RU2795220C2 - Methods for treatment of bladder cancer - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to a method for the treatment of bladder cancer, including having the REF STING allele, the WT STING allele, the AQ STING allele or the HAQ STING allele, including the administration to a patient in need of treatment of an effective amount of Compound 1 or its pharmaceutically acceptable salt or a pharmaceutical composition containing Compound 1 or its pharmaceutically acceptable salt, as well as to the use of Compound 1 or its pharmaceutically acceptable salt for the preparation of a pharmaceutical composition for the treatment of bladder cancer.

EFFECT: treatment of bladder cancer, which makes it possible to avoid or delay radical cystectomy.

(Connection 1)

17 cl, 13 dwg, 7 tbl, 112 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

Not applicable.

BACKGROUND OF THE INVENTION

STING (interferon gene stimulator) is a signaling molecule for the innate response to dsDNA in the cytosol. There are reports of STING deletion seen in some cancers. In addition, dysregulation of STING signaling in human cancers such as melanoma has also been reported (Xia T, et al., "Recurrent Loss of STING Signaling in Melanoma Correlates with Susceptibility to Viral Oncolysis" Cancer Res. 2016) and cancer large intestine. (Xia T, et al., "Deregulation of STING Signaling in Colorectal Carcinoma Constrains DNA Damage Responses and Correlates With Tumorigenesis" Cell Rep. 2016; 14:282-97). It is noteworthy that in these studies, the results of genomic analysis showed that the reason for the loss of STING expression is not a gene deletion or mutation, but epigenetic changes (Xia, Cancer Res., 2016; Xia, Cell Rep., 2016). The anti-cancer activity of STING is also supported by data from studies in mice. STING knockout mice were observed to have impaired tumor control (Woo SR, et al. "STING-dependent cytosolic DNA sensing mediates innate immune recognition of immunogenic tumors" Immunity 2014;41:830-42).

In addition, the role of STING in protecting ontogeny has been demonstrated in several spontaneous mouse models, including glioma (Ohkuri T, et al., "Protective role of STING against gliomagenesis: Rational use of STING agonist in anti-glioma immunotherapy" Oncoimmunology. 2015; 4 :e999523) and colon cancer (Zhu Q, et al., "Cutting edge: STING mediates protection against colorectal tumorigenesis by governing the magnitude of intestinal inflammation" J. Immunol. 2014; 193:4779-82). This antitumor effect may be related to the ability of STING to counteract excessive NF-kB and STAT3 activation (Woo 2014; Chandra D, et al. "STING ligand c-di-GMP improves cancer vaccination against metastatic breast cancer" Cancer Immunol Res. 2014; 2 :901-10; Corrales L, et al., "Direct Activation of STING in the Tumor Microenvironment Leads to Potent and Systemic Tumor Regression and Immunity" Cell Rep. 2015; 11:1018-30; Curran E, et al. "STING Pathway Activation Stimulates Potent Immunity against Acute Myeloid Leukemia" Cell Rep. 2016; 15:2357-66; Tang CH, et al. "Agonist-Mediated Activation of STING Induces Apoptosis in Malignant B Cells" Cancer Res. 2016; 76:2137- 52). This antitumor activity is likely associated with disruption of the tumor vasculature and subsequent induction of an adaptive immune response (Corrales L, et al., "The host STING pathway at the interface of cancer and immunity" J. Clin. Invest. 2016; 126:2404 -eleven). Accordingly, direct or intratumoral stimulation of STING in the tumor microenvironment by an agonist may represent a new approach to cancer treatment.

In addition, bladder cancer is the fourth and eleventh most common cancer in men and women, respectively, and is distinguished by its unique route of drug administration to patients with early-stage cancer (Kamat et al., “What is new in non-muscle-invasive bladder cancer in 2016?" Turk J Urol 2017; 43(1):9-13). Approximately 81,190 new cases of bladder cancer were diagnosed in the US in 2018, and approximately 17,240 deaths from this disease were expected (American Cancer Society website). BCG (Bacillus Calmette-Gueri) is the first-line therapy for high-risk non-muscle invasive bladder cancer (NMIBC). Unfortunately, up to 40% of patients do not respond to BCG treatment, and many of these patients need to undergo a radical cystectomy procedure to completely remove the bladder (Zlotta, A.R., et al., "The management of BCG failure in non-muscle-invasive bladder cancer: an update,” Can Urol Assoc J 2009; 3(Suppl4): S199-205). As a result, the quality of life of patients after surgery deteriorates. Consequently, there is a significant unmet medical need for effective alternative treatments for bladder cancer to avoid or delay radical cystectomy.

SUMMARY OF THE INVENTION

In embodiments, the invention provides a method of treating bladder cancer in a patient in need of treatment, comprising administering to the patient Compound 1 or a pharmaceutically acceptable salt thereof:

(Соединение 1).

(Compound 1).

In some embodiments, the pharmaceutically acceptable salt administered according to the embodiments provided herein is a diammonium salt or a triethylamine (TEA) salt. In further embodiments, treatment of bladder cancer is described by administering to a patient in need of treatment for bladder cancer a pharmaceutical composition comprising Compound 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

In additional embodiments, the invention provides a method of treating bladder cancer in a patient in need of treatment, comprising administering Compound 2 or a pharmaceutically acceptable salt thereof to the patient:

Соединение 2.

Connection 2.

In further embodiments, the invention provides the use of Compound 1, Compound 2, or a pharmaceutically acceptable salt thereof, as disclosed herein, for the preparation of a pharmaceutical composition for the treatment of bladder cancer.

Embodiments of the invention provide the use of Compound 1, Compound 2, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising Compound 1, Compound 2, or a pharmaceutically acceptable salt thereof, in the treatment of bladder cancer.

In embodiments, the invention provides a method for treating bladder cancer, comprising identifying an individual whose bladder cancer is treatable with Compound 1, Compound 2, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising Compound 1, Compound 2, or a pharmaceutically acceptable salt thereof; and administering to said individual a therapeutically effective amount of Compound 1, Compound 2, a pharmaceutically acceptable salt, or a pharmaceutical composition by which the bladder cancer is identified as being treatable.

In some embodiments, an individual is identified as having bladder cancer treatable with Compound 1, Compound 2, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing Compound 1, Compound 2, or a pharmaceutically acceptable salt thereof, by the presence of a variant allele in said individual REF STING.

In some embodiments, the invention provides a method of treating bladder cancer in a patient having a REF STING allele, comprising administering to said patient Compound 1, Compound 2, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising Compound 1, Compound 2, or a pharmaceutically acceptable salt thereof.

In some embodiments, the invention provides a method of treating cancer in a patient having the WT STING allele, comprising administering to said patient Compound 1, Compound 2, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing Compound 1 or a pharmaceutically acceptable salt thereof.

In some embodiments, the invention provides a method of treating cancer in a patient having an AQ STING allele, comprising administering to said patient Compound 1, Compound 2, a pharmaceutically acceptable salt of Compound 1, Compound 2, or a pharmaceutical composition containing Compound 1, Compound 2, or a pharmaceutically acceptable salt thereof. .

In some embodiments of the invention, a method of treating cancer in a patient having the HAQ STING allele is provided, comprising administering to said patient Compound 1, Compound 2, a pharmaceutically acceptable salt of Compound 1, Compound 2, or a pharmaceutical composition containing Compound 1, Compound 2, or a pharmaceutically acceptable salt thereof.

In some embodiments of the invention, Compound 1 is provided as the free acid. In some embodiments, the compound is provided as an NH ₄ salt or a triethylamine (TEA) salt.

In embodiments, the invention provides a method of treating bladder cancer in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of Compound 1, or a pharmaceutically acceptable salt thereof, as described above.

In embodiments, the invention provides a method of treating bladder cancer in a patient, comprising administering to said patient Compound 1, Compound 2, or a pharmaceutically acceptable salt or pharmaceutical composition thereof, as disclosed herein. Bladder cancer, the treatment of which is disclosed in the present description, may be a urothelial carcinoma.

BRIEF DESCRIPTION OF THE DRAWINGS

In FIG. 1 shows the synthesis of Compound 1a. This synthesis is also described in US Patent Application No. 15/898,533, filed Feb. 17, 2018, and incorporated herein by reference.

In FIG. 2A and FIG. 2B shows an alternative synthesis of Compound 1 and Compound 1a. This alternative synthesis is also shown in FIG. 2C - fig. 2E.

In FIG. 3 shows the ¹ H NMR spectrogram of Compound 1.

In FIG. 4A, FIG. 4B and FIG. 4C shows the results of X-ray crystallography analysis (FIG. ORTEP) of an asymmetric crystal of Compound 1, a first asymmetric crystal molecule, and a second asymmetric crystal molecule, respectively.

In FIG. 5 shows an X-ray crystal structure image of human WT STING in complex with compound 1.

In FIG. 6 shows the C-terminal domain of human REF STING in complex with Compound 1.

In FIG. 7 shows tumor volumes quantified by MRI on days 20-21.

In FIG. 8 shows survival curves for all of the bladder cancer treatment groups described in Example 4.

In FIG. 9 shows an expression vector map for WT STING (pLenti-WT human STING-Puro).

Fig. 10 and FIG. 11 refer to example 108 and represent the therapeutic activity of compound 1a in the CT26 twin tumor model.

Fig. 12 refers to Example 109 and is a graph of the volume of tumors that have been treated and the survival curve.

Fig. 13 refers to example 110 is a graph of the volume of tumors that were treated and the survival curve.

DETAILED DESCRIPTION OF EMBODIMENTS

Provides a compound (Compound 1) and pharmaceutically acceptable salts thereof, as well as pharmaceutical compositions that may be useful in the treatment of bladder cancer. Compound 2 or a pharmaceutically acceptable salt thereof, as well as pharmaceutical compositions comprising Compound 2 or a pharmaceutically acceptable salt thereof, may also be useful in the treatment of bladder cancer. The compounds can activate the interferon gene stimulator (STING).

Compound 1 is:

.

.

Compound 2 is:

.

.

In some embodiments, Compound 1 is provided as the free acid. In some embodiments, the compound is present, for example, as an NH ₄ salt. Reference to "Compound 1a" refers to the diammonium salt of Compound 1.

In embodiments, the invention provides a method for treating bladder cancer in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of Compound 1, Compound 2, or a pharmaceutically acceptable salt thereof.

Also provided are pharmaceutical compositions for the treatment of bladder cancer comprising a compound disclosed herein, or a pharmaceutically acceptable salt thereof, as well as a pharmaceutically acceptable excipient. The embodiments of the invention described herein may be used in the treatment of bladder cancer or in the preparation of medicaments useful in the treatment of bladder cancer.

It is known to those skilled in the art that substituents attached to phosphorus atoms (P ₁ , P ₂ ) having both single and double bonds can be subject to tautomerization. For example, tautomerization of a compound may occur under equilibrium conditions. One example is shown below:

.

.

Such tautomers are to be considered as being within the scope of the claims. A structural representation of any tautomer of a given compound will represent the same compound.

Treatment Methods

In embodiments of the invention, there is provided a method for treating bladder cancer in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of Compound 1, or a pharmaceutically acceptable salt thereof.

In some embodiments, Compound 1 is provided as the free acid or a pharmaceutically acceptable salt thereof. In some embodiments, the administered compound is in the form of an NH ₄ salt, the free acid, or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is provided as an NH ₄ salt.

In some embodiments, the invention provides a method of treating bladder cancer comprising administering to a patient in need of treatment a therapeutically effective amount of a STING agonist. In such embodiments, administration may optionally be intravesical. Examples of potential STING agonists that can be used in the treatment of bladder cancer, and which may optionally be administered intravesically, include, but are not limited to, the compounds presented in US Patent Application 15/898,533, filed Feb. 17, 2018; PCT Application PCT/US2018/018556, filed February 17, 2018; PCT Application PCT/US2018/018561, filed February 17, 2018; US 2014/0205653A1; US 2014/0329889 A1; US 2014/0341976A1; US 2007/0149462A1; WO 2018/098203A1; WO 2018/198076 Al; WO 2018/198084A1; WO 2018/140831A2; US 2014/0341976A1; WO 2015/185565 Al; US 7,709,458 B2; US 7,592,326; US 7,569,555 B2; US 2014/0205653A1; US 2014/0341976A1; US 2015/0056224A1; US 2016/0362441 A1; US 2017/0158724A1; US 2017/044206A1; US 5,547,941; US 7,569,555 B2; US 7,592,326 B2; US 7,709,458 B2; US 9,549,944 B2; WO 2009/133560 Al; WO 2015/074145A1; WO 2015/077354A1; WO 2015/185565 Al; WO 2016/100261A1; WO 2016/120305 A1; WO 2016/145102 A1; WO 2017/027645A1; WO 2017/027646A1; WO 2017/075477A1; WO 2019/043634A2; WO 2019/046496A1; WO 2019/046498A1; WO 2019/046500A1; WO 2019/046511A1; WO 2017/093933 A1; WO 2017/123657 A1; WO 2017/175156A1; EP 1740.192 B1; CN 102199183A; Fu, J., et al., "STING Agonist Formulated Cancer Vaccines Can Cure Established Tumors Resistant to PD-1 Blockade," Sci. Translational Med., 7(283):283ra52, (April 15, 2015); Corrales, L. et al., "Direct Activation of STING in the Tumor Microenvironment Leads to Potent and Systemic Tumor Regression and Immunity," Cell Reports, 11:1018-1030 (2015); and Lioux, T. et al., "Design, Synthesis, and Biological Evaluation of Novel Cyclic Adenosine-Inosine Monophosphate (cAIMP) Analogs That Activate Stimulator of Interferon Genes (STING)," J. Med. Chem. 59:10253-10267 (2016). All of these documents, including the compounds mentioned therein, are incorporated into the present description by reference; if any component of any of these documents conflicts or is otherwise inconsistent with the present disclosure, then the present disclosure takes precedence.

Dosage

The optimal dose for the treatment of bladder cancer can be determined empirically for each individual using known methods and will depend on many factors, including the activity of the agents; age, body weight, general health, sex and diet of the individual; time and method of administration; and other medicines the person is taking. Optimal doses can be determined by routine testing and by routine procedures well known in the art. The above compounds can be administered by any suitable route.

A "pharmaceutically acceptable salt" as used herein refers to salts resulting from an addition reaction between an acid or a base and the compounds of the present invention. A pharmaceutically acceptable salt is any salt that retains the activity of the parent compound and does not have any unduly deleterious or undesirable effect on the body of the subject to which it is administered and in the context in which it is administered. Pharmaceutically acceptable salts include, without limitation, metal complexes and salts of both inorganic and carboxylic acids. Pharmaceutically acceptable salts also include metal salts such as aluminum, calcium, iron, magnesium, manganese, and complex salts. In addition, pharmaceutically acceptable salts include, without limitation, acid salts such as salts of acetic, aspartic, alkylsulfonic, arylsulfonic, axetyl, benzenesulfonic, benzoic, bicarbonate, bisulfonic, divinic, butyric, calcium edetate, camsylic, carbonic, chlorobenzoic, citric , edetic, 1,2-ethandisulfonic (edisylic), laurylseric (estolic), ethanesulfonic (esyl), ethanesulfinic (esylic), formic, fumaric, glucoheptonic (gluceptic), gluconic, glutamic, glycolic, glycolylarsanilic, hexamine, hexylresorcinol, hydrabamine, hydrobromic, hydrochloric acid, hydrochloride, hydroiodic, hydroxynaphthoic, isethic, lactic, lactobionic, maleic, malic, malonic, mandelic, methanesulfonic, methylnitric, methylsulfuric, mucic, muconic, napsilic, nitric, oxalic, p-nitromethanesulfonic, pamoic, pantothenic, phosphoric , monohydrophosphoric, dihydrophosphoric, phthalic, polygalacturonic, propionic, salicylic, stearic, succinic, sulfanilic, sulfonic, sulfuric, tannic, tartaric, chlorotheophyllic (teoclic), toluenesulfonic acid, etc. Sodium salts and potassium salts can also be prepared.

Embodiments may be diammonium salts. Pharmaceutically acceptable salts may be derived from amino acids, including but not limited to cysteine. Methods for preparing compounds in the form of salts are known to those skilled in the art (see, for example, Stahl et al., Handbook of Pharmaceutical Salts: Properties, Selection, and Use, Wiley-VCH; Verlag Helvetica Chimica Acta, Zurich, 2002; Berge et al. ., J. Pharm. Sci. 66:1, 1977).

An "effective amount" or "therapeutically effective amount" of a therapeutic agent is an amount sufficient to produce an observable therapeutic improvement over bladder cancer in an untreated subject or patient.

The active agents disclosed herein may be combined with a pharmaceutically acceptable carrier to form pharmaceutical compositions based thereon. The specific choice of carrier and composition will depend on the particular route of administration for which the composition is intended.

In the context of the present description, the term "pharmaceutically acceptable carrier" refers to a non-toxic carrier, adjuvant or carrier medium that does not interfere with the pharmacological activity of the compound with which it forms the composition. Pharmaceutically acceptable carriers, adjuvants, or carriers that can be used in the compositions of the present invention include, without limitation, sorbic acid, potassium sorbate, mixtures of partial glycerides of saturated vegetable fatty acids, water, salts or electrolytes, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride. , zinc salts, colloidal silicon dioxide, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene glycol and lanolin.

The compositions of the present invention may be suitable for parenteral, oral, inhalation spray, topical, rectal, nasal, buccal, intravesical, vaginal, or implanted reservoir administration, and the like. In some embodiments, the implementation of the composition contains ingredients that are derived from natural or non-natural sources. In some embodiments, the formulation or carrier may be provided in sterile form. Non-limiting examples of a sterile carrier include endotoxin-free water or pyrogen-free water. The compositions can be administered intravesically.

In the context of the present description, the term "parenteral" includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injections or infusions. In specific embodiments, the compounds are administered intravenously, orally, subcutaneously, or intramuscularly. Sterile injectable forms of the compositions of the present invention may be in the form of an aqueous or oily suspension. These suspensions may be formulated according to methods known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic diluent or solvent suitable for parenteral administration. Acceptable vehicles and solvents that may be used include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are usually used as the solvent or suspending medium. In some embodiments, the compositions are administered intravesically.

Any bland fixed oil can be used for this purpose, including synthetic mono- or diglycerides. Fatty acids and their glyceride derivatives are also useful for the preparation of injectables, as well as natural pharmaceutically acceptable oils such as olive oil or castor oil, especially in polyoxyethylated versions. These oily solutions or suspensions may also contain a long chain alcohol diluent or dispersing agent such as carboxymethyl cellulose or similar dispersing agents commonly used in the preparation of pharmaceutically acceptable dosage forms, including emulsions and suspensions. Other commonly used surfactants such as Tweens, Spans and other emulsifying agents that are commonly used in the manufacture of pharmaceutically acceptable solid, liquid or other dosage forms can also be used to prepare the formulation.

For oral administration, the compound or salt may be presented in a suitable oral dosage form, including, without limitation, capsules, tablets, aqueous suspensions, or solutions. In the case of tablets for oral use, carriers commonly used include lactose and cornstarch. Lubricants such as magnesium stearate may also be added. For oral administration in capsule form, suitable diluents include lactose and dried cornstarch. If aqueous suspensions are required for oral administration, the active ingredient may be combined with emulsifying and suspending agents. Certain sweetening, flavoring or coloring agents may also be added, if desired. Preservatives may also be added. Suitable examples of pharmaceutically acceptable preservatives include, without limitation, various antibacterial and antifungal agents such as solvents such as ethanol, propylene glycol, benzyl alcohol, chlorobutanol, quaternary ammonium salts, and parabens (such as methylparaben, ethylparaben, propylparaben, etc.).

By "immediate release" is meant normal release, in which the release of the drug begins immediately after administration. As used herein, the term "immediate release" includes dosage forms that provide dissolution of the drug substance in the contents of the gastrointestinal tract without delaying or prolonging the process of dissolution or absorption of the drug substance. The goal is to quickly release the drug after administration, for example, with the ability to release at least 80% of the drug within about 30 minutes after the start of dissolution in the dissolution test.

"Sustained release" or "extended release" refers to dosage forms whose drug release characteristics are chosen depending on time and/or location to achieve therapeutic goals or benefits that are not achieved by conventional dosage forms, such as a solution or dosage form with immediate release.

The term "sustained" means that a plasma level is reached for a given active agent that is maintained by subsequent doses of the active agent at the same level or at a level that is above the minimum effective therapeutic level and below the minimum toxic plasma level for this active agent.

The term "dose range", as used in the present description, refers to the upper and lower limits of acceptable variation in the amount of the specified agent. In general, any amount of the agent within the indicated range may be administered to patients undergoing treatment.

