J. Med. Chem. 1995, 38, 24-63-2471
`
`2463
`
`Novel Steroidal Inhibitors of Human Cytochrome P450170. (17o.-Hyd1-oxylase-
`C1730-lyase): Potential Agents for the Treatment of Prostatic Cancer
`
`Gerard A. Potterf‘ S. Elaine Barrie, Michael Jarman,* and Martin G. Rowlands
`
`Cancer Research Campaign Centre for Cancer Therapeutics at the Institute of Cancer Research,
`
`Cancer Research Campaign Laboratory, 15 Cotswold Road, Sutton, Surrey SM2 SNG, U.K.
`
`Received January 19, 19.953
`
`Steroidal compounds having a 17-(3~pyridy1) substituent together with a 16,17-double bond
`have been synthesized, using a palladium-catalyzed cross-coupling reaction of a 17-enol triflate
`with diethyl(3-pyridyllborane, which are potent inhibitors of human testicular 1’7oL-hydroxylase-
`017,20-lyase. The requirement for these structural features is stringent: compounds having
`2-pyridyl (9), 4-pyfiayl (10), or 2-pyridylmethyl (11) substituents instead of the 3-pyridyl
`substituent were either poor inhibitors or noninhibitory. Reduction of the 16,17-double bond
`to give 17;‘!-py1'idyl derivatives diminished potency with 3-pyridyl substitution (3 -—~- 2'7; IC5()
`for lyase, 2.9 -r 23 11M) but increased it with a 4-pyridyl substituent present (10 -~ 28; lC5o 1
`,uM -* 53 nM). In contrast, a variety of substitution patterns in rings A-C of the steroid skeleton
`afforded inhibitors having potencies similar to those most closely related structurally to the
`natural substrates pregnenolone and progesterone, respectively 17-(3-pyridyl)androsta-5,16-
`dien-316-01 (3, Kim < 1 nM; IC5o for lyase, 2.9 nM) and 17-(3-pyridyl)androsta-4,16-dien-3-one
`(15; IC5g for lyase, 2.1 ILM). Thus compounds having variously aromatic ring A (18), saturated
`rings A/B (21, 22), and oxygenated ring C (26) exhibited IC50 values for lyase (1.8--3.0 nM)
`falling within a 2-fold range. The most potent compounds are candidates for development as
`drugs for the treatment of hormone-dependent prostatic carcinoma.
`
`Carcinoma of the prostate is now the most prevalent
`cancer in men in the USA. In 1993, 165 000 new cases
`were expected to be diagnosed, of which 35 000 will die
`of metastatic prostatic cancer} The most widely ac-
`cepted drug treatment is the use of GnRH agonists,
`which act by interfering with the production of test-
`osterone by the testes and represent a medical alterna-
`tive to orchiectomyfi However neither GnRH agonists
`nor orchiectomy deplete the synthesis of androgens
`through the adrenal route, and levels of testosterone and
`dihydrotestosterone in the prostate are respectively still
`25% and 10% of pretreatment levels even after 3 months
`treatment with a GnRI-I agonist.3 The importance of
`androgen synthesis by the adrenal route in maintaining
`tumor growth is suggested by the improved therapeutic
`benefit, both in terms of increase in progression-free
`survival time and survival advantage, seen in patients
`treated with the combination of GnRH agonist or
`orchiectomy with an antiandrogen, compared with those
`given GnRH agonist or orchiectozny a.lonc.‘=5
`It is
`proposed that the role of the antiandrogen is to coun-
`teract
`the stimulant action of residual androgens,
`synthesized through the adrenal route, on androgen
`receptors in the prostate cancer cells.
`In principle, the effects of the combined therapy could
`be realized by a single drug which inhibits the enzyme
`steroidal 1'70.-hydroxylase-Cn,2o~lyase. This enzyme is
`responsible for androgenic hormone biosynthesis which
`produces dehydroepiandrosterone and androstenedione,
`immediate precursors of testosterone, from their respec-
`tive precursors pregnenolone and progesterone, in both
`testes and adrenals. The irnidazole antifungal agent
`ketoconazole inhibits this enzyme when given in high
`
`"‘ To whom enquiries should be addressed.
`* Present address: Chiroscience Ltd, Mitten Rd, Cambridge CB4
`1-WE, U.K.
`‘‘ Abstract published in Advance ACS Abstracts, May 15, 1995.
`
`doses to male patients and produces the symptoms of
`androgen suppression. This drug has been used to treat
`prostate cancer}; and although success has been re-
`ported in some studies,” it proved less promising in
`others.“” The undesirable side effects, coupled with
`the inconvenience of the three times daily schedule
`which is dictated by its short half-life, limit its potential
`clinical usefulness. Nevertheless the clinical results
`
`obtained, coupled with a very recent report that careful
`scheduling of ketoconazole can produce prolonged re-
`sponses in previously hormone-refractory prostate can-
`cer,“ lend credence to the selection of this enzyme target
`and impetus to the design and development of a more
`enzyme-selective, less toxic, and less metabolically labile
`inhibitor.
