U.S. patent application number 10/647639 was filed with the patent office on 2004-03-04 for peptide-based gemini compounds.
This patent application is currently assigned to SmithKline Beecham p.l.c.. Invention is credited to Camilleri, Patrick, Kremer, Andreas, Rice, Simon Quentyn John.
Application Number | 20040043939 10/647639 |
Document ID | / |
Family ID | 10823370 |
Filed Date | 2004-03-04 |
United States Patent
Application |
20040043939 |
Kind Code |
A1 |
Camilleri, Patrick ; et
al. |
March 4, 2004 |
Peptide-based gemini compounds
Abstract
New peptide-based gemini compounds comprising two linked chains:
1 each chain having: (1) a positively charged hydrophilic head,
Q.sup.1 or Q.sup.2, formed from one or more amino acids and/or
amines (2) a central portion, P.sup.1 or P.sup.2, having a
polypeptide backbone, and (3) a hydrophobic tail, R.sup.1 or
R.sup.2, the central sections of each chain being linked together
by bridge Y through residues in P.sup.1 and P.sup.2, are disclosed.
Methods for their preparation and uses are also disclosed. Such
uses include transfection of polynucleotides into cells in-vivo and
in-vitro.
Inventors: |
Camilleri, Patrick;
(Stevenage, GB) ; Kremer, Andreas; (Turnhout,
BE) ; Rice, Simon Quentyn John; (Buntingford,
GB) |
Correspondence
Address: |
GLAXOSMITHKLINE
Corporate Intellectual Property-UW2220
P.O. Box 1539
King of Prussia
PA
19406-0939
US
|
Assignee: |
SmithKline Beecham p.l.c.
|
Family ID: |
10823370 |
Appl. No.: |
10/647639 |
Filed: |
August 25, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10647639 |
Aug 25, 2003 |
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09581068 |
Jun 8, 2000 |
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09581068 |
Jun 8, 2000 |
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PCT/GB98/03652 |
Dec 8, 1998 |
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Current U.S.
Class: |
514/21.8 ;
530/330 |
Current CPC
Class: |
A61K 47/641 20170801;
C07K 5/0606 20130101; C07K 5/1019 20130101; C12N 15/88 20130101;
A61K 48/00 20130101; C07K 5/0815 20130101; A61P 31/04 20180101 |
Class at
Publication: |
514/017 ;
530/330; 514/018 |
International
Class: |
A61K 038/06; C07K
007/06; C07K 005/06 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 9, 1997 |
GB |
9726073.1 |
Claims
1. A peptide-based gemini compound comprising two linked chains:
10each chain having: (1) a positively charged hydrophilic head,
Q.sup.1 or Q.sup.2, formed from one or more amino acids and/or
amines (2) a central portion, P.sup.1 or P.sup.2, having a
polypeptide backbone, and (3) a hydrophobic tail, R.sup.1 or
R.sup.2, the central sections of each chain being linked together
by bridge Y through residues in P.sup.1 and P.sup.2.
2. A peptide-based gemini compound according to claim 1 which has
the formula (I): 11where: A.sup.1 and A.sup.5 ,which may be the
same or different, is a positively charged group formed from one or
more amino acids or amines joined together in a linear or branched
manner; A.sup.2/A.sup.6CH(NH)CO, which may be the same or
different, is derived from an amino acid; p and q, which may be the
same or different, is 0 or 1; X.sup.1/X.sup.2CH.sub.2CH(NH)CO,
which may be the same or different, is derived from cysteine
(X.sup.1/X.sup.2.dbd.S), serine or threonine
(X.sup.1/X.sup.2.dbd.O); A.sup.4/A.sup.8CH(NH)CO, which may be the
same or different, is derived from serine or threonine; Y is a
linker group or a disulphide bond when X.sup.1 and X.sup.2 is each
S; R.sup.1 and R.sup.2 are C.sub.(10-20) saturated or unsaturated
alkyl groups, and W and Z are NH, O, CH.sub.2 or S; or a salt
thereof.
3. A peptide-based gemini compound according to claim 2 wherein the
A.sup.1 and A.sup.5 groups are bonded by an amide (CONH) bond.
4. A compound according to claims 2 or 3 wherein A.sup.1/A.sup.5
are D- or L-amino acids selected from arginine, lysine, ornithine
and histidine.
5. A compound according to claims 2 to 4 wherein A.sup.1/A.sup.5
have up to 7 amino acids linked in a linear or branched chain.
6. A compound according to claim 5 wherein A.sup.1/A.sup.5 have two
or three lysines or ornithines or a combination of lysine,
ornithine, arginine and histidine.
7. A compound according to any one of claims 2 to 6 wherein the
amino acid from which the A.sup.2/A.sup.6CH(NH)CO is derived is
serine.
8. A compound according to any one of claims 2 to 7 wherein Y is
(CH.sub.2).sub.m, where m is an integer from 1 to 6.
9. A compound according to any one of claims 2 to 7 wherein Y is a
disulphide bond when X.sup.1 and X.sup.2 is each S.
10. A compound according to claim 8 or 9 wherein m is 2.
11. A compound according to any one of claims 2 to 10 wherein R is
C.sub.12 alkyl.
12. A compound according to any one of claims 2 to 11 wherein W and
Z are NH.
13. A compound according to any one of claims 2 to 12 wherein the
salt is a pharmaceutically acceptable salt.
14. A compound according to any one of claims 1 to 13 which is
symmetrical, that is A.sup.1 and A.sup.5 are the same, A.sup.2 and
A.sup.6 are the same, A.sup.4 and A.sup.8 are the same, R.sup.1 and
R.sup.2 are the same, and W and Z are the same.
15. Compound 39: 2-amino-3-{2-[2-amino-2-(
1-dodecylcarbamoyl-2-hydroxy-et-
hylcarbamoyl)-ethylsulphanyl]-ethylsulphonyl}-N-(1-dodecylcarbamoyl-2-hydr-
oxy-ethyl-)-propionamide, and derivatives thereof, compounds 40 to
58. 12
16. The compound: 13
17. The compound: 14
18. The compound: 15
19. The compound: 16
20. The compound: 17
21. The use of a gemini-based peptide compound as defined in any
one of claims 1 to 20 in enabling transfection of DNA or RNA or
analogs thereof into a eukaryotic or prokaryotic cell in vivo or in
vitro.
22. The use of a peptide-based gemini compound according to claim
21 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.
23. The use according to claim 22 wherein the neutral carrier is
dioleyl phosphatidylethanolamine (DOPE).
24. The use according to claim 22 wherein the complexing reagent is
PLUS reagent.
25. The use according to claim 22 wherein the complexing reagent is
a peptide comprising mainly basic amino acids.
