JP2008519053A - Synthesis of polyamine conjugates of small interfering RNA (siRNA) and conjugates formed thereby - Google Patents

Abstract

  Conjugates comprising polyamines covalently linked to ribonucleic acid (RNA), preferably short interfering RNA, are described.

Description

Introduction The present invention relates generally to the production of conjugates containing ribonucleic acid (RNA), and in particular short interfering RNA (siRNA), and conjugates as therapeutic agents ( Fill gaps when using conjugates. Short interfering RNAs (siRNAs) have been referred to in the popular scientific literature as “one of the most popular in target validation over the last five years”. This enthusiasm is due to the fact that siRNA has been shown to be effective in specifically suppressing the expression of targeted genes.

  Post-transcriptional silence specific to this sequence of gene expression through the action of siRNA is a naturally occurring process first discovered in fungi and plants and can occur in bacterial and animal cells. It was shown later. Short interfering RNAs are fragments of short (approximately 21-25 nucleotides) RNA obtained either enzymatically or by chemical synthesis. Short interfering RNAs function by including specific degradation in the sequence of targeted messenger RNA (mRNA). The potential of siRNA as a potent antiviral and anticancer drug is widely accepted in the scientific community. However, before this potential becomes a reality in the form of pharmaceutical compositions, the fundamental problem in delivering siRNA to their intended target must be solved.

  The root of the delivery problem is that siRNA is a polyanion. Thus, penetration without siRNA assistance across the lipid bilayer is negligible. Traditionally, siRNAs are delivered to cells using cationic liposomes or polyplexes with polyethyleneimines. Although the use of liposomes to deliver siRNA has shown some success, a major disadvantage of liposome delivery vehicles is that many cell types cannot be transfected using liposomes. That's it. Also, several cell types cannot be transfected with liposomes, with the efficiency of producing significant biological effects. Furthermore, experiments using liposome delivery vehicles require several different procedures for cells. In short, the process is cumbersome.

  Similarly, polyethyleneimines are difficult to pump into cells because they are high molecular weight polymers. Thus, in vivo delivery of polyethylenimine-siRNA polyplexes is plagued by all obstacles inherent in systemic delivery of high molecular weight cationic complexes: the complexes were intended You have to make those paths to the site, move out of the tube into the targeted tissue, and so on. Thus, methods for transferring siRNA molecules from the extracellular environment into the cytoplasm of a target cell are in vitro (eg, in cultured and trunk cells) and in vivo (eg, as a drug). This will be a significant breakthrough in the therapeutic use of siRNA to silence genes (with systemic delivery of siRNA).

SUMMARY OF THE INVENTION Thus, the present invention is a method for creating an effective cell delivery system for siRNA that mimics a naturally occurring process, and the resulting siRNA delivery system. A number of antibiotics and inhibitors of low molecular weight enzymes have been conjugated to amines. The amine site is often critical to increasing biological activity. In many of these compounds, the amine moiety provides the structural element required to transport the conjugate into the cell. In particular, the antimicrobial activity of many antibiotics is due to (partly) amines or polyamines conjugated to glycoside, aromatic or polyketide sites. Cationic polyamine residues function to facilitate transport of antibiotics into the cell. A suitable example is streptomycin (see FIG. 4), which is an aminoglycoside, where the transporting residue is an aminoguanidino group. Similarly, in the antibiotic bleomycin-phleomycin group (see FIG. 2), the modified spermine and superimidine groups are transporting residues. Antibiotics and anti-tumor agents conjugated to sparimin (such as spagarin, laterosporamine, edein, glisperin A, B, and C, and glycocinnamoyl spermidines) In (see FIG. 3), the conjugated spermidine site is a transporter of biologically active agents into the cell. References to aminoglycoside antibiotics called kanamycins should also be made. In kanamycins, the amino moiety transports glycosides into the cell. This is also true for steroidal antibiotics isolated from squalamine (see FIG. 7), broad spectrum, shark shark, shark shark or shark shark tissue. In these compounds, the sulfated bile acid is fused to the aminopropyl primary amine of spermidine. The spermidine site of the molecule acts to carry the rest of the molecule across the cell membrane.

  In the present invention, the natural and / or synthetic polyamine groups are: 1) disrupted in the cytoplasm and set the siRNA site free; or 2) the siRNA portion of the conjugate Intact while still allowing to bind to an RNA-inducing silencing complex (RISC) molecule and thereby cleaving a specific mRNA targeted by siRNA Leave as it is: covalently attached to the molecule of the siRNA via any linkage (see Dykxhorn et al., Nature Reviews, RNAi Collection, p. 7, December 2003).

  Suitable polyamines for use in the present invention are preferably spermidine and its derivatives, spermine and its derivatives, and hirudonine and its derivatives. See FIGS. 1 and 6. Most cells take up polyamines by an energy-dependent mechanism mediated by a carrier. Many cells (eg, human fibroblasts, murine leukemia cells, rat Morris lung cancer cells, etc.) appear to have a single transporter for all polyamines. Thus, as a general suggestion, the specificity of the transporter is not very strict. For example, it is known from previous work that derivatives of polyamines substituted with alkyl substituents are also efficiently transported into cells by the same transporter system that triggers natural polyamines. That is.

  Polyamines are also ideal carriers for siRNA because of the high affinity of polyamines for ribonucleic acid. Polyamines, in particular spermine, are strongly bound to ribosomes and are a part of transfer RNA (t-RNA). Strongly basic polyamines bind to t-RNA not only by electrostatic charges but also by hydrogen bonding (Frydman et al., Proc. Natl. Acad. Sci (USA) (1992) 89: 9186; Fernandez et al., (1994) Cell. Mol. Biol. 40:93). Transfer RNA is roughly twice the size of siRNA ribonucleotides, but the strands of ribonucleotides have a similar structure. Without being bound by any particular biological mechanism, in a t-RNA complex, spermine or spermidine binds to a ribonucleotide loop; in siRNA, polyamines are siRNA-derived RNAs. It will be possible to bind to and stabilize the annealed duplex strand of the siRNA until it binds to the silenced complex.

DETAILED DESCRIPTION OF THE INVENTION A first embodiment of the invention is directed to a conjugate comprising a polyamine covalently linked to ribonucleic acid (RNA). It is preferred that the RNA conjugate is an oligo RNA, and most preferably the RNA is a short interfering RNA (siRNA).

