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Cyclophosphamide stereochemistry. I. Synthesis and configurational stability of diastereomeric cyclophosphamide derivatives
TetrahedronLettersNo. 36, PP 3139 - 314% 1975. Pergamonhens.
Printed in Great Britain.
CYCLOPHOSPHMlIDE STEREOCHEMISTRY. I. SYNTHESISAND CONFIGURATIONAL STABrLrTY OF DIASTEREOMERIC CYCLOPHOSPHAMIDE DERIVATIVES Gerald Zon Maloney Chemical Laboratory, The Catholic University of America Washington, D.C.
20064
(Beceivedin USA 11 July 1975; received in UK for publication24 July'1975) Cyclophosphamide
(l) is a clinically useful anticancer drug which is believed to require
enzymatic C,+-hydroxylation before exhibiting its chemotherapeutic effects.l elucidating the stereospecificity
Our interest in
of this metabolic activation process toward (E)- and &)-I
has led to preliminary evaluation of -in vitro configurational stability at the chiral phosphorus center in ,l, by studies utilizing suitable diasterecmeric derivatives, which we now wish to report. H
5
c
4
"lpHO
6
b/l
N(CH2CH$1)3
Addition of optically pure (+)-(Ii)-a-methylbenzylamine to ethyl acrylate in refluxing absolute EtOH afforded 5 ,2 which was reduced with LiAlH,, to 2 in 59% overall isolated yield (see Scheme).
Treatment (4 days, 25') of this presumably optically pure amino alcohol with
43 in EtOAc containing 2 equiv. Et3N yielded (63%), upon careful chromatography (silica gel; e Et,0 : CHCl, = 1 : l), near equal amounts of the desired cyclophosphamide diastereomers, $$ (Rf = 0.74) and @,
(Rf = 0.57).
Use of optically pure (-)-(S)-a-methylbenzylamine
synthetic Scheme led to isolation of the respective enantiomers of 2, Q,,$and a.
in the
To our know-
ledge, this stereofscmer set represents the first example of cyclophosphamide derivatives which can be used as amodel
system for probing chemotherapeutic
consequences of configurational home-
geniety at phosphorus in k.4 Spectroscopic assessment of diastereomeric purity of x
and #
was readily accomplished by
monitoring their individual methyl group 'H nmr (60 MHz, CDC13) doublets (J = 7 Hz) at 6 ,1.55 and 1.68, respectively.
At higher magnetic field strength (220 MHz), configurationally pure
(2 99%, tic) samples of g failed to show additional methyl group proton signals at 20'. evidence and the broad band decoupled high resolution 13C nmr spectrum of i (68.9
3'139
MHZ,
This CDC13),5
3141
No. 36
which exhibits only one set of absorption peaks at 40", together with reported6 conformational analyses of analogous ring systems, are consistent with our assumption that the six-membered ring in 2 is conformationally mobile at room temperature. Rapid interconversion of (R)- and (2)-l under physiological conditions can, in principle, Occur by reversible coordination of water to initiate fonation
of a sytmretrical phosphorane,
or by attack at phosphorus of scme other nucleophilic species with subsequent intramolecular ligand exchange in phosphorane intermediates .7
The likelihood of such events, which if opera-
tive would preclude differential antitumor activity between enantiomers of 1, was gauged indirectly via 2. Initial 'H rxnr experiments were performed with 2 (0.34 3 as co-solvent.
in DsO having 75 v/v% DMSO-&
Mildly acidic (pH c 5.6) or basic (pH 'Ir 8.4) samples of pure @
or pure a
showed no detectable (5 5%) diastereomer crossover after 15 hr at 5D", as did diastereomer samples containing 0.5 - 1.0 equiv. of either NaCl, NaOAc, or MgCl,.* was found that neither diastereomer of 2 underwent stereomutation
in human
More significantly, it
(5 l%, tic) while dissolved
blood plasma (1 mg/ml) at 37' for a period of 14 hr.
The presently reported configurational
stability at phosphorus in model substrate 2 sug-
gests that, in the absence of special in -vivo effects, enantiomerically pure cyclophosphamide should not suffer racemization during its relatively rapid transport (-<15 ming) to the hepatic enzyme system which effectuates Ck-hydroxylation. advantages of administering enantiomerically conclusion extends to isophosphamide,1°
Consequently, the conceivable therapeutic
pure 1 remain as viable research goals.
A similar
which is a chiral antitumor agent structurally related
Studies concerning the resolution of ,l, will be reported in the future.
to 1.
