HK1260250A1

HK1260250A1 - 5′-phosphorothiolate mrna 5′-end (cap) analogs, mrna comprising the same, method of obtaining and uses thereof

Info

Publication number: HK1260250A1
Application number: HK19120087.2A
Authority: HK
Inventors: Jacek Jemielity; Kaja FAC-DĄBROWSKA; Błażej WOJTCZAK; Marek Baranowski; Anna NOWICKA; Joanna Kowalska; Paweł SIKORSKI; Marcin WARMIŃSKI
Original assignee: Uniwersytet Warszawski
Priority date: 2016-01-29
Filing date: 2017-01-27
Publication date: 2019-12-13

Description

5 '-phosphorothioate mRNA 5' -end (cap) analogs, mRNAs comprising said analogs, methods of obtaining and uses thereof

A description is given.

Technical Field

The present invention relates to analogs of the 5 '-end (cap) of mRNA containing a 5' -phosphorothioate moiety, methods of their preparation, intermediates, and uses thereof.

5' -phosphorothioate cap analogs are useful as inhibitors of the DcpS enzyme, which makes their use as pharmaceuticals, particularly for the treatment of Spinal Muscular Atrophy (SMA). The invention also relates to mrnas modified at the 5' end with 5' -end (cap) analogues of mrnas according to the invention containing a 5' -phosphorothioate moiety, wherein the modification is aimed at obtaining mRNA transcripts with increased stability and translational activity under cellular conditions. Transcripts having such properties are applicable to novel mRNA-based gene therapy.

Background

Chemically derived 5' -terminal analogs of mRNA have a variety of uses, and modifications made within this structure can significantly alter the biological properties of these compounds (Ziemniak, Strenkowska et al, 2013). Among the various applications of cap analogs, the most common include their use as small molecule inhibitors of cap-dependent processes for therapeutic purposes (e.g., inhibition of DcpS enzyme-spinal muscular atrophy therapy). On the other hand, suitably modified dinucleotide cap analogs are used to modify messenger mRNA by in vitro co-transcription to obtain transcripts with improved stability and translational activity under cellular conditions. Transcripts with such properties are being increasingly studied in the context of novel mRNA-based gene therapy. In the latter case, cap structure resistance to another uncapping enzyme, Dcp2, is a critical issue.

there are two major mRNA degradation pathways in eukaryotic cells, 5'→ 3' degradation and 3'→ 5' degradation (Rydzik, Lukaszewicz et al, 2009). the two degradation pathways initiate by deadenylation degradation in the 5'→ 3' direction followed by mRNA decapping due to cleavage of the bond between the alpha and β phosphates, and degradation by 5 '-exonucleases, 3' → 5 'degradation involves mRNA degradation from the 3' -end through exosomespyrophosphatase belonging to the HIT family and hydrolysing the cap between the gamma and beta phosphates, releasing 7-methylguanosine 5' -monophosphate (m)⁷GMP) and a second product, which is a nucleoside 5' -diphosphate or a short oligonucleotide, respectively. The longer cap-capped mRNA is not a substrate for DcpS. 7-methylguanosine 5′-Diphosphonic acid (m)⁷GDP), which is a product of 5'→ 3' mRNA degradation, and is not a substrate for DcpS. The activity of the DcpS enzyme is thought to be essential for cellular homeostasis, as unnecessary cap residues released from mRNA during 3'→ 5' degradation can adversely affect other cap-dependent cellular processes. DcpS is located in both the cytoplasm and the nucleus, where it may be involved in splicing regulation (Shen, Liu et al, 2008). It is therefore proposed that the role of DcpS in cells exceeds its well-characterized function in 3'→ 5' mRNA degradation (Bail and Kiledjian 2008).

It was reported in 2008 that DcpS inhibition could provide a therapeutic effect in spinal muscular atrophy. SMA is a common neurodegenerative disease that occurs on average once every 6000 births (Akagi and Campbell 1962). It is caused by low levels of SMN proteins (live motor neurons) consisting ofSMNThe gene encodes. There are two SMN genes in humans, i.e.,SMN1andSMN2. The main difference between them is a sequence change in exon 7 that affects pre-mRNA splicing. As a result of which it is possible to,SMN1expression of the gene results in a stable and functional protein, therebySMN2The expressed protein is shortened. In two copiesSMN1Mutations in genes, including deletions, transformationsSMN2Like genes and point mutations, leading to SMA diseases. Having only one defectSMN1The person to whom the copy is made is the SMA carrier, but does not show any symptoms of the disease.

The homologous SMN2 gene did not provide sufficient amounts of functional SMN protein, but higher amounts of copies of the SMN2 gene were observed with more benign disease processes. It is therefore believed that compounds that increase the amount of protein encoded by the SMN2 gene in cells may be therapeutic agents against SMA. Some 5-substituted quinazolines were found to increase SMN2 gene expression even two-fold (Akagi and Campbell, 1962). In an attempt to reverse this molecular mechanism underlying activation, in another study using radiolabeling, the authors identified DcpS as a protein that binds 5-substituted quinazolines.

These experiments allowed the identification of DcpS as a therapeutic target for SMA treatment.

Further studies have shown that various C5-substituted quinazolines are potent inhibitors of the DcpS enzyme (already at nanomolar concentrations), and that the potential of the inhibitors is related to that of the enzymeSMN2The level of gene promoter activation is relevant. The therapeutic potential of these compounds was then demonstrated in vivo in a mouse model (Butchbach, Singh et al, 2010). Recently, a DcpS inhibitor, RG3039 compound, was reported to improve motor function in SMA bearing mice (Van Meerbeke, Gibbs et al).

Despite ongoing preclinical and clinical trials, there is no effective treatment of SMA, and thus there is a continuing need for new compounds with therapeutic potential.

Dinucleotide cap analogs with modifications in the triphosphate bridge and 7-methylguanosine ribose are useful for in vitro synthesis of capped RNA molecules. This method is useful because it allows to obtain RNA molecules with improved biological properties in cells, in particular increased translational activity and prolonged half-life (Grudzien, Kalek et al, 2006). Both of these features result in significantly greater amounts of protein being obtained while utilizing the same amount of mRNA. This may find widespread use in research and for commercial production of peptides and proteins, including therapeutic applications, for example in cancer immunotherapy (Sahin, Kariko et al, 2014).

The most common method for obtaining capped mRNA in vitro is at all four ribonucleoside triphosphates and a cap dinucleotide, e.g., (m)⁷Gppppg) was used to synthesize mRNA on a DNA template using bacterial or phage RNA polymerase. Polymerase through m⁷the nucleophilic attack of the 3' -OH of the Guo moiety in GpppG on the α -phosphate of the next transcribed nucleoside triphosphate initiates transcription, resulting in m⁷GpppGpN as initial product (Contreras and Fiers 1981, Konarska, Padgett et al, 1984).

in vivo mRNA degradation is initiated primarily by the removal of the cap from the 5' -end of the mRNA by specific pyrophosphatase Dcp1/Dcp2, which cleaves the bond between α and beta phosphates (Mildvan, Xia et al, 2005). Dcp2 enzyme, which forms a complex with the regulatory protein Dcp1, responsible for the excision of the cap structure from the full-length transcript or at least 20 nucleotide fragments thereof (Lykke-Andersen 2002), Dcp1/2 complex plays a key role in gene expression regulation, resistance of mRNA transcripts within the cap to this enzyme activity, leading to increased expression of proteins encoded by such modified mrnas (ziemiak, Strenkowska et al, 2013) when modification does not compromise the interaction with translation initiation factors (zienk, Strenkowska et al, 2013) when modification does not compromise the interaction with such modified mRNA, this leads to increased translational activity for therapeutic applications, including the modification of mRNA at sites where the modified mRNA is deficient in vivo mRNA, the expression of mRNA, which is not due to the increased by the substitution of the mRNA by endoenzyme in vivo mRNA, e-endoenzyme, or the like, the increased resistance of mRNA in the cell-endoenzyme, which is not due to the increased uptake of the modified mRNA (yakayak) in the cell, or the increased uptake of mRNA, the cell-mediated protein, the increased uptake of the cell-mediated by the mRNA-mediated protein, which is not shown by the increased uptake of the mRNA-endoenzyme, or the mRNA-mediated protein, the cell-endoenzyme-mediated protein, or its affinity, the increased uptake, including increased uptake, increased resistance to the increased uptake, increased uptake of mRNA-mediated by the cell-mediated protein, increased uptake.

Disclosure of the invention

In view of the state of the art, it is an object of the present invention to overcome the indicated disadvantages and to provide a new class of nucleotide mRNA 5' -terminal analogs that affect DcpS activity, their uses, including use in SMA treatment, and methods for their synthesis.

It is another object of the present invention to provide mrnas modified at the 5' end with 5' -end (cap) analogs of mrnas containing 5' -phosphorothioate moieties, thereby increasing mRNA stability and the efficiency of biosynthesis of proteins encoded by the mrnas in cells. It is another object of the invention to provide mrnas modified at the 5 'end with 5' -phosphorothioate moiety containing 5 '-monophosphate 5' -end (cap) analogues of the mrnas intended for use as medicaments, including for novel mRNA-based gene therapy.

The invention relates to a novel nucleotide mRNA 5' -end analogue. The novel analogs comprise a sulfur atom at the 5 '-nucleoside position, i.e., at least one oxygen atom at the 5' position is replaced with a sulfur atom. We have surprisingly found that novel analogs containing a modification of the sulfur atom at the 5' position on the 7-methylguanosine side are resistant to the hydrolytic activity of the DcpS enzyme and are inhibitors of the DcpS enzyme, thereby affecting the expression of SMN protein, which is of therapeutic relevance in SMA treatment. Such compounds that stabilize and/or affect the activity of DcpS would also be useful for modulating mRNA degradation and splicing regulation and modulation. From the viewpoint of inhibitory properties, the following analogs were found to be particularly preferred: m is⁷GSpppG (number 24), m⁷GSpppSG (No. 32), m⁷GSpp_spG D1 (number 30), m⁷GSpp_spG D2 (number 31), m⁷GSpp_spSG D1 (No. 33), m⁷GSpp_spSG D2 (No. 34), and most preferably m⁷GSpp_spSG D2 (accession number 34). Of equal advantage is the analogue m⁷GSpp (No. 12), m⁷GSppG (number 23), m⁷GSppCH ₂pG (No. 25), m^7,2’OGSpppG (number 26), m⁷GpCH ₂ppSG (No. 37).

The invention also relates to mrnas modified at the 5' end with 5' -end (cap) analogs of mrnas containing 5' -phosphorothioate moieties, thereby increasing mRNA stability and the efficiency of biosynthesis of proteins encoded by the mrnas in cells. The invention also relates to mrnas modified at the 5' end with 5' -end (cap) analogs of mrnas containing 5' -phosphorothioate moieties, said modified mrnas being intended for use as medicaments, including for novel mRNA-based gene therapy.

Surprisingly, the inventors found that the novel analogs according to the invention containing a modification of the sulfur atom in the 5' position on the 7-methylguanosine side become resistant to the hydrolytic activity of the enzyme Dcp1/2 after incorporation into the mRNA by in vitro transcription methods, and therefore they influence the stability of the mRNA and the efficiency of the biosynthesis of the protein encoded by this mRNA in cells, including the HeLa cell line. This is the first finding that modifications located away from the triphosphate bridge cleavage site by Dcp1/2 in the cap make the cap structure resistant to its removal process, resulting in an increased half-life of the mRNA. This surprising finding is of significant therapeutic importance in gene therapy involving the expression of desired proteins based on the provided synthetic mrnas, as is the case in cancer immunotherapy for the specific activation of the immune system. Thus, a modified mRNA transcript, e.g., encoding a protein specific for a given cancer type, can be used to activate the immune system against cancer cells containing that specific antigen. From the viewpoint of the translation properties of the modified mRNA, the following analogs were found to be particularly preferable: m is⁷GSpppG (number 24), m^7,2’OGSpppG (number 26), m⁷GSpppSG (No. 32), m⁷GSpp_spG D1 (number 30), m⁷GSpp_spG D2 (number 31), m⁷GSpp_spSG D1 (No. 33), m⁷GSpp_spSG D2 (No. 34), and most preferably m^7,2′OGSpppG (number 26).

The present invention relates to 5' -phosphorothioate cap analogs according to formula 1

Wherein

L¹And L²Independently selected from O and S, wherein L₁And L₂Is not O;

n = 0, 1 or 2;

X₁、X₂、X₃independently selected from O, S;

R¹is selected from CH₃、C₂H₅、CH₂Ph, alkyl or substituted alkyl;

R²and R³Independently selected from H, OH, OCH₃、OC₂H₅、-COOH、CH₂COOH、N₃、CH₂N₃Alkyl, alkenyl or alkynyl;

R⁴and R⁵Independently selected from H, OH, OCH₃、OC₂H₅、-COOH、CH₂COOH、N₃、CH₂N₃Alkyl, alkenyl or alkynyl;

Y₁、Y₂independently selected from CH₂、CHCl、CCl₂、CF₂、CHF、NH、O；

And B is a radical according to formula 3, 4, 5, 6 or 7

。

Preferred 5' -phosphorothioate cap analogs are selected from:

even more preferred 5' -phosphorothioate cap analog compounds are selected from:

the invention also relates to 5' -phosphorothioate analogues according to formula 2

Wherein

m=0、1

n = 0, 1 or 2;

L¹is S

X₁、X₂、X₃Independently selected from O, S;

R¹is selected from CH₃、C₂H₅、CH₂Ph, alkyl or substituted alkyl;

R²and R³Independently selected from H, OH, OCH₃、OC₂H₅、-COOH、N₃Alkyl or substituted alkyl;

Y₁、Y₂independently selected from CH₂、CHCl、CCl₂、CHF、CF₂NH and O;

a preferred 5 '-phosphorothioate analogue is 7-methylguanosine 5' -deoxy-5 '-thioguanosine 5' -dithiophosphate of formula 13 below

。

The invention also relates to 5' -phosphorothioate cap analogues according to the invention for use as medicaments.

