U.S. patent application number 16/958244 was filed with the patent office on 2020-10-22 for cationic lipids.
The applicant listed for this patent is Takeda Pharmaceutical Company Limited. Invention is credited to Yusutaka Hoashi, Satoru Matsumoto, Masahiro Mineno, Yoshimasa Omori.
Application Number | 20200331841 16/958244 |
Document ID | / |
Family ID | 1000004975861 |
Filed Date | 2020-10-22 |
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United States Patent
Application |
20200331841 |
Kind Code |
A1 |
Matsumoto; Satoru ; et
al. |
October 22, 2020 |
CATIONIC LIPIDS
Abstract
The present invention provides a technique that enables
introduction of active ingredients, in particular, nucleic acids,
into cells with superior efficiency; and cationic lipids, etc., for
use in the technique. The compound or a salt thereof according to
the present invention is a compound represented by formula (I) or a
salt thereof. In formula (I), n represents an integer of 2 to 5, R
represents a linear C.sub.1-5 alkyl group, a linear C.sub.7-11
alkenyl group, or a linear C.sub.11 alkadienyl group, and wavy
lines each independently represent a cis-bond or a trans-bond.
##STR00001##
Inventors: |
Matsumoto; Satoru;
(Kanagawa, JP) ; Omori; Yoshimasa; (Osaka, JP)
; Mineno; Masahiro; (Osaka, JP) ; Hoashi;
Yusutaka; (Kanagawa, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Takeda Pharmaceutical Company Limited |
Osaka |
|
JP |
|
|
Family ID: |
1000004975861 |
Appl. No.: |
16/958244 |
Filed: |
December 27, 2018 |
PCT Filed: |
December 27, 2018 |
PCT NO: |
PCT/JP2018/048054 |
371 Date: |
June 26, 2020 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 9/1617 20130101;
C07C 69/533 20130101; C12N 15/113 20130101 |
International
Class: |
C07C 69/533 20060101
C07C069/533; C12N 15/113 20060101 C12N015/113; A61K 9/16 20060101
A61K009/16 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 28, 2017 |
JP |
2017-254667 |
Claims
1. A compound represented by formula (I): ##STR00018## wherein n
represents an integer of 2 to 5, R represents a linear C.sub.1-5
alkyl group, a linear C.sub.7-11 alkenyl group, or a linear
C.sub.11 alkadienyl group, and wavy lines each independently
represent a cis-bond or a trans-bond, or a salt thereof.
2.
3-((4-(Dimethylamino)butanoyl)oxy)-2,2-bis(((9Z)-tetradec-9-enoyloxy)m-
ethyl)propyl (9Z)-tetradec-9-enoate or a salt thereof.
3.
3-((5-(Dimethylamino)pentanoyl)oxy)-2,2-bis(((9Z)-tetradec-9-enoyloxy)-
methyl)propyl (9Z)-tetradec-9-enoate or a salt thereof.
4.
3-((6-(Dimethylamino)hexanoyl)oxy)-2,2-bis(((9Z)-tetradec-9-enoyloxy)m-
ethyl)propyl (9Z)-tetradec-9-enoate or a salt thereof.
5. A lipid particle comprising the compound or a salt thereof
according to claim 1.
6. A composition for nucleic acid introduction comprising a nucleic
acid and the lipid particle according to claim 5.
7. The composition according to claim 6, wherein the nucleic acid
is an RNA.
8. The composition according to claim 7, wherein the RNA is an mRNA
or an siRNA.
Description
TECHNICAL FIELD
[0001] The present invention relates to a cationic lipid that
enables introduction of nucleic acids as an active ingredient into
many types of cells, tissues, or organs. The present invention
further relates to a lipid particle containing the cationic lipid,
and a composition containing the lipid particle and a nucleic
acid.
BACKGROUND OF INVENTION
[0002] Intensive research and development of nucleic acid drugs,
which contain a nucleic acid as an active ingredient, have been
made in recent years. For example, a number of studies have been
conducted for nucleic acid drugs having decomposition effect or
function-inhibitory effect on target mRNAs including nucleic acids
such as siRNAs, miRNAs, miRNA mimics, and antisense nucleic acids.
In addition, studies have been carried out for nucleic acid drugs
containing an mRNA or the like encoding a protein of interest to
express the protein of interest in cells. In relation to such
research and development, techniques to introduce nucleic acids
into cells, tissues, or organs with high efficiency have been
developed as drug delivery system (DDS) techniques.
[0003] Conventionally known as such a DDS technique is such a
technique that a nucleic acid and a lipid are mixed to form a
complex and cells are allowed to incorporate the nucleic acid via
the complex. Conventionally known as lipids for use in formation of
the complex are cationic lipids, hydrophilic polymer lipids, helper
lipids, and so on. As such cationic lipids, for example, compounds
described in prior art documents, as shown below, are known.
[0004] Patent Literature 1 describes a compound represented by the
following formula or a salt thereof, and so on.
##STR00002##
[0005] It is specified for the formula that R.sup.1 is
independently selected from the group consisting of optionally
substituted C.sub.8-C.sub.24 alkyl and optionally substituted
C.sub.8-C.sub.24 alkenyl; each R.sup.2 and R.sup.3 is independently
selected from the group consisting of hydrogen, optionally
substituted C.sub.1-C.sub.8 alkyl, optionally substituted
arylalkyl, and so on; each Y.sup.1 and Y.sup.2 is independently
selected from the group consisting of hydrogen, optionally
substituted C.sub.1-C.sub.6 alkyl, optionally substituted
arylalkyl, and so on; each Y.sup.3, if present, is independently
selected from the group consisting of hydrogen, optionally
substituted C.sub.1-C.sub.8 alkyl, optionally substituted
arylalkyl, and so on; m is any integer of 1 to 4; n is any integer
of 0 to 3; p is 0 or 1; the sum of m, n, and p is 4; k is any
integer of 1 to 5; and q is 0 or 1.
[0006] Patent Literature 2 describes a compound represented by the
following formula or a salt thereof, and so on.
##STR00003##
[0007] In the formula, W denotes formula --NR.sup.1R.sup.2 or
formula --N.sup.+R.sup.3R.sup.4R.sup.5(Z.sup.-); R.sup.1 and
R.sup.2 denote, each independently, a C.sub.1-4 alkyl group or
hydrogen atom; R.sup.3, R.sup.4, and R.sup.5 denote, each
independently, a C.sub.1-4 alkyl group; Z.sup.- denotes a negative
ion; X denotes a C.sub.1-6 alkylene group which may be substituted;
Y.sup.A, Y.sup.B, and Y.sup.c denote, each independently, a methine
group which may be substituted; L.sup.A, L.sup.B, and L.sup.c
denote, each independently, a methylene group which may be
substituted or a bond; R.sup.A1, R.sup.A2, R.sup.B1, R.sup.B2,
R.sup.C1 and R.sup.C2 denote, each independently, a C.sub.4-10
alkyl group which may be substituted.
CITATION LIST
Patent Literature
[0008] Patent Literature 1: International Publication No. WO
2003/102150 [0009] Patent Literature 2: International Publication
No. WO 2016/021683
SUMMARY OF INVENTION
Technical Problem
[0010] Cationic lipids that enable introduction of nucleic acids
into cells with high efficiency are expected to contribute to
creation of nucleic acid drugs that are superior in terms of
manifestation of drug action, safety (low toxicity), and so on and
exhibit therapeutically superior effect. Cationic lipids that
enable introduction of nucleic acids into various cells are
expected to enable creation of nucleic acid drugs for diseases that
occur in various tissues. Currently, however, no cationic lipid
that sufficiently satisfies those requirements has been found.
[0011] An object of the present invention is to provide a technique
that enables introduction of nucleic acids into cells with superior
efficiency; and cationic lipids, etc., for use in the
technique.
[0012] An object of the present invention from another viewpoint is
to provide a technique that enables introduction of nucleic acids
into various cells; and compounds, etc., for use in the
technique.
Solution to Problem
[0013] The present inventors have diligently examined to solve the
problem and found that the problem is successfully solved by using
a compound represented by the formula below or a salt thereof, thus
completing the present invention.
[0014] Specifically, the present invention relates at least to the
followings.
[0015] [1] A compound represented by formula (I):
##STR00004##
wherein
[0016] n represents an integer of 2 to 5,
[0017] R represents a linear C.sub.1-5 alkyl group, a linear
C.sub.7-11 alkenyl group, or a linear C.sub.11 alkadienyl group,
and
[0018] wavy lines each independently represent a cis-bond or a
trans-bond, or a salt thereof.
[0019] [2]
3-((4-(Dimethylamino)butanoyl)oxy)-2,2-bis(((9Z)-tetradec-9-eno-
yloxy)methyl)propyl (9Z)-tetradec-9-enoate or a salt thereof.
[0020] [3]
3-((5-(Dimethylamino)pentanoyl)oxy)-2,2-bis(((9Z)-tetradec-9-en-
oyloxy)methyl)propyl (9Z)-tetradec-9-enoate or a salt thereof.
[0021] [4]
3-((6-(Dimethylamino)hexanoyl)oxy)-2,2-bis(((9Z)-tetradec-9-eno-
yloxy)methyl)propyl (9Z)-tetradec-9-enoate or a salt thereof.
[0022] [5] A lipid particle containing the compound or a salt
thereof according to item 1.
[0023] [6] A composition for nucleic acid introduction containing a
nucleic acid and the lipid particle according to item 5.
[0024] [7] The composition according to item 6, wherein the nucleic
acid is an RNA.
[0025] [7a] The composition according to item 6, wherein the
nucleic acid is a DNA.
[0026] [8] The composition according to item 7, wherein the RNA is
an mRNA or an siRNA.
[0027] Herein, "the compound represented by formula (I)" is
occasionally referred to as "compound (I)". "The compound
represented by formula (I) or a salt thereof" is occasionally
called "the compound of the present invention". A "lipid particle
containing the compound represented by formula (I) or a salt
thereof (the compound of the present invention)" is occasionally
called "the lipid particle of the present invention". A
"composition for nucleic acid introduction containing a nucleic
acid and the lipid particle of the present invention" is
occasionally called "the composition of the present invention".
Advantageous Effects of Invention
[0028] The present invention enables introduction of nucleic acids
into cells, tissues, or organs with superior efficiency. The
present invention enables introduction of nucleic acids into
various types of cells, tissues, or organs (e.g., cancer cells).
The present invention enables acquisition of drugs or reagents for
research to introduce a nucleic acid into various types of cells,
tissues, or organs. Moreover, if a nucleic acid is introduced into
cells, a tissue, or an organ through the present invention, the
efficiency of manifestation of the activity (e.g., drug action)
possessed by the nucleic acid is high.
DETAILED DESCRIPTION OF INVENTION
[0029] Now, definitions of substituents used herein will be
described in detail. Substituents have the following definitions,
unless otherwise specified.
[0030] Examples of the "linear C.sub.1-5 alkyl group" herein
include methyl, ethyl, propyl, butyl, and pentyl.
[0031] Examples of the "linear C.sub.7-11 alkenyl group" herein
include 1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5-heptenyl,
6-heptenyl, 1-octenyl, 2-octenyl, 3-octenyl, 4-octenyl, 5-octenyl,
6-octenyl, 7-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 4-nonenyl,
5-nonenyl, 6-nonenyl, 7-nonenyl, 8-nonenyl, 1-decenyl, 2-decenyl,
3-decenyl, 4-decenyl, 5-decenyl, 6-decenyl, 7-decenyl, 8-decenyl,
9-decenyl, 1-undecenyl, 2-undecenyl, 3-undecenyl, 4-undecenyl,
5-undecenyl, 6-undecenyl, 7-undecenyl, 8-undecenyl, 9-undecenyl,
and 10-undecenyl. While each of these linear C.sub.7-11 alkenyl
groups has one carbon-carbon double bond, and hence the
carbon-carbon double bond can form a cis-structure and a
trans-structure, the carbon-carbon double bond may form any of the
structures.
[0032] Examples of the "linear C.sub.11 alkadienyl group" herein
include 1,3-undecadienyl, 1,4-undecadienyl, 1,5-undecadienyl,
1,6-undecadienyl, 1,7-undecadienyl, 1,8-undecadienyl,
1,9-undecadienyl, 1,10-undecadienyl, 2,4-undecadienyl,
2,5-undecadienyl, 2,6-undecadienyl, 2,7-undecadienyl,
2,8-undecadienyl, 2,9-undecadienyl, 2,10-undecadienyl,
3,5-undecadienyl, 3,6-undecadienyl, 3,7-undecadienyl,
3,8-undecadienyl, 3,9-undecadienyl, 3,10-undecadienyl,
4,6-undecadienyl, 4,7-undecadienyl, 4,8-undecadienyl,
4,9-undecadienyl, 4,10-undecadienyl, 5,7-undecadienyl,
5,8-undecadienyl, 5,9-undecadienyl, 5,10-undecadienyl,
6,8-undecadienyl, 6,9-undecadienyl, 6,10-undecadienyl,
7,9-undecadienyl, 7,10-undecadienyl, and 8,10-undecadienyl. While
each of these linear C.sub.11 alkadienyl groups has two
carbon-carbon double bonds, and hence the carbon-carbon double
bonds can each independently form a cis-structure and a
trans-structure, each carbon-carbon double bond may form any of the
structures.
[0033] Preferred examples of n and the wavy lines in formula (I)
are as follows.
[0034] n is preferably an integer of 3 to 5, and more preferably
3.
[0035] The wave lines are preferably each a cis-bond.
[0036] Specific preferred examples of compound (I) are as
follows.
[0037] Compound (A): such a compound that n is an integer of 3 to
5, R is a linear C.sub.7-11 alkenyl group in a cis-structure, and
the wavy lines are each a cis-bond.
[0038] Compound (B): such a compound that n is 4, R is a linear
C.sub.11 alkadienyl group in which two carbon-carbon double bonds
each form a cis-structure, and the wavy lines are each a
cis-bond.
[0039] Compound (C): such a compound that n is 2 or 3, R is a
linear C.sub.1-5 alkyl group, and the wavy lines are each a
cis-bond.
[0040] Specific, more preferred examples of compound (I) are as
follows.
[0041] Compound (A1): such a compound that n is an integer of 3 to
5, R is 5-heptenyl, 7-nonenyl, or 9-undecenyl in the cis-structure,
and the wavy lines are each a cis-bond.
