Cancer Research Campaign Centre for Cancer Therapeutics at the Institute of Cancer Research,
`
`Cancer Research Campaign Laboratory, 15 Cotswold Road, Sutton, Surrey SM2 5N6. UK.
`
`Gerard A. Potter,f S. Elaine Barrie, Michael Jarman.* and Martin G. Rowlands
`
`Received January 19, 1995“
`
`Steroidal compounds having a 17-(3—pyridyl) substituent together with a 16,17-double bond
`have been synthesized, using a palladium—catalyzed cross-coupling reaction of a 17-cnol tridate
`with diethyl(3-pyridyl)bomne, which are potent inhibitors of human testicular 17o-hydrcxylase-
`017‘20-13'353. The requirement for these structural features is stringent: compounds having
`2-pyridyl (9), 4-pyridyl (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 17fi—pyridyl derivatives diminished potency with 3-pyridyl substitution (3 "- 2'7; 1ng
`for lyase, 2.9 -- 23 11M) but increased it with a 4-pyridyl subotituent present (10 - 28; 1059 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-
`dicn-3fl-ol (3, K1,” < 1 nM; I050 for lyase, 2.9 nM) and 17~(3~pyridyl)androsta-4,16-dien-3-one
`(15; 1050 for lyase, 2.1 nM). Thus compounds having variously aromatic ring A (18), saturated
`rings MB (21, 22), and oxygenated ring C (26) exhibited 1050 values for lyase (1.8‘30 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.1 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 orclriiectomy.2 Howaver neither GnRH agonists
`nor orchiectomy deplete the synthesis of androgens
`through the adrenal route, and levels of testosterone and
`dihydrotestostorone in the protate are respectively still
`25% and 10% of pretreatment levels even alter 3 months
`treatment with a GnRH 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 on antiandrogen, compared with those
`given GnRH agonist or orchiectomy alone.”5 It is
`proposed that the role of the antiandrogon 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 efi‘ects of the combined therapy could
`be realized by a single drug which inhibits the enzyme
`steroidal 17u-hydroxylase-Cnlzo-lyase. This enzyme is
`responsible for androgenic hormone biosynthesis which
`produces dehydroepiandrosterone and androstenedione.
`immediate precursors of testosterone, from their rospec»
`tive precursors pregnenolone and progesterone, in both
`testes and adrenals. The imidazole antifungal agent
`ketoconacole inhibits this enzyme when given in high
`"‘ To whom enquiries should be addressed.
`‘ Present address: Chimscience Ltd. Milton Rd, Cambridge C134
`4WE, UK.
`° 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,6 and although success has been re-
`ported in some {studio-$73E it proved less promising in
`tad-nerd?“J 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,11 lend credence to the selection ofthis 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
`l7u—hydroxylase and
`017,2n-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) substitucnt together with a 18,17-clouble bond
`in the steroids] skeleton. We have previously explored
`nonsteroidel inhibitors containing a pyridyl residue,
`starting from the serendipitous discovery that certain
`esters of 4-pyridylacetic acid were effective inhibitors
`of the bydroxylase-lyase enzyme from rat testis,12 find-
`ings which have in part been rationalized by crystal-
`lographic and molecular modeling studiL-u-J.13 More
`recently. esters of 3-py1idylacetic acid have been evalu-
`ated, using enzyme from human testis.14
`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
`do new mechanism-based design approach was to
`postulate a complete catalytic cycle for the enzyme
`
`0022~2623l95l1838-2463$09.00/0
`
`© 1995 American Chemical Society
`
`MYLAN PHARMS. INC. EXHIBIT 1085 PAGE 1
`
`

`

`2464 Journal of Medicinal Chemistry, 1995, Vol. 38, No. 13
`
`Potter et a1.
`
`O
`
`.7/ jg
`
`o.
