58
`Interactions,
`Chem-Biol
`Elsevier Scientific Publishers
`
`277-288
`(1986)
`Ireland Ltd.
`
`277
`
`QUANTITATIVE
`MICROSOMAL
`
`BETWEEN STRUCTURE AND
`RELATIONSHIPS
`OXIDATION
`RATE OF 1,4-DIHYDROPYRIDINES
`
`CATHARINA
`
`BAARNHIELM*
`
`and CHRISTER WESTERLUND
`
`Hiissle
`
`Cardiovascular
`
`Research
`
`Laboratories,
`
`S-413
`
`83 Mijlndal
`
`(Sweden)
`
`lOth, 1986)
`(Received February
`(Revision
`received May 9th. 1986)
`(Accepted May 12th, 1986)
`
`SUMMARY
`
`1,4-dihydropyridine
`substituted
`rate of a series of 4-phenyl
`The oxidation
`esters was investigated
`in dog
`liver microsomes
`and was evaluated
`in relation
`to physiochemical
`properties
`as well as to antihypertensive
`effect. The
`relative
`rate of microsomal
`oxidation
`was mainly
`dependent
`on the substi-
`tuents of the aromatic
`ring and almost unaffected
`by small changes
`in
`the
`ester substituents.
`A 20-fold
`variation
`in the microsomal
`oxidation
`rate was
`observed within
`a group of dichloro-substituted
`analogues. Generally,
`the
`highest oxidation
`rate was found with 2’,6’-disubstituted
`derivatives, while
`compounds
`with substituents
`in position
`4’ exhibited
`longer half-lives.
`The
`oxidation
`rate
`increased with
`increased
`steric bulk,
`increased
`lipophilicity
`and
`increased
`electronwithdrawal
`of
`the substituents
`in position
`2’. The
`energies of the highest occupied molecular
`orbital
`(HOMO)
`were calculated
`and correlated
`with
`the oxidation
`rate of some of
`the dihydropyridine
`analogues.
`The antihypertensive
`effect appeared
`to be restricted
`to com-
`pounds with oxidation
`rates within
`a narrow
`range,
`indicating
`the unlikeli-
`hood of increasing
`the duration
`of the pharmacologic
`effect by stabilisation
`of the dihydropyridine
`system.
`
`1,4-Dihydropyridines
`Ke?l words:
`structure
`- QSAR
`
`- Microsomal
`
`oxidation
`
`- Chemical
`
`molecular
`occupied
`highest
`HOMO,
`Abbreviations:
`refraction;
`MW, molecular
`Verloop
`[ 201; MR, molar
`SHR, spontaneously
`hypertensive Okamoto
`strain
`rats.
`
`length
`L,
`orbital;
`weight; PLS, partial
`
`to
`according
`least squares;
`
`0009-2’i97186/$03.50
`Publishers
`i 1986 Elsevier Scientilic
`Printed
`and Published
`in Ireland
`
`Ireland
`
`Ltd.
`
`Ex. 1052-0001
`
`

`
`278
`
`INTRODUCTION
`
`and
`nifedipine
`diesters,
`1,4-dihydropyridine
`substituted
`The 4-phenyl
`antagonist
`type. These
`agents of
`the calcium
`felodipine,
`are vasodilating
`low bioavailability
`and a relatively
`short elimina-
`drugs are known
`to have
`tion
`half-life,
`findings which are attributed
`to high presystemic
`biotrans-
`formation
`in the
`liver
`[1,2].
`The metabolism
`of these 1,4-dihydropyridines
`has been studied
`both
`in vivo and
`in vitro. No unchanged
`drug has been
`recovered
`in urine
`in vivo, and
`identified
`metabolites
`have been
`in
`the
`oxidized
`pyridine
`form
`[3-6].
`In vitro,
`in liver microsomes
`from
`rat, dog
`and man,
`the oxidation
`of felodipine
`to
`its corresponding
`pyridine was the
`primary metabolic
`step and
`this oxidation
`was shown
`to be catalyzed
`by
`both
`the NADPH
`and the peroxide
`supported
`action of cytochrome
`P-450
`[7-g].
`August0
`et al.
`[lo]
`has demonstrated
`the
`involvement
`of cyto-
`chrome P-450
`in the oxidation
`of various alkylsubstituted
`1,4-dihydropyri-
`dines.