26. The use according to claim 25 wherein the peptide consists of
basic amino acids.
27. The use according to claim 25 or 26 wherein the basic amino
acids are selected from lysine and arginine.
28. The use according to claim 26 wherein the peptide is polylysine
or polyornithine.
29. A method of transfecting polynucleotides into cells in vivo for
gene therapy, which method comprises administering peptide-based
gemini compounds of any one of claims 1 to 20 together with, or
separately from, the gene therapy vector.
30. The use of a peptide-based gemini compound of any one of claims
1 to 20 to facilitate the transfer of a polynucleotide or an
anti-infective compounds into prokaryotic or eukaryotic organism
for use in anti-infective therapy.
31. The use of a peptide-based gemini compound of any one of claims
1 to 20 to facilitate the adhesion of cells in culture to each
other or to a solid or semi-solid surface.
32. A process for preparing peptide-based gemini compounds of claim
1 or 2 which process comprises adding amino acids or peptides to
2-amino-3-{2-[2-amino-2-(1-dodecylcarbamoyl-2-hydroxy-ethylcarbamoyl)-eth-
ylsulphanyl]-ethylsulphonyl}-N-(1-dodecylcarbamoyl-2-hydroxy-ethyl-)-propi-
onamide.
Description
[0001] This invention relates to newly identified peptide-based
gemini surfactant compounds, to the use of such compounds and to
their production. The invention also relates to the use of the
peptide-based gemini 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, 39-46, 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 the peptide-based gemini compounds
comprising two linked chains: 2
[0007] each chain having:
[0008] (1) a positively charged hydrophilic head, Q.sup.1 or
Q.sup.2, formed from one or more amino acids and/or amines;
[0009] (2) a central portion, P.sup.1 or P.sup.2, having a
polypeptide backbone; and
[0010] (3) a hydrophobic tail, R.sup.1 or R.sup.2;
[0011] the central sections of each chain being linked together by
bridge Y through residues in P.sup.1 and P.sup.2.
[0012] Preferably the central portion is made up of two or three
amino acids, P.sup.a (optional), P.sup.b and P.sup.c, in which:
[0013] P.sup.a is a D- or L-amino acid, preferably hydrophilic,
such as threonine or serine,
[0014] P.sup.b is preferably D- or L-cysteine, serine or threonine,
and
[0015] P.sup.c is preferably D- or L-serine or threonine and is
linked to R.sup.1 or R.sup.2.
[0016] Preferred compounds of the present invention include
compounds of the formula (I): 3
[0017] where:
[0018] A.sup.1 and A.sup.5 ,which may be the same or different, is
a positively charged group formed from one or more amino acids or
amines joined together in a linear or branched manner and
preferably bonded by an amide (CONH) bond;
[0019] A.sup.2/A.sup.6CH(NH)CO, which may be the same or different,
is derived from an amino acid, preferably serine;
[0020] p and q, which may be the same or different, is 0 or 1;
[0021] X.sup.1/X.sup.2CH.sub.2CH(NH)CO, which may be the same or
different, is derived from cysteine (X.sup.1/X.sup.2.dbd.S), serine
or threonine (X.sup.1/X.sup.2.dbd.O);
[0022] A.sup.4/A.sup.8CH(NH)CO, which may be the same or different,
is derived from serine or threonine;
[0023] Y is a linker group, preferably (CH.sub.2).sub.m where m is
an integer from 1 to 6, most preferably 2, and may be a disulphide
bond when X.sup.1 and X.sup.2 is each S;
[0024] R.sup.1 and R.sup.2 are C.sub.(10-20) saturated or
unsaturated alkyl groups, and W and Z are NH, O, CH.sub.2 or S;
or
[0025] a salt, preferably a pharmaceutically acceptable salt
thereof.
[0026] Preferably, the compound is symmmetrical, that is A.sup.1
and A.sup.5 are the same, A.sup.2 and A.sup.6 are the same, A.sup.4
and A.sup.8 are the same, R.sup.1 and R.sup.2 are the same, and W
and Z are the same.
[0027] Representative examples of A.sup.1/A.sup.5 include D- or
L-amino acids selected from arginine, lysine, ornithine and
histidine, preferably lysine, or amines such as spermine and
spermidine. Up to seven amino acids and /or amines may be linked in
a linear or branched chain. Prefered examples include groups having
two or three lysines or ornithines or a combination of lysine,
ornithine, arginine and histidine, for instance:
COCH(NHR)(CH.sub.2).sub.4NHCO(NH.sub.2)(CH.sub.2).sub.4NH.sub.2
[0028] or
COCH(NHR)(CH.sub.2).sub.3NHCO(NH.sub.2)(CH.sub.2).sub.3NH.sub.2
[0029] or
COCH(NHR)(CH.sub.2).sub.4NHCO(NH.sub.2)(CH.sub.2).sub.3NH.sub.2
[0030] in which R is H or NHCO(NH.sub.2)(CH.sub.2).sub.4NH.sub.2 or
NHCO(NH.sub.2)(CH.sub.2).sub.3NH.sub.2
[0031] Preferably, --X.sup.1--Y--X.sup.2-- is
--SCH.sub.2CH.sub.2S-- or --OCH.sub.2CH.sub.2O--
[0032] Preferably, R.sup.1 and R.sup.2 is each a C.sub.12-C.sub.20
alkyl group, for instance C.sub.12.
[0033] Preferably, W and Z is NH, thereby forming a further amide
(CONH) bond.
[0034] Compounds of the present invention may be prepared from
readily available starting materials using synthetic peptide
chemistry well known to the skilled person. For prefered compounds
of the present invention a useful intermediate is the compound:
4
[0035] which is synthesised in a multi-stage process beginning, for
instance, with the construction of the di-cysteine part and
subsequently building up the hydrophilic head by attaching a serine
moiety at the carboxyl group of each cysteine moiety, using
standard peptide chemistry, and then attaching the hydrocarbon
chains to the carboxyl group of the serine moiety using a standard
an-ide forming reaction well known to those skilled in the art.
This intermediate can then be taken through to compounds of formula
(I) by further reaction at the nitrogens of the cysteine
residues.
[0036] Another aspect of the invention relates to methods for using
the peptide-based gemini compounds. Such uses include facilitating
the transfer of oligonucleotides and polynucleotides into cells for
antisense, 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] The following definitions are provided to facilitate
understanding of certain terms used frequently herein.
[0042] "Amino acid" refers to dipolar ions (zwitterions) of the
form +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. 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.
[0043] "Polynucleotide" generally refers to any polyribonucleotide
or polydeoxribonucleotide, 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.
[0044] "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.
