U.S. patent application number 10/014432 was filed with the patent office on 2002-10-03 for analogs of biologically active, naturally occurring polyamines, pharmaceutical compositions and methods of treatment.
Invention is credited to Bergeron, Raymond J. JR..
Application Number | 20020143068 10/014432 |
Document ID | / |
Family ID | 27535720 |
Filed Date | 2002-10-03 |
United States Patent
Application |
20020143068 |
Kind Code |
A1 |
Bergeron, Raymond J. JR. |
October 3, 2002 |
Analogs of biologically active, naturally occurring polyamines,
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.6 may be
the same or different and are alkyl, aryl, aryl alkyl, cycloalkyl,
optionally having an alkyl chain interrupted by at least one
etheric oxygen atom, or hydrogen; N.sup.1, N.sup.2, N.sup.3 and
N.sup.4 are nitrogen atoms capable of protonation at physiological
pH's; a and b may be the same or different and are integers from 1
to 4; A, B and C may be the same or different and are bridging
groups which effectively maintain the distance between the nitrogen
atoms such that the polyamines: (i) are capable of uptake by a
target cell upon administration thereof to a human or non-human
animal; and (ii) upon uptake by the target cell, competitively bind
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; the
polyamines, upon binding to the biological counter-anion in the
cell, function in a manner biologically different than the
intracellular polyamines, the polyamines not occurring in nature;
as well as pharmaceutical compositions embodying the polyamines and
methods of treating patients requiring anti-neoplastic therapy.
Inventors: |
Bergeron, Raymond J. JR.;
(Gainesville, FL) |
Correspondence
Address: |
Miles & Stockbridge
Suite 500
1751 Pinnacle Drive
McLean
VA
22102-3833
US
|
Family ID: |
27535720 |
Appl. No.: |
10/014432 |
Filed: |
December 14, 2001 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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10014432 |
Dec 14, 2001 |
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09688386 |
Oct 17, 2000 |
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6342534 |
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09688386 |
Oct 17, 2000 |
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08080642 |
Jun 22, 1993 |
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6184232 |
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08080642 |
Jun 22, 1993 |
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07834345 |
Feb 12, 1992 |
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5342945 |
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07834345 |
Feb 12, 1992 |
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07210520 |
Jun 23, 1988 |
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5091576 |
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07210520 |
Jun 23, 1988 |
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07066227 |
Jun 25, 1987 |
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07066227 |
Jun 25, 1987 |
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06936835 |
Dec 2, 1986 |
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Current U.S.
Class: |
514/674 ;
514/649; 564/372; 564/512 |
Current CPC
Class: |
A61K 31/785 20130101;
C07C 211/14 20130101; C07C 209/62 20130101; A01N 43/54 20130101;
A61K 31/13 20130101; A61K 31/506 20130101; A61K 31/131 20130101;
C07C 209/08 20130101; A01N 33/04 20130101; A61K 31/505 20130101;
C07D 239/04 20130101; A61K 31/135 20130101 |
Class at
Publication: |
514/674 ;
564/512; 514/649; 564/372 |
International
Class: |
A61K 031/135; A61K
031/13; C07C 211/13 |
Goverment Interests
[0002] Research leading to the completion of the invention was
supported, in part, by Grant No. ______ awarded by the National
Institutes of Health (NIH). The United States Government has
certain rights in and to the invention claimed herein.
Claims
I claim:
1. A polyamine having the formula: 15or a salt thereof with a
pharmaceutically acceptable acid wherein: R.sub.1-R.sub.6 may be
the same or different and are alkyl, aryl, aryl alkyl, cycloalkyl,
optionally having an alkyl chains interrupted by at least one
etheric oxygen atom, or hydrogen N.sup.1, N.sup.2, N.sup.3 and
N.sup.4 are nitrogen atoms capable of protonation at physiological
pH's ; a and b may be the same or different and are integers from 1
to 4; A, B and C may be the same or different and are bridging
groups which effectively maintain the distance between said
nitrogen atoms such that said polyamine: (i) is capable of uptake
by a target cell upon administration of said 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 intra-cellular natural polyamines
in the target cell; said polyamine, upon binding to said biological
counter-anion in the cell, functions in a manner biologically
different than said intracellular polyamines, said polyamine not
occurring in nature.
2. A polyamine of claim 1, upon binding to said biological
counter-anion in said cell, exerting an anti-neoplastic
function.
3. A polyamine of claim 1 wherein said bridging groups A, B and C
may be the same or different and are alkylene, branched alkylene,
cycloalkylene, arylalkylene or a heterocyclic bridging group
wherein one of said N.sup.1, N.sup.2, N.sup.3 or N.sup.4 atoms is
incorporated in the ring as the hetero atom.
4. A pharmaceutical composition comprising an anti-neoplastic
effective amount of a polyamine of claim 1 or a salt thereof with a
pharmaceutically acceptable acid and a pharmaceutically acceptable
carrier therefor.
5. A method of treating a human or non-human animal in need thereof
comprising administering thereto an anti-neoplastic effective
amount of a polyamine of claim 1 or a salt thereof with a
pharmaceutically acceptable acid.
6. A polyamine of claim 1 having the formula: 16
7. A polyamine of claim 1 having the formula: 17
8. A polyamine of claim 1 having the formula: 18
Description
RELATED APPLICATIONS
[0001] This is a continuation-in-part of application Ser. No.
07/834,345 filed Feb. 12, 1992, which is a division of application
Ser. No. 07/210,520 filed Jun. 23, 1988 (now U.S. Pat. No.
5,091,576 issued Feb. 25, 1992), which is a continuation-in-part of
application Ser. No. 07/066,227 filed Jun. 25, 1987 (now
abandoned), which is a continuation-in-part of application Ser. No.
06/936,835 filed Dec. 2, 1986 (now abandoned).
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates to analogs of biologically
active, naturally occurring polyamines having a wide variety of
therapeutic properties, as well as pharmaceutical compositions
containing the analogs and their use in methods of therapeutic
treatment.
