13. A. Stoesse, G. H. Umoin, and J. B. Stathers, Can. J. Chem., 61, 372-375 (1983). 14. T. R. Kelly, J. K. Saha, and R. R. Whittle, J. Org. Chem., 50, 3679-3685 (1985). 15. K. Krohn, H. Markus, A. P. Kraemer, and W. Frank, Liebigs Ann. Chem., 1033-1041 (1988). 16. B. M. Trost, W. C. Vladuchick, and A. J. Bridges, J. Am. Chem. Soc., 102, No. i0, 3554-

3572 (1980).

AMIDES OF ANTHRACYCLINE ANTIBIOTICS

AND N-GARBOXYMETHYLASCORBIGEN

A. M. Korolev, E. I. Lazhko, M. N. Preobrazhenskaya, Ya. Bal'zarini, and E. De Klerk

UDC 615.33:577.182.540].012.6

Compounds with high antitumor activity occur among the N-acylated derivatives of an- thracycline antibiotics, in spite of a significant decrease of affinity of these materials of DNA [3].

Some of the ascorbigens, i.e., indole containing derivatives of L-ascorbic acid, are biologically-active compounds possessing antitumor properties or immunomodulating activity, capable of undergoing diverse transformations in the organism. They may interact with dif- ferent nucleophilic (thio-, amino-) groups or cleave nonenzymatically into L-ascorbic acid and 3-hydroxymethylindole or their derivatives [i, 7]. An increase in the level of L-ascorbic acid after introduction of the preparations may promote the sensitivity of the cells to chemotherapeutic agents [6], and 3-hydroxymethylindole, being an active alkylating frag- ment, may interact with diverse targets [2, 5, 7]. The wide spectrum of biological activ- ity of these compounds induced an interest in the synthesis and study of the antitumor activity of derivatives of anthracycline antibiotics covalently bonded to ascorbigen, i.e.; potential cytostatic agents and the depo-form of ascorbic acid.

The. present work undertook the synthesis of derivatives of ascorbigen and anthracycline antibiotics, to produce acylation of the amino groups of the antibiotic by N-carboxymethyl- ascorbigen. N-carboxymethylascorbigen (IV) was synthesized in three stages: alkylation of 3-formylindole with chloroacetic acid in tert-butanol in the presence of potassium tert- butoxide to give l-(carboxymethyl)-3-formylindole (II). Reduction of II with NaBH 4 gave the 3-hydroxymethyl derivative (III), and subsequent condensation of III with L-ascorbic acid in a phosphate-citrate buffer to give IV, which was transformed into the N-hydroxy- succinimide ester (V) with N-hydroxysuccinimide in the presence of dicyclohexylcarbodiimide.

I. t - B u O K ~ C ~ O f ClCH~,OOK ~---~-CIIO ~aBn~_

z ? ~ I 2

" " 0 " o

0 I - I

C I I ~ C K J O S u

Scientific-Research Institute for Research on New Antibiotics, Academy of Medical Sciences of the USSR, Moscow. Rega Institute, Catholic University of Louvain, Louvain~ Belgium. Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 25, No. ii, pp. 42-45, November, 1991. Original article submitted February 26, 1991.

0091-150X/91/2511-0805512.50 �9 1992 Plenum Publishing Corporation 805

TABLE i. ~H NMR Spectra of Compounds II-VII: 6, ppm; J, Hz

Protons of the ascorbic Protons of the indole ring cH~

Com- acid residue

411 5}1 6a H 66 tt 2 "H IJs,6a) (Js. 4; b (Jea6 b a a , b

IV*

V*

VII a

Vllb ~*

1 4"H I 5"1i 6 " H 7" I t

I

3,96 4,20 3,99 4,11 7,21 7,18 7,24 7,09 7,13 3.32 4,93 (5,8) (3,6) (9,7)

4,95 4,21 3,98 4,12 7,22 7,28 7,02 7,13 7,63 3,30 5,48 (6,0) (3,8) (9,6)

4,76 4,6t 4,27 4,45 7,74 8,14 *** *** 7,47 3,98 3,88 4,99 4,93 (6,0) (3,8) (9,4) (14,4) (16.9)

4,74 4 ,() 1 4,27 4,43 7,69 8,12 7,02 7,13 7,61 3,98 3,88 4,9!) 4,93 (6,0) (3,8) (9,4) (14,4) (16,9)

CH~-- CO

~ a ] 6b

~Ja~b)

NH JE l l , ,l"

7.88 (8,30) 7.88

(8,30)

2,81

C o l l l -

pound

IV* V* Vl!a**

Vl~**

Protons of t h e anthracycline aglycone Protons of t he aminosugar

t iilii ( I (J ( j ~ I 1411 I ' l l ' ' T b H 3"1] 4'H 5'H 5-Ct t , lJh'2) ( J I ) (J2. tP 7,~ a 7.,cl aa. j lobmc)

8,10 7,71 7,39 3,94 5,39 2,45 2,75 3,46 3,51 2,57 5,70 2.26 2,19 4,75 3,63 4,59 1,39 (7,6) ( < 1 ) (8,3} (5,3) (3.4) (14,3) (18,4) 7,9'2 7,65 7,38 3,94 5,35 2,44 2,75 3,47 3,50 2,56 5,67 2,23 2,29 4,74 3,65 4.59 1,40 (7,6) (I,7) (8,4) (5,5) (2,9) (14,4) 118,4)

*Spectra obtained in CD3OD. **Spectra obtained in CsDsN. ***Signals overlapped by solvent signals.

