Total Synthesis in Search of Potent Antibody-Drug Conjugate Payloads. From the Fundamentals to the Translational.

The emergence and evolution of antibody-drug conjugates (ADCs) as targeted cancer therapies in recent years is a living example of the "magic bullet" concept of Paul Ehrlich, introduced by him more than a century ago. Consisting of three components, the antibody serving as the delivery system, the payload drug that kills the cancer cell, and the chemical linker through which the payload is attached to the antibody, ADCs represent a currently hotly pursued paradigm of targeted cancer therapies. While the needed monoclonal antibody falls in the domains of biology and biochemistry, the potent payload and the linker belong to the realm of chemistry. Naturally occurring molecules and their derivatives endowed with high cytotoxic properties have proven to be useful payloads for the first approved ADCs (i.e., Mylotarg, Adcetris, Kadcyla, and Besponsa). The latest approaches and intensifying activities in this new paradigm of cancer therapy demands a variety of payloads with different mechanisms of action in order to address the medical needs for the various types of cancers, challenging synthetic organic chemists to enrich the library of potential payloads. Total synthesis of natural and designed molecules not only provides a powerful avenue to replicate rare naturally occurring compounds in the laboratory but also offers a unique opportunity to rationally design and synthesize analogues thereof for biological evaluation and optimization of ADC payloads. In this Account, we describe our efforts in this area highlighting a number of total synthesis endeavors through which we rendered scarce naturally occurring molecules readily available for biological evaluations and, most importantly, employed the developed synthetic strategies and methods to construct, otherwise unavailable or difficult to reach, designed analogues of these molecules. Specifically, we summarize the total syntheses of natural and designed molecules of the calicheamicin, uncialamycin, tubulysin, trioxacarcin, epothilone, shishijimicin, namenamicin, thailanstatin, and disorazole families of compounds and demonstrate how these studies led to the discovery of analogues of higher potencies, yet some of them possessing lower complexities than their parent compounds as potential ADC payloads. The highlighted examples showcase the continuing impact of total synthesis of natural products and their analogues on modern medicine, including the so-called biologics and should prove useful and inspirational in advancing both the fields of total synthesis and biomedical research and the drug discovery and development process.

A brief history of antibiotics and select advances in their synthesis.

The advent of modern antibiotics contributed enormously to the dramatic extension of human lifespan since their discovery by virtue of their lethal and selective action against pathogenic microbes. And yet despite our powerful arsenal of weapons against these pathogens, the war against them has not been won. And it may never be. Drug resistance is still menacing the society with many lives being lost due to deadly infections caused by continuously evolving strains spread beyond our means to eradicate them or prevent their spreading. Herein, the emergence and evolution of antibiotics is briefly reviewed, and a select number of total syntheses of naturally occurring antibiotics from the authors' laboratories are highlighted. The article concludes with a strong endorsement of the current efforts to intensify our fight against these dangerous pathogens with the hope that, this time, these initiatives will be sufficiently focused and serious enough so as to achieve our set goals of, at least, being prepared and ahead of them as part of our drive to improve humanity's healthcare and wellbeing.

Synthesis and Biopharmaceutical Evaluation of Imatinib Analogues Featuring Unusual Structural Motifs.

A convenient synthesis of imatinib, a potent inhibitor of ABL1 kinase and widely prescribed drug for the treatment of a variety of leukemias, was devised and applied to the construction of a series of novel imatinib analogues featuring a number of non-aromatic structural motifs in place of the parent molecule's phenyl moiety. These analogues were subsequently evaluated for their biopharmaceutical properties (e.g., ABL1 kinase inhibitory activity, cytotoxicity). The bicyclo[1.1.1]pentane- and cubane-containing analogues were found to possess higher themodynamic solubility, whereas cubane- and cyclohexyl-containing analogues exhibited the highest inhibitory activity against ABL1 kinase and the most potent cytotoxicity values against cancer cell lines K562 and SUP-B15. Molecular modeling was employed to rationalize the weak activity of the compounds against ABL1 kinase, and it is likely that the observed cytotoxicity of these agents arises through off-target effects.

