Verification of the Major Metabolic Oxidation Path for the Naphthoyl Group in Chemoattractant Receptor-Homologous Molecule Expressed on Th2 Cells (CRTh2) Antagonist 2-(2-(1-Naphthoyl)-8-fluoro-3,4-dihydro-1H-pyrido[4,3-b]indol-5(2H)-yl)acetic Acid (Setipiprant/ACT-129968)

Article abstract of DOI:10.1021/acs.jmedchem.5b00824

Various racemic and enantioenriched (trans)-X,Y-dihydroxy-X,Y-dihydronaphthoyl analogues as well as X-hydroxy-naphthoyl analogues of CRTh2 antagonist 2-(2-(1-naphthoyl)-8-fluoro-3,4-dihydro-1H-pyrido[4,3-b]indol-5(2H)-yl)acetic acid (1, Setipiprant/ACT-129968) were synthesized in order to gain insight into regio- and enantioselectivity of the metabolic oxidation of 1 and to verify the structures of four metabolites that were proposed earlier in a clinical ADME study. Analytical data of the synthetic standards were compared with data from samples of biological origin. The two major metabolites M7 and M9 were unambiguously verified as 2-(2-((trans)-3,4-dihydroxy-3,4-dihydronaphthalene-1-carbonyl)- and 2-(2-((trans)-5,6-dihydroxy-5,6-dihydronaphthalene-1-carbonyl)-8-fluoro-3,4-dihydro-1H-pyrido[4,3-b]indol-5(2H)-yl)acetic acid, respectively, each composed of two enantiomers with 68% and 44% ee in favor of (+)-(3S,4S)-M7 and (+)-(5S,6S)-M9, respectively. Likewise, minor metabolites M3 and M13 were identified as 2-(8-fluoro-2-(5-hydroxy-1-naphthoyl)- and 2-(8-fluoro-2-(4-hydroxy-1-naphthoyl)-1,2,3,4-tetrahydro-5H-pyrido[4,3-b]indol-5-yl)acetic acid, respectively.

Full text of DOI:10.1021/acs.jmedchem.5b00824

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Article
Verification of the major metabolic oxidation path for the naphthoyl
group in chemoattractant receptor-homologous molecule expressed
on Th2 cells (CRTh2) antagonist 2-(2-(1-naphthoyl)-8-fluoro-3,4-
dihydro-1H-pyrido[4,3-b]indol-5(2H)-yl)acetic acid (Setipiprant/ACT-129968)
Philippe Risch, Thomas Pfeifer, Jerome Segrestaa, Heinz Fretz, and Julien Pothier
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00824 • Publication Date (Web): 23 Sep 2015
Downloaded from http://pubs.acs.org on September 28, 2015
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Verification of the major metabolic oxidation path
for the naphthoyl group in chemoattractant
receptorꢀhomologous molecule expressed on Th2
cells (CRTh2) antagonist 2ꢀ(2ꢀ(1ꢀnaphthoyl)ꢀ8ꢀ
fluoroꢀ3,4ꢀdihydroꢀ1Hꢀpyrido[4,3ꢀb]indolꢀ5(2H)ꢀ
yl)acetic acid (Setipiprant/ACTꢀ129968)
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†
Philippe Risch, Thomas Pfeifer , Jerome Segrestaa, Heinz Fretz*, Julien Pothier
Actelion Pharmaceuticals Ltd., Drug Discovery Unit, Gewerbestrasse 16, CHꢀ4123 Allschwil
Switzerland.
KEYWORDS: Metabolite identification, (trans)ꢀdihydroxyꢀdihydronaphthalene, metabolism
of naphthalene, cytochrome Pꢀ450 oxidation, epoxide hydrolase, regioselectivity,
enantioselectivity, epoxidation
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ABSTRACT. Various racemic and enantioenriched (trans)ꢀX,YꢀdihydroxyꢀX,Yꢀ
dihydronaphthoyl regioisomers as well as Xꢀhydroxyꢀnaphthoyl regioisomers of CRTh2
antagonist
2ꢀ(2ꢀ(1ꢀnaphthoyl)ꢀ8ꢀfluoroꢀ3,4ꢀdihydroꢀ1Hꢀpyrido[4,3ꢀb]indolꢀ5(2H)ꢀyl)acetic
acid (1, Setipiprant / ACTꢀ129968) were synthesized in order to gain insight into regioꢀ and
enantioselectivity of the metabolic oxidation of 1, and to verify the structures of four
metabolites that were proposed earlier in a clinical ADME study. Analytical data of the
synthetic standards were compared with data from samples of biological origin. The two
major metabolites M7 and M9 were unambiguously verified as 2ꢀ(2ꢀ((trans)ꢀ3,4ꢀdihydroxyꢀ
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3,4ꢀdihydronaphthaleneꢀ1ꢀcarbonyl)ꢀ
and
2ꢀ(2ꢀ((trans)ꢀ5,6ꢀdihydroxyꢀ5,6ꢀ
dihydronaphthaleneꢀ1ꢀcarbonyl)ꢀ8ꢀfluoroꢀ3,4ꢀdihydroꢀ1Hꢀpyrido[4,3ꢀb]indolꢀ5(2H)ꢀyl)acetic
acid, respectively, each composed of two enantiomers with 68% and 44% ee in favor of (+)ꢀ
(3S,4S)ꢀM7 and (+)ꢀ(5S,6S)ꢀM9, respectively. Likewise, minor metabolites M3 and M13
were identified as 2ꢀ(8ꢀfluoroꢀ2ꢀ(5ꢀhydroxyꢀ1ꢀnaphthoyl)ꢀ and 2ꢀ(8ꢀfluoroꢀ2ꢀ(4ꢀhydroxyꢀ1ꢀ
naphthoyl)ꢀ1,2,3,4ꢀtetrahydroꢀ5Hꢀpyrido[4,3ꢀb]indolꢀ5ꢀyl)acetic acid, respectively.
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Introduction
Prostaglandin D2 (PGD2), a major metabolite of arachidonic acid, is predominantly
involved in the physiological response to allergy. It is released at high concentrations by
immunoglobulin E activated mast cells upon allergen challenge and was demonstrated to
activate two Gꢀprotein coupled receptors (GPCRs), the DP1 receptor and the chemoattractant
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1
,2
receptorꢀhomologous molecule expressed on Tꢀhelper 2 cells (CRTh2), more recently titled
3
DP2 receptor. CRTh2 activation leads to the recruitment of granulocytes and Th2 cells by
2
,4,5
chemotaxis to the inflammation site and has been demonstrated to be proꢀinflammatory.
