Patent Application
20060148734
This application claims the benefit of UK priority application No. GB0207283.3 filed 27 Mar. 2002 and GB0213646.3 filed 13 Jun. 2002, whose contents are incorporated herein by reference.
This invention relates to newly identified diaminoacid-polyamine:peptide and diaminoacid-aminoacid-polyamine based gemini surfactant compounds, to the use of such compounds and to their production. The invention also relates to the use of the diaminoacid-polyamine:peptide based gemini compounds to facilitate the transfer of compounds into cells for drug delivery.
Surfactants are substances that markedly affect the surface properties of a liquid, even at low concentrations. For example surfactants will significantly reduce surface tension when dissolved in water or aqueous solutions and will reduce interfacial tension between two liquids or a liquid and a solid. This property of surfactant molecules has been widely exploited in industry, particularly in the detergent and oil industries. In the 1970s a new class of surfactant molecule was reported, characterised by two hydrophobic chains with polar heads which are linked by a hydrophobic bridge (Deinega, Y et al., Kolloidn. Zh. 36, 649, 1974). These molecules, which have been termed “gemini” (Menger, F M and Littau, C A, J. Am. Chem. Soc. 113, 1451, 1991), have very desirable properties over their monomeric equivalents. For example they are highly effective in reducing interfacial tension between oil and water based liquids and have a very low critical micelle concentration (Menger, F M and Keiper, J S, Angewandte. Chem. Int. Ed. Engl., 2000, 39, 1906).
Cationic surfactants have been used inter alia for the transfection of polynucleotides into cells in culture, and there are examples of such agents available commercially to scientists involved in genetic technologies (for example the reagent Tfx™-50 for the transfection of eukaryotic cells available from Promega Corp. WI, USA).
The efficient delivery of DNA to cells in vivo, either for gene therapy or for antisense therapy, has been a major goal for some years. Much attention has concentrated on the use of viruses as delivery vehicles, for example adenoviruses for epithelial cells in the respiratory tract with a view to corrective gene therapy for cystic fibrosis (CF). However, despite some evidence of successful gene transfer in CF patients, the adenovirus route remains problematic due to inflammatory side-effects and limited transient expression of the transferred gene. Several alternative methods for in vivo gene delivery have been investigated, including studies using cationic surfactants. Gao, X et al. Gene Ther. 2, 710-722, 1995 demonstrated the feasibility of this approach with a normal human gene for CF transmembrane conductance regulator (CFTR) into the respiratory epithelium of CF mice using amine carrying cationic lipids. This group followed up with a liposomal CF gene therapy trial which, although only partially successful, demons the potential for this approach in humans (Caplen, N J. et al., Nature Medicine, 1, 39-46, 1995). More recently other groups have investigated the potential of other cationic lipids for gene delivery (Miller, A, Angew. Int. Ed. Engl., 37, 1768-1785, 1998), for example cholesterol derivatives (Oudrhiri, N et al. Proc. Natl. Acad. Sci. 94, 1651-1656, 1997). This limited study demonstrated the ability of these cholesterol based compounds to facilitate the transfer of genes into epithelial cells both in vitro and in vivo, thereby lending support to the validity of this general approach.
These studies, and others, show that in this new field of research there is a continuing need to develop novel low-toxicity surfactant molecules to facilitate the effective transfer of polynucleotides into cells both in vitro for transfection in cell-based experimentation and in vivo for gene therapy and antisense treatments. Gemini surfactants based on cysteine (WO99/29712) or on spermine (WO00/77032) or diamine (WO/00/76954) have previously been made. Other examples of gemini surfactants are found in WO/00/27795, WO02/30957 and WO02/50100.
The present invention seeks to overcome the difficulties exhibited by existing compounds.
The invention relates to diaminoacid-polyamine:peptide based gemini compounds having a diaminoacid-polyamine or a diaminoacid-aminoacid-polyamine backbone and conforming to the general structure of formula (I):
where:
Preferably, the compound is symmetrical, that is R1 and R2 are the same as each other, R3 and R4 are the same as each other, R5 and R6 are the same as each other, R7 and R8 are the same as each other.
In a preferred embodiment A1 is lysine, serine or threonine, preferably lysine. Preferably A3 and A4 are lysine, ornithine, histidine or arginine.
In a further preferred embodiment the hydrocarboxyl group is selected from:
In a preferred embodiment m is 0, n is 2 to 4, X is (CH2) or (CH2)2, Y is a bond and p is 0 to 4.
In a further preferred embodiment m is 0, n is 2 to 4, X is NH(CH2)qNH, where q is 2 to 5, Y is a bond and p is 2 to 5.
In another preferred embodiment m is 0, n is 2 to 4, X is
where R9, R10, R11 and R12 are all H, Y is a bond and p is 2 to 5.
In a still further preferred embodiment m is 0, n is 2 to 4, X is (CH2) or (CH2)2, p is 0 to 4 and Y is
In a yet further preferred embodiment m is 0, n is 2 to 4, X is NH(CH2)qNH where q is 2 to 5, p is 2 to 5 and Y is
In a yet still further preferred embodiment m is 0, n is 2 to 4, X is
where R9, R10, R11 and R12 are all H, p is 2 to 5 and Y is
A further preferred embodiment is where X is
Y is a bond, p is 1 to 6 and n is 1 to 7.
Compounds of the present invention may be prepared from readily available starting materials using synthetic peptide chemistry well known to the skilled person. The scheme shown in
Another aspect of the invention relates to methods for using the diaminoacid-polyamine:peptide based gemini compounds. Such uses include facilitating the transfer of oligonucleotides and polynucleotides into cells for antisense, gene therapy and genetic immunisation (for the generation of antibodies) in whole organisms. Other uses include employing the compounds of the invention to facilitate the transfection of polynucleotides into cells in culture when such transfer is required, in, for example, gene expression studies and antisense control experiments among others. Protocols for the preparation of such polynucleotides and antisense molecules are well known in the art (for example Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), Cohen, J S ed. Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1989)). The polynucleotides can be mixed with the compounds, added to the cells and incubated to allow polynucleotide uptake. After further incubation the cells can be assayed for the phenotypic trait afforded by the transfected DNA, or the levels of mRNA expressed from said DNA can be determined by Northern blotting or by using PCR-based quantitation methods for example the Taqman® method (Perkin Elmer, Connecticut, USA). Compounds of the invention offer a significant improvement, typically between 3 and 6 fold, in the efficiency of cellular uptake of DNA in cells in culture, compared with compounds in the previous art. In the transfection protocol, the gemini compound may be used in combination with one or more supplements to increase the efficiency of transfection. Such supplements may be selected from, for example:
In still another aspect, the invention relates to the transfer of genetic material in gene therapy using the compounds of the invention. For example the skilled person can develop gene delivery methodologies for use in gene therapy, involving the use of gemini surfactant compounds of the present invention, using protocols that are well known in the art. For example the use of surfactants for delivery of gene transfer vectors to the lung is reviewed in Weiss, D J (2002) Molecular Therapy 6(2) pp 148 to 152.
Yet another aspect of the invention relates to methods to effect the delivery of non-nucleotide based drug compounds into cells in vitro and in vivo using the compounds of the invention.
The following definitions are provided to facilitate understanding of certain terms used frequently herein.
“Amino acid” refers to dipolar ions (zwitterions) of the form +H3NCH(R)CO2−. They are differentiated by the nature of the group R, and when R is different from hydrogen can also be asymmetric, forming D and L families. There are 20 naturally occurring amino acids where the R group can be, for example, non-polar (e.g. alanine, leucine, phenylalanine) or polar (e.g. glutamic acid, histidine, arginine and lysine). In the case of un-natural amino acids R can be any other group which is not found in the amino acids found in nature.
“Polynucleotide” generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. “Polynucleotides” include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, “polynucleotide” refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNA's or RNA's containing one or more modified bases and DNA's or RNA's with backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications have been made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. “Polynucleotide” also embraces relatively short polynucleotides, often referred to as oligonucleotides.
“Transfection” refers to the introduction of polynucleotides into cells in culture using methods involving the modification of the cell membrane either by chemical or physical means. Such methods are described in, for example, Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). The polynucleotides may be linear or circular, single-stranded or double-stranded and may include elements controlling replication of the polynucleotide or expression of homologous or heterologous genes which may comprise part of the polynucleotide.
The invention will now be described by way of the following examples.
