Note: Descriptions are shown in the official language in which they were submitted.
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1082S99
BACKGROUN~ OF THE INVENTION
1. Field of the Invention
_ _ .
This invention is in the field of biocl~emistry.
2 DescriPtion of the Prior Art
Cytol~inins are a generic class of chemical sub-
stances which promote cell division and growth and which
occur at the purine, ribonucleoside and ribonucleotide
levels in plants, as well as in the transfer ~`~A's of most
forms of life. See Skoog, F. and Armstrong, D. J.,
Ann. Rev. Plant PhYsiol. 21, 359 (1970). In general, com-
pounds exhibiting cytokinin activity can be identified asadenine derivatives, preferably with the purine ring
intact, and particularly the group of compounds identified
as N6-adenine derivatives with the purine ring intact.
These can be represented by the structural formula,
RNH
H
'
wherein R is an alkyl or alkenyl group having between 1
and l0 carbon atoms with the highest cytokinin activity
being shown by those having 4 to 7 carbon atoms.
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108Z599
In plants, cytokinins play an important role in
all phases of plant development from cell division and en-
largement to the formation o~ flowers and fruits. They
affect metabolism including the activity of enzymes and
the biosynthesis of growth factors. They also influence
the appearance of organelles and the flow of assimilates
and nutrients through the plant, as well as enhancing re-
sistance to aging and to adverse environments. Cytokinins
have additionally been found to promote certain effects
in animal cells, and more specifically in mammalian cells.
For example, it has been found that phytohemagglutinin
(PHA)-transformed human lymphocyte cells can be both
stimulated and inhibited by the cytokinin 6-(3-methyl-2-
butenylamino)-9-(~-D-ribofuranosyl)purine with respect to
DNA synthesis, transformation, and mitosis. See Gallo,
R. C., Whang-Peng, J. and Perry, S., Science, 165, p. 400 ~ -
(1969). This specific effect was found to depend upon the
stage of the cell cycle and the concentration of cytokinin
employed. Additionally, this same cytokinin has been shown
to have antitumor activity in experimental animals and in
preliminary clinical trials. See Grace, Jr., J. T., Hakala,
M. T., Hall, R. H., Blakeslee, J., Proc. Am. Assoc. Cancer
Res., 8, 23 (1967)j Suk, D., Simpson, C. L., Mihick, E.,
Proc. Am; Assoc. Cancer Res., 9, 35 (1968); and Jones, Jr.,
25 R., Grace, Jr., J.-TMittelman, A., Gerner, R. E., Proc.
Am. Assoc. Cancer Res., 9, 35 (1968).
Some cytokinins are known to exhibit chemothera-
peutic properties. Two such cytokinins are N6-(~2-isopen-
tenyl)adenosine and N6-benzyladenosine. See Mittelman, A.,
--3--
.. . . ~ . . ~
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10 8 2 59~9
1 Evans, J. T., and Chheda, G. B., "Cytokinins as Chemo-
therapeutic Agents", Annals, N. Y. Acad. Sci., 255, pp.
225-234 (1975). In fact, the chemotherapeutic properties
of N6-(42-isopentenyl)adenosine have been sufficiently sig- ¦1
nificant to prompt its usage as an experimental drug in
a leukemia clinic.
It has been postulated that cytokinins mediate
their effects in phytohemagglutinin-treated human lympho-
cytes by involvement in cyclic adenosine monophosphate
(cyclic AMP) metabolism. See Gallo, R. C., Hecht, S. M.,
Whang-Peng, J. and O'Hopp, S., Biochem. Biophys. Acta.,
281, 488 (1972). Cyclic AMP is a regulatory agent which
controls the rate of a number of cellular processes. It
is formed from adenosine triphosphate by the action of
adenyl cyclase, and affects the function of several enzymes,
including protein kinases, glycogen synthetase, phospho-
fruct~inase and possibly triglyceride lipase. Cyclic AMP
is also capable of stimulating the synthesis of a number of
enzymes including tyrosine transaminase, PEP carboxykinase, `
glucose-6-phosphatase, serine dehydratase, ~-galactosidase,
and tryptophanase. This cyclic nucleotide also affects
potassium ion and calcium ion transport, lipogenesis, permea-
bility, lipolysis,steroidogenesis, oxygen consumption,
insulin release,ACTH release, GH release,HCl secretion,
muscular contraction and many other processes in diverse
organisms. See Robinson, G. A., Butcher, R. W., Sutherland,
E. W., Cyclic AMP, Academic Press, N. Y., pp. 93-97 ~1971).
10~2S9g
These effects are known in animals to cause such problems as hyperglycemia,
inhibition of ovulation, fall in blood pressure, drowsiness and enzyme
induction in reproductive organs.