`
`We report here on the synthesis and inhibitory
`activity toward the individual 17o.-hydroxylase and
`C17_2g~lyase components of the target enzyme, obtained
`from human testis, of a variety of steroidal compounds
`having as their common structural feature a 17-(3-
`pyridyl) substituent together with a 16,17-double bond
`in the steroidal skeleton. We have previously explored
`nonsteroidal inhibitors containing a pyridyl residue,
`starting from the serendipitous discovery that certain
`esters of 4-pyridylacetic acid were effective inhibitors
`of the hydroxylase-lyase enzyme from rat testis,” find-
`ings which have in part been rationalized by crystal-
`lographic and molecular modeling studies.” More
`recently, esters of 3-pyridylacetic acid have been evalu-
`ated, using enzyme from human testis.“
`The design concept used here was to consider how a
`pyridyl substituent could be incorporated into the actual
`steroid skeleton such that the pyridyl nitrogen lone pair
`would coordinate to the iron atom of the heme cofactor
`in the active site of the enzyme. The initial step of the
`de novo mechanism-based design approach was to
`postulate a. complete catalytic cycle for the enzyme
`
`0022-2623/95/1838-24633509.00/0
`
`© 1995 American Chemical Society
`
`

`

`2464 Journal of Medicinal Chemistry, 1995, Vol. 38, No. 13
`
`Potter et al.
`
`4'0
`
`WW
`
`0.
`
`0
`
`O=Fo*
`
`H0~Fa
`
`H00-Fe
`
`A’ O=Fe
`
`9.
`
`H‘
`
`.,_
`-)———-4%
`
`ET: electron transport system
`
`,2
`
`H
`
`o
`
`,F
`0-0 9
`
`OJH
`
`Figure 1. Postulated complete catalytic cycle for the 170.-hydroxylase-C17,29-lyase enzyme. For clarity, only the steroid D-ring
`and the cofactor iron atom are shown.
`
`0
`
`it
`
`(Figure 1) and then to consider the juxtaposition be-
`tween the steroid D—ring and the heme cofactor from
`the putative transition state geometry. For this pur-
`pose, three-dimensional molecular models were con-
`structed of the putative transition states using the
`Cochrane orbit molecular modeling system. From this
`analysis, it was postulated that a teroid incorporating
`a 16,17-double bond with the 17-position substituted by
`a 2-pyridyl group may inhibit the hydroxylase step and
`a 3-pyridyl derivative may inhibit the lyase step, while
`a 4-pyridyl analog should not inhibit either step. How-
`ever, the enzyme may not tolerate an aromatic ring
`attached to the 1'7-position, and all three compounds
`may be inactive, even if the coordination geometry is
`correct.
`
`The steroidal skeleton chosen for the first compound
`which was synthesized on the basis of this concept,
`namely the novel steroid 3, was that of pregnenolone,
`which appears to be the preferred substrate for the
`hydroxylase activity of the human enzyme in the
`testis}-" Alternative orientations of the pyridyl ring
`relative to the steroidal framework were explored by
`synthesizing the 2- (9) and 4- (10) pyridyl analogs, as
`was the effect of a spacer group between a 2-pyridyl
`residue and a C-17 (compound 11). The second 17-(3-
`pyridyl) derivative synthesized was 15, analogously
`related to progesterone, the alternative substrate for the
`hydroxylae activity of the target enzyme. Further
`molecules synthesized retained the ring D substitution
`pattern of 3 and 15 while further exemplifying the efi‘ect
`on enzyme inhibition of structural variations in rings
`
`‘
`
`Scheme 1“
`
`Or
`
`.Cj:§b—
`
`Aw
`dnr-ydroloiundrsntprooc
`
`
`
`5
`
`__E._
`
`\ \
`
`I
`
`3 -nccloll
`
`
`on
`
`\
`
`_
`
`,
`
`E
`,
`E
`w/'\/§/
`5
`
`°‘ (8) Tf2O, base; (b) 3-PyBEtg, Pd(PPh3)2Cl2, THF, H20, NazC03;
`(c) NaOH, H20, M901-I.
`
`A, B, and C. Finally, the effect of reducing the 16,17-
`double bond in 3 and 10 was explored.
`
`Results
`
`Chemistry. A general method for introducing the
`required 17—pyridy1 16,17-ene functionality into ring D
`was by palladium-catalyzed cross-coupling of steroidal
`17-enoi triflates with suitable pyridyl-containing nu-
`cleophilic coupling partners. For the synthesis of 3
`(Scheme 1), dehydroepiandrosterone 3-acetate was con-
`vetted into its 17-enol triflate 1 by base-catalyzed
`reaction with triflic anhydride in the presence of the
`hindered base 2,6-di-tert-butyl-4-methylpyridine. This
`reaction also produced the 3,5-diene 4 in 10% yield. The
`3-pyridyl group was then introduced into the 17-position
`by reacting 1 with diethyl(3-pyridyl)borane in THF,
`
`

`

`Novel Steroidal Inhibitors of Human Cytochrome P4501.-ra
`
`Journal of Medicinal Chemistry, 1995, Vol. 38, No. 13
`
`2435
`
`Chart 1
`
`Scheme 2“
`
`
`
`R0
`
`E
`
`E
`
`\‘
`
`,_ §/
`
`R=Ac
`R-IH
`
`6
`3
`
`\> < ”\> f0
`
`7
`I0
`
`E
`‘ll
`
`using bis(triphenylphosphine)palladium(Il) chloride as
`catalyst (0.01 equiv) and aqueous Na2C03 a nucleo-
`philic activator. The reaction proceeded remarkably
`efficiently, without the potential side reactions of triflate
`hydrolysis or ethyl coupling, to give the acetate 2 in 84%
`isolated yield. From 4, the 3~pyridyl derivative 5 was
`similarly obtained. The acetyl group of 2, which was
`stable to the mildly basic conditions of the coupling
`reaction, was easily removed with aqueou methanolic
`NaOH to afford the target 3-pyridyl steroid 3.
