U.S. patent application number 10/434091 was filed with the patent office on 2004-02-05 for lipids.
This patent application is currently assigned to CELLTECH R & D LIMITED. Invention is credited to Baker, Terence Seward, Catterall, Catherine Fiona, Eaton, Michael Anthony William, Norman, Timothy John, Parker, David, Weir, Andrew Neil Charles.
Application Number | 20040024220 10/434091 |
Document ID | / |
Family ID | 26313458 |
Filed Date | 2004-02-05 |
United States Patent
Application |
20040024220 |
Kind Code |
A1 |
Eaton, Michael Anthony William ;
et al. |
February 5, 2004 |
Lipids
Abstract
Bipolar lipids are described which are able to form complexes
with polyanions. The lipids comprise a cationic head linked to a
hydrophobic backbone and a hydrophilic tail and are capable of self
assembly to form stable complexes in aqueous solutions. The lipids
are of particular use for the delivery of bioactive substances such
as nucleic acids to cells in vitro and especially in vivo.
Inventors: |
Eaton, Michael Anthony William;
(Oxon, GB) ; Norman, Timothy John; (Buckingshire,
GB) ; Parker, David; (Durham, GB) ; Baker,
Terence Seward; (Middlesex, GB) ; Weir, Andrew Neil
Charles; (Berkshire, GB) ; Catterall, Catherine
Fiona; (Bucks, GB) |
Correspondence
Address: |
SUGHRUE MION,PLLC
2100 Pennsylvania Avenue, N.W.
Washington
DC
20037-3213
US
|
Assignee: |
CELLTECH R & D LIMITED
|
Family ID: |
26313458 |
Appl. No.: |
10/434091 |
Filed: |
May 9, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10434091 |
May 9, 2003 |
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09646766 |
Nov 20, 2000 |
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6583301 |
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09646766 |
Nov 20, 2000 |
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PCT/GB99/01076 |
Apr 8, 1999 |
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Current U.S.
Class: |
546/329 ;
548/557; 554/105 |
Current CPC
Class: |
A61K 47/6929 20170801;
A61K 31/685 20130101; A61K 47/543 20170801; A61K 31/225 20130101;
C07C 237/10 20130101; A61P 43/00 20180101; C07C 235/12 20130101;
A61K 9/1272 20130101; C07C 271/20 20130101; C07C 235/10
20130101 |
Class at
Publication: |
546/329 ;
548/557; 554/105 |
International
Class: |
C07D 211/82; C07D
213/54; C07D 207/34; C07C 229/00 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 1, 1998 |
GB |
9821407.5 |
Apr 8, 1998 |
GB |
9807727.4 |
Claims
1. A bipolar lipid comprising a cationic head (1) a hydrophobic
backbone (2) and a hydrophilic tail (3) in which: (A) the cationic
head comprises two or more cationic centres, each centre being
covalently linked to one or more others by one or more carbon
containing spacer groups; (B) the hydrophobic backbone comprises
one or more hydrocarbon chains; and (C) the hydrophilic tail
comprises one or more hydrophilic hydrocarbons each containing two
or more atoms or groups capable of being solvated by water; each of
said components (1) to (3) being covalently linked head (1) to
backbone (2) to tail (3) and arranged such that at least one
hydrocarbon chain in the hydrophobic backbone (2) is covalently
linked to a carbon atom of a spacer group in the cationic head (1)
and each hydrophilic hydrocarbon in the hydrophilic tail (3) is
covalently linked to a chain in the backbone (2) to achieve at
least a ten atom spacing along the chain between the tail (3) and
the head (1)
2. A lipid according to claim 1 wherein each cationic centre is an
amino group.
3. A lipid according to claim 1 or claim 2 wherein the number of
cationic centres in the cationic head is from three to six.
4. A lipid according to any one of claim 1 to claim 3 wherein each
carbon containing spacer group is an optionally substituted
aliphatic, cycloaliphatic, heteroaliphatic, heterocycloaliphatic,
aromatic or heteroaromatic group.
5. A lipid according to any one of claim 1 to claim 4 wherein each
hydrocarbon chain in the hydrophobic backbone is an optionally
substituted straight or branched aliphatic or heteroaliphatic chain
containing from ten to around one hundred chain-linked atoms.
6. A lipid according to claim 5 wherein the hydrophobic backbone
has one or two hydrocarbon chains indirectly linked through a
linker atom or group to a carbon atom in a spacer group connecting
two cationic centres in the cationic head (1).
7. A lipid according to any of claim 1 to claim 5 wherein each
hydrophilic hydrocarbon in the hydrophilic tail (3) is attached to
a hydrocarbon chain of the hydrocarbon backbone (2) at the terminal
carbon atom of said chain distal to the chain carbon atom attached
to the cationic head (1).
8. A lipid according to any of the preceding claims which has the
formula
(1):[R.sup.1].sub.m--(L.sup.1).sub.n--[--C(R.sup.2)(R.sup.3)(R.sup.4)]
(1)wherein R.sup.1 is a hydrocarbon chain optionally substituted by
one or more hydrophilic hydrocarbons each containing two or more
atoms or groups capable of being solvated by water, provided that
at least one hydrocarbon chain is substituted by at least one
hydrophilic hydrocarbon and each hydrophilic hydrocarbon is
attached to the hydrocarbon chain to achieve at least a ten atom
spacing along the chain between the hydrophilic hydrocarbon and the
group --(L.sup.1).sub.n--[--C (R.sup.2)(R.sup.3)(R.sup.4)]; m is an
integer from 1 to 6; L.sup.1 is a linker atom or group; n is zero
or the integer 1; --[--C(R.sup.2)(R.sup.3- )(R.sup.4)] is a
cationic head in which R.sup.2 is a hydrogen atom or an optionally
substituted aliphatic, cycloaliphatic, heteroaliphatic,
hetero-cycloaliphatic, aromatic or heteroaromatic group optionally
containing one or more cationic centres, and R.sup.3 and R.sup.4
which may be the same or different is each an optionally
substituted aliphatic, cycloaliphatic, heteroaliphatic,
heterocycloaliphatic, aromatic or heteroaromatic group containing
one or more cationic centres, or R.sup.3 and R.sup.4 together with
the carbon atom to which they are attached form a cycloaliphatic,
heterocycloaliphatic, aromatic or heteroaromatic group containing
two or more cationic centres; and the salts, solvates and hydrates
thereof.
9. A lipid according to claim 8 which has the formula
(1a):[R.sup.7].sub.p--(L.sup.3).sub.q--[R.sup.6].sub.m--(L.sup.1).sub.n---
[--C(R.sup.2)(R.sup.3)(R.sup.4)] (1a)wherein R.sup.2, R.sup.3,
R.sup.4, L.sup.1, m and n are as defined for formula (1); R.sup.6
is a hydrocarbon chain; L.sup.3 is a linker atom or group; R.sup.7
is a hydrophilic hydrocarbon containing two or more atoms or groups
capable of being solvated by water; q is zero or an integer from
one to six; p is an integer from one to six; and the salts,
solvates and hydrates thereof, provided that each R.sup.7 or
L.sup.3 group, when present, is attached to a group R.sup.6 to
achieve at least a ten atom spacing along R.sup.6 between R.sup.7
or L.sup.3 and the group --(L.sup.1).sub.n--[C(R.sup.2)(R-
.sup.3)(R.sup.4)].
10. A lipid according to claim 9 wherein R.sup.2 is a hydrogen atom
and R.sup.3 and R.sup.4 is each a group
Sp.sup.1[WSp.sup.2].sub.bWSp.sup.3 or --Sp.sup.1[WSp.sup.2].sub.bWH
in which Sp.sup.1, Sp.sup.2 and Sp.sup.3, which may be the same or
different, is each a spacer group, W is a cationic centre and b is
zero or an integer from one to six.
11. A lipid according to claim 10 where Sp.sup.1, Sp.sup.2 and
Sp.sup.3 is each an optionally substituted aliphatic,
cycloaliphatic, aromatic or heteroaromatic group.
12. A lipid according to claim 11 wherein Sp.sup.1, Sp.sup.2 and
Sp.sup.3 is each an optionally substituted C.sub.1-6alkylene
chain.
13. A lipid according to any one of claim 9 to claim 12 wherein W
is a --NH-- group.
14. A lipid according to any one of claim 9 to claim 13 wherein b
is an integer from 1 to 3.
15. A lipid according to claim 9 wherein the group
--C(R.sup.2)(R.sup.3)(R- .sup.4) is a group
--CH[Sp.sup.1NHSp.sup.2NH.sub.2].sub.2,
--CH[Sp.sup.1NHSp.sup.2NHSp.sup.2NH.sub.2].sub.2 or
--CH[Sp.sup.1NHSp.sup.2NHSp.sup.2NHCH.sub.3].sub.2 wherein Sp.sup.1
is --CH.sub.2-- and each Sp.sup.2 is --(CH.sub.2).sub.3-- or
--(CH.sub.2).sub.4--.
16. A lipid according to any one of claim 9 to claim 15 wherein n
in --(L.sup.1).sub.n-- is the integer 1.
17. A lipid according to claim 16 wherein L.sup.1 is a group
--X.sup.1Alk.sup.2-- or
--[X.sup.1].sub.2Alk.sup.1X.sup.1Alk.sup.2-- in which X.sup.1 is an
--O-- or --S-- atom or a --C(O)--, --C(O)O--, --C(S)--, --S(O),
--S(O).sub.2-- --N(R.sup.5)--, [where R.sup.5 is a hydrogen atom,
straight or branched alkyl group such as a methyl or ethyl group or
an --Alk.sup.1X.sup.1-- chain], --CON(R.sup.5)--,
--OC(O)N(R.sup.5)--, --CSN(R.sup.5)--, --N(R.sup.5)CO--,
N(R.sup.5)C(O)O--, --N(R.sup.5)CS--, --S(O)N(R.sup.5)--,
--S(O).sub.2N(R.sup.5)--, --N(R.sup.5)S(O)--,
--N(R.sup.5)S(O).sub.2--, --N(R.sup.5)CON(R.sup.5)--, or
--N(R.sup.5)SO.sub.2N(R.sup.5)-- group [where any of these groups
contains two R.sup.5 substituents these may be the same or
different]; and Alk.sup.1 and Alk.sup.2 which may be the same or
different is each an optionally substituted straight or branched
C.sub.1-6alkylene, C.sub.2-6alkenylene or C.sub.2-6alkynylene chain
optionally interrupted or terminated by one or more, e.g. one, two
or three, carbocyclic or heterocarbocyclic groups and/or
heteroatoms or heteroatom containing groups X.sup.1 as just
defined.
18. A lipid according to claim 17 wherein X.sup.1 is a --CONH--
group, Alk.sup.1 is a --CH.sub.2--CH< chain and Alk.sup.2 is a
--(CH.sub.2).sub.4--, --(CH.sub.2)5-- or --(CH.sub.2).sub.6--
chain.
19. A lipid according to any one of claim 9 to claim 18 wherein m
is an integer 1 or 2.
20. A lipid according to any one of claim 9 to claim 19 wherein
R.sup.6 is an optionally substituted C.sub.10-60aliphatic
chain.
21. A lipid according to claim 20 wherein R.sup.6 is a linear,
optionally substituted C.sub.16-38alkylene chain.
22. A lipid according to any one of claim 9 to claim 21 wherein q
is the integer 1 and p is the integer 1 or 2.
23. A lipid according to any one of claim 9 to claim 22 wherein
L.sup.3 is an atom or group --X.sup.1--,
--X.sup.1Alk.sup.1X.sup.1-- or
[X.sup.1Alk.sup.1].sub.1X.sup.1Alk.sup.2X.sup.1 in which X.sup.1 is
an --O-- or --S-- atom or a --C(O)--, --C(O)O--, --C(S)--, --S(O),
--S(O).sub.2-- --N(R.sup.5)--, [where R.sup.5 is a hydrogen atom,
straight or branched alkyl group such as a methyl or ethyl group or
an --Alk.sup.1X.sup.1-- chain], --CON(R.sup.5)--,
--OC(O)N(R.sup.5)--, --CSN(R.sup.5)--, --N(R.sup.5)CO--,
N(R.sup.5)C(O)O--, --N(R.sup.5)CS--, --S(O)N(R.sup.5)--,
--S(O).sub.2N(R.sup.5)--, --N(R.sup.5)S(O)--,
--N(R.sup.5)S(O).sub.2--, --N(R.sup.5)CON(R.sup.5)--, or
--N(R.sup.5)SO.sub.2N(R.sup.5)-- group [where any of these groups
contains two R.sup.5 substituents these may be the same or
different]; and Alk.sup.1 and Alk.sup.2 which may be the same or
different is each an optionally substituted straight or branched
C.sub.1-6alkylene, C.sub.2-6alkenylene or C.sub.2-6alkynylene chain
optionally interrupted or terminated by one or more, e.g. one, two
or three, carbocyclic or heterocarbocyclic groups and/or
heteroatoms or heteroatom containing groups X.sup.1 as just
defined.
24. A lipid according to claim 23 wherein L.sup.3 is a --NHCO--,
--CONH--, --CONH(CH.sub.2).sub.2NHCO--, or
--[CONH(CH.sub.2).sub.2--].sub.2NCO(CH.s- ub.2).sub.2CONH
group.
25. A lipid according to any one of claim 9 to claim 24 wherein
R.sup.7 is a synthetic or naturally occurring polyol or a
poly(alkylene oxide) or a derivative thereof.
26. A lipid according to claim 25 wherein R.sup.7 is a
poly(alkylene oxide) or a derivative thereof.
27. A lipid according to claim 26 wherein R.sup.7 is a
poly(ethylene oxide).
28. A lipid according to any one of the Examples herein.
29. A lipid complex comprising a bipolar lipid according to any one
of the preceding claims in association with one or more bioactive
substances.
30. A complex according to claim 29 wherein each bioactive
substance is a bioactive protein, peptide, polysaccharide, nucleic
acid, oligonucleotide or a derivative thereof, lipid, glycolipid,
lipoprotein, lipopolysaccharide or viral, bacterial, protozoal,
cellular or tissue fraction.
31. A complex according to claim 30 wherein the bioactive substance
is a polyanion.
32. A complex according to claim 31 wherein the bioactive substance
is a nucleic acid.
33. A complex according to any one of claims 29 to 32 containing
two or more different bipolar lipids.
34. A complex according to claim 33 wherein one bipolar lipid has a
hydrophilic tail formed by a poly(alkylene oxide) or a derivative
thereof and each of the others has a hydrophilic tail formed by a
synthetic or naturally occurring polyol.
35. A complex according to claim 34 wherein the poly(alkylene
oxide) is poly(ethylene oxide).
36. A composition comprising a complex according to any one of
claim 29 to claim 35 and one or more other lipids.
37. A composition according to claim 36 wherein each other lipid is
a neutral or cationic lipid.
38. A composition comprising a complex according to any one of
claim 29 to claim 35 and one or more pharmaceutically acceptable
carriers, excipients or diluents.
39. A method of preparing a lipid as claimed in any one of claims 1
to 29, comprising coupling: (A) a cationic head comprising two or
more cationic centres, each centre being covalently linked to one
or more others by one or more carbon containing spacer groups; (B)
a hydrophobic backbone comprising one or more hydrocarbon chains;
and (C) a hydrophilic tail comprising one or more hydrophilic
hydrocarbons each containing two or more atoms or groups capable of
being solvated by water, wherein starting materials A), B) and C)
contain one or more reactive functional groups suitable for
facilitating coupling.
40. A method of preparing a lipid as claimed in any one of claims 1
to 29, comprising the step of deprotecting a protected derivative
of said lipid.
41. A method of preparing a complex as claimed in any one of claims
30 to 35 comprising mixing a lipid as claimed in any one of claims
1 to 29 with a bioactive substance.
42. Use of a complex as claimed in any one of claims 30 to 35 for
delivering a bioactive substance to cells in vitro.
43. A composition as claimed in any one of claims 36 to 38 for use
in delivering a bioactive substance to cells in vivo.
44. A composition as claimed in any one of claims 36 to 38 for use
as a medicament.
45. Use of a composition as claimed in any one of claims 36 to 38
for the preparation of a medicament for the delivery of a bioactive
substance, preferably a therapeutic, diagnostic or immunomodulatory
agent.
46. Method of delivering a bioactive substance to a human or
non-human animal wherein said bioactive substance, preferably a
therapeutic, diagnostic or immunomodulatory agent, is administered
in the form of a complex as defined in any one of claims 30 to 35.
Description
[0001] This invention relates to a series of bipolar lipids and to
their use to deliver bioactive substances to cells.
[0002] To be effective, many pharmaceutical agents need to be
efficiently delivered to the cytoplasm of a eucaryotic cell. For
many low molecular weight compounds of low to moderate polarity
this is not a problem since such molecules can pass directly
through the plasma membrane of the cell and into the cytoplasm.
Direct passage is not available to other compounds of greater
polarity or high molecular weight and these generally enter the
cell by receptor mediated endocytosis or phagocytosis. These
mechanisms are not efficient however with all sizes and types of
molecule. In particular, large, polyanionic compounds are not
readily taken up by cells when delivered to them in aqueous
solution.
[0003] One general solution to this problem is to couple any poorly
transported pharmaceutical agent to a carrier which itself is
readily taken up into the cytoplasm of a cell. This is not always
satisfactory however, since coupling to the carrier may have an
undesirable effect on the metabolism and/or antigenicity of the
pharmaceutical agent and/or it may be difficult to recover the
desired biological activity from the resulting conjugate once
inside the cell.
[0004] An alternative solution is to formulate the pharmaceutical
agent with a delivery vehicle which is soluble in aqueous solutions
but which can also mimic naturally occurring cell membrane
constituents. This encourages fusion of the vehicle with a cell
membrane and subsequent delivery of any associated pharmaceutical
agent to the cytoplasm.
[0005] Amphiphilic lipids have frequently been used for this
purpose. These typically have a hydrophobic backbone composed of
one or more hydrocarbons and a hydrophilic polar head group
containing one or more ionisable groups, to facilitate the
transport of macromolecules to and across the plasma membrane of
cells and into the cytoplasm The polarity of the head group may be
controlled by the selection of the number and/or type of ionisable
groups to achieve a range of negatively charged (anionic), neutral
or positively charged (cationic) lipids.
[0006] For the delivery of polyanions it is generally advantageous
to use cationic lipids. The advent of gene therapy and the need to
deliver anionic molecules such as nucleic acids to mammalian cells
has provided much impetus to the development of this class of
lipids. First generation compounds include those with a monocation
head group such as
N-[1(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride
[DOTMA; Feigner, P L and Ringold, G M, Nature, 337 387-388 (1989)],
1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide
[DMRIE; Zabner, J et al J. Biol. Chem, 270, 18997-19007 (1995)] and
3.beta.[N-(N.sup.1,N.sup.1-dimethylaminoethane)carbamoyl]cholesterol
[DC-Chol; Farhood, H et al, Biochim. Biophys. Acta. 1111, 239-246
(1992)] and those with a polycation head group such as
dioctadecyl-amidoglycylspe- rmine [DOGS; Behr, J-P, et al, Proc.
Natl. Acad. Sci. 86, 6982-4986 (1989)].
[0007] In an effort to improve the properties of these early
compounds for in vivo delivery of polyanions many more cationic
lipids have been developed in which the nature and size of the
hydrophobic backbone and/or the cationic head group have been
varied (see for example International Patent Specifications Nos.
WO95121931, WO96/10038, WO96/17823, WO96/18273, WO96/25508,
WO96/26179, WO96/41606, WO97/18185, WO97/25339, WO97/3010 and
WO97/31934).
[0008] The goal in the development of cationic lipids for in vivo
use is to provide a molecule which is simple to use in a clinical
setting; which is robust; which forms small stable complexes over
wide pH and ionic strength ranges; which is non-toxic; and which is
capable of delivering a high concentration of polyanion to a
cell.
[0009] We have now developed a class of lipid which meets these
requirements. Importantly, our lipids are capable of self-assembly
and will form stable complexes in aqueous solutions. The lipids are
able to efficiently compact polyanions to give defined particle
sizes of less than 500 nm. The lipid-polyanion complex remains
associated over wide pH and ionic strength ranges and is able to
efficiently deliver high concentrations of polyanions to cells.
[0010] Thus according to one aspect of the invention we provide a
bipolar lipid comprising a cationic head (1) a hydrophobic backbone
(2) and a hydrophilic tail (3) in which:
[0011] (A) the cationic head comprises two or more cationic
centres, each centre being covalently linked to one or more others
by one or more carbon containing spacer groups;
[0012] (B) the hydrophobic backbone comprises one or more
hydrocarbon chains; and
[0013] (C) the hydrophilic tail comprises one or more hydrophilic
hydrocarbons each containing two or more atoms or groups capable of
being solvated by water;
[0014] each of said components (1) to (3) being covalently linked
head (1) to backbone (2) to tail (3) and arranged such that at
least one hydrocarbon chain in the hydrophobic backbone (2) is
covalently linked to a carbon atom of a spacer group in the
cationic head (1) and each hydrophilic hydrocarbon in the
hydrophilic tail (3) is covalently linked to a chain in the
backbone (2) to achieve at least a ten atom spacing along the chain
between the tail (3) and the head (1).
[0015] In the lipids according to the invention, each cationic
centre in the cationic head (1) may be provided by one or more
heteroatoms capable of retaining a positive charge at a pH in the
range from around pH 2.0 to around pH 10.0. In practice, whether a
heteroatom will retain a positive charge in this pH range will
depend on the nature and number of any other atoms or groups
attached to it. Thus particular examples of suitable cationic
centres include primary, secondary, tertiary and quaternary amino
groups, sulphonium and phosphonium groups.
[0016] The number of cationic centres may be varied as desired
depending on the intended use of the lipid of the invention. At
least two centres will be present, but three, four, five, six,
seven, eight or more may be optionally incorporated. More than one
type of centre may be present, for example mixtures of amino groups
may be accommodated, and/or sulphonium and/or phosphonium
groups.
[0017] In one general preference each cationic centre is an amino
group. Particularly useful amino groups include primary and
secondary amino groups. The number of cationic centres in the
cationic head (1) will preferably be from three to six.
[0018] Each cationic centre will in general be separated from any
other centre by spacer groups arranged to link the centres in a
linear (straight and/or branched) or cyclic fashion. The overall
effect may be a cationic head (1) which has a straight and/or
branched linear structure, a cyclic structure, or a mixture of
straight and/or branched linear and cyclic structures. More than
one type of spacer group may be present in a cationic head (1).
Where desired a spacer group may form a terminal group on the
cationic head (1), acting as a substituent on a cationic centre
rather than a group connecting centres together.
[0019] Each spacer group will in general be non-ionic and contain
at least one carbon atom. Suitable groups include optionally
substituted aliphatic, cycloaliphatic, heteroaliphatic,
heterocycloaliphatic, aromatic or heteroaromatic groups.
[0020] Particular examples of optionally substituted aliphatic
spacer groups include optionally substituted C.sub.1-10aliphatic
chains such as optionally substituted straight or branched
C.sub.1-6alkylene, C.sub.2-6alkenylene or C.sub.2-6alkynylene
chains.
[0021] Heteroaliphatic spacer groups include the aliphatic chains
just described but with each chain additionally containing one,
two, three or four heteroatoms or heteroatom-containing groups.
Particular heteroatoms or groups include atoms or groups L.sup.2
where L.sup.2 is as defined below for L.sup.1 when L.sup.1 is a
linker atom or group. Each L.sup.2 atom or group may interrupt the
aliphatic chain, or may be positioned at its terminal carbon atom
to connect the chain to the atom or group R.sup.1.
[0022] Particular examples of aliphatic spacer groups include
optionally substituted --CH.sub.2--, --CH.sub.2CH.sub.2--,
--CH(CH.sub.3)--, --C(CH.sub.3).sub.2--,
--(CH.sub.2).sub.2CH.sub.2--, --CH(CH.sub.3)CH.sub.2--,
--(CH.sub.2).sub.3CH.sub.2--, --CH(CH.sub.3)CH.sub.2CH.sub.2--,
--CH.sub.2CH(CH.sub.3)CH.sub.2--, --C(CH.sub.3).sub.2CH.sub.2--,
--(CH.sub.2).sub.4CH.sub.2--, --(CH.sub.2).sub.5CH.sub.2--,
--CHCH--, --CHCHCH.sub.2--, --CH.sub.2CHCH--,
--CHCHCH.sub.2CH.sub.2--, --CH.sub.2CHCHCH.sub.2--,
--(CH.sub.2).sub.2CHCH--, --CC--, --CCCH.sub.2--, --CH.sub.2CC--,
--CCCH.sub.2CH.sub.2--, --CH.sub.2CCCH.sub.2--, or
--(CH.sub.2).sub.2CC-- chains. Where appropriate each of said
chains may be optionally interrupted by one or two atoms and/or
groups L.sup.2 to form an optionally substituted heteroaliphatic
spacer group. Particular examples include optionally substituted
--L.sup.2CH.sub.2--, --CH.sub.2L.sup.2CH.sub.2--,
--L.sup.2(CH.sub.2).sub.2--, --CH.sub.2L.sup.2(CH.sub.2).sub.2--,
--(CH.sub.2).sub.2L.sup.2CH.sub.2--, --L.sup.2(CH.sub.2).sub.3--
and --(CH.sub.2).sub.2L.sup.2(CH.sub.2)2-- chains. The optional
substituents which may be present on aliphatic or heteroaliphatic
spacer groups include one, two, three or more substituents selected
from halogen atoms, e.g. fluorine, chlorine, bromine or iodine
atoms, or hydroxyl, C.sub.1-6alkoxy, e.g. methoxy or ethoxy,
haloC.sub.1-6alkoxy, e.g. halomethoxy or haloethoxy such as
difluoromethoxy or trifluoromethoxy, thiol, or C.sub.1-6alkylthio
e.g. methylthio or ethylthio. Particular examples of substituted
spacer groups include those specific chains just described
substituted by one, two, or three halogen atoms such as fluorine
atoms, for example chains of the type --CH(CF.sub.3)--,
--C(CF.sub.3).sub.2--, --CH.sub.2CH(CF.sub.3)--,
--CH.sub.2C(CF.sub.3).sub.2--, --CH(CF.sub.3)-- and
--C(CF.sub.3).sub.2CH.sub.2--.
[0023] Optionally substituted cycloaliphatic spacer groups in the
cationic head (1) include optionally substituted C.sub.3-10
cycloaliphatic groups. Particular examples include optionally
substituted C.sub.3-10cycloalkylene, e.g. C.sub.3-7cycloalkylene,
C.sub.3-10cycloalkenylene e.g. C.sub.3-7cycloalkenylene or
C.sub.3-10cycloalkynylene e.g. C.sub.3-7cycloalkynylene groups.
[0024] Particular examples of cycloaliphatic spacer groups include
optionally substituted cyclopropylene, cyclobutylene,
cyclopentylene, cyclohexylene, cycloheptylene,
2-cyclobuten-1-ylene, 2-cyclopenten-1-ylene and
3-cyclopenten-1-ylene groups.
[0025] Optionally substituted heterocycloaliphatic spacer groups
include the optionally substituted cycloaliphatic groups just
described but with each group additionally containing one, two,
three or four heteroatoms or heteroatom-containing groups L.sup.2
as just defined.
[0026] The optional substituents which may be present on the
cycloaliphatic or heterocycloaliphatic spacer groups include one,
two, three or more substituents selected from halogen atoms
C.sub.1-6alkyl, e.g. methyl or ethyl, haloC.sub.1-6alkyl, e.g.
halomethyl or haloethyl such as difluoromethyl or trifluoromethyl,
hydroxyl, C.sub.1-6alkoxy, e.g. methoxy or ethoxy,
haloC.sub.1-6alkoxy, e.g. halomethoxy or haloethoxy such as
difluoromethoxy or trifluoromethoxy, thiol, or C.sub.1-6alkylthio
e.g. methylthio or ethylthio groups.
[0027] Optionally substituted aromatic spacer groups include for
example monocyclic C.sub.6-12 aromatic groups, such as optionally
substituted phenylene.
[0028] Optionally substituted heteroaromatic spacer groups, include
for example optionally substituted monocyclic C.sub.1-9
heteroaromatic groups containing for example one, two, three or
four heteroatoms selected from oxygen, sulphur or nitrogen atoms.
Monocyclic heteroaromatic groups include for example five- or
six-membered heteroaromatic groups containing one, two, three or
four heteroatoms selected from oxygen, sulphur or nitrogen
atoms.
[0029] Optional substituents which may be present on the aromatic
or heteroaromatic spacer groups include one, two, three or more
substituents selected from those just described in relation to
cycloaliphatic and heterocycloaliphatic spacer groups.
[0030] In one general preference each spacer group in the cationic
head (1) is preferably an optionally substituted straight or
branched C.sub.1-6alkylene chain. Particularly useful chains
include --(CH.sub.2).sub.2--, --(CH.sub.2).sub.3-- and
--(CH.sub.2).sub.4--chains- .
[0031] In the lipids of the invention at least one spacer group
connecting two cationic centres is covalently linked through one of
its carbon atoms to a hydrocarbon chain of the hydrophobic backbone
(2). Where desired any other available carbon atom or heteroatom in
the, or any other, spacer group, or any available atom in a
cationic centre, may be additionally linked to the same or other
hydrocarbon chains making up the backbone (2). It is generally
preferred however to link the backbone (2) and cationic head (1) at
one carbon atom in one spacer group.
[0032] The hydrophobic backbone (2) in the lipids according to the
invention may comprise one or more hydrocarbon chains. Each
hydrocarbon may be for example an optionally substituted straight
or branched aliphatic or heteroaliphatic chain containing a minimum
of ten up to a maximum of around one hundred chain-linked atoms as
described in more detail below. The hydrocarbon may be attached
either directly or indirectly through a linker atom or group to the
cationic head (1). Particular examples of suitable linker groups
are those represented by the group L.sup.1 described below. As
explained above, more than one hydrocarbon chain may be attached to
the head group but a preferred class of lipids according to the
invention has one or two hydrocarbon chains as just described
indirectly linked through a linker atom or group to a carbon atom
in a spacer group connecting two cationic centres in the cationic
head (1).
[0033] The hydrophilic tail (3) in the lipids according to the
invention may in general be one or more hydrophilic hydrocarbons
having little or no overall positive or negative charge and
containing a minimum of two up to a maximum of around one hundred
atoms or groups capable of being solvated by water. Each
hydrophilic hydrocarbon in the hydrophilic tail (3) may be attached
directly or indirectly through a linker atom or group to a
hydrocarbon chain of the hydrocarbon backbone (2). The attachment
point may be anywhere on the hydrocarbon chain provided that it is
at least ten atoms along the chain, excluding branches, from the
terminal carbon atom connecting the hydrophobic backbone (2) to the
cationic head (1). In one general preference the attachment point
may be at a terminal carbon atom of a hydrocarbon chain distal to
the chain carbon atom attached to the cationic head (1). Particular
examples of suitable hydrophilic hydrocarbons which constitute the
hydrophilic tail (3) are described in more detail below.
[0034] A particularly useful group of lipids according to the
invention may be represented by the formula (1):
[R.sup.1].sub.m--(L.sup.1).sub.n--[--C(R.sup.2)(R.sup.3)(R.sup.4)]
(1)
[0035] wherein R.sup.1 is a hydrocarbon chain optionally
substituted by one or more hydrophilic hydrocarbons each containing
two or more atoms or groups capable of being solvated by water,
provided that at least one hydrocarbon chain is substituted by at
least one hydrophilic hydrocarbon and each hydrophilic hydrocarbon
is attached to the hydrocarbon chain to achieve at least a ten atom
spacing along the chain between the hydrophilic hydrocarbon and the
group --(L.sup.1).sub.n--[--C(R.sup.2)(R.- sup.3)(R.sup.4)];
[0036] m is an integer from 1 to 6;
[0037] L.sup.1 is a linker atom or group;
[0038] n is zero or the integer 1;
[0039] --[--C(R.sup.2)(R.sup.3)(R.sup.4)] is a cationic head in
which R.sup.2 is a hydrogen atom or an optionally substituted
aliphatic, cycloaliphatic, heteroaliphatic, hetero-cycloaliphatic,
aromatic or heteroaromatic group optionally containing one or more
cationic centres, and R.sup.3 and R.sup.4 which may be the same or
different is each an optionally substituted aliphatic,
cycloaliphatic, heteroaliphatic, heterocycloaliphatic, aromatic or
heteroaromatic group containing one or more cationic centres, or
R.sup.3 and R.sup.4 together with the carbon atom to which they are
attached form a cycloaliphatic, heterocycloaliphatic, aromatic or
heteroaromatic group containing two or more cationic centres;
[0040] and the salts, solvates and hydrates thereof.
