Patent Application
20130231307
DNA viruses and retroviruses cause severe diseases spreading among the world population. Current therapies (i.e. HIV treatment) often need permanent drug administration to maintain low or undetectable viral levels in patients. However drug-resistant viruses frequently appear, so new antiviral derivatives are still needed. In the last ten years, acyclic nucleoside phosphonates (ANPs) like adefovir (9-(2-Phosphonylmethoxyethyl)-adenine or PMEA), tenofovir [(1R)-9-(2-Phosphonylmethoxypropyl)-adenine or (R)-PMPA] and cidofovir [(S)-CDV] became major antiviral nucleotide derivatives that are approved for the treatment of severe virus infections. They mimic natural nucleotides (dAMP or dCMP) and thus bypass the first phosphorylation step by a nucleoside kinase. Their activities depend on metabolic conversion by two salvage pathway kinases (NMP and NDP kinases) to their diphosphates (ANP-PPs), followed by a specific interaction with the virus DNA polymerase. ANP-PPs, like the other antiviral nucleotide analogs compete with the endogenous dNTP pools for binding to the virus DNA polymerase and incorporation into the growing virus DNA chain, inhibiting viral (i.e. in HIV or vaccinia) replication. ANPs and their phosphorylated forms are resistant to nucleotidases and have long half-lifes in the body, for example >15H for PMEA-PP. However antiviral treatment involving nucleoside or nucleotide analogs frequently result in the selection of resistant virus strains, with precise point mutations in the DNA polymerase gene. The only acyclic pyrimidine nucleoside phosphonate analogs found to be effective to date is cidofovir (CDV) and the acyclic 2,4-diaminopyrimidine nucleoside phosphonates (i.e. PMEO-DAPym); Balzarini, J et al. Antimicrob. Agents Chemother. 2002, 46, 2185-93; Hocková, D. et al. J. Med. Chem. 2003, 46, 5064-5073.
CDV has a broad antiviral activity and has been approved by the FDA for use against cytomegalovirus infections. The PMEO-DAPym derivatives are currently not subject of clinical trials. The first phosphorylation step of antiviral nucleoside analogs is usually the rate-determining step in the salvage pathway. In a similar way, the efficacy of ANPs should depend on their intracellular phosphorylation and the amounts of their active form, ANP-PP. All studied ANPs are slowly phosphorylated by human NMP kinases: AMP kinases 1 and 2 for PMEA and (R)-PMPA and UMP-CMP kinase (hUCK) for cidofovir. The last phosphorylation step is performed by several enzymes, including NDP kinases and creatine kinases.
WO 2006/137953 discloses only phosphono-Pent-2-En-1-yl Nucleosides under a trans and cis-forms which are useful as antiviral agents. In these compounds the phosphonopentenyl group corresponds to the acyclic sugar moiety and said phosphonopentenyl side chain design was chosen so that the resulting analogs would resemble the corresponding portion of 2′,3′-didehydro-2′,3′-dideoxynucleosides, such as stavudine, 2′,3′-didehydro-2′,3′-dideoxyadenosine, and abacavir. Said compounds mimic the C1′, C2′, C3′, C4′ and C5′ bounds of the ribofuranose moiety or analogs especially with the cis double bound such the one found in abacavir.
The inventors have synthesised several [E]-3-(N1-uracil)-propenyl phosphonic acid corresponding to formula (6a-e)
which were found with affinities for nucleotide kinases that are similar to dUMP and dCMP, the natural substrates, and higher than that of cidofovir which is not a substrate for hUCK (Topalis, D et al. FEBS J. 2007, 274, 3704-14).
But, there is a need for compounds which could be efficient substrates for hUCK, potentially resulting in better antiviral activities.
In course of their work, the inventors have defined the structural requirements for intracellular activation leading to new compounds with better bioavalaibility. They found that by mimicking the chaining of C1′, O, C4′ and C5′ of a ribofuranose structure, the length of the alkyl chain may be reduced. The double bound is preferably in the trans form.
Thus an object of the instant invention is compounds of formula (I)
wherein
According to the invention the term nucleobase stands for natural or non natural purines and pyrimidines bases selected from, but not limited to, the group comprising uracil, thymine, cytosine, guanine, purine, hypoxanthine and adenine and analogs thereof. The term analogs thereof stands for natural derivatives or synthetic derivatives of said nucleobases such as but not limited to dihydrouridine, inosine, hypoxanthine and xanthine or nucleobase optionally substituted by an halogen atom, a (C1-C6)alkyl group or an aryl group, like for example theophylline, theobromine and caffeine.
The term halogen stands for fluorine, chlorine, bromine and iodine.
The term straight or branched (C1-C6)alkyl group stands for a straight-chain or branched hydrocarbon residue containing 1-6 C-atoms, such as, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl, isopentyl, n-hexyl.
The term (C1-C6)alkoxy group stands for alkyl-O— with alkyl as defined above, e.g. methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, isobutoxy, t-butoxy, n-pentoxy, isopentoxy and hexoxy.
The term lipophilic chain or long-chain refers to the cyclic, branched or straight chain chemical groups that when covalently linked to a phosphonic acid to form a phosphonate ester increase oral bioavailability and enhance activity as for instance for some nucleoside phosphonates when compared with the parent nucleoside. These lipophilic groups include, but are not limited to, aryl, alkyl, alkoxyalkyl, and alkylglyceryl (such as hexadecyloxypropyl (HDP)-, octadecyloxyethyl-, oleyloxypropyl-, and oleyloxyethyl-esters).
The term aromatic ring stands for, but is not limited to, aryl, e.g. phenyl, benzyl, naphtyl or indanyl, said aryl group being optionally substituted.
As used herein, pharmaceutically acceptable derivatives of the compounds according to the present invention include salts, esters, enol ethers, enol esters, acetals, ketals, orthoesters, hemiacetals, hemiketals, acids, bases, solvates, hydrates or prodrugs thereof. Such derivatives may be readily prepared by those of skill in this art using known methods for such derivatization. The compounds produced may be administered to animals or humans without substantial toxic effects and either are pharmaceutically active or are prodrugs.
Pharmaceutically acceptable salts include, but are not limited to, amine salts, such as but not limited to N,N′-dibenzylethylenediamine, chloroprocaine, choline, ammonia, diethanolamine and other hydroxyalkylamines, ethylenediamine, N-methylglucamine, procaine, N-benzylphenethylamine, 1-para-chlorobenzyl-2-pyrrolidin-1′-ylmethyl-benzimidazole, diethylamine and other alkylamines, piperazine and tris(hydroxymethyl)aminomethane; alkali metal salts, such as but not limited to lithium, potassium and sodium; alkali earth metal salts, such as but not limited to barium, calcium and magnesium; transition metal salts, such as but not limited to zinc; and other metal salts, such as but not limited to sodium hydrogen phosphate and disodium phosphate; and also including, but not limited to, nitrates, borates, methanesulfonates, benzenesulfonates, toluenesulfonates, salts of mineral acids, such as but not limited to hydrochlorides, hydrobromides, hydroiodides and sulfates; and salts of organic acids, such as but not limited to acetates, trifluoroacetates, maleates, oxalates, lactates, malates, tartrates, citrates, benzoates, salicylates, ascorbates, succinates, butyrates, valerates and fumarates. Pharmaceutically acceptable esters include, but are not limited to, alkyl, alkenyl, alkynyl, and cycloalkyl esters of acidic groups, including, but not limited to, carboxylic acids, phosphoric acids, phosphinic acids, sulfonic acids, sulfinic acids and boronic acids. Pharmaceutically acceptable enol ethers include, but are not limited to, derivatives of formula C═C(OR) where R is hydrogen, alkyl, alkenyl, alkynyl, and cycloalkyl. Pharmaceutically acceptable enol esters include, but are not limited to, derivatives of formula C═C(OC(O)R) where R is hydrogen, alkyl, alkenyl, alkynyl, or cycloalkyl. Pharmaceutically acceptable solvates and hydrates are complexes of a compound with one or more solvent or water molecules, or 1 to about 100, or 1 to about 10, or one to about 2, 3 or 4, solvent or water molecules.
According to the invention the compounds of formula (I) may be used in the treatment of virus pathologies, for example, but not limited to, pathologies due to Herpes virus, Vaccinia virus, Varicella-zoster virus, Cytomegalovirus, Vesicular stomatis virus, Influenza A, Coxsackie virus B4, respiratory syncytial virus, Feline corona virus, Feline herpes virus, Punta Toro Virus, HIV, Hepatitis B and Hepatitis C.
