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To address the key challenges in the development of next-generation drug delivery systems (DDS) with desired physicochemical properties to overcome limitations regarding safety, in vivo efficacy, and solid tumor penetration, an ultrasmall folate receptor alpha (FRα) targeted silica nanoparticle (C'Dot) drug conjugate (CDC; or folic acid CDC) was developed. A broad array of methods was employed to screen a panel of CDCs and identify a lead folic acid CDC for clinical development. These included comparing the performance against antibody-drug conjugates (ADCs) in three-dimensional tumor spheroid penetration ability, assessing in vitro/ex vivo cytotoxic efficacy, as well as in vivo therapeutic outcome in multiple cell-line-derived and patient-derived xenograft models. An ultrasmall folic acid CDC, EC112002, was identified as the lead candidate out of500 folic acid CDC formulations evaluated. Systematic studies demonstrated that the lead formulation, EC112002, exhibited highly specific FRα targeting, multivalent binding properties that would mediate the ability to outcompete endogenous folate

The total synthesis of 4-methylenegermine is described.\ud \ud

We recently described several highly potent, triazine (1) and triazolopyrimidine (2) scaffold-based, dual PI3K/mTOR-inhibitors (e.g., 1, PKI-587) that were efficacious in both in vitro and in vivo models. In order to further optimize these compounds we devised a novel series, the 2-oxatriazines, which also exhibited excellent potency and good metabolic stability. Some 2-oxatriazines showed promising in vivo biomarker suppression and induced apoptosis in the MDA-MB-361 breast cancer xenograft model.

The design and synthesis of a series of 6-methylidene penems containing [6,5]-fused bicycles (thiophene, imidazole, or pyrazle-fused system) as novel class A, B, and C beta-lactamase inhibitors is described. These penems proved to be potent inhibitors of the TEM-1 (class A) and AmpC (class C) beta-lactamases and less so against the class B metallo-beta-lactamase CcrA. Their in vitro and in vivo activities in combination with piperacillin are discussed. On the basis of the crystallographic structures of a serine-bound reaction intermediate of 2 with SHV-1 (class A) and GC1 (class C) enzymes, compounds 14a-l were designed and synthesized. Penems are proposed to form a seven-membered 1,4 thiazepine ring in both class A and C beta-lactamases. The interaction energy calculation for the enzyme-bound intermediates favor the formation of the C7 R enantiomer over the S enantiomer of the 1,4-thiazepine in both beta-lactamases, which is consistent with those obtained from the crystal structure of 2 with SHV-1 and GC1.

A novel and mild method was established to synthesize 6-methylidene penem compounds. This method entails a MgBr(2)/Et(3)N-promoted aldol-type condensation on 6-bromopenem 12 with an appropriately substituted aldehyde to yield the intermediate acetylated bromohydrin, which was smoothly converted to the final product with simultaneous deprotection of C3 carboxylic acid ester using activated zinc dust and phosphate buffer at pH 6.5. This process provides a useful variation of C-C bond formation method for penem derivatives and also serves as a practical synthetic method to prepare 6-exomethylenepenem derivatives without racemization at the C5 position.

A new beta-lactamase inhibitor, a methylidene penem having a 5,6-dihydro-8H-imidazo[2,1-c][1,4]oxazine heterocyclic substituent at the C6 position with a Z configuration, irreversibly inhibits both class A and class C serine beta-lactamases with IC(50) values of 0.4 and 9.0 nM for TEM-1 and SHV-1 (class A), respectively, and 4.8 nM in AmpC (class C) beta-lactamases. The compound also inhibits irreversibly the class C extended-spectrum GC1 beta-lactamase (IC(50) = 6.2 nM). High-resolution crystallographic structures of a reaction intermediate of (5R)-(6Z)-6-(5,6-dihydro-8H-imidazo[2,1-c][1,4]oxazin-2-ylmethylene)-7-oxo-4-thia-1-azabicyclo[3.2.0]hept-2-ene-3-carboxylic acid 1 with the SHV-1 beta-lactamase and with the GC1 beta-lactamase have been determined by X-ray diffraction to resolutions of 1.10 and 1.38 A, respectively. The two complexes were refined to crystallographic R-factors (R(free)) of 0.141 (0.186) and 0.138 (0.202), respectively. Cryoquenching of the reaction of 1 with each beta-lactamase crystal produced a common, covalently bound intermediate. After acylation of the serine, a nucleophilic attack by the departing thiolate on the C6' atom yielded a novel seven-membered 1,4-thiazepine ring having R stereochemistry at the new C7 moiety. The orientation of this ring in each complex differs by a 180 degrees rotation about the bond to the acylated serine. The acyl ester bond is stabilized to hydrolysis through resonance stabilization with the dihydrothiazepine ring and by low occupancy or disorder of hydrolytic water molecules. In the class A complex, the buried water molecule on the alpha-face of the ester bond appears to be loosely bound or absent. In the class C complex, a water molecule on the beta-face is disordered and poorly activated for hydrolysis. Here, the acyl intermediate is unable to assist its own hydrolysis, as is thought to occur with many class C substrates.

