C!. /.
`
`Journal o(Medicinat Che1J1istry, 1977, Vot: 20, No. 2 2~3
`
`References and Notes
`. (1) K. E. Kinnamon and W. E. Rothe,' Am. J. Trap. Med. Hyg·.,
`24, 174 (1975) .
`(2) J. Lascelles and D. D. Woods, Br. ·J. Exp. Pathoi., 33, 288
`(1952).
`(3) E. L. R. Stokstad and J. Koch, Physiol. Reu.; 47, &1 {1967).
`(4) H. E. Sauberlich and C. A. Baumann, J. Biol. Chem., 176,
`165 (1948).
`.
`(5) C. C. Smith and C. S. Genther, Antimicrob. Agents Che-
`·
`mother., 2, 103 (1972).
`(6) L. Rane and D.S. Rane, Abstr., 9th Int. Congr. Trop. Med.
`·Malaria, l, 281 (Abstr. No. 406) (1973).
`(7) T. S. Osdeiie, and P. B. Russell, and L. Rane, J. Med. Chem::'
`10, 431 (1967).
`'
`'
`·.
`.
`.
`.
`.''
`(8) A. F. Crowther and A. A. Levi, Br. J. Phatmacol., 8, 93.
`(1953) ..
`(9) Report of a WHO Scientific Group, W.H.0., Tech. Rep. Ser:,
`.No~ 375 (1967):
`.
`.
`.
`.
`.
`(10) C. J, Canfield and R. S. Rozman;· Bull. W.H.0., 50, 203
`.
`(1974).
`'
`:
`. ' .
`(l l) C .. C. Smith aild C. S. Genther, A11n. N. Y. Acad. Sci:, 186,
`185 (197i). .
`.
`.
`.
`.
`:
`'
`. '
`(12) .i.::tt. Schmidt and C. S. Genther, J. Pharmacol. Exp. Ther.; ·
`.
`107, 61 (1953).
`. ' '
`.
`.
`(l:J) S. R. M. Bushby and G. H. Hitchings, Br. J. Pliarinacol;
`Chemother .. , 33, 72 (1968).
`·
`(14) .L.·H. Schmidt, J. Harrison, R. Elliwn, and P. Worcester,
`Proc: Soc. Eip, Biol. Med., 131, 294 (1969).
`. ·
`.'
`·(15) G ... H. Hitchings and ..J. J. ~urchall, Acfo,.Enzymol.,.21; 417
`"
`(i965).
`:
`:
`.·
`.• ' .
`.
`. . .
`'
`(lS) 'i'. W. Sheehy and Ii Kempscy, J Am. Med~ Assoc., 214,
`109 (i970).
`'
`. .
`.
`.
`.
`(17) L."H. Schmidt, Ul)published results.
`(18) R. L. Kisliuk, M. Friedkin, L. H. Schmidt, and R. N. Ro$an;.
`Science,'156, _1616 (1967);
`·
`· .-
`·
`·.
`'
`(19) G. H: Hitchings, 'J'rans. R. Soc. Trap. Med. Hyg., 46, 467
`.
`(1952). •
`.
`. . . .
`.
`.
`. . . '
`. . .
`(2Q) .. J. 0. Lampen and M. J. Jones, J. Biol.· Chem.; 166, •35
`.
`(1946).
`.
`:
`.
`.
`.
`'
`(21) ·M. Sahyun,P. Beard;E. W. SchUJtz, J. Snow, and E. Cr<isS;.
`J. Infect.: Dis.; 58, 28 (1936).
`·
`·
`·
`
`The incidence of atherosclerotic disorders, a major cause
`of death in· the United States and other industrial societies,
`
`'.Wa.;nc;yl'Pyroph.osphate Analogues ..
`.l":~as that 9f Sahyun et al.21 to which 0.6% acid hydrolyzed casein
`Lwas added. Growth of the. test organisms wa.5 read as turbidity
`• .... }ii~ the Klett-Sununersori photoelectric colorinieter. The results
`. · ftM!f.l/!lre expressed as the micrornolar coneentration required to produce
`::i;rn·:::ii:?:5<l% growth inhibition (rC50) of the s.train under the conditions
`·%6ed. Where sufficient drug was available~ concentrations u1> to
`\JOOO µg/ml were tested; when this was not p0ssible the highest
`i.~ol)centration used was 100 µg/ml.
`{{:·In addition to antibacterial activity, tests were made for the
`'.?;:'teversal of the drug inhibition by PABA in the case of E. coli,
`g-:rolic acid with S. faecium and L. casei, and folinic acid with P.
`'/'eeievisiae'. The reversal was indicated as posit.ive when addition
`}'bf either 10 µg/ml of PABA or ten times the amounts of folic or
`((olfuic acids normally used produced at least a fourfold decrease
`/In activity of the compound.
`·
`·
`.
`/X .. Strains of S. faeciwn, L. caJJei, and P. cerevisiae were made
`:'/i~istant to CGT by serial transfer in increasing amounts of the
`}driig.5 The most highly ·resistant strains obtained with each
`:r;organisin were used in the ~ests reported here. From these data
`Di.lie fold increa.<re in resistance (FIR) of the CGT R strains compared
`('>to:the respective sensitive strains for each compound has been
`X:?Jculated (ICliO R/IC00 S). · Thus1 for ·most. compounds ten
`'('quantitative and seven qualitative (reversal) pBronictern have been
`t~~rn:'::j~etermined .. ·
`. · .
`.
`.
`. .
`,
`.
`.
`7·"·"">. Ack~owledgment. 'This work was supported in part
`..... ::by Army Contract No. DADA 17-67~C-706~ and DADA
`.FJW:Vt7-71-C-1011. This paper is Contribution No. 1385 to the
`··
`:Army Research Program on malilria. The compounds used
`:·'were supplied by the Division of Medicinal Chemistry,
`:/Walter Reed Army Institute of Medical.Research .. We
`'· .