The term "treat" is used herein to refer to the alleviation, reduction or amelioration of at least one symptom of a disease in a subject. For example, in relation to bladder cancer, the term "treat" can mean reversing, delaying onset (ie, the period before clinical manifestation of a disease or symptom of a disease), and/or reducing the risk of developing or worsening a symptom of bladder cancer. The term "protect" is used herein to mean prophylaxis to delay or treat, or both, depending on the situation, the onset, continuation, or worsening of symptoms of bladder cancer in the subject.

The term "subject" or "patient" includes animals that are capable of suffering from or suffering from bladder cancer. Examples of subjects or patients include mammals, such as humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and non-human transgenic animals. In some embodiments, the subject is a human, such as a human suffering from, at risk of, or potentially suffering from, bladder cancer.

The term "about" or "approximately" generally means within 20%, more preferably within 10%, and most preferably within 5% of a given value or range. Alternatively, especially in biological systems, the term "about" means approximately within a logarithm (ie, an order of magnitude), preferably within two times the specified value.

The use of terms in the singular in the context of the description of the invention (especially in the context of the following claims) should be construed as embracing the plural as well, unless the context clearly implies otherwise or otherwise clearly contradicts the context. The terms "comprising", "having", "including", and "consisting of" are to be construed as open-ended terms (i.e., meaning "including, without limitation"), unless otherwise indicated. The listing of ranges of values in this specification is used solely for the purpose of abbreviated reference to each individual value falling within the specified range, unless otherwise indicated in this specification, and each individual value is included in the description as if it were indicated separately in this description.

Exemplary cell proliferation disorders that can be treated with one or more of the compounds disclosed herein include, without limitation, cancer, precancerous stage or precancerous condition, and metastatic lesions in tissues and organs of the body. Cell proliferation disorders may include hyperplasia, metaplasia, and dysplasia.

The compound disclosed herein, or a pharmaceutically acceptable salt thereof, may be used to treat or prevent a cell proliferation disorder, or to treat or prevent cancer in a subject at an increased risk of developing cancer compared to the general population, or for use in identifying suitable candidates for such goals.

Pharmaceutical formulations and routes of administration

Pharmaceutical formulations containing Compound 1 or a pharmaceutically acceptable salt thereof are provided for the treatment of bladder cancer. Pharmaceutical compositions may additionally contain a carrier or excipient, a stabilizer, a flavoring agent and/or a coloring agent.

Compound 1, or a pharmaceutically acceptable salt thereof, can be administered by various routes known to those skilled in the art. Routes of administration include oral administration and intravesical administration. In some embodiments, the pharmaceutical formulation containing the compound or a pharmaceutically acceptable salt thereof may be taken orally in the form of a liquid, syrup, tablet, capsule, powder, coated particle capsule, chewable tablet, or dissolvable disc. Alternatively, the pharmaceutical compositions of the present invention may be administered intravenously or transdermally. Additional routes of administration are known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, Gennaro A. R., Ed., 20.sup.th Edition, Mack Publishing Co., Easton, Pa).

In some embodiments, the compound or pharmaceutically acceptable salt is formulated as a paste, jelly, or suspension. For example, the drug is dissolved, captured, or suspended in the form of drug particles, microencapsulated particles, or polymeric drug particles in a gel-like solution or semi-solid form. The advantage of an oral jelly formulation is that it is easier to administer to patients who have difficulty swallowing tablets, capsules or pills. In some embodiments, the compound is thoroughly mixed and suspended in a suitable medium to form a paste or gel. In the case of oral administration, additional flavoring agents may optionally be added. Peanut butter or alginate flavored with raspberries and a sweetener are examples of many suitable flavor masking agents. In various embodiments, paste or jelly formulations may also be formulated with suitable binders or excipients known in the art for topical application.

Methods for preparing sustained release formulations in the form of tablets, capsules or pills are known in the art. In some embodiments, a sustained release formulation is prepared by coating an active drug ingredient with a polymer, preferably a water-insoluble polymer. An example of a water-insoluble polymer used as a sustained release coating agent in the pharmaceutical field is an enteric coating agent or a gastroprotective coating agent. The water insoluble polymer may include, for example, ethyl cellulose, purified shellac, white shellac, RS aminoalkyl methacrylate copolymer, hydroxypropyl methyl cellulose phthalate, hydroxypropyl methyl cellulose acetate succinate, carboxymethyl ethyl cellulose, cellulose acetate phthalate, methacrylic acid copolymer L, methacrylic acid LD copolymer, methacrylic acid copolymer S , E aminoalkyl methacrylate copolymer, or diethylaminoacetate polyvinyl acetal.

The type, degree of substitution, and molecular weight of the water-insoluble polymers may depend on the solubility of the active ingredient in water or alcohol, the desired sustained release rate, and the like. Water-insoluble polymers can be used alone or in combination. Hydrogenated oil, stearic acid or cetanol as a co-coating agent and medium chain triglyceride, triacetin, triethyl citrate or cetanol as a plasticizer may also be included.

In some embodiments, the sustained release formulation is a matrix tablet or granule. The active ingredient can be coated with 3 different types of polymers. These polymers of three different types may include: 1) a water-insoluble polymer such as ethyl cellulose; 2) a pH-independent gelling polymer such as hydroxypropyl methylcellulose; and 3) a pH dependent gelling polymer such as sodium alginate. These polymers of three different types can be used together to reduce the release rate of drugs.

Dosage Forms: Release Properties

Sustained release formulations can provide a long lasting effect. However, the effect and/or bioavailability of the active ingredient may vary depending on several factors such as, for example, the absorption window, the carriers or excipients used in the formulation, the mode of delivery of the formulation, and/or the transit time of the active ingredient through the patient's gastrointestinal tract.

The therapeutic agent may contain at least one sustained release portion to perform a sustained release function and one immediate release portion to perform an immediate release function. In some embodiments, when the therapeutic agent is a unit dose, said dose may be in the form of tablets formed from a mixture of sustained release granules constituting the sustained release portion and immediate release granules constituting the immediate release portion, the formulation as a capsule obtained by filling a capsule with sustained-release beads and immediate-release beads, or a compressed-coated tablet in which the outer layer constituting the immediate-release portion is formed on the inner core constituting the sustained-release portion. However, the above embodiments are not limited.

Moreover, there are no specific restrictions on the state of the content of the medicinal substance in the composition, either in the immediate release portion or in the sustained release portion; the compound may be uniformly dispersed in the composition, immediate release portion, or sustained release portion, or may be contained in only one composition portion, immediate release portion, or sustained release portion, or may be distributed in such a way as to create a concentration gradient.

The sustained release portion of the composition of the present invention may contain at least one pH independent polymeric substance or pH dependent polymeric substance to control drug release.

The pH-independent polymeric substance used in the present invention may include a polymeric substance, the charge of which is practically unchanged at pH values typical of the gastrointestinal tract, in particular at pH from 1 to 8. This means, for example, that the polymeric substance does not has functional groups whose charge changes with pH, such as basic functional groups, such as amino groups, or acidic functional groups, such as carboxylic acid groups. It should be noted that the pH-independent polymeric material may be included to impart a sustained release function to the composition of the present invention, but may also be included for another purpose. Moreover, the pH independent polymer material used in the present invention may be insoluble in water, or may swell in water or dissolve in water to form a gel.

Examples of water-insoluble pH-independent polymeric materials include, without limitation, cellulose ethers, cellulose esters, and copolymers of methacrylic acid and acrylic acid (trade name Eudragit, manufactured by Rohm GmbH & Co. KG, Darmstadt, Germany). Examples include, without limitation, cellulose alkyl ethers such as ethyl cellulose (trade name Ethocel, manufactured by Dow Chemical Company, USA), ethyl methyl cellulose, ethyl propyl cellulose or isopropyl cellulose and butyl cellulose, aralkyl cellulose ethers such as benzyl cellulose, cyanoalkyl cellulose ethers such as cyanoethyl cellulose esters of cellulose and organic acids such as cellulose acetate butyrate, cellulose acetate, cellulose propionate or cellulose butyrate and cellulose acetate propionate, ethyl acrylate-methyl methacrylate copolymers (trade name Eudragit NE, manufactured by Rohm GmbH & Co. KG, Darmstadt, Germany ) and an aminoalkyl methacrylate copolymer RS (trade names Eudragit RL, Eudragit RS). There is no particular limitation on the average particle diameter of the water-insoluble polymer used in the present invention, but in general, the lower the average particle diameter, the better the performance, and the average particle diameter is preferably 0.1 to 100 µm, more preferably 1 to 50 µm, particularly preferably 3 to 15 µm, most preferably 5 to 15 µm. In addition, examples of water-soluble or water-swellable pH-independent polymeric materials include, without limitation, polyethylene oxide (trade name Polyox, manufactured by Dow Chemical Company, molecular weight 100,000 to 7,000,000), low-substituted hydroxypropyl cellulose (trade name L-HPC, manufactured by Shin- Etsu Chemical, Japan), hydroxypropyl cellulose (trade name HPC, manufactured by Nippon Soda, Co., Ltd, Japan), hydroxypropyl methylcellulose (trade name Metolose 60SH, 65SH, 90SH, manufactured by Shin-Etsu Chemical, Japan) and methyl cellulose (trade name Metolose SM , manufactured by Shin-Etsu Chemical, Japan).

In some embodiments, the implementation of the composition may contain one pH-independent polymeric substance or may contain several pH-independent polymeric substances. The pH-independent polymeric material, when used in the disclosed embodiments, may be a water-insoluble polymeric material, more preferably ethyl cellulose, an ethyl acrylate-methyl methacrylate copolymer (trade name Eudragit NE), or an aminoalkyl methacrylate copolymer RS (trade name Eudragit RL, Eudragit RS). Particularly preferred is at least one of the following: ethyl cellulose and RS aminoalkyl methacrylate copolymer. Most preferred is ethyl cellulose. There are no specific restrictions on the amount of pH-independent polymeric material in the composition; its amount can be appropriately selected depending on the purpose, such as the control of the sustained release of the drug.

The pH-dependent polymeric substance that can be used in the embodiments presented herein can be a polymeric substance whose charge changes at pH values typical of the gastrointestinal tract, in particular at pH 1 to 8. This means, for example, a polymeric substance containing functional groups whose charge changes with pH, such as basic functional groups, such as amino groups, or acidic functional groups, such as carboxylic acid groups. The pH-dependent functional groups of the pH-dependent polymeric substance are preferably acidic functional groups, with the pH-dependent polymeric substance most preferably containing carboxylic acid groups.

The pH-dependent polymeric substance used in the present invention may be insoluble in water or may swell in water or dissolve in water to form a gel. Examples of pH-dependent polymeric substances used in the present invention include, without limitation, enteric polymeric substances. Examples of enteric polymeric materials include, without limitation, methacrylic acid-methyl methacrylate copolymers (Eudragit L100, Eudragit S100, manufactured by Rohm GmbH & Co. KG, Darmstadt, Germany), methacrylic acid-ethyl acrylate copolymers (Eudragit L100-55, Eudragit L30D-55, manufactured by Rohm GmbH & Co. KG, Darmstadt, Germany), hydroxypropyl methylcellulose phthalate (HP-55, HP-50, manufactured by Shin-Etsu Chemical, Japan), hydroxypropyl methylcellulose acetate succinate (AQOAT, manufactured by Shin-Etsu Chemical, Japan), carboxymethyl ethyl cellulose (CMEC, manufactured by Freund Corporation, Japan) and cellulose acetate phthalate.

Examples of pH-dependent polymeric substances that swell in water or dissolve in water to form a gel include, without limitation, alginic acid, pectin, carboxyvinyl polymer, and carboxymethyl cellulose. In the present invention, the composition may contain one pH-dependent polymeric substance or may contain several pH-dependent polymeric substances. The pH-dependent polymeric substance used in the present invention is preferably an enteric polymeric substance, more preferably a methacrylic acid-ethyl acrylate copolymer, a methacrylic acid-methyl methacrylate copolymer, a hydroxypropyl methylcellulose phthalate or a hydroxypropyl methylcellulose succinate acetate, and a methacrylic acid-ethyl acrylate copolymer is particularly preferred.

When a pH-dependent polymeric substance is used in the production process of the composition of the present invention, the commercially available product can be used directly in the form of a powder or granules, or in the form of a suspension in which the pH-dependent polymeric substance is already dispersed in a solvent, or such a commercially available product. can be dispersed in water or organic solvent for its use. The smaller the particle diameter of the pH-dependent polymeric substance, the better the performance, while the pH-dependent polymeric substance is preferably in the form of a powder. In the case of a copolymer of methacrylic acid and ethyl acrylate, an example is Eudragit L100-55. There is no particular limitation on the average particle diameter of the pH-dependent polymeric substance used in the present invention, but the average particle diameter is preferably 0.05 to 100 µm, more preferably 0.05 to 70 µm, most preferably 0.05 to 50 µm. Moreover, there are no particular restrictions on the amount of pH-dependent polymeric substance; for example, in the case of an enteric polymeric substance, the amount is usually from 0.1 to 90 wt. parts, preferably from 1 to 70 wt. parts, more preferably from 5 to 60 wt. parts, particularly preferably from 10 to 50 wt. parts per 100 wt. parts of the composition.

The therapeutic agent according to the present embodiments may optionally further comprise any of various additives, such as any pharmacologically acceptable carrier such as diluent, lubricant, binder, and disintegrant, as well as any preservative, colorant, sweetener, plasticizer, film coating agent, and so on. . Examples of diluents include, without limitation, lactose, mannitol, dibasic calcium phosphate, starch, pregelatinized starch, crystalline cellulose, uncompacted silica, synthetic aluminosilicate, magnesium aluminate metasilicate, and the like. Examples of lubricants include, without limitation, magnesium stearate, calcium stearate, talc, sodium stearyl fumarate, and the like. Examples of binders include, without limitation, hydroxypropylcellulose, methylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, and the like. Examples of disintegrants include, without limitation, carboxymethyl cellulose, carboxymethyl cellulose calcium, croscarmellose sodium, sodium carboxymethyl starch, low substituted hydroxypropyl cellulose, and the like.

Examples of preservatives include, without limitation, paraoxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenylethyl alcohol, dehydroacetic acid, sorbic acid, and the like. Preferred examples of colorants include, without limitation, water-insoluble color pigments, natural pigments (eg, β-carotene, chlorophyll, iron oxide red), iron oxide yellow, iron oxide red, iron oxide black, and the like. Preferred examples of sweeteners include, without limitation, sodium saccharin, potassium glycyrrhizate, aspartame, stevia, and the like. Examples of plasticizers include, without limitation, glycerol fatty acid esters, triethyl citrate, propylene glycol, polyethylene glycol, and the like. Examples of film coating agents include, without limitation, hydroxypropyl methylcellulose, hydroxypropyl cellulose, and the like.

Manufacturing methods

To obtain the presented embodiments, you can use one traditional method or a combination of traditional methods. For example, in the manufacture of drug-containing granules as a sustained release portion or an immediate release portion, granulation is the main operation, but it can be combined with other operations such as mixing, drying, sieving and classifying. As a granulation method, for example, a wet granulation method in which a binder and a solvent are added to the powder and granulation is performed, a dry granulation method in which the powder is pressed and granulated, a melt granulation method in which a binder is added that melts when heated, heating and granulation are performed, and the like.

In addition, depending on the granulation method, an appropriate working method can be used, such as a stirring granulation method using a planetary mixer, a screw mixer, etc., a high-speed stirring granulation method using a Henschel mixer, a Super mixer, etc., extrusion and granulation method using a cylindrical granulator, a rotary granulator, a screw extruding granulator, a pellet granulator or the like, a high shear wet granulation method, a fluid bed granulation method, a post-compression granulation method, a crushing and granulation method, or spray granulation method. After granulation, drying with a dryer, fluid bed method, etc., grinding and screening can be performed to obtain granules or fine granules for use. In addition, during the preparation of the composition of the present invention, a granulation solvent may be used. There is no particular limitation on the granulation solvent, which may be water or any of various organic solvents such as water, a lower alcohol such as methanol or ethanol, a ketone such as acetone or methyl ethyl ketone, methylene chloride, or mixtures thereof.

For sustained release embodiments, granules are prepared by co-mixing at least one drug substance and at least one substance selected from pH-independent polymeric substances and pH-dependent polymeric substances, optionally adding a diluent and a binder, followed by granulation with obtaining granular material. The resulting granular material is dried with a tray dryer, fluid bed dryer, or the like, and sieved using a mill or an oscillator, thereby obtaining sustained release granules. Alternatively, at least one drug substance, at least one substance selected from pH-independent polymeric substances and pH-dependent polymeric substances, and optionally a diluent and binder, and using a dry compactor such as a roller compactor or a piston tableting machine, perform agitation compression and then granulation by grinding to a suitable size. The granular material produced by such a granulator can be used in the form of granules or fine granules according to the present invention, or can be further ground with a power mill, a roller granulator, a speed mill, or the like. and screen to obtain sustained release granules. It should be noted that immediate release beads can be made in the same manner as sustained release beads.

The compression molded product can be made into a sustained release portion that contains a drug substance or an immediate release portion, or into a composition disclosed herein, using one conventional method or a combination of conventional methods. For example, using at least one drug substance, at least one substance selected from pH-independent polymeric substances and pH-dependent polymeric substances, a diluent such as mannitol or lactose, a binder such as polyvinylpyrrolidone or crystalline cellulose, a disintegrant , such as sodium carmellose or crospovidone, and a lubricant, such as magnesium stearate or talc, perform tabletting by a conventional method, by which a compressed product can be obtained. In this case, tabletting is the main operation in the process for producing a compressed product, but it can be combined with other operations such as mixing, drying, sugar coating and coating.

Examples of the tableting process include, without limitation, direct compression, in which at least one drug substance and pharmacologically acceptable additives are mixed together, and then the mixture is directly compressed into tablets using a tableting machine, and dry granule compression or wet granule compression, in which the sustained release or immediate release granules of the present invention are compressed after the optional addition of a lubricant or disintegrant. There are no special restrictions on the tableting machine used in the compression; for example, a single-punch tableting machine, a rotary tableting machine, or a press-coating tableting machine can be used.

The sustained release drug-containing bead or immediate release bead or compression molded product according to the disclosed embodiments can be used naturally in bead or tablet form as a composition, but can also be further processed to form a composition. For example, the compression molded product or pellets can be film coated using a film coating base material such as ethylcellulose, casein, methylcellulose, hydroxypropylmethylcellulose, methacrylic acid copolymer L, cellulose acetate phthalate, shellac, etc., or sugar coating using a sugar coating liquid containing sucrose, sugar alcohol, gum arabic powder, talc or the like to obtain film-coated tablets or sugar-coated tablets. One of the solvents in this coating method may be purified water, but an organic solvent such as an alcohol, a ketone, an ether, or a chlorinated hydrocarbon, or a mixture thereof, may also be used. For example, ethanol, acetone, methylene chloride, and the like can be used as the organic solvent. In addition, as the coating apparatus, a coating apparatus commonly used for coating in the manufacture of medicines can be used, for example, including a spray coating apparatus in which coating is carried out by spraying a coating liquid or the like, and a rotary fluid bed granulator for coating layer.

In the case of capsule formulations, capsules can be made by filling hard gelatin or HPMC capsules with sustained release beads or immediate release beads as above or mini-tablets using an automatic capsule filling machine. Alternatively, in the case of preparations administered per ampoule, or in the case of a dry syrup which is administered mixed with water or a similar liquid, the sustained release granules or immediate release granules as above may be mixed with a thickener or dispersing agent to disperse these granules, after which the mixture is converted into granules or tablets. In addition, the liquid or jelly may be prepared using water and substances selected from dispersants, emulsifiers, thickeners, preservatives, pH adjusters, sweeteners, flavors, flavors, and the like. However, with regard to other manufacturing methods, their use is also not limited.

For a more complete understanding of the embodiments disclosed herein, examples are given below. It should be understood that these examples are for illustrative purposes only and should not be construed as limiting the invention.

EXAMPLES

The following abbreviations may be used in the examples:

All: allyl

DMT: 4,4'-Dimethoxytrityl

(DMTO-:

)

)

Bz: benzoyl

Hunig's base: i-Pr ₂ NEt (diisopropylethylamine)

AllylOH: allyl alcohol

OAll: -OCH ₂ CHCH ₂

ACN: acetonitrile

All: -CH ₂ CHCH ₂

2-nitroBnBr: 2-nitrobenzyl bromide

Bz: benzoyl

i-Pr: isopropyl

CE: cyanoethyl

()

DEAD: diethyl azodicarboxylate

DIAD: diisopropyl azodicarboxylate

DCM: dichloromethane

DDTT: N,N-dimethyl-N'-(3-thioxo-3H-1,2,4-dithiazol-5-yl)formamide

DMOCP: 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphinane 2-oxide

TBS: t-butyldimethylsilyl

3H-benzo[c][1,2]dithiol-3-one:

Example 1 Synthesis of Compound 1a

The complete synthesis scheme is shown in Fig. 1.

