HK1229263A1

HK1229263A1 - Design of short oligonucleotides as vaccine adjuvants and therapeutic agents

Info

Publication number: HK1229263A1
Application number: HK17103200.1A
Authority: HK
Inventors: R．P．艾依尔
Original assignee: 春堤制药公司
Priority date: 2013-02-18
Filing date: 2014-02-18
Publication date: 2017-11-17

Description

Design of short oligonucleotides as vaccine adjuvants and therapeutic agents

CROSS-REFERENCE TO RELATED APPLICATIONS

The present invention claims the benefit of priority from U.S. provisional patent application serial No. 61/766,011 filed on 2013, 2, 18, the disclosure of which is incorporated herein by reference in its entirety.

Background

Innate immunity plays a key role in the defense of the body against prokaryotic and eukaryotic pathogens, such as viruses, bacteria, fungi, parasites, and the like. In fact, acute and chronic infections caused by viruses constitute a major worldwide public health crisis, with a clear unmet medical need (1, 2). In addition to infectious diseases, viruses cause 15-20% of all cancers worldwide, including liver cancer, cervical cancer and pancreatic cancer, each leading to significant mortality and morbidity.

In addition to human suffering, viral diseases result in overwhelming health care costs and loss of productivity. For example, 500,000,000 to 600,000,000 people worldwide are chronically infected with HBV and HCV, and 1,000,000 to 2,000,000 deaths occur annually due to virus-induced cirrhosis and liver cancer. Liver transplantation is urgently required by tens of thousands of patients worldwide. Cervical cancer is caused by infection with human papillomavirus, and the incidence of Kaposi's sarcoma associated with HIV infection is well documented. Pandemic influenza is characterized by high levels of morbidity and mortality in humans and is associated with increased levels of infection and pathogenesis due to lack of pre-existing immunity against its novel antigenic subtypes. Antiviral agents not only potentially slow down the spread of pandemic influenza, but may ultimately be a solution. Today, tuberculosis caused by Mycobacterium tuberculosis (Mtb) kills more people than any other bacterial infection. In more than 90 countries, nearly one third of the population is infected with Mtb, and 2,000,000 die annually from the disease.

Although vaccines are available as prophylactics against a limited number of viruses, they have no real therapeutic benefit for those already infected. Furthermore, vaccines against certain viruses (e.g., influenza vaccines) are unlikely to have a significant impact on mortality in a pandemic due to the time required to generate a sufficient dose of a suitable vaccine against a new human strain after it has been identified. The use of adjuvants can increase the efficacy of the vaccine and provide protection against a wide range of viruses.

Therefore, our antiviral defenses rely almost exclusively on the use of antiviral drugs. Unfortunately, many medically important viruses, particularly RNA viruses, are dangerous, cannot be tested in model systems, or cannot be propagated for testing potential drug candidates.

Many of the current antiviral drugs have been developed as viral polymerase, protease, integrase, and entry inhibitors. However, drugs designed to inhibit viral growth may also adversely affect host cells because the viral life cycle is linked to normal host cell function. The limited viral targets amenable to antiviral intervention further hamper antiviral drug development. Thus, despite recent 50 years of antiviral research, our antiviral drug library is still as small as dangerous, with only about 34 antiviral drugs on the international market, mostly against HIV and herpes viruses.

Further, current treatment options for several chronic viral diseases, including HCV and HBV, remain very limited and challenging. In fact, viral rebound after cessation of therapy, drug-induced toxicity, and emergence of resistant strains under selective pressure of antiviral drugs remain serious problems in current antiviral therapies. Complete eradication of the virus is rarely achieved and at best is in a minimal patient cohort because current antiviral therapies produce an inadequate and unsustainable antiviral response. The development of drugs targeting host coding functions provides an alternative strategy for antimicrobial development.

Viruses have also continued to evolve smarter strategies to evade host immune responses and develop resistance to drugs through a variety of mechanisms. Cells protect themselves from microbial infection via cell sensors that include the retinoic acid-inducing gene (RIG-I) and other RIG-like proteins (RLR), MDA5, nucleo-oligo domain protein-2 (NOD2 and other NOD-like proteins (NLR). activation of these proteins via interaction of pathogen recognition receptors with microbial nucleic acids or peptides causes interferon signaling pathways, resulting in production of IFNs that protect cells from infection in fact, it is recognized that both DNA and RNA viruses inhibit type I Interferon (IFN) production, suggesting that control of IFN responses is essential for a wide range of viral survival (3, 4). thus, development of effective antiviral therapies must involve the use of combinations of new classes of drugs, each with novel mechanisms of action, including those that stimulate host immune responses for eradication of the virus, bacteria have evolved unique mechanisms that lead to resistance to antibacterial agents. Thus, the development of effective antibacterial therapies must involve the use of combinations of new classes of drugs, each with novel multiple mechanisms of action, including those that stimulate host immune responses for eradication of bacteria. Many different types of cancer have evolved different mechanisms of developing resistance to anticancer agents. Thus, the development of effective anti-cancer therapies must involve the use of combinations of new classes of drugs, each with novel multiple mechanisms of action, including those that stimulate the host immune response for eradication of cancer.

Summary of The Invention

In one aspect, the present invention provides a nucleoside, a short oligonucleotide compound or an analog thereof, or a pharmaceutically acceptable salt, racemate, enantiomer, diastereomer, geometric isomer or tautomer thereof (hereinafter referred to as "the compound of the present invention") for use as a prophylactic agent. In another aspect, the invention provides nucleosides, short oligonucleotide compounds or analogs thereof, or pharmaceutically acceptable salts, racemates, enantiomers, diastereomers, geometric isomers or tautomers thereof, for use as vaccine adjuvants.

The compounds of the present invention include, for example, compounds of formula (I):

or a pharmaceutically acceptable salt, racemate, enantiomer, diastereomer, geometric isomer or tautomer thereof,

wherein:

R₁and R₂Each independently is H, OH, O-alkyl, substituted alkyl, cycloalkyl, aryl, substituted arylAralkyl, heterocycle, O-aryl, O-heteroarylaryl, or heterocycle;

R₃selected from the group consisting of hydrogen, alkyl, substituted alkyl, C (O) -alkyl, C (O) O-alkyl, C (O) -aryl, C (O) O-aryl, C (O) NH-alkyl, and C (O) NH-aryl;

y and Z are each independently O or S;

B₁and B₂Each independently is adenine, guanine, thymine, cytosine, uracil, or a modified nucleoside;

m＝1-6。

R₄independently is H, alkyl, substituted alkyl, C (O) -alkyl, C (O) O-alkyl, C (O) -aryl, C (O) O-aryl, C (O) NH-alkyl and C (O) NH-aryl, monophosphate, diphosphate or triphosphate.

In one embodiment, the compounds of the invention are of formula (II):

wherein:

x ═ absence, O, NH, NR, or S;

X₁absent, O or NH;

a ═ absence, aryl, or aralkyl;

n is 0, 1,2, 3,4 or 5;

r ═ alkyl, substituted alkyl, cycloalkyl, aryl, substituted aryl, aralkyl, heterocycle, O-alkyl, O-heteroaryl, or steroid;

R₁and R₂Each independently is H,OH, O-alkyl, substituted alkyl, cycloalkyl, aryl, substituted aryl, aralkyl, heterocycle, O-aryl, O-heteroarylaryl, or heterocycle;

y and Z are each independently O or S;

m＝1-6；

In one embodiment, the compounds of the present invention are compounds having the structure:

or a pharmaceutically acceptable salt, racemate, enantiomer, diastereomer, geometric isomer, or tautomer thereof.

In another embodiment, the compounds of the present invention are compounds having the structure:

The invention also provides a method of treating a microbial infection in a subject (or host) by administering to the subject (or host) identified as in need thereof an effective amount of a compound of the invention.

In another aspect, the invention provides methods of improving an immune system response in a subject (or host) against a disease, condition, infection, or virus. The method comprises administering to the subject an effective amount of a compound of the invention as a vaccine adjuvant.

In certain embodiments of this method, the compound of the invention is a short oligonucleotide compound of formula (I) or an analog thereof, or a pharmaceutically acceptable salt, racemate, enantiomer, diastereomer, geometric isomer, or tautomer thereof.

In other embodiments, the compounds of the invention are short oligonucleotide compounds of formula (II) or analogs thereof, or pharmaceutically acceptable salts, racemates, enantiomers, diastereomers, geometric isomers or tautomers thereof.

In one embodiment, the compounds of the invention are

The invention also provides methods for preventing and treating viral infections in a host (or subject), including a human. The method comprises administering an effective amount of a compound of the invention.

In a further aspect, the present invention provides a method for the prevention and treatment of bacterial infections in a host (or subject), including a human, comprising administering an effective amount of a compound of the invention.

Further, the present invention provides a method for the prevention and treatment of parasitic infections in a host (or subject), including a human, wherein said method comprises administering an effective amount of a compound of the present invention.

Still further, the present invention provides methods for preventing and treating fungal infections in a host (or subject), including a human, by administering to the host an effective amount of a compound of the present invention.

In accordance with the methods of the present invention, the compounds of the present invention may be administered in conjunction with a vaccine (e.g., BCG vaccine) or one or more additional agents. That is, according to the methods of the invention, the compounds of the invention may be administered alone or in combination or sequence with a vaccine or another agent or agents.

In one embodiment, the compounds and vaccines of the present invention are administered for the treatment or prevention of microbial infections.

In another embodiment, the compounds of the present invention are administered in combination with other antimicrobial agents for the treatment or prevention of microbial infections.

In another embodiment, the methods and compounds of the invention are used against viruses.

In further embodiments, the methods and compounds are used to treat or prevent cancer.

The invention further provides pharmaceutical compositions and kits for treating or preventing a condition, disease, infection, or virus described herein. Pharmaceutical compositions comprise a therapeutically effective amount of a compound of the invention, and a pharmaceutically acceptable excipient. The kits of the invention comprise a therapeutically effective amount of a compound of the invention, and written instructions for administering the compound for treating or preventing a condition, disease, infection, or virus described herein. The invention also provides packaged medicaments and articles for treating or preventing a condition, disease, infection, or virus herein.

In addition, the present invention also provides the design and synthesis of compounds useful in a variety of therapeutic applications as mentioned herein. Other features and advantages of the invention will be apparent from the detailed description and from the claims.

Brief Description of Drawings

The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention.

Figure 1 shows the activation of interferon regulatory factor-3 (IRF3) -luciferase reporter genes in Untreated (UT) and SMNH compound-treated human lung epithelial cells (HLE a549 cells).

FIG. 2 demonstrates that IRF3 activation by SMNH compounds (SB 40 and SB1B [ SB 44-1]) is mediated via NOD2 activation.

FIG. 3 shows IRF3 induction following RIG-I activation by SB44 in HLE cells.

FIG. 4 demonstrates the induction of the transcription factor NF-KB upon activation of RIG-I and NOD2 by SB 44.

Figure 5 shows that induction of IRF3 results in the formation of phosphorylated IRF3, a key intermediate in the IFN signaling cascade, of phosphorylated IRF 3.

FIG. 6 shows IRF3 induction by SB1B mediated via NOD2 activation; SB40 is an active metabolite of SB 44.

FIG. 7 shows that SMNH compounds (SB44 and SB 302, a, oxalate form) induce interferon- β (IFN- β) production from human lung epithelial A549 cells.

FIG. 8 shows that SB1B (shown as "SB-44-1-D") treatment resulted in IFN- β production from human lung epithelial A549 cells.

FIG. 9 shows induction by the responder genes RIG-I and NOD2 of SB1B (shown as "SB-44-1-D"); SB50 did not activate NOD2 and was used as a negative control.

FIG. 10(a-d) shows that SMNH compounds enhance the in vitro presentation of peptide antigen-85B in macrophages, which is associated with increased MHC-II expression (SB44 is shown as SB 1A).

FIG. 11 (a-e): a) experimental models of vaccines in mice are shown; b-e) is a graph showing that NOD2 activates compounds SB1A (also shown as SB 44) and SB1B better than Muramyl Dipeptide (MDP) as an adjuvant that enhances the efficacy of BCG vaccines against tuberculosis in mice.

FIG. 12 shows that NOD2 activates the compounds SB1A (also shown as SB 44) and SB1B (Rp isoform of SB 44) to induce IL-1 β in combination with MDP or whole BCG bacilli alone by a caspase-dependent mechanism in macrophages.

Fig. 13(a-B) shows that NOD2 activating SB1A (also shown as SB 44) and SB1B (Rp isomer of SB 44) in combination with BCG vaccine induced protection against re-challenge of tuberculosis in mice, suggesting long-term protection.

Figure 14 shows a model developed to validate that SB1A (also shown as SB 44) and SB1B (Rp isomer of SB 44) induced MPEC can lead to long-term protection by regeneration into strong effector T cells in mice.