Furthermore, it is now well established that antagonizing selectively the CRTh2 receptor
could be useful in the treatment of asthma and other inflammatory diseases of the airways,
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,6,7
such as allergic rhinitis.
More recently, mouse and human studies suggested that inhibition
of PGD2 signaling has the potential for the prevention and treatment of hairloss in man, often
8
presenting as male pattern baldness, or androgenetic alopecia.
CRTh2 antagonist 2ꢀ(2ꢀ(1ꢀnaphthoyl)ꢀ8ꢀfluoroꢀ3,4ꢀdihydroꢀ1Hꢀpyrido[4,3ꢀb]indolꢀ5(2H)ꢀ
yl)acetic acid (1, Chart 1), originally discovered and developed for the treatment of asthma
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and seasonal allergic rhinitis, now under development to treat androgenic alopecia in male
1
0
subjects (KYTHꢀ105), was reported a low clearance compound with a Clint value of 5 ꢁL
−1
−1
−1
6
min (mg protein) in both, human and mouse liver microsomes, Clint of 3.1 ꢁL min (10
−
1
−1
−1
11
cells) in rat hepatocytes, and CL 1.3 mL min kg after iv application to rats. Recently,
iv
data were published from a study investigating the absorption, disposition, metabolism, and
elimination (ADME) of 1 in healthy male subjects. A single oral dose of 1,000 mg of 1,
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spiked with [ C]1 as radiotracer (mixture of [ C]1/ nonlabeled 1 in a ratio of approximately
1:2,200), was administered with a total radioactivity of 2.60ꢀ2.62 MBq (approximately 71
12
µCi). In this study, 1 appeared to be metabolized to only a moderate extent. Two
metabolites M7 and M9 (Chart 1) accounted for 9.3% and 20% of the administered
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radioactive dose in the excreta (urine and feces). With respect to the administered dose, the
recovered mean radioactivity of M7 and M9 in urine accounted for 4.78% and 1.04%,
respectively. Moreover, M7 was the only metabolite quantifiable in plasma, but at
concentrations consistently below 10% of those of the parent drug. Structurally, M7 and M9
were proposed as two distinct dihydroxyꢀdihydronaphthoyl isomers. In addition, various
metabolites W1, W2, W3 and V (Chart 1, Footnote), most likely monoꢀhydroxylated at the
naphthoyl moiety, were identified in feces and urine, however in much lower concentrations.
Herein, the syntheses of various (trans)ꢀX,YꢀdihydroxyꢀX,Yꢀdihydronaphthoyl
regioisomers in their racemic and enantioenriched form as well as regioisomeric Xꢀhydroxyꢀ
naphthoyl analogues of 1 are described, and their use as synthetic standards is described to
assign and verify the structures of metabolites M3, M7, M9, and M13 (Chart 1). The two
major metabolites M7 and M9 were isolated from human urine and the structures of the
proposed (trans)ꢀX,YꢀdihydroxyꢀX,Yꢀdihydronaphthoyl moieties were unequivocally
assigned. Additionally, the structures of the two minor metabolites M3 and M13, both
containing a Xꢀhydroxynaphthoyl group, were elucidated. With the data provided, insight
was gained into the regioꢀ and enantioselective process of the metabolic naphthyl ring
oxidation in a more complex molecule.
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Chart 1. Structure of 1 (Setipiprant, ACTꢀ129968), proposed generic structures for
metabolites M7 and M9 (2) and M3 and M13 (3). The asterix (*) denotes the position of the
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C atom in [ C]1.
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+
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2H
2O
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O
O
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O
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N
N
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F
F
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N
COOH
COOH
COOH
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Metabolites identified in in vitro metabolism assays were termed M1, M2, M3, etc., a letter
code (C, D, E, etc.) was used in aboveꢀquoted clinical study. Therein, the two most dominant
metabolites were correlated with M7 and M9 of the original in in vitro studies, and hence the
terms were adopted.
Results
Metabolite Identification in Human Urine. Recently pooled human urine samples
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became available that were collected in a phase I in vivo human ADME study of 1. A total
1
4
volume of 500 mL human urine containing excreted 1 and metabolic products ( Cꢀlabeled,
Figure 3c in reference 12), was lyophilized and the lyophilisate was extracted with EtOAc.
After preparative HPLC separation, the two metabolites M7 and M9 were isolated in mg
amounts (7.3 mg, and 2.3 mg, respectively) as powder, and were characterized by means of
1
LC/MS, LCꢀMS/MS, and HꢀNMR spectroscopy. Despite the high dilution with unlabeled
1
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product (approx.1:2,200), the respective Cꢀlabeled M7 ([ C]M7) and M9 ([ C]M9) were
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detectable with high resolution MS, however, since Cꢀlabeled products only differ in +2
amu (Table S1, Supporting Information) and otherwise display identical fragmentation
pattern, only data of the unlabeled portion will be presented and discussed within the
following paragraph.
Presented in Figure 1 are the positive ion ESI LCꢀMS of M7 and M9 with high resolution
accurate mass measurements, and with the respective MS/MS fragmentation pattern obtained
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upon collision induced dissociation (CID) and highꢀenergy collision dissociation (HCD).
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Eluting at t = 32.98 min and 34.59 min, protonated molecules were detected at m/z 437.1513
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[
M+H] corresponding to a theoretical formula with the elemental composition of
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C H22FN O (ꢀM +1.4 ppm). Compared with 1, the observed molecular mass of both, M7
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and M9, differed by +34 amu, and likely corresponded to the addition of two hydrogen and
two oxygen atoms.
+
For both metabolites, fragment ions of m/z 419 [M+H-H
2
O] were detected upon CID of
+
the [M+H] ion at m/z 437.1 in the ion trap (CID 30% relative collision energy), indicating
loss of one molecule of H O. A key fragment for both, M7 and M9, was found at m/z 232
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which is typical for the tetrahydropyridoindoleꢀacetic acid core of 1. Due to the fact that the
core remained unchanged, it was concluded that the two putative hydroxyl groups are located
at the naphthalene ring, and that both, M7 and M9, are representatives of generic structure 2
(Chart 1). The observed fragment ions m/z 171 of M7 and M9, and m/z 189 of M9 were
attributed to the naphthoyl part, and are in agreement with proposed X,YꢀdihydroxyꢀX,Yꢀ
dihydronaphthaleneꢀ1ꢀcarbonyl cationic fragments, m/z 171 is indicative for a characteristic
2
loss of another H O from fragment m/z 189.