In an advantageous embodiment according to the invention the Varicella-zoster virus does not necessarily need to show any encoded thymidine kinase activity (so called VZV−TK(−))
Another object of the invention is compounds of formula (I) wherein A is selected from a natural or non natural pyrimidine and purine bases said base being selected from the group comprising:
a)
wherein U represents a nitrogen atom or C—R11 with R11 selected from the group comprising
b)
wherein U represents a nitrogen atom or C—R12 with R12 selected from the group comprising
c)
wherein X represents an oxygen or a sulphur atom and R13 represents a group selected from
d)
wherein
V, W, X, Y, Z represents each independently of the other C or N, and in particular wherein W, X and Y or W, Y and V or W and Y are N
e)
wherein
W, X, Y, Z represents each independently of the other C or N,
means a single or double bond according to the meaning of W, X, Y, Z and R15,
R15 is selected from the group comprising
groups with R representing a straight or branched (C1-C4)alkyl group, preferably an isopropyl group and n being an integer equal to 0, 1, 2 or 3,
R16 is a hydrogen atom or an amino group,
R17 is selected from the group comprising a hydrogen atom, a halogen atom, (C1-C4)alkyl group and a phenyl group and
R18 is selected from the group comprising a hydrogen atom, a halogen atom, (C1-C4)alkyl groups, ester groups and aromatic groups for their use as antiviral agents, for the treatment of virus pathologies.
Another object of the invention are compounds of formula (I) wherein R′ and R″ independently from each other are selected from the group comprising O-methyl, O-benzyl, or a bio labile group such as an oxymethylcarbonyl group (such as OPOM, OPOC) or an alcoxyalkyl ester prodrugs (such as OHDP), for their use as antiviral agents, for the treatment of virus pathologies. POM states for pivaloyl oxymethyl [(CH3)3C—CO—O—CH2—], POC for isopropyloxymethylcarbonate [(CH3)2—C—O—C(O)—O—CH2—], and HDP for hexadecyloxypropyl [CH3 (CH2)15—O—(CH2)3—].
Another object of the invention is compounds of formula (I) wherein n is equal to 1, for their use as antiviral agents, for the treatment of virus pathologies.
Another object of the invention are compounds of formula (I) wherein:
R10 is a hydrogen atom,
n is equal to 1,
R′ and R″ represent each independently OH, OPOC, OPOM and OHDP, with the proviso that they are never simultaneously OH, and
A is as defined above.
In a preferred embodiment of the invention, the compounds are those of formula (I′).
wherein
with R11 being H, F, Cl, Br, CH3 or a group
with R13 selected from
O, Cl, OCH3, NH2 and a group
with R representing a straight or branched (C1-C4)alkyl group, preferably an isopropyl group and n being an integer equal to 0, 1, 2 or 3,
R15 being H and R14 being H or NH2,
c)
wherein X represents an oxygen atom and R12 represents a group selected from
Another object of the invention is compounds of formula (I) wherein R10 is H or of formula R′, which are under the E form, for their use as antiviral agents, for the treatment of virus pathologies.
for their use as antiviral agents, for the treatment of virus pathologies.
Another object of the invention is compounds of formula (I) selected from the group comprising:
for their use as antiviral agent, for the treatment of virus pathologies.
The compounds used according to the invention may be prepared by any methods known in the art or described in the literature from compounds disclosed in the literature or commercially available.
For example uracil phosphonates derivatives may be prepared according to procedures previously described (Kumamoto, H.; Broggi, J.; Topalis, D.; Pradere, U.; Roy, V.; Berteina-Raboin, S.; Nolan, S. P.; Deville-Bonne, D.; Snoeck, R.; Garin, D.; Agrofoglio, L. Preparation of acyclo nucleoside phosphonate analogues based-on cross-metathesis. Tetrahedron 2008, 64, 3517-3526; Roy, V.; Kumamoto, H.; Berteina-Raboin, S.; Nolan, S. P.; Topalis, D.; Deville-Bonne, D.; Balzarini, J.; Neyts, J.; Andrei, G.; Snoeck, R.; Agrofoglio, L. A. Cross-metathesis mediated synthesis of new acyclic nucleoside phosphonates. Nucleosides Nucleotides Nucleic Acids 2007, 26, 1399-1402).
Another object of the invention is pharmaceutical compositions comprising at least one compound of formula (I) in association with a pharmaceutically acceptable carrier. Pharmaceutical carriers suitable for administration of the compounds provided herein include any such carriers known to those skilled in the art to be suitable for the particular mode of administration.
According to the invention, the pharmaceutical composition may further comprise an other active ingredients like for example antiviral compounds selected from, but not limited to, the group comprising Abacavir, Acyclovir, Adefovir, Amprenavir, Atazanavir, Cidofovir, Darunavir, Delavirdine, Didanosine, Docosanol, Éfavirenz, Elvitégravir, Emtricitabine, Enfuvirtide, Étravirine, Famciclovir, Foscarnet, Fomivirsen, Ganciclovir, Indinavir, Idoxuridine, Lamivudine, Lopinavir Maraviroc, MK-2048, Nelfinavir, Névirapine, Penciclovir, Raltégravir, Rilpivirine, Ritonavir, Saquinavir, Stavudine, Ténofovir Trifluridine, Valaciclovir, Valganciclovir, Vidarabine, Ibacitabine, Amantadine, Oseltamivir, Rimantidine, Tipranavir, Zalcitabine, Zanamivir et Zidovudine.
The pharmaceutical compositions contain one or more compounds and the compounds may be formulated into suitable pharmaceutical preparations such as solutions, suspensions, tablets, dispersible tablets, pills, capsules, powders, sustained release formulations or elixirs, for oral administration or in sterile solutions or suspensions for parenteral administration, as well as transdermal patch preparation and dry powder inhalers. All the techniques and procedures used are well known in the art.
The active compound is included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the patient treated. The therapeutically effective concentration may be determined empirically by testing the compounds in vitro and in vivo systems well known to those of skill in the art and then extrapolated there from for dosages for humans.
The concentration of active compound in the pharmaceutical composition will depend on absorption, inactivation and excretion rates of the active compound, the physicochemical characteristics of the compound, the dosage schedule, and amount administered as well as other factors known to those of skill in the art.
In one embodiment, a therapeutically effective dosage should produce a serum concentration of active ingredient of from about 0.1 ng/ml to about 50-100 μg/ml. The pharmaceutical compositions, in another embodiment, should provide a dosage of from about 0.001 mg to about 2000 mg of compound per kilogram of body weight per day. Pharmaceutical dosage unit forms are prepared to provide from about 0.01 mg, 0.1 mg or 1 mg to about 500 mg, 1000 mg or 2000 mg, and in one embodiment from about 10 mg to about 500 mg of the active ingredient or a combination of essential ingredients per dosage unit form. The active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at intervals of time. It is understood that the precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.
Another object according to the invention is pharmaceutical compositions comprising at least one compound of formula (I) for their use as antiviral agent.
Still another object of the invention is the use of at least one compound of formula (I) for the preparation of a medicament useful as antiviral agent.
Finally another object of the invention is a method for treating virus pathologies, for example, but not limited to, pathologies due to Herpes virus, Vaccinia virus, Varicella-zoster virus, Cytomegalovirus, Vesicular stomatis virus, Influenza A, Coxsackie virus B4, respiratory syncytial virus, Feline corona virus, Feline herpes virus, Punta Toro Virus, HIV, Hepatitis B and Hepatitis C, said method comprising a step of administering in a patient in need thereof a therapeutically effective amount of at least one compound of formula (I).
The examples 1 to 4 and
The synthesis is illustrated in the following scheme
To a CH2Cl2 (25 mL/mmol) solution of N1-crotyl-5-substituted uracil (1 equiv.) and bis(POM), bis(POC) or (HDP/POC) allylphosphonate (1.3 equiv.) was added metathesis catalyst (0.05 equiv.) then this solution was stirred under positive pressure of dry air. After evaporation of all volatiles, the residue was purified by silica gel column chromatography to give the desired products.
According to said procedure, the following compounds are obtained and characterised
IR: 2977, 1751, 1685, 1459, 1242, 1138, 956, 855 cm−1.
1H NMR (400 MHz, CDCl3) δ 8.73 (s, 1H, NH), 7.17 (d, J=7.9 Hz, 1H, H6), 5.75-5.61 (m, 7H, O—CH2—O, H2′, H3′, H5), 4.32 (t, J=4.1 Hz, 2H, H1′), 2.72 (dd, J=22.4, 5.0 Hz, 2H, H4′), 1.23 (s, 18H, tBu).
13C NMR (100 MHz, CDCl3) δ 176.8 (C═O), 163.2 (C═O), 150.5 (C═O), 143.4 (C6), 129.5, 129.3 (C2), 124.1, 124.0 (C3′), 102.6 (C5), 81.6, 81.5 (O—CH2—O), 49.1, 49.0 (C1′), 38.7 (C(CH3)3), 31.5, 30.1 (C4′), 26.8 (C(CH3)3).
31P NMR (162 MHz, CDCl3) δ 26.4.
HRMS (ESI): m/z [M+Na]+ calcd for C20H31N2NaO9P: 497.1665, found: 497.1674.