`:mWfii'wish .to acknowledge the assistance 9f Mr. Walter Weigel
`·/ff~:'''.dn preparation oftables. _:Lt. Col.David E. Davidson, Jr.,
`'
`/of the Oepartment of Biology, Division of l\,1edfomal
`'.(.Chemistry, .Walter. ~ecJ Anny .hl.Stitute of Medical Re(cid:173)
`.. /search; supplied the P. bergflei mOU!>e. test data. The
`W':(i@;::,:';'c:hoice of the-majority of the 40 compounds was made by
`r?' .. iMf:!{.Dr: .. D~Vid P. Jac.obus, then Director of the Div_ision of
`lNh.'.\'Medicmal Chemistry, Walter Reed Army Institute of
`f~~l{~K : Medical Research. ·
`·
`:~~Ilm::
`!1[~~{~'1nhibition of Squalene.Synthetase by FarnesyLPyrophosphate Analogues 1
`1•>Paul R. Orlfa de Montellano,' J•ng Shu Wei, Raloel C..tmo, Ciwt0SK. fuu, and Amrit Bopa<ai
`.
`.
`::;;:@;:; ;~Depart.",nent of Pha;maceuticat Chemistry, Schoo;, of Pha~macy, U~ivc;sit; ofCatiforma, Sa~ Frorn:is~o. Califori~a 94143.·.
`tlll]i';;:· 'Received June 16, 1976
`.
`. .
`·
`.
`.
`:
`... ·
`. .
`. .
`. ·.
`·.
`.
`.
`l@F: :
`· The pyrophosphates of. the ,following -farnesol analogues have. been synthesized: 2-methylfarnesol; 7,11-di-
`I\#;:: ·
`.•. -f{'..
`niethyl-3~ethyl-2,6,l0-dodecatrien·l-ol; 3-demethylfarnesol; 4-methylthiofarnesol; 7 ,l l-dimethyl'3-iodo-2,6, 10-
`dodecat~ien:I-ol; 7;11-dimeth. yl'2-iod. o-2,~,10-dodeca. trien-1-ol;. 7,ll-dim~thyldodeca-6,10-dien-2-yn-l~o!; phytol;
`l
`·;wt
`3,7,ll-trimethyl-2-dodecen-1-ol; 3,7,11-tnmethy!dodecan-l-ol; and geramol. The double. bonds mall the above
`·compounds were in 'the E configuration, except phytol, which was a 7:3 mixture of 2E and .2Z isomers. Each of
`:§/.:
`\tL ·
`U1e pyrophosphates inhibitirthe incorporation of labeled farnesyl pyrophosphate into squalene by a yeast enzyme
`:::IV
`preparatiOn. Free alcohols and monophosphates are inactive. The analogues, listed in order of deereasing inhibitory
`·:"''"
`st.rength; are, by.kinetic analysis, oompetitive or mixed inhibitors. Irreversible inhibition is not observed. The reaults. ·
`:,;,.~_:.;,,! __ :_.:'..:
`suggest that binding to the enzyme is primarily mediated by the p)'rophosphate moiety assisted by relatively nonspeeilic
`lipuphilic interactions. Decreasing·the chain length and saturating double bonds severely reduces binding, while
`:.
`. substitution at the 2, 3, and 4 positions,. and lengthening of the chain, is well tolerated.
`the sterol, by enhancingits ~etabolism and elimi~e.tion:
`·and by decreQsing its· rate of bi1?9ynthesis. Chol~sterol
`synthesis, howe_ver, is sul:ijeet to fee~back regulat~on,4 so·
`that decr~ases in ·cholesterol levels tend to be .compensated
`for by increased biosynthesis.· Removal ofsterols in rat.S·
`due to cholestyramine .feeding, for example, causes a
`20(t-300% increase in hepatic cholesterol. synthesis.5 The
`most effective approaches to lowering physiological chQ-
`
`ilk
`:···',_ .. ·'{_.'•:;·;_:~,·,· .. :'.:;·',;·;.''.;_._'.. · · ~~~~~!~ot0f;:!i!J1 ~~h~d:u~~~n:rr::t~!v:!i~~if :!mor
`
`.:
`
`plaSina cholesterol levels are therefore of high interest, .even
`though the role of this sterol in the etiology of the disease
`
`I ~ti'~~'=1~~~b:\'.,~'::,~· .i.':J:::;;';::~=~,
`
`Smith
`
`ory,
`1lum
`d
`:ied
`1-
`,e
`ays
`cure
`·e
`
`ivities
`;lates.
`lCtive,
`1d low
`in the
`
`3 with
`in the
`stap(cid:173)
`ion.
`
`ecium
`1casei
`~richia
`
`1uiring
`·econ-
`in and
`:0.001
`~used
`
`l __
`
`1 of 7
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`
`244 ·Journal of Medicinal Chemistry, I9i7, Vol. 20,-No. 2
`
`Ortiz de.MonteUano r.t al. ·
`
`....................... ··············-·····--·-·--·---·---:---:----:-.,..--,-.,......,..,.......,..,......,..._ .....
`
`Table I. Physical Properties of Analogues
`GC retention time (min) of
`free alcohol isomers
`2E
`15.03
`20.29
`10.05
`20.36
`20.93
`21.94
`24.94
`4.91
`9.19
`15.56
`
`Phosphorus
`analysis
`(%oftheory)
`101
`114
`106
`93
`100
`93
`103
`102
`107
`95·
`91
`93
`
`Com pd
`1
`2
`3
`4
`.5
`6 ...
