Strategies to develop selective CB2 receptor agonists from indole carboxamide synthetic cannabinoids

Article abstract of DOI:10.1016/j.ejmech.2019.07.036

Activation of the CB₂ receptor is an attractive therapeutic strategy for the treatment of a wide range of inflammatory diseases. However, receptor subtype selectivity is necessary in order to circumvent the psychoactive effects associated with activation of the CB₁ receptor. We aimed to use potent, non-selective synthetic cannabinoids designer drugs to develop selective CB₂ receptor agonists. Simple structural modifications such as moving the amide substituent of 3-amidoalkylindole synthetic cannabinoids to the 2-position and bioisosteric replacement of the indole core to the 7-azaindole scaffold are shown to be effective and general strategies to impart receptor subtype selectivity. 2-Amidoalkylindole 16 (EC₅₀ CB₁ > 10 μM, EC₅₀ CB₂ = 189 nM) and 3-amidoalkyl-7-azaindole 21 (EC₅₀ CB₁ > 10 μM, EC₅₀ = 49 nM) were found to be potent and selective agonists with favourable physicochemical properties. Docking studies were used to elucidate the molecular basis for the observed receptor subtype selectivity for these compounds.

Full text of DOI:10.1016/j.ejmech.2019.07.036

Accepted Manuscript
Strategies to develop selective CB receptor agonists from indole carboxamide
2
synthetic cannabinoids
Michael Moir, Samuel Lane, Felcia Lai, Mark Connor, David E. Hibbs, Michael
Kassiou
PII:
S0223-5234(19)30657-9
DOI:
https://doi.org/10.1016/j.ejmech.2019.07.036
EJMECH 11533
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 June 2019
Revised Date: 10 July 2019
Accepted Date: 10 July 2019
Please cite this article as: M. Moir, S. Lane, F. Lai, M. Connor, D.E. Hibbs, M. Kassiou, Strategies to
develop selective CB receptor agonists from indole carboxamide synthetic cannabinoids, European
2
Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.07.036.
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ACCEPTED MANUSCRIPT
Simple structural
modifications
Non-selective cannabinoid
designer drugs
Selective CB agonists as potential
2
therapeutics

ACCEPTED MANUSCRIPT
Strategies to Develop Selective CB
2
Receptor Agonists from Indole
Carboxamide Synthetic Cannabinoids
a
b
c
d
c
Michael Moir , Samuel Lane , Felcia Lai , Mark Connor , David E. Hibbs , and Michael
Kassiou
a*
a
School of Chemistry, The University of Sydney, NSW 2006 Australia
Faculty of Health Sciences, The University of Sydney, NSW 2006 Australia
Sydney Pharmacy School, The University of Sydney, NSW 2006, Australia
Department of Biomedical Sciences, Macquarie University, NSW 2109, Australia
b
c
d
Corresponding Author
*
E-mail: michael.kassiou@sydney.edu.au Phone: +612 9351 2745
Abstract
Activation of the CB receptor is an attractive therapeutic strategy for the treatment of a
2
wide range of inflammatory diseases. However, receptor subtype selectivity is
necessary in order to circumvent the psychoactive effects associated with activation of
the CB
1
receptor. We aimed to use potent, non-selective synthetic cannabinoids
receptor agonists. Simple structural
designer drugs to develop selective CB
2
modifications such as moving the amide substituent of 3-amidoalkylindole synthetic
cannabinoids to the 2-position and bioisosteric replacement of the indole core to the 7-
azaindole scaffold are shown to be effective and general strategies to impart receptor
subtype selectivity. 2-Amidoalkylindole 16 (EC50 CB
1
> 10 μM, EC50 CB
2
= 189 nM) and
3
-amidoalkyl-7-azaindole 21 (EC50 CB > 10 μM, EC50 = 49 nM) were found to be potent
1
and selective agonists with favourable physicochemical properties. Docking studies
were used to elucidate the molecular basis for the observed receptor subtype selectivity
for these compounds.
Keywords: Cannabinoid; Cannabinoid type 2 receptor; Synthetic cannabinoid; Indole;
Azaindole; Docking.
1
. Introduction
In recent decades the endocannabinoid system has emerged as an important
modulatory system that is involved in a whole range of physiological processes,
1
including appetite, pain-sensation, mood and memory. The system comprises of
endogenous cannabinoids (endocannabinoids), the enzymes responsible for their
synthesis and degradation and two G protein coupled receptors which are their primary
2
targets (although other receptors may also be involved). The cannabinoid type 1 (CB
1
)
receptor is found in high levels in the central nervous system, but also in a number of
3
peripheral tissues. Conversely, the cannabinoid type 2 (CB
2
) receptor is expressed
mainly on cells of the immune system, which in the CNS includes astrocytes and
microglia.3
The discovery of the CB
immunomodulatory properties of cannabinoids. The broad distribution of the
2
receptor in 1993 provided an explanation for the established
4
1

ACCEPTED MANUSCRIPT
endocannabinoid system throughout the body means that its manipulation can have
5
implications in a wide range of diseases and disorders. It is believed that in response to
injury or damage, activation of CB
resolution of inflammation and its associated symptoms. Demonstration that CB
2
receptors triggers protective mechanisms for the
6
2
receptor activation produces desirable effects in a range of preclinical models has
motivated the design of agonists for the treatment of a wide range of diseases and
7
pathological conditions involving an inflammatory component. A requirement for the
development of CB
effects associated with activation of the CB
been devoted to the development of selective CB
circumvent psychoactive side effects. A number of compounds developed by academic
2
-based therapeutics is the avoidance of unwanted psychotropic
receptor. In light of this, much effort has
receptor agonists, in order to
1
2
labs and pharmaceutical companies have been shown to selectively activate the CB
2
8-14
receptor (1-6, Figure 1).
Despite this, CB
2
agonists have fared poorly in the clinic.
Potential reasons for the lack of translation from preclinical to clinical efficacy include,
species differences in receptors and signalling pathways, a lack of receptor subtype
selectivity in older generation agonists, issues pertaining to biased agonism or
15
functional selectivity and concerns surrounding drug tolerance. Therefore, there is a
need to develop more potent, effective and safe CB agonists.
2
Figure 1: Representative examples of CB selective agonists reported in the literature (1-6).
2
In the early 2000’s, smokable herbal products, adulterated with synthetic cannabinoids
reported in the scientific literature, appeared as legal alternatives to marijuana. In
recent years we have synthesized and characterized the pharmacological activity of
many such high concern synthetic cannabinoids, particularly 3-amidoalkyl indoles and
16-20
indazoles (Figure 2).
2

ACCEPTED MANUSCRIPT
Figure 2: Evolution of synthetic cannabinoids from APICA 7. 3-Amidoalkylindole and indazole synthetic
cannabinoids (general structure shown) are generally potent, full agonists at both the CB
1
and CB receptors.
2
The research has established simple and reliable methods to access such synthetic
cannabinoids and their analogues for pharmacological evaluation. These compounds
are generally highly potent, high efficacy agonists at both the CB
1
and CB receptors, and
2
have been shown to exhibit potent in vitro activity, cannabimimetic phenotypes in vivo,
21
and anecdotal cannabimimetic activity in humans.
Recently it was demonstrated that CB
2
selectivity could be realized for non-selective 3-
adamantylamidoalkylindoles by simply moving the amide from the 3-position to the 2-
22
position of the indole. The 2-substituted adamantyl amide compounds (for example 8)
discovered exhibited low to moderate potencies, however are generally quite lipophilic,
owing to the non-polar adamantyl group. Previous research also found that 3-
substituted bulky amino acid derived amides (from
L-valinamide, methyl-L-valinate,
tert-leucinamide and tert-leucinate) as well as cumyl amides are amongst the most
16-17, 19
potent synthetic cannabinoids known.
We have applied this strategy to such
amides in an effort to obtain more potent and selective compounds (9, 11, 14, 16 and
1
8) with favorable physicochemical properties, namely reduced lipophilicity (Figure
3). In addition, it is also reported that incorporation of a polar group, particularly a
hydroxyl group, at the terminal position of the N-pentyl chain can improve CB
selectivity. With this in mind we prepared the 5-hydroxyl analogues (10, 12, 15, 17
and 19).
2
20
3

ACCEPTED MANUSCRIPT
Figure 3: Strategies to develop CB
2
selective agonists from non-selective synthetic cannabinoids such as APICA 7.
Our recent study involving the pharmacological evaluation of azaindole derived
synthetic cannabinoids revealed that the 7-azaindole scaffold may impart some CB
2
selectivity.2 Again, we sought to determine whether this is a general strategy to
3
improve the CB selectivity of potent synthetic cannabinoids (20-29, Figure 3). In
2
addition to improving receptor subtype selectivity, bioisosteric replacement of the
indole with a 7-azaindole moiety may improve physicochemical properties.
A hybrid strategy was also explored by moving the amide group from the 3-position to
the 2-position of the 1-alkyl-7-azaindole moiety (30). Furthermore, azaindole analogues
were investigated including 3-amidoalkylpyrazolo[3,4-b]pyridine 31 and the influence
of the position of nitrogen alkylation by the preparation of pseudo-azulene compound
32.
Molecular modelling based on X-ray crystal structures of the CB
1
and CB receptors was
2
performed to rationalise the key binding interactions that impart receptor subtype
selectivity, and inform future development of cannabinoid therapeutics.
4

ACCEPTED MANUSCRIPT
2
2
2
. Results and Discussion
.1 Chemistry
-Amidoalkylindole analogues 8-19 were prepared from commercially available ethyl
indole-2-carboxylate. Sodium hydride mediated deprotonation followed by reaction
with alkyl bromides, 1-bromopentane or 34, which was prepared from mono-benzyl
protected 1,5-pentanediol 33 via an Appel reaction, gave the requisite carboxylic acid
intermediates 40 and 41 after base mediated ester hydrolysis (Scheme 1).
Operationally simple and high yielding EDCI-HOBt amide coupling using the
appropriate amines gave the 11 amides 8, 9, 11, 14, 16, 18, 42-46. Amine 36 was
synthesized from commercially available Boc-
amide coupling and subsequent Boc group deprotection under acidic conditions.
Likewise, amine 37 was prepared from commercially available -tert-leucine via the
corresponding acid chloride. Hydrogenolysis of the benzyl protecting groups from 42-
L-tert-leucine via an EDCI-HOBt mediated
L
46 afforded the 5-hydroxyl analogues 10, 12, 15, 17 and 19. Difluoromethyl analogue
13 was prepared in a two-step procedure from alcohol 17 via a Dess-Martin
periodinane (DMP) mediated oxidation followed by treatment of the aldehyde with
nucleophilic fluorinating reagent diethylaminosulfur trifluoride (DAST).
5

ACCEPTED MANUSCRIPT
Scheme 1: Synthesis of 2-amidoalkylindoles (8-19). Reagents and conditions: (a) NaH, appropriate alkyl bromide (1-
bromopentane or 34), DMF, 0 °C-rt, 16 hours; (b) NaOH (aq.), EtOH, reflux, 2 hours; (c) Appropriate amine (1-
adamantylamine, 2-phenylpropan-2-amine,
EDCI, HOBt, iPr EtN, DMF, rt, 16 hours; (d) Pd/C, H
DAST, CH Cl , rt, 24 hours; (f) NaH, BnBr, DMF, 0 °C-rt, 16 hours; (g) PPh
NH Cl, EDCI, HOBt, Et N, DMF, rt, 16 hours; (i) HCl, dioxane, EtOAc, rt, 16 hours; (j) SOCl
L
-valinamide hydrochloride, methyl-
, EtOAc, rt, 16 hours; (e) i) DMP, CH
, Br , pyridine, CH
, MeOH, reflux, 16 hours.
L
-valinate hydrochloride, 36 or 37),
Cl , THF, rt, 16 hours, ii)
Cl , 0 °C-rt, 4 hours; (h)
2
2
2
2
2
2
3
2
2
2
4
3
2
3
-Amidoalkyl-7-azaindole derivatives 20-29 were prepared following previously
23
reported procedures. Commercially available 7-azaindole was deprotonated using
sodium hydride and then reacted with alkyl bromides, 1-bromopentane or 34 to give
the N1-alkyl intermediates 47 and 48 (Scheme 2). Aluminium chloride mediated
Friedel Crafts acylation gave the 3-subsituted trifluoromethyl ketones 49 and 50, which
were hydrolysed under basic conditions after extended reaction times to afford
carboxylic acids 51 and 52. EDCI-HOBt mediated amide coupling gave the requisite
amides 20, 22-26, 28 and 53-56 and hydrogenolysis to remove the benzyl protecting
groups from 53-56 furnished the 5-hydroxyl analogues 21, 24, 27 and 29.
6

ACCEPTED MANUSCRIPT
Scheme 2: Synthesis of 3-amidoalkyl-7-azaindoles (20-29). Reagents and conditions: (a) NaH, appropriate alkyl
bromide (1-bromopentane or 34), DMF, 0 °C-rt, 16 hours; (b) AlCl
MeOH, reflux, 48 hours; (d) Appropriate amine (1-adamantylamine, 2-phenylpropan-2-amine,
hydrochloride, methyl- -valinate hydrochloride, 36 or 37), EDCI, HOBt, iPr EtN, DMF, rt, 16 hours; (e) Pd/C, H
EtOAc, AcOH, rt, 16-48 hours.
3
, (CF
3
CO)
2
O, DMF, 0 °C-rt, 3 hours; (c) LiOH (aq.),
L-valinamide
L
2
2
,
2
-Amidoalkyl-7-azaindole derivative 30 was prepared using a similar procedure, from
ethyl 7-azaindole-2-carboxylate 59 (Scheme 3). The ester was synthesized using a
procedure reported by Cai and co-workers via copper mediated
condensation/coupling/deformylation cascade process from 2-bromonicotinaldehyde
a
24
(
57) with ethyl isocyanoacetate (58). N1-Alkylation using potassium carbonate and 1-
bromopentane and base mediated ester hydrolysis gave the carboxylic acid 61. Finally
an EDCI-HOBt mediated amide coupling gave the desired amide 30.
7

