`
`Prus_tag|andinsa_nd
`Bardmvascularfllseases
`
`Edited by
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`JAPAII SCIEIITIFIC SOCIETIES PRESS Tokyo
`IIIYLCII & FRANCIS lTI]. Landon am PII'hdaIplIia
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`© JAPAN SCIENTIFIC SOCIETIES PRESS, 1986
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`

`Fluorodeoxy Prostaglandins, Synthesis and Perspectives
`
`VLADIMIR V. BEZUGLOV, EFIM M. MANEVICH,
`
`LEV D. BERGELSON, AND YURI A. OVCHINNIKOV
`
`Shemyakin Institute of Bz'oorgam‘c Chemistry, USSR Academy of Sciences,
`117871 GSP Moscow V—437, U.S.S.R.
`
`Fluorination is extensively applied to change the properties of naturaliy oc-
`curring compounds; it enhances and modifies the biological activity of natural
`bioregulators as, for example, in the case of fluorosteroids. It is not surprising
`
`that fluorination is also widely used in the prostaglandin field where the prob-
`
`lems of stability, selectivity and side effects are as great as for the steroids (1 ,2)
`
`Up to now researchers have focused their attention mainly on the substitution
`of hydrogen for fluorine in various positions of the prostane skeleton. Fluorodeoxy
`prostaglandins, containing a fluorine atom instead of a hydroxyl group are prac-
`
`tically unstudied yet . The only known examples are 9- and 1 1 -fluorodeoxy pro-
`staglandins F2 (PGF2) whose synthesis was performed by “Syntex” several years
`ago;rhowever, only preliminary results of biological testing are available (3).
`In order to fill this gap, we started a systematic investigation of the syn-
`
`thesis and biological activity of fluorodeoxy prostaglandins.
`
`FLUORINE AS AN OXYGEN SUBSTITUENT
`
`According to its physicochernical parameters, fluorine resembles oxygen
`
`(Table I). Like oxygen, fluorine functions as a hydrogen bond acceptor and
`
`mimics a hydroxyl group. But in contrast to hydroxyl, fluorine cannot be a
`
`hydrogen bond donor. Systematic substitution of various hydroxyls for fluorine
`
`in biologically active compounds permits the location of positions in the molecule
`
`which interact with the receptor proteins as hydrogen bond acceptors and (or)
`
`donors. In addition, such substitution increases lipophility of the molecule.
`
`Abbreviations: PG - prostaglandin, TX — thromboxane, ADP ~ adenosinediphosphate, HDL — human
`high density lipoproteins, PAF - phospholipid platelet activating factor
`
`191
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`192
`
`TABLE I
`
`V.V. BEZUGLOV ET AL.
`
`
`Properties of Fluorine and Oxygen Atoms (2, 4)
`
`Parameter
`F
`0
`
`
`1.4
`1.35
`Van der Waals radius (A)
`3 .5
`4 . 0
`Electronegativity
`1.43
`1.39
`OX bond length (A)
`
`
`X-CH2 bond energy (kcal/mol) 91.5 (OH) 106
`
`Noteworthy is one more important feature of fluoro compounds. High
`energy of C-F bond (Table I) strongly hinders, and in many cases even prevents,
`enzyme attacks at this site of the molecule, thus increasing the metabolic stability
`of fluorinated compounds. Thus, 15-fluorodeoxy prostaglandins promising
`stability towards 15-prostaglandin dehydrogenase are of special interest.
`Selective introduction of fluorine into prostaglandin molecules raises two
`main problems: first, selection of mild conditions for fluorination and, second,
`determination of optimal combinations of protective groups for functions which
`should be retained in the final structure. We succeeded in overcoming these
`difficulties and, as a result, a series of 15-fluorodeoxy analogs of prostaglandins
`A2, B2, E1, E2, F205, and I2 were synthesized by modification of natural pros-
`taglandins (5—7). Special attention was directed to the development of ap-
`proaches which permitted retention of natural configuration of all asymmetric
`centers in the prostaglandin molecule. Figure 1 shows the synthesis of some
`15 ~fluorodeoxy prostaglandins.
`
`o
`\\
`
`PGA2
`
`
`1.0m,
`l_\
`2. OWN-SFS
`
`t
`s\=/\/\coo~\e I.H202.OH‘
`W
`-
`- 9
`
`'I'IIII
`
`O
`\\
`
`‘
`-‘\=—/\/\COOMe
`
`0..., I
`
`'I'IIII
`
`_
`+ ll eplmer
`
`lSF-PGAz, methyl ester
`
`lSF-PGEZ, melhyl ester
`
`COONo
`
`#2
`
`O
`W
`6H
`3
`
`1. |2,NoHC03
`2. DBU
` 3. NaOH
`
`NaBHA
`
`9”
`‘ §\=/V\COOMe
`
`5H
`
`5
`
`-
`
`_
`+ 9 eplmer
`
`15F~PGl2, sodium soli
`
`lSF-PGan, methyl esler
`
`Fig. 1.