`Although these coupling reactions were developed
`independently, the palladium-catalyzed cross-coupling
`of organoboron reagents with an enol triflate has been
`reported recently by Suzuki and co-workers.” Their
`reactions employed arylboronic acids and 9-alkyl-9-BBN
`reagents and the mild base K.aPO4 as the nucleophilic
`activator under strictly anhydrous conditions. Our use
`of diethyl(3-pyridyl)bors.ne was prompted by its com-
`mercial availability (it is also easily synthesized”) and
`its previous use in palladium-catalyzed cross-coupling
`reactions with aryl iodides.“ Some features of our
`reaction compared with that of Suzuki are noteworthy.
`We found that the catalyst Pd(PPh3}2Cl2 was superior
`to Pd(PPh3)4 and consistently gave better yields of
`coupled product. The catalyst could also he used at
`much lower levels, and even at 0.001 equiv, good yields
`were obtained with prolonged reaction times.
`Impor-
`tantly our reaction did not require anhydrous condi-
`tions, and indeed an aqueous TI-IF solvent system was
`employed. Our method of introducing the 17—pyridyl
`16,17—ene functionality was more efiicient and higher
`yielding than a previous route,19'”° reaction of 3—pyiidyl-
`iithium with a 17-keto steroid and dehydration of the
`resulting tertiary alcohol.
`The 2—pyridyl (6), 4-pyridyl (7), and 2-picolyl (8)
`steroidal acetates (Chart 1) were synthesized similarly
`to 2 but employing diiferent nucleophilic coupling
`partners and modifying the conditions accordingly. The
`reagents used to prepare 8 and 8 were 2-pyridy1- and
`2-picolylzinc chloride, respectively.
`In the latter case
`the intermediate 8 was converted without isolation
`directly into 11 in good overall yield (79%). An attempt
`to prepare the 3-picolyl analog of 11 using 3-picolylzinc
`chloride was unsuccessful due to homocoupling of this
`reagent. In the synthesis of the 2- (6) and 4- (7)pyridy1
`steroid acetates, the novel palladium catalyst bron1o—
`(isopropenyl)bis(triphenylphosphine)palladium(II} was
`employed. Its use enabled the coupling reaction to be
`carried out at ambient temperature, thereby avoiding
`side reactions, and 6 was obtained in 74% yield from
`which hydrolysis gave the required 2—pyridyl analog 9.
`The catalyst had been developed to enable lo_w~_temper—
`ature cross-coupling reactions for the stereoselective
`synthesis of (E)-4-hydroxytamoxifenzlvm and was pre-
`pared from 2-hromopropene and tetra.kis(triphenylphos~
`
` sndrnltlnudianu
`
`
`12
`
`:3
`
`C
`_.....
`
`,
`
`C1,, \ \ 14
`
`" (3) C7F3, CsF, DMF; (bl Tf20, base; (C) 3-PyBEtg, Pd(P?h3)2Cl2,
`THF, H20, Na2C0a; (Ci) HC1, H20, EtOI-I; (e) Al(O-i-Pr)3.
`
`Chart 2
`
`o
`
`ms
`
`_.
`
`,(}§
`
`‘I.
`
`I‘!
`
`,
`‘
`E
`
`R0
`
`/
`
`so“‘
`
`-w. R-Ac;19
`
`11;
`EH
`
`0
`
`-.
`
`....-
`
`I
`I
`20
`A:
`R
`R-H‘.2l
`
`22
`
`pb.ine)palladiurn(0) by a procedure analogous to that
`used to make benzylchlorobis(triphenylphosphine)-
`palladium(Il).23 When the coupling reaction was per-
`formed using 4-pyridylzinc chloride, prepared from
`4-bromopyrldine, only a low yield (18%) of the 4-pyridyl
`steroid acetate 7 was obtained. Instability of 4-halopy-
`ridines can restrict the use of 4-pyridylznagnesium and
`«zinc halides in palladium cross-coupling reactions, and
`diethylié-py1idyl)borane has been used as an alternative
`reagent.“ Here, lithium triInethoxy(4-pyridyl)boronate,
`an intermediate in the synthesis” of 4-pyridylboronic
`acid, was the organoboron reagent used, and the coupled
`product thus obtained, 7, was hydrolyzed directly to give
`the 4—pyridyl steroid 10 in 53% yield overall from 1.