[0041] In the compounds of formula (1), the optionally substituted
aliphatic, cycloaliphatic, heteroaliphatic, heterocycloaliphatic,
aromatic or heteroaromatic group represented by R.sup.2, R.sup.3
and R.sup.4 may each be an optionally substituted C.sub.1-30
aliphatic, C.sub.3-10 cycloaliphatic, C.sub.1-30 heteroaliphatic,
C.sub.3-10 heterocycloaliphatic, C.sub.6-12 aromatic or C.sub.1-9
heteroaromatic group, each containing one or more cationic centres.
Particular examples of such groups include those generally and
specifically described above in relation to the spacer groups
present in the cationic head (1) with the additional presence of
one or more cationic centres as defined herein.
[0042] In general in the lipids of the invention when the
hydrophobic backbone (2) and cationic head (1) are joined
indirectly by a linker atom or group, as represented by L.sup.1 in
compounds of formula (1) when n is 1, then the linker atom or
group-may be any multivalent atom or group. Particular examples of
suitable linker atoms or groups include those of formula
--(Alk.sup.1).sub.r(X.sup.1).sub.s(Alk.sup.2).sub.t-- where X.sup.1
is an --O-- or --S-- atom or a --C(O)--, --C(O)O--, --C(S)--,
--S(O), --S(O).sub.2-- --N(R.sup.5)--, [where R.sup.5 is a hydrogen
atom, straight or branched alkyl group such as a methyl or ethyl
group or an --Alk.sup.1X.sup.1-- chain], --CON(R.sup.5)--,
--OC(O)N(R.sup.5)--, --CSN(R.sup.5)--, --N(R.sup.5)CO--,
N(R.sup.5)C(O)O--, --N(R.sup.5)CS--, --S(O)N(R.sup.5)--,
--S(O).sub.2N(R.sup.5)--, --N(R.sup.5)S(O)--,
--N(R.sup.5)S(O).sub.2--, --N(R.sup.5)CON(R.sup.5)--, or
--N(R.sup.5)SO.sub.2N(R.sup.5)-- group [where any of these groups
contains two R.sup.5 substituents these may be the same or
different]; Alk.sup.1 and Alk.sup.2 which may be the same or
different is each an optionally substituted straight or branched
C.sub.1-6alkylene, C.sub.2-6alkenylene or C.sub.2-6alkynylene chain
optionally interrupted or terminated by one or more, e.g. one, two
or three, carbocyclic or heterocarbocyclic groups and/or
heteroatoms or heteroatom containing groups X.sup.1 as just
defined, and r, s, and t, which may be the same or different, is
each zero or the integer 1, provided that when one of r, s or t is
zero at least one of the remainder is the integer 1.
[0043] Carbocyclic groups which may interrupt the groups Alk.sup.1
and Alk.sup.2 include for example optionally substituted
C.sub.4-8cycloalkyl, e.g. optionally substituted cyclopentyl or
cyclohexyl groups, or optionally substituted C.sub.4-8cycloalkenyl,
e.g. optionally substituted cyclopentenyl or cyclohexenyl groups.
Heterocarbocyclic groups include for example carbocyclic groups of
the types just mentioned containing one or more heteroatoms or
heteroatom-containing groups X.sup.1 as defined above. Optional
substituents which may be present on the chains represented by
Alk.sup.1 and Alk.sup.2 and the carbocyclic or heterocarbocyclic
groups which can interrupt or terminate them include one, two or
three substituents selected from halogen atoms, e.g. fluorine,
chlorine, bromine or iodine atoms or C.sub.1-3alkyl, e.g. methyl or
ethyl, or C.sub.1-3alkoxy e.g. methoxy or ethoxy groups.
[0044] It will be appreciated that the linker atom or group will be
at least divalent in the instance where one hydrocarbon chain in
the hydrophobic backbone (2) is attached to it. Where it is desired
to attach more than one hydrocarbon chain to the linker the latter
will need to be selected with an appropriate valency and this will
generally mean that at least one of Alk.sup.1 or Alk.sup.2 will
need to be present in a branched form and with the requisite number
of X.sup.1 atoms or groups to achieve the desired coupling.
[0045] Particular examples of linker groups include groups of
formula --X.sup.1Alk.sup.2- where X.sup.1 is as defined above and
Alk.sup.2 is an optionally substituted --CH.sub.2--,
--(CH.sub.2).sub.2--, --(CH.sub.2).sub.3--, --(CH.sub.2).sub.4--,
--(CH.sub.2).sub.5-- or --(CH.sub.2).sub.6-- chain; groups of
formula [X.sup.1].sub.2Alk.sup.1X.s- up.1Alk.sup.2 where Alk.sup.1
is a --CH.sub.2CH< group and X.sup.1 and Alk.sup.2 are as just
defined or a group of formula [X.sup.1].sub.2Alk.sup.1Alk.sup.2
where X.sup.1, Alk.sup.1 and Alk.sup.2 are as just defined.
[0046] Each hydrocarbon chain in the hydrophobic backbone (2) of
the lipids according to the invention and as represented by R.sup.1
in compounds of formula (1) may be a C.sub.10 up to about a
C.sub.60 hydrocarbon chain, for example a C.sub.16 to C.sub.60
hydrocarbon chain such as a C.sub.18 to C.sub.48 hydrocarbon
chain.
[0047] In particular, the chain may be an optionally substituted
C.sub.10-60 aliphatic chain such as an optionally substituted
straight or branched C.sub.10-60alkylene, C.sub.10-60alkenylene or
C.sub.10-60alkynylene chain. Optional substituents which may be
present on such chains include one or more halogen atoms, e.g.
fluorine, chlorine, bromine or iodine atoms, or haloC.sub.1-6alkyl,
e.g. --CF.sub.3 groups. Where desired each alkylene, alkenylene or
alkynylene chain may be interrupted by one or more oxygen or
sulphur atoms or optionally substituted C.sub.5-7cycloalkyl, e.g.
cyclopentyl or cyclohexyl, C.sub.5-7cycloalkenyl, e.g.
cyclopentenyl or cyclohexenyl, --C(O)--, --C(S)--,
--C(O)N(R.sup.5)--, --C(S)N(R.sup.5)--, --N(R.sup.5)C(O)--,
--N(R.sup.5)C(S)--, --C(O)O--, --C(O)S--, --OC(O)N(R.sup.5)--,
--S(O)--, --S(O.sub.2)--, --S(O)N(R.sup.5)--,
--S(O).sub.2N(R.sup.5)--, --N(R.sup.5)S(O)--,
--N(R.sup.5)S(O).sub.2--, --N(R.sup.5)C(O)N(R.sup.5)-- -,
--N(R.sup.5)C(S)N(R.sup.5)--, --N(R.sup.5)S(O)N(R.sup.5)-- or
--N(R.sup.5)S(O).sub.2N(R.sup.5)-- groups. Optional substituents
which may be present on cycloalkyl or cycloalkenyl groups of this
type include one or more halogen atoms or haloalkyl groups as just
described. It will be appreciated that when the hydrocarbon chain
in the hydrophobic backbone (2) is an alkenylene or alkynylene
chain it may have more than one unsaturated group.
[0048] As generally explained above, the hydrophilic tail (3) in
the lipids according to the invention may be formed by one or more
hydrophilic hydrocarbons, each attached to a hydrocarbon chain in
the hydrophobic backbone (2), for example as generally represented
by R.sup.1 in compounds of formula (1). Each hydrophilic
hydrocarbon may be an aliphatic, heteroaliphatic, cycloaliphatic,
polycycloaliphatic, heterocycloaliphatic or
polyheterocycloaliphatic group. Particular examples of aliphatic
groups include alkyl, alkenyl or alkynyl groups. Cycloaliphatic
groups include cycloalkyl or cycloalkenyl groups.
Polycycloaliphatic groups include two or more cycloalkyl or
cycloalkenyl groups either joined directly or indirectly through a
linker atom or group, for example a linker atom or group L.sup.2
where L.sup.2 is an atom or group as described above for the group
L.sup.1. Each of these aliphatic, cycloaliphatic or
polycycloaliphatic groups may be optionally interrupted by one or
more heteroatoms or heteroatom-containing groups, for example of
the type described above in relation to the group L.sup.1 to yield
heteroaliphatic, heterocycloaliphatic or polyheterocycloaliphati- c
hydrocarbon groups. In general, each hydrophilic hydrocarbon group
forming the hydrophilic tail (3) may contain from one carbon atom
to around two hundred carbon atoms.
[0049] Each hydrophilic hydrocarbon contains two or more atoms or
groups capable of being solvated by water. Examples of such groups
include oxygen atoms (--O--) or oxygen-containing groups. Oxygen
atoms may form part of a heteroaliphatic, heterocycloaliphatic or
polycyclo-heteroaliphatic group as just described.
Oxygen-containing groups may be substituents present on the various
hydrocarbons just mentioned and include for example hydroxyl, amide
and alkoxy groups such as methoxy or ethoxy groups. In general the
number of groups capable of being solvated by water in each
hydrocarbon will range from two to around two hundred.
[0050] Particular examples of suitable hydrophilic hydrocarbons
include polyols. Suitable polyols include naturally occurring
polyols such as sugars and derivatives thereof, and synthetic
polyols. Particular sugars include mono- and oligosaccharides.
Sugar derivatives include glycosides in which a non-ionic aliphatic
or heteroaliphatic group (for example of the type described herein)
is joined to a sugar by a glycosidic linkage. Monosaccharides
include for example open-chain or cyclic compounds containing three
to eight, e.g. five or six, carbon atoms and at least two hydroxyl
substituents. Oligosaccharides include for example at least two
monosaccharides as just defined linked together by a glycosidic
linkage. More than one type of monosaccharide may be present to
yield a homo- or heterooligosaccharide.
[0051] Alternatively the hydrophilic hydrocarbon may be a
polyether, for example a poly(alkylene oxide) and derivatives
thereof, such as poly(ethylene oxide), poly(propylene oxide) or
methoxy poly(ethylene oxide), a poly(oxyalkylated alcohol) or a
poly(alkenylene alcohol) or poly(alkynylene alcohol) such as
poly(vinyl alcohol). The hydrocarbons may in general be straight or
branched. Where desired co-polymers of these hydrocarbons may be
used.
[0052] Each hydrophilic hydrocarbon may be linked directly or
indirectly to a hydrocarbon chain in the hydrophobic backbone (2).
For indirect linkage a linker atom or group may be employed, for
example an atom or group L.sup.3 where L.sup.3 is as defined above
as for the linker atom or group L.sup.1. Where the group L.sup.3 is
multivalent, for example when it is a branched alkylene chain
containing more than one X.sup.1 atom or group, more than one
hydrophilic hydrocarbon may be attached to it.
[0053] A particularly useful group of compounds according to the
invention has the formula (1a):
[R.sup.7].sub.p--(L.sup.3).sub.q--[R.sup.6].sub.m--(L.sup.1).sub.n--[--C(R-
.sup.2)(R.sup.3)(R.sup.4)] (1a)
[0054] wherein R.sup.2, R.sup.3, R.sup.4, L.sup.1, m and n are as
defined for formula (1);
[0055] R.sup.6 is a hydrocarbon chain;
[0056] L.sup.3 is a linker atom or group;
[0057] R.sup.7 is a hydrophilic hydrocarbon containing two or more
atoms or groups capable of being solvated by water;
[0058] q is zero or an integer from one to six;
[0059] p is an integer from one to six;
[0060] and the salts, solvates and hydrates thereof, provided that
each R.sup.7 or L.sup.3 group, when present, is attached to a group
R.sup.6 to achieve at least a ten atom spacing along R.sup.6
between R.sup.7 or L.sup.3 and the group
--(L.sup.1).sub.n--[C(R.sup.2)(R.sup.3)(R.sup.4)].
[0061] In the compounds of formula (1a) the hydrocarbon chain
represented by R.sup.6 may be a C.sub.10 up to about a C.sub.60
hydrocarbon chain as generally and more particularly described
above in relation to the group R.sup.1. The hydrophilic hydrocarbon
R.sup.7 may similarly be a hydrophilic hydrocarbon as described
previously in relation to the group R.sup.1. The group L.sup.3 may
be a linker atom or group as just defined.
[0062] The cationic head (1) in the lipids according to the
invention will preferably be a group --C(R.sup.2)(R.sup.3)(R.sup.4)
as described above in relation to the compounds of formulae (1) and
(1a). In groups of this type, R.sup.2 is preferably a hydrogen
atom, and R.sup.3 and R.sup.4 is each preferably a group
--Sp.sup.1[WSp.sup.2].sub.bWSp.sup.3 or
--Sp.sup.1[WSp.sup.2].sub.bWH in which Sp.sup.1, Sp.sup.2 and
Sp.sup.3, which may be the same or different, is each a spacer
group as defined above, W is a cationic centre as defined herein
and b is zero or an integer from one to six.
[0063] In particular groups of this type, the cationic centre W is
preferably a --NH-- group. Sp.sup.1, Sp.sup.2 and Sp.sup.3, which
may be the same or different, is each preferably an optionally
substituted C.sub.1-6alkylene chain. b is preferably an integer
from one to three.
[0064] Particularly useful cationic heads (1) in compounds of the
invention include those of formula
--CH[Sp.sup.1NHSp.sup.2NH.sub.2].sub.2- ,
--CH[Sp.sup.1NHSp.sup.2NHSp.sup.2NH.sub.2].sub.2 or
--CH[Sp.sup.1NHSp.sup.2NHSp.sup.2NHCH.sub.3].sub.2 where each
Sp.sup.1 and Sp.sup.2 group is the same or different and is an
optionally substituted C.sub.1-6alkylene chain, particularly
wherein Sp.sup.1 is --CH.sub.2-- and each Sp.sup.2 is
--(CH.sub.2).sub.3-- or --(CH.sub.2).sub.4--.
[0065] In general in the lipids according to the invention the
hydrophobic backbone (2) preferably comprises two or, especially
one hydrocarbon chain as defined herein. Thus m in formulae (1) and
(1a) is preferably an integer 2 or, especially, an integer 1. Each
hydrocarbon chain, for example as represented by R.sup.1 and
R.sup.6 in formulae (1) and (1a) respectively, is preferably linear
and in particular is a linear, optionally substituted
C.sub.16-38alkylene chain. Optionally substituted
C.sub.18-24alkylene chains are particularly useful.
[0066] In general each hydrocarbon chain in the hydrophobic
backbone (2) is preferably linked indirectly to the cationic head
(1) through a linker atom or group. The linker atom or group may be
for example an atom or group L.sup.1 as defined herein and thus in
the compounds of formulae (1) and (1a) for example n is preferably
the integer 1.
[0067] Preferred linkers include those of formula
--X.sup.1Alk.sup.2-- or
--[X.sup.1].sub.2Alk.sup.1X.sup.1Alk.sup.2-- where X.sup.1,
Alk.sup.1 and Alk.sup.2 are as defined previously. Particularly
useful linkers of these types are those wherein Alk.sup.2 is a
--(CH.sub.2).sub.4--, --(CH.sub.2).sub.5-- or, especially,
--(CH.sub.2).sub.6-- chain. X.sup.1 in these linkers is preferably
a --CONH-- group. Alk.sup.1 when present is preferably a
--CH.sub.2--CH< chain.
[0068] In another general preference each hydrocarbon chain in the
hydrocarbon backbone (2) has two, or especially one, hydrophilic
hydrocarbon attached to it. Each hydrophilic hydrocarbon is
preferably attached to the terminal carbon atom of the hydrocarbon
chain distal to the chain carbon atom attached to the cationic head
(1). Preferably the hydrophilic hydrocarbon and hydrocarbon chain
are indirectly linked through a linker atom or group. Thus in one
particular preference in compounds of formula (1a) q is the integer
1 and p is the integer 1 or 2.
[0069] In compounds of this type and in general the group L.sup.3
may preferably be an atom or group --X.sup.1--,
--X.sup.1Alk.sup.1X.sup.1-- or
[X.sup.1Alk.sup.1].sub.2X.sup.1Alk.sup.2X.sup.1--. Particularly
useful L.sup.3 groups include --NHCO--, --CONH--,
--CONH(CH.sub.2).sub.2NHCO--, or
--[CONH(CH.sub.2).sub.2--].sub.2NCO(CH.sub.2).sub.2CONH--
groups.
[0070] In general, the hydrophilic hydrocarbon, for example as
represented by R.sup.7 in formula (1a) is preferably a synthetic
polyol, a naturally occurring polyol such as mono- or disaccharide,
or a poly(alkylene oxide) as defined herein. In particular R.sup.7
may be a poly(alkylene oxide) or a derivative thereof, especially a
poly(ethylene oxide).
[0071] Particularly useful lipids according to the invention are
those described in the Examples hereinafter, especially in Sections
H and I.
[0072] The lipids according to the invention may generally be
prepared by coupling appropriately functionalised cationic heads
(1), hydrophobic hydrocarbons (2) and hydrophilic hydrocarbons (3)
in a predetermined order. Standard chemical coupling techniques may
be employed utilising starting materials containing one or more
reactive functional groups such as acids, thioacids, anhydrides,
acid halides, esters, imides, aldehydes, ketones and amines.
Illustrative reactions are described in detail in the Examples
hereinafter for the preparation of a number of lipids according to
the invention and these may be readily adapted using different
starting materials to provide other compounds of the invention.
[0073] Thus in one general approach a homo- or heterobifunctional
hydro-carbon chain may first be coupled to a hydrophilic
hydrocarbon or cationic head and the resulting product coupled as
necessary to the remaining component to provide the lipid of the
invention.
[0074] The homo- or heterobifunctional hydrocarbon chain may be any
hydro-carbon chain described herein containing two different
reactive functional groups of the types just described.
Particularly useful groups include acids and thioacids and reactive
derivatives thereof, and amines. These can be used to participate
in acylation or thioacylation reactions to couple the hydrocarbon
chain to an amine or acid as appropriate in any suitable
hydrophilic hydrocarbon and/or cationic head.
[0075] Acylation or thioacylation may be achieved using standard
conditions for reactions of this type. Thus, for example the
reaction may be carried out in a solvent, for example an inert
organic solvent such as an amide, e.g. a substituted amide such as
dimethylformamide, an ether, e.g. a cyclic ether such as
tetrahydrofuran, or a halogenated hydrocarbon, such as
dichloromethane, at a temperature from around ambient temperature
to the reflux temperature, optionally in the presence of a base
such as an amine, e.g. triethylamine, or a cyclic amine, such as
1,8-diazabicyclo[5.4.0] undec-7-ene pyridine,
dimethylaminopyridine, or N-methylmorpholine.
[0076] Where an acid is used the acylation may additionally be
performed in the presence of a condensing agent, for example a
diimide such as 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide or
N,N'-dicyclohexylcarbodi- -imide, advantageously in the presence of
a catalyst such as a N-hydroxy compound e.g. a N-hydroxytriazole
such as 1-hydroxybenzotriazole or a N-hydroxyimide such as
N-hydroxysuccinimide. Alternatively, the acid may be reacted with a
chloroformate, for example ethylchloroformate, prior to reaction
with the amine.
[0077] In the heterobifunctional hydrocarbon chain one of the
reactive functional groups may need to be in a protected form prior
to any coupling reaction to avoid its unwanted participation in the
reaction. Similarly other functional groups when present in the
hydrocarbon chain, or the intermediates used to generate the
hydrophilic hydrocarbon and/or the cationic head may need to be in
a protected form before these reagents can be used. Conventional
protecting groups may be used in accordance with standard practice
[see, for example, Green, T. W. in "Protective Groups in Organic
Synthesis", John Wiley & Sons, 1991 and the Examples
hereinafter].
[0078] Suitable heterobifunctional hydrocarbon chains are either
known, readily available materials or may be obtained by synthesis
using conventional techniques for example as described in the
Examples hereinafter. Thus generally a heterobifunctional
hydrocarbon chain of any desired length may be synthesised in one
or more reactions using appropriately functionalised shorter
chains. Thus in one example a shorter chain aldehyde may be reacted
with a shorter chain phosphonium salt to yield a longer chain
olefin of the desired length. In this particular example the
reaction may be carried out in the presence of a base, for example
an organometallic base such as an organolithium compound, a hydride
such as sodium or potassium hydride or an alkoxide such as a sodium
alkoxide e.g. sodium methoxide. The reaction may be performed in a
suitable solvent, for example a polar aprotic solvent such as an
alkyl sulphoxide, e.g. dimethylsulphoxide at a low temperature, for
example around 0.degree. C. The starting aldehyde and phosphonium
salt may be obtained from known starting alcohols and halides
respectively using conventional procedures. Where desired, the
olefin obtained above may be hydrogenated using hydrogen and a
catalyst, for example Pearlman's catalyst, to yield the
corresponding saturated hydrocarbon chain.
[0079] Where it is desired to obtain hydrocarbon chains containing
one or more heteroatoms or heteroatom-containing groups these may
be synthesised from smaller chains containing functional groups
which can be chemically coupled, for example by acylation or
thioacylation as generally described above.
[0080] Suitable functionalised hydrophilic hydrocarbons or cationic
heads for coupling to the heterobifunctional hydrocarbon chain are
either readily available or may be synthesised from known materials
by conventional methods for example as described in the Examples
hereinafter.
[0081] The advantageous properties of the lipids according to the
invention may be demonstrated using the small scale tests described
hereinafter in the Examples. In these the lipids can be shown to
efficiently compact any bioactive substance, and to self-assemble
with the substance in aqueous solution to yield stable complexes
which remain associated over wide pH and ionic strength ranges and
which will efficiently deliver the substance to eucaryotic
cells.
[0082] The lipids can thus be expected to be of use for the
delivery of bioactive substances to cells, particularly eucaryotic
cells, in vitro and especially in vivo. Particular general uses to
which the lipids may be put thus include for the delivery of
bioactive substances to cells in culture, and in human medicine for
the delivery of therapeutic or diagnostic agents, or agents which
can generate a host immune response for vaccine or other
immuno-modulatory purposes. The lipids are particularly well suited
for delivering bioactive polyanions, especially nucleic acids, and
are of particular use to modify a host's genotype or its
expression.
[0083] Thus, in another aspect of the invention we provide a lipid
complex characterised in that it comprises a bipolar lipid
comprising a cationic head (1) a hydrophobic backbone (2) and a
hydrophilic tail (3) in which:
[0084] (A) the cationic head comprises two or more cationic
centres, each centre being covalently linked to one or more others
by one or more carbon containing spacer groups;
[0085] (B) the hydrophobic backbone comprises one or more
hydrocarbon chains; and
[0086] (C) the hydrophilic tail comprises one or more hydrophilic
hydrocarbons each containing two or more atoms or groups capable of
being solvated by water;
[0087] each of said components (1) to (3) being covalently linked
head (1) to backbone (2) to tail (3) and arranged such that at
least on e hydrocarbon chain in the hydrophobic backbone (2) is
covalently linked to a carbon atom of a spacer group in the
cationic head (1) and each hydrophilic hydrocarbon in the
hydrophilic tail (3) is covalently linked to a chain in the
backbone (2) to achieve at least a carbon atom spacing along the
chain between the tail (3) and the head (1), in association with
one or more bioactive substances.
[0088] In the complexes according to the invention, each bioactive
substance may be for example a pharmacologically active agent,
including an endosomolytic agent, a diagnostic agent or any agent
able to modify the genotype and/or phenotype of a cell.
[0089] Particular examples of such substances include bioactive
proteins, peptides, polysaccharides, nucleic acids including
synthetic poly-nucleotides, oligonucleotides and derivatives
thereof, lipids, glycolipids, lipoproteins, lipopolysaccharides and
viral, bacterial, protozoal, cellular or tissue fractions.
[0090] Where desired the complexes according to the invention may
contain two or more different bipolar lipids of the invention and
such lipid mixtures form a further particular aspect of the
invention. Especially useful mixtures include those containing two
or more bipolar lipids of the invention which differ from each
other in the nature of the hydrophilic tail present in each. The
proportion of each lipid in complexes of this type may be
manipulated to obtain complexes with different physio-chemical
properties, for example overall surface charge and/or particle
size, tailored to meet the intended use of the complex. Thus for
example in one advantageous lipid complex containing two or more
bipolar lipids, one of the lipids has a hydrophilic tail formed by
a poly(alkyene oxide) or a derivative thereof as defined herein,
while each of the others has a hydrophilic tail formed by a
synthetic or naturally occurring polyol as described previously.
The proportion of the first poly(alkylene oxide)-containing lipid
may be varied in such complexes so that the mole ratio of first
lipid to second and other lipids is from 1:10000 to 1:1,
advantageously from around 1:1000 to around 1:20, especially around
1:10. Complexes of these types, particularly where the
poly(alkylene oxide) is poly(ethylene oxide), may be obtained which
advantageously have zero surface charge and do not aggregate when
left in solution and which additionally are able to compact a
bioactive substance to give small particles of 150 nm and below,
particularly 100 nm and below, especially around 80-85 nm.
[0091] The lipids according to the invention are particularly
suited for delivering polyanions to cells and preferred lipid
complexes of the invention thus include lipid-polyanion complexes
in which the polyanion may be any of the above-mentioned bioactive
substances possessing a net negative charge. Particular polyanions
include nucleic acids, for example single or double stranded,
circular or supercoiled DNA or RNA and derivatives thereof. Where
desired the DNA may be part of a structure such as a plasmid.
[0092] The lipid complexes will in general comprise a lipid
according to the invention and a bioactive substance in a weight
ratio of around 0.1:1 to around 100:1, for example around 1:1 to
around 50:1. The complexes may be formed as liquids, by initially
mixing one or more bipolar lipids according to the invention, and a
bioactive substance together advantageously in an aqueous solvent
using conventional procedures. Where desired the solvent may be
removed, for example by lyophilisation, to obtain a solid lipid
complex.
[0093] The lipid complexes according to the invention may be
formulated with other materials such as one one or more other
lipids or other pharmaceutically acceptable carriers, excipients or
diluents and the invention extends to such compositions. In this
aspect of the invention the "other" lipid may be for example
selected from any known neutral and/or cationic lipid, for example
selected from those described herein in the introduction to the
invention (see page 2) and also especially including DOPE and other
cholesterol derivatives such as cholesterol hemisuccinate.
Particularly useful formulations of this type are those wherein the
bipolar lipid of the invention has a poly(alkylene oxide) tail,
especially a poly(ethylene oxide) tail.
[0094] Particular compositions include liposome formulations,
prepared using conventional liposome technology. Otherwise, the
compositions may take any other supermolecular form suitable for
oral, buccal, parenteral, nasal, topical or rectal administration,
or a form suitable for administration by inhalation or
insufflation.
[0095] For oral administration, the compositions may take the form
of, for example, tablets, lozenges or capsules prepared by
conventional means with pharmaceutically acceptable excipients such
as binding agents (e.g. pregelatinised maize starch,
polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers
(e.g. lactose, microcrystalline cellulose or calcium hydrogen
phosphate); lubricants (e.g. magnesium stearate, talc or silica);
disintegrants (e.g. potato starch or sodium glycollate); or wetting
agents (e.g. sodium lauryl sulphate). The tablets may be coated by
methods well known in the art. Liquid preparations for oral
administration may take the form of, for example, solutions, syrups
or suspensions, or they may be presented as a dry product for
constitution with water or other suitable vehicle before use. Such
liquid preparations may be prepared by conventional means with
pharmaceutically acceptable additives such as suspending agents,
emulsifying agents, non-aqueous vehicles and preservatives. The
preparations may also contain buffer salts, flavouring, colouring
and sweetening agents as appropriate.
[0096] Preparations for oral administration may be suitably
formulated to give controlled release of the active compound.
[0097] For buccal administration the compositions may take the form
of tablets or lozenges formulated in conventional manner.
[0098] The complexes of the invention may be formulated for
parenteral administration by injection, including bolus injection
or infusion or particle mediated injection. Formulations for
injection may be presented in unit dosage form, e.g. in glass
ampoule or multi dose containers, e.g. glass vials or a device
containing a compressed gas such as helium for particle mediated
administration. The compositions for bolus injection or infusion
may take such forms as suspensions, solutions or emulsions in oily
or aqueous vehicles, and may contain formulatory agents such as
suspending, stabilising, preserving and/or dispersing agents.
Alternatively, the complex may be in powder form for constitution
with a suitable vehicle, e.g. sterile pyrogen-free water, before
use. For particle mediated administration the complex may be coated
on particles such as microscopic gold particles.
[0099] In addition to the formulations described above, the
complexes may also be formulated as a depot preparation. Such long
acting formulations may be administered by implantation or by
intramuscular injection.
[0100] For nasal administration or administration by inhalation,
the complexes may be conveniently delivered in the form of an
aerosol spray presentation for pressurised packs or a nebuliser,
with the use of suitable propellant, e.g. dichlorodifluoromethane,
trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide
or other suitable gas or mixture of gases.
[0101] The complexes may, if desired, be presented in a pack or
dispenser device which may contain one or more unit dosage forms
containing the active ingredient. The pack or dispensing device may
be accompanied by instructions for administration.
[0102] The quantity of lipid complex required for any particular
application will to a large extent depend on the nature of the
bioactive substance being delivered. Another important factor will
include whether the lipid complex is intended for in vitro or in
vivo use. If the latter the route of administration and particular
formulation chosen as well as factors such as the age and condition
of the subject will govern the quantity of lipid complex used. In
general however up to around 50 mg of lipid complex can be used for
every kilogram of body weight.
[0103] The following Examples illustrate the invention. In these,
the preparation of the lipids of the invention and the
intermediates thereto has been divided into sections for ease of
understanding as follows:
INTERMEDIATES
[0104] Section
[0105] B. Polyamine Intermediates
[0106] C. Disugar Intermediates
[0107] D. Long Chain Amino Acid Syntheses
[0108] E. Lipid Polyamines
[0109] F. Glycolipid Syntheses
[0110] G. Two Lipid Chain Syntheses
Lipids of the Invention
[0111] Section
[0112] H. Carbohydrate Lipid Tetramines and Hexamines
[0113] I. PEG Lipids
[0114] Compounds are referred to throughout the text by their
Section numbering B1, B2, B3 . . . etc. The following abbreviations
are also used:
1 BOC--t-butoxycarbonyl; DCM--dichloromethane;
EDC--1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
TFA--trifluoroacetic acid; LDA--lithium diisopropylamide;
DBU--1,8-diazabicyclo[5.4.O]un-dec-7-ene; PDC--pyridinium
dichromate; Me--CH.sub.3, Ph--phenyl; Ac--acetate; Bn--benzyl
tlc--thin layer chromatography THF--tetrahydrofuran;
DMF--dimethylformamide DMSO--dimethylsulphoxide
DMAP--dimethylaminopyridine;
[0115] B. Polyamine Intermediates
[0116] This section contains the syntheses of:
[0117] BOC Protected Tetramine
[0118] (B8)
N-(t-Butyloxycarbonyl)-N-(t-butyloxycarbonylaminobutyl)-2-[t-b-
utyloxycarbonylaminobutyl(t-butyloxycarbonyl)-aminomethyl]octane-1,8-diami-
ne 1
[0119] BOC Protected Hexamine
[0120] (B16)
11-Aminohexyl-4,9,13,18-tetrakis(t-butyloxy-carbonyl)-4,9,13,-
18-tetraazaeicosane-1,21-diamine 2
[0121] BOC Protected Dimethylated Hexamine
[0122] (B21)
N-[Methyl(t-butyloxycarbonyl)aminopropyl(t-butyloxy-carbonyl)-
aminobutyl(t-butyloxycarbonyl)]-2-[methyl(t-butyloxycarbonyl)aminopropyl(t-
-butyloxycarbonyl)-amino
butyl(t-butyloxycarbonyl)aminomethyl]-1,8-octaned- iamine 3
[0123] BOC Protected Tetramine
[0124] (B1) 1-Benzyloxy-6-chlorohexane. 4
[0125] To a stirred solution of 6-chloro-1-hexanol (15.0 g, 0.110
mol) and benzyl bromide (18.8 g, 0.110 mol) in dry THF (200 ml) at
room temperature under argon was slowly added (over 30 min.) sodium
hydride powder (2.899 g, 0.121 mol). The solution was left
overnight and the solids filtered off. The solvent was removed
under reduced pressure, the residues taken up into dichloromethane
(100 ml) and washed (2.times.20 ml water). The solution was dried
over MgSO.sub.4 and the solvent removed to yield an orange oil
which was subsequently distilled (160.degree. C., 0.5 mBar) to
yield the title compound (17.5 g, 70%) as a colourless oil.
C.sub.13H.sub.19OCl requires 226. Found DCl: MNH.sub.4.sup.+, 244.