`7
`8
`9
`10
`11
`12
`
`2Z
`13.13
`17.40
`9.26
`18.76
`
`4.39
`8.06
`13.58
`
`15.03
`7.01
`
`Temp, ¢c
`160
`150
`150
`150
`"i76
`176
`175
`125
`· 150
`175
`150
`160
`
`GC retention ti inc (min) of isom;~-s----­
`of ethyl ester precursor
`·Temp, •c
`2E
`11:0
`160
`21.0
`160
`
`2Z
`18.0
`17.o
`
`160
`
`176
`
`°150
`
`150
`
`18.8
`
`20.8
`
`8.5
`
`11.1
`
`6.11
`
`x
`R~opp
`z
`y
`
`x
`Me
`
`Me
`H
`·e1
`I
`·tt
`
`Me
`
`I
`2
`1.
`.!.
`·1.
`.i
`L
`
`y
`
`H
`·Me
`H
`H
`H
`I
`H
`
`.z
`H
`
`H
`H
`H
`H
`H
`Mes·
`
`R'~.
`.~opp
`
`lesterol levels, therefore, are likely ·to include inhibition
`of its biosinthesis as a vital component.
`· .
`. The normal rate-controlling step in the biosynthesis of
`cholesterol is the· formation of mevalonic acid from 3-
`, hyd,roxy~3-metl).ylglut.aryl cOeiizyme A. 4 Mevalonic acid,
`. :however;. is the precursor of all isoprenyl derivatives,
`.il)clrii:ling in anima1~'c1>enzyme·Q, 4eme A,·and the doli-
`, . cbols .... The fir&t.biosyri~ticstep which leadS exclusively
`... t:.o·s.terols; .the ·oondenSatiQn of tWo farn~yl py'toj:>hOspha~
`~~
`··~· · . · (1) to give squalene, is a possible site of secondary regu-
`}~ ·•
`· Jatory mectianisms. 6 Inhibition of chOlesterql biosynthesis
`.. at. this _stage i~ therefore attraciive, not only _because
`f~:;l
`.·. ·,. no~teroidal pathwa)'s will be:miriiinally affected but also
`7"
`i \ ·
`:. because.dtigriidative processes eXist. for remoVal of famesyl
`._ pyrophosph~te.7 The synthesis of squalene fromfamesyl
`~fj·
`~r: ' · · quenee is catalyzed by squalene synthetase, a complex,
`· ·· pyrophosphate involves an· isolable · intermediate,
`·
`~f.11
`· · presqualene pyrophosphate.8 The entire synthetic se-
`'~l ·
`· 1*t!mbrane-b">Und enzym,e.9 As part of our current efforts
`~m .
`fo define' the mechanism and active site topology' of this
`'·m
`. elizyme, IO.ll .and to develop effective inhibit.ors of it, we now
`. · · deSerjbe a study of structure-actiVity·relationships which
`· ·:govern inhibition of the enzyme by substrate aJ.lalogues.
`. SY»ihesis. Pyrophosphates 1-12 were obtained from
`: · the corresponding farnescil analogues by pyrophospho(cid:173)
`·. rylation with di(triethyhu:p.moniuin) phosplia~ and tri(cid:173)
`.. chloroacetooitrile~ 12.33 The formatfon of pyrophosphates
`·was verified by quantitative phosphorus analysis (Table
`· I), 13 ~ltbough traces of mono- or polyphosphate con(cid:173)
`taminants were apparent in some cruies. The integrity of
`.the phosphorylated struCt\lreB was confirmed by regeli(cid:173)
`eratfon of the starting alcohols on treatment with bacterial
`· alkaline phosphat.ase} 4
`·
`: · Geraniol and phyful, :.the precur00rs of 8 and 10, re(cid:173)
`. spectively, were· pmchased commereially. The farnesol
`.· anaJogu~ r~riired in the synthesis of 3, 5; 6,'. and 11 were
`
`. .. .
`i~
`
`·~.;_,:::
`
`··
`
`:-~v·· ·.
`
`~.i ••. ~., •.• ~.·1n:·.·.···
`
`~>:~ .... .,. ~ ·.
`
`_!
`.1_
`lO
`
`!CH3hC =CH CH2 -
`ICHJ12CHCH1CH,CH2CH(C~)CH,CH.i-
`(CHJhCHCH2C~CH2CH(CHJ!CHiCH,CHzCl:l(CHJICH;iCHr
`11 ~·=_/opp
`ll I
`I
`·
`I
`.
`~opp
`.
`.
`.
`prepared from genmyl bromide and ethyl.3-ketopentaiioate
`by base-promoted condensation, ester hydrolysis, and
`decarboxylation. Finally, 4-methylthiofamesol (7 alcohol)
`was obt.ai.Ded by red\lction. of the corresponding ethyl ester
`17. Condensation of 3-niethylthiogeranyl acetone (16)-and
`. triethyl phosphonoacetatefumished 17, essentially as the
`•piJre 2E isomer •. The reqUired gerariyl aeetone deriV!ltive
`16 was· made from the sodium salt of l~methylthio-2-
`_propa1ione20- and geranylbromide.
`. . • .
`.
`.
