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The Modernization Act of 1997 is the result of a partnership between the US Food and Drug Administration (FDA) and the pharmaceutical industry. Highlights of the Act, including agreements on user fees, timely review, pediatric studies, and national registry of clinical trials, are presented. Although progress has been made in each of these areas, this paper concerns the profound impact of combinatorial chemistry, genetic research, and pharmacoeconomics on the FDA, the industry, and the drug development process.

A new route has been developed for the preparation of 3'-N-protected doxorubicin analogues. 4-Demethoxy-3'-N-trifluoroacetyldoxorubicin (5) was synthesized in an approach to an orally active anthracycline analogue. Tested against the B-16 murine solid tumor in mice, this compound increased life span by 133% when it was administered intraperitoneally at 25 mg/kg, and by 52% when it was given orally at 50 mg/kg.

The inhibition of chymotrypsin activity by pig, dog, human and rat blood plasma was studied. N-Benzoyl-L-tyrosyl-p-amino-benzoate (PABA-peptide) was used as the substrate. Crystalline bovine chymotrypsin as well as activated lyophilized human, pig, rat and chicken pancreatic secretions were used as enzyme sources. Certain species differences were noted. Swine plasma had no effect on bovine or human chymotrypsin while it inhibited that of the chicken by 67%. Dog plasma was a potent inhibitor of chymotrypsin activity from all sources tested. An acute pancreatitis model using the rat was also developed in which pancreatic juice flow was blocked without interference with biliary flow. After 24 h, these animals had increased plasma amylase activity, decreased plasma protease inhibitor and decreased hematocrit. By 72 h, amylase, trypsin inhibitor and hematocrit had nearly recovered while chymotrypsin inhibitor had actually increased above control levels. In rats subjected to hepatectomy or to hepatectomy plus pancreatic blockage, plasma protease inhibitor was even more severely depressed and remained so.

Previously we reported that reductive alkylation of doxorubicin with 2,2'-oxybis[acetaldehyde] and NaBH3CN to form the 4''-morpholinyl derivative also gave the intensely potent 3''-cyano-4''-morpholinyl as a byproduct, by addition of CN- to an iminium intermediate in place of hydride. We now find that sugar 4'-OH is a third nucleophile that can add to the iminium intermediate in this reaction. Bridging of the 4'-OH to the morpholine ring at C.5'' formed a novel byproduct with an oxazolidino ring fused to the sugar and morpholine. The new product was minor at neutral pH but predominant at an acidic pH. When tested against tumors in mice it was 4-6 times more potent than doxorubicin. Hence, in comparison with the 3''-cyano-4''-morpholinyl, potency was reduced up to 100-fold by the O bridge. Analytical HPLC showed the presence of three of the four possible diastereoisomers, and two were isolated. The diastereoisomers appeared to differ in stability. In vitro tests suggested that biological potency varied inversely with stability.

The novel anthracycline analogue 4-demethoxy-10,10-dimethyldaunomycin was prepared in nine chemical steps from 5,8-dimethoxy-2-tetralone. It proved to be inactive as an antitumor agent in the mouse P388 lymphocytic leukemia model.

The possibility of a new approach to diagnosis of intestinal bacterial overgrowth has been evaluated in laboratory animals. The diagnostic test involves oral administration of an enzyme-labile substrate consisting of para-aminobenzoic acid (PABA) conjugated to a bile acid. In the presence of enteric bacteria, PABA is split from the bile acid and is rapidly absorbed and excreted in the urine. The amount of PABA recovered during the 6 hr following the administered dose of the conjugate may be used as an index of bacterial overgrowth in the upper-gastrointestinal tract. The procedure has been shown to be a reliable index of this condition in laboratory animal models.

Studies to determine the feasibility of using lightly cross-linked carboxyvinyl polymers as gastrointestinal sorbents in the treatment of uremia were conducted in normal and uremic rats. In normal rats, a highly swellable bead-form resin (150-2190) was found to decrease urinary N excretion and to increase fecal excretion of NH3, urea, total N, Na, K, Ca and Mg when fed at a level of 5 g/100g diet. There was also an increase in fecal water content and a marked decrease in fecal phosphate excretion. In rats made uremic by treatment with uranyl nitrate, 150-2190 decreased the rate of increase in BUN.

