[CANCER RESEARCH 42, 3769-3775, September 1982]
0008-5472/82/0042-0000$02.00
Partial Circumvention of Resistance to 6-Mercaptopurine by Acylated
P1,P2-Bis(6-mercaptopurine-9-/?-D-ribofuranoside-5')
Pyrophosphate
Derivatives1
David M. Tidd,2 Ian Gibson, and Peter D. G. Dean
School of Biological Sciences. University of East Anglia, Norwich, NR4 7TJ [D. M. T., I. G.], and Department of Biochemistry,
3BX ¡P.D. G. D.]. United Kingdom
ABSTRACT
P1,P2-Bis(6-mercaptopurine-9-/?-D-ribofuranoside-5')
pyro-
phosphate [trivial name, bis(thioinosinate) (bis(MPRP))] and its
butyryl and hexanoyl derivatives [P\P2-bis(O2,
O3-diacyl-6mercaptopurine-9-/J-D-ribofuranoside-5')
pyrophosphate] were
synthesized by established procedures. The inhibitory effects
of bis(MPRP) on cell proliferation in cultures of D98, Chinese
hamster lung, L1210 cells, and thiopurine-resistant
sublines
were determined. Bis(MPRP) exhibited greater activity than did
6-mercaptopurine against resistant D98 cells but was approx
imately equivalent to 6-mercaptopurine
or 6-mercaptopurine
riboside (MPR) with all other lines. Extracellular degradation of
bis(MPRP) by L1210 cells was monitored by high-performance
liquid chromatography. The compound was cleaved slowly to
MPR and MPR diphosphate in contrast to the rapid extracel
lular dephosphorylation
of MPR monophosphate. Acylated
bis(MPRP) derivatives were developed on the basis that
bis(nucleotide) compounds capable of entering cells and re
leasing nucleoside diphosphates might conceivably overcome
resistance due to both loss of hypoxanthine-guanine phosphoribosyltransferase and increase in alkaline phosphohydrolase
activities. Unlike bis(MPRP), P1,P2-bis(O2,O3-dibutyryl-6-mercaptopurine-9-/8-D-ribofuranoside-5')
pyrophosphate
and
P1,P2-bis(O2,O3-dihexanoyl-6-mercaptopurine-9-/?-D-ribof
ur
anoside-5') pyrophosphate were less inhibitory than MPR to
wards parent L1210 cells but were far more active than the
riboside against an MPR-resistant subline. The culture growthinhibitory activity of the acylated derivatives was considerably
greater than could possibly be accounted for by the release of
the carboxylic acids. Therefore, it is suggested that partial
circumvention of thiopurine resistance may have resulted from
cellular uptake of intact acylated bis(MPRP) molecules.
INTRODUCTION
MP,3 like most of the antitumor purine and pyrimidine base
and nucleoside analogs, is converted intracellularly to a nucleotide as the first obligatory step in its mechanism of action
' Supported by a grant from the Cancer Research Campaign.
2 To whom requests for reprints should be addressed.
3 The abbreviations used are: MP, 6-mercaptopurine; MPRP, 6-mercaptopu
rine riboside 5'-monophosphate;
HGPRT, hypoxanthine-guanine
phosphoribosyltransferase; MPR, 6-mercaptopurine
riboside; bis(MPRP), P1,P2-bis(6-mercaptopurine-9-/J-D-ribofuranoside-5')
pyrophosphate; HPLC, high-performance
liquid chromatography;
bis(dibutyryl-MPRP),
P',P2-bis(O2,O3-dibutyryl-6-mercaptopurine-9-/3-D-ribofuranoside-5')
pyrophosphate;
bis(dihexanoyl-MPRP),
P',P2-bis(O2,O3-dihexanoyl-6-mercaptopurine-9-/8-D-ribofuranoside-5')
pyrophosphate; diacyl-MPRP. O2,O3-diacyl-6-mercaptopurine-9-i8-D-ribofuranoside 5'-monophosphate; ECso. concentration of drug required for 50% inhibition
of growth.
Received December 28. 1981 ; accepted May 18. 1982.
SEPTEMBER
1982
University of Liverpool, Liverpool, L69
(17). Cellular resistance to MP most commonly involves reduc
tion in the net accumulation of its riboside monophosphate,
MPRP, through one or more of a variety of mechanisms,
including decrease in HGPRT (17) and increase in alkaline
phosphohydrolase (9) activities. MP acts through further me
tabolism of its riboside monophosphate to 6-thioguanine nucleotides and its subsequent incorporation into DMA as 6thioguanine deoxyribonucleotide
(16, 22). Therefore, resist
ance through effects at other stages of MP anabolism is at
least theoretically possible but has not as yet been positively
identified. Resistance to MP may not be circumvented by
treatment with preformed MPRP, since cell membranes are
relatively impermeable to nucleotides, and in the case of MPRP
extracellular dephosphorylation at the cell membrane readily
converts the drug nucleotide to its nucleoside (MPR). Analo
gous situations are found to exist for most purine and pyrimi
dine antimetabolites, although there is evidence that 9-/?-oarabinofuranosyladenine
5'-monophosphate and certain other
less readily hydrolyzed nucleotides may be taken up slowly by
cells as the intact molecules (1, 11).
