title: in vitro metabolism of diarylpyrazoles, a novel...

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Title:

In Vitro Metabolism of Diarylpyrazoles, a Novel Group of Cannabinoid Receptor Ligands

Authors:

Qiang Zhang, Peng Ma, Weiqun Wang, Richard B. Cole, Guangdi Wang 1. Department of Chemistry, Xavier University of Louisiana, New Orleans, LA70125 (QZ, PM,

GW) 2. Department of Chemistry, University of New Orleans, New Orleans, LA 70148 (WW, RBC)

Primary laboratory of origin:

Department of Chemistry, Xavier University of Louisiana

QZ, PM, GW

DMD Fast Forward. Published on December 22, 2004 as doi:10.1124/dmd.104.001974

Copyright 2004 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on December 22, 2004 as DOI: 10.1124/dmd.104.001974

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a) Running Title

In Vitro Metabolism of Diarylpyrazoles b) Corresponding author:

Professor Guangdi Wang Department of Chemistry Xavier University of Louisiana 1 Drexel Drive New Orleans, LA 70125 Phone: (504)5205076 Fax: (504)5207942 Email: [email protected]

c) Number of text pages: 21

Number of tables: 5 Number of figures: 11 Number of references: 21 Number of words in the Abstract: 241 Number of words in the Introduction: 513 Number of words in the Discussion: 611

d) List of nonstandard abbreviations used in the paper: Abbreviations used are: CB1, cannabinoid receptor I, CID, collision induced dissociation; ESI, electrospray ionization; MS, mass spectrometry, COSY, correlation spectroscopy, AM281, 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide; AM251, N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide; SR141716A, N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide; m/z ratio, mass to charge ratio; J, coupling constant, UV-Vis, ultraviolet-visible; MS/MS, mass spectrometry operated in tandem.


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ABSTRACT Diarylpyrazoles are a group of 1,5-diphenylpyrazole analogues of which several have been found

to exhibit antagonist properties towards the cannabinoid receptors. SR141716A, the first reported

antagonist, is a highly potent and selective CB1 receptor ligand that prevents or reverses CB1-

mediated effects. Other analogues such as AM251 and AM281 have also shown high binding

affinities to the central cannabinoid receptor and behave as antagonists/inverse agonists. There has

been no report on the metabolism of any of the diarylpyrazoles and it is unknown whether their

metabolites retain any receptor binding properties. We report a study of the in vitro metabolisms of

three diarylpyrazole analogues, SR141716A, AM251, and AM281 in rat liver microsomes. The

metabolic profile was obtained using high performance liquid chromatography (HPLC) with UV

and mass spectrometry detectors. All identified metabolites are characterized by structural

modifications on the terminal group of the 3-substituent. Thus, three pairs of isomeric metabolites

were identified from the microsomal incubation of SR141716A, which are products of

hydroxylation, hydroxylation followed by dehydration, and a combination of the two. For AM251,

only four metabolic products were detected, with two resulting from monohydroxylation of the

piperidine ring and the other two being products of dehydration of the first pair of metabolites.

For AM281 where the terminal group of the 3-substituent is a morpholine ring, dehydration of the

first two metabolites yielded a single third metabolite due to only one possible position for the

carbon-carbon double bond on the morpholinyl ring.


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INTRODUCTION

The discovery of the cannabinoid receptors, CB1 (Matsuda et al 1990) and CB2 (Munro et

al 1993) represents a significant milestone in cannabinoid research. CB1 is the central receptor

found in brain and neuronal cells and mediates such physiological responses as analgesia and

catalepsy. The peripheral receptor, CB2, is primarily expressed in spleen and immune cells. The role

of CB2 in mediating physiological effects has not been fully determined, but it is believed that CB2

may be involved in cannabinoid-mediated immune responses (Bouaboula et al 1998, Portier et al

1999). A large variety of compounds have been found to bind to the receptors, including natural

and synthetic cannabinoids (Razdan 1986, Keimowitz et al 2000), aminoalkylindoles (Bell et al

1991, Shim et al 1998), endogenous ligands, and diarylpyrazoles (Compton et al 1996, Wiley et

al 2001).

The first synthetic compound reported to have antagonist/inverse agonist properties

toward the cannabinoid receptor is SR141716A (Rinaldi-Carmona et al 1994), a diarylpyrazole

analogue (Figure 1). SR141716A exhibits strong (low nanomolar) affinity for CB1 but rather

low affinity (approaching micromolar) for CB2 (Rinaldi-Carmona et al 1994, Showalter et al

1996, Felder et al 1995, 1998). Two less potent CB1 selective analogues, AM251 and AM281

(Figure 1) have also been reported (Gatly et al 1997, 1998, Lan et al 1999, Cosenza et al 2000).

It is believed that the antagonistic activity of the diarylpyrazoles is attributable to the structural

properties of 1- and 5-substituents whereas the 3-substituent appears to be involved in agonism

and receptor activation (Wiley et al 2001). Thus, even small modifications on the 1- and 5-

substituents result in decreased affinity and loss of antagonism, indicating the essential role of

the area in conferring receptor recognition and antagonist activity to the diarylpyrazole

compounds. On the other hand, replacement of the 3-substituent of the pyrazole ring with


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functional groups such as ethers and alkyl amides generally leads to dramatic decreases in CB1

binding affinities (Lan et al 1999). For 3-substituent analogs that show significant receptor

affinity, partial agonist properties have been observed (Wiley et al 2001).

Structural modifications to the diarylpyrazoles as a result of metabolism, however, have

not been reported. In light of the sensitivity of the receptor recognition and antagonistic efficacy

of diarylpyrazoles to structural variations on the 1-, 3-, and 5-subtituents (Figure 1), it can be

hypothesized that potential metabolites of these ligands may lose much of their receptor binding

affinity and/or antagonist properties. It is also possible that some of the metabolic products may

retain a significant amount of antagonism of the parent compounds if structural modifications

occur at less crucial sites. Thus, knowledge of the metabolic transformation of the

diarylpyrazoles is required to evaluate the role of metabolites in the overall efficacy of the

antagonists. The current study investigates the in vitro metabolism of SR141716A, AM251, and

AM281 using rat liver microsomes. High performance liquid chromatography (HPLC) was used

for the separation of complex metabolic mixtures, and tandem mass spectrometry (MS/MS)

along with high-resolution nuclear magnetic resonance (NMR) spectroscopy techniques were

employed in the structural elucidation of various metabolic products of the diarylpyrazole

analogs.

