The present invention relates to peptide nucleic acid derivatives chemically modified to show good cell penetration and strong affinity for nucleic acid.

• Figure 1 provides HPLC chromatograms before and after purification of Oligo 17 by reverse phase HPLC.
• Figure 2 provides a MALDI-TOF mass spectrum for a purified batch of Oligo 17.
• Figure 3 provides graphs of absorbance changes with temperature for Oligo 17 against complementary or mismatch DNA.
• Figure 4(a) and 4(b) provide confocal microscopy images (at 63x objective) 1, 2, 3 and 24h after HeLa cells were treated with Oligo 1 and Oligo 2 at 5µM, respectively.
• Figure 5(a) and 5(b) provide confocal microscopy images (at 63x objective) 0.5 and 1h after MCF-7 cells were treated with Oligo 6 and Oligo 7 at 2.5µM, respectively.
• Figure 6(a) and 6(b) provide confocal microscopy pictures (at 40x objective) 6 or 24h after HeLa cells were treated with Oligo 1 and Oligo 6 at 1µM, respectively.
• Figure 7(a) and 7(b) provide confocal microscopy pictures (40x objective) 24h after JAR cells were treated with Oligo 21 and Oligo 28 at 2µM, respectively.
• Figure 7(c) and 7(d) provide confocal microscopy pictures (at 40x objective) 24h after A549 cells were treated with Oligo 21 and Oligo 28 at 2µM, respectively.
• Figure 7(e) and 7(f) provide confocal microscopy pictures (at 40x objective) 12h after HeLa cells were treated with Oligo 21 and Oligo 28 at 2µM, respectively.
• Figure 7(g) provides confocal microscopy pictures (at 40x objective) 24h after HeLa cells were treated with Oligo 21 at 2µM.
• Figure 8(a), 8(b) and 8(c) provide confocal microscopy images (40x objective) 24h after HeLa, A549, and JAR cells were treated with 2µM Oligo 22, respectively.
• Figure 9 provides western blotting results for JAR cells treated with 5µM or 10µM Oligo 9, 5µM or 10µM Oligo 10, cotreatment with the oligomers at 5µM or 10µM each, and blank (no oligomer treatment).
• Figure 10 is the representative structure for the PNA oligomers of this invention.

Oligonucleotides have been used for diverse biological purposes including antisense inhibition of gene expression, PCR (polymerase chain reaction), diagnostic analysis by gene chips, and so on. Since oligonucleotides interact in a sequence specific manner with nucleic acids such as DNA and RNA, they are quite useful to predictably modulate biological processes involving DNA or RNA within cell. Unlike small molecule drugs, however, oligonucleotides do not readily penetrate mammalian cell membrane, and therefore hardly affect biological processes within cell unless properly modified or formulated to readily penetrate plasma membrane.

Proteins as Drug Targets: Proteins mediate diverse cellular functions. It would not be surprising to find that most of currently marketed drugs show therapeutic activity through modulating functions of protein(s). For example, non-steroidal anti-inflammatory drug aspirin inhibits enzymes called cyclooxygenases for its anti-inflammatory activity. Losartan binds to and antagonize the function of a trans-membrane receptor called angiotensin II receptor for its antihypertensive activity. Rosiglitazone selectively activates an intracellular receptor called peroxisome proliferator-activated receptor γ (PPARγ) to elicit its antidiabetic activity. Etanercept is a fusion protein which binds to a cytokine called tumor necrosis factor-α (TNF-α), and neutralizes the biological activity of TNF-α for its anti-rheumatic activity. Herceptin is a monoclonal antibody to treat breast cancer by selectively binding to erbB2 over-expressed in certain types of breast cancer cells.

Antisense Inhibition of Protein Synthesis: Proteins are encoded by DNA (2-deoxyribose nucleic acid). In response to cellular stimulation, DNA is transcribed to produce pre-mRNA (pre-messenger ribonucleic acid) in the nucleus. The intron portion(s) of pre-mRNA is enzymatically spliced out yielding mRNA (messenger ribonucleic acid), which is then translocated to the cytosolic compartment. In the cytosol, a complex of translational machinery called ribosome binds to mRNA and carries out the protein synthesis as it scans the genetic information encoded along the mRNA. (Biochemistry vol 41, 4503-4510, 2002; Cancer Res. vol 48, 2659-2668, 1988)

An oligonucleotide binding to mRNA or pre-mRNA in a sequence specific manner is called antisense oligonucleotide (AO). AO may tightly bind to an mRNA and inhibit the protein synthesis by ribosome along the mRNA in the cytosol. AO needs to be present within cell in order to inhibit the synthesis of its target protein. AO may tightly bind to a pre-mRNA in the nucleus and affect the splicing of the pre-mRNA, producing an mRNA of altered sequence and consequently an altered protein.

Unnatural Oligonucleotides: Oligonucleotides of DNA or RNA are susceptible to degradation by endogenous nucleases, limiting their therapeutic utility. To date, many types of unnatural oligonucleotides have been developed and studied intensively. (Clin. Exp. Pharmacol. Physiol. vol 33, 533-540, 2006) Some of them show extended metabolic stability compared to DNA and RNA. Provided above are chemical structures for some of representative unnatural oligonucleotides. Such oligonucleotides predictably bind to a complementary nucleic acid as DNA or RNA does.

Phosphorothioate oligonucleotide (PTO) is a DNA analog with one of the backbone phosphate oxygen atoms replaced with sulfur atom per monomer. Such a small structural change made PTO comparatively resistant to degradation by nucleases. (Ann. Rev. Biochem. vol 54, 367-402, 1985)

Reflecting the structural similarity of PTO and DNA, they both poorly penetrate cell membrane in most mammalian cell types. For some types of cells abundantly expressing transporter(s) for DNA, however, DNA and PTO show good cell penetration. Systemically administered PTOs are known to readily distribute to the liver and kidney. (Nucleic Acids Res. vol 25, 3290-3296, 1997)

In order to facilitate PTO's cell penetration in vitro, lipofection has been popularly practiced. However, lipofection physically alters cell membrane, causes cytotoxicity, and therefore would not be ideal for long term therapeutic use.

Over the past 20 years, antisense PTOs and variants of PTOs have been clinically evaluated to treat cancers, immunological disorders, metabolic diseases, and so on. (Biochemistry vol 41, 4503-4510, 2002; Clin. Exp. Pharmacol. Physiol. vol 33, 533-540, 2006) Many of such antisense drug candidates have not been successful partly due to PTO's poor cell penetration. In order to overcome the poor cell penetration, PTO needs to be administered at high dose for therapeutic activity. However, PTOs are known to be associated with dose dependent toxicities such as increased coagulation time, complement activation, tubular nephropathy, Kupffer cell activation, and immune stimulation including splenomegaly, lymphoid hyperplasia, mononuclear cell infiltration. (Clin. Exp. Pharmacol. Physiol. vol 33, 533-540, 2006)

Many antisense PTOs have been found to show due clinical activity for diseases with a significant contribution from the liver or kidney. ISIS-301012 (mipomersen) is a PTO analog which inhibits the synthesis of apoB-100, a protein involved in LDL cholesterol transport. Mipomersen manifested due clinical activity in a certain population of atherosclerosis patients most likely due to its preferential distribution to the liver. (www.medscape.com/viewarticle/556073: Accessed on Feb 19, 2009) ISIS-113715 is an antisense PTO analog inhibiting the synthesis protein tyrosine phosphatase 1B (PTP1B), and was found to show therapeutic activity in type II diabetes patients. (Curr. Opin. Mol. Ther. vol 6, 331-336, 2004)

In phosphoroamidite morpholino oligonucleotide (PMO), the backbone phosphate and 2-deoxyribose of DNA are replaced with phosphoamidite and morpholine, respectively. (Appl. Microbiol. Biotechnol. vol 71, 575-586, 2006) While the DNA backbone is negatively charged, the PMO backbone is not charged. Thus the binding between PMO and mRNA is free of electrostatic repulsion between the backbones, and tends to be stronger than that between DNA and mRNA. Since PMO is structurally very different from DNA, PMO wouldn't be recognized by the hepatic transporter(s) recognizing DNA. However, PMO doesn't readily penetrate cell membrane.

Peptide nucleic acid (PNA) is a polypeptide with N-(2-aminoethyl)glycine as the unit backbone, and was discovered by Nielsen and colleagues. (Science vol 254, 1497-1500, 1991) Like DNA and RNA, PNA also selectively binds to complementary nucleic acid [Nature (London) vol 365, 566-568, 1992] Like PMO, the backbone of PNA is not charged. Thus the binding between PNA and RNA tends to be stronger than that between DNA and RNA. Since PNA is structurally markedly different from DNA, PNA wouldn't be recognized by the hepatic transporter(s) recognizing DNA, and would show a tissue distribution profile very different from that of DNA or PTO. However, PNA also poorly penetrates mammalian cell membrane. (Adv. Drug Delivery Rev. vol 55, 267-280, 2003)

In locked nucleic acid (LNA), the backbone ribose ring of RNA is structurally constrained to increase the binding affinity for RNA or DNA. Thus, LNAs may be regarded as high affinity DNA or RNA derivatives. (Biochemistry vol 45, 7347-7355, 2006)

Antisense Mechanisms: Antisense mechanism differs depending on types of AOs. RNAse H recognizes a duplex of mRNA with DNA, RNA, or PTO, and degrades the duplex portion of mRNA. Thus, the antisense activity of PTO is significantly amplified by RNAse H. In the meantime, RNAse H does not recognize a duplex of mRNA with PMO, PNA, or LNA. In other words, PMO, PNA and LNA must rely purely on the steric blocking of mRNA for their antisense activity. (Biochemistry vol 41, 4501-4510, 2002)

For oligonucleotides with the same binding affinity for mRNA, PTO should therefore show stronger antisense activity than PMO, PNA, and LNA. For steric block AOs such as PMO, PNA, and LNA, strong affinity for mRNA is desired for antisense activity.

Antisense Activity of PNA: The binding affinity of PNA for mRNA would increase as the length of PNA increases to a certain point. However, the antisense activity of PNA doesn't seem to always increase to the length of PNA. There were cases that the antisense activity of PNA reached the maximum activity at 12 to 13-mer and decreases thereafter. (Nucleic acids Res. vol 32, 4893-4902, 2004) On the other hand, optimum antisense activity was reached with 15 to 18-mer PNAs against a certain mRNA, reflecting that the structural accessibility of the target binding site of the mRNA would be important. (Biochemistry vol 40, 53-64, 2001)

In many cases, PNAs have been reported to inhibit protein synthesis by ribosome at micromolar level under good cell penetrating conditions. (Science vol 258, 1481-85, 1992; Biochemistry vol 40, 7853-7859, 2001; Nucleic acids Res. vol 32, 4893-4902, 2004) However, PNAs targeting a highly accessible position of mRNA were found to show antisense activity at sub-micromolar level (Neuropeptides vol 38, 316-324, 2004; Biochemistry vol 40, 53-64, 2001) or even at sub-nanomolar level (Nucleic Acids Res. vol 36, 4424-4432, 2008) under good transfection conditions.

In addition to targeting a highly accessible site in mRNA, strong binding affinity of PNA for mRNA would be very required for good antisense activity. Unlike DNA, PTO, and LNA, the backbone of PNA is not charged. PNA tends to aggregate and become less suitable for binding to mRNA as its size increases. It is desired to improve PNA's binding affinity for mRNA without increasing the length of PNA. Incorporation of PNA monomers with a point charge would be beneficial in preventing PNA from aggregating.

Cell Penetration Strategies for PNA: PNAs do not readily penetrate cell membrane and tend to show poor antisense activity unless properly transfected. In early years, the antisense activity of PNA was assessed by microinjection (Science vol 258, 1481-85, 1992) or electroporation (Biochemistry vol 40, 7853-7859, 2001). Microinjection and electroporation are invasive and inappropriate to be applied for therapeutic purposes. In order to improve the cell penetration, various strategies have been developed. (Adv. Drug Delivery Rev. vol 55, 267-280, 2003; Curr. Top. Med. Chem. vol 7, 727-737, 2007)

PNAs have been effectively delivered into cell by covalent incorporation of cell penetrating peptides (Neuropeptides vol 38, 316-324, 2004), lipofection following duplex formation with a complementary DNA (Biochemistry vol 40, 53-64, 2001), lipofection of PNAs with a covalently attached 9-aminoacridine (Nucleic Acids Res. vol 32, 2695-2706, 2004), lipofection of PNAs with covalently attached phosphonate anions (Nucleic Acids Res. vol 36, 4424-4432, 2008), and so on. Also cell penetration was improved by attaching to PNA a lipophilc moiety such as adamantane (Bioconjugate Chem. vol 10, 965-972, 1999) or amphiphilic group such as tetraphenyl phosphonium. (Nucleic Acids Res. vol 29, 1852-1863, 2001) Nevertheless, such a covalent modification is unlikely to increase the binding affinity for mRNA despite marked improvement in the cell penetration.

PNAs with a Covalently Attached CPP: Cell penetrating peptides (CPPs) are polypeptides showing good cell penetration, and have multiple positive charges from arginine or lysine residues. To date many CPPs such as transportan, penetratin, NLS (nuclear localization signal), and Tat have been discovered. CPPs are known to efficiently carry a covalently attached cargo into cell. PNAs with a covalently attached CPP also showed good cell penetration.

Although some PNAs with a covalently attached CPP showed antisense IC₅₀s around 100nM (Neuropeptides vol 38, 316-324, 2004), micromolar antisense IC₅₀s are rather prevalent for such PNAs.

PNAs with a covalently linked CPP are composed of two portions, the hydrophobic PNA domain and the positively charged CPP domain. Such a PNA tends to aggregate and be trapped in endosomes within cell, and would not be available for the antisense inhibition of protein synthesis. (Curr. Top. Med. Chem. vol 7, 727-737, 2007; Nucleic Acids Res. vol 33, 6837-6849, 2005) Furthermore, such a covalently attached CPP hardly increases the binding affinity of PNA for mRNA.

PNAs with a Chiral Backbone: There have been attempts to introduce a chiral substituent on the PNA backbone of 2-aminoethyl-glycine (Aeg). For example, the aqueous solubility of PNA was significantly improved by incorporating PNA monomer(s) with a backbone of 2-aminoethyl-lysine in place of Aeg. (Angew. Chem. Int. Ed. Engl. vol 35, 1939-1941, 1996)

By introducing the backbone of L-(2-amino-2-methyl)ethyl-glycine in place of Aeg, the binding affinity of PNA for DNA and RNA was significantly improved. A 10-mer PNA with all of the backbone of L-(2-amino-2-methyl)ethyl-glycine in place of 2-aminoethyl-glycine showed an increase of 19°C and 10°C in T_m against complementary DNA and RNA, respectively. Such an increase doesn't seem to be proportional to the number of substitution with L-(2-amino-2-methyl)ethyl-glycine, though. (J. Am. Chem. Soc. vol 128, 10258-10267, 2006)

GPNA: The cell penetration of PNA was reported to be markedly improved by incorporating PNA monomers with a backbone of 2-aminoethyl-arginine in place of Aeg. (J. Am. Chem. Soc. vol 125, 6878-6879, 2003) Such PNAs have been termed 'GPNA' since they have guanidinium moiety on the backbone.

