, THF, 1.5 h, 25 °C, then 16, 3 h; (b) (Bu)4NF, THF, 0 °C, then 25 °C, 1 h; (c) MsCl,
pyridine, CH2Cl2, 25 °C, 5 h, then (i-Pr)2NEt, 14a , CH3CN, reflux 4 h; (d) n-BuLi, THF, -78 °C, 35 min, then 20, -78 °C, 40 min, 25 °C,
15 min; (e) BH3‚SMe2, THF, 25 °C, 18 h; (f) SOCl2, DMF, 70 °C, 17 h; (g) K2CO3, 14a , DMF, 85 °C, 4 h.
Sch em e 5. Method E, Preparation of Target
Compounds 28a -c (Table 4)a
poorly soluble in water. When protonated, the resulting
salts were generally isolated as solids (many of the free
bases were viscous gums) with greatly improved water
solubility. After removing the silyl protecting group of
15w , reaction of the resulting phenol 36 with chloro-
ethyl tosylate produced chloride 37a . Subsequent reac-
tion of 37a with imidazole provided the target compound
37b (method G). Similarly, 15l,d ′ and isoindoline 15b′
were converted to the ethylimidazole derivatives 39a -c
(method G′). Target compound 40 was obtained by the
reaction of 15l with chloroethyl dimethylamine under
basic conditions (method H).
Resu lts a n d Discu ssion
In Vit r o Biologica l R esu lt s a n d St r u ct u r e-
Activity Rela tion sh ip s (SAR). Candidate agents were
examined for their ability to restore sensitivity to
antitumor drugs in tissue culture. To do this, a subclone
of human colon carcinoma cells were selected for resist-
ance to bisantrene (S1-B1-20). Such cells exhibit a pro-
found overexpression of P-gp, are completely resistant
to 20 µM bisantrene (100% survival), and are cross-
resistant to drugs in the MDR phenotype (e.g., vinblas-
a
Reagents and reaction conditions: (a) NaI, acetone, reflux 15
h, then potassium phthalimide, DMF, 100 °C for h; (b)
2
H2NNH2‚H2O, EtOH, reflux 1.5 h; (c) toluene, (Bu)4NCl, aq NaOH,
25 °C, 48 h.
additional basic amine groups to the core structures.
Because of the high lipophilicity of these compounds
(clog P values are listed in Tables 4 and 5), they were

2148 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12
Berger et al.
Sch em e 6. Method F, Preparation of Compounds 35a -e (Table 4)a
a
Reagents and reaction conditions: (a) AlCl3, CH2Cl2, 0 °C, 4 h, 25 °C, 15 h; (b) 10% NaOH/EtOH, 25 °C, 3 h; (c) (EtO)2P(O)CN, DMF,
0 °C, then 25 °C, 15 h; (d) NaBH4, MeOH, 25 °C, 15 h; (e) CH2ClCH2Cl, ZnI2, R5SH, 25 °C, 1 h; (f) BH3‚Me2S, THF, reflux 2 h; (g) EtMgBr,
THF, 25 °C, 15 h.
Sch em e 7. Method G, Preparation of Target Compounds 37a ,b (Table 4), Method G′, Preparation of Target
Compounds 38a -c and 39a -c (Table 4), and Method H, Preparation of Target Compound 40 (Table 4)a
a
Reagents and reaction conditions: (a) (Bu)4NF, THF, 25 °C, 15 h; (b) Cl(CH2)2OTs, NaH, MEK, reflux 48 h; (c) imidazole, NaH, NaI,
DMF, 25 °C, 48 h; (d) Cl(CH2)2OTs, 1 N NaOH, MEK, reflux 18 h; (e) NaH, DMF, 25 °C, 1 h, then Cl(CH2)2N(CH3)2‚HCl, KI, 25 °C, 18
h.
tine, doxorubicin, or paclitaxel).27 Test agents (0.1-80
µM) were added to these cells alone or in combination
with 20 µM bisantrene. After 3 days of continuous
exposure, the number of cells that survived was deter-
mined. The difference score for each compound was
determined as the difference between the number of
cells that survived in the presence of the candidate
agent alone minus those that survived in the presence
of the candidate agent plus bisantrene. Minimum and
maximum values in this assay are 0 and 100, respec-
tively, with a value of 100 indicating the maximum
possible reversal activity. The IC50’s (difference scores)
and LD50’s (for reversal agents alone) are listed in Table
6.
Six regions of the target compounds were varied
(Chart 1): (1) the substituents on ring A (R1 and R2);
(2) the sulfide substituent (R5); (3) the quaternary
substituent (R6); (4) the chain linker (X); (5) the ring
size of the cyclic amine (m); and (6) the substituents on
ring B (R3, R4 and R7, R8). On the basis of the in vitro
results (Table 6), the following trends were observed:
1. In general, alkoxy substituents on the A ring

Novel MDR Reversal Agents
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12 2149
Ta ble 1. Substituted Isoindoline (4a ,b) and Tetrahydroisoquinoline (14a -i) Starting Materials
compd
R3
R4
R7
R8
m
yield (%)
formulaa
4a
4b
OH
OMe
OMe
OMe
OMe
OMe
H
H
H
H
H
H
Me
H
H
H
H
H
OMe
H
H
H
1
1
2
2
2
2
2
2
2
2
2
87
51
C9H11NO2‚0.25H2O‚0.13EtOH
37
C
10H13NO2
14a
14b
14c
14d
14e
14f
14g
14h
14i
b
O-CH2-O
90
69
69
c38
H
OMe
OMe
OH
OMe
OMe
H
C10H13NO‚HCl39
OMe
OH
OMe
OBn
H
C12H17NO3‚HCl40
b
b
3,4-dimethoxybenzyl
H
H
H
28
65
C17H19NO2‚HCl41
H
H
b
OH
OMe
c42
a
Elemental analyses were within 0.4% of the theoretical values for the formulas given, unless otherwise stated. All compounds exhibited
NMR spectra consistent with assigned structures. b Commercially available. c Synthesized by literature procedure; structure was confirmed
by 1H NMR.
Ta ble 2. Benzyl Thioether Intermediates 6a -d and 9a -f
compd
R1
OMe
OMe
OMe
OMe
R2
R5
R6
CN
CN
CN
yield (%)
formulaa
c43
C16H21NO2S
15H14N2O2S
C18H20O4S
C15H14O2S
C15H13F3OS
6a
6b
6c
6d
9a
9b
9c
9d
9e
9f
OMe
OMe
OMe
OMe
p-tolyl
65
55
64
62
80
81
86
80
94
cyclohexyl
2-pyridyl
p-tolyl
p-tolyl
p-tolyl
p-tolyl
p-tolyl
p-tolyl
phenyl
C
CO2Me
O-CH2-O
H
H
H
H
H
H
H
H
OCF3
F
OMe
OMe
H
C
C
C
b
14H13FS
16H18O2S
21H30O2SSi
OMe
OTBDMS
H
a-c See corresponding footnotes for Table 1.
consistently provided the highest activity. With the
exception of the particularly bulky tert-butyldimethyl-
silyloxy group, the P-gp binding site appeared to be
remarkably insensitive to the nature of the alkoxy
substituent at the R1 position. This is illustrated by the
almost identical activities of 36, 15s, and 37a , which
possess an R1 hydroxy, methoxy, and chloroethoxy
group, respectively. A compound possessing the ethoxy-
imidazole (47) at R1 was a particularly active reversal
agent. A cyclic benzodioxole compound 15x was less
active. It appears that the conformational rigidity and/
or smaller size of the benzodioxole substituent reduces
binding to its target site. Compounds possessing electron-
withdrawing substituents such as fluoride (15z) and
trifluoromethoxy (15y) were far less potent as MDR
reversal agents. In summary, the greatest activity was
achieved with hydrophobic electron-donating substitu-
ents.
2. Compounds possessing aromatic and aliphatic R5
groups were synthesized. Of the aliphatic groups, cy-
clohexane provided the greatest amount of activity. With
smaller pentyl (35d ) and tert-butyl (35c) groups, the
reversal agents were significantly less active, indicating
too little contact was being made with the walls of an
important binding pocket. Overall, the best R5 group
was the p-tolyl substituent. A benzyl derivative (35b)
possessed poor MDR reversal properties. This indicated
that the steric environment of P-gp binding site for the
R5 group is wide enough to accommodate the aromatic
and cyclohexyl substituents very well but contains a
narrow apex. Substitution of a pyridine residue (15e′)
for a phenyl group (to provide greater water solubility)
produced a surprisingly inactive compound. Evidently,
a strongly hydrophobic R5 group is required for optimal
activity.
3. The quaternary substituent (R6) which provided the
greatest activity was a nitrile. Generally, similar com-
pounds possessing hydrogens at R6 were somewhat less
potent. With a larger methyl ester group compound 15e
was significantly less active than the corresponding
nitrile, 41. Similarly, a reversal agent with an ethyl
substituent (35e) possessed only marginal activity.
4. An optimal length of the linker section was impor-
tant for activity, although a surprisingly large range was
tolerated. When the linker section was X ) (CH2)n,
optimal potency was observed for n ) 5-8. Shortening
or lengthening the chain led to a decrease in activity.
Rigidification of the linker chain by a phenyl ring (24)
or an alkyne (19) led to a loss of activity. It appears that
the rigidification prevented the compounds from binding
in their most preferred conformations in the binding
pocket, with unfavorable steric interactions by the bulky
phenyl substituent of 24 possibly causing a larger
decrease in binding.

2150 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12
Ta ble 3. Alkylated Benzyl Thioethers 12a -k and 13a -h
Berger et al.
compd
R1
OMe
R2
R5
R6
CN
n
X
method
yield (%)
formulaa
12a
12b
12c
12d
12e
12f
12g
12h
12i
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
p-tolyl
p-tolyl
p-tolyl
p-tolyl
p-tolyl
p-tolyl
p-tolyl
cyclohexyl
p-tolyl
2-pyridyl
p-tolyl
p-tolyl
p-tolyl
p-tolyl
p-tolyl
p-tolyl
p-tolyl
p-tolyl
phenyl
3
4
2
11
5
8
6
5
5
5
3
3
5
6
6
5
5
6
Cl
Cl
Cl
Br
Cl
Br
Cl
Cl
Br
Br
Cl
Cl
Br
Br
Br
Br
Br
Br
Br
A
A
A
A
A
A
A
A
77
64
57
73
77
70
82
76
83
82
68
94
51
57
89
67
88
12
81
C20H22ClNO2S‚0.07C20H22BrlNO2Sb
C21H24ClNO2S‚0.01C21H24BrlNO2Sb
C19H20ClNO2S
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OTBDMS
CN
CN
CN
CN
CN
CN
CN
CN
CN
CO2Me
H
H
H
H
H
H
H
H
C
C
28H38BrNO2S
22H26ClNO2S‚0.03C22H26BrlNO2Sb
C25H32BrNO2S‚0.11C17H17NO2Sb
C23H28ClNO2S
C
21H30ClNO2S‚0.82C21H30BrlNO2Sb
A
A
A
C22H26BrNO2S
20H23BrN2O2S
C21H25ClO4S
19H23ClO2S‚0.06CHCl3
C21H27BrO2S
12j
C
12k
13a
13b
13c
13d
13e
13f
13g
13h
A′
A′
A′
A′
A′
A′
A′
A′
C
C
d
22H29BrO2S‚0.54C16H18NO2Sb
O-CH2-O
C20H23BrO2S
H
H
H
OCF3
C20H22BrF3OS‚0.33C15H13FO3Sc
F
H
d
C
6
19H23BrS‚0.03C13H12Sc
a
See corresponding footnote for Table 1. b These compounds contained varying quantities of the chromatographically inseparable bromide
analogues due to displacement of the chloride groups by Br- during the course of reaction and/or reaction of the anion to some degree
with the less reactive chloride portion of the bromochloroalkanes. c These compounds could not be chromatographically separated from
d
starting materials. Compound was synthesized and utilized in the subsequent step without purification.
5. Two series of cyclic compounds were examined:
isoindolines and substituted tetrahydroisoquinolines (m
) 1, 2). Several reversal agents possessing either
heterocyclic system were very potent, although the
oxidative instability of the isoindolines caused the
measurements of activity to be more variable.
differing from 15h only by having a hydrogen rather
than a nitrile group at R6, also showed good activity
(IC50 0.33 µM). Several similarly substituted isoindoline
compounds were exceptionally active in vitro: 44 (IC50
0.26 µM), cyclohexyl thioether 28c (IC50 0.35 µM), and
its corresponding HCl salt 46 (IC50 0.26 µM). Also,
compounds possessing the water-solubilizing side chains
(39b, 47, 48) were among the most active compounds
(IC50’s 0.50, 0.29, and 0.53 µM, respectively).
These compounds were up to 50-fold more potent than
VRP in this assay. The onset of activity was observed
at 0.2 µM for the more active compounds tested. At
concentrations between 5 and 10 µM, the most potent
MDR agents began to show signs of cytotoxicity, with
LD50’s at concentrations around 30-fold higher than the
IC50’s. Not only were most of the synthesized compounds
far more potent than VRP, they were also more effica-
cious. This is graphically illustrated in Figure 1, where
the activity of 15h was compared to VRP. At doses of
up to 2 µM, 15h was nontoxic alone but killed cells in
combination with bisantrene (at higher doses the com-
pound killed tumor cells by itself, hence the biphasic
dose-response), while VRP only began to show signs
of activity at 5 µM. Furthermore, the maximal difference
score (100) was observed with 15h . Submaximal scores
were attained with VRP because toxicity of the com-
pound alone occurred before complete cell kill in the
presence of bisantrene was achieved.
6. The B ring substituents (R3 and R4) which provided
the highest activity were the electron-donating methoxy
groups. A compound (15v) possessing an additional
methoxy group at R8 had similiar activity to the corre-
sponding dimethoxy analogue (15s). The increased steric
bulk provided by a chloroethoxy group at R3 did not have
a significant effect on activity (38a versus 15h ). How-
ever, compounds possessing ethoxyimidazole substitu-
ents at R3 were somewhat less potent (for example, 48
versus 15h ). Significantly, when the imidazole was
replaced by the less lipophilic dimethylamine group, the
resulting compound 40 was essentially inactive. Benzo-
dioxole compounds 15j,q were poor reversal agents.
Compounds with hydroxy substituents (15l, 42) were
far less active than the corresponding ethers. Also,
electron-withdrawing substituents such as chlorides
(28b) greatly reduced potency. Introduction of a bulky
substituent at the benzylic position R to the nitrogen
(R7) on the tetrahydroisoquinoline moiety (15t) led to a
diastereomeric mixture which possessed modest activity
in comparison to the R7 unsubstituted compound (15h ).