[0045] The invention will now be described by way of the following
descriptions and examples.
[0046] Descriptions
[0047] Description 1. Bisthioether 3
[0048] A IL 3-necked flask equipped with mechanical stirrer, reflux
condenser and dropping funnel was flushed with N.sub.2 directly
into the flask through the condenser. A solution of 31.3 g (0.20
mole) L-cysteine.hydrochloride.xH.sub.2O (1) in 100 ml (degassed
ultrasonic for 10 minutes) was added to the flask. A degassed
solution of 34 g (0.40 mole) NaHCO.sub.3 in 300 ml H.sub.2O was
added, followed by the dropwise addition (30 minutes) of a degassed
solution of 18.8 g (8.6 ml; 0.10 mole) 1,2-dibromoethane (2) in
100m] EtOH. After another 30 minutes the mixture was heated to
65-70.degree. C. and stirred, still under N.sub.2, for another 3
hours (within 1 minutes precipitation started). The mixture was
cooled to 20.degree. C., filtered, rinsed with 30 ml H.sub.2O and
with 100 ml acetone (2.times.). After drying 19.4 g white solid was
obtained which still contained some free cysteine (.sup.1H NMR).
The solid was suspended in 250 ml 2.5% NH.sub.4OH and 25%
NH.sub.4OH was added until a clear solution was obtained. To this
solution 15 mg KCN was added and the mixture was stirred for 30
minutes. The solution was acidified to pH 6 using HOAc and stirred
for 30 minutes whilst cooling to 5.degree. C. The solid was
collected, rinsed with H.sub.2O (100 ml), acetone (2.times.100 ml)
and dried, yielding 18.1 g (68%) 3 as a white solid.
[0049] Description 2. Boc-L-Leucine 5
[0050] 19.7 g (0.15 mole) L-Leucine 4 was suspended in 200 ml
H.sub.2O and 6.75 g (0.17 mole) NaOH was added. The clear solution
was cooled to <10.degree. C. and a solution of 36 g (0.165 mole)
(BOC).sub.2O in 100 ml THF was added dropwise keeping
T<10.degree. C. (30 minutes). After stirring for 4 hours at room
temperature the mixture was acidified to pH 2 by adding 1N HCl. The
mixture was extracted with EtOAc (250, 100 and 100 ml), the
combined organic layers were dried on Na.sub.2SO.sub.4 and
evaporated, yielding 40 g (>100%) 5 as a colorless oil which
contained some THF but was used as such.
[0051] Description 3. Boc-L-Leucine-OSuc 7
[0052] 40 g crude 5 (max. 0.15 mole) was dissolved in 400 ml THF
(distilled prior to use from LAH) under N.sub.2. After addition of
17.3 g (0.15 mole) N-hydroxysuccinimide (6) and 30.9 g (0.15 mole)
DCC the mixture was stirred for 3.5 hours at room temperature. The
mixture was filtered over a P.sub.2 glassfilter, the filter was
rinsed with 50 ml THF and the filtrate was evaporated. The residue
was dissolved in 400 ml refluxing isopropylether, the solution was
filtered hot and the filtrate was placed at 4.degree. C. for 20
hours. The solid was collected, rinsed with 50 ml IPE and dried,
yielding 32 g (65%) 7 as a white solid.
[0053] Description 4. Compound 8
[0054] 8.05 g (30mmole) 3 was suspended in 200 ml H.sub.2O and
after addition of 8.3 g (60 mmole) K.sub.2CO.sub.3 the mixture was
heated to get a clear solution. After cooling to room temperature a
solution of 19.7 g (60 mmole) 7 in 200 ml THF was added at once.
The mixture was stirred at room temperature for 20 hours, followed
by acidification to pH 6 (30% HCl). After filtration the filtrate
was acidified to pH 2-3 (30% HCl) and extracted with CHCl.sub.3
(250, 100 and 100 ml). The combined organic layers were once washed
with brine (50 ml), dried on Na.sub.2SO.sub.4 and evaporated,
yielding 23 g crude 8 as a white foam which was used as such.
[0055] Description 5. Compound 9
[0056] 23 g crude 8 was dissolved in 200 ml EtOAc, cooled to
0.degree. C. and HCl-gas was bubbled through for 1 hour, followed
by stirring at this temperature for another hour. The solid was
collected, rinsed with ether and dried under vacuo over KOH. This
yielded 15.4 g (91%) 9 as a hygroscopic nearly white solid.
[0057] Description 6. Compound 10
[0058] 17.0 g (30 mmole) 9 dissolved in 250 ml H.sub.2O and after
cooling to <10.degree. C. in an ice/waterbath 7.2 g (180 mmole)
NaOH was added. After stirring for 10 minutes at room temperature a
solution of 14.4 g (66 mmole) lauroylchloride in 50 ml THF was
added dropwise in 5 minutes. The mixture was stirred for 20 hours
and extracted with hexame (2.times.150 ml). A 3-layer system formed
and the lower 2 layers were acidified to pH 1-2 (1 N HCl) and
extracted with ether (200, 100, 100 and 50 ml). The combined ether
layers were dried on MgSO4 and evaporated, yielding 24 g (93%) 10
as an oil/foam. This material was used as such.
[0059] Description 7. Compound 11
[0060] 24 g (max 27.5 mmole) 10 dissolved in 400 ml THF (distilled
from LAH) under N.sub.2. After addition of 6.33 g (55 mmole)
N-hydroxysuccinimide (6) and 11.33 g (55 mmole) DCC the mixture was
stirred at room temperature for 20 hours. The mixture was filtered
over a large P.sub.2 glassfilter, another 250 ml THF was added to
speed up filtration. The filter was rinsed with another 100 ml THF.
The filtrate was evaporated yielding 40 g white solid and this
crude material was recristallized from 400 ml IPA. Stirred 1 hour
0.degree. C. and collected. After drying 22.3 g (77%) 11 was
obtained as white solid.
[0061] Description 8. Compound 13
[0062] 542 mg (2.2 mmole) H.Arg.NH.sub.2.2HCl (12) was dissolved in
15 ml H.sub.2O and 304 mg (2.2 mmole) K.sub.2CO.sub.3 was added. A
solution of 1.05 g (1.0 mmole) 11 in 15 ml THF was added at once
and the mixture was stirred at room temperature for 20 hours. Most
of the THF was evaporated and an oil formed in the waterlayer. This
suspension was extracted with ether (6.times.50 ml), the combined
ether layers were dried on MgSO.sub.4 and evaporated, yielding 9500
mg (82%) 13 free amine as a yellow solid. Treatment of this
material with HCl-gas in EtOAc/CH.sub.2C. 12 gave 13 as a yellow
solid.