Fig. 15(a-B) shows that the SB1A (also shown as SB 44) or SB1B (Rp isomer of SB 44) combination with BCG vaccine retained the ability to generate a better recall response against re-challenge with tuberculosis.

Figure 16 shows that NOD2 activated SB1A (also shown as SB 44) and SB1B (Rp isomer of SB 44) compounds in combination with BCG vaccine induced robust expansion of antigen-specific CD8T cells in the lungs of re-challenged tuberculosis mice.

Figure 17 shows interferon induction in PBMCs treated with BCG when combined with compounds of the invention.

Detailed Description

In one aspect, the invention features compounds described herein and methods of using such compounds for treating a microbial infection in a subject. In another aspect, the invention relates to compounds, compositions and methods for improving an immune system response against a disease, condition, infection or virus in a subject.

The present invention also provides methods for preventing and treating viral, bacterial, parasitic or fungal infections in a host (or subject), including a human, by administering to the host (or subject) an effective amount of a compound of the present invention.

Multidrug resistance Mtb (MDR-Mtb) is resistant to many of the current anti-TB drugs including isoniazid, rifampin, kanamycin, and the like. The WHO estimates that up to 50,000,000 people worldwide are likely to be infected with MDR-Mtb. Due to difficulties in drug therapy/resistance, vaccination to prevent or limit tuberculosis is becoming increasingly important. Currently, BCG vaccines are the only variable protection against childhood TB and do not provide protection against adult disease. Therefore, there is a need for more effective BCG vaccines. One way to potentiate the efficacy of BCG is to combine it with an adjuvant. Adjuvants are compounds that can promote, modulate and improve the immunogenicity of vaccines. Adjuvants may also reduce the required target antigen dose and modulate antigen-specific immune responses, such as Th1 and Th2 responses, in a quantitative manner. Recent advances in innate immunity have shown that TLRs, RIG-I and NLRs, and in particular NOD2, recognize a variety of nucleic acid ligands that stimulate the innate immune response and promote adjuvant-like activity.

Vertebrate systems are constantly under attack by invading microorganisms and have evolved immune-mediated defenses for elimination of pathogens. The mammalian immune system comprises components of innate and adaptive immunity. The innate immune system recognizes microorganisms via a limited number of germ-line encoded Pathogen Recognition Receptors (PRRs) (3, 4). Phagocytic cells such as macrophages and dendritic cells mediate the innate immune response. The adaptive immune response is characterized by the specificity involved in lymphocytes that carry antigen-specific receptors generated by mechanisms such as gene rearrangement. Innate immunity plays an important role in regulating liver damage, fibrosis and regeneration. For example, activation of natural killer cells (NK cells) by interferon may be a novel strategy for treating liver fibrosis. This is because the activation of NK cells can kill particularly activated Hepatic Stellate Cells (HSCs), thereby improving liver fibrosis and liver neoplasia. Thus, the oligonucleotide analogs disclosed in the present invention may have utility in inhibiting liver fibrosis and liver cancer progression.

Microorganisms have also evolved clever strategies to evade the immune response that protects host cells from infection. In fact, both DNA and RNA viruses suppress cellular IFN production (type I, IFN-. alpha., IFN-. beta.). Intracellular IFN is a potent antiviral cytokine whose expression/production is mediated by the transcription factor IRF3(IFN regulatory factor 3) present in the cytoplasm of uninfected cells. Once a cell is infected and viral components (also known as pathogen-associated molecular patterns (PAMPS, e.g. viral genome, viral proteins etc.) are recognized by specialized viral sensors or Pattern Recognition Receptors (PRRs), IRF3 is activated IRF3 translocates to the nucleus to activate IFN gene expression in trans IFN production induces protective antiviral effects (via paracrine and autocrine activities) through various mechanisms such as (i) activation of innate and adaptive immune responses, (ii) induction of antiviral states in cells by production of antiviral and favorable pro-inflammatory factors, and (iii) controlled apoptosis of virus-infected cells PRR is therefore an essential component of the IFN response. Like viral sensor RIG-I, activation of NOD2 also results in triggering of IFN production and NF-. kappa.induced signaling cascades that promote controlled pro-inflammatory responses to potentiate the antiviral effects of IFN. Because, NOD2 is a viral sensor that detects a wide range of ssRNA viruses, such as RSV and influenza a, that presents unique host targets for antiviral development and resistance against viruses.

Like NOD2, RIG-I is a host cytosolic protein that recognizes double-stranded viral RNA as PAMPs, which activates type I interferon immune defenses, thereby inhibiting viral replication and suppressing cellular permissivity to viral infection (6-15). RIG-I is a viral sensor that detects a wide range of RNA viruses, such as flaviviruses including hepatitis c virus, sendai virus, influenza virus, and vesicular stomatitis virus, rabies virus, and japanese encephalitis virus, which present unique host targets for broad-spectrum antiviral activity. It is noteworthy that although HBV is a DNA virus, it uses a pregenomic RNA template for initiating DNA synthesis, and potentially therefore RIG-I can be a receptor for HBV pgRNA.

It has been found that certain dinucleotide compositions have the potential to activate through the RIG-I pathway for stimulating innate immunity and inducing interferon production. Such compounds may also be useful in the prevention of viral infection and as adjuvants in vaccines.

Viral sensor RIG-I is a multimeric cytosolic protein consisting of: a C-terminal Regulatory Domain (RD), two terminal caspase (caspase) activation and recruitment domains (CARDS), and a central atpase domain. Viral double-stranded RNA (dsRNA) and 5' -triphosphate are two PAMPs that allow RIG-I to distinguish pathogen RNA (dsRNA with and without triphosphate) from host RNA (which typically has a "cap" modification at the end) (6-14). In addition, RIG-I has the ability to sense viral RNA by translocation phenomenon (Myong et al, Science 323, 1070.2009). RIG-I translocation and repeated shuffling on dsRNA of the viral genome is a trigger of RIG-I to undergo conformational changes, activate its atpase and expose CARDS for ubiquitination. In the next step, CARDS interacts with mitochondrial anti-viral signaling (MAVS) [ also known as interferon beta stimulator [ (IPS-1) or VISA ] ] to trigger downstream signaling leading to type I IFN expression (IFN-. alpha., beta.).

NOD2 and RIG-I are multimeric proteins that are very similar in structure and organization. Thus, like RIG-I, NOD2 contains the CARD domain. In addition, RIG-I and NOD2 both have nucleotide binding pockets located in the NBD (nucleotide binding domain) (for NOD2) and helicase (for RIG-I) domains. Molecular modeling studies have shown that certain dinucleotide compositions have structural similarity to the Nucleoside Triphosphate (NTP) structures, which bind to NBD. We hypothesized that the short oligonucleotides act as NTP mimetics that bind to the NBD of NOD2 (and RIG-I) and thus their activation is useful for downstream antiviral effects.

The present inventors have found that the nucleosides, short oligonucleotide compounds or analogs thereof of the present invention can modulate immune pathways involving Toll-like receptors (TLRs), non-TLR receptors such as retinoic acid-inducing gene-1 (RIG-I) and nucleoligomeric proteins (NODs), collectively referred to as TLRs, RLRs and NLRs. Activation of these pathways by their respective ligands can induce the production of a variety of cytokines and chemokines, such as interleukins, interferons, NF-KB, TNF-alpha, etc., and also induce the induction of certain cellular proteins with antimicrobial activity, thereby providing antimicrobial immunity. In contrast, the present inventors designed compounds for causing inhibition of inflammatory pathways, resulting in beneficial effects in autoimmune diseases.

The present inventors have unexpectedly found that the nucleosides, short oligonucleotide compounds and analogs thereof of the present invention can be used as adjuvants for vaccines. The compounds of the present invention have therapeutic utility against a variety of infectious diseases caused by bacterial and viral agents. The compounds of the invention are also useful in the treatment or prevention of autoimmune diseases, such as allergy, asthma, inflammatory disorders, and other diseases such as cancer.

Accordingly, the present invention provides methods for treating or preventing a variety of conditions, diseases, infections, or viruses described herein.

Definition of

In order that the present invention may be more readily understood, certain terms are first defined and collected herein for convenience.

The compounds of the invention comprise one or more modifications from a "native" nucleic acid (i.e., a native internucleoside linkage, or nucleobase G, C, T, U, A, etc.). Modifications include, for example, modifications of internucleotide linkages, bases, or sugar moieties.

The nucleoside units are represented by the internationally recognized line drawing convention. In the examples below, 2' -substituted ribonucleosides are represented in both conventional structure and corresponding line pattern.

Attached to B yielding α or β N or C nucleosides₁And B₂Including but not limited to furanose, deoxyribofuranose, ribose, and arabinose.

The term "administration" or "administering" includes the route of introducing one or more compounds into a subject to perform its intended function. Examples of routes of administration that may be used include injection (subcutaneous, intravenous, parenteral, intraperitoneal, intrathecal), oral, inhalation, rectal and transdermal. The pharmaceutical preparations are of course administered in a form suitable for each route of administration. For example, these formulations are administered topically in tablet or capsule form, by injection, inhalation, by lotion or ointment; and rectal administration by suppository. Oral administration is preferred. The injection may be a bolus injection, or may be a continuous infusion. Depending on the route of administration, the compound may be coated or deposited in the selected material so as to protect it from natural conditions that may adversely affect its ability to perform its intended function.

The compound may be administered alone or in combination with another agent (e.g., a vaccine) or a pharmaceutically acceptable carrier, or both, as described above. The compound may be administered prior to, simultaneously with, or after the administration of the other agent. In addition, the compounds may also be administered in a precursor form that is converted in vivo to its active metabolite, or more active metabolite.

As used herein, the term "aryl" refers to a monocyclic or polycyclic carbocyclic ring system having one or two aromatic rings, including but not limited to phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl (idenyl), and the like.

As used herein, the term "heteroaryl" refers to a monocyclic or polycyclic (e.g., bicyclic or tricyclic or more polycyclic) aromatic radical or ring having five to ten ring atoms, wherein one or more ring atoms are selected from, for example, S, O and N; zero, one, or two ring atoms are additional heteroatoms independently selected from, for example, S, O and N; and the remaining ring atoms are carbon, wherein any N or S contained within the ring may optionally be oxidized. Heteroaryl groups include, but are not limited to, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isoxazolyl, thiadiazole, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzoxazolyl, quinoxalinyl, and the like.

In accordance with the present invention, any of the aryl, substituted aryl, heteroaryl, and substituted heteroaryl groups described herein can be any aromatic group. The aromatic group may be substituted or unsubstituted.

As used herein, the term "alkyl" refers to a saturated, straight or branched chain hydrocarbon radical containing from one to six, or from one to twelve carbon atoms, respectively. Examples of C1-C6 alkyl radicals include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, neopentyl, and n-hexyl radicals; and examples of C1-C12 alkyl radicals include, but are not limited to, ethyl, propyl, isopropyl, n-hexyl, octyl, decyl, dodecyl radicals.

The term "aralkyl" or "aralkyl" includes aryl-substituted alkyl radicals such as benzyl, benzhydryl, trityl, phenethyl, and diphenylethyl.

As used herein, the term "heterocycle" refers to a non-aromatic 5, 6, or 7-membered ring or a bicyclic or tricyclic group fused system in which (i) each ring contains one to three heteroatoms independently selected from oxygen, sulfur, and nitrogen, (ii) each 5-membered ring has 0 to 1 double bond, and each 6-membered ring has 0 to 2 double bonds, (iii) the nitrogen and sulfur heteroatoms may optionally be oxidized, (iv) the nitrogen heteroatom may optionally be quaternized, (iv) any of the above rings can be fused to a benzene ring, and (v) the remaining ring atoms are carbon atoms, which can optionally be oxy-substituted. Representative heterocycloalkyl groups include, but are not limited to, [1,3] dioxolane, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinyl (pyridazinonyl), and tetrahydrofuranyl. Such heterocyclic groups may be further substituted.

As used herein, the term "cycloalkyl" refers to a monovalent group derived from a monocyclic or polycyclic saturated carbocyclic compound by removal of a single hydrogen atom. Examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo [2.2.1] heptyl, and bicyclo [2.2.2] octyl.