The invention also relates to 5' -phosphorothioate cap analogues according to the invention for use as medicaments for the treatment of Spinal Muscular Atrophy (SMA) and/or for alleviating the symptoms of SMA.

The invention also relates to the use of a 5' -phosphorothioate cap analogue according to the invention for the preparation of a medicament.

The invention also relates to the use of a 5' -phosphorothioate cap analogue according to the invention in the manufacture of a medicament for the treatment of Spinal Muscular Atrophy (SMA) and/or alleviating a symptom of SMA.

The invention also relates to the use of 5' -phosphorothioate cap analogues according to the invention as modulators of DcpS activity, preferably as inhibitors of DcpS enzyme activity, more preferably as inhibitors of hDcpS enzyme activity.

The invention also relates to the use of a 5' -phosphorothioate cap analogue according to the invention for modulating mRNA degradation and/or for modulating mRNA splicing.

The invention further relates to analogs of 5 '-deoxy-5' -iodoguanosine having a structure according to formulae 10, 11 and 12 shown below.

。

The present invention further relates to a process for the preparation of a compound according to formula 1, said process comprising the steps of: 5' -iodonucleosides according to formula 8

Formula 8

Wherein

R⁴And R⁵Independently selected from H, OH, OCH₃、OC₂H₅、-COOH、N₃Alkyl or substituted alkyl;

and B is a radical according to formula 3, 4, 5, 6 or 7

With a 5' -phosphorothioate analogue comprising a terminal phosphorothioate moiety according to formula 2

Wherein

m=0、1

n = 0, 1 or 2;

L¹is O or S;

X₁、X₂、X₃independently selected from O, S;

R¹is selected from CH₃、C₂H₅、CH₂Ph, alkyl or substituted alkyl

Y₁and Y₂Independently selected from CH₂、CHCl、CCl₂、CHF、CF₂NH and O;

wherein if n = 0 and m = 1, then X₃Is S; and X₁Is O;

if n = 1 and m = 0, X₂Is S; and X₁Is O;

if n = 1 and m = 1, X₃Is S; and X₁、X₂Is O;

to form a 5' -phosphorothioate cap analog according to formula 1

Wherein

L¹And L²Independently selected from O and S, wherein L₁And L₂Is not O;

n = 0, 1 or 2;

X₁、X₂、X₃independently selected from O, S;

R¹is selected from CH₃、C₂H₅、CH₂Ph, alkyl or substituted alkyl;

And B is a radical according to formula 3, 4, 5, 6 or 7

。

Preferably, the above synthesis method comprises using equimolar amounts of a compound according to formula 2, a compound according to formula 8 and DBU (1, 8-diazabicyclo (5.4.0) undec-7-ene) as a base.

The invention also relates to a process for the preparation of 5' -phosphorothioate analogues according to formula 2a, wherein the imidazolidine derivative according to formula 9

Wherein

R¹Is selected from CH₃、C₂H₅、CH₂Ph, alkyl or substituted alkyl;

with triethylammonium phosphate or sodium thiophosphate to form a 5' -phosphorothioate analogue according to formula 2a

Wherein

n = 0, 1 or 2;

L¹is O or S;

X₁and X₂Independently selected from O, S;

R¹is selected from CH₃、C₂H₅、CH₂Ph, alkaneAlkyl, substituted or unsubstituted;

Y¹independently selected from CH₂、CHCl、CCl₂、CHF、CF₂NH or O.

The present invention also relates to a process for the preparation of a compound according to formula 1, said process comprising the steps of:

imidazolidine derivatives according to formula 9

Wherein

R¹Is selected from CH₃、C₂H₅、CH₂Ph, alkyl or substituted alkyl;

with a 5' -phosphorothioate analogue comprising a terminal phosphorothioate moiety according to formula 2a

Wherein

n = 1

L¹Is O or S;

X₁and X₂Independently selected from O, S;

R¹is selected from CH₃、C₂H₅、CH₂Ph, alkyl or substituted alkyl;

Y₁independently selected from CH₂、CHCl、CCl₂、CHF、CF₂The content of the nitrogen is NH or O,

to form a 5' -phosphorothioate cap analog according to formula 1

Wherein

L¹And L²Independently selected from O and S, wherein L₁And L₂Is not O;

n = 0, 1 or 2;

X₁、X₂、X₃independently selected from O, S;

R¹is selected from CH₃、C₂H₅、CH₂Ph, alkyl or substituted alkyl;

And B is a radical according to formula 3, 4, 5, 6 or 7

。

In the synthesis process with imidazolidine derivatives, the reaction is preferably carried out in the presence of a divalent metal chloride, the preferred divalent metal chloride being zinc chloride ZnCl₂。

In the synthesis process with imidazolidine derivatives, it is preferred to use a 1.5-fold excess of the imidazolidine according to formula 9 with respect to the phosphate group, the phosphorothioate group or the compound according to formula 2a in the presence of an 8-fold excess of the divalent metal chloride.

The invention also relates to a pharmaceutical formulation comprising a 5' -phosphorothioate cap analogue according to the invention and a pharmaceutically acceptable carrier.

A pharmaceutical formulation according to the invention comprising a 5' -phosphorothioate cap analogue according to the invention and a pharmaceutically acceptable carrier has the property of inhibiting DcpS activity, preferably hDcpS activity, and is intended for SMA treatment.

The choice of a pharmaceutically acceptable carrier will depend on the method of administering the pharmaceutical formulation and the necessity of protecting the 5' -phosphorothioate analogue according to the invention from degradation inactivation prior to delivery to the cell, tissue or organism. Pharmaceutically acceptable carriers include solvents, dispersion media and adjuvants (coating materials, surfactants, fragrances and flavors, antioxidants, etc.). The pharmaceutical formulations according to the invention may be administered by different routes, including injection, oral, topical and rectal administration. The dosage of the pharmaceutical formulation is determined in consideration of the route of administration, the condition to be treated or prevented, and other relevant circumstances.

The present invention also relates to mrnas comprising at the 5 'end the novel 5' -phosphorothioate cap analogues according to the present invention.

Preferred mrnas are characterized by a 5' -phosphorothioate cap analog selected from the group consisting of: m is⁷GSpppG (number 24), m^7,2’OGSpppG (number 26), m⁷GSpppSG (No. 32), m⁷GSpp_spG D1 (number 30), m⁷GSpp_spG D2 (number 31), m⁷GSpp_spSG D1 (No. 33), m⁷GSpp_spSG D2 (No. 34), more preferably it is m^7,2’OGSpppG (number 26).

The present invention also relates to a method of preparing an mRNA comprising a 5 '-phosphorothioate cap analog at the 5' -end of the molecule, the method comprising: the 5' -phosphorothioate cap analogue according to the invention was incorporated into the mRNA molecule during synthesis.

In a preferred method of preparing mRNA, the 5' -phosphorothioate cap analogue is selected from m⁷GSpppG (number 24), m⁷ ^,2’OGSpppG (number 26), m⁷GSpppSG (No. 32), m⁷GSpp_spG D1 (number 30), m⁷GSpp_spG D2 (number 31), m⁷GSpp_spSG D1 (No. 33), m⁷GSpp_spSG D2 (No. 34), more preferably it is m^7,2’OGSpppG (number 26).

In a preferred method of preparing mRNA, the synthesis of mRNA is performed by in vitro transcription.

The present invention also relates to an mRNA prepared by the method of preparing an mRNA comprising a 5 '-phosphorothioate cap analog according to the present invention at the 5' -end of the molecule.

The invention also relates to the use of an mRNA comprising a 5 '-phosphorothioate cap analogue according to the invention at the 5' -end of the molecule for the production of a protein.

The use of mRNA for the production of proteins is preferably carried out in a cellular system or a non-cellular system.

The invention also relates to an mRNA according to the invention and an mRNA prepared according to the method for preparing an mRNA comprising a 5 '-phosphorothioate cap analogue according to the invention at the 5' end of the molecule for use as a medicament.

Such mRNA is preferably used as a medicament for treating Spinal Muscular Atrophy (SMA) and/or alleviating a symptom of SMA.

Preferably, such mRNA is used as an anti-cancer drug, more preferably as a drug for anti-cancer immunotherapy.

The invention also relates to the use of the mRNA according to the invention and the mRNA prepared according to the method for preparing the mRNA comprising a 5 '-phosphorothioate analogue according to the invention at the 5' end of the molecule for the manufacture of a medicament.

In a preferred use, the mRNA is for the manufacture of a medicament for treating Spinal Muscular Atrophy (SMA) and/or alleviating the symptoms of SMA, for use as an anti-cancer medicament, more preferably for use as a medicament for anti-cancer immunotherapy.

The invention also relates to a pharmaceutical formulation comprising an mRNA according to the invention and an mRNA prepared by the method of preparing an mRNA comprising a 5 '-phosphorothioate analogue according to the invention at the 5' end of the molecule and a pharmaceutically acceptable carrier.

Non-methylated compounds (GppSG and GpppSG) were synthesized as controls for biological studies.

Table 1 lists alkylating agents used to synthesize suitable modified nucleotides, which were first obtained by the present inventors. Tables 2 and 3 list the 5' -phosphorothioate cap analogs obtained and subsequently characterized by biophysical and biochemical methods.

Of the compounds listed in tables 2 and 3, particularly preferred for SMA treatment are 5 '-phosphorothioate analogues comprising a sulfur at the 5' position on the 7-methylguanosine side (compound numbers 12, 23, 24, 25, 26, 30, 31, 32, 33, 34 and 37), which are characterized by stability in the presence of the DcpS enzyme.

The documents cited in the specification and in the documents cited therein are also incorporated herein by reference.

Brief Description of Drawings

For a better understanding of the present invention, reference will now be made to the following examples and accompanying drawings, in which:

FIG. 1 illustrates the synthesis of 5 '-deoxy-5' -iodoguanosine analogs.

FIG. 2 illustrates the synthesis of 5' -deoxy-5 ' -thioguanine-5 ' -phosphorothioate. Synthesizing an A-guanosine derivative; synthesis of B-7-methylguanosine derivatives.

FIG. 3 illustrates the synthesis of 5' -phosphorothioate cap analogs by S-alkylation. A-terminal phosphorothioate for alkylation; b-scheme of alkylation reaction using 5 '-deoxy-5' -iodoguanosine (from FIG. 1) and terminal phosphorothioate as shown in A.

Figure 4 illustrates the synthesis of a 5' -phosphorothioate cap analog via imidazolidine. A-a compound used in the method; b-synthesis using two different activated derivative numbers 9 and 29 final compounds.

FIG. 5 illustrates stability studies of analogs modified by natural dinucleotide substrate hydrolysis and 5' -S of DcpS: subfigure A-Natural Cap analog m⁷Stability studies of gppppg against DcpS; subgraph B-cap analogue No. 20 stability study for DcpS enzyme (table 3); subgraph C-cap analogue number 21 was studied for stability of DcpS enzyme (table 3).

FIG. 7 illustrates a⁷GSpp_sCrystal structure of active site of pSG D2-complexed enzyme Δ N37 hDcpS.

FIG. 8 illustrates the susceptibility of short 26 nt RNA capped with various cap analogs (the transcript without the cap at its 5' end is 25 nt long) to Dcp1/2 enzyme activity incubated with SpDcp1/2 uncappingase. Reactions were carried out for 0, 5, 15, 30 minutes, and after termination of the reaction mixtures were resolved on a denaturing 15% polyacrylamide gel, which was stained with SYBR-Gold (Invitrogen) after completion of the electrophoretic separation. In each subgraph, the leftmost lane refers to the control, which is uncapped RNA.

FIG. 9 illustrates the relative susceptibility to Dcp1/2 enzyme activity as determined from the data of FIG. 8. The relative susceptibility to Dcp1/2 activity was calculated as the ratio of the intensity of the band corresponding to RNA capped at the 5' end to the sum of the intensities of the bands corresponding to capped and uncapped RNA. For individual RNAs, all values were normalized to time 0 minutes.

FIG. 10 illustrates the codes obtained from capping at the 5' end with various cap analogs in rabbit reticulocyte extractsRenillaRelative translational efficiency of a measurement of the translational efficiency of the luciferase's mRNA.

FIG. 11 illustrates the relative translational efficiencies measured in terms of luciferase activity at select time points in HeLa cells. The results are shown for the 5' end by m₂ ^{7, 2’-O}GSpppG or m₂ ^{7, 2’-O}Luciferase Activity measured on lysates of cells transfected with GpppSG-capped mRNA⁷Ratio of luciferase activity measured by lysates of GpppG capped mRNA transfected cells. Histograms represent the mean values from three biological replicates.

The chemical synthesis of 5' -phosphorothioate cap analogs is an inventive combination of three nucleotide synthesis methods based on the following chemistry:

1) imidazolidine nucleotide derivatives (see (Abrams and Schiff 1973); (Barnes, Waldrop et al 1983); (Kalek, Jemielite et al 2006) and (Kalek, Jemielite et al 2005))

2) By S-alkylation of halogen-containing nucleoside derivatives (see (Arakawa, Shiokawa et al 2003))

3) Synthesis of the terminal nucleosides β -thio-diphosphate and γ -thio-triphosphate (see (Zuberek, Jenieity et al 2003))

to synthesize the cap analog containing sulfur at the 5' position, the inventors developed two complementary methods that generally allowed the synthesis of all types of 5' -phosphorothioate analogs of mono-, di-, and triphosphates of nucleoside and dinucleotide cap analogs (FIGS. 2-4). method 1 (FIG. 2, and first step of FIG. 3) included S-alkylation reactions using 5' -deoxy-5 ' -iodonucleosides via nucleophilic substitution reactions of β -or γ -phosphorothioates.A second method (FIG. 4) to obtain dinucleotide compounds with a sulfur atom at the 5' position, in ZnCl as a catalyst, was used₂Coupling reactions between the appropriate imidazolidine nucleotide and the diphosphate in the previously activated form are used in the presence (in both cases, an S-alkylation reaction is used at selected stages).

the first method uses the corresponding phosphorothioate (mono, di, tri) with a phosphorothioate moiety in the terminal position the optimal conditions for this reaction are the use of equimolar amounts of phosphorothioate, 5' -iodonucleoside and DBU (1, 8-diazabicyclo (5.4.0) undec-7-ene) as base so far, using this method the inventors have obtained 9 different dinucleotide cap analogues comprising two units containing methylene modifications at positions α - β and β - γ of the triphosphate bridge (figure 3).

