U.S. patent application number 09/841602 was filed with the patent office on 2002-04-18 for biologically active spermidine analogues, pharmaceutical compositions and methods of treatment.
Invention is credited to Bergeron, Raymond J. JR..
Application Number | 20020045780 09/841602 |
Document ID | / |
Family ID | 25267859 |
Filed Date | 2002-04-18 |
United States Patent
Application |
20020045780 |
Kind Code |
A1 |
Bergeron, Raymond J. JR. |
April 18, 2002 |
Biologically active spermidine analogues, pharmaceutical
compositions and methods of treatment
Abstract
Polyamines having the formula: 1 or a salt thereof with a
pharmaceutically acceptable acid wherein: R.sub.1-R.sub.5 may be
the same or different and are alkyl, aryl, aryl alkyl, cycloalkyl
or hydrogen; at least one of R.sub.1 and R.sub.2 and at least one
of R.sub.4 and R.sub.5 are not hydrogen, and any of the alkyl
chains may optionally be interrupted by at least one etheric oxygen
atom, excluding N.sup.1,N.sup.3-diethylspermidine and
N.sup.1,N.sup.3-dipropyls- permidine; and A and B are bridging
groups which effectively maintain the distance between the nitrogen
atoms such that the polyamine: (i) is capable of uptake by a target
cell upon administration of the polyamine to a human or non-human
animal; and (ii) upon uptake by the target cell, competitively
binds via an electrostatic interaction between the positively
charged nitrogen atoms to substantially the same biological
counter-anions as the intracellular natural polyamines in the
target cell.
Inventors: |
Bergeron, Raymond J. JR.;
(Gainesville, FL) |
Correspondence
Address: |
Miles & Stockbridge
Suite 500
1751 Pinnacle Drive
McLean
VA
22102-3833
US
|
Family ID: |
25267859 |
Appl. No.: |
09/841602 |
Filed: |
April 25, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09841602 |
Apr 25, 2001 |
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08834807 |
Apr 3, 1997 |
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6235794 |
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Current U.S.
Class: |
564/512 ;
548/557 |
Current CPC
Class: |
C07C 211/14
20130101 |
Class at
Publication: |
564/512 ;
548/557 |
International
Class: |
C07C 211/13; C07D
207/10 |
Goverment Interests
[0001] The research which led to the completion and reduction to
practice of the present invention was supported in part by Grant
No. R01-DK49108 awarded by the National Institutes of Health (NIH).
The U.S. Government has certain rights in and to the invention
described and claimed herein.
Claims
I claim:
1. A polyamine which does not occur in nature having the formula:
32or a salt thereof with a pharmaceutically acceptable acid
wherein: R.sub.1-R.sub.5 may be the same or different and are
alkyl, aryl, aryl alkyl, cycloalkyl or hydrogen; at least one of
said R.sub.1 and R.sub.2 and at least one of said R.sub.4 and
R.sub.5 are not hydrogen, and any of said alkyl chains may
optionally be interrupted by at least one etheric oxygen atom,
excluding N.sup.1,N.sup.3-diethylspermidine and
N.sup.1,N.sup.3-dipropylspermidine; and A and B may be the same or
different and are bridging groups including unsubstituted
heterocyclic bridging groups which effectively maintain the
distance between the nitrogen atoms such that the polyamine: (i) is
capable of uptake by a target cell upon administration of the
polyamine to a human or non-human animal; and (ii) upon uptake by
said target cell, competitively binds via an electrostatic
interaction between the positively charged nitrogen atoms to
substantially the same biological counter-anions as the
intracellular natural polyamines in the target cell, provided that
where A or B is a heterocyclic bridging group, the bridging group
is an unsubstituted heterocyclic group incorporating said N.sup.1,
N.sup.2 or N.sup.3 atoms in the heterocyclic ring as an
unsubstituted N atom; said polyamine, upon binding to the
biological counter-anion in the cell, functions in a manner
biologically different than said intracellular polyamines.
2. A polyamine according to claim 1, upon binding to said
biological counter-anion in said cell, exerting an anti-neoplastic
function.
3. A polyamine according to claim 1, wherein said bridging groups A
and B may be the same or different and are alkylene, branched
alkylene, cycloalkylene or arylalkylene.
4. The polyamine according to claim 1 having the formula:
R.sub.1--N.sup.1H--(CH.sub.2).sub.3--N.sup.2H--(CH.sub.2).sub.3--N.sup.3H-
--R.sub.2 wherein: R.sub.1 and R.sub.2 may be the same or different
and are alkyl having, at most, 10 carbon atoms.
5. The polyamine of claim 4 wherein R.sub.1=R.sub.2=methyl.
6. The polyamine of claim 4 wherein R.sub.1=R.sub.2=ethyl.
7. The polyamine of claim 4 wherein R.sub.1=R.sub.2=n-propyl.
8. The polyamine of claim 4 wherein R.sub.1=H and
R.sub.2=ethyl.
9. The polyamine of claim 4 wherein R.sub.1=H and
R.sub.2=n-propyl.
10. The polyamine according to claim 1 having the formula:
R.sub.1--N.sup.1H--(CH.sub.2).sub.3--N.sup.2H--(CH.sub.2).sub.4--N.sup.3H-
--R.sub.2 wherein: R.sub.1 and R.sub.2 may be the same or different
and are alkyl having, at most, 10 carbon atoms.
11. The polyamine of claim 10 wherein R.sub.1=R.sub.2=methyl.
12. The polyamine of claim 10 wherein R.sub.1=R.sub.2=ethyl.
13. The polyamine of claim 10 wherein R.sub.1=R.sub.2=n-propyl.
14. The polyamine of claim 10 wherein R.sub.1=ethyl and
R.sub.2=H.
15. The polyamine of claim 10 wherein R.sub.1=H and
R.sub.2=ethyl.
16. The polyamine of claim 10 wherein R.sub.1=n-propyl and
R.sub.2=H.
17. The polyamine of claim 10 wherein R.sub.1=H and
R.sub.2=n-propyl.
18. The polyamine according to claim 1 having the formula:
R.sub.1--N.sup.1H--(CH.sub.2).sub.4--N.sup.2H--(CH
.sub.2).sub.4--N.sup.3H--R.sub.2 wherein: R.sub.1 and R.sub.2may be
the same or different and are alkyl having, at most, 10 carbon
atoms.
19. The polyamine of claim 18 wherein R.sub.1=R.sub.2=methyl.
20. The polyamine of claim 18 wherein R.sub.1=R2=ethyl.
21. The polyamine of claim 18 wherein R.sub.1=R.sub.2=n-propyl.
22. The polyamine of claim 18 wherein R.sub.1=H and
R.sub.2=ethyl.
23. The polyamine of claim 18 wherein R.sub.1=H and
R.sub.2=n-propyl.
24. The polyamine according to claim 1 having the formula:
R.sub.1--N.sup.1H--(CH.sub.2).sub.4--N.sup.2H--(CH.sub.2).sub.5--N.sup.3H-
--R.sub.2 wherein: R.sub.1 and R.sub.2 may be the same or different
and are alkyl having, at most, 10 carbon atoms.
25. The polyamine of claim 24 wherein R.sub.1=R.sub.2=methyl.
26. The polyamine of claim 24 wherein R.sub.1=R.sub.2=ethyl.
27. The polyamine of claim 24 wherein R.sub.1=R.sub.2=n-propyl.
28. The polyamine according to claim 1 having the formula:
R.sub.1--N.sup.1H--(CH.sub.2).sub.5--N.sup.2H--(CH.sub.2).sub.5--N.sup.3H-
--R.sub.2 wherein: R.sub.1 and R.sub.2 may be the same or different
and are alkyl having at most, 10 carbon atoms.
29. The polyamine of claim 28 wherein R.sub.1=R.sub.2=methyl.
30. The polyamine of claim 28 wherein R.sub.1=R.sub.2=ethyl.
31. The polyamine of claim 28 wherein R.sub.1=R.sub.2=n-propyl.
32. A pharmaceutical composition in unit dosage form comprising a
pharmaceutically acceptable carrier and a pharmaceutically
effective amount of a polyamine of claim 1 or a salt thereof with a
pharmaceutically acceptable acid.
33. The pharmaceutical composition of claim 32 comprising an amount
of said polyamine or salt pharmaceutically effective to treat a
human or non-human patient afflicted with tumor cells sensitive to
said polyamine or salt thereof.
34. A method of treating a human or non-human patient in need
thereof comprising administering thereto a pharmaceutically
effective amount of a polyamine of claim 1 or a salt thereof with a
pharmaceutically acceptable acid.
35. The method according to claim 34 comprising administering to
said patient afflicted with tumor cells sensitive to said polyamine
or salt thereof an amount of said polyamine or salt thereof
pharmaceutically effective to inhibit the growth of said tumor
cells.
Description
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to novel polyamines useful as
active ingredients in pharmaceutical compositions and therapeutic
methods of treatment.
[0004] 2. Description of the Prior Art
[0005] Because of the sustained increases in polyamine biosynthesis
in pre-neoplastic and neoplastic tissues, a great deal of attention
has been directed to the polyamine biosynthetic network as a target
in anti-neoplastic strategies [Pegg, "Polyamine Metabolism and Its
Importance in Neoplastic Growth and as a Target for Chemotherapy,"
Cancer Res., Vol. 48, pages 759-774 (1988); and Marton et al,
"Directions for Polyamine Research," J. Cell Biochem., Vol. 45,
pages 7-8 (1991)]. Initial work focused on the design and synthesis
of compounds which would inhibit L-ornithine decarboxylase (ODC)
[Bey et al, "Inhibition of Basic Amino Acid Decarboxylases Involved
in Polyamine Biosynthesis," Inhibition of Metabolism Biological
Significance and Basis for New Therapies, McCann et al, eds.;
Academic Press: Orlando, Fla., pages 1-32 (1987)] and
S-adenosyl-L-methionine decarboxylase (AdoMetDC) [Pegg, Cancer
Res., Vol. 48, supra; and Williams-Ashman et al, "Methylglyoxal
Bis(guanylhydrazone) as a Potent Inhibitor of Mammalian and Yeast
S-Adenosylmethionine Decarboxylases," Biochem. Biophys. Res.
Commun., Vol. 46, pages 288-295 (1972)]. Some success was achieved
through this approach in that difluoromethylornithine (DFMO), an
ODC inhibitor, and methylglyoxyl-bis(guanylhydrazone) (MGBG), an
AdoMetDC inhibitor, were effective against both in vivo and in
vitro tumors [Sunkara et al, "Inhibitors of Polyamine Biosynthesis:
Cellular and In Vivo Effects on Tumor Proliferation," Inhibition of
Polyamine Metabolism Biological Significant Cause and Basis for New
Therapies, McCann et al, eds.; Academic Press: Orlando, Fla., pages
121-140 (1987); and Pegg et al, "S-Adenosylmethionine Decarboxylase
as an Enzyme Target for Therapy," Pharmacol. Ther., Vol. 56, pages
359-377 (1992)]. However, clinical trials did not mirror the
success realized in the model systems; the drug either was too
toxic as with MGBG [Pegg et al, Biochem. Pharmacol., Vol. 27, pages
1625-1629 (1978)] or was unable to show significant impact on
tumors in humans as with DFMO [Schecter et al, "Clinical Aspects of
Inhibition of Ornithine Decarboxylase with Emphasis on the
Therapeutic Trials of Eflornithine (DFMO) in Cancer and Protozoan
Diseases," Inhibition of Polyamine Metabolism. Biological
Significance and Basis for New Therapies, McCann et al, eds.;
Academic Press: Orlando, FL, pages 345-364 (1987)]. One of the
problems with the target enzymes ODC and AdoMetDC is associated
[Seiler et al, "Polyamine Transport in Mammalian Cells," Int. J.
Biochem., Vol. 22, pages 211-218 (1990)] with their very short
half-lives, i.e., about 20 minutes. This can translate into a
protracted exposure requirement for patients which is a less than
desirable situation. Nonetheless, both DFMO and MGBG served well as
proof of principle that the polyamine biosynthetic network was an
excellent target in the design of anti-cancer drugs.
[0006] It would thus be desirable to design polyamine analogues
which would be incorporated via the polyamine transport apparatus
and, once in the cell, would find their way to the same subcellular
distribution sites as the normal polyamines do, but would be unable
to be further processed [Jnn et al, "Polyamines in Rapid Growth and
Cancer," Biochim. Biophys. Acta, Vol. 473, page 241 (1978); and
Porter et al, "Enzyme Regulation as an Approach to Interference
with Polyamine Biosynthesis--an Alternative to Enzyme Inhibition,"
Enzyme Regul., Vol. 27, pages 57-79 (1988)]. They would appear
enough like the natural polyamines to shut down polyamine enzymes
just as when the cells are exposed to exogenous spermine.
[0007] Thus, a series of terminally N-alkylated tetraamines, which
exhibit anti-neoplastic activity against a number of murine and
human tumor lines both in vitro and in vivo, were assembled
[Bergeron et al, "Synthetic polyamine analogues as
antineoplastics," J. Med. Chem., Vol. 31, pages 1183-1190 (1988);
Bergeron et al, "Antiproliferative Properties of Polyamine
Analogues: a Structure-Activity Study," J. Med. Chem., Vol. 37,
pages 3464-3476 (1994); Bernacki et al, "Antitumor Activity of
N,N'-Bis(ethyl)spermine Homologues Against Human MALME-3 Melanoma
Xenografts," Cancer Res., Vol. 52, pages 2424-2430 (1992); Porter
et al, "Biological Properties of N.sup.4-Spermidine Derivatives and
Their Potential in Anti-cancer Chemotherapy," Cancer Res., Vol. 42,
pages 4072-4078 (1982); and Porter et al, "Biological Properties of
N.sup.4- and N.sup.1,N.sup.8-Spermidine Derivatives in Cultured
L1210 Leukemia Cells," Cancer Res., Vol. 45, pages 2050-2057
(1985)]. These tetraamines have been shown to utilize the polyamine
transport apparatus for incorporation [Bergeron et al, J. Med.
Chem., Vol. 37, supra; and Porter et al, "Aliphatic Chain Length
Specific of the Polyamine Transport System in Ascites L1210
Leukemia Cells," Cancer Res., Vol. 44, pages 126-128 (1984)],
deplete polyamine pools [Bergeron et al, "Role of the Methylene
Backbone in the Antiproliferative Activity of Polyamine Analogues
on L1210 Cells," Cancer Res., Vol. 49, pages 2959-2964 (1989)],
drastically reduce the level of ODC [Pegg et al, "Control of
Ornithine Decarboxylase Activity in
.alpha.-Difluoromethylornithine-Resistant L1210 Cells by Polyamines
and Synthetic Analogues," J. Biol. Chem., Vol. 263, pages
11008-11014 (1988); and Porter et al, "Relative Abilities of
Bis(ethyl) Derivatives of Putrescine, Spermidine and Spermine to
Regulate Polyamines Biosynthesis and Inhibit L1210 Leukemia Cell
Growth," Cancer Res., Vol. 47, pages 2821-2825 (1987)] and AdoMetDC
activities [Pegg et al, J. Biol. Chem., Vol. 263, supra; and Porter
et al, Cancer Res., Vol. 47, supra] and in some cases to
up-regulate spermidine/spermine/N.sup.1-acetyltransf- erase (SSAT)
[Pegg et al, "Effect of N.sup.1,N.sup.12-Bis(ethyl)spermine and
Related Compounds on Growth and Polyamine Acetylation, Content and
Excretion in Human Colon Tumor Cell," J. Biol. Chem., Vol. 264,
pages 11744-11749 (1989); Casero et al, "Differential Induction of
Spermidine/Spermine N.sup.1-Acetyltransferase in Human Lung Cancer
Cells by the Bis(ethyl)polyamine Analogues," Cancer Res., Vol. 49,
pages 3829-3833 (1989); Libby et al, "Major Increases in
Spermidine/Spermine-N.sup.1-Acetyltransferase by Spermine Analogues
and Their Relationship to Polyamine Depletion and Growth Inhibition
in L1210 Cells," Cancer Res., Vol. 49, pages 6226-6231 (1989);
Libby et al, "Structure-Function Correlations of Polyamine
Analog-Induced Increases in Spermidine/Spermine Acetyltransferases
Activity," Biochem. Pharmacol., Vol. 38, pages 1435-1442 (1989);
Porter et al, "Correlations Between Polyamine Analog-Induced
Increases in Spermidine/Spermine N-Acetyltransferase Activity,
Polyamine Pool Depletion and Growth Inhibition in Human Melanoma
Cell Lines," Cancer Res., Vol. 51, pages 3715-3720 (1991);
Fogel-Petrovic et al, "Polyamine and Polyamine Analog Regulation of
Spermidine/Spermine N.sup.1-Acetyltransferase in MALME-3M Human
Melanoma Cells," J. Biol. Chem., Vol. 268, pages 19118-19125
(1993); and Shappell et al, "Regulation of Spermidine/Spermine
N.sup.1-Acetyltransferase by Intracellular Polyamine Pools-Evidence
for a Functional Role in Polyamine Homeostasis," FEBS Lett., Vol.
321, pages 179-183 (1993)]. Interestingly, on incorporation of the
tetraamine analogues, the total picoequivalents of charge
associated with the analogues, as well as the natural polyamines,
is maintained for about 24 hours. Thus, as the cell is
incorporating n picoequivalents of drug, it is excreting n
picoequivalents of natural polyamines.
[0008] Very small structural alterations in these spermine
analogues and homologues result in substantial differences in their
biological activity [Bergeron et al, Cancer Res., Vol. 49, supra].
For example, while the tetraamines N.sup.1,N.sup.12-diethylspermine
(DESPM), N.sup.1,N.sup.11-diethylnorspermine (DENSPM) and
N.sup.1,N.sup.14-diethyl- homospermine (DEHSPM) suppress ODC and
AdoMetDC to about the same level at equimolar concentrations, the
effect of both DESPM and DEHSPM on cell growth occurs earlier than
that observed for DENSPM. The K.sub.i value of DENSPM is over 10
times as great [Bergeron et al, Cancer Res., Vol. 49, supra] as
those of DESPM and DEHSPM for the polyamine transport system.
However, the most notable difference between the three analogues is
related to their ability to stimulate SSAT [Casero et al, Cancer
Res., Vol. 49, supra; Libby et al, Cancer Res., Vol. 49, supra;
Libby et al, Biochem. Pharmacol., Vol. 38, supra; and Porter et al,
Cancer Res., Vol. 51, supra]. The tetraamine DENSPM up-regulates
SSAT by 1200 fold in MALME-3 cells, while DESPM and DEHSPM
stimulate SSAT by 250- and 30-fold, respectively [Porter et al,
Cancer Res., Vol. 51, supra]. Thus, the impact of the tetraamine
compounds on cell growth was shown to be dependent on: the distance
between the nitrogens; the nature of the terminal alkyl
substituents [Bergeron et al, J. Med. Chem., Vol. 37, supra] and,
most importantly, on the charge status of the molecules [Bergeron
et al, "The Role of Charge in Polyamine Analogue Recognition," J.
Med. Chem., Vol. 38, pages 2278-2285 (1995)].
[0009] It was decided to establish whether or not a similar
structure activity relationship exists for triamines, i.e.,
analogues of spermidine. The importance of this issue is
underscored by the tremendous difference in toxicity between the
triamines and tetraamines in general. Triamines are much less
toxic, thus making them of potentially useful therapeutic value
[Bergeron et al, "Metabolism and Pharmacokinetics of
N.sup.1,N.sup.11-Diethylnorspermine," Drug Metab. Dispos., Vol. 23,
pages 1117-1125 (1995)].
[0010] It is, therefore, an object of the present invention to
provide certain novel triamines possessing biological activity, in
particular, anti-neoplastic activity.
SUMMARY OF THE INVENTION
[0011] This and other objects are realized by the present
invention, one embodiment of which relates to polyamines not
occurring in nature having the formula: 2
[0012] or a salt thereof with a pharmaceutically acceptable acid
wherein:
[0013] R.sub.1-R.sub.5 may be the same or different and are alkyl,
aryl, aryl alkyl, cycloalkyl or hydrogen; at least one of R.sub.1
and R.sub.2 and at least one of R.sub.4 and R.sub.5 are not
hydrogen, and any of the alkyl chains may optionally be interrupted
by at least one etheric oxygen atom, excluding
N.sup.1,N.sup.3-diethylspermidine and
N.sup.1,N.sup.3-dipropylspermidine; and
[0014] A and B may be the same or different and are bridging groups
including unsubstituted heterocyclic bridging groups which
effectively maintain the distance between the nitrogen atoms such
that the polyamine: (i) is capable of up-take by a target cell upon
administration of the polyamine to a human or non-human animal; and
(ii) upon uptake by the target cell, competitively binds via an
electrostatic interaction between the positively charged nitrogen
atoms to substantially the same biological counter-anions as the
intracellular natural polyamines in the target cell, provided that
where A or B is a heterocyclic bridging group, the bridging group
is an unsubstituted heterocyclic group incorporating said N.sup.1,
N.sup.2 or N.sup.3 atoms in the heterocyclic ring as an
unsubstituted N atom; the polyamine, upon binding to the biological
counter-anion in the cell, functions in a manner biologically
different than the intracellular polyamines.
[0015] A further embodiment of the invention concerns a
pharmaceutical composition in unit dosage form comprising a
pharmaceutically acceptable carrier and a pharmaceutically
effective amount of a polyamine as described above or a salt
thereof with a pharmaceutically acceptable acid.
[0016] An additional embodiment of the invention comprises a method
of treating a human or non-human patient in need thereof comprising
administering thereto a pharmaceutically effective amount of a
polyamine described above or a salt thereof with a pharmaceutically
acceptable acid.
[0017] Other embodiments of the invention will become apparent from
the following detailed description of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIGS. 1-5 depict reaction schemes for the syntheses of the
polyamines of the invention.