`. Anall'i!is. by · GC shQwed . that the .· farn.esol . analOgue
`.precursors of all the pyrcipbosphates except.10 were es(cid:173)
`sentially pure all-E isomers, contaminated in no instance
`·with more than 4 % of the 2Z iso~er .. Analogue ·to was an
`exception, com~ercial phytol consisting of a·7:3 mixture
`of 2E and 2Z isomers, respectively. Stereochemical as(cid:173)
`signments have been ri;lade i!l the literaturefor the farnesol
`. analogues· except the p~rs of 2, 4, and 7. As haS been
`· noted,21 and extensively confirmed·fo our work (Table I),
`the all-E isomers of prenyl alcohol;S ·and esters have·higber
`retention times (RJ on GC than isomers with one or more
`double bonds in the Z configuration. The·assignment of
`stereochemistry to analogues 2; 4, and ·7, sugge8ted by their
`relative GC retention times, has ·been ·independently
`
`r:;p::~ ~:a:i:i~:db~~~;~:;t~;:::::~}
`
`ethyl farnesoate, followed by LiAIH4 red\lction of the ester
`runction •. The precilrsor of 9, 6,7,10,11-tetrahydrofarnesol,
`was made by. analogous reduction of ethyl ,3,7,11-tri- ··
`' methyl-2(E}-dodecenoate.17 A a mixture of 2E and 2Z
`iSomers of ethyl 2-methylfarnesoate (13) was obtained by
`Wadsworth-Emmons condensation18 of geranyl acetone
`· With diethyl l-carb0ethoxyethyl phosphonate.19 The 2E
`isomer, i8olated by spinning-band distillation,-:was reduced
`with LiAIH4 to 2-methylfamesol,. the preeutsor of 2 .
`• Similar condensation of 7 ,ll-dimethyl-6(E),10-dodec8-
`dien-3-one (.14) and triethyl phcisphonoacetate yielded, as
`. a. 6:4 mixture of 2E aiid 2Z isomers, the 3-ethy~· analogue
`of ethyl.farnesoate (15). Spinning-band distillation and
`ester reduction gave 4 alcohol. ketone 14,- in iurn, was
`
`2 of 7
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`.
`>karnesyl Pyrophosphate Analogues
`
`.
`
`,
`
`. .
`
`·._ ._:
`
`: . ..
`
`. . . . ··:_._
`
`. ·.
`
`.
`
`journal of Medicinal Chemistry, 1977, Vol. 2o, No.- 2 245 .
`
`100
`
`80
`
`60
`
`40
`
`20
`
`>
`.......
`
`0.2 . 0.4 0.6 0.8
`1/[s) uM-1
`.
`. .
`Figure 2. Lineweaver-Burk graphic analysis of the inhibition. ·
`of squalene syn~hetase by 2·1'!"ethylfarnesyl pyrophosphate (2).
`[SJ is the concentration of I l-3H]famesyl.pyrophosphate;·initial ·
`velocity ( V) .is expressed in nimomeiles of squalene .formed per
`minute per milligram .of protein. Concentrat.icin of i1Jhibit.ot (2):
`0 (e), 1 (A), and 3. µM (0).
`·
`·
`·
`incubations With no iri.hibitors, vs. th~ corieentration :of the .
`inhibitor: The strongest it1hibitors· iire 2· and. 4, while
`geranyl pyrophosphate (8) is ~he weakest. Phytyl pyro·
`phosphate (I} is !!lightly more -poteiltthan shown, since
`no adjustme_nt has been made for the presence of t~e ·. ·
`presumablyless active 2Z·isomer. A lilnited selection ci~ · · ·,
`.the. ~cohol .: and. µi~pophosp!:iate precursors . of j>yro- · .... ::<
`. ph0sphates 2..:12 has also been evaluated as inhibitors:
`: ..
`None of these-gave significant inhibition w~en present in ·
`·
`concentratfons up tci 20 µM. ·
`·
`· · · ·
`.
`.
`The. inhibition of squalene .synthetase by pyrO(cid:173)
`phosphorylated analogues h!ls been supjected to kinet~c
`analysis .. Double reciprocal plots .of initial' reaction _ve,.
`locities vs .. substrate concentratfon, at ·fixed. inhibitor -.
`eoneentrations, ~ve been m"ade.27 Kinetic ronstaittS heve ...
`not been extracted from"the deta. because the-relatively
`crude. enzyme system· used is not amena~le to ·such·
`treatment. The farnesyl pyrophospha~.Kw ·value, for
`example, l?Oried slightly (rotn one enzyme preparati~n t0
`another. Nevertheless; our acCumulateddata indicB.ie:tbat
`2. has an appar~nt K; value of approxiri1ate_lY. P.:5 ~M. ·
`Ftirthermore, the inhibition patterns dearly est.$.µ~h that : .
`each oft.he anl1.logues is a. competitive; or at l~ast par:tially
`. competitiye (mixed),. inhibitor. 27 Com~und;_2, fore~~ :
`ample, gives the.competiti.ve irihibitfon pattern in.Figu~e
`2, while 3 is shown tO be a mjxed inhibitor. irl Figure 3'.
`. Patterns. similar to one. or the other of:these•plots·were ·
`obtained for all of t}).e analogµes. The re11ults·are sum(cid:173)
`mar~d in Table II .. The conclusiOn .th.at the aria1og\i!ls .
`compete with the substrate for a berth at the a.ctiye si~e
`is well substantiated.
`·
`The kinetic mechanism of the enzyme, involving_ a
`probable covalent intermediate,9 suggested that substrate
`analogues might cause irreversible inhibition. This would·
`be the .consequence if an analogue were accepted as a
`substrate, forming the covalent complex, but was unable
`to comple~ the synthetic sequence .. Compounds 2,5..:7,
`and ti, in particular, were prep~ed wit,h such a p0ssibility
`in mind. To determine if irre.versible inhibition was oc~
`cu.rring, the analogues were preincubated for up to 5}). at.