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Figure 1. Positive electrospray ionization (ESI) LCꢀMS chromatogram and a high resolution
accurate mass measurement of (a) metabolite M9 at t = 32.98 min, extracted ion
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chromatogram (EIC) of the protonated molecule (m/z 437.1513), and of (c) M7 at t
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= 34.59
with an
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min (m/z 437.1513), corresponding to the elemental composition of C24H22FN O
accuracy of ꢀ1.4 ppm for both, M7 and M9. Positive ion ESI tandem mass spectra
fragmentation pattern upon collision induced and highꢀenergy collision dissociation (CID,
HCD) for (b) M9, and for (d) M7. Both, M7 and M9, were isolated from human urine and
separated (see text).
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Metabolite Identification in Human Microsomes, Hepatocytes and Bactosomes. Radioꢀ
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labeled [ C]1 (61 mCi/mmol) was incubated at single substrate concentration of 10
ꢁM with
freshly prepared or cryopreserved human hepatocytes for up to 24 h. The viabilities of
cryopreserved and freshly prepared hepatocytes were 77% and 93%, respectively, the
radioactivity recovery exceeded 98 % in both cases. There was a moderate difference in the
rates of metabolism between the cryopreserved batch and the freshly prepared cells, e.g. after
24h incubation time, fourteen metabolites, denominated M2, M3, M5, M7, M9, M10, M11,
M12, M13, M15, M18, M21, M32, and M33, were detected and were accounting for 61%
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and 89% of the total initial radioactivity, whereas remaining [ C]1 accounted for 37% and
11%, respectively. After 24 h incubation time, both major metabolites, M7 and M9, were
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found at retention time (t ) = 33.8 min ([ C]M7) and t = 32.2 min ([ C]M9), accounting
together for 38% and 43% of the initial radioactivity with the cryopreserved and freshly
prepared cells, respectively (Figure 2a).
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Six metabolites were observed after incubating [ C]1 with human liver microsomes for 60
min, namely M2, M3, M7, M9, M13, and M15, altogether totalizing 16% of the initial
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radioactivity. Again, [ C]M7 and [ C]M9 were identified as the most prominent metabolites
which together were accountable for more than 60% of all metabolite radioactivity (Figure
2b).
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and [ C]M9 after incubating [ C]1 with human hepatocytes and microsomes that were
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comparable with M7 and M9 in human urine. Considering the presence of C in the
molecule, exact molecular mass, theoretical formula as well as MS/MS fragmentation pattern
were identical and corresponded to structure 2 (Table S1, Figure S1, Figure S2, Supporting
Information).
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1
4
Figure 2. Representative radioꢀchromatogram after incubation of [ C]1 with (a)
1
4
14
R R
cryopreserved human hepatocytes for 24h , [ C]M7 (t = 33.84) and [ C]M9 (t = 32.19
min) totalizing 38% of the radioactivity; (b) with human liver microsomes for 60 min; (c)
with human bactosomes 2C9 (500 pmol), for 60 min; (d) with human bactosomes 2C9 (500
pmol), in the presence of human microsomal epoxide hydrolase (hER), for 60 min.
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Two metabolites, marked as M3 and M13, were identified as two Xꢀhydroxyꢀ1ꢀnaphthoyl
1
4
regioisomers in a radioꢀchromatogram after incubating [ C]1 with hepatocytes and
microsomes (Figure 2a, 2b). In order to further investigate mechanistically the formation of
14
M7, M9, M3 and M13 and to deduce their structures, incubations of [ C]1 (61 mCi/mmol)
with bacterial membranes containing human cytochrome P450 (CYP) enzymes and coꢀ
expressed human NADPHꢀcytochrome P450 reductase (human bactosomes) were performed,
in the presence and absence of microsomal human epoxide hydrolase (hEH). Among the
studied human bactosomes with enzymes 1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6,
2
E1, 3A4 and 3A5, only human bactosome 2C8, 2C9 and 3A4 were found to produce M3
(2.2%, 13%, 3.1%), and M13 (8.4%, 4.4%, 1.4%) after 60 min incubation time. A
representative radioꢀchromatogram with bactosome 2C9 (Figure 2c) revealed that two
14
14
metabolites were corresponding to the [ C]M3 and [ C]M13 peak in the incubations with
human hepatocytes and human microsomes (Figure 2a, 2b). Accurate mass measurement by
+
LCꢀMS/MS provided at t = 42.3 min and at t = 43.5 min an m/z of 421.1439 [M+H] for
R
R
both ions, corresponding to a theoretical formula with the elemental composition of
14
C
23 CH20FN
2
O
4
(ꢀ
M ꢀ0.2 ppm). After incubating human bactosomes 2C8, 2C9 and 3A4
1
4
14
with 10 µM of [ C]1 in the presence of microsomal hEH, [ C]M3 (1.2%, 9.8%, 3.1%) and
14
14
14
[ C]M13 (3.5%, 3.9%, 1.4%) as well as [ C]M7 (4.3%, 0.97, 0.46%) and [ C]M9 (1.4%,
3.0%, 2.0%) were identified in the assay cocktail and were verified by LCꢀMS, as
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demonstrated with a representative radioꢀchromatogram from a bactosome 2C9 incubation
(Figure 2d). These data support the formation of a transient oxirene ((IIaꢀg), Scheme 1) as the
first intermediate in the metabolic naphthyl ring oxidation.
The metabolism of polycyclic aromatic hydrocarbons (PAH) in general and of naphthalene
derivatives in particular has been investigated for decades. For example, as early as in 1947,
Young was able to isolate and characterize (ꢀ)ꢀ(trans)ꢀ1,2ꢀdihydroꢀ1,2ꢀdihydroxyꢀ
0
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13
naphthalene from rat urine after subcutaneous administration of naphthalene. Later, van
14
15
Bladeren et al. and Yang reported on the enantiomeric composition of (trans)ꢀ1,2ꢀ
dihydrodiol metabolites formed from PAH. Two consecutive enzymatic steps were proposed
for this transformation: an initial regioꢀ and stereoselective epoxidation occurring at a formal
double bond of a PAH by microsomal CYP enzymes, followed by a regioꢀ and
enantioselective hydration of the formed epoxide by microsomal epoxide hydrolase.
