COMPOUND WITH ANALGESIC EFFECT FOR USE IN PREVENTION AND TREATMENT OF PAIN

Description

FIELD OF THE INVENTION

[0001] The present invention relates to a compound for use in preventing and/or treating pain. Particularly, the invention pertains to adenosine derivatives and their applications in preventing and/or treating a pain.

BACKGROUND OF THE INVENTION

[0002] Pain is an uncomfortable feeling usually caused by intense stimuli or tissue/nerve damage. Pain can be classified into acute/chronic dichotomy by the affecting time. Acute pain can last just a moment or weeks depending on the circumstance. Acute pain usually disappears after recovery of the damaged tissue. Chronic pain is defined as pain which lasts more than 3-6 months, and is the normal healing time of an injury. There is a large population having chronic pain, and about one fifth of patients reported with chronic pain have predominantly neuropathic pain. Neuropathic pain is the pain arising from the nervous system, which is affected by a lesion or disease. Unlike nociceptive pain, which can be relieved by opioids or non-steroidal anti-inflammatory drugs (NSAIDs), neuropathic pain lacks effective treatments.

[0003] Fibromyalgia (FM) syndrome is characterized by chronic widespread pain and tenderness. The symptoms of fatigue, sleep disturbance and cognitive dysfunction may also occur. The major cause of FM remains unknown, but it is characterized by a decrease in the nociceptive pain threshold. Fibromyalgia and neuropathic pain have some diversities in the pathology of the peripheral and central nervous system.

[0004] Pregabalin (Lyrica^®) and gabapentin (Neurotin^®) are recommended for the treatment of neuropathic pain. Pregabalin and gabapentin have the structures analogous to γ-aminobutyric acid (GABA), but they do not interact with GABA receptors. These two drugs act by interacting with calcium channel α₂-δ subunit, resulting in the reduction of various neurotransmitters that are released from the synapse [Taylor, C.; Angelotti, T.; Fauman, E. Pharmacology and mechanism of action of pregabalin: the calcium channel alpha2-delta (α2-δ) subunit as a target for antiepileptic drug discovery. Epilepsy Res. 2006, 73, 137-150]. The labeled use of pregabalin (25 mg/day) and gabapentin (300 mg/day) also include anticonvulsants; however, side effects including sedation, dizziness and edema have been reported.

[0005] Milnacipran (Savella^®) and duloxetine (Cymbalta^®) are also used for the treatment of neuropathic pain, as well as for FM [Derry, S.; Gill, D.; Phillips, T.; Moore, R. Milnacipran for neuropathic pain and fibromyalgia in adults. Cochrane Database Syst. Rev. 2012, doi:10.1002/14651858.CD008244.pub2; Lunn, M.; Hughes, R. and Wiffen, P. Duloxetine for treating painful neuropathy, chronic pain or fibromyalgia. The Cochrane Library, 2014, doi:10.1002/14651858.CD007115.pub3]. Milnacipran and duloxetine are serotonin-norepinephrine reuptake inhibitors (SNRI), which prolong the duration of action of serotonin and norepinephrine in the synaptic cleft to enforce the signals induced by serotonin and norepinephrine. The target of serotonin is the 5-hydroxyltryptamine (5-HT) receptor, which contains more than 15 subtypes related to both pro-nociceptive and anti-nociceptive effects, depending on the subtype activated by serotonin. The anti-nociceptive effect of norepinephrine is mediated by α₂ adrenergic receptor.

[0006] Cymbalta^®, Lyrica^® and Savella^® are the only drugs approved for FM treatment by the FDA. However, two thirds of patients taking these drugs do not consider them as effective medication, according to an online survey conducted by the National Pain Foundation on more than 1300 FM patients [National Pain Foundation, Fibromyalgia drugs: successes or failures? National Pain Report 2014, April 23]. This disappointing result may reflect our insufficient understanding of FM.

[0007] It was previously identified a novel anti-nociceptive signaling mechanism of substance P by demonstrating that targeting of SP to ASIC3-positive muscle nociceptors inhibits chronic widespread pain [Lin, C.-C.J.; Chen, W.-N.; Chen, C.-J.; Lin, Y.-W.; Zimmer, A.; Chen, C.-C. An antinociceptive role for substance P in acid-induced chronic muscle pain. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, E76; Sun, W. H.; Chen, C. C. Roles of proton-sensing receptors in the transition from acute to chronic pain. J. Dent. Res. 2016, 95, 135-142]. Substance P (SP) was first isolated from rabbit's intestine, and its amino-acid sequence was later determined as Arg-Pro-Lys-Pro-Gln-Glu-Phe-Phe-Gly-Leu-Met-CONH2. SP belongs to the class of tachykinin neuropeptides, which have a similar C-terminus sequence of Phe-X-Gly-Leu-Met-NH2 (X is either an aromatic or an aliphatic amino acid) and a rather diverse N-terminus. The target of SP is the neurokinin 1 receptor (NK1R), a G protein-coupling receptor (GPCR) expressed in the central and peripheral nervous systems. SP is thought to be a major component of pain transmission, and its action is concentration dependent. Although the NK1R (a member of GPCR) knockout mice showed the hypoalgesic effect compared to wild type mice, the use of NK1R antagonist failed to generate similar results in the clinical trial. The failure in clinical trial is likely due to the species difference of NK1R distribution in supraspinal sites for pain perception [Hill, R. NK1 (substance P) receptor antagonists - why are they not analgesic in humans? Trends Pharmacol. Sci. 2000, 21, 244-246].

[0008] Based on this mechanism of action, it was previously shown that N⁶-(4-hydroxybenzyl)adenosine, designated as T1-11 (compound 2), is an anti-nociceptive agent in ASIC3-mediated pain model [Chen, C.-C.; Lin, Y.-L.; Fang, J.-M.; Chern, Y.; Lin, C.-C. J.; Chen, W.-N. Methods and compositions for treating pain.]. T1-11 mediates an outward current in SP-sensitive ASIC3-expressing muscle nociceptors and thus can inhibit acid-induced ASIC3 activation [Deval, E.; Noël, J.; Gasull, X.; Delaunay1, A.; Alloui, A.; Friend, V.; Eschalier, A.; Lazdunski, M., Lingueglia, E. Acid-sensing ion channels in postoperative pain. J. Neurosci. 2011, 31, 6059-6066]. Intramuscular SP mediates an unconventional NK1 receptor signaling pathway to inhibit acid activation in muscle nociceptors, which results in an unexpected anti-nociceptive effect against chronic mechanical hyperalgesia induced by repeated intramuscular acid injection [Lin, C.-C.J.; Chen, W.-N.; Chen, C.-J.; Lin, Y.-W.; Zimmer, A.; Chen, C.-C. An antinociceptive role for substance P in acid-induced chronic muscle pain. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, E76-E83]. WO 2013/120078 relates to a method and pharmaceutical composition for treating pain comprising an adenosine analog that activates NKI receptor signaling. WO 2015/061426 relates to compounds for use N6 -modified adenosine analogs for use in prevention and treatment of neurodegenerative disease and pain. EP 1 352 910 relates to derivatives of nitrobenzylthioinosine and their use for the treatment of pain. WO 95/18817 relates to adenosine derivatives, preparation methods therefore and pharmaceutical compositions comprising same for the use as pain killers, antihypertensive and antiproliferative agents. WO 2005/084653 relates to adenosine derivatives for the treatment of pain or inflammation. EP 2 511 283 relates to N6 -substituted adenosine and adenine derivatives and their use for treating insomnia, convulsion, epilepsy, and Parkinson's disease, and preventing and treating dementia.

SUMMARY OF THE INVENTION

[0009] In this invention, a conjugate compound Ic-2 derived from T1-11 and substance P is synthesized. Compound Ic-2 showed analgesic effect starting from 14 µg/kg (160 pico mole) in a mouse model of fibromyalgia, in which chronic widespread pain was induced by intermittent cold stress (ICS). Furthermore, a series of compounds were prepared by conjugation with amino acids (e.g., I-a1) or peptides (e.g., I-c1) to test their analgesic activities.

[0010] In one aspect, the invention relates to a compound of formula (I):

wherein,

n is 0, 1, 2 or 3;

X and Y are each independently NH, O or S;

R¹ is OH, NH2, NO₂, halogen, alkyl, haloalkyl, hydroxyalkyl, unsubstituted or substituted alkoxy, alkoxyalkyl, alkenyl or alkynyl;

R² and R³ are each independently OH, NH2, NO₂, haloalkyl, hydroxyalkyl or alkylamino; and

R⁴ is a moiety derived from glycine, alanine, cysteine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, glutamine, asparagine, threonine, arginine, lysine, proline or from a peptide RPKPQQFFGLM-CONH₂ and the peptide is linked with C(=O) in the structure via the side chain amino group of K, and R⁴ is linked with C(=O) in the structure via the N-terminal group or a side chain amino group thereof, wherein the C-terminal of the amino acid or the peptide is optionally modified;

a tautomer or stereoisomer thereof; or a pharmaceutically acceptable salt of the foregoing.

[0011] In another aspect, the invention relates to a compound of formula (II):

wherein,

k is 1, 2 or 3;

X and Y are each independently NH, O or S;

R¹ is OH, NH2, NO₂, halogen, alkyl, haloalkyl, hydroxyalkyl, unsubstituted or substituted alkoxy, alkoxyalkyl, alkenyl or alkynyl;

R² and R³ are each independently OH, NH2, NO₂, haloalkyl, hydroxyalkyl or alkylamino; and R⁴ is selected from glycine, alanine, cysteine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, glutamine, asparagine, threonine, arginine, lysine, proline, and R⁴ is linked with C(=O) in the structure via the N-terminal group or a side chain amino group thereof, or from a peptide RPKPQQFFGLM-CONH₂ and the peptide is linked with C(=O) in the structure via the side chain amino group of K, wherein the C-terminal of the amino acid or the peptide is optionally modified;

ring A is a substituted or unsubstituted aromatic heterocycle, comprising 5- or 6-membered heterocycle and fused heterocycle; the heteroatom(s) in the ring are one or more nitrogen, oxygen, and/or sulfur heteroatoms; and the substituent(s) are selected from the group consisting of fluorine, chlorine, bromine, iodine, hydroxyl, nitro, alkyl, trifluoromethyl group and combinations thereof;

a tautomer or stereoisomer thereof; and a pharmaceutically acceptable salt of the foregoing.

[0012] In another aspect, the invention relates to a composition comprising:

(a) a therapeutically effective amount of the compound as aforementioned or a pharmaceutically acceptable salt thereof; and

(b) a pharmaceutically acceptable carrier, excipient or vehicle.

[0013] Yet in another aspect, the invention relates the compound as aforementioned for use in treating pain in a subject in need thereof.

[0014] In one embodiment of the invention, the pain may be acid-induced pain. The acid-induced pain may be acid-induced muscle pain. The acid-induced muscle pain may be acid-induced chronic muscle pain.

[0015] In one embodiment of the invention, the pain is selected from the group consisting of inflammatory pain, cancer-associated pain, chest pain, back pain, neck pain, shoulder pain, migraine, headache, myofascial pain, joint pain, muscular pain syndromes, neuropathic pain, peripheral pain, sympathetic pain, postoperative pain, post-traumatic pain, and multiple sclerosis pain.

[0016] In another embodiment of the invention, the pain may be a dysfunctional pain. The dysfunctional pain may be selected from the group consisting of fibromyalgia, myofascial pain, bladder pain syndrome, a pain caused by irritable bowel syndrome, and pain associated with temporomandibular disorders.

[0017] In this invention, compounds of formulae (I) and (II) may be used to treat postoperative pain, myofascial pain syndrome, and other chronic widespread pain, which are pain with muscular origin and associated with ASIC3 and substance P.

[0018] Without wishing to be limited by theory, it is believed that the analgesic effect of compound of formulae (I) and (II) is due to the fact that compound of formula (I) and (II) can induce a slow inactivating outward current in muscle nociceptors so that the neurons become hyperpolarized and increased the firing threshold.

[0019] The accompanying drawings illustrate one or more embodiments of the invention and, together with the written description, serve to explain the principles of the invention. Wherever possible, the same reference numbers are used throughout the drawings to refer to the same or like elements of an embodiment.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] 

Figure 1 shows some representative compounds of formula (I). Among them, Compound I-a1 (JMF3737) is a conjugate of T1-11 with glycine. Compound I-c2 is a conjugate of T1-11 with substance P (an undecapeptide RPKPQQFFGLM-CONH2).

Figure 2 shows the synthetic method for compound I-a1 via PMB-protecting intermediate compound 4a.

Figure 3 shows the synthetic method for compound I-a1 via benzoate-protecting intermediate compound 4b.

Figure 4 shows the synthesis of compound II-a1.

Figure 5 shows the synthesis of compound II-b1.

Figure 6 shows the synthesis of compound I-d1.

Figure 7 shows the synthesis of compound I-e1.

Figure 8 shows the synthesis of compounds I-f1and I-f2

Figure 9 illustrates the analgesic effects in a mouse model of fibromyalgia, in which chronic widespread pain is induced by intermittent cold stress (ICS). T1-11 shows dose-dependent analgesic effect with effective dose starting from 0.03 mg/kg via intraperitoneal injection (i.p.) and 8 mg/kg via oral gavage (p.o.). In comparison, the conjugate compound I-a1 (JMF3737) derived from T1-11 and glycine shows superior dose-dependent analgesic effect with effective dose starting from 1 mg/kg (i.p.) and 1 mg/kg (p.o.). A combination treatment by oral administration of T1-11 and glycine does not exhibit synergistic effect.

Figure 10 shows the analgesic mechanism of action of compound I-a1 (JMF3737); the concentration of RP67580 and JMF3737 is each 10 µM.

Figure 11 shows no tolerance but therapeutic effect on repeated treatment of compound I-a1 (JMF3737).

Figure 12 illustrates the analgesic effects of JMF4413 and JMF3795 in a mouse model of fibromyalgia, in which chronic widespread pain was induced by intermittent cold stress (ICS).

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the case of conflict, the present document, including definitions will control. As used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural references unless the content clearly dictates otherwise.

[0022] Throughout this specification, unless the context requires otherwise, the word "comprise," or variations such as "comprises" or "comprising," will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

[0023] As used herein, the term "halo" means -F, -Cl, -Br or -I.

[0024] As used herein, the term "substituted" means that one or more hydrogens on the designated atom is replaced with a selection from the indicated group, provided that the designated atom's normal valency under the existing circumstances is not exceeded, and that the substitution results in a stable compound.

[0025] As used herein, the term "alkyl," employed alone or in combination with other terms, refers to a saturated hydrocarbon group that may be straight-chain or branched. In some embodiments, the alkyl group contains 1 to 6, 1 to 4, or 1 to 3 carbon atoms. Examples of alkyl moieties include, but are not limited to, chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methyl-1-butyl, 3-pentyl, n-hexyl, 1,2,2-trimethylpropyl, and the like. In some embodiments, the alkyl group is methyl, ethyl, or propyl.

[0026] As used herein, the term "alkenyl," employed alone or in combination with other terms, refers to a group derived from an alkyl group where at least one carbon-carbon single bond is replaced with a carbon-carbon double bond. In some embodiments, the alkenyl moiety contains 2 to 6 or 2 to 4 carbon atoms. Example alkenyl groups include, but are not limited to, ethenyl, n-propenyl, isopropenyl, n-butenyl, sec-butenyl, pentenyls, hexenyls, and the like.

[0027] As used herein, the term "alkynyl," employed alone or in combination with other terms, refers to a group derived from an alkyl group where at least one carbon-carbon single bond is replaced with a carbon-carbon triple bond. In some embodiments, the alkynyl moiety contains 2 to 6 or 2 to 4 carbon atoms. Example alkynyl groups include, but are not limited to, ethynyl, propyn-1-yl, propyn-2-yl, butynyls, pentynyls, hexynyls, and the like.

[0028] As used herein, the term "haloalkyl" means a substituted alkyl having one or more halo substituents. For example, "haloalkyl" refers to an alkyl having at least one halogen as the substituent(s) and each substituent may be the same or different; such as mono, di, and trifluoromethyl.

[0029] As used herein, the term "alkylamino" refers to the group --NRR' where R is alkyl and R' is hydrogen or alkyl.

[0030] As used herein, the term "alkoxyalkyl" refers to an alkoxy group, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein. Representative examples of alkoxyalkyl include, but are not limited to, methoxymethyl, methoxyethyl, and methoxypropyl.

[0031] As used herein, the term "peptide" refers to short chains of amino acid monomers linked by peptide bonds, preferably 50 or less amino acid monomers, more preferably 2 to 30 amino acids, more preferably 2 to 20 amino acids, such as 9, 10, 11 or 12 amino acids. Peptides include oligopeptides and polypeptides; the former normally have 2 to 20 amino acids while the latter normally have 50 or less amino acids.

[0032] As used herein, the term "a derivative of an amino acid or a peptide" includes a salt, an ester or both of an amino acid or a peptide. The salt may derived from the C-terminal, N-terminal and/or side chain group(s) and the ester may derived from the C-terminal and/or side chain group(s).

[0033] As used herein, the description "C-terminal modification of an amino acid or a peptide" or the like refers to modifications of the terminal -COOH or replacements of the terminal -COOH with an analog of bioisostere. Examples of modifications include, but are not limited to, esterification, such as methylation, etc. Examples of analogs include, but are not limited to, an amide moiety, a phosphonate moiety, a sulfonate moiety, a hydroxamate moiety, an acyl hydrazide, a phosphonamide, a sulfonamide, etc.

[0034] As used herein, "pharmaceutically acceptable salt" refers to derivatives of the disclosed compounds wherein the parent compound is modified by making acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines, pyridine, pyrimidine and quinazoline; alkali or organic salts of acidic residues such as carboxylic acids; and the like.

[0035] As used herein, the term "stereoisomer" is a general term for all isomers of individual molecules that differ only in the orientation of their atoms in space. It includes enantiomers and isomers of compounds with more than one chiral center that are not mirror images of one another (diastereoisomers). The terms "enantiomer" and "enantiomeric" refer to a molecule that cannot be superimposed on its mirror image and hence is optically active wherein the enantiomer rotates the plane of polarized light in one direction and its mirror image compound rotates the plane of polarized light in the opposite direction.

[0036] As used herein, the term "tautomer" refers to constitutional isomers of organic compounds that readily interconvert, in particular involving relocation of a proton. Examples of tautomers include, but are not limited to, amino acid and ammonium carboxylate, ketone and enol, etc.

[0037] As used herein, the term "treating" or "treatment" refers to administration of an effective amount of a therapeutic agent to a subject in need thereof, who has a neurodegenerative disease and/or pain, or a symptom or predisposition toward such a disease and/or pain, with the purpose of cure, alleviate, relieve, remedy, ameliorate, or prevent the disease and/or pain, the symptoms of it, or the predisposition towards it. Such a subject can be identified by a health care professional based on results from any suitable diagnostic method.

[0038] As used herein, the term "effective amount" refers to the amount of an active compound that is required to confer a therapeutic effect on the treated subject. Effective doses will vary, as recognized by those skilled in the art, depending on route of administration, excipient usage, and the possibility of co-usage with other therapeutic treatment.

[0039] As used herein, the term "Guidance for Industry and Reviewers Estimating the Safe Starting Dose in Clinical Trials for Therapeutics in Adult Healthy Volunteers" published by the U.S. Department of Health and Human Services Food and Drug Administration discloses a "therapeutically effective amount" may be obtained by calculations from the following formula:

[0040] HED = animal dose in mg/kg × (animal weight in kg/human weight in kg).

Compounds of the Invention

[0041] The invention provides a compound of formula (I):

wherein,

n is 0, 1, 2 or 3;

X and Y are each independently NH, O or S;

R¹ is OH, NH2, NO₂, halogen, alkyl, haloalkyl, hydroxyalkyl, unsubstituted or substituted alkoxy, alkoxyalkyl, alkenyl or alkynyl;

R² and R³ are each independently OH, NH2, NO₂, haloalkyl, hydroxyalkyl or alkylamino; and

R⁴ is selected from glycine, alanine, cysteine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, glutamine, asparagine, threonine, arginine, lysine, proline, and R⁴ is linked with C(=O) in the structure via the N-terminal group or a side chain amino group thereof, or from a peptide RPKPQQFFGLM-CONH2 and the peptide is linked with C(=O) in the structure via the side chain amino group of K, wherein the C-terminal of the amino acid or the peptide is optionally modified;

a tautomer or stereoisomer thereof; and a pharmaceutically acceptable salt of the foregoing.

[0042] In some embodiment, R¹, R², and R³ are each independently OH.

[0043] In one embodiment, n is 1.

[0044] In some embodiments, X and Y are each independently NH, O or S.

[0045] In a further embodiment, X is NH and Y is O.

[0046] In a further embodiment, X is NH and Y is NH.

[0047] In a further embodiment, X is NH and Y is S.

[0048] The compound of claim 1, wherein X is S and Y is NH.

