WO2012065139A2 - Entpd5 inhibitors - Google Patents

Abstract

The invention provides methods and pharmaceutical compositions for treating cancer or promoting apoptosis of cancer cells, or reducing the resistance of cancer cells to chemotherapy or apoptosis-promoting therapies by contacting the cancer cells with an ENTPD5-specific inhibitor.

Description

ENTPD5 inhibitors

[001] This work was supported by grants from the National Institutes of Health (NIH Grant SPOICA09S471-09); the Government has certain rights in this invention.

[002] Introduction

[003] Class I Phosphatidylinositol 3-kinases (PDKs) and lipid phophatase PTEN

(Phosphatase and Ten sin homolog deleted from chromosome ten) balance cellular response to growth and survival signals (Reviewed by Engelman et al., 2006). In response to activation of receptor tyrosine kinases, PI3K phosphorylates phosphatidylinositol 4,5- bisphosphate (PIP2) at the 3-OH position of the inositol ring to generate phosphatidylinositol 3,4,5-trisphosphate (PIP3), which recruits phosphatidylinositol-dependent kinase 1 (PDK1), and serine/threonine kinase AKT to the plasma membrane by binding to their pleckstrin homology domains (Whitman et al., 1988; Franke et al., 1997; Alessi et al., 1997). PDK1 further phosphorylates and activates AKT (Stepjens et al., 1998; Stokoe et al., 1998). AKT subsequently phosphorylates many cellular targets including TSC2 (tuberous sclerosis 2) protein, resulting in activation of the rapamycin- sensitive mTOR complex 1 (mTORCl) (Gao et al., 2002; Inoki et al., 2002; Manning et al., 2002; Potter et al., 2002). mTORCl phosphorylates p70S6K and translation initiation factor 4E-BP1 to accelerate the translational rate thus accommodating rapid growth (Brown et al., 1995; Burnett et al., 1998; Fingar et al., 2002). PTEN, by dephosphorylating PIP3 back to PIP2, antagonizes the signal generated by PI3K (Maehama and Dixon, 1998).

[004] The importance of PI3K/PTEN pathway for cell growth and survival has been manifested by frequent PI3K gain of function, or PTEN loss of function, in human cancers. PI3K, in addition to direct activation by receptor tyrosine kinases and Ras, two of the frequently activated oncogene classes, also harbors frequent activating mutations in its catalytic subunit pi 10a, PIK3CA, in a high percentage of gastric, colon, breast, and lung cancers (Chung et al., 1994; Rodriguez- Viciana et al., 1994; Samuels et al., 2004; Reviewed by Yuan and Cantley, 2008). The loss of PTEN has been observed in a variety of human cancers as well with high frequency in endometrial, glioblastoma, prostate, and breast cancers (Li et al., 1997; Liu et al., 1997; Steck et al., 1997;Tashiro et al., 1997; Rasheed et al., 1997; Reviewed by Keniry and Parsons, 2008).

[005] Mouse models of PTEN deletion further support its tumor suppressor function.

Although homozygous deletion of PTEN gene is embryonic lethal, the heterozygous loss of PTEN demonstrates haploinsufficiency, leading to neoplastic changes in many tissues including mammary gland, prostate, thyroid, colon, and lymphatic system (Di Christofano et al., 1998; Stambolic et al., 1998). The embryonic fibroblasts from the PTEN null mice (MEFs) exhibit resistance to a variety of apoptotic stimuli including growth factor withdrawal, heat shock, and UV-irradiation compared to PTEN heterozygous MEFs (Stambolic et al., 1998).

[006] AKT activation also contributes to the elevation of aerobic glycolysis seen in tumor cells, known as the Warburg effect (Rathmell et al., 2003; Elstrom et al., 2004; Warburg, 1925; 1956). AKT promotes cell surface expression of glucose transporters while sustaining activation of hexokinase and phospho-fructose kinase- 1 (PFK1) thus accelerating influx and capture of glucose for glycolysis (Reviewed by Vander Heiden et al, 2009). Interestingly, in cancer cells, there is invariant expression of the embryonic M2 splice version of pyruvate kinase, an enzyme working in the last step of glycolysis (Christofk et al., 2008a). Compared to the Ml splice isoform expressed in most of the adult tissues, the M2 isoform is a more sluggish enzyme that can be directly inhibited by phosphotyrosine, a signal that also activates PI3K/AKT (Christofk et al., 2008b). The combined effects of more glucose entering into glycolysis pathway, and slowing down pyruvate kinase activity build up intermediate metabolites for synthesis of growth-enabling macromolecules. One noticeable example is the entry of glucose-6-phosphate to the pentose shunt pathway to generate ribose for nucleotide synthesis (Reviewed by Vander Heiden et al., 2009).

[007] Another outlet of glucose-6-phosphate is to form UDP-glucose and other nucleotide- conjugated sugars, substrates for protein glycosylation. In mammalian cells, most secreted proteins and membrane proteins including growth factors receptors are glycosylated at the asparagine (Asn) sites of Asn-X-Ser/Thr (where X is any amino acid except proline) consensus sequences (Kornfeld and Kornfeld, 1985). Interestingly, receptors promoting cell growth and proliferation such as the epidermal growth factor receptor, EGFR, are much more highly glycosylated than receptors whose functions do not (Lau et al., 2007). Most of the glycosylation reactions happen in Golgi apparatus with two known exceptions. One is the dolichol-linked 14-sugar core glycan (Glc₃Man₉GlcNAc₂) that is synthesized in cytoplasm and ER membrane before being flipped into the lumen of ER where it is transferred to Asn of nascent polypeptide chain (Reviewed by Helenius and Aebi, 2004). Another is re- glucosylation in ER after the third and second glucose on the core glycan is trimmed by glycosidase I and glycosidase II, respectively. Trimming and re-glucosylation by UDP- Glucose: glycoprotein glucosyltransferase (UGGT) generate monoglucosylated structures that are recognized by calnexin/calreticulin, an ER molecular chaperone system for N- glycosylated proteins (Reviewed by Ellgaard et al., 1999). The removal and addition of glucose allows the binding and release of calnexin/calreticulin to and from nascent polypeptide chains until the proteins are correctly folded and transferred to Golgi for additional glycosylation. If proteins are misfolded beyond repair, they are subjected to degradation by the ER-associated protein degradation system (ERAD) (Reviewed by Fewell et al., 2001).

