Prodrugs of aldehyde ketone reductase inhibitors, semimestar disulfide, its preparation, pharmaceutical compositions and uses

Abstract

The present invention relates to a novel prodrug of the disulfide of aldonolactone reductase inhibitor II based on its formula I (systematic name "disulfalane"), to a method of preparing the disulfide of formula I, to the use of the disulfide of formula I for inhibiting the aldonolactone reductases AKR1B1 and AKR1B10, to the use of the disulfide of formula I in the prevention or treatment of diseases in which the activity of the aldonolactone reductases AKR1B1 and AKR1B10 is a key cause of the formation and development of said diseases, to the use of the disulfide of formula I for the prevention or treatment of cancers derived from chronic inflammation, i.e. colon cancer, lung cancer, breast cancer, liver cancer, prostate cancer, pancreatic cancer, endometrial cancer and cervical cancer. The present invention also relates to the use of the disulfide of formula I as an adjuvant therapy drug in combination with clinically used chemotherapeutic drugs, which can also be substrates of aldonolactone reductases, such as doxorubicin and daunorubicin, for the treatment of cancer. The disulfide of formula I is not active as an inhibitor of the aldonolactone reductases AKR1B1 and AKR1B10 itself, but after oral or parenteral administration it is metabolized in the body, preferably in cancer cells characterized by an increased reducing potential due to a significantly increased GSH content compared to healthy cells, thus producing two molecules of semimestar of formula II, i.e. an inhibitor of the activity of aldonolactone reductases. The disulfide of formula I according to the present invention can also have the advantage that it is better absorbed in the acidic environment of the tumor compared to semimestar of formula II alone. The disulfide of formula I can be used alone or in a pharmaceutically acceptable form: mono- or di-salt, mono- or di-ester, amide or combinations thereof or other agents, optionally in combination with other anti-inflammatory or anticancer therapeutic drugs.(I)

Description

TECHNICAL FIELD

[0001] The present invention relates to pharmaceutically useful prodrugs of the pharmacologically active compound cemtirestat, which is an inhibitor of aldoketoreductases, in particular of aldose reductase (AR, AKR1B1), to methods for their production, to pharmaceutical compositions containing them and to their use in the treatment of human and veterinary diseases. BACKGROUND

[0002] Aldoketoreductases are NAD(P)H-dependent oxidoreductases that are best characterized as hypoglycemic agents. They are involved in the pathophysiology of diabetic complications. These enzymes also metabolize lipid peroxidation products, thus in some cases eliciting inflammatory responses.

[0003] Aldose reductase (AR, AKR1B1) is effective in reducing aldehydes formed by lipid peroxidation and their conjugates with glutathione (Ramana, BioMol Concepts 2:103-114, 2011) in addition to reducing glucose participation in diabetic complications. Aldehydes produced by lipid peroxidation, such as 4-hydroxy-trans-2-nonenal (systematic name trans-4-hydroxynon-2-en-1-al) (HNE) and its glutathione conjugate (e.g., GS-HNE), are effectively reduced by AR to the corresponding alcohol DHN (1,4-dihydroxynonenal, systematic name trans-nonen-2-ene-1,4-diol) and GS-DHN (glutathione-1,4-dihydroxynon-2-ene), which mediate inflammatory signals in the body. The reduced GS-DHN conjugate is thought to be a signaling intermediate in cell signaling triggered by reactive oxygen species, which can ultimately lead to inflammatory responses (Srivastava et al.; Chem Biol Interaction 191:330-338, 2011; Balestri et al.; Antioxidants (Basel) 8(10). pii: E502, 2019; Srivastava et al.; Free Radic. Bio1. Med. 29:642-651, 2000; Shoeb et al.; Curr. Med. Chem. 21:230-237, 2014). AR inhibition is effective in eliminating inflammatory signals induced by cytokines, growth factors, endotoxins, high glucose, allergens, and autoimmune responses in cell and animal models (Ramana and Srivastava, Int. J. Biochem. Cell Biol. 42:17-20, 2010).

[0004] There is ample documentation that chronic inflammation is associated with cancer progression (Solinas et al. Cancer Metastasis Rev. 29, 243-248, 2010; Khayami et al. J. Cell. Mol. Med. 2020; 10.1111 / jcmm.15581). Epidemiological studies have shown that >25% of all cancers are closely associated with chronic infections and chronic inflammation (Vendramini-Costa and Carvalho, Curr Pharm Des. 18:3831-52, 2012). Increased expression of aldo-keto reductase has been reported in lung, breast, prostate, cervix, testis and colon tumors (Liu et al. Recent Pat Anticancer Drug Discov. 4:246-53, 2009; Laffin and Petrash, Front Pharmacol. 3:104, 2012; Terzig et al. Gastroenterology 138:2101-2114, 2010; Reddy et al. Breast 31:137-143, 2017). Moreover, increased expression of AKR1B1 in various cancers causes resistance to doxorubicin, which can be explained by increased doxorubicin detoxification mediated by carbonyl reduction by AKR1B1 (Lee et al. Anticancer Drugs 2:129-32, 2001). Several studies have shown that inhibition of AKR1B1 as an adjunct therapy increases tumor sensitivity to anticancer therapy or reduces adverse effects (Banala et al. Biomater Sci. 7:2889, 2019; Khayami et al. J. Cell Mol. Med. 2020; 10.1111.jcmm.15581).