[0005] 2. Discussion of the Prior Art
[0006] In recent years, a great deal of attention has been focussed
on the polyamines, e.g., spermidine, norspermidine, homospermidine,
1,4-diaminobutane (putrescine) and spermine. These studies have
been largely directed at the biological properties of the
polyamines probably because of the role they play in proliferative
processes. It was shown early on that the polyamine levels in
dividing cells, e.g., cancer cells, are much higher than in resting
cells. See Janne et al, A. Biochim. Biophys. Acta., Vol. 473, page
241 (1978); Fillingame et al, Proc. Natl. Acad. Sci. U.S.A., Vol.
72, page 4042 (1975); Metcalf et al, J. Am. Chem. Soc., Vol. 100,
page 2551 (1978): Flink et al, Nature (London), Vol. 253, page 62
(1975); and Pegg et al, Polyamine Metabolism and Function, Am. J.
Cell. Physiol., Vol. 243, pages 212-221 (1982).
[0007] Several lines of evidence indicate that polyamines,
particularly spermidine, are required for cell proliferation: (i)
they are found in greater amounts in growing than in non-growing
tissues; (ii) prokaryotic and eukaryotic mutants deficient in
polyamine biosynthesis are auxotrophic for polyamines; and (iii)
inhibitors specific for polyamine biosynthesis also inhibit cell
growth. Despite this evidence, the precise biological role of
polyamines in cell proliferation is uncertain. It has been
suggested that polyamines, by virtue of their charged nature under
physiological conditions and their conformational flexibility,
might serve to stabilize macromolecules such as nucleic acids by
anion neutralization. See Dkystra et al, Science, Vol. 149, page 48
(1965); Russell et al, Polyamines as Biochemical Markers of Normal
and Malignant Growth (Raven, New York 1978); Hirschfield et al, J.
Bacteriol., Vol. 101, page 725 (1970); Hafner et al, J. Biol.
Chem., Vol. 254, page 12419 (1979); Cohn et al, J. Bacteriol., Vol.
134, page 208 (1978); Pohjatipelto et al, Nature (London), Vol.
293, page 475 (1981); Mamont et al, Biochem. Biophys. Res. Commun.,
Vol. 81, page 58 (1978); Bloomfield et al, Polyamines in Biology
and Medicine (D. R. Morris and L. J. Morton, eds., Dekker, New
York, 1981), pages 183-205; Gosule et al, Nature, Vol. 259, page
333 (1976); Gabbay et al, Ann. N.Y. Acad. Sci., Vol. 171, page 810
(1970); Suwalsky et al, J. Mol. Biol., Vol. 42, page 363 (1969);
and Liquori et al, J. Mol. Biol., Vol. 24, page 113 (1968).
[0008] However, regardless of the reason for increased polyamine
levels, the phenomenon can be and has been exploited in
chemotherapy. See Sjoerdsma et al, Butterworths Int. Med. Rev.:
Clin. Pharmacol. Thera., Vol. 35, page 287 (1984); Israel et al, J.
Med. Chem., Vol. 16, page 1 (1973); Morris et al, Polyamines in
Biology and Medicine, Dekker, New York, page 223 (1981); and Wang
et al, Biochem. Biophys. Res. Commun., Vol. 94, page 85 (1980).
[0009] Because of the role the natural polyamines play in
proliferation, a great deal of effort has been invested in the
development of polyamine analogs as anti-proliferatives [Cancer
Res., Vol. 49, "The role of methylene backbone in the
anti-proliferative activity of polyamine analogues on L1210 cells,"
Bergeron et al, pages 2959-2964 (1989); J. Med. Chem., Vol. 31,
"Synthetic polyamine analogues as antineoplastics," Bergeron et al,
pages 1183-1190 (1988); Polyamines in Biochemical and Clinical
Research, "Regulation of polyamine biosynthetic activity by
spermidine and spermine--a novel antiproliferative strategy,"
Porter et al, pages 677-690 (1988); Cancer Res., Vol. 49, "Major
increases in spermidine/spermine-N.sup.1-acetyl transferase
activity by spermine analogues and their relationship to polyamine
depletion and growth inhibition in L1210 cells," Basu et al, pages
6226-6231 (1989); Biochem. J., Vol. 267, "Induction of
spermidine/spermine N.sup.1-acetyltransferase activity in
Chinese-hamster ovary cells by N.sup.1,N.sup.11-bis(ethyl)nor-
spermine and related compounds," Pegg et al, pages 331-338 (1990);
Biochem. J., Vol. 268, "Combined regulation of ornithine and
S-adenosylmethionine decarboxylases by spermine and the spermine
analogue N.sup.1N.sup.12-bis-(ethyl)spermine," Porter et al, pages
207-212 (1990); Cancer Res., Vol. 50, "Effect of
N.sup.1,N.sup.14-bis(ethyl)-homospermine on the growth of U-87 MG
and SF-126 on human brain tumor cells," Basu et al, pages 3137-3140
(1990),: and Biochem. Biophys. Res. Commun., Vol. 152, "The effect
of structural changes in a polyamine backbone on its DNA binding
properties," Stewart, pages 1441-1446 (1988)]. These efforts have
included the design of new synthetic methods [J. Org. Chem., Vol.
