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In the search for new anticancer drugs XII. Synthesis and biological evaluation of spin labeled nitrosoureas
Life Sciences, Vol. 36, pp. 1479-1483 Printed in the U.S.A.
Pergamon Pres
IN THE SEARCH FOR NEW ANTICANCER DRUGS Xll.* SYNTHESIS AND BIOLOGICAL EVALUATION OF SPIN LABELED NITROSOUREAS George Sosnovsky** and Shu Wen Li Department of Chemistry U n i v e r s i t y of Wisconsin-Milwaukee Milwaukee, Wisconsin 53201
(Received in final form February 8, 1985)
Summary The spin labeled nitrosourea l - ( 2 - c h l o r o e t h y l ) - 3 - ( l - o x y l 2 , 2 , 6 , 6 - t e t r a m e t h y l - p i p e r i d i n y l ) - l - n i t r o s o u r e a (SLCNU, 4) and i t s analogues 5-~ were synthesized e i t h e r by a r e g i o s e l e c t i v e method or by a conventional route v i a the n i t r o s a t i o n of the spin labeled intermediates ( l l a - e ) . N i t r o s a t i o n of the ureas l l a - e with d i n i t r o g e n t e t r a o x i d e resulted in b e t t e r y i e l d s than those obtained with sodium nitrite. The nitrosoureas 4-8 were tested f o r t h e i r a n t i cancer a c t i v i t y against the lymphocytic leukemia P388 in mice. Thus, e i t h e r at the equal molar dose or at the dose of equal t o x i c i t y l e v e l , the SLCNU (4) was found to be more a c t i v e than the c l i n i c a l l y used CCNU ( ! ) . Unlike CCNU (~) whose LD50 is 56 mg/kg, the SLCNU (4) possesses a low t o x i c i t y (LD50 123 mg/kg). Therefore, SLCNU (4) is a promising new e n t r y i n t o the nitrosourea class of anticancer drugs. The c h l o r o e t h y l n i t r o s o u r e a s , such as CCNU (~), BCNU ( 2 ) , and methyl CCNU (2) have been e x t e n s i v e l y used c l i n i c a l l y f o r the combat of a v a r i e t y of cancers ( I ) . U n f o r t u a n t e l y , as almost a l l a l k y l a t i n g anticancer drugs pres e n t l y in use, the nitrosoureas e x h i b i t a wide range of t o x i c i t y e f f e c t s ( I ) . Since we have shown (2-4) t h a t the n i t r o x y l r a d i c a l moiety can e x h i b i t a b e n e f i c i a l l y modifying e f f e c t on the t o x i c i t y and a c t i v i t y of TEPA and ThioTEPA d e r i v a t i v e s , i t would be extremely rewarding i f such e f f e c t s could be found in the nitrosourea s e r i e s . Therefore, we have synthesized and tested in v i v o f o r anticancer a c t i v i t y several n i t r o x y l labeled nitrosoureas with s t r u c t u r a l features shown in formulae 4-8. By substituting the cyclohexyl group in CCNUwith the l - o x y l - 2 , 2 , 6 , 6 tetramethylpiperidinyl group, compound 4 (SLCNU) was prepared. Compounds 5-~ are the analogs of SLCNU where the N-3 hydrogen was replaced by methyl, cyclohexyl and nitroxyl radical groups. The rationale for the design of these compounds was that some N,N-disubstituted chloroethylnitrosoureas were *G. Sosnovsky and S. W. Li, In the Search for New Anticancer Drugs XI, Cancer Letters, in press. **To whom correspondence should be addressed. 0024-3205/85 $3.00 + .00 Copyright (c) 1985 Pergamon Press Ltd.