`ambient temperature with the enzym!l .. and. ~n assay
`components except the substrate. Norma) bioass.ay was
`then initiated by addition of [l-3H]-l. · Irreversibie binding
`is characterized by time-dependent increases in observed
`inhibition. 28 The time-dependent decrease in inhibition
`actually observed (Figure 4), however, is incompatible with
`formation of irreversible analogue-enzyme comple)!:es. The
`
`90 -
`
`80
`
`70
`
`60
`
`z
`0 ;:
`iii 50
`i:
`~ 40
`~
`
`30
`
`20
`. 10
`
`0
`
`1,
`
`2
`
`3
`
`i\H0'-·;··
`~!;~::;.:.
`
`It~;/::-.
`
`4 -1...}-~
`· CONCENTRATIO; OF INHIBITOR
`~~:, ~:-: Figure 1. Relative inhibition of squalene synthetase by farnesyl
`"t°"> · P.Ytophosphate analogues.· Tha incorporation of [l-3H)farnesyl
`-~('.. :·. :· . pyrophosphate (2.5 µM) into squalene in the presence of 2, 4, and
`f~)jk;;./6:µM c_oncentrations of hihibitor is plotted as a p_ercent uf.t.he
`:tn:ti'L\.iricorporotion ol?served_ \vith no inryibitor present. ·Inhibitor: 2
`!'(e); 4 (.;.), 3 (•), 7 (0), 5 (6), 6, (0), 11 (<>), IO (ul), 9 (Y); 12 fO},
`)arid s (~).
`
`~·
`
`.
`
`.
`
`::·:verified. The 2E and 2Z.isomers of 13 were distingtiished
`: ... ,
`t&(\i~\:it{1:\by corr~lat.ing t~e NMR s~ifts o_f the C-2. and C-3 me~yl
`~;:Mg:J~groups m each 1s_omer with those. of the correspondmg
`im;;;:;:;:;''~~thyls in _methyl 2,a:dinie.Uiyl~Z-butenoate,22 hi the 2E
`qr:;;..'. ~omer, the 2-methyl 1s at-.1.87 ppm and the 3-methyl at
`lfaiff.\:: ~Oo ppm, while the corresponding pOOks in 'th~ 2Z isomer
`NH : . ~methyl and the ttans-3-niethyl appear at 1.81 ppm~ while
`.Wtt'h .:'the cis-3-methyl _is at 2.00 .ppm.7. Unambiguous identi(cid:173)
`i@t~> fication of the stereoisomers·of 4 alcohol, however,.could
`%K:\::·: riot be-made by NMR correlation with model structures.23
`J.'..):'_:_J_i_::j_· •. :·.!f:h.is difficulty was Qv~rcom.e by NMR studies-using.· the
`A!(:t , shift reagent Eu(fod)a. 24 A plot was made of the shift of
`E\i': the 3-ethyl protons in each isomer as a function of in(cid:173)
`ff,';iK·:_. 'creasillg shift reagent to «o~pound ratio. A large dif(cid:173)
`f:rnffit:Y :rE!renee_was ~bsei:ved in t~e shifts of the two'isomers. At
`f-,_:•:}_%_,_;,_; :•· -~.reagent to analo~e ra~10 of o.s_. for e~phi,-the ethyl
`:WA-· methylene group m,one 1sc;imer had shift~ by 0.42 ppm
`I ~?~r:==:;r~~$'!;S;~~
`''\''":·
`.and in· the other by 0.75 ppm: The isomer in which the
`111~;:i~··· .;b8; s~:~'f :::~rb~f ~~s~~~~7!:~~~~~e1;~~~~~~~l~~
`
`IWWJ-> :are at 1.81 and 1.79 ppm. In the model compound the
`
`1"
`
`~4\: . ·.E!thyl 2(E)-famesoate was shown by GC to be a major
`\h( product, while the other isomer was absent.
`t'•::·
`· Biological Results. The relative itlhibition ofsqualene
`:':it:
`synthetase by analogues 2-12 _has been measured, while
`'$:':: .. kinetic and inhibitor-enzyme preincubation.studies have
`)1}·. ·=~:~t~ ;~~~~~~~i~~=:~;~:=:~i::~:
`,~~[)~;-: was employed. in this work, since the enzyme from this
`source ·is contaminated with much lower levels of inter-
`hi:>
`· fering·phosphataSes. 26
`3;•<= ··
`·
`
`I ;~ffiz~.~~~:lf~i~~~r~;~~
`
`Ft;~~:ut:~ :i~~r::~~~c~~l~~!~~~W~~~~:~ie:::~:~~i:i
`
`l~]K
`1r:::,;_.
`
`,,
`
`lO et al.
`
`·--
`
`1tanoate
`sis, and
`alcohol)
`iyl .. est.er
`(16) and
`:"yasthe
`:rivative
`;lthio-2-
`
`nalogue
`were es(cid:173)
`ins~ce
`)was an
`mixture
`iical as-'
`famesol
`:las been
`rable l).
`'e higher
`·or more
`imentof
`:by their
`tndently
`
`3 of 7
`
`PENN EX. 2178
`CFAD V. UPENN
`IPR2015-01835
`
`

`
`.. ·
`.
`.
`.
`. 246 Journai of Medicinal Chemistry, 1977, Vol. 20, No. 2
`
`.
`
`80
`
`60
`
`?:. 40
`
`Ortiz de Mon.tcllano·et al.
`
`··~ •
`
`60
`
`z
`0
`i= 40
`iii
`ff
`~
`~ 20
`
`·.
`
`-0.4 -0.2 0 .Q.2 0.4 0.6 0.8
`1/[s] ~M- 1
`. Figure 3. Line\Vel!ver-Burk graphic analysis of the inhibitlnn
`of squalene s)'nthet_alle by 3-dernethylfarnesyl pyrophosphate (3) .
