DESCRIPTION
Title of the Invention: MORPHOLINO OLIGONUCLEOTIDE MANUFACTURING METHOD
Technical Field
[0001] 5 The present invention relates to a production method of morpholino oligonucleotide, and morpholino nucleotide used as a starting material of the production method.
Background Art
[0002] 10 Morpholino oligonucleotide is a compound attracting attention for its use as an antisense oligonucleotide, since it shows high affinity for DNA and RNA, resistance to various nucleases, stability in vivo and low toxicity (see non-patent document 1). 15
[0003] As a production method of morpholino oligonucleotide, a solid phase synthesis and a liquid phase synthesis have been reported (see non-patent document 2, patent documents 1 - 5).
The solid phase synthesis is advantageous from the aspect 20 of speed since it enables automatic synthesis. On the contrary, it is not suitable for industrial large scale synthesis since scaling-up is limited due to facility restriction, and low reactivity requires use of an excess monomer to be the reagent in a nucleotide elongation reaction. Also, it is associated 25 with defects in that confirmation of the progress status of the reaction in an intermediate stage, analysis of intermediate structure and the like are difficult.
On the other hand, the liquid phase synthesis requires complicated treatments such as column purification and the like, 30 and a large-scale and rapid synthesis of about 20mer chain length morpholino oligonucleotide, which is utilizable as an antisense pharmaceutical product, has been difficult.
[0004] In recent years, a synthetic method using hydrophobic 35
3
group-linked nucleoside, pseudo solid phase-protected nucleoside and the like has been reported as an attempt to solve the respective defects of the liquid phase method and the solid phase method (see patent documents 6, 7). However, synthesis of morpholino oligonucleotide different in the 5 reaction pathway and the reaction itself from those of oligonucleotide synthesis is not described or suggested.
[Document List]
[patent document]
[0005] 10 patent document 1: WO 91/09033
patent document 2: WO 2008/008113
patent document 3: US 2009/0131632 A1
patent document 4: WO 2009/064471
patent document 5: WO 2012/043730 15
patent document 6: JP-A-2010-275254
patent document 7: WO 2012/157723
[non-patent document]
[0006] non-patent document 1: Summerton, J. et al., Antisense and 20 Nucleic Acid Drug Development, 1997, Vol. 7, p. 187.
non-patent document 2: Harakawa et al., Bioorganic & Medicinal Chemistry Letters, 2012, Vol. 22, p. 1445-1447
SUMMARY OF THE INVENTION
Problems to be Solved by the Invention 25
[0007] The problem of the present invention is to provide a method of producing a morpholino oligonucleotide efficiently and in a high yield by a liquid phase method, and a novel morpholino nucleotide to be a starting material of the method. 30
Means of Solving the Problems
[0008] The present inventors have found that the above-mentioned problem can be solved by protecting the 5’-hydroxy group side with a particular protecting group in liquid phase synthesis of 35
4
a morpholino oligonucleotide.
That is, using a morpholino nucleotide having the 5’-hydroxy group side protected by the protecting group as a starting material of morpholino oligonucleotide liquid phase synthesis, condensation reaction itself can be performed in a 5 liquid phase, the reactivity is remarkably improved as compared to the solid phase method, monomer equivalents to be used can be remarkably reduced and, after the reaction, morpholino oligonucleotide can be conveniently isolated and purified by a crystallization or extraction operation. Consequently, a 10 morpholino oligonucleotide having an about 20mer chain length and utilizable for pharmaceutical products can be synthesized efficiently in a high yield by a liquid phase method.
[0009] On the other hand, in the course of study of liquid phase 15 synthesis of morpholino oligonucleotide by the present inventors, a problem was found that when an activated morpholino nucleoside monomer to be used for a condensation reaction remains in a trace amount in the subsequent cycle of condensation reaction, double addition is induced, and the 20 quality of the obtained morpholino oligonucleotide is degraded. In this regard, they have found that the problem can be solved by treating the activated morpholino nucleoside monomer remaining after the condensation reaction with a quenching agent. 25
That is, the present invention includes the following.
[1] A morpholino nucleotide represented by the formula (I):
[0010]
5
[0011] [wherein
m is any integer of not less than 0,
Base in the number of m+1 are each independently an optionally 5 protected nucleic acid base,
P1 is a hydrogen atom, or a temporary protecting group removable under acidic conditions,
X in the number of m are each independently a C1-6 alkoxy group, a di-C1-6 alkylamino group, or a piperazino group wherein a 10 nitrogen atom at the 4-position is protected by a protecting group and further optionally substituted,
W in the number of m are each independently an oxygen atom or a sulfur atom,
S1 is a single bond, or a group represented by *O-S2** (wherein 15 * indicates the bonding position to L, ** indicates the bonding position to a 5’-hydroxy group, and S2 is a spacer having a main chain having an atomic number of 1 to 20),
L is a single bond, or a group represented by the formula (a1):
[0012] 20
6
[0013] [wherein * indicates the bonding position to Y;
** indicates the bonding position to S1;
L1 is an optionally substituted divalent C1-22 hydrocarbon 5 group; and
L2 is C(=O) or a group represented by ***N(R3)-R1-N(R2)C(=O)** (wherein ** indicates the bonding position to L1, *** indicates the bonding position to Y, R1 is an optionally substituted C1-22 alkylene group, R2 and R3 are each independently a hydrogen 10 atom or an optionally substituted C1-22 alkyl group, or R2 and R3 are optionally joined to form an optionally substituted C1-22 alkylene bond)],
Y is a single bond, an oxygen atom or NR (wherein R is a hydrogen atom, an alkyl group or an aralkyl group), and 15
Z is a group represented by the formula (a2):
[0014]
[0015] [wherein * indicates the bonding position to Y; 20
R4 is a hydrogen atom, or when Rb is a group represented by the following formula (a3), R4 is optionally a single bond or -O- in combination with R6 to form a fluorenyl group or a xanthenyl group together with ring B;
Q in the number of k are each independently a single bond, or -25
7
O-, -S-, -OC(=O)-, -NHC(=O)- or -NH-;
R5 in the number of k are each independently an organic group having an alkyl group having not less than 10 and not more than 300 carbon atoms and/or an alkenyl group having not less than 10 and not more than 300 carbon atoms; 5
k is an integer of 1 to 4;
ring A optionally further has, in addition to QR5 in the number of k, a substituent selected from the group consisting of a halogen atom, a C1-6 alkyl group optionally substituted by a halogen atom(s), and a C1-6 alkoxy group optionally substituted 10 by a halogen atom(s);
Ra is a hydrogen atom;
Rb is a hydrogen atom, or a group represented by the formula (a3):
[0016] 15
[0017] (wherein * indicates a bonding position;
j is an integer of 0 to 4; 20
Q in the number of j are as defined above;
R7 in the number of j are each independently an organic group having an alkyl group having not less than 10 and not more than 300 carbon atoms and/or an alkenyl group having not less than 10 and not more than 300 carbon atoms; 25
R6 is a hydrogen atom, or optionally a single bond or -O- in combination with R4 to form a fluorenyl group or a xanthenyl group together with ring A; and
ring B optionally further has, in addition to QR7 in the number of j, a substituent selected from the group consisting of a 30 halogen atom, a C1-6 alkyl group optionally substituted by a
8
halogen atom(s), and a C1-6 alkoxy group optionally substituted by a halogen atom(s)), or
Ra and Rb are joined to form an oxygen atom]].
[0018] [2] The morpholino nucleotide of [1], wherein m is 0. 5
[3] The morpholino nucleotide of [1] or [2], wherein L in the formula (I) is a succinyl group, and
R5 and/or R7 are/is an alkyl group having 10 - 40 carbon atoms.
[4] The morpholino nucleotide of [1] or [2], wherein L in the formula (I) is a succinyl group, and 10
Ra and Rb are both hydrogen atoms, and R5 is an alkyl group having 10 - 40 carbon atoms.
[5] The morpholino nucleotide of [1] or [2], wherein L in the formula (I) is a succinyl group, and
R5 and/or R7 are/is an alkyl group having 12 - 30 carbon atoms. 15
[0019] [6] The morpholino nucleotide of [1] or [2], wherein, in the formula (I), L is a succinyl group, and
Z-Y- is a group selected from the group consisting of a 3,4,5-tri(octadecyloxy)benzyloxy group, 20
a 3,5-di(docosyloxy)benzyloxy group,
a 3,5-bis[3’,4’,5’-tri(octadecyloxy)benzyloxy]benzyloxy group,
a 3,4,5-tris[3’,4’,5’-tri(octadecyloxy)benzyloxy]benzyloxy group,
a 3,4,5-tri(octadecyloxy)benzylamino group, 25
a 2,4-di(docosyloxy)benzylamino group,
a 3,5-di(docosyloxy)benzylamino group,
a di(4-docosyloxyphenyl)methylamino group,
a 4-methoxy-2-[3’,4’,5’-tri(octadecyloxy)benzyloxy]benzylamino group, 30
a 4-methoxy-2-[3’,4’,5’-tri(octadecyloxy)cyclohexylmethyloxy]benzylamino group,
a 2,4-di(dodecyloxy)benzylamino group,
a phenyl(2,3,4-tri(octadecyloxy)phenyl)methylamino group,
a di[4-(12-docosyloxydodecyloxy)phenyl]methylamino group, 35
9
a 3,5-bis[3’,4’,5’-tri(octadecyloxy)benzyloxy]benzylamino group, and
a 3,4,5-tris[3’,4’,5’-tri(octadecyloxy)benzyloxy]benzylamino group.
[0020] 5 [7] The morpholino nucleotide of [1] or [2], wherein Z-Y-L- is selected from the group consisting of
a 2-{2,4-di(2’,3’-dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl group;
a 3,5-di(2’,3’-dihydrophytyloxy)benzylsuccinyl group; 10
a 4-(2’,3’-dihydrophytyloxy)benzylsuccinyl group;
a 2-{1-[(2-chloro-5-(2’,3’-dihydrophytyloxy)phenyl)]benzylaminocarbonyl}ethylcarbonyl group;
a 3,4,5-tri(2’,3’-dihydrophytyloxy)benzylsuccinyl group; 15
a 2-{3,4,5-tri(2’,3’-dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl group;
a 2-{4-(2’,3’-dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl group;
a 2-{2-[3’,4’,5’-tri(2’’,3’’-dihydrophytyloxy)benzyloxy]-4-20 methoxybenzylaminocarbonyl}ethylcarbonyl group;
a 2-{4-(2’,3’-dihydrophytyloxy)-2-methoxybenzylaminocarbonyl}ethylcarbonyl group;
a 4-(2’,3’-dihydrophytyloxy)-2-methylbenzylsuccinyl group;
a 2-{4-(2’,3’-dihydrophytyloxy)-2-25 methylbenzylaminocarbonyl}ethylcarbonyl group;
a 4-[2,2,4,8,10,10-hexamethyl-5-dodecanoylamino]benzylsuccinyl group;
a 2-{4-[2,2,4,8,10,10-hexamethyl-5-dodecanoylamino]benzylaminocarbonyl}ethylcarbonyl group; 30
a 4-(3,7,11-trimethyldodecyloxy)benzylsuccinyl group;
a 2-{4-(3,7,11-trimethyldodecyloxy)benzylaminocarbonyl}ethylcarbonyl group;
a 2-{3,5-di(2’,3’-dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl group; 35
10
a 2-{1-[2,3,4-tri(2’,3’-dihydrophytyloxy)phenyl]benzylaminocarbonyl}ethylcarbonyl group;
a 2-{1-[4-(2’,3’-dihydrophytyloxy)phenyl]-4’-(2’,3’-dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl group; 5
a 3,4,5-tris[3,4,5-tri(2’,3’-dihydrophytyloxy)benzyl]benzylsuccinyl group; and
a 2-{3,4,5-tris[3,4,5-tri(2’,3’-dihydrophytyloxy)benzyl]benzylaminocarbonyl}ethylcarbonyl group.