GPNAs with the backbone of 2-aminoethyl-D-arginine were reported to have stronger affinity for DNA and RNA than the corresponding GPNAs with that of 2-aminoethyl-L-arginine. (Chem. Commun. 244-246, 2005) For a 10-mer GPNA with 5 GPNA monomers with the backbone of 2-aminoethyl-D-arginine there was an increase of 7°C in T_m (melting temperature) against complementary DNA compared to the corresponding unmodified PNA. (Bioorg. Med. Chem. Lett. vol 16, 4931-4935, 2006)

A 16-mer antisense GPNA against human EGFR-TK was reported to show antitumor activity upon ip (intra peritoneal) administration in athymic nude mice, although the in vitro antisense activity was not documented for the antisense GPNA in the prior art. (WO 2008/061091)

PNAs with Modified Nucleobase: Like cases with DNA, nucleobase modifications have been pursued to improve PNA's affinity for nucleic acids.

PNAs with adenine replaced with 2,6-diaminopurine were evaluated for their affinity for complementary DNA or RNA. Substitution with 2,6-diaminopurine was found to elicit an increase of 2.5 ~ 6°C in T_m per replacement. (Nucleic Acids Res. vol 25, 4639-4643,1997)

PNAs with cytosine replaced with 9-(2-aminoethoxy)phenoxazine were evaluated for their affinity for complementary DNA or RNA. A single substitution with 9-(2-aminoethoxy)phenoxazine elicited an increase of 10.7 ~ 23.7°C in T_m, although such an increase was markedly dependent on the nucleotide sequence. Nucleobase 9-(2-aminopropoxy)phenoxazine also induced a large increase in T_m. Due to a huge increase in T_m, PNA monomer with either 9-(2-aminoethoxy)-phenoxazine or 9-(2-aminopropoxy)phenoxazine as a cytosine replacement has been termed 'G-clamp'. (Org. Lett. vol 4, 4395-4398, 2002) However, cell penetration data was not reported for PNAs with G-clamp(s).

PNAs with cytosine replaced with either 6-{2-(2-aminoethoxy)phenyl}-pyrrolocytosine or 6-{2,6-di(2-aminoethoxy)phenyl}pyrrolocytosine were evaluated for their affinity for complementary DNA or RNA. A single substitution with either 6-{2-(2-aminoethoxy)phenyl}pyrrolocytosine or 6-{2,6-di(2-aminoethoxy)-phenyl}pyrrolocytosine increased T_m by 3 ~ 11.5°C. (J. Am. Chem. Soc. vol 130, 12574-12575, 2008) However, such PNAs were not evaluated for cell penetration.

Other Use of PNAs: By tightly binding to a microRNA, PNA can inhibit the regulatory function of the microRNA, leading to an increase in the expression level of the protein(s) directly regulated by the microRNA. (RNA vol 14, 336-346, 2008) By tightly binding to a ribonucleoprotein such as telomerase, PNA can modulate the cellular function of the ribonucleoprotein. (Bioorg. Med. Chem. Lett, vol 9, 1273-78, 1999) By tightly binding to a certain portion of a gene in the nucleus, PNA can modulate the transcription level of the gene. (Biochemistry vol 46, 7581-89, 2007.)

Since PNA tightly binds to DNA and RNA, and sensitively discriminates a single base pair mismatch, PNA would be suitable for high fidelity detection of single nucleotide polymorphism (SNP). Since PNA binds tightly to DNA and RNA with high sequence specificity, PNA may find various other therapeutic and diagnostic applications involving DNA or RNA. (FASEB vol 14, 1041-1060, 2000)

The present invention provides a peptide nucleic acid derivative of Formula I or a pharmaceutically acceptable salt thereof: wherein,
n is an integer equal to or larger than 5;
S₁, S₂, ..., 5_n-1, S_n, T₁, T₂, ..., T_n-1, and T_n independently represent hydrogen, deuterium, substituted or non-substituted alkyl, or substituted or non-substituted aryl radical;
X and Y independently represent hydrogen, deuterium, substituted or non-substituted alkyl, substituted or non-substituted acyl, substituted or non-substituted sulfonyl, or substituted or non-substituted aryl radical;
Z represents hydroxy, substituted or non-substituted alkyloxy, substituted or non-substituted aryloxy, or substituted or non-substituted amino radical;
B₁, B₂, ..., B_n-1, and B_n are independently selected from natural nucleobases including adenine, thymine, guanine, cytosine and uracil, and unnatural nucleobases; and,
at least one of B₁, B₂, ..., B_n-1, and B_n is independently selected from unnatural nucleobases represented by Formula II, Formula III, or Formula IV: wherein,
R₁, R₂, R₃, R₄, R₅ and R₆ are independently selected from substituted or non-substituted alkyl, and hydrogen radical; and,
L₁, L₂ and L₃ are a covalent linker represented by Formula V connecting a basic amino group to the moiety responsible for nucleobase pairing properties:
wherein Q₁ and Q_m are substituted or non-substituted methylene (-CH2-) radical, and Q_m is directly linked to the basic amino group;
Q₂, Q₃, ..., and Q_m-1 are independently selected from substituted or non-substituted methylene, oxygen (-O-), sulfur (-S-), and substituted or non-substituted amino radical [-N(H)-, or -N(substituent)-]; and,
m is an integer from 2 to 15.

A PNA oligomer of Formula I shows improved binding affinity for nucleic acid and cell penetration compared to its corresponding 'unmodified' PNA oligomer. PNA oligomers of this invention are useful to sequence specifically inhibit or modulate cellular and physiological functions mediated by nucleic acids or physiologically active molecules having a nucleic acid domain such as ribonucleoproteins. Also PNA oligomers of this invention are useful for diagnostic purposes due to their sequence specific binding capability for nucleic acids.

The present invention provides a novel class of PNA oligomers represented by Formula I, or a pharmaceutically acceptable salt thereof: wherein,
n is an integer equal to or larger than 5;
S₁, S₂, ..., S_n-1, S_n, T₁, T₂, ..., T_n-1, and T_n independently represent hydrogen, deuterium, substituted or non-substituted alkyl, or substituted or non-substituted aryl radical;
X and Y independently represent hydrogen, deuterium, hydroxy, substituted or non-substituted alkyloxy, substituted or non-substituted aryloxy, substituted or non-substituted amino, substituted or non-substituted alkyl, substituted or non-substituted acyl, substituted or non-substituted sulfonyl, or substituted or non-substituted aryl radical;
Z represents hydrogen, deuterium, hydroxy, substituted or non-substituted alkyloxy, substituted or non-substituted aryloxy, substituted or non-substituted amino, substituted or non-substituted alkyl, or substituted or non-substituted aryl radical;
B₁, B₂, ..., B_n-1, and B_n are independently selected from natural nucleobases including adenine, thymine, guanine, cytosine and uracil, and unnatural nucleobases; and,
at least one of B₁, B₂, ..., B_n-1, and B_n independently represents an unnatural nucleobase with a substituted or non-substituted amino radical covalently linked to the moiety responsible for its due nucleobase pairing properties.

A PNA oligomer of this invention shows improved cell penetration and binding to nucleic acid compared to its corresponding 'unmodified' PNA oligomer. In this invention, 'unmodified' PNA oligomer refers to a PNA oligomer of Formula I, wherein S₁, S₂, ..., S_n-1, S_n, T₁, T₂, ..., T_n-1, and T_n are hydrogen radical; and B₁, B₂, ..., B_n-1, and B_n are independently selected from natural nucleobases comprising adenine, thymine, guanine, and cytosine.

A PNA oligomer of this invention readily penetrates mammalian cell membrane, and can affect or alter cellular functions by sequence specifically binding to a nucleic acid or a nucleoprotein within cell.

A PNA oligomer of Formula I can potently inhibit ribosomal protein synthesis by tightly binding to mRNA. A PNA oligomer of the present invention can tightly bind to a pre-mRNA and alter the splicing of the pre-mRNA to mRNA. Further, a PNA oligomer of the present invention can bind tightly to a microRNA, and inhibit mRNA degradation induced by the microRNA.

A PNA oligomer of Formula I can predictably bind to the nucleic acid domain of a ribonucleoprotein, for example telomerase, and modulate its physiological function(s). A PNA oligomer of the present invention can bind to a gene and modulate the transcription of the gene. A PNA oligomer of Formula I can bind to a viral gene or its transcript, and inhibit the proliferation of the virus. A PNA oligomer of this invention can affect cellular functions other than those described above by sequence specifically binding to a nucleic acid or a nucleoprotein within mammalian cell. In addition, a PNA oligomer of the present invention can tightly bind to a bacterial mRNA, nucleic acid, or gene, and inhibit bacterial proliferation or alter bacterial biosynthesis profiles.

A PNA oligomer of this invention is highly sensitive to a base mismatch in binding to its complementary DNA counterpart, and would be appropriate for detecting single nucleotide polymorphism (SNP) with high fidelity. PNA oligomers of the present invention bind tightly to their complementary DNAs with high sequence specificity, and may be useful for gene profiling. A PNA oligomer of Formula I may be useful to probe or locate a nucleic acid bearing molecule such as telomere within cell if properly tagged with a chromophore, for example, fluorophore. PNA oligomers of this invention may be useful for a variety of diagnostic or analytical purposes other than those detailed above.

A PNA oligomer of the present invention possesses good aqueous solubility compared to the corresponding 'unmodified' PNA oligomer, and can be used as dissolved in water, saline, or a buffer solution. A PNA oligomer of Formula I can be formulated with a cationic lipid such as lipofectamine. A PNA oligomer of this invention may be duplexed with a complementary DNA and the resulting duplex can be formulated with a cationic lipid.

A PNA oligomer of this invention may be formulated in a variety of dosage forms including but not limited to injectable formulation, nasal spray, tablet, granules, hard capsule, soft capsule, liposomal formulation, oral suspension, transdemal formulation, and so on.

A PNA oligomer of the present invention can be administered to a subject at therapeutically effective doses, which would vary depending on indication, administration route, dosing schedule, situations of subject, and so on.

A PNA oligomer of the present invention can be administered to a subject by a variety of routes including but not limited to intravenous injection, subcutaneous injection, intraperitoneal injection, nasal inhalation, oral administration, transdermal application, and so on.

A PNA oligomer of Formula I can be administered to a subject in combination with a pharmaceutically acceptable adjuvant including but not limited to citric acid, hydrochloric acid, tartaric acid, stearic acid, polyethyleneglycol, polypropyleneglycol, ethanol, sodium bicarbonate, distilled water, hyaluronic acid, cationic lipid such as lipofectamine, starch, gelatin, talc, ascorbic acid, olive oil, palm oil, methylcelluose, titanium oxide, sodium carboxymethylcellulose, sweetener, preservative, and so on.

A PNA oligomer of the present invention, depending on the presence of basic or acidic functional group(s) therein, may be used as neutralized with an equivalent amount of a pharmaceutically acceptable acid or base including but not limited to sodium hydroxide, potassium hydroxide, hydrochloric acid, methanesulfonic acid, citric acid, and so on.

Preferred PNA oligomers encompass PNA oligomers of Formula I, or a pharmaceutically acceptable salt thereof:

• wherein,
• n is an integer equal to or larger than 5 but smaller than or equal to 30;
• S₁, S₂, ..., S_n-1, S_n, T₁, T₂, ..., T_n-1, and T_n are hydrogen radical;
• X and Y are independently selected from hydrogen, substituted or non-substituted alkyl, substituted or non-substituted acyl, substituted or non-substituted sulfonyl, and substituted or non-substituted aryl radical;
• Z represents hydrogen, hydroxy, substituted or nonsubstituted alkyloxy, substituted or non-substituted amino, substituted or non-substituted alkyl, or substituted or non-substituted aryl radical;
• B₁, B₂, ..., B_n-1, and B_n are independently selected from natural nucleobases including adenine, thymine, guanine, cytosine and uracil, and unnatural nucleobases; and,
• at least one of B₁, B₂, ..., B_n-1, and B_n is independently selected from unnatural nucleobases represented by Formula II, Formula III, or Formula IV: wherein,
  R₁, R₂, R₃, R₄, R₅ and R₆ are independently selected from substituted or non-substituted alkyl, hydrogen, hydroxy, and substituted or non-substituted alkyloxy radical; and,
  L₁, L₂ and L₃ are a covalent linker represented by Formula V connecting a basic amino group to the moiety responsible for nucleobase pairing properties: wherein,
  Q₁ and Q_m are substituted or non-substituted methylene (-CH₂-) radical, and Q_m is directly linked to the basic amino group;
  Q₂, Q₃, ..., and Q_m-1 are independently selected from substituted or non-substituted methylene, oxygen (-O-), sulfur (-S-), and substituted or non-substituted amino radical [-N(H)-, or -N(substituent)-]; and,
  m is an integer equal to or larger than 2 but smaller than or equal to 15.

PNA oligomers of particular interest comprise PNA oligomers of Formula I, or a pharmaceutically acceptable salt thereof:

• wherein,
• n is an integer equal to or larger than 8 but smaller than or equal to 25;
• S₁, S₂, ..., S_n-1, S_n, T₁, T₂, ..., T_n-1, and T_n are hydrogen radical;
• X and Y are independently selected from hydrogen, substituted or non-substituted alkyl, and substituted or non-substituted acyl radical;
• Z represents hydroxy, or substituted or non-substituted amino radical;
• B₁, B₂, ..., B_n-1, and B_n are independently selected from natural nucleobases including adenine, thymine, guanine, cytosine and uracil, and unnatural nucleobases;
• at least two of B₁, B₂, ..., B_n-1, and B_n are independently selected from unnatural nucleobases represented by Formula II, Formula III, or Formula IV;
• R₁, R₂, R₃, R₄, R₅ and R₆ are independently selected from substituted or non-substituted alkyl, and hydrogen radical;
• Q₁ and Q_m are substituted or non-substituted methylene radical, and Q_m is directly linked to the basic amino group;
• Q₂, Q₃, ..., and Q_m-1 are independently selected from substituted or non-substituted methylene, oxygen, and amino radical; and,
• m is an integer equal to or larger than 2 but smaller than or equal to 12.

PNA oligomers of high interest comprise PNA oligomers of Formula I, or a pharmaceutically acceptable salt thereof:

• wherein,
• n is an integer equal to or larger than 10 but smaller than or equal to 25;
• S₁, S₂, ..., S_n-1, S_n, T₁, T₂, ..., T_n-1, and T_n are hydrogen radical;
• X and Y are independently selected from hydrogen, and substituted or non-substituted acyl radical;
• Z represents hydroxy, alkyloxy, or substituted or non-substituted amino radical; and,
• B₁, B₂, ..., B_n-1, and B_n are independently selected from natural nucleobases including adenine, thymine, guanine, cytosine and uracil, and unnatural nucleobases;
• at least three of B₁, B₂, ..., B_n-1, and B_n are independently selected from unnatural nucleobases represented by Formula II, Formula III, or Formula IV;
• R₁, R₂, R₃, R₄, R₅ and R₆ are independently selected from substituted or non-substituted alkyl, and hydrogen radical;
• Q₁ and Q_m are methylene radical, and Q_m is directly linked to the basic amino group;
• Q₂, Q₃, ..., and Q_m-1 are independently selected from methylene, oxygen, and amino radical; and,
• m is an integer equal to or larger than 2 but smaller than or equal to 10.

