THE
`JOURNAL
`Copyright
`
`OF
`PHARMACOLOGY
`© 1969 by The Williams
`
`EXPERIMENTAL
`AND
`Co.
`& Wilkins
`
`THERAPEUTICS
`
`Vol.
`Printed
`
`166, No.
`in U.S.A.
`
`2
`
`CHOLINERGIC
`THE
`COXFORMATIONALLY
`
`EFFECTS
`RIGID
`
`RATES
`AND
`ANALOGS
`
`HYDROLYSIS
`OF
`ACETYLCHOLINE1
`
`OF
`
`OF
`
`C. Y. CHIOU.
`
`J.
`
`P. LONG,
`
`J. G. CANNON
`
`AND
`
`P. D. ARMSTRONG
`
`Department
`Chemistry.
`
`College
`of Pharmacology,
`College
`of Pharmacy,
`
`of Medicine
`University
`
`of
`
`and
`Iowa,
`
`Division
`Iowa
`
`of Medicinal
`City,
`Iowa
`
`Accepted
`
`for
`
`publication
`
`November
`
`18, 1968
`
`ABSTRACT
`
`J. P.
`of
`
`its
`
`its
`
`with
`potency
`to
`
`by
`poorly
`acetylcholinesterase
`(-)-trans-ACTM
`and
`hydrolysis
`isomers
`the
`These
`results
`indicate
`
`of
`the
`
`With
`acetyicholine.
`
`rates
`that
`
`the
`
`and
`Exp.
`(ACTM)
`conforma-
`pressure
`was
`associated
`have
`
`and
`
`strong
`nicotinic
`of
`the
`to
`be
`(+)-
`
`form
`was
`However,
`due
`
`effects
`cholinergic
`J.
`Pharmacol.
`iodides
`cisoici
`and
`blood
`clog
`of ACTM
`to
`expected
`negligible
`it
`had
`the
`1,3-interaction
`which
`is
`believed
`activities
`between
`D(-)-acetyl-fl-methyl-
`and
`dog
`blood
`pressure
`on
`of
`respectively),
`but
`that
`on
`enzymatic
`hydrolysis
`that
`the
`relative
`rates
`were
`96
`and
`59%
`that
`61
`and
`34% in
`relation
`of
`(+)-trans-ACTM
`
`were
`(-)-
`
`were
`activity
`
`because
`
`the
`
`former
`
`is
`
`of
`of
`of
`
`is
`
`a
`
`The
`P. D.
`J. G. CANNON
`C. V.,
`Ciiiou,
`ARMSTRONG:
`LONG,
`AND
`of
`acetylcholine.
`analogs
`rigid
`conformationally
`of
`hydrolysis
`rates
`166:
`trimethylammonium
`cyclopropyl
`1969.
`2-Acetoxy
`243-248,
`Ther.
`(ACh)
`with
`transoid
`acetylcholine
`rigid
`analogs
`of
`(onformationaily
`are
`(+)-trans-ACTM
`activities
`on
`had
`strong
`muscarinic
`The
`tions.
`transoid
`ileuni
`preparations,
`suggesting
`that
`the
`guinea-pig
`(+),
`The
`(-)-cis-ACTM
`muscarinic
`activities.
`with
`activities
`owing
`to
`cisoid
`conformation.
`nicotinic
`to
`on
`frog
`rectus
`abdominis
`muscle,
`presumably
`activity
`oxygen
`group
`of
`cyclopropane
`ring
`the
`carbonyl
`methylene
`The
`ratios
`of muscarinic
`nicotinic
`activities.
`for
`required
`those
`very
`were
`close
`between
`anti
`(-)-trans-ACTM
`L(+)-
`of ACh
`and
`choline.
`The muscarinic
`activities
`(+)-trans-ACTM
`neostigmine
`(41-fold
`and
`23-fold,
`markedly
`potentiated
`trans-ACTM
`was
`potentiated
`(3-fold).
`The
`studies
`tiaii.(cid:1)ACTM
`by
`and
`cholinesterase
`revealed
`by
`acetylcholinesterase
`hydrolysis
`(+)-
`ACh.
`by
`cholinesterase
`to
`biologic
`(-)-trans-ACTM
`
`potentiated
`better
`
`sul)strate
`
`by
`
`neostigmine
`for
`the
`
`more
`cholinesterases.
`
`than
`
`that
`
`of
`
`studies
`
`the
`
`the
`
`intensive
`acetylcholine
`of
`importance
`of
`uncertain.
`For
`the
`compounds
`as
`close
`as
`
`Despite
`features
`biologic
`remains
`problem,
`1)
`structurally
`2)
`conformationally
`Smith
`et
`al.,
`acetoxy
`cyelopropyl
`(ACTM)
`were
`
`1967).
`
`ments
`have
`moiety
`chemical
`
`on
`(ACh),
`conformational
`the
`elucidation
`reported
`as
`to ACh
`possible
`rigid
`as
`present
`the
`In
`trimethylammonium
`selected
`to
`meet
`above
`because
`these
`stated
`a cyclopropane
`ring
`in
`place
`and
`are
`considered
`to
`ACh
`structure
`among
`
`of
`
`pal)le
`The
`are
`the
`
`conferring
`of
`and
`transoid
`in figure
`shown
`cholinesterase
`
`conformational
`conformers
`cholinergic
`of ACTM
`
`cisoid
`1. The
`hydrolysis
`
`rigidity.
`of ACTM
`effects
`and
`are
`re-
`
`for
`ileceivecl
`work
`1 This
`ITealth
`Public
`and NB-06100.
`
`September
`publication
`supported
`was
`Service
`Grants
`
`16,
`part
`in
`NB-1396,
`
`1968.