A number of attempts have been made to circumvent resist
ance to MP and similar drugs through the development of socalled "prodrug" forms of their 5'-monophosphates.
Several
factors have been taken into account in the design of such
prodrugs. Esterification or derivatization of the phosphate re
duces charge-repulsive effects at cell membranes and protects
the molecules from the action of extracellular phosphohydrolases. With phosphodiesters, the desired drug nucleotide may
be released intracellularly through the action of phosphodiesterases (15, 23). In addition, membrane permeability may be
enhanced by increasing the lipophilicity of prodrug molecules
through derivatization with nonpolar groups, which are suscep
tible to spontaneous or enzyme-catalyzed removal within the
cell (13).
Montgomery ef al. (1 5) reported that bis(thioinosine)-5',5'"phosphate was active against an HGPRT-deficient human epidermoid cell subline, HEp No.2/MP, in culture, while MP, MPR,
and MPRP were ineffective. However, other antimetabolite
dinucleoside phosphates were found to be no more effective
than the parent drugs against sensitive and resistant neoplasms
(8). 3',5'-Cyclic phosphate derivatives of purine and pyrimidine
nucleoside antimetabolites have been investigated as prodrugs
of the 5'-monophosphates,
in which the charge on the phos
phate is reduced by internal esterification (1, 3, 7, 8, 10). The
results have been equivocal, although there are some reports
of enhanced effects over those of the parent drugs against
resistant cells. Meyer ef al. (13) demonstrated that, although
6-thioinosine 3',5'-cyclic monophosphate was not significantly
more effective than MP against HGPRT-deficient lymphoma
3769
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.•:&
D. M. Tidd et al.
cells, the addition of a removable lipophilic group by acylation
of the molecule gave compounds which were more active. A 1/8-D-arabinofuranosylcytosine
analog of cytidine diphosphate
diglyceride (12), 1-/?-D-arabinofuranosylcytosine
conjugates of
corticosteroids through phosphodiester (4) and pyrophosphodiester (5) bonds, and 5'-phosphorodiamidates
of 5-fluoro-2'deoxyuridine (18) have been synthesized and were shown to
have cytotoxic activity against tumor cells. However, these
compounds have not been compared with their parent drugs
for activity against specifically resistant cells.
Although prodrugs of 5'-monophosphates
may conceivably
overcome resistance involving loss of HGPRT, these derivatives
would not be expected to be superior to the parent drugs,
when resistance is associated with increases in the capacity of
cells to dephosphorylate
the monophosphates.
However,
bis(nucleoside) pyrophosphate derivatives may be cleaved intracellularly by phosphodiesterase with the release of nucleoside diphosphates which not only would represent activated
forms of the drugs, thereby circumventing HGPRT loss, but
also would not be immediately susceptible to dephosphorylation by alkaline phosphohydrolase.
The present report de
scribes the synthesis and activity of acylated derivatives of
bis(MPRP) against thiopurine-sensitive
and -resistant L1210
cell cultures. A preliminary report of part of these results was
presented at the Winter 1980 meeting of The British Associa
tion for Cancer Research (21 ).
MATERIALS
Cell Cultures.
AND METHODS
The cell culture line of parent thiopurine-sensitive
L1210 cells, L1210/0, was kindly provided by Dr. K. R. Harrap, The
Institute of Cancer Research, Sutton, Surrey, United Kingdom. A subline of L1210 cells partially resistant to thiopurines, L1210/TG, was
generously supplied by Dr. A. R. P. Paterson, University of Alberta
Cancer Research Unit, McEachern Laboratory, Edmonton, Alberta,
Canada. This subline was originally derived in vivo by Dr. G. A. LePage
and subsequently adapted to cell culture by Dr. Paterson's group. The
highly resistant subline L1210/MPR was derived in our laboratory from
an L1210/0 culture by repetitive exposure to increasing concentra
tions of MPR. The basis of the thiopurine resistance of the L1210/TG
and L1210/MPR
lines was apparently their reduced capacity relative
to L1210/0 cells to accumulate intracellular thiopurine nucleoside 5'monophosphate. When exposed to 6-thioguanosine, L1210/MPR cells
formed negligible amounts of 6-thioguanosine
5'-monophosphate
[measured by our previously reported method (20)], whereas the intra
cellular concentrations of the latter in L1210/TG cells were interme
diate between those measured in L1210/0
and L1210/MPR
cells
under comparable conditions. The extent of drug nucleotide formation
correlated with the relative sensitivity of the 3 lines to thiopurines.