1.2. (Z)—N1-(4′-bis(POM)-phosphinyl-2′-butenyOuracil (Ia2)
1H NMR (400 MHz, CDCl3) δ 8.32 (s, 1H, NH), 7.47 (d, J=7.9 Hz, 1H, H6), 5.77-5.60 (m, 7H, O—CH2—O, H2′, H3′, H5), 4.44 (t, J=4.7 Hz, 2H, H″), 2.82 (dd, J=23.4, 6.7 Hz, 2H, H4′), 1.24 (s, 18H, tBu).
13C NMR (100 MHz, CDCl3) δ 177.0 (C═O), 163.2 (C═O), 150.6 (C═O), 144.4 (C6), 129.1, 128.9 (C2′), 122.1, 122.0 (C3′), 102.5 (C5), 81.6, 81.5 (O—CH2—O), 44.8, 44.7 (C1′), 38.8 (C(CH3)3), 27.0 and 25.6 (C4′), 26.9 (C(CH3)3).
31P NMR (162 MHz, CDCl3) δ 26.6.
HRMS (ESI): m/z [M+Na]+ calcd for C20H31N2NaO9P: 497.1665, found: 497.1677.
IR: 2977, 2361, 1752, 1702, 1480, 1238, 1137, 965, 871 cm−1.
1H NMR (400 MHz, CDCl3) δ 9.58 (s, 1H, NH), 7.28 (d, J=5.5 Hz, 1H, H6), 5.77-5.61 (m, 6H, O—CH2—O, H2′, HY), 4.31 (t, J=4.9 Hz, 2H, H″), 2.72 (dd, J=22.6, 5.3 Hz, 2H, H4′), 1.22 (s, 18H, tBu).
13C NMR (100 MHz, CDCl3) δ 176.8 (C═O), 157.2, 156.9 (C═O), 149.3 (C═O), 141.7, 139.3 (C5), 129.1, 128.9 (C2′), 127.7, 127.4 (C6), 124.7, 124.6 (C3′), 81.6, 81.5 (O—CH2—O), 49.3 (2C, C1′), 38.6 (C(CH3)3), 31.4, 30.0 (C4′), 26.7 (C(CH3)3).
31P NMR (162 MHz, CDCl3) δ 26.3.
HRMS (ESI): m/z [M+Na]+ calcd for C20H30N2NaO9FP: 515.1571, found: 515.1579.
1H NMR (400 MHz, CDCl3) δ 8.59 (s, 1H, NH), 7.73 (d, J=5.8 Hz, 1H, H6), 5.75-5.61 (m, 6H, O—CH2—O, H2′, H3′), 4.44 (t, J=4.7 Hz, 2H, H″), 2.80 (dd, J=23.5, 6.8 Hz, 2H, H4′), 1.23 (s, 18H, tBu).
13C NMR (100 MHz, CDCl3) δ 177.0 (C═O), 157.0, 156.7 (C═O), 149.2 (C═O), 141.7, 139.4 (C5), 128.9, 128.7, 128.6 (2C, C2′, and C6), 122.6, 122.5 (C3′), 81.7, 81.6 (O—CH2—O), 44.9 (2C, C1′), 38.8 (C(CH3)3), 26.9 and 25.5 (C4′), 26.8 (C(CH3)3).
31P NMR (162 MHz, CDCl3 δ 26.4.
HRMS (ESI): m/z [M+Na]+ calcd for C20H30N2NaO9FP: 515.1571, found: 515.1584.
IR: 2929, 1678, 1453, 1396, 1196, 986, 751 cm−1.
1H NMR (400 MHz, CD3OD) δ 7.37 (d, J=0.9 Hz, 1H, H6), 5.85-5.74 (ttd, J=15.4, 11.2, 5.1 Hz, H2′), 5.66 (m, 5H, O—CH2—O, H3′), 4.32 (t, J=5.2 Hz, 2H, H″), 2.82 (dd, J=22.5, 7.2 Hz, 2H, H4′), 1.87 (s, 3H, CH3—U) 1.23 (s, 18H, tBu).
13C NMR (100 MHz, CDCl3) δ 178.1 (C═O), 166.8 (C═O), 152.7 (C═O), 142.4 (C6), 131.8, 131.6 (C2′), 123.7, 123.6 (C3′), 111.5 (C5), 83.2, 83.1 (O—CH2—O), 49.9 (2C, C1′), 39.7 (C(CH3)3), 31.8, 30.4 (C4′), 27.2 (C(CH3)3), 12.3 (CH3—U).
31P NMR (162 MHz, CD3OD) δ 27.3.
HRMS (ESI): m/z [M+Na]+ calcd for C21H33N2NaO9P: 511.1821, found: 511.1837.
1H NMR (400 MHz, CD3OD) δ 7.46 (d, J=1.2 Hz, 1H, H6), 5.78-5.60 (m, 6H, O—CH2—O, H2′, H3′), 4.40 (dd, J=5.5, 3.8 Hz, 2H, H1′), 3.01 (dd, J=23.2, 7.8 Hz, 2H, H4′), 1.88 (d, J=1.1 Hz, 3H, CH3—U), 1.24 (d, J=3.2 Hz, 18H, tBu).
13C NMR (100 MHz, CD3OD) δ 178.2 (C═O), 166.9 (C═O), 152.9 (C═O), 142.7 (C6), 130.6, 130.4 (C2′), 123.0, 122.8 (C3′), 111.5 (C5), 83.2, 83.1 (O—CH2—O), 45.6 (C1′), 39.8 (C(CH3)3), 27.7 and 26.3 (C4′), 27.3 (C(CH3)3), 12.3 (CH3—U).
31P NMR (162 MHz, CD3OD) δ 27.6.
HRMS (ESI): m/z [M+Na]+ calcd for C21H33N2NaO9P: 511.1821, found: 511.1835.
IR: 2976, 1752, 1692, 1452, 1236, 1135, 963, 853 cm−1.
1H NMR (400 MHz, CDCl3) δ 8.73 (s, 1H, NH), 7.42 (s, 1H, H6), 5.80-5.60 (m, 6H, O—CH2—O, H2′, H3′), 4.33 (t, J=4.5 Hz, 2H, H1′), 2.72 (dd, J=22.6, 5.7 Hz, 2H, H4′), 1.23 (s, 18H, tBu).
13C NMR (100 MHz, CDCl3) δ 176.8 (C═O), 158.8 (C═O), 149.4 (C═O), 140.3 (C6), 129.0, 128.8 (C2′), 125.0, 124.9 (C3′), 109.0 (C5), 81.6 (2C, O—CH2—O), 49.5 (2C, C1′), 38.7 (C(CH3)3), 31.5, 30.1 (C4′), 26.8 (C(CH3)3).
31P NMR (162 MHz, CDCl3) δ 26.1.
HRMS (ESI): m/z [M+Na]+ calcd for C20H30N2NaO9ClP: 531.1275, found: 531.1293.
1H NMR (400 MHz, CDCl3) δ 8.48 (s, 1H, NH), 7.79 (s, 1H, H6), 5.74-5.62 (m, 6H, O—CH2—O, H2′, H3′), 4.47 (t, J=4.6 Hz, 2H, H″), 2.82 (dd, J=23.5, 6.9 Hz, 2H, H4′), 1.24 (s, 18H).
13C NMR (100 MHz, CDCl3) δ 177.0 (C═O), 158.9 (C═O), 149.7 (C═O), 141.3 (C6), 128.6, 128.4 (C2′), 122.7, 122.6 (C3′), 108.9 (C5), 81.7, 81.6 (O—CH2—O), 45.1 (2C, C1′), 38.8 (C(CH3)3), 27.0 and 25.6 (C4′), 26.8 (C(CH3)3).
31P NMR (162 MHz, CDCl3) δ 26.4
HRMS (ESI): m/z [M+Na]+ calcd for C20H30N2NaO9ClP: 531.1275, found: 531.1276.
IR: 2976, 2361, 1751, 1693, 1441, 1235, 1137, 965, 854, 768 cm−1.
1H NMR (400 MHz, CD3OD) δ 7.98 (s, 1H, H6), 5.85-5.76 (m, 1H, H2′), 5.75-5.62 (m, 5H, O—CH2-0, H3′), 4.36 (t, J=5.1 Hz, 2H, H″), 2.82 (dd, J=22.5, 6.9 Hz, 2H, H4′), 1.23 (s, 18H, tBu).
13C NMR (100 MHz, CD3OD) δ 178.1 (C═O), 162.1 (C═O), 152.0 (C═O), 146.2 (C6), 131.4, 131.3 (C2′), 124.5, 124.4 (C3′), 96.8 (C5), 83.3, 83.2 (O—CH2—O), 50.6, 50.5 (C1′), 39.8 (C(CH3)3), 31.9, 30.5 (C4′), 27.2 (C(CH3)3).
31P NMR (162 MHz, CD3OD) δ 27.1.
HRMS (ESI): m/z [M+Na]+ calcd for C20H30N2NaO9BrP: 575.0770, found: 575.0764.
