CA2621104A1 - 2'-deoxyguanosine hybrid compound containing pyrrole amide tetramer, method of producing the same and production intermediate therefor - Google Patents
2'-deoxyguanosine hybrid compound containing pyrrole amide tetramer, method of producing the same and production intermediate therefor Download PDFInfo
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- CA2621104A1 CA2621104A1 CA002621104A CA2621104A CA2621104A1 CA 2621104 A1 CA2621104 A1 CA 2621104A1 CA 002621104 A CA002621104 A CA 002621104A CA 2621104 A CA2621104 A CA 2621104A CA 2621104 A1 CA2621104 A1 CA 2621104A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/16—Purine radicals
- C07H19/173—Purine radicals with 2-deoxyribosyl as the saccharide radical
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D207/30—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members
- C07D207/34—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D473/00—Heterocyclic compounds containing purine ring systems
- C07D473/02—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6
- C07D473/18—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6 one oxygen and one nitrogen atom, e.g. guanine
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Abstract
It is intended to provide an MGB polyamide hybrid compound which has a function of recognizing a base sequence useful in gene therapy, is efficacious in synthesizing an oligomer and has a high stability. An MGB polyamide hybrid compound represented by the following formula (38) which is characterized by being a pyrrole amide tetramer prepared by introducing a 1-methylpyrrole-2-carbonyl group as a substitute for the N-terminal formyl group in the existing case.
Description
DESCRIPTION
2'-DEOXYGUANOSINE HYBRID COMPOUND CONTAINING PYRROLE
AMIDE TETRAMER, METHOD OF PRODUCING THE SAME AND
PRODUCTION INTERMEDIATE THEREOF
Technical Field The present invention relates to a novel hybrid compound in which a pyrrole amide tetramer is introduced via a linker to N2-position of 2'-deoxyguanosine and which is useful as a medicinal agent for gene therapies, production method and production intermediates therefor.
Background Art Developments of recent molecular biology and genetic technique are remarkable. Particularly, with the development of genome science, human genome has been analyzed, and further the genetic analyses of malignant neoplasms and pathogenic viruses are in progress as post-genome research. A number of causative genes responsible for diseases whose etiology has been hitherto unknown have been identified one after another, and the onset mechanism thereof have become elucidated at molecule level. There has been an idea of such gene therapies since 1950's when existence of gene diseases came to be known, but it has gradually become realistic as feasible treatment means in consequence of the subsequent development of recombinant DNA technique and gene introduction technology.
As gene therapy methods, the three following methods are known.
(1) Method of directly operating a gene itself;
This is a method of adding a normal gene to a cell while leaving the abnormal gene untouched, and this method was realized at early time and the practical use thereof is now rapidly worked forward. The gene therapy for "ADA (adenosine deaminase) deficiency" performed in the past falls under the category of this method. In addition to that, means to add respective normal gene to a cell is under examination as gene therapies for diseases such as familial hypercholesterolemia, cystic fibrosis and Fanconi anemia.
It may be said that this method is a comparatively simple method among gene therapies but there are restrictive conditions for application that the abnormal gene does not produce a harmfulness protein, that the disease is caused by the mechanism that the gene does not perform necessary functions, and so on.
(2) Method of stopping the abnormal gene from working;
This is a so-called "antisense DNA" therapy comprising binding an artificial gene sequence to a specific target gene sequence, thereby inhibiting the transcription of the abnormal gene and inactivating the function of the abnormal gene.
AMIDE TETRAMER, METHOD OF PRODUCING THE SAME AND
PRODUCTION INTERMEDIATE THEREOF
Technical Field The present invention relates to a novel hybrid compound in which a pyrrole amide tetramer is introduced via a linker to N2-position of 2'-deoxyguanosine and which is useful as a medicinal agent for gene therapies, production method and production intermediates therefor.
Background Art Developments of recent molecular biology and genetic technique are remarkable. Particularly, with the development of genome science, human genome has been analyzed, and further the genetic analyses of malignant neoplasms and pathogenic viruses are in progress as post-genome research. A number of causative genes responsible for diseases whose etiology has been hitherto unknown have been identified one after another, and the onset mechanism thereof have become elucidated at molecule level. There has been an idea of such gene therapies since 1950's when existence of gene diseases came to be known, but it has gradually become realistic as feasible treatment means in consequence of the subsequent development of recombinant DNA technique and gene introduction technology.
As gene therapy methods, the three following methods are known.
(1) Method of directly operating a gene itself;
This is a method of adding a normal gene to a cell while leaving the abnormal gene untouched, and this method was realized at early time and the practical use thereof is now rapidly worked forward. The gene therapy for "ADA (adenosine deaminase) deficiency" performed in the past falls under the category of this method. In addition to that, means to add respective normal gene to a cell is under examination as gene therapies for diseases such as familial hypercholesterolemia, cystic fibrosis and Fanconi anemia.
It may be said that this method is a comparatively simple method among gene therapies but there are restrictive conditions for application that the abnormal gene does not produce a harmfulness protein, that the disease is caused by the mechanism that the gene does not perform necessary functions, and so on.
(2) Method of stopping the abnormal gene from working;
This is a so-called "antisense DNA" therapy comprising binding an artificial gene sequence to a specific target gene sequence, thereby inhibiting the transcription of the abnormal gene and inactivating the function of the abnormal gene.
(3) Method of cutting off the abnormal part of the gene;
This is a method of cutting off the abnormal gene using a "restriction enzyme". In the current technology, however, restriction enzymes cannot cut off a target part freely as desired and there is a limitation at the cutting off position. Therefore, the application to practical treatment is difficult at the current level.
The method of the above (2) is called antisense method or antigene method, and expected as a gene therapy method for various cancer diseases and viral diseases.
From this, the development of a compound which can specifically recognize a particular etiologic target gene sequence and inhibit the expression thereof is desired.
The characteristics demanded for such a compound are as follows.
1. Ability of discerning a base sequence of a sufficient length to specify a target gene in each cell.
2. High affinity only to the target base sequence.
3. Readiness of chemical synthesis.
This is a method of cutting off the abnormal gene using a "restriction enzyme". In the current technology, however, restriction enzymes cannot cut off a target part freely as desired and there is a limitation at the cutting off position. Therefore, the application to practical treatment is difficult at the current level.
The method of the above (2) is called antisense method or antigene method, and expected as a gene therapy method for various cancer diseases and viral diseases.
From this, the development of a compound which can specifically recognize a particular etiologic target gene sequence and inhibit the expression thereof is desired.
The characteristics demanded for such a compound are as follows.
1. Ability of discerning a base sequence of a sufficient length to specify a target gene in each cell.
2. High affinity only to the target base sequence.
3. Readiness of chemical synthesis.
4. Stability in the presence of various enzymes in the living body.
5. Low cytotoxicity.
6. Ability of readily permeating the cell membrane and nuclear membrane.
7. Ability of repeatedly acting (turnover) on a lot of cells without being deactivated at one action.
On the other hand, genes having a base sequence to be targeted include:
A. Double stranded DNA (reproduction and start of transcription is inhibited: antigene method), B. Single stranded DNA (transcription is inhibited:
antisense method), and C. mRNA (translation is inhibited: antisense method, RNAi method).
The above antisense method and antigene method are methods which exhibit inhibitory effect on the proliferation of cells and bacteria as well as viruses having a target mRNA by binding a target mRNA, single stranded or double stranded DNA with a DNA which has been subjected to some kind of modification, thereby inhibiting reproduction, transcription or translation or promoting degradation by RNase H. Among compounds used for the antisense method, phosphorothioate type oligonucleotides in which one oxygen atom in the phosphate group is replaced with its congener, a sulfur atom are particularly extensively studied. These are excellent in (1) decomposition activity by RNase H, (2) resistance to nuclease, (3) cell membrane permeability and (4) water solubility and are highly convenient in that they can be used in combination with modification at the other sugar moiety and the base region thereof.
The first antisense drug approved by FDA (American Food and Drug Administration) in the application for ~ =
retinitis by cytomegalovirus is a phosphorothioate type oligonucleotide ((21-mer) 21-meric compound) (Vitravene (registered trademark)) and the effect on muscular dystrophy has been also reported recently. The phosphorothioate type oligonucleotides as therapeutic drugs, however, have problems such as low thermodynamic stability of double strands as compared with natural DNAs, insufficient recognition ability of base sequences, and besides, they may exhibit bioactivity not based on the activity as an antisense due to their strong interaction with proteins in the living body. In addition to these, BNA (Bridged ring Nucleic Acid) in which puckering of the furanose ring in the sugar moiety is fixed to 3'-endo type (N type) to enhance affinity with mRNAs, those in which a substituent group to enhance stacking effect is introduced to the base region and PNA in which sugar is replaced with an amide bond polymer in the main chain are proposed as candidates of therapeutic drugs, but they are still in a research stage at present.
Among therapies using DNA, a gene therapy using a decoy type DNA which does not target a gene but a sequence at a recognizing site in a protein referred to as transcription modulating factor and inhibits the function thereof draws attention.
Incidentally, technology using genes which has greatly attracted attention in late years is RNA
interference (RNAi) method. This method uses a series of flow starting from a cleavage of a long double stranded RNA (dsRNA) into a short double stranded RNA (small interfering RNAs; siRNAs) of 21 to 23 base pairs by an enzyme called Dicer in the living body, formation of a complex called RISC (RNA-induced silencing complex), conversion of a taken-in double stranded siRNA to a single stranded siRNA by RNA helicase, sequence selective binding of RISC to a target mRNA using a single stranded siRNA as a guide molecule and suppression of the gene expression in consequence of cleavage of mRNA to which RISC binds by RNase H. Practical applications can be easily performed by directly introducing a dsRNA having an arbitrary sequence into the cell or expressing it in vivo using a vector but there somewhat remains a problem of RNAs' stability.
As stated above, nucleic acids or the derivatives thereof can be developed into applications as medicinal drugs by making use of their intrinsic affinity to base sequence selecting genes.
In the meantime, some compounds which base-sequence-selectively act on the genes although they are not derivatives of nucleic acids as mentioned above.
Distamycin A, an antibiotic isolated from a bacterium and represented by the following formula, improves thermal stability of double stranded DNAs having three or more consecutive bases of adenine (A) or thymine (T).
H
~__ 0 HN
N~
I\
i VN ~'N CI- *NH2 0 The bow shaped molecular structure of distamycin A
matches the curve of minor groove of a B type DNA, and the compound stabilizes the binding between the molecules by forming hydrogen bonds between the inward-looking NH
groups slightly acidic with N3-position of adenine or with the carbonyl oxygen of C2-position of thymine. When the sequence contains a guanine (G) - cytidine (C) base pair, there is caused a three-dimensional obstacle between the amino group of the C2-position of guanine protruding into the minor groove and the hydrogen of the C3-position of 1-methylpyrrole (Py), and affinity to the minor groove decreases thereby exhibiting base sequence selectivity.
Subsequent studies have revealed that antiparallelly disposed two molecules of distamycin A coordinate to a single match site, and further that all of the four base pair combinations (A-T, T-A, G-C and C-G) can be recognized by replacing 1-methylpyrrole of the antiparallelly disposed distamycin A derivative with 1-methylimidazole (Im) or 3-hydroxy-l-methylpyrrole (Hp) (for example, Non-Patent Document 1). In addition, Dervan succeeded in binding antiparallelly disposed Py-Im polyamide compounds via a linker and improving the sequence selectivity and affinity to a double stranded DNA (for example, Non-Patent Document 2).
Furthermore, now that automatic production process thereof by solid phase method has been recently established, the polyamide compounds which recognize arbitrary base sequences is enabled to be easily synthesized and they are attracting increasing attention as novel base sequence recognizing molecules due to their practical advantage that a compound which will be adaptable to a target base sequence can be readily synthesized and as well as their excellent stability against nucleases which is one of the problems associated with nucleic acid derivatives and their excellent permeating properties through cell membrane and nuclear membrane.
As for the studies using these polyamide compounds, a number of attempts have been made to have a functional molecule affecting DNA bind a sequence selective minor groove binders (MGBs) polyamide and express the function selectively on base sequence. The representative examples used bleomycin, nitrogen mustard, cyclopropyl indole, DNA intercalator and duocarmycin as functional molecules. Of these, Sugiyama et al. have succeeded in . =
On the other hand, genes having a base sequence to be targeted include:
A. Double stranded DNA (reproduction and start of transcription is inhibited: antigene method), B. Single stranded DNA (transcription is inhibited:
antisense method), and C. mRNA (translation is inhibited: antisense method, RNAi method).
The above antisense method and antigene method are methods which exhibit inhibitory effect on the proliferation of cells and bacteria as well as viruses having a target mRNA by binding a target mRNA, single stranded or double stranded DNA with a DNA which has been subjected to some kind of modification, thereby inhibiting reproduction, transcription or translation or promoting degradation by RNase H. Among compounds used for the antisense method, phosphorothioate type oligonucleotides in which one oxygen atom in the phosphate group is replaced with its congener, a sulfur atom are particularly extensively studied. These are excellent in (1) decomposition activity by RNase H, (2) resistance to nuclease, (3) cell membrane permeability and (4) water solubility and are highly convenient in that they can be used in combination with modification at the other sugar moiety and the base region thereof.
The first antisense drug approved by FDA (American Food and Drug Administration) in the application for ~ =
retinitis by cytomegalovirus is a phosphorothioate type oligonucleotide ((21-mer) 21-meric compound) (Vitravene (registered trademark)) and the effect on muscular dystrophy has been also reported recently. The phosphorothioate type oligonucleotides as therapeutic drugs, however, have problems such as low thermodynamic stability of double strands as compared with natural DNAs, insufficient recognition ability of base sequences, and besides, they may exhibit bioactivity not based on the activity as an antisense due to their strong interaction with proteins in the living body. In addition to these, BNA (Bridged ring Nucleic Acid) in which puckering of the furanose ring in the sugar moiety is fixed to 3'-endo type (N type) to enhance affinity with mRNAs, those in which a substituent group to enhance stacking effect is introduced to the base region and PNA in which sugar is replaced with an amide bond polymer in the main chain are proposed as candidates of therapeutic drugs, but they are still in a research stage at present.
Among therapies using DNA, a gene therapy using a decoy type DNA which does not target a gene but a sequence at a recognizing site in a protein referred to as transcription modulating factor and inhibits the function thereof draws attention.
Incidentally, technology using genes which has greatly attracted attention in late years is RNA
interference (RNAi) method. This method uses a series of flow starting from a cleavage of a long double stranded RNA (dsRNA) into a short double stranded RNA (small interfering RNAs; siRNAs) of 21 to 23 base pairs by an enzyme called Dicer in the living body, formation of a complex called RISC (RNA-induced silencing complex), conversion of a taken-in double stranded siRNA to a single stranded siRNA by RNA helicase, sequence selective binding of RISC to a target mRNA using a single stranded siRNA as a guide molecule and suppression of the gene expression in consequence of cleavage of mRNA to which RISC binds by RNase H. Practical applications can be easily performed by directly introducing a dsRNA having an arbitrary sequence into the cell or expressing it in vivo using a vector but there somewhat remains a problem of RNAs' stability.
As stated above, nucleic acids or the derivatives thereof can be developed into applications as medicinal drugs by making use of their intrinsic affinity to base sequence selecting genes.
In the meantime, some compounds which base-sequence-selectively act on the genes although they are not derivatives of nucleic acids as mentioned above.
Distamycin A, an antibiotic isolated from a bacterium and represented by the following formula, improves thermal stability of double stranded DNAs having three or more consecutive bases of adenine (A) or thymine (T).
H
~__ 0 HN
N~
I\
i VN ~'N CI- *NH2 0 The bow shaped molecular structure of distamycin A
matches the curve of minor groove of a B type DNA, and the compound stabilizes the binding between the molecules by forming hydrogen bonds between the inward-looking NH
groups slightly acidic with N3-position of adenine or with the carbonyl oxygen of C2-position of thymine. When the sequence contains a guanine (G) - cytidine (C) base pair, there is caused a three-dimensional obstacle between the amino group of the C2-position of guanine protruding into the minor groove and the hydrogen of the C3-position of 1-methylpyrrole (Py), and affinity to the minor groove decreases thereby exhibiting base sequence selectivity.
Subsequent studies have revealed that antiparallelly disposed two molecules of distamycin A coordinate to a single match site, and further that all of the four base pair combinations (A-T, T-A, G-C and C-G) can be recognized by replacing 1-methylpyrrole of the antiparallelly disposed distamycin A derivative with 1-methylimidazole (Im) or 3-hydroxy-l-methylpyrrole (Hp) (for example, Non-Patent Document 1). In addition, Dervan succeeded in binding antiparallelly disposed Py-Im polyamide compounds via a linker and improving the sequence selectivity and affinity to a double stranded DNA (for example, Non-Patent Document 2).
Furthermore, now that automatic production process thereof by solid phase method has been recently established, the polyamide compounds which recognize arbitrary base sequences is enabled to be easily synthesized and they are attracting increasing attention as novel base sequence recognizing molecules due to their practical advantage that a compound which will be adaptable to a target base sequence can be readily synthesized and as well as their excellent stability against nucleases which is one of the problems associated with nucleic acid derivatives and their excellent permeating properties through cell membrane and nuclear membrane.