`
`’0
`
`’0
`
`O,Fo
`
`H20
`
`02
`
`NVDHOWMSE
`
`HOD-Fe
`
`O=Fe*
`
`is
`
`o‘
`
`0=Fe T HO-Fo‘
`Ho
`
`.
`
`3
`
`ET: electron transport system
`
`j 5°
`
`Ho
`
`ohO,F9
`
`OEH
`
`Figure 1. Postulated complete catalytic cycle for the 17u-hydroxylase-Cum-lyase enzyme. For clarity, only the steroid D-ring
`and the cofactor iron atom are shown.
`
`(Figure 1) and then to consider the juxtaposition be-
`tween the steroid Doing 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 steroid incorporating
`a 16,17-double bond with the 17-position substituted by
`a 2~pyrddy1 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 17-position, and all three compounds
`may be inactive, even if the courdination 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
`tetis.l5 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-py1'idyl
`residue and s C-17 (compound 11). The second 17-(3-
`pyridyl) derivative synthesized was 15, analogously
`related to progesterone, the alternative substrate for the
`hydroxylase activity of the target enzyme. Further
`molecules synthesized retained the ring D substitution
`pattern of 3 and 15 while further exemplifying the effect
`on enzyme inhibition of structural variations in ring
`
`_
`
`Scheme 1"
`
`no
`dlhydrugp‘eundlult-fnh'
`l-osust-
`
`D
`
`a
`
`l
`
`R20
`
`\
`
`
`
`5
`
`i
`
`on
`
`a
`_—- I
`no
`
`O‘H
`
`HD-
`
`3
`
`“ (a) szO, base: (In) 3-PyBEt2, Pd(PPh3l2Clz. 'ITIF, H20, Nacha;
`(c) NaOH, H20, MeOH.
`
`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-enol triflates with suitable pyridyl-containing nu-
`cleophilic coupling partners. For the synthesis of 3
`(Scheme 1), dehydroepiandrosterone 3-scetste was con-
`verted into its 17-enol triflate 1 by base-catalyzed
`reaction with triflic anhydride in the presence of the
`hindered base 2,S-di-tert-butyl-4-methylpyridine. This
`reaction also produced the 3,5-diene 4 in 10% yield. The
`3-pyridyl group we then introduced into the 17-position
`by reacting 1 with diethyl(3-pyridyl)borane in THF,
`
`MYLAN PHARMS. INC. EXHIBIT 1085 PAGE 2
`
`

`

`Novel Steroids! Inhibitors ofHuman Cytochrome 13450:?“
`
`Journal ofMedicinol Chemistry, 1995, Vol. 38, No. 13
`
`2465
`
`Chart 1
`
`
`
`R0
`
`l
`
`i
`
`\
`
`Scheme?
`
`0
`
`1
`
`I
`
`G /
`guarantee-diam
`
`o
`
`.L.
`
`.3...
`
`.
`
`l.
`i
`l
`.
`“PM MM/
`13
`:2
`
`9"
`
`‘
`
`‘s
`
`h
`
`find:
`R'IH
`
`3
`F
`
`\> < "\> r0
`
`T
`10
`
`a
`II
`
`using bis(triphenylphosphiue)palladium(ll) chloride as
`catalyst (0.01 equiv) and aqueous Nazcoa as nucleo-
`philic activator. The reaction proceeded remarkany
`efficiently, without the potential side reactions oftriflate
`hydrolysis or ethyl coupling, to give the acetate 2 in 84%
`isolated yield. From 4, the 3-pyridy1 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 aqueous methanolic
`NaOH to afl‘ord the target 3-pyridyl steroid 3.
`Although these coupling reactions were developed
`independently, the palladium-catalyzed cross-coupling
`of organoboron reagents with on cool triflate has been
`reported recently by Suzuki and coworkers.16 Their
`reactions employed arylboronic acids and 9-allsyI-9-BBN
`reagents and the mild base K7PO4 as the nucleophilic
`activator under strictly anhydrous conditions. Our use
`of diethyll3-pyridyl)boraoe 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.18 Some features of our
`reaction compared with that of Suzuki are noteworthy.
