Preparation of spin-labeled analogs of the antitumor agents TEPA and thio-TEPA.

Full text of DOI:10.1515/znc-1973-11-1231

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Preparation of Spin-Labeled Analogs of the
Antitumor Agents TEPA and Thio-TEPA lj 2
tetramethylpiperidine-l-oxyl (1) 6 with either phos-
phorus oxychloride (2, X = 0 ) or thiochloride (2,
X = S) in the presence of triethylamine resulted in
George Sosnovsky, Younn-Ing Yeh, and Gary Karas the formation of intermediates 3 (X = 0 ) and 3
(X = S ), respectively.
Department of Chemistry, University of Wisconsin-Milwaukee,
Milwaukee, Wisconsin 53201
Without isolating them, these intermediates were al-
lowed further to react with ethyleneimine (4) in
the presence of additional triethylamine to give the
products 5 (X = 0 ) and 5 (X = S ).
(Z. Naturforsch. 28 c, 781 —782 [1973] ; received
July 19, 1973)
Spin-labeling of phosphorus compounds, cancer, EPR,
2,2,6,6-tetramethylpiperidine-l-oxyl
3 + 2H N ^| (C2H5)3N/C6He r 0 P(X) | n ^ j 2
In recent years, a considerable amount of work
has been done in order to elucidate the role of free
radical intermediates in carcinogenesis 3. Particular-
ly, with the EPR technique a series of interesting but
controversial results have been obtained 3. As a con-
sequence, to date, no conclusions can be made con-
cerning the role of free radicals and other paramag-
netig species during the induction and propagation
of tumors 3. However, there is hope that it might be
possible to develop the EPR technique into a useful
diagnostic tool for detection of tumors 3.
Both compounds are well defined red solids. The
EPR spectroscopy of these compounds shows three
equidistant lines of equal intensity with a hyperfine
splitting ay of 15 G. These spectra are characteristic
of the mononitroxyl moiety 6.
An analogous spin-labeled compound (7) was
independently prepared by Rozantsev and cowor-
kers 7.
Until present, the application of chemotherapy in
treatment of cancer has been of modest success4.
Nevertheless, in certain types of cancers a consider-
able progress has been achieved, and therefore,
there exists a justifiable optimism that with the ad-
vance in better understanding of the mechanisms of
initiation and propagation of tumors, more effective
drugs can also be devised for treatment of cancers 4.
Now we would like to report the synthesis of two
new spin-labeled analogs (5, X = 0 and 5, X = S)
of the well known antineoplastic drugs TEPA (6,
X = 0) and Thio-TEPA (6, X = S) 5. It is believed
that the new compounds will be of value in cancer
research since they possess not only an easily by
EPR-detectable label but may also exhibit through
the nitroxyl moiety an inhibitory-scavening effect on
radicals and/or other paramagnetic species in tumor
tissues 6.
N
P(X) [ n
RNP(S)
H
RNHo
8
However, the starting material, 4-amino-2,2,6,6-
tetramethylpiperidine-l-oxyl (8) is much more dif-
ficult to prepare than 1. Therefore, the present
procedure for the preparation of spin-labeled ana-
logs of antineoplastic drugs seems to be the method
of choice.
E x p e r i m e n t a l
1-Oxyl-2,2,6,6-tetramethyl-4-piperidyl-N,N ;N',N'-
bis (ethylene) -phosphorodiamidate (5, X = 0)
A solution of phosphorus oxychloride (3.06 g, 0.02
mole) in dry benzene (100 ml) was cooled to
5 —8 °C, and vigorously stirred under anhydrous
conditions. A solution of 4-hydroxy-2,2,6,6-tetra-
methylpiperidine-l-oxyl6 (1, 3.44 g, 0.02 mole)
and triethylamine (2.02 g, 0.02 mole) in dry ben-
zene (100ml) was then added during 1 hour. After
an additional 1 hour at 10 °C, the reaction mixture
was allowed to warm up to room temperature
(20 °C), and stirred overnight. The precipitated tri-
ethylamine hydrochloride was removed by filtration
(2.70g, 98% of theory). The filtrate containing
1- oxyl -2,2,6,6 -tetramethyl-4-piperidyl-phosphorodi-
chloridate (3, X = 0, 0.02 mole) in dry benzene
(250 ml) was cooled to 5 —6 °C and vigorously
stirred. A solution of triethylamine (4.04 g, 0.04
mole) in dry benzene (30 ml) was added during
The synthesis of 5 (X = 0 ) and 5 (X = S) was
achieved by a “one-batch” process. Thus, the reac-
tion of a benzene solution of 4-hydroxy-2,2,6,6-
(C2H5)3N/C6H8 •
ROH + P(X)C13 V
■- R0P(X)C12
H 3 C C H 3
X = o , s
r =
h3c c h 3
Requests for reprints should be sent to Professor G.
