The Trifluoromethyl Group as a Bioisosteric Replacement of the Aliphatic Nitro Group in CB1 Receptor Positive Allosteric Modulators

Article abstract of DOI:10.1021/acs.jmedchem.9b00252

The first generation of CB₁ positive allosteric modulators (e.g., ZCZ011) featured a 3-nitroalkyl-2-phenyl-indole structure. Although a small number of drugs include the nitro group, it is generally not regarded as being "drug-like", and this is particularly true for aliphatic nitro groups. There are very few case studies where an appropriate bioisostere replaced a nitro group that had a direct role in binding. This may be indicative of the difficulty of replicating its binding interactions. Herein, we report the design and synthesis of ligands targeting the allosteric binding site on the CB₁ cannabinoid receptor, in which a CF₃ group successfully replaced the aliphatic NO₂. In general, the CF₃-bearing compounds were more potent than their NO₂ equivalents and also showed improved in vitro metabolic stability. The CF₃ analogue (1) with the best balance of properties was selected for further pharmacological evaluation. Pilot in vivo studies showed that (±)-1 has similar activity to (±)-ZCZ011, with both showing promising efficacy in a mouse model of neuropathic pain.

Full text of DOI:10.1021/acs.jmedchem.9b00252

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Article
The Trifluoromethyl Group as a Bioisosteric Replacement of the Aliphatic
Nitro Group in CB1 Receptor Positive Allosteric Modulators (PAMs)
Chih-Chung Tseng, Gemma Baillie, Giulia Donvito, Mohammed Mustafa,
Sophie Juola, Chiara Zanato, Chiara Massarenti, Sergio Dall'Angelo, William
T A Harrison, Aron H. Lichtman, Ruth Ross, Matteo Zanda, and Iain R. Greig
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00252 • Publication Date (Web): 03 May 2019
Downloaded from http://pubs.acs.org on May 3, 2019
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The Trifluoromethyl Group as a Bioisosteric Replacement of the
Aliphatic Nitro Group in CB1 Receptor Positive Allosteric Modulators
(PAMs)
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Chih-Chung Tseng,a Gemma Baillie,b Giulia Donvito,c Mohammed A. Mustafa,c Sophie E.
Juola,c Chiara Zanato,a Chiara Massarenti,a Sergio Dall’Angelo,a William T. A. Harrison,e Aron
H. Lichtman,c,f Ruth A. Ross,b Matteo Zanda,a,d,§,* Iain R. Greiga,*
a University of Aberdeen, Kosterlitz Centre for Therapeutics, Foresterhill, Aberdeen, AB25 2ZD, Scotland, UK
b University of Toronto, Department of Pharmacology & Toxicology, Toronto M5S 1A8, Canada
c Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia, 23298
USA
d C.N.R. – I.C.R.M., via Mancinelli 7, 20131 Milan, Italy
e Department of Chemistry, University of Aberdeen, AB24 3UE, Scotland, UK
f Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, Virginia, 23298, USA
§ Current address: Loughborough University, Centre for Sensing and Imaging Science, Sir David Davies building,
LE11 3TU Loughborough (UK).
ABSTRACT
The first generation of CB1 positive allosteric modulators (PAMs; e.g., ZCZ011) featured a 3-nitroalkyl-
2-phenyl-indole structure. Although a small number of drugs include the nitro group, it is generally
not regarded as being “drug-like”, and this is particularly true for aliphatic nitro groups. There are
very few case studies where an appropriate bioisostere replaced a nitro group that had a direct role
in binding. This may be indicative of the difficulty of replicating its binding interactions. Herein we
report the design and synthesis of ligands targeting the allosteric binding site on the CB1 cannabinoid
receptor, in which a CF3 group successfully replaced the aliphatic NO2. In general, the CF3-bearing
compounds were more potent than their NO2 equivalents and also showed improved in vitro
metabolic stability. The CF3-analogue (1) with the best balance of properties was selected for further
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pharmacological evaluation. Pilot in vivo studies showed that (±)-1 has similar activity to (±)-ZCZ011,
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with both showing promising efficacy in a mouse model of neuropathic pain.
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INTRODUCTION
The endocannabinoid system plays a major role in modulating neurotransmitter release in the central
nervous system and thus its modulation elicits a broad range of physiological effects. Compounds
activating cannabinoid receptors represent a promising therapeutic opportunity for the treatment of
many disorders, including depression, anxiety and pain. However, global activation of the cannabinoid
CB1 receptor by direct agonism leads to psychoactive side effects, physical dependence and abuse
liability. There is both an opportunity and a need to provide safer and more effective ways of
harnessing the therapeutic benefits of CB1 receptor activation. Recent reviews have highlighted the
therapeutic potential of targeting the allosteric binding site on the CB1 receptor using positive
allosteric modulators (PAMs),1,2 which have the advantage of enhancing the actions of
endocannabinoids directly at the orthosteric receptors expressed within spinal and brain pain
processing centres. Thus, PAMs are predicted to be free of the psychoactive side-effects associated
with direct CB1 agonists that globally activate these receptors. The impressive efficacy of small
molecule tool CB1 PAMs in models of neuropathic pain and post-traumatic stress disorder, without
any evidence of addiction or psychotropic side-effects, support this hypothesis.
S
NO2
NO2
N
N
H
H
5
ZCZ-011 ( )
AZ-4 / GAT211
Figure 1
The activity of the first CB1 PAM - AZ-4,3 also known as F-0874 and now commonly referred to as GAT-
2115 (Figure 1), was reported in a conference poster by researchers at AstraZeneca Montreal,3 having
been first synthesised by Noland and Lange in 1959.6 The structure has since been modified to
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improve potency (ZCZ011,7 5 in Table 1) and the enantiomers of AZ-4 separated (GAT228/229)5, but
no substantial development or comprehensive structure activity relationships have been reported.
The primary problematic issue with these compounds is the presence of an aliphatic nitro group.
Despite its presence in a small number of major drugs, including nifedipine, chloramphenicol,
ranitidine and metronidazole, most academic and industrial medicinal chemists do not regard a nitro
group as being “drug-like”. However, surprisingly little published evidence exists to support this
blanket exclusion of the nitro-group from drug-like space. Justification for this exclusion may be based
upon accepted wisdom, or derived from personal experience (see e.g. Muegge and co-workers;8 Beck
and co-workers;9 and discussions on internet forums, including the well-regarded “In the
Pipeline”10,11); or based on a well-established (if only potential) toxic liability associated solely with the
aromatic nitro and its propensity for formation of an aryl nitrenium ion, which can bind to DNA.12 In
additional examples of this general dismissal of the nitro group from drug-like space, the nitro was
one of the groups eliminated prior to the studies leading to Baell’s identification of the range of pan-
assay interference compounds13; likewise, any structures containing a nitro are explicitly excluded by
Rankovic and Morphy14 as being acceptable for use in fragment-based lead discovery.
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While little information exists on the toxicological issues associated with aliphatic nitro groups, the
widely-held opinions described above usually require its removal or replacement early in the
development of a bioactive compound. However, there are only a few case studies in which a nitro
group has been successfully replaced by an appropriate bioisostere,9 which may be indicative of the
difficulty of replicating its binding interactions. Nitro groups may act as hydrogen bond acceptors and
may also stack with other groups, including ketones and alcohols.15 Additionally, Alston and co-
workers16 reported that the nitroalkyl group can serve as a one- and two-electron reductant, oxidant,
ambident nucleophile, electrophile, ligand, and leaving group in reactions with enzymes and other
biomolecules and suggested that “the richness of its reactivity may have hindered the application of
the nitroalkyl group in medicinal chemistry.” Much more commonly, nitro groups are initially present
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in bioactive compounds as aromatic substituents, where their electron-withdrawing properties may
polarise the ring, giving optimal interaction with electron-rich moieties within the biological target. In
such cases, simple replacement with other strongly-electron-withdrawing substituents, e.g. halogens
or haloalkyl groups, may be possible.
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Because of this strong electron-withdrawing nature, the presence of a nitro group also facilitates many
chemical reactions; thus, it is often found in screening collections (including the collection from which
AZ-4 was originally identified). The reaction of an indole with nitrostyrene has been widely reported
in the literature and there are a considerable range of methods available for this simple 1,4-conjugate
addition reaction.17–24 There is literature precedent for the nitro group to be replaced with a carboxylic
acid in this series of compounds, which is achieved by initial reaction of an indole with the highly-
electron deficient diethylbenzylidene malonate.21,22 The limited similarity of nitro group binding to
carboxylic acid binding, in spite of the near perfect isosterism, has been discussed by Kelly and Kim.25
A small range of derivatives can be accessed from the carboxylic acid (e.g. CH2OH, CO2H, CO2Et,
CONHNH2, CONH2 and CONHMe, Figure 2 - see Supporting Information); however, these were inactive
as CB1 PAMs.