`U.S.
`by
`NB-4431
`
`243
`
`have
`possible
`(Martin-
`work
`iodides
`require-
`the
`compounds
`the
`choline
`the
`smallest
`be
`derivatives
`
`molecular
`possible
`isomerism
`of
`
`this
`been
`and
`
`2-
`
`ca-
`
`the
`ported
`of
`tionships
`the
`role
`of
`the
`cyclopropane
`and
`muscarinic
`potentiation
`activities
`dog
`blood
`of
`enzymatic
`(AChE)
`
`in
`
`and
`
`of
`(+)
`is
`pressure
`hydrolysis
`cholinesterase
`
`by
`
`-
`
`present
`conformational
`unsubstituted
`ring
`effects
`neostigmine
`and
`(-)
`correlated
`by
`
`rela-
`the
`of
`
`The
`and
`group
`nicotinic
`The
`
`on
`rates
`
`investigation.
`variations
`methylene
`to
`of ACTM
`discussed.
`are
`also
`the
`muscarinic
`of
`-trans-ACTM
`with
`their
`acetyicholinesterase
`(ChE).
`
`METHODS.
`Mongrel
`kg, were
`sodium
`administered
`and
`the
`experiments
`with
`a
`arterial
`pressure
`Offner
`were
`
`Dog
`of
`dogs
`anesthetized
`and
`250
`iv.
`
`vagi
`
`blood
`either
`with
`mg/kg
`The
`
`13
`
`the
`
`preparations.
`pressure
`9
`to
`sex,
`weighing
`thiopental
`of
`15 mg/kg
`sodium
`of
`barbital
`cannulated,
`trachea
`was
`sectioned.
`Throughout
`were
`were
`artificially
`ventilated
`dogs
`respirator.
`The
`femoral
`was
`measured
`Stat
`11am
`and
`(P23AA)
`an
`RS).
`(type
`a
`polyethylene
`
`right
`a
`with
`on
`recorded
`All
`compounds
`catheter
`
`in-
`
`via
`
`the
`Palmer
`pressure
`transducer
`Dynograph
`injected
`
`TEVA - EXHIBIT 1021
`
`

`
`244
`
`CHIOU
`
`ET
`
`AL.
`
`Vol.
`
`166
`
`(cid:1)3I
`
`#{149}:Carbon
`:Nitrogen
`:Qxyg(cid:1)n
`zHyd(cid:1)g(cid:1)n
`
`1(cid:1)
`it
`(ACT(cid:1)\I).
`which
`
`1
`
`fhe
`ihe
`cannot
`
`tr
`
`tn(cid:1)oid
`
`(+)-
`
`be
`
`and
`superimposed.
`
`( \.)
`mtl
`(-)-isomers
`
`clsol(l
`
`(B)
`either
`
`onformations
`transoid
`
`of
`
`of 2 iceto(cid:1)v
`cisoid
`ACTM
`
`c(cid:1) clopiop(cid:1)
`constitute
`
`or
`
`1 t(cid:1)
`
`inieth(cid:1)
`two
`
`1
`mirror
`
`immonium
`images
`
`the
`
`in
`
`into
`washed
`all
`
`cases
`was
`
`In
`injected
`(loses
`used
`intervals:
`
`left
`with
`the
`kept
`
`in
`
`0.1
`
`20
`and
`
`of
`
`serted
`rapidly
`saline.
`tions
`The
`4-fold
`by
`(+)-trans-ACTM,
`(-)-trans-ACTM
`for ACh.
`The
`degree
`these
`activity
`of
`\V:LS
`neostiginine
`the
`neostigmine,
`used.
`above
`(cid:1)zg/kg
`anti
`pg/kg
`and
`and
`0.064
`ag/kg
`administrations
`domized,
`bioassay
`line
`
`were
`0.032
`32
`
`and
`
`of
`
`vein
`
`2 ml
`
`were
`and
`femoral
`isotonic
`of
`about
`solu-
`total
`of the
`volume
`at 3 ml.
`constant
`bioassay
`I lie
`and
`pg/kg
`and
`(cid:1)gkg
`(cid:1)zg/kg
`0.4
`potentiation
`
`varied
`
`for
`for
`
`were
`ag/kg
`0.4
`pg/kg
`80
`(cid:1)(cid:1)g/kg
`1.6
`and
`of musearinic
`50
`pg/kg
`addition
`as
`0.008
`
`of
`of
`
`stated
`gig/kg
`(cid:1)zg/kg
`8
`0.016
`and
`The
`used.
`ran-
`were
`parallel-
`
`were
`(hugs
`valid
`
`a
`
`for
`
`by
`compounds
`stlI(li((l. Before
`dose
`levels
`same
`After
`neostiginine,
`of
`(+)-trans-ACTM,
`(-)-trans-ACTM.
`pg/kg
`of ACh
`an(i
`of
`(loses
`criteria
`all
`were met.
`al)(10)ilii(i.(
`muscle
`was
`Burn
`by
`en(
`Is
`1)0111
`solution
`0.23:
`
`I(cid:1)!OfJ
`
`reettis
`
`pipiens
`was
`with
`020;
`
`i.(cid:1)
`rUe!
`al )(lonlinis
`as
`described
`threaded
`at
`frog
`TIingcr’s
`CaCI221LO.
`
`ill
`
`The
`prepamtion.
`iscie
`Rena
`obtained
`from
`(1952).
`The
`muscle
`was
`and
`silperfilsed
`(NaCI.
`KC1,
`glucose,
`
`6.43;
`
`026:
`
`XaHCO:.
`
`with
`rate
`
`The
`4 ml/min
`pump
`into
`not
`
`was
`
`at
`
`the
`
`in
`
`by
`
`02-.