HGPRT activities measured in cell-free extracts (6) of L1210/0,
L1210/TG, and L1210/MPR
cells were respectively 158, 7, 7 nmol
inosine 5'-phosphate produced per min per 109 cells. All L1210 cell
lines were maintained in continuous culture in Fischer's medium con
taining 10% horse serum, penicillin (100 units/ml), and streptomycin
(100 fig/ml) (Gibco Europe, Ltd., Paisley, Scotland). The L1210/TG
line was passaged in the absence of thiopurines, while L1210/MPR
stock cultures were grown in the presence of 1 HIM MPR to ensure that
a high level of insensitivity was maintained. However, parallel culture
of the L1210/MPR
line in the absence of drug demonstrated that
resistance was stable. Cells were enumerated with a Model ZB Coulter
Counter (Coulter Electronics, Ltd., Luton, Bedfordshire, United King
dom). For determination of dose-response curves, replicate 2.5-ml
cultures of leukemia cells were prepared in 100 x 14-mm Nunc sterile
plastic disposable culture tubes (Gibco Europe, Ltd.), and 0.1 ml of the
3770
appropriate stock drug solution in 0.9% NaCI solution was added to
each. Cultures were incubated at 37° in an atmosphere of 5% CO2/
95% air. Cultures of thiopurine-sensitive
cells (CH/O) and a thiopurine-resistant
V79 Chinese hamster lung
subline deficient in HGPRT
(CH/TG) were kindly donated by Dr. M. Fox, Paterson Laboratories,
Christie Hospital and Holt Radium Institute, Manchester, United King
dom. The cells were maintained in continuous culture in Eagle's minimal
essential medium with Earle's salts containing 10% fetal calf serum,
penicillin (100 units/ml), and streptomycin (100/ig/ml) (Gibco Europe,
Ltd.). For determination of dose-response curves, replicate 5-ml cul
tures of Chinese hamster cells were prepared in 25-sq cm Nunc sterile
plastic disposable tissue culture flasks (Gibco Europe, Ltd.) and 0.1 ml
of an appropriate stock drug solution in 0.9% NaCI solution was added
to each. Flasks were gassed with 5% CO2/95% air and incubated at
37°.
Initial tests on bis(MPRP) were performed under the direction of Dr.
G. B. Elion, Head, Experimental Therapy, Wellcome Research Labo
ratories, Research Triangle Park, N. C. The cell lines maintained at the
Wellcome Research Laboratories and used in these tests were Detroit
98 (D98), Detroit 98/AH (D98/AH), adenocarcinoma
755/ + , and,
adenocarcinoma
755/MP.
D98 is a cell culture line derived from
human bone marrow, and D98/AH is a subline of D98 lacking HGPRT
which is resistant to MP. The in vivo passaged murine adenocarcinoma
755/+ is a MP-sensitive line while adenocarcinoma 755/MP is a MPresistant subline of the tumor which does contain HGPRT but is
nevertheless resistant to MP for reasons not yet understood.
Chromatography.
Chemical syntheses, purification, and drug me
tabolism were monitored by HPLC using an Altex gradient Chromato
graph (Anachem Ltd., Luton, Bedfordshire, United Kingdom) and a
Whatman Partisil-10 SAX strong-anion-exchange
analytical column
(Uniscience Ltd., Cambridge, United Kingdom). Separation conditions
for nucleotides were as described by Tidd and Dedhar (20), comprising
a 15-min concave gradient from 0.005 M potassium phosphate, pH
3.5, to 0.25 M potassium phosphate/0.5
M potassium chloride, pH 4.5,
at a flow rate of 3 ml/min. The UV absorption of the column effluent
was measured simultaneously at 254 and 320 nm. The course of
acylation reactions was followed by thin-layer Chromatography
on
Merck 5- x 10-cm silica gel 60 F254precoated plates (BDH Chemicals,
Ltd., Enfield, Middlesex, United Kingdom) developed with chloroform/
methanol/water/ß-mercaptoethanol
(62/25/4/0.5,
v/v/v/v).
Chemical Syntheses. Bis(MPRP) and its butyrated and hexanoylated derivatives, bis(dibutyryl-MPRP) and bis(dihexanoyl-MPRP),
were
synthesized from MPR (Sigma London Chemical Company Ltd., Poole,
Dorset, United Kingdom) by established procedures (see Chart 1 for
chemical structures).