`was to develop
`investigation
`the present
`of
`The objective
`approach
`to
`the selection
`of new drugs with a more
`favourable
`kinetic
`profile
`than
`those
`currently
`available.
`The
`relationship
`microsomal
`oxidation
`rate and chemical
`structure
`was studied
`thirty different
`1,4-dihydropyridine
`derivatives.
`in
`of the parent drug
`The rate constant,
`k, and half-life
`of disappearance
`liver microsomes
`were chosen as relative measures of oxidation
`rate
`in vivo,
`the
`rationale
`for
`this, stemming
`from a previously
`ascertained
`correlation
`between
`in vitro and
`in vivo pharmacokinetics
`for
`felodipine
`[ 91. Since
`the
`pyridine metabolites
`formed
`are almost
`inactive
`pharmacologically
`[ 111,
`an attempt
`has also been made
`to correlate
`biological
`oxidation
`rate with
`antihypertensive
`effect.
`
`a rational
`pharmaco-
`between
`in about
`
`MATERIALS
`
`AND
`
`METHODS
`
`felodipine
`
`Materials
`4-(2,3-dichlorophenyl)-1,4-
`including
`dihydropyridines,
`Most
`ester,
`acid, 5-ethyl
`3-methyl
`dihydro-2,6-dimethyl-3,5-pyridinedicarboxylic
`of Organic Chemistry,
`AB Hassle using
`were prepared
`at
`the Department
`the Hantzsch
`synthesis
`[12]
`and starting with suitably
`substituted
`benzal-
`dehydes, P-keto esters and amino
`crotonates.
`Compound
`13 was obtained
`by condensation
`of 2,3-dichlorobenzaldehyde
`with methyl
`propiolate
`and
`ammonia,
`and 5 from 2,3-dichlorobenzaldehyde,
`methyl
`acetoacetate
`and
`amino crotononitrile.
`from Boehringer,
`and NADP were purchased
`Isocitrate
`dehydrogenase
`was obtained
`from
`and
`trisodium
`isocitrate
`Mannheim
`GmbH
`(F.R.G.)
`Sigma Chemical
`Co.
`(St. Louis, MI). All other
`chemicals
`were standard
`commercial
`products
`of analytical
`grade.
`
`Ex. 1052-0002
`
`

`
`279
`
`oxidation
`Microsomal
`to
`two male Beagle dogs, according
`from
`prepared
`Dog
`liver microsomes
`at
`Ernster et al.
`[13] with some modifications,
`were pooled
`and
`incubated
`37°C with a NADPH-generating
`system
`in 50 mM Tris-HCl
`buffer
`(pH 7.5)
`in a total
`volume
`of 2 ml, as previously
`described
`[ 7,9]. The microsomal
`protein
`concentration
`was 1 mg/ml, measured
`according
`to Lowry et al.
`[14]. The
`reaction was initiated
`with
`the substrate
`to a final concentration
`of 1 PM and
`terminated
`at various
`times by addition
`of 2 ml of toluene.
`
`Calculations
`in
`tl/, , of the substrate
`the half-life,
`k, and
`rate constant,
`The elimination
`logarithmic
`plots of the
`incubations
`were calculated
`from
`the microsomal
`of parent drug versus time and based on first order kinetics.
`disappearance
`The energies of
`the HOMO
`depicted
`in Table
`II were calculated
`using
`the
`QPCE274
`CNDO molecular
`orbital
`program
`[ 151. The values are expressed
`as atomic units,
`least squares
`data analysis was carried out using partial
`The multivariate
`of Wold et al.
`modelling
`with
`latent variables
`(PLS), according
`to the method
`position
`of
`[ 161. The physicochemical
`descriptors
`used for each substituent
`[ 171; (2) the
`the aromatic
`ring were as follows:
`(1) relative
`lipophilicity
`II
`electronic
`parameters
`u [ 181 and A, where A represent
`the substituent
`effect
`on pKa
`for substituted
`anilines
`[Arne Brandstrom,
`personal
`communica-
`tions];
`(3) the steric parameters MR
`(molar
`refraction)
`[19], MW (molecular
`weight) and L (length according
`to Verloop)
`[ 201.