[0049] The invention provides a compound of formula (II)

wherein,

k is 1, 2 or 3;

X and Y are each independently NH, O or S;

R¹ is OH, NH2, NO₂, halogen, alkyl, haloalkyl, hydroxyalkyl, unsubstituted or substituted alkoxy, alkoxyalkyl, alkenyl or alkynyl;

R² and R³ are each independently OH, NH2, NO₂, haloalkyl, hydroxyalkyl or alkylamino; and R⁴ is selected from glycine, alanine, cysteine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, glutamine, asparagine, threonine, arginine, lysine, proline, and R⁴ is linked with C(=O) in the structure via the N-terminal group or a side chain amino group thereof, or from a peptide RPKPQQFFGLM-CONH₂ and the peptide is linked with C(=O) in the structure via the side chain amino group of K, wherein the C-terminal of the amino acid or the peptide is optionally modified;

ring A is a substituted or unsubstituted aromatic heterocycle, comprising 5- or 6-membered heterocycle and fused heterocycle; the heteroatom(s) in the ring are one or more nitrogen, oxygen, and/or sulfur heteroatoms; and the substituent(s) are selected from the group consisting of fluorine, chlorine, bromine, iodine, hydroxyl, nitro, alkyl, trifluoromethyl group and combinations thereof;

a tautomer or stereoisomer thereof; and a pharmaceutically acceptable salt of the foregoing.

Chemical synthesis

[0050] In another aspect, the invention relates to a method for preparing the compound of formula (I) as aforementioned, comprising the following steps:

(a) reacting N⁶-(4-hydroxybenzyl)adenosine (2) with 2,2-dimethoxypropane in acetone in the presence of an acid to afford the (2',3'-O-isopropylidene)adenosine compound of formula (3);

(b) reacting (2',3'-O-isopropylidene)adenosine compound (3) in the presence of a base with a hydroxyl group-protecting agent to afford a (2',3'-O-isopropylidene)adenosine compound of formula (4) bearing a phenol protecting group (PG);

(c) reacting the (2',3'-O-isopropylidene)adenosine derivative (4) with 1,1'-carbonyldiimidazole (CDI) in the presence of a base to afford a of (2',3'-O-isopropylidene)adenosine compound of formula (5) having the hydroxyl activating group;

(d) performing a coupling reaction by reacting the (2',3'-O-isopropylidene)adenosine compound (5) with a derivative of amino acid or peptide (such as an α-amino ester) to afford a (2',3'-O-isopropylidene)adenosine compound of formula (6);

wherein [A']-NH- is derived from the derivative of amino acid or peptide; and

(e) removing all the protecting groups from compound (6) to afford the compound of formula (I);

wherein [A]-NH- is the amino acid or peptide in the derivative of amino acid or peptide.

[0051] In one embodiment of the invention, the acid in step (a) may be p-toluenesulfonic acid or camphorsulfonic acid.

[0052] In another embodiment of the invention, the hydroxyl protecting agent (PG-Cl) in step (b) may be 4-methoxybenzyl chloride or benzoyl chloride.

[0053] In another embodiment of the invention, the derivative of an amino acid or peptide in step (d) may be glycine methyl ester, glycine ethyl ester, L-phenylalanine ethyl ester, L-valine ethyl ester, γ-aminobutyric acid methyl ester, or undecapeptide Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-CONH₂ (substance P).

[0054] In another embodiment of the invention, the deprotecting method in step (e) may comprise using a base to cleave the benzoyl group and using an acid to remove the p-methoxybenzyl group; wherein the base may be lithium hydroxide, sodium hydroxide, potassium hydroxide, cesium carbonate or ammonium hydroxide, and the acid may be trifluoroacetic acid (TFA) or hydrochloric acid.

[0055] In another embodiment of the invention, the hydrolysis in step (f) may comprise using a base to remove the alkyl group; wherein the base may be lithium hydroxide, sodium hydroxide, potassium hydroxide, cesium carbonate or ammonium hydroxide.

[0056] Further in another embodiment of the invention, the salt of formula (IA) prepared in step (f), wherein Z⁺ is Li⁺, Na⁺, K⁺, Rb⁺, Cs⁺ or NH₄⁺, is converted to carboxylic acid using a mineral acid, wherein the mineral acid may be hydrochloric acid. R is a substituent existing in common α-amino acids.

[0057] Further in another embodiment of the invention, the carboxylic acid of formula (IA, Z⁺ = H⁺) is treated with an amine R^AR^BR^CN to form an aminium salt of formula (IA), wherein the amine may be trimethylamine, triethylamine, tri-n-butylamine, benzyldimethylamine, pyrrolidine, piperidine, morpholine, or N-methylmorpholine.

[0058] In one approach, compound I-a1 is synthesized via a PMB-protecting intermediate compound 4a as shown in Figure 2:

(a) According to the previously reported procedure [ Chen, J.-B.; Liu, E. M.; Chern, T.-R.; Yang, C.-W.; Lin, C.-I.; Huang, N.-K.; Lin, Y.-L.; Chern, Y.; Lin, J.-H. Fang, J.-M. Design and synthesis of novel dual-action compounds targeting the adenosine A2A receptor and adenosine transporter for neuroprotection. ChemMedChem 2011, 6, 1390-1400], T1-11 is obtained from the substitution reaction of 6-chloropurine riboside with 4-hydroxybenzylamine, and treated with 2,2-dimethoxypropane in acetone by catalysis of p-toluenesulfonic acid to afford the acetonide compound 3:

(b) The phenol group of 3 is protected as a p-methoxybenzyl (PMB) ether by alkylation with 4-methoxybenzyl chloride in the presence of a base K₂CO₃, giving compound 4a:

(c) The hydroxyl group of 4a is activated by treatment with CDI, giving 5a, which further reacts with glycine ethyl ester to produce the carbamate compound 6a-1:

(d) The acetonide and PMB ether protecting groups of 6a-1 are removed by treatment with trifluoroacetic acid (TFA) to give compound 7a-1, which is hydrolyzed under alkaline conditions to afford compound I-a1 after purification by chromatography:

[0059] In another approach, compound I-a1 is synthesized via a benzoate-protecting intermediate compound 4b as shown in Figure 3.

(a) The phenol group of 3 is protected as a benzoate ester by acylation with benzoyl chloride in the presence of a base Et3N, giving compound 4b:

(b) The hydroxyl group of 4b is activated by treatment with CDI, and then reacted with glycine methyl ester to afford the carbamate compound 6b-1:

(c) Deprotecting the acetonide and ester groups of 6b-1 produces compound I-a1 after purification by chromatography.

[0060] In another embodiment, compound 4a is activated as the imidazolide 5a, which reacts with L-phenylalanine ethyl ester to give compound 6a-2:

[0061] In another embodiment, compound 4b is activated by CDI and then treated with L-valine methyl ester to give compound 6b-2:

[0062] In another embodiment, compound 6a-2 is treated with TFA, followed by NaOH, to remove the protecting groups to produce compound I-a2:

[0063] In another embodiment, compound 6b-2 is treated with TFA, followed by NaOH, to remove the protecting groups to produce compound I-a3:

[0064] In one aspect, the analogous compound 8 is synthesized. Compound 4b is activated by CDI and treated with n-butylamine. The product is treated with TFA, followed by NaOH, to remove the acetonide and benzoate protecting groups to produce compound 8:

[0065] In another embodiment, the analogous compound Ib-1 containing a terminal carboxylic acid is synthesized via an intermediate compound 9. The imidazolide compound 5a is reacted with γ-aminobutyric acid methyl ester to give compound 9, which is treated with TFA, followed by NaOH, to remove the acetonide, PMB and ester protecting groups to produce compound I-b1:

[0066] In another embodiment, compound I-c1 is synthesized via an intermediate compound 10. Compound 6a-1 is hydrolyzed to give the corresponding carboxylic acid, which is subjected to a coupling reaction with L-leucyl-L-methionine amide to form compound 10. Removing the acetonide and PMB protecting groups in 10 by treatment with TFA gives compound I-c1:

[0067] In another embodiment, compound I-c2 is synthesized by the coupling reaction of imidazolide compound 5a and SP, an undecapeptide Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-CONH₂, followed by treatment with TFA to remove the acetonide and PMB protecting groups:

[0068] In another embodiment, compound of formula II-a1 (14) is an analogous compound of I-a1 by replacement of the N₆-(4-hyroxybenzyl) group with an N₆-(indol-3-yl)ethyl group. Compound 14 (II-a1) is synthesized from N⁶-(indol-3-yl)ethyladenosine (compound 11) via the intermediate compounds 12 and 13 as shown in Figure 4.

(a) reacting 6-chloropurine riboside (1) with (indol-3-yl)ethylamine in ethanol in the presence of diisopropylethylamine to give N⁶-(indol-3-yl)ethyl-adenosine (11);

(b) reacting compound 11 with 2,2-dimethoxypropane in acetone in the presence of p-toluenesulfonic acid to afford the acetonide compound of formula (12);

(c) activating the hydroxyl group in compound 12 with CDI, followed by reacting with glycine methyl ester to afford the carbamate compound of formula (13); and

(d) removing the acetonide protecting group with TFA, followed by hydrolysis in alkaline condition, to afford compound 14 (II-a1).

[0069] In another embodiment, compound of formula II-b1 is an analogous compound of I-a1 by replacement of the N₆-(4-hyroxybenzyl) group with an N₆-(5-bromothien-2-yl)methyl group. Compound II-b1 is synthesized from N⁶-(5-bromothien-2-yl)methyladenosine (compound 15) via the intermediate compounds 16 and 17 as shown in Figure 5.

[0070] In another embodiment, compound I-d1 is synthesized via intermediate compounds 18, 19 and 20 as shown in Figure 6:

(a) reacting compound 4a with diphenyl phosphoryl azide (DPPA) and sodium azide in the presence of a base to afford an azido compound of formula 18, wherein the base may be triethylamine, 4-dimethylaminopyridine (DMAP), 1,4-diazabicyclo[2.2.2]octane (DABCO), 1,5-diazabicyclo[4.3.0]non-5-ene (DBN) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU);

(b) reducing the azido compound 18 by hydrogenolysis in the presence of a catalyst, followed by a coupling reaction with a glycine methyl ester hydrochloride in the present of a promoter, to afford a compound of formula 19, wherein the catalyst is Lindlar catalyst and the promoter is 1,1'-carbonyldiimidazole;

(c) removing the PMB protecting groups by hydrogenolysis to afford a compound of formula 20.

(d) removing the protecting groups of 2',3'-diol and ester by TFA and NaOH, respectively, to afford a compound of formula I-d1.

[0071] In another embodiment, compound I-e1 is synthesized via intermediate compounds 21 and 22 as shown in Figure 7:

(a) reacting compound 4a with methanesulfonyl chloride (MsCl) in the presence of a base, followed by reacting with potassium thioacetate, to afford a compound of formula 21, wherein the base may be triethylamine and 4-dimethylaminopyridine;

(b) removing the acetyl group from compound 21 with a base, followed by coupling with 2,2,2-trichloroethylglycine in the present of a promoter, to afford a compound of formula 22, wherein the base may be sodium hydroxide and potassium hydroxide, and the promoter is 1,1'-carbonyldiimidazole;

(c) removing all the protecting groups to afford a compound of formula I-e1.

[0072] In another embodiment, compound I-f1 is synthesized via intermediate compounds 23, 24a and 25a as shown in Figure 8.

Pharmaceutical Compositions

[0073] In another aspect, the invention provides a pharmaceutical composition comprising a compound of the invention and a pharmaceutically acceptable carrier.

[0074] The compound may be formulated into pharmaceutical compositions that may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Pharmaceutically acceptable carriers and diluents are familiar to those skilled in the art. For compositions formulated as liquid solutions, acceptable carriers and/or diluents include saline and sterile water, and may optionally include antioxidants, buffers, bacteriostats and other common additives. The compositions can also be formulated as pills, capsules, granules, or tablets which contain, in addition to a compound of the invention, diluents, dispersing and surface active agents, binders, and lubricants.

[0075] In a preferred embodiment, the compound is present in the composition in an amount that is effective to achieve its intended therapeutic purpose. While individual needs may vary, a determination of optimal ranges of effective amounts of each compound is within the skill of the art.

[0076] The compounds of the present invention may be useful in combination with one or more second therapeutic agents, particularly therapeutic agents suitable for the treatment and/or prevention of the conditions and diseases presented herein.

Analgesic effects

[0077] In another aspect, the invention provides a compound of the invention for use in a method for treating and/or pain, comprising administering an effective amount of the compound of the invention to a subject. In one embodiment, the pain is chronic pain. In some embodiments, the pain is associated with tissue acidosis and/or nerve injury includes, but is not limited to, inflammatory pain, cancer-associated pain, chest pain, back pain, neck pain, shoulder pain, migraine, headache, myofascial pain, joint pain, muscular pain syndromes, neuropathic pain, peripheral pain, sympathetic pain, postoperative pain, post-traumatic pain, and multiple sclerosis pain. In one embodiment, the pain is dysfunctional pain. In further embodiments, the dysfunctional pain includes, but is not limited to, fibromyalgia, bladder pain syndrome, pain caused by irritable bowel syndrome, and pain associated with temporomandibular disorders.

[0078] In one embodiment, compound 2 (T1-11) has good solubility in dimethylsulfoxide (DMSO) and excellent analgesic effect in a mouse model of fibromyalgia, in which chronic widespread pain is induced by intermittent cold stress (ICS). In this ICS mouse model, T1-11 showed dose-dependent analgesic effect with effective dose starting from 0.03 mg/kg via i.p. route (Figure 9(a)) and 8 mg/kg via p.o. route(Figure 9(b)). However, T1-11 has a low bioavailability (F ≈ 5%) that may limit its use for treating pain.

[0079] In another embodiment, conjugate compound I-a1 (JMF3737) derived from T1-11 and glycine showed superior analgesic effect to that of T1-11. In the same ICS mouse model, compound I-a1 showed dose-dependent analgesic effect with effective dose starting from 1 mg/kg (i.p.) (Figure 9(c)) and 1 mg/kg (p.o.) (Figure 9(d)). The doses of compound I-a1 can be increased to at least 64 mg/kg (i.p. and p.o.) without causing appreciable detrimental effect.

[0080] In another embodiment, a combined treatment by oral administration of T1-11 (4 µmol/kg) and glycine (4 µmol/kg) showed no synergistic effect (Figure 9(e)), in comparison with the appreciable analgesic effect of the conjugate compound I-a1 at 2 mg (4 µmol)/kg via oral route.

[0081] In another embodiment, the analgesic mechanism of action of compound I-a1 (JMF3737) is the same as T1-11 by acting on the NK1R receptor in muscle nociceptors to induce an outward current that can be inhibited by NK1R antagonist (Figure 10).

[0082] In another embodiment, daily treatment of compound I-a1 (JMF3737) in low dose (1 mg/kg, p.o.) for 4 days showed no tolerance but therapeutic effect for fibromyalgia pain (Figures 11(a) and 11(b)).

[0083] Further in another embodiment, compound I-d1 (JMF4313) showed excellent analgesic effect in the same ICS model (Figure 12(a)). The dose of compound I-d1 can be increased to 64 mg/kg (i.p.) without causing appreciable detrimental effect.

[0084] Further in another embodiment, a conjugate compound Ic-2 derived from T1-11 and substance P showed analgesic effect starting from 14 µg/kg (160 pmol, i.p.) in the ICS mouse model (Figure 12(b)).

EXAMPLES

[0085] All reagents and solvents were of reagents grade and were used without further purification unless otherwise specified. Tetrahydrofuran and diethyl ether were distilled from Na/benzophenone and CH₂Cl₂ was distilled from CaH2. All air or moisture sensitive experiments were performed under argon. All glasses were dried in an oven for more than 2 hours and used after cooling to room temperature in desiccators.

[0086] Melting points were recorded on a Yanaco micro apparatus. Optical rotations were measured on digital polarimeter of Japan JASCO Co. DIP-1000. [α]_D values are given in units of 10^-1deg cm² g^-1. Infrared (IR) spectra were recorded on Nicolet Magna 550-II. NMR spectra were obtained on Varian Unity Plus-400 (400 MHz) and chemical shifts (δ) were recorded in parts per million (ppm) relative to δ_H 7.24/ δ_C 77.0 (central line of triplet) for CHCl₃/CDCl₃, δ_H 2.05/ δ_C 29.92 for (CH₃)₂CO/(CD₃)₂CO, δ_H 3.31/ δ_C 49.0 for CH₃OH/CD₃OD, and δ_H 2.49 (m) /δ_C 39.5 (m) for (CH₃)₂SO/(CD₃)₂SO. The splitting patterns are reported as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet) and br (broad). Coupling constants (J) are given in Hz. The ESI-MS experiments were conducted on a Bruker Daltonics BioTOF III high-resolution mass spectrometer. Analytical thin-layer chromatography (TLC) was performed on E. Merck silica gel 60 F₂₅₄ plates (0.25 mm). Compounds were visualized by UV, anisaldehyde or ninhydrin spray. Column chromatography was carried out on columns packed with 70-230 mesh silica gel.

N⁶-(4-Hydroxybenzyl)-2',3'-O-isopropylidene-adenosine (3)

[0087] 

[0088] N⁶-(4-Hydroxybenzyl)adenosine (compound 2, T1-11) was prepared from the substitution reaction of 6-chloropurine riboside with 4-hydroxybenzylamine according to the previously reported procedure [Chen et al., 2011.]. In brief, a mixture of 4-hydroxybenzylamine (as the hydrochloric salt, 2.5 equiv), 6-chloropurine riboside (1 equiv), and diisopropylethylamine (DIEA, 24 equiv) in 1-propanol was heated to 70 °C for 6 h. The mixture was concentrated under reduced pressure, and triturated with water to give white precipitates, which were filtered to yield compound 2 (81% yield).

[0089] A mixture of compound 2 (374 mg, 1 mmol), p-toluenesulfonic acid monohydrate (280 mg, 1.5 mmol) and 2,2-dimethoxypropane (3 mL, 2.5 mmol) in acetone (5 mL) was stirred at room temperature for 4 h. The mixture was concentrated by rotary evaporation under reduced pressure. The residue was diluted with CH₂Cl₂, extracted with water. The water phase was washed with CH₂Cl₂, and the combined organic layers were washed with saturated NaHCO_3(aq.) and brine successively. The organic phase was dried over MgSO₄, filtered, concentrated by rotatory evaporation under reduced pressure, and purified by column chromatography (silica gel, hexane/EtOAc, gradients from 1:1 to 0:1) to give the acetonide compound 3 (358 mg, 86% yield). C₂₀H₂₃N₅O₅; TLC (EtOAc) R_f = 0.56; [α]_D²⁵ = -113.1 (CHCl₃, c = 2); IR v_max (neat) 3367, 2991, 2933, 1622, 1516, 1454, 1381, 1340, 1217, 1082 cm^-1; ¹H NMR (CDCl₃, 400 MHz) δ 8.35 (1 H, br s), 7.81 (1 H, s), 7.04 (2 H, d, J = 8.0 Hz), 6.64 (2 H, d, J = 8.0 Hz), 6.39 (1 H, br s), 5.82 (1 H, d, J = 5.2 Hz), 5.19 (1 H, dd, J = 5.6, 5.2 Hz), 5.10 (1 H, d, J = 5.6 Hz), 4.67 (2 H, br s), 4.53 (1 H, s), 3.93-4.02 (1 H, m), 3.71-3.82 (1 H, m), 1.62 (3 H, s), 1.36 (3 H, s); ¹³C NMR (CDCl₃, 100 MHz) δ 155.8, 154.5, 153.9, 147.1, 139.4, 129.1, 129.0, 120.4, 115.5, 114.0, 94.1, 86.0, 83.0, 81.6, 63.2, 44.0, 27.5, 25.1; ESI-HRMS calcd for C₂₀H₂₄N₅O₅: 414.1777, found: m/z 414.1763 [M + H]+.

2',3'-O-Isopropylidene-N⁶-(4-(4-methoxybenyloxy)benzyl)adenosine (4a)

[0090] 

[0091] A mixture of compound 3 (1.13 g, 2.74 mmol), p-methoxybenzyl chloride (800 µL, 6 mmol) and K₂CO₃ (930 mg, 6.85 mmol) in N,N-dimethylformamide (DMF, 25 mL) was stirred at 45 °C for 3 h. The mixture was concentrated by rotary evaporation under reduced pressure. The residue was diluted with EtOAc, and washed with 1 M HCl, water and brine successively. The organic phase was dried over MgSO₄, filtered, concentrated by rotatory evaporation, and purified by column chromatography (silica gel, EtOAc/hexane, gradients from 1:3 to 4:1) to give the PMB derivative 4a (1.12 g, 76% yield). C₂₈H₃₁N₅O₆; white solid; mp 68.3-71.4 °C; TLC (EtOAc/hexane (1:1)) R_f = 0.17; [α]_D²⁵ = -66.0 (CH₂Cl₂, c = 2); IR v_max (neat) 3281, 2988, 2936, 2870, 2834, 1622, 1586, 1514, 1478, 1468, 1380, 1339, 1303, 1242, 1216, 1170, 1154, 1113, 1077 cm^-1; ¹H NMR (CDCl₃, 400 MHz) δ 8.34 (1 H, br s), 7.66 (1 H, br s), 7.32 (2 H, d, J = 8.4 Hz), 7.25 (2 H, d, J = 8.4 Hz), 6.89 (4 H, dd, J = 8.4, 5.8 Hz), 6.77 (1 H, br s), 6.42 (1 H, br s), 5.79 (1 H, d, J = 4.8 Hz), 5.18 (1 H, t, J = 5.2 Hz), 5.09 (1 H, d, J = 5.6 Hz), 4.95 (2 H, s), 4.73 (2 H, br s), 4.51 (1 H, s), 3.95 (1 H, d, J = 12.46 Hz), 3.71 - 3.83 (4 H, m), 1.62 (3 H, s), 1.35 (3 H, s); ¹³C NMR (CDCl₃, 100 MHz) δ 159.3, 158.3, 154.9, 152.7, 147.3, 139.5, 130.2, 129.1, 129.0, 128.8, 121.2, 115.0, 114.0, 113.9, 94.3, 85.9, 82.9, 81.7, 69.7, 63.4, 55.2, 43.9, 27.6, 25.2; ESI-HRMS calcd. for C₂₈H₃₂N₅O₆: 534.2353, found: m/z 534.2368 [M + H]+.