To a solution of H-Lys(Boc)-OH (5.02 g, 20.4 mmol) and 20.5 mL of NaOH 1M in 85 Ml of water-acetone (1:2 v/v) cooled at 0° C. was added dropwise 4.43 g (20.3 mmol) of dodecyl chloride and NaOH aq. 1M alternatively to maintain the pH over 9. After addition keep 10 minutes more stirring at 0° C. HCl 10% was added until pH 2. Filter the solid and wash with water until pH 7. Dry over P2O5. The solid is chromatographied on silica with CHCl3—MeOH to yield 46% of compound CR-110 as a white solid. αD20−1.0 (c 1.48, MeOH); IR(KBr)vmax 3347, 2921, 2851, 1717, 1681, 1521 cm−1; 1H NMR (300 MHz, CD3OD) 4.26 (dd, 1H, J=4.77, 8.92 Hz, CH—COOH), 2.94 (t, 2H, J=6.7 Hz, CH2N), 2.16 (t, 2H, J=7.4 Hz, CH2CON), 1.78-1.74 (m, 1H, HCH—CH(COOH), 1.63-1.45 (m, 7H, HCH—CH(COOH), CH2CH2N and CH2CH2CON), 1.35 (s, 9H, (CH3)3C), 1.22 (s, 16H, CH3(CH2)8), 0.82 (t, 3H, J=6.8 Hz, CH3); 13C (75 MHz, CD3OD) 176.32 C(O)NCH2), 175.45 (COOH), 158.42 (C(O)NO), 79.73 (C(CH3)3), 54.84 (CH), 41.16 (CH2N), 36.97, 33.04, 32.64, 30.74-29.99 (CH2), 28.83 (CH3), 26.98, 24.26, 23.71 (CH2), 14.48 (CH3).
To a solution of 2.4 g (5.6 mmol) of CR-110 in THF at −20° C. were added Et3N (0.78 mL, 5.6 mmol) and EtOCOCl (0.55 mL, 5.6 mmol). The reaction was stirring at this temperature for 30 minutes and 246 mg (2.8 mmol) of 1,4-diaminobutane were added, after 1 hour more stirring at −20° C. the reaction mixture was allowed to warm at room temperature and stirred overnight. Remove the solvent in vacuum, the residue was dissolved in CHCl3 and washed with NaHCO3 aq. saturated and brine and dried over MgSO4 anh. The obtained residue was chromatographied to give compound CR-116 (50%) as a white solid: αD20−10.06 (c 1.51, MeOH); IR(KBr)vmax 3415-3307, 2920, 2851, 1688, 1637, 1515 cm−1; 1H NMR (300 MHz, CD3OD) 4.17 (dd, 1H, J=5.5, 8.5 Hz, CH—COOH), 3.12 (m, 2H, CH2N), 2.96 (q, 2H, J=6.4 Hz, CH2N), 2.17 (t, 2H, J=7.4 Hz, CH2C(O)N), 1.69-1.64 (m, 1H, HCH—HC(COOH), 1.58-1.42 (m, 5H, HCH—HC(COOH), CH2CH2CO, CH2CH2N), 1.36 (s, 9H, (CH3)3C), 1.22 (s, 16H, CH3(CH2)8CH2), 0.88 (t, 2H, J=6.8 Hz, CH3); 13C (75 MHz, CD3OD) 176.26, 174.46 C(O)NCH2), 158.42 (OC(O)N), 79.93 (C(CH3)3), 54.82 (CH) 41.11 and 39.96 (CH2N), 36.89, 33.09, 32.92, 30.77-30.38 (CH2), 28.85 (CH3), 27.63, 26.94, 24.28, 23.74(CH2), 14.48 (CH3).
1.2299 g (1.35 mmol) of CR-116 were treated with EtOAc 4 M for 45 minutes. The solid was filtered and recrystalized from MeOH and EtOAc added to obtain the compound CR-117 as a white solid (49%): αD20−13.98 (c 1.76, MeOH); IR(KBr)vmax 3422, 3298, 3089, 2920, 2851, 1638 cm−1; 1H NMR (300 MHz, CD3OD) 4.20 (dd, 1H, J=5.6, 8.4 Hz, CH—COOH), 3.12 (m, 2H, CH2N), 2.84 (t, 2H, J=6.4 Hz, CH2N), 2.18 (t, 2H, J=7.6 Hz, CH2C(O)N), 1.74-1.72 (m, 1H, HCH—CH(COOH), 1.69-1.34 (m, 5H, HCH—CH(COOH)+CH2CH2CO+CH2CH2N), 1.22 (s, 16H, CH3(CH2)8CH2), 0.82 (t, 2H, J=6.8 Hz, CH3); 13C (75 MHz; CD3OD) 176.39, 174.22 C(O)NCH2), 54.59 (CH), 40.55, 39.99 (CH2N), 33.08, 32.57, 30.76-30.41(CH2), 28.23, 27.61, 26.93 (CH2), 14.44 (CH3); C40H80Cl2N6O4.H2O 778.56 calc C 60.94%, H 10.36%, N 10.65% found C 60.88%, H10.22%, N 10.08%
To a solution of N-ε-(tertbutoxycarbonyl)-L-lysine (1.24 g, 5.03 mmol) in THF (140 mL) were added successively a solution of K2CO3 (0.75 g, 5.43 mmol, 1.08 eq.) in water (20 mL) and oleoyl succinimidate (1.92 g, 5.06 mmol, 1 eq.). The reaction was stirred at RT for 20 h and most of THF was evaporated. Water and CHCl3 (30 mL each) were added and the organic layer was separated. The aqueous layer was acidified to pH 2 and extracted twice with CHCl3 (2×30 mL). The organic layer was washed with water and brine (20 mL each), dried (Na2SO4), filtered and evaporated to give an oil. Yield: 2.46 g (4.82 mmol, 96%). 1H NMR (400 MHz, d6-DMSO): δ 12.4 (m, 1 HOH), 7.92 (d, 1 H, J=7.8, HNα), 6.70 (t, 1 H, J=6.0, HNε), 5.29 (m, 2 CH9,10), 4.10 (dt, 1 H, J=5.0, 8.9, CHα), 2.85 (q, 2 H, J=6.2, CH2ε), 2.07 (dt, 2 H, J=2.2, 7.0, CH22), 1.95 (q, 4 H, J=6.0, CH28,11), 1.62 (m, 1 H, CHβ), 1.51 (m, 1 H, CHβ), 1.45 (m, 2 H, CH23), 1.33 (s, 9 H, C(CH3)3), 1.2 (m, 26 H, 2 CH2γ,δ and 10 CH2 oleoyl), 0.82 (t, J=6.4, 3 H, CH318).
To a solution of N-α-oleoyl-N-ε-(tert-butyloxycarbonyl)-L-Lysine (1.80 g, 3.52 mmol) in THF (80 mL) were added successively N-hydroxysuccinimde (0.41 g, 3.56 mmol, 1.01 eq.) and DCC (0.73 g, 3.54 mmol, 1.01 eq.). The reaction was stirred for 16 h at RT. The precipitate was filtered and washed with EtOAc (30 mL). The filtrate was concentrated and redissolved in EtOAc and filtered again. The residue was dissolved in CHCl3 and precipitated with Et2O to give N-α-oleate-N-ε-(tert-butyloxycarbonyl)-L-Lysinyl succinimidate as a white solid. Yield: 1.98 g (93%). NMR 1H (400 MHz, CDCl3): δ 6.11 (m, 1 H, HNα), 5.38 (m, 2 H, H9,10), 4.94 (m, 1 H, CHα), 4.65 (m, 1 H, HNε), 3.12 (m, 2 H, CH2ε), 2.79 (s, 4 H, 2 CH2Su), 2.20 (t, J=6.1, 2 H, CH22), 2.00 (m, 5 H, CHβ and 2 CH28,11), 1.84 (m, 1 H, CHβ), 1.63 (m, 2 H, CH23), 1.48 (m, 4 H, 2 CH2γ,δ), 1.37 (s, 9 H, 3 CH3), 1.27 (m, 20 H, 10 CH2 oleoyl), 0.83 (t, J=6.3 Hz, 3 H, CH318).
To a solution of N4,N9-bis-tert-butyloxycarbonyl)-1,12-diamino-4,9-diazadodecane (629 mg, 1.0 mmol) in THF (80 mL) and K2CO3 (0.29 g, 2.1 mmol, 2.1 eq.) in water (10 mL) was added a solution of N-α-oleoyl-N-ε-(tert-butyloxycarbonyl)-L-lysinyl succinimidate (1246 mg, 2.05 mmol, 2.05 eq.). The reaction was stirred overnight at RT. Most of the THF was evaporated and water (30 mL) was added. The aqueous layer was extracted with CHCl3 (2×50 mL). The organic layer was washed with water, 0.1 M HCl, water and brine (20 mL each), dried (Na2SO4), filtered, evaporated and purified by column chromatography on SiO2 (CHCl3/MeOH: 95/5, Rf=0.30) to give an oil. Yield: 1060 mg (0.76 mmol, 76%). 1H NMR (400 MHz, CDCl3): δ 7.30 (bs, 2 H, 2 NHC1′), 6.33 (bs, 2 H, 2 NHα), 5.31 (m, 4 H, 2 CH9,10), 4.71 (bs, 2 H, 2 NHε), 4.41 (m, 2 H, 2 CHα), 3.18 (m, 12 H, 2 CH21′, 2 CH23′), 3.08 (m, 4 H, 2 CH23′ and 2 CH2ε), 2.18 (t, 4 H, J=6.8, 2 CH22), 1.98 (m, 8 H, 2 CH28,11), 1.90 (m, 2 H, 2 CHβ), 1.79 (m, 2 H, 2 CHβ), 1.60 (m 10 H, 2 CH22′, 2 CH2γ and 2 CH23), 1.45 (m, 26 H, 2 CH2δ, 2 CH25′ and 2 C(CH3)3), 1.40 (s, 18 H, 2 C(CH3)3), 1.25 (m, 40 H, 2×10 CH2Tail), 0.86 (m, 6 H, J=6.6, 2 CH318). 13C NMR (100 MHz, CDCl3): δ 171.1, 174.3, 155.6, 155.7, 129.5, 129.3, 79.4, 78.5, 76.8, 52.4, 48.6, 46.4, 39.6, 36.1, 33.5, 31.4, 29.3, 29.2, 29.0, 28.8, 28.7, 28.0, 26.7, 25.3, 24.5, 22.2, 22.1, 13.7.