ACCEPTED MANUSCRIPT
Scheme 3: Synthesis of 2-amidoalkyl-7-azaindole derivative 30. Reagents and conditions: (a) LDA, DMF, THF, -78 °C-
rt, 2 hours; (b) i) Ethyl formate, Et
C, 16 hours; (d) K CO , 1-bromopentane, 50 °C, 16 hours; (e) NaOH (aq.), EtOH, reflux, 2 hours; (f) 1-
Adamantylamine, EDCI, HOBt, iPr EtN, DMF, rt, 16 hours.
3
N, 60 °C, 16 hours; ii) POCl
3
, Et
3
N, CH
2
Cl
2
, 0 °C-rt, 1 hour; (c) CuI, Cs
2
CO
3
, DMSO, 80
°
2
3
2
3-Amidoalkylpyrazolo[3,4-b]pyridine derivative 31 was prepared from commercially
available 3-acetylpyridine (Scheme 4). Regioselective chlorination at the 2-position of
the pyridine was achieved from the N-oxide 62 using phosphorus oxychloride.
Subsequent condensation with methyl hydrazine formed the pyrazolo[3,4-b]pyridine
nucleus (64). N1-Alkylation with sodium hydride and 1-bromopentane followed by
oxidation of the 3-methyl group using potassium permanganate gave carboxylic acid 66.
An EDCI-HOBt mediated amide coupling afforded the desired amide 31.
Scheme 4: Synthesis of 3-amidoalkylpyrazolo[3,4-b]pyridine derivative 31. Reagents and conditions: (a) m-CPBA,
CH
2
Cl
2
, rt, 16 hours; (b) POCl , 100 °C, 2 hours; (c) Ethylene glycol, 165 °C, 16 hours; (d) NaH, 1-bromopentane, 0 °C-
3
rt, 16 hours; (e) KMnO
hours.
4
, NaOH (aq.), H
2
O, 90 °C, 2 hours; (f) 1-Adamantylamine, EDCI, HOBt, iPr
2
EtN, DMF, rt, 16
8

ACCEPTED MANUSCRIPT
N-7-Alkyl derivative 32 was formed from 3-amido-7-azaindole 69 by a regioselective
alkylation under neutral conditions after basic workup (Scheme 5). The regiochemistry
of the alkylation was confirmed by NOESY correlation between the C6 proton of the
azaindole nucleus and the methylene protons adjacent to the nitrogen of the pentyl
chain (see Supplementary Information).
Scheme 5: Synthesis of 3-aminoalkyl-7-azaindole derivative substituted at the 7-position 32. Reagents and
conditions: (a) AlCl
3
, Cl
3
COCl, CH
2
2
Cl , 0 °C-rt, 4 hours; (b) NaOH (aq.), rt, 6 hours; (c) 1-Adamantylamine, EDCI, HOBt,
iPr EtN, DMF, rt, 16 hours; (d) 1-Bromopentane, TBAI, MeCN, PhMe, 150 °C, 16 hours.
2
2
.2 Functional activity at the CB
1
and CB
2
receptors
The synthetic cannabinoid analogues 8-32 and lead compound 7 were assessed for
their ability to activate CB and CB receptors. The activity of 7-32 was assessed in AtT-
0 neuroblastoma cells stably transfected with human CB or CB receptors using a
1
2
2
1
2
FLIPR membrane potential assay which measures activation of endogenously expressed
+
G protein-gated inwardly rectifying K channels (GIRKs) by CB
1
or CB agonists.
2
Compounds displaying more than 50% activation at 10 μM in the assay were evaluated
further in dose−response studies. Their half maximal effective concentrations (EC50
and maximal effect relative to 1 μM CP 55,940 (Emax) were calculated as listed in Tables
and 2. Further experimental details are provided in the Experimental Section.
)
1
In agreement with previously reported data, 3-amidoalkylindole APICA (7) exhibited
18
potent functional activity at both the CB
structural modification of moving the amide from the 3-position to the 2-position to
afford 2-amidoindole 8 was found to completely supress activity at the CB
receptor.22
1
and CB
2
receptors (Table 1). The simple
1
Due to its high lipophilicity, we were unable to solubilise the compound in the assay
medium at high concentrations (numerous solvent mixtures were trialled, but reliable
results were not able to be obtained). It appeared from the limited data obtained that
the compound is a partial agonist with low micromolar activity, similar to what has
22
previously been reported. The poor aqueous solubility of 8 highlights the need to
develop more drug-like compounds using this strategy. It was envisioned that amino
acid derived amides and cumyl amides 9-19 would retain not only the selectivity
9

ACCEPTED MANUSCRIPT
observed for adamantyl derivative 8, but also the high potency of their respective 3-
amidoalkylindole analogues. As suspected, the receptor subtype selectivity was retained
and improved sub-micromolar potency was observed, particularly for tert-leucinamide
analogue 16 (CB
could not be generated for activity at the CB
concentrations. The tolerance of amide substitution suggests this is a general strategy
for imparting CB selectivity and further optimisation of the amide and N-alkyl chain
could further improve potency and physicochemical properties. Interestingly, 5-
hydroxypentyl derivatives 10, 12, 15 and 17 exhibited no activity at either the CB or
CB receptors, while cumyl amide derivative 19 retained selectivity and exhibited
improved potency (CB EC50 = 217 nM) compared to the pentyl derivative 18. It is
2
EC50 = 189 nM). Unfortunately for 11 and cumyl amide 18, EC50 values
2
receptor due to poor solubility at high
2
1
2
2
possible that intramolecular hydrogen bonding between the hydroxyl group and polar
amino acid derived amide substituents (but not the non-polar cumyl or adamantyl
amides) is unfavourable for CB
2
activation. Further exploration of other polar alkyl
derivatives that would not participate in such hydrogen bonding interactions could
25
yield more potent compounds. As fluorine is a weak hydrogen bond acceptor and
based on data for similar compounds, difluoromethyl analogue 13 was synthesized.22
Introduction of the polar group reduced activity at the CB receptor, but not completely,
2
as was the case for the 5-hydroxyl analogue 12. It is possible that interactions between
the difluoromethyl group and polar amino acid derived amide still affords an
unfavourable conformation for CB
is required to determine the optimal functionality for potent CB
2
activation. Further exploration of the N-alkyl moiety
activity.
2
Table 1: Agonist activities of 2-amidoalkylindoles in AtT-20 cells expressing human CB
membrane potential assay
1
or CB receptors by a FLIPR
2
a
CB
1
CB
2
Compound
pEC50 ± SEM
EC50 μM)
7.13 ± 0.09
E
max (%)
108
pEC50 ± SEM
(EC50 μM)
7.02 ± 0.08
(0.096)
DNC
6.15 ± 0.11
E
max (%)
85
(
7
(
0.074)
NA
8
9
ND
ND
ND
74
NA
(
0.713)
NA
DNC
NA
1
1
1
1
0
1
2
3
NA
NA
NA
NA
ND
ND
ND
ND
ND
ND
ND
128
5.26 ± 0.73
5.46)
6.13 ± 0.16
(
1
4
NA
ND
74
(
0.741)
NA
1
1
5
6
NA
NA
ND
ND
ND
73
6.72 ± 0.12
(
0.189)
NA
DNC
1
1
1
7
8
9
NA
NA
NA
ND
ND
ND
ND
ND
88
6.66 ± 0.09
(
0.217)
CP 55,940
7.66 ± 0.04
0.022)
101
7.20 ± 0.04
(0.063)
106
(
a
See experimental section for more details. Data represent mean values ± SEM from at least three independent
b
experiments each performed in duplicate, with CP 55,940 used as a positive control. NA: Not active, defined as <50%
activation at 10 μM in the assay. ND: Not determined; for compounds defined as not active, their maximal effects
c
10

ACCEPTED MANUSCRIPT
d
were not determined. DNC: Did not converge; data does not fit a reliable curve to generate EC50 values (due to
solubility issues at high concentration).
Our recent report suggests 3-amidoalkyl-7-azaindole 20 is a potent, CB
2
selective
agonist. In this study receptor subtype selectivity (roughly 3.5-fold) was also observed
Table 2). This selectivity however was not observed for amino acid derived amides 22,
3, 25 and 26 and cumyl amide 28, which all exhibited potent activity at both receptor
subtypes. 3-Amidoalkylpyrazolo[3,4-b]pyridine derivative 31 exhibited improved
receptor subtype selectivity with a roughly 7-fold preference for CB activation. Hybrid
-amidoalkyl-7-azaindole 30 exhibited no activity at either receptor and pseudo-
23
(
2
2
2
azulene 32 was also inactive. Fortuitously, 5-hydroxypentyl derivative 21 exhibited
complete receptor subtype selectivity. With this positive result in hand we investigated
whether other amide substitutions would exhibit similar selectivity. We therefore
synthesized the 5-hydroxy derivatives of the two most potent 3-amidoalkyl-7-azaindole
compounds 23 and 25 and also cumyl amide derivative 28 with the expectation that
their potency at the CB
selectivity. The 5-hydroxypentyl analogues 24, 27 and 29 were potent agonists at the
CB receptor with vastly improved subtype selectivity however, exhibited some activity
at CB . Adopting this approach for the corresponding 3-amidoalkylpyrazolo[3,4-
2
receptor would be retained with improved receptor subtype
2
1
b]pyridine derivatives may yield even more selective compounds.
Table 2: Agonist activities of azaindole derivatives in AtT-20 cells expressing human CB
membrane potential assay
1
or CB receptors by a FLIPR
2
a
CB
1
CB
2
Compound
pEC50 ± SEM
EC50 μM)
6.82 ± 0.14
E
max (%)
pEC50 ± SEM
E
max (%)
(
(EC50 μM)
7.37 ± 0.08
(0.043)
2
2
2
2
2
2
2
2
2
2
0
1
2
3
4
5
6
7
8
9
75
ND
97
101
93
(
0.152)
NA
7.31 ± 0.10
(
0.049)
7.25 ± 0.10
0.056)
8.77 ± 0.06
0.0017)
6.63 ± 0.12
0.234)
6.92 ± 0.14
0.121)
8.40 ± 0.06
0.004)
6.31 ± 0.08
0.491)
7.45 ± 0.09
0.035)
6.33 ± 0.12
7.84 ± 0.07
(0.015)
8.37 ± 0.09
(0.0043)
7.72 ± 0.12
(0.019)
7.23 ± 0.12
(0.058)
8.29 ± 0.08
(0.005)
7.30 ± 0.11
(0.050)
6.98 ± 0.13
(0.104)
6.82 ± 0.18
(0.152)
NA
6.92 ± 0.10
98
(
103
99
102
96
(
(
117
110
98
103
94
(
(
96
(
106
110
96
(
95
(
0.466)
NA
3
3
0
1
ND
97
ND
104
6.08 ± 0.10
(
0.824)
NA
(0.122)
NA
3
2
ND
ND
CP 55,940
7.66 ± 0.04
0.022)
101
7.20 ± 0.04
(0.063)
106
(
a
See experimental section for more details. Data represent mean values ± SEM from at least three independent
b
experiments each performed in duplicate, with CP 55,940 used as a positive control. NA: Not active, defined as
11

ACCEPTED MANUSCRIPT
c
<
50% activation at 10 μM in the assay. ND: Not determined; for compounds defined as not active, their maximal
effects were not determined.
2
.3 In silico studies
Relatively small structural modifications can have profound effects on the binding
affinity and functional profile of GPCR ligands. We performed docking simulations to
investigate the molecular basis for the varied functional activity of the investigated
compounds at the CB
1
and CB receptors. A caveat to these calculations is that the
2
inactive cannabinoid receptor structures, solved with an antagonist bound, are not ideal
2
6-27
for predicting agonist interactions.
orthosteric site of the CB receptor has been investigated by integrating docking studies,
mutagenesis studies and SAR data.
The manner in which agonists bind to the
1
28-30
Hua and co-workers have docked known CB
1
receptor agonists, including indole JWH-018, into the inactive state crystal structure
and in combination with mutation studies suggest that interactions with Phe268 and
27
Phe379 are important for functional activity. Similar studies were conducted based on
the active state agonist-bound crystal structure of the CB receptor and found that π-π
interactions with Phe177, Phe189, Phe268 and Phe379 and hydrogen bonding with
1
31
Ser383 were conserved for the agonist-bound complexes.
It has been extensively reported that CB
1
seems to use an extended molecular toggle
switch involving a synergistic conformational change between Phe200 and Trp356 for
31
receptor activation. Agonists disrupt the π–π stacking of the side chains of Phe200and
Trp356 leading to conformational change. It is difficult however to probe the
interaction of an agonist with these residues using the inactive state of the receptor.
Compounds 7, 8, 20 and 21 were chosen as representative compounds based on their
differing receptor subtype selectivities. Docking and ∆G binding scores obtained by
combining molecular mechanics (MM) terms with a generalised Born and surface area
32
(
(
GBSA) solvent mode (MM-GBSA) are summarised in the Supplementary Information
Table S1).
APICA (7) is a non-selective agonist and this is reflected in the docking scores
-
1
(
)
difference of 0.09 kcal mol ) and MM-GBSA ∆GBind values (difference of 5.57 kcal mol-
1
. At the CB receptor the compound has π–π interactions with Phe379 and a hydrogen
1
bonding interaction with Ser383, which as previously mentioned are believed to be
important interactions for functional activity (See Supplementary Information, Figure
S1). At the CB
2
receptor the agonist has π–π interactions with Phe91 and Phe183. The
importance of these residues for binding have been confirmed by Feng and co-workers
33
via site-directed mutation studies.
Movement of the amide from the 3-positon to the 2-position of the indole moiety has
been shown to eradicate functional activity at the CB receptor. This is consistent with
this docking study where a more positive MM-GBSA ∆GBind value (-55 kcal mol ) is
1
-1
-1
observed for 8 compared to 7 (-73.35 kcal mol ). Higher receptor and ligand strain are
also observed for this regioisomer at the CB receptor. Importantly the hydrogen
bonding interaction with key residue Ser383 was not observed, potentially explaining
the eradication of functional activity (Figure 4, C). At the CB receptor similar docking
scores and MM-GBSA ∆GBind values (differences of 1.07 kcal mol and 9.95 kcal mol
1
2
-1
-1
12