1H NMR (400 MHz, CD3OD) δ 8.06 (s, 1H, H6), 5.70-5.52 (m, 6H, O—CH2—O, H2′, HY), 4.45 (dd, J=6.8, 3.7 Hz, 2H, H″), 3.01 (dd, J=23.4, 7.8 Hz, 2H, H4′), 1.24 (s, 18H, tBu).
13C NMR (100 MHz, CD3OD) δ 178.2 (C═O), 162.2 (C═O), 152.1 (C═O), 146.3 (C6), 130.1, 129.9 (C2′), 123.5, 123.4 (C3′), 96.7 (C5), 83.2, 83.1 (O—CH2—O), 46.2 (2C, C1′), 39.8 (C(CH3)3), 27.7 and 26.3 (C4′), 27.3 (C(CH3)3).
31P NMR (100 MHz, CD3OD) δ 27.5.
HRMS (ESI): m/z [M+Na]+ calcd for C20H30N2NaO9BrP: 575.0770, found: 575.0778.
NMR 1H (400 MHz, CDCl3) δ 8.35 (s, 1H, HNH), 7.38 (s, 1H, H6), 7.33 (s, 1H, HCHI), 5.79-5.61 (m, 6H, HO—CH2-O, H2′ and H3′), 4.95 (sept, J=6.3 Hz, 2H, HCHisopropyl) 4.38 (t, J=4.6 Hz, 2H, H1′), 2.78 (dd, J=23.1, 5.5 Hz, 2H, H4′), 1.33 (d, J=6.3 Hz, 12H, HCH3isopropyl)
NMR 13C (100 MHz, CDCl3) δ 159.1, 153.0, 149.7, 148.7, 140.4, 129.3, 129.2, 124.5, 124.4, 109.0, 116.58, 85.4, 84.2, 84.1, 73.4, 49.4, 31.3, 29.9, 21.6, 21.5
IR (cm−1): 2985, 1756, 1679, 1467, 1257, 1152, 1101, 982, 950, 831, 788
NMR 31P (400 MHz, CDCl3) δ 26.70
HRMS (M+Na): found 778.9355 calculated for C20H27N2O11PNa 778.9340
NMR 1H (400 MHz, CDCl3) δ 8.26 (s, 1H, HNH), 7.42 (s, 1H, H6), 7.15 (s, 1H, H═CHI), 5.85-5.61 (m, 6H, HO—CH2-O, H2′ and H3′), 4.95 (sept, J=6.3 Hz, 2H, HCHisopropyl), 4.42 (t, J=4.6 Hz, 2H, H1′), 2.76 (dd, J=23.1, 5.5 Hz, 2H, H4′), 1.36 (d, J=6.3 Hz, 12H, HCH3isopropyl)
NMR 13C (100 MHz, CDCl3) δ 159.2, 153.2, 149.7, 148.7, 140.4, 129.3, 129.2, 124.5, 124.4, 109.0, 116.58, 85.4, 84.2, 84.1, 73.4, 49.4, 31.3, 29.9, 21.6, 21.5
NMR 31P (162 MHz, CDCl3) δ 26.80
IR (cm−1): 987, 1756, 1694, 1468, 1259, 1152, 1101, 981, 949, 870, 831, 787
HRMS (M+Na): found 732.2228 calculated for C20H27N2O11PNaIBr 732.2230
NMR 1H (400 MHz, CDCl3) δ 8.63 (s, 1H, HNH), 7.45 (s, 1H, H6), 6.91 (s, 1H, H═CHI), 5.83-5.59 (m, 6H, HO—CH2-O), H2′, H3′), 4.96 (sept, J=6.3 Hz, 2H, HCHisopropyl), 4.41 (t, J=4.6 Hz, 2H, H1′), 2.80 (dd, J=23.2, 5.5 Hz, 2H, H4′), 1.35 (d, J=6.3 Hz, 12H, HCH3isopropyl),
NMR 13C (100 MHz, CDCl3) δ 159.3, 152.9, 149.7, 148.9, 140.4, 129.3, 129.0, 124.5, 124.3, 109.0, 116.58, 85.5, 84.7, 84.1, 73.6, 49.4, 31.3, 29.9, 21.4, 21.3
NMR 31P (162 MHz, CDCl3) δ 26.70
IR (cm−1): 2986, 1756, 1686, 1451, 1348, 1258, 1152, 1186, 981, 949, 903, 869, 831, 787
HRMS (M+H): found 665.0179 calculated for C20H28N2O11PClI 665.0164
NMR 1H (400 MHz, CDCl3 δ 8.54 (s, 1H, HNH), 7.40 (s, 1H, H6), 6.86 (s, 1H, H═CHBr), 5.79-5.61 (m, 6H, HO—CH2-O, H2′, H3+), 4.93 (sept, J=6.3 Hz, 2H, HCHisopropyl), 4.39 (t, J=4.6 Hz, 2H, H1′), 2.77 (dd, J=23.0, 5.6 Hz, 2H, H4′), 1.32 (d, J=6.3 Hz, 12H, HCH3isopropyl)
NMR 13C (100 MHz, CDCl3) δ 159.1, 153.0, 149.7, 148.7, 140.4, 129.3, 129.2, 124.5, 124.4, 109.0, 116.58, 85.4, 84.2, 84.1, 73.4, 49.4, 31.3, 29.9, 21.6, 21.5
IR (cm−1): 2986, 1756, 1691, 1442, 1347, 1260, 1153, 1186, 1029, 983, 951, 871, 832, 789
NMR 31P (162 MHz, CDCl3 δ 26.80
HRMS (M+Na): found 685.7984 calculated for C20H27N2O11PNaBr2 685.7988
The synthesis is illustrated in the following scheme
According to said procedure, the following compounds are obtained and characterised
To a dioxane solution (1.5 mL) of bis(POM)-1-hydroxymethyl-allylphosphonate (0.131 mmol), adenine (0.326 mmol), and triphenylphosphine (0.326 mmol) under argon was then added diisopropylazodicarboxylate (0.326 mmol). After 20 h stirring, volatiles were evaporated, and residue was purified by silica gel column chromatography to give desired compound (0.074 mmol, 57%).
1H NMR (400 MHz, CDCl3): δ 8.36 (s, 1H, H2), 7.81 (s, 1H, H8), 5.95-5.85 (m, 1H, H2′), 5.74-5.60 (m, 7H, H3′, O—CH2—O, NH2), 4.80 (t, J=5.0 Hz, 2H, H1′), 2.72 (dd, J=22.6, 7.3 Hz, 2H, H4′), 1.23 (s, 18H, C(CH3)3).
13C NMR (100 MHz, CDCl3): δ 176.8 (C═O), 155.4 (C6), 153.1 (C2), 149.9 (C4), 140.1 (C8), 130.0, 129.8 (C2′), 123.4, 123.2 (C3′), 119.6 (C5), 81.6 (2C, O—CH2—O), 44.9 (2C, C1′), 38.7 (C(CH3)3), 31.5, 30.1 (C4′), 26.8 (C(CH3)3). 31P NMR (162 MHz, CDCl3): δ 26.50
HRMS (ESI): m/z [M+H]+ calcd for C21H33N5O7P: 498.2118, found: 498.2127.
1H NMR (400 MHz, CDCl3): δ 8.73 (s, 1H, H2), 8.14 (s, 1H, H8), 5.94-5.85 (m, 1H, H2′), 5.80-5.71 (m, 1H, H3′), 5.71-5.67 (m, 4H, O—CH2—O), 4.88 (t, J=5.2 Hz, 2H, H″), 2.71 (dd, J=22.8, 7.1 Hz, 2H, H4′), 1.21 (s, 18H, C(CH3)3).
13C NMR (100 MHz, CDCl3): δ 176.8 (C═O), 152.0 (C2), 151.6, 151.1 (C4 and C6), 144.7 (C8), 131.6 (C5), 128.9, 128.7 (C2′), 124.6, 124.5 (C3′), 81.6, 81.5 (O—CH2—O), 45.5, 45.4 (C1′), 38.7 (C(CH3)3), 31.4, 30.0 (C4′), 26.8 (C(CH3)3). 31P NMR (162 MHz, CDCl3): δ 26.05.
HRMS (ESI): m/z [M+H]+ calcd for C21H30N4O7PCl: 539.1438, found: 539.1449.
1H NMR (400 MHz, CDCl3): δ 7.74 (s, 1H, H8), 5.88-5.79 (m, 1H, H2′), 5.72-5.59 (m, 5H, H3′, O—CH2—O), 5.28 (s, 2H, NH2), 4.65 (t, J=5.1 Hz, 2H, H″), 2.70 (dd, J=22.7, 7.2 Hz, 2H, H4′), 1.20 (s, 18H, C(CH3)3).
13C NMR (100 MHz, CDCl3): δ 176.8 (C═O), 159.1 (C6), 153.6 (C4), 151.3 (C2), 141.8 (C8), 129.4, 129.3 (C2′), 125.1 (C5), 123.6, 123.5 (C3′), 81.6, 81.5 (O—CH2—O), 44.9 (C1′), 38.7 (C(CH3)3), 31.4, 30.0 (C4′), 26.8 (C(CH3)3). 31P NMR (162 MHz, CDCl3): δ 26.35.