2',3'-lsopropylidene
6-thioinosine was prepared by the perchloric
acid-catalyzed condensation of MPR and acetone described by Zderic
ef a/. (24). This derivative was used in the synthesis of MPRP through
coupling to 2-cyanoethyl phosphate, using dicyclohexylcarbodiimide
as a condensing agent. The cyanoethyl group was subsequently re
moved by mild alkaline hydrolysis (19). MPRP was converted to its 4morpholine A/,W-dicyclohexylcarboxamidine
salt, and self-condensa
tion to yield bis(MPRP) was effected with dicyclohexylcarbodiimide
in
anhydrous pyridine at room temperature (14). Bis(MPRP) was prepared
as the free acid by passage through a column of Dowex 50W-H+ and
was subsequently converted to its disodium salt by the addition of
sodium hydroxide solution. The resulting solution was lyophilized.
Analysis of the disodium salt of bis(MPRP)
C20H22NeO,3P2S2Na2•
3H2O
Calculated:
Found:
C 29.70,
C 29.68,
H 3.49,
H 3.44,
N 13.86,
N 13.80,
S 7.92
S 7.79
Bis(dibutyryl-MPRP) was synthesized by reaction of bis(MPRP) pyridinium salt with n-butyric anhydride in anhydrous pyridine at room
temperature. Bis(dihexanoyl-MPRP)
was prepared by 4-(dimethylamino)pyridine-catalyzed
reaction of bis(MPRP) pyridinium salt with n-
CANCER
RESEARCH
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VOL. 42
Acylated Bis(thioinosinate)
0
o
CH,-O-P-O-P-O-CH,
1
i
t *•
OH OH
Chart 1. Chemical structure of bis(MPRP) (R
C3H7CO) and hexanoyl (R = C5H,,CO) derivatives.
H), and its butyryl (R
hexanoic anhydride
at 100° (13). Because
in W,A/-dimethylformamide
of problems of deacylation on Dowex SOW resin, the purification
scheme was modified. Bis(dihexanoyl-MPRP)
was extracted with chlo
roform from an acidified aqueous suspension of the reaction mixture at
pH 2.5. Chloroform was removed under reduced pressure, and the
product was precipitated by the addition of ethyl acetate. Further
purification was achieved by column chromatography on Merck Silica
Gel 60 (BDH Chemicals, Ltd.) with methanol/chloroform
step gra
dients. The acylated bis(MPRP) derivatives were converted to their
water-soluble disodium salts for tests on L1210 cell cultures.
The UV spectra of bis(MPRP), bis(dibutyryl-MPRP),
and bis(dihexanoyl-MPRP) at pH 4.5 were essentially identical to that of MPR with
absorbance maxima occurring at 322 nm. Chemical shifts (ppm from
ferf-butyl alcohol) for the 220-MHz proton nuclear magnetic resonance
spectra of bis(MPRP) in D2O were as follows: H5 3.04, H4 3.11, H2
3.25; H3 3.46, H1 4.76, H8 6.98, and H2 7.12. Chemical shifts (ppm
from ferf-butyl alcohol) in the corresponding nuclear magnetic reso
nance spectra of MPR were: H5 2.69, H" 3.08, H2 3.25, H3 obscured
by HDD resonance at 3.56, H' 4.92, H6 7.18, and H2 7.30.
No impurities were detected by HPLC analysis of bis(MPRP) with
simultaneous monitoring of the column effluent at 254 and 320 nm.
The compound eluted at a position in the buffer gradient which was
intermediate between the elution ranges for nucleoside 5'-monophosphates and nucleoside 5'-diphosphates and which corresponded to the
region where compounds carrying 2 negative charges are observed
normally. Mild alkaline hydrolysis degraded bis(MPRP) to MPRP, while
mild oxidation with iodine in phosphate buffer at pH 7 resulted in the
reversible formation of cyclic oligomers in which the bis(MPRP) units
were joined through disulfide linkages. Monomeric bis(MPRP) was
regenerated from the oxidized product by treatment with /8-mercaptoethanol. Acylated derivatives of bis(MPRP) gave broad peaks on HPLC
anion-exchange analysis due to a supplementary reverse-phase inter
action with the column functional groups. In the case of bis(dihexanoyl-
Derivatives
tant tumor is one which does contain HGPRT but is neverthe
less resistant to MP for reasons which are not yet understood.
