MOLECULAR
`
`PHARMACOLOGY,
`
`14,
`
`290-298
`
`Structure-Activity
`
`of Anthracyclines
`Relationships
`on Macromolecular
`Syntheses
`
`Relative
`
`to Effects
`
`S.
`
`T.
`
`CROOKE,’
`
`V. H.
`
`DUVERNAY,2
`
`L. GALVAN,
`
`AND
`
`A. W.
`
`PRESTAYKO
`
`Department
`
`of Pharmacology.
`
`Baylor
`
`College
`
`of Medicine.
`
`Houston,
`
`Texas
`
`77030,
`
`and
`
`Bristol
`
`Laboratories.
`
`Syracuse.
`
`New
`
`York
`
`13201
`
`(Received
`(Accepted
`
`June
`October
`
`29, 1977)
`19,
`1977)
`
`SUMMARY
`
`S.
`
`CROOKE,
`Structure-activity
`ular
`syntheses.
`
`T.,
`
`DUVERNAY,
`relationships
`Mol.
`Pharmacol.,
`
`V.
`
`H.,
`GALVAN,
`of anthracyclines
`14,
`290-298.
`
`& PRESTAYKO,
`L.
`relative
`to
`effects
`
`(1978)
`A. W.
`on macromolec-
`
`may
`
`two
`
`the
`
`basis
`
`of
`
`their
`
`at
`
`on
`classes
`into
`divided
`adriamycin,
`I anthracyclines
`carminomycin,
`-
`syntheses
`nucleolar
`RNA
`cell
`RNA,
`and
`Class
`anthracyclines
`aclacinomycin,
`-
`whole
`cellular
`RNA
`synthesis
`at
`inhibit
`to
`inhibit
`synthesis,
`required
`lower
`concentrations
`necessary
`relationship
`studies
`the
`RNA
`synthesis-inhibitory
`studied
`had
`demonstrable
`
`effects
`and
`approxi-
`marcello-
`lower
`6-7-fold
`RNA
`nucleolar
`synthe-
`DNA
`or
`trisacchar-
`None
`preribosomal
`
`of
`
`and
`inhibit
`to
`of di-
`presence
`on
`anthracyclines.
`processing
`of
`
`II
`
`suggest
`
`DNA
`than
`
`that
`specificity
`effects
`
`on
`
`be
`studied
`anthracyclines
`The
`Class
`syntheses.
`RNA
`on DNA
`and
`whole
`DNA,
`inhibit
`pyrromycin
`-
`concentrations.
`comparable
`mately
`and
`musettamycin
`mycin,
`concentrations
`than
`at
`synthesis
`170-1250-fold
`sis.
`Structure-activity
`ides
`confers
`nucleolar
`the
`anthracyclines
`RNA.
`
`those
`
`-
`
`INTRODUCTION
`
`to
`
`its
`
`at
`
`in
`
`variability
`Although
`and
`cell
`type
`ferences
`most
`noted
`(10),
`has
`been
`have
`exceptions,
`with
`two
`synthesis
`bulk
`RNA
`to
`inhibit
`equivalent
`approximately
`trations
`concentrations
`than
`the
`greater
`synthesis
`DNA
`to
`inhibit
`aclacinomycin
`rubin
`and
`inhibit
`RNA
`ported
`to
`10-fold
`less
`trations
`DNA
`synthesis
`inhibit
`relationships
`ture-activity
`differences
`the
`count
`for
`and
`aclacinomycin
`and
`bin
`been
`not
`have
`thracyclines
`daunomycin,
`Adriamycin,
`been
`reported
`mycin
`have
`cleolar
`preribosomal
`RNA
`5-fold
`lower
`concentrations
`to
`inhibit
`whole
`cell
`RNA
`
`from
`resulting
`conditions
`culture
`anthracyclines,
`been
`reported
`concen-
`to
`necessary
`11).
`Cine-
`(6-9,
`been
`re-
`A have
`concen-
`at
`synthesis
`required
`than
`those
`The
`(12,
`13).
`may
`that
`cineru-
`between
`other
`an-
`the
`fully.
`explored
`carmino-
`and
`to
`inhibit
`synthesis
`than
`or DNA
`
`dif-
`
`or
`
`to
`
`struc-
`ac-
`
`nu-
`2-
`
`at
`required
`synthe-
`
`These
`CA-10893
`by
`a grant
`the results
`
`studies
`from
`from
`was
`
`supported
`were
`National
`the
`Bristol
`presented
`
`tion for Cancer
`‘To
`whom
`dressed,
`at
`2 Predoctoral
`tutional
`Funds.
`
`Bristol
`
`Research,
`requests
`Laboratories.
`trainee
`supported
`
`by
`part
`Institute
`Cancer
`A portion
`Laboratories.
`to the American
`Associa-
`Denver,
`May
`for reprints
`
`in
`
`Grant
`and
`of
`
`1977.
`should
`
`be
`
`ad-
`
`by
`
`Baylor
`
`Insti-
`
`290
`
`0026-895X/78/0142-0290$02.00/0
`© 1978
`Copyright
`by Academic
`All
`rights
`of
`reproduction
`in any
`
`Press,
`form
`
`Inc.
`reserved.
`
`Anthracyclines
`intercalate
`in
`native
`alterations
`sensitivity
`and
`altered
`sure
`of
`intact
`been
`reported
`DNA
`and
`minimal
`(6-9).
`
`RNA
`inhibition
`
`been
`adjacent
`between
`resulting
`DNA,
`secondary
`its
`in
`degradation
`to
`template
`cells
`to
`
`have
`
`to
`result
`syntheses,
`of
`
`protein
`
`reported
`nucleotides
`significant
`in
`structure,
`by
`nucleases,
`(1-5).
`Expo-
`activity
`anthracyclines
`has
`in
`inhibition
`of
`only
`but
`in
`synthesis
`
`Ex. 1041-0001
`
`

`
`STUDIES
`
`ON
`
`THE
`
`MECHANISM
`
`OF
`
`ACTION
`
`OF
`
`ANTHRACYCLINES
`
`ascites
`obtained
`as
`
`cells
`
`used
`from
`previously
`
`291
`
`as
`rats
`de-
`
`hepatoma
`Novikoff
`cells
`were
`“carrier”
`ascites
`cells
`bearing
`(17).