To a solution of RG 00/250 (1.04 g, 0.75 mmol) in MOH (20 mL) was added concentrated HCl (10 mL) and the reaction was stirred at RT for 2 h. The solvent were then removed and the residue redissolved in water (80 mL), filtered on a frit and evaporated again. The residue was redissolved in a minimum volume of methanol and precipitated with Et2O to give, after filtration, a pale yellow solid. Yield: 0.734 g (0.65 mmol, 86%). 1H NMR (400 MHz, d6-DMSO): δ 9.02 (m, 4 H, 2 NH), 8.16 (t, 2 H, J=6.0, 2 NHC1′), 7.98 (s, 6 H, 2 NαH and 2 NεH2), 5.29 (m, 4 H, 2 CH9,10), 4.10 (q, 2 H, J=7, 2 CHα), 3.10 (hp, 4 H, J=6.4, 2 CH21′), 2.85 (m, 8 H, 2 CH23′ and 2 CH24′), 2.71 (m, 4 H, 2 CH2ε), 2.10 (AB, 4 H, J=6.4, 2 CH22), 1.95 (m, 8 H, 2 CH28,11), 1.76 (m, 4 H, 2 CH22′), 1.68 (m, 4 H, 2 CH25′), 1.65-1.42 (m, 12 H, 2 CH2ε, 2 CH2δ and 2 CH23), 1.25 (m, 44 H, 10 CH2O1and 2 CH2γ), 0.83 (t, 6 H, 2 CH318). MS (+ES): 999.8 [M+Na].
To a solution of N-α-oleoyl-N-ε-(tert-butyloxycarbonyl)-L-lysine (900 mg, 1.48 mmol) in THF (60 mL) were added successively a solution of potassium carbonate (225 mg, 1.63 mmol, 1.1 eq.) in water (6 mL) and N-ε-(tert-butyloxycarbonyl)-L-lysine (365 mg, 1.49 mmol, 1 eq.). The solution was then stirred for 16 h at RT. Most of THF was evaporated and pH of the aqueous solution was adjust to 2 and extra with CHCl3 (2×80 mL). The organic layer was washed with water (50 mL) and brine (40 mL), dried (Na2SO4), filtered and evaporated. The oil obtained was then dissolved in a small quantity of CHCl3 and Et2O was added. The white solid was then collected. Yield: 1008 mg (1.46 mmol, 99%). 1H NMR (400 MHz, CDCl3): δ 12.60 (m, 1 H, COOH), 8.55 (m, 1 H, NH), 7.10 (m, 1 H, 1 NH), 6.70 (m, 1 H, 1 NH), 5.32 (m, 2 H, CH9,10), 4.80 (m, 1 H, NH), 4.51 (m, 2 H, 2 CHα), 3.08 (m, 4 H, 2 CH2ε), 2.20 (t, 2 H, J=7.0, 2 CH22), 1.99 (m, 4 H, CH28,11), 1.60 (m, 4 H, 2 CH2β), 1.50-1.20 (m, 44 H), 0.87 (t, 3 H, J=6.8, CH318).
To a solution of N-α-(N-α-Oleoyl-N-ε-(tert-butyloxycarbonyl)-L-lysyl)-N-ε-(tert-butyloxycarbonyl))-L-lysine (1008 mg, 1.46 mmol) in THF (40 mL) was added N-hydroxysuccinimide (177 mg, 1.49 mmol, 1.02 eq.) and DCC (311 mg, 1.50 mmol, 1.03 eq.). The reaction was stirred overnight at RT and the DCU was then filtered and washed with EtOAc. The solvent was then removed and the residue redissolved in EtOAc, the DCU filtered again and after evaporation a white solid was isolated. Yield: 1147 mg (1.36 mmol, 93%).
To a solution of N4,N9-bis-tert-butyloxycarbonyl)-1,12-diamino-4,9-diazadodecane (241 mg, 0.36 mmol) in THF (60 mL) and K2CO3 (0.10 g, 0.73 mmol, 2.1 eq.) in water (8 mL) was added a solution of N-α-(N-α-oleoyl-N-ε-(tert-butyloxycarbonyl)-L-Lysyl)-N-ε-tert-butyloxycarbonyl))-L-lysyl succinimidate (600 mg, 0.72 mmol, 2.0 eq.) in THF (10 mL). The reaction was stirred overnight at RT. Most of the THF was evaporated and water (30 mL) was added. The aqueous layer was extracted with CHCl3 (2×60 mL). The organic layer was washed with water, 0.1 M HCl, water and brine (20 mL each), dried (Na2SO4), filtered, evaporated and purified on SiO2 (CHCl3/MeOH: 9/1, Rf=0.27) to give a white solid. Yield: 497 mg (0.27 mmol, 75%). 1H NMR (400 MHz, CDCl3): δ 8.40 (m, 2 H,2 NH), 6.90 (m, 2 H, 2 NH), 6.40 (m, 2 H, 2 NH), 5.33 (m, 4H, 2 CH9,10), 4.85 (m, 4 H, 4 NεH), 4.40 (m, 4 H, 2×2 CHα), 3.28-3.02 (m, 20 H, 2×2 CH2ε, 2 CH21′, 2 CH23′ and 2 CH24′), 2.22 (m, 4 H, 2 CH22), 1.99 (m, 8 H, 2 CH28,11), 1.80 (m, 4 H, 2 CH2β), 1.72-1.25 (m, 126 H), 0.83 (t, 6 H, J=6.8, 2 CH318).
To a solution of RG 00/384 (470 mg, 0.255 mmol) in MeOH (10 mL) was added concentrated HCl (10 mL) and the reaction was stirred at RT for 1 h. The solvents were removed under vacuum and the residue redissolved into water (80 mL), filtered and evaporated again. The residual oil was dissolved in MeOH and precipitated with Et2O to give a yellow powder. Yield: 284 mg (0.194 mmol, 76%). 1H NMR (400 MHz, d6-DMSO): δ 9.10 (m, 4 H, 2 NH2+), 8.18 (m, 4 H, 4 NHC), 8.10-7.98 (m, 16 H 2×2 NαH and 2×2 NεH3+), 5.29 (m, 4 H, 2 CH9,10), 4.18 (m, 2 H, CHα), 4.11 (m, 2 H, 2 CHα), 3.10 (m, 4 H, 2 CH21′), 2.85 (m, 8 H, 2 CH23′ and 2 CH24′), 2.71 (m, 8 H, 2×2 CH2ε), 2.10 (m, 4 H, 2 CH22),1.95 (m, 8 H, 2 CH28,11), 1.80-1.39 (m, 28 H), 1.25 (m, 48 H, 10 CH2O1 and 2×2 CH2γ), 0.83 (t, 6 H, J=6.8, 2 CH318).
To a solution of N-α-(tert-butyloxycarbonyl)-L-Lysine (779 mg, 3.16 mmol) in THF (80 mL) were added successively a solution of potassium carbonate (0.524 g, 3.79 mmol, 1.2 eq.) in water (10 mL) and oleoyl succinimidate (1.20 g, 3.16 mmol, 1 eq.). The reaction is stirred overnight at room temperature. Most of THF was evaporated and water (40 mL) was added. The aqueous layer was acidified to pH 2 and extracted with CHCl3 (3×60 mL). The combined organic layers were washed with water (30 mL) and brine (40 mL), dried over sodium sulphate, filtered and evaporated to give N-α-(tert-butyloxycarbonyl)-N-ε-oleoyl-L-lysine as a colourless oil. Yield: 1.31 g (2.56 mmol, 81%). 1H NMR (400 MHz, CDCl3): d 5.78 (t, 1 H, J=8.0, NHε), 5.33 (m, 2 H, CH9,10), 5.27 (d, 1 H, J=7.8, NHα), 4.27 (m, 1 H, CHα), 3.24 (q, 2 H, J=8.0, CH2ε), 2.26 (t, 2 H, J=6.8, CH22), 1.98 (m, 4 H, CH28,11), 1.85 (m, 1 H, CHβ), 1.70 (m, 1 H, CHβ), 1.60 (m, 2 H, CH23), 1.55 (m, 2 H, CH2δ), 1.43 (s, 9 H, C(CH3)3), 1.40 (m, 2 H, CH2γ), 1.27 (m, 20 H, 10 CH2Tail), 0.87 (m, 3 H, J=6.6, CH318). HRMS (+ES): 533.40327 calculated for C29H54O5N2Na found 533.39110.