`
`Scheme for synthesis of 15-fluoro~15vdeoxy prostaglandins from PGA2.
`
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`FLUORODEOXY PROSTAGLANDINS, SYNTHESIS AND PERSPECTIVES
`
`193
`
`ALTERATION OF PROSTAGLANDIN PHYSICAL PROPERTIES
`
`X-Ray analysis of p-brornophenacyl ester of lS-fluoro-lB-deoxy-ll-epi-
`
`PGan confirmed that fluorination of the original prostaglandin did not result
`
`in spatial changes of the molecular shape (Fig. 2). Both prostanoids have the
`
`characteristic “hairpin” conformation and mutual disposition of side chains was
`
`preserved (8). Apparently, substitution of 15(S)-OH for 15(S)-F might be con-
`
`sidered as “isogeometrical transformation”; however, this substitution induces
`
`alteration of many physicochemical parameters of prostaglandins. Conversion
`
`of PGFga into its lS-fluoro analog thus increases lipophility of the molecule more
`
`than 40-fold and critical micellar concentration decreases by one order (Table
`
`II)(9 , I 0).
`
`As known, prostaglandins in some biological and artificial systems can func-
`
`tion as membrane carriers of bivalent cations (11). We demonstrated that
`
`
`
`Fig. 2. X-ray structure of p-bromophenactyl ester of lE-fluoro-lfirdeoxy—ll—epi-PGFga
`in comparison to PGFga structure.
`
`TABLE II
`
`Partition Coefficients (P) and Critical Micellar Concentrations (CMC) of PGFga and Its
`lfi-Fluorodeoxy Analog
`
`Prostaglandins
`
`PGan
`
`lS-Fluoro—lS-deoxy PGan
`
`P (heptane~water 1 : 1)
`
`CMC (uM)
`
`0.006 :i: 0.005
`
`0.28 :i: 0.05
`
`1.4
`
`0.12
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`194
`
`V.V. BEZUGLOV ET AL.
`
`15-fluoro prostaglandins displayed higher ionophore activity than correspon-
`ding natural counterparts in inducing transport of Mn2+ across the bilayer
`membrane of phospholipid vesicles (12). Obviously, this could be attributed to
`the increase in lipophility after fluorination of the prostaglandin molecule. On
`the other hand, substitution of 1 la-hydroxyl for fluorine resulted in almost com-
`plete loss of the ionophore effect. Thus, the presence of the hydroxyl group in
`position 110: of the molecule of natural prostaglandins seems to be essential for
`their ionophore activity.
`
`BIOLOGICAL PROPERTIES OF FLUORODEOXY PROSTAGLANDINS
`
`How did substitution of a hydroxyl group for fluorine influence the biological
`properties? As expected, fluorination of prostaglandins in position 15 rendered
`them stable towards 15-prostaglandin dehydrogenase leading to prolonged ac-
`tion of lB-fluorodeoxy prostaglandins upon intravenous injection in narcotized
`animals. Substitution of hydroxyl for fluorine in prostaglandins changed also
`the character of their pharmacological action. In several cases selectivity increas-
`ed. Thus, substitution of lS-hydroxyl for fluorine in PGan enhanced pressor
`action and sharply lowered the spasmogenic effect on uterus muscles (Table III).
`At the same time, respiratory muscles proved to be more sensitive to the 15-fluoro
`derivative than to PGF2a(13).
`Some fluorodeoxy prostaglandins can selectively interact with prostaglan-
`din receptors. Analysis of the contractile action of 11-(5) fluorodeoxy PGan
`on smooth muscles showed that this prostanoid could be considered as a specific
`agonist of thromboxane A2 (TXA2)(Fig. 3). Indeed, 11-(6 )fluorodeoxy PGan
`appeared to be 30-fold as active as PGan on rat aorta, the preparation known
`to be sensitive to TXA2, and only 1/20 as active as PGan on hamster fundus,
`characterized by low sensitivity to TXAz. When tested on guinea pig trachea,
`ll-(B )fluorodeoxy PGan, like TXAz, proved highly active and, contrary to
`PGan possesses no relaxant activity. In addition, the TXA2 antagonist U-54874
`(epoxyimino PGH2), completely blocks the action of 11-(6 )fluorodeoxy PGan
`on rat aorta (14). As expected, this fluoro prostaglandin also induces platelet
`aggregation though its aggregating activity is somewhat lower than that of
`TXA2(15). Thus, 11-(B)fluorodeoxy PGFga behaves in some biological systems
`as a specific TXA2 agonist. Because of the extreme instability of TXA2,
`ll-(fi )fluorodeoxy PGan can be of much use when studying the mechanisms
`of action of TXAz in normal and pathological states.