New classes of compounds have been synthesized relatively recently
which are structurally related to cytokinins but act as cytokinin antagonists
or anticytokinins in certain plant bioassays. It is postulated that such
cytokinin antagonists could be employed to regulate plant development and
the biosynthesis of specific products such as protein, vitamins, chlorophyll
and other compounds which the plant uses, for example, in its energy
metabolism and in adjustment to its environment. Additionally, such antagon-
ists might be used to study plant cell genetics because they are an appro-
priate means to prevent mitosis or cytokinesis while manipulating cells to
cause cell fusions or differentiation. These cytokinin antagonists can be
used alone or in combination with cytokinins to interrupt, for short periods
of time, the normal cytokinin effects on growth, etc.
Several classes of related compounds have been described in the
patent and scientific literature as having cytokinin antagonist activity.
In Hecht, S.M., Bock, R.M., Schmitz, R.Y., Skoog, F., and Leonard, N.J. (1971)
Proc. Nat. Acad. Sci. U.S.A. 68, 2608-2610; Skoog, F., Schmitz, R.Y.,
Hecht, S.M., and Bock, R.M. (1973) Phytochemistry 12, 25-37, for example,
there is disclosed a class of cytokinin antagonists comprising 7-substituted
pyrazolo[4,3-~ pyrimidine compounds wherein the substituent is an alkyl or
alkenyl substituted amine group. Another class of compounds previously found
B
. . . i ..
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. . .
..
108ZS99
to have cytokinin antagonist activity are certain substituted pyrrolo-
[2,3-d~pyrimidines. See Skoogl F., Schmitz, R.Y., Hecht, S.M. and Frye, R.B.,
Proc. Nat. Acad. Sci. U.S.A. _, No. 9, pp. 3508-12 (1975).
It has also been disclosed by Hecht, S.M., Faulkner, R.~. and
Hawrelak, S.D., "Competitive Inhibition of Beef Heart Cyclic AMP Phospho-
diesterase by Cytokinins and Related Compounds", Proc. Nat. Acad. Sci. U.S.A.
71, No. 12, pp. 4670-74 (1974), that cytokinin antagonists can be used to
reguIate intracellular levels of cyclic AMP. In this article, it is shown
that cytokinin antagonists can be potent, specific, competitive inhibitors
of cyclic AMP phosphodiesterase activity from beef heart. Cyclic AMP phospho-
diesterase activity is necessary for the normal degradation of cyclic AMP.
SUMMARY OF THE INVENTION
This invention relates to the surprising finding that one cytokinin
antagonist, namely 3-methyl-7-n-pentylaminopyrazolo~4,3-~ pyrimidine, is a
.
particularly potent regulator for human cells which are growing, such as PHA-
transformed human lymphocyte cells. Because of this, this cytokinin
antagonist is useful in affecting the physiological response of such cells.
At higher dosages, the cytokinin antagonist inhibits growth, whereas at lower
concentrations it often stimulates growth. Administration of 3-methyl-7-n-
-pentylaminopyrazolo [4,3-~ pyrimidine offers potentially significant advan-
tages in chemotherapeutic treatment of abnormal human cell growth, particular-
ly abnormal lymphocyte growth.
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1 BRIEF DESCRIPTION OF THE FIGURES
FIGS. 1 and 3 are dose-response curves which both
illustrate the physiological effect of 3-methyl-7-_-pentyl-
aminopyrazolo[4,3-d]pyrimidine and N6-(~2-isopentenyl)-
adenosine on PHA-stimulated human lymphocytes measured as a
percentage of lymphocytes transformed relative to a drug-
free control in leukocytes obtained from different donors;
FIGS. 2 and 4 are dose-response curves which both
illustrate the physiological effect of 3-methyl-7-_-pentyl-
aminopyrazolol4,3-d]pyrimidine and N6-(~2_isopentenyl)-
adenosine on PHA-stimulated human lymphocytes measured as
percent DNA systhesis relative to a drug-free control for
leukocytes obtained from different donors; and
FIGS. 5-10 are dose-response curves which illus-
trate the physiological effect of a variety of cytokinin
antagonists and N6-(Q2-isopentenyl)adenosine on PHA-stimu-
- lated human lymphocytes measured as percent DNA synthesis
relative to a drug-free control.
