Hari Krishna Santhapuram

Folate-vinca alkaloid conjugates for cancer therapy: a structure-activity relationship.

Vintafolide is a potent folate-targeted vinca alkaloid small molecule drug conjugate (SMDC) that has shown promising results in multiple clinical oncology studies. Structurally, vintafolide consists of 4 essential modules: (1) folic acid, (2) a hydrophilic peptide spacer, (3) a disulfide-containing, self-immolative linker, and (4) the cytotoxic drug, desacetylvinblastine hydrazide (DAVLBH). Here, we report a structure-activity study evaluating the biological impact of (i) substituting DAVLBH within the vintafolide molecule with other vinca alkaloid analogues such as vincristine, vindesine, vinflunine, or vinorelbine; (ii) substituting the naturally (S)-configured Asp-Arg-Asp-Asp-Cys peptide with alternative hydrophilic spacers of varied composition; and (iii) varying the composition of the linker module. A series of vinca alkaloid-containing SMDCs were synthesized and purified by HPLC and LCMS. The SMDCs were screened in vitro against folate receptor (FR)-positive cells, and anti-tumor activity was tested against well-established subcutaneous FR-positive tumor xenografts. The cytotoxic and anti-tumor activity was directly compared to that produced by vintafolide. Among all the folate vinca alkaloid SMDCs tested, DAVLBH-containing SMDCs were active, while those constructed with vincristine, vindesine, or vinorelbine analogues failed to produce meaningful biological activity. Within the DAVLBH series, having a bioreleasable, self-immolative linker system was found to be critical for activity since multiple analogues constructed with thioether-based linkers all failed to produce meaningful activity both in vitro and in vivo. Substitutions of some or all of the natural amino acids within vintafolides hydrophilic spacer module did not significantly change the in vitro or in vivo potency of the SMDCs. Vintafolide remains one of the most potent folate-vinca alkaloid SMDCs produced to date, and continued clinical development is warranted.

Carbohydrate-based synthetic approach to control toxicity profiles of folate-drug conjugates.

To better regulate the biodistribution of the vinblastine-folate conjugate, EC145, a new folate-spacer that incorporates 1-amino-1-deoxy-D-glucitol-gamma-glutamate subunits into a peptidic backbone, was synthesized. Synthesis of Fmoc-3,4;5,6-di-O-isopropylidene-1-amino-1-deoxy-D-glucitol-gamma-glutamate 20, suitable for Fmoc-strategy solid-phase peptide synthesis (SPPS), was achieved in four steps from delta-gluconolactone. Addition of alternating glutamic acid and 20 moieties onto a cysteine-loaded resin, followed by the addition of folate, deprotection, and cleavage, resulted in the isolation of the new folate-spacer: Pte-gammaGlu-(Glu(1-amino-1-deoxy-D-glucitol)-Glu)(2)-Glu(1-amino-1-deoxy-D-glucitol)-Cys-OH (21). The addition of 21 to an appropriately modified desacetylvinblastine hydrazide (DAVLBH) resulted in a conjugate (25) with an improved therapeutic index. Treatment of 25 with DTT in neutral buffer at room temperature demonstrated that free DAVLBH would be released under the reductive environment of the internalized endosome.

Latent Warheads for Targeted Cancer Therapy: Design and Synthesis of pro-Pyrrolobenzodiazepines and Conjugates

Pyrrolobenzodiazepines (PBDs) and their dimers (bis-PBDs) have emerged as some of the most potent chemotherapeutic compounds, and are currently under development as novel payloads in antibody-drug conjugates (ADCs). However, when used as stand-alone therapeutics or as warheads for small molecule drug conjugates (SMDCs), dose-limiting toxicities are often observed. As an elegant solution to this inherent problem, we designed diazepine-ring-opened conjugated prodrugs lacking the imine moiety. Once the prodrug (pro-PBD) conjugate enters a targeted cell, cleavage of the linker system triggers the generation of a reactive intermediate possessing an aldehyde and aromatic amine. An intramolecular ring-closing reaction subsequently takes place as the aromatic amine adds to the aldehyde with the loss of water to give the imine and, as a result, the diazepine ring. In our pro-PBDs, we mask the aldehyde as a hydrolytically sensitive oxazolidine moiety which in turn is a part of a reductively labile self-immolative linker system. To prove the range of applications for this new class of latent DNA-alkylators, we designed and synthesized several novel latent warheads: pro-PBD dimers and hybrids of pro-PBD with other sequence-selective DNA minor groove binders. Preliminary preclinical pharmacology studies showed excellent biological activity and specificity.