ACCEPTED MANUSCRIPT
respectively) were observed for the two regioisomeric compounds. Further, for 8 the
indole nucleus exhibits π-π interactions with Phe94 and the carbonyl oxygen of the
amide has a hydrogen bonding interaction with Ser285 (Figure 4, D). Li and co-workers
2
6
suggest mutations of Phe94 greatly reduces the potency of CB
2
agonists, while Feng
33
and co-workers also suggest Ser285 is an important residue for ligand binding.
It was found that for the azaindole scaffold, terminal hydroxylation of the N-1 pentyl
chain precluded CB functional activity. Compound 20, a non-selective agonist, had a
similar docking score and MM-GBSA ∆GBind value to 7 at both the CB and CB receptors,
consistent with their similar functional activity data. Similar to 7 π–π interactions with
1
1
2
Phe91 and Phe183 were observed at the CB receptor (See Supplementary Information,
2
Figure S2).
In comparison, 21 displayed a similar docking score and MM-GBSA ∆GBind value at the
CB receptor to 20. However, the preference for CB becomes quite clear when
examining the MM-GBSA ∆GBind value (CB
particularly the receptor strain (30.51 kcal mol ) at the CB
binding mode interacts with a number of important residues at CB
Ser128 and Ser383), the high receptor strain suggests this is not a favourable pose (See
Supplementary Information, Figure S3). For the CB receptor 21 shares π–π
2
2
-1
-1
1
: -60.23 kcal mol , CB
2
-71.63 kcal mol ) and
receptor. Although this
(Phe102, Phe379,
-1
1
1
2
interactions with Phe91 and Phe183 as well as hydrogen bonding interactions with
His95 and Lys278.
13

ACCEPTED MANUSCRIPT
A
B
C
D
Figure 4: Docking mode of 2-amidoalkylindole 8 in the: (a) CB
The π-π interactions are shown with blue dashed lines and hydrogen bonding is shown with yellow dashed lines; 2D
representation of 8 in the: (c) CB receptor active site, and (d) CB receptor active site. The π-π interactions are
1
receptor active site, and (b) CB
2
receptor active site.
1
2
shown with solid green lines and hydrogen bonding is shown with purple arrows.
With the aim of developing drug-like compounds, we performed in silico predictions of
physiochemical and pharmacokinetic properties. The calculated properties of 16 and
2
1 were predominantly within the recommended values as suggested by QikProp
Schrödinger), and with similar values to 95% of known drugs (Table 3). Solubility
issues are widely recognised in cannabinoid pharmacology because of the lipophilic
(
34
nature of cannabinoid ligands. Of particular note, substitution of the adamantyl group
for a polar amino acid derived amide resulted in a significant drop in the calculated
LogP value (6.25 for 7 and 3.66 for 16). Further, the 7-azaindole scaffold with 5-
hydroxypentyl group also resulted in a significant reduction in calculated LogP (6.25 for
7
and 4.38 for 21).
14

ACCEPTED MANUSCRIPT
Table 3: Calculated physicochemical and pharmacokinetic properties of lead compound 7 and analogues 16 and 21.
Calculated
MW
LogP PSA (Å2)
Oral
Absorption
QPPCaco
(nm/sec)
QPPMDCK
(nm/sec)
QPlogBB
properties (g/mol)
a
(
%)
Recommended Values
Range for 95% of known drugs (Schrödinger)
<
450
<5
<70
<25% poor
<25 poor
>500 great
4948
<25 poor
>500 great
2786
-3.0 to -
1.2
-0.13
-0.88
-0.82
>
80% high
7
364.53
343.47
381.52
6.25
3.66
4.38
34.80
80.86
66.97
100
100
100
1
6
628
1325
568
671
21
a
Physicochemical properties (molecular weight, MW; lipophilicity, log P; polar surface area, PSA) and
pharmacokinetic properties (Oral Absorption; Predicted Apparent Caco-2 Permeability, QPPCaco; Predicted apparent
MDCK cell permeability, QPPMDCK; Predicted Brain/Blood Partition Coefficient, QPlogBB) were calculated with
QikProp, Schrödinger v5.9 software.
3
. Conclusions
We report herein the development of selective CB
2
receptor agonists from non-selective
synthetic cannabinoid designer drugs. A small library of 2-amidoalkylindoles and 7-
azaindole derivatives were synthesized, characterized and their activity at the
cannabinoid receptors evaluated. It has been demonstrated that simple structural
modifications can impart receptor subtype selectivity and here we have increased the
scope of these strategies to include analogues of some of the most potent synthetic
cannabinoids known. In addition, the compounds developed have improved
physicochemical properties, more suitable for drug development. Substitution of the
amide of 2-amidoalkylindoles has been shown to be a strategy to improve potency and
optimise physicochemical properties. Bioisosteric replacement of the indole core for the
7
-azaindole scaffold, in conjunction with the addition of a polar hydroxyl group at the
terminal position of the pentyl chain has shown to be another strategy to significantly
supress CB activity. Molecular modelling results corroborated the selectivity and
1
potency data gained from activity measurements, and this analysis recapitulated
previous literature implicating significant residues at both receptors. The structure-
activity relationships developed will facilitate the design of novel CB agonists. Future
2
work will focus on the optimisation of the N-alkyl group to further improve potency and
physical properties.
4. Experimental Section
4
4
.1 Chemistry
.1.1 General Methods
Compound 7 was previously prepared according to the procedures reported by
18
Banister et al. Compound 20 was previously prepared according to the procedures
23
reported by Longworth et al. All reactions were performed under an atmosphere of
nitrogen unless otherwise specified. Anhydrous N,N-dimethylformamide,
dichloromethane and tetrahydrofuran were obtained from a PureSolv MD 7 solvent
purification system (Innovative Technology, Inc.). All other solvents and reagents were
used as received from commercial sources. Analytical thin-layer chromatography (TLC)
was performed using Merck aluminium backed silica gel 60 F254 (0.2 mm) plates, which
were visualised with shortwave (254 nm) ultraviolet light. Products were also
15

ACCEPTED MANUSCRIPT
visualised with potassium permanganate, vanillin, cerium molybdate, bromocresol
green or ninhydrin stains. Flash column chromatography was performed using Merck
Kieselgel 60 (230-400 mesh) silica gel, with the eluent mixture reported as the
volume:volume ratio. Melting points were measured in open capillaries using a Stanford
Research System Optimelt Automated melting point apparatus. Infrared absorption
spectra were recorded on a Bruker ALPHA FT-IR spectrometer as a solid or a thin film
–
1
from ethanol, and the data are reported as vibrational frequencies (cm ). Nuclear
magnetic resonance spectra were recorded at 300 K using either a Bruker AVANCE
1
DRX300 (300 MHz) or AVANCE DRX400 (400 MHz) spectrometer. H Chemical shifts
are expressed as parts per million (ppm) with residual chloroform (δ 7.26), methanol (δ
3.31) and dimethyl sulfoxide (δ 2.50) as reference and are reported as chemical shift
(
δ); relative integral; multiplicity (s = singlet, bs = broad singlet, d = doublet, dd =
doublet of doublets, ddd = doublet of doublet of doublets, dt = doublet of triplets, t=
triplet, tt = triplet of triplets, q = quartet, quin = quintet, sept = septet, m = multiplet);
coupling constants (J) reported in Hz. C chemical shifts are expressed as parts per
million (ppm) with residual chloroform (δ 77.16), methanol (δ 49.00) and dimethyl
sulfoxide (δ 39.52) as reference and reported as chemical shift (δ); multiplicity;
coupling constants (J) reported in Hz. Proton decoupled F chemical shifts are reported
as parts per million (ppm). Low-resolution mass spectrometry (LRMS) was recorded
using electrospray ionisation (ESI) recorded on a Bruker AmaZon SL ion trap
spectrometer. High-resolution mass spectrometry was performed on a Bruker Apex Qe
13
19
7
T Fourier Transform Ion Cyclotron Resonance mass spectrometer equipped with an
Apollo II ESI/MALDI dual source. Samples were run with syringe infusion at 150 μL/hr
on a Cole Palmer syringe pump into the electrospray ionisation (ESI) source. Optical
rotation was performed on an Optical Activity AA-65 Automatic Polarimeter. Specific
rotations based on the equation [α] = (100α)/(lc) are reported as unitless numbers and
T
are in the form: [α]
D
±XX (c, solvent), where c is the concentration in g/l00 mL, l is the
path length in decimetres, T is the temperature in °C and D refers to the sodium
Fraunhofer line at 589 nm. High performance liquid chromatography (HPLC) analysis of
TM
organic purity was conducted on a Waters Alliance 2695 instrument using a SunFire
C18 column (5 μm, 2.1 x 150 mm) and detected using a Waters 2996 photodiode array
(PDA) detector set at 230 nm. Separation was achieved using water + 0.1%
trifluoroacetic acid (solvent A) and acetonitrile + 0.1% trifluoroacetic acid (solvent B) at
a flow rate of 0.2 mL/min and a gradient of 0% B to 100% B over 30 minutes. HPLC data
is reported as percentage purity and retention time (RT) in minutes.
4
.1.2 General Procedures
General Procedure A: N-1 Alkylation of indole and 7-azaindole derivatives
To a solution of the appropriate NH-aromatic heterocycle (1.0 mmol) in anhydrous N,N-
dimethylformamide (3 mL) at 0 °C was added sodium hydride (60% dispersion in
mineral oil, 60 mg, 1.5 eq., 1.5 mmol) and the mixture stirred for 5 minutes before the
appropriate alkyl halide (1.2 eq.) in N,N-dimethylformamide (1-3 mL) was added
dropwise. The mixture was stirred until the starting material was consumed, as
determined by thin-layer chromatography. The reaction mixture was quenched with
saturated aqueous ammonium chloride (20 mL) and extracted with ethyl acetate (3 x 20
mL). The combined organic extracts were washed with water (3 x 20 mL) and aqueous
16

ACCEPTED MANUSCRIPT
lithium chloride (1 M, 20 mL), dried over anhydrous magnesium sulfate and
concentrated under reduced pressure.
General Procedure B: Hydrolysis of Ethyl Esters
To a solution of the ester (1.0 mmol) in a mixture of tetrahydrofuran and methanol (1:1,
3
mL) was added aqueous sodium hydroxide (3.3 mL, 3 M, 10 eq., 10 mmol) and the
mixture was stirred at room temperature or reflux until the starting material was
consumed, as determined by thin-layer chromatography. The volatiles were removed
under a stream of nitrogen and the aqueous residue washed with dichloromethane (10
mL) and acidified with aqueous hydrochloric acid (6 M). The aqueous suspension was
extracted with dichloromethane (3 x 10 mL), dried over anhydrous magnesium sulfate
and concentrated under reduced pressure.
General Procedure C: EDCI-HOBt mediated amide coupling
The appropriate carboxylic acid (1.0 mmol), appropriate amine (1.2 eq.),
hydroxybenzotriazole
(203
mg,
1.5
eq.,
1.5
mmol),
1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride (288 mg, 1.5 eq., 1.5 mmol) and N,N-
diisopropylethylamine (871 μL, 5 eq., 5.0 mmol) were dissolved in N,N-
dimethylformamide (5 mL) and the mixture stirred for 24 hours. The reaction mixture
was diluted with saturated aqueous sodium hydrogen carbonate (30 mL), extracted
with ethyl acetate (3 x 20 mL), washed with saturated aqueous sodium hydrogen
carbonate (30 mL), saturated aqueous ammonium chloride (30 mL) and aqueous
lithium chloride (1 M, 30 mL), dried over anhydrous magnesium sulfate and
concentrated under reduced pressure.
General Procedure D: Hydrogenolysis of benzyl ether protecting groups
To a solution of the benzyl ether (1.0 mmol) in ethyl acetate (5 mL) and acetic acid (1-
1
0 drops) was added palladium on carbon (10% w/w, 106 mg, 0.1 eq., 100 μmol) under
an atmosphere of nitrogen. The reaction vessel was evacuated and flushed with
hydrogen three times and the mixture stirred for 16-48 hours. The suspension was
®
filtered through a pad of Celite and the filtrate concentrated under reduced pressure.
4
.1.3 Synthetic Methods
Ethyl 1-pentyl-1H-indole-2-carboxylate 38
General procedure A was followed using ethyl indole-2-carboxylate (3.0 g, 15.9 mmol)
and 1-bromopentane (2.4 mL, 19.1 mmol) to obtain the title compound 38 (4.2 g,
1
00%) as a light yellow oil, R
f
: 0.74 (1:4 ethyl acetate, hexane); IR (vmax (neat)): 2928,
−1 1
1707, 1518, 1464, 1245, 1222, 1188, 1135, 1092, 745 cm ; H NMR (300 MHz, CDCl
3
):
δ 0.75-0.88 (3H, m), 1.19-1.42 (4H, m), 1.33 (3H, t, J = 7.1 Hz), 1.64-1.82 (2H, m), 4.30
2H, q, J = 7.1 Hz), 4.42-4.56 (2H, m), 7.06 (1H, ddd, J = 8.0, 6.7, 1.2 Hz), 7.17-7.41 (3H,
(
1
3
m), 7.56-7.65 (1H, m) ppm; C NMR (75 MHz, CDCl
3
): 14.2, 14.5, 22.6, 29.3, 30.5, 44.9,
6
0.6, 110.5, 110.6, 120.5, 122.7, 124.9, 126.1, 127.6, 139.2, 162.1 ppm; LRMS (+ESI)
+
m/z: 282.2 ([M+Na] 100%).
Ethyl 1-(5-(benzyloxy)pentyl)-1H-indole-2-carboxylate 39
17