The polyamine portion of the conjugate can be any polyamine without limitation. Suitable polyamines are those selected from the group consisting of putrescine, spermine, spermidine, hirudonine, and derivatives thereof. Explicitly included within these derivatives are the conformationally restricted polyamine compounds disclosed in US Pat. No. 6,392,098 and US Pat. No. 6,794,545. is there. More specifically, for conformationally restricted polyamines, the polyamine portion of the conjugate is preferably of formula I:
E-NH-D-NH-B-A-B-NH-D-NH-E (I)
Selected from the compounds of
Wherein A is selected from the group consisting of C ₂ to C ₆ alkenes, and C ₃ to C ₆ cycloalkyls, cycloalkenyls, and cycloaryls;
B is independently selected from the group consisting of a single bond and C ₁ to C ₆ alkyl and alkenyl;
D is independently selected from the group consisting of C ₁ to C ₆ alkyl and alkenyl, and C ₃ to C ₆ cycloalkyl, cycloalkenyl, and cycloaryl; and
E is methyl; and the polyamine portion of the conjugate is a pharmaceutically suitable salt thereof.

As detailed in more detail below, these conformationally constrained polyamines can be made according to the general reaction scheme that follows:
Formula II:
HO-BABA-OH (II)
Is a compound of formula III:
PROT-OBABABOPROT (III)
To give a compound of the following reaction with a protective reagent, preferably mesitylenesulfonyl chloride:
There, PROT is a protecting group.

And the compound of formula III is of formula V:
E-N (PROT) -D-N (PROT) -BAB-N (PROT) -D-N (PROT) -E (V)
To give compounds of formula IV:
E-N (PROT) -D-NH- (PROT) (IV)
It is made to react with the compound.

  In both formula III and formula IV intermediates, it is highly preferred that the protecting group, PROT, be a mesitylenesulfonyl moiety.

  The compound of formula V is then deprotected to yield the compound of formula I (E-NH-D-NH-BABA-NH-D-NH-E).

  The second embodiment of the present invention is directed to a method for activating RNA in living cells. The method includes conjugating RNA to a polyamine to produce a conjugate; and contacting the conjugate to a living cell. It is preferred that the RNA is conjugated to a polyamine selected from the group consisting of putrescine, spermine, spermidine, hirudonin, and derivatives thereof. It is also preferred that the siRNA be conjugated to a polyamine.

  The third embodiment of the present invention is directed to a composition of material for initiating RNA into living cells, the composition being combined with a pharmaceutically suitable carrier in the form of ribonucleic acid. Polyamines covalently bound to (RNA).

  In the present invention, polyamine transporters are linked to siRNAs by foldable bonds that release ribonucleotides as they enter the cell. Making discontinuous conjugates of polyamines with siRNA, which has no systemic toxicity when administered in vivo, studies the function of genes by modulating processes that occur naturally in mammalian cells And ultimately leads to new gene-specific therapeutics to treat the disease. Thus, the conjugates disclosed herein are highly useful for delivering siRNA from outside the cell and into the cytoplasm and / or nucleus.

1. Polyamine transport of natural products into cells:
Not only the ability to cultivate such tissues, but also the recognition of the ubiquitous and indispensable nature of polyamines in animal tissues has resulted in numerous studies of polyamine transport in mammalian cells. Inhibition of tumor cell growth by specific inhibitors of polyamine synthesis and recovery of growth by exogenous polyamines has been a major stimulus for studies of polyamine uptake. For example, several modes of uptake of [ ¹⁴ C] -benzylamine: adsorption to the cell surface (adsorbed molecules are largely removable by washing); specific processes Saturated intracellular transports shown; and slower non-saturating uptake reminiscent of a mix of diffusion and internal binding is obvious (Cohen, A Guide to the Polyamines, Oxford, 1998, pp. .467).

  There are three natural polyamines present in mammalian cells; putrescine, spermidine, and spermine (see FIG. 1). Seiler and Dezeure (1990) Int. J. et al. As briefly described by Biochem, 22: 211, polyamine uptake in mammalian cells is generally specific, saturating, demanding energy, and mediated by a carrier. . Polyamine transport can also be regulated in RNA-requiring processes and protein synthesis. A review of the data for the three membrane receptors of mammals reveals a large number of transport proteins for amino acids and significant similarity or homology of these proteins to polyamines isolated from eubacteria and fungi Sex has been described (Reizer et al., Protein Science (1993) 2:20). These data suggest that receptor proteins from mammalian cells may be described as denatured transport proteins that share a common origin with these microbial transport proteins.

Many cells have a single transporter for putrescine, spermidine, and spermine, with K _m values in the micromolar range. Both sodium-dependent and sodium-independent systems have been detected. Emphasizing the therapeutic importance of polyamine transporters in cancer, reviewers have pointed to reduced toxicity of leukemic cell lines that lack transporter systems. Several new inhibitors of putrescine uptake inhibit the uptake of this diamine more actively than that of spermidine, Minchin et al. (1991), Eur. J. et al. See Biochem, 200: 457. This also indicates that the transport of these different polyamines in melanoma cells is different.

The uptake of putrescine in human platelets is saturating and energy dependent, but appears complex. Studies within human erythrocytes have shown that they are unable to synthesize polyamines, but into polyamine receptor sites on the surface of cells as well as primarily (> 95%) internal soluble compartments. Ingestion of putrescine, spermidine, and spermine was demonstrated. Moulinaux et al. (1984), Biochimie, 66: 385. Its uptake was minimal at 4 ° C. and this was mainly related to cell stromal attachment. At 37 ° C., the intake of putrescine and spermidine from serum was relatively rapid compared to the slow entry of spermine. The K _m values for saturable putrescine and spermidine intake from plasma were 125 and 3.6 μM, respectively.