NR1R2
COR1
N
N
H
H
Figure 2.
Likewise, simple derivatives based on reduction of the nitro to an amine (Figure 2), with or without
subsequent substitution, gave inactive compounds (see Supporting Information).
Modelling studies on GAT22826 indicate that the nitro group may form polar interactions with a serine,
a methionine and a cysteine residue in the absence of the orthosteric agonist (CP55,940). However,
this case appears to apply to an orthosteric agonist. Although not stated as a conclusion by the
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authors, the modelling suggests that, in the presence of an agonist, GAT228 moves deeper into the
receptor; accordingly, the nitro group may be too far from these binding groups to retain a strong
interaction. Thus, it remains uncertain that specific binding interactions provided by the nitro group
are required for its activity as a PAM. However, the lack of activity shown by any of the compounds in
Figure 2 suggests that such interactions will be necessary for PAM activity.
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Herein we report that the CF3 offers a viable bioisosteric replacement for an aliphatic nitro group,
demonstrating the removal of the non-drug-like nitro group from the most-commonly-studied and
utilised CB1 PAMs, to achieve compounds with improved potency and metabolic stability.
RESULTS AND DISCUSSION
Chemistry.
A library of indole derivatives 1-57 (Table 1) was synthesised using a variety of methods. Nitro-indole
derivatives 2-25 were obtained by InBr3-catalyzed Michael addition of 2-(2-nitrovinyl)-aryl/heteroaryl
derivatives to the corresponding indoles, upon microwave heating (see Supporting Information
Scheme SI-1 for details). Dicyano derivatives 29 and 32-34 having R2 = Ph and R4 = CH(CN)2 were
obtained by addition of lithium indolyl-cuprate(I) to benzylidene-malononitriles (Supporting
Information, Scheme SI-4). Compounds having R2 = Ph and R4 = CH2CN were obtained either via Fischer
indole syntheses (28, 30), or via InBr3-promoted addition of phenyl-oxirane to an indole, followed by
one-carbon homologation (31). A similar strategy, followed by one-carbon homologation using TMS-
CF3 and Barton-McCombie dehydroxylation was used to access the CF3-indoles 37 and 39 (Supporting
Information, Scheme SI-5). The cyclopropyl CF3-indole 40 was also prepared using a multi-step process
involving TMS-CF3 homologation and Barton-McCombie dehydroxylation (Supporting Information,
Scheme SI-7). However, the most versatile method for efficiently preparing a wide range of CF3-indoles
is shown in Scheme 1. The starting indole was iodinated at the 3-position with NIS to be then subjected
to Sonogashira coupling to give 79. Terminal alkyne trifluoromethylation with in-situ generated "Cu-
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CF3" under aerobic conditions gave CF3-alkynes 80,27 which were transformed into iodo-alkenes 81
with lithium iodide under mild acidic aonditions,28 which were then submitted to Stille coupling with
the corresponding aryl stannanes leading to N-MOM-protected indoles 82. The latter were first de-
protected with HCl and then reduced with Et3SiH in the presence of TFA to afford the target CF3-indoles
1, 41-50 and 57.
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CF3
R3
R1
a)-c)
d)
R3
MOM
R1
R3
MOM
R1
N
N
MOM
N
79
80
e)
CF3
R3
R2
R2
I
CF3
R3
MOM
CF3
R3
g)
f)
R1
R1
R1
N
N
H
N
MOM
1 41 50 57
82
81
,
-
,
Scheme 1. Synthesis of CF3-PAMs via Stille coupling/ionic hydride reduction strategy.
Reagents and conditions: a) NIS, 0 °C, CH2Cl2; b) TMSCCH, PdCl2(PPh3)2, CuI, ET3N, 23 °C, DMF; c) TBAF, 0 °C, THF;
d) TMSCF3, CuI, TMEDA, K2CO3, air, 23 °C, DMF; e) LiI, HOAc, CH2Cl2, 0 – 23 °C; f) R2SnBu3, PdCl2(MeCN)2, P(o-
Tol)3, CuI, 55 °C, DMF; g) 6 M HCl, 60 °C, THF then Et3SiH, TFA, 0 – 23 °C, CH2Cl2.
Further late-stage functional groups manipulations, shown in Scheme 2, expanded the library of CF3-
indole derivatives to include compounds 52-56 and 83-89.
CF3
CF3
S
S
a
)
Ph
Ph
HO
O
N
N
H
H
52
50
O
b
)
S
S
S
S
S
S
S
CF3
CF3
CF3
CF3
CF3
CF3
h
)
Ph
CF3
Ph
c
d
e
f
g
)
)
)
)
)
O
O
Ph
O
O
Ph
Ph
Ph
Ph
O
N
N
SEM
S
N
SEM
N
SEM
N
SEM
N
SEM
N
SEM
N
N
H2N
N
83
84
85
86
87
88
89
H
H
N
SEM
O
O
O
HO
i
i
i
i
)
)
)
)
CF3
Ph
S
S
S
S
CF3
Ph
CF3
Ph
CF3
Ph
O
O
O
S
N
H
N
N
N
N
N
H2N
H
H
56
H
H
H
54
53
55
O
Scheme 2. Late-stage functional group interconversions strategy.
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o
Reagents and conditions: a) Super-Hydride®, THF; b) SEMCl; c) HNMe(OMe)-HCl, iPrMgCl, THF, 0 C; d) MeLi,
THF, -78 oC; e) NH2OH-HCl; f) cyanuric chloride, DMF, 23 oC; g) N2H4 hydrate, EtOH, 100 oC; h) MeSO2Cl, Et3N, 23
oC; i) HFaq-TFA.
6
7
8
9
All the compounds above were obtained in racemic form, but pure enantiomers could be obtained for
a number of derivatives, such as 1, 8, 26 and 50, using preparative HPLC separation on chiral column
(see Supporting Information for details). Compound (+)-1 was crystallized from EtOAc/Hexane
solution and its structure was determined by X-ray crystallography,29 which showed that the
stereogenic centres C9 and C31 had (S)-configurations (Figure 3). Note, as a consequence of the
change of priority of the substituents, (R)-()-1 and (S)-()GAT229 have different descriptors but share
the same stereocentre’s topicity.
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CF3
S
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NC
H
1
(S)-(+)-
(ABD1235)
1
(S)-(+)-
(ABD1235)
Figure 3. X-ray crystal structure of (S)-(+)-1.
In vitro pharmacology. Modification of the ZCZ011 structure indicated that either a 6-Me (5), 6-Cl (6),
6-CF3 (7) or 6-CN (8) substitution led to moderately potent and efficacious CB1 PAMs, as determined
by enhancement of β-arrestin recruitment, induced by the CB1 agonist CP55,940 at its EC20 in the
PathHunter® β-Arrestin assay (Eurofins DiscoverX), by a variation of the methods described
previously.7 The results are shown in Table 1. Substitutions on the 5-position of the indole gave
compounds with poor potency [5-Me (2), 5-CN (3), 5-NO2 (4)]. However, results from the same assay
demonstrated that, alongside their PAM activity, most of these compounds were also allosteric
agonists, giving receptor activation in the absence of the orthosteric ligand. This compares with
previous reports5 that the enantiomers of AZ-4 (GAT228 and 229) show considerable differences in
their properties, with the (+) enantiomer being an allosteric agonist and the () being a pure PAM.
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Separation of the enantiomers of (±)-8 (6-CN) confirmed that ()-8 was the more potent PAM but,
contrary to GAT228/229,5 did not indicate that the (+)-8 was an agonist: both ()- and (+)-8 showed
enhancement of CP55,940-induced β-arrestin recruitment (Emax = 117% and 51% for the () and (+)
respectively, p<0.005 between enantiomers) with EC50 values that were not significantly different (664
and 863 nM, for the () and (+) respectively). However, both showed very low agonism (activation in
the absence of CP55,940) (Emax = 21 and 6% for the () and (+) respectively) with very poor potency
(EC50 >10 µM) making them both effectively pure PAMs (Table 1). Replacement of the 2-phenyl group
(R3) with different aryl or cycloalkyl groups as in 9-25 was not tolerated. Other researchers30 have
noted that, in the absence of any (R1) substitutions on the indole, 4-fluoro, 4-chloro or 3-chloro-4-
fluoro substitution on the 2-phenyl (R2) appeared to be beneficial for potency in a cAMP assay;
however, no examples featuring both an R1 and R2 substitution were shown.
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In addition to the potential toxicological concerns, the methylene group adjacent to the nitro group
was expected to be acidic and potentially a metabolic liability, contributing to the short half-life.