`scribed
`whole
`on
`Effects
`were
`. Cells
`syntheses
`x
`8
`of
`approximately
`centrifugation
`by
`harvested
`at
`10 mm
`room
`temperature,
`in
`pended
`fresh
`RPMI-1640
`density
`cell
`was
`adjusted
`and
`10(cid:1)/ml,
`the
`cells
`were
`37#{176}shaking
`water
`30-60
`tion
`for
`mm
`5-mi
`taming
`aliquots
`0.05
`Then
`of
`added,
`was
`drugs
`with
`of
`[3Hjuridine,
`to a final
`The
`cells
`with
`gentle
`The
`total
`was
`cells
`were
`aliquots
`counting
`cellular
`pension.
`collecting
`by
`determined
`filters
`fiber
`glass
`of
`cells
`radioactivity
`the
`determining
`they
`had
`after
`filters
`on
`the
`1-mi
`aliquots
`with
`times
`three
`M NaC1,
`0.005
`(0.14
`medium
`TCA3-precipitable
`M MgCl2).
`0.008
`determined
`by
`was
`activity
`suspension
`cell
`ml
`of
`which
`after
`filter,
`with
`times
`three
`with
`twice
`then
`radioactivity
`The
`then
`determined.
`was
`total
`radioactivity
`the
`radioactivity
`that
`macromolecules
`each
`sample.
`in
`triplicate.
`The
`radioactivity
`in
`10 ml
`ple was
`determined
`toluene,
`lation
`fluor
`containing
`monoethyl
`ane,
`ethylene
`glycol
`naphthalene,
`2,5-diphenyloxazole,
`1 ,4-bis[2-(4-methyl-5-phenyloxazolyl)]ben-
`
`The
`
`effects
`nucleolar
`reported.
`
`of
`
`on
`
`and
`synthe-
`
`cinerubin
`RNA
`Moreover,
`the
`processing
`RNA
`have
`
`the
`
`not
`
`14).
`(9,
`sis
`on
`aclacinomycin
`been
`not
`have
`siS
`anthracyclines
`of
`effects
`preribosomal
`of
`nucleolar
`studied
`(12).
`been
`fully
`several
`new
`Since
`similarities
`structural
`have
`and
`cinerubin
`the
`and
`purified,
`were
`publication
`the
`concentrations
`required
`dines
`protein
`syntheses,
`concentrations
`dines
`necessary
`synthesis
`relative
`(c)
`to
`determine
`thracyclines
`essing,
`activity
`anthracyclines
`synthesis
`lower
`synthesis.
`
`to
`
`of
`to
`
`at
`
`than
`
`anthracyclines
`to
`aclacinomycin
`recently
`been
`isolated
`studies
`presented
`in
`this
`designed
`(a)
`to
`determine
`of
`new
`anthracy-
`inhibit
`DNA,
`RNA,
`(b)
`determine
`to
`the
`newer
`anthracy-
`inhibit
`nucleolar
`RNA
`to
`that
`of
`adriamycin,
`an-
`effects
`of
`several
`the
`proc-
`preribosomal
`RNA
`on
`elucidate
`structure-
`to
`(d)
`and
`that
`result
`relationships
`inhibit
`that
`concentrations
`those
`required
`
`with
`
`and
`the
`
`the
`
`the
`may
`nucleolar
`significantly
`inhibit
`
`to
`
`in
`
`RNA
`
`DNA
`
`MATERIALS
`
`AND
`
`METHODS
`
`and
`
`a
`
`on
`
`(Adria
`Adriamycin
`isotopes.
`Drugs
`was
`Del.)
`Wilmington,
`Laboratories,
`Carmino-
`Lane.
`from
`Dr. M.
`gift
`generous
`musettamycin,
`marcellomycin,
`mycin,
`provided
`by
`Bristol
`pyrromycin
`were
`and
`Aclacinomycin
`was
`Laboratories
`16).
`(15,
`provided
`Sanraku-Ocean
`by
`Ltd.,
`Japan.
`Fujisawa,
`95%
`shown
`least
`to
`be
`pressure
`chromatography.
`liquid
`uridine,
`and
`thymidine,
`tained
`from
`Schwarz/Mann.
`32P1 was
`obtained
`from
`Novikoff
`hepatoma
`toma
`ascites
`cells
`tained
`in
`monolayer
`sealed
`glass
`prescription
`well
`Park
`Memorial
`medium
`supplemented
`100
`serum,
`2 m(cid:1)i
`glutamine,
`of
`penicillin,
`and
`100
`(cid:1)ig/ml
`A.
`For
`studies,
`Novikoff
`drug
`ascites
`were
`grown
`in
`shaker
`cells
`(New
`Brunswick
`Gyrorotary
`shaking
`at
`37#{176}in Gibco-type
`500-ml
`cubator)
`media
`bottles
`in RPMI-1640
`medium
`0.2%
`methylcellulose.
`
`at
`
`type
`
`All
`
`pure
`
`valine
`
`Union
`cells.
`N,S,-73,
`cultures
`bottles
`Institute
`with
`
`Company,
`drugs
`were
`high-
`by
`Tritiated
`were
`Carrier-free
`Carbide.
`Novikoff
`were
`at
`with
`type
`10% fetal
`units/ml
`streptomycin
`hepatoma
`culture
`
`ob-
`
`hepa-
`main-
`37#{176}in
`Ros-
`1640
`calf
`of
`
`in-
`liquid
`with
`
`macromolecular
`cell
`grown
`to
`a
`density
`10-1
`.0
`x
`10’/ml,
`at
`800
`x g
`for
`and
`resus-
`medium.
`The
`6
`x
`x
`to
`incubated
`a
`gentle
`with
`vessels
`closed
`suspension.
`of
`cell
`drug
`solution
`aqueous
`were
`incubated
`cells
`the
`followed
`addition
`15 mm,
`by
`13H]thymidine,
`f3H]valine
`or
`concentration
`10’
`dpm/ml.
`of 5 x
`were
`then
`for
`2
`incubated
`agitation
`on
`ice.