In one thread of mouse cells, both Na ⁺ and spermidine were shown to enter the cells in a 1: 1 relationship. Khan et al. (1990) Cell. Mol. Biol. 36: 345. The transport appeared to be ATP-independent because it was not affected by 2-deoxyglucose, which depletes ATP. Examination of many more threads of mammalian cells showed that spermidine intake was not affected by the concentration of Na ⁺ , albeit slightly different in each case. Khan et al. (1990), Pathology, 58: 172. Its uptake was generally inhibited by ion-permeable pores and some polyamines themselves. Preloading the cells with asparagine accelerated the uptake of putrescine in the two threads as well as that of uptake in the cells of neuroblastoma. Rinehart and Chen (1984) J. MoI. Biol. Chem. 259: 4570. The uptake of putrescine by human colon cancer cells is stimulated> 300 times by asparagine. McCorack and Johnson (1991) Am. J. et al. Physiol. 256: G868. The nature of asparagine's effect on putrescine intake appeared to be greatly exaggerated in some human cancer cells. It is known that asparagine activates membrane Na ⁺ / H ⁺ counter transport, induces H ⁺ extrusion, and causes Na ⁺ -dependent intracellular alkalinization. Fong and Law (1988) Biochem. Biophys. Res. Commun. 155: 937.

  Studies with a wide variety of animal cells have shown a common response to the mechanism of polyamine transport. Nevertheless, cells of several tissues and cancer cells appeared to possess a single common mechanism. In contrast, others, similar to the porcine kidney LLC-PK cell line, revealed several more discriminating systems. The latter cells contained both sodium-dependent and sodium-independent transporters localized in different cell areas. Van der Bosch et al. (1990), Biochem. J. et al. 265: 609.

  By taking advantage of the aforementioned mechanism of transport of polyamines into animal cells, different drugs and chemical agents have been covalently attached to natural polyamines and thus into the cell. Was transported. A relatively non-toxic compound, the nitroimidazole-spermidine conjugate, entered the cell and inhibited free spermidine uptake, Holley et al. (1992), Biochem. Pharmacol. 43: 763. It can also be stated that the iron chelator spermine conjugate is efficiently transported into the tumor cells, even if the terminal substituents are rather large. Bergeron et al. (2003), J. et al. Med. Chem. 46: 5478, 2003. Also, polyamine conjugates with naphthalimides are efficiently delivered into cells. Lin et al. (2003), Biochem. Soc. Trans. 31 (2): 407; Indenoisoquinolines conjugated to polyamines (topoisomerase I inhibitors) are pumped into cancer cells, Nagarayan et al. (2003), J. et al. Med. Chem. 46: 5712; a spermine conjugate of a protease inhibitor known as Bowman-Birk inhibitor ("BBI", 8000 Da polypeptide) localizes BBI in the lung and liver Kennedy et al., Which is very effective in the treatment and does not have any of the unacceptable toxicity of conjugates of polylysine. (2003), Pharm. Research, 20 (12): 1908.

  Natural products are good samplers of polyamines as vectors for intracellular delivery. Not only the spermidine conjugates spagaline and edeins (see FIG. 3), but also the mention of bleomycin (for various chemical structures, see FIG. 2), along with their broad sequence of polyamine vectors, and Reference should be made to streptomycin, a diguanidino derivative (see FIG. 4). Aminoguanidines found in plants, such as coumaroyl agmatine, and hordatines (see FIG. 5) are residues of strongly basic guanidines for transport, as well as hirudonin ( hirudonine) (see FIG. 6) illustrates the importance of residues, important bacterial and plant polyamines. Squalamine (see FIG. 7) is a spermidine-steroid, where the spermidine site is a vector for delivery into the cell. All of these natural products contain polyamine sites.

  The inventor has found that the specificity of the mechanism of polyamine transport is surprisingly permissive. After probing 24 polyamine-like compounds, including the spermine and homospermine classes themselves, pentamines, and different oligoamines, they are efficiently incorporated into human cells. It was found to be transported. Thus, the subject conjugates are expected to be efficiently transported into mammalian cells, including human cells.

2. Synthesis of small interfering RNA (siRNA):
Small interfering RNA (siRNA) is a double-stranded fragment of about 21-23 ribonucleotides. The siRNA molecule is a mediator of mRNA degradation, and the chemically synthesized duplex in the fragment pattern described above is capable of guiding mRNA cleavage. Have been shown. Elbashir et al. (2001), Genes and Development, 15: 188. A currently accepted sequence of events in siRNA-mediated cleavage of mRNA is given schematically in FIG. As shown in FIG. 8, siRNAs are paired (red, shown from 5 ′ to 3 ′ at the top of FIG. 8) with a 3 ′ overhang, usually (TT) or (UU). See the reference “a” in FIG. 8, which includes the sense strand and the antisense strand (shown 3 ′ to 5 ′ in the upper part of FIG. 8). The siRNA pathway is the process by which long double-stranded (ds) RNA is driven into siRNA in an ATP-dependent reaction by an RNase III enzyme with the insignificant name “Dicer”. Start when cleaved. These siRNAs are then incorporated into RNA-inducing silencing complexes (RISC). Once ligated, the siRNA single-stranded antisense strand guides the RISC complex to a messenger RNA (mRNA, which is single-stranded) and Target the complementary sequence of the mRNA. This results in the cleavage of the targeted mRNA nucleotide strand breaks (as indicated by the symbol “b” in FIG. 8). However, there is also data that suggests targeting both sense as well as antisense strands in chemically synthesized siRNA duplexes. This process is schematically illustrated in FIG. As shown in FIG. 9, the transfected siRNA is incorporated into the RISC and either the sense or antisense strand (they are not represented in FIG. 9) It can be useful for recognizing complementary sequences in rendered mRNAs. Duxbury et al. (2004), J.A. Surgical Res. 117: 339.

  The synthesis, purification, and annealing of siRNAs through chemical processes are becoming increasingly commonplace. For example, Micura (2002), Angew. Chem. Int. Ed. , 41 (13): 2265 and Hobartner et al. (2003), Monsatsheft fur Chemie, 134: 851,2003. Chemically synthesized RNA oligonucleotides are a key component of siRNA technology. Nucleoside coupling is achieved by conventional and well-known phosphoramidite chemistry as illustrated in FIG. Since the process is well known to those skilled in the art, it will not be described in great detail. See the references cited in the following paragraphs for the full processing. There is also a thriving commercial market for conventional RNA synthesis. For example, siRNA of any designated sequence can be obtained from a number of other companies, for example, SynGen, Inc. (San Carlos, California), Midland Certified Reagent Company Inc. (Midland, Texas), and Dharmacon, Inc. (Boulder, Colorado). Additionally, a number of university-based laboratories also sell custom RNA synthesis services to the public (among many others, eg, the University of Wisconsin Biotechnology Center [Madison, Wisconsin ] And Kansas State University [Manhattan, Kansas]).

