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`0090-9SS6/88/160S~773$02.00/0
`I>RU<J METABOLISM AND DisPOSITION
`Copyriabt C 1988 by The American Society for Pharmacology and Experimental Therapeutics
`
`Vol. 16. No.5
`Printed in U.S.A .
`
`Short Communication
`
`Urinary Metabolites of Rimantadine in Humans
`
`Rimantadine (I; table I )1 is an agent used in the treatment (I)
`and prophylaxis (2) of influenza caused by the type A strain of
`virus. Rimantadine, which bas an asymmetric center and is given
`as the racemate, is an investigational drug in the United States
`but bas been routinely prescribed for many years in the USSR
`(3). Without giving data, Van Voris eta/. (3) and Hayden eta/.
`(4) have both reported that rimantadine is extensively metabo(cid:173)
`lized in man to all possible hydroxylated metabolites (2a, lb, 3,
`4a, and 4b; table 1). Suprisingly, amantadine (5), an antiviral
`agent structurally similar to rimantadine, is virtually unmetabol(cid:173)
`ized when given to humans (5).
`This communication reports the determination of the urinary
`metabolites generated after a 200-mg single oral dose of riman(cid:173)
`tadine • HO, containing 0.25 I'Cifmg of methyl-labeled [I~]
`rimantadine, to four male volunteers. The [l~]rimantadine was
`synthesized from adamantane--1-carbonyl chloride and e~
`methyl cadmium by Dr. A. Liebman, Isotope Synthesis Group,
`Hoffmann-La Roche Inc. Conversion to rimantadine using
`standard procedures yielded a product having radiochemical
`purity >97% and a specific activity of 54.9 mCi/mmol.
`Urine samples were coUected from each subject for a minimum
`of 72 hr post-dose. Urine volumes were measured. Two tenths
`to I ml of urine were mixed with 10 ml Aquasol (New England
`Nuclear, Boston, MA) containing I% glacial acetic acid, and
`radioactivity was measured on a Beckman Model LS 380 I
`counter using the external channel ratio technique. These meas(cid:173)
`urements indicated that 88, 75, 93, and 80% of the radioactivity
`was excreted in 72 hr for subjects I, 2, 3, and 4, respectively.
`Eight ml of individual urine samples (or 8 ml of representative
`pooled urine samples) were adjusted to pH 5.5 with 2 N Ha
`and incubated at 37•c for 18 hr with 80 IIi of Glusulase (Dupont
`Pharmaceuticals, Wilmington, DE). One half ml of 5 N NaOH
`was added to 2-ml samples (individual and pooled, with and
`without the Glusulase treatment), and the samples were extrac(cid:173)
`tively benzoylated to ensure that all of the rimantadine and
`metabolites were extracted in a stable form. The procedure
`involved treatment of 2 ml of urine with 8 ml of a solution of
`cyclobexane saturated with triethanolamine/chloroform (2: I),
`foUowed by 100 1£( of cyclobexane containing 2% pentafluoro(cid:173)
`benzoyl chloride ( 6 ). Samples were shaken at 30 strokes min -•
`for 20 min and centrifuged at 1500g, and 7.5 ml of the top layer
`were transferred to 16-ml screw-capped culture tubes. The or(cid:173)
`ganic solvents were removed under a stream ofN:z(g) at so·c (N(cid:173)
`EV AP; Organomation Associates, South Berlin, MA), and the
`
`residues were reconstituted in 160 #'I of heptane/2-propanol
`(95:5). These solutions were transferred to WISP vials equipped
`with conical inserts and were analyzed by LC-radioactivity mon(cid:173)
`itoring, LC-MS, and TLC-radioactivity monitoring.
`The LC-radioactivity monitoring system consisted of two
`Waters M6000A pumps, a Waters WISP autoinjector, a Waters
`model 720 system controUer (Water Associates, Millipore Divi(cid:173)
`sion, Milford, MA), and a Ro-one/Beta Model IC Radioactivity
`Monitor (Radiomatic Instruments and Chemical Co., Inc.,
`Tampa, FL) to detect and quantitate radioactivity in the HPLC
`eluent. The analytical column was a prepacked Zorbax silica, 4.6
`mm x 25 em (Dupont Instruments, Wilmington, DE). The
`mobile phase consisted ofbexane/2-propanol (95:5) at a constant
`flow of 1.5 ml min-• for 10 min, foUowed by a linear gradient to
`bexane/2-propanol (90: I 0) over 20 min with a 5-min reequili(cid:173)
`bration at initial conditions.