45, "Synthesis of N.sup.4-acylated
N.sup.1,N.sup.8-bis(acyl)spermidines: An approach to the synthesis
of siderophores," Bergeron et al, pages 1589-1592 (1980);
Synthesis, "Reagents for the selective acylation of spermidine,
homospermidine and bis-[3-amino-propy]amine," Bergeron et al, pages
732-733 (1981); Synthesis, "Reagents for the selective secondary
functionalization of linear triamines," Bergeron et al, pages
689-692 (1982); Synthesis, "Amines and polyamines from nitrites,"
Bergeron et al, pages 782-785 (1984); J. Org. Chem., Vol. 49,
"Reagents for the stepwise functionalization of spermidine,
homospermidine and bis-[3-aminopropyl]amine," Bergeron et al, page
2997 (1984); Accts. Chem. Res., Vol. 19, "Methods for the selective
modification of spermidine and its homologues," Bergeron, pages
105-113 (1986); Bioorg. Chem., Vol. 14, "Hexahydropyrimidines as
masked spermidine vectors in drug delivery," Bergeron et al, pages
345-355 (1986); J. Org. Chem., Vol. 53, "Reagents for the stepwise
functionalization of spermine," Bergeron et al, pages 3108-3111
(1988); J. Org. Chem., Vol. 52, "Total synthesis of
(.+-.)-15-Deoxyspergualin," Bergeron et al, pages 1700-1703 (1987);
J. Org. Chem., Vol. 56, "The total synthesis of Alcaligin,"
Bergeron et al, pages 586-593 (1991); and CRC Handbook on Microbial
Iron Chelates, "Synthesis of catecholamide and hydroxamate
siderophores," Bergeron et al, pages 271-307 (1991)] for the
production of these analogs, as well as extensive biochemical
studies focussed on the mechanism by which these compounds act
[Cancer Res., Vol. 46, "A comparison and characterization of growth
inhibition by .alpha.-Difluoromethylornithine (DFMO), and inhibitor
of ornithine decarboxylase and N.sup.1,N.sup.8-bis(ethyl)spermi-
dine (BES), an apparent regulator of the enzyme," Porter et al,
pages 6279-6285 (1986); Cancer Res., Vol. 47, "Relative abilities
of bis(ethyl) derivatives of putrescine, spermidine and spermine to
regulate polyamine biosynthesis and inhibit L1210 leukemia cell
growth," Porter et al, pages 2821-2825 (1987); Cancer Res., Vol.
49, "Correlation between the effects of polyamine analogues on DNA
conformation and cell growth," Basu et al, pages 5591-5597 (1989);
Cancer Res., Vol. 49, "Differential response to treatment with the
bis(ethyl)polyamine analogues between human small cell lung
carcinoma and undifferentiated large cell lung carcinoma in
culture," Casero et al, pages 639-643 (1988); Mol. Pharm., Vol. 39,
"Selective cellular depletion of mitochondrial DNA by the polyamine
analog, N.sup.1,N.sup.12-bis(ethyl)spermine, and its relationship
to polyamine structure and function," Vertino et al, pages 487-494
(1991); Biochem. and Biophys. Res. Comm., Vol. 157, "Modulation of
polyamine biosynthesis and transport by oncogene transfection,"
Chang et al, pages 264-270 (1988); and Biopolymers, Vol. 26,
"Structural determinants of spermidine-DNA interactions," Vertino
et al, pages 691-703 (1987)]. The mechanistic investigations have
encompassed uptake studies, impact on polyamine analogs on
polyamine pools and polyamine biosynthetic enzymes, as well as
their effects on translational and transcriptional events.
[0010] Anti-neoplastic derivatives of the naturally occurring
polyamines, pharmaceutical compositions and methods of treatment
are also disclosed in the following pending patent application Ser.
No. ______ filed ______, ("Sterically Hindered Tetraamines and
Method for Their Production"), as well as in U.S. Pat. No.
5,091,576 issued Feb. 25, 1992; U.S. Pat. No. 5,128,353 issued Jul.
7, 1992; and U.S. Pat. No. 5,173,505 issued Dec. 22, 1992. The
disclosures of each of the foregoing applications and patents are
incorporated herein by reference.
[0011] It is an object of the present invention to provide novel
analogs of naturally occurring, biologically active polyamines
which possess anti-neoplastic properties and to define the
structural parameters which define compounds having such
activities.
SUMMARY OF THE INVENTION
[0012] These and other objects are realized by the present
invention, one embodiment of which relates to a polyamine having
the formula: 2
[0013] or a salt thereof with a pharmaceutically acceptable acid
wherein:
[0014] R.sub.1-R.sub.6 may be the same or different and are alkyl,
aryl, aryl alkyl, cycloalkyl, any of the foregoing wherein the
alkyl chain is interrupted by at least one etheric oxygen atom, or
hydrogen;
[0015] N.sup.1, N.sup.2, N.sup.3 and N.sup.4 are nitrogen atoms
capable of protonation at physiological pH's;
[0016] a and b may be the same or different and are integers from 1
to 4;
[0017] A, B and C may be the same or different and are bridging
groups which effectively maintain the distance between the nitrogen
atoms such that the polyamine:
[0018] (i) is capable of uptake by a target cell upon
administration of the polyamine to a human or non-human animal;
and
[0019] (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 intra-cellular natural polyamines in the target cell;
[0020] the polyamine, upon binding to the biological counter-anion
in the cell, functions in a manner biologically different than the
intracellular polyamines, the polyamine not occurring in
nature.
[0021] Another embodiment of the invention comprises a
pharmaceutical composition comprising an anti-neoplastic effective
amount of the polyamine described above or a salt thereof with a
pharmaceutically acceptable acid and a pharmaceutically acceptable
carrier therefor.
[0022] An additional embodiment of the invention relates to a
method of treating a human or non-human animal in need thereof
comprising administering thereto an anti-neoplastic effective
amount of the polyamine described above or a salt thereof with a
pharmaceutically acceptable acid.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIGS. 1-8 depict reaction schemes for preparing various of
the polyamines of the invention and intermediates therefor.
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-trimethyl-phenyl;
aryl alkyl, e.g., benzyl, .alpha.-phenethyl, .beta.3-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.3O(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 12 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, B and C 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 [eg., 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] Particularly preferred polyamines are those set forth in
Tables 1 and 2 and in the examples, as well as those having the
formula: 4
[0030] wherein R.sub.1-R.sub.6 have the meanings ascribed above and
a and b may be the same or different and are integers from 2 to
8.
[0031] At physiological pH's, the naturally occurring polyamines
and the analogs of the present invention are largely in a
protonated state [Bioorg. Chem., supra]. 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.
[0032] 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. For example, although
N.sup.1,N.sup.12-diethylspermine (DESPM) is quite active against a
variety of tumors in cell culture, when the terminal ethyl groups
are replaced by .beta.,.beta.,.beta.-trifluoroethyl groups, the
anti-neoplastic activity essentially disappears. The trifluoroethyl
group substantially reduces the pK.sub.a of the terminal nitrogens
and they are no longer protonated [J. Org. Chem., Vol. 24,
"Fluorine containing nitrogen compounds--I Trifluoroethylamines,"
Bissel et al, pages 1256-1259 (1959)]. Even though the dimensions
of bis-.beta.,.beta.,.beta.-trifluoroethylspermine and
diethylspermine are essentially the same, what was once a
tetracationic spermine analog is now only a dication. On comparing
the abilities of several polyamines and polyamine analogs- to
displace ethidium iodide from calf thymus DNA,
bis-.beta.,.beta.,.beta.-trifluoroethylspermine (BTFESPM) was found
to behave more like putrescine than spermine. The same phenomenon
is observed when one acetylates a terminal nitrogen of spermine.