Apparently substituents at R7 are tolerated but do not
enhance the activity.
To determine whether the reversal agents increased
the concentration of cytotoxic agents in the cells, ac-
cumulation of vinblastine was measured in S1-B1-20
cells (Figure 2). Compounds 15h and 28a were signifi-
cantly more effective than VRP in their ability to restore
vinblastine levels in the P-gp-expressing cells. The most
potent reversal agent (15h ) was approximately 5-fold
more potent than verapamil in this assay. No effects
were seen in cell lines that do not express P-gp.28
Practically all of the tested compounds showed greater
activity than VRP (Table 6). Among the most potent
were those that combined the optimal features of the
above-mentioned regions of the reversal agents, such
as the dimethoxytetrahydroisoquinolines 15h ,i,m (IC50’s
0.37, 0.36, and 0.30 µM, respectively). These compounds
were all R-dimethoxyphenyl,R-p-tolyl nitriles, differing
only in the length of the linker chain. Compound 15o,

Novel MDR Reversal Agents
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12 2151
Ta ble 4. Substituted Tetrahydroisoquinoline and Isoindoline R-Aryl-R-thioether-alkane, -alkanenitrile, and -alkanecarboxylic Acid
Ester Derivatives
compd
R1
R2
R3
R4
R5
R6
CN
n
m
method yield (%) log Pa
formulab,c
15a
15c
15d
15e
15f
15g
15h
15i
15j
15k
15l
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe OMe
OMe OMe
OMe OMe
OMe OMe
OMe OMe
OMe p-tolyl
OMe p-tolyl
OMe p-tolyl
OMe p-tolyl
OMe p-tolyl
3
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
1
2
2
2
2
2
2
2
1
2
2
1
1
1
2
2
2
2
2
2
2
2
2
1
2
2
1
2
2
2
2
1
2
1
2
2
B
38
42
20
20
14
40
56
41
61
46
21
52
12
58
87
92
24
38
65
82
84
70
73
43
74
79
58
29
95
28
77
83
86
82
81
99
27
16
42
70
76
37
56
69
21
87
87
58
99
30
88
90
5.12 C31H36N2O4S
CN
CN
CO2CH3
CN
CN
CN
CN
CN
CN
CN
CN
H
4
2
3
11
5
5
8
5
5
5
6
3
5
5
5
6
6
5
6
5
5
6
5
5
5
5
5
5
5
5
5
5
5
5
6
6
6
5
5
5
5
5
5
5
3
5
5
4
5
6
5
B
5.65
C32H38N2O4S‚CH2Cl2
B
B
4.59 C30H34N2O4S‚0.4H2O
5.54 C32H39NO6S
B
B
B
B
9.35
6.72
6.18
7.76
C
C
C
C
39H52N2O4S
31H36N2O2S
33H40N2O4S‚0.5H2O
36H46N2O4S‚0.25C6H14
OMe
H
H
p-tolyl
OMe OMe
OMe OMe
OMe
OMe p-tolyl
OMe p-tolyl
p-tolyl
OMe p-tolyl
OMe p-tolyl
OMe p-tolyl
OMe p-tolyl
OMe p-tolyl
OMe p-tolyl
p-tolyl
OMe p-tolyl
OMe p-tolyl
OMe cyclohexyl CN
OMe p-tolyl
OMe p-tolyl
OMe p-tolyl
OMe p-tolyl
OMe p-tolyl
OMe p-tolyl
O-CH2-O
O-CH2-O
B
B
B′
B
B
6.08 C32H36N2O4S‚2H2O
OMe
H
6.64
5.90
6.71
6.36
7.42
7.88
C
C
C
C
C
C
32H38N2O3S
OMe OH
OMe OMe
OMe OMe
OMe OMe
OMe
OMe
OMe OMe
OMe OMe
OMe OMe
OMe OMe
OMe
32H38N2O4S‚0.5H2O
34H42N2O4S‚0.13H2O
30H37NO4S‚0.25H2O
32H41NO4S‚0.5H2O
31H39NO3S
15m OMe
15n
15o
15p
15q
15r
15s
15u
OMe
OMe
OMe
OMe
OMe
OMe
OMe
H
H
H
H
B
H
B′
B′
B′
B′
B
7.31 C31H37NO4S‚0.88H2O
7.32
7.95
C
C
C
C
32H41NO4S‚H2O
33H43NO4S‚H2O
32H44N2O4S
H
6.28
15w OTBDMS
H
H
H
H
CN
CN
B′
B′
B′
B
11.68
7.31 C31H37NO4S
8.44 C31H36F3NO3S
8.63
38H55NO4SSi
15x
15y
15z
15a ′ OMe
15b′ OMe
15d ′ OMe
15e′ OMe
O-CH2-O
H
H
OCF3 OMe
F
OMe
C31H38FNO2S‚0.4EtOH
OMe OCH2Ph
OMe OH
OMe OH
B
7.95 C39H44N2O4S‚H2O
B′
B′
B′
E
5.78
6.00
4.72
5.55
7.40
C
C
C
C
C
31H36N2O4S
31H42N2O4S
31H37N3O4S
32H38N2O4S
30H32Cl2N2O2Sd
OMe cyclohexyl CN
OMe pyridyl
OMe p-tolyl
Cl
OMe OMe
OMe OMe
OMe Cl
OMe OMe
OMe OMe
OMe OMe
OMe OMe
OMe OMe
OMe OMe
OMe OMe
CN
CN
CN
28a
28b
28c
35a
35b
35c
35d
35e
36
37a
37b
38a
38b
38c
39a
39b
39c
40
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OH
p-tolyl
E
OMe cyclohexyl CN
E
5.65 C31H42N2O4S
OMe cyclohexyl
OMe benzyl
OMe t-butyl
OMe n-pentyl
OMe p-tolyl
OMe p-tolyl
OMe p-tolyl
OMe p-tolyl
OMe p-tolyl
OMe p-tolyl
H
H
H
H
F
F
F
F
7.33
6.63
6.54
5.66
C
C
C
C
31H45NO4S‚0.5EtOH
32H41NO4S
29H43NO4S
30H43NO5S‚0.3EtOAc
CH2CH3
H
H
H
CN
CN
F
8.34 C34H45NO4S‚0.5EtOH
G
G
G
G′
G′
G′
G′
G′
G′
H
B
7.67
C32H41NO4S‚0.5H2O
OCH2CH2Cl OMe OMe
OCH2CH2Im OMe OMe
8.54 C34H44ClNO4S
7.63 C37H47N3O4S‚H2O
6.77 C34H41ClN2O4S
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe OCH2CH2Cl
OMe OCH2CH2Cl
OMe OCH2CH2Cl
OMe OCH2CH2Im
OMe OCH2CH2Im
OMe OCH2CH2Im
6.14 C33H39ClN2O4S
OMe cyclohexyl CN
OMe p-tolyl
OMe p-tolyl
6.87 C33H45ClN2O4S‚0.1CH2Cl2
5.86 C37H46N4O5S‚0.6EtOAc
5.23 C36H42N4O4S‚2HCl‚1.5H2O
5.96 C36H48N4O4S‚1.75HCl‚H2O
6.10 C36H47N3O4S‚0.6Et2Oe
5.12 C31H36N2O4S‚HCl
6.18
5.55
5.65
5.65
CN
CN
OMe cyclohexyl CN
OMe OCH2CH2N(Me)2 OMe p-tolyl
CN
CN
CN
CN
CN
41
43
44
45
46
47
48
OMe OMe
OMe OMe
OMe OMe
OMe OMe
OMe OMe
OMe p-tolyl
OMe p-tolyl
OMe p-tolyl
OMe p-tolyl
OMe cyclohexyl CN
OMe p-tolyl
OMe p-tolyl
B
E
B
E
C
C
C
C
30H34N2O4S‚HCl‚H2O
32H38N2O4S‚HCl‚0.75H2O
32H38N2O4S‚HCl
31H42N2O4S‚HCl
OCH2CH2Im OMe OMe
OMe OMe OCH2CH2Im
H
CN
G
G′
7.63 C37H47N3O4S‚1.88HCl‚H2O
5.86 C37H46N4O5S‚2H2O‚1.75HCl
a
b
Log P values were generated using MacClogP, version 1.03 (Biobyte Corp.), utilizing a fragment-based approach. Elemental analyses
were within 0.4% of the theoretical values for the formulas given, unless otherwise stated. All compounds exhibited NMR spectra consistent
with assigned structures. c The majority of these compounds were isolated as thick oils or glassy, hygoscopic solids; thus a significant
number of compounds contained solvents and/or water even after prolonged evacuation. In some cases compounds were dissolved in
ethanol and then evacuated in order to remove halocarbon solvents prior to biological evaluation. Cl: calcd, 12.76; found, 12.27. e C:
d
calcd, 69.63; found, 70.66.
In Vivo Biologica l Resu lts. Indications of good
potency and/or therapeutic index were important cri-
teria in choosing compounds for in vivo testing. Com-
pounds were evaluated against vincristine-resistant
murine leukemia P388/VCR cells in CDF1 mice using
a variety of treatment schedules, comparing treatments
with VCR alone (Table 7). The drugs were introduced
by an ip route. As mentioned previously, many of the
reversal agents were poorly soluble in water. After
considerable experimentation, cremophor/ethanol solu-
tion or propylene glycol/ethanol were chosen as vehicles
for in vivo administration (it was possible to avoid the
use of these solvent systems with water-soluble ana-
logues containing additional salt-forming functional
groups). Administration of the reversal agents together
with VCR produced significant increases in life span
compared to treatment with VCR alone. The largest
increases in lifespan were observed with compounds 15h

2152 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12
Berger et al.
Ta ble 5. Substituted Alkyl Chain (X) and Highly Substituted (R3, R4, R7, R8) Tetrahydroisoquinolinealkane and -alkanenitrile
Derivatives
compd R1,R2 R3,R4
R5
p-tolyl CN CH3
OMe OMe p-tolyl CN 3,4-dimethoxybenzyl
R6
R7
R8
X
method yield (%) log Pa
formulab,c
15b
15t
15v
15c′
19
OMe OH
H
CH2
(CH2)3
OMe (CH2)4
B
18
61
76
36
30
63
73
4.92 C30H34N2O4S‚0.5H2O
7.57 C42H50N2O6S
H
B′
B′
B′
C
D
B
OMe OMe p-tolyl
OMe phenyl
H
H
H
H
H
H
7.38 C34H45NO5S‚0.25H2O
H
H
H
H
H
(CH2)4
7.99 C30H37NO2S
OMe OMe p-tolyl CN
OMe OMe p-tolyl CN
CtCCH2
m-phenyl
CH2
5.52 C33H36N2O4S‚0.1H2O
24
5.91
C36H38N2O4S‚0.7EtOH
42
OMe OH
p-tolyl CN CH3
4.92 C30H34N2O4S‚HCl‚0.75H2Od
d
a-c See corresponding footnotes for Table 4. Cl: calcd, 6.24; found, 5.80.
(33% at 12.5 mg/kg, 0.2 mg/kg VCR), 15c′ (46% at 25
mg/kg, 0.2 mg/kg VCR), 28a (41% at 25 mg/kg, 0.2 mg/
kg VCR), and the water-soluble analogue 39a (48% at
50 mg/kg, 0.2 mg/kg VCR). Of these compounds, the
activity of 15c′ was anomalous. In vitro, 15c′ was a
relatively poor reversal agent, as might be expected for
a compound lacking the appropriate substituents on
ring A. It is therefore possible that this highly lipophilic
compound (clog P 7.99) was acting via non-P-gp mech-
anisms, altering the pharmacokinetics of VCR uptake
into the cells.29 Dosed at 100 mg/kg in the absence of
VCR, both 15a ,h showed no observable signs of toxicity
to the animals, as well as no antitumor effects. Simi-
larly, the cremophor/ethanol solution or propylene
glycol/ethanol vehicles alone were not toxic to the
animals.
Further in vivo evaluation of the reversal agents was
carried out on human epidermoid carcinoma KB/8.5
implanted sc (subcutaneous) in athymic mice (Table 8).
These mice were treated with a single iv dose of
doxorubicin (DOX), bracketed by two subcutaneous
doses of placebo or reversal agent. Relative tumor
growth was then determined for each group and com-
pared with the group treated with DOX alone.
Many of the reversal agents showed some level of
activity if administered at high enough doses (100-200
mg/kg for the less active compounds). The compounds
of interest were those that showed significant activity
(p e 0.01) at 50 mg/kg or less. The most active iso-
indoline compound in this test was 28a . When admin-
istered at 50 mg/kg, together with 8 mg/kg doxorubicin,
tumor growth was 65% of that observed in mice treated
with DOX alone (44% of the tumor growth observed in
the placebo-treated mice). However, these compounds
showed significant toxicity at doses > 100 mg/kg, with
weight loss, convulsions, and several deaths observed.
Because of this toxicity, and the chemical instability of
the isoindoline analogues in general (several of the more
active isoindolines possessing electron-donating sub-
stituents were rapidly air-oxidized), these compounds
were not considered to be useful as potential drug
candidates.
the tumor growth observed in the placebo-treated mice).
No signs of toxicity were observed for these compounds
at the high dose of 200 mg/kg. The water-soluble
analogue 39a was the most active compound in this
assay. When administered at 25 mg/kg, together with
8 mg/kg DOX, tumor growth was 46% of that observed
in mice treated with DOX alone (31% tumor growth as
compared to the placebo-treated mice).
In choosing compounds for further evaluation as
potential drug candidates, the two reversal agents which
consistently displayed activity in vitro and in both
animal models were 15h and 39a . Because the synthesis
of 15h could be achieved in three steps from com-
mercially available starting materials, this reversal
agent was produced on a large scale for further testing.
An account describing the detailed biological evaluation
of 15h has been published.30 One particularly important
finding was that 15h did not alter the pharmacokinetic
disposition of DOX in mice at optimum efficacious doses.
Thus, when 15h was administered with escalating doses
of DOX, significantly less toxicity was observed in mice
as compared to those similarly treated with DOX and
an optimal dose of the reversal agent cyclosporin A.
Cyclosporin A is known to alter the pharmacokinetic
disposition of anticancer drugs in animals.31 Consistent
with this fact, cyclosporin A was shown to enhance the
tumoricidal activity of DOX when tested on mice
implanted with a drug-sensitive P-gp-negative KB cell
line, while 15h had no effect.30
Con clu sion
A series of compounds have been synthesized and
evaluated as MDR reversal agents. With few exceptions,
these compounds are far more effective than verapamil.