Bis(MPRP) was no more effective against either the MP-sensi
tive or the MP-resistant tumor than was MP itself when taking
into account the fact that a molecule of bis(MPRP) contains 2
molecules of MP.4
In contrast to the results with the human D98/AH cells, the
data of Chart 2 demonstrate that bis(MPRP) was not signifi
cantly different from MPR in terms of its effects on thiopurinesensitive CH/O cells and on thiopurine-resistant CH/TG cells
which lack HGPRT. Similarly, bis(MPRP) was roughly equiva
lent to MPR when tested for growth-inhibitory effects against
thiopurine-sensitive mouse leukemia L1210/0 cells (data not
shown) and a thiopurine-insensitive subline, L1210/TG (Chart
3). However, bis(MPRP) appeared to be relatively immune to
extracellular degradation. To accentuate extracellular metab
olism of bis(MPRP) to an extreme, L1210/TG cells in expo
nentially proliferating cultures were collected by centrifugation
and resuspended in V?othe volume of fresh medium at approx
imately 3 x 106 cells/ml, cell densities that were 10,000 times
greater than the initial cell counts used for determination of the
dose-response curves in Chart 3. Bis(MPRP) was added at a
concentration of 0.5 x 10~3 M, and the cultures were incubated
at 37°. These extremely high cell densities were chosen to
determine the limit of extracellular degradation of bis(MPRP)
under cell culture conditions. The culture medium became
depleted and acidic, and some cell lysis with the release of
cellular enzymes undoubtedly occurred during the incubation
period. Samples of the medium were removed at various times,
acid extracted, and analyzed by HPLC. Chart 4 presents the
chromatograms obtained at 24 and 73 hr after the addition of
bis(MPRP). It can be seen that most of the bis(MPRP) remained
intact at 24 hr after the start of the incubation, and an appre
ciable proportion was still present at 73 hr. The major break
down products of the drug were MPR and MPR diphosphate.
At no time was MPRP, normally eluted at 4.2 min, detected in
the culture medium. The smaller peaks immediately preceding
MPRP), this resulted in a considerable increase in retention time. It was
not possible to isolate completely pure samples of the acylated
bis(MPRP) derivatives, since some decomposition
occurred during
purification. The final products contained less than 5% of the corre
sponding diacyl-MPRP derivatives as impurities. Mild alkaline hydrol
ysis of bis(dibutyryl-MPRP)
and bis(dihexanoyl-MPRP)
resulted in the
formation of MPRP.
RESULTS
Bis(MPRP) was no more effective on a molar basis than was
MP in inhibiting growth of a human bone marrow-derived cell
line, Detroit 98 (D98), in culture. For both drugs, EC50 values
against this thiopurine-sensitive
cell line were 5 x 10~7 M."
However, bis(MPRP) inhibited an MP-resistant D98 subline,
D98/AH, lacking HGPRT with an EC50of 6 x 10~s M, whereas
MP was without effect at 10~" M. In another set of experiments,
bis(MPRP) was compared with MP for activity in vivo against
adenocarcinoma 755/4- (an MP-sensitive line) and adenocarcinoma 755/MP (an MP-resistant line). In this case, the resis4 G. B. Elion, D. M. Tidd, and P. D. G. Dean, unpublished observations.
SEPTEMBER
1982
§ 3°
3
20
10
0
-10
,-7
10
10°
10,-5
10'
MOLARITY
Chart 2. Dose-response curves for cultures of V79 Chinese hamster lung
cells (CH/O) and an HGPRT-deficient subline (CH/TG) treated with MPR and
bis(MPRP). Drugs were added 24 hr after the cells were subcultured, and cell
numbers were determined 48 hr later. O, A, CHO/TG cultures; •,A, CHO/O
cultures. •,O, MPR; A, A, bis(MPRP). Points, mean value for 2 cultures.
3771
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D. M. Tidd et al.
1Ã’~B
tance of bis(MPRP) to extracellular degradation, the effects of
the derivative on culture growth were approximately the same
as those of MPR (Charts 2 and 3).
In contrast to the results with bis(MPRP), MPRP was readily
hydrolyzed by the cells. Replicate cell cultures were incubated
with MPRP in parallel with the bis(MPRP) incubations, and
HPLC analysis of the media demonstrated that approximately
10% had been dephosphorylated to MPR at 2 hr and that all of
the nucleotide had been degraded to the nucleoside within 23
hr (data not shown). MPRP was also incubated at 37° in
Fischer's medium complete with horse serum but without cells
Ì0"b
MOLARITY
Chart 3. Dose-response curves for cultures of L1210/TG cells, a thiopurïneinsensitive subline of mouse leukemia L1210, treated with MPR and bis(MPRP).
Drugs were added immediately after the cells were subcultured, and cell numbers
were determined following incubation for 7 days at 37°.•,MPR-treated cultures;
O, bis(MPRPHreated
cultures.
ISlMPRft
0
2
¿ 6
8
10
12 U
TIME
(MINI
16 18 20 22
10 12 li
as a control. Under these conditions, hydrolysis of the nucleo
tide to MPR was very slow. The concentration of MPRP re
maining at 66 hr was approximately 73% of the initial value.