[008] Our laboratory has been studying mammalian cell apoptotic pathways using biochemical approaches. During our study of why PTEN-null MEFs are more resistant to apoptosis compared to MEFs from a heterozygous littermate, we made a surprising finding that an ER UDP hydrolysis enzyme is responsive to AKT activation. This enzyme, ENTPD5, seems to mediate many of the observed cancer-related phenotypes associated with AKT activation. Here we describe the development of an enzymatic assay to identify small- molecule inhibitors of ENTPD5, and the identification of a variety of such ENTPD5 inhibitors.

[009] Summary of the Invention

[010] ENTPD5 (ectonucleoside triphosphate diphosphohydrolase 5) is highly conserved in vertebrates, being expressed in a broad range of tissues and developmental stages. It hydrolyzes UDP, GDP and 1DP but not any other nucleoside di-, mono- or triphosphates, nor thiamine pyrophosphate. It is likely to promote reglycosylation reactions involved in glycoproteins folding and quality control in the endoplasmic reticulum. Our work has shown Entpd5 contributes to the development of PTEN loss induced invasive prostate cancer, while it is dispensible in normal prostate tissue maintenance. ENTPD5 is indispensable for the survival of AKT active cancer cells, in which fast growth depends on accelerated protein synthesis and subsequent modification/folding in ER. Inhibition of ENTPD5 in these cells results in ER stress, cell growth arrest, and eventually cell death. Accordingly small molecule inhibitors against Entpd5 provide useful anticancer agents.

[011] Aberrant activation of P13KlAkt signaling pathway is frequently observed in human cancers. Several specific inhibitors targeting AKT kinase have since been developed with subnanomolar binding affinity. However, due to the pleiotropic role of AKT in the regulation of cell survival, cell cycle progression and cellular metabolism, the Akt inhibitors induce significant metabolic toxicities, including increase in insulin secretion, malaise and weight loss, that are consistent with abnormalities in glucose metabolism. On the other hand, by targeting a novel PI3K1AKT downstream effector ENTPD5, which promotes cancer cell growth and survival by accelerating protein folding in ER, we can achieve the anti-cancer effect while circumventing the potential systematic metabolic interference. Therefore, inhibition of ENTPD5 represents a previously undescribed approach to treat cancers resulting from the activation of the oncogenic PI3K1 AKT and/or loss of PTEN tumor suppression. Furthermore, the described aureol class of natural products have not been used as cancer therapeutics previously.

[012] The invention provides methods and pharmaceutical compositions for treating cancer or promoting apoptosis of cancer cells, or reducing the resistance of cancer cells to chemotherapy or apoptosis-promoting therapies by contacting the cancer cells with an ENTPD5-specific inhibitor. In particular embodiments the invention provides:

[013] - novel ENTPD5-specific inhibitors comprising a structure of a generic or a specific formula herein, including Table 1 and Figs.1-4, and including sesquiterpenoid hydroquinone and quinone structural derivatives with variations in the stereochemistry of the angular methyl groups or ring architecture, and including tautomers, stereoisomers and

pharmaceutically-acceptable salts thereof; and

[014] - natural products and metabolites and derivatives thereof comprising the structural scaffold of the diterpene aureol (Djura et al., below) that exhibit potent activity against the enzyme target ENTPD5; and

[015] - novel pharmaceutical compositions, optionally in unit dosage form, and comprising a disclosed ENTPD5 inhibitor, including tautomers, stereoisomers and pharmaceutically- acceptable salts thereof, and one or more of the disclosed pharmaceutically acceptable excipients.

[016] The invention also provides methods for inhibiting cancer cell growth in a patient in need thereof comprising administering to the patient an effective amount of a disclosed ENTPD5 inhibitor, including tautomers, stereoisomers and pharmaceutically-acceptable salts thereof; and optionally further comprising the subsequent step of detecting a resultant growth inhibition of the cancer cells; and/or optionally further comprising the antecedent step of determining that the patient has the cancer cells by detecting the same in said patient; and/or optionally further comprising the antecedent step of detecting undesirable or pathogenic ENTPD activity of a sample of the cancer cells of the patient; and/or optionally further comprising the subsequent step of detecting ENTPD activity of a sample of the cancer cells of the patient.

[017] The invention also provides: [018] -methods of detecting ENTPD5 activity comprising a disclosed assay, and optionally further comprising the step of establishing a correlation between cell type, state, status or condition and undesirable or pathogenic ENTPD5 activity, particularly detecting ENTPD5 activity comprising coupling ENTPD5 catalyzed UDP hydrolysis to UMP Kinase catalyzed UMP phosphorylation to form a futile cycle of UMP/UDP interconversion wherein one molecule of ATP being consumed in each hydrolysis/ phosphorylation cycle;

[019] -disclosed assays for ENTPD5 inhibitors;

[020] -methods of identifying ENTPD5 inhibitors comprising use of a disclosed assay; and

[021] -methods of treating a disease associated with undesirable or pathogenic ENTPD5 activity comprising the step of treating a so afflicted person with a disclosed ENTPD5 inhibitor.

[022] Cells target by the inhibitors present undesirable or pathogenic ENTPT5 UDP/GDP hydrolase activity activity, and the methods optionally comprise the step of detecting or diagnosing said undesirable or pathogenic ENTPT5 activity, which step may be performed directly by measuring said activity a sample of the cells, indirectly by measuring an indicator of said activity of a sample of the cells, or inferentially by ascertaining an indicator, such as a disease type, correlated with said activity of the cells.

