Peripherally selective cannabinoid 1 receptor (CB1R) agonists for the treatment of neuropathic pain

Article abstract of DOI:10.1021/acs.jmedchem.6b00516

Alleviation of neuropathic pain by cannabinoids is limited by their central nervous system (CNS) side effects. Indole and indene compounds were engineered for high hCB1R affinity, peripheral selectivity, metabolic stability, and in vivo efficacy. An epithelial cell line assay identified candidates with <1% blood-brain barrier penetration for testing in a rat neuropathy induced by unilateral sciatic nerve entrapment (SNE). The SNE-induced mechanical allodynia was reversibly suppressed, partially or completely, after intraperitoneal or oral administration of several indenes. At doses that relieve neuropathy symptoms, the indenes completely lacked, while the brain-permeant CB1R agonist HU-210 (1) exhibited strong CNS side effects, in catalepsy, hypothermia, and motor incoordination assays. Pharmacokinetic findings of ～0.001 cerebrospinal fluid:plasma ratio further supported limited CNS penetration. Pretreatment with selective CB1R or CB2R blockers suggested mainly CB1R contribution to an indene's antiallodynic effects. Therefore, this class of CB1R agonists holds promise as a viable treatment for neuropathic pain.

Full text of DOI:10.1021/acs.jmedchem.6b00516

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Article
Peripherally selective cannabinoid 1 receptor (CB1R)
agonists for the treatment of neuropathic pain
Herbert H. Seltzman, Craig Shiner, Erin E. Hirt, Anne F. Gilliam, Brian F Thomas, Rangan
Maitra, Rod Snyder, Sherry L Black, Purvi R Patel, Yatendra Mulpuri, and Igor Spigelman
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00516 • Publication Date (Web): 02 Aug 2016
Downloaded from http://pubs.acs.org on August 4, 2016
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Peripherally selective cannabinoid 1 receptor (CB1R) agonists for the treatment of
neuropathic pain
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Herbert H. Seltzman , Craig Shiner , Erin E. Hirt , Anne F. Gilliam , Brian F. Thomas ,
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Rangan Maitra , Rod Snyder , Sherry L. Black , Purvi R. Patel , Yatendra Mulpuri , and Igor
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Spigelman
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Organic and Medicinal Chemistry, Research Triangle Institute, Research Triangle Park, NC,
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USA ; Division of Oral Biology & Medicine, School of Dentistry, University of California, Los
Angeles, CA, USA
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Abstract
Alleviation of neuropathic pain by cannabinoids is limited by their central nervous system
CNS) side effects. Indole and indene compounds were engineered for high hCB1R affinity,
(
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peripheral selectivity, metabolic stability, and in vivo efficacy. An epithelial cell line assay
identified candidates with <1% bloodꢀbrain barrier penetration for testing in a rat neuropathy
induced by unilateral sciatic nerve entrapment (SNE). The SNEꢀinduced mechanical allodynia
was reversibly suppressed, partially or completely, after intraperitoneal or oral administration of
several indenes. At doses that relieve neuropathy symptoms, the indenes completely lacked,
while the brainꢀpermeant CB1R agonist HUꢀ210 (1) exhibited strong CNS side effects, in
catalepsy, hypothermia, and motor incoordination assays. Pharmacokinetic findings of ~0.001
cerebrospinal fluid:plasma ratio further supported limited CNS penetration. Pretreatment with
selective CB1R or CB2R blockers suggested mainly CB1R contribution to an indene’s antiꢀ
allodynic effects. Therefore, this class of CB1R agonists holds promise as a viable treatment for
neuropathic pain.
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INTRODUCTION
Various neuropathies and chronic inflammatory conditions pose a major socioeconomic and
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clinical challenge in part because of poorly understood etiologies and mechanisms and in part
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because side effects of existing treatments greatly limit their effectiveness . This includes
synthetic and naturally occurring cannabinoids (CBs) which reduce the hyperalgesia and
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allodynia associated with persistent pain of neuropathic and inflammatory origin in humans and
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animals , yet they exhibit side effects mediated primarily by activation of central nervous
system (CNS) CB1 receptors (CB1Rs). These psychotropic CNS effects also account for the
abuse potential of plantꢀbased and synthetic CBs.
In addition to their CNS expression, CB1Rs, CB2Rs, and their endogenous ligands
(endocannabinoids, ECBs) have a diverse distribution in peripheral tissues, including primary
5
afferent neurons . Local administration of CBs into inflamed tissue attenuates hyperalgesia and
6
allodynia via peripheral CBRs, at doses that produce minimal CNSꢀmediated side effects . The
crucial role of peripheral CBRs in the antihyperalgesic actions of systemically administered CBs
was demonstrated using conditional deletion of CB1Rs located on nociceptive primary afferent
7
neurons . Also, many studies have demonstrated increases in expression of CB1Rs, CB2Rs and
ECBs, both in the peripheral tissues and the CNS, during inflammation and after development of
8
painful neuropathies, reviewed in . Increases in CBR expression result in increased potency or
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efficacy of the exogenously applied CBs , and may also account for the effectiveness of CBs in
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alleviating neuropathic pain symptoms after chronic repeated treatment , unlike opioids, which
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have only limited longꢀterm effectiveness . While selective activation of CB2Rs also inhibits
experimentally induced inflammatory pain and itch or the persistent pain of neuropathic origin
10b, 12
,
activation of both CB1R and CB2Rs appears to have synergistic effects on pain
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suppression . These studies provided a rationale for the development of peripherallyꢀacting
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endocannabinoidꢀbased therapeutic interventions .
With the aim of utilizing the demonstrated benefits of CBs to sustainably ameliorate
neuropathic pain, we sought to develop peripherally restricted CB1R agonists, which would not
penetrate the bloodꢀbrain barrier (BBB), so as to avoid the unwanted psychomimetic effects such
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as those caused by ꢀ ꢀtetrahydrocannabinol (ꢀ ꢀTHC) that are also associated with activation of
central CB1Rs. Peripheral restriction can be addressed by 1) the inclusion of charge that
typically prevents BBB penetration in the absence of active transport, 2) the presence of actively
effluxed moieties such as carboxylates, and 3) adjustment of partition coefficient and the
topological polar surface area. Other factors potentially impact peripheral restriction such as
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activation of CB2Rs on the BBB endothelial cells . We chose to examine indoles and indenes
that have been demonstrated as ligands for CB1R agonist activity as starting points for
modifications towards these ends.
RESULTS
Synthesis of Indoles
The synthesis of the target indoles where the 4ꢀsubstituent of the naphthyl ring was alkyl or
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hydrogen followed the established general approaches shown in Scheme 1 . The synthesis of
indoles with a 4ꢀacyl substituted naphthylene ring required further modifications as described
below. The acylation of indole 1-1 with the naphthoyl chloride 1-2a (X = CO Me) mediated by
1
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methyl magnesium bromide and zinc chloride afforded the corresponding 1-3b in greater than
0% yield on scales of 20 gm. Subsequent alkylation of 1-3b with nꢀpentyl bromide proceeded
in yields of about 50% of 1-4a (X = CO Me) on a 1 gm scale. Upon scale up to 10 and 15 gm,
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the alkylation step did not proceed to completion. Further, work up resulted in hydroxysis or
transesterification to the Nꢀalkylated or unalkylated acid or ethyl ester (when ethyl acetate was
employed in the work up) that can likely be addressed with modified work up conditions.
Conversion of 1-4a to other 4ꢀsubstituted naphthoyl indoles was achieved by saponification
to the acid 1-7a (X = CO H, R=Pn), which was converted to the acid chloride (X = COCl,
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R=Pn) with oxalyl chloride, and subsequently to the secondary amide 1-7b (X = CONHMe,
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R=Pn)(87%) with methylamine and to the primary amide 1-7c (X = CONH )(98%) with
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ammonium hydroxide. Conversion of the acid chloride to the methyl or ethyl ketones 1-7e,d (X2
COMe, COEt) by treatment with either methyl Grignard (no product), dimethyl zinc (trace
=
product), diethyl zinc (trace product), or triethyl aluminum (no product), was disappointing.
An alternative sequence to 1-7e,d of first preparing the 4ꢀsubstituted 1ꢀnaphthoyl chloride 1-2c,d
(
X = COMe, COEt) followed by coupling to the indole (Nꢀalkylated or not) was examined in an
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effort to improve yields. Thus, commercially available 4ꢀ(methoxycarbonyl)naphthaleneꢀ1ꢀ
carboxylic acid (1-8a) was converted to 1-2a (X = CO Me), then treated with dimethyl or
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diethyl zinc to afford 1-8b,c (X = COEt, COMe) in 63% and 37% respectively, followed by
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hydrolysis of the esters to the acids 1-8d,e in greater than 90% yield and treatment with oxalyl
chloride to afford 1-2c,d in near quantitative yield. Acylating indole 1-1 with 1-2d (X = COEt)
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mediated with MeMgBr afforded a 55% yield of 1-3d (X = COEt). Alkylation of 1-3d with nꢀ
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pentyl bromide however, afforded no target compound (the product obtained from the low yield
diethyl zinc reaction above was used for testing). Attempting synthesis of the methyl ketone
analog 1-7e (X = COMe) by reversing the sequence to acylating the preꢀalkylated 1-6a (R = Pn)
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with 1-2c (X = COMe) mediated by ethylaluminum dichloride gave a complex mixture with
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minimal 1-7e (X = COMe) that could not be isolated in pure form.