`and
`chilled
`the
`radioactivity
`which
`with
`by
`incubated
`determined
`sus-
`0.05-mi
`of
`the
`was
`Total
`radioactivity
`aliquots
`0.5-mi
`E)
`and
`(type
`remaining
`been
`washed
`of NKM
`M KC1,
`and
`radio-
`1.0
`filtering
`fiber
`a glass
`through
`washed
`was
`filter
`the
`medium,
`of NKM
`1 ml
`solution.
`of 5% TCA
`1.0 ml
`the
`filter
`remaining
`on
`The
`percentage
`and
`of
`the
`incorporated
`determined
`were
`performed
`
`bath
`in
`
`ml
`and
`for
`
`was
`then
`was
`assays
`
`All
`
`present
`using
`
`10(cid:1)-8
`
`in
`agita-
`con-
`
`hr
`
`cell
`
`of
`
`cellular
`into
`for
`
`each
`of
`
`sam-
`scintil-
`1,4-diox-
`ether,
`and
`
`The
`
`abbreviations
`
`used
`
`is: TCA,
`
`trichloracetic
`
`acid.
`
`Ex. 1041-0002
`
`

`
`003H’Uridine
`
`.
`
`.
`
`(cid:1) (cid:1)
`
`,,(cid:1)-
`
`CROOKE
`
`ET
`
`AL.
`
`98
`
`90
`
`60(cid:1)
`
`3.0
`
`3.5
`
`4
`
`0
`
`O-O
`
`3H.ThYmidIne/
`
`292
`
`(cid:1)#{241}
`
`(cid:1)(cid:1)
`
`3.0
`
`3.5
`
`4
`
`4.5
`
`5.0
`
`id1!
`
`0.1
`
`1A
`
`ADRIAMYCIN
`
`(cid:1)3H.ThYm)diJ,,,,,i,(cid:1)Z7(cid:1)(cid:1)
`
`30
`
`35
`
`4.0
`
`4.5
`
`5.0
`
`5.5
`
`0
`
`;5.0
`
`(cid:1) (cid:1)
`
`j”
`
`lB
`
`CARMINOMYCIN
`
`1.0
`
`10.0
`CONCENTRATION
`
`100
`
`)IJM)
`
`I
`10.0
`CONCENTRATION
`
`100
`
`uM)
`
`95
`
`90
`
`80
`
`70
`60
`50
`40
`30
`
`2
`
`=
`
`o
`
`98
`
`95
`
`90
`
`80
`
`70
`60
`50
`40
`30
`20
`
`10
`
`2
`
`=
`
`‘C
`
`98
`
`95
`
`90
`
`80
`70
`60=
`50(cid:1)
`40(cid:1)
`30(cid:1)
`
`20
`
`z
`0
`
`6.0
`
`65
`
`0
`
`1
`
`1.0
`
`10.0
`
`‘C
`
`20
`
`10
`
`100
`
`1C
`
`PYRROMYCIN
`
`CONCENTRATION
`
`UM)
`
`1D
`
`0. 1
`
`MARCELLOMYCIN
`
`1.0
`
`10.0
`CONCENTRATION
`
`100
`
`)IJM)
`
`3. 0
`
`3. 5
`
`4. 0
`
`4. 5
`
`50
`
`0
`
`5.5
`
`6. 0
`
`6. 5
`
`7.0
`
`1E
`
`0.1
`
`98
`
`95
`
`90
`
`85
`
`80
`
`70
`
`60
`
`50
`
`40
`
`30
`
`20
`15
`
`10
`
`0
`
`z
`
`1.0
`
`10.0
`
`100
`
`MUSETTAMYC
`
`IN CONCENTRATION
`
`1pM)
`
`FIG.
`effects
`of
`analysis
`1. Probit
`precursors
`incorporation
`of labeled
`and
`experiments
`Cells
`were
`prepared
`calculated
`percentage
`of cellular
`
`as
`
`Adriamycin.
`
`B. Carminomycin.
`
`anthracyclines
`of
`into acid-insoluble
`performed
`radioactivity
`C. Pyrromycin.
`
`on DNA
`material
`indicated
`incorporated
`D. Marcellomycin.
`
`as
`
`and
`
`RNA
`
`syntheses
`
`determined
`
`by measuring
`
`in MATERIALS
`AND
`into
`TCA-insoluble
`E. Musettamycin.
`
`METHODS.
`
`data
`
`The
`radioactivity.
`F. Aclacinomycin.
`
`10
`
`were
`A.
`
`Ex. 1041-0003
`
`(cid:1)
`(cid:1)
`(cid:1)
`(cid:1)
`(cid:1)
`(cid:1)
`(cid:1)
`(cid:1)
`(cid:1)
`(cid:1)
`(cid:1)
`(cid:1)
`

`
`STUDIES
`
`ON
`
`THE
`
`MECHANISM
`
`OF
`
`ACTION
`
`OF
`
`ANTHRACYCLINES
`
`or
`
`a Beckman
`in
`The
`gradients
`fuge.
`1-mi
`fractions,
`0.6-
`was
`at
`254
`nm
`profile
`density
`gradient
`ISCO
`radioactivity
`0. 1-mi
`crose
`gradient
`fractions
`as
`described
`above.
`
`model
`
`ultracentri-
`L2-65B
`were
`fractionated
`and
`the
`absorbance
`recorded
`with
`fractionator.
`aliquots
`was
`
`the
`of
`determined
`
`in
`
`293
`
`into
`
`an
`The
`su-
`
`98.
`
`95
`
`90
`
`80
`
`700
`60
`50
`40
`30
`20
`
`10
`
`=
`
`0
`
`iF
`
`in
`
`polyethylene
`Tri-Carb
`
`vials
`model
`
`and
`3385
`
`, and
`ascites
`in RPMI-
`hr
`mCi/ml
`40-60
`
`of
`
`mCi!
`cen-
`at
`
`by
`mm
`
`for
`
`with
`(specific
`were
`850
`
`Cells
`
`basic
`
`and
`to
`
`a
`
`added
`later,
`
`of
`
`a
`
`RESULTS
`
`on
`
`the
`
`the
`
`macromolecu-
`cellular
`u’hole
`Effects
`probit
`shows
`a
`1
`.