[023] The inhibitors inhibit ENTPD5 UDP/GDP hydrolase activity, such as determined in the disclosed biochemical assays. Preferred inhibitors do not inhibit the function of the mt- PCPH oncoprotein (e.g. ViUar et al., Cancer Res 2009; 69(1) Jan 2009, 102-110), which does not have UDP/GDP hydrolase activity.

[024] The subject methods may comprise, consist of, or consist essentially of, the recited material and steps. Inventions consisting essentially of recited material or steps are limited to the recited materials or steps and those that do not materially affect the basic and novel characteristic(s) of he claimed invention, i.e. the disclosed ENTPD5-inhibitory compounds and their formulation in compositions and use as therapeutics.

[025] Brief Description of the Drawings

[026] Figure 1: (a) Structural clustering of Confirmed Inhibitors; (b) Class II core structure and its resemblance to ENTPD5 substrate.

[027] Figure 2: Sesquiterpene hydroquinones natural products: (a) Natural product inhibitor Entpd5 inhibition; (b) Fractions A8-A10 and A12-15 Entpd5 inhibition; (c) Dose response of natural inhibitors; (d) Dose response of pure compound; (e) Sesquiterpene hydroquinones inhibitor structures. [028] Figure 3. Synthetic ENTPD5 Inhibitors induce ER stress in PTEN -/- MEF Cells

[029] Figure 4. ER stress induction correlates with in vitro potency of natural inhibitors

[030] Detailed Description of Specific Embodiments of the Invention

[031] HTS assay development and optimization

[032] Given the importance of ENTPD5 in tumorigenesis, we decided to employ high volume biochemical screening method to identify its inhibitors. We found that insect cell expressed recombinant ENTPD5 specifically hydrolyze UDP and GDP in vitro. Our initial design of primary screen assay was to directly measure inorganic phosphate (Pi) released from UDP hydrolysis by ENTPD5 using a colorimetric malachite green procedure (Baykov et al., 1988); however, this format proved unworkable.

[033] So we designed a new assay by coupling the ENTPD5 catalyzed UDP hydrolysis to the UMP Kinase catalyzed UMP phosphorylation. These two reactions form a futile cycle of UMP/UDP interconversion, and the net result is one molecule of ATP being consumed in each hydrolysis/ phosphorylation cycle. Inhibition of either enzyme will result in an increase of residual ATP at the end of the reaction, which can be readily detected by well established HTS compatible lucif erase assay. The luminescence readout not only improves sensitivity and signal/noise ratio of the assay, but is also insusceptible to the influence of compound color. Design considerations include: 1) in order to enrich ENTPD5 inhibitors, we ensure that ENTPD5 is the rate limiting enzyme in the coupled reaction; and 2) counter screens are used to filter out UMP Kinase inhibitors and non-specific inhibitors from primary hit list.

[034] To implement this idea, we first cloned the UMP kinase gene pyrH, from

enterobacteria Escherichia coli genome, which encodes a peptide of 239 amino acids, into pET28a expression vector with N-terminal polyhistidine (6xHis) tag. The E. coli UMP Kinases (UMPKeco) are hexamers regulated by GTP (allosteric activator) and UTP

(inhibitor), and have no homologs in eukaryotes (Serina et al., 1995). We introduced a D159N mutation by site directed mutagenesis on UMPKeco to increase its solubility at neutral pH (Serina et al., 1996). The pET28a-UMPKecoD159N construct was transformed into competence E. coli BL21 (DE3). After induction by IPTG for 4hr at 37°C, the recombinant UMPKeco D159N protein was extracted from the bacteria and purified using Nickel beads.

[035] To test whether the coupled reaction could indeed happen in vitro, we titrated the recombinant human ENTPD5 protein, expressed and purified from insect cell, into a mixture of 18ng of UMPKeco recombinant protein, 50μΜ of UMP and 25μΜ of GTP in a total 40μ1 volume to produce the Enzyme Mixture. The reaction was started by adding in ΙΟμΙ Substrate Solution containing 500μΜ ATP. After incubation at 37°C for one hour, reaction was stopped by adding ΙΟμΙ of luciferase based Cell Titer Glo reagent into each well to quantify the residual ATP. The data indicate that 3ng of recombinant ENTPD5 protein is enough to consume about 90% ATP in the reaction, creating a robust 10-fold readout window to assess inhibition. Further increasing recombinant ENTPD5 compromised the sensitivity of the coupled reaction.

[036] A second important variable is UMP concentration. In the coupled reaction, UMP is converted by UMPKeco into UDP, the direct substrate of ENTPD5. To determine the optimal concentration range of UMP, we then titrated UMP into the Enzyme Mixture containing 3ng ENTPD5, 18ng UMPKeco and 25μΜ GTP. Reaction is started by adding ΙΟμΙ Substrate Solution and residual ATP was measured after incubation at 37°C for indicated time. After 90 min incubation at 37°C, 33μΜ UMP could already provide satisfactory reaction speed. Since the final concentration of library compound will be 5μΜ, higher UMP concentration is not advisable, because competitive inhibitors may be missed out if excessive substrate molecules are available to ultimately displace them. The UMP concentration in Enzyme Mixture was fixed at 25μΜ.

[037] We then went on to test whether ENTPD5 is the rate limiting enzyme in the coupled reaction. Due to lack of published ENTPD5 inhibitors, we have to use enzyme dilutions to mimic inhibition. We diluted both ENTPD5 and UMPKeco by 3 fold to mimic 66%

inhibition of the enzymes, and the luminescence reading readily reflected this inhibition. And then we dilute ENTPD5 or UMPKeco respectively by 3 fold, to see which one is rate-limiting. The data shows that ENTPD5 dilution accounts for the luminescence reading increase of the double enzyme dilution. This means that UMPKeco is in excess and the assay is biased to preferentially pick up ENTPD5 inhibitors.