ZCZ011 (5) was shown to be highly unstable in the rat and mouse liver microsomal assays (t < 10 min),
½
with moderate stability in human liver microsomes (t = 41 min) (Cyprotex Ltd). No significant increase
½
in stability was seen with the 6-Cl (6) and 6-CF3 (7) derivatives or with the placement of an electron-
withdrawing 4-Cl on the 2-phenyl group (16) (see Table 1). We therefore concluded that further
development of the nitro-bearing compounds was unlikely to lead to drug-like compounds (suitable
either for preclinical development or as tool compounds for in vivo target validation studies), and set
about developing the synthetic methods required to permit replacement of the nitro group with a
range of bioisosteres.
Modelling software (FieldStere and FieldAlign, products available from Cresset Biomolecular at the
time) was used to suggest moieties that might mimic the electronic properties of the nitro group, with
the primary feature modelled being that of a hydrogen bond acceptor. Many structures were
identified, but synthetic methods were generally lacking. Various 5-membered heterocycles were
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investigated (Fig. 4); however, none of these were active and the syntheses were not straightforward.
5
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Thus, this line of investigation was terminated because of unproductivity.
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N
N
N
N
N
N
N
N
N
N
N
N
N
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N
N
N
N
H
H
H
H
Figure 4. Inactive heterocyclic analogues of ZCZ011 investigated in this work (see Supporting
Information: Het-1 – Het-4).
The cyano group is sometimes proposed as being a bioisostere for a non-aromatic nitro group;31
although perhaps the most widely known example of this is in fact the use of nitro as a bioisostere of
a cyano group, as seen in the development of ranitidine from cimetidine.32 This bioisosterism was
affirmed by the (albeit modest) potency of compounds 30 and 32, which featured replacement of the
CH2NO2 with either a CH2CN or a CH(CN)2 moiety. Compound 31 showed trends for somewhat reduced
potency and efficacy as compared to its nearest nitro analogue (not significant), and showed similarly-
poor metabolic stability in rat liver microsomes, possibly again related to the acidity of the proton
adjacent to the CN group(s).
Studies aimed at replacement of the stereogenic carbon atom with an amine led to the preparation
of a range of compounds bearing a -COCF3 substituent, some of which (such as compounds 58 and 59,
Fig. 5) showed a modest degree of enhancement (~50% at 1 µM).
O
O
N
CF3
N
CF3
N
N
H
H
59
58
Figure 5. Trifluoroacetamide derivatives tested in this work.
It was assumed that these compounds would be highly susceptible to hydrolysis and they were not
regarded as development candidates or even appropriate for use as tool compounds. However, these
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results suggested that the –CF3 group might be a suitable bioisostere. Consequently, we set about
developing synthetic methodologies to allow access to target compounds.
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R2 R4
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R3
R1
N
H
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aPAM Activity:
EC50
Emax
fT1/2 (min)
RLM
>50 %
enhancement
* at 1000 nM
** at 500 nM
Comp
R1
R2
R3
R4
bPAM
cAgo
dPAM
dAgo
HLM
MLM
*** at 200 nM
2
3
4
5
6
5-Me
5-CN
5-NO2
6-Me
6-Cl
6-CF3
6-CN
6-CN
6-CN
6-Cl
6-Cl
6-Cl
6-Cl
6-Cl
6-Cl
6-Cl
6-Me
6-Cl
6-Cl
2-Thienyl
2-Thienyl
2-Thienyl
2-Thienyl
2-Thienyl
2-Thienyl
2-Thienyl
2-Thienyl
2-Thienyl
2-Thienyl
2-Thienyl
2-Thienyl
2-Thienyl 4-CO2Me-C6H4-
2-Thienyl 4-SO2Me-C6H4-
2-Thienyl 4-COOH-C6H4-
2-Thienyl
2-Thienyl
2-Thienyl
2-Thienyl
2-Thienyl
2-Thienyl
2-Thienyl
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
CH2NO2
CH2NO2
CH2NO2
CH2NO2
CH2NO2
CH2NO2
CH2NO2
(+)CH2NO2
(-)CH2NO2
CH2NO2
CH2NO2
CH2NO2
CH2NO2
CH2NO2
CH2NO2
CH2NO2
CH2NO2
CH2NO2
CH2NO2
CH2NO2
CH2NO2
CH2NO2
*
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
NA
NA
**
**
**
*
49.4
-
41.2±3.1
32.7
30.7
-
-
-
8.2
-
6.4±1.2
13.8
15.6
-
-
-
283±140
462±363
284±211
777±363
1530±1128
974±780
106±18
154±10
163±17
-
55±24
82±48
106±47
10.4±2.1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
7
(±)-8
(+)-8
(-)-8
9
-
-
-
863±211
664±215
51±18##
117±19##
9±4#
21±6#
*
*
>10000
>10000
4-OMe-C6H4-
4-NMe2-C6H4-
4-CN-C6H4-
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
10
11
12
13
14
15
16
17
18
19
20
21
4-Ac-C6H4-
4-Cl-C6H4-
4-Cl-C6H4-
4-F-C6H4-
4-F-C6H4-
3-OMe-C6H4-
3-CN-C6H4-
-
24.2
-
9.8
-
6-Me
6-Cl
6-Cl
-
-
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6-Me
6-Me
6-Me
5-CN
-
2-Thienyl
2-Thienyl
2-Thienyl
2-Thienyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
2-Thienyl
2-Thienyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Cycloprop
-yl methyl
2-Thienyl
2-Furyl
2-Furyl
2-Thienyl
Phenyl
Phenyl
2-Thienyl
Phenyl
2-Thienyl
2-Thienyl
2-Thienyl
2-Thienyl
2-Thienyl
Cyclopropyl
Cyclopentyl
Cyclohexyl
Cyclohexyl
Ph
CH2NO2
CH2NO2
CH2NO2
CH2NO2
CH2CO2H
CH2CO2H
CH2CO2H
CH2OH
*
*
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
NA
NA
NA
NA
NA
NA
NA
NA
*
*
*
*
NA
NA
NA
*
**
**
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
25
21.4
62.7
-
-
-
42.7
26.2
45.7
43.2
39.3
31.2
29.6
51.1
39.4
85.2
267
87.2
3.8
-
-
-
-
14.5
14.8
18.7
22.5
12.4
4.9
13.5
41.4
18.2
40.7
24.8
28.2
(±)-26
(+)-26
(-)-26
27
28
29
30
31
32
33
34
35
36
37
38
39
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
-
-
-
CH2CN
CH(CN)2
CH2CN
-
-
-
-
-
-
6-Me
6-Cl
6-Me
6-Me
6-Cl
-
-
-
1113±401
2354±601
100±36
24±7
CH2CN
CH(CN)2
CH(CN)2
CH(CN)2
COCF3
CHOHCF3
CH2CF3
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
6-Me
6-Me
COCF3
CH2CF3
40
6-Me
Ph
CH2CF3
*
-
-
-
-
74.4
19.8
-
41
42
43
44
45
46
47
48
49
(±)-50
(+)-50
(-)-50
51
6-Me
6-Me
6-Cl
6-Cl
6-Cl
6-CF3
6-CF3
5-Me
5-Me
6-CO2Me
6-CO2Me
6-CO2Me
6-CO2H
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
CH2CF3
CH2CF3
CH2CF3
CH2CF3
CH2CF3
CH2CF3
CH2CF3
CH2CF3
CH2CF3
CH2CF3
CH2CF3
CH2CF3
CH2CF3
**
*
**
***
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
108
59.1
63.9
157
98.6
-
49.7
-
-
-
-
-
-
17.5
10.3
46.3
54.3
78.0
-
36.7
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
***
**
322±252
801±554
203±54
95±15
NA
NA
***
***
***
NA
-
-
-
-
-
-
-
-
-
-
-
-
160±147‡‡
113±62‡
-
682±211‡
416±167‡
-
88±28##
156±41## 51±11##
26±5##
-
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52
53
(±)-1
(S)-(+)-1
(R)-(-)-1
54
6-CH2OH
6-NHAc
6-CN
6-CN
6-CN
2-Thienyl
2-Thienyl
2-Thienyl
2-Thienyl
2-Thienyl
2-Thienyl
2-Thienyl
Ph
Ph
Ph
Ph
Ph
Ph
Ph
CH2CF3
CH2CF3
CH2CF3
CH2CF3
CH2CF3
CH2CF3
CH2CF3
CH2CF3
CH2CF3
NA
NA
***
**
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
177±66
342±142‡
363±169‡‡
158±60
-
647±270
1206±345‡
828±310‡‡
741±362
-
100±25
79±32#
125±29#
121±23
-
30±14
17±4
25±6
39±9
-
72.3±30.5 34.9±7.4 26.5±1.5
**
6-Ac
6-NH2
***
NA
NA
**
51.2
-
-
12.2
-
-
-
-
-
-
55
56
57
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
6-NHSO2Me 2-Thienyl
6-CF3 2-Thienyl
Ph
-
-
-
-
301±66
755±237
162±47
68±14
Cyclopentyl
47.1
44.4
Table 1: Enhancement of β-arrestin recruitment induced by the CB1 agonist CP55,940 and metabolic stability data from human, rat and mouse liver
microsomal stability studies (HLM, RLM and MLM). aPilot data were generated as described previously7 and represents data from a single experiment.