`. . ·1sr~·the concentration of I 1-3Hlf11rnesyl pyrophosphate; initial
`· .velocity (V) is.expressed in nanomoles of squalene formed· per
`·.-_·minute per milligrllJll of prot~in. Concentratic>n. of inhibit(lr (3):
`. 0 (e), 2 (:.}°, 4 (0), and 6 µM {4). ·
`·
`.
`. ·
`.. deci:~~se in inhibition with tiine. can be ~xpl~i~~d by slow
`::en~yfoit-.and chemical degradation of. the mhibitor ..
`...... ' ... · : .. ,..
`. .·
`.. ·.
`::~ri~1'u~ions. · . ·
`·.. · ·•
`·. .
`· .. ·..
`·
`. .
`... ·. Tlie: bmdfng (If farnesyl pyrophosphate and ita anQJogUes
`< t,O:·squ~l~ne syi:ith~tase is strongly dependent 011 the PY~
`·rophosphate moiety and, to ii.lesser extent, on relatively
`.: nonspec;ific; J.ipophilic intera~tions of ·the ·hydrocarbon
`· ··chain .. This conclusion derives from the impotence ·of free
`.·,
`· illCohol and monciphosphate analogues ·as inhibitors,
`;ii, ·
`.. co~r)le~ w i . ·.·~~ .the .!1ppreciable lnhi~ition .. exhibited by .
`~~!
`i~ . : . sti~ctures with flFl)'. gross alterations m the carbon
`~~: · .. , ·fr8Jllework. Substitution !:It C-2, C-3, and C-4 (anal?gues
`J~I .:
`. 2;4;·and 7), fonxample, ts wel.I toler,11ted. Saturation of
`: the double.bonds {9, 12) jllld shortening of tJie,h)'drocarbon
`:1~ ... '
`i!~t . . "chain. (8), on the other hand, significantly i:eduee bindi~g.
`1ilt
`·.i!1cr~asin~.the chain:len.~h app~rs to enh:an~e~iriding,
`::f~l.. . : smce JO· 1s a :better 1.nh1.~1tor than .9, .patt1culady when
`i1~r . · · · iill<?Wance ismade for the fact ~t the data ';In 10 are based
`%'ll:, . : OD·ft Iiiiiture rontaining 30% of the presUJIUlblY less 1;1ctive
`j~j .: >: ·., 2Z iSOmer~ "The pairing of.strong.pyrophosphate biri«ling
`)iK ·. . · With rel8tively nonspedfic attachment of the hydrocarbon
`chain is reminiscent of the forces involved in: binding. of
`'}'i:,1 •.
`u~; ..
`··. gernnyf ·py:rophosphate to prenyl transferase29 and of
`dli(: .·
`p:eny~ p~rophoaphates to the polypeptide antibiotic ba-
`. ci!;racm;
`i;Hl..
`mt· . . . The competitiv~ ~ature o~ the i.nhibition indicates that
`the structure-act1v1ty relationsh1ps·ohserved reflect the
`:•::.•;•;;,~; .·.
`f[if:;if; · · selectivity of the active site. A sharp contrast exists,
`however,. between the relatively ·loose requirements for
`<+=~¥1 .
`, . ~/{;i(l .. ·
`binding and the catalytic demands of the enzyme, We
`· : have sul>SequenUy round that 2 and 3 are only cosubstrates
`<-
`;;'.i!;Yi~(
`. ;;
`:nu:til :
`11 while 4, 7, and 9 are not acceptable as
`for the enzyme, 10
`•
`, \ i:L;JL
`.substrates at all.31 These results exclude all.the $alogues
`'' NXWi
`. in this paper, except.pos8ibly 5, as first substrates for the
`: ;f YFB
`enzyme. Th~ lack of irteversible inhibition is thus ra-
`'
`.· tionalized, since formation of an enzyme-substrate co-
`);{;@~.
`: i xr ;ii!. .
`e TUm . . acceptable iis a. first substrate. The seiirch for suitable
`valent cqmplex requires that an analogue be catalytically
`
`. ·Geraniol and phytol were purchas~ from Aldrich, NADPH
`from Sigma, bacterial alkaline phosphatase from Worthington,
`
`0
`
`4
`3
`2
`1
`PRElt-.ICUBATION. {Hr)
`Figure 4; Effect of incubating inhibitors (4 µM) with aqualene
`synthetase prior.ro addition of [l-3H]famesyl pyrophosphate (5
`µM). and normal bioassay. Preincubation was .at ambient
`temperature with the full assay system, excluding the substrate.
`Inhibitor: 3 (•), 5 (.6), 6 (O), 7 {O), and 11 (()).
`
`s
`
`'Thble IL Classification of fohibitors
`2, competitive 6, competitive
`7. competitive
`3, mixed
`.. 4, competjtive 8, (competitive)b
`·5; competitive 9, ~mpetitivea
`·a Thefo1es in the Linew~ver,.;Burk plot curved upward
`at n!ue1!of l/(S]greater ihiµi·o:a ~M"' . .. b .The lines.qua
`t.O this weak Inhibitor.did.riot differ suffiCiently in slope
`for imambigi1ous .allsignmen.t;
`
`10, competitive
`· 11, mixed
`12, competitive
`
`· s~:w:=·: ·
`
`· m~t ..