To a solution of N-α-(tert-butyloxycarbonyl)-N-ε-oleoyl-L-Lysine (1.80 g, 3.52 mmol) in THF (80 mL) were added successively N-hydroxysuccinimide (0.41 g, 3.56 mmol, 1.01 eq.) and DCC (0.73 g, 3.54 mmol, 1.01 eq.). The reaction was stirred for 16 h at RT. The precipitate was filtered and washed with EtOAc (30 mL). The filtrate was concentrated and redissolved in EtOAc and filtered again. The residue was dissolved in CHCl3 and precipitated with Et2O to give N-α-oleate-N-ε-(tert-butyloxycarbonyl)-L-Lysinyl succinimidate as a white solid. Yield: 1.98 g (93%). 1H NMR (400 MHz, CDCl3): d 5.80 (t, 1 H, J=8.0, NHε), 5.32 (m, 2 H, CH9,10), 5.12 (d, 1 H, J=7.8, NHα), 4.66 (m, 1 H, CHα), 3.24 (q, 2 H, J=8.0, CH2ε), 2.82 (s, 4 H, 2 CH2Su), 2.14 (t, 2 H, J=6.8, CH22), 1.98 (m, 4 H, CH28,11), 1.90 (m, 2 H, 2 CHβ), 1.60 (m, 2 H, CH23), 1.55 (m, 2 H, CH2δ), 1.44 (s, 9 H, C(CH3)3), 1.39 (m, 2 H, CH2γ), 1.25 (m, 20 H, 10 CH2Tail), 0.86 (m, 3 H, J=6.6, CH318).
To a solution of N4,N9-bis-(tert-butyloxycarbonyl)-1,12-diamino-4,9-diazadodecane (414 mg, 0.659 mmol) in THF (60 mL) and K2CO3 (200 mg, 1.2 mmol, 2.2 eq.) in water (7 mL) was added a solution of N-α-(tert-butyloxycarbonyl)-N-ε-oleoyl-L-lysinyl succinimidate (800 mg, 1.32 mmol, 2.0 eq.) in THF (35 mL). The reaction was stirred overnight at RT. Most of the THF was evaporated and water (30 mL) was added. The aqueous layer was extracted with CHCl3 (2×30 mL). The organic layer was washed with water, 0.1 M HCl, water and brine (30 mL each), dried (Na2SO4), filtered, evaporated and purified on SiO2 (CHCl3/MeOH: 95/5, Rf=0.27) to give an oil. Yield: 740 mg (0.533 mmol, 81%). 1H NMR (400 MHz, CDCl3): δ 7.20 (bs, 2 H, NHC1′), 5.72 (bs, 2 H, NHε), 5.33 (m, 4 H, 2 CH9,10), 5.25 (bs, 2 H, 2 NHα), 4.08 (m, 2 H, 2 CHα), 3.24 (m, 12 H, 2 CH21′, 2 CH24′ and 2 CH2ε), 3.12 (m, 4 H, 2 CH23′), 2.13 (t, 4 H, J=6.8, 2 CH22), 1.98 (m, 8 H, 2 CH28,11), 1.80 (m, 2 H, 2 CHβ), 1.60 (m, 10 H, 2 CH22′, 2 CHβ and 2 CH23), 1.50 (m, 4 H, 2 CH2δ), 1.47 (m, 4 H, 2 CH25′), 1.45 (s, 18 H, 2 C(CH3)3), 1.42 (s, 18 H, 2 C(CH3)3), 1.37 (m, 4 H, 2 CH2γ), 1.25 (m, 40 H, 2×10 CH2Tail), 0.86 (m, 6 H, J=6.6, 2 CH318).
To a solution of RG 00/296 (750 mg, 0.540 mmol) in MeOH (10 mL) was added concentrated HCl (10 mL). The reaction was stirred at RT for 1 h and then evaporated. The residue was redissolved in water (60 mL) and filtered. Water was evaporated and the residue dissolved in a small amount of MeOH and precipitated with Et2O to give a yellow solid. Yield: 533 mg (0.470 mmol, 90%). 1H NMR (400 MHz, d6-DMSO): δ 9.02 (m, 4 H, 2 NH2+), 8.83 (t, 2 H, J=6.0, 2 NHC1′), 8.30 (d, 6 H, J=4.0, 2 NαH3+), 8.83 (t, 2 H, J=6.0, 2 NεH), 5.30 (m, 4 H, 2 CH9,10), 3.70 (q, 2 H, J=7, 2 CHα), 3.22 (m, 2 H, 2 CH1′), 3.13 (m, 2 H, 2 CH1′), 2.97 (m, 4 H, 2 CH2ε), 2.71(m, 8 H, 2 CH23′ and 2 CH24′), 2.10 (t, 4 H, J=7.3, 2 CH22), 1.95 (q, 8 H, J=6.0, 2 CH28,11), 1.82 (h, 4 H, J=7.0, 2 CH22′), 1.68 (m, 8 H, 2 CH2β and 2 CH25′), 1.43 (qu, 4 H, J=6.2, 2 CH23), 1.35 (m, 4 H, 2 CH2δ), 1.25 (m, 44 H, 2×10 CH2O1 and 2 CH2γ), 0.82 (t, 6 H, 2 CH318). MS (+ES): 999.8 [M+Na].
To a solution of activated aminoacid RG00/366 (610 g, 1.0 mmol) in THF (45 mL) was added bis-N-aminopropyl-piperazine (0.081 mL, 0.5 mmol, 0.5 eq.) and then potassium carbonate (0.15 g, 1.1 mmol, 2.2 eq.) in water (10 mL) and the reaction was stirred at RT for 20 h. Most of the THF was removed under vacuum, CHCl3 was added and the organic layer was extracted, washed with water (20 mL), dried (Na2SO4), filtered and evaporated. The residue was purified by column chromatography on silica (CHCl3/MeOH: 8.5/1.5, Rf=0.3) to give a white solid. Yield: 490 mg (0.413 mmol, 83%). 1H NMR (400 MHz, CDCl3): δ 7.68 (m, 2 H, 2 NHC1), 6.46 (m, 2 H, 2 NαH), 5.32 (m, 4 H, 2 CH9,10), 4.86 (m, 2 H, 2 NεHboc), 4.33 (q, 2 H, J=, 2 CHα), 3.38 (m, 2 H, CH1′), 3.28 (m, 2 H, CH1′), 3.05 (m, 4 H, 2 CH2ε), 2.47 (m, 12 H, 2 CH23′ and 4 CH22′), 2.18 (t, 4 H, J=, 2 CH22), 1.99 (m, 8 H) 2 CH28,10), 1.82-1.54 (m, 12 H, 2 CH22′, 2 CH23 and 2 CH2β), 1.48 (m, 4 H, 2 CH2γ), 1.42 (s, 18 H, 2 (CH3)3), 1.21 (m, 24 H, 10 CH2O1 and 2 CH2γ), 0.87 (t, 6 H, J=6.4, 2 CH318). 13C NMR (400 MHz, CD3OD): δ 175.2, 173.4, 157.5, 129.9, 129.8, 78.8, 56.0, 53.8, 52.9, 41.3, 40.1, 37.7, 35.9, 32.1, 31.9, 29.9, 29.6, 29.5, 29.4, 29.3, 27.9, 27.2, 26.2, 26.0, 23.3, 22.8, 13.5.
To a solution of protected RG00/518 (490 mg, 0.413 mmol) in MeOH (10 mL) was added concentrated HCl (10 mL). The reaction was stirred for 1 h and the solvent was then evaporated. The residue was redissolved in water (40 mL), filtered and evaporated. In this case it was impossible to precipitate the compound using MeOH/Et2O. A white solid was collected. Yield: 381 mg (0.337 mmol, 81%). HRMS (+ES): 985.8879 calculated for C58H113N8O4, found 985.8890. Note: a similar procedure using TFA and neutralisation with K2CO3 was used to isolate the free amine in a quantiative yield. 1H NMR (400 MHz, d6-DMSO): δ 7.78 (2 d, 4 H, J=8.0, 4 NHCO), 5.29 (m, 4 H, 2 CH9,10), 4.12 (q, 2 H, J=6.2, 2 CHα), 3.04 (m, 4 H, 2 CH21′), 2.47 (m, 8 H, 4 CH24), 2.29 (m, 4 H, 2 NH2), 2.19 (t, 4 H, J=6.2, 2 CH23′), 2.05 (m, 4 H, 2 CH22), 1.95 (m, 8 H, 2 CH28,10), 1.35-1.69 (m, 12 H, 2 CH22′, 2CH23 and 2 CH2β), 1.21 (m, 26 H, 10 CH2O1 and CH2δ and CH2γ), 0.82 (t, 6 H, J=6.4, 2 CH318).