DESCRIPTION OF PREFERRED EMBODIMENTS
The compound 3-methyl-7-n-pentylaminopyrazolo-
[4,3-d~pyrimidine has been shown to be a cytokinin antagonist
or anticytokinin in appropriate plant assays. This compound,
which can be synthesized by reacting n-pentylamine with
3-methyl-7-methylthiopyrazolo[4,3-d]pyrimidine, is repre-
sented by the structural formula: .
~N~ I
~.~''`
C~3
J ~
~ \ .
108~9
1 It has now been discovered that 3-methyl-7-_-
pentylaminopyrazolo~4,3-d]pyrimidine, when administered
to human cells which are growing, acts as a potent regula-
tor of their growth. Inhibition of growth can be achieved
at dosages significantly below those required with other
compounds already found to be clinically useful as anti-
cancer drugs. At even lower dosages, this specific cyto-
kinin antagonist has been found to stimulate growth in many
cases.
In particular, 3-methyl-7-n-pentylaminopyrazolo-
[4,3-d]pyrimidine has been tested in assay systems based on
cell cultures containing human lymphocytes which have been
stimulated by phytohemagglutinin (PHA). Previous work has
shown that PHA-stimulated lymphocytes are an effective test
system for drugs having potential antimitogenic activity.
See, for example, Ling, N. R. and Kay, J. E., "Lymphocyte
Stimulation", American-Elsevier Publishing Company, N. Y.
(1975). In these assaysj the physiological effect of the
cytokinin antagonist was compared to that of N6-~ 2-isopentenyl)-
adenosine, a cytokinin having the structural formula-
C~3
~ OH - - I
N~ 2_isopentenyl)adenosine acts as an immuno-
suppressive agent, as do other known anti-cancer agents.
Additionally, it has been demonstrated that N6-~ 2-isopentenyl)-
adenosine has clinically useful activity as an anti-cancer
agent and particularly as an anti-leukemic agent. See
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1082599
1 Mittelman, A., Evans, J. T., and Chheda, G. B., "Cytokinins
as Chemotherapeutic Agents", Anals, N. Y. Acad. Sci., 255,
pp. 225-234 (1975).
Physiological response in early lymphocyte assays
was determined both by actual morphological change and by
following DNA synthesis using [3H]thymidine to label DNA
molecules as they were synthesized. Excellent correlation
between the data from both techniques was obtained, and
because of this, only DNA synthesis was followed in later
assays. These assays demonstrated that 3-methyl-7-n-pentyl-
aminopyrazolo[4,3-d]pyrimide is a more potent growth regu-
lator for PHA-stimulated'human lyrllphocytes than N6-(~2-Isopen-
tenyl)adenosine.
These assays also demonstrated that the cytokinin
antagonist has surprising activity compared to similar
homologs. Its activity is also unexpected in view of pre~
viously'published literature. For example, simply changing
the substituént in the 7-position on the pyrazolo[4,3-d]-
pyrimidine ring from n-pentyl to isopentyl, cyclopentyl or
n-hexyl significantly diminished or completely eliminated
the greater potency for inhibition. Additionally, 3-methyl-
7-n-pentylaminopyrazolo[4,3-d]pyrimidine does not contain
a ribose moiety, which had previously been suggested to
be necessary for the promotion of inhibitory activity.
'In fact, it was previously postulated that free purines
would be low''in activity relative'to their co'rres'ponding
ribonucleosides, or without inhibitory action at all.
See Gallo, R. C., Hecht, S. ~., Whang-Peng, J. and O'Hopp,
Biochim. Biophys. Acta, 281, pp. 488-500 ~1972). ~hen the
ribose analog was tested, it was found to have only low
inhibitory activity to PHA-stimulated human lymphocytes.
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108Z59~ ` -
1 Additionally, there doesn't appear to be any correlation be-
tween the relative degree of cyto~inin antagonist activity-evi-
denced by 3-methyl-7-n-pentylaminopyrazolo[4,3-d]pyrimidine
when tested against compounds from the same series and their
inhibitory effect on PHA-stimulated lymphocyte growth.
3-methyl-7-n-pentylaminopyrazolo[4,3-d]pyrimidine,
N6-(~2-isopentenyl)adenosine and various other cytokinins
or cytokinin antagonists were also tested in assays con-
taining mouse fibroblast cell lines. Cell lines utilized
included 3T3 (a normal, contact inhibited, spontaneously
transformed cell line), 3T6 (a spontaneously transformed cell
line with poor contact inhibition properties considered
to be more "transformed" in a cancerous sense than 3T3) and
SV3T3 (a Simian Virus 40 transformed cell line which is a
cancerous line).