N⁶-(4-(Bezoyloxy)benzyl)-2',3'-O-isopropylidene-adenosine (4b)

[0092] 

[0093] A mixture of compound 2 (265 mg, 0.64 mmol) and NEt₃ (267 µL, 1.9 mmol) in CH₂Cl₂ (15 mL) was stirred at 0 °C while a solution of benzoyl chloride (74 µL, 0.64 mmol) in CH₂Cl₂ (9 mL) was added via syringed pump (4 mL/h). After the addition was complete, the mixture was concentrated by rotary evaporation under reduced pressure. The residue was diluted with EtOAc, and washed with 1 M HCl, saturated NaHCO₃, water and brine successively. The organic phase was dried over MgSO₄, filtered, concentrated by rotatory evaporation, and purified by column chromatography (silica gel, hexane/EtOAc, gradients from 1:1 to 0:1) to give compound 4b (258 g, 78% yield). C₂₇H₂₇N₅O₆; white foam; TLC (EtOAc/hexane (1:1)) Rf = 0.13; [α]_D²⁵ = -74.4 (CHCl₃, c = 2); IR v_max (neat) 3279, 2989, 2934, 2860, 1735, 1622, 1508, 1481, 1357, 1340, 1265, 1202, 1081, 1065 cm^-1; ¹H NMR (CDCl₃, 400 MHz) δ 8.33 (1 H, br s), 8.15 (2 H, d, J = 7.6 Hz), 7.57 (1 H, t, J = 7.6 Hz ), 7.45 (3 H, t, J = 7.6 Hz), 7.35 (3 H, d, J = 8.0 Hz), 7.12 (2 H, d, J = 8.0 Hz), 6.63 (1 H, br s), 5.82 (1 H, d, J = 4.8 Hz), 5.17 (1 H, dd, J = 5.2, 4.8 Hz), 5.06 (1 H, d, J = 5.2 Hz), 4.81 (2 H, d, J = 18.8 Hz), 4.46 (1 H, s), 3.91 (1 H, d, J = 12.0 Hz), 3.73 (1 H, d, J = 12.0 Hz), 1.57 (3 H, s), 1.31 (3 H, s); ¹³C NMR (CDCl₃, 100 MHz) δ 165., 154.9, 152.6, 150.0, 147.3, 139.6, 136.0, 133.5, 130.0, 129.2, 128.6, 128.4, 121.8, 120.9, 113.7, 93.9, 85.9, 82.9, 81.5, 63.2, 43.4, 27.4, 25.1; ESI-HRMS calcd. for C₂₇H₂₈N₅O₆: 518.2040, found: m/z 518.2050 [M + H]+.

5'-O-(Imidazolyl-N-carbonyl)-2',3'-O-isopropylidene-N⁶-(4-(4-methoxybenzyloxy)benzyl)adenosine (5a)

[0094] 

[0095] A mixture of compound 4a (207 mg, 0.38 mmol), 4-dimethylaminopyridine (DMAP, catalytic amount) and 1,1'-carbonyldiimidazole (500 mg, 3.1 mmol) in anhydrous CH₂Cl₂ (3 mL) was stirred at room temperature under argon for 3 h. After the reaction was complete (monitored by TLC), the mixture was washed with 1 M HCl, water and brine successively. The organic phase was dried over MgSO₄, filtered, and concentrated by rotatory evaporation to give compound 5a (241 mg, 98 % yield).

[0096] C₃₂H₃₃N₇O₇; white powder; mp 68.3-71.5 °C; TLC (EtOAc/hexane (1:1)) R_f = 0.34; [α]_D²⁵ = 2.36 (CH₂Cl₂, c = 2); IR v_max (neat) 3281, 3134, 3035, 2995, 2929, 2835, 1769, 1617, 1585, 1151, 1482, 1409, 1389, 1295, 1242, 1176, 1102, 1004 cm^-1; ¹H NMR (CDCl₃, 400 MHz) δ 8.32 (1 H, br s), 8.11 (1 H, s), 7.71 (1 H, s), 7.37 (1 H, s), 7.32 (2 H, d, J = 8.8 Hz), 7.27 (2 H, d, J = 8.4 Hz), 7.02 (1 H, s), 6.89 (4 H, t, J = 8.0 Hz), 6.26 (1 H, br s), 6.02 (1 H, d, J = 1.6 Hz), 5.53 (1 H, dd, J = 6.4, 1.6 Hz), 5.22 (1 H, dd, J = 6.4, 4.0 Hz), 4.95 (2 H, s), 4.74 (1 H, br s), 4.68 (1 H, dd, J = 11.2, 4.0 Hz), 4.54-4.62 (1 H, m), 4.46-4.54 (1 H, m), 3.78 (3 H, s), 1.60 (3 H, s), 1.38 (3 H, s); ¹³C NMR (CDCl₃, 100 MHz) δ 159.2, 158.1, 154.5, 148.1, 140.9, 139.4, 139.3, 137.0, 134.0, 13.5, 13.3, 129.0, 128.9, 128.7, 117.0, 115.0, 114.7, 113.9, 90.8, 84.6, 83.8, 81.4, 69.8, 67.2, 55.3, 39.0, 27.3; ESI-HRMS calcd. for C₃₂H₃₄N₇O₇: 628.2520, found: m/z 628.2514 [M + H]+.

5'-O((Ethyl glycinate)-N-carbonyl)-2',3'-O-isopropylidene-N⁶-(4-(4-methoxybenzyloxy)benzyl)adenosine (6a-1)

[0097] 

[0098] A mixture of compound 5a (104 mg, 0.16 mmol), DMAP (catalytic amount), DIEA (110 µL, 0.64 mmol) and glycine ethyl ester hydrochloride (93 mg, 0.66 mmol) in DMF (4 mL) was stirred at room temperature under argon for 12 h. After the reaction was complete (monitored by TLC), the mixture was concentrated by rotary evaporation under reduced pressure. The residue was diluted with EtOAc and washed with 1 M HCl, saturated NaHCO₃, water and brine successively. The organic phase was dried over MgSO₄, filtered, concentrated by rotatory evaporation, and purified by column chromatography (silica gel, EtOAc/hexane, gradients from 1:3 to 9:1) to give compound 6a-1 (104 mg, 94%yield). C₃₃H₃₈N₆O₉; white solid; mp 69.6-71.2 °C; [α]_D²⁵ = -25.5 (CH₂Cl₂, c = 2); IR v_max (neat) 3367, 2987, 5938, 2835, 1724, 1618, 1581, 1511, 1483, 1466, 1381, 1303, 1246, 1217, 1172, 1078, 1029cm^-1; ¹H NMR (CDCl₃, 400 MHz) δ 8.36 (1 H, br s), 7.78 (1 H, s), 7.31 (2 H, d, J = 8.4 Hz), 7.25 (2 H, J = 8.4 Hz ), 6.88 (2 H, d, J = 8.4 Hz), 6.87 (2 H, d, J = 8.4 Hz), 6.57 (1 H, t, J = 5.3 Hz), 6.05 (1 H, s), 5.81 (1 H, br s), 5.35 (1 H, d, J = 4.8 Hz), 4.97 (1 H, dd, J = 6.0, 3.2 Hz), 4.93 (2 H, s), 4.74 (2 H, br s), 4.38-4.44 (1 H, m), 4.32 (1 H, dd, J = 11.6, 4.0 Hz), 4.08-4.20 (3 H, m), 3.79-3.96 (2 H, m), 3.77 (3 H, s), 1.57 (3 H, s), 1.34 (3 H, s), 1.22 (4 H, t, J = 7.2 Hz); ¹³C NMR (CDCl₃, 100 MHz) δ 169.9, 159.3, 158.1, 155.7, 154.6, 153.3, 148.3, 139.1, 130.7, 129.1, 129.0, 128.8, 120.1, 114.9, 114.3, 113.9, 90.7, 85.0, 84.0, 81.5, 69.7, 64.7, 61.4, 55.2, 43.8, 42.7, 27.0, 25.2, 14.0; ESI-HRMS calcd. for C₃₃H₃₉N₆O₉: 663.2779, found: m/z 663.2772 [M + H]+.

5'-O-(Ethyl L-phenylalaninate)-N-carbonyl)-2',3'-O-isopropylidene-N⁶-(4-(4-methoxybenzyloxy)benzyl)adenosine (6a-2)

[0099] 

[0100] A mixture of compound 5a (110 mg, 0.17 mmol), DMAP (catalytic amount), DIEA (125 µL, 0.68 mmol) and L-phenylalanine ethyl ester hydrochloride (160 mg, 0.68 mmol) in anhydrous DMF (13 mL) was stirred at room temperature under argon for 12 h. After the reaction was complete (monitored by TLC), the mixture was concentrated by rotary evaporation under reduced pressure. The residue was diluted with EtOAc, and washed with 1 M HCl, saturated NaHCO₃, water and brine successively. The organic phase was dried over MgSO₄, filtered, concentrated by rotatory evaporation under reduced pressure, and purified by column chromatography (silica gel, EtOAc/hexane, gradients from 1:3 to 9:1) to give compound 6a-2 (44 mg, 34% yield). C₄₀H₄₄N₆O₉; white solid; mp 80.1-83.6 °C; TLC (EtOAc) R_f = 0.75; [α]_D²⁵ = -9.55 (CH₂Cl₂, c = 1); IR v_max (neat) 3371, 3028, 2991, 2942, 2876, 2844, 1728, 1621, 1585, 1512, 1489, 1463, 1377, 1344, 1299, 1250, 1209, 1176, 1082, 1033 cm^-1; ¹H NMR (CDCl₃, 400 MHz) δ 8.36 (1 H, br s), 7.76 (1 H, s), 7.32 (2 H, d, J = 8.4 Hz), 7.26 (2 H, d, J = 8.4 Hz), 7.15-7.22 (3 H, m), 7.04 (2 H, d, J = 6.6 Hz), 6.89 (2 H, d, J = 8.4 Hz), 6.88 (2 H, d, J = 8.4 Hz), 6.39 (1 H, t, J = 5.2 Hz), 6.05 (1 H, d, J = 1.2 Hz), 5.50 (1 H, d, J = 8.0 Hz), 5.42 (1 H, dd, J = 6.4, 1.2 Hz), 4.99 (1 H, dd, J = 6.4, 3.2 Hz), 4.94 (2 H, s), 4.74 (2 H, br s), 4.55 (1 H, td, J = 8.0, 6.4 Hz), 4.39-4.47 (1 H, m), 4.26-4.34 (1 H, m), 4.17-4.24 (1 H, m), 4.10 (2 H, qd, J = 7.2, 1.5 Hz), 3.78 (3 H, s), 3.02 (2 H, qd, J = 9.6, 6.0 Hz), 1.59 (3 H, s), 1.37 (3 H, s), 1.17 (3 H, t, J = 7.2 Hz); ¹³C NMR (CDCl₃, 100 MHz) δ 171.1, 159.1, 158., 154.8, 154.4, 153.1, 139.1, 139.0, 135.5, 13.4, 129.1, 129.0, 128.9, 128.7, 128.3, 126.8, 120.1, 114.9, 114.3, 113.8, 90.8, 8.2, 84.0, 81.6, 69.8, 64.7, 61.5, 55.2, 54.9, 43.4, 38.3, 27.2, 25.5, 14.2; ESI-HRMS calcd. for C₄₀H₄₅N₆O₉: 753.3248, found: m/z 753.3228 [M + H]+.

N⁶-(4-(Benzoyloxy)benzyl)-2',3'-O-isopropylidene-5'-O-((methyl glycinate)-N-carbonyl)-adenosine (6b-1)

[0101] 

[0102] A mixture of compound 4b (93 mg, 0.18 mmol) and 1,1'-carbonyldiimidazole (117 mg, 0.72 mmol) in anhydrous tetrahydrofuran (THF, 2 mL) was stirred at room temperature under argon for 1 h. After the reaction was complete (monitored by TLC), water (9.72 uL, 0.54 mmol), glycine methyl ester (as the hydrochloric salt, 90 mg, 0.72 mmol) and DIEA (129 µL, 0.72 mmol) were added. The mixture was stirred for another 12 h, and then concentrated by rotary evaporation under reduced pressure. The residue was diluted with EtOAc, wash with 1 M HCl, saturated NaHCO₃, water and brine successively. The organic phase was dried over MgSO₄, filtered, concentrated by rotatory evaporation, and purified by column chromatography (silica gel, hexane/EtOAc, gradients from 1:1 to 0:1) to give the carbamate compound 6b-1 (69 mg, 60 % yield). C₃₁H₃₂N₆O₉; colorless oil; TLC (EtOAc/hexane (1:1)) R_f = 0.12; [α]_D²⁵ = -32.5 (CH₂Cl₂, c = 2); IR v_max (neat) 3366, 2988, 2952, 1731, 1620, 1583, 1508, 1481, 1452, 1438, 1419, 1376, 1331, 1267, 1209, 1081, 1064 cm^-1; ¹H NMR (CDCl₃, 400 MHz) δ 8.36 (1 H, br s), 8.15 (2 H, d, J = 7.6 Hz), 7.75 (1 H, br s), 7.59 (1 H, t, J = 7.2 Hz), 7.46 (2 H, dd, J = 7.6, 7.2 Hz), 7.40 (2 H, d, J = 8.4 Hz), 7.13 (2 H, d, J = 8.4 Hz), 6.89 (1 H, br s), 6.07 (1 H, s), 5.99 (1 H, br s), 5.30 (1 H, d, J = 5.2 Hz), 4.94 (1 H, dd, J = 5.6, 3.2 Hz), 4.85 (2 H, br s), 4.38-4.44 (1 H, m), 4.29-4.37 (1 H, m), 4.15 (1 H, dd, J = 11.2, 5.6 Hz), 3.80-3.98 (2 H, m), 3.66 (3 H, s), 1.56 (3 H, s), 1.32 (3 H, s); ¹³C NMR (CDCl₃, 100 MHz) δ 170.5, 165.1, 155.8, 154.6, 153.3, 150.1, 148.4, 139.3, 136.3, 133.5, 130.1, 129.4, 128.7, 128.5, 121.8, 120.1, 114.3, 90.6, 84.9, 84.0, 81.4, 64.8, 52.3, 42.5, 29.6, 27.1, 25.2; ESI-HRMS calcd. for C₃₁H₃₃N₆O₉: 633.2309, found: m/z 633.1201 [M + H]+.

N⁶-(4-(Benzoyloxy)benzyl)-2',3'-O-isopropylidene-5'-O-(methyl L-valinate)-N-carbonyl)-adenosine (6b-2)

[0103] 

[0104] A mixture of compound 4a (70 mg, 0.13 mmol) and 1,1'-carbonyldiimidazole (90 mg, 0.55 mmol) in anhydrous THF (4 mL) was stirred at room temperature under argon for 1 h. After the reaction was complete (monitored by TLC), water (4.8 µL, 0.26 mmol), L-valine methyl ester hydrochloride (90 mg, 0.55 mmol) and DIEA (96 µL, 0.55 mmol) were added. The mixture was stirred for another 12 h, and then concentrated by rotary evaporation under reduced pressure. The residue was diluted with EtOAc, washed with 1 M HCl, saturated NaHCO₃, water and brine successively. The organic phase was dried over MgSO₄, filtered, concentrated by rotatory evaporation, and purified by column chromatography (silica gel, hexane/EtOAc, gradients from 2:1 to 0:1) to give compound 6b-2 (35 mg, 40% yield). C₃₄H₃₈N₆O₉; white solid; mp 83.9-84.6 °C; TLC (EtOAc/hexane (1:1)) R_f = 0.21; [α]_D²⁵ = -17.6 (CH₂Cl₂, c = 2); IR v_max (neat) 3363, 2964, 2936, 1731, 1620, 1508, 1481, 1452, 1375, 1332, 1266, 1208, 1166, 1082, 1064, 1025 cm^-1; ¹H NMR (CDCl₃, 400 MHz) δ 8.38 (1 H, br s), 8.17 (2 H, d, J = 7.2 Hz), 7.76 (1 H, s), 7.60 (1 H, t, J = 7.6 Hz), 7.48 (2 H, dd, J = 7.2, 7.6 Hz), 7.41 (2 H, d, J = 8.4 Hz), 7.15 (2 H, d, J = 8.4 Hz), 6.60 (1 H, t, J = 5.6 Hz), 6.07 (1 H, s), 5.41-5.51 (2 H, m), 5.05 (1 H, dd, J = 6.0, 3.2 Hz), 4.86 (2 H, br s), 4.40-4.47 (1 H, m), 4.28-4.37 (1 H, m), 4.16-4.26 (2 H, m), 3.68 (3 H, s) 2.00-2.16 (1 H, m), 1.58 (3 H, s), 1.37 (3 H, s), 0.84 (6 H, dd, J = 24.0, 6.8 Hz); ¹³C NMR (CDCl₃, 100 MHz) δ 172.4, 165.1, 155.6, 154.7, 153.3, 150.2, 146.7, 139.4, 136.2, 133.6, 130.1, 129.4, 128.8, 128.5, 121.9, 120.4, 114.5, 90.9, 85.3, 84.0, 81.7, 64.8, 59.0, 52.1, 31.2, 29.6, 27.1, 25.3, 18.8, 17.5; ESI-HRMS calcd. for C₃₄H₃₉N₆O₉: 675.2779, found: m/z 675.2755 [M + H]+.

5'-O-((Ethyl glycinate)-N-carbonyl)-N⁶-(4-hydroxybenzyl)adenosine (7a-1)

[0105] 

[0106] A solution of compound 6a-1 (92 mg, 0.14 mmol) in MeOH (0.8 mL) and water (0.2 mL) was stirred at 0 °C while TFA (2 mL) was added slowly. After the addition was complete, the mixture was warmed to room temperature and stirred for another 2 h. The mixture was concentrated by rotatory evaporation under reduced pressure, diluted with MeOH/EtOAc (1:9), and washed with saturated NaHCO₃, water and brine successively. The organic phase was dried over MgSO₄, filtered, concentrated by rotatory evaporation under reduced pressure, and purified by column chromatography (silica gel, MeOH/EtOAc, gradients from 0:1 to 1:9) to give compound 7a-1 (32 mg, 37% yield). C₂₂H₂₆N₆O₈; white solid; mp 197.9-199.2 °C; [α]_D²⁵ = -36.3 (DMSO, c = 2); IR v_max (film) 3328, 2933, 1708, 1637, 1517, 1499, 1334, 1303, 1265, 1240, 1219, 1174, 1136, 1080 cm^-1; ¹H NMR (DMSO-d₆, 400 MHz) δ 9.23 (1 H, s), 8.35 (1 H, s), 8.27 (1 H, br s), 8.22 (1 H, br s), 7.75 (1 H, t, J = 6.0 Hz), 7.14 (2 H, d, J = 8.0 Hz), 6.67 (2 H, d, J = 8.0 Hz), 5.93 (1 H, d, J = 6.0 Hz), 5.53 (1 H, d, J = 6.0 Hz), 5.39 (1 H, d, J = 4.8 Hz), 4.67 (1 H, d, J = 6.0 Hz), 4.59 (2 H, br s), 4.26 (1 H, dd, J = 11.6, 2.8 Hz), 4.13-4.18 (2 H, m), 4.07-4.13 (3 H, m), 3.74 (2 H, d, J = 6.0 Hz), 1.18 (3 H, t, J = 7.2 Hz); ¹³C NMR (DMSO-d₆, 100 MHz) δ 170.1, 156.4, 156.1, 154.4, 152.7, 149.0, 139.4, 130.2, 128.5, 115.0, 87.0, 82.3, 73.0, 70.6, 64.6, 62.8, 60.4, 42.4, 42.2, 14.1; ESI-HRMS calcd. for C₂₂H₂₇N₆O₈: 503.1890, found: m/z 503.1908 [M + H]+.

5'-O-(Glycine-N-carbonyl)-N⁶-(4-hydroxybenzyl)adenosine e (I-a1)

[0107] 

[0108] Method A. To a solution of compound 7a-1 (25 mg, 0.05 mmol) in MeOH (4 mL) was added 1 M NaOH (4 mL). The mixture was stirred for was 30 min, concentrated by rotary evaporation under reduced pressure, and purified by reversed-phase column chromatography (RP-18 silica gel, MeOH/H₂O, gradients from 0:1 to 1:1) to give compound I-a1 (18 mg, 76% yield).