`Figure
`syntheses
`lar
`anthra-
`various
`of
`effects
`the
`analysis
`of
`RNA
`syntheses
`and
`DNA
`cyclines
`on
`incorporation
`of
`by
`determined
`into
`TCA-precipitable
`precursors
`active
`1 summarizes
`the
`Table
`products.
`analyses
`for
`adriamycin,
`probit
`of
`the
`pyrromycin,
`marcellomycin,
`minomycin,
`and
`aclacinomycin
`musettamycin,
`values
`determined
`compares
`other
`those
`obtained
`by
`with
`studies
`lines.
`employing
`different
`cell
`tigators
`for
`adriamycin,
`carminomycin,
`IC(cid:1)
`values
`were
`and
`pyrromycin
`for
`DNA
`synthesis
`RNA
`less
`than
`twice
`the
`IC(cid:1)
`values
`marcel-
`synthesis.
`For
`the
`anthracyclines
`aclacinomy-
`lomycin,
`musettamycin,
`and
`synthesis
`cm,
`the
`IC(cid:1),
`values
`DNA
`their
`were
`6.6-7.6
`times
`than
`synthesis.
`spective
`IC50
`values
`RNA
`None
`the
`anthracyclines
`inhibited
`of
`tein
`synthesis
`to
`a significant
`extent.
`
`as
`
`radio-
`
`results
`car-
`
`in
`
`and
`these
`inves-
`The
`
`for
`
`re-
`
`pro-
`
`for
`greater
`for
`
`1
`
`disposable
`zene
`in
`a Packard
`counting
`spectrometer.
`scintillation
`isolation
`RNA
`labeling,
`Nucleolar
`hepatoma
`. Novikoff
`fractionation
`30-40
`incubated
`cells
`were
`0.01
`1640
`medium
`activity,
`{3H]uridine
`then
`collected
`mmole).
`x
`for
`10-15
`at
`trifugation
`g
`with
`washed
`once
`temperature,
`room
`Earle’s
`modified
`“phosphate-free”
`(17),
`resuspended
`Eagle’s
`medium
`medium
`concentration
`the
`with
`same
`x
`0.6-1.0
`10’
`cells!ml.
`of approximately
`incubation
`for
`30 mm,
`After
`preliminary
`to
`the
`cell
`culture.
`drug
`was
`the
`was
`added
`to
`minutes
`Thirty
`mCi!ml.
`The
`0.2
`concentration
`final
`for
`isotope
`60
`with
`were
`incubated
`cells
`washing
`on
`ice. After
`and
`chilled
`mm
`cells
`were
`medium,
`the
`with
`NKM
`twice
`weight)
`10-20-fold
`(by
`with
`combined
`cells.
`Nucleoli
`“carrier”
`unlabeled
`described
`previously
`prepared
`from
`extracted
`RNA
`sulfate-
`dodecyl
`sodium
`cleoli
`The
`RNA
`at
`65#{176}(18,
`19).
`phenol
`in
`4-8 ml
`sterile
`deion-
`was
`reprecipitated
`with
`2 vol-
`ized
`ethanol
`with
`2% potassium
`umes
`approximately
`4
`hr
`at
`-20#{176}.
`acetate
`was
`then
`resuspended
`in
`1.0 ml
`RNA
`The
`deionized
`water
`and
`layered
`on
`sterile
`of
`linear
`sucrose
`gradient
`(5-40%)
`con-
`33-mi
`0.1
`ml
`of NaCl,
`M sodium
`taining
`(pH
`5.1),
`and
`acetate
`M disodium
`Centrifugation
`carried
`out
`EDTA.
`an
`SW 27 rotor
`at
`for
`16 hr
`at
`
`then
`
`as
`was
`
`the
`by
`method
`redissolved
`water
`of
`
`and
`95%
`for
`
`of
`
`were
`
`(18).
`isolated
`
`nu-
`
`of
`
`IC5
`
`values
`
`TABLE
`
`of anthracyclines
`syntheses
`
`for
`
`DNA
`
`and
`
`RNA
`
`The
`
`IC50
`
`(50%
`
`inhibitory
`
`concentration)
`
`values
`
`were
`Fig.
`in
`tained
`
`determined
`1. Numbers
`from
`the
`
`using
`
`in
`literature.
`
`analyses
`probit
`parentheses
`
`as
`
`indicated
`values
`
`are
`
`ob-
`
`Compound
`
`IC5
`
`Adriamycin
`Carminomycin
`Pyrromycin
`
`Marcellomycin
`Musettamycin
`Aclacinomycin
`
`DNA
`
`p.M
`
`(0.8)”
`(0.04)0
`
`(1.2)’
`
`6.1
`14.7
`5.7
`
`11.3
`10.0
`6.3
`
`RNA
`
`p.M
`
`(0.9)”
`(0.2)0
`
`(0.12)”
`
`3.2
`8.9
`4.5
`
`1.7
`1.5
`0.83
`
`From
`Studied
`
`Henry
`(12).
`in Micrococcus
`
`lvsodeikticus
`
`(20).
`
`a
`
`in
`4#{176}
`
`0.01
`0.001
`was
`rpm
`
`26,000
`
`Ex. 1041-0004
`
`(cid:1)
`(cid:1)
`(cid:1)
`

`
`294
`
`CROOKE
`
`ET
`
`AL.
`
`I
`
`E
`
`60
`
`56
`
`52
`
`48
`
`44
`
`40
`
`36
`
`32
`
`28
`
`24
`
`20
`
`16
`
`12
`
`0 2
`
`4
`
`20
`
`16
`
`12
`
`I
`
`I
`
`ADR IAMYC
`
`IN
`
`10
`
`(cid:1)0.5
`
`I:
`
`10
`
`(cid:1)RYCIN,
`
`(cid:1)o5
`
`..,(cid:1)1
`
`0
`
`0
`
`0
`
`10
`
`20
`FRACTION
`
`30
`
`40
`NUMBER
`
`50
`
`60
`Botto”
`
`0
`
`10
`
`20
`
`30
`
`40
`
`50
`
`FRACTION
`
`NUMBER
`
`60
`Bottom
`
`to
`
`et
`
`and
`
`analyzed
`
`extracted
`RNA
`as
`indicated
`
`control
`from
`in MATERIALS
`5-40%
`
`and
`
`AND
`
`cells
`RNA
`also
`
`FIG.