`The LC-MS system consisted of an ISCO ~'LC-500 pump, a
`Rheodyne injector with a 5-I£( loop, a I mm x 25 em silica
`column (Brownlee Labs, Santa Clara, CA). and a modified
`Finnigan I 0 15 mass spectrometer equipped with a high speed
`pumping system (a Varian VHS-6 diffusion pump was used in
`the source region and a Varian VHS-4 diffusion pump was used
`in the analyzer region). The emuent from the LC was connected
`to the mass spectrometer using 36 in. of 60 "m (i.d.) flexible
`fused quartz tubing from SGE (Austin, TX). The last 18 in. of
`the quartz tubing were fit through 18 in. of stainless steel tubing
`heated by passage of an electrical current of 10 A at 5 V. The
`flow of bexane/2-propanol (90:10) was 125 "I min-•. The ion
`source temperature was 300•c, and the mass spectrometer was
`tuned to give the maximum response consistent with reasonable
`peak shape and unit resolution. A hoUow (0.25 in. i.d.) stainless
`steel tube connected at one end to a Balzer 18m3 hr- 1 forepump
`was introduced into the high vacuum source region through a
`0.5-in. solid probe inlet and was coupled inside the vacuum
`housing to the ion source to provide additional pumping.
`The TLC plates (silica gel, product 60F-254; E. Merck, Darms(cid:173)
`tadt, FRG) were developed either in system I [chloroform/ethyl
`acetate/ethanol (70:30:1)] or system 2 [bexane/2-propanol
`(90: I 0)]. Radioactivity was detected using a Packard model 720 I
`radioactivity monitor.
`Analysis of the experimental data showed that four distinct
`radioactive species were present, which corresponded to riman(cid:173)
`tadine and three chemicaUy distinct metabolites. Fig. I shows
`the HPLC radiochromatogram containing the four radioactive
`peaks. The identity of each peak was discerned from the analysis
`of MS (LC-MS, EIMS,2 and GC-CIMS) and NMR data.
`ReceMicl o-nber 2. 1987; IICOIIptecl March 31 . 1988.
`Peak I (RT 6.4 miD). The material comprising the peak had an
`' The proper nomenclature (llJ»AC name) for al compooods lls18d in table 1 Is
`as follows: 1 • ...melhytlric:y [3.3.1.1 .. '] dlc:ane-1/kneiiWWnin8; 5. trleydo
`R., of 0.6 in TLC system I and bad chromatographic, mass
`(3.3.1.1 .. ") decln-1tJ.amine; 2a, 1tl-(1~ [3.3.1 .1 .. ')decln-2a-<ll;
`spectral, and NMR properties identical to authentic derivatized
`2b, 1tl-(1~ [3.3.1.1 .. ')decln-Ud; 3, 3a-i1~
`rimantadine.
`[3.3 .1.1 ... ]dec:an-1 tkJI; .... 1 tl-(1-aminoethyl)-trleydo [3.3.1.1 .. "]decan-4a-<11; 4b,
`The LC-MS data from this peak (fig. 2) showed an MH• ion
`1tl-(1 .. nlnoethyf)-trleydo [3.3.1.1 .. ']dec:en-4tkJI. Note that the llb8olute ~
`at m/ z 374, the protonated molecular weight of derivatized intact
`c:hemlstry ol the 1-an .IOelhyiiUbltilua1tls not dellgl1818d.
`rimantadine, and an ion of less intensity corresponding to the
`loss of HF (m/z 354). An ElMS (Vacuum Generators 7070) of
`
`Send ,.,mt ,..._ to: Or. E. K. Fukuda. Depertment ol Drug Metabolism,
`Hoffmann.La Roche Inc .• Nutley, NJ 07110.
`
`1 AbbnMations used are: El. electron Ionization; Cl. chemical Ionization.
`
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`774
`
`RUBIO ET AL.