[0019] FIGS. 6(a) and 6(b) depict the structure-activity
relationship between triamine analogues and tetraamine analogues,
respectively, and SSAT up-regulation.
[0020] FIG. 7 elaborates the metabolic transformation of the
triamine analogues.
[0021] FIG. 8 depicts the structure-activity relationship between
the triamine analogues and K.sub.i values.
[0022] FIG. 9(a) represents the structure-activity relationship
between the triamine analogues and the IC.sub.50 values.
[0023] FIG. 9(b) illustrates the structure-activity relationship
between the tetraamine analogues and the IC.sub.50 values.
DETAILED DESCRIPTION OF THE INVENTION
[0024] In the polyamines of the invention, as described in the
above structural formula, R.sub.1-R.sub.6 may be alkyl, e.g.,
methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl,
tert-butyl; aryl, e.g., phenyl, p-tolyl, 2,4,6-trimethylphenyl;
aryl alkyl, e.g., benzyl, .alpha.-phenethyl, .beta.-phenethyl;
cycloalkyl, e.g., cyclohexyl, cyclobutyl, cyclopentyl, cycloheptyl;
any of the foregoing wherein the alkyl chain is interrupted by
etheric oxygen, e.g., CH.sub.3O(CH.sub.2).sub.2--,
CH.sub.3O(CH.sub.2).sub.2O(CH.sub.2).sub.2--- , CH.sub.3
O(CH.sub.2).sub.2O(CH.sub.2).sub.2O(CH.sub.2).sub.2--; or
hydrogen.
[0025] Except where R.sub.1-R.sub.6 are hydrogen or etheric
substituents, each are hydrocarbyl and may have from about 1 to
about 10 carbon atoms, it being understood that the size of the
substituents will be tailored in each case to ensure that the
polyamine is capable of uptake by the target cell and, upon uptake,
will competitively bind with the intracellular counter-anions as
described above.
[0026] The bridging groups A and B may be the same or different and
may be alkylene having 1-8 carbon atoms, e.g., methylene,
trimethylene, tetramethylene, pentamethylene; branched alkylene,
e.g., --CH(CH.sub.3)CH.sub.2CH.sub.2--,
--CH.sub.2CH(CH.sub.3)CH.sub.2--, --CH(CH.sub.3)CH.sub.2CH.sub.2--,
--CH.sub.2CH(CH.sub.3)CH.sub.2CH.sub.2-- -; arylalkylene, e.g.,
--CH(Ph)CH.sub.2CH.sub.2--, --CH.sub.2CH(Ph)CH.sub.- 2--,
--CH(Ph)CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2CH(Ph)CH.sub.2--CH.sub.- 2--; cycloalkylene, e.g.,
cyclohexylene, cis- and trans-1,3-cyclohexylene, 1,4-cyclohexylene,
1,3-cyclopentylene; heterocyclic groups which incorporate within
the ring one of the nitrogen atoms of the polyamine [e.g., 3
[0027] it being understood that the heterocyclic nitrogen group may
be located at the terminal end(s) or within the interior of the
polyamine.
[0028] Those skilled in the art will appreciate that it is only
necessary that the bridging groups be selected so as to ensure
uptake by the cell and competitive binding to the intracellular
counter-anion as described above.
[0029] At physiological pH's, the naturally occurring polyamines
and the analogs of the present invention are largely in a
protonated state [Bergeron et al, "Hexahydropyrimidines as masked
spermidine vectors in drug delivery," Bioorg. Chem., Vol. 14, pages
345-355 (1986)]. At a cellular level, these polycations can bind to
a collection of single unconnected anions or to anions tethered to
a single biomolecule, e.g., the phosphates on a nucleic acid.
[0030] If there is any significance to the role of charge
interaction in the biological properties of the polyamine analogs,
alterations in the polyamine methylene backbone should have
significant impact on the compound's biological properties. In
fact, the significance of charge and the length of the methylene
bridges separating the cations in the biological properties of the
polyamine analogs has been demonstrated.
[0031] Among the most preferred polyamines of the invention are
those of the following formula:
R.sub.1--N.sup.1H--(CH.sub.2).sub.m--N.sup.2H--(CH.sub.2)--N.sup.3H--R.sub-
.2
[0032] wherein: R.sub.1 and R.sub.2 may be the same or different
and are H or alkyl; preferably having, at most, 10 carbon atoms;
most preferably, methyl, ethyl and n-propyl (with the proviso that
both R.sub.1 and R.sub.2 may not be H); and m and n may be the same
or different and are 3, 4 or 5.
[0033] Exemplary of preferred polyamines of the invention are:
[0034] dimethylnorspermidine (DMNSPD)
[0035] monoethylnorspermidine (MENSPD)
[0036] diethylnorspermidine (DENSPD)
[0037] monopropylnorspermidine (MPNSPD)
[0038] dipropylnorspermidine (DPNSPD)
[0039] dimethylspermidine (DMSPD)
[0040] monoethylspermidine [(MESPD)N1]
[0041] monoethylspermidine [(MESPD)N8]
[0042] diethylspermidine (DESPD)
[0043] monopropylspermidine (MPSPD)N1]
[0044] monopropylspermidine [(MPSPD)N8]
[0045] dipropylspermidine (DPSPD)
[0046] dimethylhomospermidine (DMHSPD)
[0047] diethylhomospermidine (DEHSPD)
[0048] monopropylhomospermidine (MPHSPD)
[0049] dipropylhomospermidine (DPHSPD)
[0050] CH.sub.3NH(CH.sub.2).sub.4NH(CH.sub.2).sub.5NHCH.sub.3
[DM(4,5)]
[0051]
CH.sub.3CH.sub.2NH(CH.sub.2).sub.4NH(CH.sub.2).sub.5NHCH.sub.2CH.su-
b.3 [DE (4, 5)]
[0052]
CH.sub.3(CH.sub.2).sub.2NH(CH.sub.2).sub.4NH(CH.sub.2).sub.5NH(CH.s-
ub.2).sub.2CH.sub.3 [DP(4, 5)]
[0053] CH.sub.3NH(CH.sub.2).sub.5NH(CH.sub.2).sub.5NHCH.sub.3
[DM(5,5)]
[0054]
CH.sub.3CH.sub.2NH(CH.sub.2).sub.5NH(CH.sub.2).sub.5NHCH.sub.2CH.su-
b.3 [DE(5,5)]
[0055]
CH.sub.3(CH.sub.2).sub.2NH(CH.sub.2).sub.5NH(CH.sub.2).sub.5NH(CH.s-
ub.2).sub.2CH.sub.3 [DP(5,5)]
[0056] It will be understood by those skilled in the art that the
polyamines of the present invention may be employed to effect any
desired biological effect mediated by the polyamine biosynthetic
network or system, e.g., anti-neoplastic, anti-viral,
anti-psoriasis, anti-inflammatory, anti-arrhythmic, etc.
[0057] For the purposes of a detailed description of a preferred
embodiment of the invention, however, the activity of a
representative number of polyamines against tumor cells sensitive
thereto will be described.
[0058] The triamines of the invention described hereinbelow can be
envisioned as belonging to one of two families of polyamines having
the structural formula: 4
[0059] One family of polyamines can be characterized as having
symmetrical methylene backbones, i.e., wherein m=n.
[0060] The other family is unsymmetrical, i.e., m.noteq.n.
[0061] Synthesis of Triamines.
[0062] The two families of triamines were synthesized: (1) those
with symmetrical methylene backbones, i.e., derived from the parent
polyamines norspermidine (3,3), homospermidine (4,4) or the longer
triamine (5,5) [wherein (3,3), (4,4) and (5,5) refer to the number
of methylene groups, i.e., (m,n)], with an alkyl group at one or
both terminal nitrogens; and (2) those with unsymmetrical methylene
backbones, i.e., from the parent polyamines spermidine (3,4) or the
(4,5) triamine, with an alkyl group at one or both terminal
nitrogens (Table 1). The numbers in parentheses refer to the number
of methylenes separating successive nitrogens. In the case of the
N.sup..alpha.,N.sup..omega.-disubstituted norspermidine (m=3, n=3)
and spermidine (m=3, n=4) analogues, the commercially available
triamines norspermidine (NSPD) (1) and spermidine (SPD) (7) were
reacted with mesitylenesulfonyl chloride (3 equiv) under biphasic
conditions (CH.sub.2Cl.sub.2/dilute NaOH) to give 30 [Bergeron et
al, Drug Metab. Dispos., Vol. 23, supra], and 31, respectively
(step e) (FIG. 1, Scheme 1). These trisulfonamides were
deprotonated with NaH in DMF and treated with an excess of the
appropriate primary alkyl iodide to make intermediates 43, 44 and
46-49 (step f). Finally, the mesitylenesulfonyl blocking groups
were cleanly removed under reductive conditions utilizing 30% HBr
in HOAc and phenol in CH.sub.2Cl.sub.2 (step g) to give terminal
dimethyl-(2, 8), diethyl-(4, 11), and dipropyl-(6, 14) NSPD and
SPD, respectively, which were isolated as their recrystallized
trihydrochloride salts.
[0063] The symmetrical triamines homospermidine (HSPD) (15) (4,4)
and 1,7,13-triazatridecane (24) (5,5), which were not commercially
available, and their terminally dialkylated derivatives were
synthesized by a segmented synthesis (FIG. 1, Scheme 1).
Mesitylenesulfonamide (35) [Bergeron et al, Drug Metab. Dispos.,
Vol. 23, supra] was dialkylated with either
N-(4-bromobutyl)phthalimide to give 37 (step i) or with
5-chlorovaleronitrile to furnish 39 (step c). Hydrogenation of the
cyano groups of 39 with Raney nickel in methanolic ammonia gave
N,N-bis(5-aminopentyl)mesitylenesulfonamide (42) (step d), which
provided 5,5-triamine 24 in good yield by treatment with 30% HBr in
HOAc (step g). Use of the aromatic imide blocking group in 37
avoided the solubility problems during attempted hydrogenation
(Raney nickel, methanolic NH.sub.3) of
N,N-bis(3-cyanopropyl)mesitylenesulfonamide. Hydrazinolysis of 37
in refluxing EtOH (step j) led to
N,N-bis(4-aminobutyl)mesitylenesu- lfonamide (40). HSPD (15) itself
resulted from reductive deprotection of monosulfonamide 40 (step
g). Terminal diamines 40 and 42 were converted to their
mesitylenesulfonamides 32 and 34, respectively, (step e) and were
alkylated with the appropriate primary halide (step f). Hydrogen
bromide-promoted deprotection of masked analogues 50, 52 and 55-57
yielded DMHSPD (16), DPHSPD (19), DM(5,5) (25), DE(5,5) (26) and
DP(5,5) (27), respectively.
[0064] N.sup.1,N.sup.9-Diethylhomospermidine (DEHSPD) (17) was made
by a convergent route (FIG. 1, Scheme 1). Alkylation of sulfonamide
35 with N--(4-bromobutyl)-N-ethylmesitylenesulfonamide (58)
[Bergeron et al, J. Med. Chem., Vol. 37, supra] (2 equiv) led to
triprotected analogue 51 (step h), which was unmasked with
HBr/HOAc, giving DEHSPD (17) (step g). 3,8,14-Triazahexadecane
[DE(4,5)] (22), the terminally diethylated analogue of the
unsymmetrical 4,5-triamine, was assembled from
N-(tert-butoxycarbonyl)-N-mesitylenesulfonamide (59), a diprotected
ammonia synthon [Bergeron et al, J. Med. Chem., Vol. 37, supra]
(FIG. 2, Scheme 2). Alkylation of reagent 59 with
N-(4-bromobutyl)-N-ethylsulfonam- ide (58) (NaH/DMF) (step a) gave
triprotected monoethylputrescine 62. The BOC group of 62 was
removed with trifluoroacetic acid (TFA) (step c). The resulting
sulfonamide 63 was alkylated with N-(5-bromopentyl)-N-ethylmesi-
tylenesulfonamide (61) (step a), which was made from
ethylsulfonamide 60 and excess 1,5-dibromopentane (NaH/DMF) (step
b), to generate fully protected triamine 64. Deprotection of the
amino groups of 64 with HBr led to the diethylated analogue 22
(step d).
[0065] The 4,5-triamine 1,6,12-triazadodecane (20) and its
dialkylated analogues 2,7,13-triazatetradecane [DM(4,5)] (21) and
4,9,15-triazaoctadecane [DP(4,5)] (23) were produced by a segmented
synthesis (FIG. 1, Scheme 1). Consecutive monoalkylation of
sulfonamide 35 with 4-bromobutyronitrile (step a) and
5-bromovaleronitrile (step b) generated dinitrile 38. The cyano
groups of 38 were reduced in a Parr shaker with Raney nickel in
methanolic ammonia (step d), resulting in primary amine 41.
Cleavage of the sulfonyl group of 41 with HBr (step g) produced the
parent 4,5-triamine 20. Treatment of 41 with mesitylenesulfonyl
chloride (2 equiv) gave 33 (step e), which was terminally
dialkylated with iodomethane to 53 or with 1-iodopropane to 54
(step f). Unmasking the amino groups led to dimethylated and
dipropylated 4,5-analogues 21 and 23, respectively (step g).
[0066] N.sup.1-Propylnorspermidine (MPNSPD) (5) was made by
treating trimesitylenesulfonyl NSPD 30 [Bergeron et al, Drug Metab.
Dispos., Vol. 23, supra] with 1-iodopropane (1 equiv/NaH/DMF), and
isolating 45 from the statistical mixture of mono- and di-alkylated
products by flash column chromatography (step f) (FIG. 1, Scheme
1). Since SPD is unsymmetrical, reaction of its trisulfonamide 31
with a primary alkyl iodide (1 equiv) would lead to N.sup.1- and
N.sup.8-monoalkylated products, which may be difficult to separate.
Thus, the synthesis of both SPD and the HSPD monopropyl analogues
required a fragment synthesis (FIG. 3, Scheme 3).
N-Propylmesitylenesulfonamide (65) was converted to 3-bromopropyl
66 or 4-bromobutyl reagent 67, with the required dibromoalkane in
excess (NaH/DMF). Triphenylmethyl chloride was stirred at room
temperature with either 1,3-diaminopropane or 1,4-diaminobutane (5
equiv) in CH.sub.2Cl.sub.2 (step c), resulting in
N.sup.1-tritylated-trimethylenediamine 68 or -putrescine 69.
Sulfonation of 68 and 69 occurred at the primary nitrogen and not
next to the bulky triphenylmethyl group to give N,N'-disubstituted
diamines 70 and 71, respectively (step a).
[0067] Reaction of the anions of 70 or 71 with the appropriate
bromide 66 or 67 resulted in regiospecific N-alkylation at the
sulfonamide terminus. Specifically, reaction of 70 with 67 gave 73,
and 71 plus 66 or 67 led to 72 or 74, respectively.
[0068] The protecting groups of 45 and 72-74 were removed
simultaneously with Hbr in HOAc/PhOH, resulting in MPNSPD (5),
MPSPD(N.sup.1) (12), MPSPD(N.sup.8) (13) and MPHSPD (18),
respectively.
[0069] Both N.sup.1- (9) and N.sup.8-ethylspermidine (10) were
obtained from reduction of the requisite monoacetylspermidine with
lithium aluminum hydride in hot THF, thus completing the synthesis
of the triamine series.
[0070] Tetraamine analogue N.sup.1,N.sup.11-dipropylnorspermine
(DPNSPM) (28) was accessed from commercially available norspermine
(FIG. 4, Scheme 4). Bis-alkylation of the tetrasulfonamide dianion
of 75 [Bergeron et al, J. Med. Chem., Vol. 37, supra] with
1-iodopropane (step a) and facile removal of the mesitylenesulfonyl
blocking groups of 76 with HBr (step b) generated DPNSPM (28).
[0071] The longer polyamine 3,9,14,20-tetraazadocosane [DE(5,4,5)]
(29), the terminally diethylated derivative of the unknown (5,4,5)
tetraamine, was synthesized in three high yield steps by the
segmenting method (FIG. 5, Scheme 5). N-Ethylmesitylenesulfonamide
(60) [Schreinemakers, Recl. Trav. Chim. Pays-Bas Belg., Vol. 16,
pages 411-424 (1897)] was deprotonated (NaH/DMF) and treated with
1,5-dichloropentane (10 equiv), resulting in alkyl chloride 77
(step a). N.sup.1,N.sup.4-Bis(mesitylenesu- lfonyl)putrescine (78)
[Bergeron et al, J. Med. Chem., Vol. 37, supra] was alkylated with
synthon 77 to give masked tetraamine 79 (step b). The four blocking
groups were removed with HBr (step c) to furnish DE(5,4,5) 29 as
its crystalline tetrahydrochloride salt. Biological Evaluations. In
summarizing the biological properties of the polyamine analogues,
the results are separated into three sets of measurements: the 48-
and 96-hour IC.sub.50 values against L1210 cells and the
corresponding K.sub.i values for the polyamine transport apparatus
(Table 1); the effect on polyamine pools (Table 2); and the impact
on ODC, AdoMetDC and SSAT (Table 3). The compounds are arranged in
sets by increasing length, e.g., norspermidine, spermidine,
homospermidine, (4,5)- and (5,5)-triamines. Each set is ordered in
terms of the size of the terminal alkyl groups. While the IC.sub.50
and K.sub.i values of DESPD and its impact on polyamine pools, ODC,
AdoMetDC and SSAT have been previously reported [Porter et al,
Cancer Res., Vol. 45, supra], the measurements on this compound
were repeated so that the appropriate positive control and not a
historical control would be in place. In order to showcase the
importance of the polyamine's overall chain length in
structure-activity relationships, there is included a brief
commentary of results of tetraamine analogues [Bergeron et al, J.
Med. Chem., Vol. 37, supra] where available. Thus, numbers included
in parentheses in the tables represent the values for the
corresponding tetraamine analogues. A brief discussion is also
presented on the metabolic profile of the triamines and on the
cationic conservation of charge the cell maintains as defined by
the polyamines. Finally, a comparison of the acute and chronic in
vivo toxicities of several key triamines and tetraamines is
presented.
[0072] Antiproliferative Activity--IC.sub.50 of L1210 cells.
[0073] As shown in Table 1, NSPD is the most active among the NSPD
family of analogues with an IC.sub.50 of 0.9 .mu.M at 48 hours and
0.5 .mu.M at 96 hours. This activity is probably related to the
fact that this triamine can easily be converted to toxic
metabolites [Alarcon et al, "Evidence for the Formation of
Cytotoxic Aldehyde Acrolein from Enzymatically Oxidized Spermine or
Spermidine," Arch. Biochem. Biophys., Vol. 137, pages 365-372
(1970)]. All of the alkylated norspermidine analogues have
IC.sub.50 values >100 .mu.M at 48 hours. At 96 hours, the
IC.sub.50 values range from 3.5 to >100 .mu.M with an order of
DMNSPD<DENSPD<DPNSPD<MENSPD and MPNSPD (most to least
active). Thus, in this family, terminal dialkylation with smaller
groups increases the compound's activity, while triamines with a
single alkyl group are less active than the corresponding compound
with bis N.sup..alpha.,N.sup..omega.-alkyl substitution. In
contrast, analogues of the tetraamine norspermine, although also
inactive at 48 hours, were more active than the corresponding
triamines at 96 hours. Moreover, whether norspermine was
symmetrically substituted with methyl or ethyl groups or had a
single ethyl fixed to one of the terminal nitrogens was
insignificant relative to the 96-hour IC.sub.50 values, which were
around 2 .mu.M.
[0074] At 48 hours, SPD and all of its analogues had an IC.sub.50
of at least 100 .mu.M. Unlike NSPD, and not surprisingly, SPD is
the least active compound in its family with IC.sub.50 values above
100 .mu.M at both 48 and 96 hours. At 96 hours, DMSPD and DESPD are
substantially more active than DPSPD. When an ethyl group was
removed from either end of DESPD, a monoalkylated analogue was
produced with lower activity than DESPD, by one to two orders of
magnitude. It is interesting that monoalkylation of SPD by ethyl or
propyl at different ends result in very different activities. At 96
hours, with an IC.sub.50 of 4 .mu.M, MESPD(N.sup.1) was about 10
times more active than MESPD(N.sup.8). The same trend was found,
although to a lesser degree, among the two monopropyl SPD analogues
in that MPSPD(N.sup.1) was more than twice as active as
MPSPD(N.sup.8). Thus, alkylation at the N.sup.1 position results in
a higher activity than alkylation at N.sup.8 (Table 1). The
spermine analogues had a significant effect on cell growth even at
48 hours and at 96 hours, the IC.sub.50 concentrations of
tetraamines ranged from 0.2 to 0.8 .mu.M, with
DESPM<DPSPM<MESPM<DMSPM. Again, in every instance, the
tetraamines were more active. It is interesting that 3.7% of
intracellular N.sup.1-MESPD and 6.1% of intracellular N.sup.1-MPSPD
are metabolically converted to the corresponding tetraamines,
ME-[3,4,3] and MP-[3,4,3] respectively (Table 4). Given the potent
antiproliferative activity of the tetraamines in general, this may
help explain the enhanced activity of the N.sup.1-alkylspermidines
in comparison to the N.sup.8-alkylspermidines since the latter are
not metabolically converted to tetraamines.
[0075] Among the HSPD analogues, DEHSPD is active at 48 hours with
an IC.sub.50 of 25 .mu.M. Other analogues' IC.sub.50s are at least
100 .mu.M at 48 hours. At 96 hours, all of the IC.sub.50s fall into
the range from 0.3.about.0.9 .mu.M, except for DPHSPD which has an
IC.sub.50 of 6 .mu.M. Compared to the norspermidine and spermidine
analogues, the homospermidine analogues as a group are more active.
With the tetraamines, the most notable differences in activity were
between the diethyl and dimethyl compounds (Table 1).
[0076] The (4,5) series are the most effective triamines
identified. As the triamine chain increases in length from (4,5) to
(5,5), the activity decreases at both 48 hours and 96 hours.