The in vitro data demonstrates activity at lower doses
for these compounds, as well as a more efficacious
response based on the difference scores obtained in the
assay. In vivo, several reversal agents restore the
activity of vincristine in mice bearing vincristine-
resistant murine tumors. At the proper doses, the
reversal agents are not cytotoxic and do not possess
anticancer activity in the absence of a chemotherapeutic
drug. These compounds also potentiate the action of
doxorubicin to reduce the tumor size in “nude” mice
bearing the doxorubicin-resistant human epidermoid
carcinoma KB/8.5.
Several tetrahydroisoquinolines possessed promising
activity as reversal agents in this model. When com-
pounds 15a ,f,h were administered at 50 mg/kg, together
with 8 mg/kg DOX, tumor growth in these mice was
63%, 61%, and 59%, respectively, of that observed in
mice treated with DOX alone (25%, 24%, and 41% of
Of this series, compounds 15h and 39a (and the
corresponding HCl salt 48) are particularly effective

Novel MDR Reversal Agents
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12 2153
Ta ble 6. In Vitro Activity of Compounds on the S1-B1-20 Cell
Line
a
b
compd
IC50 (µM)
LD50 (µM)
VRP
15d
15e
15f
15g
15h
15i
15j
15k
15l
15m
15n
15o
15p
15q
15r
15s
15t
15u
15v
15w
15x
15y
15z
15a ′
15c′
15e′
19
17.27
2.30
1.47
1.74
2.56
0.37
0.36
3.09
1.92
3.11
0.30
1.23
0.33
3.09
IA
1.84
0.90
1.06
0.46
0.70
10.68
0.50
2.07
2.70
0.85
3.82
2.12
0.97
2.19
0.81
10.49
0.35
0.73
1.98
3.58
0.50
1.23
0.84
0.96
0.69
0.37
0.69
0.50
0.96
IA
c
33.67
32.76
10.77
24.65
13.57
11.62
30.03
15.72
24.85
13.52
25.82
16.85
22.21
24.95
22.06
13.20
78.24
13.95
13.98
49.11
15.91
16.91
26.99
40.00
24.61
50.82
36.00
23.33
22.81
c
14.96
16.06
15.13
76.75
16.33
11.76
13.13
10.34
5.52
16.80
14.68
8.37
8.39
6.68
F igu r e 1. Comparison of reversal activity of 15h (9) and
verapamil (0) in S1-B1-20 MDR cells. Difference score )
(percent survival of cells treated with test agent alone) -
(percent survival of cells treated with test agent and 20 µM
bisantrene). See Experimental Section for details.
24
28a
28b
28c
35a
35b
35c
35d
35e
36
37a
37b
38a
39a
39b
39c
40
41
42
44
45
46
47
48
0.46
4.70
0.26
0.62
0.26
0.29
0.53
23.99
c
11.36
41.77
11.83
5.64
F igu r e 2. Effect of reversal agents on vinblastine accumula-
tion in S1-B1-20 cells. One million cells were grown for 2 days
in drug-free medium. After preincubation in serum-free media
(60 min), cells were incubated in the absence or presence of
20 µM test agent (90 min), followed by continued incubation
with the addition of 1 µM [3H]vinblastine (90 min). The
experiment was terminated by rapidly washing cells in ice-
cold phosphate-buffered saline, briefly trypsinizing the cells,
and determining the cell number as well as the radioactivity
in the cell suspension by liquid scintillation counting. Data is
expressed as percentage of drug-sensitive S1 cells. Values are
means ( SE; n ) 3-5 independent experiments.
11.61
a
Concentration at which (percent survival of cells treated with
test agent alone) - (percent survival of cells treated with test agent
b
and 20 µM bisantrene) ) 50. Concentration at which 50% of cells
treated with test agent alone survived. c Determinations could not
be made due to the limiting solubilities of the reversal agents. See
Experimental Section for details.
modulators of MDR resulting from the overexpression
of the MDR-1 gene. In addition to the results presented
in this work, other studies have provided strong evi-
dence that the effects of 15h are due to its interaction
with P-gp:30 Reversal agent 15h selectively restores the
cytotoxic activity of hydrophobic anticancer agents such
as vinblastine, bisantrene, colchicine, and doxorubicin
in resistant cells, by increasing drug accumulation in
such cells. Sensitivity to water-soluble drugs such as
cisplatin is not altered by 15h . No resensitization is
found in cells lacking P-gp. These results suggest that
the reversal agents inhibit the P-gp-mediated efflux of
cytotoxic agents, probably by blocking the ability of P-gp
to interact with antitumor drugs. Given the hydrophobic
nature of the P-gp substrates, it seems likely that the
hydrophobic reversal agents also bind to P-gp. Con-
sistent with this, the binding of the photoaffinity probe
[
125I]iodoarylazidoprazosin (which is believed to be a
reporter for the vinblastine binding site) to P-gp is
inhibited by 15h and verapamil. However, further
studies described in this work show that unlike ver-
apamil (and cyclosporin A), 15h is not effluxed from cells
by P-gp, which suggests that it binds more strongly to
or at a distinct site of the binding pocket. It appears
likely that the superior reversal activity of 15h versus

2154 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12
Berger et al.
Ta ble 7. Antitumor Activity of Vincristine Alone and in
Combinationa with an Optimal Dose of MDR Reversal Agents
against Vincristine-Resistant Murine Leukemia P388/VCR
Ta ble 8. In Vivo Evaluation of MDR Reversal Agents (dosed at
12.5-200 mg/kg) against Human Epidermoid Carcinoma
KB/8.5 Implanted sc in Athymic ‘Nude’ Mice
%
%
compd dose
(mg/kg)
optimal tumor growth inhibition
c
d
compd
15a f
15d
15e
15f
15g
15h
15i
15j
15k
15m
15o
15c′
28a
39a
VCRb ILSplacebo ILSVCR
t-teste
compd
DOXa
% TGIb
compd dose (mg/kg)
t-test
51
67
13
24
14
37
24
24
26
37
21
15
15
15
73
89
25
42
27
83
34
38
56
68
37
68
63
71
-1
1
15
13
11
14
11
33
8
16
24
23
13
46
41
48
50
12.5
50
25
25
12.5
6.3
25
12.5
12.5
6.3
25
25
50
0
0
100
100
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
0.03
<0.01
<0.01
<0.01
0.02
15a
15f
15h
15o
15q
15r
15s
15t
15u
15v
15x
15y
15a ′
15c′
28a
28b
35a
35b
35c
35e
36
39
39
69
58
100
75
45
64
67
64
100
100
100
59
67
57
67
77
67
77
63
61
59
68c
57
60
64
58
69c
54
39
51
71
65
65d
94
57
68
53
72
54
67
64
46
56
50
50
50
<0.01
<0.01
<0.01
0.01
0.04
<0.01
0.03
<0.01
0.09
<0.01
<0.01
0.02
100
200
100
100
200
100
100
200
100
200
100
50
200
100
100
200
100
200
100
100
25
<0.01
<0.01
<0.01
0.10
<0.01
<0.01
0.34
<0.01
0.05
PG/ET/Salg
Crm/ET/Salh
15a i
15h i
-3
-4
a
<0.01
0.06
MDR reversal agents plus 0.2 mg/kg vincristine were admin-
istered simultaneously (both ip) once daily on days 1-7, where
day 1 is 1 day after tumor implantation. Percent increase in
mean life span when 0.2 mg/kg vincristine was administered ip
once daily on days 1-7, relative to placebo control treatment.
c Percent increase in mean life span, relative to placebo control
b
75
72
75
67
<0.01
<0.01
<0.01
<0.01
<0.01
37b
38a
39a
47
d
80
100
treatment. Percent increase in mean life span, relative to treat-
ment with an equivalent dose of vincristine alone. e Student t-test
analysis of mean survival time, comparing treatment with vincris-
tine plus MDR reversal agents to treatment with vincristine alone.
f 15a was administered ip 1 h before each daily 0.4 mg/kg ip dose
of vincristine on days 1-3. g Propylene glycol/ethanol/saline vehicle
administered at a dose equivalent to a 100 mg/kg/day dose of MDR
reversal agent, together with a 0.5 mg/kg ip dose of vincristine
a
Relative tumor growth as a percentage of saline control on
b
day 14 when treated with DOX (8 mg/kg) alone. Percent tumor
growth inhibition (TGI) obtained for the combination of DOX (8
mg/kg) together with MDR reversal agent (at the specified dose),
versus DOX (8 mg/kg) alone on day 14. p (t-test) values < 0.05
are statistically significant. See Experimental Section for details.
c Toxicity was observed at a dose of 200 mg/kg MDR reversal agent,
together with 8 mg/kg DOX: 2/5 dead/total. Toxicity was ob-
served at a dose of 200 mg/kg MDR reversal agent, together with
8 mg/kg DOX: 4/5 dead/total.
h
once daily on days 1-7. Cremophor EL/ethanol/saline vehicle
d
administered at a dose equivalent to a 100 mg/kg/day dose of MDR
reversal agent, together with a 0.5 mg/kg ip dose of vincristine
i
once daily on days 1-7. MDR reversal compounds at a dose of
100 mg/kg in Cremophor/ethanol/saline vehicle administered ip
most active compounds overall were relatively lipophilic
(clog P > 5.5). Altering the steric bulk of certain
substituents (the R5 and R6 groups) appeared to strongly
affect binding to P-gp, while relatively large changes
down the long axis (the linker chain length n and the
size of the alkoxy substituents R1-R4) seemed to be
well-accommodated. The presence of certain functional
groups was critical for activity; for example, electron-
donating alkoxy substituents on the aromatic rings
provide far greater activity than halogens in the same
positions. Thus, despite the fact that P-gp acts as an
efflux pump for a variety of structurally unrelated
hydrophobic substrates, it displays a significant sensi-
tivity to structural changes within a particular series
of compounds.
without vincristine, once daily on days 1-7.
verapamil is at least in part due to the fact that it is
accumulated in greater concentrations, particularly in
cells overexpressing P-gp.
Despite the structural similarity of the compounds to
VRP, they possess significantly less calcium channel
activity. When 15h was assayed for calcium channel
antagonistic activity in rat thoracic aortic strips, it was
found to be 70-fold less potent than racemic verapamil.30
Additional tests (results not shown) for other represen-
tative compounds showed the same level of activity.
Thus, the structural differences are sufficient to provide
a significantly different activity profile. Consequently,
the calcium channel-blocking effect (hypotension) is not
expected to be a side effect of these compounds.
As in the case of verapamil, most of the active
compounds possess a chiral quaternary center. While
some studies have shown that both enantiomers of
verapamil exhibit equal activity as MDR reversal
agents,32,33 the R-enantiomer is approximately 10-fold
less active as a calcium channel blocker than the
S-enantiomer.34 Although an enantioselective synthesis
of verapamil has been carried out,35 the structural
differences of our lead compounds have necessitated a
different synthetic approach toward producing the pure
enantiomers to study their relative activities. The
results will be reported in due course.
Exp er im en ta l Section
Biologica l P r oced u r es. 1. In Vitr o Testin g. Cells: A
clonal cell line, S1, derived from human colon carcinoma cells,
LS 174 T, was subjected to increasing concentrations of
bisantrene (CL 216,942; from Lederle Laboratories, Pearl
River, NY) over a period of 12-16 weeks. The resistant cell
line was designated S1-B1-20.27
Scr een in g a ssa y for r ever sa l a gen ts: Cells, prepared by
trypsinization, were plated at 20 000 cells/well in media with
and without bisantrene (20 µM). Then, test agents at varying
concentrations were added to the cells. Test agents were
solubilized in 100% DMSO. The final DMSO concentration in
the seeded plates did not exceed 0.1%. Plates were incubated
for 3 days at 37 °C in a moisturized 7% CO2 atmosphere. Cell
survival was estimated by the sulforhodamine B assay.36 Dye
While no clear trend was observed correlating the
lipophilicity of the reversal agents with activity, the

Novel MDR Reversal Agents
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12 2155
content/well was read at 540 nm in a microtiter reader. Values
were electronically processed, percent cell survival compared
to untreated (without bisantrene) cells was computed, and the
results were expressed as the difference between these values.
The difference scores of those two experiments were deter-
mined at concentrations from 0.1 to 80 µM (typically at 0.1,
0.5, 1, 5, 10, 20, 40, and 80 mM concentrations). Two to four
sets of measurements were made for each compound, and the
results were plotted. The maximum variation of these data
points was (15%. A best fit equation was then applied (with
R2 g 0.990), and the IC50’s were determined. The LD50’s were
determined similarly by utilizing the percent cell survival data
from experiments in which test agent alone was used.
2. In Vivo Testin g. Eva lu a tion of MDR-1 r ever sa l
a gen ts a ga in st th e vin cr istin e-r esista n t m u r in e leu k e-
m ia P 388/VCR im p la n ted in m ice: The vincristine-resistant
murine leukemia P388/VCR was propagated by intraperitoneal
(ip) injection of 1 × 106 tumor cells into syngeneic DBA/2 mice
(Charles River Laboratories, Inc.). Tumor cell ascites was
harvested 5-7 days later. For drug testing, groups of 10-15
CDF1 mice (DBA/ 2 × Balb/ c F1 hybrids; Charles River
Laboratories, Inc.) were injected ip with 0.5 mL of diluted
ascitic fluid containing 1 × 106 viable P388/VCR tumor cells.
All mice were of one sex, weighing a minimum of 18 g, and all
within a 3-g weight range per test. Drugs were administered
once daily, by ip, starting 1 day after tumor implantation and
using a variety of treatment schedules. Mice were weighed
periodically, survival was recorded for up to 30 days post-
tumor implantation, and mean survival times were calculated
for all groups of mice. Statistical analyses of mean survival
times were carried out using the Student t-test. Positive
MDR-1 drug resistance reversal activity was evidenced by a
statistically significant (p < 0.05) increase in mean survival
time for groups of mice treated with vincristine plus MDR-1
compound, compared to groups of mice treated with an
equivalent dose of vincristine alone.
