Propenone oxime ethers and pharmaceutical compositions containing them

This invention relates to novel propenone oxime ethers, methods of preparing them and pharmaceutical compositions containing them. The compounds have the formula: ##STR1## wherein R.sup.1 is H, or glucuronide; R.sup.2 and R.sup.3 are independently H or methyl; R.sup.4 is 0, or glucuronide, and n is 0 or 1, provided that when R.sup.1 is H, n is 1.

BACKGROUND OF THE INVENTION 
Congy et al., U.S. Pat. No. 5,166,416 discloses propenone oxime ethers that 
are antagonists of the 5HT.sub.2 receptors. Examples 11, 12 and 23-28 
therein describe various salts of SR 46349 having the structure: 
##STR2## 
SUMMARY OF THE INVENTION 
We have discovered that SR 46349 is metabolized to a variety of different 
analogs, including various propenone oxime ethers via a number of 
different pathways including N-oxidation, N-demethylation, glucuronidation 
and sulfation. 
It is an advantageous feature of this invention that analogs of SR 46349 
are provided which find utility in pharmaceutical compositions having a 
high affinity for the 5HT.sub.2 receptor. 
It is another advantageous feature of this invention that analogs of SR 
46349 are provided which find particular utility as pro-drugs in 
pharmaceutical compositions.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
Trans 1-N,N-dimethylaminoethoxyimino 1-(2-fluorophenyl)-3-(4-hydroxyphenyl) 
2-propene hemifumarate, also known as trans-4-(3A) 
3-((dimethylaminoethyl)oxyimino)-3-(2-fluorophenyl propen-1-yl! phenol 
hemifumarate or SR 46349B is a potent 5-HT.sub.2 antagonist which is being 
clinically investigated for the treatment of depression. This particular 
salt of SR 46349 is described by Congy et al., U.S. Pat. No. 5,166,416, 
Example 12. 
According to this invention, there are provided compounds having the 
formula: 
##STR3## 
In the structural formula above, R.sup.1 is H, glucuronide or sulfate; 
R.sup.2 and R.sup.3 are independently H or methyl; 
R.sup.4 is O, or glucuronide; and 
n is 0 or 1; 
provided that when R.sup.1 is H, n is 1. 
When R.sub.4 is O, the compound is in the form of an N-oxide. 
As shown below, most of the compounds of this invention have been 
identified as mixtures in various proportions of syn and anti-isomers as 
described by Congy et al. in U.S. Pat. No. 5,166,416 cited above. 
As used herein, glucuronide, also known as glucuronoside, is intended to 
refer to a compound in which glucuronic acid, combined as a sugar 
(hexose), not as an acid, is linked by a glycosidic bond to a group e.g., 
a hydroxyl or carboxyl group, on another compound. 
The N-oxide, sulfate and glucuronide compounds described herein can be 
prepared by conventional organic chemistry synthetic techniques. 
Alternatively, the N-oxide, sulfate and glucuronide compounds can be 
enzymatically synthesized using appropriate enzyme systems, for example, 
containing the cofactors phosphoadenosinephosphosulfate or UDP-glucuronic 
acid. The compounds of this invention can also be isolated from mammalian 
samples of plasma, urine and/or feces after dosing with SR 46349. SR 46349 
can be prepared as described by Congy et al., U.S. Pat. No. 5,166,416, the 
disclosure of which is hereby incorporated by reference in it entirety. 
The compounds are generally administered in unit doses. The unit doses are 
preferably formulated in pharmaceutical compositions in which the active 
principle is mixed with a pharmaceutical excipient. 
According to another aspect, the invention relates to pharmaceutical 
compositions in which the active principle is an aforementioned compound 
or a pharmaceutically acceptable salt thereof. 
In the pharmaceutical compositions according to the invention for oral, 
sublingual, subcutaneous, intramuscular, intravenous, transdermic, local 
or rectal administration, the active ingredients can be administered in 
unit forms of administration, mixed with conventional pharmaceutical 
excipients, to animals and to man. The suitable unit forms of 
administration comprise oral forms such as tablets, capsules, powders, 
granules and oral solutions or suspensions, sublingual and buccal forms of 
administration, subcutaneous, intramuscular, intravenous, intranasal or 
intraoccular forms of administration and rectal forms of administration. 
Each unit dose can contain 0.1 to 500 mg of active ingredient, preferably 
2.5 to 125 mg, in combination with a pharmaceutical excipient. Each unit 
dose can be administered 1 to 4 times per day. 
When a solid composition is prepared in tablet form, the main active 
ingredient is mixed with a pharmaceutical excipient such as gelatine, 
starch, lactose, magnesium stearate, talc, gum arabic or the like. The 
tablets can be coated with saccharose or suitable other substances or 
treated so that they have prolonged or delayed activity and so that they 
continuously release a given quantity of the active principle. 
A preparation in capsules is obtained by mixing the active ingredient with 
a diluent and pouring the resulting mixture into soft or hard capsules. 
A preparation in syrup or elixir form can contain the active ingredient 
together with a sweetener, preferably without calories, and methyl paraben 
and propyl paraben antiseptics and suitable flavouring and dye. 
The powders or granules dispersible in water can contain the active 
ingredient mixed with dispersing agents or wetting agents or suspension 
agents such as polyvinylpyrrolidone, and with sweeteners or taste 
adjusters. 
Rectal administration is made via suppositories prepared with binders such 
as cocoa butter or polyethylene glycols, which melt at the rectal 
temperature. 