  Improvements in the structure of suitable protecting groups have taken routine RNA construction to the level of product quality and accessible oligonucleotide length, as is the case for DNA synthesis. The desire for a robust RNA construction strategy has resulted in the creation of new and upgraded protecting groups. These procedures were introduced in 1998 and covered by various patents (see, eg, Pitsch et al., US Pat. No. 5,986,084), together with the scientific literature (Pitsch). et al., (2001) Helv. Chim Acta, 84: 3773). Most common procedures (used in the synthesis of commercial automated DNA after some technical regulation) are the “TOM” protecting group (2′-O-triisopropylsilyloxymethyl). (It is a ribonucleoside protected with a TOM protecting group at the 2′-O position, see FIG. 10A). This protecting group is superior to the classical 2'-O-tert-butyldimethylsilyl (TBDMS) group used in the synthesis of DNA.

  Construction of protected nucleotides such as those shown in FIG. 10A begins with N-acetylation at the exonucleo amino group of the ribonucleoside nucleobase and 5 ′ to give 5′-O-DMT. Followed by tritylation in oxygen. This is followed by "TOMylation" in 2 'oxygen to give a derivative of 2'-O-TOM. Finally, the “phosphitylation” step at 3 ′ oxygen using 2-cyanoethyldiisopropylphosphoramido chloridite yields the 3′-O-CEPA derivative shown in FIG. 10A. . The incorporation of phosphoramidites into oligoribonucleotides is well documented in detail. Micura et al. (2001), Nucleic Acid Res. 29: 3997; Hobartner et al. (2002), Angew. Chem. Int. Ed. 41; 605; Micura et al. (2000), Angew. Chem. Int. Ed. 39: 922; Micura et al. (2001), Nucleosides, Nucleotides, Nucleic Acids, 20: 1287; and Ebert et al. (2000), Helv. Chim. Acta, 83: 2238.

  Another building block for oligoribonucleotides is depicted in FIG. 10B. See also Scaringe (2000), Methods in Enzymology, 317: 3, first reported in 1998 in US Pat. No. 6,111,086 for this protected ribonucleoside. The rationale behind the protected nucleoside shown in FIG. 10B was a requirement for mildly acidic aqueous conditions for final deprotection at the 2'-O group of the synthetic ribonucleotide. This cannot be achieved if the protecting group at 5'-O, which is itself unstable to mildly acidic conditions, is DMT. Thus, DMT is composed of 5′-O-bis (trimethyl) with 2′-O-bis (2-acetoxyethyloxy) methyl (ACE) orthoester. It was replaced with 5'-O-bis (trimethylsiloxy) cyclododecyloxysilyl ether (DOD). Here, the 3′-OH group is one in which the cyanoethyl group (used in the TOM-protected nucleoside of FIG. 10A) is unstable to the fluoride reagent required to cleave the DOD. Since it proved that, it is induced | guided | derived as methyl-N, N-diisopropyl phosphoramidite (methyl-N, N-diisopropylphosphoramidite). The yield of coupling with this protected ribonucleoside is higher than 99%.

After assembly of the oligonucleotide, the phosphate methyl protecting group was converted to 2-carbamoyl-2-cyanoethylene-1,1-dithiolate disodium trihydrate in DMF. , 1-dithiolate trihydrate) (S ₂ Na ₂ ) (see FIG. 12). Basic conditions (40% aqueous methylamine) can then be applied to the oligonucleotide from the solid support along with the removal of the acyl protecting group at the exocyclic amino group and the acetyl group at the 2'-orthoesters. Causes cleavage. The resulting 2′-O-bis (2-hydroxyethyloxy) methyl orthoesters are more than ten times more unstable than before the removal of the acetyl group. Consequently, very mild conditions (pH 3.8, 30 minutes, 60 ° C.) are all that are required for the final deprotection step. The 5′-O-DOD group is cleaved with a fluoride reagent. The published HPLC chromatogram of the crude oligoribonucleotide obtained by this procedure is impressive and presents almost no by-products.

A general scheme for automated synthesis of oligoribonucleotides is depicted in FIG. 11 (PG = protecting group). The synthesis begins at the 3 'end by attachment to the resin. Deprotection is achieved in two steps, followed by CH ₄ NH ₂ in ethanol / water followed by Bu ₄ NF in tetrahydrofuran without degradation of the RNA product. The process can be repeated up to about 150 monomer units of product before significant product degradation results.

3. Synthesis of conformationally constrained polyamines:
Hosts of naturally occurring polyamines, including putrescine, spermine, spermidine, and hirudonin, are purchased from many global suppliers such as Aldrich Chemical Co, Milwaukee, Wisconsin, and Fisher Scientific, Hampton, New Hampshire. can do. Aldrich catalog numbers are putrescine (D1, 320-8), spermine (S383-6), and spermidine (S382-8): Fisher's catalog number for hirudonin (Diguanylspermidine, CAS No. 2465-97-6) is ICN 222595.

  Conformationally constrained polyamines suitable for use in the present invention are preferably those disclosed in US Pat. No. 6,392,098 and US Pat. No. 6,794,545. Is synthesized as follows. Briefly, either various rigid sites, cyclic sites or double or triple bonded sites are introduced into the polyamine backbone.

  The first targeted location was the central 1,4-diaminobutane segment of the polyamine. In its twisted conformation, four half-overlapping conformational rotamers are possible around the diaminobutane segment. Four have an enantiomeric relationship. Introduction of a bond between the C-1 and C-3 positions or the C-2 and C-4 positions in the central diaminobutane segment generates a cyclopropane ring. . Introduction of an additional bond between the C-2 and C-3 positions generates a conformationally restricted alkene derivative. Cyclobutyl, cyclopentyl, and cyclohexyl moieties can be introduced into the structure following the same strategy.

  Using this approach, four conformationally semi-rigid structures were obtained, which mimic the structure of four semi-overlapping conformations of spermine. Two of the semi-rigid structures are the other two epimers.