[0042] Compound (B1): such a compound that n is 4, R is
2,5-undecadienyl in which two carbon-carbon double bonds each form
a cis-structure, and the wavy lines are each a cis-bond.
[0043] Compound (C1): such a compound that n is 2 or 3, R is
methyl, propyl, or pentyl, and the wavy lines are each a
cis-bond.
[0044] The salt of compound (I) is preferably a pharmacologically
acceptable salt, and examples thereof include salts with an
inorganic base, salts with an organic base, salts with an inorganic
acid, salts with an organic acid, and salts with a basic or acidic
amino acid.
[0045] Preferred examples of salts with an inorganic base include
alkali metal salts such as sodium salts and potassium salts; alkali
earth metal salts such as calcium salts and magnesium salts;
aluminum salts; and ammonium salts. Preferred are sodium salts,
potassium salts, calcium salts, and magnesium salts, and more
preferred are sodium salts and potassium salts.
[0046] Preferred examples of salts with an organic base include
salts with trimethylamine, triethylamine, pyridine, picoline,
ethanolamine, diethanolamine, triethanolamine,
tromethamine[tris(hydroxymethyl)methylamine], tert-butylamine,
cyclohexylamine, benzylamine, dicyclohexylamine, and
N,N-dibenzylethylenediamine.
[0047] Preferred examples of salts with an inorganic acid include
salts with hydrofluoric acid, hydrochloric acid, hydrobromic acid,
hydroiodic acid, nitric acid, sulfuric acid, and phosphoric acid.
Preferred are salts with hydrochloric acid and salts with
phosphoric acid.
[0048] Preferred examples of salts with an organic acid include
salts with formic acid, acetic acid, trifluoroacetic acid, phthalic
acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric
acid, succinic acid, malic acid, methanesulfonic acid,
benzenesulfonic acid, and p-toluenesulfonic acid.
[0049] Preferred examples of salts with a basic amino acid include
salts with arginine, lysine, and ornithine.
[0050] Preferred examples of salts with an acidic amino acid
include salts with aspartic acid and glutamic acid.
[0051] In the present invention, the compound of the present
invention may be used as a cationic lipid. The cationic lipid can
form a complex with a plurality of molecules in a solvent or
dispersion medium. The complex may contain an additional component
in addition to the compound of the present invention. Examples of
the additional component include an additional lipid component and
a nucleic acid.
[0052] Examples of the additional lipid component include
structural lipids capable of constituting a lipid particle. For
such a structural lipid, for example, at least one selected from
the group consisting of the following may be used:
sterols (e.g., cholesterol, cholesteryl ester, cholesteryl
hemisuccinate); phospholipids (e.g., phosphatidylcholine (e.g.,
dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine,
lysophosphatidylcholine, dioleoylphosphatidylcholine,
palmitoyloleoylphosphatidylcholine,
dilinolenoylphosphatidylcholine, MC-1010 (NOF CORPORATION), MC-2020
(NOF CORPORATION), MC-4040 (NOF CORPORATION)), phosphatidylserine
(e.g., dipalmitoylphosphatidylserine, distearoylphosphatidylserine,
dioleoylphosphatidylserine, palmitoyloleoylphosphatidylserine),
phosphatidylethanolamine (e.g.,
dipalmitoylphosphatidylethanolamine,
distearoylphosphatidylethanolamine,
dioleoylphosphatidylethanolamine,
palmitoyloleoylphosphatidylethanolamine,
lysophosphatidylethanolamine), phosphatidylinositol, phosphatidic
acid); and polyethylene glycol lipids (PEG lipids) (e.g., PEG-DAA,
PEG-DAG, PEG-phospholipid cunjugate, PEG-Cer, PEG-cholesterol,
PEG-C-DOMG, 2KPEG-CMG, GM-020 (NOF CORPORATION), GS-020 (NOF
CORPORATION), GS-050 (NOF CORPORATION)). In the present invention,
it is preferred to use all of the three, namely, a sterol (in
particular, cholesterol), a phospholipid (in particular,
phosphatidylcholine), and a polyethylene glycol lipid, as the
structural lipid.
[0053] The ratio between the compound of the present invention and
the structural lipid in the mixed lipid component constituting the
lipid particle of the present invention may be appropriately
controlled according to the purpose or use. For example, the ratio
of the structural lipid is typically 0.008 to 4 mol and preferably
0.4 to 1.5 mol per mole of the compound of the present invention.
In another definition of ratios in the mixed lipid component, the
amount of the compound of the present invention is typically 1 to 4
mol, that of the sterol is typically 0 to 3 mol, that of the
phospholipid is typically 0 to 2 mol, and that of the polyethylene
glycol lipid is typically 0 to 1 mol. In a more preferred
embodiment with use of a mixture of the compound of the present
invention and additional lipid components, with respect to ratios,
the amount of the compound of the present invention is 1 to 1.5
mol, that of the sterol is 0 to 1.25 mol, that of the phospholipid
is 0 to 0.5 mol, and that of the polyethylene glycol lipid is 0 to
0.125 mol.
[0054] The compound of the present invention may be used for
producing the lipid particle of the present invention. The lipid
particle of the present invention refers to the complex described
above but containing no nucleic acid. The shape of the lipid
particle of the present invention is not limited to a particular
shape, and the scope includes a complex in which the compound of
the present invention and so on assemble to form a sphere; a
complex in which the compound of the present invention and so on
assemble without forming a particular shape; a complex in which the
compound of the present invention and so on dissolve in a solvent;
and a complex in which the compound of the present invention and so
on homogeneously or heterogeneously disperse in a dispersion
medium.
[0055] The lipid particle of the present invention (e.g., a lipid
particle composed of the compound of the present invention and
structural lipids other than the compound) may be used, for
example, for producing the composition of the present invention
containing the lipid particle and a nucleic acid (in particular, a
nucleic acid as a substance useful for pharmaceutical applications
or applications for research). The composition of the present
invention may be used as a drug or a reagent. It is preferred for
the composition of the present invention that the lipid particle
encapsulate a nucleic acid in a ratio as high as possible (i.e., in
a high encapsulation ratio).
[0056] The "nucleic acid" may be any molecule in which molecules of
nucleotide or molecules having functions equivalent to those of the
nucleotide are polymerized, and examples thereof include RNA, which
is a polymer of ribonucleotide; DNA, which is a polymer of
deoxyribonucleotide; a polymer of a mixture of ribonucleotide and
deoxyribonucleotide; and a nucleotide polymer including a
nucleotide analog. Further, the nucleic acid may be a nucleotide
polymer including a nucleic acid derivative. The nucleic acid may
be a single-stranded nucleic acid or a double-stranded nucleic
acid. The concept of the double-stranded nucleic acid encompasses a
double-stranded nucleic acid such that one strand hybridizes with
the other strand under stringent conditions.
[0057] The nucleotide analog may be any molecule obtained by
modifying ribonucleotide, deoxyribonucleotide, RNA, or DNA to
improve nuclease resistance, to stabilize, to enhance affinity with
a complementary-strand nucleic acid or to enhance cell permeability
as compared with RNA or DNA, or for visualization. The nucleotide
analog may be a naturally-occurring molecule or a non-natural
molecule, and examples thereof include a sugar-modified nucleotide
analog and a phosphodiester bond-modified nucleotide analog.
[0058] The sugar-modified nucleotide analog may be any one obtained
by addition of or substitution with a substance having an arbitrary
chemical structure for a part or the whole of the chemical
structure of the sugar of a nucleotide. Specific examples thereof
include a nucleotide analog substituted with 2'-O-methylribose, a
nucleotide analog substituted with 2'-O-propylribose, a nucleotide
analog substituted with 2'-methoxyethoxyribose, a nucleotide analog
substituted with 2'-O-methoxyethylribose, a nucleotide analog
substituted with 2'-O-[2-(guanidinium)ethyl]ribose, a nucleotide
analog substituted with 2'-O-fluororibose, a crosslinked artificial
nucleic acid provided with two cyclic structures by introducing a
crosslinked structure to the sugar moiety (Bridged Nucleic Acid)
(BNA), more specifically, a locked artificial nucleic acid in which
the oxygen atom at position 2' and the carbon atom at position 4'
are crosslinked via methylene (Locked Nucleic Acid) (LNA) and an
ethylene-crosslinked artificial nucleic acid (Ethylene bridged
nucleic acid) (ENA) [Nucleic Acid Research, 32, e175 (2004)],
further, a peptide nucleic acid (PNA) [Acc. Chem. Res., 32, 624
(1999)], an oxypeptide nucleic acid (OPNA) [J. Am. Chem. Soc., 123,
4653 (2001)], and a peptide ribonucleic acid (PRNA) [J. Am. Chem.
Soc., 122, 6900 (2000)].
[0059] The phosphodiester bond-modified nucleotide analog may be
any one obtained by addition of or substitution with an arbitrary
chemical substance for a part or the whole of the chemical
structure of the phosphodiester bond of a nucleotide. Specific
examples thereof include a nucleotide analog substituted with a
phosphorothioate bond and a nucleotide analog substituted with an
N3'-P5' phosphoramidate bond [Saibo Kogaku (in Japanese, translated
title: Cell Engineering), 16, 1463-1473 (1997)] [RNAi and Antisense
Strategies, KODANSHA LTD. (2005)].
[0060] The nucleic acid derivative may be any molecule obtained by
adding to a nucleic acid another chemical substance to improve
nuclease resistance, to stabilize, to enhance affinity with a
complementary-strand nucleic acid, or to enhance cell permeability
as compared with the nucleic acid, or for visualization. Specific
examples thereof include a derivative with 5'-polyamine added, a
derivative with cholesterol added, a derivative with steroid added,
a derivative with bile acid added, a derivative with vitamin added,
a derivative with Cy5 added, a derivative with Cy3 added, a
derivative with 6-FAM added, and a derivative with biotin
added.
[0061] The nucleic acid in the present invention is not limited to
a particular nucleic acid, and may be a nucleic acid, for example,
for the purpose of ameliorating a disease, a symptom, a disorder,
or sickness, and mitigating a disease, a symptom, a disorder, or
pathological condition or preventing the onset thereof (herein,
occasionally referred to as "treatment or the like of a disease"),
or a nucleic acid for regulating expression of a desired protein
that is useful for research, even though the protein does not
contribute to treatment or the like of a disease.
[0062] Genes or polynucleotides related to diseases (herein,
occasionally referred to as "disease-related genes") are available,
for example, from McKusick-Nathans Institute of Genetic Medicine,
Johns Hopkins University (Baltimore, Md.) and National Center for
Biotechnology Information, National Library of Medicine (Bethesda,
Md.).
[0063] Specific examples of the nucleic acid in the present
invention include siRNAs, shRNAs, miRNAs, miRNA mimics, antisense
nucleic acids, ribozymes, mRNAs, decoy nucleic acids, aptamers,
plasmid DNAs, Cosmid DNAs, and BAC DNAs. The nucleic acid is
preferably an RNA such as an siRNA and an mRNA or an analog or
derivative obtained by artificially modifying an RNA.
[0064] In the present invention, the "siRNA" refers to a
double-stranded RNA or relative thereof consisting of 10 to 30
nucleotides, preferably of 15 to 25 nucleotides, and including
complementary sequences. The siRNA includes protruding nucleotides
preferably of one to three nucleotides, more preferably of two
nucleotides, at the 3'-end. The moiety of complementary sequences
may be completely complementary or include non-complementary
nucleotides, but preferably are completely complementary.
[0065] The siRNA in the present invention is not limited to a
particular siRNA, and, for example, an siRNA for knockdown of gene
expression against a disease-related gene may be used. A
disease-related gene refers to any gene or polynucleotide
generating a transcription or translation product at an abnormal
level or in an abnormal form in cells derived from tissue of a
patient, as compared with tissue or cells from a disease-free
control. For the siRNA in the present invention, an siRNA to
regulate expression of a desired protein useful for research may be
used.
[0066] In the present invention, the "mRNA" refers to an RNA
including a nucleotide sequence that can be translated into a
protein. The mRNA in the present invention is not limited to a
particular mRNA and may be any mRNA capable of expressing a desired
protein in cells. The mRNA is preferably an mRNA useful for
pharmaceutical applications (e.g., applications of disease
treatment) and/or applications for research, and examples of such
mRNAs include an mRNA to express a marker protein such as
luciferase in cells.
[0067] The disease is not limited to a particular disease, and
examples of the disease include diseases listed below. Contents in
each "0" are examples of the corresponding disease-related gene,
except for the case that specific disease examples are listed.
Another example of the nucleic acid in the present invention is a
nucleic acid that regulates the expression level of any of those
disease-related genes (or a protein encoded by the disease-related
gene).
