Furan-2-carboxamide derivative, a novel microtubule stabilizing agent induces mitotic arrest and potentiates apoptosis in cancer cells.

The vital role played by microtubules in the cell division process, marks them as a potential druggable target to decimate cancer. A novel furan-2-carboxamide based small molecule, is a selective microtubule stabilizing agent (MSA) with IC50 ranging from 4 µM to 8 µM in different cancer cell lines. Inhibition of tubulin polymerization or stabilization of tubulin polymers abrogates chromosomal segregation during cell division, results in cell cycle arrest and leads to cell death due to the delayed repair mechanism. A novel furan-2-carboxamide based small molecule exhibited potent anti-proliferative and anti-metastatic property In-Vitro against the panel of cancer cells. Annexin V-FITC/PI, double staining reveals potent cytotoxic effect of SH09 against HeLa cells. FACS analysis displays induction of G2/M arrest and accumulation of subG1 population of cells upon treatment with SH09. Molecular docking study unveils SH09 binding affinity to the Taxol binding pocket of tubulin proteins and MM-GBSA also confirms strong binding energies of SH09 with tubulin proteins.

Plasmid loss in plasmid-carrying strains of Escherichia coli treated with phenoxazines and an approach to study their DNA binding properties.

The effect of subinhibitory concentrations of 2-trifluoromethyl-N(10)-substituted phenoxazines on plasmid-coded antibiotic resistance in Escherichia coli was investigated. Phenoxazine treatment resulted in the loss of resistance markers to an extent of 8-63% in all the strains tested, and the disappearance of plasmid DNA in phenoxazine sensitive colonies was evidenced by agarose gel electrophoresis. The resistant strains were sensitized in the presence of phenoxazines with a concomitant reduction in the MIC (minimum inhibitory concentration) values. The UV, fluorescence spectral, and ethidium bromide displacement agarose gel assay methods revealed that phenoxazines are intercalated with plasmid DNA. Progressive addition of DNA led to a significant reduction in the peak intensity of the absorption maximum of phenoxazine derivative. Further, destabilization of ethidium bromide-DNA complex as seen from fluorescence microscopy in the presence of phenoxazines was observed. The potency of phenoxazines to sensitize the resistant organisms follows the order butyl > propyl > acetyl derivatives.

Targeting calmodulin in reversing multi drug resistance in cancer cells.

Calmodulin is a Ca2+ binding protein found in many eukaryotic cells. It is one of the most important intracellular mediators of Ca2+-dependant signaling in eukaryotic cells. It regulates diverse processes including mitosis, muscle contraction and nucleotide metabolism by modulating the activity of at least 30 different target enzymes in a calcium-dependant manner. Calmodulin plays an important role in the regulation of processes, such as the assembly and disassembly of microtubules by controlling protein kinase activities, by exerting an indirect influence upon a wide variety of cellular processes. It is observed that multi-drug resistant cells have a greater intracellular concentration of calcium than non-resistant cells which contributes to their increased sensitivity to calmodulin antagonism compared with that of non resistant cells. Calmodulin mediated processes can be effectively inhibited by a variety of pharmacological agents of different chemical structures, eg:The calcium channel blocker verapamil and antipsychotic drugs like the Phenothiazines. Many bioisosteres of phenothiazines like phenoxazines and acridones have been prepared and these have also shown very good calmodulin antagonism. These calmodulin antagonists have been shown to modulate multi-drug resistance (MDR) in cancer cells. This review highlights concepts of identification and optimization of new inhibitors of calmodulin in reversing MDR in cancer cells.

Sensitization of multidrug resistant (MDR) cancer cells to vinblastine by novel acridones: correlation between anti-calmodulin activity and anti-MDR activity.