  For the purposes of the present invention, the cis and trans isomers of the subject compounds are highly apparent three-dimensional thanks to the limited bond rotation provided by the centrally located ring structure or unsaturation. It is important to note that a conformation is assumed. All geometric isomers (including optically active or not), and mixtures thereof, including pure isolated cis and pure isolated trans forms of polyamines, are clearly Are within the scope of the present invention. In addition, all positional isomers of the subject compounds are clearly within the scope of the present invention. When A or D of Formula I is a periodic moiety, the two B substituents or amino moieties are oriented at 1, 2, or 1, 3 or 1, 4 positions with respect to each other, respectively. Sometimes.

(A) Spermine analogs containing a cyclopropyl ring:
The cis and trans cyclopropyl analogs of spermine were prepared below via the reactions illustrated in Schemes 1, 2, 3, 4, 4A, 5, and 5A.

  Referring to Schemes 1 and 2, cyclopropyl diesel 1 and 2 were first converted to their hydrazides 103 and 4, and the hydrazides were converted to diamines 5 and 6, respectively. Diamines 5 and 6 were then mesitylated to give amides 7 and 8, and the amide was alkylated with 9 to give 10 and 11, respectively. Hydrolysis of the protecting group yielded trans analog 12 and cis analog 13.

Referring now to Scheme 3, in a separate reaction, transcyclopropyl diester 1 was converted to amide 14 by reaction with benzylamine (BnNH ₂ ) and the amide was reduced to amine 15. At the same time the amine was alkylated to 16. The phthalyl residue was then cleaved with hydrazine to give 17. And compound 17 was either deprotected by hydrogenolysis to give 18; or was fully alkylated to 19 and the benzyl residue was by hydrogenolysis to give 20. Cleaved.

  Referring to Scheme 4, amine 15 was also alkylated with 21 to give 22. Compound 22 was then deprotected to yield transcyclopropyl analog 23.

  An alternative (and preferred) route to 23 is given in Scheme 4A. Here, 3-ethylaminopropionitrile 101 was converted to the corresponding amine 102, which was then mesitylated to yield 3. In a parallel synthesis, cis diester 1 was reduced to dialcohol 15 ', which was then mesitylated to give dimesityl derivative 16'. Reacting 3 and 16 'in the presence of sodium hydride yielded 22'.

そして、スキーム４と同じ様式で、２２’は、トランスシクロプロピル類似体２３を生じさせるために、脱保護された。

Then, in the same manner as in Scheme 4, 22 ′ was deprotected to yield transcyclopropyl analog 23.

  Referring now to Scheme 5, in a separate reaction, ciscyclopropyl diester 2 was reduced to dialcohol 24. The dialcohol was then converted to amine 25 and the amine was protected by mesitylation to 26. Compound 26 was then alkylated with 9 to yield 27 and deprotected to yield ciscyclopropyltetraamine 28.

  An alternative (and preferred) route to 28 is given in Scheme 5A. Here, ciscyclopropyl diester 2 is reduced to dialcohol 24 in the same manner as in Scheme 5. Compound 24 was then protected by mesitylation to give 25 '. Compound 25 'was then reacted with 3 to yield 27. Deprotection yielded tetraamine 28.

(B) Spermine analogs containing a cyclobutyl ring:
The cis and trans cyclobutyl analogs of spermine were prepared via the reactions illustrated below in Schemes 6, 7, 8, 8A, 9, and 9A.

  Referring now to Schemes 6 and 7, the synthesis of cyclobutyl derivatives began with trans and cis 1,2-diaminobutanes 29 and 30, respectively. These compounds were first converted to amides 31 and 32 and then alkylated to 33 and 34, respectively. Compounds 33 and 34 were then deprotected to yield transtetraamine 35 (Scheme 6) and cistetraamine 36 (Scheme 7).

  Referring to Schemes 8 and 9, in a separate reaction, transcyclobutyl ester 37 and ciscyclobutyl diester 38 were reduced to the respective dialcohols 39 and 40, while the dialcohol was converted to diamines 41 and 42. Converted. Diamines 41 and 42 were then protected by mesitylation to give 43 and 44, respectively. These compounds were then alkylated to give 45 and 46. The protecting group was then removed to yield transcyclobutyltetraamine 47 (Scheme 8) and cistetraamine 48 (Scheme 8).

４７及び４８への代替の（及び好適な）経路は、それぞれ、スキーム８Ａ及び９Ａに与えられる。シス及びトランスのジエステル３７及び３８は、スキーム８及び９におけるものと同じ様式で、それぞれのジアルコール３９及び４０へ還元された。そして、化合物３９及び４０は、それぞれ、４１’及び４２’を生じさせるために、メシチル化された。３との４１’及び４２’の反応は、４５（スキーム８Ａ）及び４６（スキーム９Ａ）を生じさせる。脱保護することは、所望の生産物４７及び４８を生じさせる。

Alternative (and preferred) routes to 47 and 48 are given in Schemes 8A and 9A, respectively. The cis and trans diesters 37 and 38 were reduced to the respective dialcohols 39 and 40 in the same manner as in Schemes 8 and 9. Compounds 39 and 40 were then mesitylated to give 41 ′ and 42 ′, respectively. Reaction of 41 ′ and 42 ′ with 3 gives 45 (Scheme 8A) and 46 (Scheme 9A). Deprotecting yields the desired products 47 and 48.

(C) Spermine analogs containing unsaturation:
The cis and trans unsaturated analogs of spermine were prepared via the reactions illustrated in Schemes 10, 10A, 11, and 11A.

  Referring to Scheme 10, trans diester 49 was reduced to dialcohol 50, which was then converted to transdiamine 51. Referring to Scheme 11, cis diamine 52 was obtained from commercially available cis dialcohol 43 '. Referring to both Scheme 10 and Scheme 11, compounds 51 and 52 were protected by mesitylation to give 53 and 54, respectively. Compounds 53 and 54 were alkylated to 55 and 56 and were finally deprotected to give transtetraamine 57 (Scheme 10) and cistetraamine 58 (Scheme 11).