`flow
`was
`RD
`stream
`than
`initial
`con-
`on
`
`95%
`of
`and
`(type
`the
`niore
`an
`by
`grams
`recorded
`RS).
`in
`drugs
`in-
`and
`(cid:1)g
`for
`
`40
`and
`400
`
`in
`and
`(type
`prepared
`of
`4-fold
`(cid:1)g
`800
`(cid:1)g
`
`oxygenated
`this was
`g/liter);
`0.71
`room
`temperature.
`5% CO2
`at
`to
`fluid
`of
`superfusion
`3
`was
`motor
`maintained
`a Holter
`by
`solutions
`were
`injected
`45).
`Drug
`fluid
`in
`of
`superfusion
`of
`volumes
`placed
`0.1 ml.
`rectus
`muscle
`The
`developed
`1 g.
`The
`tension
`of
`tension
`tile muscle
`was measured
`traction
`of
`transducer
`GT-03)
`ham
`a Stat
`(type
`Offner
`Dynograph
`on
`recorder
`an
`of
`Solutions
`were
`compounds
`water.
`The
`concentrations
`distilled
`used
`the
`bioassay
`wen
`spaced
`0.4
`(cid:1)g
`and
`1.6
`(cid:1)tg
`for
`tervals:
`160
`(+)-trans-ACTM,
`for
`(cid:1)g
`for
`(-)-trans-ACTM
`(+)
`, (-)-cis-ACTM.
`and
`doses
`drugs
`Guinea-pig
`weighing
`the
`head.
`
`ACh,
`200
`(cid:1)zg
`(cid:1)g
`and
`
`100
`
`administrations
`
`and
`The
`randomized.
`were
`preparations.
`ilemirn
`g were
`500
`stunned
`terminal
`cm
`g
`1
`injection
`
`to
`300
`The
`
`port
`
`ion
`
`approximately
`initial
`The
`
`tension
`methods
`
`3
`
`of
`of
`
`in
`
`was
`
`length,
`placed
`drugs
`
`of
`
`of
`
`pigs
`on
`
`Guinea
`a blow
`by
`the
`ileum,
`used.
`An
`the
`tissue.
`recor(ling
`
`of
`was
`on
`and
`
`(cid:1)
`(cid:1)
`

`
`1969
`
`PHARMACOLOGY
`
`OF ACTM
`
`245
`
`were
`95%
`
`the
`as
`were
`contractions
`of
`in
`described
`the
`with Krebs-bicar-
`superfused
`The
`ilea were
`section.
`KC1,
`0.354;
`CaCl(cid:1)
`solution
`(NaC1,
`6.93;
`bonate
`0.373;
`KH,P04,
`211,0,
`0.163;
`MgSO4,
`0.143;
`2.09;
`glucose,
`NaHCO,,
`g/liter).
`con-
`The
`1.80
`of
`bioassay
`centrations
`in
`drugs
`used
`by
`4-fold
`were
`spaced
`intervals:
`and
`(cid:1)ig
`(+),
`0.004
`for ACh
`10 (cid:1)ig
`(cid:1)g
`for
`and
`and
`The
`of
`administration
`drugs
`(-)-cis-ACTM.
`The
`relative
`p0-
`and
`doses
`randomized.
`tencies
`confidence
`of
`and
`limits
`and
`(+)-
`(-)-trans-ACTM
`were
`from
`et
`Armstrong
`cited
`al.
`(1968).
`method
`cholinesterase
`titration
`Radiometer
`hy-
`during
`the
`acid
`formed
`The
`activity.
`acetic
`0J)5 N NaOH
`titrated
`with
`of
`esters
`was
`drolysis
`on
`a Radiometer
`titrator
`and
`titri-
`TTT1c
`type
`graph
`One
`of
`milliliter
`type
`SBR2c.
`potassium
`(0.3676
`1 ml
`ml;
`hydrogen
`phthalate
`g/200
`=
`0.05 N NaOH
`j(cid:1)mo1
`of NaOH)
`was
`titrated
`with
`From
`under
`N,
`gas.
`this
`titration
`curve,
`the
`the
`calibrated
`ordinate
`of
`was
`titrigraph
`chart
`directly
`in micromoles
`of NaOH
`used,
`which
`equivalent
`to micromoles
`of
`acetic
`liberated
`acid
`from
`esters
`by
`cholinesterases.
`The
`values
`were
`expressed
`in micromoles
`hour
`per
`of
`per
`unit
`1.0 U/mI
`The
`enzyme
`was
`prepared
`used
`enzyme.
`without
`NaHCO,
`in Krebs.-bicarbonate
`solution
`(7.5 X 10’ M NaCl,
`7$ X 10’ M KG!
`4 X
`and
`10’
`M MgCl,(cid:1)6H(cid:1)0).
`The
`substrate
`solutions
`the
`were
`prepared
`with
`same
`solution
`in
`a
`con-
`5.6 X 10’ M. The
`total
`volume
`of
`centration
`of
`The
`reaction
`con-
`the
`reactants
`was
`1 ml.
`vessel
`0.8 ml
`the
`7.0.
`The
`tained
`of
`enzyme
`at
`sample
`the
`substrate
`(02
`ml)
`was
`added
`through
`and
`hole,
`the
`reaction
`mixture
`was
`titrated
`pH 7.0 at
`10 mm.
`The
`the
`38#{176}Cfor
`in
`reaction
`vessel
`replaced
`by N, gas.
`was
`in
`The
`Drugs
`used.