Multidrug resistance (MDR) of cancer cells remains to be an important cause of chemotherapy failure. Search for the new MDR reversal agents is still an unceasing challenge for the scientists. In an attempt to find clinically useful modulators of MDR, a series of 19 N(10)-substituted-2-bromoacridones has been synthesized. Parent compound 1, prepared by the Ullmann condensation of o-chlorobenzoic acid and p-bromoaniline, undergoes N-alkylation in the presence of a phase transfer catalyst. N-(omega-Chloroalkyl) analogues were subjected to iodide catalyzed nucleophilic substitution reaction with various secondary amines to get the products 3-10 and 12-19, which increased the uptake of vinblastine (VLB) in MDR KBCh(R)-8-5 cells to a greater extent (1.25 to 1.9-fold) than did a similar concentration of the standard modulator, verapamil (VRP). Results of the efflux experiment showed that each modulator significantly inhibited the efflux of VLB, suggesting that they may be competitors for P-gp. All the compounds effectively compete with [(3)H] azidopine for binding to P-gp, pointed out this transport membrane protein as their likely site of action. Compounds at IC(10) were evaluated for their efficacy to modulate the cytotoxicity of VLB in KBCh(R)-8-5 cells and found that the modulators enhanced the cytotoxicity of VLB by 3.8 to 34-fold. The study on the structure-activity relationship revealed that substitution of hydrogen atom at position C-2 in acridone nucleus by a bromine atom increased the cytotoxic and anti-MDR activities. The ability of acridones to inhibit calmodulin-dependent cyclic AMP phosphodiesterase has been determined and the results have shown a strong positive correlation between anti-calmodulin activity and cytotoxicity in KBCh(R)-8-5 cells or anti-MDR activity.

Expression of hyaluronan in human tumor progression.

BACKGROUND: The development and progression of human tumors is accompanied by various cellular, biochemical and genetic alterations. These events include tumor cells interaction with extracellular matrix molecules including hyaluronan (HA). Hyaluronan is a large polysaccharide associated with pericellular matrix of proliferating, migrating cells. Its implication in malignant transformation, tumor progression and with the degree of differentiation in various invasive tumors has well accepted. It has been well known the role HA receptors in tumor growth and metastasis in various cancer tissues. Previously we have observed the unified over expression of Hyaluronic Acid Binding Protein (HABP), H11B2C2 antigen by the tumor cells in various types progressing tumor tissues with different grades. However, the poor understanding of relation between HA and HA-binding protein expression on tumor cells during tumor progression as well as the asymmetric observations of the role of HA expression in tumor progression prompted us to examine the degree of HA expression on tumor cells vs. stroma in various types of human tumors with different grades. METHODS: In the present study clinically diagnosed tumor tissue samples of different grades were used to screen the histopathological expression of hyaluronan by using b-PG (biotinylated proteoglycan) as a probe and we compared the relative HA expression on tumor cells vs. stroma in well differentiated and poorly differentiated tumors. Specificity of the reaction was confirmed either by pre-digesting the tissue sections with hyaluronidase enzyme or by staining the sections with pre-absorbed complex of the probe and HA-oligomers. RESULTS: We show here the down regulation of HA expression in tumor cells is associated with progression of tumor from well differentiated through poorly differentiated stage, despite the constant HA expression in the tumor associated stroma. CONCLUSION: The present finding enlighten the relative roles of HA expression on tumor vs. stroma during the progression of tumors.

Interaction of 2-chloro-N10-substituted phenoxazine with DNA and effect on DNA melting.

Five N10-substituted phenoxazines having different R groups and -Cl substitution at C-2 were found to bind to calf -thymus DNA and plasmid DNA with high affinity as seen from by UV and CD spectroscopy. The effect of phenoxazines on DNA were studied using DNA-ethidium bromide complexes. Upon addition of phenoxazines, the ethidium bromide dissociated from the complex with DNA. The binding of phenoxazines to plasmid PUC18 reduced ethidium bromide binding as seen from the agarose gel electrophoresis. Butyl, and propyl substituted phenoxazines were able to release more ethidium bromide compared with that of acetyl substitution. Addition of phenoxazines also enhanced melting temperature of DNA.

Interaction of substituted phenoxazine chemosensitizers with bovine serum albumin.

The binding of 10-[3'-[N-bis(hydroxyethyl)amino]propyl]phenoxazine [BPP], 10-[3'-[N-bis(hydroxyethyl)amino]propyl]-2-chlorophenoxazine [BPCP], 10-[3'-[N-bis-(hydroxyethyl)amino]propyl]-2-trifluoromethylphenoxazin e [BPFP], 10-(3'-N-pyrrolidino propyl)-2-chlorophenoxazine [PPCP] or 10-(3'-N-pyrrolidinopropyl)-2-trifluoromethylphenoxazine [PPFP] to bovine serum albumin (BSA) has been measured by gel filtration and equilibrium dialysis methods. The binding of these modulators to bovine serum albumin based on dialysis experiments has been characterized by the following parameters: percentage (beta) of bound drug, the association constant 'K1', the apparent binding constant 'k' and the free energy deltaFdegrees. The binding of phenoxazine derivatives to bovine serum albumin is correlated with their octanol-water partition coefficient, log10P. In addition, the displacing activity of hydroxyzine and acetylsalicylic acid on the binding of phenoxazines to albumin has been studied. The results of the displacing experiments showed that the phenoxazine benzene rings and the tertiary amines attached to the side chain of the phenoxazine moiety are bound to a hydrophobic area on the albumin molecule.