(Insert Figure 1 near here)

EXPERIMENTAL

Materials AM281 and AM251 were purchased from Tocris Cookson (Ellisville, MO).

HPLC-grade solvents (acetonitrile, methanol, and water) were purchased from Sigma-Aldrich

Co. (St. Louis, MO). All other chemicals were obtained from Fisher Scientific (Fair Lawn, NJ).


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Rat liver microsomes were purchased from Gentest Corporation (Woburn, MA) and stored at -80

oC prior to use.

Synthesis of SR141716A Synthesis of SR141716A was carried out following procedures

reported by Dutta et al (1994). Briefly, bromination of 4-chloropropiophenone gave the

bromoketone intermediate. Treatment of the anion of ethyl acetoacetate with the bromoketone

furnished the acetoacetate derivative. The sodium salt of the acetoacetate derivative was allowed

to react with a solution of 2,4-dichlorodiazonium chloride, followed by base hydrolysis to give

the pyrazole carboxylic acid which was then treated with 1-aminopiperidine and triethylamine to

give the target compound. The product was purified by flash chromatography and crystallized

from methanol. Structural confirmation of the synthetic SR141716A was done by 1H and 13C

NMR spectroscopy (Tables 1-3) and HPLC-MS/MS (Figure 2).

Microsomal Incubations Stock solutions of SR141716A, AM-251, and AM-281 were

prepared in DMSO, at a concentration of 20 mM. Rat liver microsomes containing 1.8 mg/ml of

protein were pre-incubated at 37 °C for 3 minutes. The 0.2-mL incubation aliquots contained

75 mM potassium phosphate (pH 7.4), 17 mM magnesium chloride, 7 mM NADP+, 17 mM

glucose-6-phosphate, and 1.2 units/mL of glucose-6-phosphate dehydrogenase. To the incubation

aliquots were added 1 µL of the SR141716A, AM281, or AM251 stock solution. Incubation

times ranged from 0.5 to 2 hours. Incubations were halted by placing the vials in an ice bath

followed by adding an equal volume of methanol (0.2 mL). The quenched incubation mixtures

were stored at -20°C until analysis. Prior to HPLC separation, microsomal proteins were

precipitated by centrifugation (10,000g, 15 min) at room temperature, and the supernatant was

evaporated with a stream of nitrogen at 37 °C to 0.2 mL. The residual solution was applied to 6-

ml SUPELCO (Bellefonte, PA) C18 solid-phase extraction columns pretreated with water (4 x 4


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mL) and methanol (4 x 4 mL). The columns were washed with HPLC-grade water (2 x 3 mL)

and eluted with methanol (3 x 3 mL); the effluents were again concentrated by a nitrogen stream

at 37°C to 1.0 mL. For semipreparative purposes, a total of 5x10mL larger scale incubations

were carried out. In each 10-mL incubation mixture were 18 mg protein, 75 mM potassium

phosphate buffer (pH 7.4), 17 mM magnesium chloride, 7 mM NADP+, 17 mM glucose-6-

phosphate, and 1.2 units/mL of glucose-6-phosphate dehydrogenase and 50 µL of 20mM

SR141716A.

Control incubations were performed under identical conditions with heat-inactivated

microsomes (heated to 100°C for 10 min). In addition, incubations in the absence of NADPH, or

in the absence of microsomes, were carried out. To eliminate matrix interferences arising from

microsomes and buffer components, blank incubations were done where all elements were

present except the drug compounds.

HPLC-UV Analysis Initial analysis of the incubation products were performed by a

Shimadzu (Colombia, MD) HPLC system equipped with a UV-Vis SPD-10ADVP detector. A

2.1 × 150 mm; 4 µm pore size Phenomenex (Torrence CA) ODS HPLC column was used for

separation. Mobile phase flow rate was set at 0.3 mL/min, with gradient elution starting at 10%

acetonitrile and 90% water for 5 min, followed by a linear increase to 50% acetonitrile in 14 min,

and a linear change to 100% acetonitrile in 5 minutes, and a final linear change back to 10%

acetonitrile in 5 minutes. Injection volume was 10 µL. Eluted components were detected by the

UV detector (λmax, 270 nm). To ensure reproducibility, at least three injections were performed

for each incubation aliquot. No significant qualitative or quantitative differences were found

between runs (CV% =3.5).


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Semipreparative HPLC Separation of the metabolites was carried out on a Phenomenex

(Torrance, CA) ODS HPLC column (10.0×250 mm; 4 µm pore size) coupled to a Phenomenex

ODS guard precolumn (10×50 mm, 4 µm). A Model 7125 Rheodyne manual injector with 500-

µL loop volume (Rheodyne Inc., Cotati, CA) was used for sample introduction. Mobile phase

flowrate was set at 5.5 mL/min, with gradient elution starting at 10% acetonitrile and 90%

water for 5 min, followed by a linear increase of acetonitrile to 50% in 15 min, and a linear

change to 100% acetonitrile in 7 minutes. Eluent absorbance was monitored at 270 nm using a

variable wavelength detector. Samples corresponding to the metabolite were pooled and dried

under vacuum and used for NMR analysis.

LC/MS and LC/MS/MS Analysis A Phenomenex ODS HPLC column (2.1 × 150 mm;

4 µm pore size) coupled to a Supelco C18 guard column (2×18 mm, 5 µm) was used for

separation. A Shimadzu LC-MS 2010 was used for initial screening of possible metabolic

products generated from the microsomal incubations by obtaining the mass spectra of all

chromatographic peaks. HPLC mobile phase flowrate was set at 0.20 mL/min, with gradient

elution starting at 10% acetonitrile and 90% water for 5 min, followed by a linear increase of

acetonitrile composition to 100% in 16 min. MS/MS experiments were performed on a Quattro II

triple quadrupole tandem mass spectrometer equipped with an electrospray (ES) source

(Micromass Inc., Beverly, MA). The ES needle potential was set at 3.46 kV and the orifice

potential was set at 70 V for MS scans and 62-67 V for MS/MS measurements. In MS/MS

experiments the collisionally induced dissociation (CID) of selected precursor ions took place in

the central hexapole collision cell using argon as collision gas. The collision energies ranged

from 17 to 20 eV. The ion source was held at 250 °C.