[0021] 10 [8] The morpholino nucleotide of any one of [1] - [7], wherein P1 is a trityl group, a monomethoxytrityl group, or a dimethoxytrityl group.
[0022] [9] A method of producing n+p-mer morpholino oligonucleotide, 15 comprising (2) a step of condensing a p-mer morpholino oligonucleotide (p is any integer of one or more) wherein a 5’-hydroxy group is activated (thio)phosphated or activated (thio)phosphoramidated, and a morpholine ring nitrogen atom is protected by a temporary protecting group removable under 20 acidic conditions, with an n-mer morpholino oligonucleotide (n is an integer of one or more) wherein a 5’-hydroxy group or, when the 5’-hydroxy group has a substituent having a hydroxy group, the hydroxy group present on the substituent is protected by a protecting group having an alkyl group having 25 not less than 10 and not more than 300 carbon atoms and/or an alkenyl group having not less than 10 and not more than 300 carbon atoms, and the morpholine ring nitrogen atom is not protected, by a (thio)phosphoramidate bond or (thio)phosphorodiamidate bond via the morpholine ring nitrogen 30 atom.
[0023] [10] The production method of [9], wherein p is 1.
[11] The production method of [9] or [10], comprising treating the reaction mixture with a quenching agent after completion of 35
11
the reaction.
[0024] [12] The production method of any one of [9] - [11], further comprising the following step (1):
(1) a step of removing the temporary protecting group of the 5 morpholine ring nitrogen atom by reacting, before the condensation step (2), the n-mer morpholino oligonucleotide wherein the 5’-hydroxy group or, when the 5’-hydroxy group has a substituent having a hydroxy group, the hydroxy group present on the substituent is protected by a protecting group having an 10 alkyl group having not less than 10 and not more than 300 carbon atoms and/or an alkenyl group having not less than 10 and not more than 300 carbon atoms, and the morpholine ring nitrogen atom is protected by a temporary protecting group removable under acidic conditions, with an acid in a non-polar 15 solvent.
[0025] [13] The production method of [12], wherein the temporary protecting group is removed in the presence of a cation scavenger. 20
[14] The production method of [12] or [13], further comprising a step of neutralizing with an organic base in step (1), after removing the temporary protecting group of the morpholine ring nitrogen atom.
[0026] 25 [15] The production method of any one of [9] - [14], wherein the protecting group having an alkyl group having not less than 10 and not more than 300 carbon atoms and/or an alkenyl group having not less than 10 and not more than 300 carbon atoms is a group represented by the following formula (II): 30
[0027]
[0028] [L is a single bond, or a group represented by the formula 35
12
(a1):
[0029]
[0030] 5 [wherein * indicates the bonding position to Y;
** indicates the bonding position to S1;
L1 is an optionally substituted divalent C1-22 hydrocarbon group; and
L2 is C(=O) or a group represented by ***N(R3)-R1-N(R2)C(=O)** 10 (wherein ** indicates the bonding position to L1, *** indicates the bonding position to Y, R1 is an optionally substituted C1-22 alkylene group, R2 and R3 are each independently a hydrogen atom or an optionally substituted C1-22 alkyl group, or R2 and R3 are optionally joined to form an optionally substituted C1-22 15 alkylene bond)],
Y is a single bond, an oxygen atom or NR (wherein R is a hydrogen atom, an alkyl group or an aralkyl group), and
Z is a group represented by the formula (a2):
[0031] 20
[0032] [wherein * indicates the bonding position to Y;
R4 is a hydrogen atom, or when Rb is a group represented by the 25 following formula (a3), R4 is optionally a single bond or -O-
13
in combination with R6 to form a fluorenyl group or a xanthenyl group together with ring B;
Q in the number of k are each independently a single bond, or -O-, -S-, -OC(=O)-, -NHC(=O)- or -NH-;
R5 in the number of k are each independently an organic group 5 having an alkyl group having not less than 10 and not more than 300 carbon atoms and/or an alkenyl group having not less than 10 and not more than 300 carbon atoms;
k is an integer of 1 to 4;
ring A optionally further has, in addition to QR5 in the number 10 of k, a substituent selected from the group consisting of a halogen atom, a C1-6 alkyl group optionally substituted by a halogen atom(s), and a C1-6 alkoxy group optionally substituted by a halogen atom(s);
Ra is a hydrogen atom; 15
Rb is a hydrogen atom, or a group represented by the formula (a3):
[0033]
20
[0034] (wherein * indicates a bonding position;
j is an integer of 0 to 4;
Q in the number of j are as defined above;
R7 in the number of j are each independently an organic group 25 having an alkyl group having not less than 10 and not more than 300 carbon atoms and/or an alkenyl group having not less than 10 and not more than 300 carbon atoms;
R6 is a hydrogen atom, or optionally a single bond or -O- in combination with R4 to form a fluorenyl group or a xanthenyl 30 group together with ring A; and
14
ring B optionally further has, in addition to QR7 in the number of j, a substituent selected from the group consisting of a halogen atom, a C1-6 alkyl group optionally substituted by a halogen atom(s), and a C1-6 alkoxy group optionally substituted by a halogen atom(s)), or 5
Ra and Rb are joined to form an oxygen atom].
[0035] [16] The production method of [15], further comprising the following step (3):
(3) a step of obtaining morpholino oligonucleotide by adding a 10 polar solvent to the reaction mixture obtained in step (1) and/or (2) and collecting precipitates thereof by solid-liquid separation.
[0036] [17] The production method of any one of [12] - [16], wherein 15 the non-polar solvent is selected from the group consisting of a halogenated solvent, an aromatic solvent, an ester solvent, an aliphatic solvent, a non-polar ether solvent, and a combination of these.
[18] The production method of [16] or [17], wherein the polar 20 solvent is an alcohol solvent or a nitrile solvent.
[0037] [19] The production method of any one of [9] - [18], further comprising the following step (4):
(4) a step of removing all the protecting groups of the 25 obtained n+p-mer morpholino oligonucleotide.
[20] The production method of any one of [9] - [19], wherein the temporary protecting group removable under acidic conditions is a trityl group, a dimethoxytrityl group, or a monomethoxytrityl group. 30
Effect of the Invention
[0038] According to the present invention, since an advantage of a liquid phase method that monomer equivalent of a starting material of a condensation reaction can be reduced by improving 35
15
the reactivity by protecting the 5’-hydroxy group side of morpholino nucleotide with a particular protecting group, and an advantage of a solid phase method that morpholino oligonucleotide can be conveniently isolated and purified by a crystallization, washing or extraction operation in each step 5 of an elongation reaction can be combined, an industrial large-scale production of a morpholino oligonucleotide having about 20mer chain length and useful as a pharmaceutical product can be efficiently performed in a high yield.
In addition, it was found that degradation of the quality 10 of the object morpholino oligonucleotide can be prevented by treating activated morpholino nucleoside monomer remaining after condensation reaction with a quenching agent. By combining same with the above-mentioned method, an efficient production method of high quality morpholino oligonucleotide in 15 a large scale can be provided.
Description of Embodiments
[0039] The present invention relates to a novel morpholino nucleotide wherein the 5’-hydroxy group side is protected by a 20 particular protecting group, and a morpholine ring nitrogen atom is optionally protected by a temporary protecting group removable under acidic conditions.
Another embodiment of the present invention is a method of producing n+p-mer morpholino oligonucleotide, comprising a 25 step of condensing a p-mer morpholino oligonucleotide (p is any integer of one or more) wherein a 5’-hydroxy group is activated (thio)phosphated or activated (thio)phosphoramidated, and a morpholine ring nitrogen atom is protected by a temporary protecting group removable under acidic conditions, with an n-30 mer morpholino oligonucleotide (n is an integer of one or more) wherein a 5’-hydroxy group or a hydroxy group present on a substituent of the 5’-hydroxy group is protected by a protecting group having an alkyl group having not less than 10 and not more than 300 carbon atoms and/or an alkenyl group 35
16
having not less than 10 and not more than 300 carbon atoms, and the morpholine ring nitrogen atom is not protected, by a (thio)phosphoramidate bond or (thio)phosphorodiamidate bond via the morpholine ring nitrogen atom.
Explanations are given below. 5
[0040] [Explanation of terms]
Unless otherwise specified in the sentences, any technical terms and scientific terms used in the present specification, have the same meaning as those generally 10 understood by those of ordinary skill in the art the present invention belongs to. Any methods and materials similar or equivalent to those described in the present specification can be used for practicing or testing the present invention, and preferable methods and materials are described in the following. 15 All publications and patents referred to in the specification are hereby incorporated by reference so as to describe and disclose constructed products and methodology described in, for example, publications usable in relation to the described invention. 20
[0041] In the present specification, the “morpholino nucleoside” to be a constitutional unit of morpholino oligonucleotide is a compound represented by the following formula (1).
[0042] 25
[0043] (wherein Base is an optionally protected nucleic acid base).
Morpholino nucleoside (1) can be prepared by a method 30
17
known per se (e.g., the method described in WO 91/09033A1), or a method analogous thereto. Specifically, as shown in the following scheme, the corresponding ribonucleoside (2) is subjected to oxidative ring opening with sodium periodate etc. to give the corresponding 2’,3’-dialdehyde (3), the dialdehyde 5 (3) is subjected to ring closure with ammonia to give 2’,3’-dihydroxymorpholino nucleoside (4), and dihydroxymorpholino nucleoside (4) is reduced with a reducing agent (e.g., sodium cyanoborohydride, sodium triacetoxyborohydride and the like), whereby morpholino nucleoside (1) can be obtained. 10
[0044]
[0045] In the present specification, the position numbers (1’, 15 2’ and the like) of morpholino nucleoside correspond to the position numbers of carbon atoms of the ribose of the starting material ribonucleoside (2).
reduction
18
[0046] In the present specification, morpholino oligonucleotide means a compound wherein two or more morpholino nucleosides are polymerized by (thio)phosphoramidate bonding or (thio)phosphorodiamidate bonding via a 5’-hydroxy group and the 5 nitrogen atom of a morpholine ring. For example, as m’+1-mer morpholino oligonucleotide, a compound represented by the following formula (5) can be mentioned.