HRMS (ESI): m/z [M+Na]+ calcd for C21H31N5O7NaPCl: 554.1547, found: 554.1566.
2.4. N9-(4′-bis(POM)-phosphinyl-2′-butenyl)-6-cyclopropylaminopurine (Iy)
A solution of compound Iw (0.046 mmol) in a mixture of cyclopropylamine (0.2 mL) and dichloromethane (2 mL) was stirred for 20 h. After evaporation of all volatiles, the residue was purified by silica gel column chromatography to give desired compound (0.038 mmol, 82%).
1H NMR (400 MHz, CDCl3): δ 8.47 (s, 1H, H2), 7.75 (s, 1H, H8), 5.95 (s, 1H, NH), 5.93-5.84 (m, 1H, H2′), 5.69-5.61 (m, 5H, H3′, O—CH2—O), 4.78 (t, J=5.1 Hz, 2H, H″), 3.04 (d, J=3.0 Hz, 1H, NHCH), 2.70 (dd, J=22.6, 7.3 Hz, 2H, H′1′), 1.21 (s, 18H, C(CH3)3), 0.94 (td, J=8.4, 6.9 Hz, 2H, NHCHCH2), 0.68-0.64 (m, 2H, NHCHCH2).
13C NMR (100 MHz, CDCl3): δ 176.8 (C═O), 155.8 (C6), 153.3 (C2), 149.0 (C4), 139.5 (C8), 130.1, 129.9 (C2′), 123.2, 123.0 (C3′), 119.8 (C5), 81.6, 81.5 (O—CH2—O), 44.8 (2C, C1′), 38.7 (C(CH3)3), 31.5, 30.1 (C4′), 26.8 (C(CH3)3), 23.7 (NHCH), 7.4 (NHCHCH2).
31P NMR (162 MHz, CDCl3): δ26.57.
HRMS (ESI): m/z [M+H]+ calcd for C24H37N5O7P: 538.2431, found: 538.2430.
A solution of compound Ix (0.037 mmol) in mixture of cyclopropylamine (0.2 mL) and dichloromethane (2 mL) was stirred for 20 h. After evaporation of all volatiles, the residue was purified by silica gel column chromatography to give desired compound (0.029 mmol, 77%).
1H NMR (400 MHz, CDCl3): δ 7.44 (s, 1H, H2), 5.89-5.81 (m, 1H, H2′), 5.76 (s, 1H, NH), 5.67-5.57 (m, 5H, H3′, O—CH2—O), 4.79 (s, 2H, NH2), 4.61 (t, J=5.1 Hz, 2H, HY), 3.05-2.95 (m, 1H, NHCH), 2.69 (dd, J=23.0, 7.0 Hz, 2H, H4′), 1.22 (s, 18H, C(CH3)3), 0.85 (td, J=6.9, 5.4 Hz, 2H, NHCHCH2), 0.63-0.58 (m, 2H, NHCHCH2).
13C NMR (100 MHz, CDCl3): δ 176.8 (C═O), 160.1, 156.3 (C6 and C2), 151.0 (C4), 136.9 (C8), 130.6, 130.4 (C2′), 122.4, 122.3 (C3′), 114.6 (C5), 81.6 (2C, O—CH2—O), 44.4, 44.3 (2C, C″), 38.7 (C(CH3)3), 31.4, 30.0 (C4′), 26.8 (C(CH3)3), 23.7 (NHCH), 7.4 (NHCHCH2). 31P NMR (162 MHz, CDCl3): δ 26.77.
HRMS (ESI): m/z [M+H]+ calcd for C24H38N6O7P: 553.2531, found: 553.2540.
2.6. N9-(4′-bis(POM)-phosphinyl-2′-butenyphypoxanthine (Iaa)
A solution of compound by (0.046 mmol) in a mixture of water (0.75 mL) and formic acid (0.75 mL) was stirred for 20 h. After evaporation of all volatiles, the residue was purified by silica gel column chromatography to give desired compound (0.040 mmol, 86%).
1H NMR (400 MHz, CDCl3): δ 12.94 (s, 1H, NH), 8.17 (s, 1H, H2), 7.81 (s, 1H, H8), 5.94-5.83 (m, 1H, H2′), 5.78-5.60 (m, 5H, H3′, O—CH2—O), 4.78 (t, J=5.2 Hz, 2H, H″), 2.72 (dd, J=22.7, 7.2 Hz, 2H, H4′), 1.21 (s, 18H, C(CH3)3).
13C NMR (100 MHz, CDCl3): δ 176.8 (C═O), 159.1 (C6), 148.9 (C4), 145.0 (C2), 139.7 (C8), 129.7, 129.5 (C2′), 124.5 (C5), 123.8, 123.7 (C3′), 81.6 (2C, O—CH2—O), 45.3 (2C, CY), 38.7 (C(CH3)3), 31.4, 30.0 (C4′), 26.8 (C(CH3)3). 31P NMR (162 MHz, CDCl3): δ 26.40.
HRMS (ESI): m/z [M+Na]+ calcd for C24H26N4O8Na: 521.1648, found: 521.1625.
A solution of compound Ix (0.041 mmol) in a (1:1) mixture of water (0.75 mL) and formic acid (0.75 mL) was stirred for 20 h. After evaporation of all volatiles, the residue was purified by silica gel column chromatography to give desired compound (0.035 mmol, 86%).
1H NMR (400 MHz, CDCl3): δ 12.11 (s, 1H, NH), 7.60 (s, 1H, H8), 6.65 (s, 2H, NH2), 5.95-5.84 (m, 1H, H1′), 5.73-5.62 (m, 5H, H3′, O—CH2—O), 4.60 (t, J=4.7 Hz, 2H, H″), 2.72 (dd, J=22.6, 7.3 Hz, 2H), 1.20 (s, 18H, C(CH3)3).
13C NMR (100 MHz, CDCl3): δ 176.9 (C═O), 159.0 (C6), 153.9 (C2), 151.4 (C4), 137.2 (C8), 130.6, 130.4 (C2′), 122.7, 122.6 (C3′), 116.8 (C5), 81.7, 81.6 (O—CH2—O), 44.8 (C1′), 38.7 (C(CH3)3), 31.4, 30.0 (C4′), 26.8 (C(CH3)3).
31P NMR (100 MHz, CDCl3): δ 27.04.
HRMS (ESI): m/z [M+Na]+ calcd for C21H32N5O8NaP: 536.1886, found: 536.1890.
From 6-cyclopropylaminopurine by using general procedure, compound (Ia2) was obtained as a colourless oil (52%).
NMR 1H (400 MHz, CDCl3) δ 8.39 (s, 1H, H2), 7.75 (s, 1H, H8), 6.10-5.76 (m, 1H, H2′), 5.75-5.54 (m, 5H, H3′, HO—CH2-O), 5.25 (ddd, J=13.7, 11.6, 1.4 Hz, 2H, HC3), 4.78 (t, J=5.0 Hz, 2H, H1′), 4.33 (d, J=11.6 Hz, 2H, HC1), 2.71 (dd, J=22.6, 7.2 Hz, 2H, H4′), 1.21 (s, 18H, HC(CH3)3)
NMR 13C (100 MHz, CDCl3) δ 176.26, 175.83, 158.08, 152.42, 151.06, 141.07, 136.02, 133.44, 126.22, 121.87, 114.80, 83.85, 82.95, 46.24, 41.99, 39.86, 39.47, 32.31, 26.99, 26.78
NMR 31P (162 MHz, CDCl3) δ 26.57
HRMS (M+H) found 538.2440 calculated for C24H37N5O7P: 538.2431
From 2-amino-6-methyoxypurine, by using general procedure compound (Iae) was obtained as a colourless oil (51%).
NMR 1H (400 MHz, CDCl3) δ 8.04 (s, 1H, H8), 5.73-5.61 (m, 6H, H2′, H3′, HO—CH2-O), 4.76 (d, J=5.3 Hz, 1H, H1′a), 4.56 (d, J=5.4 Hz, 1H, H1′b), 3.99 (s, 3H, HOMe), 3.02 (s, 2H, HNH2), 2.66 (dd, J=11.9, 5.4 Hz, 2H, H4′), 1.26 (s, 18H, HC(CH3)3)
NMR 13C (100 MHz, CDCl3) δ 176.04, 164.04, 160.79, 149.49, 137.51, 133.43, 127.28, 121.87, 83.40, 53.95, 46.23, 39.66, 32.65, 31.97, 26.88
NMR 31P (162 MHz, CDCl3) δ 26.51
HRMS (M+H) found 528.2228 calculated for C22H35N5O8P: 528.2223
From 6-(4-isopropyl)phenylaminopurine, by using general procedure, compound (Iaf) was obtained as a colourless oil (39%).