(1) Diseases of blood system [anaemia (CDAN1, CDA1, RPS19, DBA,
PKLR, PK1, NT5C3, UMPH1, PSN1, RHAG, RH50A, NRAMP2, SPTB, ALAS2,
ANH1, ASB, ABCB7, ABC7, ASAT), bare lymphocyte syndrome (TAPBP,
TPSN, TAP2, ABCB3, PSF2, RING11, MHC2TA, C2TA, RFX5), hemorrhagic
diseases (TBXA2R, P2RX1, P2X1), factor H and factor H-like 1
deficiencies (HF1, CFH, HUS), factor V and factor VIII deficiencies
(MCFD2), factor VII deficiency (F7), factor X deficiency (F10),
factor XI deficiency (F11), factor XII deficiency (F12, HAF),
factor XIIIA deficiency (F13A1, F13A), factor XIIIB deficiency
(F13B), Fanconi's anaemia(FANCA, FACA, FA1, FA, FAA, FAAP95,
FAAP90, F1134064, FANCB, FANCC, FACC, BRCA2, FANCD1, FANCD2, FANCD,
FACD, FAD, FACE, FACE, FANCF, XRCC9, FANCG, BRIP1, BACH1, FANCJ,
PHF9, FANCL, FANCM, KIAA1596), haemophagocytic lymphohistiocytosis
(PRF1, HPLH2, UNC13D, MUNC13-4, HPLH3, HLH3, FHL3), haemophilia A
(F8, F8C, HEMA), haemophilia B (F9, HEMB), haemorrhagic disorders
(PI, ATT, F5), leucocyte deficiency (ITGB2, CD18, LCAMB, LAD,
EIF2B1, EIF2BA, EIF2B2, EIF2B3, EIF2B5, LVWM, CACH, CLE, EIF2B4),
sickle cell anaemia (HBB), thalassaemia (HBA2, HBB, HBD, LCRB,
HBA1), and so on]; (2) inflammatory/immunologic diseases [AIDS
(KIR3DL1, NKAT3, NKB1, AMB11, KIR3DS1, IFNG, CXCL12, SDF1),
autoimmune lymphoproliferative syndrome (TNFRSF6, APT1, FAS, CD95,
ALPS1A), combined immunodeficiency (IL2RG, SCIDX1, SCIDX, IMD4),
HIV infection (CCL5, SCYA5, D17S135E, TCP228, IL10, CSIF, CMKBR2,
CCR2, DMKBR5, CCCKR5, CCR5), immunodeficiency (CD3E, CD3G, AICDA,
AID, HIGM2, TNFRSF5, CD40, UNG, DGU, HIGM4, TNFSF5, CD40LG, HIGM1,
IGM, FOXP3, IPEX, AIID, XPID, PIDX, TNFRSF14B, TACI), inflammation
(IL10, IL-1, IL-13, IL-17, IL-23, CTLA4), severe combined
immunodeficiency (JAK3, JAKL, DCLRE1C, ATREMIS, SCIDA, RAG1, RAG2,
ADA, PTPRC, CD45, LCA, IL7R, CD3D, T3D, IL2RG, SCIDX1, SCIDX,
IMD4), rheumatoid arthritis, psoriasis, inflammatory bowel diseases
(e.g., Crohn's disease, ulcerative colitis), Sjogren's syndrome,
Behcet's disease, multiple sclerosis, systemic lupus erythematosus,
lupus nephritis, discoid lupus erythematosus, Castleman's disease,
ankylosing spondylitis, polymyositis, dermatomyositis,
polyarteritis nodosa, mixed connective tissue disease,
dermatosclerosis, lupus profundus, chronic thyroiditis, Graves'
disease, autoimmune gastritis, type I and type II diabetes
mellitus, autoimmune haemolytic anaemia, autoimmune neutropenia,
thrombocytopenia, atopic dermatitis, chronic active hepatitis,
myasthenia gravis, graft-versus-host disease, Addison's disease,
abnormal immune response, arthritis, dermatitis, radiodermatitis,
primary biliary cirrhosis, and so on]; (3) metabolic/liver/kidney
diseases [amyloid neuropathy (TTR, PALB), amyloidosis (APOA1, APP,
AAA, CVAP, AD1, GSN, FGA, LYZ, TTR, PALB), non-alcoholic
steatohepatitis and hepatic fibrosis (COL1A1), cirrhosis (KRT18,
KRT8, CIRH1A, NAIC, TEX292, KIAA1988), cystic fibrosis (CFTR,
ABCC7, CF, MRP7), glycogen storage disease (SLC2A2, GLUT2, G6PC,
G6PT, G6PT1, GAA, LAMP2, LAMPB, AGL, GDE, GBE1, GYS2, PYGL, PFKM),
hepatocellular adenoma (TCF1, HFN1A, MODY3), hepatic failure
(SCOD1, SCO1), hepatic lipase deficiency (LIPC), hepatoblastoma
(CTNNB1, PDFGRL, PDGRL, PRLTS, AXIN1, AXIN, TP53, P53, LFS1, IGF2R,
MPRI, MET, CASP8, MCH5), medullary cystic kidney disease (UMOD,
HNFJ, FJHN, MCKD2, ADMCKD2), phenylketonuria (PAH, PKU1, QDPR,
DHPR, PTS), multicystic kidney and liver diseases (FCYT, PKHD1,
APRKD, PDK1, PDK2, PDK4, PDKTS, PRKCSH, G19P1, PCLD, SEC63), and so
on]; (4) nervous system diseases [ALS (SOD1, ALS2, STEX, FUS,
TARDBP, VEGF), Alzheimer's disease (APP, AAA, CVAP, AD1, APOE, AD2,
PSEN2, AD4, STM2, APBB2, FE65L1, NOS3, PLAU, URK, ACE, DCP1, ACE1,
MPO, PACIP1, PAXIP1L, PTIP, A2M, BLMH, BMH, PSEN1, AD3), autism
(BZRAP1, MDGA2, GLO1, MECP2, RTT, PPMX, MRX16, MRX79, NLGN3, NLGN4,
KIAA1260, AUTSX2), fragile X syndrome (FMR2, FXR1, FXR2, mGLUR5),
Huntington's disease (HD, IT15, PRNP, PRIP, JPH3, JP3, HDL2, TBP,
SCA17), Parkinson's disease (NR4A2, NURR1, NOT, TINUR, SNCAIP, TBP,
SCA17, SNCA, NACP, PARK1, PARK4, DJ1, DBH, NDUFV2), Rett syndrome
(MECP2, RTT, PPMX, MRX16, MRX79, CDKL5, STK9), schizophrenia
(GSK3,5-HTT, COMT, DRD, SLC6A3, DAOA, DTNBP1), secretase-related
disorder (APH-1), and so on]; (5) eye diseases [age-related macular
degeneration (Abcr, Cc12, cp, Timp3, cathepsin D, Vldlr, Ccr2),
cataract (CRYAA, CRYA1, CRYBB2, CRYB2, PITX3, BFSP2, CP49, CP47,
PAX6, AN2, MGDA, CRYBA1, CRYB1, CRYGC, CRYG3, CCL, LIM2, MP19,
CRYGD, CRYG4, BSFP2, CP49, CP47, HSF4, CTM, MW, AQPO, CRYAB, CRYA2,
CTPP2, CRYBB1, CRYGD, CRYG4, CRYA1, GJA8, CX50, CAE1, GJA3, CX46,
CZP3, CAE3, CCM1, CAM, KRIT1), corneal opacity (APOA1, TGFB1, CSD2,
CDGG1, CSD, BIGH3, CDG2, TASTD2, TROP2, M1S1, VSX1, RINX, PPCD,
PPD, KTCN, COL8A2, FECD, PPCD2, PIP5K3, CFD), congenital hereditary
corneal plana (KERA, CNA2), glaucoma (MYOC, TIGR, GLC1A, JOAG,
GPOA, OPTN, GLC1E, FIP2, HYPL, NRP, CYP1B1, GLC3A, OPA1, NTG, NPG,
CYP1B1, GLC3A), Leber's congenital amaurosis (CRB1, RP12, CRX,
CORD2, CRD, RPGRIP1, LCA6, CORDS, RPE65, RP20, AIPL1, LCA4, GUCY2D,
GUC2D, LCA1, CORD6, RDH12, LCA3), macular dystrophy (ELOVL4, ADMD,
STGD2, STGD3, RDS, RP7, PRPH2, PRPH, AVMD, AOFMD, VMD2), and so
on]; and (6) neoplastic diseases [malignant tumor, neovascular
glaucoma, infantile hemangioma, multiple myeloma, chronic sarcoma,
metastatic melanoma, Kaposi's sarcoma, angioproliferation,
cachexia, metastasis or the like of breast cancer, cancer (e.g.,
colorectal cancer (e.g., familial colorectal cancer, hereditary
nonpolyposis colorectal cancer, gastrointestinal stromal tumor),
lung cancer (e.g., non-small cell lung cancer, small cell lung
cancer, malignant mesothelioma), mesothelioma, pancreatic cancer
(e.g., pancreatic ductal carcinoma), gastric cancer (e.g.,
papillary adenocarcinoma, mucinous adenocarcinoma, adenosquamous
cell carcinoma), breast cancer (e.g., invasive ductal breast
carcinoma, noninvasive ductal breast carcinoma, inflammatory breast
carcinoma), ovarian cancer (e.g., epithelial ovarian cancer,
extragonadal germ cell tumor, ovarian germ cell tumor, low-grade
ovarian tumor), prostate cancer (e.g., hormone-dependent prostate
cancer, hormone-independent prostate cancer), liver cancer (e.g.,
primary liver cancer, extrahepatic bile duct cancer), thyroid
cancer (e.g., medullary thyroid cancer), kidney cancer (e.g., renal
cell carcinoma, transitional cell carcinoma of renal pelvis and
ureter), uterine cancer, brain tumor (e.g., pineal astrocytoma,
pilocytic astrocytoma, diffuse astrocytoma, anaplastic
astrocytoma), melanoma, sarcoma, bladder cancer, hematological
cancer or the like including multiple myeloma, pituitary adenoma,
glioma, acoustic neurinoma, retinal sarcoma, pharyngeal cancer,
laryngeal cancer, tongue cancer, thymoma, esophageal carcinoma,
duodenal cancer, colon cancer, rectal cancer, hepatocellular
carcinoma, pancreatic endocrine tumor, bile duct cancer,
gallbladder cancer, penile cancer, ureteric cancer, testicular
tumor, vulvar carcinoma, cervix cancer, corpus uteri carcinoma,
uterine sarcoma, trophoblastic disease, vaginal cancer, skin
cancer, mycosis fungoides, basalioma, soft part sarcoma, malignant
lymphoma, Hodgkin's disease, myelodysplastic syndrome, adult T-cell
leukemia, chronic myeloproliferative disease, pancreatic endocrine
tumor, fibrous histiocytoma, leiomyosarcoma, rhabdomyosarcoma,
carcinoma of unknown primary), leukaemia (e.g., acute leukaemia
(e.g., acute lymphocytic leukaemia, acute myeloid leukaemia),
chronic leukaemia (e.g., chronic lymphocytic leukaemia, chronic
myeloid leukaemia), myelodysplastic syndrome), uterine sarcoma
(e.g., uterine mesodermal mixed tumor, uterine leiomyosarcoma,
endometrial stromal tumor), myelofibrosis, and so on].
[0068] The composition of the present invention as a drug may be
produced by using a method known in the art of drug formulation
with a pharmaceutically acceptable carrier. Examples of the dosage
form of the drug include formulations for parenteral administration
(e.g., a liquid such as an injection) blended with a conventional
auxiliary such as a buffering agent and/or a stabilizer; and
formulations for topical administration, such as an ointment, a
cream, a liquid, and a plaster, blended with a conventional
pharmaceutical carrier.
[0069] The composition of the present invention may be used for
introduction of an active ingredient into various types of cells,
tissues, or organs. Examples of cells to which the composition of
the present invention may be applied include mesenchymal stem
cells, neural stem cells, skin stem cells, splenocytes, nerve
cells, glial cells, pancreatic B cells, bone marrow cells,
mesangial cells, Langerhans cells, epidermal cells, epithelial
cells, endothelial cells, fibroblasts, fiber cells, muscle cells
(e.g., skeletal muscle cells, cardiac muscle cells, myoblasts,
muscle satellite cells, smooth muscle cells), fat cells, blood
cells (e.g., macrophages, T cells, B cells, natural killer cells,
mast cells, leukocytes, neutrophils, basophils, eosinophils,
monocytes, megakaryocytes, hematopoietic stem cells), synoviocytes,
chondrocytes, osteocytes, osteoblasts, osteoclasts, mammary cells,
hepatocytes or stromal cells, ova, spermatids, or precursor cells
capable of inducing differentiation into these cells, stem cells
(e.g., including induced pluripotent stem cells (iPS cells),
embryonic stem cells (ES cells)), primordial germ cells, oocytes,
and fertilized ova. Examples of tissues or organs to which the
composition of the present invention may be applied include all
tissues or organs in which the above cells are present, for
example, brain, sites of brain (e.g., olfactory bulb, amygdala,
basal ganglion, hippocampus, thalamus, hypothalamus, subthalamic
nucleus, cerebral cortex, medulla oblongata, cerebellum, occipital
lobe, frontal lobe, temporal lobe, putamen, caudate nucleus,
callosum, substantia nigra), spinal cord, pituitary gland, stomach,
pancreas, kidney, liver, gonad, thyroid, gallbladder, bone marrow,
adrenal gland, skin, lung, digestive tract (e.g., large intestine,
small intestine), blood vessel, heart, thymus, spleen,
submandibular gland, peripheral blood, peripheral blood cells,
prostate, placenta, uterus, bones, joints, and muscles (e.g.,
skeletal muscle, smooth muscle, cardiac muscle). Those cells,
tissues, or organs may be cancer cells, cancer tissues, or the like
that have undergone canceration.
[0070] The composition of the present invention is particularly
superior in efficiency to introduce a nucleic acid into cancer
cells.
[0071] The compound, the lipid particle, and the composition of the
present invention are stable and have low toxicity and can be
safely used. In using the composition of the present invention in
vivo, or using the composition as a drug, the composition is
suitably administered to a subject (e.g., a human or a non-human
mammal (preferably, a human)) so that an effective amount of the
nucleic acid can be delivered to targeted cells.
[0072] In using the composition of the present invention in vivo,
or using the composition as a drug, the composition can be orally
or parenterally (e.g., local, rectal, or intravenous
administration) administered in a safe manner in the form of a
pharmaceutical formulation such as tablets (including sugar-coated
tablets, film-coated tablets, sublingual tablets, orally
disintegrating tablets), powders, granules, capsules (including
soft capsules, microcapsules), liquids, troches, syrups, emulsions,
suspensions, injections (e.g., subcutaneous injections, intravenous
injections, intramuscular injections, intraperitoneal injections),
topical formulations (e.g., formulations for nasal administration,
transdermal formulations, ointments), suppositories (e.g., rectal
suppositories, vaginal suppositories), pellets, nasal formulations,
pulmonary formulations (inhalations), and infusions. Each
formulation may be a controlled-release formulation such as a
fast-release formulation and a sustained-release formulation (e.g.,
a sustained-release microcapsule).
[0073] Now, a method for producing the compound of the present
invention will be described.
[0074] Raw materials and reagents used in each step of the
production method below, and the resulting compound may each form a
salt. Examples of such salts are the same as the above-mentioned
salts for the compound of the present invention.
[0075] When a compound obtained in each step is a free compound,
the compound may be converted into an intended salt by using a
known method. Conversely, when a compound obtained in each step is
a salt, the compound may be converted into a free form or another
intended salt by using a known method.
