design and synthesis of allosteric modulators of …rx...other approaches such as development of cb1...
TRANSCRIPT
Design and Synthesis of Allosteric Modulators of CB1 Cannabinoid Receptor
Master’s Thesis Research Proposal
by
Abhijit R. Kulkarni
Advisor: Ganesh A. Thakur, Ph.D
Department of Pharmaceutical Sciences
Northeastern University
August 2011
1
DEDICATION
I dedicate this thesis in the memory of my loving father, Raghunath Dnyanraj Kulkarni. He
always believed in me for what I did and instilled into me the curiosity for learning science. He
played a major role in shaping me in my childhood and to be what I am today. He will be dearly
missed.
2
ACKNOWLEDGEMENTS
I would like to thank Dr. Paresh Salgaonkar and Dr. David Janero for their direction assistance
and guidance. In particular, Dr. Janero took keen interest in the whole process and whose
recommendation and insightful criticism has been very valuable in shaping my thesis altogether.
Dr. Salgaonkar’s strong chemistry background helped in the decisive steps of my reactions. Dr.
Pushkar Kulkarni, a senior, colleague, friend, was with me the whole time during my thesis and
provided his help in every aspect. His command over literature reading helped me learn to pace
up my speed in the research. I will thank him a ton for all his help. I would also like to thank Dr.
Vidyanand Shukla for his helpful comments in troubleshooting some difficult reactions.
I had saved the last for the best person, Dr. Ganesh Thakur, a dedicated scientist, researcher, a
well-wisher and my mentor and advisor on this project, and I take this opportunity to thank him
from the bottom of my heart. He gave me the confidence to do research and be what I am today.
There was a time I remember when I have nearly given up on this thesis when he gave me the
moral boost I needed. He stood behind me in everything and paved me a way through all the
difficult situations, not only related to this thesis. He has spent sleepless nights with me to insure
that the thesis was in good form. I thank him dearly for all his help and support.
My special thanks go to Dr. Roger Kautz for giving me time generously to build my NMR
basics and guiding me in the whole quest. Dr. Jim Glick helped me a lot with Mass Spectrometry
and took time to walk me through the whole process; I thank him for all his help. Dr. Mike
Pollastry deserves special thanks for his kind help for giving me access to his LC-MS instrument.
3
I would like to thank my dear friend Rohit Kapile for sticking with me during the thesis for
whatever help I needed; Abhijit Kamerkar for providing technical support and Shivan Acharya,
an undergrad in our group who provided his help in whatever I needed in the lab. Rosalee
Robinson, and Roger Avelino, our department secretaries helped me a lot in all the formalities
related to the proposal and defense. I am also very thankful to Prof. Mansoor Amiji for his
support thorough out my MS thesis work.
Finally, I would like to thank my loving mother Sadhana Raghunath Kulkarni and my dearest
sister Arundhati Kulkarni for providing me the mental support and composure that I needed in
order to finish this thesis and backing me up in all of my endeavors.
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Table of Contents
1. Abbreviation --------------------------------------------------------------------------5
2. List of figures-------------------------------------------------------------------------6
3. Abstract--------------------------------------------------------------------------------7
4. Introduction---------------------------------------------------------------------------9
5. Specific Aims------------------------------------------------------------------------17
6. Chemicals and Materials-----------------------------------------------------------18
7. Ligand Design-----------------------------------------------------------------------19
8. Retrosynthesis and Chemistry-----------------------------------------------------22
9. Experimental-------------------------------------------------------------------------28
10. Conclusions--------------------------------------------------------------------------44
11. References----------------------------------------------------------------------------45
5
1. Abbreviations:
GPCR – G protein-coupled receptors
CB1 – Subtype 1 of the cannabinoid receptor class of G protein-coupled receptors
CB2 - Subtype 2 of the cannabinoid receptor class of G protein-coupled receptors
GABA – gamma amino butyric acid
LAPS – Ligand-assisted protein structure
MS – Mass spectrometry
Org – Organon
NAM – Negative Allosteric Modulator
PAM – Positive Allosteric Modulator
AEA – (Anandamide) Arachidonoyl ethanolamine
2-AG – 2-Arachidonoyl glycerol
TM - Transmembrane
HTS – High-throughput screening
HIV – Human immunodeficiency virus
TBAI – Tetrabutyl ammonium iodide
NMP – N-Methyl-2-pyrrolidone
THF – Tetrahydrofuran
EDCI – 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
HOBT – Hydroxybenzotriazole
R.T – room temperature
h – hour
DIPEA – Diisopropylethylamine (Hunig’s base)
6
2. List of Figures:
Figure 1: Structures of 9-THC and two key endogenous cannabinoids
Figure 2: Structures of NAM and PAMs of CB1 receptor
Figure 3: Overlap of Org27569 and PSNCBAM-1using Maestro.
Figure 4: Novel covalent probes for CB1 allosteric site
Figure 5: Iodinated analog of Org27569
Figure 6: Structures of two proposed hybrid analogs
Scheme 1: Synthesis of amine part of Org27569
Scheme 2: Synthesis of 5-chloro indole ester.
Scheme 3: Synthesis of Org27569
Scheme 4: Synthesis of the nitro/amino/azido/iosthiocyanate analogs (Targets 1 and 2) of Org 27569
Scheme 5: Synthesis of iodo analog of Org 27569 (Target 3)
Scheme 6: Synthesis of hybrid analog 33 (Target 4)
Scheme 7: Synthesis of hybrid analog 39 (Target 5)
7
3. Abstract:
Cannabinoid receptors are a major class of cell-membrane receptors which belong to the super-
family of G protein-coupled receptors (GPCRs) and are targeted for the treatment of several
diseases including neurodegenerative diseases, cancer, obesity, inflammation and neuropathic
and inflammatory pain. Two subtypes of cannabinoid receptors, namely, CB1 and CB2, have
been cloned and studied more intensively. CB1 receptor is the most abundant GPCR in the brain,
and a wide range of selective and potent CB1-receptor ligands for its orthosteric site have been
developed. However, their therapeutic utility has been limited due to side effects associated with
indiscriminate cannabinoid receptor activation and propensity for receptor desensitization. This
problem is exemplified by the recent cancellation of the Phase III clinical trials of the CB1
antagonists / inverse agonists Taranabant and Otenabant and the manufacturer’s (Sanofi-Aventis)
voluntary withdrawal of the marketed drug Rimonabant in the European Union. Rimonabant
(Acomplia), which was approved as an adjunctive weight-loss drug in Europe, suffered serious-
dose related gastrointestinal and psychiatric side effects such as depression and suicidal ideation.
Other approaches such as development of CB1 neutral as well as peripherally-acting antagonists
have shown therapeutic promise and reduced side effects in recently published preclinical
studies.1-4 A promising alternative approach is the development of CB1 allosteric modulators
which by binding to a sub-type specific and topographically distinct site from the orthosteric site
can either enhance or inhibit the action of endocannabinoids and thus act more selectively to tune
the CB signaling in a site- and event-specific manner. Recently high-throughput screening
(HTS) from two different laboratories has identified two different classes of ligand (e.g.,
Org27569 and PSNCBAM-1) exhibiting negative allosteric modulation at CB1 receptors. Due to
the unavailability of the cannabinoid receptor’s crystal structure, characterization of the binding
8
site(s) of these allosteric modulators is lacking. Availability of such data will prove instrumental
in elucidating their molecular basis for activity and in developing highly selective, potent CB1
allosteric modulators. The objective of the present study is to develop covalent probes (both
photo-activatable and electrophilic) based upon the parent structure of Org27569 bearing azido
and isothiocyanate functionality at the judiciously chosen positions. Using Ligand-Assisted
Protein Structure approach (LAPS), which involves use of such probes for labeling the receptor
covalently followed by MS analysis of the protein and validating the resulting data with site-
directed mutagenesis and molecular modeling studies, the chemical nature and tertiary structure
of the active allosteric sites of CB1 can be elucidated. Additionally, we propose to synthesize an
iodinated analog of Org27569 to facilitate development of radiometric competitive binding
assays directed at CB1 allosteric site. We also propose to synthesize two hybrid analogs of
Org27569 and PSNCBAM-1 to help understand structural requirements for CB1 allosteric site
and facilitate development of future structure-activity relationship studies.