`The preparation of 15, starting from androstenedione
`(Scheme 2), required selective protection of the 3-keto
`function, to prevent the formation of a 3-dienol triflate.2’5
`Protection as the periluorotolyl enol ether 12 by reaction
`with octafluorotoluene in the presence of cesium fluoride
`has proved to be a convenient one-step procedure.” The
`perfluoroaryl group was table to the subsequent steps
`needed to insert the pyridyl substituent and was then
`cleaved by acidic hydrolysis. It was later found that 15
`was more conveniently prepared directly from 3 by
`Oppenauer oxidation using cyclohexanone and alumi-
`num isopropoxide.
`Several 3-pyridyl derivatives (18, 21, 22; Chart 2}
`exemplifying further structural variation in rings A and
`B were prepared using procedures analogou to those
`already described. Adrenosterone was the starting
`point for the synthesis of a ring-C-substituted variant,
`26 (Scheme 3). which was prepared in good overall yield
`(60%). The formation of the tert-hutyldimethylsilyl
`dienol ether 23 provided an alternative protecting
`
`

`

`2466 Journal of Medicinal Chemistry, 1995, Vol. 38, No. 13
`
`Scheme 3“
`
` KSDNSO
`
`“ (a) t-BDMSO'I‘f, base; (b) (i-Pr)2NLi, PhN'I‘f2; (c) 3-PyBEt2,
`Pd(PPl'l3}2Cl2, THF, H20, Na2C0a; (d) BLHNF, TI-IF, H20.
`
`Scheme 4“
`
`
`
`‘I (a) NgH4,, Ac0H, Et0H, air; (bl Red-Al, ZnCl2. THF.
`
`strategy for the 3-keto function. The chemical shifts for
`the two vinylic protons in this product were very similar
`to those previously reported for the silyl dienoi ether
`formed from a testosterone derivative,” and the present
`product is therefore similarly formulated as the 2,4-
`dienol other. In the following step, N—phenyltriflimide29
`was employed to prepare the enol triflate 24 since use
`of triflic anhydride resulted in deilylation and 3-dienol
`triflate formation. This also enabled selective formation
`of the 17-enol triflate without affecting the 11-keto
`function by preparing the intermediate lithium enolate
`under kinetic conditions at low temperature.
`Lastly, analogs containing a saturated D-ring were
`prepared from the corresponding 16,17-ene compounds.
`Reduction of 3 using diirnide, generated in situ from
`hydrazine hydrate, gave the 1713-(3-pyridyl) steroid 27
`(Scheme 4). Reduction of the 16,17-double bond of the
`4-pyridyl steroid (10) utilized the electron-withdrawing
`influence of the 4-pyridyl substituent under electrophilic
`activation by zinc chloride to achieve direct hydride
`reduction with Red-Al to produce the 17)3~(4—pyridyl)
`steroid 28. The l9-orientation of the pyridyl ring in
`compounds 27 and 28 was confirmed by ‘H-NMR
`spectroscopy which showed an apparent triplet with a
`coupling constant of 10 Hz for the 1'70:-proton which is
`characteristic of 17;?-substituted steroids.2°»3° Attempts
`at preparing the correponding 17:1-(4~pyridyl) analog,
`by either direct reduction of 10 or epimerization of 28,
`were unsuccessful.
`
`Inhibition of Human Testicular 17cL-Hyd.roxy1-
`use and C1730-Lyase. Structure——Activity Relation-
`ships. We have identified as potent
`inhibitors of
`human testicular steroidal 17oL—hydroxylase—C17,20-lyase
`a variety of pyridyl steroids having as their common
`structural feature the 17-(3-pyridyl) 16,17-ene moiety
`(Table 1). Although it might be expected that the most
`potent compounds would be those (3, 15) with structures
`most closely related to natural substrates, there was an
`unexpected tolerance for structural variation in this
`respect. Comparing 3 and 15 with analogs (18, 21, 22,
`
`Table 1. Enzyme Inhibition Data
`1C5o (nM)‘‘
`
`was (JIM)?
`
`Potter et al.
`
`compound C1739-lyase 17:1-hydroxylase aromatase 50.-reductase
`17
`18
`2.9
`4
`5.6
`12.5
`76
`270
`1000
`4000
`> 10 000
`> 10 000
`> 10 000
`> 10 000
`2.1
`2.8
`1.8
`2.6
`2.5
`4.3
`3
`4. 7
`2.9
`13
`23
`47
`53
`160
`26
`65
`
`> 50
`
`10
`
`> 50
`
`2
`3
`5
`9
`10
`11
`14
`15
`18”
`21
`22
`26
`27
`28
`ketoconazole
`
`> 20
`
`>20
`> 20
`
`1.8
`
`“ The standard errors were usually < 10% of the IC5o value. The
`concentration of enzyme in the assays for iyasefhydroxylase
`inhibition was estimated to be about 4-5 nM, except in the assays
`of 9, ll, [4, and ketoconazole for which the concentration was ca.
`25 and 10 nM for the lyase and hydroxylase assays, respectively.
`5 Other biological activity: estrogen receptor binding affinity
`(estradiol = 100), 4.9.