1480
Nitroxyl Labeled Nitrosoureas
Vol. 36, No. 15, 1985
recently found (5-8) to possess high activity. We have also designed a bifunctional chloroethylnitrosourea (8) since i t was found (7) that some bifunctional chloroethylnitrosoureas exhibit significant activity against Walker carcinoma 256 in rats. Recently, we found an abstract of a talk dealing with a similar topic. However, no experimental details were available (9)
R1/ CCNU ~ R=HRI=
NCNCH2CH2Cl ~O
BCNU ~ R=H, RI=cH2CH2CI
MeCCNU~ R=H R I = ~ c H 3
H3C CH3
~1--06 H3C CH3
SLCNU ~ R=H R I = < ~ N - - O • ,,----/,, H3C CH3
R=CH3, RI=
N3C CH g
H3C CH3
Z ~H3C <" 7 R=R1= <
CH3
~N O"
" -H3C ~c H3C CH3
H3
NO ,
8
R=
N --0",
H3C
R1: (CH2)2
3
"
--
NCONCH2CH2CI
i, c
H3C H3
CH3 H3
O"
Materials and Methods The nitrosoureas 4-8 were prepared as shown in Scheme I . The l - o x y l 2,2,6,6-tetramethyl-4-oxopiperidine (9) was used as the starting material. The reductive amination of 9 with ammonium acetate gave in addition to the primary amino compound lOa (lO) also the secondary amine lOd ( l l ) which was isolated by chromatography. Compounds lOb (lO), lOc and lOe were prepared by the reductive amination of 9 using methylamine, cyclohexylamine and ethylenediamine, respectively. The introduction of the chloroethyl ureido group to give compounds l l a - e was accomplished by the reaction of lOa-e with 2-chloroethyl isocyanate. Scheme 1 O
RNH
H3C
CH3
H3
CH3
L H3C
CH3
O*
aRcH
RNCNHCH2CH2CI ~ H3C
O*
O,
10a-,~e
1Ia,~e
H3C CH3
4
-
_
_
8
CH3
H3C R=CH2CH2-NH
CH3 N--Oo
H3C
3
11,.,~,.e:R=CH2CH2NCONHCH2CH2CI H3C H3
CH3 CH3 O.
Vol. 36, No. 15, 1985
Nitroxyl Labeled Nitrosoureas
1481
The nitrosation of these ureas ( l l a - e ) was carried out by the use of either dinitrogen tetraoxide or sodium n i t r i t e in diluted acetic acid. The former nitrosation method gave better results. In order to prove the position of the nitroso group in SLCNU (4) a regioselective synthetic method was used to prepare 4. Thus, N'-hydroxysuccinimide-N-(2-chloroethyl)-nitrosocarbamate (133) (12) was condensed with lOa to afford the nitrosourea 4 where the nitroso group is attached at N-l, the required position. Scheme 2 o
CICH2CH2NHCOO
CICH2CH2NCOON j.J tF O
O
The compounds 4 obtained either by the conventional route (Scheme l ) or by the regioselective method (Scheme 2) were identical. The physical properties of the nitrosoureas 4-8 are shown in TABLE I. TABLE I . Compound
Physical Properties of Spin Labeled Nitrosoureas Molecular~ Formula
m.p. [°C]
yield
M.S. m/e~
[%1
C12H22N403ClC80-83 (305.78) (dec.)
82
307(48) 306(59) 305(100) 276(62) 240(54) 183(92)
5
C13H24N403C1C68-70 (319.81)
60
321(36) 320(52) 319(62) 290(100) 255(64) 215(65) 156(84)
6
ClBH32N403Cl~128-129 (387.93) (dec.)
61
388(20) 387(25) 358(25) 254(100) 224(43)
7
C21H38N504ClC127-128 (460.01)
67
461(20) 460(22) 432(28) 396(22) 355(70) 327(82)
SLCNU (4)
253(100) 8
C26H46N806CI 2 ~ 142-143 (637.61) (dec.)
69
3 9 5 ( I 0 0 )396(64) 380(22) 309(29)
Satisfactory microanalyses were obtained: C, H, N ± 0.4. spectra were consistent with the structures.
The I . R.
Chemical ionizatons using methane as reactant gas, therefore, for mol. wt. M+÷l values are reported. ESR: three lines, aN = 16-17 G. ESR: five lines, however, intensities are not 1:2:3:2:1, more complex exchange interactions. All compounds ( ! and 4--B) were evaluated in vivo against the P388 lymphocytic leukemia in CD2Fl male mice according to the protocol of the National Cancer I n s t i t u t e (13). Approximately lO6 cells of P388 were inoculated ip into the mice at day 0 of the experiment (6 mice/group). Daily
1482
Nitroxyl Labeled Nitrosoureas
Vol. 36, No. 15, 1985
ip drug admlnistratlon was begun 24 h after the inoculation and performed once daily for 9 days. At day 5, the animals were weighed and then observed for 60 days keeping a record of deaths and survivors. Data were analyzed by the T/C method where T represents the mean survival time of the treated group, and C the mean survival time of the tumor bearing conrol group. The percent of increase in l i f e span (%ILS) parameter was evaluated by [(T-C)/C] x lO0. For the determination of the LDso four logarithmically spaced doses were injected ip into four groups of 4 Swiss male mice. The observation period for the determinations of the LD50 was 30 days. The LDso values were calculated by Well's method (14). TABLE I I .