PNA oligomers of higher interest encompass PNA oligomers of Formula I, or a pharmaceutically acceptable salt thereof:

• wherein,
• n is an integer equal to or larger than 10 but smaller than or equal to 20;
• S₁, S₂, ..., S_n-1, S_n, T₁, T₂, ..., T_n-1, and T_n are hydrogen radical;
• X and Y are independently selected from hydrogen, and substituted or non-substituted acyl radical;
• Z represents hydroxy, or substituted or non-substituted amino radical;
• B₁, B₂, ..., B_n-1, and B_n is independently selected from natural nucleobases including adenine, thymine, guanine, cytosine and uracil, and unnatural nucleobases;
• at least three of B₁, B₂, ..., B_n-1, and B_n are independently selected from unnatural nucleobases represented by Formula II, Formula III, or Formula IV;
• R₁, R₃, and R₅ are hydrogen radical, and R₂, R₄, and R₆ independently represent hydrogen, or substituted or non-substituted amidinyl radical;
• Q₁ and Q_m are methylene radical, and Q_m is directly linked to the basic amino group;
• Q₂, Q₃, ..., and Q_m-1 are independently selected from methylene, oxygen, and amino radical; and,
• m is an integer equal to or larger than 2 but smaller than or equal to 10.

PNA oligomers of highest interest comprise PNA oligomers of Formula I, or a pharmaceutically acceptable salt thereof:

• wherein,
• n is an integer equal to or larger than 10 but smaller than or equal to 20;
• S₁, S₂, ..., S_n-1, S_n, T₁, T₂, ..., T_n-1, and T_n are hydrogen radical;
• X and Y are independently selected from hydrogen, and substituted or non-substituted acyl radical;
• Z represents hydroxy, or substituted or non-substituted amino radical;
• B₁, B₂, ..., B_n-1, and B_n are independently selected from adenine, thymine, guanine, cytosine, and unnatural nucleobases;
• at least three of B₁, B₂, ..., B_n-1, and B_n are independently selected from unnatural nucleobases represented by Formula II, Formula III, or Formula IV;
• R₁, R₃, and R₅ are hydrogen radical, and R₂, R₄, and R₆ independently represents hydrogen or amidinyl radical;
• Q₁ and Q_m are methylene radical, and Q_m is directly linked to the basic amino group;
• Q₂, Q₃, ..., and Q_m-1 are independently selected from methylene, and oxygen radical; and,
• m is an integer equal to or larger than 2 but smaller than or equal to 8.

Specific PNA oligomers of strong interest comprise PNA oligomers of Formula I, or a pharmaceutically acceptable salt thereof:

• wherein,
• n is an integer equal to or larger than 8 but smaller than or equal to 20;
• S₁, S₂, ..., S_n-1, S_n, T₁, T₂, ..., T_n-1, and T_n are hydrogen radical;
• X is hydrogen radical;
• Y represents hydrogen, or substituted or non-substituted acyl radical;
• Z represents hydroxy, or substituted or non-substituted amino radical;
• B₁, B₂, ..., B_n-1, and B_n are independently selected from adenine, thymine, guanine, cytosine, and unnatural nucleobases;
• at least three of B₁, B₂, ..., B_n-1, and B_n are independently selected from unnatural nucleobases represented by Formula II, Formula III, or Formula IV;
• R₁, R₃, and R₅ are hydrogen radical, and R₂, R₄, and R₆ independently represent hydrogen or amidinyl radical;
• L₁ represents -(CH₂)₂-O-(CH₂)₂-, -CH₂-O-(CH₂)₂-, or -CH₂-O-(CH₂)₃-with the right end is directly linked to the basic amino group; and,
• L₂ and L₃ are independently selected from -(CH₂)₂-O-(CH₂)₂-, -(CH₂)₃-O-(CH₂)₂-, -(CH₂)₂-O-(CH₂)₃-, -(CH₂)₂-, -(CH₂)₃-, -(CH₂)₄-, -(CH₂)₅-, -(CH₂)₆-, - (CH₂)₇-, and -(CH₂)₈- with the right end is directly linked to the basic amino group.

The above used terms and abbreviations for the PNA oligomers of this invention are illustrated in the table below. Term/Abbreviation Illustration or definition oligomer oligonucleotide hydrogen single hydrogen atom (-H) deuterium single deuterium atom (-D) alkyl linear or branched alkyl radical aryl aromatic group such as phenyl, pyridyl, furyl, naphthyl, etc methylene -(CH₂)- acyl '-C(O)-' substituted with hydrogen, alkyl, or aryl radical sulfonyl '-S(O)₂-' substituted with alkyl, or aryl radical alkyloxy 'R-O-' where R is substituted or non-substituted alkyl radical oxygen '-O-' sulfur '-S-' amidinyl cytosine (C) thymine (T) uracil (U) adenine (A) guanine (G)

For characterization of molecules of this invention NMR spectra were recorded on a Varian Mercury 300MHz, Bruker Avance 400MHz, or Varian Inova 500MHz NMR spectrometer. Either a Bruker Daltonics Ultraflex MALDI-TOF or an Agilent LC/MS Ion Trap System was employed for determination of molecular weight. PNA oligomers were analyzed and purified by C₁₈-reverse phase HPLC either on a Hewlett Packard 1050 HPLC or a Shimazu LC-6AD HPLC. Unless noted otherwise, silica gel was used for chromatographic separation of small molecules prepared in this invention. Melting point is reported as uncorrected.

Unnatural nucleobase derivatives used for the synthesis of PNA monomers of this invention were prepared according to one of the methods (Methods A, B, and C) provided below or with minor modification(s) thereof, unless detailed otherwise in actual synthetic examples.

Method A: 6-alkyl-pyrollocytosine derivatives were synthesized as properly protected according to Scheme 1 or with minor variation(s) thereof. Such 6-alkyl-pyrollocytosine derivatives were used to synthesize PNA monomers containing a nucleobase represented by Formula II as a cytosine equivalent.

First compound a was deprotonated with NaH and then alkylated with ethylbromoacetate to obtain compound b. Compound b was subjected to a palladium catalyzed coupling with a terminal acetylene derivative, which was in situ annulated to product c according to the literature. (Nucleosides Nucleotides & Nucleic Acids vol 22, 1029-1033, 2003)

Method B: 2,6-diaminopurine derivatives were synthesized as properly protected according to Scheme 2 or with minor variation(s) thereof. Such 2,6-diamino-purine derivatives were used to synthesize PNA monomers containing a nucleobase represented by Formula III as an adenine equivalent.

First 2-haloadenine was reacted with a diamine at high temperature to obtain compound d, which was then reacted with Boc₂O to give compound e. Compound e was deprotonated with NaH, and alkylated with ethylbromoacetate to obtain compound f. The aromatic amino group of compound f was protected with either Cbz or Boc group to yield compound g.

Method C: N-alkylated guanine derivatives were synthesized as properly protected according to Scheme 3 or with minor variations thereof. Such guanine derivatives were used to synthesize PNA monomers containing a nucleobase represented by Formula IV as a guanine equivalent.

First 2-halohypoxanthine was reacted with a diamine at high temperature to obtain compound h, which was then reacted with Boc₂O to give compound i. Compound i was deprotonated with NaH, and alkylated with ethylbromoacetate to obtain compound j.

Two types of PNA monomers were synthesized according to either Method D or Method E to prepare PNA oligomers of Formula I. PNA oligomers were prepared by Panagene, Inc. (www.panagene.com, Daejon, South Korea) using PNA monomers of type o of Scheme 4 upon request of CTI Bio. Alternatively, PNA monomers of type q of Scheme 5 were used in-house for the synthesis of PNA oligomers according to the method described in the prior art or with minor modification(s) thereof. (USP 6,133,444)

Method D: PNA monomers with a modified nucleobase were prepared according to Scheme 4 or with minor variation(s) thereof as properly protected for the PNA oligomer synthesis method described in the literature. (Org. Lett. vol 9, 3291-3293, 2006) In Scheme 4, compound k may correspond to compound c of Scheme 1, compound g of Scheme 2, or compound j of Scheme 3, however, may not be necessarily limited to one of those ester compounds.

First ester k was subjected to alkaline hydrolysis to afford acid l, which was then coupled with ethyl N-[2-{N-(2-benzothiazolinyl)sulfonylamino}ethyl]-glycinate to obtain compound m. Compound m was mildly hydrolyzed with LiOH to give acid n, which was cyclized by an EDCI coupling reaction to obtain modified PNA monomer o. The chemical structure for PNA monomer o was assumed as in Scheme 4 throughout this invention, given that such assigned PNA monomers have successfully yielded PNA oligomers in the literature. (Org. Lett. vol 9, 3291-3293, 2006)

Method E: Alternatively, PNA monomers with a modified nucleobase were prepared according to Scheme 5 or with minor variation(s) thereof as properly protected for the PNA oligomer synthesis method provided in the prior art. (USP 6,133,444) In Scheme 5, compound k may correspond to compound c of Scheme 1, compound g of Scheme 2, or compound j of Scheme 3, however, may not be necessarily limited to one of those ester compounds.

First acid l was coupled with ethyl N-[2-{N-(9H-fluoren-9-yl)amino}ethyl]-glycinate to obtain compound p by an EDCI coupling reaction. Then compound p was mildly hydrolyzed with LiOH to obtain PNA monomer q with a modified nucleobase.

The following examples contain detailed descriptions of the preparation methods for compounds of this invention. The detailed descriptions of these examples are presented for illustrative purposes only and should not be interpreted as a restriction to the present invention. Most of these detailed descriptions fall within the scope, and serve to exemplify the above described GENERAL SYNTHETIC PROCEDURES which form a part of the invention. The abbreviations used in the following examples are defined in the following table. Category Denotation ¹H NMR Proton nuclear magnetic resonance. In presenting NMR data, widely accepted abbreviations were used as follows: s for singlet, d for doublet, t for triplet, q for quartet, m for multiplet, br for broad, J for coupling constant, CDCl₃ for deuterated chloroform, DMSO_d6 for hexa-deuterated DMSO, and so on. MS Mass spectroscopy. In presenting MS data, popularly accepted abbreviations were used as follows: MALDI-TOF for matrix assisted laser desorption ionization time of flight, ESI for electrospray ionization, MW for molecular weight, (m+1) for MH⁺ ion peak, (m+23) for MNa⁺ ion peak, etc. Solvents Widely accepted abbreviations were used for solvents as follows: THF for tetrahydrofuran, MC for methylene chloride, DMF for dimethylformamide, EtOH for ethanol, MeOH for methanol, DMSO for dimethylsulfoxide, EA for ethyl acetate, and so on. Reagents Popularly accepted abbreviations were used for reagents as follows: NaH for sodium hydride, HCl for hydrochloric acid, EDCI for 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, HOBT for 1-hydroxy-benzotriazole, Boc for t-butyloxycarbonyl, Boc₂O for Boc anhydride or di-t-butyl-dicarbonate, Cbz for benzyloxycarbonyl, Fmoc for (9H-fluoren-9-yl)-methoxycarbonyl, Bts for (benzo[d]thiazole-2-sulfonyl), Bts-Cl for (benzo-[d]thiazole-2-sulfonyl)chloride, TFA for trifluoroacetic acid, TEA for triethyl-amine, DIEA for N,N-diisopropylethylamine, LiOH for lithium hydroxide, Aeg for N-(2-aminoethyl)glycine, Fmoc-Aeg-OH for N-[2-{(9H-fluoren-9-yl)-methoxycarbonyl}amino-ethyl]glycine, Fmoc-Aeg-OMe for methyl N-[2-(Fmoc-amino)ethyl]-glycinate, Fmoc-Aeg-OtBu for t-butyl N-[2-(Fmoc-amino)ethyl]-glycinate, Fmoc-Aeg-OSu for N-succinyl N-[2-(Fmoc-amino)-ethyl]-glycinate, HBTU for O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluranium hexafluorophosphate, DCC for 1,3-dicyclohexylcarbodiimide, and so on. Others Widely accepted abbreviations were used for terminologies as follows: mp for melting point, °C for degree in Celcius, h for hour, min for minute, g for gram, mg for milligram, kg for kilogram, l for liter, ml for milliliter, M for mole/l, compd for compound, aq for aqueous, RT for room temperature, and so on.

To 14g of 3-amino-1-propanol dissolved in 150ml THF and 150ml water, was added drop-wise over 30min 40.7g of Boc₂O dissolved in 100ml THF. After the reaction mixture was stirred for 24h, the THF was removed under reduced pressure. The resulting aq layer was extracted with 200ml EA, and the organic layer was washed with 0.5M aq citric acid and with distilled water, and then dried over anhydrous magnesium sulfate. Magnesium sulfate was filtered off, and the resulting filtrate was concentrated in vacuo to give 25g of compd 1 as a colourless liquid. ¹H NMR (400MHz; CDCl₃): δ 4.84 (br s, 1H), 3.66 (t, J = 5.6 Hz, 2H), 3.28 (q, J = 6.0 Hz, 2H), 3.05 (br s, 1H), 1.66 (m, 2H), 1.45 (s, 9H).

To a stirred solution of 8.3g of N-benzoyl-5-iodocytosine dissolved in 60ml DMF, was added at 0°C 1.06g of 55% NaH in mineral oil, and the solution was stirred at RT for 2h. After 2.7ml ethyl bromoacetate was added to the reaction mixture, the reaction solution was stirred for another 24h at RT, which was followed by removal of the solvent under reduced pressure. The resulting residue was dissolved and the insoluble material was filtered off. The filtrate was washed two times with saturated aq ammonium chloride, dried over anhydrous magnesium sulfate, and concentrated in vacuo. The resulting residue was purified by column chromatography (1:1 hexane/EA) to yield 6.5g of compd 2 (compd b in Scheme 1) as a yellow solid. mp 154-5°C. ¹H NMR (400MHz; CDCl₃) δ 13.31 (br s, 1H), 8.37 (d, J = 7.2 Hz, 2H), 7.69 (s, 1H), 7.55 (t, J = 7.4 Hz, 1H), 7.46 (t, J = 7.6 Hz, 2H), 4.49 (s, 2H), 4.27 (q, J = 7.2 Hz, 2H), 1.32 (t, J = 7.2 Hz, 3H).