bSelected compounds were evaluated by Eurofins DiscoverX in three independent experiments, using the PathHunter® β-Arrestin assay. Positive allosteric
modulation was assessed using EC20 CP55,940. The EC50 value represents the compound concentration at which 50% of the maximal achievable EC20 CP55,940-
induced β-arrestin recruitment is seen (± standard deviation) for each specific compound. cAgonism was evaluated in the same assay and EC50 and Emax values
d
were generated as described for positive allosteric modulation, using compound alone in the absence of agonist. The Emax for each compound is the maximum
EC20 CP55,940-induced β-arrestin recruitment relative to the maximum achievable with CP55,940. eHuman, rat and mouse liver microsomal stability studies
were conducted Cyprotex Ltd. Half-life values represent data from a single experiment (5 timepoints), except where ± stdev is given (three independent
experiments). ## P<0.005; # P<0.05, between enantiomers (Student’s t-test). ‡‡ P< 0.005 ‡ P<0.05, between IC50 values as PAM and agonist (two-tailed t-
test). NA = not active (defined as <50% enhancement in CP55,940-induced β-arrestin recruitment at 1 µM).
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The unsubstituted derivatives (35, 36 and 37) featuring the CH2CF3, COCF3 and CH(OH)CF3 showed a
small degree of enhancement (30 – 50%) at 1 µM (see Table 1). Incorporation of the 6-methyl group
from ZCZ011 (compounds 38 and 39) gave a further increase in potency and efficacy (50 – 100%
enhancement at 300 nM). Compounds showed good stability in human liver microsomes, and
potential improvement in rat liver microsomes (RLM) compared to their nitro analogues (see Table 1).
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We prepared a range of CF3-derivatives (40-57) with the aim of SAR elucidation and further
improvements in potency and metabolic stability. Several compounds showed excellent potency and
efficacy, with enhancement of over 100% at <200 nM and HLM/RLM T1/2 >50 min; however, results
from these compounds tended to be variable, due to solubility issues not seen with the nitro
derivatives. In order to address these solubility issues, we prepared analogues with a range of polar
substituents on the indole. In parallel, we used the nitro series of compounds as a model system for
rapid preparation of the analogues bearing substituents on the 2-phenyl group. Unfortunately, these
studies showed an unforgiving SAR (see Supporting Information for observations on the SAR) in that
none of the substitutions on the 2-phenyl were tolerated and none of 6-CO2H (51), 6-CH2OH (52), 6-
NHAc (53), 6-NH2 (55) or 6-NHSO2Me (56) gave active compounds. The 6-Ac derivative (54) was potent
and efficacious but showed poor stability in the RLM assay (T1/2 = 12 min). Results are shown in Table
1.
Aside from those compounds with solubility issues, the most potent derivative was the 6-CO2Me (50)
for which the enantiomers were separated. As expected from GAT228/2295 and our own observations
from nitro compound 8, as shown in Table 1, the () enantiomer was significantly more efficacious
(p<0.005) than the (+) enantiomer both as a PAM (Emax = 156±41 vs 88±28%, for the () and (+)
respectively; and as an agonist (Emax = 51±11 vs 26±5%, for the () and (+) respectively). Both the ()
and (+) enantiomers were more potent (p<0.05) as PAMs than as agonists (EC50 = 113±62 / 160±147
nM for the () and (+) respectively as PAMs vs EC50 = 416±167 / 682±211 nM for the () and (+)
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8
respectively as agonists. However, as an ester, compound 50 was assumed to be unsuitable for further
evaluation, due to metabolic instability and, unfortunately, the likely metabolite (the 6-CO2H analogue
51) was inactive.
9
10
11
12
13
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The compound with the best balance of properties was the 6-CN analogue (1). As with the 6-CO2Me
analogue 50, both the () and (+) enantiomers were significantly (p<0.05) more efficacious as PAMs
(Emax = 125±29 / 79±32% for the () and (+) respectively) than as agonists (Emax = 25±6 / 17±4% for the
() and (+) respectively); with the () significantly more efficacious than the (+) enantiomer (p<0.05)
as a PAM. Likewise, both the () and (+) enantiomers were more potent (p<0.05) as PAMs than
agonists (EC50 = 363±169 / 342±142 nM as PAMs, for the () and (+) respectively; and IC50 = 828±310
/ 1206±345 nM as agonists, for the () and (+) respectively). The 6-CN derivative ()-1 was considerably
more stable than ZCZ011, with HLM/RLM/MLM T1/2 of 72.3±30.5 (N.S. in comparison with ZCZ011) /
34.9±7.4 (p<0.005 to ZCZ011) / 26.5±1.5 min (p<0.005 to ZCZ011) respectively, compared to 41.2±3.1
/ 6.4±1.2 / 10.4±2.1 min respectively for ZCZ011. Results are shown in Table 1.
Thus, compound ()-1 was selected for further pharmacological evaluation. The enantiomers of CF3-
derivative 1 and the enantiomers of its nitro analogue 8 were compared to ZCZ011 in several assays,
to assess further the bioisosterism of the -CF3 and -NO2 groups.
A similar pattern for compound potency, but not efficacy, was seen in the DiscoverX Hithunter® cAMP
assay (Eurofins DiscoverX): -CF3-bearing compounds 1 showed a trend for greater potency, than their
-NO2 equivalents 8, and the () enantiomers also showed a trend for greater potency than their (+)
analogues. The results are shown in Table 2. However, contrary to the situation seen with β-arrestin
recruitment, the compounds possessed similar efficacy, with all compounds acting as both PAMs and
full agonists, completely inhibiting forskolin-stimulated cAMP production. The significance of this
apparent difference between the effects on β-arrestin and cAMP is unclear. However, it must be noted
that, as neither ZCZ011 or (±)-1 showed any activity in the tetrad or psychoactive effects (see
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Supporting Information), agonism, with regard to cAMP signalling, does not appear to translate to
4
5
agonism at CB1 in vivo.
6
7
8
9
cAMP EC50 (nM)
cAMP Emax (%)
PAM
Agonist
32 ± 10
251 ± 75
46 ± 7
76 ± 8
29 ± 6
PAM
Agonist
94 ± 4
96 ± 2
97 ± 2
89 ± 6
84 ± 8
10
11
12
13
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18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ZCZ011
(+)-8
()-8
(S)-(+)-1
(R)-()-1
18 ± 1
181 ± 95
36 ± 10
40 ± 17
17 ± 5
98 ± 0
102 ± 5
99 ± 2
96 ± 3
90 ± 1
Table 2. Effects of compounds on cAMP generation, either alone (Agonist) or induced by EC20 CP55,940
(PAM) in the presence of EC80 forskolin in the DiscoverX Hithunter® cAMP assay, using a variation of
the methodology described previously.7 Data is the result of three independent experiments.
Binding studies were also conducted (Eurofins Cerep) using a variant of the methodology described
previously7 and demonstrated that, like ZCZ011, 1 µM (+)-1 and ()-1, showed a significant
enhancement in binding of the CB1 agonist, [3H]CP55940. At this concentration, no enhancement was
noted with either (+)-8 and ()-8. The results are shown in Table 3.
% Enhancement of
[3H]CP55,940 binding
ZCZ011
(+)-8
()-8
127.1 ± 13.3
99.0 ± 10.1
108.4 ± 8.3
144.4 ± 3.5
131.4 ± 4.3
(S)-(+)-1
(R)-()-1
Table 3. Effect of 1 µM ZCZ011, (+)-8, ()-8, (+)-(S)-1 or (R)-()-1 on [3H]CP55,940 binding, using a
variation of the methodology described previously.7 Data is the result of three independent
experiments.
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8
In vivo efficacy. Using the methodology previously described for the racemic ZCZ011,7 (±)-1 was
evaluated in several CB1 receptor-sensitive in vivo models and was shown to possess a similar activity
profile as ZCZ011.
9
10
11
12
13
14
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(±)-1 (40 mg/kg) did not elicit pharmacological effects in the tetrad assay when administered alone.