Compound
Anticancer A c t i v i t y of Spin Labeled Nitrosoureas Against P388 Leukemia in CD2Fl Male Mice Daily Dose mq/kg/d mmol/kg/d_
CCNU (1)
16
0.068
SLCNU
I0 2O 35
0.033 0.065 O.ll4
30 60 90
(4) 5
Acute LD50 5 day weight~ mq/kq ip chanqe[%1 564
ILS~ax [%]
CuresC Survival/ Total) __
-9.2
182~
0/6
123
+9.8 +4.4 -5.1
298 542 542
216 6/6 6/6
0.094 0.187 0.28
353
+2.5 +2.5 -ll.2
54 130 329
0/6 0/6 I/6
75
0.19
337
+4.7
31
0/6
60
0.13
-3.4
21
0/6
30 60
0.047 0.094
+2.7 -2.6
32 53
0/6 0/6
~The change of average percentage weight on day 5 was taken as a measure of drug t o x i c i t y . ~Results obtained on day 60. CCure means survival after 60 days. ~Reference 15. ~In reference 16, the ILS (%) is 172. Results and Discussion The anticancer effects and LD50 of nitrosoureas together with the comparative data for the positive control using CCNU (!) are summarized in TABLE I f . At an equal molar dose SLCNU (4) was found to be more active than CCNU ( ! ) . Thus, at a dose of 0.065 mmol/kg (20mg/kg), SLCNU (4) e l i c i t e d an ILS value of 542%, and a l l mice were alive after 60 days, whereas the ILS of CCNU (!) at a dose of 0.068 mmol/kg (16 mg/kg), was only 182% and a l l mice died within 30 days. In regard to t h e i r t o x i c i t i e s , there was no t o x i c i t y observed in the treated group of SLCNU, while the average weight of mice on
Vol. 36, No. 15, 1985
Nitroxyl Labeled Nitrosoureas
1483
day 5 in the treated group of CCNUdecreased by 9%. The SLCNU was also more active than the CCNUwhen compared at doses e l i c i t i n g equal t o x i c i t y . Even at the lower dose of lO mg/kg, the ILS of SLCNU (4) was found to be 298% which is 1.6 times higher than 1B2% ILS of CCNUat the dose of 16 mg/kg. In conclusion, with these results, we have shown that the SLCNU (4) possesses a higher a c t i v i t y and a much lower t o x i c i t y than the c l i n i c a l l y used drug CCNU. Acknowledgements These studies were conducted pursuant to a contract with the National Foundation for Cancer Research. The cancer lines were obtained from the DCT Tumor Repository, NCI-Frederick Cancer Research F a c i l i t y . References
(i) (2) (3) (4) (5) (6) (7) (8) (9) (I0) (II) (12) (13) (14) (15) (16)
S. CARTER, M. T. BAKOWSKI and K. HELLMANN, Chemotherapy of Cancer, p. 99, John Wiley & Sons, Inc., New York (1981). M. KONIECZNY, G. SOSNOVSKY and P. GUTIERREZ, Z. Naturforsch, 36b, 888 (1981). P. GUTIERREZ, M. KONIECZNY and G. SOSNOVSKY, Z. Naturforsch, 36b, 1612 (1981). G. SOSNOVSKY and SHU WEN LI, submitted to Life Sciences. K. TSUJIHARA, M. OZEKI, T. MORIKAWA and Y. ARAI, Chem. Pharm. B u l l . , 29, 2509 (1981). K. TSUJIHARA, M. OZEKI, T. MORIKAWA, N. TAGA, M. KAWAMORI, and Y. ARAI, Chem. pharm. B u l l . , 29, 3262 (1981). H.H. FIEBIG, G. EISENBRAND, W. J. ZELLER and T. DEUTSCH-WENZEL, Eur. J. Cancer, 13, 937 (1977). T . P . JOHNSTON, G. S. MCCALEB, W. C. ROSEand J. A. MONTGOMERY, J. Med. Chem., 27, 97 (1984). Z.D. RAIKOV, D. N. GRUEVA and I. V. DRAGNOV, The Eighth Meeting of International Society of Magnetic Resonance, Chicago (1983). G. M. ROSEN, J. Med. Chem., 17, 357 (1974). E. J. RAUCKMAN, G. M. ROSEN and W. W. HORD, Org. Prep. and Proc. I n t . 9(2), 53 (1977). J. MARTINEZ, J. OIRY, J. L. IMBACH and F. WINTERNITZ, J. Med. Chem., 25, 178 (1982). R. GERAN, N. GREENBERG, M. MACDONALD, A. SCHUMACHER and B. ABBOTT, Can. Chem. Rep., part 3, 2(2), 7 (1972). C. S. WEIL, Biometrics, 8, 249 (1952). G. R. THOMPSON and R. E. LARSON, Toxicol. Appl. Pharmacol., 21, 405 (1972). National Cancer Institute Monograph 55, Experimental Evaluation of Antitumor Drugs in the USA and USSR and clinical correlations. National Cancer Institute, Bethesda, Maryland (1980).