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NMR Spectroscopy. NMR spectra were recorded at 400 MHz or 500MHz on a Varian

Unity-400 or Varian Unity-500 spectrometer (Varian Instruments, Inc., Palo Alto, CA). Data

were processed on a SUN-5 computer using Varian VNMR software version 6.1B. Sample was

dissolved in 0.5 ml chloroform-d1 (99.8 atom % 2H) for NMR analysis. Chemical shifts are

reported on the δ scale by assigning the 7.26 (1H) and 77.0 (13C) for chloroform.

RESULTS

SR141716A (a)

Detailed LC-MS/MS and NMR spectroscopic analysis of the parent compound

SR141716A (a) were carried out to facilitate identification of the structures of its metabolites.

The full scan mass spectrum of a showed a prominent protonated molecule (MH+) at m/z 463

Da, which upon CID, yielded a base peak at m/z 363 (Figure 2). This fragment ion was proposed

to arise from the exclusion of the aminopiperidine moiety. Further fragmentation of the ion at

m/z 363 yielded at least three product ions at m/z 299, m/z 282, and m/z 164. The fragment ions

observed at m/z 99, m/z 84, and m/z 55 were proposed to be related to the aminopiperidine

moiety. The 1HNMR chemical shifts as well as the respective coupling patterns of SR141716A

and its metabolite Ma4 are summarized in table 1. All proton assignments were assisted by

COSY-experiments (Table 2). The 13 C NMR chemical shifts of SR141716A are presented in

Table 3. The 13C NMR assignments were assisted by DPET, HMQC and HMBC.

The broad single peak at around δ 7.60 (1H) can be assigned to the NH-proton at W. The

aromatic region of the 1H NMR spectrum of SR141716A indicated four protons having an

AA’BB’ system with two protons at δ 7.05 showing a correlation peak with the other two

protons at δ 7.24. However, the four protons show no correlation peak with any other protons in


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the molecule, suggesting a p-substituted aromatic ring, and they are assigned as follows: δ 7.05

for proton L and P, δ 7.24 for proton M and O. The chemical shift and splitting pattern of the

remaining three aromatic protons (δ 7.29, δ 7.31 and δ 7.43) were indicative of a 1,2,4-

trisubstituted ring. In the COSY spectrum, protons at δ 7.29 and δ 7.43 show correlation peaks

with the proton at δ 7.31. Based on the above information, the following assignments can be

made: δ 7.29 for proton R, δ 7.31 for proton S, and δ 7.43 for proton U. The coupling patterns of

the protons are consistent with the above proton assignments (JR,S=8.4Hz, ortho coupling,

JS,U=2.0Hz, meta coupling, JR,U=0.6Hz, para coupling).

Assignment of the aliphatic region (between δ 1.40 and δ 2.90) is based upon the

following. Integration of the singlet at δ 2.36 indicates the presence of three protons, linking the

single peak to the methyl protons (J) on the pyrazole ring. The signals from the ten protons on

the piperidine moiety are accounted for by peaks at δ 1.40 (2H), at δ 1.75 (4H), and at δ 2.86

(4H). The protons at δ 1.40 and at δ 2.86 all show correlation peaks with the protons at δ 1.75.

Clearly, the four protons at δ 2.86 are the two CH2 groups (A and E) next to the nitrogen atom,

the two protons at δ 1.40 belong to the CH2 (D), and the other four at δ 1.75 are the two CH2

groups (B and C).

SR141716A Metabolites

Shown in Figure 3 are the total ion chromatogram (TIC) obtained from an incubation

product mixture and the selected ion chromatograms for m/z 477, 479, and 461 that were

extracted from a single run. The selected ion chromatogram for m/z 477 is shown in Figure 3A,

in which 2 peaks are observed at retention times of 17.63 min. and 18.25 min., respectively.

These two metabolites, designated as Ma1 and Ma2, show an isobaric MH+ ion at m/z


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477, 14 amu higher than the [M+H]+ ion of SR141716A and 16 amu higher than the protonated

Ma5 and Ma6 (see also discussions on Ma5 and Ma6), suggesting that Ma1 and Ma2 are the

products of hydroxylation of the corresponding Ma5 or Ma6. The product ion spectra of Ma1

and Ma2 are identical and the spectrum is shown in Figure 4. A comparison with the product ion

spectrum of SR141716A and its proposed fragmentation pathways (Figure 2) indicates that the

fragment ion at m/z 98 is related to the SR141716A fragment ion at m/z 84. All other identical

fragment ions provide evidence that the only site of metabolic oxidation occurring in Ma1 and

Ma2 is the piperidine moiety. The proposed fragmentation pathways are also given in Figure 4

based on the tandem mass spectral details.

Figure 3B is the selected ion chromatogram for m/z 479 showing two metabolites (Ma3

and Ma4) eluting at 18.29 min. and 19.20 min., respectively. Both metabolites have the same

molecular weight of 478, indicating an increase of 16 mass units, which is consistent with

addition of one oxygen atom to the structure. The product ion spectrum of the protonated Ma3

and Ma4 is given in Figure 5. Compared to the fragment ions of a in Figure 2, a group of

fragment ions are found identical, including ones at m/z 363, m/z 299, m/z 282, and m/z 164.

The two fragment ions that differ from those of a are 461 (loss of H2O from m/z 479) and m/z 82

(2 amu lower than the fragment ion at m/z 84 in that of a), revealing structural modifications on

the piperidine ring via hydroxylation.

