Acrolein Mediated by Bifunctional Catalysts 4
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[CANCER RESEARCH 42, 830-837, March 19821 0008-5472/82/0042-0000$02.00 Conversionof 4-Hydroperoxycyclophosphamideand4- HydroxycyclophosphamidetoPhosphoramideMustard and AcroleinMediated by BifunctionalCatalysts1 Joseph E. Low, Richard F. Borch, and N. E. Sladek2 Departments of Pharmacology (J. E. L., N. E. 5.1and Chemistry (R. F. B.], University of Minnesota, Minneapolis, Minnesota 55455 ABSTRACT in the urine as well as the pH of the urine may be important with regard to the potential of cyclophosphamide to induce, via The rates at which 4-hydroperoxycyclophosphamide and 4- acrolein, hemorrhagic cystitis. hydroxycyclophosphamide are converted to phosphoramide mustard and acrolein were determined as a function of buffer INTRODUCTION composition, buffer concentration, and pH. Conversion of 4- hydroperoxycyclophosphamide to 4-hydroxycyclophospham Cyclophosphamide is a prodrug widely used as an antitumor ide in 0.5 MTris buffer, pH 7.4, 37°,wasfirst-order (k = 0.016 and immunosuppressive agent; its chemistry, metabolism, and min@), but subsequent conversion of 4-hydroxycyclophos pharmacology have been reviewed (4, 21 , 22). A metabolic phamide to phosphoramide mustard and acrolein under these scheme summarizing the current understanding of its metabo conditions was negligible. Phosphoramide mustard and acro lism is presented in Chart 1. Cyclophosphamide is first hydrox lein were readily generated from 4-hydroperoxycyclophos ylated to 4-hydroxycyclophosphamide, also a prodrug, via a phamide or 4-hydroxycyclophosphamide when either of these reaction catalyzed by the microsomal mixed-function oxygen agents was placed in phosphate buffer. Conversion of 4-hy ase system of the liver and, to a lesser extent, lung. 4-Hydrox droxycyclophosphamide to phosphoramide mustard and ac ycyclophosphamide serves as a circulating (transport) form of rolein was first-order with respect to 4-hydroxycyclophospham cyclophosphamide and gives rise to aldophosphamide, be ide (k = 0.1 26 min' in 0.5 M phosphate buffer, pH 8, 37°)as lieved to be yet another prodrug (4, 16, 21, 39). Aldophos well as first-order with respect to phosphate serving as a phamide can, in turn, undergo /1elimination to generate phos catalyst. The rate-determining step in the reaction was pH phoramide mustard and acrolein. dependent only insofar as the hydrogen ion concentration Phosphoramide mustard released within cells from 4-hydrox governed the relative amounts of monobasic and dibasic phos ycyclophosphamide is believed to account for the bulk of the phate present. Pseudo-first-order rate constants were 0.045 cytotoxic action of cyclophosphamide to those cells (4, 21, M1min1 for monobasic phosphate and 0.256 M1min' for 49); unequivocalevidencechallengingthis view has yet to be dibasic phosphate. The role of phosphate in this reaction was presented. However, the basis for the relatively favorable ther as that of a bifunctional catalyst. The reaction was not subject apeutic index of cyclophosphamide remains obscure; we and to specific or general, acid or base, catalysis. Other bifunctional others believe that it resides with 4-hydroxycyclophosphamide catalysts such as glucose-6-phosphate and bicarbonate also and/or aldophosphamide (4, 21). It may depend, at least in catalyzed the reaction, albeit less efficiently. Aldophosphamide part, on the relative rates at which 4-hydroxycyclophospham apparently exists only transiently; its presence could not be ide is converted to phosphoramide mustard within sensitive established by 31Pnuclear magnetic resonance spectroscopy. and insensitive cells. Other than its apparent pH dependence We conclude that, in the reaction sequence 4-hydroxycyclo (4), little is known about the chemistryof this reaction or the phosphamide .-* aldophosphamide —@phosphoramidemustard factors that might affect the rate at which it proceeds. + acrolein, the conversion of 4-hydroxycyclophosphamide to The first reaction in the sequence of interest is the conversion aldophosphamide is rate limiting and is subject to bifunctional of 4-hydroxycyclophosphamide, a carbinolamide, to aldophos catalysis; this reaction can proceed efficiently only in the phamide, an amidoaldehyde. Carbinolamides and carbinolam presence of a bifunctional catalyst. Assuming that the onco ines are known to be subject to the action of polyfunctional toxic specificity of cyclophosphamide resides with 4-hydroxy catalysts, e.g., inorganic phosphate (6, 13, 14, 25, 33). Re cyclophosphamide and that its cytotoxic effect at therapeutic ports that the rate at which 4-hydroxycyclophosphamide was doses is largely mediated by phosphoramide mustard released converted to phosphoramide mustard and acrolein varied con within cells, these