As for the studies using these polyamide compounds, a number of attempts have been made to have a functional molecule affecting DNA bind a sequence selective minor groove binders (MGBs) polyamide and express the function selectively on base sequence. The representative examples used bleomycin, nitrogen mustard, cyclopropyl indole, DNA intercalator and duocarmycin as functional molecules. Of these, Sugiyama et al. have succeeded in . =
having a hybrid compound of duocarmycin and the derivatives thereof with an MGB polyamide selectively act on an arbitrary target base sequence (Non-Patent Document 3). It has been thus demonstrated that the MGB polyamide can add unique base sequence recognition ability while maintaining a pharmacological activity of a functional molecule in this way, and what can be to is clarified.
The present inventors also synthesized and reported hybrid compounds in which a pyrrole polyamide trimer was introduced to N2-position of 2'-deoxyguanosine via a linker having an amide bond and hybrid compounds in which a pyrrole amide trimer was introduced via a linker by N-alkylation as shown below aiming at creating a genetic information control molecule based on genome chemistry under the above background (Non-Patent Document 4, Non-Patent Document 5).
H
~=0 HN
N", ~N : NH
HO 0 N N~NH H N
O~N~ / ~ 0 OH " N" (1) H
>=0 HN
N~
N
</ ~ ~ \
HO N N NH H N I N
~ V N' 0 OH N"
0 (20) When these hybrid compounds of a nucleoside and an MGB polyamide are taken into a DNA by a biosynthetic pathway like normal nucleic acids or modified nucleosides used as anticancer agents, it is supposed that they exhibit stabilization of the double stranded DNA by forming an extremely tight and stable binding to the minor groove thereby inhibiting replication and transcription of the DNA in the case that a site which the MGB polyamide specifically recognizes is present in the neighboring sequence.
On the other hand, in the case that a site which the MGB polyamide does not specifically recognize is present in the neighboring region, it can be expected that the compound does not exhibit stabilization of the double stranded DNA, thereby inhibiting no reproduction or transcription. In addition, when an MGB polyamide-nucleoside hybrid compound acts on a double stranded DNA
by itself, it is supposed that the nucleoside in the hybrid forms a Hoogsteen type hydrogen bond and has an antigene-like effect of exhibiting affinity and base selectivity by the MGB polyamide.
As described above, hybrid compounds of the MGB
polyamide have all of the characteristics such as high base sequence recognition ability, high stability of MGB
polyamide against enzymes in the living body and excellent membrane permeability and can be expected as candidates as genetic information control molecules.
[Non-Patent Document 1] White, S.; Baird, E. E.; Dervan, P. B. Chem. Biol., 1997, 4, 569.
[Non-Patent Document 2] Trauger, J. W.; Baird, E. E.;
Dervan, P. B. J. Am. Chem. Soc., 1996,118, 6160.
[Non-Patent Document 3] Tao, Z.; Fujiwara, T.; Sugiyama, H. J. Am. Chem.Soc., 1999,121,4961.
[Non-Patent Document 4] Y.; Ohba, Y.; Terui, K.; Kamaike, T.; Oshima, E.; Kawashima, Nucleic Acids Symposium Series No. 48, 2004, p.55-56.
[Non-Patent Document 4] Y.; Ohba, Y.; Terui, K.; Kamaike, T.; Oshima, E.; Kawashima, Nucleic Acids Symposium Series No. 48, 2004,p.55-56.
[Non-Patent Document 5] "Abstracts of 14th antisense symposium" Antisense DNA/RNA Research Group, December 2, 2004, p. 55.
Disclosure of the Invention The MGB polyamide can add unique base sequence recognition ability while maintaining the pharmacological activity of various functional molecules by forming hybrids with such molecules. Furthermore, since the MGB
polyamide has high stability against enzymes in the living body and excellent membrane permeability in addition to the high base sequence recognition ability, a hybrid compound with the MGB polyamide can be expected to have all of these characteristics.
These hybrid compounds have an effect of selectively stabilizing dsDNA containing a target base sequence (antigene type approach) . Alternatively, the hybrid compound itself is incorporated in a DNA via the biosynthetic pathway and have a selectively stabilizing effect by primarily coordinating only to the minor groove having a site which the MGB polyamide in this DNA
specifically recognizes, and therefore, they can be expected to inhibit reproduction and transcription of the target gene.
Incidentally, when the present inventors evaluated the stability against the ammonia treatment necessary in the oligomer synthesis of the above hybrid compound (20), 2'-deoxyguanosine, which was detected by the ammonia treatment of the hybrid compound (1) via a linker by amide bonding, was not detected in the examination by TLC
and 'H-NMR. It has become clear from this that the stability at the N2-position of the hybrid compound against the ammonia treatment can be enhanced by changing binding form to the linker by N-alkylation. However, a spot having an Rf value lower than that of (20) was just slightly caused on TLC. From 'H-NMR data, this was supposed to be a compound having lost a formyl group of the N-terminal end. That is, although 2'-deoxyguanosine was not detected in the ammonia treatment, dropout of a formyl group of the N-terminal end was observed, which means that the above compound (20) does not necessarily have satisfactory stability.
Therefore, an object of the present invention is to provide an MGB polyamide hybrid compound excellent in stability which enables to synthesize the oligomer effectively while maintaining the performance of the MGB
polyamide hybrid compound. Another object of the present invention is to provide a production method of the MGB
polyamide hybrid compound and the intermediates useful for that purpose.
The present inventors have conducted intensive studies for finding an MGB polyamide hybrid compound which has high base sequence recognition ability, high stability against enzymes in the living body and excellent membrane permeability and enables to synthesize the oligomer effectively, and consequently have found that it is enabled to synthesize the oligomer effectively while maintaining excellent base sequence recognition ability, excellent resistance to enzymes in the living body and excellent membrane permeability by introducing a 1-methylpyrrole-2-carbonyl group in place of a formyl group at the N-terminal end to form a pyrrole amide tetramer, and thus completed the present invention.
That is, the present invention is directed to the following.
1. A 21-deoxyguanosine hybrid compound represented by following general formula (38) characterized in that the compound contains a pyrrole amide tetramer. 0 HN
\ N~
~/ " J H \
HO N N NH H N I \
OH 0 N (38) 2. A 2'-deoxyguanosine hybrid compound having protected hydroxyl groups and represented by following general formula (37) characterized in that the compound contains a pyrrole amide tetramer, HN
\ N~
N
, ~ ~
RO O N N~!NH H N I\
N 0 (37) wherein R' and R2 each mean the same or different hydroxyl group protecting groups.
3. A protected 2'-deoxyguanosine hybrid compound represented by following general formula (36) characterized in that the compound contains a pyrrole amide tetramer, 0 HN
\ N~
/N \N 0 Rl0 0\N N5:~NH H N I \ 11 R2 0 0 N (36) wherein R', R 2 and R3 each mean the same or different hydroxyl group protecting groups.
The present inventors also synthesized and reported hybrid compounds in which a pyrrole polyamide trimer was introduced to N2-position of 2'-deoxyguanosine via a linker having an amide bond and hybrid compounds in which a pyrrole amide trimer was introduced via a linker by N-alkylation as shown below aiming at creating a genetic information control molecule based on genome chemistry under the above background (Non-Patent Document 4, Non-Patent Document 5).
H
~=0 HN
N", ~N : NH
HO 0 N N~NH H N
O~N~ / ~ 0 OH " N" (1) H
>=0 HN
N~
N
</ ~ ~ \
HO N N NH H N I N
~ V N' 0 OH N"
0 (20) When these hybrid compounds of a nucleoside and an MGB polyamide are taken into a DNA by a biosynthetic pathway like normal nucleic acids or modified nucleosides used as anticancer agents, it is supposed that they exhibit stabilization of the double stranded DNA by forming an extremely tight and stable binding to the minor groove thereby inhibiting replication and transcription of the DNA in the case that a site which the MGB polyamide specifically recognizes is present in the neighboring sequence.
On the other hand, in the case that a site which the MGB polyamide does not specifically recognize is present in the neighboring region, it can be expected that the compound does not exhibit stabilization of the double stranded DNA, thereby inhibiting no reproduction or transcription. In addition, when an MGB polyamide-nucleoside hybrid compound acts on a double stranded DNA
by itself, it is supposed that the nucleoside in the hybrid forms a Hoogsteen type hydrogen bond and has an antigene-like effect of exhibiting affinity and base selectivity by the MGB polyamide.
As described above, hybrid compounds of the MGB
polyamide have all of the characteristics such as high base sequence recognition ability, high stability of MGB
polyamide against enzymes in the living body and excellent membrane permeability and can be expected as candidates as genetic information control molecules.
[Non-Patent Document 1] White, S.; Baird, E. E.; Dervan, P. B. Chem. Biol., 1997, 4, 569.
[Non-Patent Document 2] Trauger, J. W.; Baird, E. E.;
Dervan, P. B. J. Am. Chem. Soc., 1996,118, 6160.
[Non-Patent Document 3] Tao, Z.; Fujiwara, T.; Sugiyama, H. J. Am. Chem.Soc., 1999,121,4961.
[Non-Patent Document 4] Y.; Ohba, Y.; Terui, K.; Kamaike, T.; Oshima, E.; Kawashima, Nucleic Acids Symposium Series No. 48, 2004, p.55-56.
[Non-Patent Document 4] Y.; Ohba, Y.; Terui, K.; Kamaike, T.; Oshima, E.; Kawashima, Nucleic Acids Symposium Series No. 48, 2004,p.55-56.
[Non-Patent Document 5] "Abstracts of 14th antisense symposium" Antisense DNA/RNA Research Group, December 2, 2004, p. 55.
Disclosure of the Invention The MGB polyamide can add unique base sequence recognition ability while maintaining the pharmacological activity of various functional molecules by forming hybrids with such molecules. Furthermore, since the MGB
polyamide has high stability against enzymes in the living body and excellent membrane permeability in addition to the high base sequence recognition ability, a hybrid compound with the MGB polyamide can be expected to have all of these characteristics.
These hybrid compounds have an effect of selectively stabilizing dsDNA containing a target base sequence (antigene type approach) . Alternatively, the hybrid compound itself is incorporated in a DNA via the biosynthetic pathway and have a selectively stabilizing effect by primarily coordinating only to the minor groove having a site which the MGB polyamide in this DNA
specifically recognizes, and therefore, they can be expected to inhibit reproduction and transcription of the target gene.
Incidentally, when the present inventors evaluated the stability against the ammonia treatment necessary in the oligomer synthesis of the above hybrid compound (20), 2'-deoxyguanosine, which was detected by the ammonia treatment of the hybrid compound (1) via a linker by amide bonding, was not detected in the examination by TLC
and 'H-NMR. It has become clear from this that the stability at the N2-position of the hybrid compound against the ammonia treatment can be enhanced by changing binding form to the linker by N-alkylation. However, a spot having an Rf value lower than that of (20) was just slightly caused on TLC. From 'H-NMR data, this was supposed to be a compound having lost a formyl group of the N-terminal end. That is, although 2'-deoxyguanosine was not detected in the ammonia treatment, dropout of a formyl group of the N-terminal end was observed, which means that the above compound (20) does not necessarily have satisfactory stability.
Therefore, an object of the present invention is to provide an MGB polyamide hybrid compound excellent in stability which enables to synthesize the oligomer effectively while maintaining the performance of the MGB
polyamide hybrid compound. Another object of the present invention is to provide a production method of the MGB
polyamide hybrid compound and the intermediates useful for that purpose.
The present inventors have conducted intensive studies for finding an MGB polyamide hybrid compound which has high base sequence recognition ability, high stability against enzymes in the living body and excellent membrane permeability and enables to synthesize the oligomer effectively, and consequently have found that it is enabled to synthesize the oligomer effectively while maintaining excellent base sequence recognition ability, excellent resistance to enzymes in the living body and excellent membrane permeability by introducing a 1-methylpyrrole-2-carbonyl group in place of a formyl group at the N-terminal end to form a pyrrole amide tetramer, and thus completed the present invention.
That is, the present invention is directed to the following.
1. A 21-deoxyguanosine hybrid compound represented by following general formula (38) characterized in that the compound contains a pyrrole amide tetramer. 0 HN
\ N~
~/ " J H \
HO N N NH H N I \
OH 0 N (38) 2. A 2'-deoxyguanosine hybrid compound having protected hydroxyl groups and represented by following general formula (37) characterized in that the compound contains a pyrrole amide tetramer, HN
\ N~
N
, ~ ~
RO O N N~!NH H N I\
N 0 (37) wherein R' and R2 each mean the same or different hydroxyl group protecting groups.
3. A protected 2'-deoxyguanosine hybrid compound represented by following general formula (36) characterized in that the compound contains a pyrrole amide tetramer, 0 HN
\ N~
/N \N 0 Rl0 0\N N5:~NH H N I \ 11 R2 0 0 N (36) wherein R', R 2 and R3 each mean the same or different hydroxyl group protecting groups.
4. A production method of a protected 2'-deoxyguanosine hybrid compound represented by above general formula (36) characterized by reacting a diamine compound represented by following general formula (35) with a compound represented by following general formula (23), HN
\ N~
H
~N H N I\N
Fmoc ~N 0 ~ (35) ~
/
N
Rl0 N N~Hal (23) ~
wherein Fmoc means a 9-fluorenylmethoxycarbonyl group;
and Rl, R 2 and R3 are the same as above.
5. A diamine compound represented by following general formula (35) characterized in that the compound contains a pyrrole amide tetramer, HN
\ N~
H H N N ~ \
Fmoc -N
0 (35) wherein Fmoc is the same as above.
6. A production method of a diamine compound represented by above general formula (35) characterized by reacting a carbamate compound represented by following formula (26) with a carboxylic acid compound containing a pyrrole amide tetramer represented by following formula (34). 0 HN
N~
H
N
0 N (34) \ I I /
H
0 'Y N~,_/ N*H3 -CI
0 (26) 7. A carbamate compound represented by following formula (26) for the production of a diamine compound (35) represented by above general formula (35).
H
0 'Y N,_,-,,/ N=H3 -CI
0 (26) The pyrrole amide tetramer-2'-deoxyguanosine hybrid compound of the present invention has a 1-methylpyrrole-2-carbonyl group at the N-terminal end as one of the characteristics thereof and the compound has excellent base sequence recognition ability as compared with distamycin A, excellent resistance to enzymes in the living body and excellent membrane permeability and enables to synthesize the oligomer effectively.
Therefore, the MGB polyamide-nucleoside hybrid compound of the present invention has high base sequence selecting ability, and utility as a novel genetic information control molecule is greatly expected.
\ N~
H
~N H N I\N
Fmoc ~N 0 ~ (35) ~
/
N
Rl0 N N~Hal (23) ~
wherein Fmoc means a 9-fluorenylmethoxycarbonyl group;
and Rl, R 2 and R3 are the same as above.
5. A diamine compound represented by following general formula (35) characterized in that the compound contains a pyrrole amide tetramer, HN
\ N~
H H N N ~ \
Fmoc -N
0 (35) wherein Fmoc is the same as above.
6. A production method of a diamine compound represented by above general formula (35) characterized by reacting a carbamate compound represented by following formula (26) with a carboxylic acid compound containing a pyrrole amide tetramer represented by following formula (34). 0 HN
N~
H
N
0 N (34) \ I I /
H
0 'Y N~,_/ N*H3 -CI
0 (26) 7. A carbamate compound represented by following formula (26) for the production of a diamine compound (35) represented by above general formula (35).
H
0 'Y N,_,-,,/ N=H3 -CI
0 (26) The pyrrole amide tetramer-2'-deoxyguanosine hybrid compound of the present invention has a 1-methylpyrrole-2-carbonyl group at the N-terminal end as one of the characteristics thereof and the compound has excellent base sequence recognition ability as compared with distamycin A, excellent resistance to enzymes in the living body and excellent membrane permeability and enables to synthesize the oligomer effectively.
Therefore, the MGB polyamide-nucleoside hybrid compound of the present invention has high base sequence selecting ability, and utility as a novel genetic information control molecule is greatly expected.
Best Mode for Carrying Out the Invention In the previous hybrid compound invention by the present inventors, the pyrrole polyamide trimer of the MGB polyamide moiety can be readily synthesized by purification method utilizing partition by acid-base distribution based on the report of Boger et al.
Subsequently, the present inventors have considered stability when a hybrid compound is incorporated into a DNA and performed designing and synthesis of a hybrid compound which is stable under the deprotection condition at the time of the DNA synthesis by changing the binding form at N2-position from amide bond to a binding by N-alkylation. In order to selectively protect the amino group of one side of diaminopropane used as a linker, Fmoc phenylcarbonate was synthesized as a chemical reagent for forming a mono-Fmoc compound, and just one equivalent was allowed to act on the diaminoalkane. As a result, conversion to a mono-Fmoc compound was successfully achieved with good yield by converting one amino group to a hydrochloride salt. This synthetic process can be widely used as a method for introducing a protecting group which is stable against acid in synthesis researches of polyamine compounds and the like, and therefore it is considered to highly useful.
