Enzymatic Synthesis of the C-Glycosidic Moiety of Nogalamycin R.

Carbohydrate moieties are essential for the biological activity of anthracycline anticancer agents such as nogalamycin, which contains l-nogalose and l-nogalamine units. The former of these is attached through a canonical O-glycosidic linkage, but the latter is connected via an unusual dual linkage composed of C-C and O-glycosidic bonds. In this work, we have utilized enzyme immobilization techniques and synthesized l-rhodosamine-thymidine diphosphate (TDP) from α-d-glucose-1-TDP using seven enzymes. In a second step, we assembled the dual linkage system by attaching the aminosugar to an anthracycline aglycone acceptor using the glycosyl transferase SnogD and the α-ketoglutarate dependent oxygenase SnoK. Furthermore, our work indicates that the auxiliary P450-type protein SnogN facilitating glycosylation is surprisingly associated with attachment of the neutral sugar l-nogalose rather than the aminosugar l-nogalamine in nogalamycin biosynthesis.

Identification of late-stage glycosylation steps in the biosynthetic pathway of the anthracycline nogalamycin.

Nogalamycin is an anthracycline antibiotic that has been shown to exhibit significant cytotoxicity. Its biological activity requires two deoxysugar moieties: nogalose and nogalamine, which are attached at C7 and C1, respectively, of the aromatic polyketide aglycone. Curiously, the aminosugar nogalamine is also connected through a C-C bond between C2 and C5''. Despite extensive molecular genetic characterization of early biosynthetic steps, nogalamycin glycosylation has not been investigated in detail. Here we show that expression of the majority of the gene cluster in Streptomyces albus led to accumulation of three new anthracyclines, which unexpectedly included nogalamycin derivatives in which nogalamine was replaced either by rhodosamine with the C-C bond intact (nogalamycin R) or by 2-deoxyfucose without the C-C bond (nogalamycin F). In addition, a monoglycosylated intermediate-3',4'-demethoxynogalose-1-hydroxynogalamycinone-was isolated. Importantly, when the remaining biosynthetic genes were introduced into the heterologous host by using a two-plasmid system, nogalamycin could be isolated from the cultures, thus indicating that the whole gene cluster had been identified. We further show that one of the three glycosyltransferases (GTs) residing in the cluster-snogZ-appears to be redundant, whereas gene inactivation experiments revealed that snogE and snogD act as nogalose and nogalamine transferases, respectively. The substrate specificity of the nogalamine transferase SnogD was demonstrated in vitro: the enzyme was able to remove 2deoxyfucose from nogalamycin F. All of the new compounds were found to inhibit human topoisomerase I in activity measurements, whereas only nogalamycin R showed minor activity against topoisomerase II.

Synthetic studies on nogarol anthracyclines. Enantioselective total synthesis of an aminohydroxy epoxybenzoxocin.

A chiral synthesis of the aminohydroxy expoxybenzoxocin 6 is described. Enantioselective Friedel-Crafts coupling using a chiral titanium catalyst was employed to produce the optically active atrolactic ester 16a from the phenol 11 and l-menthyl pyruvate (12). The phenolic group in 16a was protected as the benzyl ether and the t-alcohol functionality as the MEM ether to give 20, which after sequential reduction/oxidation provided the aldehyde 22. Addition of the acetylide anion of propargyl aldehyde diethyl acetal (23) to aldehyde 22, followed by oxidation of the resultant diastereoisomeric carbinols, gave the acetylenic ketone 24. Lindlar reduction of 24 afforded the trans-enone 26. Reaction of 26 with thiophenylate anion furnished 27, which was then cyclized to the alpha-methyl pyranoside 29. Oxidation of 29 to the sulfoxide and subsequent thermolysis afforded the hexenulose 30. Sequential epoxidation of 30, reduction of the keto epoxide 31, and reaction of the resultant epoxycarbinol 32 with dimethylamine produced the aminohydroxy pyranose 33a. Debenzylation of 33a to the phenol 33b, followed by intramolecular cyclization, completed the fabrication of the optically active aminohydroxy epoxybenzoxocin 6. The 17-step sequence from the phenol 11 to 6 was achieved in 22% overall yield.

Structure and dynamics of the antitumor drugs nogalamycin and disnogalamycin complexed to d(CGTACG)2: comparison of crystal and solution structures.