Parenteral, intranasal or intraocular administration is via aqueous 
suspensions, or isotonic saline solutions or sterile injectable solutions 
containing pharmacologically compatible dispersing and/or wetting agents, 
e.g. propylene glycol or butylene glycol. 
Alternatively the active principle can be formulated in microcapsules, with 
one or more excipients or additives if required. 
The compounds have low toxicity. More particularly their acute toxicity is 
compatible with use thereof as drugs, e.g. to prevent clotting of 
platelets, or as psychotropic drugs. 
For this purpose, mammals requiring this treatment are given an effective 
quantity of the compound or of one of its pharmaceutically acceptable 
salts. 
The aforementioned compounds and their pharmaceutically acceptable salts 
can be used in daily doses of 0.01 to 10 mg per kilogram body weight of 
the mammal under treatment, preferably at daily doses of 0.1 to 5 mg/kg. 
In man, the dose can preferably vary from 0.5 to 500 mg per day, more 
particularly from 2.5 to 250 mg depending on the patient's age or the type 
of treatment, i.e. whether prophylactic or curative. 
The following examples illustrate the invention. 
The objective of this study was to quantitate the biotransformation of 
.sup.14 C-SR 46349B in humans. Six healthy male volunteers were treated 
orally with a single 30 mg dose of .sup.14 C-SR 46349B (corresponding to 
35.4 mg of the salified compound), labeled on the C-1 position of the 
propenyl chain with .sup.14 C (1.375 MBq). Excretion-balance, plasma and 
blood pharmacokinetics of radioactivity, and clinical safety/tolerance 
were reported. Plasma, urine and fecal homogenates were analyzed for 
quantitation and identification of SR 46349B and metabolites. 
EXPERIMENTAL PROCEDURE 
Reagents for Metabolites Identification and Quantitation 
Scintillation cocktails (Biofluor, Ultima, M, Carbo-sorb and Permafluor 
E+), combustocones and pads were purchased from Packard Instrument Company 
(Meriden, Conn.). Bond-Elut solid phase extraction cartridges (3 mL) were 
purchased from Varian (Harbor City, Calif.). HPLC columns were purchased 
from YMC, Inc. (Wilmington, N.C.). All other reagents and solvents used in 
this study were of standard reagent grade or better. 
TEST ARTICLE 
Formulation 
1-Propenyl-.sup.14 C!-SR 46349B was synthesized by Sanofi in Alnwick, UK, 
using a process similar to that described in U.S. Pat. No. 5,166,416. 
Radiochemical purity of drug substance was determined to be 99%. Specific 
activity of drug product was determined to be 1.2375 .mu.Ci/mg. .sup.14 
C-SR 46349B was administered in a single gelatin capsule. 
Administration 
Subjects were fasted overnight prior to the morning of .sup.14 C-SR 46349B 
administration. All subjects received a single oral 30 mg dose of .sup.14 
C-SR 46349B (corresponding to 35.4 mg of the unsalified compound) in a 
single gelatin capsule. The capsule was dropped directly in the mouth and 
swallowed with 150 mL of tap water at about 8:00 AM. Ingestion of the 
capsule was verified by inspection of the oral cavity of each subject. 
SAMPLE COLLECTION 
Plasma Samples 
Blood samples were obtained at 0, 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10, 12, 
16, 24, 36, 48, 72, 96, 120, 144, 168, 192, 216, 240, 264, 288 and 312 hr 
after dosing. For each sample, approximately 7 mL of blood (22 mL at 2, 3, 
4, 8, 12, 24 and 48 hrs after dosing) was drawn into polypropylene 
collection tubes containing lithium heparin (as an anticoagulant). 
Following the removal of 1 mL of blood for radiocarbon analysis, samples 
were centrifuged (within 30 min) at 2000-3000.times.g for 10 min and the 
plasma transferred into polypropylene transport tubes. All samples were 
stored frozen (.ltoreq.-18.degree. C.) until analysis. 
Urine Samples 
All voided urine was collected in polypropylene bottles and stored 
refrigerated (2.degree.-8.degree. C.) until the collection interval was 
completed. Collection intervals were 0 (pre-dose), 0-12, 12-24, 24-28, 
48-72, 72-96, 96-120, 120-144, 144-168, 168-192, 192-216, 216-240, 
240-264, 264-288, 288-312 and 312-336 hr after dosing. The samples were 
weighed and mixed at the end of each collection interval and a 25 mL 
aliquot from each collection interval was transferred into polypropylene 
vials. All samples were stored frozen (.ltoreq.-18.degree. C.) until 
analysis. 
Fecal Samples 
All stools were collected in polypropylene bags, sealed and immediately 
frozen (.ltoreq.-18.degree. C.). The last stool was obtained at least 276 
hr after dosing. All stools within a 24 hr period were combined for 
analysis, mixed with distilled water (enough to produce a fluid 
consistency after homogenizatoin, approximately 80% (w/v) typically) and 
the mixture was homogenized for approximately 120 seconds in a stomacher. 
Following homogenization, a 25 mL aliquot from each collection interval 
was transferred into transport tubes. All samples were stored frozen 
(.ltoreq.-18.degree. C.) until analysis. 
