LU102863B1 - Small molecule-inhibitors of 6-phosphofructo-1-kinase for reducing Reactive oxygen species (ROS) generation by cancer cells - Google Patents

Abstract

The present invention relates to a compound selected from the group consisting of: (2-[(5-benzo[1,3]dioxol-5-yl-1,3,4-oxadiazol-2-yl)sulfanyl]-N-isoxazol-3-yl-acetamide) 5 (Inhibitor No. 3); (3-methoxy-6-(3-{1 -[(5-methyl-1,2,4-oxadiazol-3-yl)methyl]-1 H-pyrazol-3-yl}phenyl)pyridazine) (Inhibitor No. 9); (4-[3-(5-amino-1,3,4-oxadiazol-2-yl)isoxazol-5-yl]phenol) (Inhibitor No. 30); (N-[5-(methanesulfonamido)-1,3,4-thiadiazol-2-yl]-6,7,8,9-tetrahydro-5H-carbazole-3- 10 carboxamide) (Inhibitor No. 23); (3-(4-chlorophenyl)sulfonyl-N-(5-isoxazol-5-yl-1,3,4-oxadiazol-2-yl)propanamide) (Inhibitor No. 29); ((2R)-N-(5-isoxazol-5-yl-1,3,4-oxadiazol-2-yl)-2,3-dihydro-1,4-benzodioxine-2-carboxamide) (Inhibitor No. 31); and 15 (3-(benzenesulfonyl)-N-(5-isoxazol-5-yl-1,3,4-oxadiazol-2-yl)propanamide) (Inhibitor No. 32); or a pharmaceutically acceptable salt, solvate or derivative thereof, for use in a method of treatment of a cancer, dysplasia and/or carcinoma in situ through inhibition of ROS generation in the cells of the neoplasm associated with said cancer, dysplasia and/or carcinoma in situ.

Description

Small molecule-inkibiiors of É-phosphofrunin-T-Hinase for reducing Reactive oxygen species (ROE) generation by cancer celfs

METHODS OF TREATMENT

& Field of the Invention

The present invention relies to compounds Targeting the aivcolytio pathway, The compounds dascribed herein ara small molecule inhibitors of B-phosphofrucio--kinase (FRR) that may be useful in the treatment neoplasms in which ROR (and in particular SOX} generation is implicated, ang may therefore : te esnecialty usefu} in the treainmient of patients in the sary stages of cancer development. : ;

Background

Cancer is a disease in which cells undergo uncontroiled proliferation, resulting in formation of à mass of ses, The deviant energetic meiaboliam of cancer cells is characterized by the consumption of larger anıpunis of glucose compared to the normal cells and the conversion of majority of glucose into attic : anid. This coowrs even in the presence of ample oxygen; the process being Known as the Warkung : effec” or asrobic glycolyeis, Higher rate of glycolysis and redendant oytoscfie NADH generation is : tharafore al the root of tumor formation and growth. In cells of ail organisme, maintenance of redox : homeostasis ie of utmost iripertance, In cancer cells surplus of NADH is soivead by Rs rapid re-caidation : and concomitant reduction of pyrovate to lactic acid, {DeBarardinia et ai, 2007; :

ZU DOL 10-1018; bbapap 2018 03.005) However, mince portion of redundant oylosolio NADH can be :

Transported nde minchandrie by medate-asparate shuttle where À saparade reactions are involved and is : kineticaily much slower than pyruvate to factale conversion (Borst DOL 10,1002. 23871 : in mitochondrial matrix the electrons from NADH enter sisctron transport chain (870) and cortritute fo : increased superoxide anion Oy (BOX) formation al Cylachrome | and Cytochrome Hi positions (Turrens, :

EB 2003; DOL 10.911 physiol 2003.04847 8). SOXS are shoriived and they are rapidly disrutated (SCHZI ; is hydrogen peroxide (H:023 another, more stable form of reactive oxygen species (ROS), that are : locates both in the mitochondrial mairix and intermembrane space, Mitochondria derived SUX can partly ; diffuse across the mitochondrial membranes. increased sieady stale cytosolic concentrations of SOX : raaÿy reduce transition metals, which in lum react with HO; producing hydroxy! radicals (OH) ar may :

I react with nitric oxide to form: peroxynitrite (ONGO), Both hydroxyl radicals and peroxy-rifrife are strong : oxidants which are capable of irreversible nitration of proteins, inactivating sncymes, causing DNA : damage, and disruption of mitoohondrial integrity (Madamanchi and Runge: ;

BON TTS VOLKES.O000758450.34413.061 A diagram of Ihe reactive oXygen species (ROS) : generation and regulation is show in Figure 1 (Reczek and Chandet DOL 10 148 annursv-cancedio- ;

Q41915-D8SS0S) increased production of ROS, largely from mifochondia, may promote cancer by : increasing DNA mutations, reguisting signaling and ransoription, and promoting inflammation (Wellen : and Thompson: 2010; DOL 10.1018 mace! 2010.10.004),

Summary of the Imrantion

The invention is based on the inventors’ insight that à therapy for reducing ROS (and in particelar SOM)

B generation could be therefore used in the prevention of cancer, and in particular in the Hagiment of ; patients in the sary stages of cancer development, That is, in some aspects the vention may relate to a teaimant io prevent cancer Seveloping fram a dysplasia or carainoma #7 sifu,

The present invention relates to compounds as follows: : 1G 24{5-panzoll, Jdioxal-5-¢-1,3 S-oxadiazni-2-visulfanyi-Nasopzoh-3ad-acetamide} : {inhibitor No. 35: : {Z-methoxy-6-(3-{ 1-1 S-mathyi-1 2 4-oxadiazel-3yimathyli-1H-pyrazol-Swiiphenylipyridazing) : {Inhibitor No. 9}; : {4-13{5-amino-1 3 4-oxadiazol-2-ylisoxazoi-B-yHphenob {inhibitor No, 30% : 18 N-1S-trnathanesulfonamide)- 1.3 4 dhiadiazo}-2-411-5,7, &89-tetrahydro-5H-carbaroie-3- : carboxamide) (inhibitor No. 23% : {3-{4-chicrophenyheulfony-N-5-{soxezol-B-y1-1,3, 4-oxadiazol-2-vHproparamide {inhibiler No, : 29% :

HERUM Son Soi 2 doxadiazol-Zdh2 Sdihyaro-1 4-hereadionne-2-carhoxamide} : ai (inhibitor No. 31); and : {3-{perzenesuliomd-N-{S-isoxazol-3-yi-1,3 d-oxadiazoi-2-yhipropanamide} {inhibitor No, 32) :

These are refered lo herein as compounds of the Invention or inhibitors of the irvention, :

Accordingly, in a first aspent the Invention may provide a compound of the ihverdion or a pharmaceutically : acceptable sait, solvate or derivative thereof, for use In a method of treatment of à cancer, dysplasia : and/or carcinoma In sit through inhibition of ROS generation in the cells of the neoplasm associated with : said cancer, dysplasia and/or carcinoma in sifu in à patient. :

Suitably, the ROD is or includes SOX, That is, the treatment inhibits SOX generation in Ihe cells. will be : urdersinod that redundant cytosolic NADH in cancer cells enfers mitochondria and contibutes only fo ;

SOX formation, However, SGX, which is ang example of the group “reactive oxygen species” (ROS), is shon-ved and is rapidly enzymatically changed fo other BOS. Therefore, by inhibiting FFKT enzyme activity in the cancer calls NADH level is reduced in oyfosol and SOX formation and concomitantly ROS : formation is dimirdshed, Accordingly, if will be understood that by redusing SEX formation the metho : 38 redunes ROG formation, :

LU102863 :

As desoribed herein, the inhibition of ROS, and in particular SOX, generation In calls may make the : compounds of the invention especially useful for the treatment? of patients in Ihe exdy siages of cancer : devetopment, :

Accordingly, in à further aspect the invention may provide a compound of the invention pr : pharmaceutically acceptabie sait, solvate ar derivative thereof, for use In à method of treatment of : dysplasia and/or carcinoma in situ in 3 patient, :

Ascordingiy, in a further aspect the invention may provide & compound of he invertion ara : phanviacentioally acceptable sait, solvate or derivative therad!, for use in à mathod of treatment of stage © : cancer {in à patient nr the freaiment of stage 1 or stage € cancer in a patient. X will be appreciated that In : the context of such early stage cancer, treatment may de considered pravention of cancer development, : in Giher words, the treatment may prevent of inhibit cancer progression, :

In some embodiments of hese aspecis, the treatment is freatment of stage Ö cancer. Stage D cancer ; refers to the different stages of dysplasia and carcinoma in situ, Therapy may Le referred to as canoer : prevention, or a method of prophyiznis of cancer. ; in some embodiments, the treatment is treatment of stage 1 cancer, Stage 1 cancer refers fo early stage : invasive cancer. The treatment may also ba of stage 2 cancer, The treatment may prevent or inhibit ;

Cancer progression. Ë

That is, in some embodiments the invention may provide 8 compaunı of the invention of a Ë pharmacautically acceptable sait, solvate or derivative thergo!, for use in a method of prevention of : cancer develppment in he early stapes of invasive cancer progression it à patient, Ë

In some embodiments, the neoplnem comprises acute lymphoblastic leukemia cells. In some : ernbodiments, the cancer, dysplasia and/or carcinoma in su comprises acute ymphokdsstic leukaemia : calls. : in some ambhodiments, the neoplasm comprises colorectal adenoma carcinoma cells. In some : 23 ambodiments, the cancer, dysplasia and/or parcinama in sit comprises colomciad adeaoma cargos : cela. Ë in some embodiments, the neoplasm comprises melanama cells, In some embodiments, the cancer, : dysplasia andfor carcinoma i su comprises melanoma cells, :

In some embodiments, the neoplasm comprises marmmandtesast cancer cells. In some ambodiments, ; the cancer, dysplasia andior carcinoma In siti comprises mammany/oraast cancer cafe, : ft will however ve understocd that the invention is not fimited to these conditions. The usefulness of the : compounds and methods in the treatment of à wide variety of cancer and pre-cancercus diseases is Ë envisaged and encompassed. Preliminary diagnostic tests may be carviad out as is appropriate and : understood in the art. : 38 in some embodiments, the compound IS used in combination with other anticancer freaimants andine : medicaments. ;

The invention includes the combination uf the aspects and preferred features dasoribad except where : such a combination is clearly impermissible or axprassly avoided. :

The invention relates to seven compounds that donk to the ATP binding site of the cancer specifis & phosphofmicie-1-kinuss (RFK), inhibit its activity and decrsase Reantive oxygen species (ROE) : formation by the cannes colls. The targeted enzyme is believed te be the pivotal factor of deragulated : vesiyte Bux that results in abundant NADH formation: In order do meatnlain redox potential, he cells : reduce redundant NADH by reducing pyruvale Ho lactats in the oytosol white minor portion «f radundant :