Eva lu a tion of MDR-1 r ever sa l a gen ts a ga in st th e
h u m a n epider m oid ca r cin om a KB/8.5 im p la n ted in a th y-
m ic “n u d e” m ice: The MDR human epidermoid carcinoma
KB/8.5 was propagated in RPMI 1640 medium supplemented
with 5% fetal calf serum, gentamicin (50 µg/mL), ITS culture
supplement (Collaborative Medical Products, Bedford, MA),
and colchicine (10 ng/mL) to maintain drug resistance. Colchi-
cine was then removed from the medium, for two in vitro
passages, prior to subcutaneous (sc) implant of 8 × 106 KB/
8.5 tumor cells into athymic “nude” mice (Harlan Sprague-
Dawley, Inc., Indianapolis, IN). Approximately 7-10 days post-
tumor implant, the sc tumors attained a mass of 100-300 mg.
At this time (day 0 of the test period) mice were allocated to
treatment groups of 5-10 mice/group so that the groups had
mean tumor masses as comparable as possible. Drug treat-
ment was initiated 1 day later, according to the following
treatment schedule: doxorubicin was administered as a single
intravenous dose (8 mg/kg), and either placebo or MDR-1
reversal agent was administered in two sc doses (12.5-200
mg/kg/dose) given 2 h before and 2 h after the single iv dose
of doxorubicin. The tumor mass of each mouse and the mean
tumor mass of each treatment group were determined on days
+14 and +21 of the test period. For each treatment group,
the relative tumor growth (RTG) was calculated as follows:
doxorubicin plus MDR-1 reversal compound, compared to
treatment with an equivalent dose of doxorubicin alone.
Ch em istr y. Melting points were determined on a Mel-Temp
apparatus and are uncorrected. Fast atom bombardment
(FAB) mass spectra were determined on a VG-ZAB SE mass
spectrometer. Electron impact (EI) and chemical ionization (CI)
mass spectra were determined on a Finnigan MAT-90 mass
spectrometer. IR spectra were recorded on a Nicolet 20SXB
FT-IR spectrometer. 1H NMR spectra were determined at 300
MHz using a Nicolet QE-300 WB spectrometer; chemical shifts
(δ) given are in parts per million relative to tetramethylsilane.
Where analyses are indicated only by symbols of the elements,
analytical results obtained for these elements were within
0.4% of the theoretical value. Unless otherwise noted all
reagents and solvents obtained from commercial suppliers
were used without further purification.
1,2-Bis(ch lor om eth yl)-4-m eth oxy-5-(p h en ylm eth oxy)-
ben zen e (2). Into a mixture of 12.0 g (56.0 mmol) of 1-meth-
oxy-2-(phenylmethoxy)benzene (1), 18.0 mL (240.0 mmol) of
37% formaldehyde, 2.0 g (6.27 mmol) of zinc iodide, and 200
mL of ether was bubbled HCl gas at a rate such that a gentle
reflux was maintained. The addition of HCl gas was stopped
after 1.5 h, and the solution was stirred for an additional 1.5
h. The solution was poured into ice water and extracted with
ether. The organic layer was washed with brine, dried with
anhydrous Na2SO4, and filtered and the filtrate was evapo-
rated to yield 15.0 g of an oil. This oil was taken up in ether
and passed through a short pad of magnesol with 400 mL of
ether. The volatiles were removed, and the residual oil was
purified by flash chomatography (4:96 ethyl acetate/hexane).
This afforded 1.36 g (8%) of 2 as a white fluffy solid, mp 114-
116 °C: 1H NMR (CDCl3) δ 7.46-7.31 (m, 5H), 6.91 (s, 1H),
6.90 (s, 1H), 5.15 (s, 2H), 4.69 (s, 1H), 4.46 (s, 1H), 3.88 (s,
3H). Anal. Calcd for C16H16Cl2O2‚0.18C15H13ClO2: C, H, Cl.
2,3-Dih yd r o-5-m eth oxy-6-(p h en ylm eth oxy)-2-(p h en yl-
m et h yl)-1H-isoin d ole (3). To a stirred solution of 2.5 mL
(25.0 mmol) of 10 N NaOH, 0.61 mL (0.6 g, 5.6 mmol) of
benzylamine, and 0.1 g (0.36 mmol) of n-tetrabutylammonium
chloride was added a solution of 1.0 g (3.2 mmol) of 2 in 10
mL of toluene under argon, and the resulting mixture was
stirred vigorously for 3 days. The mixture was poured into
water, and the two layers were separated. Following extraction
of the aqueous layer with ethyl acetate, the combined organic
layers were washed with brine. The crude product was dried
with anhydrous Na2SO4 and evaporated to yield an oil, which
was purified by flash chromatography on silica gel eluting with
a gradient of 0.2-1% CH3OH/CH2Cl2 to yield 0.74 g (67%) of
3, as a beige solid, mp 94-96 °C: 1H NMR (CDCl3) δ 7.43-
7.24 (m, 10H), 6.75 (s, 1H), 6.72 (s, 1H), 5.10 (s, 2H), 3.88 (s,
4H), 3.85 (s, 3H), 3.82 (s, 2H); MS (FAB) m/z 346 (M + H+).
Anal. (C23H23NO2‚0.03CH2Cl2) C, H, N.
2,3-Dih yd r o-6-m eth oxy-1H-isoin d ol-5-ol (4a ). A 0.5-g
(1.45-mmol) portion of 3 was warmed in 35 mL of ethanol until
dissolution. After cooling to room temperature, 0.13 g of 35%
palladium hydroxide on carbon was added. The resulting
mixture was shaken on a Parr apparatus at 50 psi of hydrogen
for 21 h. The solution was filtered through a pad of Celite,
and the pad was washed with ethanol. Evaporation of the
volatiles afforded 0.209 g (87%) of 4a as a tan wax: 1H NMR
(CDCl3) δ 6.80 (s, 1H), 6.72 (1H), 4.10 (d, 4H), 3.80 (s, 2H).
Anal. (C9H11NO2‚0.25H2O‚0.125EtOH) C, H, N.
RTG ) mean tumor mass on day +14 or day +21
P r ep a r a tion of Com p ou n d s 6a ,b (Ta ble 2). Rep r esen t-
a tive Exa m p le: 3,4-Dim eth oxy-r-[(4-m eth ylp h en yl)th io]-
ben zen ea ceton itr ile (6a ). A mixture of 4.12 g (19.5 mmol)
of R-chloro-3,4-dimethoxybenzeneacetonitrile (5),44 2.48 g (20.0
mmol) of p-thiocresol, 2.76 g (18.0 mmol) of K2CO3, and 100
mL of CH3CN was stirred at 65 °C for 12 h. After the salts
were filtered, the reaction mixture was concentrated under
vacuum, leaving a golden oil. The oil was dissolved in a
minimum amount of ethyl acetate and diluted with 3 volumes
of hexane. On standing, 3.81 g (65%) of 6a as light-yellow
crystals were obtained: 1H NMR (CDCl3) δ 7.37 (d, 2H, J ) 8
Hz), 7.15 (d, 2H, J ) 8 Hz), 6.82 (m, 3H), 4.87 (s, 1H), 3.88 (s,
Statistical analysis of log RTG was carried out using the
Student t-test. Transformation of the data from absolute mean
tumor mass (mg of tumor mass) to log RTG prior to statistical
analysis has two beneficial effects: (1) it corrects the data for
differences in mean tumor mass of the different treatment
groups at the start of the test (day 0); and (2) the log
transformation of the data more adequately reflects the
exponential growth pattern of sc implanted tumors and makes
the use of the Student t-test more appropriate. Positive drug
resistance reversal activity is indicated by a statistically
significant (p < 0.05) reduction in RTG for groups treated with

2156 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12
Berger et al.
3H), 3.82 (s, 3H), 2.36 (s, 3H); MS (EI) 299 (M+). Anal. (C17H17
NO2S) C, H, N.
-
gradient elution progressing from hexane to CHCl3 followed
by recrystallization from 2-propanol afforded 70.52 g (76%
yield) of 11 as white crystals, mp 62-63.5 °C: 1H NMR (CDCl3)
δ 7.22 (d, 2H, J ) 8 Hz), 7.06 (d, 2H, J ) 8 Hz), 6.88 (s, 1H),
6.74 (s, 2H), 5.56 (s, 1H), 3.99 (s, 2H), 3.85 (s, 3H), 2.30 (s,
3H); MS (CI) 278 (M+). Anal. (C15H16O2S) C, H, S.
(1,1-Dim et h ylet h yl)[2-m et h oxy-5-[[(4-m et h ylp h en yl)-
th io]m eth yl]p h en oxy]d im eth ylsila n e (9e). A mixture of
11.1 g (42.6 mmol) of 11, 7.46 g (49.5 mmol) of tert-butyldi-
methylsilyl chloride, and 0.3 g (2.45 mmol) of 4-(dimethyl-
amino)pyridine was heated under reflux in 40 mL of pyridine.
After cooling, the mixture was poured into water and extracted
with 3 × 75 mL of ether. The ether extract was washed with
brine, dried with anhydrous sodium sulfate, and filtered and
the filtrate was evaporated to yield a brown oil. The crude
residue was distilled at 161-165 °C/0.03 mmHg, affording
15.06 g (94% yield) of 9e as a clear oil: 1H NMR (CDCl3) δ
7.20 (d, 2H, J ) 8 Hz), 7.05 (d, 2H, J ) 8 Hz), 6.75 (m, 3H),
3.96 (s, 2H), 3.77 (s, 3H), 2.30 (s, 3H), 0.97 (s, 9H), 0.11 (s,
6H); MS (CI) 392 (M+). Anal. (C21H30O2SSi) C, H, S.
P r ep a r a tion of Com p ou n d s 6c,d (Ta ble 2). Rep r esen t-
a tive Exa m p le: (3,4-Dim eth oxyp h en yl)(2-p yr id ylth io)-
a ceton itr ile (6c). To a 5 °C solution of 2.66 g (15.0 mmol) of
(3,4-dimethoxyphenyl)acetonitrile (7a ) in 40 mL of THF was
added dropwise a solution of 30.0 mL (30.0 mmol) of a 1.0 M
solution of sodium bis(trimethylsilyl)amide in THF over a
period of 20 min. The resulting mixture was stirred at 5 °C
for 50 min and then added dropwise via cannula to a solution
of 3.33 g (15.0 mmol) of 2,2′-dipyridyl disulfide in 10.0 mL of
THF. After stirring at 5 °C for 40 min and then at room
temperature for 1.5 h, the mixture was poured into ice water.
Following extraction with ethyl acetate, the organic layer was
washed with brine, dried, and concentrated in vacuo to yield
an oil which solidified on standing. The solid was recrystallized
from ethyl acetate/hexane to yield 2.75 g (64%) of 6c, as an
orange solid, mp 108-110 °C: 1H NMR (CDCl3) δ 8.56-8.54
(m, 1H), 7.62-7.56 (m, 1H), 7.24-7.11 (m, 3H), 7.05 (d, 1H, J
) 2 Hz), 6.87 (d, 1H, J ) 8.3 Hz), 6.06 (s, 1H), 3.90 (s, 3H),
3.89 (s, 3H). Anal. (C15H14N2SO2) C, H, N, S.
P r ep a r a tion of Com p ou n d s 12a -k (Ta ble 3): Meth od
A. R ep r esen t a t ive E xa m p le: r-(4-Ch lor ob u t yl)-3,4-d i-
m et h oxy-r-[(4-m et h ylp h en yl)t h io]b en zen ea cet on it r ile
(12b). To a solution of 3.79 g (12.7 mmol) of 13a in 40 mL of
DMSO under an argon atmosphere was added 0.516 g (12.9
mmol) of 60% sodium hydride in oil with stirring. After the
mixture stirred for 1.5 h, 1.50 mL (13.0 mmol) of 1-bromo-4-
chlorobutane was added and the reaction mixture was further
stirred for 5 h. The reaction mixture was poured into 200 mL
of ice water and twice extracted with ether, and the combined
organic extracts were dried with MgSO4. The ethereal solution
was filtered and concentrated in vacuo to give 3.18 g (64%) of
12b as a colorless oil: 1H NMR (CDCl3) δ 7.25 (d, 2H, J ) 8
Hz), 7.07 (d, 2H, J ) 8 Hz), 6.95 (d, 1H, J ) 8 Hz), 6.89 (s,
1H), 6.78 (d, 1H, J ) 8 Hz), 3.88 (s, 3H), 3.84 (s, 3H), 3.48 (t,
2H, J ) 6 Hz), 2.32 (s, 3H), 2.22 (m, 2H), 1.80 (m, 2H), 1.68
(m, 1H), 1.45 (m, 1H); MS (CI) m/z 407 (M + NH4+). Anal.
Calcd for C21H24ClNO2S‚0.01C21H24BrNO2S: C, H, N, S, Cl,
Br.
P r ep a r a tion of Com p ou n d s 9a -c (Ta ble 2). Rep r esen t-
a tive Exa m p le: 5-[[(4-Meth ylp h en yl)th io]m eth yl]-1,3-
ben zod ioxole (9a ). A mixture of 28.5 g (230 mmol) of
p-thiocresol, 250 mL of water, and 8.8 g (220 mmol) of NaOH
was warmed on a steam bath and then stirred vigorously while
cooling to room temperature. Next, 250 mL of toluene, 39.71
g (116.4 mmol) of 4-(methylenedioxy)benzyl chloride (8a ) in
30 mL of CH2Cl2, and 3.29 g (11.0 mmol) of tetrabutylammo-
nium chloride were added to the reaction. The mixture was
stirred at room temperature for 1 h; then 4.4 g (11.0 mmol) of
NaOH was added. After stirring for 12 h, the layers were
separated and the organic layer was washed with first with 1
N NaOH and then with brine. The organic layer was dried
with Na2SO4 and concentrated under vacuum to a brown oil.
Flash chromatography on silica gel, using a hexane/CHCl3 and
then a CH3OH/CHCl3 gradient, gave 24.01 g (80%) of 9a as a
clear oil: 1H NMR (CDCl3) δ 7.20 (d, 2H, J ) 8 Hz), 7.06 (d,
2H, J ) 8 Hz), 6.79 (s, 1H), 6.68 (s, 2H), 5.92 (s, 2H), 3.98 (s,
2H), 2.30 (s, 3H); MS (EI) 259 (M+). Anal. (C15H14O2S) C, H,
S.
1,2-Dim eth oxy-4-[[(4-m eth ylp h en yl)th io]m eth yl]ben -
zen e (9d ). To a solution of 0.745 g (6.00 mmol) of p-thiocresol
in 5 mL of DMF was added 0.24 g (6.0 mmol) of 60% sodium
hydride in oil. After the effervescence had stopped, 1.33 g (5.76
mmol.) of 3,4-dimethoxybenzyl bromide45 (8d ) was added. The
reaction was left to stand for 12 h and then evaporated under
vacuum and the residue partitioned between CHCl3 and water.