[0047] 10
[0048] (wherein
m’ is any integer of one or more,
Base in the number of m’+1 are each independently an optionally 15 protected nucleic acid base,
X in the number of m’ are each independently a C1-6 alkoxy group, a di-C1-6 alkylamino group, or a piperazino group wherein a nitrogen atom at the 4-position is protected by a protecting group, and further optionally substituted, and the like, and 20
W in the number of m’ are each independently an oxygen atom or a sulfur atom).
[0049]
19
In the present specification, the “piperazino group wherein a 4-position nitrogen atom is protected by a protecting group, and further optionally substituted” means that the 4-position nitrogen atom of the piperazino group is protected by a protecting group, and a piperazino group protected by a 5 protecting group sustainable under the deprotection conditions of the morpholine ring nitrogen atom of morpholino nucleotide is preferable. As the ”protecting group of the 4-position nitrogen atom of the piperazino group”, an acyl group is preferable and, for example, an acyl group having a fluoro 10 group in the carbon chain such as monofluoroacetyl group, difluoroacetyl group, trifluoroacetyl group, 2-fluoropropionyl group, 2,2-difluoropropionyl group, 3,3,3-trifluoropropionyl group, 2,3,3,3-tetrafluoropropionyl group, pentafluoropropionyl group and the like is more preferable (see WO 2008/008113). In 15 the piperazino group, a hydrogen atom bonded to the carbon atom of the piperazino group may be substituted, and examples of the substituent include an alkyl group (preferably having 1 – 3 carbon atoms) such as methyl group and the like, and the like.
[0050] 20 In the present specification, morpholino nucleoside at the terminal on the side having a free hydroxy group at the 5’-position of morpholino oligonucleotide (upper left side of the above-mentioned formula (5)) is referred to as the “5’-terminus”, and morpholino nucleoside at the terminal on the 25 opposite side (lower right side of the above-mentioned formula (5)) is referred to as the “3’-terminus”, according to the usual practice in the nucleic acid chemistry.
[0051] In the present specification, the “nucleic acid base” is 30 not particularly limited as long as it can be used for the synthesis of nucleic acid and includes, for example, a pyrimidine base such as cytosyl group, uracil group, thyminyl group and the like, and a purine base such as adenyl group, guanyl group and the like. The “optionally protected nucleic 35
20
acid base” means, for example, that an amino group may be protected in an adenyl group, a guanyl group or a cytosyl group, which is a nucleic acid base having an amino group, and a nucleic acid base wherein the amino group therein is protected by a protecting group sustainable under the deprotection 5 conditions of the morpholine ring nitrogen atom of the morpholino nucleotide is preferable. The “amino-protecting group” is not particularly limited, and examples thereof include the protecting groups described in Greene’s PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, 4th edition, Wiley-Interscience, 10 2006 and the like. Specific examples of the “amino-protecting group” include a pivaloyl group, a pivaloyloxymethyl group, a trifluoroacetyl group, a phenoxyacetyl group, a 4-isopropylphenoxyacetyl group, a 4-tert-butylphenoxyacetyl group, an acetyl group, a benzoyl group, an isobutyryl group, a 15 dimethylformamidinyl group, a 9-fluorenylmethyloxycarbonyl group and the like. Among them, a phenoxyacetyl group, a 4-isopropylphenoxyacetyl group, an acetyl group, a benzoyl group, an isobutyryl group and a dimethylformamidinyl group are preferable. In addition, the carbonyl group of the nucleic 20 acid base is optionally protected, and can be protected, for example, by reacting phenol, 2,5-dichlorophenol, 3-chlorophenol, 3,5-dichlorophenol, 2-formylphenol, 2-naphthol, 4-methoxyphenol, 4-chlorophenol, 2-nitrophenol, 4-nitrophenol, 4-acetylaminophenol, pentafluorophenol, 4-pivaloyloxybenzyl 25 alcohol, 4-nitrophenethyl alcohol, 2-(methylsulfonyl)ethanol, 2-(phenylsulfonyl)ethanol, 2-cyanoethanol, 2-(trimethylsilyl)ethanol, dimethylcarbamoyl chloride, diethylcarbamoyl chloride, ethylphenylcarbamoyl chloride, 1-pyrrolidinecarbonyl chloride, 4-morpholinecarbonyl chloride, 30 diphenylcarbamoyl chloride and the like. In some cases, the carbonyl-protecting group does not need to be particularly introduced. Moreover, in addition to the above-mentioned groups, a modified nucleic acid base (e.g., a 8-bromoadenyl group, a 8-bromoguanyl group, a 5-bromocytosyl group, a 5-35
21
iodocytosyl group, a 5-bromouracil group, a 5-iodouracil group, a 5-fluorouracil group, a 5-methylcytosyl group, a 8-oxoguanyl group, a hypoxanthinyl group etc.), which is a nucleic acid base substituted by any 1 to 3 substituents (e.g., a halogen atom, an alkyl group, an aralkyl group, an alkoxy group, an 5 acyl group, an alkoxyalkyl group, a hydroxy group, an amino group, monoalkylamino, dialkylamino, carboxy, cyano, nitro etc.) at any position(s), are also encompassed in the “nucleic acid base”.
[0052] 10 In the present specification, the “halogen atom” means a fluorine atom, a chlorine atom, a bromine atom or iodine atom.
[0053] In the present specification, examples of the “alkyl (group)” include a linear or branched chain alkyl group having 15 one or more carbon atoms. When the carbon number is not particularly limited, it is preferably a C1-10 alkyl group, more preferably a C1-6 alkyl group. When the carbon number is not particularly limited, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, 20 hexyl and the like are preferable, and methyl and ethyl are particularly preferable.
[0054] In the present specification, the “aralkyl (group)” means a C7-20 aralkyl group, preferably a C7-16 aralkyl group (C6-10 25 aryl-C1-6 alkyl group). Specific preferable examples include benzyl, 1-phenylethyl, 2-phenylethyl, 1-phenylpropyl, naphthylmethyl, 1-naphthylethyl, 1-naphthylpropyl and the like, and benzyl is particularly preferable.
[0055] 30 In the present specification, examples of the “alkoxy (group)” include an alkoxy group having one or more carbon atoms. When the carbon number is not particularly limited, it is preferably a C1-10 alkoxy group, more preferably a C1-6 alkoxy group. When the carbon number is not particularly limited, 35
22
methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, hexyloxy and the like are preferable, and methoxy and ethoxy are particularly preferable.
[0056] In the present specification, examples of the “acyl 5 (group)” include a linear or branched chain C1-6 alkanoyl group, a C7-13 aroyl group and the like. Specific examples thereof include formyl, acetyl, n-propionyl, isopropionyl, n-butyryl, isobutyryl, pivaloyl, valeryl, hexanoyl, benzoyl, naphthoyl, levulinyl and the like, each of which is optionally substituted. 10
[0057] In the present specification, examples of the “alkenyl (group)” include a linear or branched chain C2-6 alkenyl group and the like. Examples thereof include vinyl, 1-propenyl, allyl, isopropenyl, butenyl, isobutenyl and the like. Among 15 them, a C2-C4 alkenyl group is preferable.
[0058] In the present specification, preferable examples of the “alkynyl (group)” include a C2-6 alkynyl group and the like. Examples thereof include ethynyl, 1-propynyl, 2-propynyl, 1-20 butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl and the like. Among them, a C2-C4 alkynyl group is preferable.
[0059] 25 In the present specification, the “cycloalkyl (group)” means a cyclic alkyl group, and examples thereof include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and the like. Among them, a C3-C6 cycloalkyl group such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and 30 the like is preferable, and cyclohexyl is particularly preferable.
[0060] In the present specification, the “aryl (group)” means a monocyclic aromatic or polycyclic (fused) aromatic hydrocarbon 35
23
group. Specific examples thereof include a C6-14 aryl group such as phenyl, 1-naphthyl, 2-naphthyl, biphenylyl, 2-anthryl and the like, and the like. Among them, a C6-10 aryl group is more preferably and phenyl is particularly preferable.
[0061] 5 In the present specification, examples of the “hydrocarbon group” include an aliphatic hydrocarbon group, an aromatic-aliphatic hydrocarbon group, a monocyclic saturated hydrocarbon group, an aromatic hydrocarbon group and the like, and specific examples thereof include monovalent groups such as 10 an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, an aryl group, an aralkyl group and the like and a divalent group induced therefrom.
[0062] In the present specification, the “organic group having a 15 hydrocarbon group” means a group having the aforementioned “hydrocarbon group”, and the moiety other than the “hydrocarbon group” of the “organic group having a hydrocarbon group” can be determined freely. For example, the organic group optionally has, as a linker, a moiety such as -O-, -S-, -COO-, -OCONH-, -20 CONH- and the like.
[0063] In the present specification, the “substituent” of the “optionally substituted” encompasses the aforementioned halogen atom, alkyl group, aralkyl group, alkoxy group, acyl group, 25 alkenyl group, alkynyl group, cycloalkyl group, aryl group, as well as hydroxy group, nitro group, cyano group, guanidyl group, carboxy group, alkoxycarbonyl group (the alkoxy moiety is the same as that in the aforementioned alkoxy group), sulfo group, phospho group, alkylthio group (the alkyl moiety is the same as 30 that in the aforementioned alkyl group), alkylsulfinyl group (the alkyl moiety is the same as that in the aforementioned alkyl group), alkylsulfonyl group (the alkyl moiety is the same as that in the aforementioned alkyl group), amino group, monoalkylamino group (the alkyl moiety is the same as that in 35
24
the aforementioned alkyl group), dialkylamino group (the alkyl moiety is the same as that in the aforementioned alkyl group), oxo group and the like.
[0064] [morpholino nucleotide wherein 5’-hydroxy group or hydroxy 5 group present on the substituent of the 5’-hydroxy group is protected by a particular protecting group, and morpholine ring nitrogen atom is optionally protected by a temporary protecting group removable under acidic conditions]
Using the morpholino nucleotide wherein 5’-hydroxy group 10 or hydroxy group present on the substituent of the 5’-hydroxy group is protected by a particular protecting group used in the present invention, a production method of a morpholino oligonucleotide suitable for liquid phase synthesis can be provided. 15
Since high efficiency and high yield can be achieved in the production method of the object morpholino oligonucleotide, a morpholino nucleotide wherein 5’-hydroxy group or hydroxy group present on the substituent of the 5’-hydroxy group is protected by a protecting group having an alkyl group having 20 not less than 10 and not more than 300 carbon atoms and/or an alkenyl group having not less than 10 and not more than 300 carbon atoms is preferable.