To 6.5g of 55% NaH in mineral oil dispersed in 150ml THF at 0°C, was added dropwise over 15 min 25g of compd 1, and the mixture was stirred for 1h. After 17.5ml propargyl bromide (80% toluene solution) was added drop-wise over 30min, the reaction mixture was stirred at RT for 20h. The reaction was quenched by slowly adding 250ml water and THF was removed under reduced pressure. Then the resulting aq mixture was extracted with 250ml EA, which was washed 3 times with 250ml water. The organic layer was dried over anhydrous magnesium sulfate, and magnesium sulfate was filtered off. The resulting filtrate was concentrated in vacuo and subjected to column chromatography (5:1 Hexane/EA) to afford 22.7g of compd 3 as a yellow liquid. ¹H NMR (400 MHz; DMSO_d6) δ 6.78 (t, J = 5.2 Hz, 1H), 4.09 (d, J = 2.4 Hz, 2H), 3.43-3.39 (m, 3H), 2.95 (q, J = 6.4 Hz, 2H), 1.60 (m, 2H), 1.37 (s, 9H).

To 3.8g of 4-(2-azidoethoxy)-1-butyne dissolved in 17ml THF, were added 7.2g of triphenylphosphine and 0.7ml water, and the reaction mixture was stirred for 8h, which was followed by removal of the solvent under reduced pressure. Then the resulting residue was dissolved in 20ml EA and extracted twice with 10ml 1M aq HCl. Aq sodium carbonate was added to the aq layer to adjust pH to 9 ~ 10. 5.96g of Boc₂O dissolved in 15ml THF was added to the solution, and the reaction mixture was stirred for 12h. After THF was removed in vacuo, the resulting solution was extracted with EA. The organic layer was washed with 0.5M aq citric acid, and dried over anhydrous magnesium sulfate. The organic layer was concentrated and purified by column chromatography (9:1 Hexane/EA) to afford 3.4g of compd 4 as a yellow oil. ¹H NMR (400MHz; CDCl₃) δ 4.95 (s, 1H), 3.58 (t, J = 6.8 Hz, 2H), 3.53 (t, J = 5.0 Hz, 2H), 3.32 (m, 2H), 2.46 (m, 2H), 2.00 (t, J = 2.8 Hz, 1H), 1.45 (s, 9H).

20g of 2-{(t-butoxycarbonyl)amino}-1-ethanol was reacted and purified by similarly following the procedure described in Example 3 to afford 23.7g of compd 5 as a pale yellow oil. ¹H NMR (400MHz; DMSO_d6) δ 6.81 (t, 1H), 4.11 (d, J = 2.4 Hz, 2H), 3.41 (m, 3H), 3.07 (q, J = 6.0 Hz, 2H), 1.38 (s, 9H).

To a stirred solution of N-[3-(t-butoxycarbonylamino)propyl]-N-(2-propynyl)amine dissolved in 83ml THF and 95ml water, was added drop-wise 42g of Boc₂O at RT. The reaction solution was stirred for 1.5h, and concentrated in vacuo. The resulting aq layer was extracted with EA. The EA layer was washed in series with 0.5M aq citric acid and brine, dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and purified by column chromatography (1:1 Hexane/EA) to give 19g of compd 6 as a yellow oil. ¹H NMR (400MHz; CDCl₃) δ 5.26 (br s, 0.6H), 4.74 (br s, 0.4H), 4.07 (br s, 1H), 3.98 (br s, 1H), 3.40 (t, J = 6.4 Hz, 2H), 3.13 (m, 2H), 2.21 (t, 1H), 1.73 (m, 2H), 1.49 (s, 9H), 1.45 (s, 9H).

To a stirred solution of 10.9g of compd 5 dissolved in 110ml MC, was added 110ml TFA at 0°C drop-wise over 2h, and the reaction mixture was stirred for another 3h. The reaction solution was concentrated under reduced pressure and the resulting residue was dissolved in 40ml MC at 0°C, to which was added 12.3ml TEA and then 8.8g of 1,3-bis(benzyloxycarbonyl)-2-(methylthio)pseudourea at RT. The reaction solution was stirred for 4h and washed twice with water. The organic layer was dried over anhydrous magnesium sulfate, concentrated in vacuo, and subjected to column chromatography (5:1 hexane/EA) to afford 9.8g of compd 7 as a white solid. ¹H NMR (400MHz; DMSO_d6) δ 11.72 (s, 1H), 8.58 (s, 1H), 7.40-7.35 (m, 10H), 5.18 (s, 2H), 5.12 (s, 2H), 4.18 (d, 2H), 3.67-3.66 (m, 4H), 2.43 (t, 1H).

10.8g of t-butyl (R)-1-hydroxypropan-2-ylcarbamate was reacted and purified by similarly following the procedure described in Example 3 to afford 10.1g of compd 8 as a yellow oil. ¹H NMR (500MHz; DMSO_d6) δ 6.63 (d, 1H), 4.11 (d, 2H), 3.60 (m, 1H), 3.37-3.33 (m, 2H), 3.26-3.23 (m, 1H), 1.38 (s, 9H), 1.05 (d, 3H).

To a stirred solution of 5g of 2-{(3-butynyl)-1-oxy}ethyl methanesulfonate and 5.32g of 2-[2-{2-(t-butoxycarbonyl)amino}ethyl-1-oxy]ethylamine in 60ml acetonitrile, was added drop-wise 3.6g of potassium carbonate dissolved in water at 0°C. The reaction solution was allowed to slowly warm to RT and stirred for another 24h, and then concentrated under reduced pressure. The resulting residue was dissolved in MC and washed with water. The organic layer was concentrated and dissolved in 80ml THF and 80ml water, to which was added 8.4g of Boc₂O dissolved in 50ml THF. The reaction mixture was stirred at RT for 16h, which was followed by removal of THF in vacuo and extraction with EA. The organic layer was washed in series with 0.5M aq citric acid, water, and brine. The organic layer was dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (hexane → 1:4 EA/hexane) to obtain 2.45g of compd 9 as a pale yellow oil. ¹H NMR (400MHz; CDCl₃) δ 5.08 (br s, 0.5H), 4.93 (br s, 0.5H), 3.61-3.46 (m, 12H), 3.31 (m, 2H), 2.48 (m, 2H), 1.99 (t, 1H), 1.48 (s, 9H), 1.46 (s, 9H).

To a stirred solution of 6.5g of compd 2 dissolved in 120ml DMF, were added in series 580mg of CuI, 4.2ml TEA, 9.74g of compd 3, and 1.76g of Pd(PPh₃)₄. Then the reaction mixture was stirred for 24h at 50°C with light shielded, and concentrated under reduced pressure. The resulting residue was dissolved in 250ml EtOH and stirred at reflux for 18h. Then the solution was concentrated in vacuo and subjected to chromatographic separation (95:5 EA/EtOH) to obtain 2.3g of compd 10 as a dark red foam/solid. ¹H NMR (400MHz; DMSO_d6) δ 11.30 (br s, 1H), 8.37 (s, 1H), 6.78 (m, 1H), 6.19 (s, 1H), 4.70 (s, 2H), 4.37 (s, 2H), 4.14 (q, J = 7.2 Hz, 2H), 3.42 (t, J = 6.4 Hz, 2H), 2.98 (m, 2H), 1.63 (m, 2H), 1.36 (s, 9H), 1.20 (t, J = 7.2 Hz, 3H).

To 3.3g of compd 10, were added 15ml THF, 30ml water, and then 760mg LiOH, and the mixture was stirred at RT for 20 min. After THF was removed under reduced pressure, the resulting aq solution was washed with diethyl ether. The aq layer was acidified to pH 3 with 1M aq HCl and extracted with EA. The organic layer was dried over anhydrous sodium sulfate and concentrated in vacuo to yield 2.46g of compd 11 as a white solid. ¹H NMR (400MHz; DMSO_d6) δ 11.05 (s, 1H), 8.16 (s, 1H), 6.79 (t, 1H), 6.12 (s, 1H), 4.35 (s, 2H), 4.23 (s, 2H), 3.41 (t, 2H), 2.97 (q, J = 6.4 Hz, 2H), 1.64 (m, 2H), 1.36 (s, 9H).

To 4.0g of compd 11 and 3.6g of ethyl N-[2-{(benzo[d]thiazole-2-sulfonyl)amino}ethyl]glycinate dissolved in 30ml DMF, were added at RT 2.42g of EDCI and 1.70g of HOBt. The reaction mixture was stirred for 8h. After the solvent was removed in vacuo, the resulting residue was dissolved in MC, and washed with 1M aq HCl and then with water. The MC layer was concentrated under reduced pressure and purified by column chromatography (95:5 MC/MeOH) to obtain 4.6g of compd 12 as a yellow foam/solid. ¹H NMR (400MHz; DMSO_d6) δ 11.09 (br s, 1H), 8.74 (s, 0.6H), 8.58 (s, 0.4H), 8.27 (m, 1H), 8.20-8.14 (m, 2H), 7.66 (m, 2H), 6.56 (br s, 1H), 6.16 (m, 1H), 4.91 (s, 1.2H), 4.73 (s, 0.8H), 4.38 (s, 2.6H), 4.17 (m, 0.9H), 4.07 (m, 2.5H), 3.67 (m, 1.1H), 3.49-3.44 (m, 4H), 3.26 (m, 0.9H), 3.01 (m, 2H), 1.66 (m, 2H), 1.38 (s, 9H), 1.24 (t, J = 7.0 Hz, 1.2H), 1.17 (t, J = 7.0 Hz, 1.8H).

4.5g of compd 12 and 670mg of LiOH were dispersed in 20ml THF and 20ml water, and stirred at RT for 20min. THF was removed in vacuo, and the resulting aq solution was washed with diethyl ether. The aq layer was acidified to pH 3 with 1M aq HCl, and extracted with EA. The EA layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford 4.4g of compd 13 as a dark yellow solid. ¹H NMR (400MHz; DMSO_d6) δ 11.32 (br s, 1H), 8.36 (m, 1H), 8.28 (m, 1.6H), 8.22 (s, 0.4H), 7.73 (m, 2H), 6.78 (m, 1H), 6.20 (s, 1H), 4.94 (s, 1.2H), 4.84 (s, 0.8H), 4.52 (s, 0.8H), 4.37 (s, 2H), 4.30 (s, 1.2H), 4.26 (m, 1.2H), 4.07 (m, 2H), 3.87 (m, 0.8H), 3.43 (m, 2H), 2.99 (m, 2H), 1.63 (m, 2H), 1.37 (s, 9H).

4.4g of compd 13 and 1.49g of EDCI in 50ml DMF were stirred at RT for 16h. After the reaction mixture was concentrated in vacuo, the resulting residue was dissolved in 50ml MC. The MC solution was washed in series with 1M aq HCl and water, concentrated in vacuo, and then purified by column chromatography (acetone) to obtain 1.5g of compd 14 as a brown foam/solid. ¹H NMR (400MHz; DMSO_d6) δ 11.25 (br s, 1H), 8.36 (m, 1H), 8.29 (m, 1H), 8.25 (s, 0.6H), 8.19 (0.4H), 7.72 (m, 2H), 6.78 (t, J = 5.2 Hz, 1H), 6.18 (s, 1H), 4.92 (s, 1.2H), 4.82 (s, 0.8H), 4.51 (s, 0.8H), 4.37 (s, 2H), 4.29 (s, 1.2H), 4.23 (m, 1.2H), 4.06 (m, 2H), 3.87 (m, 0.8H), 3.41 (t, J = 6.4 Hz, 2H), 2.98 (q, J = 6.8 Hz, 2H), 1.62 (m, 2H), 1.36 (s, 9H). MS/ESI (m+23/MNa⁺) = 682.2 (observed), MW = 659.8 (C₂₈H₃₃N₇O₈S₂).

Starting from acetylene derivatives 4 ~ 9, pyrollocytosine derivatives 15 ~ 20 were prepared by similarly following the procedure described in Example 10. Spectral and physical data for compds 15 ~ 20 are provided in the table below.

Compd Starting Material X Spectral & Physical Data 15 4 ¹H NMR (400MHz; DMSO_d6) δ 11.12 (s, 1H), 8.27 (s, 1H), 6.79 (t, J = 5.4 Hz, 1H), 6.00 (s, 1H), 4.68 (s, 2H), 4.14 (q, J = 7.2 Hz, 2H), 3.65 (t, J = 6.6, 2H), 3.39 (t, J = 6.2 Hz, 2H), 3.08 (m, 2H), 2.78 (t, J = 6.6 Hz, 2H), 1.37 (s, 9H), 1.20 (t, J = 7.2 Hz, 3H). Pale green foam/solid. 16 5 ¹H NMR (400MHz; DMSO_d6) δ 11.35 (s, 1H), 8.39 (s, 1H), 6.87 (t, J = 5.2 Hz, 1H), 6.21 (s, 1H), 4.70 (s, 2H), 4.41 (s, 2H), 4.15 (q, J = 7.2 Hz, 2H), 3.43 (m, 2H), 3.12 (m, 2H), 1.38 (s, 9H), 1.21 (t, J = 7.2 Hz, 3H). Pale yellow foam/solid. 17 6 ¹H NMR (400MHz; DMSO_d6) δ 11.20 (br s, 0.6H), 8.86 (br s, 0.4H), 8.57 (s, 0.2H), 8.35 (s, 0.8H), 6.83-6.76 (m, 1H), 6.00 (s, 0.8H), 5.76 (s, 0.2H), 4.75 (s, 0.3H), 4.70 (s, 1.7H), 4.55 (s, 0.3H), 4.30 (s, 1.7H), 4.14 (q, J = 7.2 Hz, 2H), 3.18 (m, 2H), 2.90-2.88 (m, 2H), 1.58 (m, 2H), 1.40-1.36 (m, 18H), 1.20 (t, 3H). Brown foam/solid. 18 7 ¹H NMR (400MHz; DMSO_d6) δ 11.57 (s, 1H), 11.33 (s, 1H), 8.50 (m, 1H), 8.37 (s, 1H), 7.44-7.31 (m, 10H), 6.22 (s, 1H), 5.22 (s, 2H), 5.03 (s, 2H), 4.70 (s, 2H), 4.44 (s, 2H), 4.14 (q, 2H), 3.57-3.53 (m, 4H), 1.21 (t, 3H). Pale brown solid. 19 8 ¹H NMR (500MHz; DMSO_d6) δ 11.32 (s, 1H), 8.38 (s, 1H), 6.71 (d, 1H), 6.20 (s, 1H), 4.70 (s, 2H), 4.41 (m, 2H), 4.14 (q, 2H), 3.65 (m, 1H), 3.37-3.34 (m, 1H), 3.26-3.22 (m, 1H), 1.37 (s, 9H), 1.20 (t, 3H), 1.02 (d, 3H). Pale brown solid. 20 9 ¹H NMR (500MHz; DMSO_d6) δ 11.13 (s, 1H), 8.25 (s, 1H), 6.73 (s, 1H), 5.99 (s, 1H), 4.68 (s, 2H), 4.12 (q, 2H), 3.67 (t, 2H), 3.48~3.27 (m, 10H), 3.04 (q, 2H), 2.78 (t, 2H), 1.38 (s, 9H), 1.36 (s, 1H), 1.19 (t, 3H). Brown solid.