Stage A

To a mixture of (2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-fluorotetrahydrofuran- 3-yl(2-cyanoethyl)diisopropylphosphoramidite (compound 100) (mixture of phosphorus diastereomers; 80.0 g, 91.332 mmol, 1 eq., ChemGenes Corporation catalog # ANP-9151), allyl alcohol (9.63 ml, 142 mmol ) and triphenylphosphine (38.3 g, 146 mmol, 1.60 eq.) in THF (1.1 L) was added DEAD (40 wt.% solution in toluene; 54.2 ml, 137 mmol, 1.5 eq. ) at ambient temperature (rt). Stirring was continued at ambient temperature and the reaction was monitored by LC/MS. At the end (19 h) the mixture was concentrated in vacuo (35° C.) and the resulting mixture was purified by silica gel column chromatography (800 g x2 column, 40 to 60% EtOAc in n-heptane buffered with 0.5% triethylamine) to give Compounds 101 as a white foam (yield 84.2 g as a mixture of phosphorus diastereomers).

¹ H NMR (3:2 mixture of phosphorus diastereomers 400 MHz, CDCl ₃ ) δ 1.14-1.21 (m, 12H) 2.40 (t, J=6.2 Hz, 1.2H) 2.59 ( t, J=6.2 Hz, 0.8H) 3.27 (d, J=8.6 Hz, 1H) 3.52-3.66 (m, 5H) 3.78 (s, 2.4H) 3.79 (s, 3.6H) 4.28-4.34 (m, 1H) 4.84-4.96 (m, 0.4 H) 4.99 (d, J=5.5 Hz, 2H) 4.95-5.10 (m, 0.6H) 5.05 (d, J=10.9 Hz, 1H) 5.22 (br.d, J=17.6 Hz, 1H) 5, 64 (br.d, J=53.2 Hz, 0.6H) 5.70 (br.d, J=51.6 Hz, 0.4H) 5.96-6.75 (m, 1H) 6, 20 (d, J=16.0 Hz, 0.6H) 6.24 (d, J=17.2 Hz, 0.4H) 6.74-6.79 (m, 4H) 7.02-7, 06 (m, 2H) 7.17-7.24 (m, 8H) 7.32-7.34 (m, 2H) 7.41-7.44 (m, 2H) 8.11 (s, 1H) 8.52 (s, 0.4H) 8.54 (s, 0.6H).

Stage B

To a solution of Compound 101 (3.00 g, 3.28 mmol, 1 eq.) in acetonitrile (30 ml) was added water (0.118 ml, 6.55 mmol, 2.0 eq.) and pyridine trifluoroacetate salt (0.759 g, 3.93 mmol, 1.2 eq.). After stirring at ambient temperature for 1 minute, tert-butylamine (14.5 g, 21.0 ml, 0.20 mol, 60 eq.) was added. After complete elimination of the cyanoethyl group (monitored by LC/MS), the reaction mixture was concentrated in vacuo and azeotropically distilled twice with acetonitrile. The crude mixture was dissolved in DCM (45.0 ml) and treated with water (0.118 ml, 6.55 mmol, 2.0 eq.) and NaHSO ₄ -SiO ₂ (1.18 g, 6.55 mmol, 2 eq.) at ambient temperature. After complete elimination of the DMT group (monitoring by LC/MS, approx. 1 hour), the reaction mixture was filtered and washed twice with a mixture of DCM/MeOH (9/1, 20 ml). The combined filtrates were concentrated in vacuo and treated with 1:1 n-heptane/toluene (~30 ml). The top layer was removed by decanting. The same operation was repeated once more with n-heptane/toluene (1/1, 30 ml) and the bottom layer was azeotropically distilled twice with acetonitrile to give compound 102 (assumed theoretical yield 100%). The product was used in the next step without further purification.

Stage C

To a mixture of Compound 102 (1.56 g, 3.27 mmol, 1 eq.) and Compound 101 (3.00 g, 3.28 mmol, 1 eq.) in acetonitrile (30 ml) was added pyridine trifluoroacetate salt (azeotropically dried with pyridine, 0.760 g, 3.94%, mmol, 1.25 eq.). After 5 minutes, DDTT (0.840 g, 4.09 mmol, 1.30 eq, ChemGenes Corporation catalog # RN-1588) was added and after complete sulfurization (monitoring by LC/MS), the reaction mixture was concentrated in vacuo. The residue was dissolved in DCM (30 ml) and treated with water (0.57 ml, 32 mmol, 10 eq.) and 6% dichloroacetic acid (1.56 ml, 18.9 mmol, 6.0 eq.) in DCM (30 ml). After 20 minutes the reaction was stopped by adding pyridine (20 ml) and concentrated in vacuo. The residue was azeotropically distilled with pyridine to give compound 103 (3.22 g, assumed theoretical yield 100%). The product was used in the next step without further purification.

Stage D

To a solution of Compound 103 (3.22 g, 3.15 mmol, 1 eq.) in pyridine (100 ml) was added DMOCP (1.45 g, 7.88 mmol, 2.50 eq.) at ambient temperature. After complete macrocyclization (monitoring by LC/MS), water (1.7 ml, 94.5 mmol, x10 times DMOCP) was added followed by 3H-benzo[c][1,2]dithiol-3-one (0.795 g, 4.73 mmol, 1.5 eq.). After complete sulfurization (about 40 minutes) the reaction mixture was partially concentrated in vacuo to about 15 ml and poured into a mixture of saturated aqueous NaHCO ₃ (50 ml) and water (30 ml). After 10 min stirring at ambient temperature, the mixture was extracted with a mixture of EtOAc/MTBE (1:1) (60 ml x3 times). The organic layers were combined, washed with brine (25 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by silica gel column chromatography (0-20% MeOH in DCM) to give Compound 104 (3.31 g, 3.20 mmol, assumed theoretical yield 100%) as a brown oil. The product was used in the next step without further purification.

Stage E

To a solution of Compound 104 (3.31 g, 3.20 mmol, 1 eq.) in acetonitrile (66.2 ml) was added 2-nitrobenzyl bromide (2.42 g, 11.2 mmol, 3.50 eq.) and triethylamine (1.78 ml, 12.8 mmol, 4.00 eq.). After completion of the reaction (monitoring by LC/MS, approximately 20 hours at ambient temperature), the reaction mixture was concentrated in vacuo and purified by silica gel column chromatography (60% ethyl acetate/n-heptane to 100% ethyl acetate) to give 0.568 g of product as mixtures of phosphorus diastereomers. Separation of the diastereomers by preparative HPLC gave Compound 105 (SR isomer; 0.225 g, 0.180 mmol, 5.6% overall yield from Compound 101) and Compound 106 (RR isomer; 0.187 g, 0.149 mmol, 4.7% overall yield from Compound 1).

Compound 105 (SpRp) ¹ H NMR (400 MHz, CDCl ₃ ) δ=8.63 (s, 1H), δ=8.61 (s, 1H), 8.04-8.00 (m, 2H), 7.99 (s, 1H), 7.90 (s, 1H), 7.65-7.44 (m, 8H), 7.40-7.31 (m, 4H), 7.25-7. 21 (m, 4H), 6.15-5.89 (m, 5H), 5.61 (dd, J=52.0, 5.1 Hz, 1H), 5.55 (dd, J=51, 2, 4.7, 2.7 Hz, 1H) 5.51-5.42 (m, 1H), 5.31-5.22 (m, 2H), 5.11 (dd, J=3.9 , 9.8 Hz, 2H), 5.04-4.95 (m, 4H), 4.55-4.37 (m, 7H), 4.29-4.12 (m, 3H)

Compound 106 (RpRp) ¹ H NMR (400 MHz, CDCl ₃ ) δ=8.65 (s, 2H), 8.06 (dd, J=1.4, 8.0 Hz, 2H), 7.98 ( s, 2H), 7.57-7.52 (m, 6H), 7.47-7.32 (m, 6H), 7.25-7.21 (m, 4H), 6.15 (d, J=18.7 Hz, 2H), 6.09-5.99 (m, 2H), 5.82-5.76 (m, 2H), 5.60 (dd, J=51.8, 4, 9 Hz, 2H), 5.27 (dd, J=1.2, 17.2 Hz, 2H), 5.12 (dd, J=1.0, 10.4 Hz, 2H), 5.06- 4.96 (m, 4H), 4.55-4.40 (m, 4H), 4.36-4.24 (m, 4H), 4.21-4.02 (m, 2H)

Preparative HPLC conditions:

Tool Agilent 1200 HPLC column Preparative column C18 OBD Waters Sunfire, 5 µm, 30x250 mm, #186003969 Flow rate 50 ml/min mobile phase A: water, B: acetonitrile Gradient time (min) 0 8 9.9 10 12 B% 50 99 99 50 50 Chromatogram recording time 12 min Injection volume 150 µl (0.08 g/ml in acetonitrile) Detection UV, 254 nm retention time compound 105 (SpRp) 7.7 min connection 106 (RpRp) 8.0 min

Stage F

To a heated (90°C) solution of Compound 105 (519 mg, 0.414 mmol, 1 eq.) in toluene (519 ml) was added Hoveid-Grubbs™ 2nd generation catalyst ((1,3-bis-(2,4, 6-trimethylphenyl)-2-imidazolidinylidene)dichloro(o-isopropoxyphenylmethylene)ruthenium, available from SIGMA-ALDRITCH® Cat. No. 569755; CAS 301224-40-8; 91 mg, 0.15 mmol, 0.35 eq) and quinone (0.102 ml, 1.243 mmol, 3.0 eq.). The mixture was refluxed and the progress of the reaction was followed by LC/MS. After 3 hours, additional catalyst (91 mg, 0.15 mmol, 0.35 eq.) was added and the reaction was continued for another 3 hours. After cooling, the mixture was treated with DMSO (0.59 ml, 8.3 mmol, 20 eq.) at ambient temperature for 15 hours, concentrated in vacuo and purified by silica gel column chromatography (SiO ₂ 25 g, 66% ethyl acetate in n- heptane to 100% ethyl acetate) to give Compound 107 (200 mg, 0.163 mmol, 39% yield) as a brown dry foam.

¹ H NMR (400 MHz, CDCl ₃ ) δ=8.19 (s, 1H), 8.12 (dd, J=7.8 Hz, 1.9 Hz, 1H), 8.10 (s, 1H) , 8.02 (d, J=8.2 Hz, 1H), 7.89 (s, 1H), 7.63 (br.d, J=7.0 Hz, 1H), 7.53-7, 41 (m, 10H), 7.35-7.30 (m, 2H), 7.25-7.20 (m, 4H), 6.23 (d, J=17.6 Hz, 1H), 6 .14 (d, J=18.8 Hz, 1H), 5.86-5.75 (m, 1H), 5.75 (dt, J=15.3, 5.0 Hz, 1H), 5.67 ( dt, J=15.3, 4.7 Hz, 1H), 5.60 (dd, J=52.0, 3.9 Hz, 1H), 5.48 (dd, J=50.4, 3, 9 Hz, 1H), 5.50-5.39 (m, 1H), 4.91-4.64 (m, 4H), 4.57-4.25 (m, 9H), 4.15 (d , J=7.03 Hz, 1H), 4.11 (d, J=7.03 Hz, 1H)

Stage G

To a solution of Compound 107 (88 mg, 0.072 mmol, 1 eq.) in 1,4-dioxane (1.76 ml) was added thiophenol (0.88 ml, 8.55 mmol, 119 eq.) and triethylamine (0.88 ml, 6.31 mmol, 88 eq.). The resulting mixture was stirred at ambient temperature. After completion of the reaction (monitored by LC/MS, 13 hours), methanol (5.28 ml) and 28% ammonium hydroxide (3.52 ml) were added and the resulting mixture was heated to 50°C. After completion of the reaction (monitoring by LC/MS, 5 hours), the mixture was cooled to ambient temperature, and the resulting brownish suspension was filtered and washed with water (15 ml). The filtrate was again filtered to remove additional solids. The final filtrate was extracted twice with 1:1 mixture of toluene and heptane (30 ml). The aqueous layer was concentrated in vacuo and then resuspended in water (6 ml). The resulting solid was filtered and the filtrate was subjected to preparative HPLC to give the diammonium salt of Compound 1 (also referred to as Compound 1a) (39 mg, 0.050 mmol, 70% yield) as a white solid.

Compound 1a (SpRp, trans) ¹ H NMR (400 MHz, CD ₃ OD) δ=9.05 (s, 1H), 8.33 (s, 1H), 8.25 (s, 1H), 8.12 (s, 1H), 6.34 (br.s, 2H), 5.88 (br.s, 2H), 5.66 (br.d, J=51.6 Hz, 1H), 5.59 ( br.d, J=52.2 Hz, 1H) 5.01 (br.s, 2H), 4.68-4.34 (m, 6H), 4.07-3.82 (m, 2H), 3.79-3.55 (m, 2H); ³¹ P NMR (162 MHz, CD ₃ OD) δ=55.48 (s, 1P), 55.16 (s, 1P).

Compound 1a. Preparative HPLC conditions:

Tool Agilent 1200/1260 AS/FC HPLC column Waters XBridge C18, 10x100mm, No. 1413 Flow rate 3.0 ml/min Column temperature 35°C mobile phase A: 0.1% NH ₄ OH in water, B: 0.1% NH ₄ OH in acetonitrile Gradient (B%) 0 → 50 Chromatogram recording time 20 minutes Injection volume 50 µl (4 mg/ml in water) Detection UV 260 nm retention time 6.5 min

Example 1.1 - Alternative Synthesis of Compound 1a

An alternative synthesis route for Compound 1a is shown in FIGS. 2A and 2B, as well as in FIG. 2C, and is described below.

Stage 1

Compound 129 (570 g, 1.53 mol, 1 wt%, 1 vol, 1 eq) was dissolved in pyridine (2.85 L, 35.2 mol, 4.89 wt %, 5.0 vol, 23 equiv.). The mixture was cooled to 2.6° C. and treated with 4,4'-dimethoxytrityl chloride (DMTCl; 543 g, 1.60 mol, 0.953 wt%, 1.05 eq.). The mixture was stirred at 0-5° C. for 2 hours and then allowed to warm to ambient temperature. The reaction was monitored by LC/MS and complete conversion was confirmed after overnight stirring. The reaction mixture was cooled to below 5°C and quenched by treatment with MeOH (124 ml, 3.05 mol, 0.172 wt.%, 0.217 vol., 2.0 eq.) for 15 minutes. The mixture was co-evaporated with toluene (2.00 L, 3.04 wt%, 3.51 vol) in vacuo and then diluted with EtOAc (2.850 L, 4.5 wt %, 5.0 vol) and n-heptane (2.85 L, 3.42 wt.%, 5.0 vol.). The organic layer was washed with saturated NaHCO ₃ (9 wt.% solution in water; 2.0 l, 3.5 vol.). Additional EtOAc (2.85 L, 4.5 wt%, 5.0 vol) was added to completely dissolve the crude product. After stirring for 5 minutes, the two layers were separated. The organic layer was washed with water (2.0 L, 3.5 wt.%, 3.5 vol.). The solid began to slowly precipitate from the organic layer. The aqueous layer was separated. The organic layer was then concentrated to approximately 1 volume. The crude product was suspended in a mixture of n-heptane (2.00 L, 2.40 wt%, 3.51 v/v) and toluene (0.50 L, 0.76 wt%, 0.88 v/v). After stirring for 15 minutes, a pale yellow solid was collected by vacuum filtration. The filter cake was washed successively with: (1) a mixture of n-heptane (0.60 L, 0.72 wt.%, 1.05 vol.) and toluene (0.30 L, 0.46 wt.%, 0.53 vol.), and then (2) n-heptane (3.00 l, 3.6 wt.%, 5.26 vol.). The solid was dried without heat for 30 minutes and then transferred to drying trays at 50°C in a vacuum oven overnight to give Compound 130 as a pale yellow solid (996.7 g, 1.47 mol, 1. 75 wt%, 97% yield.

¹ H NMR (400 MHz, CHLOROFORM-d) δ=8.99 (s, 1H), 8.76 (s, 1H), 8.21 (s, 1H), 8.04-8.00 (m, 2H), 7.64-7.59 (m, 1H), 7.57-7.50 (m, 2H), 7.41-7.36 (m, 2H), 7.32-7.15 ( m, 7H), 6.83-6.76 (m, 4H), 6.31 (dd, J=2.5, 17.0 Hz, 1H), 5.68 (dd, J=2.3, 4.7, 52.7 Hz, 1H), 4.88-4.77 (m, 1H), 4.26-4.21 (m, 1H), 3.77 (s, 6H), 3.57 (dd, J=3.1, 10.9 Hz, 1H), 3.43 (dd, J=4.1, 10.7 Hz, 1H), 2.60 (br.s, 1H)

Stage 1'

Compound 129 (430 g, 1.15 mol, 1 wt%, 1 vol, 1 eq) and imidazole (118 g, 1.73 mol, 0.274 wt%, 1.50 eq) were dissolved in DMF ( 1.72 l, 3.78 wt.%, 4.0 vol.), and the resulting mixture was cooled to 5°C. TBS-Cl (191 g, 1.27 mol, 0.444 wt%, 1.10 eq) was added. The mixture was stirred at 0-11°C for 2 hours, left to warm slowly to ambient temperature (the progress was followed by LC/MS). The reaction was completed 6 hours after the addition of TBS-Cl, left to stir at ambient temperature for an additional 20 hours. The mixture was cooled to 2°C and treated with methanol (93 ml, 74 g, 2.3 mol, 0.17 wt.%, 0.22 wt.%, 2.0 eq.) for 10 minutes. The reaction mixture was diluted with a mixture of MTBE (1.72 L, 1.23 kg, 2.96 wt.%, 4.0 vol.) and EtOAc (1.72 L, 1.55 kg, 3.60 wt.%, 4 .0 vol.), and then saturated NH ₄ Cl (28 wt.% solution in water; 2.15 l, 5.0 vol.). The solid began to slowly fall out of solution. The mixture was allowed to warm to 24° C. and water (1.08 L, 1.08 kg, 2.5 wt%, 2.5 vol) was added thereto (internal temperature=22° C.). New solid particles began to fall out of the mixture. Water (1.08 L, 1.08 kg, 2.5 wt%, 2.5 vol) and MTBE (1.40 L, 1.04 kg, 2.4 wt%, 3 , 3 rev.). An off-white solid was collected by vacuum filtration. The reactor was flushed with water (320 mL, 0.74 vol) followed by MTBE (1.80 L, 1.33 kg, 3.10 wt%, 4.19 vol) to transfer any remaining solid to the filter. The filter cake was washed successively with: (1) water (1.80 L, 1.80 kg, 4.2 wt.%, 4.2 vol.), (2) water (1.80 L, 1.80 kg, 4.2 wt.%, 4.2 vol.), (3) a mixture of MTBE (0.90 l, 0.67 kg, 1.5 wt.%, 2.1 vol.) and n-heptane (0. 90 L, 0.62 kg, 1.4 wt.%, 2.1 vol.), (4) MTBE mixture (0.90 L, 0.67 kg, 1.5 wt.%, 2.1 vol. ) and n-heptane (0.90 L, 0.62 kg, 1.4 wt%, 2.1 vol). The recovered solid was dried in vacuo at 40° C. for 2 days to give Compound 133 as a white solid (483 g, 0.991 mol, 1.12 wt%, 86% yield).

¹ H NMR (400 MHz, CHLOROFORM-d) δ=8.97 (s, 1H), 8.82 (s, 1H), 8.36 (s, 1H), 8.04-8.00 (m, 2H), 7.64-7.58 (m, 1H), 7.56-7.51 (m, 2H), 6.40 (dd, J=2.3, 16.0 Hz, 1H), 5 .45 (ddd, J=2.7, 4.3, 53.1 Hz, 1H), 4.75-4.66 (m, 1H), 4.22-4.17 (m, 1H), 4 .07 (dd, J=2.3, 11.7 Hz, 1H), 3.91 (dd, J=2.7, 11.7 Hz, 1H), 2.38 (dd, J=2.7 , 7.0 Hz, 1H), 0.92 (s, 9H), 0.11 (s, 3H), 0.11 (s, 3H).