Specifically, DM(4,5) and DE(4,5) have IC.sub.50 values in the 2-6
.mu.M range, while the DP(4,5) has an IC.sub.50 around 100 .mu.M at
48 hours. At 96 hours, the IC.sub.50 values of both series
substantially decrease; even DP(4,5) has an IC.sub.50<2 .mu.M.
The numbers are uniformly higher for the (5,5) triamines even at 96
hours. The corresponding tetraamine analogues DE(4,5,4) and
DE(5,4,5) are more active at both 48 and 96 hours.
[0077] Competitive Uptake Determinations in L1210 cells.
[0078] The ability of the norspermidines, spermidines,
homospermidines, 4,5- and 5,5-triamines to compete with
radiolabeled SPD for uptake was evaluated (Table 1). The general
trend is that the terminally alkylated triamines have higher
K.sub.i values than the unalkylated triamines and are thus less
easily taken up by the cell. In the dialkylated series of
spermidines, homospermidines and 4,5-triamines, K.sub.i values
increase as the size of the terminal group increases. This is not
completely true with the norspermidines and the 5,5 triamines. The
relationship holds with methyl and ethyl but not for the propyl of
the latter two systems. Finally, the number of methylenes
separating the amines plays a role in determining polyamine uptake
properties. In general the effectiveness with which the analogues
compete for uptake is spermidines.apprxeq.homosp- ermidines>4,5
triamines>5,5 triamines>norspermidine. Interestingly, this
same trend is observed with the ethylated tetraamines,
spermines.apprxeq.homospermines>DE(3,4,4).apprxeq.DE(4,5,-
4)>norspermine.
1TABLE 1 TRIAMINE ANALOGUE STRUCTURES, ABBREVIATIONS, L1210 GROWTH
INHIBITION AND TRANSPORT IC.sub.50 (.mu.M) Structure Abbreviation
48 hour 96 hour K.sub.i(.mu.M) Norspermidines 1 5 NSPD 0.9 0.5 7.2
2 6 DMNSPD >100 (>100) 3.5-6.0 (2.5) 60 (5.6) 3 7 MENSPD
>100 (>100) >100 (2.5) 34 (7.7) 4 8 DENSPD >100
(>100) 10 (2) 250 (17) 5 9 MPNSPD >100 .about.100 33 6 10
DPNSPD >100 (>100) 60 (18) 125 (11) Spermidine 7 11 SPD
>100 >100 2.2 8 12 DMSPD >100 (>100) 1.5-1.8 (0.75) 5.1
(1.1) 9 13 MESPD(N1) >100 (99) 3.0-5.0 (0.33) 8.6 (1.7) 10 14
MESPD(N8) >100 40 7 11 15 DESPD .about.100 (30) 0.6-0.8 (0.18)
19.3 (1.6) 12 16 MPSPD(N1) >100 20-35 3.0 13 17 MPSPD(N8)
>100 50-60 8.5 14 18 DPSPD >100 (3) 30-35 (0.2) 25.6 (2.3)
Homospermidines 15 19 HSPD >100 1.7-4.0 3.4 16 20 DMHSPD >100
(>100) 0.9 (0.32) 5.5 (0.97) 17 21 DEHSPD 18-25 (0.2) 0.3-0.4
(0.07) 19 (1.4) 18 22 MPHSPD 100 0.5-0.7 5.0 19 23 DPHSPD >100
6.0 67 4,5-triamines 20 24 4,5-Triamine >100 0.15-0.20 1.4 21 25
DM(4,5) 2.0 0.11-0.12 21 22 26 DE(4,5) 3.0-6.0 (0.3) 0.19-0.2
(0.035) 64 (6.0) 23 27 DP(4,5) .about.100 1.0-1.4 75 5,5-triamines
24 28 5,5-Triamine .about.100 0.3-0.5 13.8 25 29 DM(5,5) 15 0.4 133
26 30 DE(5,5) 10-12 (0.4) 0.65-0.7 (0.03) 174 (16) 27 31 DP(5,5)
>100 6.0 87 K.sub.i values and IC.sub.50 concentrations at 48
and 96 hours. K.sub.i determinations were made by following
analogue inhibition of spermidine transport. The IC.sub.50 and
K.sub.i values of corresponding tetraamine analogues are shown in
parentheses. The tetraamine corresponding to DE(4,5) (22) is
DE(4,5,4), and the tetraamine corresponding to DE(5,5) (26) is
DE(5,4,5) (29).
[0079] Polyamine Pools.
[0080] The following guidelines were adopted for studying the
impact of the analogues on polyamine pools (Table 2). The
measurements were made after a 48-hour exposure to the analogue,
and two different concentrations of analogue were evaluated in each
case. For analogues whose IC.sub.50 concentration exceeded 100
.mu.M at 48 hours, the polyamine pools were determined at 100 and
500 .mu.M. For the other analogues, the effect on polyamine pools
was evaluated at the 48-hour IC.sub.50 concentration and at 5 times
this number.
[0081] At 500 .mu.M, the effects of DMNSPD, DENSPD and MPNSPD on
polyamine pools were similar (Table 2), i.e., PUT was depleted
below detectable limits and spermidine was reduced to 6-15% of
controls, while spermine levels were diminished to below 50%.
DPNSPD was not as effective as the other norspermidine analogues in
depletion of polyamine pools, e.g., at 500 .mu.M, PUT was only
lowered to 68%, SPD to 71% and no effect on SPM level. The dipropyl
analogue was similar in behavior to the parent norspermidine. The
corresponding norspermines were again more effective. At 100 .mu.M,
the effect of DMNSPM and DENSPM on polyamine pools was similar,
i.e., putrescine was depleted to below detectable limits and
spermidine was reduced to around 5% of controls, while spermine
levels were diminished to 27-36%.
[0082] DMSPD and DESPD at 100 .mu.M depleted PUT to below
detectable limits, SPD to 5%, SPM to 58% and 74% of control,
respectively. The monoalkylated SPD analogues MESPD(N.sup.1),
MESPD(N.sup.8) and MPSPD(N.sup.1) gave a similar pattern of
polyamine pool depletion. At 100 .mu.M, putrescine was depleted to
below detectable levels, spermidine to 25% and spermine to 80%, 84%
and 90% of control value. MPSPD(N.sup.8) was slightly less active
than MPSPD(N.sup.1). At 500 .mu.M, DPSPD reduced PUT to below
detectable level and SPD to around 10% of control. Like DPNSPD,
DPSPD showed little suppression of SPM levels and possibly even
some up-regulation at 100 .mu.M. Interestingly, at the level of PUT
and SPD suppression, MPSPD (N.sup.1) and MPSPD (N.sup.8) behave
very much like their MESPD counterparts. However, the propyl
analogues are slightly less effective at spermine suppression. The
parent amine SPD suppresses PUT, but not SPD or SPM. Again, the
corresponding spermines are more effective than the triamines. At
100 or 500 .mu.M DMSPM or MESPM or at 30 and 150 .mu.M DESPM,
putrescine was reduced to below detectable limits, spermidine
diminished to under 2% of control and spermine to under 25%. At 3
.mu.M, DPSPM reduced putrescine to below detection and spermidine
to 18%, while the spermine level remained at 64% of control. At 15
.mu.M DPSPM, spermidine was further reduced to 9% and spermine to
43%. Among the homospermidine analogues, the parent triamine, HSPD,
was the most active at polyamine suppression. At 100 .mu.M, PUT was
depleted to again below detectable levels, SPD to 4% and SPM to
32%. With all of the HSPD analogues, at 500 .mu.M, the level of
putrescine was diminished to below detectable limits and the SPD
level below 10% of control. DMHSPD and DEHSPD had little impact on
SPM level, while MPHSPD produced a mild decrease. In the case of
cells grown in 100 .mu.M and 500 .mu.M DPHSPD, the level of SPM
seemed to be increased compared to the controls. As is usual, the
homospermines were more effective than the corresponding triamine
counterparts. At 100 .mu.M, the homospermine analogue DMHSPM was
similar to the corresponding alkyl spermine in its ability to
deplete the polyamines. However, DEHSPM was somewhat less effective
at suppressing spermine pools in comparison to DESPM.
[0083] Similar results were observed with homospermidine
homologues, the (4,5) and (5,5) triamines. At 500 .mu.M, the (4,5)
and (5,5) parent amines depleted both PUT and SPD below detectable
level and SPM to 35% and 20% of control, respectively. DM(4,5) at
10 .mu.M and DE(4,5) at 15 .mu.M reduce PUT below detectable limits
and SPD to 18% of control. However, neither is very effective at
reducing SPM levels. DP(4,5) even at 500 .mu.M, while it depletes
the cell of PUT, only reduces SPD to 31% of control with possible
stimulation of SPM. Finally, DP(5,5) is only marginally active,
requiring a 500 .mu.M concentration to even reduce PUT by 50% and
SPD by 30% and with no impact on SPM. However, the homospermine
homologues DE(4,5,4) and DE(5,4,5), both of which demonstrated low
48-hour IC.sub.50 values, 0.3 and 0.4 .mu.M, respectively, were
similar to the corresponding triamines at reducing polyamines.
2TABLE 2 IMPACT OF TRIAMINE ANALOGUES ON POLYAMINE POOLS.sup.a
Compd Conc.(.mu.M) Put Spd Spm Analogue.sup.b Norspermidines 1 NSPD
0.9 38 44 113 1.09 4.5 0 12 83 2.14 2 DMNSPD 100 (100) 0 (0) 9 (5)
58 (36) 5.00 (2.14) 500 (500) 0 (0) 6 (3) 48 (27) 5.51 (1.84) 4
DENSPD 100 (10) 0 (30) 17 (14) 74 (31) 3.67 (1.59) 500 (100) 0 (0)
7 (6) 47 (30) 3.77 (2.44) 5 MPNSPD 100 0 29 56 3.07 500 0 15 49
4.78 6 DPNSPD 100 (100) 70 (61) 76 (35) 96 (77) 0.49 (0.89) 500
(500) 68 (0) 71 (19) 102 (56) 1.24 (1.28) Spermidines 7 SPD 100 0
117 118 -- 500 0 145 108 -- 8 DMSPD 100 (100) 0 (0) 5 (0) 58 (21)
4.96 (1.26) 500 (500) 0 (0) 0 (0) 54 (24) 4.89 (1.24) 9 MESPD(N1)
100 (100) 0 (0) 25 (1) 80 (21) 4.20 (1.24) 500 (500) 0 (0) 14 (1)
53 (19) 4.73 (1.23) 10 MESPD(N8) 100 0 26 84 4.41 500 0 15 61 4.96
11 DESPD 100 (30) 0 (0) 5 (0) 74 (12) 4.61 (0.40) 500 (150) 0 (0) 0
(0) 55 (14) 4.20 (1.13) 12 MPSPD(N1) 100 0 25 90 3.97 500 0 15 72
4.95 13 MPSPD(N8) 100 0 33 103 3.52 500 0 16 95 5.16 14 DPSPD 100
(3) 6 (0) 35 (18) 135 (64) 3.26 (1.12) 500 (15) 0 (0) 12 (9) 99
(43) 3.69 (1.09) Homospermidines 15 HSPD 100 0 4 32 3.58 500 0 2 21
4.44 16 DMHSPD 100 (100) 0 (0) 3 (0) 106 (30) 5.51 (1.49) 500 (500)
0 (0) 0 (0) 106 (27) 5.85 (1.03) 17 DEHSPD 25 (10) 0 (0) 6 (0) 114
(61) 4.61 (2.94) 125 0 3 97 4.69 18 MPHSPD 100 0 2 82 5.16 500 0 0
66 5.86 19 DPHSPD 100 0 19 144 3.12 500 0 7 111 3.68 4,5-Triamines
20 4,5 100 0 1 53 3.02 500 0 0 35 3.20 21 DM(4,5) 2 0 33 112 2.79
10 0 18 111 5.36 22 DE(4,5) 3 (0.3) 0 (44) 47 (61) 99 (70) 1.20
(0.26) 15 (1.5) 0 (0) 18 (5) 98 (31) 3.40 (0.72) 23 DP(4,5) 100 0
40 119 1.40 500 0 31 121 2.42 5,5-Triamines 24 5,5 100 0 0 33 2.58
500 0 0 20 2.50 25 DM(5,5) 15 0 33 115 2.56 75 0 20 101 3.61 26
DE(5,5) 15 (0.15) 0 (37) 55 (55) 97 (88) 1.09 (0.34) 75 (0.75) 0
(0) 23 (10) 73 (58) 1.59 (1.48) 27 DP(5,5) 100 59 73 97 0.86 500 51
69 103 1.33 .sup.aPutrescine (Put), spermidine (Spd) and spermine
(Spm) levels after 48 hours of treatment are given as % polyamine
found in untreated controls. Typical control values in
pmol/10.sup.6 L1210 cells are Put = 260 .+-. 59, Spd = 3354 .+-.
361, Spm = 658 .+-. 119. .sup.bAnalogue amount is expressed as
nmol/10.sup.6 cells. Untreated L1210 cells (10.sup.6) correspond to
about 1 .mu.L volume; therefore, concentration can be estimated as
nmol/mM.
[0084] Impact of Analogues on ODC and AdoMetDC Activities.
[0085] A comparison of the effects of the triamine versus
tetraamine polyamine analogues on ODC and AdoMetDC clearly
demonstrates that the tetraamines are more effective at suppressing
these enzymes than the corresponding triamines [Bergeron et al, J.
Med. Chem., Vol. 37, supra]. Previous studies [Porter et al,
"Regulation of Ornithine Decarboxylase Activity by Spermidine and
the Spermine Analogue N.sup.1,N.sup.8-Bis(ethy- l) spermidine,"
Biochem. J., Vol. 242, pages 433-440 (1987); and Porter et al,
"Combined Regulation of Ornithine and S-Adenosylmethionine
Decarboxylase by Spermine and the Spermine Analogue
N.sup.1,N.sup.2-Bis(ethyl)-spermine," Biochem. J., Vol. 268, pages
207-212 (1990)] suggested that the effect of the polyamine
analogues on ODC and AdoMetDC is fairly rapid. For example, DESPM
induced reduction in ODC activity plateaued at 4 hours and AdoMetDC
at 6 hours. On the basis of these studies, it was elected to
evaluate the impact of the triamines on ODC and AdoMetDC at 4 and 6
hours, respectively.
[0086] The parent triamine norspermidine reduced ODC activity to
11% of control, the corresponding dimethyl, DMSPD, to 17%, the
diethyl analogue, DENSPD, to 80% and the dipropyl compound, DPNSPD,
had no effect on this enzyme (Table 3). Monoethylnorspermidine,
MENSPD, was more active than the corresponding dialkyl analogue,
DENSPD, with reduction to 42 versus 80% of control, as was the
monopropyl, MPNSPD, relative to its dipropyl counterpart DPNSPD,
with reduction to 33 versus 100% of control. In 4 hours, 1 .mu.M
DMNSPM, MENSPM or DENSPM reduced ODC activity to nearly the same
extent, to approximately 7% of control. The triamines are generally
less effective than the corresponding tetraamines at suppressing
AdoMetDC, although the differences are not as profound with the
norspermidines versus the norspermines. The norspermidines reduce
the AdoMetDC to 41-58% of control and the norspermines to 33-49% of
control, except the dipropyls, which are at best marginally
effective.
[0087] The spermidine analogues are less active than the
corresponding spermines but more effective at reducing ODC activity
than the norspermidines. At 1 .mu.M DMSPM, MESPM or DESPM, ODC
activity was reduced to 10% or less of control, while ODC in
DPSPM-treated cells was only lowered to 52% of controls. The parent
triamine spermidine reduces ODC to 16% of control while the
alkylated analogues except for DPSPD, diminish ODC activity to
between 10-30% of control. Again, the monoalkyl analogues are more
effective than the corresponding dialkyl compounds. MESPD(N.sup.1)
and MESPD(N.sup.8) reduce ODC to 10 and 17% versus 30% for DESPD.
This property of the monoalkylated analogue is even further
accentuated with the propylated spermidines MPSPD(N.sup.1) and
MPSPD(N.sup.8) versus DPSPD lowering ODC to 18 and 14% versus 75%
of control. Again, when comparing dialkylated compounds, the larger
the alkyl substituent the less active the analogue.
[0088] The spermidine analogues were less effective than the
corresponding norspermidines and spermines at reducing AdoMet
activity. The spermidine analogues, except for DPSPD, reduce
AdoMetDC to under 70% of control activity. DPSPD has no impact on
the enzyme. Again, as with ODC, the monoalkylated spermidine
compounds were generally more active than the dialkylated
compounds. DMSPM, MESPM or DESPM at 1 .mu.M almost paralleled the
ability of the corresponding norspermine analogues to suppress
AdoMetDC with an average reduction to 33% of control, slightly more
active than the spermidines. DPSPM at 1 .mu.M reduced AdoMetDC
activity to 72% of that seen in untreated cells.
[0089] The homospermidines were less active than the corresponding
homospermines at reducing ODC activity, but similar in behavior to
the spermidines. Also, consistent with the norspermidine and
spermidine results, the triamines with the larger substituent,
propyl, were least effective and the monoalkyl compounds were more
active than the corresponding dialkyl ones. Finally, the
homospermidine analogues, except for MPHSPD, were not effective at
AdoMetDC inhibition and certainly less active than the
corresponding tetraamines.
[0090] Interestingly, adding a methylene unit to DEHSPM to produce
DE(4,5,4) resulted in a decrease in ODC suppressing activity. ODC
was lowered to only 7% of control with DEHSPM and to 20% of control
with DE(4,5,4). This methylene addition had little effect on
reduction of AdoMetDC activity, to about 40% for both. The same
phenomenon was observed on moving from lower alkylhomospermidines
to the dialkyl (4,5) and dialkyl (5,5) compounds, the
ODC-suppressing capacity substantially decreased while the AdoMetDC
properties were similar to those of the homospermidines. It is
noteworthy, however, that the parent (4,5)-triamine demonstrates
reasonably effective suppression of ODC and AdoMetDC. The (5,5)
parent triamine is an effective and highly selective ODC
antagonist, reducing ODC to 16% of control with little effect on
AdoMetDC. Other than this, there is little effect on either ODC or
AdoMetDC by (5,5) analogues. The tetraamine analogue DE(5,4,5) is
far more active against both ODC and AdoMetDC than DE(5,5).
[0091] Impact of Triamine Analogues on SSAT Activity.
[0092] While the influence of chain length and terminal
substituents are more monotonic regarding their effect on the
analogues' suppression of ODC and AdoMetDC, there are nevertheless
some notable structure-activity relationships for SSAT stimulation.
The ability of triamine analogues to up-regulate SSAT in L1210
cells is remarkably sensitive to small structural changes (Table 3;
FIG. 6a). For example, the diethyl triamines stimulate SSAT 780%
for DE(3,3) to a peak of 1380% for DE(3,4), with a decrease to 640%
for DE(4,4) and falling to essentially control values for DE(4,5),
120%, and DE(5,5), 90%. The DE triamine structure activity curve
appears to be shifted to the right from the corresponding DE
tetraamine curve (FIG. 6b). Thus, the DE tetraamine curve is
maximal at 1500% of control for DE(3,3,3) and falls to nearly
control value for DE(4,4,4), DE(4,5,4) and DE(5,4,5).
[0093] Substituent changes on the triamines have a profound effect
on SSAT stimulation only with the (3,3) and (3,4) compounds. The
differences are more compressed for the (4,4) and (4,5) triamines
and absent with (5,5) triamines. In the case of the tetraamines,
the (3,3,3) system is the only framework in which a marked effect
in SSAT stimulation is observed with substituent changes. While
there are some changes with the (3,4,3) backbone, these are again
compressed.
[0094] With both the triamines and tetraamines, unlike with ODC and
AdoMetDC, there is no relationship between substituent size and
SSAT up-regulation. However, when clear differences exist between
stimulatory abilities, i.e., (3,3), (3,4), (3,3,3), the ethyl group
is clearly the superior.
3TABLE 3 EFFECT OF POLYAMINE HOMOLOGUES ON ORNITHINE DECARBOXYLASE
(ODC), S-ADENOSYLMETHIONINE DECARBOXYLASE (AdoMetDC) AND
SPERMIDINE/SPERMINE ACETYLTRANSFERASE (SSAT) IN L1210 CELLS Compd
ODC AdoMetDC SSAT Norspermidines 1 NSPD 11 62 150 2 DMNSPD 17 (6)
41 (49) 250 (200) 3 MENSPD 42 (5) 58 (33) 390 (410) 4 DENSPD 80
(10) 45 (42) 780 (1500) 5 MPNSPD 33 38 470 6 DPNSPD 100 (79) 99
(70) 220 (460) Spermidines 7 SPD 16 43 160 8 DMSPD 22 (3) 68 (40)
270 (300) 9 MESPD(N1) 10 (10) 58 (27) 430 (150) 10 MESPD(N8) 17 54
400 11 DESPD 30 (3) 68 (28) 1380 (460) 12 MPSPD(N1) 18 56 1200 13
MPSPD(N8) 14 64 500 14 DPSPD 75 (52) 107 (72) 1030 (500)
Homospermidines 15 HSPD 11 54 430 16 DMHSPD 20 (4) 86 (45) 510
(140) 17 DEHSPD 47 (7) 90 (41) 640 (110) 18 MPHSPD 20 59 570 19
DPHSPD 86 123 420 4,5-triamines 20 4,5-triamine 19 57 410 21
DM(4,5) 56 71 130 22 DE(4,5) 100 (20) 70 (39) 120 (120) 23 DP(4,5)
83 86 80 5,5-triamines 24 5,5-triamine 16 88 90 25 DM(5,5) 105 97
90 26 DE(5,5) 100 (19) 109 (54) 90 (190) 27 DP(5,5) 73 123 90
Enzyme activity is expressed as percent of untreated control for
ODC (1 .mu.M at 4 hours), AdoMetDC (1 .mu.M at 6 hours) and SSAT
(10 .mu.M at 48 hours for triamine analogues, and 2 .mu.M at 48
hours for tetraamine analogues). The ODC, AdoMetDC and SSAT levels
of corresponding tetraamine analogues are shown in parentheses.