Naphthalene (I), R substituted at position C(1), offers seven formal double bonds for
epoxidation that would lead to isomeric dihydronaphthalene epoxide ((IIaꢀg), Scheme 1). The
two sterically more hindered sites, the C(1)ꢀC(2) and the C(7)ꢀC(8) double bonds, were
thought to be less prone to the enzyme catalyzed oxidation leading to (IIa) and (IIf),
respectively. Epoxides (IIb) and (IIe) as well as dihydrodiols (IIIb) and (IIIe) were expected
to be unstable, mainly due to the nonꢀaromatic character of the apparent tetraene structures.
Once formed, epoxides (IIb) and (IIe) were anticipated to rearrange to the respective
benzo[d]oxepine rather than to undergo hydration to form the ringꢀopened 2,3ꢀdihydroxyꢀ2,3ꢀ
16ꢀ18
dihydronaphthalene (IIIb) and 6,7ꢀdihydroxyꢀ6,7ꢀdihydroꢀnaphthalene (IIIe).
To date, no
precedent has been reported on nonꢀaromatic naphthalene oxide (IIg) and 4a,8aꢀ
dihydronaphthaleneꢀ4a,8aꢀdiol (IIIg), neither as putative naphthalene metabolites, nor on
their synthetic access and properties. Due to the vast steric hindrance, enzyme catalyzed
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epoxidation and hydrolaseꢀcatalyzed hydration of the epoxide was expected to occur rather
unlikely at this position.
Based on both, the aforementioned mass spectroscopy results and the above discussed
literature, metabolism of 1 to M7 and M9 was hypothesized to occur in two consecutive
enzymatic steps, with the initial CYP catalyzed epoxidation at the naphthyl ring, followed by
the EH catalyzed hydration of the epoxide, thus opening the oxirane ring to form two out of
seven hypothetical vicinal substituted (trans)ꢀX,YꢀdihydroxyꢀX,Yꢀdihydronaphthoyl
regioisomers.
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In concordance with the results of Jerina et al. who demonstrated 1,2ꢀnaphthalene oxide as
the sole intermediate in the microsomal metabolism of naphthalene, epoxides (IIa), (IIc), (IId)
19
and (IIf) were considered potential intermediates (Scheme 1) in the oxidation process of (I).
Taking into consideration that the CYP catalyzed epoxidation might take place preferentially
at the two least hindered and more alkeneꢀlike C(3)ꢀC(4) and C(5)ꢀC(6) double bonds,
formation of dihydronaphtho[1,2ꢀb]oxirene (IIc) and (IId), respectively, was anticipated.
Therefore, in the particular case of the metabolic oxidation of 1, (trans)ꢀ3,4ꢀdihydroxyꢀ3,4ꢀ
dihydronaphthoyl 2b and (trans)ꢀ5,6ꢀdihydroxyꢀ5,6ꢀdihydronaphthoyl 2c were proposed to
be formed as major metabolites (Chart 2). However, in the absence of EH, oxirene (IIc) and
(IId) were expected to rearrange with a subsequent hydride migration (NIH shift) leading to
the reꢀaromatized 4ꢀhydroxyꢀ and 5ꢀhydroxynaphthyl regioisomers (IVa) and (IVc),
respectively (Scheme 1). Therefore, structures 3a and 3b were anticipated to represent monoꢀ
hydroxylated metabolites of 1 (Chart 2).
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6
Scheme 1. Proposed twoꢀstep mechanism of (trans)ꢀX,YꢀdihydroxyꢀX,Yꢀ
1
3,14
dihydronaphthalene metabolite formation based on literature precedence.
Epoxidation of
naphthalene (I) to seven hypothetically oxirene regioisomers (IIaꢀg), followed by the epoxide
hydrolase catalyzed ring opening to form (trans)ꢀX,YꢀdihydroxyꢀX,Yꢀdihydronaphthalene
(IIIaꢀg), and the corresponding rearranged (NIH shift) Xꢀhydroxynaphthalene derivatives
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0
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9
0
(
IVaꢀd).
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Chart 2. Structures of (trans)ꢀX,YꢀdihydroxyꢀX,Yꢀdihydronaphthalene isomers 2aꢀd and of
Xꢀhydroxynapthalene isomers 3a and 3b.
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Two studies were supportive regarding the preferred sites of epoxidation. Data of an in
vitro biotransformation study with antifungal 5 (Chart 3) using human liver microsomes and
20
recombinant human CYPs, and an in vivo metabolism study where calcimimetic agent 6
2
1
(
Chart 3) was administered to animals and human volunteers, led to the conclusion that the
respective metabolites comprised a (trans)ꢀ3,4ꢀdihydroxyꢀ3,4ꢀdihydronaphthalene and a
trans)ꢀ5,6ꢀdihydroxyꢀ5,6ꢀdihydronaphthalene modification. However, as indicated with
(
arrows in Chart 3, inconsistent conclusions concerning the site of oxidative attack were
provided by three other reports on the metabolism of drugs or drug candidates containing
naphthyl as a structural feature. For example, isomeric (trans)ꢀ3,4ꢀdihydroxyꢀ3,4ꢀdihydroꢀ
and (trans)ꢀ7,8ꢀdihydroxyꢀ7,8ꢀdihydroꢀnaphthalene derivatives were proposed as main
metabolites in human urine samples from pharmacokinetic studies with 4, suggesting that
enzymatic attack occurred at one of the preferred and at one of the sterically more hindered
22
double bond. In two in vitro microsomal metabolism studies the authors claimed that both,
2
3
24
cannabinoid receptor CB
1
2
agonist 7, and peripheral cannabinoid receptor CB agonist 8,
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4
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5
5
5
5
5
5
5
6
were leading to the respective nonꢀaromatic 2,3ꢀdihydroxyꢀ2,3ꢀdihydronaphthalene
metabolites. However, in both cases a second dihydroxyꢀdihydronaphthalene isomer was
observed and characterized. A favorable (trans)ꢀ5,6ꢀdihydroꢀ5,6ꢀdihydroxyꢀnaphthalene
structure was assigned for 7, and again a sterically less favored (trans)ꢀ7,8ꢀdihydroꢀ7,8ꢀdiol
for 8.