[038] To accommodate as many plates as possible in each four-hour screen session assigned by HTS laboratory, and to simplify the incubation condition, we finally optimized the screen assay to perform at room temperature for 3 hours. Hence our primary assay protocol comprised steps: dispense Enzyme Mixture 40ul/well ... add 0.5ul compound

(0.5mM) in DMSO ... (incubate for 5 min RT) ... add substrate solution lOul ... (incubate for 180 min RT) ... add lOul cell titer glo ... (shake for 5 min RT) ... read plates with luminometer.

[039] Counter screen design

[040] In order to filter out potential false positive hits from primary screen result, we split the coupled reaction apart, as secondary (counter screen) UMPKeco assay and tertiary (confirmatory) malachite green assay. The secondary assay is based on UMPKeco catalyzed UMP phosphorylation, during which ATP will be consumed. Secondary Enzyme Mixture consists of 18ng UMPKeco protein, 25μΜ GTP (allosteric activator), and 500μΜ of UMP in a total 40μ1 volume. Assay was started by adding ΙΟμΙ of 250μΜ ATP into the Enzyme Mixture. After one hour incubation at room temperature, reaction was stopped by ΙΟμΙ Cell Titer Glo reagent, and residual ATP was quantified 5 minutes later by luminometer.

[041] For the tertiary assay, the Enzyme Mixture contains 3ng ENTPD5 protein in 40μ1 buffer, and the reaction was started by addition of ΙΟμΙ of 500μΜ UDP. After one hour incubation at room temperature, the released inorganic phosphate was quantified by malachite green reagent.

[042] We rehearsed the optimized primary screen assay on a chemical library at National Institute of Biological Sciences (NIBS, Beijing, CHINA), and got a handful of hits. Our top hit, cmpd4, did not inhibit UMPKeco in secondary assay, but completely inhibited ENTPD5 as indicated by tertiary malachite green assay. Such results of secondary and tertiary assays combined form a profile for each primary hits, based on which one can tell whether a particular primary hits is a specific inhibitor, a false positive inhibitor, or non-specific inhibitor. Cmpd4 clearly exemplified a true inhibitor of ENTPD5 and later served as positive compound control in our UT Southwestern HTS library screen.

[043] The robustness and reproducibility of all three assays was examined quantitatively using a simple statistic parameter, Z' factor, formulated by Zhang et al (Zhang et ah, 1999). Plate Z' factor reflects the signal/noise ratio of given assay and the consistency of data

[044] quality among plates, and is defined as:

[045] In order to assess the data quality within the sample region of the plate, we also calculated Z'_sampi_ej

[046] where sample wells data are used instead of negative control data to calculate the Z' factor. According to these equations, a perfect noise-free assay yields a Z' of 1.0; assays having values > 0.5 are generally considered robust and suitable for single replicate high- throughput screening. We determined Z' and Z'_sampi_e of all three assays to be around 0.8 in mock plate tests, as being in the "excellent assay" range. During the large scale screen, the raw plate data was batch processed on a daily basis by HTS personnel using PERL (Practical Extraction and Reporting Language) scripts. Those plates with a Z' factor below 0.7 will be discarded and redone on the next day, as a quality control procedure.

[047] Identification of a diverse collection of inhibitors

[048] With the optimized primary assay we screened the 200,000 compounds from UT Southwestern HTS library, consisting of commercially available drug-like small molecule compounds and a marine natural products collection laboratory. Each compound was tested at a 5 μΜ final concentration in singlet along with DMSO control wells in each screening plate.

[049] The activity of each screening compound was calculated as normalized fold increase over negative control average:

Criteria for selecting hits are that sample reading be at least three times standard deviation of the daily sample collection above the daily sample reading average (zScore >= 3), where zScore for each compound was calculated as:

[050] We identified about 1,500 potential hits in the primary screen according to the above criteria. We ranked the hit list by inhibition potency, reflected by NormData, and cherry- picked the top 640 compounds for counter screen confirmation.

[051] The selected 640 compounds were first serial diluted into 1 μΜ, 2.5 μΜ, 5 μΜ and 10 μΜ final concentrations and then tested by primary screen assay, and secondary/tertiary counter screen assays in triplicate respectively. The data collected for each compound was used to construct a dose response profile of this compound in all three screen assays. By analysis these profiles, one can distinguish verified positive hits from false positive and nonspecific inhibitors. With real ENTPD5 inhibitors, one expects to see a dose dependent increase of inhibition in primary assay (increase of residual ATP readings) and in tertiary malachite green assay (decrease of free Pi production), while the activity of coupling enzyme was not affected as indicated in secondary UMPKeco assay. In contrast, false positive inhibitors will give a similar positive primary assay readout, which was due to inhibition of UMPKeco activity (increase of residual ATP readings in secondary assay), but rather than ENTPD5 inhibition, as evidenced by no decrease of free phosphate formation in tertiary assay. At last, the non-specific or promiscuous inhibitors kills both UMPKeco and ENTPD5 activity to generate positive primary assay readout. We eventually verified 160 compounds out of the 640 cherry-picked according to this scheme as true in vitro ENTPD5 inhibitors. We ranked the 160 compounds according to their primary assay readout at 10 μΜ, and assigned them PID# as future identifier.

[052] Structural classification of verified compounds

[053] The 160 verified inhibitors were clustered based on Tanimoto similarity into eight structural classes (Figure la). Each class of compounds shares some distinctive common structural features.

[054] Class II compounds represented about one third of all verified hits. They share a common core structure of uracil-like ring, including barbiturate and 2-thiobarbiturate rings, with 5-Z-olefin bond (Figure lb).

[055] This Z-configuration of the olefin bond seems to be stereo specific for ENTPD5, because E-configuration isomers do exist in the library, but were not picked up by our screen assay. Since the preferred substrate of ENTDP5 is UDP, the highly enriched uracil-like core structure implies that this class of compounds may occupy the catalytic site of ENTPD5 and function as competitive inhibitors. Class Γ compounds share urea or thiourea substructure, which may also compete for uracil ring interacting residuals within the catalytic pocket of ENTPD5.