`drugs
`used
`sulfate,
`ACh
`bromide,
`atropine
`methyl
`sulfate
`d-tubocurarine
`
`last
`
`9
`
`is
`
`at
`
`the
`0.001
`ig
`
`40
`
`for
`
`pH
`
`air
`
`and
`
`this
`
`were
`study
`neostigmine
`chloride.
`The
`
`et
`
`as
`
`(+),
`and
`(-)-trans-ACTM
`(+)-trans-ACTM,
`by
`synthesized
`iodides
`were
`(-)-cis-ACTM
`doses
`(1968).
`All
`at.
`of
`used
`drugs
`Armstrong
`ob-
`ChE
`form.
`AChE
`salt
`the
`were
`refer
`to
`and
`Nutritional
`Biochemicals
`Corpora-
`from
`tained
`tion with
`specific
`activities
`of
`1000 U/mg
`of
`pro-
`and
`4 U/mg
`tein
`of protein,
`respectively.
`The
`relative
`Statistical
`analysis.
`degree
`of potentiation
`the
`the
`of
`from
`a
`four-point
`calculated
`assay
`described
`by
`Finney
`with
`were
`servations
`evaluated
`(Snedecor,
`1956).
`A probability
`less was
`considered
`to be
`significant.
`
`potencies
`compounds
`parallel-line
`(1955).
`Paired
`Student’s
`value
`
`of
`
`and
`were
`bio-
`ob..
`test
`or
`
`t
`.05
`
`The
`muscarinic
`The
`pressure.
`blood
`fall
`onset
`of
`
`a
`
`of ACTM
`activities
`elicited
`by
`response
`that
`pressure
`in
`was
`duration.
`The
`brief
`and
`95% confidence
`limits
`and
`the
`shown
`are
`(-)-trans-ACTM
`4.7
`(+)-trans-ACTM
`was
`whereas
`than
`ACh,
`(-)-trans-
`as ACh.
`Direct
`only Ms
`potent
`as
`and
`of
`(-)-trans-ACTM
`(+)-
`192
`times
`former
`the
`was
`95% confidence
`latter
`(the
`effects
`of
`The
`depressor
`(+)-
`were
`completely
`and
`ACh
`sulfate.
`of atropine
`of ACTM
`actimties
`1 shows
`the
`relative
`and
`(-)-trans-ACTM
`
`RESULTS.
`dog
`blood
`on
`ACTM
`was
`immediate
`relative
`of
`(+)-
`table
`more
`ACTM
`comparison
`that
`showed
`than
`active
`105-385).
`were
`(-)-trans-ACTM
`2 mg/kg
`blocked
`by
`The muscarinic
`Table
`ileum.
`pig
`of
`activities
`(+)-
`(-)-cis-ACTM
`on
`guinea-pig
`Mo.ooo
`, (-)-cis-ACTM
`was
`significant
`There
`no
`was
`on guinea-pig
`activity
`(+)-trans-ACTM.
`
`in
`potencies
`and
`1. The
`potent
`was
`
`the
`
`(+),
`
`(+)
`ACh.
`muscarinic
`ACh
`and
`
`in
`times
`
`more
`limits
`and
`
`guinea-
`on
`muscarinic
`and
`The
`as
`in
`
`ilea.
`active
`as
`difference
`ileum
`between
`The
`(-)-trans-
`
`The
`
`relative
`
`muscarinic
`
`activities
`
`of
`
`(+)-
`
`1
`TABLE
`(-)-trans-ACTM#{176}
`and
`to acetykholine
`(ACh)
`
`and
`
`(+),
`
`(-)-cis-ACTM
`
`with
`
`respect
`
`Dog Blood
`
`Pressure
`
`Guinea-Pig
`
`fleum
`
`Compound
`
`of
`
`No.
`animals
`
`Relative
`potency
`
`95% Confidence
`limits
`
`of
`
`No.
`animals
`
`Relative
`potency
`
`95% Confidence
`limits
`
`0.81-1.46
`0.0019-0.0025
`0.00004-0.00029
`
`1 1
`
`.136
`0.0O22(cid:1)’
`0.00010
`
`10
`10
`5
`
`3.21-9.79
`0.021-0.025
`
`1 4
`
`.70
`0.023
`
`10
`10
`
`ACh
`(+)-trans-ACTM
`(-)-trans-ACTM
`(+),(-)-cis-ACTM
`
`#{176}ACTM,
`Cited
`
`2-acetoxy
`from
`Armstrong
`
`cyclopropyl
`et al.
`
`trimethylammonium
`(1968).
`
`iodide.
`
`

`
`(cid:1)oo
`
`as
`
`active
`
`as ACh
`
`(Arm-
`
`about
`was
`1968).
`at.,
`et
`of
`activities
`nicotinic
`The
`muscle.
`abdominis
`abdominis
`frog
`rectus
`abolished
`which
`was
`As
`chloride.
`357
`times
`77
`and
`(-)-trans-ACTM,
`and
`comparison
`of
`the
`made,
`and
`was
`4.6
`be
`found
`to
`was
`95% confidence
`limits
`, (-)-cis-ACTM
`was
`
`ACTM
`effect
`muscle
`by
`indicated
`times
`
`5
`
`on
`frog
`of ACTM
`was
`con-
`10-’ M
`x
`table
`2,
`in
`active
`more
`respectively.
`and
`(-)-
`(+)-trans-
`more
`active
`3.3-6.5).
`The
`(cid:1)so
`as
`active
`
`(+)-
`the
`times
`were
`about
`
`246
`
`ACTM
`
`by
`cated
`ACh
`
`strong
`The
`rectus
`the
`on
`traction,
`d-tubocurarine
`ACh
`was
`than
`(+)-
`A
`direct
`trans-ACTM
`ACTM
`(the
`(-F)
`asACh.