`
`drug
`of unchanged
`determination
`Quantitative
`by
`studied were determined
`the 1,4-dihydropyridine
`derivatives
`Most of
`gas chromatography
`with electron
`capture detection,
`after solvent extraction
`as described
`previously
`for
`felodipine,
`1,
`[7,21]
`but with slight modifica-
`tions
`in
`the
`temperature
`programming.
`The minimum
`measurable
`concentra-
`tion was <15
`nmol/l
`except
`for
`the nitrosubstituted
`analogues
`(5 nmol/l).
`The standard deviation was 3% at 1 PM and 10% at the lower
`limit
`of deter-
`mination.
`Compounds
`3, 14 and 30 were determined
`using a gas chromato-
`graph with a mass selective detector
`and a micro
`computer working
`station
`(Helwett-Packard).
`
`registration
`after oral admini-
`potency was determined
`in vivo antihypertensive
`of compound
`to rats of
`the spontaneously
`hypertensive
`Okamoto
`(SHR),
`as described
`previously
`for
`felodipine
`[22; B. Karlsson,
`pers.
`
`Effect
`The
`stration
`strain
`comm.].
`
`RESULTS
`
`oxidation
`The microsomal
`the half-life
`of disappearance
`
`and
`rate was expressed as the rate constant
`of unchanged
`drug. Felodipine,
`1, was used as
`
`Ex. 1052-0003
`
`

`
`280
`
`I
`
`TABLE
`OF ALTERATION
`EFFECT
`ON THE MICROSOMAL
`
`IN SUBSTITUENT
`OXIDATION
`RATE
`
`IN THE DIHYDROPYRIDINE
`
`RING
`
`Corn-
`pound
`,-lo.
`
`Position
`
`of the substituents
`
`2’
`
`3’
`
`2
`
`3
`
`4
`
`5
`
`1
`2
`3
`-4
`5
`6
`7
`8
`9
`10
`11
`12
`13
`
`Cl
`Cl
`Cl
`Cl
`Cl
`NO,
`NO,
`NO,
`H
`H
`Cl
`Cl
`. Cl
`
`Cl
`Cl
`Cl
`Cl
`Cl
`H
`H
`H
`NO,
`NO,
`Cl
`Cl
`Cl
`
`CH,
`CH,
`CH,
`W
`W
`CH,
`CH,
`CH,
`CH,
`CH,
`CH,
`CH,
`H
`
`COOCH,
`COOC,H,
`COOCH,
`COOCH,
`COOCH,
`COOCH,
`COOCH,
`COOC,H,
`COOCH,
`COOC,H,
`COOCH,
`COO&H,
`COOCH,
`
`a Low or no antihypertensive
`
`effect.
`
`H COOC,H,
`H COOC,H,
`D COOC,H,
`H COOCH(CH,),
`H CN
`H COOCH,
`H COOC,H,
`H COOC,H,
`H COOC,H,
`H COOC,H,
`H COOC,H,
`H COOC,H,
`H COOC,H,
`
`fY*
`(min)
`
`k
`(min.‘)
`
`7
`7
`7
`7
`3
`7
`6
`6
`6
`7
`7
`128
`I
`
`0.099
`0.099
`0.099
`0.099
`0.231
`0.099
`0.116
`0.116
`0.116
`0.099
`0.099
`0.058
`0.099
`
`6
`
`CH,
`CK
`CH,
`W
`CH,
`CH,
`W
`CH,
`CH,
`CH,
`CWCH,),
`CH(CH,),
`H
`
`was
`liver microsomes
`in dog
`the mean half-life
`and
`compound
`a reference
`repro-
`recorded
`to be 7 f 1 min
`(3c + S.D.; n = 30). The results were highly
`ducible
`and
`the half-life
`was not affected
`by storage of
`the microsomal
`preparations
`for up
`to 6 months
`at
`--80°C. The 1,4-dihydropyridine
`analogues were divided
`into
`two different
`groups based on
`the chemical
`structure.
`The
`structures
`of derivatives
`with
`various
`substituents
`in
`the
`dihydropyridine
`ring and
`their
`corresponding
`h and
`tlh values are listed
`in
`Table
`I. Table
`II shows the effect on oxidation
`rate of different
`substituents
`in
`the aromatic
`ring. The values given are a mean of two experiments.
`The
`numbering
`of substituent
`positions
`is shown
`in the formula
`at the top of the
`tables.