(±)-1 did not affect either the distance travelled or immobility time during assessment of locomotor
behavior, did not produce antinociception in the tail withdrawal test, did not produce hypothermia or
cause immobility in the catalepsy bar test (see Supporting Information). Thus, even at a high dose,
(±)-1 did not elicit in vivo effects indicative of CB1 receptor orthosteric agonism. In contrast, (±)-1 (40
mg/kg) augmented a subset of pharmacological effects of the CB1 receptor orthosteric agonist
CP55,940. Importantly, (±)-1 produced significant leftward shifts in the dose-response relationships of
CP55,940 in producing antinociception in the tail withdrawal test (see Supporting Information) and
thus was further evaluated for its potential in the mouse chronic constrictive injury of the sciatic nerve
(CCI) model of neuropathic pain.
As shown in Figure 6, mice displayed profound mechanical allodynia seven days post CCI surgery in
both the ipsilateral (Panel A) and contralateral (Panel B) paws. At 40 mg/kg (the same dose at which
ZCZ011 produces anti-allodynic effects7) (±)-1 significantly reversed CCI-induced mechanical allodynia
in a time-dependent manner in the ipsilateral [Ftime(6, 168)=6.411; Fdrug(3, 28)= 101.5; P<0.0001;
Finteraction(18, 168) = 7.394; P<0.0001, Figure 6A] and contralateral [Ftime(6, 168)=14.81; Fdrug(3, 28) =
182.2; Finteraction(18, 168) = 4.901; P<0.0001, Figure 6B] paws. In particular, (±)-1 produced a significant
anti-allodynic effect at 0.5 h post-administration and achieved maximal effectiveness by 1 h.
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Figure 6. Acute administration of 40 mg/kg (±)-1 time-dependently reverses CCI-induced mechanical
sensitivity in ipsilateral and contralateral paw. Data are expressed as mean ± SEM (n = 8/group).
****p<0.0001, *p<0.05 vs sham+vehicle; ###p<0.001 ##p<0.01, #p<0.05 vs CCI+vehicle. pre-sx (pre-
surgery); post-sx (post-surgery).
CONCLUSION
In conclusion, we have demonstrated that a -CF3 serves as a viable bioisosteric replacement for an
aliphatic nitro group. Thus, we have shown that it is possible to remove the “non-drug-like” nitro group
from the most-commonly-studied CB1 PAMs to give compounds with improved potency and metabolic
stability, which retain the activity profile of the parent nitro compound. These findings offer major
assistance in the development of a preclinical candidate suitable for evaluation of the therapeutic
potential of CB1 PAMs in a range of unmet medical needs. We hypothesise that the CF3 group might
be a bioisosteric replacement for a nitro group also in other classes of bioactive compounds, especially
when the nitro group is not involved in highly stabilizing hydrogen-bonds with the receptor, as the CF3
group is known to be a weak hydrogen-bond acceptor.
EXPERIMENTAL SECTION
General Methods: All reactions were carried out under a nitrogen atmosphere, with dry solvents
under anhydrous conditions, unless otherwise mentioned. Solvents and reagents: Anhydrous
tetrahydrofuran (THF), diethyl ether (Et2O), methylene chloride (CH2Cl2), and toluene were purchased
from commercial suppliers. Reagents were purchased at the highest commercial quality, used without
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further purification and handled in accordance with COSHH regulations. Chromatography: Flash
chromatography (FC) was carried out on Silica gel (Merck Silica gel Si 60, 40-63 µm). Thin-layer
chromatography (TLC) was carried out on glass-based 0.25 mm Merck silica gel plates (60F-254) which
were developed with UV irradiation (254 nm and 365 nm), an aqueous solution of KMnO4 and an
ethanolic solution of ammonium molybdate, and heat as developing agents. 1H NMR spectra: These
were recorded at 400 MHz on a Bruker ADVANCEIII 400 instrument. Chemical shifts (δH) are given in
parts per million (ppm) as referenced to the appropriate residual solvent peak. 13C NMR spectra: These
were recorded at 100.6 MHz on a Bruker ADVANCE III 400 instrument. 19F NMR spectra: These were
recorded at 376 MHz on a Bruker ADVANCE III 400 instrument without 1H decoupling. Chemical shifts
(δC) are given in parts per million (ppm) as referenced to CFCl3 as 0 ppm. All NMR spectra were
recorded at 298 K unless otherwise stated. Chemical shifts (δC) are given in parts per million (ppm) as
referenced to CHCl3. The following abbreviations were used to explain the multiplicities: s = singlet, d
= doublet, t = triplet, q = quartet, quint = quintet, m = multiplet, br = broad. Purity analysis: purities
of all final compounds were determined by reverse phase HPLC-MS using an Agilent 1200 series
chromatograph system coupled with an Agilent G6120 signal quadrupole detector equipped with an
electrospray ionisation source and detection in positive mode. HPLC conditions for purity analysis:
Phenomenex Luna C18(2) 100Å column, 4.6 × 250 mm, 5 μm; mobile phase: 75% MeOH / 25% H2O with 0.1%
formic acid; flow rate: 1mL/min. All tested compounds were found to be at least 96% purity. Chiral separation:
semi-preparative enantiomeric separations were carried out using an Agilent Technologies 1200
Series HPLC system equipped with a DAD and a normal phase ChiralPak® IA (10.0 × 250 mm, 5 µm)
chiral column from Daicel Chemical Industries Ltd. Optical rotations: These were measured on an AA-
65 Angular Scale automatic polarimeter from Optical Activity Limited with a 1 dm cell at the sodium D
line.
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The following procedures for the preparation of CF3-PAMs 1, 41 and 52-56 are representative
examples.
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2-Phenyl-3-(3,3,3-trifluoro-1-(thiophen-2-yl)propyl)-1H-indole-6-carbonitrile (1)
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To a solution of compound 82l (147 mg, 0.335 mmol) in THF (5 mL) was added 6 M HCl. (0.5 mL). The
mixture was stirred at 60 °C for 16 h prior to quenching with a saturated solution of NaHCO3 (10 mL)
and diluting with CH2Cl2 (10 mL). The layers were separated and the aqueous layer was extracted with
CH2Cl2 (4 × 5 mL). The combined organic layers were dried over Na2SO4 and concentrated in vacuo.
The residue was passed through a short plug of silica gel (CH2Cl2) and concentrated in vacuo prior to
use.
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To a solution of the crude product in CH2Cl2 (5 mL) was added Et3SiH (233 mg, 320 µL, 2.0 mmol) and
trifluoroacetic acid (193 mg, 130 µL, 1.689 mmol) at 0 °C. The resulting red solution was stirred at 23
°C for 16 h before it was quenched with a saturated solution of NaHCO3 (10 mL) and diluted with CH2Cl2
(10 mL). The layers were separated and the aqueous layer was extracted with CH2Cl2 (4 × 5 mL). The
combined organic layers were dried over Na2SO4 and concentrated in vacuo. The residue was purified
by flash column chromatography (silica gel, hexanes:EtOAc 10:1) to give compound 1 (102 mg, 77%
1
for 2 steps) as pale yellow solid. H NMR (400 MHz, CDCl3) δ = 8.58 (s, 1H), 7.77 (dd, J = 1.3, 0.6 Hz,
1H), 7.61 – 7.46 (m, 6H), 7.34 (dd, J = 8.3, 1.4 Hz, 1H), 7.22 (dd, J = 5.1, 0.8 Hz, 1H), 6.98 (dd, J = 5.1,
3.6 Hz, 1H), 6.93 (dt, J = 3.6, 1.2 Hz, 1H), 4.96 (dd, J = 9.5, 4.8 Hz, 1H), 3.35 – 2.93 (m, 2H); 13C NMR
(101 MHz, CDCl3) δ = 146.9, 140.2, 134.9, 131.2, 129.8, 129.3, 129.1, 128.8, 127.0, 126.0 (q, J = 277.9
Hz), 124.5, 124.2, 123.0, 120.7, 120.5, 116.0, 113.6, 104.6, 39.2 (q, J = 37.5 Hz), 31.8 (q, J = 3.1 Hz); 19F
NMR (376 MHz, CDCl3) δ = -64.34 (t, J = 10.2 Hz, 3F). m/z (ESI) 397.1 [M+H+].