.delta. H(CDCl.sub.3) 1.49 (4H, m,
(CH.sub.2).sub.2(CH.sub.2).sub.2Cl), 1.70 (2H, p,
CH.sub.2CH.sub.2O), 1.83 (2H, t, CH.sub.2CH.sub.2Cl), 3.54, 3.56
(4H, 2.times.t, CH.sub.2CH.sub.2O, CH.sub.2Cl), 4.56 (2H, s,
CH.sub.2Ph), 7.40 (5H, m, Ph). .delta. C (CDCl.sub.3) 25.3 (1C,
CH.sub.2(CH.sub.2).sub.2Cl), 26.4 (1C, CH.sub.2(CH.sub.2).sub.3Cl,
29.3 (1C, CH.sub.2(CH.sub.2).sub.2O), 32.3 (1C,
CH.sub.2CH.sub.2Cl), 44.8 (1 C, CH.sub.2Cl), 69.9, 72.6 (2C,
CH.sub.2O), 127.2 (1C, CH(CH.sub.2).sub.2C), 127.3 (2C, CHC), 128.1
(2C, CHCHC), 138.4 (1C, CCH.sub.2O).
[0126] (B2) Diethyl 2-(benzyloxyhexyl)malonate 5
[0127] To sodium metal (1.32 g, 0.0573 mol) dissolved in dry
ethanol (150 ml) under argon at room temperature was added diethyl
malonate (14.13 g, 0.088 mol) over a period of 10 minutes and the
solution left for 2 hours. B1 (10.0 9, 0.044 mol) was then added
dropwise over 2 hours and this solution heated at reflux overnight.
Excess salts were quenched by the addition of 50 ml of water and
the total solvent volume reduced to 50-100 ml. Diethyl ether (100
ml) and water (50 ml) were added and the aqueous layer extracted 3
times with diethyl ether. The organic fractions were combined,
dried (K.sub.2CO.sub.3), and the solvent removed to yield the crude
product as a yellow oil. Distillation of the oil (0.5 mBar,
220.degree. C.) gave the title compound (10.52 g, 68%) as a
colourless viscous oil. .delta. H (CDCl.sub.3) 1.32 (6H, t, Me),
1.43 (6H, br s, (CH.sub.2).sub.3CH.sub.2CH), 1.69 (2H, p,
CH.sub.2CH.sub.2O), 1.99 (2H, q, CH.sub.2CH), 3.39 (1H, t, CH),
3.53 (2H, t, CH.sub.2OCH.sub.2Ph), 4.25, 4.28 (4H, 2.times.q,
OCH.sub.2Me), 4.57 (2H, s, CH.sub.2Ph), 7.3-7.4 (5H, m, Ph).
.delta. C (CDCl.sub.3) 13.9 (2 C, Me), 25.7 (1 C,
CH.sub.2(CH.sub.2).sub.2CH), 27.1 (1 C, CH.sub.2(CH.sub.2).sub.2O),
28.5 (1 C, CH.sub.2CH.sub.2CH), 28.9 (1 C, CH.sub.2CH.sub.2O), 29.5
(1 C, CH.sub.2CH), 51.8 (CHCO.sub.2Et), 61.0 (2C, OCH.sub.2Me),
70.1, 72.7 (2C, CH.sub.2OCH.sub.2), 127.3 (1C, CH(CH).sub.2C),
127.4 (2C, CHC), 128.1 (2C, CHCHC), 138.5 (1C, CCH.sub.2O) 169.3
(2C, CO.sub.2).
[0128] (B3) N,N'-bis(Aminobutyl)-2-(benzyloxyhexyl)malonamide 6
[0129] To a large excess of 1,4-diaminobutane (85 g, 0.964 mol) at
90.degree. C. under argon was slowly added (over 2 hours) B2 (10.50
g, 0.030 mol). The solution was heated at 90.degree. C. overnight
and the excess diamine distilled off under reduced pressure (0.1
mBar, 28.degree. C.) to quantitatively yield the title compound as
a pale yellow low melting point solid.
C.sub.24H.sub.42N.sub.4O.sub.3 requires 434. Found DCl: M.sup.++1
435. I.R. 1664 cm.sup.-1 (CO.sub.2). .delta. H (CDCl.sub.3)
1.15-1.40 (10H, br m, (CH.sub.2).sub.3CH.sub.2CH, NH.sub.2),
1.40-1.60 (10H, br m, (CH.sub.2).sub.2CH.sub.2NH.sub.2,
CH.sub.2CH.sub.2O), 1.76 (2H, q, CH.sub.2CH), 2.63 (4H, t,
CH.sub.2NH.sub.2), 2.93 (2H, t, CH.sub.2CH), 3.16 (4H, q,
CH.sub.2NH), 3.37,(2H, t, CH.sub.2OCH.sub.2Ph), 4.42 (2H, s,
CH.sub.2Ph), 7.26 (5H, m, Ph), 7.61 (2H, t, CONH). .delta. C
(CDCl.sub.3) 25.7 (1C, CH.sub.2(CH.sub.2).sub.2CH), 26.6 (2C,
CH.sub.2CH.sub.2NH.sub.2), 28.8 29.4 (2C, CH.sub.2CH.sub.2CH,
CH.sub.2CH.sub.2O), 30.6 (2C, CH.sub.2CH.sub.2NH), 32.7 (1C,
CH.sub.2CH), 39.0 (2C, CH.sub.2NHCO), 41.4 (2C, CH.sub.2NH.sub.2),
54.7 (1C, CHCO), 70.1, 72.6 (2C, CH.sub.2OCH.sub.2), 127.2 (1C,
CH(CH).sub.2C), 127.3 (2C, CHC), 128.0 (2C, CHCHC), 138.5 (1C,
CCH.sub.2O), 171.0 (2C, CONH).
[0130] (B4)
N,N'-bis(Aminobutyl)-2-benzyloxyhexyl-1,3-propanediamine 7
[0131] To B3 (0.430 g, 0.989 mmol) under argon was added an excess
(25 ml) of 1.0 M BH.sub.3-THF, and the solution heated at
85.degree. C. overnight. Excess borane was slowly quenched with
methanol (10 ml) and the solvents removed under reduced pressure.
The residues were taken up into 0.1 M HCl (100 ml), heated at
60.degree. C. for 1 hour, and the solvent removed under reduced
pressure. The residues were entrained with methanol (4.times.20
ml), and the tetraamine hydrochloride salt taken up into water (30
ml). The solution was basified to pH.about.14 with sodium hydroxide
and exhaustively extracted with dichloromethane. The organic
fractions were combined, dried (K.sub.2CO.sub.3), and the solvent
removed to yield the title compound (0.352 g, 88%) as a pale yellow
oil/gum. C.sub.24H.sub.46N.sub.4O requires 406. Found DCl:
M.sup.++1, 407. .delta. H (CDCl.sub.3) 1.0-1.6 (25H, br m,
CH(CH.sub.2).sub.5, NH, NH.sub.2,
(CH.sub.2).sub.2CH.sub.2NH.sub.2), 2.2-2.6 (12H, br m, CH.sub.2N),
3.28 (2H, t, CH.sub.2OCH.sub.2Ph), 4.31 (2H, s, CH.sub.2Ph), 7.14
(5H, m, Ph). .delta. C (CDCl.sub.3) 25.3 (1C, CH.sub.2CH.sub.2CH),
26.2 (1C, CH.sub.2(CH.sub.2).sub.2CH), 26.6 (2C
CH.sub.2CH.sub.2NH.sub.2), 28.9 (2C,
CH.sub.2(CH.sub.2).sub.2CH.sub.2CH), 30.7 (3 C, CH.sub.2CH.sub.2NH,
CH.sub.2CH.sub.2O), 37.6 (1C, CH), 41.2 (2C, CH.sub.2NH.sub.2),
49.3 (2C, (CH.sub.2).sub.3CH.sub.2NH), 53.5 (2C, CHCH.sub.2NH),
69.5, 71.9 (2C, CH.sub.2OCH.sub.2), 126.5 (1C, CH(CH).sub.2C),
126.6 (2C, CHC), 127.4 (2 C, CHCHC), 137.8 (1C, CCH.sub.2O).
[0132]
(B5)N,N'-bis(t-Butyloxycarbonyl)-N-[2-t-butyloxycarbonyl-aminobutyl-
(t-butyloxycarbonyl)aminomethyl]benzyloxyoctyl-1,4-diaminobutane.
8
[0133] B4 (10 g, 24.6 mmol) was dissolved in aqueous sodium
hydroxide (100 ml, 2M). To this solution was added
t-butyidicarbonate (27 g, 123.3 mmol) portionwise with stirring at
room temperature. The reaction was stirred overnight, water added
and the aqueous phase extracted with ethyl acetate. The combined
organic extracts were washed with water, brine, dried &
evaporated to give the crude product. Chromatography (silica--20%
ethyl acetate in hexane) gave the title compound as a glass (11 g,
55%). C.sub.44H.sub.78N.sub.4O.sub.9 requires 806. Found DCl:
M.sup.++1, 807.8. .delta. H (CDCl.sub.3) 7.74(5H, m, ArH), 4.49
(2H, s, CH.sub.2Ar), 3.45(2H, t, CH.sub.2OCH.sub.2Ph), 2.9-3.3 (12
H, m, CH.sub.2N), 1.95 (1H,brm,CH.sub.2CH(CH.sub.2).sub.2), 1.2-1.7
(52H,m,CH.sub.2).
[0134] (B6)
8-[t-Butyloxycarbonylaminobutyl(t-butyloxycarbonyl)-amino]-7-[-
t-butyloxycarbonylaminobutyl(t-butyloxy-carbonyl)aminomethyl]octanol
9
[0135] To B5 (11 g) in methanol (70 ml) under argon was added
palladium on carbon catalyst (1 g). The stirred suspension was put
under a hydrogen atmosphere for two days at atmospheric pressure
and room temperature. The mixture was filtered through Celite which
was washed with dichloromethane, Evaporation yielded the title
compound (9.67 g, 87%) which was used for the next stage without
purification.
[0136] (B7)
8-[t-Butyloxycarbonylaminobutyl(t-butyloxycarbonyl)-amino]-7-[-
t-butyloxycarbonylaminobutyl(t-butyloxy-carbonyl)aminomethyl]octylmethanes-
ulphonate 10
[0137] Methanesulphonyl chloride (1.25 ml, 16.21 mmol) was added to
a stirred solution of B6 (9.67 g, 13.5 mmol) in dry dichloromethane
(100 ml) containing triethylamine (2.82 ml, 20.26 mmol) at
0.degree. C. The flask was stirred at 0.degree. C. for 1 hr, then
overnight at room temperature. The solvent was removed and the
residue chromatographed (silica--50% ethyl acetate in hexane) to
yield the title compund (8.5 g, 79%.
C.sub.38H.sub.74N.sub.4O.sub.11S requires 794. Found DCl:
M.sup.++1, 795.6. .delta. H (CDCl.sub.3) 3.45(2H, t, CH.sub.2O),
2.9-3.3 (12H, m, CH.sub.2N), 2.99 (3H, s, MeS), 1.95 (1
H,brm,CH.sub.2CH(CH.sub.2).sub.2), 1.2-1.8 (50H,m,CH.sub.2).
[0138] (B8)
N-(t-Butyloxycarbonyl)-N-(t-butyloxycarbonyiaminobutyl)-2-[t-b-
utyloxycarbonylaminobutyl(t-butyloxycarbonyl)-aminomethyl]octane-1,8-diami-
ne 11
[0139] Sodium azide (2.05 g, 31.6 mmol) was added to a stirred
solution of B7 (8.36 g, 10.53 mmol) in dry DMF (35 ml) at room
temperature. The reaction was stirred for 48 hr and water added.
The aqueous solution was extracted with ethyl acetate. The organic
phase was washed with brine, dried (MgSO.sub.4) and evaporated to
dryness. The product was chromatographed (silica--up to 50% ethyl
acetate in hexane) and the resulting azide was dissolved in
methanol (100 ml) under argon and palladium on charcoal added. The
atmosphere was changed to hydrogen and the reaction stirred
overnight. The catalyst was filtered off and the product
chromatographed (silica--up to 20% methanol in dichloromethane
containing triethylamine to yield the title compound (4.5 g).
C.sub.37H.sub.73N.sub.5O.sub.8 requires 715.55. Found DCl:
M.sup.++1, 716.7. .delta. H (CDCl.sub.3) 4.71(2H, br, NHBoc),
2.95-3.3(12H, m, CH.sub.2N), 2.85 (2H, br, NH.sub.2), 2.75 (2H, t,
CH.sub.2NH.sub.2), 1.95 (1H,brm,CH.sub.2CH(CH.sub.2).sub.2),
1.2-1.7 (54H,m,CH.sub.2).
[0140] BOC Protected Hexamine Synthesis
[0141] (B9)
N,N'-bis(p-Methoxyphenylsulphonyl)-N-{2-[p-methoxy-phenylsulph-
onylaminobutyl(p-methoxyphenyl-sulphonyl)aminomethyl]benzyloxyoctyl}-1,4-b-
utane-diamine 12
[0142] To B4 (301 mg, 0.740 mmol) and triethylamine (749 mg, 7.40
mmol) in dry THF (30 ml) under argon at -50.degree. C. was slowly
added p-methoxybenzenesulphonyl chloride (627 mg, 3.03 mmol). The
solution was allowed to slowly warm to room temperature and left
overnight. The product (tlc r.f. 0.7, 2% methanol in
dichloromethane) was purified by gradient alumina column
chromatography (0-2% methanol in dichloromethane) to yield the
title compound (610 mg, 76%) as a colourless gumisolid. .delta. H
(CDCl.sub.3) 1.15-1.70 (18 H, br m, (CH.sub.2).sub.5CH.sub.2O,
(CH.sub.2).sub.2CH.sub.2NH), 2.00 (1H, t, CH), 2.75-3.15 (12H, br
m, CH.sub.2N), 3.45 (2H, t, CH.sub.2OCH.sub.2Ph), 3.81, 3.84 (12H,
2.times.s, OMe), 4.47 (2H, s, CH.sub.2Ph), 5.25 (2H, t, NH), 6.92,
6.97 (8H, 2.times.d, CHCSO.sub.2), 7.29 (5H, m, Ph), 7.69, 7.75
(8H, 2.times.d, CHCOMe). .delta. C (CDCl.sub.3) 25.2-26.2 (5C,
CH(CH.sub.2).sub.5), 29.2, 29.3 (4C, (CH.sub.2).sub.2CH.sub.2NH),
36.0 (1C, CH), 42.1 (2C, CH.sub.2NH), 48.8 (2C,
(CH.sub.2).sub.3CH.sub.2NH), 51.2 (2C, CHCH.sub.2N), 55.2 (4 C,
OMe), 70.0, 72.3 (2 C, CH.sub.2OCH.sub.2), 113.8, 114.0 (8C,
CHCSO.sub.2), 127.0, 127.2, 127.9 (5C, Ph), 128.7, 128.9 (8C,
CHCOMe), 129.8, 131.0 (4C, CSO.sub.2), 138.2 (1C, CCH.sub.2O),
162.3, 162.5 (4 C, COMe).
[0143] (B10)
11-Benzyloxyhexyl-4,9,13,18-tetrakis(p-toluene-sulphonyl)-4,9-
,13,18-tetraazaeicosane-1,21-dinitrile 13
[0144] To B9 (588 mg, 0.541 mmol) and mesh potassium carbonate (523
mg, 3.784 mmol) in 20 ml of dry DMF under argon was added freshly
distilled acrylonitrile (95 mg, 1.787 mmol) and the reaction left
stirring at room temperature for 34 days. T.l.c (alumina 2% MeOH in
CH.sub.2Cl.sub.2) indicated the presence of two compounds r.f 0.9
and 0.8, later shown to be the desired di-nitrile and the
mono-nitrile respectively. Gradient alumina column chromatography
(as for B9) yielded the title compound as a colourless gum/solid in
52% (337 mg) yield. C.sub.58H.sub.76N.sub.6O.sub.- 13S.sub.4
requires 1193 Found ES+: MNa.sup.+ 1216. .delta. H (CDCl.sub.3)
1.15-1.40 (8H, br m, CH(CH.sub.2).sub.4), 1.40-1.70 (10H, br,
(CH.sub.2).sub.2CH.sub.2N, CH.sub.2CH.sub.2O), 2.02 (1 H, t, CH),
2.63 (4H, t, CH.sub.2CN), 2.80-3.16 (12H, br m, CH.sub.2N), 3.24
(4H, t CNCH.sub.2CH.sub.2N), 3.47 (2H, t, CH.sub.2OCH.sub.2Ph),
3.81, 3.82 (12H, 2.times.s, OMe), 4.46 (2H, s, CH.sub.2Ph), 6.95,
6.96 (8 H, 2.times.d, CHCSO.sub.2), 7.29 (5H, m, Ph), 7.69, 7.70 (8
H, 2.times.d, CHCOMe). .delta. C (COCl.sub.3) 18.9 (2C,
CH.sub.2CN), 25.3-28.3 (5C, CH(CH.sub.2).sub.5), 29.6, 29.7 (4C,
(CH.sub.2).sub.2CH.sub.2N), 36.4 (1C, CH), 44.5 (2C,
CNCH.sub.2CH.sub.2), 48.7 (2C, CH.sub.2N(CH.sub.2).sub.2CN), 49.0
(2C, CH.sub.2NCH.sub.2CH), 51.5 (2C, CHCH.sub.2N), 55.5 (4C, OMe),
70.3, 72.7 (2C, CH.sub.2OCH.sub.2), 114.2, 114.4 (8C, CHCSO.sub.2),
117.7 (2C, CN), 127.3, 127.5, 128.2 (5C, Ph), 129.2 (8C, CHCOMe),
129.5, 130.2 (4C, CSO.sub.2), 138.5 (1C, CCH.sub.2O), 162.7, 163.0
(4C, COMe).
[0145] (B11)
11-Benzyloxyhexyl-4,9,13,18-tetrakis(p-toluene-sulphonyl)-4,9-
,13,18-tetraazaeicosane-1,21-diamine 14
[0146] To B10 (337 mg, 0.282 mmol) under argon was added an excess
(30 ml) of 1.0 M BH.sub.3-THF, and the solution heated at
85.degree. C. overnight. Excess borane was slowly quenched with
methanol (10 ml) and the solvents removed under reduced pressure.
The residues were taken up into 0.1 M HCl (100 ml), heated at
60.degree. C. for 1 hour, and the solvent removed under reduced
pressure. The residues were entrained with methanol (4.times.20
ml), and the hydrochloride salt taken up into water (30 ml). The
solution was basified to pH>14 with sodium hydroxide and
exhaustively extracted with dichloromethane. The organic fractions
were combined, dried (K.sub.2CO.sub.3), and the solvent removed to
yield the title compound (285 mg, 84%) as a colourless gum/solid.
C.sub.58H.sub.84N.sub.6O.sub.13S.sub.4 requires 1201. Found ES+:
MH.sup.+ 1202. .delta. H (CDCl.sub.3) 1.2-1.7 (26H, br m,
CH(CH.sub.2).sub.5, CH.sub.2(CH.sub.2).sub.2CH.sub.2N,
CH.sub.2CH.sub.2NH.sub.2), 2.20 (1H, t, CH), 2.70 (4H, t,
CH.sub.2NH.sub.2), 2.85-3.35 (16H, br m, CH.sub.2N), 3.50 (2H, t,
CH.sub.2OCH.sub.2Ph), 3.86, 3.88 (12H, 2.times.s, OMe), 4.52 (2H,
s, CH.sub.2Ph), 6.99, 7.02 (8 H, 2.times.d, CHCSO.sub.2), 7.75,
7.76 (8H, 2.times.d, CHCOMe). .delta. C (CDCl.sub.3) 25.1-26.2 (5C,
CH(CH.sub.2).sub.5), 29.4, 29.6 (4C,
CH.sub.2(CH.sub.2).sub.2CH.sub.2N), 32.0 (2C,
CH.sub.2CH.sub.2NH.sub.2), 36.2 (1C, CH), 38.8 (2C,
CH.sub.2NH.sub.2), 45.9 (2C, CH.sub.2(CH.sub.2).sub.2NH.sub.2),
47.7 (2C, CH.sub.2N(CH.sub.2).sub.3NH.sub.2), 48.7 (2C,
CHCH.sub.2NCH.sub.2), 51.1 (2C, CHCH.sub.2N), 55.3 (4C, OMe), 70.1,
72.5 (2C, CH.sub.2OCH.sub.2), 113.9, 114.0 (8C, CHCSO.sub.2),
127.1, 127.3, 128.0 (5C, Ph), 128.8, 129.0 (8C, CHCOMe), 130.2,
130.7 (4C, CSO.sub.2), 138.5 (1C, CCH.sub.2O), 162.4, 162.5 (4C,
COMe).
[0147] (B12)
8-(Aminopropylaminobutylamino)-7-(aminopropyl-aminobutylamino-
methyl)octanol 15
[0148] To B11 (743 mg, 0.618 mmol) in THF (30 ml) and ethanol (2
ml) at -78.degree. C. was condensed liquid ammonia (75 ml). To this
was then added 300 mg of lithium metal (turned blue), and the
solution stirred for 2 hours. The solution was then allowed to
slowly warm to room temperature overnight, boiling off the ammonia
as it did. Ethanol (2 ml) followed by water (70 ml) were added to
the now yellow solution and the organic solvents removed under
reduced pressure. The pH of the remaining aqueous solution was
lowered to 2 (concentrated HCl), washed (4.times.20 ml) with
diethyl ether, and basified to pH 10-12 (NaOH). The solvent was
removed to yield the title compound in the presence of a large
excess of salts. This material was used for the next step without
purification
[0149] (B13)
8-[t-Butyloxycarbonylaminopropyl(t-butyloxy-carbonyl)aminobut-
yl(t-butyloxycarbonyl)amino]-7-[t-butyloxycarbonylaminopropyl(t-butyloxyca-
rbonyl)-aminobutyl(t-butyloxycarbonyl)aminomethyl]octanol 16
[0150] To B12 dissolved in methanol (5 ml) was added 10 equivalents
of ditertbutyl dicarbonate (1.349 g, 6.183 mmol) and the solution
left overnight. The solvent was removed, the residues taken up into
water (20 ml), and extracted with dichloromethane (5.times.30 ml).
The organic fractions were combined, dried (K.sub.2CO.sub.3), and
the solvent removed to yield a pale yellow gum. Purification
required alumina column chromatography (0-3% methanol in
dichloromethane) to yield the desired BOC protected title compound
(alumina tlc r.f. 0.7, 5% methanol in dichloromethane) as a
colourless gum (281 mg, 44%). C.sub.53H.sub.102N.sub.6O.sub.13
requires 1031. Found ES+: MH.sup.+ 1032, ES-: M.sup.- 1031,
MCl.sup.- 1066. .delta. H (CDCl.sub.3) 1.1-2.0 (77H, br m,
C(Me).sub.3, CH(CH.sub.2).sub.5, CH.sub.2(CH.sub.2).sub.2CH.sub.2N,
CH.sub.2CH.sub.2NH), 2.7-3.4 (20H, br, CH.sub.2N), 3.53 (2H, t,
CH.sub.2O), 5.26, 5.45 (2H, br, NHBOC). .delta. C (CDCl.sub.3)
partial 43.5, 46.5, 48.9 (10C, CH.sub.2N), 62.1 (1C, CH.sub.2OH),
78.5, 79.1 (6C, C(Me).sub.3), 155.3, 155.7 (6 C, CO).
[0151] (B14)
8-[t-Butyloxycarbonylaminopropyl(t-butyloxycarbonyl)-aminobut-
yl(t-butyloxycarbonyl)amino]-7-[t-butyloxy-carbonylaminopropyl(t-butyloxyc-
arbonyl)aminobutyl(t-butyloxycarbonyl)aminomethyl]octylmethane-sulphonate
17
[0152] To B13 (555 mg, 0.538 mmol) and triethylamine (163 mg, 1.614
mmol) in dry dichloromethane (20 ml) at -20 .degree. C. under argon
was added mesyl chloride (124 mg, 1.076 mmol) dropwise in
dichloromethane (5 ml) over a period of 30 minutes. The solution
was allowed to warm to room temperature and left overnight. The
solvent was removed to quantitatively yield the title compound, tlc
r.f. 0.35 (5% methanol in dichloromethane on alumina). .delta. H
(CDCl.sub.3) 1.1-2.0 (77H, br m, C(Me).sub.3, CH(CH.sub.2).sub.5,
CH.sub.2(CH.sub.2).sub.2CH.sub.2N, CH.sub.2CH.sub.2NH), 2.7-3.4
(20H, br, CH.sub.2N), 3.01 (3H, s, MeS), 4.18 (2H, t, CH.sub.2O),
5.26, 5.45 (2H, br, NHBOC). .delta. C (CDCl.sub.3) partial 38.4
(MeS), 43.5, 46.5, 48.9 (10C, CH.sub.2N), 69.3 (C, CH.sub.2O) 78.5,
79.1 (6C, C(Me).sub.3), 155.3, 155.7 (6C, CO).
[0153] (B15)
11-Azidohexyl-N,N',4,9,13,18-hexa(t-butyloxycarbonyl)-4,9,13,-
18-tetraaza-1,21-eicosanediamine 18
[0154] To the crude mesylate B14 in dry DMF (15 ml) under argon was
added excess sodium azide (600 mg) and the solution/suspension
stirred overnight. The volume was reduced to approximately 5 ml and
added to 70 ml of water. This aqueous phase was extracted with
ethyl acetate (10.times.30 ml) and the organics combined, dried
(MgSO.sub.4) and the solvent removed to yield the title compound as
a pale yellow gum (570 mg, 100%). I.R. 2095 cm.sup.-1 (N.sub.3).
C.sub.53H.sub.101N.sub.9O.sub.12 requires 1056. Found ES+: MH.sup.+
1057, MNa.sup.+ 1079. .delta. H (CDCl.sub.3) 1.1-2.0 (77H, br m,
C(Me).sub.3, CH(CH.sub.2).sub.5, CH.sub.2(CH.sub.2).sub.2CH.sub.2N,
CH.sub.2CH.sub.2NH), 2.7-3.4 (20H, br, CH.sub.2N), 3.31(2H, t,
CH.sub.2N.sub.3), 5.26, 5.45 (2H, br, NHBOC). .delta. C
(CDCl.sub.3) partial 43.5, 46.5, 48.9 (10C, CH.sub.2N), 51.3 (1C,
CH.sub.2N.sub.3), 78.5, 79.1 (6C, C(Me).sub.3), 155.3, 155.7 (6C,
CO).
[0155] (B16)
11-Aminohexyl-4,9,13,18-tetrakis(t-butyloxycarbonyl)-4,9,13,1
8-tetraazaeicosane-1,21-diamine 19
[0156] To B15 (134 mg, 0.127 mmol) in methanol (15 ml) was added
10% Pd/C (40 mg) and the suspension stirred overnight under an
atmosphere of hydrogen. Removal of the catalyst by filtration
through Celite followed by removal of the solvent gave the desired
title compound as a colourless gum in quantitative yield (131 mg).
C.sub.53H.sub.103N.sub.7O.sub.12 requires 1030. Found ES+: MH.sup.+
1031, MHNa.sup.2+ 527. .delta. H (CDCl.sub.3) 1.1-2.0 (79H, br m,
NH.sub.2, C(Me).sub.3, CH(CH.sub.2).sub.5,
CH.sub.2(CH.sub.2).sub.2CH.sub.2N, CH.sub.2CH.sub.2NH), 2.7-3.4
(22H, br, CH.sub.2N), 5.26, 5.45 (2H, br, NHBOC). .delta. C
(CDCl.sub.3) partial 39.8 (1C, CH.sub.2NH.sub.2), 43.5, 46.5, 48.9
(10C, CH.sub.2N), 78.5, 79.1 (6C, C(Me).sub.3), 155.3, 155.7 (6C,
CO).
[0157] BOC Protected Dimethylated Hexamine
[0158] (B17)
N,N'-bis(t-Butyloxycarbonylaminopropanoyl-aminobutyl)-2-(benz-
yloxyhexyl)malonamide 20
[0159] To a solution of BOC-.beta.-alanine (2.5 g, 13.22 mmol) in
dry dichloromethane (20 ml) was added N-methylmorpholine (1.6 ml,
14.55 mmol) followed by N-hydroxysuccinimide (91.67 g, 14.55 mmol).
The flask was left stirring at 20.degree. C. for 5 mins under argon
before adding EDC (2.79 g, 14.55 mmol). The reaction was stirred
overnight. TIc showed that all the acid had been converted to the
active ester. A solution of the B3 (2.86 g, 6.61 mmol) and
triethylamine (4.6 ml, 33.05 mmol) in dichloromethane was added and
the reaction stirred for 1 hr. A precipitate formed. The product
was purified by chromatography (silica--5-10% methanol in
dichloromethane) to give the title compound as a glass (2.31 g,
46%). The product on Tlc is green when sprayed with ninhydrin and
contains bis-BOC-1,4-diaminobutane, an impurity in the starting
material. C.sub.40H.sub.60N.sub.6O.sub.9 requires 776.5. Found ES:
M.sup.++1 777.6. .delta. H (CDCl.sub.3) 7.31 (5H, m, PhCH.sub.2O),
4.47 (2H, s, PhCH.sub.2O), 3.45 (2H, t, PhCH.sub.2OCH.sub.2),
3.4-3.1 (12H, dm, CH.sub.2N), 3.02 (1H, t, CHR.sub.3), 2.34 (4H, t,
COCH.sub.2), 1.9-1.2 (26H, m, CH.sub.2+Me).
[0160] (B18)
13-Benzyloxyhexyl-2,6,11,15,20,24-hexa(t-butyloxy-carbonyl)-2-
,6,11,15,20,24-hexaazapentacosane 21
[0161] B17 (15.36 g, 15.36 mmol) was suspended in THF (240 ml) and
borane methylsulphide complex (10M, 32 ml, 320 mmol) in THF (50 ml)
added dropwise. Hydrogen was evolved and the tetraamide dissolved
over 1 hr. The reaction was refluxed for 48 hr and carefully
quenched with methanol. Solvent was removed in vacuo and
hydrochloric acid added (6M, 100 ml). The reaction was refluxed for
1 hr at 60.degree. C. and the hydrochloric acid removed in vacuo.
The product was entrained in methanol and the solvent removed. The
residue was dissolved in methanol containing 20% water and basified
with solid sodium hydroxide and tert-butyl dicarbonate (32.7 g, 150
mmol) was added whilst maintaining the pH at 12. The basic solution
was extracted with ethyl acetate, washed with brine, dried
(MgSO.sub.4) and evaporated to dryness. The residue was columned on
silica (33% ethyl acetate in hexane) to give the title compound as
an oil. C.sub.60H.sub.106N.sub.6O.sub.13 requires 1142. .delta. H
(CDCl.sub.3) 7.31 (5H, m, PhCH.sub.2O), 4.48 (2H, s, PhCH.sub.2O),
3.44 (2H, t, PhCH.sub.2OCH.sub.2), 3.4-3.1 (20H, m, CH.sub.2N),
3.02 (1H, t, CHR.sub.3), 2.82 (6H, s, NMe), 1.9-1.2 (76H, m,
CH.sub.2+Me).
[0162] (B19)
8-[Methyl(t-butyloxycarbonyl)aminopropyl(t-butyloxy-carbonyl)-
aminobutyl(t-butyloxycarbonyl)amino]-7-[methyl(t-butyloxycarbonyl)aminopro-
pyl(t-butyloxy-carbonyl)aminobutyl(t-butyloxycarbonyl)aminomethyl]-octanol
22
[0163] B18 (10.23 g, 89.6 mmol) was dissolved in t-butanol (100 ml)
to which was added Raney nickel (8 ml) under argon. The atmosphere
was changed to hydrogen and the reaction stirred for 48 hr. The
Raney nickel was removed by filtration (glass fibre) and the
product purified by chromatography on silica (50-75% ethyl acetate
in hexane) to yield the title compound (6.75 g, 70%).
C.sub.55H.sub.106N.sub.6O.sub.13 requires 1058.8. Found ES:
MNa.sup.+ 1081.8 .delta. H (CDCl.sub.3). 3.62 (2H, t, HOCH.sub.2),
3.3-3.0 (20+1H, m, CH.sub.2N+CHR.sub.3), 2.84 (6H, s, NMe), 1.9-1.2
(76H, m, CH.sub.2+Me).
[0164] (B20)
8-[Methyl(t-butyloxycarbonyl)aminopropyl(t-butyloxy-carbonyl)-
aminobutyl(t-butyloxycarbonyl)amino]-7-[methyl(t-butyloxycarbonyl)aminopro-
pyl(t-butyloxy-carbonyl)aminobutyl(t-butyloxycarbonyl)aminomethyl]-octyl
methanesulphonate 23
[0165] B19 ( 6.75 g, 6.54 mmol) was dissolved in dichloromethane
(40 ml) containing triethylamine (1.2 ml) at 0.degree. C. Methane
sulphonyl chloride (0.6 ml, 7.8 mmol) was added in dichloromethane
(10 ml) under argon. After 2 hr Tlc showed complete reaction
(silica, 66% ethyl acetate in hexane). The title compound was
evaporated to dryness and used in the next step without further
purification. C.sub.56H.sub.108N.sub.6O.sub.15S requires 1136.76.