9
4. Introduction:
The endocannabinoid system (ECS) is comprised of two well-characterized cannabinoid
receptors (CB1 and CB2), endogenous cannabinoids (endocannabinoids) and several enzymes
and proteins involved in their biosynthesis, transport and metabolism.5-7
4.1 Cannabinoid Receptors and Their Endogenous Ligands: The first milestone in
endocannabinoid research was the isolation and characterization of the major psychoactive
component of Cannabis, the phytocannabinoid 9-tetrahydrocannabinol (9-THC; 2; Fig.1) that
served as prototype for the synthesis of numerous analogs as potential pharmacological agents.8
The next milestone was the discovery that the cannabinergic agents produce most of their
biochemical and pharmacological effects by interacting with two cannabinoid receptors, CB1
and CB2.7,9-13 These two cannabinoid receptors belong to class A (rhodopsin-like) of the
superfamily of GPCRs and are Gi/o coupled. CB1, but not CB2 under certain conditions can
also be Gs coupled. They share 44% overall homology and 68% homology in their
transmembrane domain.13 The rat, mouse and human CB1 receptors have been cloned and show
97-99% sequence identity across species, whereas the mouse CB2 exhibits 82% sequence
identity with the human clone.12,14 CB1 and CB2 share some common signaling pathways, such
as inhibition of adenylyl cyclase and stimulation of mitogen-activated protein kinase. Activation
of CB1, but not CB2 mediates inhibition of N- and P/Q –type calcium channels and stimulation
of potassium channels.5,7
The CB1 is the most abundant GPCR in the brain with high density in the cerebellum,
hippocampus and striatum.10,15 It is also found in a variety of other organs including the heart,
vascular endothelium, vas deferens, testis, small intestine, sperum and uterus.11,16-18 Conversely,
10
CB2 receptor is mainly expressed in the cells and tissues of the immune system19 and to a limited
extent in healthy brain.20 However, CB2 is upregulated ‘on demand’ during early inflammatory
events in both CNS and peripheral tissues. Recent data from CB1 and CB2 knockout mice
suggest the presence of additional cannabinoid receptors such as GPR55, which is expressed in
brain and various peripheral tissues of human and rats and is activated by several cannabinoid-
like ligands.21-23 There is also evidence for yet another cannabinoid receptor referred to as
‘endothelial cannabinoid receptor’ or ‘abnormal-CBD receptor’.24
Soon after the discovery of cannabinoid receptors, the quest for endogenous cannabinoids
resulted in identification of two key endogenous cannabinoids, N-arachidonoylethanolamine25
(anandamide, AEA; 2; Fig.1) and 2-arachidonoylglycerol (2-AG; 3; Fig.1).26 AEA is a highly
lipophilic compound with sensitivity to both oxidation and hydrolysis and exhibits moderate
affinity at CB1 (Ki = 61 nM) and lower affinity at CB2 (Ki = 1930 nM) and functionally behaves
as partial agonist at both CB receptors. On the other hand, 2-AG which is present in higher
concentration in brain (~170-fold) behaves as full agonist at both CB receptor but exhibits
reduced affinities (Ki at CB1 = 472 nM, and Ki at CB2 = 1400 nM). These endocannabinoids are
not stored into the storage vesicles like other neurotransmitters, but are biosynthesized upon
demand in both CNS and PNS. Extensive studies on the ECS have revealed a number of
cannabinergic proteins involved in the inactivation and biosynthesis of endocannabinoids. The
inactivating enzymes include fatty acid amide hydrolase (FAAH) , which hydrolyses both AEA
and 2-AG, and monoacyl glycerol lipase (MGL), which hydrolyses 2-AG.
11
Figure 1: Chemical structures of 9-THC and two key endogenous cannabinoids.
Pathological ECS activity has been identified in the etiology of important disease state (e.g.
obesity/metabolic syndrome, substance abuse, and neurodegenerative diseases) that represents
important medical problems worldwide. As the biological role of the ECS is being elucidated,
medicinal chemistry efforts are directed towards producing high-potency ligands selective for
either CB1 or CB2 receptor as well as inhibitors of FAAH and MGL. Thus, the endocannabinoid
system holds a promise for the future development of important drugs.6,7
4.2 Therapeutic Potential of CB1 Receptor Antagonists: Thirteen years ago, Colombo et al.
demonstrated appetite suppression and weight loss in adult rats after the administration of CB1
selective antagonist/inverse agonist, Rimonabant (SR141716A).27 Because obesity has become a
major health problem in recent years28 this observation stimulated a worldwide search for CB1
receptor antagonists as anti-obesity drugs. Rimonabant has recently been registered in Europe for
the treatment of obesity. However, its approval in the USA has been suspended. Side effects such
as dizziness, nausea, diarrhea, joint pain, anxiety and depression have been reported during
clinical studies.29 Taranabant is another CB1 antagonist, whose clinical development program for
obesity has been abandoned by the manufacturer due to side effects. Rimonabant has been found
active in many preclinical drug addiction models wherein it suppressed the reinforcing and
12
rewarding properties of cocaine, nicotine, heroin and alcohol.30 CB1 receptor antagonist also
shows good prospects for the treatment of cognitive disorders, based on results from animal
models.31,32 Recently, expression of the CB1 receptor was shown to be induced in human
cirrhotic samples and in liver fibrogenic cells. Rimonabant inhibited the progression of fibrosis
in three models of chronic liver injury.33 Also, CB1 antagonists have been reported34 to inhibit
human breast cancer cell proliferation and exerted a significant action by reducing the volume of
induced tumors in vivo in mice. The treatment of inflammation and arthritis are two new
potential indications for CB1 receptor antagonists as was recently proposed by researchers from
Pfizer.35
4.3 Allosteric Modulators of the CB1 Cannabinoid Receptor and their Therapeutic
Potential: Although allosteric modulators are well established as research tools and therapeutic
modulators of ligand-gated ion channels, they have not been a traditional focus of drug discovery
efforts for GPCRs.36,37 Until recently, the main principle underlying GPCR-based drug discovery
has invariably been the targeting and optimization of small molecules to GPCR orthosteric sites
to obtain selectivity and high efficiency of binding and action. More recently, progress has been
made in recognizing GPCR allosterism, and numerous GPCRs have been shown to possess
allosteric binding sites for endogenous and/or synthetic ligands.37 The binding of an allosteric
modulator induces a conformational change in the receptor which affects the affinity and/or
efficacy of an orthosteric ligand, thereby fine-tuning the pre-existing action of the orthosteric
ligand. In the case of the CB receptors, the endocannabinoids AEA and 2-AG represent the best-
studied, physiological orthosteric ligands. An important feature of an allosteric modulator is that
it exerts an effect only in the presence of orthosteric ligand, as has been demonstrated for several
GPCR allosteric modulators. Benzodiazepines perhaps best exemplify successful therapeutic
13
allosteric modulation as positive allosteric enhancers of gamma-aminobutyric acid (GABA)
binding at the GABA receptor. A more recent example is the use of galantamine, an allosteric
enhancer of nicotinic acetylacholine receptors, for the treatment of cognitive dysfunctions.
Recently, two GPCR allosteric modulators have entered the market, generating further
excitement in this field. The first such drug, cinacalcet [spesipar/Mimpara (Amgen)], a positive
allosteric modulator (PAM) of the calcium sensing receptor (CaSR), is used to treat
hyperparathyroidism.38 The other, maraviroc [Celsentri (Pfizer)], a negative allosteric modulator
(NAM) of chemokine receptor CCR5, was launched for the treatment of HIV disease.39 Several
GPCRs allosteric modulators have been reported, and some associated (pre)clinical data has been
summarized.37 The first novel allosteric modulators of the CB1 receptor (e.g. Org27569, Fig. 2)
were reported in 2005 by Organon.40 These compounds displayed several characteristics
commonly associated with allosteric modulators. However, they exhibited markedly divergent
effects on orthosteric ligand affinity versus efficacy, a characteristic not commonly associated
with allosteric modulators. Thus, they behaved as allosteric enhancers of agonist binding affinity,
but allosteric inhibitors of its signaling efficacy. In general, most GPCR allosteric modulators
have profound inhibitory effects on orthosteric ligand efficacy, but minimal effects on orthosteric
ligand binding.
Figure 2: Structures of NAM and PAMs of CB1 receptor
14
Recently, a strikingly similar pharmacological profile for PSNCBAM-1 has been reported (Fig.
