Spin-labeled phorbol esters and their interaction with cellular membranes. I. Synthesis of spin-labeled phorbol-12, 13-diesters and related compounds

Chemistry and Phystcs of Lipids, 35 (1984) 151-159 Elsevier Scientific Publishers Ireland Ltd.

151

SPIN-LABELED PHORBOL ESTERS AND THEIR INTERACTION WITH CELLULAR MEMBRANES. I. SYNTHESIS OF SPIN-LABELED PHORBOL-12, 13-DIESTERS AND RELATED COMPOUNDS

S. PE(~ARa, B. SORGb, M. SCHARAc and E. HECKERb abDnepartment of Pharmacy, E. Kardel/ University of Ljubl/ana, 61000 L/ubl/ana {Yugoslavia), stitute of Biochemistry, German Cancer Research Center, D-6900 Heidelberg {F:R. G.} and cj. Stefan Institute, E. Kardel/ University of L/ubl/ana, 6100 L/ubl/arm /Yugoslavia} Received April 20th, 1983 accepted April 24th, 1984

revision received April 16th, 1984

The synthesis of analogs of the cocarcinogen and tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) containing spin labeled acid residues in position 12 or 13 of the phorbol moiety is described. Chemical and spectroscopic characteristics of the new compounds are reported. The stability of the nitroxide groups of the spin probes and their isotropic hyperfine splittings in different solvents are determined.

Keywords: cocarcinogens; phorbol esters; spin labeling; tumor promoters.

I. Introduction A great number of molecularly defined diterpene esters with tigliane, ingenane or daphnane structures were established as the most active class o f cocarcinogens of the initiation (or tumor) promoter-type. They exhibit their biological and biochemical effects in doses similar to those of proliferation stimulating hormones [1,2]. As a prototype representing this class the tigliane derivative 12-O-tetradecanoylphorbol-13-acetate (TPA) is widely used for investigations in experimental cell biology and in cancer research [3]. TPA is readily available by partial synthesis from phorbol [ 4 - 6 ] . At the tissue and cell level TPA was shown to exhibit biochemical pleiotropism in that it induces a large number of biological events (for recent reviews, see Refs. 3 and 7). There are many indications that they are triggered by interaction of TPA (and corresponding other diterpene ester type promoters) with receptors in cellular membranes [ 2 , 7 - 1 2 ] . Spin labeled analogs of TPA may be informative new tools to study the interaction of initiation-promoters with membranes and the microenvironment of corresponding receptors using electron spin resonance (ESR) spectroscopic methods [ 13].

II. Materials and Methods The infrared spectra (i.r.) were recorded on Perkin Elmer spectrometers models 0009-3084/84/$03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland

152 257 and 521. The ultraviolet spectra (u.v.) were taken on a Beckman spectrophotometer DK 2. The mass spectra (ms) were done on a Varian MAT 711. The ESR spectra were registered on a Varian E-9 spectrometer, operated at 0.053 mT modulation amplitude and 20 mW resonator power input (approx. 10-s-10 -4 M :olution of spin labeled compounds in different solvents). The proton magnetic resonance spectra (NMR) (90 MHz) were recorded on a Bruker HX-90. Thin layer chromatography (TLC) was done on silica gel 60 F2s4 aluminium sheets (Merck). Spots were detected by ultraviolet light (254 nm) and by spraying with vanillin-sulfuric acid followed by heating for some minutes at llO°C. For column chromatography silica gel 60 (200-400 mesh, Merck) was used. Preparative TLC was performed using 2-mm plates made from silica gel 60 P F ~ (Merck). Elemental analyses were performed by the Department of Chemistry and Chemical Technology, Faculty of Natural Science and Technology, E. Kardelj university of Ljubljana, Ljubljana, Yugoslavia. The starting materials phorbol-13,20-diacetate and 4-O-methylphorbol-13,20diacetate were prepared according to known procedures [ 14,15 ]. Phorbol- 12-acetate20-trityl ether was synthesized from phorbol-12-acetate [ 14] by using trityl chloride/ pyridine, p-Toluene sulfonyl chloride was purified according to Fieser et al. [16]. Pyridine was freshly distilled from potassium hydroxide. The esters (n,m)OMe* and (n,m)OEt of spin labeled acids (n,m)OHwere prepared from the corresponding ketoesters via oxazolidines to doxyl-nitroxides using 3chloroperoxybenzoic acid as the oxidant [17-20]. They were purified using column chromatography [ 17,19]. Hitherto unknown doxyl esters were characterized by mass, i.r. and ESR spectroscopy as well as elemental analyses (Table I). The corresponding data for the (l,O)OEt, (3,10)OMe and (5,8)OMe are given in Refs. 17, 18 and 20. As a representative some characteristic data of (5,4)OMe are given: ms, m/z = 314(M÷), 2 8 4 , 2 5 4 , 2 4 3 , 2 2 8 , 1 8 6 ; i.r. (CC14), v = 1730 cm -1 (COOCH3); Rf(ether/petroleum ether, 1:3), 0.25; aN(Tris-HCl, pH 7.4), 1.55 mT. Esters (n,m)OMe or (n,m)OEt were hydrolyzed in dioxane with 1 M aqueous sodium hydroxide according to known procedures [ 18,19]. The isolated spin labeled acids (n,m)OH were dried in vacuum over P205 (8 h) before use.