ACCEPTED MANUSCRIPT
General procedure A was followed using ethyl indole-2-carboxylate (3.0 g, 15.9 mmol)
and alkyl bromide 34 (4.1 g, 15.9 mmol). The crude product was purified by flash
column chromatography using ethyl acetate, hexane (1:19 to 1:9) as an eluent to obtain
the title compound 39 (4.6 g, 76%) as a light yellow oil, R
hexane); IR (vmax (neat)): 2928, 1707, 1454, 1246, 1178, 1092, 745 cm ; H NMR (300
MHz, CDCl ): δ 1.31 (3H, t, J = 7.1 Hz), 1.30-1.43 (2H, m), 1.50-1.63 (2H, m), 1.67-1.80
2H, m), 3.35 (2H, t, J = 6.4 Hz), 4.27 (2H, q, J = 7.1 Hz), 4.38 (2H, s), 4.43-4.52 (2H, m),
f
: 0.55 (1:4 ethyl acetate,
−1 1
3
(
1
3
7
.04 (1H, ddd, J = 8.0, 6.6, 1.3 Hz), 7.14-7.33 (8H, m), 7.53-7.62 (1H, m) ppm; C NMR
): 14.5, 23.8, 29.6, 30.6, 44.8, 60.6, 70.3, 73.0, 110.5, 110.6, 120.5, 122.7,
24.9, 124.9, 126.1, 127.6, 127.6, 127.7, 128.4, 138.7, 139.1, 162.1 ppm; LRMS (+ESI)
(75 MHz, CDCl
3
1
+
+
+
m/z: 388.2 ([M+Na] 100%); HRMS (ESI) Cald for C23
H
27NO [M+Na] : 388.18886,
3
found 388.18814.
1
-Pentyl-1H-indole-2-carboxylic acid 40
General procedure C was followed using ethyl ester 38 (4.0 g, 15.4 mmol) to obtain the
title compound 40 (3.4 g, 94%) as a white solid, mp: 108.2-111.4 °C; IR (vmax (neat)):
−
1 1
3
(
032, 2952, 2859, 2601, 1677, 1519, 1426, 1265, 1225, 1133, 910, 740 cm ; H NMR
300 MHz, CDCl ): δ 0.70-0.90 (3H, m), 1.16-1.37 (4H, m), 1.65-1.83 (2H, m), 4.50 (2H, t,
J = 7.4 Hz), 7.00-7.12 (1H, m), 7.22-7.45 (3H, m), 7.54-7.66 (1H, m) ppm; C NMR (75
3
1
3
MHz, CDCl
3
): 14.1, 22.6, 29.2, 30.5, 45.0, 110.5, 112.9, 120.8, 123.1, 125.7, 126.1, 126.3,
-
139.8, 167.2 ppm; LRMS (-ESI) m/z: 230.0 ([M-H] 100%).
1
-(5-(Benzyloxy)pentyl)-1H-indole-2-carboxylic acid 41
General procedure B was followed using ethyl ester 39 (4.0 g, 10.9 mmol) at reflux for 3
hours. The crude product was purified by flash column chromatography using
methanol, dichloromethane (0:1 to 1:19) as an eluent to obtain the title compound 41
(
(
3.7 g, 100%) as a viscous yellow oil, R
neat)): 2921, 2859, 2588, 1677, 1519, 1237, 1093, 903, 728 cm ; H NMR (300 MHz,
): δ 1.39-1.61 (2H, m), 2.84 (2H, quin, J = 6.7 Hz), 1.89 (2H, quin, J = 7.7 Hz), 3.51
2H, t, J = 6.3 Hz), 4.54 (2H, s), 4.58-4.68 (2H, m), 7.20 (1H, ddd, J = 7.9, 6.3, 1.5 Hz),
f
: 0.30 (1:49 ethyl acetate, hexane); IR (vmax
−1 1
CDCl
(
7
3
1
3
.26-7.54 (8H, m), 7.75 (1H, d, J = 8.1 Hz) ppm; 13C NMR (75 MHz, CDCl
0.5, 44.8, 70.2, 73.0, 110.8, 112.9, 120.8, 123.1, 125.7, 126.0, 126.3, 127.6, 127.8, 128.5,
3
): 23.7, 29.6,
+
+
38.6, 139.8, 167.0 ppm; LRMS (+ESI) m/z: 360.2 ([M+Na] 100%); HRMS (ESI) Cald
+
for C21
H
23NO
3
[M+Na] : 360.15756, found 360.15681.
N-(-Adamantan-1-yl)-1-pentyl-1H-indole-2-carboxamide 8
General procedure C was followed using carboxylic acid 40 (120 mg, 520 μmol) and 1-
adamantylamine (94 mg, 620 μmol). The crude product was purified by flash column
chromatography using ethyl acetate, hexane (0:1 to 1:19) as an eluent and
recrystallization from dichloromethane, hexane to obtain the title compound 8 (190 mg,
7
7%) as a white solid, the spectroscopic data of which corresponded to previously
22
described. mp: 163.3-163.7 °C; R
f
: 0.41 (1:19 ethyl acetate, hexane); IR (vmax (neat)):
−1
3
290, 2906, 2853, 1632, 1535, 1454, 1343, 1280, 1220, 808, 747, 730 cm ; LRMS
+
+
+
(+ESI) m/z: 387.3 ([M+Na] 100%), 751.4 ([2M+Na] 24%); HRMS (ESI) Cald for
+
C
24
H
32
N
2
O [M+Na] : 387.24123, found 387.24047; HPLC: 99.8%, RT: 33.2 mins.
(
S)-Methyl 3-methyl-2-(1-pentyl-1H-indole-2-carboxamido)butanoate 9
General procedure C was followed using carboxylic acid 40 (200 mg, 870 μmol) and
methyl- -valinate hydrochloride (170 mg, 1.0 mmol). The crude product was purified by
L
18

ACCEPTED MANUSCRIPT
flash column chromatography using ethyl acetate, hexane (1:9) as an eluent and
recrystallization from hexane to obtain the title compound 9 (190 mg, 64%) as a
colourless solid, mp: 72.9-73.8 °C; R : 0.69 (2:3 ethyl acetate, hexane); IR (vmax (neat)):
f
−1 1
3
365, 3282, 3195, 2957, 1650, 1630, 1530, 1460, 1303, 748 cm ; H NMR (400 MHz,
CDCl ): δ 0.83-0.91 (3H, m), 1.00 (3H, d, J = 6.9 Hz), 1.04 (3H, d, J = 6.9 Hz), 1.25-1.37
3
(
4H, m), 1.74-1.86 (2H, m), 2.24-2.36 (1H, m), 3.79 (3H, s), 4.48-4.56 (2H, m), 4.76 (1H,
dd, J = 8.9, 5.0 Hz), 6.66 (1H, d, J = 8.9 Hz), 6.95 (1H, s), 7.14 (1H, ddd, J = 7.9, 6.9, 1.0 Hz),
7
.31 (1H, ddd, J = 8.4, 6.9, 1.2 Hz), 7.37-7.42 (1H, m), 7.65 (1H, dt, J = 8.0, 1.0 Hz) ppm;
1
3
3
C NMR (100 MHz, CDCl ): δ 14.1, 18.1, 19.2, 21.6, 29.3, 30.5 , 31.8, 44.7, 52.4, 57.2,
1
04.5, 110.6, 120.6, 122.1, 124.2, 126.2, 131.4, 138.6, 162.5, 172.6 ppm; LRMS (+ESI)
+
+
+
m/z: 352.2 ([M+Na] 100%); HRMS (ESI) Cald for C20
found 367.19903; [α]
H
28
N
2
O
3
[M+Na] : 367.19976,
2
7
D
: +35.2 (0.62, CHCl
3
); HPLC: 95.8%, RT: 31.1 mins.
(
S)-Methyl 3,3-dimethyl-2-(1-pentyl-1H-indole-2-carboxamido)butanoate 11
General procedure C was followed using carboxylic acid 40 (250 mg, 1.1 mmol) and
amine hydrochloride salt 37 (230 mg, 1.3 mmol). The crude product was purified by
flash column chromatography using ethyl acetate, hexane (0:1 to 1:19) as an eluent to
obtain the title compound 11 (210 mg, 54%) as a colourless oil, R
f
: 0.42 (1:19 ethyl
acetate, hexane); IR (vmax (neat)): 3256, 2952, 1737, 1633, 1622, 1536, 1251, 1014, 741
−
1 1
cm ; H NMR (400 MHz, CDCl
.79 (2H, quin, J = 7.7 Hz), 3.78 (3H, s), 4.49-4.55 (2H, m), 4.67 (1H, d, J = 9.6 Hz), 6.70
1H, d, J = 9.5 Hz), 6.93 (1H, d, J = 0.8 Hz), 7.14 (1H, ddd, J = 7.9, 6.9, 1.0 Hz), 7.31 (1H,
3
): δ 0.83-0.89 (3H, m), 1.07 (9H, s), 1.26-1.37 (4H, m),
1
(
1
3
ddd, J = 8.4, 6.9, 1.2 Hz), 7.37-7.42 (1H, m), 7.65 (1H, dt, J = 8.0, 1.0 Hz) ppm; C NMR
100 MHz, CDCl ): δ 14.1, 22.6, 26.8, 29.3, 30.5, 35.4, 44.8, 52.1, 60.0, 104.4, 110.6,
20.6, 122.0, 124.2, 126.2, 131.5, 138.6, 162.3, 172.2 ppm; LRMS (+ESI) m/z: 381.2
[M+Na] 100%), 739.3 ([2M+Na] 43%); HRMS (ESI) Cald for C21
81.21541, found 381.21466; [α]
(
3
1
(
3
+
+
+
+
H
30
N
O
2 3
[M+Na] :
2
7
D
: +21.1 (1.42, CHCl
3
); HPLC: 99.3%, RT: 31.7 mins.
(
S)-N-(1-Amino-3-methyl-1-oxobutan-2-yl)-1-pentyl-1H-indole-2-carboxamide 14
General procedure C was followed using carboxylic acid 40 (120 mg, 520 μmol) and
L
-
valinamide hydrochloride (95 mg, 620 μmol). The crude product was purified by
recrystallization from ethyl acetate to obtain the title compound 14 (170 mg, 100%) as
a white solid, mp: 246.1-248.9 °C; R : 0.55 (9:1 ethyl acetate, hexane); IR (vmax (neat)):
f
−1 1
3
365, 3282, 3195, 2957, 1650, 1630, 1530, 1460, 1303, 748 cm ; H NMR (400 MHz,
DMSO-d ): δ 0.80 (3H, t, J = 7.1 Hz), 0.93 (3H, d, J = 6.8 Hz), 0.95 (3H, d, J = 6.8 Hz), 1.11-
6
1
7
1
=
4
.29 (4H, m), 1.65 (2H, quin, J = 7.4 Hz), 2.11 (1H, sept, J = 6.9 Hz), 4.26 (1H, dd, J = 8.9,
.5 Hz), 4.51 (2H, t, J = 7.3 Hz), 7.05-7.13 (2H, m), 7.16 (1H, s), 7.25 (1H, ddd, J = 8.3, 6.9,
.2 Hz), 7.44 (1H, bs), 7.53 (1H, dd, J = 8.4, 0.9 Hz), 7.64 (1H, d, J = 7.9 Hz), 8.14 (1H, d, J
13
8.9 Hz) ppm; C NMR (100 MHz, DMSO-d
6
): δ 13.8, 18.5, 19.5, 21.9, 28.4, 29.9, 30.1,
3.5, 58.3, 105.0, 110.5, 120.0, 121.6, 123.4, 125.7, 131.7, 137.7, 161.8, 172.9 ppm;
+
+
+
LRMS (+ESI) m/z: 352.2 ([M+Na] 100%); HRMS (ESI) Cald for C19
H
27
N
O
3 2
[M+Na] :
2
7
352.20010, found 352.19950; [α]
D
: +70.2 (0.57, DMSO); HPLC: 98.8%, RT: 26.9 mins.
(S)-N-(1-Amino-3,3-dimethyl-1-oxobutan-2-yl)-1-pentyl-1H-indole-2-carboxamide 16
General procedure C was followed using carboxylic acid 40 (200 mg, 870 μmol), and
amine hydrochloride salt 36 (170 mg, 1.0 mmol). The crude product was purified by
flash column chromatography using ethyl acetate, hexane (3:7 to 2:3) as an eluent to
obtain the title compound 16 (190 mg, 64%) as a colourless solid, mp: 92.9-96.6 °C; R
f
:
0.31 (1:1 ethyl acetate, hexane); IR (vmax (neat)): 3370, 3196, 2955, 1638, 1523, 1399,
19