[0109] Method B. A solution of ester 6b-1 (125 mg, 0.19 mmol) in MeOH (0.9 mL) and water (0.1 mL) was stirred at 0 °C while TFA (2 mL) was added slowly. After the addition was complete, the mixture was warmed to room temperature and stirred for another 2 h. The reaction was concentrated by rotatory evaporation under reduced pressure, diluted with MeOH/EtOAc (1:9), and washed with saturated NaHCO₃, water and brine successively. The organic phase was dried over MgSO₄, filtered and concentrated by rotatory evaporation under reduced pressure. The residue was diluted with MeOH (3 mL), added 1 M NaOH (3 mL), and stirred at room temperature for 30 min. The mixture was concentrated by rotary evaporation under reduced pressure, and purified by normal-phase column chromatography (silica gel, AcOH/MeOH/EtOAc, gradients from 0:1:9 to 1:1:8) then reversed-phase column chromatography (RP-18 silica gel, MeOH/H₂O, gradients from 0:1 to 1:1) to give compound I-a1 (67 mg, 74% yield). The purity of product I-a1 was 98.1% as shown by HPLC on Chromolith RP-18 high-resolution column (Merck, 100 mm × 4.6 mm), t_R = 11.05 min ((CH₃CN/0.5% TFA aqueous solution, gradients from 0% to 20% in 15 min). C₂₀H₂₂N₆O₈; white solid; mp 172.4-174.6 °C; [α]_D²⁵ = -19.5 (H₂O, c = 1); IR v_max (film) 3192, 2951, 1708, 1686, 1641, 1595, 1521, 1491, 1447, 1410, 1356, 1310, 1262, 1230, 1196, 1169, 1159, 1142, 1068, 1032 cm^-1; ¹H NMR (CD₃OD, 400 MHz) δ 8.29 (1 H, br s), 8.27 (1 H, br s), 7.21 (2 H, d, J = 8.4 Hz), 6.74 (2 H, d, J = 8.4 Hz), 6.05 (1 H, d, J = 5.4 Hz), 4.66-4.73 (3 H, m), 4.34-4.40 (2 H, m), 4.28-4.33 (1 H, m), 4.22-4.27 (1 H, m), 3.61-3.72 (2 H, m); ¹³C NMR (CD₃OD, 100 MHz) δ 177.3, 158.6, 158.0, 156.1, 154.2, 150.2, 140.6, 131.0, 130.2, 120.8, 116.5, 89.6, 84.6, 75.7, 72.1, 65.3, 45.9, 45.0; ESI-HRMS (negative mode) calcd. for C₂₀H₂₁N₆O₈: 473.1421, found: m/z 473.1416 [M - H]^-.

N⁶-(4-Hydroxybenzyl)-5'-O-(L-phenylalanine-N-carbonyl)adenosine (I-a2)

[0110] 

[0111] A solution of compound 6a-2 (72 mg, 0.1 mmol) in MeOH (0.8 mL) and water (0.2 mL) was stirred at 0 °C while TFA (2 mL) was added slowly. After the addition was complete, the reaction was warmed to room temperature and stirred for another 2 h. The reaction was concentrated by rotatory evaporation under reduced pressure. The residue was diluted with MeOH (4 mL) and stirred at room temperature while 1 M NaOH (4 mL) was added. The mixture was stirred for was 30 min, concentrated by rotary evaporation under reduced pressure, and purified by reversed-phase column chromatography (RP-18 silica gel, water/MeOH, gradients from 1:0 to 1:1) to give compound I-a2 (43 mg, 76% yield). The purity of product I-a2 was 95.1% as shown by HPLC on Chromolith RP-18 high-resolution column (Merck, 100 mm × 4.6 mm), t_R = 11.0 min (CH3CN/0.5% TFA aqueous solution, gradients of 5% to 40% aqueous CH3CN in 15 min). C₂₇H₂₈N₆O₈; white solid; mp 180.2-182.3 °C; [α]_D²⁵ = -7.40 (DMSO, c = 1); IR v_max (film) 3409, 2940, 1702, 1642, 1517, 1498, 1453, 1401, 1340, 1248, 1178, 1082, 1056 cm^-1; ¹H NMR (CD₃OD, 400 MHz) δ 8.25 (1 H, s), 8.22 (1 H, s), 7.19 (2 H, d, J = 8.4 Hz), 7.15-7.17 (2 H, m), 7.10-7.14 (2 H, m), 7.04-7.08 (1 H, m), 6.73 (2 H, d, J = 8.4 Hz), 6.02 (1 H, d, J = 4.4 Hz), 4.67 (2 H, br s), 4.63 (1 H, dd, J = 4.4, 4.4 Hz), 4.30-4.34 (1 H, m), 4.23-4.29 (2 H, m), 4.17-4.21 (2 H, m), 3.17 (1 H, dd, J = 14.0, 4.8 Hz), 2.83-2.92 (1 H, m); ¹³C NMR (CDCl₃, 100 MHz) δ 177.4, 157.8, 157.7, 155.9, 154.0, 149.9, 140.5, 139.2, 130.8, 130.4, 130.0, 129.2, 127.4, 120.7, 116.3, 89.4, 84.4, 75.5, 71.8, 65.1, 58.1, 44.8, 39.2; ESI-HRMS (negative mode) calcd. for C₂₇H₂₇N₆O₈: 563.1890 found: m/z 563.1871 [M - H]^-.

N⁶-(4-Hydroxybenzyl)-5'-O-(L-valine-N-carbonyl) adenosine (I-a3)

[0112] 

[0113] A solution of compound 6b-2 (35 mg, 0.05 mmol) in MeOH (0.4 mL) and water (0.1 mL) was stirred at 0 °C while TFA (1 mL) was added slowly. After the addition was complete, the reaction was warmed to room temperature and stirred for another 2 h. The mixture was concentrated by rotatory evaporation under reduced pressure. The residue was diluted with MeOH (4 mL) and stirred at room temperature while 1 M NaOH (4 mL) was added and react for 30 min. The mixture was concentrated by rotary evaporation under reduced pressure, purified by normal phase column chromatography (silica gel, AcOH/MeOH/EtOAc, gradients from 0:1:9 to 1:1:8) and reversed-phase column chromatography (RP-18 silica gel, water/MeOH, gradients from 1:0 to 1:1) to give compound I-a3 (21 mg, 80% yield). The purity of product I-a3 was 98.9% as shown by HPLC on Chromolith RP-18 high-resolution column (Merck, 100 mm × 4.6 mm), t_R = 3.78 min (CH3CN/0.5% TFA aqueous solution, gradients of 15% to 40% in 15 min). C₂₃H₂₈N₆O₈; white solid; mp 166.6-168.8 °C; TLC (MeOH/AcOH/EtOAc (1:1:8)) R_f = 0.5; [α]_D²⁵ = -2.2 (DMSO, c = 1); IR v_max (film) 3332, 2965, 1702, 1625, 1517, 1466, 1406, 1340, 1240, 1173, 1107, 1087, 1050 cm^-1; ¹H NMR (CD₃OD, 400 MHz) δ 8.27 (2 H, s), 7.24 (2 H, d, J = 8.8 Hz), 6.75 (2 H, d, J = 8.8 Hz), 6.05 (1 H, d, J = 5.6 Hz), 4.72-4.74 (1 H, m), 4.70 (2 H, br s), 4.38-4.43 (1 H, m), 4.29-4.36 (2 H, m), 4.24-4.27 (1 H, m), 4.02 (1 H, d, J = 5.2 Hz), 2.09-2.20 (1 H, m), 0.93 (7 H, dd, J = 16.4, 6.8 Hz); ¹³C NMR (CD₃OD, 100 MHz) δ 176.5, 158.5, 157.8, 155.9, 154.0, 150.0, 14.6, 130.8, 130.0, 120.7, 116.3, 89.5, 84.3, 75.3, 72.0, 65.5, 61.7, 44.8, 31.9, 19.8, 18.2; ESI-HRMS calcd. for C₂₃H₂₉N₆O₈: 517.2047, found: m/z 517.2040 [M + H]+.

5'-O-(n-Butylaminocarbonyl)-N⁶-(4-hydroxybenzyl)adenosine (8)

[0114] 

[0115] A mixture of compound 4b (70 mg, 0.14 mmol) and 1,1'-carbonyldiimidazole (90 mg, 0.56 mmol) in anhydrous THF (4 mL) was stirred at room temperature under argon for 2 h. After the reaction was complete (monitored by TLC), water (4.8 µL, 0.28 mmol) and butylamine (110 µL, 1.1 mmol) was added successively. The mixture was stirred for another 12 h, and then concentrated by rotary evaporation under reduced pressure. The residue was diluted with EtOAc, washed with 1 M HCl, water and brine successively. The organic phase was dried over MgSO₄, filtered, and concentrated by rotatory evaporation to give a crude product.

[0116] A solution of the crude product in MeOH (0.8 mL) and water (0.2 mL) was stirred at 0 °C while TFA (2 mL) was added slowly. After the addition was complete, the mixture was warmed to room temperature and stirred for another 2 h. The mixture was concentrated by rotatory evaporation under reduced pressure, diluted with MeOH/EtOAc (1:9), and extracted with saturated NaHCO₃, water and brine successfully. The organic phase was dried over MgSO₄, filtered and concentrated by rotatory evaporation. The residue was diluted with MeOH (2 mL) and stirred at room temperature while 1 M NaOH (2 mL) was added. The mixture was stirred at room temperature for 30 min, and concentrated by rotary evaporation under reduced pressure. The residue was diluted with MeOH/EtOAc (1:9), washed with water and brine successively. The organic phase was dried over MgSO₄, filtered, concentrated by rotatory evaporation, and purified by column chromatography (silica gel, MeOH/CH₂Cl₂, gradients from 0:1 to 1:9) to give compound 8 (34 mg, 51% yield). The purity of product 8 was 98.2% as shown by HPLC on Chromolith^® RP-18 high-resolution column (Merck, 100 mm × 4.6 mm), t_R = 6.6 min (CH3CN/0.5% TFA aqueous solution, gradients of 15% to 40% in 15 min). C₂₂H₂₈N₆O₆; white solid; mp 187.4-189.0 °C; TLC (MeOH/CH₂Cl₂ (1:19)) Rf = 0.08; [α]_D²⁵ = -31.3 (DMSO, c = 2); IR v_max (film) 3339, 3149, 2958, 2933, 2872, 1696, 1620, 1519, 1465, 1372, 1337, 1293, 1248, 1168, 1115, 1079, 1049 cm^-1; ¹H NMR (CD₃OD, 400 MHz) δ 8.26 (1 H, s), 8.20 (1 H, s), 7.21 (2 H, d, J = 8.4 Hz), 6.74 (2 H, d, J = 8.4 Hz), 6.03 (1 H, d, J = 4.8 Hz), 4.61-4.75 (3 H, m), 4.37-4.43 (1 H, m), 4.33 (1 H, t, J = 4.4 Hz), 4.20-4.29 (2 H, m), 3.08 (2 H, t, J = 6.8 Hz), 1.38-1.51 (2 H, m), 1.25-1.37 (2 H, m), 0.90 (3 H, t, J = 7.3 Hz); ¹³C NMR (CD₃OD, 100 MHz) δ 158.7, 158.0, 156.2, 154.2, 140.5, 131.1, 130.1, 121.0, 116.5, 90.2, 84.4, 75.7, 72.2, 65.3, 41.8, 33.1, 21.0, 14.1; ESI-HRMS calcd. for C₂₂H₂₉N₆O₆: 473.2149, found: m/z 473.2145 [M + H]+.

2',3'-O-Isopropylidene-5'-O-((3-methoxycarbonyl-propylamino)carbonyl)-N⁶-(4-(4-methoxybenzyloxy)benzyl)adenosine (9)

[0117] 

[0118] A mixture of compound 5a (90 mg, 0.14 mmol), DMAP (catalytic amount), DIEA (110 µL, 0.6 mmol) and γ-aminobutyric acid methyl ester (as the hydrochloride salt, 180 mg, 1.2 mmol) in DMF (3 mL) was stirred at room temperature under argon for 12 h. After the reaction was complete (monitored by TLC), the mixture was concentrated by rotary evaporation under reduced pressure. The residue was diluted with EtOAc, and washed with 1 M HCl, saturated NaHCO₃, water and brine successively. The organic phase was dried over MgSO₄, filtered, concentrated by rotatory evaporation under reduced pressure, and purified by column chromatography (silica gel, EtOAc/hexane, gradients from 1:3 to 9:1) to give compound 9 (97 mg, 99%). C₃₄H₄₀N₆O₉; oil; [α]_D²⁵ = -32.6 (CH₂Cl₂, c = 2); IR v_max (neat) 3359, 2989, 2938, 1727, 1614, 1584, 1514, 1479, 1467, 1377, 1330, 1297, 1243, 1174, 1089, 1033 cm^-1; ¹H NMR (CDCl₃, 400 MHz) δ 8.37 (1 H, br s), 7.73 (1 H, s), 7.31 (2 H, d, J = 8.4 Hz), 7.25 (2 H, d, J = 8.4 Hz), 6.89 (2 H, d, J = 8.4 Hz), 6.88 (2 H, d, J = 8.4 Hz), 6.47 (1 H, br s), 6.05 (1 H, s), 5.42-5.47 (1 H, m), 5.01 (1 H, dd, J = 5.6, 2.8 Hz), 4.94 (2 H, s), 4.74 (2 H, br s), 4.42 (1 H, d, J = 2.9 Hz), 4.25-4.32 (1 H, m), 4.13-4.21 (1 H, m), 3.77 (3 H, s), 3.61 (3 H, s), 3.12 (2 H, q, J = 6.3 Hz), 2.28 (2 H, t, J = 7.2 Hz), 1.74 (2 H, quin, J = 6.9 Hz), 1.58 (3 H, s), 1.36 (3 H, s); ¹³C NMR (CDCl₃, 100 MHz) δ 173.5, 159.3, 158.1, 155.7, 154.6, 153.3, 139.0, 130.6, 129.1, 129.0, 128.8, 120.2, 114.9, 114.4, 113.9, 91.0, 85.2, 84.0, 81.6, 69.7, 64.4, 55.2, 51.6, 43.8, 40.3, 31.0, 27.1, 25.3, 24.9; ESI-HRMS calcd. for C₃₄H₄₁N₆O₉: 677.2935, found: m/z 677.2933 [M + H]⁺.

5'-O-((3-Carboxypropylamino)carbonyl)-N⁶-(4-hydroxybenzyl)adenosine (I-b1)

[0119] 

[0120] A solution of compound 9 (92 mg, 0.14 mmol) in MeOH (0.8 mL) and water (0.2 mL) was stirred at 0 °C while TFA (2 mL) was added slowly. After the addition was complete, the mixture was warmed to room temperature and stirred for another 2 h. The mixture was concentrated by rotatory evaporation under reduced pressure, diluted with MeOH/EtOAc (1:9), and washed with saturated NaHCO₃, water and brine successfully. The organic phase was dried over MgSO₄, filtered, concentrated by rotatory evaporation under reduced pressure. The residue was diluted with MeOH (4 mL), added 1 M NaOH (4 mL), and stirred for 30 min. The mixture was concentrated by rotary evaporation under reduced pressure, diluted with H₂O, and washed with EtOAc. The aqueous phase was concentrated by rotatory evaporation under reduced pressure, and purified by reversed phase column chromatography (RP-18 silica gel, water/MeOH, gradients from 1:0 to 1:1) to give compound I-b1 (32.1 mg, 47%). The purity of product I-b1 was 98.0% as shown by HPLC on Chromolith RP-18 high-resolution column (Merck, 100 mm × 4.6 mm), t_R = 10.2 min (CH3CN/0.5% TFA aqueous solution, gradients of 0% to 30% in 15 min). C₂₂H₂₆N₆O₈; white solid; mp 226.1-229.0 °C; [α]_D²⁵ = -29.7 (H₂O, c = 1); IR v_max (film) 3482, 3414, 2929, 1701, 1619, 1560, 1546, 1517, 1459, 1438, 1340, 1259, 1129, 1087, 1053 cm^-1; ¹H NMR (CD₃OD, 400 MHz) δ 8.26 (1 H, s), 8.23 (1 H, s), 7.21 (2 H, d, J = 8.4 Hz), 6.74 (2 H, d, J = 8.4 Hz), 6.05 (1 H, d, J = 5.4 Hz), 4.82-4.84 (2 H, m), 4.68 (1 H, t, J = 5.2 Hz), 4.33-4.41 (2 H, m), 4.20-4.31 (2 H, m), 3.05-3.17 (2 H, m), 2.18 (2 H, t, J = 7.2 Hz), 1.76 (2 H, quin, J = 7.2 Hz); ¹³C NMR (CD₃OD, 100 MHz) δ 183.2, 158.7, 158.0, 156.1, 154.2, 150.1, 140.5, 131.0, 130.2, 120.9, 116.5, 89.7, 84.5, 75.6, 72.2, 65.2, 45.0, 52.0, 36.0, 27.6; ESI-HRMS calcd. for C₂₂H₂₇N₆O₈: 503.1890, found: m/z 503.1894 [M + H]+.

N-[2',3'-O-Isopropylidene-N⁶-(4-(4-methoxybenzyloxy)benzyl)adenosine-5'-O-carbonyl]glycyl-L-leucyl-L-methioninamide (10)

[0121] 

[0122] A solution of compound 6a-1 (130 mg, 0.17 mmol) in 1 M NaOH (2 mL) and MeOH (2 mL) was stirred at room temperature for 30 min. The mixture was concentrated by rotary evaporation under reduced pressure. The residue was diluted with EtOAc, washed with 1 M HCl, water and brine successively. The organic phase was dried over MgSO₄, filtered, concentrated by rotatory evaporation under reduced pressure. To the residue in DMF (3 mL) were added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 57 mg, 0.29 mmol) and hydroxybenzotriazole (HOBt, 40 mg, 0.30 mmol). The mixture was stirred at 0 °C for 30 min. After the activation procedure was complete (monitored by TLC), a solution of L-leucyl-L-methionine amide hydrochloride (223 mg, 0.75 mmol) and DIEA (100 µL, 0.56 mmol) in DMF (3 mL) was added. The mixture was stirred at room temperature for 16 h, and then concentrated by rotary evaporation under reduced pressure. The residue was diluted with MeOH/EtOAc (1:9), washed with 1 M HCl (twice), saturated NaHCO₃, water and brine successively. The organic phase was dried over MgSO₄, filtered, concentrated by rotatory evaporation, and purified by column chromatography (silica gel, AcOH/MeOH/EtOAc, gradients from 0:0:1 to 1:1:8) to give a coupling compound 10 (115 mg, 77% yield). C₄₂H₅₅N₉O₁₀S; colorless oil; TLC (AcOH/EtOAc (1:9)) R_f = 0.26; [α]_D²⁵ = -38.1 (acetone, c = 1); IR v_max (neat) 3309, 2954, 2925, 2872, 2361, 2337, 1716, 1663, 1618, 1585, 1516, 1467, 1381, 1242, 1172, 1086, 1033 cm^-1; ¹H NMR (CDCl₃, 400 MHz) δ 8.35 (1 H, br s), 8.08 (1 H, br s), 7.93 (1 H, s), 7.71 (1 H, br s), 7.29 (2 H, d, J = 8.4 Hz), 7.23 (2 H, d, J = 8.0 Hz), 7.07 (1 H, br s), 6.98 (1 H, br s), 6.85 (2 H, d, J = 8.4 Hz), 6.84 (2 H, d, J = 8.0 Hz), 6.57 (1 H, br s), 6.47 (1 H, br s), 6.09 (1 H, br s), 5.33 (1 H, d, J = 4.4 Hz), 4.95 (1 H, br s), 4.89 (2 H, s), 4.74 (2 H, br s), 4.57 (1 H, d, J = 5.6 Hz), 4.50 (1 H, br s), 4.28 (1 H, d, J = 8.8 Hz), 4.06-4.16 (1 H, m), 3.82 (1 H, br s), 3.75 (3 H, s), 2.43 (2 H, br s), 1.97-2.09 (1 H, m), 1.95 (3 H, s), 1.91 (1 H, br s), 1.55 (5 H, s), 1.48 (1 H, br s), 1.32 (3 H, s), 0.74-0.86 (6 H, m); ¹³C NMR (CDCl₃, 100 MHz) δ 174.0, 172.5, 169.8, 159.4, 158.1, 156.4, 154.6, 138.9, 134.2, 130.9, 129.4, 129.2, 129.0, 128.9, 119.9, 114.8, 114.5, 113.9, 90.5, 84.8, 84.3, 8.2, 697, 64.8, 55.2, 52.5, 52.1, 44.3, 41.0, 1.0, 30.5, 30.2, 28.9, 27.1, 25.3, 24.7, 22.7, 22.0, 15.2, 14.0, 11.0; ESI-HRMS calcd. for C₄₂H₅₆N₉O₁₀S: 878.3874, found: m/z 878.3866 [M + H]+.