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0
Chart 3. Proposed sites of metabolic conversion at the naphthyl ring, epoxide formation and
2
2
20
EH catalyzed hydration, as reported for antifungal naftifine (4) and terbinafine (5),
21
23
calcimimetic agent cinacalcet (6), cannabinoid receptor (CB
1
) agonist WIN55212ꢀ2 (7),
24
2
and peripheral cannabinoid receptor (CB ) agonist JWHꢀ015 (8), are indicated with arrows.
Consequently, the syntheses of the four regioisomers 2aꢀd (Chart 2), in racemic and in
enantioenriched form, were envisaged. These synthetic standards were expected to be useful
tools for assigning the correct structure to the (trans)ꢀX,YꢀdihydroxyꢀX,Yꢀdihydronaphthoyl
group in M7 and M9, by comparing the analytical data with those obtained from biological
samples. Hence, further insight into the regioꢀ and enantioselective course of the metabolic
oxidation of 1 was anticipated. Herein, no attempts have been undertaken towards the
syntheses of unprecedented and unlikely to be generated regioisomers of structural type 2,3ꢀ
dihydroxyꢀ2,3ꢀdihydronaphthalene (IIIb), 6,7ꢀdihydroxyꢀ6,7ꢀdihydroꢀnaphthalene (IIIe) and
4
a,8aꢀdihydronaphthaleneꢀ4a,8aꢀdiol (IIIg).
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Generally, dihydronaphthoꢀoxirenes like (IIc) and (IId) (Scheme 1) are difficult to detect by
means of mass spectrometric analysis, especially in biological samples. This is due to the
short lifetime of (IIc) and (IId) and the propensity to immediately rearrange with a subsequent
hydride migration (NIH shift) in aqueous media or under the ionization conditions in the
mass spectrometer, preferentially leading to respective reꢀaromatized Xꢀhydroxyꢀ1ꢀnaphtyl
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25
regioisomers (IVa) and (IVc). An effort was directed towards the synthesis of 3a and 3b
(Chart 2) in order to verify the structures of M3 and M13 and to futher prove the preferred
site of oxidative attack.
Chemistry
Retrosynthetic Analysis. Key to the synthetic access of (trans)ꢀX,YꢀdihydroxyꢀX,Yꢀ
dihydronaphthalene isomers 2aꢀd was the preparation of the respective isomeric (trans)ꢀX,Yꢀ
dihydroxyꢀX,Yꢀdihydronaphthaleneꢀ1ꢀcarboxylic acids 9aꢀd (Scheme 2), modified with an
appropriate protecting group (PG) for the vicinal (trans)ꢀdiol. Epoxides 10aꢀd should be
obtained in multiple steps starting from αꢀtetralone (11a), 5ꢀbromoꢀ1ꢀtetralone (11b), and 8ꢀ
bromoꢀ1ꢀtetralone (11c), respectively. Diol protection was expected to be important for the
successful final amide bond forming step between ethyl 2ꢀ(8ꢀfluoroꢀ3,4ꢀdihydroꢀ1Hꢀ
pyrido[4,3ꢀb]indolꢀ5(2H)ꢀyl)acetate (12) and building block 9a-d. In any case, pure 13a-d
was provided in reasonable yields and without any hydroxyꢀnaphthoyl contaminants (Scheme
3).
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6
Scheme 2. Retrosynthetic analysis of (trans)ꢀX,YꢀdihydroxyꢀX,Yꢀdihydronaphthalene
analogues 2aꢀd.
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Scheme 3. Amide bond formation and final deprotection.
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a
Reagents and conditions: (a) Carboxylic acid 9aꢀd, HATU, DIPEA, DMF, rt, 20ꢀ73% (2
steps); (b) 30% 1N NaOH in EtOH or THF, rt, workꢀup with aqueous 1N HCl, 17ꢀ59%.
Synthesis of (trans)-1,2-dihydroxy-1,2-dihydronaphthalene-1-carbonyl)-8-fluoro-3,4-
dihydro-1
H-pyrido[4,3-b]indol-5(2H)-yl)acetic acid (2a). As depicted in Scheme 4, the
synthesis of building block 9a started with the conversion of αꢀtetralone (11a) to vinyl
2
6
bromide 14 using bromotriphenoxyphosphonium bromide (TPPBr ). Lithiation of 14 with
2
1
.3 equivalent of nꢀBuLi at ꢀ78°C followed by addition of solid CO
2
(dry ice) as the
27
electrophile gave 3,4ꢀdihydronaphthaleneꢀ1ꢀcarboxylic acid (15) in 68% yield.
Esterification of acid 15 was performed under acidic condition to provide the corresponding
ethyl ester 16 in 79% yield. Epoxide 10a was obtained by mꢀCPBA epoxidation of olefin 16
in 99% yield. A highly regioꢀ and stereoselective epoxide ringꢀopening process was reported
2
8
by Harvey and Tang. Under acidꢀcatalyzed hydrolytic conditions, using catalytic amounts
of 10ꢀcamphorsulfonic acid (CSA) in aqueous acetone at room temperature, (trans)ꢀdiol was
formed with inverted stereochemistry at the benzylic carbon atom of the epoxide ring.
However, no epoxide hydrolysis was observed for 10a under those conditions, until the
reaction temperature was raised to 60°C for 48h. Hence, two epimers of 1,2ꢀdihydroꢀ1,2ꢀdiol
1
7 were formed in 70% yield, and a mixture of (cis)ꢀ/(trans)ꢀdiol in a 18:82 ratio was
identified by LCꢀMS, inseparable in a preparative scale. The presence of the two epimers
indicated that the presumed diastereoselective hydrolysis of epoxide 10a was compromised.
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9
As concluded in an earlier study by Chini et al., the tertiary benzylic oxirane carbon atom
facilitates syn addition of the nucleophile (H O) due to the stabilizing effect of the
2
carbocationic centre at C(1). This carbocationic centre is additionally stabilized by the
adjacent carboxylate group in the present case of epoxide 10a (Scheme 2). The neighboring
group effect of the carboxylate was supposedly responsible for the increased resistance to
hydrolysis of the oxirane ring. As a consequence, enhanced reaction temperature and
prolonged reaction time were necessary to drive the reaction to completion. In contrast to the
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reported 1ꢀmethyl substituted 1,2ꢀepoxide, no rearrangement to the ethyl 2ꢀoxoꢀ1,2,3,4ꢀ
tetrahydronaphthaleneꢀ1ꢀcarboxylate 18 was observed in the reaction mixture.