`Cells
`extracted
`indicated
`absorbance
`Radioactivity
`gradients
`
`2.
`Sedimentation
`were
`prepared,
`according
`in MATERIALS
`at
`254
`was
`right
`
`to
`
`the
`
`profiles
`treated,
`Steele
`
`of
`and
`al.
`
`nucleolar
`labeled
`(19)
`
`AND
`
`METHODS.
`
`monitored
`as
`described
`pattern.
`The
`
`Gradients
`by
`
`four
`
`were
`
`of
`text.
`
`means
`in
`the
`patterns
`
`on
`linear
`fractionated,
`a
`flow
`Patterns
`represent
`
`cell
`
`are
`the
`
`adriamycin-treated
`Nucleolar
`METHODS.
`gradients,
`density
`sucrose
`fractions
`were
`collected,
`0.6-ml
`recording
`ultraviolet
`and
`bottoms
`displayed
`the
`results
`experiment.
`
`of
`
`a
`
`with
`single
`
`was
`as
`and
`
`monitor.
`of
`
`the
`
`was
`nm
`determined
`of
`each
`
`basis
`the
`On
`anthracyclines
`the
`1,
`into
`two
`divided
`and
`adriamycin,
`dines
`-
`RNA
`and
`inhibited
`pyrromycin
`-
`equivalent
`at
`approximately
`syntheses
`anthracyclines
`Class
`II
`concentrations.
`and
`musettamycin,
`marcellomycin,
`synthesis
`inhibited
`RNA
`cinomycin
`than
`concentrations
`7-fold
`synthesis.
`inhibit
`DNA
`quired
`of
`ant
`hracyclines
`Effects
`Since
`very
`RNA
`synthesth.
`of
`newer
`anthracyclines
`ble,
`double-label
`technique
`ployed
`to
`assess
`specific
`S RNA
`in
`each
`density
`Because
`the
`cells
`labeled
`
`Table
`in
`tentatively
`I anthracy-
`
`-
`
`ada-
`at
`
`6-
`re-
`
`those
`
`on
`limited
`were
`
`nucleolar
`amounts
`availa-
`was
`em-
`activity
`of
`45
`gradient.
`for
`30
`
`hr
`
`of
`
`results
`
`classes.
`
`shown
`be
`can
`Class
`carminomycin,
`DNA
`
`-
`
`lower
`to
`
`the
`
`a
`
`the
`sucrose
`were
`
`[3Hjuridine,
`represents
`RNA.
`To
`of
`newly
`the
`RNA,
`[3H]uridine
`at
`
`with
`tivity
`lar
`bition
`somal
`to
`peak
`determined
`and
`compared
`untreated
`RNA
`somal
`probit
`from
`Figure
`absorbance
`nucleolar
`with
`cm.
`cm
`of
`
`various
`Increasing
`progressively
`into
`45
`
`32p
`
`radioac-
`nucleo-
`of
`inhi-
`preribo-
`the
`45
`in
`of
`peak
`was
`5
`28
`the
`concentration
`drug
`observed
`value
`the
`45
`S preribo-
`for
`IC50
`then
`estimated
`was
`ratios.
`these
`density
`
`[3H]uridine
`the
`uniformly
`labeled
`determine
`degree
`synthesized
`45
`ratio
`
`the
`
`32(cid:1)
`
`5
`
`in
`
`each
`with
`The
`cells.
`synthesis
`analyses
`2 shows
`and
`RNA
`
`of
`sucrose
`radioactivity
`obtained
`concentrations
`concentrations
`inhibited
`5 preribosomal
`
`from
`
`gradient
`profiles
`cells
`treated
`of
`adriamy-
`of
`adriamy-
`incorporation
`RNA.
`
`S
`
`in
`
`of
`
`Ex. 1041-0005
`
`

`
`STUDIES
`
`ON
`
`THE
`
`MECHANISM
`
`OF
`
`ACTION
`
`OF
`
`ANTHRACYCLINES
`
`295
`
`0
`
`E
`
`E
`
`60
`
`50
`
`40
`
`30
`
`20
`
`10
`
`0 6
`
`0
`
`50
`
`41
`
`30
`
`20
`
`10
`
`a
`
`50
`
`40
`
`e30
`
`20
`
`10
`
`0
`
`a
`
`50
`
`z
`
`40
`
`30
`
`20
`
`:o
`
`10
`
`0
`
`1.0
`
`(cid:1)0.5
`
`0
`
`1.0
`
`Q0(cid:1)5
`
`0
`
`4
`
`8
`
`12
`
`16
`
`20
`
`24
`
`2B
`
`32
`
`0
`
`4
`
`8
`
`12
`
`16
`
`20
`
`24
`
`28
`
`32
`
`FIG.
`3. Sedimentation
`are
`Procedures
`
`indicated
`
`in
`
`Fig.
`
`2,
`
`described
`except
`
`0
`
`profiles
`in
`
`FRACTION
`
`NUMBER
`
`36
`Bottom
`
`FRACTION
`
`NUMBER
`
`36
`Bottom
`
`of
`
`nucleolar
`
`RNA
`
`extracted
`
`from
`
`control
`
`and
`
`pyrromycin-treated
`
`cells
`
`MATERIALS
`
`AND
`
`that
`
`1.0-ml
`
`fractions
`
`METHODS.
`were
`collected.
`
`Gradients
`
`were
`
`fractionated
`
`and
`
`displayed
`
`as
`
`of
`
`sucrose
`the
`nucleolar
`with
`
`density
`RNA
`
`Figure
`dient
`from
`tions
`RNA
`served
`The
`lar
`
`data
`to
`the
`Figure
`iments
`which
`tained
`mycin.
`necessary
`thesis
`quired
`quantitative
`studies
`
`to
`
`were
`to
`
`were
`
`3 shows
`patterns
`treated
`cells
`pyrromycin.