[056] These structural classes define genera well represented in our screening hits:

[057] The genera include, inter alia, alkyl, aryl and acyl substituents. Preferred substituents represented in the inhibitors are C1-C8 alkyls, such as methyl, ethyl, n-propyl, isopropyl, n- butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl and the like;

preferred acyls include acetyl, propionyl, butyryl, decanoyl, pivaloyl, benzoyl and the like; and preferred aryls include include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl and 1,2,3,4- tetrahydronaphthalene .

[058] The alkyl, aryl and acyl substituents may optionally comprise one or more

heteroatoms such as oxygen (O), phosphorus (P), sulfur (S), nitrogen (N), silicon (S), arsenic (As), selenium (Se), and halogens, and preferred heteroatom functional groups are haloformyl, hydroxyl, aldehyde, amine, azo, carboxyl, cyanyl, thocyanyl, carbonyl, halo, hydroperoxyl, imine, aldimine, isocyanide, iscyante, nitrate, nitrile, nitrite, nitro, nitroso, phosphate, phosphono, sulfide, sulfonyl, sulfo, and sulfhydryl.

[059] The optionally hetero alkyl, aryl and acyl functional groups include both substituted and unsubstituted forms of the indicated radical. Preferred substituents include -OR', =0, =NR', =N-OR', -NR'R", -SR', halogen, -SiR'R"R"', -OC(0)R', -C(0)R', -C0₂R', -CONR'R", - OC(0)NR'R", -NR"C(0)R', -NR'-C(0)NR"R'", -NR'-S02NR'", -NR"C0₂R', -NH- C(NH₂)=NH, -NR'C(NH₂)=NH, -NH-C(NH₂)=NR', -S(0)R', -S0₂R', -S0₂NR'R", -NR"S0₂R, -CN and -N0₂, wherein R', R" and R'" each independently refer to hydrogen, unsubstituted (Cl-C8)alkyl and heteroalkyl, unsubstituted aryl, aryl substituted with one to three halogens, unsubstituted alkyl, alkoxy or thioalkoxy groups, or aryl-(Cl-C4)alkyl groups.

Bioactivity directed purification of natural compounds [061] Among the 160 verified inhibitors (Table 2), three were from the natural product collection. The isolation and biological activity of these will be summarized below. Initial screening results of inhibitors of the enzyme target Entpd5 revealed that the hexane and dichloromethane (07-092-DCM) soluble extracts from the sponge Smenospongia aurea exhibited a 6 fold increase in luminescence at a concentration of 10 mg/mL (figure 2a), an indication of Entpd5 inhibition. Following the process of bioassay guided fractionation we began pursuing the active components from these active fractions.

[062] Fraction 07-092-hex was further purified using flash Si0₂ chromatography using a gradient from 100:0 hexane: EtOAc to 50:50 hexane:EtOAc over 2.5 L of solvent to give a total of 17 fractions that were tested for their ability to inhibit Entpd5 in an enzyme based assay. A number of these fractions, A8-A10 and A12-15 showed strong inhibitory effects at a concentration of -20 μg/mL in DMSO (figure 2b). Based on NMR and MS profiles it was determined that these fractions were enriched in small diterpene compounds - as indicated in the NMR spectra by a series of methyl singlets from 1.2-1.7 ppm. Further analysis my MS indicated molecular weights in the range of m/z 310 -360, indicative of a terpene with 2 - 4 oxygen atoms. Based on the complexity of the NMR spectra, we initially pursued fraction A 10, as it appeared to be predominately composed of a single compound. Normal phase HPLC purification using isocratic conditions of 95:5 «-hexane:IPA gave a single pure compound that was subsequently analyzed with the Entpd5 enzyme assay to obtain dose- response data, revealing an IC₅₀ of 20 μΜ for fraction AlO-1 (figure 2c).

[063] Using NMR and MS data we were able to determine the structure of this compound as aureol (1) (figure 2e), a previously reported diterpene (Djura 1980), that is characterized by the fusion of the diterpene core to a an aromatic ring through a furan ring. The NMR data for aureol matches the literature reports for this compound.

[064] After establishing the active component of fraction A 10, we took a more in depth analysis of fraction A9, a more complex mixture of compounds. Normal phase HPLC purification using isocratic conditions of 95:5 w-hexane ^'-PrOH gave three additional pure compounds, in addition to aureol. The three additional compounds are all sesquiterpene hydroquinone metabolites related to aureol. BA07-092-A10-9-8 (2) (IC₅₀ = 30 μΜ) lacks the C ring furan, BA07-092-A10-9-11 (3) (IC₅₀ = 30 μΜ) has a rearranged terpene skeleton giving an exocyclic double bond in the A ring, and BA07-092-A10-9-10 (4) (IC₅₀ = 10 μΜ) has an alternate cyclization forming the C ring furan. Analysis of the NMR data for 4 revealed the known compound 8-epichromazonarol, previously reported by the Faulkner lab (Djura 1980). The IC₅₀ curves for these three compounds can be seen in figure 2d. In addition to sesquiterpene hydroquinone analogs 1 - 4, we isolated additional analogs that are undergoing further biological evaluation.

[065] Aureol (1) has received considerable attention for broad biological activity, including anti-viral, anti-tumor and neurological (US 5051519; US 5204367; US 20090093513 and US 120090409). However, there have been no reports of a specific target for aureol or analogs. We are aware of no reports on the biological activity of molecules with the carbon backbone of 4. Additionally, the hydrocarbon nature of 1 - 4, makes significant functionalization and SAR projects challenging. The few analogs of aureol are simple derivatives on the phenol group or naturally occurring halogenated analogs on the aromatic ring. We have designed a semi- synthetic route that provides access to more functionalized analogs, derivatized at the unfunctionalized aromatic positions. This involves the formation of nitro substitution, which provides a further handle for chemistry. Additional compounds suitable in the subject methods and compositions include sesquiterpenoid hydroquinone and quinone structural families that have been isolated with variations in the stereochemistry of the angular methyl groups as well as the ring architecture (Djura, 1980; Minale 1974; Ravi 1979).