`Fluorodeoxy analogs considerably differ from the corresponding natural
`prostaglandins in their action on the blood clotting system. Fluorination in posi-
`tion 15 increases, as a rule,
`the antiaggregating potency of prostaglandins,
`natural inhibitors of aggregation. So, methyl ester of 15-fluoro-15-deoxy PGE]
`
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`FLUORODEOXY PROSTAGLANDINS, SYNTHESIS AND PERSPECTIVES
`
`195
`
`TABLE III
`
`Pharmacological Spectrum of 15-Fluoro-15-de0xy PGFga v5. PGFQU
`
`Substance
`
`Biological test
`.
`.
`
`Guinea plg trachea
`Rat blood pressure
`Rat uterus
`(contraction)
`(increase)
`Contraction Relaxation
`
`
`1
`l
`1
`1
`PGFga
`
`
`
`
`2.6 210.005lB-Fluoro—lfi-deoxy PGan 250
`
`
`
`Raf aorta Guinea-pig Hamster
`
`irachea fundus
`_v_
`\
`_d \
`v—l
`
`TXAz-sensiiive
`
`TXAz-insensifive
`
`TXAg—like contractile activity of ll-fluoro-ll-deoxy PGFga on smooth muscle.
`Fig. 3.
`- 11-(5) HUOTOdEOXY PGFfla; El PGFZa;
`standard TXA2 agonist U-46619
`Insert: Chemical structure of ll-(B) fluorodeoxy PGFga
`
`possessed rather high antiaggregating activity, inhibiting platelet aggregation
`induced with ADP by 15-fold compared to methyl ester of PGE1 (Fig. 4).
`Moreover, this modification gives rise to antiaggregating properties of other pros—
`taglandins (15).
`In experiments with ADP induced platelet aggregation, the
`15-fluoro derivative of 11,9-epoxymethano PGH2 methyl ester appeared to be
`a potent inhibitor unlike the nonmodified prostaglandin (Fig. 5). At the same
`time, both prostanoids were active inductors of platelet aggregation (16).
`Noteworthy, prostacyclin, the most potent natural inhibitor of platelet ag-
`gregation whose antiaggregating properties are rather sensitive to chemical
`modification, essentially retained its activity after fluorination in position 15
`(Fig. 4). Some prolongation of the antiaggregation effect was observed (15, 16).
`Substitution of the 11—hydroxyl group for fluorine in PGSE also leads to
`analogs with high antiaggregating activity, and fluorination of two hydroxyl
`
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`

`196
`
`V.V. BEZUGLOV ET AL.
`
`30—
`
` 40-
`
`lOF
`
`i2
`
`15'F'l2
`
`Ii 5'F'E1 E1
`\fl—J
`
`Methyl esters
`
`Fig. 4.
`
`Inhibition of ADP-induced platelet aggregation by 15-fluor'inated prostaglandins.
`
`induction
`
`
`
`Inhibition A0!” a“?
`100
`o —"'" prelncubahon of
`/o
`platelets with 11
`
`Fig. 5. Aggregating properties of methyl ester of PGH2 analog (U 46619) (I) and its
`15-fluoro derivative (II).
`
`groups in PGE1 and PGE2 is not accompanied by considerable loss of the an-
`tiaggregation effect as compared to monofluoro derivatives (1'6) Thus, fluorine
`in some cases can successfully replace the hydroxyl group of prostaglandins
`without loss of biological activity. Moreover, substitution of hydroxyl for fluorine
`allows modification of biological properties of prostaglandins enhancing the selec-
`tivity and duration of action.
`
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`FLUORODEOXY PROSTAGLANDINS, SYNTHESIS AND PERSPECTIVES
`
`197
`
`MOLECULAR MECHANISMS OF INTERACTION
`
`Though pharmacological properties of prostaglandins have been extensively
`studied, the mechanism of their action on the molecular level is still obscure.
`One proven approach to investigate molecular aspects of the action of prostaglan-
`dins on biological membranes is the fluorescence study of prostaglandin - mem-
`brane interactions. Especially promising is
`the application of fluorescent
`phospholipid (17) and prostaglandin probes.