The organic layer was dried (sodium sulfate) and evaporated
again. Chromatography (silica gel with gradient elution pro-
gressing from hexane/CHCl3 to CH3OH/CHCl3) gave 1.35 g of
crystalline solid, mp 49-51 °C. Recrystallization from hexane,
with filtration of the hot solution through diatomaceous silica,
gave 1.29 g (82%) of white needles, mp 50-52 °C: 1H NMR
(CDCl3) δ 7.21 (d, 2H, J ) 8 Hz), 7.06 (d, 2H, J ) 8 Hz), 6.76
(m, 3H), 4.02 (s, 2H), 3.84 (s, 3H), 3.80 (s, 3H), 2.30 (s, 3H);
MS (EI) 274 (M+). Anal. (C16H18O2S) C, H, S.
2-Meth oxy-5-[[(4-m eth ylph en yl)th io]m eth yl]ph en ol (11).
A mixture of 53.34 g (430 mmol) of p-thiocresol, 1 L of water,
24.08 g (430 mmol) of KOH, 100 mL of toluene, and 2.54 g
(7.5 mmol) of tetrabutylammonium hydrogen sulfate was
stirred for 1 h. Meanwhile, a suspension of 22.10 g (143 mmol)
of 3-hydroxy-4-methoxybenzyl alcohol (10) in 720 mL of
toluene was cooled and stirred in an ice bath while hydrogen
bromide was bubbled in at a rapid rate for 30 min. The clear
reddish supernatant, containing 3-hydroxy-4-methoxybenzyl
bromide, was decanted from a small quantity of gum into the
thiocresol solution. The 3-hydroxy-4-methoxybenzyl bromide
formation was repeated twice more until a total of 54.97 g
(356.7 mmol) of 17 had been consumed. After the reaction was
stirred for 12 h, the layers were separated, and the organic
layer was washed with dilute HCl. After drying with Na2SO4,
the solvent was evaporated under vacuum. On standing, the
residue crystallized. Chromatography on silica gel with a
P r ep a r a tion of Com p ou n d s 13a -h (Ta ble 3): Meth od
A′. Rep r esen ta tive Exa m p le: 4-[4-Ch lor o-1-[(4-m eth yl-
p h en yl)th io]bu tyl]-1,2-d im eth oxyben zen e (13a ). To a so-
lution of 13.72 g (50.0 mmol) of 9d and a crystal of phenan-
throline in 250 mL of dry THF under nitrogen at -78 °C was
added 21.0 mL (52.5 mmol) of 2.5 M n-butyllithium in hexane
via syringe. After 10 min, the brown solution was warmed to
0 °C for 1 h, then it was recooled to -78 °C, and 8.02 mL (75.0
mmol) of 1-bromo-3-chloropropane was added via syringe. The
reaction was warmed to room temperature and left for for 12
h. To the light-yellow reaction was added 10 mL of ethyl
acetate, and the resulting solution was concentrated in vacuo.
The residue was partitioned between ethyl acetate and water
and the organic phase dried (Na2SO4) and concentrated under
vacuum to a yellow oil. Purification by chromatography on
silica gel with gradient elution progressing from hexane/CHCl3
to CHCl3/CH3OH afforded 16.47 g (94% yield) of 13a as a
yellow oil: 1H NMR (CDCl3) δ 7.15 (d, 2H, J ) 8 Hz), 7.02 (d,
2H, J ) 8 Hz), 6.72 (m, 3H), 4.01 (m, 1H), 3.86 (s, 3H), 3.82 (s,
3H), 3.49 (t, 2H, J ) 7 Hz), 2.29 (s, 3H), 2.05 (m, 2H), 1.82 (m,
2H); MS (CI) m/z 227, 229 (M + H+ - C7H7S). Anal. (C19H23
ClO2S‚0.055CHCl3) C, H, Cl, S.
-
P r ep a r a tion of Com p ou n d s 15a -e′ (Ta bles 4-6):
Meth od B. Rep r esen ta tive Exa m p le: r-(3,4-Dim eth oxy-
p h en yl)-3,4-d ih yd r o-6,7-d im eth oxy-r-[(4-m eth ylp h en yl)-
th io]-2(1H)-isoqu in olin eh ep ta n en itr ile (15h ). To a solu-
tion of 4.65 g (11.5 mmol) of 19e in 60 mL of DMF, under
argon, were added 4.24 g (30.7 mmol) of K2CO3, 0.511 g (3.1
mmol) of KI, and 4.25 g (18.5 mmol) of 14a . The reaction
mixture was then heated to 95 °C for 4 h and cooled to ambient
temperature. After 12 h, the solvent was evaporated and the
residue was partitioned between ethyl ether and water. The
ether layer was dried with MgSO4, filtered, and evaporated
to give a brown oil. Silica gel chromatography (ethyl acetate)
afforded 3.67 g (56%) of 15h , as a beige gum: 1H NMR (CDCl3)

Novel MDR Reversal Agents
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12 2157
δ 7.24 (d, 2H, J ) 8 Hz), 7.07 (d, 2H, J ) 8 Hz), 6.95 (d, 1H,
J ) 8 Hz), 6.77 (d, 1H, J ) 8 Hz), 6.58 (s, 1H), 6.51 (s, 1H),
3.88 (s, 3H), 3.84 (s, 9H), 3.51 (br s, 2H), 2.80 (t, 2H, J ) 6
Hz), 2.68 (t, 2H, J ) 6 Hz), 2.44 (t, 2H, J ) 7 Hz), 2.32 (s, 3H),
2.20 (t, 2H, J ) 7 Hz), 1.55 (m, 3H), 1.36 (m, 3H); HR FAB
(MS) calcd for C33H41N2O4S (M + H) 561.2287, found 561.2287.
Anal. Calcd for C33H40N2O4S‚0.5H2O: C, H, N, S.
Hz), 7.01 (d, 1H, J ) 2 Hz), 6.82 (d, 1H, J ) 8 Hz), 3.89 (s,
3H), 3.87 (s, 3H), 3.61 (t, 2H, J ) 6 Hz), 3.04 (t, 2H, J ) 2 Hz),
2.34 (s, 2H), 1.83 (br s, 1H); HR MS (EI) calcd for C22H23NO3S
381.1398, found 381.1386. Anal. (C22H23NO3S) C, H, N, S.
(c) r-[5-(3,4-Dih yd r o-6,7-d im eth oxy-2(1H)-isoqu in olin -
yl)-2-p en tyn yl]-3,4-d im eth oxy-r-[(4-m eth ylp h en yl)th io]-
ben zen ea ceton itr ile (19). To a solution containing 0.18 g
(0.47 mmol) of 18 in 5 mL of CH2Cl2 was added 0.060 g (0.52
mmol) of mesyl chloride. The solution was cooled to 0 °C, and
0.1 mL (0.57 mmol) of diisopropylethylamine was added. The
mixture was warmed to room temperature and stirred for 5
h. The solvent was removed with a stream of argon; then 5
mL of acetonitrile, 0.25 mL (2.61 mmol) of diisopropylethyl-
amine, and 0.087 g (1.41 mmol) of 14a were added. After
heating to reflux under argon for 4 h, the reaction mixture
was cooled and evaporated and the residue partitioned be-
tween ethyl acetate and water. The organic layer was washed
with saturated brine, dried, and concentrated in vacuo. Silica
gel chromatography (CH2Cl2/CH3OH (95:5)) afforded 0.065 g
(25%) of 19 as a yellow oil: 1H NMR (CDCl3) δ 7.38 (d, 2H, J
) 8 Hz), 7.12 (d, 2H, J ) 8 Hz), 7.09 (dd, 1H, J ) 8 Hz, J ) 2
Hz), 7.02 (d, 1H, J ) 2 Hz), 6.79 (d, 1H, J ) 8 Hz), 6.56 (s,
1H), 6.50 (s, 1H), 3.87 (s, 3H), 3.86 (s, 3H), 3.83 (s, 3H), 3.81
(s, 3H), 3.55 (s, 2H), 3.03 (t, 2H, J ) 2 Hz), 2.78 (t, 2H, J ) 5
Hz), 2.70 (d, 2H, J ) 5 Hz) 2.62 (d, 2H, J ) 7 Hz) 2.34 (s, 2H);
HR MS (EI) calcd for C26H29N2O4 (M - C7H7S) 433.2127, found
433.2120. Anal. (C33H36N2O4S‚0.1H2O) C, H, N, S.
Meth od D. (a ) 3-[2-Cya n o-2-(3,4-d im eth oxyp h en yl)-2-
[(4-m eth ylp h en yl)th io]eth yl]ben zoic Acid (21). To a -78
°C solution of 1.86 g (6.2 mmol) of 13a in 15 mL of THF was
added dropwise a solution of 2.9 mL (6.34 mmol) of n-
butyllithium (2.2 M) in hexane. This solution was stirred at
-78 °C for 35 min. A solution of 0.67 g (3.1 mmol) of
m-carboxybenzyl bromide (20) in 5 mL of anhydrous THF was
added dropwise; the mixture was stirred at -78 °C for 40 min
and then at room temperature for 15 min. The mixture was
quenched with water and extracted with ethyl acetate. The
extract was washed with brine, dried with MgSO4, and
evaporated in vacuo to yield a yellow oil. Flash chromatogra-
phy (95:5 CH2Cl2/CH3OH) afforded 1.15 g (86%) of 21, as a
yellow foam: 1H NMR (CDCl3) δ 7.95 (d, 1H, J ) 7.5 Hz), 7.73
(s, 1H), 7.37 (d, 2H, J ) 8 Hz), 7.36-7.2 (m, 2H), 7.1 (d, 2H,
J ) 8 Hz), 6.98-6.92 (m, 2H), 6.75 (d, 1H, J ) 8 Hz), 3.87 (s,
3H), 3.83 (s, 3H), 3.49 (s, 2H), 2.34 (s, 3H). Anal. (C25H23NSO4‚
0.3H2O) C, H, N, S.
(b) r-(3,4-Dim eth oxyp h en yl)-3-(h yd r oxym eth yl)-r-[(4-
m eth ylp h en yl)th io]ben zen ep r op a n en itr ile (22). A mix-
ture of 1.0 g (2.3 mmol) of 21 and 0.7 mL (7.0 mmol) of borane-
methyl sulfide complex (10 M) in 50 mL of THF was stirred
at room temperature for 18 h. The mixture was quenched with
CH3OH, treated with HCl gas for 5 min, and stirred, and the
solvent was evaporated to yield a white foam. After the foam
was dissolved in water, the solution was made basic with 5 N
sodium hydroxide and extracted with ethyl acetate. The extract
was washed with brine, dried with MgSO4, filtered, and
evaporated in vacuo to yield a yellow oil. This was purified by
flash chromatography (98:2 CH2Cl2/CH3OH) to afford 0.55 g
(52%) of 22 as a colorless oil: 1H NMR (CDCl3) δ 7.35 (d, 1H,
J ) 2 Hz), 7.34 (d, 1H, J ) 2 Hz), 7.22-7.08 (m, 4H), 6.95-
6.87 (m, 4H), 6.74 (d, 1H, J ) 8 Hz), 4.57 (s, 2H), 3.87 (s, 3H),
3.82 (s, 3H), 3.42 (s, 2H), 2.33 (s, 3H). Anal. (C25H25NO3S) C,
H, N, S.
(c) 3-(Ch lor om et h yl)-r-(3,4-d im et h oxyp h en yl)-r-[(4-
m eth ylp h en yl)th io]ben zen ep r op a n en itr ile (23). A mix-
ture of 0.1 mL (1.37 mmol) of thionyl chloride and 0.12 mL
(1.42 mmol) of DMF was stirred at room temperature for 30
min. To this was added a solution of 0.34 g (0.81 mmol) of 22
in 1.0 mL of DMF. The resulting mixture was heated at 70 °C
for 17 h, cooled to room temperature, and poured into water.
Following extraction with ether, the ether extract was washed
with brine, dried with MgSO4, filtered, and evaporated in
vacuo to yield an oil. This was purified by flash chromatog-
raphy (9:1 hexane/ethyl acetate) to afford 0.27 g (76%) of 23
as a colorless oil: 1H NMR (CDCl3) δ 7.38 (d, 2H, J ) 8 Hz),
Meth od B′. Rep r esen ta tive Exa m p le: 2-[6-(3,4-Di-
m eth oxyp h en yl)-6-[(4-m eth ylp h en yl)th io]h exyl]-1,2,3,4-
tetr a h yd r o-6-m eth oxyisoqu in olin e (15p ). To a solution of
4.23 g (10 mmol) of 13b in 40 mL of CH3CN were added 3.88
g (20 mmol) of 14c, 1.74 mL (10 mmol) of diisopropylethyl-
amine, and 0.01 g (0.067 mmol) of NaI. The solution was
heated at reflux for 43 h and cooled to room temperature. The
reaction mixture was partitioned between CHCl3 and satu-
rated K2CO3 and the organic layer dried with Na2SO4. Fol-
lowing evaporation in vacuo, the residue was chromatographed
on silica gel using a gradient elution of hexane/CHCl3 to CHCl3/
CH3OH. The residue from the chromatography was dissolved
in ether and passed through diatomaceous silica. This afforded
4.4 g (87% yield) of 15p as a yellow oil: 1H NMR (CDCl3) δ
7.14 (d, 2H, J ) 8 Hz), 7.01 (d, 2H, J ) 8 Hz), 6.72 (m, 6H),
4.00 (m, 1H), 3.85 (s, 3H), 3.81 (s, 3H), 3.77 (s, 3H), 3.52 (s,
2H), 2.86 (m, 2H), 2.66 (m, 2H), 2.43 (m, 2H), 2.28 (s, 3H),
1.90 (m, 2H), 1.54 (m, 2H), 1.33 (m, 4H); MS (FAB) m/z 506
(M + H+). Anal. (C31H39NO3S) C, H, N, S.
[(5-Br om o-3-p en tyn yl)oxy](1,1-d im eth yleth yl)d im eth -
ylsila n e (16). A solution of 0.20 g (0.93 mmol) of 5-[[(1,1-
dimethylethyl)dimethylsilyl]oxy]-2-pentyn-1-ol46 in 6 mL of
CH2Cl2 under argon was cooled to -23 °C. To this were added
0.183 g (1.03 mmol) of N-bromosuccinimide and 0.294 g (1.12
mmol) of triphenylphosphine. After stirring at -23 °C for 1 h,
the reaction was quenched with 30 mL of saturated NaHCO3.