NMR 1H (400 MHz, CDCl3) δ 8.67 (s, 1H, H2), 8.19 (s, 1H, H8), 7.22 (d, J=7.5 Hz, 2H, Harom), 6.93 (d, J=7.5 Hz, 2H, Harom), 5.72-5.59 (m, 6H, H2′, H3′, HO—CH2-O), 4.96 (d, J=5.4 Hz, 1H, H1′a), 4.48 (d, J=5.2 Hz, 1H, H1′b), 3.86 (s, 1H, HNH), 3.04 (hept, J=6.4 Hz, 1H, HCHisopropyl), 2.66 (dd, J=11.9, 5.4 Hz, 2H, H4′), 1.34 (d, J=6.4 Hz, 6H, HCH3isopropyl), 1.30 (s, 18H, HC(CH3)3)
NMR 13C (100 MHz, CDCl3) δ 176.04, 153.12, 151.64, 150.82, 143.37, 141.06, 138.38, 133.43, 127.76, 123.52, 123.04, 121.87, 83.40, 46.23, 39.66, 34.19, 32.65, 31.97, 26.88, 23.37
NMR 31P (162 MHz, CDCl3) δ 26.48
HRMS (M+H) found 616.2909 calculated for C30H43N5O7P: 616.2900
From 6-cyclohexylaminopurine, by using general procedure, compound (Iag) was obtained as a colourless oil (42%).
NMR 1H (400 MHz, CDCl3) δ 8.39 (s, 1H, H2), 8.14 (s, 1H, H8), 5.73-5.59 (m, 6H, H2′, H3′, HO—CH2-O), 4.97 (d, J=5.4 Hz, 1H, H1′a), 4.48 (d, J=5.4 Hz, 1H, H1′b), 3.78 (ft, J=7.9, 3.8 Hz, 1H, HCHcyclohexyl), 2.66 (dd, J=11.9, 5.4 Hz, 2H, H4′), 1.92 (dqd, J=11.4, 5.7, 2.1 Hz, 2H, HCH2cyclohexyl), 1.78 (s, 1H, HNH), 1.72 (tdd, J=11.5, 5.7, 2.2 Hz, 2H, HCH2cyclohexyl), 1.67-1.52 (m, 6H, HCH2cyclohexyl), 1.28 (s, 18H, HC(CH3)3)
NMR 13C (100 MHz, CDCl3) δ 176.04, 157.04, 151.05, 150.21, 141.06, 133.43, 124.13, 121.87, 83.40, 52.78, 46.23, 39.66, 33.31, 32.65, 31.97, 26.88, 25.91, 24.72
NMR 31P (162 MHz, CDCl3) δ 26.54
HRMS (M+H) found 580.6410 calculated for C27H43N5O7P: 580.6417
From 6-n-butylaminopurine, by using general procedure, compound (Iah) was obtained as a colourless oil (49%).
NMR 1H (400 MHz, CDCl3) δ 8.32 (s, 1H, H2), 8.02 (s, 1H, H8), 5.71-5.58 (m, 6H, H2′, H3′, HO—CH2-O), 4.85 (d, J=5.3 Hz, 1H, H1′a), 4.74 (d, J=5.4 Hz, 1H, H1′b), 3.44 (t, J=4.9 Hz, 1H, HC1a), 3.35 (t, J=4.9 Hz, 1H, HC1b), 2.66 (dd, J=11.9, 5.4 Hz, 2H, H4′), 2.01 (s, 1H, HNH), 1.58 (ddt, J=12.8, 7.6, 5.0 Hz, 2H, HC2), 1.46-1.36 (m, 2H, HC3), 1.28 (s, 18H, HC(CH3)3), 0.99 (t, J=6.6 Hz, 3H, HC4)
NMR 13C (100 MHz, CDCl3) δ 176.04, 157.21, 152.47, 151.00, 141.06, 133.43, 125.05, 121.87, 83.40, 46.23, 43.71, 39.66, 32.65, 31.97, 30.87, 26.88, 20.22, 14.01
NMR 31P (162 MHz, CDCl3) δ 26.48
HRMS (M30 H) found 554.2739 calculated for C25H41N5O7P: 554.2744
From 6-phénylthiopurine, by using general procedure, compound (Iai) was obtained as a colourless oil (38%).
NMR 1H (400 MHz, CDCl3) δ 8.60 (s, 1H, H2), 7.96 (s, 1H, H8), 7.18 (dd, J=7.5, 1.5 Hz, 2H, Harom), 7.12 (t, J=7.4 Hz, 2H, Harom), 7.06-7.00 (m, 1H, Harom), 5.75-5.62 (m, 6H, H2′ H3′, HO—CH2-O), 4.74 (d, J=5.3 Hz, 1H, H1′), 4.53 (d, J=5.3 Hz, 1H, H8), 2.66 (dd, J=11.9, 5.4 Hz, 2H, H4′), 1.29 (s, 18H, HC(CH3)3)
NMR 13C (100 MHz, CDCl3) δ 176.04, 166.24, 150.60, 144.49, 142.89, 136.74, 135.37, 133.43, 131.46, 129.78, 129.43, 121.87, 83.40, 46.23, 39.66, 32.65, 31.97, 26.88
NMR 31P (162 MHz, CDCl3) δ 26.49
HRMS (M+H) found 591.6321 calculated for C27H36N4O7PS: 591.6329
From 2,6-diaminopurine, by using general procedure compound (Iaj) was obtained as a colourless oil (41%).
NMR 1H (400 MHz, CDCl3) δ 7.50 (s, 1H, H8), 5.94-5.76 (m, 1H, H2′), 5.71-5.58 (m, 5H, H3′ and HO—CH2-O), 5.54 (s, 2H, HNH2), 4.80 (s, 2H, HNH2), 4.61 (t, J=5.2 Hz, 2H, H1′), 2.69 (dd, J=22.6, 7.2 Hz, 2H, H4′), 1.21 (s, 18H, HC(CH3)3)
NMR 13C (100 MHz, CDCl3) δ 176.26, 175.83, 164.27, 159.42, 149.75, 140.52, 133.44, 122.34, 121.87, 83.85, 82.95, 46.24, 39.86, 39.47, 32.31, 26.99, 26.78
NMR 31P (162 MHz, CDCl3) δ 26.42
HRMS (M+H) found 513.2225 calculated for C21H34N6O7P: 513.2227
From 2-amino-6-allylaminopurine, by using general procedure compound (Iak) was obtained as a colourless oil (40%).
NMR 1H (400 MHz, CDCl3) δ 8.08 (s, 1H, H8), 5.79 (ddt, J=16.4, 10.1, 6.2 Hz, 1H, HC2), 5.72-5.60 (m, 6H, H2′, H3′, HO—CH2-O), 5.12 (ddd, J=8.8, 4.7, 1.0 Hz, 2H, HC3), 4.98 (d, J=5.5 Hz, 1H, H1′a), 4.48 (d, J=5.4 Hz, 1H, H1′b), 4.22 (d, J=6.2 Hz, 1H, HC1a), 4.12 (d, J=6.0 Hz, 1H, HC1b), 2.66 (dd, J=11.9, 5.5 Hz, 2H, H4′), 2.12 (s, 1H, HNH), 1.29 (s, 18H, HC(CCH3)3)
NMR 13C (100 MHz, CDCl3) δ 176.26, 175.83, 161.42, 160.16, 150.97, 141.07, 136.02, 133.44, 123.91, 121.87, 114.80, 83.85, 82.95, 46.24, 41.99, 39.86, 39.47, 32.31, 26.99, 26.78
NMR 31P (162 MHz, CDCl3) δ 26.52
HRMS (M+H) found 553.4687 calculated for C24H38N6O7P: 553.4688
From 2-amino-6-(4-isopropyl)phenylaminopurine, by using general procedure, compound (Ial) was obtained as a colourless oil (45%).
NMR 1H (400 MHz, CDCl3) δ 7.88 (s, 1H, H2), 7.22 (d, J=7.5 Hz, 2H, Harom), 6.93 (d, J=7.5 Hz, 2H, Harom), 5.71-5.58 (m, 6H, H2′, H3′, HO—CH2-O), 4.91 (d, J=5.2 Hz, 1H, H1′a), 4.49 (d, J=5.4 Hz, 1H, H1′b), 3.89 (s, 1H, HNH), 3.04 (hept, J=6.3 Hz, 1H, HCHisopropyl), 2.66 (dd, J=11.9, 5.4 Hz, 2H, H4′), 2.11 (s, 2H, HNH2), 1.33 (d, J=6.4 Hz, 6H, HCH3isopropyl), 1.30 (s, 18H, HC(CH3)3)
NMR 13C (100 MHz, CDCl3) δ 176.04, 161.65, 155.21, 150.38, 143.37, 141.06, 138.38, 133.43, 127.76, 123.52, 122.63, 121.87, 83.40, 46.23, 39.66, 34.19, 32.65, 31.97, 26.88, 23.37
NMR 31P (162 MHz, CDCl3) δ 26.42
HRMS (M+H) found 631.6754, calculated for C30H44N6O7P: 631.6748
From 2-amino-6-cyclohexylaminopurine by using general procedure, compound (Iam) was obtained as a colourless oil (50%).