[0076] A compound obtained in each step may be used for the
subsequent reaction directly as a reaction solution, or a crude
product may be obtained therefrom and used for the subsequent
reaction. Alternatively, a compound obtained in each step may be
isolated and/or purified from a reaction mixture according to a
conventional method using a separation means such as concentration,
crystallization, recrystallization, distillation, solvent
extraction, fractional distillation, and chromatography.
[0077] If a compound as a raw material or reagent in each step is
commercially available, the commercially available product may be
directly used.
[0078] Reaction time for reaction in each step, which may vary
depending on reagents and solvents to be used, is typically 1
minute to 48 hours, and preferably 10 minutes to 8 hours, unless
otherwise specified.
[0079] Reaction temperature for reaction in each step, which may
vary depending on reagents and solvents to be used, is typically
-78.degree. C. to 300.degree. C., and preferably -78.degree. C. to
150.degree. C., unless otherwise specified.
[0080] Pressure for reaction in each step, which may vary depending
on reagents and solvents to be used, is typically 1 atm to 20 atm,
and preferably 1 atm to 3 atm, unless otherwise specified.
[0081] A microwave synthesis apparatus such as an Initiator
produced by Biotage is occasionally used in reaction in a step. The
reaction temperature, which may vary depending on reagents and
solvents to be used, is typically room temperature to 300.degree.
C., preferably room temperature to 250.degree. C., and more
preferably 50.degree. C. to 250.degree. C., unless otherwise
specified. The reaction time, which may vary depending on reagents
and solvents to be used, is typically 1 minute to 48 hours, and
preferably 1 minute to 8 hours, unless otherwise specified.
[0082] In reaction in each step, a reagent is used in an amount of
0.5 equivalents to 20 equivalents, preferably in an amount of 0.8
equivalents to 5 equivalents, to the amount of a substrate, unless
otherwise specified. When a reagent is used as a catalyst, the
reagent is used in an amount of 0.001 equivalents to 1 equivalent,
preferably in an amount of 0.01 equivalents to 0.2 equivalents, to
the amount of a substrate, unless otherwise specified. If a reagent
serves as a reaction solvent in combination with its own role, the
reagent is used in an amount as solvent.
[0083] In reaction in each step, the reaction is performed without
solvent, or in an appropriate solvent dissolving or suspending
reactants therein, unless otherwise specified. Examples of the
solvent include solvents described in Examples and the following
solvents.
[0084] Alcohols: methanol, ethanol, isopropanol, isobutanol,
tert-butyl alcohol, 2-methoxyethanol, and so on;
[0085] ethers: diethyl ether, diisopropyl ether, diphenyl ether,
tetrahydrofuran, 1,2-dimethoxyethane, and so on;
[0086] aromatic hydrocarbons: chlorobenzene, toluene, xylene, and
so on;
[0087] saturated hydrocarbons: cyclohexane, hexane, heptane, and so
on;
[0088] amides: N,N-dimethylformamide, N-methylpyrrolidone, and so
on;
[0089] halogenated hydrocarbons: dichloromethane, carbon
tetrachloride, and so on;
[0090] nitriles: acetonitrile and so on;
[0091] sulfoxides: dimethylsulfoxide and so on;
[0092] aromatic organic bases: pyridine and so on;
[0093] acid anhydride: acetic anhydride and so on;
[0094] organic acids: formic acid, acetic acid, trifluoroacetic
acid, and so on;
[0095] inorganic acids: hydrochloric acid, sulfuric acid, and so
on;
[0096] esters: ethyl acetate, isopropyl acetate, and so on;
[0097] ketones: acetone, methyl ethyl ketone, and so on; and
[0098] water.
[0099] Two or more of these solvents may be mixed for use with an
appropriate ratio.
[0100] When a base is used in reaction in each step, for example,
any of bases listed below or bases described in Examples is
used.
[0101] Inorganic bases: sodium hydroxide, potassium hydroxide,
magnesium hydroxide, and so on;
[0102] basic salts: sodium carbonate, calcium carbonate, sodium
hydrogen carbonate, and so on;
[0103] organic bases: triethylamine, diethylamine, pyridine,
4-dimethylaminopyridine, N,N-dimethylaniline,
1,4-diazabicyclo[2.2.2]octane, 1,8-diazabicyclo[5.4.0]-7-undecene,
imidazole, piperidine, and so on;
[0104] metal alkoxides: sodium ethoxide, potassium tert-butoxide,
sodium tert-butoxide, and so on;
[0105] alkali metal hydrides: sodium hydride and so on;
[0106] metal amides: sodium amide, lithium diisopropylamide,
lithium hexamethyldisilazide, and so on; and
[0107] organolithiums: n-butyllithium, sec-butyllithium, and so
on.
[0108] When an acid or an acidic catalyst is used in reaction in
each step, for example, any of acids and acidic catalysts listed
below and acids and acidic catalysts described in Examples is
used.
[0109] Inorganic acids: hydrochloric acid, sulfuric acid, nitric
acid, hydrobromic acid, phosphoric acid, and so on;
[0110] organic acids: acetic acid, trifluoroacetic acid, citric
acid, p-toluenesulfonic acid, 10-camphorsulfonic acid, and so on;
and
[0111] Lewis acids: boron trifluoride-diethyl ether complex, zinc
iodide, anhydrous aluminum chloride, anhydrous zinc chloride,
anhydrous iron chloride, and so on.
[0112] Unless otherwise specified, reaction in each step is
performed in accordance with a known method such as a method
described in The Fifth Series of Experimental Chemistry, Vol. 13 to
19 (The Chemical Society of Japan (ed.)); Shin Jikken Kagaku Koza
(in Japanese, translated title: New Experimental Chemistry), Vol.
14 and 15 (The Chemical Society of Japan (ed.)); Seimitsu Yuki
Kagaku (in Japanese, translated title: Precise Organic Chemistry,
original title: Reaktionen und Synthesen im organisch-chemischen
Praktikum und Forschungslaboratorium) Revised 2nd Edition (L. F.
Tietze, Th. Eicher, Nankodo Co., Ltd.); Organic Name Reaction; The
Reaction Mechanism and Essence Revised Edition (TOGO, Hideo,
KODANSHA LTD.); ORGANIC SYNTHESES Collective Volume I to VII (John
Wiley & Sons Inc.); Modern Organic Synthesis in the Laboratory
A Collection of Standard Experimental Procedures (Jie Jack Li,
Oxford University Pres); Comprehensive Heterocyclic Chemistry III,
Vol. 1 to Vol. 14 (Elsevier Japan K.K.); Strategic Applications of
Named Reactions in Organic Synthesis (translation supervisor:
TOMIOKA, Kiyoshi, publisher: Kagaku-Dojin Publishing Company,
INC.); Comprehensive Organic Transformations (VCH Publishers Inc.)
(1989); or the like, or in accordance with a method described in
Examples.
[0113] Protection or deprotection reaction for a functional group
in each step is performed in accordance with a known method such as
a method described in "Protective Groups in Organic Synthesis, 4th
Ed." (Theodora W. Greene, Peter G. M. Wuts) published by
Wiley-Interscience Publication, 2007; "Protecting Groups 3rd Ed."
(P. J. Kocienski) published by Thieme Medical Publishers, 2004; or
the like, or in accordance with a method described in Examples.
[0114] Examples of protective groups for a hydroxy group of
alcohols or the like and phenolic hydroxy groups include ether-type
protective groups such as methoxymethyl ether, benzyl ether,
p-methoxybenzyl ether, t-butyldimethylsilyl ether,
t-butyldiphenylsilyl ether, and tetrahydropyranyl ether;
carboxylate-type protective groups such as acetate; sulfonate-type
esters such as methanesulfonate; and carbonate-type protective
groups such as t-butylcarbonate.
[0115] Examples of protective groups for a carbonyl group of
aldehydes include acetal-type protective groups such as
dimethylacetal; and cyclic acetal-type protective groups such as
cyclic 1,3-dioxane.
[0116] Examples of protective groups for a carbonyl group of
ketones include ketal-type protective groups such as dimethyl
ketal; cyclic ketal-type protective groups such as cyclic
1,3-dioxane; oxime-type protective groups such as O-methyloxime;
and hydrazone-type protective groups such as
N,N-dimethylhydrazone.
[0117] Examples of protective groups for a carboxy group include
ester-type protective groups such as methyl ester; and amide-type
protective groups such as N,N-dimethylamide.
[0118] Examples of protective groups for thiol include ether-type
protective groups such as benzyl thioether; and ester-type
protective groups such as thioacetate, thiocarbonate, and
thiocarbamate.
[0119] Examples of protective groups for an amino group and
aromatic heterocycles such as imidazole, pyrrole, and indole
include carbamate-type protective groups such as benzylcarbamate;
amide-type protective groups such as acetamide; alkylamine-type
protective groups such as N-triphenylmethylamine; and
sulfonamide-type protective groups such as methanesulfonamide.
[0120] Removal of a protective group may be performed by using a
known method such as a method using an acid, a base, ultraviolet
light, hydrazine, phenylhydrazine, sodium N-methyldithiocarbamate,
tetrabutylammonium fluoride, palladium acetate, or
trialkylsilylhalide (e.g., trimethylsilyl iodide, trimethylsilyl
bromide), or by using a reduction method.
[0121] Examples of reductants to be used when reduction reaction is
performed in each step include metal hydrides such as lithium
aluminium hydride, sodium triacetoxyborohydride, sodium
cyanoborohydride, diisobutylaluminium hydride (DIBAL-H), sodium
borohydride, and tetramethylammonium triacetoxyborohydride; boranes
such as a borane-tetrahydrofuran complex; Raney nickel; Raney
cobalt; hydrogen; and formic acid. For example, Raney nickel or
Raney cobalt may be used in the presence of hydrogen or formic
acid. When a carbon-carbon double bond or triple bond is reduced, a
method using a catalyst such as palladium-carbon and Lindlar's
catalyst may be used.
[0122] Examples of oxidants to be used when oxidation reaction is
performed in each step include peracids such as m-chloroperbenzoic
acid (MCPBA), hydrogen peroxide, and t-butylhydroperoxide;
perchlorates such as tetrabutylammonium perchlorate; chlorates such
as sodium chlorate; chlorites such as sodium chlorite; periodates
such as sodium periodate;
[0123] hypervalent iodine reagents such as iodosylbenzene;
manganese-containing reagents such as manganese dioxide and
potassium permanganate; leads such as lead tetraacetate;
chromium-containing reagents such as pyridinium chlorochromate
(PCC), pyridinium dichromate (PDC), and the Jones reagent; halogen
compounds such as N-bromosuccinimide (NBS); oxygen; ozone; a sulfur
trioxide-pyridine complex; osmium tetroxide; selenium dioxide; and
2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ).
[0124] Examples of radical initiators to be used when radical
cyclization reaction is performed in each step include azo
compounds such as azobisisobutyronitrile (AIBN); water-soluble
radical initiators such as 4-4'-azobis-4-cyanopentanoic acid
(ACPA); triethylboron in the presence of air or oxygen; and benzoyl
peroxide. Examples of radical reaction reagents to be used include
tributylstannane, tristrimethylsilylsilane,
1,1,2,2-tetraphenyldisilane, diphenylsilane, and samarium
iodide.
[0125] Examples of Wittig reagents to be used when the Wittig
reaction is performed in each step include alkylidenephosphoranes.
Alkylidenephosphoranes may be prepared by using a known method such
as reaction of a phosphonium salt and a strong base.
[0126] Examples of reagents to be used when the Horner-Emmons
reaction is performed in each step include phosphonoacetates such
as methyl dimethylphosphonoacetate and ethyl
diethylphosphonoacetate; and bases such as alkali metal hydrides
and organolithiums.
[0127] Examples of reagents to be used when the Friedel-Crafts
reaction is performed in each step include a Lewis acid with an
acid chloride or an alkylating agent (e.g., a halogenated alkyl, an
alcohol, an olefin). Alternatively, an organic acid or an inorganic
acid may be used instead of a Lewis acid, and an acid anhydride
such as acetic anhydride may be used instead of an acid
chloride.
[0128] When aromatic nucleophilic substitution reaction is
performed in each step, a nucleophile (e.g., an amine, imidazole)
and a base (e.g., a basic salt, an organic base) are used as
reagents.
[0129] Examples of bases used to generate a carbanion when
nucleophilic addition reaction with a carbanion, nucleophilic
1,4-addition reaction with a carbanion (Michael addition reaction),
or nucleophilic substitution reaction with a carbanion is performed
in each step include organolithiums, metal alkoxides, inorganic
bases, and organic bases.
[0130] Examples of Grignard reagents to be used when the Grignard
reaction is performed in each step include arylmagnesium halides
such as phenylmagnesium bromide; and alkylmagnesium halides such as
methylmagnesium bromide and isopropylmagnesium bromide.
[0131] Grignard reagents may be prepared by using a known method
such as reaction of a halogenated alkyl or halogenated aryl and
metal magnesium in a solvent of an ether or tetrahydrofuran.
[0132] When the Knoevenagel condensation reaction is performed in
each step, an active methylene compound sandwiched between two
electron-withdrawing groups (e.g., malonic acid, diethyl malonate,
malononitrile) and a base (e.g., an organic base, a metal alkoxide,
an inorganic base) are used as reagents.
[0133] When the Vilsmeier-Haack reaction is performed in each step,
phosphoryl chloride and an amide derivative (e.g.,
N,N-dimethylformamide) are used as reagents.
[0134] Examples of azidating agents to be used when azidation
reaction is performed for an alcohol, an alkyl halide, or a
sulfonate in each step include diphenylphosphorylazide (DPPA),
trimethylsilylazide, and sodium azide. When an alcohol is azidated,
for example, a method using diphenylphosphorylazide and
1,8-diazabicyclo[5,4,0]undec-7-ene (DBU) or a method using
trimethylsilylazide and a Lewis acid may be used.
[0135] Examples of reductants to be used when reductive amination
reaction is performed in each step include sodium
triacetoxyborohydride, sodium cyanoborohydride, hydrogen, and
formic acid. Examples of carbonyl compounds to be used when the
substrate is an amine compound include aldehydes such as
paraformaldehyde as well as acetaldehyde, and ketones such as
cyclohexanone. Examples of amines to be used when the substrate is
a carbonyl compound include ammonia; primary amines such as methyl
amine; and secondary amines such as dimethylamine.