`We found that the catalyst Pd(PPh3)2012 was superior
`to PdiPPhah and censistentiy gave better yields of
`coupled product. The catalyst could also be 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 THF solvent system was
`employed. Our method of introducing the 17-pyridyl
`16,17-ene functionality was more efficient and higher
`yielding than a previous route,1MB reaction of 3-py'ridyl-
`lithium with a 17-keto steroid and dehydration of the
`resulting tertiary alcohol.
`The 2-pyridy1 (6), 4-pyridyl (7), and 2-picolyl (8)
`steroidal acetates (Chart 1) were synthesized similarly
`to 2 but employing different nucleophilic coupling
`partners and modifying the conditions accordingly. The
`reagents used to prepare 6 and 8 were 24pyridy1- and
`2-picolylzinc chloride, respectively.
`In the latter case
`the intermediate 8 was converted without iolation
`directly into 11 in good overall yield (79%). An attempt
`to prepare the 3-picoly1 analog of 11 uing 3-picclylzinc
`chloride was unsuccessful due to homocoupling of this
`reagent. In the synthesis of the 2- (6) and 4- (7) pyridyl
`steroid acetates, the novel palladium catalyst bromo-
`(isopropenyllbi5(triphenylphosphinelpalladium(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 2vpyridyl analog 9.
`The catalyst had been developed to enable low-temper-
`ature cross-coupling reactions for the sterecselective
`synthesis of (E)-4daydrmrytamourii‘en'l1‘22 and was pre-
`pared from 2-bromopropene and tetrakis(tr'iphenylphos-
`
`
`
` is
`
`" (Bl CiFe, CSF. DMF; (13) rN20, base: (c) 3-PyBEt2, Pd(PPh,-,)ZCIR,
`THF, H20, N51200:; (ill HCI, H20, EtOH; (e) AHO-t—Prla.
`
`Chart 2
`
`O
`
`D"
`
`/ \
`——
`
`,‘K‘
`
`RUM:!7
`R-v-f‘la
`
`‘
`ao'“
`
`.‘c... R-Ac:l9
`'
`‘-
`
`Rim”
`R I H121
`
`m
`m)
`E
`
`a
`
`22
`
`phine)palladiurn(0) by a procedure analogous to that
`used to make benzylchlorobia(triphenylphosphine)—
`palladiumfll)?a When the coupling reaction was per-
`formed using 4-pyridylzinc chloride, prepared from
`4-bromopyridine, only a low yield (18%) of the 4-pyridyl
`steroid acetate 7 was obtained. Instability of 4-halopy-
`ridine can restrict the use of 4-pyridylmagnesium and
`-zinc halides in palladium cross-coupling reactions, and
`diethylié-pyridyllborane has been used as an alternative
`reagent.“ Here, lithium trimethoxyt4—pyiidyl)boronate,
`an intermediate in the synthesis25 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 ofa 3-dienoi tritium.26
`Protection as the perfluorotolyl encl ether 12 by reaction
`with octafluorotoluene in the presence of cesium fluoride
`has preved to be a cenvenient one-step procedure.” The
`perfluoroaryl group was stable 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 B-pyridyl derivatives (18, 21, 22; Chart 2.)
`exemplifying further structural variation in rings A and
`B were prepared using procedures analogous 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 tart-butyldimethylsilyl
`dienol ether 23 provided an alternative protecting
`
`MYLAN PHARMS. INC. EXHIBIT 1085 PAGE 3
`
`

`

`24-66
`
`Journal ofMedicinsi Chemistry, 1995, Vol. 38, No. 13
`
`Schme 3“
`
` tDMW
`
`° (a) t-BDMSOTI', base; (b) (i-PrhNLi, PhNch'. (c) S-PyBEoz.