Having established that extracellular degradation of bis(MPRP) proceeded at a comparatively slow rate and that cells
were capable of cleaving the compound to release MPR di
phosphate, we attempted to enhance cellular uptake of the
intact molecule by increasing its lipophilicity through acylation
of the sugar hydroxyl groups. Two amphiphilic compounds
were synthesized, the n-butyrated derivative, bis(dibutyrylMPRP), and the n-hexanoylated derivative, bis(dihexanoylMPRP). Both nucleotides were soluble in organic solvents (e.g.,
chloroform) as the protonated species but dissolved in water
as their sodium salts. The compounds were converted to the
16 18 20
Chart 4. HPLC separation of bis(MPRP) and its metabolites present in culture
media during incubation of the drug with L1210/TG cells at 37°24 hr (A) and 73
hr (B) after addition of 0.5 mM bis(MPRP). The cell density was 3 X 106 cells/ml.
bis(MPRP) and MPR diphosphate may represent S-substituted
derivatives of the compounds since the 320 nm/254 nm HPLC
peak height ratios (not shown) were lower than for MPR deriv
atives and were similar to those observed with known S-sub
stituted derivatives [e.g., 6-methylthioinosinate, a metabolite of
MPRP (17)]. S-substituted derivatives of MPR would not be
separated from MPR under the Chromatographie conditions
used, and these may be present within the MPR peak. A similar
result was obtained with cultures of the highly MPR-resistant
L1210/MPR
subline in which approximately
70% of the
bis(MPRP) remained at 23 hr after the addition of the drug.
Again MPR and MPR diphosphate were the breakdown prod
ucts detected in the culture media. When bis(MPRP) was
incubated at 37°in Fischer's tissue culture medium complete
with 10% horse serum but without cells, slow hydrolysis of the
compound to MPRP and MPR was observed. No MPR diphos
phate was detected in this case. At 66 hr after the start of the
incubation, the concentration of unchanged bis(MPRP) was
approximately 57% of the initial value, while 25% of the drug
had been hydrolyzed to MPRP and 18% had become degraded
to MPR. It is noteworthy that, despite the comparative resis
3772
Charts. Dose-response curves for cultures of L1210/0
cells, the parent
thiopurine-sensitive
line, treated
with MPR, bis(dibutyryl-MPRP),
and
bis(dihexanoyl-MPRP). A, comparison of MPR (•)and bis(dibutyryl-MPRP) (O);
B, comparison of MPR (•)and bis(dihexanoyl-MPRP)
(A). Drugs were added
immediately after the cells were subcultured, and cell numbers were determined
following incubation for 3 days at 37°. Points, mean value for 2 cultures.
CANCER
RESEARCH
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VOL. 42
Acylated Bis(thioinosinate)
sodium salts for evaluation of their inhibitory effects on cultures
of L1210 cells. Chart 5 presents comparisons of dose-re
sponse data for MPR and the acylated bis(MPRP) derivatives
versus thiopurine-sensitive L1 210/0 cells. It can be seen that
these cells are less sensitive to the acylated nucleotides than
to the parent drug. The EC50 of 1.2 x 10~7 M for bis(dibutyrylMPRP) was approximately 4 times that for MPR (EC50 3 x
10~8 M) versus replicate L1210/0 cultures or 8 times greater
in terms of MPR equivalents. Similarly, the EC50 of 2.2 x 10~7
M for bis(dihexanoyl-MPRP) was 27 times greater than that for
MPR (ECso 8.2 x 10~9 M) versus replicate L1210/0 cultures
or 54 times higher in terms of MPR content. The data of Chart
5 demonstrate that a small drift in the sensitivity of L1210/0
cells to MPR had occurred during the time between experi
ments. The curves of Chart 6 represent a comparison of doseresponse data for the highly MPR-resistant L1210 subline,
L1210/MPR. The effects of the acylated bis(MPRP) derivatives
are shown in conjunction with the results for MPR and the
corresponding fatty acid salts. MPR itself had no effect on
these cells even at a concentration of 10~3 M, whereas for
bis(dibutyryl-MPRP)
the EC50 was 3.8 x 10~5 M and for
120
MPR
110
100
0
E 80
o
g 70
Ã- 60
BISIDIBUTMPRP)
£ Ì0
O
120
110
_ 100
90
E 80
o
g 70
f- 60
S 50
o
u tQ
BISlOIHEXMPRPI
¡30
0 20
10
O
,-6
10
n-5
10"
n-3
MOLARITY
Charte. Dose-response curves for cultures of L1210/MPR
cells, a highly
MPR-resistant
L1210
subline,
treated
with MPR, bis(dibutyryl-MPRP),
bis(dihexanoyl-MPRP),
sodium butyrate, and sodium hexanoate. A, comparison
of MPR (•).bis(dibutyryl-MPRP) (O), and sodium butyrate (0); ß.comparison of
MPR (•),bis(dihexanoyl-MPRP)
(A), and sodium hexanoate (A). Drugs were
added immediately after the cells were subcultured, and cell numbers were
determined following incubation for 3 days at 37°. Points, mean value for 2
SEPTEMBER
1982
experiments of Charts 5 and 6 were repeated with different
batches of the acylated bis(MPRP) derivatives and the same
results as those presented were obtained.