[0065] “When the 5’-hydroxy group has a substituent” means that 25 the hydrogen atom of the 5’-hydroxy group is substituted by a substituent having a hydroxy group. The “substituent” of the “substituent having a hydroxy group” is not particularly limited as long as the main chain is constituted of 1 to 20 atoms. Here, the “main chain” means a shortest atom chain 30 linking the oxygen atom of the 5’-hydroxy group and the oxygen atom of the hydroxy group on the substituent, and the atom chain is optionally further substituted. The atom constituting the main chain is selected from carbon atom, oxygen atom, nitrogen atom, sulfur atom, phosphorus atom and the like. 35
25
Specific examples of the “substituent having a hydroxy group ” include organic groups having a hydrocarbon group such as alkyl group, aralkyl group, acyl group, alkenyl group, alkynyl group, cycloalkyl group, aryl group, alkoxycarbonyl group and the like, wherein the hydrogen atom on the hydrocarbon group is 5 substituted by a hydroxy group and the like. In addition, for example, the substituent of the following 5’-hydroxy group disclosed in WO 2008/008113 and the like can be mentioned.
[0066] 10
[0067] Examples of the “alkyl group having not less than 10 and not more than 300 carbon atoms and/or alkenyl group having not less than 10 and not more than 300 carbon atoms” include 15 monovalent groups and divalent groups induced therefrom. Among them, alkyl group having 10 – 40 carbon atoms is preferable, and alkyl group having 10 – 30 carbon atoms is particularly preferable. The alkyl group and the alkenyl group of the “alkyl group having not less than 10 and not more than 300 20 carbon atoms and/or alkenyl group having not less than 10 and not more than 300 carbon atoms” include linear or branched chain alkyl group, and linear or branched chain alkenyl group. In the production method of the present invention, linear alkyl group and linear alkenyl group are preferable, and linear alkyl 25 group is particularly preferable. Specific preferable examples of the “alkyl group having not less than 10 and not more than
26
300 carbon atoms and/or alkenyl group having not less than 10 and not more than 300 carbon atoms” include monovalent aliphatic hydrocarbon group such as decyl group, dodecyl group, tridecyl group, myristyl group, cetyl group, stearyl group, oleyl group, linolyl group, arachyl group, behenyl group, 5 isostearyl group and the like, and divalent groups induced therefrom.
[0068] As the morpholino oligonucleotide wherein a 5’-hydroxy group or a hydroxy group present on a substituent of the 5’-10 hydroxy group is protected by a protecting group having an alkyl group having not less than 10 and not more than 300 carbon atoms and/or an alkenyl group having not less than 10 and not more than 300 carbon atoms, and the morpholine ring nitrogen atom is optionally protected by a temporary protecting 15 group removable under acidic conditions, a morpholino nucleotide wherein the 5’-hydroxy group or the hydroxy group present on the substituent of the 5’-hydroxy group is protected by a protecting group represented by the formula (II): Z-Y-L- is preferable. Specifically, a novel compound represented by 20 the following formula (I) (hereinafter sometimes to be referred to as the compound of the present invention) can be mentioned.
The formula (I):
[0069]
27
[0070] [wherein
m is any integer of not less than 0,
Base in the number of m+1 are each independently an optionally 5 protected nucleic acid base,
P1 is a hydrogen atom, or a temporary protecting group removable under acidic conditions,
X in the number of m are each independently a C1-6 alkoxy group, a di-C1-6 alkylamino group, or a piperazino group wherein a 10 nitrogen atom at the 4-position is protected by a protecting group and further optionally substituted,
W in the number of m are each independently an oxygen atom or a sulfur atom,
S1 is a single bond, or a group represented by *O-S2** (wherein 15 * indicates the bonding position to L, ** indicates the bonding position to 5’-hydroxy group, and S2 is a spacer having a main chain having an atomic number of 1 to 20), and
L is a single bond, or a group represented by the formula (a1):
[0071] 20
28
[0072] [wherein * indicates the bonding position to Y;
** indicates the bonding position to S1;
L1 is an optionally substituted divalent C1-22 hydrocarbon 5 group; and
L2 is C(=O) or a group represented by ***N(R3)-R1-N(R2)C(=O)** (wherein ** indicates the bonding position to L1, *** indicates the bonding position to Y, R1 is an optionally substituted C1-22 alkylene group, R2 and R3 are each independently a hydrogen 10 atom or an optionally substituted C1-22 alkyl group, or R2 and R3 are optionally joined to form an optionally substituted C1-22 alkylene bond)],
Y is a single bond, an oxygen atom or NR (wherein R is a hydrogen atom, an alkyl group or an aralkyl group), and 15
Z is a group represented by the formula (a2):
[0073]
[0074] 20 [wherein * indicates the bonding position to Y;
R4 is a hydrogen atom, or when Rb is a group represented by the following formula (a3), R4 is optionally a single bond or -O- in combination with R6 to form a fluorenyl group or a xanthenyl group together with ring B; 25
Q in the number of k are each independently a single bond, or -
29
O-, -S-, -OC(=O)-, -NHC(=O)- or -NH-;
R5 in the number of k are each independently an organic group having an alkyl group having not less than 10 and not more than 300 carbon atoms and/or an alkenyl group having not less than 10 and not more than 300 carbon atoms; 5
k is an integer of 1 to 4;
ring A optionally further has, in addition to QR5 in the number of k, a substituent selected from the group consisting of a halogen atom, a C1-6 alkyl group optionally substituted by a halogen atom(s), and a C1-6 alkoxy group optionally substituted 10 by a halogen atom(s);
Ra is a hydrogen atom;
Rb is a hydrogen atom, or a group represented by the formula (a3):
[0075] 15
[0076] (wherein * indicates a bonding position;
j is an integer of 0 to 4; 20
Q in the number of j are as defined above;
R7 in the number of j are each independently an organic group having an alkyl group having not less than 10 and not more than 300 carbon atoms and/or an alkenyl group having not less than 10 and not more than 300 carbon atoms; 25
R6 is a hydrogen atom, or optionally a single bond or -O- in combination with R4 to form a fluorenyl group or a xanthenyl group together with ring A; and
ring B optionally further has, in addition to QR7 in the number of j, a substituent selected from the group consisting of a 30 halogen atom, a C1-6 alkyl group optionally substituted by a halogen atom(s), and a C1-6 alkoxy group optionally substituted
30
by a halogen atom(s)), or
Ra and Rb are optionally joined to form an oxygen atom.]]
[0077] In the below-mentioned production method of the morpholino oligonucleotide of the present invention, the 5’-5 hydroxy group is activated (thio)phosphated or activated (thio)phosphoramidated, and the morpholine ring nitrogen atom is bonded to p-mer morpholino oligonucleotide (p is any integer of one or more) protected by a temporary protecting group removable under acidic conditions, whereby the compound of the 10 present invention can form m+1+p-mer morpholino oligonucleotide (p is any integer of one or more).
When m is 0, the compound of the present invention is understood to be a “morpholino nucleoside”, which is a starting compound of the 5’-terminal in the synthesis of the morpholino 15 oligonucleotide of the present invention. In addition, the compound of the present invention also encompasses one wherein the morpholine ring nitrogen atom on the 3’-terminus side is unprotected (P1 is a hydrogen atom) in a broad sense.
[0078] 20 In the above-mentioned formula (I), m is any integer of not less than 0, preferably, 0. While the upper limit of m is not particularly limited, it is preferably 49 or less, more preferably 29 or less, further preferably 19 or less.
[0079] 25 Base in the number of m+1 in the above-mentioned formula (I) are each independently an optionally protected nucleic acid base. The “optionally protected nucleic acid base” means, for example, that an amino group may be protected in an adenyl group, a guanyl group or a cytosyl group, which is a nucleic 30 acid base having an amino group, or imide group is optionally protected in a thymyl group, an uracil group having a cyclic imide group, and a nucleic acid base wherein the amino group therein is protected by a protecting group sustainable under the deprotection conditions of the morpholine ring nitrogen 35
31
atom is preferable. The protecting group of the “amino-protecting group” and the “imido-protecting group” is not particularly limited and, for example, any protecting groups described in Greene’s PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, 4th ed., JOHN WILLY&SONS (2006) and the like can be mentioned. 5 Specific examples of such “amino-protecting group” and “imide-protecting group” include a pivaloyl group, a pivaloyloxymethyl group, a trifluoroacetyl group, a phenoxyacetyl group, a 4-isopropylphenoxyacetyl group, a 4-tert-butylphenoxyacetyl group, an acetyl group, a benzoyl group, an isobutyryl group, a 10 dimethylformamidinyl group, a 9-fluorenylmethyloxycarbonyl group and the like. Among them, a phenoxyacetyl group, a 4-isopropylphenoxyacetyl group, an acetyl group, a benzoyl group, an isobutyryl group and a dimethylformamidinyl group are preferable. In addition, the carbonyl group of the nucleic 15 acid base is optionally protected, and can be protected, for example, by reacting phenol, 2,5-dichlorophenol, 3-chlorophenol, 3,5-dichlorophenol, 2-formylphenol, 2-naphthol, 4-methoxyphenol, 4-chlorophenol, 2-nitrophenol, 4-nitrophenol, 4-acetylaminophenol, pentafluorophenol, 4-pivaloyloxybenzyl 20 alcohol, 4-nitrophenethyl alcohol, 2-(methylsulfonyl)ethanol, 2-(phenylsulfonyl)ethanol, 2-cyanoethanol, 2-(trimethylsilyl)ethanol, dimethylcarbamoyl chloride, diethylcarbamoyl chloride, ethylphenylcarbamoyl chloride, 1-pyrrolidinecarbonyl chloride, 4-morpholinecarbonyl chloride, 25 diphenylcarbamoyl chloride and the like. In some cases, the carbonyl-protecting group does not particularly need to be introduced.
[0080] The temporary protecting group P1 that can be used as the 30 protecting group of the morpholine ring nitrogen atom at the 3’-terminus of the present invention is not particularly limited as long as it can be deprotected under acidic conditions and can be used as a hydroxy-protecting group. Examples thereof include a trityl group, 9-(9-phenyl)xanthenyl 35
32
group, a 9-phenylthioxanthenyl group, di(C1-6 alkoxy)trityl groups such as a 1,1-bis(4-methoxyphenyl)-1-phenylmethyl group, a dimethoxytrityl group and the like, mono(C1-18 alkoxy)trityl groups such as 1-(4-methoxyphenyl)-1,1-diphenylmethyl group, monomethoxytrityl group and the like, and the like can be 5 mentioned. Among these, a trityl group, a monomethoxytrityl group and a dimethoxytrityl group are preferable, and a dimethoxytrityl group is more preferable, in view of easiness of deprotection and easy availability. Among these, a trityl group, a monomethoxytrityl group and a dimethoxytrityl group 10 are preferable, and a trityl group and a dimethoxytrityl group are more preferable, in view of easiness of deprotection and easy availability.
[0081] In the above-mentioned formula (I), X in the number of m 15 are each independently a C1-6 alkoxy group, a di-C1-6 alkylamino group, or a piperazino group wherein a nitrogen atom at the 4-position is protected by a protecting group and further optionally substituted, preferably a di-C1-6 alkylamino group.