A. Syntheses era series of (n,m)PA To a solution of 0.88 mmol of phorbol-13,20-diacetate and 0.96 mmol of the spin labeled acid in 1.1 ml of pyridine, 1.77 mmol of tosyl chloride was added. The

*Terminology of spin labeled compounds: spin labeled carboxylic acids HOOC(CH2)n-C(Iabel)(CH~)mCH3(see Fig. 2) are abbreviated by (n,m)OH, methyl or ethylestersthereofby (n,m)OMe and (n,m)OEt, respectively. (n,m)PA means phorbol-12,13-diesters where the spin labeled acid moiety is in position 12 of phorbol-13-acetate. 4-O-Me(n,m)PA is the 4-methyl ether of a (n,rn)PA. AP(n,m) is the abbreviation for isomeric (n,m)PA with the spin labeled acid moiety in position 13 of phorbol-12-acetate.

153 TABLE 1 ELEMENTAL ANALYSIS OF THE DOXYL ESTERS SYNTHESIZED Compound

(5,0)OMe (4,2)OMe (5,2)OEt (5,4)OMe (5,6)OMe (11,2)OMe (2,11)OMe (12,1)OMe (1,12)OMe

C

H

N

Calcd.

Found

Calcd.

Found

Calcd.

l:ou nd

60.44 61.74 63.97 64.94 66.63 68.07 68.07 68.07 68.71

60.48 61.62 63.85 64.89 66.73 68.48 68.26 68.27 68.94

9.36 9.62 10.07 10.26 10.59 10.88 10.88 10.88 11.01

9.60 9.93 10.11 10.32 10.45 10.59 10.93 10.73 10.89

5.42 5.14 4.66 4.45 4.09 3.78 3.78 3.78 3.64

5.61 5.0 I 4.32 4.33 3.89 3.66 3.51 3.49 3.53

mixture was stirred at 50°C for 24 h. The brown viscous mixture was diluted by adding 2 - 3 ml of methanol and the solution was transferred into 200 ml of ethyl acetate. The organic phase was successively extracted with phosphate buffer (pH = 6.5, 1 ×50 ml), 1 M HCI (1 ×50 ml), phosphate buffer (1 × 5 0 ml), 0.38 M K2CO3 (1 ×50 ml) and finally with brine. After drying (Na2SO,) the e.thyl acetate was evaporated and the residue was used in the next step without further purification. The crude triester was dissolved in 16 mM HCIO4 in methanol (0.5 ml of solution per mg of crude material) and kept for 2 5 - 3 0 h at room temperature. The reaction was stopped by adding 10 mg of sodium acetate per ml of solution. The methanol was evaporated and the residue was suspended in 200 ml of ethyl acetate. The solution was washed with a 0.52 M solution of KHCOa (1 × 50 ml), brine and dried (Na2SO4). After evaporation of the ethyl acetate the (n,m)PA was purified on a silica gel column (ethyl acetate/petroleum ether, 2:1) followed by TLC (ethyl acetate/ether, 1 : 2). Table 11 shows the yields and Rf-values of the (n,m)PA synthesized according to this general procedure. All compounds were light yellow resins. NMR, u.v. and i.r. spectroscopical data are given for (1,12)PA as a representative. NMR (CDCI3), 8 = 7.58 (1H, l-H), 5.8-5.2 (3H, 7-H, 12-H, 9-OH), 4.03 (2H, 20-H2), 3.0-3.4 (2H, 8-H, 10-H), 1.88 (19-H3), 2.1 (CH3CO), 1.27 ppm ((CH2)n); uv (CH3OH), hmax (e) = 230 nm (8640); i.r. (KBr), v = 1725, 1715, 1631 cm -1. For mass spectroscopical data compare 4-O-Me(5,4)PA (below).