Starting from pyrollocytosine derivatives 15, 16, 17, and 20, modified cytosine PNA monomers 21 ~ 24 were prepared by similarly following the procedures described in Examples 11 ~ 14. Spectral and physical data for compds 21 ~ 24 are provided in the table below.

Compd Starting Material X Spectral & Physical Data 21 15 ¹H NMR (400MHz; DMSO_d6) δ 11.06 (s, 1H), 8.36 (m, 1H), 8.28 (m, 1H), 8.14 (s, 0.6H), 8.08 (2, 0.4H), 7.72 (m, 2H), 6.78 (t, 1H), 5.98 (s, 1H), 4.91 (s, 1.2H), 4.80 (s, 0.8H), 4.51 (s, 0.8H), 4.29 (s, 1.2H), 4.24 (m, 1.2H), 4.06 (m, 2H), 3.86 (m, 0.8H), 3.64 (t, J = 6.4 Hz, 2H), 3.38 (t, J = 6.0 Hz, 2H), 3.07 (m, 2H), 2.78 (m, 2H), 1.37 (s, 9H). MS/ESI (m+1) = 660.2 (observed), MW = 659.8 (C₂₈H₃₃N₇O₈S₂). Brown foam/solid. 22 16 ¹H NMR (400MHz; DMSO_d6) δ 11.31 (s, 1H), 8.36 (m, 1H), 8.30-8.27 (m, 1.6H), 8.22 (s, 0.4H), 7.73 (m, 2H), 6.87 (t, J = 5.6 Hz, 1H), 6.20 (m, 1H), 4.94 (s, 1.2H), 4.83 (s, 0.8H), 4.52 (s, 0.7H), 4.41 (s, 2.1H), 4.30 (s, 1.1 H), 4.25 (m, 1.2H), 4.06 (m, 2H), 3.87 (m, 0.8H), 3.42 (t, 2H), 3.12 (m, 2H), 1.38 (s, 9H). MS/ESI (m+1) = 646.2 (observed), MW = 645.7 (C₂₇H₃₁N₇O₈S₂). Red foam/solid. 23 17 ¹H NMR (400MHz; DMSO_d6) δ 11.16 (br s, 1H), 8.36 (m, 1H), 8.28 (m, 1H), 8.21 (s, 0.6H), 8.15 (s, 0.4H), 7.73 (m, 2H), 6.77 (br s, 1H), 6.00 (br s, 1H), 4.92 (s, 1.2H), 4.82 (s, 0.8H), 4.52 (s, 0.9H), 4.30 (s, 3.1H), 4.25 (m, 1.2H), 4.07 (m, 2H), 3.87 (m, 0.8H), 3.19 (m, 2H), 2.89 (m, 2H), 1.59 (m, 2H), 1.41-1.36 (m, 18H); MS/ESI (m+23/MNa⁺) = 781.3 (observed), MW = 758.9 (C₃₃H₄₂N₈O₉S₂). Red foam/solid. 24 20 ¹H NMR (500MHz; DMSO_d6) δ 11.10 (m,1H), 8.35 (m, 1H), 8.28 (m, 1H), 8.14 (s, 0.6H), 8.08 (s, 0.4H), 7.72 (m, 2H), 6.76 (m, 1H), 5.97-5.96 (s, 1H), 4.90 (s, 1.2H), 4.80 (s, 0.8H), 4.51 (s, 0.8H), 4.29 (s, 1.2H), 4.25 (t, 1.2H), 4.08-4.04 (m, 2H), 3.86 (t, 0.8H), 3.66 (m, 2H), 3.47 (m, 2H), 3.41 (m, 2H), 3.32-3.30 (m, 4H), 3.27 (m, 2H), 3.04 (m, 2H), 2.77 (m, 2H), 1.37 (s, 9H), 1.35 (s, 9H). MS/ESI (m+23/MNa⁺) = 869.3 (observed), MW = 847.0 (C₃₇H₅₀N₈O₁₁S₂). Yellow solid.

6.8g of 2-chloroadenine dissolved in 68ml 1,3-diaminopropane and 68ml monomethoxyethanol was stirred at reflux for 24h, and the reaction mixture was concentrated in vacuo. The resulting residue was dissolved in 100ml THF and 100ml water, to which was slowly added 60g of BoC₂O dissolved in 70ml THF. The reaction mixture was stirred at RT for 6h, and then the organic solvent was removed under reduced pressure. The resulting aq layer was extracted twice with 100ml EA. The organic layer was washed with 0.5M aq citric acid and with brine, and dried over anhydrous magnesium sulfate. The organic layer was concentrated under reduced pressure and subjected to chromatographic separation (1:10 MeOH/MC) to obtain 4.07g of a compd protected with two Boc groups. This compound was dissolved in 100ml MeOH, to which was added slowly 45ml saturated aq sodium carbonate. The reaction solution was stirred at 50°C for 1h, and then concentrated in vacuo. The resulting residue was dissolved in 50ml MeOH and the insoluble material was filtered off. Then the filtrate was concentrated to afford 3.16g of compd 25 as a white solid. ¹H NMR (400MHz; DMSO_d6) δ 12.11 (br s, 1H), 7.63 (s, 1H), 6.78 (t, 1H), 6.55 (s, 2H), 6.07 (t, 1H), 3.20 (q, 2H), 2.96 (q, 2H), 1.60 (m, 2H), 1.37 (s, 9H).

Starting from 2-chloroadenine and a proper diamine, 2,6-diaminopurine derivatives 26 ∼ 30 were prepared by similarly following the procedure described in Example 25. Spectral and physical data for compounds 26 ~ 30 are provided in the table below.

Compd Starting Diamine L2 Spectral & Physical Data 26 -(CH₂)₂- ¹H NMR (400MHz; DMSO_d6) δ 12.20 (br s, 1H), 7.66 (s, 1H), 6.84 (t, 1H), 6.62 (s, 2H), 6.10 (t, 1H), 3.25 (q, 2H), 3.08 (q, 2H), 1.36 (s, 9H). Pale yellow solid. 27 -(CH₂)₄- ¹H NMR (500MHz; DMSO_d6) δ 12.07 (br s, 1H), 7.63 (s, 1H), 6.75 (s, 1H), 6.50 (s, 2H), 6.02 (s, 1H), 3.18 (q, 2H), 2.91 (q, 2H), 1.48-1.36 (m, 13H). Yellowish green solid. 28 -(CH₂)₅- ¹H NMR (400MHz; DMSO_d6) δ 12.14 (br s, 1H), 7.65 (s, 1H), 6.77 (t, 1H), 6.55 (s, 2H), 6.01 (s, 1H), 3.17 (m, 2H), 2.89 (q, 2H), 1.48 (m, 2H), 1.41-1.36 (m, 11H), 1.26 (m, 2H). Pale yellow solid. 29 -(CH₂)₇- ¹H NMR (500MHz; DMSO_d6) δ 12.11 (br s, 1H), 7.64 (s, 1H), 6.78 (t, J = 5.6 Hz, 1H), 6.56 (s, 2H), 6.04 (t, J = 5.5 Hz, 1H), 3.17 (td, J = 6.3, 6.3 Hz, 2H), 2.88 (td, J = 6.7, 6.7 Hz, 2H), 1.49-1.47 (m, 2H), 1.36-1.31 (m, 11H), 1.29-1.22 (m, 6H). Yellowish green solid. 30 ¹H NMR (500MHz; DMSO_d6) δ 12.15 (s, 1H), 7.64 (s, 1H), 6.84 (t, 1H), 6.56 (s, 2H), 6.05 (t, 1H), 3.48 (t, 2H), 3.39-3.34 (m, 4H), 3.07 (q, 2H), 1.37 (s, 9H). Yellow foam.

11g of 2-chlorohypoxanthine and 4.96ml 1,2-diaminopropane (racemic) were dispersed in 33ml monomethoxyethanol, and stirred for 24h at 130°C. The solvent was removed in vacuo, and the resulting residue was dissolved in 97ml THF and 97ml water, to which was slowly added 22.8g of Boc₂O dissolved in 64ml THF. The reaction mixture was stirred at RT for 6h, and EA was added to the solution. The resulting precipitate was collected by filtration to obtain compd 31 as a grey solid. ¹H NMR (500MHz; DMSO_d6) δ 12.42 (s, 1H), 10.44 (br s, 1H), 7.61 (s, 1H), 6.76 (d, 1H), 6.27 (m, 1H), 3.67 (m, 1H), 3.32 (m, 1H), 3.14 (m, 1H), 1.36 (s, 9H), 1.02 (d, 3H).

To a stirred solution of 3.16g of compd 25 dissolved in 100ml DMF, was added 480mg of 55% NaH in mineral oil. The reaction solution was stirred for 2h, after which was slowly added 1.98ml ethyl bromoacetate. 2h later, the reaction mixture was concentrated in vacuo, and purified by column chromatography (1:10 EtOH/EA) to give 2.92g of diaminopurine analog 32 as a pale yellow solid. ¹H NMR (400MHz; DMSO_d6) δ 7.67 (s, 1H), 6.80 (t, 1H), 6.71 (s, 2H), 6.28 (t, 1H), 4.85 (s, 2H), 4.15 (q, 2H), 3.20 (q, 2H), 2.94 (q, 2H), 1.57 (m, 2H), 1.37 (s, 9H), 1.21 (t, 3H).

To a stirred solution of 4.68g of compd 32 dissolved in 100ml DMF, was added at RT 13.2g of N-(benzyloxycarbonyl)-N'-methyl-imidazolium triflate. 12h later the reaction mixture was concentrated under reduced pressure, and subjected to column chromatography (5% MeOH in MC) to yield 5.4g of compd 33 as a white solid. ¹H NMR (400MHz; DMSO_d6) δ 10.19 (s, 1H), 7.92 (s, 1H), 7.45-7.33 (m, 5H), 6.88 (t, 1H), 6.77 (t, 1H), 5.18 (s, 2H), 4.94 (s, 2H), 4.16 (q, 2H), 3.25 (q, 2H), 2.95 (q, 2H), 1.60 (m, 2H), 1.36 (s, 9H), 1.21 (t, 3H).

5.4g of compd 33 and 950mg of LiOH were dissolved in 40ml THF and 40ml water, and stirred at RT for 1h. THF was removed in vacuo, and the resulting aq solution was acidified to pH 3 with 1M aq HCl, and then extracted with EA. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting residue and 2.92g of ethyl 2-N-[2-{(benzo[d]thiazole-2-sulfonyl)amino}ethyl]glycinate were dissolved in 240ml DMF, to which were added at RT 1.95g of EDCI and 1.38g of HOBt. The reaction mixture was stirred for 20h, concentrated under reduced pressure, and dissolved in MC. The MC solution was washed with 1M aq HCl, concentrated in vacuo, and then purified by column chromatography (5% MeOH/MC) to obtain 2.7g of compd 34 as a pale yellow foam. ¹H NMR (400MHz; DMSO_d6) δ 10.18 (m, 1H), 8.97 (br s, 0.6H), 8.80 (br s, 0.4H), 8.28 (d, 1H), 8.18 (m, 1H), 7.80 (s, 0.6H), 7.76 (s, 0.4H), 7.66 (m, 2H), 7.46-7.32 (m, 5H), 6.77 (m, 2H), 5.18 (s, 2H), 5.10 (s, 1.2H), 4.89 (s, 0.8H), 4.45 (s, 0.8H), 4.17 (q, 0.8H), 4.07-4.00 (m, 2.4H), 3.68 (m, 1.2H), 3.47 (m, 1.2H), 3.41 (m, 0.9H), 3.22 (m, 2.7H), 2.94 (m, 2H), 1.59 (m, 2H), 1.36 (s, 9H), 1.31-1.12 (m, 3H).

2.7g of compd 34 and 340mg of LiOH were dispersed in 15ml THF and 20ml water, and stirred for 30 min at RT. THF was removed under reduced pressure. Then the resulting aq layer was acidified to pH 3 with 1M aq HCl, and extracted with EA. The EA layer was dried over anhydrous sodium sulfate and concentrated in vacuo to obtain 2.48g of a crude product. The crude product and 716mg of EDCI dissolved in 70ml DMF were stirred at RT for 20h. The solvent was removed under reduced pressure, and the resulting residue was dissolved in MC and washed with 1M aq HCl and then with water. The organic layer was concentrated in vacuo and purified by column chromatography (acetone) to obtain 1.4g of compd 35 as a white foam. ¹H NMR (400MHz; DMSO_d6) δ 10.16 (s, 1H), 8.35 (m, 1H), 8.26 (m, 1H), 7.81 (s, 0.6H), 7.77 (s, 0.4H), 7.72 (m, 2H), 7.45-7.31 (m, 5H), 6.78 (m, 2H), 5.18 (s, 2H), 5.12 (s, 1.2H), 5.01 (s, 0.8H), 4.55 (s, 0.8H), 4.29-4.27 (m, 2.4H), 4.09 (m, 2H), 3.88 (m, 0.8H), 3.26 (m, 2H), 2.95 (m, 2H), 1.61 (m, 2H), 1.36 (s, 9H); MS/ESI (m+1) = 779.2 (observed), MW = 778.9 (C₃₄H₃₈N₁₀O₈S₂).

Starting from 2,6-diaminopurine derivatives 26 ∼ 30, modified adenine PNA monomers 36 ∼ 40 were prepared by similarly following the procedures described in Examples 32 ∼ 35. Spectral and physical data for compds 36 ∼ 40 are provided in the table below.