MADH enters the mitochondria. Electrons from transferred NADH enter the électron transport chain Gut : 19 some preiiminari escape the chain that generates superodds anion (SOX), Mat is rapidly converted to : other ROS, increased levels of highly re-acfive SOX and ROS oxidize polymeric molecules inCiuding : nucleic acids causing mutations thai drive cancer progression, ;

For curing cancers and other diseases such as some inflammatory diseases, nsurclogival dissases, ; vascular diseases, diabetes, aging and inferdifité, à would be of utmost importance to roinimize ghvoolylic : flux and consequently decrease ROS generation {Yang and Lian, bilpsididol org 10100775 11090-8018 03867-51, Thus, the aim of the vention is to provide compounds that interfere with highly active cancer : specific forms of PRK is and to reduce thelr activities io the level of the naive PFK is enzymes found in the normal cells. Partial inhibition of modified cancer specific PFICIS might result in decressed glycolytic : flux while redex potential remains balanced. Because no redundant NADH is formed any more, ovicsnie :

MADH transfer into mitochondrial compartment by malale-aspantate shuffle (Borst, :

DOI 19.100%[ub 2387) is prevented. Consequentiy, mitochondrial SOX formation at tha loved of :

Cytochrome ! and Cytochrome IH and is ransformation inte other ROS js reduced, Such treatment is : tateved not to intarere with other celular mechantients, it ahould not be cylôtosée, while the possible side : effects might ba negligible. :

Description of the Figures :

Embodiments and experiments Bustrating the principles of the invention will nou be discussed with : reference to the sccompanyine figuras in which: [ 30 Figure 4: À diagram to show the reactive oxygen specias (RUS) generation and regulation (Reczek and ;

Changed; DOE 10,1 M46/annurev-cahcertis-04 19176-065808}. :

Figure 2: ai ROE, ana b SOX levels detected after 50 hours of incubation in Jurkat cells In he presences ; of inhibitors that were sequentisiy added fo he mediam in 10 gM concentration at 24 hours intervals.

Selected inhibilers are marked with darker color. KO, untreated self; Ke, calls reated with pyoovanin : 38 {positive controls Ku, calls treated with N-anefyi-L-cysteine {negative aontrod). The results are : representative of three or more Independent measurements and are presented as means + standard ; deviation, ;

LU102863 : 5/30 ;

Figure 3 a) ROS, and b} SOX levels detected after 80 hours of incubation in Caco-2 ces in Ihe presence of inhibitors that ware sequentially achten Ic the medium in 10 uM conceritration af 24 hows : intervals. Selected inhibitors are marked with the darker color, KO, untreated call Ks, nells tested with : pycoyanin {positive central), Ko, calls bealad with Neavehd-L.oyataine (negative control), The resuits are 8 representative of thea or more independent measurements and ars proserdad as means à standard deviation,

Figure 4; a) HOS, and ©) SOX levels detected after 80 hours of incubation in Cole 839 ondis in the : presence of inhibitors that were sequantialiiy added to the medium in 15 ui concentration at 34 hours : intervals. Selected inhibilees are marked with the darker calor, KO, untreated cell; K+, calls treated with : 1 pyooyanin (positive controll; K-, cells treated with N-acetyl-L-cysteins (negative contraly, Tha results are : representative of thres or more independent messuramerds and are presented as means + standard : deviation. :

Figure § a) ROS, and kb} SDX levels detected after 80 hours of incubation in MDAMBA231 cells in the : presence of inhibitors that wera sequentially added to the medium in 10 uM concentration at 24 hours : intervals Selected inhiilers are marked with the darker calor, KO, untreated cell: K+, cells treated with ; pyocyaniri {positive control); K-, cells treated with N<acetsd-l-cysteirs (negative control, The results are : reprasemiative of three or more independent measurements and are presented as means à standard : deviation,

Figure § 2) ROS, and hi SOM levais were tested after 80 fours of incubation in Jurkat cells In the ; presence of inh, No, 3 at different concentrations ranging from 32.510 25 pM that were added sequeniially : to the medium at 24 Hours intervals, KO. untreated call; K+, cells treated with pyocyanin {positive control); :

Ka, calls treated with N-acelyl-L-cysioine (negative control The results are representative of threes or : moors independent measurements and ats presented as means £ standard deviation, :

Figure Toa) BOS, and b} SOX levels were tested after 80 hours of incubation in Jurkat cells In the : pracence of Inh, Na. 3, 9, 93, 28, 30, 34, and 32 at different concentrations rangiog from 5 to 20 pid that : were added sequentially io the medium at 34 hours intervals. KO, untreated cell; K+, cells treated with : pyooyanis (positive control); K- cells treated with N-acebd-L-cysieine {negative control), The results are ; representative of three or more independent measurements and are presented as means à standarg : deviation,

Figure § a) BOS, and bi SOX levels were tested after 80 hours of incubation in Caco-é cells in the : presence of inh. No, 3, 8 and 30 at different concentrations ranging from 5 fo 20 uM that were added : sequentially to the medium af 34 hours intervals, KO, uniroaied cell, Kt, cells treated with pyocyanin : {positive controll, Ke, calls reatad with N-anetyi-L-oystiaine (negative control], The results are ; representative of three or more independent measurements and are presented as means à standard deviaton.

Figure 8a) HOS, and bi SOM levels ware tested after 30 hours of incubation in Colo 828 cells In the : presence of inh, Na. 3, 8, and 30 at different concentrations ranging from 510 20 gb thal were added : sequentially to Ihe medium st 24 hours intervals, KO, untreated cell, K+, cells treated with pyocyani :

{positive nontralh: Ke, cells tread with M-acetyl-L-cyateine {negative control}, The results are representative of ihres or more independant measureraents and are presented as means + slandand : deviation. :

Figure 10: a) ROS, and b) SOX levels were fasted after 80 fours of incubator: in MDAMB-231 oells In : the presence of inh, No. 3, 5, and 30 af different concentrations ranging from 5 to 20 uM that were added : seguartialy to the medium af 24 hours Intervals. KO, unfresled call; K+, cells treated with pyocyanin : {positive sonteed): Ko, calls beaiad with N-acetyll-oysieine (negative control). The results are : representative of three or more independant MESSurements and are presented as means + standard : deviation. :

Detailed Description of the invention :

Aspects and embodiments of the present invention will now be discussed with reference to the : accompanying figures. Further aspacis and embodiments will be apparent to those skilled in the art All ; documents mentioned in this text are incorgorated herein by reference, :

The present innovation presents an upgrade of previous Bring that selentes inribitore of highly active : cerner speoific modified PPK enzymes suppress lectate generation In the cancer cells {LegiSa et al,

WO 2020/0308613 A1), Specifically, the same inhibitors were found io concurrently decrease the superoxide anions (SOX; and reactive oxygen species (ROE) in the cells, The suppression: nf SOX and :

ROR may be useful for cancer prevention in pafients with the early stages of cancer development, such as patients with dysplasia andfor carcinoma in safe, :

Early stages of cancer development :

The development of cancer is a slow, stepwise process, yploatly taking many years te several decades. ;

The process starts with ariniflation mutation in à capital gene in à stem cell of à normal cell. The initatirig : mutation typically makes the sell more suscantible to additional mutations, This sisaddy continues over ; tre as the nascent cancer grows, This lengthened phase of cancer deveiogmert is designated as : progression, The soquisition of new mutations atcelersies as the mechanisms of mutation repair ; machinery become mutaiad and dysfunollenal, Simultanaously, the growth rate is also accelerated due to : the riutations in ihe signaling systems of the cells, During the early development, a cancer is invisible and undetectable, unless cells or Hssues are soguired and examined microscopically, Therefore, the : progression from normal fissue 16 invasive cancer is characterized by genetic and spigenelie changes : that ocour at the diffarant points along the carcinogenic continuum, The early changes in cell morphiniogy ; are Knows: as dyspiasia, The aberration batome progressively more pronounced, from mild fo severe : dysplasia, uni! they acquire the morphological characteristics of a malignant necplesm, AU ks stage, tha ; neoplasm is confined to Rs original ansalomic location In the epithellur and is calles carcinoma insite. :

With further progression, the neoplashy develops new traits and capabifiies that allow the nenphæsir io : overiake and destroy the normal Essue, It invades the tissue beneath ths epithelium of origin and invades :

FIR info blood and for vranhatis vessels, Oriy ai the invasive stage of cancer all typical halimarks (Haoaban : and Weinberg 2014, DOE 10.1016 col 2011 02.043) are fully developed. Different upgraced stages of : invasive cancer are characterized by the type of cancer a person has, tha size of the initial tumor, and : whether ar nel the cancer has spread to other areas of the body (Dompion (2020) :

Hitne//doi.org/10.1007/678-3-030-40681-02) Accordingly. ail the halimarks that ara generated by : mutations, such as: genomie instability and mutation, recisfing os death, derequiated cellular energetics, : sustaining profiferals signaling, evading growth suppressors, enabling replicative immuoitality, slowly : develop during the early phases of cancer developraeni. In particular, the "gename inslabilily and : midafion” halimark is reostly biggered by the increased cellular levels of ROS :

Mosicommonty the oncologicsl changes in a patient are Hirst observed when the invasive canner has : already developed. Medical treatment in general requires surgical removal of Ihe canceragentc calls and : the surrounding ssues. However, In more advanced stages of the disease radiation: therapy, largeted : therapy, and aggressive treatment with chemotherapy is nesded apart to surgery. ;

Recently more attention has been drawn to cancer prevention al the sary stages of cancer development. :

Prevention of cancer development rernains the most promising strategy for reducing both its incidence : and the mortality due ta his disease. Mow the risk factors and causes for the cancer initiation and ; development are well understoud, Most commen risk factors include ageing. alcohot, tobacco, fancerogenie substances in the environment, chronic infammaton, infectious agants, obesity {Curie 2021; 12 Cancer Risk Factors and Causes {hips /ÄAvwwrnedicinenel com/ cancer causes/article him}. An ; important risk factor are alse genetic predispositions for cancer development in healthy persons Mat can : row be detested and recognized hy risk biomarkers (Umar etal, 2012, BOL 10 HE38mrcl387). :

Moreuver, the patiants with recuperate cancer disease might be alas included in a group with high risk of

CANcET rE-aplvaraniea. :

Fisk asssesment can be done by specific shinical rials by detecting specific pre-cancer biomarkers of biepsy (Umar ei al, 2012, BOR 10103803387) :

Although the cancer development can be offen prevented by healthy lifestyle the number of : pharmaceutical agents Tor cancer prevention is increaaing (Umar etal, 2012, DOI 11038 wed 387). :

The use of drugs that are agproved and in widespread use for diseases cher then sancer is another tested and seliuhiy practice. For instance, mefforrain, which is widely used for first-line management of : type 2 diahetes educes cancer rates. Various spidemiclogical studies point Ic several Cancers as sxhitiing inverse associations with metformin use: pancreatic cancer, hepatocellular carcinoma, ; edloractal cancer, lung cancer and brags! cancer. Other cancer prevention, the seiecied agents or :

