U.S. patent application number 10/990819 was filed with the patent office on 2005-04-28 for novel compounds.
This patent application is currently assigned to SmithKline Becham p.I.c.. Invention is credited to Camilleri, Patrick, Guedat, Philippe, Kirby, Anthony John, Kremer, Andreas.
Application Number | 20050089493 10/990819 |
Document ID | / |
Family ID | 10855486 |
Filed Date | 2005-04-28 |
United States Patent
Application |
20050089493 |
Kind Code |
A1 |
Camilleri, Patrick ; et
al. |
April 28, 2005 |
Novel compounds
Abstract
Spermine:peptide-based surfactant compounds are disclosed. The
compounds are based on a spermine backbone with peptide groups and
optionally hydrocarbyl groups linked thereto. Uses of the
spermine:peptide-based surfactant compounds and methods for their
production are also disclosed.
Inventors: |
Camilleri, Patrick; (Harlow,
GB) ; Guedat, Philippe; (Lyon, FR) ; Kirby,
Anthony John; (Cambridge, GB) ; Kremer, Andreas;
(Leverkusen, DE) |
Correspondence
Address: |
GLAXOSMITHKLINE
Corporate Intellectual Property - UW2220
P.O. Box 1539
King of Prussia
PA
19406-0939
US
|
Assignee: |
SmithKline Becham p.I.c.
Cambridge University Technical Services Ltd.
|
Family ID: |
10855486 |
Appl. No.: |
10/990819 |
Filed: |
November 17, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10990819 |
Nov 17, 2004 |
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10018547 |
Jul 19, 2002 |
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6835712 |
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10018547 |
Jul 19, 2002 |
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PCT/GB00/02364 |
Jun 16, 2000 |
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Current U.S.
Class: |
424/70.14 ;
530/329; 530/330; 530/331 |
Current CPC
Class: |
C07K 5/06086 20130101;
C07K 5/06069 20130101; C07K 5/0215 20130101; C07C 233/36 20130101;
C07C 237/10 20130101; C12N 15/87 20130101; C07D 209/48 20130101;
C07C 211/11 20130101; A61K 48/00 20130101 |
Class at
Publication: |
424/070.14 ;
530/331; 530/329; 530/330 |
International
Class: |
A61K 007/06; A61K
007/11; C07K 007/08; C07K 007/06; C07K 005/06 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 16, 1999 |
GB |
9914045.1 |
Claims
1. A spermine:peptide-based surfactant compound having the general
structure of formula (1): 21where R.sub.1 and R.sub.3 are hydrogen
and R.sub.2 and R.sub.4, which may be the same or different, are
peptide groups formed from one or more amino acids linked together,
in a linear or branched manner, by amide (CONH) bonds and further
linked to the spermine backbone by amide bonds, having the general
formula (II): 22where p1 is 0 to 5 and p2 is 1 to 5; and the values
for p3 and p4, which may be the same or different, are from 0 to 5;
A1, A3 and A4, which may be the same or different, are amino acids
selected from serine, lysine, ornithine, threonine, histidine,
cysteine, arginine and tyrosine; and A2 is an amino acid selected
from lysine, ornithine and histidine; and R.sub.5 and R.sub.6 are
saturated or unsaturated hydrocarbyl groups having up to 24 carbon
atoms and linked to the spermine backbone by an amide or an amine
(NCH.sub.2) linkage; or where R.sub.1 and R.sub.3 are hydrogen,
R.sub.2 and R.sub.4, which may be the same or different are
saturated or unsaturated hydrocarbyl groups having up to 24 carbon
atoms and linked to the spermine backbone by amide or amine bonds,
and R.sub.5 and R.sub.6, which may be the same or different, are
peptide groups of formula (II) linked to the spermine backbone by
amide bonds; or a salt, preferably a pharmaceutically acceptable
salt thereof.
2. A spermine:peptide-based surfactant compound according to claim
1 which is symmmetrical, that is R.sub.1 and R.sub.3 are the same,
R.sub.2 and R.sub.4 are the same, and R.sub.5 and R.sub.6 are the
same.
3. A spermine:peptide-based surfactant compound according to claim
1 or 2 wherein in the peptide group of formula (I) p1 is 1 and p2,
p3 and p4 are all 0.
4. A spermine:peptide-based surfactant compound according to claim
1 or 2 wherein in the peptide group of formula (11).sub.p1 and p2
are both 1 and p3 and p4 are both 0.
5. A spermine:peptide-based surfactant compound according to claim
1 or 2 wherein in the peptide group of formula (I) p1 is 0 and p2,
p3 and p4 are all 1.
6. A spermine:peptide-based surfactant compound according to claim
1 or 2 wherein in the peptide group of formula (I) p1 and p3 are 0,
p2 is 1 and p4 is 2.
7. A spermine:peptide-based surfactant compound according to any
one of claims 1 to 6 wherein the A1 is serine.
8. A spermine:peptide-based surfactant compound according to any
one of claims 1 to 6 wherein the A2 is lysine.