As the C1-6 alkoxy group, a methoxy group or an ethoxy 20 group is preferable, and a methoxy group is more preferable.
As the di-C1-6 alkylamino group, a dimethylamino group, a diethylamino group, an N-ethyl-N-methylamino group and the like are preferable, and a dimethylamino group is preferable.
[0082] 25 As the protecting group of the 4-position nitrogen atom of the piperazino group, an acyl group is preferable and, for example, an acyl group having a fluoro group in the carbon chain, such as a monofluoroacetyl group, a difluoroacetyl group, a trifluoroacetyl group, a 2-fluoropropionyl group, a 2,2-30 difluoropropionyl group, a 3,3,3-trifluoropropionyl group, a 2,3,3,3-tetrafluoropropionyl group, a pentafluoropropionyl group and the like, is more preferable. While the protecting group is generally deprotected after completion of the elongation reaction, the amino group of the piperazino group 35
33
may be further modified, after deprotection, by a modifying group according to the method described in WO 2008/008113. Examples of the modifying group include a halogen atom, an alkyl group, an aralkyl group, an alkoxy group, an acyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, an aryl 5 group, a hydroxy group, a nitro group, a cyano group, a guanidyl group, a carboxy group, an alkoxycarbonyl group, a sulfo group, a phospho group, an alkylthio group, an alkylsulfinyl group, an alkylsulfonyl group and the like. As the modifying group of the piperazino group, an optionally 10 substituted acyl group is preferable, and an acyl group (e.g., 6-guanidinohexanoyl group) optionally substituted by a guanidyl group is more preferable. In the piperazino group, the hydrogen atom bonded to the carbon atom of the piperazino group may be substituted, and examples of the substituent include an 15 alkyl group (preferably having 1 - 3 carbon atoms) such as a methyl group and the like, and the like.
[0083] W in the number of m is each independently an oxygen atom or a sulfur atom, preferably an oxygen atom. 20
[0084] S1 in the above-mentioned formula (I) is a single bond, or a group represented by *O-S2** (wherein * indicates the bonding position to L, ** indicates the bonding position to 5’-hydroxy group, and S2 is a spacer having a main chain having an 25 atomic number of 1 to 20), preferably a single bond.
The “spacer having a main chain having an atomic number of 1 to 20” for S2 is, for example, a divalent group formed by removing a hydroxy group from the aforementioned “substituent having a hydroxy group” when the 5’-hydroxy group has a 30 substituent.
[0085] A preferable embodiment of the linker L represented by the above-mentioned formula (a1) is a group wherein, in the formula (a1), 35
34
L1 is an ethylene group or CH2-O-1,4-phenylene-O-CH2; and
L2 is C(=O), or a group represented by ***N(R3)-R1-N(R2)C(=O)** (wherein ** indicates the bonding position to L1, *** indicates the bonding position to Y, R1 is a C1-6 alkylene group, and R2 and R3 are each independently a hydrogen atom or an optionally 5 substituted C1-6 alkyl group, or R2 and R3 are optionally joined to form an optionally substituted C1-6 alkylene bond).
[0086] Another preferable embodiment of the linker L represented by the above-mentioned formula (a1) is a group wherein, in the 10 formula (a1),
L1 is an ethylene group; and
L2 is C(=O).
[0087] Another preferable embodiment of the linker L represented 15 by the above-mentioned formula (a1) is a group wherein, in the formula (a1),
L1 is an ethylene group; and
the moiety of N(R3)-R1-N(R2) for L2 is a piperazinylene group.
[0088] 20 Another preferable embodiment of the linker L represented by the above-mentioned formula (a1) is a group wherein, in the formula (a1),
L1 is an ethylene group; and
L2 is a group represented by ***N(R3)-R1-N(R2)C(=O)** (wherein ** 25 indicates the bonding position to L1, *** indicates the bonding position to Y, R1 is a pentylene group or a hexylene group, and R2 and R3 are each independently a hydrogen atom or a methyl group).
[0089] 30 A particularly preferable example of the above-mentioned linker L is a single bond or a succinyl group since it is easily available and economical.
[0090] Y in the above-mentioned formula (I) is a single bond, an 35
35
oxygen atom, or NR (wherein R is a hydrogen atom, an alkyl group or an aralkyl group).
[0091] In the present specification, the “alkyl group” for R is a C1-30 alkyl group, preferably a C1-10 alkyl group, more 5 preferably a C1-6 alkyl group. Specific preferable examples thereof include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl and the like, and methyl and ethyl are particularly preferable.
[0092] 10 In the present specification, the “aralkyl group” for R is a C7-30 aralkyl group, preferably a C7-20 aralkyl group, more preferably a C7-16 aralkyl group (C6-10 aryl-C1-6 alkyl group). Specific preferable examples thereof include benzyl, 1-phenylethyl, 2-phenylethyl, 1-phenylpropyl, α-naphthylmethyl, 15 1-(α-naphthyl)ethyl, 2-(α-naphthyl)ethyl, 1-(α-naphthyl)propyl, β-naphthylmethyl, 1-(β-naphthyl)ethyl, 2-(β-naphthyl)ethyl, 1-(β-naphthyl)propyl and the like, and benzyl is particularly preferable.
[0093] 20 R is preferably a hydrogen atom, a C1-6 alkyl group or a C7-16 aralkyl group, more preferably a hydrogen atom, methyl, ethyl or benzyl, particularly preferably a hydrogen atom.
[0094] Y is preferably a single bond, an oxygen atom or NH. 25
[0095] A preferable embodiment of Z is a group represented by the formula (a2).
[0096] The preferable embodiment for Z in the above-mentioned 30 formula (I), that is, a group represented by the formula (a2) for Z in the above-mentioned formula (I) is a particular benzyl group (in the formula (a2), both Ra and Rb are hydrogen atoms, and R4 is a hydrogen atom); a particular benzoyl group (in the formula (a2) wherein Ra and Rb are joined to form an oxygen 35
36
atom, and R4 is a hydrogen atom); a particular diphenylmethyl group (in the formula (a2), Ra is a hydrogen atom, R4 is a hydrogen atom, k is 1 to 3, and Rb is a group represented by the formula (a3) (wherein R6 is a hydrogen atom, and j is 0 or 1)); a particular fluorenyl group (in the formula (a2), Ra is a 5 hydrogen atom, k is 1, Rb is a group represented by the formula (a3) (wherein j is 0)), and R6 is a single bond together with R4 to form a fluorene ring together with ring A); a particular xanthenyl group (in the formula (a2), Ra is a hydrogen atom, k is 1, Rb is a group represented by the formula (a3) (wherein j 10 is 0), and R6 is -O- together with R4 to form a xanthine ring together with ring A).
[0097] In the QR5 group in the number of k in the above-mentioned formula (a2), and the QR7 group in the number of j in 15 the formula (a3), Q is a single bond, or -O-, -S-, -OC(=O)-, -NHC(=O)- or -NH-, preferably -O-. The QR5 group in the number of k, and QR7 group in the number of j may be the same or different.
[0098] 20 In the above-mentioned formula (a2), the “Ra and Rb are joined to form an oxygen atom” means that Ra and Rb are joined to form a carbonyl group (C(=O)).
[0099] The “organic group having an alkyl group having not less 25 than 10 and not more than 300 carbon atoms and/or an alkenyl group having not less than 10 and not more than 300 carbon atoms” for R5 or R7 is a monovalent organic group having an alkyl group having not less than 10 and not more than 300 carbon atoms and/or an alkenyl group having not less than 10 30 and not more than 300 carbon atoms in the molecule structure thereof.
[0100] The carbon number of the “alkyl group having not less than 10 and not more than 300 carbon atoms and/or alkenyl group 35
37
having not less than 10 and not more than 300 carbon atoms” of the “organic group having an alkyl group having not less than 10 and not more than 300 carbon atoms and/or an alkenyl group having not less than 10 and not more than 300 carbon atoms” is preferably 14 - 40, more preferably 14 - 30. 5
The moiety of the “alkyl group having not less than 10 and not more than 300 carbon atoms and/or alkenyl group having not less than 10 and not more than 300 carbon atoms” of the “organic group having an alkyl group having not less than 10 and not more than 300 carbon atoms and/or an alkenyl group 10 having not less than 10 and not more than 300 carbon atoms” is not particularly limited, and it may be present at the terminus (monovalent group), or the other site (e.g., divalent group).
[0101] As the “alkyl group having not less than 10 and not more 15 than 300 carbon atoms and/or alkenyl group having not less than 10 and not more than 300 carbon atoms”, a monovalent group and a divalent group induced therefrom can be mentioned. Among them, an alkyl group having 14 – 40 carbon atoms is preferable, and an alkyl group having 14 – 30 carbon atoms is particularly 20 preferable. Specific examples of the “alkyl group having not less than 10 and not more than 300 carbon atoms and/or alkenyl group having not less than 10 and not more than 300 carbon atoms” include monovalent linear aliphatic hydrocarbon groups such as a decyl group, a dodecyl group, a tridecyl group, a 25 myristyl group, a cetyl group, a stearyl group, an oleyl group, a linolyl group, an arachyl group, a behenyl group and the like, monovalent branched chain aliphatic hydrocarbon groups such as a 3,7,11,15-tetramethylhexadecyl group, a 3,7,11-trimethyldodecyl group, a 2,2,4,8,10,10-hexamethyl-5-dodecanoyl 30 group and the like, and divalent groups induced therefrom.
[0102] In the “organic group having an alkyl group having not less than 10 and not more than 300 carbon atoms and/or an alkenyl group having not less than 10 and not more than 300 35
38
carbon atoms”, the moiety other than the “alkyl group having not less than 10 and not more than 300 carbon atoms and/or alkenyl group having not less than 10 and not more than 300 carbon atoms” can be determined freely. For example, it optionally has a moiety such as -O-, -S-, -COO-, -OCONH-, and -5 CONH-, and a hydrocarbon group (monovalent group or divalent group) and the like as a linker. Examples of the “hydrocarbon group” include an aliphatic hydrocarbon group, an aromatic aliphatic hydrocarbon group, a monocyclic saturated hydrocarbon group, an aromatic hydrocarbon group and the like. 10 Specifically, for example, monovalent groups such as an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, an aryl group, an aralkyl group and the like, and divalent groups derived therefrom are used. As “alkyl group”, “alkenyl group”, “alkynyl group”, “cycloalkyl group”, “aryl group”, or 15 “aralkyl group” as the moiety other than “aliphatic hydrocarbon group”, those similar to the aforementioned groups can be mentioned. The “hydrocarbon group” is optionally substituted by a substituent selected from a halogen atom (chlorine atom, bromine atom, fluorine atom, iodine atom), a C1-6 alkyl group 20 optionally substituted by one or more halogen atoms, an oxo group and the like.