`
`26) synthesized from other naturally occurring steroid
`precursors, there was little variation (from 1.8 to 3.0
`nM) in the IC5o values for inhibition of the iyase
`component. The absence of any functionality at the
`3-position in the steroid skeleton leads to a modest drop
`in potency (compound 5). The markedly lower potency
`of the acetoxy derivative 2 compared with 3 could reflect
`a limited bulk tolerance at the 3-position, as indicated
`by the total loss in activity for the much more statically
`demanding perfluorotolyl derivative 14 of the potent
`inhibitor 15. The stringent requirement for the 17-(3-
`pyridyl} 16,17-one functionality for good inhibition was
`in marked contrast to the relative flexibility in relation
`to other features discussed and is reflected in the
`
`marked reduction, or abolition of activity, on relocating
`the pyridyl nitrogen (compounds 9, 10) or on reducing
`the 16,17-double bond of 3 to give the 1716-pyridyl
`derivative 27.
`In contrast, reduction of the 4-pyridyl
`derivative 10 gave a product, 28, with markedly im-
`proved inhibitory potency over its parent.
`The most inhibitory compounds in the present study
`were far more potent than any inhibitor of hydroxylasel
`lyase for which comparable data have been previously
`described. The K,,, D for 3 was < 1 nM, whereas the most
`potent inhibitor, also steroidal, reported to date is 1716-
`(cyclopropylaminolandrost-5-en-3,6-0131 with a Kim of 90
`nM. Another steroidal compound, 4-pregnen-3-one-20,fL
`carboxaldehyde oxime has been developed as a com—
`bined inhibitor of this enzyme and testosterone 5on-
`reductase.” Though a potent inhibitor (K; = 16 nM) of
`the reductase, it was much less inhibitory toward the
`rat hydroxylase/lyase, being comparable to ketoconazole.
`175-Ureidmsubstituted steroids with potent activity
`toward the rat hydroxylase/lyase enzyme have been
`described.33-3‘ Though the data are presented in a way
`not easily comparable with the results of the present
`study, one of these compounds, 17,6-ureido-1,4-andros
`tadien-3-one, markedly suppressed testosterone levels
`and ablated androgen-dependent organs in the rat.
`Liarozole is a nonsteroidal irnidazole derivative having
`activity toward the rat testicular enzyme very similar“
`to that of ketoconazole. No example among our previ-
`ously mentionedm-14 esters of 4- and 3-pyridylacetic acid
`comp ares in potency with the best of the present
`steroidal derivatives.
`
`

`

`Novel Steroidal Inhibitors of Human Cytochrome P450,-7,,
`
`Journal of Medicinal Chemistry, 1995, Vol. 38, No. 13 2467
`
`Other Biological Activities. While inhibition of
`other targets was not explored in detail in the present
`study, limited evaluations have been carried out (Table
`I), particularly where such activity might be antici-
`pated, from structural analogy with compounds known
`to interact with the target in question. Thus 15,
`structurally related to androstenedione, a substrate for
`aromatase, was a moderate inhibitor of aromatase.
`Likewise the inhibition by 15 of testosterone 5u-reduc-
`tae might reflect its structural resemblance to the
`natural substrate testosterone, whereas 22, correspond-
`ingly related to the product 5o.-dihydrotestosterone, was
`not an inhibitor. Notably, compound 3 inhibited neither
`aromatase nor testosterone 5o.—reductase at the highest
`concentration tested, respectively 20 and 50 ,uM. Lastly,
`the estradiol-related analog 18 had an appreciable
`binding affinity for the estrogen receptor, 5% of that of
`estradiol itself.
`
`Concluding Remarks
`
`Two of the compounds described here, namely 2 (as
`a prodrug for 3) and 15, have been evaluated in vivo in
`the WHT mouse.” Each markedly reduced the weights
`of androgen-dependent organs, and 2 depressed test-
`osterone to undetectable levels. The adrenals were
`
`unaffected, implying that 3 and 15, unlike ketoconazole,
`do not inhibit enzymes in the pathway leading to
`corticosterone. This evidence for selective inhibition of
`
`testosterone biosynthesis, together with the further
`evidence for selectivity of action provided here for 3 in
`particular, makes 3 a strong candidate for further
`development as a potential drug for the treatment of
`prostatic carcinoma in humans.
`
`Experimental Section
`
`Chemical Methods. ‘H-NMR spectra (250 MHZ) (internal
`Me;Si = 6 0) were determined in CDCl:; (unless otherwise
`indicated) using a Broker AC 250 spectrometer.
`Infrared
`spectra were determined with a Perkin-Elmer 1720X spec-
`trometer. Mass spectra (electron impact, 70 eV) were obtained
`by direct insertion with a VG 70701-I spectrometer and VG
`2235 data system. Melting points were determined with a
`Reichert micro hot stage apparatus and are uncorrected.
`Chromatography refers to column chromatography on silica
`gel (Merck Art. 15111) with the solvent indicated applied
`under positive pressure. Light petroleum refers to the fraction
`with hp 60-80 °C. 3-Pyridyl(diethyl)borane was purchased
`from Aldrich Chemical Co., Gillinghaun, Dorset, U.K. Elemen-
`tal analyses were determined hy CHN Analysis Ltd., South
`Wigston, Leicester, England.