  Alternative (and preferred) routes to 57 and 58 are given in Schemes 10A and 11A, respectively. Cis and trans dialcohols 50 'and 50 were obtained in the same manner as in Schemes 10 and 11. Compounds 50 'and 50 were then mesitylated to give 51' and 52 ', respectively. 51 'and 52' reaction with 3 gives 55 (Scheme 10A) and 56 (Scheme 11A). Deprotecting yields the desired products 57 and 58.

Following the above general protocol, and using appropriate and well-known starting reagents, A and D are independently C ₅ or C ₆ cycloalkyl, cycloalkenyl, or All of the compounds of formula I, including those that are cycloaryl, can be readily obtained. An exemplary list of compounds of formula I is given in Table 1.

純粋な共役体は、それらの薬学的に適切な塩のみならず、明白に、本発明の範囲内にある。用語“薬学的に適切な塩”によって、主題の共役体のいずれの塩の形態をも、意味されると共に、それは、それらを、選ばれた経路による投与に対してより受け入れられるものにする。広い範囲のこのような塩は、薬学的な技術における業者には周知のものである。好適な薬学的に適切な塩は、塩化物、臭化物、ヨウ化物、及び同様のもののような酸付加塩である。

Pure conjugates are clearly within the scope of the present invention as well as their pharmaceutically suitable salts. By the term “pharmaceutically suitable salts” is meant any salt form of the subject conjugates, which makes them more acceptable for administration by the chosen route. A wide range of such salts are well known to those skilled in the pharmaceutical arts. Suitable pharmaceutically suitable salts are acid addition salts such as chloride, bromide, iodide, and the like.

4). Synthesis of polyamine conjugates of siRNA Based on the data described above, the three nucleosides shown in FIG. 13 can be prepared. Nucleoside A is a 2′-O-TOM nucleoside, nucleoside B is a 2′-O-ACE nucleoside, and nucleoside C is a 5′-thiol nucleoside. Nucleoside C is prepared from a 5'-thioriboside that is acetylated at its nucleobase and is converted to its 2'-O-ACE derivative and finally phosphitylated at the 3 'position. .

  Nucleoside B is attached to the polyamine chain at 5'-O. The linker is preferably a carbamate bond (as shown in FIG. 14). This linker can be attached either through the chloroformate at 5'-O or by adding an alcohol to the alkyl isocyanate. In either case, the polyamine residue will be attached to the ribose by a carbamate linkage. The polyamine chains are protected with alkali labile protecting groups such as FMOC, trifluoroacetate, and the like. As the oligonucleotide strand grows from the 3 'end to the 5' end (as shown in Figure 11), the polyamine-conjugated nucleoside will be attached in the last step of the synthesis. Release of the oligonucleotide from the resin under mildly alkaline conditions (as shown in FIG. 10) will also cleave the protecting group in the polyamine chain. The mild acid conditions required to liberate 2'-O will not affect the carbamate linkage and an oligonucleotide covalently linked to the polyamine residue is obtained. It will be. The resulting polyamine-RNA conjugate is transported into living cells in the same manner as that of other polyamine conjugates.

  For annealing, duplex siRNA strands are constructed independently; the sense strand is constructed of nucleotides of the desired sequence, and the antisense strand is constructed of the corresponding complementary bases. Single strands are incubated with each other (pH 7.4, 1 min, 90 ° C.) to form a duplex. This pairing is known as annealing siRNA. In a preferred embodiment of the invention, the polyamine site is attached to 5'-O of the sense strand (see Figure 8).

  Nucleoside C is constructed using 5'-thioribose. Residues derived from polyamines (see FIG. 15 for an exemplary listing of suitable residues) are attached to a short thioalkyl linker. The residue of the N-thioethylpolyamine illustrated in FIG. 15 can be obtained by starting with S-benzylcysteineamine and by subsequent alkylation following the established procedure of the polyamine. Strengthen the chain. Valasinas et al. (2003) and references therein, and finally by deprotecting the thiol group by using hydrogenolysis to cleave the benzyl group.

  Synthesis of disulfide linked polyamines to 5'-S-oligoribonucleosides is accomplished by treatment of the mixture with a diamine, a known thiol oxidant. The conjugated nucleoside is then attached to the sense oligoribonucleotide chain as discussed above. The sense oligoribonucleotide strand is deprotected at the 2'-O position, the polyamine protecting group is cleaved, and the strand is annealed to the complementary antisense strand. While not limited to any particular biological mechanism or phenomenon, the rationale behind this approach is as follows: Polyamines are the protozoa of siRNA duplexes As well as facilitating transport across the membrane, the conjugate will be freely translocated into the cytoplasm. The disulfide bond will then be reduced in the cytoplasm by thiols, thereby releasing the siRNA portion of the conjugate. The released siRNA will proceed to cause degradation of the sequence specific mRNA, which was designed to be achieved based on its predetermined sequence. .

  Even though the conjugate remains intact after delivery into the cell, the two strands of siRNA will be partially dissociated at RISC after delivery of the conjugate to the cytoplasm. This means that if the single-stranded antisense siRNA can silence the expression of the endogenous gene in the cell, it will not affect the function of the siRNA duplex.

  A third approach to the synthesis of oligoribonucleotide polyamine conjugates is to successfully mimic peptidases and establish linkers that set free amides and esters attached to them by an intramolecular catalyzed cleavage scheme. use. Polyamine chains and nucleosides are linked through Kemp's triacid (Kemp and Petrakis (1981), J. Org. Chem. 46: 5140). This remarkable triacid has three carboxylates in all axial orientations. One carboxyl is linked to the amine (as an amide) and the second carboxyl forms an ester with the alcohol. At a pH of about 6, the molecule will first release the amine residue via intramolecular formation of the anhydride. The alcohol is then released by intramolecular anhydride rearrangement (see FIG. 16). Upon entering the cytoplasm, the conjugate will be faced by various peptidases and lysosomal enzymes that will cleave both amide and ester bonds. This cleavage is aided by the axial geometry of the carboxylate. The reduction of internal compression during the formation of the anhydride thus contributes to the enzyme-driven acceleration of the hydrolysis. The contact distance of van der Waals in Kemp's acid is very short, and an energetically expensive desolvation process can thus be avoided.