To a solution of bis aminocompound (150 mg, 0.152 mmol) in THF (40 mL) was added successively a solution of K2CO3 (42 mg, mmol, 2.1 eq.) in water (2 mL) and N,N-bis-(tertbutoxycarbonyl)-L-lysinyl succinimidate (140 mg, 0.304 mmol, 2.0 eq.) in THF (10 mL). The reaction was then stirred for 16 h at RT. Most of THF was evaporated and the residue redissolved in CHCl3. Water (10 mL) was added and the organic layer extracted, washed with water (2×10 mL) and brine (20 mL). After drying (Na2SO4), filtration and evaporation, the residue is purified on SiO2 (eluent: CHCl3/MeOH/NH4OH: 87/12/1, Rf=0.28). Et2O is then added and the resulting white solid filtered off. Yield: 0.124 g (0.076 mmol, 50%). 1H NMR (400 MHz, d6-DMSO): δ 7.75 (m, 4 H, 2 NHα1 and 2 NHC1), 7.68 (t, 2 H, J=, 2 NHε1), 6.69 (t, 2 H, J=, 2 NHε2 ), 6.63 (d, 2 H, J=, 2 NHα2), 5.29 (m, 4 H, 2 CH9,10), 4.10 (q, 2 H, J=, 2 CHα1), 3.78 (q, 2 H, J=2 CHα2), 3.00 (m, 6 H, 2 CH2ε1 and 2 CH1′), 2.95 (m, 2 H, 2 CH1′), 2.84 (m, 4 H, 2 CH2ε2), 2.29 (m, 8 H, 4 CH24′), 2.19 (m, 4 H, 2 CH23′), 2.06 (t, 4 H, J=, 2 CH22), 1.95 (m, 8 H, 2 CH28,10), 1.55-1.4 (m, 16 H), 1.32 (s, 36 H, 4 C(CH3)3), 1.20 (m, 48 H, 0.82 (t, 6 H, J=6.4, 2 CH318).
To a solution of RG00/794 (124 mg, 0.0755 mmol) in MeOH (5 mL) was added concentrated HCl (5 mL). The reaction was stirred at RT for 1 h and the solvent were then removed under vacuum. The residue was dissolved in water, filtered and evaporated. The compound was dissolved in a minimum amount of MeOH and precipitated with Et2O. The resulting solid was filtered and collected. Yield: 0.102 g (0.070 mmol, 93%). 1H NMR (400 MHz, d6-DMSO): δ 8.66 (d, 2 H, J=7.8, 2 NHε1), 8.28 (m, 6 H, 2 NαH3+), 8.09 (m, 2 H, 2 NHC1), 8.05 (m, 6 H, 2 NεH3+), 7.98 (d, 2 H, J=7.0, 2 NαH), 5.29 (m, 4 H, 2 CH9,10), 4.09 (m, 2 H, 2 CHα1), 3.72 (m, 2 H, 2 CHα2), 3.65 (m, 2 H, 2 NH+), 3.10 (m, 12 H, 2 CH2ε1, 2 CH23′ and 2 CH21′), 2.74 (m, 8 H, 2 CH2ε2), 2.11 (t, 4 H, J=7.2, 2 CH22), 1.95 (m, 8 H, 2 CH28,10), 1.82 (m, 2 H, 2 CH2δ1), 1.70 (m, 2 H, 2 CH2β2), 1.57 (m, 6 H, 2 CH2δ2 and 2 CHβ1), 1.50-1.15 (m, 66 H), 0.84 (t, 6 H, J=6.4, 2 CH318). MS (+ES): 1264.9 [M+Na].
To a solution of 1,6-diaminohexane (72 mg, 0.62 mmol) in THF (60 mL) and K2CO3 (180 mg, 1.30 mmol, 2.1 eq.) in water (10 mL) was added a solution of N-α-oleoyl-N-ε-(tert-butyloxycarbonyl)-L-lysinyl succinimidate (750 mg, 1.23 mmol, 2 eq.). The reaction was stirred overnight at RT. Most of the THF was evaporated and water (30 mL) was added. The aqueous layer was extracted with CHCl3 (2×50 mL). The organic layer was washed with water, 0.1 M HCl, water and brine (20 mL each), dried (Na2SO4), filtered, evaporated and purified by column chromatography on SiO2 (CHCl3/MeOH: 9/1, Rf=0.33) to give an oil. Yield: 650 mg (0.59 mmol, 95%). 1H NMR (400 MHz, d6-DMSO): δ 7.73 (m, 4 H, 2 NαH and 2 N1′H), 6.68 (t, 2 H, J=5.0, 2 NεH), 5.28 (m, 4 H, 2 CH9,10), 4.12 (m, 2 H, 2 CHα), 2.99 (q, 4 H, J=6.4, 2 CH1′), 2.83 (q, 4 H, J=6.6, 2 CH2ε), 2.07 (dt, 4 H, J=3.2, 7.0, 2 CH22), 1.95 (m, 8 H, 2 CH28,11), 1.52 (m, 2 H, 2 CHβ), 1.42 (m, 6 H, 2 CH23 and 2 CHβ), 1.32 (s, 18 H, 2 C(CH3)3), 1.31-1.15 (m, 56 H, 2×10 CH2Tail, 2 CH2δ, 2 CH2γ, 2 CH22′ and 2 CH23′), 0.82 (t, 6 H, J=6.8, 2 CH318).
To a solution of protected compound (640 mg, 0.581 mmol) in CH2Cl2 (10 mL) was added TFA (10 mL). The reaction was stirred at RT for 1 h and then evaporated (using several Et2O (10 mL) to coevaporate). The oily residue was then dissolved in CH2Cl2, washed with 10% aqueous K2CO3 (10 mL), water and brine. The organic phase was dried (Na2SO4), filtered and evaporated to give a pale brown solid which was triturated with Et2O, filtered and dried to give a white solid. Yield: 460 mg (0.510 mmol, 88%). The deprotection can be carried out using concentrated HCl in methanol giving the hydrochloric salt named GSN 14. 1H NMR (400 MHz, d6-DMSO): δ 7.80 (m, 4 H, 2 NαH and 2 N1′H), 5.28 (m, 4 H, 2 CH9,10), 4.16 (m, 2 H, 2 CHα), 3.20 (bs, 4 H, 2 NH2), 2.99 (q, 4 H, J=6.4, 2 CH1′), 2.53 (m, 4 H, 2 CH2ε), 2.10 (dt, 4 H, J=3.2, 7.0, 2 CH22), 1.91 (m, 8 H, 2 CH28,11), 1.52 (m, 2 H, 2 CHβ), 1.48 (m, 2 H, 2 CHβ), 1.42 (m, 4 H, 2 CH23), 1.31-1.15 (m, 56 H, 2×10 CH2Tail, 2 CH2δ, 2 CH2γ, 2 CH22′ and 2 CH23′), 0.81 (t, 6 H, J=6.8, 2 CH318).
To a 1/9 mixture of water and THF (20 mL) containing RG 00/873 (100 mg, 0.111 mmol) and potassium carbonate (32 mg, 0.232 mmol, 2.1 eq.) was added N,N-bis-tert-butyloxycarbonyl)-L-lysinyl succinimidate (103 mg, 0.232 mmol, 2.1 eq.). The reaction was stirred for 20 h at RT. Most of THF was removed and the residue diluted with water (10 mL) and CHCl3 (40 mL). The organic layer was decanted and washed successively with water (10 mL), 0.1 M HCl (20 mL), water (10 mL) and brine (25 mL). The organic layer was dried over sodium sulphate, filtered and evaporated. The resulting oil was crystallised from Et2O. The white solid was collected. Yield: 164 mg (0.105 mmol, 95%).
To a solution of RG 00/874 (160 mg, 0.103 mmol) in methanol (5 mL) is added concentrated HCl (5 mL). The reaction is stirred for 1 h and then evaporated to dryness. The residue is then dissolved in water (30 mL), filtered on sintered frit funnel (No 3), evaporated to dryness using EtOH to coevaporate. The residue is dissolved in a small amount of methanol and precipitated with Et2O to give the desired compound as a pale brown solid. Yield: 124 mg (0.951 mmol, 95%). 1H NMR (400 MHz, d6-DMSO): δ 8.59 (t, 2 H, J=5.0, 2 N1′H), 8.22 (m, 6 H, 2 Nα2H3+), 7.96 (m, 6 H, 2 Nε2H3+), 7.89 (d, 2 H, J=8.0, 2 Nα1H), 7.89 (t, 2 H, J=5.8, 2 Nε1H), 5.29 (m, 4 H, 2 CH9,10), 4.14 (dt, 2 H, J=5.4, 8.0, 2 CHα1), 3.70 (m, 2 H, 2 CHα2), 3.05 (m, 4 H, 2 CH21′), 2.98 (q, 4 H, J=5.8, 2 CH2ε1), 2.72 (m, 4 H, 2 CH2ε2), 2.09 (t, 4 H, J=7.0, 2 CH22), 1.94 (m, 8 H, 2 CH28,11), 1.69 (m, 2 H, 2 CH2β2), 1.55 (m, 6 H, 2 CH2δ2 and 2 CHβ1), 1.48-1.15 (m, 56 H), 0.81 (t, 6 H, J=6.6, 2 CH318).