`What are the advantages of this method? First, high sensitivity that allows
`registration of changes in the membrane organization caused by one effector
`molecule per cell (18). Second, weak disturbance of the membrane by probes
`when they are correctly chosen. In addition, the method permits determination
`of the lifetime of the ligand—receptor complex and the number of binding sites
`on the membrane surface. The following examples illustrate this fruitful ap-
`proach. We investigated the influence of various prostaglandins on the molecular
`organization of lipids in HDL (19) and rabbit erythrocytes (18). In these ex-
`periments a subphysiological amount of PGE1 appeared to change dramatically
`the state of human high density lipoproteins (HDL) globules or the erythrocyte
`membrane.
`
`to
`This effect can be revealed at prostaglandin concentrations equal
`10— 12M (Fig. 6) and strongly depends on the prostaglandin structure. Thus,
`in the case of HDL it is observed with PGE1, but with neither PGE2 nor PGan.
`PGE1 also was active in erythrocytes; its effects are much more expressed in
`hypercholesterinemic animals. Taking into account the antiatherogenic activi-
`ty of PGE1 and HDL, we suppose that the interaction of PGE1 with HDL and
`
`15
`
`10
`
`0.037 0.37
`
`3.7
`
`37
`
`370
`
`[PGE1]
`
`(Xl0_11M)
`
`
`AP
`
`Fig. 6.
`
`PGE1 induced changes in fluorescence polarization P (P =
`
`Po
`fluorescence polarization without effectors) of HDL labeled with fluorescent sphingomyelin.
`
`%, where P0 is the
`
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`198
`
`V.V. BEZUGLOV ET AL‘
`
`12
`
`PGEI
`
`iS-F-PGFZD
`
`PG F20
`
`PAF H-F-PGan
`
`—12
`
`—16
`
`—20
`
`@6969
`
`ED
`9
`
`Fig. 7. Changes in fluorescence polarization P of labeled human platelets induced by
`prostaglandins and PAF. Platelets labeled with fluorescent. III phosphatidylcholine;
`sphingomyelin; I phosphatidylethanolamine; GB and 9 indicate induction or inhibi-
`tion of ADP induced platelet aggregation. Concentration of PGs was 10’9 M.
`
`erythrocytes is of importance in the genesis of atherosclerosis and other car-
`diovascular diseases.
`Another example shows how the character of the low-weight effector ac—
`tion on the platelet membrane can be determined by comparing fluorescence
`polarization of various phospholipid probes.
`Figure 7 shows that effectors with pro- or antiaggregating activity cause
`opposite changes in fluorescence polarization in platelets labeled by fluorescent
`sphingomyelin and phosphatidylcholine, reflecting changes in microfluidity of
`the membrane. Noteworthy is the similarity in the action of phospholipid platelet
`activating factor (PAF), a potent natural aggregant and the TXA2 agonist
`11-(8 )fluorodeoxy PGFZQ.
`At the same time, effects of the 15-fluoro analog of PGan on the state
`of platelet lipids are more expressed than those of PGan, which was reported
`to compete for the receptor of TXA2 on platelets (20). Since all these effectors
`induce no changes in parameters of fluorescent probes in artificial phospholipid
`vesicles, one may conclude that the observed effects are mediated by membrane
`proteins. Of importance is the fact that these membrane effects were detected
`at low effector concentrations (10—9M or less).
`
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`FLUORODEOXY PROSTAGLANDINS, SYNTHESIS AND PERSPECTIVES
`
`199
`
`CONCLUSION
`
`Substitution of hydroxyl for fluorine in prostaglandins leads to substantial
`changes in their physiological properties. Apparently this is connected, to a cer-
`tain degree, with changes in the molecular mechanism of interaction of fluoro
`prostanoids with receptor components of the cell membrane.
`As years of fluoro prostanoid research have turned into a decade; successes
`in this field are not so impressive yet as for steroids. However, the study of fluoro
`prostaglandins paves the way for better understanding the mechanism of action
`of naturally occurring prostaglandins in biological systems.
`
`SUMMARY
`
`limited by their rapid
`The clinical application of prostaglandins is
`catabolism and broad spectrum of physiological activities. A way to overcome
`these shortcomings is modification of prostaglandins by selective fluorination.