The major metabolite (Ma4) at 19.20 min was successfully isolated in sufficient amount

for further structural analysis by NMR. Compared to the parent compound a, the 1H NMR

spectrum of Ma4 in CDCl3 showed only minor variations in proton chemical shift in the

dichlorophenyl, chlorophenyl, and methyl substituents of the pyrazole ring, but significant changes

in chemical shift were observed in the region corresponding to the piperidine ring (Table 1). The


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piperidine ring is therefore the site of oxidation. Four possible structures can be envisioned, the

tertiary N-oxide (i), or a hydroxylated structure (ii, iii, iv), as illustrated in Chart 1.

The tertiary N-oxide i would be expected to have 10 proton signals in the piperidine ring.

In the 1H NMR spectrum of Ma4, only 9 proton signals were observed in this region (Table 1),

indicating one of the 10 protons has been substituted, suggesting that Ma4 can be a hydroxylated

structure (ii, iii or iv). One proton of the piperidine ring shifted down field significantly to δ 4.26,

indicative of a hydroxyl group formed on one of the five carbons (A, B, C, D or E), thus

eliminating structure i as a possibility. The second possible structure ii in which carbon B is the

site of hydroxylation, would require the remaining proton on carbon B to have correlation peaks

with four other protons (two on carbon A and two on carbon C). The third possible structure iii

where carbon C has the hydroxyl substituent would also require the remaining proton on carbon

C to have correlation peaks with four other protons (two on carbon B and two on carbon D). In

the 1H- 1H cosy spectrum of Ma4 (Table2), the remaining proton on the hydroxylated carbon

yields only correlation peaks with two protons, thus eliminating ii and iii as possible structures.

Overall, the NMR data points to the fourth structure (iv) as the structure of Ma4 with

Chart 1

OH

O OH

OH

(B=D)

(A=E)

i ii

iii iv

NA B

CDE

NA B

CDE

NA B

CDE

NA B

CDE


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hydroxylation occurring on carbon E. The proton on carbon E (δ 4.26) gives correlation peaks

with the two protons on carbon D (δ1.93 and δ2.08). Additional evidence includes correlation

peaks between the two protons on C and the two protons on B and the two protons on D, and

those between the two protons on B and the two protons on A (Table 2).

Figure 3C represents two other metabolites designated as Ma5 and Ma6 that have the

same molecular weight of 460. The protonated molecule at m/z 461 is 2 amu lower than the

[M+H]+ ion of SR141716A. A comparison of the product ion spectrum of Ma5 and Ma6

(Figure 6) and SR141716A (Figure 2) reveals that the fragment ions at m/z 97 and m/z 82 in

Figure 6 are also 2 amu lower than the fragment ion at m/z 99 and m/z 84, respectively, in

Figure 2, while all other fragment ions are identical. The above mass spectral information

indicates that the piperidine ring of SR141716A has been dehydrogenated to yield Ma5 and

Ma6. The proposed fragmentation pathways of Ma5 and Ma6 are given in the same figure. As

discussed earlier, the fragment ion of the protonated Ma1 and Ma2 at m/z 98 corresponds to the

fragment ion of the protonated Ma5 (or Ma6) at m/z 82. The mass difference of 16 and the

fragmentation from m/z 98 to m/z 80 via loss of H2O suggests the involvement of a hydroxyl

group on the dihydropiperidine moiety.

AM251 (b)

The product ion spectrum of AM251 (b) was obtained before HPLC-MS/MS analysis of

the incubation products. All tandem mass spectral data for AM251 and its metabolites are

summarized in Table 4. The total ion chromatogram of an incubation product mixture and the

selected ion chromatograms for the metabolites are shown in Figure 7. The selected ion

chromatogram for m/z 571 in Figure 7A shows two peaks (Mb1 and Mb2) at retention times of


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18.90 min. and 19.88 min., respectively. The m/z value for the protonated Mb1 and Mb2

indicates an increase of 16 mass units compared to the parent compound AM251, consistent with

the addition of a single oxygen atom to the structure. The tandem mass spectra data (Table 4)

show that Mb1 and Mb2 both share several identical fragment ions with AM251, i.e., Fb1 to Fb4

at m/z 455, 391, 328, 374, and 256, indicating no metabolic modification on these fragments.

The two fragment ions, Fb5 and Fb6, are each at 16 mass units higher than their counterparts of

the parent compound AM251. This information indicates that the piperidine ring of AM251 has

been hydroxylated. The fragmentation of the ion at m/z 100 via a dehydration pathway is

responsible for the ion observed at m/z 82, suggesting the involvement of a hydroxyl group on

the dihydropiperidine moiety.

Mb3 and Mb4 are two isomeric metabolites observed at retention times of 19.96 min. and

20.34 min., respectively. The two metabolites show a common [M+H]+ ion at m/z 553, 2 amu

lower than the [M+H]+ ion of AM251. A comparison of their product ion spectrum and AM251

(Table 4) reveals that the fragment ion at m/z 82 for the metabolites is also 2 amu lower than the

fragment ion at m/z 84 of AM251, while all other fragment ions are identical. The mass spectral

evidence suggests that the piperidine ring of AM251 has been dehydrogenated to yield Mb3 and

Mb4.

AM281 (c)

The full scan mass spectrum of the parent compound showed a prominent protonated

peak at m/z 557 Da., which upon CID results in a product ion at m/z 455 (Table 5) via loss of

the aminomorpholine moiety. Further fragmentation of the ion at m/z 455 yields peaks observed


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at m/z 328, and m/z 256. The fragment ions at m/z 101, m/z 86, and m/z 56 are related to the

aminomorpholine moiety.

Shown in Figure 8 are the total ion chromatogram (TIC) obtained from an incubation

product mixture of AM281 and the selected ion chromatograms for m/z 573 and 555, all

reconstructed from a single run. The selected ion chromatogram for m/z 573 (Figure 8A) shows

two peaks (Mc1 and Mc2) at retention times of 17.96 min. and 18.38 min., respectively,

indicating a pair of isobaric metabolites. The increase of 16 mass units compared to the parent

compound AM281 is consistent with the introduction of a hydroxyl group in replacing a

hydrogen atom. LC/MS/MS data summarized in Table 5 show that these two metabolites yield,

indicating no metabolic modification in these fragments of the molecule. Another fragment ion at

m/z 555, two amu less than the protonated AM281, is apparently the result of losing a H2O

molecule from the precursor ion at m/z 573. Further fragmentation of the ion at m/z 555 yields an

ion of the morpholinyl moiety that is also two amu lower than the corresponding fragment ion of

the protonated AM281. Clearly, hydroxylation has occurred on the morpholine ring.