Mitrients are: SERA tamoxifen and ralkıylane, aromatase inhibitors anastrozole and exermasiane, aspidn, : combination of sulindac and DMSO, Sw-reductase inhibitors finasteride and dutasteride, soy isoflavones, : lvcopene, resveratrol, omegs-3-atiy adds, pomegranate, curcumin. These substances ar being tested ; at various cancer types, Pharmaceutical use of some have been already approved by FDA (US Fond and ;

Drug Administration) white the irnlals of others are at diferent stages of clinics! tests (Umar et al, 2012, :

BOL 10.1038/nrc3397), :

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Strong oxidants induced muülallans affect genome stability abd dynamics of gene exprasaics and are generally achepted as a cause for cancer initlation (Waris of al, DOV 11159147 7-S162-5-14), Most : mutations target GC bases, prevalentiÿ causing base pair substitutions rather than deletions or ingariions,

À well andersitod mechanism ia the G-to-T conversion found fa the lumorsuppressar gene TRI (Tubek at al, wwwalinorg/AJSTRTO04001). He product p5S can act as a attass sensor protein, and iis antin:idant role emerges from its ability to enhance DNA repair and Io regulate the expression of a subset of : antioxidant genes, Thus, logs of pid function induces further Increase of infracatiuiar RDS, provoke : abnorme! mitosis and promote cancer developraent Sablina at al, ROL 10, 1038/Ami1320) Regular : exposure of mitochondrial DRA 16 SOX and HC. Induces oxidative hgions in genes encoding respiratory : 19 chain complexes {IV and V {Ralph ef al, DOL 10.1016. mam 2010.02.008). Mutations in comple | : which tigner high production of ROE, have been reported to enfance ouf proliferation {Ishikawa at al, :

BO 1003 128/a0ience. 1156906). All these observations indicate that HOS-4nduçad DNA damages, and metabolic adaptations are crucial for cancer inflation and promotion. Therefore, inhibition of ROS may : siow and/or inorease the lafency of spontaneous earb<-stage tumor development {Sabling et al, :

DOE 10,1038mm 1320), ‘

ROE as signalling motecuies

Hydrogen peroxide concentration in milochondrie is spprximately T00-daid higher than that of BOX. This : property renders H,O, an ideal signalling molecule in the cells, thal promote many aspects of tumor development and progression (Liat al, DOE 10. 11R88/{756-8723-6-19}. Increased ROS levels found in pancer cells promote tie activation of Ras and have been shown to up-reguiate MAPK and :

PI3SR/AKUNTOR sigraling pathways but inactivate PTEN signalling cascade. Moreover, ROE promoie : angiogenesis and métastases by stabilizing MIF transcriptional factor activity and activate B'acsnosiné : moncphosphete-activated protein kinase (AMPK) (Kumari ef ai, DOR 10, 1177/117727 1918755201) In ; 28 order io maintuin oxidative homeostasis, tumor cells alsa express increased loveis of antioxidant pradeing :

Io defoxify ROS, sugoesting that a delicate balance of intracellular ROS levels is required for cancer ca ; function {Liou and Stroz, DOL 10:3109/10715761003687554), :

Late stages of cancer ; in The evolution from normal cells inte in sife carcino and mvasive coronoma is, respectively gasociated with moderate, high and excessive increases in ROS levels {Gordnd et al, DOI 101038 nrd3002) This is : particularly dus fo the increased need of energy that enhances tumor metabolism and the subsequently

ROE production {Paver at al, DOL 10 1038/cddis 20161051 Although BOS are crucial for the transition : of normal cells inte cancerous and further cancer development the accumulation of oxidative damages at : later-stages of cancer development promote the apopfosis of cancer cefs {Wu el al, :

DOL 131002400 21550), Malignant cancer cells are known to produce excessive levels of Hull new the : toxic Hreshoid thos giving antiexidents an important role in reducing ROS and promoting proliferation. For : his reason, cancer calls have implemented a mechanism trough which some derivatives of the :

LU102863 :

SG : glycolysis pathway are shutiled to the pentose phosphate pathway te synthesize NADPH and regenenais : dlutathione (GSH), an important antioxidant that prevenis the gocumulation of ROS {Rea et al,

DOE 10.5821 58-83 00. OO 11-0334), However, Bar cancer frsalæant at the lake stage of cancer development reduned antioxidants levels may enable ROS rise over the threshold needed far triggering £5 gpoplosis and concomitantly causing cell death. it has been four that silencing of S4ructa-2- :

Kinaca/Fructo-46-bisphosphatose (PFKFB4) leads to à decrease of its product fruslose-2 8-disphosphate {F.2 588 and consequently to a decrease of several pentose phosphate pathway intermediates, Among others ihe levels of NADPH are reduced that are required to maintain cellular stores of GBH (Ros et al,

DOL 10.11858/215¢-8280.C0-11-0234), F-2,8-8F is à strong activator of 8-ghosghafructa- kinase {FFKI the most important regulator of glycolyfic flux which reduced activity is obvicusly responsible for : the reduction of pentose phosphate pathway in the cells with siencad PFRFB4. Therefore, reduced : antioxidant formation in a form of GSH can be expected by inhibited cancer specific forms of PFRT as wall, 45 Antioxidants :

The following enzymes ant as naturat antoidants: Superaxide dismutase family (SOD). catalase, gluiathiche peroxidase {GP}, Peroxiredoxin family and Thicradoxin small proteins, :

Besides, a number of synihetio ROS scavengers have boen developer. These compounds easily pass : through iniogical membranes. One of them is MÉOVISE In which vitamin £ is covalendly attached in 2 tiphenyphosphoniuns cation, MROVILE decreases ROS wroductien and apoptosis in aortic endothelial : cells induce by oxidative stress, but Xi inaffective against hyposic-Aschemic stradt injury in reonatal rats, :

Ancîhes ROS scavenger is MHOQUD which consists of ipoghific riphanviphasphonium cation covalently : attached via an aliphatic fioker to a ubiquinone derivative, MitoQ TO inhiblis mitochondrial oxidative : damage ir rodent models of cardiac ischamia and repedusion injury. 28 Furthermore, blocking ROS production rather than ROS neulralizalion was shown ti be another approach : for decreasing cancer cell invasion and increased call death that was sdoely shown by siudying ocidative : stress in prostais cancer (Kumar etal, DON 10.1 188/0008-54 72 CAN-07-5259) The authors focused on : alternative way of RUE generation in the oytosoi by NADPH oxidase (NOX) system that is critical for : malignant phenotype of prostate cancer cells. Diphentfodoniuns, à specific NOK inhibitor blocked : proliferation and has modulated the activity of growth signalling cascade that caused the cyclin B- : dependent GA cell arrest. In fact, it was stown that ROS generation is orfinal for migratory and : invasivanass phenclyoes. if was revealed that reduced ROS production might offer effective means of ; combating prostate cancer in particular, and perhaps other malignancies in general, ;

On the other Hand, mitochondrial sirtuii SETA that act as 8 taylor suppressor, has an ability io suppress : 38 ROS and dowreregulats hypoxia inducible factor (HIF-to) activity, SETS knnchdown increasas : tumorigenesis in xanogratt models bal # is sbolished by giving mice the ant-cxidant N-acetybL-cysising ) {Belt et al, BOL 10.1038/0nc 2011,37) Sirkuing are NAD-dependant deacyiazey whose enzymatic : activities are raguisted by the ratio of NADY te NADH, high NAD“ levels aclivate sitiulng and conversaly ;

high NADH levels inhibit activiies. In contrast miiochondrial SOX and ROS are formes at high NADH : levels. In audition, agents Hat scavenge ROS or reverse Warburg effect prevent the formation and : malignant phenciyas cheered in the cells lacking SHER Thus, mice lacking Set3 provide a model that : meshanistically connects aberrant ROS, the Warburg effect, and carcinogenesis (Maga ef al, :

DOE 10,1156/0008-5472 CANS 1.3833) :

H is important to realise that chronic avarprodustion of mitochendeat ROS leads also to destrugtion of : pencreatic foslls increased oxidation of kw-dansity ipeprolein and dysfunction of endothelial celle ~ : factors that promote atheresciernais (Madamanchi and Runge, :

BOE 10118101 RES (000258450. 444 13.96). ;

Yat to the inventors’ kaowledge, there were no aftempls to dacrease SDX and ROS generation by : reduning deregulated glycolytic flux (Warburg affect) in the cancer cells, so far, The most prominent : change of an allosieric glycolytic enzyme in cancer call searns to be posttranstationa] modification of 8- : ghosphofructo-1-kinage (BFK) (Sere et al, DOL 10.137 tournal pons 0019645). This enzyme is the ; key regulatory enzyme of glycolysis that catalyses the phosphorylation of fruciose-S-phosphale to

Suckose-1.6-bisphosphate (FBP) using Mg-ATP as a phosphoryl donos is stimulated by Fucte-2,8- bisphosphate (F2E8P), ADP/AMP, and ammaniom ions, whereas cifrate and ATF act as strong inhibitors : {Dunaway et at, Biochem. 281: 8577-83) Three isoforms of PFK1 enzyme are present in manag, ; muscle type (PFK-M), ver age (PFIGL) and platelet type (PFICP). Their expression in cancer and normal cote differs sionificanth, We were first io describe postransiational modification of human PERRY : enzymes in the cancer colis, Namely, the native muscle type 85 kDa form {FF KA) pan be cleaved by specific proigase forming 2 45 KDa shorter fragment {mers et al, DOE 10,137 {journal pone DG19645; :

Andree et at, BOP 10.108 16tbapap.2018.03.006) white 85 KDa fiver type (PFR-L} is cleaved to 70 kDa : fragment {Krist et al, 2081). Newly produced fragments retain activity but have altered kinstic : charactersfios. The truncated enzime is resistant ic fesdbank inhibition by citrate and ATP, whereas : 28 some effectors, such as fructo-P, E-bisphosphats (F2 8BP), increase enzymes activity te à level higher thar that of the native enzyme, Such significant change of an allosteric enzyme leads 10 deregulated : glycolytie Aus and increased oviosolic NADH production. However, in the cells of all organisms, : maintenance of radox homeostasis which is main) controles by pyridine nucleotide ratios, is of utmast : imgorlance. In cancer cells the redundant NADH is rapiifly re-oxidized by reduction of pyruvic to lactic : ach in the cyiozol. However, some NADH molecules enter mitochondrial compartiment where, after the : re-pxdation the electrons originating from NADH, enter the électron transport chair of cidative : phosphorylation (OXPHOS). :

Reduced control of the glynolylic ux at the level of PFKT has been previousty confioned alse by a contrat : aratyses by woe fast growing tumorigenic cell types; human Hela cells and rodent As-200 seits, Kinetic : 38 analyses of bolsted PFK1s showed low sencifivity towards He alloslarkı inhibitors sitrate and ATF {Marine :