`
`TABLE I
`Struaures of rimonlOiline, rimanlodine metabolites, and amantadine
`R1
`
`R3
`
`R2
`H
`H
`OH
`H
`H
`H
`H
`
`Rl
`
`CH(CH1)NH2
`NH2
`CH(CH1)NH2
`CH(CH3)NH2
`CH(CH1)NH2
`CH(CH3)NH2
`CH(CH1)NH2
`
`R3
`H
`H
`H
`OH
`H
`H
`H
`
`R4
`H
`H
`H
`H
`OH
`H
`H
`
`R5
`H
`H
`H
`H
`H
`H
`OH
`
`R6
`H
`H
`H
`H
`H
`OH
`H
`
`Name
`Rimantadine
`Amancadine
`See footnote I
`See footnote I
`See footnote I
`See footnote I
`See footnote I
`
`Desipation
`I
`5
`2a
`:Zb
`
`3 ...
`
`4b
`
`500r-------------------------------~
`
`l Q
`
`0~~~~~ . . ~~~~~~~~~~~
`25
`0
`20
`30
`10
`ltETfNTION Tl ME ( ml11uteal
`
`FIG. I. HPLC-rodiochromoJogram of extrocted experimenllll urine
`qft~ Glusula.se IIWJII'I'Iml from a vohmt«r givm radioacti"11~
`rimtlllllldine.
`
`an ethyl acetate extract of the TLC spot showed ions at m/z 373
`+
`(Mt, 2% ofbasepeak), m/z 238 (CF~=O)NH-CH-CH3, 1%),
`+
`m/z 19S (c.Fs-C-o, IS%), m/z 13S (adaiDantyl cation, 100%),
`m/z 107 (m/z 13S- C21L. 7%), and m/z 93 (m/z 13S- CJi6,
`IS%). The formation of the latter three ions from tbe adamantyl
`cation bas been described (7). A GC-CIMS analysis (Fmnigan
`lOIS, methane PCI at 0.5 torr, 4 feet by 2 mm glass column
`packed with 3% SP 22SO on 100-120 mesh GCQ at 21s•q gave
`peaks at m/z 374 (MH+, 100%), m/z 372 (MH•- H2, 13%), m/
`+
`z 3S4 (MH• - HF, 6%), m/z 163 (adamantyl-CH-CH), 78%),
`and m/z 13S (adamantyl cation, 90%).
`The NMR spectrum (Varian XL400, CDCh, 400 MHz. 2TC
`temperature, pulse width equivalent to a 40• flip angle, 2-sec
`pulse-repetition rate) of the material in the peak showed a
`partially resolved doublet at clS.66 (IH, J • 10 Hz; NH), a
`partially resolved eight-peak pattern (doublet of quartets) cen(cid:173)
`tered at &3.92 (IH, J = 7 and 10Hz; HN-CH-CH3), a singlet at
`&2.00 (3H, R4 protons), a complex series of peaks at &1.44 to
`&1.72 (remaining methylene protons plus impurities), and a
`doublet centered at &1.12 (AX, 3H,J• 8Hz; HN-CH-CH3). For
`reference, the &1.2 to &1 .8 region in the NMR spectrum of
`authentic derivatized rimantadine consists of three singlets at
`&1.68, cli.S8, and &1.48 (6H; R2, and R3 protons) and a quartet
`
`centered at &1.58 [AB, 6H, J = 36Hz (geminal coupling); RS
`and R6 protons].
`Based on the above, the material comprising the peak was
`identified as derivatized rimantadine. In the four subjects, intact
`rimantadine acx:ounted for 22 ± 8% of the dose (mean ± SD;
`table 2). Another 10 ± 3% of the dose was conjugated intact
`rimantadine (table 2).
`Peak D (Ry 15.5 Dlin). The material comprising this peak bad
`an R.: of0.3 in TLC system I and an RF of0.2 in TLC system 2
`and bad chromatographic, mass spectral, and NMR properties
`different than either derivatized authentic 3 obtained from Dr.
`P. Mancband, Hoffmann-La Roche (Nutley, NJ) or derivatized
`authentic 21/lb (mixture)3 obtained from Dr. I. Sims, Hoffmann(cid:173)
`La Roche (Nutley, NJ). Authentic 4a/4b was not available. The
`purity of the pentaOuorobenzoyl derivatives of authentic 3 and
`21/lb were checked by TLC.