9. A spermine:peptide-based surfactant compound according to claim
1 wherein the hydrocarbyl group is selected from:
--(CH.sub.2).sub.11CH.sub- .3 --(CH.sub.2).sub.13CH.sub.3
--(CH.sub.2).sub.15CH.sub.3 --(CH.sub.2).sub.17CH.sub.3
--(CH.sub.2).sub.19CH.sub.3 --(CH.sub.2).sub.23CH.sub.3
--(CH.sub.2).sub.8CH.dbd.CH(CH.sub.2).sub.5CH- .sub.3
--(CH.sub.2).sub.8CH.dbd.CH(CH.sub.2).sub.7CH.sub.3
--(CH.sub.2).sub.8CH.dbd.CHCH.sub.2CH.dbd.CH(CH.sub.2).sub.4CH.sub.3
--(CH.sub.2).sub.8(CH.dbd.CHCH.sub.2).sub.3CH.sub.3
--(CH.sub.2).sub.4CH.dbd.CH(CH.sub.2CH.dbd.CH).sub.3(CH.sub.2).sub.4CH.su-
b.3 --(CH.sub.2).sub.8CH.dbd.CH(CH.sub.2).sub.5CH.sub.3 Trans
--(CH.sub.2).sub.8CH.dbd.CH(CH.sub.2).sub.7CH.sub.3 Trans
--(CH.sub.2).sub.9CHCH.sub.3(CH.sub.2).sub.7CH.sub.3
10. A spermine:peptide-based surfactant compound according to claim
1 wherein the hydrocarbyl group is selected from:
--CO(CH.sub.2).sub.10CH.s- ub.3 --CO(CH.sub.2).sub.12CH.sub.3
--CO(CH.sub.2).sub.14CH.sub.3 --CO(CH.sub.2).sub.16CH.sub.3
--CO(CH.sub.2).sub.18CH.sub.3 --CO(CH.sub.2).sub.22CH.sub.3
--CO(CH.sub.2).sub.7CH.dbd.CH(CH.sub.2).sub- .5CH.sub.3
--CO(CH.sub.2).sub.7CH.dbd.CH(CH.sub.2).sub.7CH.sub.3
--CO(CH.sub.2).sub.7CH.dbd.CHCH.sub.2CH.dbd.CH(CH.sub.2).sub.4CH.sub.3
--CO(CH.sub.2).sub.7(CH.dbd.CHCH.sub.2).sub.3CH.sub.3
--CO(CH.sub.2).sub.3CH.dbd.CH(CH.sub.2CH.dbd.CH).sub.3(CH.sub.2).sub.4CH.-
sub.3 --CO(CH.sub.2).sub.7CH.dbd.CH(CH.sub.2).sub.5CH.sub.3 Trans
--CO(CH.sub.2).sub.7CH.dbd.CH(CH.sub.2).sub.7CH.sub.3 Trans
--CO(CH.sub.2).sub.8CHCH.sub.3(CH.sub.2).sub.7CH.sub.3
--COCHOH(CH.sub.2).sub.21CH.sub.3 23
11. The compound: 24
12. The compound: 25
13. The compound GSC1 of formula: 26
14. The compound GSC4 of formula: 27
15. The compound GSC40 of formula: 28
16. The compound GSC42 of formula: 29
17. The compound GSC2 of formula: 30
18. The compound GSC12 of formula: 31
19. The use of a spermine:peptide-based surfactant compound as
defined in any one of claims 1 to 15 in facilitating transfection
of DNA or RNA polynucleotides or analogs thereof into a eukaryotic
or prokaryotic cell in vivo or in vitro.
20. The use of a spermine:peptide-based surfactant compound
according to claim 19 wherein the compound is used in combination
with one or more supplements selected from the group consisting of:
(i) a neutral carrier; or (ii) a complexing reagent.
21. The use according to claim 20 wherein the neutral carrier is
dioleoyl phosphatidylethanolamine (DOPE).
22. The use according to claim 20 wherein the complexing reagent is
PLUS reagent.
23. The use according to claim 20 wherein the complexing reagent is
a peptide comprising mainly basic amino acids.
24. The use according to claim 23 wherein the peptide consists of
basic amino acids.
25. The use according to claim 23 or 24 wherein the basic amino
acids are selected from lysine and arginine.
26. The use according to claim 24 wherein the peptide is polylysine
or polyornithine.
27. The use according to any one of claims 19 to 26 wherein the
oligonucleotides or polynucleotides are transferred into cells to
achieve an antisense knock-out effect.
28. The use according to claim 19 wherein the oligonucleotides or
polynucleotides are transferred into cells for gene therapy.
29. The use according to claim 19 wherein the oligonucleotides or
polynucleotides are transferred into cells for genetic immunisation
(for the generation of antibodies) in whole organisms.
30. The use according to any one of claims 19 to 26 wherein the
oligonucleotides or polynucleotides are transferred into cells in
culture.
31. The use of a spermine:peptide-based surfactant compound of any
one of claims 1 to 18 to facilitate the transfer of a
polynucleotide or an anti-infective compounds into prokaryotic or
eukaryotic organism for use in anti-infective therapy.
32. The use of a spermine:peptide-based surfactant compound of any
one of claims 1 to 18 to facilitate the adhesion of cells in
culture to each other or to a solid or semi-solid surface.
33. A process for preparing spermine:peptide-based surfactant
compounds of claim 1 which process comprises adding amino acids or
peptides to a hydrocarbylated spermine backbone.
Description
[0001] This invention relates to newly identified
spermine:peptide-based surfactant compounds, to the use of such
compounds and to processes for their preparation. The invention
also relates to the use of the spermine:peptide-based surfactant
compounds to facilitate the transfer of compounds into cells for
drug delivery.