Compd Starting Material L₂ Spectral & Physical Data 36 26 -(CH₂)₂- ¹H NMR (400MHz; DMSO_d6) δ 10.17 (s, 1H), 8.36 (m, 1H), 8.26 (m, 1H), 7.82 (s, 0.6H), 7.78 (s,0.4H), 7.72 (m, 2H), 7.45-7.31 (m, 5H), 6.79 (2H), 5.18 (s, 2H), 5.12 (s, 1.2H), 5.01 (s, 0.8H), 4.55 (s, 0.8H), 4.29-4.25 (m, 2.4H), 4.09 (m, 2H), 3.87 (m, 0.8H), 3.29 (m, 2H), 3.11 (m, 2H), 1.33 (d, 9H). MS/ESI (m+1) = 765.2 (observed), MW = 764.8 (C₃₃H₃₆N₁₀O₈S₂). White foam. 37 27 -(CH₂)₄- ¹H NMR (400MHz; DMSO_d6) δ 10.10 (s, 1H), 8.36 (m, 1H), 8.26 (m, 1H), 7.80 (s, 0.6H), 7.76-7.71 (m, 2.4H), 7.46-7.31 (m, 5H), 6.81-6.73 (m, 2H), 5.18 (s, 2H), 5.12 (s, 1.2H), 5.01 (s, 0.8H), 4.55 (s, 0.8H), 4.30-4.25 (m, 2.4H), 4.09 (m, 2H), 3.88 (m, 0.8H), 3.26 (m, 2H), 2.90 (m, 2H), 1.50-1.36 (m, 13H); MS/ESI (m+1) = 793.3 (observed), MW = 792.9 (C₃₅H₄₀N₁₀O₈S₂). Yellowish red foam/solid. 38 28 -(CH₂)₅- ¹H NMR (400MHz; DMSO_d6) δ 10.09 (s, 1H), 8.35 (m, 1H), 8.26 (m, 1H), 7.80 (s, 0.6H), 7.76 (s, 0.4H), 7.74-7.72 (m, 2.0H), 7.46-7.31 (m, 5H), 6.79-6.72 (m, 2H), 5.18 (s, 2H), 5.12 (s, 1.2H), 5.01 (s, 0.8H), 4.56 (s, 0.8H), 4.30-4.27 (m, 2.4H), 4.09 (m, 2H), 3.88 (m, 0.8H), 3.25 (m, 2H), 2.89 (m, 2H), 1.49 (m, 2H), 1.36 (m, 11H), 1.25 (m, 2H); MS/ESI (m+1) = 807.3 (observed), MW = 806.9 (C₃₆H₄₂N₁₀O₈S₂)- Yellow foam/solid. 39 29 -(CH₂)₇- ¹H NMR (500MHz; DMSO_d6) δ 10.11 (d, J = 3.1 Hz, 1H), 8.37-8.34 (m, 1H), 8.28-8.24 (m, 1H), 7.80 (s, 0.6H), 7.76 (s, 0.4 Hz), 7.75-7.70 (m, 2H), 7.75-7.31 (m, 5H), 6.82-6.74 (m, 2H), 5.18 (s, 2H), 5.12 (s, 1.2H), 5.01 (s, 0.8H), 4.58 (s, 0.8H), 4.29 (m, 1.2H), 4.27 (q, J = 4.9 Hz, 1H), 4.06-4.03 (m, 2H), 3.88 (t, J = 5.2 Hz, 1H), 3.26-3.20 (m, 2H), 2.88-2.85 (m, 2H), 1.51-1.45 (m, 2H), 1.39-1.32 (m, 11H), 1.28-1.15 (m, 6H). MS/ESI (m+1) = 834.8 (observed), MW = 835.0 (C₃₈H₄₆N₁₀O₈S₂). Reddish yellow foam/solid. 40 30 ¹H NMR (400MHz; DMSO_d6) δ 10.14 (s, 1H), 8.35 (m, 1H), 8.26 (m, 1H), 7.82 (s, 0.6H), 7.78 (s, 0.4H), 7.73 (m, 2H), 7.46-7.31 (m, 5H), 6.81-6.74 (m, 2H), 5.18 (s, 2H), 5.13 (s, 1.2H), 5.02 (s, 0.8H), 4.55 (s, 0.8H), 4.30-4.26 (m, 2.4H), 4.09 (m, 2H), 3.88 (m, 0.8H), 3.50 (m, 2H), 3.43-3.38 (m, 4H), 3.07 (m, 2H), 1.36 (s, 9H); MS/ESI (m+1) = 809.3 (observed), MW = 808.9 (C₃₅H₄₀N₁₀O₉S₂). Pale yellow foam.

To a stirred solution of 4.69g of compd 31 in 47ml DMF, was added 790mg of 55% NaH in mineral oil and the reaction solution was stirred for 2h. After 1.85ml ethyl bromoacetate was slowly added, the reaction solution was stirred for another 2h. The reaction mixture was concentrated in vacuo and purified by column chromatography (5:95 MeOH/MC) to obtain 5.04g of compd 41 as a pale yellow solid. ¹H NMR (500MHz; DMSO_d6) δ 10.55 (s, 1H), 7.67 (s, 1H), 6.74 (d, 1H), 6.40 (m, 1H), 4.87 (s, 2H), 4.17 (q, 2H), 3.65 (m, 1H), 3.28 (m, 1H), 3.16 (m, 1H), 1.36 (s, 9H), 1.21 (t, 3H), 1.01 (d, 3H).

To 146g of [2-{2-(t-butoxycarbonylamino)ethoxylethyl]methane sulfonate was dissolved in 500ml DMF, was added 134g of sodium azide. The reaction mixture was stirred at 70°C for 20h, and then concentrated under reduced pressure. The resulting residue was dissolved in 1,200ml water and extracted with EA. The organic layer was dried over anhydrous sodium sulfate and concentrated in vacuo. The resulting residue was dissolved in 2,000ml THF, to which was added 162g of triphenylphosphine. The reaction mixture was stirred at RT for 2h, after which was added 200ml water. The reaction mixture was stirred at RT for 18h and concentrated to 500ml under reduced pressure. Then the resulting precipitate was filtered off. The filtrate was further concentrated under reduced pressure to remove THF, and washed with MC. The aq layer was concentrated to obtain 86.2g of compd 42 as a liquid. ¹H NMR (400MHz; CDCl₃) δ 4.96 (br s, 1H), 3.54-3.48 (m, 4H), 3.34 (q, 2H), 2.88 (t, 2H), 1.48-1.46 (m, 11H).

6.3g of compd 42 and 2.0g of 2-bromohypoxanthine were dispersed in 55ml monomethoxyethanol and 17.5ml water. The reaction mixture was stirred at reflux for 16h, and the solvent was removed under reduced pressure. Then the concentrate was stirred in 20ml MC and 10ml water for 30 min, and the resulting precipitate was collected by filtration to obtain 2.1g of compd 43 as a pale yellow solid. ¹H NMR (500MHz; DMSO_d6) δ 12.43 (br s, 1H), 10.45 (br s, 1H), 7.89 (s, 0.2H), 7.61 (s, 0.8H), 6.77 (m, 1H), 6.34 (s, 0.8H), 6.12 (s, 0.2H), 3.52 (t, 2H), 3.41 (m, 4H), 3.09 (q, 2H), 1.36 (s, 9H).

2-{3-(t-butoxycarbonylamino)propyloxy}ethylamine and 2-bromohypoxanthine were reacted by similarly following the procedure described in Example 43 to yield compound 44 as a white solid. ¹H NMR (500MHz; DMSO_d6) δ 12.43 (br s, 1H), 10.45 (br s 1H), 7.61 (m, 1H), 6.80 (t, 1H), 6.30 (s, 0.7H), 6.08 (s, 0.3H), 3.49 (t, 2H), 3.41 (t, 4H), 2.99 (q, 2H), 1.61 (m, 2H), 1.37 (s, 9H).

A mixture of 10g of chlorohypoxanthine and 19.6ml 1,3-diaminopropane dispersed in 40ml monomethoxyethanol was stirred at 130°C for 10h. Then the solvent was removed under reduced pressure and the resulting residue was dissolved in 150ml THF and 150ml water, to which was added slowly 19.2g of Boc₂O dissolved in 100ml THF. The mixture was stirred at RT for 6h. After EA was added, the resulting precipitate was collected by filtration to obtain 6.31g of compd 45 as a dark green solid. ¹H NMR (400MHz; DMSO_d6) δ 11.13 (br s, 1H), 7.64 (s, 1H), 6.87 (s, 1H), 6.31 (s, 1H), 3.23 (q, 2H), 2.98 (m, 2H), 1.62 (m, 2H), 1.38 (s, 9H).

Guanine derivatives 46 ∼ 47 were prepared using a proper diamine by similarly following the procedure described in Example 45. Spectral and physical data for compds 46 ∼ 47 are provided in the table below.

Compd Diamine used n Spectral & Physical Data 46 Ethylene diamine 2 ¹H NMR (500MHz; DMSO_d6) δ 12.43 (br s, 1H), 10.61 (br, 1H), 7.62 (s, 1H), 6.93 (t, 1H), 6.32 (s, 1H), 3.29 (q, 2H),3.10 (q, 2H), 1.37 (s, 9H). Grey solid. 47 Pentylene diamine 5 ¹H NMR (500MHz; DMSO_d6) δ 12.44 (s, 1H), 10.35 (s, 1H), 7.60 (s, 1H), 6.80 (m, 1H), 6.29 (m, 1H), 3.21 (m, 2H), 2.90 (m, 2H), 1.49 (m, 2H), 1.39-1.35 (m, 11H), 1.27-1.23 (m, 2H). Pale brown solid.

Compds 43 ∼ 46 were transformed into compds 48 ∼ 51 by similarly following the procedure described in Example 32. Spectral and physical data for compounds 48 ∼ 51 are provided in the table below.

Compd Starting Material L₃ Spectral & Physical Data 48 43 ¹H NMR (500MHz; DMSO_d6) δ 10.67 (s, 1H), 7.69 (s, 1H), 6.78 (m, 1H), 6.15 (t, 1H), 4.87 (s, 2H), 4.15 (q, 2H), 3.51 (m, 2H), 3.41 (m, 4H), 3.10 (m, 2H), 1.37 (s, 9H), 1.20 (t, 3H). White foam/solid. 49 44 ¹H NMR (500MHz; DMSO_d6) δ 10.57 (s, 1H), 7.69 (s, 1H), 6.79 (m, 1H), 6.44 (m, 1H), 4.87 (s, 2H), 4.16 (q, 2H), 3.48 (t, 2H), 3.40 (m, 4H), 2.99 (q, 2H), 1.61 (m, 2H), 1.37 (s, 9H), 1.21 (t, 3H). Yellow foam/solid. 50 45 ¹H NMR (500MHz; DMSO_d6) δ 10.64 (s, 1H), 7.68 (s, 1H), 6.91 (t, 1H), 6.47 (s, 1H), 4.88 (s, 2H), 4.16 (q, 2H), 3.28 (q, 2H), 3.08 (q, 2H), 1.36 (s, 9H), 1.21 (t, 3H). Dark red solid. 51 46 ¹H NMR (400MHz; DMSO_d6) δ 10.44 (br s, 1H), 7.66 (s, 1H), 6.77 (m, 1H), 6.41 (m, 1H), 4.86 (s, 2H), 4.16 (q, 2H), 3.21 (q, 2H), 2.89 (q, 2H), 1.48 (m, 2H), 1.41-1.36 (m, 11H), 1.28-1.19 (m, 5H). Dark grey solid.

Starting from guanine derivatives 48, 49 and 51, modified guanine PNA monomers 52 ∼ 54 were prepared by similarly following the procedures described in Examples 34 ∼ 35. Spectral and physical data for compds 52 ∼ 54 are provided in the table below.

Compd Starting Material L₃ Spectral & Physical Data 52 48 ¹H NMR (400MHz; DMSO_d6) δ 10.61 (m, 1H), 8.36 (m, 1H), 8.25 (m, 1H), 7.76-7.65 (m, 3H), 6.78 (t, 1H), 6.54 (m, 1H), 5.07 (s, 1.2H), 4.96 (s, 0.8H), 4.54 (s, 0.8H), 4.30 (s, 1.2H), 4.25 (m, 1.2H), 4.07 (m, 2H), 3.88 (m, 0.8H), 3.49 (m, 2.4H), 3.40 (m, 3.6H), 3.09 (m, 2H), 1.36 (s, 9H); MS/ESI (m+1) = 676.1 (observed), MW = 675.8 (C₂₇H₃₃N₉O₈S₂). Dark brown foam/solid. 53 49 ¹H NMR (400MHz; DMSO_d6) δ 10.69 (s, 1H), 8.36 (m, 1H), 8.25 (m, 1H), 7.73 (m, 2H), 7.64-7.60 (m, 1H), 6.80 (t, 1H), 6.65 (br s, 1H), 5.05 (s, 1.2H), 4.94 (s, 0.8H), 4.54 (s, 0.8H), 4.29 (s, 1.2H), 4.24 (m, 1.2H), 4.07 (m, 2H), 3.87 (m, 0.8H), 3.46∼3.39 (m, 6H), 2.97 (m, 2H), 1.60 (m, 2H), 1.36 (s, 9H); MS/ESI (m+1) = 689.8 (observed), MW = 689.8 (C₂₈H₃₅N₉O₈S₂). Yellow foam/solid. 54 51 ¹H NMR (400MHz; DMSO_d6) δ 10.42-10.40 (m, 1H), 8.37-8.32 (m, 1H), 8.28-8.25 (m, 1H), 7.73-7.70 (m, 2H), 7.58-7.54 (m, 1H), 6.76 (t, 1H), 6.39-6.38 (m, 1H), 5.03 (s, 1.2H), 4.92 (s, 0.8H), 4.54 (s, 0.8H), 4.29 (s, 1.2H), 4.25 (m, 1.2H), 4.08-4.07 (m, 2H), 3.87 (m, 0.8H), 3.18 (m, 2H), 2.89 (m, 2H), 1.47 (m, 2H), 1.40-1.30 (m, 11H), 1.24 (m, 2H). MS/ESI (m+23/MNa⁺) = 696.2 (observed), MW = 673.8 (C₂₈H₃₅N₉O₇S₂). Red foam/solid.

Compd 55 was prepared from compd 26 by similarly following the procedure for Example 32. Pale yellow solid. ¹H NMR (400MHz; DMSO_d6) δ 7.70 (s, 1H), 6.84 (t, 1H), 6.79 (s, 2H), 6.30 (t, 1H), 4.87 (s, 2H), 4.16 (q, 2H), 3.25 (q, 2H), 3.08 (q, 2H), 1.37 (s, 9H), 1.22 (t, 3H).

To 4.42g of compd 55 dissolved in 22ml MC, was slowly added 22ml TFA at 0°C, and the solution was stirred for 2.5h. The reaction solution was concentrated under reduced pressure, to which was added 100ml diethyl ether. The resulting precipitate was collected by filtration to obtain 5.79g of a pale brown solid intermediate product. 3.9g of the intermediate was dissolved in 39ml MC, to which was added slowly 6.9ml TEA at 0°C. The solution was stirred for 15min at RT, to which was added 2.48g of 1,3-bis(benzyloxycarbonyl)-2-(methylthio)pseudourea. Then the reaction mixture was stirred for another 24h, and washed with 0.5M aq HCl The organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure to yield 4.58g of compd 56 as a pale yellow solid. ¹H NMR (500MHz; DMSO_d6) δ 11.59 (s, 1H), 8.56 (t, 1H), 7.69 (s, 1H), 7.39-7.29 (m, 10H), 6.75 (s, 2H), 6.53 (s, 1H), 5.15 (s, 2H), 5.02 (s, 2H), 4.86 (s, 2H), 4.13 (q, 2H), 3.50 (q, 2H), 3.37 (m, 2H), 1.19 (t, 3H).

4.54g of compd 56 and 8.22g of N-(benzyloxycarbonyl)-N'-methylimidazolium triflate were dissolved in 90ml DMF, and stirred for 29h at RT. The solvent was removed under reduced pressure, and the resulting residue was purified by column chromatography (1:3 hexane/EA) to afford 3.06g of compd 57 as a white foam/solid. ¹H NMR (500MHz; DMSO_d6) δ 11.60 (s, 1H), 10.25 (s, 1H), 8.57 (t, 1H), 7.95 (s, 1H), 7.45-7.29 (m, 15H), 7.14 (t, 1H), 5.18 (s, 2H), 5.14 (s, 2H), 5.02 (s, 2H), 4.95 (s, 2H), 4.15 (q, 2H), 3.54 (q, 2H), 3.42 (q, 2H), 1.19 (t, 3H).