Subsequently, an MGB polyamide was condensed with the mono-Fmoc-diaminopropane and after the removal of the Fmoc group, it was reacted with a 2-fluoroinosine derivative to achieve the synthesis of the hybrid compound (N-terminal-l-methylpyrrole-2-carbonyl compound/pyrrole amide tetramer) linked by N-alkylation.
Ammonia treatment of a hybrid compound (pyrrole amide tetramer) linked by N-alkylation, and as a result of the examination, it was revealed that the compound was stable and that oligomer synthesis was possible.
The "hydroxyl group protecting group" in R' and R2, which may be the same or different, is acyl, carbamoyl or aralkyl. The said acyl group is preferably formyl, C1-lo alkylcarbonyl, C1-6 alkoxycarbonyl, C6_14 arylcarbonyl, C7-13 aralkylcarbonyl, aromatic heterocycle-carbonyl, 5- to 7-membered non-aromatic heterocycle-carbonyl, C3-lo cycloalkylcarbonyl (e.g., cyclopentane carbonyl, cyclohexane carbonyl) ; C8-13 arylalkenylcarbonyl (e.g., styrylcarbonyl); C8-13 arylalkynylcarbonyl (e.g., phenylethynylcarbonyl); C6-14 arylsulfonyl (e.g., phenylsulfonyl); mono- or di-C1-6 alkylcarbamoyl (e.g., methylcarbamoyl, tert-butyl carbamoyl); C3-10 cycloalkylcarbamoyl (e.g., cyclopropylcarbamoyl, cyclopentylcarbamoyl, cyclohexylcarbamoyl); C6-14 arylcarbamoyl ( e. g., phenylcarbamoyl); C7-14 aralkylcarbamoyl (e.g., benzylcarbamoyl, phenethylcarbamoyl, diphenylethylcarbamoyl); C4-13 cycloalkylalkylcarbamoyl (e.g., cyclohexylmethyl carbamoyl), aromatic heterocycle-carbamoyl (e.g., isoxazolyl carbamoyl, benzothiazolylcarbamoyl); non-aromatic heterocycle-carbamoyl (e.g., pyrrolidinyl carbamoyl ) , C7_14 aralkyloxycarbamoyl (e . g . , benzyloxycarbamoyl); etc. Examples of the "acyl group having 1 to 15 carbon atoms" include C1_6 alkoxy-carbamoyl (e.g., methoxycarbamoyl), C1_6 alkoxycarbonylcarbamoyl (e.g., methoxycarbonylcarbamoyl, ethoxycarbonylcarbamoyl).
Preferably the "hydroxyl group protecting groups" are the same, and particularly preferably it is an acetyl group.
The "hydroxyl group protecting group" may be TIPDS in which R' and R 2 bind together.
The production method of the compound of the present invention is as follows. Refer to Examples for details.
In the previously synthesized hybrid compounds (1) and (20), the amide bond between the pyrrole rings in itself was stable against ammonia treatment. This is supposed to be attributable to stabilization of n-bonds in the amide by resonance with the pyrrole ring.
Therefore, an attempt was made to achieve stabilization against ammonia treatment by binding a 1-methylpyrrole-2-carbonyl group via an amide bond as the N-terminal end group in place of a formyl group so that stability of the hybrid compound against ammonia treatment performed in the subsequent oligomer synthesis might be improved.
At first, compound 3 was converted to carboxylic acid 32 with aqueous sodium hydroxide and the latter was condensed with a compound in which a Boc group was removed from pyrrole amide trimer 8 to obtain compound 33.
Subsequently, the present inventors have considered stability when a hybrid compound is incorporated into a DNA and performed designing and synthesis of a hybrid compound which is stable under the deprotection condition at the time of the DNA synthesis by changing the binding form at N2-position from amide bond to a binding by N-alkylation. In order to selectively protect the amino group of one side of diaminopropane used as a linker, Fmoc phenylcarbonate was synthesized as a chemical reagent for forming a mono-Fmoc compound, and just one equivalent was allowed to act on the diaminoalkane. As a result, conversion to a mono-Fmoc compound was successfully achieved with good yield by converting one amino group to a hydrochloride salt. This synthetic process can be widely used as a method for introducing a protecting group which is stable against acid in synthesis researches of polyamine compounds and the like, and therefore it is considered to highly useful.
Subsequently, an MGB polyamide was condensed with the mono-Fmoc-diaminopropane and after the removal of the Fmoc group, it was reacted with a 2-fluoroinosine derivative to achieve the synthesis of the hybrid compound (N-terminal-l-methylpyrrole-2-carbonyl compound/pyrrole amide tetramer) linked by N-alkylation.
Ammonia treatment of a hybrid compound (pyrrole amide tetramer) linked by N-alkylation, and as a result of the examination, it was revealed that the compound was stable and that oligomer synthesis was possible.
The "hydroxyl group protecting group" in R' and R2, which may be the same or different, is acyl, carbamoyl or aralkyl. The said acyl group is preferably formyl, C1-lo alkylcarbonyl, C1-6 alkoxycarbonyl, C6_14 arylcarbonyl, C7-13 aralkylcarbonyl, aromatic heterocycle-carbonyl, 5- to 7-membered non-aromatic heterocycle-carbonyl, C3-lo cycloalkylcarbonyl (e.g., cyclopentane carbonyl, cyclohexane carbonyl) ; C8-13 arylalkenylcarbonyl (e.g., styrylcarbonyl); C8-13 arylalkynylcarbonyl (e.g., phenylethynylcarbonyl); C6-14 arylsulfonyl (e.g., phenylsulfonyl); mono- or di-C1-6 alkylcarbamoyl (e.g., methylcarbamoyl, tert-butyl carbamoyl); C3-10 cycloalkylcarbamoyl (e.g., cyclopropylcarbamoyl, cyclopentylcarbamoyl, cyclohexylcarbamoyl); C6-14 arylcarbamoyl ( e. g., phenylcarbamoyl); C7-14 aralkylcarbamoyl (e.g., benzylcarbamoyl, phenethylcarbamoyl, diphenylethylcarbamoyl); C4-13 cycloalkylalkylcarbamoyl (e.g., cyclohexylmethyl carbamoyl), aromatic heterocycle-carbamoyl (e.g., isoxazolyl carbamoyl, benzothiazolylcarbamoyl); non-aromatic heterocycle-carbamoyl (e.g., pyrrolidinyl carbamoyl ) , C7_14 aralkyloxycarbamoyl (e . g . , benzyloxycarbamoyl); etc. Examples of the "acyl group having 1 to 15 carbon atoms" include C1_6 alkoxy-carbamoyl (e.g., methoxycarbamoyl), C1_6 alkoxycarbonylcarbamoyl (e.g., methoxycarbonylcarbamoyl, ethoxycarbonylcarbamoyl).
Preferably the "hydroxyl group protecting groups" are the same, and particularly preferably it is an acetyl group.
The "hydroxyl group protecting group" may be TIPDS in which R' and R 2 bind together.
The production method of the compound of the present invention is as follows. Refer to Examples for details.
In the previously synthesized hybrid compounds (1) and (20), the amide bond between the pyrrole rings in itself was stable against ammonia treatment. This is supposed to be attributable to stabilization of n-bonds in the amide by resonance with the pyrrole ring.
Therefore, an attempt was made to achieve stabilization against ammonia treatment by binding a 1-methylpyrrole-2-carbonyl group via an amide bond as the N-terminal end group in place of a formyl group so that stability of the hybrid compound against ammonia treatment performed in the subsequent oligomer synthesis might be improved.
At first, compound 3 was converted to carboxylic acid 32 with aqueous sodium hydroxide and the latter was condensed with a compound in which a Boc group was removed from pyrrole amide trimer 8 to obtain compound 33.
Then, after ester 33 was converted to compound 34 by hydrolyzation, the latter was condensed with mono-Fmoc diaminopropane 26 to obtain compound 35.
CI3C /\ 2 M NaOH aq. HO /\
N MeOH,60 C
O O
3 HN Boc 32 89% N
HN O HN
N
\ 1) AcCI, MeOH, AcOEt, 0 C N- 2 M NaOH aq. N-HN
O 2) 32, EDCI, DMAP HN MeOH, 60 C HN
O
DMF, R to 80 C 0 N N H ~~ H ~~
Me0 Me0 N
/ O N
8 Fmoc-NH 33 73% N\ 34 quant.
26, DCC, HOBT, DIEA ~ HN 0 NH H ~
DMF, rt O /N ~ NH N I
0 N\
O N
35 86%
Here, pyrrole amide trimer 8 protected with a Boc group was synthesized by condensation of 4-(tert-butoxycarbonyl)amino-l-methylpyrrole-2-carboxylic acid 6 with the amino compound removed the Boc group of pyrrole amide dimer 7 under acidic condition. The pyrrole amide dimer 7 was prepared by condensation of the carboxylic acid 6 with the amino compound derived by the catalytic reduction of the nitro group of compound 5.
CI3C /\ 2 M NaOH aq. HO /\
N MeOH,60 C
O O
3 HN Boc 32 89% N
HN O HN
N
\ 1) AcCI, MeOH, AcOEt, 0 C N- 2 M NaOH aq. N-HN
O 2) 32, EDCI, DMAP HN MeOH, 60 C HN
O
DMF, R to 80 C 0 N N H ~~ H ~~
Me0 Me0 N
/ O N
8 Fmoc-NH 33 73% N\ 34 quant.
26, DCC, HOBT, DIEA ~ HN 0 NH H ~
DMF, rt O /N ~ NH N I
0 N\
O N
35 86%
Here, pyrrole amide trimer 8 protected with a Boc group was synthesized by condensation of 4-(tert-butoxycarbonyl)amino-l-methylpyrrole-2-carboxylic acid 6 with the amino compound removed the Boc group of pyrrole amide dimer 7 under acidic condition. The pyrrole amide dimer 7 was prepared by condensation of the carboxylic acid 6 with the amino compound derived by the catalytic reduction of the nitro group of compound 5.
NOz 1) H2, 10% Pd/C, AcOEt, rt MeO N-Boc Me0 ~~ 2) 6, EDCI, DMAP, DMF, rt N
O I ' N O 1 7 99% HN-Boc OMe 1) AcCI, MeOH, AcOEt, rt O NH N I N
2) 6, EDCI, DMAP, DMF, rt O
O N
~ 8 98%
The above synthetic process uses l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) as a condensation agent, and therefore purification of the reaction product can be performed only by liquid separation operation by acid-base, and therefore, target pyrrole polyamide trimer 8 can be synthesized at a high yield without purification by column chromatography.
Subsequently, compound 36 was obtained by removing a Fmoc group of compound 35 with triethylamine and performing substitution reaction with a fluorine atom of 2'-deoxyinosine derivative 23. Finally, 2'-deoxyguanosine hybrid compounds 38 containing a pyrrole amide tetramer can be obtained by removing 2-(4-nitrophenyl)ethyl group at 06-position, and acetyl groups at 3'-position and 5'-position of compound 36.
O I ' N O 1 7 99% HN-Boc OMe 1) AcCI, MeOH, AcOEt, rt O NH N I N
2) 6, EDCI, DMAP, DMF, rt O
O N
~ 8 98%
The above synthetic process uses l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) as a condensation agent, and therefore purification of the reaction product can be performed only by liquid separation operation by acid-base, and therefore, target pyrrole polyamide trimer 8 can be synthesized at a high yield without purification by column chromatography.
Subsequently, compound 36 was obtained by removing a Fmoc group of compound 35 with triethylamine and performing substitution reaction with a fluorine atom of 2'-deoxyinosine derivative 23. Finally, 2'-deoxyguanosine hybrid compounds 38 containing a pyrrole amide tetramer can be obtained by removing 2-(4-nitrophenyl)ethyl group at 06-position, and acetyl groups at 3'-position and 5'-position of compound 36.
O
p HN
HN
N-p HN O
HN triethylamine \ N DMF, rt or 60 C \ N
H \ HN
O
H H O H2N,N N
Fmoc'N,N N p i O
HN p 23, triethylamine N ~ DBU
DMF, rt to 60 C N N~NH \ N
~ pyridine, rt AcO HN
NH N O
~ N
Aco p N p 36 79% / -~ N-O
HN O
NH
N N"~NH \ N
AcO ~-NH HN N 0 AcO p N/ N ~N-i p 0 37 80% N NH HN O
NaOMe ~/ ~ ~
MeOH, pyridine, rt N N NH N
HO p L-L HN \
~
HO p N/ p 38 97%
Hereinbelow, the present invention is specifically described by way of Examples.
p HN
HN
N-p HN O
HN triethylamine \ N DMF, rt or 60 C \ N
H \ HN
O
H H O H2N,N N
Fmoc'N,N N p i O
HN p 23, triethylamine N ~ DBU
DMF, rt to 60 C N N~NH \ N
~ pyridine, rt AcO HN
NH N O
~ N
Aco p N p 36 79% / -~ N-O
HN O
NH
N N"~NH \ N
AcO ~-NH HN N 0 AcO p N/ N ~N-i p 0 37 80% N NH HN O
NaOMe ~/ ~ ~
MeOH, pyridine, rt N N NH N
HO p L-L HN \
~
HO p N/ p 38 97%
Hereinbelow, the present invention is specifically described by way of Examples.
The analytical instruments and so on used in this experiment are as follows.(1) 1H nuclear magnetic resonance spectrum (1H-NMR), * Bruker DPX 400 NMR spectrometer * Measurement solvent; heavy chloroform, heavy dimethylsulfoxide, heavy methanol.
(2) 13C nuclear magnetic resonance spectrum (13C-NMR), * Bruker DPX 400 NMR spectrometer * Measurement solvent; heavy chloroform, heavy dimethylsulfoxide, heavy methanol.
(3) 31P nuclear magnetic resonance spectrum (31P-NMR), * Bruker DPX 400 NMR spectrometer * Measurement solvent; heavy chloroform, heavy dimethylsulfoxide.
(4) Mass spectrum (MS), * VG Auto SpecE (Micro Mass) * TSQ-700 (Thermoquest) (5) Elemental analysis, * Elemental Vavio EL
(6) Thin-layer chromatography (TLC), * Merck Kieselgel 60 F254 (developed by ascending method) * Detection method: UV absorption (5% sulfuric acid -methanol solution was atomized and heated at 100 to 150 C. After immersed in a mixture of anisic aldehyde, ethanol, sulfuric acid, water and acetic acid the mixture was heated at 100 to 150 C) .
(7) Column chromatography, * Wako Pure Chemical Industries Wakogel C-300 * Kanto Chemical silica gel 60N
(8) Melting point measurement, * Micro-melting-point apparatus of Yanagimoto Mfg. Co., Ltd. (uncorrected values are described) Example 1 [Pre-step 1]
Synthesis of 2-trichloroacetyl-l-methylpyrrole (3);
O
CI3CCI CIgC YQ
I Et20, rt O
Trichloroacetyl chloride (130 mL, 1.16 mol) was dissolved in ether (250 mL) under argon atmosphere and cooled to 0 C. 1-methylpyrrole (100 mL, 1.12 mol), which was dissolved in ether (250 mL) beforehand, was added dropwise using a dripping funnel, while taking care of heat generation. After the dropwise addition was completed, the mixture was stirred for one hour while gradually elevating the temperature to room temperature.
The reaction was terminated by adding 2 M potassium carbonate aqueous solutions (300 mL) and the organic layer was removed by liquid separation operation. After extracted from the aqueous layer with ether (300 mLx2), all the organic layers were combined and washed with a saturated sodium chloride aqueous solution (300 mL). The organic layer was dried over anhydrous magnesium sulfate and the anhydrous magnesium sulfate was removed by filtration, and 244 g(96%) of compound 3 was obtained by concentrating the filtrate under reduced pressure.
1H-NMR (DMSO-d6) :8 7.44 (s, 1H, Py-H) , 7.43 (d, 1H, J
=1 . 18Hz, Py-H) , 6. 30 (dd, 1H, J1',2'=2 . 57Hz, J2',2"=1 . 63 Hz, Py-H),3.91(s,3H,Py-CH3) 13C-NMR(DMSO-d6):5171.84,135.30, 123.68, 120.68, 109.10, 96.04, 37.93 FAB-MS:m/z 225 (M+H) Molecular weight (theoretical value) C7H6N1O1C13 : C, 37.13, H, 2.67, N, 6.18.
Molecular weight (observed value): C, 37.14: H, 2.82: N, 6.15.
[Pre-step 2]
Synthesis of 2-trichloroacetyl-l-methyl-4-nitropyrrole (4) ;
N Oz C13C ~ HN03 C13C
O I Ac2O, 40 C
The above compound 3 (2.14 g, 9.46 mmol) was dissolved in acetic anhydride (12 mL) and cooled to -40 C. Fuming nitric acid (0.83 mL) was added dropwise to this solution while keeping the temperature at -40 C.