`Fluorine resembles hydrogen in its shape and the hydroxyl group in its charge;
`in the past its introduction proved beneficial for steroid hormones, antitumor
`drugs and other pharmaceuticals. In order to substitute fluorine for various
`hydroxyl groups in prostaglandins we developed a selective method of fluorina—
`tion for use under mild conditions. Special attention was directed towards
`substitution of the I5-hydroxyl group, since biological deactivation of prostaglan-
`dins was induced by the action of 15-prostaglandin dehydrogenase. Using this
`method we prepared 9-, ll-, and IS-fluorodeoxy prostaglandins of the A2, E2,
`E1, F2“, and 12 series. In a number of tests many of these derivatives showed
`higher and/or more selective activity than their natural counterparts. Thus, in-
`troduction of fluorine into position 15 of PGE1 and some other prostaglandins
`led to a sharp increase of the antiaggregating activity, without significant in-
`fluence on the muscle relaxation. Introduction of fluorine into position 15 of
`PGan caused a selective increase of the pressor effect with concomitant decrease
`of the muscle contractile activity on the gastrointestinal tract. An interesting
`feature of fluorodeoxy prostaglandins is their ability to compete with natural
`prostaglandins for receptor binding. One example is 11-(8) fluorodeoxy PGan
`which is a highly active TXA2 agonist. Using fluorescence measurements it was
`shown that prostaglandins as well as fluorodeoxy prostaglandins at physiological
`concentrations specifically alter the state of membrane lipids of erythrocytes,
`platelets and high density lipoproteins.
`
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`200
`
`REFERENCES
`
`V.V. BEZUGLOV ET AL.
`
`«round:-
`
`9"
`
`1. Schlosser, M. Tetrahedron, 34,3 (1978).
`Barnette, W.E. CRC Cn't.Rev.Bz‘ochem., 15,201 (1984).
`(JUN
`Arroniz, C.E., Gallina,]., Martinez, 13., Muchowski,J.M., Velarde, E., and Rooks,
`W.H. Prostaglandz'ns, 16,47 (1978).
`Benson, S.W.].Chem.Ed., 42,502 (1965).
`Bezuglov, V.V. and Bergelson, L.D. Bz'oorgan.Khimz'ya, 5,1531 (1979).
`Bezuglov, V.V. and Bergelson, L.D. D0kl.AN SSSR, 250,458 (1980).
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`277,1400 (1984).
`_
`Smirnova, V.I., Nazirnova, N.V., Titshenko, G.N., Bezuglov, V.V., and Bergelso‘n,
`L.D. In “Prostaglandins. Synthesis and Research," ed. Ya.F.Freimanis, p. 21 (1982).
`AN Latv SSR, Riga.
`9. Bezuglov, V.V., Voznuk, T.P., Shevchenko, V.P., Myasoedov, N.F., Gafurov, R.G.,
`and Bergelson, L.D. Dokl. AN SSSR, 268,224 (1983).
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`11. Ovchinnikov, Yu.A. In “Frontiers in Bioorganic Chemistry and Molecular Biology,”
`ed. Yu.A. Ovchinnikov and M.N. Kolosov, p. 129 (1979). Elsevier/North-Holland
`Biomedical Press, Amsterdam, Oxford, and New York.
`12. Viktorov, A.V., Bezuglov, V.V., zind Bergelson, L.D. Biol. Membr., 1,478 (1984).
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`Bergelson, p. 88 (1982). FAN, Tashkent.
`14. Gafurov, R.G., Nigamatov, I.M., Serkov, I.V., Bezuglov, V.V., and Bergelson,
`L.D. Abstr. 16th FEBS Meet., p. 222 (1984).
`15. Lakin, K.M., Makarov, V.A., Novikova, N.V., and Tretjak, V.M.
`makol.Toksz'kol., p. 113 (1983).
`16. Lakin, K..,M Makarov, V.A., Bezuglov, V.V., Kovalev, 5.6., and Bergelson, L.D.
`In “Prostaglandins. Synthesis and Research," ed. Ya.F. Freimanis, p. 48 (1982). AN
`Latv SSR, Riga.
`17. Bergelson, L.D., Molotkovsky, Yu1.G., and Manevich, E.M. Chem.Phys.Lz'pids, 37,
`165 (1985).
`18. Manevich, E.M., Lakin, K.M., Archakov, A.I., Li, V.S., Molotkovsky, Yul. G.,
`Bezuglov, V.V., and Bergelson, L.D. Bz'ochz'm. Biophys. Acta., 815,455 (1985).
`19. Manevich, E.M, Muzya, G.I., Prokazova, N .V., Molotkovsky, Yul. (3., Bezuglov,
`V.V., and Bergelson, L.D. FEBS Lett., 173,291 (1984).
`20. Lakin, K.M., Manevich, E.M., Makarov, V.A., Bezuglov, V.V., and Bergelson,
`L.D. Abstr. 16th FEBS Meet., p. 353 (1984).
`
`Far—
`
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