[0136] When the Mitsunobu reaction is performed in each step, an
azodicarboxylate (e.g., diethyl azodicarboxylate (DEAD),
diisopropyl azodicarboxylate (DIAD)) and triphenylphosphine are
used as reagents.
[0137] Examples of reagents to be used when esterification
reaction, amidation reaction, or urea formation reaction is
performed in each step include halogenated acyl forms such as acid
chlorides and acid bromides; and activated carboxylic acids such as
acid anhydrides, activated ester forms, and sulfate forms. Examples
of activators for carboxylic acids include carbodiimide condensing
agents such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride (WSCD); triazine-based condensing agents such as
4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium
chloride-n-hydrate (DMT-MM); carbonate condensing agents such as
1,1-carbonyldiimidazole (CDI); diphenylphosphoryl azide (DPPA);
benzotriazol-1-yloxy-trisdimethylaminophosphonium salt (BOP
reagent); 2-chloro-1-methyl-pyridinium iodide (Mukaiyama reagent);
thionyl chloride; lower alkyl haloformates such as ethyl
chloroformate;
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HATU); sulfuric acid; and any combination of
them. When a carbodiimide condensing agent is used, an additive
such as 1-hydroxybenzotriazol (HOBt), N-hydroxysuccinimide (HOSu),
and dimethylaminopyridine (DMAP) may be further added to the
reaction.
[0138] Examples of metal catalysts to be used when coupling
reaction is performed in each step include palladium compounds such
as palladium(II) acetate, tetrakis(triphenylphosphine)palladium(0),
dichlorobis(triphenylphosphine)palladium(II),
dichlorobis(triethylphosphine)palladium(II),
tris(dibenzylideneacetone)dipalladium(0),
1,1'-bis(diphenylphosphino)ferrocenepalladium(II) chloride, and
palladium(II) acetate; nickel compounds such as
tetrakis(triphenylphosphine)nickel(0); rhodium compounds such as
tris(triphenylphosphine)rhodium(III) chloride; cobalt compounds;
copper compounds such as copper oxide and copper(I) iodide; and
platinum compounds. A base may be further added to the reaction,
and examples of the base include inorganic bases and basic
salts.
[0139] When thiocarbonylation reaction is performed in each step,
diphosphorus pentasulfide is typically used as a thiocarbonylating
agent; however, not only diphosphorus pentasulfide, but also a
reagent having 1,3,2,4-dithiadiphosphetane-2,4-disulfide structure
such as
2,4-bis(4-methoxyphenyl)-1,3,2,4-dithiadiphosphetane-2,4-disulfide
(Lowesson reagent) may be used.
[0140] Examples of halogenating agents to be used when the
Wohl-Ziegler reaction is performed in each step include
N-iodosuccinimide, N-bromosuccinimide (NBS), N-chlorosuccinimide
(NCS), bromine, and sulfuryl chloride. Further, the reaction may be
accelerated by addition of heat, light, or a radical initiator such
as benzoyl peroxide and azobisisobutyronitrile.
[0141] Examples of halogenating agents to be used when halogenation
reaction is performed for a hydroxy group in each step include
hydrohalic acids and acid halides of inorganic acids, specifically,
hydrochloric acid, thionyl chloride, phosphorus oxychloride for
chlorination, and 48% hydrobromic acid for bromination. A method
may be used in which a halogenated alkyl form is obtained from an
alcohol by the action of triphenylphosphine and carbon
tetrachloride, carbon tetrabromide, or the like. Alternatively, a
method may be used in which a halogenated alkyl form is synthesized
through two-step reaction such that an alcohol is converted into a
sulfate and then reacted with lithium bromide, lithium chloride, or
sodium iodide.
[0142] Examples of reagents to be used when the Arbuzov reaction is
performed in each step include halogenated alkyls such as
bromoethyl acetate; and phosphites such as triethylphosphite and
tri(isopropyl)phosphite.
[0143] Examples of sulfonating agents to be used when sulfonation
reaction is performed in each step include methanesulfonyl
chloride, p-toluenesulfonyl chloride, methanesulfonic anhydride,
p-toluenesulfonic anhydride, and trifluoromethanesulfonic
anhydride.
[0144] When hydrolysis reaction is performed in each step, an acid
or a base is used as a reagent. When acid hydrolysis reaction is
performed for a t-butyl ester, formic acid or triethylsilane is
added in some cases to reductively trap t-butyl cations produced as
byproducts.
[0145] Examples of dehydrating agents to be used when dehydration
reaction is performed in each step include sulfuric acid,
diphosphorus pentoxide, phosphorus oxychloride,
N,N'-dicyclohexylcarbodiimide, alumina, and polyphosphoric
acid.
[0146] Compound (I) may be produced, for example, by using a
production method shown below. Among the compounds (I), a compound
in which each of the wavy lines forms a cis-structure and a
compound in which one or both of the wave lines forms a
trans-structure can be both produced by using the same production
method as the production method shown below. In the present
invention, compound (I) with a desired structure can be synthesized
by using an appropriate raw material for the intended structure of
compound (I) particularly in esterification. A salt of compound (I)
can be obtained through appropriate mixing with an inorganic base,
an organic base, an organic acid, or a basic or acidic amino
acid.
##STR00005## ##STR00006##
[0147] Now, a method for producing a lipid particle containing the
compound of the present invention and that for producing a
composition containing the lipid particle and a nucleic acid for
nucleic acid introduction will be described.
[0148] The lipid particle of the present invention can be produced
by mixing the compound of the present invention (cationic lipid)
and, as necessary, an additional lipid component, and then applying
a known method to prepare a lipid particle from a lipid component.
For example, the lipid particle can be produced as a lipid particle
dispersion by dissolving the (mixed) lipid component in an organic
solvent and mixing the resulting organic solvent solution with
water or a buffer (e.g., through an emulsifying method). The mixing
may be performed by using a microfluid mixing system (e.g., the
apparatus NanoAssemblr (Precision NanoSystems)). The lipid particle
obtained may be subjected to desalting or dialysis and sterile
filtration. As necessary, pH adjustment or osmotic pressure
adjustment may be performed.
[0149] Compound (I) can form different structures depending on
combination of the definitions of n, R, and the wavy lines of
formula (I). To produce the lipid particle, one compound having a
specific structure may be used alone as compound (I), and a mixture
of a plurality of compounds of different structures may be used as
compound (I).
[0150] Examples of the "additional lipid component" include the
above-mentioned structural lipids such as sterols, phospholipids,
and polyethylene glycol lipids. The "additional lipid component" is
used, for example, in an amount of 0.008 to 4 mol per mole of the
compound of the present invention. The compound of the present
invention is preferably used as a mixture with the additional lipid
component (in particular, cholesterol, phosphatidylcholine, and a
polyethylene glycol lipid). In a preferred embodiment using a
mixture of the compound of the present invention and the additional
lipid component, the mixture is a mixture of 1 to 4 mol of the
compound of the present invention, 0 to 3 mol of a sterol, 0 to 2
mol of a phospholipid, and 0 to 1 mol of a polyethylene glycol
lipid. In a more preferred embodiment using a mixture of the
compound of the present invention and the additional lipid
component, the mixture is a mixture of 1 to 1.5 mol of the compound
of the present invention, 0 to 1.25 mol of a sterol, 0 to 0.5 mol
of a phospholipid, and 0 to 0.125 mol of a polyethylene glycol
lipid.
[0151] The concentration of the compound of the present invention
or the mixture of the compound of the present invention and the
additional lipid component in the organic solvent solution is
preferably 0.5 to 100 mg/mL.
[0152] Examples of the organic solvent include methanol, ethanol,
1-propanol, 2-propanol, 1-butanol, tert-butanol, acetone,
acetonitrile, N,N-dimethylformamide, dimethylsulfoxide, and
mixtures of them. The organic solvent may contain 0 to 20% of water
or a buffer.
[0153] Examples of the buffer include acidic buffers (e.g., acetate
buffer, citrate buffer, 2-morpholinoethanesulfonate (MES) buffer,
phosphate buffer), and neutral buffers (e.g.,
4-(2-hydroxyethyl)-1-piperazineethanesulfonate (HEPES) buffer,
tris(hydroxymethyl)aminomethane (Tris) buffer, phosphate buffer,
phosphate-buffered saline (PBS)).
[0154] If mixing is performed by using a microfluid mixing system,
it is preferred to mix 1 to 5 parts by volume of water or the
buffer per part by volume of the organic solvent solution. The flow
rate of the mixed solution (mixed solution of the organic solvent
solution and water or the buffer) in the system is preferably 0.1
to 10 mL/min, and the temperature is preferably 15 to 45.degree.
C.
[0155] The composition of the present invention can be produced as
a lipid particle dispersion containing an active ingredient by
adding in advance a nucleic acid as an active ingredient to water
or the buffer in production of the lipid particle or a lipid
particle dispersion. The active ingredient is preferably added so
that the active ingredient concentration of water or the buffer
reaches 0.05 to 2.0 mg/mL.
[0156] In addition, the composition of the present invention can be
produced as a lipid particle dispersion containing an active
ingredient by admixing the lipid particle or a lipid particle
dispersion and an active ingredient or an aqueous solution of the
active ingredient through a known method. The lipid particle
dispersion can be prepared by dispersing the lipid particle in an
appropriate dispersion medium. The aqueous solution of the active
ingredient can be prepared by dissolving the active ingredient in
an appropriate solvent.
[0157] The content of the compound of the present invention in the
composition of the present invention with the dispersion medium and
solvent excluded is preferably 40 to 70% by weight.
[0158] The content of the active ingredient in the composition of
the present invention with the dispersion medium and solvent
excluded is preferably 1 to 20% by weight.
[0159] The dispersion medium of the lipid particle dispersion or
the dispersion containing the composition can be replaced with
water or a buffer through dialysis. The dialysis is performed with
an ultrafiltration membrane having a molecular weight cutoff of 10
to 20K at 4.degree. C. to room temperature. The dialysis may be
repeatedly performed. For replacement of the dispersion medium,
tangential flow filtration (TFF) may be used. After replacement of
the dispersion medium, pH adjustment or osmotic pressure adjustment
may be performed, as necessary.
[0160] Now, methods for analyzing a lipid particle containing the
compound of the present invention, and a composition containing the
lipid particle and a nucleic acid as an active ingredient will be
described.
[0161] The particle size of the lipid particle (in the composition)
can be measured by using a known means. For example, a Zetasizer
Nano ZS (Malvern Instruments Limited), a particle size analyzer
based on an NIBS (non-invasive backscatter) technique, can be used
to calculate the particle size as a z-average particle size through
cumulant analysis of the autocorrelation function. The particle
size (average particle size) of the lipid particle (in the
composition) is preferably 10 to 200 nm.
[0162] The concentration and encapsulation ratio of a nucleic acid
(e.g., an siRNA, an mRNA) as an active ingredient in the
composition of the present invention can be measured by using a
known means. For example, after the nucleic acid is
fluorescence-labeled with Quant-iT (TM) RiboGreen (R) (Invitrogen),
the concentration and the encapsulation ratio can be determined by
measuring the fluorescence intensity. The concentration of the
nucleic acid in the composition can be calculated by using a
standard curve prepared from aqueous solutions of the nucleic acid
with known concentrations, and the encapsulation ratio can be
calculated on the basis of difference in fluorescence intensity
depending on the presence or absence of addition of Triton-X100 (a
surfactant to disintegrate the lipid particle). The concentration
of the nucleic acid in the composition refers to the total
concentration of molecules of the nucleic acid encapsulated in the
lipid particle and molecules of the nucleic acid not encapsulated
in the lipid particle, and the encapsulation ratio refers to the
fraction of molecules of the nucleic acid encapsulated in the lipid
particle to all the molecules of the nucleic acid in the
composition.
EXAMPLES
[0163] The present invention will be further described in detail
with reference to Examples, Test Examples, and Formulation
Examples; however, these do not limit the present invention, and
modification may be made without departing from the scope of the
present invention.
[0164] "Room temperature" in Examples below typically indicates
approximately 10.degree. C. to approximately 35.degree. C. Each
ratio shown for mixed solvent indicates a volume ratio, unless
otherwise stated. % indicates % by weight, unless otherwise
stated.
[0165] Elution in column chromatography was performed under
observation with TLC (thin-layer chromatography), unless otherwise
described. In TLC observation, a 60 F.sub.254 produced by Merck
KGaA was used as a TLC plate, and a solvent used as an elution
solvent in column chromatography was used as an eluent. A UV
detector was employed for detection, and observation was performed
with a TLC coloring reagent, as necessary. In description of silica
gel column chromatography, NH indicates that
aminopropylsilane-bonded silica gel was used, and Diol indicates
that 3-(2,3-dihydroxypropoxy)propylsilane-bonded silica gel was
used. In description of preparative HPLC (high-performance liquid
chromatography), C18 indicates that octadecyl-bonded silica gel was
used. Each ratio shown for elution solvent indicates a volume
ratio, unless otherwise stated.
[0166] .sup.1H NMR was measured by using a Fourie
transformation-NMR. The software ACD/SpecManager (product name) and
so on were used for .sup.1H NMR analysis. Description is
occasionally omitted for peaks for a hydroxy group, an amino group,
and so on with a very broad proton peak.
[0167] MS was measured through an LC/MS and an MALDI/TOFMS. For the
ionization method, an ESI method, an APCI method, or an MALDI
method was used. CHCA was used for the matrix. Measured values
(found) were reported as data. In typical cases, some molecular ion
peaks are observed as fragment ions. In the case of a salt, a
molecular ion peak for the free form, or cationic, anionic, or
fragment ion peaks are typically observed.
[0168] In Examples below, the following abbreviations are used.