Conditions that were successfully applied earlier to form the bisꢀsilyl ether of related
vicinal diols involved two equivalents of tertꢀbutyldimethylsilyl chloride (TBSCl) and
30
imidazole in DCM, at room temperature for 8h. However, attempts to protect both hydroxyl
groups in 17 as bisꢀsilyl ether failed, even after heating at reflux for 24 h, presumably due to
steric hindrance caused by the adjacent carboxylate group. Selective TBS protection at C(2)ꢀ
OH provided 19 in 92% yield as a mixture of epimeric (cis)ꢀ and (trans)ꢀ19. These epimers
could be separated by flash chromatography on silica gel. The structure was verified for each
1
1
epimer by NMR studies including selective gradient Hꢀ H 2D ROESY experiments. Two
x
2
significant ROE correlations were observed between H (
δ
= 5.58 ppm) and H (
= 1.87 ppm). Together with the observed coupling
constants JH2,H3 = 4.3 Hz and JH2,H3’ = 9.8 Hz a (trans)ꢀ1,2ꢀdiol configuration could be
δ = 4.00
2
3
ppm), H (δ = 4.00 ppm) and H (δ
R
established for the second eluting epimer at t = 1.08 min ((trans)ꢀ19, Figure S3, Supporting
Information).
For the first eluting epimer at t = 1.05 min, two ROE correlations were observed between
R
x
3’
2
3
H (
δ
= 4.79 ppm) and H (
δ
= 2.01 ppm), and H (
δ
= 4.32 ppm) and H (
δ
= 1.78 ppm), but
x
2
no ROE correlation was observed between H (
δ
= 4.79 ppm) and H (
δ
= 4.32 ppm).
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Together with the measured coupling constant JH2,H3 = 3.5 Hz and JH2,H3’ = 11.9 Hz a (cis)ꢀ
1,2ꢀdiol configuration could be demonstrated ((cis)ꢀ19, Figure S3, Supporting Information).
Scheme 4. Synthesis of building block 9a.
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25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a
Reagents and conditions: (a) (PhO)
3 2
P, Br , Et
3
N, DCM, ꢀ60°C to rt, 18h, reflux for 3h,
(dry ice), 68% ; (c) 4N HCl in
dioxane, EtOH, 18h at reflux, 79%; (d) mꢀCPBA, DCM, 1h at ꢀ15°C then room temperature
for 12h, 99%; (e) CSA, acetone/ H O (4:1), 60°C, 48 h, 70%; (f) TBSCl, imidazole, in THF,
at reflux, 30h, 92%; (g) NaClO , tꢀBuOOH, MeCNꢀH O (3:1), reflux, 24h, 43%; (h) pꢀ
7
7%; (b) 2.5M nꢀBuLi in hexane, THF, ꢀ78°C, then CO
2
2
2
2
TsNHNH , toluene, reflux, 2 h, quantitative yield; (i) NaH, toluene, 0°C to reflux, 2.5h, 18%;
(k) 1N NaOH in aqueous THF, rt, 24h, 92%.
2
Benzylic oxidation at C(4) in (trans)-19 was achieved according to the mild method
30
developed by Suresh et al, applying a mixture of NaClO
reflux, to give tetralone 20 in 43% yield. Treatment with pꢀTsNHNH
tosylhydrazone 21. Olefin 22 was obtained after treatment of tosylhydrazone 21 with sodium
2
and tꢀBuOOH in MeCN in H
2
O at
2
furnished
31,32
hydride in low 18% yield as a BamfordꢀStevens degradation product.
Saponification of
ethyl 1,2ꢀdihydroꢀnaphthaleneꢀ1ꢀcarboxylate 22 gave the envisaged building block 9a in
excellent yield. The final steps towards 2a are depicted in Scheme 3. Ethyl 2ꢀ(8ꢀfluoroꢀ3,4ꢀ
dihydroꢀ1Hꢀpyrido[4,3ꢀb]indolꢀ5(2H)ꢀyl)acetate 12 and carboxylic acid 9a were coupled
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under peptide bond forming condition using coupling reagent HATU and
diisopropylethylamine (DIPEA) in THF to provide 13a in low 20% yield. The ethyl ester in
13a was saponified with 2N aqueous NaOH in EtOH and the TBS group was cleaved under
acidic workꢀup conditions, providing 2a in 52% yield.
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Synthesis of (trans)-X,Y-dihydroxy-X,Y-dihydronaphthalene-1-carbonyl)-8-fluoro-
3,4-dihydro-1H-pyrido[4,3-b]indol-5(2H)-yl)acetic acid (2b-d). The syntheses of building
blocks 9bꢀd started with commercially available αꢀtetralone (11a), 5ꢀbromoꢀ1ꢀtetralone (11b)
and 8ꢀbromoꢀ1ꢀtetralone (11c) via the key intermediate epoxides 10bꢀd, respectively
(Scheme 5). Olefins 23aꢀc were obtained in two steps by means of a high yielding (94ꢀ98%)
sodium borohydride reduction of the ketones 11aꢀc and subsequent pꢀtoluenesulfonic acid
30
(
PTSA) catalyzed dehydration of the respective benzylic alcohols 23aꢀc. In order to obtain
23d, olefin 23c was first lithiated with nꢀBuLi in THF at ꢀ78°C, and then reacted with CO
2
,
added as dry ice to the reaction mixture. The intermediate carboxylic acid was isolated and
subsequently esterified to the ethyl ester 23d in good 93% yield for the two steps. The desired
respective oxiranes 10bꢀd were produced by applying mꢀCPBA epoxidation reaction
conditions to olefins 23aꢀd. Crude oxirene products 10bꢀd were submitted without further
purification to a regioꢀ and stereoselective acid catalyzed epoxide hydrolysis in the presence
of H
based on published data. HꢀNMR spectroscopic evidence confirmed a (trans)ꢀ1,2ꢀdiol
arrangement for all three cases. For example, coupling constants at 4.27, 4.28 and 4.48 ppm
J= 5.85, 5.7, and 5.5 Hz) for HꢀC(1) in 24aꢀc, respectively, were found to correlate well with
2
O and CSA. Solely (trans)ꢀ1,2ꢀdiols 24aꢀc were generated in 51ꢀ83% yield, as expected
28 1
δ
(
33
published data for 24a. The two hydroxy groups in 24a-c were protected using TBSCl and
imidazole at room temperature to provide 25aꢀc in 59ꢀ83% yield. The benzylic methylene
position at C(4) was oxidized to the respective αꢀtetralones 26aꢀc in 31ꢀ83% yield, again by
means of the aforementioned mixture of oxidizing reagents NaClO
2
and tꢀBuOOH.