2).41 PSNCBAM-1 increased the equilibrium binding of [3H]CP-55,940, but inhibited CP55,940-
stimulated [35S]GTPS binding. A Shield analysis of these compounds characterized the
functional antagonism of PSNCBAM-1 as noncompetitive, thereby confirming their binding to
an allosteric site of CB1 receptor. The Org27569 and PSNCBAM-1 display a ligand-dependent
effect, whereby they enhance the specific binding of the non-selective CB1/CB2 agonist [3H]
CP-55,940 but inhibit the binding of the inverse agonist [3H]SR141716A. In the absence of
agonist, the Organon compounds did not affect CB1 receptor transmission (as [35S]GTPS
binding in brain membranes and mouse isolated vas deferens, cAMP formation). Thus, the
Organon compounds are neither agonists nor inverse agonists of the CB1 receptor. Similarly,
PSNCBAM-1 was found to be inactive in yeast cells expressing constitutively active human CB1
(hCB1) receptors; however, it behaved as an inverse agonist in the [35S]GTPS binding assay
with significantly lower efficacy than SR141716A.41-43
Very recently, some 3-phenyltropane analogs which also inhibit the dopamine transporter (DAT)
were shown to behave as positive allosteric modulators (functional enhancers) of the hCB1
receptor.44 A representative example, RTI-371, is shown in Fig. 2. RTI-371 and structurally
related tropane analogs RTI-370, JHW007, and GBR12909 increase the intrinsic activity of the
CB1 agonist CP55,940 with variable effects on its potency in stimulating calcium mobilization.44
However, the effect of these modulators on orthosteric agonist affinity was not reported. This
structural diversity among these CB1 allosteric modulators suggests the presence of more than
one allosteric binding site in the CB1 receptor.
Discovery of allosteric CB1-receptor modulators invites the design and profiling of newer
generation, novel small molecules directed at the CB1-receptor allosteric site as potential
15
medications. Initial evidence has emerged indicating that CB1 allosteric inhibitors can suppress
appetite: in rats, PSNCBAM-1 (30 mg/kg, i.p.) elicited a significant reduction in food intake
equivalent to that observed with a 10 mg/kg dose of the CB1-receptor antagonist/inverse agonist
rimonabant.
4.4 LAPS Approach:
The structural characterization of the CB1 receptor’s allosteric site(s) is critical.45 The CB1
receptor is the most abundant CNS GPCR and remains a focus of intensive scrutiny as an
important therapeutic target in drug discovery. However, solving the three-dimensional structure
of GPCRs such as the CB1 receptor constitutes a significant obstacle. Several reasons, including
their substantial molecular weight, intricate interhelical packing, and membrane-associated
topology, have hindered efforts aimed at purification of structurally and functionally intact CB1
receptor.45 In the absence of sufficient quantities of pure CB1 receptor in its native conformation,
classical methods of structural analysis such as X-ray crystallography and nuclear magnetic
resonance spectroscopy cannot be utilized. Alternative methods must therefore be explored to
gain insight into the structural features involved in ligand/drug–CB1 receptor interactions. A
highly successful approach termed “Ligand-Assisted Protein Structure” (LAPS) utilizes probes
or affinity labels capable of binding covalently to a target GPCR at its binding site(s).45-48
Various reactive moieties may be incorporated in different regions of the covalent probe, each of
which is purposely designed to react in a chemically specific manner with a distinct amino acid.
Information obtained from this approach can be validated by the use of site-directed mutagenesis
and extended into ligand-receptor modeling to enable three-dimensional mapping of the GPCR
binding site and characterization of the site’s ligand-binding requirements. Photoaffinity labeling
16
is a remarkably efficient method for studying the interaction of biologically significant
compounds (ligands) with their target macromolecules. The method allows the identification of
the target as well as the binding domain within the target protein.
Therefore, the suitability of the photoactive labeling group for the desired reactivity and the
position of its incorporation into the parent ligand’s structure are important to the successful
development of photoaffinity ligands. Thus, convenient syntheses enabling generation of diverse
probes which can then be screened for their labeling capabilities is an experimentally attractive
approach, especially when the three-dimensional binding site of the target receptor/protein is not
known. We propose here the rational derivatization of Org27569 to yield photoaffinity labels to
be used in a photo cross-linking approach to identify and characterize the allosteric ligand-
binding site(s) of the CB1 receptor.
17
5. Specific Aims:
The specific aims for my MS thesis research work are as follows:
1. Development of novel route for the synthesis of Org27569.
2. Synthesis of two covalent probes [aryl 5-azido (photoactivatable) and 5-isothicyanate
(electrophilic)] of Org27569 template.
3. Synthesis of 5-iodo analog of Org27569.
4. Synthesis of two hybrid analogs of PSNCBAM-1 and Org27569.
18
6. Chemicals and Methods:
All reagents and solvents were purchased from Aldrich and Alfa Aesar, unless otherwise
specified, and used without further purification. All anhydrous reactions were performed under
an argon or nitrogen atmosphere in flame-dried glassware using scrupulously dry solvents. Flash
column chromatography employed silica gel 60 (230-400 mesh) and was performed on Interchim
Puriflash450. All compounds were demonstrated to be homogeneous by analytical TLC on
precoated silica gel TLC plates (Merck, 60 F245 on glass, layer thickness 250 μm), and
chromatograms were visualized by phosphomolybdic acid or anisaldehyde reagent staining.
Melting points were determined on a micromelting point apparatus and are uncorrected. IR
spectra were recorded on a Perkin-Elmer Spectrum One FT-IR spectrometer. NMR spectra were
recorded in CDCl3, CD3OD, DMSO-d6 or in Acetone-d6, on Varian 500 MHz and Varian 400
Mz spectrometers, and chemical shifts are reported in units of δ relative to internal TMS.
Multiplicities are indicated as br (broadened), s (singlet), d (doublet), t (triplet), q (quartet), m
(multiplet), and coupling constants (J) are reported in hertz (Hz). Low resolution mass spectra
were performed in the Department of Chemistry and Chemical Biology at Northeastern
University.
19
7. Ligand Design and Synthesis:
7.1 Rational Design of Photoaffinity Probes:
There are a total of three structural
templates (i.e., Org27569,
PSNCBAM-1 and RTI-371) (Figure 3)
reported in the literature for ligands
that interact with the CB1 cannabinoid
receptor’s allosteric site. Org27569
was superimposed to PSNCBAM-1
using the flexible ligand alignment tool in Maestro (Fig. 3). Org27569 is shown with grey
carbons and PSNCBAM-1 with cyan carbons. A mesh representation of the surface area for both
compounds is also shown. Org27569 and PSNCBAM-1 have some commons features: a) Both
adopt a U-shape and their pharmacophores show good overlap; b) both are positive allosteric
modulators of CP-55,940 binding and negative allosteric modulator of its functional potency
([35S] GTPS binding) and (c) other biochemical characterizations also appear similar. These
compounds are not yet reported to interact with any other target. Whereas RTI-371, shows
distinct structural features compared to Org and PSN compounds and also exhibits positive
allosteric modulation of CP-55,940’s functional potency. Additionally, RTI-371 is also a potent
inhibitor of dopamine transporter (DAT). These results suggest that the binding domain of RTI-
371 may not be identical to those of the Org and PSN compounds. For these reasons we have
focused on PSNCBAM-1 and Org27569 templates. Overlap of both Org27569 and PSNCBAM-1
shows (Figure 3) that both adopt a U-type molecular shape and arguably may target the same
binding site. The “head group” (5-chloroindole) of Org27569 and the amide group nearly
Figure 3: Overlap of Org27569 and PSNCBAM-1
20
overlap, respectively, with the “head group” (4-chlorophenyl) and urea moiety of PSNCBAM-1.
We have designed two covalent probes of Org27569 that incorporate reactive moiety at the C-5
position to map allosteric site of CB1 using LAPS. Two photoaffinity-labeling moieties chosen
are: 1) aryl azides (Target 1); 2) aryl isothiocyanate (Target 2). The aryl azide group is selected
as photoactivatable group, whereas, the isothiocyanate group is selected as an electrophilic
reactive group for incorporation into this molecule. The proposed covalent probes derived from
Org27569 are shown in (Fig. 4).
The rationale behind using
different functional groups is
multifold: 1) They vary in
their steric and electronic
contributions for tolerance at
the receptor site. 2) They also vary in their reactivity and specificity towards different functional
groups. Since no structure-activity relationship data have been published on any of these
allosteric ligands, the present design attempts to incorporate photoreactive group(s) and
electrophilic group at the overlapping and important areas of the molecule. As the synthetic route
for Org27569 is not yet published, we have first
developed a novel route for its synthesis and using
the same synthetic strategy we have synthesized
Target-1 and Target-2.
7.2 Iodinated Analog of Org27569:
In order to develop SAR of Org27569 and facilitate characterization of the allosteric site through
competitive radiometric assays, it is important to have radio-labeled Org27569. As of today, no
Figure 4: Novel covalent probes for CB1 allosteric site.
Figure 5: Iodinated analog of Org27569
21
SAR studies around both allosteric modulators have been updated. As the original molecule has
a chloro functionality, we hypothesized that replacing it with iodo group (Target 3, Fig. 5),
which is slightly bulkier than chloro group, will not markedly affect its binding affinity and
potency at the allosteric site. The iodo analog can be easily converted to the stannylated analog
by treatment with hexabutylditin and then can be iododestannylation with Na125I. The synthesis
of iodo analog has been successfully executed.