MS: Mass spectrum M: Molar concentration
N: Normality
[0169] CDCl.sub.3: Deuterated chloroform DMSO-d.sub.6: Deuterated
dimethylsulfoxide .sup.1H NMR: Proton nuclear magnetic resonance
LC/MS: Liquid chromatograph/mass spectrometer ESI: Electrospray
ionization APCI: Atmospheric pressure chemical ionization MALDI:
Matrix-assisted laser desorption/ionization TOFMS: Time-of-flight
mass spectrometry CHCA: .alpha.-Cyano-4-hydroxycinnamic acid
DMF: N,N-dimethylformamide
THF: Tetrahydrofuran
DMAP: 4-Dimethylaminopyridine
[0170] TBAF: Tetrabutylammonium fluoride
[Example 1]
3-((4-(Dimethylamino)butanoyl)oxy)-2,2-bis(((9Z)-tetradec-9-enoyloxy)meth-
yl)propyl (9Z)-tetradec-9-enoate
A) 2-(((tert-Butyl dim ethyl
silyl)oxy)methyl)-2-(hydroxymethyl)propane-1,3-diol
[0171] To a mixture of 2,2-bis(hydroxymethyl)propane-1,3-diol (5.45
g), 1H-imidazole (2.72 g) and DMF (190 mL), a solution of
tert-butylchlorodimethylsilane (3.01 g) in DMF (10 mL) was added at
room temperature. After stirring for 24 hours, the reaction mixture
was concentrated under reduced pressure. The residue was diluted
with ethyl acetate, washed three times with water and once with
saturated brine, and then dried over anhydrous sodium sulfate, and
the solvent was distilled off under reduced pressure. The residue
was purified by silica gel column chromatography (ethyl
acetate/hexane) to afford the title compound (2.25 g).
[0172] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. ppm 0.08 (6H, s),
0.90 (9H, s), 2.53 (3H, t, J=5.5 Hz), 3.66 (2H, s), 3.73 (6H, d,
J=5.5 Hz)
B)
3-((tert-Butyl(dimethyl)silyl)oxy)-2,2-bis(((9Z)-tetradec-9-enoyloxy)me-
thyl)propyl (9Z)-tetradec-9-enoate
[0173] To a solution of
2-(((tert-butyldimethylsilyl)oxy)methyl)-2-(hydroxymethyl)propane-1,3-dio-
l (258 mg), (9Z)-tetradec-9-enoic acid (769 mg) and DMAP (126 mg)
in DMF (3 mL), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride (790 mg) was added at room temperature. After
stirring for 18 hours, the reaction mixture was diluted with ethyl
acetate, washed twice with water and once with saturated brine, and
then dried over anhydrous sodium sulfate, and the solvent was
distilled off under reduced pressure. The residue was purified by
silica gel column chromatography (NH, ethyl acetate/hexane) to
afford the title compound (860 mg).
[0174] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. ppm 0.03 (6H, s),
0.81-0.96 (18H, m), 1.18-1.41 (36H, m), 1.53-1.67 (6H, m),
1.91-2.10 (12H, m), 2.29 (6H, t, J=7.6 Hz), 3.58 (2H, s), 4.08 (6H,
s), 5.27-5.43 (6H, m)
C) 3-Hydroxy-2,2-bis(((9Z)-tetradec-9-enoyloxy)methyl)propyl
(9Z)-tetradec-9-enoate
[0175] To a solution of
3-((tert-butyl(dimethyl)silyl)oxy)-2,2-bis(((9Z)-tetradec-9-enoyloxy)meth-
yl)propyl (9Z)-tetradec-9-enoate (5.91 g) in THF (120 mL), a
mixture of a THF solution of TBAF (1 M, 14.85 mL) and acetic acid
(4.91 mL) was added at room temperature. After stirring for 3 days,
the reaction mixture was concentrated under reduced pressure. The
residue was diluted with ethyl acetate, washed once with saturated
aqueous solution of sodium hydrogen carbonate and once with
saturated brine, and then dried over anhydrous sodium sulfate, and
the solvent was distilled off under reduced pressure. The residue
was purified by silica gel column chromatography (ethyl
acetate/hexane) to afford the title compound (4.96 g).
[0176] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. ppm 0.82-0.97 (9H,
m), 1.16-1.42 (36H, m), 1.52-1.68 (6H, m), 1.90-2.12 (12H, m), 2.32
(6H, t, J=7.6 Hz), 2.52 (1H, t, J=7.0 Hz), 3.49 (2H, d, J=7.0 Hz),
4.11 (6H, s), 5.26-5.42 (6H, m)
D)
3-((4-(Dimethylamino)butanoyl)oxy)-2,2-bis(((9Z)-tetradec-9-enoyloxy)me-
thyl)propyl (9Z)-tetradec-9-enoate
[0177] To a solution of
3-hydroxy-2,2-bis(((9Z)-tetradec-9-enoyloxy)methyl)propyl
(9Z)-tetradec-9-enoate (4.96 g), DMAP (796 mg) and
4-(dimethylamino)butanoic acid hydrochloride (2.19 g) in DMF (20
mL), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
(2.50 g) was added at room temperature. After stirring for 18
hours, the reaction mixture was diluted with ethyl acetate, washed
once with saturated aqueous solution of sodium hydrogen carbonate
and once with saturated brine, and then dried over anhydrous sodium
sulfate, and the solvent was distilled off under reduced pressure.
The residue was purified by silica gel column chromatography (NH,
ethyl acetate/hexane) to afford the title compound (5.31 g).
[0178] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. ppm 0.82-0.94 (9H,
m), 1.20-1.42 (36H, m), 1.50-1.66 (6H, m), 1.69-1.83 (2H, m),
1.90-2.10 (12H, m), 2.20 (6H, s), 2.23-2.41 (10H, m), 4.11 (8H, s),
5.23-5.44 (6H, m)
[Example 4]
3-((4-(Dimethylamino)butanoyl)oxy)-2,2-bis(((9Z)-tetradec-9-enoyloxy)meth-
yl)propyl (9Z)-hexadec-9-enoate
A)
2-(((tert-Butyl(diphenyl)silyl)oxy)methyl)-2-(hydroxymethyl)propane-1,3-
-diol
[0179] To a mixture of 2,2-bis(hydroxymethyl)propane-1,3-diol (5.0
g), 1H-imidazole (2.5 g) and DMF (200 mL), a solution of
tert-butylchlorodiphenylsilane (5.1 g) in DMF (10 mL) was added at
room temperature. After stirring for 18 hours, the reaction mixture
was concentrated under reduced pressure. The residue was diluted
with ethyl acetate, washed three times with water and once with
saturated brine, and dried over anhydrous sodium sulfate, and the
solvent was distilled off under reduced pressure. The residue was
purified by silica gel column chromatography (ethyl acetate/hexane)
to afford the title compound (6.4 g).
[0180] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. ppm 1.07 (9H, s),
2.34 (3H, t, J=5.5 Hz), 3.67 (2H, s), 3.74 (6H, d, J=5.5 Hz),
7.39-7.48 (6H, m), 7.63-7.67 (4H, m)
B)
(5-(((tert-Butyl(diphenyl)silyl)oxy)methyl)-2,2-dimethyl-1,3-dioxan-5-y-
l)methanol
[0181] To a solution of
2-(((tert-butyl(diphenyl)silyl)oxy)methyl)-2-(hydroxymethyl)propane-1,3-d-
iol (3.5 g) and 2,2-dimethoxypropane (1.5 g) in acetone (35 mL),
p-toluenesulfonic acid monohydrate (88.9 mg) was added at room
temperature. After stirring for 2 hours, diluted aqueous ammonia
was added to the reaction mixture to neutralize the reaction
mixture, and the solvent was then distilled off under reduced
pressure. The residue was purified by silica gel column
chromatography (ethyl acetate/hexane) to afford the title compound
(2.7 g).
[0182] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. ppm 1.07 (9H, s),
1.27 (3H, s), 1.41 (3H, s), 2.12-2.18 (1H, m), 3.69-3.78 (8H, m),
7.38-7.47 (6H, m), 7.65-7.69 (4H, m)
C)
(5-(((tert-Butyl(diphenyl)silyl)oxy)methyl)-2,2-dimethyl-1,3-dioxan-5-y-
l)methyl (9Z)-hexadec-9-enoate
[0183] To a solution of
(5-(((tert-butyl(diphenyl)silyl)oxy)methyl)-2,2-dimethyl-1,3-dioxan-5-yl)-
methanol (910 mg), DMAP (215 mg) and (9Z)-hexadec-9-enoic acid (838
mg) in DMF (10 mL), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride (757 mg) was added at room temperature. After
stirring for 6 hours, ethyl acetate was added to the reaction
mixture, the resultant was washed twice with water and once with
saturated brine, and then dried over anhydrous sodium sulfate, and
the solvent was distilled off under reduced pressure. The residue
was purified by silica gel column chromatography (ethyl
acetate/hexane) to afford the title compound (1.43 g).
[0184] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. ppm 0.84-0.91 (3H,
m), 1.03-1.07 (9H, m), 1.22-1.35 (16H, m), 1.40 (6H, d, J=17.0 Hz),
1.49-1.63 (2H, m), 2.01 (4H, q, J=6.5 Hz), 2.24 (2H, t, J=7.6 Hz),
3.65 (2H, s), 3.73 (2H, d, J=11.7 Hz), 3.80 (2H, d, J=12.0 Hz),
4.17(2H, s), 5.29-5.39 (2H, m), 7.35-7.46 (6H, m), 7.65 (4H, d,
J=6.9 Hz)
D) (5-(Hydroxymethyl)-2,2-dimethyl-1,3-dioxan-5-yl)methyl
(9Z)-hexadec-9-enoate
[0185] To a solution of
(5-(((tert-butyl(diphenyl)silyl)oxy)methyl)-2,2-dimethyl-1,3-dioxan-5-yl)-
methyl (9Z)-hexadec-9-enoate (1.43 g) in THF (4 mL), a THF solution
of TBAF (1 M, 2.64 mL) was added at room temperature. After
stirring for 3 hours, the reaction mixture was concentrated under
reduced pressure. The residue was diluted with ethyl acetate,
washed once with saturated aqueous solution of sodium hydrogen
carbonate and once with saturated brine, and then dried over
anhydrous sodium sulfate, and the solvent was distilled off under
reduced pressure. The residue was purified by silica gel column
chromatography (ethyl acetate/hexane) to afford the title compound
(0.82 g).
[0186] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. ppm 0.85-0.91 (3H,
m), 1.24-1.36 (16H, m), 1.42 (6H, s), 1.58-1.66 (2H, m), 2.01 (4H,
q, J=6.5 Hz), 2.30 (1H, t, J=6.6 Hz), 2.35 (2H, t, J=7.6 Hz), 3.48
(2H, d, J=6.6 Hz), 3.69-3.75 (4H, m), 4.25 (2H, s), 5.31-5.38 (2H,
m)
E)
(5-(((4-(Dimethylamino)butanoyl)oxy)methyl)-2,2-dimethyl-1,3-dioxan-5-y-
l)methyl (9Z)-hexadec-9-enoate
[0187] To a solution of
(5-(hydroxymethyl)-2,2-dimethyl-1,3-dioxan-5-yl)methyl
(9Z)-hexadec-9-enoate (410 mg), DMAP (97 mg) and
4-(dimethylamino)butyric acid hydrochloride (250 mg) in DMF (4 mL),
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (343
mg) was added at 50.degree. C. After stirring for 4 hours, ethyl
acetate was added to the reaction mixture, the resultant was washed
twice with water and once with saturated brine, and then dried over
anhydrous sodium sulfate, and the solvent was then distilled off
under reduced pressure. The residue was purified by silica gel
column chromatography (NH, ethyl acetate/hexane) to afford the
title compound (430 mg).
[0188] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. ppm 0.86-0.91 (3H,
m), 1.24-1.36 (16H, m), 1.42 (6H, s), 1.53-1.69 (2H, m), 1.78 (2H,
m), 1.98-2.04 (4H, m), 2.21 (6H, s), 2.29 (4H, m), 2.37 (2H, t,
J=7.6 Hz), 3.74 (4H, s), 4.11 (4H, d, J=5.7 Hz), 5.31-5.38 (2H,
m)
F)
3-((4-(Dimethylamino)butanoyl)oxy)-2,2-bis(((9Z)-tetradec-9-enoyloxy)me-
thyl)propyl (9Z)-hexadec-9-enoate
[0189] To
(5-(((4-(dimethylamino)butanoyl)oxy)methyl)-2,2-dimethyl-1,3-dio-
xan-5-yl)methyl (9Z)-hexadec-9-enoate (430 mg), acetic acid (2 mL)
and water (1 mL) were added, and the resultant was stirred at
75.degree. C. for 2 hours, and the solvent was then distilled off
under reduced pressure. Ethyl acetate and saturated aqueous
solution of sodium hydrogen carbonate were added to the residue,
and the resultant was stirred for 2 hours. After washing with water
was performed twice, the resultant was dried over anhydrous sodium
sulfate, and the solvent was then distilled off under reduced
pressure. To a solution of the resulting residue, DMAP (201 mg) and
(9Z)-tetradec-9-enoic acid (466 mg) in DMF (4 mL),
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (458
mg) was added at 50.degree. C. After stirring for 4 hours, ethyl
acetate was added to the reaction mixture, the resultant was washed
twice with water and once with saturated brine, and then dried over
anhydrous sodium sulfate, and the solvent was then distilled off
under reduced pressure. The residue was purified by silica gel
column chromatography (NH, ethyl acetate/hexane) to afford the
title compound (604 mg).
[0190] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. ppm 0.85-0.92 (9H,
m), 1.24-1.36 (40H, m), 1.55-1.64 (6H, m), 1.73-1.80 (2H, m),
1.98-2.05 (12H, m), 2.20 (6H, s), 2.24-2.33 (8H, m), 2.36 (2H, t,
J=7.6 Hz), 4.09-4.13 (8H, m), 5.31-5.38 (6H, m)
[Example 8]
2-(((N,N-Dimethyl-.beta.-alanyl)oxy)methyl)-2-((octanoyloxy)methyl)propan-
e-1,3-diyl (9Z,9'Z)bis-tetradec-9-enoate
A) (2-(4-Methoxyphenyl)-1,3-dioxane-5,5-diyl)dimethanol
[0191] A solution of 2,2-bis(hydroxymethyl)propane-1,3-diol (506 g)
in water (2.0 L) was stirred at 50.degree. C. Concentrated
hydrochloric acid (18 mL) was added thereto, to which
p-methoxybenzaldehyde (474 mL) was added dropwise at around
30.degree. C. over 3 hours. Thereafter, the temperature of the
reaction solution was adjusted to 25.degree. C., and the reaction
solution was stirred for 5 hours. Thereto, 2 N aqueous solution of
sodium hydroxide (120 mL) was added, and the resultant was stirred
for 1 hour. Crystals were collected through filtration and washed
with water, and then recrystallization was performed with ethyl
acetate/hexane to afford the title compound (769 g).