NMR 1H (400 MHz, CDCl3) δ 7.81 (s, 1H, H8), 5.73-5.60 (m, 6H, H2′, H3′, HO—CH2-O), 4.92 (d, J=5.3 Hz, 1H, H1′a), 4.51 (d, J=5.3 Hz, 1H, H1′b), 3.71-3.62 (m, 1H, HCH2cyclohexyl), 2.66 (dd, J=11.9, 5.4 Hz, 2H, H4′), 2.14-2.03 (m, 3H, HNH and HCH2cyclohexyl), 1.75-1.56 (m, 8H, HCH2cyclohexyl), 1.30 (s, 18H, HC(CH3)3)
NMR 13C (100 MHz, CDCl3) δ 176.04, 159.80, 159.56, 151.27, 141.06, 133.43, 122.02, 121.69, 83.40, 52.78, 46.23, 39.66, 33.31, 32.65, 31.97, 26.88, 25.91, 24.72
NMR 31P (162 MHz, CDCl3) δ 26.51
HRMS (M+H) found 595.6322 calculated for C27H44N6O7P: 595.6333
Linear compound is solubilized in methanol and triethylamine mixture (7/3). The solution is heated at 70° C. for 5 hours. After completion of the reaction (checked by TLC), solvent are removed under reduced pressure and the obtained crude product is purified on silica gel (eluent: petroleum ether ethyl acetate 4/6). To a CH2Cl2 (25 mL/mmol) solution of N1-crotyl-4,5-substituted furano-uracil (1 equiv.), bis(POC) allylphosphonate (1.3 equiv.) and catalyst (0.05 eq) was added. This solution was stirred under positive pressure of dry argon. After evaporation of all volatiles, the residue was purified by silica gel column chromatography (EtOAc/Petroleum ether).
According to said procedures the following compounds are obtained and characterized
NMR 1H (400 MHz, CDCl3) δ 7.97 (s, 1H, H6), 7.69 (d, J=8.2 Hz, 2H, Harom), 7.35-7.18 (m, 2H, Harom), 6.71 (s, 1H, Hfurano), 5.98-5.58 (m, 6H, HO—CH2-O, H2′, H3′), 4.95 (sept, J=6.3 Hz, 2H, HCHisopropyl), 4.38 (t, J=4.6 Hz, 2H, H1′), 2.81 (dd, J=22.7, 6.6 Hz, 2H, H4′), 2.64 (t, J=7.6 Hz, 2H, HC1), 1.68 (dq, J=14.6, 7.3 Hz, 2H, HC2), 1.32 (d, J=6.3 Hz, 12H, HCH3isopropyl), 0.97 (t, J=7.3 Hz, 3H, HC3)
NMR 13C (100 MHz, CDCl3) δ 171.44, 162.33, 155.38, 154.95, 151.54, 144.56, 143.62, 131.59, 130.92, 127.57, 127.18, 124.39, 124.18, 122.27, 102.19, 85.18, 84.27, 72.53, 72.22, 51.10, 38.12, 32.31, 24.48, 22.65, 22.44, 12.98
NMR 31P (162 MHz, CDCl3) δ 27.00
HRMS (M+Na): found 643.2049 calculated for C29H37N2O11NaP 643.2033
NMR 1H (400 MHz, CDCl3) δ 7.96 (s, 1H, H6), 7.69 (d, J=8.2 Hz, 2H, Harom), 7.27 (d, J=8.1 Hz, 2H, Harom), 6.70 (s, 1H, Hfurano), 5.97-5.61 (m, 6H, HO—CH2-O, H2′, H3′), 4.93 (kept, J=6.3 Hz, 2H, HCHisopropyl), 4.72-4.62 (t, J=4.6 Hz, H1′), 2.81 (dd, J=22.7, 6.6 Hz, 2H, H4′), 2.71-2.59 (m, 2H, HC1), 1.65 (dt, J=15.1, 7.5 Hz, 2H, HC2), 1.41-1.24 (m, 16H, HC3, HC4, HCH3isopropyl), 0.92 (t, J=6.7 Hz, 3H, HC5)
NMR 13C (100 MHz, CDCl3) δ 171.44, 162.33, 155.38, 154.95, 151.54, 143.94, 143.62, 131.59, 131.04, 128.08, 127.87, 124.31, 124.10, 122.27, 102.19, 85.18, 84.27, 72.53, 72.22, 51.10, 36.41, 32.31, 30.64, 30.02, 22.94, 22.65, 22.44, 14.02
NMR 31P (162 MHz, CDCl3) δ 27.04
HRMS (M+Na): found 671.6524 calculated for C31H41N2O11NaP 671.6533
NMR 1H (400 MHz, CDCl3) δ 7.81 (s, 1H, H6), 7.37-7.15 (m, 5H, Harmo), 6.09 (s, 1H, Hfurano), 5.95-5.58 (m, 6H, HO—CH2-O, H2′, H3′), 4.90 (hept, J=6.2 Hz, 2H, HCHisopropyl), 4.67 (t, J=4.7 Hz, H1′), 3.10-2.93 (m, 4H, HC1, HC2), 2.80 (dd, J=22.7, 6.7 Hz, 2H, H4′), 1.34 (d, J=6.3 Hz, 12H, HCH3isopropyl)
NMR 13C (100 MHz, CDCl3) δ 163.72, 162.33, 155.38, 154.95, 148.01, 141.00, 138.22, 131.59, 129.05, 128.65, 126.53, 122.27, 108.20, 103.63, 85.18, 84.27, 72.53, 72.22, 51.10, 33.25, 28.37, 22.65, 22.44
NMR 31P (162 MHz, CDCl3) δ 27.10
HRMS (M+Na): found 643.2049 calculated for C29H37H2O11NaP 643.2033
NMR 1H (400 MHz, CDCl3) δ 7.79 (s, 1H, H6), 6.09 (s, 1H, Hfurano), 5.88-5.57 (m, 6H, HO—CH2-O, H2′, H3′), 4.89 (hept, J=6.3 Hz, 2H, HCHisopropyl), 4.74 (t, J=4.5 Hz, H1′), 2.75 (dd, J=22.7, 7.2 Hz, 2H, H4′), 2.61 (t, J=7.4 Hz, 2H, HC1), 1.70-1.59 (m, 2H, HC2), 1.27 (m, 22H, HC3, HC4, HC5, HC6, HC7, HCH3isopropyl), 0.85 (t, J=6.8 Hz, 3H, HC8)
NMR 13C (100 MHz, CDCl3) δ 171.95, 160.08, 155.36, 153.09, 137.92, 130.28, 123.88, 108.21, 98.54, 84.11, 73.43, 52.01, 31.77, 31.44, 30.05, 29.28, 28.82, 28.26, 26.73, 22.59, 21.60, 14.05
NMR 31P (162 MHz, CDCl3) δ 27.07
HRMS (M+H): found 615.2682 calculated for C28H44N2O11P 615.2683
NMR 1H (400 MHz, CDCl3) δ 7.79 (s, 1H, H6), 6.09 (s, 1H, Hfurano), 5.88-5.56 (m, 6H, HO—CH2-O+H2′+H3′), 4.89 (hept, J=6.3 Hz, 2H, HCHisopropyl), 4.63-4.55 (m, 2H, H1′), 2.74 (dd, J=22.7, 7.1 Hz, 2H, H4′), 2.61 (t, J=7.4 Hz, 2H, HC1), 1.71-1.60 (m, 2H, HC2), 1.37-1.24 (m, 16H, HC3+HC4+HCH3isopropyl), 0.87 (dd, J=8.5, 5.6 Hz, 3H, HC5)
NMR 13C (100 MHz, CDCl3) δ 171.94, 160.04, 155.36, 153.09, 137.97, 130.27, 123.87, 108.21, 98.57, 84.11, 73.42, 52.00, 31.44, 31.13, 30.05, 28.21, 26.40, 22.26, 21.59, 13.88
NMR 31P (162 MHz, CDCl3) δ 27.06
HRMS (M+H): found 573.2208 calculated for C25H38N2O11P 573.2213
To a CH2Cl2 (15 mL/mmol) solution of N1-2-methyl-allyl uracil (0.60 mmol., 99.7 mg, 1.5 equiv.) and bis(POM) allylphosphonate (0.40 mmol., 140.1 mg, 1.0 equiv.) was added RuCl2(PCy3)IMesBenzylidene catalyst (0.020 mmol., 17 mg, 0.05 equiv.) then this solution was stirred at 40° C. for 16 h under positive pressure of dry air. After evaporation of all volatiles, the residue was purified by silica gel column chromatography to give the desired product
1H NMR (400 MHz, MeOD) δ 7.46 (d, J=7.9, 1H), 5.69-5.61 (m, 5H), 5.34 (q, J=7.8, 1H), 4.33 (d, J=4.8, 2H), 2.82 (dd, J=22.9, 7.8, 2H), 1.69 (d, J=4.8, 3H), 1.23 (s, 18H).