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Nꢀωꢀcarboxyalkylꢀindoles (butanoates and pentanoates, 1-4f and g respectively) were
prepared by acylation of indole 1-1a with 1-2e mediated by methylmagnesium bromide affording
the naphthoylindole 1-3a, which was then alkylated with ethyl ωꢀbromoalkanoate and sodium
hydride to yield the corresponding ethyl butanoate and pentanoate and followed by hydrolysis to
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provide the corresponding acids 1-4f,g (R = ꢀ(CH ) ꢀCO H, n = 3,4) .
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Fluoroindole analogs were prepared from commercially available fluoroindole substituted in
the 4, 5, 6, or 7ꢀposition (1-1b-e, Z = F) via acylation with 1-2e (X = H) in the presence of
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MeMgBr and ZnCl to afford 1-3e-h in 70 ꢀ 85% yields followed by alkylation with nꢀpentyl
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bromide/ NaH to give 1-4h-k (Z = F ) in >80% yields. The 4ꢀfluoro indole 1-3e was also
alkylated with 5ꢀfluoroꢀ1ꢀbromopentane/ NaH to give the corresponding 1-4l in 27% yield.
Similar alkylation with iodoethylmorpholine/ NaH afforded 1-4m (X = H, Z = 4ꢀF, R =
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morpholinoethyl) in 68% yield after chloroethylmorpholine did not alkylate 1-3e (Z = 4ꢀF). The
analog 1-4n (X = nꢀpropyl, Z = 4ꢀF, R = 5ꢀFꢀpentyl) was prepared from 4ꢀfluoroindole 1-1b by
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acylation with 4ꢀnꢀpropylꢀ1ꢀnaphthoyl chloride (1-2b, X = nꢀpropyl) (MeMgBr, ZnCl ) (58%),
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followed by alkylation with 5ꢀfluoroꢀ1ꢀbromopentane/ NaH to give the corresponding 1-4n in
24% yield.
Synthesis of Indenes
Synthesis of the target 4ꢀsubstituted naphthylideneꢀ or substituted benzylideneꢀ3ꢀ
morpholinoethylꢀ2ꢀindenes (2-5) with the Eꢀolefin geometry was achieved as shown in Scheme 2
by alkylation of lithiated indene 2-1 with 1ꢀchloroꢀ2ꢀ(4ꢀmorpholino)ethane (2-2) to afford a
mixture of the 1ꢀ and 3ꢀalkylated 1Hꢀindenes. Treatment of the mixture with sodium hydroxide
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induced isomerization to the more stable 3ꢀ(4ꢀmorpholinoethane)ꢀ1Hꢀindene
(2-3) in 45%
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yield (Z=H). Treatment of 2-3 with the appropriate 1ꢀnaphthaldehyde, benzaldehyde or aryl
aldehyde in the presence of sodium methoxide gave the target Eꢀolefin 2-5a-s after either 18 h of
heating at reflux (in 66% yield 2-5a) or microwave heating. Microwave heating provided the
same product more expeditiously in 15 min at 105° C but in lesser yield (53% 2-5a); a process
that we employed for the majority of the analogs, which were obtained in >90% purity (HPLC).
Phenyl acetaldehyde, 4ꢀhydroxyꢀ1ꢀnaphthaldehyde, 2ꢀnitroꢀ, and 2ꢀcyanoꢀbenzaldehyde were not
ammenable to this method of synthesis. But, particular interest in preparing and testing the
phenyl acetaldehyde derived product, (1E)ꢀ3ꢀhexylꢀ1ꢀ(2ꢀphenylethylidene)ꢀ1Hꢀindene (2-7) with
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only a distal aromatic ring (see above), was achieved HornerꢀWittig chemistry . The Eꢀ
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geometry was established for the 2-5a-s analogs by comparison of the H NMR on the architype
Eꢀ(1ꢀnaphthylidene)ꢀ3ꢀmorpholinoethylꢀ2ꢀindenes prepared via the condensation chemistry from
2-3 with that prepared by HornerꢀWittig chemistry that was characterized by nOe NMR
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spectroscopy .
Neuropathy testing
We examined the effectiveness of select indenes in alleviating the painful symptoms of
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neuropathy induced by unilateral sciatic nerve entrapment (SNE) . SNE was demonstrated to
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produce consistent pain behaviors
, a transient loss of varicosities in nociceptive fibers , and
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increased excitability of sciatic nerve . The hyperexcitability and ectopic burst discharge of
primary sensory neurons are widely considered as major contributors to pain symptomatology of
peripheral neuropathy models. The SNEꢀinduced mechanical allodynia models the most common
complaint of human neuropathy patients of dynamic mechanical allodynia. Fig. 1 illustrates that
systemic injection of 0.3 mg/kg of PrNMI (2-5u) or MoNMI (2-5j) results in large, reversible
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decreases in mechanical allodynia. By contrast, ENMI (2-5a) has a much smaller effect,
consistent with its lower CBR affinity and faster metabolism (Table 2).
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Pharmacokinetics
Analysis of plasma samples after 2-5u injections yielded its initial pharmacokinetic profile
(Fig. 1E) which was in good agreement with its antiꢀallodynic effects (Fig. 1B). Measurements
of drug brain penetration include drug partitioned into brain lipids + unbound drug in
equilibrium with extracellular fluid. The cerebrospinal fluid (CSF)/plasma ratios are considered
to be more precise estimates of a drug’s brain penetration because of the continuity of CSF with
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extracellular space . However, both measures are needed to confirm minimal CNS access and
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to compare with other reported peripherallyꢀrestricted CB1R ligands
. Analysis of plasma,
brain and CSF samples confirmed the minimal penetration of 2-5u into the CNS after systemic
administration (Fig. 1E).
In subsequent experiments, we demonstrated that 2-5u was also effective in suppressing
neuropathy symptoms after oral administration, which is more representative of future
therapeutic uses. The high oral dose of 2-5u (3 mg/kg) likely accounts for its continued antiꢀ
allodynic effectiveness at the 24 hr time point (Fig. 1F).
Tetrad testing
The CNSꢀmediated psychotropic actions of CB1R ligands represent their most troubling side
effects. The catalepsy, motor performance, hypothermia, and analgesia tests are classically
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predictive of CNS CB1R activation . Effects in all 4 tests have been thought to be mediated by
the activation of central CB1Rs, but it is now well established that peripheral CBRs make a
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major contribution to the analgesic effects of CBs
. We used the “tetrad” to determine
whether the novel ligands have antinociceptive effects and sideꢀeffect profile consistent with
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central CB1R activation. We also studied the potent CB1R agonist, 1 (HUꢀ210, Scheme 3)
Tocris Bioscience, Ellisville, MO), to allow comparisons of this positive control with the
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(
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putatively brainꢀimpermeant analogs. The systemic doses of 1 and of novel indenes (e.g., Fig.
1
) were consistent with their demonstrated effectiveness in alleviating painful neuropathy
symptoms. The “tetrad” tests were modified for rats, with rotarod substituting for the
spontaneous activity test. Unlike 1, 2-5u and other indenes lack effects in the catalepsy, rotarod
or hypothermia assays, although a small effect in the tailꢀflick assay is observed, as expected for
analgesic peripherallyꢀacting CB1R ligands (Fig. 2).
CB1Rs mediate anti-allodynic effects of 2-5u
Despite similar affinities for the CB1R and CB2R subtypes, indene PRCBs are full agonists
at hCB1R, but only partial agonists at hCB2R (Table 2). To determine which receptor subtype is
responsible for the antiꢀallodynic effects of the novel CBR ligands in the SNE neuropathy, we
measured the ability of a representative ligand, 2-5u, to suppress mechanical allodynia in SNE
rats in the presence of either CB1R or CB2R selective antagonists. 2-5u was administered alone
or 30 min after pretreatment with CBR blockers in SNE rats at 3ꢀday intervals (Fig. 3A).
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Pretreatment with the CB1R inverse agonist, SR141716 (2, Scheme 3) , completely blocked the
antiꢀallodynic effect of 2-5u (Fig. 3B, E), whereas pretreatment with a CB2R selective inverse
agonist, SR144528 (3, Scheme 3) had little effect on suppression of allodynia by 2-5u (Fig. 3D).
In the same rats, pretreatment with a peripherallyꢀrestricted analogue of 2, 18A, recently
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developed by our group , also prevented the antiꢀallodynic effect of 2-5u (Fig. 3C, E). These
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studies demonstrated the CB1R dependence of 2-5u’s antiꢀallodynic effects in the SNE
neuropathy.