7.3 Hybrid Analogs of Org27569 and PSNCBAM-1:
As shown in our computational studies, both Org27569 and PSNCBAM-1 show good overlap
and may be interacting with the same allosteric site of CB1 receptor (Fig. 3). We therefore
designed two
hybrid analogs of
Org27569 and
PSNCBAM-1, as
shown in Fig. 6.
We hypothesized that availability of these hybrid analogs and their biochemical evaluation data
will provide information about the preferred motif for the CB1 allosteric site and guide future
SAR studies.
Figure 6: Structures of two proposed hybrid analogs.
22
8. Retrosynthetic Strategy for Org27569 and Chemistry:
The retrosynthesis of Org27569 was planned as shown below. The molecule can be divided into
two major fragments: a) 5-chloro indole acid, and b) 4-piperidinyl phenethyl amine. The indole
moiety was planned to be constructed from alkyl pentanoate by Japp-Klingeman method. The
amine part was planned to be obtained starting from 4-bromobenzaldehyde by Henry’s reaction
followed by reduction.
The amine part of Org27569 was synthesized as shown in the Scheme-1. The 4-
(piperidinyl)benzaldehyde (2) was prepared in 78% yield by treating commercially available 4-
bromobenzaldehyde with piperidine under basic conditions.49 Nitrostyrene 3 was obtained by in
68% yield by refluxing a mixture of aldehyde 2 and nitromethane in the presence of ammonium
acetate (Henry’s reaction).50 We then tried various approaches to transform 3 to the desired
amine 5 (refluxing in LiAlH4 or reduction using Pd/C and Raney Nickel), but none yielded 5 in
23
satisfactory yield and purity. Alternatively, we first selectively reduced the double bond with
sodium borohydride in 85% yield.50 Reduction of the nitro group to an amino was accomplished
by in situ generated nickel borohydride in 81% yield.51 Interestingly, the direct exposure of
nitrostyrene 3 to nickel borohydride yielded a mixture of partially reduced products.
Scheme 1: Synthesis of amine part of Org27569
The synthesis of indole part of Org27569 was carried out as shown in Scheme-2. The indole 13
is a new chemical entity, and its synthesis has not been reported in the literature. Alkylation of
ethyl acetoacetate (6) with 1-iodopropane in presence of sodium ethoxide yielded 8 in 78% yield
together with dialkylated product (16%).52 This mixture proved difficult to separate by fractional
distillation and was effectively separated by flash column chromatography. Treatment of
diazonium salt of 4-chloroaniline 10 with sodium acetate-treated ethyl 2-acetylpenanoate
yielded a mixture of azo compound 11 and hydrozone 12 in 4:1 ratio in 70% yield.53 Careful
monitoring of the reaction revealed that 11 was initially formed almost exclusively and that it
slowly converted to 12, depending upon reaction time and temperature. Fischer cyclization of the
mixture of azo and hydrazone was accomplished in 50% yield by treatment with 20% H2SO4 in
ethanol.54
24
Scheme 2: Synthesis of 5-Chloro Indole Ester
The synthesis of Org27569 was carried out as shown in Scheme-3. Direct amidation of indole
ester 13 with amine 5 under NaCN-catalyzed conditions was unsuccessful. Alternatively,
alkaline hydrolysis of ester 13 gave acid 14 in nearly quantitative yields, which, upon coupling
with amine 5, yielded desired Org27569 in 70% yield.
The azido analog 24 was synthesized as shown in Scheme 4. Treatment of diazonium salt of 4-
nitroaniline with sodium acetate treated ethyl 2-acetylpentanoate gave a mixture of azo (18) and
hydrazone (19) in 80% yields. Fischer cyclization of this mixture in 20% H2SO4 in ethanol
yielded nitroindole ester 20 in 57% yield. Base hydrolysis gave acid 21 in 72% yield. Coupling
Scheme 3: Synthesis of Org27569
25
of acid 21 with amine 5 gave 5-nitro analog of Org27569 (22) in 92% yield. Reduction of 22
under Pd/C conditions at atmospheric pressure of hydrogen was slow an incomplete even after
24 h. Alternatively, in situ generated nickel borohydride efficiently and quickly reduced aromatic
nitro group to amino in high yield (86%). With this procedure, compound 23 was prepared on
gram quantities. Exposure of aromatic amine 23 to a mixture of tert-butyl nitrite and
azidotrimethylsilane yielded aromatic azide 24 (Target 1) in 41% yield. We synthesized aryl
isothiocyanate analog (Target 2) in 53% yield by treatment of 24 with triphenylphosphine
followed by exposure to CS2 (Staudinger/Aza-Wittig reaction).
Scheme 4: Synthesis of the nitro/amino/azido/isothiocyanate analogs of Org 27569(Target 1&2)
26
The synthesis of iodinated analog (Target 3) is shown in Scheme 5. Direct diazotization of 23
using NaNO2 followed by exposure to KI yielded the desired 31 only in 7% yield. Alternatively,
we synthesized azo isomer 28 from 4-iodoaniline in 72% yield. It was then cyclized to indole 29
in 20% ethanolic sulfuric acid (68% yield). Hydrolysis of indole ester 29 yielded acid 30 (87%
yield). Coupling of acid 30 with amine 5 yielded desired iodo analog (Target 3) in 70% yield.
I
NH2
I
N2+
Cl-
I
NN
O
OO
I
NH O
O
NaNO2, HClO - 5ºC, 1h OºC, 6h
72%
20% EtOH / H2SO4
reflux, 12h68%
26 27
29
28
8, EtOHNaOAc,
I
NH
OH
O
dioxane,KOH,
I
NH O
HNN
5, EDCl, HOBT,NMP, DIPEA,
30
31
R.T, 14 h70%
reflux, 3h87.5%
Scheme 5: Synthesis of aryl iodo analog of Org 27569 (Target 3)
Our proposed hybrid analog 33 (Target 4) was synthesized as shown in Scheme 6. Coupling of
amine 5 with commercially available 4-chlorophenyl isocyanate 32 yielded in 70% yield.
Scheme 6: Synthesis of hybrid analog 33 (Target 4)
27
The synthesis of hybrid analog 39 (Target 5) was accomplished as shown in Scheme 7.
Intermediate 38 was synthesized by reported patent procedure.55 Thus, the treatment of
pyrrolidine with 2,6-dibromopyridine gave mono substituted pyridine 36 in 95% yield.
Microwave accelerated Suzuki-coupling of 36 with 2-nitrophenyl boronic acid gave tricyclic
intermediate 37 (76% yield) which was reduced under hydrogenation conditions to give amine
38 in 82% yield. Coupling of amine 38 with acid 14 under EDCI conditions yielded the desired
amide 39 in 68% yield.
Scheme 7: Synthesis of hybrid analog 39 (Target 5):
28
9. Experimental:
4-(Piperidin-1-yl)benzaldehyde (2)
To a solution of 4-bromobenzaldehyde 1 (20 g, 108.1 mmol), piperidine (14 g, 165 mmol) in dry
NMP (40 mL) was added anhydrous K2CO3 (44 g) and the resulting solution was stirred at
135°C under an argon atmosphere for 48 h. Reaction was quenched by addition of water (50 mL)
and diluted with EtOAc (200 mL). Organic layer was separated and aqueous layer extracted with
EtOAc (4 x 100 mL). Combined organic layer was washed with water, brine and dried (MgSO4)
and solvent evaporated under reduced pressure. Purification of the crude by flash column
chromatography (0%15% EtOAc: Hexane) gave 2 as a pale yellow solid (16.27 g, 78.2%
yield). M.p. = 64-66°C; Rf = 0.5 (EtOAc/Hexane = 20/80). 1H NMR (500 MHz, CDCl3): δ 9.75
(s, 1H), 7.72 (d, J = 9.5 Hz, 2H), 6.89 (d, J = 9.0 Hz, 2H), 3.40 (m as br s, 4H), 1.67 (br s, 6H).