The second method for effective yield requires the presence of a divalent metal chloride, such as ZnCl₂It also improves solubility in organic media, protects the imidazolidine derivative from hydrolysis and accelerates the reaction rate by bringing the imidazole derivative and the phosphate ester of other molecules close to each other. The optimal conditions for this reaction are an 8-fold excess of ZnCl in DMF₂When present, 1.5 equivalents of imidazole derivative to diphosphate was used. Using the second method, the inventors obtainedthe other 95 ' -phosphorothioate cap analogues containing two sulfur at the 5' position and one sulfur at the β -non-bridging position of the triphosphate chain (fig. 4.) so far, the use of 5' -phosphorothioate analogues of nucleotides in this type of reaction has not been described, each analogue containing a β -S-sulfur atom is obtained as a mixture of diastereomers due to the presence of a stereocenter located at the phosphorus atom (referred to as D1 and D2 according to their elution order from the RP-HPLC column).

The obtained cap analogue was purified by ion exchange chromatography, DEAE Sephadex A-25, and if not pure enough, by preparative HPLC. The purified compounds were then tested for biochemical and biological properties.

Synthetic routes to generate cap analogs containing a sulfur atom at the 5' position are shown in FIGS. 1-4.

The resulting cap analog was then tested as a substrate for the human enzyme DcpS (hDcpS). As determined using Reverse Phase HPLC (RPHPLC), only 4 analogs: m is⁷GppSG (No. 21), m⁷GpppSG (No. 22), m⁷,^2'-OGpppSG (No. 38) and m⁷Gpp_spSG D1/D2 (Nos. 35-36) was hydrolyzed by DcpS. Other analogs containing a sulfur atom at the 5' position on the 7-methylated guanosine side resisted hydrolysis of hDcpS (comparison of the stability of the two different analogs (No. 22) and (No. 24) -fig. 5, table 4). In contrast to Compound Nos. 21, 22, 38, and 35-36, analog 37 (m)⁷GpCH₂ppSG) was additionally modified with methylene bisphosphonate moieties and also resistant to hydrolysis by the enzyme hDcpS (table 5). Then, the ability of these compounds to inhibit the enzyme hDcpS was determined using a fluorescence method and a fluorescent probe, while the parameter IC of the compound against the enzyme activity was determined₅₀(see patent application PL 406893). After the study, the resulting compounds were found to be very good inhibitors of the human enzyme DcpS.

Analogue No. 34, showing the best inhibitory properties for the hDcpS enzyme from all cap analogues tested, co-crystallizing with shortened form of the enzyme (Δ N37 hDcpS; full length enzyme does not form crystals), and complexing2.05A-resolved Structure of the material was determined by X-ray crystallography (FIG. 7.) the conformation of analog number 34 observed in the complex structure is clearly different from the unmodified cap analog m complexed with the catalytically inactive H277N hDcpS mutant (Gu, Fabrega et al 2004)⁷Conformation of gppppg (compound No. 0). A particularly significant difference between these two ligands was observed in the alignment of the triphosphate bridges, resulting in the exclusion of the gamma phosphate of analogue number 34 from the catalytic center. In addition, in addition to interacting with the typical cap of the C-terminal domain/DcpS enzyme complex, analog number 34 interacts with lysine 142 and tyrosine 143 residues through hydrogen bonds. These amino acids are located in the so-called hinge region, which connects the C-and N-terminal domains, which move relative to each other during the catalytic cycle.

The structure and purity of the obtained compound was determined by mass spectrometry and¹h and³¹p NMR confirmed.

m⁷The observation that GSpppG (compound No. 24) and its analogues were resistant to hDcpS was unexpected because the hydrolysis of the resulting compound proceeds via nucleophilic attack on the phosphate group of the adjacent 7-methylguanosine, consistent with a defined catalytic mechanism for the natural substrate.

In summary, the present invention describes structures and methods for synthesizing various analogs of the 5 'end of an mRNA (cap) containing a 5' -phosphorothioate moiety. The cap analogs, their properties against the enzyme DcpS, and methods of their use, particularly for treating Spinal Muscular Atrophy (SMA) and/or alleviating the symptoms of SMA, have not been previously described in the literature.

The analogs selected were used for mRNA synthesis using an in vitro transcription method with RNA SP6 polymerase (New England BioLabs). What percentage of the pool of transcripts having a length of 35 nucleotides was examined to have cap structures, and then these transcripts were examined for expression in Schizosaccharomyces pombe (Schizosaccharomyces pombe)Schizosaccharomyces pombe) The susceptibility to degradation of recombinase Dcp1/2 (example 2, experiment 4, FIG. 8, FIG. 9, Table 6). Full-length transcripts encoding luciferase (as reporter genes) in rabbit reticulocyte lysates (FIG. 1)0, example 2, experiment 5) and in HeLa cells transfected with modified mRNA (fig. 11, example 2, experiment 6). In both cases, mRNA translation efficiency was determined by examining the activity of the synthetic protein (luciferase) in both translation systems (table 6).

The terms used in the specification have the following meanings. In light of the present disclosure and the background of the description of the present patent application, terms not defined herein have the meanings that are set forth and understood by those skilled in the art. The following conventions, unless otherwise indicated, are used in the present specification, the terms having the meanings indicated in the definitions below.

Term "Alkyl radical"refers to a saturated straight or branched chain hydrocarbyl substituent having the specified number of carbon atoms. Examples of alkyl substituents are-methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, -n-hexyl, -n-heptyl, -n-octyl, -n-nonyl and-n-decyl. Representative branched- (C1-C10) alkyls include-isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, -neopentyl, -1-methylbutyl, -2-methylbutyl, -3-methylbutyl, -1, 1-dimethylpropyl, -1, 2-dimethylpropyl, -1-methylpentyl, -2-methylpentyl, -3-methylpentyl, -4-methylpentyl, -1-ethylbutyl, -2-ethylbutyl, -3-ethylbutyl, -1, 1-dimethylbutyl, -1, 2-dimethylbutyl, 1, 3-dimethylbutyl, -2, 2-dimethylbutyl, -2, 3-dimethylbutyl, -3, 3-dimethyl-butyl, -1-methylhexyl, 2-methylhexyl, -3-methylhexyl, -4-methylhexyl, -5-methylhexyl, -1, 2-dimethylpentyl, -1, 3-dimethylpentyl, -1, 2-dimethylhexyl, -1, 3-dimethylhexyl, -3, 3-dimethylhexyl, 1, 2-dimethylheptyl, -1, 3-dimethylheptyl, and-3, 3-dimethylheptyl, and the like.

Term "Alkenyl radical"refers to a saturated straight or branched chain acyclic hydrocarbyl substituent having the specified number of carbon atoms and containing at least one carbon-carbon double bond. Examples of alkenyl substituents are-vinyl, -allyl, -1-butenyl, -2-butenyl, -isobutenyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl, -1-hexenyl, -2-hexenyl, -3-hexenyl, -1-heptenyl, -2-heptenyl, -3-heptenyl, -1-octenyl, -2-octenyl, -3-octenyl, -1-nonenyl, -2-nonenyl, -3-nonenyl, -1-decenyl, -2-decenyl, -3-decenyl, and the like.

Term "Alkynyl radical"refers to a saturated straight or branched chain acyclic hydrocarbyl substituent having the specified number of carbon atoms and containing at least one carbon-carbon triple bond. Examples of alkynyl substituents are ethynyl, propynyl, -1-butynyl, -2-butynyl, -1-pentynyl, -2-pentynyl, -3-methyl-1-butynyl, 4-pentynyl, -1-hexynyl, -2-hexynyl, -5-hexynyl, and the like.

Term "Hetero atom"refers to an atom selected from oxygen, sulfur, nitrogen, phosphorus, and the like.

Term "HPLC"refers to high performance liquid chromatography, and a solvent designated as a solvent for" HPLC "refers to a solvent of suitable purity for HPLC analysis (high performance liquid chromatography).

Term "NMR"refers to nuclear magnetic resonance.

The term "cell system" refers to a cell capable of undergoing a protein biosynthetic process on an RNA template.

The term "acellular system" refers to a biological mixture, usually a lysate of animal or plant cells, containing all the components necessary for the biosynthesis of proteins based on an RNA template.

Modes for carrying out the invention

The following examples are provided only to illustrate the invention and explain various aspects thereof, are not provided to limit the same, and should not be equated with all the scope thereof, which is defined in the appended claims. The following examples, unless otherwise indicated, refer to the use of standard materials and methods used in the art or procedures recommended by the manufacturer for specific materials and methods.

Examples

General information relating to the Synthesis, isolation and characterization of novel Cap analogs

The nucleotide used as an intermediate is in DEAE Sephadex A-25 (HCO)₃ ^-Form) was purified by ion exchange chromatography using a linear triethylammonium bicarbonate (TEAB)/deionized water gradient. After evaporation under reduced pressure, 96% ethanol was added several times during this time to decompose the TEAB buffer and the intermediate was isolated as the triethylammonium salt. The final product (cap analogue) was purified in the same way, then by semi-preparative HPLC, and lyophilized several times for isolation as the ammonium salt. Analytical Reverse Phase HPLC (RPHPLC) was performed on an Agilent Technologies Series 1200 instrument with a Supelcosil LC-18 RP-T column (4.6X 250 mm, flow rate 1.3 ml/min) with a linear gradient of 0% to 25% methanol (procedure A)/0.05M ammonium acetate (pH 5.9) or 0% to 50% methanol (procedure B)/0.05M ammonium acetate (pH 5.9). Eluted compounds were detected using a UV-VIS detector (at 260 nm) and a fluorescence detector (excitation 260 nm, emission 370 nm). Preparative RP HPLC was performed on the same instrument using a Discovery RP Amide C16 column (21.2 mm × 250 mm, flow rate 5.0 ml/min) using a linear gradient of acetonitrile/0.05M ammonium acetate (pH 5.9) as the mobile phase.¹H NMR and³¹p NMR spectra were recorded at 25 ℃ on VarianUNITY-plus at frequencies 399.94 MHz and 161.90 MHz, respectively.¹H NMR chemical shift vs TSP (3-trimethylsilyl [2,2,3, 3-D4)]Sodium propionate)/D₂O (internal standard) report.³¹P NMR chemical shifts to 20% phosphoric acid/D₂O (external standard) report. With a negative [ MS ESI (-)]Or positive ion mode [ MS ESI (+)]The high-resolution mass spectra of (a) were recorded on a Micromass QToF 1 MS. Read out fluorescent plate readers were performed on a Tecan Infinit 200 PRO with 480 nm excitation and 535 nm emission. The samples were placed in black 96-well plates (Greiner). Crystallization was performed on 96-well plates with 3-lens wells (Swissci) using a pipetting robot Mosquito Crystal (TTp Labtech). Solvents and other reagents were purchased from Sigma-Aldrich and used without further purification unless otherwise stated below. Commercially available on Dowex 50 WX8 using ion exchange chromatographyThe resulting GMP and GDP sodium salts are converted to the triethylammonium salt. Triethylammonium salt and m⁷GMP and m⁷GDP sodium salt, m⁷GMP-Im and m⁷GDP-Im was obtained as described in the literature (Kalek, Jemielite et al 2005), (Jemielite, Fowler et al 2003). 5' -deoxy-5 ' -iodoguanosine, 5' -deoxy-5 ' -thioguanine-5 ' -monothiophosphate and triethylamine phosphorothioate were obtained as described in the literature ((Arakawa, Shiokawa et al 2003), (Zuberek, Jenieity et al 2003)). m is⁷GpCH₂The p-triethylammonium salt was prepared as described in the literature (Kalek, Jemielite et al 2006). GpCH₂ppS was prepared as described (Kowalska, Ziemniak et al 2008).

In the following examples, specific compounds are given reference to the figures in parentheses and indicate the number of the indicated substituent which corresponds to the specific number of the specific cap analogue.

Example 1 Synthesis and isolation of novel Cap analogs

General procedure for the Synthesis of 5' -iodonucleoside derivatives (FIG. 1, Nos. 3 and 4)

Iodine (3 mmol, M =253.81 g/mol) was added over 5 minutes to a magnetically stirred suspension of the corresponding nucleoside (1mmol), triphenylphosphine (3 mmol, M =262.29 g/mol) and imidazole (6 mmol, M =68.08 g/mol) in N-methyl-2-pyrrolidone (to a nucleoside concentration of 0.25 mol/l) at room temperature. The reaction was carried out over 3 h and the progress of the reaction was monitored using RP HPLC. The reaction mixture was then poured into CH₂Cl₂:H₂O solution (3:1, v/v), the reaction mixture was diluted 12 times. A white crystalline precipitate formed at the interface of the two layers during 24 h at 4 ℃. The precipitate is filtered off under reduced pressure, washed with dichloromethane and filtered over P₂O₅And (5) drying in vacuum.