The amide nitrogen is not protonated at physiological pH, and the
compound has no anti-neoplastic properties and behaves very much
like spermidine in displacing ethidium iodide from DNA.
[0033] Perhaps the most impressive finding regarding the separation
of charge is. associated with S-acetyltransferase (SAT), an enzyme
responsible for the N-terminal acetylation of spermine and
spermidine. It has been determined that very slight changes in the
methylene backbone of the polyamine analogs have pronounced effects
on up-regulating the production of this protein, diethylnorspermine
(DENSPM)>diethylspermin- e (DESPM)>diethyl-homospermine
(DEHSPM) [Proc. Am. Assoc. Cancer Res., Vol. 49, "Differential
induction of spermidine/spermine N.sup.1-acetyltransferase in human
lung cancer cells by the bis(ethyl)polyamine analogues," Casero et
al, pages 3829-3833 (1989); Proc. Am. Assoc. Cancer Res., Vol. 30,
"Potent induction of spermidine/spermine N.sup.1-acetyltransferase
(SSAT) activity and its relationship to inhibition of cell growth,"
Libby et al, page 586 (1989); Biochem. Pharma., Vol. 38, No. 9,
"Structure-function correlations of polyamine analog-induced
increases in spermidine/spermine acetyltransferase activity," Libby
et al, pages 1435-1442 (1989); Cancer Res., Vol. 49, Basu et al,
supra; Biochem. J., Vol. 268, Porter et al, supra; Biochem. J.,
Vol. 267, Pegg et al, supra; Arch. Biochem. Biophys., Vol. 284,
"Characterization of human spermidine/spermine
N.sup.1-acetyltransferase purified from cultured melanoma cells,
Libby et al, pages 238-244 (1991); and Cancer Res., Vol. 51,
"Correlations between polyamine analog-induced increases in
spermidine/spermine N.sup.1-acetyl-transferase activity and growth
inhibition in human melanoma cell lines," Porter et al, pages
3715-3720 (1991)]. Extracting a single methylene from each of the
three methylene bridges of DEHSPM substantially increases the
ability of the analog to stimulate SAT up-regulation, DEHSPM has
little impact on SAT levels, while DESPM up-regulates the enzyme by
a factor of 200 and DENSPM by 1,200 fold. Although each of the
tetraamines is a closely related linear tetracation at
physiological pH, each provides a different signal to the cell.
While DEHSPM has three 4-methylene bridges and little
SAT-stimulating activity, DESPN which has a single central
4-methylene bridge and two terminal 3-methylene bridges is a more
active SAT-stimulating factor. DENSPM has three 3-methylene bridges
and is the most active SAT stimulator. Presumably, the cell then
"reads the charge distribution" on the polyamines. This suggests
that the key issue in the polyamine analogue's activity is the
distance between the charged centers and not the nature of the
groups separating these centers, "the insulators." However, that
this was indeed the case required proof.
[0034] The total concentration of polyamines in cation equivalents,
as well as the ratios of the various polyamines, is very tightly
regulated and, when this equilibrium is disturbed, the cell acts
quickly to re-adjust. For example, when polyamine analogs are
incorporated, these dynamics are disrupted and the cell responds by
either excreting polyamines in the free state or by first
acetylating them, followed by excretion of the N.sup.1-acetyl
compounds [J. Biol. Chem., Vol. 264, No. 20, "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," Pegg et al, pages 11744-11749 (1989); and Cancer Res.,
Vol. 51, Porter et al, supra]. On analog incorporation, the cell
disposes of the appropriate number of polyamine cation equivalents
in order to maintain the total polyamine charge balance.
[0035] The analogs appear to the cell as normal polyamines and
provide many of the same regulatory messages that the natural
polyamines do. As analog concentration increases in the cell, the
following events ensue. Polyamine biosynthesis is down-regulated,
just as when cells are grown in exogenous polyamines. The levels of
ornithine decarboxylase (ODC) and S-adenosylmethionine
decarboxylase (AdoMETDC) are drastically reduced just as with the
normal negative polyamine feedback control. Analogs with
aminopropyl groups cause a marked increase in S-acetyl transferase
(SAT). Once again, the issues are how the cell reads the charge
distribution on the polyamines and how does it translate this
information into regulatory events. The answers to both of these
questions will allow one to define the structural boundary
conditions for the general design of polyamine anti-neoplastics.
Two fundamental issues needed to be addressed regarding the general
structural requirements for anti-neoplastic polyamines: (1) Are the
cationic centers really key to the compound's anti-neoplastic
activity or are the nitrogen centers simply enough, and (2) what
are the boundary conditions on the insulators, e.g., bridging
groups separating the nitrogen or cationic centers?
[0036] The first question can be answered by comparing two
polyamines with essentially the same distance between the
nitrogens, but with different pKa values on spatially equivalent
nitrogens. While the bis-.beta.,.beta.,.beta.-trifluoroethyl
analogue of N.sup.1,N.sup.12-diethylspermine strongly suggested
that charge was significant in polyamine analogue activity,
additional examples had to be developed. In particular, examples
were utilized in which other than simple methylene insulators were
employed. This experiment is typified by the compounds and their
activities as set forth in Table 1.
1TABLE 1 Piperidine and Pyridine Bicyclic Tetraamines IC.sub.50
(.mu.M) of L1210 cells at 48/96 h 5 2/0.2 6 >100/90 7
>100/0.25 8 >100/>100
[0037] The results of Table 1 clearly indicate that the nitrogen
cationic centers are required for anti-neoplastic activity and not
simply a nitrogen center. The pyridine compounds have their
nitrogen separated by distances very similar to those in the
corresponding piperidine systems. The major difference is, at
physiological pH, the pyridine compounds are not substantially
protonated at the pyridine nitrogens. This means that the
piperidine compounds are tetracations at physiological pH, while
the pyridines are dications. The dications, unlike the
corresponding reduced and acyclic linear molecules, are not active
against L1210 cells.