[0002] Surfactants are substances that markedly affect the surface
properties of a liquid, even at low concentrations. For example
surfactants will significantly reduce surface tension when
dissolved in water or aqueous solutions and will reduce interfacial
tension between two liquids or a liquid and a solid. This property
of surfactant molecules has been widely exploited in industry,
particularly in the detergent and oil industries. In the 1970s a
new class of surfactant molecule was reported, characterised by two
hydrophobic chains with polar heads which are linked by a
hydrophobic bridge (Deinega, Y et al., Kolloidn. Zh. 36, 649,
1974). These molecules, which have been termed "gemini" (Menger, F
M and Littau, C A, J. Am. Chem. Soc. 113, 1451, 1991), have very
desirable properties over their monomeric equivalents. For example
they are highly effective in reducing interfacial tension between
oil and water based liquids and have a very low critical micelle
concentration.
[0003] Cationic surfactants have been used inter alia for the
transfection of polynucleotides into cells in culture, and there
are examples of such agents available commercially to scientists
involved in genetic technologies (for example the reagent
Tfx.TM.-50 for the transfection of eukaryotic cells available from
Promega Corp. WI, USA).
[0004] The efficient delivery of DNA to cells in vivo, either for
gene therapy or for antisense therapy, has been a major goal for
some years. Much attention has concentrated on the use of viruses
as delivery vehicles, for example adenoviruses for epithelial cells
in the respiratory tract with a view to corrective gene therapy for
cystic fibrosis (CF). However, despite some evidence of successful
gene transfer in CF patients, the adenovirus route remains
problematic due to inflammatory side-effects and limited transient
expression of the transferred gene. Several alternative methods for
in vivo gene delivery have been investigated, including studies
using cationic surfactants. Gao, X et al. (1995) Gene Ther. 2,
710-722 demonstrated the feasibility of this approach with a normal
human gene for CF transmembrane conductance regulator (CFTR) into
the respiratory epithelium of CF mice using amine carrying cationic
lipids. This group followed up with a liposomal CF gene therapy
trial which, although only partially successful, demonstrated the
potential for this approach in humans (Caplen, N J. et al., Nature
Medicine, 1, 3946, 1995). More recently other groups have
investigated the potential of other cationic lipids for gene
delivery, for example cholesterol derivatives (Oudrhiri, N et al.
Proc. Natl. Acad. Sci. 94, 1651-1656, 1997). This limited study
demonstrated the ability of these cholesterol based compounds to
facilitate the transfer of genes into epithelial cells both in
vitro and in vivo, thereby lending support to the validity of this
general approach.
[0005] These studies, and others, show that in this new field of
research there is a continuing need to develop novel low-toxicity
surfactant molecules to facilitate the effective transfer of
polynucleotides into cells both in vitro for transfection in
cell-based experimentation and in vivo for gene therapy and
antisense treatments. The present invention seeks to overcome the
difficulties exhibited by existing compounds.
[0006] The invention relates to spermine:peptide-based surfactant
compounds having a spermine backbone and having the general
structure of formula (1): 1
[0007] where R.sub.1 and R.sub.3 are hydrogen and R.sub.2 and
R.sub.4, which may be the same or different, are peptide groups
formed from one or more amino acids linked together, in a linear or
branched manner, by amide (CONH) bonds and further linked to the
spermine backbone by amide bonds, having the general formula (II):
2
[0008] where p1 is 0 to 5 and p2 is 1 to 5, preferably 1; and the
values for p3 and p4, which may be the same or different, are from
0 to 5, preferably 0;
[0009] A1, A3 and A4, which may be the same or different, are amino
acids selected from serine, lysine, ornithine, threonine,
histidine, cysteine, arginine and tyrosine; and
[0010] A2 is an amino acid selected from lysine, ornithine and
histidine;
[0011] and R.sub.5 and R.sub.6 are saturated or unsaturated
hydrocarbyl groups having up to 24 carbon atoms and linked to the
spermine backbone by an amide or an amine (NCH.sub.2) linkage;
or
[0012] where R.sub.1 and R.sub.3 are hydrogen, R.sub.2 and R.sub.4,
which may be the same or different are saturated or unsaturated
hydrocarbyl groups having up to 24 carbon atoms and linked to the
spermine backbone by amide or amine bonds, and R.sub.5 and R.sub.6,
which may be the same or different, are peptide groups of formula
(11) linked to the spermine backbone by amide bonds; or
[0013] a salt, preferably a pharmaceutically acceptable salt
thereof.
[0014] When used herein, the term "hydrocarbyl" refers to a group
having from 1 to 24 carbon atoms which may be in a straight chain
or a branched chain and include a saturated carbocyclic ring having
from 3 to 6 carbon atoms and which chain may contain unsaturation
(double and/or triple carbon-carbon bonds).
[0015] The amide linkages between the amino acids A, A2 and A3 in
the peptide group of formula (II) are standard peptide bonds (a
bonds), unless the amino acid is a diamine, for example lysine or
ornithine, where the linkage may involve either of the two amine
groups. For example, where A1 is lysine, the linkage to the amino
acid A2 may be a standard alpha amide bond, or an epsilon (F) amide
bond involving the amine of the lysine side chain. Similarly where
A1 is ornithine the amide bond linking A1 to A2 may be an alpha
bond or a delta (8) bond that is created using the amine on the
side chain of the ornithine amino acid residue.