`rate when
`oxidation
`the microsomal
`effect was observed on
`isotope
`No
`1, was replaced with deuterium,
`3,
`in position
`4 of felodipine,
`the hydrogen
`is not
`involved
`in
`the rate limiting
`removal
`of this hydrogen
`indicating
`that
`step of the oxidation.
`The oxidation
`rate was generally
`unaltered when
`the
`ester substituents
`were changed
`from dimethyl
`to diethyl
`to methyl-ethyl
`
`Ex. 1052-0004
`
`

`
`281
`
`TABLE
`
`II
`
`OF
`
`EFFECT
`OXIDATION
`DINE
`DIESTERS
`
`SUBSTITUENTS
`RATE
`OF
`
`IN
`
`AROMATIC
`THE
`4-PHENYL
`
`RING
`SUBSTITUTED
`
`ON
`
`MICROSOMAL
`THE
`1,4-DIHYDROPYRI-
`
`VARIOUS
`
`The
`
`calculated
`
`HOMO
`
`energies
`
`are also shown
`
`for
`
`some
`
`compounds.
`
`4+--q
`
`Com-
`pound
`no.
`
`1
`7
`9
`14a
`15
`
`16
`17
`18
`19
`20
`21
`22
`23
`24
`25
`26
`21
`28
`29
`30
`31
`
`Position
`
`of
`
`the
`
`substituents
`
`2’
`
`Cl
`NO,
`H
`H
`OCH,
`phenyl
`
`Cl
`H
`H
`Cl
`Cl
`Cl
`H
`H
`Cl
`NO,
`Cl
`Br
`Br
`Cl
`CH,
`Cl
`
`3’
`
`Cl
`H
`NO,
`H
`H
`
`H
`Cl
`H
`H
`H
`H
`Cl
`Cl
`O-I.\
`Cl
`CH,
`Cl
`Br
`Br
`CH,
`OCH,
`
`4’
`
`H
`H
`H
`H
`H
`
`H
`H
`Cl
`H
`H
`Cl
`Cl
`H
`H
`H
`H
`H
`H
`H
`H
`H
`
`5’
`
`H
`H
`H
`H
`H
`
`H
`H
`H
`H
`Cl
`H
`H
`Cl
`H
`H
`H
`H
`H
`H
`H
`H
`
`6’
`
`H
`H
`H
`H
`H
`
`H
`H
`H
`Cl
`H
`H
`H
`H
`Cl
`H
`H
`H
`H
`H
`H
`H
`
`HOMO
`(atomic
`units)
`
`-0.3886
`
`-0.3859
`
`-0.3823
`-0.3897
`-0.3902
`-0.3915
`-0.3909
`-0.3805
`
`%
`t min)
`
`k
`(mine’
`
`)
`
`7
`6
`6
`7b
`3
`
`5
`5
`lib
`lb
`4
`llh
`13b
`19b
`lb
`3
`‘I
`7
`6
`6
`5
`9b
`
`0.099
`0.116
`0.116
`0.099
`0.231
`
`0.138
`0.138
`0.063
`0.693
`0.173
`0.063
`0.053
`0.036
`0.693
`0.231
`0.173
`0.099
`0.116
`0.116
`0.139
`0.077
`
`* Diethyl
`b Low
`
`not
`and
`ester
`or no antihypertensive
`
`included
`
`in
`effect.
`
`the PLS
`
`analysis.
`
`of an ester with
`replacement
`I). However,
`esters (Table
`or methyl-isopropyl
`a cyan0 group decreased
`the half-life
`from 7, 1, to 3 min, 5. Small changes
`in the substituents
`in positions
`2 and 6 had little effect on the oxidation
`rate,
`with
`the exception
`of the 6-isopropyl
`derivative,
`12.
`in this respect
`important
`The substituents
`of the aromatic
`ring were more
`(Table
`II). The oxidation
`rate varied significantly
`with
`the position
`of chloro
`
`Ex. 1052-0005
`
`

`
`282
`
`TIME min
`of felodipine,
`1. ( .), the 2’,6’-dichloro
`23,
`(- ) in
`incubations
`with
`dog
`
`19, (0)
`derivative,
`substituted
`liver microsomes
`(described
`in
`
`Fig. 1. Elimination
`and
`the 3’,5’-isomer,
`Methods).
`
`19, was
`derivative,
`the 2’,6’-dichlorosubstituted
`For example,
`substituents.