Stage 2

Compound 130 (993 g, 1.47 mol, 1 wt%, 1 vol, 1 eq) and imidazole (150 g, 2.20 mol, 0.151 wt%, 1.5 eq) were dissolved in DMF ( 3.48 l, 3.28 kg, 3.3 wt.%, 3.5 vol.), and the mixture was cooled to 5°C. TBS-Cl (244 g, 1.62 mol, 0.245 wt%, 1.10 eq) was added. The reaction mixture was stirred at 0-5° C. for 2 hours, allowed to warm slowly to ambient temperature and monitored by LC/MS. After 17 hours, additional imidazole (100 g, 1.47 mol, 0.10 wt%, 1.0 eq) and TBS-Cl (111 g, 735 mmol, 0.112 wt%, 0.50 eq) were added. and stirring was continued at ambient temperature for 2 hours and at 35°C for 2 hours. The resulting mixture was cooled to 13.6° C. and treated with MeOH (119 ml, 2.94 mol, 2 eq.) for 10 minutes. Ice (5 kg, 5 wt%) and saturated NH ₄ Cl (28 wt% solution in water; 5.0 L, 5 vol) were added to a separate reactor. The reaction mixture was added to ice/NH ₄ Cl. An off-white solid immediately began to precipitate out of the solution. An additional 2 kg of ice (2 kg, 2 wt%) and water (3.0 L, 3 vol) were added to the mixture. The reaction flask was rinsed with water (0.50 L, 0.5 vol) and rinse aid was added to the mixture. n-Heptane (2.00 L, 2 vol) was added to the mixture and stirring was continued for 10 minutes. An off-white solid was collected by vacuum filtration. The filter cake was washed with: (1) water (4.0 L, 4.0 vol.), (2) water (4.0 L, 4.0 vol.), (3) n-heptane (4.0 L , 4.0 vol.), (4) n-heptane (4.0 L, 4.0 vol.). The recovered solid was dried in vacuo at 45° C. for 4 days to give Compound 131 as an off-white solid (1.095 kg, 1.39 mol, 1.10 wt%, 94% yield).

¹ H NMR (400 MHz, CHLOROFORM-d) δ=9.09 (s, 1H), 8.78 (s, 1H), 8.28 (s, 1H), 8.02 (d, J=7, 4 Hz, 2H), 7.63-7.59 (m, 1H), 7.55-7.50 (m, 2H), 7.37 (d, J=7.1 Hz, 2H), 7, 29-7.17 (m, 7H), 6.79 (d, J=7.9 Hz, 4H), 6.29 (dd, J=2.9, 16.2 Hz, 1H), 5.60 (ddd, J=2.7, 3.9, 53.1 Hz, 1H), 4.78 (ddd, J=4.7, 6.4, 15.8 Hz, 1H), 4.26-4 .22 (m, 1H), 3.77 (s, 6H), 3.58 (dd, J=3.1, 10.9 Hz, 1H), 3.26 (dd, J=3.7, 10 .7 Hz, 1H), 0.85 (s, 9H), 0.10 (s, 3H), 0.02 (s, 3H)

Stage 3

Compound 131 (1000 g, 1.27 mol, 1 wt%, 1 vol, 1 eq) and trans-2-butene-1,4-diol (olefin geometry confirmed by ¹ H-NMR; 335 g, 3, 80 mol, 0.335 wt%, 3.0 eq.) was azeotropically distilled twice with THF (3.0 L, 3.0 vol.). The residue was dissolved in a mixture of THF (10 L, 10 vol.) and toluene (15 L, 15 vol.). Triphenylphosphine (432 g, 1.65 mol, 0.432 wt%, 1.3 eq.) was added and then the reaction mixture was cooled to -5°C. DIAD (0.320 L, 1.65 mol, 333 g, 0.333 wt%, 0.320 vol, 1.3 eq.) was added slowly over 20 minutes keeping T-internal below 5°C. The reaction was stirred at 0-5°C for 1 h and monitored by LC/MS. The ice bath was removed and the mixture was allowed to warm to room temperature. After stirring overnight (17 h), triphenylphosphine (83 g, 0.32 mol, 0.083 wt.%, 0.25 eq.) and DIAD (62 ml, 0.32 mol, 64 g, 0.064 wt.%, 0.062 vol., 0.25 eq.). After an additional 1 hour at room temperature, the reaction mixture was diluted with MTBE (10 L, 10 vol), washed twice with half-saturated NaCl (18 wt.% solution in water; 2×4 L) and concentrated in vacuo to a thick oil. The mixture was redissolved in a mixture of MTBE (4.00 L, 4 vol) and n-heptane (0.50 L, 0.5 vol) and then cooled to 0°C. The solution was seeded with triphenylphosphine oxide. Solids began to slowly precipitate out of the solution and the solution was stirred overnight. The white solid was collected by vacuum filtration and washed with MTBE (2 L, 2 vol) to isolate 540 g of triphenylphosphine oxide. The filtrate was concentrated and purified through Biotage 150L KP-Sil (SiO ₂ 5 kg; presoaked with 1% TEA in Hep/etOAc; eluents: heptane/EtOAc (48 L 33% EtOAc with 1% TEA, 24 L 50% EtOAc with 1% TEA, 24 L 66% EtOAc with 1% TEA) → 100% EtOAc with 1% TEA). The column was monitored by TLC (2:1 EtOAc/n-heptane). Pure product fractions were combined and concentrated in vacuo to give compound 132 as a pale white solid foam (634 g, containing 14 wt% by-product derived from DIAD, net 545 g, 0.63 mol, corrected yield 50%). Mixture fractions were combined and concentrated in vacuo to give a solid pale yellow foam (750 g) which was repurified through a Biotage 150M HP-Sphere (2.5 kg SiO ₂ ; pre-soaked with 1% TEA in Hep/EtOAc; sample loaded with toluene eluents: Hep/EtOAc/1% TEA (12 L 50% EtOAc with 1% TEA, 16 L 66% EtOAc with 1% TEA) → EtOAc with 1% TEA). The column was monitored by TLC (2/1/0.03 EtOAc/n-heptane/TEA). Pure product fractions were combined and concentrated in vacuo to give additional compound 132 as a pale white solid foam (206 g, 0.24 mol, 18% yield).

¹ H NMR (400 MHz, CHLOROFORM-d) δ=8.58 (s, 1H), 8.10 (s, 1H), 7.43-7.37 (m, 2H), 7.32-7, 28 (m, 2H), 7.24-7.15 (m, 8H), 7.03-6.98 (m, 2H), 6.78-6.73 (m, 4H), 6.18 ( dd, J=2.7, 17.2 Hz, 1H), 5.88 (td, J=5.5, 15.6 Hz, 1H), 5.77 (td, J=5.1, 15, 6 Hz, 1H), 5.60 (ddd, J=2.7, 4.3, 53.1 Hz, 1H), 5.03-4.96 (m, 2H), 4.91 (ddd, J =4.5, 6.6, 16.6 Hz, 1H), 4.18-4.14(m, 1H), 3.88-3.82(m, 2H), 3.78(s, 6H ), 3.52 (dd, J=2.7, 10.9 Hz, 1H), 3.14 (dd, J=3.5, 10.9 Hz, 1H), 0.85 (s, 9H) , 0.10 (s, 3H), 0.01 (s, 3H).

Stage 4

Compound 132 (800 g, 0.930 mol, 1 wt.%, 1 vol., 1 eq.) and compound 133 (522 g, 1.07 mol, 0.652 wt.%, 1.15 eq.) were azeotropically dried with THF ( 2×3 L, 2×3.8 vol) and redissolved in THF (9.60 L, 8.45 kg, 12.0 vol) at room temperature. Triphenylphosphine (317 g, 1.21 mol, 0.396 wt%, 1.30 eq.) was added and the mixture was cooled below -5°C. DIAD (226 ml, 1.16 mol, 235 g, 0.294 wt.%, 0.283 vol., 1.25 eq.) was added at T-internal at a temperature below 7°C. The reaction mixture was allowed to warm slowly to room temperature. The reaction was monitored by LC/MS. After 21 hours, the reaction mixture was concentrated in vacuo to a thick oil, azeotropically distilled with n-heptane (2.00, 1.37 kg, 1.71 wt%, 2.50 vol) and then redissolved in MTBE ( 2.40 L, 1.78 kg, 2.2 wt%, 3.0 v/v) and n-heptane (800 ml, 547 g, 0.68 wt%, 1.0 v/v). The solution was seeded with triphenylphosphine oxide and cooled to 5° C., diluted with n-heptane (400 ml, 274 g, 0.34 wt.%, 0.50 vol.), and stirred at 5° C. for 30 minutes. A white solid precipitate was collected by vacuum filtration and washed with 2:1 (v/v) with a mixture of MTBE and n-heptane (1.8 L) to give triphenylphosquine oxide (455 g). The filtrate was concentrated in vacuo and purified through Biotage 150L KP-Sil (SiO ₂ 5 kg; presoaked with 1% TEA; sample was loaded by dissolving in eluents of toluene: 9:1 heptane/EtOAc (16 L) and 15 TEA, 3.6: 1 (46 L), 2:1 (20 L) and 1% TEA, 1:1 (30 L) and 1% TEA, and 100% EtOAc (16 L) and 1% TEA). The combined pure product fractions were concentrated in vacuo to give Compound 134 as an off-white solid foam (662.2 g). Fractions of the mixture were combined and concentrated in vacuo (480 g). A white insoluble solid formed upon dilution with toluene (300 ml) prior to loading into Biotage 150L was removed by vacuum filtration. Toluene soluble material was purified with Biotage 150M HP-Sphere (SiO ₂ 2.5 kg (pretreated with 1% TEA); sample loading with toluene; eluents: 2:1 heptane/EtOAc (26 L) with 1% TEA, 1 :1 (25 L) with 1% TEA, 1:4 (34 L) with 1% TEA). The column was monitored by TLC (heptane/EtOAc, 1:1). The combined pure product fractions were concentrated in vacuo to give additional compound 134 as an off-white solid foam (165.5 g. .%, yield 67%).

¹ H NMR (400 MHz, CHLOROFORM-d) δ=8.47 (s, 1H), 8.39 (s, 1H), 8.20 (s, 1H), 8.01 (s, 1H), 7.38 -7.31 (m, 5H), 7.27-7.19 (m, 6H), 7.14-7.06 (m, 3H), 6.93-6.87 (m, 2H), 6 .76 (d, J=8.6 Hz, 4H), 6.26 (dd, J=2.0, 16.0 Hz, 1H), 6.15 (dd, J=2.7, 17.2 Hz, 1H), 5.86 (dd, J=4.7, 15.2 Hz, 1H), 5.80 (dd, J=4.7, 15.2 Hz, 1H), 5.51 (ddd , J=2.7, 4.3, 52.8 Hz, 1H), 5.31 (ddd, J=2.0, 4.3, 52.8 Hz, 1H), 4.87 (d, J =4.7 Hz, 2H), 4.85-4.81 (m, 1H), 4.79 (d, J=4.3 Hz, 2H), 4.71-4.59 (m, 1H) , 4.20-4.13 (m, 2H), 4.06 (dd, J=2.7, 11.3 Hz, 1H), 3.90 (dd, J=2.7, 11.7 Hz , 1H), 3.77 (s, 6H), 3.52 (dd, J=3.1, 10.9 Hz, 1H), 3.18 (dd, J=3.9, 10.9 Hz, 1H), 0.92 (s, 9H), 0.84 (s, 9H), 0.10 (s, 3H), 0.09 (s, 6H), 0.07 (s, 3H)

Stage 5-6

To a solution of Compound 134 (410.7 g, 309 mmol, 1 wt.%, 1 vol., 1 eq.) in pyridine (1.23 L, 1.21 kg, 15.2 mol, 2.9 wt.% , 3.0 vol., 49 eq.) was added diphenyl phosphite (90 ml, 109 g, 0.46 mol, 0.26 wt.%, 0.22 vol., 1.5 eq.). The reaction was stirred at room temperature and monitored by LC/MS. After 2 hours (80% conversion) additional diphenyl phosphite (29.9 mL, 36.2 g, 155 mmol, 0.088 wt%, 0.073 vol, 0.50 eq) was added. After an additional 1 hour, additional diphenyl phosphite (6.0 ml, 7.2 g, 31 mmol, 0.018 wt.%, 0.015 vol., 0.10 eq.) was added and the reaction was continued for another 0.5 hours (98% conversion) . The reaction mixture was added to a mixture of saturated NaHCO ₃ (9 wt.% solution in water; 2.1 l, 5 vol.) and water (1.0 l, ml, 2.5 vol.), maintaining T-internal from 4, 7 to 12°C. The reactor was washed with a small volume of EtOAc. Stirring was continued at room temperature for 30 minutes and the reaction was monitored by LC/MS (100% conversion). The reaction mixture was extracted twice with a 1:1 mixture of EtOAc and MTBE (2×8.2 L, 2×20 vol.). The combined organic layers were washed with water (4.1 L, 10 vol.), concentrated in vacuo and azeotropically distilled with toluene (3 x 4.1 L, 3 x 10 vol.; continuous feed) to remove pyridine to give Compound 135 ( 0.55 equivalents of pyridine left).

Step 6: Crude compound 135 was dissolved in dichloromethane (3.08 L, 4.07 kg, 9.9 wt%, 7.5 vol) at ambient temperature. Water (55.7 ml, 0.136 vol., 10 eq.) was added followed by dichloroacetic acid solution (77 ml, 120 g, 0.93 mol, 0.29 wt.%, 0.19 vol., 3.0 eq. .) in DCM (3.08 L, 7.5 vol.) while maintaining internal T below 25°C. (Turn to orange solution). Triethylsilane (Et ₃ SiH; 494 mL, 359 g, 3.09 mol, 0.875 wt%, 1.20 vol, 10.0 eq) was added after 30 min (T-int. changed from 18.2°C to 17°C) and stirring was continued for 20 minutes. Triethylamine (431 ml, 313 g, 3.09 mol, 0.762 wt.%, 1.05 vol., 10.0 eq.) was added (T-internal changed from 17.8°C to 22°C). The mixture was concentrated to 1.55 kg (3.8 wt%), redissolved in EtOAc (6.2 L, 5.5 kg, 14 wt%, 15 vol), washed successively with: (1) water (1 .0 l, 2.5 vol.) and saturated NaHCO ₃ (9 wt.% solution in water, 0.82 l, 2.0 vol.). A solution of the crude EtOAc product was stored at -20°C overnight; 0.82 L, 2.0 vol.), and the next day the solution was concentrated in vacuo at 25°C. The crude mixture thus obtained (654 g) was triturated with: (1) n-heptane (3.01 L, 7.5 vol.), (2) n-heptane mixture (2.46 L, 6.0 vol.) and toluene (0.82 l, 2.0 vol.). Part of the solution (supernatant) was discarded and the solid remaining at the bottom was dissolved in acetonitrile (4.1 L, 10 vol). The mixture was concentrated in vacuo at 25° C. and azeotropically distilled twice with acetonitrile to give compound 136. The product was used in the next step without purification (assumed theoretical yield 100%).

Stage 7

Stage 7a. Compound 136 (337 g, 309 mmol, 1 wt%, 1 v/v, 1 eq) was dissolved in anhydrous pyridine (13.5 L, 13.2 kg, 39 wt%, 40 v/v) at room temperature. Triethylamine (129 mL, 927 mmol, 94 g, 0.28 wt%, 0.38 vol, 3.0 eq) was added followed by 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphinan 2-oxide (DMOCP; 103 g, 556 mmol, 0.31 wt%, 1.80 eq.). The resulting mixture was stirred at ambient temperature for 30 minutes and monitored by LC/MS (100% conversion) to give Compound 137.

Stage 7b. To the above mixture of Compound 137 was added TEA (129 ml, 927 mmol, 94 g, 0.28 wt.%, 0.38 vol., 3.0 eq.), water (100 ml, 5.56 mol, 0. 30 wt.%, 0.30 wt.%, 18 eq.) and sulfur (34.7 g, 1.08 mol, 0.10 wt.%, 3.5 eq.). After 90 minutes (100% conversion), NaHCO ₃ (9 wt.% solution in water; 3.37 L, 10 vol.) was added, keeping the T-internal below 30°C (from 16.6°C to 27°C) . The resulting mixture was filtered to remove salts. The filtrate was concentrated in vacuo, diluted with MTBE (5.1 L, 15 vol) and washed twice with NaCl (30 wt% solution in water; 2 x 1.35 L, 2 x 4 vol). Insoluble solids were filtered off, the filtrate was concentrated in vacuo and azeotropically distilled with toluene (4.0 L, 12 vol). The resulting solid was removed by filtration and the crude mixture was dissolved in toluene and purified through Biotage 150L KP-Sil (SiO ₂ 5 kg; pretreated with Hep/EtOAc/TEA (1.5/1.5/0.03 CV); eluted: EtOAc/TEA (3/0.03 CV), EtOAc/MeOH/TEA (4/0.2/0.04 CV), EtOAC/MeOH/TEA (2/0.2/0.02 CV) column was monitored with by TLC (EtOAC/MeOH/TEA=9/1/0.1) Fractions containing the Sp isomer were combined and concentrated in vacuo to give Compound 138 as a light pink solid foam (Sp isomer; 154 g, 128 mmol, 0.46 wt%, yield 41.3%) Fractions containing the Rp isomer were combined and concentrated in vacuo to give compound 240 as a light pink solid foam (Rp isomer; 64 g, 53 mmol, 0.19 wt%, yield 17%).

Compound 138 (Sp-isomer):

¹ H NMR (400 MHz, CHLOROFORM-d) δ=8.51 (s, 1H), 8.50 (s, 1H), 8.22 (s, 1H), 8.14 (s, 1H), 7 .49-7.44 (m, 2H), 7.38-7.27 (m, 4H), 7.25-7.21 (m, 2H), 7.14 (t, J=7.1 Hz , 2H), 6.44 (dd, J=2.5, 13.9 Hz, 1H), 6.18 (d, J=15.2 Hz, 1H), 5.78 (td, J=6, 3, 15.6 Hz, 1H), 5.69 (td, J=4.7, 15.6 Hz, 1H), 5.56 (dd, J=3.9, 50.8 Hz, 1H), 5.20-5.06 (m, 1H), 4.95-4.79 (m, 4H), 4.69 (dd, J=4.3, 16.0 Hz, 1H), 4.54- 4.38 (m, 3H), 4.35 (d, J=5.5 Hz, 1H), 4.32-4.29 (m, 1H), 4.05 (dd, J=1.6, 11.7 Hz, 1H), 3.91 (dd, J=3.1, 11.7 Hz, 1H), 3.14-3.06 (m, 6H), 1.30 (t, J=7 .4 Hz, 9H), 0.91 (s, 9H), 0.90 (s, 9H), 0.12 (s, 3H), 0.08 (s, 3H), 0.06 (s, 3H ), 0.05 (s, 3H)

Compound 240 (Rp isomer):

¹ H NMR (400 MHz, CHLOROFORM-d) δ=8.54 (s, 1H), 8.38 (s, 1H), 8.33 (s, 1H), 8.01 (s, 1H), 7 .39-7.09 (m, 10H), 6.39 (dd, J=2.3, 14.1 Hz, 1H), 6.13 (d, J=17.2 Hz, 1H), 5, 72 (d, J=3.1 Hz, 2H), 5.68 (dd, J=4.3, 51.2 Hz, 1H), 5.43-5.29 (m, 1H), 5.10 -4.96 (m, 3H), 4.90-4.83 (m, 2H), 4.78-4.72 (m, 1H), 4.52 (ddd, J=3.9, 6, 6, 17.2 Hz, 1H), 4.44-4.35 (m, 2H), 4.31-4.26 (m, 1H), 4.20-4.12 (m, 2H), 3 .87 (dd, J=3.5, 11.7 Hz, 1H), 3.79-3.77 (m, 1H), 3.15-3.09 (m, 6H), 1.33 (t , J=7.4 Hz, 9H), 0.94 (s, 9H), 0.89 (s, 9H), 0.13 (s, 3H), 0.12 (s, 3H), 0.10 (s, 3H), 0.09 (s, 3H)

Stage 8

Compound 138 (221 g, 183 mmol, 1 wt%, 1 v/v, 1 eq) was dissolved in a mixture of pyridine (530 ml, 6.56 mol, 519 g, 2.3 wt%, 2.4 v/v). ) and TEA (2.65 L, 19.0 mol, 1.93 kg, 8.7 wt%, 12 vol., 104 eq.). Triethylamine trihydrofluoride (264 ml, 1.62 mol, 262 g, 1.2 wt%, 1.2 vol., 8.9 eq. as complex, 27 eq. HF) was added and the mixture was stirred at room temperature, monitoring while the degree of conversion using LC/MS. After 3 hours (97% conversion), methoxytrimethylsilane (™SOMe; 1.40 L, 10.2 mol, 1.06 kg, 4.8 wt%, 6.3 vol, 55 eq) was added and stirring was continued within 30 minutes. The reactor was covered with a sticky solid. Part of the solution (supernatant) was decanted. The solid was triturated twice with toluene (2 x 2.2 L, 2 x 10 vol; supernatant discarded). The crude solid remaining in the reactor was dissolved in dichloromethane (2.2 L, 10 vol.) and washed with NH ₄ Cl (28 wt.% solution in water; 2.2 L, 10 vol.). The aqueous layer was back extracted with dichloromethane (2.2 L, 10 vol). The combined organic layers were washed with a mixture of NaCl (36 wt.% solution in water; 1.1 L, 5 vol.) and water (1.1 L, 5 vol.), and then concentrated under vacuum to obtain Compound 139 as a yellow- brown dry foam (152 g, 155 mmol, 0.70 wt%, 85% yield). The crude product was used in the next step without purification.