ASSAY METHODOLOGIES 
Plasma Concentrations of SR 46349 
The concentration of SR 46349 in plasma samples was determined using a 
validated HPLC method with UV detection at 320 nm. Briefly, the procedure 
involved adding 1 mL of plasma to 0.05 mL of a 10 mg/L solution of 
internal standard, followed by vortex mixing. After storing in the dark 
for approximately 15 min, 2.0 mL of titrisol buffer (pH 10) was added and 
samples were immediately applied to Extrelut 3 columns. After 
approximately 15 minutes in the dark, columns were eluted with 14 mL of 
methylene chloride. The samples were dried at room temperature and 
resuspended in 0.15 mL of mobile phase and 0.05 mL was analyzed. The HPLC 
system utilized a Hewlett-Packard Model 1050 HPLC system and a Lichrospher 
100-RP18 (4.0.times.125 mm; 5.mu.) column with a solvent system of TEA 
buffer (pH 3.7): acetonitrile (77:23, v/v) at a flow rate of 1.2 mL/min. 
Unknown concentrations of SR 46349 were quantified by reference to a 
1/y.sup.2 weighted regression analysis of the peak-area ratio (SR 
46349/internal standard) versus concentration of the calibration curve 
standards. The LOQ of SR 46349 in plasma was 0.0025 mg/L. 
Plasma, Urine and Fecal Concentrations of Radioactivity 
Aliquots of plasma and urine samples were added directly to Ready-Safe 
scintillant (9 mL), hand shaken for a few seconds and left for at least 2 
hours prior to counting. Aliquots of fecal homogenates were weighed in a 
Combusto-Cone fitted with a CombustoPad. The samples were allowed to dry 
overnight before combustion in a Packard Sample Oxidizer, Model 307 
(Meriden, Conn.). Carbo-Sorb and Permafluor E.sup.+ were used as 
scintillants (10 mL). Samples mixed with scintillant were left for at 
least 4 hours prior to counting. Radioactivity assays were performed with 
a Packard Instruments Tri-Carb, Model 1600TR Liquid Scintillation System. 
The LOQ was set at 12 cpm above background. 
Pharmacokinetic Parameter Analysis for SR 46349 
Model-independent pharmacokinetic parameters for plasma concentrations of 
SR 46349 were calculated for each subject. The maximum observed plasma 
concentration (C.sub.max) of SR 46349 and its corresponding time 
(t.sub.max) were determined by visual inspection of individual 
concentration-time profiles. Half-life (t 1/2) was calculated from the 
ratio of ln(2)/.lambda..sub.Z, where .lambda..sub.Z is the regressed slope 
of the terminal phase. The area under the plasma concentration of SR 
46349-versus-time-curves (AUC.sub.(0-t)) was calculated using the 
trapezoidal rule. AUC was calculated for the sum of AUC.sub.(0-t) and 
Cp(1)/.lambda..sub.Z wherein Cp(1) is the last measurable concentration 
time point. Nominal sample times were used to calculate pharmacokinetic 
parameters. 
METABOLISM 
Determinations of Metabolite Profiles 
Quantitation of Urinary Metabolites 
A representative pool of urine was prepared for each subject by mixing 
aliquots from each collection interval in proportion to the percent of 
radioactivity present in each collection interval. Pools were prepared to 
represent approximately 95% of total collected urinary radioactivity. 
Aliquots of pooled urine samples (1 mL) were applied to 3 mL Bond-Elut 
solid-phase extraction cartridges (pre-conditioned with 2 mL of methanol 
followed by 2 mL of water), washed with 2 mL of water followed with 2 mL 
of methanol:water (1:4 v/v), and then eluted with 3 mL of methanol. 
Eluates were dried under nitrogen using a TurboVap evaporator at 
40.degree. C. Dried samples were reconstituted in 0.25 mL of 20 mM aqueous 
ammonium acetate (pH 3.0): acetonitrile (9:1, v/v) by vigorous mixing and 
sonication for 5 min and then 0.18 mL was injected onto the HPLC. Pools 
for each subject were extracted and analyzed in triplicate. 
Quantitation of Fecal Metabolites 
A representative pool of fecal homogenate was prepared for each subject by 
mixing aliquots from each collection interval in proportion to the percent 
of radioactivity present in each collection interval. Pools were prepared 
to represent approximately 95% of total collected fecal radioactivity. 
Aliquots of pooled fecal homogenates (approximately 3 grams) were mixed 
with 6 mL of methanol in 15 mL polypropylene centrifuge tubes for 3 
minutes on a multi-tube vortexer, and then centrifuged 
(4500.times.g.times.10 min). The pellets were extracted twice more with 5 
mL of methanol and supernatants from the three extraction were combined 
and dried under nitrogen for approximately 15 hr using a TurboVap 
evaporator at 35.degree. C. 
Dried samples were reconstituted in 0.75 mL of methanol:water (3:1, v/v) 
with vigorous mixing and sonication for 10 min, followed by centrifugation 
(15000 g.times.5 min). The supernatant was concentrated under nitrogen at 
40.degree. C. using a TurboVap evaporator for approximately 30 min. The 
concentrated supernatants were diluted to approximately 0.5 mL (final 
volume of the diluted solution was measured with an Eppendorf pipetman) 
with methanol:water (1:1, v/v), and then 0.18 mL was injected onto the 
HPLC. Pools for each subject were extracted and analyzed in triplicate, as 
described below. 
