The Discovery of Thiazole-4-Carboxamide Adenine Dinucleotide (TAD

Downloaded by UNIV OF GUELPH LIBRARY on July 27, 2012 | http://pubs.acs.org Publication Date: March 3, 2003 | doi: 10.1021/bk-2003-0839.ch010

Chapter 10

The Discovery of Thiazole-4-Carboxamide Adenine Dinucleotide (TAD) and a Recent Synthetic Approach for the Construction of a Hydrolytically Resistant Surrogate Victor E. Marquez National Cancer Institute, National Institutes of Health, Laboratory of Medicinal Chemistry, Center for Cancer Research, Building 376 Boyles Street, Room 104, NCI-Frederick, P.O. Box B, Frederick, MD 21702 ([email protected])

A short account of the mechanism of action of the oncolytic nucleoside, tiazofurin, and the discovery of its active metabolite, TAD, is described. The chapter also includes a brief history of the early chemical approaches used for the synthesis of T A D and other analogues, including the phosphodiesterase-resistant β-methylene TAD phosphonate. A new synthetic approach to the latter compound is highlighted as a viable way to obtain this class of compounds in larger quantities.

The C -nucleoside tiazofurin (2-p-D-ribofuranosylthiazole-4-carboxamide, la, X = S) was first synthesized ^ by ICN chemists as one in a series of potential antiviral compounds (1,2). Although the compound exhibited moderate antiviral activity, it was not sufficient to warrant further development as an antiviral agent (3). However, initial results from tests conducted at the National Cancer Institute revealed that tiazofurin was an effective antitumor agent as judged by the percent increase in life span (ILS) in murine models (Table 1). Furthermore, tiazofurin proved to be effective and curative against an extremely resistant tumor line such as Lewis Lung carcinoma (Table 2) (4). 198

U.S. government work. Published 2003 American Chemical Society

In Inosine Monophosphate Dehydrogenase; Pankiewicz, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.


199

l b , X = Se (selenazofurin) Table 1. Effect of tiazofurin (la) on several mouse tumor models Tumor System %ILS Treatment schedule (median) and route 134 IPL1210 QD 1-9 (IP) leukemia 127 IPL1210 QD 1-9 (PO) leukemia QD 1-5 (IV) IPL1210 126 leukemia IP P388 127 QD 1-9 (IP) leukemia 224 IV Lewis Lung QD 1-9 (IP) IC Lewis Lung p D 1-9 (IP) 108 tumor implated IP = intraperitoneal, IV ^intravenous treatment IP = intraperitoneal, PO = oral, IV = intravenous 1

a

6

Table 2. Effect of tiazofurin on the life span of mice inoculated Drug Control Tiazofurin (la) QD 1-9 (IP)

Dose (mg/Kg) 400 200 100 50 25

60-day survivors 0/40 7/10 10/10 9/10 10/10 10/10



200 The complex studies that ultimately deciphered the mechanism of action of tiazofurin have been summarized in two excellent reviews (5,6). Briefly, two laboratories almost simultaneously confirmed that the oncolytic effect of tiazofurin was dependent on its conversion to a phosphodiester analogue of N A D , the ordinary cofactor in the inosine monophosphate dehydrogenase (IMPD) reaction (7,8). Formation of this metabolite, thiazole-4-carboxamide adenine dinucleotide (2, TAD), was responsible for the profound dosedependent fall in the intratumoral concentration of GTP resulting from the potent inhibition of IMPD (5,6). IMPD catalyzes a unique step in the de novo biosynthesis of guanine nucleotides, which is considered essential for supporting cell proliferation. This NAD-dependent reaction is responsible for the conversion of IMP to XMP, which is then aminated to GMP by GMP synthetase (Figure 1). Through the successive action of several enzymes, GMP is then converted to some of the building blocks of DNA (dGTP) and R N A (GTP) synthesis. 5-phosphoribosyl1-pyropyosphate

Guanosine PNP

IMP

IMPD

NAD

XMP

GMP Synthase

GMP

HGPRT

Guanine

NADH

f GDP dGDP

DNA synthesis

dGTP

GTP

RNA synthesis

Figure 1. Schematic representation of the de novo and salvage pathways of purine nucleotide biosynthesis converging on GMP The isolation and characterization of TAD (7,8) was soon followed by its first total synthesis, which firmly confirmed the identity of the metabolite (9).


201


With N A D as the variable substrate, TAD showed non-competitive inhibition of IMPD with a K , in the submicromolar range (Ki = 0.12 uM). T A D did not have substrate activity as a coenzyme, i.e. the molecule did not undergo reduction, and its inhibitory activity was very specific as shown by its inability to inhibit other dehydrogenases at concentrations capable of inhibiting IMPD (5,6).

Figure 2. Biosynthesis of TAD

The biosynthesis of TAD starts with the generation of the 5'monophosphate of tiazofurin (la-MP) by adenosine kinase and/or by 5'nucleotidase (10). The ensuing formation of TAD from la-MP and ATP is mediated by N A D pyrophosphorylase acting in the synthetic direction (Figure 2) (5,6,11). Since levels of IMPD are higher in tumors as compared to normal tissues (12-16), inhibition of this enzyme might account for the observed antitumor activity of tiazofurin commensurate with the formation of TAD. Human IMPD exists as two isoforms (I and II) of which type I is predominant in normal cells and type II is upregulated in tumor cells (17-19). Unfortunately TAD and structurally similar analogues showed equal effective inhibitory activity against both isotypes (20,21). The recently solved X-ray structures of IMPD, particularly that of IMPD type II complexed with 6-chloropurine riboside 5'-monophosphate and with the selenium analogue of TAD (selenazole4-carboxamide adenine dinucleotide, SAD) (22) suggest important strategies for the design of isoform-specific inhibitors (23). The respective formation of T A D and SAD from tiazofurin and selenazofurin (lb, X = Se) is unique since other structurally related compounds such as ribavirin (3,R) and the natural purine precursor 5-amino-4imidazolecarboxamide ribonucleoside (4, Z) do not form the corresponding dinucleotides (RAD and ZAD) in vivo (Table 3). Furthermore, synthetically made nicotinamide adenine dinucleotide analogues of 3 and 4, prepared in the same manner as TAD and SAD, failed to show any inhibition of IMPD (Table 4) (24).


202 HN 2

HO-%

_

* N N

The Discovery of Thiazole-4-Carboxamide Adenine Dinucleotide (TAD

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