Spin labeled amino acid nitrosourea derivatives

27 Bulka, E.; Pfeiffer, W. D.; Troltsch, C.; Dilk, E.; G artner, H.; Daniel, D.: Collect, Czech. Chem. Com m un. 43, 1227 (1978) 28 Kiihle, E.: Angew. Chem. 81, 18 (1969) 29 K laym an, D. L.: Griffin, T. S.: J. Amer. Chem. Soc. 95, 197 (1973)

Eingegangen am 6. M ai 1994 Angenommen am 20. M ai 1994

Prof. Dr. A. Hetzheim Priv.-Doz. Dr. W.-D. Pfeiffer Soldtm annstr. 16 D-17489 Greifswald Doz. D r. P. Pazdera K otlarska 2 61137 Brno

Higher Medical Institute1, Stara Zagora, Bulgaria, National Centre of O nco log y2, Sofia, Bulgaria

Spin labeled amino acid nitrosourea derivatives - synthesis and antitumour activity A N T O A N E T A Z H E L E V A 1, Z. R A IK O V 1, M A Y A IL A R I0 N 0 V A 2 and D. T 0 D 0 R 0 V 2

T he synthesis of three spin labeled derivatives of N -[N '(2-chloroethyl)-N '-nitrosocarbam oyl] am in o acids is reported. T he new n itro so u rea s are o b tain ed by co n d en ­ sation of th e co rresp o n d in g N -[N '-(2-chloroethyl)-N 'nitrosocarbam o y l] am in o acid w ith 2,2,6,6-tetram ethylf-oxyl-4-am in o p ip erid in e using dicyclohexylcarbodiimide. T heir chem ical stru ctu res are confirm ed by ele­ m ental analysis, IR , M S, an d E P R spectroscopy. All newly synthesized c o m p o u n d s show ed high a n titu m o u r activity against the ly m p h o id leukem ia L1210 in B DF1 mice.

2. Investigation, results and discussion A nalysis o f th e above d a ta led us to synthesize and test several nitroxyl labeled am ino acid n itro so u reas in vivo fo r anticancer activity. T he co m p ou nd s have been synthesized according to the Scheme. Scheme

/

R —CH — NH —CO —N —CH2—CH2CI + H2N —( |

I

COOH

\

^

N -0‘

/

П РР

----- — ---- ► CH2C I2, 0 - 5 °C

NO (4-Am ino-TEM PO )

/ CH3 1 R=H

2 R = CH2- C H 2- S - C H 3

Synthese und zytostatische Wirkung neuer spinmarkierter Nitrosocarbamoyl-Aminosauren D ie Synthese dreier sp in m a rk ierte r N -[N '-(2 -C h lo rethyl)-N '-nitrosocarbam oyl]-A m inosaure-D erivate w ird beschrieben. D ie neuen P ro d u k te w u rd en d u rch K onden satio n von N -[N '-(2 -C h lo reth y l)-N '-n itro socarb am oyl]-A m inosauren m it 2,2,6,6-Tetram ethyl-4-am inopiperidin-l-oxid u n ter Beteiligung von Dicyclohexylcarbodiim id erh alten . Ih re chem ischen S tru k tu ren w urden d urch E lem en taran aly se sow ie d u rch IR , M S u n d E P R Spektroskop ie verifiziert. Alle neuen V erbindungen zeigten hohe A k tiv ita t gegen L 1 2 l0 -L eu k am ien bei B D F l-M a u se n .

1. Introduction N -(2 -C h lo ro eth y l)-N -n itro so u re a s are a class o f alkylating a n titu m o u r agents w ith a b ro a d spectrum o f activity in experim ental system s an d find ap p licatio n fo r tre a tm en t o f h u m an cancer. Such as ly m p h o m as, m elanom as, gliom as and som e solid tu m o u rs [1 -4 ]. M o st w idely used are B C N U , C C N U , M e C C N U ; som e new n itro so u reas as fotem ustine [5, 6], tau ro m u stin e [7, 8] a n d perim u stin e [9] are u n d e r clinical trials. T heir clinical a p p licatio n , how ever, is lim ited because they show delayed a n d cum ulative toxic effects, b one m arro w suppression being prev alen t an d dose lim iting [10-12]. In ord er to achieve a m o re selective cytotoxic effect o f the 2-ch lo ro eth y l-N -n itro so carb am o y l g ro u p various carrier m o ­ lecules have been selected fo r syntheses o f new n itro so u rea derivatives [13-17]. M o reo v er, it has been established th a t the nitroxyl radical m oiety (spin label) can exhibit a b e­ neficially m odifying effect o n th e toxicity a n d activity o f T E P A an d T h io -T E P A derivatives [18, 19]. Based on these d a ta a spin labeled derivative o f C C N U has been synthesized [20, 21] a n d show ed a high a n titu m o u r activity ag ain st a wide spectrum o f experim ental tu m o u rs [20]. P harm azie 50 (1995), H. 1

3 R = C H2- C H , CH3

—

NO I R, — CH — NH —CO — N —CH2—CH2CI

I

/ vN - 0 ‘

C O - N H ------(

l a R ,= H

2a R ,= C H 2- C H z - S - C H 3

/CH 3 3 a R , = C H 2- C H ^ CH3

T he chem ical structures o f the n itro so u reas l a , 2a and 3a w ere confirm ed by elem ental analysis, IR , M S, an d E P R spectroscopy (Fig.). T he new spin labeled n itro so u reas w ere tested fo r a n titu m o u r activity again st ascitic lym phoid leukem ia L1210 in BDF1 mice by m ethods described in the literatu re [22]. The results are p resented in the T able. All the n itro so u reas w ere rem ark a­ bly active ag ain st the experim ental tu m o u r m odel used. A well m anifested dose effect relatio n sh ip w as observed. C om ­ p o u n d 1 a show ed b etter oncopharm aco lo g ical pro p erties th an

Fig.: EPR spectrum of spin labeled nitrosourea 2a. Spectrom eter settings: microwave pow er, 6.32 mW ; m odulation am plitude, 0.920 G ; time con­ stant, 40.96 msc; field intensity, 3474 G ; gain 1 x 105.