Compd 58 was prepared from compd 27 as a reddish yellow foam/solid by similarly following the procedure described in Example 32. ¹H NMR (500MHz; DMSO_d6) δ 7.67 (s, 1H), 6.79 (t, 1H), 6.69 (s, 2H), 6.30 (m, 1H), 4.85 (s, 2H), 4.15 (q, 2H), 3.22-3.17 (m, 2H), 2.93-2.89 (m, 2H), 1.45 (m, 2H), 1.40-1.36 (m, 11H), 1.21 (t, 3H).

Compd 59 was prepared from compd 58 as a pale yellow foam/solid by similarly following the procedures described in Examples 56 ∼ 57. ¹H NMR (500MHz; DMSO_d6) δ 11.49 (s, 1H), 10.12 (s, 1H), 8.28 (t, 1H), 7.91 (s, 1H), 7.45-7.31 (m, 5H), 6.95 (t, 1H), 5.17 (s, 2H), 4.93 (s, 2H), 4.16 (q, 2H), 3.28 (m, 4H), 1.51 (m, 4H), 1.46 (s, 9H), 1.38 (s, 9H), 1.21 (t, 3H).

Compd 60 was prepared from compd 28 as a reddish yellow foam/solid by similarly following the procedure described in Example 32. ¹H NMR (500MHz; DMSO_d6) δ 7.67 (s, 1H), 6.78 (t, 1H), 6.69 (s, 2H), 6.28 (m, 1H), 4.85 (s, 2H), 4.15 (q, 2H), 3.18 (q, 2H), 2.89 (q, 2H), 1.47 (m, 2H), 1.40-1.34 (m, 11H), 1.25 (m, 2H), 1.21 (t, 3H).

To 6.98g of compd 60 dissolved in 100ml THF, were added 7.95g of Boc₂O and 186mg of 4-(N,N-dimethylamino)pyridine, and the solution was stirred for 10min. Then the solution was mixed with 4.62ml TEA, stirred for 30min, slowly heated to 50°C, and then stirred for another 24h at the temperature. The reaction solution was concentrated in vacuo, and the resulting residue was dissolved in 170ml EA and washed in series with 0.5M aq HCl and water. The organic layer was dried over anhydrous sodium sulfate, concentrated, and subjected to chromatographic separation (1:1 hexane/MC → MC) to obtain compd 61 as a yellow foam/solid. ¹H NMR (500MHz; DMSO_d6) δ 8.05 (s, 1H), 7.23 (t, 1H), 6.77 (t, 1H), 5.00 (s, 2H), 4.19 (q, 2H), 3.25 (q, 2H), 2.91 (q, 2H), 1.53 (m, 2H), 1.40-1.39 (m, 29H), 1.28 (m, 2H), 1.22 (t, 3H).

Compd 50 was converted to compd 62 as a white solid by similarly following the procedure described in Example 57. ¹H NMR (500MHz; DMSOd₆) δ 11.59 (s, 1H), 10.68 (s, 1H), 8.50 (t, 1H), 7.68 (s, 1H), 7.42-7.29 (m, 10H), 6.58 (m, 1H), 5.13 (s, 2H), 5.02 (s, 2H), 4.86 (s, 2H), 4.12 (q, 2H), 3.50 (m, 2H), 3.46 (m, 2H), 1.18 (t, 3H).

To 2.57g of compd 57 dissolved in 7.1ml THF and 7.1ml water, was added 340mg of LiOH at 0°C, and the solution was stirred at RT for 40min. The reaction solution was acidified to pH 5~6 with 1N aq HCl at 0°C, and the resulting solid was collected by filtration to yield 2.33g of compd 63 as a white solid. ¹H NMR (500MHz; DMSO_d6) δ 11.59 (s, 1H), 10.21 (s, 1H), 8.57 (t, 1H), 7.93 (s, 1H), 7.45-7.28 (m, 15H), 7.12 (t, 1H), 5.17 (s, 2H), 5.13 (s, 2H), 5.02 (s, 2H), 4.83 (s, 2H), 3.53 (q, 2H), 3.42 (q, 2H).

To 1.6g of compd 63 dissolved in 30ml DMF, were added at 0°C 660mg of EDCI and 910mg of Fmoc-Aeg-OtBu. The reaction solution was stirred for 2h at RT and then concentrated under reduced pressure. The resulting residue was dissolved in 50ml MC and washed with 0.5M aq HCl, and the organic layer was dried over anhydrous sodium sulfate. Then the organic layer was concentrated and subjected to chromatographic separation (65:1 MC/MeOH) to obtain 500mg of compd 64 as a white solid. ¹H NMR (500MHz; DMSO_d6) δ11.59 (s, 0.4H), 11.58 (s, 0.6H), 10.21 (s, 1H), 8.55 (m, 1H), 7.47-7.28 (m, 20H), 7.06 (br, 1H), 5.17-4.89 (m, 8H), 4.34-4.28 (m, 2.8H), 4.20 (m, 1H), 3.95 (s, 1.2H), 3.52 (m, 3.4H), 3.43 (m, 2.2H), 3.34 (m, 1.7H), 3.12 (m, 0.7H), 1.43 (s, 3H), 1.34 (s, 6H).

To 460mg of compd 64 dissolved in 3.6ml MC, was slowly added 3.6ml TFA at 0°C. The reaction solution was stirred at RT for 3.5h, and then 50ml diethyl ether was added. The resulting precipitate was collected by filtration to yield 430mg of compd 65 as a white solid. ¹H NMR (400MHz; DMSO_d6) δ 11.57 (s, 1H), 10.77 (br s, 1H), 8.66 (s, 1H), 8.54 (s, 1H), 7.87 (m, 2H), 7.63 (m, 2H), 7.50-7.28 (m, 21H), 5.26-4.96 (m, 8H), 4.34-4.18 (m, 4H), 4.03 (s, 1H), 3.52-3.36 (m, 7H), 3.13 (m, 1H). MS/ESI (m+1) = 1019.4 (observed), MW = 1018.0 (C₅₃H₅₁N₁₁O₁₁).

Compd 59 was converted to compd 66 as a white foam/solid by similarly following the procedures described in Examples 63 ∼ 65. ¹H NMR (500MHz; DMSO_d6) δ 12.84 (br s, 1H), 11.50 (s, 1H), 10.14-10.13 (m, 1H), 8.28 (m, 1H), 7.88 (m, 2H), 7.80-7.77 (m, 1H), 7.68-7.66 (m, 2H), 7.49 (t, 1H), 7.45-7.29 (m, 9H), 6.90 (m, 1H), 5.17 (s, 2H), 5.07 (s, 1.2H), 4.89 (s, 0.8H), 4.35-4.18 (m, 3H), 4.00 (s, 1H), 3.52 (m, 1H), 3.35-3.25 (m, 6H), 3.12 (m, 1H), 1.49 (m, 4H), 1.44 (d, 9H), 1.37 (d, 9H). MS/ESI (m+1) = 978.4 (observed), MW = 978.1 (C₄₉H₅₉N₁₁O₁₁).

Compd 18 was converted to compd 67 as a pale yellow solid by similarly following the procedures described in Examples 63 ∼ 65. ¹H NMR (500MHz; DMSO_d6) δ 11.99 (br s, 1H), 11.57 (br, 1H), 8.56 (m, 1H), 8.48-8.45 (m, 1H), 7.89-7.87 (m, 2H), 7.70-7.65 (m, 2H), 7.49-7.26 (m, 15H), 6.36-6.33 (m, 1H), 5.20 (s, 2H), 5.03-5.01 (m, 3.3H), 4.83 (s, 0.7H), 4.49-4.17 (m, 5.7H), 4.01 (m, 1.3H), 3.57-3.11 (m, 8H); MS/ESI (m+1) = 899.7 (observed), MW = 898.9 (C₄₇H₄₆N₈O₁₁).

Compd 62 was converted to compd 68 as a white foam/solid by following the procedures described in Examples 63 ∼ 65. ¹H NMR (500MHz; DMSO_d6) δ 11.58 (s, 1H), 10.88 (s, 1H), 8.51 (m, 1H), 7.93 (m, 1H), 7.87 (m, 2H), 7.64 (m, 2H), 7.47 (t, 1H), 7.41-7.26 (m, 14H), 6.66 (br, 1H), 5.16-4.89 (m, 8H), 4.34-4.18 (m, 3.8H), 4.00 (m, 1.2H), 3.50-3.35 (m, 7H), 3.13 (m, 1H); MS/ESI (m+1) = 885.3 (observed), MW = 884.9 (C₄₅H₄₄N₁₀O₁₀).

Compound 16 was hydrolyzed to compound 69 as a pale brown solid by similarly following the procedure described in Example 11. ¹H NMR (500MHz; DMSO_d6) δ 13.03 (br s, 1H), 11.31 (s, 1H), 8.37 (s, 1H), 6.85 (t, 1H), 6.19 (s, 1H), 4.63 (s, 2H), 4.40 (s, 2H), 3.42 (t, 2H), 3.11 (q, 2H), 1.37 (s, 9H).

3.6g of compd 69, 3.6g of Fmoc-Aeg-OMe, 2.5g of EDCI 1.73g of HOBt, and 2.24ml DIEA were dissolved in 70ml DMF, and stirred at RT for 1.5h. The reaction solvent was removed under reduced pressure, and the resulting residue was dissolved in 100ml MC and washed in series with 1M aq HCl, distilled water, and brine. The organic layer was dried over anhydrous sodium sulfate, concentrated in vacuo, and purified by column chromatography (100:2 MC/MeOH) to afford 2.5g of compd 70 as a yellow foam/solid. ¹H NMR (500MHz; DMSO_d6) δ 11.30 (s, 1H), 8.24 (s, 0.65H), 8.21 (s, 0.35H), 7.89-7.87 (m, 2H), 7.71-7.67 (m, 2H), 7.48-7.25 (m, 5H), 6.87 (t, 1H), 6.17 (s, 0.7H), 6.15 (s, 0.3H), 4.93 (s, 1.3H), 4.74 (s, 0.7H), 4.40-4.39 (m, 2.7H), 4.35-4.21 (m, 3H), 4.08 (s, 1.3H), 3.73 (s, 0.8H), 3.62 (s, 2.2H), 3.51 (t, 1.4H), 3.43-3.30 (m, 3.6H), 3.13-3.10 (m, 3H), 1.37 (s, 9H).

To 5.0g of compd 70 dissolved in 75ml 1:1:1 acetonitrile/acetone/water, was slowly added at 0°C 28.5ml 2.5N aq LiOH. The reaction solution was stirred for 10min and neutralized with 20% aq citric acid. After the solution pH was adjusted to 8 with saturated aq sodium bicarbonate, 516mg of Fmoc-OSu was added to the solution and the solution was stirred for 2h at RT. Then the solution was acidified to pH 3 with 20% aq citric acid and stirred for 90min at 0°C. The resulting precipitate was collected by filtration to give 4.0g of compd 71 as a yellowish green solid. ¹H NMR (500MHz; DMSO_d6) δ 12.02 (br, 1H), 8.51-8.49 (m, 1H), 7.89-7.88 (d, 2H), 7.70-7.50 (m, 2H), 7.49 (t, 1H), 7.42-7.28 (m, 4H), 6.87 (t, 1H), 6.36 (s, 0.7H), 6.33 (s, 0.3H), 5.02 (s, 1.2H), 4.84 (0.8H), 4.43-4.42 (m, 2.4H), 4.34-4.19 (m, 3.2H), 4.01 (s, 1.4H), 3.48 (t, 1.2H), 3.44-3.41 (m, 2.1H), 3.37-3.29 (m, 2H), 3.12-3.10 (m, 2.7H), 1.37 (s, 9H); MS/ESI (m+1) = 689.3 (observed), MW = 688.7 (C₃₅H₄₀N₆O₉).

Compd 51 was converted to compd 72 as a white foam/solid by similarly following the procedures described in Examples 69 ∼ 71. ¹H NMR (500MHz; DMSO_d6) δ 13.01 (br, 1H), 10.52-10.46 (m, 1H), 7.88 (d, 2H), 7.65 (d, 2H), 7.54 (s, 0.5H), 7.50 (s, 0.5H), 7.48 (m, 1H), 7.40 (t, 2H), 7.31 (m, 2H), 6.81 (t, 0.5H), 6.72 (t, 0.5H), 6.52-6.48 (m, 1H), 4.98 (s, 1H), 4.77 (s, 1H), 4.33 (d, 1H), 4.23-4.21 (m, 2H), 4.05 (m, 1H), 3.96 (s, 1H), 3.50 (m, 1H), 3.35 (m, 2H), 3.21 (m, 2H), 3.14 (q, 1H), 2.88 (m, 2H), 1.46 (q, 2H), 1.39-1.35 (m, 11H), 1.23 (m, 2H); MS/ESI (m+1) = 717.4 (observed), MW = 716.8 (C₃₆H₄₄N₈O₈)

Compd 61 was converted to compd 73 as a white foam/solid by similarly following the procedures described in Examples 69 ∼ 71. ¹H NMR (500MHz; DMSO_d6) δ 12.71 (br s, 1H), 7.90-7.87 (m, 3H), 7.67 (m, 2H), 7.44-7.39 (m, 3H), 7.31 (m, 2H), 7.07 (m, 1H), 6.69 (m, 1H), 5.11 (s, 1.2H), 4.93 (s, 0.8H), 4.37-4.21 (m, 3.8H), 4.01 (s, 1.2H), 3.52 (m, 1H), 3.36 (m, 2H), 3.23 (m, 2H), 3.13 (m, 1H), 2.88 (m, 2H), 1.49 (m, 2H), 1.38-1.35 (m, 27H), 1.27-1.25 (m, 4H); MS/ESI (m+1) = 916.5 (observed), MW = 916.0 (C₄₆H₆₁N₉O₁₁).

Preparation of PNA Oligomers: PNA monomers o, which were synthesized according to Scheme 4, were sent to Panagene, Inc (www.panagene.com, Daejon, South Korea) to prepare PNA oligomers of Formula I by Panagene according to the method described in the literature or with minor modification(s) thereof. (Org. Lett. vol 9, 3291-3293, 2006) PNA oligomers were received from Panagene as characterized by MALDI-TOF and analyzed by C₁₈-reverse phase HPLC. PNA oligomers received from Panagene were used without further purification.

PNA monomers q of Scheme 5 were used to synthesize PNA oligomers of Formula I according to the method disclosed in the prior art or with minor modification(s) thereof. (USP 6,133,444) Those PNA oligomers were purified by C₁₈-reverse phase HPLC (aq acetonitrile with 0.1% TFA) and characterized by MALDI-TOF. Figure 1 provides HPLC chromatograms before and after purification of Oligo 17 by reverse phase HPLC. Figure 2 provides a MALDI-TOF mass spectrum for a purified batch of Oligo 17. Figures 1 and 2 are provided for illustrative purposes only and should not be interpreted as a restriction to this invention.