Hernandez et ai. FERS 3 273:1575-88; and Mann-Hernandez et at, DOI 10, 10164 bbabie 201011006

State of the art :

Simaltancousiy with the progress In the understanding of cancer metabolism, a number of new possible : drug targets were identified, Unfortunately, only a rekafively low number of metabaly inhibiore that have : been developed so far are in regular use for cancer frraiment, Mis worl noting that there is à fot of : uniformity between the meiabolierm of malignant cells and that of highly profiferating nomad cells, : therefore such inhibilors nan cause significant side affecte, The most promising metaboie targets for : cancer therapy have baen recanty discussed by Gallurzi ef al (2013; DOL 10, 103@mr0d 145), inthe : review, the compounds which treatment is in the phase of preciinal frais (VV258117, SPTES) some that : have promoted to clinical deveinpmiant bul were discontinuad (3-bramepyruvate, Methyl-jasmonate, : iG Thalfon), and few with a clinical experience (Metformin, Phenformin, Dichlornanéfate) are fisted, Among : the compounds reviewed, some compounds farget Hycohdio enzymes such as cancer specti : hexokinase (HKZ) (3-bromopyeuvates and pyrovate kinase (FRM2) (Thallion} and are In advanced level of : anti-cancer tals (oinical studies), Some other compounds are in the initial stage of lasing. :

PFKI iso-enzymes have been proposad as à larget for anti-cancer drugs several times, bul only a , relatively low number of PRK inhibitors have bean discovered and studied. Mostly ail compounds : affecling tha activities of native PFICT iso-erzymes were reported, The only accagiion the inventors are ; aware of is à patent application, fad by the same Inventors, describing small-molecule inhibilors of : cancer-specific modified human PFKT isc-enzymes that suppress lactate generation in the cancer cells : (WO 2020/050813 AT),

The drugs known to diminish PRICE activities so far are cisplatin {Thou et ai, :

Hip: nebinim bib gowpubaed/ 12087288), clotarmagote (Fuviado sf al, :

DOE 10.137 Vlournaipone ODSDABE; and DOL 10 417021810444 1000810), stoposide {Thou et al, : 2002, btn scbinim.nih qev/pubmed/12067988), and laxoi (Gless-Marmor et ai, DOL :

ID, 10 1A/SOU14-26DG(SDIONTES-7), Taxal induces datachiment of PFK1 from cytoskeleton and was : reported to influence PRT activity by reducing the gene expression, However, these substances act by : reducing the transcription of not only the pf genes Sul also otfier glycolytic genes. For instance, cisplatin : that is used for a clinical treatment of testicular, ovarian, cervical head and neck, non-small ceff lung : cancers and relapsed lymphomas, impairs transcdption and replication that promotes apoptosis (Zhou et : al, Big fan. robin ih. govioukmed! 1 2087898). Similarly, the tue inhibilian of S-bromopyruvate for :

HK2 is imited, as this agent has been shown le inhibit agdiional enzymes that are involved in glycolysis, : including dlyceraktehyle-S-phosphale dehydrogenase (GAPDH) and mitochondrial succinate : dehvdrogenase {Shosan; DOL 10.1007 18501 2-8418-2), Apparently, Such compounds Hkely inhibit : biosnergatio metabolism ales in the normal cells at the same doses assayed 10 arrest himour progression : and pause significant side effects. Similar systemic toxicity was observed also with other substances :

Hotes above that exhibit multi target inhibitory effects (Gaflozzi et al, DOE 301008041145; Ganapathy- :

Kanniappan and Geschwind, DIE 111884764583 12-152). :

Smai-moieoufe inhibition of S-phosphofrucio-éKinasefructose-6-bischosphatase (PFRFS3) proved tb : te a proraising target for reducing enhanced glycolytic Sux In the cancer Calis. In neoplastic calls, the :

PERERA is the most abundant form among 4 soanzymes since À is constitutively expressed and is :

required for the high glycolytie rate. Although is nol a ghreolytie Enzyme, de product Fructose S- : biephoephate [F-2,5-8P) acts as a sirong activator of PFRT, For drug design, PREFERS 3D- : shristalographic struciure model was prepared and three characterisiio amino acid residues from the : substrate binding site were selected for the vhtualscrearting runs, Afler examining 13 different : gormpounds for thelr ability Io inhibi recombinant PFKFRS isoenzyme autivity, a single compound 3-{3- : pyridinyh-1-<d-nyridinyl}é-propen-1-cne GPO) way found Io suppress basal enzymatic activity, 3P0 : decrsased Ihe irdraceliular concentration of F-2,6-BF, suppressed glucose uptake, lowered ATP, NADH,

NAD levels and diminished lactate excretion in tumorigenio Jurkat cells, 3P0 also markedly attenuated : the proliferation of several other human malignant call fnac (LS Patent No. 3,088,385}, However, 3R0 : was found to cause an arcest in the cell oycle progression in the Jurkai calls and therefore its : pharmacokinetic properties were sigaificantiy below those required to justify testing in human subjects. In : thin raspeot 73 derivatives of SPC were synthesized and screened for inhibition of PFKFBS, One : moietule, M{é-pyridihel-3-02-quinolingh-2-propen-tone {PRR1S) was selected for further pre-clinical évaluation. Positive initial results promotad advanced phase 1 clinical fials of te efficacy in advanced : cancer patients in 2013 as giver in US Patent Mo. 8,088,385. Later, in 2014, another 3F0 derivative, ;

PFK158 was discovered, that is mors potent than APG, has improved pharmacokinetic properties for testing in clinical triads and causes ~80% growth inhibition in several mouse models and human-derived tumors (htp;/www.cancerandmetabolisn.comicontent 25 1/814], | has been shown that inhibition of ,

PFKFBS using PFKTSS inhibitor resulted in reduced glucose uptake, ATR production, lactate release as : wel as induction of apoptosis In ovhaecological cancer cells {Mondai 8. atal, DOL 10.100280 31885). :

Document WO 2017/065751 describes some possible inhibitors of the FFK enzyme: however, the : proposed inhibitors do not appear in be tested thal in vitec and in vive activity. Further, they aiffer from the present venticn, Also, the following articles have proposed inhibition of PER enzymes and is

Importance in treating cancer = Yietal (2013) PRET Giyoceylation Is à Key Regulator of Cancer Cell Growth and Central :

Metabolic Pathways ; - Granchl and Minutgle (F013) Anti-cancer agents pourileractHng tora giyooiysie ; - Hasawi ef al (20141 htps Hwa crob-ontine convarticle/S 1040-848 14 100088-D/pdf ; - Prasad ef al. 12017 Redusen production and untake of lactate are essential for the ability of

WINTES signalling to inhibit breast cancer cell migration and invasion ;

These articles do not propose the same inhibitors as the present invention, ;

In patent application WO 2020/030613 AT Had in 2078, we have shown thal cancer specific, highiy-actve :

PERT fragments can be inhibited by smal-molecule inhibitors and preventing lactate generation by the : various harorigenic sells. However, there is significant difference between suppressing lactate and RDB : and BOX generation. By reducing lactate levels, extracellular aoidosds Is neutralized therefore hwnune : celis can efficiently attack and destroy cancer cells in tumors. Therefore, suppressing lactate generation ; in the fumors has à therapeutic effect and inhibiters can be used for curative of different cancers, such as : invasive cancers, Un the other hand, increased ROS and SOX levels In cancers are the most Important A

LU102863 : drivers of onoc-ufations. Nonmally, damaged DNA can be repairad in human cells, bul elevated levais of :

ROS enhance the formation of mutations that start ic accumulate fo cancer cells are become crucial for : canoes development and progression, Several additional halımarka of cancer depend an generation of Ë onco-mulations, such as: “sustaining proifferais signaling”, "evading growth suppressors”, “evading : replicative mortal”, “resisting cell death’. i is important fo realize that if is practically impossible to : remove mutations by therapeutic teatments however thay can ba avoided by preventalive measures. :

The use of PFE Inhibitors far suppressing laciate generation is of ourailve nature more appropriate for : cancer therapy of the later stages of cancers, such as invasive cancers, while the use of FFIST inhibitors : to suppress redundant ROS and SOX formation act as à praveniafive action af the early stages of cancer : development In the present patent application wa show that the same small-molecide inbitdtors also : féduce ROS formation in tested umarigenic cells. :

Compourds of the invention :

Descoribed herein are seven compounds which bind to the ATF binding site on human Sphosphelracio- Te : kinase (PERI), namaly ; - inhibitor No. 3 02-K5-bDenzoft, 3eioxol-S-yi- 1,3, 4-oxadiarol-Z-vÜüsufanyfl-N-isoxarol-S-ylanetamide), ; - inhibitor No, § 3-mathoxy-8-{3-{1-{{E-methyi-1,2 4-oradiazol-2-imethyil- 1 H-pyrazol-3- ; vhohenylioyridazinet. : - inhibitor No. 23 (NS (msthanesidionamido}- 1.0 4hfadtazcl-2-v156,7 8, B-tetrahyoro-SH-carbazoie-3- Ë carboxamide), ê ~ inhibitor Ne, 25 (3-04-chiorophenyhautfonyi-N-{6<soxaroi-S-4113 {-oxadtazoi-2-yRhoropanarmidet, Ë - inhibitor No, 30 {4-3-(Bamine 1,3 4-oxaitiazol-2viisonazo!-5-f{pheriot, : ~ inhibitor No. 31 {ERIN SBonaeohSad- 1 3 d-cadiaent- 290-2 B-dibydro-1 4-benzodioxing-3- Ë carboxarniie) ans : 28 - inhibitor No. 32 (3-{benzenasulfonyli-N-{B-isoxarol-B-yi- 1,3 4-oxadiazob-2yiipropanamidi). :

The structures of the compounds are shown in Table 1,

Table 1 : inhibiter No, Structure : eve ; 3 pe AS Sons oF a N 2 :

FE fT 5 ka SR Ë ce Ë sets +

Inhibitor No. Structure

Oy at, :

INTRA, or :

K ad ee. An ON Ts, :

CATT Ne eT RE : 8 tie Yi Sr A PE : i i SR : © 0e nN ‘+ :

Sa Wa :

CaN

Rey : 3 :

One aa :

ESSONNE : iad “3 = . :

SEEN Le N : - ao à 2 : à R# : = 4 : i : > z si 7 23 JN

Smt” ge :

AT iF : 1 x dé >, ]

X KA i :

Fond m :

LIN 2 :

Hana ;

N Ë

3 i 3 ~~ 3

See i i = i 3 a 25 3 : À A poe] 3

MN Si 3

NEAT TN : ~~ 3 #3 STOUR es NA QE AUS Ë 47 TNT Sag et # 4 i ex 3 $ Neon 3 3

Li i BONS 3 oT wy CF ENS 5 gi Neng i Ë

IR 3 na LO : a Sep 5

Sond ; 7 5

HN, A Ho : ee ES N Ad 5

Ti 3 Ne a EE : 38 PT : id A È =

IN. RE A À =

RG bre 42 5 i = 3 À AN Ë ~ i Ho Wen 3 i

RS SS a ON = a rN ge 5e i = 34 ° te a x ee Nd ê i : | i Ë : 2 } + Ë

N ee = =

Ne : * ko =

EN 3

ES Ë ÿ i D Ë

NE 22 4 : 3 Ne 2 aa ra pre © 32 3 SN FR. GG HP © fa eg a SR Ë en Y 1 2 i Ë {2 it Boh SN : 3 NT oie 8 2 Tes N ÈS 3