[0095] Metabolism.
[0096] In an experiment focused on the impact of DPNSPD (DP-[3,3]
in Table 4) on polyamine pools, a substantial unexpected peak
appeared in the chromatogram of treated cells. The suspicious peak
was shown to correspond to MPNSPD (MP-[3,3] in Table 4) as
confirmed by co-elution with an authentic sample. The intracellular
levels of MPNSPD after a 48-hour exposure to DPNSPD was about 50%
of intracellular level of the parent compound. Although
N-dealkylation had been shown to be an important step in the
metabolism of the alkylated tetraamines DENSPM [Bergeron et al,
Drug Metab. Dispos., Vol. 23, supra] and DEHSPM [Bergeron et al,
"Metabolism and Pharmacokinetics of
N.sup.1,N.sup.14-Diethylhomospermine," Drug Metab. Dispos., Vol.
24, pages 334-343 (1996)] in vivo in rodents, dogs and man,
previous in vitro studies with DEHSPM or DESPM [Bergeron et al, J.
Med. Chem., Vol. 37, supra] in L1210 cells revealed either little
or no N-dealkylation under the conditions of the experiments. The
observation of N-depropylation of DPNSPD compelled a closer look at
the metabolism of the polyamine analogues in L1210 cells. In
particular, the significance of the nature of the N-alkyl groups on
N-dealkylation was evaluated, in addition to the length and
symmetry of the polyamine backbone.
4TABLE 4 METABOLIC TRANSFORMATION OF POLYAMINE ANALOGUES BY L1210
CELLS Analogue# N-Monodealkylation Deaminopropylation Elaboration
DM-[3,3] (100 .mu.M) 5000 (100%) no N-demethylation MM-[3,3] (100
.mu.M) 2633 (82.9%) no N-demethylation MM-[3,3] 523 (16.5%)
MM-[3]** 20 (0.6%) DE-[3,3,3] (500 .mu.M) 2440(95.3%) ME-[3,3,3]
194 (4.7%) DE-[3,3] (500 .mu.M) 3761 (90.7%) ME-[3,3] 194 (4.7%)
ME-[3] 192 (4.6%) ME-[3,3] (100 .mu.M) 3051 (78.2%) [3,3] 20 (0.5%)
ME-[3] 831 (21.3%) no elaboration of N-Monoalkyl [3,3] DP-[3,3,3]
(100 .mu.M) 893 (78.2%) MP-[3,3,3]* 160 (14.0%) MP-[3,3] 89 (7.8%)
DP-[3,3] (100 .mu.M) 404 (57.7%) MP-[3,3] 214 (30.6%) [3,3]*** 64
(9.1%) MP-[3]* 18 (2.6%) MP-[3,3] (100 .mu.M) 3019 (94.3%) [3,3]
144 (4.5%) MP-[3] 37 (1.2%) no elaboration of N-Monoalkyl [3,3]
DM-[3,4] (100 .mu.M) 5000 (100%) no N-demethylation DE[3,4] (100
.mu.M) 4041 (96.3%) N8-ME-[4,3] 32 (0.8%) N1-ME-[3,4] 101 (2.4%)
N1-ME-[3,4] (100 .mu.M) 3946 (96.3%) [3,4] not determined
ME-[3,4,3] 150 (3.7%) N8-ME-[4,3] (100 .mu.M) 4825 (99.4%) [3,4]
not determined ME-[4] 29 (0.6%) no elaboration of N8-Alkyl [4,3]
DP-[3,4,3] (15 .mu.M) 1568 (79.0%) MP-[3,4,3]* 361 (18.2%)
N1-MP-[3,4] 57 (2.9%) DP-[3,4] (100 .mu.M) 3260 (90.1%) N8-MP-[4,3]
192 (5.3%) N1-MP-[3,4] 166 (4.6%) N1-MP-[3,4] (100 .mu.M) 4238
(93.9%) [3,4] not determined MP-[3,4,3] 275 (6.1%) N8-MP-[4,3] (100
.mu.M) 3549 (99.3%) [3,4] not determined MP-[4]* 26 (0.7%) no
elaboration of N8-Alkyl [4,3] DM-[4,4] (100 .mu.M) 5500 (100%) no
N-demethylation no exposed aminopropyl terminal segment DE-[4,4,4]
(100 .mu.M) 4215 (100%) no N-deethylation " DE-[4,4] (100 .mu.M)
4215 (100%) no N-deethylation " DP-[4,4] (500 .mu.M) 3149 (87.7%)
MP-[4,4] 441 (12.3%) " elaboration of N-Monoalkyl [4,4] DM-[4,5] (2
.mu.M) 2790 (100%) no N-demethylation no exposed primary
aminopropyl terminal segment DE-[4,5] (100 .mu.M) 4215 (100%) no
N-deethylation " DP-[4,5] (100 .mu.M) 1325 (69.7%) N10-MP-[5,4]*
576 (30.3%) " no elaboration of N-Monoalkyl [5,4] DM-[6,5] (15
.mu.M) 2560 (100%) no N-demethylation no exposed primary
aminopropyl terminal segment DE-[6,6] (75 .mu.M) 1585 (100%) no
N-deethylation " DP-[6,5] (500 .mu.M) 1300 (100%) no
N-depropylation " # L1210 cells were grown 48 hours in medium
containing polyamine analogue at the indicated concentration. Then
the polyamine contents of the cells were analyzed by HPLC of the
fluorescent DANSYL derivatives. Concentrations of parent drug and
metabolites in L1210 cells are in pmols/10.sup.6 and as % of total
drug in the cell. *An authentic sample of these presumed
metabolites was not available for analytical reference. All other
metabolites were identified and quantitated by comparison to
authenticated reference compounds. **Formed by deaminopropylation
of primary metabolite MM-[3,3]. ***Formed by N-depropylation of
MP-[3,3].
[0097] In order to assure that the observation was not some
artifact of the experimental conditions, it was assessed whether or
not components of the culture media itself were responsible for
dealkylation (Table 5). Fetal bovine serum (FBS), for example, is
well known to contain amine oxidases [Morgan, "Polyamine Oxidases
and Oxidized Polyamines," Chapter 13 in The Physiology of
Polyamines, Vol. I; Bachrach et al, eds., CRC Press: Boca Raton,
Fla. (1989), pages 203-229]. Indeed, 1 mM aminoguanidine, an
inhibitor [Gahl et al, "Reversal by Aminoguanidine of the
Inhibition of Proliferation of Human Fibroblasts by Spermidine and
Spermine," Chem.-Biol. Interactions, Vol. 22, pages 91-98 (1978)]
of bovine serum amine oxidase present in the standard L1210 cell
culture media, did not totally eliminate such FBS-related amine
oxidase activity. When the "complete" RPMI-40 medium containing FBS
and 1 mM aminoguanidine was incubated in the presence of 100 or 500
.mu.M DPNSPD, a small amount (<3%) of the DPNSPD was metabolized
to MPNSPD in the absence of L1210 cells. This corresponds to a
comparatively low extracellular concentration of MPNSPD (.about.3
.mu.M) and, given its relatively poor affinity (K.sub.i=33 .mu.M)
for the polyamine transport apparatus, argues against the
extracellular medium as a major source of the high levels of MPNSPD
(264 .mu.M) seen intracellularly. This conclusion is further
supported by experiments which partially or totally eliminate the
source of extracellular metabolism. For example, when FBS was
replaced with either NuSerum, a semi-synthetic substitute, or
purified bovine serum albumin, a high level of intracellular
metabolite (50% of parent analogue, Table 5) was still observed.
The chelator bathophenanthroline disulfonic acid is a well-known
inhibitor of the Cu-dependent amine oxidases present in plasma
[Frieden, "Complex Copper of Nature," Metamorphosis, A Problem in
Developmental Biology, 2nd ed., Gibert et al, eds., Plenum Press:
New York, N.Y., pages 478-483 (1981)] and, given its comparatively
high MW and anionic charge, does not cross the cell membrane
[Alcain et al, "Iron Reverses Impermeable Chelator Inhibition of
DNA Synthesis in CCl 39 cells," Proc. Natl. Acad. Sci. U.S.A., Vol.
91, pages 7903-7906 (1994); and Glahn et al, "Bathophenanthroline
Disulfonic Acid and Sodium Dithionite Effectively Remove
Surface-Bound Iron from CaCO.sub.2 Cell Monolayers," J. Nutr., Vol.
125, pages 1833-1840 (1995)]. As expected, bathophenanthroline
disulfonic acid completely abolished the ability of RPMI-40+10% FBS
to convert DPNSPD to MPNSPD. However, when cells were grown in
RPMI-containing FBS and bathophenanthroline disulfonic acid, high
intracellular concentrations of MPNSPD corresponding to ca. 50% of
the intracellular DPNSPD content were observed. These results are
in keeping with the idea that the dealkylation indeed takes place
within L1210 cells.
5TABLE 5 METABOLISM OF DPNSPD IN DIFFERENT CULTURE SYSTEMS
Metabolites Assay # Experiment Treatments (% of DPNSPD) 1 FBS.sup.a
+ L1210 (48 hours) MPNSPD (50%) 2 NuSerum.sup.a + L1210 (48 hours)
MPNSPD (50%) 3 FBS (48 hours) MPNSPD (3%) 4 Albumin.sup.b + L1210
(4 hours) MPNSPD (50%) 5 FBS + bathophenanthroline disul- MPNSPD
(0%) fonic acid (0.1 mM) (48 hours) 6 FBS + L1210 +
bathophenanthroline MPNSPD (50%) disulfonic acid (0.1 mM) (48
hours) In all of the assays, RPMI-1640 was used as culture media.
NuSerum IV is a semi-synthetic FBS substitute, containing 25% of
FBS. .sup.aAt concentration of 10%. .sup.bAt concentration of
1.5%.
[0098] Assured that what was being seen were the results of
intracellular metabolic transformation of bisalkylated triamines,
an examination of the influence of polyamine analogue structure on
the metabolite pattern observed in L1210 cells was undertaken.
These results are detailed in Table 4 for the bisalkyl triamines
and a number of their primary metabolites. Several representative
tetraamine analogues are also included to demonstrate their similar
metabolic fate to the corresponding triamines. Note, too, that the
structures are depicted with the polyamine backbone described as
Arabic numerals separated by commas so that the numeral represents
the number of methylenes in the linear alkane sections separating
amine centers, thus [3,3]=NSPD, [3,4,3]=SPM, [4]=putrescine, and so
forth.
[0099] Three types of metabolic transformations explain the
particular patterns observed (FIG. 7). First, bisalkyl polyamines
must undergo N-dealkylation before any further metabolism can
occur. If this N-dealkylation results in exposure of a primary
aminopropyl segment, the primary metabolite(s) may undergo
deaminopropylation by the SSAT/PAO polyamine degradation pathway.
If this N-dealkylation results in exposure of a primary aminobutyl
segment, then the triamine might undergo elaboration into a
tetraamine by serving as a substrate for spermine synthase, which
anneals an aminopropyl segment derived from S-adenosylmethionine
(AdoMet) to the free aminobutyl end of the molecule. Below, the
evidence as revealed in the metabolite patterns that support these
three types of metabolic transformations is detailed, along with
comments on the implications these data have with respect to the
structural requirements of the corresponding enzyme systems in
vivo.
[0100] A careful inspection of chromatograms from cells treated
with DM-[3,3], DM-[3,4], DM-[4,4], DM-[4,5] and DM-[5,5] revealed
no N-demethylation (Table 4). The dimethyl tetraamines DM-[3,3,3],
DM-[3,4,3] and DM-[4,4,4] also showed no evidence of N-dealkylation
(data not shown), and the unsymmetric tetraamine MM-[3,3,3] is only
metabolized by deaminopropylation at the primary amine terminus end
of the molecule.
[0101] Treatment of cells with DE-[3,3] or the corresponding
tetraamine, DE-[3,3,3], each resulted in the monodeethylated
metabolite, ME-[3,3] or ME-[3,3,3], respectively, in similar
amounts: 4.7% on a mole percent basis of the total (parent drug
+identified metabolites) in the cell. Interestingly, cells treated
with the unsymmetric DE-[3,4] contain each of the two possible
monodeethylated metabolites, N1-ME-[3,4] and N8-ME-[4,3] with the
total amount representing about 4% of the drug in the cell. Among
the diethylated triamine analogues, only DE-[3,3] and DE-[3,4]
showed N-deethylated metabolite(s); the analogues with longer
backbones, i.e., DE-[4,4], DE-[4,5] and DE-[5,5], do not show
significant N-deethylation at all.
[0102] Of the five different dipropyl triamines which were
evaluated, DP-[3,3], DP-[3,4], DP-[4,4], DP-[4,5] and DP-[5,5], all
but DP-[5,5] showed significant N-depropylation. As suggested from
the quantity of analogue present in cells as monodealkylated
metabolite (Table 4), N-depropylation in general occurs to a
greater extent than N-deethylation. For example, in L1210 cells
treated with DP-[3,3], 57.7% is present as the parent drug,
DP-[3,3], 30.6% as the mono-N-dealkylated metabolite, MP-[3,3],
9.1% as the di-N-dealkylated metabolite, [3,3], and 2.6% as the
secondary metabolite, MP-[3], formed by deaminopropylation of
MP-[3,3]. The same general pattern holds for cells treated with the
corresponding tetraamine, DP-[3,3,3], where 14.0% of the total is
present as MP-[3,3,3] and 7.8% as MP-[3,3] formed by secondary
deaminopropylation of MP-[3,3,3]. Cells treated with the
corresponding diethyl analogues contain substantially lower amounts
of metabolites by comparison so that 90.7% of DE-[3,3] or 95.3% of
DE-[3,3,3] is present as the unmetabolized parent compound.
[0103] The dipropyl triamine with the shortest backbone DP-[3,3]
seemed most sensitive to metabolism with MP-[3,3] representing 31%
of the total drug. With DP-[3,4], both possible monoalkylated
products N1-MP-[3,4] and N8-MP-[4,3] were detected at levels
corresponding to 4.6% and 5.3%, respectively, of the total drug in
the cell. Cells exposed to DP-[4,4] contained the
mono-N-dealkylated metabolite, MP-[4,4], representing 12.3% of the
total drug in the cell. Interestingly, only one of the two possible
N-dealkylated metabolites was apparent in cells treated with the
unsymmetric triamine DP-[4,5], and this MP-[5,4] metabolite
represented 32.9% of the total drug in the cell. When DP-[5,5] was
evaluated, no metabolic products were found, suggesting that the
aminobutyl end of DP-[4,5] system was selectively dealkylated to
form the monodealkylated metabolite, N10-MP-[5,4].
[0104] If mono-N-dealkylation exposes a primary aminopropyl
terminus, this compound is subject to further metabolism by the
SSAT/PAO system present in all cells. First, SSAT acetylates the
exposed primary amine end, then PAO oxidatively deaminates at the
interior secondary amino nitrogen of the acetamidopropylamine
segment to give acetamidopropanal, i.e., net deaminopropylation of
the substrate. PAO actively deaminopropylates
N.sup.1-acetylspermine and N.sup.1-acetylspermidine, the native
substrates, but does not recognize the acetamidobutyl segment of
N8-acetylspermidine or N-acetylputrescine as substrate. Table 4
demonstrates that, in L1210 cells, there is a strict adherence to
this specificity for a primary aminopropyl segment for further
metabolism of the monoalkylated triamines, i.e., only examples of
deaminopropylation are observed. For example, the tetraamine
MM-[3,3,3] shows a substantial amount of the deaminopropylation
metabolite, MM-[3,3], representing 16.5% of the total drug in the
cell and even some MM-[3] (0.6%), the product of deaminopropylation
of MM-[3,3]. No examples of deaminobutylation are seen, e.g.,
N8-alkyl-[4,3], monoalkyl-[4,4] and monoalkyl-[5,4] do not give
rise to such metabolites. In the case of cells treated with
ME-[3,3] or MP-[3,3], both N-dealkylation and deaminopropylation
are available paths of primary metabolism. The deaminopropylation
metabolite, ME-[3], represented 21.3% of the total drug in the
ME-[3,3] treated cells compared to only 0.5% for the N-deethylation
product, [3,3]. In contrast, the N-depropylation product, [3,3]
(4.5%), predominated compared to the deaminopropylation metabolite,
MP-[3], in MP-[3,3] treated cells.
[0105] In cells treated with the N.sup.1-monoalkylated spermidines,
N1-ME-[3,4] or N1-MP-[3,4], peaks corresponding to the respective
tetraamines ME-[3,4,3] (3.7% of total drug in cell) and MP-[3,4,3]
(6.1% of total drug in cell) were observed in the HPLC
chromatograms of the dansylated cell extract (Table 4). In the case
of cells containing substantial amounts of triamine analogues with
a free primary aminopropyl end (i.e., ME-[3,3], MP-[3,3],
N8-ME-[4,3] and N8-MP-[4,3]), no evidence of a tetraamine
elaboration metabolite was observed. only in those cases where a
free aminobutyl end was available on a spermidine, [3,4], backbone
was a tetraamine metabolite produced. No such metabolite was
produced from triamines with a free aminobutyl end on a longer
backbone (i.e., MP-[4,4] or N10-MP-[5,4]).
[0106] Thus, it is likely that at least two of the pathways
responsible for metabolic transformation involve enzymes of the
polyamine metabolic cycle present in all cells. Spermine synthase
is responsible for elaboration of a spermidine analogue to the
corresponding N-alkylspermine by annealing an aminopropyl segment
to an exposed primary aminobutyl end of the triamine. The
deaminopropylation observed in L1210 cells treated with triamine
and tetraamine analogues is readily explained as a consequence of
action by the SSAT/PAO polyamine degradative enzymes. The
possibility that the N-dealkylation step required for further
metabolic transformation of bis-alkylpolyamines may also involve
PAO is an interesting question raised by the metabolic patterns
observed.
[0107] N-Dealkylation of analogues with a hydrophobic segment
shorter than N-propyl appears to occur much less efficiently in the
case of N-ethyl, or not at all in the case of N-methyl. Among the
reported amine oxidases, polyamine oxidase (PAO) is the only one
which usually attacks at a secondary amine center, three
hydrophobic methylene carbons internal to the neutral
N.sup.1-acetamido nitrogen terminus of N.sup.1-acetylspermidine,
for example. The corresponding acetamidobutyl segment of
N.sup.8-acetylspermidine is not recognized and, therefore, not
deaminobutylated.
[0108] Conservation of Charge.
[0109] In two earlier studies, it was noted that there was a
conservation of charge with respect to the total tetraamine
cationic picoequivalence in the cell [Bergeron et al, Cancer Res.,
Vol. 49, supra; and Porter et al, Cancer Res., Vol. 51, supra]. For
example, if, after 24 hours of exposure to an alkylated polyamine,
each of the equivalent concentrations associated with charge on the
amines of both the analogues and natural polyamine is added
together, the numbers are fairly constant. For example, each
picoequivalent of putrescine is associated with two picoequivalents
of cationic charge, each picoequivalent of spermidine or its
analogues with three, and each picoequivalent of spermine with
four. In order to maintain this balance of charge, the cell
processes the natural polyamines, e.g., exports them as it
incorporates the analogues. The maintenance of total cellular
charge holds for all of the triamines examined, except the 5,5
triamines (Table 6). The implication is that the cell will not
incorporate the analogue beyond a point where the charge balance is
disrupted, at which time cell death may occur. In the case of the
tetraamines, the conservation of charge behavior seems to hold for
24 hours, but erodes after a period of time [Bergeron et al, Cancer
Res., Vol. 49, supra]. With the triamines, the conservation of
charge continues even at 48 hours.
6TABLE 6 SUMMATION OF INTRACELLULAR LEVELS OF ANALOGUES AND
POLYAMINES ANALYZED FOR AMINE EQUIVALENCE AFTER EXPOSURE TO
POLYAMINE ANALOGUES Polyamine Picoequivalents of Amine/ Average
.+-. Standard Analogues 10.sup.6 cells (.times. 10.sup.3) Deviation
Control Cell 13.21 2 DMNSPD 18.40 4 DENSPD 13.70 5 MPNSPD 17.71 6
DPNSPD 15.01 16.21 .+-. 2.22 8 DMSPD 16.09 9 MESPD(N1) 16.99 10
MESPD(N8) 17.99 11 DESPD 14.05 12 MPSPD(N1) 18.25 13 MPSPD(N8)
19.59 14 DPSPD 15.33 16.90 .+-. 1.89 16 DMHSPD 20.34 17 DEHSPD
16.92 18 MPHSPD 20.55 19 DPHSPD 15.99 18.45 .+-. 2.34 21 DM(4,5)
20.81 22 DE(4,5) 14.59 23 DP(4,5) 15.15 16.85 .+-. 3.44 24 DM(5,5)
15.5 25 DE(5,5) 9.01 26 DP(5,5) 13.91 12.81 .+-. 3.38 All Analogues
Mean 16.47 .+-. 2.08 The L1210 cells were treated with polyamine
analogues at 500 .mu.M, except DEHSPD (125 .mu.M), DM(4,5) (10
.mu.M), DE(4,5) (15 .mu.M), DM(5,5) (75 .mu.M) and DE(5,5) (75
.mu.M), for 48 hours. Levels of amine equivalence for every
analogue treated cell are averages from analysis of triplicate cell
samples. Values are obtained by multiplying the number of moles of
spermine by four, spermidine by three, putrescine #by two and
analogue by three. The typical control values in nmol/million L1210
cells are PUT = 0.260 .+-. 0.059, SPD = 3.354 .+-. 0.361, SPM =
0.658 .+-. 0.119.