Characterization of 2-chloro-N10-substituted phenoxazines for reversing multidrug resistance in cancer cells.

Twenty-one 2-chloro-N10-substituted phenoxazines have been synthesized and characterized as potential modulators of multidrug resistance (MDR). Many of the compounds, at a concentration of 100 microM, enhanced accumulation of vinblastine (VLB) in drug-resistant KB8-5 cells to a greater extent than the same concentration of verapamil (VRP). However, the effects on VLB accumulation were specific, because these derivatives had little activity in the parental drug-sensitive line KB3-1. The compounds slowed the efflux of VLB from KB8-5 cells, suggesting that the chlorophenoxazines, like VRP, can inhibit P-glycoprotein (P-gp)-mediated efflux of VLB from this cell line. Two of the chlorophenoxazine derivatives, and also VRP, were able to stimulate the vanadate-sensitive ATPase activity attributable to P-gp in membranes isolated from MDR1 baculovirus-infected Sf9 cells. This result suggests that these modulators exert their effect by directly interacting with P-gp. Apart from the parent unsubstituted molecule, 2-chlorophenoxazine, there was a good correlation between log10P and the ability of the compounds to enhance VLB accumulation in KB8-5. This suggests that lipophilicity of a modulator is important, but is not the sole determinant of potency. Within this series of compounds, the optimal structural features for MDR modulation include a hydrophobic phenoxazine ring with a -Cl atom in the C-2 position and a tertiary amine group four carbons from the tricyclic ring. Many of the agents at the IC10 concentration completely reversed the 37-fold VLB resistance in KB8-5 cells. The most active agents in KB8-5 were able to partially reverse VLB resistance in an MDR colon carcinoma cell line GC3/c1 and completely reversed the 86-fold VLB resistance in the MDR1-overexpressing breast carcinoma cell line BC19/3. These same agents could only partially sensitize BC19/3 cells to taxol and doxorubicin, suggesting that the chlorophenoxazine derivatives show some specificity for modulating VLB resistance.

Liquid secondary ionization mass spectrometry and collision-induced dissociation study of 2-chloro-N10-substituted phenoxazines.

Positive-ion liquid secondary ionization mass spectrometry in combination with 3-nitrobenzyl alcohol as the liquid matrix was used to investigate the mass spectral features of a set of 21 N10-substituted derivatives of 2-chlorophenoxazine. The N-10 substitution included propyl, butyl and acetyl groups containing various secondary amines (N,N-diethylamine, N,N-diethanolamine, morpholine, piperidine, pyrrolidone or beta-hydroxyethylpiperazine) or a chloro group. These compounds are potent multi-drug resistance modulators. The molecular ions are observed as M+ and [M + H]+ ions. In general, the fragmentation pathways of these molecules are similar and very straightforward. The phenoxazine ring system remains stable under the Cs+ ion beam bombardment conditions, while fragmentations are observed along the length of the alkyl and acetyl side-chains. The fragmentation reactions were corroborated by acquiring product ion and constant neutral loss tandem mass spectrometric scans of the pertinent ions.

Characterization of a novel bisacridone and comparison with PSC 833 as a potent and poorly reversible modulator of P-glycoprotein.

Novel compounds, composed of two acridone moieties connected by a propyl or butyl spacer, were synthesized and tested as potential modulators of P-glycoprotein (P-gp)-mediated multidrug resistance. The propyl derivative 1,3-bis(9-oxoacridin-10-yl)-propane (PBA) was extremely potent and, at a concentration of 1 microM, increased steady state accumulation of vinblastine (VLB) approximately 9-fold in the multidrug-resistant cell line KB8-5. In contrast to the readily reversible effects of VRP and cyclosporin A on VLB uptake and similar to the effects of the cyclosporin analog PSC 833, this modulation by PBA was not fully reversed 6-8 hr after transfer of cells to PBA-free medium. Continuous exposure to 3 microM PBA was nontoxic and could completely reverse VLB resistance in KB8-5 cells. Consistent with its effects on VLB transport, the drug resistance-modulating effect of PSC 833 was significantly more persistent than that of VRP. However, the effect of PBA was, like that of VRP, rapidly reversed once the modulator was removed from the extracellular environment. PBA was able to compete with radiolabeled azidopine for binding to P-gp and to stimulate P-gp ATPase activity. However, both the steady state accumulation of PBA and the rate of efflux of PBA were similar in drug-sensitive KB3-1 and drug-resistant KB8-5 cells, suggesting that this compound is not efficiently transported by P-gp. These results indicate that PBA represents a new class of potent and poorly reversible synthetic modulators of P-gp-mediated VLB transport.