Quantitation of Plasma Metabolites 
Pools of plasma for each collection time were prepared by mixing equal 
amounts of plasma from all subjects. This made available enough plasma at 
each time point to quantitate metabolites. Aliquots of plasma pools (1 mL) 
were applied to 3 mL Bond-Elut solid phase extraction cartridges 
(pre-conditioned with 2 mL of methanol followed by 2 mL of water), washed 
with 2 mL of water and eluted with 3 mL of ethanol. Eluates were 
concentrated under nitrogen using a TurboVap evaporator at 40.degree. C. 
until approximately 0.3 mL of solution remained. Due to low amounts of 
radioactivity in plasma, five concentrated extracts from the same time 
point were combined. Tubes that contained extract were rinsed twice with 
0.2 mL of methanol and rinses combined with the extract. Samples were 
dried under nitrogen at 40.degree. C. using a TurboVap evaporator. Dried 
samples were reconstituted in 0.25 mL of 20 mM aqueous ammonium acetate 
(pH 3.00):acetonitrile (9:1, v/v) by vigorous mixing and sonication for 5 
min. After centrifugation (4500 g.times.5 min), 0.18 mL of supernatant was 
injected onto the HPLC. Pools for each collection time were analyzed in 
triplicate. 
Chromatographic Methods 
Metabolite quantitation was conducted with an HP1090 HPLC system (Hewlett 
Packard, Wilmington, Del. HPLC eluant was fraction collected (0.33 
mL/fraction using a FOXY.TM. Model 200 fraction collector; Isco, Lincoln, 
Nebr.). Fractions were mixed with 5 mL of Biofluor cocktail and then 
counted using a Tri-Carb 2700TR liquid scintillation counter (Packard, 
Meriden, Conn.). 
Prior to profiling urine, fecal or plasma extract, a urine pool extract was 
chromatographed as a reference standard to determine that the HPLC system 
was working properly and to compare retention times of metabolites in 
feces and plasma to urine. HPLC eluant for the urine reference standard 
was directed through a Packard Radiomatic 500TR Flowone/Beta radioactivity 
detector. Eluant was mixed with Ultima M scintillation cocktail using a 
1:3 mixture (eluant:cocktail). 
Calculation of Metabolite Concentrations and Excretion 
Sample extraction and HPLC column recoveries were calculated using measured 
radioactivity in samples, sample extracts and HPLC eluants. Recoveries of 
100% were based on the expected amount of radioactivity in a sample prior 
to extraction or HPLC analysis. 
Urinary and fecal metabolites are expressed as a percentage of the 
radioactivity in each matrix and as a percentage of excreted (recovered) 
radioactivity. Values were corrected for total experimental recoveries. 
METABOLITE STRUCTURAL ELUCIDATION 
Characterization of Urine and Fecal Metabolites 
Urine samples were prepared by pooling equal volumes across subjects from 
the 0-48 hr collection intervals. Aliquots of pooled urine samples (1 mL) 
were applied to 3 mL Bond-Elut solid-phase extraction cartridges 
(pre-conditioned with 2 mL of methanol followed by 2 mL of water), washed 
with 2 mL of water and 2 mL of methanol:water (1:4, v/v), and then eluted 
with 3 mL of methanol. Eluates were dried under nitrogen at 40.degree. C. 
using a TurboVap evaporator. Dried samples were reconstituted in 0.25 mL 
of 20 mM aqueous ammonium acetate (pH 3.0):acetonitrile (9:1, v/v) by 
vigorous mixing and sonication for 5 min and analyzed by LC-MS/MS. 
Sometimes extracts were pooled after reconstitution to increase the amount 
of material available for analysis. 
Fecal homogenate samples were prepared and extracted as described above. 
Liquid Chromatography-mass Spectrometry 
LC-MS/MS was performed using a Hewlett-Packard HP1090 HPLC system 
interfaced with a Finnigan MAT TSQ 7000 (Finnigan MAT, San Jose, Calif.). 
The mass spectrometer was optimized in the positive ion, electrospray 
(ESI) mode using an unlabeled SR 46349B standard. Source parameters were 
optimized using the HPLC with approximately 40% of the column eluant 
directed to the ESI source. The remaining eluant was split to a fraction 
collector. The parent molecular ion (m/z 329) was scanned in the selected 
ion monitoring (SIM) mode for source optimization. The collision energy 
and collision gas pressure were optimized by scanning MS/MS product ions 
of m/z 329. 
Radiochromatograms of pooled samples were generated from fractions obtained 
from the HPLC system used for metabolite quantitation or after splitting 
the eluant for LC-MS/MS analysis. These chromatograms were compared to 
confirm that the retention times of SR 46349 and metabolites on the system 
used for LC-MS/MS analysis corresponded to those obtained on the system 
used for metabolite quantitation. 
RESULTS 
Pharmacokinetic Results 
Plasma Concentration of SR 46349 and Radioactivity 
Following oral administration of .sup.14 C-SR 46349B, plasma concentrations 
of SR 46349 reached a mean (.+-.SD) C.sub.max of 0.05 (.+-.0.01) mg/L 
between 1.5 and 4 hours after dosing. After this time, the concentrations 
declined rapidly with a terminal elimination half-life of 22.9 (.+-.7.3) 
hr. The mean (.+-.SD) AUC was 1.2 (.+-.0.15) mg.circle-solid.hr/L. 
Maximal plasma concentrations of radioactivity were higher than those 
observed for intact drug, reaching a mean (.+-.SD) C.sub.max of 0.16 
(.+-.0.02) mg eq/L between 2 and 12 hours after dosing. The terminal 
elimination half-life for plasma radioactivity was 63.3 (.+-.5.6) hr. The 
mean (.+-.SD) AUC was 13.7 (.+-.2. 1) mg eq.circle-solid.hr/L, 
approximately 11 times greater than the mean plasma AUC for SR 46349. 