As used herein, the terms "substituted aryl", "substituted alkyl", "cycloalkyl" refer to previously defined aryl, alkyl and cycloalkyl groups wherein one, two or three or more hydrogen atoms are replaced by substituents including, but not limited to, -F, -Cl, -Br, -I, -OH, protected hydroxy, -NO2, -CN, -NH2, protected amino, -NH-C1-C12-alkyl, -NH-C2-C12-alkenyl, -NH-C2-C12-alkenyl, -NH-C3-C12-cycloalkyl, -NH-aryl, -NH-heteroaryl, -NH-heterocycloalkyl, -dialkylamino, -diarylamino, or, -diheteroarylamino, -O-C1-C12-alkyl, -O-C2-C12-alkenyl, -O-C2-C12-alkenyl, -O-C3-C12-cycloalkyl, -O-aryl, -O-heteroaryl, -O-heterocycloalkyl, -C (O) -C1-C12-alkyl, -C (O) -C2-C12-alkenyl, -C (O) -C2-C12-alkenyl, -C (O) -C3-C12-cycloalkyl, -C (O) -aryl, -C (O) -heteroaryl, -C (O) -heterocycloalkyl, -CONH2, -CONH-C1-C12-alkyl, -CONH-C2-C12-alkenyl, -CONH-C2-C12-alkenyl, -CONH-C3-C12-cycloalkyl, -CONH-aryl, -CONH-heteroaryl, -CONH-heterocycloalkyl, -OCO 2-C1-C12-alkyl, -OCO 2-C2-C12-alkenyl, -OCO 2-C2-C12-alkenyl, -OCO 2-C3-C12-cycloalkyl, -OCO 2-aryl, -OCO 2-heteroaryl, -OCO 2-heterocycloalkyl, -OCONH2, -OCONH-C1-C12-alkyl, -OCONH-C2-C12-alkenyl, -OCONH-C2-C12-alkenyl, -OCONH-C3-C12-cycloalkyl, -OCONH-aryl, -OCONH-heteroaryl, -OCONH-heterocycloalkyl, -NHC (O) -C1-C12-alkyl, -NHC (O) -C2-C12-alkenyl, -NHC (O) -C2-C12-alkenyl, -NHC (O) -C3-C12-cycloalkyl, -NHC (O) -aryl, -NHC (O) -heteroaryl, -NHC (O) -heterocycloalkyl, -NHCO 2-C1-C12-alkyl, -NHCO 2-C2-C12-alkenyl, -NHCO 2-C2-C12-alkenyl, -NHCO 2-C3-C12-cycloalkyl, -NHCO 2-aryl, -NHCO 2-heteroaryl, -NHCO 2-heterocycloalkyl, -NHC (O) NH2, -NHC (O) NH-C1-C12-alkyl, -NHC (O) NH-C2-C12-alkenyl, -NHC (O) NH-C2-C12-alkenyl, -NHC (O) NH-C3-C12-cycloalkyl, -NHC (O) NH-aryl, -NHC (O) NH-heteroaryl, -NHC (O) NH-heterocycloalkyl, NHC (S) NH2, -NHC (S) NH-C1-C12-alkyl, -NHC (S) NH-C2-C12-alkenyl, -NHC (S) NH-C2-C12-alkenyl, -NHC (S) NH-C3-C12-cycloalkyl, -NHC S) NH-aryl, -NHC (S) NH-heteroaryl, -NHC (S) NHC (NH) -heterocycloalkyl, -NHC (NH) NH2, -NHC (NH) NH-C1-C12-alkyl, -NHC (NH) NH-C2-C12-alkenyl, -NHC (NH) NH-C2-C12-alkenyl, -NHC (NH) NH-C3-C12-cycloalkyl, -NHC (NH) NH-aryl, -NHC (NH) NH-heteroaryl, -NHC (NH) NH-heterocycloalkyl, -NHC (NH) -C1-C12-alkyl, -NHC (NH) -C2-C12-alkenyl, -NHC (NH) -C2-C12-alkenyl, -NHC (NH) -C3-C12-cycloalkyl, -NHC (NH) -aryl, -NHC (NH) -heteroaryl, -NHC (NH) -heterocycloalkyl, -C (NH) NH-C1-C12-alkyl, -C (NH) NH-C2-C12-alkenyl, -C (NH) NH-C2-C12-alkenyl, -C (NH) NH-C3-C12-cycloalkyl, -C (NH) NH-aryl, -C (NH) NH-heteroaryl, -C (NH) NH-heterocycloalkyl, -S (O) -C1-C12-alkyl, -S (O) -C2-C12-alkenyl, -S (O) -C2-C12-alkenyl, -S (O) -C3-C12-cycloalkyl, -S (O) -aryl, -S (O) -heteroaryl, -S (O) -heterocycloalkyl-SO 2NH2, -SO2 2-C2-C2-alkyl, -SO2 2-C2-C2-alkenyl, -SO2 2-C2-cycloalkyl, -SO2 2-aryl, -SO2 2-heteroaryl, -SO2 2-heterocycloalkyl, -NHSO 2-C2-C2-alkyl, -NHSO 2-C2-C2-alkenyl, -NHSO 2-C2-C2-cycloalkyl, -NHSO 2-aryl, -NHSO 2-heteroaryl, -NHSO 2-heterocycloalkyl, -CH2NH2, -CH2SO2CH 2, -aryl, -SO 2-C2-C2-cycloalkyl, -NHSO 2-heteroaryl, -CH2NH2, -CH 3, -arylalkyl, -heteroaryl, -heteroarylalkyl, -heterocycloalkyl, -C3-C12-cycloalkyl, polyalkoxyalkyl, polyalkoxy, -methoxymethoxy, -SH, -S-C1-C12-alkyl, -S-C2-C12-alkenyl, -S-C2-C12-alkenyl, -S-C3-C12-cycloalkyl, -S-aryl, -S-heteroaryl, -S-heterocycloalkyl, or methylthiomethyl. It is understood that aryl, heteroaryl, alkyl, and the like may be further substituted.

As used herein, the term "steroid" refers to any of a number of naturally occurring or synthetic fat-soluble organic compounds having 17 carbon atoms arranged in four rings as a basis and including sterols and bile acids, adrenal glands and sex hormones, certain natural drugs such as digitalis compounds, and precursors of certain vitamins. Examples of steroidal structures include, but are not limited to, cholesterol, cholestanol, 3 α -cyclo 5- α -cholestan-6- β -ol, cholic acid, cholesteryl formate, cholestene formate.

As used herein, the term "modified nucleoside" refers to any nucleoside that includes a modified heterocyclic base, a modified sugar moiety, or a combination thereof. In some embodiments, the modified nucleoside is a non-natural pyrimidine or purine nucleoside, as described herein. Examples of modified nucleosides include, but are not limited to, 2' -substituted ribonucleosides, arabinosides (arabinanucleosides) or 2' -deoxy-2 ' -fluoroarabinosides, deazaadenines, deazaguanines.

For the purposes of the present invention, the term "one or more short oligonucleotides" refers to a mono-, di-or polynucleoside formed from 1 to about 6 linked nucleoside units. Such short nucleotides may be obtained from existing nucleic acid sources, including genomic or cDNA, but are preferably produced by synthetic methods. The nucleoside residues may be coupled to each other by any of a number of known internucleoside linkages. Such internucleoside linkages may be modified or unmodified and include, but are not limited to, phosphodiester, phosphorothioate, phosphorodithioate, alkylphosphonate, alkylphosphonothioate, phosphotriester, phosphoramidate, siloxane, carbonate, alkoxycarbonyl, acetamidate, carbamate, morpholino, boron (borano), thioether, bridged phosphoramidate, bridged methylenephosphonate, bridged phosphorothioate and sulfone internucleoside linkages. The term "short nucleotide" also encompasses a polynucleotide having one or more stereospecific internucleoside linkages, such as (RP) -or (SP) -phosphorothioate, alkylphosphonate, or phosphotriester linkages. The short nucleotides of the present invention include any such internucleoside linkage, whether or not the linkage comprises a phosphate group. In certain preferred embodiments, these internucleoside linkages may be modified or unmodified, and include, but are not limited to, phosphodiester, phosphorothioate or phosphorodithioate linkages, or combinations thereof.

The term "one or more short nucleotides" also encompasses additional substituents including, but not limited to, protein groups, lipophilic groups, intercalators, diamines, folic acid, cholesterol, and adamantane.

The term "one or more short nucleotides" also encompasses any other nucleobase containing polymer, including but not limited to Peptide Nucleic Acids (PNA), peptide nucleic acids with phosphate groups (PHONA), Locked Nucleic Acids (LNA).

Examples of PNAs and LNAs are shown below:

"nucleotide" refers to a nucleic acid subunit (whether DNA or RNA or analogs thereof, such as Peptide Nucleic Acids (PNA) and Locked Nucleic Acids (LNA)) that includes internucleotide linkages, sugar groups, and heterocyclic bases, as well as analogs of such subunits. "nucleoside" refers to a nucleic acid subunit comprising a sugar group and a heterocyclic nucleobase. It will be understood that, as used herein, the terms "nucleoside" and "nucleotide" include moieties that contain not only naturally occurring internucleotide linkages (in the case of "nucleotide"), such as phosphodiester internucleotide linkages; naturally occurring sugar moieties such as ribose and deoxyribose moieties; and naturally occurring nucleobases such as purine and pyrimidine bases, e.g., adenine (a), thymine (T), cytosine (C), guanine (G), or uracil (U), further containing modified internucleotide linkages, modified sugar moieties, and modified purine and pyrimidine bases or analogs thereof, or any combination of modified and unmodified internucleotide linkages, sugar moieties, and purine and pyrimidine bases. Other examples of modified nucleosides include acyclic nucleosides, which consist of a ring-opened form of ribose and deoxyribose moieties. Accordingly, such open-ring nucleosides can be used to form modified nucleotides. Other examples of modified nucleosides include C-nucleosides such as pseudoisocytosine, and nucleoside mimetics include nucleoside isosteres such as peptide nucleic acid monomers and locked nucleic acid monomers.

Nucleobases include naturally occurring purine and pyrimidine nucleobases as well as modified nucleobases including, but not limited to, methylated purines or pyrimidines, acylated purines or pyrimidines, and the like, or the addition of protecting groups such as acetyl, difluoroacetyl, trifluoroacetyl, isobutyryl, benzoyl, and the like. The purine or pyrimidine base may also be an analogue of the foregoing; suitable analogues will be known to those skilled in the art and are described in the relevant textbooks and literature. Common analogs include, but are not limited to, 1-methyladenine, 2-methyladenine, N6-methyladenine, N6-isopentyladenine, 2-methylthio-N-6-isopentyladenine, N-dimethyladenine, 8-bromoadenine, 2-thiocytosine, 3-methylcytosine, 5-ethylcytosine, 4-acetylcytosine, 1-methylguanine, 2-methylguanine, 7-methylguanine, 2-dimethylguanine, 8-bromoguanine, 8-methoxyguanine, 8-methoxyadenine, 8-chloroguanine, 8-aminoguanine, 8-methylguanine, 8-thioguanine, N6-methyladenine, N6-isopentyladenine, 2-methylcytosine, 3-methylcytosine, 5-ethylcytosine, 4, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, 5-ethyluracil, 5-propyluracil, 5-methoxyuracil, 5-hydroxymethyluracil, 5- (carboxyhydroxymethyl) uracil, 5- (methylaminomethyl) uracil, 5- (carboxymethylaminomethyl) -uracil, 2-thiouracil, 5-methyl-2-thiouracil, 5- (2-bromovinyl) uracil, uracil-5-oxyacetic acid methyl ester, pseudouracil, 1-methylpseudouracil, Q nucleoside (queosine), inosine, 1-methylinosine, hypoxanthine, xanthine, 2-aminopurine, 6-hydroxyaminopurine, 6-thiopurine, 2, 6-diaminopurine-5-trifluoromethyl thymine, 6-chloro-adenine and 7-deazaadenine. Other examples include, but are not limited to, 5-fluoro-uracil, 5-trifluoromethyl thymine, 6-chloro-adenine, 2-cyclopentyloxy adenine, 7-deazaadenine.

In certain embodiments, base B may be a non-natural nucleobase, including a universal nucleobase. Such examples of base B include, but are not limited to, difluorotolyl, nitropyrrolyl, and nitroimidazolyl, among others.

It is also understood that "modified base" also referred to as "modified nucleobase" includes nitrogen-containing compounds, which may or may not be heterocyclic. Such preferred nitrogen-containing compounds include, but are not limited to, -NHR18, wherein R18 is hydrogen, butoxycarbonyl (Boc), benzyloxycarbonyl, allyl, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted aralkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or a heterocycle.

The term "modified nucleobase" is further intended to include heterocyclic compounds which are not nucleobases in the most general sense, but can serve as nucleobases. Such compounds include "universal bases" as known in the art. The universal base may include an aromatic ring moiety, which may or may not contain a nitrogen atom. In some embodiments, the universal base may be covalently attached to the C-1' carbon of the pentose sugar of the nucleoside. Examples of universal bases include 3-methylpropenylquinolone (PIM), 3-Methylisoquinolones (MICS), and 5-methylisoquinolones moieties. Further examples include inosine derivatives, pyrrole carboxamide analogues, nitropyrroles and nitroimidazoles.