The reaction solution was gradually warmed to room temperature and stirred for another hour. The reaction solution was cooled to -20 C again and after added with isopropanol (12 mL), stirred for 15 minutes. Then the solution was allowed to stand still for 15 minutes while keeping the temperature at -20 C and the resulted crystals in the solution were separated by suction filtration. After the crystals were washed with isopropanol and dried under reduced pressure to obtain white crystals. The filtrate was further concentrated under reduced pressure, added with isopropanol again, stirred for 10 minutes at -20 C and the resulted crystals were separated by suction filtration. After the crystals were washed with isopropanol and dried under reduced pressure to obtain 2.01 g of compound 4 (78%) together with the crystals obtained above in total.
1H-NMR (DMSO-d6) : 88.55 (d, 1H, J= 1.44 Hz, Py-H), 7.80 (d, 1H, J= 1.91 Hz, Py-H), 4.00 (s, 3H, Py-CH3) 13C-NMR (DMSO-d6) : 8172.79, 134.25, 132.57, 120.60, 116.30, 94.54 m.p.: 134-136 C
[Pre-step 31 Synthesis of methyl 1-methyl-4-nitropyrrole-2-carboxylate (5) ;
NaH
CI3C Me0 N MeOH, rt N
(2) 13C nuclear magnetic resonance spectrum (13C-NMR), * Bruker DPX 400 NMR spectrometer * Measurement solvent; heavy chloroform, heavy dimethylsulfoxide, heavy methanol.
(3) 31P nuclear magnetic resonance spectrum (31P-NMR), * Bruker DPX 400 NMR spectrometer * Measurement solvent; heavy chloroform, heavy dimethylsulfoxide.
(4) Mass spectrum (MS), * VG Auto SpecE (Micro Mass) * TSQ-700 (Thermoquest) (5) Elemental analysis, * Elemental Vavio EL
(6) Thin-layer chromatography (TLC), * Merck Kieselgel 60 F254 (developed by ascending method) * Detection method: UV absorption (5% sulfuric acid -methanol solution was atomized and heated at 100 to 150 C. After immersed in a mixture of anisic aldehyde, ethanol, sulfuric acid, water and acetic acid the mixture was heated at 100 to 150 C) .
(7) Column chromatography, * Wako Pure Chemical Industries Wakogel C-300 * Kanto Chemical silica gel 60N
(8) Melting point measurement, * Micro-melting-point apparatus of Yanagimoto Mfg. Co., Ltd. (uncorrected values are described) Example 1 [Pre-step 1]
Synthesis of 2-trichloroacetyl-l-methylpyrrole (3);
O
CI3CCI CIgC YQ
I Et20, rt O
Trichloroacetyl chloride (130 mL, 1.16 mol) was dissolved in ether (250 mL) under argon atmosphere and cooled to 0 C. 1-methylpyrrole (100 mL, 1.12 mol), which was dissolved in ether (250 mL) beforehand, was added dropwise using a dripping funnel, while taking care of heat generation. After the dropwise addition was completed, the mixture was stirred for one hour while gradually elevating the temperature to room temperature.
The reaction was terminated by adding 2 M potassium carbonate aqueous solutions (300 mL) and the organic layer was removed by liquid separation operation. After extracted from the aqueous layer with ether (300 mLx2), all the organic layers were combined and washed with a saturated sodium chloride aqueous solution (300 mL). The organic layer was dried over anhydrous magnesium sulfate and the anhydrous magnesium sulfate was removed by filtration, and 244 g(96%) of compound 3 was obtained by concentrating the filtrate under reduced pressure.
1H-NMR (DMSO-d6) :8 7.44 (s, 1H, Py-H) , 7.43 (d, 1H, J
=1 . 18Hz, Py-H) , 6. 30 (dd, 1H, J1',2'=2 . 57Hz, J2',2"=1 . 63 Hz, Py-H),3.91(s,3H,Py-CH3) 13C-NMR(DMSO-d6):5171.84,135.30, 123.68, 120.68, 109.10, 96.04, 37.93 FAB-MS:m/z 225 (M+H) Molecular weight (theoretical value) C7H6N1O1C13 : C, 37.13, H, 2.67, N, 6.18.
Molecular weight (observed value): C, 37.14: H, 2.82: N, 6.15.
[Pre-step 2]
Synthesis of 2-trichloroacetyl-l-methyl-4-nitropyrrole (4) ;
N Oz C13C ~ HN03 C13C
O I Ac2O, 40 C
The above compound 3 (2.14 g, 9.46 mmol) was dissolved in acetic anhydride (12 mL) and cooled to -40 C. Fuming nitric acid (0.83 mL) was added dropwise to this solution while keeping the temperature at -40 C.
The reaction solution was gradually warmed to room temperature and stirred for another hour. The reaction solution was cooled to -20 C again and after added with isopropanol (12 mL), stirred for 15 minutes. Then the solution was allowed to stand still for 15 minutes while keeping the temperature at -20 C and the resulted crystals in the solution were separated by suction filtration. After the crystals were washed with isopropanol and dried under reduced pressure to obtain white crystals. The filtrate was further concentrated under reduced pressure, added with isopropanol again, stirred for 10 minutes at -20 C and the resulted crystals were separated by suction filtration. After the crystals were washed with isopropanol and dried under reduced pressure to obtain 2.01 g of compound 4 (78%) together with the crystals obtained above in total.
1H-NMR (DMSO-d6) : 88.55 (d, 1H, J= 1.44 Hz, Py-H), 7.80 (d, 1H, J= 1.91 Hz, Py-H), 4.00 (s, 3H, Py-CH3) 13C-NMR (DMSO-d6) : 8172.79, 134.25, 132.57, 120.60, 116.30, 94.54 m.p.: 134-136 C
[Pre-step 31 Synthesis of methyl 1-methyl-4-nitropyrrole-2-carboxylate (5) ;
NaH
CI3C Me0 N MeOH, rt N
The above compound 4 (15.0 g, 55.3 mmol) was dissolved in methanol (20 mL) under argon atmosphere, and sodium hydride (60%) (0.2 g, 5.5 mmol)dissolved in methanol (5.0 mL) beforehand was added dropwise thereto.
After the reaction solution was stirred at room temperature for one hour, strong sulfuric acid (0.3 mL, 5.5 mmol) was added to terminate the reaction. After neutralization with a saturated sodium hydrogen carbonate aqueous solution (10 mL), the residue was diluted with ethyl acetate (200 mL) and the reaction solution was washed with a saturated sodium hydrogen carbonate aqueous solution (100 mLx2) and a saturated sodium chloride aqueous solution (100 mL) The organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure after the anhydrous magnesium sulfate was removed by filtration. The residue was crystallized form ethyl acetate and 6.02 g (61%) of compound 5 was obtained by collecting crystals by suction filtration.
The filtrate was further concentrated under reduced pressure, the residue was purified by silica gel column chromatography (hexane:ethyl acetate =5:1) to obtain 9.78 g(96%) of compound 5 in total.
1H-NMR (DMSO-d6) : 58.27 (d, 1H, J= 1.97 Hz, Py-H), 7.30 (d, 1H, J= 2.07 Hz, Py-H), 3.92 (s, 3H, Py-CH3), 3.80 (s, 3H, -OCH3) 13C-NMR (DMSO-d6) : 6159.83, 134.16 129.42, 122.59, 111.52, 51.77, 37.44 [Pre-step 4]
Synthesis of 4-(tert-butoxycarbonyl)amino-l-methylpyrrole-2-carboxylic acid (6);
H
N0z N
Me0 1) H2, 10% Pd/C, AcOEt, rt' HO ' Boc TN 2)(Boc)20, Et20,rt N
1 3) 2 M NaOH aq., MeOH, 60 C O O
The above compound 5 (6.00 g, 32.6 mmol) was dissolved in ethyl acetate (200 mL) and added with 10%
palladium carbon (2.00 g) . The suspension was vigorously agitated under hydrogen atmosphere at room temperature for 12 hours and then palladium carbon was removed by filtration with celite. Immediately after concentrating the filtrate under reduced pressure, the residue was dissolved in ether (30 mL) under argon atmosphere. Di-tert-butyldicarbonate (7.46 g, 34.2 mmol) which was dissolved in ether (20 mL) beforehand was added dropwise and the mixture was stirred at room temperature for two hours. After concentrated under reduced pressure, the reaction solution was dissolved in methanol (100 mL), added with 2 M aqueous sodium hydroxide (100 mL) and stirred at 60 C for two hours. After methanol was removed by concentration under reduced pressure, remaining aqueous layer was washed with ether (50 mLx2).
After the aqueous layer was adjusted to pH 3 with 10%
hydrochloric acid, extraction with ethyl acetate (200 mLx3) was performed. The organic layers were collected all together, washed with a saturated sodium chloride aqueous solution (200 mL) and dried over anhydrous magnesium sulfate. After the anhydrous magnesium sulfate was removed by filtration, the organic layer was concentrated under reduced pressure and the residue was crystallized from ethyl acetate-hexane to obtain 6.97 g of compound 6 (89%).
1H-NMR (DMSO-d6) : 812 . 08 (s, 1H, -COOH) , 9.03 (s, 1H, Boc-NH), 7.04 (s, 1H, Py-H), 6.58 (d, 1H, J = 1.04 Hz, Py-H), 3.77 (s, 3H, Py-CH3) , 1.44 (s, 9H, CH3- of Boc group) 13C-NMR (DMSO-d6) : 5161.86, 152.70, 122.80, 119.64, 118.72, 107.45, 78.39, 36.01, 28.14 [Pre-step 5]
Synthesis of methyl 4-{[4-(tert-butyloxycarbonyl)amino-l-methylpyrrol-2-yl]carbonyl}amino-l-methylpyrrole-2-carboxylate (7);
NOz 1) H2110% Pd/C, AcOEt, rt Me0 NH
-goc N
Me0 Z) 6, EDCI, DMAP, DMF, rt O YI/~
The above compound 5 (3.00 g, 16.3 mmol) was dissolved in ethyl acetate (100 mL) and added with 10%
palladium carbon (1.00 g) The suspension was agitated vigorously under hydrogen atmosphere at room temperature for 12 hours and then palladium carbon was removed by filtration with celite. Immediately after concentrating the filtrate under reduced pressure, carboxylic acid 6 (3.92 g, 16.3 mmol), EDCI (6.25 g, 32.6 mmol) and DMAP
(3.98 g, 32.6 mmol) were added thereto and the mixture was dissolved in DMF (100 mL) under argon atmosphere and stirred at room temperature for three hours. The reaction solution was concentrated under reduced pressure to about one-third and diluted with a mixed solvent of ethyl acetate (200 mL) and methanol (20 mL). The organic layer was washed with 10% hydrochloric acid (100 mLx3), a saturated sodium hydrogen carbonate aqueous solution (100 mLx3), a saturated sodium chloride aqueous solution (100 mL) and dried over anhydrous magnesium sulfate. The anhydrous magnesium sulfate was removed by filtration and the organic layer was concentrated under reduced pressure to obtain 6.07 g of compound 7(99%).
1H-NMR (DMSO-d6) : 59.85 (s, 1H, -CONH-), 9.09 (s, 1H, Boc-NH), 7.45 (d, 1H, J = 1.86 Hz, Py-H), 6.90 (d, 1H, J
= 1.90 Hz, Py-H), 6.89 (s, 1H, Py-H), 6.84 (s, 1H, Py-H), 3.83 (s, 3H, Py-CH3), 3.81 (s, 3H, Py-CH3), 3.73 (s, 3H, -OCH3) , 1.45 (s, 9H, CH3- of Boc group) 13C-NMR (DMSO-d6) : 6160.80, 158.42, 152.85, 122.98, 122.60, 122.42, 120.72, 118.49, 117.15, 108.37,103.82, 78.30, 50.90, 36.13, 36.13, 36.01, 28.19 [Pre-step 61 Synthesis of methyl 4-[(4-{[4-(tert-butyloxycarbonyl)amino-l-methylpyrrol-2-yl]carbonyl}amino-l-methylpyrrol-2-yl]carbonyl]amino-l-methylpyrrole-2-carboxylate (8);
H N- Boc MeO N-goc OMe H
N 1) AcCI, MeOH, ACOEt, rt 0 N I N
N O i 2) 6, EDCI, DMAP, DMF, rt NH O ~
~
7 g Compound 7 (3.00 g, 7.97 mmol) was dissolved in ethyl acetate (20 mL) under argon atmosphere and cooled to 0 C. Methanol (4.1 mL, 100 mmol) was added and subsequently acetyl chloride (5.7 mL, 80 mmol) was added dropwise while taking care of heat generation. After stirred at 0 C for 30 minutes, the solvent and the reaction reagents were removed by concentration under reduced pressure. Carboxylic acid 6(2.11 g, 8.77 mmol), EDCI (3.06 g, 15.94 mmol) and DMAP (2.92 g, 23.91 mmol) were added to the residue and dissolved in DMF (50 mL) under argon atmosphere and stirred at room temperature for three hours. The reaction solution was concentrated under reduced pressure to about a one-third and diluted with a mixed solvent of ethyl acetate (200 mL) and methanol (20 mL) . The organic layer was washed with 10%
hydrochloric acid (100 mLx3), a saturated sodium hydrogen carbonate aqueous solution(100 mLx3) and a saturated sodium chloride aqueous solution (100 mL) and dried over anhydrous magnesium sulfate. The anhydrous magnesium sulfate was removed by filtration and the organic layer was concentrated under reduced pressure to obtain 3.90 g of compound 8 (98%).
1H-NMR (DMSO-d6) :89. 92 (s, 1H, -CONH-) , 9. 87 (s, 1H, -CONH-),9.08(s,1H,Boc-NH), 7.47 (d,1H,J =1.85 Hz,Py-H),7.22(d,1H,J=1.61 Hz,Py-H),7.08(d,1H,J =1.59 Hz,Py-H), 6.92(d,1H,J=1.93 Hz,Py-H),6.90(s,1H,Py-H),6.85(s,1H,Py-H),3.85(s,3H,Py-CH3),3.84(s,3H,Py-CH3),3.82(s,3H,Py-CH3) , 3. 74 (s, 3H, -OCH3) , 1.46 (s, 9H, CH3-of Boc group) 13C-NMR(DMSO-d6):8160.81, 158.52, 158.44, 152.88, 123.01, 122.83, 122.52, 122.34, 20.74, 118.52, 117.08, 108.38, 104.83, 103.85, 78.29, 50.91, 36.15, 36.04, 28.20 [Pre-step 7]
Synthesis of 3',5'-di-O-acetyl-2-fluoro-06-[2-(4-nitrophenyl)ethyl]-2'-dioxyguanosine (23);
[Pre-step 7-1]
Synthesis of 31,5'-di-O-acetyl-2'-dioxyguanosine (21);
N N:]
N \ N I i\
N NH2 Ac20, DMAP N NHZ
HO 0 DMF, rt Ac0 0 HO AcO 21 2'-Deoxyguanosine 2'-Deoxyguanosine (8.02 g, 30 mmol) was azeotropically dried with pyridine three times and dissolved in DMF (24 mL) under argon atmosphere.
Subsequently, 4-dimethylaminopyridine (0.37 g, 3.0 mmol), pyridine (24 mL) and acetic anhydride (22.1 mL, 240 mmol) were added and the mixture was stirred at room temperature for three hours. Then, water (10 mL) was added to the reaction solution to terminate the reaction and the mixture was stirred with ethanol (200 mL) for 30 minutes and allowed to stand still at room temperature.
The resulted crystals were suction filtered and the crystals ware washed with ethanol (10 mLx3), and after further washed with 2-propanol (100 mL), dried under reduced pressure to obtain 8.92 g of compound 21 (85%).
1H-NMR (DMSO-d6) :81 . 87 and 2. 03 (2s, 6H, CH3COx2) , 2.45 (ddd, 1H, J1',Z"= 6.0 z, JZ',Z"= 2.3 Hz, J2",3,=4 . 0 Hz,H-2"), 2.91(m,1H,H-2'), 4.17-4.29(m,3H,H-4',5',5"), 5.29(m,1H,H-3' ), 6.13 (dd, 1H, J1',2'8.6 Hz, J1',2"=6.0 Hz,H-1' ), 6.52 (brs, 2H,N2-H2) 7. 90 (s, 1H,H-8), 10.74 (brs, 1H,N1-H) .
[Pre-step 7-2]
Synthesis of 3' , 5' -di-O-acetyl-O5- [2- (4-nitrophenyl)ethyl]-2'-dioxyguanosine (22);
O
N O
~ NH
I 2-(4-nitrophenyl)ethanol N N
N N H2 Ph3P, DEAD I N I ~
O
Ac0 1,4-dioxane, rt ' N NHZ
Ac0 O
AcO 21 AcO 22 Compound 21 (1.75 g, 5.0 mmol) was azeotropically dried by 1,4-dioxan three times, added with triphenylphosphine (2.62 g, 10.0 mmol) and 2-(4-nitrophenyl)ethanol (1.67 g, 10.0 mmol) and the mixture was dissolved in 1,4-dioxan under argon atmosphere (50 mL) and stirred at room temperature for 15 minutes.