Stage 9

Compound 139 (150 g, 153 mmol, 1 wt%, 1 v/v, 1 eq) was azeotroped with acetonitrile (4 L, 27 v/v) and then redissolved in acetonitrile (1.05 L, 0. 83 kg, 5.5 wt.%, 7.0 vol.) at rt (room temperature). 2-Nitrobenzyl bromide (44.4 g, 205 mmol, 0.30 wt%, 1.34 eq) was added at room temperature and the reaction was monitored by LC/MS. After 23 hours (100% conversion), EtOAc (1.50 L, 10 vol.), NH ₄ Cl (28 wt.% solution in water; 300 ml, 2 vol.), and water (300 ml, 2 vol.) were added. ) (pH=6), and the resulting mixture was partially concentrated under vacuum at 25°C to 1.11 kg. EtOAc (2.25 L, 15 vol) was added and the mixture was stirred for 5 minutes. The two layers were separated. The aqueous layer was extracted with ethyl acetate (750 ml, 5 vol). The combined organic layers were washed successively with: (1) a mixture of NaCl (36 wt.% solution in water; 300 ml, 2 vol.) and water (300 ml, 2 vol.) and (2) water (600 ml, 4 vol.) . The organic layer was then concentrated in vacuo and azeotropically distilled with n-heptane (1.50 L, 10 vols). MTBE (0.95 L, 6.3 vol) was added to the crude solid and the mixture was heated at 40°C. The mixture was diluted with EtOAc (300 ml, 2 vol) and slowly cooled to 0°C. The dense solid was allowed to settle, and the supernatant was pumped out through a tube with a filter frit. The solid was washed twice with MTBE (2 x 300 ml, 2 x 2 volumes; the supernatant was siphoned off through a fritted tube each time) and dried in vacuo at 40° C. overnight to give Compound 140 as a pale yellow solid (156 g). The filtrate was concentrated in vacuo to give a brown oil (17.8 g) which was purified through Biotage Snap-Ultra 340 g (eluents: 0 to 5% MeOH in EtOAc) to give additional compound 140 as a pale yellow solid (5 .8 g). Total Yield: 156g+5.8g=161.8g (net 152 mmol, 95% purity, 99% yield)

¹ H NMR (400 MHz, CHLOROFORM-d) δ=8.46 (s, 1H), 8.15 (s, 1H), 8.10 (s, 1H), 8.09-8.06 (m, 1H), 7.89 (s, 1H), 7.54-7.51 (m, 1H), 7.49-7.45 (m, 4H), 7.37-7.28 (m, 3H) , 7.24-7.19 (m, 3H), 7.16-7.11 (m, 2H), 6.22 (d, J=16.8 Hz, 1H), 6.14 (dd, J =2.7, 17.2 Hz, 1H), 5.83-5.61 (m, 3H), 5.60-5.48 (m, 1H), 5.07 (dd, J=3.5 , 51.6 Hz, 1H), 5.06-4.96 (m, 1H), 4.79 (dd, J=4.9, 15.8 Hz, 1H), 4.69 (d, J= 5.9 Hz, 2H), 4.67-4.56 (m, 1H), 4.48-4.40 (m, 3H), 4.37-4.30 (m, 1H), 4.27 (d, J=5.9 Hz, 2H), 4.19-4.13 (m, 1H), 3.93-3.85 (m, 1H), 3.85-3.78 (m, 1H )

Stage 10-11

Step 10 Compound 140 (purity 95%, net 73.2 g, 72.3 mmol, 1 wt%, 1 v/v, 1 eq) and 2-cyanoethyl N, N, N', N'-tetraisopropyl phosphorodiamidite ( 25.3 ml, 79.5 mmol, 0.33 wt.% 0.35 vol., 1.10 eq.) was azeotropically distilled with anhydrous acetonitrile three times (3×2 L), redissolved in dichloromethane (0. 73 l, 10 vol.) and cooled to 0-5°C. Diisopropylammonium tetrazolide (6.19 g, 36.1 mmol, 0.085 wt%, 0.50 eq) was added. The resulting reaction mixture was stirred at 0°C for 10 hours, heated to 10°C for 2 hours, kept at 10°C for 10 hours and warmed to room temperature for 2 hours. The reaction was followed by LC/MS and TLC (EtOAc with 0.5% TEA). After 18 hours, anhydrous acetonitrile (0.73 L, 10 vol) was added and the mixture was stored at -20° C. for 3 days.

Stage 11a. The mixture obtained in step 10 was warmed to ambient temperature and added via addition funnel in portions (100 ml every 30 minutes, for 9 hours) to a mixture of pyridine trifluoroacetate salt (previously twice azetropine-distilled with pyridine; 41.9 g, 217 mmol, 0.57 wt.%, 3.0 eq.) and acetonitrile (5.85 L, 80 vol.). The reaction was monitored by LC/MS. After 13 hours, a solution of 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (5.8 ml, 18 mmol, 0.25 eq.) in acetonitrile (24 ml) was added over 4 hours. The amount of additional reagent was determined based on the transamination of Compound 140 (~30% by LC/MS). A higher degree of conversion of the diol was observed after 6 hours.

Stage 11b. (Dimethylaminomethylidene)amino)-3H-1,2,4-dithiazolin-3-thione (DDTT; 20.8 g, 101 mmol, 0.28 wt%, 1.4 eq) was added and stirring was continued for 1 hour. The reaction mixture was partially concentrated to ~800 ml and diluted with MTBE (1.46 L, 20 vol.), NaHCO ₃ (9 wt.% solution in water; 1.1 L, 15 vol.) and water (0.37 L, 20 vol.). 5 vol.), pH=8. The layers were separated and the aqueous layer was extracted with a mixture of MTBE (1.46 L, 20 vol) and EtOAc (1.10 L, 15 vol). The combined organic layers were washed twice with 30% aqueous NaCl (2×0.73 L, 2×10 vol.), concentrated in vacuo at 35°C and azeotropically distilled with toluene (1.46 L, 20 vol.). The results of LC/MS and TLC (EtOAc) showed that the ratio of compound 143 (SpRp, desired) to compound 241 (SpSp) is 5:1.

The crude product was purified through Biotage 150M KP-Sil, (SiO ₂ 2.5 kg; eluents: EtOAc/Hep: 2:1 (4 CV), 3:1 (2.5 CV), 4:1 (2.5 CV ), 100% EA (3 CV), 5-10% MeOH in EA 4 CV) to give Compound 143 (36 g, 31.5 mmol, 44% yield).

Compound 143 (SpRp): ¹ H NMR (400 MHz, CHLOROFORM-d) δ=8.59 (s, 1H), 8.10 (s, 1H), 8.03-7.99 (m, 1H), 7.91 (s, 1H), 7.56-7.53 (m, 2H), 7.49-7.40 (m, 5H), 7.35-7.28 (m, 2H), 7. 24-7.16 (m, 4H), 6.92 (s, 1H), 6.29 (d, J=14.9 Hz, 1H), 6.08 (d, J=20.7 Hz, 1H ), 5.97-5.83 (m, 1H), 5.76 (td, J=4.7, 15.6 Hz, 1H), 5.61-5.51 (m, 2H), 5. 40 (d, J=4.3 Hz, 1H), 5.29-5.17 (m, 1H), 4.91 (dd, J=7.4, 14.9 Hz, 1H), 4.86 -4.75 (m, 3H), 4.63 (dd, J=3.7, 9.2 Hz, 1H), 4.58-4.43 (m, 5H), 4.34-4.19 (m, 4H), 2.79 (td, J=5.9, 16.8 Hz, 1H), 2.66 (td, J=6.3, 16.8 Hz, 1H).

Compound 241 (SpSp) ¹ H NMR (400 MHz, CHLOROFORM-d) δ=8.11 (s, 1H), 8.03 (d, J=8.2 Hz, 1H), 7.94 (s, 1H ), 7.90 (s, 1H), 7.61 (s, 1H), 7.56-7.40 (m, 7H), 7.33-7.28 (m, 2H), 7.23- 7.17 (m, 4H), 6.22 (d, J=17.6 Hz, 1H), 6.15 (d, J=18.8 Hz, 1H), 5.85 (dd, J=3 .5, 51.2 Hz, 1H), 5.75-5.45 (m, 5H), 4.95-4.23 (m, 14H), 2.82 (t, J=6.1 Hz, 2H).

Stage 12

Compound 143 (71.6 g, 62.6 mmol, 1 wt%, 1 v/v, 1 eq) was dissolved in 1,4-dioxane (0.43 L, 6 v/v). Thiophenol (215 ml, 2.09 mol, 230 g, 3.2 wt%, 3 vol, >30 eq.) was added followed by triethylamine (215 ml, 1.54 mol, 156 g, 2.2 wt. %, 3 vol.). Some exotherm was observed (T-internal increased by ~7°C), so a water/ice bath was used to cool and maintain T-intrinsic below 27°C. The reaction was monitored by LC/MS. After 2 hours, MeOH (0.57 L, 8 vol.) and NH ₄ OH (28 wt%; 15 mol, 0.57 L, 8 vol., >200 eq.) were added. The resulting mixture was heated at 50°C for 5 hours, cooled to room temperature and stirred overnight. After 14 hours, water (0.72 L, 10 vol) was added (no solid precipitation was observed) and the mixture was extracted three times with a mixture of n-heptane and toluene (3 x 0.86 L, 3 x 12) 1:1 (v ./vol.), and then toluene (0.57 l, 8 vol.). The aqueous layer was concentrated in vacuo at 40-50° C. and diluted with water (1.07 L, 15 vol). The resulting suspension was left overnight at room temperature. The resulting solid was filtered off, washing with water (0.36 L, 5 vol). The filtrate was still cloudy and was filtered through celite and a Kuno filter. The turbidity was still present. HCl (1.0M solution in water; 132 ml, 132 mmol, 2.1 eq.) was added over 1 hour and the pH was checked (pH<2). Stirring was continued at room temperature for 1 hour and the mixture was filtered. The filter cake was washed with water (8×0.20 L), dried in a vacuum oven at 35°C for 2 days and without heat for 1 day to give Compound 1 as a pale orange solid (44.88 g, 60.1 mmol, 0.63 wt%, 96% yield.

Stage 13

To Compound 1 in the free acid form (22.42 g, 30.03 mmol, 1 wt.%, 1 vol., 1 eq.) was added ammonia (2.0 M solution in MeOH; 220 ml, 440 mmol, 10 vol. ., 15 eq.). EtOH (55 mL, 2.5 vol) was added and the resulting solution was filtered through a Kuno filter (0.45 micron; PTFE), washing 1:1 with a mixture (v/v) of MeOH and EtOH (90 mL, 4 vol). .). The filtrate was concentrated in vacuo at 30° C. to give an off-white solid which was dried at room temperature overnight, ground with a spatula (easily crumbled) and dried in vacuo at room temperature. The isolated solid was then suspended in toluene (250 ml) and stirred at room temperature for 30 minutes. Thereafter, the solid was collected by vacuum filtration and washed twice with toluene (2×50 ml). The solid was then dried under vacuum in a vacuum oven to give 22.4 g of compound 1a (diammonium salt of compound 1).

Recrystallization: Compound 1a (22.14 g, 28.36 mmol, 1 wt%, 1 v/v, 1 eq) was dissolved in a mixture of water (664 ml, 30 v/v) and ammonium hydroxide (28 wt%; 2 .5 ml, 18 mmol, 0.63 eq.) (pH=9-10) and extracted three times with toluene (3×300 ml, 3×14 vol.), three times with EtOAc (3×200 ml, 3×9 vol. ) and three times with toluene (3×300 ml, 3×14 vol.). The resulting aqueous layer was treated with HCl (1.0 M solution in water; 90 ml, 90 mmol, 3.2 eq.) for 3.5 hours (pH≤2). The mixture was stirred for 30 minutes and then the solid precipitate was collected by vacuum filtration. The filter cake was washed three times with water (3×200 ml, 3×9 vol.) and dried in vacuum overnight. Ammonia (2.0 M solution in MeOH; 250 ml, 500 mmol, 17.6 eq.) and ethanol (100 ml) were added to the solid, and the resulting mixture was concentrated in vacuo until crystals appeared (~100 ml), after which the concentration process was stopped and the mixture was stirred for 20 minutes. Ethanol (45 ml) was added and the mixture was partially concentrated (45 ml removed). This operation was repeated two more times, after which the mixture was cooled to 0°C and stirred for 3.5 hours. The white solid was collected by vacuum filtration and washed with cold ethanol (20 ml) and then with ethyl acetate (2x50 ml). The white solid was dried in vacuo at room temperature for 3 days to give Compound 1a as a white solid (16.6 g, 21.3 mmol, 0.75 wt%, 75% yield). The filtrate was concentrated in vacuo and dried in vacuo at room temperature for 3 days to give compound 1a as an off-white solid (4.16 g, 5.3 mmol, 18% yield).

Example 1.2- ¹ H NMR analysis of compound 1

¹ H NMR spectrogram of compound 1a is shown in Fig.3. The resulting spectrum was as follows:

¹ H NMR spectrum (400 MHz, DMSO-d6, δH 2.49 ppm, 80°C)

δ (ppm): 3.05-3.13 (4H, m), 3.70 (1H, dd, J=13.5 Hz), 3.78 (1H, dd, J=12.4 Hz), 4.21-4.24(2H, m), 4.28(1H, m), 4.38(1H, m), 4.53-4.68(2H, m), 5.22(1H, m), 5.76(2H, s), 5.78(1H, m), 6.26(1H, m), 6.29(1H, m), 8.13(1H, s), 8, 14 (1H, s), 8.36 (1H, br.s), 8.59 (1H, br.s).

Example 1.3 - X-Ray Analysis of Compound 1

Approximately 2 mg of compound 1 was dissolved in 600 μl of water. 120 μl of this solution was placed in another glass vial, and then this vial was stored in a closed container with 3 ml of MeCN at room temperature for 1 week. The sample preparation method was the H ₂ O/MeCN vapor diffusion method.

A colorless blocky (cellular) single crystal (0.1 x 0.1 x 0.1 mm) found in the crystallization solution was dispersed in Parabar 10312 liquid and mounted on a Dual-Thickness MicroMounts™ (MiTeGen). Diffraction data were collected at -160°C with an XtaLAB PRO P200 MM007HF (Rigaku) ω oscillation method using monochromatized Cu-Kα radiation using multilayer coated mirrors.

In FIG. 4A shows an ORTEP image of the molecules of Compound 1 as an asymmetric unit along with disordered water molecules. In FIG. 4B shows the crystal structure of one of the molecules of Compound 1 shown in FIG. 4A. In FIG. 4C shows the crystal structure of another molecule of Compound 1 shown in FIG. 4A.

The crystal structure of Compound 1 was obtained with a final R-factor of 0.1354. The flack parameter was almost zero (0.083 (17)), indicating that the absolute configuration of Compound 1 is (R, S). Crystal structure analysis also showed that many water molecules were present in the large channel of compound 1, indicating that water molecules can easily slip out of the channel. The analysis also confirmed that the conformations of both crystallographically independent molecules of the asymmetric unit are practically the same.

Additional parameters of X-ray diffraction analysis are given below:

Temperature 113K Wavelength 1.54184 Å Crystal system, space group monoclinic, P2 ₁ Crystal lattice parameter a=8.1584(3) Å b=35.451(2) Å c=15.9146(6) Å β=91.313(3)° Volume 4601.7(4) Å ³ Z value, calculated density 4, 1.127 g/ ^cm3 Crystal size 0.1×0.1×0.1mm Total number of reflections/number of unique reflections 52006/17198 [R(intensity)=0.0876] completeness 92.2% Phase detection Direct methods (SHELXT version 2014/5) refinement method Full Matrix Least Squares by F ² (SHELXL version 2014/7) Data/parameter 17198/1116 Goodness Criteria 1.545 Remainders: R(I>2σ(I)) 0.1354 Remains: Rw 0.3886 Flack parameter 0.083(17) Difference between maximum and minimum peaks 1.17 and -0.88 e ^- / Å ³

Example 2 X-Ray Structure Confirming Complex with WT STING

In order to better understand the mechanism by which the proposed new compounds bind to targets, the crystal structure of WT STING in complex with the compounds was determined using X-ray diffraction analysis.

A. Expression and purification of the C-terminal domain of WT STING (residues 155-341)

The DNA sequence encoding the human WT STING protein from amino acid 155 to amino acid 341 (SEQ ID NO: 4) was cloned into the pET21b vector, after a His-TEV-Sumo tag at the N-terminus (SEQ ID NO: 5). The pET21b sequence has been deposited with addgene and is available at addgene.org/vector-database/2550/; this sequence is included in the present description by reference.

E. coli BL21 (DE3)-Codon Plus cells were transformed with this plasmid and expression of the recombinant protein was induced with 0.1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG). The protein was purified from the soluble fraction of the cell lysate by affinity chromatography on a Ni-NTA column. The His-TEV-Sumo label was removed with Sumo protease and separated from unlabeled WT STING 155-341 using a second Ni-NTA affinity column. The protein was further purified by anion exchange and size exclusion chromatography and stored in a buffer containing 20 mM Tris-HCl pH 7.5 and 150 mM NaCl at a concentration of 35 mg/ml.

C. Crystallization and determination of the structure of the C-terminal domain of WT STING in complex with compound 1

For co-crystallization of WT STING 155-341 with compound 1, the WT STING protein was diluted to 10 mg/ml with storage buffer (20 mM Tris-HCl pH 7.5 and 150 mM NaCl) and mixed with compound 1 (100 mM stock solution in DMSO ) in a molar ratio of 1:5. The mixture was incubated for 4 hours at 4°C and centrifuged at 13,000 rpm for 20 minutes before crystallization. Set mesh trays for crystallization using the method of vapor diffusion in the form of a hanging drop at 18°C. Crystals were grown by mixing 1 μl of WT STING/Compound 1 solution with an equal volume of well solution containing 100 mM HEPES (pH=7.5), 200 mM CaCl ₂ , and when the crystals were flash frozen in liquid nitrogen, as cryoprotectant 15% (w/v) PEG 8000, 20% (w/v) PEG 400 were used. Diffraction data sets were collected using a Pilatus detector on the SSRF BL19U1 beam channel and processed using the HKL3000 and the SCALEPACK2MTZ program in the software package CCP4.

The structure of WT STING 155-341 associated with Compound 1 was determined by molecular replacement using the PHASER (Maximum Likelihood Molecular Replacement) program with PDB ID 4F9E as the initial search model. The presence of Compound 1 between the contact areas of the WT STING dimer was confirmed on the Fo-Fc difference map calculated with model phases. The model was built and supplemented manually using the Coot program and finalized using the Refmac5 program in the CCP4 software package. The final refined structure is shown at 2.38 Å resolution in space group P212121 with unit cell measured at a=33.820, b=78.110, c=132.212, α=90.00, β=90.00, γ=90.00. In each asymmetric unit, two copies of WT STING 155-341 were identified, binding to one molecule of compound 1 at the dimer contact area.

C. Reaction of compound 1 with WT STING observed in the crystal structure obtained by X-ray diffraction analysis

In FIG. 5 shows an X-ray diffraction image of the crystal structure of human STING WT complexed with Compound 1. The X-ray diffraction analysis of the crystal structure of human WT STING complexed with Compound 1, which was co-crystallized from a sample of Compound 1a, was examined. The compound binds to the contact region of the pocket formed by the dimer of the WT STING protein. The two faces of the compound's adenine base are in π-π stacking interaction with Tyr240 and the guanidine group of Arg238, respectively. The trans-olefin linker is in van der Waals interaction with the aliphatic part of the side chain of Arg 238. The fluorine substituent at the C2'-ribose position of the compound is in a hydrophobic cavity defined by Thr263, Pro264 and Tyr163. The negatively charged thiophosphate group of the compound forms a salt bridge with Arg238 and hydrogen bonds with Ser162 and Thr267, respectively. In addition, the thiophosphate group also interacts electrostatically with the guanidine group of Arg232. The LID loop region of WT STING, consisting of 226-243 residues, is wrapped around two main groups and a trans-olefin linker.

Example 3 Crystal Structure Determination of REF STING Complexed with Compound 1 by X-Ray Analysis

A. Expression and purification of the REF STING C-terminal domain (residues 155-341, SEQ ID NO: 6)

The DNA sequence encoding the human REF STING protein amino acid 155 to amino acid 341 (SEQ ID NO: 6) was cloned into the pET21b vector after a His-TEV-Sumo tag at the N-terminus (SEQ ID NO: 7). The pET21b sequence has been deposited with addgene and is available at addgene.org/vector-database/2550/; this sequence is included in the present description by reference.

E. coli BL21 (DE3)-Plus Codon cells were transformed with this plasmid and expression of the recombinant protein was induced with 0.1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG). The protein was purified from the soluble fraction of the cell lysate by affinity chromatography on a Ni-NTA column. The His-TEV-Sumo tag was removed with Sumo protease and separated from the unlabeled REF STING_155-341 using a second Ni-NTA affinity chromatography column. The protein was further purified by anion exchange and size exclusion chromatography and stored in a buffer containing 20 mM Tris-HCl pH 7.5 and 150 mM NaCl at a concentration of 24 mg/ml.