Mass spectrum m/z – 190.12 [M+H]+
(E)-1-[4-(2-Nitrovinyl)phenyl]piperidine (3)
To a stirred solution of 2 (4 g, 211 mmol.) in 20 mL anhydrous nitromethane was added
ammonium acetate (8 g, 140 mmol.) and the resulting mixture was refluxed under an argon
atmosphere for 2 h. Solvent was removed under reduced pressure and reaction mixture was
diluted with ethyl acetate and water. Organic layer was separated and aqueous layer extracted
with EtOAc (6 x 30 mL). Combined organic layer was washed with water, brine and dried
(MgSO4). Evaporation of volatiles under reduced pressure gave crude which was purified by
flash column chromatography (5%20% EtOAc: Hexane) to give 3 as a dark orange crystalline
29
solid (2.9 g, 60% yield). M.p. = 110 - 112 °C; Rf = 0.48 (EtOAc/Hexane = 20/80). 1H NMR (500
Mz, CDCl3): δ 7.95 (d, J = 13.5 Hz, 1H), 7.50 (d, J = 13.5 Hz, 1H), 7.42 (d, J = 9.0 Hz, 2H),
6.86 (d, J = 9.0 Hz, 2H), 3.40-3.36 (m, 4H), 1.67 (m as br s, 6H). Mass spectrum m/z – 233.12
[M+H]+
1-[4-(2-Nitroethyl)phenyl]piperidine (4)
To a solution of 3 (5 g, 21.5 mmol) in dry nitromethane (100 mL) and anhydrous methanol (50
mL) at room temperature under an argon atmosphere was added NaBH4 (2.5 g, 65 mmol) portion
wise. The reaction mixture was stirred at same temperature for 2h and then quenched by the
addition of saturated solution of ammonium chloride. The reaction mixture was concentrated
under reduced pressure and the crude was dissolved in EtOAc and water. Organic layer was
separated and aqueous layer extracted with EtOAc (4 x 25 mL). Combined organic layer was
washed with brine and dried (MgSO4). Evaporation of volatiles under reduced pressure gave
crude which was purified by flash column chromatography (10%40% EtOAc: Hexane) to give
pure 4 as a pale yellow oil (4.27 g, 85% yield). Rf = 0.42 (EtOAc/Hexane = 20/80). 1H NMR
(400 MHz, CDCl3): δ 7.07 (d, J = 8.8 Hz, 2H), 6.88 (d, J = 8.8 Hz, 2H), 4.55 (t, J = 7.2 Hz, 2H),
3.23 (t, J = 7.2 Hz, 2H), 3.16-3.08 (m, 4H), 1.74-1.65 (m, 4H), 1.62-1.52 (m, 2H). ). Mass
spectrum m/z – 234.14 [M+H]+
30
2-(4-(Piperidin-1-yl)phenyl)ethanamine (5)
To a solution of 4 (1 g, 4.2 mmol) in anhydrous THF (25 mL) and methanol (2 mL) under an
argon atmosphere at room temperature was added NiCl.6H2O (1.96 g, 8.2 mmol) and reaction
mixture was stirred for 45 min at room temperature. It was then cooled to -5ºC and NaBH4 (0.9
g, 25.3 mmol) was added in small portions to result in a black solution, the reaction was then
gradually warmed to room temperature and stirred for 1 h. Reaction was quenched by addition of
saturated aqueous solution of ammonium chloride and concentrated under reduced pressure. The
residue was diluted with EtOAc and water and filtered. The organic layer was separated and
aqueous layer was extracted with EtOAc (6 x 40 mL). Combined organic layer was washed with
brine and dried (MgSO4) and evaporated under vacuum to yield crude (695 mg, 81% yield)
which was taken for the next reaction without further purification. Rf = 0.81 (MeOH/DCM =
20/80). 1H NMR (500 MHz, CDCl3): δ 7.08 (d, J = 8.0 Hz, 1H), 6.89 (d, J = 8.0 Hz, 1H), 3.17-
3.08 (m, 4H), 2.92 (t, J = 7.0 Hz, 2H), 2.66 (t, J = 7.0 Hz, 2H), 1.76-1.66 (m, 4H), 1.61-1.52 (m,
2H), 1.31 (br s, 2H). Mass spectrum m/z – 205.16 [M+H]+
Ethyl-2-acetyl pentanoate (8)
To a flask containing anhydrous ethanol (200 mL) under an argon atmosphere at room
temperature was added sodium metal (6 g, 260 mmol) portion wise and was stirred till it
completely dissolves (30 min). To this was added ethyl acetoacetate (6) (30 g, 230 mmol) and
the resulting solution was refluxed for 30 min. This was followed by addition of Propyl iodide
(7) (44.44 g, 241.5 mmol), over a period of 30 min through dropping funnel and the reaction was
refluxed for 20 h. The reaction mixture was cooled to room temperature and filtered. The filtrate
31
was neutralized by adding 1N HCl and concentrated under reduced pressure and partitioned in
EtOAc and water. The organic layer separated and aqueous layer extracted with EtOAc (2 x 200
mL). Combined organic layer was washed with brined and dried (Na2SO4). The product was
purified by flash column chromatography (5%20% EtOAc: Hexane) to give 8 as a clear liquid
(30.87 g: 78% yield). Rf = 0.45 (EtOAc/Hexane = 20/80).1H NMR (400 MHz, CDCl3): δ 4.20 (q,
J = 7.2 Hz, 2H), 3.42 (t, J = 7.4 Hz, 1H), 2.22 (s, 3H), 1.92-1.76 (m, 2H), 1.40-1.20 (m, 5H,
especially 1.28, t, J = 7.2 Hz, 3H), 0.93(t, J = 7.2 Hz, 3H). Mass spectrum m/z – 172.10 [M+H]+
4-Chlorobenzenediazonium chloride (10)
A solution of sodium nitrite (1.7 g, 23 mmol) in water (5 mL) cooled to 0ºC was added to a
suspension of finely powdered 4-chloro aniline (9) (2.54 g, 20 mmol) in 10 mL of 24% aq.
hydrochloric acid at 0ºC and the resulting solution was stirred for 45 min keeping the
temperature between 0º- 5ºC. The resuling pale yellow solution of diazonium salt was directly
used for the next reaction.
(E)-Ethyl 2-[(4-chlorophenyl)diazenyl]pentanoate (11) and (E)-ethyl 2-acetyl-2-((4-
chlorophenyl)diazenyl)pentanoate (12)
To a solution of 8 (0.5 g, 2.9 mmol) in 30 mL ethanol under an argon atmosphere at room
temperature was added sodium acetate trihydrate (0.83 g, 6.12 mmol) and the resulting mixture
was stirred at same temperature for 45 min. It was then cooled to -5ºC and the above diazonium
salt (10) was added to this together with additional sodium acetate to maintain the pH at 5 and
32
the resulting solution was stirred for 3 h keeping the temperature between 0ºC to 5ºC. The
reaction was quenched by adding a saturated aqueous NaHCO3 solution. The volatiles were
removed under reduced pressure and the mixture was extracted with EtOAc (4 x 40 mL). The
organic layer was washed with water, brine and dried over sodium sulfate. The solvent was
removed over vacuum to give crude as red oil (0.55 g, 70.6% yield) as a 4:1 mixture of 11 and
12. The crude was purified by flash column chromatography (5%30% EtOAc: Hexane) to give
azo compound 11 (0.44 g) as a yellow solid which was first eluted followed by 12 (0.11 g) which
was obtained as a brown solid. Compound 11: Rf = 0.78 (EtOAc/Hexane = 20/80) 1H NMR
(500 MHz, CDCl3): δ 7.71, (d, J = 9.0 Hz, 2H), 7.47 (d, J = 9.0 Hz, 2H), 4.31-4.20 (m, 2H), 2.30
(s, 3H), 2.21-2.06 (m, 2H), 1.48-1.28 (m, 2H), 1.26 (t, J = 7.0 Hz, 3H), 0.91 (t, J = 7.5 Hz, 3H).