Subsequently, the reaction solution was added dropwise in azodicarboxylic acid diphenyl ester 40% toluene solution (4.5 mL, 10.0 mmol) and stirred at room temperature for 30 minutes. After concentrated under reduce pressure and added with ethyl acetate (50 mL), the reaction solution was washed with a saturated solution of sodium hydrogen carbonate (50 mL) twice, water (50 mL) and a saturated brine (50 mL) once respectively and after the organic layer was dried over anhydrous magnesium sulfate, the anhydrous magnesium sulfate was removed by filtration and the filtrate was concentrated under reduced pressure.
The residue was purified by silica gel column chromatography (chloroform: ethyl acetate =1:3) to obtain 2.30 g of compound 22 (92%).
1H-NMR (CDC13) :52.08 and 2.13 (2s, 6H, CH3COx2) , 2.52 (ddd, 1H, J1',2 = 6.1 Hz, J2',2" = 14.1 Hz, J2",3, = 2.5 Hz, H-2") , 2.98(ddd, 1H, J1',2' = 7.8 Hz, Jz,,Zõ = 14.1 Hz, J2,,3,=4.9 Hz, H-2' 1) 3.27 (t, 2H, J = 6. 8 Hz, CH2CH2PhNO2) , 4. 31-4.47 (m, 3H, H-4',51,5"), 4.73(t, 2H, J= 6.8 Hz, CHZCHzPhNOz), 4. 89 (brs, 2H, N2-H2) , 5.42 (m, 1H, H-3' ), 6.27 (dd, iH, J1,,2, = 7.8 Hz, Jl',Z = 6. 1 Hz, H-1' ), 7.48 (d, 2H, J 8. 6 Hz, Ph-H of 2-(4-nitrophenyl)ethyl group), 7.74(s, 1H, H-8), 8.16 (d, 2H, J = 8.6 Hz, Ph-H of 2-(4-nitrophenyl)ethyl group).
[Pre-step 7-3]
Synthesis of 3',5'-di-O-acetyl-2-fluoro-OS-[2-(4-nitrophenyl)ethyl]-2'-dioxyguanosine (23);
NOz NO
\ I \ I
N ~ N 45% HF-pyridine N
t-butyl nitrite _ N N NH2 -45 C to rt N N!\F
Ac0 O Ac0 O
Ac0 22 AcO 23 Compound 22 (0.50 g, 1.00 mmol) was azeotropically dried with pyridine three times and added with 45%
hydrogen fluoride/pyridine solution (4.8 mL) at -30 C
and added with t-butyl nitrite (0.36 mL, 3.00 mmol) under argon atmosphere and stirred for 30 minutes after resumed to room temperature. Then the mixture was neutralized with a saturated sodium hydrogen carbonate aqueous solution (15 mL), extracted with chloroform (30 mL) three times and washed with water (50 mL) twice. The organic layer was dried over anhydrous magnesium sulfate and the anhydrous magnesium sulfate was removed by filtration, and the filtrate was concentrated under reduced pressure.
The residue was purified by silica gel column chromatography (chloroform: ethyl acetate =1:3) to obtain 0.44 g of compound 23 (87%).
After the reaction solution was stirred at room temperature for one hour, strong sulfuric acid (0.3 mL, 5.5 mmol) was added to terminate the reaction. After neutralization with a saturated sodium hydrogen carbonate aqueous solution (10 mL), the residue was diluted with ethyl acetate (200 mL) and the reaction solution was washed with a saturated sodium hydrogen carbonate aqueous solution (100 mLx2) and a saturated sodium chloride aqueous solution (100 mL) The organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure after the anhydrous magnesium sulfate was removed by filtration. The residue was crystallized form ethyl acetate and 6.02 g (61%) of compound 5 was obtained by collecting crystals by suction filtration.
The filtrate was further concentrated under reduced pressure, the residue was purified by silica gel column chromatography (hexane:ethyl acetate =5:1) to obtain 9.78 g(96%) of compound 5 in total.
1H-NMR (DMSO-d6) : 58.27 (d, 1H, J= 1.97 Hz, Py-H), 7.30 (d, 1H, J= 2.07 Hz, Py-H), 3.92 (s, 3H, Py-CH3), 3.80 (s, 3H, -OCH3) 13C-NMR (DMSO-d6) : 6159.83, 134.16 129.42, 122.59, 111.52, 51.77, 37.44 [Pre-step 4]
Synthesis of 4-(tert-butoxycarbonyl)amino-l-methylpyrrole-2-carboxylic acid (6);
H
N0z N
Me0 1) H2, 10% Pd/C, AcOEt, rt' HO ' Boc TN 2)(Boc)20, Et20,rt N
1 3) 2 M NaOH aq., MeOH, 60 C O O
The above compound 5 (6.00 g, 32.6 mmol) was dissolved in ethyl acetate (200 mL) and added with 10%
palladium carbon (2.00 g) . The suspension was vigorously agitated under hydrogen atmosphere at room temperature for 12 hours and then palladium carbon was removed by filtration with celite. Immediately after concentrating the filtrate under reduced pressure, the residue was dissolved in ether (30 mL) under argon atmosphere. Di-tert-butyldicarbonate (7.46 g, 34.2 mmol) which was dissolved in ether (20 mL) beforehand was added dropwise and the mixture was stirred at room temperature for two hours. After concentrated under reduced pressure, the reaction solution was dissolved in methanol (100 mL), added with 2 M aqueous sodium hydroxide (100 mL) and stirred at 60 C for two hours. After methanol was removed by concentration under reduced pressure, remaining aqueous layer was washed with ether (50 mLx2).
After the aqueous layer was adjusted to pH 3 with 10%
hydrochloric acid, extraction with ethyl acetate (200 mLx3) was performed. The organic layers were collected all together, washed with a saturated sodium chloride aqueous solution (200 mL) and dried over anhydrous magnesium sulfate. After the anhydrous magnesium sulfate was removed by filtration, the organic layer was concentrated under reduced pressure and the residue was crystallized from ethyl acetate-hexane to obtain 6.97 g of compound 6 (89%).
1H-NMR (DMSO-d6) : 812 . 08 (s, 1H, -COOH) , 9.03 (s, 1H, Boc-NH), 7.04 (s, 1H, Py-H), 6.58 (d, 1H, J = 1.04 Hz, Py-H), 3.77 (s, 3H, Py-CH3) , 1.44 (s, 9H, CH3- of Boc group) 13C-NMR (DMSO-d6) : 5161.86, 152.70, 122.80, 119.64, 118.72, 107.45, 78.39, 36.01, 28.14 [Pre-step 5]
Synthesis of methyl 4-{[4-(tert-butyloxycarbonyl)amino-l-methylpyrrol-2-yl]carbonyl}amino-l-methylpyrrole-2-carboxylate (7);
NOz 1) H2110% Pd/C, AcOEt, rt Me0 NH
-goc N
Me0 Z) 6, EDCI, DMAP, DMF, rt O YI/~
The above compound 5 (3.00 g, 16.3 mmol) was dissolved in ethyl acetate (100 mL) and added with 10%
palladium carbon (1.00 g) The suspension was agitated vigorously under hydrogen atmosphere at room temperature for 12 hours and then palladium carbon was removed by filtration with celite. Immediately after concentrating the filtrate under reduced pressure, carboxylic acid 6 (3.92 g, 16.3 mmol), EDCI (6.25 g, 32.6 mmol) and DMAP
(3.98 g, 32.6 mmol) were added thereto and the mixture was dissolved in DMF (100 mL) under argon atmosphere and stirred at room temperature for three hours. The reaction solution was concentrated under reduced pressure to about one-third and diluted with a mixed solvent of ethyl acetate (200 mL) and methanol (20 mL). The organic layer was washed with 10% hydrochloric acid (100 mLx3), a saturated sodium hydrogen carbonate aqueous solution (100 mLx3), a saturated sodium chloride aqueous solution (100 mL) and dried over anhydrous magnesium sulfate. The anhydrous magnesium sulfate was removed by filtration and the organic layer was concentrated under reduced pressure to obtain 6.07 g of compound 7(99%).
1H-NMR (DMSO-d6) : 59.85 (s, 1H, -CONH-), 9.09 (s, 1H, Boc-NH), 7.45 (d, 1H, J = 1.86 Hz, Py-H), 6.90 (d, 1H, J
= 1.90 Hz, Py-H), 6.89 (s, 1H, Py-H), 6.84 (s, 1H, Py-H), 3.83 (s, 3H, Py-CH3), 3.81 (s, 3H, Py-CH3), 3.73 (s, 3H, -OCH3) , 1.45 (s, 9H, CH3- of Boc group) 13C-NMR (DMSO-d6) : 6160.80, 158.42, 152.85, 122.98, 122.60, 122.42, 120.72, 118.49, 117.15, 108.37,103.82, 78.30, 50.90, 36.13, 36.13, 36.01, 28.19 [Pre-step 61 Synthesis of methyl 4-[(4-{[4-(tert-butyloxycarbonyl)amino-l-methylpyrrol-2-yl]carbonyl}amino-l-methylpyrrol-2-yl]carbonyl]amino-l-methylpyrrole-2-carboxylate (8);
H N- Boc MeO N-goc OMe H
N 1) AcCI, MeOH, ACOEt, rt 0 N I N
N O i 2) 6, EDCI, DMAP, DMF, rt NH O ~
~
7 g Compound 7 (3.00 g, 7.97 mmol) was dissolved in ethyl acetate (20 mL) under argon atmosphere and cooled to 0 C. Methanol (4.1 mL, 100 mmol) was added and subsequently acetyl chloride (5.7 mL, 80 mmol) was added dropwise while taking care of heat generation. After stirred at 0 C for 30 minutes, the solvent and the reaction reagents were removed by concentration under reduced pressure. Carboxylic acid 6(2.11 g, 8.77 mmol), EDCI (3.06 g, 15.94 mmol) and DMAP (2.92 g, 23.91 mmol) were added to the residue and dissolved in DMF (50 mL) under argon atmosphere and stirred at room temperature for three hours. The reaction solution was concentrated under reduced pressure to about a one-third and diluted with a mixed solvent of ethyl acetate (200 mL) and methanol (20 mL) . The organic layer was washed with 10%
hydrochloric acid (100 mLx3), a saturated sodium hydrogen carbonate aqueous solution(100 mLx3) and a saturated sodium chloride aqueous solution (100 mL) and dried over anhydrous magnesium sulfate. The anhydrous magnesium sulfate was removed by filtration and the organic layer was concentrated under reduced pressure to obtain 3.90 g of compound 8 (98%).
1H-NMR (DMSO-d6) :89. 92 (s, 1H, -CONH-) , 9. 87 (s, 1H, -CONH-),9.08(s,1H,Boc-NH), 7.47 (d,1H,J =1.85 Hz,Py-H),7.22(d,1H,J=1.61 Hz,Py-H),7.08(d,1H,J =1.59 Hz,Py-H), 6.92(d,1H,J=1.93 Hz,Py-H),6.90(s,1H,Py-H),6.85(s,1H,Py-H),3.85(s,3H,Py-CH3),3.84(s,3H,Py-CH3),3.82(s,3H,Py-CH3) , 3. 74 (s, 3H, -OCH3) , 1.46 (s, 9H, CH3-of Boc group) 13C-NMR(DMSO-d6):8160.81, 158.52, 158.44, 152.88, 123.01, 122.83, 122.52, 122.34, 20.74, 118.52, 117.08, 108.38, 104.83, 103.85, 78.29, 50.91, 36.15, 36.04, 28.20 [Pre-step 7]
Synthesis of 3',5'-di-O-acetyl-2-fluoro-06-[2-(4-nitrophenyl)ethyl]-2'-dioxyguanosine (23);
[Pre-step 7-1]
Synthesis of 31,5'-di-O-acetyl-2'-dioxyguanosine (21);
N N:]
N \ N I i\
N NH2 Ac20, DMAP N NHZ
HO 0 DMF, rt Ac0 0 HO AcO 21 2'-Deoxyguanosine 2'-Deoxyguanosine (8.02 g, 30 mmol) was azeotropically dried with pyridine three times and dissolved in DMF (24 mL) under argon atmosphere.
Subsequently, 4-dimethylaminopyridine (0.37 g, 3.0 mmol), pyridine (24 mL) and acetic anhydride (22.1 mL, 240 mmol) were added and the mixture was stirred at room temperature for three hours. Then, water (10 mL) was added to the reaction solution to terminate the reaction and the mixture was stirred with ethanol (200 mL) for 30 minutes and allowed to stand still at room temperature.
The resulted crystals were suction filtered and the crystals ware washed with ethanol (10 mLx3), and after further washed with 2-propanol (100 mL), dried under reduced pressure to obtain 8.92 g of compound 21 (85%).
1H-NMR (DMSO-d6) :81 . 87 and 2. 03 (2s, 6H, CH3COx2) , 2.45 (ddd, 1H, J1',Z"= 6.0 z, JZ',Z"= 2.3 Hz, J2",3,=4 . 0 Hz,H-2"), 2.91(m,1H,H-2'), 4.17-4.29(m,3H,H-4',5',5"), 5.29(m,1H,H-3' ), 6.13 (dd, 1H, J1',2'8.6 Hz, J1',2"=6.0 Hz,H-1' ), 6.52 (brs, 2H,N2-H2) 7. 90 (s, 1H,H-8), 10.74 (brs, 1H,N1-H) .
[Pre-step 7-2]
Synthesis of 3' , 5' -di-O-acetyl-O5- [2- (4-nitrophenyl)ethyl]-2'-dioxyguanosine (22);
O
N O
~ NH
I 2-(4-nitrophenyl)ethanol N N
N N H2 Ph3P, DEAD I N I ~
O
Ac0 1,4-dioxane, rt ' N NHZ
Ac0 O
AcO 21 AcO 22 Compound 21 (1.75 g, 5.0 mmol) was azeotropically dried by 1,4-dioxan three times, added with triphenylphosphine (2.62 g, 10.0 mmol) and 2-(4-nitrophenyl)ethanol (1.67 g, 10.0 mmol) and the mixture was dissolved in 1,4-dioxan under argon atmosphere (50 mL) and stirred at room temperature for 15 minutes.
Subsequently, the reaction solution was added dropwise in azodicarboxylic acid diphenyl ester 40% toluene solution (4.5 mL, 10.0 mmol) and stirred at room temperature for 30 minutes. After concentrated under reduce pressure and added with ethyl acetate (50 mL), the reaction solution was washed with a saturated solution of sodium hydrogen carbonate (50 mL) twice, water (50 mL) and a saturated brine (50 mL) once respectively and after the organic layer was dried over anhydrous magnesium sulfate, the anhydrous magnesium sulfate was removed by filtration and the filtrate was concentrated under reduced pressure.
The residue was purified by silica gel column chromatography (chloroform: ethyl acetate =1:3) to obtain 2.30 g of compound 22 (92%).
1H-NMR (CDC13) :52.08 and 2.13 (2s, 6H, CH3COx2) , 2.52 (ddd, 1H, J1',2 = 6.1 Hz, J2',2" = 14.1 Hz, J2",3, = 2.5 Hz, H-2") , 2.98(ddd, 1H, J1',2' = 7.8 Hz, Jz,,Zõ = 14.1 Hz, J2,,3,=4.9 Hz, H-2' 1) 3.27 (t, 2H, J = 6. 8 Hz, CH2CH2PhNO2) , 4. 31-4.47 (m, 3H, H-4',51,5"), 4.73(t, 2H, J= 6.8 Hz, CHZCHzPhNOz), 4. 89 (brs, 2H, N2-H2) , 5.42 (m, 1H, H-3' ), 6.27 (dd, iH, J1,,2, = 7.8 Hz, Jl',Z = 6. 1 Hz, H-1' ), 7.48 (d, 2H, J 8. 6 Hz, Ph-H of 2-(4-nitrophenyl)ethyl group), 7.74(s, 1H, H-8), 8.16 (d, 2H, J = 8.6 Hz, Ph-H of 2-(4-nitrophenyl)ethyl group).
[Pre-step 7-3]
Synthesis of 3',5'-di-O-acetyl-2-fluoro-OS-[2-(4-nitrophenyl)ethyl]-2'-dioxyguanosine (23);
NOz NO
\ I \ I
N ~ N 45% HF-pyridine N
t-butyl nitrite _ N N NH2 -45 C to rt N N!\F
Ac0 O Ac0 O
Ac0 22 AcO 23 Compound 22 (0.50 g, 1.00 mmol) was azeotropically dried with pyridine three times and added with 45%
hydrogen fluoride/pyridine solution (4.8 mL) at -30 C
and added with t-butyl nitrite (0.36 mL, 3.00 mmol) under argon atmosphere and stirred for 30 minutes after resumed to room temperature. Then the mixture was neutralized with a saturated sodium hydrogen carbonate aqueous solution (15 mL), extracted with chloroform (30 mL) three times and washed with water (50 mL) twice. The organic layer was dried over anhydrous magnesium sulfate and the anhydrous magnesium sulfate was removed by filtration, and the filtrate was concentrated under reduced pressure.