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DISCUSSION
Structure Activity Relationships (SAR)
The introduction of charge to CB1R ligands to impart peripheral selectivity was explored via
quaternization of morpholinoethylꢀindoles and ꢀindenes of demonstrated CB1R agonists. Thus,
15a
morpholinoethyl indoles 1ꢀ[2ꢀ(4ꢀmorpholino)ethyl]ꢀ3ꢀ(1ꢀnaphthoyl)indole (1-9a, JWHꢀ200),
15a
1ꢀ[2ꢀ(4ꢀmorpholino)ethyl]ꢀ3ꢀ(4ꢀmethoxyꢀ1ꢀnaphthoyl)indole (1-9c, JWHꢀ198),
and 1ꢀ[2ꢀ(4ꢀ
1
5a
morpholino)ethyl]ꢀ3ꢀ(4ꢀmethylꢀ1ꢀnaphthoyl)indole (1-9e, JWHꢀ193)
respectively and
2
8
pravadoline
1-9g were quaternized to 1-9b,d,f,h. Also, conformationally constrained
indenes, (2-5a, Eꢀ4ꢀ[2ꢀ[1ꢀ(1ꢀnaphthalenylmethylene)ꢀ1Hꢀindenꢀ3ꢀ
and its 2ꢀmethyl analog 2-5v, were quaternized to afford the
morpholinoethyl
1
7
yl]ethyl]morpholine)
corresponding charged quaternary ammonium analogs (2-6a and 2-6b). Both of these changes
resulted in a reduction of hCB1R binding affinity of between one and two orders of magnitude
(see Table 1). Similarly, alkyl carboxy chains linked to the indole nitrogen as ωꢀbutanoic (1-4f)
and –pentanoic (1-4g) acids, as putative effluxed moieties, also exhibited no hCB1R affinity (Ki
=
>10 ꢁM) in contrast to the unmodified pentyl chain of 3ꢀ(naphthaleneꢀ1ꢀcarbonyl)ꢀ1ꢀpentylꢀ
15a
1Hꢀindole, JWHꢀ018 (1-4p) (Ki = 4.3 nM) . The 4ꢀcarboxy substituted naphthoyl analog 1-7a
also showed no receptor affinity (Ki = >10 ꢁM). These results steered our efforts away from
charged analogs.
Screening results of peripheral selectivity and hCB1R affinity on our early analogs redirected
ligand design. The peripheral selectivity of the high affinity compounds was tested in the
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MadinꢀDarby canine kidney (MDCK) epithelial cell line assay as a model of the BBB and
showed an association with the nꢀpentyl indoles and morpholinoethyl indenes respectively. Thus,
the Nꢀpentyl 4ꢀcarboxy methyl ester indole 1-4a was compared to the Nꢀmorpholinoethyl 4ꢀ
carboxy methyl ester indole analog 1-4o. The basolateral : apical ratio (B:A) of the nꢀpentyl
analog 1-4a was 0.00 while that of the Nꢀmorpholinoethyl analog 1-4o was 1.02 indicating nonꢀ
permeability of 1-4a and equal central:peripheral distribution of 1-4o in the MDCK model. This
would suggest similar permeability for these indole analogs across the BBB.
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1
7
The morpholinoethyl indene 2-5a and the nꢀpentyl indene 1ꢀ{[(1E)ꢀ3ꢀpentylꢀ1Hꢀindenꢀ1ꢀ
15a
ylidene]methyl}naphthalene, JWHꢀ176
, 2-7a were also chosen as candidates for
modification as they had high affinity for the CB1R (Ki = 4.69 and 17.2 nM respectively).
2
9
Surprisingly, both tested in the MDCK assay as peripherally restricted (B:A = 0.00 and 0.04
respectively) with the morpholinoethyl moiety 2-5a showing slightly greater preferral restriction
over the pentyl moiety (2-7a). This is the opposite order of the indole morpholinoethyl and the
indole pentyl side chain pair tested (above) and of significant difference of B:A of the
morpholinoethyl substituted indole 1-4o. Thus, subsequent indene modifications were evolved
from the morpholinoethyl indene analog 2-5a and subsequent indole modifications were evolved
from nꢀpentyl indoles to examine SAR trends in affinity, MDCK permeability, and metabolic
stability in order to select candidates for in vivo testing.
The effect upon hCB1R affinity of substituting the naphthoyl 4ꢀposition on the indoles,
15b
which has been associated with high receptor affinity in reported analogs , was examined in
the Nꢀpentyl family. Thus, in contrast to the 4ꢀcarboxy substituted naphthoyl analog 1-7a (Ki =
>
10 ꢁM), the corresponding ethyl ester (1-4c) exhibited a Ki = 115 nM and the shorter methyl
ester (1-4a) exhibited a Ki = 20 nM. The three atomꢀlong chain 4ꢀpropanoylꢀnaphthyl analog (1-
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d) exhibited a Ki = 5.7 nM. The 4ꢀNꢀmethylamido analog 1-7b (Ki = 127 nM) was less active
than its isosteric oxygen or carbon analogs (1-4a and 1-7d). The 4ꢀamido analog (1-7c), however,
had higher affinity for hCB1R (Ki = 96 nM) than the corresponding acid 1-7a.
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A similar effect of the 4ꢀposition substituent was observed for the indenes in the
morpholinoethyl family. The hCB1R affinity progressed from 4ꢀH (4.69 nM)(2-5a) to 4ꢀOMe
(2.43 nM)(2-5j) to 4ꢀnꢀPr (1.18 nM)(2-5u) to 4ꢀEt (0.86 nM)(2-5t), all of which were
subsequently tested in vivo.
Modelling and SAR studies of indole CB1R agonists, and similarly for the indene mimics of
the indoles, support that the favored conformation of the naphthoyl ring for binding/activation in
2
8
the analogs of 1-4p and 5 (WIN 55,212ꢀ2) in the CB1R site is one in which the naphthoyl ring
is nearly parallel to the XZ plane when the indole ring (pentyl chain pointing down) is in the XY
15a, 17, 30
plane
. Further, for the indoles, it is the distal ring of the naphthoyl bicyclic system that
3
1
has the role of binding stabilization by aromatic stacking interactions with the receptor pocket .
Hence, we prepared and tested the 2ꢀphenylethylidene indene analog (2-7c) wherein the
naphthylidene ring was replaced with 2ꢀphenylethylidene that retains only the distal aromatic
3
2
ring in analogy with similar active indole analogs . This change lost 10ꢀfold affinity versus the
corresponding naphthylidene analog 2-7b (both with an nꢀhexyl pendant group on indenꢀ3ꢀyl)
and 256 fold versus 2-7a, which carries a pentyl pendant group on indenꢀ3ꢀyl).
Reported studies on indenes that are conformationally restrained by an arylidene double bond
indicate that it is the orientation defined by the Eꢀgeometry that is active in the indenes and, by
1
7
extension, in the more conformationally mobile naphthoyl indoles . Designing forward from
these factors, other arrangements of arylidene rings on the indenes were synthesized and tested to
evolve an SAR that mapped the regions proximal to the 1ꢀnaphthyl ring. Thus, fusing an
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additional phenyl ring to naphthylene gave the 5ꢀphenanthrylidene ring (2-5s)(Ki = 22.9 nM)
versus the naphylidene 2-5a (4.69 nM) indicating a tolerence for the extra ligand volume. In
contrast, an alternate fusing of a phenyl ring to a naphthyl ring in indoles to give the symmetrical
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9
ꢀanthranoyl ring is reported to result in a significant loss of binding affinity . This suggests
that the volume available to the naphthoyl is not equivalent on both sides of the 9ꢀanthranoyl
bond to the indole and by extension to the indene.
Displacing the distal phenyl ring of the naphthylidene group of 2-5a from the fused to a
pendant arrangement as in a 2ꢀ or 3ꢀphenylbenzylidene group (2-5k and 2-5e respectively)
results in a reasonably tolerated receptor binding for hCB1R for 2-5k (Ki = 82.9 nM) but not for
2
-5e (Ki = 2603 nM). Reorienting the 1ꢀnaphylidene ring to the 2ꢀnaphylidene ring (2-5r) (Ki =
134 nM) reduces binding affinity by a moderate extent.
Orthoꢀsubstituted benzylidene morpholinoethyl indenes were also synthesized and screened
15b
for hCB1R affinity based on the reported activity of 2ꢀsubstituted benzoyl indoles . Versus the
unsubstituted 2ꢀprotio analog 2-5b (Ki = 1297 nM), the 2ꢀhalogeno analogs (2-5d,f,g,c) (2ꢀF, Cl,
Br, I respectively) showed increasing affinity (Ki = 1000, 862, 647, 607 nM respectively) with
larger halogen atoms, but never rising to the effect of the much larger 2ꢀphenylbenzylidene
analog (2-5k, Ki = 82.9 nM). The 2ꢀmethoxy substituent (2-5h, Ki = 149 nM) gave a large
increase in binding affinity (vs 2-5b) that was likely due to electronic issues given the similar
enhancement in affinity seen for the 4ꢀmethoxynaphthylidene 2-5j vs 2-5a.