`of
`synthesis
`in
`cells
`were
`analysis
`4 shows
`performed
`are
`representative
`with
`musettamycin
`The
`concentrations
`inhibit
`markedly
`inhibit
`results
`readily
`
`gra-
`isolated
`concentra-
`various
`on
`45
`The
`effects
`those
`similar
`to
`with
`adriamycin.
`in
`a manner
`adriamycin
`of
`similar
`aclacinomycin,
`of
`results
`and
`marcello-
`of
`these
`agents
`RNA
`syn-
`those
`re-
`The
`these
`
`the
`to
`and
`RNA
`studied.
`synthesis
`DNA
`preribosomal
`714,
`and
`
`1256
`
`and
`
`the
`
`of
`for
`for
`
`all
`the
`for
`
`re-
`1.12,
`
`IC50
`ratios
`2 compares
`Table
`whole
`the
`IC50
`synthesis
`DNA
`for
`nucleo-
`the
`IC50
`synthesis
`RNA
`cell
`for
`synthesis
`preribosomal
`lar
`of
`The
`ratios
`anthracyclines
`IC50
`to
`the
`IC50
`for
`synthesis
`RNA
`nucleolar
`aclacinomycin,
`for
`170,
`were
`marcellomycin,
`musettamycin,
`0.93,
`ratios
`were
`Similar
`spectively.
`for
`pyrromycin,
`carminomycin,
`and
`1.02
`and
`adriamycin,
`respectively.
`Effects
`on
`preribosomal
`The
`[3H]uridine
`ing.
`in
`the
`S RNA
`synthesized
`prior
`liminary
`incubation)
`to
`drug.
`The
`
`45
`
`RNA
`radioactivity
`peak
`represents
`(during
`the
`and
`during
`radioactivity
`
`to
`
`process-
`present
`RNA
`pre-
`30-hr
`exposure
`represents
`
`32(cid:1)
`
`5
`ob-
`
`simi-
`data.
`exper-
`
`ob-
`
`in
`
`were
`treated
`analyzed
`the
`of
`results
`with
`
`nucleolar
`than
`less
`synthesis.
`DNA
`obtained
`reproducible.
`
`Ex. 1041-0006
`
`(cid:1)
`(cid:1)
`(cid:1)
`(cid:1)
`

`
`296
`
`CROOKE
`
`ET
`
`AL.
`
`120
`
`100
`
`-
`
`80
`
`60
`
`40
`
`20
`
`0
`
`120
`
`100
`
`80
`
`60
`
`40
`
`20
`
`0
`
`I
`
`2
`E
`
`0
`
`24
`
`2B
`
`32
`
`36
`
`0
`
`4
`
`B
`
`12
`
`16
`
`20
`
`24
`
`28
`
`32
`
`36
`
`I
`
`Cc
`
`(cid:1)50
`
`(cid:1)40
`
`230
`
`I
`
`)0
`
`z
`
`(cid:1)50
`
`Z40
`
`o (cid:1)3
`
`0
`
`20
`
`10
`
`0
`
`1.0
`
`(cid:1)0.5
`
`0
`
`1.0
`
`,,05
`
`,..,#{176} 20
`
`10
`
`0
`
`0
`
`0
`
`4
`
`B
`
`12
`
`16
`
`20
`
`FRACTION
`
`NUMBER
`
`BOSOm
`
`FRACTION
`
`NUMBER
`
`Bottom
`
`4.
`FIG.
`Procedures
`indicated
`
`Sedimentation
`are
`described
`in Fig.
`except
`
`2,
`
`profiles of nucleolar
`in MATERIALS
`1.0-ml
`fractions
`
`that
`
`RNA
`
`extracted
`
`AND
`
`METHODS.
`were
`collected.
`
`from
`Gradients
`
`control
`
`and
`
`were
`
`aclacinomycin-treated
`fractionated
`and
`
`cells
`
`displayed
`
`as
`
`clear
`is
`it
`ever,
`step
`first
`the
`45
`S RNA
`of
`components.
`
`in
`to
`
`5
`
`there
`that
`processing,
`smaller
`
`no
`is
`i.e.,
`molecular
`
`delay
`cleavage
`weight
`
`in
`
`DISCUSSION
`
`to drug.
`of
`each
`45
`inhi-
`these
`were
`would
`S peak.
`observed.
`observed
`from
`
`exposure
`during
`synthesized
`RNA
`concentrations
`high
`relatively
`At
`into
`of
`incorporation
`na
`drug,
`total
`indicating
`observed,
`was
`RNA
`Under
`synthesis.
`of 45 5 RNA
`bition
`if processing
`of 45 S RNA
`conditions,
`[3H}uridine
`radioactivity
`inhibited,
`observed
`the
`45
`been
`have
`experiment
`was
`this
`in
`no
`fact,
`radioactivity
`was
`[3H]uridine
`No
`peak
`in
`RNA
`isolated
`the
`45
`in
`with
`high
`concentrations
`treated
`cells
`that
`the
`[3Hjuridine-la-
`suggesting
`drugs,
`been
`processed
`during
`had
`45 5 RNA
`beled
`to smaller
`molecular
`weight
`exposure
`drug
`(Figs.
`2-4).
`Additional
`studies
`components
`necessary
`to
`prove
`conclusively
`will
`be
`that
`processing
`totally
`unaffected.
`How-
`
`32(cid:1)
`
`in
`
`S
`
`is
`
`In
`
`of
`
`4
`
`the
`
`is
`
`C-7.
`
`and
`3
`Tables
`anthracyclines
`the
`several
`additional
`and
`adriamycin
`group
`13
`carbonyl
`group,
`ent
`on C-b,
`and
`sugar
`daunosamine,
`at
`sidic
`linkage
`characterized
`group
`is
`function
`carbonyl
`a
`carboxymethyl
`presence
`of
`
`at C-13,
`group
`amino
`
`at
`sugar
`
`the
`
`structures
`show
`in
`this
`employed
`anthracyclines.
`characterized
`the
`lack
`presence
`attached
`The
`by
`
`the
`
`of
`
`a
`
`study
`The
`a C-
`by
`substitu-
`of
`the
`amino
`of
`via
`a glyco-
`aclacinomycin
`the
`lack
`the
`presence
`C-b,
`and
`rhodosamine.