`Pd(PPhs)2Cl-z, THF, H20, M2003; (d) BUMNF, THF, HgO.
`
`Scheme 4“
`
`
`
`“ (a) NaHh AcOH. EtOH. air; (b) Red-Al, ZnClz. 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 siiyl dienol ether
`formed from a testosterone derivative,23 and the present
`product is therefore similarly formulated as the 2,4-
`dienol ether. In the following step, N-phenyltriflimidega
`was employed to prepare the anal triflate 24 since use
`of triflic anhydride resulted in desilylation and 3-disnol
`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 diimide, generated in situ from
`hydrazine hydrate, gave the 17fi-(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-pyridy1 substituent under electrophilic
`activation by zinc chloride to achieve direct hydride
`reduction with Red-Al to produce the 17fi-(4-pyridyl)
`steroid 28. The fi-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 1701-proton which is
`characteristic of I‘m-substituted steroidsflh30 Attempts
`at preparing the corresponding 17u-(4~pyridy1) analog,
`by either direct reduction of 10 or epimerization of 28,
`were unsuccessful.
`
`Inhibition of Human Testicular 17a—Hydroxyl—
`use and Cnao-Lyase. Structure—Activity Relation-
`ships. We have identified as potent
`inhibitors of
`human testicular steroidal l7u-hydroxylase-C17,2g-lyass
`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 naturai substrates, there was an
`unexpected tolerance for structural variation in this
`respect. Comparing 3 and 15 with analogs (18, 21, 22,
`
`Potter at al.
`
`Table 1. Enzyme Inhibition Data
`1C“ {HM}I
`1050 MM)“
`
`compound Cum-lyase lTu-hydroxylase aromatase ou-peductase
`2
`17
`18
`h
`3
`2.9
`4
`5
`56
`12.5
`9
`76
`270
`10
`1000
`4000
`11
`3 10 000
`7' 10 000
`14
`>10 000
`>10 000
`15
`2.1
`2.3
`18"
`1.8
`2.6
`21
`2.5
`4,3
`22
`3
`4"?
`26
`2.9
`13
`2'1
`23
`47
`28
`53
`160
`
`‘26kstoconazois 85
`
`
`3'20
`
`>20
`>20
`
`1.3
`
`>50
`
`1.0
`
`> 50
`
`“ The standard errors were usually < 10% oi‘the ICso value. The
`concentration of enzyme in the assays for iyasefhydroxylase
`inhibition was estimated to be about 4—5 11M. except in the assays
`of 9, 11, 14, and ketoconazole for which the concentration was ca.
`25 and 10 111“ for the lyase and hydroxylase assays, respectively.
`b Other biological activity: estrogen receptor binding affinity
`(estradiol = 100), 4.9.
`
`28) synthesized from other naturally occurring steroid
`precursors, there was little variation (from 1.8 to 3.0
`nM) in the IC59 values for inhibition of the lyase
`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
`ofthe acetoxy derivative 2 compared with 3 could reflect
`a limited bulk tolerance at the 3-position, as indicated
`by the total 10 in activity for the much more sterically
`demanding perfluorotolyl derivative 14 of the potent
`inhibitor 15. The stringent requirement for the 17-(3-
`pyridyl} 16,17-ene 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 173-pyridyl
`derivative 27.
`In contrast, reduction of the 4-pyridyl
`derivative 10 gave a product, 23, with markedly im-
`proved inhibitory potency over it parent.
`The most inhibitory compounds in the present study
`were far more potent than any inhibitor of hydroxylase/
`lyase for which comparable data have been previously
`described. The Ki, 9 for 3 was < 1 11M, whereas the most
`potent inhibitor, also steroidal, reported to date is 175-
`[cyclopropylamino)androst-5-en-3fi-ol31 with a Kim of 90
`nM. Another steroidal compound, 4-pregnen-3-one-203-
`carboxaldehyde oxime has been developed as a com-
`bined inhibitor of this enzyme and testosterone 50.-
`reductaee.32 Though a potent inhibitor (K, = 16 nM) of
`the reductase, it was much less inhibitory toward the
`rat hydroxylase/lyase, being comparable to ketooonazole.