Found ES: M.sup.++1 1138.0 .delta. H(CDCl.sub.3). 4.21(2H, t,
HOCH.sub.2), 3.3-3.0 (20+1H, m, CH.sub.2N+CHR.sub.3), 2.84 (6H, s,
NMe), 2.99 (3H, s, SMe), 1.9-1.2 (76H, m, CH.sub.2+Me.
[0166] (21)
13-Aminohexyl-2,6,11,15,20,24-hexakis(t-butyloxy-carbonyl)-2,6-
,11,15,20,24-hexaazaeicosane 24
[0167] B20 ( 6.7 g, 6.5 mmol) was dissolved in DMF with heating,
cooled and sodium azide (2.5 g) added portionwise. After 24 hr
stirring the reaction was poured into brine (1.vertline.) and
extracted with ethyl acetate. The organic phase was washed with
water, dried and evaporated to dryness. This material was used for
the reduction without purification. The crude azide was dissolved
in t-butanol (50 ml) and 10% palladium on carbon (2 g) added under
argon. The atmosphere was changed to hydrogen and the reaction
stirred for 48 hr. The catalyst was removed and the product
evaporated to dryness. The product was purified by chromatography
(silica saturated with triethylamine, 1-10% methanol in DCM
containing 0.1% triethylamine) to yield the title compound (3 g) as
a glassy solid. C.sub.55H.sub.107N.sub.7O.sub.12 requires 1057.8.
Found ES: M.sup.++1 1058.7 .delta. H(CDCl.sub.3). 3.3-2.9(20+1H, m,
CH.sub.2N+CHR.sub.3), 2.83 (6H, s, NMe), 2.72 (2H, t,
NH.sub.2CH.sub.2), 1.9-1.1 (76H, m, CH.sub.2+Me).
[0168] C. Disugar Intermediates
[0169] This section contains the synthesis of the following:
[0170] (C4) N,N'-bis(Peracetylglucuronylaminoethyl)succinamic acid
25
[0171] (C1) bis(Glucuronylaminoethyl)amine 26
[0172] To a solution of d-gluconolactone (10.000 g, 56.14 mmol) in
dry methanol (200 ml) under argon was added diethylenetriamine
(2.825 g, 27.38 mmol) and the reaction stirred at room temperature
overnight resulting in the formation of white precipitates. The
solvent was removed from the resulting suspension giving a
quantitative yield of the title compound as a pure white solid.
C.sub.16H.sub.33N.sub.3O.sub.12 requires 459.2. Found ES.sup.+:
MH.sup.+, 460.2. .delta..sub.H (D.sub.2O) 2.79 (4H, t,
CH.sub.2NHCH.sub.2), 3.40 (4H, dt, CH.sub.2NHCO), 3.6-3.9 (8H, m,
CHOH), 4.09 (2H, d, CH.sub.2OH), 4.33 (2H, d, CH.sub.2OH).
[0173] (C2) N,N-bis(Glucuronylaminoethyl)-O-t-butylcarbamate 27
[0174] To C1 (7.000 g, 15.23 mmol) dissolved in methanol (235 ml)
and water (90 ml) were added di-tert-butyl dicarbonate (3.990 g,
18.28 mmol) and triethylamine (1.542 g, 15.23 mmol) and the
reaction stirred overnight at room temperature. The solvent was
removed to quantitatively yield the BOC protected title compound
which was used crude in the next synthetic step.
C.sub.20H.sub.37N.sub.3O.sub.15 requires 559.2. Found ES.sup.+:
MH.sup.+, 560.4. .delta..sub.H (D.sub.2O) 1.47 (9H, s,
C(Me).sub.3), 3.3-3.6 (8H, br, CH.sub.2N), 3.6-3.8 (8H, m, CHOH),
4.09 (2H, br s, CH.sub.2OH), 4.30 (2H, d, CH.sub.2OH).
[0175] (C3)
N,N'-bis(Peracetylglucuronylaminoethyl)-O-t-butyl-carbamate 28
[0176] To a solution of crude C2 (15.23 mmol) in dry pyridine (50
ml) at 0.degree. C. under argon was slowly added acetic anhydride
(120 ml) and the solution allowed to warm to room temperature
overnight. The majority of the solvent was removed and to the
residues was added water (100 ml) and ethyl acetate (150 ml). The
organic layer was separated off and the remaining aqueous layer
extracted with more ethyl acetate (5.times.50 ml). The organics
were combined, washed (1.times.citric acid, 4.times.water) and
dried (MgSO.sub.4) to yield on removal of the solvent a pale yellow
gum. Gradient silica column chromatography (80% ethyl acetate in
hexane to ethyl acetate) yielded the desired polyacetylated title
compound (13.1 g, 88% as a white solid.
C.sub.41H.sub.61N.sub.3O.su- b.24 requires 979.4. Found ES.sup.+:
MNa.sup.+, 1002.4. .delta..sub.H (CDCl.sub.3) 1.45 (9H, s,
C(Me).sub.3), 2.02, 2.03, 2.04, 2.06, 2.09 (30H, 5.times.s, MeCO),
3.34 (8H, br, CH.sub.2N), 4.10, 4.31 (4H, 2.times.dd, CH.sub.2OAc),
5.06 (2H, m, CHOAC), 5.23 (2H, br, CHOAc), 5.45 (2H, q, CHOAc),
5.61 (2H, t, CHOAc), 6.61, 7.18 (2H, 2.times.br, NHCO).
[0177] (C4) N,N'-bis(Peracetylglucuronylaminoethyl)succinamic acid
29
[0178] To C3 (3.000 g, 3.06 mmol) was added 1:1 trifluoroacetic
acid: dichloromethane (15 ml) and the solution left at room
temperature for 15 minutes. The solvent was removed and the
residues dissolved in dry dichloromethane (40 ml). To this solution
was added succinic anhydride (613 mg, 6.12 mmol) and triethylamine
(1.549 g, 15.31 mmol) and the reaction stirred overnight at room
temperature under argon. The solvent was removed, the residues
taken up into dichloromethane (100 ml) and 1M aqueous HCl (50 ml)
added. The solution was rapidly stirred for 5 hours, the aqueous
layer removed, and the remaining organic layer washed
(3.times.water). This was then dried (MgSO.sub.4) and the solvent
removed to yield the title compound as a white solid (2.945 g,
98%). C.sub.40H.sub.57N.sub.3O.sub.25 requires 979.3 Found
ES.sup.+: MH.sup.+, 980.2, MNa.sup.+, 1002.2, ES.sup.-:
(M-H.sup.+).sup.- 978.2. .delta..sub.H (CDCl.sub.3) 2.03-2.23 (30H,
10.times.s, Me), 2.4-2.8 (4H, m, CH.sub.2CO), 3.3-3.9 (8H, brm,
CH.sub.2N), 4.11 (2H, m, CH.sub.2OAc), 4.35 (2H, dt, CH.sub.2OAc),
5.09 (2H, 2.times.q, CHCH.sub.2OAc), 5.18 (1H, d, CHCO), 5.27 (1H,
d, CHCO), 5.44, 5.46, 5.58, 5.59 (4H, 4.times.t, CHOAc), 7.07, 7.15
(2H, 2.times.t, CONH).
[0179] D. Long Chain Aminoacids
[0180] This section contains the syntheses of:
[0181] C.sub.24 Aminoacid
[0182] (D6) 24-Aminotetracosanoic acid
NH.sub.2(CH.sub.2).sub.23CO.sub.2H
[0183] C.sub.18 Aminoacid
[0184] (D10) 18-Aminooctadecanoic acid
H.sub.2N--(CH.sub.2).sub.17--CO.sub.2H
[0185] C.sub.20 Reduced Bixin Aminoacid
[0186] (D15) N-Aminoethyl-4,8,13,17-tetramethyl-1,20-dodecanamoic
acid trifluoroacetate salt 30
[0187] C24 Aminoacid Containing Mid-Chain Amide
[0188] (D18) 12-(Aminododecanoylamino)dodecanoic acid
NH.sub.2--(CH.sub.2).sub.11--CONH--(CH.sub.2).sub.11CO.sub.2H
[0189] (D19) 24-(Benzyloxycarbonylamino)tetracosanoic acid 31
[0190] C.sub.24 Aminoacid
[0191] (D1) 12-Aminododecanol hydrochloride
NH.sub.2(CH.sub.2).sub.11CH.sub.2OH.HCl
[0192] 12-Aminododecanoic acid (21.52 g 100 mmol) was suspended in
100 ml THF and borane THF complex (500 mmol, 1M solution) added.
The reaction was left overnight and carefully quenched with
methanol before evaporation to small bulk. The residue was
suspended in 1M HCl (500 ml) and heated at 40.degree. C. for 1 hr
and left overnight. The white solid was filtered off and washed
with cold 1M HCl. The product was recrystallised from 1M HCl,
filtered off and dried over P.sub.2O.sub.5 in vacuo to yield the
title compound (18.70 g, 79%). Mp 120.degree. C. softens,
169.degree. C. liquid. C.sub.12H.sub.28N.sub.1O.sub.1Cl.1/5
H.sub.2O requires C: 59.70%, H: 11.86%, N: 5.80%. Found: C: 59.65%,
H: 11.82%, N: 5.76%. C.sub.12H.sub.27N.sub.1O.sub.1 requires 201.
Found ES+: MH.sup.+ 202.1 (100%). .delta..sub.H (CD.sub.3CO.sub.2D)
3.64 (2H, t, CH.sub.2O), 3.06 (2H, t, NCH2), 1.73 (2H, m,
CH.sub.2CH.sub.2O), 1.57 (2H,m, NCH.sub.2CH.sub.2), 1.2-1.5 (16H,
m, CH2).
[0193] (D2) 12-(Dibenzylamino)dodecanol
Bn.sub.2N(CH.sub.2).sub.11CH.sub.2OH
[0194] D1 (15 g, 63.2 mmol) was suspended in a mixture of
dichloromethane (150 ml) and saturated sodium carbonate in water
(150 ml). Benzyl bromide (189.6 mmol, 33.7 g, 23.5 ml) was added
slowly. The suspension cleared and reaction was complete after 4
hr, aqueous ammonia (0.880, 30 ml) was added & the reaction
left overnight. The organic layer was dried (MgSO.sub.4) and
evaporated to dryness. The product was stirred vigorously in
refluxing hexane. The flask was left at -20.degree. C. when
crystals of the title compound slowly appeared. The crystals (Mp
45.degree. C.) were collected by filtration (18.03 g, 75%).
C.sub.26H.sub.39N.sub.1O.sub.1 requires C: 81.84%, H: 10.30%, N:
3.67%. Found: C: 81.64%, H: 10.24%, N: 3.54%.
C.sub.26H.sub.39N.sub.1O.sub.1 requires 381. Found ES+: MH.sup.+
382 (100%). .delta..sub.H (CDCl.sub.3) 7.1-7.6 (10 H, m, Ar), 3.64
(2H, t, CH.sub.2O), 3.56 (4H, s, ArCH2), 2.41 (2H, t, NCH2),
1.1-1.8 (22H, dm, CH2).
[0195] (D3) 12-(Dibenzylamino)dodecanal
Bn.sub.2N(CH.sub.2).sub.11CHO
[0196] To a solution of anhydrous DMSO (30 mmol, 2.13 ml) in
dichloromethane (200 ml) at -78.degree. C. was added carefully
oxalyl chloride (2.6 ml, 30 mmol) in dichloromethane (60 ml). After
15 mins D1 (10 g, 26 mmol) was added in dichloromethane (60 ml) and
the reaction stirred for 20 mins at -78.degree. C. Triethylamine
(28 ml) was added dropwise to the cold reaction. A precipitate
formed and after 15 mins the reaction was allowed to reach room
temperature. Water (100 ml) was added to the reaction which was
extracted with dichloromethane. The organic layers were washed with
water, dried (MgSO.sub.4) and evaporated to dryness. The residue
was chromatographed (SiO.sub.2, hexane -10% ethyl acetate in
hexane) to give the title compound as an oil (7.97 g, 80%). This
compound is unstable and should be used on the day of
preparation.
[0197] I.R. 1725 cm.sup.-1 (COH). C.sub.26H.sub.37NO requires
379.29. Found ES+: MH.sup.+ 380.29. .delta..sub.H (CDCl.sub.3) 1.32
(14H, br, (CH.sub.2).sub.7(CH.sub.2).sub.2N), 1.61 (4H, 2.times.p,
CH.sub.2CH.sub.2N, CH.sub.2CH.sub.2CO), 2.43, 2.44 (4H, 2.times.t,
CH.sub.2N, CH.sub.2CO), 3.60 (4H, s, CH.sub.2Ph), 7.2-7.5 (10H, m,
Ph), 9.78 (1H, t, COH). d.sub.C (CDCl.sub.3) 22.0, 26.9, 27.1,
29.0, 29.3, 29.4, 29.5 (9C, (CH.sub.2).sub.9CH.sub.2N), 43.8 (1C,
CH.sub.2COH), 53.3 (1C, CH.sub.2N), 58.2 (2C, CH.sub.2Ph), 126.6
(2C, CH(CH).sub.2C), 128.0 (4C, CHC), 128.6 (4C, CHCHC), 140.0 (2C,
CCH.sub.2N), 202.3 (1C, COH).
[0198] (D4) 11-(Carboxyundecyl)triphenylphosphoniumbromide
Ph.sub.3P--(CH.sub.2).sub.11CO.sub.2H.Br.sup.-
[0199] To 12-bromododecanoic acid (3.000 g, 10.7 mmol) suspended in
acetonitrile (12 ml) was slowly added triphenylphosphine (2.818 g,
10.7 mmol). The reaction was heated at 100.degree. C. (no
condenser) with argon blowing over the flask until the reaction was
a fusion, then maintained at 100.degree. C. (with condenser) for 24
hrs. The warm residues were dissolved in acetonitrile (18 ml) and
added dropwise to rapidly stirred cold (dry ice) diethyl ether. The
white precipitate formed was then filtered off and the title
compound dried (5.353 g, 92%). Mp 110-112.degree. C.
C.sub.30H.sub.38O.sub.2PBr requires C: 66.54%, H: 7.07%. Found: C:
66.42%, H: 7.10%. .delta..sub.P (CDCl.sub.3) 24.3 (s).
.delta..sub.H (CDCl.sub.3) 1.05-1.30 (12 H, br,
(CH.sub.2).sub.6(CH.sub.2- ).sub.2CO.sub.2H), 1.53 (6H, br,
(CH.sub.2).sub.2CH.sub.2P, CH.sub.2CH.sub.2CO.sub.2H), 2.28 (2H, t,
CH.sub.2CO.sub.2), 3.55 (2H, br, CH.sub.2P), 7.6-7.8 (15H, m, Ph).
d.sub.C (CDCl.sub.3) 22.1, 22.3, 22.8, 24.5, 28.8, 28.9, 30.0, 30.2
(10C, (CH.sub.2).sub.10CO.sub.2H), 34.2 (1 C, CH.sub.2P), 117.3,
118.7 (3 C, CP), 130.3, 130.5 (6C, CHCHCP), 133.3, 133.5 (6C,
CHCP), 134.9 (3C, CH(CH).sub.2CP), 177.4 (1C, CO.sub.2H).
[0200] (D5) 24-(Dibenzylamino)-12-tetracosenoic acid
[0201]
Bn.sub.2N--(CH.sub.2).sub.11--CH.dbd.CH--(CH.sub.2).sub.10--CO.sub.-
2H
[0202] The phosphonium salt D4 (13.52 g, 25 mmol) was dissolved in
dry DMSO (40 ml) under argon at .about.0.degree. C. (no DMSO
solidification). 2.2 Equivalents of 2.0M LDA(25 ml) were added, the
solution turning orange. The reaction was left at 0.degree. C. for
1/2 hour, and to the now dark orange solution was added a solution
of D3 (7.97 g, 21 mmol) in dry THF (30 ml). The solution was
maintained at 0.degree. C. for 4 hours then added to 2M HCl (50
ml). The aqueous layer was extracted with dichloromethane, the
fractions combined, dried (MgSO.sub.4) and the solvent removed to
yield the crude material as a pale yellow gum. Silica column
chromatography (30-100% ethyl acetate in hexane) yielded the title
compound (6.20 g, 53%), as a pale yellow gum.
C.sub.38H.sub.59NO.sub.2 requires 561.46. Found ES+: MH.sup.+
562.53, ES-: (M-H.sup.+).sup.- 560.55. .delta..sub.H (CDCl.sub.3)
1.26 (30H, br, (CH.sub.2).sub.8CH.sub.-
2CH.dbd.CHCH.sub.2(CH.sub.2).sub.7), 1.42-1.72 (4H, m,
CH.sub.2CH.sub.2CO.sub.2H, CH.sub.2CH.sub.2N), 2.02 (4 H,
d.times.t, CH.sub.2CH.dbd.CHCH.sub.2), 2.34 (2H, t,
CH.sub.2CO.sub.2H), 2.46 (2H, t, CH.sub.2N), 3.65 (4H, s,
CH.sub.2Ph), 5.36 (2H, t, CH.dbd.CH), 7.2-7.4 (10H, m, Ph).
.delta..sub.C (CDCl.sub.3) 25.0, 26.4, 27.2, 29.3, 29.6 (19C,
(CH.sub.2).sub.10CH.dbd.CH(CH.sub.2).sub.9), 34.5 (1C,
CH.sub.2CO.sub.2H), 52.9 (1C, CH.sub.2N), 57.7 (2C, CH.sub.2Ph),
127.0 (2C, CH(CH).sub.2C, 128.2 (4C, CHC), 129.1 (4C, CHCHC), 129.9
(2C, CH.dbd.CH), 138.6 (2C, CCH.sub.2N), 179.2 (1C, CO.sub.2H).
[0203] (D6) 24-Aminotetracosanoic acid
NH.sub.2(CH.sub.2).sub.23CO.sub.2H
[0204] D5 (6.2 g) under an atmosphere of hydrogen was heated at
60.degree. C. (to avoid the monobenzyl product) overnight in
glacial acetic acid using Pearlman's catalyst (10% wlw). The
reaction was filtered through glass fibre and evaporated to
dryness. The title compound was crystallised from acetic acid/ether
(4.2 g, 100%) and subjected to high vacuum to remove traces of
acetic acid. Mp 151-155.degree. C. C.sub.24H.sub.49NO.sub.2.0.75
MeCO.sub.2H requires C: 71.44%, H: 12.23%, N: 3.27%. Found: C:
71.43%, H: 12.15%, N: 3.26%. C.sub.24H.sub.49NO.sub.2 requires
383.38. Found ES+: MH.sup.+ 384.29. .delta..sub.H (CD.sub.3OD+TFA)
1.32 (38H, br, (CH.sub.2).sub.19(CH.sub.2).sub.2 NH.sub.2), 1.65
(4H, br, CH.sub.2CH.sub.2NH.sub.2, CH.sub.2CH.sub.2CO.sub.2H), 2.33
(2H, t, CH.sub.2CO.sub.2H), 2.74 (2H, m, CH.sub.2NH.sub.2).
.delta..sub.C (CD.sub.3OD+TFA) partial 33.8 (1C,
CH.sub.2CO.sub.2H), 35.3 (1C, CH.sub.2NH.sub.2).
[0205] C.sub.18 Aminoacid
[0206] (D7) 6-(Dibenzylamino)-1-hexanol
(Bn).sub.2N(CH.sub.2).sub.5CH.sub.2OH
[0207] Benzyl bromide (61 ml, 511 mmol) was added to a stirred
solution of 6-amino-1-hexanol (20 g, 170 mmol) and triethylamine
(142 ml, 1.02 mol) in acetonitrile (500 ml) at room temperature for
two days. The acetonitrile solution was concentrated to 100 ml and
diluted with water. The aqueous phase was extracted with ethyl
acetate, washed with brine, dried (MgSO.sub.4) and evaporated to
dryness to yield an orange oil. The product was chromatographed on
silica (hexane--50% ethyl acetate/hexane) to yield the title
compound as a colourless oil (25 g, 50%). .delta..sub.H
(CDCl.sub.3) 7.23-7.39 (10H, m, (ArH), 3.59 (6H, t+ds,
CH.sub.2OH+ArCH.sub.2), 2.42(2H, t, CH.sub.2N), 1.47-1.56 (4H, m,
CH.sub.2CH.sub.2NH.sub.2+CH.sub.2CH.sub.2OH), 1.24-1.32 (4H, m,
2.times.CH.sub.2).
[0208] (D8) 6-(Dibenzylamino)hexanal
(Bn).sub.2N(CH.sub.2).sub.5CHO
[0209] To a stirred solution of DMSO (20 mmol, 1.41 ml) in
dichloromethane (100 ml) at -78.degree. C. was carefully added
oxalyl chloride (1.7 ml, 20 mmol) in dichloromethane (30 ml). After
15 mins D7 (5 g, 16.83 mmol) was added in dichloromethane (30 ml)
maintaining the temperature at -78.degree. C. The reaction was
stirred for 20 mins and triethylamine (14 ml) added dropwise. A
precipitate formed, after 15 mins the reaction was allowed to reach
room temperature. Water (100 ml) was added to the reaction which
was extracted with dichloromethane. The organic layers were washed
with water, dried (MgSO.sub.4) and evaporated to dryness. The
residue was chromatographed (SiO.sub.2, hexane-20% ethyl acetate in
hexane) to give the title compound as an oil (4.10 g, 83%).
C.sub.20H.sub.25NO requires C: 81.31%, H: 8.53%, N: 4.74%. Found:
C: 81.00%, H: 8.49%, N: 4.63%. C.sub.20H.sub.25NO requires 295.
Found ES+: MH.sup.+ 296 .delta..sub.H (CDCl.sub.3) 9.71 (1H, s,
CHO), 7.2-7.5 (10H,m, ArH,), 3.57(4H, s, ArCH.sub.2), 2.3-2.5 (4H,
dt, CH.sub.2), 1.2-1.7 (6H, dm, CH.sub.2).
[0210] (D9) 18-(Dibenzylamino)-12-octadecenoic acid
Bn.sub.2N--(CH.sub.2).sub.5--CH.dbd.CH--(CH.sub.2).sub.10--CO.sub.2H
[0211] D4 (1.082 g, 2 mmol) was dissolved in dry DMSO (5 ml) under
argon at .about.0.degree. C. (no DMSO solidification). 2.2
equivalents of 2.0M LDA (4 ml) was added, the solution turning
orange. The reaction was left at 0.degree. C. for 1/2 hour, and to
the now dark orange solution was added a solution of D8 (0.7 g, 2
mmol) in dry THF (10 ml). The solution was maintained at 0.degree.
C. for 4 hours then added to 2M HCl (50 ml). The aqueous layer was
extracted with ethyl acetate, the fractions combined, dried
(MgSO.sub.4) and the solvent removed to yield the crude material as
a pale yellow gum. Silica column chromatography (30% ethyl acetate
in hexane or 5% methanol in dichloromethane) yielded the title
compound (453 mg, 53%), as a low melting (Mp 21.degree. C.) white
solid. C.sub.32H.sub.47NO.sub.2 requires C: 80.45%, H: 9.92%, N:
2.93%. Found: C: 80.20%, H: 9.92%, N: 2.74%.
C.sub.38H.sub.59NO.sub.2 requires 477. Found ES+: MH.sup.+ 478.
.delta..sub.H (CDCl3) 8.6-9.2 (1 H, vbr, (CO.sub.2H), 7.39-7.21 (10
H, m, ArH), 5.37-5.29 (2H, m, trans HC.dbd.CH), 3.63 (4 H, s,
PhCH.sub.2), 2.48-2.43 (2H, t, NCH.sub.2), 2.36-2.31(2H, t,
CH.sub.2CO.sub.2H), 2.01-1.97 (2H, t, CH.sub.2CH.dbd.CH), 1.66-1.55
(4H, m, CH.sub.2), 1.29-1.24 (18 H, m, CH.sub.2).
[0212] (D10) 18-Aminooctadecanoic acid
H.sub.2N--(CH.sub.2).sub.17--CO.sub.2H
[0213] D8 (13 g) under an atmosphere of hydrogen was heated at
60.degree. C. overnight in glacial acetic acid with Pearlman's
catalyst (10% w/w). The reaction was filtered hot through glass
fibre and evaporated to dryness. The product was crystallised from
acetic acid/ether (8.2 g, 100%). The title compound was subjected
to high vacuum to remove traces of acetic acid. Mp 162-163.degree.
C. C.sub.24H.sub.49NO.sub.2.0.25H.sub.- 2O requires C: 71.12%, H:
12.43%, N: 4.61%. Found: C: 71.20%, H: 12.35%, N: 4.49%.
C.sub.24H.sub.49NO.sub.2 requires 299. Found ES+: MH.sup.+ 300.
d.sub.H (CD.sub.3CO.sub.2D) 3.06 (2H, t, CH.sub.2NH.sub.2), 2.38
(2H, t, CH.sub.2CO.sub.2H), 1.63-1.73 (4H, m,
CH.sub.2CH.sub.2CO.sub.2H+CH.sub.2C- H.sub.2NH.sub.2), 1.33 (26H,
m, CH.sub.2).
[0214] C.sub.20 Reduced Bixin Aminoacid
[0215] (D11) Hydrogen Methyl
4,8,13,17-tetramethyl-1,20-dodecanedioate 32
[0216] A suspension of bixin (9.959 g, 24.59 mmol) was stirred
overnight in methanol (200 ml) in the presence of a hydrogen
atmosphere and 10% Pd/C catalyst (1 g). The catalyst and solvent
were removed to yield an opaque yellow viscous oil, silica tlc r.f.
0.4 (25% ethyl acetate in hexane) purified by gradient (20-35%
ethyl acetate in hexane) silica chromatography to yield the title
compound as a very pale yellow clear viscous oil (7.181 g, 71%).
I.R. 1710 cm.sup.-1 (CO.sub.2H), 1743cm.sup.-1 (CO.sub.2Me).
C.sub.24H.sub.48O.sub.4 requires 412.36. Found ES+: MNa.sup.+
435.38. .delta..sub.H (CDCl.sub.3) 0.85-1.0 (12 H, m, CHMe),
1.0-1.8 (28 H, br, CH.sub.2, CHCH.sub.3), 2.35 (4H, m, CH.sub.2CO),
3.70 (3H, s, OMe)
[0217] (D12) N-aminoethyl-O-t-Butylcarbamate 33
[0218] BOC-ON (16.4 g, 0.066 mmol) was added to a stirred mixture
of ethylenediamine (13.4 ml, 0.2 mmol) and triethylamine (28 ml,
0.2 mmol) at room temperature under argon and left overnight. Ethyl
acetate was added and the product extracted into potassium
dihydrogen orthophosphate solution. The extracts were combined and
basified to pH12 with sodium hydroxide. The aqueous basic solution
was extracted with ethyl acetate, the combined organic phases
washed with brine, dried and evaporated to dryness to yield the
title compound which analysed without further purification. Yield
6.4 g. C.sub.7H.sub.16N.sub.2O.sub.2 requires 160. Found ES+:
MH.sup.+ 161. .delta..sub.H (CDCl.sub.3) 5.12 (1H, br, CONH),
3.19(2H, dt, OCONHCH.sub.2), 2.93(2H, s, NH.sub.2), 2.82 (2H, t,
CH.sub.2N), 1.42 (9H, s, Me).
[0219] (D13) Methyl
N-(t-butyloxycarbonylaminoethyl)-4,8,13,17-tetramethyl-
-1,20-dodecanamoate 34
[0220] To D11 (7.181 g, 17.4 mmol) in dry dichloromethane (50 ml)
under argon was added triethylamine (1.761 g, 17.4 mmol), EDC
(5.171 g, 17.4 mmol) and N-hydroxysuccinimide (2.002 g, 17.4 mmol).
The solution was left for three hours, during which time the
formation of the slower moving NHS active ester was followed by
silica tlc (r.f. 0.25, 25% ethyl acetate in hexane). Once complete
ester formation was achieved, D12 (3.067 g, 19.1 mmol) was added in
dichloromethane (20 ml) and the reaction left 48 hrs. Purification
on silica (50% ethyl acetate in hexane) yielded the title compound
(4.285 g, 44%), silica tlc r.f. 0.15 (25% ethyl acetate in hexane).
C.sub.32H.sub.62N.sub.2O.sub.5 requires 554.47. Found ES+: MH.sup.+
555.48, MNa.sup.+ 557.41. .delta..sub.H (CDCl.sub.3) 0.7-0.9 (12 H,
m, CHMe), 0.8-1.8 (28 H, br m, CHMe, CH.sub.2), 1.41 (9 H, s,
C(Me).sub.3), 2.15 (2H, d.times.t, CH.sub.2CONH), 2.27 (2H, m,
CH.sub.2CO.sub.2Me), 3.15-3.35 (4 H, m, CH.sub.2NH), 3.63 (3 H, s,
OMe), 5.33 (1 H, t, NHCO.sub.2), 6.65 (1 H, t, NHCO). .delta..sub.C
(CDCl.sub.3) 19.1, 19.5, 24.2, 27.3, 28.2, 31.7, 32.2, 32.6, 34.3,
37.0 (25 C, CH.sub.2, CHMe, C(CH.sub.3).sub.3), 40.2, 40.4 (2 C,
CH.sub.2N), 51.2 (1 C, OMe), 79.2 (1 C, C(Me).sub.3), 156.8 (1 C,
NHCO.sub.2), 174.2, 174.4 (2 C, CO.sub.2Me, CONH).
[0221] (D14)
N-(t-Butyloxycarbonylaminoethyl)-4,8,13,17-tetramethyl-1,20-d-
odecanamoic acid 35
[0222] To D13 (4.285 g, 7.722 mmol) in methanol (35 ml) was added a
suspension/solution of lithium hydroxide (3.239 g, 77.22 mmol) in
water (10 ml). This was stirred for 2 hours then poured into 10%
citric acid solution and extracted with ethyl acetate. These
organic fractions were combined, washed with water and the solvent
removed to yield the title compound. With dichloromethane:
methanol:water 6:1:1 on silica the tlc of D13 and D14 have R.sub.f
of 0.35 and 0.05 respectively. C.sub.31H.sub.60N.sub.2O.sub.5
requires 540.45. Found ES+: MH.sup.+ 541.48, MNa.sup.+ 563.45.
.delta..sub.H (CDCl.sub.3) 0.85-1.0 (12 H, m, Me), 1.0-1.8 (28 H,
br m, CH.sub.2, CH), 1.46 (9 H, s, C(Me).sub.3), 2.20 (2H, m,
CH.sub.2CONH), 2.36 (2H, m, CH.sub.2CO.sub.2), 3.15-3.45 (4 H, m,
CH.sub.2N), 5.16 (1 H, t, NHCO.sub.2), 6.55 (1 H, t, NHCO).
.delta..sub.C (CDCl.sub.3) 19.3, 19.7, 24.2, 27.2, 28.3, 31.8,
32.3, 32.6, 34.4, 36.9 (25 C, CH.sub.2, CHMe, C(CH.sub.3).sub.3),
40.0, 40.6 (2 C, CH.sub.2N), 79.6 (1 C, C(Me).sub.3), 157.0 (1 C,
NHCO.sub.2), 174.5 (1 C, CONH), 179.5 (1 C, CO.sub.2H).
[0223] (D15) N-Aminoethyl-4,8,13,17-tetramethyl-1,20-dodecanamoic
acid trifluoroacetate salt 36
[0224] D14 was taken up into 96% TFA and left for 30 minutes. The
solvent was removed to give the amino acid as a viscous oil which
after silica chromatography (15% methanol in dichloromethane+0.1%
acetic acid) gave the title compound as a colourless solid/gum,
silica tlc r.f. 0.25, ninhydrin +ve (6:1 dichloromethane:methanol).
The yield for the two steps D14 to D15 was 2.044 g, 60%.
C.sub.26H.sub.52N.sub.2O.sub.3 requires 440.40. Found ES+: MH.sup.+
441.36. .delta..sub.H (CD.sub.3OD) 0.95-1.1 (12 H, m, Me), 1.1-1.9
(28 H, br m, CH, CH.sub.2), 2.38 (4 H, m, CH.sub.2CO), 3.20 (2H, t,
CH.sub.2NH.sub.3.sup.+), 3.60 (2H, t, CH.sub.2NHCO). .delta..sub.C
(CD.sub.3OD) 20.1, 20.5, 22.0, 25.6, 28.6, 33.6, 33.8, 34.0, 35.0,
38.4, 38.6 (22 C, CH, CH.sub.2, Me), 40.9 (2 C, CH.sub.2N), 177.9
(1 C, CONH), 179.4 (1 C, CO.sub.2H).