5 '-deoxy-5' -iodoguanosine(FIG. 1, No. 3)

5 '-deoxy-5' -iodoguanosine (FIG. 1, No. 3) (10.4 g, 26.5 mmol, 75%) was obtained according to the general procedure starting from guanosine (FIG. 1, No. 1) (10 g, 35.3 mmol). t is t_R(B) = 12.36 min;

¹ ₆H NMR (400 MHz, DMSO-d) δ ppm10.65 (s, 1H, H-1), 7.89 (s, 1H, H-8),6.47 (bs, 2H, NH2), 5.68 (d, 1H, J = 6.26 Hz, H-1’), 5.51 (d, 1H, J = 6.26Hz, 2’-OH), 5.35 (d, 1H, J = 4.70 Hz, 3’-OH), 4.59 (q, 1H, J = 5.48 Hz, H-2’), 4.03 (q, 1H, J= 5.09, 3.13 Hz, H-3’), 3.90 (dt, 1H, J = 6.26, 3.13 Hz,H-4’), 3.53 (dd, 1H, J = 6.26, 5.87 Hz, H-5’), 3.39 (dd, 1H, J = 10.17, 6.65Hz, H-5’);HRMS ESI(-) to C₁₀H₁₁IN₅O₄ ^-, (M-H)^-Computingm/z391.9861, found 391.98610

5 '-deoxy-5' -iodo-2-O-methyl-guanosine(FIG. 1, No. 4)

2’-O-methyl-5 ' -deoxy-5 ' -iodo-guanosine (figure 1, No. 4) (328.8 mg, 0.81 mmol, 80%) was obtained following the general procedure starting from 2 ' -O-methylguanosine (figure 1, No. 2) (300 mg, 1.0 mmol). t is t_R(B) = 14.44 min;

¹ ₆H NMR (400 MHz, DMSO-d) δ ppm7.95 (s, 1H, H-8), 6.50 (bs, 2H, NH2),5.81 (d, 1H, J = 6.41 Hz, H-1’), 5.50 (d, 1H, J = 5.34 Hz, 3’-OH), 4.41, 4.40(2d, 1H, J = 6.26, 6.41 Hz, H-2’), 4.28-4.25 (m, 1H, H-3’), 3.97, 3.96 (2t,1H, J = 6.56, 3.05 Hz, H-4’), 3.56 (dd, 1H, J = 6.41, 10.38 Hz, H-5’), 3.43(dd, 1H, J = 10.53, 6.87, 6.71 Hz, H-5’), 3.30 (s, 3H, CH₃);

HRMS ESI (-)To C₁₁H₁₃IN₅O₄ ^-[M-H]^-Computingm/z406.0090, found 406.0021.

5 '-deoxy-5' -iodo-7-methylguanosine(FIG. 1, No. 5)

5 '-deoxy-5' -iodoguanosine (FIG. 1, number 3) (2g, 5.09 mmol) was dissolved in anhydrous DMF (20 mL) and Mel (2.5 mL, 40.7 mmol) was added. Subjecting the reaction mixture to magnetic forceStir at room temperature on a stirrer. The reaction progress was monitored by RP HPLC. When no starting material was observed, the reaction was stopped by addition of water (10 mL), and the excess methyl iodide was evaporated under vacuum and the reaction mixture was concentrated under reduced pressure. Then, CH was added to the remaining crude product₂Cl₂(100 mL) and a yellow precipitate formed. The precipitate is filtered off under reduced pressure and taken up with CH₂Cl₂(3X 20 mL) washing and passage through P₄O₁₀Vacuum drying for 24 h. Yield 1.6g (77.0%). t is t_R(B) = 11.94 min;

¹ ₂H NMR (400 MHz, DO) δ ppm5.98 (d, 1H, J = 3.91 Hz, H-1’), 4.81 (dd,1H, J = 4.70 Hz, H-2’), 4.31 (t, 1H, J = 5.09, H-3’), 4.15 (q, 1H, J = 5.48,H-4’), 4.07 (s, 3H, CH₃), 3.50-3.62 (m, 2H, J = 4.70, 5.87 Hz, H-5’);

HRMS ESI (+)Calculate m/z C₁₁H₁₅IN₅O₄ ⁺[M+H]⁺408.01687, found 408.01163.

5' -deoxy-5 ' -iodo-2 ' -O-methyl-7-methylguanosine(FIG. 1, No. 6)

5' -deoxy-5 ' -iodo-2 ' -O-methylguanosine (FIG. 1, No. 4) (200.8 mg, 0.49 mmol) was dissolved in anhydrous DMSO (3.3 mL) and Mel (0.25 mL, 3.9 mmol) was added. The reaction mixture was stirred on a magnetic stirrer at room temperature. The reaction progress was monitored by RP HPLC. When no starting material was observed, the reaction was quenched with water (10 mL), and NaHCO was used₃The pH was adjusted to neutral, excess methyl iodide was extracted with diethyl ether, and the aqueous phases were combined, followed by concentration of the mixture and purification by preparative HPLC to yield 45.8 mg of compound (77%). t is t_R(B) = 11.94 min;

¹ ₆H NMR (400 MHz, DMSO-d) δ ppm9.03 (s, 1H, H-8), 6.39 (bs, 2H, NH₂),5.95 (d, 1H, J = 4.27 Hz, H-1’), 4.40 (t, 1H, J = 4.58 Hz, H-2’), 4.24 (t,1H, J = 4.88 Hz, H-3’), 4.08-4.06 (m, 1H, H-4’), 4.02 (s, 3H, CH₃), 3.59 (dd,1H, J = 5.19, 4.88, 10.68 Hz, H-5’), 3.50 (dd, 1H, J = 7.93, 7.63, 10.68 Hz,H-5’), 3.41 (s, 3H, CH₃);

HRMS ESI (-)Calculate m/z C₁₂H₁₅IN₅O₄ ^-[M-H]^-420.01741, found 420.01758.

Guanosine 5' -deoxy-5 ' -thioguanine-5 ' -monothiophosphate (FIG. 2, number 7)

To 5 '-deoxy-5' -iodoguanosine (FIG. 2, No. 3) (2.0 g, 5.1 mmol) in 100 mL of DMF: H₂To the suspension in O mixture (1:1, v/v) was added sodium trithiophosphate (4.6 g, 25.5 mmol). The reaction mixture was stirred at room temperature for 24 h. The precipitate was removed by filtration and the filtrate was evaporated under reduced pressure. The residue was dissolved in 50 mL of water, and the excess sodium trithiophosphate was precipitated by adding 100 mL of methanol. After separation, the crude product was purified by ion exchange chromatography on Sephadex. The product was lyophilized. Yield 1.9 g (64%). t is t_R(B) = 4.24 min;

¹ ₂H NMR (400 MHz, DO) δ ppm8.05 (s, 1H, H-8), 5.89 (d, 1H, J = 5.73 Hz,H-1’), 4.85 (dd, 1H, J = 5.48 Hz, H-2’), 4.51 (2d, 1H, J = 4.98, 4.23 Hz, H-3’), 4.33-4.39 (m, 1H, H-4’), 3.16-3.08 (m, 2H, Hz, H-5’);³¹P NMR (162 MHz,D₂O) δ ppm 15.42 (s, 1P);

HRMS ESI (-)Calculate m/z C₁₀H₁₃N₅O₇PS^-[M-H]^-378.02788, found 378.02828.

Guanosine 5' -deoxy-5 ' -thio-7-methylguanosine-5 ' -monothiophosphate (FIG. 2, number 8)

To a suspension of 5 '-deoxy-5' -iodo-7-methylguanosine (FIG. 2, No. 6) (2.0 g, 4.92 mmol) in 100 mL of DMF was added sodium trithiophosphate (4.43 g, 24.6 mmol). The reaction mixture was stirred at room temperature for 48 h. The precipitate was removed and the filtrate was evaporated under reduced pressure. The residue was dissolved in 50 mL of waterAnd precipitating excess sodium trithiophosphate by adding methanol (100 mL). After separation, the crude product was purified by ion exchange chromatography on Sephadex. The product was lyophilized. Yield 1.55 g (53%). t is t_R(B) = 4.64 min;

¹ ₂H NMR (400 MHz, DO) δ ppm7.85 (s, 1H, H-8), 5.89 (d, 1H, J = 3.74 Hz,H-1’), 4.78-4.75 (m, 1H, H-2’), 4.43-4.39 (m, 2H, H-3’, H-4’), 4.09 (s, 3H,CH₃), 3.08-2.94 (m, 2H, Hz, H-5’);³¹P NMR (162 MHz, D₂O) δ ppm 14.45 (s, 1P);

HRMS ESI (-)Calculate m/z C₁₁H₁₅N₅O₇PS^-[M-H]^-392.04353, found 392.04378.

General procedure for the Synthesis of guanosine 5' -deoxy-5 ' -thioguanine-5 ' -monothiophosphate imidazolidine (FIG. 2, Nos. 9 and 10)

The appropriate starting compound (nucleotide TEA salt) (1mmol) was mixed with imidazole (10 mmol) and 2, 2' -dithiodipyridine (3 mmol) in DMF (to a nucleotide concentration of 0.15M). Then triethylamine (3 mmol) and triphenylphosphine (3 mmol) were added and the mixture was stirred at room temperature for 24 h. NaClO in anhydrous acetone (10 times the volume of DMF added) was added₄An anhydrous solution (4 mmol for each phosphoric acid moiety) resulted in precipitation of the product from the reaction mixture. Cooling to 4 deg.C, filtering out precipitate, washing with cold anhydrous acetone, and passing through P₄O₁₀And (5) drying in vacuum.

Guanosine 5' -deoxy-5 ' -thioguanine-5 ' -monothiophosphate imidazolidine (FIG. 2, number 9)

Guanosine 5 '-deoxy-5' -thioguanine-5 '-monothiophosphate imidazolidine (fig. 2, No. 9) (352 mg, 0.75mmol, 89%) was obtained according to the general procedure starting from 5' -deoxy-5 '-thioguanine-5' -monothiophosphate (fig. 2, No. 7) (500mg, 0.86 mmol). t is t_R(B) = 8.27 min;

³¹ ₂P NMR (162 MHz, DO) δ ppm11.69 (m, 1P);

HRMS ESI (-)To C₁₃H₁₅N₇O₆PS^-[M-H]^-428.05476 is calculated, 428.05452 is found.

5' -deoxy-5 ' -thioguanine-7-methylguanine-5 ' -monothiophosphate imidazolidine (FIG. 2, number 10)

5 '-deoxy-5' -thioguanine-7-methylguanine-5 '-monothiophosphate imidazolidine (FIG. 2, No. 10) (321 mg,0.69 mmol, 82%) was obtained according to the general procedure starting from 5' -deoxy-5 '-thio-7-methylguanine-5' -monothiophosphate (FIG. 2, No. 8) (500mg, 0.84 mmol). t is t_R(B)=8.39 min;

HRMS ESI (-)To C₁₄H₁₇N₇O₆PS^-[M-H]^-442.07041 is calculated, 442.07070 is found.

Guanosine 5' -deoxy-5 ' -thioguanine-5 ' -dithiophosphate (FIG. 2, number 11)

Guanosine 5' -deoxy-5 ' -thioguanine-5 ' -monothiophosphate imidazolidine (FIG. 2, No. 9) (100 mg, 0.22mmol) was dissolved in anhydrous DMF (2 mL), and tris (triethylammonium) phosphate (100 mg, 0.26 mmol) was added, followed by ZnCl₂(235.84 mg, 1.76 mmol). The progress of the reaction was controlled by RP-HPLC. The reaction mixture was stirred at room temperature until the starting material disappeared. The reaction was then stopped by the addition of aqueous EDTA (513.92 mg, 1.76 mmol, 50 mL) and 1M NaHCO₃And (4) neutralizing. The crude product was purified by ion exchange chromatography on DEAE-Sephadex and isolated as TEA salt. Yield: 108.5 mg (0.14 mmol, 65%);

HRMS ESI (-)to C₁₀H₁₄N₅O₁₀P₂S^-[M-H]^-457.99421 is calculated, 457.99481 is found.

5 '-deoxy-5' -thioguanine-7-methylguanine-diphosphate (FIG. 2, number 12)

5' -deoxy-5 ' -thioguanine-7-methylguanine-5 ' -monothiophosphate imidazolidine (FIG. 2, number 10) (100 mg,0.21 mmol) was dissolved in anhydrous DMF (2 mL), and tris (triethylammonium) phosphate (100 mg, 0.26 mmol) was added, followed by ZnCl₂(224.54 mg, 1.68 mmol). The progress of the reaction was controlled by RP-HPLC. The reaction mixture was stirred at room temperature until the starting material disappeared. The reaction was then stopped by the addition of aqueous EDTA (490.56 mg, 1.68 mmol, 50 mL) and 1M NaHCO₃And (4) neutralizing. The crude product was purified by ion exchange chromatography on DEAE-Sephadex and isolated as TEA salt. Yield: 91 mg (0.12 mmol, 54%), t_R(B) = 5.07 min,

¹ ₂H NMR (400 MHz, DO) δ ppm8.11 (s, 1H, H-8 slowly exchangeable), 5.97(d, 1H, J = 3.91 Hz, H-1’), 4.50, 4.49 (2d, 1H, J = 5.09 Hz, H-2’), 4.41 (q,1H, J = 5.48, 5.09 Hz, H-3’), 4.07 (s, 3H, CH₃), 3.34-3.13 (m, 3H, H-4’, H-5’);³¹P NMR (162 MHz, D₂O) δ ppm 6.71 (d, 1P, J = 30.81 Hz), 8.21 (d, 1P, J =30.81 Hz);

HRMS ESI (-)To C₁₁H₁₆N₅O₁₀P₂S- [M-H]^-472.00986 is calculated, 472.00967 is found.