[0103] The “organic group having an alkyl group having not less than 10 and not more than 300 carbon atoms and/or an alkenyl 25 group having not less than 10 and not more than 300 carbon atoms” indicated as “R5 (group)” and/or “R7 (group)” constituting Z in the above-mentioned formula (I) may contain plural “alkyl groups having not less than 10 and not more than 300 carbon atoms and/or alkenyl groups having not less than 10 30 and not more than 300 carbon atoms” due to branching and the like. When a plurality of “alkyl group having not less than 10 and not more than 300 carbon atoms and/or alkenyl group having not less than 10 and not more than 300 carbon atoms” is present in the “organic group having an alkyl group having not less 35
39
than 10 and not more than 300 carbon atoms and/or an alkenyl group having not less than 10 and not more than 300 carbon atoms”, they may be the same or different.
[0104] The lower limit of the total carbon number of the 5 “organic group having an alkyl group having not less than 10 and not more than 300 carbon atoms and/or an alkenyl group having not less than 10 and not more than 300 carbon atoms” for “R5 (group)” and/or “R7 (group)” constituting Z in the above-mentioned formula (II) is preferably 10 or more, more 10 preferably 12 or more, further preferably 14 or more, still more preferably 18 or more, and particularly preferably 30 or more. On the other hand, the upper limit of the total carbon number of the “organic group having a an alkyl group having not less than 10 and not more than 300 carbon atoms and/or an 15 alkenyl group having not less than 10 and not more than 300 carbon atoms” for “R5(group)” and/or “R7(group)” is preferably 200 or less, more preferably 150 or less, further preferably 120 or less, still more preferably 100 or less, especially preferably 80 or less, and particularly preferably 60 or less. 20 When the carbon number is higher, the crystallinity or solubility of the compound of the present invention in a polar solvent is fine even when the morpholino oligonucleotide has a long chain.
[0105] 25 A preferable embodiment of Z represented by the above-mentioned formula (a2) is a group represented by the formula (a2), wherein, in the formula (a2),
Ra and Rb are both hydrogen atoms;
R4 is a hydrogen atom, 30
Q in the number of k is -O-,
R5 in the number of k are each independently an organic group having an alkyl group having not less than 10 and not more than 300 carbon atoms and/or an alkenyl group having not less than 10 and not more than 300 carbon atoms (e.g., C10-40 alkyl 35
40
group); and
k is an integer of 1 - 3.
[0106] Another preferable embodiment of Z represented by the above-mentioned formula (a2) is a group wherein, in the formula 5 (a2),
k is an integer of 1 – 3;
Ra and Rb are both hydrogen atoms;
R4 is a hydrogen atom,
Q in the number of k is -O-, 10
R5 in the number of k are each independently a benzyl group having 1 - 3 alkyl groups having not less than 10 and not more than 300 carbon atoms and/or alkenyl groups having not less than 10 and not more than 300 carbon atoms, or a cyclohexyl group having 1 - 3 alkyl groups having not less than 10 and not 15 more than 300 carbon atoms and/or alkenyl groups having not less than 10 and not more than 300 carbon atoms; and
ring A optionally further has, in addition to QR5 in the number of k, substituent(s) selected from the group consisting of a halogen atom, a C1-6 alkyl group optionally substituted by a 20 halogen atom, and a C1-6 alkoxy group optionally substituted by a halogen atom.
[0107] Another preferable embodiment of Z represented by the above-mentioned formula (a2) is a group wherein, in the formula 25 (a2),
Ra is a hydrogen atom; and
Rb is a group represented by the above-mentioned formula (a3) (wherein * indicates a bonding position; j is an integer of 0 to 3; Q in the number of j is –O-; R7 in the number of j are 30 each independently a C10-40 alkyl group; and R4 and R6 are both hydrogen atoms.
[0108] A still another preferable embodiment of Z represented by the above-mentioned formula (a2) is a group wherein, in the 35
41
formula (a2),
Ra is a hydrogen atom;
Rb is a group represented by the above-mentioned formula (a3) (wherein * indicates a bonding position; j is an integer of 0 to 3; Q in the number of j is –O-; R7 in the number of j are 5 each independently a C10-40 alkyl group;
R6 is joined with R4 of ring A to form a single bond or -O-, and therefore, ring A and ring B form a fluorenyl group or a xanthenyl group in combination.
[0109] 10 Another preferable embodiment of Z represented by the above-mentioned formula (a2) is a group wherein, in the formula (a2),
Ra and Rb are joined to form an oxygen atom;
R4 is a hydrogen atom, 15
Q in the number of k is -O-,
R5 in the number of k are each independently an organic group having an alkyl group having not less than 10 and not more than 300 carbon atoms and/or an alkenyl group having not less than 10 and not more than 300 carbon atoms (e.g., C10-40 alkyl 20 group); and
k is an integer of 1 - 3.
[0110] Another preferable embodiment of Z represented by the above-mentioned formula (a2) is a group wherein, in the formula 25 (a2),
k is an integer of 1 - 3;
Ra and Rb are joined to form an oxygen atom;
R4 is a hydrogen atom;
Q in the number of k is -O-, 30
R5 in the number of k are each independently a benzyl group having 1 - 3 alkyl groups having not less than 10 and not more than 300 carbon atoms and/or alkenyl groups having not less than 10 and not more than 300 carbon atoms, or a cyclohexyl group having 1 - 3 alkyl groups having not less than 10 and not 35
42
more than 300 carbon atoms and/or alkenyl groups having not less than 10 and not more than 300 carbon atoms; and
ring A optionally further has, in addition to QR5 in the number of k, substituent(s) selected from the group consisting of a halogen atom, a C1-6 alkyl group optionally substituted by a 5 halogen atom(s), and a C1-6 alkoxy group optionally substituted by a halogen atom(s).
[0111] As the protecting group represented by the formula (II): Z-Y-L-, a group not easily cleaved under acidic conditions 10 under which the protecting group P1 of the morpholine ring nitrogen atom at the 3’-terminus can be removed, and cleaved under basic conditions is preferable.
Representative examples of the protecting group include a group wherein, for example, 15
L is a group represented by the above-mentioned formula (a1) (preferably a succinyl group etc.), and
Z-Y is the following group:
a 3,4,5-tri(octadecyloxy)benzyloxy group,
a 3,5-di(docosyloxy)benzyloxy group, 20
a 3,5-bis[3’,4’,5’-tri(octadecyloxy)benzyloxy]benzyloxy group,
a 3,4,5-tris[3’,4’,5’-tri(octadecyloxy)benzyloxy]benzyloxy group,
a 3,4,5-tri(octadecyloxy)benzylamino group,
a 2,4-di(docosyloxy)benzylamino group, 25
a 3,5-di(docosyloxy)benzylamino group,
a di(4-docosyloxyphenyl)methylamino group,
a 4-methoxy-2-[3’,4’,5’-tri(octadecyloxy)benzyloxy]benzylamino group,
a 4-methoxy-2-[3’,4’,5’-30 tri(octadecyloxy)cyclohexylmethyloxy]benzylamino group,
a 2,4-di(dodecyloxy)benzylamino group,
a phenyl(2,3,4-tri(octadecyloxy)phenyl)methylamino group,
a di[4-(12-docosyloxydodecyloxy)phenyl]methylamino group,
a 3,5-bis[3’,4’,5’-tri(octadecyloxy)benzyloxy]benzylamino group, 35
43
or
a 3,4,5-tris[3’,4’,5’-tri(octadecyloxy)benzyloxy]benzylamino group.
[0112] As another embodiment of the protecting group Z-Y-L-, the 5 following benzylsuccinyl groups and diphenylmethylsuccinyl groups can be mentioned.
a 2-{2,4-di(2’,3’-dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl group;
a 3,5-di(2’,3’-dihydrophytyloxy)benzylsuccinyl group; 10
a 4-(2’,3’-dihydrophytyloxy)benzylsuccinyl group;
a 2-{1-[(2-chloro-5-(2’,3’-dihydrophytyloxy)phenyl)]benzylaminocarbonyl}ethylcarbonyl group;
a 3,4,5-tri(2’,3’-dihydrophytyloxy)benzylsuccinyl group; 15
a 2-{3,4,5-tri(2’,3’-dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl group;
a 2-{4-(2’,3’-dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl group;
a 2-{2-[3’,4’,5’-tri(2’’,3’’-dihydrophytyloxy)benzyloxy]-4-20 methoxybenzylaminocarbonyl}ethylcarbonyl group;
a 2-{4-(2’,3’-dihydrophytyloxy)-2-methoxybenzylaminocarbonyl}ethylcarbonyl group;
a 4-(2’,3’-dihydrophytyloxy)-2-methylbenzylsuccinyl group;
a 2-{4-(2’,3’-dihydrophytyloxy)-2-25 methylbenzylaminocarbonyl}ethylcarbonyl group;
a 4-[2,2,4,8,10,10-hexamethyl-5-dodecanoylamino]benzylsuccinyl group;
a 2-{4-[2,2,4,8,10,10-hexamethyl-5-dodecanoylamino]benzylaminocarbonyl}ethylcarbonyl group; 30
a 4-(3,7,11-trimethyldodecyloxy)benzylsuccinyl group;
a 2-{4-(3,7,11-trimethyldodecyloxy)benzylaminocarbonyl}ethylcarbonyl group;
a 2-{3,5-di(2’,3’-dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl group; 35
44
a 2-{1-[2,3,4-tri(2’,3’-dihydrophytyloxy)phenyl]benzylaminocarbonyl}ethylcarbonyl group;
a 2-{1-[4-(2’,3’-dihydrophytyloxy)phenyl]-4’-(2’,3’-dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl group; 5
a 3,4,5-tris[3,4,5-tri(2’,3’-dihydrophytyloxy)benzyl]benzylsuccinyl group; and
a 2-{3,4,5-tris[3,4,5-tri(2’,3’-dihydrophytyloxy)benzyl]benzylaminocarbonyl}ethylcarbonyl group.
[0113] 10 Another embodiment of the protecting group represented by the formula (II): Z-Y-L- is a group wherein
L and Y are each a single bond,
Z shows the formula (a2),
Ra and Rb are joined to form an oxygen atom; 15
R4 is a hydrogen atom,
Q in the number of k is -O-,
R5 in the number of k are each independently an organic group having an alkyl group having not less than 10 and not more than 300 carbon atoms and/or an alkenyl group having not less than 20 10 and not more than 300 carbon atoms (e.g., C10-40 alkyl group); and
k is an integer of 1 - 3.
[0114] Another embodiment of the protecting group represented by 25 the formula (II): Z-Y-L- is a group wherein
L shows the formula (a1),
L2 is ***N(R3)-R1-N(R2)C(=O)** (wherein ** indicates the bonding position to L1, *** indicates the bonding position to Y, R1 is an optionally substituted C1-22 alkylene group, R2 and R3 are 30 each independently a hydrogen atom or an optionally substituted C1-22 alkyl group, or R2 and R3 are optionally joined to form an optionally substituted C1-22 alkylene bond),
Y is a single bond,
Z shows the formula (a2), 35
45
Ra and Rb are joined to form an oxygen atom;
R4 is a hydrogen atom,
Q in the number of k is -O-,
R5 in the number of k are each independently an organic group having an alkyl group having not less than 10 and not more than 5 300 carbon atoms and/or an alkenyl group having not less than 10 and not more than 300 carbon atoms (e.g., C10-40 alkyl group); and
k is an integer of 1 - 3.
