Tong, Henry, Acton
36 Journal of Mcdicinai Chr.niistr>, 1979, V O ~22, . No. 1
5-Iminodaunorubicin, Reduced Cardiotoxic Properties in an Antitumor Anthracycline George L. Tong, David
W.Henry, and Edward M. Acton*
Rio-Organic. Chcniistr>' Ilcpnrtnient, SRI International, Menlo Park, California 94025. Rewired Ma), 8, 1978
Treatment of daiinorubicin with methanolic ammonia affords S-iminodaunoruhicin, the first quinone-modified analogue of either daunorubicin or adriamycin. This product retains antileukemic activity in mice, is less cardiotoxic by electrocardiographic measurements in rats. and is nonmutagenic in Salmonella typhimurium (Ames test). Development of t h e important anticancer drugs adriamycin (1) a n d daunorubicin (2) has brought about
q$pJ-x
v0
CH30
0
v0
HO HO
I
CH,O
R
HO HO
"2.
"2.
HCI
1, X - OH 2,X=H
major advances in t h e treatment of a variety of solid tumors a n d leukemias in humans.' However, these agents produce cardiotoxic side effects t h a t are dose dependent a n d cumulative. so that dose levels must be limited, a n d sometimes successful courses of treatment must even be halted t o avoid the risk of fatal heart failPerhaps t h e best solution to this problem would be development of structural analogues in which antitumor efficacy is retained while cardiotoxic effects are minimized, b u t a rational approach t o such analogues requires much more t h a n is presently known about the mechanisms of biological action of 1 and 2. T h e interaction of these drugs with DNA is important a n d has been studied widely,'' particularly t h e intercalation of 1 and 2 into t h e helical form of DNA, b u t various biochemical mechanisms are accessible to these molecules. Several current studies point t o t h e quinone unit of 1 and 2 as a key site of biological action t h a t might involve t h e redox properties of t h e quinone function6 or possibly its alkylating potential.1° Analogues modified a t the quinone would be important for further mechanistic studies that might lead t o a separation of t h e antitumor and cardiotoxic effects. Several hundred analogues of 1 and 2 have been synbut thesized for evaluation of antitumor properties, surprisingly it appears that none has provided a structural change a t t h e quinone function. So far as we are aware, this report is t h e first of such an analogue. Chemistry. Derivatization of the quinone was first observed during ammonolysis of the ester 3 (obtained'? by side-chain degradation of 1) t o form t h e amide 4. Thin-layer chromatographic analysis showed t h a t t h e product 4, reddish orange like 1 and 2 and other anthracyclines, was accompanied by one or two violet byproducts. T h e byproducts appeared t o arise from ring amination, according t o mass spectral a n d 'H N M R analyses and a shift in the Ci'-visible absorption spectrum t o longer wavelength (Table I). When the reaction was conducted in cold methanol solution saturated with ammonia, ring amination appeared t o be favored, whereas conversion t o t h e amide appeared to be favored in liquid ammonia. T h e amide 4 was best obtained if ester 3, a5 a thin film on t h e walls of a flask, was exposed to an atJ
HCI
3, R = 0; X = OCH, 4,R=O;X=NH, 5, R = NH; x = OCH, 6,R = NH; X = NH,
mosphere of ammonia a t -10 t o -15 "C for several days, followed by chromatographic purification. T h e violet byproducts were not fully purified and characterized, and they were assumed to be the 5-imino ester *5 and t h e 5imino amide 6 hy analogy with t h e product (7) from
8
k O \I
"2'
HCI