`activities
`of muscarinic
`Potentiation
`pressure.
`on
`dog
`blood
`neostigmine
`3,
`the
`muscarinic
`in
`table
`(+)-trans-ACTM
`were
`and
`
`CRIOU
`
`ET
`
`AL.
`
`Vol.
`
`166
`
`and
`
`50 pg/kg
`by
`both
`compounds
`cholinesterases.
`was
`potentiated
`it was
`a
`poor
`
`The
`
`of neostigmine,
`were
`good
`activity
`of
`3-fold
`only,
`substrate
`for
`
`that
`
`41-fold
`23-fold
`that
`indicating
`for
`substrates
`(-)-trans-ACTM
`suggesting
`cholinesterases.
`The
`hydrolysis
`eneymic
`The
`relative
`cholinesterases.
`at
`trans-ACTM
`of ACh
`and
`of 5.6 x 10’ M are
`centration
`indicated
`that
`The
`results
`were
`good
`(+)-trans-ACTM
`(-)-trans-ACTM
`AChE,
`whereas
`than
`(+)-trans-ACTM
`substrate
`The
`rate
`of hydrolysis
`was measured
`concentrations
`of
`x
`10-’ M,
`5.6
`x
`10-’ M,
`32
`10-’ M,
`1.8
`x
`1
`10’
`M.
`substrate
`activity
`were
`curves
`optimum
`rates
`
`of
`
`trans-ACTM
`rates
`of hydrolysis
`the
`substrate
`con-
`shown
`in table
`4.
`both
`ACh
`and
`substrates
`for
`a poorer
`was
`for
`AChE.
`at
`substrate
`1 x
`10’ M,
`x
`10-s M and
`concentration-
`bell-shaped
`a substrate
`
`by
`
`with
`con-
`
`of ACTM
`As
`indi-
`activities
`of
`potentiated
`
`1.8
`x
`The
`obtained
`of hydrolysis
`
`at
`
`TABLE
`activity
`(-F)(cid:1)
`
`nicotinic
`relative
`The
`trans-ACTM#{176}
`and
`respect
`to
`acetylcholine
`abdominis
`muscle
`
`2
`
`of
`(+)-
`(-)-cis-ACTM
`(ACh)
`on
`
`and
`
`frog
`
`(-)-
`with
`rectus
`
`Compound
`
`of
`No.
`Animals
`
`Relative
`Potency
`
`95% Confidence
`Limits
`
`degree
`The
`of
`(+)-
`(ACh)
`
`and
`by
`
`of
`
`3
`TABLE
`activities
`of muscarinic
`potentiation
`acetyicholine
`(-)-trans-ACTM#{176}
`and
`neostigmine”
`on
`blood
`pressure
`
`dog
`
`Compound
`
`of
`
`No.
`Ani.
`,,(cid:1),
`
`Degree
`Potentiation
`
`of
`
`95%
`Confidence
`Limits
`
`41-fold
`23-fold
`2.8-fold
`
`22-100
`11-79
`1.6-5.4
`
`7 7
`
`0.008-0.021
`0.0018-0.0046
`0.0039-0.0047
`
`ACh
`(+)-trans-ACTM
`(-)-tran8-ACTM
`
`ACh
`(+)-trans-ACTM
`(-)-trans-ACTM
`(-)-cis-
`(+),
`ACTM
`
`1
`0.013
`0.0028
`0.0042
`
`10
`10
`5
`
`#{176}ACTM,
`monium
`
`2-acetoxy
`iodide.
`
`cyclopropyl
`
`trimethylam-
`
`2-acetoxy
`#{176}ACTM,
`iodide.
`monium
`b 50 pg/kg
`of neostigmine
`
`cyclopropyl
`
`trimethylam-
`
`methyl
`
`sulfate.
`
`The
`
`rates
`
`of hydrolysis
`
`of
`
`(+)-
`
`and
`
`TABLE
`(-)-lrans-ACTM#{176}
`
`4
`
`and
`
`acetylcholine
`
`(AC/i)
`
`by AChE”
`
`and
`
`ChEC
`
`AChE
`
`ChE
`
`of
`
`No.
`expts.
`
`of
`Rate
`(mean
`
`hydrolysis
`±
`S.E.)
`
`Relative
`hydrolysis
`(mean
`±
`
`rate
`
`of
`
`S.E.)
`
`of
`
`No.
`expts.
`
`of hydrolysis
`Rate
`±
`S.E.)
`(mean
`
`rate
`Relative
`hydrolysis
`(mean
`±
`
`S.E.)
`
`of
`
`pmol/Isr/U
`enzyme
`
`of
`
`33.8
`20.5
`11.3
`
`0.6
`±
`± 0.2
`0.3
`±
`
`100.3
`60.8
`33.5
`
`1.8
`±
`0.7’
`±
`± 0.8’
`
`55 5
`
`99.8
`96.0
`58.9
`
`5.4
`±
`± 4.4(cid:1)’
`± 7.9’
`
`pmol/kr/U
`enzyme
`
`of
`
`9.2
`8.8
`6.2
`
`±
`
`0.5
`0.4
`±
`± 0.9
`
`5 55
`
`Substrate
`
`ACh
`(+)-trans-ACTM
`(-)-trans-ACTM
`
`#{176}ACTM,
`b AChE,
`ChE,
`d P>
`#{149}P <
`
`trimethylammonium
`1 U/nil.
`
`cyclopropyl
`2-acetoxy
`acetyicholinesterase,
`cholinesterase,
`1 U/mi.
`.05 compared
`with
`acetyicholine.
`.05 compared
`with
`acetyicholine.
`
`iodide
`
`at 5.62 X 10’ M.