`The LC-MS analysis of this peak (fig. 2) indicated that it was
`a hydroxylated rimantadine metabolite. An intense MH• - H20
`ion at m/z 372 and a very small MH+ ion at m/z 390 (protonated
`molecular ion of derivatized hydroxylated rimantadine) were
`observed. An EI mass spectrum of an ethyl acetate extract of the
`TLC spot showed ions at m/z 389 (Mt, 3%), m/z 238 (c,F5 -
`+
`+
`q-o)NH- CH-CHJ, 4%), m/z 19S (c.Fs- C • 0, 20%), m/z
`167 (c.Fs•, S%), mfz 1S1 (hydroxy-adamantyl cation, 100%),
`m/z 133 (m/z lSI - H20, 20%), m/z 101 (m/z 133- C2H2,
`9%), m/z lOS (m/z 133 - C2~ 10%), m/z 93 (m/z 133 -
`
`a Properties of pentaftuorobenz Z./2tlare u follows: R,. In TLC sys18m
`1 • 0.4, ElMS llho!Ned Ions (>30'11. Allatl¥e lbundlnce) at m/z 371 (Mt -HaO. 70%
`oft.. peak), mJz 239 (70%), m/z 221 (100'11.), and m/z 195 (80%). The OC-CIMS
`lfllllylia (/ri/Z >100 and > 10% Allatl¥e lbundlnce) g8W two c:hoomatogl aphlc
`peeks. The t1rst c:hoornatognlphlc peak gaw mass spec:lnll peeks at mJz 372
`(100'11.). mJz 178 (12%), and m/z 161 (10%). The seconct chromatogrlphk: peek
`gaw mass spec:lnll peeks at m/z 372 (MW - H,,, 100'11.), mJz 222 {90%), m/z
`178 (13%), mJz 189 (40%), and m/z 161 (25%). TheN~ spectrum, rwllectlng the
`dlut8l eomertc mlxtln, showed two parlilly AIIIOived doublets c:enlllnld at a5.64
`a1d 66.8 (AX, 1 H: NH), two parlilly AIIIOived elghtiJ81k patterns (doublet of quartet)
`centered at M.O and M.31 (1H; HN-CH-CH.), two singlets at a3.74 and a3.64 (H(cid:173)
`C-OH), • complex . . . . of peeks at 61.4 10 42.2 (rwnalnlng methylenu and the
`three ~ proiOns at 61.93), a1d • complex . . . . of peeks centered at
`61.22 (side chlln methyl).
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`ns
`The NMR spectrum of the material in the peak showed a
`partially resolved doublet at 65.70 (IH; NH), a partially resolved
`eight-peak pattern centered at 63.92 ( IH; HN-CH-CH3), a singlet
`at 63.82 (IH; HO-C-H), a singlet at 62.0 (3H; R4 protons), a
`complex series of peaks at 61.2 to 62.0 (remaining methylene
`protons plus impurities), and a doublet centered at 61.14 (AX,
`3H, J = 8 Hz; NH-CH-CHJ). Included in the complex series of
`peaks was a quartet centered at 61.68 (AB, 4H, J = 12 Hz; R5
`and R6 protons) and three doublets at 61.96-, 61.88, and 61.34
`(AB, 2H, J = 12Hz; R2 and R3 protons). The integrated signals
`for the bridgehead and methylene protons were less than ex(cid:173)
`pected, probably due to interferences from residual water and
`ethyl acetate resonances. Based on the above and the data that
`will be presented on peaks m and IV, the material comprising
`peak U was identified as 4b. In the four subjects, this metabolite
`accounted for 13 ± 7% of the dose (table-2);- Another 1.6 ± 0.5%
`of the dose was conjugated 4b.
`Peak m (Ry 19.2 miD). The material comprising the peak had
`an It, of 0.2 in TLC system I and had chromatographic, mass
`spectral, and NMR properties identical to derivatized authentic
`3 .
`The LC-MS analysis of this peak (fig. 2) indicated that it was
`a hydroxylated rimantadine metabolite. An intense MH+ ion at
`mjz 390 and ions corresponding to [MH+- H20) and [MH+(cid:173)
`HrH20] at m/z 372 and m/z 370, respectively, of about a third
`the intensity of the MH+ ion were observed. An EI mass spectrum
`of an ethyl acetate extract of the spot showed ions at m/z 389
`(Mt, 2%), m/z 239 (cy,-{:(OH)NH.CH-CHn 100%), m/z 220
`+
`(m/z239-F,20%),m/z 195(cy5-C=0, 40%),m/z 167(cyt,
`15%), m/z lSI (hydroxy adamantyl cation, SS%), m/z 133 (m/
`z 151 - H20, 30%), m/z 107 (m/z 151 - C21L. 15%), m/z 93
`(m/z lSI - C3~, 35%), and m/z 79 (m/z lSI - C.H,, 19%).