BIOTRANSFORMATION 
Metabolite Profiling 
Urinary Metabolites 
The cumulative urinary excretion of radioactivity up to 336 hr post-dose 
represented 70.3 (.+-.4.6)% (mean (.+-.SD), range 63.1 to 77.1%) of the 
administered does. Extraction recovery for urine was 95.4 (.+-.4.8)%. 
Column recovery for urine extracts was 102.5 (.+-.6.0)%. The peak labeled 
HU17 had a similar retention time with SR 46349. Twenty-four metabolites 
were evident by radiochromatography or LC-MS/MS in extracted urine 
samples. Approximately 82% of excreted urinary radioactivity was accounted 
for by SR 46349 and metabolites. Of the 24 metabolites noted in urine , 17 
have been structurally characterized using LC-MS/MS. Also, based on HPLC 
retention times, metabolites HU16 and HU19 corresponded to N-demethlyated 
SR 46349 and the N-demethylated isomer of SR 46349. 
Fecal Metabolites 
The cumulative fecal excretion of radioactivity up to 276 hr post-dose 
represented 21.6 (.+-.3.5)% (mean (.+-.SD), range 16.4 to 26.5%) of the 
administered does. Extraction recovery for fecal homogenates was 79.6 
(.+-.1.5)%. Column recovery for fecal homogenate extracts was 103.9 
(.+-.2.8)%. The peak labeled HF17 was identified as SR 46349 based on 
similar HPLC retention times and mass spectral data with standard SR 
46349. Nine metabolites and the isomer of SR 46349 (HF16, HF18&19, 
HF20&21, HF22, HF25A, HF25B and HF25C) were evident by radiochromatography 
or LC-MS/MS in extracted fecal samples. These metabolites corresponded to 
metabolites with the same identification number in urine based on HPLC 
retention time, and were also confirmed using LC-MS/MS (except for HF18, 
HF21 and HF25). It appeared that HF25 consisted of 3 compounds in feces. 
Metabolite H25 was a minor component of urine but represented a 
significant proportion (approximately 12%) of fecal radioactivity. 
Approximately 55% of excreted fecal radioactivity was accounted for by SR 
46349 and metabolites. 
Plasma Metabolites 
Extraction recovery for plasma ranged from approximately 85 to 95% over the 
different time points investigated. Column recovery for plasma extracts 
ranged from approximately 103 to 120%. Radiochromatograms of plasma 
contained at least 16 peaks consistent with metabolites identified in 
urine. Plasma metabolites were identified by comparison of HPLC retention 
times with urinary metabolites, using chromatograms obtained on the same 
day and by LC-MS/MS. The plasma concentrations of SR 46349B (HP17) were 
higher than any single metabolite, except at the 48 hr time point. 
METABOLITE IDENTIFICATION 
Identification of Urinary Metabolites 
Experimental results for identification of SR 46349 are presented first, 
followed by results for oxidative metabolites, and then conjugated 
metabolites. 
Mass Spectral Characterization of an Authentic SR 46349B Standard 
The positive full scan electrospray spectrum of an authentic standard of SR 
46349B showed a base peak at m/z 329 (MH!.sup.+) indicating a molecular 
weight of 328. The product ion spectrum of the m/z 329 ion contained 
several characteristic fragment ions for this compound. The major fragment 
produced was at m/z 240, corresponding to cleavage of the N--O bond of the 
oxime. The 240 fragment contained both substituted phenyl rings connected 
by the C.sub.3 H.sub.2 N. The 240 fragment underwent further fragmentation 
and produced characteristic ions at 119 (phenol+HC=CH), 144 (240-FC.sub.6 
H.sub.4), and 91 (tropylium ion). The remaining (CH.sub.3).sub.2 NCH.sub.2 
CH.sub.2 O fragment produced characteristic ions at m/z 44, 58 and 72. The 
characteristic ions described were used to identify potential metabolites 
and to assist in determining sites of metabolism. 
Identification of SR 46349 (HU17) and its Isomer, HU20 
The positive full scan electrospray spectrum of urine component HU17 showed 
a base peak at m/z 329 (MH!.sup.+) indicating a molecular weight of 328. 
The product ion spectrum of the m/z 329 ion contained several 
characteristic ions of the parent drug. The major fragment produced was at 
m/z 240, corresponding to cleavage of the N--O bond of the oxime. The 240 
fragment contained both substituted phenyl rings connected by the C.sub.3 
H.sub.2 N. The 240 fragment underwent further fragmentation and produced 
characteristic ions at 119 (phenol+HC=CH), 144 (240 -FC.sub.6 H.sub.4), 
and 91 (tropylium ion). The remaining (CH.sub.3).sub.2 NCH.sub.2 CH.sub.2 
O fragment was evident at m/z 88. The tertiary amine fragmented further 
and produced characteristic ions at m/z 44, 58, and 72. These data, 
combined with evidence of similar retention time with an authentic SR 
46349 standard, indicated that HU17 was the parent drug. Similar data were 
obtained for HU20 and indicated that HU20 was an isomer of HU17. 
Corresponding components were identified in human fecal samples and were 
designated HF17 and HF20. 
