Sungsook Lee

Identification of CKD-516: A Potent Tubulin Polymerization Inhibitor with Marked Antitumor Activity against Murine and Human Solid Tumors

Tubulin polymerization inhibitors had emerged as one of promising anticancer therapeutics because of their dual mechanism of action, i.e. apoptosis by cell-cycle arrest and VDA, vascular disrupting agent. VDAs are believed to be more efficient, less toxic, and several of them are currently undergoing clinical trials. To identify novel tubulin inhibitors that possess potent cytotoxicity and strong inhibition of tubulin polymerization as well as potent in vivo antitumor efficacy, we have utilized benzophenone scaffold. Complete SAR analysis of newly synthesized analogues that were prepared by incorporation of small heterocycles (C2, C4, and C5 position) into B-ring along with the evaluation of their in vitro cytotoxicity, tubulin polymerization inhibition, and in vivo antitumor activity allowed us to identify 22 (S516). Compound 22 was found to have potent cytotoxicity against several cancer cells including P-gp overexpressing MDR positive cell line (HCT15). It also induced cell cycle arrest at G(2)/M phase, which is associated with strong inhibition of tubulin polymerization. Its in vivo efficacy was improved by preparing its (l)-valine prodrug, 65 (CKD-516), which together with greatly improved aqueous solubility has shown marked antitumor efficacy against both murine tumors (CT26 and 3LL) and human xenogratfs (HCT116 and HCT15) in mice.

The AcbC protein from Actinoplanes species is a C7-cyclitol synthase related to 3-dehydroquinate synthases and is involved in the biosynthesis of the alpha-glucosidase inhibitor acarbose.

The putative biosynthetic gene cluster for the α-glucosidase inhibitor acarbose was identified in the producerActinoplanes sp. 50/110 by cloning a DNA segment containing the conserved gene for dTDP-d-glucose 4,6-dehydratase,acbB. The two flanking genes were acbA(dTDP-d-glucose synthase) and acbC, encoding a protein with significant similarity to 3-dehydroquinate synthases (AroB proteins). The acbC gene was overexpressed heterologously in Streptomyces lividans 66, and the product was shown to be a C7-cyclitol synthase using sedo-heptulose 7-phosphate, but not ido-heptulose 7-phosphate, as its substrate. The cyclization product, 2-epi-5-epi-valiolone ((2S,3S,4S,5R)-5-(hydroxymethyl)cyclohexanon-2,3,4,5-tetrol), is a precursor of the valienamine moiety of acarbose. A possible five-step reaction mechanism is proposed for the cyclization reaction catalyzed by AcbC based on the recent analysis of the three-dimensional structure of a eukaryotic 3-dehydroquinate synthase domain (Carpenter, E. P., Hawkins, A. R., Frost, J. W., and Brown, K. A. (1998) Nature 394, 299–302).

CKD712, (S)-1-(α-naphthylmethyl)-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, inhibits the lipopolysaccharide-stimulated secretion of HMGB1 by inhibiting PI3K and classical protein kinase C.

CKD712, (S)-1-(α-naphthylmethyl)-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, was considered as a new effective drug candidate to sepsis, based on its anti-inflammatory activity. It was reported that CKD712 inhibited various signal pathways which play a key role in production of proinflammatory cytokines. Here, we examined the effect of CKD712 on the secretion of high mobility group box 1 (HMGB1), which is one of the proinflammatory cytokines. CKD712 can reduce Gram-negative lipopolysaccharide (LPS)- and Gram-positive lipoteichoic acid (LTA)-stimulated HMGB1 secretion in RAW264.7 and human peripheral blood monocytes (PBMo), and also reduce LPS-induced nucleocytoplasmic translocation of HMGB1 1h before or after LPS treatment. CKD712 could dose-dependently inhibit the activation of PI3K and PI3K-dependent kinase 1 (PDK1), which are involved in HMGB1 secretion signaling pathway. In addition, CKD712 inhibited classical protein kinase C (cPKC), the effective kinase for phosphorylation of HMGB1 for secretion, however, had no effect on histone acetyl-transferase activity, which is another mechanism known for HMGB1 secretion. Thus, we suggest that CKD712 could inhibit LPS- and LTA-stimulated HMGB1 secretion through the inhibition of HMGB1 phosphorylation by inhibiting PI3K-PKC signaling pathway.