[0016] Preferably, the compound is symmetrical, that is R.sub.1 and
R.sub.3 are the same, R.sub.2 and R.sub.4 are the same, and R.sub.5
and R.sub.6 are the same. Symmetrical spermine:peptide-based
surfactant compounds of the invention are "gemini" surfactants.
[0017] In a preferred embodiment A1 in the group of formula (II) is
serine or threonine, prefereably serine. Preferably A3 and A4 in
the group of formula (II) are lysine, ornithine, histidine or
arginine.
[0018] In a further preferred embodiment the hydrocarbyl group is
selected from: 3
[0019] In another preferred embodiment the hydrocarbyl group is
selected from:
[0020] --(CH.sub.2).sub.11CH.sub.3
[0021] --(CH.sub.2).sub.13CH.sub.3
[0022] --(CH.sub.2).sub.15CH.sub.3
[0023] --(CH.sub.2).sub.17CH.sub.3
[0024] --(CH.sub.2).sub.19CH.sub.3
[0025] --(CH.sub.2).sub.23CH.sub.3
[0026] --(CH.sub.2).sub.8CH.dbd.CH(CH.sub.2).sub.5CH.sub.3
[0027] --(CH.sub.2).sub.8CH.dbd.CH(CH.sub.2).sub.7CH.sub.3
[0028]
--(CH.sub.2).sub.8CH.dbd.CHCH.sub.2CH.dbd.CH(CH.sub.2).sub.4CH.sub.-
3
[0029] --(CH.sub.2).sub.8(CH.dbd.CHCH.sub.2).sub.3CH.sub.3
[0030]
--(CH.sub.2).sub.4CH.dbd.CH(CH.sub.2CH.dbd.CH).sub.3(CH.sub.2).sub.-
4CH.sub.3
[0031] --(CH.sub.2).sub.8CH.dbd.CH(CH.sub.2).sub.5CH.sub.3
Trans
[0032] --(CH.sub.2).sub.8CH.dbd.CH(CH.sub.2).sub.7CH.sub.3
Trans
[0033] --(CH.sub.2).sub.9CHCH.sub.3(CH.sub.2).sub.7CH.sub.3
[0034] Compounds of the present invention may be prepared from
readily available starting materials using synthetic peptide
chemistry well known to the skilled person. The scheme shown in
FIGS. 1a and 1b shows a general process for the synthesis of the
compounds of the invention wherein the hydrocarbyl groups are
linked to the spermine moiety by amine bonds and the scheme shown
in FIGS. 2a and 2b shows a general process for the synthesis of the
compounds of the invention wherein the carbonyl groups are linked
to the spermine moiety by amide bonds.
[0035] The processes shown in FIGS. 1 and 2 are for the synthesis
of symmetrical, that is "gemini", spermine:peptide-based
surfactants. Non-symmetrical spermine:peptide-based surfactants of
the invention can be prepared by introducing asymmetry, for example
at the primary amines of spermine, by using different protecting
groups. Suitable nitrogen protecting groups are well known in the
art and are described in, for example, "Protective Groups in
Organic Chemistry" (T. W. Greene, Wiley-Interscience, New York, 2nd
Edition, 1991).
[0036] Another aspect of the invention relates to methods for using
the spermine:peptide-based surfactant compounds. Such uses include
facilitating the transfer of DNA or RNA polynucleotides, or analogs
thereof, into a eukaryotic or prokaryotic cell in vivo or in vitro.
These uses include facilitating transfection of polynucleotides to
achieve an antisense knock-out effect, for gene therapy and genetic
immunisation (for the generation of antibodies) in whole organisms.
Other uses include employing the compounds of the invention to
facilitate the transfection of polynucleotides into cells in
culture when such transfer is required, in, for example, gene
expression studies and antisense control experiments among others.
The polynucleotides can be mixed with the compounds, added to the
cells and incubated to allow polynucleotide uptake. After further
incubation the cells can be assayed for the phenotypic trait
afforded by the transfected DNA, or the levels of mRNA expressed
from said DNA can be determined by Northern blotting or by using
PCR-based quantitation methods for example the Taqman.RTM. method
(Perkin Elmer, Connecticut, USA). Compounds of the invention offer
a significant improvement, typically between 3 and 6 fold, in the
efficiency of cellular uptake of DNA in cells in culture, compared
with compounds in the previous art. In the transfection protocol,
the gemini compound may be used in combination with one or more
supplements to increase the efficiency of transfection. Such
supplements may be selected from, for example:
[0037] (i) a neutral carrier, for example dioleyl
phosphatidylethanolamine (DOPE) (Farhood, H., et al (1985) Biochim.
Biophys. Acta 1235 289);
[0038] (ii) a complexing reagent, for example the commercially
available PLUS reagent (Life Technologies Inc. Maryland, USA) or
peptides, such as polylysine or polyornithine peptides or peptides
comprising primarily, but not exclusively, basic amino acids such
as lysine, ornithine and/or arginine. The list above is not
intended to be exhaustive and other supplements that increase the
efficiency of transfection are taken to fall within the scope of
the invention.
[0039] In still another aspect, the invention relates to the
transfer of genetic material in gene therapy using the compounds of
the invention.
[0040] Yet another aspect of the invention relates to methods to
effect the delivery of non-nucleotide based drug compounds into
cells in vitro and in vivo using the compounds of the
invention.
[0041] In a further aspect, the invention relates to methods to
facilitate the transfer of a polynucleotide or an anti-infective
compounds into prokaryotic or eukaryotic organism for use in
anti-infective therapy.
