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The Skaggs Institute
for Chemical Biology

Prodrug Activation, Targeting Therapy, and Organic Synthesis

S.C. Sinha, R.A. Lerner, S. Das, S. Abraham, F. Guo, Z. Chen

In the past year, we focused on the use of aldolase antibodies in prodrug activation and the development of antibody constructs that target the αvβ3vβ5 integrins. We also investigated insulin prodrugs that can be activated by light. In addition, we made significant progress in the synthesis of sorangiolides and the library of annonaceous acetogenins; we prepared 2 new nonnatural analogs of the bis-tetrahydrofuran acetogenin bullatacin.

Prodrug Activation

Aldolase antibodies 38C2 and 93F3 produced by the reactive immunization technique against diketone haptens can catalyze the activation of the prodrugs of numerous chemotherapeutic agents. These monoclonal antibodies most likely will be highly useful as the enzyme component in the antibody-directed abzyme prodrug therapy approach. Using a catalytic monoclonal antibody is better than using the endogenous or exogenous enzymes because the endogenously expressed enzymes can activate a prodrug nonselectively and the nonhuman enzyme can be highly immunogenic to the host. In contrast, a catalytic monoclonal antibody can have unique substrates that are not acted on by natural enzymes, thereby increasing the diversity of chemicals available for prodrug design, and it can be humanized to reduce the immunogenicity to patients.

Using aldol or aldol-Michael linkers, we have synthesized the prodrugs of enediynes, doxorubicin and doxorubicin analogs, and CBI analogs. Activation of these prodrugs is catalyzed by antibodies 93F3 and 38C2 to produce the labile intermediates, which undergo degradation under physiologic conditions, yielding free drugs. Among all the prodrugs, those of doxorubicin analogs seem the most promising (Fig. 1). These prodrugs are activated by antibody 93F3 at a comparatively high rate. Nevertheless, our endeavor to develop more promising prodrugs continues.

Fig. 1. Structure of the prodrugs of doxorubicin analogs.

Integrin-Targeting 38C2 Constructs

Using antibody 38C2 and antagonists of αvβ3vβ5 integrins, we produced several antagonist-38C2 conjugates. These conjugates bound efficiently to cells expressing αvβ3vβ5 and inhibited the growth of several cancerous tumors in animal models. This research was done in collaboration with C.F. Barbas, the Skaggs Institute. In continuation, we produced additional 38C2 constructs that target αvβ3 integrins and evaluated the constructs in the breast cancer cell lines MDA-MB-435 and MDA-MB-231. The new constructs are more selective that the older ones and bind to αvβ3 integrins. We are evaluating the features of new constructs. This research was done in collaboration with B. Mueller, La Jolla Institute for Molecular Medicine, San Diego, California.

Insulin Prodrugs

Hyperglycemia is the cause of most complications associated with diabetes mellitus. To prevent or reduce the complications, patients with diabetes often require multiple insulin injections daily. Alternatively, a closed-loop system of regulated insulin delivery in response to elevated blood glucose levels can be constructed by using insulin prodrugs. We synthesized 2 insulin prodrugs (Fig. 2) and evaluated their ability to stimulate glucose uptake in an in vitro system.

Fig. 2. Protection of the primary amines in insulin by using the photolabile groups to produce the prodrugs insulin-2P and insulin-3P.

The prodrugs were minimally active in the protected state but become active after photolysis with 365-nm light. Thus, they have potential for use in an implantable closed-loop device, coupling a glucose sensor to a small ultraviolet lamp to photoactivate and release the correct dose of insulin in response to elevated glucose levels. This work was done in collaboration with J. Olefsky, University of California, San Diego.

Synthesis of Sorangiolides and Bis-Tetrahydrofuran Acetogenins

Sorangiolides A and B, naturally occurring macrocyclic molecules (Fig. 3), are weakly active against gram-positive bacteria. As a first step to producing more active analogs of these molecules, we synthesized the naturally occurring macrolides. The key steps in the synthesis of these compounds include the Suzuki-Fu reaction, the 1,5-induction in the stereoselective aldol reaction developed by Evans, and the macrolactinization reaction. To synthesize a complete library of bis-tetrahydrofuran annonaceous acetogenins, we have developed a bidirectional approach for the synthesis of all 64 diastereomers of the adjacent bis-tetrahydrofuran acetogenins (Fig. 3).

Fig. 3. Structure of sorangiolides A and B and a general structure of bis-tetrahydrofuran annonaceous acetogenins.

Starting with 8 diene lactones, we synthesized 36 bifunctional adjacent bis-tetrahydrofuran lactones by using 5 key reactions: (1) monooxidative or bis-oxidative cyclization mediated by rhenium(VII) oxides, (2) Shi monoasymmetric or bis-asymmetric epoxidation, (3) Sharpless asymmetric dihydroxylation, (4) Williamson-type etherification, and (5) Mitsunobu inversion. Starting with a bis-tetrahydrofuran lactone, we synthesized 2 nonnatural bullatacin analogs.


Das, S., Li, L.-S., Abraham, S., Chen, Z., Sinha, S.C. A bidirectional approach to the synthesis of a complete library of adjacent-bis-THF annonaceous acetogenins. J. Org. Chem. 70:5922, 2005.

Li, L.-S., Babendure, J.L., Sinha, S.C., Olefsky J.M., Lerner, R.A. Synthesis and evaluation of photolabile insulin prodrugs. Bioorg. Med. Chem. Lett. 15:3917, 2005.

Saphier, S., Hu, Y., Sinha, S.C., Houk, K.N., Keinan, E. Origin of selectivity in the antibody 20F10-catalyzed Yang cyclization. J. Am. Chem. Soc. 132:127, 2005.


Subhash C. Sinha, Ph.D.
Associate Professor