Guanosine 5' -deoxy-5 ' -thio-7-methylguanosine-2 ' -dithiophosphate (FIG. 2, number 13)

5' -deoxy-5 ' -thioguanine-7-methylguanine-5 ' -monothiophosphate imidazolidine (FIG. 2, number 10) (100 mg,0.21 mmol) was dissolved in anhydrous DMF (2 mL), and sodium thiophosphate (47 mg, 0.26 mmol) was added, followed by ZnCl₂(224.54 mg, 1.68 mmol). The progress of the reaction was controlled by RP-HPLC. The reaction mixture was stirred at room temperature until the starting material disappeared, then stopped by adding aqueous EDTA (490.56 mg, 1.68 mmol, 50 mL) and 1M NaHCO₃And (4) neutralizing. The crude product was purified by ion exchange chromatography on DEAE-Sephadex and the isolated TEA salt was used directly in the coupling reaction. Yield: 110 mg (0.13 mmol, 64 %);

HRMS ESI (-)To C₁₁H₁₆N₅O₉P₂S₂ ^-[M-H]^-487.98702 is calculated, 487.98724 is found.

Synthesis of 5' -S-cap analogs by S-alkylation

General procedure

The nucleoside-terminal phosphorothioate TEA salt (1 eq) was suspended in DMSO (to a concentration of approximately 0.1-0.2M). Then, DBU (1, 8-diazabicyclo (5.4.0) undec-7-ene) (1 equivalent) and the derivative of 5' -iodoguanosine (1 equivalent) were added. The reaction progress was monitored by RP HPLC. The reaction was stopped after no signal from the terminal phosphorothioate by adding 1% acetic acid to pH =7, the reaction mixture was diluted with water and washed with ethyl acetate. The product was purified by ion exchange chromatography on DEAE-Sephadex and isolated as triethylammonium salt. The product was purified by semi-preparative RP-HPLC.

P1- (guanosine-5 '-yl) -P2- (5' -deoxy-5 '-thioguanine-5' -yl) diphosphate-GppSG (FIG. 3, numbering) 19)

GppSG (207 mOD, 0.009 mmol, 24%) starting from GDP β S (FIG. 3, number 14), (506 mOD, 0.042mmol) was obtained according to the general procedure RP-HPLC: t_R(A) = 6.9 min;

¹ ₂H NMR (400 MHz, DO) δ ppm7.96 (s, 1 H), 7.81 (s, 1 H), 5.77 (d, 1 H,J= 5.48 Hz), 5.70 (d, 1 H,J= 5.87 Hz), 4.80-4.70(m, 2H, overlap with water signal), 4.64(t, 1H,J= 5.48 Hz), 4.43 (t, 1 H, t,J= 3.91 Hz), 4.37 (t, 1 H,J= 3.91Hz), 4.30-4.15 (m, 4H), 3.30-3.13 (m, 2 H);

³¹ ₂P NMR (162 MHz, DO) δ ppm7.63 (d, 1P, J = 32.28, 12.5 Hz), -12.02 (d,1P, J = 30.81 Hz);

HRMS ESI (-) pair C₂₀H₂₅N₁₀O₁₄P₂S^-[M-H]^-723.07531 is calculated, 723.07546 is found.

P1- (7-methyl-guanosine-5 '-yl) -P2- (5' -deoxy-5 '-thioguanine-5' -yl) diphosphate-m ⁷ GppSG (FIG. 3, number 21)

m⁷GppSG (1028 mOD, 0.045 mmol, 9%) from m⁷GDP β S (FIG. 3, No. 17, 5830 mOD, 0.51mmol) obtained following the general procedure RP-HPLC: t_R(A) = 5.9 min;

¹ ₂H NMR (400 MHz, DO) δ ppm8.98 (s, 1 H, H-8 m⁷G), 7,87 (s, 1 H, H-8 G),5.88 (d, 1 H,J= 2.0 Hz, H-1’ m⁷G), 5.73 (d, 1 H,J= 5.7 Hz, H-1’ G), 4.69(t, 1 H,J= 5.5 Hz, H-2’ G), 4.51 (bs., 1 H, H-2’ m⁷G), 4,32 – 4.44 (m, 5 H,H-3’ G, H-3’ m⁷G, H-4’ G, H-4’ m⁷G, H-5’ m⁷G), 4.24 (dd, 1 H,J=11.3, 5.4 Hz,H5” m⁷G), 4.04 (s, 3 H, CH₃), 3.24 – 3.41 (m, 2 H, H5’, 5” G);

³¹ ₂P NMR (162 MHz, DO) δppm 7.38 (dt, 1P, J = 29.0, 11.5 Hz), -12.00 (d,1P, J = 32.23 Hz);HRMS ESI (-)To C₂₁H₂₇N₁₀O₁₄P₂S^-[M-H]^-737.09096 is calculated, 737.09052 is found.

P1- (7-methyl-5 '-deoxy-5' -thioguanin-5 '-yl) -P2-guanin-5' -yl diphosphate m ⁷ GSppG (graph) 3, number 23)

m⁷GSppG (1660 mOD, 0.073 mmol, 35%) starting from GDP β S (FIG. 3, No. 14; 2532 mOD, 0.21mmol) was obtained following the general procedure RP-HPLC: t_R(A) = 7.75 min;

¹ ₂H NMR (400 MHz, DO) δ ppm7.99 (s, 1 H, H-8 G), 5.83 (d, 1 H, J = 4,2Hz, H-1’ m⁷G), 5.80 (d, 1 H, J = 6.0 Hz, H-1’ G), 4.65 – 4.70 (2 H, m, H-2’G, H-2’ m⁷G), 4.45 (t, 1 H, J = 4.1 Hz, H-3’ G), 4.19 – 4.41 (5 H, m, H-3’m⁷G, H-4’ G, H-4’ m⁷G, H5’, 5” G), 4.05 (s, 3 H, CH₃), 3.35 – 3.43 (m, 2 H,H5’, 5” m⁷G);

³¹ ₂P NMR (162 MHz, DO) δ ppm7.32 (dt, 1P, J = 29.0, 11.0 Hz), -11.84 (d,1P, J = 29.00 Hz);HRMS ESI(-) to C₂₁H₂₇N₁₀O₁₄P₂S^-[M-H]^-737.09096 is calculated, 737.09146 is found.

P1- (guanosine-5 '-yl) -P3- (5' -deoxy-5 '-thioguanin-5' -yl) triphosphate-GpppSG (FIG. 3, numbering) 20)

GpppSG (1149 mOD, 0.051 mmol, 51%) from GTP_γS (FIG. 3, number 15; 1233 mOD, 0.10mmol) was obtained following the general procedure. RP-HPLC t_R(A) = 5.50 min;

¹ ₂H NMR (400 MHz, DO) δ ppm8.02 (s, 1 H, H-8 G), 7.90 (s, 1 H, H-8 G),5.82 (d, 1 H, J = 6.0 Hz, H-1’ G), 5.78 (d, 1 H, J = 6.2 Hz, H-1’ G), 4.84(t, 1 H, J = 5.7 Hz, H-2’ G), 4.74 (t, 1 H, J = 5.7 Hz, H-2’ G),4.52 (t, 1H, J = 4.2 Hz, H-3’ G), 4.47 (t, 1 H, J = 4.3 Hz, H-3’ G), 4.30 – 4.38 (m, 2H, H-4’, 5’ G), 4.27 (m, 2 H, H-4’, 5” G), 3.25 – 3.35 (m, 2 H, H5’, 5” G);

³¹ ₂P NMR (162 MHz, DO) δ ppm8.21 (dt, 1P, J = 27.00, 13.3 Hz), -11.34 (d,1P, J = 19.30 Hz), --23.78 (dd, 1P, J = 27.00, 19.30 Hz);

HRMS ESI(-) to C₂₀H₂₆N₁₀O₁₇P₃S^-[M-H]^-803.04164 is calculated, 803.04135 is found.

P1- (7-methyl-5' -deoxy)Oxy-5 ' -thioguanin-5 ' -yl) -P3-guanosine-5 ' -yl triphosphate-m ⁷ GSpppG (Panel) 3, number 24)

m⁷GSpppG (729 mOD, 0.032 mmol,13%) from GTP_γS (FIG. 3, number 15; 3000 mOD, 0.25mmol) and was obtained according to the general procedure. RP-HPLC t_R(A) = 5.36 min;

¹ ₂H NMR (400 MHz, DO) δ ppm8.92 (s, 1 H, H-8 m⁷G), 7.96 (s, 1 H, H-8 G),5.78 (d, 1 H, J = 4.30 Hz, H-1’ m⁷G), 5,74 (d, 1 H, J = 5.87 Hz, H-1’ G),4.63 (m, 2 H, H-2’ G, H2’ m⁷G), 4,48 (dd, 1 H, J = 4.43, 3.52 Hz, H-3’ m⁷G),4.36 – 4.26 (m, 4 H, H-3’ G, H-4’ G, H-4’ m⁷G, H-5’ G), 4.24 -4.19 (m, 1H, H-5” G), 4.00 (s, 3 H, CH₃), 3.33 – 3.24 (2 H, m, H-5’, 5” m⁷G);

³¹ ₂P NMR (162 MHz, DO) δ ppm7.57 (d, 1P, J = 27.88 Hz), -11.68 (d, 1P, J= 20.54 Hz), -24.00 (dd, 1P, J = 29.35, 22.01 Hz);

HRMS ESI(-) to C₂₁H₂₈N₁₀O₁₇P₃S^-[M-H]^-M/z 817.05729 was calculated and 817.05494 was found.

P1- (7-methyl-guanosine-5 '-yl) -P3- (5' -deoxy-5 '-thioguanol-5' -yl) triphosphate-m ⁷ GpppSG (FIG. 3, number 22)

m⁷GpppSG (1582 mOD, 0.07mmol, 32%) from m⁷GTP_γS (FIG. 3, number 18; 2616 mOD, 0.23mmol) was obtained following the general procedure. RP-HPLC t_R(A) = 6.06 min;

¹ ₂H NMR (400 MHz, DO) δ ppm9.02 (s, 1 H, H-8 m⁷G), 7.87 (s, 1 H, H-8 G),5.84 (d, 1 H, J = 3.52 Hz, H1’ m⁷G), 5.70 (d, 1 H, J = 6.65 Hz, H-1’ G), 4.80– 4.67 (m, 1 H, H-2’ G), 4.52 (t, 1 H, J = 4.30 Hz, H-2’ m⁷G), 4.41 (dd, 2 H,J=4.70, 4.30 Hz, H3’ G, H3’ m⁷G), 4.38 – 4.30 (m, 2 H, H-4’ G, H-4’ m⁷G),4.36-4.31 m, 2H, H5’ m⁷G), 4.02 (s, 3 H, CH₃), 3.30-3.20 (m, 2 H, J = 12.6,6.3 Hz, H5’, 5” G);

³¹ ₂P NMR (162 MHz, DO) δ ppm7.66 (d, 1P, J = 29.35 Hz), -11.73 (d, 1P, J= 22.01 Hz), -23.95 (dd, 1P, J = 22.01, 27.88 Hz);

HRMS ESI(-) to C₂₁H₂₈N₁₀O₁₇P₃S^-[M-H]^-817.05729 is calculated, 817.05748 is found.

P1- (2 ' -O-methyl-7-methyl-5 ' -deoxy-5 ' -thioguanin-5 ' -yl) -P3-guanosine-5 ' -yl triphosphate- m ₂ ^7,2’-O GSpppG (FIG. 3, number 26)

m₂ ^7,2’-OGSpppG (140 mOD, 0.006 mmol, 5%) from GTP_γS (FIG. 3, number 15; 1500 mOD, 0.12mmol) and was obtained according to the general procedure. RP-HPLC t_R(A) = 7.89 min;

¹ ₂H NMR (400 MHz, DO) δ ppm7.93 (s, 1H, G), 5.81 (d, 1H, J = 3.91 Hz, H-1’ m⁷G), 5.72 (d, 1H, J = 6.26 Hz, H-1’ G), 4.65 (t, 1H, J = 5.48 Hz, H-2’m⁷G), 4.43-4.40 (m, 1H, H-2’, G, H-3’ m⁷G), 4.32-4.18 (m, 6H, H-3’ G, H-4’, H-5’, G, m⁷G), 4.01 (s, 3H, CH₃), 3.52 (s, 3H, OCH₃);

³¹ ₂P NMR (162 MHz, DO) δ ppm7.35 (d, 1P, J = 26.41 Hz), -11.68 (d, 1P, J= 19.07 Hz), -24.02, -24.18 (2d, 1P, J = 26.41, 19.07 Hz);

HRMS ESI(-) to C₂₂H₃₀N₁₀O₁₇P₃S^-[M-H]^-M/z 831.07294 was calculated and 831.07477 was found.

P1- (7-methyl-5 '-deoxy-5' -thioguanidin-5 '-yl) -P3-guanosine-5' -yl-2, 3-methylene triphosphate- m ⁷ GSppCH ₂ pG (FIG. 3, number 25)

m⁷GSppCH₂pG (353 mOD, 0.016mmol, 27%) from m⁷GpCH₂pp γ S (FIG. 3, number 16; 717 mOD,0.06 mmol) was obtained following the general procedure. RP-HPLC t_R(A) = 6.36 min;

¹ ₂H NMR (400 MHz, DO) δ ppm9.03 (s, 1H, H-8, m⁷G), 8.17 (s, 1H, G), 5.83(d, 1H, J = 4.30 Hz, H-1’ m⁷G), 5.78 (d, 1H, J = 4.48 Hz, H-1’ G), 4.70-4.66(m, 2H, H-2’ m⁷G, H-2’, G), 4.46 (d, 1H, J = 3.91, 5.09 Hz, H-3’, G), 4.38-4.33 (m, 2H, H-3’, m⁷G, H-4’, G), 4.32-4.28 (m, 1H, H-4’, m⁷G), 4.26-4.20 (m,1H, H-5’, G), 4.19-4.13 (m, 1H, H-5” G), 4.02 (s, 3H, CH₃), 3.34-3.22 (m, 4H,H-5’, G, m⁷G);³¹P NMR (162 MHz, D₂O) δ ppm

³¹ ₂P NMR (162 MHz, DO) δ ppm17.03 (d, 1P, J = 10.27 Hz), 7.47-6.97 (m,2P);

HRMS ESI(-) to C₂₂H₃₀N₁₀O₁₆P₃S^-[M-H]^-M/z 815.07803 was calculated and 815.07923 was found.