To a solution of RG00/794 (110 mg, 0.052 mmol) in MeOH (7 mL) was added concentrated HCl (7 mL). The reaction was stirred at RT for 1 h and the solvent were then removed under vaccuum using EtOH to coevaporate. The residue was dissolved in water, filtered and evaporated. The compound was dissolved in a minimum amount of MeOH and precipitated with Et2O. The resulting solid was filtered and collected as a white powder. Yield: 88 mg (0.049 mmol, 94%). 1H NMR (400 MHz, d6-DMSO): δ 8.69 (d, 2 H, J=7.8, 2 NHα2), 8.30 (m, 2 H, 2 NHα1), 8.12 (m, 2 H, 2 NHC1), 7.95 (m, 12 H, 2 NHε3 , 2 NHε3 and NHα3), 5.29 (m, 4 H, 2 CH9,10), 4.20 (m, 2 H, 2 CHα1), 4.08 (m, 2 H, 2 CHα3), 3.84 (m, 2 H, 2 CHα2), 3.65 (m, 2 H, 2 NH+), 3.10 (m, 12 H, 2 CH23′ and 2 CH24′), 3.05 (m, 2 H, 2 CH1′), 2.95 (m, 2 H, 2 CH1′), 2.74 (m, 8 H, 2 CH2ε1 and 2 CH2ε2), 2.11 (t, 4 H, J=7.2, 2 CH22), 1.95 (m, 8 H, 2 CH28,10), 1.80-1.12 (m, 82 H), 0.84 (t, 6 H, J=6.4, 2 CH318). MS (+ES): m/z [M+H]2+ 750.1.
To a solution of bis aminocompound (140 mg, 0.142 mmol) in THF (48 mL) was added successively a solution of K2CO3 (40 mg, mmol, 2.1 eq.) in water (10 mL) and (Boc)4-KKKOSu (256 mg, 0.284 mmol, 2.0 eq.) in THF (10 mL). The reaction was then stirred for 16 h at RT. Most of THF was evaporated and the residue redissolved in CHCl3. Water (10 mL) was added and the organic layer was extracted, washed with 5% K2CO3, water (10 mL) and brine (20 mL). After drying (Na2SO4), filtration and evaporation, the residue was purified on SiO2 (eluent: CHCl3/MeOH/NEt3: 85/15/1, Rf=0.32). Et2O is then added and the resulting white solid filtered off. Yield: 0.310 g (0.121 mmol, 85%). 1H NMR (400 MHz, d6-DMSO): δ 8.00 (m, 2 H, 2 NHα), 7.85 (m, 2 H, 2 NHα), 7.75 (m, 4 H, 2 NHα and 2 NHC1), 6.85 (m, 2 H, 2 NαH), 6.68 (m, 6 H, 2×3 NεH), 5.29 (m, 4 H, 2 CH9,10), 4.18 (m, 2 H, 2 CHα), 4.09 (m, 4 H, 2×2 CHα), 3.82 (m, 4 H, 2×2 CHα), 3.00 (m, 6 H, 2 CH2ε and 2 CH1′), 2.75 (m, 2 H, 2 CH1′), 2.84 (m, 12 H, 2×3 CH2ε), 2.47 (m, 8 H, 2×2 CH24′), 2.29 (m, 4 H, 2 CH23′), 2.09 (t, 4 H, J=9.0, 2 CH22), 1.95 (m, 8 H, 2 CH28,10), 1.65-1.15 (m, 168 H), 0.82 (t, 6 H, J=6.4, 2 CH318).
To a solution of RG00/797 (110 mg, 0.052 mmol) in MeOH (7 mL) was added concentrated HCl (7 mL). The reaction was stirred at RT for 1 h and the solvent were then removed under vaccuum using EtOH to coevaporate. The residue was dissolved in water (40 mL), filtered and evaporated. The compound was dissolved in a minimum amount of MeOH and precipitated with Et2O. The resulting solid was filtered and collected as a pale brown powder. Yield: 88 mg (0.049 mmol, 94%). 1H NMR (400 MHz d6-DMSO): δ 8.80 (d, 2 H, J=7.8, 2 NHα), 8.30 (m, 6 H, 2×3 NHα), 8.03 (m, 14 H, 2 NHC1 and 2×3 NεH3+), 5.30 (m, 4 H, 2 CH9,10), 4.28 (m, 2 H, 2 CHα), 4.18 (m, 2 H, 2 CHα), 4.08 (m, 2 H, 2 CHα), 3.85 (m, 2 H, 2 CHα), 3.65 (m, 2 H, 2 NH+), 3.10 (m, 16 H, 2 CH2ε1, 2 CH23′ and 2×2 CH24′), 3.02 (m, 2 H, 2 CH1′), 2.95 (m, 2 H, 2 CH1′), 2.74 (m, 8 H, 2×3 CH2ε), 2.10 (t, 4 H, J=7.2, 2 CH22), 1.95 (m, 8 H, 2 CH28,10), 1.71 (m, 4 H, 2 CH2β), 1.60-1.17 (m, 108 H), 0.84 (t, 6 H, J=6.4, 2 CH318). MS (+ES): m/z [M+H]2+ 750.1.
To a solution of bis aminocompound (76 mg, 0.077 mmol) in THF (40 mL) was added successively a solution of K2CO3 (22 mg, 0.159 mmol, 2.06 eq.) in water (2 mL) and Boc3(K-ε-K)-OSu (105 mg, 0.156 mmol, 2.0 eq.) in THF (8 mL). The reaction was then stirred for 16 h at RT. Most of THF was evaporated and the residue redissolved in CHCl3. Water (10 mL) was added and the organic layer extracted, washed with water (2×10 mL) and brine (20 mL). After drying (Na2SO4), filtration and evaporation, the residue was purified on SiO2 (eluent: CHCl3/MeOH/NEt3: 91/8/1, Rf=0.30). Et2O is then added and the resulting white solid filtered off. Yield: 0.124 g (0.059 mmol, 77%). 1H NMR (400 MHz, d6-DMSO): δ 7.79 (m, 4 H, 2 NHα and 2 NHε), 7.67 (m, 4 H 2 NHC1 and 2 NHε), 6.69 (m, 2 H, 2 NHε), 8.28 (m, 8 H, 2×2 NHα), 5.28 (m, 4 H, 2 CH9,10), 4.10 (m, 2 H, 2 CHα1), 3.78 (m, 4 H, 2×2 CHα), 3.00 (m, 6 H, 2 CHε and 2 CH1′), 2.98 (m, 2 H, 2 CH1′), 3.13 (m, 12 H, 2×2 CH2ε), 2.47 (m, 4 H, 2 CH23′), 2.25 (m, 8 H, 2×2 CH24′), 2.08 (t, 4 H, J=7.2, 2 CH22), 1.95 (m, 8 H, 2 CH28,10), 1.80-1.14 (m, 138 H), 0.82 (t, 6 H, J=6.4, 2 CH318).
To a solution of RG00/823 (124 mg, 0.059 mmol) in MeOH (10 mL) was added concentrated HCl (6 mL). The reaction was stirred at RT for 1 h and the solvent were then removed under vaccuum using EtOH to coevaporate. The residue was dissolved in water (40 mL), filtered and evaporated. The compound was dissolved in a minimum amount of MeOH and precipitated with Et2O. The resulting solid was filtered and collected as a pale pink powder. Yield: 101 mg (0.056 mmol, 96%). 1H NMR (400 MHz, d6-DMSO): δ 8.73 (t, 2 H, J=7.8, 2 NHε), 8.67 (m, 2 H 2 NHε1), 8.28 (m, 8 H, 4 NH2α), 8.15 (m, 6 H, 2 NHC1, 2 NHε2 and 2 NHε3), 7.99 (m, 2 H, 2 NHα1), 5.29 (m, 4 H, 2 CH9,10), 4.09 (m, 2 H, 2 CHα1), 3.72 (m, 4 H, 2 CHα3 and 2 CHα2), 3.65 (m, 2H, 2 NH+), 3.08 (m, 10 H, 2 CH2ε1, 2 CH2ε2, 2 CH23′ and 2 CH24′), 2.74 (m, 2 H, 2 CH21′), 2.11 (t, 4 H, J=7.2, 2CH22), 1.95 (m, 8 H 2 CH28,10), 1.80-1.14 (m, 82 H), 0.83 (t, 6 H, J=6.4, 2 CH318).
Examples 29 to 40 describe alternative routes for the synthesis of GSC170 and derivatives thereof.
To a solution of N-ε-(t-butoxycarbonyl)-L-lysine (446 mg, 1.89 mmol) in THF (53 mL) were added successively a solution of K2CO3 (288 mg, 2.08 mmol) in water (8 mL) and oleoyl succinimidate (754 mg, 1.99 mmol). The reaction was stirred at RT for 15 h and the most of THF was evaporated. Water and CHCl3 (25 mL each) were added and the mixture was acidified with 1 M HCl to pH: 2. The organic layer was separated, and the aqueous layer was extracted with CHCl3 (2×20 ml). The organic layer was washed with brine (15 mL), dried (Na2SO4), filtrated and evaporated to give a white solid. Yield: 940 mg (1.84 mmol, 97%). Rf(SiO2): 0.28 (CHCl3—MeOH 92:8).