`36-Acetoxyandrosta-5,16-.dien-17-yl 'l‘rlfluororaetl1ane-
`sulfonate (1) and Androsta-3,5,16«trien-17-yl 'I‘rifluo-
`romethanesulfonate (4). To a stirred solution of dehydro-
`epiandrosterone 3-acetate (24.8 g, 75 mmol) in dry CH2Cl2 (500
`mL) containing 2,6-di-tert—butyl-4-methylpyridine (18.5 g, 90
`mmol) was added trifluoromethanesulfonic anhydride (12.6
`mL, 75 mmoll. After 12 h the mixture was filtered, washed
`with water (50 mL), and dried (MgSO4} and the solvent
`evaporated. Chromatography, on elution with CI-IgCl;;-light
`petroleum (1:6), gave first 4 (3.02 g, 10%) as an oil: vmn for
`CI-O str absent; ‘H NMR 6 0.99 (s, 3, 1-1-18), 102 (s, 3, I-I-1.9),
`5.39 (m, 1, H-6), 5.59 (m, 1, H-16), 5.62 (in, 1, H-3), 5.93 (dm,
`1, J = 9.4 Hz, H-4); m/z 402 091*).
`Further elution with CH2Cl3-light petroleum (1:3) afforded
`I (20.1 g, 58%}: mp 75-76 °C (from hexane); v,,,,,, 1734 cm'1
`(CH0 str); ‘H NMR 6 1.00 (s, 3, I-I-18), 1.06 (s, 3, H-19), 2.04
`(s, CH3CO), 4.59 (In, 1, 11-311), 5.39 (dm, 1, J = 4.9 Hz, 1-1-6),
`5658 (m, 1, I-I-16); m/z 402 (M* ~— Ac0l-1}. Anal. {C22H2g05F3S)
`, H, F, S.
`3fl-Acetoxy-17-(3-pyridyllandrosta-5,18~d_iene (2). Di-
`ethy1(3-pyridyl)borane (3.3 g, 23 mmol) was added to a stirred
`
`solution of 1 (6.94 g, 15 mmol) in THF (75 mL) containing bis-
`(tripheny1phosphine)palladium(II) chloride (0.105 g, 0.15 mmol).
`An aqueous solution of Na-.»C03 (2 M, 30 mL) was then added
`and the stirred mixture heated at 80 °C for 1 h and then
`partitioned between Et-,0 and H20. The organic phase was
`dried (NazCOa), filtered through a short column of silica gel,
`and concentrated. Chromatography, on elution with Et2O-
`light petroleum (1:2), afforded 2 (4.95 g, 84%): mp 144-145
`“C (from hexane); :r,,,,,,, 1732 cm” (C-O str); ‘H NMR (5 1.05
`(S, 3, H-19), 1.08 (8, 3, H-18), 2.04 (S, 3, CH3CO), 4.60 (In, 1,
`H-30.), 5.42 (dm, 1, J = 4.7 Hz, H-6), 5.99 (m, 1, 11-16), 7.23
`(dd, 1, J5_4 = 8.1 Hz, J53 = 3.9 Hz, pyriclyl H-5), 7.65 (ddd, 1,
`J4,2 = 2.0 Hz, J.,_;.; = 1.6 Hz, pyridyl H-4), 8.46 (dd, 1, pyridyl
`H-6), 8.62 (d, 1, pyridyl I-I-2); m/z 392 (M’' + H). Anal.
`(C25H33-
`N02) C, H, N.
`17-(3-Pyrldyllandrosta-«5,18-dien-3,8-ol (3). To a solution
`of 2 (4.90 g, 12.5 mmol) in methanol (50 mL) was added 2.5 M
`N303 (10 mL), and the mixture was stirred at 80 °C for 5
`min and then allowed to cool, poured into water, neutralized
`with 1 M HCl, rebasified with saturated aqueous NsHCOa,
`and extracted with hot toluene (3 x 100 mL). The toluene
`extracts were dried (Na2C0a) and concentrated. Chromatog-
`raphy, on elution with Et2O-toluene (1:2), gave 3 (3.45 g,
`79%): mp 228-229 "C (from toluene); am“ 3351 (OH str); ‘H
`NMR :5 1.05 (s, 3, H-19), 1.07 (s, 3, H-18), 3.54 (m, 1, H-30.),
`5.40 (dm, 1, J = 5.0 Hz, H-6), 5.99 (m, 1, H-16), 7.22 (dd, 1,
`pyridyl H-5). 7.65 (ddd, 1, pyrldyl H-4), 8.46 (dd, 1, pyridyl
`H-6), 8.62 (d, 1, pyridyl H-2); m/z 349 (M*). Anal.
`(C24H31-
`NO) C. H, N.
`17-(3-Pyrldyllandrosta-3.5,18-triene (5). The method
`followed that described for 2 but used 4 (2.01 g, 5.0 mmol).
`Chromatography, on elution with CH2C12, gave 5 (1.39 g,
`84%): mp 110'-112 “C {from hexane); ‘H NMR :5 1.02 (S, 3,
`H-19), 1.07 (s, 3, H-18), 5.44 (m, 1, H-6), 5.61 (m, 1, H-3), 5.95
`(dm, 1, J‘-''- 9.8 Hz, H-4), 6.01 (m, 1, H-16), 7.23 (dd, 1, pyridyl
`H-5), 7 .66 (ddd, 1, pyridyl H-4), 8.46 (dd, 1, pyridyl H-6), 8.63
`(d, 1, pyridyl H-2); m/z 331 (M+). Anal.