The residue was purified by silica gel column chromatography (chloroform: ethyl acetate =1:3) to obtain 0.44 g of compound 23 (87%).
1H-NMR (CDC13) :52.08 and 2.11 (2s, 6H, CH3COx2) , 2.60 (ddd, 1H, J1',2" = 5.9 Hz, J2,,2" = 14.1 Hz, J2,,,3' = 2.5 Hz, H-2") , 2.87 (ddd, 1H, J1',2' = 7.9 Hz, J2',2" = 14.2 Hz, J2',3' = 6.4 Hz, H-2'), 3.30 (t, 2H, J= 6.7 Hz, CH2CH2PhNO2) , 4.30-4.39 (m, 3H, H-4' , 5' , 5") , 4. 82 (t, 2H, J= 6. 7 Hz, CH2CH2PhNO2) 5.39 (m, 1H, H-3' ), 6.35 (dd, 1H, J1,,2' = 7. 9 Hz, J1',2" =
5.9 Hz, H-1'), 7.48 (d, 2H, J = 8.7 Hz, Ph-H of 2-(4-nitrophenyl)ethyl group), 8.06 (s, 1H, H-8), 8.15 (d, 2H, J = 8.7 Hz, Ph-H of 2-(4-nitrophenyl)ethyl group).
[Pre-step 8]
Synthesis of 9-fluorenylmethyl 3-aminopropyl-i-carbamate hydrochloride (26);
/1,3-diaminopropane(1.0 eq.) ONH
u O then HCI-pyridine, rt II
O ~ N'H3 CI
Compound 25 (3.80 g, 12.0 mmol) was suspended in methanol (50 mL) Immediately after that, 1,3-diaminopropane (1.00 mL, 12.0 mmol) was added and the mixture was stirred at room temperature for four hours.
After pyridine hydrochloride (3.00 g, 26.0 mmol) was added to the reaction solution which became slightly transparent, the mixture was stirred for 10 minutes and then concentrated under reduced pressure. The residue was suspended in eluent (chloroform:methanol =4:1) for silica gel column chromatography and purified by column chromatography shortly loaded with silica gel (chloroform:methanol =4:1) to obtain 3.25 g of compound 26 (820). Although it was usually difficult to protect one of the diamine with a protecting group, this purpose was able to be achieved with the above compound.
1H-NMR (DMSO-d6) :57.79 (d, 2H, J = 7.52 Hz, Fmoc-H) , 7.63 (d, 2H, J = 7.39 Hz, Fmoc-H), 7.39 (t, 2H, J = 7.38 Hz Ph-H), 7.31 (t, 2H, J = 7.45 Hz Ph-H), 4.40 (d, 2H, J
6.61 Hz, Fmoc-H), 4.20 (t, 1H, J = 6.44 Hz, Fmoc-H), 3.20 (d, 2H, J = 6.44 Hz, CH2NH), 2.91 (t, 2H, J = 7.34 Hz, NHCH2) , 1.82. (tt, 2H, J = 7.11 Hz, J = 6.83 Hz, -CH2CH2-) 13C-NMR (DMSO-d6) :6159.49, 145.41, 142.80, 128.94, 128.27, 126.21, 121.10, 67.83, 48.64, 38.42, 29.32 HRESIMS m/z : 297. 1615 (Calcd for C1aH2102N2:297.1603) Anal. Calcd for C18H2ON20zCl + H20 : C, 61 . 62 ; H, 6. 61 ; N, 7.98 Found. C, 61.29; H, 6.83; N, 7.27 m.p.: 129-130 C
[Step 11 Synthesis of 1-methylpyrrole-2-carboxylic acid (32);
CI3C 2 M NaOH aq. HO
N
N MeOH, 60 C O
O
Compound 3 (5.00 g, 22.0 mmol) synthesized in Pre-step 1 was dissolved in methanol (50 mL) and added with 2.0 M aqueous sodium hydroxide (50 mL) . The reaction solution was warmed to 60 C and stirred for one hour.
Methanol was removed under reduced pressure and the remaining solution was diluted with water (50 mL) and washed with ether (50 mL). The aqueous layer was adjusted to approximately pH 3 with 10% hydrochloric acid and extracted with ethyl acetate (100 mLx3) . After the organic layers were collected all together, washed with a saturated sodium chloride aqueous solution (150 mL) and dried over anhydrous magnesium sulfate, the anhydrous magnesium sulfate was removed by filtration. The filtrate was concentrated under reduced pressure and the residue was dried under reduced pressure to obtain 2.45 g of compound 32 (89%).
1 H-NMR (DMSO-d6) :512 .23 (br. , 1H, -COOH) , 6. 99 (d, 1H, J =
1.94 Hz, Py-H), 6.80 (d, 1H, J = 1.80 Hz, Py-H), 6. 04 (d, 1H, J = 2.58 Hz, Py-H), 3.84 (s, 3H, Py-CH3), 13C-NMR (1JMSO-d6):6162.14, 129.82, 122.67, 117.37, 107.42, 36.34 FAB-MS m/z:125.1 Anal. Calcd for C6H7N102: C, 57 . 59; H, 5. 64 ; N, 11.19 Found. C.57.59; H,5.64; N,11.06 m.p.: 127-130 C
[Step 21 Synthesis of methyl 1-methyl-4-{[4-({4-[(1-methylpyrrol-2-yl)carbonyl)amino-l-methylpyrrol-2-yl}carbonyl)amino-l-methylpyrrol-2-yl]carbonyl}aminopyrrole-2-carboxylate (33) ;
5.9 Hz, H-1'), 7.48 (d, 2H, J = 8.7 Hz, Ph-H of 2-(4-nitrophenyl)ethyl group), 8.06 (s, 1H, H-8), 8.15 (d, 2H, J = 8.7 Hz, Ph-H of 2-(4-nitrophenyl)ethyl group).
[Pre-step 8]
Synthesis of 9-fluorenylmethyl 3-aminopropyl-i-carbamate hydrochloride (26);
/1,3-diaminopropane(1.0 eq.) ONH
u O then HCI-pyridine, rt II
O ~ N'H3 CI
Compound 25 (3.80 g, 12.0 mmol) was suspended in methanol (50 mL) Immediately after that, 1,3-diaminopropane (1.00 mL, 12.0 mmol) was added and the mixture was stirred at room temperature for four hours.
After pyridine hydrochloride (3.00 g, 26.0 mmol) was added to the reaction solution which became slightly transparent, the mixture was stirred for 10 minutes and then concentrated under reduced pressure. The residue was suspended in eluent (chloroform:methanol =4:1) for silica gel column chromatography and purified by column chromatography shortly loaded with silica gel (chloroform:methanol =4:1) to obtain 3.25 g of compound 26 (820). Although it was usually difficult to protect one of the diamine with a protecting group, this purpose was able to be achieved with the above compound.
1H-NMR (DMSO-d6) :57.79 (d, 2H, J = 7.52 Hz, Fmoc-H) , 7.63 (d, 2H, J = 7.39 Hz, Fmoc-H), 7.39 (t, 2H, J = 7.38 Hz Ph-H), 7.31 (t, 2H, J = 7.45 Hz Ph-H), 4.40 (d, 2H, J
6.61 Hz, Fmoc-H), 4.20 (t, 1H, J = 6.44 Hz, Fmoc-H), 3.20 (d, 2H, J = 6.44 Hz, CH2NH), 2.91 (t, 2H, J = 7.34 Hz, NHCH2) , 1.82. (tt, 2H, J = 7.11 Hz, J = 6.83 Hz, -CH2CH2-) 13C-NMR (DMSO-d6) :6159.49, 145.41, 142.80, 128.94, 128.27, 126.21, 121.10, 67.83, 48.64, 38.42, 29.32 HRESIMS m/z : 297. 1615 (Calcd for C1aH2102N2:297.1603) Anal. Calcd for C18H2ON20zCl + H20 : C, 61 . 62 ; H, 6. 61 ; N, 7.98 Found. C, 61.29; H, 6.83; N, 7.27 m.p.: 129-130 C
[Step 11 Synthesis of 1-methylpyrrole-2-carboxylic acid (32);
CI3C 2 M NaOH aq. HO
N
N MeOH, 60 C O
O
Compound 3 (5.00 g, 22.0 mmol) synthesized in Pre-step 1 was dissolved in methanol (50 mL) and added with 2.0 M aqueous sodium hydroxide (50 mL) . The reaction solution was warmed to 60 C and stirred for one hour.
Methanol was removed under reduced pressure and the remaining solution was diluted with water (50 mL) and washed with ether (50 mL). The aqueous layer was adjusted to approximately pH 3 with 10% hydrochloric acid and extracted with ethyl acetate (100 mLx3) . After the organic layers were collected all together, washed with a saturated sodium chloride aqueous solution (150 mL) and dried over anhydrous magnesium sulfate, the anhydrous magnesium sulfate was removed by filtration. The filtrate was concentrated under reduced pressure and the residue was dried under reduced pressure to obtain 2.45 g of compound 32 (89%).
1 H-NMR (DMSO-d6) :512 .23 (br. , 1H, -COOH) , 6. 99 (d, 1H, J =
1.94 Hz, Py-H), 6.80 (d, 1H, J = 1.80 Hz, Py-H), 6. 04 (d, 1H, J = 2.58 Hz, Py-H), 3.84 (s, 3H, Py-CH3), 13C-NMR (1JMSO-d6):6162.14, 129.82, 122.67, 117.37, 107.42, 36.34 FAB-MS m/z:125.1 Anal. Calcd for C6H7N102: C, 57 . 59; H, 5. 64 ; N, 11.19 Found. C.57.59; H,5.64; N,11.06 m.p.: 127-130 C
[Step 21 Synthesis of methyl 1-methyl-4-{[4-({4-[(1-methylpyrrol-2-yl)carbonyl)amino-l-methylpyrrol-2-yl}carbonyl)amino-l-methylpyrrol-2-yl]carbonyl}aminopyrrole-2-carboxylate (33) ;
N-Boc HN
HN O
N~
HN 1) AcCI, MeOH, AcOEt, 0 C N--O 2) 32, EDCI, DMAP HN
H DMF, rt to 80 C 0 ~ \
MeO N 0 N MeO N N
p N" \~ J O \
O
/ N
Compound 8 (100 mg, 0.20 mmol) synthesized in Pre-step 6 was dissolved in methanol (5.0 mL) under argon atmosphere and cooled to 0 C. Subsequently, acetyl chloride (1.7 mL, 23.9 mmol) was added dropwise while taking care of heat generation and after stirred at 0 C
for 30 minutes, the solvents and the reaction reagents were removed by performing concentration under reduced pressure. The above compound 32 (27.7 mg, 0.22 mmol), EDCI(84.5 mg, 0.44 mmol) and DMAP (53.4 mg, 0.44 mmol) were added to the residue, dissolved in DMF (2.0 mL) under argon atmosphere and stirred at 80 C for one hour.
The reaction solution was concentrated under reduced pressure and the residue was purified by silica gel column chromatography (chloroform:methanol =19:1) to obtain 71.4 mg of compound 33 (73%).
1H-NMR (DMSO-d6):59.96 (s, 1H, Py-NH) , 9.95 (s, 1H, Py-NH), 9.85 (s, 1H, Py-NH), 7.48 (d, 1H, J = 1.89 Hz, Py-H), 7.25 (d, 1H, J = 1.74 Hz, Py-H), 7.24 (d, 1H, J = 1.73 Hz, Py-H), 7.07 (d, 1H, J = 1.77 Hz, Py-H), 7.05 (d, 1H, J
1.76 Hz, Py-H), 6.95 (d, 1H, J = 1.89 Hz, Py-H), 6.93 (d, 1H, J = 1.66 Hz, Py-H), 6.91 (d, 1H, J = 1.66 Hz, Py-H), 6.06 (d, 1H, J = 2.58 Hz, Py-H), 3.88 (s, 3H, Py-CH3), 3.86 (s, 3H, Py-CH3), 3.85 (s, 3H, Py-CH3), 3.84 (s, 3H, Py-CH3) , 3.74 (s, 3H, O-CH3) 13C-NMR (DMSO-d6):5160.82, 158.61, 158.50, 128.16, 125.45, 122.99, 122.73, 122.49, 122.32, 122.11, 120.76, 118.57, 118.48, 112.67, 108.34, 106.66, 104.77, 104.69, 50.97, 36.27, 36.20, 36.14 HRESIMS m/z :506.2156 (Calcd for C25H28N705 :506.2152) Anal Calcd for C25H27N705: C, 59.40; H, 5.38; N, 19.40 Found. C, 59.22; H, 5.45; N,19.38 m.p.: 229-231 C
[Step 31 Synthesis of 1-methyl-4-{[4-({4-[(l-methylpyrrol-2-yl)carbonyl)amino-l-methylpyrrol-2-yl}carbonyl)amino-l-methylpyrrol-2-yl]carbonyl}aminopyrrole-2-carboxylic acid (34) ;
\
2 M NaOH aq. N-\
HN MeOH, 60 C HN 0 MeO N I N N N
HN O
N~
HN 1) AcCI, MeOH, AcOEt, 0 C N--O 2) 32, EDCI, DMAP HN
H DMF, rt to 80 C 0 ~ \
MeO N 0 N MeO N N
p N" \~ J O \
O
/ N
Compound 8 (100 mg, 0.20 mmol) synthesized in Pre-step 6 was dissolved in methanol (5.0 mL) under argon atmosphere and cooled to 0 C. Subsequently, acetyl chloride (1.7 mL, 23.9 mmol) was added dropwise while taking care of heat generation and after stirred at 0 C
for 30 minutes, the solvents and the reaction reagents were removed by performing concentration under reduced pressure. The above compound 32 (27.7 mg, 0.22 mmol), EDCI(84.5 mg, 0.44 mmol) and DMAP (53.4 mg, 0.44 mmol) were added to the residue, dissolved in DMF (2.0 mL) under argon atmosphere and stirred at 80 C for one hour.
The reaction solution was concentrated under reduced pressure and the residue was purified by silica gel column chromatography (chloroform:methanol =19:1) to obtain 71.4 mg of compound 33 (73%).
1H-NMR (DMSO-d6):59.96 (s, 1H, Py-NH) , 9.95 (s, 1H, Py-NH), 9.85 (s, 1H, Py-NH), 7.48 (d, 1H, J = 1.89 Hz, Py-H), 7.25 (d, 1H, J = 1.74 Hz, Py-H), 7.24 (d, 1H, J = 1.73 Hz, Py-H), 7.07 (d, 1H, J = 1.77 Hz, Py-H), 7.05 (d, 1H, J
1.76 Hz, Py-H), 6.95 (d, 1H, J = 1.89 Hz, Py-H), 6.93 (d, 1H, J = 1.66 Hz, Py-H), 6.91 (d, 1H, J = 1.66 Hz, Py-H), 6.06 (d, 1H, J = 2.58 Hz, Py-H), 3.88 (s, 3H, Py-CH3), 3.86 (s, 3H, Py-CH3), 3.85 (s, 3H, Py-CH3), 3.84 (s, 3H, Py-CH3) , 3.74 (s, 3H, O-CH3) 13C-NMR (DMSO-d6):5160.82, 158.61, 158.50, 128.16, 125.45, 122.99, 122.73, 122.49, 122.32, 122.11, 120.76, 118.57, 118.48, 112.67, 108.34, 106.66, 104.77, 104.69, 50.97, 36.27, 36.20, 36.14 HRESIMS m/z :506.2156 (Calcd for C25H28N705 :506.2152) Anal Calcd for C25H27N705: C, 59.40; H, 5.38; N, 19.40 Found. C, 59.22; H, 5.45; N,19.38 m.p.: 229-231 C
[Step 31 Synthesis of 1-methyl-4-{[4-({4-[(l-methylpyrrol-2-yl)carbonyl)amino-l-methylpyrrol-2-yl}carbonyl)amino-l-methylpyrrol-2-yl]carbonyl}aminopyrrole-2-carboxylic acid (34) ;
\
2 M NaOH aq. N-\
HN MeOH, 60 C HN 0 MeO N I N N N
Compound 33 (127 mg, 0.25 mmol) was dissolved in methanol (2.0 mL) and added with 2.0 M aqueous sodium hydroxide (2 mL) . The reaction solution was warmed to 60 C and stirred for two hours. Methanol was removed under reduced pressure and the remaining solution was diluted with water (5 mL) and washed with ether (3 mL). The aqueous layer was adjusted to approximately pH 3 with 10%
hydrochloric acid and extracted with ethyl acetate (10 mLx3) . After the organic layers were collected all together, washed with a saturated sodium chloride aqueous solution (15 mL) and dried over anhydrous magnesium sulfate, the anhydrous magnesium sulfate was removed by filtration. The filtrate was concentrated under reduced pressure and the residue was dried under reduced pressure to obtain 123 mg of compound 34 (quant.).