Analysis of phenoxazine chemosensitizers: an electron ionization and keV-ion beam bombardment mass spectrometry study.

The mass spectral behavior of a set of eight 2- and 10-disubstituted phenoxazines putatively possessing anticancer drug enhancer properties was investigated. Both electron ionization (EI) and keV-ion beam bombardment (liquid secondary ion mass spectrometry, LSIMS) were used. As expected, EI led to extensive fragmentation to produce structurally characteristics ions. Except in one example, the molecular ions were reasonably abundant. Two different liquid matrices--sulfolane and 3-nitrobenzyl alcohol--were used to obtain LSIMS data. The use of the latter produced more stable molecular ions. Ion beam bombardment also produced several structure-specific fragments. A unique feature of the LSI spectra obtained using either of the above matrices is production of both M+. and [M + H]+ ions, with the former being more abundant in most cases. Adduct formation with the liquid matrices was also observed for many compounds.

Pharmacological characterization of N-substituted phenoxazines directed toward reversing Vinca alkaloid resistance in multidrug-resistant cancer cells.

Previously we reported the synthesis and partial characterization of 21 N10-substituted phenoxazines in reversing Vinca alkaloid resistance. Here we report on a subset of these compounds; we have compared their activities in increasing Vinca alkaloid accumulation and reversing drug resistance in KB-ChR8-5 and GC3/c1 (human colon carcinoma) cell lines. Results demonstrated that 1) N-substituted phenoxazines increase accumulation of vinblastine; 2) within this series, there is little correlation or ranking of activity between the two cell lines when Vinca alkaloid accumulation is compared at equal concentrations of modulator; 3) N-substituted phenoxazines demonstrate both quantitative and qualitative differences, compared with verapamil, a standard modulator; and 4) the series includes at least two compounds, 10-[3'-[N-bis(hydroxyethyl)amino]propyl]phenoxazine and 10-(N-piperidinoacetyl)phenoxazine, which increase Vinca alkaloid accumulation but do not significantly inhibit efflux. Additionally, certain of these multidrug resistance modulators significantly enhance accumulation (8-50-fold) of Vinca alkaloids in cell lines with very low or undetectable P-glycoprotein levels, where verapamil has little activity. It is concluded that at least part of the activity of some of these N-substituted phenoxazine modulators may be mediated through a P-glycoprotein-independent mechanism.

Synthesis and chemical characterization of N-substituted phenoxazines directed toward reversing vinca alkaloid resistance in multidrug-resistant cancer cells.

A series of 21 N-substituted phenoxazines has been synthesized in an effort to find more specific and less toxic modulators of multidrug resistance (MDR) in cancer chemotherapy. Thus, N-(omega-chloroalkyl)- and N-(chloroacyl)phenoxazines were found to undergo iodide-catalyzed nucleophilic substitution on reaction with various secondary amines, including N,N-diethylamine, N,N-diethanolamine, morpholine, piperidine, pyrrolidine and (beta-hydroxyethyl)piperazine. Products were characterized by UV, IR, 1H-, and 13C-NMR, mass spectral data, and elemental analyses. All of the compounds were examined for cytotoxicity and for their ability to increase the accumulation of the vinca alkaloids, vincristine (VCR) and vinblastine (VLB) in multidrug-resistant GC3/Cl (human colon adenocarcinoma) and KBChR-8-5 (HeLa variant) cell lines. Compounds were compared to the standard modulator verapamil (VRP). Substitutions on the phenoxazine ring at position 10 were associated with an increase in antiproliferative and anti-MDR activities. Modification of the length of the alkyl bridge and the type of amino side chain also influenced the potency of these effects. From among the compounds examined, 10 derivatives were found to increase the accumulation of VCR and VLB in GC3/Cl and KBChR-8-5 cells relative to the effect of VRP, suggesting that with the exception of pyrrolidinyl, the tertiary amine attachments to the phenoxazine nucleus linked through a three- or four-carbon alkyl chain resulted in enhanced anti-MDR activity. On the basis of their 50% growth inhibitory (IC50) values, five of the ten compounds, namely, 10-(3'-chloropropyl)phenoxazine, 10-[3'-[N-bis(hydroxyethyl)- amino]propyl]phenoxazine, 10-(3'-N-morpholinopropyl)phenoxazine, 10-(4'-N-morpholinobutyl)phenoxazine and 10-(N-piperidinoacetyl)phenoxazine were selected as relatively nontoxic chemosensitizers. These modulators, at nontoxic concentrations, potentiated the cytotoxicity of VCR and VLB in GC3/Cl and KBChR-8-5 cells. Further, two compounds 10-(3'-N-morpholinopropyl)phenoxazine, and the butyl derivative, enhanced accumulation of VLB in GC3/Cl, KBChR8-5 and highly resistant KB-V1 cells to a level significantly greater than the maximal level achieved with VRP. Additional experiments to understand the mechanism of action of these agents in modulating MDR are in progress.