`(C24H29N) H, N; C:
`calcd 86.96; found, 86.24.
`3]}-Acetoxy-17-(2-pyridybandrosta-5,16-diene (6). To
`E1220 (6 mL), at -18 °C, was added 71-butyllithiuru (0.96 mL,
`2.5 M solution in hexanes) followed dropwise by 2-bromopy-
`ridine (0.228 mL, 2.4 mmoi) in Et2O (2 mL). The resulting
`blood-red solution of 2-pyridyllithium was added dropwise to
`a solution of Z1101: (382 mg, 2.8 mmol) in THF, cooled to -18
`°C, and the orangedorown solution of 2-pyridylzinc chloride was
`stirred for a further 30 min. For the preparation of the
`palladium catalyst, 11 solution of tetrakis(triphenylphosphine)-
`pa.lladium(0) (1.16 g, 1 mmol) in benzene (10 mL) was treated
`with 2-bromopropene (0.13 mL, 242 mg, 2 mmol) and the
`mixture stirred for 16 h at ambient temperature, whereupon
`the initially orange suspension became a yellow solution. The
`solvent was removed under vacuum, the residue was triturated
`with Et20, and the pale yellow product (0.70 g) bromo-
`(isoprope11yl)bis(triphenylphosphine)palladium(II) was recov-
`ered by filtration: 1H NMR (5 0.81 (5, CH3), 4.6 (m, C=CH2),
`7.2-7.8 (m, arom H).
`To a solution of 1 (926 mg, 1 mmol) in THF (10 mL)
`containing the palladium catalyst (76 mg, ca. 0.1 mmol) was
`added the solution of 2-pyridylzinc chloride, and the mixture
`was stirred at ambient temperature. After 1 h, the mixture
`was partitioned between E1220 and H20 and the organic phase
`was dried (Na2C03) and concentrated. Chromatography, on
`elution with Etz0-light petroleum (1:4). gave 6 (0.583 g,
`74%): mp 189-190 ‘C (from light petroleum); v,,,,,,, 1734 cm")
`(C-0 str); ‘H NMR :5 1.09 (S, 3, H-19), 1.15 (3, 3, H-18), 2.04
`(3, s, Cl-I300), 4.62 (m, 1, 11-311), 5.42 (dm, 1, H-6), 6.37 (m, 1,
`11-16), 7.09 (dd, 1, J5‘; = 7.9 Hz, Jg,5 2 4.1 1-1:, pyridyl H-5),
`7.38 (d, 1, J:a.4 W 7.9 Hz, pyridyl H-3), 7.59 (t, J = 7.7 Hz, 1,
`pyridyl H-4), 8.55 (d, 1, pyridyl I-I-6); m/z 391 (M*). Anal.
`(CssHs3N02) C. H. N-
`3fl-Acetoxy-17-(4-pyridyllandrosta-5,16-diene (7). 4-Bro-
`mopyridine (4.5 g) was liberated from its hydrochloride (5 g,
`26 mmol) using the procedure previously applied to 4-cl1loro-
`pyridine” but keeping solutions below 10 “C during concentra-
`tion to prevent polymerization. The free base was twice
`concentrated from Et2O (to remove residual CHC13), and then
`a solution of the freshly prepared 4-bromopyridine (1.58 g, 10
`
`

`

`2468 Journal of Medicinal Chemistry, 1995, Vol. 38, No. 13
`
`mmol) in Et20 (8 mL) was added dropwise during 30 min to a
`mixture of n-butyllithium (2.5 M in hexanes, 4 mL, 10 mmol)
`and Et2O (20 mL) at -20 “C. Of the resulting solution of
`4-pyridyllithium, 15 mL (4.5 mmol) was added to a stirred
`solution of anhydrous ZnC1g (681 mg, 5 mmol) in dry THF (25
`mL). After 1 h at ambient temperature, 30 mL of the red
`solution of 4-pyridylzinc chloride was added to a solution of 1
`(925 mg, 2 mmol) in THF containing bromo(isopropeny1)bis-
`(triphenylphosphine)palladium(II) (see 8 above; 75 mg, 0.1
`mmol) and the mixture tirred at ambient
`temperature
`overnight and then filtered through Celite. Chromatography,
`on elution with Et2O—light petroleum (1:2), gave '7 (140 mg,
`18%): mp 175-177 “C (from light petroleum); vm, 1732 cm“
`(C-0 str); ‘H NMR d 1.08 (s, 3, H-19), 1.63 (s, 3, H-18), 2.05
`(s, 3, CH3CO), 4.63 (m, 1, l-I-30.), 5.42 (dm, 1, H-6), 6.18 (m, 1,
`H-16), 7.26 (d, 2, J = 6.0 Hz, pyridyl H-3, H-5), 8.50 (d, 2,
`pyridyl H-2, H-6); m/z 331 (M* — Ac0l-I). Anal.
`(C2eH3sN02)
`C, H, N.