[0042] The following definitions are provided to facilitate
understanding of certain terms used frequently herein.
[0043] "Amino acid" refers to dipolar ions (zwitterions) of the
form .sup.+H.sub.3NCH(R)CO.sub.2--. They are differentiated by the
nature of the group R, and when R is different from hydrogen can
also be asymmetric, forming D and L families. Amino acids may be
natural or un-natural amino acids. There are 20 naturally occurring
amino acids where the R group can be, for example, non-polar (e.g.
alanine, leucine, phenylalanine) or polar (e.g. glutamic acid,
histidine, arginine and lysine). In the case of un-natural amino
acids R can be any other group which is not found in the amino
acids found in nature.
[0044] "Polynucleotide" generally refers to any polyribonucleotide
or polydeoxyribonucleotide, which may be unmodified RNA or DNA or
modified RNA or; DNA. "Polynucleotides" include, without limitation
single- and double-stranded DNA, DNA that is a mixture of single-
and double-stranded regions, single- and double-stranded RNA, and
RNA that is mixture of single- and double-stranded regions, hybrid
molecules comprising DNA and RNA that may be single-stranded or,
more typically, double-stranded or a mixture of single- and
double-stranded regions. In addition, "polynucleotide" refers to
triple-stranded regions comprising RNA or DNA or both RNA and DNA.
The term polynucleotide also includes DNAs or RNAs containing one
or more modified bases and DNAs or RNAs with backbones modified for
stability or for other reasons. "Modified" bases include, for
example, tritylated bases and unusual bases such as inosine. A
variety of modifications have been made to DNA and RNA; thus,
"polynucleotide" embraces chemically, enzymatically or
metabolically modified forms of polynucleotides as typically found
in nature, as well as the chemical forms of DNA and RNA
characteristic of viruses and cells. "Polynucleotide" also embraces
relatively short polynucleotides, often referred to as
oligonucleotides.
[0045] "Transfection" refers to the introduction of polynucleotides
into cells in culture using methods involving the modification of
the cell membrane either by chemical or physical means. Such
methods are described in, for example, Sambrook et al., MOLECULAR
CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. (1989). The
polynucleotides may be linear or circular, single-stranded or
double-stranded and may include elements controlling replication of
the polynucleotide or expression of homologous or heterologous
genes which may comprise part of the polynucleotide.
[0046] The invention will now be described by way of the following
examples.
EXAMPLES
Example 1
Synthesis of GSC1
[0047] Step 1: 4
[0048] A solution of N-carbethoxyphthalimide (4 mmol, 439 mg) in
chloroform (5 ml) was added as one portion at room temperature to a
stirred solution of spermine (2 mmol, 406.7 mg) in chloroform (5
ml). The mixture was stirred for 1 hour and the solvent was
removed, the crude was dried under good vacuum to get the title
compound without further purification (Sosnovsky, G. (1986)
Zeitschrift fur Naturforschung 41b: 122-129).
[0049] Step 2: 5
[0050] The protected spermine 350 mg (0.76 mmol) was dissolved in
10 ml of dry THF, the Lauroyl chloride 3 eq (2.28 mmol) and 2 ml of
dry pyridine was added. After refluxing 30 min, the mixture was
stirred at room temperature overnight. Solvent was removed, and the
crude was dissolved in AcOEt (25 ml) and was washed with saturated
bicarbonate (2.times.20 ml). The organic phase was dried over
anhydrous sodium sulfate and evaporated. The resulting oil was
chromatographied on silica gel with AcOEt/hexane (1:1 to 4:1)
rf=0.20 (AcOEt/hexane 1:1) to yield 490 mg (84%) of bis-dodecyl as
oil which crystallized as white semi-solid crystal. .sup.1H-NMR
(CDCl.sub.3): 7.9-7.6 m (8H (--C.sub.6H.sub.4--).sub- .2); 3.7-3.6
m (4H); 3.4-3.2 m (8H); 2.3-2.1 m (4H); 2.0-1.8 m (4H); 1.6-1.4 m
(8H); 1.3-1.1 m (32H); 0.9-0.8 m (6H). .sup.13C-NMR (CDCl.sub.3):
173.1, 173.0, 172.9, 172.8, 168.3, 168.2, 134.2, 134.1, 133.9,
133.8, 132.2, 132.1, 132.0, 131.9, 123.4, 123.2, 47.6, 45.7, 45.5,
45.4, 43.5, 43.4, 35.9, 33.9, 33.2, 33.1, 30.9, 29.6, 29.5, 29.4,
29.3, 29.1, 28.3, 28.2, 27.1, 26.7, 26.4, 25.5, 25.4, 25.2, 25.1,
24.9, 22.7, 14.1.
[0051] Step 3: 6
[0052] The bis-phthalimide (400 mg, 0.48 mmol) was dissolved in 20
ml of MeOH, hydrazine monohydrate 0.4 ml was added and the mixture
was refluxed 6H. Solvent was removed, and the crude redissolved in
MeOH (20 ml) and 2 ml of concentrated HCl was added. The mixture
was refluxed for 30 min, and solvent was removed. The residue was
dissolved in aqueous NaOH 10%, the precipitate was filtrated and
wash with 3 amount of cold water. The residue was dried to give 112
mg (41%) of white powder. .sup.1H-NMR (MeOH): 3.21 t (.sup.3J=6.8
Hz, 4H); 2.6-2.5 m (8H); 2.76 t (.sup.3J=7.5 Hz, 4H); 1.7-1.5 m
(12H); 1.4-1.2 m (32H); 0.89 t (.sup.3J=6.9 Hz, 6H
(--CH.sub.3).sub.2).