1H-NMR (DMSO-d6) :812 . 13 (brs, 1H, COOH) , 9. 93 (s, 1H, Py-NH), 9.89 (s, 1H, Py-NH), 9.82 (s, 1H, Py-NH), 7.43 (d, 1H, J = 1.91 Hz, Py-H), 7.24 (d, 2H, J = 2.10 Hz, Py-H), 7.07 (d, 1H, J = 1.83 Hz, Py-H), 7.04 (d, 1H, J = 1.84 Hz, Py-H), 6.95 (d, 1H, J = 1.81 Hz, Py-H), 6.92 (d, 1H, J =
1.73 Hz, Py-H), 6.85 (d, 1H, J = 1.94 Hz, Py-H), 6.06 (d, 1H, J = 2.55 Hz, Py-H), 6.06 (d, 1H, J = 2.58 Hz, Py-H), 3.88 (s, 3H, Py-CH3) , 3.86 (s, 3H, Py-CH3) , 3.85 (s, 3H, Py-CH3) , 3 .83 (s, 3H, Py-CH3) , 13C-NMR (DMSO-d6):5161.94, 158.59, 158.47, 158.43, 128.09, 125.45, 122.75, 122.68, 122.58, 122.26, 122.08, 120.24, 117.50, 118.45, 112.63, 108.38, 106.62, 104.69, 36.20, 36.08 HRESIMS m/z :492.1989 (Calcd for C24H26N705 : 492.1995) Anal Calcd for C24H25N705 + MeOH : C, 57. 35; H, 5.58;
N,18.73 Found. C,56.95; H, 5.64; N, 18.56 m.p.: 180-182 C
[Step 41 Synthesis of 9-fluorenylmethyl 3-[(4-{[4-{4-[(1-methylpyrrol-2-yl)carbonyl]amino-l-methylpyrrol-2-yl}carbonyl)amino-l-methylpyrrol-2-yl]carbonyl}amino-l-methylpyrrol2-yl)carbonyl]aminopropyl-l-carbamate (35);
N Fmoc-NH
~
HN HN
O O
HO 26, DCC, HOBT, DIEA NH H
N ~ N N
O N~ NH O \ DMF, rt N NH O
O N O N
Compound 34 (1.00 g, 2.04 mmol) and compound 26 (0.74 g, 2.24 mmol) synthesized in the above Pre-step 8 were dissolved in DMF (10 mL) along with dicyclohexylcarbodiimide (0.63 g, 3.06 mmol) and 1-hydroxybenztriazole (0.41 g, 3.06 mmol) under argon atmosphere and after added with diisopropylethylamine (0.39 ml, 2.24 mmol), stirred at room temperature for 12 hours. Subsequently, the reaction solution was diluted with ethyl acetate (200 mL) and washed with 10%
hydrochloric acid and extracted with ethyl acetate (10 mLx3) . After the organic layers were collected all together, washed with a saturated sodium chloride aqueous solution (15 mL) and dried over anhydrous magnesium sulfate, the anhydrous magnesium sulfate was removed by filtration. The filtrate was concentrated under reduced pressure and the residue was dried under reduced pressure to obtain 123 mg of compound 34 (quant.).
1H-NMR (DMSO-d6) :812 . 13 (brs, 1H, COOH) , 9. 93 (s, 1H, Py-NH), 9.89 (s, 1H, Py-NH), 9.82 (s, 1H, Py-NH), 7.43 (d, 1H, J = 1.91 Hz, Py-H), 7.24 (d, 2H, J = 2.10 Hz, Py-H), 7.07 (d, 1H, J = 1.83 Hz, Py-H), 7.04 (d, 1H, J = 1.84 Hz, Py-H), 6.95 (d, 1H, J = 1.81 Hz, Py-H), 6.92 (d, 1H, J =
1.73 Hz, Py-H), 6.85 (d, 1H, J = 1.94 Hz, Py-H), 6.06 (d, 1H, J = 2.55 Hz, Py-H), 6.06 (d, 1H, J = 2.58 Hz, Py-H), 3.88 (s, 3H, Py-CH3) , 3.86 (s, 3H, Py-CH3) , 3.85 (s, 3H, Py-CH3) , 3 .83 (s, 3H, Py-CH3) , 13C-NMR (DMSO-d6):5161.94, 158.59, 158.47, 158.43, 128.09, 125.45, 122.75, 122.68, 122.58, 122.26, 122.08, 120.24, 117.50, 118.45, 112.63, 108.38, 106.62, 104.69, 36.20, 36.08 HRESIMS m/z :492.1989 (Calcd for C24H26N705 : 492.1995) Anal Calcd for C24H25N705 + MeOH : C, 57. 35; H, 5.58;
N,18.73 Found. C,56.95; H, 5.64; N, 18.56 m.p.: 180-182 C
[Step 41 Synthesis of 9-fluorenylmethyl 3-[(4-{[4-{4-[(1-methylpyrrol-2-yl)carbonyl]amino-l-methylpyrrol-2-yl}carbonyl)amino-l-methylpyrrol-2-yl]carbonyl}amino-l-methylpyrrol2-yl)carbonyl]aminopropyl-l-carbamate (35);
N Fmoc-NH
~
HN HN
O O
HO 26, DCC, HOBT, DIEA NH H
N ~ N N
O N~ NH O \ DMF, rt N NH O
O N O N
Compound 34 (1.00 g, 2.04 mmol) and compound 26 (0.74 g, 2.24 mmol) synthesized in the above Pre-step 8 were dissolved in DMF (10 mL) along with dicyclohexylcarbodiimide (0.63 g, 3.06 mmol) and 1-hydroxybenztriazole (0.41 g, 3.06 mmol) under argon atmosphere and after added with diisopropylethylamine (0.39 ml, 2.24 mmol), stirred at room temperature for 12 hours. Subsequently, the reaction solution was diluted with ethyl acetate (200 mL) and washed with 10%
hydrochloric acid (50 mLx3), a saturated sodium hydrogen carbonate aqueous solution (50 mLx3) and a saturated sodium chloride aqueous solution (50 mL) and dried over anhydrous magnesium sulfate. After the anhydrous magnesium sulfate was removed by filtration and the filtrate was concentrated under reduced pressure, the residue was purified by silica gel column chromatography (chloroform:methanol =19:1) to obtain 1.35 g of compound 35 (86%) ) .
1H-NMR (DMSO-d6):89.93 (s, 1H, Py-NH) , 9.89 (s, 1H, Py-NH), 9.82 (s, 1H, Py-NH), 8.31 (t, 1H, J = 5.55 Hz, Boc-NH), 7.90 (d, 2H, J = 7.48 Hz, Fmoc-H), 7.70 (d, 2H, J =
7.31 Hz, Fmoc-H), 7.41 (t, 2H, J = 7.37 Hz, Fmoc-H), 7.33 (t, 2H, J = 7.42 Hz, Fmoc-H), 7.28 (t, 1H, J = 5.67 Hz, Py-CONH), 7.25 (d, 2H, J = 1.65 Hz, Py-H), 7.19 (d, 1H, J
= 1.17 Hz, Py-H), 7.06 (d, 2H, Py-H), 6.95 (d, 1H, J =
1.79 Hz, Py-H), 6.93 (d, 1H, J = 1.64 Hz, Py-H), 6.88 (d, 1H, J = 1.32 Hz, Py-H), 6.06 (d, 1H, J = 2.57 Hz, Py-H) 4.32 (d, 2H, J = 6.84 Hz, Fmoc-CH2), 4.22 (t, 1H, J =
6.64 Hz, Fmoc-CH), 3.89 (s, 3H, Py-CH3), 3.86 (s, 3H, Py-CH3) , 3.85 (s, 3H, Py-CH3) , 3.80 (s, 3H, Py-CH3) , 3.17 (dt, 2H, J = 8.15 Hz, J = 5.27 Hz, -NHCH2-), 3.02 (dt, 2H, J
6.44 Hz, J = 6.28 Hz, -CH2NH-), 1.62 (tt, 2H, J = 6.68 Hz, -CH2CH2CH2-) 13C-NMR (DMSO-d6):8161.27, 158.50, 158.46, 156.12, 143.93, 140.74, 128.11, 127.58, 128.04, 125.47, 125.11, 122.91, 122.79, 122.19, 122.16, 122.09, 120.10, 118.43, 117.80, 112.64, 106.64, 104, 71, 104.13, 65.21, 48.59, 46.78, 38.05, 36.21, 36.07.35.99, 29.66 HRESIMS m/z :770.3400 (Calcd for C42H44N9O6 :770.3415) Anal Calcd for C42H43N9O6 + MeOH : C, 62 . 99; H, 6. 02 ; N, 15 . 37 Found. C,62.92; H, 5.79; N, 15.19 [Step 5]
Synthesis of 3' , 5' -di-O-acetyl-N2- {3- [ (4- { [4- ( {4- [ (1-methylpyrrol-2-yl)carbonyl]amino-i-methylpyrrol-2-yl}carbonyl)amino-l-methylpyrrol-2-yl]carbonyl}amino-l-methylpyrrol - 2 -yl ) carbonyl ] aminopropyl } - O6- [2 - ( 4 -nitrophenyl)ethyl-2'-deoxyguanosine (36);
~ N~
-O N-HN NOZ O
N_ I HN
\ -O 1) triethylamine O N_ HN
DMF, rt or 60 C N ~ N HN
N 2) 23, triethylamine 0 HN DMF, rt to 60 N N NlH H I N
H H O Ac0 O N N \
~ 0 Fmoc NN N
O Ac0 O
Compound 35 (1.00 g, 1.30 mmol) and compound 23 (0.69 g, 1.36 mmol) synthesized in the above Pre-step 7-3 were dissolved in DMF (5.0 mL) under argon atmosphere.
Subsequently, the mixture was added with triethylamine (1.0 mL) and stirred at 60 C for 12 hours. After the reaction solution was concentrated under reduced pressure, the residue was purified by silica gel column ~
1H-NMR (DMSO-d6):89.93 (s, 1H, Py-NH) , 9.89 (s, 1H, Py-NH), 9.82 (s, 1H, Py-NH), 8.31 (t, 1H, J = 5.55 Hz, Boc-NH), 7.90 (d, 2H, J = 7.48 Hz, Fmoc-H), 7.70 (d, 2H, J =
7.31 Hz, Fmoc-H), 7.41 (t, 2H, J = 7.37 Hz, Fmoc-H), 7.33 (t, 2H, J = 7.42 Hz, Fmoc-H), 7.28 (t, 1H, J = 5.67 Hz, Py-CONH), 7.25 (d, 2H, J = 1.65 Hz, Py-H), 7.19 (d, 1H, J
= 1.17 Hz, Py-H), 7.06 (d, 2H, Py-H), 6.95 (d, 1H, J =
1.79 Hz, Py-H), 6.93 (d, 1H, J = 1.64 Hz, Py-H), 6.88 (d, 1H, J = 1.32 Hz, Py-H), 6.06 (d, 1H, J = 2.57 Hz, Py-H) 4.32 (d, 2H, J = 6.84 Hz, Fmoc-CH2), 4.22 (t, 1H, J =
6.64 Hz, Fmoc-CH), 3.89 (s, 3H, Py-CH3), 3.86 (s, 3H, Py-CH3) , 3.85 (s, 3H, Py-CH3) , 3.80 (s, 3H, Py-CH3) , 3.17 (dt, 2H, J = 8.15 Hz, J = 5.27 Hz, -NHCH2-), 3.02 (dt, 2H, J
6.44 Hz, J = 6.28 Hz, -CH2NH-), 1.62 (tt, 2H, J = 6.68 Hz, -CH2CH2CH2-) 13C-NMR (DMSO-d6):8161.27, 158.50, 158.46, 156.12, 143.93, 140.74, 128.11, 127.58, 128.04, 125.47, 125.11, 122.91, 122.79, 122.19, 122.16, 122.09, 120.10, 118.43, 117.80, 112.64, 106.64, 104, 71, 104.13, 65.21, 48.59, 46.78, 38.05, 36.21, 36.07.35.99, 29.66 HRESIMS m/z :770.3400 (Calcd for C42H44N9O6 :770.3415) Anal Calcd for C42H43N9O6 + MeOH : C, 62 . 99; H, 6. 02 ; N, 15 . 37 Found. C,62.92; H, 5.79; N, 15.19 [Step 5]
Synthesis of 3' , 5' -di-O-acetyl-N2- {3- [ (4- { [4- ( {4- [ (1-methylpyrrol-2-yl)carbonyl]amino-i-methylpyrrol-2-yl}carbonyl)amino-l-methylpyrrol-2-yl]carbonyl}amino-l-methylpyrrol - 2 -yl ) carbonyl ] aminopropyl } - O6- [2 - ( 4 -nitrophenyl)ethyl-2'-deoxyguanosine (36);
~ N~
-O N-HN NOZ O
N_ I HN
\ -O 1) triethylamine O N_ HN
DMF, rt or 60 C N ~ N HN
N 2) 23, triethylamine 0 HN DMF, rt to 60 N N NlH H I N
H H O Ac0 O N N \
~ 0 Fmoc NN N
O Ac0 O
Compound 35 (1.00 g, 1.30 mmol) and compound 23 (0.69 g, 1.36 mmol) synthesized in the above Pre-step 7-3 were dissolved in DMF (5.0 mL) under argon atmosphere.
Subsequently, the mixture was added with triethylamine (1.0 mL) and stirred at 60 C for 12 hours. After the reaction solution was concentrated under reduced pressure, the residue was purified by silica gel column ~
chromatography (chloroform:methanol =29:1) to obtain 1.07 g of compound 36 (79%).
1H-NMR (DMSO-d6):89.93(s, 1H, Py-NH) , 9.88 (s, 1H, Py-NH) 9.82 (s, 1H, Py-NH), 8.18 (d, 2H, J = 8.50 Hz, NPE-H), 8.03 (s, 1H, H-8), 8.02 (t, 1H, J = 5.42 Hz, -CH2NH-CO-) 7.60 (d, 2H, J = 8.05 Hz, NPE-H), 7.24 (d, 2H, J = 1.69 Hz, Py-H), 7.17 (d, 1H, J = 1.59 Hz, Py-H), 7.06 (s, 1H, Py-H), 7.05 (s, 1H, Py-H), 7.03 (t, 1H, J = 5.9 Hz, NZ-H), 6.95-6.92 (m, 3H, Py-H), 6.25 (t, 1H, J = 6.78 Hz, H-1'), 6.07 (dd, 1H, J = 2.65 Hz, J = 3.86 Hz, Py-H), 5.43 (m, 1H, H-3'), 4.69 (t, 2H, J = 6.59 Hz, NPE-H), 4.31 (t, 1H, J = 7.59 Hz, H-4'), 4.22 (m, 2H, H-5'), 3.89 (s, 3H, Py-CH3) , 3.86 (s, 3H, Py-CH3) , 3.85 (s, 3H, Py-CH3) , 3. 80 (s, 3H, Py-CH3), 3.37 (m, 2H, -CH2NH-CO-), 3.27 (m, 4H, -COCH2, NPE-CH2), 3.18 (m, 1H, H-2' ), 2.46 (m, 1H, H-2") , 2.08 (s, 3H, OAc), 1.99 (s, 3H,OAc),1.77 (tt, 2H, J = 6.75 Hz,-CH2CH2CH2) 13C-NMR (DMSO-d6):5170.01, 161.33, 160.01, 158.72, 158.60, 158.50, 157.86, 146.72, 146.24, 130.22, 128.10, 125.47, 123.39, 122.96, 122.79, 122.20, 122.09, 118.43, 117.75, 112.64, 106.63, 104.71, 104.16, 81.37, 74.24, 65.42, 63.65, 36.21, 36.07, 35.91, 34.33, 20.78, 20.48 HRESIMS m/z :1031.4148 (Calcd for C49H55N14012 : 1031.4124) [Step 6]
Synthesis of 3' , 5' -di-O-acetyl-NZ-{3- [ (4-{ [4-{4- [ (1-methylpyrrol-2-yl)carbonyl]amino-l-methylpyrrol-2-yl}carbonyl)amino-l-methylpyrrol-2-yl]carbonyl}amino-l-methylpyrrol-2-yl)carbonyl]aminopropyl}-21-deoxyguanosine (37) ;
O
N- H N
iN NO z -~ HN 0 I DBU HN O
0 N\ pyridine, rt ~
N ~ N
~~ ~ N HN O N~NH H
N N NH H ~ N N N NH N I N O
Ac0 O ~ N N 0 AcO N
Ac0 0 36 AcO 37 Compound 36 (22.8 mg, 22 mol) was dissolved in 0.5 M
DBU/pyridine solution (1.0 mL) under argon atmosphere.
After stirring at room temperature for 12 hours, ammonium chloride (50.0 mg, 0.94 mmol) was added thereto to terminate the reaction. The reaction solution was concentrated under reduced pressure and the residue was purified by silica gel column chromatography (chloroform:methanol =4:1) to obtain 15.6 mg of compound 37 (80%).