These seven compounds individually partially fohibit Ihe aclivities of highly active cancer specific PFROI enzymes, te the level of activities of native PRK isc-enzymes with tha aim of reducing glynalytio Aux and abundant cytosolic NADH formation. In the cancer cells reduridant oytasclic NADH is re-axidized by reducing pyruvate into lactate while a minor portion of redundant ovtosolic NADH enters mitochondrial

S oompariment felling electron transport pathway and ROS generation, idention compounds can be therefore used for the suppression of lactate and ROS gensration the nancer cals. inthe methods of reducing ROS levels in the cancer cells described herein equal experimental methods {kmorigenic cells and growth conditions} were applied as for previous conducted innovation for :

D suppressing haclate generation In the cancer celis Legi$a at al, WO 2020/0230812 AT) ;

For both inventions, drug design based on the crystal structure of the human PHISF isocanzyme published : by Webb et al (2015, DOE 10, 1038mature 14405) was conducted. The atomic model of the human PFK-P : ispercyrse was designed on Ihe base of ihe said crystal structure in combination with ATP-Mg™ ai 33 À resolution. The mode! was used ia virtually screen ihe ZINC Drugs NOW database by docking the : 15 compounds to the ATP binding site of the enzyme. Initially 4.5 million compounds In the database were : fiterad to exclue expecied aggregators and poorly soluble compounds, By using an n- house : supercomputer {CROW TS} with approximately 3000 processor coves, 38 compounds were selected and : 18 potential FFKP inhibitors ware purchased from Enamine (Kyl, Ukraine) (1-81, ChemBridge {San :

Diago, CA, USA) (8-18) and ChemDiv (San Diego, CA, USA) {17 and 18) for in-depth testing (15 : 20 generation of inhibitors), The said inhibHors are listed in the Table 2 balow. :

Table 2: tested compounds that are possible SEK-P ang PREM inhibitors {1% generation} : : Inhisiter No. | IPAC name ; ;

Ï 1 i SASRITA23-dihvaro-1,4-Benzodiban-8-451}-5-0x0-Dyroidhe-23- ; : i darborsianminelmethyffisoxazols-5 i : by | NS Metrain- Tih 334M 1,2 d-trlazob- Gib 1, 2 d-oxadiazol Swylfpropadarnive | : a i =-{{E-ponzoit 3igiaxob-S-yi-1,3 d-oxadiazoi-2-ylisulfanyl]-MNusoxazob-3-v i : i acetamide : : 4 i 1-{2.35-gihydro-1 4-bengndinking-S-gulfonyll-Me{S-mathyl 1,3 d-oxadiazol-2- | ; i i yhpineridine-d-carhoxanide ; : 5 | N<{5-methyl-1,3,4-oxadiaroi-2-y{}-1-{2-naphthylsuifomtpiparidina-4-cerboxamice | : 8 N(E-meihyl-1,3, d4-oxadiazot-244}1-tetralin-2-plsuonÿd-phheridine--carboxamide : Ë i 7 i 4~{1,3 S-oxadiazeb 2 whphenyl 2, S-dimathylguinoradine-S-cartoxylaie | : ; 8 i (B-methyl-1,3,4-oxadiazoi-2-yfmettyf I-phenyl-21-berzocezole-S-cerbonyiale | : i & ; 3-methony- S-311-0S-methyl- 1,2.4-oxadlazolSeylimethyfi- TH-Dyrazal-3- | : ı 7 i yiphenyfipyridazine | : : 49 i 4-13-01 R-bergodionnt-Baylk 1.4.8, 7detrabydro-6H-purevolois, S-cipwidind- | ; : . ! viimethyl-1Hpyrazole-3-carhowytic acid | :

TT SSSR CS SSSR ES SR ES ESS SSSR ETES : inhibilor No. | IUPAC name ; i + | 3-3-3 fucrophenyl-6, Zufihydroisekazolold, S-olovridin-S(€H imethyilN- : i a ! mattyd-1, 2 d-oxadiancie-Soarboxamine i : ; 45 | 543-110 2-benrodionst-5-41)-6, T-ditydroisoxazolofd, S-cipyridin-S(sH-vimethyl- | : ; oe | 12 4-oxadiazole-I-varhoxamide i :

Ü Lmercetccaneencancententeneenmnnd[enmenmenneneenentinnneenaneanen ane ann nn nn A Rm en re] : 23 | 3A3-{2-Huoropheny)-6 T-dihygroisoxazaiol S-clpyridin- Sa Hpyimethyil-ie : :

Ë > | meihyi-1,2,4-cxadiazoie-S-carboxamide ; : ; 34 | 3-mathyhS8-{ 3-01 Surmathyl- 1,3 d-oxariezcl-dHmethyl}-1H-pyrazoi-2- i : : | yliohenyhoyridarine i : ; 4 : S{j2<Barethoxy-2-naphihylimorpholin-dyiimethyll- 1.2 4-oxadiazois-2- ; : | ; à : i { carboxamide i : : | 343-041 S-benrodioxel-Sy 8 ?-dihyimisoxazoio(d Solpyridin Bab wlimethyl | : : 4€ i {= Vis y t yi : i or ! 1.2. doxatiazole-S-carhoxamide ; : : 47 | N-(2-hydroxy-5-mietry;-phenylh-4-{3-nitro-12 4-itacoi-1 4h butyracices : ; : 38 | Ne{d~sutfamoyiphenyl-dH-chromens{ 4-diiscxarole-S-suforarmide i ;

Ag ateady mentioned previously, human cells express three types of PERS, namely PERF, PFA and :

PRL, While M and Pisoforme have iderdical ATP binding site, PFK-Us differs from the former in one Ë amino acid, Thérafors, a model with modified ATF binding site (TIGER) was used to virtuaiy screen the : 8 ZING Drugs NOW database by the Supercompuier {27 generation of inhibitors), Additional 15 potoniiai Ë

PFK-L inhibitors were purchased from Enarmine (Kyhs Ukraine) (21-28) and ChemBridge {San Diego, CA, ;

USA} (29-88; The said inhibitors are listed in the Table 3 below, Ë

Table À tested compounds that are possible PFKAL iihitdios {2% genenation) :

Inhibitor No. | FUPAC name : ê : 21 | MMi-methyl-1,2,4-nxetiazci-2-ylimethylsuffonyl- MH Ri-tetrafin-{-yliacelamide | Ë i 23 | 2,4-dimethyl-N-[3-{methylsulfonyimelhy1} 1,2 é-oxadiazol-5-4i}-1H-indule-5- i Ë i 7 i carboxamide ; ; ; 7 | N-{B-irnathanesulfonamido 1,34 -thindinzol 218,78 S-detrahydro-SH-carbazole- | Ë : 7 | Icarboxamide i : i 24 | 2{G-methvtiaonaral-3adioxy-N- 3 {-methyitetazo!-Boyliphanyilacetaride i : ; 28 | {31 1,3,4-oacdiazol-<-Honenyl} i : ; 26 { N-(R-mathyl-1.3.4-5xaslarcb-Z40 1m deulfenyinigeridine-4-carboramide ; ; i 27 | 2-{3-{4H1 2 4 -friozol-3-pisuifonylipropyh 1. 3 Denzokazole ; Ê i 28 i M d-dimethyl-M-4-01,3 d-cradiazol-2-vDbensyibenzenssuifenamide ; Ê ; 28 | R-4-chiorophamdisutionyb- No Sisonarol8-y1, 3 4d-onadiazol-2-yijpropanamide | Ê

LU102863 : 4720 ; inhibitor No. | HARALD name ; : i 38 | dJM5-amino 1,3 A-oxadiazoi-2-v{isoxanoh8-{fpherot ; : 24 | (DRIN4B-Isoxarol-5-y1-1,3,d-cxadiarol-d-yB-2.3-dihydrc-1 4-bertodioxine-2- : : i ol ; x { : carboxamide { 22 | 3-({benzanasulfonyi-MN-{8-lsoxarol-E-4i-13 4-oxadiazol-<#Dproparamide { : { 34 | N4{5-isoxazoi-5-4k-13 doxadineol 24-3 { 2 naphthyliacelamide ; : 38 | 2-14 -Auographenyisulfonyi- AH Bwsoxazgl-S-yi- 1,3 A-cadazol-2--Hacetaraide : :

A | (NZ N-A5-AmethoxymethyD-3H-13,4hhadhazot-2-yldenai-2-{3R 2 -Dxniroii- : : ; ; ; ylacetamide : :

Preliminary scraening of alt compounds for the prevention of lantale generation revealed seven : compounds with was à dietinet inbivitory activity. Two of compounds, namely Inhibitor No. 3 (3-({S- : benzo(1,9idioxob By. 1,3 d-oxscdiazoh 24 Daulfenyl-N-isoxezof- 3-vi-acetamide), and Inhibilor No. 8 {3- ; 8 methogeB-(341-{G-methyl-1,2 A-cxadiazol-3-Hmethyf{-1H-ovracol--HohenyDpyeidezine) orlainate from : the 1 generation of soreening were preliminary tested on acute Tost leukemia Jurkat cells, In : confinuation five compounds of the 277 generation of screening inhibitor Ne, ZB (NGIS- Ë {mathanesuffonamidos-t 3.4 thiadiazal-2yil-8, 7 & Gtetrahvdro-SH-carbazole-3-cerboxamide}, inhibitor :

Na. 29 {3-{4-chinrophenyiisuifonyl-N-{S-soxazol-3-yi-1,3 d-madiazob-2-yhipropangmide). inhibitor No. 20 : {$3 {Saraino-1.3 d-0xadiazol-2y{itoxazol-5-Aphenol, inhiblior No. 31 {ZRMN-{Gasoxkanal Bei 1.3.4- : oxadiezoi-2-y1}-2,3-dihydro-1 4-henzodinxiné-2-cerboximide) and inhibitor No. 32 (3-{benzenesulfonyl}- :

N-5-isquazol-5-44.1,3 d-oxadiszobIwhpropanamide) were primarily tested on coloreciat adenocarcinoma Ë cells (Cace-2}, matanonia (Calo 825) aoû metastatic breast cancer cells (MDA-ME-251) More detailed : analyses of inhibitfons showed dose dependent affects of compounds on reduced lactate generation, vel Ë some differences of thei inhibitory abilities were observed in disfinot tumorigenin call fines, However, later : tests with consecutive addition of siiqueis of a compound to the culls revealed high efficacy of lactate and ;