Examples of modified nucleotide and nucleoside sugar moieties include, but are not limited to: trehalose, arabinose, 2' -deoxy-2 ' -substituted pentose moieties, 2' -O-substituted pentose moieties, ribose, lyxose and xylose, or hexose groups. For the purposes of the present invention, the term "2 '-substituted" such as "2' -substituted ribonucleoside" or "2 '-substituted arabinoside" in any of the named sugar groups includes ribonucleosides or arabinosides in which the hydroxyl group at the 2' -position of the pentose moiety is substituted to produce a 2 '-substituted or 2' -O-substituted ribonucleoside or arabinoside. Preferably, such substitution uses lower alkyl groups containing 1 to 6 saturated or unsaturated carbon atoms, or uses aryl groups having 6 to 10 carbon atoms, wherein such alkyl or aryl groups may be unsubstituted, or may be substituted, for example, with halogen, hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxy, alkoxycarbonyl, or amino. Examples of 2' -O-substituted ribonucleosides or 2' -O-substituted arabinosides include, but are not limited to, 2' -O-methyl ribonucleosides (also indicated herein as 2' -OMe) or 2' -O-methyl arabinosides and 2' -O-methoxyethyl ribonucleosides or 2' -O-methoxyethyl arabinosides. The term "2 ' -substituted ribonucleoside" or "2 ' -substituted arabinoside" also includes ribonucleosides or arabinosides in which the 2' -hydroxyl group is replaced by a lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, or an amino or halogen group. Examples of such 2 '-substituted ribonucleosides or 2' -substituted arabinosides include, but are not limited to, 2 '-amino, 2' -fluoro, 2 '-allyl, and 2' -propargyl ribonucleosides or arabinosides.

Examples of modified internucleotide linkages include, but are not limited to: substituted and unsubstituted phosphorothioate, phosphorodithioate, alkylphosphonate, alkylphosphonothioate, phosphotriester, phosphoramidate, siloxane, carbonate, alkoxycarbonyl, acetamidate, carbamate, morpholino, boron, thioether, bridged phosphoramidate, bridged methylenephosphonate, bridged phosphorothioate and sulfone internucleoside linkages.

The compounds of the present invention contain one or more asymmetric centers and thus may occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. The invention is intended to include short nucleotide compounds having a beta-D stereochemical configuration for the five-membered furanose ring, i.e., wherein the substituents at C-1 and C-4 of the five-membered furanose ring have a beta-stereochemical configuration ("up" orientation, which is generally indicated by the bold line in some of the formulae described herein).

The term "prodrug" includes compounds having moieties that can be metabolized in vivo. Generally, prodrugs are metabolized in vivo by esterases or by other mechanisms to the active drug. Examples of prodrugs and uses thereof are well known in the art (see, e.g., Berge et al (1977) "Pharmaceutical Salts", J.pharm.Sci.66: 1-19). Prodrugs can be prepared in situ during the final isolation and purification of the compound, or by separately reacting the purified compound in its free acid form or a hydroxy group with a suitable esterifying agent. The hydroxyl group can be converted to an ester via treatment with a carboxylic acid. Examples of prodrug moieties include substituted and unsubstituted, branched or unbranched lower alkyl ester moieties (e.g., propionates), lower alkenyl esters, di-lower alkyl-amino lower alkyl esters (e.g., dimethylaminoethyl ester), acylamino lower alkyl esters (e.g., acetoxymethyl ester), acyloxy lower alkyl esters (e.g., pivaloyloxymethyl ester), aryl esters (phenyl esters), aryl-lower alkyl esters (e.g., benzyl esters), substituted (e.g., with methyl, halogen, or methoxy substituents) aryl and aryl-lower alkyl esters, amides, lower alkylamides, di-lower alkylamides, and hydroxyamides. Some prodrug moieties are, for example, propionates and acyl esters. Prodrugs that are converted to the active form in vivo by other mechanisms are also included.

Abbreviations

Abbreviations that may be used in the description of the schemes and examples below are:

AcOH for acetic acid;

Boc₂o for di-tert-butyl dicarbonate;

boc for t-butyloxycarbonyl;

bpoc for 1-methyl-1- (4-biphenylyl) ethylcarbonyl;

bz for benzoyl;

bn for benzyl;

BocNHOH for tert-butyl N-hydroxycarbamate;

t-BuOK for potassium tert-butoxide;

Bu₃SnH is used for tributyltin hydride;

CDI for carbonyldiimidazole;

CH₂Cl₂for dichloromethane;

CH₃for methyl;

CH₃CN for acetonitrile;

DMSO for dimethyl sulfoxide;

EtOAc in ethyl acetate;

EtOH for ethanol;

Et₂o for diethyl ether;

HCl for hydrogen chloride;

MeOH for methanol;

MOM for methoxymethyl;

ms for mesyl or-SO₂-CH₃；

Ms₂O for methanesulfonic anhydride or methanesulfonyl anhydride;

NaCl for sodium chloride;

NaH for sodium hydride;

NaHCO₃for sodium bicarbonate (sodium bicarbonate) or sodium hydrogenocarbonate (sodium hydrogenocarbonate);

Na₂CO₃sodium carbonate;

NaOH is used for sodium hydroxide;

Na₂SO₄for sodium sulfate;

NaHSO₃for sodium bisulfite or sodium hydrogensulfiteite)；

Na₂S₂O₃For sodium thiosulfate;

NH₂NH₂for hydrazine;

NH₄HCO₃for ammonium bicarbonate;

NH₄cl for ammonium chloride;

OH for hydroxyl;

OMe for methoxy;

OEt for ethoxy;

TEA or Et₃N is used for triethylamine;

TFA trifluoroacetic acid;

THF for tetrahydrofuran;

TPP or PPh₃For triphenylphosphine;

ts for tosyl or-SO₂-C₆H₄CH₃；

Ts₂O for toluenesulfonic anhydride or tosyl anhydride;

TsOH for p-toluenesulfonic acid;

ph is for phenyl;

TBS for tert-butyldimethylsilyl;

TMS for trimethylsilyl;

TMSCl is used for trimethylchlorosilane.

Compounds of the invention

The present invention provides nucleosides, short oligonucleotide compounds or analogs thereof, or pharmaceutically acceptable salts, racemates, enantiomers, diastereomers, geometric isomers or tautomers thereof. The invention also includes compounds in the form of their prodrugs and metabolites.

In certain embodiments, the invention provides di-and trinucleotides, including but not limited to 3-dApsu_2'-OMe、3'dApsA_{7 denitrification}And 3' -dApsTpsC and analogs thereof, wherein "ps" refers to phosphorothioate internucleotide linkages.

wherein:

R₁and R₂Each independently is H, OH, O-alkyl, substituted alkyl, cycloalkyl, aryl, substituted aryl, aralkyl, heterocycle, O-aryl, O-heteroarylaryl, or heterocycle;

y and Z are each independently O or S;

m＝1-6。

In one embodiment, the compounds of the present invention include compounds of formula (II):

wherein:

x ═ absence, O, NH, NR, or S;

X₁absent, O or NH;

a ═ absence, aryl, or aralkyl;

n is 0, 1,2, 3,4 or 5;

y and Z are each independently O or S;

m＝1-6；

Other embodiments of the present invention provide the following compounds or a pharmaceutically acceptable salt, racemate, enantiomer, diastereomer, geometric isomer or tautomer thereof:

further, the compounds of the invention may comprise one or more modifications from the "native" nucleic acid, i.e., a native internucleoside linkage, or the nucleobase G, C, T, U, A, etc. Modifications include, for example, modifications of internucleotide linkages, bases, or sugar moieties.

The present invention also provides tautomers, stereoisomers, optical isomers, N-oxides, hydrates, solvates, polymorphs, pharmaceutically acceptable esters, amides, salts, prodrugs, and isotopic derivatives of the compounds described herein.

The structures of some of the compounds of the present invention include asymmetric carbon atoms. Accordingly, isomers arising from such asymmetries (e.g., all enantiomers and diastereomers) are included within the scope of the invention unless otherwise indicated. Such isomers may be obtained in substantially pure form by conventional separation techniques and/or by stereochemically controlled synthesis.

Naturally occurring or synthetic isomers can be isolated in several ways known in the art. Methods for separating racemic mixtures of two enantiomers include chromatography using Chiral stationary phases (see, e.g., "Chiral LiquidChromatography," w.j.lough, ed.chapman and Hall, New York (1989)). Enantiomers can also be separated by conventional resolution techniques.

Methods of obtaining the compounds of the invention include purchasing, synthesizing, or otherwise obtaining the compounds. The synthesis of the compounds of the present invention is within the chemical methods of one of ordinary skill in the art; exemplary methods for preparing the compounds of the present invention are described herein. Methods for optimizing reaction conditions, if desired to minimize competing by-products, are known in the art. The methods may further comprise steps before or after the steps specifically described herein to add or remove suitable protecting groups in order to ultimately allow synthesis of the compounds herein. In addition, the various synthetic steps may be performed in alternating order or sequence to obtain the desired compound.

Method and use

The compounds of the invention are useful in a wide range of therapeutic areas involving host immune components, including but not limited to: allergy, inflammation, autoimmune disease, COPD, asthma, and the like. Due to the fact that these compounds can act as immune response modifiers through a number of mechanisms, they have therapeutic utility in autoimmune diseases. For example, because compounds have the potential to stimulate an innate immune response, they can be used alone or in combination with other agents to treat a variety of cancers, including but not limited to melanoma, myeloma, carcinoma, glioblastoma, and sarcoma. For example, because certain oligonucleotide compositions have the potential to suppress immune responses, they can be used alone or in combination with other agents to treat a variety of autoimmune diseases, including but not limited to allergy, asthma, COPD, and multiple sclerosis.

Because many interferon-related gene products are capable of inducing apoptosis, compounds can induce selective cell death of cancer cells. Thus, the compositions of the invention may be used alone or in combination or sequence with a vaccine or another agent or agents.

The present invention thus provides a method of treating a microbial infection in a subject (or host) by administering to the subject (or host) identified as in need thereof an effective amount of a compound of the present invention.

In another aspect, the invention provides methods of improving an immune system response against a disease, condition, infection, or virus in a subject. The method comprises administering to the subject an effective amount of a compound of the invention as a vaccine adjuvant.

The invention also provides methods for preventing and treating viral infections in a host (or subject) by administering an effective amount of a compound of the invention. The compounds of the invention may be administered alone or in combination or sequence with vaccines or other agents.

In another aspect, the invention provides methods for preventing and treating bacterial infections in a host (or subject) by administering an effective amount of a compound of the invention. The compounds of the invention may be administered alone or in combination or sequence with vaccines or other agents.

Further, the present invention provides methods for preventing and treating parasitic infections in a host (or subject) by administering an effective amount of a compound of the present invention. The compounds of the invention may be administered alone or in combination or sequence with vaccines or other agents.

Also disclosed are methods for preventing and treating fungal infections in a host (or subject). The method comprises administering an effective amount of a compound of the invention, alone or in combination or sequence with a vaccine or another agent or agents.

In one embodiment, the compounds of the invention are

Further, the present invention provides the use of a compound provided in the following diagram, or a pharmaceutically acceptable salt, racemate, enantiomer, diastereomer, geometric isomer or tautomer thereof:

according to the methods of the invention, the compounds of the invention may be administered alone or in combination with a vaccine (e.g., BCG vaccine) or one or more additional agents.

In another embodiment, the methods of the invention are used against viruses.

In a further embodiment, the method is for treating or preventing cancer.

The methods of the invention comprise administering to the subject a therapeutically effective or inhibitory amount of a compound of the invention, in an amount and for a time necessary to achieve the desired result. Another method of the invention is to treat a biological sample with an inhibitory amount of a compound of the composition of the invention, in an amount and for a time necessary to achieve the desired result.

Another aspect of the invention includes the use of a compound of the invention in combination with one or more agents useful in the treatment of a disease. For example, such agents directed against viral activity include lamivudine (3TC), adefovir, tenofovir, ganciclovir, acyclovir, interferon, ribavirin, telbivudine; other agents directed to COPD, asthma, allergy, allergic rhinitis include, but are not limited to, theophylline, Alvesco (ciclesonide), Patanase (olopatadine hydrochloride), litaris (ambrisentan) (gilead), Xyzal (levocetirizine hydrochloride), Brovana (arformoterol tartrate), sreful (tiotropium bromide), Clarinex, beclomethasone propionate, remodellanil (treprostrostinil), xpenex, Duoneb (salbutamol and tiotropium bromide), formoterol fumarate, pancreligiol (bosentan), triamcinolone acetonide, budesonide, cisnine, sertraline, nidogen, nedocromil (inhaler and nebulizer), Zyflo, ancolol, anceol, clementine, herceptin. Among the anticancer agents are, for example, but not limited to, taxol, cisplatin, herceptin, gleevec, interferon, and the like.

In accordance with this method of the invention, the individual components of the combination may be administered separately at different times during the course of therapy or concurrently in divided or single combination forms. The invention is thus to be understood as embracing all such regimes of simultaneous or alternating treatment and the term "administering" is to be interpreted accordingly. It is to be understood that the scope of combinations of the compounds of the present invention with other agents includes in principle any combination with any pharmaceutical composition for the treatment of viral, bacterial, parasitic, fungal infections and the like. When a compound of the present invention or a pharmaceutically acceptable salt thereof is used in combination with a second therapeutic agent, the dose of each compound may be the same as or different from the dose of the compound when administered alone.