[0110] Acute and Chronic Toxicity of Triamines.
[0111] In early studies of polyamine toxicity in laboratory
animals, triamines were found less toxic than tetraamines.
Spermidine was approximately one-twentieth as nephrotoxic as
spermine, and putrescine was the least toxic [Tabor et al,
"Pharmacology of Spermine and Spermidine. Some Effects on Animals
and Bacteria," J. Pharmacol. Exp. Ther., Vol. 116, pages 139-155
(1956); and Shaw, "Some Pharmacological Properties of the Polyamine
Spermine and Spermidine - a Re-appraisal," Arch. Int. Pharmacodyn.
Ther., Vol. 198, pages 36-48 (1972)].
[0112] In the current study, the acute toxicity of six analogues
and the chronic toxicity of two triamines were measured (Table 7).
The value of all polyamine LD.sub.50s are shown in both mg/kg and
mmol/kg for comparison. For acute toxicities, the polyamine
analogues were administered as a single i.p. injection to groups of
five or six animals at each dose. The animals were scored two hours
after administration of the drug. It is clear that the acute
LD.sub.50s for triamine analogues are approximately twice the acute
LD.sub.50s for the corresponding tetraamine analogues.
[0113] In the chronic toxicity regimen, mice were administered the
polyamine analogue in three doses per day (t.i.d.) for six days for
a total of eighteen injections per animal and observed for 10 days
after the final dose for lethalities. The most active triamine
DE(4,5) against L1210 cells in vitro and the spermidine analogue
DE(3,4) demonstrated much less toxicity in mice than the related
tetraamines DE(4,5,4), DE(5,4,5) and DE(3,4,3). In the early study
of tetraamines, a preliminary investigation suggested a direct
ratio relationship between the IC.sub.50 and the chronic LD.sub.50
values [Bergeron et al, J. Med. Chem., Vol. 37, supra]. However, in
the triamine systems, the 96-hour IC.sub.50 values of DE(4,5)
suggested that this triamine should be about 5 times less toxic
than corresponding tetraamine analogue, DE(4,5,4), and six times
less toxic than DE(5,4,5) (Table 1), but, in fact, they are about
eight-fold less toxic than the DE(4,5,4) and greater than ten-fold
less toxic than DE(5,4,5) (Table 7). A similar difference is also
observed in the chronic toxicity of DE(3,4). The ratio of the
triamine to the tetraamine 96-hour IC.sub.50s suggests that DE(3,4)
should be approximately four times less toxic than DE(3,4,3), but,
in fact, DE(3,4) is about five times less toxic than DE(3,4,3) in
vivo. These results suggest a potential widening of the therapeutic
window, which renders the triamine analogues as promising
anti-neoplastics of lower toxicity and encourages further pursuit
of animal studies.
7TABLE 7 COMPARISON OF THE ACUTE AND CHRONIC TOXICITY OF TETRAAMINE
AND TRIAMINE ANALOGUES ON MICE TETRAAMINES TRIAMINES Acute.sup.a
LD.sub.50 mg/kg Chronic.sup.b LD.sub.50 mg/kg- Acute LD.sub.50
mg/kg Chronic LD.sub.50 mg/kg- Compound (mmol/kg) day (mmol/kg-day)
Compound (mmol/kg) day (mmol/kg-day) DE-[3,4,3] 340 (0.842)
87.sup.e (0.215) DE-[3,4] >650.sup.d (>3.22) 426 (1.37)
DE-[4,5,4] 285 (0.638) 48 (0.104) DE-[4,5] 555.sup.e (1.64) 375
(1.11) DE-[5,4,5] 195 (0.424) 36 (0.078) DE-[5,5] 500 (1.42) nd All
of the polyamine analogues were administered in the form of
hydrochloride salts. .sup.aSingle dose i.p. .sup.bMultiple dose
i.p. (t.i.d. .times. 6 days) .sup.cAt a single dose of 250 mg/kg,
no death within the initial 2 hours, but all six animals were
expired within seven days. .sup.d15 mg/kg (t.i.d. .times. 3 days),
4/5 died on day 6 and 5/5 died on day 7. .sup.eAt a single dose of
600 mg/kg, no death within the initial 2 hours, but 5/5 died within
seven days.
[0114] The study serves to define the similarities and differences
between triamines and tetraamine analogue antineoplastics. With
both tetraamine and triamine analogues, K.sub.i values are
sensitive to the size of the terminal substituents and the length
of the backbone. This is illustrated for triamines in FIG. 8.
Generally, the larger the terminal substituent, the more poorly the
analogues are transported. In the triamine family, spermidine
analogues are the best transport competitors. Interestingly, the
(3,3) and (5,5) triamine analogues are most sensitive to N-terminal
substituent changes. With regards to uptake, the triamines are more
effectively accumulated in L1210 cells than the corresponding
tetraamines. Once in the cell, tetraamine analogues have a greater
impact on lowering overall polyamine pools; however, the triamines
are more selective at reducing spermidine. The total intracellular
charge in picoequivalents associated with polyamines, both native
and analogues, is maintained by cells exposed to both tetraamines
and triamines. However, cells treated with triamines are able to
maintain this charge balance for a more prolonged period of time.
Both tetraamine and triamine analogues, except for DENSPD and
DE(4,5), reduce ODC more effectively than AdoMetDC activity, and
tetraamines are more active at this. It was demonstrated that
triamine analogue dealkylation was very specific for triamines with
backbones of less than or equal to four methylenes and most
effective for triamines and tetraamines with
N.sup..alpha.,N.sup..omega.-dipropyl substituents.
[0115] The tetraamine analogues are uniformly more active against
L1210 cells than their triamine counterparts. With both the
triamine and tetraamine analogues the compounds' IC.sub.50 values
are also sensitive to the size of the terminal substituent and the
length of the backbone. However, the overall length between the
terminal nitrogens is the most critical issue in assessing this
activity; FIG. 9a illustrates the triamine case. When comparing
N.sup.1- with N.sup.8-monoalkylspermidines, the N.sup.1 compounds,
both ethyl and propyl were more active than the N.sup.8 compound.
The fact that the N.sup.1 compounds are elaborated by the cell to
the corresponding and more active N.sup.1-alkylspermines is in
keeping with this observation. It is recalled that the
N.sup.8-alkylspermidines cannot be and are not further processed in
the polyamine biosynthetic network. While the optimum length for
the tetraamine activity has not yet been determined (FIG. 9b),
evidence would suggest that the optimum length for the triamines
has been determined, as seen in the terminally dialkylated
(4,5)-methylene backbone series.
[0116] The triamine analogues are less toxic than the corresponding
tetraamines. Furthermore, and most important when comparing the
ratio of the 96-hour IC.sub.50/chronic LD.sub.50 values of the two
triamines, DE(3,4) and DE(4,5), with the corresponding tetraamines,
a kind of therapeutic window, the triamines appear more favorable.
This is a critical issue in the choice of the best polyamine
therapeutic.
[0117] The invention is illustrated by the following non-limiting
examples wherein parenthetical reference numerals correspond to
those in Schemes 1-5.
[0118] MENSPD (3) [Bergeron et al, Drug Metab. Dispos., Vol. 23,
supra] and tetraamine analogues [Bergeron et al, J. Med. Chem.,
Vol. 31, supra; and Bergeron et al, J. Med. Chem., Vol. 37, supra],
except for DPNSPM and DE(5,4,5), were previously synthesized.
N.sup.1- and N.sup.8-Acetylspermidine dihydrochlorides were
purchased. N--(3-aminopropyl)-1,3-propanediamine (1) was converted
to its trihydrochloride salt and recrystallized from aqueous
ethanol. Sodium hydride reactions were run in distilled DMF under
an inert atmosphere. THF was distilled from sodium and
benzophenone. Fisher Optima grade solvents were routinely used, and
organic extracts were dried with sodium sulfate. Silica gel 32-63
(40 .mu.M "flash") was used for flash column chromatography.
Melting points were determined on a Fisher-Johns melting point
apparatus and are uncorrected. Proton NMR spectra were run at 90 or
300 MHz in CDCl.sub.3 (not indicated) or D.sub.2O with chemical
shifts in parts per million downfield from tetramethylsilane or
3-(trimethylsilyl)propionic-2,2,3,3-d.sub.4 acid, sodium salt,
respectively. Coupling constants (J) are in hertz. FAB mass spectra
were run in a glycerol/trifluoroacetic acid matrix. Elemental
analyses were also performed.
[0119] Cell culture materials, RPMI-1640 medium, fetal bovine
serum, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES),
and 3-(N-morpholino)propanesulfonic acid (MOPS) were purchased.
Cell numbers were determined by electronic particle analysis
(Coulter Counter, Model ZF). The solid phase extraction columns
(SPE-3 mL/500 mg) were used. Murine L1210 leukemia cells were
obtained from the American Type Tissue Corporation.
[0120] [.sup.3H]Spermidine for uptake determinations and acetyl
coenzyme A (acetyl-1-.sup.14C) were purchased.
L-[Carboxyl-.sup.14C]-ornithine and
S-adenosyl-L-[carboxyl-.sup.14C]methionine for enzyme assays were
also purchased.
[0121] Cell Culture and IC.sub.50 Determination.
[0122] Murine L1210 leukemia cells (ATCC CCL 219) were maintained
in logarithmic growth in RPMI-1640 medium containing 10% fetal calf
serum or a semi-synthetic equivalent, NuSerum, 2% HEPES-MOPS buffer
and 1 mM aminoguanidine. The IC.sub.50s, the concentration of
compound which reduces cell growth to 50% of untreated control cell
growth, was determined after 48 hours and 96 hours of exposure to
polyamine analogue as detailed elsewhere [Bergeron et al, J. Med.
Chem., Vol. 37, supra].
[0123] Polyamine Pool Analysis.
[0124] L1210 cells in logarithmic growth were treated with
polyamine analogue at the concentrations indicated in Table 4 for
48 hours. The cells were washed twice with cold RPMI-1640, and the
pellet was treated with 0.6 N HClO.sub.4 (1 ml/10.sup.7 cells).
Polyamine contents of the perchloric acid extracts were quantitated
by HPLC of the DANSYL derivatives [Bergeron et al, J. Med. Chem.,
Vol. 37, supra].
[0125] Uptake Determinations.
[0126] The polyamine derivatives were studied for their ability to
compete with [.sup.3H]-SPD for uptake into L1210 cells [Bergeron et
al, J. Med. Chem., Vol. 37, supra]. Lineweaver-Burk plots indicated
a simple competitive inhibition with respect to SPD.
[0127] Enzyme Assays.
[0128] ODC and AdoMetDC activities were determined as
.sup.14CO.sub.2 released from [.sup.14C]-carboxyl-labeled
L-ornithine [Seely et al, "Ornithine Decarboxylase (Mouse Kidney),"
Methods Enzymol., Vol. 94, pages 158-161 (1983)] or
S-adenosyl-L-methionine [Pegg et al, "S-Adenosylmethionine
Decarboxylase (Rat Liver)," Methods Enzymol., Vol. 94, pages
234-239 (1983)], respectively. Included in each assay were
untreated L1210 cells as controls, as well as cells treated with
DEHSPM, a drug having a known reproducible effect on each enzyme,
as positive controls.
[0129] Spermidine/spermine N.sup.1-acetyltransferase activity was
based on quantitation of [.sup.14C]-N.sup.1-acetylspermidine formed
by acetylation of SPD with [.sup.14C] acetyl coenzyme A according
to the method of Libby et al [Biochem. Pharmacol., Vol. 38, supra].
Cells treated with DENSPM were positive controls.
[0130] Toxicity in Mice.
[0131] Acute and chronic toxicities were assessed in 10-12 week old
CD-1 female mice from Harlan Sprague-Dawley. For acute toxicities,
the polyamine analogues were administered in a single i.p.
injection to groups of five or six animals at each dose. The
animals were scored two hours after administration of the dose. All
survivors were further observed for 10 days to assess late onset of
toxicity from the single acute dose. In the chronic toxicity
regimen, mice were administered polyamine analogue in three i.p.
doses per day (t.i.d.) for six days, for a total of eighteen doses
per animal. Appetite, weight and overall appearance were monitored
daily. Animals were observed for 10 days following the final dose,
at which time the final score was registered. At least three test
groups of 5-6 animals each, representing three different dose
levels, were evaluated for each analogue tested. These dose levels
were chosen so that at least two groups presented with lethalities,
one with a high fraction of lethalities (>0.50, but
<1.00).
EXAMPLE 1
[0132] N.sup.1,N.sup.4,N.sup.7-Tris
(mesitylenesulfonyl)-N.sup.1,N.sup.7-d- imethylnorspermidine
(43).
[0133] NaH (60% in oil, 0.44 g, 11 mmol) was added to a solution of
30 [Bergeron et al, Drug Metab. Dispos., Vol. 23, supra] (3.39 g, 5
mmol) in DMF (70 mL) at 0.degree. C. After hydrogen evolution
ceased (30 minutes), iodomethane (1.63 g, 11.5 mmol) was slowly
added to the mixture. After stirring for 12 hours at room
temperature, the reaction mixture was quenched with distilled water
(10 mL). The solvents were removed under high vacuum, and the
residue was combined with H.sub.2O (30 mL) and extracted with
CHCl.sub.3 (4.times.40 mL). The organic portion was washed with
brine (80 mL) and evaporated by rotary evaporation. Purification by
column chromatography (8:1 toluene/EtOAc) gave 2.47 g (70%) of 43
as an oil: NMR .delta. 1.73 (quintet, 4H), 2.29 (s, 6H), 2.54-2.55
(2 s, 18H), 2.60 (s, 6H), 2.99-3.06 (2 t, 8H, J=7), 6.94 (s, 6H).
Anal. (C.sub.35H.sub.51N.sub.3O.sub.6S.sub.3) C, H, N.
EXAMPLE 2
[0134] N.sup.1,N.sup.7-Dimethylnorspermidine Trihydrochloride
(2).
[0135] HBr (30% in HOAc, 30 mL) was added slowly to a mixture of 43
(2.13 g, 3.02 mmol) and phenol (12.27 g, 0.13 mol) in
CH.sub.2Cl.sub.2 at 0.degree. C. After stirring for 1 day at room
temperature, H.sub.2O (20 mL) was added to the reaction mixture
followed by extraction with CH.sub.2Cl.sub.2 (3.times.30 mL). The
aqueous portion was concentrated under high vacuum, and the residue
was basified to pH 14 with 1 N NaOH (4 mL) and 19 N NaOH (2 mL) and
extracted with CHCl.sub.3 (14.times.10 mL). The organic extracts
were concentrated, and the residue was taken up in absolute EtOH
(40 mL) and acidified with concentrated HCl (2 mL). After the
solvents were removed, the solid was recrystallized from aqueous
EtOH to generate 0.298 g (37%) of 2 as plates: NMR (D.sub.2O)
.delta. 2.08-2.19 (m, 4H), 2.75 (s, 6H), 3.13-3.22 (m, 8H). Anal.
(C.sub.8H.sub.24Cl.sub.3N.sub.3) C, H, N.
EXAMPLE 3
[0136] N.sup.1,N.sup.4,N.sup.7-Tris
(mesitylenesulfonyl)-N.sup.1,N.sup.7-d- iethylnorspermidine
(44).
[0137] NaH (60%, 9.0 g, 0.23 mol), 30 [Bergeron et al, Drug Metab.
Dispos., Vol. 23, supra] (70.0 g, 0.103 mol), and iodoethane (19
mL, 0.24 mol) in DMF (400 mL) were reacted and worked up by the
method of 43. Column chromatography (1:4 EtOAc/pet ether) afforded
63.7 g (84%) of 44 as a viscous oil: NMR .delta. 0.97 (t, 6H, J=7),
1.60-1.72 (m, 4H), 2.29 (s, 9 H), 2.54 and 2.55 (2 s, 18H),
2.95-3.15 (m, 12H), 6.92 (s, 6H). Anal.
(C.sub.37H.sub.55N.sub.3O.sub.6S.sub.3) C, H, N. A sample was
recrystallized from EtOAc/pet ether, mp 97.degree. C.
EXAMPLE 4
[0138] N.sup.1,N.sup.7-Diethylnorspermidine Trihydrochloride
(4).
[0139] HBr (30% in HOAc, 300 mL), 44 (23.0 g, 30.7 mmol), and
phenol (116 g, 1.23 mol) in CH.sub.2Cl.sub.2 (300 mL) were reacted,
and product was isolated using the procedure of 2 to give 6.45 g
(71%) of 4 as colorless plates: NMR (D.sub.2O) .delta. 1.30 (t, 6H,
J=7), 2.05-2.18 (m, 4H), 3.08-3.22 (m, 12H). Anal.
(C.sub.10H.sub.28Cl.sub.3N.sub.3) C, H, N.
EXAMPLE 5
[0140]
N.sup.1-Propyl-N.sup.1,N.sup.4,N.sup.7-tris(mesitylenesulfonyl)nors-
permidine (45).
[0141] NaH (60%, 0.52 g, 13 mmol), 30 (2.57 g, 3.8 mmol), and
1-iodopropane (0.46 mL, 4.7 mmol) in DMF were reacted and worked up
by the method of 43. Column chromatography (3:1 hexane/EtOAc)
afforded 1.16 g (23%) of 45 as an oil: NMR 66 0.66 (t, 3H, J=7),
1.23-1.31 (m, 2H), 1.58-1.62 (m, 4H), 2.26-2.27 (2 s, 9H),
2.51-2.52 (2 s, 12H), 2.59 (s, 6H), 2.82-2.99 (m, 8H), 3.21 (t, 2H,
J=7), 4.85 (br t, 1H), 6.89-6.92 (m, 6H). Anal.
(C.sub.36H.sub.53N.sub.3O.sub.6S.sub.3) C, H, N.
EXAMPLE 6
[0142] N.sup.1-Propylnorspermidine Trihydrochloride (5).
[0143] HBr (30% in HOAc, 30 mL), 45 (1.14 g, 1.58 mmol), and phenol
(6.4 g) in CH.sub.2Cl.sub.2 were reacted, and product was isolated
using the procedure of 2 to give 87 mg (20%) of 5 as crystals: NMR
(D.sub.2O) .delta. 0.98 (t, 3H, J=7), 1.67-1.75 (m, 2H), 2.07-2.16
(m, 4H), 3.01-3.21 (m, 10H). Anal. (C.sub.9H.sub.26Cl.sub.3N.sub.3)
C, H, N.
EXAMPLE 7
[0144]
N.sup.1,N.sup.7-Dipropyl-N.sup.1,N.sup.4,N.sup.7-tris(mesitylenesul-
fonyl)-norspermidine (46).
[0145] NaH (60%, 0.44 g, 11 mmol), 30 [Bergeron et al, Drug Metab.
Dispos., Vol. 23, supra] (3.39 g, 5 mmol), and 1-iodopropane (1.95
g, 11.5 mmol) in DMF (70 mL) were reacted and worked up by the
method of 43. Column chromatography (3:1 hexane/EtOAc) afforded
3.54 g (93%) of 46 as an oil: NMR .delta. 0.7 (s, 6H), 1.20-1.65
(m, 8H), 2.25 (s, 9H), 2.50 (s, 18H), 2.80-3.05 (m, 12H), 6.87 (s,
6H). Anal. (C.sub.39H.sub.59N.sub.- 3O.sub.6S.sub.3) C, H, N.
EXAMPLE 8
[0146] N.sup.1,N.sup.7-Dipropylnorspermidine Trihydrochloride
(6).
[0147] HBr (30% in HOAc, 80 mL), 46 (3.475 g, 4.57 mmol), and
phenol (15.8 g) in CH.sub.2Cl.sub.2 (30 mL) were reacted, and
product was isolated by the procedure of 2 to provide 1.26 g (85%)
of 6 as plates: NMR (D.sub.2O) .delta. 0.87 (t, 6H, J 7), 1.60 (m,
4H), 2.01 (m, 4H), 2.93 (t, 4H, J=7), 3.06 (m, 8H). Anal.
(C.sub.12H.sub.32Cl.sub.3N.sub.3) C, H, N.
EXAMPLE 9
[0148] N.sup.1,N.sup.4,N.sup.8-Tri (mesitylenesulfonyl) spermidine
(31).
[0149] Mesitylenesulfonyl chloride (6.87 g, 31.4 mmol) in CH
Cl.sub.2 (30 mL) was added to spermidine trihydrochloride (2.5 g,
9.8 mmol) in 1 N NaOH (35 mL) at 0.degree. C., and the mixture was
efficiently stirred at room temperature overnight. The layers were
separated, and the aqueous phase was extracted with CHCl.sub.3
(3.times.50 mL). The organic phase was the washed with brine (100
mL), evaporated, and purified by column chromatography (4:3
hexane/EtOAc) to give 3.73 g (55%) of 31 as a white foam: NMR 67
1.30 (m, 2H), 1.44 (m, 2H), 1.66 (m, 2H), 2.30 (s, 9H), 2.46 (s,
6H), 2.60 (s, 12H), 2.76 (q, 2H), 2.84 (q, 2H), 3.04 (t, 2H, J=7),
3.24 (t, 2H, J=7), 4.56 (br t, 1H), 4.92 (br t, 1H), 6.90 (s, 2H),
6.95 (s, 4H). Anal. (C.sub.34H.sub.49N.sub.3O.sub.6S.sub.3) C, H,
N.
EXAMPLE 10
[0150] N.sup.1,N.sup.8-Dimethyl-N.sup.1,N.sup.4,N.sup.8-tris
(mesitylenesulfonyl) spermidine (47).
[0151] NaH (60%, 0.41 g, 10 mmol), 31 (2.15 g, 3.11 mmol), and
iodomethane (0.62 mL, 10 mmol) in DMF (60 mL) were reacted and
worked up as was 43. Column chromatography (5:3 hexane/EtOAc)
furnished 2.24 g (100%) of 47 as an oil: NMR .delta. 1.39-1.43 (m,
4H), 1.69-1.78 (m, 2H), 2.28 (s, 3H), 2.30 (s, 6H), 2.55 (s, 12H),
2.57 (s, 6H), 2.60 (s, 3H), 2.62 (s, 3H), 2.96-3.13 (m, 8H).