Discovery of a potent tubulin polymerization inhibitor: Synthesis and evaluation of water-soluble prodrugs of benzophenone analog

Prodrugs have proven to be very useful in enhancing aqueous solubility of sparingly water-soluble drugs, thereby increasing in vivo efficacy without a need of special excipients. In vitro and in vivo evaluations of a number of amino acid prodrugs of 1, a previously identified potent tubulin polymerization inhibitor and cytotoxic against various cancer cell lines led to the discovery of 3·HCl (l-valine attached) which is highly efficacious in mouse xenografts bearing human cancer. Pharmacokinetic analysis in rats revealed that compound 1 was released immediately upon administration of 3·HCl intravenously, with rapid clearance of 3·HCl indicating the effective cleavage of prodrug. Compound 3·HCl (CKD-516) has now been progressed to phase 1 clinical trial.

Enzymatic synthesis of [7-14C, 7-3H]- and [1-13C]sedoheptulose 7-phosphate and [1-13C]ido-heptulose 7-phosphate

Abstract The enzymatic synthesis of isotopically labeled d -sedoheptulose 7-phosphate from either labeled d -glucose or labeled l -serine is described. [7- 14 C , 7- 3 H ] Sedoheptulose 7-phosphate is prepared with transketolase from xylulose 5-phosphate, as C 2 donor, and d - [5- 14 C , 5- 3 H ] ribose 5-phosphate, which in turn is generated from d - [6- 14 C , 6- 3 H ] glucose with the glycolytic enzymes. [1- 13 C ] Sedoheptulose 7-phosphate is obtained in a one-pot reaction in 69% yield from unlabeled ribose 5-phosphate and l - [3- 13 C ] serine via hydroxypyruvate as the C 2 donor using the enzymes alanine racemase, d -amino acid oxidase, catalase and transketolase. By the latter route labeled [1- 13 C ] ido -heptulose 7-phosphate was also prepared when xylose 5-phosphate was substituted for the ribose 5-phosphate.

Stereochemistry of conversion of the suicide substrates β-chloro-D-alanine-and D- and L-serine O-sulfates into pyruvate by D-amino acid aminotransferase and by L-aspartate aminotransferase

β-Chloro-D-alanine and D-serine O-sulfate are converted into a putative aminoacrylate intermediate by D-amino acid aminotransferase. This either reacts with pyridoxal phosphate to form a reactive inhibitor of the enzyme or it is protonated and hydrolysed to give pyruvate. The protonation reaction is shown to occur with modest stereoselectivity, indicating overall retention of stereochemistry in replacement of the leaving group by hydrogen. The corresponding reaction of L-serine O-sulfate using L-aspartate aminotransferase shows little or no stereosetectivity.

(S)-Tetrahydroisoquinoline Alkaloid Inhibits LPS-Induced Arachidonic Acid Release through Downregulation of cPLA2 Expression

Sepsis, a systemic inflammatory response syndrome, remains a potentially lethal condition. (S)-1-α-Naphthylmethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (CKD712) is noted as a drug candidate for sepsis. Many studies have demonstrated its significant anti-inflammatory effects. Here we first examined whether CKD712 inhibits lipopolysaccharide (LPS)-induced arachidonic acid (AA) release in the RAW 264.7 mouse monocyte cell line, and subsequently, its inhibitory mechanisms. CKD712 reversed LPS-associated morphological changes in the RAW 264.7 cells, and inhibited LPS-induced release of AA in a concentrationdependent manner. The inhibition was apparently due to the diminished expression of a cytosolic form of phospholipase A2 (cPLA2) by CKD712, resulting from reduced NF-κB activation. Furthermore, CKD712 inhibited the activation of ERK1/2 and SAP/JNK, but not of p38 MAPK. CKD712 had no effect on the activity or phosphorylation of cPLA2 and on calcium influx. Our results collectively suggest that CKD712 inhibits LPS-induced AA release through the inhibition of a MAPKs/NF-κB pathway leading to reduced cPLA2 expression in RAW 264.7 cells.

Reaction of Nitrile Oxides with 3(2H)- and 2(5H)-Furanones.

1,3-Dipolar cyclization of various nitrile oxides with 3(2H)- and 2(5H)-furanones regioselectively furnished the corresponding syn-addition products of 3-oxotetrahydro-furano[4,5-d]isoxazolines and 2-oxotetrahydrofurano[3,4-d]isoxazolines.