`
`and cake baker's yeast from a·loeal bakery. Eastman (No .. 1381)
`or.Analtech silica gel preooated plat.es were used for TLC analysis,
`·while Merck silica, gel 60 (70-2.30 mesh) was used for column
`chromaiography after qeactivation with IQ%· water. A Varian
`Moder 2100 GC instrument ·with flame ionization detectors,
`nitrogen carrier gas (18 ml/min). and .6 ft x 2 ·mm i.d. giass
`colum11s packed.with 3% OV-225 oli 100-120 mesh Vai'oport 30
`was. used for all aiial)rtical gas chromatography. Infrared spectl'a
`were obtained as thin mms on"a Perkin-Elmer Model 337, while
`a Karl Zeiss PMQU sp·ectrophotometer was \1sed to measure
`optiCa.l densities. NMR spectra were taken on· a Varian A-60 as
`approximately 25% vfv°deut.erfochloroform solutions. Shifts are
`reported iri parts per.million downfi~ld from Me~Si. The NMR
`sWt reagent Eu(fotih W.as ~btairied from Willow:~~k I.;abo(cid:173)
`ratori.es .. ~ spec~a were .. measured on an AEI ·MS-~ adap~
`to· a ch~m.ical ionization.mode (isobui.ane gas). Curve fitting was
`accomplished by ..Ifoear regression, analysis; using either. a
`· Hewiett~Packard Model9100 cali:Ulalor (program no. 7o8oaf or
`the PROPHE'(system sponsor(ld by NIH (or .chemical-biological
`lnfom:iation handiing. Micrbanalylienvere performed by ·the
`Berkeley 'Microanalytical Laboratory.
`·
`Preparation of Enzyme.9 Baker's yeast (200 g) was silspended
`in ZOO ml of 100 '.mM potassium phosphate buffer (pH" 7.0)
`wntaining 5· rnM · MgCl2, 1 mM sodiµm EDTA, and 10 mM 2-
`mercaptoethanol. The S\JSpension was srinica!ed Jn 100-ml aliquots
`with a Branson \V-i85D sonifier (90-W output). Each aliquot
`was sonicated· with ice c:Ooling for two 5-mm periods: The.crude
`mixture was centrifuged at 0· 0c and 7o0og for ·10 min. The
`supernatant waK centrifuged at 73000g for 45 inin. The·fraction
`of-I.he resulting high~speed supernatat)t which precipitated be(cid:173)
`tween 3o and 553 of ammonium sulfate saturation WaS lli turn
`i&o!ated by eeptri(ugutiori at 12000g for ·10 min. The pellet.
`resuspended in i5 ml of lOQ.inM potassium phQ!!phate buffer (pH
`7.0) containing 20 mM 2-mercaptoethanol, was dialyzed against
`'l'he ·resulti~g enzyme
`.1100 ml of the. saIJ!e buffer mixture.
`preparatiOn .was divided into small aliquots which were se~tely
`stored in ~ freezer until individually used. PrQtein concentrations
`were assl,\yed by the pro.ced.ure of Sutherland.32
`·
`lnhibiticin BiOassli.y. The incorparatiOn of [1·9H]farnesyl
`· pyropho5phate into. squalene in the presence ·or inhibitors was
`assayed in standard I-ml incubatiOn mixttires, each·containing
`(ooncentration units): [1-3H]farnesyl pyrophosphate (6. mCi/
`.mmol), 1-10 µM; MgC12> IO mM; NADPH, 1.6 mM; NR40H, 0.8
`mM (due to storage of.pyrophosphates in this medium); protein,
`
`1,:.
`
`.1:-. a I
`
`i1'
`1:·
`I':
`
`~i
`~·:
`-i:
`l
`1:
`F
`t
`~: {:O.
`
`4 of 7
`
`PENN EX. 2178
`CFAD V. UPENN
`IPR2015-01835
`
`

`
`,:.
`C·et al.
`
`~ualene
`•hate (6
`mbient
`bstrate.
`
`;ive
`
`;ive
`
`ward
`1 due
`ope
`
`) .. 1381)
`nalysis,
`ooluinn
`:varian
`tectors,
`:!. glass
`:port30
`spectra
`7;while
`leilli'Ure
`
`A•60 as
`1ifts ·are
`aNMR
`[Labo(cid:173)
`ldapted
`ing.wiis
`ither a
`•Soo) or
`>logical
`··by the
`
`.pended
`IH 7.0)
`'mM 2-
`iliquots
`:aliquot
`ecrude
`~- The
`'raction
`t.ed"be(cid:173)
`·in tum
`·pellet,
`fer·(p}:I
`against
`mz.yme,
`18I'8tely ·
`trations
`
`arnesyl
`ors was
`fuiriing
`s mCi/
`~H,0.8
`>rotein,
`
`.:££.~rnesyl Pyrophosphate Aoologues
`=:·:··::·····
`;!(b';34 mg/ml; and potassium phosphate buffer (pH 7.5), 50 mM.
`.::&the:inhibitor concentration varied between 0 and 6 µM. Each
`~F~~ndard incubation was initiated by addition of the enzyme to
`i~):''if°niixt.ure of the other components prewarmed to 37 °C. After
`. ,,,,,,~,,,,,_/Hfrilin at 31 °c in a reciprocating bat'h, th~ incubation waa
`-;e~~~!W')~rminated by a?dition of ethano.l (2 ml). The a1:1ueous mixture
`
`it!!fo);;.;:was extracted with two 1.5-ml and two 3.0-ml aliquots of 3o-60
`
`"!.~.;)l·:,~.i ... '~./.;j.'~.:~.\.~.'~ .. petroleum ether, each aliquot being adde~ dire~?' to a
`~ii~f)) disposable 0.6 x 5 cm column of 10% water deactivated silica gel.
`fh;{.~\/f.l1e minicolumn effluent was directly collected in a scintillation
`. '!i:l~t~i-~,#~~J!~ ~~'C1!::f~:ii;.~~~W~~iJ~~i:fu~9!i:=:!:~
`WmW\/after addition of New England Nuclear Aquasol (10 ml). Control
`W@WF e~P.erimenta demonstrated that squa!ene was cleanly etuled under
`· ifii'i'::NMthese conditions, whereas polar products like farnesol remained
`ll~rt1:·t:;'.1t;:~et~~1~::t:~~~~:tb::;:~r~~~t)~\t'hS: ~~=~.:!~!