[0225] C24 Aminoacid Containing Mid-Chain Amide
[0226] (D16) 12-(t-Butyloxycarbonylamino)dodecanoic acid
BocNH--(CH.sub.2).sub.11--CO.sub.2H
[0227] 12-Aminododecanoic acid (2.15 g, 10 mmol) was dissolved in
1M NaOH (50 ml) at 50.degree. C. BOC anhydride (2.33 g, 10 mmol)
was added to the reaction which was stirred for 30 mins. The
reaction was poured into stirred 10% citric acid (100 ml) and the
white solid filtered off. The solid was washed with citric
acid,water and dried in vacuo. The product was dissolved in ether
and filtered before evaporating to dryness. The title compound was
crystallised from hexane (2.33 g, 74%). Mp 72-76.degree. C.
C.sub.17H.sub.33NO.sub.4 requires C: 64.73%, H: 10.55%, N: 4.44%.
Found: C: 64.78%, H: 10.58%, N: 4.41%. C.sub.24H.sub.49NO.sub.2
requires 315. Found ES+: MH.sup.+ 316. .delta..sub.H (CDCl3) 3.08
(2H, t, CH.sub.2NH.sub.2), 2.33 (2H, t, CH.sub.2CO.sub.2H), 1.6(2H,
m, CH.sub.2CH.sub.2NH.sub.2), 1.44 (11 H, s+m,
CH.sub.2CH.sub.2CO.sub.2H+Boc- ), 1.26 (14 H, m, CH.sub.2).
[0228] (D17) 12-(t-B utyloxycarbonylaminododecanoylamino)
dodecanoic acid
BocNH--(CH.sub.2).sub.11--CONH--(CH.sub.2).sub.11CO.sub.2H
[0229] To D16 (1.59 g, 5.05 mmol) in stirred dichloromethane (20
ml) was added DBU (0.755 ml, 5.05 mmol), followed by
N-hydroxysuccinimide (0.581 g, 5.05 mmol) and EDC (0.968 g, 5.05
mmol). The reaction was left overnight to go to completion.
Chloroform (30 ml) containing 12-aminododecanoic acid (1.087 g,
5.05 mmol) and DBU (1.51 ml, 10.1 mmol) was added. After 3 hours
the reaction was poured into 10% citric acid (100 ml) and extracted
with dichloromethane (3.times.50 ml). The organic phases were dried
(MgSO.sub.4) and evaporated to dryness. The solid residue was
triturated with refluxing hexane & dissolved in refluxing ethyl
acetate. The solution was filtered hot and allowed to crystallise
to yield the title compound (2.33 g,90%). Mp 85-87.degree. C.
C.sub.29H.sub.56N.sub.2O.sub.5 requires 512. Found ES+: MH.sup.+
513. .delta..sub.H (CDCl.sub.3) 5.74 (1 H, brt, CONH), 4.57(1 H,
br, CONH), 3.0-3.3 (2.times.2 H, q+m, CH.sub.2NH), 2.30(2H, t,
CH.sub.2CO), 2.15 (2H, t, CH.sub.2CO), 1.60 (4 H, m, CH.sub.2),
1.2-1.5(9+32H, m, CH.sub.2).
[0230] (D18) 12-(Aminododecanoylamino)dodecanoic acid
NH.sub.2--(CH.sub.2).sub.11--CONH--(CH.sub.2).sub.11CO.sub.2H
[0231] 96% TFA (4% water, 7 ml) was added to D17 (2.3 g, 4.5 mmol)
and the resulting solution stirred for 30 min. The reaction was
evaporated to dryness and azeotroped with toluene/methanol before
submitting to high vacuum. The title compound was recrystallised
from ether containing acetic acid as a white solid (2.12 g). Mp
66-68.degree. C. C.sub.24H.sub.48N.sub.2O.sub.3 requires 412. Found
ES+: MH.sup.+ 413. .delta..sub.H (CD.sub.3CO.sub.2D) 3.26 (2H, t,
CH.sub.2NH), 3.09 (2H, t, CH.sub.2NH), 2.38(2H,t, CH.sub.2CO),
2.28(2H,t, CH.sub.2CO), 1.5-1.9(4 H,m, CH.sub.2CH.sub.2NH),
1.5-1.25(32 H, m, CH.sub.2).
[0232] (D19) 24-(Benzyloxycarbonylamino)tetracosanoic acid 37
[0233] To D6 (0.500 g, 1.30 mmol) and DBU (794 mg, 5.21 mmol) in
refluxing methanol (100 ml) was added neat benzyichloroformate and
the reaction refluxed for 3 hours until complete conversion of D6
to either the protected amino acid D19 or its methyl ester was
observed. The solvent was removed and the residues taken up into
dioxane (60 ml) and water (2 ml) and lithium hydroxide (1 g) added.
The reaction was then refluxed for 2 hours until hydrolysis of the
methyl ester derivative to D19 was complete. The solvent was
removed, the residues suspended in 1M HCl (100 ml) and extracted
with hot ethyl acetate (300 ml). The hot ethyl acetate was dried
(MgSO.sub.4) and the solvent volume reduced to 40 ml. The
solution/suspension was left at -10.degree. C. for 1 hour, allowed
to warm to room temperature and the resulting white precipitates
filtered off and dried under vacuum to yield the title compound
(585 mg, 87%). M.Pt 102-104.degree. C. C.sub.32H.sub.55NO.sub.4
requires 517.4. Found ES.sup.-: MCl.sup.-, 552.6. .delta..sub.H
(d.sub.6DMSO), 1.32 (38H, s, (CH.sub.2).sub.19(CH.sub.2).sub.2N),
1.52 (2H, p, CH.sub.2CH.sub.2CO.sub.- 2H), 1.65 (2H, p,
CH.sub.2CH.sub.2NH), 2.35 (2H, t, CH.sub.2CO.sub.2H), 3.18 (2H, t,
CH.sub.2N), 5.14 (2H, s, CH.sub.2Ph), 7.35 (5H, m, Ph).
[0234] E. Lipid Polyamine Intermediates
[0235] This section contains the synthesis of:
[0236] (E2)
N-{8-[t-Butyloxycarbonylaminobutyl(t-butyloxycarbonyl)-amino]--
7-[t-butyloxycarbonylaminobutyl(t-butyloxy-carbonyl)aminomethyl]octyl}-23--
aminotetracosanamide 38
[0237] (E4)
N-{8-[Methyl(t-butyloxycarbonyl)aminopropyl(t-butyloxy-carbony-
l)aminobutyl(t-butyloxycarbonyl)amino]-7-[methyl(t-butyloxycarbonyl)aminop-
ropyl(t-butyloxy-carbonyl)aminobutyl(t-butyloxycarbonyl)aminomethyl]-octyl-
}-23-aminotetracosanamide 39
[0238] (E1)
N-{8-[t-Butyloxycarbonylaminobutyl(t-butyloxycarbonyl)-amino]--
7-[t-butyloxycarbonylaminobutyl(t-butyloxy-carbonyl)aminomethyl]octyl}-23--
benzyloxycarbonyl-amino)tetracosanamide 40
[0239] D19 (448 mg, 0.865 mmol), EDC hydrochloride (249 mg, 1.30
mmol), N-hydroxysuccinimide (149 mg, 1.30 mmol) and DBU (263 mg,
1.73 mmol) were dissolved in anhydrous dichloromethane (10 ml) and
activated ester formation left overnight at room temperature under
argon. B8 (589 mg, 0.82 mmol) was added and the reaction left for a
further five hours. The solvent was removed and the residues
purified by gradient silica column chromatography (40-60% ethyl
acetate in hexane) to yield the title compound as a colourless
glass (887 mg, 88%). C.sub.69H.sub.126N.sub.6O.s- ub.11 requires
1214.9. Found ES.sup.+: MH.sup.+, 1215.9. .delta..sub.H
(CDCl.sub.3) 1.24 (48H, br, (CH.sub.2).sub.20(CH.sub.2).sub.2N,
(CH.sub.2).sub.4CH), 1.43 (48H, br, Me, CH.sub.2CH.sub.2N), 2.03
(1H, br, CH), 2.18 (2H, t, CH.sub.2CO), 3.00-3.35 (16H, brm,
CH.sub.2N), 4.44.6 (3H, br, NHCO.sub.2), 5.09 (2H, s, CH.sub.2O),
5.90 (1H, br, CONH), 7.34 (5H, m, Ph).
[0240] (E2)
N-{8-[t-Butyloxycarbonylaminobutyl(t-butyloxycarbonyl)-amino]--
7-[t-butyloxycarbonylaminobutyl(t-butyloxy-carbonyl)aminomethyl]octyl}-23--
aminotetracosanamide 41
[0241] To E1 (877 mg) dissolved in tert-butanol (60 ml) was added
Pearlmans catalyst (500 mg), ammonium formate (3 g) and Raney
nickel (approximately 1 ml). The reaction was heated at 45.degree.
C. overnight and to maintain a hydrogen atmosphere the reaction was
fitted with a bubbler. The catalysts were filtered off and the
solvent removed to give a colourless gum which was purified by
silica column chromatography (10% methanol in dichloromethane +0.1%
triethylamine) to yield the title compound as a colourless
glass/solid (744 mg, 95%). C.sub.61H.sub.120N.sub.6O.sub.9 requires
1080.9. Found ES.sup.+: MH.sup.+, 1082.1, ES.sup.-: MCl.sup.-,
1116.1. .delta..sub.H (CDCl.sub.3) 1.24 (46H, br,
(CH.sub.2).sub.19(CH.sub.2).sub.2CO), 1.42 (50H, br, Me,
CH.sub.2CH.sub.2N, CH.sub.2CH.sub.2CO), 1.95 (1H, br, CH), 2.14
(2H, t, CH.sub.2CO), 2.95 (2H, t, CH.sub.2NH.sub.2), 3.0-3.3 (14H,
m, CH.sub.2N), 4.69 (2H, br, NHCO.sub.2), 5.64 (1H t, NHCO).
[0242] (E3)
N-{8-[Methyl(t-butyloxycarbonyl)aminopropyl(t-butyloxy-carbony-
l)aminobutyl(t-butyloxycarbonyl)amino]-7-[methyl(t-butyloxycarbonyl)aminop-
ropyl(t-butyloxy-carbonyl)aminobutyl(t-butyloxycarbonyl)aminomethyl]-octyl-
}-23-(benzyloxycarbonylamino)tetracosanamide 42
[0243] D19 (580 mg, 1.12 mmol), EDC hydrochloride (429 mg, 2.24
mmol), N-hydroxysuccinimide (193 mg, 1.68 mmol) and DBU (341 mg,
2.24 mmol) were dissolved in anhydrous dichloromethane (30 ml) and
activated ester formation left overnight at room temperature under
argon. B21 (1.186 g, 1.12 mmol) in anhydrous dichloromethane (20
ml) was added and the reaction left for a further five hours. The
solvent was removed and the residues purified by gradient silica
column chromatography (40-60% ethyl acetate in hexane) to yield the
title compound as a colourless glass (1.261 g, 72%).
C.sub.87H.sub.160N.sub.8O.sub.15 requires 1557.2. Found ES.sup.+:
MH.sup.+, 1558.3. .delta..sub.H (CDCl.sub.3) 1.24 (46H,
(CH.sub.2).sub.19(CH.sub.2).sub.2CO, (CH.sub.2).sub.4CH), 1.4-1.8
(72H, br+m, (Me).sub.3C, CH.sub.2CH.sub.2CO, CH.sub.2CH.sub.2N),
2.03 (1H, br, CH), 2.21 (2H, t, CH.sub.2CO), 2.83 (6H, s, MeN),
3.0-3.35 (24H, m, CH.sub.2N), 4.73 (1H, br, NHCO.sub.2), 5.3 (2H,
s, CH.sub.2O), 6.08 (IH, br, NHCO), 7.34 (5H, m, Ph).
[0244] (E4)
N-{8-[Methyl(t-butyloxycarbonyl)aminopropyl(t-butyloxy-carbony-
l)aminobutyl(t-butyloxycarbonyl)amino]-7-[methyl(t-butyloxycarbonyl)aminop-
ropyl(t-butyloxy-carbonyl)-aminobutyl(t-butyloxycarbonyl)aminomethyl]-octy-
l}-23-aminotetracosanamide 43
[0245] To E3 (1.260 g) dissolved in tert-butanol (60 ml) was added
Pearimans catalyst (500 mg), ammonium formate (3 g) and Raney
nickel (approximately 1 ml). The reaction was heated at 45.degree.
C. overnight and to maintain a hydrogen atmosphere the reaction was
fitted with a bubbler. The catalysts were filtered off and the
solvent removed to give a colourless gum which was purified by
silica column chromatography (10% methanol in dichloromethane+0.1%
triethylamine) to yield the title compound as a colourless
glass/solid (1.088 g, 94%). C.sub.79H.sub.154N.sub.8O.sub.13
requires 1423.2. Found ES.sup.+: MH.sup.+, 1424.3 .delta..sub.H
(CDCl.sub.3) 1.24 (48H, (CH.sub.2).sub.20CH.sub.2CO,
(CH.sub.2).sub.4CH), 1.43 (70H, br+m, (Me).sub.3C,
CH.sub.2CH.sub.2N), 1.95 (1H, br, CH), 2.14 (2H, t, CH.sub.2CO),
2.80 (2H, t, CH.sub.2NH.sub.2), 2.83 (6H, s, NMe), 3.0-3.35 (22H,
m, CH.sub.2N), 5.65 (1H, br, CONH).
[0246] (E5) 36-Chlorohexatriaconta-12,24-dienoic acid
[0247]
Cl--(CH.sub.2).sub.11--CH.dbd.CH--(CH.sub.2).sub.10--CH.dbd.CH--(-
CH.sub.2).sub.10--CO.sub.2H
[0248] To a rapidly stirring suspension of silica (200 ml) and 50%
dichloromethane in hexane (200 ml) was slowly added tosic acid (2
g) in water (6 ml). The suspension was stirred for 10 minutes and
used to pack a column. After washing the column with 50%
dichloromethane in hexane
35-Chloro-1-(1,3-dioxalan-2-yl)pentatriaconta-12,24-diene (4.04 g)
was loaded and eluted over 2 hours with 50% dichloromethane in
hexane to give the aldehyde 2.5264 g, 66% as a white waxy solid. To
PDC (3.792 g, 10.080 mmol) in anhydrous DMF (30 ml) was added the
aldehyde (2.5264 g, 4.582 mmol) in anhydrous DMF (30 ml) and the
reaction stirred at ambient temperature under argon for 2 days. The
reaction was poured into water (500 ml), extracted with hexane and
the combined hexane fractions washed with water (3.times.150 ml).
The solution was dried (MgSO.sub.4), and the solvent removed to
yield a brown oil which was purified by silica gradient
chromatography eluting with 10-20% ethyl acetate in hexane to give
the title compound as a white waxy solid, 1.528 g, 50%.
C.sub.36H.sub.67O.sub.2Cl requires 566.6. Found ES.sup.-:
(M-H.sup.+).sup.-, 565.6. .delta..sub.H (CDCl.sub.3) 1.27 (46H, br,
Cl(CH.sub.2).sub.2(CH.sub.2).sub.8CH.sub.2CH.dbd.CHCH.sub.2(CH.sub.2).sub-
.8 CH.sub.2CH.dbd.CHCH.sub.2(CH.sub.2).sub.7), 1.63 (2H, p,
CH.sub.2CH.sub.2CO.sub.2H), 1.76 (2Hp, CH.sub.2CH.sub.2Cl), 2.01
(8H, m, CH.sub.2CH.dbd.), 2.34 (2H, t, CH.sub.2CO.sub.2H), 3.52
(2H, t, CH.sub.2Cl), 5.34 (4H, m, CH.dbd.CH).
[0249] (E6) 36-Azidohexatriaconta-12,24-dienoic acid
N.sub.3--(CH.sub.2).sub.11--CH.dbd.CH--(CH.sub.2).sub.10--CH.dbd.CH--(CH.s-
ub.2).sub.10--CO.sub.2I
[0250] To E5 (1.528 g, 2.693 mmol) in anhydrous DMF (70 ml) was
added sodium azide (1.226 g, 18.851 mmol) and the reaction heated
for 5 days at 50.degree. C. under argon. The solvent was reduced to
almost dryness and the residues taken up into water (150 ml) and
ethyl acetate (150 ml). The aqueous layer was further extracted
with ethyl acetate (4.times.150 ml), the fractions combined, washed
(2.times.150 ml water), dried (MgSO.sub.4) and the solvent removed
to quantitatively yield the title compound as a pale yellow waxy
solid 1.465 g, 95%. C.sub.36H.sub.67O.sub.2N.sub.3 requires 573.5.
Found ES.sup.-: (M-H.sup.+).sup.-, 572.6. d.sub.H (CDCl.sub.3) 1.27
(46H, br, N.sub.3(CH.sub.2).sub.2(CH.sub.2).sub.8
CH.sub.2CH.dbd.CHCH.sub.2(CH.sub.2).sub.8CH.sub.2CH.dbd.CHCH.sub.2
(CH.sub.2).sub.7), 1.63 (4H, m, CH.sub.2CH.sub.2N.sub.3),
CH.sub.2H.sub.2CO.sub.2H), 2.02 (8H, m, CH.sub.2CH.dbd.), 2.34 (2H,
t, CH.sub.2CO), 3.35 (2H, t, CH.sub.2N.sub.3), 5.35 (4H, m,
CH.dbd.CH).
[0251] (E7)
N-{8-[Methyl(t-butyloxycarbonyl)aminopropyl(t-butyloxy-carbony-
l)aminobutyl(t-butyloxycarbonyl)amino]-7-[methyl(t-butyloxycarbonyl)aminop-
ropyl(t-butyloxy-carbonyl)aminobutyl(t-butyloxycarbonyl)aminomethyl]-octyl-
}-36-azidohexatriaconta-12,24-dienamide 44
[0252] To E6 (299 mg, 0.521 mmol) in anhydrous dichloromethane (10
ml) were added EDC hydrochloride (200 mg, 1.043 mmol),
N-hydroxysuccinimide (90 mg, 0.782 mmol) and the reaction left for
4 hours at room temperature. To the reaction were then added B21
(607 mg, 0.573 mmol) and triethylamine (211 mg, 2.085 mmol) in
anhydrous dichloromethane (10 ml). The reaction was left for a
further 3 hours and the solvent removed. The residues were purified
by gradient silica chromatography eluting with 30-60% ethyl acetate
in hexane to yield the title compound as a colourless viscous oil,
720 mg, 86%. C.sub.91H.sub.172N.sub.10O.sub.13 requires 1613.3.
Found ES.sup.+: MH.sup.+, 1614.3, MH.sub.2.sup.2+, 807.9.
.delta..sub.H (CDCl.sub.3) 1.27 (54H, br, N.sub.3(CH.sub.2).sub.2(-
COH.sub.2).sub.8CH.sub.2CH.dbd.CHCH.sub.2(CH.sub.2).sub.8CH.sub.2CH.dbd.CH-
CH.sub.2(CH.sub.2).sub.7), (CH.sub.2).sub.4CH), 1.35-1.80 (72H, m,
(Me).sub.3C, CH.sub.2CH.sub.2N.sub.3, CH.sub.2CH.sub.2CO,
CH.sub.2CH.sub.2N), 2.00 (8H, m, CH.sub.2CH.dbd.), 2.05 (1H, br,
CH), 2.23 (2H, t, CH.sub.2CO), 2.84 (6H, s, NMe), 2.95-3.35 (22H,
m, CH.sub.2N), 3.25 (2H, t, CH.sub.2N.sub.3), 5.34 (4H, m,
CH.dbd.CH), 6.12 (11H, br, CONH).
[0253] (E8)
N-{8-[Methyl(t-butyloxycarbonyl)aminopropyl(t-butyloxy-carbony-
l)aminobutyl(t-butyloxycarbonyl)amino]-7-[methyl(t-butyloxycarbonyl)aminop-
ropyl(t-butyloxy-carbonyl)aminobutyl(t-butyloxycarbonyl)aminomethyl]-octyl-
}-36-aminohexatriacontanamide 45
[0254] To E7 (184 mg) dissolved in tert-butanol at 40.degree. C.
was added Pd/C (50 mg) and the atmosphere changed to hydrogen. The
hydrogenation was heated at 40.degree. C. for 3 days, the catalyst
filtered off and the solvent removed. The residues were purified by
silica chromatography eluting initially with 100:10:0
CH.sub.2Cl.sub.2:MeOH:NH.sub.4OH to remove faster running
impurities then with 100:10:1 to remove the title compound as a
colourless gum, 61 mg, 34%. C.sub.91H.sub.178N.sub.8O.sub.1- 3
requires 1591.3. Found ES.sup.+: MH.sup.+, 1592.3. .delta..sub.H
(CDCl.sub.3) 1.24 (72H, br,
H.sub.2N(CH.sub.2).sub.2(CH.sub.2).sub.32,
CONH(CH.sub.2).sub.2(CH.sub.2).sub.4), 1.44 (66H, br, (Me).sub.3C,
CH.sub.2CH.sub.2N), 1.72 (4H, p, NCH.sub.2CH.sub.2CH.sub.2N), 1.98
(1H, br, CH), 2.14 (2H, t, CH.sub.2CO), 2.67 (2H, t,
CH.sub.2NH.sub.2), 2.83 (6H, s, NMe), 2.95-3.30 (22H, m,
NCH.sub.2), 5.62 (1H, br, CONH(CH.sub.2).sub.6CH).
[0255] F. Glycoaminolipid Syntheses
[0256] This section contains the syntheses of:
[0257] (F4) 18-(Peracetylglucuronylamino)octadecanoic acid 46
[0258] (F5)
N-(Peracetylglucuronylaminoethyl)-4,8,13,17-tetramethyl-dodeca-
namic acid 47
[0259] (F6)
(12-(Peracetylglucuronylaminododecanoylamino)-dodecanoic acid
48
[0260] (F8) 12-(Peracetylglucuronylamino)dodecanoic acid 49
[0261] C.sub.24 Glycoaminolipid
[0262] (F1) 24-(Glucuronylamino)tetracosanoic acid 50
[0263] A suspension of D6 (792 mg, 2.064 mmol), d-gluconolactone
(1.839 g, 10.32 mmol) and DBU (4.2 g, 30.9 mmol) in dry methanol
(90 ml) were heated at 60.degree. C. for approximately 10 minutes
until all solids had dissolved. The solution was left at room
temperature overnight, then the solvent removed. The residues were
taken up into water (5 ml) and acidified to pH 1 with 1M HCl to
precipitate out the desired compound. This was filtered off and
dried to yield the title compound as a white solid (765 mg, 66%).
Silica tlc R.sub.f 0.35, ninhydrin negative (1:1:1 methanol:acetic
acid:dichloromethane). I.R. 1581 cm.sup.-1 (CO.sub.2.sup.-), 1639
cm.sup.-1 (CONH). .delta..sub.H (DMSO) 1.32 (42 H, br,
(CH.sub.2).sub.21CH.sub.2CO.sub.2H), 2.27 (2H, t,
CH.sub.2CO.sub.2H), 3.15 (2H, m, CH.sub.2N), 3.3-3.8 (4 H, m,
CHOH), 4.0-4.1 (2H, m, CH.sub.2O).
[0264] (F2) 24-(Peracetylglucuronylamino)tetracosanoic acid 51
[0265] To F1 (765 mg, 1.362 mmol) dissolved in dry pyridine (20 ml)
was added acetic anhydride (20 ml). The solution was stirred under
argon overnight and water (50 ml) added slowly. The solution was
extracted with dichloromethane and the dichloromethane then washed
with HCl pH 3 (2.times.20 ml) and water (5.times.30 ml). The
organics were dried (MgSO.sub.4) and the solvent removed to yield
the title compound as a white solid (940 mg, 89%). Alumina tlc
R.sub.f 0.15 (15% methanol in dichloromethane).
C.sub.40H.sub.69NO.sub.13 requires 771.48. Found ES+: MH.sup.+
772.07, MNa.sup.+ 794.25. ES-: (M-H.sup.+).sup.- 770.65.
.delta..sub.H (CDCl.sub.3) 1.26 (38 H, br,
(CH.sub.2).sub.19(CH.sub.2).su- b.2CO.sub.2H), 1.64 (4 H, m,
CH.sub.2CH.sub.2CO.sub.2H, CH.sub.2CH.sub.2NH), 2.07, 2.11, 2.13,
2.21 (15 H, s, MeCO), 2.35 (2H, t, CH.sub.2CO.sub.2H), 3.24 (2H, m,
CH.sub.2NH), 4.30 (2H, 2.times.d.times.d, CH.sub.2OAc), 5.05 (1 H,
q, CH(OAc)CH.sub.2OAc), 5.32 (1 H, d, CH(OAc)CONH), 5.46 (1 H, t,
CH(OAc)CH(OAc) CH.sub.2OAc), 5.70 (1 H, t, CH(OAc)CH(OAc)CONH),
6.42 (1 H, t, NH). .delta..sub.C (CDCl.sub.3) 20.4, 24.5, 26.6,
28.8-29.5 (26 C, (CH.sub.2).sub.21CH.sub.2CO.sub.2H, MeCO), 33.8 (1
C, CH.sub.2CO.sub.2H), 39.3 (1 C, CH.sub.2NH), 61.3 (1 C,
CH.sub.2OAc), 68.5, 68.9, 69-1, 71.5 (4 C, CHOAc), 165.8 (1 C,
CONH), 160.0, 169.5, 169.7, 170.4 (5 C, MeCO), 178.6 (1 C,
CO.sub.2H).
[0266] C.sub.18 Glycolipid
[0267] (F3) 18-(Glucuronylamino)octadecanoic acid 52
[0268] To a suspension of D10 ( 850 mg, 2.8 mmol) in methanol (100
ml) at 50.degree. C. was added DBU (1.27 g, 8.4 mmol) When the
aminoacid had dissolved d-gluconolactone (748 mg, 4.2 mmol) was
added After 3 hrs no ninhydrin positive material was seen on tlc
and the reaction was evaporated to dryness. Cold 0.5 M HCl was
added and the resulting buff precipitate filtered off, washed with
water and dried in vacuo to yield the title compound which was used
without further purification.
[0269] (F4) 18-(Peracetylglucuronylamino)octadecanoic acid 53
[0270] Crude F3 was dissolved in 50 ml 20% acetic anhydride in
pyridine containing DMAP (100 mg). The reaction was left overnight,
water added (10 ml) with cooling and evaporated to dryness. Dilute
hydrochloric acid (50 ml. 0.5M) was added and the aqueous phase
extracted with ethyl acetate The organic phase was dried
(MgSO.sub.4 and evaporated to dryness to give a brown tar. This
material was dissolved in dichloromethane and chromatographed
(Silica eluted with ethyl acetate in hexane (30% to 50%), all
containing 0.1% acetic acid. Solvent removal yielded the title
compound as a white solid (826 mg, 49% 2 steps).
C.sub.34H.sub.56NO.sub.1- 3 requires C: 59.46%, H: 8.22%, N: 2.04%.
Found: C: 59.24%, H: 8.35%, N: 1.97%. C.sub.24H.sub.49NO.sub.2
requires. 687. Found ES+: MH.sup.+ 688. .delta..sub.H
(CD.sub.3CO.sub.2D) 6.10 (1 H, brt, CONH, 5.70 (1 H, t, CH
(OAc)CH(OAc)CONH), 5.45 (1 H, t, CH(OAc)CH(OAc)CH--(OAc)CONH), 5.30
(1 H, m,CH(OAc)CONH), 5.05 (1 H, m AcOCH.sub.2(OAc)CH), 4.31 (1 H,
dd[J.sub.1=4 Hz,J.sub.2=12 Hz] AcOCH.sub.2(OAc), 4.13 (1 H,
dd[J.sub.1=6 Hz,J.sub.2=12 Hz] AcOCH.sub.2(OAc), 3.22 (2H, m,
NCH.sub.2), 2.33 (2H, t, CH.sub.2CO.sub.2H), 2.0-2.25(15 H,
5.times.s, acetate), 1.61 (2H, t, NCH.sub.2CH.sub.2), 1.45(1H, t,
CH.sub.2CH.sub.2CO.sub.2H), 1.25(26H,s, CH.sub.2).
[0271] Reduced Bixin Glycolipid
[0272] (F5)
N-(Peracetylglucuronylaminoethyl)-4,8,13,17-tetramethyidodecan-
amic acid 54
[0273] D15 (4.91 g, 11.1 mmol) was dissolved in methanol (50 ml)
containing DBU (5.02 ml, 33.6 mmol). .differential.-Gluconolactone
(2.4 g, 13.3 mmol) was added to the stirred solution, which was
left at room temperature for 5 hrs. The reaction was evaporated to
dryness and 10% cold citric acid added (50 ml). The precipitant was
kept cold whilst it was filtered off and washed with cold water.
The step can be slow. The solid was dried in vacuo then dissolved
in pyridine and dried by evaporation of solvent. The residue was
dissolved in pyridine (100 ml) containing acetic anhydride (20 ml)
and DMAP (200 mg) and left stirring overnight. Water was added (30
ml) with cooling and the reaction evaporated to dryness. The
resulting black tar was chromatographed (silica, 30% ethyl acetate
in hexane containing 0.1% acetic acid) to yield the title compound
as a glassy solid (1.83 g, 20%). C.sub.42H.sub.72N.sub.2O.sub.14
requires 828. Found ES+: MH.sup.+ 829. d.sub.H (CD.sub.3CO.sub.2D)
7.1 (1 H, brt, CONH), 6.17 (1 H, brt, CONH), 5.60 (1 H, t, CH
(OAc)CH(OAc)CONH), 5.41 (1 H, t, CH(OAc)CH (OAc)CH(OAc)CONH), 5.24
(1 H, m,CH(OAc)CONH), 5.02 (1 H, m, AcOCH.sub.2(OAc)CH), 4.29 (1 H,
dd[J.sub.1=4 Hz,J.sub.2=12 Hz] AcOCH.sub.2(OAc), 4.11 (1 H,
dd[J.sub.1=6 Hz,J.sub.2=12 Hz] AcOCH.sub.2(OAc), 3.2-3.62 (4 H, m,
NCH.sub.2), 2.33-0.8 (57H, mm, CH.sub.2 & CH.sub.3).
[0274] C24 Amide Containing Glycolipid
[0275] (F6)
(12-(Peracetylglucuronylaminododecanoylamino)-dodecanoic acid
55
[0276] D18 (2.12 g, 5.1 mmol) was dissolved in methanol (50 ml)
containing DBU (2.3 ml, 15.3 mmol). d-gluconolactone (1.09 g, 6.12
mmol) was added to the stirred solution, which was left at room
temperature for 3 hrs. The reaction was evaporated to dryness &
10% cold citric acid added (50 ml). The precipitant was kept cold
whilst it was filtered off and washed with cold water. This step
can be slow. The solid was dried in vacuo then dissolved in
pyridine and dried by evaporation of solvent. The residue was
dissolved in pyridine (100 ml) containing acetic anhydride (20 ml)
& DMAP (200 mg) and left stirring overnight. Water was added
(30 ml) with cooling and the reaction evaporated to dryness. Dilute
hydrochloric acid (1M, 100 ml) was added and the aqueous phase
extracted with dichloromethane, dried and evaporated to dryness.
The resulting black tar was chromatographed (silica, 5% methanol in
dichloromethane) to yield the title compound as a glass (2.44 g,
53%). C.sub.40H.sub.68N.sub.2O.sub.14 requires 800. Found ES+:
MH.sup.+ 801. .delta..sub.H (CDCl.sub.3) 7.1 (1 H, brt, CONH), 5.68
(1 H, br, CONH), 5.66 (1 H, t, CH (OAc)CH(OAc)CONH), 5.43 (1 H, t,
CH(OAc)CH(OAc)CH(OAc)CONH), 5.29 (1 H, m,CH(OAc) CONH), 5.04 (1 H,
m, AcOCH.sub.2(OAc)CH), 4.33 (1 H, dd[J.sub.1=4 Hz,J.sub.2=12 Hz]
AcOCH.sub.2(OAc), 4.15 (1 H, dd[J.sub.1=6 Hz,J.sub.2=12 Hz]
AcOCH.sub.2(OAc), 3.27-3.18 (4 H, m, NCH.sub.2), 2.33 (2H, t,
CH.sub.2CO), 2.19-2.04 (2H+15H, t+5s, CH.sub.2CO+acetates), 1.6-1.1
(36H, tm, CH.sub.2).