N6- ~ 2-isopentenyljadenosine was found capable of
regulating growth of all three lines. It exhibited inhibitory
; effects at very low concentrations and pronounced cytotoxicity
effects at moderate concentrations. It was most cytotoxic
; 20 to Sv3T3, less cytotoxic to 3T6 and least cytotoxic to
3T3, implying a favorable differential cytotoxicity toward
transformed cells as compared to normal cells.
All cytokinin antagonists tested were found to
regulate growth of the mouse fibroblast cell lines. Although
3-methyl-7-n-pentylaminopyrazolo~4,3-d]pyrimide was found
to be the best of those tested, its activity was not so
remarkable in view of the others that its surprising potency
in regard to human cells would be predicted. This can be seen
by referring to Table II, Example 4, infra.
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108Z599
.
1 A cell line made resistant to N6-(~2-isopentenyl)- -
adenosine was not resistant to 3-methyl-7-n-pentylamino-
pyrazolo[4,3-d]pyrimidine. Attempts to generate a mouse
fibroblast cell line resistant to the cytokinin antagonist
failed. Although the reasons for this are not fully under-
stood, it is anticipated that this inability to develop
resistance to the inhibitory effect of the cytokinin'antag-
onist would also hold true in human cells which are growing.
Administration of the cytokinin antagonist can
be made using art-recognized techniques. In testing its
effectiveness in cell cultures, the compound was simply
added into the culture medium. For administration to human
cells, the drug could be administered by intravenous
techniques, orally, or by using any othe'r established tech-
nique. In most cases, the compound would undoubtedly be
combined with suitable carriers or diluents as is'common
practice in administering drugs.
The exact dosage to be administered will very "
with many factors, including the particular human cells
to which the drug is to be administered as well as the
desired effect to be obtained. In chemotherapeutic applica-
tions, it has been suggested that lOmg/kg/day of N6-(~2_
isopentenyl)adenosine be used in certain pharmacological
' studies, but others have used as much as 50-60 mg/kg/day.
See Mittelman, A., Evans, J. T., and Chheda, G. B., "Cyto-
kinins as Chemotherapeutic Agents", Anals, N. Y. Acad.
Sci., 255, pp. 225-234 (1975).
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~082S99
1 ' From this description, it can be appreciated by
those skilled in the art that administration of 3-methyl-7-n-
pentylaminopyrazolo[4,3-d]pyrimidine is useful in regulating
the physiological response of human cells which are growing.
These applications are generally known as chemotherapy, and
include, for example, the inhibition of growth of human
lymphocytes as is desired in the treatment of leukemia.
The following examples further illustrate and
describe this invention.
EXAMPLE 1
Synthesis of 3-methyl-7-n-pentylaminopyrazolo[4,3-d]pyrimidine
Ten ml. of n-pentylamine was ad~ed to 1.0 g.
(5.6 mmoles) of 3-methyl-7-methylthiopyrazolo[4,3-dl-
pyrimidine. The resulting solution was heated at reflux
under nitrogen for 10 hours and the cooled solution was
concentrated under diminished pressure. The residue was
purified by chromatography on 30 g. of Sephadex*LH-20
elutiontwith a water-ethanol gradient. The solid
product obtained was recrystallized from ether to produce
white crystals of 3-methyl-7-_-pentylaminopyrazolo-
[4,3-d]pyrimidine, yield 808 mg. (67%), m.p. 155-156;
CllHi7N5 (M~ calculated 219.1492; found 219.148);
tOH (pH 1)311 nm (~13,100) and 263 (6,600),Amin 280
(5,200) and 226 (2,400); ~EmOaX (pH 7) 319 (sh), 307 (10,100),
296 (12,700) and 238 (6,200.~ min 304 (9,900). 258 (3,900)
and 224 (3,500);~ EmOH (pH 12) 308 (8,100), 265 (sh) and
245 (15,400),~ min 281 (4,000) and 229 (9,200); mass spectrum:
m/e 219.148 (M~), 190.110, 176.095, 162.079, 149.073.
* Trade Mark
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1EXAMPLE 2
Transformed Lymphoc~te Assays Containing
3-methyl-7 -n- pentylaminop~razolo[4,3-dJ pyrimidine
Compared to Similar Assays Containing
5N6- ~2-isopenteny~)adenosine
Two normal healthy blood donors were used.