`of 1, compared
`to
`the 19 min of the 3’,5’-isomer,
`shown
`to have a half-life
`23
`(Fig. 1). There was a linear
`relationship
`(r = 0.89, P < 0.02)
`for
`the
`dichloro-substituted
`compounds
`between
`rate constant,
`k, and the electronic
`descriptor
`A
`(Fig. 2).
`If, however,
`the corresponding
`values
`for all
`the
`dihydropyridine
`analogues of Table
`II were considered,
`the correlation
`was
`weak
`.(r = 0.64).
`As an alternative
`approach,
`the energy of
`the highest
`occupied molecular
`orbital, HOMO, was calculated
`for a limited
`number
`of
`chloro-substituted
`analogues,
`I,1 7,19-24,
`(Table
`II) and correlated
`against
`k (r = 0.93, P< 0.001; Fig. 3).
`on biologi-
`structure
`of chemical
`influence
`In order
`to
`further
`clarify
`the
`data analysis was applied.
`The descriptors
`cal oxidation
`rate, a multivariate
`of each substituent
`in the various positions
`of the aromatic
`ring
`(2’,3’,4’,5’
`and 6’; Table
`II) were
`the
`relative
`lipophilicity,
`the electronic
`descriptors,
`u, A, and
`the steric parameters MW, MR and L, as stated
`in the calculations.
`The substituent
`positions
`2’ and 6’, and also 3’ and 5’, are treated as non-
`equivalent,
`although
`the
`free rotation
`around
`the peri bond
`[23] makes
`this
`approach,
`of course, somewhat
`ambiguous.
`The rule
`followed
`for assignment
`is that with only one ortho-substituent
`or only one meta-substituent,
`the
`substituent
`is assigned
`to the 2’ or the 3’ position,
`respectively.
`Similarly,
`for
`disubstituted
`derivatives,
`the positions
`3’,4’ and 2’,5’ are those used instead
`
`Ex. 1052-0006
`
`

`
`Y
`
`01
`0
`1
`
`o.o-
`
`\
`
`-0.5
`
`-1.
`
`o-
`
`-1.
`
`s--
`-4
`
`log
`
`A
`
`Fig. 2. The electronic
`some
`dichlorosubstituted
`structures
`listed
`in Table
`
`II.
`
`A, versus the microsomal
`descriptor,
`1,4-dihydropyridine
`analogues.
`
`283
`
`rate constant,
`oxidation
`The numbers
`refer
`
`h, for
`to
`the
`
`0.8,
`
`0.6
`
`0.4
`
`0.2
`
`T-3
`I
`C
`'2
`
`Y
`
`0.0
`-0.392
`
`-0.388
`
`-0.384
`
`-0.380
`
`HOMO
`1,4-dihydropyridine
`of some chloro-substituted
`HOMO
`energies
`Fig. 3. The calculated
`derivatives
`versus
`the microsomal
`oxidation
`rate constant,
`h. The numbers
`refer
`to the
`structures
`listed
`in Table
`II.
`
`Ex. 1052-0007
`
`

`
`284
`
`OBSERVED
`
`log
`
`k
`
`constant
`k, versus the predicted
`rate constant,
`oxidation
`Fig. 4. The observed microsomal
`of some 1,4-dihydropyridine
`analogues.
`The
`obtained
`after multivarite
`PLS analysis
`numbers
`refer
`to the structures
`listed
`in Table
`II. *Compound
`19 and 24 have the same
`values.
`
`A PLS analysis of log k versus the descrip-
`of 4’, 5’ and 3’, 6’, respectively.
`in one highly
`significant
`equation,
`which
`tors mentioned
`above
`resulted
`reduced
`the sum of variance
`to 79%, comparable
`to
`r = 0.9. This
`result
`is
`presented
`as measured
`versus predicted
`rate constants
`(Fig. 4). The para-
`meters
`(loadings)
`describing
`the
`influence
`of the various variables are listed
`in Table
`III.
`These parameters
`cannot
`be used as regular
`regression
`coef-
`
`III
`TABLE
`THE PLS ANALYSIS
`FROM
`CALCULATED
`PARAMETERS
`THE DESCRIPTOR
`ring, 0.03; x A for all substituents
`of the aromatic
`of the aromatic
`x A for all substituents
`ring, -0.22;
`log k for oxidation
`to pyridine,
`1.0.