`17-(2-Pyridyflandrosta-5,16-dien-3-5-ol (9). The method
`followed that described for 3 but used 6 (392 mg, 1 mmol),
`except that on completion of the reaction the product was
`extracted with Et20 followed by benzene and crystallized
`without prior chromatography giving 9 (273 mg, 78%): mp
`206-207 “C (from benzene—light petroleum); v,,,.,. 3390 cm“
`(OH str); ‘H NMR 6 1.08 (s, 3, H-18), 1.15 (, 3, H-19). 3.56
`(m, 1, H—3Cl.), 5.41 (m, 1, H-6), 6.38 (in, 1, H-16), 7.10 (m, 1,
`pyridyl H-5). 7.38 (d, 1, J = 7.8 Hz, pyridyl H-3}, 7.59 (m, 1,
`pyridyl H-4), 8.55 (d, 1, J = 4.2 Hz, pyridyl H-6); mfz 349 (M*).
`Anal.
`(C24,H31N0) C, H, N.
`17-(4-Pyridyl)androsta-5,16-diam17-ol (10)- A solution
`of 4-bromopyridine (from the hydrochloride; 25 g, 129 mmol;
`see '7 above) in Et20 (80 mL) was added dropwise to a stirred
`mixture of n-butyllithium (51.6 mL, 2.5 M in hexanes, 129
`mmol) and E220 (200 mL) at —-76 °C. The resulting solution
`of 4-pyridyllithium was added by transfer needle to a cooled
`(~76 °C) solution of trimethyl borate (13.4 g, 14.6 mL, 129
`mmol) in Etg0 (75 mL); the mixture was stirred for 20 min
`and then allowed to reach ambient temperature. Water (10
`mL) was added, and the resulting light brown precipitate of
`lithium trimethoxy(4-pyridyl)boronate (22.04 g, ca. 90%) was
`collected by filtration, washed with Et-30, and dried in vacuo.
`This product (2.83 g, ca. 15 mmol) was added to a solution of
`1 (1.21 g, 5 mmol) in THF (30 mL) containing bis(tripheny1-
`phophine)palladium(II) chloride (175 mg, 0.25 mmol), followed
`by 2 M aqueous Na2CO3 (12.5 mL), and the mixture heated at
`80 °C, for 6 h, and then partitioned between Etg0 and H20.
`The organic phase was dried (Na2CO3) and filtered through a
`short column of silica gel to give crude 7 which was used for
`the next step without further purification. The method for
`converting 7 into 10 followed that described for 2 —‘ 3.
`Chromatography, eluting with Et20~toluene (1:2), gave 10
`(928 mg, 53% from 1): mp 226-228 “C (from toluene); ‘H NMR
`(5 1.08 (s, 3, 11-19), 1.62 (s, 3, H-18), 3.55 (m, 1, H-30.), 5.40
`(dm, 1, H-6), 6.18 (in, 1, H-16), 7.26 (d, 2, J = 6.1 Hz, pyridyl
`H-3, H-5), 8.51 (d, 2, pyridyl H-2, H-6). Anal.
`(C2=iHs1NO) C.
`H, N.
`I7-(2-Pyridylrnethylhndrosta-5,16-diam17-ol (11). To
`a solution of 2-picoline (7.45 g, 7.9 mL, 80 mmol) in THF (42
`mL) at -20 "C was added n-butyllithium (50 mL, 1.6 M in
`hexanes, 80 mmol) during 30 min. The red solution of
`2-picolyllithium“ (10 mL) was added with vigorous stirring
`under argon to anhydrous ZnClg (1.09 g, 8 mmol), followed by
`benzene (10 mL). The resulting homogeneous solution of‘
`2-picolylzinc chloride (15 mL) was added to a solution of 1 (925
`mg, 2 mmol) in THF (6 mL) containing bis(triphenylphos-
`phine)palladium(II) chloride (70 mg, 0.1 mmol), and the
`resulting yellow solution was heated at 70 “C for 2 h and then
`partitioned between Et2O and H20. The organic phase was
`concentrated and the crude 3,6-acetoxy-17-(2-pyridylmethyl}
`androsta-5,16-diene (8) (650 mg) used directly for the next step.
`The method followed that described for 2 -~-* 3. Chromatog-
`raphy, eluting with EtgO-light petroleum (1:1). Save 11 (460
`mg, 79%): mp 86-88 °C (from light petro1eum—to1uene); Vmu
`3330 cm“ (OH str); ‘H NMR 6 0.82 (s, 3, H-19), 1.04 (s, 3,
`H-18), 3.5 (S + m, 3, benzyl H + H-30.), 5.10 (m, 1, H-16), 5.35
`(In, 1, H-6), 7.12 (dd, 1, J5_4 = 6.5 Hz, J55 -7- 4.7 Hz, pyridyl
`H-5), 7.24 (cl, 1, Ja,.; = 7.8 Hz, pyridyl H-3), 7.62 (dd, 1, pyridyl
`
`Potter et cal.
`
`(C25H3a-
`
`H-4). 8.54 (d, 1, pyridyl H-6); m/z 363 (M+). Anal.
`NO-H20) C, H, N.
`3-[2,3.5,6-Tetrafluoro-4w(trifluorom