Bis(dibutyryl-MPRP)andbis(dihexanoyl-MPRP)(0.5
x 10~3
M) were incubated at 37° in Fischer's medium complete with
horse serum but without cells. Samples were removed at var
ious times, acid extracted, and analyzed by HPLC. No deacylation of the bis compounds to give bis(MPRP) was observed
over the course of 66 hr. However, slow hydrolysis to the
corresponding diacyl-MPRP and diacyl-MPR derivatives oc
curred. No MPR diphosphates were formed. The rates of
hydrolysis of bis(dihexanoyl-MPRP)
and bis(dibutyryl-MPRP)
were such that approximately 63 and 58%, respectively, had
been degraded by 66 hr. Deacylation of the diacyl-MPR and
diacyl-MPRP breakdown products was demonstrated by the
subsequent appearance of MPRP and MPR peaks on the
chromatograms. Deacylation of diacyl-MPRP was slower than
that of diacyl-MPR derivatives. Consequently, release of car-
It is interesting to note that bis(thioinosine)-5',5'"-phosphate
10
cultures.
derivatives tested. Each of the fatty acids as their sodium salts
was found to inhibit the growth of L1210/0 (data not shown)
and L1 21 0/MPR cells (Chart 6) to the same extent. From the
data of Chart 6, sodium butyrate had an EC60 of 5.2 x 10"4
M, and for sodium hexanoate the EC50 was 3.0 x 10~" M. The
DISCUSSION
1 3°
3 20
S
the EC50 was 1.9 x 10 5 M. However,
limited cell proliferation was observed in L1 21 0/MPR cultures
at the highest concentrations (5 x 10"" M) of the acylated
boxylates occurred to a greater extent during the latter stages
of the incubations. At 66 hr, the deacylation products MPRP
and MPR accounted for approximately 5 and 18%, respec
tively, of the initial content of bis(dibutyryl-MPRP) and 1 1 and
17% of the original concentration of bis(dihexanoyl-MPRP).
90
§ 50
bis(dihexanoyl-MPRP)
Derivatives
(15) and bis(MPRP) (this paper) had very significant activity
against MP-resistant human cell lines deficient in HGPRT,
whereas the latter compound and presumably also the former
were ineffective against thiopurine-resistant animal cells. This
might conceivably reflect differences between the cell lines in
their ability to take up the intact molecules. Extracellular deg
radation of bis(MPRP) to MPR and MPR diphosphate occurred
at a comparatively slow rate. Therefore, it is difficult to account
for the apparent equivalence of MPR and bis(MPRP) in their
dose-response curves with thiopurine-sensitive and -insensitive
Chinese hamster and L1210 cell lines. This result contrasted
with the rapid extracellular dephosphorylation
of MPRP by
L1 21 0 cells, presumably mediated by cell surface-bound phosphohydrolase. It is apparent that any MPRP formed in the
culture media from bis(MPRP) would be rapidly degraded by
cells to MPR. Indeed, MPRP was not detected as an extracel
lular breakdown product of bis(MPRP), but this nucleotide was
formed very slowly as a decomposition product when the latter
was incubated in Fischer's medium at 37° without cells. It
would seem that 2 extracellular processes were occurring
simultaneously during incubation of leukemia cell cultures with
bis(MPRP): (a) slow spontaneous chemical and possibly enzymic hydrolysis to MPRP; and (b) enzyme-catalyzed hydroly
sis to MPR and MPR diphosphate. However, MPRP formed by
process (a) was rapidly converted to MPR by cellular phosphohydrolases. This would account for the fact that the
3773
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D. M. Tïddef al.
amounts of M PR present in the chromatograms of Chart 4 are
demonstrably greater than are the quantities of MPR diphosphate. Bis(MPRP) is probably metabolized by a cellular phosphodiesterase, and the MPR diphosphate released is not a
substrate for phosphohydrolases.
Therefore, it is likely that
derivatives of this type with additional modification to enhance
membrane permeability would be effective in circumventing
resistance due to elevated phosphohydrolase levels. Esterification of the sugar hydroxyls of bis(MPRP) with n-butyric and
n-hexanoic acids gave compounds which were active against
the thiopurine-resistant
L1210 subline, L1210/MPR,
to the
extent that partial circumvention of resistance was achieved.