About 30 ml of blood was withdrawn from each donor in
heparinized tubes, capped and allowed to stand at 37O
for 2-3 hours to allow separation of the red blood cells.
White cell plasma, in the form of a yellowish layer, rose
to the top of these tubes. The yellowish layer was collected
using sterile pipettes and placed in sterile plastic tubes.
After mixing the whi~e cell suspension by turning these
tubes upside down two or three times, 20 ~1 of the suspen-
sion was diluted~in one ml of sterile phosphate bufferedsaline solution (1:50 dilution). Tel ~1 of the diluted
suspension was used to count white cells in a hematocy-
tometer to determine the amount of white cells per~ml.
The red blood~cell suspension previously left
behind was then placed in a clinical centrifuge and spun
to produce cell-free serum. A mixture was prepared by
taking a sufficient amount of the white blood cell serum
to provide 100 million white cells and diluting this serum
to 10 ml with cell-free serum. To this, 20 ml of minimum
essential medium (MEM~ with Earle's salts and glutamine
was added together with 50~ul of penicillin-streptomycin
-13- r
. . . , . . ; -.. ,, : . : .: ,
1082Sg~
1 solution containing 5,000 units pencillin and 5,000 ~g
streptomycin per ml. Two 0.6 ml aliquots were withdrawn
at this point to serve as controls.
Subsequently, 460 ~I of a solution containing
16.4 mg/ml of M form phytohemagglutinin (PHA) after re-
constitution with 10 ml of water was added to the mixture
containing the white cells. After phytohemagglutinin
addition, 0.6 ml aliquots were taken for assay, with a
typical assay consisting of 2 controls without PHA, 4 con-
trols with PHA, 6 different concentrations (0.2, 1, 2, 4, 10
and 25 ~iM) of N6-~2-isopentenyl)adenosine and 7 different
concentrations (0.2, 1, 2, 4, 10, 25 and 50 ~ of 3-methyl-
7-n-pentylaminopyrazolo[4,3-d]pyrimidine. Each of the con-
centrations for N6-(~2_isopentenyl)adenosine and 3-methyl-
7-n-pentylaminopyrazolo[4,3-d~pyrimidine was determined in
triplicate. Therefore, this assay procedure required a
total of forty-five 0.6 ml cultures: two control cultures
without PHA, four control cultures with PHA, eighteen cul-
tures with N6- ~ 2-isopentenyl)adenosine and twenty-one
cultures with 3-methyl-7-_-pentylaminopyrazolol4,3-d]pyrimidine.
Each culture, prior to drug addition, was care-
fully capped and incubated for 24 hours at 37C. At this
point, [3Hl-thymidine with a specific activity of 53.9 Ci/mmole
(5 mCi/5 ml) was added so that DMA synthesis in the
transformed leukocytes could be monitored. In order to
10~2599
1 add one ~Ci to each culture accurately, 50 ~1 were taken
directly from the container and diluted to one ml (1:20
dilution) with sterile phosphate buffered saline solution,
thereby allowing the use of a 20 yl Eppendorf pipette for
each addition. Then, one or the other of N6~ isopentenyl)-
adenosine or 3-methyl-7-_-pentylaminopyrazolo[4,3-d]-
pyrimidine were used in appropriate dilutions with sterile
phosphate-buffered saline solution so that the desired
concentrations were reached with ~0-~1 additions. Each~
tube was care~ully capped and incubated for another
24 hours at 37~ C and at the end of this incubation, each
culture was centrifuged for 10 minutes at maximum speed
on a clinical centrifuge. Supernatant was removed by
vacuum aspiration and the remaining precipitate was re-
15 - suspended in 100 ul of sterile phosphate-buffered
saline solution and 10-~ul aliquots were withdrawn from
each tube and set on glass slides for staining. After
allowing them to dry, the cells were stained by covering
the slide with a giemsa-based stain (Camko Quik Stain) for
30 seconds; following staining the cells were washed with
distilled water for one minute. The lymphoblast count
was calculated in a total of 500 cells counted.
To-isolate labeled DNA, two ml of sterile phos-
phate-buffered saline solution was added to the remaining
contents of each tube,which were then centrifuged again.
After removing the supernatant, the precipitate was re-
suspended in 0.5 ml of sterile phosphate buffered saline
solution and frozen and thawed three times by alternate
immersion in a dry ice-ethanol mixture and a 37C water
bath. The contents of each tube was then precipitated by the
-15-
~V8ZS99
1 addition of 1.5 ml of 5% ice cold perchloric acid whi~h
precipitated all nucleic acids. After 20 minutes in ice,
each tube was centrifuged again. The supernatant was re-
moved and discarded and the precipitate was washed with
1 ml of 5% ice cold perchloric acid and centrifuged again.