[0192] .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta. ppm 3.24 (2H, d,
J=5.0 Hz), 3.67 (2H, d, J=5.4 Hz), 3.74 (3H, s), 3.77 (2H, d,
J=11.3 Hz), 3.88 (2H, t, J=11.3 Hz), 4.53 (1H, t, J=5.4 Hz), 4.62
(1H, t, J=5.0 Hz), 5.34 (1H, s), 6.90 (2H, d, J=8.9 Hz), 7.33 (2H,
d, J=8.9 Hz)
B-1)
2-(Hydroxymethyl)-2-(((4-methoxybenzyl)oxy)methyl)propane-1,3-diol
[0193] To a suspension solution of
(2-(4-methoxyphenyl)-1,3-dioxane-5,5-diyl)dimethanol (10.0 g) in
toluene (100 mL), 1.5 M DIBAL-H solution (105 mL) was added
dropwise at room temperature, and the resultant was stirred for 5
hours. Methanol (30 mL) was added thereto, and 2 N hydrochloric
acid (20 mL) and 4 N aqueous solution of sodium hydroxide (240 mL)
were then added thereto and the resultant was stirred for 2 hours,
and thereafter the toluene layer was removed. After the aqueous
layer was neutralized with hydrochloric acid, extraction was
performed with ethyl acetate, and the extract was washed twice with
saturated brine and filtered through a Celite. The solvent was
distilled off under reduced pressure, and the residue was
recrystallized with ethyl acetate/hexane to afford the title
compound (6.6 g).
[0194] .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta. ppm 3.32 (2H,
s), 3.39 (6H, d, J=5.4 Hz), 3.74 (3H, s), 4.21 (3H, t, J=5.4 Hz),
4.36 (2H, s), 6.90 (2H, d like, J=7.8 Hz), 7.23 (2H, d like, J=7.8
Hz)
C-1)
(5-(((4-Methoxybenzyl)oxy)methyl)-2,2-dimethyl-1,3-dioxan-5-yl)methan-
ol
[0195] To a solution of
2-(hydroxymethyl)-2-(((4-methoxybenzyl)oxy)methyl)propane-1,3-diol
(1.00 g) and 2,2-dimethoxypropane (1.22 g) in DMF (5 mL),
pyridinium p-toluenesulfonate (10 mg) was added at room
temperature. After stirring for 2 hours, ethyl acetate was added to
the reaction mixture, the resultant was washed once with saturated
aqueous solution of sodium hydrogen carbonate and twice with 5%
saline, and then dried over anhydrous sodium sulfate, and the
solvent was distilled off under reduced pressure. The residue was
purified by silica gel column chromatography (ethyl acetate/hexane)
to afford the title compound (426 mg).
[0196] .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta. ppm 1.29 (3H,
s), 1.29 (3H, s), 3.35 (2H, s), 3.39 (2H, d, J=5.1 Hz), 3.61 (4H,
s), 3.74 (3H, s), 4.38 (2H, s), 4.59 (1H, t, J=5.1 Hz), 6.90 (2H, d
like, J=7.5 Hz), 7.24 (2H, d like, J=7.5 Hz)
B-2)
9-(4-Methoxyphenyl)-3,3-dimethyl-2,4,8,10-tetraoxaspiro[5.5]undecane
[0197] To a solution of
(2-(4-methoxyphenyl)-1,3-dioxane-5,5-diyl)dimethanol (2.00 g) and
2,2-dimethoxypropane (2.46 g) in DMF (8 mL), pyridinium
p-toluenesulfonate (20 mg) was added at room temperature. After
stirring for 4 hours, the reaction mixture was diluted with ethyl
acetate, washed twice with saturated aqueous solution of sodium
hydrogen carbonate and twice with saturated brine, and then dried
over anhydrous magnesium sulfate, and the solvent was then
distilled off under reduced pressure. The residue was
recrystallized with ethyl acetate/hexane to afford the title
compound (1.62 g).
[0198] .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta. ppm 1.34 (6H,
s), 3.33 (2H, s), 3.63 (2H, d, J=11.7 Hz), 3.74 (3H, s), 3.99 (2H,
s), 4.12 (2H, d, J=11.7 Hz), 5.37 (1H, s), 6.90 (2H, d, J=8.8 Hz),
7.34 (2H, d, J=8.8 Hz)
C-2)
(5-(((4-Methoxybenzyl)oxy)methyl)-2,2-dimethyl-1,3-dioxan-5-yl)methan-
ol
[0199] To a suspension solution of
9-(4-methoxyphenyl)-3,3-dimethyl-2,4,8,10-tetraoxaspiro[5.5]undecane
(22.0 g) in toluene (200 mL), 1.5 M DIBAL-H solution (60 mL) was
added dropwise at 5 to 20.degree. C., and the resultant was stirred
at 15.degree. C. for 3 hours. Methanol (22 mL) was added thereto,
and 2 N aqueous solution of sodium hydroxide (100 mL) and 4 N
aqueous solution of sodium hydroxide (200 mL) were then added
dropwise thereto in the order presented. After stirring for 1.5
hours, the toluene layer was separated and washed with 5% saline.
The solvent was distilled off under reduced pressure. The residue
was purified by silica gel column chromatography (ethyl
acetate/hexane) to afford the title compound (14.7 g).
[0200] .sup.1H NMR (500 MHz, DMSO-d6) .delta. ppm 1.29 (3H, s),
1.29 (3H, s), 3.35 (2H, s), 3.39 (2H, d, J=5.1 Hz), 3.61 (4H, s),
3.74 (3H, s), 4.38 (2H, s), 4.59 (1H, t, J=5.1 Hz), 6.90 (2H, d
like, J=7.5 Hz), 7.24 (2H, d like, J=7.5 Hz)
D)
(5-(((4-Methoxybenzyl)oxy)methyl)-2,2-dimethyl-1,3-dioxan-5-yl)methyl
octanoate
[0201] To a solution of
(5-(((4-methoxybenzyl)oxy)methyl)-2,2-dimethyl-1,3-dioxan-5-yl)methanol
(2.00 g), DMAP (412 mg) and octanoic acid (1.27 g) in DMF (20 mL),
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.94
g) was added at 50.degree. C. After stirring for 4 hours, ethyl
acetate was added to the reaction mixture, the resultant was washed
twice with water and once with saturated brine, and then dried over
anhydrous sodium sulfate, and the solvent was then distilled off
under reduced pressure. The residue was purified by silica gel
column chromatography (ethyl acetate/hexane) to afford the title
compound (2.78 g).
[0202] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 0.84-0.91 (3H, m),
1.22-1.33 (8H, m), 1.40 (6H, s), 1.53-1.61 (2H, m), 2.26 (2H, t,
J=7.6 Hz), 3.39 (2H, s), 3.68-3.74 (2H, m), 3.76-3.80 (2H, m), 3.80
(3H, s), 4.15 (2H, s), 4.42 (2H, s), 6.87 (2H, d, J=7.8 Hz),
7.20-7.24 (2H, m)
E) (5-(Hydroxymethyl)-2,2-dimethyl-1,3-dioxan-5-yl)methyl
octanoate
[0203] To a solution of
(5-(((4-methoxybenzyl)oxy)methyl)-2,2-dimethyl-1,3-dioxan-5-yl)methyl
octanoate (2.78 g) in ethanol (30 mL), Pd-carbon (840 mg) was added
at room temperature, and the resultant was stirred under a hydrogen
atmosphere for 5 hours. After the reaction, Pd-carbon was removed
through filtration, and the solvent was then distilled off under
reduced pressure. The residue was purified by silica gel column
chromatography (ethyl acetate/hexane) to afford the title compound
(1.35 g).
[0204] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 0.85-0.91 (3H, m),
1.23-1.34 (8H, m), 1.38-1.44 (6H, m), 1.63 (2H, m), 2.30-2.38 (3H,
m), 3.48 (2H, d, J=6.6 Hz), 3.68-3.75 (4H, m), 4.25 (2H, s)
F)
(5-(((N,N-Dimethyl-.beta.-alanyl)oxy)methyl)-2,2-dimethyl-1,3-dioxan-5--
yl)methyl octanoate
[0205] To a solution of
(5-(hydroxymethyl)-2,2-dimethyl-1,3-dioxan-5-yl)methyl octanoate
(400 mg), DMAP (129 mg) and 3-(dimethylamino)propanoic acid
hydrochloride (305 mg) in DMF (4 mL),
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (456
mg) was added at room temperature. After stirring for 4 hours,
ethyl acetate was added to the reaction mixture, the resultant was
washed twice with water and once with saturated brine, and then
dried over anhydrous sodium sulfate, and the solvent was then
distilled off under reduced pressure. The residue was purified by
silica gel column chromatography (NH, ethyl acetate/hexane) to
afford the title compound (320 mg).
[0206] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 0.84-0.92 (3H, m),
1.21-1.33 (8H, m), 1.42 (6H, s), 1.61 (2H, br), 2.23 (6H, s), 2.32
(2H, t, J=7.6 Hz), 2.47-2.51 (2H, m), 2.57-2.61 (2H, m), 3.75 (4H,
s), 4.13 (4H, d, J=11.7 Hz)
G)
2-(((N,N-Dimethyl-.beta.-alanyl)oxy)methyl)-2-((octanoyloxy)methyl)prop-
ane-1,3-diyl (9Z,9'Z)bis-tetradec-9-enoate
[0207] To
(5-(((N,N-dimethyl-.beta.-alanyl)oxy)methyl)-2,2-dimethyl-1,3-di-
oxan-5-yl)methyl octanoate (320 mg), acetic acid (1.6 mL) and water
(0.8 mL) were added, and the resultant was stirred at 65.degree. C.
for 3.5 hours, and the solvent was then distilled off under reduced
pressure. Ethyl acetate and saturated aqueous solution of sodium
hydrogen carbonate were added to the residue, and the resultant was
stirred for 2 hours. After washing was performed twice with water
and once with saturated brine, the resultant was dried over
anhydrous sodium sulfate, and the solvent was then distilled off
under reduced pressure. To a solution of the resulting residue (150
mg), DMAP (101 mg), and (9Z)-tetradec-9-enoic acid (235 mg) in DMF
(4.5 mL), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride (231 mg) was added at 50.degree. C. After stirring
for 8 hours, ethyl acetate was added to the reaction mixture, the
resultant was washed twice with water and once with saturated
brine, and then dried over anhydrous sodium sulfate, and the
solvent was then distilled off under reduced pressure. The residue
was purified by silica gel column chromatography (NH, ethyl
acetate/hexane) to afford the title compound (230 mg).
[0208] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 0.84-0.93 (9H, m),
1.24-1.36 (30H, m), 1.53-1.65 (8H, m), 1.98-2.05 (8H, m), 2.22 (6H,
s), 2.30 (6H, t, J=7.6 Hz), 2.48 (2H, t, J=6.9 Hz), 2.58 (2H, t,
J=6.8 Hz), 4.06-4.22 (8H, m), 5.31-5.38 (4H, m)
[Example 11]
2-(((4-(Dimethylamino)butanoyl)oxy)methyl)-2-((dodecanoyloxy)methyl)propa-
ne-1,3-diyl (9Z,9'Z)bis-tetradec-9-enoate
A) 3-Hydroxy-2,2-bis(hydroxymethyl)propyl dodecanoate
[0209] To a solution of 2,2-bis(hydroxymethyl)propane-1,3-diol
(5.00 g), DMAP (2.24 g) and lauric acid (3.68 g) in DMF (150 mL),
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (7.04
g) was added at room temperature. After stirring for 20 hours,
ethyl acetate was added to the reaction mixture, the resultant was
washed twice with water and once with saturated brine, and then
dried over anhydrous sodium sulfate, and the solvent was then
distilled off under reduced pressure. The residue was purified by
silica gel column chromatography (ethyl acetate/hexane) to afford
the title compound (1.79 g).
[0210] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 0.85-0.91 (3H, m),
1.22-1.33 (16H, m), 1.59-1.66 (3H, m), 2.36 (2H, t, J=7.6 Hz), 2.51
(2H, t, J=5.8 Hz), 3.65 (6H, d, J=5.7 Hz), 4.23 (2H, s)
B) 2-((Dodecanoyloxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl
(9Z,9'Z)bis-tetradec-9-enoate
[0211] To a solution of 3-hydroxy-2,2-bis(hydroxymethyl)propyl
dodecanoate (400 mg), DMAP (153 mg) and (9Z)-tetradec-9-enoic acid
(569 mg) in DMF (4 mL),
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (602
mg) was added at room temperature. After stirring for 8 hours,
ethyl acetate was added to the reaction mixture, the resultant was
washed twice with water and once with saturated brine, and then
dried over anhydrous sodium sulfate, and the solvent was then
distilled off under reduced pressure. The residue was purified by
silica gel column chromatography (ethyl acetate/hexane) to afford
the title compound (230 mg).
[0212] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 0.85-0.93 (9H, m),
1.22-1.36 (40H, m), 1.58-1.65 (6H, m), 1.99-2.05 (8H, m), 2.32 (6H,
t, J=7.6 Hz), 2.52 (1H, t, J=7.1 Hz), 3.48 (2H, d, J=6.9 Hz),
4.09-4.14 (6H, m), 5.31-5.38 (4H, m)
C)
2-(((4-(Dimethylamino)butanoyl)oxy)methyl)-2-((dodecanoyloxy)methyl)pro-
pane-1,3-diyl (9Z,9'Z)bis-tetradec-9-enoate
[0213] To a solution of
2-((dodecanoyloxy)methyl)-2-(hydroxymethyl)propane-1,3-diyl
(9Z,9'Z)bis-tetradec-9-enoate (230 mg), DMAP (38 mg) and
4-(dimethylamino)butyric acid hydrochloride (105 mg) in DMF (4 mL),
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (150
mg) was added at room temperature. After stirring for 3 hours,
ethyl acetate was added to the reaction mixture, the resultant was
washed twice with water and once with saturated brine, and then
dried over anhydrous sodium sulfate, and the solvent was then
distilled off under reduced pressure. The residue was purified by
silica gel column chromatography (NH, ethyl acetate/hexane) to
afford the title compound (100 mg).