Structural determinants of phenoxazine type compounds required to modulate the accumulation of vinblastine and vincristine in multidrug-resistant cell lines.

Phenoxazine and seven other structurally related compounds were investigated to determine whether they would increase accumulation of Vinca alkaloids in multidrug-resistant (MDR) GC3/C1 (human colon adenocarcinoma) and KB-ChR-8-5 (HeLa variant) cell lines. Among eight compounds examined, phenoxazine caused greater accumulations of vincristine (VCR) and vinblastine (VLB) than the other chemosensitizers. The structure-activity relationship of these compounds for anti-MDR activity suggested an ideal tricyclic ring structure with a basic nitrogen atom at position 10 for modulating the accumulation of Vinca alkaloids. Addition of oxygen to position 5 of the tricyclic ring system further increased the activity, implying that a highly electronegative element with one, or more, lone pair of electrons in the nucleus opposite to heterocyclic nitrogen was a requirement for better anti-MDR activity. The relationship between the concentration of phenoxazine and the potentiation of Vinca alkaloid accumulation in comparison to verapamil was examined. For VCR in GC3/C1 cells, maximal modulation indices were: for verapamil, 1.8; phenoxazine, 8.6; and for VLB, 1.3 for verapamil compared to 3.3 for phenoxazine. In KB-ChR-8-5 cells, for VCR the maximal modulating index values were 9.0 and 4.3, respectively, and for phenoxazine and verapamil and for VLB were 5.0 and 3.7, respectively. Accumulations of VLB in GC3/C1 cells were similar in the presence of 1 microM phenoxazine or 10 mM sodium azide plus 10 mM 2-deoxyglucose. The effects of verapamil and phenoxazine on the accumulation of Vinca alkaloid were additive. Further, phenoxazine decreased the efflux of VLB by 30% in KB-ChR-8-5 cell line and 10% in GC3/C1 cells. In addition to enhancing the cytotoxicities of VCR and VLB, phenoxazine competed relatively weakly for binding to P-glycoprotein with [3H]azidopine and moderately with [3H]azidoverapamil, at equal concentrations, suggesting that the multidrug transporter may be the primary target for phenoxazine.

Modulation by verapamil of vincristine pharmacokinetics and toxicity in mice bearing human tumor xenografts.

The effect of the calcium channel blocker verapamil (VRP) on the accumulation and retention of vincristine (VCR) has been examined in mice bearing xenografts of human rhabdomyosarcomas. The tumors were Rh18, moderately sensitive to VCR, and its subline, Rh18/VCR3, selected in vivo for primary resistance to VCR. Administration of VRP by i.p. bolus at dose levels above 75 mg/kg was limited by acute lethality. At this dose, the maximal concentration in plasma was 24 microM, with rapid elimination such that plasma concentrations reported to modulate resistance in vitro (approximately 5-10 microM) were maintained for less than 60 min. To sustain a 10 microM plasma concentration, mice were infused with VRP at 6.25 mg/kg/hr (150 mg/kg/day) for up to 7 days using osmotic pumps implanted in the peritoneal cavity. Steady-state plasma levels were greater than or equal to 10 microM for at least 96 hr, and this schedule demonstrated minimal toxicity. Administration of VCR 20 hr after the start of VRP infusion produced significant lethality, requiring an 8-fold reduction in the VCR dose. Pharmacokinetic studies showed that VRP markedly increased the uptake and retention of VCR in small intestine, liver and kidney of mice. In small intestine, 8-fold greater levels of VCR were determined 24 hr after VCR administration, and this was associated with in increase in T1/2 for elimination from 350 to 913 min. HPLC analysis of extracts from small intestine showed that greater than 90% of the radiolabel eluted with VCR or 4-desacetyl-VCR. Modulation of VCR retention was also related to the dose of VCR administered. The VRP-sensitive efflux pathway appeared more effective in certain tissues only at higher concentrations of VCR. In contrast, VRP did not alter significantly the uptake and retention of VCR in either the parent or VCR-resistant human xenografts. The data demonstrated that, in the mouse, VRP modulates the uptake and retention of VCR in several tissues, and this may indicate that drug efflux mediated by a VRP-sensitive mechanism (e.g. GP-170, associated with the multiple drug resistance phenotype) has a protective function against xenobiotics in these tissues.