`17fl-Ureido-substituted 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, 17fl-ureido-1,4-andros-
`tadien-B-one, markedly suppressed testosterone levels
`and ablated androgen-dependent organs in the rat.
`Liarozole is a nonsteroidal imidazole derivative having
`activity toward the rat testicular enzyme very sirnila1~35
`to that of ketoconazole. No example among our previ-
`ously mentionedms” esters of 4- and 3-pyridylacetic acid
`compares in potency with the best of the present
`steroidal derivatives.
`
`MYLAN PHARMS. INC. EXHIBIT 1085 PAGE 4
`
`

`

`Novel Steroidal Inhibitors of Hunion Cylochrome P450m.
`
`Journal ofMedicina! 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
`1). 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 Ba-reduc-
`tase might reflect its structural resemblance to the
`natural substrate testosterone, whereas 22, correspond-
`ingly related to the product 5u-dihydrotestosterone, was
`not an inhibitor. Notably, compound 3 inhibited neither
`aromatase nor testosterone 5u-reductase at the highest
`concentration tested, respectively 20 and 50 11M. 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 prong 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 biosyntheis, 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
`proatatic carcinoma in humans.
`
`Experimental Section
`
`Chemical Methods. 1H-NMR spectra (250 MHz) (internal
`MerSi = (S 0) were determined in CD013 (unless otherwise
`indicated) using a Bruker AC 250 spectrometer.
`Infrared
`spectra were determined with a Perkin-Elmer 1720K spec-
`trometer. Mass spectra (electron impact, 70 eV) were obtained
`by direct insertion with a VG 7070K 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., Gillingham, Dorset, U.K. Elemen~
`tal analyses were determined by CHN Analysis Ltd, South
`Wigston, Leicester, England.
`afl-Acetoxyandrosta-B, lB—djen- 17-yl Trifluoromethane-
`sulfonate (1) and Androsta-3,5,16-tfien-17-yl 'l‘rifluo-
`romethanesulfonate (4). To a stirred solution of dehydro-
`epiandrosterone 3-acetate (24.8 g, 75 mmol) in dry CHzclg (500
`mL) containing 2,6-di-ter:-butyl~4—methylpyridlne (18.5 g, 90
`mmol) was added trifluorometbanesulfonic anhydride (12.6
`mL, 75 mmol). After 12 h the mixture was filtered, washed
`with water (50 mL), and dried (MgSOd and the solvent
`evaporated. Chromatography, on elation with CHgClg~light
`petroleum (1:6), gave first 4 (3.02 g, 10%) as an oil: vm for
`CII-O str absent; lH NMRd 0.99 (a, 3, H—lB), 1.02 (s, 3, H-19),
`5.39 (m, 1, 11-6), 5.59 (m, 1, 111-16), 5.62 (m, 1, 11-3), 5.93 (dm,
`1, J = 9.4 Hz, Hal); m/z 402 (114*).
`Further elution with Clinch—light petroleum (1:3) afforded
`1 (20.1 g, 58%): mp 75*76 “C (from hexane}; 17...... 1734 cm'1
`(CH-0 str); 1H NMR 6 1.00 (s, 3, 11-18), 1.06 (s, 3. H-19), 2.04
`(3, (31-1300), 4.59 (m, I, 11-30.). 5.39 (dm, 1, J :7 4.9 Hz, 3-6).
`2.58 (m, H-16): m/z 402 (M+ ~— AcOH}. Anal. (CRHBOstS)
`, H, F.
`.
`afl-Acetoxy'17«(3-pyridyl)androsta-5,16~dlene (2). Di-
`ethyl(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-
`(triphenylphospbinemalladiumfll) chloride (0.105 g, 0.15 mmol).