Acknowledgments.
This investigation was supported by NIH research grant nunber CA-16158,
awarded by the National Cancer Institute, PHS/DHEW.
We thank Drs. William Egan and Sanford P.
Markey (NIH) for stimulating discussions, and Ms. Maria L. Thomas for preparation of this manuscript. REFERENCES AND FOOTNOTES A. Takamizawa, aal.,
J. Med. Chem., ,l$,,376 (1975); A. Takamizawa, S. Matsumoto, and T.
Iwata, Tetrahedron Lett., 517 (1974); R. F. Struck, Cancer Res., $4, 2933 (1974); J. A. Montgomery and R. F. Struck, Arzneim.-Forsch.,
K,
322 (1973).
New compounds gave acceptable elemental analyses (* 0.4%) and exhibited spectral data (pmr, ir) consistent with their assigned structures. 0. M. Friedman and A. M. Seligman, J. Amer. Chem. Sot., a,
655 (1954).
Absolute configurations about phosphorus in $, and Q are presently unkncwn; comparative
No. 36
3142
antitunor activity of 5, and & will be published at a later date.
We thank Ms. Joan A.
Brandt for preparation of 6. 5.
W. Egan and 6. Zon, unpublished results.
6.
J. Durrieu, R. Kraemer, and J. Navech, Org. Magn. Resonance, 2, 407 (1973); J. A. Mosbo and J. G. Verkade, J. Amer. Chem. Sot., a,
7.
4659 (1973).
M. Mikolajczyk, J. Kryzywanski, and B. Ziemnicka, Tetrahedron Lett., 1607 (1975); 6. Zon and K. Mislow, Fortsch. them. Forsch., R, and H. S. Aaron, Chem. Ccmun.,
8.
Occurrence ofstereospecific
61 (1971); L. P. Reiff, L. J. Szafraniec,
366 (1971).
oxygen-exchange between the phosphoryl group of R
and water
under the saline conditions was ruled out by substituting HzlsO containing 10.1 atom % l*O/mole for normal water. 9. 10.
Mass spectral analyses were performed by Dr. S. P. Markey.
N. Brock, Cancer Chemotherapy Rep., A. Takamizawa, J. Med. Chem., u,
$,,J,,315 (1967).
1237 (1974) and refs. cited therein.
Printed in Great Britain.
CYCLOPHOSPHMlIDE STEREOCHEMISTRY. I. SYNTHESISAND CONFIGURATIONAL STABrLrTY OF DIASTEREOMERIC CYCLOPHOSPHAMIDE DERIVATIVES Gerald Zon Maloney Chemical Laboratory, The Catholic University of America Washington, D.C.
20064
(Beceivedin USA 11 July 1975; received in UK for publication24 July'1975) Cyclophosphamide
(l) is a clinically useful anticancer drug which is believed to require
enzymatic C,+-hydroxylation before exhibiting its chemotherapeutic effects.l elucidating the stereospecificity
Our interest in
of this metabolic activation process toward (E)- and &)-I
has led to preliminary evaluation of -in vitro configurational stability at the chiral phosphorus center in ,l, by studies utilizing suitable diasterecmeric derivatives, which we now wish to report. H
5
c
4
"lpHO
6
b/l
N(CH2CH$1)3
Addition of optically pure (+)-(Ii)-a-methylbenzylamine to ethyl acrylate in refluxing absolute EtOH afforded 5 ,2 which was reduced with LiAlH,, to 2 in 59% overall isolated yield (see Scheme).
Treatment (4 days, 25') of this presumably optically pure amino alcohol with
43 in EtOAc containing 2 equiv. Et3N yielded (63%), upon careful chromatography (silica gel; e Et,0 : CHCl, = 1 : l), near equal amounts of the desired cyclophosphamide diastereomers, $$ (Rf = 0.74) and @,
(Rf = 0.57).
Use of optically pure (-)-(S)-a-methylbenzylamine
synthetic Scheme led to isolation of the respective enantiomers of 2, Q,,$and a.
in the
To our know-
ledge, this stereofscmer set represents the first example of cyclophosphamide derivatives which can be used as amodel
system for probing chemotherapeutic
consequences of configurational home-
geniety at phosphorus in k.4 Spectroscopic assessment of diastereomeric purity of x
and #
was readily accomplished by
monitoring their individual methyl group 'H nmr (60 MHz, CDC13) doublets (J = 7 Hz) at 6 ,1.55 and 1.68, respectively.