observations offer the possibility that the siderably with the nature of the buffer used (Ref. 43 quoted in intracellular concentration of bifunctional catalysts, whether in Ref. 4; 46), and our own preliminary observation that 4-hy the form of inorganic phosphates, organic phosphates, en droxycyclophosphamide was highly stable in Tris buffer, known zymes, or other species, serve as important determinants with to be incapable of polyfunctional catalysis, strongly suggested regard to the oncotoxic potential and specificity of cyclophos-•that the first step in the reaction sequence was rate limiting phamide. Similarly, the concentration of bifunctional catalysts and that it was subject to polyfunctional, but not to specific or general, acid or base, catalysis. This hypothesis was pursued ,SupportedbyUSPHSGrantsCA26357andCA21737.Adescriptionof in the present investigation. parts of this investigation has appeared in abstract form (38). This is Paper 10 in @ a series on ‘CyclophosphamideMetabolism.― 2 To whom requests for reprints should be addressed, at 3-260 Millard Hall, MATERIALS AND METHODS 435 Delaware Street SE., Minneapolis, Minn. 55455. Received August 13, 1981 ; accepted November 30, 1981. Materials. Triethyl phosphite, acrolein, m-aminophenol, and hydrox 830 CANCERRESEARCHVOL. 42 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1982 American Association for Cancer Research. Activation of 4-Hydroxycyclophosphamide CICH2CH2 P*4.O.@2 \/ \ tation wavelength was 346 nm; the emission wavelength was 500 nm. N-P.O CH2 Authentic 4-hydroperoxycyclophosphamide was used as the standard. CIC@42CPl2/\/ O@CH2 The lower limit of sensitivity was approximately 1 nmol of 4-hydrope cva..ot'[email protected]€ roxycyclophosphamide per ml of water. NADPH Acrolein Assay.The releaseof acroleinfrom 4-hydroperoxycyclo PARENT r@@[email protected]$e phosphamide or 4-hydroxycyclophosphamide was monitored in a Gil ford Model 2400 automatic recording spectrophotometer equipped -- --@@-@-----@ 0 with a Haake constant temperature circulator to maintain a reaction @ CICH2cM2 NH-CPI OcH2cH2 NH-C temperature of 37°(2).The reaction mixture routinely contained 70 to @ @@d-P'@ oldehyde @idose \/ \ @ /\/@ 100 nmol of 4-hydroperoxycyclophosphamide or 4-hydroxycyclophos @ OCH@O12 @0@CH2 C1CH2CM2 0-CH@ phamide per ml of buffer; NaCI was added to maintain a constant ionic @ 4-HVDRCX'tt'@ti@0PH0SPHAMI0( 4-KETocYCLOPHOSPHAMIDE strength of 1.6 (32). Buffers studied included phosphate, bicarbonate, NAD borate, and glucose-6-phosphate; buffer concentrations and pH are TRANSPORT j N@@@?.@.,CICHarH21 NN@ indicated for each experiment. Reaction mixtures were sealed in 1-cm quartz cuvets and monitored at 210 nm. Authentic acrolein was used @ C;CH@CH@ NH@ [email protected]/massspectrometrywasusedto N-P-0 confirm the identity of the acrolein formed. The lower limit of UV CICH2CH2/\?I 0CH@CH@-C-H I sensitivity was approximately 5 nmol of acrolein per ml. Initial rates of 0*,_. ALDOPHOSPHAMIDE acrolein appearance were used to obtain the half-life of 4-hydroxycy clophosphamide; this was necessary because acrolein slowly disap @@iç peared (k = 0.0003 to 0.002 min ‘depending on the pH and buffer composition of the reaction medium), presumably because it polymer OCH@CH2 N2 I CARSOXYPHOSPHAMIDE ized under the reaction conditions. 31pNMR@'Spectrometry.3'P NMR spectra were obtainedwith a CIDCH;)@@Z@oH 7 Varian XL-100 muitinuclear spectrometer operating in the Fourier PHOSPHORAMIDEMUSTARD transform mode with external ‘9Flockand proton decoupling; pulse acquisition time was 400 msec, and 500 transients were collected @ TOX@ t INACTIVE giving an elapsed acquisition time of 3.3 mm. Hexamethylphosphor CICH2CH2 I amide (1 % in water) was used as a coaxial standard, and both chemical \ shifts and peak intensities were measured from this standard. The RF frequency was 40.5066 MHz with a spectral width of 5000 Hz; the norHN2 reference peak appeared at 361 1 Hz, and the compounds of interest Chart 1. Proposed route of cyclophosphamide metabolism. appeared in the region 2300 to 3200 Hz. Identification was based on signals obtained with authentic compounds. The chemical shifts of ylamine hydrochloridewere purchasedfrom Aldrich ChemicalCo., phosphate and phosphoramide mustard were highly dependent upon Milwaukee, Wis. Acrolein was distilled before use. Glucose-6-phos PH;largervalueswereobtainedasthe pHwasdecreased. phatedisodiumwas purchasedfrom SigmaChemicalCo., St. Louis, Data Analysis. Values were submitted to linear regression analysis Mo.AmericanChemicalSocietyreagent-gradeacetonewaspurchased to obtainall linearfunctions. fromSpectrumChemicalManufacturingCorp.,RedondoBeach,Calif., and was dried and distilled immediatelybeforeuse. Linearhigh-per RESULTS formancethin-layerchromatography(LHP-K)plateswere