Sosnovsky, Department of Chemistry, University of Wis-
consin-Milwaukee, M ilwaukee, Wisconsin 53201, U.S.A.
Unauthenticated
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782
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0.5 hour, followed by a solution of ethyleneimine triethylamine (2.02 g, 0.02 mole) in dry benzene
(100 ml) was then added during 1.0 hour. After
was added during 0.8 hour at 6 —8 °C. After stir- stirring at 10 °C for 1.5 hour, the reaction mixture
(1.72 g, 0.04 mole) in dry benzene (40 ml) which
ring at 10 °C for 1 hour, the mixture was stirred at
room temperature for 7 days. The suspension was
then filtered to remove the triethylamine hydro-
chloride (5.23 g, 95% of theory), and the filtrate
was concentrated on a rotating evaporator at 40 °C
(25 mmHg). The remaining red oil was dissolved
in benzene, and the solution chromatographed on an
alumina column (1 g oil/lO g A120 3) using a 4:1
(v/v) solution of benzene and ethyl acetate as
eluent. Concentration of the eluted solution gave a
red oil which solidified on strorage in a refrigerator
overnight to a red solid, 3.7 g (61%); m.p. 56 —
60 °C (dec.). EPR: 3 equidistant lines of equal
was allowed to warm up to room temperature, and
the stirring continued for 24 days. The precipitated
triethylamine hydrochloride was removed by filtra-
tion (2.75 g, 100% of theory). The filtrate con-
taining
O- (l-oxyl-2,2,6,6-tetramethyl-4-piperidyl)
phosphorodichloridothioate (3, X = S, 0.02 mole)
in dry benzene (200 ml) was cooled to 5 —8 °C
and vigorously stirred. A solution of ethyleneimine
(1.72 g, 0.04 mole) and triethylamine (4.04 g,
0.04 mole) in dry benzene (70 ml) was added
during 1.5 hours. After stirring for 2 hours at
10 °C, the reaction mixture was allowed to warm
up to room temperature, and the stirring continued
for 6 days. The reaction mixture was then filtered to
remove the triethylamine hydrochloride (5.28 g,
96% of theory). After removal of the solvent on a
rotating evaporator at 40 °C (25 mmHg), the re-
maining red oil was dissolved in dry benzene, and
the solution chromatographed on a alumina column
(1 .0 g oil/10g A120 3) using a 4 : 1 (v/v) solution
of benzene and ethyl acetate as eluent. Concentra-
tion of the eluted solution gave a red solid, 3.0 g
(50%); m.p. 91 —93 °C (dec.). EPR: 3 equidistant
intensity,
= 15 G.
Analysis
C13H25N30 3P :
Calcd: C 51.64 H 8.33 N 13.90 Mol. wt. 302.32,
Found: C 51.43 H8.01 N 13.60 Mol. wt. 309.
0-(l-O x yl-2 ,2,6,6-tetramethly-4-piperidyl) -N,N ;
N',N'-bis (ethylene) -phosphorodiamidothioate
(5, X = S)
lines of equal intensity,
= 15 G.
A solution of phosphorus thiochloride (3.39 g,
0.02 mole) in dry benzene (100ml) was cooled to
6 —8 °C, and vigorously stirred under anhydrous
conditions. A solution of 4-hydroxy-2,2,6,6-tetra-
Analysis.
C13H25N30 2PS:
Calcd: C 49.06 H 7.86 N 13.20 Mol. wt. 318.38,
methylpiperidine-l-oxyl6 (1, 3.44 g, 0.02 mole) and Found: C 49.07 H 7.76 N 13.02 Mol. wt. 315.
1 This investigation was supported by a grant from the
Public Health Service, U.S. Department of Health, Edu-
cation and Welfare (GM 16741).
* Mention of commercial or proprietory names in this paper
does not constitute an endorsement or quality preference
of these products over other commercial products of the
same chemical composition by the authors, editors, or pub-
lishers.
4 C. G. Schmidt, Krebsforschung und Krebsbekämpfung, eds.
H. E. Bock and U. Dold, vol. 6, p. 309, Urban & Schwar-
zenberg, München 1967, and references therein.
5 The Merck Index, ed. P. G. Stecher, 8th edition, p. 1073,
Merck & Co., Inc., Rahway, N.J., USA 1968.
• E. G. Rozantsev, Free Nitroxyl Radicals, pp. 185, 214, and
references therein, Plenum Publ., New York 1970.
7 A. B. Shapiro, A. A. Kropaeheva, V. I. Suskina, B. V.
Rozynov, and E. G. Rozantsev, Izv. Akad. Nauk SSSR,
Ser. Khim. 1965, 864.
* H. M. Swartz, Advances Cancer Res. 15, 227 [1972], and
references therein.
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