[0277] (F7) 12-(Glucuronylamino)dodecanoic acid 56
[0278] To a solution of d-gluconolactone (1.654 g, 9.288 mmol) in
dry methanol (50 ml) under argon was added 12-aminolauric acid
(2.000 g, 9.288 mmol) in dry methanol (50 ml), followed by dry
triethylamine (9.398 g, 92.88 mmol). The solution/suspension was
heated at 80.degree. C. for two hours until all solids dissolved
then left at room temperature overnight. Removal of the solvent
yielded the desired product as a white insoluble powder, a
suspension of which was washed in water (100 ml) at 90.degree. C.
for two hours. Filtration and subsequent drying under vacuum
yielded the title compound (3.817 g, 83%) as a pure white solid.
I.R. 1561cm.sup.-1 (CO.sub.2.sup.-), 1626 cm.sup.-1 (CONH,
CO.sub.2.sup.-). .delta..sub.H(CD.sub.3OD) 1.0-1.7 (18 H, br m,
(CH.sub.2).sub.9CH.sub.2CO.sub.2), 2.11 (2 H, t, CH.sub.2CO.sub.2),
3.20 (2 H, t, CH.sub.2N), 3.5-3.9 (4 H, m, CH.sub.2OH), 4.05, 4.25
(2 H, 2.times.br, CH.sub.2OH). d.sub.C(CD.sub.3OD) 11.6, 29.4, 32.0
(9 C, (CH.sub.2).sub.9CH.sub.2CO.sub.2), 41.0, 42.5 (2 C,
CH.sub.2NH, CH.sub.2CO.sub.2), 66.0 (1 C, CH.sub.2OH), 73.7, 74.4,
75.6, 76.8 (4 C, CHOH).
[0279] (F8) 12-(Peracetylglucuronylamino)dodecanoic acid 57
[0280] To F7 (1.739 g, 3.515 mmol) dissolved in dry pyridine (10
ml) under argon was added acetic anhydride (10 ml). The solution
was left overnight at room temperature and water (10 ml) slowly
added to decompose any anhydride. The solution was extracted
exhaustively with dichloromethane and this organic fraction washed
(4.times.20 ml HCl, pH3, and 4.times.20 ml water), dried
(MgSO.sub.4) and the solvent removed to quantitatively yield the
title compound as a pale yellow solid/gum, alumina tlc r.f. 0.1
(10% methanol in dichloromethane). C.sub.28H.sub.45NO.sub.13
requires 603.29. Found ES+: MH.sup.+ 604.31, ES-: (M-H.sup.+).sup.-
602.37. .delta..sub.H(CDCl.sub.3) 1.08 (15 H, br, Me), 1.27, 1.42
(4 H, 2.times.m, (CH.sub.2).sub.2(CH.sub.2).sub.4
.delta..sub.H(CDCl.sub.3) 1.08 (15 H, br, Me), 1.27, 1.42 (4 H,
2.times.m, (CH.sub.2).sub.2(CH.sub.- 2).sub.4 CO.sub.2H), 1.75-2.05
(14 H, br, (CH.sub.2).sub.4(CH.sub.2).sub.2-
(CH.sub.2).sub.3CO.sub.2H), 2.14 (2 H, m, CH.sub.2CO.sub.2H), 3.00
(2 H, m, CH.sub.2NH), 3.90-4.15 (2 H, br, CH.sub.2O), 4.90, 5.11,
5.26, 5.48 (4 H, 4.times.br, CHO), 6.95 (1 H, NHCO).
.delta..sub.C(CDCl.sub.3) 19.6, 19.9 (5 C, Me), 24.1, 26.1, 28.3,
28.5, 28.6, 28.7, 28.8, 33.3 (9 C,
(CH.sub.2).sub.9CH.sub.2CO.sub.2H), 38.9 (1 C, CH.sub.2CO.sub.2H),
53.1 (1 C, CH.sub.2N), 60.8 (1 C, CH.sub.2O), 68.1, 68.4, 68.7,
71.3 (4 C, CHO), 165.8 (1 C, CONH), 168.6, 169.1, 169.2, 169.9,
170.0 (5 C, COMe), 176.7 (1 C, CO.sub.2H).
[0281] G. Two Lipid Chain Syntheses
[0282] This section contains the synthesis of:
[0283] (G4)(RS)-N-{1-[8-aminobutylamino-7-aminobutylaminomethyl)
octylaminocarbonyl)-2-(peracetylglucuronylaminotetra-cosanoylamino)ethyl}-
-24-(peracetylglucuronylamino)-tetracosanamide
tetra(trifluoroacetate) salt 58
[0284] (G1)
(RS)-2,3-bis(Peracetylglucuronylaminotetracosanoyl-amino)propa-
noic acid 59
[0285] To F2 (250 mg, 0.324 mmol) dissolved in anhydrous
dichloromethane (10 ml) were added EDC hydrochloride (68 mg, 0.356
mmol) and N-hydroxysuccinimide (41 mg, 0.356 mmol) and the reaction
left overnight under argon at room temperature. To this was added a
solution of 2,3 diaminopropionic acid hydrochloride (228 mg, 0.162
mmol) and DBU (148 mg, 0.972 mmol) and the reaction stirred rapidly
overnight. The solvent was removed and the residues taken up into
hot water (20 ml). The solution was allowed to cool and acidified
to pH 1 with 1M HCl. The resulting precipitate was filtered off,
washed with water and dried under vacuum to yield the title
compound as a buff coloured precipitate (249 mg, 92%).
C.sub.83H.sub.142N.sub.4O.sub.26 requires 1611.0. Found ES.sup.+:
MNa.sup.+, 1633.4, MH.sup.+, 1611.6, ES.sup.- (M-H.sup.+).sup.-
1609.6. .delta..sub.H (CDCl.sub.3) 1.24 (76H, br,
(CH.sub.2).sub.19(CH.sub.2).sub- .2CO), 1.46 (4H, p,
CH.sub.2CH.sub.2CO), 1.62 (4H, br, CH.sub.2CH.sub.2N), 2.04-2.19
(30H, m, MeCO), 2.24 (4H, m, CH.sub.2CO), 3.23 (4H, m, CH.sub.2N),
3.45, 3.85 (2H, 2.times.m, CHCH.sub.2N), 4.12, 4.31 (4H, 2.times.m,
CH.sub.2OAc), 4.38 (1H, m, CHCO.sub.2), 5.04 (2H, q,
CHOAcCH.sub.2OAc), 5.28 (2H, d, CHOAcCONH), 5.43, 5.66 (4H,
2.times.t, (CHOAc).sub.2CHOAcCH.sub.2OAc), 6.09 (2H, t,
CONH(CH.sub.2).sub.23), 6.65 (1H, t,
CH.sub.2NHCO(CH.sub.2).sub.23), 7.76 (1H, d, CONHCHCO.sub.2H).
[0286] (G2)
(RS)-N-(1-{B-[t-butyloxycarbonylaminobutyl(t-butyloxy-carbonyl-
)amino]-7-[t-butyloxycarbonylaminobutyl(t-butyloxycarbonyl)aminomethyl]oct-
ylaminocarbonyl}-2-(peracetylglucuronylaminotetracosanoylamino)ethyl)-24-(-
peracetylglucuronylamino)tetracosanamide 60
[0287] To G1 (240 mg, 0.149 mmol) in anhydrous dichloromethane (20
ml) were added EDC hydrochloride (40 mg, 0.208 mmol) and
N-hydroxysuccinimide (19 mg, 0.164 mmol) and the reaction left
overnight under argon at room temperature. To this was added B8
(112 mg, 0.156 mmol) and triethylamine (90 mg, 0.893 mmol) and the
reaction left for a further five hours. The solvent was removed and
the residues purified by silica column chromatography (80% ethyl
acetate in hexane) to yield the title compound (163 mg, 47%) as a
colourless solid. C.sub.120H.sub.213N.sub.9O.sub.33 requires
2308.5. Found ES.sup.+: MHNa.sup.2+, 1167.5, MNa.sub.2.sup.2+,
1177.5. .delta..sub.H (CDCl.sub.3) 1.24 (84H, br,
(CH.sub.2).sub.19(CH.su- b.2).sub.2CO, (CH.sub.2).sub.4CH), 1.43
(54H, m, CH.sub.2CH.sub.2N, C(Me).sub.3, CH.sub.2CH.sub.2CO), 2.0
(1H, br, CH), 2.0-2.3 (30H, m, MeCO), 2.24 (4H, m, CH.sub.2CO),
2.95-3.35 (18H, m, CH.sub.2N), 3.45-3.80 (2H, m, CHCH.sub.2N),
4.1-4.35 (4H, m, CH.sub.2OAc), 4.40 (1H, m, CHCH.sub.2N), 5.03 (2H,
q, CHOAcCH.sub.2OAc), 5.16 (2H, d, CHOAcCO), 5.44, 5.54 (4H,
2.times.t, (CHOAc).sub.2CHOAcCO), 6.10 (3H, br, CHCONH, NHCOCHOAc),
7.45, 7.60 (2H, 2.times.br, CONHCHCH.sub.2NHCO).
[0288] (G3)
(RS)-N-(1-{8-[t-butyloxycarbonylaminobutyl(t-butyloxy-carbonyl-
)amino]-7-[t-butyloxycarbonylaminobutyl(t-butyloxycarbonyl)aminomethyl]oct-
ylaminocarbonyl}-2-(glucuronylaminotetracosanoylamino)ethyl)-24-(glucurony-
lamino)tetracosanamide 61
[0289] To G2 (163 mg) dissolved in methanol (30 ml) was added
concentrated ammonium hydroxide until the solution started to
become cloudy (approximately 3 ml). The reaction was left for three
hours but had not given total deacetylation and was precipitating
out of solution. The solvent was removed, the residues taken up
into 2:1 dichloromethane:methanol (60 ml) with heating and
concentrated ammonium hydroxide added until the solution started to
become cloudy (approx 10 ml). The reaction was left for a further
six hours and the solvents removed to yield the title compound as
an off white solid too insoluble for analysis and used crude in the
next step.
[0290] (G4)
(RS)-N-{1-[8-aminobutylamino-7-(aminobutylamino-methyl)octylam-
inocarbonyl)-2-(peracetylglucuronyl-aminotetracosanoylamino)ethyl}-24-(per-
acetylglucuronyl-amino)tetracosanamidetetra(trifluoroacetate) salt
62
[0291] G3 (120 mg) was dissolved in 96:4 trifluoroacetic
acid:dichloromethane (8 ml) and left for 20 minutes at room
temperature. The solvent was removed, the residues taken up into
water, filtered (0.45 mm polypropylene) and freeze dried to give
the title compound as a pale yellow solid (126 mg).
C.sub.80H.sub.161N.sub.9O.sub.15 requires 1488.2. Found ES.sup.+:
MH.sub.2.sup.2+, 745.4, MH.sup.+, 1489.3. .delta..sub.H (D.sub.2O)
1.7-2.25 (94H, br, (CH.sub.2).sub.21CH.sub.2CO,
(CH.sub.2).sub.5CH), 2.38 (8H, br,
NCH.sub.2(CH.sub.2).sub.2CH.sub.2N), 2.80 (5H, br, CH, CH.sub.2CO),
3.65-3.95 (18H, m, NCH.sub.2), 4.11 (2H, br, CHCH.sub.2NH), 4.2-4.5
(8H, m, CHOH), 4.6 (1H, m, COCHNH), 4.7, 4.85 (4H, 2.times.br,
CH.sub.2OH).
[0292] H. Syntheses of Protected & Unprotected Carbohydrate
Lipid Tetramines and Hexamines
[0293] This section contains the syntheses of:
[0294] Carbohydrate Lipid Tetramines
[0295] (H3)
N-[8-(aminobutylamino)-7-(aminobutylaminomethyl)octyl]-24-(glu-
curonylamino)tetracosanamidetetra(trifluoroacetate) salt 63
[0296] (H6)
N-[8-(Aminobutylamino)-7-(aminobutylaminomethyl)-octyl]-18-(gl-
ucuronylamino)octadecanamidetetra(trifluoroacetate) salt 64
[0297] (H12)
N-[8-Aminobutylamino-7-(Aminobutylamin-o-methyl)octyl]-18-(gl-
ucuronylaminododecanoylamino)-dodecanamidetetra(trifluoroacetate)
salt 65
[0298] (H9)
N-[8-(aminobutylamino)-7-(aminobutylaminomethyl)-octyl]-N'-(gl-
ucuronylaminoethyl)-4,8,13,17-tetramethyl-1,20-docosadiamidetetra(trifluor-
oacetate) salt 66
[0299] (H15)
N-[8-(aminobutylamino)-7-(aminobutylamino-methyl)octylaminoca-
rbonyltricosanyl]-N',N'-bis(glucuronylaminoethyl)succinamidetetra(trifluor-
oacetate) salt 67
[0300] Carbohydrate Lipid Hexamines
[0301] (H18)
N-[8-(Aminopropylaminobutylamino)-7-(amino-propylaminobutylam-
inomethyl)octyl]-24-(glucuronyl-amino)tetracosanamide.
hexa(trifluoroacetate) salt 68
[0302] (H21)
N-[8-(aminopropylaminobutylamino)-7-(aminopropyl-aminobutylam-
inomethyl)octyl]-18-(glucuronylamino)-octadecanamide.
hexa(trifluoroacetate) salt 69
[0303] (H24)
N-[8-(aminopropylaminobutylamino)-7-(aminopropyl-aminobutylam-
inomethyl)octyl]-N'-(glucuronyl-aminoethyl)-4,8,13,17-tetramethyl-1,20-doc-
osadiamidehexa(trifluoroacetate) salt 70
[0304] (H27)
N-[8-(Aminopropylaminobutylamino)-7-(aminopropyl-butylaminome-
thyl)octyl]-12-(glucuronylamino-dodecanoylamino)dodecanamidehexa(trifluoro-
acetate) salt 71
[0305] (H20)
N-[8-(Methylaminopropylaminobutylamino)-7-(methyl-aminopropyl-
aminobutylaminomethyl)octylaminocarbonyl
tricosanyl}-N',N'-bis(glucuronyla- minoethyl)succinamide 72
[0306] (H33)
N-[8-(methylaminopropylaminobutylamino)-7-(methylaminopropyla-
minobutylaminomethyl)octyl]-24-(glucuronylamino)tetracosanamide
73
[0307] (H36)
N-[8-(Methylaminopropylaminobutylamino)-7-(methylaminopropyla-
minobutylaminomethyl)octyl]-24-(glucuronylaminododecanoylamino)tetracosana-
mide 74
[0308] (H38) 75
[0309] Carbohydrate Lipid Tetramines
[0310] C24 Tetramine
[0311] (H1)
N-{8-[t-Butyloxycarbonylaminobutyl(t-butyloxy-carbonyl)amino]--
7-[t-Butyloxycarbonylaminobutyl(t-butyloxycarbonyl)aminomethyl]octyl}-24-(-
peracetylglucuronylamino)tetracosanamide 76
[0312] N-Methylmorpholine (0.12 ml, 1.1 mmol) was added to a
stirred solution of F2 (0.771 mg, 1 mmol) in dry dichloromethane
(10 ml) at room temperature under argon. After a few minutes
N-hydroxysuccinimide (127 mg, 1.1 mmol) was added followed by EDC
(270 mg, 1.4 mmol), the reaction was stirred overnight (tlc showed
conversion to the active ester). A solution of B8 (860 mg, 1.3
mmol) and triethylamine (0.7 ml, 5.5 mmol) in dry dichloromethane
(5 ml) was added dropwise and stirring continued for two hours. Tlc
showed the active ester had been converted to a slower moving
product (silica-60% ethyl acetate in hexane). The solvent was
removed and the product chromatographed (silica-60% ethyl acetate
in hexane) to yield the title compound (1.25 g, 70%).
C.sub.77H.sub.140N.sub.6O.sub.20.H.sub.2O requires C:62.53%, H:
9.54%, N: 5.61%. Found: C: 62.53%, H: 9.52%, N: 5.69%.
C.sub.77H.sub.140N.sub.6O.su- b.20 requires 1469. Found ES+:
MH.sup.+ 1470. .delta..sub.H (CDCl.sub.3) 6.07 (1 H, brt, CONH),
5.67 (1 H, t, CH (OAc)CH(OAc)CONH), 5.43 (1 H, t,
CH(OAc)CH(OAc)CH(OAc)CONH), 5.30 (1 H, m,CH(OAc)CONH), 5.05 (1 H, m
AcOCH.sub.2(OAc)CH), 4.31 (1 H, dd[J.sub.1=4 Hz,J.sub.2=12 Hz]
AcOCH.sub.2(OAc), 4.13 (1 H, dd[J.sub.1=6 Hz,J.sub.2=12 Hz]
AcOCH.sub.2(OAc), 2.9-3.4 (16 H, m, NCH.sub.2), 2.0-2.3 (17 H, m,
CH.sub.2CO), 1.2-1.7 (96 H, dm, CH.sub.2),
[0313] (H2)
N-{8-[t-Butyloxycarbonylaminobutyl(t-butyloxy-carbonyl)-amino]-
-7-[t-Butyloxycarbonylaminobutyl(t-butyloxycarbonyl)aminomethyl]octyl}-24--
(glucuronyl-amino)tetracosanamide 77
[0314] A solution of potassium carbonate (431 mg, 3.12 mmol) in
water (2 ml) was added dropwise to a stirred solution of H1 (905
mg, 0.61 mmol) in methanol (15 ml) at room temperature. The flask
was stirred for 20 min whereupon tlc showed no starting material
was present. Water was added and the precipitate filtered, washed
and dried. The product was purified by chromatography (silica-15%
methanol in dichloromethane) to yield the title compound (445
mg,57%). C.sub.67H.sub.130N.sub.6O.sub.15 requires 1259. Found ES+:
MH.sup.+ 1260. .delta..sub.H (CD.sub.3OD) 3.6-4.2 (6 H, m, sugar),
3.0-3.4 (16H, m, CH.sub.2N), 2.15 (3 H, t, CH.sub.2CO), 2.05 (1 H,
m, CH.sub.2CH(CH.sub.2).sub.2), 1.2-1.7 (96 H, m,CH.sub.2),
[0315] (H3)
N-[8-(aminobutylamino)-7-(aminobutylaminomethyl)-octyl]-24-(gl-
ucuronylamino)tetracosanamidetetra-(trifluoroacetate) salt 78
[0316] A solution of the H2 (445 mg, 0.353 mmol) in 96% TFA (4%
water) was stirred for 30 mins at room temperature. The TFA was
removed under vacuum, and traces removed by evaporation of water.
The title compound was obtained as a colourless solid by
lyophilisation. 400 mg, 86%.
C.sub.55H.sub.102N.sub.6O.sub.15F.sub.12. 1.6H.sub.2O requires
C:46.95%, H: 7.34%, N: 5.76%. Found: C: 46.99%, H: 7.17%, N: 5.82%.
The free base C.sub.47H.sub.98N.sub.6O.sub.7 requires 858.7. Found
ES+: MH.sup.+ 859.7. .delta..sub.H (CD.sub.3OD) 3.6-4.2 (6 H, m,
sugar), 3.0-3.4 (16H, m, CH.sub.2N), 2.0-2.2 (2 H, t, CH.sub.2CO),
1.2-1.7 (96 H, m,CH.sub.2).
[0317] C18 Tetramine
[0318] (H4)
N-{8-[t-Butyloxycarbonylaminobutyl(t-butyloxycarbonyl)-amino]--
7-[t-Butyloxycarbonylaminobutyl(t-butyloxy-carbonyl)aminomethyl]octyl}-18--
(peracetylglucuronyl-amino)octadecanamide 79
[0319] N-Methylmorpholine (0.12 ml, 1.1 mmol) was added to a
stirred solution of F4 (687 mg, 1 mmol) in dry dichloromethane (20
ml) at room temperature under argon. A few minutes later
N-hydroxysuccinimide (127 mg, 1.1 mmol) was added followed by EDC
(270 mg, 1.4 mmol), the reaction was stirred overnight (tlc showed
conversion to the active ester). A solution of B8 (394 mg, 0.55
mmol) and triethylamine (0.35 ml, 5.5 mmol) in dry dichloromethane
(4 ml) was added dropwise and stirring continued for two hours. Tlc
showed the active ester had been converted to a slower moving
product (silica-60% ethyl acetate in hexane). The solvent was
removed and the product chromatographed (silica60% to 80% ethyl
acetate in hexane) to yield the title compound (533 mg, 80%).
C.sub.61H.sub.128N.sub.6O.sub.15.2/3H.sub.2O requires C: 61.01%, H:
9.33%, N: 6.01%. Found: C: 61.05%, H: 9.32%, N: 5.87%.
C.sub.61H.sub.128N.sub.6O.sub.20 requires 1384.9. Found ES+:
MH.sup.+ 1386.3. .delta..sub.H (CDCl.sub.3) 6.07 (1 H, brt, CONH),
5.85 (1 H, br, CONH), 5.67 (1 H, t, CH (OAc)CH(OAc)CONH), 5.43 (1
H, t, CH(OAc)CH(OAc)CH(OAc)CONH), 5.30 (1 H, m,CH(OAc)CONH), 5.05
(1 H, m AcOCH.sub.2(OAc)CH), 4.65 (2H, br, 2.times.NH), 4.31 (1 H,
dd [J.sub.1=4 Hz,J.sub.2=12 Hz] AcOCH.sub.2(OAc), 4.13 (1 H,
dd[J.sub.1=6 Hz,J.sub.2=12 Hz] AcOCH.sub.2(OAc), 2.9-3.4 (16 H, m,
NCH.sub.2), 2.0-2.3 (17 H, m, MeCO+CH.sub.2CO), 1.2-1.7(84 H, dm,
CH.sub.2),
[0320] (H5)
N-{8-[t-Butyloxycarbonylaminobutyl(t-butyloxycarbonyl)-amino]--
7-[t-Butyloxycarbonylaminobutyl(t-butyloxy-carbonyl)aminomethyl]octyl}-18--
(glucuronylamino)-octadecanamide 80
[0321] A solution of potassium carbonate (207 mg, 1.5 mmol) in
water (1 ml) was added dropwise to a stirred solution of H4 (415
mg, 0.3 mmol) in methanol (12 ml) at room temperature. The flask
was stirred for 30 mins whereupon tlc showed no starting material
was present. Amberlite CG50 (4 g wet) was added and the resin
filtered, washed with methanol and dried. The product was purified
by chromatography (silica-10% methanol in dichloromethane) to yield
the title compound (190 mg, 54%).
C.sub.61H.sub.118N.sub.6O.sub.15.3/5H.sub.2O requires C: 61.75%, H:
10.13%, N: 7.081%. Found: C: 61.755%, H: 10.06%, N: 6.95%.
C.sub.61H.sub.118N.sub.6O.sub.15 requires 1174.9. Found ES+:
MH.sup.+ 1176.0. .delta..sub.H (CD.sub.3OD) 3.6-4.2 (6 H, m,
sugar), 3.0-3.4 (16H, m, CH.sub.2N), 2.15 (2 H, t, CH.sub.2CO),
2.06 (1 H, m, CH.sub.2CH(CH.sub.2).sub.2, 1.2-1.7 (84 H,
m,CH.sub.2).
[0322] (H6)
N-[8-(Aminobutylamino)-7-(aminobutylaminomethyl)-octyl]-18-(gl-
ucuronylamino)octadecanamidetetra-(trifluoroacetate) salt 81
[0323] A solution of H5 (190 mg, 0.16 mmol) in 96% TFA (4% water)
was stirred for 30 mins at room temperature. The TFA was removed
under vacuum, and traces removed by evaporation of water. The title
compound was obtained as a colourless hygroscopic solid (190 mg,
95%) by lyophilisation.
C.sub.49H.sub.90N.sub.6O.sub.15F.sub.122H.sub.2O
(Mwtanhydrous=1230) requires C:46.95%, H: 7.34%, N: 5.76%. Found:
C: 46.99%, H: 7.17%, N: 5.82%. The free base
C.sub.41H.sub.86N.sub.6O.sub.7 requires 774. Found ES+: MH.sup.+
775. .delta..sub.H (CD.sub.3OD) 3.6-4.25 (6 H, m, sugar), 2.9-3.4
(16H, m, CH.sub.2N), 2.15 (2H, t, CH.sub.2CO), 2.23 (1 H, m,
CH.sub.2CH(CH.sub.2).sub.2), 1.2-1.9 (84 H, m, CH.sub.2).
[0324] Bixin Tetramine
[0325] (H7)
N-{8-[t-Butyloxycarbonylaminobutyl(t-butyloxycarbonyl)-amino]--
7-[t-butyloxycarbonylaminobutyl(t-butyloxy-carbonyl)aminomethyl]octyl}-N'--
(peracetylglucuronyl-aminoethyl)-4,8,13,17-tetramethyl-1,20-docosadiamide
82
[0326] N-Methylmorpholine (0.16 ml, 1.47 mmol) was added to a
stirred solution of F5 (1.04 g, 1.33 mmol) in dry dichloromethane
(20 ml) at room temperature under argon. A few minutes later
N-hydroxysuccinimide (170 mg, 1.47 mmol) was added followed by EDC
(282 mg, 1.47 mmol), the reaction was stirred overnight (tlc showed
conversion to the active ester). A solution of B8 (520 mg, 1.45
mmol) and triethylamine (0.46 ml, 3.35 mmol) in dry dichloromethane
(10 ml) was added dropwise and stirring continued for two hours.
Tlc showed the active ester had been converted to a slower moving
product (silica-60% ethyl acetate in hexane). The solvent was
removed and the product chromatographed (silica-5% methanol in
dichloromethane) to yield the title compound (810 mg, 60%).
C.sub.79H.sub.143N.sub.7O.sub.21 requires C: 62.14%, H: 9.44%, N:
6.42%. Found: C: 61.76%, H: 9.42%, N: 6.35%.
C.sub.79H.sub.143N.sub.7O.sub.21 requires 1526. Found ES+: MH.sup.+
1527.1. .delta..sub.H (CDCl.sub.3) 7.15 (1 H, br, CONH), 6.47 (1 H,
br, CONH), 5.95 (1 H, br, CONH), 5.67 (1H, t, CH(OAc)CH(OAc)CONH),
5.43 (1 H, t, CH(OAc)CH(OAc)CH(OAc)CONH), 5.30 (1 H,
m,CH(OAc)CONH), 5.05 (1 H, m AcOCH.sub.2(OAc)CH), 4.65 (2 H, br,
2.times.NH), 4.31 (1 H, dd[J.sub.1=4 Hz,J.sub.2=12 Hz]
AcOCH.sub.2(OAc), 4.13 (1 H, dd[J.sub.1=6 Hz,J.sub.2=12 Hz]
AcOCH.sub.2(OAc), 4.70(2H, br, CONH), 2.9-3.5 (18 H, m, NCH.sub.2),
2.0-2.3 (21 H, m, MeCO+CH.sub.2CO), 1.0-1.7(82 H, dm, CH.sub.2),
0.8-40.9(12 H, 4.times.s, Me).
[0327] (H8)
N-{8-[t-Butyloxycarbonylaminobutyl(t-butyloxycarbonyl)-amino]--
7-[t-butyloxycarbonylaminobutyl(t-butyloxy-carbonyl)aminomethyl]octyl}-N'--
(glucuronylaminoethyl)-4,8,13,17-tetramethyl-1,20-docosadiaamide
83
[0328] A solution of potassium carbonate (372 mg, 2.7 mmol) in
water (1 ml) was added dropwise to a stirred solution of H7 (790
mg, 0.52 mmol) in methanol (12 ml) at room temperature. The flask
was stirred for 30 min whereupon tlc showed no starting material
was present. Amberlite.RTM. CG50 (5 g wet) was added and the resin
filtered, washed with methanol & dried. The product was
purified by chromatography (silica-10%-20% methanol in
dichloromethane) to yield the title compound (230 mg, 72%).
C.sub.69H.sub.133N.sub.7O.sub.16 requires 1315.9. Found ES+:
MH.sup.+ 1317.0. .delta..sub.H (CD.sub.3OD) 3.6-4.3 (6 H, m,
sugar), 3.0-3.4 (18H, m, CH.sub.2N.), 2.0-2.3 (6 H, m, CH.sub.2CO),
1.0-1.7 (81 H, m, alkane, 0.8-1.0 (12H, 4.times.s,Me).
[0329] (H9)
N-[8-(aminobutylamino)-7-(aminobutylamino-methyl)octyl]-N'-(gl-
ucuronylaminoethyl)-4,8,13,17-tetramethyl-1,20-docosadiamidetetra(trifluor-
oacetate) salt 84
[0330] A solution of H8 (180 mg, 0.137 mmol) in 96% TFA (4% water)
was stirred for 30 min at room temperature. The TFA was removed
under vacuum, and traces removed by evaporation of water. The title
compound was obtained as to a colourless hygroscopic solid (170 mg,
91%) by lyophilisation. The free base
C.sub.49H.sub.101N.sub.7O.sub.8 requires 915.8 (salt
C.sub.57H.sub.105F.sub.12N.sub.7O.sub.16=1371). Found ES+: MH.sup.+
917.1. .delta..sub.H (CD.sub.3OD) 3.6-4.25 (1+1+4H, 3.times.m,
sugar), 2.9-3.4 (4+8+4H, 3.times.m, CH.sub.2N), 2.1-2.35 (5 H, m,
CH.sub.2CO+CH.sub.2CH(CH.sub.2).sub.2), 1.0-2.0 (44 H, m, alkane),
0.8-1.0 (12 H, 4.times.s, Me).
[0331] C24 Amide Tetramine
[0332] (H10) N-{8-[t-Butyloxycarbonylaminobutyl(t-butyloxycarbonyl)
amino]-7-[t-butyloxycarbonylaminobutyl(t-butyloxy-carbonyl)aminomethyl]oc-
tyl}-12-(peracetylglucuronyl-aminododecanoylamino)dodecanamide
85
[0333] DBU (0.05 ml, 0.36 mmol) was added to a stirred suspension
of F6 (264 mg, 0.33 mmol) in dry dichloromethane (4 ml) at room
temperature under argon. After a few minutes N-hydroxysuccinimide
(38 mg, 0.33 mmol) was added followed by EDC (63 mg, 0.33 mmol),
the reaction was stirred overnight (tlc showed conversion to the
active ester). A solution of B8 (260 mg, 0.36 mmol) and DBU(0.1 ml,
0.66 mmol) in dry dichloromethane (4 ml) was added dropwise and
stirring continued for two hours. Tlc showed the active ester had
been converted to a slower moving product (silica-60% ethyl acetate
in hexane). The solvent was removed and the product chromatographed
(silica-2% to 5% methanol in dichloromethane) to yield the title
compound (376 mg, 76%). C.sub.77H.sub.139N.sub.7O.sub.21 requires
1498.0. Found ES+: MH.sup.+ 1499.0 .delta..sub.H (CDCl.sub.3) 6.15
(1 H, brt, CONH), 5.92 (1 H, br, CONH), 5.75 (1 H, br, CONH), 4.7
(2H, br, 2.times.CONH) 5.67 (1 H, t, CH (OAc)CH(OAc)CONH), 5.43 (1
H, t, CH(OAc)CH(OAc)CH(OAc)CONH), 5.30 (1 H, m,CH(OAc)CONH), 5.05
(1 H, m AcOCH.sub.2(OAc)CH), 4.31 (1 H, dd[J.sub.1=4 Hz,J.sub.2=12
Hz] ACOGH.sub.2(OAc), 4.13 (1 H, dd[J.sub.1=6 Hz,J.sub.2=12 Hz]
AcOCH.sub.2(OAc), 3.0-3.3 (20 H, m, NCH.sub.2), 2.0-2.3 (18 H, m,
MeCO+CH.sub.2CO+CH.sub.2CH(CH.sub.2).sub.2), 1.2-1.7(90 H, dm,
CH.sub.2+Me).
[0334] (H11)
N-{8-[t-Butyloxycarbonylaminobutyl(t-butyloxy-carbonyl)amino]-
-7-[t-butyloxycarbonylaminobutyl(t-butyloxycarbonyl)aminomethyl]octyl}-12--
(glucuronylaminododecanoylamino)dodecanamide 86
[0335] Ammonia solution (20 ml, 0.880) was added to a stirred
solution of H10 (370 mg, 0.25 mmol) in methanol (20 ml) at room
temperature. The flask was stirred for 30 min and evaporated to
dryness. The white solid was suspended in water, filtered off and
dried. The product was chromatographed (Reverse phase silica,
Merck-Lichroprep eluted with dichloromethane/methanol/water
[2:6:1]). The product was evaporated to dryness and triturated with
ether. High vacuum yielded the title compound (234 mg) as a white
solid. C.sub.67H.sub.129N.sub.7O.sub.16 requires 1287.9. Found ES+:
MH.sup.+ 1288.9. .delta..sub.H (CD.sub.3OD) 3.6-4.2 (6 H, m,
sugar), 3.0-3.4 (18H, m, CH.sub.2N), 2.15 (4 H, t, CH.sub.2CO),
2.06 (1 H, m, CH.sub.2CH(CH.sub.2).sub.2, 1.2-1.7 (90 H, m,
CH.sub.2+Me).