7 daunorubicin. There was no evidence t h a t t h e violet byproducts could be hydrolyzed back t o t h e parent quinones nor were they converted with excess 30% hydrogen peroxide a t room temperature. I t was of interest t o extend t h e amination t o daunorubicin (2) because the reaction appeared to give negligible cleavage of t h e glycosidic bond or other types of degradation. Daunorubicin hydrochloride was best treated with methanolic ammonia a t 0-5 "C for 39 h t o give a single blue-violet product 7 t h a t was chromatographically purified as t h e hydrochloride in 57% yield. A minor blueviolet byproduct was observed ( 1 2 % ) but not isolated and identified. T h e selectivity of t h e amination to produce a single major product seemed surprising a t first. Attack by NH, was expected t o occur a t either of the quinone carbonyls t o produce two isomeric products. However, there are reasons why amination a t C-5 to give the &imino derivative 7 might be preferred. First, quaternization of t h e quinone carbon a t t h e intermediate stage of t h e amination, as in 8, should be favored a t C-5 since it permits relief of t h e steric strain from t h e three peri substituents a t C-4, C-5, a n d C-6. Furthermore, in 7 t h e imino group a t t h e 5 position can be stabilized by H bonding t o t h e 4-OCH1 as shown in the structure, as well as by H bonding
0022-2fi23/79/1822-00~fi~01.00/0 C, 1978 American Chemical Society
5- Iminodaunoru bicin
Table I . Comparison of UV-Visible Spectraa hmau, nm ( e x lo-')), in MeOH compd 233 (38.4). 252 (26.0), 288 (8.87), 478 (12.4), 4 494 (12.3), 530 (6.64) crude 220 (26.2), 233 (23.0), 252 (27.7), 303 (6.15), 310 sh. 518 sh. 550 (13.9). 591 (16.1) 5 6 26 234 (37.5), 252 (26.1j, 290 (8.60), 480 (12.11, 495 (12.3), 532 (6.30) 7 220 (29.8), 233 sh, 252 (32.0), 307 (6.93), 335 sh, 357 sh, 520sh, 551 (16.6), 592 (19.8) Obtained on Cary 11 o r Cary 1 4 recording spectroData from G. Cassinelli and P. Orezzi, G. Micro meters. b i d . , 11, 167 (1963).
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of the 6-OH to N.A precedent for this type of stabilization is found in t h e observation13 t h a t treating l-methoxyanthraquinone with methanolic ammonia produced in high yield a single quinone imine which was weakly H bonded t o the methoxyl, whereas 2-methoxyanthraquinone did not form a n imine even a t 100 OC. T h e elemental analysis of product 7 confirmed t h a t a second N had been introduced into the molecule. A shift in UV-visible absorption t o longer wavelengths in going from 2 to 7 is shown in Table I and corresponds to the shift from 4 t o 5 a n d 6. Data in support of structure 7 as 5iminodaunorubicin were observed in t h e N M R spectra, which are reported in Table 11. T h e 'H N M R spectrum of 7 in dimethyl-d, sulfoxide (Me2SO)solution showed two H-bonded protons a t 6 13.47 and 9.54. Both signals were broad singlets, unlike t h e sharp singlets typical of t h e H-bonded phenolic OH'S in anthracyclines such as 1 and 2. Clearly the proton at 6 9.54 was only weakly H bonded, but as is characteristic of H bonding both protons kept the same chemical shift and signal broadening in all samples and M e 2 S 0 solutions of 7. Addition of a small amount (0.2 molar equiv) of trifluoroacetic acid'$ to t h e 'H N M R solution did not produce sharpening of these signals as we expected, but it did cause the appearance of a third proton as a very sharp singlet, strongly H bonded a t 6 15.80. Sharpening of the other two protons was achieved only by irradiation, first a t d 13.47 and then a t 6 9.54, showing that these protons were coupled and thus indicating t h a t both were H bonded about t h e N. T h e only arrangement of atoms in this product t h a t can accommodate three H bonds is structure 7, having t h e imino group a t C-5. If alternatively the imino group had been introduced a t C-12, only two of t h e three protons could be H bonded. Therefore, we have assigned the 5-NH to the signal a t d 9.54 (weak H bond to 4-OCH3), t h e 6-OH to 6 13.47 (normal H bond t o N ) , a n d the 11-OH to 6 15.80 (strong