Chemical characterization of the degradation products of vinblastine dihydrogen sulfate.

Vinblastine was incubated in 0.2 M glycine buffer (pH 7.4 or 8.8) containing bovine serum albumin (1%) at 37 degrees C for 72 h. The reaction mixture was extracted with CH2Cl2, and the extract gave 6 major peaks (A, B, C, D, E, and F) with retention times of 5.0, 7.5, 11.0, 13.0, 23.0, and 30.0 min, respectively, in a high-performance liquid chromatography system (muBondapak C18 column; solvent, 50% MeOH in 10 mM KH2PO4, pH 4.5; flow rate, 1.2 ml/min; detector, 254 nm). Vinblastine in this system corresponded with peak C, and its spectral data were identical to those of the parent compound. The UV, infrared, and mass spectral properties of these peaks were as follows [UV (lambda max); infrared (cm-1); mass spectrum (m/z)]: peak A: 214, 266, and 315 nm; 3464, 2850, and 1730; and 769 (MH+); peak B: 213, 258, 285, and 295 nm; 3457, 2951, 2580, and 1734; and 809 (MH+); peak C: 214, 266, 292, and 312 nm; 3457, 2951, and 1734; and 811 (MH+); peak D: 212, 266, 285, and 312 nm; 3467, 2915, and 1734; and 811 (MH+); peak E: 212, 260, 285, 294, and 313 nm; 3479, 2850, and 1734; and 825 (MH+); and peak F: 212, 265, 283, and 312 nm; 3407, 2857, and 1734; and 807 (MH+). These data suggest the following tentative structures for the degradation products: peak A, 4-deacetylvinblastine; peak B, 19'-hydroxy-3',4'-dehydrovinblastine; peak D, an isomer of vinblastine; peak E, 19'-oxovinblastine; and peak F, 3',4'-dehydro-19'-oxovinblastine. The structure of peak A as 4-deacetylvinblastine was further confirmed by chemical synthesis.

Structural studies on the degradation products of vincristine dihydrogen sulfate.

Vincristine was incubated at 37 degrees C for 72 h in 0.2 M glycine buffer (pH 7.4 or 8.8) containing 1% bovine serum albumin. The reaction mixture was extracted with CH2Cl2. High-performance liquid chromatography analysis (muBondapak C18 reverse-phase 10-micron steel column; solvent, 50% MeOH in 10 mM KH2PO4, pH 4.5; flow rate, 1.2 ml/min) of the CH2Cl2 extract gave 6 peaks, A, B, C, D, E, and F, with retention times of 4.8, 6.5, 10.0, 12.5, 17.5, and 23.5 min, respectively. Peak C corresponded with vincristine. Spectroscopic data for these peak fractions were as follows [UV (lambda max); infrared (cm-1); mass spectrum (m/z)]: peak A: 220, 256, and 295 nm; 3457, 2922, 1730, and 1669; and 783 (MH+); peak B: 218, 255, and 296 nm; 3435, 2922, 1731, and 1673; and 783 (MH+); peak C: 220, 255, and 296 nm; 3457, 2922, 1738, and 1680; and 825 (MH+); peak D: 218, 252, and 296 nm; 3385, 2922, 1734, and 1677; and 825 (MH+); peak E: 208, 218, 252, and 298 nm; 3371, 2922, 1727, and 1665; and 768 (MH2+); and peak F: 209, 222, 255, and 296 nm; 3392, 2922, 1734, and 1673; and 823 (MH+). These data suggest the following tentative structures for the degradation products: peak A, 4-deacetylvincristine; peak B, an isomer of 4-deacetylvincristine; peak D, an isomer of vincristine; peak E, 4-deacetyl-3-deoxyvincristine; and peak F, N-formylleurosine. The structure of peak A as 4-deacetylvincristine was confirmed by chemical synthesis.