Identification of HU22 and its Isomer. HU23 
The positive full scan electrospray spectrum of urine metabolite HU22 
showed a base peak at m/z 345 (MH!.sup.+) indicating a molecular weight 
of 344. This molecular weight corresponded to an addition of 16 amu and 
suggested an oxidation of SR 46349. The product ion spectrum of the m/z 
345 ion contained characteristic ions of the parent drug. The major 
fragment produced was at m/z 240, corresponding to cleavage of the N--O 
bond of the oxime. The 240 fragment contained both substituted phenyl 
rings connected by the C.sub.3 H.sub.2 N. The 240 fragment underwent 
further fragmentation and produced characteristic ions at 119 (phenol 
+HC=CH), 144 (240 - FC.sub.6 H.sub.4), and 91 (tropylium ion). The 
remaining (CH.sub.3).sub.2 NCH.sub.2 CH.sub.2 O fragment was evident at 
m/z 88. An additional mass peak was observed at m/z 106 and corresponded 
to the oxidation of alkylamine moiety. Although this represented an 
addition of 18 amu to this fragment, the mechanism for the formation of 
the m/z 88 fragment has an inherent loss of two protons. This mechanism 
was altered by the presence of the oxide and accounted for the two 
additional protons observed. This oxidized tertiary amine fragmented 
further and produced characteristic ions at m/z 44, 58, and 72. These 
data, combined with evidence of similar HPLC retention time and mass 
spectral data, indicated that HU22 was the N-oxide of SR 46349. Similar 
data were obtained for HU23 and indicated that HU23 was an isomer of HU22. 
Corresponding metabolites were identified in human fecal samples and were 
designated HF22 and HF23. 
Identification of Metabolite HU2 and its Isomer, HU3 
The positive full scan electrospray spectrum of urine metabolite HU2 showed 
a base peak at m/z 505 (MH!.sup.+) indicating a molecular weight of 504. 
This molecular weight corresponded to an addition of 176 amu and suggested 
glucuronidation of SR 46349. The product ion spectrum of the m/z 505 ion 
contained characteristic ions of the parent drug. The major fragment 
produced was at m/z 240, corresponding to cleavage of the N--O bond of the 
oxime and the loss of 176 amu. The 240 fragment contained both substituted 
phenyl rings connected by the C.sub.3 H.sub.2 N. The 240 fragment 
underwent further fragmentation and produced the characteristic ions at 
119 (phenol+HC=CH). The fragment at m/z 329 corresponded to the parent 
drug and resulted from the facile loss of 176 amu from the metabolite 
molecular ion. The fragment at 416 corresponded to the loss of 
(CH.sub.3).sub.2 NCH.sub.2 CH.sub.2 O resulting from cleavage of the oxime 
and indicated that conjugation occurred on the m/z 240 fragment. The most 
likely site of conjugation was on the phenolic oxygen. The 
(CH.sub.3).sub.2 NCH.sub.2 CH.sub.2 O fragment produced characteristic 
ions at m/z 58 and 72. These data indicated that HU2 was the phenolic 
glucuronide of the parent drug. Similar data were obtained for HU3 and 
indicated that HU3 was an isomer of HU2. 
Identification of Metabolite HU4 and its Isomer, HU6 
The positive full scan electrospray spectrum of urine metabolite HU4 showed 
a base peak at m/z 521 (MH!.sup.+) indicating a molecular weight of 520. 
This molecular weight corresponded to an addition of 192 amu and suggested 
an oxidation and glucuronidation of SR 46349. The product ion spectrum of 
the m/z 521 ion contained characteristic ions of the parent drug. The 
major fragment produced was at m/z 240, corresponding to cleavage of the 
N--O bond of the oxime and the loss of 192 amu. The 240 fragment contained 
both substituted phenyl rings connected by the C.sub.3 H.sub.2 N. The 
fragment at m/z 345 corresponded to the addition of 16 amu to the parent 
drug and resulted from the facile loss of 176 amu from the metabolite 
molecular ion. The fragment at 416 corresponded to the loss of 
(CH.sub.3).sub.2 NCH.sub.2 CH.sub.2 O and an additional 16 amu resulting 
from cleavage of the oxime and indicated that conjugation occurred on the 
m/z 240 fragment and oxidation occurred on the (CH.sub.3).sub.2 NCH.sub.2 
CH.sub.2 O moiety. The most likely site of conjugation was on the phenolic 
oxygen. The oxidated (CH.sub.3).sub.2 NCH.sub.2 CH.sub.2 O fragment 
produced characteristic ions at m/z 58, 72, 88 and 106. These data 
indicated that HU4 was the phenolic glucuronide of the N-oxide of the 
parent drug. Similar data were obtained for HU6 and indicated that HU6 was 
an isomer of HU4. 
Identification of Metabolite HU1 
The positive full scan electrospray spectrum of urine metabolite HU1 showed 
a base peak at m/z 491 (MH!.sup.+) indicating a molecular weight of 490. 