Journal of Medicinal Chemistry, 1979, Vol 22, No. J
37
H bond t o C=O). T h e stronger nature of t h e H bond t o t h e (2-12 carbonyl in 7 is attributed t o t h e effect a t (2-12 of the more electron-releasing imine a t C-5 (relative to the carbonyl at C-5 in 2). By similar reasoning we assign t h e stronger H bond in 2 t o t h e 6-OH, based on t h e electron-releasing effect of the 4-methoxyl on the carbonyl a t C-5. I t appears t h a t t h e phenolic OH'S in 1 a n d 2 have not been individually assigned in previous literature, and this no doubt reflects the lack of study of quinone modifications. T h e '?C N M R data supplement the above observations. In particular, placement of the imino function on one of t h e quinone carbons was clearly confirmed. In 2 t h e quinone carbons were indistinguishable a t 6 185.9 and 186.1, but on going to 7 they were shifted strongly upfield and became well separated a t 6 172.7 and 179.3. T h e shift to 6 172.7 is the largest for any carbon and clearly involves the site of amination, although it does not permit a choice between C-5 and C-12 that would confirm the assignment based on 'H N M R data. T h e site of amination might independently be defined from t h e changes a t C-4a a n d C-12a, since these sites were already distinguished in t h e starting material 2. Indeed, the larger upfield shift a t C-4a (3.3 ppm) than a t C-12a (1.9 ppm) in going from 2 to 7 does support the assignment of 7 as the 5-imino compound, but t h e effects were not large enough for this to be used as an independent criterion. Other changes in t h e I3C N M R spectra were also small, except a t C-6 a n d C-6a where appreciable downfield shifts occurred. Perhaps this can be attributed to t h e existence in some proportion of t h e tautomer 7a with a carbonyl a t C-6 a n d an amino group
7a a t C-5. Also, t h e observed coupling of t h e protons about the N might be better explained in t h e tautomer 7a t h a n in structure 7. All of the data are consistently explained only by the 5-imino structure 7 or the tautomer 7a. More detailed N M R studies of 7 that confirm these assignments and explore the substituent effects and tautomerism of the aglycon will be t h e subject of a separate report. I t is possible t h a t the presence of the adjacent methoxyl group and t h e resultant arrangement of hydrogen bonds in structure 7 are essential for t h e formation of 7 and for
Table 11. Comparison of NMR Data 'HNMRa chemical shift, h (Me,SO-d, soln, Me,Si internal ref)e compd H-l H-2 H-3 4-OCH3 COCH, H-1' H-5' 5'-CH, 6-OH 11-OH =NH 2 7.83, m 7.59, m 7.83, m 3.96,s 2.30,s 5 . 2 9 , b r s 4.22,bq 1 . 1 7 , b d 1 4 . 0 5 , s 13.26,s 7 7 . 9 5 , m 7 . 7 9 , m 7 . 5 8 , m 4.10,s 2 . 3 1 , s 5 . 4 7 , b r s 4 . 2 0 , C m 1 . 1 9 , b d 1 3 . 4 7 , b r s 15.80,ds 9 . 5 4 , b r s 'T NMRa chemical shift, 6 ( D 2 0soln, dioxane internal ref) com45'pd C-1 C-2 C-3 C-4 OCH, C-4a C-12a C-5 C-12 C-5a C - l l a C-6 C-11 C-6a C-loa C-13 C-14 CH, 2 120.0 137.2 120.1 161.0 57.1 119.1 134.0 185.9 186.1 110.9 110.9 156.4 154.7 134.9 135.0 215.8 25.1 16.6 or or or or or or 120.1 120.0 135.0 134.9 186.1 185.9 7 119.7 134.3 117.1 159.8 57.2 115.8 132.1 172.7 179.3 103.9 109.4 160.9 155.4 141.2 135.4 216.0 25.2 16.6 diff 3.3 1.9 13.2 6.8 3.5 6.1 a Varian XL-100 spectrometer. Shift assignments are based on proton noise decoupling and o n previous assignments by A Arnone et and L. H. Wright, J. A. Chan, J. A. Schroer, and A. A. Aszalos, J. Org. Chem., 42, 2344 (1977). Jish,= Sharp singlet appeared after adding trifluoroacetic acid t o the solution. e m = multiplet, 6-7 Hz. Quartet obscured. br = broad, s = singlet, d = doublet, q = quartet.