[0063] Description 9. Compound 15
[0064] 5.25 g (5.0 mmole) 11 dissolved in 50 ml THF (some heating
was needed) and a solution of 1.31 g (10.5 mmole) Taurine (14) and
1.45 g (10.5 mmole) K.sub.2CO.sub.3 in 50 ml H2O was added at once.
After stirring at room temperature for 20 hours most of the THF was
removed by evaporation and 300 ml MeOH was added. Mixture placed at
-20.degree. C. for 20 hours, solid collected, rinsed with MeOH and
dried. This yielded 3.3 g 15 contaminated with
N-hydroxysuccinimide., which was combined with 700 mg impure
material of an other run. This 4.0 g was recrystallized from 200 ml
MeOH+50 ml H.sub.2O. Some solid was removed by filtration and the
clear filtrate was placed at -20.degree. C. for 2 hours, the solid
was colected, rinsed with MeOH and dried. This yielded 2.0 g 15 as
an off white solid. A 2.sup.nd crop of 800 mg was obtained from the
filtrate.
[0065] Description 10. Compound 16
[0066] 1.05 g (1.0 mmole) 11 was dissolved in 20 ml THF and a
solution of 275 mg (2.2 mmole) 2-aminoethylphosphonic acid and 300
mg (2.2 mmole) K.sub.2CO.sub.3 in 20 ml H.sub.2O was added at once.
After stirring at room temperature for 20 hours most of the THF was
removed by evaporation and the aqueous solution was freeze dried.
This yielded a white solit which was recrystallized from 20 ml MeOH
and placed at -20.degree. C. for 20 hours. The solid was collected
and dried yielding 150 mg. .sup.1H NMR showed it to be
N-hydroxysuccinimide. The filtrate was evaporated and the remaining
yellow oil was dissolved in 10 ml refluxing MeOH and after addition
of 20 ml IPA placed at -20.degree. C. for 4 hours. Solid was
collected and dried, yielding 260 mg.
[0067] The filtrate was evaporated, dissolved in 20ml EtOAc and
placed at -20.degree. C. for 20 hours. The solid was collected.
[0068] Description 11. Compound 17
[0069] 1.05 g (1.0 mmole) 11 was dissolved in 20 ml THF and a
solution of 310 mg (2.2 mmole) O-phosphocolamine and 300 mg (2.2
mmole) K.sub.2CO.sub.2 in 20 ml H.sub.2O was added at once. After
stirring at room temperature for 20 hours most of the THF was
removed by evaporation and the aqueous solution was freeze dried.
The white solid was recrystallized from 20 ml MeOH and placed at
-20.degree. C. for 20 hours. The solid was collected, rinsed with
MeOH and dried. This yielded 310 mg white solid. No product.
[0070] The filtrate was evaporated and the remaining yellow solid
was dissolved in 10 ml MeOH, 20 ml IPA was added and the mixture
was placed at -20.degree. C. for 20 hours. Solid collected, 20-30
mg
[0071] Filtrate evaporated, residue dissolved in 25 ml EtOAc,
placed at -20.degree. C. and after 20 hours the solid was
collected.
[0072] Description 12. Boc-Glycine Boc-Glycine 19
[0073] 18.8 g (0.25 mole) Glycine 18 was suspended in 250 ml
H.sub.2O and 11 g (0.275 mole) NaOH was added. The clear solution
was cooled to <10.degree. C. and a solution of 60 g (0.165 mole)
(BOC).sub.2O in 250 ml THF was added dropwise keeping
T<10.degree. C. (20 minutes). After stirring for 20 hours at
room temperature the mixture was acidified to pH 1 by adding 1N
HCl. The mixture was extracted with EtOAc (250, 100 and 100 ml),
the combined organic layers were dried on MgSO.sub.4 and
evaporated, yielding 47.5 g (>100%) 5 as a colourless oil which
contained some THF but was used as such.
[0074] Description 13. Boc-Glycine-OSuc 20
[0075] 47.5 g crude 19 (max. 0.25 mole) was dissolved in 500 ml THF
(distilled prior to use from LAH) under N.sub.2. After addition of
30 g (0.26 mole) N-hydroxysuccinimide (6) and 53.5 g (0.26 mole)
DCC at <10.degree. C., the mixture was stirred for 20 hours at
room temperature. Now the mixture was filtered over 1 cm celite on
a P2 glassfilter, the filter was rinsed with 200 ml THF and the
filtrate was evaporated. The crude material (56 g) was
recrystallized from refluxing isopropylether/THF (600 ml 1:1), the
solution was filtered hot and the filtrate was stirred at 0.degree.
C. for 3 hours. The solid was collected, rinsed with 50 ml IPE and
dried, yielding 16.4 g (24%) 20 as a white solid. Filtrate
evaporated and stored.
[0076] Description 14. Compound 21
[0077] 8.05 g (30 mmole) 3 was suspended in 200 ml H.sub.2O and
after addtion of 8.3 g (60 mmole) K.sub.2CO.sub.3 the mixture was
heated to get a clear solution. After cooling to <40.degree. C.
a solution of 16.3 g (60 mmole) 20 in 200 ml THF was added in 4
portions within 2 minutes. The mixture was stirred at room
temperature for 72 hours, followed by acidification to pH 6 (30%
HCl). After filtration the filtrate was acidified to pH 2-3 (30%
HCl) and extracted with CHCl.sub.3 (250, 100 and 100 ml). The
combined organic layers were once washed with brine (50 ml), dried
on MgSO.sub.4 and evaporated, yielding 15.8 g (90%) 21 as a white
foam which was used as such.
[0078] Description 15. Compound 22
[0079] 15.8 g (27 mmole) crude 21 was dissolved in 300 ml EtOAc and
HCl-gas was bubbled through for 1 hour, followed by stirring at
0.degree. C. in an ice/waterbath and a solution of 12.2 g (56
mmole) lauroylchloride in 50 ml THF was added at once. The mixture
was stirred for 20 hours and extracted with hexane (2.times.100
ml). The water layer was acidified to pH 1-2 (1 N HCl) and
extracted with ether (3.times.150 ml). A solid formed during
extraction, which was collected and dried. This yielded 7.5 g (39%)
23 white solid.
[0080] The filtrate was evaporated and the remaining slurry was
stirred in 250 ml Et.sub.2O. An attempt to collect the solid failed
and addition of 25 ml MeOH gave a clear solution. This solution was
placed at -20.degree. C. for 20 hours. The solid was collected,
rinsed with ether and dried.