ACCEPTED MANUSCRIPT
−
1 1
1
1
6
228, 741 cm ; H NMR (400 MHz, CDCl ): δ 0.86 (3H, t, J = 6.8 Hz), 1.12 (9H, s), 1.25-
3
.37 (4H, m), 1.72-1.82 (2H, m), 4.41-4.56 (2H, m), 4.61 (1H, d, J = 9.4 Hz), 5.99 (1H, bs),
.42 (1H, bs), 6.96 (1H, s), 7.00 (1H, d, J = 9.5 Hz), 7.13 (1H, ddd, J = 7.9, 6.9, 1.1 Hz), 7.30
1H, ddd, J = 8.4, 6.9, 1.2 Hz), 7.35-7.40 (1H, m), 7.61 (1H, dt, J = 8.0, 1.0 Hz) ppm; C
NMR (100 MHz, CDCl ): δ 14.1, 22.6, 26.8, 29.3, 30.5, 35.0, 44.7, 60.0, 104.9, 110.5,
20.7, 122.1, 124.2, 126.3, 131.4, 138.5, 162.6, 172.8 ppm; LRMS (+ESI) m/z: 366.2
[M+Na] 100%); HRMS (ESI) Cald for C20
66.21501; [α]
1
3
(
3
1
(
3
+
+
+
H
N O
29 3 2
[M+Na] : 366.21575, found
2
7
D
: +48.2 (0.52, DMSO); HPLC: 99.6%, RT: 28.1 mins.
1
-Pentyl-N-(2-phenylpropan-2-yl)-1H-indole-2-carboxamide 18
General procedure C was followed using carboxylic acid 40 (150 mg, 650 μmol) and 1-
methyl-1-phenylethylamine (110 μL, 780 μmol). The crude product was purified by
flash column chromatography using ethyl acetate, hexane (1:19 to 1:9) as an eluent and
recrystallization from dichloromethane, hexane to obtain the title compound 40 (130
mg, 58%) as a white solid, mp: 122.6-124.1 °C; R
max (neat)): 3412, 3253, 2928, 1635, 1538, 1457, 729 cm ; H NMR (400 MHz, CDCl
δ 0.86 (3H, t, J = 6.8 Hz), 1.12 (9H, s), 1.20-1.36 (4H, m), 1.74 (2H, quin, J = 7.5 Hz), 1.83
6H, s), 4.47-4.54 (2H, m), 6.50 (1H, bs), 6.87 (1H, d, J = 0.8 Hz), 7.14 (1H, ddd, J = 8.0,
f
: 0.55 (1:4 ethyl acetate, hexane); IR
−1 1
(v
3
):
(
6
1
1
.9, 1.0 Hz), 7.23-7.33 (2H, m), 7.33-7.40 (3H, m), 7.46-7.51 (2H, m), 7.64 (1H, dt, J = 8.1,
1
3
3
.0 Hz) ppm; C NMR (100 MHz, CDCl ): δ 14.1, 22.6, 26.8, 29.3, 30.5, 35.0, 44.7, 60.0,
04.9, 110.5, 120.7, 122.1, 124.2, 126.3, 131.4, 138.5, 162.6, 172.8 ppm; LRMS (+ESI)
+
+
+
m/z: 371.3 ([M+Na] 100%); HRMS (ESI) Cald for C23
H
28
N O [M+Na] : 371.20993,
2
found 371.20918; HPLC: 99.8%, RT: 31.8 mins.
(
S)-Methyl 2-(1-(5-(benzyloxy)pentyl)-1H-indole-2-carboxamido)-3-methylbutanoate 42
General procedure C was followed using carboxylic acid 41 (300 mg, 930 μmol) and
methyl- -valinate hydrochloride (190 mg, 1.1 mmol). The crude product was purified
by flash column chromatography using ethyl acetate, hexane (1:9) as an eluent to obtain
the title compound 42 (260 mg, 62%) as a light yellow oil, R : 0.15 (1:9 ethyl acetate,
hexane); IR (vmax (neat)): 3278, 2932, 1738, 1656, 1523, 1455, 1204, 1090, 743 cm ; H
NMR (300 MHz, CDCl ): δ 1.02 (3H, d, J = 6.9 Hz), 1.06 (3H, d, J = 6.9 Hz), 1.38-1.53 (2H,
L
f
−1 1
3
m), 1.60-1.74 (2H, m), 1.85 (2H, quin, J = 7.7 Hz), 2.20-2.42 (1H, m), 3.46 (2H, t, J = 6.5
Hz), 3.81 (3H, s), 4.49 (2H, s), 4.52-4.62 (2H, m), 4.77 (1H, dd, J = 8.9, 4.9 Hz), 6.68 (1H,
d, J = 8.9 Hz), 6.97 (1H, s), 7.17 (1H, ddd, J = 7.9, 6.8, 1.1 Hz), 7.25-7.48 (7H, m), 7.68 (1H,
1
3
dt, J = 8.0, 1.0 Hz) ppm; C NMR (75 MHz, CDCl
3
): δ 18.1, 19.2, 23.8, 29.6, 30.6, 31.8,
4
4.7, 52.4, 57.2, 70.3, 73.0, 104.6, 110.5, 120.6, 122.1, 123.2, 126.2, 127.6, 127.7, 128.5,
+
1
31.3, 138.6, 138.8, 162.4, 172.6 ppm; LRMS (+ESI) m/z: 473.3 ([M+Na] 100%); HRMS
ESI) Cald for C27
+
+
(
H
N O
34 2 4
[M+Na] : 473.24163, found 473.24102.
(
4
S)-Methyl 2-(1-(5-(benzyloxy)pentyl)-1H-indole-2-carboxamido)-3,3-dimethylbutanoate
3
General procedure C was followed using carboxylic acid 41 (300 mg, 930 μmol) and
amine hydrochloride salt 37 (200 mg, 1.1 mmol). The crude product was purified by
flash column chromatography using ethyl acetate, hexane (0:1 to 1:9) as an eluent to
obtain the title compound 43 (170 mg, 40%) as a colourless oil, R : 0.25 (1:9 ethyl
f
−1
acetate, hexane); IR (vmax (neat)): 3260, 2939, 1736, 1639, 1534, 1453, 1085, 742 cm ;
1
H NMR (300 MHz, CDCl
.75 (2H, quin, J = 7.7 Hz), 3.36 (2H, t, J = 6.5 Hz), 3.69 (3H, s), 4.39 (2H, s), 4.41-4.50
2H, m), 4.58 (1H, d, J = 9.5 Hz), 6.62 (1H, d, J = 9.6 Hz), 6.85 (1H, s), 7.07 (1H, ddd, J =
3
): δ 0.99 (9H, s), 1.27-1.43 (2H, m), 1.56 (2H, quin, J = 6.7 Hz),
1
(
20

ACCEPTED MANUSCRIPT
8
CDCl
.0, 6.8, 1.1 Hz), 7.14-7.35 (7H, m), 7.57 (1H, d, J = 7.9 Hz) ppm; 13C NMR (75 MHz,
3
): δ 23.8, 26.8, 29.7, 30.6, 35.3, 44.7, 52.1, 60.0, 70.3, 73.0, 104.5, 110.6, 120.6,
1
22.1, 124.3, 126.2, 127.6, 127.7, 128.5, 131.4, 138.6, 138.8, 162.2, 172.1 ppm; LRMS
+
+
+
(+ESI) m/z: 487.3 ([M+Na] 100%); HRMS (ESI) Cald for C28
H
36
N
2
O [M+Na] :
4
487.25728, found 487.25661.
(
S)-N-(1-Amino-3-methyl-1-oxobutan-2-yl)-1-(5-(benzyloxy)pentyl)-1H-indole-2-
carboxamide 44
General procedure C was followed using carboxylic acid 41 (350 mg, 1.1 mmol) and
L-
valinamide hydrochloride (200 mg, 1.3 mmol). The crude product was purified by
recrystallization from ethyl acetate, hexane to obtain the title compound 44 (320 mg,
6
(
8%) as a white solid, mp: 179.1-182.5 °C; R
neat)): 3404, 3278, 2856, 1673, 1623, 1532, 1455, 1255, 1088 cm ; H NMR (300
MHz, DMSO-d ): δ 0.94 (6H, d, J = 6.8 Hz), 1.19-1.38 (2H, m), 1.51 (2H, quin, J = 6.7 Hz),
.67 (2H, quin, J = 7.1 Hz), 2.12 (1H, sept, J = 6.8 Hz), 3.35 (2H, t, J = 6.7 Hz), 4.28 (1H, t, J
8.1 Hz), 4.49 (2H, s), 4.51 (2H, t, J = 7.2 Hz), 7.03-7.37 (9H, m), 7.45 (1H, bs, NH), 7.53
f
: 0.46 (4:1 ethyl acetate, hexane); IR (vmax
−1 1
6
1
=
(
1H, d, J = 8.4 Hz), 7.64 (1H, d, J = 7.9 Hz), 8.13 (1H, d, J = 8.9 Hz) ppm; 13C NMR (75
): δ 18.5, 19.4, 23.0, 28.9, 30.1, 43.6, 58.3, 69.4, 71.7, 115.0, 110.6, 120.0,
21.6, 123.4, 125.7, 127.2, 127.3, 128.2, 131.6, 137.7, 138.7, 161.8, 172.9 ppm; LRMS
MHz, DMSO-d
1
6
+
+
+
(+ESI) m/z: 458.3 ([M+Na] 100%); HRMS (ESI) Cald for C26
H
33
N
3
O [M+Na] :
3
458.24196, found 458.24111.
(S)-N-(1-Amino-3,3-dimethyl-1-oxobutan-2-yl)-1-(5-(benzyloxy)pentyl)-1H-indole-2-
carboxamide 45
General procedure C was followed using carboxylic acid 41 (300 mg, 930 μmol) and
amine hydrochloride salt 36 (190 mg, 1.1 mmol). The crude product was purified by
flash column chromatography using ethyl acetate, hexane (2:3 to 1:1) as an eluent to
obtain the title compound 45 (290 mg, 70%) as a white foam, R
hexane); IR (vmax (neat)): 3251, 2939, 1638, 1536, 1453, 1084, 742 cm ; H NMR (300
MHz, CDCl ): δ 1.13 (9H, s), 1.35-1.50 (2H, m), 1.65 (2H, quin, J = 6.7 Hz), 1.83 (2H, quin,
f
: 0.35 (1:1 ethyl acetate,
−1 1
3
J = 7.6 Hz), 3.45 (2H, t, J = 6.4 Hz), 4.46-4.57 (4H, m), 4.60 (1H, d, J = 9.4 Hz), 6.00 (1H,
bs), 6.40 (1H, bs), 6.95-7.04 (2H, m), 7.16 (1H, ddd, J = 7.9, 6.7, 1.2 Hz), 7.25-7.45 (7H,
13
m), 7.66 (1H, d, J = 8.0 Hz) ppm; C NMR (75 MHz, CDCl
3
): δ 23.8, 26.8, 29.6, 30.6, 35.0,
4
1
4.6, 60.0, 70.2, 73.0, 104.9, 110.5, 120.6, 122.1, 124.3, 126.2, 127.6, 127.7, 128.5, 131.3,
+
+
38.6, 138.7, 162.6, 172.8 ppm; LRMS (+ESI) m/z: 472.3 ([M+Na] 100%); HRMS (ESI)
+
Cald for C27
H
N O
35 3 3
[M+Na] : 472.25761, found 472.25680.
1
-(5-(Benzyloxy)pentyl)-N-(2-phenylpropan-2-yl)-1H-indole-2-carboxamide 46
General procedure C was followed using carboxylic acid 41 (220 mg, 695 μmol) and 1-
methyl-1-phenylethylamine (120 μL, 830 μmol). The crude product was purified by
flash column chromatography using ethyl acetate, hexane (1:9) as an eluent to obtain
the title compound 46 (260 mg, 62%) as a white solid, mp: 77.1-80.2 °C; R : 0.52 (1:4
f
−1
ethyl acetate, hexane); IR (vmax (neat)): 3259, 2937, 1638, 1536, 1452, 1270, 747 cm ;
1
H NMR (300 MHz, CDCl ): δ 1.32-1.48 (2H, m), 1.55-1.72 (2H, m), 1.74-1.86 (2H, m),
3
1
6
.84 (6H, s), 3.44 (2H, t, J = 6.5 Hz), 4.48 (2H, s), 4.53 (2H, t, J = 7.4 Hz), 6.50 (1H, bs),
.89 (1H, s), 7.13-7.21 (1H, m), 7.23-7.54 (12H, m), 7.66 (1H, d, J = 7.9 Hz) ppm; 1
3
C
3
NMR (75 MHz, CDCl ): δ 23.7, 29.5, 29.6, 30.6, 44.3, 56.4, 70.3, 73.0, 103.7, 110.5, 120.5,
121.8, 123.9, 124.8, 126.2, 126.9, 127.6, 127.7, 128.5, 128.7, 132.7, 138.4, 138.8, 146.9,
21