`An aqueous solution of Na2COa (2 M, 30 mL) was then added
`and the stirred mixture heated at 80 “C for 1 h and then
`partitioned between EtQO and H20. The organic phase was
`dried (NazCOa), filtered through a short column of silica gel,
`and concentrated. Chromatography, on elation with ano-
`light petroleum (1:2). afi‘orded 20195 g. 84%): mp 144—145
`“C (from hexane); rm, 1732 cm“ ((3-0 str); 1H NMR :5 1.05
`(s, 3, H-19), 1.08 (s, 3, H-IB), 2.04- (s, 3, 01-1300), 4.60 (m, 1,
`14-30.), 5.42 ((1111, l, J = 4.7 Hz, 11-6), 5.99 (m. 1, H-lfl), 7.23
`(dd, 1. J“. = 8.1 Hz, J53 = 3.9 Hz, pyridyl 11-5), 7.65 (ddd, 1,
`J“ = 2.0 Hz, J... a» 1.6 Hz, pyridyl 7-1—4), 5.46 (dd, 1, pyridyl
`H-S), 8.62(d, 1, pyridyl 11-2): m/z 392 (M+ + H). Anal.
`(0251133-
`N01) 0,11, N.
`17-(3-P3u'idyl)androsta-5,16-dien-3fl-ol (3). To a solution
`of 2 (4.90 g, 12.5 mmol) in methanol (50 mL) was added 2.5 M
`NaOH (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 NaHCOa,
`and extracted with hot toluene (3 x 100 mL). The toluene
`extracts were dried (N11260:) and concentrated. Chromatog-
`raphy, on elation with Etzowtoluene (1:2), gave 8 (3.45 g,
`79%): mp 228—229 “C (from toluene); vmu 3351 (OH ctr); 1H
`NMR 6 1.05 (s, 3, H-19), 1.07 (s, 3, 11-13), 3.54 (m, 1, 8430.),
`5.40 (dm, 1, J = 5.0 Hz, H-6). 5.99 (m, 1, 11-16), 7.22 (dd, 1.
`pyridyl H-5), 7.65 (ddd, 1, pyridyl H-4), 8.46 (dd, 1, pyridyl
`H-6), 8.62 (d. 1, pyridyl 11-2); mjz 349 (Ml). Anal.
`(Cad-la;-
`N0) C, H, N.
`17-(3-Pyrldyl)androsta-3,5,ld-triene (5). The method
`followed that described for 2 but used 4 (2.01 g, 5.0 mmol).
`Chromatography, on elution with CH2Cl2, gave 5 (1.39 g,
`84%): mp 110-112 c‘C (from hexane); 1H NMR d 1.02 (s, 3,
`H-19), 1.07 (s, 3, H-IS), 5.44 (m, 1, 11-6), 561011, 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, pyrldyl H-4), 8.46 (dd, 1, pyridyl H-G), 8.63
`(d, 1, pyridyl 11-2): m/z 331 (Mt). Anal.
`(024112914) H, N; C:
`calcd 36.96; found, 86.24.