Synthesis of 5' -S-cap analogs via imidazolidines

General procedure

5' S-GMP-Im, (FIG. 2, No. 9) (Na salt, 50 mg, 0.11 mmol) and the appropriate diphosphate (1mmol): m₂ ^O7,2’-GDP (FIG. 4, number 28), m⁷GpCH₂p (FIG. 4, number 27), m⁷The 5' S GDP (figure 2,number 12), m⁷GSpp β S (FIG. 2, number 13) or m⁷GDP β S (FIG. 3, number 17) was suspended in anhydrous DMF (1.0 mL) followed by addition of anhydrous ZnCl₂(95 mg, 10 eq, 0.7 mmol). The reaction mixture was shaken vigorously until the reagents were dissolved. The progress of the reaction was monitored by RP-HPLC. After completion (24 h), the appropriate amount of EDTA solution (Na) was added₂EDTA, 237 mg, 0.7 mmol), with solid NaHCO₃The pH was adjusted to 6, followed by ion exchange chromatography, purification of the crude product by DEAE-Sephadex and isolation as TEA salt (or direct purification by preparative HPLC). The product was then further purified by RP-HPLC.

P1- (2 ' -O-methyl-7-methyl-guanosine-5 ' -yl) -P3- (5 ' -deoxy-5 ' -thioguanine-5 ' -yl) triphosphate- m ₂ ^7,2’-O GpppSG (FIG. 4, number 38)

m₂ ^7,2’-OGpppSG (122 mOD, 0.005 mmol, 6%) fromm ₂ ^7,2’-O GDP (FIG. 4, number 28; 912 mOD,0.08 mmol) and was obtained by the general procedure. RP-HPLC t_R(A)=6.29 min;

¹ ₂H NMR (400 MHz, DO) δ ppm9.00 (s, 1H, H-8 m⁷G), 7.88 (s, 1H, G), 5.87(d, 1H, J = 2.74 Hz, H-1’ m⁷G), 5.69 (d, 1H, J = 6.65 Hz, H-1’ G), 4.64 (t,1H, J = 5.48 Hz, H-2’ m⁷G), 4.48 (dd, 1H, J = 4.48 Hz, H-2’, G), 4.43-4.38(m, 2H, H-3’,G, H-3’, m7G), 4.36-4.32 (m, 1H, H-4’, G), 4.30-4.26 (m, 1H, H-4’, m⁷G), 4.25-4.16 (m, 2H, H-5’, G), 4.03 (s, 3H, CH3), 3.53 (s, 3H, OCH₃),3.30-3.22 (m, 2H, H-5’, m⁷G);

³¹ ₂P NMR (162 MHz, DO) δ ppm7.68(d, 1P, J = 27.88 Hz), -11.68 (d, 1P, J =20.54 Hz), -23.78, -23.94 (2d, 1P, J = 27.88, 19.07Hz);

HRMS ESI(-) to C₂₂H₃₀N₁₀O₁₇P₃S^-[M-H]^-M/z 831.07294 was calculated and 831.07350 was found.

P1- (7-methyl-5 '-deoxy-5' -thioguanidin-5 '-yl) -P3-guanosine-5' -yl 1, 2-methylene triphosphate- m ⁷ GpCH ₂ PPSG (figure 4, number 37)

m⁷GpCH₂ppSG (1002 mOD, 0.044 mmol, 25%) from m⁷GpCH₂p (FIG. 4, No. 27; 2052 mOD,0.18 mmol) and 5' -S-GMP-Im (122 mg, 0.27 mmol) were obtained by the general procedure. RP-HPLC t_R(A) = 6.26 min,

¹ ₂H NMR (400 MHz, DO) δ ppm9.31 (s, 1H, H-8, m⁷G), 8.02 (s, 1H, G), 5.90(d, 1H, J = 3.13 Hz, H-1’ m⁷G) 5.75 (d, 1H, J = 5.87 Hz, H-1 'G), 4.80-4.70(m, 2H, overlap with solvent signal, H-2'm)⁷G, H-2’, G), 4.58 (dd, 1H, J = 3.91, 3.48 Hz,H-3’, G),4.48 (t, 1H, H-3’, m⁷G), 4.40 (dd, 1H, J = 3.91, 4.06, H-4’, G),4.37-4.29 (m, 3H, H-4’, m⁷G, H-5’, G), 4.19-4.13 (m, 2H, H-5’, G), 4.03 (s,3H, CH₃), 3.30-3.19 (m, 2H, H-5’, G, m⁷G), 2.40 (t, 2H, J = 20.35 Hz, CH₂);

³¹ ₂P NMR (162 MHz, DO) δ ppm17.11 (d, 1P, J = 8.80 Hz), 7.64-6.76 (m,2P);

HRMS ESI(-) to C₂₂H₃₀N₁₀O₁₆P₃S^-[M-H]^-M/z 815.07803 was calculated and 815.07906 was found.

P1- (7-methyl-5 '-deoxy-5' -thioguanosin-5 '-yl) -P3- (5' -deoxy-5 '-thioguanosin-5' -yl) triphosphates Acid-m ⁷ GSpppSG (FIG. 4, number 32)

m⁷GSpppSG (768 mOD, 32 mg, 0.028 mmol, 40%) from m⁷Starting from 5 'S-GDP (57 mg, 0.07mmol) and 5' S-GMP-Im (50 mg, 0.11 mmol)General procedure was obtained. RP-HPLC t_R(A) =6.80 min;

¹ ₂H NMR (400 MHz, DO) δ ppm8.38 (s, 1H, H-8 m⁷G slowly exchangeable), 7.84 (s, 1H, G), 5.78 (d, 1H, J = 4.70 Hz, H-1'm)⁷G), 5.69 (d, 1H, J = 6.65 Hz, H-1’ G),4.64 (t, 1H, J = 4.70 Hz, H-2’ m⁷G), 4.40, 4.39 (2d, 1H, J = 2.74, 3.52, 4.40Hz, H-3’, G), 4.36-4.29 (m, 3H, H-4’ G, H-5’, G), 3.99 (s, 3H, CH₃), 4.37-4.28 (m, 3H, H-4’, H-5’, m⁷G);

³¹ ₂P NMR (162 MHz, DO) δ ppm7.74 (t, 2P, J = 27.88), -24.61 (t, 1P, J =29.35 Hz);

HRMS ESI(-) to C₂₁H₂₈N₁₀O₁₆P₃S₂ ^-[M-H]^-M/z 833.03445 was calculated and 833.03550 was found.

P1- (7-methyl-5 '-deoxy-5' -thioguanidin-5 '-yl) -P3-guanosine-5' -yl 2-thiotriphosphate- m ⁷ GSpp _s pG D1/D2 (FIG. 4, numbered 30 and 31, respectively)

m⁷GSpp_spG (1080 mOD, 45 mg, 0.039 mmol, 56%) from m⁷GSpp β S (56 mg, 0.07mmol) and 5' S-GMP-Im (50 mg, 0.11 mmol) starting with the general procedure, obtained as a mixture of diastereomer D1/D2 the diastereomer was separated using RP-HPLC and isolated as ammonium salt D1 (FIG. 4, number 30): 438 mOD, 18 mg, 0.016mmol, 23%) RP-HPLC: t_R(A) = 6.56 min;

¹ ₂H NMR (400 MHz, DO) δ ppm8.98 (s, 1H, H-8, m⁷G), 8.08 (s, 1H, G), 5.82(d, 1H, J = 4.27 Hz, H-1’ m⁷G), 5.77 (d, 1H, J = 5.80Hz, H-1’ G), 4.67-4.65(m, 2H, H-2’ m⁷G, H-2’, G), 4.49-4.47 (m, 1H, H-3’, G), 4.39-4.35 (m, 1H, H-3’, m⁷G, H-4’, G), 4.33-4.23 (m, 3H, H-4’, m⁷G, H-5’, G), 4.02 (s, 3H, CH₃),3.38-3.25 (m, 2H, H-5’, m⁷G);

³¹ ₂P NMR (162 MHz, DO) δ ppm29.18 (dd, 1P, J = 34.83, 27.37 Hz), 6.96(dt, 1P, J = 34.83, 12.44 Hz), -12.37 (d, 1P, J = 27.37 Hz);

HRMS ESI(-) to C₂₁H₂₈N₁₀O₁₆P₃S₂ ^-[M-H]^-833.03445, calculating m/z, and 833.03549 actually measured;

d2 (FIG. 4, number 31) m⁷GSpp_spGD2(380 mOD, 16 mg, 0.014 mmol, 20%) RP-HPLC:t_R(A) = 6.71 min;

¹ ₂H NMR (400 MHz, DO) δ ppm8.98 (s, 1H, H-8, m⁷G), 8.14 (s, 1H, G), 5.82(d, 1H, J = 4.27 Hz, H-1’ m⁷G), 5.77 (d, 1H, J = 5.49 Hz, H-1’ G), 4.69-4.65(m, 2H, H-2’ m⁷G, H-2’, G), 4.49-4.45 (m, 1H, H-3’, G), 4.40-4.35 (m, 1H, H-3’, m⁷G, H-4’, G), 4.34-4.21 (m, 3H, H-4’, m⁷G, H-5’, G), 4.03 (s, 3H, CH₃),3.39-3.24 (m, 2H, H-5’, m⁷G);

³¹ ₂P NMR (162 MHz, DO) δ ppm29.44-28.67 (m, 1P), 7.17-6.54 (m, 1P), -12.09-(-12.72) (m, 1P);

HRMS ESI(-) to C₂₁H₂₈N₁₀O₁₆P₃S₂ ^-[M-H]^-833.03445 is calculated, 833.03606 is found.

P1- (7-methyl-5 '-deoxy-5' -thioguanin-5 '-yl) -P3- (5' -deoxy-5 '-thioguanin-5' -yl) 2-thio Substituted triphosphate-m ⁷ GSpp _s pSG D1/D2 (FIG. 4, numbered 33, 34, respectively)

m⁷GSpp_spSG (942 mOD, 39 mg, 0.003 mmol, 48%) from m⁷GSpp β S (56 mg, 0.07mmol) and 5' S-GMP-Im (50 mg, 0.11 mmol) starting with the general procedure, obtained as a mixture of diastereomer D1/D2 the diastereomer was separated using RP-HPLC and isolated as ammonium salt D1 (FIG. 4, number 33) (510 mOD, 21 mg, 0.018mmol, 26%) RP-HPLC: t_R(A) = 7.53 min;

¹ ₂H NMR (400 MHz, DO) δ ppm9.00 (s, 1H, H-8, m⁷G), 7.99 (s, 1H, G), 5.83(d, 1H, J = 4.27 Hz, H-1’ m⁷G) 5.75 (d, 1H, J = 6.10 Hz, H-1 'G), 4.79-4.68(m, 2H, overlap with solvent signal, H-2'm)⁷G, H-2’, G), 4.44 (dd, 1H, J = 4.58 Hz, H-3’,G), 4.41-4.33 (m, 3H, H-3’, m⁷G, H-4’, G, m⁷G), 4.03 (s, 3H, CH₃), 3.39-3.26(m, 4H, H-5’, G, m⁷G);

³¹ ₂P NMR (162 MHz, DO) δ ppm28.25 (t, 1P, J = 34.83 Hz), 7.31-6.74 (m,2P);

HRMS ESI(-) to C₂₁H₂₈N₁₀O₁₅P₃S₃ ^-[M-H]^-849.01161 is calculated, 849.01213 is found.

D2 (FIG. 4, number 34) (274 mOD, 11 mg, 0.0098 mmol, 14%), RP-HPLC: t_R(A)= 7.62 min;

¹ ₂H NMR (400 MHz, DO) δ ppm8.99 (s, 1H, H-8, m⁷G), 8.04 (s, 1H, G), 5.83(d, 1H, J = 4.58 Hz, H-1’ m⁷G) 5.75 (d, 1H, J = 6.10 Hz, H-1 'G), 4.78-4.66(m, 2H, overlap with solvent signal, H-2'm)⁷G, H-2’, G), 4.46-4.42 (m, 1H, H-3’, G), 4.41-4.34 (m, 3H, H-3’, m⁷G, H-4’, G, m⁷G), 4.04 (s, 3H, CH₃), 3.39-3.24 (m, 4H, H-5’, G, m⁷G);

³¹ ₂P NMR (162 MHz, DO) δ ppm28.29 (t, 1P, J = 34.83 Hz), 7.32-6.68 (m,2P);

HRMS ESI(-) to C₂₁H₂₈N₁₀O₁₅P₃S₃ ^-[M-H]^-849.01161 is calculated, 849.01217 is found.