The third metabolite, Mc3 is observed at a retention time of 19.21 min, which gives an

[M+H]+ ion at m/z 555, 2 amu lower than the [M+H]+ ion of AM281. A comparison of the

fragment ions of Mc3 and those of AM281 (Table 5) reveals that the fragment ion at m/z 84 of

the metabolite is also 2 amu lower than the fragment ion at m/z 86 of AM281, with all other

fragment ions being identical. Thus, the mass spectral evidence points to Mc3 as a metabolite

containing a double bond on the morpholine ring.


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DISCUSSION

Based upon the chromatographic and mass spectrometric data on the metabolic products

of the three diarylpyrazole compounds, the metabolic pathways for SR141716A, AM251, and

AM281 are illustrated in Figure 9, 10, and 11, respectively. All detected metabolites result from

structural modifications on the terminal ring of the 3-substituent. For SR141716A, a total of six

metabolites have been identified, which are designated as Ma1 through Ma6, in the order of

increasing retention time during chromatographic elutions. Hydroxylation on the piperidine ring

gives rise to the pair of isomeric metabolites Ma3 and Ma4 due to different possible sites of

hydroxylation. However, with the isolation and purification of Ma4, the only metabolite obtained

in pure form, it was possible to determine by NMR analysis that the hydroxyl group in Ma4 is

located at the alpha carbon on the piperidine ring. Dehydration of Ma3 and Ma4 leads to another

pair of isomeric metabolites, Ma5 and Ma6, each representing one of the two possible positions

of the double bond introduced on the piperidine ring. While it is likely that Ma5 and Ma6 are

preceded by Ma3 and Ma4, they may also be formed via direct dehydrogenation of the

piperidine ring (Sai et al 2001). The next two metabolites, Ma1 and Ma2 may be formed via two

possible routes. First, further oxidation of Ma5 and Ma6 on the piperidine ring introduces a

hydroxyl group that can have several different sites of hydroxylation. However, it is also

plausible that the monohydroxyl metabolites (Ma3 and Ma4) can be oxidized to first form

dihydroxylated intermediates (Figure 9), which upon dehydration leads to Ma1 and Ma2.

In AM251 the p-chlorophenyl group is replaced by a p-iodophenyl substituent, which is

the only structural difference between it and SR141716A. It appears that such a structural

difference may be responsible for the observation of only four metabolites for AM251 (Figure

10), compared to six total for SR141716A. The metabolic pathways of AM251 involving


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hydroxylation on the piperidine ring followed by dehydration, however, is essentially the same

as proposed for SR141716A. Compared with AM 251, the third diarylpyrazole antagonist

examined in this study, AM281, has an additional structural modification in that the piperidine

ring is replaced by a morpholine group. As a result, only three metabolites of AM281 have been

identified, of which two are an isomeric pair (Mc1 and Mc2) resulting from the

monohydroxylation of the morpholine ring and the third a product of dehydration of Mc1 and

Mc2. It is noted that dehydration of both Mc1 and Mc2 leads to the same metabolite, Mc3, due to

only one possible position for the carbon-carbon double bond (Figure 11).

In conclusion, it has been demonstrated that three diarylpyrazole analogues that behave

as, cannibinoid receptor antagonists, undergo unique metabolic pathways that are shared neither

by traditional cannabinoids nor by synthetic agonists such as aminoalkylindoles (AAIs). Unlike

AAIs where extensive hydroxylations were observed at various sites of the parent compounds

(Zhang et al 2002, 2004), the only site vulnerable to metabolic activities appears to be the

terminal group of the 3-substituent on the pyrazole ring (Figure 1), whereas no metabolic

modification has been detected at other sites of the diarylpyrazoles. Given the sensitivity of

antagonist properties to structural variations on the 3-substituent (Wiley et al 2001, Lan et al

1999), it will be of great interest to understand whether the metabolites still bind to the receptors

with considerable affinity, and furthermore, whether they behave as antagonists or agonists after

structural modifications on the 3-substituents. With one major metabolite of SR141716A (Ma1)

isolated in pure form, further studies are now possible to determine the receptor binding affinities

and physiological efficacies of the metabolite for both CB1 and CB2.


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Reference: Bell MR, D’Ambra TE, Kumar V, Eissenstat MA, Hermann JL (1991): Antinociceptive

(aminoalkyl)-indoles. J Med Chem 34:1099-1110. Bouaboula M, Dussossoy D, Casellas P (1999): Regulation of peripheral cannabinoid receptor

CB2 phosphorylation by the inverse agonist SR144528 J Biol Chem 274:20397-20405. Compton DR, Rice KC, De Costa BR, Razdan RK, Melvin LS, Johnson MR, Martin BR (1993):

Cannabinoid structure-activity relationships: correlation of receptor binding and in vivo activities. J Pharm Exp Therap 265:218-226.

Cosenza M, Gifford AN, Gatley SJ, Pyatt B, Liu Q, Makriyannis A, Volkow ND (2000): Locomotor activity and occupancy of brain cannabinoid CB1 receptors by the antagonist/inverse agonist AM281. Synapse 38: 477-82.

Dutta AK, Sard H, Ryan W, Razdan RK, Compton DR, Martin BR (1994): The synthesis and pharmacological evaluation of the cannabinoid antagonist SR141716A. Med Chem Res 5: 54-62.

Felder CC, Joyce KE, Briley EM, Mansouri J, Mackie K, Blond O, Lai Y, Ma AL, Mitchell RL (1995): Comparison of the pharmacology and signal transduction of the human cannabinoid CB1 and CB2 receptors. Mol Pharmacol. 48: 443-50.