ACCEPTED MANUSCRIPT
+
+
1
61.9 ppm; LRMS (+ESI) m/z: 477.3 ([M+Na] 100%); HRMS (ESI) Cald for C30
H
34 2 2
N O
+
[M+Na] : 477.25180, found 477.25092.
(
S)-Methyl 2-(1-(5-hydroxypentyl)-1H-indole-2-carboxamido)-3-methylbutanoate 10
General procedure D was followed using benzyl ether 42 (190 mg, 420 μmol). The
crude residue was purified by flash column chromatography using ethyl acetate, hexane
(
3:7 to 2:3) as an eluent to obtain the title compound 10 (100 mg, 67%) as a white solid,
mp: 87.1-90.8 °C; R : 0.10 (3:7 ethyl acetate, hexane); IR (vmax (neat)): 3395, 3290,
945, 1745, 1637, 1529, 1459, 1203, 1147, 1007, 748 cm ; H NMR (400 MHz, CDCl
δ 1.01 (3H, d, J = 6.9 Hz), 1.04 (3H, d, J = 6.8 Hz), 1.33-1.43 (2H, m), 1.50-1.60 (2H, m),
.70 (1H, bs), 1.83 (2H, quin, J = 7.4 Hz), 2.22-2.35 (1H, m), 3.59 (2H, t, J = 6.4 Hz), 3.79
3H, s), 4.45-4.65 (2H, m), 4.72 (1H, dd, J = 8.9, 5.0 Hz), 6.66 (1H, d, J = 8.9 Hz), 6.95 (1H,
d, J = 0.8 Hz), 7.14 (1H, ddd, J = 8.0, 6.9, 1.0 Hz), 7.31 (1H, ddd, J = 8.4, 6.9, 1.2 Hz), 7.36-
f
−1 1
2
3
):
1
(
1
3
7
.41 (1H, m), 7.65 (1H, dt, J = 8.0, 1.0 Hz) ppm; C NMR (100 MHz, CDCl ): δ 18.1, 19.2,
3
2
3.3, 30.4, 31.6, 32.5, 44.5, 52.5, 57.3, 62.8, 104.7, 110.5, 120.7, 122.1, 124.3, 126.3,
+
+
131.4, 138.5, 162.6, 172.8 ppm; LRMS (+ESI) m/z: 383.2 ([M+Na] 100%); HRMS (ESI)
+
27
Cald for C20
H
N O
28 2 4
[M+Na] : 383.19467, found 383.19401; [α]
D
: +6.8 (0.75, CHCl
3
);
HPLC: 98.6%, RT: 25.1 mins.
(S)-Methyl 2-(1-(5-hydroxypentyl)-1H-indole-2-carboxamido)-3,3-dimethylbutanoate 12
General procedure D was followed using benzyl ether 43 (120 mg, 260 μmol). The
crude residue was purified by flash column chromatography using ethyl acetate, hexane
(
1:9 to 3:7) as an eluent to obtain the title compound 12 (84 mg, 87%) as a colourless
oil, R : 0.09 (3:7 ethyl acetate, hexane); IR (vmax (neat)): 3357, 2935, 1732, 1647, 1523,
458, 1320, 1215, 1163, 741 cm ; H NMR (400 MHz, CDCl
2H, m), 1.51-1.65 (3H, m), 1.83 (1H, quin, J = 7.4 Hz), 3.60 (2H, t, J = 6.4 Hz), 3.78 (3H,
f
−1 1
1
(
3
): δ 1.07 (9H, s), 1.33-1.43
s), 4.47-4.62 (2H, m), 4.63 (1H, d, J = 9.5 Hz), 6.70 (1H, d, J = 9.5 Hz), 6.93 (1H, d, J = 0.8
Hz), 7.15 (1H, ddd, J = 7.9, 6.9, 1.0 Hz), 7.31 (1H, ddd, J = 8.3, 6.9, 1.2 Hz), 7.36-7.41 (1H,
1
3
m), 7.65 (1H, dt, J = 8.0, 1.0 Hz) ppm; C NMR (100 MHz, CDCl
3
): δ 23.3, 26.8, 30.4, 32.5,
3
1
2.5, 44.6, 52.1, 60.1, 62.8, 104.6, 110.5, 120.7, 122.1, 124.3, 126.3, 131.5, 138.5, 162.4,
+
+
72.3 ppm; LRMS (+ESI) m/z: 397.2 ([M+Na] 100%); HRMS (ESI) Cald for C21
H
30 2 4
N O
+
27
[M+Na] : 397.21033, found 397.20957; [α]
D
: +13.3 (0.58, CHCl ); HPLC: 97.8%, RT:
3
26.4 mins.
(
1
S)-N-(1-Amino-3-methyl-1-oxobutan-2-yl)-1-(5-hydroxypentyl)-1H-indole-2-carboxamide
5
A modified version of general procedure D was followed using benzyl ether 44 (250 mg,
70 μmol) in a mixture of methanol (5 mL) and tetrahydrofuran (5 mL). The crude
residue was purified by recrystallization from isopropanol, diethyl ether to obtain the
title compound 15 (170 mg, 83%) as a white solid, mp: 220.8-221.2 °C; R : 0.10 (4:1
ethyl acetate, hexane); IR (vmax (neat)): 3382, 3263, 2934, 1622, 1536, 1265, 1253, 741
5
f
−
1 1
cm ; H NMR (400 MHz, DMSO-d
6
): δ 0.88-0.99 (6H, m), 1.17-1.30 (2H, m), 1.39 (2H,
quin, J = 6.9 Hz), 1.65 (2H, quin, J = 7.4 Hz), 2.12 (1H, sept, J = 6.8 Hz), 3.28-3.40 (2H, m),
4
7
.23-4.35 (2H, m), 4.50 (2H, t, J = 7.3 Hz), 7.05-7.14 (2H, m), 7.17 (1H, s), 7.25 (1H, t, J =
.7 Hz), 7.46 (1H, bs), 7.53 (1H, d, J = 8.4 Hz), 7.64 (1H, d, J = 7.9 Hz), 8.15 (1H, d, J = 8.9
1
3
Hz) ppm; C NMR (100 MHz, DMSO-d
6
): δ 18.5, 19.5, 22.9, 30.1, 30.2, 32.3, 43.7, 58.3,
6
0.6, 105.0, 110.6, 120.0, 121.6, 123.5, 125.7, 131.6, 137.7, 161.8, 172.9 ppm; LRMS
+
+
+
(
+ESI) m/z: 368.3 ([M+Na] 100%); HRMS (ESI) Cald for C19
68.19501, found 368.19431; [α]
H
27
N
3
O [M+Na] :
3
2
7
3
D
: +33.3 (0.75, DMSO); HPLC: 95.2%, RT: 21.5 mins.
22

ACCEPTED MANUSCRIPT
(S)-N-(1-Amino-3,3-dimethyl-1-oxobutan-2-yl)-1-(5-hydroxypentyl)-1H-indole-2-
carboxamide 17
A modified version of general procedure D was followed using benzyl ether 45 (240 mg,
5
40 μmol) in a mixture of methanol (5 mL) and tetrahydrofuran (5 mL). The crude
residue was purified by flash column chromatography using ethyl acetate, hexane (3:2
to 7:3) as an eluent and recrystallization from dichloromethane, hexane to obtain the
title compound 17 (160 mg, 84%) as a white solid, mp: 140.8-144.7 °C; R
f
: 0.08 (3:2
ethyl acetate, hexane); IR (vmax (neat)): 3415, 3331, 3189, 2933, 1632, 1527, 1457,
−
1 1
1
1
5
7
8
3
1
C
9
403, 1354, 1314 cm ; H NMR (400 MHz, DMSO-d ): δ 1.02 (9H, s), 1.18-1.29 (2H, m),
6
.39 (2H, quin, J = 6.9 Hz), 1.65 (1H, quin, J = 7.5 Hz), 3.29-3.37 (2H, m), 4.31 (1H, t, J =
.1 Hz), 4.41 (1H, d, J = 9.6 Hz), 4.46-4.53 (2H, m), 7.09 (1H, ddd, J = 7.9, 6.9, 0.9 Hz),
.13-7.20 (2H, m), 7.26 (1H, ddd, J = 8.3, 7.0, 1.2 Hz), 7.50-7.56 (2H, m), 7.64 (1H, dt, J =
.0, 0.9 Hz), 7.80 (1H, d, J = 9.6 Hz) ppm; 13C NMR (100 MHz, DMSO-d
): δ 23.3, 27.4,
0.7, 32.7, 34.6, 44.3, 60.6, 61.0, 105.4, 111.0, 120.5, 122.1, 124.0, 126.2, 132.1, 138.2,
6
+
+
62.1, 172.3 ppm; LRMS (+ESI) m/z: 382.2 ([M+Na] 100%); HRMS (ESI) Cald for
[M+Na] : 382.21066, found 382.20991; [α]
8.2%, RT: 22.7 mins.
+
27
20
H
N O
29 3 3
D
: +48.4 (0.62, DMSO); HPLC:
1
-(5-Hydroxypentyl)-N-(2-phenylpropan-2-yl)-1H-indole-2-carboxamide 19
General procedure D was followed using benzyl ether 46 (140 mg, 310 μmol). The
crude residue was purified by flash column chromatography using ethyl acetate, hexane
(
3:7 to 2:3) as an eluent to obtain the title compound 19 (90 mg, 80%) as a white solid,
mp: 103.1-106.2 °C; R : 0.12 (3:7 ethyl acetate, hexane); IR (vmax (neat)): 3290, 2935,
632, 1529, 1447, 1354, 1271, 981, 759 cm ; H NMR (400 MHz, CDCl
2H, m), 1.49 (2H, m), 1.60 (1H, bs), 1.72-1.83 (2H, m), 1.82 (6H, s), 3.56 (2H, t, J = 6.5
Hz), 4.45-4.53 (2H, m), 6.53 (1H, bs), 6.88 (1H, d, J = 0.8 Hz), 7.14 (1H, ddd, J = 8.0, 6.9,
.0 Hz), 7.23-7.32 (2H, m), 7.33-7.39 (3H, m), 7.45-7.50 (2H, m), 7.64 (1H, dt, J = 8.1, 1.0
f
−1 1
1
3
): δ 1.29-1.39
(
1
1
3
Hz) ppm; C NMR (100 MHz, CDCl
3
): δ 23.2, 29.5, 30.4, 32.4, 44.3, 56.4, 62.7, 103.8,
1
10.5, 120.5, 121.9, 124.0, 124.8, 126.2, 126.9, 128.7, 132.6, 138.3, 146.9, 161.9 ppm;
+
+
+
LRMS (+ESI) m/z: 387.3 ([M+Na] 100%); HRMS (ESI) Cald for C23
87.20485, found 387.20411; HPLC: 98.1%, RT: 26.8 mins.
H
28
N
2
O [M+Na] :
2
3
(S)-N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-(5,5-difluoropentyl)-1H-indole-2-
carboxamide 13
To a solution of alcohol 17 (70 mg, 196 μmol) in a mixture of dichloromethane (1 mL)
and tetrahydrofuran (1 mL) at 0 °C was added Dess-Martin periodinane (100 mg, 236
μmol) and the mixture was warmed to room temperature and stirred for 16 hours. The
solids were removed by filtration through a pad of silica and the filtrate concentrated
under reduced pressure to obtain the crude aldehyde intermediate, which was used
immediately without further purification or characterization.
The crude aldehyde (50 mg, 140 μmol) was taken up in dichloromethane (2 mL) and
cooled to 0 °C. Diethylaminosulfur trifluoride (43 μL, 322 μmol) was added slowly and
the mixture was warmed to room temperature and stirred for 24 hours. The reaction
mixture was quenched with saturated aqueous sodium hydrogen carbonate (10 mL)
and the mixture extracted with dichloromethane (3 x 10 mL). The combined organic
extracts were dried over anhydrous magnesium sulfate, concentrated under reduced
pressure and the crude product purified by flash column chromatography using ethyl
acetate, hexane (1:9 to 1:1) as an eluent to obtain the title compound 13 (30 mg, 40%)
23

ACCEPTED MANUSCRIPT
as a colourless foam, R
f
: 0.70 (7:3 ethyl acetate, hexane); IR (vmax (neat)): 3323, 3196,
−1 1
2
961, 2928, 1641, 1525, 1455, 1403, 1123, 1026, 735 cm ; H NMR (400 MHz, CDCl
δ 1.11 (9H, s), 1.42-1.57 (2H, m), 1.78-1.94 (4H, m), 4.45 (1H, d, J = 9.3 Hz), 4.51-4.60
2H, m), 5.56 (1H, bs), 5.73 (1H, bs), 5.77 (1H, tt, J = 56.8, 4.5 Hz), 6.91 (1H, d, J = 9.1 Hz),
.97 (1H, s), 7.15 (1H, ddd, J = 8.0, 6.7, 1.0 Hz), 7.29-7.40 (2H, m), 7.65 (1H, d, J = 8.0 Hz)
3
):
(
6
13
ppm; C NMR (100 MHz, CDCl
3
): δ 19.8 (t, J = 5.7 Hz), 26.8, 30.0, 33.9 (t, J = 20.9 Hz),
3
1
4
4
5.0, 44.3, 60.2, 105.0, 110.3, 117.3 (t, J = 238.9 Hz), 120.8, 122.3, 124.5, 126.3, 131.2,
1
9
38.5, 162.5, 172.4 ppm; F NMR (376 MHz, CDCl
3
): δ -115.9 ppm; LRMS (+ESI) m/z:
+
+
+
02.2 ([M+Na] 100%); HRMS (ESI) Cald for C20
H
27
F
N O
2 3 2
[M+Na] : 402.19690, found
2
7
02.19702; [α]
D
: +10.3 (0.66, CHCl ); HPLC: 96.8%, RT: 22.4 mins.
3
1
-Pentyl-1H-pyrrolo[2,3-b]pyridine 47
General procedure A was followed using 7-azaindole (2.0 g, 16.9 mmol) and 1-
bromopentane (2.5 mL, 20.5 mmol) to obtain the title compound 47 (3.2 g, 100%) as a
35
yellow oil, the spectroscopic data of which corresponded to previously described. R :
f
0.58 (2:3 ethyl acetate, hexane).
1
-(5-(Benzyloxy)pentyl)-1H-pyrrolo[2,3-b]pyridine 48
General procedure A was followed using 7-azaindole (1.5 g, 12.7 mmol) and alkyl
bromide 34 (3.9 g, 15.2 mmol). The crude residue was purified by flash column
chromatography using ethyl acetate, hexane (1:9 to 1:4) as an eluent to obtain the title
compound 48 (3.1 g, 85%) as a light yellow oil, R
max (neat)): 2935, 2859, 1509, 1425, 1306, 1094, 796, 773 cm ; H NMR (400 MHz,
CDCl ): δ 1.41-1.51 (2H, m), 1.63-1.73 (2H, m), 1.93 (2H, quin, J = 7.4 Hz), 3.47 (2H, t, J =
f
: 0.39 (1:4 ethyl acetate, hexane); IR
−1 1
(v
3
6
.5 Hz), 4.32 (2H, t, J = 7.2 Hz), 4.50 (2H, s), 6.47 (1H, d, J = 3.5 Hz), 7.07 (1H, dd, J = 7.8,
.7 Hz), 7.23 (1H, d, J = 3.5 Hz), 7.27-7.40 (5H, m), 7.93 (1H, dd, J = 7.8, 1.6 Hz), 8.35 (1H,
4
1
3
dd, J = 4.7, 1.6 Hz) ppm; C NMR (100 MHz, CDCl
3
): δ 23.7, 29.5, 30.3, 44.6, 70.2, 73.0,
9
9.4, 115.6, 120.7, 127.6, 127.7, 128.1, 128.4, 128.8, 138.7, 142.7, 147.5 ppm; LRMS
+
+
+
(+ESI) m/z: 295.2 ([M+H] 62%), 317.2 ([M+Na] 100%); HRMS (ESI) Cald for
+
C
19
H
N
22 2
O [M+Na] : 317.16298, found 317.16232.
2
,2,2-Trifluoro-1-(1-pentyl-1H-pyrrolo[2,3-b]pyridin-3-yl)ethanone 49
To a solution of 47 (3.0 g, 15.9 mmol) in N,N-dimethylformamide (100 mL) at 0 °C was
added aluminium chloride (10.6 g, 79.7 mmol) portionwise and the mixture stirred for
1
hour. Trifluoroacetic anhydride (3.4 mL, 23.9 mmol) was added dropwise and the
mixture stirred at room temperature for 3 hours. The reaction mixture was quenched
with ice, diluted with water (150 mL) and extracted with ethyl acetate (3 x 100 mL).
The combined organic extracts were washed with water (3 x 100 mL) and aqueous
lithium chloride (1 M, 100 mL), dried over anhydrous magnesium sulfate and
concentrated under reduced pressure to obtain the title compound 49 (3.5 g, 78%) as a
23
yellow oil, the spectroscopic data of which corresponded to previously described. R :
f
0.40 (1:4 ethyl acetate, hexane).
1
-(1-(5-(Benzyloxy)pentyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)-2,2,2-trifluoroethanone 50
To a solution of 48 (2.4 g, 8.2 mmol) in anhydrous N,N-dimethylformamide (60 mL) at 0
°
C was added aluminium chloride (5.4 g, 40.8 mmol) portionwise and the mixture
stirred for 1 hour. Trifluoroacetic anhydride (1.7 mL, 12.2 mmol) was added dropwise
and the mixture was warmed to room temperature and stirred for 4 hours. The reaction
mixture was poured on to ice-water (100 mL) and extracted with ethyl acetate (3 x 50
24