Condensation of V and daunomycin (Via) or carminomycin (Vlb) was carried out with an equi- molar ratio of reagents in dry dioxane at room temperature for 8 h. The compounds obtained were isolated by preparative TLC and column chromatography. The structure of compounds II, IV, VIIa and VIIb was verified by IR, UV, and IH NMR spectroscopy and elemental analysis. The physico-chemical characteristics of the new materials are presented in Table 1 and the Experimental Section.

The U~r spectra compounds VIIa and VIIb were the sums of the spectra of the starting

compounds.

The IR spectra of compounds VIIa and VIIb showed characteristic absorption bands for the amide linkages at 1640-1650 cm -I and the absence of the absorption of the carboxyl group of the starting antibiotic at 1745 cm -~. An absorption band at 1790 cm -I indicated the preservation of the lactone ring of the ascorbigen molecule.

]RO 0 HO H 0 ~ x . J

I1 / Cj-IzO00- Su

ot-~tz~, r tIO

ETa,b

H 014 0 0 it0 0 ~'~.~

Z I~ 6 ,,

a e"CYrI CH5 ASC=

~ o o . o ~ o ~'~-~'-KV ,~

a' ~ I' a) Hl=Ol % b) Rz=H

I ~ra- c o c ~ A s c HO

~Z a,b

806

TABLE 2. Suppression of Cell Prolifera- tion, IDa0* (~Molar)

I Cell type Compound

1-1210 j J .~IOLT-4 F

IV >100 >100 Via 0,04 0,044 V Ib 0,005 0,005 VlJa 3,10 2,90 V I Ib 0,02 0,03

MT 4

>1oo

1,98 0,13

*Concentration of the preparation suppress- ing the proliferation of cells by 50%.

All signals characteristic of both constituents of the molecules were identified in the iH NMR spectra of compounds VIIa and VIIb. It should be noted that the signals for 3'-H, 2'a-H and 2'b-H undergo a displacement to weaker field compared with the starting anti- biotics, more significant for the 3'-H signal, which indicates N-acylation. The signals of these complex molecules were treated by the methods of double resonance and KOSY~

EXPERIMENTAL (CHEMISTRY)

TLC was carried out using Silufol UF-254 plates, and for preparative chromatography, 200 • 200 mm plates with thickness of the fixed layer of Merck HF2s ~ silica gel of 0.5 mm. Materials on the chromatogram were visualized with UV-light or by heating the plates. The silica gel for column chromatography was Kieselgel 60 (Merck), and the solvent systems were chloroform/methanol 7:1 (A) or 9:1 (B). The iH NMR spectra were recorded of a Varian VXR-400 spectrometer using tetramethyl silane as internal standard. The IR spectra in KBr were obtained with a Pye Unicam SP-IIO0 (England), UV spectra were recorded on Beckman UV- 5260 instrument (Austria) in cm quartz cuvettes in ethanol. Melting points were deter- mined with a Buchi SMP-20 apparatus (Switzerman). Specific optical rotations were obtained with a Perkin Elmer 241 polarimeter (France) in 1 cm ~ • i0 cm cuvettes. Elemental analy- sis data agreed with the calculated values.

N-Carboxymethyl-3-formylindole (II). Powdered 3-formylindole I (0.5 g, 3.40 mmoles) was added with stirring to a solution of potassium t-butoxide prepared from 0.13 g of potas- sium in 15 ml of t-butanol under nitrogen. The solution was stirred for 1 h, then a sus- pension of 0.45 g (3.40 mmoles) of potassium chloroacetate in t-butanol and i ml of dimethy]- sulfoxide was added and the mixture was heated at 90~ for 5 h. The reaction mixture was poured into water, IN NaHCO 3 was added to pH 8.5, and the starting 3-formylindole was ex- tracted with ethyl acetate, after which the water layer was acidified with I'N HCI to pH 2.0 and the product was extracted with ethyl acetate in 92% yield, mp = 176-177~ CIIH~" NO 3. ~H NMR spectral data (6, ppm; CD~OD) 5.10 s (2H, CH~COO) 7 19 t (IH, 5-H), 7.28 t (IH, 6-H), 7.42 d (IH, 4-H), 7.61 d (IH, 7-H), 9.88 s (IH, CHO); spectrum (Vma x, cm-1): 1746 (COOH), 1650 (CHO). UV spectrum (imax, nm; e): 213 (24,500), 245 (15,600).