[0053] Step 4: 7
[0054] The diamine 100 mg (0.176 mmol), HOBt 2 eq (0.353 mmol, 54
mg), BOC.sub.2Lys 2.4 eq (0.423 mmol, 135 mg) and DIPEA 2 eq (0.353
mmol, 62 ml) was dissolved in CH.sub.2Cl.sub.2 (20 ml). The mixture
was cooled at -10.degree. C. and DCC 2.4 eq (0.423 mmol, 87 mg) was
added. The mixture was stirred at -10.degree. C. and allowed to
reach room temperature very slowly and set aside overnight. The DCU
was removed by filtration, and solvent was evaporated off. The
crude was redissolved in AcOEt (30 ml), was washed successively 4%
NaHCO.sub.3 (2.times.15 ml), 4% citric acid (2.times.15 ml), water
(20 ml) and finally brine (20 ml). Organic phase was dried over
anhydrous sodium sulfate and was evaporated to dryness. The residue
was chromatographied on silica gel and was eluated with
hexane/AcOEt 1/1 (rf=0.01) to AcOEt/MeOH 9/1 (rf=0.6) yield 152 mg
(71%) as oil. .sup.1H-NMR (CDCl.sub.3): 7.05 sbroad (1H); 6.69
sbroad (1H); 5.6-4.7 m (6H); 4.9-4.1 m (4H); 3.7-3.0 m (16H);
2.2-2.0 m (6H); 2.0-1.0 m (88H); 0.88 t (.sup.3J=6.8 Hz, 6H
(--CH.sub.3).sub.2).
[0055] Step 5: 8
[0056] The tetra-protected Gemini 150 mg (0.12 mmol) was dissolved
in CH.sub.2Cl.sub.2/TFA 1/1 (25 ml) and was stirred at room
temperature for 0.5H. Solvent was removed to give the deprotected
compounds as a TFA salt which was exchanged on anionic Dowex
1.times.8 to get the tetrahydrochloride salt as a sticky oil. The
compound was redissolved in small amount of MeOH and precipitate
with ether, solvent was removed by decantation, this was repeated
three times, to give GSC1. .sup.1H-NMR (MeOH): 3.8-3.1 m (8H);
3.0-2.8 m (4H); 2.2-2.1 m (4H); 2.0-1.4 m (26H); 1.4-1.2 m (36H);
0.90 t (.sup.3J=6.8 Hz, 6H (--CH.sub.3).sub.2). M/z(H.sub.+):
823.75; (2H.sup.+): 412.38.
Example 2
Synthesis of GSC4
[0057] Step 1: 9
[0058] The diamine 150 mg (0.265 mmol) was dissolved in 5% aqueous
acetonitrile (10 ml) and triethylamine 2.2 eq (0.6 mmol, 80 .mu.L)
was added. The N.alpha.,N.epsilon.-bis-ter-butyl-carbamate-L
lysine-L-serine-O-succinimidate (was built by the usual peptide
synthesis) 2 eq (0.528 mmol, 280 mg) in acetonitrile (8 ml) was
added slowly. The mixture was stirred 48H at room temperature, and
solvent was removed. The crude was redissolved in AcOEt (30 ml),
was washed successively water (2.times.20 ml), 3% HCl (2.times.20
ml), water (20 ml) and finally brine (20 ml). Organic phase was
dried over anhydrous sodium sulfate and was evaporated to dryness,
to yield 252 mg (68%). .sup.1H-NMR (CDCl.sub.3): 4.9-4.1 m (4H);
3.6-3.0 m (20H); 2.2-2.0 m (8H); 2.0-1.0 m (80H); 0.88 t
(.sup.3J=6.8 Hz, 6H (--CH.sub.3).sub.2).
[0059] Step 2: 10
[0060] The tetra-protected Gemini 248 mg (0.177 mmol) was dissolved
in THF/HClconc 1/1 (25 ml) and was stirred at room temperature for
1H. Solvent was removed to give the deprotected compounds as a
sticky dark oil which was redissolved in small amount of MeOH and
precipitate with ether, solvent was removed by decantation, this
was repeated several times, to yield 120 mg (60%) GSC4 as pink
powder. .sup.1H-NMR (MeOH): 4.0-3.9 m (2H); 3.5-3.2 m; 3.1-2.9 m
(4H); 2.5-2.3 m (4H); 2.3-2.1 m (1H); 2.0-1.5 m (20H); 1.4-1.2 m
(32H); 0.90 t (.sup.3J=6.8 Hz, 6H (--CH.sub.3).sub.2).
Example 3
Synthesis of GSC40
[0061] Step 1: 11
[0062] The diamine 120 mg (0.212 mmol), HOBt 2.5 eq. (0.53 mmol, 72
mg), (BOC.sub.2Lys)Lys 2.5 eq. (0.53 mmol, 426 mg) and DIPEA 2.5
eq. (0.53 mmol, 92 .mu.l) was dissolved in CH.sub.2Cl.sub.2/THF 1/1
(20 ml). The mixture was cooled at -10.degree. C. and DCC 2.4 eq.