It will be appreciated that the scope of combinations of the compounds of the invention with other agents includes in principle any combination with any pharmaceutical composition for the treatment of COPD, asthma, allergic rhinitis, cancer and the like. When a compound of the present invention or a pharmaceutically acceptable salt thereof is used in combination with a second therapeutic agent, the dose of each compound may be the same as or different from the dose of the compound when administered alone.

For the purposes of the present invention, antimicrobial agents are intended to indicate compounds that are effective against viral, bacterial, fungal and parasitic infections.

As used herein, the term "therapeutically effective amount" of a compound of the present invention means a sufficient amount of the compound to produce a beneficial biological response in a biological sample or subject. As is well understood in the medical arts, a therapeutically effective amount of a compound of the present invention will be at a reasonable benefit/risk ratio applicable to any medical treatment.

The inhibitory amount or dose of the compounds of the invention may range from about 0.1mg/Kg to about 500mg/Kg, alternatively from about 1 to about 50 mg/Kg. The amount or dosage of inhibition will also vary depending on the route of administration and the possibility of co-use with other agents.

As used herein, the term "one or more biological samples" means a substance of biological origin intended for administration to a subject. Examples of biological samples include, but are not limited to, blood and its components such as plasma, platelets, subpopulations of blood cells, and the like; organs such as kidney, liver, heart, lung, brain, etc.; sperm and eggs; bone marrow and its components; or a stem cell. Thus, another embodiment of the invention is a method of treating a biological sample by contacting said biological sample with an inhibitory amount of a compound or pharmaceutical composition of the invention.

Upon improvement of the subject's condition, a maintenance dose of a compound, composition or combination of the invention can be administered, if desired. Subsequently, depending on the symptoms, the dosage and frequency of administration, or both, may be reduced to a level at which the improved condition is maintained, and treatment should be discontinued when the symptoms have been alleviated to the desired level. However, following any recurrence of disease symptoms, the subject may require intermittent treatment on a long-term basis. However, it will be understood that the total daily use of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific inhibitory dose for any particular patient will depend upon a variety of factors, including the disorder to be treated and the severity of the disorder; the activity of the particular compound employed; the specific composition employed; the age, weight, general health, sex, and diet of the patient; time of administration, route of administration, and time of rate of excretion of the particular compound employed; the duration of the treatment; drugs used in combination or concomitantly with the specific compound employed; and similar factors well known in the medical arts.

Pharmaceutical composition

The present invention provides pharmaceutical compositions comprising the compounds of the present invention and derivatives thereof, in combination with a pharmaceutically acceptable carrier. Another example of the invention is a pharmaceutical composition prepared by combining any of the above compounds with a pharmaceutically acceptable carrier.

The pharmaceutical compositions of the invention comprise at least one compound of the invention or a pharmaceutically acceptable salt thereof as active ingredient and may also contain pharmaceutically acceptable carriers and excipients and optionally other therapeutic ingredients. By "pharmaceutically acceptable" it is meant that the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. Compositions include those suitable for oral, rectal, topical, parenteral (including subcutaneous, intramuscular, and intravenous), ocular (ophthalmic), pulmonary (nasal or buccal inhalation), or nasal administration, although the most suitable route in any given case will depend on the nature and severity of the condition to be treated and on the nature of the active ingredient. They may be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy.

In actual use, the compounds of the present invention may be combined as the active ingredient in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical formulation techniques. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral (including intravenous). In preparing the compounds for oral dosage forms, any of the usual pharmaceutical media may be employed, such as water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like in the case of oral liquid preparations such as suspensions, elixirs and solutions; or carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations such as powders, hard and soft capsules and tablets, with solid oral preparations preferably being more than liquid preparations.

Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage unit form in which case solid pharmaceutical carriers are obviously employed. If desired, the tablets may be coated by standard aqueous or non-aqueous techniques. Such compositions and formulations should contain at least 0.1 percent of the active compound. The percentage of active compound in these compositions may of course vary and may conveniently be from about 2 to about 60 percent of the unit weight. The amount of active compound in such therapeutically useful compositions is such that an effective dose will be obtained. The active compounds may also be administered intranasally as, for example, liquid drops or sprays.

Tablets, pills, capsules and the like may also contain binders such as tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; disintegrating agents such as corn starch, potato starch, alginic acid; lubricants such as magnesium stearate; and sweetening agents such as sucrose, lactose or saccharin.

When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier such as a fatty oil. Various other materials may be present as coatings or to modify the physical form of the dosage unit. For example, the tablets may be coated with shikonin, sugar, or both. A syrup or elixir may contain, in addition to the active ingredient, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Various stabilizers may be added which will stabilize the active pharmaceutical ingredient against degradation, such as amino acids or polyamines. Other excipients may include, but are not limited to, PEG 400, glycine, vitamin E derivatives, sorbitan monooleate, chitosan, choline citrate, sorbitan monostearate, Tween 80, Igepal CA 630, Brij 35, NP-40, and similar derivatives thereof.

The compounds of the invention may also be administered parenterally. Solutions or suspensions of these active compounds can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form is preferably sterile and preferably fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.

Any suitable route of administration may be employed to provide a therapeutically effective dose of a compound of the invention to a mammal, particularly a human. The terms "administration" and "administering" of a compound are understood to mean providing a compound of the invention to an individual in need thereof. Routes of administration include, for example, but are not limited to, oral, sublingual, transmucosal, intravenous, subcutaneous, intranasal, topical, vaginal, and the like.

Regardless of the route of administration chosen, one or more compounds used in a suitable hydrated form and/or the pharmaceutical compositions of the present invention may be formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

The actual dosage level and time course of administration of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for the particular patient, composition and mode of administration, and which is non-toxic to the patient. Exemplary dosage ranges are about 0.1 μ g to 20 mg/kg body weight/day (mg/kg/day) (e.g., 0.1 μ g/kg to 2mg/kg, 0.3-3 μ g/kg, 0.18-0.54 mg/kg). In other embodiments, the amount varies from about 0.1 mg/kg/day to about 100 mg/kg/day. In still other embodiments, the amount varies from about 0.001 μ g to about 100 μ g/kg (e.g., body weight). Ranges intermediate to the above values are also contemplated as part of the invention.

The total daily inhibitor amount of a compound of the invention administered to a subject in a single, multiple or divided dose may be in an amount of, for example, 0.01-50mg/kg body weight, or more typically 0.1-25mg/kg body weight.

A single dosage composition may contain such amounts, or submultiples thereof, to make up a daily dose. The multiple doses may be a single dose taken at different time intervals. Generally, a treatment regimen according to the present invention comprises administering to a patient in need of such treatment from about 10mg to about 1000mg of one or more compounds of the present invention per day in single or multiple doses.

Further, the compounds of the present invention may be administered by multiple delivery routes such as oral, intravenous, sublingual, intranasal, topical, and the like.

Reagent kit

The invention also provides kits for treating or preventing a disease, condition, or disorder described herein. In one embodiment, the kit comprises a therapeutic or prophylactic composition comprising an effective amount of a compound of the invention in unit dosage form. In some embodiments, the compounds of the invention are provided in combination with a vaccine or conventional therapeutic agent. In other embodiments, the kit comprises a sterile container containing the therapeutic or prophylactic composition; such containers may be in the form of boxes, ampoules, bottles, vials, tubes, bags, pouches, blister packs, or other suitable containers known in the art. Such containers may be made of plastic, glass, laminated paper, metal foil, or other material suitable for containing a medicament.

Where desired, the compounds of the invention are provided along with instructions for administering the compounds to a subject having or at risk of a particular disease, condition, or disorder described herein. The instructions generally include information about using the composition for treating or preventing a disease, condition, disorder, infection, or virus. In other embodiments, the instructions include at least one of: instructions for a therapeutic agent; a dosage schedule and administration for the treatment or prevention of ischemia or a symptom thereof; matters to be noted; a warning; indications; contraindications; overdose information; adverse reactions; animal pharmacology; clinical studies; and/or a reference. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate leaflet, booklet, card or folder supplied in or with the container.

Examples

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the assays, screens, and therapeutic methods of the invention are made and used, and are not intended to limit the scope of what the inventors regard as their invention.

Synthesis of Compounds

A pooled library of nucleotide analogues as described in the present invention as an illustrative example such asPreviously synthesized (17-22). Both solid phase synthesis and liquid phase strategies are used in complementary fashion to prepare a pooled library as exemplified. The method can be used to synthesize libraries having modifications at sugar, nucleobase, and internucleotide linkages. Several technological innovations in our laboratory facilitate the synthesis of nucleotide libraries: a) we have previously developed strategies for the synthesis of dinucleotide libraries using solid phase phosphoramidite and H-phosphonate techniques (17-22). Recent technological innovations from our laboratory have also been used for solid phase synthesis of dinucleotide compounds and analogues (17-22). (i) Ultra-rapid preparation of amino-and carboxy-functionalized solid supports using microwave-assisted methods, (ii) novel methods for loading nucleosides onto solid supports. An improved process for large scale preparation of nucleoside loaded supports was developed which involved the use of Dimethylformamide (DMF) as a solvent. (ii) high nucleoside loadings to obtain 80-300. mu. moles/g support, (iii) for loading solid supports and solid phase synthesis, termedThe novel reactor of (1), which facilitates large scale synthesis of dinucleotides.Are multi-purpose reactors (20-22) equipped with pneumatic valves for controlled delivery of reactants.

Example 1

Using specially constructed LOTUS reactors(Padmanabhan, S.; Coughlin, J.E.; Iyer, R.P. tetrahedron Lett.2005, 46, 343; Iyer, R.P.; Coughlin, J.E.; Padmanabhan, S.org.Prep.Proc.Intl.2005, 37, 205) Synthesis of phosphorothioate analogs 3-dApsu2' -OM by Large Scale (1 millimole nucleoside loaded Controlled Pore Glass (CPG) support) solid phase phosphoramidite chemistry (Beaucage, S.L.; Iyer, R.P. tetrahedron 1993, 49, 1925)e (1) mixture of Rp and Sp. The dA-linked CPG supports were prepared using our recently developed ultra-rapid functionalization and loading procedure for solid supports. For sulfurization of the internucleotide dinucleotide phosphite coupled product, 3H-1, 2-benzothiophen-3-one-1, 1, -dioxide (0.4M in dry CH3 CN) (Iyer, R.P.; Regan, J.B.; Egan, W.; Beaucage, S.L.J.Am.chem.Soc.1990, 112, 1253) was used.

After processing, chromatographic purification and lyophilization, obtain>96% pure Rp, Sp 5 (. about.60: 40 mixture) sodium salt by³¹P and¹h NMR was carried out for characterization. Thus, solid phase synthesis of a pooled library of dinucleotide compounds and analogs is readily performed. In a complementary strategy, liquid phase synthesis was also used for the synthesis of 1. These methods allow the synthesis of compounds with a variety of chemical modifications.

Example 2: synthesis of Compound 3

The target compound 3 was prepared in two steps.

And (1). Preparation of iodomethyl isopropyl carbonate: to a solution of anhydrous sodium iodide (6g, 40mmol) in anhydrous acetonitrile (20mL) was added dropwise a solution of chloromethyl isopropyl carbonate (2.9g, 19mmol) in anhydrous acetonitrile (10mL) over 20 minutes. The reaction mixture covered with aluminum foil (protected from light) was stirred at room temperature overnight. The prepared solid was filtered, washed with acetonitrile and the filtrate was concentrated under reduced pressure. The residue was dissolved in water (10mL) and the organics were extracted in ether (25 mL). The ether extract was washed with sodium bisulfite (5%, 10mL), followed by brine (10 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, concentrated and dried under high drying vacuum. Yield 2.72g (58%);¹H-NMR1.3(d，6H)，4.95(m，1H)，5.95(s，2H)。

and 2. step 2. Alkylation of compound 5: to a solution of dinucleotide 1(60mg, 0.098mmol) in water (HPLC, 400mL) was added a solution of iodomethyl isopropyl carbonate (80mg, 0.0166mmol, 3.33 equiv.) in acetone (1mL) with stirring. Additional acetone (1mL) was added to obtain a clear solution to avoid any separation of the oily globules of alkylating agent. The reaction mixture covered with aluminum foil was stirred for 3 hours, concentrated under rotary evaporation conditions, and then concentrated in high vacuum to obtain the reaction mixture as a white solid. This was purified by silica column chromatography, initially using chloroform and slowly using chloroform containing 2% to final 8% methanol. The fractions containing the major component were combined, concentrated and dried under high vacuum overnight. The desired pure product 3 was isolated in almost quantitative yield (68 mg);³¹P-NMR(MeOH-d₄)27.7，28.6。

example 3: discovery and Activity of SB40, SB44, and SB1B as NOD2/RIG-I ligands

The evaluation of SMNH compounds for NOD2 activation was performed by primary, secondary and tertiary assays. In a primary assay, SMNH compounds were tested for induction of IRF3 expression in HLE a549 cells, which are known to endogenously express NOD 2. As shown in fig. 1-2, the dinucleotide analogs SB40, SB 43, SB44, and SB1B, but not the related analogs such as the dinucleotides SB50, SB 110, and SB 60, induced IRF3 activation. SB44, a mixture of the two isomers Rp and Sp, is a prodrug of SB 40. The Rp isomer of SB 1B-SB 44, shown as SB 44-1 in FIG. 1-2.