`The intense ion at m/z 239 in the EI mass spectrum can be
`rationalized as the result of direct ionization at the hydroxy
`group with subsequent elimination of oxygenated adamantyl.
`Direct ionization would be made possible by the lower ionization
`potential of the tertiary hydroxy group (8) compared with a
`carbonyl adjacent to a strongly electron-withdrawing group. The
`GC-CIMS analysis gave peaks at m/z 390 (MH+, 80%), m/z 372
`(m/z 390 - H~. 80%), m/z 239 (CF5-t(OH)NH-CH-CHn
`+
`90%), m/z 179 (hydroxy actamantyi-CH(CH3), 100%), m/z 161
`(m/z 179 - H20, 55%), and m/z lSI (hydroxy adamantyl
`cation, 45%).
`The NMR spectrum of the material in the peak showed a
`partially resolved doublet at 65.68 (AX, IH, J • 8Hz; NH), a
`partially resolved eight-peak pattern centered at 64.02 (I H; HN(cid:173)
`CH-CH)), a singlet at 62.25 (2H; R4 protons), a complex series
`of peaks at 61.4 to 61.8 (remaining methylene protons plus
`impurities), and a doublet centemi at 61 .1 S (AX, JH, 8 Hz; HN(cid:173)
`CH-CH3). Included in the complex series of peaks is a quartet
`centered at 61.66 (AB, 4H, J- 12 Hz; RS and R6 protons). An
`unresolved multiplet adjacent to the H20 peak at 6 • 1.45 was
`presumably due to four methylene protons. The remaining
`.. missing" resonances due to another four methylene protons
`were presumed to be underneath the H20 peak. NMR assign(cid:173)
`ments were aided by comparison with authentic derivatized 3
`and with the published NMR spectra of I ,3-disubstituted ada(cid:173)
`mantanes (9).
`Based on the above, the material comprising the peak was
`identified as derivatized 3. In the four subjects, 3 accounted for
`
`RIMANTAOINE URINARY METABOUTES
`
`100
`
`__. \.......-..i
`400
`
`-
`
`0
`
`:so.ooo
`
`20,000
`
`....
`10,000
`
`~
`
`0
`
`~ ...
`iii
`z ... ...
`... > 5 20,000
`..I ... c
`
`""
`
`TIC
`
`.,, 374
`
`.,, 3 72
`
`\.._}\
`
`-
`
`.. ,, 3,0
`
`10,000
`1-
`
`0
`
`500 0
`
`250 0
`
`.f\..-J "-
`
`20
`
`0
`
`15
`10
`RETENTION TIME ( •inulesl
`FIG. 2. Total ion curmr1 (TIC) fl1lli recon3tructed ion currenl profile
`tracings from the LC-MS analysis of a portion of an extrocti11e
`pentqf/uorobenzoylation mklue from urine of a IIOIUIIleer administered
`ri1MIIltldine.
`CJ4 9%), m/z 91 (m/z 133- CJI., 10%), m/z 81 (m/z 133-
`CJL. 10%), and m/z 79 (m/z 133- ~ 8%). The GC-CIMS
`analysis gave peaks at m/z 390 (MH+, 10%), m/z 372 (MH+-
`+
`H20, 100%), m/z 195 (cy,..C-o, 6%), m/z 179 (hydroxy
`+
`adamantyi-CH-CH3, 56%), m/z 161 (m/z 179- H~, 82%), m/
`z 151 (hydroxy adamantyl cation, 98%), and m/z 133 (m/z 151
`- H20, 20%).
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`Rt..eiO ET AL.
`
`TABLE 2
`Urinary Excretiotf of rimantadine and its metabolites in four healthy male volunteers
`3
`RiiiWitadine
`oft
`
`Subject
`
`I
`2
`3
`4
`Mean±SD
`• 0-72 br post~.
`• Number in parentheses is the additional percentaae of the dOIJC released on treatment with Glusulase.