[0038] Whether the cations can be insulated from each other by
other than a methylene bridge, e.g., a more rigid cyclic backbone,
while still maintaining the activity of the compound is a question
important not only to an understanding of how cells process
information on the polycations, but also to the design of improved
therapeutics. If a variety of other aliphatic systems can be
substituted for the methylene bridges in the polyamines, it is
possible to alter not only the metabolic properties of the
polyamine anti-neoplastics, but also their organ distribution and
clearance properties. The data set forth in Table 2 further
indicates that simple linear insulators are not a strict
requirement for polyamine analogue antineoplastic activity. The
results suggest that the polyamine nitrogens can be incorporated
into a cyclic backbone. This observation was further investigated
to verify that nitrogen insulators could also be cyclic alkyl
groups. Table 2 clearly indicates that this is true. The cyclohexyl
fragment works well in a variety of different arrangements as an
insulator.
2 TABLE 2 L1210 I.C. .sub.50 (.mu.M) Analogue 48 h 96 h Ki (uM) 9
70 0.08 4.1 10 >100 0.3 1.5 11 >100 0.2 7.9 12 >100 0.6
1.8 13 >100 3 30 14 >100 40 n.a.
[0039] The intention is that the cell should incorporate these
compounds via the polyamine's transport apparatus and that these
analogues should find their way to the same sub-cellular
distribution sites that the naturally occurring polyamines do, but
once there, because of subtle alterations in the molecules, they
should be unable to be further processed. The inability of the
polyamines to be further processed is largely related to the fact
that the terminal nitrogens in the active compounds are alkylated
and unable to be acetylated by SAT.
[0040] Linear Polyamine, Methylene Insulators
[0041] Previous synthetic methods [J. Org. Chem., Vol. 45, Bergeron
et al, supra; Synthesis, Bergeron et al (1981), supra; Synthesis,
Bergeron et al (1982), supra; Synthesis, Bergeron et al (1984),
supra; J. Org. Chem., Vol. 49, Bergeron et al, page 2997 (1984);
Accts. Chem. Res., Vol. 19, Bergeron, supra; Bioorg. Chem., Vol.
14, Bergeron et al, supra; J. Org. Chem., Vol. 53, Bergeron et al,
supra; J. Org. Chem., Vol. 52, Bergeron et al, supra; J. Org.
Chem., Vol. 56, Bergeron et al, supra; and CRC Handbook on
Microbial Iron Chelates, Bergeron et al, supra] were not designed
for polyamine bridge expansions, but for the introduction of
different alkyl groups at different nitrogens in the triamines or
tetraamines. However, it has been recently shown that many of the
simple terminally dialkylated polyamine analogs of interest can be
accessed via the appropriate tosylamide. For example, to synthesize
DESPM, spermine is first tosylated and then monoalkylated at each
terminal tosylamide by treatment with sodium hydride and ethyl
iodide. The tosyl groups are then removed under conditions of
dissolving metal reduction. The shortcomings of the procedure are
three-fold: (1) the alkylation must be symmetrical, i.e., the same
alkyl group must be fixed to both terminal nitrogens, (2) the
methylene insulators between the nitrogens are regulated by the
availability of the starting polyamine, and (3) removal of the
tosyl protecting group proceeds in low yield.
[0042] A more satisfactory alternative involves formation of the
appropriate tetramesitylenesulfonamides, which can be alkylated in
high yields and the tetramesitylenesulfonyl (MES) protecting groups
quantitatively removed by treatment with 30% HBr in acetic acid and
phenol (FIG. 1). This approach eliminates the low yield problem
associated with removal of the tosyl protecting group under
dissolving metal reduction conditions. The MES methodology has been
extended to a symmetrical "segmented synthesis" which allows for
the facile alteration of the methylene backbone (FIG. 2).
[0043] The segmented approach to constructing polyamine analogs
offers numerous advantages in terms of flexibility and high yields.
The procedure begins with mesitylenesulfonation of a primary amine
providing (1), followed by alkylation of (1) with an excess of the
appropriate dihalide (FIG. 2). The resulting halosulfonamide (2)
can then be utilized to alkylate the disulfonamides, e.g., (3). The
tetrasulfonamide (4) is treated with HBr/HOAc/PhOH to remove the
mesitylenesulfonamide protecting groups. The resulting bromide is
converted to the corresponding HCl salts. Thus, the limitations
associated with the availability of the starting tetraamine have
been eliminated, as well as removal of the tosyl protecting group.
In addition, the earlier method was limited to terminal primary
alkyl groups because of poor yields when alkylating with secondary
and tertiary halides.
[0044] Linear Polyamine, Non-Methylene Insulators
[0045] The above methodology has been successfully applied to the
synthesis of, e.g., trans-1,4-diaminocyclohexane DESPM analog (FIG.
3). The cyclic disulfonamide (2) which is obtained by sulfonation
of trans-1,4-cyclohexanediamine (1) is alkylated with two
equivalents of halosulfonamide (3, n=3). Finally, the amine
protecting groups of tetrasulfonamide (4) are easily removed by
treatment with phenol and HBr as above to provide cyclic spermine
analog (5).
[0046] Piperidine Polyamine Anti-Neoplastic Synthesis
[0047] Tetraamines in which the terminal nitrogens are incorporated
into piperidine rings can also be prepared using
mesitylenesulfonyl-protected segments, as shown in FIGS. 4 and
5.
[0048] 4-(Aminomethyl)piperidine (1) was converted into
bis-sulfonamide (2) which was alkylated with 1,4-dibromobutane (0.5
equivalent)/NaH/DMF to complete the polyamine framework (3).
Reductive removal of the sulfonamide protecting groups in (3) was
accomplished with 30% HBr in HOAc/PhOH, generating bicyclic DEHSPM
analog (4) (FIG. 4).
[0049] The corresponding 5-4-5 bicyclic polyamine was synthesized
from the inside out (FIG. 5). Crystalline
N,N'-bis-(mesitylenesulfonyl)putrescine (1) was alkylated at both
ends with mesitylenesulfonate (2), a solid derived from
4-piperidineethanol and mesitylenesulfonyl chloride (MesSO.sub.2Cl)
in pyridine. Deprotection of sulfonamide (3) with HBr as usual gave
the larger bicyclic spermine homolog (4).