[0081] Description 16. Compound 24
[0082] 7.47 g (10 mmole) 23 dissolved in 200 ml THF (distilled from
LAH) under N.sub.2. After addition of 2.53 g (22 mmole)
N-hydroxysuccinimide (6) and 4.53 g (22 mmole) DCC the mixture was
stirred at room temperature for 72 hours. The mixture was filtered
over a 1 cm layer celite on a large P.sub.2 glassfilter (very
slow). The filtrate was evaporated yielding 1.90 g 24 as a
foam.
[0083] The filer was rinsed with 300 ml dioxane and the filtrate
was evaporated yielding 6.9 g 24 as a foam. Total yield 8.8 g
(94%).
[0084] Description 17. Compound 25
[0085] 542 mg (2.2 mmole) H.Arg.NH.sub.2.2HCl (12) was dissolved in
15 ml H.sub.2O and 750 mg (5.4 mmole) K.sub.2CO.sub.3 was added. A
solution of 941 mg (1.0 mmole) 24 in 15 ml THF was added at once
and the mixtrue was stirred at room temperature for 20 hours. Most
of the THF was evaporated and the waterlayer was extracted with
EtOAc (2.times.50 ml), the combined EtOAc layers were dried on
MgSO.sub.4 and evaporated, yielding 150 mg 25 free amine as a foam.
Both portions were combined and dissolved in CH.sub.2Cl.sub.2 and
after the addition of 75 ml EtOAc, HCl-gas was bubbled through for
1.5 hour. Now the mixture was cooled to 0.degree. C. and stirred
for another 2 hours. Attempts to collect the solid failed, so 100
ml ether was added and the mixture was stirred at room temperature
for 20 hours. The solid was collected, rinsed with ehter and dried,
yielding 520 mg 25 as a slightly brown solid.
[0086] Description 18. Compound 26
[0087] 250 mg (2.0 mmole) taurine (14) was dissolved in 15 ml
H.sub.2O and 280 mg (2.0 mmole) K.sub.2CO.sub.3 was added. Now a
solution of 941 mg (1.0 mmole) 24 in 15 ml THF was added at once
and the mixture was stirred at room temperature for 20 hours. Most
of the THF was evaporated and the aqueous solution was freeze
dried. The resulting white solid was recrystallized from 30 ml
MeOH, stirred 3 hours at 0.degree. C. and the solid was collected,
rinsed with ether and dried.
[0088] This yielded 225 mg 26 as a white solid.
[0089] The filtrate was partly evaporated and placed at -20.degree.
C. for 20 hours. The solid was collected, rinsed with ether and
dried, yieleing 210 mg 26 as a white solid. Both portions were
combined.
[0090] Description 19. Compound 27
[0091] 26.8 g (0.1 mole) 3 was suspended in 300 ml H.sub.2O and 9.6
g (0.24 mole) NaOH was added. A clear solution formed within 5
minutes, the mixture was cooled to <10.degree. C. and a solution
of 43.6 g (0.2 mole) BOC.sub.2O in 300 ml THF was added dropwise in
30 minutes. The mixture was stirred at room temperature overnight.
After addition of a solution of 2.5 g (0.06 mole) NaOH in 25 ml
H.sub.2O and 15 g (0.07 mole) BOC.sub.2O in 75 ml THF the mixture
was stirred for another 18 hours.
[0092] The mixtue was acidified to pH 2 by adding 2N HCl and after
addition of 300 ml brine, extracted with THF (3.times.400 ml) and
EtOAc (2.times.300 ml) The combined organic layers were dried on
MgSO.sub.4 and evaporated, yielding 42 g white solid. This solid
was recrystallized from MEK/pentane, stirred at room temperature
for 2 hours and placed at -20.degree. C. for 2 hours. The solid was
collected and dried, yielding 37.8 g (81%) 27 as a white solid.
[0093] Description 20. Compound 28
[0094] 8.9 g (19 mmole) 27 was dissolved in 300 ml THF (from LAH)
under N.sub.2 and 4.37 g (38 mmole) 6 and 7.38 g (38 mmole) DCC
were added. After stirring at room temperature for 18 hours the
mixture was filtered over 1 cm celite, the filter was rinsed with
another 300 ml THF and the filtrate was evaporated yielding 11.1 g
(88%) 28 as a white solid.
[0095] Description 21. Compound 29
[0096] 2.7 g (20.6 mmole) L-leusine (4) and 2.8 g (20.3 mmole)
K.sub.2CO.sub.2 were dissolved in 100 ml H.sub.2O and a suspension
of 6.6 g (10 mmole) 28 in 50 ml dioxane was added. The mixture was
stirred at room temperature for 20 hours and most of the dioane was
removed by evaporation. The aqueous solution was extracted with 50
ml ether and acidified to pH 1 by addition of 30% HCl. Now the
mixture was extracted with CHCl.sub.3 (150, 100 and 50 ml), the
combined organic layers were washed with brine (200 ml), dried on
MsSO4 and evaporated, yielding a white foam which was stripped with
THF to get 7.5 g (>100%) 29 as a solid white foam.
[0097] Description 22. Compound 30
[0098] 7.5 g crude 29 (max. 10 mmole) was dissolved in 100 ml THF
(from LAH) under N.sub.2, 2.3 g (20 mmole) N-hydroxysuccinimide (6)
and 4.12 g (20 mmole) DCC were added and the mixture was stirred at
room temperature for 20 hours. The mixture was filtered over 1 cm
celite, the filter was rinsed with THF and the filtrate was
evaporated, yielding 9.5 g white foam which was recrystallized from
75 ml IPA and placed at -20.degree. C. for 3 hours. The solid was
collected but liquified immediately on the glass filter. The oily
material was dissolved in 20 ml THF and evaporated, yielding 6.5 g
(73%) 30 as a solid white foam.
[0099] Description 23. Compound 31
[0100] 6.5 g (7.3 mmole) 30 was dissolved in 100 ml THF and after
addition of 2.78 g (15 mmole) dodecylamine the mixture was stirred
at room temperature for 18 hours. After evaporation a foam was
obtained which was dissolved in 100 ml CHCl.sub.3. The solution was
washed with H.sub.2O (2.times.75 ml), dried on MgSO4 and
evaporated, yielding 7.5 g (100%) 31 as a solid foam.
[0101] Description 24. Compound 32
[0102] 7.5 g (7.3 mmole) crude 31 was dissolved in 250 ml EtOAc
under heating and after cooling to room temperature HCl-gas was
bubbled through for 2 hours. Stirring was continued at 0.degree. C.
for 3 hours. The solid was collected, rinsed with ether and dried
under vacuo over KOH. This yielded 4.0 g (60%) 32 as a white
solid.