A series of mono-morpholino 1,3,5-triazine derivatives (8a-8q) bearing a 3-oxa-8-azabicyclo[3.2.1]octane were prepared and evaluated for PI3-kinase/mTOR activity. Replacement of one of the bis-morpholines in lead compound 1 (PKI-587) with 3-oxa-8-azabicyclo[3.2.1]octane and reduction of the molecular weight yielded 8m (PKI-179), an orally efficacious dual PI3-kinase/mTOR inhibitor. The in vitro activity, in vivo efficacy, and PK properties of 8m are discussed.

The partnering of a β-lactam with a β-lactamase inhibitor is a highly effective strategy that can be used to combat bacterial resistance to β-lactam antibiotics mediated by serine β-lactamases (EC 3.2.5.6). To this end, we tested two novel penem inhibitors against OXA-1, a class D β-lactamase that is resistant to inactivation by tazobactam. The K i of each penem inhibitor for OXA-1 was in the nM range ( K i of penem 1, 45 ± 8 nM; K i of penem 2, 12 ± 2 nM). The first-order rate constant for enzyme and inhibitor complex inactivation of penems 1 and 2 for OXA-1 β-lactamase were 0.13 ± 0.01 s −1 and 0.11 ± 0.01 s −1 , respectively. By using an inhibitor-to-enzyme ratio of 1:1, 100% inactivation was achieved in ≤900 s and the recovery of OXA-1 β-lactamase activity was not detected at 24 h. Covalent adducts of penems 1 and 2 (changes in molecular masses, +306 ± 3 and +321 ± 3 Da, respectively) were identified by electrospray ionization mass spectrometry (ESI-MS). After tryptic digestion of OXA-1 inactivated by penems 1 and 2, ESI-MS and matrix-assisted laser desorption ionization-time-of-flight MS identified the adducts of 306 ± 3 and 321 ± 3 Da attached to the peptide containing the active-site Ser67. The base hydrolysis of penem 2, monitored by serial 1 H nuclear magnetic resonance analysis, suggested that penem 2 formed a linear imine species that underwent 7-endo-trig cyclization to ultimately form a cyclic enamine, the 1,4-thiazepine derivative. Susceptibility testing demonstrated that the penem inhibitors at 4 mg/liter effectively restored susceptibility to piperacillin. Penem β-lactamase inhibitors which demonstrate high affinities and which form long-lived acyl intermediates may prove to be extremely useful against the broad range of inhibitor-resistant serine β-lactamases present in gram-negative bacteria.

The design and synthesis of a series of seven tricyclic 6-methylidene penems as novel class A and C serine beta-lactamase inhibitors is described. These compounds proved to be very potent inhibitors of the TEM-1 and AmpC beta-lactamases and less so against the class B metallo-beta-lactamase CcrA. In combination with piperacillin, their in vitro activities enhanced susceptibility of all class C resistant strains from various bacteria. Crystallographic structures of a serine-bound reaction intermediate of 17 with the class A SHV-1 and class C GC1 enzymes have been established to resolutions of 2.0 and 1.4 A, respectively, and refined to R-factors equal 0.163 and 0.145. In both beta-lactamases, a seven-membered 1,4-thiazepine ring has formed. The stereogenic C7 atom in the ring has the R configuration in the SHV-1 intermediate and has both R and S configurations in the GC1 intermediate. Hydrophobic stacking interactions between the tricyclic C7 substituent and a tyrosine side chain, rather than electrostatic or hydrogen bonding by the C3 carboxylic acid group, dominate in both complexes. The formation of the 1,4- thiazepine ring structures is proposed based on a 7-endo-trig cyclization.