N-[(N⁶-(4-Hydroxybenzyl)adenosine)-5'-O-carbonyl]glycyl-L-leucyl-L-methioninamide (I-c1)

[0123] 

[0124] A solution of compound 10 (115 mg, 0.13 mmol) in MeOH (2 mL) and water (0.5 mL) was stirred at 0 °C while TFA (5 mL) was added slowly. After the addition was complete, the mixture was warmed to room temperature and stirred for another 2 h. The mixture was concentrated by rotatory evaporation under reduced pressure, diluted with saturated NaHCO₃, and extracted successively with EtOAc and n-BuOH. The organic phase was collected, and concentrated by rotatory evaporation under reduced pressure. The residue was purified by reversed-phase column chromatography on RP-18 gel (water/MeOH, isocratic 1:3), and repeatedly on another RP-18 gel column (water/acetone, gradients from 1:0 to 1:1), to give the tripeptide conjugated compound I-c1 (46.7 mg, 50% yield). The purity of product I-c1 was 96.9% as shown by HPLC on Chromolith RP-18 high-resolution column (Merck, 100 mm × 4.6 mm), t_R = 6.3 min (CH3CN/0.5% TFA aqueous solution, gradients of 15% to 40% in 15 min). C₃₁H₄₃N₉O₉S; white powder; mp 181.2-184.3 °C; [α]_D²⁵ = -34.0 (DMSO, c = 2); IR v_max (film) 3297, 2958, 2917, 1703, 1679, 1626, 1544, 1516, 1446, 1417, 1250, 1172, 1127, 1082, 1046 cm^-1; ¹H NMR (DMSO-d₆, 400 MHz) δ 9.23 (1 H, s), 8.36 (1 H, s), 8.27 (1 H, br s), 8.22 (1 H, br s), 8.05 (1 H, d, J = 7.8 Hz), 7.92 (1 H, d, J = 8.0 Hz), 7.57 (1 H, t, J = 5.2 Hz), 7.17 (1 H, br s), 7.14 (2 H, d, J = 8.4 Hz), 7.07 (1 H, br s), 6.67 (2 H, d, J = 8.4 Hz), 5.92 (1 H, d, J = 6.0 Hz), 5.52 (1 H, d, J = 6.0 Hz), 5.39 (1 H, d, J = 4.8 Hz), 4.67 (1 H, dd, J = 6.0, 6.0 Hz), 4.59 (2 H, br s), 4.19-4.32 (3 H, m), 4.10-4.19 (2 H, m), 4.03-4.09 (1 H, m), 3.63 (2 H, d, J = 3.2 Hz), 2.29-2.47 (2 H, m), 2.02 (3 H, s), 1.87-1.98 (1 H, m), 1.72-1.85 (1 H, m), 1.59 (1 H, d septet, J = 6.4, 6.4 Hz), 1.45 (2 H, dd, J = 6.4, 3.2 Hz), 0.87 (7 H, d, J=6.4 Hz), 0.83 (7 H, d, J = 6.4 Hz); ¹³C NMR (DMSO-d₆, 100 MHz) δ 172.3, 171.9, 169.2, 156.4, 156.1, 154.3, 152.6, 137.3, 13.2, 129., 128.5, 114.9, 86.9, 82.3, 73.0, 70.6, 64.5, 51.7, 51.2, 43.4, 40.6, 31.5, 29.7, 24.1, 23.0, 21.5, 14.6; ESI-HRMS calcd. for C₃₁H₄₄N₉O₉S: 718.2983, found: m/z 718.2972 [M + H]+.

Substance-P conjugate compound (I-c2)

[0125] 

[0126] A mixture of compound 5a (130 mg, 200 µmol) and an undecapeptide Arg-Pro-Lys-Pro-Glu-Glu-Phe-Phe-Gly-Leu-Met-CONH₂ (substance P, 10 mg, 7 µmol) in a two-phase system of CH₂Cl₂ (5 mL) and PBS solution (5 mL) was stirred vigorously at room temperature under argon for 4 h. The two phases were separated using a separation funnel. The aqueous layer was centrifuged (8000 rpm, 20 min, 4 °C), and the supernatant was decanted. The residue was washed with water, centrifuged (8000 rpm, 20 min, 4 °C) again to give a crude product after removal of supernatant.

[0127] A solution of the crude product (10 mg, 5.2 µmol) in MeOH (0.4 mL) and water (0.1 mL) was stirred at 0 °C while TFA (1 mL) was added slowly. After the addition was complete, the mixture was warmed to room temperature and stirred for another 2 h. The mixture was concentrated by rotatory evaporation under reduced pressure, and purified by reversed-phase column chromatography (RP-18 gel, water/MeOH, gradients from 1:0 to 1:1) to give compound I-c2 (1.6 mg, 12% yield). The purity of product I-c2 was 96.2% as shown by HPLC on Chromolith RP-18 high-resolution column (Merck, 100 mm × 4.6 mm), t_R = 1.8 min (CH3CN/0.5% TFA aqueous solution, gradients of 10% to 20% in 15 min). C₈₁H₁₁₅N₂₃O₁₉S; white powder; RP-18 TLC (AcOH/MeOH/H₂O (2:9:9)) R_f = 0.45; MALDI-TOF calcd. for C₈₁H₁₁₆N₂₃O₁₉S: 1746.8, found: m/z 1746.7 [M + H]+.

N⁶-(Indol-3-yl)ethyl-2',3'-O-isopropylidene-adenosine (12)

[0128] 

[0129] N⁶-(Indol-3-yl)ethyladenosine (compound 11) was prepared from the substitution reaction of 6-chloropurine riboside with (indol-3-yl)ethylamine according to the previously reported procedure [Chen, J.-B.; Liu, E. M.; Chern, T.-R.; Yang, C.-W.; Lin, C.-I.; Huang, N.-K.; Lin, Y.-L.; Chern, Y.; Lin, J.-H. Fang, J.-M. Design and synthesis of novel dual-action compounds targeting the adenosine A2A receptor and adenosine transporter for neuroprotection. ChemMedChem 2011, 6, 1390-1400]. In brief, a mixture of tryptamine (2.5 equiv), 6-chloropurine riboside (1 equiv), and diisopropylethylamine (DIEA, 4.5 equiv) in ethanol was irradiated at 150 W for 10 min in a focused monomode microwave reactor. The mixture was concentrated under reduced pressure, and purified by flash chromatography (silica gel; MeOH/EtOAc (1:9)) to give compound 11 in 83% yield.

[0130] A mixture of compound 11 (147 mg, 0.36 mmol), p-toluenesulfonic acid monohydrate (75 mg, 0.48 mmol) and 2,2-dimethoxypropane (1 mL, 0.83 mmol) in acetone (2 mL) was stirred at room temperature for 4 h. The mixture was concentrated by rotary evaporation under reduced pressure. The residue was diluted with CH₂Cl₂, extracted successively with water and saturated NaHCO₃. The organic phase was dried over MgSO₄, filtered, concentrated by rotatory evaporation under reduced pressure, and purified by column chromatography (silica gel, hexane/EtOAc (1:1)) to give the acetonide compound 12 (131 mg, 81% yield). C₂₃H₂₆N₆O₄; [α]_D²⁵ = -103.4 (CHCl₃, c = 1); IR ν_max (neat) 3421, 1624, 1458, 1340, 1215, 1155, 1080 cm^-1; ¹H NMR (400 MHz, CDCl₃) δ 8.59 (1 H, br s), 8.33 (1 H, br s), 7.57 (1 H, d, J = 8.4 Hz), 7.50 (1 H, d, J = 2.0 Hz), 7.15-7.11 (1 H, m), 7.09-7.05 (1 H, m), 6.88 (1 H, d, J = 2.0 Hz), 6.82 (1 H, br s), 6.49 (1 H, br s), 5.74 (1 H, br s), 5.18 (1 H, t, J = 5.2 Hz ), 5.08 (1 H, dd, J = 6.0, 0.8 Hz), 4.51 (1 H, s), 3.95 (2 H, m), 3.88-3.76 (2 H, m), 3.06 (2 H, t, J = 6.8 Hz),1.62 (3 H, s) , 1.35 (3 H, s); ¹³C NMR (100 MHz, CDCl₃) δ 155.2, 139.4, 136.5, 127.3, 122.2, 119.5, 118.8, 113.9, 112.8, 111.2, 94.4, 86.1, 82.9, 81.8, 63.5, 50.4, 27.7, 25.2; ESI-HRMS calcd. for C₂₃H₂₇N₆O₄: 451.2094, found: m/z 451.2099 [M + H]+.

N⁶-(Indol-3-yl)ethyl-2',3'-O-isopropylidene-5'-O-((methyl glycinate)-N-carbonyl) adenosine (13)

[0131] 

[0132] A mixture of compound 12 (78 mg, 0.17 mmol) and CDI (113 mg, 0.77 mmol) in anhydrous THF (4 mL) was stirred at room temperature under nitrogen for 2 h. Another batch of CDI (113 mg, 0.77 mmol) was added, and the mixture was stirred for 2 h. After the reaction was complete (monitored by TLC), a small amount of water was added to quench excess CDI, followed by adding glycine methyl ester hydrochloride (347 mg, 2.8 mmol) and DIEA (0.5 mL, 2.8 mmol). The mixture was stirred for another 24 h, and then concentrated by rotary evaporation under reduced pressure. The residue was diluted with CH₂Cl₂, washed with 1 M HCl, water and brine successively. The organic phase was dried over MgSO₄, filtered, concentrated by rotatory evaporation, and purified by column chromatography (silica gel, hexane/EtOAc (2:3)) to give compound 13 (48 mg, 50% yield). C₂₇H₃₁N₇O₇; oil; [α]_D²⁵= -34.2 (CHCl₃, c = 1); IR ν_max (neat) 3406, 1724, 1620, 1536, 1460, 1379, 1211, 1076 cm^-1; ¹H NMR (CD₃OD) δ 8.28 (1 H, br s), 8.16 (1 H, br s), 7.59 (1 H, d, J = 8.0 Hz), 7.31 (1 H, d, J = 7.6 Hz), 7.10-7.04 (2 H, m), 6.99-6.95 (1 H, m), 6.18 (1 H, d, J = 2.8 Hz), 5.43-5.41 (1 H, m), 5.08-5.06 (1 H, m), 4.59 (2 H, br s), 4.55 (1 H, dd, J = 8.0, 4.8 Hz), 4.30 (1 H, dd, J = 11.6, 4.8 Hz), 4.19 (1 H, dd, J = 11.6, 4.8 Hz), 3.89 (1 H, br s), 3.86 (1 H, s), 3.69 (3 H, s), 3.12 (2 H, t, J = 7.2 Hz), 1.61 (3 H, s), 1.33 (3 H, s); ¹³C NMR (100 MHz, CD₃OD) δ 172.4, 158.7, 154.3, 140.8, 138.4, 129.0, 125.2, 123.8, 122.5, 119.7, 119.5, 115.7, 113.3, 112.3, 91.8, 86.0, 85.6, 83.2, 66.0, 52.8, 49.6, 49.4, 43.3, 42.6, 27.6, 26.6, 25.7; ESI-HRMS calcd. for C₂₇H₃₂N₇O₇: 566.2363, found: m/z 566.2336 [M + H]+.

N⁶-(Indol-3-yl)ethyl-5'-O-(glycine-N-carbonyl)adenosine (14, II-a1)

[0133] 

[0134] A solution of compound 13 (21 mg, 0.04 mmol) in MeOH (1 mL) and water (0.05 mL) was stirred at 0 °C while TFA (1.3 mL) was added slowly. After the addition was complete, the mixture was warmed to room temperature and stirred for another 2 h. The mixture was concentrated by rotatory evaporation under reduced pressure. The residue was diluted with MeOH (0.5 mL), added 1 M NaOH (2.5 mL), and stirred at room temperature for 15 min. The mixture was concentrated by rotary evaporation under reduced pressure, and purified by reversed-phase column chromatography (RP-18 silica gel, MeOH/H₂O, gradients from 1:9 to 1:1) to give compound 14 (II-a1) (14 mg, 74% yield). The purity of product II-a1 was 91.6% as shown by HPLC on Chromolith RP-18 high-resolution column (Merck, 100 mm × 4.6 mm), t_R = 15.4 min ((CH₃CN/0.1% TFA aqueous solution, gradients from 5% to 20% in 22 min). C₂₃H₂₅N₇O₇; [α]_D²⁵ = -44.0 (DMSO, c = 1); IR ν_max (neat) 3447, 1793, 1624, 1559, 1456, 1299, 1245, 1096 cm^-1; ¹H NMR (400 MHz,CD₃OD) δ 8.27 (2 H, br s), 7.59 (1 H, d, J = 7.6 Hz), 7.32 (1 H, d, J = 8.0 Hz), 7.10-7.04 (2 H, m), 6.99-6.95 (2 H, m), 6.04 (1 H, d, J = 5.2 Hz), 4.68 (2 H, t, J = 5.2 Hz), 4.38-4.34 (2 H, m), 4.31 (1 H, d, J = 3.6 Hz), 4.25 (1 H, t, J = 4.0 Hz), 3.90 (2 H, br s), 3.15-3.11 (2 H, m); ¹³C NMR (100 MHz,CD₃OD) δ 177.3, 158.6, 156.3, 154.2, 140.5, 138.3, 128.9, 123.8, 122.4, 120.9, 120.3, 119.7, 119.5, 113.2, 112.4, 89.6, 84.6, 75.6, 72.1, 65.3, 45.9, 42.7, 26.5; ESI-HRMS calcd for C₂₃H₂₄N₇O₇: 510.1737, found: m/z 510.1753 [M - H]^-.

N⁶-((5-Bromothien-2-yl)methyl)adenosine (15)

[0135] 

[0136] A mixture of 6-chloropurine riboside (287 mg, 1 mmol), 2-aminomethyl-5-bromothiophene (as the hydrochloric salt, 384 mg, 2 mmol) and DIEA (3 mL, 17 mmol) in i-PrOH (8 mL) was heated at 50 °C for 48 h. The mixture was concentrated by rotary evaporation under reduced pressure, added an appropriate amount of MeOH, and centrifugated (8000 rpm, 4 °C, 20 min). The precipitate was collected and recrystallized from MeOH to give compound 15 (375 mg, 85% yield). C₁₅H₁₆N₅O₄SBr; white solid; mp 153.1-154.7 °C; TLC (i-propanol/hexane (2:3)) R_f = 0.36; [α]_D²⁵ = -152.8 (acetone, c = 1); IR v_max (neat) 3309, 2929, 2869, 1709, 1626, 1584, 1483, 1441, 1347, 1298, 1230, 1125, 1084, 1053 cm^-1; ¹H NMR (DMSO-d₆, 400 MHz) δ 8.54 (1 H, br s), 8.40 (1 H, s), 8.29 (1 H, br s), 7.02 (1 H, d, J = 3.6 Hz), 6.87 (1 H, d, J = 3.6 Hz), 5.90 (1 H, d, J = 6.0 Hz), 5.46 (1 H, d, J = 6.0 Hz), 5.36 (1 H, dd, J = 6.8, 4.8 Hz), 5.20 (1 H, d, J = 4.8 Hz), 4.77 (2 H, br s), 4.61 (1 H, ddd, J = 6.8, 6.0, 6.0 Hz), 4.15 (1 H, d, J = 2.8 Hz), 3.97 (1 H, d, J = 2.8 Hz), 3.62-3.73 (1 H, m), 3.52-3.60 (1 H, m); ¹³C NMR (DMSO-d₆, 100 MHz) δ 154.0, 152.3, 145.0, 140.2, 129.6, 126.4, 122.9, 109.8, 88.0, 85.9, 73.6, 70.6, 61.6; ESI-HRMS calcd for C₁₅H₁₇N₅O₄S⁷⁹Br and C₁₅H₁₇N₅O₄S⁸¹Br: 442.0185 and 444.0164, found: m/z 442.0190 and 444.0167 [M + H]+.

N⁶-(5-Bromothien-2-yl)methyl-2',3'-O-(isopropylidene)adenosine (16)

[0137] 

[0138] A mixture of compound 15 (150 mg, 0.34 mmol), p-toluenesulfonic acid monohydrate (70 mg, 0.48 mmol) and 2,2-dimethoxypropane (1 mL, 0.83 mmol) in acetone (2 mL) was stirred at room temperature for 4 h. The mixture was concentrated by rotary evaporation under reduced pressure. The residue was diluted with CH₂Cl₂, extracted successively with water and saturated NaHCO₃. The organic phase was dried over MgSO₄, filtered, concentrated by rotatory evaporation under reduced pressure, and purified by column chromatography (silica gel, hexane/EtOAc (2:3)) to give the acetonide compound 16 (131 mg, 80% yield). C₁₈H₂₀BrN₅O₄S; colorless oil; [α]_D²⁵= -126.3 (CHCl₃, c = 1); IR ν_max (neat) 3422, 1618, 1478, 1382, 1341, 1297, 1264, 1215, 1110, 1081 cm^-1; ¹H NMR (400 MHz, CDCl₃) δ 8.33 (1 H, br s), 7.67 (1 H, br s), 6.82(1 H, d, J = 3.6 Hz), 6.71 (1 H, d, J = 4.0 Hz), 5.80(1 H, d, J = 4.8 Hz), 5.16 (1 H, t, J = 5.2 Hz), 5.07 (1 H, dd, J = 6.0, 0.8 Hz), 4.85 (2 H, s), 4.49 (1 H, s) , 3.92 (1 H, d, J = 12.0Hz), 3.76 (1 H, d, J = 12.0 Hz), 1.60 (3 H, s), 1.33 (3 H,s); ¹³C NMR (100 MHz, CDCl₃) δ 171.1, 154.4, 152.6, 142.9, 139.7, 129.3, 126.2, 121.1, 113.9, 111.6, 109.2, 94.2, 86.0, 82.9, 81.6, 63.3, 27.6, 25.2; ESI-HRMS calcd for C₁₈H₂₁⁷⁹BrN₅O₄S: 482.0485, found: m/z 482.0498 [M + H]+

N⁶-(5-Bromothien-2-yl)methyl-2',3'-O-isopropylidene-5'-O-((methyl glycinate)-N-carbonyl)adenosine (17)

[0139] 

[0140] A mixture of compound 16 (208 mg, 0.44 mmol) and CDI (81 mg, 0.55 mmol) in anhydrous THF (17 mL) was stirred at room temperature under nitrogen for 2 h. Another batch of CDI (81 mg, 0.55 mmol) was added, and the mixture was stirred for 2 h. After the reaction was complete (monitored by TLC), a small amount of water was added to quench excess CDI, followed by adding glycine methyl ester hydrochloride (864 mg, 7 mmol 347 mg, 2.8 mmol) and DIEA (1.25 mL, 7 mmol). The mixture was stirred for another 24 h, and then concentrated by rotary evaporation under reduced pressure. The residue was diluted with CH₂Cl₂, washed successively with 1 M HCl, water and brine. The organic phase was dried over MgSO₄, filtered, concentrated by rotatory evaporation, and purified by column chromatography (silica gel, hexane/EtOAc (1:1)) to give compound 17 (118 mg, 45% yield). C₂₂H₂₅BrN₆O₇S; colorless oil; [α]_D²⁵ = -29.7 (MeOH, c = 1); IR ν_max (neat) 3422, 1735, 1719, 1618, 1214, 1076 cm^-1; ¹H NMR (400 MHz, CDCl₃) δ 8.38 (1 H, br s), 7.84 (1 H, br s), 6.82 (1 H, d, J = 3.6 Hz), 6.74 (1 H, d, J = 4.0 Hz), 6.06 (1 H, d, J = 2.0 Hz), 5.94 (1 H, s), 5.07 (1 H, dd, J = 6.0, 2.0 Hz),4.94 (1 H, dd, J = 6.4, 3.2 Hz), 4.88 (2 H, s),4.43-4.39 (1 H, m), 4.33 (1 H, dd, J = 11.6, 4.0 Hz), 4.19-4.15 (1 H, m), 3.97-3.83 (2 H, m), 3.69 (3 H,s), 1.57 (3 H,s), 1.33 (3 H,s); ¹³C NMR (100 MHz, CDCl₃) δ 170.4, 155.7, 154.1, 153.2, 148.4, 143.2, 139.4, 129.4, 126.2, 120.2, 114.5, 111.5, 109.4, 90.9, 85.1, 84.1, 81.5, 64.8, 52.4, 42.6, 27.1, 25.3; ESI-HRMS calcd for C₂₂H₂₆⁷⁹BrN₆O₇S: 597.0767, found: m/z 597.0739 [M + H]+.