[0103] Description 25. Compound 33
[0104] 903 mg (1.0 mmole) 32 was dissolved in 10 ml H.sub.2O under
heating (gel formed), after cooling to room temperature, 80 mg (2.0
mmole) NaOH dissolved in 2 ml H.sub.2O was added. A suspension was
formed and THF was added until a clear solution was obtained. Now a
solution of 572 mg (2 mmole) BOC-.beta.-alaOSuc (42) in 5 ml THF
was added and the mixture was stirred at room temperature for 5
hours. Most of the THF was removed by evaporation, another 30 ml
H.sub.2O was added and after stirring for another hour the solid
was collected, rinsed with 10 ml H.sub.2O and dried. This yielded
1.0 g (85%) 33 as an off white solid.
[0105] Description 26. Compound 34
[0106] 1.0 g (0.85 mmole) 33 suspended in 25 ml EtOAc and 25 ml
CH.sub.2Cl.sub.2 added to get a clear solution. HCl-gas bubbled
through for 1.5 hour and stirred at 0.degree. C. for another 2
hours. No solid had formed so most of the CH.sub.2Cl.sub.2 was
removed by evaporation and stirring at 0.degree. C. was continued
for anothe 30 minutes. The solid was collected, partly by
filtration (very slow), mainly be centrifugation. Total yield after
drying 810 mg (91%) 34 as a yellow solid.
[0107] Description 27. Compound 36
[0108] 4.2 g (40 mmole) L-serine 35 and 5.53 (40 mmole)
K.sub.2CO.sub.3 were disslved n 300 ml H.sub.2O and a suspension of
12.8 g (max. 19 mmole) 28 in 300 ml THF was added. The mixture was
stirred at room temperature for 72 hours and most of the THF was
removed by evaporation. The aqueous solution was acidified to pH 1
by addition of 1N HCl. The mixture was extracted with
CH.sub.2Cl.sub.2+15% MeOH (250, 100 and 100 ml), the combined
organic layers were dried on MgSO.sub.4 and evaporated, yielding
8.5 g (70%) 36 as a white solid foam which was used as such.
[0109] Description 28. Compound 37
[0110] 8.5 g (max. 13.2 mmole) 36 was dissolved in 200 ml THF (from
LAH) under N.sub.2 and after addition of 3.46 g (30 mmole)
N-hydroxysuccinimide (6) and 6.2 g (30 mmole) DCC the mixture was
stirred at room temperature for 24 hours. The mixture was filtered
over 1 cm celite, the filter was rinsed with 50 ml THF and
evaporated. This yielded 12.5 g (>100%) 37 as a white foam which
was used as such.
[0111] Description 29. Compound 38
[0112] 12.5 g crude (max 13.2 mmole) 37 was dissolved in 200 ml THF
and stirred with 5.0 g (27 mmole) dodecylamine at room temperature
for 48 hours. The THF was removed by evaporation and the residue
was dissolved in 250 ml CHCl.sub.3 and extracted with brine
(2.times.150 ml). The combined brine layers were extracted with 50
ml CHCl.sub.3 and the combined CHCl.sub.3 layers were dried on
MgSO.sub.4 and evaporated. This yielded 15.4 g (>100%) 38 as a
nearly white solid which was used as such.
EXAMPLES
Example 1
[0113] Compound 39
2-amino-3-{2-[2-amino-2-(1-dodecylcarbamoyl-2-hydroxy-e-
thylcarbamoyl)-ethylsulphanyl]-ethylsulphonyl}-N-(1-dodecylcarbamoyl-2-hyd-
roxy-ethyl-)-propionamide
[0114] 15.4 g (max. 13.2 mmole) 38 dissolved in 400 ml EtOAc and
HCl-gas was bubbled through for 1.5 hour. The mixture was stirred
at 0.degree. C. for 2 hours, the solid was collected, rinsed with
ether and dried, yielding 9.9 g (88%) 39 as a white solid. 5
Example 2
[0115] Compound 40
[0116] 4.25 g (5 mmole) 39 was dissolved in 100 ml H.sub.2O with
heating and after cooling to <40.degree. C. a solution of 460 mg
(10 mmole) NaOH in 10 ml H.sub.2O was added. A suspension formed
and THF was added until a clear solution was obtained (150 ml).
Next 2.86 g (10 mmole) BOC-.beta.-alaOSu (42) was added and the
mixture was stirred at room temperature for 20 hours. Most of THF
was removed by evaporation, another 100 ml H.sub.2O was added and
the mixture was stirred at 0.degree. C. for 3 hours. The solid was
collected, rinsed with 20 ml H.sub.2O and dried. This yielded 5.2 g
(93%) 40 as a nearly white solid.
Example 3
[0117] Compound 41
[0118] 5.2 g (4.6mmole) 40 was dissolved in 100 ml CH.sub.2Cl.sub.2
and 200 ml EtOAc was added. HCl-gas was bubbled through for 1.5
hour and stirring was continued at 0.degree. C. for 2 hours. The
solid was collected, rinsed with ether and dried, yielding 4.7 g
(100%) 41 as off white solid.
Example 4
[0119] Compounds 42 and 43
[0120] After neutralization of compounds 34 and 41 using 2 eq. of
NaOH in MeOH both compounds were treated with (CH.sub.2O).sub.n and
NaCNBH.sub.3 under N.sub.2 for 18 hours. In both reactions complex
mixtures were formed, probably due to alkylation on amide nitrogen
as well.
Example 5
[0121] Compound 44
[0122] 332 mg (0.39 mmole) 39 was dissolved in 15 ml H.sub.2O under
heating and after cooling to <40.degree. C. a solution of 33 mg
(0.83 mmole) NaOH in 1 ml H.sub.2O was added. A white suspension
formed and THF was added until a clear solution was obtained (25
ml). To this solution 499 mg (0.78 mmole) BOC-Arg(Z).sub.2--OSu
(47) was added and the mixture was stirred at room temperature for
20 hours. Most of the THF was evaporated and another 15 ml H.sub.2O
was added. After 2 hours stirring the solid was collected, rinsed
with H.sub.2O and dried, yielding 700 mg (98%) 44 as a white
solid.
Example 6
[0123] Compound 45
[0124] 100 mg (0.05 mmole) 44 was dissolved in 20 ml HOAc and 500
mg 10% Pd on Carbon (0.5 mmole Pd) was added. The mixture was
stirred under H.sub.2 (5 bar) for 48 hours. The mixture was
filtered over 1 cm celite, the filter was rinsed with 10 ml HOAc
and the filtrate was evaporated. This yielded 100 mg crude 45 as a
green oil.