act. vs. leukemia P388 in mice:" C., TIC (dosr, m:;/k, How well t h e nciiiriiutape~~icity of 7 i i i ;ulmonella .-trains will predict for human therapy is. again, n c i t t'ztablished. This compound was found t o he about as mutagenic as 1. though less mutagenic than 2, \vhen tested i t i V79 Chinese hamster cells,?' which suggest> that some process for trarisforination to a mutagen m a y I ) t ' present in mammalian cells that is absent in tiacteria. h i any case. norimutagenicity of 7 in t h e Ames test a t least indicate. t h e loss o f some f o r m o f interaction tvith DN.4 t h a t is hr,)iight ahout b y the ininor nicidif'ic~itioni n itrrict\ire a t
5 - Iminodaunorubicirz
the quinone. Lf'hether this is related to the reduced cardiotoxicity in the Zbinden test may be an interesting subject for further study. Table 111 presents d a t a from other in vitro tests of interaction with DNA. T h e increase (AT,) in thermal denaturation temperature'? of helical DNA in the presence of t h e test compound is a measure of its stabilization of t h e helical form of DNA, presumably by intercalative binding. This effect is significantly weaker for 7 . On the other hand, 7 retains the potency of 1 and 2 as an inhibitor of DNA and KNA synthesis" in L1210 lymphoid leukemia. T h u s . apart from its potential as an anthracycline anticancer agent with reduced cardiotoxicity, 7 is a useful subject for mechanism of action studies, and its properties suggest to us that hitherto overlooked modifications of the qui none should continue to be explored . Experimental Section Solutions !\'ere evaporated in vacuo using a rotary evaporator. Melting points are uncorrected. IR spectra were taken on a Perkin-Elmer 137 spectrometer. Mass spectra were recorded on an LKB Model 9000 spectrometer at 18 ei,'. The R, values are from thin-layer chromatography on silica gel GF 250-pm plates (Analtech). 5-Iminodaunorubicin (7). To a stirred solution of 1700 mL of methanolic ammonia (saturated at 0 "Cj in an ice bath was added a solution of 28.8 g (51.0 mmol) of daunorubicin hydrochloride in 500 mL of methanol. The cold solution was stirred briefly (1 h), stored at 0-5 " C for 39 h, and evaporated. To remove traces of ammonia, the violet residue was thrice dissolved in 400 mL of CHCl,-CH,OH (41) and the solution evaporated, yielding 33.9 g: R, 0.12 (violet. trace impurity),0.14 (violet, 71,0.21 (orange, trace 2 ) , and 0.71. 0.75 (magenta, trace aglycons) in CHC13-CH,0H-H20 (40:lU:l). The product was isolated by chromatography of the residue in two equal batches. A solution of 16.9 g in 200 mL of CHClj CHtIOH(6:li was added to a column (6.7 x 86 cm) of silica gel (20+325 mesh. Mallinckrodt SilicAR CC-71, which was eluted with the same solvent. After collection (100-mL fractions, monitored by T I X ) of 13.7L of initial eluate, a 5.4-L fraction was evaporated to yield 9.67 g. which was precipitated by dissolving in 100 mL of CHC13-CH,0H (4:l) and adding 100 mL of ether dropwise. The precipitate was washed with ether and dried in vacuo at 25 " C to yield 8.79 g of 7 as a homogeneous blue-violet powder: mp 175- 178 "C dec (preheated to 160 Ti; IR (Nujol) 2.92 (OH), 5.82 (C=O),6.31 pm (C=O of chelated quinone, C-ru'). The combined yield was 16.8 g (57% 1. Anal. (C?;H;ioN2OqHCl.H20) C. H, C1,N. (7s, 9 S ) - 7 - [(3-Amino-2,3,6-trideoxy-~u-~-lyxo -hexopyranosyl)oxy]-7,8,9,l0-tetrahydro-6,9,1 l-trihydroxy-4methoxy-5,12-dioxonaphthacene-9-carboxamideHydrochloride ( 4 ) . A solution of 0.478 g (0.88 nimol) of the ester 3 as free base (isolated before conversion to the HC1 salt'') and 0.054 g (1.0 mmol) of NH,Cl was evaporated to leave a film on the walls of a 1-L flask. (The NH,CI is required for disproportionation to give