[0005] In addition to AKR1B1, the related enzyme AKR1B10 is implicated in several types of cancer (Penning, Clin Cancer Res. 11: 1687-90, 2005; Fukumoto et al; Clin Cancer Res. 11: 1776-1785, 2005; Yan et al; Int. J. Cancer 121: 2301-6, 2007; Zhao et al; Eur. J. Med. Chem. 45(9): 4354-7, 2010; Matsunaga et al; Front. Pharmacol. 3: 5. 2012; Laffin and Petrash, Front. Pharmacol. 3: 104, 2012; Liu et al; Biochem J. 442: 273-82, 2012). AKR1B10 is a 36-kDa cytosolic reductase with an amino acid sequence (71% identity) and tertiary structure with a "(a / B)8 barrel" topology similar to AKR1B1. Like AKR1B1, the AKR1B10 enzyme uses NADPH as a coenzyme to reduce various aromatic and aliphatic aldehydes, dicarbonyl compounds, and some carbonyl-containing drugs. AKR1B10 is normally produced in the gastrointestinal tract. Overexpression occurs in many tumors, such as liver, lung, and breast cancers. AKR1B10 can participate in tumor development through multiple mechanisms and can be a useful biomarker for cancer diagnosis and a potential therapeutic target (Huang et al; Recent Patents on Anti-Cancer Drug Discovery 11: 184-196, 2016). Silencing of AKR1B10 expression causes inhibition of colorectal cancer cell proliferation (Yan et al Int. J. Cancer 121: 2301-6 2007).

[0006] The role of aldoketoreductases in the etiology of colorectal cancer has been established (Tammali et al; Cancer Res. 66, 9705-9713, 2006; Tammali et al; Cancer Lett. 252, 299-306, 2007). Analysis of colorectal cancer cell lines showed that high expression levels of AKR1B1 and AKR1B10 were significantly associated with disease progression (Taskoparan et al; Cell Oncol. (Dordr). 40:563-578, 2017). Thus, aldose reductase inhibition appears to be a promising therapeutic target in the treatment of colorectal cancer (Grewal et al; Mini Rev. Med. Chem. 16, 120-162, 2016; Saxena et al Cancer Lett. 355, 141-147, 2014; Saxena et al, Eur. J. Cancer. 49(15):3311-9, 2013; Tammali et al; Carcinogenesis, 32:1259-1267, 2011; Shoeb et al; Free Radic. Biol. Med. 63:280-290, 2013, Tammali et al; Carcinogenesis 8:1259-67, 2011; Tammali et al; Curr Cancer Drug Targets. 11:560-71, 2011; Ramana et al; Mol Cancer Ther. 9:813-24, 2010; Shukla et al, Cancer Lett. 28;411:57-63, 2017).

[0007] In HeLa cell culture studies (Ji et al; Mol. Biol. Rep. 47:6091-6103, 2020), the aldose reductase inhibitor epalrestat inhibited tumor progression by inhibiting AKR1B1 and was considered a new drug for the treatment of cervical cancer. In a study on SCID mice (Wu x et al; J. Exp. Med. 214:1065-1079, 2017), epalrestat significantly inhibited the progression of basal breast cancer.

[0008] The aldose reductase inhibitor fidarestat enhances sensitivity of cancer cells to oxidative stress and inhibits their growth in vivo and in vitro (Shukla et al.; Cancer Lett. 411:57, 2017), inhibits angiogenesis and tumor proliferation (Tammali et al.; Angiogenesis 14:209, 2011).

[0009] U.S. Patent No. 20110925666 relates to methods of treating lung, breast and prostate cancers and inhibiting colon cancer related metastasis using aldose reductase inhibitors.

[0010] Banala et al. (Biomaterials Sci. 7:2889, 2019) developed a micellar carrier for the aldose reductase inhibitor epalrestat (EPR) based on a conjugate of a disulfide (systematic name “disulfane”) with tocopheryl-polyethylene glycol-succinate (TPGS). In a reducing environment, epalrestat is released controlled by cleavage of the disulfide (systematic name “disulfane”) bond of the EPR-S-S-TPGS conjugate.

[0011] Patent P 285588 describes a mixture of biologically acceptable disulfides of lipoic acid, tetranorlipoic acid, bisonorlipoic acid and 8-hydroxy-bisonorlipoic acid with essential fatty acids for the treatment and prevention of diabetes, insulin resistance or complications of diabetes.

[0012] WO2019040825 and WO2017064675 describe the use of chemotherapeutic agents in disulfide-based conjugates for the treatment of cancer.

[0013] U.S. Patent No. 20010036926 describes labile disulfide-bonded substances that are activated by the action of free thiols under physiological conditions.