Example 7
[0125] Compound 46
[0126] 100 mg crude 45 (max 0.05 mmole) was disslved in 10 ml
CH.sub.2Cl.sub.2 and 10 ml EtOAc was added. HCl-gas was bubbled
through for 1 hour and the mixture was stirred 18 hours at room
temperature. Most of the CH.sub.2Cl.sub.2 was removed by
evaporation, 30 ml ether was added and the mixture was stirred at
0.degree. C. for 1 hour. No crystalline material had formed so the
mixture was evaporated, yielding 75 mg crude 46 as a yellow
oil.
Example 8
[0127] Compound 49
[0128] 850 mg (1.0 mmole) 39 was dissolved in 20 ml H.sub.2O and
after 88 mg (2.2 mmole) NaOH was added a suspension formed. Now THF
was added until a clear solution was obtained (30 ml) and 974 mg
(2.2 mmole) BOC.sub.2LysOSuc (compound 48) was added. After
stirring at room temperature for 20 hours most of the THF was
removed by evaporation, another 20 ml H.sub.2O was added and the
mixtrue was stirred for 2 hours. The solid was collected, rinsed
with H.sub.2O and dried, yielding 1.35 g (90%) 49 as a white
solid.
Example 9
[0129] Compound 50
[0130] 500 mg 49 was dissolved in 25 ml CH.sub.2Cl.sub.2 and after
addition of 25 ml EtOAc HCl-gas was bubbled through for 1 hour, the
mixture was stirred at 0.degree. C. for 1.5 hour. An attempt to
collect the solid failed, 40 ml ether was added and stirring was
continued for 18 hours. The solid was collected, rinsed with ehter
and dried, yielding 290 mg 50 as a white solid.
Example 10
[0131] Compound 51
[0132] 425 mg (0.43 mmole) 41 was dissolved in 20 ml H.sub.2O and
44 mg (1.1 mmole) NaOH was added. A suspension formed and THF was
added until a clear solution was obtained (25 ml). After addition
of 487 mg (1.1 mmole) 48 the mixture was stirred at room
temperature for 20 hours. Most of the THF was evaporated, another
20 ml H.sub.2O was added and after stirring for 1.5 hour the solid
was collected, rinsed with H.sub.2O and dried. This yielded 750 mg
51 as a white solid.
Example 11
[0133] Compound 52
[0134] 250 mg 51 was dissolved in 30 ml CH.sub.2Cl.sub.2 and after
addition of 30 ml EtOAc, HCl-gas was bubbled through for 1 hour,
the mixture was stirred at 0.degree. C. for 1.5 hour. The solid was
collected, rinsed with ether and dried, yielding 120 mg 52 as a
white salt.
Example 12
[0135] Compound 57 (SucOSerLysBOC.sub.2)
[0136] 4.43 g (10 mmole) BOC.sub.2LysOSuc (48) was dissolved in 50
ml THF and a solution of 1.16 g (11 mmole) L-serine and 1.52 g (11
mmole) K.sub.2CO.sub.3 in 50 ml H.sub.2O was added immediately. The
mixture was stirred at room temperature for 72 hours. Most of the
THF was removed by evaporation and the remaining slurry was
acidified to pH 2 by the addition of 1M HCl and extracted with
CHCl.sub.2 (2.times.75 ml). The combined organic layers were dried
(Na.sub.2SO.sub.4) and evaporated, yielding 57 as a white solid
foam which was used as such in example 43.
Example 13
[0137] Compound 54
[0138] 850 mg (1.0 mmole) 39 was dissolved in 30 ml H.sub.2O and 88
mg (2.2 mmole) NaOH was added, followed by the addition of 30 ml
THF to get a clear solution. A solution of 1.5 g (max. 2.3 mmole)
57 in 30 ml THF was added immediately and the solution was stirred
at room temperature for 48 hours. Most of the THF was removed by
evaporation, another 30 ml H.sub.2O was added and stirring was
continued for 1 hour. Because no solid had formed, the mixture was
extracted twice with 75 ml EtOAc/ether (2:1). The combined organic
layers were dried (Na.sub.2SO.sub.4) and evaporated yielding the
BOC-protected intermediate as a solid foam.
[0139] This foam was dissolved in 25 ml CH.sub.2Cl.sub.2 and 50 ml
EtOAc was added. HCl gas was bubbled through the clear solution for
1 hour and stirring was continued at 0.degree. C. for another hour.
The salt was collected, rinsed with ether and dried under vacuum,
yielding 1.15 g (85%) 54 as a slightly brown solid.
Example 14
[0140] Compound 55
[0141] 1.18 g (1.0 mmole) 50 was dissolved in 25 ml H.sub.2O and
176 mg (4.4 mmole) NaOH was added, followed by the addition of 30
ml THF to get a clear solution. 974 mg (2.2 mmole) 48 were added
and the mixture stirred at room temperature for 48 hours. Most of
the THF was removed by evaporation, another 50 ml H.sub.2O was
added and the mixture was stirred for 2 hours. Because no solid was
formed the mixture was extracted with ether (2.times.100 ml), the
combined organic layers were dried (Na.sub.2SO.sub.4) and
evaporated, yielding 2 g of the BOC-protected intermediate as a
solid foam. The foam was dissolved in 50 ml EtOAc and HCl-gas was
bubbled through the solution for 1 hour and stirring was continued
at 0.degree. C. for another hour. The salt was collected, rinsed
with ether and dried under vacuum, yielding 1.15 g (76%) 55 as a
nearly white solid. 6
Example 15
[0142] Compound 56
[0143] Compound 56 was synthesised as for compound 55 except that
1.95 g (4.4 mmole) 48 was used. This yielded 1.1 g (60%) 56 as an
off white solid. 7
Example 16
[0144] Compounds 57, 58 and 59
[0145] Compounds 57, 58 and 59 are synthesised in a similar manner
to the compounds described above. Compound 39, or an intermediate
equivalent to compound 39 but having different saturated or
unsaturated hydrocarbon chains, is combined with an ornithine
compound using synthetic peptide chemistry well known to the
skilled person. 89
[0146] It will be appreciated by a person skilled in the art that
in the formulae shown in the examples above, the hydrogen atoms
have been omitted from the N, C and O atoms, where appropriate, for
clarity.
Example 17
[0147] Transfection of Recombinant Plasmid Expressing Luciferase
into HEK293 Cells Using Peptide-based Gemini Compounds.