[066] Cellular effects of in vitro confirmed compound inhibitors

[067] We have shown that ENTPD5 is essential for maintaining ER homeostasis in AKT hyperactive cancer cells. Knockdown of ENTPD5 by siRNA leads to ER stress, GRP78/BiP upregulation and degradation of EGF receptor. Our newly identified chemical inhibitors targeting ENTPD5 provide a similar phenotype.

[068] We tested representative members from each structural class of synthetic compounds on PTEN knockout MEF cells at 30 μΜ for 24 hours, and monitored GRP78/BiP expression level by Western Blotting as an indicator of ER stress. Six tested compounds induced strong GRP78/BiP upregulation. Five out of the six belong to the Class II structural group featuring Uracil-like ring, and the other one falls into the Class IV containing a urea/thiourea substructure. Most confirmed synthetic inhibitors have limited cellular effect due to their poor cell permeability.

[069] The two most potent synthetic inhibitors, PID#38 and PID#51 were then tested for cellular dose response on PTEN knockout MEF cells. Both compounds could induce ER stress evidenced by GRP78/BiP increase and EGF receptor decrease in a dose dependent manner, recapitulating ENTPD5 siRNA knockdown.

[070] On the other hand, the purified natural compounds from sea sponge are highly lipophilic, and readily cell permeable. They could also induce ER stress in PTEN knockout MEF cells in a dose dependent manner. Furthermore, the strength of ER stress induction is correlating with their in vitro potency. Aureol at 30 μΜ starts to show cytotoxicity.

[071] We then asked whether ENTPD5 inhibitors would distinguish between normal tissue and cancer cells. When treated with the same dosage, would cancerous tissue be more sensitive to these inhibitors. To answer these questions, we compared inhibitor dose response of PTEN knockout MEF cells, which is considered a model of PTEN deficient, AKT hyperactive cancer, with that of PTEN heterozygous MEF cells. PTEN knockout MEF cells are more sensitive to synthetic inhibitor induced ER stress. Similar results were observed with purified natural compound inhibitors.

[072] ENTPD5 is important for cancer cell growth

[073] To verify the functional significance of ENTPD5 expression in human cancer cells, we generated a cell line from the original LNCaP cells in which a shRNA against human ENTPD5 could be induced by Dox. In these cells, knockdown ENTPD5 by adding Dox to the culture media also lowered of N-glycosylation and induced BiP expression. These phenotypes were rescued by the expression of a wild type shRNA resistant ENTPD5 transgene but not by the active site mutant ENTPD5. Several growth factor receptors were also checked in these cell lines after Dox treatment. EGFR, and Her2/ErbB-2 were significantly down and IGFR was slightly down. They were restored to the normal level by the expression of shRNA-resistant ENTPD5 transgene but not the active site mutant.

Consistently, when the cell number was measured after 4-day knockdown of ENTPD5, only about half of LNCaP cells were there compared to a control knockdown cell line, and the defect was rescued by expression of wild type ENTPD5 transgene but not active site mutant.

[074] To test whether knocking down ENTPD5 in LNCaP cells also has an effect on their growth in vivo, we implanted the LNCaP cells bearing a Dox-inducible shRNA targeting ENTPD5 in matrigel in nude mice. As a control, LNCaP cells with a Dox-inducible shRNA targeting GFP were also implanted. After the xenograft tumors reached the size of 500 mm , a cohort of 7 mice were fed with Dox-containing water. The level of ENTPD5 in these tumors was measured after 6 weeks. Compared with mice fed with normal water, the

ENTPD5 levels in ENTPD5-targeting shRNA containing tumors from mice fed with Dox- containing water were significantly lower. While ENTPD5-targeting shRNA containing tumors in mice fed with normal water continued to grow, the tumors in mice fed with Dox- containing water shrank. When these tumor samples were analyzed under a microscope after fixing and staining with hematoxylin and eosin, there were very few tumor cells left in the matrigel in tumors grown in Dox-fed mice while in mice fed with normal water, the matrigel was filled with tumor cells. The GFP shRNA containing tumors did not respond to Dox treatment and continued to grow during the period of experiment.

[075] Similar tumor shrinkage and reduction in cells left in the matrigels are observed with both synthetic and purified natural compound ENTPD5 inhibitors (supra). Inhibitors are administered IP, 3 mice per treatment group in each of 1, 10 and 100 mg dosages (200ul of 5, 50 and 500mg/ml solution administered IP), every other day. After 6 weeks turmor size and tumor cell survival are measured. Consistent with our knockdown results, while tumors of sham injected mice show continued growth, the tumors in mice treated with inhibitors consistently shrink, and when these tumor samples are analyzed under a microscope after fixing and staining with hematoxylin and eosin, there are very few tumor cells left in the matrigel in tumors of treated mice while in sham-treated mice, the matrigel is rich with tumor cells.

[076] Inhibitor Forms, Pharmaceutical Compositions, Dosages, Formulations, Delivery

[077] Pharmaceutically acceptable salts include salts of the active compounds which are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein. When compounds contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt. When compounds contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric,

dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, oxalic, maleic, malonic, benzoic, succinic, suberic, fumaric, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like (see, for example, Berge et al. (1977) J. Pharm. Sci.66: l-19). Certain compounds of the invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts.

[078] The neutral forms of the compounds may be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the compound for the purposes of the invention.