The nuclear magnetic resonance (NMR) solution structures of the 2:1 complexes of nogalamycin-d(CGTACG)2 (Ng-CGTACG) and disnogalamycin-d(CGTACG)2 (DNg-CGTACG) have been determined by a quantitative treatment of two-dimensional nuclear Overhauser effect (2D-NOE) crosspeak intensities. The 1.3 A resolution crystal structure of the 2:1 complex of Ng-CGTACG was used as a starting model for refinement using the procedure, SPEDREF [Robinson and Wang, Biochemistry 31 (1992) 3524-3533], which incorporates full matrix relaxation theory and simulated annealing minimization. The refined solution structures have R-factors of 16.1 and 19.6% between the observed and simulated NOEs for Ng-CGTACG and DNg-CGTACG, respectively. The refined NMR structures retain major features of the crystal structure in which the elongated aglycone chromophore is intercalated between the CpG steps with its nogalose and aminoglucose lying in the minor and major grooves, respectively. The root mean square deviation between the solution and crystal structure for the complexes is 1.01 A (Ng-CGTACG) and 1.20 A (DNg-CGTACG) for the drug, plus the three base pairs surrounding the drug, indicating a very similar local structure at the intercalation site. In the NMR structure, the two G:C Watson-Crick base pairs (C1:G12 and G2:C11) that wrap around the aglycone have large buckles, as do those seen in the crystal structure. There is a 22 degree bend at the T3-A4 step in the refined solution structure. This rearrangement of the solution conformation is likely due to the absence of crystal packing. Specific hydrogen bonds between the drug and G:C bases in both grooves of the helix are preserved in the solution structure. A separate study of the 2:1 complex at low pH showed that the terminal G-C base pairing is destabilized.

[Pharmacokinetic studies of menogaril (TUT-7) with rats].

Menogaril (TUT-7) is a novel antitumor antibiotic belonging to anthracyclines. The pharmacokinetic parameters derived from plasma concentration-time profiles after repeated (for 14 days) or single oral administration of TUT-7 to rats were found to be not significantly different by either administration schedule. The rats with artificial liver dysfunction were obtained by subcutaneous application of carbon tetrachloride (CCl4, 1 ml/kg) for 3 days. After oral administration of TUT-7 to the rats with CCl4-induced liver toxicity (3 daily administrations of 1mg/kg, S.C.), the maximum plasma concentrations (Cmax) and AUC of both the unchanged drug and its metabolite N-Demethyl menogaril, were increased. Also over all elimination was slower in animals with liver dysfunction.

Menogaril in the treatment of malignant mesothelioma: a phase II study.

Menogaril is a new semisynthetic anthracycline agent derived from the antitumor antibiotic Nogalomycin. Compared to doxorubicin it has similar or improved activity in anti-tumor cell line screening; human tumor cloning assays suggest modest anti-tumor activity as well. Menogaril is much less cardiotoxic than doxorubicin. We performed a phase II trial of this agent in 22 patients with advanced malignant mesothelioma. At a dose of 200 mg/m2 iv every 4 weeks (160 mg/m2 in previously radiated patients) only 1 of 22 (5%) evaluable patients had a partial remission lasting 4 months. (95% confidence limits 0.1-23%). The major toxic effects included pain at the site of infusion and granulocytopenia. While well tolerated, Menogaril has minimal activity in malignant mesothelioma. We do not plan further studies with Menogaril in this disease.

Justification for evaluating new anticancer drugs in selected untreated patients with extensive-stage small-cell lung cancer: an Eastern Cooperative Oncology Group randomized study.

BACKGROUND: Studies have shown that response to a given chemotherapy in previously untreated patients with extensive-stage small-cell lung cancer is superior to that in patients previously treated with other regimens. This finding raises the question of whether it is necessary and ethical to study the effects of new anticancer agents in untreated patients. Such studies appear to be the best test for drug development, but there has been no evaluation of whether survival of untreated patients, whose cancer is sensitive to established drugs, is adversely affected in trials of new drugs. PURPOSE: This randomized study of untreated patients with extensive-stage small-cell lung cancer was designed (a) to compare the survival of patients treated with either effective standard chemotherapy or an investigational anticancer drug as initial therapy and (b) to evaluate response rates and toxic effects of such therapies. METHODS: Eighty-six patients were randomly assigned to receive, as initial therapy, either the standard CAV regimen--cyclophosphamide (1000 mg/m2), doxorubicin (50 mg/m2), and vincristine (1.4 mg/m2) every 3 weeks--or the phase II drug menogaril (200 mg/m2) every 4 weeks. Treatment after induction therapy varied, depending on patient response, but nonresponders and those with disease progression received salvage chemotherapy--etoposide (120 mg/m2 on days 1, 2, and 3) and cisplatin (60 mg/m2 on day 1), repeated every 3 weeks. RESULTS: Of the 43 patients on CAV, 42% responded (eight complete responses and 10 partial responses); 5% of the 43 on menogaril responded (two partial responses) (P = .0001). Twelve (22%) of 54 patients responded to salvage chemotherapy (five complete responses and seven partial responses). Within 3 months from start of treatment, twelve patients died--3 patients in the CAV group and nine patients in the menogaril group (P = .12). The estimated median survival was 37 weeks with menogaril and 45 weeks with CAV (P = .28). At 6 months, survival was 76.7% for the CAV group and 67.4% for the menogaril group. At 12 months, survival rates were 24.4% and 27.9%, respectively. Confidence intervals (95%) for the differences between the proportions surviving in the two groups were -9%-28% at 6 months and -25%-14% at 12 months. Use of CAV resulted in significantly higher occurrence of severe and life-threatening treatment-related complications (P = .002). CONCLUSION: The confidence intervals for the differences in survival are too wide to conclude that evaluation of a new drug in untreated patients with extensive-stage small-cell lung cancer is or is not harmful. The data do suggest, however, that use of this study design may have no adverse effect on survival.