Abstract 5435: A novel HDAC inhibitor, CKD-581, demonstrates a potent in vivo efficacy in various human tumor xenograft models

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Histone deacetylases (HDAC) inhibition is now well established as a new approach for solid and hematological tumor therapy. Studies with different HDAC inhibitors showed broad activity toward cancer cells, i.e. activation of proapoptotic pathway and inhibition of antiapoptotic pathway, induction of cell differentiation, antiangiogenic activity and synergism with other cancer therapeutics. Several HDAC inhibitors have demonstrated therapeutic benefits and are under clinical investigations as mono- or combi-therapy in various cancer cell lines such as cutaneous T-cell lymphoma (CTCL) with SAHA launched in 2006. Highly water-soluble CKD-581 is a potent and novel pan HDAC inhibitor developed in our institute. In this study, we analyzed first the in vitro growth inhibitory effect of CKD-581 on various cancer cells by MTT assay and inhibition of HDAC enzymes as well as its antitumor efficacy in human colon, prostate, and lung xenograft models. In addition, western blotting analysis for acH3, acH4 and p21 (WAF-1/CIP-1) and fluorescence-activated cell sorting analysis were performed to verify the associated molecular mechanisms involved in CKD-581 mediated cell death and cell cycle progression. CKD-581 showed strong cytotoxicity against several cancer cell lines including HCT-116, PC-3, A549 and H460 with IC50 values ranging from 10 nM to 100 nM. Its cytotoxicity was closely related with potent inhibitory activity against human HDAC1 and HDAC6 enzymes at single nMs. In various cancer cell lines, CKD-581 induced acetylation of histone and expression of tumor suppressor proteins involved in apoptosis pathway. CKD-581 significantly inhibited the in vivo growth of human tumor xenografts (HCT-116, PC-3, A549 and H460) in nude mice in a dose-dependent manner. Treatment of CKD-581 (60, 80, 100 mg/kg, b.i.wk, i.p.) caused 50-70% reduction in the mean tumor volume compared with controls. It is of note to observe that CKD-581 was highly efficacious when orally administered to nude mice bearing HCT 116. To correlate the pharmacodynamics with pharmacokinetics of CKD-581 in nude mice (HCT-116), we analyzed the kinetics of histone acetylation in tumors and plasma drug exposure at the end of 3 weeks of treatment (80 mg/kg, b.i.wk, i.p.), where Tmax of CKD-581 in tumor and in plasma were 4 h and 0.25 h after dosing, respectively and AUClast in tumor was 2.6 times higher than that of in plasma. CKD-581 is a potent HDAC inhibitor and showed a strong cytotoxicity against many cancer cell lines, demonstrated through the various in vitro and in vivo studies. Moreover, significant antitumor efficacy was shown in nude mice bearing several human tumors. Its potency together with excellent pharmacokinetic properties warrants further clinical investigations of CKD-581. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5435.

Abstract 4425: Oral efficacy and preclinical ADME evaluation of CKD-516, a novel tubulin polymerization inhibitor

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC CKD-516 is a potent anticancer agent that inhibits microtubule assembly and disrupts tumor vasculature (VDA). CKD-516 is an amino acid prodrug of S516 releasing S516 in vivo. Previously we reported the discovery and its antitumor efficacy of CKD-516 as well as various in vitro studies (Abstract 5561, 2009 AACR). As a continuation of the investigation, a detailed ADME evaluation of CKD-516 in both rats and dogs and its potential as an oral agent is described in this report. Following intravenous administration to rats and dogs, CKD-516 rapidly became the active metabolite S516 in plasma and the half-lives of S516 were 1.5 hrs in rats, and 8 hrs in dogs. Protein binding was high with an unbound fraction less than or equal to 20% in all species tested. In distribution studies in rats dosed intravenously, S516 was mainly distributed in thymus, lung and stomach and had a short half-life less than 2 hrs in all tissues tested. In rats, the excretion of CKD-516 in urine, bile and feces amounted to 0.22%, 8.22% and 4.92%, respectively. S516 was found to have comparable metabolic stabilities both in dog and human liver microsomes where there was no difference between male and female microsomes. However, the rate of metabolism in rats from male and female liver microsomes were markedly different. In general, S516 is expected to have low drug-drug interaction with IC50 values of >10 μM against most CYP450 enzymes but had a moderate inhibition against CYP450 1A2 and 2C8. The intestinal absorption of CKD-516 was predicted to be moderate to high as the apparent permeabilities (×106 cm/sec) of CKD-516 and its active metabolite S516 in Caco-2 permeability assay were 15.1 and 35.4, respectively. Thus, CKD-516 was found to have an acceptable oral bioavailability in rats, supporting its potential efficacy as an oral agent. In vivo antitumor experiments were performed in the HCT116 human tumor xenograft model. When the established tumor reached the size of 150 mm3, CKD-516 was administered to mice orally. Groups of animals were received CKD-516 with doses of 10 mg/kg or 20 mg/kg (Q4d × 5). The treated group showed a tumor growth suppression (IR = 50∼60%). Drug related toxicity and body weight decrease were not detected at doses tested. In summary, a complete ADME characterization of CKD-516, a novel tubulin polymerization inhibitor and VDA, both in vitro and in vivo was described. Moreover, CKD-516 was found to have a good potential as an oral agent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4425.