An additional 50 mL of ether was added to the mixture, and
the layers were separated. The organic layer was washed with
30 mL of brine, dried with MgSO4, and concentrated in vacuo.
Following purification by silica gel chromatography (98:2
hexane/ethyl acetate), 0.12 g (46%) of 16 was obtained as a
clear oil: 1H NMR (CDCl3) δ 3.91 (t, 2H, J ) 3 Hz), 3.72 (t,
2H, J ) 7 Hz), 2.49-2.43 (m, 2H), 0.90 (s, 9H), 0.08 (s, 6H);
MS (CI) m/z 277. Anal. (C11H21BrOSi‚0.1C6H14) C, H, Br.
Meth od C. (a ) r-[5-[[(1,1-Dim eth yleth yl)d im eth ylsilyl]-
oxy]-2-pen tyn yl]-3,4-dim eth oxy-r-[(4-m eth ylph en yl)th io]-
ben zen ea ceton itr ile (17). To a solution of 0.108 g (0.36
mmol) of 6a in 3 mL of anhydrous THF under an argon
atmosphere was added 0.017 g (0.43 mmol) of 60% sodium
hydride in mineral oil. After the mixture stirred for 1 h, 0.10
g (0.36 mmol) of 16 was added to the reaction mixture. The
reaction was stirred for 1 h and then poured into 10 mL of
water. The aqueous layer was extracted with 3 × 30 mL of
ether. The combined ether layers were dried with MgSO4, and
solvent was removed in vacuo. Silica gel chromatography (5:1
hexane/ethyl acetate) provided 0.14 g (78%) of 17 as a clear
oil: 1H NMR (CDCl3) δ 7.38 (d, 2H, J ) 8 Hz), 7.12 (d, 2H, J
) 8 Hz), 7.08 (dd, 1H, J ) 8 Hz, J ) 2 Hz), 7.02 (d, 1H, J ) 2
Hz), 6.81 (d, 1H, J ) 8 Hz), 3.89 (s, 3H), 3.87 (s, 3H), 3.59 (t,
2H, J ) 7 Hz), 3.02 (t, 2H, J ) 2 Hz), 2.34 (s, 2H), 0.86 (s,
9H), 0.02 (s, 6H); HR MS (FAB) calcd for C28H37NO3SSiNa (M
+ Na) 518.2161, found 518.2166. Anal. (C28H37NO3SSi) C, H,
N, S.
(b) r-(5-Hydr oxy-2-pen tyn yl)-3,4-dim eth oxy-r-[(4-m eth -
ylp h en yl)th io]ben zen ea ceton itr ile (18). A 0.289-g (0.58
mmol) portion of 17 was dissolved in 5 mL of THF and cooled
to 0 °C. To this was added 0.90 mL (0.90 mmol) of 1.0 M
tetrabutylammonium fluoride (in THF) while stirring. The
reaction mixture was warmed to room temperature and stirred
for 3 h. The reaction was quenched with 5 mL of saturated
aqueous NaHCO3 solution and 5 mL of water. Following
extraction of the aqueous phase with 3 × 10 mL of ether, the
combined organic layers were washed with 10 mL of brine and
dried with MgSO4. The solvent was evaporated in vacuo. Silica
gel chromatography (1:1 hexane/ethyl acetate) afforded 0.184
g (83%) of 18 as a clear oil: 1H NMR (CDCl3) δ 7.37 (d, 2H, J
) 8 Hz), 7.12 (d, 2H, J ) 8 Hz), 7.08 (dd, 1H, J ) 8 Hz, J ) 2

2158 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12
Berger et al.
7.29-7.09 (m, 4H), 6.96-6.91 (m, 4H), 6.73 (d, 1H, J ) 8 Hz),
4.49-4.44 (m, 2H), 3.90 (s, 3H), 3.8 (s, 3H), 3.42 (s, 2H), 2.33
(s, 3H). Anal. (C25H24ClNO2S) C, H, N, Cl, S.
system (150 mL of ammonium hydroxide in water was
adjusted to a pH of 4.00 with acetic acid and then diluted to
7.875 L with water, 5700 mL of acetonitrile, and 1425 mL of
CH3OH). Concentration in vacuo afforded 0.85 g of 28a as a
yellow oil: 1H NMR (CDCl3) δ 7.25 (d, 2H, J ) 8 Hz), 7.06 (d,
2H, J ) 8 Hz), 6.96 (d, 1H, J ) 8 Hz), 6.90 (s, 1H), 6.77 (d,
1H, J ) 8 Hz), 6.73 (s, 2H), 3.85 (m, 16H), 2.67 (t, 2H, J ) 6
Hz), 2.31 (s, 3H), 2.20 (t, 2H, J ) 6 Hz), 1.55 (m, 3H), 1.40 (m,
3H); HR MS (EI) calcd for C32H38N2O4S 546.2553, found
546.2569. Anal. (C32H38N2O4S) C, H, N, S.
P r ep a r a tion of Com p ou n d s 35a -e (Ta ble 4): Meth od
F . R ep r esen t a t ive E xa m p le (35a ): (a ) E t h yl 6-(3,4-
Dim eth oxy-(E)-oxoben zen e)h exa n oa te (31a ). To a 0 °C
solution of 1.52 g (11.0 mmol) of veratrole (29) in 15 mL of
CH2Cl2 was added 2.66 g (20.0 mmol) of anhydrous aluminum
chloride in portions so as to maintain the temperature at 0
°C. A solution of 1.93 g (10.0 mmol) of adipic acid chloride
monoethyl ester (30) in 10 mL of CH2Cl2 was added dropwise.
The resulting mixture was stirred at 0 °C for 4 h and warmed
to room temperature for 12 h. The crude product was poured
into a mixture of ice/concentrated HCl. The aqueous layer was
further extracted with CH2Cl2. The combined organic layers
were washed with brine and dried (MgSO4). The filtrate was
evaporated to an oil, which was purified by silica gel chroma-
tography (hexane/ethyl acetate, 4:1) to afford 2.76 g (94%) of
31a as a white solid, mp 50-52 °C: 1H NMR (CDCl3) δ 7.58
(dd, 1H, J ) 2 Hz, J ) 8 Hz), 7.53 (d, 1H, J ) 2 Hz), 6.90 (d,
1H, J ) 8 Hz), 4.13 (q, 2H, J ) 7 Hz), 3.95 (s, 3H), 3.94 (s,
3H), 2.96 (t, 2H, J ) 7 Hz), 2.36 (t, 2H, J ) 7 Hz), 1.78-1.72
(m, 4H), 1.26 (t, 3H, J ) 7 Hz). Anal. (C16H22O5) C, H.
(d ) 3-(3,4-Dih yd r o-6,7-d im eth oxy-2(1H)-isoqu in olyl)-r-
(3,4-d im e t h oxyp h e n yl)-r-[(4-m e t h ylp h e n yl)t h io]b e n -
zen ep r op a n en itr ile (24). A mixture of 0.21 g (0.47 mmol) of
32, 0.091 g (0.47 mmol) of 14a , and 0.3 g (2.16 mmol) of
anhydrous K2CO3 in 2.0 mL of DMF was heated at 85 °C for
4 h, cooled, poured into water, and extracted with ethyl
acetate. The extract was washed with brine and dried with
MgSO4. The filtrate was evaporated in vacuo to yield an oil,
which was purified by flash chromatography (98:2 CH2Cl2/
CH3OH) and then precipitated from cold EtOH to afford 0.18
g (63%) of 24 as a yellow glassy solid: 1H NMR (CDCl3) δ 7.35-
6.88 (m, 10H), 6.70 (d, 1H, J ) 8.5 Hz), 6.58 (s, 1H), 6.48 (s,
1H), 3.86-3.79 (m, 12H), 3.58 (s, 2H), 3.50 (s, 2H), 3.43 (s,
2H), 2.78-2.71 (m, 2H), 2.67-2.55 (m, 2H), 2.32 (s, 3H); MS
(FAB) 595 (M + H+). Anal. (C36H38N2O4S‚0.7EtOH) C, H, N,
S.
P r ep a r a tion of Com p ou n d s 28a -c (Ta bles 4 a n d 5):
Meth od E. Rep r esen ta tive Exa m p le (28a ): (a ) r-(3,4-
Dim eth oxyph en yl)-1,3-dih ydr o-r-[(4-m eth ylph en yl)th io]-
1,3-d ioxo-2H-isoin d ole-2-h ep ta n en itr ile (25a ). A mixture
of 4.50 g (11.1 mmol) of 12e, 48 g (320 mmol) of NaI, and 155
mL of acetone was heated at reflux for 12 h, protected from
light. The volatiles were evaporated in vacuo, and 500 mL of
ether was added to the residue. This was filtered and evapo-
rated. Silica gel chromatography (hexane/ethyl acetate, 2:1)
afforded 6.24 g of R-(5-iodopentyl)-3,4-dimethoxy-R-[(4-meth-
ylphenyl)thio]benzeneacetonitrile as a clear oil.
(b ) 6-(3,4-Dim et h oxy-(E)-oxob en zen e)h exa n oic Acid
(31b). A mixture of 2.5 g (8.5 mmol) of 31a and 16 mL of 10%
ethanolic NaOH was stirred at room temperature for 3 h. The
mixture was diluted with water, and the ethanol was evapo-
rated in vacuo. Concentrated HCl was added to acidify the
solution. The solid obtained was collected by filtration, washed
with water, and dried in vacuo to afford 2.2 g (97%) of 31b, as
a beige solid, mp 123-125 °C: 1H NMR (CDCl3) δ 7.58 (dd,
1H, J ) 2 Hz, J ) 8.5 Hz), 7.53 (d, 1H, J ) 2 Hz), 6.89 (d, 1H,
J ) 8.5 Hz), 3.95 (s, 3H), 3.94 (s, 3H), 2.97 (t, 2H, J ) 7 Hz),
2.43 (t, 2H, J ) 7 Hz), 1.81-1.74 (m, 4H). Anal. (C14H18O5‚
0.1H2O) C, H.
(c) 2-[6-(3,4-Dim eth oxyp h en yl)-1,6-d ioxoh exyl]-1,2,3,4-
tetr a h yd r o-6,7-d im eth oxyisoqu in olin e (32). To a suspen-
sion of 1.73 g (7.5 mmol) of 14a in 20 mL of DMF was added
4.2 mL (3.05 g, 30.13 mmol) of triethylamine. The solution was
stirred for 5 min and then cooled with an ice bath. To this
solution were added 2.0 g (7.5 mmol) of 31b followed by 1.5 g
(9.2 mmol) of diethyl cyanophosphonate. The mixture was
stirred at room temperature for 12 h. The crude product
mixture was poured into water, extracted with ethyl acetate,
washed with 2 N HCl, a saturated solution of NaHCO3, and
brine, and dried (MgSO4). Following evaporation of the solvent,
3.15 g (95%) of 32 was obtained as a white solid, mp 101-103
°C: 1H NMR (CDCl3) δ 7.60 (dd, 1H, J ) 8 Hz, J ) 2 Hz), 7.53
(br s, 1H), 6.90-6.86 (m, 1H), 6.63-6.60 (m, 2H), 4.65 (s, 1H),
4.56 (s, 1H), 3.95-3.93 (m, 6H), 3.87-3.85 (m, 6H), 3.83 (t,
1H, J ) 6 Hz), 3.67 (t, 1H, J ) 6 Hz), 2.99 (t, 2H, J ) 7 Hz),
2.82 (t, 1H, J ) 6 Hz), 2.76 (t, 1H, J ) 6 Hz), 2.47 (t, 2H, J )
7 Hz), 1.88-1.76 (m, 4H). Anal. (C25H31NO6‚0.2H2O) C, H, N.
(d ) 2-[6-(3,4-Dim eth oxyp h en yl)-6-h yd r oxy-1-oxoh exyl]-
1,2,3,4-tetr ah ydr o-6,7-dim eth oxyisoqu in olin e (33a). A mix-
ture of 1.4 g (3.1 mmol) of 32 and 0.28 g (7.4 mmol) of sodium
borohydride in 25 mL of CH3OH was stirred at room temper-
ature for 12 h. The mixture was poured into water and
extracted with ethyl acetate. The ethyl acetate extract was
washed with brine and dried (Na2SO4). Evaporation of the
filtrate afforded 1.4 g of 33a as a colorless oil, which was used
in the subsequent step without further purification.
(e) 2-[6-(Cycloh exylt h io)-6-(3,4-d im et h oxyp h en yl)-1-
oxoh e xyl]-1,2,3,4-t e t r a h yd r o-6,7-d im e t h oxyisoq u in o-
lin e (34a ). To a solution of 1.0 g (2.2 mmol) of crude 33a in
10 mL of 1,2-dichloroethane were added 0.72 g (2.2 mmol) of
zinc iodide followed by the addition of 0.30 g (2.6 mmol) of
A mixture of 6.12 g (11.1 mmol) of this intermediate, 2.22 g
(12.0 mmol) of potassium phthalimide, and 50 mL of DMF was
heated on a steam bath for 2 h. After evaporation of solvent,
the residue was partitioned between ether and water, with
agitation. The organic layer was washed with 1 N NaOH,
followed by aqueous K2CO3. The solution was dried with
Na2SO4, and evaporated in vacuo. Chromatography on silica
gel, with gradient elution progressing from hexane/CHCl3 to
CHCl3/CH3OH, afforded 4.77 g (83% yield) of 25a as a
fluorescent oil: 1H NMR (CDCl3) δ 7.84 (m, 2H), 7.71 (m, 2H),
7.23 (d, 2H, J ) 8 Hz), 7.06 (d, 2H, J ) 8 Hz), 6.88 (m, 3H),
3.88 (s, 3H), 3.83 (s, 3H), 3.63 (t, 2H, J ) 7 Hz), 1.65 (m, 2H),
1.34 (m, 4H,); MS (FAB) m/z 515 (M + H+). Anal. (C30H30N2O4S‚
0.375H2O) C, H, N, S.
(b) r-(5-Am in open tyl)-3,4-dim eth oxy-r-[(4-m eth ylph en -
yl)th io]ben zen ea ceton itr ile (26a ). A mixture of 4.72 g (9.17
mmol) of 25a , 100 mL of ethanol, and 7.0 mL (225.0 mmol) of
hydrazine hydrate was heated at reflux. After 30 min, a heavy
precipitate formed. After 1.3 h, the reaction was cooled to room
temperature and concentrated in vacuo. The residue was
warmed on a steam bath with 2 N HCl for 10 min. Diatoma-
ceous silica and CHCl3 were added; the mixture was shaken
thoroughly and then filtered through more diatomaceous silica.