IRF3 luciferase transfected cells were incubated with dmso (ut) only or SMNH compound (μ Μ). After 12 hours incubation, Luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's protocol. Transfection efficiency was normalized by measuring the expression of renilla luciferase. Luciferase units (i.e., fold induction) were measured by standard methods (mean ± sd; 3 independent experiments). The results are depicted in fig. 1.

HEK293 cells were transfected with NOD2, pcDNA and IRF3 luciferase. The cells were then incubated with either ssRNA (0.5mg/ml) or SMNH compound (1 μm). After 12 hours incubation, luciferase activity was measured as previously described. Luciferase assay results are presented as mean ± s.d. from three independent experiments (see reference (28) for detailed materials and methods employed in these assays).

FIG. 3 shows RIG-I activation by SB 44. HEK293 cells were transfected with RIGI, pcDNA and IRF 3-luciferase. Cells were then incubated with SB44 (100 or 500 nM). After 8 or 16 hours incubation, luciferase activity was measured as previously described. Luciferase assay results are presented as mean ± s.d. from three independent experiments.

FIG. 4 demonstrates the induction of NF-kB activity by SB44 via RIGI and Nod2 activation. HEK293 cells were transfected with RIGI, pcDNA, Nod2 and NF-kB-luciferase. The cells were then incubated with SB44 (10. mu.M). After 12 hours incubation, luciferase activity was measured as previously described. Luciferase assay results are presented as mean ± s.d. from three independent experiments.

Fig. 5 shows detection of activated IRF3 (phosphorylated IRF3 or phospho-IRF 3) in SB44 treated Human Lung Epithelial Cells (HLECs). Human lung epithelial a549 cells were incubated with DMSO or SB44 (500 nM). At the indicated post-treatment time points, western blots using antibodies specific for phosphorylated IRF3 (phospho-IRF 3) (cell signaling) were performed on the cell lysates.

Fig. 6 shows detection of activated IRF3 (phosphorylated IRF3 or phospho-IRF 3) in SB44 treated Human Lung Epithelial Cells (HLECs). Human lung epithelial a549 cells were incubated with DMSO or SB44 (500 nM). At the indicated post-treatment time points, western blots using antibodies specific for phosphorylated IRF3 (phospho-IRF 3) (cell signaling) were performed on the cell lysates.

Further test results are depicted in fig. 7-9.

Conclusion and discussion: based on the above results, compounds SB40, SB44 and SB1B were evaluated for adjuvant-like activity in combination with BCG vaccine. When used in conjunction with BCG, the dinucleotide compounds SB40, SB44 and SB1B, which activate NOD2/RIG-I, were also found to enhance antigen presentation in vitro, increase MHC-II in Μ Φ and cause a dramatic increase in anti-TB T cell function in mice. As previously mentioned, SB44 is a mixture of two isomers (Rp and Sp), with SB1B being the Rp isomer making up about 55-60% of the mixture.

Example 4: design of optimal ligands for NOD2 and RIG-I

To find the best ligands for NOD2 and RIG-I, the dinucleotide structures SB40 and SB44 were used as initial lead compounds for the synthesis of additional compounds involving both base and sugar modifications as shown below as illustrative examples. Both solid-phase and liquid-phase synthesis strategies, chemically coupled to phosphoramidites and H-phosphonates, are used for their synthesis. Many building blocks are commercially available that will be synthesized internally.

Representative examples of compounds related to SB40 and SB44 are shown below:

example 5: study of NLR ligands as adjuvants with BCG vaccine using macrophages and mice

Principle of adjuvant development on SB 1B: adjuvants are known to enhance cytokine secretion in m Φ, but their effect on m Φ and DC ability to present antigen to activate T cells is unknown. Likewise, it is unclear whether T cell function can be modulated for long term efficacy. BCG is today the most widely used vaccine for the prevention of tuberculosis worldwide. It protects children from tuberculosis, but has limited efficacy against adult TB, possibly due to lack of long-term efficacy. In this section, we demonstrated that SB1A and SB1B enhance μ Φ function when infected by BCG vaccine, and that they potentiate BCG activity when administered as a primary vaccine to mice, and have the potential to protect against re-infection of tuberculosis as a booster vaccine.

C57B1/6 mouse bone marrow-derived macrophages (μm. phi.) were treated with 10 μ g/well of SMNH compound 1-4 and LPS positive control (1 μ g/mL) for 4 hours followed by 4 hours of bacterial infection with Mycobacterium bovis (Mycobacterium bovis) BCG (BCG). BB7T cells specific for the antigen 85B epitope were overlaid and supernatants collected after 18 hours were tested for IL-2 using a sandwich ELISA (p-value, t-test).

FIG. 10(a-d) shows: a) initial screening for the most active SB compounds, SB 1(NOD2 agonist) and SB3 and SB44 (SB 1-associated dinucleotides) were evaluated along with LPS, a known TLR4 agonist. a) SB1 and 2 enhance antigen presentation; b) SB1A (SB 44) and SB1B (SB 1B, Rp isomer of SB 44) were compared to SB 2 and LPS. The Rp isomer of SB44 (SB 1B) retained full activity; c) μ m Φ were treated with 5 μm each of the signaling molecule inhibitors (AP-1, CREB and MAPK (p38, ERK1/2 and JNK) for 2 hours, followed by 2 hours of activation with SB compound and 2 hours of infection with BCG. μ m Φ was fixed and surface MHC-II was analyzed using flow cytometry. SB1A enhances surface expression of MHC-II, which is inhibited by blocking MAPK or AP-1/CREB (> 0.5log 10 transition compared to the SB1A combination with BCG). SB1B has similar activity (not shown); d) inhibitors of MAPK or AP1/CREB alone had no effect on MHC-II.

Example 6: NOD2-RIG-I activating SB1A and 1B Compounds enhance the efficacy of BCG vaccines against experimental aerosol-induced tuberculosis

Vaccine experimental design in mice: C57B1/6 mice were vaccinated with BCG alone or mixed with SB1A, SB1B and MDP; this was followed 4 weeks later by challenge with aerosol of mycobacterium tuberculosis. Mice were sacrificed after 4 weeks for assessment of protection (decreased CFU count for Mtb). The phenotype and function of T cells of the spleen or lung were measured using flow cytometry. FIG. 11(a)

Fig. 11(B) shows that SB1A and SB1B are potent adjuvants that reduce the Mtb count of the lung better than BCG or MDP + BCG (p value by two-factor ANOVA). The numbers above the bars indicate the log10 bacterial load (MDP vs. SB 1A/SB 1B + BCG; p < 0.02). SB1A and SB1B were also better than the BCG vaccine in reducing Mtb counts in the spleen. Compared to the BCG vaccine, SB1B enhanced the number of multifunctional T cells, but both SB1A and SB1B induced comparable levels of tetramer-specific CD8T cells (fig. 11 (c-d)).

SB1A induced better memory precursor T cells (MPEC) than the BCG vaccine (fig. 11 e). The histograms illustrated below show flow cytometric analysis of T cell populations expressing memory markers in the spleen.

Primary macrophages derived from C57B1/6 mouse bone marrow were treated with SB1A or SB1B alone, MDP alone or the indicated combinations for 4 hours. BCG was used for infection for 4 hours and the supernatant collected after 18 hours of titration for mature IL1 β using ELISA. Macrophages were first treated with ZVAD-fmk for caspase blocking (2 hours), followed by activation with SB1A and SB1B (4 hours) and BCG infection (4 hours). Cytokines were measured after 18 hours. The results are presented in fig. 12, showing that by a caspase-dependent mechanism in macrophages, NOD2 activating compounds SB1A and SB1B induced IL-1 β in combination with MDP or whole BCG bacilli only.

NOD2 activated SB 1A/1B combination with BCG vaccine induced protection against re-challenge of tuberculosis in mice, suggesting long-term protection. The BCG vaccine is the only vaccine approved for the prevention of TB, although it is weakly protective against aerosol-induced TB and induces weak central memory in mice (29). This is believed to be due to the inability of BCG to induce MPEC. The SB compound was found to induce elevated MPEC (see fig. 11 (e)). However, MPEC producing central memory is present in low numbers in mice, and its function can only be determined by its strong recall expansion effect CD8T cells.

Therefore, a new model was developed to demonstrate that SB1A and SB 1B-induced MPEC can lead to long-term protection by regeneration into strong effector T cells in mice. This model is shown in fig. 14, where Mtb as well as vaccine were cleared by drug therapy after primary vaccination and challenge, allowing MPEC to quiesce. Subsequently, mice were re-challenged with virulent Mtb for assessment of protection.

C57B1/6 mice were vaccinated and challenged and isoniazid and rifampicin were used for 3 weeks to clear vaccine and Mtb organisms, followed by rest and re-challenge with aerosol doses of virulent Mtb. After four weeks, protection was assessed by CFU counts and T cell analysis of lungs and spleen from 3 separate mice/group. Most importantly, the SB1B (SB 1B) combination with BCG was twice as well protected (p-value by two-factor ANOVA; 5 mice/group/time point) after both primary and secondary re-challenge.

Fig. 15(a-B) shows that SB1A or SB1B combinations with BCG vaccine retained the ability to generate a better recall response against re-challenge with tuberculosis.

After re-challenge (week 16), the lungs of the mice shown in figure 11 were analyzed for CD8T cells using flow cytometry. The histogram example shows tetramer staining for CD8T cells. Tetramer + IFN γ stain was used to measure functional T cells, and tetramer + CD44 stain was used to measure memory CD8T cells against tuberculosis in the lung. (FIGS. 16a-b)

The data show that the lungs of vaccinated mice contain comparable numbers of CD62L + CD44+ central memory T cells and functional CD8T cells expressing perforin + granzyme. (FIG. 16c-d) however, the lungs contained elevated levels of tetramer + IFN γ CD8T cells, which also expressed memory markers CD62L and CD 44. This suggests that SB1A and SB1B potentiate the magnitude of antigen-specific memory T cells, which is critical for long-term suppression of tuberculosis.

In summary, these separate studies in mice have shown that SMNH combinations with BCG vaccine enhance protection against both primary and secondary challenge with tuberculosis.

Example 7: preclinical study of SB44

A number of preclinical studies have been performed using SB44 and SB40, and these studies illustrate results with respect to the lead compound adjuvant candidate SB 1B.

As exemplified by studies using BCG, the mechanism of action of SB44 and SB40 involves the induction and activation of RIG-I and NOD2 in the presence of "foreign" nucleic acids. Most exogenous nucleic acids have a marker pattern (PAMP, pathogen-associated molecular pattern) in their structure that underlies the selective recognition of these nucleic acids as "exogenous" by RIG-I and NOD 2. To illustrate this unique selectivity in mechanism of action, several in vitro studies were conducted and summarized below: 1) by multiplexing assays for 28 cytokines, SB40 and SB44 were evaluated for their ability to induce cytokines in Peripheral Blood Mononuclear Cells (PBMCs): IFN-alpha, IFN-beta, IFN-gamma, IL-1a, IL-1b, IL-1RA, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12p40, IL-12p70, IL-13, IL-17A, TNF alpha, TNF beta, G-CSF, GM-CSF IL-8, MCP-1, Eotaxin, Mm pi-1 alpha, Mpi-1 beta and RANTES. Compounds up to 50 μ Μ did not cause cytokine induction in human PBMC cultures.

2) To assess whether SB40 and SB44 can potentiate IFN production in cells in the presence of bacterial nucleic acids, PBMCs were measured with BCG alone and with BCG with SB44 or SB40 at 25 μ Μ. BCG is used as a source of bacterial nucleic acid. PBMCs were incubated with compounds alone or in the presence of BCG for 24 hours and supernatants were harvested. IFN levels were determined using ELISA. In the presence of BCG, the compounds caused a modest boost in IFN production. (according to the same experiment, BCG did not induce other cytokines such as IL-1, 6, 8, 10 and TNF-. alpha.).

3) An experiment was performed using an ISG56 luciferase reporter to determine whether exogenous RIG-I and NOD2 expression could be induced in receptor deficient cells. For this purpose, poly I-poly C was used as a positive control. The addition of SB40 and SB44 to these cells caused a modest 2-fold increase in ISG-56 expression over that induced by poly I-poly C due to activation of RIG-I and NOD 2.