[079] In addition to salt forms, the invention provides compounds which are in a prodrug form. Prodrugs of the compounds described herein are those compounds that undergo chemical changes under physiological conditions to provide the compounds of the invention. Additionally, prodrugs can be converted to the compounds of the invention by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to the compounds of the invention when placed in a transdermal patch reservoir with a suitable enzyme or chemical reagent. Prodrugs are often useful because, in some situations, they may be easier to administer than the parent drug. They may, for instance, be more bioavailable by oral administration than the parent drug. The prodrug may also have improved solubility in pharmacological compositions over the parent drug. A wide variety of prodrug derivatives are known in the art, such as those that rely on hydrolytic cleavage or oxidative activation of the prodrug. An example, without limitation, of a prodrug would be a compound of the invention which is administered as an ester (the "prodrug"), but then is metabolically hydrolyzed to the carboxylic acid, the active entity. Additional examples include peptidyl derivatives of a compound of the invention.

[080] Certain compounds of the invention can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are intended to be encompassed within the scope of the invention. Certain compounds of the invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the invention and are intended to be within the scope of the invention.

[081] Certain compounds of the invention possess asymmetric carbon atoms (optical centers) or double bonds; the racemates, diastereomers, geometric isomers and individual isomers are all intended to be encompassed within the scope of the invention.

[082] The compounds of the invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may be radiolabeled with radioactive isotopes, such as for example tritium ( H), iodine- 125 ( I) or carbon- 14 ( C). All isotopic variations of the compounds of the invention, whether radioactive or not, are intended to be encompassed within the scope of the invention.

[083] Therapeutically effective amount refers to the amount of the subject compound that will elicit, to some significant extent, the biological or medical response of a tissue, system, animal or human that is being sought by the researcher, veterinarian, medical doctor or other clinician, such as when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the symptoms of the condition or disorder being treated. The therapeutically effective amount will vary depending on the compound, the disease and its severity and the age, weight, etc., of the mammal to be treated.

[084] The invention also provides pharmaceutical compositions comprising the subject compounds and a pharmaceutically acceptable excipient, particularly such compositions comprising a unit dosage of the subject compounds.

[085] The compositions for administration can take the form of bulk liquid solutions or suspensions, or bulk powders. More commonly, however, the compositions are presented in unit dosage forms to facilitate accurate dosing. The term "unit dosage forms" refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient. Typical unit dosage forms include prefilled, premeasured ampules or syringes of the liquid

compositions or pills, tablets, capsules, losenges or the like in the case of solid compositions.

[086] In such compositions, the compound is usually a minor component (from about 0.1 to about 50% by weight or preferably from about 1 to about 40% by weight) with the remainder being various vehicles or carriers and processing aids helpful for forming the desired dosing form.

[087] Suitable excipients or carriers and methods for preparing administrable compositions are known or apparent to those skilled in the art and are described in more detail in such publications as Remington's Pharmaceutical Science, Mack Publishing Co, NJ (1991). In addition, the compounds may be advantageously used in conjunction with other therapeutic agents as described herein or otherwise known in the art, particularly other anti-cancer agents. Hence the compositions may be administered separately, jointly, or combined in a single dosage unit.

[088] The amount administered depends on the compound formulation, route of

administration, etc. and is generally empirically determined in routine trials, and variations will necessarily occur depending on the target, the host, and the route of administration, etc. Generally, the quantity of active compound in a unit dose of preparation may be varied or adjusted from about 1, 2, 5, 10 or 20, 50, 100, 200 or 500 to about 2, 5, 10, 20, 50, 100, 200, 500 or 1000 mg, according to the particular application. In a particular embodiment, unit dosage forms are packaged in a multipack adapted for sequential use, such as blisterpack, comprising sheets of at least 6, 9 or 12 unit dosage forms. The actual dosage employed may be varied depending upon the requirements of the patient and the severity of the condition being treated. Determination of the proper dosage for a particular situation is within the skill of the art. Generally, treatment is initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small amounts until the optimum effect under the circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions during the day if desired.

[089] The following are examples (Formulations 1-4) of carbazole capsule formulations.

[090] Table 2. Capsule Formulations

[091] Preparation of Solid Solution

[092] Crystalline inhibitor (80 g/batch) is dissolved in methylene chloride (5000 mL). The solution is dried using a suitable solvent spray dryer and the residue reduced to fine particles by grinding. The powder is then passed through a 30 mesh screen and confirmed to be amorphous by x-ray analysis. [093] The solid solution, silicon dioxide and magnesium stearate are mixed in a suitable mixer for 10 minutes. The mixture is compacted using a suitable roller compactor and milled using a suitable mill fitted with 30 mesh screen. Croscarmellose sodium, Pluronic F68 and silicon dioxide are added to the milled mixture and mixed further for 10 minutes. A premix is made with magnesium stearate and equal portions of the mixture. The premix is added to the remainder of the mixture, mixed for 5 minutes and the mixture encapsulated in hard shell gelatin capsule shells.

[094] The compounds can be administered by a variety of methods including, but not limited to, parenteral, topical, oral, or local administration, such as by aerosol or

transdermally, for prophylactic and/or therapeutic treatment. Also, in accordance with the knowledge of the skilled clinician, the therapeutic protocols (e.g., dosage amounts and times of administration) can be varied in view of the observed effects of the administered therapeutic agents on the patient, and in view of the observed responses of the disease to the administered therapeutic agents.

[095] The therapeutics of the invention can be administered in a therapeutically effective dosage and amount, in the process of a therapeutically effective protocol for treatment of the patient. For more potent compounds, microgram (ug) amounts per kilogram of patient may be sufficient, for example, in the range of about 1, 10 or 100 ug/kg to about 0.01, 0.1, 1, 10, or 100 mg/kg of patient weight though optimal dosages are compound specific, and generally empirically determined for each compound.

[096] Routine experimentation in clinical trials will determine specific ranges for optimal therapeutic effect, for each therapeutic, each administrative protocol, and administration to specific patients will also be adjusted to within effective and safe ranges depending on the patient condition and responsiveness to initial administrations. However, the ultimate administration protocol will be regulated according to the judgment of the attending clinician considering such factors as age, condition and size of the patient as well as compounds potency, severity of the disease being treated. For example, a dosage regimen of the compounds can be oral administration of from 10 mg to 2000 mg/day, preferably 10 to 1000 mg/day, more preferably 50 to 600 mg/day, in two to four (preferably two) divided doses. Intermittent therapy (e.g., one week out of three weeks or three out of four weeks) may also be used.