N⁶-(5-Bromothien-2-yl)methyl-5'-O-(glycine-N-carbonyl)adenosine (II-b1)

[0141] 

[0142] A solution of compound 17 (19 mg, 31 µmol) in MeOH (0.5 mL) and water (0.05 mL) was stirred at 0 °C while TFA (1 mL) was added slowly. After the addition was complete, the mixture was warmed to room temperature and stirred for another 1 h. The mixture was concentrated by rotatory evaporation under reduced pressure. The residue was diluted with MeOH (1 mL), added 1 M NaOH (5 mL), and stirred at room temperature for 10 min. The mixture was concentrated by rotary evaporation under reduced pressure, and purified by reversed-phase column chromatography (RP-18 silica gel, MeOH/H₂O, gradients from 1:9 to 6:4) to give compound II-b1 (16 mg, 95% yield). The purity of product II-b1 was 95.5% as shown by HPLC on Chromolith RP-18 high-resolution column (Merck, 100 mm × 4.6 mm), t_R = 4.1 min ((CH₃CN/0.1% TFA aqueous solution, gradients from 5% to 20% in 22 min). C₁₈H₁₉BrN₆O₇S; white solid, mp = 171.6-174.0 °C; [α]_D²⁵ = -16.1 (DMSO, c = 1); IR ν_max (neat) 3448, 1623, 1278, 1119, 1084, 1060 cm^-1; ¹H NMR (400 MHz, CDCl₃) δ 8.31 (2 H, d, J = 4.0 Hz), 6.90(1 H, d, J = 4.0 Hz), 6.85 (1 H, d, J = 3.6 Hz), 6.06 (1 H, d, J = 5.2 Hz), 4.90 (2 H, s), 4.69(1 H, t, J = 5.2 Hz), 4.36-4.40 (2 H, m), 4.24-4.30 (2 H, m), 3.66 (2 H, s); ¹³C NMR (100 MHz, CDCl₃) δ 177.6, 158.7, 155.7, 154.1, 145.7, 141.0, 130.8, 127.6, 121.0, 112.1, 107.6, 89.6, 84.6, 75.7, 72.2, 65.4, 45.8, 30.9. ESI-HRMS calcd for C₁₈H₁₈⁷⁹BrN₆O₇S: 541.0141, found: m/z 541.0140 [M - H]^-.

5'-Azido-5'-deoxy-2',3'-O-isopropylidene-N⁶-(4-(4-methoxybenzyloxy)benzyl)adenosine (18)

[0143] 

[0144] A mixture of compound 4a (500 mg, 0.94 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 420 µL, 2.81 mmol) in 1,4-dioxane (3 mL) was stirred at 0 °C while diphenyl phosphoryl azide (DPPA, 404 µL, 1.88 mmol) was added in one portion. After 10 min, the mixture was allowed to warm to room temperature, and stirred for another 2.5 h. Sodium azide (304 mg, 4.67 mmol) and 15-crown-5 ether (18.5 µL, 0.09 mmol) were added. The mixture was then stirred at 80 °C for 16 h. The mixture was cooled, and concentrated by rotary evaporation under reduced pressure. The residue was diluted with EtOAc, and washed successively with 1 M HCl, NaHCO₃ (sat.), water and brine. The organic phase was dried over MgSO₄, filtered, concentrated under reduced pressure, and purified by column chromatography (silica gel, EtOAc/CH₂Cl₂ gradients from 1:19 to 2:3) to give compound 18 (368 mg, 70% yield). C₂₈H₃₀N₈O₅; white foamy solid; mp 49.2-50.8 °C; TLC (EtOAc/CH₂Cl₂ (1:3)) R_f = 0.42; [α]D²⁵ = +7.21 (CHCl₃, c = 2); IR ν_max (neat) 2929, 2101, 1615, 1583, 1513, 1479, 1464, 1375, 1330, 1295, 1240, 1215, 1173, 1155, 1093, 1034 cm^-1; ¹H NMR (CDCl₃, 600 MHz) δ 8.42 (1 H, br s) 7.72 (1 H, br s) 7.36 (2 H, d, J = 8.2 Hz) 7.29 (2 H, d, J = 8.2 Hz) 6.93 (4 H, t, J = 7.4 Hz) 6.54 (1 H, t, J = 5.6 Hz) 6.09 (1 H, s) 5.47 (1 H, d, J = 6.7 Hz) 5.08 (1 H, dd, J = 5.9,3.3 Hz) 4.99 (2 H, s) 4.79 (2 H, br. s.) 4.43-4.35 (1 H, m) 3.82 (3 H, s) 3.64-3.51 (2 H, m) 1.63 (3 H, s) 1.41 (3 H, s); ¹³C NMR (CDCl₃, 150 MHz) δ 159.4, 158.2, 154.7, 153.3, 139.2, 130.5, 129.1, 129.0, 128.9, 120.3, 115.0, 114.6, 113.9, 90.6, 85.7, 84.0, 82.1, 69.8, 55.2, 52.3, 43.9, 27.0, 25.3; ESI-HRMS calcd. for C₂₈H₃₁N₈O₅: 559.2417, found: m/z 559.2443 [M + H]+.

5'-Deoxy-2',3'-O-isopropylidene-5'-((methylglycinate)-N-amido)-N⁶-(4-(4-methoxybenzyloxy)benzyl)adenosine (19)

[0145] 

[0146] A mixture of compound 18 (368 mg, 0.66 mmol) and Lindlar catalyst (280 mg, 0.13 mmol) in MeOH (3 mL) and EtOAc (3 mL) was stirred at room temperature under an atmosphere of hydrogen (in balloon) for 24 h to complete hydrogenolysis of azide as shown by TLC. The mixture was diluted with MeOH, filtered through a pad of Celite, and rinsed with MeOH and CH₂Cl₂. The filtrate was concentrated under reduced pressure to give a crude amine product. Under an atmosphere of nitrogen, the crude amine product was dissolved in anhydrous THF (10 mL), cooled to 0 °C, and a solution of 1,1'-carbonyldiimidazole (330 mg, 2.04 mmol) in anhydrous THF (10 mL) was added dropwise with stirring. The mixture was stirred for 1 h, warmed to room temperature, and stirred for another 2 h. Glycine methyl ester hydrochloride (512 mg, 4.08 mmol), Et₃N (568 µL, 4.08 mmol) and DMAP (25 mg, 0.20 mmol) were added. The mixture was stirred at room temperature for 16 h, and then concentrated by rotary evaporation under reduced pressure. The residue was diluted with EtOAc, washed successively with 1 M HCl water and brine. The organic phase was dried over MgSO₄, filtered, concentrated by rotatory evaporation under reduced pressure, and purified by column chromatography (silica gel, MeOH/EtOAc, gradients from 0:1 to 3:97) to give the urea derivative 19 (379 mg, 86 % yield). C₃₂H₃₇N₇O₈; white foamy solid; mp 84.6-85.8 °C; TLC (MeOH/DCM (1:9)) R_f=0.68; [α]D²⁵ = -118.2 (CHCl₃, c = 2); IR ν_max (neat) 1750, 1616, 1578, 1559, 1513, 1375, 1297, 1240, 1214, 1175, 1097, 1082, 1034 cm^-1; ¹H NMR (CDCl₃, 600 MHz) δ 8.49 (1 H, br s), 7.67 (1 H, br s), 7.45 (1 H, d, J = 8.2 Hz), 7.34 (2 H, d, J = 8.7 Hz), 7.27 (2 H, d, J = 8.7 Hz), 6.91 (10 H, dd, J = 8.7, 3.6 Hz), 6.70 (1 H, t, J = 5.6 Hz), 5.77 (1 H, d, J = 5.1 Hz), 5.42-5.34 (2 H, m), 4.96 (2 H, s), 4.88 (1 H, dd, J = 6.0, 1.8 Hz), 4.76 (2 H, br s), 4.46 (1 H, dd, J = 4.6, 2.0 Hz), 4.13 (1 H, dd, J = 18.2, 6.4 Hz), 3.98 (1 H, ddd, J=14.1, 9.5, 2.0 Hz), 3.90 (1 H, dd, J = 18.2, 4.9 Hz), 3.80 (3 H, s), 3.72 (3 H, s), 3.30-3.24 (1 H, m), 1.62 (3 H, s), 1.37 (3 H, s); ¹³C NMR (CDCl₃, 150 MHz) δ 171.7, 159.4, 158.7, 158.2, 155.0, 152.9, 140.1, 130.4, 129.1, 129.0, 128.8, 121.2, 115.0, 114.4, 113.9, 92.9, 84.0, 81.9, 81.7, 69.7, 55.2, 52.1, 42.1, 42.0, 27.5, 25.1; ESI-HRMS calcd for C₃₂H₃₈N₇O₈: 648.2782, found: m/z 648.2780 [M + H]+.

5'-Deoxy-2',3'-O-isopropylidene-5'-((methyl glycinate)-N-amido)-N⁶-(4-hydroxybenzyl)adenosine (20)

[0147] 

[0148] A mixture of compound 19 (100 mg, 0.15 mmol) and 10% Pd/C (82 mg, 0.07 mmol) in MeOH (2.7 mL) and acetic acid (0.3 mL) was stirred at room temperature under an atmosphere of H2 for 3 days. The mixture was diluted with EtOAc, and filtered through a pad of Celite. The filtrate was concentrated by rotatory evaporation under reduced pressure, diluted with EtOAc, and washed successively with saturated NaHCO₃, water and brine. The organic phase was dried over MgSO₄, filtered, concentrated by rotatory evaporation under reduced pressure, and purified by column chromatography (silica gel, MeOH/CH₂Cl₂, gradients from 1:99 to 7:93) to give the product 20 (72 mg, 89% yield). C₂₄H₂₉N₇O₇; TLC (MeOH/DCM (1:19)) R_f = 0.35; ¹H NMR (CDCl₃, 600 MHz) δ 8.91 (1 H, br s), 8.42 (1 H, br s), 7.72 (1 H, br s), 7.27 (1 H, br s), 6.98 (2 H, d, J = 8.2 Hz), 6.74 (1 H, br s), 6.61 (2 H, d, J = 8.2 Hz), 5.77 (1 H, d, J = 4.6 Hz) ,5.44 (1 H, t, J = 5.4 Hz), 5.32-5.26 (1 H, m), 4.82 (1 H, dd, J = 6.1, 1.5 Hz), 4.52-4.71 (2 H, m), 4.38 (1 H, d, J = 2.0 Hz), 4.05 (1 H, dd, J = 18.0, 6.4 Hz), 3.85 (2 H, dd, J = 18.0, 5.1 Hz), 3.64 (3 H, s), 3.24 (1 H, d, J = 14.3 Hz), 1.56 (3 H, s), 1.30 (3 H, s); ¹³C NMR (CDCl₃, 150 MHz) δ 171.7, 158.9, 156.0, 154.6, 153.1, 147.7, 139.8, 129.0, 120.5, 115.5, 114.5, 92.5, 84.1, 82.1, 81.6, 52.2, 43.9, 42.1, 41.9, 27.4, 25.1; ESI-HRMS calcd for C₂₄H₃₀N₇O₇: 528.2207, found: m/z 528.2207 [M + H]+.

5'-Deoxy-5'-(glycine-N-amido)-N⁶-(4-hydroxybenzyl)adenosine (I-d1)

[0149] 

[0150] A solution of compound 20 (70 mg, 0.13 mmol) in MeOH (0.8 mL) and water (0.2 mL) was stirred at 0 °C while TFA (2 mL) was added slowly. The mixture was warmed to room temperature and stirred for 2 h. The mixture was concentrated by rotatory evaporation under reduced pressure. The residue was diluted with MeOH (5 mL), 1 M NaOH (5 mL) was added, and stirred at room temperature for 30 min. The mixture was concentrated by rotary evaporation under reduced pressure, purified by reversed-phase column chromatography (RP-18 silica gel, MeOH/water gradients from 0:1 to 3:7) to give compound I-d1 (61.8 mg, 98% yield). The purity of product I-d1 was 99.2% as shown by HPLC on Chromolith RP-18 column (Merck, 100 mm × 4.6 mm), t_R = 6.82 min (CH3CN/0.1% TFA aqueous solution, gradients of 0% to 30 % in 10 min). C₂₀H₂₃N₇O₇; white solid, mp >200 °C (decomposed); [α]D²⁵ = -19.5 (H₂O, c = 1); IR ν_max (film) 2924, 1623, 1598, 1577, 1398, 1337, 1250, 1171, 1133, 1080 cm^-1; ¹H NMR (D₂O, 600 MHz) δ 8.05 (1 H, s), 8.00 (1 H, s), 7.00 (2 H, d, J = 8.2 Hz), 6.54 (2 H, d, J = 8.7 Hz), 5.87 (1 H, d, J = 5.1 Hz), 4.62 (1 H, t, J = 5.1 Hz), 4.39 (2 H, br. s.), 4.25 (1 H, t, J = 5.1 Hz), 4.12 (1 H, q, J = 4.6 Hz), 3.58-3.50 (2 H, m), 3.45-3.37 (2 H, m); ¹³C NMR (D₂O, 150 MHz) δ 178.1, 165.4, 160.3, 160.1, 153.9, 152.6, 139.2, 128.8, 126.2, 118.9, 117.0, 112.5, 87.6, 83.6, 73.3, 70.9, 43.8, 41.1; ESI-HRMS (negative mode) calcd for C₂₀H₂₄N₇O₇: 474.1731, found: m/z 474.1737 [M + H]⁺.

5'-Acetylthio-5'-deoxy-N⁶-(4-(4-methoxybenzyloxy)benzyl)-2',3'-(O-isopropylidene)adenosine (21)

[0151] 

[0152] A mixture of N⁶-(4-hydroxybenzyl)-2',3'-O-isopropylidene-adenosine (3) (291 mg, 0.55 mmol), Et₃N (442 µL, 3.2 mmol) and DMAP (2.75 mg, 0.03 mmol) in CH₂Cl₂ (4 mL) was stirred at 0 °C while a solution of methanesulfonyl chloride (MsCl, 125 µL, 1.62 mmol) in CH₂Cl₂ (1.5 mL) was added dropwise. The mixture was stirred at 0 °C for 10 min, warmed to room temperature, and stirred for another 2 h. The mixture was concentrated by rotatory evaporation under reduced pressure, diluted with EtOAc, and washed successively with saturated NH₄Cl, saturated NaHCO₃, water and brine. The organic phase was dried over MgSO₄, filtered, concentrated by rotatory evaporation under reduced pressure to give a crude mesylation product. To a solution of the mesylation product in DMF (5 mL) were added KSAc (358 mg, 3.19 mmol) and KI (4.4 mg, 0.03 mmol). The mixture was stirred at room temperature for 20 h, and then concentrated by rotary evaporation under reduced pressure. The residue was diluted with EtOAc, and washed successively with saturated NaHCO₃, water and brine. The organic phase was dried over MgSO₄, filtered, concentrated by rotatory evaporation under reduced pressure, and purified by column chromatography (silica gel, EtOAc/CH₂Cl₂ gradients from 0:1 to 3:7) to give compound 21 (242 mg, 74% yield). C₃₀H₃₃N₅O₆S; white foamy solid; mp 85.2-86.7 °C; TLC (EtOAc/DCM (1:3)) R_f=0.53; [α]_D²⁵ = -20.39 (CHCl₃, c = 2); IR ν_max (neat) 1695, 1616, 1583, 1513, 1465, 1422, 1375, 1330, 1297, 1241, 1216, 1174, 1155, 1091, 1035 cm^-1; ¹H NMR (400 MHz, CDCl₃) δ 8.38 (1 H, s), 7.75 (1 H, s), 7.32 (2 H, d, J = 8.6 Hz), 7.27 (2 H, d, J = 8.6 Hz), 6.90 (4 H, dd, J = 8.6, 7.2 Hz), 6.16 (1 H, br. s.), 6.02 (1 H, d, J = 2.0 Hz), 5.49 (1 H, dd, J = 6.4, 2.0 Hz), 4.97 (1 H, d, J = 3.2 Hz), 4.95 (2 H, s), 4.31 (1 H, td, J = 6.4, 3.2 Hz), 3.79 (3 H, s), 3.31-3.22 (1 H, m), 3.21-3.11 (1 H, m), 2.32 (3 H, s), 1.57 (3 H, s), 1.36 (3 H, s); ¹³C NMR (CDCl₃, 100 MHz) δ 194.5, 159.4, 158.3, 154.7, 153.3, 139.3, 130.5, 129.2, 128.9, 115.1, 114.4, 114.0, 90.9, 86.1, 84.2, 83.7, 69.8, 55.3, 31.3, 30.5, 27.1, 25.3; ESI-HRMS calcd for C₃₀H₃₄N₅O₆S: 592.2230, found: m/z 592.2243 [M + H]+.

5'-Deoxy-5'-((2,2,2-trichloroethyl glycinate)-N-(carbonyl)thio)-N⁶-(4-(4-methoxybenzyloxy)benzyl)-2',3'-O-(isopropylidene)adenosine (22)

[0153] 

[0154] Under an atmosphere of argon, compound 21 (120 mg, 0.20 mmol) was treated in an deoxygenated solution of 1 M KOH_(aq) (5 mL), THF (5 mL) and MeOH (5 mL)_. The mixture was stirred at room temperature for 2 h. A deoxygenated solution of citric acid (1.05 g) in water (10 mL) was added to neutralize the excess base. The mixture was concentrated under reduced pressure to less than 10 mL, diluted with EtOAc, and washed successively with saturated NH₄Cl, water and brine. The organic phase was dried over MgSO₄, filtered, concentrated by rotatory evaporation under reduced pressure, and purified by column chromatography (silica gel, EtOAc/CH₂Cl₂ gradients from 0:1 to 1:4) to give a deacetylation product (66 mg, 60% yield).

[0155] Under an atmosphere of nitrogen, a solution of 2,2,2-trichloroethyl glycinate (as the hydrochloride salt, 140 mg, 0.58 mmol) and 1,1'-carbonyldiimidazole (140 mg, 0.86 mmol) in CH₂Cl₂ (0.8 mL) and THF (0.2 mL) was stirred at room temperature for 4 h. The mixture was concentrated under reduced pressure, diluted with CH₂Cl₂, and washed with brine. The organic phase was dried over MgSO₄, filtered, and concentrated by rotatory evaporation under reduced pressure to give a crude imidazolide product.

[0156] Under an atmosphere of argon, a THF solution (4 mL) containing the above-prepared deacetylated thiol product (66 mg, 0.12 mmol) and imidazolide was stirred at room temperature for 20 h. The mixture was concentrated by rotary evaporation under reduced pressure, diluted with EtOAc, and washed successively with saturated NH₄Cl, water and brine. The organic phase was dried over MgSO₄, filtered, concentrated by rotatory evaporation under reduced pressure, and purified by column chromatography (silica gel, EtOAc/CH₂Cl₂ gradients from 1:9 to 1:4) to give compound 22 (57 mg, 60% yield). C₃₃H₃₅Cl₃Ne₆O₈S; TLC (EtOAc/DCM (1:3)) R_f=0.35; [α]D²⁵ = -51.1 (CHCl₃, c = 2); IR ν_max (neat) 1768, 1723, 1669, 1612, 1514, 1457, 1382, 1302, 1241, 1216, 1174, 1157, 1082, 1034, 1008 cm^-1; ¹H NMR (CDCl₃, 600 MHz) δ 8.38 (1 H, br. s.), 7.80 (1 H, s), 7.34-7.31 (2 H, m), 7.29-7.26 (2 H, m), 6.94-6.86 (4 H, m), 6.20 (1 H, br s), 6.09 (1 H, t, J = 5.4 Hz), 6.04 (1 H, d, J = 2.6 Hz), 5.46 (1 H, dd, J=6.4, 3.0 Hz), 5.00 (1 H, dd, J = 6.1, 3.0 Hz), 4.95 (2 H, s), 4.82-4.71 (4 H, m), 4.43-4.36 (1 H, m), 4.18 (2 H, d, J = 5.1 Hz), 3.79 (3 H, s), 3.34-3.20 (2 H, m), 1.58 (3 H, s), 1.36 (3 H, s); ¹³C NMR (CDCl3, 150 MHz) δ 168.0, 159.4, 158.3, 154.7, 153.4, 139.2, 129.2, 128.9, 115.1, 114.5, 114.0, 94.2, 90.8, 86.4, 84.2, 83.5, 74.7, 74.4, 69.8, 55.3, 46.5, 42.5, 39.1, 32.2, 27.1, 25.4; ESI-HRMS calcd for C₃₃H₃₆³⁵Cl₃N₆O₈S: 783.1353, found: m/z 783.1363 [M + H]+.

5'-Deoxy-5'-(glycine-N-(carbonyl)thio)-N⁶-(4-hydroxybenzyl)adenosine (I-e1)

[0157] 

[0158] A solution of compound 22 (55 mg, 0.07 mmol) in MeOH (0.8 mL) and water (0.2 mL) was stirred at 0 °C while TFA (2 mL) was added slowly. The mixture was warmed to room temperature, stirred for another 2 h, and then concentrated by rotatory evaporation under reduced pressure. The residue was diluted with MeOH (3 mL) and stirred at room temperature while NaOAc•3H₂O (816 mg, 6 mmol), acetic acid (343 µL, 6 mmol) and zinc dust (46 mg, 0.7 mmol) were added. The mixture was stirred at room temperature for 2 h, concentrated by rotary evaporation under reduced pressure, and purified by reversed-phase column chromatography (RP-18 silica gel, MeOH/water gradients from 0:1 to 1:1) to give compound I-e1 (6.0 mg, 17% yield). C₂₀H₂₂N₆O₇S; ¹H NMR (DMSO-d₆, 600 MHz) δ 9.22 (1 H, br s), 8.38 (1 H, br s), 8.35 (1 H, s), 8.25 (1 H, br s), 8.22 (1 H, br s), 7.14 (2 H, d, J = 8.2 Hz), 6.67 (2 H, d, J = 8.2 Hz), 5.88 (1 H, d, J = 6.1 Hz), 5.47 (1 H, d, J = 5.1 Hz), 5.38-5.30 (1 H, m), 4.78 (1 H, d, J = 4.6 Hz), 4.58 (2 H, br s), 4.09 (1 H, br s), 3.94 (1 H, d, J = 2.0 Hz), 3.72 (2 H, d, J = 4.6 Hz), 3.10 (1 H, dd, J = 13.8, 7.2 Hz); ESI-HRMS calcd for C₂₀H₂₃N₆O₇S: 491.1343, found: m/z 491.1350 [M + H]+.