The supernatant was again discarded and the final pre-
cipitate was digested with 0.5 ml of Protosol tissue
solubilizer (New England Nuclear) by incubation overnight
at room temperature in a shaker at high speed. The
following morning, the contents of each tube were placed
in 5 ml of scintillation fluid and counted for 5 minutes.
Data obtained from these assay procedures are
plotted in FIGS. 1-4.
FIG. 1 is a dose-response curve illustrating the
physiological effect of N6-~2-isopentenyl)adenosine and
3-methyl-7-n-pentylaminopyrazolo~4,3-d]pyrimidine, respec-
tively, on PHA-transformed lymphocytes from the first donor,
, measured as percent of lymphocytes transformed at different
drug concentrations and relative to drug-free controls.
FIG. 2 is also a dose-response curve, but it is based upon
DNA synthesis relative to a drug-free control. These FIGS.
illustrate that there i8 excellent correlation between data
obtained by both the staining technique which produced the
data plotted in FIG. 1 and the DMA labeling which produced
the data plotted in FIG. 2. In both FIGS., it can be seen
that low concentrations of each drug produced stimulation
of PHA-transformed leukocyte growth, whereas both drugs
produce inhibition at higher drug concentration. The
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1082S99
1 greater potency of 3-methyl-7-n-pentylaminopyrazolo[4,3-d]-
pyrimidine contrasted to the potency of N6-(~2-isopentenyl~-
adenosine is clearly demonstrated.
FIGS. 3 and 4 are analogous to FIGS. 1 and 2,
respectively, and the data plotted in these FIGS. was ob-
tained from assays prepared using blood from the second
donor. The general trend is the same as in FIGS. 1 and 2
except that no stimulation of growth was seen for low
concentrations of 3-methyl-7-_-pentylaminopyrazolo~4,3-d]-
pyrimidine; however, such stimulation might have been present
at lower drug concentrations for which data was not obtained.
The greater potency of 3-methyl-7-n-pentylaminopyrazolo-
[4,3-d]pyrimidine is clearly demonstrated once again.
As illustrated by FIGS. 1 and 2 as compared to
FIGS. 3 and 4, the absolute data obtained in these assays
varies from donor to donor. In fact, the data can vary
for the same donor when the blood used is withdrawn at
different times. Such variation in the absolute data is
expected. It i8, of course, the relative data for the
effect of different drugs on the same donor's blood
withdrawn at the same time which i9 important.
EXAMPLE 3
Transformed LymPhocyte Assays
Contai~ing Various_Anticytokinins
Compared to Similar Assays Containing
N6-(a2-isopentenyl)adenosine
.
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I . . .
10~2599
1 The apparatus, materials and procedures of
Example 2 were used except that other anticytokinin
compounds were substituted for 3-methyl-7-n-pentylamino-
pyrazolol4,3-d]pyrimidine to measure their relative physio-
logical effects on transformed lymphocytes compared to that
of N~ 2-isopentenyl)adenosine. Only DNA synthesis was
followed because of its convenience and because it was
found to correlate so well with actual morphological
change. The data obtained from these assays are plotted
in FIGS. 5-10. A summary of this data in tabular form is
also presented in Table I.
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1 EXAMPLE 4
Mouse Fibroblast Cell Cultures
Containin~ A Cytokinin or
Anticytokinin
Mouse fibroblast cells (3T6) were cultured in
Dulbecco's modified medium, supplemented with penicillin-
G(6 mg/lO0 ml), streptomycin (10 mg/lO0 ml) and horse
serum. Tissue culture dishes containing 4 ml of medium
were inoculated with 2 x 104 cells. After 24 hours of incu-
bation, the medium was replaced with fresh medium contain-
ing either a cytokinin or anticytokinin at one of the six
concentrations tested in duplicate for each compound. The
dishes were incubated at 37 and each was observed for
growth relative to drug-free controls at five days. In-
hibition was regarded as fewer cells than in the drug-
free controls, and toxicity was indicated by ceIl detach-
ment from the monolayers. The data obtained is summarized
in Table II.
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-23-
~082599
1 Those skilled in the art will recognize many
equivalents to the specific embodiments of the invention
described herein. Such equivalents are considered part
of this invention and are intended to be encompassed by
the scope of the following claims.
-24-
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