[0115] 10 A preferable embodiment of the compound of the present invention represented by the formula (I) is a compound of the formula (I), wherein
m is 0,
Base is a cytosyl group, a uracil group, a thyminyl group, an 15 adenyl group, or a guanyl group, each of which is optionally protected;
P1 is a trityl group, a di(C1-6 alkoxy)trityl group or a mono(C1-6 alkoxy)trityl group;
S1 is a single bond; and 20
Z-Y-L is the combination of each group shown as a preferable embodiment in the aforementioned formula (I).
[0116] Another preferable embodiment of the compound of the present invention represented by the formula (I) is a compound 25 of the formula (I), wherein
m is 0,
Base is a cytosyl group, a uracil group, a thyminyl group, an adenyl group, or a guanyl group, each of which is optionally protected; 30
P1 is a trityl group, a dimethoxytrityl group or a monomethoxytrityl group;
S1 is a single bond; and
Z-Y-L is the combination of each group shown as a preferable embodiment in the aforementioned formula (I). 35
46
[0117] A still another preferable embodiment of the compound of the present invention represented by the formula (I) is a compound of the formula (I), wherein
m is 0, 5
Base is a cytosyl group, a uracil group, a thyminyl group, an adenyl group, or a guanyl group, each of which is optionally protected;
P1 is a trityl group;
S1 is a single bond; and 10
Z-Y-L is the combination of each group shown as a preferable embodiment in the aforementioned formula (I).
[0118] [Production method of the compound of the present invention]
A production method of the compound of the present 15 invention represented by the formula (I) wherein m is 0, and S1 is a single bond (hereinafter to be referred to as “the formula (Ia)”) is not particularly limited, and it can be produced by a method known per se (Richard T. Pon et al., Nucleic Acids Research 2004, 32, 623-631) or a method analogous thereto. 20
A general production method of a compound of the above-mentioned formula (Ia) wherein L is a succinyl group is shown below.
[0119] 25
47
[0120] (wherein each symbol is as defined above.)
Morpholino nucleoside (a) wherein the 3’-terminus morpholine ring nitrogen atom is protected by a protecting 5 group P1 is reacted with succinic anhydride in the presence of a base to give compound (b) wherein succinic acid is introduced into the 5’-hydroxy group. Compound (b) is subjected to a dehydration condensation with a precursor (Z-Y-H) (alcohol or amine) of the protecting group in the presence of a condensing 10 agent, whereby a compound represented by the formula (Ia) can be obtained.
[0121] The conversion step of the above-mentioned morpholino nucleoside (a) to compound (b) is advantageously performed in a 15 solvent inert to the reaction. While such solvent is not particularly limited as long as the reaction proceeds, halogenated hydrocarbon solvents such as dichloromethane, 1,2-dichloroethane, chloroform, carbon tetrachloride and the like, aromatic hydrocarbon solvents such as benzene, toluene, xylene 20 and the like, aliphatic hydrocarbon solvents such as pentane,
condensing agent
48
hexane, heptane, octane and the like, ether solvents such as diethyl ether, tetrahydrofuran, cyclopentyl methyl ether and the like, and mixed solvents thereof are preferable. Of these, dichloromethane and chloroform are particularly preferable.
[0122] 5 While the base is not particularly limited, for example, an organic base mentioned below can be used, with preference given to N,N-dimethylaminopyridine, triethylamine and the like.
[0123] The above-mentioned dehydration condensation step is 10 advantageously performed in a solvent inert to the reaction. While such solvent is not particularly limited as long as the reaction proceeds, halogenated hydrocarbon solvents such as dichloromethane, 1,2-dichloroethane, chloroform, carbon tetrachloride and the like, aromatic hydrocarbon solvents such 15 as benzene, toluene, xylene and the like, or aliphatic hydrocarbon solvents such as pentane, hexane, heptane, octane and the like, and mixed solvents thereof are preferable. Of these, dichloromethane and chloroform are particularly preferable. 20
[0124] Examples of the condensing agent used for the condensation reaction of compound (b) with Z-Y-H include dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), N-ethyl-N’-(3-dimethylaminopropyl)carbodiimide and 25 hydrochloride thereof (EDC HCl), (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBop), O-(benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium tetrafluoroborate (TBTU), 1-[bis(dimethylamino)methylene]-5-chloro-1H-benzotriazolium-3-oxide hexafluorophosphate (HCTU), 30 O-benzotriazole-N,N,N’,N’-tetramethyluronium hexafluorophosphate (HBTU) and the like. Of these, HBTU, HCTU, N-ethyl-N’-(3-dimethylaminopropyl)carbodiimide and hydrochloride thereof (EDC HCl) are preferable.
[0125] 35
49
The amount of the condensing agent to be used is 1 to 10 mol, preferably 1 to 5 mol, per 1 mol of compound (b). The amount of Z-Y-H to be used is 1 to 10 mol, preferably 1 to 5 mol, per 1 mol of compound (b). While the reaction temperature is not particularly limited as long as the reaction proceeds, 5 it is preferably -10C to 50C, more preferably 0C to 30C. The reaction time is 30 min to 70 hr.
[0126] A compound of the above-mentioned formula (Ia) wherein L is other than a succinyl group can also be produced by 10 performing a reaction similar to the above-mentioned production method except that a corresponding acid anhydride, a corresponding dicarboxylic acid halide, an activated ester of corresponding dicarboxylic acid and the like is used instead of succinic anhydride. 15
A compound wherein S1 is *O-S2** (wherein each symbol is as defined above) can be produced by introducing the aforementioned “substituent having a hydroxy group” into the 5’-hydroxy group of the morpholino nucleoside (a) by a known method (e.g., the method described in WO 2008/008113) and 20 thereafter following the above-mentioned method.
A compound wherein Y is a single bond can be produced by reacting an activated derivative (halide, acid halide, activated carboxy group etc.) of Z-Y-H with morpholino nucleoside (a) by a method known per se, or reacting Z-Y-H with 25 morpholino nucleoside (a) in the presence of a condensing agent. The condensation reaction of Z-Y-H and morpholino nucleoside (a) can be performed in the same manner as in the condensation reaction of Z-Y-H and compound (b).
A compound of the above-mentioned formula (I) wherein m 30 is one or more can be produced by repeating the 5’-terminus elongation process according to the following production method of the present invention and using a compound represented by the formula (Ia) as a starting material.
[0127] 35
50
While the production method of precursor (Z-Y-H) (alcohol, amine or carboxylic acid) of the aforementioned protecting group is not particularly limited, it can be produced from a starting compound according to a method known per se (e.g., Bull. Chem. Soc. Jpn. 2001, 74, 733-738, JP-A-2000-44493, WO 5 2006/104166, WO 2007/034812, WO 2007/122847, WO 2010/113939, JP-A-2010-275254, WO 2012/157723 etc.) or a method analogous thereto.
A compound to be used as a starting compound, for example, a halide corresponding to R5 and R7 constituting Z in the 10 formula (I) and the like is a commercially available product, or can be produced according to a method known per se or a method analogous thereto.
The precursor (Z-Y-H) of the protecting group can be produced by a method known per se or a method analogous thereto, 15 as mentioned above. When a starting compound has a substituent (e.g., hydroxy group, amino group, carboxy group) that influences the reaction, the starting compound is generally protected in advance by a suitable protecting group according to a known method and then subjected to the reaction. Such 20 protecting group can be removed after the reaction by a known method such as an acid treatment, an alkali treatment, a catalytic reduction and the like.
[0128] While the production method of morpholino nucleoside (a) 25 wherein the 3’-terminus morpholine ring nitrogen atom is protected by protecting group P1 is not particularly limited, it can be produced from morpholino nucleoside (1) by a method known per se (e.g., see WO 91/09033A1) or a method analogous thereto. 30
For example, when P1 is a trityl group, morpholino nucleoside (1) is reacted with trityl chloride in the presence of a base such as triethylamine and the like, whereby compound (a) can be obtained.

claim:
1. A morpholino nucleotide represented by the formula (I):
[wherein 5
m is any integer of not less than 0,
Base in the number of m+1 are each independently an optionally protected nucleic acid base,
P1 is a hydrogen atom, or a temporary protecting group removable under acidic conditions, 10
X in the number of m are each independently a C1-6 alkoxy group, a di-C1-6 alkylamino group, or a piperazino group wherein a nitrogen atom at the 4-position is protected by a protecting group and further optionally substituted,
W in the number of m are each independently an oxygen atom or a 15 sulfur atom,
S1 is a single bond, or a group represented by *O-S2** (wherein * indicates the bonding position to L, ** indicates the bonding position to a 5’-hydroxy group, and S2 is a spacer having a main chain having an atomic number of 1 to 20), 20
L is a single bond, or a group represented by the formula (a1):
116
[wherein * indicates the bonding position to Y;
** indicates the bonding position to S1;
L1 is an optionally substituted divalent C1-22 hydrocarbon group; and 5
L2 is C(=O) or a group represented by ***N(R3)-R1-N(R2)C(=O)** (wherein ** indicates the bonding position to L1, *** indicates the bonding position to Y, R1 is an optionally substituted C1-22 alkylene group, R2 and R3 are each independently a hydrogen atom or an optionally substituted C1-22 alkyl group, or R2 and R3 10 are optionally joined to form an optionally substituted C1-22 alkylene bond)],
Y is a single bond, an oxygen atom or NR (wherein R is a hydrogen atom, an alkyl group or an aralkyl group), and
Z is a group represented by the formula (a2): 15
[wherein * indicates the bonding position to Y;
R4 is a hydrogen atom, or when Rb is a group represented by the 20 following formula (a3), R4 is optionally a single bond or -O- in combination with R6 to form a fluorenyl group or a xanthenyl group together with ring B;
Q in the number of k are each independently a single bond, or -O-, -S-, -OC(=O)-, -NHC(=O)- or -NH-; 25
R5 in the number of k are each independently an organic group
117
having an alkyl group having not less than 10 and not more than 300 carbon atoms and/or an alkenyl group having not less than 10 and not more than 300 carbon atoms;
k is an integer of 1 to 4;
ring A optionally further has, in addition to QR5 in the number 5 of k, a substituent selected from the group consisting of a halogen atom, a C1-6 alkyl group optionally substituted by a halogen atom(s), and a C1-6 alkoxy group optionally substituted by a halogen atom(s);
Ra is a hydrogen atom; 10
Rb is a hydrogen atom, or a group represented by the formula (a3):
15
(wherein * indicates a bonding position;
j is an integer of 0 to 4;
Q in the number of j are as defined above;
R7 in the number of j are each independently an organic group having an alkyl group having not less than 10 and not more than 20 300 carbon atoms and/or an alkenyl group having not less than 10 and not more than 300 carbon atoms;
R6 is a hydrogen atom, or optionally a single bond or -O- in combination with R4 to form a fluorenyl group or a xanthenyl group together with ring A; and 25
ring B optionally further has, in addition to QR7 in the number of j, a substituent selected from the group consisting of a halogen atom, a C1-6 alkyl group optionally substituted by a halogen atom(s), and a C1-6 alkoxy group optionally substituted by a halogen atom(s)), or 30
Ra and Rb are joined to form an oxygen atom]].