Felder CC, Joyce KE, Briley EM, Glass M, Mackie KP, Fahey KJ, Cullinan GJ, Hunden DC, Johnson DW, Chaney MO, Koppel GA, Brownstein M. (1998): LY320135, a novel cannabinoid CB1 receptor antagonist, unmasks coupling of the CB1 receptor to stimulation of cAMP accumulation. J Pharmacol Exp Ther. 284:291-7.

Gatley SJ, Lan R, Pyatt B, Gifford AN, Volkow ND, Makriyannis A (1997): Binding of the non-classical cannabinoid CP 55,940, and the diarylpyrazole AM251 to rodent brain cannabinoid receptors. Life Sci. 61:PL 191-7.

Gatley SJ, Lan R, Volkow ND, Pappas N, King P, Wong CT, Gifford AN, Pyatt B, Dewey SL, Makriyannis A (1998): Imaging the brain marijuana receptor: development of a radioligand that binds to cannabinoid CB1 receptors in vivo. J Neurochem. 70:417-423.

Keimowitz AR, Martin BR, Razdan RK, Crocker PJ, Mascarella SW, Thomas BF (2000): QSAR analysis of Delta 8-THC analogues: relationship of side-chain conformation to cannabinoid receptor affinity and pharmacological potency. J Med Chem 43:59-70.

Lan R, Liu Q, Fan P, Lin S, Fernando SR, McCallion D, Pertwee R, Makriyannis A (1999): Structure-activity relationships of pyrazole derivatives as cannabinoid receptor antagonists. J Med Chem. 42:769-76.

Matsuda LA, Lolait SJ, Brownstein MJ, Young AC, Bonner TI. (1990): Structure of a cannabinoid receptor and functional expression of the cloned cDNA. Nature. 346:561-564

Munro S, Thomas KL, Abu-Shaar M (1993): Molecular characterization of a peripheral receptor for cannabinoids. Nature 365:61-65.

Portier M, Rinaldi-Carmona M, Pecceu F, Combes T, Poinot-Chazel C, Calandra B, Barth F, Le Fur G, Casellas P. (1999): SR144528, an antagonist for the peripheral cannabinoid receptor that behaves as an inverse agonist. J Pharmacol Exp Ther 288:582-589.

Rinaldi-Carmona M, Barth F, Heaulme M, Shire D, Calandra B, Congy C, Martinez S, Maruani J, Neliat G, Caput D, Ferrara P, Soubrie P, Breliere J-C, Le Fur G. (1994): SR141719A, a potent and selective antagonist of the brain cannabinoid receptor. FEB Letts 350:240-244.

Sai K, Kaniwa N, Ozawa S, Sawada J. (2001): A New Metabolite of Irinotecan in Which Formation Is Mediated by Human Hepatic Cytochrome P-450 3a4. Drug Metab Dispos 29:1505-1513.


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Shim JY, Collantes ER, Welsh WJ, Subramaniam B, Howlett AC, Eissenstat MA, Ward SJ (1998): Three-dimensional quantitative structure-activity relationship study of the cannabimimetic (aminoalkyl)indoles using comparative molecular field analysis. J Med Chem 41:4521-4532.

Showalter VM, Compton DR, Martin BR, Abood ME (1996) Evaluation of binding in a transfected cell line expressing a peripheral cannabinoid receptor (CB2): identification of cannabinoid receptor subtype selective ligands. J Pharmacol Exp Ther 278: 989-999.

Wiley JL, Jefferson RG, Grier MC, Mahadevan A, Razdan RK, Martin BR (2001): Novel pyrozole cannabinoids: insights into CB1 receptor recognition and activation. . J Pharmacol Ex Ther 296: 1013-1022.

Zhang Q, Ma P, Iszard M, Cole RB, Wang W, Wang G (2002): In vitro metabolism of R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo [1,2,3-de]1,4-benzoxazinyl]-(1-naphthalenyl) methanone mesylate, a cannabinoid receptor agonist. Drug Metab Dispos 30:1077-1086.

Zhang Q, Ma P, Wang W, Cole RB, Wang G (2004): Characterization of rat liver microsomal metabolites of AM-630, a potent cannabinoid receptor antagonist, by high performance liquid chromatography/electrospray ionization tandem mass spectrometry. J Mass Spectrom 39:672-681.


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Footnote

a) Footnote to the title:

Financial support was provided by NIH-MBRS through GM08008, and in part by the National Science Foundation through CHE-9981948.

b) Person to whom reprint requests should be made:

Professor Guangdi Wang Department of Chemistry Xavier University of Louisiana 7325 Palmetto Street New Orleans, LA 70125

c) Numbered footnotes: 1. Department of Chemistry, Xavier University of Louisiana, New Orleans, LA70125 2. Department of Chemistry, University of New Orleans, LA 70148


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Figure Legend Figure 1. Structures of the diarylpyrazole antagonists SR141716A (a), AM251 (b), and AM281 (c). Figure 2. MS/MS spectrum obtained by collision-induced dissociation of the protonated SR141716A (a) and its proposed fragmentation pathway. Figure 3. HPLC-MS chromatograms of SR141716A (a) metabolites from rat microsomal incubation: (A) SIM chromatogram of m/z 477; (B) SIM chromatogram of m/z 479; (C) SIM chromatogram of m/z 461; (D) SIM chromatogram of m/z 463; and (E) total ion chromatogram (TIC). Figure 4. MS/MS spectrum obtained by collision-induced dissociation of the protonated Ma1 and Ma2 and their proposed fragmentation pathways. Figure 5. MS/MS spectrum obtained by collision-induced dissociation of the protonated metabolites at m/z 479 (Ma3, Ma4) and their proposed fragmentation pathways. Figure 6. MS/MS spectrum obtained by collision-induced dissociation of the protonated Ma5 and Ma6 at m/z 461 and its proposed fragmentation pathways. Figure 7. HPLC-MS chromatograms of AM251 (b) metabolites from rat microsomal incubation: (A) SIM chromatogram of m/z 571; (B) SIM chromatogram of m/z 553; (C) SIM chromatogram of m/z 555; and (D) total ion chromatogram (TIC). Figure 8. HPLC-MS chromatograms of AM281(c) metabolites from rat microsomal incubation: (A) SIM chromatogram of m/z 573; (B) SIM chromatogram of m/z 555; (C) SIM chromatogram of m/z 557; and (D) total ion chromatogram (TIC). Figure 9. Proposed metabolic pathways of a (SR141716A).