P1- (7-methyl-guanosine-5 '-yl) -P3- (5' -deoxy-5 '-thioguanine-5' -yl) 2-thiotriphosphate- m ⁷ Gpp _s pSG D1/D2 (FIG. 4, numbered 35, 36, respectively)

m⁷GppspSG (1941 mOD, 0.086 mmol, 28%) from m⁷GDP β S (3492 mOD, 0.31 mmol) and 5' S-GMP-Im (5550 mOD, 0.46 mmol) were obtained as a mixture of diastereomers D1/D2 following the general procedure, the diastereomers were separated using RP-HPLC and isolated as ammonium salts D1 (FIG. 4, number 35): 888 mOD, 0.039 mmol,13%) RP-HPLC: t_R(A) = 7.12 min;

¹ ₂H NMR (400 MHz, DO) δ ppm9.07 (s, 1H, H-8, m⁷G), 7.95 (s, 1H, G), 5.88(d, 1H, J = 3.52 Hz, H-1’ m⁷G), 5.74 (d, 1H, J = 6.26 Hz, H-1’ G), 4.80-4.70(m, 2H, H-2’ m⁷G, H-2', G and D₂O signal overlap), 4.56 (dd, 1H, J = 4.70, 3.52 Hz, H-3 ', G), 4.47-4.40 (m, 2H, H-3', m)⁷G, H-4’, G), 4.39-4.33 (m, 3H, H-4’,m⁷G, H-5’, G), 4.03 (s, 3H, CH₃), 3.35-3.20 (m, 2H, H-5’, m⁷G);

³¹ ₂P NMR (162 MHz, DO) δ ppm29.00 (dd, 1P, J = 33.75, 26.41Hz), 6.98 (d,1P, J = 33.75, Hz), -12.56 (d, 1P, J = 24.94 Hz);

HRMS ESI(-) to C₂₁H₂₈N₁₀O₁₆P₃S₂ ^-[M-H]^-833.03445, measured 833.03514;

d2 (FIG. 4, number 36) m⁷GppspG D2 (1053 mOD, 0.046 mmol, 15%) RP-HPLC: t_R(A)= 7.42 min;

¹ ₂H NMR (400 MHz, DO) δ ppm9.04 (s, 1H, H-8, m⁷G), 7.95 (s, 1H, G), 5.85(d, 1H, J = 3.52 Hz, H-1’ m⁷G), 5.73 (d, 1H, J = 6.26 Hz, H-1’ G), 4.80-4.70(m, 2H, H-2’ m⁷G, H-2', G and D₂O signal overlap), 4.54 (dd, 1H, J = 4.30, 3.91 Hz, H-3 ', G), 4.45 (t, 1H, J = 5.09 Hz, H-3', m)⁷G), 4.43-4.40 (m, 1H, H-4’, G),4.39-4.32 (m, 3H, H-4’, m⁷G, H-5’, G), 4.03 (s, 3H, CH₃), 3.37-3.21 (m, 2H, H-5’, m⁷G);

³¹ ₂P NMR (162 MHz, DO) δ ppm28.99 (dd, 1P, J = 33.75, 26. 41, 24.94Hz),6.94 (d, 1P, J = 35.21, Hz), -12.48 (d, 1P, J = 24.94 Hz);

HRMS ESI(-) to C₂₁H₂₈N₁₀O₁₆P₃S₂ ^-[M-H]^-833.03445 is calculated, 833.03494 is found.

EXAMPLE 2 characteristics of the novel Cap analogs

Test 1 study of the susceptibility of the analogs to DcpS enzymatic degradation

The purpose of this experiment was to examine whether the novel 5' -phosphorothioate cap analogue was hydrolysed by the human DcpS enzyme (hDcpS). Recombinant human proteins encoding the DcpS enzyme were expressed as described previously (Kowalska, Lewdorowicz et al 2008). The susceptibility of the novel analogs to hydrolysis of hDcpS was tested in 50 mM Tris-HCl buffer containing 200 mM KCl and 0.5 mM EDTA. The reaction mixture included the cap analogue tested (20. mu.M) and the hDcpS enzyme (100 nM)/400. mu.l buffer. At appropriate intervals, 100 μ l samples were collected from the reaction mixture. The samples were incubated at 98 ℃ for 2.5 min, then cooled to 0 ℃ and analyzed on RP-HPLC under the conditions described in the general information. In the experiments, commercially available DcpS inhibitors, compound RG3039 (No. 000) (https:// www.mda.org/quest/fda-aproves-phase-1-clinical-trial-RG 3039-sma), GppSG (No. 19), GpppSG (No. 20) and m⁷GpppG (number 0) and m⁷Gpp (number 00) was used as a control. Exemplary results obtained are shown in fig. 5 and table 5.

Test 2. IC of selected inhibitors₅₀Measurement of

The objective of this assay is to determine the concentration at which a given inhibitor inhibits DcpS activity to 50% of the maximum under specific conditions. The buffer was the same for this assay and assay 1. 10 mixture reactions were prepared simultaneously, and they each contained m in 200. mu.l buffer⁷GMPF (60. mu.M), hDcpS enzyme (50 nM) and test compound at a concentration range of 0-50. mu.M. After a suitable time, the reaction was stopped by mixing with 100. mu.l ACN when 30% of the substrate was converted to product without inhibitor. A25 μ l sample was taken for analysis, then mixed with 90 μ l of a 2.5 μ M TBDS-fluorescein in DMSO solution, and incubated for 60 min. Next, 100 μ l of 200 mM HEPES buffer pH = 7.0 was added to the sample and fluorescence was measured as described in the general information. Based on the results, the correlation of inhibitor concentration to fluorescence was plotted, and IC was determined by fitting a theoretical curve to the data₅₀The value is obtained. The results obtained are provided in table 5 and fig. 5.

TABLE 5 IC of selected Compounds₅₀Value and susceptibility to DcpS enzymatic degradation

Numbering	Compound (I)	DcpS susceptibility	IC₅₀ [μM]
				0	m⁷GpppG	Hydrolyzable	nd
00	m⁷Gpp	Resistant/inhibitory agent	4.30 ± 0.78
				000	RG3039	Resistant/inhibitory agent	0.041 ± 0.012
12	m⁷GSpp	Resistant/inhibitory agent	1.93 ± 0.38
				19	GppSG	Hydrolyzable	Higher than 100
20	GpppSG	Hydrolyzable	Higher than 100
				21	m⁷GppSG	Hydrolyzable	nd
22	m⁷GpppSG	Hydrolyzable	nd
				23	m⁷GSppG	Resistant/inhibitory agent	2.81 ± 0.51
24	m⁷GSpppG	Resistant/inhibitory agent	0.84 ± 0.07
				25	m⁷GSppCH ₂pG	Resistant/inhibitory agent	6.25 ± 1.22
26	m^7,2’OGSpppG	Resistant/inhibitory agent	12.57 ± 5.22
				30	m⁷GSpp_spG D1	Resistant/inhibitory agent	0.23 ± 0.04
31	m⁷GSpp_spG D2	Resistant/inhibitory agent	0.17 ± 0.02
				32	m⁷GSpppSG	Resistant/inhibitory agent	0.33 ± 0.09
33	m⁷GSpp_spSG D1	Resistant/inhibitory agent	0.26 ± 0.04
				34	m⁷GSpp_spSG D2	Resistant/inhibitory agent	0.051 ± 0.008
35	m⁷Gpp_spSG D1	Hydrolyzable	nd
				36	m⁷Gpp_spSG D2	Hydrolyzable	nd
37	m⁷GpCH ₂ppSG	Resistant/inhibitory agent	5.67 ± 1.01
				38	m^7,2’OGpppSG	Hydrolyzable	72 ± 17

Run 3. with analogous number 34 (m)⁷GSpp_spSGD2) Structure determination of Complex human DcpS enzyme (. DELTA.N 37hDcpS)

The purpose of this experiment was to investigate the interaction mechanism of analogue number 34 with the human DcpS enzyme. Recombinant human DcpS enzyme truncated at the N-terminus (. DELTA.N 37-residues Ala38 to Ser337) was obtained as described previously (Singh et al 2008). Crystallization was performed by sitting-drop vapor diffusion using 0.2 uL of a sample containing 0.1M analog 34 and 7.3 mg/mL DcpS enzyme (incubated on ice for 15 min prior to crystal set-up) and 0.2 uL of the stock solution. After about one week, complex crystallization occurred in a mixture containing 29% PEG 4000 and 0.1MTris · HCl pH 7.6. A mixture of the storage solution and glycerol (1:1 v/v) was added to the droplets containing the crystals, which were then harvested and flash frozen in liquid nitrogen. Diffraction data were collected at 100K with a synchrotron source (Beamline 14.1, Bessy II, Helmholtz-Zentrum Berlin, Germany) using a Dectripilates 6M detector, and then processed using XDS software (Kabsch 2010). The structure was resolved by Molecular Replacement using Phaser software (McCoy, Grosse-Kunstleve et al 2007) to give the structure of DcpS (pdb: 3BL9) (Singh, Salcius et al 2008) bound to DG157493 inhibitor as a study model. Ligand models and codebases were generated using ProDRG (Schuttelkopf and van Aalten 2004). Model construction and ligand fitting were performed with Coot software (Emsley & Cowtan 2004). Refinishing of structures was performed using phenix.

Test 4. study of the susceptibility of short RNA molecules comprising a cap analogue at the 5' end to degradation by Dcp1/2 enzyme

The objective of this study was to examine whether incorporation of a selected 5 '-phosphorothioate cap analogue into the 5' end of the RNA could affect the susceptibility of the transcripts thus prepared to Dcp1/2 decapping enzyme activity. Recombinant protein of Schizosaccharomyces pombe in the form of heterodimers Dcp1/2 was obtained as described previously (Floor, Jones et al 2010). The transcripts used in this experiment were obtained by in vitro transcription using RNA SP6 polymerase (New England BioLabs). The annealed oligonucleotides ATACGATTTAGGTGACACTATAGAAGAAGCGGGCATGCGGCCAGCCATAGCCGATCA (SEQ ID NO.: 1) and TGATCGGCTATGGCTGGCCGCATGCCCGCTTCTTCTATAGTGTCACCTAAATCGTAT (SEQ ID NO: 2), which comprise the promoter sequence of SP6 polymerase (ATTTAGGTGACACTATAGA (SEQ ID NO: 3)), allowing to obtain 35 nt long RNA with sequence GAAGAAGCGGGCAUGCGGCCAGCCAUAGCCGAUCA (SEQ ID NO: 4), whereas the 5' end-capped RNA is 36 nt long, were used as templates for in vitro transcription. A typical in vitro transcription reaction was performed in a volume of 20 μ l and incubated at 40 ℃ for 2 hours and comprised the following: 1U SP6 polymerase, 1U RiboLock RNase inhibitor (ThermoFisher S)cientific), 0.5 mM ATP/CTP/UTP, 0.125 mM GTP, 1.25 mM dinucleotide cap analogue and 0.1. mu.M template. After 2 hours incubation, 1U of DNase I (Ambion) was added to the reaction mixture and incubation was continued for 30 min at 37 ℃ before EDTA was added to a final concentration of 25 mM. RNA Clean was used for the obtained RNA&Concentrator-25 (ZymoResearch). The quality of the synthesized RNA was then determined on a denaturing 15% polyacrylamide gel. The concentration of RNA was further evaluated spectrophotometrically. The RNA thus obtained was characterized by significant heterogeneity at the 3' end, and therefore to eliminate this problem, the obtained RNA was incubated with DNAzyme 10-23 (TGATCGGCTAGGCTAGCTACAACGAGGCTGGCCGC (SEQ ID NO: 5)), which resulted in obtaining RNA 25 nt long. RNA with a cap at the 5' end was 26 nt long. The reaction to cleave the 3' end is as follows: 1 μ M RNA and 1 μ M DNazyme 10-23 in the presence of 50 mM MgCl₂And 50 mM Tris-HCl pH 8.0 for 1 hour at 37 ℃ (Coleman et al, 2004).

For the enzymatic assay, 20 ng of each RNA was used, with 3.5 nM Dcp1/2 enzyme in 50 mM Tris-HClpH 8.0, 50 mM NH₄Cl, 0.01% NP-40, 1mM DTT and 5mM MgCl₂Is incubated in the buffer of (1). The reaction was carried out at 37 ℃ in a final volume of 25. mu.l. The reaction was stopped after 0, 5, 15 and 30 min by adding an equal amount of a mixture of 5M urea, 44% formamide, 20 mm edta, 0.03% bromophenol blue, 0.03% xylene blue. The reaction products were resolved on a denaturing 15% polyacrylamide gel, and after completion of the electrophoretic separation, the gel was stained with SYBR Gold (Invitrogen) and visualized using Storm 860PhosphorImager (GE Healthcare). The results obtained were quantified using ImageQuant software (Molecular Dynamics). Representative results of this assay are provided in fig. 8, 9, and table 6.

TABLE 6 biological Properties of mRNAs comprising selected cap analogs at the 5' end

	Efficiency of capping^a	Dcp1/2 susceptibility^b	Relative translation efficiency^c
				GpppG	0.91	0	0.05 ± 0.01
m⁷GpppG	0.93	0.69	1.00
				m₂ ^{7, 2’-O}GpppG	0.84	0.52	1.56 ± 0.14
m₂ ^{7, 2’-O}Gpp_SpG D2	0.82	0.43	3.45 ± 0.42
				m₂ ^{7, 2’-O}GSpppG	0.70	0.07	1.73 ± 0.24
m₂ ^{7, 2’-O}GpppSG	0.76	0.52	2.23 ± 0.31

^aThe data of fig. 8 (time point 0') was used to calculate capping efficiency.

^bThe data of FIG. 8 was used to calculate susceptibility to Dcp1/2 activity, provided as the ratio of capped RNA to the sum of uncapped and capped RNA at 15 min for each RNA and after normalization to 0' time.

^cRelative translation efficiency was shown normalized to m for the 5' end⁷Biological triplicates after values obtained for GpppG capped mRNARenillaAverage translation efficiency of luciferase mRNA.