6-Methyl-2-phenyl-3-(3,3,3-trifluoro-1-(thiophen-2-yl)propyl)-1H-indole (41)
Using the method analogous to prepare compound 1, with Et3SiH (176 mg, 242 µL, 1.51 mmol),
compound 82a (108 mg, 0.25 mmol) and trifluoroacetic acid (144 mg, 97 µL, 1.26 mmol) were
employed. Purification by flash column chromatography (silica gel, hexanes:EtOAc 10:1) to give
compound 41 (64 mg, 66% for 2 steps) as a white solid. 1H NMR (400 MHz, CDCl3) δ = 7.95 (s, 1H), 7.62
– 7.36 (m, 6H), 7.23 (s, 1H), 7.20 (dd, J = 4.2, 2.4 Hz, 1H), 7.04 – 6.91 (m, 3H), 4.97 (dd, J = 9.2, 4.8 Hz,
1H), 3.30 – 3.16 (m, 1H), 3.16 – 3.00 (m, 1H), 2.53 (s, 3H); 13C NMR (101 MHz, CDCl3) δ = 148.0, 136.7,
135.4, 132.6, 132.3, 128.9 128.7, 128.3, 126.8, 126.3 (q, J = 278.1 Hz), 124.5, 124.2, 124.1, 121.7, 119.9,
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112.8, 111.3, 39.6 (q, J = 27.1 Hz), 32.1 (q, J = 3.2 Hz), 21.7; 19F NMR (376 MHz, CDCl3) δ = -64.42 (t, J =
10.7 Hz, 3F). m/z (ESI) 386.1 [M+H+].
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(2-Phenyl-3-(3,3,3-trifluoro-1-(thiophen-2-yl)propyl)-1H-indol-6-yl)methanol (52)
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To a solution of compound 50 (15 mg, 0.035 mmol) in THF (2 mL) was added a solution of Super-
Hydride® (105 µL, 0.105 mmol, 1.0 M in THF) at 0 °C. The resulting mixture was stirred at 0 °C and
allowed to warm to 23 °C for 16 h before it was quenched with a saturated solution of NH4Cl (10 mL)
and diluted with CH2Cl2 (10 mL). The layers were separated and the aqueous layer was extracted with
CH2Cl (4 x 5 mL). The combined organic layers were dried over Na2SO4 and concentrated in vacuo. The
residue was purified by flash column chromatography (silica gel, hexanes:EtOAc 5:1  1:1) to give
compound 52 (13 mg, 94%) as a colourless oil. 1H NMR (400 MHz, CDCl3) δ = 8.13 (s, 1H), 7.69 – 7.35
(m, 7H), 7.19 (dd, J = 4.7, 1.5 Hz, 1H), 7.12 (d, J = 8.1 Hz, 1H), 7.02 – 6.83 (m, 2H), 4.95 (dd, J = 9.3, 4.8
Hz, 1H), 4.81 (s, 2H); 19F NMR (376 MHz, CDCl3) δ = -64.54 (t, J = 10.6 Hz, 3F). m/z (ESI) 424.1 [M+Na+].
N-(2-Phenyl-3-(3,3,3-trifluoro-1-(thiophen-2-yl)propyl)-1H-indol-6-yl)acetamide (53)
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To a solution of compound 87 (15 mg, 0.027 mmol)in acetonitrile (2 mL) in a PTFE container was added
an aqueous solution of HF (1 mL, 48 % wt) at 0 °C. The resulting solution was allowed to warm to 23
°C and stirred for 16 h before it was quenched with a saturated solution of NaHCO3 (20 mL). The
mixture was stirred at 23 °C for 10 min before EtOAc (20 mL) was added. The layers were separated
and the organic layer was extracted with H2O (2 x 10 mL). The combined organic layers were dried
over Na2SO4 and concentrated in vacuo.
The residue was re-dissolved in MeOH (2 mL) and K2CO3 was added at 0 °C. The mixture was stirred at
0 °C for 30 min prior to quenching with a saturated solution of NH4Cl and diluted with EtOAc (30 mL).
The layers were separated and the organic layer was extracted with H2O (2 x 10 mL). The combined
organic layers were dried over Na2SO4 and concentrated in vacuo. The residue was purified by flash
column chromatography (silica gel, hexanes:EtOAc 3:1) to give compound 53 (9 mg, 78% for 2 steps)
as pale yellow solid. 1H NMR (400 MHz, CDCl3) δ = 8.56 (brs, 1H), 8.14 (d, J = 1.5 Hz, 1H), 7.57 – 7.45
(m, 4H), 7.45 – 7.36 (m, 2H), 7.31 (s, 1H), 7.18 (dd, J = 5.0, 1.1 Hz, 1H), 6.97 – 6.89 (m, 2H), 6.79 (dd, J
= 8.5, 1.8 Hz, 1H), 4.92 (dd, J = 8.9, 4.9 Hz, 1H), 3.22 – 2.93 (m, 2H), 2.17 (s, 3H); 13C NMR (101 MHz,
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CDCl3) δ = 168.3, 148.2, 142.3, 137.8, 134.2, 132.9, 129.0, 128.6, 128.2, 126.9, 126.4 (q, J = 278.5 Hz),
124.3, 121.1, 120.4, 113.1, 110.9, 97.0, 39.1 (q, J = 26.8 Hz), 32.2 (q, J = 2.8 Hz), 24.9; 19F NMR (376
MHz, CDCl3) δ = -64.42 (t, J = 10.4 Hz, 3F). m/z (ESI) 429.1 [M+H+].
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1-(2-Phenyl-3-(3,3,3-trifluoro-1-(thiophen-2-yl)propyl)-1H-indol-6-yl)ethan-1-one (54)
Using the method analogous to prepare compound 53, with compound 85 (22 mg, 0.04 mmol) was
employed. Purification by flash column chromatography (silica gel, hexanes:EtOAc 3:1) to give
compound 54 (12 mg, 72% for two steps) as a colourless oil. 1H NMR (400 MHz, CDCl3) δ = 8.59 (s, 1H),
8.13 (s, 1H), 7.74 (dd, J = 8.4, 1.3 Hz, 1H), 7.59 – 7.42 (m, 6H), 7.20 (d, J = 4.8 Hz, 1H), 7.02 – 6.86 (m,
2H), 4.96 (dd, J = 9.5, 4.6 Hz, 1H), 3.18 – 3.03 (m, 2H), 2.67 (s, 3H); 13C NMR (101 MHz, CDCl3) δ = 198.2,
147.3, 140.1, 135.7, 131.7, 131.6, 130.4, 129.1, 128.7, 126.9, 126.2 (q, J = 276.2 Hz), 124.4, 124.2,
120.4, 119.7, 113.3, 112.2, 39.4 (q, J = 27.1 Hz), 31.9 (q, J = 2.8 Hz), 26.8; 19F NMR (376 MHz, CDCl3) δ
= -64.39 (t, J = 10.4 Hz, 3F). m/z (ESI) 414.1 [M+H+].
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2-Phenyl-3-(3,3,3-trifluoro-1-(thiophen-2-yl)propyl)-1H-indol-6-amine (55)
Using the method analogous to prepare compound 53, with compound 88 (30 mg, 0.058 mmol) was
employed. Purification by flash column chromatography (silica gel, hexanes:EtOAc 1:1) gave
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compound 55 (18 mg, 80% for 2 steps) as pale yellow solid. H NMR (400 MHz, CDCl3) δ = 7.86 (brs,
1H), 7.53 – 7.42 (m, 4H), 7.41 (dd, J = 5.2, 3.6 Hz, 1H), 7.31 (s, 1H), 7.20 – 7.13 (m, 1H), 6.94 (d, J = 3.6
Hz, 2H), 6.72 (d, J = 1.8 Hz, 1H), 6.56 (dd, J = 8.4, 2.0 Hz, 1H), 4.91 (dd, J = 8.8, 5.1 Hz, 1H), 3.28 – 2.55
(m+brs, 4H); 13C NMR (101 MHz, CDCl3) δ = 148.0, 142.2, 137.6, 134.0, 132.7, 128.9, 128.5, 128.0,
126.7, 126.3 (q, J = 277.1 Hz), 124.1, 120.9, 120.2, 112.9, 110.7, 96.8, 39.6 (q, J = 26.8 Hz), 32.0 (q, J =
3.2 Hz); 19F NMR (376 MHz, CDCl3) δ = -64.45 (t, J = 10.6 Hz, 3F). m/z (ESI) 387.1 [M+H+].
N-(2-Phenyl-3-(3,3,3-trifluoro-1-(thiophen-2-yl)propyl)-1H-indol-6-yl)methanesulfonamide (56)
Using the method analogous to prepare compound 53, with compound 89 (32 mg, 0.054 mmol) was
employed. Purification by flash column chromatography (silica gel, hexanes:EtOAc 1:1) gave
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compound 56 (20 mg, 81% for 2 steps) as pale yellow solid. H NMR (400 MHz, CDCl3) δ = 8.29 (brs,
1H), 7.64 – 7.40 (m, 7H), 7.19 (dd, J = 5.0, 1.2 Hz, 1H), 7.02 – 6.91 (m, 2H), 6.89 (dd, J = 8.5, 2.0 Hz, 1H),
6.67 (brs, 1H), 4.94 (dd, J = 9.1, 4.9 Hz, 1H), 3.22 – 3.01 (m, 2H), 3.00 (s, 3H); 13C NMR (101 MHz, CDCl3)
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δ = 147.5, 137.1, 136.5, 132.0, 131.1, 129.0, 128.7, 126.8, 126.3 (q, J = 276.8 Hz), 125.0, 124.3, 124.1,
120.9, 115.3, 112.8, 105.7, 39.4 (q, J = 27.2 Hz), 39.0, 31.9 (q, J = 3.1 Hz); 19F NMR (376 MHz, CDCl3) δ
= -64.38 (t, J = 10.4 Hz, 3F). m/z (ESI) 464.1 [M+H+].