[0014] The above findings suggest that AKR1B1 and AKR1B10 inhibitors are promising as new therapeutic drugs for cancer treatment. AKR1B1 inhibitors, such as acetic acid derivatives (epalrestat, tolrestat, zenarestat), spirohydantoins (sorbinil), or succinimide compounds (ranirestat), have been investigated mainly for their role in the prevention of diabetic complications (Hotta, Biomed Pharmacother. 5, 244-250 1995.; Costantino et al.; Expert Opin Ther Patents. 10, 1245-1262, 2000; Miyamoto, Expert Opin Ther Patents. 2002, 12, 621-631 2002; Srivastava et al.; Endocr Rev. 26, 380-392 2005). In recent years, interest has shifted to new chemical types in the search for better AKR inhibitors (Alexiou et al.; Curr Med Chem. 16:734-52, 2009; Chatzopoulou et al.; Expert Opin Ther Patents. 11:1303-23, 2012).

[0015] Despite the fact that many aldo-keto reductase inhibitors have been extensively studied, none of them has shown sufficient efficacy without side effects in human clinical trials. Therefore, there is still a need to develop new, effective and safe aldo-keto reductase AKR1B1 and AKR1B10 inhibitors as potential drugs for the treatment of diabetic complications, inflammatory diseases and cancer.

[0016] 3-mercapto-5H-1,2,4-triazino[5,6-b]indole-5-acetic acid (semimestar of Formula II, systematic name 2-(3-sulfoxy-2,3-dihydro-5H-[1,2,4]triazino[5,6-b]indol-5-yl)acetic acid) has been proposed and patented as a potent and selective aldose reductase inhibitor with antioxidant properties (Stefek et al.; Patent 288508; Stefek et al., J. Med. Chem. 58:2649-57, 2015; Prnova et al.; Redox Rep. 20:282-82 2015; Soltesova Prnova et al.; Physiol. Res. 64:587-91, 2015; Stefek et al.; IJASEAT 4:41 -44, 2016; Zhan et al.; J. Biomol. Struct. Dyn. 37:1724-1735, 2018, Soltesova Prnova et al.; Neuroscience 443:206-217, 2020; Valachova et al.; Int J Mol Sci: E5609, 2020). The ability of semimestar of Formula II to alleviate symptoms of peripheral neuropathy in ZDF and STZ-induced diabetic rats was recently described (Soltesova Prnova et al.; Neurochem Res. 44:1056-1064, 2019; Prnova et al.; Naunyn Schmiedebergs Arch Pharmacol. 393:651 -661 2020). Semimestar of Formula II was proven to have very low cytotoxicity in vitro and in vivo and, moreover, no significant changes were observed in Wistar rats in a 120-day toxicology test (Soltesova Prnova et al.; Interdiscip. Toxicol. 12:120-128 2019).

[0017]

[0018] Two tautomeric forms of semimestar of Formula II

[0019] While semagacestat of formula II shows potent inhibition of the aldo-keto reductase enzymes AKR1B1 and AKR1B10 with IC50 values = 0.48 ± 0.29 and 3.69 ± 0.53 μΜ, respectively (Stefek et al.; J. Med. Chem. 58:2649-57, 2015), it would be advantageous to improve the pharmacokinetic properties of both oral and parenteral administration of semagacestat and such desired improvement includes inter alia improved absorption from the gastrointestinal tract, prolonged duration of action of the active compound and inter alia targeted distribution to cancer cells. Oral and parenteral administration of active aldo-keto reductase inhibitors can lead to adverse effects due to high local concentrations in healthy tissues, such as hepatotoxicity, malabsorption, kidney, sperm and other diseases.

[0020] The present invention proposes to solve these problems by using a therapeutic strategy based on the administration of active drug-based prodrugs. In such a prodrug-based strategy, the active drug is released at the desired site of action after chemical or metabolic activation of the inactive prodrug. This approach can significantly increase drug availability at the site of action and reduce toxicity compared to direct drug administration.

[0021] Semimepaytate disulfide of Formula I was synthesized and tested to obtain an effective aldose reductase inhibitor. Given the key role of aldoketoreductases AKR1B1 and AKR1B10 in the etiology of several chronic inflammatory cancer types, as mentioned above, it was hypothesized that the release of semimepaytate of Formula II within tumor cells by reduction of its precursor with glutathione GSH would increase efficacy and drug action. This concept is based on the fact that reduced glutathione levels are 10-1000 times higher in tumors compared to healthy tissues or extracellular environment (Saito et al.; Adv. Drug Deliv. Rev. 55:199, 2003; Wang et al.; Current Organic Chemistry 20:1477, 2016; Turell et al.; Free Radic. Biol. Med. 65:244, 2013). Due to the high levels of GSH in tumor cells, the disulfide bond is cleaved upon entry into tumor tissue, and is expected to remain stable in the blood circulation. Prodrug strategies using redox-sensitive disulfides have been widely developed, especially in cancer therapy, as a means of targeting the distribution of chemotherapeutic drugs to cancer cells (Meng et al.; Biomaterials 30:2180, 2009; Yang et al.; J. Phys. Chem. B 118:12311, 2014 Brülisauer et al.; J. Control Release 195:147, 2014; Li et al.; Asian J. Pharm. Sci. 15:311, 2020).