  The synthesis uses Kemp's acid chloride-anhydride, see FIG. A polyamide is formed by reaction with a polyamine. Ring opening of the anhydride with 5'-O alcohol of nucleoside A yields Kemp's acid substituted with amides and esters. The Kemp's acid thus substituted with an amide and an ester of nucleoside A is then coupled to the growing edge of the sense ribonucleotide through the 3'-O-CEPA group (see, eg, Figure 11). ). Protecting groups in TOM protecting groups, N-acetyl and polyamines are cleaved in mild alkali. The ester amide ribonucleotide is then annealed to the antisense strand and transported to the cytoplasm.

  It is expected that the polyamide chain will be first cleaved by a cytoplasmic peptidase that will release the oligoribonucleotides from the polyamine chain. Cleavage of the ester group by cellular esterases will follow with the release of the siRNA duplex. It should be pointed out that even if the latter hydrolysis is slow (in time), the siRNA linger in the cell is not degraded in a few hours. Thus, there is sufficient time to achieve hydrolysis of both the amide linkage and the ester linkage. The dispensed Kemp acid is stable in plasma at pH 7.4. Nucleoside A is suitable in this synthetic sequence and is also conjugated due to the sensitivity of the ortho ester protecting group at 2'-O in nucleoside B to low pH conditions (and also conjugated). Nucleoside B is not, in order to avoid cleavage in the acid of Kemp substituted during deprotection of the oligonucleotide).

  A fourth approach for conjugating oligoribonucleotides to polyamines will be based on the construction of a connector chain that breaks after the sequence of hydrolysis steps; the first is enzymatic The second with cleavage involves solvolysis that proceeds spontaneously after the first step occurs. The connector chain is constructed as shown in FIG. Polyamine units (acid-stable groups; eg, protected with trifluoroacetate) will be linked to the α-amino group of lysine through a carbamate linkage (PG-1 must be ε-FMOC) Lysine is then converted to its corresponding amide by treatment with p-aminobenzyl alcohol. Addition of benzyl alcohol to p-nitrophenyl isocyanate results in p-nitroanilide 1a. Cleavage of the protecting group at the α-amino residue gives 1b.

  Compound 1b will undergo rapid hydrolysis in the presence of trypsin with the release of p-nitroaniline. In the present invention, the residue of p-nitroaniline is replaced with nucleoside A via a simple eviction reaction (see FIG. 15). After deprotection of PG1 at the α-amino group of the lysine residue to give 2a, the amide bond at the lysine residue is hydrolyzed by trypsin and / or lysosomal proteolytic enzymes cathepsins B and L Can be released, thereby releasing benzyl = carbamate 3 (see FIG. 17), which will undergo spontaneous solvolysis to nucleoside A and p-aminobenzyl alcohol.

  Briefly, compound 2a is used as the last step in the construction of an oligonucleotide to obtain a sense ribonucleotide chain attached through a foldable linker to the polyamine chain. Cleavage of the protecting group of TOM as well as that of the polyamine protecting group and P1 in α-lysine, followed by annealing with complementary RNA strands, was sent to living cells and after hydrolysis Provides a conjugated siRNA complex that decays into the component sites of

5. Administration and dosage form of the pharmaceutical unit:
Since the conjugates described above are effective at triggering RNA into living cells, the conjugates can be used for therapeutic treatment of mammals in vivo, including humans, and mammals. Any treatment regimen that requires activation of RNA into the cells is appropriate for treating mammalian cells in vitro. Briefly, conjugates are useful for moving RNA, including siRNA, from the extracellular space into the cytoplasm of mammalian cells.

  Administration of the subject conjugates to human or non-human patients can be accomplished by any means known in the pharmaceutical arts. The preferred route of administration is either pure or in combination with a pharmaceutical carrier suitable for the chosen route of administration, intravenous administration, intraarterial administration, intratumoral administration, intramuscular. Administration, intraperitoneal administration, and subcutaneous administration. The method of treatment is also acceptable for oral administration.

  As with all pharmaceuticals, the concentration or amount of polyamine-RNA conjugate administered depends on the severity of discomfort treated, the mode of administration, the condition and age of the subject being treated, and the particular polyamine used It has to be noted that it will vary depending on the RNA conjugate or the combination of conjugates.

  The conjugates described herein are in the form of tablets, pills, powder mixtures, capsules, injections, solutions, suppositories, emulsions, dispersions, food premixes, and other suitable forms It can be administered. Pharmaceutical dosage forms containing the conjugates described herein are conveniently mixed with a non-toxic pharmaceutical organic carrier or a non-toxic pharmaceutical inorganic carrier. Typical pharmaceutically acceptable carriers are, for example, mannitol, urea, dextran, lactose, potato and corn starch, magnesium stearate, talc, vegetable oil, polyalkylene glycol, ethyl cellulose, poly (vinyl pyrrolidone), calcium carbonate Ethyl oleate, isopropyl myristate, benzyl benzoate, sodium carbonate, gelatin, potassium carbonate, silicic acid, and other conventionally used acceptable carriers. Pharmaceutical dosage forms may also contain nontoxic auxiliary substances such as emulsifiers, preservatives, or wetting agents, and the like.

  Solid forms, such as tablets, capsules and powders, can be made using conventional tablets and capsule filling machinery, which are well known in the art. is there. A solid dosage form may be any number known in the art, including excipients, lubricants, desiccants, binders, colorants, disintegrants, dry flow modifiers, preservatives, and the like. It may also contain additional inert inclusions.

  Liquid forms for ingestion use known liquid carriers, including aqueous and non-aqueous carriers, suspensions, oil-in-water and / or water-in-oil emulsions, and the like It can be prepared. Liquid formulations also contain any number of additional inert ingredients, including colorants, fragrances, seasonings, viscosity modifiers, preservatives, stabilizers, and the like. Sometimes.

  For parenteral administration, the subject conjugates are conjugated in a physiologically acceptable diluent or sterile liquid carrier, such as water or oil, with or without additional surfactants or adjuvants. It may be administered as an injectable dose of a solution or suspension of the body. Exemplary listings of carrier oils would include animal and vegetable oils (peanut oil, soybean oil), oils derived from petroleum (mineral oil), and synthetic oils. Generally, for injectable unit dosages, water, saline, aqueous dextroglucose and related sugar solutions, and ethanol and glycols such as propylene glycol or polyethylene glycol The solution is a suitable liquid carrier.