25

2a an d За at this exp erim en tal conditions. In conclusion it can be stated th a t spin labeled am in o acids are suitable carriers for the cytotoxic g ro u p of th e N -(2-chloroethyl)-N -nitrosoureas. F u rth e r preclinical o n co p h arm aco lo g ical studies of n i­ tro so u reas la , 2a an d 3a are in progress.

3a : Yield: 64%. m.p. 118-120 °C (dec). R f = 0.73 [iso-C3H 7O H /C H 3O H /N H 3 (10:5:3)]. MS m/e: 418 (M +), 388 (M + -NO). IR (KBr): amide 1 (1650cm -1 ), amide 11 (1530 c m -1 ), N N O (1490 c m -1 and 1305 c m - [ ), N -O (1360 cm -1 ). C 18H 33N s0 4C1 (418.62) Calcd.: C 51.70 H 7.80 N 16.40 Cl 8.46 F ound: C 51.64 H 7.88 N 16.72 Cl 8.75

T a b le: A ntitum our activity o f nitrosoureas l a , 2 a and 3 a on lym phoid leukem ia L 1 2 1 0 a

Acknowledgements: The authors wish to thank Dr. B. G. C arpenter (School of Pharm acy and Biomedical Sciences, U niversity of Portsm outh, U K ) and Mr. N. J. A rm strong (Science Faculty M ass Spectroscopy and N M R Unit, University of Portsm outh, UK ) for the help in earring out the IR and MS com pounds analyses.

C om pd.

Control la

2

3a

Dose [mg/kg] —

33.3

66.6 100.0 150.0 33.3 66.6 100.0 150.0 33.3 66.6 100.0 150.0

M TS”

T /C *

И

[%1

8.4

S u rv iv al [60 days]

-

-

27.0 52.5 50.3

264.4 625.0 599.0

2.8

33.3

i/6 5/6 5/6 0/6

34.3 14.2 13.8

571.7

3/6

169.0 164.0

0/6 1/6 0/6 2/6 3/6 0/6 0/6

8.2

97.6

25.2 32.7 6.5 4.0

300.0 389.3 77.4

47.6

References 1 2 3 4 5 6

7

8 a 1 x 105 t u m o u r cells in 0.1 m l, i.p. tra n s p la n te d b T /C [% ]: (M S T tre a te d /M S T c o n tro l) x 100; T /C ^ a c tiv ity ; T / C ^ 8 5 % -“ to x ic ity ” v alu e s [22] c M S T : M e a n s u rv iv a l tim e -d e te rm in e d a t t h e d 6 0

1 2 5 % -m in im a l c rite ria fo r a n titu m o u r

9 10 11 12

3. Experimental 3.1. N - [ N '- ( 2 - c h l o r o e t h y l ) - N '- n i t r o s o c a r b a m o y l ] a m in o a c id s 1. 2 and 3 The products were prepared according to [13].

13 14 15 16

3.2. N - [ N '- ( - c h l o r o e t h y l ) - N '- n i t r o s o c a r b a m o y l ] a m in o a c id s — [ 2 , 2 , 6 , 6 - t e t r a m e t h y l - 1 - o x y l- 4 - im in o p ip e r i d in y l] — l a , 2 a a n d 3 a Com pounds 1, 2 o r 3 (2 mmol) and 2.2 mmol D C C were dissolved into 15 ml C H 2C12 at 0 -5 °C. The m ixture was stirred vigorously for 15 min and 2 mmol of 4-A m ino-TE M PO dissolved in 10 ml C H 2C12 were added dropwise. After 14 h stirring the precipitated. N.N '-dicyclohexyl urea was filtered off and the mixture was concentrated under reduced pressure. The residue was crystallized from C H 2Cl2/n -C 6H 14. la : Yield 62% , m.p. 122-124 °C (dec). R r = 0.71 [iso-C3H vO H /C H 3O H /N H 3 (10:5:3)]. MS m/e: 362 (M +), 332 (M + -NO). IR (K B r): amide I (1680 cm “ 1), amide II (1535 c m -1 ), N N O (1490 c m -1 and 1300 cm " ‘i N - O 0 (1350 c m -1 ). C ,4H 25N 50 4C1 (362.59) Calcd.: C 46.80 H 6.60 N 1 8 .1 0 Cl 9.77 F ound: C 46.37 H 6.89 N 1 8 .3 0 Cl 9.20 2 a : Yield: 58% m.p. 135 °C (dec). Rf = 0.70 [iso-C3H vO H /C H 3O H /N H ,

(10:5:3)]. MS m/e: 436 (M +), 406 (M +-NO). IR (KBr): amide I (1650cm - 1 ), amide II (1 5 3 0 cm -1 ), N N O (1 4 9 0 cm -1 and 13 1 0 cm -1 ), N -O ' (1360cm -1 ). C 17H 31N 50 4C1 (436.67) Calcd.: C 46.71 H 7.70 N 1 6.40 Cl 8.11 F ound: C 46.75 H 7.02 N 16.03 Cl 7.98

26

17 18 19 20 21 22

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Received April 15, 1994 Accepted J uly 24, 1994

A ntoaneta M. Zheleva D epartm ent Chem istry and Biochemistry H igher M edical Institute 11 A rm eiska St. 6000 Stara Zagora Bulgaria

P h arm azie 50 (1995), H. 1