EXAMPLE 11
[0152] N.sup.1,N.sup.8-Dimethylspermidine Trihydrochloride (8).
[0153] HBr (30% in HOAc, 60 mL), 47 (2.24 g, 3.11 mmol), and phenol
(12.3 g) in CH.sub.2Cl.sub.2 (30 mL) were reacted, and product was
isolated by the procedure of 2 to give 0.658 g (75%) of 8 as
crystals: NMR (D.sub.2O) .delta. 1.77-1.82 (m, 4H), 2.06-2.18 (m,
2H), 2.73 (s, 3H), 2.75 (s, 3H), 3.06-3.19 (m, 8H). Anal.
(C.sub.9H.sub.26Cl.sub.3N.sub.3) C, H, N.
EXAMPLE 12
[0154]
N.sup.1,N.sup.8-Diethyl-N.sup.1,N.sup.4,N.sup.8-tris(mesitylenesulf-
onyl)spermidine (48).
[0155] NaH (80%, 0.68 g, 23 mmol), 31 (7.06 g, 10.2 mmol), and
iodoethane (2.5 mL, 31 mmol) in DMF (75 mL) were combined as in 43.
The mixture was heated at 65.degree. C. for 12 hours, cooled and
cautiously quenched with water (70 mL) and brine (100 mL), followed
by extraction with EtOAc (5.times.100 mL). Combined organic
extracts were washed with 100 mL of 1% Na.sub.2SO.sub.3, H.sub.2O
(2.times.), and brine. The solvents were removed, and the residue
was purified by column chromatography (4.5% EtOAc/CH.sub.2Cl.sub.2)
to produce 7.21 g (94%) of 48 as an oil: NMR .delta. 0.8-1.8 (m,
12H), 2.28 (s, 9H), 2.54 (s, 18H), 2.8-3.3 (m, 12H), 6.90 (s, 6H).
Anal. (C.sub.38H.sub.57N.sub.3O.sub.6S.sub.3) C, H, N.
EXAMPLE 13
[0156] N.sup.1,N.sup.8-Diethylspermidine Trihydrochloride (11).
HBr
[0157] (30% in HOAc, 150 mL), 48 (7.16 g, 9.57 mmol), and phenol
(28 g, 0.30 mol) in CH.sub.2Cl.sub.2 (125 mL) were reacted, and
product was isolated utilizing the procedure of 2 to give 2.17 g
(73%) of 11 as white plates: NMR (D.sub.2O) .delta. 1.28 and 1.30
(2 t, 6H, J=7), 1.73-1.85 (m, 4H), 2.06-2.18 (m, 2H), 3.04-3.21 (m,
12 H). Anal. (C.sub.11H.sub.30Cl.sub.3N.sub.3) C, H, N.
EXAMPLE 14
[0158]
N.sup.1,N.sup.8-Dipropyl-N.sup.1,N.sup.4,N.sup.8-tris(mesitylenesul-
fonyl)-spermidine (49).
[0159] NaH (80%, 0.80 g, 27 mmol) was added to 31 (8.13 g, 11.7
mmol) in DMF (75 mL) at 0.degree. C. The mixture was stirred at
room temperature for 1 hour, and 1-iodopropane (3.5 mL, 36 mmol)
was added by syringe. The mixture was stirred at 80.degree. C. for
12 hours and worked up as was 48. Purification by column
chromatography (3.5% EtOAc/CH.sub.2Cl.sub.2) resulted in 8.42 g
(93%) of 49 as an oil: NMR .delta. 0.55-1.72 (m, 16H), 2.25 (s, 9
H), 2.50 (s, 18H), 2.7-3.3 (m, 12H), 6.87 (s, 6H). Anal.
(C.sub.40H.sub.61N.sub.3O.sub.6S.sub.3) C, H, N.
EXAMPLE 15
[0160] N.sup.1,N.sup.8-Dipropylspermidine Trihydrochloride
(14).
[0161] HBr (30% in HOAc, 150 mL), 49 (8.32 g, 10.7 mmol), and
phenol (28 g, 0.29 mol) in CH.sub.2Cl.sub.2 (125 mL) were reacted,
and product was isolated by the procedure of 2 to produce 2.58 g
(71%) of 14 as white plates: NMR (D.sub.2O) .delta. 0.97 and 0.98
(2 t, 6H, J=7), 1.63-1.83 (m, 8H), 2.06-2.19 (m, 2H), 2.97-3.21 (m,
12H). Anal. (C.sub.13H.sub.34Cl.sub.3N.sub.3) C, H, N.
EXAMPLE 16
[0162] N,N'-Bis(4-Phthalimidobutyl)mesitylenesulfonamide (37).
[0163] NaH (60%, 1.6 g, 40 mmol) was added to 35 [Schreine-makers,
Recl. Trav. Chim. Pays-Bas Belg., Vol. 16, supra] (2.72 g, 13.5
mmol) in DMF (60 mL) at 0.degree. C. After the mixture was stirred
at 0.degree. C. for 30 minutes, N-(4-bromobutyl)phthalimide (11.51
g, 40 mmol) in DMF (20 mL) was introduced. The mixture was stirred
at room temperature for 1 hour and at 60.degree. C. over-night.
Following the workup procedure of 43, column chromatography (25:1
CHCl.sub.3/acetone) gave 3.77 g (46%) of 37 as a white powder: NMR
.delta. 1.51-1.54 (m, 8H), 2.18 (s, 3H), 2.57 (s, 6H), 3.18-3.24
(m, 4H), 3.55-3.60 (m, 4H), 6.86 (s, 2 H), 7.69-7.25 (m, 4H),
7.82-7.85 (m, 4H). HRMS calcd. for C.sub.33H.sub.36N.sub.3O.sub.6S
602.2325 (M+H), found 602.2320 (M+H).
EXAMPLE 17
[0164] N,N'-Bis(4-aminobutyl)mesitylenesulfonamide (40).
[0165] Hydrazine monohydrate (0.82 g, 16 mmol) was added to a
suspension of 37 (3.5 g, 5.8 mmol) in absolute EtOH (100 mL), and
the mixture was stirred at 65.degree. C. for 24 hours. After
cooling, the solid was filtered and washed with EtOH (2.times.10
mL). The combined filtrate was concentrated and purified by column
chromatography (6:1 MeOH/concentrated NH.sub.4OH) to produce 1.50 g
(76%) of 40 as a viscous oil: NMR .delta. 1.37 (quintet, 4H), 1.52
(quintet, 4H), 2.30 (s, 3H), 2.54 (t, 4H, J=7), 2.58 (s, 6H), 3.20
(t, 4H, J=7), 7.04 (s, 3H).
EXAMPLE 18
[0166] Homospermidine Trihydrochloride (15).
[0167] HBr (30% in HOAc, 30 mL), 40 (1.50 g, 4.39 mmol) and phenol
(4.49 g, 48 mmol) in CH.sub.2Cl.sub.2 (20 mL) were reacted, and
product was isolated by the procedure of 2 to afford 0.86 g (73%)
of 15 as white crystals: NMR (D.sub.2O) .delta. 1.73-1.80 (m, 8H),
3.03-3.14 (m, 8H). Anal. (C.sub.8H.sub.24Cl.sub.3N.sub.3) C, H,
N.
EXAMPLE 19
[0168]
N.sup.1,N.sup.5,N.sup.9-Tris(mesitylenesulfonyl)homospermidine
(32).
[0169] Mesitylenesulfonyl chloride (6.71 g, 30.7 mmol) and 40 (4.76
g, 14 mmol) in CH.sub.2Cl.sub.2 (30 mL) and 1 N NaOH (35 mL) were
combined and worked up by the method of 31. Column chromatography
(4:1 toluene/EtOAc) produced 3.06 g (31%) of 32 as a white foam:
NMR .delta. 1.32-1.38 (m, 4H), 1.44-1.54 (m, 4H), 2.28-2.29 (2 s,
9H), 2.54 (s, 6H), 2.60 (s, 12H), 2.79 (quartet, 4H), 3.09 (t, 4H,
J 7), 4.70-4.80 (br s, 2H), 6.90 (s, 2H), 6.92 (s, 4H). Anal.
(C.sub.35H.sub.5 N.sub.3O.sub.6S.sub.3) C, H, N.
EXAMPLE 20
[0170] N.sup.1,N.sup.9-Dimethyl-N.sup.1,N.sup.5,N.sup.9-tris
(mesitylenesulfonyl)homospermidine (50).
[0171] NaH (60%, 0.17 g, 4.2 mmol), 32 (1.28 g, 1.8 mmol), and
iodomethane (0.25 mL, 4.0 mmol) in DMF (50 mL) were reacted and
worked up as was 43. Column chromatography (2:1 toluene/EtOAc) gave
1.14 g (86%) of 50 as an oil: NMR .delta. 1.38-1.44 (m, 8H), 2.28
(s, 3H), 2.30 (s, 6H), 2.57 (s, 18 H), 2.62 (s, 6H), 3.03-3.14 (m,
8H), 6.93-6.94 (2 s, 6H). Anal.
(C.sub.37H.sub.55N.sub.3O.sub.6S.sub.3) C, H, N.
EXAMPLE 21
[0172] N.sup.1,N.sup.9-Dimethylhomospermidine Trihydrochloride
(16).
[0173] HBr (30% in HOAc, 30 mL), 50 (1.12 g, 1.52 mmol), and phenol
(5.4 g, 57 mmol) in CH.sub.2Cl.sub.2 (25 mL) were reacted, and
product was isolated by the procedure of 2 to provide 354 mg (79%)
of 16 as plates: NMR (D.sub.2O) .delta. 1.78 (m, 8H), 2.73 (s, 6H),
3.08-3.12 (m, 8H). Anal. (C.sub.10H.sub.28Cl.sub.3N.sub.3) C, H,
N.
EXAMPLE 22
[0174] N.sup.1,N.sup.9-Diethyl-N.sup.1,N.sup.5,N.sup.9-tris
(mesitylenesulfonyl) homospermidine (51).
[0175] NaH (80%, 0.264 g, 8.8 mmol) was added to 35
[Schreinemakers, Recl. Trav. Chim. Pays-Bas Belg., Vol. 16, supra]
(0.796 g, 4 mmol) in DMF (60 mL) at 0.degree. C. After the mixture
was stirred at 0.degree. C. for 30 minutes, 58 [Bergeron et al, J.
Med. Chem., Vol. 37, supra]. (3.19 g, 8.8 mmol) in DMF (15 mL) was
added. The mixture was heated at 75.degree. C. overnight and worked
up by the procedure of 43. Column chromatography (3:1 hexane/EtOAc)
gave 2.82 g (93%) of 51 as an oil: NMR .delta. 0.96 (t, 6H),
1.20-1.40 (m, 8H), 2.25 (s, 9H), 2.55 (s, 18H), 2.85-3.20 (m, 12H),
6.90 (s, 6H). Anal. (C.sub.39H.sub.59N.sub.3O.sub.6S.sub.3) C, H,
N.
EXAMPLE 23
[0176] N.sup.1,N.sup.9-Diethylhomospermidine Trihydrochloride
(17).
[0177] HBr (30% in HOAc, 20 mL), 51 (1.87 g, 2.45 mmol), and phenol
(4.4 g, 49 mmol) in CH.sub.2Cl.sub.2 (20 mL) were reacted, and
product was isolated by the procedure of 2 to give 493 mg (62%.) of
17 as plates: NMR (D.sub.2O) .delta. 1.30 (s, 6H), 1.55-1.90 (m,
8H), 2.95-3.20 (m, 12H). Anal. (C.sub.12H.sub.32Cl.sub.3N.sub.3) C,
H, N.
EXAMPLE 24
[0178]
N.sup.1,N.sup.9-Dipropyl-N.sup.1,N.sup.5,N.sup.9-tris(mesitylenesul-
fonyl)homospermidine (52).
[0179] NaH (60%, 0.17 g, 4.2 mmol), 32 (1.28 g, 1.8 mmol), and
1-iodopropane (0.39 mL, 4.0 mmol) in DMF (50 mL) were reacted and
worked up using the procedure of 43. Column chromatography (4:1
hexane/EtOAc) gave 1.26 g (89%) of 52 as an oil: NMR .delta. 0.74
(t, 6H, J=7), 1.26-1.45 (m, 12H), 2.29 (s, 9H), 2.55 (s, 18H),
2.98-3.13 (m, 12H), 6.87 (s, 6H). Anal.
(C.sub.41H.sub.63N.sub.3O.sub.6S.sub.3) C, H, N.
EXAMPLE 25
[0180] N.sup.1,N.sup.9-Dipropylhomospermidine Trihydrochloride
(19).
[0181] HBr (30% in HOAc, 30 mL), 52 (1.24 g, 1.56 mmol), and phenol
(5.4 g, 57 mmol) in CH.sub.2Cl.sub.2 (25 mL) were reacted, and
product was isolated by the procedure of 2 to give 430 mg (78%) of
19 as plates: NMR (D.sub.2O) .delta. 0.98 (t, 6H, J=7), 1.70 (m,
4H), 1.76-1.80 (m, 8H), 3.02 (t, 4H, J=7), 3.08-3.12 (m, 8H). Anal.
(C.sub.14H.sub.36Cl.sub.3N.su- b.3) C, H, N.
EXAMPLE 26
[0182] N-(3-Cyanopropyl)mesitylenesulfonamide (36).
[0183] NaH (60%, 2.0 g, 50 mmol), 35 [Schreinemakers, Recl. Trav.
Chim. Pays-Bas Belg., Vol. 16, supra] (10.0 g, 50 mmol), and
4-bromobutyronitrile (4 mL, 40 mol) in DMF (100 mL) were combined.
The mixture was heated at 80.degree. C. overnight and worked up by
the procedure of 43. Column chromatography (4:3 hexane/EtOAc) gave
5.04 g (38%) of 36 as an oil: NMR .delta. 1.78 (s, 3H), 2.25 (s,
3H), 2.35 (t, 2H, J=7), 2.95 (q, 2H), 5.05 (br t, 1H), 6.90 (s,
2H). Anal. (C.sub.13H.sub.18N.sub.2O.sub.2S) C, H, N.
EXAMPLE 27
[0184] N--(4-Cyanobutyl)-N--(3-cyanopropyl) mesitylenesulfonamide
(38).
[0185] NaH (60%, 0.90 g, 23 mmol), 36 (5.02 g, 18.85 mmol), and
5-bromovaleronitrile (2.4 mL, 21 mmol) in DMF were combined and
worked up by the method of 43. Column chromatography (1:1
hexane/EtOAc) provided 5.20 g (79%) of 38 as an oil: NMR .delta.
1.49-1.66 (m, 4H), 1.82 (m, 2H), 2.22 (t, 2H, J=7), 2.25 (t, 2H,
J=7), 2.29 (s, 3H), 2.57 (s, 6H), 3.19 (t, 2H, J=7), 3.29 (t, 2H,
J=7), 6.95 (s, 2H). Anal. (C.sub.18H.sub.25N.sub.3O.sub.2S) C, H,
N.
EXAMPLE 28
[0186] 6-(Mesitylenesulfonyl)-1,6,12-Triazadodecane (41).
[0187] Raney nickel (W-2 grade, 7.60 g) and concentrated NH.sub.4OH
(10 mL) were successively added to 38 (5.06 g, 14.6 mmol) in
CH.sub.3OH (30 mL) and THF (30 mL) in a 200 mL Parr bottle, and a
slow stream of NH.sub.3 was bubbled through the mixture for 30
minutes at 0.degree. C. After hydrogenation in a Parr bottle was
carried out at 50-55 psi for 8 hours, the suspension was filtered
through Celite, and the solvents were removed in vacuo to give 4.70
g (91%) of 41 as an oil: NMR .delta. 1.14-1.24 (m, 10H), 2.25 (s, 3
H), 2.6 (s, 6H), 3.05-3.25 (m, 8H), 3.45 (s, 4H), 6.9 (s, 2H).
EXAMPLE 29
[0188] 1,6,12-Triazadodecane Trihydrochloride (20).
[0189] HBr (30% in HOAc, 33 mL), 41 (2.43 g, 6.83 mmol), and phenol
(6 g, 60 mmol) in CH.sub.2Cl.sub.2 were reacted, and product was
isolated by the procedure of 2 to give 0.97 g (50%) of 20 as a
hygroscopic solid: NMR (D.sub.2O) .delta. 1.47 (m, 2H), 1.70-1.80
(m, 8H), 3.00-3.10 (m, 8H). Anal. (C.sub.9H.sub.26Cl.sub.3N.sub.3)
C, H, N.
EXAMPLE 30
[0190] 1,6,12-Tris(mesitylenesulfonyl)-1,6,12-Triazadodecane
(33).
[0191] Mesitylenesulfonyl chloride (4.29 g, 19.6 mmol) and 41 (3.17
g, 8.92 mmol) in CH.sub.2Cl.sub.2 (40 mL) and 1 N NaOH (20 mL) were
combined and worked up by the method of 31. Column chromatography
(4:3 hexane/EtOAc) generated 5.66 g (88%) of 33 as an oil: NMR
.delta. 1.12-1.17 (m, 2H), 1.34-1.51 (m, 8H), 2.29 (s, 3H), 2.30
(s, 6H), 2.55 (s, 6H), 2.60-2.62 (2s, 12H), 2.77-2.81 (m, 4H), 3.06
(t, 2H, J =7), 3.11 (t, 2H, J=7), 4.50-4.60 (m, 2H), 6.92 (s, 2H),
6.95 (s, 2H). Anal. (C.sub.36H.sub.53N.sub.3O.sub.6S.sub.3) C, H,
N.
EXAMPLE 31
[0192] 2,7,13-Tris(mesitylenesulfonyl)-2,7,13-Triazapentadecane
(53).
[0193] NaH (60%, 0.28 g, 6.9 mmol), 33 (2.16 g, 3.0 mmol), and
iodomethane (6.1 mL, 9.8 mmol) in DMF (30 mL) were combined and
worked up by the method of 43. Column chromatography (7:3
hexane/EtOAc) gave 1.90 g (85%) of 53 as an oil: NMR .delta.
1.08-1.16 (m, 2H), 1.38-1.50 (m, 8H ), 2.28-2.29 (2s, 9H),
2.57-2.58 (2 s, 18H), 2.63 (s, 3H), 2.65 (s, 3H), 3.02-3.14 (m,
8H), 6.95 (s, 6H); HRMS calcd. for C.sub.38H.sub.58N.sub.3O-
.sub.6S.sub.3 748.3487 (M+H), found 748.3483 (M+H).
EXAMPLE 32
[0194] 2,7,13-Triazatetradecane Trihydrochloride (21).
[0195] HBr (30% in HOAc, 45 mL), 53 (1.85 g, 2.47 mmol), and phenol
(8.5 g) in CH.sub.2Cl.sub.2 (20 mL) were reacted, and product was
isolated by the procedure of 2 to give 529 mg (69%) of 21 as
crystals: NMR (D.sub.2O) .delta. 1.42-1.52 (m, 2H), 1.69-1.81 (m,
8H), 2.73-2.74 (2s, 6H), 3.03-3.12 (m, 8H). Anal.
(C.sub.11H.sub.30Cl.sub.3N.sub.3) C, H, N.
EXAMPLE 33
[0196] 4,9,15-Tris(mesitylenesulfonyl)-4,9,15-Triazaheptadecane
(54).
[0197] NaH (60%, 0.273 g, 6.84 mmol), 33 (2.24 g, 3.11 mmol), and
1-iodopropane (0.67 mL, 6.9 mmol) in DMF (30 mL) were combined and
worked up by the method of 43. Column chromatography (3:1
hexane/EtOAc) provided 2.01 g (80%) of 54 as an oil: NMR .delta.
0.71-0.78 (m, 6H), 1.01-1.11 (m, 2H), 1.34-1.48 (m, 12H), 2.29 (s,
6H), 2.57-2.58 (2 s, 18H), 2.98-3.13 (m, 12H), 6.92 (s, 6H). Anal.
(C.sub.42H.sub.65N.sub.3O.sub.6S.- sub.3H.sub.2O ) C, H, N.
EXAMPLE 34
[0198] 4,9,15-Triazaoctadecane Trihydrochloride (23).
[0199] HBr (30% in HOAc, 45 mL), 48 (1.99 g, 2.47 mmol), and phenol
(8.5 g) in CH.sub.2Cl.sub.2 (20 mL) were reacted, and product was
isolated by the procedure of 2 to give 852 mg (83%) of 23 as
plates: NMR (D.sub.2O) .delta. 0.97 (s, 6H), 1.40-1.51 (m, 2H),
1.66-1.80 (m, 12 H), 2.98-3.15 (m, 12H). Anal.
(Cl.sub.5H.sub.38Cl.sub.3N.sub.3) C, H, N.
EXAMPLE 35
[0200] N,N-Bis(4-cyanobutyl)mesitylenesulfonamide (39).
[0201] NaH (80%, 1.22 g, 51 mmol), 35 [Schreinemakers, Recl. Trav.
Chim. Pays-Bas Belg., Vol. 16, supra] (5.0 g, 25 mmol), and
5-chlorovaleronitrile (6.5 g, 55 mmol) in DMF (50 mL) were
combined. The mixture was heated at 60.degree. C. overnight and
worked up by the procedure of 43. Column chromatography (7:3
hexane/EtOAc) yielded 6.31 g (70%) of 39 as an oil: NMR .delta.
1.57 (m, 4H), 1.66 (m, 4H), 2.26 (t, 4H, J=7), 2.60 (s, 6H), 3.22
(t, 4H, J=7), 6.98 (s, 2H). Anal. (C.sub.19H.sub.27N.sub.3O.sub.2S)
C, H, N.