(0.508 mmol, 105 mg) was added. The mixture was stirred at
-10.degree. C. and allowed to reach room temperature very slowly
and set aside overnight. The DCU was removed by filtration, and the
solvent was evaporated off. The crude product was redissolved in
EtOAc (30 ml), was washed successively with 4% NaHCO.sub.3
(2.times.15 ml), 4% citric acid (2.times.15 ml), water (20 ml) and
finally brine (20 ml). the organic phase was dried over anhydrous
sodium sulfate and was evaporated to dryness. The residue was
chromatographed on silica gel and was eluated with hexane/EtOAc 1/1
(rf=0.01) to EtOAc/MeOH 9/1 (rf=0.55) yield 210 mg (46%) as oil.
.sup.1H-NMR (CDCl.sub.3): 4.9-4.6 m (2H); 4.1-3.9 m (4H); 3.5-2.9 m
(16H); 2.3-2.1 m (2H); 1.9-1.0 m (164H); 0.88 t (.sup.3J=6.8 Hz, 6H
(--CH.sub.3).sub.2).
[0063] Step 2: 12
[0064] The octa-protected gemini 200 mg (0.094 mmol) was dissolved
in THF/HClconc 1/1 (20 ml) and was stirred at room temperature for
1H. The solvent was removed to give the deprotected compounds as a
yellow oil which was redissolved in small amount of MeOH and
precipitate with ether, the solvent was removed by decantation,
this was repeated several times, to yield 130 mg (85%) GSC40 as
yellow powder.
Example 4
Synthesis of GSC41
[0065] Step 1: 13
[0066] The diamine 200 mg (0.35 mmol) was dissolved in 5% aqueous
acetonitrile (20 ml) and triethylamine 2 eq. (0.7 mmol, 0.1 ml) was
added. The
bis-(N.alpha.,N.epsilon.-bis-ter-butyl-carbamate-L-lysine)-L-l-
ysine-L-serine-O-succinimidate 2 eq (0.71 mmol, 700 mg) in
acetonitrile (10 ml) was added slowly. The mixture was stirred 48 H
at room temperature, and solvent was removed. The crude product was
redissolved in AcOEt (30 ml), was washed successively water
(2.times.20 ml), 3% HCl (2.times.20 ml), water (20 ml) and finally
brine (20 ml). The organic phase was dried over anhydrous sodium
sulfate and was evaporated to dryness, to yield 509 mg (63%).
.sup.1H-NMR (CDCl.sub.3): 4.9-4.6 m; 4.1-3.9 m (4H); 3.6-2.9 m
(22H); 2.3-2.1 m (4H); 2.0-1.0 m; 0.88 t (.sup.3J=6.8 Hz, 6H
(--CH.sub.3).sub.2).
[0067] Step 2: 14
[0068] The tetra-protected gemini 499 mg (0.216 mmol) was dissolved
in THF/HClconc 1/1 (30 ml) and was stirred at room temperature for
1H. the solvent was removed to give the deprotected compounds as a
sticky brawn oil which was redissolved in small amount of MeOH and
precipitated with ether, solvent was removed by decantation, this
was repeated several times, to yield 350 mg (90%) GSC41 as brown
powder.
Example 5
Synthesis of GSC42
[0069] Step 1: 15
[0070] The diamine 200 mg (0.35 mmol) was dissolved in 5% aqueous
acetonitrile (20 ml) and triethylamine 2 eq. (0.7 mmol, 0.1 ml) was
added. The
bis-(N.alpha.,N.epsilon.-bis-ter-butyl-carbamate-L-lysine)-L-l-
ysine-L-serine-O-succinimidate 2 eq (0.71 mmol, 700 mg) in
acetonitrile (10 ml) was added slowly. The mixture was stirred 48 H
at room temperature, and solvent was removed. The crude product was
redissolved in AcOEt (30 ml), was washed successively water
(2.times.20 ml), 3% HCl (2.times.20 ml), water (20 ml) and finally
brine (20 ml). The organic phase was dried over anhydrous sodium
sulfate and evaporated to dryness, to yield 522 mg (70%).
.sup.1H-NMR (CDCl.sub.3): 6.7-6.6 m (2H); 5.5-5.3 m (2H); 4.9-4.6 m
(4H); 4.54.3 m (4H); 4.1-3.9 m (4H); 3.5-2.9 m (20H); 2.3-2.1 m
(4H); 1.9-1.0 m; 0.88 t (.sup.3J=6.8 Hz, 6H
(--CH.sub.3).sub.2).
[0071] Step 2: 16
[0072] The tetra-protected Gemini 517 mg (0.242 mmol) was dissolved
in THF/HClconc 1/1 (30 ml) and was stirred at room temperature for
1H. The solvent was removed to give the deprotected compounds as a
sticky dark oil which was redissolved in small amount of MeOH and
precipitate with ether, solvent was removed by decantation, this
was repeated several times, to yield 346 mg (88%) GSC42 as brown
powder.
Example 6
Synthesis of GSC2
[0073] Step 1: 17
[0074] The protected spermine 1.23 g (2.65 mmol) was dissolved in
1,2-dichloroethane (25 ml) and dodecyl aldehyde 2.5 eq. (6.6 mmol,
1.5 ml) was added. After 10 nm, the sodium cyanoborohydride 6 eq.
(15.9 mmol, 1 g) was added and the mixture was stirred at room
temperature overnight. Then sodium cyanoborohydide 4.5 eq. (750 mg)
was added again and mixture was stirred overnight at room
temperature. This solution was partitioned between dichloromethane
(150 ml) and water (150 ml) and the pH was adjusted to 9. The
resulting emulsion was left in a separating funnel for 12 H. Then,
the organic phase was dried over sodium sulfate and concentrated.
