Methods of synthesis and use

Details Methods of synthesis and use Methods of synthesis and use

1995-03-09

2001-05-22

Fredman, Jeffrey (Department: 1655)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S025300, C435S006120

Reexamination Certificate

active

06235886

ABSTRACT:

FIELD OP THE INVENTION
This invention relates to novel amine-containing compounds useful for therapeutics and methods of making and using the same.
BACKGROUND OF THE INVENTION
It is well known that most of the bodily states in mammals including most disease states, are effected by proteins. Such proteins, either acting directly or through their enzymatic functions, contribute in major proportion to many diseases in animals and man. Classical therapeutics has generally focused upon interactions with such proteins in efforts to moderate their disease causing or disease potentiating functions. Recently, however, attempts have been made to moderate the actual production of such proteins by interactions with molecules that direct their synthesis, intracellular RNA. These interactions involved the binding of complementary “antisense” oligonucleotides or their analogs to the transcellular RNA in a sequence specific fashion such as by Watson-Crick base pairing interactions.
The pharmacological activity of antisense compounds, as well as other therapeutics, depends on a number of factors that influence the effective concentration of these agents at specific intracellular targets. One important factor is the ability of antisense compounds to traverse the plasma membrane of specific cells involved in the disease process.
Cellular membranes consist of lipid protein bilayers that are freely permeable to small, nonionic, lipophilic compounds and inherently impermeable to most natural metabolites and therapeutic agents. Wilson,
Ann. Rev. Biochem.
1978, 47, 933. The biological and antiviral effects of natural and modified oligonucleotides in cultured mammalian cells have been well documented, so it appears that these agents can penetrate membranes to reach their intracellular targets. Uptake of antisense compounds into a variety of mammalian cells, including HL-60, Syrian Hamster fibroblast, U937, L929, CV-1, and ATH8 cells has been studied using natural oligonucleotides and nuclease resistant analogs, such as alkyl triesters, Miller, et al.,
Biochemistry
1977, 16, 1988; methylphosphonates, Marcus-Sekura, et al.,
Nuc. Acids Res.
1987, 15, 5749 and Miller, et al.,
Biochemistry
1981, 20, 1874; and phosphorothioates, Ceruzzi, et al.,
Nucleosides
&
Nucleotides
1989, 8, 815; Miller, et al.,
Biochemistry
1987, 16, 1988; and Loke, et al.,
Curr. Top. Microbiol. Immunol.
1988, 141, 282.
Enhanced cellular uptake has previously been achieved by attachment of functional groups to the 3′ and 5′ end of oligonucleotides to enhance cellular uptake in specific cell types. Previous studies have shown that plasmid DNA complexed with an (asialo) glycoprotein-poly(L-lysine) conjugate, could be targeted to hepatocytes, which contain unique cell surface receptors for galactose-terminal (asialo) glycoproteins. Wu, et al.,
Biochemistry
1988, 27, 887. Other groups have synthesized oligodeoxyribonucleotides that have a 5′-attached alkylating agent and a 3′ attached cholesterol moiety and determined that these modified oligonucleotides were taken up into cells more efficiently than control compounds without the steroid moiety. Zon, G. in
Oligodeoxynucleotides: Antisense Inhibitors of Gene Expression
234-247, ed. J. S. Cohen (CRC Press, Boca Raton Fla., 1989). Letsinger, et al.,
Proc. Natl. Acad. Sci. U.S.A.
1989, 86, 653, have also synthesized cholesteryl-conjugated phosphorothioates whose anti-HIV activity is significantly greater than natural oligonucleotides with the same sequence. Additional modifications include conjugation of oligonucleotides to poly(L-lysine) alone. Stevenson, et al.,
J. Gen. Virol
1989, 70, 2673 and Lemaitre, et al.,
Proc. Natl. Acad. Sci. U.S.A.
1987, 84, 648. This modification enhanced the antiviral activity of the compound studied presumably due to increased cellular uptake imparted by the polycationic poly(L-lysine).