Synthesis and biological evaluation of new paclitaxel analogs and discovery of potent antitumor agents.

Reaction of 10-deacetylbaccatin III (III) and its 7-TES derivative (IV) with DAST under various conditions resulted in the formation of an array of new fluorinated and non-fluorinated 13-keto taxoid compounds (2a­4a) through a vinylogous pinacol­pinacolone rearrangement. Further fluorination of some of these products (2a, 3a) with NFSi or Selectfluor gave additional derivatives. Sodium borohydride reduction of the 13-keto group of these products (2a, 2b, 3a, 3b, 4a, 8, 9, 11­14) led to a series of 9α-hydroxy taxoid derivatives, which were esterified using the docetaxel side chain employing the corresponding protected ß-lactam, followed by deprotection to furnish a library of docetaxel analogs and related compounds. A selected number of synthesized compounds (7, 10, 19a, 19b, 21a, 21b, 23, 27, 29, 34­36) were submitted to the National Cancer Institute (NCI) 60 cell line screening program and tested for cytotoxic properties. Taxoids 19a, 19b, 21a, 21b, 23, 27, 29, 34 and 35 were found to exhibit significant anticancer activity against various cancerous cell lines with 23, 27, and 29 being the most potent compounds, demonstrating GI50 values of ≤5 nM in several assays.

Aldehyde dehydrogenase inhibitors: a comprehensive review of the pharmacology, mechanism of action, substrate specificity, and clinical application.

Aldehyde dehydrogenases (ALDHs) belong to a superfamily of enzymes that play a key role in the metabolism of aldehydes of both endogenous and exogenous derivation. The human ALDH superfamily comprises 19 isozymes that possess important physiological and toxicological functions. The ALDH1A subfamily plays a pivotal role in embryogenesis and development by mediating retinoic acid signaling. ALDH2, as a key enzyme that oxidizes acetaldehyde, is crucial for alcohol metabolism. ALDH1A1 and ALDH3A1 are lens and corneal crystallins, which are essential elements of the cellular defense mechanism against ultraviolet radiation-induced damage in ocular tissues. Many ALDH isozymes are important in oxidizing reactive aldehydes derived from lipid peroxidation and thereby help maintain cellular homeostasis. Increased expression and activity of ALDH isozymes have been reported in various human cancers and are associated with cancer relapse. As a direct consequence of their significant physiological and toxicological roles, inhibitors of the ALDH enzymes have been developed to treat human diseases. This review summarizes known ALDH inhibitors, their mechanisms of action, isozyme selectivity, potency, and clinical uses. The purpose of this review is to 1) establish the current status of pharmacological inhibition of the ALDHs, 2) provide a rationale for the continued development of ALDH isozyme-selective inhibitors, and 3) identify the challenges and potential therapeutic rewards associated with the creation of such agents.

Involvement of caspase activation in azaspiracid-induced neurotoxicity in neocortical neurons.

Azaspiracids (AZAs) are a novel group of marine phycotoxins that have been associated with severe human intoxication. We found that AZA-1 exposure increased lactate dehydrogense (LDH) efflux in murine neocortical neurons. AZA-1 also produced nuclear condensation and stimulated caspase-3 activity with an half maximal effective concentration (EC(50)) value of 25.8 nM. These data indicate that AZA-1 triggers neuronal death in neocortical neurons by both necrotic and apoptotic mechanisms. An evaluation of the structure-activity relationships of AZA analogs on LDH efflux and caspase-3 activation demonstrated that the full structure of AZAs was required to produce necrotic or apoptotic cell death. The similar potencies of AZA-1 to stimulate LDH efflux and caspase-3 activation and the parallel structure-activity relationships of azaspiracid analogs in the two assays are consistent with a common molecular target for both responses. To explore the molecular mechanism for AZA-1-induced neurotoxicity, we assessed the influence of AZA-1 on Ca(2+) homeostasis. AZA-1 suppressed spontaneous Ca(2+) oscillations (EC(50) = 445 nM) in neocortical neurons. A distinct structure-activity profile was found for inhibition of Ca(2+) oscillations where both the full structure as well as analogs containing only the FGHI domain attached to a phenyl glycine methyl ester moiety were potent inhibitors. The molecular targets for inhibition of spontaneous Ca(2+) oscillations and neurotoxicity may therefore differ. The caspase protease inhibitor Z-VAD-FMK produced a complete elimination of AZA-1-induced LDH efflux and nuclear condensation in neocortical neurons. Although the molecular target for AZA-induced neurotoxicity remains to be established, these results demonstrate that the observed neurotoxicity is dependent on a caspase signaling pathway.