The affinity and possibly the functional bias of the receptor with its binding ligand can be
influenced by the interaction of the arylidene or aroyl moieties of the indenes or the indoles
respectively. Modelling suggests that this interaction is one of aromatic stacking, which is
effected by the electronic character of the interacting aromatic rings. Varying the arylidene ring
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systems to introduce electronꢀrich and electronꢀpoor proximal or distal rings, we examined the
effect on binding affinity. Thus, comparing the monoꢀcyclic electronꢀrich furanylidene 2-5n (Ki
=
>10 ꢁM) to the electronꢀpoor thiophenylidene 2-5m (Ki = 8.5 ꢁM), binding is favored by the
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electronꢀpoor proximal ring. Similarly, comparing the biꢀcyclic electronꢀpoor 4ꢀquinolidene 2-
5p (Ki = 23.3 nM) to the electronꢀneutral naphthylidene 2-5a (Ki = 4.69 nM), binding is favored
by the electronꢀneutral (or relatively electronꢀrich) proximal ring. Even though the binding by
the electronꢀpoor proximal ring was quite good, other factors such as interaction with the basic
nitrogen atoms could contribute to the overall binding preference change towards electron
density. Comparing the biꢀcyclic electronꢀrich 7ꢀindolydene 2-5l (Ki = 107 nM) to the electronꢀ
poor 5ꢀisoquinolidene 2-5o (Ki = 454 nM), binding is favored by the electronꢀrich distal ring.
Given the ready metabolism of indoles, which not only leads to the consumption of the drug
33
candidate but also to multiple metabolites that exhibit mixed pharmacologies , the issue of
stabilizing lead compounds was also addressed by introducing fluorine substitution on the indole
ring to develop improved resistance to metabolism. The 4ꢀ, 5ꢀ, 6ꢀ, and 7ꢀF indole analogs of 1-
4p (1-4h-k) all had Ki’s in the single digit nM range. Tested for stability in rat plasma, where
they exhibited stability of 69 – 99% after 1 h (see Table 3) and S9 plasma fraction, where
stability increased from 3% to 50% in 1 h with F substitution going from positions 4ꢀ7. In our
analogs, the indenes were more metabolically stable than the indoles.
In vivo efficacy of indenes
We show that systemic (0.3 mg/kg, i.p.) administration of 2-5u (2-5u) or 2-5j (2-5j)
produced complete suppression of mechanical allodynia symptoms (Fig. 1B, C), while the same
and 3ꢀfold higher doses of these compounds had no effect in the assays of CNS CB1R activation,
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compared to the brainꢀpermeant positive control, 1 (Fig. 2). Similarly, an oral dose of 3 mg/kg 2-
5u produced complete suppression of allodynia symptoms at peak effect, while a 10 mg/kg oral
dose had no significant effect on central CB1Rs (Fig. 2). These data support the high antiꢀ
allodynic efficacy of indeneꢀbased peripherally restricted cannabinoids (PRCBs) at doses that do
not produce any CNS side effects.
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The pharmacokinetic data, which showed a CSF:plasma ratio of 0.001 at ~75 min after 2-5u
(0.3 mg/kg, i.p.) administration (Fig. 1E) supports its relative lack of brain permeability.
Generally, increasing aqueous solubility decreases the likelihood of a drug gaining access to
brain tissue. However, numerous lipidꢀsoluble molecules, among them many useful therapeutic
drugs have lower brain permeability than would be predicted from a determination of their lipid
3
4
solubility . Given the very low aqueous solubility of PRCBs, their relative lack of BBB
permeability suggests that they may be substrates for active drug efflux transporters such as Pꢀ
glycoprotein or multidrug resistance proteins. Future studies using selective inhibitors of such
3
5
36
transporters (e.g., ) or transporter knockout rodents (e.g., ) should help identify precisely the
efflux transporters for which PRCBs serve as substrates.
We also demonstrated that PRCBs such as 2-5U have small effects on acute nociception
compared to the brainꢀpermeable 1 in the tailꢀflick assay in naïve rats (Fig. 2d, h, i), yet, at the
same doses, exhibit potent antiꢀallodynic effects in SNE neuropathy. Previous studies have
demonstrated increases in expression of both CB1R and CB2R in sensory ganglia after
6a, 37
inflammation and peripheral nerve injuries
. Increases in CBR expression result in increased
9
potency or efficacy of the exogenously applied CBs ; such increases may also account for the
1
0
effectiveness of CBs in alleviating neuropathic pain symptoms after chronic repeated treatment .
Thus, increases in CB1R and CB2R expression may potentially account for the increased
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potency/efficacy of PRCBs after SNE injury. An alternative explanation for the increased
efficacy of 2-5u in SNE involves alterations in bloodꢀnerve barrier (BNB) function. Many of the
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8
BBB efflux transporters are also involved in maintaining BNB function . In normal conditions,
these transporters may limit PRCB access to CBRs on nociceptors, which would account for the
weak antiꢀnociceptive efficacy of PRCBs. However, there is growing evidence that chronic pain
syndromes exhibit tissue abnormalities caused by microvasculature dysfunction in the blood
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vessels of skin, muscle, or nerve . Such dysfunction, e.g., loosening of the tight junctions
3
8
between the endoneurial endothelial cells , may increase PRCB access to CBRs on sensory
neurons, thereby increasing their effectiveness in suppressing painful neuropathy symptoms.
Our binding studies revealed similar affinities of 2-5u and related indenes for the CB1R and
2
+
CB2R subtypes. However, subsequent Ca flux assays revealed that indene PRCBs are full
agonists at hCB1R, but only partial agonists at hCB2R (Table 2). In vivo, pretreatment with the
CB2Rꢀselective inverse agonist, 3 (Scheme 3), had only a small effect on suppression of
allodynia by 2-5u, while pretreatment with the brainꢀpermeable CB1Rꢀselective inverse agonist,
27
rimonabant, or it’s peripherally restricted analogue, 18A , prevented the antiꢀallodynic effect of
2-5u (Fig. 4). These studies suggested that CB1Rs are mainly responsible for the antiꢀallodynic
effects of PRCBs. Previous studies with the brainꢀpermeant synthetic CB, 4 (Scheme 3), which
has full agonist activity at both CB1R and CB2R, demonstrated that its antiꢀallodynic effects in
6
c, 10a, 40
some neuropathy models were mediated primarily by CB1R activation
, whereas in other
12d, 41
models both CB1R and CB2R were involved
. Further, our conclusions must be tempered
by the fact that in vivo effects of inverse agonists such as rimonabant may be affected by
changes in levels of endocannabinoids and their activation of CBRs in SNE neuropathy. Future
studies using transgenic mice with deletions of CB1Rs and CB2Rs should be able to determine
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more precisely the relative contribution of CBR subtypes to the antiꢀallodynic effects of PRCBs
in different neuropathy models.
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CONCLUSIONS
The hydrophobic PRCB compounds, which we developed, are the first in their class of high
peripheral selectivity CB1R agonists to exhibit, after systemic or oral administration, potent and
repeated suppression of neuropathy symptoms with a lack of side effects mediated by activation
of central CB1 receptors. The potency, peripheral selectivity, in vivo efficacy, and absence of
CNS side effects of the PRCBs hold promise as a viable treatment for neuropathic pain states.
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METHODS
General
1
13
H and C NMR spectra were run on a Bruker Avance 300 MHz or a Varian Unity Inova
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500 MHz NMR spectrometer. Mass spectra (MS) were run on a PerkinꢀElmer Sciex API 150
EX mass spectrometer. High resolution mass spectra (HRMS) were run on a Waters Synapt G2
QꢀTOF mass spectrometer in highꢀresolution mode. Column chromatography was carried out
using a Teledyne Isco Combiflash Rf system with RediSep Rf silica cartridges. Preparative thin
layer chromatography was carried out using Analtech TLC Uniplates (silica gel, 1000 mm,
20×20 cm). High pressure liquid chromatography was performed using a system consisting of a
Waters 1525 pump unit, driven by Empower software, and a Waters 2487 detector. Microwave
chemistry was carried out using a CEM Discover SP microwave with 10 mL irradiation tubes.
4-Propylnaphthalene-1-carbonyl chloride (1-2b)
4
ꢀpropylnaphthaleneꢀ1ꢀcarboxylic acid (500 mg, 2.33 mmol) was dissolved in
dichloromethane (15 mL) and cooled to 0°C in an ice bath. Oxalyl chloride (1.49 g) was then
added dropwise over 5 min. Once the addition was complete, the mixture was allowed to stir at
room temperature for 45 min and then under reflux for 45 min. The solution was then cooled to
room temperature. Removal of the solvent afforded 4ꢀpropylnaphthaleneꢀ1ꢀcarbonyl chloride (1-
2b), which was used in the preparation of 1-3f without further purification (assuming 542 mg,
100%).