[097] Assay plate setup and results analysis

[098] The setup of 384-well plate for the primary screen is as following: 1) The first column of 16 wells (Al-Pl) were set as positive control (PosCtrl), containing reaction buffer and Substrate Solution in the presence of 1% DMSO but no Enzyme Mixture, corresponding to theoretical 100% inhibition. 2) The second and 23 rd column (A2-P2, A23-P23) were used as negative control (NegCtrl), which contain both Enzyme Mixture and Substrate Solution plus 1% DMSO, reflecting null inhibition. And 3) the 24^th column (A24-P24) was the positive compound control (CmpdCtrl), where 0.5μ1 of 0.5mM Cmpd4 in DMSO was added into the complete enzymatic reaction. 4) The rest of the plate, 320 wells (C1-P22), was designated for the library compounds (Sample) to be assayed in complete enzymatic reaction. The secondary and tertiary assay plate setups were similar, with their respective Enzyme Mixture and Substrate Solution used. The structure clustering of cherry picked compounds was performed by pipeline pilot software from Accelrys (San Diego).

[099] References

Baykov, A. A., Evtushenko, O.A., and Avaeva, S.M. (1988). A malachite green procedure for orthophosphate determination and its use in alkaline phosphatase-based enzyme immunoassay. Anal Biochem 171, 266-270.

Serina, L., Blondin, C, Krin, E., Sismeiro, O., Danchin, A., Sakamoto, H., Gilles, A.M., and Barzu, O. (1995). Escherichia coli UMP-kinase, a member of the aspartokinase family, is a hexamer regulated by guanine nucleotides and UTP. Biochemistry 34, 5066-5074.

Serina, L., Bucurenci, N., Gilles, A.M., Surewicz, W.K., Fabian, H., Mantsch, H.H., Takahashi, M., Petrescu, I., Batelier, G., and Barzu, O. (1996). Structural properties of UMP- kinase from Escherichia coli: modulation of protein solubility by pH and UTP. Biochemistry 35, 7003-7011.

Zhang, J.H., Chung, T.D., and Oldenburg, K.R. (1999). A Simple Statistical

Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. J Biomol Screen 4, 67-73.

Djura, P., Stierle, D.N., Sullivan, D.J., Faulkner, D.J., Arnold, E., Clardy, J. (1980) Some Metabolites of the Marine Sponges Smenospongia aurea and Smenospongia (=

Polyfibrospongia) echina. J. Org. Chem. 45, 1435-1441.

Minale, L., Riccio, R., Sodano, G. (1974) Avarol, a Novel Sesquiterpenoid

Hydroquinone with a Rearranged Drimane Skeleton from the Spinge Disidea Avara.

Tetrahedron Letters 38. 3401-3404.

Ravi, B.N., Perzanowski, H.P., Ross, R.A., Erdman, T.R., Scheur, P.J., Finer, J., Clardy, J. (1979) Recent Research in Marine Natural Products: Puupehenones. Pure and Applied Chemistry 51, 1893-1900. [0100] The foregoing examples and detailed description are offered by way of illustration and not by way of limitation. All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0101] Table 1. Verified inhibitors PID#1-162 - all demonstrating significant luminescence increase at luM, and dose response increases to lOuM, and ENTPD5-specificity per UMPK assay and malachite green assay.

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Claims

WHAT IS CLAIMED IS:

1. A novel ENTPD5-specific inhibitor comprising a structure of a generic or a specific formula disclosed herein, including Table 1 and Figs.1-4, and including sesquiterpenoid hydroquinone and quinone structural derivatives with variations in the stereochemistry of the angular methyl groups or ring architecture, and including tautomers, stereoisomers and pharmaceutically-acceptable salts thereof.

2. A novel pharmaceutical composition in unit dosage comprising a disclosed ENTPD5 inhibitor, including tautomers, stereoisomers and pharmaceutically-acceptable salts thereof, and one or more of the disclosed pharmaceutically acceptable excipients.

3. A method for inhibiting cancer cell growth in a patient in need thereof comprising administering to the patient an effective amount of a disclosed ENTPD5 inhibitor, including tautomers, stereoisomers and pharmaceutically-acceptable salts thereof; and

optionally further comprising the subsequent step of detecting a resultant growth inhibition of the cancer cells; and/or

optionally further comprising the antecedent step of determining that the patient has the cancer cells by detecting the same in said patient; and/or

optionally further comprising the antecedent step of detecting undesirable or pathogenic ENTPD activity of a sample of the cancer cells of the patient.

optionally further comprising the subsequent step of detecting ENTPD activity of a sample of the cancer cells of the patient.

4. A method of detecting ENTPD5 activity comprising the step of coupling ENTPD5 catalyzed UDP hydrolysis to UMP Kinase catalyzed UMP phosphorylation to form a futile cycle of UMP/UDP interconversion wherein one molecule of ATP being consumed in each hydrolysis/ phosphorylation cycle.

5. The method of claim 4 further comprising the step of establishing a correlation between cell type, state, status or condition and undesirable or pathogenic ENTPD5 activity

6. The method of claim 4 wherein inhibition of either enzyme results in a detectable increase of residual ATP at the end of the reaction, wherein the detectable increase is detected by luciferase assay, and wherein to enrich ENTPD5 inhibitors, ENTPD5 is the rate limiting enzyme in the coupled reaction.

7. The method of claim 6 wherein to further enrich ENTPD5 inhibitors counter screens are used to filter UMP Kinase inhibitors and non-specific inhibitors .

8. A method of treating a disease associated with undesirable or pathogenic ENTPD5 activity comprising the step of treating a so afflicted person with a disclosed ENTPD5 inhibitor.