EXAMPLE 36
[0202] 7-Mesitylenesulfonyl-1,7,13-triazatridecane (42).
[0203] Raney nickel (W-2 grade, 2.9 g) and 39 (5.69 g, 15.8 mmol)
in concentrated NH.sub.4OH (10 mL) and CH.sub.3OH (60 mL) were
saturated with NH.sub.3 as 41. The mixture was shaken with hydrogen
at 50-55 psi in a 200 mL Parr bottle for 42 hours. The suspension
was filtered through Celite, and the solvents were removed in
vacuo. The residue was passed through a short silica gel column
(EtOH then 5% concentrated NH.sub.4OH/EtOH) to give 5.71 g (98%) of
42 as a light yellow oil: NMR .delta. 1.17 (m, 4H), 1.47 (m, 8H),
2.27 (s, 3H), 2.57 (s, 6H), 2.61 (m, 4H), 3.13 (t, J=7.5, 4H), 6.91
(s, 2H); HRMS calcd. for C.sub.19H.sub.36N.sub.3O.sub.2S 370.2528
(M+H), found 370.2530 (M+H).
EXAMPLE 37
[0204] 1,7,13-Triazatridecane Trihydrochloride (24).
[0205] HBr (30% in HOAc, 26 mL), 42 (2.0 g, 5.42 mmol), and phenol
(4.8 g, 51 mmol) in CHCl.sub.3 (40 mL) were reacted, and product
was isolated by the method of 2 to give 0.97 (61%) of 24 as a white
solid: NMR (D.sub.2O) .delta. 1.45 (m, 4H), 1.70 (m, 8H), 3.01 (m,
8H). Anal. (C.sub.10H.sub.28Cl.sub.3N.sub.3) C, H, N.
EXAMPLE 38
[0206] 1,7,13-Tris(mesitylenesulfonyl)-1,7,13-triazatridecane
(34).
[0207] Mesitylenesulfonyl chloride (4.52 g, 20.7 mmol) and 42 (3.47
g, 9.4 mmol) in CH.sub.2Cl.sub.2 and 1 N NaOH (30 mL) were combined
and worked up by the method of 31. Column chromatography (3:2
hexane/EtOAc) gave 6.44 g (93%) of 34 as a white solid: NMR .delta.
1.16 (m, 4H), 1.39 (m, 8H), 2.30 (s, 3H), 2.31 (s, 6H), 2.57 (s,
6H), 2.62 (s, 12H), 2.81 (d of t, 4 H), 3.10 (t, 4H, J=7), 4.49 (br
t, 2H), 6.95 (s, 2H), 6.97 (s, 4H); HRMS calcd. for
C.sub.37H.sub.56N.sub.3O.sub.6S.sub.3 734.3331 (M+H), found
734.3351 (M+H).
EXAMPLE 39
[0208] 2,8,14-Tris(mesitylenesulfonyl)-2,8,14-triazapentadecane
(55).
[0209] NaH (80%, 0.207 g, 6.9 mmol), 34 (1.58 g, 2.16 mmol), and
iodomethane (0.30 mL, 4.8 mmol) in DMF (30 mL) were reacted and
worked up as was 43. Column chromatography (5:2 hexane/EtOAc) gave
1.51 g (92%) of 55 as an oil: NMR .delta. 1.06-1.18 (m, 4H),
1.40-1.52 (m, 8H), 2.29 (s, 9H), 2.59 (s, 18H), 2.66 (s, 6H),
3.03-3.14 (m, 8H), 6.95 (s, 6H). Anal.
(C.sub.39H.sub.59N.sub.3O.sub.6s.sub.3) C, H, N.
EXAMPLE 40
[0210] 2,8,14-Triazapentadecane Trihydrochloride (25).
[0211] HBr (30% in HOAc, 30 mL), 55 (1.48 g, 1.94 mmol), and phenol
(5.2 g, 55 mmol) in CH.sub.2Cl.sub.2(30 mL) were reacted, and
product was isolated by the method of 2 to produce 480 mg (76%) of
25 as needles: NMR (D.sub.2O) .delta. 1.4-1.5 (quintet, 4H),
1.7-1.8 (quintet, 8H), 2.7 (s, 6H), 3.05 (t, 8H, J=7). Anal.
(C.sub.12H.sub.32Cl.sub.3N.sub.3) C, H, N.
EXAMPLE 41
[0212] 3,9,15-Tris(mesitylenesulfonyl)-3,9,15-triazaheptadecane
(56).
[0213] NaH (80%, 0.52 g, 17 mmol), 34 (3.2 g, 4.36 mmol), and
iodoethane (1.5 g, 9.6 mmol) in DMF (20 mL) were reacted and worked
up by the method of 43. Column chromatography (4:1 hexane/EtOAc)
gave 2.91 g (85%) of 56 as a white solid: mp 60-62.degree. C.; NMR
.delta. 1.01 (t, 6H, J=7), 1.08 (m, 4H), 1.42 (m, 8H), 2.29 (s,
9H), 2.57 (s, 6H), 2.58 (s, 12H), 3.07 (t, 4H, J=7), 3.11 (t, 4H,
J=7), 3.17 (q, 4H), 6.92 (s, 6H). Anal.
(C.sub.41H.sub.63N.sub.3O.sub.6S.sub.3) C, H, N.
EXAMPLE 42
[0214] 3,9,15-Triazaheptadecane Trihydrochloride (26).
[0215] HBr (30% in HOAc, 20 mL), 56 (2.9 g, 3.67 mmol), and phenol
(3.25 g, 34.5 mmol) in CHCl.sub.3 (27 mL) were reacted, and product
was isolated by the method of 2 to give 1.0 g (77%) of 26 as white
crystals: NMR (D.sub.2O) .delta. 1.28 (t, 6H, J=7), 1.45 (m, 4H),
1.73 (m, 8H), 3.08 (m, 12H). Anal.
(C.sub.14H.sub.36Cl.sub.3N.sub.3) C, H, N.
EXAMPLE 43
[0216] 4,10,16-Tris(mesitylenesulfonyl)-4,10,16-triazanonadecane
(57).
[0217] NaH (80%, 198 mg, 6.6 mmol), 34 (1.51 g, 2.06 mmol), and
1-iodopropane (0.44 mL, 4.5 mmol) in DMF (40 mL) were reacted and
worked up by the method of 43. Column chromatography
(7:2/hexane/EtOAc) provided 1.61 g (95%) of 57 as an oil: NMR
.delta. 0.75 (t, 6H, J=7), 1.02-1.14 (m, 4H), 1.36-1.52 (m, 12H),
2.30 (s, 9H), 2.60 (s, 18H), 3.02-3.16 (m, 12H), 6.95 (s, 6H).
Anal. (C.sub.43H.sub.67N.sub.3O.sub.6S.sub.3) C, H, N.
EXAMPLE 44
[0218] 4,10,16-Triazanonadecane Trihydrochloride (27).
[0219] HBr (30% in HOAc, 30 mL), 57 (1.58 g, 1.93 mmol), and phenol
(5.2 g, 55 mmol) were reacted, and product was isolated by the
method of 2 to give 579 mg (79%) of 27 as plates: NMR (D.sub.2O)
.delta. 0.95 (t, 6H, J=7), 1.38-1.49 (m, 4H), 1.62-1.77 (m, 12 H),
2.95-3.05 (m, 12H). Anal. (C.sub.16H.sub.40Cl.sub.3N.sub.3) C, H,
N.
EXAMPLE 45
[0220] N-(5-Bromopentyl)-N-ethylmesitylenesulfonamide (61).
[0221] NaH (80%, 1.26 g, 42 mmol), 60 [Schreinemakers, Recl. Trav.
Chim. Pays-Bas Belg., Vol. 16, supra] (6.82 g, 30.0 mmol), and
1,5-dibromopentane (49 mL, 0.36 mol) in DMF (100 mL) were combined.
The mixture was heated at 74.degree. C. overnight and worked up by
the procedure of 43. Column chromatography (7:1 hexane/EtOAc)
produced 7.87 g (70%) of 61 as an oil: NMR .delta. 1.00 (t, 3H,
J=7), 1.30-1.75 (m, 6H), 2.20 (s, 3H), 2.50 (s, 6H), 3.02-3.30 (m,
6H), 6.80 (s, 2H); HRMS calcd. for C.sub.16H.sub.27BrNO.sub.2S
376.0946 (M+H), found 376.0960 (M+H).
EXAMPLE 46
[0222] N.sup.1,N.sup.4-Bis(mesitylenesulfonyl)-N.sup.1-
(tert-butoxycarbonyl)-N.sup.4-ethyl-1,4-diaminobutane (62).
[0223] NaH (80%, 0.45 g, 23 mmol) was added to 59 [Bergeron et al,
J. Med. Chem., Vol. 37, supra]. (3.45 g, 11.5 mmol) in DMF (100 mL)
at 0.degree. C. After the mixture was stirred at 0.degree. C. for
40 minutes, 58 [Bergeron et al, J. Med. Chem., Vol. 37, supra].
(5.00 g, 13.8 mmol) in DMF (10 mL) was added. The mixture was
heated at 60.degree. C. for 18 hours and then worked up by the
method of 43. Column chromatography with (20:1 toluene/EtOAc) gave
6.44 g (96%) of 62 as an oil: NMR .delta. 1.12 (t, 3H, J=7), 1.20
(s, 9H), 1.55-1.65 (m, 4H), 2.29 (s, 2H), 2.30 (s, 2H), 2.52 (s,
4H), 2.62 (s, 4H), 3.16-3.24 (m, 2H), 3.30 (q, 2H), 3.70 (m, 2H),
6.94 (s, 4H).
EXAMPLE 47
[0224] N.sup.1,N.sup.4-Bis
(mesitylenesulfonyl)-N.sup.4-ethyl-1,4-diaminob- utane (63).
[0225] TFA (70 mL) was slowly dripped into a solution of 62 (6.20
g, 10.6 mmol) in CH.sub.2Cl.sub.2 (30 mL) at 0.degree. C. After the
solution was stirred at 0.degree. C. for 20 minutes and at room
temperature for 30 minutes, solvents were removed by rotary
evaporation. The residue was basified to pH >8 with saturated
NaHCO.sub.3 and extracted with CH.sub.2Cl.sub.2 (4.times.100 mL).
Removal of organic extracts led to 5.10 g (100%) of 63 as a foam:
NMR .delta. 0.97 (t, 3H, J=7), 1.20-1.50 (m, 4H), 2.25 (s, 6H),
2.50-2.55 (2 s, 12H), 2.95-3.25 (m, 6H), 4.45 (t, 1H), 6.85 (s,
4H). Anal. (C.sub.24H.sub.36N.sub.2O.sub.4S.sub.2) C, H, N.
EXAMPLE 48
[0226] 3,8,14-Tris(mesitylenesulfonyl)-3,8,14-Triazahexadecane
(64).
[0227] NaH (80%, 0.41 g, 14 mmol) was added to 63 (5.10 g, 10.6
mmol) in DMF (50 mL) at 0.degree. C. After the mixture was stirred
at 0.degree. C. for 30 minutes, 61 (4.80 g, 12.7 mmol) in DMF (10
mL) was added. The mixture was heated at 89.degree. C. overnight
and then worked up by the method of 43. Column chromatography (12:1
toluene/EtOAc) gave 5.43 g (66%) of 64 as an oil: NMR .delta.
0.9-1.1 (m, 6H), 1.2-1.5 (m, 10H), 2.25 (s, 6H), 2.30 (s, 3H), 2.55
(s, 18H), 2.9-3.2 (m, 12H), 6.85 (s, 6H). Anal.
(C.sub.40H.sub.61N.sub.3O.sub.6S.sub.3) C, H, N.
EXAMPLE 49
[0228] 3,8,14-Triazahexadecane Trihydrochloride (22).
[0229] HBr (30% in HOAc, 100 mL), 64 (5.4 g, 7.0 mmol), and phenol
(25 g, 0.28 mol) were reacted, and product was isolated by the
method of 2 to give 1.63 g (69%) of 22 as plates: NMR (D.sub.2O)
.delta. 1. 38 (t, 3H, J 7), 1.39 (t, 3H, J=7), 1.60-1.70 (m, 10H),
3.02-3.15 (m, 12H). Anal. (C.sub.13H.sub.34Cl.sub.3N.sub.3) C, H,
N.
EXAMPLE 50
[0230] N.sup.1-Triphenylmethyl-1,3-diaminopropane (68).
[0231] A solution of triphenylmethyl chloride (6.97 g, 25 mmol) in
CH.sub.2Cl.sub.2 (100 mL) was added dropwise to a rapidly stirred
solution of 1,3-diaminopropane (9.86 g, 133 mmol) in
CH.sub.2Cl.sub.2 (100 mL). After stirring at room temperature for 2
days, 1 N NaOH (50 mL) was added to the mixture, which was
extracted with CHCl.sub.3 (3.times.50 mL). Organic extracts were
washed with 100 mL of H.sub.2O and brine. After solvent removal,
column chromatography (3% concentrated NH.sub.4OH/MeOH) gave 6.32 g
(80%) of 68 as a white solid: mp 59-61.degree. C. [Parg et al, "A
Semiconducting Langmuir-Blodgett Film of a Non-amphiphilic
Bis-tetrathiofulvalene Derivative," J. Mater. Chem., Vol. 5, pages
1609-1615 (1995); mp 59-61.degree. C.]; NMR .delta. 1.35-1.65 (m,
6H), 2.18 (t, 2H, J=7), 2.77 (s, 1H), 7.13-7.24 (m, 9H), 7.44-7.46
(m, 6H). Anal. (C.sub.22H.sub.24N.sub.2) C, H, N.
EXAMPLE 51
[0232]
N.sup.1-Mesitylenesulfonyl-N.sup.3-triphenylmethyl-1,3-diaminopropa-
ne (70).
[0233] Mesitylenesulfonyl chloride (5.25 g, 24 mmol) and 68 (6.30
g, 20 mmol) in CH.sub.2Cl.sub.2 (30 mL) and 1 N NaOH (27 mL) were
combined and worked up by the method of 31. Column chromatography
(7:2 hexane/EtOAc) gave 8.37 g (84%) of 70 as a white solid: NMR
.delta. 1.56-1.66 (m, 3H), 2.17 (t, 2H, J=7), 2.29 (s, 3H), 2.60
(s, 6H), 3.08 (q, 2H), 5.25 (br t, 1H), 6.93 (s, 2H), 7.15-7.28 (m,
9H), 7.35-7.39 (m, 6H). Anal. (C.sub.31H.sub.34N.sub.2O.sub.2S) C,
H, N.
EXAMPLE 52
[0234] N.sup.1-Triphenylmethyl-1,4-diaminobutane (69).
[0235] A solution of triphenylmethyl chloride (59.94 g, 0.215) in
CH.sub.2Cl.sub.2 (500 mL) was added dropwise to a rapidly stirred
solution of 1,4-diaminobutane (96.47 g, 1.094 mol) in
CH.sub.2Cl.sub.2 (1.1 L) over a period of 2 hours. The reaction
mixture was stirred at room temperature for 3 days and was worked
up following the method of 68 to give a quantitative yield of 69 as
an oil which was used directly in the next step: NMR .delta.
1.39-1.54 (m, 7H), 2.12 (t, 2H, J=7), 2.62 (t, 2H, J=7), 7.13-7.28
(m, 9 H), 7.41-7.47 (m, 6H).
EXAMPLE 53
[0236]
N.sup.1-Mesitylenesulfonyl-N.sup.4-triphenylmethyl-1,4-diaminobutan-
e (71).
[0237] Mesitylenesulfonyl chloride (3.1 g, 14 mmol) and 69 (3.39 g,
10.3 mmol) in CH.sub.2Cl.sub.2 (20 mL) and 1 N NaOH (15 mL) were
combined and worked up by the method of 31. Column chromatography
(1:3 hexane/EtOAc) furnished 3.79 g (72%) of 71 as a white solid:
NMR .delta. 1.41-1.50 (m, 5H), 2.03-2.08 (t, 2H, J=7), 2.27 (s,
3H), 2.62 (s, 6H), 2.87 (q, 2H), 4.41 (br t, 1H), 6.93 (s, 2H),
7.15-7.28 (m, 9H), 7.40-7.44 (m, 6H). Anal.
(C.sub.32H.sub.36N.sub.2O.sub.2S) C, H, N.
EXAMPLE 54
[0238] N-Propylmesitylenesulfonamide (65).
[0239] Mesitylenesulfonyl chloride (12.0 g, 55 mmol) and
propylamine (2.96 g, 50 mmol) in CH.sub.2Cl.sub.2 (60 mL) and 1 N
NaOH (60 mL) were combined and worked up by the method of 31.
Column chromatography (3:1 hexane/EtOAc) afforded 8.44 g (85%) of
65 as a crystalline solid: mp 53-54.degree. C.; NMR .delta. 0.86
(t, 3H, J=7), 1.44-1.51 (m, 2H), 2.30 (s, 3H), 2.64 (s, 6H), 2.86
(q, 2H), 4.40 (br t, 1H), 6.96 (s, 2H). Anal.
(C.sub.12H.sub.19NO.sub.2S) C, H, N.
EXAMPLE 55
[0240] N-(3-Bromopropyl)-N-propylmesitylenesulfonamide (66).
[0241] NaH (60%, 0.34 g, 8.4 mmol), 65 (1.7 g, 7.0 mmol), and
1,3-dibromopropane (17.0 g, 84 mmol) in DMF (30 mL) were combined
and worked up by the method of 43. Column chromatography (6:1
hexane/EtOAc) produced 1.82 g (80%) of 66 as an oil: NMR .delta.
0.79 (s, 3H), 1.48-1.56 (m, 2H), 2.04-2.10 (m, 2H), 2.30 (s, 3H),
2.60 (s, 6H), 3.09-3.14 (m, 2H), 3.29-3.36 (m, 4H). Anal.
(C.sub.15H.sub.24BrNO.sub.2S) C, H, N.
EXAMPLE 56
[0242] N-(4-Bromobutyl)-N-propylmesitylenesulfonamide (67).
[0243] NaH (60%, 0.70 g, 17 mmol), 65 (3.5 g, 14.5 mmol), and
1,4-dibromobutane (37.6 g, 174 mmol) in DMF (40 mL) were combined
and worked up by the method of 43. Excess 1,4-dibromobutane was
removed by a Kugelrohr apparatus under high vacuum. Column
chromatography (7:1 hexane/EtOAc) produced 5.21 g (95%) of 67 as an
oil: NMR .delta. 0.79 (t, 3H, J=7), 1.46-1.54 (m, 2H), 1.64-1.78
(m, 4H), 2.30 (s, 3H), 2.60 (s, 6H), 3.11 (t, 2H, J=7), 3.21 (t,
2H, J=7), 3.31 (t, 2H, J=7), 6.93 (s, 2H). Anal.
(C.sub.16H.sub.26BrNO.sub.2S) C, H, N.
EXAMPLE 57
[0244]
6,10-Bis(mesitylenesulfonyl)-1-triphenylmethyl-1,6,10-triazatrideca-
ne (72).
[0245] NaH (60%, 0.24 g, 5.96 mmol) was added to 71 (2.55 g, 4.97
mmol) in DMF (40 mL) at 0.degree. C. After the mixture was stirred
at 0.degree. C. for 20 minutes, 66 (1.80 g, 4.97 mmol) in DMF (20
mL) was added. The mixture was stirred at room temperature for 1
day and then worked up following the method of 43. Column
chromatography (20:1 toluene/EtOAc) produced 3.72 g (94%) of 72 as
an oil: NMR .delta. 0.73 (t, 3H, J=7), 1.26-1.70 (m, 8H), 2.01 (t,
2H, J=7), 2.26 (s, 3 H), 2.27 (s, 3H), 2.54 (s, 12H), 2.96-3.04 (m,
8H), 6.90 (s, 4H), 7.18-7.45 (m, 15H). Anal.
(C.sub.47H.sub.59N.sub.3O.sub.4S.sub.2) C, H, N.
EXAMPLE 58
[0246]
5,10-Bis(mesitylenesulfonyl)-1-triphenylmethyl-1,5,10-triazatrideca-
ne (73).
[0247] NaH (60%, 0.22 g, 5.42 mmol), 70 (2.25 g, 4.52 mmol) in DMF
(40 mL), and 67 (1.70 g, 4.52 mmol) in DMF (20 mL) were reacted and
worked up following the method of 72. Column chromatography (25:1
toluene/EtOAc) produced 2.87 g (80%) of 73 as an oil: NMR .delta.
0.75 (t, 3H, J=7), 1.41-1.46 (m, 9H), 1.97 (t, 2H, J=7), 2.26 (s,
6H), 2.53 (s, 6H), 2.56 (s, 6H), 3.04 (t, 2H, J=7), 3.10-3.15 (m,
6H), 6.88 (s, 2H), 6.90 (s, 2H), 7.15-7.38 (m, 15H). Anal.
(C.sub.47H.sub.59N.sub.3O.sub.4S.sub.2) C, H, N.
EXAMPLE 59
[0248]
6,11-Bis(mesitylenesulfonyl)-1-triphenylmethyl-1,6,11-triazatetrade-
cane (74).
[0249] NaH (60%, 0.12 g, 2.90 mmol), 71 (1.24 g, 2.42 mmol) in DMF
(30 mL), and 67 (0.91 g, 2.42 mmol) in DMF (10 mL) were reacted and
worked up following the method of 72. Column chromatography (25:1
toluene/EtOAc) gave 1.67 g (85%) of 74 as an oil: NMR .delta. 0.74
(t, 3H, J=7), 1.35-1.45 (m, 11H), 2.00 (t, 2H, J=7), 2.25 (s, 3H),
2.28 (s, 3H), 2.55 (s, 6H), 2.56 (s, 6H), 2.98-3.08 (m, 8H), 6.90
(s, 2H), 6.91 (s, 2H), 7.15-7.44 (m, 15H). Anal.