The organic phase was washed first with aqueous ammonia
and then with brine. After drying with Na2SO4, the filtrate
was concentrated in vacuo to provide 3.57 g (99.8%) of 26a as
a yellow oil: 1H NMR (CDCl3 + TFA) δ 7.21 (d, 2H, J ) 8 Hz),
7.09 (d, 2H, J ) 8 Hz), 6.88 (m, 3H), 3.89 (s, 3H), 3.84 (s, 3H),
3.10 (m, 2H), 2.33 (s, 3H), 2.23 (m, 2H), 1.70 (m, 2H), 1.43 (m,
4H); MS (CI) m/z 385 (M + H+). Anal. (C22H28N2O2S‚0.58H2O)
C, H, N, S.
(c) r-(3,4-Dim eth oxyph en yl)-1,3-dih ydr o-5,6-dim eth oxy-
r-[(4-m eth ylp h en yl) th io]-2H-isoin d ole-2-h ep ta n en itr ile
(28a ). To a solution of 3.03 g (7.88 mmol) of 26a , 1.85 g (7.88
mmol) of 27a ,24 20 mL of toluene, and 0.22 g (0.79 mmol) of
tetrabutylammonium chloride was added 2.0 g (50 mmol) of
NaOH in 40 mL of water. The mixture was stirred for 48 h.
After the pH of the aqueous layer was adjusted to 9 with solid
NaHCO3, the layers were separated and the aqueous layer was
further extracted with CHCl3. The combined organic extracts
were dried with NaSO4, passed through a pad of hydrous
magnesium silicate, and concentrated in vacuo to a brown
gum. This gum was purified by HPLC using a reverse-phase
C-18 radial-pak column (6 cm × 30 cm) with a buffered solvent

Novel MDR Reversal Agents
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12 2159
cyclohexyl mercaptan.26 The resulting mixture was stirred at
room temperature for 1 h, poured into water and extracted
with CH2Cl2. The CH2Cl2 layer was washed with NaOH and
brine. After drying (MgSO4), the solvent was evaporated in
vacuo to afford 1.06 g (89%) of 34a , as a glassy solid: 1H NMR
(CDCl3) δ 6.88 (d, 1H, J ) 1.5 Hz), 6.79-6.78 (m, 2H), 6.63-
6.56 (m, 2H), 4.65 (s, 1H), 4.56 (s, 1H), 3.90-3.85 (m, 12H),
3.75 (m, 2H), 3.62 (t, 1H, J ) 5.9 Hz), 2.85-2.70 (m, 2H), 2.34
(t, 3H, J ) 7.6), 2.00-1.20 (m, 16H). Anal. (C31H43NO5S) C,
H, N, S.
(f) 2-[6-(Cycloh exylth io)-6-(3,4-dim eth oxyph en yl)h exyl]-
1,2,3,4-tetr ah ydr o-6,7-dim eth oxyisoqu in olin e (35a). A mix-
ture of 0.95 g (1.75 mmol) of 34a and 0.35 mL (3.5 mmol) of
borane-methyl sulfide complex (10 M) in 10 mL of THF was
heated at reflux for 2 h, cooled, and quenched with CH3OH.
Volatiles were evaporated in vacuo, and the residue was taken
up in 10 mL of ethanol. To this was added 8 mL of 1 N NaOH,
and the resulting mixture was heated at reflux for 2 h. The
solution was cooled, diluted with water, and extracted with
ethyl acetate. The extract was washed with brine, dried
(MgSO4), and filtered. Evaporation of the filtrate yielded an
oil, which was purified by flash chromatography (2% CH3OH/
CH2Cl2) to afford 0.71 g (77%) of 35a as a light-yellow oil: 1H
NMR (CDCl3) δ 6.90 (s, 1H), 6.82 (m, 2H), 6.58 (s, 1H), 6.51
(s, 1H), 3.89 (s, 3H), 3.88 (s, 3H), 3.83 (s, 3H), 3.82 (s, 3H),
3.51 (s, 2H), 2.80 (t, 2H, J ) 6.5 Hz), 2.68 (t, 2H, J ) 6.5 Hz),
2.44 (t, 2H, J ) 8.5 Hz), 2.33 (m, 1H), 2.20-1.00 (m, 19H).
Anal. (C31H45O4NS‚0.5EtOH) C, H, N, S.
3.75 (t, 2H, J ) 7 Hz), 3.54 (s, 2H), 2.83 (m, 2H), 2.71 (m, 2H),
2.47 (m, 2H), 2.28 (s, 3H), 1.90 (m, 2H), 1.55 (m, 2H), 1.30 (br
s, 6H); MS (CI) m/z 598 (M + H+). Anal. (C34H44ClNO4S) C,
H, Cl, N, S.
(b) 1,2,3,4-Tetr a h yd r o-2-[7-[3-[2-(1H-im id a zol-1-yl)eth -
oxy]-4-m eth oxyp h en yl]-7-[(4-m eth ylp h en yl)th io]h ep tyl]-
6,7-d im eth oxyisoqu in olin e (37b). To 0.76 g (11.0 mmol) of
imidazole in 5 mL of dry DMF was added 0.18 g (4.5 mmol) of
60% sodium hydride in oil. When the effervescence subsided,
0.26 g (1.7 mmol) of NaI was added, followed by 1.12 g (1.87
mmol) 37a in 7 mL of DMF. After stirring for 48 h, the solution
was concentrated in vacuo and the residue partitioned between
ethyl acetate and water. The organic layer was extracted with
1 N HCl, the acid layer basified with aqueous ammonia, and
the resulting oil extracted into ethyl acetate. After drying with
Na2SO4, the extract was concentrated in vacuo to 0.53 g of a
tan oil. Flash chromatography on silica gel (gradient elution:
hexane to CHCl3 to CH3OH) gave a tan oil which was taken
up in ethyl acetate and passed through a pad of hydrous
magnesium silicate. This afforded 0.194 g (16% yield) of 37b
as a yellow gum: 1H NMR (CDCl3) δ 7.62 (s, 1H), 7.12 (s, 1H),
7.08 (d, 2H, J ) 8), 7.07 (s, 1H), 6.99 (d, 2H, J ) 8), 6.76 (m,
2H), 6.64 (m, 1H), 6.59 (s, 1H), 6.51 (s, 1H), 4.3 (m, 2H), 4.17
(m, 2H), 3.95 (m, 1H), 3.83 (m, 9H), 3.53 (s, 2H), 2.81 (m, 2H),
2.71 (m, 2H), 2.45 (m, 2H), 2.26 (s, 3H), 1.88 (m, 2H), 1.55 (m,
2H), 1.29 (m, 6H); MS (CI) m/z 630 (M + H+). Anal.
(C37H47N3O4S‚H2O) C, H, N, S.
P r ep a r a tion of Com p ou n d s 38a -c a n d 39a -c (Ta ble
4): Meth od G′. Rep r esen ta tive Exa m p le: (a ) 7-(2-
Ch lor oet h oxy)-r-(3,4-d im et h oxyp h en yl)-3,4-d ih yd r o-6-
m et h oxy-r-[(4-m et h ylp h en yl)t h io]-2(1H )-isoq u in olin e-
h ep ta n en itr ile (38a ). A mixture of 0.93 g (1.70 mmol) of 15l,
10 mL of 1 N NaOH, 0.92 mL (5.1 mmol) of 2-chloroethyl
p-toluenesulfonate, and 25 mL of 2-butanone was heated at
reflux for 18 h under argon. The mixture was cooled and
concentrated in vacuo, and the residue was partitioned be-
tween ethyl acetate and water. After the organic layer was
washed with brine and dried (MgSO4), concentration in vacuo
afforded an oil. Flash chromatography (ethyl acetate) afforded
0.433 g (76%) of 38a as a colorless gum: 1H NMR (CDCl3) δ
7.24 (d, 2H, J ) 8 Hz), 7.06 (d, 2H, J ) 8 Hz), 6.95 (d, 1H, J
) 8 Hz), 6.89 (s, 1H), 6.77 (d, 1H, J ) 8 Hz), 6.61 (s, 1H), 6.57
(s, 1H), 4.24 (t, 2H, J ) 6 Hz), 3.88 (s, 3H), 3.81 (m, 8H), 3.49
(s, 2H), 2.79 (t, 2H, J ) 6 Hz), 2.67 (t, 2H, J ) 6 Hz), 2.44 (t,
2H, J ) 8 Hz), 2.32 (s, 3H), 2.19 (t, 2H, J ) 8 Hz), 1.54 (m,
In ter m ed ia te for Syn th esis of 35e: (g) 2-[6-(3,4-Di-
m et h oxyp h en yl)-6-h yd r oxy-1-oxooct yl]-1,2,3,4-t et r a h y-
d r o-6,7-d im eth oxyisoqu in olin e (33b). To a solution of 3.0
g (6.8 mmol) of 32 in 20 mL of anhydrous THF was added 15.0
mL (15.0 mmol) of ethylmagnesium bromide (1 M in THF).
The resulting mixture was stirred at room temperature for
12 h. After pouring the mixture into ice and saturated
ammonium chloride, the aqueous layer was extracted with
ether. The extract was washed with brine, dried (MgSO4), and
evaporated to afford 3.0 g (94%) of 33b as a colorless oil, which
was subsequently used without further purification: 1H NMR
(CDCl3) δ 7.00-6.55 (m, 5H), 4.63 (s, 1H), 4.49 (s, 1H), 3.89-
3.85 (m, 12H), 3.80 (t, 1H, J ) 6 Hz), 3.60 (t, 1H, J ) 6 Hz),
2.76 (m, 2H), 2.32 (m, 2H), 1.83-1.70 (m, 3H), 1.69-0.80 (m,
5H), 0.76 (t, 3H, J ) 7.5 Hz).
5-[7-(3,4-Dih yd r o-6,7-d im eth oxy-2(1H)-isoqu in olin yl)-
1-[(4-m eth ylp h en yl)th io]h ep tyl]-2-m eth oxyp h en ol (36).
Compound 36 was prepared by the procedure of compound 18.
A 10.28-g (15.8 mmol) portion of 15x was reacted with 49 mL
(49.0 mmol) of 1.0 M tetrabutylammonium fluoride/THF in 150
mL of THF. The crude product was dissolved in ether and
passed through a short hydrous magnesium silicate pad to
afford, after solvent removal, 8.75 g (99.5%) of 36 as a brown
oil: 1H NMR (CDCl3) δ 7.16 (d, 2H, J ) 8 Hz), 7.01 (d, 2H, J
) 8 Hz), 6.85 (d, 1H, J ) 2 Hz), 6.70 (m, 2H), 6.58 (s, 1H),
6.51 (s, 1H), 5.6 (br s, 1H), 3.96 (m, 1H), 3.83 (m, 9H), 3.52 (s,
2H), 2.81 (m, 2H), 2.70 (m, 2H), 2.44 (m, 2H), 2.28 (s, 3H),
1.87 (m, 2H), 1.55 (m, 2H), 1.28 (m, 6H); MS (CI) m/z 536 (M
+ H+). Anal. (C32H41NO4S‚0.5H2O) C, H, N, S.
Met h od G. (a ) 2-[7-[3-(2-Ch lor oet h oxy)-4-m et h oxy-
p h en yl]-7-[(4-m eth ylp h en yl)th io]h ep tyl]-1,2,3,4-tetr a h y-
d r o-6,7-d im eth oxyisoqu in olin e (37a ). To a stirred solution
of 5.57 g (1.02 mmol) of 36 and 3.05 g (13.0 mmol) of
2-chloroethyl tosylate in 50 mL of 2-butanone was added 0.44
g (11.0 mmol) of 60% sodium hydride in oil. The solution was
heated at reflux for 48 h and then cooled to room temperature.
The crude product mixture was partitioned between CHCl3 and
brine and the organic layer dried (Na2SO4). Following evapora-
tion of the solvent in vacuo, the residue was chromatographed
on silica gel with a gradient elution going from hexane/CHCl3
to CHCl3/CH3OH. The product was dissolved in ether and
passed through a short hydrous magnesium silicate pad. The
solvent was evaporated in vacuo to afford 1.6 g (27% yield) of
37a as a light-tan gum: 1H NMR (CDCl3) δ 7.11 (d, 2H, J )
8 Hz), 7.03 (d, 2H, J ) 8 Hz), 6.77 (m, 2H), 6.71 (m, 1H), 6.58
(s, 1H), 6.51 (s, 1H), 4.17 (m, 2H), 3.97 (m, 1H), 3.83 (m, 9H),
3H), 1.31 (m, 3H); EI (HRMS) calcd for
C34H41N2O4SCl
608.2476, found 608.2483. Anal. (C34H41ClN2O4S) C, H, Cl, N,
S.
(b) r-(3,4-Dim eth oxyph en yl)-3,4-dih ydr o-7-[2-(1H-im id-
a zol-1-yl)eth oxy]-6-m eth oxy-r-[(4-m eth ylp h en yl)th io]-2-
(1H)-isoqu in olin eh ep ta n en itr ile (39a ). Compound 39a was
prepared by the procedure of compound 37b. A 0.366-g (0.60
mmol) portion of 38a was reacted with 0.062 g (0.91 mmol) of
imidazole and 0.037 g (0.91 mmol) of 60% sodium hydride in
oil in 16 mL of DMF. Flash chromatography (98:2 CH2Cl2/
CH3OH), followed by precipitation of a polar impurity from
ether/ethyl acetate, afforded 0.144 g (37%) of a colorless oil:
1H NMR (CDCl3) δ 7.63 (s, 1H), 7.23 (d, 2H), 7.08 (m, 4H),
6.95 (d, 1H, J ) 8 Hz), 6.89 (s, 1H), 6.79 (d, 1H, J ) 8 Hz),
6.60 (s, 1H), 6.43 (s, 1H), 4.32 (t, 2H, J ) 5 Hz), 4.20 (t, 2H, J
) 5 Hz), 3.88 (s, 3H), 3.83 (s, 3H), 3.80 (s, 3H), 3.46 (s, 2H),
2.80 (t, 2H, J ) 6 Hz), 2.68 (t, 2H, J ) 6 Hz), 2.43 (t, 2H, J )
7 Hz), 2.32 (s, 3H), 2.19 (t, 2H, J ) 7 Hz), 1.54 (m, 3H), 1.35
(m, 3H). Anal. (C37H44N4O4S‚0.6EtOAc) C, H, N, S.