[0022] It is understood that this is the first example of a disulfide-based prodrug of an aldose reductase inhibitor. To date, patented and published prodrugs of aldose reductase inhibitors are based on esters (Bruno et al.; Bioorg. Med. Chem. 10:1077, 2002; Da Settimo et al.; J. Med. Chem. 46:1419-28, 2003; Da Settimo et al., Med. Chem. 48:6897-907, 2005; Rakowitz et al.; Eur. J. Med. Chem. 15:11-20, 2002; Sunkara et al.; J. Pharm. Pharmacol. 52:1113, 2000) or amides (Wrobel et al. J. Med. Chem. 34:2504, 1991). SUMMARY

[0023] The present invention provides a compound of formula I semimebarbital disulfide 2,2'-(dithioxalylbis(5H-[1,2,4]triazino[5,6-b]indole-3,5-diyl))diacetic acid:

[0024]

[0025] or a pharmaceutically acceptable form thereof: a mono- or di- salt, mono- or di- ester, amide or a combination thereof.

[0026] In addition to the structure of semimebarbital disulfide of formula I, the present invention relates to a process for its preparation, said process comprising the following steps:

[0027] - treating the compound of formula II semimebarbital with a mild oxidizing agent NaNC in acetic acid at room temperature

[0028]

[0029] wherein the product of formula I has been precipitated from the reaction mixture during the reaction;

[0030] - filtering, washing the product with ice water and cold methanol;

[0031] - drying the product of formula I by progressive vacuum from 3000 Pa to 0.1 Pa obtained by water pump and then by oil pump.

[0032] In one embodiment of the present invention, the compound of formula I semimebarbital disulfide or a pharmaceutically acceptable form thereof as mentioned above is used as a medicament.

[0033] In another embodiment of the present invention, the compound of formula I semimebarbital disulfide or a pharmaceutically acceptable form thereof as mentioned above is used for the treatment of a condition wherein inhibition of AKR1B1 and / or AKR1B10 aldo-keto reductases in cells / tissues with high GSH (glutathione reduced) is desired.

[0034] In another embodiment of the present invention, the compound of formula I semimebarbital disulfide or a pharmaceutically acceptable form thereof as mentioned above is used for the treatment of cancers derived from chronic inflammation, i.e. colon and rectum cancer, lung cancer, breast cancer, liver cancer, pancreatic cancer, prostate cancer, endometrial cancer and cervical cancer.

[0035] In another embodiment of the present invention, the compound of formula I semimebarbital disulfide or a pharmaceutically acceptable form thereof as mentioned above is used as an adjuvant therapy drug in combination with chemotherapeutic drugs used clinically as substrates for aldo-keto reductases such as doxorubicin and daunorubicin for the treatment of cancer.

[0036] In yet another embodiment of the present application, a semimeisilate prodrug of Formula II is used as a medicament, as the semimeisilate disulfide compound of Formula I or a pharmaceutically acceptable form thereof as mentioned above.

[0037] In yet another embodiment of the present application, a pharmaceutical composition comprising, as an active ingredient, the compound semimeisilate disulfide of Formula I or a pharmaceutically acceptable form thereof as mentioned above, in admixture with a pharmaceutically acceptable agent, diluent or carrier (excipient).

[0038] The present application also relates to a compound of Formula I as defined for use as a medicament.

[0039] Preparation

[0040] The compounds of Formula I of the present application can be prepared by various methods well known to one skilled in the art of organic synthesis, including the methods described below or variations thereof. The methods described are preferred, but the possible methods of preparation are not limited to these.

[0041] In general, symmetrical aromatic disulfides (systematic name disulfanes) can be prepared by oxidation of the appropriate aromatic thiol according to the reaction outlined in Scheme 1, according to procedures well known to one skilled in the art. Peroxydisulfate salts, such as (NH4)2S2O8 or K2S2O8, can be used as oxidizing agents at elevated temperatures in acetonitrile, iodine, H2O2 or NaNO2, at room temperature in ethanol or in weak acid or solid NaIO4(Montazerozohori et al., Molecules 12, 694-702, 2007; Ramadas et al., Organic Preparations and Procedures Int. 28, 352-355, 1996; Phakdeeyothin and Yotphan, Org. & Biomol. Chem. 17, 6432-40, 2019; Firouzabadi et al.; Synth. Comm. 28, 1179-87, 1998; Parida et al.; Chem. Select 1, 490-4, 2016; Carbonnel et al.; Chem. Comm. 53, 5706-9, 2017; Owen et al.; US6566384, 2003; Teplyakov et al.; Org. Lett. 15:4038-41, 2013; Branc 2011 et al.; PCT International Application 209125191, 2009; Rubino et al.; ChemMedChem. 6, 1258-68).