`
`Descriptor
`
`Positions’
`
`2’
`
`0.14
`0.08
`-0.16
`0.13
`0.16
`0.15
`
`3’
`
`-0.10
`-0.09
`0.09
`0.0002
`-0.04
`0.02
`
`4’
`
`-0.19
`-0.19
`0.19
`-0.19
`-0.19
`-0.19
`
`5’
`
`-0.10
`-0.09
`0.09
`-0.09
`-0.09
`-0.09
`
`6’
`
`0.30
`0.30
`-0.30
`0.30
`0.30
`0.30
`
`ll
`
`CJ
`A
`MR
`MW
`L
`
`Ex. 1052-0008
`
`

`
`relative
`their
`[16] but
`ficients
`rate.
`oxidation
`biological
`the rate of oxidation
`When
`compounds
`could
`be divided
`potency;
`intermediate
`half-life
`
`size is proportional
`
`to
`
`their
`
`influence
`
`on the
`
`the
`effect,
`to the antihypertensive
`was related
`low
`into
`three
`groups:
`short half-life
`.-
`- high potency;
`long half-life
`-
`low potency.
`
`DISCUSSION
`
`conditions
`to oxidative
`are quite sensitive
`In general, 1,4-dihydropyridines
`in
`has been described as being very unstable
`[12] and
`the parent
`compound
`air. Electron withdrawing
`substituents
`in the 3- and 5-positions
`are thought
`to have a stabilising
`effect by means of conjugative
`interactions.
`The exact
`mechanism
`of oxidation,
`especially
`for NADH
`and related
`compounds,
`has
`been much disputed
`[24,25].
`Thus,
`it
`is not clear whether
`two consecutive
`one electron
`transfers
`or one
`two electron
`transfer
`is involved
`[&lo].
`Efforts
`to correlate
`the oxidation
`of 1,4-dihydropyridines
`with chemical
`structure
`have been reported
`[26-281.
`Thus
`for electrochemical
`oxidation,
`the electronic
`properties
`of
`the 3- and 5-substituents
`and
`the steric effects
`of the 4-substituent
`appear
`to be important.
`In biological
`systems, however,
`the results are more ambiguous
`and
`it has not been possible
`to predict
`such
`results
`from electrochemical
`data.
`varia-
`limited
`that
`is evident
`it
`this study,
`From
`the
`results presented
`in
`oxidation.
`influence
`the rate of microsomal
`tion
`in
`the ester groups does not
`This agrees with previous data of electrochemical
`oxidations
`[ 271. However,
`in contrast,
`exchange of COOEt
`by CN (1 vs. 5) increases
`the rate of oxida-
`tion
`by a
`factor
`of 2. This
`variation
`of substituents
`would have been
`expected
`to give a decreased oxidation
`rate when based on purely electronic
`considerations.
`of
`influence
`the
`towards elucidating
`directed
`This study has been mainly
`oxidation.
`It was
`substituents
`of the aromatic
`ring on the rate of microsomal
`isomers were
`found
`that
`the
`rate constant
`of some dichloro-substituted
`A. The
`reason
`for
`reasonably well correlated
`to
`the electronic
`descriptor,
`electronic
`descrip-
`using A in this context,
`instead of the more conventional
`It was found by
`tor u, is the
`lack of tabulated
`values
`for ortho substituents.
`in a more
`rapid
`these means
`that an increased electron-withdrawal
`resulted
`rate of oxidation,
`which again
`is contrary
`to what one would expect. How-
`ever,
`in
`this
`limited
`series,
`the steric
`influence
`of the substituents
`probably
`co-varies
`to some extent with A. The results
`indicate,
`however,
`that
`factors
`other
`than electronic
`ones are important
`when considering
`a larger group of
`compounds.
`has been stated as being a measure of
`energy of a compound
`The HOMO
`its ease of oxidation
`[29]. As is evident
`from Fig. 3, a correlation
`although
`with somewhat
`poor distribution
`of points, was found
`between biological
`oxidation
`rate and
`the calculated HOMO
`energies
`for some chlorine
`contain-
`ing derivatives.
`Efforts
`to extend
`this
`limited
`series, failed, however,
`indicat-
`ing
`the
`restricted
`applicability
`of
`this parameter
`for predictive
`purposes.