Figure 10. Proposed metabolic pathways of b (AM251). Figure 11. Proposed metabolic pathways of c (AM281).


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Table 1. 1H NMR assignments for SR141716A (a) and its metabolite Ma4

A E B D C J L,P M,O R S U W a

2.86 4H m

2.86 4H m

1.75 4H m

1.75 4H m

1.43 2H m

2.36 3H s

7.05 2H d J=8.8Hz

7.24 2H d J=8.8Hz

7.29 1H dd JR,S=8.4Hz JR,U=0.6Hz

7.31 1H dd JR,S=8.4Hz JS,U=2.0Hz

7.43 1H dd JS,U=2.0Hz JR,U=0.6Hz

7.63 1H br-s (D2O exchangeable)

Ma4 3.11 1H m 2.81 1H m

4.26 1H m

1.75 2H m

1.93 1H m 2.08 1H m

1.50 1H m 1.62 1H m

2.27 3H s

6.98 2H d J=8.8Hz

7.18 2H d J=8.8Hz

7.21 1H dd JR,S=8.4Hz JR,U=0.6Hz

7.23 1H dd JR,S=8.4Hz JS,U=2.0Hz

7.34 1H dd JS,U=2.0Hz JR,U=0.6Hz

7.92 1H br-s (D2O exchangeable)

Abbreviations: br-s, broad singlet; d, doublet, dd, doublet of doublets, J, coupling constant in Hz; m, multiplet, s, singlet Reference: TMS

OH

Cl

Cl

NN

HNO

N

Cl

AB C

DE

F

GH

I

J

KL

M

N OP

QR

ST

UV

W

Cl

Cl

NN

HNO

N

Cl

AB C

DE

F

GH

I

J

KL

M

N OP

QR

ST

UV

W This article has not been copyedited and form

atted. The final version m

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Table 2. 1H-1H COSY assignment for SR141716A (a) and its metabolite Ma4

A E B D C J L,P M,O R S U W SR141716A (a) δ a 2.86 2.86 1.75 1.75 1.43 2.36 7.05 7.24 7.29 7.31 7.43 7.63 (in CDCl3) COSY b 1.75 1.75 1.43

2.86 1.43 2.86

1.75 NO 7.24 7.05 7.31 7.43

7.29 7.43

7.31 7.29

NO

Ma4 δ a 3.11 2.81

4.26

1.75 1.93 2.08

1.50 1.62

2.27 6.98 7.18 7.21 7.23 7.34 7.92

(in CDCl3) COSY b 1.75 1.93 2.08

3.11 2.81 1.50 1.62

1.50 1.62 4.26

1.93 2.08 1.75

NO 7.18 6.98 7.23 7.34

7.34 7.21

7.21 7.23

NO

a Proton chemical shift δ in ppm relative to methanol or chloroform b COSY, correlation spectroscopy; homonuclear spin coupling observed to the indicated resonance. c NO, not observed with the acquisition parameters used in the experiment.

OH

Cl

Cl

NN

HNO

N

Cl

AB C

DE

F

GH

I

J

KL

M

N OP

QR

ST

UV

W

Cl

Cl

NN

HNO

N

Cl

AB C

DE

F

GH

I

J

KL

M

N OP

QR

ST

UV

W

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Table 3. 13C Assignment for SR-141716A (a)

C13 HMQC HMBC DEPT A 57.29 2.86 1.75; 1.43; 7.63 CH2 B 25.68 1.75 2.86; 1.43 CH2 C 23.60 1.43 2.86; 1.75 CH2 D 25.67 1.75 2.86; 1.43 CH2 E 57.29 2.86 1.75; 1.43; 7.63 CH2 F 160.21 - 7.63 C G 144.68 - 2.36 C H 118.47 - 2.36 C I 143.16 - 2.36; 7.05 C J 9.54 2.36 - CH3 K 127.45 - 7.24 C L 131.04 7.05 7.24; 7.05 CH M 129.12 7.24 7.05; 7.24 CH N 135.16 - 7.05; 7.24 C O 129.12 7.24 7.05; 7.24 CH P 131.04 7.05 7.24; 7.05 CH Q 136.21 - 7.29; 7.31 C R 130.83 7.29 7.31 CH S 128.11 7.31 7.29; 7.43 CH T 136.17 - 7.43 C U 130.56 7.43 7.31 CH V 133.21 - 7.43 C

Cl

Cl

NN

HNO

N

Cl

AB C

DE

F

GH

I

J

KL

M

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ST

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m/z 571

Cl

Cl

NN

HNO

N

I

OH

391

100-H2O

256

455

-I

328

115

82 55

m/z 555

84 55

391

99

455

374

NN

256

Cl

Cl

I

HNO

N

-I

328

m/z 553

82 55

391

98

455

374

NN

256

I

Cl

Cl

HNO

N

-I

328

Table 4. [M+H]+ and the Characteristic Fragment Ions of AM251 (b) and Its Metabolites

MH Fb1 Fb2 Fb1-127 Fb3 Fb4 Fb5 Fb6 Fb7 other

AM251 555 455 391 328 374 256 99 84 55

Mb1,Mb2 571 455 391 328 - 256 115 100 55 82

Mb3,Mb4 553 455 391 328 374 256 98 82 55




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m/z 557 m/z 555m/z 573

86 56

99

455N

N

256

Cl

Cl

I

HNO

N

O

-I

328

84

Cl

Cl

NN

HNO

N

O

I

OH-H2O

455

-I

328

555-H2O

84 54

455N

N

I

Cl

Cl

HNO

N

O

-I

328

Table 5. [M+H]+ and the Characteristic Fragment Ions of AM281(c) and Its Metabolites