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3-Ethynyl-1-(methoxymethyl)-6-methyl-2-phenyl-1H-indole (79a)
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To a solution of 1-(methoxymethyl)-6-methyl-2-phenyl-1H-indole (575 mg, 2.29 mmol) in CH2Cl2 (10
mL) was added N-iodosuccinimide (525 mg, 2.33 mmol) at 0 °C. The reaction mixture was stirred for 3
h before it was quenched with a saturated solution of Na2SO3 (5 mL) and diluted with H2O (10 mL).
The layers were separated and the aqueous layer was extracted with CH2Cl2 (3 × 10 mL). The combined
organic layers were dried over Na2SO4 and concentrated in vacuo to give the corresponding 3-iodo-
indole, which was used without further purification.
To a solution of the crude 3-iodo-indole in DMF (5 mL) was added trimethylsilylacetylene (337 mg, 475
µL, 3.43 mmol), PdCl2(PPh3)2 (80 mg, 0.114 mmol), CuI (26 mg, 0.137 mmol) and Et3N (695 mg, 957 µL,
6.87 mmol) at 23 °C. The reaction mixture was stirred for 6 h at 23 °C before it was quenched with a
saturated solution of NH4Cl (10 mL) and diluted with EtOAc (50 mL). The layers were separated, and
the organic layer was extracted with H2O (3 x 10 mL). The combined organic layers were dried over
Na2SO4 and concentrated in vacuo. The residue was passed through a short plug of silica gel (CH2Cl2)
and concentrated in vacuo prior to use.
To a solution of the crude product was added a solution of tetra-n-butylammonium fluoride (2.8 mL,
2.8 mmol, 1.0 M in THF) at 0 °C. The reaction mixture was stirred for 2 h at 23 °C before it was it was
quenched with a saturated solution of NH4Cl (10 mL) and diluted with EtOAc (50 mL). The layers were
separated, and the organic layer was extracted with H2O (3 x 10 mL). The combined organic layers
were dried over Na2SO4 and concentrated in vacuo. The residue was purified by flash column
chromatography (silica gel, hexanes:EtOAc 5:1) to give compound 79a (566 mg, 90% for 3 steps). 1H
NMR (400 MHz, CDCl3) δ = 7.76 – 7.66 (m, 1H), 7.59 – 7.44 (m, 5H), 7.40 – 7.33 (m, 1H), 7.15 (d, J = 8.0
Hz, 1H), 5.39 (s, 2H), 3.28 (s, 3H), 3.18 (s, 1H), 2.56 (s, 3H); 13C NMR (101 MHz, CDCl3) δ = 144.4, 137.1,
133.6, 131.2, 130.5, 130.3, 128.8, 128.5, 127.2, 123.4, 119.7, 110.5, 79.7, 78.00, 74.9, 56.0, 22.0. m/z
(ESI) 276.1 [M+H+].
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3-Ethynyl-1-(methoxymethyl)-2-phenyl-1H-indole-6-carbonitrile (79g)
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Using the method analogous to prepare compound 79a, with compound 94d (700 mg, 2.67 mmol), N-
iodosuccinimide (616 mg, 2.74 mmol), trimethylsilylacetylene (394 mg, 555 µL, 4.01 mmol),
PdCl2(PPh3)2 (94 mg, 0.134 mmol), CuI (25 mg, 0.131 mmol), Et3N (813 mg, 1.12 mL, 8.03 mmol) and
tetra-n-butylammonium fluoride (3.40 mL, 3.40 mmol, 1.0 M in THF) were used. Purification by flash
column chromatography (silica gel, hexanes:EtOAc 5:1) gave compound 79g (596 mg, 78% for 3 steps)
as a colourless oil. 1H NMR (400 MHz, CDCl3) δ = 7.88 (d, J = 0.5 Hz, 1H), 7.85 (dd, J = 8.2, 0.5 Hz, 1H),
7.72 (d, J = 1.8 Hz, 1H), 7.70 (d, J = 1.4 Hz, 1H), 7.61 – 7.49 (m, 4H), 5.41 (s, 2H), 3.30 (s, 3H), 3.19 (s,
1H); 13C NMR (101 MHz, CDCl3) δ = 147.8, 135.5, 132.5, 130.2, 129.7, 129.1, 129.0, 128.8, 124.6, 120.9,
120.2, 115.5, 106.1, 80.9, 76.2, 75.3, 56.3. m/z (ESI) 309.1 [M+Na+].
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1-(Methoxymethyl)-6-methyl-2-phenyl-3-(3,3,3-trifluoroprop-1-yn-1-yl)-1H-indole (80a)
Using a method described by Blanchard,27 to a solution of N,N,N´,N´-tetramethylethylenediamine (324
mg, 418 µL, 2.79 mmol) in DMF (5 mL) was added CuI (531 mg, 2.79 mmol) and K2CO3 (771 mg, 5.58
mmol) at 23 °C. The resulting blue mixture was stirred vigorously at 23 °C under air (1 atm) for 20 min
before trimethyl(trifluoromethyl)silane (529 mg, 550 µL, 3.72 mmol) was added and the resulting
mixture was stirred for further 15 min under air. After which time, the deep green mixture was cooled
to
0 °C prior to adding a mixture of compound 79a (512 mg, 1.86 mmol) and
trimethyl(trifluoromethyl)silane (529 mg, 550 µL, 3.72 mmol) in DMF (2 mL). The reaction mixture was
stirred at 0 °C for 30 min and allowed to warm to 23 °C for 6 h before it was quenched with a saturated
solution of NH4Cl (10 mL) and diluted with EtOAc (50 mL). The layers were separated, and the organic
layer was extracted with H2O (3 x 10 mL). The combined organic layers were dried over Na2SO4 and
concentrated in vacuo. The residue was purified by flash column chromatography (silica gel,
hexanes:EtOAc 4:1) to give compound 80a (556 mg, 87%) as a pale yellow oil. 1H NMR (400 MHz, CDCl3)
δ = 7.75 – 7.64 (m, 3H), 7.64 – 7.47 (m, 3H), 7.39 (s, 1H), 7.19 (d, J = 8.0 Hz, 1H), 5.40 (s, 2H), 3.32 (s,
3H), 2.58 (s, 3H); 13C NMR (101 MHz, CDCl3) δ = 147.0 (q, J = 1.7 Hz), 137.1, 134.3, 130.1, 129.50,
129.47, 128.8, 126.5, 124.1, 119.4, 115.5 (q, J = 256.2 Hz), 110.9, 93.6 (q, J = 2.5 Hz), 83.3 (q, J = 6.2
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Hz), 78.7 (q, J = 52.2 Hz), 75.0, 56.1, 22.0; 19F NMR (376 MHz, CDCl3) δ = -48.59 (s, 3F). m/z (ESI) 344.1
[M+H+].
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Using the method analogous to prepare compound 80a, with compound 79g (300 mg, 1.05 mmol),
N,N,N´,N´-tetramethylethylenediamine (185 mg, 255 µL, 1.57 mmol), CuI (300 mg, 1.58 mmol), K2CO3
(435 mg, 3.15 mmol) and trimethyl(trifluoromethyl)silane (596 mg, 620 µL, 4.19 mmol) were used.
Purification by flash column chromatography (silica gel, hexanes:EtOAc 6:1) gave compound 80g (352
mg, 95%) as a pale yellow oil. 1H NMR (400 MHz, CDCl3) δ = 7.92 (s, 1H), 7.83 (d, J = 8.2 Hz, 1H), 7.72 –
7.65 (m, 2H), 7.64 – 7.59 (m, 3H), 7.56 (dd, J = 8.2, 1.2 Hz, 1H), 5.44 (s, 2H), 3.33 (s, 3H); 13C NMR (101
MHz, CDCl3) δ = 150.5 (q, J = 1.7 Hz), 135.6, 131.8, 130.4, 130.0, 129.0, 128.2, 125.3, 120.7, 119.7,
115.9, 115.1 (q, J = 256.4 Hz), 106.9, 94.5 (q, J = 2.2 Hz), 81.1 (q, J = 6.2 Hz), 79.5 (q, J = 52.4 Hz), 75.4,
56.4. 19F NMR (376 MHz, CDCl3) δ = -49.13 (s, 3F). m/z (ESI) 355.1 [M+H+].