ROS formation pravention ai low 10 or 20 uM concentrations. :

Based or the inhibitory effect of ail selected compounds on lautale and ROS generation in wmndgenis : cal fines, their effects were ala lesled on cell growth proliferation and cytotoxicity. Despite reduced lactic : ack generation due to treatment of tested iumorigenis cell ines with the above-rmantines inhibitors : similar call numbers and survivals were observed in treated and untreated tumorigenic call fines at the : end of experiments. These data suggested thal minor negative side affects of compounds on the normal : human cells might be expected. : inthe ned step all compounds were tested in vitro Tor inhibitions of tha purified haven native enzyme Ë 28 {98 kDa) and modified cancer specific shorter fagrments (47 ka) of muscle type PREM, followed by : rosasurements of the purified native enzyme (85 kDa) and shorier fragments (70 kh of ver type PRL ; isoform. Boh recombinant human PERM enzymes (Andreic and LegiSa, : doLora/10,1018h.bapap 2048 03.0058) and both recombinant human PERL enzymes were synthesized : and isolaied from the enginenrad yeast & cerevisiae trañsfurmant {Keistl at al, 2021), The compounds :

LU102863 : 38/50 :

No. 3 and 8 were shown jo strongly inhibit the isolated recombinant human modified PERM with Kiss : values of approcimately 15 and 17 ub respectively, Other inhibifors that were selected Io inhibit fiver type :

PFK-L iso-anzyme, the ATP binding site of PRIGL differs in one amine acs in respect to the PEM ATH : bindirug site, also showed stung inhibilions with 10s values belwesn 8-27 URL Characterdstically stronger : inhibitory effects were cbservad in cancer specific shorter PRCA or FFICL fragments in respect io the : native PFK-4 and PFK-L enzymes (ICs beiwern 15 fo >80 pid). :

The inhibitors No. 3 and 9 were tested also by the deferminativn of glycolytie capacities in acute : wmphoblosti leukemia tells édurkat}, For this purpose, Seahorses XF assay (Agilent Technologies) was : performed that enabled non-destructive measurements of extracefhder acidification sats (ECAR) and : oxygen consumption rate (OCR) of tested cells, Untreated Jurkat cells revealed higher ECAR and OCR : values in respect to the cells treated with int, No. 2 or inh, No. 8. These results implisd that inhibRed :

PERT enzymes might efficiently redune glvenlyic flux suggesting that PFICT enzymes might indead be the : target of inhibition. The figures for the above prelindnary experiments are provided in WOR020/020813, : the contents of which is incorporated herein by reference. : 35 The compounds that reduce ROS formation might significantly alter the cancer signalling that drives ; proiifaratis phenotype. Reduced levels of ROS are belleven fn deorense the number of fransformed cells : by depriving them of fuel, Lowered ROS levels also result in & decragse of free radical damage in the : cefis which is linked fo the formation of many degenerative dissases, including cancer, cardiovasouler : diseases, cataracts, and aging {Let a. 2043) :

Gr the other hand, the selected compounds showed negiioibie cytostatic and oytoionie effects on tested : turnorigenie cell lines (Jurkat, Cacn-2, COLO 628, MDA-MB-231) and nan-lumdrigenio cells {such as HEK and normal MOF 104 cells), therefore mild or no side effects on normal cells might be expected, These : results suggest that inhitifors No, 3, 8, 28, 28, 30, 31, 32 significantly reduce lactats and ROS expration : 28 by cancer cells, consequently leading ic higher susceptibility of calls ic immune system, while Cancer : development driven by successive mutations is diminished by the reduced level of highly reactive free : radicals. These seven compounds are thus promising inhibitors In be used in dancer therapy and ; treatment or perhaps some other diseases characterized by deregulated alyooiyic tux Their use, either ; alone, either in any possible combination of inhibitors of the 19 and 8 generation that inhibit differant : 30 human PFK isoenzymes is the prefered ambodiresnt of the invention, as combinations of inhibitors of the : and 27 generation could inbibit all three types of tha cancer specific modified PFKT enzymes. it has : beer observed that inhibitor efficiency decreases with time, bence the seleciad inhibitor ar thelr ; combination is preferably administered in regular time inlarvals fo ensure permanent proper dosage. The ; results shown in the examples below sanfim success of such interval reaimant, as lactate and ROS generation has been successfully inhibited with fow inhibitor concentrations, Desoribed inhitligrs can aise : be used in combination with offer anticancer treatments and/or medicaments in any possibile way : depending on interactions batwaen compounds. Such use may be aimuflansous or sequential :

The present invention relates to use of the atove-mentioned compounds as medicamants to reduce ROE ; and SOX generation by the cancer cells, thereby treating cancer. The compounds of the vention are : especialy useful in the tmalmant of the early stages of cancer, :

The compounds may be admiiistered with Ihe aim of draatically reducing ROS and SOX generation by : the cancer cells that lose proffferating signalling while the DNA damages that enable cancer progression : are diminished, His also possible to use any pharmaceutically suflable Salt, solvate, Sfructural analogue : and/or dedeaiive of inhibitors No. 3. 8, 23, 25, 30, 31 and 32 which retain the capebility of dcoking into the :

ATR binding site of PFK-1 and show inhibition of ovaraclive cancer specific PEK enzymes, The : expression derivative in this application is considered to be 2 compound thal is derfved from inhibitors Na. ; 3.9, 33,29 30, 2%, and 32 by a chemical reaction, The expression derivative in also considere to cover : structural analogues of the said inhibitors, :

Pharmaceutical compositions :

Tha compounds may be provided in a pharmaceutical composition. : 45 Pharmaceutical compositions may be prapared using a pharmaneutically acceptable "carre" composed : of materials that are considered safe and affective. “Pharmacaudically asceptatie” refers to moipculer ; entities and compositions that are "generally regarded as safe”, 2.9, That are physiologically tolerable and do not typical produce an allergic of similar untoward reaction, such as gastric upset ard the Ike, when administered to a human. In some ernbodiments, this term refers io molecular antilles and compositions ; approved by a regulafory agency of the US federal ar a slate goverment, as the GRAS fist under section : 204 (a) and 409 of the Feveral Food, Drug and Cosmetic Act, that is subject to pramarket review and , approval by the FDA or similar fists, the LLS. Phasnacopela or another generally recognized : pharmiacopeia for use in animals, and more parlicularly in humans, :

The tem “carrier” refers to diiuents, binders, libricants and disintegranta, Those with skill in the art are : 28 familiar with such pharmaceutical carriers sod methods of compounding phaomacautivel covpusiions :

USING such caroes., :

The pharmaceutical compositions provided haralı may include one or more exciplents. 5.8. SONS, : ‚Aolubility snhancers, suspending agents, buffering agents, isoloninity agents, antioxidants or antimicrobial : preservatives. When used, the excipients of the compasitions will not adversely affect the stability, : bieavellability, safely, and/or efficacy of the active ingredients, Le, the compounds described herein used : it the composition. Thus, the skilled person will appreciate that compositions are provided wherein thers : is ne Incompatibility between any of the components of the dosage form, Excipients may be selected : from thé group consisting of buffering sgenis, solubilizing agents, lonicity agents, chelating agents, ; ariiaxidants, artinicroblal agents, and preservativas, :

I may be nonverdent or desirable to prepare, pay, and/or handle a corresponding sait of the active : compound, for example, a phammaceufionii-anceptable sait. Examples of pharmaceutically acceptable : salts are discussed in Berge of al, 1977, "Fharmaceulically Acceptable Salts,” J. Pharm, Bol, Vol. 68, pp. ; 118, :

Far example, # ihe compound is anionic, or has a functions! group which may be anlonic {8.g., OCR : i tha compound is cationic, or has a functional group which may be cationic {8.0 NH may be NH" ; then à sait may be formed with a suitable anion. Examples of seilable inorganic anions include, but are : hot limited te, those derived fram the following romanic acide: hydrochiorie, hydrobromie, hydeoiondis, ; sulfuric, sufurous, ndlag, nitrous, phosphoric, and phosphoraus, :

Examples of suitable organic anions include, but are not fimited te, those derived from the following ; organic acids: S-acelyoxybenzois, acelic, ascorbic, aspartic, barzote, campharsulfonio, CINNamic, citric, ; edétic, eihanedisuifonie, athanesulfonie, fumarie, glusheptonic, gluconie, glutamic, giventic, : hydronymeadaic, hydmanaphthalens carboxylic, isethionic, lactic, lactabienie, laurio, maleic, malin, : methanesulfonic, mucis, oleic, oxalic, palmitio, pamoic, pantothenic, phenylacetic, phenylautonie, ; {5 pronionis, pyruvic, salisylic, stearic. succinic, suifaniiic, tartare, ivfuenesulfonic, and valerie. Examples of :

PER y : suitable polymeric organic anions include, but are not limited to, those darives Fam the following ; polymads acids: anne sold, carboxymethyl Celluidse, :

Liniess otherwise specified, a referanos io à particular compound also chide sait forme thereal, ;

Soivates : it may be convenient or desiratls io prepare, purify, and/or handle a narrespanding solvate of He active ; compound. The term “solvate” is used harein in the conventional sense 10 refer to à complex of solute : (8.5, aove compound, eat of active compound} and solvent. if the solvent is water, the solvate may be : convenientiy referred to as a hydrate, for axarnple, à mano-hydrate, a di-tivcirate, à bhydate, elo, : 35 Unless otherwise specified, à reference to à paricular compound also include solvate forms thereof, :

Method of treatment :

The compos described herein, or their phammaceutically acceptable salts, solvates or derivatives à thereof, are suitadie for use in a method of treatment of the early stages of cancer developreant, Such as : dysplasia and/or carcinoma in sity. Treatment in fds corded may be considered prevention of cancer Ë development or inhibition of cancer development. The treatment may also be prevention of cancer : development during early siages of invasive cancer grogmesion. Ë

The compounds described herein, or their pharmaceutically acceptable saffs, solvaiss or dethvatives : thereof, are inhibitors of §-ghosphofracie-{-kinase (FEKT) enzyme. Sultabiy the compounds described : herein, or their pharmaceutioally acceptable sale, solvates or derivatives theraof, are also suitable For use ; in a method of reairment of a disease wherein the modulation or signalling of PFRT is implicated. :

LU102863 : 24/30 :