The crude product was chromatographed on silica gel, eluated with
EtOAc/Hexane 1/4 to 1/2 (rf: 0.3) containing 0.1% of NEt.sub.3, to
yield 540 mg of semi-solid yellow compound (26%).
[0075] Step 2: 18
[0076] The protected spermine 200 mg (0.25 mmol) was dissolved in
methanol (10 ml) and hydrazine monohydrate 10 eq (2.5 mmol, 0.13
ml) was added. The mixture was refluxed for 8H, and the solvent was
removed. The crude product was redissolved in 10 ml of methanol and
conc. HCl (2 ml) was added, the mixture was refluxed for 30 min,
cooled in ice and filtered. The solvent was removed and the crude
product redissolved in dichloromethane (30 ml), stirred 30 min at
room temperature and the precipate was removed by filtration. The
solution was evaporated and dried under reduce pressure to yield
150 mg GSC2 as a yellow semi-solid compound (88%).
Example 7
Synthesis of GSC12
[0077] Step 1: 19
[0078] The diamine 143 mg (0.265 mmol) was dissolved in 5% aq. THF
(10 ml) and NEt.sub.3 0.6 mmol (80 ml), activated peptide 2 eq
(0.528 mmol, 282 mg) in THF (5 ml) were added. The mixture was
stirred overnight and concentrated. Then, the crude product was
dissolved in EtOAc (60 ml) and was washed successively with water
(2.times.20 ml) 3% HCl (2.times.20 ml) water (20 ml) brine (20 ml).
The organic phase was dried over sodium sulfate and the solvent was
evaporated to yield 210 mg of a sticky white compound (58%).
[0079] Step 2: 20
[0080] The tetra-Boc 200 mg (0.146 mmol) was dissolved in THF/conc.
HCl 1/1 (20 ml) and was stirred at room temperature for 1H. The
solvent was removed and the crude product was redissolved in small
amount of MeOH and precipitated with ether, cooled to -20.degree.
C., the solvent was removed by decantation, this was repeated
several times, to yield 150 mg (87%) GSC12 as a yellow powder.
Example 8
Transfection of Recombinant Plasmid Expressing Luciferase into
Cells using Spermine:Peptide-Based Surfactant Compounds
[0081] Transfection studies were performed using the adherent cell
line CHO-K1 (Puck et al. 1958). Complete medium consisted of MEM
alpha medium supplemented with 10% v/v foetal bovine serum and
1.times.L-Glutamine. All media and supplements were obtained from
Life Technologies.
[0082] Stable transfected cell lines expressing
.beta.-galactosidase were generated by cotransfection of the
plasmid pSV-.beta.-Galactosidase Control Vector (Promega) with the
plasmid Selecta Vecta-Neo (R & D Systems) in a 10:1 ratio.
Following G418 (Life Technologies) selection (0.8 mg ml.sup.-1);
candidate cell lines were tested for .beta.-galactosidase activity
(.beta.-Gal Reporter Gene Assay, chemiluminescent; Roche
Diagnostics).
[0083] In Vitro Gene Transfection.
[0084] Cells were seeded into 96-well MTP plates (Beckton
Dickinson) 16-18 hours prior to transfection at an approximate
density of 1.times.10.sup.4 cells per well. For transfection, 64 ng
of the luciferase reporter gene plasmid, pGL3-Control Vector
(Promega) per well, was incubated with various concentrations of
the spermine:peptide-based surfactant compounds and complexing
agents. After 30 minutes incubation at RT, OPTI-MEM.RTM. medium
(Life Technologies) was added to the transfection mixture and the
solution placed on the cells (final volume per well: 100 .mu.l).
Following a 3 hour or over night incubation at 37.degree. C., the
transfection solution was replaced with complete medium and the
cells incubated further at 37.degree. C. Reporter gene assays were
performed according to the manufacturer's guidelines (Roche
Diagnostics) approximately 48 hours post transfection. Luminescence
was measured in a Packard TopCount NXT Microplate Scintillation and
Luminescence Counter. For normalization purpose,
.beta.-galactosidase activity (.beta.-Gal Reporter Gene Assay,
chemiluminescent; Roche Diagnostics) was measured and luciferase
activity per .beta.-galactosidase activity was calculated.
BRIEF DESCRIPTION OF THE DRAWINGS
[0085] FIG. 1a and FIG. 1b. General sheme for synthesis of
spermine:peptide-based surfactant compounds wherein the carbonyl
groups (in this instance dodecanoyl) are linked to the spermine
moiety by amide bonds. The final compound in FIG. 1a is the first
compound in FIG. 1b.
[0086] FIG. 2a and FIG. 2b. General sheme for synthesis of
spermine:peptide-based 110 surfactant compounds wherein the
carbonyl groups (in this instance dodecanoyl) are linked to the
spermine moiety by amine bonds. The final compound in FIG. 2a is
the first compound in FIG. 2b.
[0087] FIG. 3. Transfection of CHO-K1 cells (stable transfected
with beta-galactosidase) with spermine:peptide-based gemini
surfactants GS-C-1, GS-C-2, GS-C-3, GS-C-4 and GS-C-12. Bars
represent the mean cps (counts per second) of 8 experiments.+-.the
standard error of the mean.
* * * * *