B. Crystallization and Structure Determination of the C-Terminal Domain of REF STING in Complex with Compound 1

For co-crystallization of REF STING_155-341 with Compound 1, REF STING protein was diluted to 10 mg/ml with storage buffer (20 mM Tris-HCl pH 7.5 and 150 mM NaCl) and mixed with Compound 1 (100 mM stock solution in DMSO ) in a molar ratio of 1:5. The mixture was incubated for 4 hours at 4°C and centrifuged at 13,000 rpm for 20 minutes before crystallization. Set mesh trays for crystallization using the method of vapor diffusion in the form of a hanging drop at 18°C. Crystals were grown by mixing 1 μl of REF STING/Compound 1 solution with an equal volume of well solution containing 100 mM HEPES (pH=7.5), 200 mM CaCl ₂ and, when the crystals were flash frozen in liquid nitrogen, as a cryoprotectant 15% (w/v) PEG 8000, 20% (w/v) PEG 400 were used. Diffraction data sets were collected with a Pilatus detector on a SSRF BL18U1 beam channel and processed with HKL3000 and SCALEPACK2MTZ software in CCP4 software package . This structure is shown in Fig. 6.

The structure of REF STING 155-341 bound to Compound 1 was determined by molecular replacement using the PHASER (Maximum Likelihood Molecular Replacement) program, using the previously determined structure of WT STING 155-341 (as described above) as the initial search model. The presence of Compound 1 between the contact areas of the WT STING dimer was confirmed on the Fo-Fc difference map calculated with model phases. The model was built and supplemented manually using the Coot program and finalized using the Refmac5 program in the CCP4 software package. The final refined structure is shown at 2.76 Å resolution in space group P212121 with unit cell measured at a=33.733, b=77.831, c=131.689, α=90.00, β=90.00, γ=90.00. In each asymmetric unit, two copies of REF STING 155-341 were identified, binding to one molecule of Compound 1 at the dimer interface.

C. Reaction of compound 1 with REF STING observed in the crystal structure obtained by X-ray diffraction analysis

In FIG. 6 shows the X-ray diffraction crystal structure of human REF STING complexed with Compound 1, which was co-crystallized from a sample of Compound 1a. The compound binds to the contact region of the pocket formed by the dimer of the STING protein. The two faces of the compound's adenine base are in π-π stacking interaction with Tyr240 and the guanidine group of Arg238, respectively. The trans-olefin linker is in a van der Waals interaction with the aliphatic part of the Arg238 side chain, while the guanidine part of the Arg238 side chain is in π-π stacking interaction with the outward imidazole group of the His232 side chain. The olefin linker is in contact with an interacting pair of Arg238 and His232 side chains. The fluorine substituent at the C2'-ribose position of the compound is located in a hydrophobic cavity defined by Thr263, Pro264 and Tyr163. The negatively charged thiophosphate group of the compound forms a salt bridge with Arg238 and hydrogen bonds with Ser162 and Thr267, respectively. The loop region of the LID REF STING, consisting of residues 226 to 243, is wrapped around two base groups and a trans-olefin linker.

Example 4 In Vivo Evaluation of Compound 1a in an Intravesical Mouse Model of Bladder Cancer

The MBT-2 orthotopic bladder cancer model in mice was created and characterized by Lee et al. and this model has been shown to have similar histology to human bladder cancer. Therefore, this model was chosen to evaluate the activity of Compound 1 against bladder cancer. The HCl pretreatment protocol described by Lee, et al. was used to create the model. Briefly, 30 µl 0.1 N. HCl solution was injected into the mouse bladder through a catheter, and this HCl solution was left in the bladder for 15 seconds. Then the HCl solution was replaced with 30 μl of 0.1 N. NaOH solution followed by a washing step with 1x PBS (pH 7.4). The procedure for subsequent tumor cell implantation has been modified from that published. Briefly, after the washing step, MBT-2 tumor cells (2×10 ⁶ cells in 50 μl of RPMI1640 medium) were injected into the bladder and left for 45 minutes. At the end of this 45 minute period, the bladder was emptied.

Three days after tumor cell implantation, mice were treated with various doses of compound 1a, BCG (OncoTICE®, Merck Canada Inc.), mouse anti-PD-1 antibody (clone no. RMP1-14, Bioxcell), and a combination of compound 1a with anti-PD- 1 antibody according to the scheme shown in Table 1. Compound 1a and BCG were administered intravesically, while the anti-PD1 antibody was administered by intraperitoneal injection.

On each dosing day, BCG was diluted in 0.9% NaCl (Lavoisier, France) to a final concentration of 16.875 mg/mL for dosing. Any remaining dosing solution was discarded after use. The stock solution of anti-PD-1 antibodies was diluted with 1x PBS (Lonza, France) to obtain a stock solution of 1 mg/ml used for dosing. For Compound 1a, a 10 mg/ml stock solution was first prepared by dissolving the dried powder in 1x PBS. Various concentrations of Compound 1a were then obtained, including 5 mg/ml (400 μg/group of mice), 2.5 mg/ml (200 μg/group of mice) and 1.25 mg/ml (100 μg/group of mice) by further dilution stock solution in 1x PBS.

Table 1. Therapeutic agents and dosing regimens

Group Number of animals Treatment Dose Route of administration Treatment schedule 1 10 Carrier - IVe ¹ Q7Dx3 2 10 BCG 1.35 mg/mouse IVe Q7Dx3 3 10 PD1 antibody 10 mg/kg/inject. IP ² TWx2 4 10 Compound 1a 100 µg/mouse IVe Q7Dx3 5 10 Compound 1a 200 µg/mouse IVe Q7Dx3 6 10 Compound 1a 400 µg/mouse IVe Q7Dx3 7 10 Compound 1a 400 µg/mouse IVe Q7Dx3 PD1 antibody 10 mg/kg/inject. IP TWx2

1. IVe, intravenous administration

2. IP, intraperitoneal injection

From day 20 to day 21, tumor growth in the bladder was quantified by MRI (magnetic resonance imaging), and tumor sizes in all mice were plotted as shown in FIG. 7. In FIG. 7 shows the quantification of tumor volume by MRI from days 20 to 21 in all study animals. As shown in FIG. 7, all groups treated with compound 1a (groups 4 to 7) showed much lower tumor volumes than the vehicle group (group 1), the BCG group (group 2) and the anti-PD1 antibody group (group 3).

Compound 1a also showed a statistically significant improvement in survival compared to vehicle-treated animals, as shown in FIG. 8.

Both BCG and anti-PD1 antibody did not show significant anti-cancer activity in this orthotopic mouse bladder cancer model, suggesting that the model used is a BCG/PD1 antibody resistance/refractory model.

Example 103 Reporter Assay for Activity of a HAQ STING Agonist Construct

THP1-Dual™ cells (InvivoGen, cat. no. thpd-nfis) were used to determine EC ₅₀ . THP1 Dual™ cells were characterized by the supplier Invivogen (Insight 201402-1) as carriers of the HAQ STING genotype. Cells were grown and maintained under conditions recommended by the manufacturer. In order to determine the EC _{50 ,} the interferon regulatory factor (IRF) pathway was induced as described in the manufacturer's manual. Briefly, cells were seeded and treated with various concentrations of the compound for 20 hours under incubation conditions at 37°C, 5% CO ₂ . Cells were resuspended and QUANTI-Luc™ solution (Cat. No: rep-qlc1) was added. The resulting light emission was measured with a luminometer (Envision, Perkin Elmer). The received signals were plotted and EC ₅₀ was calculated using the GraphPad Prism7 program.

EC ₅₀ human STING values (µM) for Compound 1a are shown in Table 2 below.

Example 104 - Reporter analysis of specific STING variants

Human STING has 4 main variants including WT, HAQ, REF and AQ variants. REF-STING, also referred to as R232H, for example, occurs in approximately 14% of the human population. Compared to the wild-type allele, R232H has a reduced response to bacterial and metazoan cyclic dinucleotides. Details of these 4 major variants, as well as other rare variants, are available from Yi G, et al., "Single nucleotide polymorphisms of human STING can affect innate immune response to cyclic dinucleotides" PLoS One 2013; 8:e77846. STING variant-specific reporter cell lines were generated using THP1-Dual™ KO-STING cells (InvivoGen, Cat # thpd-kostg) and three STING variant protein expression vectors. The expression vector map for WT STING is shown in FIG. 9. For the other two expression vectors, different STING variant sequences were used in this vector, with the WT STING replaced with the corresponding nucleotide sequence.

Vectors expressing variants of STING, WT-STING, REF-STING and AQ-STING were prepared and stably transfected into THP1-Dual™ KO-STING cells for use in reporter analysis of specific variants of STING, WT-STING, REF-STING and AQ-STING. STING, respectively. EC ₅₀ values were determined as described above in Example 103 regarding the reporter analysis of the activity of the HAQ STING agonist construct. The results are shown in Table 2 below. The DNA sequences used for these STING variants are shown in SEQ ID NO: 1 (nucleotide sequence of human WT STING), SEQ ID NO: 2 (nucleotide sequence of human REF STING) and SEQ ID NO: 3 ( nucleotide sequence of human AQ STING).

Human WT STING:

atgccccactccagcctgcatccatccatcccgtgtcccaggggtcacggggcccagaaggcagccttggttctgctgagtgcctgcctggtgaccctttgggggctaggagagccaccagagcacactctccggtacctggtgctccacctagcctccctgcagctgggactgctgttaaacggggtctgcagcctggctgaggagctgc gccacatccactccaggtaccggggcagctactggaggactgtgcgggcctgcctgggctgccccctccgccgtggggccctgttgctgctgtccatctatttctactactccctcccaaatgcggtcggcccgcccttcacttggatgcttgccctcctgggcctcgcaggcactgaacatcctcctgggcctcaagggcctggcccca gctgagatctctgcagtgtgtgaaaaagggaatttcaacgtggcccatgggctggcatggtcatattacatcggatatctgcggctgatcctgccagagctccaggcccggattcgaacttacaatcagcattacaacaacctgctacggggtgcagtgagccagcggctgtattctcctcccattggactgtggggtgcctgataacctga gtatggctgaccccaacattcgcttcctggataaactgccccagcagaccggtgaccgggctggcatcaaggatcgggtttacagcaacagcatctatgagcttctctggagaacgggcagcgggcgggcacctgtgtcctggagtacgccacccccttgcagactttgtttgccatgtcacaatacagtcaagctggctttagccgggaggatagg cttgagcaggccaaactcttctgccggacacttgaggacatcctggcagatgcccctgagtctcagaacaactgccgcctcattgcctaccaggaacctgcagatgacagcagcttctcgctgtcccagggaggttctccggcacctgcggcaggaggaaaaggaagaggttactgtgggcagcttgaagacctcagcggtgcccagtacc tccacgatgtcccaagagcctgagctcctcatcagtggaatggaaaagcccctccctctccgcacggatttctcttga (SEQ ID NO: 1).

Human REF STING:

atgccccactccagcctgcatccatccatcccgtgtcccaggggtcacggggcccagaaggcagccttggttctgctgagtgcctgcctggtgaccctttgggggctaggagagccaccagagcacactctccggtacctggtgctccacctagcctccctgcagctgggactgctgttaaacggggtctgcagcctggctgaggagctgc gccacatccactccaggtaccggggcagctactggaggactgtgcgggcctgcctgggctgccccctccgccgtggggccctgttgctgctgtccatctatttctactactccctcccaaatgcggtcggcccgcccttcacttggatgcttgccctcctgggcctcgcaggcactgaacatcctcctgggcctcaagggcctggcccca gctgagatctctgcagtgtgtgaaaaagggaatttcaacgtggcccatgggctggcatggtcatattacatcggatatctgcggctgatcctgccagagctccaggcccggattcgaacttacaatcagcattacaacaacctgctacggggtgcagtgagccagcggctgtattctcctcccattggactgtggggtgcctgataacctga gtatggctgaccccaacattcgcttcctggataaactgccccagcagaccggtgaccatgctggcatcaaggatcgggtttacagcaacagcatctatgagcttctctggagaacgggcagcgggcgggcacctgtgtcctggagtacgccacccccttgcagactttgtttgccatgtcacaatacagtcaagctggctttagccgggaggatagg cttgagcaggccaaactcttctgccggacacttgaggacatcctggcagatgcccctgagtctcagaacaactgccgcctcattgcctaccaggaacctgcagatgacagcagcttctcgctgtcccagggaggttctccggcacctgcggcaggaggaaaaggaagaggttactgtgggcagcttgaagacctcagcggtgcccagtacc tccacgatgtcccaagagcctgagctcctcatcagtggaatggaaaagcccctccctctccgcacggatttctcttga (SEQ ID NO: 2)

Human AQ STING:

atgccccactccagcctgcatccatccatcccgtgtcccaggggtcacggggcccagaaggcagccttggttctgctgagtgcctgcctggtgaccctttgggggctaggagagccaccagagcacactctccggtacctggtgctccacctagcctccctgcagctgggactgctgttaaacggggtctgcagcctggctgaggagctgc gccacatccactccaggtaccggggcagctactggaggactgtgcgggcctgcctgggctgccccctccgccgtggggccctgttgctgctgtccatctatttctactactccctcccaaatgcggtcggcccgcccttcacttggatgcttgccctcctgggcctcgcaggcactgaacatcctcctgggcctcaagggcctggcccca gctgagatctctgcagtgtgtgaaaaagggaatttcaacgtggcccatgggctggcatggtcatattacatcggatatctgcggctgatcctgccagagctccaggcccggattcgaacttacaatcagcattacaacaacctgctacggggtgcagtgagccagcggctgtattctcctcccattggactgtggggtgcctgataacctga gtatggctgaccccaacattcgcttcctggataaactgccccagcagaccgctgaccgagctggcatcaaggatcgggtttacagcaacagcatctatgagcttctggagaacgggcagcgggcgggcacctgtgtcctggagtacgccacccccttgcagactttgtttgccatgtcacaatacagtcaagctggctttagccgggaggatagg cttgagcaggccaaactcttctgccagacacttgaggacatcctggcagatgcccctgagtctcagaacaactgccgcctcattgcctaccaggaacctgcagatgacagcagcttctcgctgtcccagggaggttctccggcacctgcggcaggaggaaaaggaagaggttactgtgggcagcttgaagacctcagcggtgcccagtacc tccacgatgtcccaagagcctgagctcctcatcagtggaatggaaaagcccctccctctccgcacggatttctcttga (SEQ ID NO: 3)

Example 105 - Reporter Assay for Murine STING Agonist Activity

RAW-Lucia™ ISG cells (InvivoGen, cat. no. rawl-isg) were used for reporter analysis of the mouse STING agonist. EC ₅₀ values were determined as described in Example 103 above in a HAQ STING agonist activity reporter assay. The results are shown below in Table 2.

Example 106 Differential Scanning Fluorimetry (DSF) analysis.

The DSF assay was used to measure the physical interaction between the compounds and the recombinant STING protein. The truncated recombinant STING protein (a.k. 155-341) (SEQ ID NO: 4) was expressed in E. coli and isolated for analysis as described below. The assay matrix was prepared in 384-well plates with a final volume of 10 μl per well, consisting of 1 μM recombinant STING protein (a.a. 155-341) (SEQ ID NO: 4), 100 mM PBS (pH 7.4) , supplemented with 100 mM KCl, 5x SYPRO orange dye, and 50 μM compound (0-1% final DMSO concentration). Analyzes were performed on a QuantStudio 12K Flex real-time PCR system using a temperature gradient from 25°C to 95°C at a rate of 0.05°C/min and excitation and emission filters of 470 and 586 nm, respectively. From derived fluorescence curves generated using Applied Biosystems® Protein Thermal Shift software (algorithm version 1.3), the melting point (Tm) of unbound and ligand bound recombinant STING protein and the melting point difference (dTm D) were calculated.

Compounds with ΔTm values greater than 0 are generally considered to physically interact with the protein under test, with the ΔTm value being directly related to the binding affinity of the compound. In this case, compound 1a showed a ΔTm of 17.6 (Table 2), indicating a physical interaction with the STING protein.

Table 2. Characterization of compound 1a in vitro

Compound EC ₅₀ human STING (µM) EC ₅₀ mouse STING (µM) ∆Tm DSF WT STING (°C) wt HAQ REF AQ 1a 0.9 4.1 4.8 1.2 3.4 17.6

Example 107 Human PBMC Stimulation Ex Vivo Assay

Human blood from 5 healthy donors was collected in 10.0 ml BD Vacutainer sodium heparin tubes (p/n 367874). Isolation of peripheral blood mononuclear cells (PBMCs) was performed in 50 ml SIGMA ACCUSPIN (cat. no. A2055) and SIGMA ACCUSPIN System-HISTOPAQUE-1077 (cat. no. A7054) tubes according to the manufacturer's protocol. The PBMC layer was collected and washed with 1x phosphate buffered saline (PBS) as suggested by Sigma. PBMCs were counted and finally suspended at 1×10 ⁶ /ml in RPMI (Corning cat. no. 10-041-CV) supplemented with 10% fetal bovine serum (FBS) (Gibco cat. no. 20140.79). One ml of cells (1×10 ⁶ ) was transferred to a 5 ml Falcon round bottom polypropylene tube (cat. no. 352063) and stimulated with various concentrations (0, 0.1, 1, 10 μM) for 24 hours in a 5% incubator _CO2 at 37°C.

24 hours after incubation, the tubes were centrifuged at 1400 rpm for 5 minutes and the supernatants were collected. The supernatant was stored at -80°C until the next measurement of IFNβ. IFNβ measurement was performed using a human IFN-β core kit (Meso Scale Diagnostics, cat. no. K151ADA) and using the protocol provided by the manufacturer. IFN-beta was assessed by reading the assay plate on a MESO SECTOR Imager 2400 using the MSD Discovery Workbench 4.0 software. IFNβ protein analysis was performed 24 hours later. The results showed that compound 1a can induce the production of primary human IFNβ protein in PBMCs in a dose-dependent manner.

The results presented in Table 3 represent the average of measurements taken from five different donors.

Table 3 Human PBMC stimulation ex vivo assay

PBS (control) Compound 1a 0.1 µM 1 µM 10 µM IFNβ (pg/ml) 0 21.3±17.8 227.5±62.4 540.2±215.0

To quantify IFNβ mRNA, total RNA was isolated using the RNeasy Mini Kit (Qiagen, Germany) according to the manufacturer's protocol. IFNβ mRNA was quantified by quantitative PCR. Briefly, total RNA (400 to 1000 ng) was converted to cDNA in a 60 μl reaction volume using the SuperScript VILO MasterMix (Life Technologies, USA). The resulting cDNAs (10 ng) were subsequently amplified using TaqMan expression assays from Applied Biosystems using RNA specific primers for IFNB1 (Hs01077958_s1) and GAPDH (Hs99999905_m1). Quantitative PCR analysis was performed using TaqMan Fast Advanced Master Mix (Life Technologies, USA) on an Applied Biosystems Quantstudio 12K Flex real-time PCR system with an initial 2-minute step at 50°C, then at 95°C for 2 sec, and followed by 40 cycles at 95°C for 1 sec and 60°C for 20 sec. Relative gene expression was calculated after normalization to the reference GAPDH gene using the 2-δδCT method. Calculations were performed using Applied Biosystems Quantstudio 12K Flex v1.2.2 software. The fold changes in IFNβ mRNA compared to vehicle treated samples are summarized in Table 4. The results showed that Compound 1a can induce IFNβ mRNA in primary PBMCs in a dose and time dependent manner. Table 4 shows the average calculated for five different donors.

Table 4 - Analysis of 3-hour and 24-hour stimulation of human PBMCs (mRNA) ex vivo

IFNβ mRNA (fold changes relative to vehicle-treated samples) Compound 1a 0.1μM 1 µM 10 µM 3 hour processing 51.0 ± 21.7 219.8 ± 69.8 1973.3 ± 1023.0 24 hour processing 28.1 ± 28.9 10652.3±4992.4 24157.3 ± 9224.2

Example 108 Anticancer effect of Compound 1a in the CT26 twin tumor model.

Compound 1a was tested for anticancer activity in the CT26 twin tumor model, which is a mouse model of colon cancer. Each 5-6 week old female Balb/cJ mouse (Jackson Labs, Bar Harbor, Maine) was subcutaneously implanted with CT26 tumor cells on both sides, 10 ⁵ cells per side. In study A, treatment was started 5 days (1.25 mg/kg, 2.5 mg/kg and 5 mg/kg) after tumor implantation, when the average tumor size reached approximately 100 mm ³ . In study B, treatment was started 8 days (0.6 mg/kg and 10 mg/kg) after tumor implantation, when the average tumor size reached approximately 120 mm ³ . The treatment regimen is presented in tables 5 and 6.