1 H-NMR (DMSO-d6) :610. 67 (br, 1H,H-1), 9.93 (s, 1H, Py-NH) 9.88 (s, 1H, Py-NH),9.82 (s, 1H, Py-NH), 8.05 (t, 1H, J
5.61 Hz, Py-CONH), 7.87 (s, 1H, H-8), 7.24 (d, 1H2H, J =
1.52 Hz, Py-H), 7.18 (d, 1H, J = 1.62 Hz, Py-H), 7.05 (dm, 1H2H, J = 1.58, Py-H), 6.95-6.90 (m, 3H, Py-H), 6.53 (brs, 1H,N2-H),6.17 (d, 1H, J = 7.00 Hz, H-11), 6.06 (dd, 1H, J
1H-NMR (DMSO-d6):89.93(s, 1H, Py-NH) , 9.88 (s, 1H, Py-NH) 9.82 (s, 1H, Py-NH), 8.18 (d, 2H, J = 8.50 Hz, NPE-H), 8.03 (s, 1H, H-8), 8.02 (t, 1H, J = 5.42 Hz, -CH2NH-CO-) 7.60 (d, 2H, J = 8.05 Hz, NPE-H), 7.24 (d, 2H, J = 1.69 Hz, Py-H), 7.17 (d, 1H, J = 1.59 Hz, Py-H), 7.06 (s, 1H, Py-H), 7.05 (s, 1H, Py-H), 7.03 (t, 1H, J = 5.9 Hz, NZ-H), 6.95-6.92 (m, 3H, Py-H), 6.25 (t, 1H, J = 6.78 Hz, H-1'), 6.07 (dd, 1H, J = 2.65 Hz, J = 3.86 Hz, Py-H), 5.43 (m, 1H, H-3'), 4.69 (t, 2H, J = 6.59 Hz, NPE-H), 4.31 (t, 1H, J = 7.59 Hz, H-4'), 4.22 (m, 2H, H-5'), 3.89 (s, 3H, Py-CH3) , 3.86 (s, 3H, Py-CH3) , 3.85 (s, 3H, Py-CH3) , 3. 80 (s, 3H, Py-CH3), 3.37 (m, 2H, -CH2NH-CO-), 3.27 (m, 4H, -COCH2, NPE-CH2), 3.18 (m, 1H, H-2' ), 2.46 (m, 1H, H-2") , 2.08 (s, 3H, OAc), 1.99 (s, 3H,OAc),1.77 (tt, 2H, J = 6.75 Hz,-CH2CH2CH2) 13C-NMR (DMSO-d6):5170.01, 161.33, 160.01, 158.72, 158.60, 158.50, 157.86, 146.72, 146.24, 130.22, 128.10, 125.47, 123.39, 122.96, 122.79, 122.20, 122.09, 118.43, 117.75, 112.64, 106.63, 104.71, 104.16, 81.37, 74.24, 65.42, 63.65, 36.21, 36.07, 35.91, 34.33, 20.78, 20.48 HRESIMS m/z :1031.4148 (Calcd for C49H55N14012 : 1031.4124) [Step 6]
Synthesis of 3' , 5' -di-O-acetyl-NZ-{3- [ (4-{ [4-{4- [ (1-methylpyrrol-2-yl)carbonyl]amino-l-methylpyrrol-2-yl}carbonyl)amino-l-methylpyrrol-2-yl]carbonyl}amino-l-methylpyrrol-2-yl)carbonyl]aminopropyl}-21-deoxyguanosine (37) ;
O
N- H N
iN NO z -~ HN 0 I DBU HN O
0 N\ pyridine, rt ~
N ~ N
~~ ~ N HN O N~NH H
N N NH H ~ N N N NH N I N O
Ac0 O ~ N N 0 AcO N
Ac0 0 36 AcO 37 Compound 36 (22.8 mg, 22 mol) was dissolved in 0.5 M
DBU/pyridine solution (1.0 mL) under argon atmosphere.
After stirring at room temperature for 12 hours, ammonium chloride (50.0 mg, 0.94 mmol) was added thereto to terminate the reaction. The reaction solution was concentrated under reduced pressure and the residue was purified by silica gel column chromatography (chloroform:methanol =4:1) to obtain 15.6 mg of compound 37 (80%).
1 H-NMR (DMSO-d6) :610. 67 (br, 1H,H-1), 9.93 (s, 1H, Py-NH) 9.88 (s, 1H, Py-NH),9.82 (s, 1H, Py-NH), 8.05 (t, 1H, J
5.61 Hz, Py-CONH), 7.87 (s, 1H, H-8), 7.24 (d, 1H2H, J =
1.52 Hz, Py-H), 7.18 (d, 1H, J = 1.62 Hz, Py-H), 7.05 (dm, 1H2H, J = 1.58, Py-H), 6.95-6.90 (m, 3H, Py-H), 6.53 (brs, 1H,N2-H),6.17 (d, 1H, J = 7.00 Hz, H-11), 6.06 (dd, 1H, J
= 2.61 Hz, J = 3.88 Hz, H-1'), 5.41 (m, 1H, H-3'), 4.30 (m, 1H, H-4'), 4,18 (m, 2H, H-5', H-5"), 3.89 (s, 3H, Py-CH3), 3.86 (s, 3H, Py-CH3), 3.85 (s, 3H, Py-CH3), 3.81 (s, 3H, Py-CH3) 3.36 (m, 2H, -CH2NH-) , 3.25 (m, 4H, -COCH2, ), 3.06 (m, 1H, H-2'), 2.47 (m, 1H, H-2"), 2.08 (s, 3H, OAc)11.99(s, 3H, OAc), 1.75 (s, 3H,OAc).
13C-NMR (DMSO-d6):8170.12, 170.01, 161.43, 158.61, 158.50, 158.46, 156.73, 152.58, 150.45, 136.25, 128.10, 125.46, 122.89, 122.78, 122.18,146.72, 146.24, 130.22, 128.10, 125.47, 123.39, 122.96, 122.79, 122.20, 122.09, 118.42, 117.28, 112.63, 106.64, 104.72, 104.22, 83.28, 81.33, 79.16, 74.12, 63.67, 38.18, 36.21, 36.07, 35.90, 35.05, 29.18, 20.76, 20.47 HRESIMS m/z :882 .3661 (Calcd for C41H48N13010 :881.89) [Step 7]
Synthesis of N2-{3- [ (4-{ [4-{4- [ (1-methylpyrrol-2-yl)carbonyl]amino-l-methylpyrrol-2-yl}carbonyl)amino-l-methylpyrrol-2-yl]carbonyl}amino-l-methylpyrrol-2-yl)carbonyl]aminopropyl}-2'-deoxyguanosine (38);
HN HN
NaOMe MeOH, pyridine, rt HN O
O ~ 0 \ N ~ N
~
I NH HN N I NH H
N N~NH H O N N~NH H I\ O
AcO ~-O N ~N HO O N N
AcO 37 HO 38 Compound 37 (60.0 mg, 76 mol) was dissolved in pyridine (0.2 mL) under argon atmosphere. Subsequently, the mixture was added with 0.0 M sodium methoxide/methanol solution (0.8 mL) and stirred at room temperature for two hours. The reaction solution was adjusted to pH 6 with Dowex-50(H+ form) and the resin was removed by filtration. After the filtrate was concentrated under reduced pressure, the residue was added with methanol to perform recrystallization. The resulted crystals were collected by suction filtration to obtain 51.6 mg of compound 38 (97%).
1H-NMR (DMSO-d6) :510.56 (brs, 1H, H-1) , 9.93 (s, 1H, Py-NH), 9.89 (s, 1H, Py-NH), 9.84 (s, 1H, Py-NH), 8. 08 (s, 1H, H-8), 7.90 (t, 1H, J = 5.49 Hz, -CH2NH-) , 7.24(d, 1H, J
1.66 Hz, Py-H), 7.20 (m, 2H, Py-H), 7.05(d, 1H, J = 1.65 Hz, Py-H), 6.95-6.89 (m, 3H, Py-H), 6.52 (brs, 1H, N2-H), 6.15 (t, 1H, J 6.67 Hz, H-i'), 6.06(d, 1H, J = 1.65 Hz, Py-H) 5.27 (br, 1H, 5'-OH), 4.86(t, 1H, J 5.4 Hz, H-3'), 4.36 (br, iH, 3'-OH), 3.88 (s, 3H, Py-CH3), 3.86 (s, 3H, Py-CH3') , 3 .85 (s, 3H, Py-CH3' ) , 3. 82 (s, 3H, Py-CH3' ) , 3.81 (m, 1H, H-4'), 3.55 (m, 1H, H-5'), 3.51 (m, H1, H-5"), 3.32 (dt, 2H, J = 5.47 Hz, J = 6.49 Hz, -CH2NHCO-), 3.25 (m, 2H, -CH2N2H), 2.60 (m, 1H, H-2'), 2.21 (m, iH, H-2 " ) , 1.75 (m, 2H, -CH2CH2CH2-) 13C-NMR (DMSO-d6) :8161.44, 158.60, 158.46, 156.75, 152.54, 150.50, 135.75, 128.11, 125.45, 122.75, 122.08, 118.42, 117.84, 116.87, 112.63, 106.64, 104.71, 104.24, 87.57, 82.76, 70.86, 61.83, 38.18, 36.21, 36.07, 35.92, 29.18 HRESIMS m/z :798.3445 (Calcd for C37H44N1308 : 798.3436) Anal. Calcd for C37H43N13O8 + 3H20 + MeOH : C, 51 . 64 ; H, 6.04; N, 20.60 Found. C, 51.85; H,5.88;N,20.48 As above, the present inventors have synthesized MGB
polyamide-nucleoside hybrid compounds for which utility as novel genetic information control molecules were expected.
Industrial Applicability Pyrrole amide-2'-deoxyguanosine hybrid compound 38 of the present invention forms a stable double bond to ssDNA having a site which a pyrrole polyamide specifically recognizes when it is incorporated in ssDNA
by biosynthesis. In addition, the compound has an extremely high base sequence selection ability. Thus, the MGB polyamide-nucleoside hybrid compound of the present invention is a novel genetic information control molecule having high base sequence selection ability and effective applications to antisense drugs can be expected.
13C-NMR (DMSO-d6):8170.12, 170.01, 161.43, 158.61, 158.50, 158.46, 156.73, 152.58, 150.45, 136.25, 128.10, 125.46, 122.89, 122.78, 122.18,146.72, 146.24, 130.22, 128.10, 125.47, 123.39, 122.96, 122.79, 122.20, 122.09, 118.42, 117.28, 112.63, 106.64, 104.72, 104.22, 83.28, 81.33, 79.16, 74.12, 63.67, 38.18, 36.21, 36.07, 35.90, 35.05, 29.18, 20.76, 20.47 HRESIMS m/z :882 .3661 (Calcd for C41H48N13010 :881.89) [Step 7]
Synthesis of N2-{3- [ (4-{ [4-{4- [ (1-methylpyrrol-2-yl)carbonyl]amino-l-methylpyrrol-2-yl}carbonyl)amino-l-methylpyrrol-2-yl]carbonyl}amino-l-methylpyrrol-2-yl)carbonyl]aminopropyl}-2'-deoxyguanosine (38);
HN HN
NaOMe MeOH, pyridine, rt HN O
O ~ 0 \ N ~ N
~
I NH HN N I NH H
N N~NH H O N N~NH H I\ O
AcO ~-O N ~N HO O N N
AcO 37 HO 38 Compound 37 (60.0 mg, 76 mol) was dissolved in pyridine (0.2 mL) under argon atmosphere. Subsequently, the mixture was added with 0.0 M sodium methoxide/methanol solution (0.8 mL) and stirred at room temperature for two hours. The reaction solution was adjusted to pH 6 with Dowex-50(H+ form) and the resin was removed by filtration. After the filtrate was concentrated under reduced pressure, the residue was added with methanol to perform recrystallization. The resulted crystals were collected by suction filtration to obtain 51.6 mg of compound 38 (97%).
1H-NMR (DMSO-d6) :510.56 (brs, 1H, H-1) , 9.93 (s, 1H, Py-NH), 9.89 (s, 1H, Py-NH), 9.84 (s, 1H, Py-NH), 8. 08 (s, 1H, H-8), 7.90 (t, 1H, J = 5.49 Hz, -CH2NH-) , 7.24(d, 1H, J
1.66 Hz, Py-H), 7.20 (m, 2H, Py-H), 7.05(d, 1H, J = 1.65 Hz, Py-H), 6.95-6.89 (m, 3H, Py-H), 6.52 (brs, 1H, N2-H), 6.15 (t, 1H, J 6.67 Hz, H-i'), 6.06(d, 1H, J = 1.65 Hz, Py-H) 5.27 (br, 1H, 5'-OH), 4.86(t, 1H, J 5.4 Hz, H-3'), 4.36 (br, iH, 3'-OH), 3.88 (s, 3H, Py-CH3), 3.86 (s, 3H, Py-CH3') , 3 .85 (s, 3H, Py-CH3' ) , 3. 82 (s, 3H, Py-CH3' ) , 3.81 (m, 1H, H-4'), 3.55 (m, 1H, H-5'), 3.51 (m, H1, H-5"), 3.32 (dt, 2H, J = 5.47 Hz, J = 6.49 Hz, -CH2NHCO-), 3.25 (m, 2H, -CH2N2H), 2.60 (m, 1H, H-2'), 2.21 (m, iH, H-2 " ) , 1.75 (m, 2H, -CH2CH2CH2-) 13C-NMR (DMSO-d6) :8161.44, 158.60, 158.46, 156.75, 152.54, 150.50, 135.75, 128.11, 125.45, 122.75, 122.08, 118.42, 117.84, 116.87, 112.63, 106.64, 104.71, 104.24, 87.57, 82.76, 70.86, 61.83, 38.18, 36.21, 36.07, 35.92, 29.18 HRESIMS m/z :798.3445 (Calcd for C37H44N1308 : 798.3436) Anal. Calcd for C37H43N13O8 + 3H20 + MeOH : C, 51 . 64 ; H, 6.04; N, 20.60 Found. C, 51.85; H,5.88;N,20.48 As above, the present inventors have synthesized MGB
polyamide-nucleoside hybrid compounds for which utility as novel genetic information control molecules were expected.
Industrial Applicability Pyrrole amide-2'-deoxyguanosine hybrid compound 38 of the present invention forms a stable double bond to ssDNA having a site which a pyrrole polyamide specifically recognizes when it is incorporated in ssDNA
by biosynthesis. In addition, the compound has an extremely high base sequence selection ability. Thus, the MGB polyamide-nucleoside hybrid compound of the present invention is a novel genetic information control molecule having high base sequence selection ability and effective applications to antisense drugs can be expected.
Claims (7)
1. A 2'-deoxyguanosine hybrid compound represented by following general formula (38) characterized in that the compound contains a pyrrole amide tetramer.
2. A 2'-deoxyguanosine hybrid compound having protected hydroxyl groups and represented by following general formula (37) characterized in that the compound contains a pyrrole amide tetramer, wherein R1 and R2 each mean the same or different hydroxyl group protecting groups.
3. A protected 2'-deoxyguanosine hybrid compound represented by following general formula (36) characterized in that the compound contains a pyrrole amide tetramer, wherein R1, R2 and R3 each mean the same or different hydroxyl group protecting groups.
4. A production method of a protected 2'-deoxyguanosine hybrid compound represented by above general formula (36) characterized by reacting a diamine compound represented by following general formula (35) with a compound represented by following general formula (23), wherein Fmoc means a 9-fluorenylmethoxycarbonyl group; R1, R2 and R3 are the same as above; and Hal means a halogen atom.
5. A diamine compound represented by following general formula (35) characterized in that the compound contains a pyrrole amide tetramer, wherein Fmoc is the same as above.
6. A production method of a diamine compound represented by above general formula (35) characterized by reacting a carbamate compound represented by following formula (26) with a carboxylic acid compound containing a pyrrole amide tetramer represented by following formula (34).
7. A carbamate compound represented by following formula (26) for the production of diamine compound (35) represented by above general formula (35).
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2005257045 | 2005-09-05 | ||
| JP2005-257045 | 2005-09-05 | ||
| PCT/JP2006/304666 WO2007029364A1 (en) | 2005-09-05 | 2006-03-03 | 2’-deoxyguanosine hybrid compound containing pyrrole amide tetramer, method of producing the same and production intermediate therefor |
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|---|---|
| CA2621104A1 true CA2621104A1 (en) | 2007-03-15 |
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ID=37835499
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| CA002621104A Abandoned CA2621104A1 (en) | 2005-09-05 | 2006-03-03 | 2'-deoxyguanosine hybrid compound containing pyrrole amide tetramer, method of producing the same and production intermediate therefor |
Country Status (3)
| Country | Link |
|---|---|
| JP (1) | JPWO2007029364A1 (en) |
| CA (1) | CA2621104A1 (en) |
| WO (1) | WO2007029364A1 (en) |
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| US9504680B2 (en) | 2013-06-17 | 2016-11-29 | Lupin Limited | Pyrrole derivatives as alpha 7 nAChR modulators |
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2006
- 2006-03-03 CA CA002621104A patent/CA2621104A1/en not_active Abandoned
- 2006-03-03 JP JP2007534250A patent/JPWO2007029364A1/en active Pending
- 2006-03-03 WO PCT/JP2006/304666 patent/WO2007029364A1/en active Application Filing
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| WO2007029364A1 (en) | 2007-03-15 |
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