Phase I study of oral menogaril administered daily for 14 consecutive days.

Twenty-eight patients were treated with oral menogaril daily x 14 every 4 weeks. Granulocytopenia was dose-limiting at 50-60 mg/m2 per day. Neutropenic fever occurred in one patient. Thrombocytopenia occurred in 3 of 6 patients treated with menogaril 60 mg/m2/day. Nausea and vomiting and other toxic effects were generally mild. No cardiac toxicity was seen. One partial remission and three minor responses were noted among 11 previously treated patients with carcinoma of the bladder. One minor response was noted among 3 patients with colorectal cancer. The dose recommended for phase II studies is 50 mg/m2 per day for 14 days. Phase II studies are recommended in carcinoma of the bladder. Gastrointestinal toxicity is substantially less on this schedule than with oral menogaril administered on a weekly or q4wk schedule, but thrombocytopenia may be more common.

Menogaril in the treatment of relapsed multiple myeloma. A phase II trial of the Cancer Center of Wake Forest University.

Fifteen patients with relapsed multiple myeloma (MM) were treated with menogaril 160 mg/m2 intravenously (IV) every 28 days. No responses were seen: 8 patients had stable disease, 4 progressed after one course of therapy, and 3 patients were removed from study after 1 course for other reasons. Four of the 8 patients with stable disease had an improved performance status, and 3 had a decrease in analgesic use. The major toxicity was myelosuppression. The median progression-free interval was 3.0 months with a range of 0.7 to 22 months and median survival was 11.3 months with a range of 0.7 to 39+ months. Menogaril displays little activity in patients with previously treated MM.

Phase II study of oral menogaril as first line chemotherapy for advanced breast cancer: a National Cancer Institute of Canada Clinical Trials Group study.

The National Cancer Institute of Canada (NCIC) Clinical Trials Group conducted a phase II study of weekly oral menogaril as first-line therapy in 51 patients with incurable, metastatic or locally advanced breast cancer. While no prior chemotherapy for metastatic disease was permitted, prior adjuvant chemotherapy was allowed provided that no anthracycline or anthracene had been given. Forty-eight patients were evaluable for response. Two patients (4%) achieved complete remissions, 9 patients (19%) achieved partial remissions, 26 patients (54%) were stable and 11 patients (23%) failed. At the initial menogaril dose of 275 mg/m2 per week, 13 of 14 patients required a dose reduction and/or a treatment delay of one or more weeks. Therefore, the menogaril dose was reduced to 225 mg/m2 per week for the last 37 patients. At that those, 20 of 37 patients developed grade 3 or 4 granulocytopenia and 22 required dosage delays. At the initial starting dose, the average dose intensity actually delivered was 169 mg/m2 per week. At 225 mg/m2 the average dose intensity actually delivered was 197 mg/m2 per week. Toxic effects included mild to moderate nausea and vomiting, diarrhea, hair loss and occasional hyperpigmentation. In summary, menogaril is an anthracycline derivative that has modest activity when administered orally to minimally pretreated patients with breast cancer.

Phase II evaluation of menogaril in patients with advanced cervical carcinoma. A collaborative trial of the North Central Cancer Treatment Group and Mayo Clinic.

Fourteen patients with advanced/recurrent squamous cell carcinoma of the uterine cervix received menogaril, 200 mg/m2 by one hour intravenous infusion at four-week intervals. No objective regressions were observed. Median time to progression was less than two months and median survival was seven months. All patients experienced neutropenia. Platelet toxicity was negligible. Venous irritation and phlebitis occurred at the infusion site in 43% of patients. Menogaril as administered in this protocol is ineffective in treating previously irradiated advanced/recurrent squamous cell carcinoma of the uterine cervix and warrants no further investigation in this disease at the dosage and administration schedule used in this protocol.