Table 5 Dosing schedule in study A

Group Number of animals Treatment Way and scheme of administration A 6 Carrier (1x PBS) I.T.*; single dose B 6 5 mg/kg Compounds 1a I.T.; single dose C 6 2.5 mg/kg Compounds 1a I.T.; single dose D 6 1.25 mg/kg Compounds 1a I.T.; single dose

*I.T. - inside the tumor

Table 6 Dosing schedule in study B

Group Number of animals Treatment Way and scheme of administration A 5 Carrier (1x PBS) I.T.*; single dose B 5 10 mg/kg Compounds 1a I.T.; single dose C 5 0.6 mg/kg Compounds 1a I.T.; single dose

*I.T. - inside the tumor

All mice in this study had two subcutaneous CT26 tumors. "Treated tumor" indicates a tumor with direct administration of the compound, while "untreated tumor" indicates a tumor without direct administration of the compound. Tumor volume was monitored throughout the experiment. Tumor volume was measured twice a week after the start of treatment. Tumor burden was calculated from caliper measurements using the formula for the volume of an elongated ellipsoid (LxW ² )/2, where L and W are the respective orthogonal measurements of length and width (mm).

Compound 1a showed high therapeutic activity in the CT26 twin tumor model (FIG. 10 and FIG. 11). Even at the lowest dose tested in the study, the cure rate of treated tumors was 20% (FIG. 8, 0.6 mg/kg dose). At the same time, by the end of the study, the highest dose made it possible to cure 100% of the animals from this tumor (10 mg/kg). A dose-dependent antitumor effect was also observed for untreated tumors. The highest dose group (10 mg/kg) showed 80% treatment effect; ever lower doses also showed tumor growth inhibiting activity. Therefore, compound 1a showed a therapeutic window ranging from 0.6 mg/kg to 10 mg/kg, with antitumor activity not only local, but also systemic, as evidenced by the presence of effects in the distal site of the tumor, not subjected to injection. In conclusion, these results show that topical administration of Compound 1a can induce both local and systemic (occult) anti-cancer activity.

Example 109 Anticancer Effect of Compound 1a in the CT26 Liver Metastasis Model

Compound 1a was tested for anticancer activity in the CT26 liver metastasis model. Anesthetized 5-6 week old female BALB/cJ mice (Jackson Labs, Harbor, Maine) were implanted in the spleen with luciferase-expressing CT26 tumor cells (5 x 10 ⁵ cells per mouse). A subsequent 10-minute waiting period allowed the tumor cells to circulate in the liver of the animals. The spleens were then removed, the animals were sutured, and allowed to recover. Three days later, CT26 tumor cells (10 ⁵ cells per mouse) were again implanted, this time subcutaneously (sc) under the region of the right forelimb, to allow the development of a tumor mass for compound administration. Nine days after injection into the spleen, the compound was injected once into the subcutaneous tumor (10 mg/kg).

The topical anti-cancer effect of a compound was measured by its effect on the subcutaneous tumor, while the latent effect of the compound was assessed by overall survival of treated mice compared to vehicle-treated control mice based on the detrimental effect of a growing tumor mass in the liver of each mouse. Compound 1a showed both potent activity against local SC tumors and therapeutic systemic activity in 9 out of 10 treated animals (FIG. 12). These results indicate that topical administration of Compound 1a can induce both local and systemic (absorptive) anti-cancer activity, including deep damage, for example, in the liver.

Example 110 Anti-Cancer Effect of Compound 1a on GL261 Orthotopic Brain Model

Compound 1a was tested for anticancer activity in the GL261 orthotopic brain model. GL261 is a mouse glioma cell line. Luciferase-expressing GL261 mouse glioma cells (2×10 ⁴ cells/mouse) were implanted intracranially in female B6 albino mice at 5-6 weeks of age (Jackson Labs, Bar Harbor, Maine). Three to four days later, GL261 cells were implanted subcutaneously (10 ⁶ cells/mouse) under the region of the right forelimb to allow the development of a tumor mass for compound administration. Ten days after implantation of the intracranial tumor cells, the compound was injected once into the subcutaneous tumor (10 mg/kg). The local anti-cancer effect of the compound was measured by its effect on the subcutaneous tumor, while the latent effect of the compound was assessed by the overall survival of treated mice compared to vehicle-treated control mice, based on the detrimental effect of the growing tumor mass in the brain of each mouse. Compound 1a showed both potent activity against local subcutaneous tumors and therapeutic systemic activity in 5 out of 8 treated animals (FIG. 13). These results indicate that topical administration of Compound 1a can induce both local and systemic (absorptive) anti-cancer activity, including deep damage, for example, in the brain.

Example 111 - Comparisons

EC ₅₀ values were calculated for human STING assays: WT STING, HAQ STING, AQ STING and REF STING, in head-to-head comparison using compound 1a of the invention, natural STING ligand (2'3'cGAMP) and putative STING agonist - ML RR-S2 The CDAs described by Corrales, et al. in "Direct Activation of STING in the Tumor Microenvironment Leads to Potent and Systemic Tumor Regression and Immunity," Cell Reports (2015) 11:1018-1030, which is incorporated herein by reference. Analyzes were performed as described in the examples above. It should be noted that the assay results reported in Table 7 are limited to those performed by direct comparison and may not reflect average values determined from more trials as indicated above or elsewhere. Also note that "2'3'cGAMP" is the same as "ML cGAMP" as stated in the Cell Reports publication.

Table 7

Compound Kd(WT)
(µM) Human EC ₅₀ (mM) (20 hour analysis) wt HAQ AQ REF 2'3' cGAMP 0.07 61.7 57.9 56.5 33-100 ML RR-S2 CDA 0.4 5.9 8.4 5.5 >100 Compound 1a 0.04 0.6 1.9 1.0 3.9

Table 7 also lists the dissociation (binding) constants (Kd) for the binding of wild-type human STING to each of the three test compounds, as measured by isothermal titration calorimetry (ITC). ITC is a microcalorimetic titration technique that measures the thermodynamic properties associated with intermolecular interactions. Based on the results obtained in these tests, Compound 1a appears to form the strongest bond with WT STING compared to the other compounds tested.

materials

Recombinant human wild-type STING protein (aa, 139-379, H232R) was generated by expression in E. coli of a construct encoding the human STING WT cytosolic domain containing amino acids 139-379.

Reagents

The sources of reagents used in this study are listed below:

Reagent Source Cat. No. 1X PBS without calcium or magnesium Corning 21-040-CV MilliQ deionized water MilliQ Z000Q0V0T0 Liquinox Alconox 1201 NaOH EM SXO593-1 methanol EMD Millipore MX0475-1 Connections: Compound 1a 2'3'cGAMP ML RR-S2 CDA

Protein Buffer Preparation

STING protein was stored at -60°C in aliquots of 90 μl and 100 μl each at a concentration of 3.0 mg/ml and 20 mg/ml, respectively, in PBS, pH 7.5, containing 5% glycerol. On the day of analysis, protein aliquots were thawed, diluted to 400 µl, and buffer exchanged with PBS using an Amicon Ultra centrifugal filter (cut-off 10 kDa, 0.5 ml), centrifuging at least four times for 10 min at 14,000 g in an Eppendorf microcentrifuge, and then finally diluted to 20-30 μM (depending on the experiment) using 1X PBS. The protein concentration was determined using a Nanodrop 2000 spectrophotometer with a protein extinction coefficient of 22140 (M ^-1 cm ^-1 ).

Sample preparation

In 1.5 ml microcentrifuge tubes, 200 μl of 1 mM stock solutions of Compound 1a, 2'3'cGAMP and ML RR-S2CDA were added. Before each experiment, the samples were diluted to concentrations from 200 μM to 500 μM (depending on the experiment).

Methods

Analyzes were performed on an Affinity ITC instrument (TA Instruments no. 609003.901) equipped with an ITC cleaner (TA Instruments no. 601800.901). The STING protein solution (approximately 400 μl containing 20 to 30 μM STING protein) was pipetted into a 185 μl well of the calorimeter, allowing for some acceptable excess. The control well contained an equivalent amount of Milli-Q water. Incubation was performed at 25°C with 20 injections of 2.5 μl of compounds at a concentration of 100 to 300 μm. ITC Run control software version 3.3.0.0 (TA Instruments) was used to obtain thermograms consisting of several peaks of heat release (µcal/sec) representing the rate of heating at each injection. Nano Analyze version 3.70 analysis software (TA Instruments) was used to adjust the baseline, correct for heat from blank or dilute (at saturation), and integrate thermal peak levels to produce "Q" plots. The obtained isotherms were adjusted using an independent model to obtain thermodynamic parameters.

Kd and n values (molar ratios at the inflection points of the curve) were obtained and recorded. Optimal conditions for protein and ligand concentrations were obtained from preliminary experiments.

Results

Thermograms of the heating rate were obtained and from them the binding isotherms of test compounds with wild-type recombinant human STING (a.k. 139-379, H232R) were obtained. The binding of each compound to STING was endothermic, as indicated by the negative rate of heating with the exothermic (positive direction) heat of dilution (observed after saturation of the compound with protein). 2',3'cGAMP has been shown to elicit a similar endothermic response to various STING variants. Compound 1a provides the lowest Kd=0.04 µM followed by 2'3'cGAMP with Kd=0.07 µM and then ML RR-S2 CDA with Kd=0.40 µM. All compounds showed n values close to 0.5, suggesting that the STING protein is present as a dimer and binds at a ratio of 1 mol compound per 2 mol STING.

Example 112 Identification of Potential Metabolites

Compound 1a was incubated in hepatocytes of CD-1 mice, Sprague Dawley rats, beagle dogs, Cynomolgus monkeys and humans to assess the formation of major metabolites.

materials

Cryopreserved pooled hepatocytes were purchased from ThermoFisher Scientific (Waltham, MA), Xenotech, LLC (Kansas City, KS) and In Vitro ADMET Laboratories (Columbia, MD), and appropriate media were purchased from In Vitro ADMET Laboratories (Columbia, MD) and Life Technologies (Carlsbad, CA). AOPI stain and phosphate buffer were purchased from Corning Life Sciences (Tewksbury, MA) and Nexcelom Bioscience (Lawrence, MA), respectively. All chemicals, reagents, and solvents used in the assay were of either analytical grade or HPLC grade.

Scheme and procedures of the experiment

Incubation of hepatocytes

Compound 1a was weighed and dissolved in HPLC water containing 0.12% PBS with formic acid to a concentration of 1020 mmol/l. The solution was then diluted separately 2.5-fold to 4 mmol/L, and then further diluted 21,000-fold using Williams E medium containing 0.1% human serum albumin and 2 mmol/L L-glutamine to give a stock solution with concentration of 20 µmol/l.

Before incubation, cryopreserved hepatocytes were thawed in a water bath at 37°C. The contents of one tube of cryopreserved hepatocytes were added to each 50 ml conical tube of cryopreserved hepatocyte recovery medium (UCRM) purchased from In Vitro ADMET Laboratories (Columbia, MD). Cells were centrifuged in a Beckman centrifuge (Brea, CA) with a GH 3.8 rotor at 740 rpm for 10 minutes at 4°C. The supernatant was removed and the cells were resuspended for enumeration in seeding medium. After the cells were resuspended in seeding medium, 20 µl of the suspension was transferred and mixed with 20 µl of the AOPI staining solution. The solution was gently stirred and cells were counted using a cellometer (Nexcelom, Lawrence, MA). After counting, the cells were resuspended at 1 or 2 million viable cells/ml in Williams E medium containing 2 mmol/l L-glutamine (pH 7.4).

A suspension of hepatocytes (50 μl/well) was added to a 48-well plate. Fifty microliters of working stock solution containing compound 1a (20 µmol/l) was added to start the reaction. The plate was placed in a tissue culture incubator (5% CO ₂ /95% humidity and 37°C) and the reaction was stopped by adding 200 μl of a stop solution consisting of a mixture of 100% methanol/acetonitrile (1/1, v/v. ) with 2010 ng/ml furosemide and 0.2 µmol/l (R)-propranolol, at 5, 30, 60, 120, 180 and 240 minutes. The mixture was centrifuged and filtered and the supernatant was collected for analysis. Final concentrations of cryopreserved hepatocytes were 1×10 ⁶ cells/ml. The final incubation concentration of Compound 1a was 10 µmol/l.

LC-MS/MS Conditions for Metabolite Identification

The LC/MS/MS system consisted of a Shimadzu HPLC system and an AB-SCIEX Triple TOF 5600 TOF hybrid quadrupole mass spectrometer (Framingham, MA). The Shimadzu HPLC system (Kyoto, Japan) consists of a communication bus module (CBM-20A), an autosampler (SIL-30AC) with an attached rack changer (Rack Changer II), two pumps (LC-30AD) and a column heater (CTO -30A). The mass spectrometer was calibrated using AB-SCIEX APCI negative and positive calibration solutions (Framingham, MA). Samples resulting from incubation with hepatocytes were analyzed in both negative and positive scanning modes. Below is a brief description of the main analytical methods and instrument settings. Changing the spectrometer settings depended on the type of analyte.

LC-MS/MS conditions:

Chromatography Setup Options: Shimadzu LC30AD Column type Agilent Eclipse XDB-C8, 5 µm, 2.1x150 mm, lot # 993700-906, serial # USSN002817 mobile phases A: water/methanol=95/5 (v/v) with 5 mM ammonium acetate
B: methanol/water=95/5 (v/v) with 5 mM ammonium acetate Gradient 0-1 min at 1%B; 1-5 min linear in 10%B; 5-13 min linear in 95%B; 13-19.9 min at 95%B; 19.9-20 min linear in 1%B; 20-24 min at 1%B Flow rate 0.4 ml/min Analysis time 24 min Sample tray temperature 4°C Injectable volume 15 µl Mass spectrometer
Installation options: AB Sciex Hybrid Quadrupole-TOF LC-MS/MS Triple TOF 5600 Ion source DuoSpray ion source Polarity negative or positive Ion Sputter Voltage (ISVF) 4500V or 5500V Temperature (TEM) 550°C Gas mist (CUR) thirty GS1 50 GS2 50 TOF-MS:
Installation options CE -5,000 or 5,000
DP -100,000 or 100,000
Scan range: 120.0-1000.0 TOF MS^2:
Installation options CE -60,000 or 60,000
CES 5,000
DP -100,000 or 100,000
Scan range: 100.0-1000.0

Activity data analysis

Mass spectrometry data were obtained using AB-Sciex Analyst TF software (version 1.5.1; Framingham, MA). Chromatograms and spectra were obtained using AB-Sciex PeakView software (version 2.2.0.1; Framingham, MA). The comparison of the relative peak areas of the extracted ion chromatograms is based on ±0.0002 Da expected exact mass-to-charge ratio (m/z) for each analyte of interest.

Results

After incubation with hepatocytes, the metabolite was not detected. Under the analytical conditions shown, Compound 1a showed a retention time of approximately 7.8 minutes. In negative scan mode, compound 1a showed a deprotonated molecular ion m/z 745 (C ₂₄ H ₂₅ F ₂ N ₁₀ O ₈ P ₂ S ₂ ^- ) and a doubly deprotonated molecular ion m/z 372 (C ₂₄ H ₂₄ F ₂ N ₁₀ O ₈ P ₂ S ₂₂ ^- ). The main observed MS/MS product ions had m/z 533 (C ₁₉ H ₁₉ FN ₁₀ O ₄ PS ^- ) and m/z 186 (C ₉ H ₈ N ₅ ^- ). In the positive scanning mode, Compound 1a had a protonated molecular ion m/z 747 (C ₂₄ H ₂₇ F ₂ N ₁₀ O ₈ P ₂ S ₂ ⁺ ) and the main MS/MS ions of shallow products m/z 651 (C ₂₄ H ₂₆ F ₂ N ₁₀ O ₆ PS ⁺ ), m/z 252 (C ₁₀ H ₁₁ FN ₅ O ₂ ⁺ ) and m/z 188 (C ₉ H ₁₀ N ₅ ⁺ ). MS and MS/MS data confirm the structure of Compound 1a.

Compound 1a was stable when incubated with mouse, rat, dog, monkey and human hepatocytes. No detectable metabolite of Compound 1a was identified in this study. In the samples obtained as a result of incubation with hepatocytes, only compound 1a itself was detected, which was confirmed by fragments of tandem mass spectrometry (MS/MS).

All documents referenced in this specification are incorporated herein by reference, and if any document included conflicts with the description provided, the present description shall prevail. Those skilled in the art will appreciate that various changes and modifications may be made to the material described herein, and that such material is within the scope and spirit of the invention.

SEQUENCE LISTS

SEQ ID NO: 1 (wild-type human STING):

atgccccactccagcctgcatccatccatcccgtgtcccaggggtcacggggcccagaaggcagccttggttctgctgagtgcctgcctggtgaccctttgggggctaggagagccaccagagcacactctccggtacctggtgctccacctagcctccctgcagctgggactgctgttaaacggggtctgcagcctggctgaggagctgc gccacatccactccaggtaccggggcagctactggaggactgtgcgggcctgcctgggctgccccctccgccgtggggccctgttgctgctgtccatctatttctactactccctcccaaatgcggtcggcccgcccttcacttggatgcttgccctcctgggcctcgcaggcactgaacatcctcctgggcctcaagggcctggcccca gctgagatctctgcagtgtgtgaaaaagggaatttcaacgtggcccatgggctggcatggtcatattacatcggatatctgcggctgatcctgccagagctccaggcccggattcgaacttacaatcagcattacaacaacctgctacggggtgcagtgagccagcggctgtattctcctcccattggactgtggggtgcctgataacctga gtatggctgaccccaacattcgcttcctggataaactgccccagcagaccggtgaccgggctggcatcaaggatcgggtttacagcaacagcatctatgagcttctctggagaacgggcagcgggcgggcacctgtgtcctggagtacgccacccccttgcagactttgtttgccatgtcacaatacagtcaagctggctttagccgggaggatagg cttgagcaggccaaactcttctgccggacacttgaggacatcctggcagatgcccctgagtctcagaacaactgccgcctcattgcctaccaggaacctgcagatgacagcagcttctcgctgtcccagggaggttctccggcacctgcggcaggaggaaaaggaagaggttactgtgggcagcttgaagacctcagcggtgcccagtacc tccacgatgtcccaagagcctgagctcctcatcagtggaatggaaaagcccctccctctccgcacggatttctcttga

SEQ ID NO: 2 (human REF STING):

atgccccactccagcctgcatccatccatcccgtgtcccaggggtcacggggcccagaaggcagccttggttctgctgagtgcctgcctggtgaccctttgggggctaggagagccaccagagcacactctccggtacctggtgctccacctagcctccctgcagctgggactgctgttaaacggggtctgcagcctggctgaggagctgc gccacatccactccaggtaccggggcagctactggaggactgtgcgggcctgcctgggctgccccctccgccgtggggccctgttgctgctgtccatctatttctactactccctcccaaatgcggtcggcccgcccttcacttggatgcttgccctcctgggcctcgcaggcactgaacatcctcctgggcctcaagggcctggcccca gctgagatctctgcagtgtgtgaaaaagggaatttcaacgtggcccatgggctggcatggtcatattacatcggatatctgcggctgatcctgccagagctccaggcccggattcgaacttacaatcagcattacaacaacctgctacggggtgcagtgagccagcggctgtattctcctcccattggactgtggggtgcctgataacctga gtatggctgaccccaacattcgcttcctggataaactgccccagcagaccggtgaccatgctggcatcaaggatcgggtttacagcaacagcatctatgagcttctctggagaacgggcagcgggcgggcacctgtgtcctggagtacgccacccccttgcagactttgtttgccatgtcacaatacagtcaagctggctttagccgggaggatagg cttgagcaggccaaactcttctgccggacacttgaggacatcctggcagatgcccctgagtctcagaacaactgccgcctcattgcctaccaggaacctgcagatgacagcagcttctcgctgtcccagggaggttctccggcacctgcggcaggaggaaaaggaagaggttactgtgggcagcttgaagacctcagcggtgcccagtacc tccacgatgtcccaagagcctgagctcctcatcagtggaatggaaaagcccctccctctccgcacggatttctcttga

SEQ ID NO: 3 (human AQ STING):

atgccccactccagcctgcatccatccatcccgtgtcccaggggtcacggggcccagaaggcagccttggttctgctgagtgcctgcctggtgaccctttgggggctaggagagccaccagagcacactctccggtacctggtgctccacctagcctccctgcagctgggactgctgttaaacggggtctgcagcctggctgaggagctgc gccacatccactccaggtaccggggcagctactggaggactgtgcgggcctgcctgggctgccccctccgccgtggggccctgttgctgctgtccatctatttctactactccctcccaaatgcggtcggcccgcccttcacttggatgcttgccctcctgggcctcgcaggcactgaacatcctcctgggcctcaagggcctggcccca gctgagatctctgcagtgtgtgaaaaagggaatttcaacgtggcccatgggctggcatggtcatattacatcggatatctgcggctgatcctgccagagctccaggcccggattcgaacttacaatcagcattacaacaacctgctacggggtgcagtgagccagcggctgtattctcctcccattggactgtggggtgcctgataacctga gtatggctgaccccaacattcgcttcctggataaactgccccagcagaccgctgaccgagctggcatcaaggatcgggtttacagcaacagcatctatgagcttctggagaacgggcagcgggcgggcacctgtgtcctggagtacgccacccccttgcagactttgtttgccatgtcacaatacagtcaagctggctttagccgggaggatagg cttgagcaggccaaactcttctgccagacacttgaggacatcctggcagatgcccctgagtctcagaacaactgccgcctcattgcctaccaggaacctgcagatgacagcagcttctcgctgtcccagggaggttctccggcacctgcggcaggaggaaaaggaagaggttactgtgggcagcttgaagacctcagcggtgcccagtacc tccacgatgtcccaagagcctgagctcctcatcagtggaatggaaaagcccctccctctccgcacggatttctcttga

SEQ ID NO: 4 (residues 155-341 WT STING):

VAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDRAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEV

SEQ ID NO: 5 (His-TEV-Sumo-WT STING 155-341)

MHHHHHHSSGVDLGTENLYFQSNAMSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGGGSVAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQ TGDRAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEV

SEQ ID NO: 6 (residues 155-341 REF STING):

VAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQTGDHAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSSFSLSQEVLRHLRQEEKEEV

SEQ ID NO: 7 (His-TEV-Sumo-REF STING 155-341)

MHHHHHHSSGVDLGTENLYFQSNAMSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGGGSVAHGLAWSYYIGYLRLILPELQARIRTYNQHYNNLLRGAVSQRLYILLPLDCGVPDNLSMADPNIRFLDKLPQQ TGDHAGIKDRVYSNSIYELLENGQRAGTCVLEYATPLQTLFAMSQYSQAGFSREDRLEQAKLFCRTLEDILADAPESQNNCRLIAYQEPADDSFSLSQEVLRHLRQEEKEEV

--->

SEQUENCE LISTS

<110> Eisai R&D Management Co., Ltd.