Methyl 4-[(1H-Indol-3-yl)carbonyl]naphthalene-1-carboxylate (1-3b)
Indole (1-1) (8.12 g, 98%, 0.068 mol) was dissolved in dichloromethane (150 mL), purged
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with nitrogen and cooled to 0°C in an ice bath. Methylmagnesium bromide (0.023 L, 3M in
ether, 0.069 mol) was added to the solution over 10 min and mixture stirred for an additional 10
min at 0°C. Powdered ZnCl (31.3 g, solid addition funnel) and anhydrous ether (80 mL,
2
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addition funnel) were then added over 10 min and the mixture stirred for an additional 10 min at
0°C. The solution was allowed to warm to room temperature and stir for 30 min. Methyl 4ꢀ
(
carbonochloridoyl)naphthaleneꢀ1ꢀcarboxylate (1-2a) (17.24 g, 0.069 mol) in CH CL (80 mL)
2 2
was then added over several minutes and the solution stirred at room temperature overnight.
Subsequently, saturated aqueous ammonium chloride solution (80 mL) was added and the
mixture stirred for 15 min. The solid was removed by filtration and washed with water and ether.
This solid was vacuumed dried to give the title product (11.97 g). The organic and aqueous
layers of the filtrate were then separated. The organic layer was then washed with water, dried
over sodium sulfate and the solvent removed under reduced pressure. The residue was then
triturated with a small amount of ether. The solid was removed by filtration and vacuumed dried
1
to give additional product (8.36 g, 20.33 g total, 91%). H NMR (300 MHz, CDCl ) δ 4.05 (s,
3
3H), 7.31ꢀ7.52 (m, 5H), 7.58ꢀ7.67 (m, 2H), 8.12 (d, J=8.4 Hz, 1H), 8.18 (d, J=7.4 Hz, 1H), 8.45ꢀ
8.52 (m, 1H), 8.78 (br s, 1H), 8.91 (d, J=8.6 Hz, 1H).
X-Fluoro-3-[(naphthalen-1-yl)carbonyl]-1H-indole (X = 4-7)(1-3e-h)
These analogs were prepared by a similar method described for 1-3b with variations of
additional further extractions of the aqueous layer with ether or dichloromethane, drying and
trituration in dichloromethane or ether.
4-Fluoro-3-[(naphthalen-1-yl)carbonyl]-1H-indole (1-3e )
4
ꢀFluoroindole (1-1b) (2.03 g, 0.0147 mol) was dissolved in dichloromethane (60 mL) and
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cooled to 0°C in an ice bath. Methylmagnesium bromide (0.005 L, 3M in ether, 0.015 mol) was
added to the solution, under nitrogen, over 5 min. The mixture was then stirred for 10 min at 0°C,
ZnCl solution (0.0049 L, 1M in ether, 0.0496 mol) added dropwise over 10 min and the mixture
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stirred for an additional 10 min at 0°C. The solution was allowed to warm to room temperature
and stir for 30 min. 1ꢀNaphthoyl chloride (1-2e) (0.002 L, 97%, 0.015 mol) was then added over
several minutes and the solution stirred at room temperature overnight. Subsequently, saturated
aqueous ammonium chloride solution (120 mL) was added and the mixture stirred for 30 min.
The precipitate was removed by filtration, washed well with water and dried under vacuum to
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give the title compound as an offꢀwhite solid (3.40 g, 80%). H NMR (300 MHz, DMSO-d6) δ
6.95ꢀ7.05 (m, 1H), 7.23ꢀ7.33 (m, 1H), 7.38 (d, J=8.0 Hz, 1C), 7.48ꢀ7.62 (m, 3H), 7.65ꢀ7.73 (m,
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H), 8.00ꢀ8.13 (m, 3H), 8.08ꢀ8.17 (m, 2H), 12.30 (s, 1H). C NMR (75.5 MHz, DMSO-d6) δ
107.51 (d, J=20.8 Hz, 1C), 108.82 (d, J=3.8 Hz, 1C), 113.37 (d, J=21.2 Hz, 1C), 117.20 (d, J=5.9
Hz, 1C), 124.15 (d, J=7.5 Hz, 1C), 124.80, 125.28, 126.20 (2C), 126.75, 128.28, 129.91, 130.25,
33.25, 137.22, 138.81 (d, J=2.3 Hz, 1C), 140.03 (d, J=11.4 Hz, 1C), 155.88 (d, J=251.0 Hz, 1C),
189.48.
1
5-Fluoro-3-[(naphthalen-1-yl)carbonyl]-1H-indole (1-3f)
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Offꢀwhite solid (3.37 g, 85%). H NMR (300 MHz, DMSO-d6) δ 7.11ꢀ7.22 (m, 1H), 7.48ꢀ
1
3
7
.67 (m, 4H), 7.68ꢀ7.75 (m, 1H), 7.78 (s, 1H), 7.94ꢀ8.14 (m, 4H), 12.20 (s, 1H). C NMR (75.5
MHz, DMSO-d6) δ 106.28 (d, J=24.6 Hz, 1C), 111.39 (d, J=26.0 Hz, 1C), 113.71 (d, J=9.8 Hz,
1C), 117.17 (d, J=4.4 Hz, 1C), 124.88, 125.22, 125.73, 126.25, 126.44 (d, J=11.0 Hz, 1C),
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26.74, 128.27, 129.78, 129.99,133.29, 133.56, 138.01, 138.23, 158.83 (d, J=234.9 Hz, 1C),
91.17.
-Fluoro-3-[(naphthalen-1-yl)carbonyl]-1H-indole (1-3g)
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Light pink solid (3.42 g total, 80%). H NMR (300 MHz, DMSO-d6) δ 7.11ꢀ7.22 (m, 1H),
7.30ꢀ7.38 (m, 1H), 7.48ꢀ7.66 (m, 3H), 7.67ꢀ7.76 (m, 2H), 7.97ꢀ8.06 (m, 2H), 8.10 (d, J=8.1 Hz,
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1
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H), 8.25ꢀ8.36 (m, 1H), 12.12 (s, 1H). C NMR (75.5 MHz, DMSO-d6) δ 98.67 (d, J=25.8 Hz,
C), 110.44 (d, J=23.9 Hz, 1C), 117.10, 122.52 (d, J=1.7 Hz, 1C), 122.66, 124.86, 125.23,
25.75, 126.24, 126.74, 128.27, 129.76, 130.01, 133.28, 137.08 (d, J=12.5 Hz, 1C), 137.35 (d,
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J=1.9 Hz, 1C), 138.25, 159.46 (d, J=237.4 Hz, 1C), 191.23.
7-Fluoro-3-[(naphthalen-1-yl)carbonyl]-1H-indole (1-3h)
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White solid (1.51 g total, 72%). H NMR (300 MHz, DMSO-d6) δ 7.11ꢀ7.21 (m, 1H), 7.22ꢀ
7
.32 (m, 1H), 7.49ꢀ7.68 (m, 3H), 7.70ꢀ7.78 (m, 2H), 8.00ꢀ8.07 (m, 2H), 8.08ꢀ8.17 (m, 2H), 12.68
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(
s, 1H). C NMR (75.5 MHz, DMSO-d6) δ 108.30 (d, J=15.8 Hz, 1C), 117.56 (d, J=3.5 Hz, 1C),
17.91 (d, J=1.3 Hz, 1C), 122.80 (d, J=6.0 Hz, 1C), 124.76 (d, J=13.3 Hz, 1C), 124.89, 125.19,
125.92, 126.26, 126.78, 128.30, 129.47 (d, J=4.6 Hz, 1C), 129.92, 129.99, 133.31, 137.06,
1
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38.18, 149.12 (d, J=245.1 Hz, 1C), 191.34.
4
-Fluoro-3-[(4-propylnaphthalen-1-yl)carbonyl]-1H-indole (1-3i)
4
ꢀFluoroindole (1-1b) (1.47 mmol), methylmagnesium bromide (2.4 mmol), ZnCl solution
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(
1M in ether, 8.0 mmol), 4ꢀpropylnaphthaleneꢀ1ꢀcarbonyl chloride (1-2b) (2.33 mmol) were
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similarly processed as for 1-3b. White solid (440 mg total, 58%). H NMR (300 MHz, DMSO-
d6) δ 1.02 (t, J=7.3Hz, 3C), 1.67ꢀ1.82 (m, 2H), 3.09 (t, J=7.4Hz, 2C), 6.92ꢀ7.02 (m, 1H), 7.20ꢀ
7
8
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.31 (m, 1H), 7.36 (d, J=8.0 Hz, 1H), 7.42 (d, J=7.2 Hz, 1H), 7.47ꢀ7.63 (m, 3H), 7.66 (s, 1H),
1
3
.09 (d, J=8.1 Hz, 1H), 8.17 (d, J=8.3 Hz, 1H), 12.30 (s, 1H). C NMR (75.5 MHz, DMSO-d6) δ
3.99, 23.45, 34.47, 107.41 (d, J=20.8 Hz, 1C), 108.80 (d, J=3.7 Hz, 1C), 113.44 (d, J=21.1 Hz,
1C), 117.31 (d, J=5.9 Hz, 1C), 124.03 (d, J=7.7 Hz, 1C), 124.12, 124.73, 126.07 (3C), 126.22,
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30.75, 131.58, 137.00, 137.28 (d, J=2.4 Hz, 1C), 140.02 (d, J=11.4 Hz, 1C), 140.60, 155.90 (d,
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J=251.1 Hz, 1C), 189.63.