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Subsequent treatment with either hydrazine hydrate in refluxing EtOH or pꢀTsNHNH in
refluxing toluene furnished hydrazone 27a and tosylhydrazones 27b and 27c,
34,35
respectively.
2
Reacting 27a with CuBr in the presence of tꢀBuOLi gave vinyl bromide 28a
36
in 73% yield. Olefins 28b and 28c were obtained as the BamfordꢀStevens degradation
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
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59
60
products in 69% and 49% yield after exposure of tosylhydrazones 27b and 27c, respectively,
31,32
to a large excess of NaH in toluene at reflux.
Halogenꢀlithium exchange in 28a and 28b
with 1.3 equivalent of nꢀBuLi at ꢀ78°C in THF was followed by the addition of solid CO
2
(dry ice) as the electrophile, affording envisaged dihydronaphthaleneꢀ1ꢀcarboxylic acid 9b in
73% and 9c as a crude product in 86% yield. Final saponification of the ethyl ester 28c
afforded building block 9d albeit in a low 24% yield. The syntheses of 2bꢀd were completed
by condensing 12 with carboxylic acids 9bꢀd using HATU as peptide coupling reagent in the
presence of DIPEA in DMF to give ethyl esters 13bꢀd in 51ꢀ73% yield (Scheme 3).
Saponification with aqueous 2N NaOH in EtOH and subsequent cleavage of the two TBS
groups during acidic workꢀup provided the final products 2bꢀd in 17ꢀ73% yield.
Enantioenriched syntheses of
derivatives (3 ,4 )-2b, (3 ,4 )-2b, (5
preparation of (trans)ꢀX,YꢀdihydroxyꢀX,Yꢀdihydronaphthalene (3R,4R)ꢀ2b, (3S,4S)ꢀ2b,
5R,6R)ꢀ2c and (5S,6S)ꢀ2c is reported in the following paragraph (Scheme 6). Jacobsenꢀ
(
trans)-X,Y-dihydroxy-X,Y-dihydronaphthalene
R
R
S
S
R
,6 )-2c and (5 ,6 )-2c. An enantioselective
R
S S
(
Katsuki type epoxidation of 23a and 23b, using 5% Jacobsen’s catalyst (+)ꢀ(1S,2S)ꢀ29 in the
presence of NaOCl and NaH PO in H O at room temperature, afforded (1aR,7bS)ꢀ10b and
2
4
2
37,38
(1aR,7bS)ꢀ10c in 23% and 40% yield, respectively.
Using the antipodal catalyst (ꢀ)ꢀ
(1R,2R)ꢀ29, (1aS,7bR)ꢀ10c was isolated in 37% yield. Due to poor chemical stability,
epoxides (1aR,7bS)ꢀ10b, (1aR,7bS)ꢀ10c as well as its enantiomer (1aS,7bR)ꢀ10c were
obtained with only moderate purities of 70%, 83% and 88%, respectively, but nevertheless
were subjected without any further purification to the next reaction step. Consequently, no
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5
6
enantiomeric ratio (er) could be determined for the epoxidation step. As already described
above, ringꢀopening of the epoxides (1aR,7bS)ꢀ10b, (1aR,7bS)ꢀ10c and its enantiomer
(1aS,7bR)ꢀ10c was achieved under acid catalyzed hydrolysis with a catalytic amount of CSA,
to producing an enantiomerically enriched mixture of (1R,2R)ꢀ24a and (1S,2S)ꢀ24a with
90:10 er in 79% isolated yield, a mixture of (1R,2R)ꢀ24b and (1S,2S)ꢀ24b with 88:12 er in
0
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9
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7
8
9
0
75% yield, and a mixture of (1S,2S)ꢀ24b and (1R,2R)ꢀ24b with 87:13 er in 76% yield,
respectively. The absolute configuration of (1R,2R)ꢀ24a was proven upon comparison with
commercially available enantiopure samples by analytical chiral HPLC. Enantioenriched
(1R,2R)ꢀ24a and (1R,2R)ꢀ24b were converted to their respective acids (3R,4R)ꢀ9b and
(5R,6R)ꢀ9c, applying the same reaction sequence as described in Scheme 5 for racemic 24a
and 24b. The two acids (3R,4R)ꢀ9b and (5R,6R)ꢀ9c were coupled without further purification
with tetrahydropyridoindole 12 under the same peptide coupling conditions mentioned above
(Scheme 3). Subsequent saponification and desilylation of (3R,4R)ꢀ13b and (5R,6R)ꢀ13c
provided finally enantioenriched (3R,4R)ꢀ2b and (5R,6R)ꢀ2c, respectively. If compared with
the respective precursors (1R,2R)ꢀ24a and (1R,2R)ꢀ24b, an unchanged 90:10 er for the
(3R,4R)ꢀ2b / (3S,4S)ꢀ2b and of 88:12 for the (5R,6R)ꢀ2c / (5S,6S)ꢀ2c mixture was observed,
indicating that no racemization occurred in the course of this multistep reaction sequence.
Enantiopure (>99% ee) reference samples (3R,4R)ꢀ2b, (3S,4S)ꢀ2b, (5R,6R)ꢀ2c, and (5S,6S)ꢀ
2c were obtained from racemic (trans)ꢀdiol 2b and 2c after preparative chiral separation.