ACCEPTED MANUSCRIPT
mL). The combined organic extracts were washed with water (3 x 100 mL) and aqueous
lithium chloride (1 M, 80 mL), dried over anhydrous magnesium sulfate and
concentrated under reduced pressure to obtain the title compound 50 (2.8 g, 88%) as a
yellow oil, R : 0.28 (1:4 ethyl acetate, hexane); IR (vmax (neat)): 2940, 2861, 1670, 1527,
f
−1 1
1
385, 1287, 1136, 882, 802, 714 cm ; H NMR (400 MHz, CDCl
3
): δ 1.44-1.55 (2H, m),
1
.65-1.75 (2H, m), 1.94-2.04 (2H, m), 3.49 (2H, t, J = 6.3 Hz), 4.40 (2H, t, J = 7.4 Hz), 4.50
(2H, s), 7.25-7.38 (6H, m), 8.09 (1H, d, J = 1.7 Hz), 8.48 (1H, dd, J = 4.7, 1.7 Hz), 8.66 (1H,
1
3
dd, J = 7.9, 1.6 Hz) ppm; C NMR (100 MHz, CDCl
3
): δ 23.6, 29.3, 29.9, 46.0, 69.9, 73.1,
1
08.0, 117.0 (q, J = 290.6 Hz), 119.6, 119.8, 127.7, 127.7, 128.5, 131.2, 137.4 (q, J = 4.2
1
9
Hz), 138.5, 145.6, 148.1, 174.9 (q, J = 35.4 Hz) ppm; F NMR (376 MHz, CDCl
ppm; LRMS (+ESI) m/z: 391.2 ([M+H] 52%), 413.2 ([M+Na] 100%); HRMS (ESI) Cald
for C21
3
): δ -72.4
+
+
+
+
H
21
F
N O
3 2 2
[M+Na] : 413.14528, found 413.14448.
1
-Pentyl-1H-pyrrolo[2,3-b]pyridine-3-carboxylic acid 51
To a solution of 49 (3.5 g, 12.3 mmol) in methanol (100 mL) was added aqueous lithium
hydroxide (3 M, 10.3 mL, 30.8 mmol) and the mixture stirred at reflux for 48 hours. The
volatiles were removed under a stream of nitrogen and the aqueous residue washed
with dichloromethane (20 mL), acidified with aqueous hydrochloric acid (6 M) and
extracted with dichloromethane (3 x 50 mL). The combined organic extracts were dried
over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain
the title compound 51 (2.4 g, 84%) as an off-white solid, the spectroscopic data of
23
which corresponded to previously described. R : 0.07 (1:4 ethyl acetate, hexane).
f
1
-(5-(Benzyloxy)pentyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxylic acid 52
A solution of 50 (2.5 g, 6.4 mmol) in a mixture of methanol and tetrahydrofuran (1:1,
0 mL) was added aqueous lithium hydroxide (2 M, 16 mL, 32 mmol) and the mixture
3
was heated at reflux for 72 hours. The volatiles were removed under a stream of
nitrogen and the aqueous residue washed with dichloromethane (30 mL), acidified with
aqueous hydrochloric acid (6 M) and extracted with dichloromethane (3 x 50 mL). The
combined organic extracts were dried over anhydrous magnesium sulfate and
concentrated under reduced pressure to afford the title compound 52 (1.6 g, 74%) as a
light yellow solid, mp: 108.1-110.8 °C; R : 0.28 (1:4 ethyl acetate, hexane); IR (vmax
f
−1 1
(
(
neat)): 3102, 2061, 2855, 2502, 1686, 1533, 1260 1095, 800, 758, 730 cm ; H NMR
400 MHz, CDCl ): δ 1.43-1.53 (2H, m), 1.69 (2H, quin, J = 6.6 Hz), 1.97 (2H, quin, J = 7.4
3
Hz), 3.47 (2H, t, J = 6.4 Hz), 4.38 (2H, t, J = 7.2 Hz), 4.50 (2H, s), 7.24-7.38 (6H, m), 8.06
1
3
(
1H, s), 8.43 (1H, dd, J = 4.7, 1.7 Hz), 8.50 (1H, dd, J = 7.9, 1.6 Hz) ppm; C NMR (100
MHz, CDCl
3
): δ 23.7, 29.4, 30.0, 45.5, 70.0, 73.1, 105.0, 118.4, 119.6, 127.7, 127.8, 128.5,
+
130.3, 135.4, 138.6, 144.2, 148.0, 169.3 ppm; LRMS (+ESI) m/z: 361.2 ([M+Na] 100%);
+
+
HRMS (ESI) Cald for C20
H
22
N
2
O [M+Na] : 361.15281, found 361.15205.
3
(
S)-Methyl 3-methyl-2-(1-pentyl-1H-pyrrolo[2,3-b]pyridine-3-carboxamido)butanoate 22
General procedure C was followed using carboxylic acid 51 (250 mg, 1.1 mmol) and
methyl- -valinate hydrochloride (220 mg, 1.3 mmol). The crude product was purified by
L
flash column chromatography using ethyl acetate, hexane (1:9 to 2:3) as an eluent and
recrystallization from dichloromethane, hexane to obtain the title compound 22 (240
mg, 65%) as a white solid, mp: 86.9-89.8 °C; R : 0.20 (1:4 ethyl acetate, hexane); IR (vmax
f
−
1
(
neat)): 3286, 2961, 2931, 1743, 1619, 1541, 1523, 1400, 1286, 1196, 1142, 776 cm ;
1
H NMR (400 MHz, CDCl
3
): δ 0.87 (3H, t, J = 6.9 Hz), 1.01 (3H, d, J = 6.9 Hz), 1.04 (3H, d, J
=
6.9 Hz), 1.26-1.41 (4H, m), 1.88 (2H, quin, J = 7.4 Hz), 2.22-2.37 (1H, m), 3.78 (3H, s),
25

ACCEPTED MANUSCRIPT
4
.26-4.34 (2H, m), 4.84 (1H, dd, J = 8.6, 4.9 Hz), 6.42 (1H, d, J = 8.6 Hz), 7.20 (1H, dd, J =
.0, 4.7 Hz), 7.83 (1H, s), 8.32 (1H, dd, J = 8.0, 1.6 Hz), 8.37 (1H, dd, J = 4.7, 1.6 Hz), 8.48
8
1
3
(
1H, s) ppm; C NMR (100 MHz, CDCl
3
): δ 14.0, 18.2, 19.2, 21.4, 29.0, 30.0, 31.8, 45.2,
5
2.4, 57.0, 108.9, 117.7, 118.3, 128.8, 131.0, 143.9, 147.8, 164.4, 173.3 ppm; LRMS
+
+
+
(+ESI) m/z: 368.2 ([M+Na] 100%), 713.3 ([2M+Na] 43%); HRMS (ESI) Cald for
+
27
C
19
H
N O
27 3 3
[M+Na] : 368.19501, found 368.19429; [α]
D
: +34.5 (0.58, CHCl ); HPLC:
3
99.5%, RT: 25.9 mins.
(
2
S)-Methyl 3,3-dimethyl-2-(1-pentyl-1H-pyrrolo[2,3-b]pyridine-3-carboxamido)butanoate
3
General procedure C was followed using carboxylic acid 51 (200 mg, 860 μmol) and
amine hydrochloride salt 37 (190 mg, 1.0 mmol). The crude product was purified by
flash column chromatography using ethyl acetate, hexane (1:4 to 3:7) as an eluent to
obtain the title compound 23 (140 mg, 45%) as a white solid, mp: 68.3-70.1 °C; R
f
: 0.37
(
1
2:3 ethyl acetate, hexane); IR (vmax (neat)): 3334, 3101, 2956, 1738, 1614, 1537, 1515,
−1 1
3
426, 1267, 1217, 1140, 777 cm ; H NMR (400 MHz, CDCl ): δ 0.88 (3H, t, J = 6.9 Hz),
1.08 (9H, s), 1.28-1.41 (4H, m), 1.89 (2H, quin, J = 7.4 Hz), 3.76 (3H, s), 4.27-4.34 (2H,
m), 4.76 (1H, d, J = 9.4 Hz), 6.42 (1H, d, J = 9.3 Hz), 7.21 (1H, dd, J = 7.9, 4.7 Hz), 7.83 (1H,
1
3
s), 8.31 (1H, dd, J = 8.0, 1.5 Hz), 8.38 (1H, dd, J = 4.7, 1.6 Hz) ppm; C NMR (100 MHz,
CDCl
3
): δ 14.1, 22.4, 26.9, 29.0, 30.1, 35.3, 45.3, 52.1, 59.8, 108.9, 117.7, 118.2, 128.7,
+
1
31.1, 143.9, 147.8, 164.3, 172.9 ppm; LRMS (+ESI) m/z: 382.3 ([M+Na] 100%), 741.4
+
+
+
([2M+Na] 32%); HRMS (ESI) Cald for C20
H
N O
29 3 3
[M+Na] : 382.21066, found
2
7
382.20991; [α]
D
3
: +30.5 (1.15, CHCl ); HPLC: 99.8%, RT: 27.4 mins.
(S)-N-(1-Amino-3-methyl-1-oxobutan-2-yl)-1-pentyl-1H-pyrrolo[2,3-b]pyridine-3-
carboxamide 25
General procedure C was followed using carboxylic acid 51 (230 mg, 1.0 mmol) and L-
valinamide hydrochloride (180 mg, 1.2 mmol). The crude product was purified by
recrystallization from ethyl acetate to obtain the title compound 25 (240 mg, 73%) as a
white solid, mp: 210.0-213.4 °C; R
193, 2934, 1649, 1621, 1530, 1516, 1426, 1297, 1144 cm ; H NMR (400 MHz, DMSO-
): δ 0.84 (3H, t, J = 7.1 Hz), 0.96 (3H, d, J = 6.7 Hz), 0.98 (3H, d, J = 6.7 Hz), 1.20-1.38
f
: 0.19 (ethyl acetate); IR (vmax (neat)): 3372, 3293,
−1 1
3
d
6
(
(
4H, m), 1.84 (2H, quin, J = 7.2 Hz), 2.09 (1H, sept, J = 6.8 Hz), 4.19-4.38 (3H, m), 7.07
1H, bs), 7.20 (1H, dd, J = 7.9, 4.7 Hz), 7.49 (1H, bs), 7.69 (1H, d, J = 8.8 Hz), 8.30 (1H, dd,
1
3
J = 4.7, 1.7 Hz), 8.41 (1H, dd, J = 7.9, 1.7 Hz), 8.48 (1H, s) ppm; C NMR (100 MHz,
DMSO-d
6
): δ 13.8, 18.5, 19.5, 21.7, 28.3, 29.2, 30.3, 44.1, 57.5, 107.9, 117.1, 118.9, 129.4,
+
1
31.2, 143.2, 147.1, 163.5, 173.4 ppm; LRMS (+ESI) m/z: 353.3 ([M+Na] 100%); HRMS
ESI) Cald for C18
+
+
27
(
H
N O
26 4 2
[M+Na] : 353.19534, found 353.19461; [α]
D
: +22.8 (0.88,
DMSO); HPLC: 96.3%, RT: 21.8 mins.
(S)-N-(1-Amino-3,3-dimethyl-1-oxobutan-2-yl)-1-pentyl-1H-pyrrolo[2,3-b]pyridine-3-
carboxamide 26
General procedure C was followed using carboxylic acid 51 (250 mg, 1.1 mmol) and
amine hydrochloride salt 36 (220 mg, 1.3 mmol). The crude product was purified by
flash column chromatography using ethyl acetate, hexane (9:1 to 1:0) as an eluent to
obtain the title compound 26 (310 mg, 84%) as a white solid, mp: 90.1-94.3 °C; R
f
: 0.21
(
1
ethyl acetate); IR (vmax (neat)): 3334, 3187, 2954, 1674, 1620, 1535, 1513, 1398, 1367,
−1 1
261, 1140 cm ; H NMR (400 MHz, DMSO-d
6
): δ 0.84 (3H, t, J = 7.1 Hz), 1.01 (9H, s),
1.19-1.38 (4H, m), 1.85 (2H, quin, J = 7.3 Hz), 4.19-4.36 (2H, m), 4.48 (1H, d, J = 9.5 Hz),
26