Pergamon Pres
IN THE SEARCH FOR NEW ANTICANCER DRUGS Xll.* SYNTHESIS AND BIOLOGICAL EVALUATION OF SPIN LABELED NITROSOUREAS George Sosnovsky** and Shu Wen Li Department of Chemistry U n i v e r s i t y of Wisconsin-Milwaukee Milwaukee, Wisconsin 53201
(Received in final form February 8, 1985)
Summary The spin labeled nitrosourea l - ( 2 - c h l o r o e t h y l ) - 3 - ( l - o x y l 2 , 2 , 6 , 6 - t e t r a m e t h y l - p i p e r i d i n y l ) - l - n i t r o s o u r e a (SLCNU, 4) and i t s analogues 5-~ were synthesized e i t h e r by a r e g i o s e l e c t i v e method or by a conventional route v i a the n i t r o s a t i o n of the spin labeled intermediates ( l l a - e ) . N i t r o s a t i o n of the ureas l l a - e with d i n i t r o g e n t e t r a o x i d e resulted in b e t t e r y i e l d s than those obtained with sodium nitrite. The nitrosoureas 4-8 were tested f o r t h e i r a n t i cancer a c t i v i t y against the lymphocytic leukemia P388 in mice. Thus, e i t h e r at the equal molar dose or at the dose of equal t o x i c i t y l e v e l , the SLCNU (4) was found to be more a c t i v e than the c l i n i c a l l y used CCNU ( ! ) . Unlike CCNU (~) whose LD50 is 56 mg/kg, the SLCNU (4) possesses a low t o x i c i t y (LD50 123 mg/kg). Therefore, SLCNU (4) is a promising new e n t r y i n t o the nitrosourea class of anticancer drugs. The c h l o r o e t h y l n i t r o s o u r e a s , such as CCNU (~), BCNU ( 2 ) , and methyl CCNU (2) have been e x t e n s i v e l y used c l i n i c a l l y f o r the combat of a v a r i e t y of cancers ( I ) . U n f o r t u a n t e l y , as almost a l l a l k y l a t i n g anticancer drugs pres e n t l y in use, the nitrosoureas e x h i b i t a wide range of t o x i c i t y e f f e c t s ( I ) . Since we have shown (2-4) t h a t the n i t r o x y l r a d i c a l moiety can e x h i b i t a b e n e f i c i a l l y modifying e f f e c t on the t o x i c i t y and a c t i v i t y of TEPA and ThioTEPA d e r i v a t i v e s , i t would be extremely rewarding i f such e f f e c t s could be found in the nitrosourea s e r i e s . Therefore, we have synthesized and tested in v i v o f o r anticancer a c t i v i t y several n i t r o x y l labeled nitrosoureas with s t r u c t u r a l features shown in formulae 4-8. By substituting the cyclohexyl group in CCNUwith the l - o x y l - 2 , 2 , 6 , 6 tetramethylpiperidinyl group, compound 4 (SLCNU) was prepared. Compounds 5-~ are the analogs of SLCNU where the N-3 hydrogen was replaced by methyl, cyclohexyl and nitroxyl radical groups. The rationale for the design of these compounds was that some N,N-disubstituted chloroethylnitrosoureas were *G. Sosnovsky and S. W. Li, In the Search for New Anticancer Drugs XI, Cancer Letters, in press. **To whom correspondence should be addressed. 0024-3205/85 $3.00 + .00 Copyright (c) 1985 Pergamon Press Ltd.