The results demonstrate that IFN induction by SB40 and SB44 (mediated by the NOD2/RIG-I signaling cascade) can occur in the presence of exogenous nucleic acids. There is therefore less potential for these compounds to cause non-specific stimulation of the immune pathway, which produces a "cytokine storm" and associated systemic toxicity. These mechanisms of action were observed to be consistent with the adjuvant activity of SB44 on BCG and should be equally applicable to ITS ISOMERS.

Example 8: metabolism of SB44 in the S9 fraction from human liver

SB44 (10 μ M) was incubated with a mixture containing S9 fractions (pooled by humans) (1mg/ml), NADPH (1.3mM), UDPGA (5mM), doxorubicin (10 μ g/ml) in 1ml of 1 XPBS buffer, S- (5' -adenosine) -L-methionine iodide (0.1mM), adenosine 3' -phosphate 5' -lithium phosphate sulfate hydrate (0.1mM), and acetyl CoA (1 mM). Metabolite samples were evaluated by HPLC and LC/MS analysis.

Prodrug R_p，S_pExposure of SB44 to liver microsomes for up to eight hours results in a stereospecific conversion to dinucleotide R_p，S_pSB 40. LC/MS assessment of microsomal incubation revealed that one or more major products of metabolism are R_p，S_pThe dinucleotide SB 40. Also small amounts of desulfurization products were detected as determined by MS analysis: (<5%). A few minor metabolites are also found: (<10%). However, the identity of these metabolites cannot be adequately determined based on the molecular ions. In all cases, R of the prodrug SB44 is involved in microsomal metabolism_pAnd S_pAll isomers underwent stereospecific conversion to active SB40, with only minor amounts of desulfurization products being observed [ corresponding to SB40]. In thatIndividual R of SB44_pAnd S_pConversion of isomers to R of SB40, respectively_pAnd S_pIn isomers, there was no significant rate difference.

Similar to the serum-mediated SB44 to SB40 switch, in the microsomal case, liver esterases appear to be the major metabolic enzymes involved in the hydrolytic switch of SB44 to SB 40. In the early stage of serum use_p，S_pIn the bioreversibility study of SB44, the individual isomers are stereospecifically converted to R at nearly equal rates_p-SB 44，S_pSB 40. The mild biotransformation of SB44 to SB40 in liver and plasma is also consistent with the broad substrate specificity of ubiquitous esterases.

Example 9: in vitro metabolism of SB44 by the S9 fraction

Similar to the purified human liver microsome study, exposure of the prodrug SB44 to the human liver S9 fraction resulted in the stereospecific conversion of SB44 to the dinucleotide SB 40. Evaluation of the incubation by LC/MS revealed that in addition to the main product SB40, a small amount of desulfurization product (< 5%) corresponding to SB40 was formed. Minor secondary products were also observed. The metabolite pattern for the S9 fraction was similar to that observed with purified liver microsomes. Thus, there was no clear evidence of any phase II conjugation reaction of SB44 or the initially formed active metabolite SB 40.

Example 10: in vitro preclinical toxicology study of SB44

SB44 was subjected to cytotoxicity assessment against a panel of cell lines including Madin-Darby bovine kidney (MDBK), Vero and HFF (human foreskin fibroblasts) to determine their CC₅₀. The standard microculture Tetrazolium Assay (MTT) was performed in 96-well plates using MDBK, Vero and HFF cell lines (from american type culture center). Controls included nucleoside analogs 3TC, AZT and ddC, as well as drug-free medium. SDS used as a positiveSex cytotoxicity control. All prodrugs were tested in triplicate at concentrations of 100, 300 and 1000 μm. After 24 hours incubation of the cells with the test substance, the MTT assay was performed.

CC of SB40₅₀Is composed of>1000μΜ。

Example 11: bacterial mutation test (screening format) of SB44

The aim of this study was to evaluate the genotoxicity of the test article using a screening (non-GLP) version of the bacterial mutation test.

Research and design: the test is performed in only one occasion. A subset of standard test strains (tabulated strains) was used to evaluate the genotoxic potential of the test article.

Test articles were formulated in Dimethylsulfoxide (DMSO) and tested at a maximum concentration of 5000 μ g/plate (the standard limiting dose for this assay) along with approximately half-log dilutions, using bacterial mutation testing in preincubated format. The absence of colonies on the sterility check plate confirms the absence of microbial contamination. The average revertant colony count for the vehicle control was close to, or within, the laboratory historical control data. The increase in the number of revertant colonies induced by the appropriate positive control compound (+/-S9 mixture) was at least twice (1.5X for strain TA 100) the simultaneous vehicle control level with the appropriate bacterial strain, confirming that the same sensitivity and activity of the S9 mixture was tested. After exposure to SB44, no significant thinning of the background lawn of the non-revertant bacteria was obtained, indicating that the test article was non-toxic to the bacteria at the level tested. No precipitation was observed.

In the absence or presence of the S9 mixture, no substantial increase in colony count of the revertants was obtained in any of the strains after exposure to the test article. It was therefore concluded that SB44 did not show any evidence of genotoxic activity in this in vitro mutagenesis assay.

Example 12: postural cardiovascular safety assay (hERG test).

The purpose of this study was to examine the in vitro effects of SB40 and SB44 on the hERG (human ether-a-go-go related gene) potassium channel current (IKr, a surrogate for rapidly activating delayed rectifier cardiac potassium current).

In this study, the hERG channel was expressed in a human embryonic kidney (HEK293) cell line lacking endogenous IKr. HEK293 cells were stably transfected with hERG cDNA and evaluated at room temperature using QPatch(automated parallel patch clamp system). Each test article was evaluated at 10, 50, 100 and 200 μ Μ, with each concentration tested in three cells (n-3). The duration of exposure to each test article concentration was 3 minutes.

Since the highest concentration of hERG current inhibition passing the test was less than 50%, the IC of both test items could not be determined₅₀The value is obtained. The positive control E-4031 confirms the sensitivity of the test system to hERG inhibition.

In summary, SB40 and SB44 did not show any activity in the hERG assay, and IC₅₀Is composed of>200μΜ。

Example 13: in vivo preclinical toxicology study of SB44

SB44 is a mixture of two diastereomers (Rp, Sp), where SB1B is the Rp isomer, present in the mixture to an extent of 55-60%. Many preclinical studies were performed using SB44 administered via oral gavage, and the study results are illustrative of possible results when SB1B was used alone. Studies have laid a solid foundation for preclinical studies of SB1B, as analytical and bioanalytical methods, formulations for toxicological studies, toxicological protocols, etc. can be transferred to SB 1B.

Example 14: dose Range finding study of SB44 in Sprague-Dawley rats

The objective of this study was to determine the potential toxic effects, identify potentially toxic target organs, and determine the Maximum Tolerated Dose (MTD) and the level at which no adverse effects were observed for the end-point of the examination (NOAEL), after 7 consecutive days of daily oral gavage of SB44 in adult male and female Sprague-Dawley rats. The information from this study was used to design a subsequent toxicity study and to determine the suitability of the proposed human dose. The study consisted of phase a and phase B. Phase a is a dose range finding study. Two rats (1 male and 1 female) were included in this study. Animals were given a single dose of SB44 at 1000mg/kg by oral gavage (po) and observed for 3 days. Both animals had the expected weight gain and appeared normal throughout the study until the time of their scheduled sacrifice. Autopsy was not performed.

In phase B, rats (3/sex/group) were given daily oral doses of SB44 at 50, 250 and 1000 mg/kg/day for 7 consecutive days. The control group (3/gender) was also given a daily oral dose of equal volumes of vehicle, 50% PEG 400 plus 50% HPMCT (0.1% hydroxypropyl methylcellulose and 0.2% Tween 80 in sterile water) for 7 days. Animals were sacrificed on day 8. The following parameters were evaluated: mortality/morbidity, clinical observations, body weight, clinical pathology (hematology and serum chemistry), organ weight, and at necropsy, macroscopic and microscopic histopathology of liver tissue.

All animals in stage B survived until their scheduled necropsy. Dose-dependent "shoveling" patterns were noted in all animals in the treatment group at different days after dose administration, except males in the low dose group. Since there are no other adverse signs associated with nervous system function or other toxicological parameters, this observation most likely indicates that the rat perceives the taste and/or texture of the test article as unpleasant. No other drug related effects were found for body weight, clinical pathology, organ weight, and macroscopic and microscopic evaluation. In summary, daily oral gavage of SB44 administered to male and female Sprague-Dawley rats for 7 consecutive days produced no significant biologically or toxicologically significant adverse effects. NOAEL was considered to be at least 1000 mg/kg/day when SB44 was administered by daily oral administration for 7 consecutive days.

Example 15: 14-day toxicity study of SB44 in rats with two-week recovery phase and bone marrow micronucleus assessment

The objective of this study was to determine the potential toxic effects of SB44 after 14 days of daily oral dose administration, and to assess the genotoxicity of SB44 as determined by micronucleus assessment. Male and female Sprague-Dawley rats were administered SB44 daily at doses of 50, 200 and 500 mg/kg/day for 14 consecutive days. The satellite set was similarly processed for the purpose of pharmacokinetic (TK) assessment, and plasma was analyzed for SB40 (active metabolite of SB 44) levels. Micronucleus evaluation was performed to determine the potential genotoxicity of SB 44. Necropsies were performed at day 15 for rats in the main study group and at day 28 for rats in the recovery group.

All rats survived to the end of the study. SB44 caused no significant toxicity as determined by clinical observations, clinical pathology, body weight, urine parameters, autopsy, and organ weight. However, microscopic evaluation of the tissues revealed changes in the thymus associated with the test article. A minimal to mild increase in thymophagocytic macrophages was observed in both males and females at 200 and 500 mg/kg/day, indicating increased thymic lymphocyte destruction. This thymus change is present in both the primary and recovery groups, but is not associated with any histopathological evidence of thymus atrophy or increased thymus deterioration. There was no evidence of increased lymphocyte destruction in other organs. This observation may be GI stress related and is therefore considered to have limited toxicological implications. In the micronucleus assay part of this study, SB44 was not found to induce micronuclei in rat bone marrow erythrocytes.

Example 16: pharmacokinetic analysis

Only the maximum plasma concentration (C) after oral administration is reported_max) And the area under the plasma concentration time curve (AUC) up to the last sampling time point_last) Since most samples have SB40 plasma concentrations at or near LLOQ, the determination of all TK parameters is problematic. At both days 1 and 14, for C in male and female rats_maxAnd AUC_lastThere is no non-linear dose relationship. At day 14, the highest systemic exposure was measured at the highest dose (500mg/kg) for both males and females, and for each dose administered, day 14C for SB40_maxAnd AUC_lastHigher than those on day 1. This accumulation of SB40 after 14 days of daily administration of SB44 can be caused by a significant distribution to the liver combined with a slow release back into the plasma. Females generally had greater systemic exposure than males, with the greatest difference being about twice as great at day 14 in the 500mg/kg dose group. The level of No Observed Effect (NOEL) in this study was determined to be 50 mg/kg/day based on the microscopic findings in the thymus and the absence of restored evidence of these effects from SB 44.

Example 17: dose range finding study of SB44 in cynomolgus monkeys

The objective of this study was to determine the Maximum Tolerated Dose (MTD) and the level of no adverse effect observed for the endpoint of the examination (NOAEL) after 4 consecutive days of daily oral gavage of SB44 on cynomolgus monkeys. The information from this study was used to design a subsequent dose range toxicity study and to determine the suitability of the proposed human dose.

SB44 at 500 mg/kg/dayThrough the mouthApplied to cynomolgus monkeys for 4 consecutive days. This dose was well tolerated clinically and was accompanied by about a 2-fold increase in ALT and about a 6-fold increase in AST, which returned to baseline about one week after treatment cessation. All hematological parameters appeared normal and there was no evidence of other apparent toxicity.

And (4) conclusion: as mentioned above, SB44 is a mixture of two isomers (Rp, Sp), where SB1B is the Rp isomer, present at greater than 55%. SB1B appears to be more effective as an adjuvant than SB44 in short-term primary and long-term Mtb challenge experiments in mice. In addition, little is known about the efficacy of the second isomer of SB44 (Sp-isomer) as an adjuvant. Many preclinical studies were performed using SB 44. The results of the study are illustrative of the excellent safety of the dinucleotide composition represented by SB 44. Thus, an excellent safety profile for subcutaneously administered SB1B is expected.

Example 18: induction of interferon in PBMC treated with BCG and compounds

For this assay, PBMCs were plated at 1 million cells/ml in 24-well plates. One set of plates was treated with BCG, BCG + compound and incubated for 24 hours. Cells were lysed and supernatants harvested. Cytokine and type I interferon production was assessed using ELISA assays with standard kits. According to the same experiment, BCG did induce other cytokines (IL-1, IL-6, IL-8, IL-10 and TNF). Cells treated with BCG and compound induced increased production of IFN compared to BCG alone. The results are provided in fig. 17.