[0042]

[0043] Scheme 1

[0044] For example, the well-established procedure for the preparation of the compound of formula I shown in Scheme 2 can be used for the synthesis of various disulfides under the conditions used by other authors on different types of substrates (Abazid et al.; Phosphorus, Sulfur, and Silicon and the Related Elements 1994, 88(1-4), 195-206).

[0045]

[0046] Scheme 2: Preparation of semimustite disulfide of formula I

[0047] Semimustite of formula II, which is used as starting material for the synthesis of the compound of formula I, can be prepared by synthesis procedures well known to the person skilled in the art, which are described in standard works, such as Romanchick and Joullie, Heterocycles 9, 1631, 1978; Neunhoeffer, H., Wiley, P.E. Chem. Heterocycl. Compd.; Wiley-Interscience: New York, 1978, 33, 749; El Ashry, E.S.H.; Rashed, N.; Taha, M. Advances in Heterocyclic Chemistry; Katritzky, A.R., Ed.; Academic Press: New York, 59 (1994).

[0048] For the preparation of semimustite of formula II, the procedure previously published using starting indigo (a, Scheme 3) can be used (El Ashry, E.S.H.; Rashed, N.; Taha, M. Advances in Heterocyclic Chemistry; Katritzky, A.R., Ed.; Academic Press: New York, 59 (1994). et al.; J. Med. Chem. 63:369-381, 2020. The reaction proceeds by deprotonation with calcium hydride, where compound a is alkylated in excess ethyl chloroacetate in DMF by stirring at 100 °C for 5 hours to b. Subsequent condensation with thiosemicarbazide in DMF at 100 °C gives the semicarbazone c. Finally, intermediate c is refluxed in aqueous potassium carbonate for two days to give semimustite of formula II in 39% overall yield.

[0049]

[0050] Scheme 3: Synthesis of semimestar from indigo (a) to formula II

[0051] Use in medicine and pharmacy

[0052] The present application also relates to the use of the disulfide of formula I, in that this substance is metabolized in the body to form two molecules of semimestar of formula II, which has pharmacological activity. It is therefore indicated as a drug and, in particular, as a prodrug of the active compound of formula II.

[0053] Although the compound of formula I of the present application does not interact with aldose reductase (AKR1B1) and other aldo-keto reductases per se, it is metabolized in tissues characterized by an increased reducing potential, such as tumor cells, to semimestar of formula II, which is an effective inhibitor of the aldo-keto reductases AKR1B1 and AKR1B10. The statement "the compound of formula I of the present application does not interact with the aldo-keto reductases AKR1B1 and AKR1B10 per se" is to be understood as meaning that the IC50 value of the inhibition of AKR1B1 or AKR1B10 by the compound of formula I is higher than 100 μmol / 1.

[0054] The compound of formula I of the present application is therefore expected to be useful in pathologies characterized by an increased reducing potential that requires the inhibition of AKR1B1 or AKR1B10, such as tumor cells. These pathologies are characterized by a significant increase in the levels of reduced glutathione (GSH) compared to physiologically healthy cells and, in particular, compared to the extracellular space (gastrointestinal tract, central blood chamber, etc.). The compound of formula I of the present application can be used in therapeutic and / or preventive regimens of pathologies comprising colon cancer, lung cancer, breast cancer, liver cancer, prostate cancer, pancreatic cancer, endometrial cancer, cervical cancer, etc.

[0055] Another aspect of the present application is a method for treating pathologies in which the inhibition of AKR1B1 or AKR1B10 aldo-keto reductases targets tissues characterized by an increased reducing potential, such as tumor cells, which are characterized by a significant increase in the levels of reduced glutathione (GSH) compared to healthy cells or the extracellular space (gastrointestinal tract, central blood chamber, etc.). This method comprises administering to a subject suffering from or susceptible to such pathologies a therapeutically effective amount of the disulfide of formula I or a pharmaceutically acceptable form thereof.

[0056] Another aspect of the present application is the use of the disulfide of formula I as an adjuvant drug in cancer therapy in combination with a chemotherapeutic drug or a similar type of chemotherapeutic drug used clinically as a substrate for aldo-keto reductases. This method comprises administering to a subject suffering from or susceptible to such pathologies a therapeutically effective amount of the disulfide of formula I or a pharmaceutically acceptable form thereof, as described above, in combination with a therapeutic dose of a chemotherapeutic drug used clinically, such as doxorubicin, daunorubicin, etc.

[0057] The disulfides of formula I according to the present application are inactive compounds against AKR1B1 and other aldoketoreductases. Thus, the compounds of formula I remain inactive in healthy cells and in extracellular spaces (gastrointestinal tract, central blood chamber, etc.), characterized by a reduced reduction potential (low GSH levels), and thus avoid the known potential side effects of orally administered active aldose reductase inhibitors, such as hepatotoxicity, indigestion, kidney, sperm and other disorders.