RMN-1H (500 MHz, CDCl3), δ (ppm): 5.33 (m, 2 H, CH9,10), 4.15 (m, 1 H, CHα), 3.05 (m, 2 H, CH2ε), 2.15 (m, 2 H, J=6.0 Hz, CH22), 1.98 (m, 4 H, CH28,11), 1.80-1.48 (m, 4 H, CH23, CH2β), 1.46-1.20 (m, 33 H, CH2γ, CH2δ, C(CH3)3, 10 CH2ol), 0.84 (t, 3 H, J=6.8 Hz, CH318). RMN-13C (125 MHz, CDCl3), δ (ppm): 178.6, 174.4, 156.2, 129.9, 129.7, 78.9, 54.5, 40.0, 36.4, 31.9, 31.3, 29.8, 29.5, 29.4, 29.3, 28.5, 27.3, 27.2, 25.9, 25.0, 22.9, 22.7, 14.1. HRMS (+ESI): C29H54N2O5Na [M+Na], calcd.: m/z=533.3930, found: m/z=533.3903
To a solution of aminoacid RG00/781 (410 mg, 0.80 mmol) in CH2Cl2 (4 ml) were added benzotriazole-1-yl-oxi-tris-pyrrolidino-phosphonium hexafluorphosphate (418 mg, 0.80 mmol), DIEA (280 μl, 1.61 mmol) and 1,4-Bis(3-aminopropyl)piperazine (64 μl, 0.31 mmol). The mixture was stirred at RT for 16 h. The solvent was removed, CHCl3 (40 mL) was added and the organic layer was washed with 5% NaHCO3 (3×12 mL) and brine (30 mL), dried (Na2SO4), filtrated and evaporated. The residue was purified by column chromatography on reverse phase (C-18) to give a syrup. Yield: 295 mg (0.25 mmol, 81%), Rf (SiO2): 0.46 (CHCl3—MeOH 85:15), Rf (C-18): 0.15 (MeOH). RMN-1H (500 MHz, CDCl3), δ (ppm): 7.49 (m, 2 H, NHCO), 6.52 (m, 2 H, NαHCO), 5.32 (m, 4 H, 2 CH9,10), 4.81 (m, 2 H, NHBOC), 4.29 (m, 2 H, CHα), 3.30 (m, 4 H, 2 CH21′), 3.05 (m, 4 H, J=6.0 Hz, 2 CH2ε), 2.76 (s broad, 8 H, 4 CH24′), 2.61 (s broad, H, 2 CH23′), 2.19 (t, 4 H, J=7.4 Hz, 2 CH22), 1.97 (m, 8 H, 2 CH28,11), 1.72 (m, 6 H, 2 CH22′, 2 CHaβ), 1.68 (m, 6 H, 2 CH23, 2 CHbβ), 1.52-1.15 (m, 66 H, 2 CH2γ, 2 CH2δ, 2×10 CH2o1, 2 C(CH3)3), 0.85 (t, 6 H, J=6.7 Hz, 2 CH318). RMN-13C (125 MHz, CDCl3), δ (ppm): 173.6, 172.3, 156.2, 130.0, 129.7, 79.0, 55.7, 53.1, 52.0, 40.0, 38.0, 36.5, 32.0, 31.9, 29.7, 29.5, 29.3, 29.2, 28.4, 27.2, 25.8, 25.7, 24.8, 22.7, 22.6, 14.1. HRMS (+ESI): C68H129N8O8 [M+H], calcd.: m/z=1185.9928, found: m/z=1186.0006.
To a solution of RG00/518 (710 mg, 0.60 mmol) in EtOAc (50 mL) was added 4.9 N HCl-EtOAc (50 ml). The reaction was stirred at RT for 2 h. The precipitate was filtrated and washed with ether to give a white solid. Yield 542 mg (0.48 mmol, 80%), Rf (SiO2): 0.63 (MeOH—NH4OH 80:20).
RMN-1H (500 MHz, DMSO-d6), δ (ppm): 8.12 (m, 2 H, NHCO), 8.05-7.87 (m, 8 H, NαHCO, 2 NεH3+), 5.31 (m, 4 H, 2 CH9,10), 4.10 (m, 2 H, J=5.0 Hz, 8.4 Hz, 2 CHα), 3.70 (s broad, 4 H, 4 CHaxial4′), 3.44 (s broad, 4 H, 4 CHecuat.4′), 3.10 (m, 8 H, 2 CH21′, 2 CH23′), 2.72 (m, 4 H, J=6.2 Hz, 2 CH2ε), 2.12 (m, 4 H, J=6.8 Hz, 2 CH22), 1.95 (m, 8 H, 2 CH28,11), 1.84 (m, 4 H, J=6.3 Hz, 2 CH22′), 1.65-1.40 (m, 12 H, 2 CH2β, 2 CH2δ, 2 CH23), 1.35-1.15 (m, 44 H, 2 CH2γ, 2×10 CH2o1), 0.83 (t, 6 H, J=6.7 Hz, 2 CH318).
RMN-13C (125 MHz DMSO-d6), δ (ppm): 172.5, 172.1, 129.7, 55.0, 53.7, 52.7, 48.1, 40.1, 39.9, 39.8, 39.6, 39.4, 39.3, 39.1, 38.5, 31.4, 29.3, 29.2, 28.9, 28.8, 28.7, 28.7, 26.7, 25.3, 22.2, 14.1. HRMS (+ESI): C58H113N8O4 [M+H], calcd.: m/z=985.8879, found: m/z=985.8805.
To a solution of N-δ-(t-butoxycarbonyl)-L-ornitine (440 mg, 1.89 mmol) in THF (53 mL) were added successively a solution of K2CO3 (288 mg, 2.08 mmol) in water (8 mL) and oleoyl succinimidate (755 mg, 1.99 mmol). The reaction was stirred at RT for 15 h and the most of THF was evaporated. Water and CHCl3 (25 mL each) were added and the mixture was acidified with 1 M HCl to pH: 2. The organic layer was separated, and the aqueous layer was extracted with CHCl3 (2×20 ml). The organic layer was washed with brine (15 mL), dried (Na2SO4), filtrated and evaporated to give a white solid. Yield: 898 mg (1.81 mmol, 96%). Rf(SiO2): 0.16 (CHCl3—MeOH 92:8).
RMN-1H (500 MHz, CDCl3), δ (ppm): 7.03 (m, 1 H, NHCO), 5.32 (m, 2 H, CH9,10), 4.40 (m, 1 H, CHα), 3.11 (m, 2 H, CH2δ), 2.19 (t, 2 H, J=7.4 Hz, CH22), 1.99 (m, 4 H, CH28,11), 1.85 (m, 1 H CHaβ), 1.70-1.20 (m, 34 H, CHbβ, CH23, CH2γ, C(CH3)3, 10 CH2o1), 0.87 (t, 6 H, J=6.7 Hz, CH318). RMN-13C (125 MHz, CDCl3), δ (ppm): 176.6, 174.4, 156.5, 130.0, 129.7, 79.4, 53.0, 39.8, 36.4, 33.9, 31.9, 29.8, 29.5, 29.4, 29.3, 29.1, 28.9, 28.4, 27.2, 26.4, 25.7, 25.6, 24.8, 22.7, 14.1. HRMS (+ESI): C28H52N2O2 [M+H], calcd.: m/z=519.3774, found: m/z=519.3767.
To a solution of aminoacid 4 (548 mg, 1.10 mmol) in CH2Cl2 (5.5 ml) were added benzotriazole-1-yl-oxi-tris-pyrrolidino-phosphonium hexafluorphosphate (574 mg, 1.10 mmol), DIEA (385 μl, 2.20 mmol) and 1,4-Bis(3-aminopropyl)piperazine (84 μl, 0.41 mmol). The mixture was stirred at RT for 18 h. The solvent was removed, CHCl3 (40 mL) was added and the organic layer was washed with 5% NaHCO3 (3×10 mL) and brine (25 mL), dried (Na2SO4), filtrated and evaporated. The residue was purified by column chromatography on reverse phase (C-18) to give a syrup. Yield: 360 mg (0.31 mmol, 76%), Rf (SiO2): 0.46 (CHCl3—MeOH 85:15), Rf (C-18): 0.22 (MeOH).
RMN-1H (500 MHz, CDCl3), δ (ppm): 7.60 (m, 2 H, 2 NHCO), 6.50 (m, 2 H, J=6.5 Hz, 2 NαHCO), 5.36 (m, 4 H, 2 CH9,10), 4.85 (m, 2 H, 2 NHBOC), 4.48 (m, J=5.7 Hz, 2 H, 2 CHα), 3.38 (m, J=13.2 Hz, J=6.6 Hz, 2 H, 2 CHa1′), 3.28 (m, 4 H, 2 CHb1′, 2 CHaδ), 3.11 (m, 2 H, J=13.2 Hz, J=6.3 Hz, 2 CHbδ), 2.48 (m, 12 H, 2 CH23′, 4 CH24′), 2.23 (t, 4 H, J=7.8 Hz, 2 CH22), 2.03 (m, 8 H, 2 CH28,11), 1.87-1.40 (m, 34 H, 2 CH2β, 2 CH22′, 2 CH23, 2 CH2γ, 2 C(CH3)3), 1.30-1.22 (m, 40 H, 2×10 CH2o1), 0.91 (t, 6 H, J=6.4 Hz, 2 CH318).