MH Fc1 Fc1 -127 Fc6 Fc7 Other

AM281 557 455 328 86 56 101 265 256

Mc1, Mc2 573 455 328 84 - 555

Mc3 555 455 328 84 54




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VU

TS

RQ

PO

N

ML

K

J

I

H G

F

ED

CBA

Cl

N

HNO

NN

Cl

Cl

W

Cl

Cl

I

NN

HN

O

N

O

Cl

Cl

I

NN

HN

O

N

51

4

2

3

(a) (b) (c)

Figure 1




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25 50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550m/z0

100

%

463

363

29999

84

55 164282

Cl

Cl

NN

O

Cl

363m/z

N

84m/z

55m/z

N

HN99m/z

164m/z

Cl NH

Cl

Cl

N

Cl

282m/z

299m/z

Cl

Cl

Cl

N NH

SR141716A

[H ]

Cl

Cl

NN

HNO

N

Cl

463m/z

Figure 2




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Figure 3

13 14 15 16 17 18 19 20 21 22 23 24 min0

5000

10000

15000

20000

25000

30000

35000

40000

Int.

13 14 15 16 17 18 19 20 21 22 23 24 min0e3

250e3

500e3

750e3

1000e3

Int.

13 14 15 16 17 18 19 20 21 22 23 24 min0e3

50e3

100e3

150e3

200e3

250e3

300e3

350e3

400e3

Int.

13 14 15 16 17 18 19 20 21 22 23 24 min0e3

100e3

200e3

300e3

400e3

500e3

600e3

700e3

800e3

Int.

13 14 15 16 17 18 19 20 21 22 23 24 min

1.0e6

2.0e6

3.0e6

4.0e6

5.0e6

6.0e6

7.0e6

Int.

E TIC

D m/z 463

C m/z 461

B m/z 479

A m/z 477Ma1

Ma2

Ma4

Ma6

SR141716( a)

0

0100%

0100%

0100%

Ma3

Ma5

Time (min.)

13 15 17 19 21 23 25

100%

0100%




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Figure 4

25 50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550m/z0

100

%

477

363

299

98

80

70 164282

299m/z

Cl

Cl

Cl

N NH

- CO

- H2O

Cl

Cl

NN

O

Cl

363m/z

Cl

Cl

N

Cl

282m/zN

70m/z

N OH

98m/z

164m/z

Cl NH

Cl

Cl

NN

HNO

N

Cl

477m/z

OH

[H ]

80m/zN




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Figure 5

25 50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550m/z0

100

%

479

363

299

99

8255

164282

461

Cl

Cl

NN

HNO

N

Cl

Cl

Cl

NN

O

Cl

479m/z

363m/z

OH

Cl

Cl

NN

HNO

N

Cl

461m/z

H

H2O

Cl

Cl

N

Cl

282m/z164m/z

Cl NH

[H ]

299m/z

Cl

Cl

Cl

N NH

99m/zH2N

N

H2N

N

55m/z

N

82m/z




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Figure 6

25 50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550m/z0

100

% 461

55

82

97

299

363

164 282

Cl

Cl

N

Cl

282m/z

164m/z

Cl NH

55m/z

299m/z

Cl

Cl

Cl

N NH

Cl

Cl

NN

O

Cl

m/z 363

N

HN97m/z

N

82m/z

Cl

Cl

Cl

NN

HNO

N

m/z 461

[H ]




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Figure 7

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 min

1.0e6

2.0e6

3.0e6

4.0e6

5.0e6

6.0e6

7.0e6

Int. 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 min0e3

100e3

200e3

300e3

400e3

500e3

600e3

700e3

Int. 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 min0

25000

50000

75000

Int. 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 min0e3

50e3

100e3

150e3

200e3

250e3

300e3

350e3

400e3

Int.

D TIC

C m/z 555

B m/z 553

A m/z 571

Mb1

Mb2

Mb4

AM-251(b)

0100%

0

0100%

Mb3

Time (min.)10 13 16 19 22 25 27

100%

0100%




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Figure 8

D TIC

C m/z 557

B m/z 555

A m/z 573Mc1

Mc2

AM281(c)

0

Mc3

10 11 12 13 14 15 16 17 18 19 20 21 22 23 min

500e3

1000e3

1500e3

2000e3

Int.

10 11 12 13 14 15 16 17 18 19 20 21 22 23 min0

25000

50000

75000

100000

125000

150000

175000

200000

Int.

10 11 12 13 14 15 16 17 18 19 20 21 22 23 min0e3

50e3

100e3

150e3

200e3

250e3

300e3

Int.

10 11 12 13 14 15 16 17 18 19 20 21 22 23 min0e3

50e3

100e3

150e3

200e3

250e3

Int.

Time (min.)

10 12 14 16 18 20 22 24

100%

100%

100%

100%

0

0

0




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Figure 9

OHOH

ClCl

- H2O

m/z 477

Cl

Cl

NN

Cl

Cl

NN

OH

OH

Cl

Cl

Cl

NN

ClClCl

- H2O

m/z 463 m/z 479 m/z 461

Cl

Cl

NN

Cl

Cl

NN

SR141716A

NN

Cl

Cl

N

HNO

N

HN

O

N

HNO

N

HN

O

N

HNO

N

HNO

Ma5, Ma6

Ma1, Ma2

Cl

Cl

Cl

NN

N

OHHN

O

OH

Cl

Cl

Cl

NN

N

HNO

Ma3 Ma4

- H2




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Cl

Cl

I

NN

HNO

AM251

m/z 555

NN

I

Cl

Cl

NN

I

Cl

Cl

m/z 553

NN

I

Cl

Cl

m/z 571

OHN

- H2O

Mb1, Mb2 Mb3, Mb4

HNO

NHN

O

N

HNO

N

Figure 10




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Figure 11

Cl

Cl

I

NN

HNO

AM281

m/z 557

NN

I

Cl

Cl

m/z 555

HN

O

N

O

NN

I

Cl

Cl

m/z 573

N

O

- H2O

OH

Mc1, Mc2 Mc3

HNO

N

O




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title: in vitro metabolism of diarylpyrazoles, a novel...

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