[0058] The advantage of the disulfides of formula I therapy over the semimestar of formula II or other aldose reductase inhibitors is the targeted distribution of the active drug to the tumor cells, which can achieve higher efficacy and fewer side effects compared to semimestar of formula II.

[0059] The disulfides of formula I according to the present application can also have the advantage that in the acidic environment of the tumor, this compound is better absorbed compared to semimestar of formula II alone, as documented in Example 3, which describes that in an acidic environment at pH 4.6, the water / octanol partition ratio of the compound of formula I is almost 4 times that of semimestar of formula II.

[0060] According to the present application, the disulfides of formula I can also have the advantage that due to their molecular symmetry, they provide two molecules of the effective AR inhibitor semimestar of formula II at the same time, and thus this treatment can be more effective.

[0061] According to the present application, the disulfides of formula I can also have other useful pharmacological properties that are more advantageous than the known aldoketoreductase inhibitors, which are active as such.

[0062] The disulfides of formula I of the present application will generally be administered orally, buccally, rectally, dermally, nasally, tracheally, bronchially or by any other parenteral route or by inhalation, or in the form of a pharmaceutically acceptable non-toxic organic or inorganic salt, or in the form of a pharmaceutically acceptable compound of formula I as defined above. Depending on the disease and the patient to be treated, as well as the route of administration, the composition can be administered in different doses.

[0063] The present application also relates to a pharmaceutical composition comprising a compound of formula I as defined above or a pharmaceutically acceptable form thereof in admixture with a pharmaceutically acceptable agent, stabilizer, diluent or carrier. BRIEF DESCRIPTION OF DRAWINGS

[0064] Figure 1The cleavage kinetics of Formula I disulfide forming Formula II cesimistat (CMTI) due to increased GSH concentration are presented. Typical results from individual experiments are given. Formula I disulfide, 50 μM; GSH 0 (▲); GSH 25 μM (■); GSH 50 (M (○); GSH 100 μM (·); Experiments were consistently conducted at pH 7.4 and 37 °C in 20 mM phosphate buffer. Detailed Implementation

[0065] This invention is illustrated by the following examples, which present the preparation of disulfides of Formula I, the measurement of the reduction kinetics of compounds of Formula I in the presence of various concentrations of GSH, the measurement of the inhibition of aldose reductase (AKR1B1) by disulfides of Formula I, and the determination of the water / octyl-1-ol partition ratio. The examples of disulfides given do not limit the scope of the claims. It will be apparent to those skilled in the art that many modifications can be made to the materials and methods without departing from the spirit and scope of the invention.

[0066] General experimental procedures

[0067] Performed on a Varian Gemini spectrometer in CDCl3 or DMSO-d6. 1 H-NMR and 13 C-NMR measurements (300MHz or 600MHz for) 1 H, and 75MHz or 150MHz for 13 C-NMR. Chemical shifts are expressed in ppm relative to the selected TMS standard, and the interaction constant J is expressed in Hz. IR spectra were measured in solid phases on a solvent-free and air-free diamond detector on an Agilent Technologies Cary 630FTIR instrument, ranging from 650 to 4002 cm⁻¹. -1 UV spectra were recorded on an Agilent Technologies Cary 8454UV-Vis instrument. Liquid chromatography-mass spectrometry (LC-MS) analysis was performed using electrospray ionization (ESI) on an Agilent Technologies 1200 series instrument equipped with an Agilent Technologies 6100 quadrupole mass spectrometer. Melting points were determined using the Büchi melting point M-565 without correction. Elemental analysis (C, H, N) was performed using a Carlo-Erba Strumentazione Model 1106.

[0068] Preparation of starting materials

[0069] Semaxanib (2-(3-sulfoxy-2,3-dihydro-5H-[l,2,4]triazino[5,6-b]indol-5-yl)acetic acid) of Formula II was prepared according to the method described in the following publication: Semaxanib (2-(3-sulfoxy-2,3-dihydro-5H-[l,2,4]triazino[5,6-b]indol-5-yl)acetic acid) of Formula II was prepared according to the method described in the following publication:

[0070] Example 1

[0071] Synthesis of Disulfide-2,2'-(disulfanediylbis(5H-[l,2,4]triazino[5,6-b]indole-3,5-diyl)) diacetic acid of Formula I

[0072] To 200 mg (0.768 mmol, 1.00 mol equiv) of Semaxanib of Formula II in 20 mL of glacial acetic acid was added an excess of anhydrous sodium nitrite 138 mg (2.000 mmol, 2.60 mol equiv) by stirring at room temperature. The color of the reaction mixture changed from yellow to brick red during the addition of NaNO2, releasing a smoke containing red nitroxide. The solid material precipitated out of the reaction mixture within ten minutes. The mixture was allowed to stand for another hour. The product of Formula I was isolated from the reaction mixture by filtration. The precipitate on the filter was washed with ice water and then with ice-cold CH3OH. The solid residue was dried in vacuum to give 163 mg (0.314 mmol, 82%) of disulfide of Formula I as a light brown solid; M.p.: 220-250 °C (Celsius);