mass spectrum m/z – 311.11 [M+H]+. Compound 12: Rf = 0.6 (EtOAc/Hexane = 20/80) 1H
NMR (500 MHz, CDCl3): δ 7.93 (s, 1H, NH), 7.29 (d, J = 9.0 Hz, 2H), 7.18 (d, J = 9.0 Hz, 2H),
4.36 (q, J = 7.0 Hz, 2H), 2.6 (t, J = 8.0 Hz, 2H), 1.68-1.58 (m, 2H), 1.42 (t, J = 7.0 Hz, 3H), 1.06
(t, J = 8.0 Hz, 3H). Mass spectrum m/z – 269.11 [M+H]+
Ethyl 5-chloro-3-ethyl-1H-indole-2-carboxylate (13)
A mixture of 11 (0.4 g, 1.28 mmol) and 12 (0.1 g, 0.37 mmol) was taken in 20% ethanolic
(anhydrous) H2SO4 (30 mL) and the resulting solution was refluxed for 4h under an argon
atmosphere. The reaction mixture was cooled to room temperature and neutralized by adding
saturated NaHCO3 solution and extracted with EtOAc (4 x 50 mL). Combined organic layer was
washed with water, brine and dried (MgSO4). The volatiles were removed under vacuum to yield
crude which was purified by flash column chromatography on silica gel (0%15% EtOAc:
33
Hexane) to give pure 13 as a white crystalline solid (250 mg, 50% yield). Rf = 0.35
(EtOAc/Hexane = 20/80). 1H NMR (400 MHz, CDCl3): δ 8.74 (br s, 1H, NH), 7.65 (d, J = 1.2
Hz, 1H), 7.30 (d, J = 8.8 Hz, 2H), 7.25 (dd, J = 8.8 Hz, J = 1.2 Hz, 1H), 4.42 (q, J = 7.2 Hz, 2H),
3.07 (q, J = 7.2 Hz, 2H), 1.43 (t, J = 7.2 Hz, 3H), 1.26 (t, J = 7.2 Hz, 3H). Mass spectrum m/z –
251.07 [M+H]+
5-Chloro-3-ethyl-1H-indole-2-carboxylic acid (14)
To a solution of 13 (330 mg, 1.31 mmol) in dioxane (30 mL) was added a solution of KOH (440
mg, 7.7 mol) in water (5 mL) and the resulting solution was refluxed for 2 h. It was then cooled
to room temperature, concentrated under reduced pressure and neutralized by addition of 1N
HCl. The precipitated acid was filtered, washed with cold water and air dried to give pure acid
14 (306 mg, 98% yield) as white solid. 1H NMR (400 MHz, DMSO-d6): δ 11.65 (br s, 1H,
COOH), 7.72 (s, 1H), 7.41 (d, J = 8.8 Hz, 1H), 7.18 (d, J = 8.8 Hz, 1H), 3.35 (br s, 1H, NH),
3.02 (q, J = 7.0 Hz, 2H), 1.18 (t, J = 7.0 Hz, 3H). Mass spectrum m/z – 224.04 [M+H]+
5-Chloro-3-ethyl-N-[4-(piperidin-1-yl) phenethyl]-1H-indole-2-carboxamide (15)
To a solution of acid 14 (25 mg, 0.0996 mmol), amine 5 (50 mg, 0.2447 mmol), HOBT (14 mg,
0.1083 mmol), DIPEA (50 mg, 0.3868 mmol) in anhydrous NMP (5 mL) was added EDCI (40
mg, 0.257mmol) under an argon atmosphere and at room temperature and the resulting mixture
was stirred overnight. Reaction mixture was diluted with ethyl acetate (25 mL) and water (10
mL). The organic layer was separated and aqueous layer extracted with EtOAc (3 x 20 mL).
34
Combined organic layer was washed with water, brine and dried (Na2SO4). Evaporation of
volatiles under reduced pressure gave crude which was purified by flash column chromatography
on silica gel (10%40% EtOAc: Hexane) to give 15 as a white crystalline solid (17.5 mg 70%
yield). Rf = 0.7 (EtOAc/Hexane = 50/50). 1H NMR (500 MHz, CDCl3): 9.10 (br s, 1H, NH
indole), 7.54 (d, J = 2.0 Hz, 1H), 7.29 (d, J = 8.5 Hz, 1H),7.20 (dd, J = 8.5 Hz, J = 2.0 Hz, 1H),
7.14 (d, J = 8.5 Hz, 2H), 6.92 (d, J = 8.5 Hz, 2H), 5.97 (br s, 1H, NH amide), 3.78 (q, J = 6.5 Hz,
2H), 3.13 (t, J = 5.5 Hz, 4H), 2.88 (t, J = 7.0 Hz, 2H), 2.69 (q, J = 7.5 Hz, 2H), 1.76-1.66 (m,
4H), 1.62-1.54 (m, 2H), 1.08 (t, J = 7.5 Hz, 3H, -CH3). Mass spectrum m/z – 410.18 [M+H]+
4-Nitrobenzenediazonium chloride (17)
A solution of sodium nitrite (0.7 g, 9.5 mmol) in water (5 mL) cooled to 0ºC was added to a
suspension of finely powdered 4-nitro aniline (16) (1 g, 7.25 mmol) in 10 mL of 24% aq.
hydrochloric acid at 0ºC and the resulting solution was stirred for 45 min keeping the
temperature between 0º- 5ºC. The resuling pale yellow solution of diazonium salt (17) was
directly used for the next reaction.
(E)-ethyl 2-[2-(4-nitrophenyl)hydrazono]pentanoate (18) and (E)-ethyl 2-acetyl-2-[(4-
nitrophenyl)diazenyl] pentanoate (19)
To a solution of 8 (1.3 g, 5.4 mmol) in 30 mL ethanol under an argon atmosphere at room
temperature was added sodium acetate trihydrate (6.5 g) and the resulting mixture was stirred at
same temperature for 45 min. It was then cooled to -5ºC and the above diazonium salt (17) was
35
added to this together with additional sodium acetate to maintain the pH at 5 and the resulting
solution was stirred for 3 h keeping the temperature between 0ºC to 5ºC. The reaction was
quenched by adding a saturated aqueous NaHCO3 solution. The volatiles were removed under
reduced pressure and the mixture was extracted with EtOAc (4 x 40 mL). The organic layer was
washed with water, brine and dried over sodium sulfate. The solvent was removed over vacuum
to give crude as red oil (1.39 g, 80.6% yield) as a 4:1 mixture of 18 and 19. The crude was
purified by flash column chromatography (5%30% EtOAc: Hexane) to give azo compound 18
(1.12 g) as a yellow solid which was first eluted followed by 19 (0.27 mg) which was obtained as
a brown solid. Compound 18: Rf = 0.46 (EtOAc/Hexane = 20/80). 1H NMR (500 MHz, CDCl3):
δ 8.37 (d, J = 9.0 Hz, 2H), 7.87 (d, J = 9.0 Hz, 2H), 4.35-4.24 (m, 2H), 2.33 (s, 3H), 2.26-2.12
(m, 2H), 1.52-1.35 (m, 2H), 1.28 (t, J = 7.0 Hz, 3H), 0.93 (t, J = 7.5 Hz, 3H). Mass spectrum m/z
– 308.12 [M+H]+. Compound 19: Rf = 0.23 (EtOAC/Hexane = 20/80). 1H NMR (500 MHz,
CDCl3): δ 8.22 (d, J = 9.0 Hz, 2H), 8.08 (s, 1H, NH), 7.25 (d, J = 9.0 Hz, 2H), 4.34 (q, J = 7.0
Hz, 2H), 2.60 (t, J = 8.0 Hz, 2H), 1.67-1.58 (m, 2H), 1.40 (t, J = 7.0 Hz, 3H), 1.05 (t, J = 7.5 Hz,
3H). Mass spectrum m/z – 280.12 [M+H]+
Ethyl 3 ethyl-5-nitro-1H-indole-2-carboxylate (20)
A mixture of 18 (0.8 g, 2.48 mmol) and 19 (0.2 g, 0.716 mmol) was taken in 20% ethanolic
(anhydrous) H2SO4 (30 mL) and the resulting solution was refluxed overnight under an argon
atmosphere. The reaction mixture was cooled to room temperature and neutralized by adding
saturated NaHCO3 solution and extracted with EtOAc (4 x 50 mL). Combined organic layer was
washed with water, brine and dried (Na2SO4). The volatiles were removed under vacuum to yield
36
crude which was purified by flash column chromatography on silica gel (0%15% EtOAc:
Hexane) to give pure 20 as a white crystalline solid (370.5 mg, 57% yield). Rf = 0.29
(EtOAc/Hexane = 20/80). 1H NMR (400 MHz, CDCl3): δ 9.02 (s, 1H, NH), 8.69 (d, J = 2.0 Hz,
1H), 8.22 (dd, J = 8.8 Hz, J = 2.4 Hz, 1H), 7.43 (d, J = 9.6 Hz, 1H), 4.46 (q, J = 7.6 Hz, 2H),
3.16 (q, J = 7.2 Hz, 2H), 1.45 (t, J = 7.6 Hz, 3H), 1.31 (t, J = 7.2 Hz, 3H). Mass spectrum m/z –
263.11 [M+H]+
Ethyl 3-ethyl-5-nitro-1H-indole-2-carboxylic acid (21)
To a solution of 20 (170 mg, 0.648 mmol) in dioxane (10 mL) was added a solution of KOH
(200 mg, 3.54 mmol.) in water (3 mL) and the resulting solution was refluxed for 2 h. It was then
cooled to room temperature, concentrated under reduced pressure and neutralized by addition of
1N HCl. The precipitated acid was filtered, washed with cold water and air dried to give pure
acid 21 (104 mg, 72% yield) as white solid.1H NMR (400 MHz, DMSO): δ 12.14 (s, 1H), 8.67
(d, J = 2.0 Hz, 1H), 8.11 (dd, J = 8.8 Hz, J = 2.5 Hz, 1H), 7.54 (d, J = 8.8 Hz, 1H), 3.12 (q, J =
8.0 Hz, 2H), 1.22 (t, J = 7.6 Hz, 3H). Mass spectrum m/z – 235.06 [M+H]+
3-Ethyl-5-nitro-N-[4-(piperidin-1-yl) phenethyl]-1H-indole-2-carboxamide (22)
To a solution of acid 21 (40 mg, 0.172 mmol), amine 5 (60 mg, 0.29 mmol), HOBT (50 mg, 0.37
mmol), DIPEA (100 mg, 0.775 mmol) in anhydrous NMP (5 mL) was added EDCI (100 mg,
0.645 mmol) under an argon atmosphere and at room temperature and the resulting mixture was
stirred overnight. Reaction mixture was diluted with ethyl acetate (25 mL) and water (10 mL).