B. Synthesis of 4-O-Me(5,4)PA The compound was synthesized analogously to the procedure described for the preparation of the (n,m)PA by using 4-O-methylphorbol-13,20-diacetate as starting material. Table II shows the yield and Rf-value of the 4-O-Me(5.4)PA. ms (electron impact): m/z (%, based on rn/z = 43 (100%)) = 703 (1.3), 702 (2.5), 687 (1.7), 685 (1.5), 684 (1.8), 642 (0.9), 632 (1.9), 631 (2.4), 630 (1.4), 628 (1.6)627(2.1),

154 TABLE 11 YIELDS AND Rf-VALUES OF THE SPIN LABELED PHORBOL DERIVATIVES SYNTHESIZED Compound

Yielda (%)

Rb

(1,0)PA (5,0)PA (4, 2)PA (5,2)PA (5,4)PA (5,6)PA (5,8)PA (3,10)PA (ll,2)PA (2,11)PA (12,1)PA (1,12)PA

74 75 68 50 59 56 69 59 75 59 60 48

0.07 0.10 0.12 0.13 0.18 0.19 0.21 0.20 0.19 0.20 0.22 0.22

4-O-Me(5,4)PA

60

0.22

AP(5,4) AP(I,I 2)

60 60

0.24 0.58

aBased upon the starting material of the corresponding synthetic path. bTLC on silica gel with ether as the mobile phase.

618 ( 1.0), 617 (3.6), 616 (9.0), 615 ( 1.3), 614 (3.0), 613 (0.8), 594 (1.3), 593 (2.6), 588 (2.1), 572 (1.8), 570 (1.3), 556 (3.0), 538 (2.4), 524 (2.6), 522 (1.4), 507 (3.7), 506 (7.8), 405 (2.3), 404 (11), 403 (42).

C Synthesis oftypicalAPfn, m) To a solution of 0.39 mmol of phorbol-12-acetate-20-trityl ether and 0.44 mmol of the spin labeled acid (5.4)OH or (1,12)OH, respectively in 0.4 ml of pyridine, 0.79 mmol of tosyl chloride was added. After staying overnight at room temperature the product was isolated as described for the synthesis of a (n,m)PA. The crude material was dissolved in 16 mM HC104 in methanol (1 ml/10 mg of material). After 60 min the reaction was stopped by adding 10 mg of sodium acetate per ml. The methanol was removed by rotary evaporation, the semisolid residue dissolved in ethyl acetate (100 ml) and washed with phosphate buffer (pH 7, 1 × 30 ml), brine and dried (NaaSO4). After evaporation of the ethyl acetate the reaction mixture was purified on a silica gel column (ether/laexane, 3 : 1) followed by TLC (ethyl acetate/ether, 1:2). Table II shows the yields and Rt~values of the two AP(n,m) synthesized. Spectroscopical data were similar to those of the (n,m)PA types (above).