Without wishing to be bound by theory, the compounds desoribed herein, or their pharmaceutically : achantuble sais, solvates or derivatives thereet, inhibit PFKT to decrease reactive oxygen Species (ROS) : generation andior superadde anion (SOX) generation by calls that ars precursurs fo cancer, The : reduction in ROBISON generation reduces DNA damage and mutations that initiate and promote cancer ; development This reduction in ROS/SOX generation may be used as a prophylactic treatment against : cancer for cancer prevention, : tn some embodiments. the cells that are precursors to cancer, such as calls in dysplasia and/or : carcinoma in situ, may comprise of acute lymphoblastic leukemia cells (Jurkaf}, colorectal adenoma Ë carcinoma cancer cells {Cane-2}, melanoma cancer alls {Tole 838), or marmrmary/breast Cancer cells : (MUA-MB-2374 : # will be understood that cancer development is divided in is two phases: cancer progression and cancer Ë or invasive cancer. Therapy in the first phase (Stage 0) is typically designated as cancer prevention while : the therapy for invasive cancers is typically designated as cancer reaiment, Without wishing fo be bound : by theory, tha inventors believe that because the inhibitors of fhe present inventions should prevent : 35 formation of raw mutations only, Hey can be used for the wrevention of cancer development during early : stages of invasive cancer progression, Ë in some embodiments, the compounds describe herein, or their pharmaceutically acceptable sais, Ë sotvates or derivatives hereof, are administerad sequential at regular time intervals, in some Ë emibodimants, the regular time inierval is very 10 to 48 hours, In some embodiments, the regular ime : aterealis every 18 to 24 hours, In some srebadiments, the regular me interval is avery 24 hours. Ë in other words, in some embodiments the administration is once per day {QD :

Combination therapies :

The compounds described hetein, or thelr pharmassutically acceplable salls, soivaies or derivatives : theeaof, are suitable for use in a method of treatment wherein he compound is used alone or in any : combination iheract. :

The compounds described herein, of their pharmaceulically aocepiable salts. solvates or derivatives : fhereof, are suitable for use in a method of treatment in combination with other anticancer treatments Ë aration medicaments, : avr : es Ï

The features disclosed in tha faragaine description, or in the following CREME, or In the accompanying Ë drawings, expressed in their spécifie forme or In terms of a means for pedurming the disciosed function, : or a method of process for shtaining the disclosed results, as appropriate, may, separalely, Or in aay Ë combination of such features, be utiised for realising the invention in diverse forms thereof Ê

While Ihe invention has been described in conjunction with the exemplary erpbodimants descrihed above, : many equivalent modifications and variations will be apparent to those skilled in the arf when given this :

LU102863 i

QUA : discioeurs. Accordingiy, the exsmplary ernbodiments of the Invention set forth above arp considered to be :

Hhasirative and not firming. Various changes to the described embodiments may be made without : departing from the spirit and scope of the invention, :

For tha avoidance of any doubt, any theoretical explanations provided hersin are provided for the : purposes of improving the understanding of a reader. The Inventors do not wish to be bound by any of : these theoretical explanations, :

Any section headings used herein ave for organizations! purposes onfy and are not fo be construed as : finiting the subject matier describes. Ë

Throughout this spécification, including the claims which follow, unless the context requires otherwise, the : 36 word “comprise” and include”, and variations such as “comprises”, “comprising”, and “including” will be : undersicod to imply the inclusion of a stated integer or step ne group of infegers or steps but nat the : exclusion of any other integer pr sieg or group of infegers or steps. : it must be notad that, as used in the specification and the appended claims, fie singular forms "a" “an,” : ang “the” include plural referents unless the context clearly dictates otherwise, Ranges may ba exprassad Ë iS herein as from “shoul” one particular value, and/or to “about” ancther partiouler value, When such a range : is expressed, another embodiment includes from the one particular value and/or to the other partinuler Ë value. Similarly, when values are expressed as approximations, by the use of the antecedent “about” it : will be pndersivod that the particular value forms another enbodiment. The term “about” in relationio a : nurerioad value is options! and means for examipie + 10% :

Examples :

EXAMPLE 1 :

Al smali-moiecule inhibliore listed inn the description were previously tested for suppressing lactais : generation by the identical timorigenic call nes and under He essentially identical growth conditions : {POT/EP201V/0T105R, WO 2020/030613) as used in the present experiments, :

PFK-P inhibitors were purchased from Engraihe (Kylv, Ukraine) {inhibitor No, 1-8 in Table 23, Ë

ChemBridge {Ban Diego, CA, USA) (Inhibitor No. 9-16 in Table 2) and ChemDiv San Diago, CA, USA) : trihititer No. 17 and 18 in Table 2), 5

PFKL inhibitors were purchased from Enamine (Kyiv, Ukraine) {inhibitor No, 21.28 in Tabie 3} and :

ChemBridge {San Diego, CA, USA) (inhibitor No. 25-36 in Table 3). :

All purchased compounds wars supplied as 5 mg lyophilized powder and ware diseolved in DMEO to the : final concentrations of 10 mi, ; 38 :

AH the compounds were preliminary {ested for the prevention of Reactive Oxygen Species (ROE) and :

Superaiide (SOX) generation by acute lymphoblastic leukemia cells (Jurkat}, colorectal adenoma [ carcinoma oells (Caco-23, melanoma cells (Colo 829), and manimeary/breast cancer cells (MOA-MS-Z37) :

Aff lumorigente call fines were grown it the RPMI 1840 GlutaMAX medium (Theme Fisher) :

E supplemeriad with 10% of feta! bovine serum (FEE) The cells were incubated in. 4 mé of medium (38 : weit plates) at 5% CO; at 37°C Individuel Inhibilos was fret added to the medium in 10 uM concentration at 8 hours after inanudetion, followed by sequential ré-ingertion of inhibitor {10 pM} at 24 hours Iniervels, if : not spectied differently. As à control, equivalent amount of DME, as used for the addition of inhibitors, was supplementes La he medium, The tests started with 1x 105 calls per mb. For the SCAROS : 30 measurements the cells ware washed by senttifugation after 72 hours of incubation with sequeriibal : addian of inhibitor and immersed into the 500 pl. MEME medium supplemented with adequate : concentration of inhibitor for 3 hours. For the detection of ROS and SOX lavels in the cells, 100 ut of : medium with cells was placed inl à wall on the 36 well black waïtiear bottom plate and 100 pl ROS : detection reagent (Green) or SOX detection reagent (Orange) was added as specified by the Cellular : 18 ROSSuperoxide Detection Assay Kit {ab136478 Abcarn, Cambridge, UK), Relative Suorescence was : measured by micro plate reader (Biotek, Vermont, USA} using Exim 400/626 nm for the Green and ;

ExEm 550/620 for the Orange reagent. For the positive control (K+) ROS inducer Pyouyanin was used : arıd for magative (K-} ROS inhibitor N-acetybL-cysteine according fo the manufachrers specifications, faysevar, positive control sould not be applied for assessing maximal SOX Formation in the cells. : 359 As shown previously, partial inhibition of the modified cancer spacifis PRR enzymes by smalh-moiecule : inhibitors led to reduced NADH formation in the cancer cells. Consequently, there is ni need for rapid re : ésidation of redundant NADH by reducing pyruvate into lactale and is excretion out of the cancer cells. :

However, in the untesaied cancer cells part of abundant NADH enter mitochondsal compartment Jhrough : the aspariate-malate shuttle. In mitochondrial matrix NADH re-oxidised io NAD" while électrons enter : electron transfer pathway and contribute te the superoxide {SOX} formation al the Cytochrome | and :

Cévohrome Hl eveis, SOX is then tramsporied into cytoeot and reduced to more stable other raactive : oxygen specias (ROS) superoxids dismutase and related enzymes. Hycothetinaiy, the inhitilion of : modified cancer spenifis FFKT snaymes that prevents the lactate generation in cancer cells might restrici :

HOS and BOK femmationt as weil, : 39 To condi theelect of PEKT inhibition on reduction of cellular ROS and SOX levels in tumorigenic cells : al available Inhibitors tested before for reducing lactate generation were texted also for reducing ROS : and SOX levels. The growth of wnorigenic cell fines and reeasuremends of ROS/SQX were perfomaed : essentially as described above. : in Jurkat cells relatively low ROS levels were recorded where the actual level of ROB in untreated cells : (KO) was similar to that of the calls salad ty N-acelyl-L-oysteine (K-} the substances known as @ song :

ROS shinier, Inhibitors No, 25, 29 and 30 reduced ROS formation most efficiently (Figure 2a}, However, : strong reduction of the SOX formation was detected by seven inhibitors out of 32 tested. Indesd, the ; sirongest inhibition was observed by Inhibitors No, 3, 9, 23, while Inhibitors No, 28, 30, 31, 32 showed ; jess strong inhibition but some inhibition could be observed also by the inh. No, 16 and 18 (Figure 2h), :

LU102863 :

S4/30 :

These inhibitors were previously selacind as promising ala for he prevanlıan of lactate gonstation and : are marken with dark colour on all further diagrams. :

By testing Caco-2 calls with idenlical inhibitors again Inh, No, 23 reduced the level of HOS nicaly and : some reduction was observad slap with Inh, Ne, 31, 36 and 36 (Figure 3a). Again, selected inhibitors : § were more efficient for suppressing SOX formation in Caco-Z cells, All inhibitors that proved previously to : be efficient laciaia suppressors showed strong reduction of SGX formation, Beaides, Inh, No. 35 and 38 : proved similarly efficiency (Figure 3b}. :

Unfortunately, none of ssiscted compounds showed remarkable Inhibitory effect on ROS formation in :

Cole 629 cells, Again, the basic level (KO) of ROS proved to be vary low in untreated ces {Figure 43). ;

However, Inh. No, 3,9, and 23 reduced SOX formation mors efficiently in Cold 829 cells, with Inh, No, 3 ; showing the strongest inhibilor (Figure 48), ; inhibitors Mo, 23, 28 and 38 were the only inhibitors to show substaniial reduction of ROS In MDA-MD. 3 231 sells (Figure 5a) and the same inhibitors were efficient alse for reducing SOX levels. However, SOX : formation was nicely suppressed gen by the Inh. 3, ©, 15, 2% and 31 in the MUAME-231 cells (Figure :

Sh) ;

Tu conclude, he inhibitors No, 3, 9, 23, 28, 30, 31, and 32 that were pravipusly recogrizsd as strong Ë

PRR infibitors thal revente lactate generation in selected tumorigenic calla, proved Io be among the : most effective compounds ala for the reducing of ROS/SD in lumarigenic cells. :

À decrease of ROS and SOX levels was observed ina dose dependent manner by Inh, Ne, Sir Jurkat Ë cells, The exporimert was esserdiafly conducted as described in Example 1 however, different : concentrations of an inhibitor were added to the sells sequentially ai 28 hours intervals. After 72 hours of ê incubation the lavet of BUS and SON were determined. Total ROS values were reduced by half at very : 25 tow dS uM concentration of iniibiter while 90% inhibition was reached at 5 ul concentration, Al higher : concentrations of inh, No, 3, the levels similar to that recorded in the Jurkat celia treated with N-acetyt-L- : cysisige (K-), were observed. For reducing the levels of mitochondrial SQX, slightly Higher ini, Na. 2 : concentrations were needed, Virkselly no inhibition could be delaclad at 2.8 uM concentration while ; approxieately 30% reduction was observed with 5 uM concentration, However, nearly complete inhibition : was detected at 10 uM or higher concentrations, As à vehicle untreated Jurkat ces were incubated at : idertioat conditions as the cells treated with Inh, No, 3 with sequentially addifions of cormresponding DMSO Ë amounts {KO} {Figure 8), As a positive controi {K+} the Jurkat celle were Yrealad by Pyocyanin, a strong :