13C NMR (101 MHz, MeOD) δ 178.53, 146.64, 102.89, 83.28, 55.04, 39.90, 27.39.
31P NMR (162 MHz, MeOD) δ 27.65.
HRMS (ESI): m/z [M+Na]+ calcd for C21H33N2NaO9P: 511.18159, found: 511.18159
To a DMF (1 mL) solution of (E)-4-bromomethyl-bis(POM)-allylphosphonate (0.25 mmol., 180 mg, 1.25 equiv.) was added to a suspension of sodium hydride (0.22 mmol., 5.3 mg, 1.1 equiv.) of cytosine (0.20 mmol., 18.5 mg, 1.0 equiv.) in DMF (1 mL) then this solution was stirred at 50° C. for 24 h under dry air. After evaporation of all volatiles, the residue was purified by silica gel column chromatography to give the desired products
1H NMR (400 MHz, Acetone) δ 7.34 (d, J=6.8, 1H), 5.87 (d, J=6.8, 1H), 5.86-5.54 (m, 8H), 4.67 (t, J=4.9, 2H), 2.74 (dd, J=24.7, 9.3, 2H), 1.22 (s, 18H).
31P NMR (162 MHz, MeOD) δ 26.82.
HRMS (ESI): m/z [M+Na]+ calcd for C20H32N3NaO8P: 496.18192, found: 496.18214
To a 1,4-dioxane/H2O (2 mL, 1/1, v/v) solution of N9-(4′-bis(POM)-phosphinyl-2′-butenyl)-2-amino-6-chloropurine (116 mg, 0.218 mmol) was added 2-chloroacetaldehyde (2.5 mL, 50 wt. % in H2O). The resulting mixture was stirred 6 h at 70° C. and was extracted with ethyl acetate. Combined organic layers were dried over MgSO4 and concentrated in vacuo. Chromatography over silica gel in CH2Cl2/MeOH 98:2 afforded 56 mg (48%) of desired product as a white solid.
1H NMR (400 MHz, CDCl3) δ 11.63 (s, 1H, NH), 7.66 (d, J=2.8 Hz, 1H, H7), 7.64 (s, 1H, H2), 7.25 (d, J=7.9 Hz, 1H, H7), 5.85 (dq, J=16.0, 5.6 Hz, 1H, H2′), 5.70-5.60 (m, 5H, HO—CH2-O, H3′), 4.65 (t, J=4.8 Hz, 2H, H1′), 2.72 (dd, J=22.4, 7.2 Hz, 2H, H4′), 1.18 (s, 18H).
NMR 13C (100 MHz, CDCl3) δ 176.92, 152.28, 150.32, 146.12, 138.07, 130.32 (d, J=15 Hz), 122.52 (d, J=11.6 Hz), 115.91 (d, J=18.2 Hz), 107.35, 81.67 (d, J=6.2 Hz), 38.66, (d, J=2.1 Hz), 31.28, 29.89, 26.76.
31P NMR (162 MHz, CDCl3) δ 26.81
HRMS (ESI): m/z [M+Na]+ calcd for C23H33N5O8P: 538.20613, found: 538.20578
To a solution of alkyne (0.11 mmol., 14.4 mg, 1.3 equiv.) and (E,Z)-4-azido-bis(POM)-allylphosphonate (0.08 mmol., 34.1 mg) in H2O/t-BuOH (1:1, 100 μL) were added Cu powder (0.40 mmol., 11.6 mg, 5.0 equiv.) and CuSO4 (0.02 mmol., 5.0 mg, 0.25 equiv.). The resulting suspension was stirred 8 h at room temperature, then the mixture was diluted with EtOAc (1 mL), and purified by preparative thin layer chromatography to give (E)-Triazol-1-yl-bis(POM)-allylphosphonate (26 mg, 59%)
1H NMR (400 MHz, CDCl3) δ 7.75 (s, 1H, Htriazol), 7.73 (d, J=8.0 Hz, 2H, Harom), 7.22 (d, J=8.0 Hz, 2H, Harom), 5.95-5.87 (m, 1H, CH═), 5.82-5.70 (m, 1H, CH═), 5.69-5.63 (m, 4H, HO—CH2-O, H3′), 4.99 (t, J=5.2, 2H, H1′), 2.74 (dd, J=22.8, 7.2, 2H, H4′), 2.60 (t, J=7.6, 2H, CH2), 1.65 (t, J=7.6, 3H, CH3), 1.21 (s, 18H), 0.94 (t, J=7.6, 3H, CH3).
NMR 13C (100 MHz, CDCl3) δ 176.84, 148.22, 142.78, 129.20 (d, J=15.1 Hz), 128.87 127.94, 126.62, 124.46 (d, J=11.6 Hz), 118.95, 88.57 (d, J=6.2 Hz), 51.75 (d, J=2.1 Hz), 38.71, 37.79, 31.51, 30.11, 24.43, 13.75.
31P NMR (162 MHz, CDCl3) δ 26.35
HRMS (ESI): m/z [M+Na]+ calcd for C27H41N3O7P: 550.26766, found: 550.26727
4.1. Material and Methods
4.1.1. Antiviral Assays
The herpes and vaccinia virus assays were based on inhibition of virus-induced cytopathicity in HEL cell cultures [herpes simplex virus type 1 (HSV-1) (KOS), HSV-2 (G), HSV-1 TK− (KOS acyclovir resistant, ACV) and vaccinia virus (VV). Confluent cell cultures in microtiter 96-well plates were inoculated with 100 CCID50 of virus (1 CCID50 being the virus dose to infect 50% of the cell cultures) and the infected cell cultures were incubated in the presence of varying concentrations (200, 40, 8, . . . μM) of the test compounds. Viral cytopathogenicity was recorded as soon as it reached completion in the control virus-infected cell cultures that were not treated with the test compounds.
Confluent human embryonic lung (HEL) fibroblasts were grown in 96-well microtiter plates and infected with the human cytomegalovirus (HCMV) strains Davis and AD-169 at 100 PFU per well. After a 2-h incubation period, residual virus was removed and the infected cells were further incubated with medium containing different concentrations of the test compounds (in duplicate). After incubation for 7 days at 37° C., virus-induced cytopathogenicity was monitored microscopically after ethanol fixation and staining with Giemsa. Antiviral activity was expressed as the EC50 or compound concentration required to reduce virus-induced cytopathogenicity by 50%. EC50 values were calculated from graphic plots of the percentage of cytopathogenicity as a function of concentration of the compounds.
The laboratory wild-type VZV strain OKA and the thymidine kinase-deficient VZV strain 07/1 were used. Confluent HEL cell cultures grown in 96-well microtiter plates were inoculated with VZV at an input of 20 PFU per well. After a 2-h incubation period, residual virus was removed and varying concentrations of the test compounds were added (in duplicate). Antiviral activity was expressed as the 50%-effective concentration required to reduce viral plaque formation after 5 days by 50% as compared with untreated controls.
4.1.2. Cytotoxicity Assays
Cytotoxicity measurements were based on the inhibition of HEL cell growth. HEL cells were seeded at a rate of 5×103 cells/well into 96-well microtiter plates and allowed to proliferate for 24 h. Then, medium containing different concentrations of the test compounds was added. After 3 days of incubation at 37° C., the cell number was determined with a Coulter counter. The 50%-cytostatic concentration (CC50) was calculated as the compound concentration required to reduce cell growth by 50% relative to the number of cells in the untreated controls. CC50 values were estimated from graphic plots of the number of cells (percentage of control) as a function of the concentration of the test compounds. Cytotoxicity was also expressed as the minimum cytotoxic concentration (MCC) or the compound concentration that causes a microscopically detectable alteration of cell morphology.
4.2. Results
They are given in
The compounds were evaluated against a variety of DNA viruses including herpes simplex virus type 1 (HSV-1) (strain KOS), HSV-2 (strain G), thymidine kinase (TK)-deficient HSV-1 TK−, varicella-zoster virus (VZV) (strain OKA), the TK-deficient VZV TK− (07/1), human cytomegalovirus (HCMV) and vaccinia virus (VV) in HEL cell cultures.
All derivatives according to the invention showed pronounced inhibitory activity against HSV-1, HSV-2 and VZV. In contrast with the [E] enantiomers (Ia1) and (Ie1), the corresponding [Z] enantiomers (Ia2 and Ie2) were clearly less, or not antivirally active.
The compounds were not significantly cytotoxic at 200 μM, but slightly cytostatic at 34-70 μM against HEL cell proliferation.
The [E] derivative (Iel) proved most inhibitory against HSV-1, HSV-2 and VZV. The independence of cellular TK activity is testified by the pronounced antiviral activity of the compound against mutant TK-deficient HSV-1 TK− and VZV TK.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10305983.8 | Sep 2010 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP11/59090 | 6/1/2011 | WO | 00 | 5/15/2013 |