155 TABLE lIl STABILITY OF SPIN PROBES IN ETHANOL (E) AND IN TRIS-HCI BUFFER SOLUTION, pH 7.4 (T) Residual paramagnetism after 21 days at various temperatures. Values expressed as % of initial paramagneti~n. Temperature - 10°C

4"C

50°C

20°C

Solvent

(1,0)OEt (5,4)OMe (1,0)PA (5,4)PA

E

T

E

T

E

T

E

T

100 100 100 100

60 70 90 70

100 100 100 100

10 55 100 75

100 90 90 90

5 60 50 85

10 100 65 100

20 50 0 5

D. Stability of the nitroxide radical in the compounds synthesized The stability of the nitroxide radical in spin labeled TPA analogs was tested in ethanol solution as well as in Tris-HCl buffer solution (pH 7.4). The samples were stored as 7.4 × 10 -6 M solutions in sealed capillaries at - 10°C, 4°C, 20°C and 50°C. The ESR intensity measurements were performed at various time intervals at room temperature. The final intensities of the samples after 21 days of storage in percentage of the initial intensities of the solutions are given in Table III. E. Reduction with ascorbate and oxidation with potassium hexacyanoferrate (III)

of (5,4)PA For detailed experimental conditions see legend Fig. 3. nl. Results and Discussion

Spin labeled carboxylic acids of different chain length (from C4 to C t6) and with various positions of the 4,4-dimethyloxazolidine.N-oxyl ('doxyl') moiety were used for acylation (Fig. 1) of phorbol-13,20-diacetate as weU as 4-O-methylphorbol-13, 20-diacetate and phorbol-12-acetate-20.trityl ether. Acylations were brought about by using a standard procedure: mixed earboxylic-p-toluene sulfonic acid anhydrides in pyridine [21,22]. The resulting compounds, after liberation of 20-OH with weak perchloric acid/methanol [14], yielded the spin labeled analogues of TPA, Fig. 2. The overall acyl chain length increases from N = 4 to N = 16. The position of the spin label within the acyl chain varies from 'close to the carbonyl group' (n = 1,2,3)

156

OH

lI 0..p-O" OC(CH2}nC(CH2)mCH3

0 if

~,~.-- OC,CH3

.....

HO-,.

4~

q

u

OH

I II CoH2OCCH3

1. (n.m)OH/TsCl/pyridine 2. MeOH/HCIOL,

Fig. 1. General scheme of synthesis of the spin labeled phorbol derivatives. In case of 4-O-Me (5,4)PA 4-O-methylphorbol-12,13-diacetate,in caseofAP(n,m)phorbol-12-acetate-20-trttylether is the starting material. over 'medium positioned' to 'close to the CH3 end' (m = 0,1,2). With regard to the acyl chain the (5,6)PA is the direct spin labeled analogue of TPA. The products could not be characterized by elemental analysis due to their resinous nature a property, inherent in most of the many well known phorbol esters (see e.g. Ref. 5). The starting materials, i.e. phorbol derivatives and the spin labeled acids, used for the well known and mild acylation procedure, were well defined (for the acids: see Table I). The products were pure according to TLC in different solvent systems. All compounds synthesized were of radical nature as shown by ESR spectroscopy (for examples, see Table Ill). All other spectroscopic data are in support of the proposed structures. In the mass spectra (electron impact) of (5,8)PA, (ll,2)PA, (12,1)PA and 4-O-Me(5,4)PA the molecular ions were detectable but had very low intensity. As a representative, 4-O-Me(5,4)PA shows reasonable fragmentations: m/z = 684 (m/z = 702-H20), m/z = 642 (702-acetic acid), m/z = 616 (702-isobutene-NO, characteristic of [25 26] 'doxyl' compounds) and m/z = 403 (702-long chain carboxylate). The ions mentioned support the presence of a methylphorbol acetate acylate bearing a 'doxyl' moiety. Though in the mass spectra of the other compounds the corresponding parent ions could not be registered similar fragment ions were indicative for analogous parts of the respective molecules. The u.v. spectrum of, for example, (1,12)PA shows a maximum at 230 nm with e = 8640 (phorbol-12,13-diacetate: e233 = 5200 [ 14] ), indicating an increase in e corresponding to the nitroxide (e23o 3000 [25] ) introduced additionally. The NMR spectra were in general similar to other phorbol-12,13-diesters. However, the resolution of the individual signals often was lowered due to paramagnetic broadening. The stability test (Table 111) for four spin probes: (1,0)OEt, (5,4)OMe, (1,0)PA and (5,4)PA showed that they are ESR stable in ethanol solution up to 4°C and reasonably stable up to room temperature during a period of 3 weeks. In Tris-HC1 buffer solution their stability is less pronounced and more temperature sensitive. The position of the label in the acid chain appears to play an important role in this regard, too (data not shown/.