[0073] 1 H-NMR (600 MHz, DMSO-d6): δ 13.23 (br, 2H, 2x-COOH), 8.06 (dd, 2H, J(8,9)=7.6 Hz, J(7,9)=1.4 Hz, 2x H-C(9)), 7.68-7.65 (m, 4H, 2x H-C(6) and 2x H-C(7)), 7.41 (ddd, 2H, J(7,8)=8.2 Hz, J(8,9)=7.6 Hz, J(6,8)=1.4 Hz, 2x H-C(8)), 5.06 (s, 4H, 2x NCH2COOH);

[0074] 13 C-NMR (150 MHz, DMSO-d6): δ 168.9 and 164.6 (2x C-S and 2x-COOH), 145.9, 142.9, 142.1, 132.1, 123.8, 122.3, 117.3, 112.1 and 42.9 (2x-CH2-).

[0075]

[0076] FT-IR (measured as solid, cm -1 ): 1730 (m), 1619 (w), 1577 (m), 1508 (w), 1467 (m), 1438 (m), 1413 (w), 1369 (m), 1334 (m), 1210 (m), 1174 (s), 1149 (m), 1089 (m), 1033 (w), 968 (m), 932 (m), 886 (w), 802 (m), 748 (s), 702 (w), 667 (m), 615 (m), 562 (w), 514 (w), 447 (m).

[0077] UV-Vis (DMSO): λ max = 269 nm; HPLC-MS purity 98%; elemental analysis C 22 H 14 N8O4S2 (518.526), calculated: C, 50.96; H, 2.72; N, 21.61, found: C, 50.87; H, 3.05; N, 21.80.

[0078] Example 2

[0079] Test A

[0080] Reduction of the disulfide of Formula I by GSH

[0081] The kinetics of cleavage of the disulfide of Formula I to form semimevinshe of Formula II as the GSH concentration was increased was investigated as follows: To a solution of the disulfide of Formula I (50 μΜ) in 20 mM phosphate buffer (pH 7.4) was added GSH at increasing concentrations ranging from 0 to 100 μΜ. Immediately after the addition of GSH, the absorbance of the resulting semimevinshe of Formula II at 302 nm was initiated. Using the extinction coefficient ε = 38,4 mM -1 .cm -1 The concentration of semimevinshe of Formula II was calculated.

[0082] The kinetics of reduction of the disulfide of Formula I to the monomer semimevinshe of Formula II in the presence of increasing GSH concentrations was tested for the compound of Formula I of Example 1 in Test A, and it was found that the dimer of the compound of Formula I split to form the monomer (semimevinshe of Formula II) in the presence of GSH, such that the rate and yield of the reaction increased as the GSH concentration was increased Figure 1 , Table 1).

[0083] Table 1: Initial rate of cleavage of the disulfide of Formula I to form semimevinshe of Formula II and final degree of reduction of the disulfide of Formula I in the presence of increasing GSH concentrations.

[0084]

[0085] * Calculated after reaching maximum at 60 seconds. Disulfide of Formula I, 50 μM; 20 mM phosphate buffer (pH 7.4), at 37°C. Results are arithmetic means ± SD of three parallel experiments. The abbreviation n.d. means "not detectable low value".

[0086] Example 3

[0087] Test B

[0088] Inhibition of ALR2 by disulfides of Formula I in the presence of GSH

[0089] Preparation of ALR2. ALR2 enzyme was isolated from rat lens by the method of Hayman and Kinoshita (J. Biol. Chem. 240: 877-882, 1965). The homogenate was centrifuged at 10,000 g for 20 minutes at 0-4°C. The supernatant was progressively precipitated with saturated ammonium sulfate solution at 40%, 50% and finally 75% saturation. After the first two precipitations, the supernatant was used. The precipitate from the last step containing ALR2 activity was resuspended in 75% ammonium sulfate and stored in small aliquots in liquid nitrogen containers.

[0090] Enzyme test. ALR2 activity was measured by determining NADPH consumption at 340 nm spectrophotometrically (Stefek et al.; Bioorg. Med. Chem. 16: 4908-4920, 2008) and expressed as decrease in optical density (O.D.) / sec / mg protein. The reaction mixture contained 4.67 mM D,L-glyceraldehyde as substrate, 67 mM phosphate buffer (pH 7.4) containing 0.11 mM NADPH, the desired concentration of GSH and 0.05 ml of enzyme preparation in a total volume of 1.5 ml. Reference (blank) samples contained all of the above components except the D,L-glyceraldehyde substrate and were used to correct for NADPH oxidation independent of substrate reduction. Enzyme catalysis was initiated by the addition of D,L-glyceraldehyde and the reaction was monitored after an initial 1 minute at 37°C for 4 minutes. Enzyme activity was adjusted by diluting the enzyme preparation with distilled water so that 0.05 ml of the preparation gave an average reaction rate in the control samples in the range of 0.020 ± 0.005 absorbance units / minute. The effect of inhibitors on enzyme activity was determined by adding a stock solution of inhibitor-containing DMSO to the reaction mixture to a final concentration of 100 μM, resulting in a final DMSO concentration of 1%. Inhibitors were added to the reference samples at the same concentration. Enzyme activity was started by the addition of D,L-glyceraldehyde as substrate 2 minutes after the addition of GSH to the reaction mixture and the I(%) values were determined from the measured absorbance decrease.