[0050] Synthesis of Unsymmetrical Polyamine Anti-Neoplastics
[0051] The development of a tri-protected diamine reagent (FIG. 6)
permits the efficient synthesis of tetraamines which are
unsymmetrical with respect to both their outer methylene chains and
terminal alkyl substituents. This methodology eliminates the
limitation in earlier routes that the terminal alkyl substituents
must be identical. Furthermore, terminal monoethyl polyamines (FIG.
8), which are useful as standards in studies of diethyl analog
metabolism, can also be generated in a systematic way.
[0052] N-(tert-Butoxycarbonyl)-N-mesitylenesulfonylamide
(BOCNHSO.sub.2Mes) (1), a di-protected ammonia, was alkylated with
4-chlorobutyronitrile (NaH/DMF) to give (2). The cyano group of (2)
was hydrogenated with Raney nickel in methanolic ammonia, resulting
in primary amine (3). Both the tertbutoxycarbonyl and
mesitylenesulfonyl amine protecting groups were stable to these
reduction conditions. Attachment of a second mesitylenesulfonyl
functionality to amine (3) under bi-phasic conditions generated the
reagent, N-(tert-butoxy-carbonyl-
)-N,N'-bis(mesitylenesulfonyl)putrescine (4). It is noteworthy that
this route is flexible, as well, in that an
.omega.-chloroalkanenitrile of any length can be employed in the
alkylation of BOCNHSO.sub.2Mes (1).
[0053] Applications of the tri-protected diamine reagent to
unsymmetrical polyamine preparation are shown in FIGS. 7 and
Reagent (1) was deprotonated and alkylated with
N-(ethyl-amino)tetramethylene unit (2), providing (3) (FIG. 7).
After removal of the tert-butoxycarbonyl group under mild acidic
conditions (TFA, CH.sub.2Cl.sub.2) to produce (4), the other
nitrogen of the putrescine reagent was elaborated with
N-(ethylamino)-trimethylene segment (5). Unmasking of the amino
groups of (6) generated tetraamine (7), in which the outer chains
are unequal.
[0054] N-Ethyl trisulfonamide (3) (FIG. 8) was alkylated with
N-(4-bromobutyl)-N-(tert-butyl)mesitylenesulfonamide (2) to afford
masked polyamine (4). Treatment of (4) with HBr/HOAc/PhOH cleanly
removed the tert-butyl group, as well as the sulfonamides, giving
N.sup.1-ethylhomospermine (5), a polyamine analog metabolite. Only
the sulfonamides were cleaved using sodium and liquid ammonia to
provide the unsymmetrically dialkylated homospermine derivative
(6).
[0055] The invention is illustrated by the following non-limiting
examples.
EXAMPLE 1
Bis-N.N'-(2-mesitylenesulfonyl)-trans-1.4-cyclohexanediamine [(2a)
FIG. 3]
[0056] A solution of 2-mesitylenesulfonyl chloride (12.15 g, 55.0
mmol) in 100 ml CH.sub.2C1.sub.2 was slowly dripped into a solution
of trans-1,4-diaminocyclohexane (2.92 g, 25.0 mmol) in 100 ml 1 N
NaOH solution, which had been cooled to 0.degree. C. The mixture
was stirred at 0.degree. C. for 30 min. and at room temperature
overnight. The solid was filtered out and washed with water and
ethanol, to give 10 g (2a) (82%): mp >300.degree. C.; NMR
(DMSO-d.sub.6) .delta.0.97-1.23 (m, 4H), 1.40-1.63 (m, 4H), 2.20
(s, 6H), 2.50 (s, 12H), 2.63-2.83 (m, 2H).
N-Ethyl-N- (4 -bromobutyl mesitylenesul fonamide [(3b) FIG. 3]
[0057] Nail (0. 792 g, 80%, 26.4 mmol) was added into a solution of
N-ethyl-(2-mesitylenesulfonylamide) (5 g, 22.0 timol) in 60 ml DMF,
which had been cooled to 0.degree. C. The mixture was stirred at
0.degree. C. for 30 min., and 1,4-dibromobutane (31.5 ml, 261.4
mmol) was added. The solution was warmed to room temperature for 30
min. and then heated to 80.degree. C. overnight. The DMF was
removed, and the residue was treated with 40 ml water, followed by
the extraction with CH.sub.2Cl.sub.2 (50 ml.times.4). The
extractions were dried over anhydrous sodium sulfate, and the
solvent was rotovapped. The crude oil was purified by silica gel
column chromatography with 10/1 hexanes/EtOAc as an eluant, to
provide 5.85 g (3b) (73%) as an oil; NMR (CDCl.sub.3) .delta.1.07
(t, J=12, 3H), 1.60-1.83 (m, 4H), 2.27 (s, 3H), 2.57 (s, 6H),
3.07-3.37 (m, 6H), 6.90 (s, 2H). Anal. calcd. for
C.sub.15H.sub.24BrNO.sub.2: C-49.73; H-6.68; N-3.87. Found:
C-49.78; H-6.72; N-3.88.
N,N'-Bis{4-[N"-ethyl,N"(2-mesitylenesulfonyl)]-aminobutyl}-trans-1,4-N,N'--
bis[(2-mesitylenesulfonyl)-amino]cyclohexane [(4a) FIG. 3]
[0058] NaH (206.6 mg, 80%, 6.89 mmol) was added into a solution of
(2a) (1.5 g, 3.13 mmol) in 40 ml DMF, which had been cooled to
0.degree. C. The solution was stirred at 0.degree. C. for 30 min.,
and the solution of (3b) (2.5 g, 6.89 mmol) in 20 ml DMF was slowly
added at 0.degree. C. Then the mixture was stirred at 0.degree. C.
for 20 min., room temperature for 30 min. and 70.degree. C.
overnight, respectively, following the procedure of (3b) above, the
residue of which was purified by column chromatography with 5%
ethanol in chloroform as an eluant, to give 0.93 g (4a) (29%) as an
oil; NMR (CDCl.sub.3) 1.00 (t, J=12, 6H), 1.13-1.43 (m, 4H),
1.73-1.93 (m, 4H), 2.27 (s, 12H), 2.57 (s, 24H), 2.83-3.23 (m,
12H), 3.35-3.67 (m, 2H), 6.90 (s, 8H). Anal. calcd. for
C.sub.52H.sub.76N.sub.4O.sub.8S.sub.4: C-61.63; H-7.56, N -5.53.