the evaporated product as the HC1 salt; alternatively, NH4C1 may be omitted if the HCI salt o f 3 is used.) The flask was cooled to -60 "C and 150 rnL of' ammonia was condensed into it with stirring. The solution was allowed to evaporate while the flask was rotated at room temperature to leave a violet residue on the wall under an atmosphere of ammonia. The flask was stoppered immediately, stored at -10 to --15 " C for 6 days, and flushed with N?. The residue was dissolved several times in CHCI:,-CH,OH ~ l : l and ) , the solution was evaporated to afford an amber residue, 0.55 g [R, 0.17 (violet) and 0.23 ( 4 , reddish orange) in CHCI,-CH30H-H2O (20:10:1)]. A solution in 40 mL of CHC1,-CH30H (1:l)was added to a column ( 2 X 48 cm) of 200-325 mesh silica gel (50 g: Bio-Si1 A), which was eluted with CHC1,--CH,30H-H,0 (40:lO:l). An initial 100 mL of eluate was collected. The reddish orange product was then collected in a 200-mL fraction, which was evaporated. The residue was dissolved in 15 mL of CHCl,--CH,,OH i1:l) and 2 mL of 0.1 M HCI in CH30H (some dissociation to the free base appeared to occur during chromatography) and reprecipitated with 80 niL of ether to yield 0.307
Journal of Medicinal Chemistry, 1979, Vol. 22, No. 1 39 g (59%) of 4: mp 189-192 "C dec; IR (Nujol) 2.90 (OH), 6.00 (amide C=O), 6.20, 6.31 Fm (C=O, chelated quinone): MS [as the (Me,Si), derivative] m j e 945 (M CH,), 930 (M - 2CHJ. Anal. (C28H2sN2010.HC1.1.5Hz0) C, H, C1, N. When 4 was treated with methanolic ammonia, as described for 7 , the product was a mixture of 5 and 6 containing 'I.Iwaizumi, K. Handa, and Y.Tamura, Gann, 68, 603 (1977). J. Goodman and P. Hochstein, Biochem. Biophys. Res. Commun., 77, 797 (1977). J. W.Lown, S. Sim, K. C. Majumdar, and R. Chang, Biochem. Biophys. Res. Commun., 7 7 , 705 (1977). H. R. Moore, Science, 197, 527 (1977). F. Arcamone, Lloydia, 40, 45 (19771, G. L. Tong. R. If'. Lee, D. R. Black, and D. R. Henry, J. Med. Chem., 19,395 (1976). A. Muller, K. Kormendy. F. Ruff, P. Sohar, and M. Vajda, Acta Chim. Acad. Sci. Hung.. 59,99 (1969): A. Muller, K. K6rmendv. and F. Ruff. ibid.. 58. 453 (1968). A . Arnonkl ~ G. Fronza,~K. hbndelli, 'and-A . Vigevani, Tetrahedron Lett., 3349 (1976j. Screening for antitumor properties was done under auspices of the National Cancer Institute, Division of Cancer Treatment, Developmental Therapeutics Program, according to its protocols; R. I. Geran, K. H. Greenberg, M. M. MacDonald, A. M. Schumacher, and B. J. Abbott, Cancer Chemother. Rep., Part 3 , 3 (2),1-103 (1972). R e thank Dr. Harry B. Wood of NCI for providing the data. G. Zbinden and E. Brandle, Cancer Chemother. Rep., Part I , 59, 707 (1975). We are indebted to Dr. Zbinden for carrying out the tests. (a) J. McCann, E. Choi, E. Yamasaki, and B. N. Ames, Proc. Natl. Acad. Sci. l'.S.A., 72, 5135 (1975); (b) B. N. Ames, J. McCann, and E. Yaniasaki, Mutat. Res., 31, 347 (1975). For the determinations and helpful discussions we are indebted to Dr. D. G. Kleid (under NIH Grant CA 21593) and to Dr. V. F. Simmon. F. S. Philips, A. Gilladoga. H. hlarquardt, S. S. Sternberg, and P. M. Vidal, Cancer Chemother. Rep., Part 3. 6 (21, 177 (1975). H. Marquardt, F. S. Philips. and S. S. Sternberg, Cancer Res., 36, 2065 (1976). W. F. Benedict, M. S. Baker, L. Haroun, E. Choi, and €3. N. Ames, Cancer Res., 37, 2209 (1977). H. Marquardt and H. Marquardt, Cancer (Philadelphia), 40, 1930 (1977). E. K. Weissburger, Cancer (Philadelphia), 40, 1935 (1977). Method of ref 19. H. Marquardt, personal communication, courtesy of Dr. Harry B. Wood. Judging from the percent survival of the cells, 7 was considerably less cytotoxic than either 1 or 2 in this test. -
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