PNA oligomers synthesized for this invention are provided in Table 1 along with their molecular weight data by MALDI-TOF. Of the abbreviations used in Table 1, A, T, G, and C refer to unmodified nucleobase adenine, thymine, guanine, and cytosine, respectively. Modified nucleobases C(mXn), C(mXn_g), A(mXn), A(m), A(m_g), and G(m) are as defined below Table 1 along with Lys, Fam, L(1), and L(2). These PNA oligomers are presented for illustrative purposes only and should not be interpreted as a restriction to the present invention. Entry Sequence (N → C) MW (m+1)^b Oligo 1 Fam-L(1)L(1)-TGC(1O3)-TAC(1O3)-TAC(1O3)-TG-Lys-NH₂ 4079.0 4078.3 Oligo 2 Fam-L(1)L(1)-TGC-TAC-TAC-TG-Lys-NH₂ 3745.6 3745.5 Oligo 3 TGC(1O3)-TAC-TAC(1O3)-TG-Lys-NHz 3319.4 3318.5 Oligo 4 TGC-TAC(1O3)-TAC-TG-Lys-NH₂ 3208.3 3208.3 Oligo 5 TGC-TAC-TAC-TG-Lys-NH₂ 3097.2 3097.8 Oligo 6 Fam-L(1)L(1)-TC(1O3)T-CC(1O3)C-AGC(1O3)-GTG-C(1O3)GC-C(1O3)AT-Lys-NH2 6140.1 6141.8 Oligo 7 Fam-L(1)L(1)-TCT-CCC-AGC-GTG-CGC-CAT-Lys-NH₂ 5584.4 5583.1 Oligo 8 TGC(2O2)-TAC-TAC(202)-TG-Lys-NH₂ 3319.4 3318.9 Oligo 9 GC(2O2)A-C(2O2)AT-TTG-C(2O2)CT-NH₂ 3553.7 3552.7 Oligo 10 GC(1O2)A-C(1O2)AT-TTG-C(1O2)CT-NH₂ 3511.6 3511.1 Oligo 11 GCA-CAT-TTG-CCT-Lys-NH₂ 3348.3 3345.8 Oligo 12 CA(3)T-A(3)GT-A(3)TA-A(3)GT-NH₂ 3580.8 3580.9 Oligo 13 CA(4)T-A(4)GT-A(4)TA-A(4)GT-NH₂ 3636.9 3634.9 Oligo 14 CA(5)T-A(5)GT-A(5)TA-A(5)GT-NH₂ 3693.0 3691.5 Oligo 15 CA(7)T-A(7)GT-A(7)TA-A(7)GT-NH₂ 3805.0 3803.4 Oligo 16 CAT-AGT-ATA-AGT-Lys-NH₂ 3420.3 3418.3 Oligo 17 CA(5)T-A(5)GT-A(5)TA-A(5)GT-Lys-NH₂ 3820.9 3819.8 Oligo 18 CA(2O2)T-A(2O2)GT-A(2O2)TA-A(2O2)GT-NH₂ 3700.7 3701.4 Oligo 19 L(1)-TAG(2O3)-CTG(2O3)-CTG-ATT-Lys-NH₂ 3746.9 3748.9 Oligo 20 TG(5)G-C(1O2)AA-C(1O2)TG-A(5)T-Lys-NH₂ 3525.6 3523.8 Oligo 21 Fam-L(2)-TG(5)G-C(102)AA-C(102)TG-A(5)T-Lys-NH₂ 3997.0 3996.1 Oligo 22 Fam-L(2)-TT-C(1O2)AT-A(5)GT-A(5)TA-AG(5)T-Lys-NH₂ 4806.9 4806.7 Oligo 23 Fam-L(2)L(2)-TC(1O2)A-GA(5)A-C(1O2)TT-A(5)T-Lys-NH₂ 4084.2 4083.8 Oligo 24 Fam-L(2)-CA(5)T-A(4_g)GT-A(4_g)TA(5)-AGT-Lys-NH₂ 4348.5 4347.4 Oligo 25 TT-C(1O2_g)AT-A(5)GT-A(5)TA-AG(5)T-Lys-NH₂ 4377.4 4375.6 Oligo 26 GC(1N3)A-C(1N3)AT-TTG-C(1N3)CT-NH₂ 3550.8 3550.9 Oligo 27 CAT-AGT-ATA-AGT-NH₂ 3292.3 3292.5 Oligo 28 Fam-L(2)-TGG-CAA-CTG-AT-Lys-NH₂ 3617.5 3616.3 a. The employed abbreviations for monomers are defined as below.
b. Observed ion peak for MH⁺ unless noted otherwise.

Binding Affinity for DNA: PNA oligomers of this invention were evaluated for their binding affinity for DNA by measuring T_m values as follows.

4µM PNA oligomer and 4µM DNA were mixed in aq buffer (pH 7.16, 10mM sodium phosphate, 100mM NaCl), and incubated at 90°C for a few minutes and slowly cooled down to RT. Then the solution was transferred into a 4ml quartz cuvette and the cuvette was tightly sealed. The cuvette was mounted on an Agilent 8453 UV/Visible spectrophotometer and absorbance changes at 260nm were recorded with increasing the temperature of the cuvette by either 0.5 or 1.0°C per minute. From the absorbance vs temperature curve, the temperature showing the largest increase rate in absorbance was read out as the melting temperature T_m between PNA and DNA. DNAs for T_m measurement were purchased either from Bioneer, Inc. (www.bioneer.com, Daejon, South Korea) or from Ahram Biosystems (www.ahrambio.com, Seoul, South Korea), and used without further purification.

Figure 3 provides graphs of absorbance changes with temperature for Oligo 17 against complementary or mismatch DNA. For sequences of the mismatch DNAs against Oligo 17, refer to Table 2. In Figure 3, there is a transition temperature in each curve, which was read out as the T_m value for the curve.

T_m values are provided in Table 2 for PNA oligomers of this invention. These T_m values are presented for illustrative purposes only and should not be interpreted as a restriction to this invention. Entry DNA Sequence (5' → 3') T_m, °C Remark Oligo 5 CAG-TAG-TAG-CA 55 unmodified PNA oligomer Oligo 3 65 C(1O3) x 2 Oligo 4 61 C(1O3) x 1 Oligo 8 68 C(2O2) x 2 Oligo 10 AGG-CAA-TTG-TGC > 85 C(1O2) x 3 Oligo 11 59 unmodified PNA oligomer Oligo 12 ACT-TAT-ACT-ATG 60 A(3) x 4 Oligo 13 64 A(4) x 4 Oligo 14 69 A(5) x 4 Oligo 15 71 A(7) x 4 Oligo 18 66 A(2O2) x 4 Oligo 27 55 unmodified PNA oligomer Oligo 16 ACT-TAT-ACT-ATG 56 unmodified PNA oligomer Oligo 17 ACT-TAT-ACT-ATG 72 complementary ACT-TAC-ACT-ATG 61 mismatch (T → C) ACT-TAA-ACT-ATG 59 mismatch (T → A) ACT-TAG-ACT-ATG 58 mismatch (T → G) Oligo 24 ACT-TAT-ACT-ATG 70 A(5) x 2 plus A(4_g) x 2 Oligo 20 ATC-AGT-TGC-CA 84 complementary ATC-ATT-TGC-CA 62 mismatch (G → T) ATC-AAT-TGC-CA 65 mismatch (G → A)

Replacement of cytosine with an unnatural nucleobase pyrrolocytosine derivative of this invention markedly increased PNA oligomer's affinity for complementary DNA. For example, Oligo 10 having three 'modified' cytosine 'C(1O2) monomers showed a T_m exceeding 85°C, while the corresponding 'unmodified' Oligo 11 showed a T_m of 58°C. Other modified cytosine monomers such as 'C(1O3) or 'C(2O2)' also significantly increased PNA oligomer's affinity for complementary DNA, as exemplified with Oligo 3 and Oligo 8.

'Modified' adenine nucleobases of this invention also significantly increased PNA oligomer's affinity for complementary DNA. For example, Oligo 15 having four 'modified' adenine A(7) monomers showed a T_m of 71°C, which is significantly higher than the T_m of 55°C observed with 'unmodified' Oligo 27. Other 'modified' adenine monomers such as A(4), and A(5) also markedly increased affinity for complementary DNA.

'Modified' PNA monomers of this invention were found to be quite sensitive to base mismatch. For example, decreases of 11 ∼ 14°C in T_m were observed with single base mismatches for an A(5) monomer in Oligo 17. Single base mismatches for a C(102) monomer in Oligo 20 resulted in decreases of 19 ∼ 22°C in T_m.

Cell Penetration: In order to evaluate the cell penetration ability of PNA oligomers of this invention, cancer cells of human origin were treated with PNA oligomers covalently tagged with fluorescein. The applied method is provided in brief as follows.

To each cover glass (autoclaved) placed in each well of a 24-well plate, were seeded 20,000 ∼ 100,000 cells depending on the growth rate of the cell line used, and the cells were cultured at 37°C and 5% CO₂ for 16 to 24h. Then the medium was replaced with 500µl fresh Opti-MEM medium (with or without 1% FBS), to which was added an aliquot of aq stock solution of a PNA oligomer covalently tagged with fluorescein. After cells were cultured for a designated interval, the cells were washed with PBS, and fixed by incubating in 3.7% or 4% paraformaldehyde. The cells were thoroughly washed several times with PBS or PBS containing 0.1% Tween-20. Then the cover glass was mounted onto a slide glass using a drop of mounting solution and sealed with nail polish for confocal fluorescence microscopy. Fluorescence images were taken either on a Zeiss LSM 510 confocal microscope (Germany) at 63X objective or on a Nikon C1Si confocal microscope at 40X objective.

The cell penetration images in Figures 4∼8 are provided for illustrative purposes only and should not be interpreted as a restriction to the present invention.

In Figure 4(a) and 4(b), are provided confocal microscopy images (at 63x objective) 1, 2, 3 and 24h after HeLa cells were treated with Oligo 1 and Oligo 2 at 5µM, respectively (without FBS). While fluorescence intensity is clear and becomes intense over 24h in Figure 4(a), fluorescence intensity is faint in Figure 4(b), indicating that Oligo 1 penetrates HeLa cells significantly faster than 'unmodified' Oligo 2.

In Figure 5(a) and 5(b), are provided confocal microscopy images (at 63x objective) 0.5 and 1h after MCF-7 cells were treated with Oligo 6 and Oligo 7 at 2.5µM, respectively (without FBS). While fluorescence intensity is clear and becomes intense over 1h in Figure 5(a), fluorescence intensity is faint in Figure 5(b), indicating that Oligo 6 penetrates MCF-7 cells significantly faster than 'unmodified' Oligo 7.

In Figure 6(a) and 6(b), are provided confocal microscopy pictures (at 40x objective) 6 or 24h after HeLa cells were treated with Oligo 1 and Oligo 6 at 1µM, respectively (with 1% FBS). While fluorescence intensity is faint even at 24h in Figure 6(a), fluorescence intensity is clear and becomes intense over 24h in Figure 6(b), suggesting that Oligo 6 penetrate HeLa Cells significantly faster than Oligo 1.

In Figure 7(a) and 7(b), are provided confocal microscopy pictures (40x objective) 24h after JAR cells were treated with Oligo 21 and Oligo 28 at 2µM, respectively (without FBS). While fluorescence intensity is strong in Figure 7(a), there is no significant fluorescence intensity in Figure 7(b), suggesting that Oligo 21 penetrate JAR cells significantly faster than 'unmodified' Oligo 28.

In Figure 7(c) and 7(d), are provided confocal microscopy pictures (at 40x objective) 24h after A549 cells were treated with Oligo 21 and Oligo 28 at 2µM, respectively (without FBS). While fluorescence intensity is strong in Figure 7(c), there is no significant fluorescence intensity in Figure 7(d), suggesting that Oligo 21 penetrate A549 cells significantly faster than 'unmodified' Oligo 28.

In Figure 7(e) and 7(f), are provided confocal microscopy pictures (at 40x objective) 12h after HeLa cells were treated with Oligo 21 and Oligo 28 at 2µM, respectively (without FBS). While fluorescence intensity is apparent in Figure 7(e), there is no significant fluorescence intensity in Figure 7(f), suggesting that Oligo 21 penetrate HeLa cells significantly faster than 'unmodified' Oligo 28.

In Figure 7(g), are provided confocal microscopy pictures (at 40x objective) 24h after HeLa cells were treated with Oligo 21 at 2µM (without FBS). Given that the celluar fluorescence in Figure 7(g) is significantly stronger than that in Figure 7(e), Oligo 21 appears to penetrate over 24h rather than 12h.

Figure 8(a), 8(b) and 8(c) provide confocal microscopy images (40x objective) 24h after HeLa, A549, and JAR cells were treated with 2µM Oligo 22, respectively (without FBS). All the images are associated with fluorescence within cell, indicating that Oligo 22 possesses good cell penetration in the tested cells.

Antisense Example: Oligo 9 and Oligo 12 possess the same base sequences as T1-12 and T5-12, respectively, which were reported to inhibit the ribosomal synthesis of mdm2 in the literature. (Nucleic Acids Res. vol 32, 4893-4902, 2004) Oligo 9 and Oligo 12 were evaluated for their ability to inhibit the ribosomal synthesis of mdm2 in JAR cells as follows. The following antisense example is presented for illustrative purposes only and should not be interpreted as a restriction to the present invention.

JAR cells (ATCC catalog # HTB-144) were grown in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C and 5% CO₂. Cells were then seeded into each well of a 12-well plate containing 1ml of the same medium, and treated with an aliquot of an aqueous stock solution of Oligo 9 or Oligo 12 of a designated concentration. Then the cells were incubated at 37°C and 5% CO₂ for 15h.

The cells in each well were washed with cold PBS and treated with 80µl RIPA buffer containing 1% protease inhibitors cocktail, and the plate was incubated at 4°C and agitated slowly for 15min. The content of each well was scraped out into a microtube. The microtube was incubated in ice for 10min and centrifuged at 10,000g. The resulting supernatant was collected and subjected to protein quantification by Bradford assay and western blot analysis. For electrophoresis, 20µg of protein was loaded onto each lane of the gel in a minigel apparatus, separated and transferred onto a PVDF membrane (0.45µ, Millipore). The primary mdm2 antibody used for western blotting was SC-965 (Santa Cruz Biotechnology).

Figure 9 provides western blotting results for JAR cells treated with 5µM or 10µM Oligo 9, 5µM or 10µM Oligo 10, cotreatment with the oligomers at 5µM or 10µM each, and blank (no oligomer treatment). In Figure 9, treatment with Oligo 9 or Oligo 10, or cotreatment with Oligo 9 and Oligo 10 significantly inhibited ribosomal synthesis of mdm2 in JAR cells both at 5µM and 10µM.