Methyl 4-[(1-pentyl-1H-indol-3-yl)carbonyl]naphthalene-1-carboxylate (1-4a)
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DMF (15 mL) was cooled to 0°C, and sodium hydride (245 mg, 60% in mineral oil, 6.1
mmol) added over a few minutes. After the addition was complete, the mixture was stirred for
an additional 10 min before adding a solution of methyl 4ꢀ[(1HꢀIndolꢀ3ꢀyl)carbonyl]naphthaleneꢀ
1ꢀcarboxylate (1-3b) (1 g, 0.003 mol) in DMF (15 mL) dropꢀwise. The solution was then stirred
for 30 min at 0°C. A solution of pentyl bromide (0.415 mL) in DMF (8 mL) was added and the
mixture allowed to warm to room temperature and stirred overnight. The solvent was removed
under reduced pressure and EtOAc (25 mL) and water (25 mL) added to the residue. The mixture
was shaken, the organic layer removed and washed with water. The solvent was removed under
reduced pressure and residue was columned over silica gel (Isco, gradient from 100% hexane to
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30% EtOAc/ 70% hexane) to give the title product as an offꢀwhite solid (630 mg, 52%). H
NMR (300 MHz, CDCl ) δ 0.84 (t, J=6.6 Hz, 3H), 1.17ꢀ1.37 (m, 4H), 1.72ꢀ1.88 (m, 2H), 3.98ꢀ
3
4.10 (m, 5H), 7.23ꢀ7.29 (m, 1H), 7.32ꢀ7.43 (m, 3H), 7.44ꢀ7.54 (m, 1H), 7.68ꢀ7.78 (m, 2H), 8.09ꢀ
8.17 (m, 1H), 8.20 (d, J=7.4 Hz, 1H), 8.42ꢀ8.50 (m, 1H), 8.92 (d, J=8.5 Hz, 1H).
Ethyl 4-[(1H-Indol-3-yl)carbonyl]naphthalene-1-carboxylate (1-4b) and Ethyl 4-[(1-pentyl-
1H-indol-3-yl)carbonyl]naphthalene-1-carboxylate (1-4c)
DMF (150 mL) was cooled to 0°C and sodium hydride (3.68 g, 60% in mineral oil, 0.092
mol) was added over several minutes. The mixture was then stirred for an additional 10 min and
a solution of methyl 4ꢀ[(1HꢀIndolꢀ3ꢀyl)carbonyl]naphthaleneꢀ1ꢀcarboxylate (1-3b) (15 g, 0.046
mol) in DMF (150 mL) was added drop wise. The solution was then stirred for 30 min at 0°C.
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A solution of pentyl bromide (7.56 g, 0.05 mol) in DMF (75 mL) was added and the mixture
allowed to warm to room temperature and stir overnight. The solvent was removed, EtOAc (100
mL) added to the residue and the inorganic solid removed by filtration. Water (100 mL) was
then added to the filtrate and the mixture stirred for 10 min. The organic layer was removed,
dried over sodium sulfate, and the solvent removed under reduced pressure. The residue was
purified over silica gel (Isco, gradient from 100% hexane to 30% EtOAc/70% hexane) to give
the title compounds via transesterification as light yellow solids.
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Ethyl 4-[(1H-Indol-3-yl)carbonyl]naphthalene-1-carboxylate (1-4b) (3.0 g, 19%) H NMR
(
300 MHz, CDCl ) δ 1.49 (t, J=7.1 Hz, 3H), 4.52 (q, J=7.1 Hz, 2H), 7.26 (s, 1H), 7.28ꢀ7.49 (m,
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4H), 7.54ꢀ7.65 (m, 2H), 8.09 (d, J=8.5 Hz, 1H), 8.15 (d, J=7.4 Hz, 1H), 8.89 (d, J=8.7 Hz, 1H),
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1
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.89 (d, J=8.7 Hz, 1H), 9.08 (broad s, 1H). C NMR (75.5 MHz, CDCl ) δ 14.40, 61.41, 11.60,
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18.93, 122.56, 123.23, 123.77, 124.28, 125.73, 125.93, 126.29, 126.95, 127.93, 128.50, 129.23,
31.06, 131.52, 135.25, 136.60, 142.23, 167.40, 192.11.
Ethyl 4-[(1-pentyl-1H-indol-3-yl)carbonyl]naphthalene-1-carboxylate (1-4c) (6.7 g, 36%).
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H NMR (300 MHz, CDCl ) δ 0.84 (t, J=6.8 Hz, 3H), 1.16ꢀ1.36 (m, 4H), 1.49 (t, J=7.1 Hz, 3H),
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3
8
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.71ꢀ1.85 (m, 2H), 4.04 (t, J=7.2 Hz, 2H), 4.52 (q, J=7.1 Hz, 2H), 7.27 (s, 1H), 7.32ꢀ7.45 (m,
H), 7.46ꢀ7.53 (m, 1H), 7.58ꢀ7.69 (m, 2H), 8.14 (d, J=8.2 Hz, 1H), 8.19 (d, J=7.1 Hz, 1H), 8.43ꢀ
1
3
.53 (m, 1H) 8.92 (d, J=8.6 Hz, 1H). C NMR (75.5 MHz, CDCl ) δ 13.84, 14.43, 22.15, 28.91,
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9.45, 47.26, 61.36, 110.08, 117.41, 122.91, 123.10, 123.72, 123.81, 125.92, 126.48, 126.79,
126.87, 127.91, 128.59, 129.01, 131.18, 131.57, 137.12, 138.09, 143.65, 167.47, 191.31. HPLC
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7 % (Waters XꢀBridge Cꢀ18 5 µm, 4.6 × 100 mm column, 10 mM aqueous NH OAcꢀCH CN,
4 3
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0:60, UV detection at 254 nm). HRMS: Calculated for C H NO (M + H): 414.2069. Found:
27 28 3
414.2063 (M + H).
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N-(3-Ethoxycarbonylpropyl)-3-(1-naphthoyl)-1H-indole (1-4d)
To a vacuum dried flask under N was added NaH (60% dispersion in oil, 9.1 mmol, 0.37
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mg) and the flask was cooled to 0 °C and dry DMF (29 mL) was added. The mixture was stirred
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for 10 min and a solution of 3ꢀ(1ꢀnaphthoyl)indole (2.0 g, 0.00729 mol) in DMF (28 mL) was
added dropwise over 30 min. After complete addition the mixture was stirred an additional 30
min and then a solution of ethyl bromobutyrate (3.55 g, 0.0182 mol) in 14 mL of DMF was
added over 15 min and the reaction mixture was allowed to warm to room temperature overnight.
TLC (SiO ; hexane/EtOAc (10:1)) showed the consumption of starting material. The DMF was
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removed under reduced pressure and the resulting residue was partitioned between water (100
mL) and EtOAc (3 × 100 mL) followed by CH Cl (2 × 100mL). The organics were combined,
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the solvent removed in vacuo and the residual material was chromatographed on an Isco 80 g
silica column eluting with a gradient from hexanes (100%) to hexane/EtOAc (7:3) that afforded
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the desired compound in 92% yield. H NMR (300 MHz, CDCl ) δ 1.08 (t, J=7.13 Hz, 3H),
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1.93ꢀ2.08 (m, 2H), 2.10ꢀ2.22 (m, 2H), 3.91ꢀ4.11 (m, 4H), 7.19ꢀ7.48 (m, 7H), 7.55 (d, J=6.9 Hz,
1H), 7.80 (d, J=7.5 Hz, 1H), 7.86 (d, J=8.2 Hz, 1H), 8.09 (d, J=8.0 Hz, 1H), 8.37ꢀ8.47 (m, 1H).
13
C NMR (75.5 MHz, CDCl ) δ 14.11, 24.97, 30.77, 46.05, 60.68, 109.92, 117.89, 122.98,
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123.01, 123.80, 124.54, 125.82, 125.95, 126.28, 126.76, 127.01, 128.17, 130.01, 130.79, 133.77,
137.00, 137.80, 139.00, 172.31, 192.00.