6-Chloropurine-2',3'-O-(isopropylidene)riboside (23)

[0159] 

[0160] A mixture of 6-chloropurine riboside (1) (1.0 g, 3.5 mmol), p-toluenesulfonic acid monohydrate (1.0 g, 5.3 mmol) and 2,2-dimethoxypropane (10 mL) in acetone (20 mL) was stirred at room temperature under N2 for 3 h. Another portion of 2,2-dimethoxypropane (10 mL) was added, and the mixture was stirred for another 1 h. The mixture was concentrated by rotary evaporation under reduced pressure. The residue was diluted with CH₂Cl₂, and extracted with water. The aqueous phase was washed with CH₂Cl₂, and the combined organic layers were washed with saturated NaHCO₃ and brine. The organic phase was dried over MgSO₄, filtered, concentrated by rotatory evaporation under reduced pressure, and purified by column chromatography (silica gel, EtOAc/hexane gradients from 3:7 to 1:0) to give compound 23 (870 mg, 76% yield). C₁₃H₁₅ClN₄O₄; [α]D²⁵ = -112.6 (CHCl₃, c = 2); IR ν_max (neat) 1592, 1563, 1490, 1438, 1419, 1400, 1384, 1337, 1259, 1202, 1154, 1136, 1108, 1080 cm^-1; ¹H NMR (CDCl₃, 600 MHz) δ 8.79 (1 H, s), 8.25 (1 H, s), 6.00 (1 H, d, J = 4.6 Hz), 5.24-5.21 (1 H, m), 5.14 (1 H, dd, J = 5.6, 1.5 Hz), 4.93 (1 H, dd, J = 10.6, 2.0 Hz), 4.57 (1 H, d, J = 1.5 Hz), 4.00 (1 H, dt, J = 12.7, 2.0 Hz), 3.84 (1 H, ddd, J = 12.7, 10.6, 2.3 Hz), 1.68 (3 H, s), 1.41 (3 H, s); ¹³C NMR (CDCl₃, 150 MHz) δ 152.4, 151.7, 150.4, 144.7, 133.4, 114.5, 94.1, 86.3, 83.2, 81.5, 63.2, 27.6, 25.2; ESI-HRMS calcd. for C₁₃H₁₆³⁵ClN₄O₄: 327.0855, found: m/z 327.0868 [M + H]+.

2',3'-O-Isopropylidene-6-[(4-methoxybenzylthio)purine]riboside (24a)

[0161] 

[0162] A solution of compound 23 (163 mg, 0.5 mmol) and S-(4-methoxybenzyl) thioacetate (106 mg, 0.55 mmol) in MeOH (8 mL) and THF (2 mL) was stirred at 0 °C while K₂CO₃ (76 mg, 0.55 mmol) was added in one portion. The mixture was stirred at 0 °C for 2 h, diluted with EtOAc, and washed with water and brine. The organic phase was dried over MgSO₄, filtered, concentrated by rotatory evaporation under reduced pressure, and purified by column chromatography (silica gel, EtOAc/CH₂Cl₂ gradients from 0:1 to 1:2) to give compound 24a (168 mg, 76% yield). C₂₁H₂₄N₄O₅S; white foam; TLC (EtOAc/DCM (1:3)) R_f=0.33; ¹H NMR (600 MHz, CDCl₃) □ 8.71 (1 H, s), 8.13 (1 H, s), 7.36 (2 H, d, J = 8.4 Hz), 6.82 (2 H, d, J = 8.4 Hz), 5.96 (1 H, d, J = 5.4 Hz), 5.19 (1 H, t, J = 5.4 Hz), 5.10 (1 H, d, J = 5.4 Hz), 4.56 - 4.64 (2 H, m), 4.53 (1 H, s), 3.97 (1 H, d, J = 12.0 Hz), 3.81 (1 H, dd, J = 12.0, 2.0 Hz), 3.77 (3 H, s), 1.64 (3 H, s), 1.37 (3 H, s); ¹³C NMR (CDCl₃, 100 MHz) δ 162.2, 158.8, 151.3, 146.9, 142.2, 132.0, 130.2, 128.8, 114.1, 113.9, 93.9, 86.3, 83.3, 81.5, 63.1, 55.2, 32.5, 27.5, 25.1; ESI-HRMS calcd. For C₂₁H₂₅N₄O5S: 445.1540, found: m/z 445.1543 [M + H]+.

2',3'-O-Isopropylidene-6-[(4-(4-methoxybenzyloxy)benzylthio)purine]riboside (24b)

[0163] 

[0164] A solution of compound 23 (61.8 mg, 0.19 mmol) and S-[4-(4-methoxybenzyloxy)benzyl] thioacetate (63 mg, 0.21 mmol) in MeOH (4 mL) and THF (1 mL) was stirred at 0 °C while K₂CO₃ (34.6 mg, 0.25 mmol) was added in one portion. The mixture was stirred at 0 °C for 2 h, diluted with EtOAc, and washed with water and brine. The organic phase was dried over MgSO₄, filtered, concentrated by rotatory evaporation under reduced pressure, and purified by column chromatography (silica gel, EtOAc/CH₂Cl₂ gradients from 0:1 to 2:3) to give compound 24a (85 mg, 81% yield). C₂₈H₃₀N₄O₆S; white foam; TLC (EtOAc/hexane (1:1)) R_f = 0.24; ¹H NMR (600 MHz, CDCl₃) δ 8.72 (1 H, s), 8.06 (1 H, s), 7.38 (2 H, d, J = 8.7 Hz), 7.34 (2 H, d, J = 8.7 Hz), 6.91 (4 H, d, J = 8.7, 2.0 Hz), 5.94 (1 H, d, J = 4.6 Hz), 5.23-5.19 (1 H, m), 5.13 (1 H, dd, J = 5.9, 1.4 Hz), 4.97 (2 H, s), 4.65-4.58 (2 H, m), 4.55 (1 H, d, J = 1.4 Hz), 3.98 (1 H, dd, J = 12.5, 1.8 Hz), 3.83 (1 H, d, J = 1.8 Hz), 3.81 (3 H, s), 1.66 (3 H, s), 1.39 (3 H, s); ¹³C NMR (CDCl₃, 100 MHz) δ 162.4, 159.3, 158.1, 146.9, 142.2, 132.4, 130.2, 129.09, 129.06, 128.8, 114.8, 113.9, 93.9, 86.1, 83.1, 81.5, 69.7, 63.2, 55.2, 32.4, 27.5, 25.1; ESI-HRMS calcd for C₂₈H₃₁N₄O₆S: 551.1959, found: m/z 551.2983 [M + H]⁺.

2',3'-O-Isopropylidene-6-(4-methoxybenzylthio)purine-5'-O-((methyl glycinate)-N-carbonyl)riboside (25a)

[0165] 

[0166] A mixture of compound 24a (88 mg, 0.2 mmol) and CDI (97 mg, 0.6 mmol) in anhydrous THF (3 mL) was stirred at room temperature under argon for 3 h. Another batch of CDI (50 mg, 0.3 mmol) was added, and the mixture was stirred for 1 h. After the reaction was complete (monitored by TLC), glycine methyl ester hydrochloride (175 mg, 1.4 mmol), DMAP (1.2 mg, 0.01 mmol) and TEA (194 µL, 1.4 mmol) were added. The mixture was stirred for another 20 h, and then concentrated by rotary evaporation under reduced pressure. The residue was diluted with EtOAc, washed with saturated NH₄Cl_(aq), water and brine successively. The organic phase was dried over MgSO₄, filtered, concentrated by rotatory evaporation, and purified by column chromatography (silica gel, EtOAc/CH₂Cl₂ gradients from 0:1 to 1:1) to give compound 25a (96 mg, 85% yield). C₂₅H₂₉N₅O₈S; white foam; TLC (EtOAc/DCM (1:9)) R_f = 0.07; ¹H NMR (CDCl3, 400 MHz) δ 8.75 (1 H, s), 8.23 (1 H, s), 7.36 (2 H, d, J = 8.7 Hz), 6.82 (2 H, d, J = 8.7 Hz), 6.18 (1 H, d, J = 2.4 Hz), 5.81 (1 H, t, J = 5.1 Hz), 5.43 (1 H, dd, J = 6.0, 2.4 Hz), 5.05 (1 H, dd, J = 6.0, 4.2 Hz), 4.59 (2 H, s), 4.49 (1 H, m), 4.35 (1 H, dd, J = 11.8, 4.2 Hz), 4.26 (1 H, dd, J = 11.8, 5.4 Hz), 3.89 (2 H, m), 3.76 (3 H, s), 3.69 (3 H, s), 1.61 (3 H, s), 1.38 (3 H, s); ¹³C NMR (CDCl3, 100 MHz) δ170.2, 161.0, 158.8, 155.7, 151.9, 147.6, 141.9, 131.0, 130.2, 128.9, 114.5, 113.8, 90.9, 85.1, 84.1, 81.4, 64.6, 55.1, 52.2, 42.5, 32.4, 27.0, 25.2; ESI-HRMS calcd. for C₂₅H₂₉N₅O₈S: 560.1810, found: m/z 560.1817 [M + H]+.

5'-O-(Glycine-N-carbonyl)-6-[(4-methoxybenzylthio)purine]riboside(I-f1)

[0167] 

[0168] A solution of compound 25a (10 mg, 0.01 mmol) in MeOH (0.8 mL), THF (0.1 mL) and water (0.1 mL) was stirred at room temperature while formic acid (2 mL) was added slowly. After the addition was complete, the reaction was heated to 40 °C and stirred for another 24 h. The mixture was concentrated by rotatory evaporation under reduced pressure. The residue was diluted with MeOH (2 mL) and H₂O (1 mL), stirred at room temperature while K₂CO₃ (28 mg, 0.2 mmol)) was added. The mixture was stirred for 2 h, and then quenched with saturated NH₄Cl (1 mL). The mixture was concentrated by rotary evaporation under reduced pressure, and purified by reversed-phase column chromatography (RP-18 silica gel, MeOH/water gradients from 0:1 to 9:1) to give compound I-f1 (3.5 mg, 39%). C₂₁H₂₃N₅O₈S; TLC (MeOH/AcOH/EtOAc (1:1:8)) R_f = 0.50; ¹H NMR (DMSO-d6, 600 MHz) δ 8.80 (1 H, s), 8.67 (1 H, s), 7.44 (1 H, br s), 7.38 (2 H, d, J = 8.7 Hz), 6.87 (2 H, d, J = 8.7 Hz), 6.02 (1 H, d, J = 5.6 Hz), 4.69 (1 H, t, J = 5.6 Hz), 4.57 - 4.65 (2 H, m), 4.25 (1 H, dd, J = 11.8, 2.6 Hz), 4.19 (1 H, d, J = 4.1 Hz), 4.09 - 4.17 (2 H, m), 3.72 (3 H, s), 3.59 (2 H, d, J = 5.1 Hz); ¹³C NMR (DMSO-d₆, 150 MHz) δ 159.4, 158.5, 156.2, 151.7, 149.5, 148.5, 143.2, 130.8, 130.2, 129.4, 113.9, 87.3, 82.7, 73.1, 70.5, 64.2, 55.1, 42.7, 31.3; ESI-HRMS calcd for C₂₁H₂₄N₅O₈S: 506.1340, found: m/z 506.1345 [M + H]+.

Intermittent cold stress model

[0169] Female C57BL/6JNarl mice aged 8-12 weeks were purchased from the National Laboratory Animal Center (Taipei, Taiwan). The fibromyalgia model was developed by Ueda's group, in which mice were treated with intermittent cold stress for 2 days ( Nishiyori, M.; Ueda, H. Prolonged gabapentin analgesia in an experimental mouse model of fibromyalgia. Mol. Pain 2008, 4, 52). Mice treated with intermittent cold stress developed long-lasting (>2 weeks) mechanical and thermal hyperalgesia. Analgesic effects of compound I-a1 (JMF3737) via i.p. (in 0.5% HPβCD) or p.o. (in 1% HPβCD) routes and compound I-d1 (JMF4413) via i.p. (in 0.5% HPβCD) were tested in these mice 5 days after intermittent cold stress. Mechanical hyperalgesia was assayed by testing the withdrawal response of mouse hindpaws to 0.2-mN von Frey filament stimulation. The experiment results are shown in Figures 9-12.

[0170] Figure 9 reveals that T1-11 shows dose-dependent analgesic effect with effective dose starting from 0.03 mg/kg via intraperitoneal injection (i.p.) and 8 mg/kg via oral gavage (p.o.). In comparison, the conjugate compound I-a1 (JMF3737) derived from T1-11 and glycine shows superior dose-dependent analgesic effect with effective dose starting from 1 mg/kg (i.p.) and 1 mg/kg (p.o.). A combination treatment by oral administration of T1-11 and glycine does not exhibit synergistic effect.

[0171] Figure 10 shows a representative whole-cell patch clamp recording, which reveals the analgesic mechanism of action of compound I-a1 (JMF3737); the mechanism is the same as the T1-11 by acting on the NK1R signaling. In muscle nociceptors, JMF3737 induces an outward current (I_JMF3737) that can be reversibly inhibited (from 40pA down to 15pA) by NK1R antagonist (RP67580) in whole-cell patch clamp recording on muscle nociceptors. The JMF3737-induced outward current (I_JMF3737) can cause nociceptor hyperpolarization and thus counteract the acid-induced depolarization in muscle nociceptors and thus inhibit pain associated with tissue acidosis.

[0172] Figures 11(a) and 11(b) reveal that no tolerance but therapeutic effect can be obtained by repeated treatments of compound I-a1 (JMF3737). In the fibromyalgia model, mice received oral treatment of 1 mg/kg JMF3737 once a day for 4 consecutive days from day 5 after pain is induced by intermittent cold stress. (a) Repeated treatment of JMF3737 resulted in decreased pain sensitivity to von Frey filament stimulation since day 8 (vehicle, n = 2; JMF3737, n = 4). (b) Repeated treatment of JMF3737 does not cause tolerance. JMF3737 shows acute analgesic effect from day 5 to day 8 (n = 4).

[0173] Figure 12(a) reveals that Conjugate compound of I-d1 (JMF4413) shows good analgesic effect with dose of 64 mg/kg via i.p. route (n=2). Figure 12(b) reveals that Conjugate compound of Ic-2 (JMF3795) shows good analgesic effect starting from 14 µg/kg (160 pmol) via i.p. route (n=3).

[0174] As a result, the conjugate compounds show (1) a better analgesic effect over T1-11 via oral route (e.g., JMF3737); (2) better solubility (up to 64 mg/kg) than T1-11 (e.g., JMF3737, JMF3795, JMF4413); (3) wider safety ranges (up to 64 mg/kg) than T1-11 (e.g., JMF3737, JMF4413).

Claims

1. A compound of formula (I):

wherein,

n is 0, 1, 2 or 3;

X is NH, O or S

Y is O, NH or S;

R¹ is OH, NH₂, NO₂, halogen, alkyl, haloalkyl, hydroxyalkyl, unsubstituted or substituted alkoxy, alkoxyalkyl, alkenyl or alkynyl;

R² and R³ are each independently OH, NH₂, NO₂, haloalkyl, hydroxyalkyl or alkylamino; and

R⁴ is selected from glycine, alanine, cysteine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, glutamine, asparagine, threonine, arginine, lysine, proline, and R⁴ is linked with C(=O) in the structure via the N-terminal group or a side chain amino group thereof, or R⁴ is a peptide RPKPQQFFGLM-CONH₂ and the peptide is linked with C(=O) in the structure via the side chain amino group of K, wherein the C-terminal of the amino acid or the peptide is optionally modified;

a tautomer or stereoisomer thereof; and a pharmaceutically acceptable salt of the foregoing.

2. The compound of claim 1, wherein R¹, R², and R³ are each independently OH.

3. The compound of claim 1 or 2, wherein n is 1.

4. The compound of any of claims 1-3, wherein X is NH and Y is O, NH or S.

5. The compound of any of claims 1-3, wherein X is S and Y is NH.

6. The compound of any of claims 1-5, wherein

R⁴ is selected from glycine, valine, cysteine, phenylalanine, or glutamine, or

R⁴ is the peptide RPKPQQFFGLM-CONH₂ and the peptide is linked with C(=O) in the structure via the side chain amino group of K.

7. The compound of any of claims 1-6, wherein the compound is of formula (IA):

wherein,

R is a substituent existing in common α-amino acids; and

Z⁺ is H⁺, Li⁺, Na⁺, K⁺, Rb⁺, Cs⁺, NH₄⁺ or aminium ion;

wherein,

aminium ion is R^AR^BR^CNH⁺ including acyclic and cyclic aminium ions; and R^A, R^B and R^C are independently H, C₁-C₄ alkyl, or benzyl;

a tautomer or stereoisomer thereof; and a pharmaceutically acceptable salt of the foregoing.

8. The compound of any of claims 1-7, which is selected from the group consisting of:

9. A method for preparing a compound of formula (I) according to any of claims 1-4,6-8,

wherein X is NH and Y is O, the method comprising:

(a) reacting N⁶-(4-hydroxybenzyl)adenosine (2) with 2,2-dimethoxypropane in acetone in the presence of an acid to afford a compound of formula (3):

wherein,
the acid is p-toluenesulfonic acid or camphorsulfonic acid;

(b) reacting compound (3) in the presence of a base with an agent to afford a compound of formula (4):

wherein,
PG is 4-methoxybenzyl or benzoyl; the agent is 4-methoxybenzyl chloride or benzoyl chloride; and the base is potassium carbonate or triethylamine;

(c) reacting compound (4) with 1,1'-carbonyldiimidazole (CDI) in the presence of a base to afford a compound of formula (5):

wherein,
the base is 4-dimethylaminopyridine (DMAP);

(d) reacting compound (5) with a derivative of amino acid or peptide to afford a compound of formula (6):

wherein,
[A']-NH- is derived from the derivative of amino acid or peptide; and

(e) removing all the protecting groups to afford a compound of formula (IB):

wherein,
[A]-NH- is the amino acid or the peptide in the derivative of an amino acid or a peptide.

10. A method for preparing a compound of formula (I) according to any of claims 1-4,6,8,

wherein X and Y are independently NH, the method comprising:

(a) reacting compound (4a) with diphenyl phosphoryl azide (DPPA) and sodium azide in the presence of a base to afford a compound of formula (18):

wherein,
the base is 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU);

(b) reducing compound (18) by hydrogenolysis in the presence of a catalyst, followed by coupling with glycine methyl ester hydrochloride in the present of a promoter, to afford a compound of formula (19):

wherein,
the catalyst is Lindlar catalyst and the promoter is 1,1'-carbonyldiimidazole;

(c) removing all the protecting groups and the ester group derived from the glycine methyl ester to afford a compound of formula (I-d1):

11. A method for preparing a compound of formula (I) according to any of claims 1-4,6,8,

wherein X is NH and Y is S, the method comprising:

(a) reacting compound (4a) with methanesulfonyl chloride (MsCl) in the presence of a base, followed by reacting with potassium thioacetate, to afford a compound of formula (21):

wherein,
the base is triethylamine and 4-dimethylaminopyridine;

(b) removing the acetyl group from compound (21) with a base, followed by coupling with 2,2,2-trichloroethylglycine in the present of a promoter, to afford a compound of formula (22):

wherein,
the base is potassium hydroxide and the promoter is 1,1'-carbonyldiimidazole;

(c) removing all the protecting groups and the ester group derived from 2,2,2-trichloroethylglycine to afford a compound of formula (I-e1):

12. A compound of formula (II):

wherein,

k is 1, 2 or 3;

X and Y are each independently NH, O or S;

R¹ is OH, NH₂, NO₂, halogen, alkyl, haloalkyl, hydroxyalkyl, unsubstituted or substituted alkoxy, alkoxyalkyl, alkenyl or alkynyl;

R² and R³ are each independently OH, NH₂, NO₂, haloalkyl, hydroxyalkyl or alkylamino; and

R⁴ is selected from glycine, alanine, cysteine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, glutamine, asparagine, threonine, arginine, lysine, proline, and R⁴ is linked with C(=O) in the structure via the N-terminal group or a side chain amino group thereof, or from a peptide RPKPQQFFGLM-CONH₂ and the peptide is linked with C(=O) in the structure via the side chain amino group of K, wherein the C-terminal of the amino acid or the peptide is optionally modified;

ring A is a substituted or unsubstituted aromatic heterocycle, comprising 5- or 6-membered heterocycle and fused heterocycle; the heteroatom(s) in the ring are one or more nitrogen, oxygen, and/or sulfur heteroatoms; and the substituent(s) are selected from the group consisting of fluorine, chlorine, bromine, iodine, hydroxyl, nitro, alkyl, trifluoromethyl group and combinations thereof;

a tautomer or stereoisomer thereof; and a pharmaceutically acceptable salt of the foregoing.