[0091] The compound of formula I of Example 1 was tested in Test B and the I(%) values were found to vary with the GSH concentration, as shown in Table 2.

[0092] Table 2: Inhibition of aldose reductase isolated from rat lens by disulfides of formula I in the presence of increasing concentrations of GSH

[0093]

[0094] * The ALR2 preparation used had a natural GSH content < 0.1 nmol / ml. Disulfides of formula I were used at a concentration of 100 μM; the phosphate buffer was at a concentration of 67 mM (pH 7.4) and was used at 37°C. The enzyme activity was measured 2 minutes after the addition of GSH to the reaction mixture containing all the components except D, L-glyceraldehyde used as substrate. The results are the arithmetic mean ± SD of three parallel experiments.

[0095] Example 4

[0096] Test C

[0097] Determination of the partition ratio in a buffer / octan-1-ol system

[0098] The partition ratio in a buffer / 1-octanol system (system name octan-1-ol) defined as the ratio of the total solute concentration in the organic phase to the total solute concentration in the aqueous phase was determined by the shake flask method at room temperature. Octan-1-ol saturated with the desired buffer was used as the organic phase for the determination. Disulfides of formula I or semimearlstat of formula II were dissolved in the appropriate buffer (4 mL) at a final concentration of 100 μM in the presence of DMSO (1%) as a reference substance. The aqueous solution was shaken with octan-1-ol (20 mL) for 3 hours. Subsequently, the phases were separated in a separatory funnel and the concentration of the solute in both phases was determined spectrophotometrically by means of a calibration curve.

[0099] The disulfides of formula I of Example 1 were tested in Test C and the partition ratio in a phosphate buffer (pH 7.4) / octan-1-ol system was determined as shown in Table 3.

[0100] Table 3: Partition ratio of disulfides of formula I in a buffer / octan-1-ol system compared with semimearlstat of formula II

[0101]

[0102] * phosphate buffer (0.1 M; pH 7.4) + 0.15 M KCl; ** acetate buffer (0.1 M; pH 4.6) + 0.15 M KCl; disulfides of formula I (100 μM); room temperature. The results are the arithmetic mean ± SD of three parallel experiments.

[0103] Industrial applicability

[0104] Industrial applicability is the compound of formula I is a prodrug suitable for the production of efficient aldo-keto reductase inhibitors of formula II, which targets distribution in cancer cells, is expected to have higher efficacy and fewer side effects compared with semaglutide of formula II alone, but is more easily absorbed in the acidic tumor environment compared with semaglutide of formula II alone.

Claims

1. A compound, cemtirestat, disulfide, which is 2,2'-(dithioalkyldiylbis(5H-[1,2,4]triazinco[5,6-b]indol-3,5-diyl)) diacetic acid of formula I: Or a pharmaceutically acceptable mono- or di-salt, monoester or diester, amide or combination thereof.

2. A method for preparing the compound of formula I according to claim 1, characterized in that the method comprises the following steps: -The cesmidat compound of formula II The mixture was treated with a mild oxidizing agent, NaNO2, in acetic acid at room temperature; wherein the product of formula I had already precipitated from the reaction mixture during the reaction. - Filter out the product of Formula I and wash with ice water and cold methanol; - The product of Formula I is dried under a gradually decreasing pressure from 3000 Pa to 0.1 Pa.

3. Use of the compound of formula I according to claim 1, or a pharmaceutically acceptable mono- or di-salt, monoester or diester, amide or combination thereof, in the preparation of a medicament for treating symptoms of aldehyde-ketone reductase AKR1B1 and / or AKR1B10 in cells / tissues having high concentrations of reduced glutathione GSH.

4. Use of the compound of formula I according to claim 1, or a pharmaceutically acceptable mono- or di-salt, monoester or diester, amide or combination thereof, in the preparation of a medicament for treating cancers originating from chronic inflammation, namely colon and rectal cancer, lung cancer, breast cancer, liver cancer, pancreatic cancer, prostate cancer, endometrial cancer and cervical cancer.

5. The use of the compound of formula I according to claim 1, or a pharmaceutically acceptable mono- or di-salt, monoester or diester, amide or combination thereof, in the preparation of a medicament for the treatment of cancer in combination with a chemotherapeutic agent used clinically as an aldehyde-ketone reductase substrate, such medicament as an adjuvant therapy, and doxorubicin and daunorubicin.

6. A pharmaceutical composition comprising, as an active ingredient, a compound of formula I according to claim 1, semisterat disulfide or a pharmaceutically acceptable mono- or di-salt thereof, monoester or diester, amide or combination thereof, and a mixture of a pharmaceutically acceptable agent, diluent or carrier, and optionally another excipient.

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