`
`. !#~:hi?iireparation; equivalent data pomta were essentially superim·
`· !fafa(~pa"oiable, r11rely differing by more than 5%.
`. @Wf:J~'(;.Inhibitor Preincubations. The complete.standard incubation
`):;\~ix.tore, excluding the substrate but including the enzyme, was
`~\{,=focubated with 4 µM concentrations of inhibitor for 2.5 or 5 h
`;M#i;'\ ilL ambient temperature: Control incubations. were .treated
`~fr'.%'='?idimtically, except no inhibitor w.as added. The mixtures were
`ll1:En;·;i>~ewarmed to 37 °c.at ih.e end of the.preinc?bation period.
`h#V:::'[l-3H]farnesyl pyrophosphate (5 µM-concentration) was added,
`l/%iS:\iind the.assay was comple4id as deacribed.
`fi@'f/'.''· Synthesis of Pyrophosphates.12 Di(triethylammonium)
`~,..,. · ·}j)Jipsphate33 (450 J'!lg, 1.5 lllJ!lOl) in 30 ml of dry CH3CN was added
`i,9v.er 4 h at ambient temperature lo a stirred .solu~i()n of the
`,
`t::n)\farnesol anillogue (o.5 mmol) and trichloroacetonitrile (650 mg,
`~!\Wtnt4-~ "inm()I) in 5 ml of CH3CN. The mixture was stirred 24 h and
`[N'j}·)~~ncentrated on a ro~y evaporato~.. The yellow residue was
`hi&>·:·:transferred to.:a centrifuge tube w1.th .10 ml of acetone and
`t¥W~'./i:conce~trated N~OH (0.S-1 ml) was added. The precipitated
`'iM%t'·a,iiimonium salts, isolated by i:e.ntrifugation, were washed· twice
`NP,{fby.resuspension in 5-ml aliquots·of ac"etone .containijig 0.01 N
`%Wi:,':': N.H40H .. Purification was accomplished by one of the following.
`1M/:::,-:
`·Procedure A. A solution of the resulting white solid. in 5 ml
`g%f ;: of 0.01 N NH40H was added to a stirred slurry of 100 g of
`@%N·."'prewashed Amberlite XAD-2 resin33 inlOO ml of0.01 N NH~OH.
`:i\Hi:'.J The-resin, collected on a sintered funn~l after stirring overnight,
`:itlC.: was washed with the same solvent (5 x 60 ml). The organic
`%1ib) phosphates were eluted from the resin with methanol containing
`Wif:::· ~ few drops of NH.OH (5 X 60 ml). Solvent removal (rotary
`\f,J): :.evaporator below 50 °C) gave a solid which was transferred lo
`Wm' ... a centrifuge tube with 2-3 ml of methanol, The pyrophosphates,
`......
`·contaminated with traces of moilophoophat.es, were precipitated
`·by addition of 4·-8 ml of acetone to which.a few drops of NH 40H
`:.had been· added. Repetition of the precipitation sequence gave
`. essentially pure pyrophosphates (hy TLC). These were dried
`under high vacuum.
`.
`Procedure B.34 The crude product obtained from 150 mg of
`starting ·alcohol was dissolved in a minimal amount of 1·
`·propanol-ammonia-water (9:4:1)- and was added t.o a 2.5 x 18 cm
`column containing 40 g of silica gel. The silica gel·had been
`preconditioned by stirring in 300 ml of 1:1 concentrated HCl-water
`for 2 h, standing overnight, decantation or the aqueous layer,
`washing witl1 water (6 X 300 ml), and drying at 150 °C for 48 h.
`The column was prewashed and eluted with a 9:4:1 1-propa-
`
`, .. ;.
`
`to 2 ml on a rotary.evaporator (below 25 °C), lyophylized, and
`
`by TLC on silica gel plates using l-propanol-NH40H-H20 (6:3:1)
`
`'f~tt·· ~r~~J!~0:0~=:~~r~~:=~-i: C:~12; :~t~i:i:=
`.. '.·,:.t:1.~.t.~.·.:.,:.·,·.· !!~2~i!~~ :;~~1::::i~~rr:~~:;s-~~;:~:r~~°!n:~tr~~
`'.'..','=l,~·'·f.~_-_:·'.·.i.:· :~:;e:~~~~~~~~?:~o~Yo~i~fy;=t~~~~~~~
`
`.. :',.~-.;'·.·;,_:,:i.,·1 .. l.::•_.:::·····:.·,·· .. ~~~t~;~~;~;~;;
`
`as described by Goodman and Popjak.14 The recovered alcohols
`
`·:.::_:
`
`. :
`;:*;).,
`:@)
`
`Journal of Medicina.l Chemistry, 1977, Vol. 20, No. 2 241
`were compared With authentic samples by TLC and GC. In each
`case the recovered alcohol was. e5sentially- identical with the
`corresponding starting material
`.
`.
`·
`.
`.
`Ethyl 2,3,7,11·Tetramethyl-2(E),6(E),10-dodecatrienoale
`(13). Sod.ium hydride (3.62 g of 57% oil suspension, S!) mmol),
`washed with petroleum ether, was stirred in 400 ml of dry THF
`(nitrogen atmosphere). Diethyl 1-<'arboethoxyethyl phosphonate19
`(20. 78 g, 87 mmol) in l 00 ml of THF was added at 0 °C over 30
`min: The mixture was stirred at 30-40 °C ~til hydrogen ev(cid:173)
`olution .ceased (1 h). After cooling to 0. °C, 16. 7 g (86 mmol) of
`. geranylacetone in 100 ml THF was added and the mix