This molecular weight corresponded to an addition of 162 amu and suggested 
a glucuronidation and demethylation of SR 46349. The product ion spectrum 
of the m/z 491 ion contained characteristic ions of the parent drug. The 
major fragment produced was at m/z 240, corresponding to cleavage of the 
N--O bond of the oxime and the loss of 176 amu. The 240 fragment contained 
both substituted phenyl rings connected by the C.sub.3 H.sub.2 N. The 
fragment at m/z 315 corresponded to the loss of 14 amu to the parent drug 
and resulted from the facile loss of 176 amu from the metabolite molecular 
ion. The fragment at 416 corresponded to the loss of the alkylamine moiety 
resulting from cleavage of the oxime. This indicated that conjugation 
occurred on the m/z 240 fragment and demethylation occurred on the 
(CH.sub.3).sub.2 NCH.sub.2 CH.sub.2 O moiety of the parent drug. The most 
likely site of conjugation was on the phenolic oxygen. The alkylamine 
fragment produced characteristic ions at m/z 44 and 58. These data 
indicated that HU1 was the phenolic glucuronide of N-demethylated SR 
46349. It was likely that the corresponding isomer of HU1 co-eluted under 
the chromatographic conditions used. 
Identification of Metabolite HU9 and its Isomer, HU14 
The positive full scan electrospray spectrum of urine metabolite HU9 showed 
a base peak at m/z 409 (MH!.sup.+) indicating a molecular weight of 408. 
This molecular weight corresponded to an addition of 80 amu and suggested 
sulfation of SR 46349. The product ion spectrum of the m/z 409 ion 
contained characteristic ions of the parent drug. The major fragment 
produced was at m/z 240, corresponding to cleavage of the N--O bond of the 
oxime and the loss of 80 amu. The 240 fragment contained both substituted 
phenyl rings connected by the C.sub.3 H.sub.2 N. The 240 fragment 
underwent further fragmentation and produced the characteristic ions at 
119 (phenol+HC=CH) and 144 (240 - FC.sub.6 H.sub.4). The fragment at m/z 
329 corresponded to the parent drug and resulted from the facile loss of 
80 amu from the metabolite molecular ion. The fragment at 320 corresponded 
to the loss of the (CH.sub.3).sub.2 NCH.sub.2 CH.sub.2 O resulting from 
cleavage of the oxime and indicated that conjugation occurred on the m/z 
240 fragment. The most likely site of conjugation was on the phenolic 
oxygen. The (CH.sub.3).sub.2 NCH.sub.2 CH.sub.2 O fragment produced 
characteristic ions at m/z 58, 72 and 88. These data indicated that HU9 
was the phenolic sulfate conjugate of the parent drug. Similar data were 
obtained for HU14 and indicated that HU14 was an isomer of HU9. 
Identification of Metabolite HU12 and its Isomer, HU15 
The positive full scan electrospray spectrum of urine metabolite HU12 
showed a base peak at m/z 425 (MH!.sup.+) indicating a molecular weight 
of 424. This molecular weight corresponded to an addition of 96 amu and 
suggested an oxidation and sulfation of SR 46349. The product ion spectrum 
of the m/z 425 ion contained characteristic ions of the parent drug. The 
major fragment produced was at m/z 240, corresponding to cleavage of the 
N--O bond of the oxime and the loss of 96 amu. The 240 fragment contained 
both substituted phenyl rings connected by the C.sub.3 H.sub.2 N. The 
fragment at m/z 345 corresponded to the addition of 16 amu to the parent 
drug and resulted from the facile loss of 80 amu from the metabolite 
molecular ion. The fragment at 320 corresponded to the loss of 
(CH.sub.3).sub.2 NCH.sub.2 CH.sub.2 O and an additional 16 amu resulting 
from cleavage of the oxime and indicated that conjugation occurred on the 
m/z 240 fragment and oxidation occurred on the (CH.sub.3).sub.2 NCH.sub.2 
CH.sub.2 O moiety. The most likely site of conjugation was on the phenolic 
oxygen. The oxidized (CH.sub.3).sub.2 NCH.sub.2 CH.sub.2 O fragment 
produced characteristic ions at m/z 58, 72, 88 and 106. These data 
indicated that HU12 was the phenolic sulfate of the N-oxide of the parent 
drug. Similar data were obtained for HU15 and indicated that HU15 was an 
isomer of HU12. 
Identification of Metabolite HU8 and its Isomer, HU13 
The positive full scan electrospray spectrum of urine metabolite HU8 showed 
a base peak at m/z 395 (MH!.sup.+) indicating a molecular weight of 394. 
This molecular weight corresponded to an addition of 66 amu and suggested 
a sulfation and demethylation of SR 46349. The product ion spectrum of the 
m/z 425 ion contained characteristic ions of the parent drug. The major 
fragment produced was at m/z 240, corresponding to cleavage of the N--O 
bond of the oxime and the loss of 80 amu. The 240 fragment contained both 
substituted phenyl rings connected by the C.sub.3 H.sub.2 N. The fragment 
at m/z 315 corresponded to the loss of 14 amu from the parent drug and 
resulted from the facile loss of 80 amu from the metabolite molecular ion. 
The fragment at 320 corresponded to the loss of the alkylamine moiety 
resulting from cleavage of the oxime. This indicated that conjugation 
occurred on the m/z 240 fragment and demethylation occurred on the 
(CH.sub.3).sub.2 NCH.sub.2 CH.sub.2 O moiety of the parent drug. The most 
likely site of conjugation was on the phenolic oxygen. The alkylamine 
fragment produced a characteristic ion at m/z 58. These data indicated 
that HU8 was the phenolic sulfate conjugate of N-demethylated SR 46349. 
Similar data were obtained for HU13 and indicated that HU13 was an isomer 
of HU8. 