Kim, Dae Shik

<120> BLADDER CANCER TREATMENT

<130> 0080171-000500

<160> 7

<170>PatentIn version 3.5

<210> 1

<211> 1140

<212> DNA

<213> Homo sapiens

<400> 1

atgccccact ccagcctgca tccatccatc ccgtgtccca ggggtcacgg ggcccagaag 60

gcagccttgg ttctgctgag tgcctgcctg gtgacccttt gggggctagg agagccacca 120

gagcacactc tccggtacct ggtgctccac ctagcctccc tgcagctggg actgctgtta 180

aacggggtct gcagcctggc tgaggagctg cgccacatcc actccaggta ccggggcagc 240

tactggagga ctgtgcgggc ctgcctgggc tgccccctcc gccgtggggc cctgttgctg 300

ctgtccatct atttctacta ctccctccca aatgcggtcg gcccgccctt cacttggatg 360

cttgccctcc tgggcctctc gcaggcactg aacatcctcc tgggcctcaa gggcctggcc 420

ccagctgaga tctctgcagt gtgtgaaaaa gggaatttca acgtggccca tgggctggca 480

tggtcatatt acatcggata tctgcggctg atcctgccag agctccaggc ccggattcga 540

acttacaatc agcattacaa caacctgcta cggggtgcag tgagccagcg gctgtatatt 600

ctcctcccat tggactgtgg ggtgcctgat aacctgagta tggctgaccc caacattcgc 660

ttcctggata aactgcccca gcagaccggt gaccgggctg gcatcaagga tcgggtttac 720

agcaacagca tctatgagct tctggagaac gggcagcggg cgggcacctg tgtcctggag 780

tacgccaccc ccttgcagac tttgtttgcc atgtcacaat acagtcaagc tggctttagc 840

cgggaggata ggcttgagca ggccaaactc ttctgccgga cacttgagga catcctggca 900

gatgcccctg agtctcagaa caactgccgc ctcattgcct accaggaacc tgcagatgac 960

agcagcttct cgctgtccca ggaggttctc cggcacctgc ggcaggagga aaaggaagag 1020

gttactgtgg gcagcttgaa gacctcagcg gtgcccagta cctcccgat gtcccaagag 1080

cctgagctcc tcatcagtgg aatggaaaag cccctccctc tccgcacgga tttctcttga 1140

<210> 2

<211> 1140

<212> DNA

<213> Homo sapiens

<400> 2

atgccccact ccagcctgca tccatccatc ccgtgtccca ggggtcacgg ggcccagaag 60

gcagccttgg ttctgctgag tgcctgcctg gtgacccttt gggggctagg agagccacca 120

gagcacactc tccggtacct ggtgctccac ctagcctccc tgcagctggg actgctgtta 180

aacggggtct gcagcctggc tgaggagctg cgccacatcc actccaggta ccggggcagc 240

tactggagga ctgtgcgggc ctgcctgggc tgccccctcc gccgtggggc cctgttgctg 300

ctgtccatct atttctacta ctccctccca aatgcggtcg gcccgccctt cacttggatg 360

cttgccctcc tgggcctctc gcaggcactg aacatcctcc tgggcctcaa gggcctggcc 420

ccagctgaga tctctgcagt gtgtgaaaaa gggaatttca acgtggccca tgggctggca 480

tggtcatatt acatcggata tctgcggctg atcctgccag agctccaggc ccggattcga 540

acttacaatc agcattacaa caacctgcta cggggtgcag tgagccagcg gctgtatatt 600

ctcctcccat tggactgtgg ggtgcctgat aacctgagta tggctgaccc caacattcgc 660

ttcctggata aactgcccca gcagaccggt gaccatgctg gcatcaagga tcgggtttac 720

agcaacagca tctatgagct tctggagaac gggcagcggg cgggcacctg tgtcctggag 780

tacgccaccc ccttgcagac tttgtttgcc atgtcacaat acagtcaagc tggctttagc 840

cgggaggata ggcttgagca ggccaaactc ttctgccgga cacttgagga catcctggca 900

gatgcccctg agtctcagaa caactgccgc ctcattgcct accaggaacc tgcagatgac 960

agcagcttct cgctgtccca ggaggttctc cggcacctgc ggcaggagga aaaggaagag 1020

gttactgtgg gcagcttgaa gacctcagcg gtgcccagta cctcccgat gtcccaagag 1080

cctgagctcc tcatcagtgg aatggaaaag cccctccctc tccgcacgga tttctcttga 1140

<210> 3

<211> 1140

<212> DNA

<213> Homo sapiens

<400> 3

atgccccact ccagcctgca tccatccatc ccgtgtccca ggggtcacgg ggcccagaag 60

gcagccttgg ttctgctgag tgcctgcctg gtgacccttt gggggctagg agagccacca 120

gagcacactc tccggtacct ggtgctccac ctagcctccc tgcagctggg actgctgtta 180

aacggggtct gcagcctggc tgaggagctg cgccacatcc actccaggta ccggggcagc 240

tactggagga ctgtgcgggc ctgcctgggc tgccccctcc gccgtggggc cctgttgctg 300

ctgtccatct atttctacta ctccctccca aatgcggtcg gcccgccctt cacttggatg 360

cttgccctcc tgggcctctc gcaggcactg aacatcctcc tgggcctcaa gggcctggcc 420

ccagctgaga tctctgcagt gtgtgaaaaa gggaatttca acgtggccca tgggctggca 480

tggtcatatt acatcggata tctgcggctg atcctgccag agctccaggc ccggattcga 540

acttacaatc agcattacaa caacctgcta cggggtgcag tgagccagcg gctgtatatt 600

ctcctcccat tggactgtgg ggtgcctgat aacctgagta tggctgaccc caacattcgc 660

ttcctggata aactgcccca gcagaccgct gaccgagctg gcatcaagga tcgggtttac 720

agcaacagca tctatgagct tctggagaac gggcagcggg cgggcacctg tgtcctggag 780

tacgccaccc ccttgcagac tttgtttgcc atgtcacaat acagtcaagc tggctttagc 840

cgggaggata ggcttgagca ggccaaactc ttctgccaga cacttgagga catcctggca 900

gatgcccctg agtctcagaa caactgccgc ctcattgcct accaggaacc tgcagatgac 960

agcagcttct cgctgtccca ggaggttctc cggcacctgc ggcaggagga aaaggaagag 1020

gttactgtgg gcagcttgaa gacctcagcg gtgcccagta cctcccgat gtcccaagag 1080

cctgagctcc tcatcagtgg aatggaaaag cccctccctc tccgcacgga tttctcttga 1140

<210> 4

<211> 187

<212> Protein

<213> Homo sapiens

<400> 4

Val Ala His Gly Leu Ala Trp Ser Tyr Tyr Ile Gly Tyr Leu Arg Leu

1 5 10 15

Ile Leu Pro Glu Leu Gln Ala Arg Ile Arg Thr Tyr Asn Gln His Tyr

20 25 30

Asn Asn Leu Leu Arg Gly Ala Val Ser Gln Arg Leu Tyr Ile Leu Leu

35 40 45

Pro Leu Asp Cys Gly Val Pro Asp Asn Leu Ser Met Ala Asp Pro Asn

50 55 60

Ile Arg Phe Leu Asp Lys Leu Pro Gln Gln Thr Gly Asp Arg Ala Gly

65 70 75 80

Ile Lys Asp Arg Val Tyr Ser Asn Ser Ile Tyr Glu Leu Leu Glu Asn

85 90 95

Gly Gln Arg Ala Gly Thr Cys Val Leu Glu Tyr Ala Thr Pro Leu Gln

100 105 110

Thr Leu Phe Ala Met Ser Gln Tyr Ser Gln Ala Gly Phe Ser Arg Glu

115 120 125

Asp Arg Leu Glu Gln Ala Lys Leu Phe Cys Arg Thr Leu Glu Asp Ile

130 135 140

Leu Ala Asp Ala Pro Glu Ser Gln Asn Asn Cys Arg Leu Ile Ala Tyr

145 150 155 160

Gln Glu Pro Ala Asp Asp Ser Ser Phe Ser Leu Ser Gln Glu Val Leu

165 170 175

Arg His Leu Arg Gln Glu Glu Lys Glu Glu Val

180 185

<210> 5

<211> 311

<212> Protein

<213> Artificial sequence

<220>

<223> Design His-TEV-Sumo-WT STING

<400> 5

Met His His His His His His Ser Ser Gly Val Asp Leu Gly Thr Glu

1 5 10 15

Asn Leu Tyr Phe Gln Ser Asn Ala Met Ser Asp Ser Glu Val Asn Gln

20 25 30

Glu Ala Lys Pro Glu Val Lys Pro Glu Val Lys Pro Glu Thr His Ile

35 40 45

Asn Leu Lys Val Ser Asp Gly Ser Ser Glu Ile Phe Phe Lys Ile Lys

50 55 60

Lys Thr Thr Pro Leu Arg Arg Leu Met Glu Ala Phe Ala Lys Arg Gln

65 70 75 80

Gly Lys Glu Met Asp Ser Leu Arg Phe Leu Tyr Asp Gly Ile Arg Ile

85 90 95

Gln Ala Asp Gln Thr Pro Glu Asp Leu Asp Met Glu Asp Asn Asp Ile

100 105 110

Ile Glu Ala His Arg Glu Gln Ile Gly Gly Gly Ser Val Ala His Gly

115 120 125

Leu Ala Trp Ser Tyr Tyr Ile Gly Tyr Leu Arg Leu Ile Leu Pro Glu

130 135 140

Leu Gln Ala Arg Ile Arg Thr Tyr Asn Gln His Tyr Asn Asn Leu Leu

145 150 155 160

Arg Gly Ala Val Ser Gln Arg Leu Tyr Ile Leu Leu Pro Leu Asp Cys

165 170 175

Gly Val Pro Asp Asn Leu Ser Met Ala Asp Pro Asn Ile Arg Phe Leu

180 185 190

Asp Lys Leu Pro Gln Gln Thr Gly Asp Arg Ala Gly Ile Lys Asp Arg

195 200 205

Val Tyr Ser Asn Ser Ile Tyr Glu Leu Leu Glu Asn Gly Gln Arg Ala

210 215 220

Gly Thr Cys Val Leu Glu Tyr Ala Thr Pro Leu Gln Thr Leu Phe Ala

225 230 235 240

Met Ser Gln Tyr Ser Gln Ala Gly Phe Ser Arg Glu Asp Arg Leu Glu

245 250 255

Gln Ala Lys Leu Phe Cys Arg Thr Leu Glu Asp Ile Leu Ala Asp Ala

260 265 270

Pro Glu Ser Gln Asn Asn Cys Arg Leu Ile Ala Tyr Gln Glu Pro Ala

275 280 285

Asp Asp Ser Ser Phe Ser Leu Ser Gln Glu Val Leu Arg His Leu Arg

290 295 300

Gln Glu Glu Lys Glu Glu Val

305 310

<210> 6

<211> 187

<212> Protein

<213> Homo sapiens

<400> 6

Val Ala His Gly Leu Ala Trp Ser Tyr Tyr Ile Gly Tyr Leu Arg Leu

1 5 10 15

Ile Leu Pro Glu Leu Gln Ala Arg Ile Arg Thr Tyr Asn Gln His Tyr

20 25 30

Asn Asn Leu Leu Arg Gly Ala Val Ser Gln Arg Leu Tyr Ile Leu Leu

35 40 45

Pro Leu Asp Cys Gly Val Pro Asp Asn Leu Ser Met Ala Asp Pro Asn

50 55 60

Ile Arg Phe Leu Asp Lys Leu Pro Gln Gln Thr Gly Asp His Ala Gly

65 70 75 80

Ile Lys Asp Arg Val Tyr Ser Asn Ser Ile Tyr Glu Leu Leu Glu Asn

85 90 95

Gly Gln Arg Ala Gly Thr Cys Val Leu Glu Tyr Ala Thr Pro Leu Gln

100 105 110

Thr Leu Phe Ala Met Ser Gln Tyr Ser Gln Ala Gly Phe Ser Arg Glu

115 120 125

Asp Arg Leu Glu Gln Ala Lys Leu Phe Cys Arg Thr Leu Glu Asp Ile

130 135 140

Leu Ala Asp Ala Pro Glu Ser Gln Asn Asn Cys Arg Leu Ile Ala Tyr

145 150 155 160

Gln Glu Pro Ala Asp Asp Ser Ser Phe Ser Leu Ser Gln Glu Val Leu

165 170 175

Arg His Leu Arg Gln Glu Glu Lys Glu Glu Val

180 185

<210> 7

<211> 311

<212> Protein

<213> Artificial sequence

<220>

<223> Design His-TEV-Sumo-REF STING 155-341

<400> 7

Met His His His His His His Ser Ser Gly Val Asp Leu Gly Thr Glu

1 5 10 15

Asn Leu Tyr Phe Gln Ser Asn Ala Met Ser Asp Ser Glu Val Asn Gln

20 25 30

Glu Ala Lys Pro Glu Val Lys Pro Glu Val Lys Pro Glu Thr His Ile

35 40 45

Asn Leu Lys Val Ser Asp Gly Ser Ser Glu Ile Phe Phe Lys Ile Lys

50 55 60

Lys Thr Thr Pro Leu Arg Arg Leu Met Glu Ala Phe Ala Lys Arg Gln

65 70 75 80

Gly Lys Glu Met Asp Ser Leu Arg Phe Leu Tyr Asp Gly Ile Arg Ile

85 90 95

Gln Ala Asp Gln Thr Pro Glu Asp Leu Asp Met Glu Asp Asn Asp Ile

100 105 110

Ile Glu Ala His Arg Glu Gln Ile Gly Gly Gly Ser Val Ala His Gly

115 120 125

Leu Ala Trp Ser Tyr Tyr Ile Gly Tyr Leu Arg Leu Ile Leu Pro Glu

130 135 140

Leu Gln Ala Arg Ile Arg Thr Tyr Asn Gln His Tyr Asn Asn Leu Leu

145 150 155 160

Arg Gly Ala Val Ser Gln Arg Leu Tyr Ile Leu Leu Pro Leu Asp Cys

165 170 175

Gly Val Pro Asp Asn Leu Ser Met Ala Asp Pro Asn Ile Arg Phe Leu

180 185 190

Asp Lys Leu Pro Gln Gln Thr Gly Asp His Ala Gly Ile Lys Asp Arg

195 200 205

Val Tyr Ser Asn Ser Ile Tyr Glu Leu Leu Glu Asn Gly Gln Arg Ala

210 215 220

Gly Thr Cys Val Leu Glu Tyr Ala Thr Pro Leu Gln Thr Leu Phe Ala

225 230 235 240

Met Ser Gln Tyr Ser Gln Ala Gly Phe Ser Arg Glu Asp Arg Leu Glu

245 250 255

Gln Ala Lys Leu Phe Cys Arg Thr Leu Glu Asp Ile Leu Ala Asp Ala

260 265 270

Pro Glu Ser Gln Asn Asn Cys Arg Leu Ile Ala Tyr Gln Glu Pro Ala

275 280 285

Asp Asp Ser Ser Phe Ser Leu Ser Gln Glu Val Leu Arg His Leu Arg

290 295 300

Gln Glu Glu Lys Glu Glu Val

305 310

<---

Claims (43)

1. A method of treating bladder cancer comprising administering to a patient in need of treatment an effective amount of Compound 1 or a pharmaceutically acceptable salt thereof:

(Compound 1). 2. The method of claim 1 wherein said pharmaceutically acceptable salt is a diammonium salt. 3. A method for treating bladder cancer, comprising administering to a patient in need of treatment an effective amount of a pharmaceutical composition containing Compound 1 or a pharmaceutically acceptable salt thereof:

(Compound 1) and a pharmaceutically acceptable excipient. 4. A method for treating bladder cancer, including: identifying an individual whose bladder cancer is treatable with the compound

(Compound 1) a pharmaceutically acceptable salt of Compound 1 or a pharmaceutical composition containing Compound 1 or a pharmaceutically acceptable salt of Compound 1; And administering to said individual an effective amount of the compound, pharmaceutically acceptable salt or pharmaceutical composition by which bladder cancer has been identified as treatable. 5. The method of claim 4, wherein said individual is identified as having bladder cancer treatable with Compound 1, a pharmaceutically acceptable salt of Compound 1, or a pharmaceutical composition containing Compound 1 or a pharmaceutically acceptable salt of Compound 1, by the presence of the allele in the patient REF STING option. 6. A method for treating bladder cancer in a patient having the REF STING allele, comprising administering to said patient an effective amount of a compound

(Compound 1) a pharmaceutically acceptable salt of Compound 1 or a pharmaceutical composition containing Compound 1 or a pharmaceutically acceptable salt of Compound 1. 7. A method of treating bladder cancer in a patient having the WT STING allele, comprising administering to said patient an effective amount of a compound

(Compound 1) a pharmaceutically acceptable salt of Compound 1 or a pharmaceutical composition containing Compound 1 or a pharmaceutically acceptable salt of Compound 1. 8. A method of treating bladder cancer in a patient having an AQ STING allele, comprising administering to said patient an effective amount of a compound

(Compound 1) a pharmaceutically acceptable salt of Compound 1 or a pharmaceutical composition containing Compound 1 or a pharmaceutically acceptable salt of Compound 1. 9. A method of treating bladder cancer in a patient having the HAQ STING allele, comprising administering to said patient an effective amount of a compound

(Compound 1) a pharmaceutically acceptable salt of Compound 1 or a pharmaceutical composition containing Compound 1 or a pharmaceutically acceptable salt of Compound 1. 10. The method according to any one of claims 1 to 9, wherein the administration of Compound 1, a pharmaceutically acceptable salt of Compound 1, or a pharmaceutical composition containing Compound 1 or a pharmaceutically acceptable salt of Compound 1 is performed intravesically. 11. The method of any one of claims 1-9, wherein the bladder cancer is non-muscle invasive bladder cancer. 12. The method according to any one of claims 1 to 11, wherein the administration of Compound 1, a pharmaceutically acceptable salt of Compound 1, or a pharmaceutical composition containing any of the above is performed intravesically. 13. Use of compound 1 or its pharmaceutically acceptable salt for the preparation of a pharmaceutical composition for the treatment of bladder cancer:

(Compound 1). 14. Use of Compound 1, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing Compound 1 or a pharmaceutically acceptable salt thereof, in the treatment of bladder cancer:

(Compound 1). 15. Use according to claim 13 or 14, wherein the administration of Compound 1, a pharmaceutically acceptable salt of Compound 1 or a pharmaceutical composition containing Compound 1 or a pharmaceutically acceptable salt of Compound 1 is performed intravesically. 16. Use according to claim 13 or 14, wherein the bladder cancer is non-muscle invasive bladder cancer. 17. Use according to any one of claims 13-16, wherein the administration of Compound 1, a pharmaceutically acceptable salt of Compound 1, or a pharmaceutical composition containing any of the above is performed intravesically.

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