`Sfi-Acetoxy-l7-(2-pyridyllandrosta'5,Ill-cliche (6). To
`Eth (6 mL), at -18 “C, was added n-butyllithium (0.96 mL,
`2.5 M solution in hexanes) followed dropwise by 2-bromopy-
`ridino (0.228 mL, 2.4 mmol) in 111220 (2 mL). The resulting
`blood-red solution of 2-pyridyllithium was added dropwise to
`a solution of ZnClz (382 mg, 2.8 mmol) in THF, cooled to ~18
`“C, and the orange-brown solution of 2-pyridylzinc chloride was
`stirred for a further 30 min. For the preparation of the
`palladium catalyst, a solution of tetrakis(triphenylpbosphine)—
`palladium(0) (1.16 g, 1 mmol) in benzene (10 mL) was treated
`with 2-bromopropene (0.18 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 retrieved under vacuum, the residue was triturated
`with Et20, and the pale yellow product (0.70 g) bromov
`(isopmpenyl)bidtnphenylphosphine}palladium(II) was recov-
`ered by filtration: 1H NMR 6 0.81 (s, CH3), 4.5 (m, C=CH2),
`7.2-7.8 (m, arorn 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-pyriclylzinc chloride, and the mixture
`was stirred at ambient temperature. After 1 h. the mixture
`was partitioned between Eth and H90 and the organic phase
`was dried (Nachs) and concentrated. Chromatography, on
`elution with EtzO—light petroleum (1:4). gave 8 (0.583 g,
`74%): mp 189—190 °C (from light petroleum); v...u 1734 cm"
`(C-O str): 1H NMR (5 1.09 (s. 3. H—19). 1.15 (s, 3, H-18), 2.04
`(3, s, CHaCO), 4.62 (m, 1, H—3a), 5.42 (dm, 1, 1:16), 6.37 (m, 1,
`5-16). 7.09 (dd. 1. J._, = 7.9 Hz, J... = 4.1.Hz,pyridy1 3—5).
`7.33 (d, 1. .13.. = 7.9 Hz, pyridyl 11-3), 7.59 (t, J = 7.7 Hz, 1,
`pyridyl H-4), 8.55 (d, 1, pyridyl Hod); mlz 39!. (31*). Anal.
`(CssHsoNCls) C. H: N-
`3fi-Acetoxy-17-(4-pyridy1hndmsta-5JB-diene (1'). d—Bro-
`mopyridine (4.5 g) was liberated from its hydrochloride (5 g.
`26 mmol) using the procedure previously applied to 4-chloro-
`pyridine” but keeping solutions below 10 "C during concentra—
`tion to prevent polymerization. The free base was twice
`concentrated From EteO (to remove residual 011013), and then
`a solution of the freshly prepared 4-bromopyridine (1.53 g, 10
`
`MYLAN PHARMS. INC. EXHIBIT 1085 PAGE 5
`
`

`

`2483 Journal of Medicinal Chemistry. 1995, Vol. 38, No. 13
`
`mmol) in Elth (8 mL) was added dropwise during 30 min to a.
`mixture of n—butyllithium (2.5 M in homes. 4 mL. 10 mmol)
`and M20 (20 ml.) at —20 “C. 0f the resulting solution of
`4-pyridyllithium, 15 mL (4.5 mmol) was added to a stirred
`solution of anhydrous ZnClg (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 bmmdisopropenylmis-
`(triplienylphosphins)palludium(ll) (see 6 above; 75 mg, 0.1
`mmol) and the mixture stirred at ambient temperature
`overnight and then filtered through Celite. Chromatography,
`on eiution with EtaO—light petroleum (1:2). gave 7 (140 mg,
`18%): mp 175—177 “C (from light petroleum); 1a.... 1732 cm“
`(C-O str); 11-! NMR 6 1.08 (s, 3. 1-1-19). 1.63 (s. 3, 11-18), 2.05
`(s, 3, CHSCO), 4.63 (m, 1, H-au), 5.42 (dm. 1. 1-1-6). 6.18 (m, 1.
`1-1-16), 7.26 (d. 2, J = 6.0 Hz, pyridyl H-a. H-s), 8.50 (d, 2,
`pyridyl 1-1-2, 1-1—6); m/z 331 (M‘ — AcOH). Anal.
`(CzeHmNOfl
`C. H, N.