  The selected pharmaceutical unit dose is preferably fabricated and administered to provide a concentration of the conjugate at the point of contact with the target cell, eg, from about 1 μM to about 10 mM. The More preferred is a concentration from about 1 μM to about 100 μM. This concentration will, of course, depend on the chosen route of administration and the mass of the subject being treated. Concentrations above and below the ranges stated above are within the scope of the invention.

Cross-reference to related applications Priority is hereby claimed for US provisional application serial number 60 / 624,906 filed November 4, 2004, which is hereby incorporated by reference.

Incorporation of literature All of the papers and prior patents cited below are hereby incorporated by reference.

FIG. 1 depicts the chemical structure of putrescine, spermidine, and spermine. FIG. 2 depicts the chemical structure of a compound of the bleomycin class. FIG. 3 depicts the chemical structure of spagalin and edyne A and B. FIG. 4 depicts the chemical structure of streptomycin. FIG. 5 depicts the chemical structure of 4-coumaroyl agmatine and holdertin A, B, and M. FIG. 6 depicts the chemical structure of hirudonin. FIG. 7 depicts the chemical structure of squalamine. FIG. 8 is a schematic illustrating the cleavage specific to siRNA-mediated sequences of mRNA. FIG. 9 is a schematic illustrating a siRNA-mediated sequence specific for mRNA and a silenced complex (RISC) that induces RNA. FIGS. 10A and 10B depict the chemical structure of protected ribonucleosides that can be used to make oligoribonucleotide siRNAs. FIG. 11 is a reaction scheme illustrating the synthesis of oligoribonucleotides based on phosphoramadites. FIG. 12 is a reaction scheme illustrating the deprotection of the ribonucleoside shown in FIG. 10B. FIG. 13 depicts the chemical structure of three different protected ribonucleosides for use in the present invention. FIG. 14 depicts a series of polyamine-siRNA conjugates according to the present invention. FIG. 15 depicts another series of polyamine-siRNA conjugates according to the present invention. FIG. 16 depicts yet another series of polyamine-siRNA conjugates according to the present invention. FIG. 17 depicts a general reaction scheme for making polyamine-siRNA conjugates according to the present invention.

Claims (14)

  A conjugate comprising a polyamine covalently linked to ribonucleic acid (RNA).   The conjugate according to claim 1, wherein the RNA is an oligo RNA.   The conjugate of claim 1, wherein the RNA is a short interfering RNA (siRNA).   The conjugate according to claim 1, 2, or 3, wherein the polyamine is selected from the group consisting of putrescine, spermine, spermidine, hirudonin, and derivatives thereof. The polyamine has the formula (I):
E-NH-D-NH-B-A-B-NH-D-NH-E (I)
Selected from the group consisting of:
A is selected from the group consisting of C ₂ to C ₆ alkenes, and C ₃ to C ₆ cycloalkyls, cycloalkenyls, and cycloaryls;
B is independently selected from the group consisting of a single bond and C ₁ to C ₆ alkyl and alkenyl;
D is independently selected from the group consisting of C ₁ to C ₆ alkyl and alkenyl, and C ₃ to C ₆ cycloalkyl, cycloalkenyl, and cycloaryl;
and,
4. A conjugate according to claim 1, 2 or 3, wherein E is methyl. Putrescine, spermine, spermidine, hirudonin, and derivatives thereof, and formula (I):
E-NH-D-NH-B-A-B-NH-D-NH-E (I)
A conjugate comprising a polyamine selected from the group consisting of:
A is selected from the group consisting of C ₂ to C ₆ alkenes, and C ₃ to C ₆ cycloalkyl, cycloalkenyl, and cycloaryl;
B is independently selected from the group consisting of a single bond and C ₁ to C ₆ alkyl and alkenyl;
D is independently selected from the group consisting of C ₁ to C ₆ alkyl and alkenyl, and C ₃ to C ₆ cycloalkyl, cycloalkenyl, and cycloaryl;
and,
E is methyl; and
The conjugate, wherein the polyamine is covalently attached to siRNA.   7. The conjugate of claim 6, wherein the polyamine is bound to the siRNA by a bond that is hydrolysable by a cytosolic enzyme. A method of activating RNA into a living cell,
The method is
Conjugating RNA to a polyamine to produce the conjugate; and contacting the conjugate to the living cell. The RNA includes putrescine, spermine, spermidine, hirudonin, derivatives thereof, and formula (I):
E-NH-D-NH-B-A-B-NH-D-NH-E (I)
And conjugated to a polyamine selected from the group consisting of:
A is selected from the group consisting of C ₂ to C ₆ alkenes, and C ₃ to C ₆ cycloalkyl, cycloalkenyl, and cycloaryl;
B is independently selected from the group consisting of a single bond and C ₁ to C ₆ alkyl and alkenyl;
D is independently selected from the group consisting of C ₁ to C ₆ alkyl and alkenyl, and C ₃ to C ₆ cycloalkyl, cycloalkenyl, and cycloaryl;
and,
The method of claim 8, wherein E is methyl.   10. The method of claim 8 or 9, wherein siRNA is conjugated to the polyamine. A composition of materials for activating RNA into living cells,
The composition comprises a polyamine covalently linked to ribonucleic acid (RNA) in combination with its pharmaceutically suitable carrier.   The composition according to claim 11, wherein the RNA is an oligo RNA.   12. The composition of claim 11, wherein the RNA is a short interfering RNA (siRNA). The polyamines include putrescine, spermine, spermidine, hirudonin, derivatives thereof, and formula (I):
E-NH-D-NH-B-A-B-NH-D-NH-E (I)
Selected from the group consisting of:
A is selected from the group consisting of C ₂ to C ₆ alkenes, and C ₃ to C ₆ cycloalkyl, cycloalkenyl, and cycloaryl;
B is independently selected from the group consisting of a single bond and C ₁ to C ₆ alkyl and alkenyl;
D is independently selected from the group consisting of C ₁ to C ₆ alkyl and alkenyl, and C ₃ to C ₆ cycloalkyl, cycloalkenyl, and cycloaryl;
and,
14. A composition according to claim 11, 12 or 13, wherein E is methyl.

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