`
`Ex. 1052-0009
`
`

`
`286
`
`the
`and
`rate constant
`the oxidation
`between
`correlation
`A significant
`by the
`ring, was obtained
`the aromatic
`descriptors
`for each substituent
`in
`to inter-
`somewhat
`difficult
`PLS analysis. The results
`(Fig. 4) are, however,
`pret due to
`the
`lack of variation
`in positions
`4’,5’ and 6’. Thus,
`the descrip-
`tors
`function mainly
`as indicator
`variables
`in these positions,
`i.e. describing
`only
`if
`the
`relevant
`position
`is substituted
`or not. Substitution
`at
`the 6’-
`position
`appears
`to give a high
`rate of oxidation,
`probably
`due to unfavour-
`able steric
`interactions
`with
`the dihydropyridine
`ring, while substituents
`in
`the 4’-position
`reduce
`the oxidation
`rate. With
`regard
`to
`the 2’-position,
`all
`descriptors
`appear
`to be of some
`importance,
`indicating
`that
`the
`rate of
`oxidation
`increases with
`increasing
`electron withdrawal,
`increasing
`steric
`bulk and
`increasing
`lipophilicity.
`The nature of the substituents
`in the 3’- or
`5’-positions,
`however, does not seem
`to be important.
`The slow oxidation
`of the 3’,5’-dichloro
`derivative,
`23, can possibly be interpreted
`as the result
`of a stacking of steric bulk close to the more sensitive 4’-position.
`These con-
`clusions were based on
`the PLS-analysis
`(Table
`III)
`despite
`the possible
`objection
`that
`the correlation
`shown
`in Fig. 4 only describes a relationship
`between
`three groups of compounds,
`i.e. those with 6’-substituents
`(high h),
`those with
`4’-substituents
`(low h) and
`those with
`substituents
`in other
`positions
`of the aromatic
`ring (intermediate
`k).
`electron
`that an oxidative
`Recently, Marinov
`and Saxon
`[30] proposed
`with
`transfer
`is involved
`in the mechanism
`of action
`for 1,4-dihydropyridines
`although
`antihypertensive
`effect. The
`results
`from
`the present
`investigation,
`since an
`with a limited
`number
`of compounds,
`may support
`this hypothesis
`interesting
`pattern
`emerges when comparing
`the oxidation
`rate with pharma-
`cological
`activity.
`The
`two compounds
`exhibiting
`very rapid oxidation,
`19
`and 24, showed only a low
`in vivo antihypertensive
`effect
`in SH-rats. This
`could be expected when
`the metabolic
`turnover
`into
`inactive pyridines
`is an
`extremely
`rapid process. More
`intriguing,
`however, was the additional
`fact
`that every compound
`with a slow rate of oxidation,
`12, 18, 21, 22, 23 and
`31, demonstrated
`a low
`in vivo potency. This would
`imply
`that oral activity
`in this
`type of compound
`is restricted
`to a certain
`range of oxidative
`ability.
`Such an implication
`could have
`far reaching
`consequences
`for
`the develop-
`ment of new drugs of this category.
`It,
`thus, seems difficult
`to
`increase
`the
`duration
`of effect by increasing
`the biological
`half-life
`through
`stabilisation
`of the dihydropyridine
`system,
`since
`this would also ultimately
`result
`in a
`lowering of the antihypertensive
`potency.
`on the microsomal
`structure
`In conclusion,
`the
`influence
`of the chemical
`oxidation
`rate of 4-phenyl
`substituted
`1,4-dihydropyridines
`have been
`studied. A 20-fold
`variation
`in microsomal
`oxidation
`rate was observed and
`was attributable
`to
`the positioning
`of chloro-substituents
`in
`the aromatic
`ring.
`It
`is evident
`that
`for a series of rather
`closely
`related
`compounds,
`the
`biological
`oxidation
`rates can be predicted
`from one physiochemical
`para-
`meter, e.g. the electron
`descriptor
`A or HOMO, while
`the multivariate
`data
`analysis
`is more useful
`for a more differentiated
`group of analogues.
`
`Ex. 1052-0010
`
`

`
`ACKNOWLEDGEMENTS
`
`for
`acknowledged
`are greatfully
`and Erik Johansson
`Drs. Peder Berntsson
`their kind
`interest
`in
`this work and Dr Inger K5llsson
`for calculation
`of the
`HOMO
`energies.
`
`287
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