[0336] (H12)
N-[8-(Aminobutylamino)-7-(aminobutylaminomethyl)-octyl]-12-(g-
lucuronylaminododecanoylamino)-dodecanamidetetra(trifluoroacetate)
salt 87
[0337] A solution of H11 (260 mg, 0.18 mmol) in 96% TFA (6 ml, 4%
water) was stirred for 30 min at room temperature. The TFA was
removed under vacuum, and traces removed by evaporation of
toluene/water. The compound was subjected to high vacuum overnight,
dissolved in water (5 ml) and filtered through a 0.45 .mu.M
(Whatman PP) filter The compound was converted to a colourless
solid by lyophilisation. The lyophilised hygroscopic solid was
triturated with ether and dried in vacuo to yield the title
compound (287 mg, 100%). C.sub.47H.sub.97N.sub.7O.sub.8.
C.sub.8H.sub.4O.sub.8.F.sub.12. 3.25H.sub.2O (Mwt anhydrous=1344.4)
requires C:47.09%, H: 7.72%, N: 6.99%. Found: C: 49.14%, H: 7.57%,
N: 7.29%. The free base C.sub.47H.sub.97N.sub.7O.sub.8 requires
887.7. Found ES+: MH.sup.+ 888.7. .delta..sub.H (D.sub.2O) 3.6-4.25
(6 H, m, sugar), 2.9-3.4 (18H, m, CH.sub.2N), 2.15 (4 H, dt,
CH.sub.2CO), 2.23 (1 H, m, CH.sub.2CH(CH.sub.2).sub.2), 1.2-1.9 (54
H, m, CH.sub.2).
[0338] Disugar Tetramines
[0339] (H13)
N-{8-[t-Butyloxycarbonylaminobutyl(t-butyloxy-carbonyl)amino]-
-7-[t-butyloxycarbonylaminobutyl(t-butyloxycarbonyl)aminomethyl]octylamino-
carbonyltricos anyl}-N',N'-bis(peracetylglucuronylaminoethyl)
succinamide 88
[0340] C4 (259 mg, 0.254 mmol), EDC hydrochloride (101 mg, 0.528
mmol) and N-hydroxysuccinimide (46 mg, 0.396 mmol) were dissolved
in anhydrous dichloromethane (20 ml) and activated ester formation
left at room temperature for two hours under argon. To the solution
was added E2 (300 mg, 0.277 mmol) and triethylamine (80 mg, 0.792
mmol) and the reaction left overnight at room temperature under
argon. The solvents were removed, and the resulting residues
purified by gradient silica column chromatography (3-7% methanol in
dichloromethane) to yield the title compound as a colourless solid
(350 mg, 65%). C.sub.101H.sub.175N.sub.9O.- sub.33 requires 2042.2.
Found ES.sup.+: MH.sup.+, 2043.4, MNa.sup.+, 2065.5, MK.sup.+,
2081.4. .delta..sub.H (CDCl.sub.3) 1.24 (48H, br,
(CH.sub.2).sub.20CH.sub.2CO, (CH.sub.2).sub.4CH), 1.43 (48H, br,
(Me).sub.3C, CH.sub.2CH.sub.2N), 2.03-2.23 (30H, 10.times.s, MeCO),
2.19 (2H, t, (CH.sub.2).sub.22CH.sub.2CO), 2.3-2.8 (4H, brm,
(CH.sub.2).sub.2CO), 3.0-3.4 (24H, brm, CH.sub.2N), 4.10 (2H, m,
CH.sub.2OAc), 4.31 (2H, m, CH.sub.2OAc), 4.65 (2H, br, NHCO.sub.2),
5.05 (2H, m, CHCH.sub.2OAc), 5.21, 5.25 (2H, 2.times.d, CHCON),
5.44, 5.66 (4H, 2.times.m, (CHOAc).sub.2CHOAcCH.sub.2OAc), 5.75
(1H, br, (CH.sub.2).sub.23CONH), 6.48 (1H, br,
(CH.sub.2).sub.2CONH), 7.21, 7.70 (2H, 2.times.t,
N(CH.sub.2CH.sub.2NHCO).sub.2).
[0341] (H14)
N-{8-[t-Butyloxycarbonylaminobutyl(t-butyloxy-carbonyl)amino]-
-7-[t-butyloxycarbonylaminobutyl(t-butyloxycarbonyl)aminomethyl]octylamino-
carbonyltricos anyl}-N',N'-bis(glucuronylaminoethyl)succinamide
89
[0342] To H13 (349 mg) in methanol (20 ml) was added concentrated
ammonium hydroxide (5 ml). The cloudy solution/suspension was
rapidly stirred for two hours whereupon the solvent was removed and
the resulting residues purified by reverse phase silica
chromatography eluting with 2:6:1 dichloromethane:methanol:water to
yield the title compound as a colourless solid (238 mg, 86%).
C.sub.81H.sub.155N.sub.9O.sub.23 requires 1622.1 Found ES.sup.+:
MH.sub.2.sup.2+, 812.4, MHNa.sup.2+, 823.7, ES.sup.-: MCl.sup.-,
1657.3. .delta..sub.H (CD.sub.3OD) 1.28 (46H, br,
(CH.sub.2).sub.19(CH.sub.2).sub.2CO, (CH.sub.2).sub.4CH), 1.44
(50H, m, (Me).sub.3C, CH.sub.2CH.sub.2N), 2.03 (1H, br, CH), 2.16
(2H, t, (CH.sub.2).sub.22CH.sub.2CO), 2.49 (2H, t, CH.sub.2CONH),
2.70 (2H, m, CH.sub.2CON), 3.0-3.3 (16H, m, CH.sub.2NBOC), 3.4-3.6
(8H, m, N(CH.sub.2).sub.2N), 3.6-3.85 (8H, m, CHOH), 4.11 (2H, m,
CH.sub.2OH), 4.19, 4.22 (2H, 2.times.d, CH.sub.2OH).
[0343] (H15)
N-[8-(aminobutylamino)-7-(aminobutylaminomethyl)-octylaminoca-
rbonyltricosanyl]-N',N'-bis(glucuronyl-aminoethyl)succinamidetetra(trifluo-
roacetate) salt 90
[0344] To H14 (234 mg) was added 96:4 trifluoroacetic
acid:dichloromethane (10 ml). The solution was left for 20 minutes
at room temperature, the solvents removed and the residues taken up
into MilliQ water. The solution was filtered (0.45 mm
polypropylene) and freeze dried to give a white solid. This was
suspended in diethyl ether, left for 20 minutes and the ether
decanted off. The remaing white solids were dried under vacuum to
give the title compound as a white, hydroscopic solid (226 mg).
C.sub.61H.sub.123N.sub.9O.sub.15 requires 1221.9 Found ES.sup.+:
MH.sub.2.sup.2+, 612.0, MH.sup.+, 1222.9. .delta..sub.H (D.sub.2O)
1.29 (44H, br, (CH.sub.2).sub.19(CH.sub.2).sub.2CO,
(CH.sub.2).sub.3CH.sub.2CH- ), 1.49 (8H, brm, CH.sub.2CH,
CH.sub.2CH.sub.2CO, CH.sub.2CH.sub.2NHCO), 1.78 (8H, br,
NCH.sub.2(CH.sub.2).sub.2CH.sub.2N), 2.21 (3H, br, CH), 2.51,
2.72(4H, 2.times.t, CO(CH.sub.2).sub.2CO), 3.0-3.3 (16H, m,
CH.sub.2N), 3.3-3.65 (8H, m, N(CH.sub.2).sub.2N), 3.65-3.95 (8H, m,
CHOH), 4.10, 4.30 (4H, 2.times.br, CH.sub.2OH).
[0345] Carbohydrate Lipid Hexamines
[0346] C24 Hexamine
[0347] (H16)
N-{8-[t-Butyloxycarbonylaminopropyl(t-butyloxy-carbonyl)amino-
butyl(t-butyloxycarbonyl)amino]-7-[t-butyloxycarbonylaminopropyl(t-butylox-
ycarbonyl)-aminobutyl(t-butyloxycarbonyl)aminomethyl]octyl}-24-(peracetylg-
lucuronylamino)tetracosanamide 91
[0348] N-Methylmorpholine (0.06 ml, 0.57 mmol) was added to a
stirred solution of F2 (400 mg, 0.52 mmol) in dry dichloromethane
(10 ml) at room temperature under argon. After a few minutes
N-hydroxysuccinimide (66 mg, 0.57 mmol) was added followed by EDC
(109 mg, 0.57 mmol), the reaction was stirred overnight (tlc showed
conversion to the active ester). A solution of B16 (640 mg, 0.622
mmol) and triethylamine (0.36 ml, 2.6 mmol) in dry dichloromethane
(5 ml) was added dropwise and stirring continued for two hours. Tlc
showed the active ester had been converted to a slower moving
product (silica-60% ethyl acetate in hexane). The solvent was
removed and the product chromatographed (silica-66% ethyl acetate
in hexane) to yield the title compound (400 mg, 58%).
C.sub.93H.sub.170N.sub.8O.sub.24 requires 1783.23. Found ES+:
MH.sup.+ 1785.4. C.sub.93H.sub.170N.sub.8O.sub.24 requires
C:63.33%, H: 10.24%, N: 7.12%. Found: C: 63.41%, H: 10.57%, N:
6.98%. .delta..sub.H (CDCl.sub.3) 6.07 (1 H, brt, CONH), 5.6 (1 H,
brt, CONH), 5.67 (1 H, t, CH (OAc)CH(OAc)CONH), 5.43 (1 H, t,
CH(OAc)CH(OAc)CH(OAc)CONH), 5.30 (1 H, m,CH(OAc)CONH), 5.05 (1 H, m
AcOCH.sub.2(OAc)CH), 4.31 (1 H, dd[J.sub.1=4 Hz,J.sub.2=12 Hz]
AcOCH.sub.2(OAc), 4.13 (1 H, dd[J.sub.1=6 Hz,J.sub.2=12 Hz]
AcOCH.sub.2(OAc), 2.9-3.4 (24 H, m, NCH.sub.2), 2.0-2.3 (17 H, m,
CH.sub.2CO+5.times.acetate), 2.23 (1 H, m,
CH.sub.2CH(CH.sub.2).sub.2), 1.2-1.8 (118 H, dm, CH.sub.2+Me).
[0349] (H17)
N-{8-[t-Butyloxycarbonylaminopropyl(t-butyloxy-carbonyl)amino-
butyl(t-butyloxycarbonyl)amino]-7-[t-butyloxycarbonylaminopropyl(t-butylox-
ycarbonyl)-aminobutyl(t-butyloxycarbonyl)aminomethyl]octyl}-24-(glucuronyl-
amino)tetracosanamide 92
[0350] A solution of potassium carbonate (186 mg, 1.26 mmol) in
water (1 ml) was added dropwise to a stirred solution of H16 (380
mg, 0.21 mmol) in methanol (7 ml) at room temperature. The flask
was stirred for 30 mins whereupon tlc showed no starting material
was present. Water (20 ml) was added and the precipitate filtered,
washed and dried. The product was purified by chromatography
(Reverse phase silica, Merck Lichroprep-15% methanol in
dichloromethane) to yield the title compound (445 mg,57%).
C.sub.83H160N.sub.8O.sub.14 requires C:63.33%, H: 10.24%, N: 7.12%.
Found: C: 63.41%, H: 10.57%, N: 6.98%.
C.sub.67H.sub.130N.sub.6O.sub.15 requires 1573.18. Found ES+:
MH.sup.+ 1574.3. .delta..sub.H (CD.sub.3OD) 3.64.2 (6 H, m, sugar),
3.0-3.4 (24H, m, CH.sub.2N), 2.15 (3 H, t, CH.sub.2CO), 2.05 (1 H,
m, CH.sub.2CH(CH.sub.2).sub.2), 1.2-1.7 (118 H, m,
CH.sub.2+Me),
[0351]
(H18)N-[8-(Aminopropylaminobutylamino)-7-(aminopropyl-aminobutylami-
nomethyl)octyl]-24-(glucuronyl-amino)tetracosanamidehexa(trifluoroacetate)
salt 93
[0352] A solution of H17 (245 mg, 0.353 mmol) in 96% TFA (10 ml, 4%
water) was stirred for 30 min at room temperature. The TFA was
removed under vacuum, and traces removed by evaporation of water.
The title compound was cobtained as a colourless hygroscopic solid
(400 mg, 86%) by lyophilisation.
C.sub.65H.sub.118N.sub.8O.sub.19F.sub.18.2CF.sub.3CO.sub.- 2H
requires C:43.95%, H: 6.42%, N: 5.94%. Found: C: 47.10%, H: 7.18%,
N: 6.76%. The free base C.sub.53H.sub.112N.sub.8O.sub.7 requires
972.86. Found ES+: MH.sup.+ 973.9. .delta..sub.H (CD.sub.3OD)
3.6-4.2 (6 H, m, sugar), 3.0-3.4 (24H, m, CH.sub.2N), 2.0-2.2 (7
H,m,
CH.sub.2CO+CH.sub.2CH(CH.sub.2).sub.2+CH.sub.2CH.sub.2NH.sub.2),1.2-1.7
(60 H, m,CH.sub.2).
[0353] C18 Hexamine
[0354] (H19)
N-{8-[t-Butyloxycarbonylaminopropyl(t-butyloxy-carbonyl)amino-
butyl(t-butyloxycarbonyl)amino]-7-[t-butyloxycarbonylaminopropyl(t-butylox-
ycarbonyl)-aminobutyl(t-butyloxycarbonyl)aminomethyl]octyl}-18-(peracetylg-
lucuronylamino)octadecanamide 94
[0355] N-Methylmorpholine (0.05 ml, 0.42 mmol) was added to a
stirred solution of F4 (260 mg, 0.38 mmol) in dry dichloromethane
(8 ml) at room temperature under argon. A few minutes later
N-hydroxysuccinimide (48 mg, 0.42 mmol) was added followed by EDC
(100 mg, 0.42 mmol), the reaction was stirred overnight (tlc showed
conversion to the active ester). A solution of B16 (506 mg, 0.49
mmol) and triethylamine (0.3 ml, 1.89 mmol) in dry dichloromethane
(6 ml) was added dropwise and stirring continued for two hours. Tlc
showed the active ester had been converted to a slower moving
product (silica-60% ethyl acetate in hexane). The solvent was
removed and the product chromatographed (silica-70% ethyl acetate
in hexane) to yield the title compound (420 mg, 65%).
C.sub.87H.sub.158N.sub.8O.sub.24.2H.sub.2O requires C:60.18%, H:
9.41%, N: 6.45%. Found: C: 60.18%, H: 9.25%, N: 6.55%.
.delta..sub.H (CDCl.sub.3) 6.07 (1 H, brt, CONH), 5.67 (1 H, t, CH
(OAc)CH(OAc)CONH), 5.43 (1 H, t, CH(OAc)CH(OAc)CH(OAc)CONH), 5.30
(1 H, m,CH(OAc) CONH), 5.05 (1 H, m AcOCH.sub.2(OAc)CH), 4.31 (1 H,
dd[J.sub.1=4 Hz,J.sub.2=12 Hz] AcOCH.sub.2(OAc), 4.13 (1 H,
dd[J.sub.1=6 Hz,J.sub.2=12 Hz] AcOCH.sub.2(OAc), 2.9-3.4 (24 H, m,
NCH.sub.2), 2.0-2.3 (17 H, m, CH.sub.2CO+5.times..acetate), 1.95 (1
H, m, CH.sub.2CH(CH.sub.2).sub.2), 1.2-1.8 (106 H, tm,
CH.sub.2+Me).
[0356] (H20)
N-{8-[t-Butyloxycarbonylaminopropyl(t-butyloxy-carbonyl)amino-
butyl(t-butyloxycarbonyl)amino]-7-[t-butyloxycarbonylaminopropyl(t-butylox-
ycarbonyl)-aminobutyl(t-butyloxycarbonyl)aminomethyl]octyl}-18-(glucuronyl-
amino)octadecanamide 95
[0357] A solution of potassium carbonate (178 mg, 1.28 mmol) in
water (1 ml) was added dropwise to a stirred solution of H19 (420
mg, 0.25 mmol) in methanol (7 ml) at room temperature. The flask
was stirred for 30 min whereupon tlc showed no starting material
was present. Water (20 ml) was added and the precipitate filtered,
washed and dried. The product was purified by chromatography
(Reverse phase silica, Merck
Lichroprep--dichloromethane/methanol/water [2:6:1]) to yield, after
trituration, with ether the title compound (220 mg, 60%).
C.sub.67H.sub.130N.sub.6O.sub.15 requires 1489. Found ES+: MH.sup.+
1490
[0358] (H21)
N-[8-(aminopropylaminobutylamino)-7-(aminopropyl-aminobutylam-
inomethyl)octyl]-18-(glucuronylamino)-octadecanamide.
hexa(trifluoroacetate) salt 96
[0359] A solution of H20 (200 mg, 0.134 mmol) in 96% TFA (10 ml, 4%
water) was stirred for 30 min at room temperature. The TFA was
removed under vacuum, and traces removed by evaporation of
toluenetmethanol, then high vacuum. The compound was dissolved in
water (8 ml) and filtered through a 0.45 .mu.m filter (Whatman PP),
the compound was converted to a colourless solid by lyophilisation.
The product was triturated with ether to give the title compound as
a hygroscopic white solid (183 mg, 88%).
C.sub.59H.sub.106N.sub.8O.sub.19F.sub.18. 2H.sub.2O(anhydrous M.Wt
1572) requires C:44.03%, H: 6.89%, N; 6.96%. Found: C: 43.99%, H:
6.67%, N: 6.89%. The free base C.sub.47H.sub.100N.sub.8O.sub.7
requires 888.7. Found ES+: MH.sup.+ 890. .delta..sub.H (CD.sub.3OD)
3.6-4.2 (6 H, m, sugar), 3.0-3.4 (24H, m,
CH.sub.2N+CH.sub.2NH.sub.2), 2.0-2.2 (7 H, m,
CH.sub.2CO+CH.sub.2CH(CH.sub.2).sub.2+CH.sub.2CH.sub.2NH.sub.2),
1.2-1.7 (48 H, m,CH.sub.2).
[0360] Bixin Hexamine
[0361] (H22)
N-{8-[t-Butyloxycarbonylaminopropyl(t-butyloxy-carbonyl)-amin-
obutyl(t-butyloxycarbonyl)amino]-7-[t-butyloxycarbonylaminopropyl(t-butylo-
xycarbonyl)-aminobutyl(t-butyloxycarbonyl)aminomethyl]octyl}-N'-(peracetyl-
glucuronylaminoethyl)-4,8,13,17-tetramethyl-1,20-docosadiamide
97
[0362] N-Methylmorpholine (0.04 ml, 0.34 mmol) was added to a
stirred solution of F5 (282 mg, 0.34 mmol) in dry dichloromethane
(10 ml) at room temperature under argon. After a few minutes
N-hydroxysuccinimide (39 mg, 0.34 mmol) was added followed by EDC
(65 mg, 0.34 mmol), the reaction was stirred overnight (tlc showed
conversion to the active ester). A solution of B16 (318 mg, 0.34
mmol) and triethylamine (0.237 ml, 1.7 mmol) in dry dichloromethane
(10 ml) was added dropwise and stirring continued for two hours.
Tlc showed the active ester had been converted to a slower moving
product (silica-10% methanol in dichloromethane). The solvent was
removed and the product chromatographed (silica-5% methanol in
dichloromethane) to yield the title compound (430 mg, 69%).
C9.sub.5H.sub.173N.sub.9O.sub.- 25 requires 1840.25. Found ES+:
MH.sup.+ 1841.2. .delta..sub.H (CDCl.sub.3) 7.10 (1 H, br, CONH),
6.27 (1 H, br, CONH), 5.75 (1 H, br, CONH), 5.67 (1H, t,
CH(OAc)CH(OAc)CONH), 5.43 (1 H, t, CH(OAc)CH(OAc)CH(OAc) CONH),
5.30 (1 H, m,CH(OAc)CONH), 5.05 (1 H, m AcOCH.sub.2(OAc)CH), 4.31
(1 H, dd[J.sub.1=4 Hz,J.sub.2=12 Hz] AcOCH.sub.2(OAc), 4.13 (1 H,
dd[J.sub.1=6 Hz,J.sub.2=12 Hz] AcOCH.sub.2(OAc), 2.9-3.5 (26H, m,
NCH.sub.2), 2.0-2.3 (15+9 H, m,
MeCO+CH.sub.2CO+CH.sub.2CH(CH.sub.2).sub.2+MeCH), 1.0-1.8(54+12+30
H, ms, CH.sub.2), 0.84-0.91(12 H, 4.times.s, Me).
[0363] (H23)
N-{8-[t-Butyloxycarbonylaminopropyl(t-butyloxy-carbonyl)amino-
butyl(t-butyloxycarbonyl)amino]-7-[t-butyloxycarbonylaminopropyl(t-butylox-
ycarbonyl)-aminobutyl(t-butyloxycarbonyl)aminomethyl]octyl}-N'-(glucuronyl-
aminoethyl)-4,8,13,17-tetramethyl-1,20-docosadiamide 98
[0364] Ammonia solution (0.880, 7 ml) was added to a stirred
solution of H22 (600 mg, 0.33 mmol) in methanol (15 ml, or until in
solution when ammonia added) at room temperature. The flask was
stirred for 1 hr whereupon tlc showed no starting material was
present. The reaction was evaporated to dryness and the product
purified by chromatography (Reverse phase silica, Merck
Lichroprep--dichloromethane/methanol/water [2:6:1]) to yield the
title compound (416 mg, 78%). C.sub.85H.sub.163N.sub.9O.sub.-
20.3/4H.sub.2O (anhydrous M.Wt 1630) requires C: 62.07%, H: 10.08%,
N: 7.66%. Found: C: 62.08%, H: 10.14%, N: 7.69%.
C.sub.85H.sub.163N.sub.9O.s- ub.20 requires 1630. Found ES+:
MH.sup.+ 1631.2. .delta..sub.H (CD.sub.3OD), 3.6-4.3(6H, ms,
sugar), 2.9-3.5 (26H, m, NCH.sub.2), 2.0-2.3 (9 H, m,
CH.sub.2CO+CH.sub.2CH(CH.sub.2).sub.2+4.times.CH.sub.3CH- ),
1.0-1.8(54+12+30 H, ms, Me+CH.sub.2), 0.84-0.91(12 H, 4.times.s,
Me).
[0365] (H24)
N-[8-(aminopropylaminobutylamino)-7-(aminopropyl-aminobutylam-
inomethyl)octyl]-N'-(glucuronyl-aminoethyl)-4,8,13,17-tetramethyl-1,20-doc-
osadiamidehexa(trifluoroacetate) salt 99
[0366] A solution of H23 (400 mg, 0.25 mmol) in 96% TFA (10 ml, 4%
water) was stirred for 30 min at room temperature. The TFA was
removed under vacuum, and traces removed by evaporation of water.
The title compound was obtained as to a colourless hygroscopic
solid (382 mg, 90%) by lyophilisation.
C.sub.55H.sub.115N.sub.9O.sub.8.C.sub.12H.sub.6F.sub.18O.-
sub.12.2.5H.sub.2O (anhydrous M.Wt 1713 requires C: 45.73%, H:
7.22%, N: 7.16%. Found: C: 45.73%, H: 7.14%, N: 6.86%. The free
base C.sub.49H.sub.101N.sub.7O.sub.8 requires 1029.89. Found ES+:
MH.sup.+ 1031.0. .delta..sub.H (CD.sub.3OD), 3.6-4.3(6H, ms,
sugar), 2.9-3.5 (26H, m, NCH.sub.2), 2.0-2.3 (9 H, m,
CH.sub.2CO+CH.sub.2CH(CH.sub.2).sub.2+4.t- imes.MeCH),
1.0-1.8(12+H, ms, CH.sub.2), 0.84-0.91(12 H, 4.times.s, Me).
[0367] C24 Amide Hexamine
[0368] (H25)
N-{8-[t-Butyloxycarbonylaminopropyl(t-butyloxy-carbonyl)-amin-
obutyl(t-butyloxycarbonyl)-amino]-7-[t-butyloxycarbonylaminopropyl(t-butyl-
oxycarbonyl)butyl(t-butyloxycarbonyl)aminomethyl]-octyl}-12-(peracetylgluc-
uronylaminododecanoylamino)-dodecanamide 100
[0369] DBU (0.06 ml, 0.4 mmol) was added to a stirred suspension of
F6 (320 mg, 0.4 mmol) in dry dichloromethane (5 ml) at room
temperature under argon. A few minutes later N-hydroxysuccinimide
(46 mg, 0.4 mmol) was added followed by EDC (76 mg, 0.4 mmol), the
reaction was stirred overnight (tlc showed conversion to the active
ester). A solution of B16 (411 mg, 0.4 mmol) and DBU(0.12 ml, 0.4
mmol) in dry dichlotomethane (5 ml) was added dropwise and stirring
continued for five hours. Tlc showed the active ester had been
converted to a slower moving product (silica-60% ethyl acetate in
hexane). The reaction was poured into 10% citric acid and extracted
with dichloromethane, washed with brine, dried, and the solvent
removed. The product was purified by chromatography (silica-2% to
5% methanol in dichloromethane) to yield the title compound (538
mg, 74%). C.sub.93H.sub.169N.sub.9O.sub.25. 3/4H.sub.2O requires C:
61.14%, H: 9.41%, N: 6.90%. Found: C: 61.16%, H: 9.37%, N: 6.82%.
C.sub.93H.sub.169N.sub.9O.sub.25 requires 1812.2. Found ES+:
MH.sup.+ 1814.3 .delta..sub.H (CDCl.sub.3) 6.13 (1 H, brt, CONH),
5.93 (1 H, br, CONH), 5.75 (1 H, br, CONH), 5.67 (1 H, t, CH
(OAc)CH(OAc)CONH), 5.43 (1 H, t, CH(OAc)CH(OAc)CH(OAc)CONH), 5.30
(1 H, m,CH(OAc)CONH), 5.05 (1 H, m AcOCH.sub.2(OAc)CH), 4.31 (1 H,
dd[J.sub.1=4 Hz,J.sub.2=12 Hz] AcOCH.sub.2(OAc), 4.13 (1 H,
dd[J.sub.1=6 Hz,J.sub.2=12 Hz] AcOCH.sub.2(OAc), 2.9-3.3 (26 H, m,
NCH.sub.2), 2.0-2.3 (20 H, m,
MeCO+CH.sub.2CO+CH.sub.2CH(CH.sub.2).sub.2), 1.2-1.7(112 H, dm,
CH.sub.2+Me).
[0370] (H26)
N-(8-[t-Butyloxycarbonylaminopropyl(t-butyloxy-carbonyl)amino-
butyl(t-butyloxycarbonyl)amino]-7-[t-butyloxycarbonylaminopropyl(t-butylox-
ycarbonyl)butyl(t-butyloxycarbonyl)aminomethyl]octyl)-12-(glucuronylaminod-
odecanoylamino)dodecanamide 101
[0371] Ammonia solution (10 ml, 0.880) was added to a stirred
solution of H25 (530 mg, 0.3 mmol) in methanol (10 ml) at room
temperature, (extra methanol can be added to ensure a solution).
The flask was stirred for 1 hr and evaporated to dryness. The white
solid was suspended in water, filtered off and dried. The product
was purified by chromatography (Reverse phase silica,
Merck-Lichroprep eluted with dichloromethane/methanol/water
[2:6:1]). The product was evaporated to dryness, suspended in
water, filtered off, dried and triturated with ether. High vacuum
yielded the title compound (368 mg, 77%) as a white solid.
C.sub.83H.sub.159N.sub.9O.sub.20 requires C: 62.18%, H: 10.00%, N:
7.86%. Found: C: 62.04%, H: 9.93%, N: 7.77%.
C.sub.83H.sub.159N.sub.9O.su- b.20 requires 1602.17. Found ES+:
MH.sup.+ 1603.4 .delta..sub.H (CD.sub.3OD) 3.5-4.2(6H, m, sugar),
2.9-3.3 (26 H, m, NCH.sub.2), 2.15(4 H, t, CH.sub.2CO), 2.07(1 H,
br, CH.sub.2CH(CH.sub.2).sub.2), 1.2-1.8(112 H, dm,
CH.sub.2+Me).
[0372] (H27)
N-[8-(Aminopropylaminobutylamino)-7-(aminopropyl-butylaminome-
thyl)octyl]-12-(glucuronylamino-dodecanoylamino)dodecanamidehexa(trifluoro-
acetate) salt 102
[0373] A solution of H26 (360 mg, 0.23 mmol) in 96% TFA (6 ml, 4%
water) was stirred for 30 min at room temperature. The TFA was
removed under vacuum, and traces removed by evaporation of
toluene/methanol. The compound was subjected to high vacuum
overnight, dissolved in water (5 ml) and filtered through a 0.45
.mu.M (Whatman PP) filter The compound was converted to a
colourless solid by lyophilisation. The lyophilised hygroscopic
solid was triturated with ether and dried in vacuo to yield the
title compound (370 mg, 98%). C.sub.53H.sub.111N.sub.9O.sub.20.
C.sub.12H.sub.6O.sub.12F.sub.18. 2.8 H.sub.2O (Mwt
anhydrous=1686.67) requires C:44.94%, H: 7.11%, N: 7.26%. Found: C:
44.93%, H:6.92%, N: 7.12%. The free base
C.sub.53H.sub.111N.sub.9O.sub.8 requires 1001.86. Found ES+:
MH.sup.+ 1003.9. .delta..sub.H (CD.sub.3OD) 7.92 (1 H, br, CONH),
7.80 (1 H, br, CONH), 3.6-4.25 (6 H, m, sugar), 2.9-3.4 (26H, m,
CH.sub.2N), 2.0-2.3 (9 H, m,
CH.sub.2CO+CH.sub.2CH(CH.sub.2).sub.2+CH.sub- .2CH.sub.2NH.sub.2),
1.2-1.9 (50 H, m, CH.sub.2).
[0374] Disugar Hexamine
[0375] (H28)
N-{8-[Methyl(t-butyloxycarbonyl)aminopropyl(t-butyloxycarbony-
l)aminobutyl(t-butyloxycarbonyl)amino]-7-[methyl(t-butyloxycarbonyl)aminop-
ropyl(t-butyloxycarbonyl)aminobutyl(t-butyloxycarbonyl)-aminomethyl]octyla-
minocarbonyltricosanyl}-N',N'-bis(peracetylglucuronylaminoethyl)succinamid-
e 103
[0376] C4 (197 mg, 0.201 mmol), EDC hydrochloride (77 mg, 0.401
mmol) and N-hydroxysuccinimide (35 mg, 0.301 mmol) were dissolved
in anhydrous dichloromethane (30 ml) and activated ester formation
left at room temperature for two hours under argon. To the solution
was added E4 (300 mg, 0.211 mmol) and triethylamine (61 mg, 0.602
mmol) and the reaction left overnight at room temperature under
argon. The solvents were removed, and the resulting residues
purified by gradient silica column chromatography (3-7% methanol in
dichloromethane) to yield the title compound as a colourless solid
(378 mg, 78%). C.sub.119H.sub.209N.sub.11O- .sub.37 requires
2384.5. Found ES.sup.+: MH.sub.2.sup.2+, 1193.9, MH.sub.3.sup.3+,
796.4. .delta..sub.H (CDCl.sub.3) 1.23 (46H,
(CH.sub.2).sub.19(CH.sub.2).sub.2CO, (CH.sub.2).sub.4CH), 1.44
(72H, br+m, (Me).sub.3C, CH.sub.2CH.sub.2N, CH.sub.2CH.sub.2CO),
1.95 (1H, br, CH), 2.02-2.23 (30H, 10.times.s, MeCO), 2.17 (2H, t,
(CH.sub.2).sub.22CH.sub.2CO), 2.4-2.8 (4H, brm,
CO(CH.sub.2).sub.2CO), 2.83 (6H, s, MeN), 2.9-3.5 (32H, m,
CH.sub.2N), 4.12 (2H, m, CH.sub.2OAc), 4.30 (2H, dt, CH.sub.2OAc),
5.20, 5.25 (2H, 2.times.d, CHOAcCO), 5.43, 5.60 (4H, 2.times.m,
(CHOAc).sub.2CHOAcCO), 5.71 (1H, br, (CH.sub.2).sub.23CONH), 6.11
(1H, br, CO(CH.sub.2).sub.2CONH), 7.18, 7.65 (2H, 2.times.t,
N(CH.sub.2CH.sub.2NH).sub.2.