ROS but rot SOX inducer, therefore the positive control was determined anly at the ROS tesis. Ê

In Jurkat cols all inhibitors that were previously shown fo suppress isolate generation, were testad for : reducing ROS ang SOX levels, Again, the cells were incubated for 3 days with consequential addition of Ê spenified inbibitors ai different concentrations. The analyses have shown slightly different reduction : strength of ROE formation by specific inhibilors, The most potent inhibition was observed by Inh, No. 38 whinh effectively reduced ROS formation already at low concantration (5 pb) ROE iofréations wars : proficientiy reduced also by Inh. No. 3 and Ne, ©. Although ihe reduced ROE levels wera observed also : by resting the Jurkat cells in a dose dependent way by other Inhibitors (Ne. 23, 20, 31, 32), thelr : inhibition seamed to be less powerful, Sirdar resulis ware obtained by measuring SOX levels in Jurkat : cells treated by the same inhibitors. Again Inh, Ne, 20 effectively reduces SOX formation af low : concentrations and similar results were obtained by Inh. No. Sand 8. À strong prevention of SOX : generation was observed also by other inhibitors (No, 23, 29, 31, 32) yat higher concentrations : 19 {10-20 uM) were needed (Figure 7). :

EXAMPLE 4 :

Three most powerful inbibitors that supprassad ROS and SOX generation in Jurkat cells were tested also ; an colorectal adenoma carcinoma peffs {Tace-2), melanoma cells (Cole 826), and mammany/braast : cancer sells (MDA-ME-231) All wee Inhibitors No. 3, 8 and 30 nicely reduced ROE formation by Caco-2 : tumeriganie cote, About 50% reduction of ROS was obssrved with 5 gM concentrations of all three : inhibitors while at 10 ub concentration the same levels of ROS ware observed in the cells as with the celts treated with à strong inhibitor (N-acetylcysteine). Similar inhitlicay effects of selected inhibitors : were racorded by measuring reduction of SOX generation in Cacc-2 cells, However, tha most potent inhibiter proved to be Inh, No, 9 thaf reduced SOX production for approximately 70% at & uM : concentration, Other two inhibiters reached the same effectiveness at 10 uM while complete Inhibition : was observed at 20 ub concentration {Figure 8}. :

By testing melangma Coin 888 calls, ayaln Ihe Inh. No. 5 proved to be very efficient In preventing ROS : and BOX formation, However, Inh, No, 30 also showed à strong ability of ROS reduction while nh, No. 3 : appeared to be equaliy effective inhibitor of SOX formation as the other BWG inhibitors. Again 10 gM ; concentration of ail inhibitors proved io be sufficient for substantial diminish of ROS and SOX formation in :

Code 828 cells if supplied sequentially at 24 hours intervals (Figure 8), :

No significant differences were observed among three inhibilgra in thelr abilities fo repress SCX formation : by MDA-MB-23.1 calle. AN three Inhibiions showed distinet dose dependent effectiveness in reducing SOX, ;

Similar resuls have been obtained by tasting ROS formation however, ini, No. 8 showed slightly stronger : ability to reduce ROB generation than the other ve inhibitors in this particular fumorigenic cell ine ; (Figure 10} 3

To conclude, all three inhibitors prove io be potent inhitilors in the variety of differant lumorigenic cote : that originate from different human tumors. Nevertheless, also the remaining inhibitors No, 23, 39, 31, : and 32 that shower significant ability for raducing ROS and SOX formation in tha cancer cells, might ; prove io be sucoosafui drugs for preventing the formation of highly reactive mnlecules in the cancer cells ;

LU102863 : 25/30 :

References

A number of publications are oiled above in order to more fully describe and disclose the Ivantiorr and : the state of the art fo which the wention pertains. Fui calins for these references are provided below, :

The entirety of each of these referenons is Incorporated herein, :

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Claims (10)

Claims:

1. A compound selected from the group consisting of: (2-[(5-benzo[1,3]dioxol-5-yl-t,3,4-oxadiazol-2-yl)sulfanyl]-N-isoxazol-3-yl-acetamide} (Inhibitor No, 3); (3-methoxy-6-(3-{1-[(5-methyl-1,2 4-oxadiazol-3-yl)methyi]- 1H-pyrazol-3-yl}phenyl)pyridazine) {Inhibitor No. 9); {4-[3-(5-amino-1,3,4-oxadiazol-2-yl)isoxazol-5-yl}phenol) (Inhibitor No. 30); {N-[5-(methanesuffonamido)-1,3,4-thiadiazoi-2-yi]-6,7,8,9-tetrahydro-5H-carbazole-3- carboxamide) {Inhibitor No. 23); (3-(4-chlorophenyl)sulfonyl-N-(5-isoxazoi-5-yl-1,3,4-oxadiazol-2-yl}propanamide) (inhibitor

No. 29}; ((2R)-N-(5-isoxazol-5-yl-1,3,4-oxadiazol-2-yl)-2,3-dihydro-1,4-benzodioxine-2-carboxamide) (inhibitor No. 31}; and (3-(benzenesulfonyl)-N-{5-isoxazol-5-yl-1,3,4-oxadiazol-2-yl)propanamide) {Inhibitor No. 32}; or a pharmaceutically acceptable salt, solvate or derivative thereof, for use in a method of treatment of a cancer, dysplasia and/or carcinoma in situ through inhibition of ROS generation in the cells of the neoplasm associated with said cancer, dysplasia and/or carcinoma in situ.

2. À compound seiected from the group consisting of: {2-[(5-benzo{1,3]dioxoi-5-yt-1,3,4-oxadiazol-2-yl}sulfanyl}-N-isoxazol-3-yl-acetamide) {inhibitor No. 3); ; (3-methoxy-6-(3-{1-[{5-methyl-1,2,4-oxadiazol-3-yi)methyl]- 1H-pyrazol-3-yl}phenyl)pyridazine) (Inhibitor No. 9}; ; (4-[3-(5-amino-1,3,4-oxadiazol-2-yhisoxazol-5-yl|phenot} (Inhibitor No, 30} ; (N-[5-(methanesulfonamido)-1,3,4-thiadiazol-2-yl}-6,7,8,9-tetrahydro-5SH-carbazole-3- carboxamide) (Inhibitor No. 23); : (3-(4-chlorophenyl)sulfonyi-N-(5-isoxazol-5-yi-1,3 4-oxadiazol-2-yl)propanamide) {inhibitor

No. 29); ((2R)-N-{5-isoxazoi-5-yl-1,3,4-oxadiazol-2-yl)-2,3-dihydro-1,4-benzodioxine-2-carboxamide) : (inhibitor No. 31); and } (3-(benzenesulfonyl)-N-(5-isoxazol-5-yi-1,3 4-oxadiazol-2-yipropanamide} (Inhibitor No. 32}; ; or a pharmaceutically acceptable salts, solvate or derivative thereof, for use in a method of treatment of dysplasia and/or carcinoma in situ. ;

3. The compound for use according to claim 1 or claim 2, selected from the group consisting of: ; (2-[(5-benzoft,3]dioxol-5-yl-1,3,4-oxadiazol-2-yl)sulfanyl}-N-isoxazol-3-yl-acetamide) ; (Inhibitor No. 3); {3-methoxy-6-(3-{1-[(5-methyl-1,2,4-0xadiazol-3-yiimethyll- 1H-pyrazol-3-yl}phenyl}pyridazine) ; (inhibitor No. 9); ; (4-[3-{5-amino-1,3,4-oxadiazol-2-yl)isoxazol-5-yl}phenol) (Inhibitor No. 30); ;

(3-(4-chlorophenyl)sulfonyI-N-(5-isoxazol-5-yl-1,3,4-oxadiazol-2-yl)propanamide) {Inhibitor

No. 29); ((2R)-N-(5-isoxazol-5-yl-1,3,4-oxadiazol-2-yl)}-2,3-dihydro-1,4-benzodioxine-2-carboxamide) (Inhibitor No. 31%; and (3-(benzenesulfonyl)-N-(5-Isoxazol-5-yl-1,3,4-oxadiazol-2-y)propanamide) (Inhibitor No. 32}; or a pharmaceutically acceptabie salts, solvate or derivative thereof.

4. The compound for use according to claim 1 or 2, selected from the group consisting of: {2-[(5-benzo[1,3]dioxol-5-yi-1,3,4-oxadiazol-2-yl)sulfanyl}-N-isoxazol-3-yl-acetamide) {Inhibitor No. 3}; (4-[3-(5-amino-1,3,4-oxadiazol-2-yl)isoxazol-5-yljphenol) {Inhibitor No. 30); (3-(4-chiorophenyl)sulfonyl-N-(5-isoxazol-5-yl-1,3,4-oxadiazol-2-y!)propanamide} {Inhibitor

No. 29}; ((2R)-N-(5-isoxazol-5-yt-1,3,4-oxadiazol-2-y1)-2,3-dihydro-1,4-benzodioxine-2-carboxamide} (Inhibitor No. 31); and (3-(benzenesulfonyl)-N-(5-isoxazol-5-yl-t,3,4-oxadiazol-2-yl)propanarnide) (inhibitor No. 32}; or a pharmaceutically acceptable salts, solvate or derivative thereof.

5. The compound for use according to claim 1 or 2, selected from the group consisting of: (2-[(5-benzof1,3]diox0i-5-y1-1,3 4-0oxadiazol-2-yl)sulfanyl}-N-isoxazol-3-yl-acetamide) {inhibitor No. 3); (3-methoxy-6-(3-{1-[{5-methyl-1,2,4-oxadiazol-3-yl)methyl]- 1H-pyrazol-3-yi}phenyl)pyridazine) (Inhibitor No. 9} and {4-{3-(5-amino-1,3,4-oxadiazol-2-yhisoxazol-5-yl] phenol) {Inhibitor No. 30); or a pharmaceutically acceptable salts, solvate or derivative thereof, :

6. The compound for use according to any one of the preceding claims, or a pharmaceutically acceptable salt, solvate or derivative thereof; wherein the neoplasm comprises acute lymphoblastic : leukemia cells,

7. The compound for use according to any one of claims 1 to 5, or a pharmaceutically acceptable salt, ; solvate or derivative thereof; wherein the neoplasm comprises colorectal adenoma carcinoma cells,

8. The compound for use according to any one of claims 1 to 5, or a pharmaceutically acceptable salt, solvate or derivative thereof; wherein the neoplasm comprises melanoma cells.

9. The compound for use according to any one of claims 1 to 5, or a pharmaceutically acceptable salt, solvate or derivative thereof; wherein the neoplasm comprises mammary/breast cancer cells,

10. The compound for use according to any one of the preceding claims, or a pharmaceutically : acceptable sait, solvate or derivative thereof; wherein the compound is used in combination with other ; anticancer treatments and/or medicaments.