Treatment of doxorubicin with formaldehyde and NaCN afforded the N-(cyanomethyl) derivative as a stable alpha-cyanoamine with but moderate antitumor activity in mice, although it was prototypal to the intensely potent alpha-cyanomorpholine derivative. 2-Methoxyacetaldehyde and NaCN afforded the N-(2-methoxy-1-cyanoethyl) derivative as an open-chain analogue of the cyanomorpholine. This analogue underwent rapid hydrolysis to doxorubicin and appeared to act as a prodrug, giving increased antitumor efficacy although with decreased potency. N-(Carboxymethyl)daunorubicin was a highly water-soluble but inactive analogue, synthesized by N-alkylation with ethyl iodoacetate and saponification. The similar N-alkylation of N-(cyanomethyl) daunorubicin demonstrated the combining of N-alkyl chains having different functional substituents.

The susceptibility of 3'-deamino-3'-(3-cyano-4-morpholinyl)doxorubicin to hydrolysis at pH 7, 4, and 2 has been compared with that of the typically stable morpholine analogue. At pH 7, 74% of the cyanomorpholine was unchanged after 24 h at room temperature, but at pH 2 only 10% remained. Products identified were aglycon (8%) and N-(2-hydroxyethyl)doxorubicin (7%). Most of the losses were to unidentified polar products not eluted from HPLC. Authentic hydroxyethyl was synthesized from doxorubicin by reductive alkylation with glycolaldehyde. Antitumor potency was comparable to that of doxorubicin rather than of cyanomorpholine.

The 9-aza analogue of N-(trifluoroacetyl)-4-demethoxydaunomycin has been synthesized from 2,5-dimethoxybenzaldehyde. Pomeranz-Fritsch condensation followed by borohydride reduction and acid-catalyzed cyclization led smoothly to 4-hydroxy-5,8-dimethoxy-1,2,3,4-tetrahydroisoquinoline. Selective N-acetylation and subsequent Friedel-Crafts acylation with phthalic anhydride produced 2-acetyl-5,12-dihydroxy-1,2-dihydro-2-azanaphthacene-6,11-dione, which was protected as its dimethyl ether and epoxidized to an acylated aza Brigl's anhydride. This was converted to (+/-)-2-acetyl-4-hydroxy-5,12-dimethoxy-1,2,3,4-tetrahydro-2- azanaphthacene-6,11-dione by dehydration to the 4-keto analogue followed by cyanoborohydride reduction either stepwise or in situ. The protecting groups were removed with boron trichloride and the resulting aglycone glycosidated with optically active N,O-bis(trifluoroacetyl)daunosamine bromide and silver trifluoromethanesulfonate. The resulting diastereoisomers were separated by column chromatography and their structures established by CD and NMR spectroscopy. Unexpectedly it was not possible to remove the N-trifluoroacetyl blocking group without aromatization to the azanaphthaquinone. Both (R)- and (S)-acetyl-4-O-[N-(trifluoroacetyl)daunosaminyl]-5,12-dihydroxy-2- azanaphthacene-6,11-dione were inactive ip in mice carrying the P388 tumor. Drugs were given at various concentrations on days 0, 5, and 9.

3'-Deamino-3'-(3-cyano-4-morpholinyl)doxorubicin is a new analogue that is 100 to 1000 times more potent than doxorubicin against tumors in cell culture or in mice, that is active by intraperitoneal, intravenous, or oral dosing, and that does not produce chronic myocardial lesions in mice. This analogue was encountered in studies on the reductive alkylation of doxorubicin and daunorubicin with 2,2' - oxybis [acetaldehyde], which constructs a morpholino ring incorporating the amino N. The morpholinyl nitrile byproducts are separated by virtue of their nonbasicity from the expected morpholino derivatives. The 13-dihydro and 5-imino derivatives are also described in this important new class of anthracyclines.