`
`

`
`1969
`
`PHARMACOLOGY
`
`OF
`
`ACTM
`
`247
`
`x 10-i M for ACh
`as well
`5.6
`(-)-trans-ACTM,
`indicating
`of
`concentration
`(+)-
`inhibits
`of
`
`61
`
`as
`that
`and
`relative
`The
`AChE.
`and
`(-)-trans-
`(+)-
`34% of
`that
`and
`concentration-activity
`of
`by
`ChE
`
`of
`
`high
`
`of
`centration
`and
`for
`(+)-
`a
`high
`substrate
`(-)-trans-ACTM
`hydrolysis
`rates
`of
`were
`ChE
`by
`ACTM
`substrate
`The
`ACh.
`no
`inhibition
`showed
`curves
`concentrations.
`substrate
`It
`is
`that
`DIscussIoN.
`reasonable
`con-
`ACh
`molecule
`flexible
`the
`the
`thus
`is
`capable
`formations
`and
`been
`has
`types
`of ACh
`receptors.
`different
`is
`that
`the
`cisoid
`form
`suggested
`asso-
`with
`its
`nicotinic
`activity
`transoid
`ciated
`1956;
`muscarinic
`activity
`with
`form
`1966).
`1962;
`Archer
`Smissman
`at.,
`et
`this
`on
`expected
`hypothesis,
`it would
`Based
`(fig.
`1A)
`transoid
`form
`of
`ACTM
`the
`that
`mainly
`muscarinic
`responses
`and
`elicit
`would
`form
`(fig.
`1B)
`mainly
`nicotinic
`re-
`cisoid
`the
`The
`results
`indicate
`that
`this
`true
`sponses.
`(table
`1)
`but
`for
`for muscarinic
`responses
`nicotinic
`responses
`(table
`2).
`Therefore,
`some
`factors
`other
`than
`isomerism
`be
`cis-trans
`involved
`in determining
`nicotinic
`activity.
`Structurally,
`ACTM
`acetyl
`is
`similar
`to
`acetyl-
`methyicholine
`and
`resembles
`a hybrid
`of
`acetyl-
`a-methylcholine
`(A-a-MCh)
`and
`which
`(A-/3-MCh),
`both
`of
`/3-methyicholine
`Simonart
`by
`have
`been
`synthesized
`and
`studied
`The
`pre-
`(1932)
`and Major
`and Bonnett
`(1935).
`is
`dominant
`muscarinic
`activity
`of A-f3-MCh
`presumably
`due
`the
`to
`the
`1,3-interaction
`of
`/3-methyl
`group
`with
`the
`carbonyl
`oxygen,
`which
`the
`nicotinic
`activity
`is
`required
`for
`and Holland,
`1961a,b;
`Sekul
`1963;
`et
`al,
`(Sekul
`Coleman
`at.,
`1965;
`Triggle,
`1968),
`whereas
`et
`the
`predominant
`nicotinic
`activity
`of A-a-MCh
`the
`is
`probably
`to
`the
`1,3-interaction
`of
`a-methyl
`with
`ether
`which
`the
`oxygen
`is
`required
`the
`muscarinic
`activity
`(Ing
`at.,
`et
`at.,
`1952;
`Waser,
`1961;
`Beckett
`Triggle,
`1961;
`1965).
`shown
`in
`figure
`the
`methylene
`group
`of
`cyclopropane
`of
`oxy-
`ACTM
`would
`interact
`with
`the
`carbonyl
`gen
`not
`with
`the
`ether
`oxygen.
`Therefore,
`but
`the
`methylene
`group
`abolishes
`nicotinic
`activity
`of
`ACTM.
`Accordingly,
`ACTM
`structurally
`similar
`to A-/3-MCh
`not
`MCh.
`studies
`on
`cholinergic
`effects
`ACTM
`the
`present
`work
`support
`this
`
`to
`
`has
`
`to
`
`of
`
`assume
`different
`fitting
`It
`of ACh
`and
`the
`(Schueler,
`et
`at.,
`be
`
`is
`not
`
`must
`
`due
`group
`for
`
`The
`in
`
`As
`the
`
`et
`1,
`ring
`
`the
`
`but
`
`is
`
`A-a-
`of
`con-
`
`1)
`
`1)
`
`is
`
`(table
`to
`
`the
`
`2).
`
`a
`
`is
`
`is
`
`a
`
`of
`
`In
`
`hydrolysis
`that
`for
`
`(+)-
`AChE
`as ACh.
`fast
`for
`substrate
`is
`hydrolysis
`hy-
`rates
`of
`by CItE
`4).
`These
`the musca-
`(-)-trans-
`41-fold,
`3).
`
`strong
`has
`(+)-trans-ACTM
`because
`clusion
`weak
`very
`but
`(table
`activity
`muscarinic
`words,
`other
`In
`2).
`(table
`activity
`nicotinic
`muscarinic
`predominant
`has
`(+)-trans-ACTM
`transoid
`con-
`to
`owing
`activity
`(table
`its
`proper
`fitting
`for
`favorable
`formation,
`which
`addition,
`the
`In
`receptor.
`with
`the
`muscarinic
`1 , 3-interaction
`group
`of
`the
`methylene
`of
`oxygen
`elimi-
`the
`carbonyl
`with
`trans-ACTM
`cis-ACTM
`activity.