Met h od H . r-(3,4-Dim et h oxyp h en yl)-7-[2-(d im et h yl-
amino)ethoxy]-3,4-dihydro-6-methoxy-r-[(4-methylphenyl)-
th io]-2(1H)-isoqu in olin eh ep ta n en itr ile (40). To a solution
of 0.583 g (1.07 mmol) of 15l in 20 mL of DMF was added 0.051
g (1.3 mmol) of 60% sodium hydride in oil, and the solution
was stirred for 1 h. To this solution were added 3.50 mg (3.2
mmol) of freshly prepared 2-(dimethylamino)ethyl chloride and
0.06 g (0.36 mmol) of KI. The solution was stirred at room
temperature for 18 h, and the volatiles were evaporated at in
vacuo. The residue was dissolved in a minimum amount of

2160 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12
Berger et al.
(12) Slater, L. M.; Sweet, P.; Stupecky, M.; Gupta, S. Cyclosporine
A reverses vincristine and daunorubicin resistance in acute
lymphocytic leukemia in vitro. J . Clin. Invest. 1986, 77, 1405-
1408.
ethyl acetate, and 5 volumes of ether was added. After the
solution was passed through a short pad of magnesol, the
volatiles were evaporated in vacuo. Silica gel chromatography
(gradient of ether/ethyl acetate to ethyl acetate/CH3OH) af-
forded 0.140 g (21%) of 40 as a clear glass: 1H NMR (CDCl3)
δ 7.24 (d, 2H, J ) 8 Hz), 7.06 (d, 2H, J ) 8 Hz), 6.97 (d, 1H,
J ) 8 Hz), 6.89 (s, 1H), 6.77 (d, 1H, J ) 8 Hz), 6.58 (s, 1H),
6.54 (s, 1H), 4.06 (t, 2H, J ) 6 Hz), 3.88 (s, 3H), 3.83 (s, 3H),
3.81 (s, 3H), 3.49 (s, 2H), 2.77 (m, 4H), 2.66 (t, 2H, J ) 6 Hz),
2.42 (t, 2H, J ) 7 Hz), 2.34 (s, 6H), 2.32 (s, 3H), 2.19 (t, 2H, J
) 7 Hz), 1.54 (m, 3H), 1.31 (m, 3H). Anal. (C36H47N3O4S‚
0.6Et2O) C, H, N, S.
(13) Kuntebommanahalli, N. T.; Horton, J . K.; Seshadri, R.; Israel,
M.; Houghton, J . A.; Harwood, F. C.; Houghton, P. J . Synthesis
and chemical characterization of N-substituted phenoxazines
directed toward reversing vinca alkaloid resistance in multidrug-
resistant cancer cells. J . Med. Chem. 1992, 35, 3358-3364.
(14) Tsuruo, T.; Iida, H.; Tsukagoshi, S.; Sakurai, Y. Increased
accumulation of vincristine and adriamycin in drug-resistant
P388 tumor cells following incubation with calcium antagonists
and calmodulin inhibitors. Cancer Res. 1982, 42, 4730-4733.
(15) Dhainaut, A.; Regnier, G.; Atassi, G.; Pierre, A.; Leonce, S.;
Kraus-Berthier, L.; Prost, J .-F. New triazine derivatives as
potent modulators of multidrug resistance. J . Med. Chem. 1992,
35, 2481-2496.
P r ep a r a tion of Hyd r och lor id e Sa lts (41-48). Rep -
r esen ta tive Exa m p le: r-(3,4-Dim eth oxyp h en yl)-3,4-d ih y-
dr o-7-[2-(1H-im id a zol-1-yl)eth oxy]-6-m eth oxy-r-[(4-m eth -
ylp h en yl)th io]-2(1H)-isoqu in olin eh ep ta n en itr ile Hyd r o-
ch lor id e (48). To a solution of 0.8 g (1.25 mmol) of 39a in
100 mL of ether and 5 mL of ethanol was added 0.83 mL (3.74
mmol) of 4.5 M HCl in ethanol. The solution was stirred for
20 min. A white solid precipitate was collected, washed three
times with small portions of ether, and dried in vacuo to afford
0.84 g (90%) of 48 as a white solid, mp 70 °C dec. 1H NMR
(DMSO-d6) δ 9.06 (br s, 1H), 7.76 (m, 1H), 7.63 (m, 1H), 7.22
(d, 2H, J ) 8 Hz), 7.17 (d, 2H, J ) 8 Hz), 6.97-6.86 (m, 3H),
6.83 (s, 1H), 6.80 (s, 1H), 4.58 (t, 2H, J ) 5 Hz), 4.31-4.02 (m,
4H), 3.75 (m, 9H), 3.45-3.32 (m, 4H), 3.06 (t, 2H, J ) 6 Hz),
2.41 (t, 1H, J ) 7 Hz), 2.30 (s, 3H), 2.16 (t, 1H, J ) 7 Hz), 1.76
(m, 2H), 1.37 (m, 3H), 1.21 (m, 1H). Anal. (C37H44N4O4S‚
1.75HCl‚2.0H2O) C, H, N, S, Cl.
(16) Hyafil, F.; Vergely, C.; Duvignaud, P.; Grandperret, T. In vitro
and in vivo reversal of multidrug resistance by GF-120918, an
acridonecarboxamide derivative. Cancer Res. 1993, 53, 4595-
4602.
(17) Germann, U. A.; Zelle, R. E.; Shlyakhter, D.; et al. VX-710: A
novel agent for reversal of P-glycoprotein-mediated multidrug
resistance. Proc. Am. Assoc. Cancer Res. 1995, 36, 346.
(18) Shinoda, H.; Inaba, M.; Tsuruo, T. In vivo circumvention of
vincristine resistance in mice with P388 leukemia using a novel
compound, AHC-52. Cancer Res. 1989, 49, 1722-1726.
(19) Pirker, R.; Keilhauer, G.; Raschack, M.; Lechner, C.; Ludwig,
H. Reversal of multidrug resistance in human KB cell lines by
structural analogues of verapamil. Int. J . Cancer 1990, 45, 916-
919.
(20) Toffoli, G.; Simone, F.; Corona, G.; Raschack, M.; Cappelleto,
B.; Gigante, M.; Boiocchi, M. Structure-activity relationship of
verapamil analogues and reversal of multidrug resistance.
Biochem. Pharmacol. 1995, 50, 1245-1255.
(21) Boesch, D.; Gaveriaux, C.; J achnes, B.; Poutier-Manzanedo, A.;
Bollinger, P.; Loor, F. In vivo circumvention of P-glycoprotein-
mediated multidrug resistance of tumors with SDZ PSC 833.
Cancer Res. 1991, 51, 4226-4233.
(22) Robert, J . Multidrug resistance reversal agents. Drugs Future
1997, 22, 149-158.
(23) Carhart, R. E.; Smith, D. H.; Venkataraghavan, R. Atom Pairs
as Molecular Features in Structure-Activity Studies: Definition
and Applications. J . Chem. Inf. Comput. Sci. 1985, 25, 64-73.
(24) Wood, J . H.; Perry, M. A.; Tung, C. C. The Bis-chloromethylation
of Aromatic Compounds. J . Am. Chem. Soc. 1950, 72, 2989.
(25) Levy, L. A. The Synthesis of 2,3,6,7 Tetrasubstituted Naphtha-
lenes: 2,3,6,7 Tetrachloronaphthalene. Synth. Commun. 1983,
13, 639-648.
Su p p or tin g In for m a tion Ava ila ble: Analytical data for
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
Refer en ces
(1) Young, R. C. Drug resistance: the clinical problem. In Drug
Resistance in Cancer Therapy; Ozols, R. F., Ed.; Kluwer Aca-
demic Press: Boston, 1989; pp 1-12.
(2) Pratt, W. B. In Principles of Drug Action. The Basis of Phar-
macology; Pratt, W. B., Ed.; Churchill Livingstone: New York,
1990; pp 565-638.
(3) Endicott, J . A.; Ling, V. The biochemistry of P-glycoprotein-
mediated multidrug resistance. Annu. Rev. Biochem. 1989, 58,
137-171.
(4) Gottesman, M. M.; Pastan, I. Biochemistry of multidrug resist-
ance mediated by the multidrug transporter. Annu. Rev. Bio-
chem. 1993, 62, 385-427.
(26) Guindon, Y.; Frenette, R.; Fortin, R.; Rokach, J . Direct Synthesis
of Thio Ethers from Thiols and Alcohols. J . Org. Chem. 1983,
48, 1357-1359.
(5) Goldstein, L. J .; Galski, H.; Fojo, A.; Willingham, M.; Lai, S. L.;
Gazdak, A.; Pirker, R.; Cossman, J .; Gottesman, M. M.; Pastan,
I. Expression of multidrug resistance gene in human cancers.
J . Natl. Cancer Inst. 1989, 81, 116-124.
(27) Zhang, X. P.; Ritke, M. K.; Yalowich, J . C.; Slovak, M. L.; Ho, J .
P.; Collins, K. I.; Annable, T.; Arceci, R. J .; Greenberger, L. M.
P-glycoprotein mediates profound resistance to bisantrene.
Oncol. Res. 1994, 6, 291-301.
(6) Tsuruo, T.; Iida, H.; Tsukagoshi, S.; Sakurai, Y. Overcoming of
vincristine resistance in P388 leukemia in vivo and in vitro
through enhanced cytotoxicity of vincristine and vinblastine by
verapamil. Cancer Res. 1981, 41, 1967-1972.
(7) Tsuruo, T. Circumvention of drug resistance with calcium
channel blockers and monoclonal antibodies. In Drug Resistance
in Cancer Therapy; Ozols, E. R. F., Ed.; Kluwer Academic
Press: Boston, 1989; pp 73-95.
(8) Nogae, I.; Kohno, K.; Kikuchi, J .; Kuwano, M.; Akiyama, S.; Kiue,
A.; Suzuki, K.; Yoshida, Y.; Cornwell, M. M.; Pastan, I.; Gottes-
man, M. M. Analysis of structural features of dihydropyridine
analogues needed to reverse multidrug resistance and to inhibit
the photoaffinity labeling of P-glycoprotein. Biochem. Pharmacol.
1989, 38, 519-528.
(9) Shudo, N.; Mizoguchi, T.; Arita, M.; Yoshimura, A.; Seto, K.;
Sakoda, R.; Akiyama, S. Two pyridine analogues with more
effective ability to reverse multidrug resistance and with lower
calcium channel blocking activity than their dihydropyridine
counterparts. Cancer Res. 1990, 50, 3055-3061.
(10) Zamora, J . M.; Pearce, H. L.; Beck, W. T. Physical-chemical
properties shared by compounds that modulate multidrug
resistance in human leukemic cells. Mol. Pharmacol. 1988, 33,
454-462.
(11) Tsuruo, T.; Iida, H.; Kitatani, Y.; Yokota, K.; Tsukagoshi, S.;
Sakurai, Y. Effects of quinidine and related compounds on
cytotoxicity and cellular concentration of vincristine and adria-
mycin in drug-resistant tumor cells. Cancer Res. 1984, 44, 4303-
4307.
(28) Data not shown.
(29) Relling, M. V. Ther. Drug Monit. 1996, 18, 350-356.
(30) Greenberger, L. M.; Collins, K. I.; Annable, T.; Boni, J . P.; May,
M. K.; Lai, F. M.; Kramer, R.; Citerella, R. V.; Hallett, W. A.;
Powell, D. CL 329,753: a novel chemosensitizing agent for
P-glycoprotein-mediated resistance with improved biological
properties compared to verapamil and cyclosporine A. Oncol. Res.
1996, 8, 207-218.
(31) Colombo, T.; Zucchetti, M.; D’Incalci, M. Cyclosporine A mark-
edly changes the distribution of doxorubicin in mice and rats.
J . Pharmacol. Exp. Ther. 1994, 269, 22-27.
(32) Gruber, A.; Peterson, C.; Reizenstein, P. D-verapamil and
L-verapamil are equally effective in increasing vincristine ac-
cumulation in leukemic cells in vitro. Int. J . Cancer 1988, 41,
224-226.
(33) Ho¨llt, V.; Kouba, M.; Dietel, M.; Vogt, G. Stereoisomers of
calcium antagonists which differ markedly in their potencies as
calcium blockers are equally effective in modulating drug
transport by P-glycoprotein. Biochem. Pharmacol. 1992, 43,
2601-2608.
(34) Mannhold, R. Calcium antagonists: basic chemical and phara-
cological properties. Drugs Today 1984, 20, 69-90.
(35) Theodore, L. J .; Nelson, W. L. Stereospecific Synthesis of the
Enantiomers of Verapamil and Gallopamil. J . Org. Chem. 1987,
52, 1309-1315.

Novel MDR Reversal Agents
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12 2161
(36) Skehan, P.; Stornet, R.; Scudiero, D.; Monks, A.; McMahon, J .;
Vistica, D.; Warren, J .; Bokbosch, H.; Kenny, S.; Boyd, M. New
colorimetric cytotoxicity assay for anticancer-drug screening. J .
Natl. Cancer Inst. 1990, 82, 1107-1112.
(37) Ushujima, R.; Nakagawa, S.; Susumu, M. Isoindole derivatives,
processes for their preparation, and their use as intermediates
for cephalosporin intermediates. European Patent 227986;
Chem. Abstr. 1987, 108, 186564.
(38) Ruchirawat, S.; Chaisupakitsin, M.; Patranuwatana, N.; Cashaw,
J . L.; Davis, V. E. A Convenient Synthesis of Simple Tetrahy-
droisoquinolines. Synth. Commun. 1984, 14, 1221-1228.
(39) Skraup, Z. H. U¨ ber das Parachinanisol, Monatsh. Chem. 1885,
6, 760-770.
(40) Chrisey, L. A.; Brossi, A. N,N′,N′′-Tris(3′,4′,5′-trimethoxyphen-
ethyl)-1,3,5-hexahydrotriazine, a methylenemescaline trimer:
characterization and selective cyclization to anhalinine under
nonaqueous conditions. Heterocycles 1989, 29, 1179-83.
(41) Meyers, A. I.; Guiles, J . The Asymmetric Total Synthesis of (+)-
Reticuline. Heterocycles 1989, 28, 295-301.
(42) Bobbitt, J . M.; McNew-Kiely, J .; Khanna, K. L.; Ebermann, R.
Synthesis of Isoquinolines III. A New Synthesis of 1,2,3,4-
Tetrahydroisoquinolines. J . Org. Chem. 1965, 30, 2247-2250.
(43) Regnier, G.; Gargouil, Y. M.; Vilaine, J . P. Preparation of