At higher magnetic field strength (220 MHz), configurationally pure
(2 99%, tic) samples of g failed to show additional methyl group proton signals at 20'. evidence and the broad band decoupled high resolution 13C nmr spectrum of i (68.9
3'139
MHZ,
This CDC13),5
3141
No. 36
which exhibits only one set of absorption peaks at 40", together with reported6 conformational analyses of analogous ring systems, are consistent with our assumption that the six-membered ring in 2 is conformationally mobile at room temperature. Rapid interconversion of (R)- and (2)-l under physiological conditions can, in principle, Occur by reversible coordination of water to initiate fonation
of a sytmretrical phosphorane,
or by attack at phosphorus of scme other nucleophilic species with subsequent intramolecular ligand exchange in phosphorane intermediates .7
The likelihood of such events, which if opera-
tive would preclude differential antitumor activity between enantiomers of 1, was gauged indirectly via 2. Initial 'H rxnr experiments were performed with 2 (0.34 3 as co-solvent.
in DsO having 75 v/v% DMSO-&
Mildly acidic (pH c 5.6) or basic (pH 'Ir 8.4) samples of pure @
or pure a
showed no detectable (5 5%) diastereomer crossover after 15 hr at 5D", as did diastereomer samples containing 0.5 - 1.0 equiv. of either NaCl, NaOAc, or MgCl,.* was found that neither diastereomer of 2 underwent stereomutation
in human
More significantly, it
(5 l%, tic) while dissolved
blood plasma (1 mg/ml) at 37' for a period of 14 hr.
The presently reported configurational
stability at phosphorus in model substrate 2 sug-
gests that, in the absence of special in -vivo effects, enantiomerically pure cyclophosphamide should not suffer racemization during its relatively rapid transport (-<15 ming) to the hepatic enzyme system which effectuates Ck-hydroxylation. advantages of administering enantiomerically conclusion extends to isophosphamide,1°
Consequently, the conceivable therapeutic
pure 1 remain as viable research goals.
A similar
which is a chiral antitumor agent structurally related
Studies concerning the resolution of ,l, will be reported in the future.
to 1.
Acknowledgments.
This investigation was supported by NIH research grant nunber CA-16158,
awarded by the National Cancer Institute, PHS/DHEW.
We thank Drs. William Egan and Sanford P.
Markey (NIH) for stimulating discussions, and Ms. Maria L. Thomas for preparation of this manuscript. REFERENCES AND FOOTNOTES A. Takamizawa, aal.,
J. Med. Chem., ,l$,,376 (1975); A. Takamizawa, S. Matsumoto, and T.
Iwata, Tetrahedron Lett., 517 (1974); R. F. Struck, Cancer Res., $4, 2933 (1974); J. A. Montgomery and R. F. Struck, Arzneim.-Forsch.,
K,
322 (1973).
New compounds gave acceptable elemental analyses (* 0.4%) and exhibited spectral data (pmr, ir) consistent with their assigned structures. 0. M. Friedman and A. M. Seligman, J. Amer. Chem. Sot., a,
655 (1954).
Absolute configurations about phosphorus in $, and Q are presently unkncwn; comparative
No. 36
3142
antitunor activity of 5, and & will be published at a later date.
We thank Ms. Joan A.
Brandt for preparation of 6. 5.
W. Egan and 6. Zon, unpublished results.
6.
J. Durrieu, R. Kraemer, and J. Navech, Org. Magn. Resonance, 2, 407 (1973); J. A. Mosbo and J. G. Verkade, J. Amer. Chem. Sot., a,
7.
4659 (1973).
M. Mikolajczyk, J. Kryzywanski, and B. Ziemnicka, Tetrahedron Lett., 1607 (1975); 6. Zon and K. Mislow, Fortsch. them. Forsch., R, and H. S. Aaron, Chem. Ccmun.,
8.
Occurrence ofstereospecific
61 (1971); L. P. Reiff, L. J. Szafraniec,
366 (1971).
oxygen-exchange between the phosphoryl group of R
and water
under the saline conditions was ruled out by substituting HzlsO containing 10.1 atom % l*O/mole for normal water. 9. 10.
Mass spectral analyses were performed by Dr. S. P. Markey.
N. Brock, Cancer Chemotherapy Rep., A. Takamizawa, J. Med. Chem., u,
$,,J,,315 (1967).
1237 (1974) and refs. cited therein.