N-(3-Ethoxycarbonylbutyl)-3-(1-naphthoyl)-1H-indole (1-4e)
Prepared as for the ethoxycarbonylpropyl analog 1-4d from ethyl bromovalerate (0.160 mL,
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1
.014 mmol) afforded 85% of the title compound. H NMR (300 MHz, CDCl ) δ 1.09 (t, J=7.1
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Hz, 3H), 1.41ꢀ1.55 (m, 2H), 1.65ꢀ1.81 (m, 2H), 2.16 (t, J=7.2 Hz, 3H), 3.90ꢀ4.06 (m, 4H), 7.21ꢀ
7.48 (m, 7H), 7.55 (d, J=7.0 Hz, 1H), 7.79 (d, J=7.7 Hz, 1H), 7.85 (d, J=8.2 Hz, 1H), 8.09 (d,
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J=8.1 Hz, 1H), 8.36ꢀ8.44 (m, 1H). C NMR (75.5 MHz, CDCl ) δ 14.17, 22.14, 29.21, 33.52,
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46.82, 60.43, 109.87, 117.77, 122.91, 123.02, 123.70, 124.56, 125.84, 125.98, 126.28, 126.75,
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27.05, 128.17, 129.98, 130.81, 133.78, 136.99, 137.69, 139.07, 172.81, 191.97
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N-(3-Carboxypropyl)-3-(1-naphthoyl)-1H-indole (1-4f)
0 mg of the ester 1-4d was then treated with sodium hydroxide and water:methanol (1:1)
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mixture and heated to 50 °C for 2 hrs. Cooling and treatment with 2N HCl, extraction with ethyl
acetate, and drying with magnesium sulfate followed by filtration and concentration provided the
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desired acid for testing purposes in yields of 96%. H NMR (300 MHz, Acetonitrileꢀd3) δ 1.96ꢀ
2.10 (m, 2H), 2.17ꢀ2.29 (m, 2H), 4.19 (d, J=7.1 Hz, 2H), 7.30ꢀ7.43 (m, 2H), 7.46ꢀ7.63 (m, 5H),
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.67ꢀ7.75 (m, 1H), 7.95ꢀ8.14 (m, 3H), 8.36ꢀ8.46 (m, 1H). C NMR (75.5 MHz, Acetonitrileꢀd3)
δ 24.44, 29.66, 45.47, 110.35, 116.70, 121.84, 122.35, 123.23, 124.55, 125.26, 125.64, 126.01,
26.41, 126.64, 127.97, 129.46, 130.27, 133.46, 136.91, 138.69, 138.75, 172.89, 191.34. EIMS:
Calculated for C H NO : 357.40. Found: 358.4 (M + H).
1
23 19
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N-(3-Carboxybutyl)-3-(1-naphthoyl)-1H-indole (1-4g)
Prepared as for the above carboxypropyl analog from the corresponding ester in 78% yield.
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H NMR (300 MHz, CDCl ) δ 1.45ꢀ1.58 (m, 2H), 1.71ꢀ1.84 (m, 2H), 2.24 (t, J=7.2 Hz, 2H),
3
3.97ꢀ4.05 (m, 2H), 7.24ꢀ7.48 (m, 7H), 7.57 (d, J=6.7 Hz, 1H), 7.78ꢀ7.92 (m, 2H), 8.10 (d, J=8.1
1
3
Hz, 1H), 8.37ꢀ8.44 (m, 1H). C NMR (75.5 MHz, CDCl ) δ 21.84, 29.11, 33.14, 46.75, 109.85,
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117.77, 122.97, 123.01, 123.74, 124.56, 125.87, 125.94, 126.26, 126.76, 127.00, 128.17, 130.03,
130.77, 133.74, 136.97, 137.77, 138.96, 178.13, 192.14. EIMS: Calculated for C H NO :
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4
21
3
371.43. Found: 372.1 (M + H).
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X-Fluoro-3-[(naphthalen-1-yl)carbonyl]-1-alkyl-1H-indole (X = 4-7)(1-4h-m)
These analogs were prepared by the same method described for 1-4h on the same or 55, 55,
59, 25% scale (1-4k-n).
4-Fluoro-3-[(naphthalen-1-yl)carbonyl]-1-pentyl-1H-indole (1-4h )
Sodium hydride (207 mg, 60% in oil, 5.18 mmol) was added to DMF (20 mL) at 0°C and the
mixture stirred for 10 min. A solution of 4ꢀfluoroꢀ3ꢀ[(naphthalenꢀ1ꢀyl)carbonyl]ꢀ1Hꢀindole (1-
e) (750 mg, 2.59 mmol) in DMF (10 mL) was added dropwise over 5 min and the resulting
solution stirred at 0 °C for 30 min. A solution of 1ꢀbromopentane (431 mg, 2.85 mmol) in DMF
1 mL) was then added dropwise to the stirred mixture. Cooling was continued for an additional
0 min before allowing the solution to warm to room temperature and stir overnight. The
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(
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reaction was quenched with water (75 mL), and EtOAc (50 mL) was added. The mixture was
shaken and the organic layer removed. The aqueous layer was then extracted with additional
EtOAc (50 mL). The organic layers were combined, dried over Na SO and the solvent removed
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under reduced pressure. The residue was purified over silica gel (Isco, 120 g column, gradient
from 100% hexane to 30% EtOAc/ 70% hexane) to give the title compound as a colorless resin
1
(760 mg, 82%). H NMR (300 MHz, CDCl ) δ 0.85 (t, J=6.8 Hz, 3H), 1.15ꢀ1.38 (m, 4H), 1.72ꢀ
3
1.87 (m, 2H), 4.04 (t, J=7.2 Hz, 2H), 6.93ꢀ7.03 (m, H), 7.16 (d, J=8.0 Hz, 1H), 7.22ꢀ7.30 (m, 1H),
7.33 (s, 1H), 7.43ꢀ7.55 (m, 3H), 7.63ꢀ7.70 (m, 1H), 7.85ꢀ7.93 (m, 1H), 7.97 (d, J=8.3 Hz, 1H),
13
8
.23ꢀ8.31 (m, 1H). C NMR (75.5 MHz, CDCl ) δ 13.83, 22.14, 28.86, 29.32, 47.43, 106.11 (d,
3
J=4.1 Hz, 1C), 108.48 (d, J=21.5 Hz, 1C), 114.91 (d, J=21.2 Hz, 1C), 117.64 (d, J=5.8 Hz, 1C),
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Journal of Medicinal Chemistry
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24.33 (d, J=8.0 Hz, 1C), 124.41, 126.08, 126.26, 126.75, 126.86, 128.18, 130.40, 131.07,
33.74, 138.12, 139.09 (d, J=2.7 Hz, 1C), 139.91 (d, J=11.0 Hz, 1C), 157.03 (d, J=254.0 Hz, 1C),
90.34. HPLC 99% (Waters XꢀBridge Cꢀ18 5 µm, 4.6 × 100 mm column, H OꢀCH CN, 35:65,
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10
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UV detection at 254 nm). HRMS: Calculated for C H NOF (M + H): 360.1764. Found:
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360.1760 (M + H).
5-Fluoro-3-[(naphthalen-1-yl)carbonyl]-1-pentyl-1H-indole (1-4i)
1
Colorless resin (760 mg, 81%). H NMR (300 MHz, CDCl ) δ 0.85 (t, J=6.8 Hz, 3H), 1.17ꢀ
3
1
7
8
.38 (m, 4H), 1.71ꢀ1.88 (m, 2H), 4.05 (t, J=7.2 Hz, 2H), 7.04ꢀ7.14 (m, 2H), 7.37 (s, 1H), 7.43ꢀ
.57 (m, 3H), 7.61ꢀ7.68 (m, 1H), 7.87ꢀ7.94 (m, 1H), 7.98 (d, J=8.2 Hz, 1H), 8.13ꢀ8.21 (m, 1H),
1
3
.39ꢀ8.48 (m, 1H). C NMR (75.5 MHz, CDCl ) δ 13.83, 22.14, 28.89, 29.48, 47.46, 108.33 (d,
3
J=24.8 Hz, 1C), 110.76 (d, J=9.8 Hz, 1C), 112.00 (d, J=26.7 Hz, 1C), 117.50 (d, J=4.5 Hz, 1C),
124.54, 125.82, 125.91, 126.36, 126.84, 127.75 (d, J=11.7 Hz, 1C), 128.21, 130.12, 130.76,
133.57, 133.80, 138.68, 138.84, 159.92 (d, J=238.7 Hz, 1C), 191.73. HPLC 99% (Waters Xꢀ
Bridge Cꢀ18 5µm, 4.6 × 100 mm column, H OꢀCH CN, 35:65, UV detection at 254 nm).
2
3
HRMS: Calculated for C H NOF (M + H): 360.1764. Found: 360.1763 (M + H).