N-Carboxymethylascorbigen (IV). To a solution of 0.3 g (1.47 mmoles) of II in 25 ml of ethanol was added with stirring 0.055 g (1.46 mmoles) of NaBH~, after which the stirring was continued for 1 h at room temperature. The solvent was evaporated under vacuum and to the residue was added 2 ml of acetone and the solvent was again evaporated. The residue (0.45 g) containing 3-hydroxymethyl-N-carboxymethylindole III was dissolved in i0 ml of alcohol and slowly added with stirring to a solution of 0.6 g of L-ascorbic acid in i00 ml of phosphate-citrate buffer at pH 4.1 and the stirring was continued for an additional 3 h. The reaction mixture was extracted with ethyl acetate, the extract was washed with water, dried with Na2SO 4 and the solvent was evaporated under reduced pressure. The resi- due was chromatographed on a silica gel column using eluent system B. The amorphous , material was dried under vacuum to give IV in 54% yield. CITHITNOs. IR spectrum (Vmax, cm-i): 1746 (COOH), 1790 (CO). UV spectrum (imax, nm; s): 223 (33,000), 280 (6500). [~]22 = + I0.2oc (c 2.0, ethanol).

Succinimide Ester of N-Carboxymethylascorbi~en (V). To a solution of 0.05 g (0.14 mmole) of acid IV and 0.016 g (0.14 mmole) of N-hydroxysuccinimide in i0 ml of ethyl acetate was added with stirring a solution of 0.028 g (0.14 mmole) of dicyclohexylcarbodiimide in

807

3 ml of ethyl acetate, and the mixture was stirred magnetically for 3 h. The urea pre- cipitate was separated, the solution was washed with water, dried with N~2SO ~ , and the sol- vent was evaporated under vacuum to give a 90% yield of V, C21H20N2Ol0. IR spectrum (Vma x, cm-~): 1790 (CO).

Amide of l-(Carboxymethyl)ascorbigen and Daunomycin (VIIa). To a solution of 0.i g (0.19 mmole) of the antibiotic daunomycin (free base) in i0 ml of dioxane was added at room temperature 0.087 g (0.19 mmole) of N-carboxymethylascorbigen VI in 5 ml of dioxane, and the reaction mixture was stirred for 8 h at the same temperature. The solvent was evaporated under vacuum and the residue was separated on silica gel plates using eluent system A. One band was selected (Rf 0.35), eluted with acetone, and dried under vacuum to give Vlla in 67% yield, mp 185-187~ (dec). C44H~N2OI7.

Amide of l-(carboxymethyl)ascorbigen and carminomycin (Vllb) was obtained analogously to Vlla. The material was isolated on silica gel plates, selecting the band with Rf 0.037 after elution with System A. Yield of Vllb = 65%, mp = 208-210~ C43H42N2OI7.

EXPERIMENTAL (BIOLOGICAL)

Table 2 shows the action of these compounds compared to the starting l-carboxymethyl- ascorbigen IV and daunomycin Via or carbinomycin V!5 on the proliferation of cells of mouse cell Leucosis L-1210, and human cell T-lymphobiast Molt-4F and T-lymphocyte MT-4. Intro- duction of the N-carboxymethylascorbigen residue IV leads to a decrease of the cytostatic activity, particularly in the case of derivatives of rubomycin Vlla. The compounds sup- pressed the cytopathogenic viruses HIV-I and HIV-2 in MT-4 cells only in nearly cytotoxic concentrations. The cytostatic and anti-HIV activity studies were carried out according to the method of [4].

1.

2. 3.

4.

5.

6. 7.

LITERATURE CITED

I. L. Plikhtyak, I. V. Yartseva, Zy En Khan, and M. N. Preobrazhenskaya, Bioorg. Khim., 14, No. i0, 1437-1443 (1988). F. Amat-Guerri, R. Martinex-Utrilla, and C. Rascual, J. Chem. Res. (S), 160-161 (1984). J. W. Lowen (ed)., Anthracycline and Anthracenedione-Based Anticancer Agents, Vol. 6, (1984), p. 447. J. Balzarini, M. Bada, R. Pauwels, et al., Mol. Pharmacol., 33, 243-249 (1988). A. T. Fong, H. I. Swanson, R. H. Dashwood, et al., Biochem. Pharmacaol., 39, 19-26 (1990). S. Marcus, Cancer Res., 47, 4208-4213 (1987). M. N. Preobrazhenskaya, A. M. Korolev, I. L. Plikhtyak, et al., Heterocycles in Bio- organic Chemistry, H. van der Plas, et al., (eds.), New York (1991).

808