1480
Nitroxyl Labeled Nitrosoureas
Vol. 36, No. 15, 1985
recently found (5-8) to possess high activity. We have also designed a bifunctional chloroethylnitrosourea (8) since i t was found (7) that some bifunctional chloroethylnitrosoureas exhibit significant activity against Walker carcinoma 256 in rats. Recently, we found an abstract of a talk dealing with a similar topic. However, no experimental details were available (9)
R1/ CCNU ~ R=HRI=
NCNCH2CH2Cl ~O
BCNU ~ R=H, RI=cH2CH2CI
MeCCNU~ R=H R I = ~ c H 3
H3C CH3
~1--06 H3C CH3
SLCNU ~ R=H R I = < ~ N - - O • ,,----/,, H3C CH3
R=CH3, RI=
N3C CH g
H3C CH3
Z ~H3C <" 7 R=R1= <
CH3
~N O"
" -H3C ~c H3C CH3
H3
NO ,
8
R=
N --0",
H3C
R1: (CH2)2
3
"
--
NCONCH2CH2CI
i, c
H3C H3
CH3 H3
O"
Materials and Methods The nitrosoureas 4-8 were prepared as shown in Scheme I . The l - o x y l 2,2,6,6-tetramethyl-4-oxopiperidine (9) was used as the starting material. The reductive amination of 9 with ammonium acetate gave in addition to the primary amino compound lOa (lO) also the secondary amine lOd ( l l ) which was isolated by chromatography. Compounds lOb (lO), lOc and lOe were prepared by the reductive amination of 9 using methylamine, cyclohexylamine and ethylenediamine, respectively. The introduction of the chloroethyl ureido group to give compounds l l a - e was accomplished by the reaction of lOa-e with 2-chloroethyl isocyanate. Scheme 1 O
RNH
H3C
CH3
H3
CH3
L H3C
CH3
O*
aRcH
RNCNHCH2CH2CI ~ H3C
O*
O,
10a-,~e
1Ia,~e
H3C CH3
4
-
_
_
8
CH3
H3C R=CH2CH2-NH
CH3 N--Oo
H3C
3
11,.,~,.e:R=CH2CH2NCONHCH2CH2CI H3C H3
CH3 CH3 O.
Vol. 36, No. 15, 1985
Nitroxyl Labeled Nitrosoureas
1481
The nitrosation of these ureas ( l l a - e ) was carried out by the use of either dinitrogen tetraoxide or sodium n i t r i t e in diluted acetic acid. The former nitrosation method gave better results. In order to prove the position of the nitroso group in SLCNU (4) a regioselective synthetic method was used to prepare 4. Thus, N'-hydroxysuccinimide-N-(2-chloroethyl)-nitrosocarbamate (133) (12) was condensed with lOa to afford the nitrosourea 4 where the nitroso group is attached at N-l, the required position. Scheme 2 o
CICH2CH2NHCOO
CICH2CH2NCOON j.J tF O
O
The compounds 4 obtained either by the conventional route (Scheme l ) or by the regioselective method (Scheme 2) were identical. The physical properties of the nitrosoureas 4-8 are shown in TABLE I. TABLE I . Compound
Physical Properties of Spin Labeled Nitrosoureas Molecular~ Formula
m.p. [°C]
yield
M.S. m/e~
[%1
C12H22N403ClC80-83 (305.78) (dec.)
82
307(48) 306(59) 305(100) 276(62) 240(54) 183(92)
5
C13H24N403C1C68-70 (319.81)
60
321(36) 320(52) 319(62) 290(100) 255(64) 215(65) 156(84)
6
ClBH32N403Cl~128-129 (387.93) (dec.)
61
388(20) 387(25) 358(25) 254(100) 224(43)
7
C21H38N504ClC127-128 (460.01)
67
461(20) 460(22) 432(28) 396(22) 355(70) 327(82)
SLCNU (4)
253(100) 8
C26H46N806CI 2 ~ 142-143 (637.61) (dec.)