Test 5 investigation of the Effect of the Presence of novel Cap analogs in Rabbit reticulocyte lysates on the translation efficiency of mRNA

The aim of this study was to examine the effect of introducing a novel cap analogue at the 5' end of the mRNA on translation efficiency. For this purpose, a series of preparations were preparedRenillaLuciferase encodes mRNA, which differs in the cap structure at the 5' end. Transcripts for this assay were obtained by an in vitro transcription reaction using SP6 RNA polymerase. As a template for in vitro transcription, PCR products were used, which were prepared using primers ATTTAGGTGACACTATAGAACAGATCTCGAGCTCAAGCTT (SEQ ID NO: 6) and GTTTAAACATTTAAATGCAATGA (SEQ ID NO: 7) and hRLuc-pRNA2(A)128 plasmid (Williams et al 2010). The PCR reaction thus carried out allows to encodeRenillaThe promoter sequence of SP6 polymerase was introduced upstream of the sequence of luciferase. The transcription reaction itself is analogous to the short RNA synthesis described above (experiment 4). The reaction was carried out for 2 hours at 40 ℃ in 20 μ l, and included the following: 1U SP6 polymerase, 1U RiboLock RNase inhibitor (ThermoFisher Scientific), 0.5 mM ATP/CTP/UTP, 0.125 mM GTP, 1.25 mM dinucleotide cap analogue and 100 μ g of template. After 2 hours incubation, 1U DNase I (Ambion) was added and continued at 37 ℃Incubation was for 30 min, after which EDTA was added to a final concentration of 25 mM. The obtained mRNA was purified using NucleoSpin RNA Clean-up XS (Macherey-Nagel). The quality of the synthesized RNA was checked on a denaturing 15% polyacrylamide gel. The RNA concentration was determined spectrophotometrically.

In vitro translation reactions were performed in rabbit reticulocyte lysates (RRL, Promega) under conditions determined for cap-dependent translation (Rydzik et al, 2009). A typical reaction mixture (10 μ l) contains: 40% RRL lysate, 0.01mM amino acid mixture (Promega), 1.2 mM MgCl₂170 mM potassium acetate and a suitable cap analog at the 5' endRenillaLuciferase-encoding mRNA, the mixture was incubated at 37 ℃ for 1 hour. Four different concentrations of mRNA: 0.1 ng/mul, 0.25 ng/mul, 0.5 ng/mul, 0.75 ng/mul were used for the experiment. The activity of the synthesized luciferase was measured in a microplate reader Synergy H1 (BioTek) using the dual-luciferase reporter assay system (Promega). The results obtained were analyzed in Origin (Gambit) software and a theoretical curve was fitted to the experimental data, where the slope of the curve obtained represents the translation efficiency. Representative data are provided in fig. 10, while the average translational efficiencies obtained for the biological three replicates are provided in table 6.

Test 6 investigation of the Effect of the Presence of novel Cap analogs on the translation efficiency of mRNA in HeLa cells

Human cervical cancer HeLa cells in DMEM (Gibco) supplemented with 10% FBS (Sigma-Aldrich), 1% penicillin/streptomycin (Gibco) and L-glutamine at a final concentration of 2mM in 5% CO₂And 37 ℃ growth. 10 μ l of antibiotic-free medium suspended in 100 μ l of medium the day before the planned experiment⁴Individual cells were seeded into each well of a 96-well plate. Cell Transfection was performed by adding 0.3. mu.l Lipofectamine MessengerMAX Transfection Reagent (Invitrogen), 0.1. mu.g mRNA and 10. mu.l Opti-MEM (Gibco) to each well. Transfection was performed in an incubator for 1 hour. After transfection, cells were washed three times with PBS and supplemented with fresh medium without antibiotics. After 2,3, 4.5, 6.5, 10.5 and 24 hours from the start of transfection, cells were washed three times with PBS and lysedLysis, and measurement of luciferase activity using the luciferase reporter assay system (Promega) using a Synergy H1 microplate reader. Exemplary data is shown in fig. 11.

mRNA encoding firefly luciferase and two repeats of β -globin 3' UTR at the 3' end and a poly (A) tail of 128 adenines was used for transfection, this mRNA, containing different cap analogs at the 5' end, obtained by in vitro transcription A pJET _ luc _128A plasmid digested with AarI (ThermoFisher scientics) was used as the synthesis template A typical in vitro transcription reaction was performed at 40 ℃ for 2 hours in a 20 μ l volume and contained 1U SP6 polymerase, 1 URiboLock RNase inhibitor (ThermoFisher Scientific), 0.5 mM ATP/CTP/UTP, 0.125 mMGTP, 1.25 mM dinucleotide cap analog, and 0.1 μ g template.

Sequence listing

<110> university of Huasha

<120>5 "-phosphorothioate mRNA 5' -end (cap) analogs, mrnas comprising said analogs, methods of obtaining and uses thereof

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atttaggtga cactatagaa cagatctcga gctcaagctt 40

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<211>23

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<213> Artificial

<220>

<223> oligonucleotide 4

<400>7

gtttaaacat ttaaatgcaa tga

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Sequence listing

<110> university of Huasha

<120>5 '-phosphorothioate mRNA 5' -end (cap) analogs, mRNAs comprising said analogs, methods of obtaining and uses thereof

<130>PZ/3566/AGR/PCT

<160>7

<170>PatentIn version 3.5

<210>1

<211>57

<212>DNA

<213> Artificial

<220>

<223> oligonucleotide 1

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tgatcggcta tggctggccg catgcccgct tcttctatag tgtcacctaa atcgtat 57

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gaagaagcgg gcaugcggcc agccauagcc gauca 35

<210>5

<211>35

<212>DNA

<213> Artificial

<220>

<223>DNazym 10-23

<400>5

tgatcggcta ggctagctac aacgaggctg gccgc 35

<210>6

<211>40

<212>DNA

<213> Artificial

<220>

<223> oligonucleotide 3

<400>6

atttaggtga cactatagaa cagatctcga gctcaagctt 40

<210>7

<211>23

<212>DNA

<213> Artificial

<220>

<223> oligonucleotide 4

<400>7

gtttaaacat ttaaatgcaa tga 23

Claims

1. 5' -phosphorothioate cap analogs according to formula 1

Wherein

L¹And L²Independently selected from O and S, wherein L₁And L₂Is not O;

n = 0, 1 or 2;

X₁、X₂、X₃independently selected from O, S;

R¹is selected from CH₃、C₂H₅、CH₂Ph, alkyl or substituted alkyl;

R²and R³Independently selected from H, OH, OCH₃、OC₂H₅、-COOH、N₃Alkyl, alkenyl or alkynyl;

And B is a radical according to formula 3, 4, 5, 6 or 7

。

2. The compound of claim 1 selected from:

。

3. a compound according to claim 1 or 2 selected from:

。

4. 5' -phosphorothioate analogues according to formula 2

Wherein

m = 0、1；

n = 0, 1 or 2;

L¹is S;

X₁、X₂、X₃independently selected from O, S;

R¹is selected from CH₃、C₂H₅、CH₂Ph, alkyl or substituted alkyl;

Y₁、Y₂independently selected from CH₂、CHCl、CCl₂、CHF、CF₂NH and O.

5. The compound of claim 4 which is 5' -deoxy-5 ' -thioguanine-7-methylguanine 5' -dithiophosphate according to formula 13

And (3) formula 13.

6. A compound according to any one of claims 1 to 5 for use as a medicament.

7. A compound according to any one of claims 1 to 5 for use as a medicament for the treatment of Spinal Muscular Atrophy (SMA) and/or for alleviating a symptom of SMA.

8. Use of a compound according to any one of claims 1 to 5 in the manufacture of a medicament.

9. Use according to claim 6, characterized in that the compound is used for the preparation of a medicament for the treatment of Spinal Muscular Atrophy (SMA) and/or for alleviating the symptoms of SMA.

10. Use of a compound according to any one of claims 1 to 5 as a modulator of DcpS activity, preferably as an inhibitor of DcpS enzyme activity, more preferably as an inhibitor of hDcpS enzyme activity.

11. Use of a compound according to any one of claims 1 to 5 for modulating mRNA degradation and/or modulating mRNA splicing.

12. A pharmaceutical formulation comprising a compound of any one of claims 1-5 and a pharmaceutically acceptable carrier.

13.5 '-deoxy-5' -iodoguanosine analogs having structures according to formulae 10, 11 and 12 shown below

。

14. A process for the preparation of a compound according to formula 1, said process comprising the steps of: 5' -iodonucleosides according to formula 8

Formula 8

Wherein

and B is a radical according to formula 3, 4, 5, 6 or 7

Wherein

m=0、1

n = 0, 1 or 2;

L¹is O or S;

X₁、X₂、X₃independently selected from O, S;

R¹is selected from CH₃、C₂H₅、CH₂Ph, alkyl or substituted alkyl;

wherein if n = 0 and m = 1, then X₃Is S; and X₁Is O;

if n = 1 and m = 0, X₂Is S; and X₁Is O;

if n = 1 and m = 1, X₃Is S; and X₁、X₂Is O;

to form a 5' -phosphorothioate cap analog according to formula 1

Wherein

L¹And L²Is independently selected from the group consisting of O and S,wherein L is₁And L₂Is not O;

n = 0, 1 or 2;

X₁、X₂、X₃independently selected from O, S;

R¹is selected from CH₃、C₂H₅、CH₂Ph, alkyl or substituted alkyl;

And B is a radical according to formula 3, 4, 5, 6 or 7

。

15. The process according to claim 14, characterized in that equimolar amounts of the compound according to formula 2 and the compound according to formula 8 and DBU (1, 8-diazabicyclo (5.4.0) undec-7-ene) as base are used.

16. A process for the preparation of 5' -phosphorothioate analogues according to formula 2a wherein the imidazolidine derivative according to formula 9

Wherein

R¹Is selected from CH₃、C₂H₅、CH₂Ph, alkyl or substituted alkyl;

Wherein

n = 0, 1 or 2;

L¹is O or S;

X₁and X₂Independently selected from O, S;

R¹is selected from CH₃、C₂H₅、CH₂Ph, alkyl or substituted alkyl;

Y¹is selected from CH₂、CHCl、CCl₂、CHF、CF₂NH and O.

17. A process according to claim 16, characterized in that the reaction is carried out in the presence of a divalent metal chloride.

18. The method of claim 17, wherein the divalent metal chloride is zinc chloride (ZnCl)₂。

19. Process according to claim 17 or 18, characterized in that a 1.5-fold excess of the imidazolidine derivative according to formula 9 relative to the phosphate or phosphorothioate group is used in the presence of an 8-fold excess of the divalent metal chloride.

20. A process for the preparation of a compound according to formula 1 comprising the steps of:

imidazolidine derivatives according to formula 9

Wherein

R¹Is selected from CH₃、C₂H₅、CH₂Ph, alkyl or substituted alkyl;

Wherein

n = 1

L¹Is O or S;

X₁and X₂Independently selected from O, S;

R¹is selected from CH₃、C₂H₅、CH₂Ph, alkyl or substituted alkyl;

Y₁independently selected from CH₂、CHCl、CCl₂、CHF、CF₂And (c) NH and O,

to form a 5' -phosphorothioate cap analog according to formula 1

Wherein

L¹And L²Independently selected from O and S, wherein L₁And L₂Is not O;

n = 0, 1 or 2;

X₁、X₂、X₃independently selected from O, S;

R¹is selected from CH₃、C₂H₅、CH₂Ph, alkyl or substituted alkyl;

And B is a radical according to formula 3, 4, 5, 6 or 7

。

21. A process according to claim 20, characterized in that the reaction is carried out in the presence of a divalent metal chloride.

22. The method according to claim 21, characterized in that the divalent metal chloride is zinc chloride ZnCl₂。

23. Process according to claim 21 or 22, characterized in that a 1.5-fold excess of the imidazolidine derivative according to formula 9 relative to the compound according to formula 2a is used in the presence of an 8-fold excess of the divalent metal chloride.

24. An mRNA comprising at the 5 'end a 5' -phosphorothioate cap analogue according to any one of claims 1 to 3.

25. The mRNA of claim 24, wherein the 5' -phosphorothioate cap analog is selected from m⁷GSpppG (number 24), m^7,2’OGSpppG (number 26), m⁷GSpppSG (No. 32), m⁷GSpp_spG D1 (number 30), m⁷GSpp_spG D2 (number 31), m⁷GSpp_spSG D1 (No. 33), m⁷GSpp_spSG D2 (No. 34), more preferably it is m^7,2’OGSpppG (number 26).

26. method for the preparation of an mRNA comprising a 5' -phosphorothioate cap analogue at the 5' end of the mRNA molecule, characterized in that the 5' -phosphorothioate cap analogue of any one of claims 1-3 is incorporated during synthesis of the mRNA molecule.

27. The method for the preparation of the mRNA of claim 26, characterized in that the 5' -phosphorothioate cap analog is selected from the group consisting of m⁷GSpppG (number 24), m^7,2’OGSpppG (number 26), m⁷GSpppSG (No. 32), m⁷GSpp_spG D1 (number 30), m⁷GSpp_spG D2 (number 31), m⁷GSpp_spSG D1 (No. 33), m⁷GSpp_spSG D2 (No. 34), more preferably it is m^7,2’OGSpppG (number 26).

28. Method for the preparation of mRNA according to claim 26 or 27, characterized in that the synthesis of mRNA is performed by in vitro transcription.

29. An mRNA prepared by the method of any one of claims 26-28.

30. Use of an mRNA comprising a 5 '-phosphorothioate cap analogue at the 5' -end of any one of claims 24-25 and 29 for the production of a protein.

31. Use of an mRNA according to claim 30, characterized in that the protein production is carried out in a cellular system or a non-cellular system.

32. The mRNA of any one of claims 24 to 25 and 29 for use as a medicament.

33. The mRNA of any one of claims 24-25 and 29 for use as a medicament for treating Spinal Muscular Atrophy (SMA) and/or alleviating a symptom of SMA.

34. The mRNA of any one of claims 24 to 25 and 29 for use as an anti-cancer medicament, more preferably for use as a medicament for anti-cancer immunotherapy.

35. Use of the mRNA of any one of claims 24-25 and 29 for the manufacture of a medicament.

36. Use according to claim 33, characterized in that the mRNA is used for the preparation of a medicament for the treatment of Spinal Muscular Atrophy (SMA) and/or alleviating the symptoms of SMA, as an anti-cancer medicament, more preferably as a medicament for anti-cancer immunotherapy.

37. A pharmaceutical formulation comprising the mRNA of any one of claims 24-25 and 29 and a pharmaceutically acceptable carrier.