`17-(2-Pyridyl)androsta—5,16-dien-3-fi-ol (9). The method
`followed that described for 8 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, 73%): mp
`206—207 °C (from benzene-light petroleum): 11...... 3390 cm‘1
`(0H str); ‘H NMR (5 1.08 (s, 3, 1-1-18), 1.15 (s, 3, 11-19), 3.56
`(In, 1, 11-30.), 5.41 (m, 1, 1-1-6), 6.38 (m. 1, 11-16), 7.10 (m. 1,
`pyridyl H-s). 7.38 (d. 1, J = 7.3 Hz, pyridyl 11-3), 7.59 (m. 1,
`pyridyl H-4). 8.55 (d, 1, J = 4.2 Hz, pyridyl H—B); m/z 349 (011*).
`Anal. (CafiuNO) C, H, N.
`17-(4—Pyridyl)androsta-5,16-dien-17—ol (10). A solution
`of 4-bromopyridine (from the hydrochloride; 25 g, 129 mmol;
`see 7 above) in H20 (80 mL) was added dropwise to a stirred
`mixture of n-butyllithium (51.6 mL, 2.5 M in hexanes. 129
`mmol) and E1220 (200 mL) at -76 °C. The resulting solution
`of 4-pyridyllithium was added by transfer needle to a cooled
`(-76 "0) solution of trimethyl borate (13.4 g. 14.6 mL, 129
`mmol) in 11th (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 tfimethom‘l—pyfidylmoronate (22.04 3, ca. 90%) was
`collected by filtration, washed with E610, and dried in vacuo.
`This product (2.83 g. cs. 15 mmoi) was added to a solution of
`1 (1.21 g, 5 mmol) in THF (30 mL) containing bis(triphenyl-
`phosphine)palladium(ll) chloride (175 mg, 0.25 mmol), followed
`by 2 M aqueous N21200:; (12.5 mL), and the mixture heated at
`60 “C, for 6 h, and then partitioned between Etzo and H20.
`The organic phase was dried (NegCOa) 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 Enos-toluene (1:2). gave 10
`(928 mg. 53% from 1): mp 226-228 “C (from toluene); 1H NMR
`6 1.08 (s, 3, 1-1-19), 1.62 (s. 3, 1-1-18). 3.55 (m. 1, 11-30.), 5.40
`(dm, 1, 1-1-6), 6.13 (m, 1. 11-16). 7.26 (d. 2, J = 6.1 Hz. pyridyl
`H-3, 1-1-5). 8.51 (d, 2. pyridyl H-2, H-6). Anal. (CzrflalNO) C.
`1-1, N.
`17-(2-Pyridylmethylmndrosta-5,16-dien-17-01 (11). To
`a solution of 2-picoline (7.45 g. 7.9 mL. BO 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-picolyllithium38 (10 mL) was added with vigorous stirring
`under argon to anhydrous 21101: (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(tripheny1phos—
`phine)pallsdium(ll) chloride (70 mg. 0.1 mmol). and the
`resulting yellow solution was heated at 70 “C for 2 h and then
`partitioned between Eth and H20. The organic phase was
`concentrated and the crude afi-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 Et20~light petroleum (1:1). gave 11(460
`mg. 79%): mp 86—88 “C (from light petroleum—toluene); v...“
`3330 cm" (OH str); 1H NMR 6 0.82 (a. 3, 1-1-19). 1.04 (s, 3,
`H—18), 3.5 (s + m. 3. bensyl H + 11-37:), 5.10 (m. 1, 1-1-16), 5.35
`(m, 1, 11-6). 7.12 (dd, 1, J54 = 6.5 Hz, J55 = 4.7 Hz, pyridyl
`11-5). 7.24 (d. 1. .133 = 7.8 Hz, pyridyl 11-3), 7.62 (dd. 1. pyridyl
`
`Potter et a1.
`
`(Casi-{33-
`
`11-4). 8.54 ((1, ll pyridyl H-B); m/z 363 (M*). Anal.
`NO-H20) C. H, N.
`S-[2,3.5,6-Tetrafluoro-4-(tfifluoromethylmhenoxw}an-
`drosta-3,5,18-trien-17-yl Trifl