RMN-13C (125 MHz, CDCl3), δ (ppm): 173.2, 171.5, 156.4, 130.0, 129.8, 79.2, 57.0, 53.3, 52.2, 39.7, 39.2, 36.7, 31.9, 30.6, 29.8, 29.5, 29.3, 29.2, 28.5, 27.2, 26.5, 25.7, 25.2, 22.7, 14.1. HRMS (+ESI): C58H113N8O4 [M+H], calcd.: m/z 1157.9615, found: m/z=1157.9531.
To a solution of compound 5 (138 mg, 0.12 mmol) in EtOAc (10 mL) was added 4.9 N HCl-EtOAc (10 ml). The reaction was stirred at RT for 2 h. The precipitate, was filtrated and washed with ether to give a white solid. Yield 116 mg (0.10 mmol, 83%), Rf (SiO2): 0.61 (MeOH—NH4OH 80:20).
RMN-1H (500 MHz, CDCl3), δ (ppm): 8.24 (s broad, 2 H, 2 NHCO), 8.04 (d, 2 H, J=7.8 Hz, 2 NHαCO), 7.94 (s broad, 6 H, 2 NH3+), 5.33 (m, 4 H, 2 CH9,10), 4.20 (m, J=5.5 Hz, 2 H, 2 CHα), 3.76 (m 4 H, 4 CHaxial4′), 3.48 (m, 4 H, 4 CHecuat4′), 3.12 (m, 8 H, 2 CH21′, 2 CH23′), 2.76 (m, 4 H, J=5.7 Hz, 2 CH2δ), 2.14 (t, 4 H, J=7.4 Hz, 2 CH22), 1.98 (m, 8 H, 2 CH28,11), 1.84 (m, 4 H, 2 CH22′), 1.70 (m, 2 H, 2 CHaβ), 1.64-1.40 (m, 10 H, 2 CHbβ, 2 CH23, 2 CH2γ), 1.24 (m, 40 H, 2×10 CH2o1), 0.86 (t, 6 H, J=6.7 Hz, 2 CH318).
RMN-13C (125 MHz, CDCl3), δ (ppm): 172.5, 171.8, 129.7, 79.3, 53.8, 51.9, 48.1, 38.3, 35.3, 31.4, 29.3, 29.2, 28.9, 28.8, 28.7, 26.7, 25.3, 22.2, 14.1. HRMS (+ESI): C58H113N8O4 [M+H], calcd.: m/z 957.8572, found: m/z=957.8575.
To a solution of N-γ-(t-butoxycarbonyl)-L-diaminobutyric acid (203 mg, 0.93 mmol) in THF (27 mL) were added successively a solution of K2CO3 (141 mg, 1.02 mmol) in water (4 mL) and oleoyl succinimidate (371 mg, 0.98 mmol). The reaction was stirred at RT for 16 h and the most of THF was evaporated. Water and CHCl3 (10 mL each) were added and the mixture was acidified with 1 M HCl to pH: 2. The organic layer was separated, and the aqueous layer was extracted with CHCl3 (2×10 ml). The organic layer was washed with brine (8 mL), dried (Na2SO4), filtrated and evaporated to give a syrup. Yield: 441 mg (0.91 mmol 98%). Rf (SiO2): 0.35 (CHCl3—MeOH 85:15).
To a solution of aminoacid 7 (410 mg, 0.85 mmol) in CH2Cl2 (4 ml) were added benzotriazole-1-yl-oxi-tris-pyrrolidino-phosphonium hexafluorphosphate (442 mg, 0.85 mmol), DIEA (296 μl, 1.70 mmol) and 1,4-Bis(3-aminopropyl)piperazine (67 μl, 0.33 mmol). The mixture was stirred at RT for 16 h. The solvent was removed, CHCl3 (40 mL) was added and the organic layer was washed with 5% NaHCO3 (3×12 mL) and brine (30 mL), dried Na2SO4), filtrated and evaporated. The residue was purified by column chromatography on reverse phase (C-18) to give a syrup. Yield: 316 mg (0.28 mmol, 85%), Rf (SiO2): 0.51 (CHCl3—MeOH 85:15), Rf (C-18): 0.14 (MeOH).
To a solution of compound 8 (244 mg, 0.22 mmol) in EtOAc (15 mL) was added 4.9 N HCl-EtOAc (15 ml). The reaction was stirred at RT for 2 h. The precipitate was filtrated and washed with ether to give a white solid. Yield 198 mg (0.18 mmol, 82%), Rf (SiO2): 0.52 (MeOH—NH4OH 85:15).
To a solution of N-β-(t-butoxycarbonyl)-L-diaminopropionic acid (560 mg, 2.74 mmol) in THF (77 mL) were added successively a solution of K2CO3 (416 mg, 3.02 mmol) in water (11 mL) and oleoyl succinimidate (1.04 g, 2.74 mmol). The reaction was stirred at RT for 18 h and the most of THF was evaporated. Water and CHCl3 (30 mL each) were added and the mixture was acidified with 1 M HCl to pH: 2. The organic layer was separated, and the aqueous layer was extracted with CHCl3 (2×30 ml). The organic layer was washed with brine (20 mL), dried (Na2SO4), filtrated and evaporated to give a white solid. Yield: 1.25 g (2.67 mmol, 97%). Rf(SiO2): 0.35 (CHCl3—MeOH 85:15).
To a solution of aninoacid 10 (1.22 g, 2.60 mmol) in CH2Cl2 (14 ml) were added benzotriazole-1-yl-oxi-tris-pyrrolidino-phosphonium hexafluorphosphate (1.35 g, 2.60 mmol), DIEA (910 μl, 5.21 mmol) and 1,4-Bis(3-aminopropyl)piperazine (206 μl, 1.00 mmol). The mixture was stirred at RT for 15 h. The solvent was removed, CHCl3 (75 mL) was added and the organic layer was washed with 5% NaHCO3 (3×30 mL) and brine (60 mL), dried (Na2SO4), filtrated and evaporated. The residue was purified by column chromatography on reverse phase (C-18) to give a syrup. Yield: 940 mg (0.85 mmol, 85%), Rf(SiO2): 0.52 (CHCl3—MeOH 85:15), Rf(C-18): 0.16 (MeOH).
To a solution of compound 11 (355 mg, 0.32 mmol) in EtOAc (30 mL) was added 4.9 N HCl-EtOAc (30 ml). The reaction was stirred at RT for 2 h. The precipitate was filtrated and washed with ether to give a white solid. Yield 280 mg (0.27 mmol, 84%), Rf (SiO2): 0.34 (MeOH—NH4OH 99:1).
Transfection of recombinant plasmid expressing luciferase into cells using lysine-polyamine-based gemini compounds. Transfection studies were performed using the adherent cell line CHO-DG44. Complete medium consisted of MEM alpha medium supplemented with 10% v/v foetal bovine serum and 1× L-Glutamine. All media and supplements were obtained from Life Technologies. In Vitro Gene Transfection. Cells were seeded into Biocat poly-D-lysine 96-well black plates (BD) 16-18 hours prior to transfection at an approximate density of 3×104 cells per well. For transfection, 0.1 mg of the luciferase reporter gene plasmid, pGL3-Control Vector (Promega) per well, was incubated with various concentrations of the diaminoacid-polyamine:peptide-based gemini compounds and complexing agents in a final volume of 100 μl. After 30 minutes incubation at RT, OPTI-MEM® medium (Life Technologies) was added to the transfection mixture and the solution placed on the cells (final volume per well: 100 μl). Following a 3 hour or over night incubation at 37° C., the transfection solution was replaced with complete medium and the cells incubated further at 37° C. Reporter gene assays were performed according to the manufacturer's guidelines (Roche Diagnostics) approximately 48 hours post transfection. Luminescence was measured in a Packard TopCount NXT Microplate Scintillation and Luminescence Counter.
GSC170 (1 mg/ml in water) was diluted to a 10× solution with Optimem serum free media. A FITC-tagged oligonucleotide was similarly diluted in Optimem at 10× final concentration. The GSC170 and oligonucleotide were then mixed 1:1 and incubated for fifteen minutes at room temperature. The adherent cell lines: RBL-2H3, J774 and 16HBE14o were plated out the day before transfection. Murine primary T cells were transfected either inactivated or after differentiation into T helper 2 cells. GSC170:oligo complexes were diluted to 1× in Optimem and added to adherent cells that had been washed once in Optimem then all media removed. Nuclear delivery of the oligonucleotide was oserved over a period of 24 hours and compared to the commercial reagent, Lipofectamine 2000 (LF2K).
Transfection efficiencies are shown in Table 1:
# = too low to estimate
All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein as though fully set forth.
| Number | Date | Country | Kind |
|---|---|---|---|
| 0207283.3 | Mar 2002 | GB | national |
| 0213646.3 | Jun 2002 | GB | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/GB03/01291 | 3/26/2003 | WO | 6/7/2005 |