[0377]
(H29)N-{8-[Methyl(t-butyloxycarbonyl)aminopropyl(t-butyloxy-carbony-
l)aminobutyl(t-butyloxycarbonyl)amino]-7-[methyl(t-butyloxycarbonyl)aminop-
ropyl(t-butyloxy-carbonyl)aminobutyl(t-butyloxycarbonyl)aminomethyl]-octyl-
aminocarbonyltricosanyl}-N',N'-bis(glucuronylaminoethyl)succinamide
104
[0378] To H28 (377 mg) in methanol (15 ml) was added concentrated
ammonium hydroxide (5 ml). The cloudy solution/suspension was
rapidly stirred for two hours whereupon the solvent was removed and
the resulting residues purified by reverse phase silica
chromatography eluting with 2:6:1 dichloromethane:methanol:water to
yield the title compound as a colourless solid (266 mg, 86%).
C.sub.99H.sub.189N.sub.11O.sub.27 requires 1964.4 Found ES.sup.+:
MH.sub.2.sup.2+, 983.7. .delta..sub.H (CD.sub.3OD) 1.28 (46H, br,
(CH.sub.2).sub.19(CH.sub.2).sub.2CO, (CH.sub.2).sub.4CH), 1.46
(68H, br, (Me).sub.3C, CH.sub.2CH.sub.2N, CH.sub.2CH.sub.2CO), 1.76
(4H, p, NCH.sub.2CH.sub.2CH.sub.2N), 2.10 (1H, br, CH), 2.16 (2H,
t, (CH.sub.2).sub.22CH.sub.2CO), 2.49, 2.70 (4H, t+dt,
CO(CH.sub.2).sub.2CO), 2.85 (6H, s, NMe), 3.0-3.4 (24H, m,
CH.sub.2N), 3.4-3.65 (8H, m, N(CH.sub.2).sub.2N, 3.65-3.85 (8H, m,
CHOH), 4.11, 4.20 (4H, brs+dd, CH.sub.2OH). n
[0379] (H30)
N-[8-(Methylaminopropylaminobutylamino)-7-(methyl-aminopropyl-
aminobutylaminomethyl)octylaminocarbonyl
tricosanyl}-N',N'-bis(glucuronyla- minoethyl)succinamide 105
[0380] To H29 (266 mg) was added 96:4 trifluoroacetic acid :
dichloromethane (10 ml). The solution was left for 20 minutes at
room temperature, the solvents removed and the residues taken up
into MilliQ water. The solution was filtered (0.45 mm
polypropylene) and freeze dried to give a white solid. This was
suspended in diethyl ether, left for 20 minutes and the ether
decanted off. The remaing white solids were dried under vacuum to
give the title compound as a white, hydroscopic solid (269 mg).
C.sub.69H.sub.141N.sub.11O.sub.15 requires 1364.1 Found ES.sup.+:
MH.sub.2.sup.2+, 683.4, MH.sup.+, 1365.4. .delta..sub.H
(CD.sub.3OD) 1.28 (44H, br, (CH.sub.2).sub.19(CH.sub.2).sub.2CO,
(CH.sub.2).sub.3CH.sub.2CH), 1.50 (8H, m, CH.sub.2CH,
CH.sub.2CH.sub.2NH, CH.sub.2CH.sub.2CO), 1.82 (8H, br,
NCH.sub.2(CH.sub.2).sub.2CH.sub.2N), 2.05-2.25 (6H, m, CH.sub.2CO,
NCH.sub.2CH.sub.2CH.sub.2N), 2.28 (1H, m, CH), 2.49, 2.72 (4H,
2.times.t, CO(CH.sub.2).sub.2CO), 2.73 (6H, s, NMe), 3.05-3.30
(24H, m, CH.sub.2N), 3.35-3.65 (8H, m, NCH.sub.2CH.sub.2N),
3.65-3.85 (8H, m, CHOH), 4.10, 4.22 (4H, brs+dd, CH.sub.2OH).
[0381] (H31
)N-{8-[Methyl(t-butyloxycarbonyl)aminopropyl(t-butyloxy-carbon-
yl)aminobutyl(t-butyloxycarbonyl)amino]-7-[methyl(t-butyloxycarbonyl)amino-
propyl(t-butyloxycarbonyl)aminobutyl(t-butyloxycarbonyl)-aminomethyl]octyl-
}-24-(peracetylglucuronylamino)-tetracosanamide 106
[0382] To F2 (131 mg, 0.170 mmol) in anhydrous dichloromethane (15
ml) were added EDC hydrochloride (46 mg, 0.238 mmol),
N-hydroxysuccinimide (27 mg, 0.238 mmol) and the reaction left
overnight. To the reaction were then added B21 (198 mg, 0.187 mmol)
and triethylamine (68 mg, 0.679 mmol) and the reaction left for 3
hours at which point the solvent was removed. The residues were
purified by gradient silica chromatography eluting with 60-80%
ethyl acetate in hexane to yield the title compound as a colourless
gum, (241 mg, 78%). C.sub.95H.sub.174N.sub.8O.sub.24 requires
1811.3 Found ES.sup.+: MH.sub.2.sup.2+, 907.0. .delta..sub.H
(CDCl.sub.3) 1.24 (48H, br, (CH.sub.2).sub.19(CH.sub.2).sub.2N,
CH.sub.2CH.sub.2CO, (CH.sub.2).sub.4CH), 1.3-1.8 (70H, m,
(Me).sub.3C, CH.sub.2CH.sub.2N), 2.0 (1H, br, CH), 2.04-2.19 (15H,
5.times.s, MeCO), 2.22 (2H, t, CH.sub.2CO), 2.83 (6H, s, NMe),
2.9-3.35 (24H, m, CH.sub.2N), 4.10-4.40 (2H, m, CH.sub.2OAc), 5.03
(1H, m, CHOAcCH.sub.2OAc), 5.28 (1H, d, CHOAcCO), 5.43, 5.67 (2H,
2.times.t, (CHOAc).sub.2CHOAcCH.sub.2OAc), 6.05 (2H, 2.times.t,
NHCO).
[0383]
(H32)N-{8-[Methyl(t-butyloxycarbonyl)aminopropyl(t-butyloxy-carbony-
l)aminobutyl(t-butyloxycarbonyl)amino]-7-[methyl(t-butyloxycarbonyl)aminop-
ropyl(t-butyloxy-carbonyl)aminobutyl(t-butyloxycarbonyl)-aminomethyl]octyl-
}-24-(glucuronylamino)-tetracosanamide 107
[0384] To H31 (235 mg) dissolved in methanol (10 ml) was slowly
added with stirring NH.sub.4OH (4 ml) until the solution started to
become cloudy. After approximately 20 minutes an additional 3 ml of
methanol was added to dissolve some of the forming white
precipitate. The solution/suspension was stirred for a total of one
hour at which point all solvents were removed. The residues were
purified by reverse phase chromatography eluting with 2:6:1
CH.sub.2Cl.sub.2:MeOH:NH.sub.4OH to yield the title compound as a
colourless solid, (206 mg, 99%). C.sub.85H.sub.164N.sub.8O.sub.19
requires 1601.2 Found ES.sup.+: MH.sup.+, 1601.9. .delta..sub.H
(CDCl.sub.3) 1.24 (46H, br, (CH.sub.2).sub.19(CH.sub.2).sub.2N,
(CH.sub.2).sub.4CH), 1.35-1.80 (72H, brm, (Me).sub.3C,
CH.sub.2CH.sub.2N, CH.sub.2CH.sub.2CO), 2.05 (1H, br, CH), 2.14
(2H, t, (CH.sub.2).sub.22CH.sub.2CO), 2.82 (6H, s, NMe), 3.0-3.4
(24H, brm, NCH.sub.2), 3.5-4.1 (4H, br, CHOH), 4.17, 4.30 (2H,
2.times.br, CH.sub.2OH), 5.85, 7.21 (2H, 2.times.br, NHCO).
[0385] (H33)
N-[8-(methylaminopropylaminobutylamino)-7-(methyl-aminopropyl-
aminobutylaminomethyl)octyl]-24-(glucuronylamino)tetracosanamide
108
[0386] H32 (185 mg) was dissolved in 96:4 TFA:DCM and left for 20
minutes. The solvent was removed and the residues taken up in
MilliQ water, filtered through a 0.2 mm filter, and the solution
freeze dried to quantitatively give the title compound as a white
solid. C.sub.55H.sub.116N.sub.8O.sub.7 requires 1000.9 Found
ES.sup.+: MH.sup.+, 1001.9. .delta..sub.H (CD.sub.3OD) 1.32 (44H,
br, (CH.sub.2).sub.19(CH.su- b.2).sub.2CO,
(CH.sub.2).sub.3CH.sub.2CH), 1.52 (8H, m, CH.sub.2CH,
CH.sub.2CH.sub.2N, CH.sub.2CH.sub.2CO), 1.82 (8H, m,
NCH.sub.2(CH.sub.2).sub.2CH.sub.2N), 2.08 (1H, br, CH), 2.12 (4H,
m, NCH.sub.2CH.sub.2CH.sub.2N), 2.23 (2H, m, CH.sub.2CO), 2.73 (6H,
s, NMe), 3.0-3.3 (24H, m, CH.sub.2N), 3.58-3.82 (4H, m, CHOH),
4.08, 4.22 (2H, 2.times.m, CH.sub.2OH).
[0387] (H34)
N-{8-[Methyl(t-butyloxycarbonyl)aminopropyl(t-butyloxycarbony-
l)aminobutyl(t-butyloxycarbonyo)amino]-7-[methyl(t-butyloxycarbonyl)aminop-
ropyl(t-butyloxycarbonyl)aminobutyl(t-butyloxycarbonyl)-aminomethyl]octyl}-
-24-(peracetylglucuronylamino-dodecanoylamino)tetracosanamide
109
[0388] To F8 (67 mg, 0.111 mmol) in anhydrous dichloromethane (20
ml) were added EDC hydrochloride (30 mg, 0.155 mmol),
N-hydroxysuccinimide (18 mg, 0.155 mmol) and the reaction left
overnight. To this were then added E4 (174 mg, 0.122 mmol) and
triethylamine (45 mg, 0.444 mmol) and the reaction left for a
further four hours. The solvent was removed and the residues
purified by gradient silica chromatography eluting with 50-90%
ethyl acetate in hexane to yield the title compound, (90 mg, 40%)
as a colourless gum. C.sub.107H.sub.197N.sub.9O.sub.25 requires
2008.4 Found ES.sup.+: MNH.sub.4.sup.+, 2027.8. .delta..sub.H
(CDCl.sub.3) 1.25 (56H, br, NH(CH.sub.2).sub.2(CH.sub.2).sub.20,
NH(CH.sub.2).sub.2(CH.sub.2).sub- .8), 1.3-1.8 (72H, brm, (Me)3C,
CH.sub.2CH.sub.2N), 1.96 (1H, br, CH), 2.04, 2.05, 2.09, 2.20, 2.20
(15H, 5.times.s, MeCO), 2.23 (4H, m, CH.sub.2CO), 2.84 (6H, s,
NMe), 3.0-3.3 (26H, m, NCH.sub.2), 4.14, 4.31 (2H, m, CH.sub.2OAc),
5.04 (1H, dt, CH.sub.2CHOAc), 5.29 (1H, d, CHOAcCO), 5.44, 5.66
(2H, 2.times.t, (CHOAc).sub.2CHOAcCO), 5.92, 6.09, 6.29 (3H,
br+t+br, NHCO).
[0389] (H35)
N-{8-[Methyl(t-butyloxycarbonyl)aminopropyl(t-butyloxycarbony-
l)aminobutyl(t-butyloxycarbonyl)amino]-7-[methyl(t-butyloxycarbonyl)aminop-
ropyl(t-butyloxycarbonyl)aminobutyl(t-butyloxycarbonyl)-aminomethylioctyl}-
-24(glucuronylaminododecanoyl-amino)tetracosanamide 110
[0390] To a solution of H34 (90 mg) in methanol (12 ml) was added
NH.sub.4OH (4 ml) and the reaction left for 1 hour at which point
the solvent was removed. The residues were purified by reverse
phase silica chromatography eluting with 2:6:1 DCM:MeOH:H.sub.2O to
yield the title compound (80 mg, 99%) as a white solid.
C.sub.97H.sub.187N.sub.9O.sub.20 requires 1798.4 Found ES.sup.+:
MH.sub.2.sup.2+, 900.9, MH.sup.+, 1799.6. .delta..sub.H
(CDCl.sub.3) 1.25 (52H, br, N(CH.sub.2).sub.2(CH.sub.2).sub- .19,
N(CH.sub.2).sub.2(CH.sub.2).sub.7), 1.40-1.85 (76H, brm,
(Me).sub.3C, CH.sub.2CH.sub.2N, CH.sub.2CH.sub.2CO), 1.95 (1H, br,
CH), 2.16 (2H, t, CH.sub.2CO), 2.84 (6H, s, NMe), 2.9-3.4 (26H, m,
NCH.sub.2), 3.82 (4H, br, CHOH), 4.17, 4.32 (2H, 2.times.br,
CH.sub.2OH), 5.92, 6.55, 7.22 (3H, 3.times.br, NHCO).
[0391] (H36)
N-[8-(Methylaminopropylaminobutylamino)-7-(methyl-aminopropyl-
aminobutylaminomethyl)octyl]-24-(glucuronylaminododecanoylamino)tetracosan-
amide 111
[0392] H35 (80 mg) was treated as in the synthesis of H33 to give
the title compound in quantitative yield as a white solid.
C.sub.67H.sub.139N.sub.9O.sub.8 requires 1198.1 Found ES.sup.+:
MH.sub.2.sup.2+, 600.2, MH.sup.+, 1198.8. .delta..sub.H
(CD.sub.3OD) 28 (58H, br, (CH.sub.2).sub.7(CH.sub.2).sub.2CO,
(CH.sub.2).sub.19(CH.sub.2)- .sub.2CO, (CH.sub.2).sub.3CH.sub.2CH),
1.51 (12H, m, CH.sub.2CH, CH.sub.2CH.sub.2N, CH.sub.2CH.sub.2CO),
1.83 (8H, m, NCH.sub.2(CH.sub.2).sub.2CH.sub.2N), 2.14 (9H, m,
CH.sub.2CO, NCH.sub.2CH.sub.2CH.sub.2N, CH), 2.73 (6H, s, NMe),
3.0-3.3 (26H, m, NCH.sub.2), 3.6-3.8 (4H, m, CHOH), 4.07, 4.19 (2H,
2.times.m, CH.sub.2OH).
[0393] (H37)
N-{8-[Methyl(t-butyloxycarbonyl)aminopropyl(t-butyloxycarbony-
l)aminobutyl(t-butyloxycarbonyl)amino]-7-[methyl(t-butyloxycarbonyl)aminop-
ropyl(t-butyloxycarbonyl)aminobutyl(t-butyloxycarbonyl)-aminomethyl]octyl}-
-36-(glucuronylamino)-hexatriacontanamide 112
[0394] To EB (612 mg, 0.038 mmol) in anhydrous methanol (15 ml)
were added d-gluconolactone (14 mg, 0.077 mmol) and triethylamine
(16 mg, 0.153 mmol) and the reaction stirred overnight at ambient
temperature under argon. The solvents were removed and the residues
purified by reverse phase chromatography eluting with 2:6:1 then
2:6:0.5 DCM:MeOH:H.sub.2O to give the title compound (34 mg, 51%)
as a colourless solid. C.sub.97H.sub.188N.sub.8O.sub.19 requires
1769.4 Found ES.sup.+: MNa.sup.+, 1793.2. .delta..sub.H
(CDCl.sub.3) 1.24 (72H, br,
(CH.sub.2).sub.32CH.sub.2CONH(CH.sub.2).sub.2(CH.sub.2).sub.4),
1.44 (66H, m, (Me).sub.3C, CH.sub.2CH.sub.2N), 1.72 (4H, p,
NCH.sub.2CH.sub.2CH.sub.2N), 2.0 (1H, br, CH), 2.14 (2H, t,
CH.sub.2CO), 2.83 (6H, s, NMe), 3.0-3.4 (24H, m, CH.sub.2N),
3.6-3.9 (4H, br, CHOH), 4.10, 4.25 (2H, 2.times.br, CH.sub.2OH),
5.80 (1H, br, CONH(CH.sub.2).sub.6CH), 7.30 (1 H, br, CONH).
[0395] (H38)
N-[8-(Methylaminopropylaminobutylamino)-7-(methyl-aminopropyl-
aminobutylaminomethyl)octyl]-36-(glucuronylamino)hexatriacontanamide
113
[0396] H37 (34 mg) was treated as in the synthesis of H33 to give
the title compound in quantitative yield as a white solid.
C.sub.67H.sub.140N.sub.8O.sub.7 requires 1169.1 Found ES.sup.+:
MH.sub.2.sup.2+, 585.7, MH.sup.+, 1169.7. .delta..sub.H
(CD.sub.3OD) 1.29 (68H, br, (CH.sub.2).sub.31(CH.sub.2).sub.2CO,
(CH.sub.2).sub.3CH.sub.2CH- ), 1.51 (8H, m, CH.sub.2CH,
CH.sub.2CH.sub.2N, CH.sub.2CH.sub.2CO), 1.83 (8H, brm,
NCH.sub.2(CH.sub.2).sub.2CH.sub.2N), 2.16 (7H, m, CH.sub.2CO,
NCH.sub.2CH.sub.2CH.sub.2N, CH), 2.73 (6H, s, NMe), 3.0-3.3 (24H,
m, NCH.sub.2), 3.05-3.35 (4H, m, CHOH), 4.08-4.20 (2H, m+d,
CH.sub.2OH), 7.8-7.9 (2H, 2.times.br, NHCO).
[0397] I. PEG Lipids
[0398] This section contains the synthesis of:
[0399] (I2)
N-[8-(Aminobutylamino)-7-(aminobutylaminomethyl)-octyl]-24-[me-
thoxypoly(ethyleneoxy)propanoylamino]-tetracosanamidetetra(trifluoroacetat-
e) salt 114
[0400] (I4)
N-[8-(Methylaminopropylaminobutylamino)-7-(methylaminopropylam-
inobutylaminomethyl)octyl]-24-[methoxypoly(ethyleneoxy)propanoylamino]tetr-
a-cosanamidehexa(trifluoroacetate) salt 115
[0401] (I1)
N-{8-[t-Butyloxycarbonylaminobutyl(t-butyloxycarbonyl)-amino]--
7-[t-butyloxycarbonylaminobutyl(t-butyloxy-carbonyl)aminomethyl]octyl}-24--
[methoxypoly-(ethyleneoxy)propanoylamino]tetracosanamide 116
[0402] To methoxyPEGpropionic acid (MW approx 1800) (282 mg, 0.157
mmol) in anhydrous dichloromethane (20 ml) were added EDC
hydrochloride (38 mg, 0.199 mmol) and N-hydroxysuccinimide (23 mg,
0.199 mmol) and the reaction left overnight under argon at room
temperature. To this was added E2 (154 mg, 0.142 mmol) and
triethylamine (57 mg, 0.570 mmol) and the reaction left for a
further six hours. To this was then added water (0.2 ml) and the
hydrolysis of any remaining activated ester left overnight. The
solvent was removed and the residues purified by gradient silica
chromatography (5-10% methanol in dichloromethane) to yield the
title compound (369 mg, 90%) as a white solid. For n=38
C.sub.143H.sub.282N.sub- .6O.sub.50 requires 2884.0 Found ES.sup.+:
MNa.sub.2.sup.2+, 1465.6. .delta..sub.H (CDCl.sub.3) 1.24 (44H, br,
(CH.sub.2).sub.19(CH.sub.2).sub- .2CO, (CH.sub.2).sub.3CH.sub.2CH),
1.43 (52H, m, (Me).sub.3C, CH.sub.2CH, CH.sub.2CH.sub.2N,
CH.sub.2CH.sub.2CO, NCH.sub.2(CH.sub.2).sub.2CH.sub.2N- ), 1.97
(1H, br, CH), 2.22 (2H, t, (CH.sub.2).sub.22CH.sub.2CO), 2.60 (2H,
t, OCH.sub.2CH.sub.2CO), 3.05-3.15 (16H, m, NCH.sub.2), 3.37 (3H,
s, OMe), 3.63 (.about.170 H, m, OCH.sub.2).
[0403] (I2)
N-[8-(Aminobutylamino)-7-(aminobutylaminomethyl)-octyl]24-[met-
hoxypoly(ethyleneoxy)propanoylamino]-tetracosanamidetetra(trifluoroacetate-
) salt 117
[0404] I1(369 mg) was dissolved in 96:4 trifluoroacetic
acid:dichloromethane (9 ml) and left for 15 minutes at room
temperature. The solvent was removed, the residues taken up into
water, filtered (0.45 mm polypropylene) and freeze dried to give
the title compound a pale yellow viscous oil (370 mg). For n=38
C.sub.123H.sub.250N.sub.6O.sub.42 requires 2483.8. Found ES.sup.+:
MH.sub.2.sup.2+, 1243.5. d.sub.H (CDCl.sub.3) 1.24 (52H, brm,
(CH.sub.2).sub.21CH.sub.2CO, (CH.sub.2).sub.5CH), 1.85 (8H, br,
NCH.sub.2(CH.sub.2).sub.2CH.sub.2N), 2.21 (2H, t,
(CH.sub.2).sub.22CH.sub.2CO), 2.35 (1H, br, CH), 2.51 (2H, t,
OCH.sub.2CH.sub.2CO), 2.8-3.35 (16H, brm, NCH.sub.2), 3.37 (3H, s,
OMe), 3.63 (.about.170H, br, OCH.sub.2). (I3)
N-{8-[Methyl(t-butyloxycarb-
onyl)aminopropyl(t-butyloxycarbonyl)aminobutyl(t-butyloxycarbonyl)amino]-7-
-[methyl(t-butyloxycarbonyl)aminopropyl(t-butyloxy-carbonyl)aminobutyl(t-b-
utyloxycarbonyl)aminomethyl]-octyl}-24-[methoxypoly(ethyleneoxy)propanoyla-
mino]-tetracosanamide 118
[0405] To methoxyPEGpropionic acid (MW approx 1800) (210 mg, 0.117
mmol) in anhydrous dichloromethane (20 ml) were added hydrochloride
(29 mg, 0.148 mmol) and N-hydroxysuccinimide (17 mg, 0.148 mmol)
and the reaction left overnight under argon at room temperature. To
this was added E4 (151 mg, 0.106 mmol) and triethylamine (32 mg,
0.318 mmol) and the reaction left for a further six hours. To this
was then added water (0.2 ml) and the hydrolysis of any remaining
activated ester left overnight. The solvent was removed and the
residues purified by gradient silica chromatography (5-10% methanol
in dichloromethane) to yield the title compound (205 mg, 60%) as an
off white solid. For n=38 C.sub.161H.sub.316N.sub.8O.sub.54
requires 3226.2 Found ES.sup.+: MNa2.sup.2+, 1636.8. .delta..sub.H
(CDCl.sub.3) 1.24 (48H, brs, (CH.sub.2).sub.20CH.sub.2CO,
(CH.sub.2).sub.4CH), 1.43 (70H, brs+m, (Me).sub.3C,
CH.sub.2CH.sub.2N), 1.98 (1H, br, CH), 2.19 (2H, t,
(CH.sub.2).sub.22CH.sub.2CO), 2.54 (2H, t, OCH.sub.2CH.sub.2CO),
2.83 (6H, s, NMe), 3-3.5 (24H, brm, NCH.sub.2), 3.36 (3H, s, OMe),
3.6 (.about.170H, s, OCH.sub.2), 5.95, 6.95 (2H, br, CONH).
[0406] (I4)
N-[8-(Methylaminopropylaminobutylamino)-7-(methylaminopropylam-
inobutylaminomethyl)octyl]-24-[methoxypoly(ethyleneoxy)propanoylamino]tetr-
acos-anamidehexa(trifluoroacetate) salt 119
[0407] I3 (206 mg) was dissolved in 96:4 trifluoroacetic acid :
dichloromethane (8 ml) and left for 15 minutes at room temperature.
The solvent was removed, the residues taken up into water, filtered
(0.45 mm polypropylene) and freeze dried to give the title compound
as an off white solid (213 mg). For n=38
C.sub.131H.sub.268N.sub.8O.sub.42 requires 2625.9 Found ES.sup.+:
MH.sub.2.sup.2+, 1314.5. .delta..sub.H (D.sub.2O) 1.87 (44H, br,
(CH.sub.2).sub.19(CH.sub.2).sub.2CO, (CH.sub.2).sub.3CH.sub.2CH),
2.06 (8H, br, CH.sub.2CH, CH.sub.2CH.sub.2NH, CH.sub.2CH.sub.2CO),
2.38 (8H, br, NCH.sub.2(CH.sub.2).sub.2CH.sub.2N), 2.72 (7H, br,
(CH.sub.2).sub.22CH.sub.2CO, NCH.sub.2CH.sub.2CH.sub.2N, CH), 3.05
(2H, t, OCH.sub.2CH.sub.2CO), 3.34 (6H, s, NMe), 3.72 (24H, br,
CH.sub.2N), 3.95 (3H, s, OMe), 4.25 (.about.170H, br,
OCH.sub.2).
[0408] The advantageous properties of the lipids according to the
invention may be demonstrated in the following tests:
[0409] PRONASE and DNase Treatment
[0410] One of the ultimate goals of current gene therapy research
is to develop a delivery system which will remain stable and
effective in vivo, since this would remove the need for expensive
and time-consuming ex-vivo manipulations. Since intravenous
administration offers the possibility of delivery to the largest
number of tissue sites, survival of the gene delivery complex in
the presence of serum could be an important feature of any
effective technology. Many published studies have demonstated the
susceptibility of gene delivery complexes to inactivation by serum
even at levels as low as 10%. This effect is due, at least in part,
to the destabilisation of complexes by a poorly understood
mechanism, and this can lead to degradation of the DNA within the
complex by serum-associated nucleases. We have therefore formed
complexes between the lipids of the invention and plasmid DNA and
subjected these to treatment with purified DNase and pronase as
well as with 50% foetal calf serum. Integrity of the plasmid DNA
was then measured.
[0411] Methods
[0412] Plasmid DNA (pEG/acZ) was prepared at a concentration of 120
.mu.g/ml in water. A lipid according to the invention [for example
the hexamine H18 described in the Example above] was prepared in a
range of concentrations such that charge ratios of lipid:DNA of
0.25:1, 1:1, 2:1, 4:1, 8:1 would be obtained (based on the
assumption that DNA at 120 .mu.g/ml is equivalent to 0.387 mM of
negative charge, and the hexamine H18 for example at 10 mg/ml is
equivalent to 36.23 mM of positive charge). An equal volume of DNA
was added dropwise to a vortexing tube containing the lipid in
water. For DNase treatment, DNase I (FPLCpure, Pharmacia) was added
at a concentration of 1 unit/1 .mu.g of DNA and tubes were
incubated at 37.degree. C. for 10 minutes. To inhibit further
action of DNase, EGTA was added to a final concentration of 25 mM.
For pronase treatment, protease XIV (Sigma) was added to samples to
a final concentration of 150 .mu.g/ml, and samples incubated for 30
min at 37.degree. C. Complexes were disrupted in 0.5% SDS with
incubation at 55.degree. C. for 20 min. Serum treatment involved
incubating the samples in the presence of 50% foetal calf serum
(final concentration) for 30 min at 37.degree. C. EGTA was added to
a final concentration of 50 mM in an attempt to prevent subsequent
action of serum-associated nucleases. Finally, all samples were
analysed by electrophoresis on 0.8% agarose gels.
[0413] Results
[0414] Analysis of the mobility of plasmid DNA through gels
demonstrated that, as the amount of lipid increased, the DNA tended
to be retarded in the wells. Thus, for example, at a charge ratio
of 2:1 (H18:DNA), no DNA entered the gel, and at higher ratios, the
plasmid DNA was no longer visible by ethidium bromide fluorescence
(see below), suggesting that the DNA had become fully condensed.
The H18/DNA condensates were resistant to treatment by pronase. In
addition, DNA condensed with H18 a charge ratio of at least 2:1 was
resistant to treatment by DNase. At charge ratios of 2:1 or
greater, addition of serum to 50% did not lead to an increase in
the amount of DNA degradation, suggesting that lipids according to
the invention are stable in serum.
[0415] Physical Chemical Assays
[0416] Two physical chemical assays can be used to assess the
ability of the lipids of the invention to compact supercoiled DNA
and to determine the stability of the condensed particles.
[0417] Assay 1
[0418] The first assay involves the use of ethidium bromide, a
molecule which fluoresces when intercalated into the DNA helix.
Solutions containing DNA and a lipid according to the invention are
prepared so that the charge ratio between the negatively charged
phosphate groups of the DNA and the postively charged polyamines of
the lipids varies between zero and three [see the "Methods" in the
previous section]. After ethidium bromide has been added to each
solution the fluorescence reading is measured. As the charge ratio
increases towards charge neutrality, because of the increasing
amounts of lipid present, there is a progressive decrease in the
fluorescence of the ethidium bromide when this molecule is excluded
from binding to the DNA as compaction occurs. The point at which
compaction is complete corresponds to the point at which the
fluorescence reading levels-off at a minimum. In the case of
thelipids of the invention the fluorescence minimum is reached at
charge ratios of lipid to DNA in the range of 0.8 to 2.5. This
assay has been used to demonstrate that lipid are compaction
competent under conditions of physiological salt (150 mM NaCl) and
at acidic conditions down to pH 3.0. Repeating the assay also shows
that the compacted DNA particles are stable for many hours both in
physiological salt and under low pH conditions.
[0419] Assay 2
[0420] The second assay involves gel electrophoresis using ethidium
bromide as a stain. Samples of lipid and DNA are prepared as before
and loaded into separate lanes in a polyacrylamide gel. After
electrophoresis the fluorescence reading of each lane is
determined. Two effects are observed. First, as the charge ratio
increases towards neutrality the distance that the DNA/lipid
complex travels through the gel decreases progressitvely. This is a
result of two physical processes; compaction, which renders the DNA
less able to move through the viscous gel and neutralisation of DNA
negative charge, which reduces the electrostatic attraction between
the complex and the cathode. Second, as the charge ratio increases
beyond neutrality the brilliance of the fluorescent response
decreases as the ethidium bromide stain is excluded from the DNA
helix. This assay has been used to confirm that the lipids
according to the invention cause DNA compaction close to the point
of charge neutrality, in agreement with theory.
[0421] Transfection of Mammalian Cells with Lipid Condensed DNA
Complexes
[0422] Condensation of DNA
[0423] Plasmid DNA (pEGlacZ) was prepared at concentrations of
typically 60 or 120 .mu.g/ml in water. Solutions of lipids
according to the invention were prepared in water over a range of
concentrations (typically 30 to 960 .mu.g/ml). An equal volume of
DNA was added dropwise to a tube containing a solution of the
lipids whilst vortexing the tube.
[0424] CHO Transfection Protocol
[0425] Chinese Hamster Ovary (CHO) cells were seeded in to 24 well
plates at 100,000 cells per well 24 h before experiment. Cells were
washed once in Optimem.TM. medium prior to transfection. Wash
medium was removed and replaced with 0.5 ml of Optimem.TM. to which
the required amount of lipid condensed DNA was added (typically 1
to 5 .mu.g DNA equivalent). Usually three replicate transfection
wells were set up per condensed DNA sample tested. Cells were
incubated for a further 3-4 h at 37.degree. C., 5% CO.sub.2 before
removal of the complex and additon of 1 ml of fresh medium
(Iscoves.TM. medium: modified DMEM plus glutamate, asparagine,
adenosine, guanosine, cytidine, uridine, thymidine and 10% dialysed
foetal calf serum). Cells were cultured for a further 48-72 hours
before harvesting and assay. Levels of Beta galactosidase reporter
gene activity were determined using an enzyme assay system from
Promega as follows. Cells were washed twice with 1 ml of phosphate
buffered saline and solubilised in 200 ul of 1.times.cell lysis
buffer. 50 ul of cell extract was incubated with the provided
buffer and substrate o-nitrophenyl-.beta.-D-galactopyranoside and
the optical density measured spectrophoto-metrically. Levels of
.beta.-gal expression were quantitated by reference to the standard
curve and related to the amount of protein in the extract (measured
using the BCA assay kit from Pierce) to give a final value
expressed as mU of .beta.-gal per mg of protein.
[0426] Results
[0427] Typical transfection results for lipid DNA samples are shown
in the following table:
2 .beta.-galactosidase in Lipid Lipid:DNA cell extract mUnits (see
Examples) mass ratio per mg protein H3 8:1 11 .+-. 4 H18 8:1 40
.+-. 9
[0428] Non-condensed DNA control resulted in an undetectable level
of .beta.-galactosidase, whereas the tetramine (H3) and hexamine
(H18) condensed DNA achieved significant .beta.-galactosidase
expression in the CHO cells.
* * * * *