`
`15.89 (10.70)b
`33.40 (7.30)
`21.12 (14.57)
`17.94(7.71)
`22 ± 8 (10 ± 3)
`
`14.26 (7.77)
`5.23 (7.37)
`12.43 (8.37)
`12.27 (5.66)
`11 ±4(7± I)
`
`%ofdtJse
`17.60 ( 1.47)
`2.09 (1.02)
`16.61 ( 1.62)
`16.59 (2.31)
`13 ± 7 (1.6 ± 0.5)
`
`6.56 (5.75)
`0.87 (3.53)
`5.51 (4.05)
`5.63 (4.37)
`4.6 ± 2.6 (4.4 ± 0.9)
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`II± 4% of the dose (table 2). Another 7 ± 1% of the dose was
`conjugated 3.
`Peak IV (RT 22.7 min). The material comprising the peak bad
`an ~ of 0.3 in system I, an ~ of 0.1 in system 2, and
`chromatographic, mass spectral, and NMR properties different
`than either derivatized authentic 3 or derivatized authentic 2a/
`lb.
`The LC-MS analysis of this peak (fig. 2) indicated that it was
`hydroxylate<! rimantadine metabolite. An intense MH+ ion at
`m/z 390 and less intense ions corresponding to the loss of H20
`(m/z 372) and H20 and H2 (m/z 370) were observed. An EI
`mass spectrum of an ethyl acetate extract of the spot showed
`+
`ions at m/z 389 M:, 5%), m/z 238 (c,F,-C(=O)NH-CH-CH3,
`+
`15%), m/z 195 (Cf',- c-o, 40%), m/z 167 (Cf'?, 10%), m/
`z 151 (hydroxy adamantyl cation, 100%), m/z 133 (m/z 151 -
`H20, 80%), m/z 105 (m/z 133- C21L. 15%), m/z 91 (m/z 133
`- C31L., 40%), and m/z 79 (m/z 133 - CJ{3, 20%). The GC(cid:173)
`OMS analysis gave peaks at m/z 390 (MH+, 100%), m/z 372
`+
`(MH+- H20, 50%), m/z 195 (c,F,-C=O, 5%), m/z 179 (by-
`•
`droxyadamantyl-C-CH3, 70%), m/z 161 (m/z 179- H20, 30%),
`m/z 151 (hydroxy adamantyl cation, 75%), and m/z 133 (m/z
`151 - H20. 35%).
`The NMR spectrum of the material in the peak showed a
`partially resolved doublet at &5.65 (IH; NH), a partially resolved
`eight-peak pattern centered at 63.98 ( IH; HN-CH-CH3), a singlet
`at 63.82 (I H; HO-C-H), two doublets, centered at &2.06 and
`61.45 (AX, 4H, 12 Hz; methylene protons), a singlet at &1.96
`(3H; R4 protons), a complex series of peaks at &1.2 to ol.8
`(remaining methylene protons plus impurities), and a doublet
`centered at ol.l4 (3H; HN-CH-CH3). The integrated signals for
`the methylene protons were less than expected, probably due to
`interferences from residual water and ethyl acetate resonances.
`The EI and GC-OMS data suggest that peak II and IV are
`both ring-hydroxylate<! metabolites. By elimination, they must
`be 4a and 4b. A striking difference between the epimers is their
`retention times. In the nonpolar HPLC solvent system used,
`peak II elutes over 1 min earlier than peak IV. A possible
`explanation for this is gained by inspecting molecular models. If
`peak II is 4b, the hydroxyl proton can hydrogen bond to the
`carbonyl adjacent to the pentafluorophenyl ring, and the hydro(cid:173)
`gen-bonded species would be expected to be less polar than the
`nonhydrogen-bonded species. Such hydrogen bonding would not
`be possible with 4a. Consistent with this assignment is the fact
`that the hydroxy metabolite comprising peak II loses water in
`the LC-MS and GC-MS analyses much more readily than that
`comprising peak IV. The hydrogen-bonded epimer would be
`expected to have a higher proton affinity than the nonhydrogen(cid:173)
`bonded epimer ( I 0), and protonation at this site will lead to
`
`water loss. For 4a, protonation will occur predominately at the
`amide carbonyl, a site remote from the hydroxy. The NMR also
`supports the assignment. The 4b isomer has a quartet (remaining
`R5 and R6 protons) centered at ol.69. In the 4a isomer, this
`quartet has collapsed to two separate doublets because of the
`chemical inequivalence of the R5 and R6 protons resulting from
`the interaction of the R6 protons with the R6 hydroxy (II). In
`addition, the substance comprising peak IV, with 4.6 ± 2.6% of
`the dose excreted intact (table 2) and 4.4 ± 0.9% of dose excreted
`conjugated (table 2), is conjugated to greater extent than that in
`peak II, which would be expected considering the greater steric
`crowding offered to the conjugating enzyme system by 4b com(cid:173)
`pared with 4a.