157

OR2

~[ X: C -

-

(CH2)n-~C-(C H2)~C H 3 (n,m)PA

R: = CH3CO

R== X

R~= H

Abbreviation

N

(1,0) (5.0) (4,2) (5,2) (5,4) (5,6)

(I,0)PA (5,0)PA (4,2)PA (5,2)PA (5,4)PA (5,6)PA

4 8 9 10 !2 14

(5,8)

(5,8)PA

16

(3,10) (11,2) (2,11) (12,1) (1,12)

(3,10)PA (11,2)PA (2,11)PA (12,1)PA (1,12)PA

16 16 16 16 16

4-O-Me(5,4)PA

N = 12

Abbreviation

N

AP(5,4) AP(1,12)

12 16

(n,m)

R ~ = CH3CO

(5,4)

R 3 = CH 3 AP(n,m)

Rl = X

R 2 = CH3CO

R~ = H

(n,m) (5,4) (1,12)

Fig. 2. Overview of the spin labeled phorbol derivatives synthesized. Overall length of acyl chain: N = n + m + 3. For further definitions o f abbreviations used see f o o t n o t e in Materials and Methods.

The isotropic hyperfine splittings depend on the polarity of the microenvironment of the nitroxide group. The data (1.33 mT for hexane to 1.54 mT for TrisHCI buffer solutions) for (n,m)PA are qualitatively in agreement with the published data for di-t-butylnitroxide, although the former relate to five membered oxazolidine rings [26]. It should be stressed that the splittings aN for the compounds as well as the differences shown between the extreme hydrophobic (hexane) and hydrophilic (water, Tris-buffer) environment are smaller than those found for di-t-butylnitroxide. The response to polarity of these oxazolidine type spin probes is important to gain information on the location of the nitroxide group of (n,m)PA within the micro-

158

environment in the membrane-fluidity systems. The coupling constants a N are within the experimental error (-+2%) independent of the length of the hydrocarbon chain and of the position of the oxazolidine ring. Their value increa~s with the increasing polarity of the solvent. In aqueous solution the spin probes may be reduced by ascorbate or oxidized by potassium hexacyanoferrate(IIl) (Fig. 3) to yield the non-paramagnetic hydroxylamine derivative and a non-paramagnetic, probably oxonium, derivative, respectively. It should be mentioned that the reduction mid-point potential for the oxazolidine labels was found lower than that of the piperidine labels [27]. Therefore as found previously in our work [ 13,19 ] the oxazolidine nitroxide group is chemically transformed by the oxidation with potassium hexacyanoferrate(lII) in contrast to the piperidine labels where only the broadening is observed [28]. After oxidation of (4,2)OMe with an 1.7 molar excess of potassium hexacyanoferrate(l'lt) in water the corresponding ketocster (methyl-6-oxononanoate) was isolated and identified. The double peak in Fig. 3 pertains to the superimposed free radical of the semihydroquinone ascorbate radical, which appears visible under some conditions as an intermediate in the oxidation of ascorbate by the nitroxide radicals [29]. Reduction and oxidation of the spin probes together with the paramagnetic broadening have been shown to be useful tools for ESR investigations of interactions with biological materials [13].

Fig. 3. Typical ESR spectra of (a) (5,4)PA (1.5 × 10-s M) in Tris-HCI buffer solution, (b) the same solution as in (a) 10 rain after addition of sodium ascorbate (6.6 x 10 -3 M) and (c) 10 rain after addition of potassium hexacyanoferrate(III) (5.25 X 10 -2 M). All spectra are taken at room temperature.

159 Acknowledgement It is gratefully a c k n o w l e d g e d that one o f us (S.P.) was recipient o f grants by the Visiting Scientist Program o f the Deutsches Krebsforschungszentrum, Heidelberg.