13. The compound of claim 12, which is selected from the group consisting of:

and

14. A pharmaceutical composition comprising:

(a) a therapeutically effective amount of a compound as claimed in any one of claims 1 to 8, 12 and 13, a tautomer or stereoisomer thereof, or a pharmaceutically acceptable salt of the foregoing; and

(b) a pharmaceutically acceptable carrier, excipient or vehicle.

15. A compound as claimed in any one of claims 1 to 9, 12 and 13, a tautomer or stereoisomer thereof, or a pharmaceutically acceptable salt of the foregoing for use for treating pain in a subject in need thereof, wherein

preferably the pain is associated with tissue acidosis, nerve injury or both, and/or

preferably the pain is dysfunctional pain, preferably wherein the dysfunctional pain is selected from the group consisting of fibromyalgia, bladder pain syndrome, pain caused by irritable bowel syndrome and pain associated with temporomandibular disorders, and/or

preferably the pain is selected from the group consisting of inflammatory pain, cancer-associated pain, chest pain, back pain, neck pain, shoulder pain, migraine, headache, myofascial pain, joint pain, muscular pain syndromes, neuropathic pain, peripheral pain, sympathetic pain, postoperative pain, post-traumatic pain, and multiple sclerosis pain, and/or

preferably wherein the pain is chronic pain.

Ansprüche

1. Verbindung von Formel (I):

wobei

n 0, 1, 2 oder 3 ist;

X NH, O oder S ist;

Y O, NH oder S ist;

R¹ OH, NH₂, NO₂, Halogen, Alkyl, Haloalkyl, Hydroxyalkyl, unsubstituiertes oder substituiertes Alkoxy, Alkoxyalkyl, Alkenyl oder Alkinyl ist;

R² und R³ jeweils unabhängig voneinander OH, NH₂, NO₂, Halogenalkyl, Hydroxyalkyl oder Alkylamino sind; und

R⁴ ausgewählt ist aus Glycin, Alanin, Cystein, Valin, Leucin, Isoleucin, Methionin, Phenylalanin, Tryptophan, Glutamin, Asparagin, Threonin, Arginin, Lysin, Prolin, und R⁴ mit C(=O) in der Struktur über die N-terminale Gruppe oder eine Seitenketten-Aminogruppe davon verbunden ist, oder R⁴ ein Peptid RPKPQQFFGLM-CONH₂ ist und das Peptid mit C(=O) in der Struktur über die Seitenketten-Aminogruppe von K verbunden ist, wobei der C-Terminus der Aminosäure oder des Peptids optional modifiziert ist;

ein Tautomer oder Stereoisomer davon; und ein pharmazeutisch annehmbares Salz des Vorstehenden.

2. Verbindung nach Anspruch 1, wobei R¹, R² und R³ jeweils unabhängig OH sind.

3. Verbindung nach Anspruch 1 oder 2, wobei n 1 ist.

4. Verbindung nach einem der Ansprüche 1-3, wobei X NH ist und Y O, NH oder S ist.

5. Verbindung nach einem der Ansprüche 1-3, wobei X S ist und Y NH ist.

6. Verbindung nach einem der Ansprüche 1-5, wobei

R⁴ ausgewählt ist aus Glycin, Valin, Cystein, Phenylalanin oder Glutamin, oder

R⁴ das Peptid RPKPQQFFGLM-CONH₂ ist, und das Peptid in der Struktur über die Seitenketten-Aminogruppe von K mit C(=O) verbunden.

7. Verbindung nach einem der Ansprüche 1-6, wobei die Verbindung von Formel (IA) ist:

wobei

R ein Substituent ist, der in gewöhnlichen α-Aminosäuren vorkommt; und

Z⁺ is H⁺, Li⁺, Na⁺, K⁺, Rb⁺, Cs⁺, NH₄⁺ oder Aminiumion ist;

wobei

Aminiumion R^AR^BR^CNH⁺ ist, einschließlich azyklischer und zyklischer Aminiumionen; und R^A, R^B und R^c unabhängig H, C₁-C₄-Alkyl oder Benzyl sind; ein Tautomer oder Stereoisomer davon; und ein pharmazeutisch annehmbares Salz des Vorstehenden.

8. Verbindung nach Anspruch 1-7, ausgewählt aus der Gruppe bestehend aus:

















und

9. Verfahren zum Herstellen einer Verbindung von Formel (I) nach einem der Ansprüche 1-4, 6-8,

wobei X NH ist und Y O ist, das Verfahren umfassend:

(a) Reagieren von N⁶-(4-Hydroxybenzyl)adenosin (2) mit 2,2-Dimethoxypropan in Aceton in Anwesenheit einer Säure zum Bilden einer Verbindung von Formel (3):

wobei
die Säure ist p-Toluolsulfonsäure oder Camphersulfonsäure;

(b) Reagieren von Verbindung (3) in Anwesenheit einer Base mit einem Mittel, um eine Verbindung von Formel (4) zu ergeben:

wobei
PG 4-Methoxybenzyl oder Benzoyl ist; das Mittel 4-Methoxybenzylchlorid oder Benzoylchlorid ist; und die Base ist Kaliumcarbonat oder Triethylamin ist;

(c) Reagieren von Verbindung (4) mit 1,1'-Carbonyldiimidazol (CDI) in Anwesenheit einer Base, um eine Verbindung von Formel (5) zu ergeben:

wobei
die Base 4-Dimethylaminopyridin (DMAP) ist;

(d) Reagieren von Verbindung (5) mit einem Derivat einer Aminosäure oder eines Peptids, um eine Verbindung von Formel (6) zu ergeben:

wobei
[A']-NH- abgeleitet ist von dem Derivat der Aminosäure oder des Peptids; und

(e) Entfernen aller Schutzgruppen, um eine Verbindung von Formel (IB) zu ergeben:

wobei
[A]-NH- die Aminosäure oder das Peptid in dem Derivat einer Aminosäure oder eines Peptids ist.

10. Verfahren zum Herstellen einer Verbindung von Formel (I) nach einem der Ansprüche 1-4, 6, 8,

wobei X und Y unabhängig NH sind, das Verfahren umfassend:

(a) Reagieren von Verbindung (4a) mit Diphenylphosphorylazid (DPPA) und Natriumazid in Anwesenheit einer Base, um eine Verbindung von Formel (18) zu ergeben:

wobei
die Basis 1,8-Diazabicyclo[5.4.0]undec-7-en (DBU) ist;

(b) Reduzieren einer Verbindung (18) durch Hydrogenolyse in Anwesenheit eines Katalysators, gefolgt von einem Koppeln mit Glycinmethylesterhydrochlorid in Anwesenheit eines Promotors, um eine Verbindung von Formel (19) zu ergeben:

wobei
der Katalysator ein Lindlar-Katalysator ist und der Promotor 1,1'-Carbonyldiimidazol ist;

(c) Entfernen aller Schutzgruppen und der von dem Glycinmethylester abgeleiteten Estergruppe, um eine Verbindung von Formel (I-d1) zu ergeben:

11. Verfahren zum Herstellen einer Verbindung von Formel (I) nach einem der Ansprüche 1-4, 6, 8,

wobei X NH ist und Y S ist, das Verfahren umfassend:

(a) Reagieren von Verbindung (4a) mit Methansulfonylchlorid (MsCI) in Anwesenheit einer Base, gefolgt von Reagieren mit Kaliumthioacetat, um eine Verbindung von Formel (21) zu ergeben:

wobei
die Base Triethylamin und 4-Dimethylaminopyridin ist;

(b) Entfernen der Acetylgruppe von Verbindung (21) mit einer Base, gefolgt von einer Kupplung mit 2,2,2-Trichlorethylglycin in Anwesenheit eines Promotors, um eine Verbindung von Formel (22) zu ergeben:

wobei
die Base Kaliumhydroxid ist und der Promotor 1,1'-Carbonyldiimidazol ist;

(c) Entfernen aller Schutzgruppen und der von 2,2,2-Trichlorethylglycin abgeleiteten Estergruppe, um eine Verbindung von Formel (I-e1) zu ergeben:

12. Verbindung von Formel (II)

wobei

k 1, 2 oder 3 ist;

X und Y jeweils unabhängig NH, O oder S sind;

R¹ OH, NH₂, NO₂, Halogen, Alkyl, Haloalkyl, Hydroxyalkyl, unsubstituiertes oder substituiertes Alkoxy, Alkoxyalkyl, Alkenyl oder Alkinyl ist;

R² und R³ jeweils unabhängig voneinander OH, NH2, NO2, Halogenalkyl, Hydroxyalkyl oder Alkylamino sind; und

R⁴ ausgewählt ist aus Glycin, Alanin, Cystein, Valin, Leucin, Isoleucin, Methionin, Phenylalanin, Tryptophan, Glutamin, Asparagin, Threonin, Arginin, Lysin, Prolin, und R⁴ mit C(=O) in der Struktur über die N-terminale Gruppe oder eine Seitenketten-Aminogruppe davon verbunden ist, oder von einem Peptid RPKPQQFFGLM-CONH2 ist und das Peptid mit C(=O) in der Struktur über die Seitenketten-Aminogruppe von K verbunden ist, wobei der C-Terminus der Aminosäure oder des Peptids optional modifiziert ist;

Ring A ein substituierter oder unsubstituierter aromatischer Heterozyklus ist, umfassend einen 5- oder 6-gliedrigen Heterozyklus und einen kondensierten Heterozyklus; das/die Heteroatom(e) in dem Ring ein oder mehrere Stickstoff-, Sauerstoff- und/oder Schwefel-Heteroatom(e) ist/sind; und der/die Substituent(en) ausgewählt ist/sind aus der Gruppe, bestehend aus Fluor, Chlor, Brom, Jod, Hydroxyl, Nitro, Alkyl, einer Trifluormethylgruppe und Kombinationen davon;

ein Tautomer oder Stereoisomer davon; und ein pharmazeutisch annehmbares Salz des Vorstehenden.

13. Verbindung nach Anspruch 12, wobei die Verbindung ausgewählt ist aus der Gruppe bestehend aus:

und

14. Pharmazeutische Zusammensetzung, umfassend:

(a) eine therapeutisch wirksame Menge einer Verbindung nach einem der Ansprüche 1 bis 8, 12 und 13, eines Tautomers oder Stereoisomers davon oder eines pharmazeutisch annehmbaren Salzes des Vorstehenden; und

(b) einen pharmazeutisch annehmbaren Träger, Hilfsstoff oder Vehikel.

15. Verbindung nach einem der Ansprüche 1 bis 9, 12 und 13, ein Tautomer oder Stereoisomer davon oder ein pharmazeutisch annehmbares Salz des Vorstehenden zur Verwendung bei der Behandlung von Schmerzen bei einem Patienten, der diese benötigt, wobei

vorzugsweise steht der Schmerz mit einer Gewebeazidose, einer Nervenverletzung oder beidem assoziiert ist, und/oder

die Schmerzen vorzugsweise dysfunktionale Schmerzen sind, vorzugsweise wobei die dysfunktionalen Schmerzen ausgewählt sind aus der Gruppe, bestehend aus Fibromyalgie, Blasenschmerzsyndrom, durch Reizdarmsyndrom verursachten Schmerzen und Schmerzen, die mit temporomandibulären Störungen assoziiert sind, und/oder

Revendications

1. Composé de formule (I) :

dans lequel,

n vaut 0, 1, 2, ou 3 ;

X est NH, O ou S

Y est O, NH ou S ;

R¹ est OH, NH₂, NO₂, halogène, alkyle, halogénoalkyle, hydroxyalkyle, alcoxy non substitué ou substitué, alcoxyalkyle, alcényle ou alcynyle ;

R² et R³ sont chacun indépendamment OH, NH₂, NO₂, halogénoalkyle, hydroxyalkyle ou alkylamino ; et

R⁴ est choisi parmi glycine, alanine, cystéine, valine, leucine, isoleucine, méthionine, phénylalanine, tryptophane, glutamine, asparagine, thréonine, arginine, lysine, proline, et R⁴ est lié à C(=O) dans la structure via le groupe N-terminal ou un groupe amino de chaîne latérale de celui-ci, ou R⁴ est un peptide RPKPQQFFGLM-CONH₂ et le peptide est lié à C(=O) dans la structure via le groupe amino de chaîne latérale de K, dans lequel l'extrémité C-terminale de l'acide aminé ou du peptide est éventuellement modifiée ;

tautomère ou stéréoisomère de celui-ci ; et sel pharmaceutiquement acceptable de ce qui précède.

2. Composé selon la revendication 1, dans lequel R¹, R², et R³ sont chacun indépendamment OH.

3. Composé selon la revendication 1 ou 2, dans lequel n vaut 1.

4. Composé selon l'une quelconque des revendications 1 à 3, dans lequel X est NH et Y est O, NH ou S.

5. Composé selon l'une quelconque des revendications 1 à 3, dans lequel X est S et Y est NH.

6. Composé selon l'une quelconque des revendications 1 à 5, dans lequel R⁴ est choisi parmi glycine, valine, cystéine, phénylalanine ou glutamine, ou R⁴ est le peptide RPKPQQFFGLM-CONH₂ et le peptide est lié avec C(=O) dans la structure via le groupe amino de chaîne latérale de K.

7. Composé selon l'une quelconque des revendications 1 à 6, dans lequel le composé est de formule (IA):

dans lequel,

R est un substituant existant dans des acides α-aminés communs ; et

Z⁺ est H⁺, Li⁺, Na⁺, K⁺, Rb⁺, Cs⁺, NH₄⁺ ou ion aminium ;

dans lequel,

l'ion aminium est R^AR^BR^CNH⁺ comportant des ions aminium acycliques et cycliques ; et R^A, R^B et R^c sont indépendamment H, alkyle en C₁-C₄ ou benzyle;

tautomère ou stéréoisomère de celui-ci ; et sel pharmaceutiquement acceptable de ce qui précède.

8. Composé selon l'une quelconque des revendications 1 à 7, qui est choisi dans le groupe constitué par :

















et

9. Procédé de préparation d'un composé de formule (I) selon l'une quelconque des revendications 1 à 4, 6 à 8,

dans lequel X est NH et Y est O, le procédé comprenant :

(a) la réaction de N⁶-(4-hydroxybenzyl)adénosine (2) avec du 2,2-diméthoxypropane dans l'acétone en présence d'un acide pour donner un composé de formule (3) :

dans lequel,
l'acide est l'acide p-toluènesulfonique ou l'acide camphorsulfonique ;

(b) la réaction du composé (3) en présence d'une base avec un agent pour donner un composé de formule (4) :

dans lequel,
PG est 4-méthoxybenzyle ou benzoyle ; l'agent est le chlorure de 4-méthoxybenzyle ou le chlorure de benzoyle ; et la base est le carbonate de potassium ou la triéthylamine ;

(c) la réaction du composé (4) avec du 1,1'-carbonyldiimidazole (CDI) en présence d'une base pour donner un composé de formule (5) :

dans lequel,
la base est la 4-diméthylaminopyridine (DMAP) ;

(d) la réaction du composé (5) avec un dérivé d'acide aminé ou de peptide pour donner un composé de formule (6) :

dans lequel,
[A']-NH- est dérivé du dérivé d'acide aminé ou de peptide ; et

(e) l'élimination de tous les groupes protecteurs pour donner un composé de formule (IB):

dans lequel,
[A]-NH- est l'acide aminé ou le peptide dans le dérivé d'un acide aminé ou d'un peptide.

10. Procédé de préparation d'un composé de formule (I) selon l'une quelconque des revendications 1 à 4, 6, 8,

dans lequel X et Y sont indépendamment NH, le procédé comprenant :

(a) la réaction du composé (4a) avec l'azide de diphénylphosphoryle (DPPA) et l'azide de sodium en présence d'une base pour donner un composé de formule (18) :

dans lequel,
la base est le 1,8-diazabicyclo[5.4.0]undéc-7-ène (DBU) ;

(b) la réduction du composé (18) par hydrogénolyse en présence d'un catalyseur, suivie d'un couplage avec du chlorhydrate d'ester méthylique de glycine en présence d'un promoteur, pour donner un composé de formule (19) :

dans lequel,
le catalyseur est le catalyseur de Lindlar et le promoteur est le 1,1'-carbonyldiimidazole ;

(c) l'élimination de tous les groupes protecteurs et du groupe ester dérivé de l'ester méthylique de glycine pour donner un composé de formule (I-d1):

11. Procédé de préparation d'un composé de formule (I) selon l'une quelconque des revendications 1 à 4, 6, 8,

dans lequel X est NH et Y est S, le procédé comprenant :

(a) la réaction du composé (4a) avec du chlorure de méthanesulfonyle (MsCI) en présence d'une base, suivie d'une réaction avec du thioacétate de potassium, pour donner un composé de formule (21) :

dans lequel,
la base est la triéthylamine et la 4-diméthylaminopyridine ;

(b) l'élimination du groupe acétyle du composé (21) avec une base, suivie d'un couplage avec de la 2,2,2-trichloroéthylglycine en présence d'un promoteur, pour donner un composé de formule (22) :

dans lequel,
la base est l'hydroxyde de potassium et le promoteur est le 1,1'-carbonyldiimidazole ;

(c) l'élimination de tous les groupes protecteurs et du groupe ester dérivé de la 2,2,2-trichloroéthylglycine pour donner un composé de formule (l-e1):

12. Composé de formule (II):

dans lequel,

k vaut 1, 2, ou 3 ;

X et Y sont chacun indépendamment NH, O ou S ;

R¹ est OH, NH₂, NO₂, halogène, alkyle, halogénoalkyle, hydroxyalkyle, alcoxy non substitué ou substitué, alcoxyalkyle, alcényle ou alcynyle ;

R² et R³ sont chacun indépendamment OH, NH₂, NO₂, halogénoalkyle, hydroxyalkyle ou alkylamino ; et

R⁴ est choisi parmi glycine, alanine, cystéine, valine, leucine, isoleucine, méthionine, phénylalanine, tryptophane, glutamine, asparagine, thréonine, arginine, lysine, proline, et R⁴ est lié à C(=O) dans la structure via le groupe N-terminal ou un groupe amino de chaîne latérale de celui-ci, ou un peptide RPKPQQFFGLM-CONH₂, et le peptide est lié à C(=O) dans la structure via le groupe amino de chaîne latérale de K, dans lequel l'extrémité C-terminale de l'acide aminé ou du peptide est éventuellement modifiée ;

le cycle A est un hétérocycle aromatique substitué ou non substitué, comprenant un hétérocycle à 5 ou 6 chaînons et un hétérocycle fusionné ; le ou les hétéroatomes dans le cycle sont un ou plusieurs hétéroatomes d'azote, d'oxygène et/ou de soufre ; et le ou les substituants sont choisis dans le groupe constitué par fluor, chlore, brome, iode, hydroxyle, nitro, alkyle, un groupe trifluorométhyle et leurs combinaisons ;

tautomère ou stéréoisomère de celui-ci ; et sel pharmaceutiquement acceptable de ce qui précède.

13. Composé selon la revendication 12 qui est choisi dans le groupe constitué par :

et

14. Composition pharmaceutique comprenant :

(a) une quantité thérapeutiquement efficace d'un composé tel que revendiqué dans l'une quelconque des revendications 1 à 8, 12 et 13, un tautomère ou un stéréoisomère de celui-ci, ou un sel pharmaceutiquement acceptable de ce qui précède ; et

(b) un support, excipient ou véhicule pharmaceutiquement acceptable.

15. Composé selon l'une quelconque des revendications 1 à 9, 12 et 13, tautomère ou stéréoisomère de celui-ci, ou sel pharmaceutiquement acceptable de ce qui précède, pour une utilisation dans le traitement de la douleur chez un sujet qui en éprouve le besoin, dans lequel de préférence, la douleur est associée à une acidose tissulaire, à une lésion nerveuse ou aux deux, et/ou

de préférence, la douleur est une douleur dysfonctionnelle, de préférence dans lequel la douleur dysfonctionnelle est choisie dans le groupe constitué par la fibromyalgie, le syndrome de la douleur vésicale, la douleur causée par le syndrome du côlon irritable et la douleur associée à des troubles temporomandibulaires, et/ou

de préférence, la douleur est choisie dans le groupe constitué par les douleurs inflammatoires, les douleurs associées au cancer, les douleurs thoraciques, les douleurs dorsales, les douleurs cervicales, les douleurs d'épaule, les migraines, les céphalées, les douleurs myofasciales, les douleurs articulaires, les syndromes de douleurs musculaires, les douleurs neuropathiques, les douleurs périphériques, les douleurs sympathiques, les douleurs postopératoires, les douleurs post-traumatiques et les douleurs liées à la sclérose en plaques, et/ou

de préférence dans lequel la douleur est une douleur chronique.

Drawing