69
3 9 5 ( I 0 0 )396(64) 380(22) 309(29)
Satisfactory microanalyses were obtained: C, H, N ± 0.4. spectra were consistent with the structures.
The I . R.
Chemical ionizatons using methane as reactant gas, therefore, for mol. wt. M+÷l values are reported. ESR: three lines, aN = 16-17 G. ESR: five lines, however, intensities are not 1:2:3:2:1, more complex exchange interactions. All compounds ( ! and 4--B) were evaluated in vivo against the P388 lymphocytic leukemia in CD2Fl male mice according to the protocol of the National Cancer I n s t i t u t e (13). Approximately lO6 cells of P388 were inoculated ip into the mice at day 0 of the experiment (6 mice/group). Daily
1482
Nitroxyl Labeled Nitrosoureas
Vol. 36, No. 15, 1985
ip drug admlnistratlon was begun 24 h after the inoculation and performed once daily for 9 days. At day 5, the animals were weighed and then observed for 60 days keeping a record of deaths and survivors. Data were analyzed by the T/C method where T represents the mean survival time of the treated group, and C the mean survival time of the tumor bearing conrol group. The percent of increase in l i f e span (%ILS) parameter was evaluated by [(T-C)/C] x lO0. For the determination of the LDso four logarithmically spaced doses were injected ip into four groups of 4 Swiss male mice. The observation period for the determinations of the LD50 was 30 days. The LDso values were calculated by Well's method (14). TABLE I I .
Compound
Anticancer A c t i v i t y of Spin Labeled Nitrosoureas Against P388 Leukemia in CD2Fl Male Mice Daily Dose mq/kg/d mmol/kg/d_
CCNU (1)
16
0.068
SLCNU
I0 2O 35
0.033 0.065 O.ll4
30 60 90
(4) 5
Acute LD50 5 day weight~ mq/kq ip chanqe[%1 564
ILS~ax [%]
CuresC Survival/ Total) __
-9.2
182~
0/6
123
+9.8 +4.4 -5.1
298 542 542
216 6/6 6/6
0.094 0.187 0.28
353
+2.5 +2.5 -ll.2
54 130 329
0/6 0/6 I/6
75
0.19
337
+4.7
31
0/6
60
0.13
-3.4
21
0/6
30 60
0.047 0.094
+2.7 -2.6
32 53
0/6 0/6
~The change of average percentage weight on day 5 was taken as a measure of drug t o x i c i t y . ~Results obtained on day 60. CCure means survival after 60 days. ~Reference 15. ~In reference 16, the ILS (%) is 172. Results and Discussion The anticancer effects and LD50 of nitrosoureas together with the comparative data for the positive control using CCNU (!) are summarized in TABLE I f . At an equal molar dose SLCNU (4) was found to be more active than CCNU ( ! ) . Thus, at a dose of 0.065 mmol/kg (20mg/kg), SLCNU (4) e l i c i t e d an ILS value of 542%, and a l l mice were alive after 60 days, whereas the ILS of CCNU (!) at a dose of 0.068 mmol/kg (16 mg/kg), was only 182% and a l l mice died within 30 days. In regard to t h e i r t o x i c i t i e s , there was no t o x i c i t y observed in the treated group of SLCNU, while the average weight of mice on
Vol. 36, No. 15, 1985
Nitroxyl Labeled Nitrosoureas
1483
day 5 in the treated group of CCNUdecreased by 9%. The SLCNU was also more active than the CCNUwhen compared at doses e l i c i t i n g equal t o x i c i t y . Even at the lower dose of lO mg/kg, the ILS of SLCNU (4) was found to be 298% which is 1.6 times higher than 1B2% ILS of CCNUat the dose of 16 mg/kg. In conclusion, with these results, we have shown that the SLCNU (4) possesses a higher a c t i v i t y and a much lower t o x i c i t y than the c l i n i c a l l y used drug CCNU. Acknowledgements These studies were conducted pursuant to a contract with the National Foundation for Cancer Research. The cancer lines were obtained from the DCT Tumor Repository, NCI-Frederick Cancer Research F a c i l i t y . References
(i) (2) (3) (4) (5) (6) (7) (8) (9) (I0) (II) (12) (13) (14) (15) (16)
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