According to the same experiment, BCG did induce other cytokines (IL-1, IL-6, IL-8, IL-10 and TNF). Cells treated with BCG and compound induced increased production of IFN compared to BCG alone.

In summary, the compounds claimed in the present invention act as activators of intracellular microbial sensors and cause activation of immune responses. When used in conjunction with a vaccine, the compound acts as an adjuvant and boosts the immune response induced by the vaccine.

Example 19 in vitro cytotoxicity Studies

In vitro cytotoxicity studies of one or more lead compounds using a panel of cell lines (internal) predictive of liver, kidney, bone marrow and mitochondrial toxicity.

Compounds 1 and 3 have an excellent safety profile with CC in many cell lines₅₀>1000 micromolar. Standard MTT assays were performed in 96-well plates using Promega Cell titer96 Non-radioactive Cell Proliferation Assay Kit in combination with 96-well plate readers (ThermoMax, Molecular devices), and MDBK, Vero, and HFF Cell lines (from ATCC). Several controls were used, including nucleoside analogs 3TC, AZT and ddC, and drug-free medium. SDS was used as a positive cytotoxicity control. Compounds were tested in triplicate at concentrations of 100, 300 and 1000 μ Μ. After 24 hours incubation of the cells with the test substance, the MTT assay was performed. All compounds tested showed CC50>1000 micromoles, indicating a high safety index for the compound.

Cited references

1.(a)WHO REPORT:Global prevalence of hepatitis A,B,C.WeeklyEpidemiological Record,Vol 77,6,2002；(b)Delwaide,J.,Gerard,C.Evidence-basedmedicine treatment of chronic hepatitis C.Liege study Group on Viralhepatitis.Revue medicale de Liege 55,337,2000.

2.(a)Sorrell,M.F.,Belongia,E.A.,Costa,J.,Gareen,I.F.,Grem,J.L.,Inadomi,J.M.,Kern,E.R.,McHugh,J.A.,Peterson,G.M.,Rein,M.F.,Strader,D.B.,Trotter,H.T.National Institutes of Health Consensus Development ConferenceStatement:Management of Hepatitis B.Ann.Int.Med.150,204,2009.

(b)Wild,C.P.,Hall,A.J.Primary prevention of hepatocellular carcinomain developing countries.Mutation Res.462,381,2000.Liang,T.J.,Rehermann,B.,Seeff,L.B.,Hoofnagle,J.H.Pathogenesis,natural history,treatment,andprevention of hepatitis C.Ann.Intern.Med.132,296,2000.

3.Akira,S.,Uematsu,S.,Takeuchi,O.Pathogen recognition and Innateimmunity.Cell,24,783-801,2006.

4.Katze,M.G.,He,Y.,Gale,M.Viruses and interferon:A fight forsupremacy.Nature Reviews,2,675,Sept 2002.

5.Takeda,K.,Akira,S.Toll-like receptors in innateimmunity.International Immunology,17,1-14,2005.

6.Saito,T.,Hirai,R.,Loo,Y-M.,Owen,D.,Johnson,C.L.,Sinha,S.C,Akira,S.,Fujita,T.,Gale,M.Regulation of innate antiviral defenses through a sharedrepressor domain in RIG-I and LGP2.Proc.Natl.Acad.Sci.USA,10,No.2,582-587,2007.

7.Meylan,E.,Curran,J.,Hofmann,K.,Moradpour,D.,Binder,M.,Bartenschlager,R.,Tschopp,J.Cardif is an acceptor protein in the RIG-Iantiviral pathway and is targeted by hepatitis C virus.Nature,437,1167-1172,2005.

8.Hiscott,J.,Lin,R.,Nakhaei,P.,Paz,S.MasterCARD:A priceless link toinnate immunity.TRENDS in Molecular Medicine,12,53-56,2006.

9.Cui,S.,Eisenacher,K.,Kirchhofer,A.,Brozka,K.,Lammens,A.,Lammens,K.,Fujita,T.,Conzelmann,K-K.,Krug,A.,Hopfner,K-P.The C-terminal regulatorydomain is the RNA 5'-triphosphate sensor of RIG-I.Molecular Cell,29,169-179,2008.

10.Yoneyama,M.,Kikuchi,M.,Natsukawa,T.,Shinobu,N.,Imaizumi,T.,Miyagishi,M.,Taira,K.,Akira,S.,Fujita,T.The RNA helicase RIG-I has anessential function in double-stranded RNA-induced innate antiviralresponses.Nat.Immunol.5,730-737,2004.

12.Hornung,V.,Ellegast,J.,Kim,S.,Brzozka,K.,Jung,A.,Kato,H.,Poeck,H.,Akira,S.,Conzelmann,K-K.,Schlee,M.,Endres,S.,and Hartmann,G.5'-triphosphateRNA is the ligand for RIG-I.Science,314,994-997,2006.

13.Pichlmair,A.,Schulz,O.,Tan,C.P.,Naslund,T.I.,Liljestrom,P.,Weber,F.,Reis e Sousa,C.RIG-I mediated antiviral responses to single-stranded RNAbearing 5'-phosphates.Science,314,997-1001,2006.

14.Myong,S.,Cui,S.,Cornish,P.V.,Kirchhofer,A.,Gack,M.U.,Jung,J.U.,Hopfner,K-P.,Ha,T.Cytosolic viral sensor RIG-I is a5'-triphosphate-dependenttranslocase on double-stranded RNA.Science,323,1070,2009.

15.Gack,M.U.,Kirchhofer A.,Shin,Y.C,Inn K.S.,Liang C,Cui S.,Myong S.,Ha T.,Hopfner,K-P.,Jung,J.U.Roles of RIG-I N-terminal tandem CARD and splicevariant in TRIM25-mediated antiviral signal transduction.Proc Natl Acad Sci US A.105(43):16743-8,2008.

15.(a)Sabbah,A.,Chang,T.H.,Harnack,R.,Frohlich,V.,Tominaga,K.,Dube,P.H.,Xiang,Y.,Bose,S.Activation of innate immune antiviral responses byNOD2.Nat.Immunol.10,1073,2009.(b)Franchi,L.,Warner,N.,Viani,K.,Nunez,G.Function of NOD-like receptors in microbial recognition and hostdefense.Immunol.Rev.227,106,2009.

16.(a)Iyer,R.P.,Pandey,R.,Kuchimanchi,S.RNA interference:An excitingnew approach for target validation,gene expression analysis andtherapeutics.Drugs of the Future,2003,28,51-59.(b)For reviews on antisenseapproach see:(i)Mirabelli,C.T.,Crooke,S.T.Antisense Oligonucleotides in thecontext of modern molecular drug discovery and development.In:AntisenseResearch and Applications,S.T.Crooke and B.Lebleu(Eds.,)CRC Press,New York1993,7-35.(ii)Szymkowski,D.E.Developing antisense oligonucleotides from thelaboratory to clinical trials.Drug.Disc.Today,1996,1,415-28

17.Iyer,R.P.,Jin,Y.,Roland A.,Morrey,J.D.,Mounir,S.,Korba,B.E.Phosphorothioate Di-and Tri-nucleotides as a novel class of anti-HBVagents.Antimicrob.Agents Chemother.48(6):2199-2205,2004.

18.Iyer,R.P.,Roland A.,Jin,Y.,Mounir,S.,Korba,B.E.,Julander,J.,Morrey,J.D.Anti-hepatitis B virus activity of ORI-9020,a novelphosphorothioate dinucleotide,in a transgenic mouse model.Antimicrob AgentsChemother.48(6):2318-20,2004.

19.For examples of nucleotide combinatorial synthesis and antiviralevaluation,see:(a)Jin,Y.,Roland,A.,Zhou,W.,Fauchon,M.,Lyaku,J.,Iyer,R.P.Bioorg.Med.Chem.Lett.10,1921-25,2000.(b)Jin,Y.,X.Chen,M.-E.Cote,A.Roland,B.Korba,S.Mounir,Iyer,R.P.Bioorg.Med.Chem.Lett.11,2057-2060,2001.

20.Iyer,R.P.,Coughlin,J.,Padmanabhan,S.Microwave-assistedfunctionalization of solid supports for rapid loading of nucleosides.CurrentProtocols Unit 3.13,(Beaucage et al Eds,)John Wiley and Sons,2006.

21.Padmanabhan,S.,Coughlin,J.,Iyer,R.P.Microwave-assistedfunctionalization of solid supports.Application in the rapid loading ofnucleosides on controlled-pore-glass(CPG).Tet.Lett.46,343-347,2005.

22.Iyer,R.P.,Coughlin,J.,Padmanabhan,S.Rapid functionalization andloading of solid supports.Organic Preparations&Procedure International,37,205-212,2005.

Claims

1. A method of treating a microbial infection by administering to a subject identified as in need thereof an effective amount of a prophylactic agent, wherein the prophylactic agent is a short oligonucleotide compound or an analog thereof, or a pharmaceutically acceptable salt, racemate, enantiomer, diastereomer, geometric isomer, or tautomer thereof.

2. A method of improving an immune system response against a disease, condition, infection, or virus in a subject, the method comprising administering to the subject an effective amount of a vaccine adjuvant, wherein the vaccine adjuvant is a short oligonucleotide compound or an analog thereof, or a pharmaceutically acceptable salt, racemate, enantiomer, diastereomer, geometric isomer, or tautomer thereof.

3. The method of claim 1 or 2, wherein the short oligonucleotide compound or analog thereof is a compound of formula (I):

wherein:

y and Z are each independently O or S;

m＝1-6；

4. The method of claim 1 or 2, wherein the short oligonucleotide compound or analog thereof is a compound of formula (II):

wherein

X ═ absence, O, NH, NR, or S;

X₁absent, O or NH;

a ═ absence, aryl, or aralkyl;

n is 0, 1,2, 3,4 or 5;

y and Z are each independently O or S;

m＝1-6；

5. The method of claim 1 or 2, wherein the short oligonucleotide compound or analog thereof is a compound of the structure:

6. The method of claim 1 or 2, wherein the short oligonucleotide compound or analog thereof is a compound of the structure:

7. The method of claim 1 or 2, wherein the method comprises administering a vaccine.

8. The method of claim 7, wherein the vaccine is a BCG vaccine.

9. The method of claim 8, wherein the prophylactic agent or vaccine adjuvant is a compound of formula (I), or a pharmaceutically acceptable salt, racemate, enantiomer, diastereomer, geometric isomer, or tautomer thereof.

10. The method of claim 8, wherein the prophylactic agent or vaccine adjuvant is a compound of formula (II), or a pharmaceutically acceptable salt, racemate, enantiomer, diastereomer, geometric isomer, or tautomer thereof.

11. The method of claim 8, wherein the prophylactic agent or vaccine adjuvant is a compound of the following structure:

12. The method of claim 8, wherein the prophylactic agent or vaccine adjuvant is a compound of the following structure:

13. The method of claim 2, wherein the vaccine adjuvant is administered with a vaccine for treating or preventing a microbial infection.

14. The method of claim 7, wherein the method is for improving an immune system response against a virus in the subject.

15. The method of claim 7, wherein the method is for improving an immune system response against cancer.

16. A method for preventing and treating a viral infection in a subject, said method comprising administering to said subject an effective amount of a compound, wherein said compound is a compound of formula (I) or (II), or a pharmaceutically acceptable salt, racemate, enantiomer, diastereomer, geometric isomer, or tautomer thereof.

17. The method of claim 16, wherein the method comprises administering a vaccine or one or more additional agents in combination or sequentially with the compound.

18. The method of claim 16, wherein said compound is

19. A method for preventing and treating a bacterial infection in a subject, said method comprising administering to said subject an effective amount of a compound, wherein said compound is a compound of formula (I) or (II), or a pharmaceutically acceptable salt, racemate, enantiomer, diastereomer, geometric isomer, or tautomer thereof.

20. The method of claim 19, wherein the method comprises administering a vaccine or one or more additional agents in combination or sequentially with the compound.

21. The method of claim 19, wherein said compound is

22. A method for preventing and treating a fungal infection in a subject, said method comprising administering to said subject an effective amount of a compound, wherein said compound is a compound of formula (I) or (II), or a pharmaceutically acceptable salt, racemate, enantiomer, diastereomer, geometric isomer, or tautomer thereof.

23. The method of claim 22, wherein the method comprises administering a vaccine or one or more additional agents in combination or sequentially with the compound.

24. The method of claim 22, wherein said compound is

25. A method for preventing and treating a parasitic infection in a subject, comprising administering to the subject an effective amount of a compound, wherein the compound is a compound of formula (I) or (II), or a pharmaceutically acceptable salt, racemate, enantiomer, diastereomer, geometric isomer, or tautomer thereof.

26. The method of claim 25, wherein the method comprises administering a vaccine or one or more additional agents in combination or sequentially with the compound.

27. The method of claim 25, wherein said compound is