`The
`the
`nicotinic
`nates
`cisoid
`to
`due
`not
`a muscarinic
`its
`stimulant
`nicotinic
`it
`1),
`is
`nor
`conformation
`of
`the
`1,3-interaction
`the
`due
`stimulant
`oxygen
`the
`carbonyl
`group
`methylene
`with
`suggesting
`hypothesis
`opposite
`The
`(table
`nicotinic
`of ACh
`favors
`form
`transoid
`the
`that
`form
`muscarinic
`ac-
`cisoid
`activities
`and
`Canepa
`1966)
`1957;
`(Jellineck,
`tivities
`et
`at.,
`present
`study
`the
`in
`because
`is unlikely
`(+)-
`muscarinic
`activities.
`strong
`had
`trans-ACTM
`there
`that
`is about
`to
`note
`It
`is
`interesting
`in
`the muscarinic
`activities
`250-fold
`difference
`D(-)-A-/3-MCh
`on
`guinea-
`and
`between
`L(+)-
`blood
`pressure
`(Beckett
`cat
`pig
`ileum
`and
`et
`at.,
`It
`1963).
`been
`1961;
`Beckett
`et
`has
`at.,
`difference
`in
`activity
`due
`suggested
`that
`the
`group
`in
`to
`the
`/3-methyl
`D(-)-A-/3-MCh,
`the
`proper
`interaction
`with
`its
`which
`prevents
`This
`hypothesis
`is
`further
`muscarinic
`receptor.
`a
`present
`work,
`there
`is
`as
`supported
`in
`the
`in muscarinic
`be-
`difference
`activities
`similar
`(-)-trans-ACTM
`(192-fold
`tween
`and
`(+)-
`and
`330-fold
`differences
`in
`activities
`on
`dog
`blood
`and
`guinea-pig
`ileum,
`respec-
`pressure
`tively).
`The
`ACTM
`AChE
`is
`trans-ACTM
`is hydrolyzed
`it
`since
`(-)-Trans-ACTM
`AChE
`since
`59%
`that
`of
`drolysis
`and
`are
`61
`explain
`results
`activities
`rinic
`are
`ACTM
`and
`23-fold
`words,
`other
`trans-ACTM
`than
`more
`the
`former
`
`studies
`by
`
`on
`
`a
`
`as
`
`of
`
`enzymic
`the
`and
`reveal
`CItE
`substrate
`good
`by AChE
`poorer
`a
`is
`rate
`relative
`its
`relative
`of ACh.
`The
`and
`(-)-trans-ACTM
`(+)-
`(table
`34% that
`of ACh
`that
`the
`observation
`and
`of ACh
`and
`(+)-
`neostigmine
`potentiated
`by
`3-fold,
`respectively
`(table
`the
`muscarinic
`activity
`is
`potentiated
`by
`of
`(-)-trans-ACTM
`better
`substrate
`
`that
`is
`
`a
`
`of
`(+)-
`neostigmine
`because
`the
`cho-
`
`for
`
`

`
`248
`
`CHIOU
`
`ET
`
`AL.
`
`Vol.
`
`166
`
`Since
`
`the
`
`the
`than
`more
`are much
`enzyme
`preparations,
`that
`the
`degrees
`activities
`of
`in
`the
`of
`
`of
`
`intact
`latter.
`linesterases
`iso-
`animals
`than
`complex
`the
`lated
`ex-
`not
`would
`one
`bio-
`of
`potentiation
`of
`pect
`neostigmine
`by
`the
`logic
`drugs
`in magni-
`the
`difference
`and
`measured
`vivo
`rates
`of
`relative
`tude
`enzymatic
`hy-
`these
`compounds
`measured
`drolysis
`vitro
`in
`the
`It
`should
`be
`true,
`however,
`be
`would
`same.
`relative
`rates
`of
`hydrolysis
`of
`these
`the
`that
`cholinesterases
`and
`the
`degrees
`of
`by
`esters
`the
`of
`muscarinic
`activities
`of
`potentiation
`these
`compounds
`by
`neostigmine
`are
`the
`in
`in
`same
`order
`this
`study:
`that
`and
`the
`case
`ACh
`(-I-)-trans-ACTM>
`(-)-trans-ACTM.
`The
`activities
`of
`2-acetoxy
`CONCLUSIONS.
`cyclopropyl
`trimethylammonium
`iodide
`(ACTM)
`been
`studied
`pharmacologic
`and
`enzymologic
`had
`strong
`muscarinic
`trans-ACTM
`but
`The
`weak
`nicotinic
`activities.
`had
`negligible
`muscarinic
`nicotinic
`The
`these
`ties.
`reasons
`of
`possible
`The
`potency
`have
`been
`discussed.
`muscarinic
`activities
`between
`(+)-
`to
`were
`fairly
`close
`trans-ACTM
`n(-)
`-acetyl-/3-methylcholine.
`L (+)-
`relative
`hydrolysis
`of
`by
`ACh
`and
`tentiation
`order:
`ACTM.
`
`a
`of
`number
`The
`systems.
`activities
`css-ACTM
`activi-
`findings
`ratios
`and
`(-)-
`between
`The
`and
`po-
`same
`
`ACTM
`degrees
`had
`
`the
`(-)-trans-
`
`have
`
`in
`
`is
`
`and
`
`and
`of
`rates
`cholinesterases
`biologic
`of
`their
`(+)-trans-ACTM>
`
`ACh
`
`the
`effects
`
`those
`
`of
`
`of
`
`REFERENCES
`Lzwis,
`A. M.
`S., L(cid:1)s,
`AND
`ARCHER,
`methiodides.
`2-acetoxytropine
`meric
`Chem.
`5:
`1962.
`423-430,
`Pharm.
`J. G. AND
`ARMSTRONG, P. D.,
`CANNON,
`of
`analogs
`Conformationally
`rigid
`65-66,
`Nature
`(London)
`220:
`1968.
`A. H., HANPER, N.