`
`of
`
`a
`of
`the
`
`Ex. 1041-0007
`
`(cid:1)
`

`
`STUDIES
`
`ON
`
`THE
`
`MECHANISM
`
`OF
`
`ACTION
`
`OF
`
`ANTHRACYCLINES
`
`297
`
`RNA
`hibited
`pyrro-
`and
`carminomycin,
`Adriamycin,
`syntheses
`DNA
`and
`mycin
`inhibited
`ascites
`toma
`than
`trations
`concentra-
`equivalent
`at
`approximately
`DNA synthesis,
`of the
`IC(cid:1)0 for DNA syn#{149}
`tions. The
`ratio
`DNA
`synthesis
`for
`nucleolar
`RNA
`syn-
`the
`thesis
`to
`IC50
`0.93
`to
`for
`these
`thesis
`varied
`from
`1.12
`synthesis
`varied
`compounds.
`In
`contrast,
`inarcellomycin,
`two
`classes
`of
`musettamycin,
`and
`aclacinomycin
`
`RNA
`
`in-
`
`TABLE
`
`2
`
`Inhibition
`whole
`
`nucleolar
`of
`cellular
`
`DNA
`
`RNA
`and
`
`synthesis
`RNA
`
`relative
`syntheses
`
`to
`
`hepa-
`in Novikoff
`synthesis
`concen-
`6-7-fold
`lower
`cells
`at
`inhibit
`to
`required
`those
`of IC(cid:1)0 for
`and the ratios
`to
`IC50
`for
`nucleolar
`RNA
`from
`170
`to
`1256.
`Thus
`anthracyclines
`are
`defined.
`
`TABLE
`
`4
`
`Structural
`aclacinomycin
`
`modifications
`class
`
`in
`ant
`
`cinerubin
`hracyclines
`
`of
`
`A-
`
`The
`column
`cose
`
`and
`
`abbreviations
`headed
`cinerulose,
`
`“R.2”
`
`and
`DF
`for
`stand
`respectively.
`
`C
`the
`
`indicated
`sugars
`
`in
`2-deoxyfu-
`
`the
`
`R
`
`0
`
`R3
`
`COOCH3
`
`:(cid:1)2(cid:1)x(cid:1)b0(cid:1)4
`
`CH3
`
`N(CH3)2
`
`F 20
`
`A-aclacinomycin
`
`for
`values
`The
`by
`determining
`estimated
`the
`45
`5
`tivity
`radioactivity
`[3H]uridine
`drug
`concentration
`ratios
`against
`in
`a manner
`
`IC50
`
`in
`
`drug
`similar
`
`nucleolar
`
`peak
`
`the
`(see
`the
`
`in
`plotting
`and
`concentration
`to
`that
`
`RNA
`ratio
`Figs.
`28
`the
`for
`in
`
`32p,
`
`were
`synthesis
`of
`radioac-
`to
`
`the
`each
`
`2-4)
`at
`S peak
`32P:L3Hluridine
`probit
`analysis
`Fig.
`
`1.
`
`shown
`
`Anthracycline
`
`IC50
`No-RNA#{176}
`
`IC50
`DNAb:
`IC50 WC-
`RNA0
`
`IC50
`DNA0:1C5
`No-RNA
`
`p.M
`
`Adriamycin
`Carminomycin
`Pyrromycin
`
`Marcellomycin
`Musettamycin
`Aclacinomycin
`“No-RNA,
`
`6.0
`13.06
`6.15
`
`0.009
`0.014
`0.037
`
`1.89
`1.64
`1.26
`
`6.53
`6.66
`7.65
`
`1.02
`1.12
`0.93
`
`1256
`714
`170
`
`nucleolar
`
`RNA.
`
`WC-RNA,
`
`whole
`
`cell
`
`RNA.
`
`Values
`
`were
`
`ob-
`
`tained
`
`from
`
`Table
`
`1.
`
`General
`
`structure
`
`of
`of
`
`cinerubin
`anthracyclines
`
`class
`
`Compound
`
`Pyrromycin
`Musettamycin
`Marcellomycin
`Cinerubin
`Aclacinomycin
`
`A
`
`R,
`
`OH
`OH
`OH
`OH
`H
`
`R2
`
`H
`DF
`DF-DF
`DF-C
`DF-C
`
`R3
`
`H
`H
`H
`H
`H
`
`Structural
`
`modifications
`
`in
`
`adriamycin-daunomycin
`
`class
`
`of anthracyclines
`
`TABLE
`
`3
`
`:(cid:1)i(cid:1)2(cid:1)’#{176}9;t(cid:1)2
`
`CH3
`
`H0(cid:1)
`
`NR3R4
`
`General
`
`structur
`
`e of
`
`adriamycin-dauno
`
`mycin
`
`cla
`
`ss
`
`of
`
`Compound
`
`Adriamycin
`Daunomycin
`Carminomycin
`Rubidazone
`AD-32
`AD-41
`
`R,
`
`OCH3
`OCH3
`OH
`OCH3
`OCH3
`OCH3
`
`R2
`
`CH2OH
`CH3
`CH3
`CH3
`CH2OCOC4H5
`CH2OH
`
`R3
`
`H
`H
`H
`H
`H
`H
`
`anthracyclines
`K
`
`H
`H
`H
`H
`COCF3
`COCF3
`
`X -
`
`0
`0
`0
`NNHCOCRH5
`0
`0
`
`Ex. 1041-0008
`
`

`
`298
`
`CROOKE
`
`ET
`
`AL.
`
`I
`
`anthracyclines
`RNA
`synthesis
`concentrations.
`inhibit
`significantly
`those
`
`at
`
`and
`
`an-
`syn-
`
`DNA
`inhibit
`at
`approximately
`Class
`II
`RNA
`concentra-
`they
`inhibit
`
`nucleolar
`lower
`which
`
`same
`
`the
`musettamy-
`induced
`effects
`comparable
`carminomycin,
`apparently
`molecu-
`Rather,
`appears
`or
`third
`agly-
`
`but
`syntheses
`and
`aglycone
`the
`observed
`differences.