Found: C-61.72; H-7.59; N-5.56.
N,N'-Bis[4-(N"-ethylamino)butyl]-trans-1,4-cyclohexanediamine
tetrahydrochloride [(5a) FIG. 3]
[0059] Phenol (2 g, 21.3 mmol) and 20 ml 30% HBr-HOAc were added
into a solution of (4a) (720 mg, 0.69 mmol) in 25 ml
CH.sub.2Cl.sub.2, and the solution was stirred at room temperature
for 24 hours. The solution was diluted with 60 ml H.sub.2O, and the
CH.sub.2Cl.sub.2 layer was separated from the aqueous layer and the
aqueous layer was washed by CH.sub.2Cl.sub.2 (40 ml.times.5). The
water was removed, and the residue was dissolved in 10 ml H.sub.2O,
basified to pH>12 by the NaOH solution, extracted by CHCl.sub.3
(40 ml.times.5) and dried over sodium sulfate. The salt was
filtered out and the solvent was rotovapped. The oil was dissolved
in 50 ml EtOH, and 1 ml concentrated HCl acid was added. The EtOH
was removed and 320 mg crude solid was recrystallized from the
mixture of H.sub.2O and EtOH to produce 127 mg (5a) (40%) as nice
crystal. NMR (D.sub.2O) 1.30 (t, J=12, 6H), 1.50-1.67 (m, 4H),
1.67-1.97 (m, 8H), 2.07-2.40 (m, 4H), 2.90-3.40 (m, 14H). Anal.
calcd. for C.sub.18H.sub.44Cl.sub.4N.sub.4: C-47.17; H-9.68;
N-12.22. Found: C-47.01; H-9.67; N-12.13.
EXAMPLE 2
N,N'-Bis(2,4,6-trimethylbenzenesulfonyl)-4-(aminomethyl)piperidine
[(2) FIG. 4]
[0060] A solution of 2-mesitylenesulfonyl chloride (19.49 g, 89.1
mmol) in CH.sub.2Cl.sub.2 (100 ml) was added to
4-(aminomethyl)-piperidine (1) (5.15 g, 45.1 mmol) in 1 N NaOH (100
ml) at 0.degree. C. After the addition was complete, the biphasic
mixture was stirred for 24 hours (0.degree. C. to room
temperature). The layers were separated and the aqueous portion was
extracted with CHCl.sub.3 (2.times.). The combined organic phase
was washed with 0.5 N HCl (200 ml) and H.sub.2O (100 ml), dried
with sodium sulfate and evaporated in vacuo. Recrystallization from
aqueous ethanol produced 18.72 g (88%) of (2) as plates: mp
158.5-160.degree. C.; NMR (CDCl.sub.3/TMS) .delta.0.8-2.0 (m, 5H),
2.25 (s, 6H), 2.46-2.93 (m+2s, 16H), 3.37-3.65 (m, 2H), 4.67 (t,
1H, J=6), 6.90 (s, 4H). Anal. calcd. for
C.sub.24H.sub.34N.sub.2O.sub.4S.sub.2: C-60.22; H-7.16; N-5.85.
Found: C-60.31; H-7.19; N-5.86.
N,N'-1,4-Bis(2,4,6-trimethylbenzenesulfonyl)-butanediylbis[4-(2,4,6-trimet-
hylbenzenesulfonyl)-piperidinemethanamine [(3) FIG. 4]
[0061] Sodium hydride (80% in oil, 1.411 g, 47.0 mmol) was added to
(2) (18.43 g, 38.5 mmol) and NaI (0.146 g, 0.97 mmol) in DMF (165
ml) at 0.degree. C. The suspension was stirred for 13/4 hours at
room temperature under nitrogen. 1,4-Dibromobutane (2.2 ml, 18.4
mmol) was added by syringe, and the reaction mixture was heated at
84.degree. C. for 19 hours. After cooling to 0.degree. C., H.sub.2O
(200 ml) was cautiously added to quench residual NaH, followed by
extraction with CHCl.sub.3 (300 ml, 2.times.100 ml). The combined
organic phase was washed with 1% Na.sub.2SO.sub.3 (100 ml) and
H.sub.2O (2.times.100 ml), dried with sodium sulfate and evaporated
under high vacuum. Recrystallization from EtOAc/CHCl.sub.3 gave
13.00 g (70%) of (3) as an amorphous solid: mp 202-203.5.degree.
C.; NMR (CDCl.sub.3/TMS) .delta.0.75-1.90 (m, 14H), 2.25 (s, 12H),
2.40-3.18 (m+2s, 36H), 3.3-3.6 (m, 4H), 6.87 (s, 8H). Anal. calcd.
for C.sub.52H.sub.74N.sub.4O.sub.8S.s- ub.4: C-61.75; H-7.37;
N-5.54. Found: C-61.49; H-7.39; N-5.43.
N,N'-1,4-Butanediylbis(4-piperidine-methanamine) [(4) FIG. 4]
[0062] 30% HBr in acetic acid (100 ml) was added over 10 min. to a
solution of (3) (5.34 g, 5.28 mmol) and phenol (18.97 g, 0.202 mol)
in CH.sub.2C1.sub.2 (75 ml) at 0.degree. C. The reaction was
stirred for 24 hours (0.degree. C. to room temperature) and cooled
to 0.degree. C. Distilled H.sub.2O (120 ml) was added, followed by
extraction with CHC.sub.2 (3.times.100 ml). The aqueous layer was
evaporated under high vacuum. The residue was basified with 1 N
NaOH (12 ml) and 50% (w/w) NaOH (20 ml) with ice cooling, followed
by extraction with CHCl.sub.3 (10.times.50 ml), while adding NaCl
to salt out the aqueous layer. Organic extracts were dried with
sodium sulfate and evaporated. The residue was taken up in ethanol
(200 ml), acidified with concentrated HCl (3.5 ml) and solvents
were removed under vacuum. Tetrahydrochloride salt was
recrystallized with 7% aqueous EtOH to furnish 1.318 g (58%) of (4)
as a white solid. NMR (D.sub.2O)/TSP) .delta.1.19-2.23 (m, 14H),
2.8-3.6 (m, 16H). Anal. calcd. for C.sub.16H.sub.38Cl.sub.4N.sub.4:
C-44.87; H-8.94; N-13.08. Found: C-44.77; H-9.00; N-13.00.