Androgens are a major driver of prostate cancer growth. As prostate cancer progresses, it can escape standard androgen deprivation therapy by relying on extra-testicular CYP17 enzyme activity for androgen synthesis. EN3356 is a novel non-steroidal CYP17 inhibitor that reduces androgen synthesis and is designed to treat castration-resistant prostate cancer. EN3356 provides potent inhibition of CYP17 lyase activity in rat and human testicular microsomes, and in human adrenocortical carcinoma H295R cells it also inhibits testosterone synthesis potently. In contrast to abiraterone, EN3356 is selective for inhibiting testosterone synthesis over cortisol synthesis. This finding suggests that EN3356 may not require co-administration of prednisone to mitigate mineralocorticoid excess. In pre-clinical studies, EN3356 exhibited high oral bioavailability. EN3356 inhibited serum testosterone levels in the rat and caused a robust decrease of seminal vesicle and prostate weights following 14-days of oral administration. GLP safety studies were completed in two preclinical species and identified a high safety margin. A combination Phase I/II study in metastatic castration-resistant prostate cancer is currently under initiation in the US and is anticipated to start enrolling subjects in the fourth quarter of 2013. Based on the potent inhibitory activity of EN3356 against CYP17 lyase in vitro, selective inhibition of testosterone synthesis versus cortisol synthesis, and robust weight reduction of androgen sensitive organs in vivo, EN3356 has the potential to be a selective and efficacious therapy for the treatment of castration-resistant prostate cancer. Disclosure: Funding for this research was provided by Endo Pharmaceuticals Inc. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C134. Citation Format: Nicholas J. Laping, Dhanalakshmi Sivanandhan, Raghava Reddy Kethiri, Purushottam M. Dewang, Ajith Vijayendra, Mohd Zainuddin, Ramesh Mullangi, Niranjan Rao, Aranapakam M. Venkatesan, Sanjeeva Reddy, Sandeep Gupta, Roger A. Smith. EN3356, a novel CYP17A inhibitor for the treatment of castration resistant prostate cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C134.

Temsirolimus, a rapamycin analog, is a selective mTor inhibitor that has been successfully used in the clinic for the treatment of renal cancer. mTOR integrates signals from multiple upstream growth and survival regulatory pathways including the LKB1/AMPK and PI3K (Phosphatidylinositol-3-kinase)/Akt pathways. Among these, Phosphatidylinositol-3-kinase (PI3-K) is a lipid kinase which phophorylates phosphatidylinositol diphosphate (PIP-2) to phosphatidylinositol triphosphate. This pathway regulates cell proliferation, growth, survival, and apoptosis. The aberrant activation of PI3K-α and its downstream effectors including Akt and mTOR, has been linked to the initiation and maintenance of numerous tumor phenotypes. During tumorigenesis, PI3K/Akt/mTOR pathway activation occurs through various mechanisms, including loss of PTEN (the phosphatase that regulates PI3K signaling), over-expression or activation of certain receptor tyrosine kinases (e.g. EGFR, HER-2), interaction with activated Ras, overexpression of the PI3K-α gene (PIKC3A), or mutations in PIKC3A that cause elevated PI3K kinase activity. Deregulated PI3K/Akt/mTOR pathway signaling has been implicated in poor prognosis and low survival rate in patients with various lymphatic tumors, glioblastomas, melanomas as well as breast, prostate, lung, colon, and ovarian cancers. Numerous pre-clinical and clinical studies indicate that PI3K-α plays a key role in the biology of human cancer. Hence, it is likely that a small molecule inhibitor of both PI3K and mTOR will have clinical utility. Our project team has identified small molecules that potently inhibit the PI3K/mTor pathway. The current clinical candidate, PKI-587 (whose structure will be disclosed) is a pan PI3K (class I)/mTOR inhibitor. PKI-587 decreases tumor cell survival and proliferation in vitro. Additionally, PKI-587 has shown compelling efficacy in multiple human tumor nude mouse xenograft models such as MDA 361 (Breast), U87 (Glioma), H1975, A549 (NSCLC) in nude mice when administered as a single agent. The design of this novel potent inhibitor, synthesis, structure-activity relationships, PK and in vivo efficacy data will be presented. 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 723.