`No 'la/lb metabolites were found, although the techniques
`used in this study were sufficiently sensitive to detect these
`metabolites at a level of I% of the dose. This finding is contrary
`to that reported by Hayden and co-workers (3, 4). Although the
`introduction of an hydroxy substituent at the 2-position results
`in diastereomers that do have different chromatographic prop(cid:173)
`erties, no metabolite having identical chromatographic properties
`to authentic pentaOuorobenzoyl 'la/lb (~ = 0.4, solvent system
`I) was observed. In solvent system I, the la and lb diastereomers
`do not separate.
`Coaclusloas. In summary, the urinary metabolites of riman(cid:173)
`tadine have been identified as coqjugated and unconjugated
`rimantadine, 3, 4a, and 4b. Contrary to previous reports, no 2a/
`lb was found. In the 72-br post-dose urine of four human
`volunteers, rimantadine and its metabolites account for a mean
`± SD of 74 ± 10% of the dose.
`
`Department of Drug Metabolism,
`Hoffmann-LaRoche. Inc.
`
`F. R. RUBIO
`E. K. FuKUDA
`W. A. GARLAND
`
`Rerereaces
`I. L. P. Van Voris, R. F. Betts, F. G. Hayden, W. A. Cbristmas, and
`R. G. DouaJas, Jr.: SuccessfuJ treatment of naturally occurring
`influenza A/USSR/17 HINI. J. Am. Med. Assoc. 245, 1128-1131
`(1981).
`2. R. Dolin, R. C. Reichman, H. P. Madore, R. Maynard, P. N. Linton,
`and J. Webber-Jones: A controlled trial of amantadine and riman(cid:173)
`tadine in the prophylaxis of influenza A infection. N. Engl. J.
`Med. 307, 580-584 (1982).
`3. L P. Van Voris, J. Bartram, and F. G. Hayden: Pharmacokinetics
`of amantadine and rimantadine. In "Respiratory Virus Infections"
`(D. Scblessigner, ed.). Microbiology-1984. pp. 421-423. American
`Society for Microbiology, Washington, D.C., 1984.
`4. F. G. Hayden, A. Minocba, D. A. Spyker, and H. E. Hoffman:
`Comparative single-dose pharmacokinetics of amantadine hydro(cid:173)
`chloride and rimantadine hydrochloride in young and elderly
`adults. Antimicrob. Agents Chemother. 28, 216-221 (1983).
`S. C. Koppel and J. Tenczer: A revision of the metabolic disposition
`
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`RIMANTADINE URINARY METABOUTES
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`777
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`of amantadine. Biomed. Mass Spearom. 1%,499-501 (1985).
`6. F. T. Delbeke, M. DeBackere, J. A. A. Jonckbeere, and A. P.
`DeLc:enbeer: Pentafluorobenzoyl derivatives of dopina agents. I.
`Extractive benzoylation and gas chromatopapby with electron(cid:173)
`capture detection of primary and secondary amines. J. Chroma(cid:173)
`togr. 273, 141-149 (1983).
`7. J. Yinon and S. Bulusu: Mass spectral fragmentation pathways in
`nitroad•mantanes. A tandem mass spectrometric collisionally in(cid:173)
`duced dissociation study. Org. Mass Spectrom. 21, 529-533
`(1986).
`
`8. J. B. Peel and G. D. Willen: Photoelectron spectrosCopic studies of
`the higher alcohols. Aust. J. Chem. 28, 2357-2364 ( 1975).
`9. R. C Fon and P. von R. Scbleyer: The proton magnetic resonance
`spectra of adamantane and its derivatives. Org. Chem. Ser. Mon(cid:173)
`ogr. 30,789-796 (1965).
`10. A. G. Harrison: "Chemical Ionization Mass Spectrometry," p. 135.
`CRC Press, Boca Raton, FL, 1983.
`II. R. M. Silverstein, G. C. Bassler, and T. C. Morrill: "Spectrometric
`Identification of Orpnic Compounds," p. 206. Fourth ed. John
`Wiley and Sons, 1981.
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