Phase II evaluation of menogaril in patients with advanced hypernephroma.

Fifteen patients with advanced renal cell carcinoma were treated with Menogaril, 200 mg/m2 by one-hour, intravenous infusion at four-week intervals. No objective regressions were observed. Median time to progression was two months, and median survival was seven months. All patients experienced neutropenia. Platelet toxicity was negligible. Venous irritation and phlebitis at the infusion site was seen in 47% of patients. Menogaril as administered in this protocol is ineffective in advanced renal cell carcinoma.

A phase II evaluation of menogaril in cisplatin-refractory advanced ovarian carcinoma. A collaborative trial of the North Central Cancer Treatment Group and the Mayo Clinic.

Forty-one women with advanced, recurrent epithelial ovarian carcinoma (in whom prior chemotherapy with a platinum-based regimen failed) were treated with menogaril 200 mg/m2 intravenously every 4 weeks in a Phase II trial. Partial responses were seen in two of 19 (10.5%) measurable disease patients and three of 12 (25%) nonmeasurable but evaluable patients, an overall objective response rate of 16.1% (95% confidence interval, 5% to 34%). Median time to progression for all patients was 2 months and median survival, 5 months. Toxicities were acceptable and consisted primarily of leukopenia and gastrointestinal toxicity. Twenty-nine percent of the patients had venous irritation or painful phlebitis at the intravenous injection site. Menogaril, as administered in this protocol, had modest antineoplastic activity in previously treated ovarian carcinoma patients. The responses were of short duration, and there appeared to be no survival advantage with menogaril treatment.

Phase II evaluation of menogaril (NSC-269148) in non-small cell lung carcinoma. A Southwest Oncology Group study.

Forty-five patients with non-small cell lung cancer were treated in a phase II trial with menogaril 200 mg/m2 IV every twenty-eight days by a one-hour infusion. One partial response was noted while twenty-two patients had stable disease (51%). Progressive disease was noted in the remaining twenty-two patients. There was one fatal complication due to pancytopenia and pneumonia. Otherwise, the drug was reasonably well tolerated. At this dosage and schedule, menogaril has no substantial anti-tumor activity for patients with non-small cell lung cancer.

A phase II study of menogaril in low-grade non-Hodgkin's lymphoma. An NCI Canada Clinical Trials Group study.

The NCI Canada Clinical Trials Group conducted a phase II study of menogaril given intravenously every 4 weeks in low-grade non-Hodgkin's lymphoma. Fifteen of 26 eligible patients had had no prior therapy. Partial responses were seen in 9 patients (35%). Toxicity was moderate including myelosuppression, nausea, phlebitis, alopecia, and lethargy. This drug has only modest activity in this potentially responsive group of patients.

New anthracycline antitumor antibiotics.

Doxorubicin is an essential component of the treatment of aggressive lymphoma, childhood solid tumors, bone and soft tissue sarcomas, and breast cancer and additional indications are emerging. On the other hand, daunorubicin has occupied the central position of interest in the treatment of acute leukemia. Epirubicin has a spectrum very similar to doxorubicin but lesser toxicity. The ability to protect against cardiotoxicity with ICRF-187 further enhances clinical interest in exploiting modifications in doze intensity to therapeutic advantage. Idarubicin has at least equivalent activity to daunorubicin and doxorubicin in leukemia. New areas of research in relation to anthracycline antibiotics include introduction of new the analogs, insight into mechanisms of resistance, the reversal of multidrug resistance in vitro, the protection of cardiac toxicity, and the study of other important biochemical reactions relevant to cytotoxicity. Orally active anthracyclines such as idarubicin and compounds which lack cross-resistance with the parent drugs or have other mechanisms for cytotoxicity are being developed. It is likely that these modifications will lead to an expanding therapeutic spectrum for these already widely useful drugs.

The biochemical pharmacology of nogalamycin and its derivatives.

This review assimilates up-to-date information on the biochemical pharmacology of nogalamycin and selected derivatives that have shown good biological activities and/or received a relatively detailed investigation. The structure and chemical preparation of these derivatives from nogalamycin is described and the nomenclature which has been rather perplexing in the literature is clarified. The interaction of this class of compounds, particularly nogalamycin, with DNA is extensively reviewed. The biochemical mechanism of action of nogalamycin and its structurally closely-related derivatives is described. Among nogalamycin derivatives, menogaril showed distinct biochemical effects as well as superior cytotoxicity and antitumor activity and also proved to be effective against breast cancer clinically.