37
The organic layer was separated and aqueous layer extracted with EtOAc (3 x 20 mL).
Combined organic layer was washed with water, brine and dried (Na2SO4). Evaporation of
volatiles under reduced pressure gave crude which was purified by flash column chromatography
on silica gel (10%40% EtOAc: Hexane) to give 22 as a white crystalline solid (66.4 mg, 92%
yield.) M.p. = 208– 211ºC. Rf = 0.8 (MeOH/DCM = 20/80. 1H NMR (500 MHz, CDCl3): δ 9.97
(s, 1H, indole NH), 8.59 (d, J = 2.0 Hz, 1H), 8.16 (dd, J = 9.5 Hz, J = 2.0 Hz, 1H), 7.44 (d, J =
9.5 Hz, 1H), 7.15 (d, J = 8.5 Hz, 2H), 6.93 (d, J = 8.5 Hz, 2H), 6.12 (br t, J = 6.0 Hz, 1H, NH of
amide), 3.82 (q, J = 6.0 Hz, 2H), 3.14 (t, J = 5.5 Hz, 4H), 2.91 (t, J = 7.0 Hz, 2H), 2.79 (q, J =
7.5 Hz, 2H), 1.76-1.68 (m, 4H), 1.62-1.54 (m, 2H), 1.13 (t, J = 7.5 Hz, 3H). Mass spectrum m/z –
421.22 [M+H]+
5-Amino-3-ethyl-N-[4-(piperidin-1-yl)phenethyl]-1H-indole-2-carboxamide (23)
To a solution of 22 (75 mg, 0.178 mmol) in anhydrous THF (25 mL) and methanol (2 mL) under
an argon atmosphere at room temperature was added NiCl.6H2O (200 mg, 0.841 mmol) and
reaction mixture was stirred for 45 min at room temperature. It was then cooled to -5ºC and
NaBH4 (100 mg, 2.64 mmol) was added in small portions to result in a black solution, the
reaction was then gradually warmed to room temperature and stirred for 1 h. Reaction was
quenched by addition of saturated aqueous solution of ammonium chloride and concentrated
under reduced pressure. The residue was diluted with EtOAc and water and filtered. The organic
layer was separated and aqueous layer was extracted with EtOAc (6 x 40 mL). Combined
organic layer was washed with brine and dried (MgSO4) and evaporated under vacuum to yield
crude (60 mg, 86% yield) which was taken for the next reaction without further purification.
38
M.p. = 208 - 209 ºC Rf = 0.8 (MeOH/DCM = 20/80). 1H NMR (500 MHz, DMSO-d6): δ 10.60
(s, 1H, NH of indole), 7.74 (t, J = 5.5 Hz, 1H), 7.08 (d, J = 9.0 Hz, 3H, two overlapping
doublets), 6.85 (d, J = 9.0 Hz, 2H), 6.69 (d, J = 2.0 Hz, 1H), 6.62 (dd, J = 9.0 Hz, J = 2.0 Hz,
1H), 4.56 (br s, 2H, NH2), 3.44 (q, J = 6.5 Hz, 2H), 3.06 (t, J = 5.5 Hz, 4H), 2.90 (q, J = 7.5 Hz,
2H), 2.74 (t, J = 7.5 Hz, 2H), 1.64-1.56 (m, 4H), 1.54-1.46 (m, 2H), 1.11 (t, J = 7.5 Hz, 3H).
Mass spectrum m/z – 390.24 [M+H]+
5-Azido-3-ethyl-N-[4-(piperidin-1-yl)phenethyl]-1H-indole-2-carboxamide (24)
To a solution of 23 (100 mg, 0.256 mmol) in THF (20 mL) was added tertiary butyl nitrite (1.6 g,
15.5 mmol) and tetramethylsilyl azide (1.2 g, 10.42 mmol), and the reaction was stirred
overnight at 0ºC under argon atmosphere. The solvent evaporated under reduced pressure at
room temperature to give crude which was purified using flash column chromatography on silica
gel to yield pure 24 as a white solid (44 mg, 41.26% yield). Mp = 170 – 173ºC. Rf = 0.81
(MeOH/DCM = 20/80) 1H NMR (500 MHz, CDCl3): δ 9.14 (s, 1H, NH of indole), 7.35 (d, J =
9.0 Hz, 1H), 7.20 (d, J = 2.0 Hz, 1H), 7.14 (d, J = 9.0 Hz, 2H), 6.95 (dd, J = 9.0 Hz, J = 2.0 Hz,
1H), 6.92 (d, J = 9.0 Hz, 2H), 5.98 (br t, J = 6.0 Hz, 1H, NH of amide), 3.78 (q, J = 6.5 Hz, 2H),
3.13 (t, J = 5.5 Hz, 4H), 2.89 (t, J = 6.5 Hz, 2H), 2.70 (q, J = 8.0 Hz, 2H), 1.75-1.68 (m, 4H),
1.64-1.54 (m, 2H), 1.08 (t, J = 8.0 Hz, 3H). Mass spectrum m/z – 417.23 [M+H]+
39
3-Ethyl-5-isothiocyanato-N-[4-(piperidin-1-yl)phenethyl]-1H-indole-2-carboxamide (25)
To a solution of 24 (27 mg, 0.065 mmol) in benzene (5 mL) was added triphenyl phosphine
(17.04 mg, 0.065 mmol) under an argon atmosphere and the reaction mixture was refluxed for 4h
with constant stirring. CS2 (1 mL) was added to this and the reaction mix was refluxed for 12 h.
CS2 and benzene were evaporated under reduced pressure to obtain crude 25 which was purified
using flash column chromatography to obtain pure 25 as a white solid (15 mg, 53.5%). Rf = 0.78
(MeOH/DCM = 20/80). 1H NMR (400 MHz, CDCl3): δ 9.79 (s, 1H), 7.46 (s, 1H), 7.35 (d, J =
8.8 Hz, 1H), 7.17-7.09 (m, 3H, especially 7.14, d, J = 8.0 Hz, 2H), 6.92 (d, J = 8.0 Hz, 2H), 6.03
(br t, J = 6.4 Hz, 1H, NH of amide), 3.80 (q, J = 6.0 Hz, 2H), 3.13 (br t, J = 5.6 Hz, 4H), 2.90 (t,
J = 6.4 Hz, 2H), 2.70 (q, J = 7.6 Hz, 2H), 1.76-1.67 (m, 4H), 1.62-1.54 (m, 2H), 1.08 (t, J = 7.6
Hz, 3H). Mass spectrum m/z – 433.21 [M+H]+
4-Iodobenzene diazonium chloride (27)
A solution of sodium nitrite (0.75 g, 10.79 mmol) in water (5 mL) cooled to 0 ºC was added to a
suspension of finely powdered 4-iodo aniline (26) (1 g, 4.56 mmol) in 10 mL of 24% aq.
hydrochloric acid at 0ºC and the resulting solution was stirred for 45 min keeping the
temperature between 0º- 5ºC. The resuling pale yellow solution of diazonium salt (27) was
directly used for the next reaction.