(C.sub.48H.sub.61N.sub.3O.sub.4S.sub.2) C, H, N.
EXAMPLE 60
[0250] N.sup.1-Propylspermidine Trihydrochloride (12).
[0251] HBr (30% in HOAc, 45 mL), 72 (3.70 g, 4.66 mmol), and phenol
(8.4 g, 89 mmol) in CH.sub.2Cl.sub.2 (50 mL) were reacted, and
product was isolated by the method of 2 to give 1.02 g (74%) of 12
as plates: NMR (D.sub.2O) .delta. 0.98 (t, 3H, J=7) 1.66-1.79 (m,
6H), 2.06-2.17 (m, 2H), 3.01-3.19 (m, 10H). Anal.
(C.sub.10H.sub.28Cl.sub.3N.sub.3) C, H, N.
EXAMPLE 61
[0252] N.sup.8-Propylspermidine Trihydrochloride (13).
[0253] HBr (30% in HOAc, 35 mL), 73 (2.85 g, 3.59 mmol), and phenol
(6.5 g, 69 mmol) in CH.sub.2Cl.sub.2 (30 mL) were reacted, and
product was isolated by the method of 2 to give 810 mg (76%) of 13
as plates: NMR (D.sub.2O) .delta. 0.98 (t, 3H, J=7), 1.66-1.79 (m,
6H), 2.01-2.12 (m, 2H), 2.99-3.14 (m, 10H). Anal.
(C.sub.10H.sub.28Cl.sub.3N.sub.3) C, H, N.
EXAMPLE 62
[0254] N.sup.1-Propylhomospermidine Trihydrochloride (18).
[0255] HBr (30% in HOAc, 20 mL), 74 (1.65 g, 2.0 mmol), and phenol
(3.6 g, 38 mmol) in CH.sub.2Cl.sub.2 (20 mL) were reacted, and
product was isolated by the method of 2 to give 268 mg (43%) of 18
as plates: NMR (D.sub.2O) .delta. 0.98 (t, 3H, J=7), 1.66-1.80 (m,
10 H), 2.99-3.11 (m, 10H). HRMS calcd. for C.sub.11H.sub.28N.sub.3
202.2283 (free amine, M+H), found 202.2296 (M+H).
EXAMPLE 63
[0256] N.sup.1-Ethylspermidine Trihydrochloride (9).
[0257] Lithium aluminum hydride (1.6 g, 42 mmol) was added to
N.sup.1-acetylspermidine dihydrochloride (0.50 g, 1.9 mmol) in THF
(300 mL) at 0.degree. C., and the mixture was heated at reflux for
17 hours.
[0258] The reaction was quenched at 0.degree. C. with H.sub.2O (1.6
mL), 15% NaOH (1.6 mL), and H.sub.2O (4.8 mL). Salts were filtered
and washed with THF, and solvent was removed by rotary evaporation.
The residue was distilled in a Kugelrohr apparatus under high
vacuum (T.ltoreq.60.degree. C.), and the distillate was dissolved
in EtOH (5 mL) and treated with concentrated HCl (0.5 mL).
Recrystallization from aqueous EtOH gave 0.096 g (18%) of 9 as
crystals: NMR (D.sub.2O) .delta. 1.30 (t, 3H, J=7), 1.72-1.83 (m,
4H), 2.05-2.16 (m, 2H), 3.02-3.19 (m, 10H). Anal.
(C.sub.9H.sub.26Cl.sub.3N.sub.3) C, H, N.
EXAMPLE 64
[0259] N.sup.8-Ethylspermidine Trihydrochloride (10).
[0260] Lithium aluminum hydride (1.73 g, 45.6 mmol) and
N.sup.8-acetylspermidine dihydrochloride (0.54 g, 2.1 mmol) in THF
(300 mL) were reacted, and product was isolated by the method of 9
to furnish 0.164 g (28%) of 10 as crystals: NMR (D.sub.2O) .delta.
1.29 (t, 3H, J=7), 1.73-1.83 (m, 4H), 2.03-2.16 (m, 2H), 3.05-3.20
(m, 10H). Anal. (C.sub.9H.sub.26Cl.sub.3N.sub.3) C, H, N.
EXAMPLE 65
[0261] N.sup.1,N.sup.4
N.sup.8,N.sup.11-Tetrakis(mesitylenesulfonyl)-N.sup-
.1,N.sup.11-dipropylnorspermine (76).
[0262] NaH (60%, 3.60 g, 90.0 mmol), 75 [Bergeron et al, J. Med.
Chem., Vol. 37, supra]. (27.5 g, 30.0 mmol), and 1-iodopropane (7.5
mL, 77 mmol) in DMF (200 mL) were combined, and the reaction was
worked up by the method of 48. Column chromatography (5:1
toluene/EtOAc) resulted in 27.79 g (92%) of 76 as a white foam: NMR
.delta. 0.72 (t, 6H, J=7), 1.2-1.7 (m, 10H), 2.30 (s, 12H), 2.55
(s, 24H), 2.92-3.03 (m, 16H), 6.93 (s, 8H). Anal.
(C.sub.51H.sub.76N.sub.40.sub.8S.sub.4) C, H, N.
EXAMPLE 66
[0263] N.sup.1,N.sup.11-Dipropylnorspermine Tetrahydrochloride
(28).
[0264] HBr (30% in HOAc, 500 mL), 76 (27.54 g, 27.5 mmol), and
phenol (105 g, 1.12 mol) in CH.sub.2Cl.sub.2 (250 mL) were reacted,
and product was isolated by the method of 2 to give 7.82 g (68%) of
28 as white plates: NMR (D.sub.2O) .delta. 0.98 (t, 6H, J=7),
1.64-1.78 (m, 4H), 2.07-2.21 (m, 6H), 3.00-3.25 (m, 16H). Anal.
(C.sub.15H.sub.40Cl.sub.4N.sub.4) C, H, N.
EXAMPLE 67
[0265] N-(5-Chloropentyl)-N-ethylmesitylenesulfonamide (77).
[0266] NaH (80%, 1.34 g, 44.7 mmol) was added to 60 [Bergeron et
al, J. Med. Chem., Vol. 37, supra]. (7.68 g, 33.8 mmol) in DMF (130
mL) at 0.degree. C. The mixture was stirred at room temperature for
1 hour, and cooled to 0.degree. C. 1,5-Dichloropentane (45 mL, 0.35
mol) was added all at once. The reaction was stirred at 55.degree.
C. for 12 hours and was worked up by the method of 48. Column
chromatography (11.5% EtOAc/hexane) gave 10.54 g (94%) of 77 as an
oil: NMR .delta. 1.07 (t, 3H, J=8), 1.25-1.37 (m, 2H), 1.47-1.71
(m, 4H), 2.30 (s, 3H), 2.60 (s, 6H), 3.14-3.28 (m, 4H), 3.44 (t,
2H, J=7), 6.94 (s, 2H). Anal. (C.sub.16H.sub.26ClNO.sub.2S) C, H,
N.
EXAMPLE 68
[0267]
3,9,14,20-Tetrakis(mesitylenesulfonyl)-3,9,14,20-tetraazadocosane
(79).
[0268] NaH (80%, 1.14 g, 38.0 mmol) was added to 78 [Bergeron et
al, J. Med. Chem., Vol. 37, supra]. (5.77 g, 12.7 mmol) in DMF (75
mL) at 0.degree. C. The mixture was stirred at room temperature for
1 hour, and 77 (10.51 g, 31.7 mmol) in DMF (55 mL) was added by
cannula. The reaction was stirred at 55.degree. C. for 16 hours and
was worked up by the method of 48. Column chromatography (30%
EtOAc/hexane) afforded 12.67 g (96%) of 79 as an oil: NMR .delta.
0.97-1.12 (m, 10H), 1.30-1.47 (m, 12H), 2.29 (s, 12H), 2.56 and
2.58 (2 s, 24H), 2.97-3.22 (m, 16H), 6.93 (s, 8H). Anal.
(C.sub.54H.sub.82N.sub.4O.sub.8S.sub.4) C, H, N.
EXAMPLE 69
[0269] 3,9,14,20-Tetraazadocosane Tetrahydrochloride (29).
[0270] HBr (30% in HOAc, 195 mL), 79 (12.66 g, 12.1 mmol), and
phenol (33.61 g, 0.357 mol) in CH.sub.2Cl.sub.2 (135 mL) were
reacted, and product was isolated by the method of 2 to provide
4.50 g (81%) of 29 as white crystals: NMR (D.sub.2O) .delta. 1.28
(t, 6H, J=7), 1.40-1.52 (m, 4H), 1.66-1.80 (m, 12H), 3.00-3.14 (m,
16H). Anal. (C.sub.18H.sub.46Cl.sub.4N.sub.4).
ANALYTICAL DATA
[0271] 2 Anal. calcd. for C.sub.8H.sub.24Cl.sub.3N.sub.3: C,
35.77;H, 9.00; N, 15.64. Found: C, 35.91;H, 8.96; N, 15.69.
[0272] 4 Anal. calcd. for C.sub.10H.sub.28Cl.sub.3N.sub.3: C,
40.48;H, 9.51; N, 14.16. Found: C, 40.31;H, 9.36; N, 14.12.
[0273] 5 Anal. calcd. for C.sub.9H.sub.26Cl.sub.3N.sub.3: C,
38.24;H, 9.27; N, 14.87. Found: C, 38.15;H, 9.32; N, 14.75.
[0274] 6 Anal. calcd. for C.sub.12H.sub.32Cl.sub.3N.sub.3: C,
44.38;H, 9.93; N, 12.94. Found: C, 44.42;H, 9.89; N, 12.88.
[0275] 8 Anal. calcd. for C.sub.9H.sub.26Cl.sub.3N.sub.3: C,
38.24;H, 9.27; N, 14.86. Found: C, 38.19;H, 9.28; N, 14.79.
[0276] 9 Anal. calcd. for C.sub.9H.sub.26Cl.sub.3N.sub.3: C,
38.24;H, 9.27; N, 14.86. Found: C, 38.31;H, 9.23; N, 14.90.
[0277] 10 Anal. calcd. for C.sub.9H.sub.26Cl.sub.3N.sub.3: C,
38.24;H, 9.27 N, 14.86. Found: C, 38.28;H, 9.31; N, 14.95.
[0278] 11 Anal. calcd. for C.sub.11H.sub.30Cl.sub.3N.sub.3: C,
42.52;H, 9.73; N, 13.52. Found: C, 42.59;H, 9.79; N, 13.47.
[0279] 12 Anal. calcd. for C.sub.10H.sub.28Cl.sub.3N.sub.3: C,
40.48;H, 9.51; N, 14.16. Found C, 40.55;H, 9.45; N, 14.18.
[0280] 13 Anal. calcd. for C.sub.10H.sub.28Cl.sub.3N.sub.3: C,
40.48;H, 9.51; N, 14.16. Found C, 40.52;H, 9.52; N, 14.09.
[0281] 14 Anal. calcd. for C.sub.13H.sub.34Cl.sub.3N.sub.3: C,
46.09;H, 10.12; N, 12.40. Found: C, 46.15;H, 10.17; N, 12.43.
[0282] 15 Anal. calcd. for C.sub.8H.sub.24Cl.sub.3N.sub.3: C,
35.77;H, 9.00; N, 15.64; S, 39.59. Found: C, 35.88;H, 8.91; N,
15.68; S, 39.48.
[0283] 16 Anal. calcd. for C.sub.10H.sub.28 Cl.sub.3N.sub.3: C,
40.48;H, 9.51; N, 14.16. Found: C, 40.45;H, 9.44; N, 14.09.
[0284] 17 Anal. calcd. for C.sub.12H.sub.32Cl.sub.3N.sub.3: C,
44.38;H, 9.93; N, 12.94. Found: C, 44.49;H, 9.98; N, 12.96.
[0285] 19 Anal. calcd. for C.sub.14H.sub.36Cl.sub.3N.sub.3: C,
47.66;H, 10.29; N, 11.91. Found: C, 47.70;H, 10.21; N, 11.86.
[0286] 20 Anal. calcd. for C.sub.9H.sub.26Cl.sub.3N.sub.3: C,
38.24;H, 9.27; N, 14.87. Found: C, 38.28;H, 9.22; N, 14.82.
[0287] 21 Anal. calcd. for C.sub.11H.sub.30Cl.sub.3N.sub.3: C,
42.52;H, 9.73; N, 13.52. Found: C, 42.52;H, 9.69; N, 13.58.
[0288] 22 Anal. calcd. for C.sub.13H.sub.34Cl.sub.3N.sub.3: C,
46.09;H, 10.12; N, 12.40. Found: C, 46.20;H, 10.08; N, 12.47.
[0289] 23 Anal. calcd. for C.sub.15H.sub.38Cl.sub.3N.sub.3: C,
49.11;H, 10.44; N, 11.45. Found: C, 49.02;H, 10.40; N, 11.42.
[0290] 24 Anal. calcd. for C.sub.10H.sub.28Cl.sub.3N.sub.3: C,
40.48;H, 9.51; N, 14.16; Cl, 35.85. Found: C, 40.63;H, 9.44; N,
14.16; Cl, 35.70.
[0291] 25 Anal. calcd. for C.sub.12H.sub.32Cl.sub.3N.sub.3: C,
44.38;H, 9.93; N, 12.94. Found: C, 44.33;H, 9.90; N, 12.89.
[0292] 26 Anal. calcd. for C.sub.14H.sub.36Cl.sub.3N.sub.3: C,
47.66;H, 10.28; N, 11.91; Cl, 30.15. Found: C, 47.69;H, 10.24; N,
11.96; Cl, 30.08.
[0293] 27 Anal. calcd. for C.sub.16H.sub.40Cl.sub.3N.sub.3: C,
50.46;H, 10.59; N, 11.03. Found: C, 50.49;H, 10.55; N, 11.07.
[0294] 28 Anal. calcd. for C.sub.15H.sub.40Cl.sub.4N.sub.4: C,
43.07;H, 9.64; N, 13.39. Found: C, 43.24;H, 9.57; N, 13.44.
[0295] 29 Anal. calcd. for C.sub.18H.sub.46Cl.sub.4N.sub.4: C,
46.96;H, 10.07; N, 12.17. Found: C, 47.08;H, 9.98; N, 12.18.
[0296] 30 Anal. calcd. for C.sub.33H.sub.47N.sub.3O.sub.6S.sub.3:
C, 58.47;H, 6.99; N, 6.20. Found: C, 58.36;H, 6.95; N, 6.18.
[0297] 31 Anal. calcd. for C.sub.4H.sub.49N.sub.3O.sub.6S.sub.3: C,
59.02;H, 7.14; N, 6.07. Found: C, 58.74;H, 7.12; N, 5.99.
[0298] 32 Anal. calcd. for C.sub.35H.sub.51N.sub.3O.sub.6S.sub.3:
C, 59.55;H, 7.28; N, 5.95. Found: C, 59.34;H, 7.29; N, 5.92.
[0299] 33 Anal. calcd. for C.sub.36H.sub.53N.sub.3O.sub.6S.sub.3:
C, 60.05;H, 7.42; N, 5.84. Found: C, 59.88;H, 7.41; N, 5.80.
[0300] 36 Anal. calcd. for C.sub.13H.sub.18N.sub.2O.sub.2S: C,
58.62;H, 6.81; N, 10.52. Found: C, 58.52;H, 6.86; N, 10.46.
[0301] 38 Anal. calcd. for C.sub.18H.sub.25N.sub.3O.sub.2S: C,
62.22;H, 7.25; N, 12.09; S, 8.87. Found: C, 62.24;H, 7.28; N,
11.99.
[0302] 39 Anal. calcd. for C.sub.19H.sub.27N.sub.3O.sub.2S: C,
63.13;H, 7.53; N, 11.62; S, 8.87. Found: C, 63.31;H, 7.68; N,
11.43; S, 8.97.
[0303] 43 Anal. calcd. for C.sub.35H.sub.51N.sub.3O.sub.6S.sub.3:
C, 59.55;H, 7.28; N, 5.95. Found: C, 59.50;H, 7.33; N, 5.88.
[0304] 44 Anal calcd. for C.sub.37H.sub.55N.sub.3O.sub.6S.sub.3: C,
60.54;H, 7.55; N, 5.72. Found: C, 60.64;H, 7.54; N, 5.73.
[0305] 45 Anal. calcd. for C.sub.36H.sub.53N.sub.3O.sub.6S.sub.3:
C, 60.05;H, 7.42; N, 5.84. Found: C, 59.79;H, 7.32; N, 5.70.
[0306] 46 Anal. calcd. for C.sub.39H.sub.59N.sub.3O.sub.6S.sub.3:
C, 61.55;H, 7.68; N, 5.52. Found: C, 61.52;H, 7.79; N, 5.55.
[0307] 48 Anal. calcd. for C.sub.38H.sub.57N.sub.3O.sub.6S.sub.3:
C, 61.01;H, 7.68; N, 5.62. Found: C, 61.22;H, 7.76; N, 5.56.
[0308] 49 Anal. calcd. for C.sub.40H.sub.61N.sub.3O.sub.6S.sub.3:
C, 61.90;H, 7.92; N, 5.41. Found: C, 61.71;H, 7.86; N, 5.35.
[0309] 50 Anal. calcd. for C.sub.37H.sub.55N.sub.3O.sub.6S.sub.3:
C, 60.54;H, 7.55; N, 5.72. Found: C, 60.26;H, 7.61; N, 5.63.
[0310] 51 Anal. calcd. for C.sub.39H.sub.59N.sub.3O.sub.6S.sub.3:
C, 61.47;H, 7.80; N, 5.51. Found: C, 61.27;H, 7.89; N, 5.44.
[0311] 52 Anal. calcd. for C.sub.41H.sub.63N.sub.3O.sub.6S.sub.3:
C, 62.32;H, 8.04; N, 5.32. Found: C, 62.19;H, 8.00; N, 5.33.
[0312] 54 Anal. calcd. for
C.sub.42H.sub.65N.sub.3O.sub.6S.sub.3.H.sub.2O: C, 61.35;H, 8.21;
N, 5.11. Found: C, 61.34;H, 8.07; N, 5.05.
[0313] 55 Anal. calcd. for C.sub.39H.sub.59N.sub.3O.sub.6S.sub.3:
C, 61.47;H, 7.80; N, 5.51. Found: C, 61.54;H, 7.79; N, 5.51.
[0314] 56 Anal. calcd. for C.sub.41H.sub.63N.sub.3O.sub.6S.sub.3:
C, 62.32;H, 8.04; N, 5.32; S, 12.17. Found: C, 62.40;H, 8.08; N,
5.25; S, 12.07.
[0315] 57 Anal. calcd. for C.sub.43H.sub.67N.sub.3O.sub.6S.sub.3:
C, 63.12;H, 8.25; N, 5.14. Found: C, 63.21;H, 8.23; N, 5.04.
[0316] 63 Anal. calcd. for C.sub.24H.sub.36N.sub.2O.sub.4S.sub.2:
C, 59.97;H, 7.55; N, 5.83. Found: C, 59.83;H, 7.56; N, 5.76.
[0317] 64 Anal. calcd. for C.sub.40H.sub.61N.sub.3O.sub.6S.sub.3:
C, 61.90;H, 7.92; N, 5.41. Found: C, 62.03;H, 7.97; N, 5.33.
[0318] 65 Anal. calcd. for C.sub.12H.sub.19NO.sub.2S: C, 59.72;H,
7.93; N, 5.80. Found: C, 59.69;H, 7.88; N, 5.80.
[0319] 66 Anal. calcd. for C.sub.15H.sub.24BrNO2S: C, 49.72;H,
6.68; N, 3.87. Found C, 49.97;H, 6.76; N, 3.83.
[0320] 67 Anal. calcd. for C.sub.16H.sub.26BrNO.sub.2S: C, 51.06;H,
6.96; N, 3.72. Found: C, 51.17;H, 6.95; N, 3.74.
[0321] 68 Anal. calcd. for C.sub.22H.sub.24N.sub.2: C, 83.50;H,
7.64; N, 8.85. Found C, 83.42;H, 7.67; N, 8.86.
[0322] 70 Anal. calcd. for C.sub.31H.sub.34N.sub.2O.sub.2S: C,
74.67;H, 6.87; N, 5.62. Found: C, 74.62;H, 6.89; N, 5.54.
[0323] 71 Anal. calcd. for C.sub.32H.sub.36N.sub.2O.sub.2S: C,
74.97;H, 7.08; N, 5.46. Found: C, 74.71;H, 7.12; N, 5.51.
[0324] 72 Anal. calcd. for C.sub.47H.sub.59N.sub.3O.sub.4S.sub.2:
C, 71.09;H, 7.49; N, 5.29. Found C, 71.35;H, 7.53; N, 5.18.
[0325] 73 Anal. calcd. for C.sub.47H.sub.59N.sub.3O.sub.4S.sub.2:
C, 71.09;H, 7.49; N, 5.29. Found C, 71.16;H, 7.46; N, 5.33.
[0326] 74 Anal. calcd. for C.sub.48H.sub.61N.sub.3O.sub.4S.sub.2:
C, 71.34;H, 7.61; N, 5.20. Found C, 71.22;H, 7.64; N, 5.10.
[0327] 76 Anal. calcd. for C.sub.51H.sub.76N.sub.4O.sub.8S.sub.4:
C, 61.17;H, 7.65; N, 5.59. Found: C, 61.21;H, 7.67; N, 5.58.
[0328] 77 Anal. calcd. for C.sub.16H.sub.26ClNO.sub.2S: C, 57.90;H,
7.90; N, 4.22. Found: C, 57.98;H, 7.82; N, 4.27.
[0329] 79 Anal. calcd. for C.sub.54H.sub.82N.sub.4O.sub.8S.sub.4:
C, 62.16;H, 7.92; N, 5.37. Found: C, 62.30;H, 7.86; N, 5.37.
* * * * *