Optical rotatory power measurements revealed negative [
α]
D
values for enantiopure (3R,4R)ꢀ
2
5
24
2
b ([α]
positive values for (3S,4S)ꢀ2b ([α]
46.7, c=0.39, MeOH), which are opposite the reported [
trans)ꢀdihydroxyꢀdihydro configured starting materials (1R,2R)ꢀ24a ([α] D= +100, c=0.32,
D
= ꢀ61.8, c=0.54, MeOH) and (5R,6R)ꢀ2c ([α]
D
= ꢀ48.6, c=0.38, MeOH), and
25
24
D
= +65.8, c=0.055, MeOH) and (5S,6S)ꢀ2c ([α]
D
=
+
D
α] values of the enantiopure
25
(
28
39,40
3 D 3
CHCl ), and (1S,2S)ꢀ24a ([α] = ꢀ103, c=1, CHCl ).
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Syntheses
of
2-(8-fluoro-2-(X-hydroxy-1-naphthoyl)-1,2,3,4-tetrahydro-5H-
pyrido[4,3-b]indol-5-yl)acetic acid 3a and 3b. Commercially available intermediate
building block 4ꢀhydroxyꢀ and 5ꢀhydroxyꢀ1ꢀnaphthoic acids (30a and 30b, respectively) were
used in the amide coupling reaction with tetrahydropyridoindole 12 (Scheme 7), delivering
31a and 31b in reasonable 54% and 73% yield, respectively. Concluding saponification of
the ethyl ester using NaOH in EtOH afforded expected 3a and 3b in moderate 45% and good
10
11
12
13
14
15
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17
18
19
20
21
22
23
24
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54
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56
57
58
59
60
7
9% yield, respectively (Scheme 7).
Scheme 5. Synthesis of building blocks 9bꢀd.
a
Reagents and conditions: (a) NaBH
4
, THF, MeOH, rt, 2h, quantitative yield; (b) PTSA,
, ꢀ78°C to rt, 73ꢀ86%; (d) 4N
toluene, reflux, 1h, 94ꢀ98%; (c) nꢀBuLi, THF, ꢀ78°C, then CO
HCl in 1,4ꢀdioxane, 10% in EtOH, reflux, 1h, 93%; (e) mꢀCPBA, DCM, ꢀ15°C, 1h, no
purification; (f) CSA, acetone / H O (4:1), rt, 2h, 51ꢀ83% over 2 steps; (g) TBSCl, imidazole,
2
2
THF, rt, 24h, 59ꢀ83%; (h) NaClO
NH NH , EtOH, reflux, 18h, 81%; (j) pꢀTsNHNH
BuOLi, THF, 0°C then rt, 24h, 73%; (l) NaH (50 equivalent), toluene, reflux, 1h, 49ꢀ69%;
m) KOH 15 equivalent, THF / H O / EtOH (1:1:1), 60h, 17% (47% recovered 28c).
2
, tꢀBuOOH, MeCNꢀH
2
O (3:1), reflux, 24h, 31ꢀ83%; (i)
2
2
2
, toluene, reflux, 2h, 51ꢀ60%; (k) CuBr
2
, tꢀ
(
2
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6
Scheme 6. Synthesis of enantioenriched building blocks.
0
1
2
3
4
5
6
7
8
9
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6
7
8
9
0
a
Reagents and conditions: (a
1
) 5% (+)ꢀ(1S,2S)ꢀ29, NaOCl, NaH
) 5% (ꢀ)ꢀ(1R,2R)ꢀ29, NaOCl, NaH
O (4:1), rt, 2h, 75ꢀ79%.
2
PO
4
, H
2
O, 0°C, rt, 18h,
23% (1aR,7bS)ꢀ10b, (1aR,7bS)ꢀ10c, 40%; (a
0°C, rt, 18h, 37%; (b) CSA, acetone / H
2
2
PO , H O,
4
2
2
Scheme 7. Synthesis of the Xꢀhydroxyꢀ1ꢀnaphthoyl standards 3a and 3b.
a
Reagents and conditions: (a) DIPEA, HATU, DCM or DMF, rt, 54% (31a) and 73%
(31b); (b) 30% 1N NaOH (10 equivalent) in EtOH, rt, 45% (3a) and 79% (3b).
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Metabolite Structure Verification
The analytical data of metabolites M7, M9, M3 and M13 were compared with the data
obtained from the synthetic standards 2aꢀd, enantioenriched (3R,4R)ꢀ2b and (5R,6R)ꢀ2c, and
3
a and 3b.
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Human Urine. Metabolite M7, eluting at t = 35.9 min with m/z 437.1506 for formula
R
C
24
H
22FN
2
O
5
, matched the synthetic standard 2b eluting at t
identical LCꢀMS tandem mass spectra (trace M7 and 2b, Figure 3) and coꢀelution of M7 and
b after coꢀinjection of equal concentrations (Figure S4a, Supporting Information) the
R
= 36.0 min. Based on the
2
structure of M7 was unambiguously verified as 2ꢀ(2ꢀ((trans)ꢀ3,4ꢀdihydroxyꢀ3,4ꢀ
dihydronaphthaleneꢀ1ꢀcarbonyl)ꢀ8ꢀfluoroꢀ1,2,3,4ꢀtetrahydroꢀ5Hꢀpyrido[4,3ꢀb]indolꢀ5ꢀ
yl)acetic acid. Likewise, M9 was demonstrated to coꢀelute with 2c during LCꢀMS/MS
(
R
Figure S4b, Supporting Information), and the structure of M9 (t = 34.3 min, m/z of
4
37.1506) was verified as 2ꢀ(2ꢀ((trans)ꢀ5,6ꢀdihydroxyꢀ5,6ꢀdihydronaphthaleneꢀ1ꢀcarbonyl)ꢀ
8ꢀfluoroꢀ3,4ꢀdihydroꢀ1Hꢀpyrido[4,3ꢀb]indolꢀ5(2H)ꢀyl)acetic acid by comparing the retention
time (t = 34.3 min for 2c) and fragmentation pattern of the metabolite with the synthetic
R
standard (trace M9 and 2c, Figure 3). Although accurate mass measurement of 2a and 2d
gave identical m/z of 437.1506 confirming the same elemental composition as C24
2 5
H22FN O ,
neither retention time nor fragmentation pattern matched with M7 and M9 (Figure 3).
NMR spectra were recorded under identical experimental conditions with similar sample
1
concentrations for 2aꢀd, M7 and M9. A clear mismatch was observed if the H NMR spectra
of M7 and M9 were compared with those of synthetic standard 2a and 2d. However, the
1
identity of 2b and M7, and 2c and M9 could be confirmed by assessing their respective H
NMR spectra (Figure 4).
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