[0148] All tissue culture reagents were obtained from Life
Technologies Inc. HEK 293 cells were seeded at 2-3.times.10.sup.5
cells per well in Nunc six-well culture plates, 24 hours prior to
transfection. The cells were seeded in 2 mls Dulbeccos Modified
Eagle medium containing Earles salts and supplemented with 10% v/v
foetal bovine serum (=complete medium). The cells were grown at
37.degree. C. in 5% CO.sub.2 in a humidified atmosphere. 6 ug DNA
("luciferase control plasmid" from Promega Corp.) were dissolved in
100 ul serum free medium (OPTI-MEMO). The peptide-based gemini
compounds were made up at 1 mg/ml in tissue culture grade water and
then diluted in OPTI-MEM.RTM. to the appropriate concentration to a
final volume of 100 ul. The DNA and gemini solutions were mixed (to
a total volume of 200 ul; final concentrations of 5, 25, 50, 100,
150, 200, 250 and 300 ug/ml) and left at room temperature for 15
minutes. The DNA/gemini mix was placed onto the cells in each well
and left in contact for 18-20 hours. The cells were then washed
twice with phophate buffered saline prior to 1 ml of fresh complete
medium being added. Cells were incubated for a further 24 hours
prior to lysis and luciferase activity assayed.
[0149] All luciferase activity assays were performed using the
Canberra Packard (Berkshire, UK) Luclite kit according to the
manufacturer's instructions with the exception that the cells in
each well were resuspended in 1 ml lysis buffer and 100 ul aliquots
mixed with 100 ul of the luciferase substrate. The reaction mix was
left for a 15 minutes adaptation period in the dark before counting
for 5 minutes in a Top Count scintillation counter. Luciferase
activity is measured as counts per second (CPS) from the
scintillation counter. Four independent counts were taken per
well.
[0150] Control transfections were set up with no DNA, CaPO.sub.4,
an anionic gemini compound (1) and the commercially available
lipofection reagents LipofectAmine.TM. and Lipotaxi.TM. at the
manufacturers recommended concentrations (10, 25, 50, 75, 125 ug/ml
and 175, 250, 325, 400 and 500 ug/ml respectively).
[0151] The results (FIG. 1) clearly show that the cationic
peptide-based gemini compounds (54), (55) and (56) are very
efficient agents for facilitating the transfection of the
luciferase plasmid into HEK293 cells at concentrations above 150
ug/ml. In particular compound (54) peaks at 250 ug/ml with a mean
count (of 4 independent counts) of over 70,000 cps. Compound (55)
is most effective at 300 ug/ml with an average count of about
45,000 cps. Compound (56) is most effective at 200 ug/ml with an
average count of about 50,000 cps. In contrast the `no DNA`
negative control gives a background count as do the anionic gemini
(1) and the cationic geminis (50 and 52). The CaPO.sub.4
transfection shows a very low count of about 2,000 cps. In
comparison FIG. 2 shows the results for the Lipofectamine
transfections which at peak efficiency gave only 12,500 cps (125
ug/ml) and Lipotaxi 2,500 cps (at 175 ug/ml and 325 ug/ml).
Example 18
[0152] Transfection of Recombinant Plasmid Expressing Luciferase
into CHO-K1 Cells Using Peptide-Based Gemini Compounds.
[0153] CHO-K1 cells (ATCC: CRL-9618) were seeded into
T.sub.25-culture flasks (Corning-Costar Buckinghamshire, UK), at
7.times.105 cells per flask, 24 hours prior to transfection. The
CHO-K1 cells were seeded in 5 ml MEM alpha medium with
ribonucleosides and deoxyribonucleosides and supplemented with
1.times. L-glutamine and 10% v/v foetal bovine serum (complete
medium). The cells were grown at 37.degree. C. in 5% CO.sub.2 in a
humidified atmosphere.
[0154] For transfection, 5 ug DNA (luciferase control plasmid) was
incubated with the gemini compounds in water (final volume 400
.mu.l). The peptide-based gemini compounds were made up at 1 mg
ml.sup.-1 in tissue culture grade water and then diluted to the
appropriate concentration to a total volume of 200 ul. Following a
30 minute room temperature incubation, 2.6 ml OPTI-MEM.RTM. medium
was added and the solution placed on the cells. Following an
overnight incubation at 37.degree. C., the transfection solution
was replaced with complete medium and the cells incubated at
37.degree. C. 24 hours post transfection the cells were detached
from the flask and seeded into 96-well plates at a density of
0.5.times.10.sup.5 cells per well and incubated for a further 24
hours at 37.degree. C. Luciferase reporter gene assays were
performed according to the manufacturers instructions (Roche
Diagnostics, Mannheim, Germany) approximately 48 hours post
transfection. The plates were left for a 15 minutes adaption period
in the dark before counting for 60 seconds in a TopCount NXT
counter (Canberra Packard). An average of eight wells were counted
per transfection.
[0155] Control transfections were set up with no DNA, an anionic
gemini compound and the commercially available reagent
LipofectAmine PLUS.TM..
[0156] The results, shown in FIG. 3, demonstrate that the cationic
peptide-based compounds 54, 55, and 56 are very efficient agents
for facilitating the transfection of the luciferase plasmid into
CHO-K1 cells. Using the conditions described above, compound 54
peaks at 30 mM with a mean count in excess of 1.4.times.10.sup.5
counts per second (cps). Compound 55 is most effective at 30 mM
with an average count of about 2.4.times.10.sup.5 cps. Compound 56
is most effective at 40 mM with an average count of about
1.9.times.10.sup.5 cps. In contrast negative controls gave a
negligable count.
Example 19
[0157] Transfection of Recombinant Plasmid Expressing Luciferase
into CHO-K1 Cells Using Peptide-Based Gemini Compounds in
Combination with Various Supplements.
[0158] The transfection ability of the gemini compounds could be
further enhanced by the addition of a neutral carrier, for example,
dioleyl phosphatidylethanolamine (DOPE) (Farhood, H., et al (1985)
Biochim. Biophys. Acta 1235 289) or a complexing reagent, for
example, PLUS compound (Life Technologies Inc.).
[0159] FIG. 4 shows, for example, a 9-fold increase of luciferase
activity at a 2:1 ratio of compound 55 and DOPE. Transfection
mediated by compound 55 with DOPE in a 2:1 ratio and the addition
of 11.6 ul of PLUS compound lead to a mean count of
6.5.times.10.sup.5 cps representing a 12-fold increase of
luciferase activity in comparison to compound 55 alone. Incubation
of the PLUS compound with the DNA and combination with compound 55
alone also lead to a 4-fold increase.
Example 20
[0160] Use of Peptide-Based Gemini Compounds to Facilitate Adhesion
of Cells in Culture to the Culture Flask.
[0161] Using normal growth medium and culture conditions (RPMI plus
10% foetal bovine serum; 37.degree. C., 5% CO2 ) but with the
addition of 50-60 ug peptide-based gemini compound per well, it was
observed that with the suspension cell line Jurkat, cells could
attach to the bottom surface of the plastic culture vessel. In the
absence of gemini compounds, the Jurkat cells grew in
suspension.
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