`difference
`a
`second
`anthracycline
`
`of
`
`the
`to
`
`the
`
`also
`
`45
`
`5
`
`suggest
`studied
`preribosomal
`weight
`studies
`that
`
`these
`
`none
`
`that
`inhibits
`
`RNA
`
`RNA
`species.
`be
`
`of
`the
`to
`
`re-
`is
`
`II
`
`1. Waring,
`2. DiMarco,
`
`M.
`A.
`Antibiotics
`in
`Antimicrobial
`
`3.
`
`6.
`
`7.
`
`10.
`
`11.
`
`Hahn,
`&
`J. W.
`coran,
`Berlin.
`128,
`Springer
`W. & Hamilton,
`, Fuller,
`J.
`Pigram,
`W.
`35,
`17-19.
`(1972)Nat.
`NewBiol.,
`4. Gabbay,
`D.,
`Fingele,
`J.,
`Grier,
`E.
`S. W. & Wilson,
`, Pearce,
`R.
`15,
`2062-2069.
`Biochemistry,
`5. Tsou,
`K. C. & Yip,
`K. F. (1976)CancerRes.,
`
`Dr. I. Daskal,
`manuscript.
`
`and
`
`Dr.
`
`Y. C. Choi
`
`for
`
`reviewing
`
`the
`
`REFERENCES
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`
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`
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`
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`
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`
`Class
`nucleolar
`equivalent
`thracyclines
`at
`thesis
`than
`tions
`synthesis.
`DNA
`contains
`pyrromycin
`Since
`as
`marcellomycin,
`aglycone
`aclacinomycin
`and
`din,
`and
`DNA
`on
`RNA
`of
`adriamycin
`to
`those
`the
`differences
`in
`do
`not
`contribute
`lar
`pharmacological
`the
`critical
`structural
`to
`be
`the
`presence
`sugar
`linked
`to
`cone.
`results
`These
`anthracyclines
`the
`conversion
`of
`lower
`molecular
`will
`However,
`additional
`processing
`quired
`demonstrate
`to
`anthracyclines.
`by
`totally
`unaffected
`studies
`confirmed
`Electron
`microscopic
`class
`I and
`class
`differences
`between
`the
`II
`anthracyclines
`anthracyclines.
`Class
`in marked
`nucleo-
`were
`observed
`result
`lar
`morphological
`alterations,
`similar
`those
`induced
`actinomycin
`D at
`low
`concentrations
`(21).
`Thus
`morphologi-
`cal
`observations
`correlate
`with
`molecular
`pharmacological
`Research
`in
`progress
`the
`structure-activity
`fine
`responsible
`for
`these
`delineate
`reasons
`the
`in
`activity
`of class
`I and
`dines.
`
`well
`studies.
`should
`further
`relationships
`and
`should
`differences
`anthracy-
`
`findings
`the
`for
`class
`
`II
`
`to
`
`by
`
`to
`very
`
`the
`
`de-
`
`3367-3373.
`. Yarbro,
`A.
`Theologides,
`21,
`J.
`(1968)Cancer,
`A. & DiMarco,
`Rusconi,
`29, 1507-1511.
`& Bachur,
`D.
`8. Meriwether,
`W.
`1137-1142.
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`, Frederiksen,
`9. Dano,
`K.
`P.
`(1972)CancerRes.,
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`L.
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`D. W.
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`(Philadelphia).
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`Y.,
`Oki,
`T.,
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`I., Hori,
`S.,
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`K.,
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`A.,
`Ishizuka,
`M.,
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`H.,
`H.,
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`Hamada,
`T.
`(1975)
`14. DiMarco,
`Dasdia,
`Nettleton,
`A.,
`W.,
`Vulcano,
`
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`
`15.
`
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`
`A.,
`Takamatsu,
`Naganawa,
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`
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`
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`
`&
`
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`
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`T.,
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`A. B., Mosely,
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`
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`Chemical
`Chemistry
`
`R. & Henry,
`395-398.
`Chemotherapy
`169th
`Meet-
`Society,
`Divi-
`Symposium
`
`ACKNOWLEDGMENTS
`
`The
`
`authors
`
`are
`
`indebted
`
`to Ms.
`
`Ms.
`and
`preparation
`T.
`Doyle
`
`Polly
`of
`
`for
`
`for
`
`Wischmeier
`this
`manuscript.
`numerous
`discussions
`
`Durantini
`in
`
`Julie
`assistance
`thank
`We
`also
`regarding
`
`the
`Dr.
`the
`
`of
`
`chemistry
`ration
`helpful
`manifestations
`and
`guidance
`ated.
`The
`
`the
`
`of
`of Dr.
`authors
`
`of the anthracyclines
`Tables
`3
`and
`4,
`and
`discussions
`regarding
`anthracyclines.
`Harris
`Busch
`also
`wish
`to
`
`and
`Dr.
`the
`
`are
`
`thank
`
`in prepa-
`help
`I.
`Daskal
`for
`morphological
`The
`support
`most
`appreci-
`Dr.
`Strong,
`
`J.
`
`19.
`
`20.
`
`21.
`
`16. Bradner,
`in
`press.
`17. Mauritzen,
`(1970)
`18. Muramatsu,
`
`W.
`
`T. & Misiek,
`
`M.
`
`(1978)J.
`
`Antibiot.,
`
`Choi,
`C. M.,
`Methods
`Cancer
`M.
`& Busch,
`
`Y.
`Res.,
`H.
`
`& Busch,
`C.
`6, 253-282.
`(1967)
`
`Methods
`
`H.
`
`Cancer
`Steele,
`
`Res.,
`J.,
`
`W.
`
`2, 303-359.
`Okamura,
`240,
`
`N. & Busch,
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`J. Biol. Chem.,
`L. N.,
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`Y.,
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`Gause,
`G.
`F.
`(1974) Antibiotiki,
`Daskal,
`I., Woodard,
`C.,
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`in
`H. (1977)
`Cancer
`Res.,
`press.
`
`H.
`
`(1965)
`
`Kozmyan,
`
`J. &
`L.
`514-517.
`19,
`S. T. & Busch,
`
`Ex. 1041-0009