Interpretation of these results is complicated by the observed
growth-inhibitory
effects of the sodium salts of fatty acids
themselves. Sodium butyrate has been shown to inhibit the
growth of neoplastic cells reversibly and to induce a better
differentiated or more benign phenotype (2). Sodium hexanoate may possibly act in a similar manner. It would appear that
mouse leukemia L1210 cells were more sensitive to growth
inhibition by carboxylic acids than were the S49 murine lymphoma cells used by Meyer et al. (1 3) to evaluate the cytotoxicity of 2'-O-acyl-6-thioinosine
cyclic
3',5'-phosphates.
Release of fatty acids undoubtedly contributed to the inhibitory
effects of the acylated bis(MPRP) derivatives on L1210/MPR
cell cultures. However, the EC50 values for butyrate and hexanoate were approximately 14 and 16 times greater, respec
tively, than were those observed for bis(dibutyryl-MPRP) and
bis(dihexanoyl-MPRP). If acylated bis(MPRP) derivatives were
rapidly and completely deacylated in L1210 cultures and their
effects on MPR-resistant cells were due solely to the released
carboxylic acids, EC60 values of only one-fourth of those for
the corresponding carboxylic acid would be observed, since 4
mol of acid are associated with 1 mol of bis(MPRP). Further
more, the data for thiopurine-sensitive L1210/0 cells suggest
that deacylation is slow, since these cells were less sensitive
to the acylated derivatives than to MPR and bis(MPRP). The
EC50 values of bis(dibutyryl-MPRP) and bis(dihexanoyl-MPRP)
in terms of MPR content were approximately 8 and 54 times
greater, respectively, than the corresponding EC60 values for
MPR in replicate L1210/0 cultures. The carboxylic acids were
without activity at the concentrations of the derivatives which
were effective against L1210/0 cells, and therefore the ob
served cytotoxic effects could be due only to the thiopurine
portions of the molecules. The lower efficacy of bis(dibutyrylMPRP) and bis(dihexanoyl-MPRP) against L1 21 0/0 cells com
pared with MPR and bis(MPRP) may be explained if it is
assumed that deacylation is a rate-limiting step in the action of
these agents on thiopurine-sensitive cells.
Deacylation of bis(dibutyryl-MPRP)
and bis(dihexanoylMPRP) did not occur at 37°in Fischer's medium in the absence
of cells. However, the compounds were slowly hydrolyzed to
diacylated MPRP and MPR derivatives. Carboxylic acids were
released slowly from these breakdown products and after 3
days amounted to approximately 23 and 28% of the total bound
to bis(dibutyryl-MPRP)
and bis(dihexanoyl-MPRP),
respec
tively, at the start of the experiments. Hydrolysis of the MPR
diesters was more rapid than deacylation of the MPRP deriva
tives, and consequently the bulk of free carboxylic acid for
mation occurred during the latter stages of the incubations.
Bis(dibutyryl-MPRP)
and bis(dihexanoyl-MPRP)
were con
siderably more active against parent L1210/0 cells than they
3774
were against the L1210/MPR subline. This would suggest that
these agents are unlikely to be of practical use in the chemo
therapy of thiopurine-resistant disease, since they would prob
ably be more toxic to normal cells than to resistant tumor cells.
However, it is hoped that using the present results as a starting
point, further modifications in prodrug design will lead to the
development of dinucleoside pyrophosphates with greater ac
tivity against resistant cells.
The greater part of the observed activities of the acylated
bis(MPRP) derivatives against L1210/MPR cells may possibly
have resulted from cellular uptake of the intact molecules. The
extracellular and intracellular metabolism and mechanism of
action of these compounds are currently being investigated
and will form the subject of a subsequent publication.
ACKNOWLEDGMENTS
The authors thank Dr. M. Gait of the MRC Laboratory of Molecular Biology,
Cambridge. United Kingdom, for his advice on chemical synthetic matters.
Bis(MPRP) was originally synthesized by Dr. H. Potuzak at the Department of
Biochemistry, University of Liverpool, as part of a research project funded by the
North West Cancer Research Fund. We also extend our thanks to Heather P.
Johnston of the School of Biological Sciences, University of East Anglia, who
performed HGPRT assays on cell-free extracts of our LI 210 lines, to Naomi
Cohn of Burroughs Wellcome Co. for her work with bis(MPRP) in D98 and O98/
AH cell cultures, and to Vincent Knick of Burroughs Wellcome Co. who undertook
the animal testing of bis(MPRP) on Ad755 and Ad755/MP.
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Partial Circumvention of Resistance to 6-Mercaptopurine by
Acylated P1,P2-Bis(6-mercaptopurine-9-β-d-ribofuranoside-5′)
Pyrophosphate Derivatives
David M. Tidd, Ian Gibson and Peter D. G. Dean
Cancer Res 1982;42:3769-3775.
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