`J. AND CLITHEBOw,
`BECKZrT,
`The
`W.:
`absolute
`configuration
`the
`and
`a-
`fi-methylcholine
`acetyl
`isomers
`and
`their
`15:
`succinylesters.
`Pharm.
`Pharmacol.
`361,
`1963.
`A. H.,
`N.
`HARPER,
`BECKETT,
`LERszit, W.:
`Muscarinic
`AND
`189:
`(London)
`671-673,
`1961.
`
`Iso-
`T. R.:
`J. Med.
`
`J. P.:
`LoNG,
`acetylcholine.
`
`J.
`and
`and
`349-
`
`J.,
`
`CLITHEROW
`receptors.
`
`J. W.
`(cid:1)ature
`
`J.
`
`Pharmacology,
`Oxford,
`1952.
`P.
`AND
`substrates
`other
`(London)
`
`Blackwell
`
`SORUM,
`of
`
`H.:
`cholin-
`
`210:
`
`907-909,
`
`Hou(cid:1),
`of
`J.
`
`W. C.:
`several
`sub-
`Pharmacol.
`
`Design
`of Chicago
`
`and
`Its
`Press,
`
`Sta-
`Chi-
`
`P.
`
`D. P.
`Wn(cid:1)u(cid:1)ss,
`TUDoR
`AND
`relationship
`structure-action
`J. Pharmacol.
`Chem-
`Brit.
`
`Acta
`
`muscarine.
`
`of
`1957.
`Preparation
`T.:
`H.
`acetyl
`and
`dextro-
`J. Amer.
`Chem.
`
`F.
`
`10:
`
`The
`
`: Practical
`J. H.
`BURN,
`Scientific
`Publications,
`G.,
`CANEPA,
`PAULINO
`Structure
`of ACh
`and
`Nature
`ergic
`systems.
`1966.
`(cid:1)n
`M. E., Htmrs, A. H.
`Cotmi(cid:1)x,
`Nicotine-like
`stimulant
`actions
`stituted
`phenylcholine
`ethers.
`66-70,
`Ther.
`148:
`Exp.
`1965.
`FINNEY,
`J.:
`D.
`Experimental
`tistical
`Basis,
`University
`cago,
`1955.
`ING, H.
`R., KORDIK,
`H.:
`on
`the
`Studies
`of
`the
`group.
`choline
`other.
`7:
`103-116,
`1952.
`F.:
`Structure
`JELLINECK,
`Crystallogr.
`277-280,
`T.
`MAJOR,
`R.
`BONNETT,
`of
`acetyl
`and
`properties
`levo-p-methylcholine
`chloride.
`57:
`2125-2130,
`Soc.
`1935.
`G. A.
`M.,
`S(cid:1)tm,
`MARTIN-SMITH,
`STENLAK.E,
`AND
`conformational
`role
`possible
`of
`J.
`B.:
`the
`biological
`actions
`of
`acetyl-
`in
`isomerism
`1967.
`choline.
`J. Pharm.
`Pharmacol.
`561-589,
`19:
`F. W.:
`analogs
`Two
`cyclic
`of
`acetyl-
`SCHUELER,
`J.
`Amer.
`Pharm.
`Ass.
`Ed.
`45:
`choline.
`Sci.
`197-199,
`1956.
`Snxur(cid:1),
`A. A.
`(cid:1)eo
`HOLLAND,
`certain
`of
`pressor
`effect
`of
`choline.
`J. Pharmacol.
`318, 1961a.
`A. A.
`SEKUL,
`AND
`of
`senecioylcholine.
`171-175,
`132:
`A. A.,
`SEKuL.,
`Relationship
`intensity
`Arch.
`1963.
`SIMONART,
`of choline.
`1932.
`L.,
`W.
`E.
`SMISs(cid:1),
`NELSON,
`DAY,
`L.:
`Conformational
`AND
`receptor
`sites.
`The
`acetylcholine
`methylammonium-2-acetoxy-trans-decalin
`lides
`and
`the
`isomeric
`a,fl-dimethylacetylcholine
`J. Med.
`9:
`halides.
`Chem.
`458-465,
`1966.
`SNEDECOR, G. W.:
`Statistical
`Methods,
`1956.
`Iowa
`State
`College
`Press,
`Tiuaoi(cid:1),
`D.
`J.:
`of Autonomic
`Chemical
`Nervous
`System,
`pp.
`Academic
`Press,
`New
`York,
`1965.
`WASER,
`P. G.:
`muscarine,
`pounds.
`
`W. C.:
`unsaturated
`Ther.
`Exp.
`
`Comparative
`esters
`acid
`313-
`133:
`
`HOLLAND,
`J.
`
`W. C.:
`Pharmacol.
`
`Pharmacology
`Exp.
`
`Ther.
`
`1961b.
`HOLLAND,
`between
`of nicotine
`like
`Pharmacodyn.
`
`Int.
`
`(cid:1)xi
`
`HOUSE,
`C.
`W.
`structure
`electronic
`choline
`of
`action
`Th#{233}r.141:
`
`R.:
`and
`esters.
`404-411,
`
`A.: On
`the
`J. Pharmacol.
`
`action
`
`of
`Exp.
`
`certain
`Ther.
`
`derivatives
`46:
`157-193,
`
`E.,
`J.
`
`LAPnrns,
`aspects
`isomeric
`
`J. B.
`of
`
`3-tn-
`ha-
`
`5th
`
`ed.,
`
`Ames,
`Aspects
`74-165,
`
`Chemistry
`muscarone
`Pharmacol.
`
`Rev.
`
`and
`
`and
`13:
`
`pharmacology
`some
`related
`465-515,
`1961.
`
`of
`com-
`
`(cid:1)
`(cid:1)
`(cid:1)