ACCEPTED MANUSCRIPT
7
.11 (1H, bs), 7.21 (1H, dd, J = 7.9, 4.7 Hz), 7.40 (1H, d, J = 9.5 Hz), 7.55 (1H, bs), 8.31
1
3
(1H, dd, J = 4.7, 1.6 Hz), 8.39 (1H, dd, J = 7.9, 1.6 Hz), 8.55 (1H, s) ppm; C NMR (100
MHz, DMSO-d
6
): δ 13.8, 21.7, 26.9, 28.4, 29.2, 34.1, 44.1, 59.3, 107.8, 117.1, 118.8, 129.2,
+
1
31.4, 143.2, 147.2, 163.3, 172.4 ppm; LRMS (+ESI) m/z: 367.2 ([M+Na] 100%), 711.3
+
+
+
([2M+Na] 20%); HRMS (ESI) Cald for C19
H
N O
28 4 2
[M+Na] : 367.21100, found
2
7
367.21025; [α]
D
: +25.5 (1.57, DMSO); HPLC: 98.4%, RT: 23.2 mins.
1
-Pentyl-N-(2-phenylpropan-2-yl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide 28
General procedure C was followed using carboxylic acid 51 (110 mg, 470 μmol) and 1-
methyl-1-phenylethylamine (70 mg, 520 μmol). The crude product was purified by flash
column chromatography using ethyl acetate, hexane (1:4 to 3:7) as an eluent to obtain
the title compound 28 (84 mg, 51%) as a white solid, mp: 171.8-174.3 °C; R
f
: 0.30 (2:3
ethyl acetate, hexane); IR (vmax (neat)): 3328, 3093, 2923, 1620, 1538, 1516, 1426,
−
1 1
1
1
7
=
2
1
4
9
270, 1198 cm ; H NMR (400 MHz, CDCl ): δ 0.91 (3H, t, J = 6.8 Hz), 1.29-1.46 (4H, m),
3
.84-1.97 (8H, m), 4.33 (2H, t, J = 7.3 Hz), 6.13 (1H, bs), 7.20 (1H, dd, J = 7.9, 4.7 Hz),
.26-7.31 (1H, m), 7.38 (2H, t, J = 7.7 Hz), 7.50-7.56 (2H, m), 7.77 (1H, s), 8.29 (1H, dd, J
7.9, 1.6 Hz), 8.39 (1H, dd, J = 4.8, 1.5 Hz) ppm; C NMR (100 MHz, CDCl
9.1, 29.7, 30.1, 45.2, 56.4, 110.2, 117.4, 118.3, 124.9, 126.9, 128.7, 128.8, 130.6, 143.8,
47.4, 147.8, 163.8 ppm; LRMS (+ESI) m/z: 372.3 ([M+Na] 100%), 721.4 ([2M+Na]
6%); HRMS (ESI) Cald for C22
9.9%, RT: 27.4 mins.
1
3
3
): δ 14.1, 22.4,
+
+
+
+
H
27
N O [M+Na] : 372.20518, found 372.20444; HPLC:
3
N-(Adamantan-1-yl)-1-(5-(benzyloxy)pentyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide
53
General procedure C was followed using carboxylic acid 52 (120 mg, 35 μmol) and 1-
adamantylamine (64 mg, 43 μmol). The crude residue was purified by flash column
chromatography using ethyl acetate, hexane (1:4 to 2:3) as an eluent to obtain the title
compound 53 (110 mg, 65%) as a white gummy solid, R
f
: 0.29 (2:3 ethyl acetate,
hexane); IR (vmax (neat)): 3365, 2907, 2848, 1620, 1536, 1516, 1422, 1278, 1251, 1095,
−
1 1
7
54 cm ; H NMR (400 MHz, CDCl
3
): δ 1.37-1.48 (2H, m), 1.59-1.80 (8H, m), 1.90 (2H,
quin, J = 7.4 Hz), 2.10-2.21 (9H, m), 3.44 (2H, t, J = 6.4 Hz), 4.30 (2H, t, J = 7.2 Hz), 4.47
(
2H, s), 5.55 (1H, bs), 7.17 (1H, dd, J = 7.9, 4.7 Hz), 7.23-7.36 (5H, m), 7.68 (1H, s), 8.27
1
3
(1H, dd, J = 8.0, 1.5 Hz), 8.35 (1H, dd, J = 4.7, 1.6 Hz) ppm; C NMR (100 MHz, CDCl
3
): δ
2
1
1
3.7, 29.4, 29.7, 30.2, 36.6, 42.3, 45.1, 52.3, 70.1, 73.0, 110.7, 117.3, 118.4, 127.7, 127.8,
+
28.5, 128.9, 130.1, 138.6, 143.7, 147.7, 163.9 ppm; LRMS (+ESI) m/z: 494.3 ([M+Na]
+
+
00%); HRMS (ESI) Cald for C30
H
37
N
3
O [M+H] : 494.27835, found 494.27749.
2
(
S)-N-(1-Amino-3,3-dimethyl-1-oxobutan-2-yl)-1-(5-(benzyloxy)pentyl)-1H-pyrrolo[2,3-
b]pyridine-3-carboxamide 55
General procedure C was followed using carboxylic acid 52 (420 mg, 1.3 mmol) and
amine hydrochloride salt 36 (250 mg, 1.5 mmol). The crude product was purified by
flash column chromatography using methanol, dichloromethane (0:1 to 1:19) as an
eluent to obtain the title compound 55 (510 mg, 91%) as a colourless foam, R
f
: 0.29
(
1
1:19 methanol, dichloromethane); IR (vmax (neat)): 3103, 2938, 2856, 1685, 1535,
−1 1
260, 1096 cm ; H NMR (400 MHz, CDCl ): δ 1.16 (9H, s), 1.38-1.48 (2H, m), 1.66 (2H,
3
quin, J = 6.7 Hz), 1.90 (2H, quin, J = 7.4 Hz), 3.45 (2H, t, J = 6.4 Hz), 4.29 (2H, td, J = 7.1,
.9 Hz), 4.48 (2H, s), 4.79 (1H, d, J = 9.2 Hz), 5.98 (1H, bs), 6.81 (1H, d, J = 9.3 Hz), 6.94
1H, bs), 7.19 (1H, dd, J = 8.0, 4.7 Hz), 7.24-7.37 (5H, m), 7.84 (1H, s), 8.33 (1H, dd, J =
1
(
8
1
3
.0, 1.6 Hz), 8.37 (1H, dd, J = 4.7, 1.6 Hz) ppm; C NMR (100 MHz, CDCl ): δ 23.6, 26.9,
3
27

ACCEPTED MANUSCRIPT
2
9.4, 30.1, 34.9, 45.1, 59.9, 70.0, 73.0, 108.9, 117.7, 118.3, 127.6, 127.7, 128.5, 128.8,
+
130.9, 138.6, 143.9, 147.7, 164.5, 173.5 ppm; LRMS (+ESI) m/z: 473.3 ([M+Na] 100%);
+
+
HRMS (ESI) Cald for C26
H
N O
34 4 3
[M+Na] : 473.25286, found 473.25201.
1
-(5-(Benzyloxy)pentyl)-N-(2-phenylpropan-2-yl)-1H-pyrrolo[2,3-b]pyridine-3-
carboxamide 56
General procedure C was followed using carboxylic acid 52 (250 mg, 740 μmol) and 1-
methyl-1-phenylethylamine (250 mg, 890 μmol). The crude product was purified by
flash column chromatography using ethyl acetate, hexane (3:7 to 3:2) as an eluent to
obtain the title compound 56 (290 mg, 85%) as a white solid, mp: 137.8-138.8 °C; R :
f
(
1
0.19 2:3 ethyl acetate, hexane); IR (vmax (neat)): 3337, 2926, 2854, 1621, 1537, 1275,
−1 1
094, 760 cm ; H NMR (400 MHz, CDCl ): δ 1.38-1.48 (2H, m), 1.60 (2H, m), 1.86 (6H,
3
s), 1.86-1.95 (2H, m), 3.44 (2H, t, J = 6.4 Hz), 4.30 (2H, t, J = 7.2 Hz), 4.46 (2H, s), 6.13
1H, s), 7.16 (1H, dd, J = 8.0, 4.7 Hz), 7.22-7.38 (8H, m), 7.48-7.53 (2H, m), 7.73 (1H, s),
.27 (1H, dd, J = 8.0, 1.6 Hz), 8.36 (1H, dd, J = 4.7, 1.6 Hz) ppm; 13C NMR (100 MHz,
CDCl ): δ 23.7, 29.4, 29.7, 30.2, 45.1, 56.3, 70.1, 73.2, 110.2, 117.4, 118.3, 124.9, 126.8,
27.7, 127.7, 128.5, 128.6, 128.9, 130.4, 138.7, 143.8, 147.4, 147.8, 163.7 ppm; LRMS
(
8
3
1
+
+
+
(+ESI) m/z: 456.3 ([M+H] 31%), 478.3 ([M+Na] 100%); HRMS (ESI) Cald for
+
C
26
H
N O
34 4 3
[M+Na] : 473.25286, found 473.25201.
N-(Adamantan-1-yl)-1-(5-hydroxypentyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamide 21
General procedure D was followed using benzyl ether 53 (70 mg, 148 μmol). The crude
residue was purified by flash column chromatography using ethyl acetate, hexane (1:1
to 3:2) as an eluent to obtain the title compound 21 (49 mg, 86%) as a white solid, mp:
1
10.9-113.4 °C; R
f
: 0.16 (3:2 ethyl acetate, hexane); IR (vmax (neat)): 3320, 2904, 2849,
−
1 1
1618, 1534, 1517, 1425, 1290, 1160, 776 cm ; H NMR (400 MHz, CDCl
3
): δ 1.34-1.45
(
(
(
2
1
C
2H, m), 1.53-1.63 (2H, m), 1.66-1.79 (6H, m), 1.85-1.94 (3H, m), 2.09-2.20 (9H, m), 3.61
2H, t, J = 6.4 Hz), 4.29 (2H, t, J = 7.1 Hz,), 5.62 (1H, bs), 7.16 (1H, dd, J = 7.9, 4.7 Hz), 8.26
1
3
1H, dd, J = 7.9, 1.5 Hz), 8.33 (1H, dd, J = 4.7, 1.5 Hz) ppm; C NMR (100 MHz, CDCl
3
): δ
3.1, 29.7, 30.1, 32.4, 36.6, 42.3, 45.0, 52.4, 62.5, 110.6, 117.4, 118.4, 129.0, 130.3, 143.6,
+
+
47.6, 164.0 ppm; LRMS (+ESI) m/z: 404.3 ([M+Na] 100%); HRMS (ESI) Cald for
+
23
H
N O
31 3 2
[M+Na] : 404.23140, found 404.23067; HPLC: 99.0%, RT: 24.5 mins.
(
S)-Methyl
2-(1-(5-hydroxypentyl)-1H-pyrrolo[2,3-b]pyridine-3-carboxamido)-3,3-
dimethylbutanoate 24
General procedure C was followed using carboxylic acid 52 (36 mg, 106 μmol) and
amine hydrochloride salt 37 (23 mg, 127 μmol). The crude residue contained an
inseparable mixture of products and hence was taken on to the next step without
further purification or characterization.
General procedure D was followed using the crude benzyl protected intermediate. The
crude residue was purified by flash column chromatography using ethyl acetate, hexane
(1:1 to 1:4) as an eluent to obtain the title compound 24 (16 mg, 40% over 2 steps) as a
colourless foam, R : 0.08 (1:1 ethyl acetate, hexane); IR (vmax (neat)): 3323, 2999, 2946,
f
−1 1
2
927, 1739, 1547, 1520, 1476, 1426, 1331, 1214, 1162, 1055 cm ; H NMR (400 MHz,
CDCl ): δ 1.08 (9H, s), 1.40-1.51 (2H, m), 1.59-1.68 (2H, m), 1.92-2.02 (2H, m), 3.65 (2H,
3
t, J = 6.2 Hz), 3.77 (3H, s), 4.34-4.50 (2H, m), 4.76 (1H, d, J = 9.3 Hz), 6.48 (1H, d, J = 9.3
1
3
Hz), 7.23-7.32 (1H, m), 7.87 (1H, s), 8.35-8.51 (2H, m) ppm; C NMR (100 MHz, CDCl
3
):
δ 22.9, 26.9, 29.9, 31.9, 35.4, 52.1, 59.8, 62.5, 77.4, 109.9, 117.7, 117.8, 131.4, 131.4,
+
+
1
43.3, 146.9, 163.8, 172.9 ppm; LRMS (+ESI) m/z: 398.5 ([M+Na] 100%); HRMS (ESI)
28