40
(E)-Ethyl 2-acetyl-2-[(4-iodophenyl)diazenyl]pentanoate (28)
To a solution of 8 (960 mg, 2.386 mmol) in 40 mL ethanol under an argon atmosphere at room
temperature was added sodium acetate trihydrate (2 g, 14.69 mmol) and the resulting mixture
was stirred at same temperature for 4h. It was then cooled to -5ºC and the above diazonium salt
(27) was added to this together with additional sodium acetate to maintain the pH at 5 and the
resulting solution was stirred for 3 h keeping the temperature between 0ºC to 5ºC. The reaction
was quenched by adding a saturated aqueous NaHCO3 solution. The volatiles were removed
under reduced pressure and the mixture was extracted with EtOAc (4 x 40 mL). The organic
layer was washed with water, brine and dried over sodium sulfate. The solvent was removed
over vacuum to give crude as red oil (690 mg, 72% yield). Rf = 0.6 (EtOAc/Hexane = 20/80). 1H
NMR (500 MHz, CDCl3): δ 7.85 (d, J = 8.5 Hz, 2H), 7.48 (d, J = 8.5 Hz, 2H), 4.32-4.21 (m,
2H), 2.30 (s, 3H), 2.22 – 2.07 (m, 2H), 1.50 – 1.32 (m, 2H) 1.26 (t, J = 7.0 Hz, 3H), 0.91 (t, J =
7.5 Hz, 3H). Mass spectrum m/z – 403.04 [M+H]+
Ethyl 5-iodo-3-ethyl-1H-indole-2-carboxylate (29)
A solution of 28 (997 mg, 2.48 mmol) was taken in 20% ethanolic (anhydrous) H2SO4 (30 mL)
and the resulting solution was refluxed overnight under an argon atmosphere. The reaction
mixture was cooled to room temperature and neutralized by adding saturated NaHCO3 solution
and extracted with EtOAc (4 x 50 mL). Combined organic layer was washed with water, brine
and dried (Na2SO4). The volatiles were removed under vacuum to yield crude which was
purified by flash column chromatography on silica gel (10%40% EtOAc: Hexane) to give pure
20 as a white crystalline solid (578 mg, 68% yield). Rf = 0.3 (EtOAc/Hexane = 20/80). 1H NMR
41
(500 MHz, CDCl3): δ 8.73 (br s, 1H, NH of indole), 8.03 (d, J = 1.5 Hz, 1H), 7.54 (dd, J = 8.5
Hz, J = 2.0 Hz, 1H), 7.16 (d, J = 8.5 Hz, 1H), 4.42 (q, J = 7.5 Hz, 2H), 3.06 (q, J = 7.5 Hz, 2H),
1.43 (t, J = 7.0 Hz, 3H), 1.26 (t, J = 7.0 Hz, 3H). Mass spectrum m/z – 344.01 [M+H]+
5-Iodo-3-ethyl-1H-indole-2-carboxylic acid (30)
To a solution of 29 (155 mg, 0.451mmol.) in dioxane (15 mL) was added a solution of KOH
(350 mg, 6.14 mmol) in water (3 mL) and the resulting solution was refluxed for 45 min. It was
then cooled to room temperature, concentrated under reduced pressure and neutralized by
addition of 1N HCl. The precipitated acid was filtered, washed with cold water and air dried to
give pure acid 21 (124.4 mg, 87.5 % yield) as a white solid. 1H NMR (400 MHz, Acetone-d6): δ
10.57 (s, 1H), 7.96 (d, J = 1.2 Hz, 1H), 7.41 (dd, J = 8.8 Hz, J = 1.2 Hz, 1H), 7.22 (d, J = 8.8 Hz,
1H), 3.00 (q, J = 7.2 Hz, 2H), 1.95 – 1.90 (m, 1H) 1.12 (t, J = 7.2 Hz, 3H). Mass spectrum m/z –
315.98 [M+H]+
3-Ethyl-5-iodo-N-[4-(piperidin-1-yl)phenethyl]-1H-indole-2-carboxamide (31)
To a solution of acid 30 ( 54 mg, 0.172 mmol), amine 5 (60 mg, 0.29 mmol), HOBT (50 mg,
0.37 mmol), DIPEA (100 mg, 0.775 mmol) in anhydrous NMP (5 mL) was added EDCI (100
mg, 0.645 mmol) under an argon atmosphere and at room temperature and the resulting mixture
was stirred overnight. Reaction mixture was diluted with ethyl acetate (25 mL) and water (10
mL). The organic layer was separated and aqueous layer extracted with EtOAc (3 x 20 mL).
Combined organic layer was washed with water, brine and dried (Na2SO4). Evaporation of
42
volatiles under reduced pressure gave crude which was purified by flash column chromatography
on silica gel (10%40% EtOAc: Hexane) to give 31 as a white crystalline solid (60.4 mg, 70%
yield). M.p. = 180 - 181°C. Rf = 0.83 (MeOH/DCM = 20/80). 1H NMR (400 MHz, CDCl3): δ
9.30 (br s, 1H, NH indole), 7.91 (br s, 1H), 7.49 (dd, J = 8.8 Hz, J = 1.6 Hz, 1H), 7.16 (d, J = 8.8
Hz, 1H), 7.13 (d, J = 8.0 Hz, 2H), 6.92 (d, J = 8.0 Hz, 2H), 5.98 (br t, J = 5.2 Hz, 1H, NH
amide), 3.78 (q, J = 6.8 Hz, 2H), 3.13 (t, J = 5.6 Hz, 4H), 2.89 (t, J = 6.4 Hz, 2H), 2.68 (q, J =
7.2 Hz, 2H), 1.76-1.67 (m, 4H), 1.63-1.54 (m, 2H), 1.07 (t, J = 7.6 Hz, 3H, -CH3). Mass
spectrum m/z – 502.13 [M+H]+
1-(4-Chlorophenyl)-3-[4-(piperidin-1-yl)phenethyl]urea (33)
To a solution of amine 5 (102 mg, 0.499 mmoles) in dichloromethane, was added the
commercially available 4-chlorophenyl isocyanate (32) (80mg, 0.52 mmol) and stirred at 0 ºC for
2 h and then overnight at room temperature. The reaction mixture was evaporated under reduced
pressure to obtain the crude which was purified by flash chromatography on silica gel
(10%40% EtOAc: Hexane) to give 33 as a pink solid (125 mg, 70% yield). M.p. = 188 -
190°C. Rf = 0.65 (EtOAc/Hexane = 50/50). 1H NMR (500 MHz, CD3OD): δ 7.35 (d, J = 9.0 Hz,
2H), 7.23 (d, J = 9.0 Hz, 2H), 7.15 (d, J = 8.5 Hz, 2H), 6.97 (d, J = 8.5 Hz, 2H), 3.41 (t, J = 7.5
Hz, 2H), 3.10 (br t, J = 5.5 Hz, 4H), 2.76 (t, J = 7.0 Hz, 2H), 1.80-1.70 (m, 4H), 1.66-.156 (m,
2H), 1.31 (br s, 2H). Mass spectrum m/z – 376.16 [M+H]+
43
5-Chloro-3-ethyl-N-[3-(6-(pyrrolidin-1-yl)pyridin-2-yl)phenyl]-1H-indole-2-carboxamide
(39)
To a solution of acid 14 (38.5 mg, 0.172 mmol), amine 38 (69.40 mg, 0.29 mmol) prepared from
a patented route, HOBT (50 mg, 0.37 mmol), DIPEA (100 mg, 0.775 mmol) in anhydrous NMP
(5 mL) was added EDCI (100 mg, 0.645 mmol) under an argon atmosphere and at room
temperature and the resulting mixture was stirred overnight. Reaction mixture was diluted with
ethyl acetate (25 mL) and water (10 mL). The organic layer was separated and aqueous layer
extracted with EtOAc (3 x 25 mL). Combined organic layer was washed with water, brine and
dried (Na2SO4). Evaporation of volatiles under reduced pressure gave crude which was purified
by flash column chromatography on silica gel (10%40% EtOAc: Hexane) to give 31 as a
white solid (52.0 mg, 68% yield). Rf = 0.8 (EtOAc/Hexane = 50/50). 1H NMR (500 MHz,
CDCl3): δ 9.34 (br s, 1H), 8.22 (dd as t, J = 2.0 Hz, 1H), 7.87 (s, 1H), 7.83 (dd, J = 8.5 Hz, J =
1.5 Hz, 1H), 7.76 (dd, J = 7.5 Hz, J = 1.5 Hz, 1H), 7.63 (d, J = 2.0 Hz, 1H), 7.51 (dd, J = 8.5 Hz,
J = 7.0 Hz, 1H), 7.46 (d, J = 7.5 Hz, 1H), 7.44 (d, J = 8.0 Hz, 1H), 7.21 (dd, J = 8.5 Hz, J = 2.0
Hz, 1H), 7.04 (d, J = 7.0 Hz, 1H), 6.35 (d, J = 8.0 Hz, 1H), 3.60-3.50 (m, 4H), 3.11 (q, J = 7.5
Hz, 2H), 2.06-1.97 (m, 4H), 1.45 (t, J = 7.5 Hz, 3H). Mass spectrum m/z – 445.17 [M+H]+
44
10. Conclusions:
We have successfully synthesized Org27569, a negative allosteric modulator of CB1
cannabinoid receptor by a novel synthetic route. This route permits the synthesis of this allosteric
modulator in gram quantities for its further in vivo evaluation. We have also completed the
synthesis of aryl azido as well as aryl isothicyanate analog of Org27569. These two novel
covalent probes will be evaluated for their ability to label the CB1 and CB2 receptors. The iodo
analog of Org27569 has been synthesized successfully and will be utilized for development of
radioligand for CB1 allosteric site. We have also successfully designed and synthesized two
novel hybrid analogs of Org27569 and PSNCBAM-1. All these compounds will be evaluated in
vitro for their ability to modulate binding of CP-55,940 and to alter CP-55,940 mediated cAMP
reduction.
45
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