Cell volume decrease as a link between azaspiracid-induced cytotoxicity and c-Jun-N-terminal kinase activation in cultured neurons.

Azaspiracids (AZAs) are a group of marine toxins recently described that currently includes 20 members. Not much is known about their mechanism of action, although the predominant analog in nature, AZA-1 targets several organs in vivo, including the central nervous system, and exhibits high neurotoxicity in vitro. AZA distribution is increasing globally with mussels being most widely implicated in AZA-related food poisoning events, with human poisoning by AZAs emerging as an increasing worldwide problem in recent years. We used pharmacological tools to inhibit the cytotoxic effect of the toxin in primary cultured neurons. Several targets for AZA-induced neurotoxicity were evaluated. AZA-1 elicited a concentration-dependent hyperpolarization in cerebellar granule cells of 2-3 days in vitro; however, it did not modify membrane potential in mature neurons. Furthermore, in immature cells, AZA-1 decreased the membrane depolarization evoked by exposure of the neurons to 50mM K(+). Preincubation of the neurons with 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), 4-acetamido-4'-isothiocyanato-2,2'-stilbenedisulfonic acid (SITS), 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), amiloride, or ouabain before addition of AZA-1 decreased the AZA-1-induced neurotoxicity and the increase in phosphorylated c-Jun-N-terminal kinase (JNK) caused by the toxin, indicating that disruption in ion fluxes was involved in the neurotoxic effect of AZA-1. Furthermore, short exposures of cultured neurons to AZA-1 caused a significant decrease in neuronal volume that was reverted by preincubation of the neurons with DIDS or amiloride before addition of the toxin. The results presented here indicate that the JNK activation induced by AZA-1 is secondary to the decrease in cellular volume elicited by the toxin.

Identification of distinct thiopeptide-antibiotic precursor lead compounds using translation machinery assays.

Most thiopeptide antibiotics target the translational machinery: thiostrepton (ThS) and nosiheptide (NoS) target the ribosome and inhibit translation factor function, whereas GE2270A/T binds to the elongation factor EF-Tu and prevents ternary complex formation. We have used several in vitro translational machinery assays to screen a library of thiopeptide antibiotic precursor compounds and identified four families of precursor compounds that are either themselves inhibitory or are able to relieve the inhibitory effects of ThS, NoS, or GE2270T. Some of these precursors represent distinct compounds with respect to their ability to bind to ribosomes. The results not only provide insight into the mechanism of action of thiopeptide compounds but also demonstrate the potential of such assays for identifying lead compounds that might be missed using conventional inhibitory screening protocols.

Enhanced microtubule-dependent trafficking and p53 nuclear accumulation by suppression of microtubule dynamics.

The tumor suppressor protein p53 localizes to microtubules (MT) and, in response to DNA damage, is transported to the nucleus via the MT minus-end-directed motor protein dynein. Dynein is also responsible for MT-mediated nuclear targeting of adenovirus type 2 (Ad2). Here we show that treatment with low concentrations of MT-targeting compounds (MTCs) that do not disrupt the MT network but are known to suppress MT dynamics enhanced p53 nuclear accumulation, and the activation of the p53-downstream target genes. p53 nuclear accumulation required binding of MTCs to MTs and enhanced the induction of p53-up-regulated modulator of apoptosis (PUMA) mRNA and apoptosis on challenging cells with the DNA-damaging drug adriamycin. Low concentrations of MTCs enhanced the rate of movement of fluorescent Ad2 to the nucleus and increased the nuclear targeting efficiency of Ad2. We propose that suppression of MT dynamics by low concentrations of MTCs enhances MT-dependent trafficking toward the minus ends of MTs and facilitates nuclear targeting.