References 1 E. Hecker, Pure Appl. Chem., 49 (1977) 1423-1431. 2 E. Hecker, in: T.J. Siaga, A. Sivak and R.K. Boutweil (Eds.), Carcinogenesis - A Comprehensive Survey, Vol. 2, Raven Press, New York, 1978, pp. 11-48. 3 E. Hecker, N.E. Fusenig, W. Kunz, F. Marks and H.W. Thielmann (Eds.), Carcinogenesis A Comprehensive Survey, Vol. 7, Raven Press, New York, 1982. 4 E. Hecker, in: H. Busch (Ed.), Methods in Cancer Research, Vol. 6,,Academic Press, New York, 1971, pp. 439-483. 5 E. Hecker and R. Schmidt, Chem. Org. Natur. Prod., 31 (1974) 377-467. 6 H. Bresch, G. Kreibich, H. Kubinyi, H.H. Schairer, H.W. Thielmann and E. Hecker, Z. Naturforsch., 23b (1968) 538-546. 7 I. Berenblum and V. Armuth, Biochim. Biophys. Acta, 651 (1981) 51-63. 8 K.B. Delclos,S.D. Nagle and P.M. Blumberg, Cell, 19 (1980) 1025- 1032. 9 M. Shoyab and G.J. Todaro, Nature, 288 (1980) 451-455. I0 C.L. Ashendel and R.K. Boutwell, Biochem. Biophys. Res. Commun., 99 (1981) 543-549. 11 M. Hergenhahn and E. Hecker, Carcinogenesis, 2 (1981) 1277-1281. 12 R. Schmidt, W. Adolf, A. Marston, H. Roeser, B. Sorg, H. Fujiki,T. Sugimura, R.E. Moore and E. Hecke~, Carcinogenesis,4 (1983) 77-81. 13 S. Pe~ar, M. Sentjurc, M. Schara, M. Nemec, B. Sorg and E. Hecker, in: E. Hecker et al. (Eds.), Carcinogenesis - A Comprehensive Survey, Vol. 7, Raven Press, New York, 1982, pp. 541-553. 14 C.v. Szczepanski, H.U. Schairer, M. Gschwendt and E. Hecker, Lieb~s Ann. Chem., 705 (1967) 199-210. 15 G. Kreibich and E. Hecker, Z. Naturforsch., 23b (1968) 1444-1452. 16 L.F. Fieser and M. Fieser, Reagents for Organic Synthesis, Vol. 1, John Wiley and Sons. Inc., New York, London, Sydney, 1967, pp. 1179-1180. 17 W.L. Hubbell and H.M. McConnell, J. Am. Chem. Soc., 93 (1971) 314-326. 18 B.J. Gaffney, in: L.J. Berliner (Ed.), The Chemistry of Spin Labels in Spin Labeling, Theory and Applications, Academic Press, New York, 1976, pp. 183-238. 19 S. Pe~ar, Ph.D. Thesis, University E. Kardelj of Ljubljana, Ljubljana, Yugoslavia, 1980. 20 J.C. Hsia and A. Panthananickal, Can. J. Biochem., 54 (1976) 704-706. 21 C.G. Overberger and F. Sarlo, J. Am. Chem. Soc., 85 (1%3) 2446. 22 F. Effenberger and G. Epple, Angew, Chem., 84 (1972) 294-295. 23 S. Chou, J.A. Nelson and T.A. Spencer, J. Org. Chem., 39 (1974) 2356-2361. 24 L. MaraJ, J.J. Myher, A. Kuksis, L. Stuhne-Sekalec and N.Z. Stanacev, Chem. Phys. IApids, 17 (1976) 213-221. 25 A.R. Forrester, J.M. Hay and R.H. Thomson, Organic Chemistry of Stable Free Radk'als. Academic Press, New York, 1968. 26 O.H. Griffith, P.J. Dehlinger and S.P. Van, J. Membrane Biol., 15 (1974) 159-192. 27 A.T. Quintanilha and L. Packer, Proc. Natl. Acad. Sci. U.S.A., 74 (1977) 570-574. 28 A.T. Quintanflha and R3. Mehlhorn, FEBS Lett., 91 (1978) 104-108. 29 J.E. Wertz and J.R. Bolton, Electron Spin Resonance, McGraw-Hill Book Company, New York, 1972, p. 380.