EXAMPLE 3
N,N'-Bis(2,4,6-trimethylbenzenesulfonyl)-1,4-butanediamine [(1)
FIG. 5]
[0063] 2-Mesitylenesulfonyl chloride (54.40 g, 0.249 mol) in
CH.sub.2Cl.sub.2 (300 ml) was added to 1,4-diaminobutane (11.34 g,
0.129 mol) in 1 N NaOH (300 ml) at 0.degree. C., and the biphasic
mixture was stirred for 24 hours at room temperature. Organic
solvent was evaporated and 2.4 N HCl (250 ml) was added to the
combined portions. Solid was filtered, washed with water (250 ml)
and recrystallized from aqueous ethanol to give 50.46 g (90%) of
(1) as needles: mp 156.5-157.5.degree. C.; NMR (CDCl.sub.3/TMS)
.delta.1.36-1.60 (m, 4H), 2.27 (s, 6H), 2.57 (s, 12H), 2.69-2.96
(m, 4H), 4.65 (t, 2H, J=6), 6.89 (s, 4H). Anal. calcd. for
C.sub.22H.sub.32N.sub.2O.sub.4S.sub.2: C-58.38; H-7.13; N-6.19.
Found: C-58.31; H-7.19; N-6.14.
N,O-Bis(2,4,6-trimethylbenzenesulfonyl)-4-piperidineethanol [(2)
FIG. 5]
[0064] 2-Mesitylenesulfonyl chloride (24.78 g, 0.113 mol) in
pyridine (60 ml) was added all at once to 4-piperidine-ethanol
(5.58 g, 43.2 mmol) in pyridine (25 ml) at -16.degree. C.; the
temperature rose to -11.degree. C. The flask was stored in the
refrigerator at 5.5.degree. C. for 44 hours under argon. The
reaction mixture was poured into ice (1 kg) and after 3 hours,
16.00 g (75%) of (2) as a yellow solid was filtered off: mp
93.5-94.degree. C.; NMR (CDCl.sub.3/TMS) .delta.1.4-2.1 (m, 7H1),
2.27 (s, 6H), 2.44-2.96 (m+s, 14H), 3.37-3.69 (m, 2H), 3.97 (t, 2H,
J=5), 6.90 and 6.93 (2s, 4H). Anal. calcd. for
C.sub.25H.sub.35NO.sub.5S.sub.2: C-60.82; H-7.15; N-2.84; Found:
C-60.90; H-7.13; N-2.85.
N,N'-1,4-Bis(2,4,6-trimethylbenzenesulfonyl)-butanediylbis[4-(2,4,6-trimet-
hylbenzenesulfonyl)-piperidinemethanamine [(3) FIG. 5]
[0065] Sodium hydride (80% in oil, 0.783 g, 26.1 mmol) was added to
(1) (5.15 g, 11.4 mmol) and NaI (0.376 g, 2.5 mmol) in DMF (140 ml)
at 0.degree. C. The suspension was stirred for 23 min. at room
temperature, followed by the introduction of (2) (15.84 g, 32.1
mmol). The reaction mixture was heated at 58-67.degree. C. for 18
hours and then poured into H2O (300 ml), followed by extraction
with CHCl.sub.3 (4.times.100 ml). The combined extracts were washed
with saturated NaHCO.sub.3 (100 ml), 1% NaHSO.sub.3 (100 ml) and
H.sub.2O (100 ml), dried with sodium sulfate and evaporated under
high vacuum. Column chromatography on silica gel eluting with 1 to
2% CH.sub.3OH/CHCl.sub.3 furnished 10.03 g (85%) of (3) as an
amorphous solid: NMR (CDCl.sub.3/TMS) .delta.0.8-2.0 (m, 18H),
2.08-2.71 (m+3s, 40H), 2.8-3.5 (m, 12H), 6.87 (s, 8H). Anal. calcd.
for C.sub.54H.sub.78N.sub.4O.sub.8S.sub.4.H.sub.2O: C-61.33;
H-7.62; N-5.30. Found: C-61.50; H-7.44; N-5.33.
N,N'-1,4-Butanediylbis(4-piperidineethanamine [(4) FIG. 5]
[0066] 30% HBr in acetic acid (180 ml) was added over 30 min. to a
solution of (3) (9.83 g, 9.45 mmol) and phenol (33.38 g, 0.355 mol)
in CH.sub.2Cl.sub.2 (135 ml) at 0.degree. C. The reaction was
stirred for 24 hours (0.degree. C. to room temperature) and cooled
to 0.degree. C. Distilled H.sub.2O (200 ml) was added, followed by
extraction with CH.sub.2Cl.sub.2 (2.times.100 ml). The aqueous
portion was evaporated under high vacuum. The residue was basified
with 1 N NaOH (50 ml) and then 50% (w/w) NaOH (10 ml) (ice
cooling), followed by extraction with CHCl.sub.3 (10.times.), while
adding NaCl to salt out the aqueous layer. Organic extracts were
dried over sodium sulfate and evaporated. Concentrated HCl (5 ml)
in ethanol (300 ml) was added to the residue, and solvents were
removed under vacuum. Tetrahydrochloride salt was recrystallized in
3% aqueous EtOH to give 2.91 g (68%) of (4) as a white solid: NMR
(D.sub.2O/TSP) .delta.1.15-2.09 (m, 18H), 2.75-3.59 (m, 16H). Anal.
calcd. for C.sub.18H.sub.42Cl.sub.4N.sub.4: C-47.37, H-9.28, N -
12.28. Found: C-47.25, H-9.35, N-12.17.
* * * * *