118
2. The morpholino nucleotide according to claim 1, wherein m is 0.
3. The morpholino nucleotide according to claim 1 or 2, wherein L in the formula (I) is a succinyl group, and 5
R5 and/or R7 are/is an alkyl group having 10 - 40 carbon atoms.
4. The morpholino nucleotide according to claim 1 or 2, wherein L in the formula (I) is a succinyl group, and
Ra and Rb are both hydrogen atoms, and R5 is an alkyl group 10 having 10 - 40 carbon atoms.
5. The morpholino nucleotide according to claim 1 or 2, wherein L in the formula (I) is a succinyl group, and
R5 and/or R7 are/is an alkyl group having 12 - 30 carbon atoms. 15
6. The morpholino nucleotide according to claim 1 or 2, wherein, in the formula (I), L is a succinyl group, and
Z-Y- is a group selected from the group consisting of a 3,4,5-tri(octadecyloxy)benzyloxy group, 20
a 3,5-di(docosyloxy)benzyloxy group,
a 3,5-bis[3’,4’,5’-tri(octadecyloxy)benzyloxy]benzyloxy group,
a 3,4,5-tris[3’,4’,5’-tri(octadecyloxy)benzyloxy]benzyloxy group,
a 3,4,5-tri(octadecyloxy)benzylamino group, 25
a 2,4-di(docosyloxy)benzylamino group,
a 3,5-di(docosyloxy)benzylamino group,
a di(4-docosyloxyphenyl)methylamino group,
a 4-methoxy-2-[3’,4’,5’-tri(octadecyloxy)benzyloxy]benzylamino group, 30
a 4-methoxy-2-[3’,4’,5’-tri(octadecyloxy)cyclohexylmethyloxy]benzylamino group,
a 2,4-di(dodecyloxy)benzylamino group,
a phenyl(2,3,4-tri(octadecyloxy)phenyl)methylamino group,
a di[4-(12-docosyloxydodecyloxy)phenyl]methylamino group, 35
119
a 3,5-bis[3’,4’,5’-tri(octadecyloxy)benzyloxy]benzylamino group, and
a 3,4,5-tris[3’,4’,5’-tri(octadecyloxy)benzyloxy]benzylamino group.
5 7. The morpholino nucleotide according to claim 1 or 2, wherein Z-Y-L- is selected from the group consisting of
a 2-{2,4-di(2’,3’-dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl group;
a 3,5-di(2’,3’-dihydrophytyloxy)benzylsuccinyl group; 10
a 4-(2’,3’-dihydrophytyloxy)benzylsuccinyl group;
a 2-{1-[(2-chloro-5-(2’,3’-dihydrophytyloxy)phenyl)]benzylaminocarbonyl}ethylcarbonyl group;
a 3,4,5-tri(2’,3’-dihydrophytyloxy)benzylsuccinyl group; 15
a 2-{3,4,5-tri(2’,3’-dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl group;
a 2-{4-(2’,3’-dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl group;
a 2-{2-[3’,4’,5’-tri(2’’,3’’-dihydrophytyloxy)benzyloxy]-4-20 methoxybenzylaminocarbonyl}ethylcarbonyl group;
a 2-{4-(2’,3’-dihydrophytyloxy)-2-methoxybenzylaminocarbonyl}ethylcarbonyl group;
a 4-(2’,3’-dihydrophytyloxy)-2-methylbenzylsuccinyl group;
a 2-{4-(2’,3’-dihydrophytyloxy)-2-25 methylbenzylaminocarbonyl}ethylcarbonyl group;
a 4-[2,2,4,8,10,10-hexamethyl-5-dodecanoylamino]benzylsuccinyl group;
a 2-{4-[2,2,4,8,10,10-hexamethyl-5-dodecanoylamino]benzylaminocarbonyl}ethylcarbonyl group; 30
a 4-(3,7,11-trimethyldodecyloxy)benzylsuccinyl group;
a 2-{4-(3,7,11-trimethyldodecyloxy)benzylaminocarbonyl}ethylcarbonyl group;
a 2-{3,5-di(2’,3’-dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl group; 35
120
a 2-{1-[2,3,4-tri(2’,3’-dihydrophytyloxy)phenyl]benzylaminocarbonyl}ethylcarbonyl group;
a 2-{1-[4-(2’,3’-dihydrophytyloxy)phenyl]-4’-(2’,3’-dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl group; 5
a 3,4,5-tris[3,4,5-tri(2’,3’-dihydrophytyloxy)benzyl]benzylsuccinyl group; and
a 2-{3,4,5-tris[3,4,5-tri(2’,3’-dihydrophytyloxy)benzyl]benzylaminocarbonyl}ethylcarbonyl group.
10 8. The morpholino nucleotide according to any one of claims 1 to 7, wherein P1 is a trityl group, a monomethoxytrityl group, or a dimethoxytrityl group.
9. A method of producing n+p-mer morpholino oligonucleotide, 15 comprising (2) a step of condensing a p-mer morpholino oligonucleotide (p is any integer of one or more) wherein a 5’-hydroxy group is activated (thio)phosphated or activated (thio)phosphoramidated, and a morpholine ring nitrogen atom is protected by a temporary protecting group removable under 20 acidic conditions, with an n-mer morpholino oligonucleotide (n is an integer of one or more) wherein a 5’-hydroxy group or, when the 5’-hydroxy group has a substituent having a hydroxy group, the hydroxy group present on the substituent is protected by a protecting group having an alkyl group having 25 not less than 10 and not more than 300 carbon atoms and/or an alkenyl group having not less than 10 and not more than 300 carbon atoms, and the morpholine ring nitrogen atom is not protected, by a (thio)phosphoramidate bond or (thio)phosphorodiamidate bond via the morpholine ring nitrogen 30 atom.
10. The production method according to claim 9, wherein p is 1.
11. The production method according to claim 9 or 10, 35
121
comprising treating the reaction mixture with a quenching agent after completion of the reaction.
12. The production method according to any one of claims 9 to 11, further comprising the following step (1): 5
(1) a step of removing the temporary protecting group of the morpholine ring nitrogen atom by reacting, before the condensation step (2), the n-mer morpholino oligonucleotide wherein the 5’-hydroxy group or, when the 5’-hydroxy group has a substituent having a hydroxy group, the hydroxy group present 10 on the substituent is protected by a protecting group having an alkyl group having not less than 10 and not more than 300 carbon atoms and/or an alkenyl group having not less than 10 and not more than 300 carbon atoms, and the morpholine ring nitrogen atom is protected by a temporary protecting group 15 removable under acidic conditions, with an acid in a non-polar solvent.
13. The production method according to claim 12, wherein the temporary protecting group is removed in the presence of a 20 cation scavenger.
14. The production method according to claim 12 or 13, further comprising a step of neutralizing with an organic base in step (1), after removing the temporary protecting group of the 25 morpholine ring nitrogen atom.
15. The production method according to any one of claims 9 to 14, wherein the protecting group having an alkyl group having not less than 10 and not more than 300 carbon atoms and/or an 30 alkenyl group having not less than 10 and not more than 300 carbon atoms is a group represented by the following formula (II):
35
122
[L is a single bond, or a group represented by the formula (a1):
[wherein * indicates the bonding position to Y; 5
** indicates the bonding position to S1;
L1 is an optionally substituted divalent C1-22 hydrocarbon group; and
L2 is C(=O) or a group represented by ***N(R3)-R1-N(R2)C(=O)** (wherein ** indicates the bonding position to L1, *** indicates 10 the bonding position to Y, R1 is an optionally substituted C1-22 alkylene group, R2 and R3 are each independently a hydrogen atom or an optionally substituted C1-22 alkyl group, or R2 and R3 are optionally joined to form an optionally substituted C1-22 alkylene bond)], 15
Y is a single bond, an oxygen atom or NR (wherein R is a hydrogen atom, an alkyl group or an aralkyl group), and
Z is a group represented by the formula (a2):
20
[wherein * indicates the bonding position to Y;
R4 is a hydrogen atom, or when Rb is a group represented by the following formula (a3), R4 is optionally a single bond or -O- in combination with R6 to form a fluorenyl group or a xanthenyl 25 group together with ring B;
Q in the number of k are each independently a single bond, or -
123
O-, -S-, -OC(=O)-, -NHC(=O)- or -NH-;
R5 in the number of k are each independently an organic group having an alkyl group having not less than 10 and not more than 300 carbon atoms and/or an alkenyl group having not less than 10 and not more than 300 carbon atoms; 5
k is an integer of 1 to 4;
ring A optionally further has, in addition to QR5 in the number of k, a substituent selected from the group consisting of a halogen atom, a C1-6 alkyl group optionally substituted by a halogen atom(s), and a C1-6 alkoxy group optionally substituted 10 by a halogen atom(s);
Ra is a hydrogen atom;
Rb is a hydrogen atom, or a group represented by the formula (a3):
15
(wherein * indicates a bonding position;
j is an integer of 0 to 4;
Q in the number of j are as defined above; 20
R7 in the number of j are each independently an organic group having an alkyl group having not less than 10 and not more than 300 carbon atoms and/or an alkenyl group having not less than 10 and not more than 300 carbon atoms;
R6 is a hydrogen atom, or optionally a single bond or -O- in 25 combination with R4 to form a fluorenyl group or a xanthenyl group together with ring A; and
ring B optionally further has, in addition to QR7 in the number of j, a substituent selected from the group consisting of a halogen atom, a C1-6 alkyl group optionally substituted by a 30 halogen atom(s), and a C1-6 alkoxy group optionally substituted by a halogen atom(s)), or
124
Ra and Rb are joined to form an oxygen atom].
16. The production method according to claim 15, further comprising the following step (3):
(3) a step of obtaining morpholino oligonucleotide by adding a 5 polar solvent to the reaction mixture obtained in step (1) and/or (2) and collecting precipitates thereof by solid-liquid separation.
17. The production method according to any one of claims 12 to 10 16, wherein the non-polar solvent is selected from the group consisting of a halogenated solvent, an aromatic solvent, an ester solvent, an aliphatic solvent, a non-polar ether solvent, and a combination of these.
15 18. The production method according to claim 16 or 17, wherein the polar solvent is an alcohol solvent or a nitrile solvent.
19. The production method according to any one of claims 9 to 18, further comprising the following step (4): 20
(4) a step of removing all the protecting groups of the obtained n+p-mer morpholino oligonucleotide.
20. The production method according to any one of claims 9 to 19, wherein the temporary protecting group removable under 25 acidic conditions is a trityl group, a dimethoxytrityl group, or a monomethoxytrityl group.