2
4
23
6-Fluoro-3-[(naphthalen-1-yl)carbonyl]-1-pentyl-1H-indole (1-4j)
1
Colorless resin (840 mg, 90%). H NMR (300 MHz, CDCl ) δ 0.85 (t, J=6.8 Hz, 3H), 1.18ꢀ
3
1
7
8
.40 (m, 4H), 1.72ꢀ1.87 (m, 2H), 4.00 (t, J=7.2 Hz, 2H), 7.02ꢀ7.16 (m, 2H), 7.32 (s, 1H), 7.43ꢀ
.58 (m, 3H), 7.61ꢀ7.69 (m, 1H), 7.87ꢀ7.95 (m, 1H), 7.98 (d, J=8.2 Hz, 1H), 8.14ꢀ8.22 (m, 1H),
1
3
.39ꢀ8.48 (m, 1H). C NMR (75.5 MHz, CDCl ) δ 13.82, 22.15, 28.88, 29.35, 47.32, 96.64 (d,
3
J=26.9 Hz, 1C), 111.34 (d, J=23.9 Hz, 1C), 117.70, 123.38, 124.09 (d, J=10.1 Hz, 1C), 124.51,
125.89, 125.93, 126.35, 126.83, 128.21, 130.13, 130.80, 133.79, 137.31 (d, J=11.6 Hz, 1C),
138.13 (d, J=2.9 Hz, 1C), 138.83, 160.57 (d, J=241.0 Hz, 1C), 191.88. HPLC 99% (Waters Xꢀ
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Bridge Cꢀ18 5µm, 4.6 × 100 mm column, H OꢀCH CN, 35:65, UV detection at 254 nm).
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HRMS: Calculated for C H NOF (M + H): 360.1764. Found: 360.1758 (M + H).
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7-Fluoro-3-[(naphthalen-1-yl)carbonyl]-1-pentyl-1H-indole (1-4k)
0
1
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0
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0
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0
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0
1
Colorless resin (430 mg, 83%). H NMR (300 MHz, CDCl ) δ 0.85 (t, J=6.7 Hz, 3H), 1.17ꢀ
3
1
.38 (m, 4H), 1.72ꢀ1.88 (m, 2H), 4.20 (t, J=7.2 Hz, 2H), 6.97ꢀ7.08 (m, 1H), 7.20ꢀ7.31 (m, 2H),
.43ꢀ7.58 (m, 3H), 7.61ꢀ7.68 (m, 1H), 7.87ꢀ7.94 (m, 2H), 8.13ꢀ8.21 (m, 1H), 8.27 (d, J=8.0 Hz,
7
1
3
1H). C NMR (75.5 MHz, CDCl ) δ 13.84, 22.14, 28.65, 30.83 (d, J=2.0 Hz, 1C), 50.0 (d,
3
J=5.1 Hz, 1C), 109.47 (d, J=18.2 Hz, 1C), 118.02, 118.67 (d, J=3.6 Hz, 1C), 123.31 (d, J=6.5 Hz,
1
C), 124.54, 124.88 (d, J=7.5 Hz, 1C), 125.91 (2C), 126.37, 126.86, 128.22, 130.18, 130.72,
30.76, 133.79, 138.89, 139.08, 149.89 (d, J=245.3 Hz, 1C), 191.89. HPLC 99% (Waters Xꢀ
1
Bridge Cꢀ18 5µm, 4.6 × 100 mm column, H OꢀCH CN, 35:65, UV detection at 254 nm).
2
3
HRMS: Calculated for C H NOF (M + H): 360.1764. Found: 360.1763 (M + H).
2
4
23
4-Fluoro-1-(5-fluoropentyl)-3-[(naphthalen-1-yl)carbonyl]-1H-indole (1-4l)
1
Colorless resin (150 mg, 28%). H NMR (300 MHz, CDCl ) δ 1.32ꢀ1.46 (m, 2H), 1.55ꢀ1.76
3
(m, 2H), 1.77ꢀ1.92 (m, 2H), 4.07 (t, J=7.2 Hz, 2H), 4.29 (t, J=7.2 Hz, 1H), 4.45 (t, J=5.8 Hz, 1H),
6.93ꢀ7.03 (m, 1H), 7.16 (d, J=8.1 Hz, 1H), 7.22ꢀ7.31 (m, 1H), 7.33 (s, 1H), 7.43ꢀ7.55 (m, 3H),
1
3
7.63ꢀ7.69 (m, 1H), 7.85ꢀ7.92 (m, 1H), 7.96 (d, J=8.2 Hz, 1H), 8.22ꢀ8.29 (m, 1H). C NMR
(
75.5 MHz, CDCl ) δ 22.76 (d, J=4.9 Hz, 1C), 29.31, 29.82 (d, J=19.8 Hz, 1C), 47.27, 83.51(d,
3
J=165.1 Hz, 1C), 106.04 (d, J=4.0 Hz, 1C), 108.54 (d, J=21.3 Hz, 1C), 114.92 (d, J=21.2 Hz, 1C),
117.79 (d, J=5.8 Hz, 1C), 124.43, 124.45 (d, J=7.8 Hz, 1C), 126.04, 126.28, 126.79, 126.87,
128.21, 130.46, 131.05, 133.75, 137.99, 139.00 (d, J=2.8 Hz, 1C), 139.85 (d, J=11.0 Hz, 1C),
157.05 (d, J=254.3 Hz, 1C), 190.31. HPLC 98% (Waters XꢀBridge Cꢀ18 5µm, 4.6 × 100 mm
column, H OꢀCH CN, 35:65, UV detection at 254 nm). HRMS: Calculated for C H NOF (M
2
3
24 22
2
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+
H): 378.1669. Found: 378.1671 (M + H).
4-Fluoro-1-[2-(morpholin-4-yl)ethyl]-3-[(naphthalen-1-yl)carbonyl]-1H-indole (1-4m)
1
Light yellow liquid (420 mg, 69%). H NMR (300 MHz, CDCl ) δ 2.34 (t, J=4.6 Hz, 4H),
3
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11
12
13
14
15
16
17
18
19
20
21
22
23
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2
.65 (t, J=6.2 Hz, 2H), 3.49 (t, J=4.5 Hz, 4H), 4.11 (t, J=6.2 Hz, 2H), 6.95ꢀ7.04 (m, 1H), 7.15 (d,
J=8.1 Hz, 1H), 7.21ꢀ7.32 (m, 1H), 7.42 (s, 1H), 7.39ꢀ7.55 (m,3H), 7.62ꢀ7.69 (m, 1H), 7.86ꢀ7.93
13
(m, 1H), 7.96 (d, J=8.0 Hz, 1H), 8.19ꢀ8.27 (m, 1H). C NMR (75.5 MHz, CDCl ) δ 42.10,
3
51.32 (2C), 55.01, 64.54 (2C), 103.57 (d, J=3.8 Hz, 1C), 106.39 (d, J=21.3 Hz, 1C), 112.48 (d,
J=21.3 Hz, 1C), 115.46 (d, J=5.9 Hz, 1C), 122.13, 122.25, 123.79, 124.06, 124.27, 124.62,
1
25.97, 128.07, 128.77, 131.48, 136.91 (d, J=2.7 Hz, 1C), 137.12, 137.70 (d, J=11.1 Hz, 1C),
1
54.85 (d, J=254.3 Hz, 1C), 188.03. HPLC 99% (Waters XꢀBridge Cꢀ18 5µm, 4.6 × 100 mm
column, 10 mM aqueous NH OAcꢀCH CN, 45:55, UV detection at 254 nm). HRMS: Calculated
4
3
for C H N O F (M + H): 403.1822. Found: 403.1830 (M + H).
2
5
24
2
2
4
-Fluoro-1-(5-fluoropentyl)-3-[(4-propylnaphthalen-1-yl)carbonyl]-1H-indole (1-4n)
1
Light yellow liquid (68 mg, 24%). H NMR (300 MHz, CDCl ) δ 1.06 (t, J=7.3 Hz, 3H),
3
1.33ꢀ1.46 (m, 2H), 1.56ꢀ1.75 (m, 2H), 1.76ꢀ1.91 (m, 4H), 3.11 (t, J=7.5 Hz, 2H), 4.07 (t, J=7.1
Hz, 2H), 4.30 (t, J=5.8 Hz, 1H), 4.46 ((t, J=5.9 Hz, 1H), 6.94ꢀ7.03 (m, 1H), 7.16 (d, J=8.1 Hz,
1
H), 7.23ꢀ7.29 (m, 1H), 7.30ꢀ7.37 (m, 2H), 7.43ꢀ7.57 (m, 2H), 7.58ꢀ7.62 (m, 2H), 8.11 (d, J=8.0
1
3
Hz, 1H), 8.28ꢀ8.36 (m, 1H). C NMR (75.5 MHz, CDCl ) δ 14.29, 22.77 (d, J=5.0 Hz, 1C),
3
2
9.33, 29.83 (d, J=19.7 Hz, 1C), 35.50, 47.24, 83.51 (d, J=165.1 Hz, 1C), 105.97 (d, J=3.9 Hz,
1C), 108.33, 108.61, 115.01 (d, J=21.2 Hz, 1C), 117.97 (d, J=5.8 Hz, 1C), 118.01, 124.03,
24.36 (d, J=7.9 Hz, 1C), 124.50, 126.01, 126.33, 126.80, 126.82, 131.49, 132.21, 137.35 (d,
1
J=2.6 Hz, 1C), 139.76, 139.90, 141.66, 157.08 (d, J=254.4 Hz, 1C), 190.54. HPLC 99% (Waters
XꢀBridge Cꢀ18 5µm, 4.6 × 100 mm column, H OꢀCH CN, 35:65, UV detection at 254 nm).
2
3
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