Abstract 4462: Structure-activity relationship studies for PSMA-targeted tubulysin conjugates

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Prostate-specific membrane antigen (PSMA) is a cell-surface receptor expressed on prostate cancer cells. Recent findings suggest that PSMA is also abundant on newly formed blood vessels which supply most non-prostatic solid tumors; including lung, colon, breast, renal, liver, and pancreatic carcinomas. These reports have provided solid support for the concept of designing drug conjugates for PSMA-targeted cancer therapy. Here we describe our structure-activity relationship analyses resulting in the discovery of highly potent PSMA-targeted tubulysin conjugates. In our study, more than thirty ligand-drug constructs were designed and synthesized based on strategically placing a variety of chemical groups responsible for evoking a targeted biological effect in vitro and in vivo. The optimized molecular architecture is comprised of a high affinity PSMA-binding urea-type ligand, a water soluble peptide- or carbohydrate-based spacer unit, a cleavable disulfide-containing self-immolative linker system, and the highly potent chemotherapy drug tubulysin which inhibits microtubule activity . One of the conjugates possessed superior targeted activity and was selected for further preclinical development. Citation Format: Kevin Wang, Spencer Hahn, Hari Santhapuram, Longwu Qi, Paul Kleindl, Jeremy Vaughn, Fei You, Joe Reddy, Ryan Dorton, Christopher Leamon, Iontcho Vlahov. Structure-activity relationship studies for PSMA-targeted tubulysin conjugates. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4462. doi:10.1158/1538-7445.AM2014-4462

Abstract 4499: Total synthesis of tubulysins: A new chemical reaction leads to analogues with enhanced cytotoxicity.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Tubulysins are natural products isolated from myxobacterial species and are mitotic poisons which exceed the cell growth inhibition of any clinically relevant traditional chemotherapeutic. Structurally, tubulysins are linear tetrapeptides comprised of N-methyl pipecolic acid (Mep), isoleucine (Ile), the novel amino acid tubuvaline (Tuv), and the novel tyrosine analogue tubutyrosine (Tut). All isolated tubulysins possess an acid- and base-sensitive N-acyloxymethyl substituent not previously found in nature. This N,O-acetal of formaldehyde is attached as a side chain to the amide N-atom of the Tuv fragment. The isolation of natural tubulysins from culture extracts provides only limited quantities. Recently, we reported a large scale total synthesis of natural tubulysin B. Among the multiple synthetic and stereochemical challenges, the most striking were: a) the incorporation of the labile N,O-diacyl N,O-acetal and b) the regioselective hydrolysis of the C-terminal methyl ester (OMe) in the tripeptide Mep-Ile-Tuv-OMe. Herein, we present a convergent total synthesis of tubulysin analogues incorporating an alkoxymethyl side chain. Such molecular architecture allows for compounds that are more base and esterase inert, thus providing additional metabolic/catabolic stabilization. The key step in our synthetic strategy relies on use of a novel reagent for the efficient conversion of the N-acyloxymethyl substituent to novel side chain groups with concomitant facilitated hydrolysis of the C-terminal methyl ester. Following LiOH-based hydrolysis of the C-terminal ester, the Tut fragment is added resulting in novel tubulysin analogues. This novel process opens the door to tubulysin analogs with improved potency for treating cancer. Citation Format: Iontcho Vlahov, Longwu Qi, Fei You, Spencer Hahn, Hari K. Santhapuram, Kevin Wang, Paul Kleindl, Jeremy Vaughn, Marilynn Vetzel, Joseph Reddy, Christopher Leamon. Total synthesis of tubulysins: A new chemical reaction leads to analogues with enhanced cytotoxicity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4499. doi:10.1158/1538-7445.AM2013-4499

Abstract 5515: Design and synthesis of releasable folate-drug conjugates possessing optimized carbohydrate-based spacer units

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The spacer region of releasable folate-drug conjugates may play an important role in “tailoring” the properties of the molecules for targeted therapies by: 1) modulating the affinity toward the folate receptor, 2) providing desirable hydrophilicity, and 3) changing dramatically the biological properties. Herein we describe the design and synthesis of a variety of novel carbohydrate-based spacers mimicking a peptidic backbone. After carefully optimizing the chemical structure of the carbohydrate units, we incorporated such spacers in a folate-containing molecular systems using standard solid phase synthesis (SPS). The synthetic utility of this elegant and innovative approach was exemplified by creating novel releasable folate conjugates of single cytotoxic agents, e.g. Tubulysin-Folate conjugate (currently in preclinical development) and Vinblastine-Folate conjugate, (already in clinical development). Furthermore, using the same powerful method, we synthesized several constructs containing a rational combination of two drugs (e.g. Vinblastine and Doxorubicin) attached to a single folate-targeting ligand via a dual-headed releasable Spacer-Linker system. 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 5515.