The conjugation of polyamines to oligonucleotides have been found to enhance cellular uptake of oligonucleotides, increased lipophilicity, cause greater cellular retention and increased distribution of the compound. Vasseur,
Nucleosides and Nucleotides
1991, 10, 107 prepared abasic sites at different sites of oligothymidylates by acid hydrolysis. Thereafter the abasic sites were functionalized with functionalities such as 3-amino carbazole, 9-amino elipticine and psoralen. Vasseur, et al., also refer to unpublished results in which the functionalities spermidine and proflavin were employed. The abasic site was generated by one of the following three methods: (i) selective depurination by acid treatment in a pyrimidine-rich oligonucleotide having one purine in a chosen site, (ii) incorporating 2′,3′-dideoxynebularine at the 5′-end with the nebularine phosphoramidite at the last step of the oligonucleotide synthesis, and subsequent acid treatment (30 mM HCl at 37° C.) to create an abasic site at 5′ end (in this case the open-chain structure is CHO—(CH
2
)
2
—CHOH—CH
2
O— at the 5′ end and the conjugate from the amine RNH
2
is RNH—(CH
2
)
3
—CHOH—CH
2
—O-Oligo), and (iii) incorporating a protected abasic 2′-deoxy-D-ribofuranose nucleotide synthon that has a photo-labile O-nitrobenzyl group as the anomeric hydroxyl-protecting group in oligonucleotide synthesis and removing it prior to conjugation.
Groebke and Leumann used a silyl-protecting group at the anomeric center to generate the abasic site. 2′-Deoxy-5-O-dimethoxytrityl-D-ribofluranose was silylated at the 1-O-position using TBDMSCl and the silyl group was removed later by hydrolysis at pH 2.0 to yield the abasic site. Unfortunately, fluoride-ion-mediated deprotection of the silyl group caused a &bgr;-elimination and DNA degradation.
McLaughlin's group has utilized 1-(&bgr;—D-2-deoxyribosyl)-2-pyrimidone-based phosphoramidite to generate abasic sites at pH3.0. The N-glycosyl cleavage occured, however, slower in oligonucleotides than in parent nucleosides; nearly 60 hours of acid treatment was necessary to generate 90% abasic site formation. However, conjugation chemistry via enzymatically generated abasic sites are unknown in the literature.
Le Doan, et al.,
Nucleic Acids Research
1987, 15, 8643 teaches oligothymidylates covalently linked to porphyrins at their 3′ end via one of the linkers —O—CH
2
—CO—NH—(CH
2
)
2
—NH or PO
4
—(CH
2
)
6
—NH—. Le Doan, et al., also used the linker PO
4
—(CH
2
)
6
—NH— to link porphyrins to the 5′ end of oligothymidylates. Another group, Summerton, et al., U.S. Pat. No. 5,034,506 issued Jul. 23, 1991 teaches morpholino subunits, linked together by uncharged, achiral linkages such as amides. As described in PCT/US91/04086 filed Jun. 10, 1991, polyamines have also been linked at the 5′ end of an oligonucleotide at the 5′ site of the sugar moiety of the terminal nucleoside and at the 2-position carbon of the heterocyclic base of 2′-deoxyadenosine, 2′-deoxyguanosines and other purines and purine analogs by known procedures as described in PCT/US/91/00243 filed Jan. 11, 1991.
Novel amines and methods of preparing the same are greatly needed in order to enhance cellular uptake of oligonucleotides, increase lipophilicity, cause greater cellular retention and increase distribution of the compound within the cell. The present invention fulfills this need.
OBJECTS OF THE INVENTION
It is one object of the present invention to provide novel amine-containing compounds useful in therapeutics.
It is a further object of the present invention to provide methods of producing said novel compounds.
It is another object of the present invention to provide methods of modulating the production of a protein by an organism.
It is still a further object of the present invention to provide methods of treating a mammal suffering from a disease characterized by the undesired production of a protein.
It is yet a further object of the present invention to provide methods of diagnosing the presence of an RNA in a biological sample.
These and other objects will become apparent from the foll

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