[0214] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 0.85-0.93 (9H, m),
1.23-1.35 (40H, m), 1.55-1.67 (6H, m), 1.77 (2H, m), 1.98-2.05 (8H,
m), 2.20 (6H, s), 2.24-2.33 (8H, m), 2.36 (2H, t, J=7.6 Hz),
4.09-4.13 (8H, m), 5.31-5.38 (4H, m)
[0215] Examples 2 and 3, 5 to 7, and 9 and 10 in a table below were
produced in accordance with any of the methods shown in Examples
above and methods conforming thereto. Table 1 shows compound names,
structures, and mass numbers observed in production (indicated as
MS in the table) for those Examples together with Examples 1, 4, 8,
and 11.
TABLE-US-00001 TABLE 1 Example MS: m/z number IUPAC name Structural
formula (M + H) 1 3-((4- (dimethylamino) butanoyl)
oxy)-2,2-bis(((9Z)- tetradec-9- enoyloxy)methyl) propyl
(9Z)-tetradec- 9-enoate ##STR00007## 874.75 2 3-((5-
(dimethylamino) pentanoyl) oxy)-2,2-bis(((9Z)- tetradec-9-
enoyloxy) methyl)propyl (9Z)-tetradec- 9-enoate ##STR00008## 888.73
3 3-((6- (dimethylamino) hexanoyl) oxy)-2,2-bis(((9Z)- tetradec-9-
enoyloxy)methyl) propyl (9Z)-tetradec- 9-enoate ##STR00009## 902.74
4 3-((4- (dimethylamino) butanoyl) oxy)-2,2-bis(((9Z)- tetradec-9-
enoyloxy) methyl)propyl (9Z)-hexadec- 9-enoate ##STR00010## 902.74
5 3-((5- (dimethylamino) pentanoyl) oxy)-2,2-bis(((9Z)- tetradec-9-
enoyloxy) methyl)propyl (9Z)-hexadec- 9-enoate ##STR00011## 916.76
6 3-((5- (dimethylamino) pentanoyl) oxy)-2,2-bis(((9Z)- tetradec-9-
enoyloxy)methyl) propyl (9Z)-octadec- 9-enoate ##STR00012## 944.79
7 3-((5- (dimethylamino) pentanoyl) oxy)-2,2-bis(((9Z)- tetradec-9-
enoyloxy) methyl)propyl (9Z,12Z)-octadec- 9,12-dienoate
##STR00013## 942.77 8 2-(((N,N- dimethyl-.beta.- alanyl)oxy]
methyl}-2- [(octanoyloxy) methyl)propane- 1,3-diyl (9Z,9'Z)bis-
tetradec-9-enoate ##STR00014## 778.62 9 2-(((4- (dimethylamino)
butanoyl) oxy)methyl)-2- ((octanoyloxy) methyl)propane- 1,3-diyl
(9Z,9'Z)bis- tetradec-9-enoate ##STR00015## 792.63 10
2-((decanoyloxy) methyl)- 2-(((4- (dimethylamino) butanoyl)
oxy)methyl) propane-1,3- diyl (9Z,9'Z)bis- tetradec- 9-enoate
##STR00016## 820.67 11 2-(((4- (dimethylamino) butanoyl)
oxy)methyl)-2- ((dodecanoyloxy) methyl) propane-1,3-diyl
(9Z,9'Z)bis- tetradec-9- enoate ##STR00017## 848.70
Production Example and Test Example for Composition Containing
Lipid Particle of Present Invention and Nucleic Acid for Nucleic
Acid Introduction
Production Example 1
[0216] A lipid mixture (cationic lipid produced in
Examples:DPPC:cholesterol:GM-020=60:10.6:28:1.4, in mole ratio) was
dissolved in 90% EtOH/10% RNase-free water to afford a 8.5 mg/mL
lipid solution. Luciferase mRNA (TriLink Bio Technologies) was
dissolved in 10 mM 2-morpholinoethanesulfonate (MES) buffer at pH
4.0 to afford a 0.22 mg/mL nucleic acid solution. The lipid
solution and nucleic acid solution obtained were mixed by using the
apparatus NanoAssemblr (Precision Nanosystems) at room temperature
with a flow rate ratio of 3 mL/min:6 mL/min to afford a dispersion
containing a lipid particle encapsulating the nucleic acid. By
using a Slyde-A-Lyzer (molecular weight cutoff: 20 k, Thermo Fisher
Scientific), the dispersion obtained was dialyzed with water at
room temperature for 1 hour and with PBS at 4.degree. C. for 48
hours. Subsequently, filtration was performed with a 0.2 .mu.m
syringe filter (IWAKI CO., LTD.) to prepare a composition for
nucleic acid introduction, and thereafter the composition was
stored at 4.degree. C. The particle size of the lipid particle was
measured by using a Zetasizer Nano ZS (Malvern Instruments
Limited). The mRNA concentration and encapsulation ratio of the
lipid particle were measured by using a Quant-iT (TM) RiboGreen (R)
(Invitrogen). Table 2 shows the analysis results.
TABLE-US-00002 TABLE 2 Particle mRNA Encapsulation Cationic size
Concentration ratio lipid (nm) (.mu.g/mL) (%) Example 1 71 136 96
Example 2 90 73 96 Example 3 93 87 96 Example 4 76 113 97 Example 5
91 96 98 Example 8 67 84 94 Example 9 108 129 62 Example 10 92 86
84 Example 11 83 85 92
[Test Example 1] Test of mRNA Transfection into Cultured Cells
[0217] The human colorectal cancer-derived cell line HCT116 cells
were cultured in a 96-well plate at a cell density of 6000
cells/well, and 24 hours thereafter 10 .mu.L of the composition
containing 10 ng of luciferase mRNA for nucleic acid introduction
(a solution obtained by diluting the product of Production Example
1 to an mRNA concentration of 10 ng/10 .mu.L) was added to the
medium. The level of luciferase expressed in HCT116 24 hours after
the addition of mRNA was measured by using a Picagene LT2.0 kit
(TOYOBO CO., LTD.). Tables 3 and 4 show the measurement
results.
TABLE-US-00003 TABLE 3 Cationic Mean value of luminescence lipid
(cps) for 3 wells PBS Control 560 Example 1 227720 Example 2 570027
Example 3 412187 Example 4 407547 Example 5 545507 Example 8 3853
Example 10 1203467 Example 11 953520
TABLE-US-00004 TABLE 4 Cationic Mean value of luminescence lipid
(cps) for 3 wells PBS Control 627 Example 9 549893
Production Example and Test Example for Composition Containing
Lipid Particle of Present Invention and Nucleic Acid for Nucleic
Acid Introduction
Production Example 2
[0218] A lipid mixture (cationic lipid produced in
Examples:DPPC:cholesterol:GM-020=60:10.6:28:1.4, in mole ratio) was
dissolved in 90% EtOH/10% RNase-free water to afford an
approximately 7 mg/mL lipid solution. Equal amounts of an siRNA for
Collal and an siRNA for Factor VII (FVII) were mixed together, and
the mixture was dissolved in 25 mM acetate buffer at pH 4.0 to
afford a 0.2 mg/mL nucleic acid solution. Sequence information on
the siRNAs are shown in a table below. The lipid solution and
nucleic acid solution obtained were mixed by using the apparatus
NanoAssemblr (Precision Nanosystems) at room temperature with a
flow rate ratio of 3 mL/min:9 mL/min to afford a dispersion
containing a lipid particle including the nucleic acids. By using a
Slyde-A-Lyzer (molecular weight cutoff: 20k, Thermo Fisher
Scientific), the dispersion obtained was dialyzed with water at
room temperature for 1 hour and with PBS at 4.degree. C. for 48
hours. Subsequently, filtration was performed with a 0.2 .mu.m
syringe filter (IWAKI CO., LTD.) to prepare a composition for
nucleic acid introduction, and thereafter the composition was
stored at 4.degree. C. The particle size of the lipid particle was
measured by using a Zetasizer Nano ZS (Malvern Instruments
Limited). The mRNA concentration and encapsulation ratio of the
lipid particle were measured by using a Quant-iT (TM) RiboGreen (R)
(Invitrogen). Table 6 shows the analysis results.
TABLE-US-00005 TABLE 5 Col1a1 siRNA (cited from Calvente et al.
Hepatology 2015, 62:4) Sense 5'-G[mU][mC][mU]AGA[mC]A[mU]G[mU][mU]
[mC]AG[mC][mU][mU][ts]t-3' Antisense
5'-AAGCUGAA[mC]AUGUC[mU]AGAC[ts]t-3' Factor VII siRNA (cited from
Landesman et al. Silence 2010, 1:16) Sense
5'-[mC][mU]A[mC]GAAAG[mC]A[mU][mC][mC] [mU][mU][mC]A[mU][ts]t-3'
Antisense 5'-AUGAAGGAUGCUUUCG[mU]AG[ts]t-3' N: RNA n: DNA [mN]:
2'-OMe RNA [ns]: phosphorothioate
TABLE-US-00006 TABLE 6 Particle siRNA Encapsulation size
Concentration ratio Compound (nm) (.mu.g/ml) (%) Example 2 104 251
98 Example 3 117 196 95 Example 10 88 307 90 Example 11 85 402 91
Example 4 90 355 89 Example 5 103 367 94 Example 6 87 333 98
Example 7 102 320 96
[Test Example 2] Test of siRNA Delivery to Hepatic Stellate Cells
in Model Mice with Liver Disorder Caused by Carbon
Tetrachloride
[0219] The PBS dispersion of the lipid particle including the
Collal siRNA and the FVII siRNA was diluted with PBS to respective
siRNA concentrations of 40 .mu.g/mL and 120 .mu.g/mL, and
administered to the orbital sinus of each Balb/c mouse to achieve
respective siRNA doses of 0.2 mg/kg and 0.6 mg/kg. Three hours
after the administration of the siRNAs, carbon tetrachloride was
orally administered to achieve a dose of 0.1 mL/kg. Four days after
the administration of carbon tetrachloride, each mouse was
euthanized by bleeding under anesthesia with isoflurane, and the
liver was removed. From the liver obtained, the total RNA was
extracted with an RNeasy Mini Kit (QIAGEN), and the Collal mRNA,
FVII mRNA, and GAPDH mRNA levels were measured through a
quantitative PCR method. The expression reduction rates for Collal
mRNA and FVII mRNA normalized against the GAPDH mRNA level were
calculated with reference to those for mice without siRNA
administration, and the following table shows the calculation
results.
TABLE-US-00007 TABLE 7 COL1A1 Knockdown FVII Knockdown Compound
efficiency (%) efficiency (%) Example 2 73 0 Example 3 81 0 Example
10 31 0 Example 11 60 0 Example 4 67 19 Example 5 83 14 Example 6
86 27 Example 7 85 24
INDUSTRIAL APPLICABILITY
[0220] The compound, the lipid particle, or the composition of the
present invention enables introduction of nucleic acids into
various types of cells, tissues, or organs. Accordingly, the
compound, the lipid particle, or the composition of the present
invention is available as a DDS technique for nucleic acid drugs.
In addition, the compound, the lipid particle, or the composition
of the present invention is available as a reagent for nucleic acid
introduction for research.
Sequence CWU 1
1
4121DNAArtificial SequenceCol1a1 siRNA
(Sense)misc_feature(1)..(21)The sequence comprises both RNA and DNA
bases, some of which are modified bases.modified_base(2)..(2)The
"u" stands for 2'-O-methyluridine (um)modified_base(3)..(3)The "c"
stands for 2'-O-methylcytidine (cm)modified_base(4)..(4)The "u"
stands for 2'-O-methyluridine (um)modified_base(8)..(8)The "c"
stands for 2'-O-methylcytidine (cm)modified_base(10)..(10)The "u"
stands for 2'-O-methyluridine (um)modified_base(12)..(12)The "u"
stands for 2'-O-methyluridine (um)modified_base(12)..(12)The "c"
stands for 2'-O-methylcytidine (cm)modified_base(13)..(13)The "u"
stands for 2'-O-methyluridine (um)modified_base(14)..(14)The "c"
stands for 2'-O-methylcytidine (cm)modified_base(17)..(17)The "c"
stands for 2'-O-methylcytidine (cm)modified_base(18)..(18)The "u"
stands for 2'-O-methyluridine (um)misc_feature(20)..(21)The
nucleosides (t and t) are bound with phosphorothioate. 1gucuagacau
guucagcuut t 21221DNAArtificial SequenceCol1a1 siRNA
(Antisense)misc_feature(1)..(21)The sequence comprises both RNA and
DNA bases, some of which are modified
bases.modified_base(9)..(9)The "c" stands for 2'-O-methylcytidine
(cm)modified_base(15)..(15)The "u" stands for 2'-O-methyluridine
(um)misc_feature(20)..(21)The nucleosides (t and t) are bound with
phosphorothioate. 2aagcugaaca ugucuagact t 21321DNAArtificial
SequenceFactor VII siRNA (Sense)misc_feature(1)..(21)The sequence
comprises both RNA and DNA bases, some of which are modified
bases.modified_base(1)..(1)The "c" stands for 2'-O-methylcytidine
(cm)modified_base(2)..(2)The "u" stands for 2'-O-methyluridine
(um)modified_base(4)..(4)The "c" stands for 2'-O-methylcytidine
(cm)modified_base(10)..(10)The "c" stands for 2'-O-methylcytidine
(cm)modified_base(12)..(12)The "u" stands for 2'-O-methyluridine
(um)modified_base(13)..(13)The "c" stands for 2'-O-methylcytidine
(cm)modified_base(14)..(14)The "c" stands for 2'-O-methylcytidine
(cm)modified_base(15)..(15)The "u" stands for 2'-O-methyluridine
(um)modified_base(16)..(16)The "u" stands for 2'-O-methyluridine
(um)modified_base(17)..(17)The "c" stands for 2'-O-methylcytidine
(cm)modified_base(19)..(19)The "u" stands for 2'-O-methyluridine
(um)misc_feature(20)..(21)The nucleosides (t and t) are bound with
phosphorothioate. 3cuacgaaagc auccuucaut t 21421DNAArtificial
SequenceFactor VII siRNA (Antisense)misc_feature(1)..(21)The
sequence comprises both RNA and DNA bases, some of which are
modified bases.modified_base(17)..(17)The "u" stands for
2'-O-methyluridine (um)misc_feature(20)..(21)The nucleosides (t and
t) are bound with phosphorothioate. 4augaaggaug cuuucguagt t 21
* * * * *