Identification of Metabolite HU10A and its Isomer, HU10B 
The positive full scan electrospray spectrum of urine metabolite HU10A 
showed a base peak at m/z 505 (MH!.sup.+) indicating a molecular weight 
of 504. This molecular weight corresponded to an addition of 176 amu and 
suggested a glucuronidation of SR 46349. The product ion spectrum of the 
m/z 505 ion contained characteristic ions of the parent drug. The major 
fragment produced was at m/z 329, corresponding to the facile loss of 176 
amu. The characteristic fragment at m/z 240, corresponding to cleavage of 
the N--O bond of the oxime and the loss of 176 amu, was also produced. The 
240 fragment contained both substituted phenyl rings connected by the 
C.sub.3 H.sub.2 N. The 240 fragment underwent further fragmentation and 
produced the characteristic ions at 119 (phenol+HC=CH). The fragment at 
m/z 416 corresponding to the loss of (CH.sub.3).sub.2 NCH.sub.2 CH.sub.2 O 
resulting from cleavage of the oxime was not produced and indicated that 
conjugation did not occur on the m/z 240 fragment. The most likely site of 
conjugation was on the N,N-dimethylamine moiety. The (CH.sub.3).sub.2 
NCH.sub.2 CH.sub.2 O fragment produced characteristic ions at m/z 58 and 
72. These data indicated that HU10A was the N-glucuronide of the parent 
drug. Similar data were obtained for HU10B and indicated that HU10B was an 
isomer of HU10A. 
Metabolites HU5, HU7, HU11, HU18, HU21, HU24 and HU25 
The positive full scan and precursor ion scan electrospray spectrum of 
urine metabolite HU5, HU7, HU11, HU18, HU21, HU24 and HU25 indicated that 
these peaks were potentially related to SR 46349, but limited 
concentrations of these metabolites prevented the acquisition of the 
interpretable product ion data required for positive identification. Each 
of these potential metabolites accounted for less than two percent of the 
total recorded radioactivity in urine. 
DISCUSSION OF RESULTS 
Following oral administration of a 30 mg dose of .sup.14 C-SR 46349B, 
maximal plasma concentrations of radioactivity were approximately 3 times 
higher than plasma concentrations of SR 46349 at similar t.sub.max values. 
After this point, plasma concentrations of SR46349 decline with a mean 
half-life of approximately 23 hr. In contrast, plasma concentrations of 
radioactivity decline slower with a mean terminal half-life of 
approximately 63 hr. The mean (.+-.SD) AUC for plasma radioactivity was 
13.7 (.+-.2. 1) mg eq.circle-solid.hr/L, approximately 11 times greater 
than the mean plasma AUC for SR 46349 (1.20 (.+-.0.15) mg 
eq.circle-solid.hr/L). 
Of the recovered radioactivity, approximately 82% in urine and 56% in feces 
was accounted for as SR 46349 and metabolites. In total, approximately 76% 
of the excreted radioactivity was characterized radiochromatographically. 
The remainder of the recovered radioactivity was attributable to 
extraction losses (primarily feces) or non-distinct chromatography (too 
diffuse or too minor to characterize or identify). SR 46349 accounted for 
approximately 6% of the excreted radioactivity, indicating that the 
compound was extensively metabolized. 
Altogether, 26 putative metabolites were noted in urine or feces. 
Structural information was obtained on 17 of these metabolites. Pathways 
of SR 46349 metabolism included N-oxidation, N-demethylation, sulfation 
and glucuronidation. Some metabolites were identified that had undergone 
two steps of metabolism (i.e., N-oxidation or N-demethylation, followed by 
conjugation). For almost all the metabolites studied, two isomers were 
observed. Isomerization of SR 46349 has been noted previously. Therefore, 
it is likely that the different metabolite isomers observed were the Z and 
E forms, although other possible isomers can be postulated. 
On a percentage basis, the N-oxide metabolite of SR 46349 (H22 & H23) 
accounted for the greatest percentage of excreted radioactivity 
(approximately 23% of excreted radioactivity), followed by the 
N-demethylated metabolite (H16 & H19, approximately 11% of excreted 
radioactivity). Furthermore, additional amounts of conjugated (sulfate and 
glucuronide) N-oxide and N-demethylated metabolites were noted. 
Collectively, these two pathways of metabolism accounted for nearly half 
of the excreted radioactivity. In addition to oxidative metabolites or 
conjugates of oxidative metabolites of SR 46349, SR 46349 was also 
conjugated directly with sulfate or glucuronic acid. Sulfate conjugates 
were phenolic, while both N-glucuronides and O-glucuronides were observed. 
Taken together, it appears that oxidation of SR 46349 (N-oxidation and 
N-demethylation) appears to be the major mechanism of metabolic clearance, 
with conjugation playing a lesser role. 
Many of the excreted metabolites were also observed in plasma. Assignment 
of structures for plasma metabolites was done by comparing HPLC retention 
times with urine extract and by LC-MS/MS. The larger plasma AUC and longer 
half-life of radioactivity, when compared with SR 46349 is related to the 
presence of these circulating metabolites. Those metabolites which 
represent most of the plasma radioactivity include the N-oxide and 
N-demethylated phenolic sulfates of SR 46349, the phenolic sulfate of SR 
46349 and the N-oxide of SR 46349. However, at all but the last time point 
(48 hr) plasma concentrations of SR 46349 were always higher than any 
individual metabolite. 
It should be understood by those skilled in the art that, while the 
invention has been described and illustrated above in connection with 
certain specific embodiments, many variations and modifications may be 
employed without departing from the scope of the invention.