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(E)-1-(Methoxymethyl)-6-methyl-2-phenyl-3-(3,3,3-trifluoro-1-iodoprop-1-en-1-yl)-1H-indole (81a)
Using a modified method described by Zhong,28 to a solution of compound 80a (420 mg, 1.22 mmol)
in CH2Cl2 (4 mL) was added LiI (180 mg, 1.35 mmol) and HOAc (1 mL) at 0 °C. The resulting mixture was
stirred at 23 °C for 16 h before it was quenched with a saturated solution of NaHCO3 (10 mL) and
diluted with CH2Cl2 (10 mL). The layers were separated, and the aqueous layer was extracted with
CH2Cl2 (2 × 10 mL). The combined organic layers were dried over Na2SO4 and concentrated in vacuo.
The residue was purified by flash column chromatography (silica gel, hexanes:EtOAc 10:1) to give
compound 81a (558 mg, 97%) as a pale yellow solid. 1H NMR (400 MHz, CDCl3) δ = 7.65 – 7.48 (m, 6H),
7.38 (s, 1H), 7.24 – 7.14 (m, 1H), 6.71 (q, J = 7.1 Hz, 1H), 5.38 (s, 2H), 3.25 (s, 3H), 2.58 (s, 3H); 13C NMR
(101 MHz, CDCl3) δ = 137.5, 137.0, 133.6, 132.0 (q, J = 33.9 Hz), 130.4, 130.2, 129.0, 128.6, 124.0,
123.3, 121.3 (q, J = 274.4 Hz), 119.4, 115.8, 110.6, 104.4 (q, J = 6.2 Hz), 74.8, 55.9, 22.0; 19F NMR (376
MHz, CDCl3) δ = -60.80 (d, J = 7.1 Hz, 3F). m/z (ESI) 472.0 [M+H+].
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(E)-1-(methoxymethyl)-2-phenyl-3-(3,3,3-trifluoro-1-iodoprop-1-en-1-yl)-1H-indole-6-carbonitrile
(81g)
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Using the method analogous to prepare compound 81a, with compound 80g (330 mg, 0.931mmol)
and LiI (150 mg, 1.12 mmol). The residue was passed through a short plug of silica gel (CH2Cl2) and
concentrated in vacuo to give compound 81g, which was used without further purification.
(E)-1-(Methoxymethyl)-6-methyl-2-phenyl-3-(3,3,3-trifluoro-1-(thiophen-2-yl)prop-1-en-1-yl)-1H-
indole (82a)
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To a solution of compound 81a (146 mg, 0.31 mmol) in DMF (5 mL) was added tri-n-butyl(thiophen-2-
yl)stannane (175 mg, 0.47 mmol), P(o-Tol)3 (14 mg, 0.046 mmol), PdCl2(MeCN)2 (7.8 mg, 0.03 mmol)
and CuI (9 mg, 0.046 mmol) at 23 °C. The resulting mixture was stirred at 23 °C for 20 min before it
was heated to 55 °C for 3 h. After which time, the reaction mixture was quenched with a saturated
solution of NH4Cl (10 mL) and diluted with Et2O (50 mL). The layers were separated, and the organic
layer was extracted with H2O (3 x 10 mL). The combined organic layers were dried over Na2SO4 and
concentrated in vacuo. The residue was purified by flash column chromatography (silica gel,
hexanes:EtOAc 10:1) to give compound 82a (120 mg, 90%) as a colourless oil. 1H NMR (400 MHz, CDCl3)
δ = 7.50 – 7.31 (m, 7H), 7.27 (d, J = 5.0 Hz, 1H), 7.06 (d, J = 8.1 Hz, 1H), 6.98 (d, J = 3.4 Hz, 1H), 6.91
(dd, J = 4.9, 3.8 Hz, 1H), 6.31 (q, J = 8.0 Hz, 1H), 5.46 (s, 2H), 3.24 (s, 3H), 2.57 (s, 3H); 13C NMR (101
MHz, CDCl3) δ = 144.3, 138.4 (q, J = 5.5 Hz), 137.3, 132.9, 130.8, 130.3, 128.4, 128.3, 128.1, 127.7,
127.4, 126.2, 123.1 (q, J = 270.6 Hz), 123.0, 119.4, 115.1 (q, J = 33.4 Hz),111.1, 110.5, 74.8, 55.6, 22.0;
19F NMR (376 MHz, CDCl3) δ = -58.23 (d, J = 8.0 Hz, 3F). m/z (ESI) 428.1 [M+H+].
(E)-1-(methoxymethyl)-2-phenyl-3-(3,3,3-trifluoro-1-(thiophen-2-yl)prop-1-en-1-yl)-1H-indole-6-
carbonitrile (82l)
Using the method analogous to prepare compound 82a, with compound 81g (449 mg, 0.931 mmol),
tri-n-butyl(thiophen-2-yl)stannane (522 mg, 1.40 mmol), P(o-Tol)3 (43 mg, 0.141 mmol), PdCl2(MeCN)2
(24 mg, 0.093 mmol) and CuI (18 mg, 0.095 mmol) were used. Purification by flash column
chromatography (silica gel, hexanes:EtOAc 5:1) gave compound 82l (334 mg, 82%) as a pale yellow oil.
1H NMR (400 MHz, CDCl3) δ = 7.93 (s, 1H), 7.48 (d, J = 8.3 Hz, 1H), 7.45 – 7.32 (m, 6H), 7.31 – 7.23 (m,
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1H), 6.93 – 6.83 (m, 2H), 6.30 (q, J = 8.2 Hz, 1H), 5.47 (s, 2H), 3.26 (s, 3H); 13C NMR (101 MHz, CDCl3) δ
= 143.5, 142.6, 137.1 (q, J = 6.0 Hz), 135. 6, 131.1, 130.1, 129.5, 129.3, 128.5, 128.2, 127.89, 127.86,
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124.2, 123.3 (q, J = 268.2 Hz), 121.5, 120.3, 115.9 (q, J = 34.2 Hz), 115.7, 111.4, 105.3, 75.1, 56.0; 19
NMR (376 MHz, CDCl3) δ = -58.18 (d, J = 8.2 Hz, 3F). m/z (ESI) 439.1 [M+H+].
Methyl 2-phenyl-3-(3,3,3-trifluoro-1-(thiophen-2-yl)propyl)-1-((2-(trimethylsilyl)-ethoxy)methyl)-
1H-indole-6-carboxylate (83)
F
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To a solution of compound 50 (540 mg, 1.257 mmol) in THF (3 mL) was added NaH (75 mg, 1.886
mmol, 60 % in mineral oil) at 0 °C. After stirring at 0 °C for 30 min, 2-(trimethylsilyl)ethoxymethyl
chloride (SEM-Cl, 282 mg, 300 µL, 1.51 mmol, 90% technical grade) was added. The resulting reaction
mixture was stirred at 0 °C and allowed to warm to 23 °C for 3 h before it was quenched with a
saturated solution of NH4Cl (5 mL) and diluted with EtOAc (50 mL). The layers were separated and the
organic layer was extracted with H2O (4 x 10 mL). The combined organic layers were dried over Na2SO4
and concentrated in vacuo. The residue was purified by flash column chromatography (silica gel,
hexanes:EtOAc 5:1) to give compound 83 (680 mg, 97%) as a colourless oil. 1H NMR (400 MHz, CDCl3)
δ = 8.33 (s, 1H), 7.85 (dd, J = 8.4, 1.3 Hz, 1H), 7.59 – 7.39 (m, 6H), 7.16 (d, J = 5.0 Hz, 1H), 6.93 (dd, J =
5.1, 3.6 Hz, 1H), 6.86 (d, J = 3.5 Hz, 1H), 5.41 (s, 2H), 4.65 (dd, J = 9.9, 4.3 Hz, 1H), 3.97 (s, 3H), 3.34
(ABq, J = 7.6 Hz, 2H), 3.26 – 3.08 (m, 1H), 3.08 – 2.92 (m, 1H), 0.80 (ABq, J = 7.6 Hz, 2H), -0.07 (s, 9H);
13C NMR (101 MHz, CDCl3) δ = 167.9, 147.3, 142.0, 136.7, 130.8, 130.2, 129.6, 129.3, 128.6, 126.7,
126.1 (q, J = 277.1 Hz), 124.2, 124.0, 123.8, 121.5, 119.4, 114.7, 113.1, 94.3, 72.9, 65.8, 52.0, 39.0 (q,
J = 27.5 Hz), 32.1 (q, J = 2.6 Hz), 17.8, -1.5; 19F NMR (376 MHz, CDCl3) δ = -64.20 (t, J = 10.4 Hz, 3F). m/z
(ESI) 560.2 [M+H+].
N-Methoxy-N-methyl-2-phenyl-3-(3,3,3-trifluoro-1-(thiophen-2-yl)propyl)-1-((2-
(trimethylsilyl)ethoxy)methyl)-1H-indole-6-carboxamide (84)
Using a modified method described by Williams,33 to a solution of compound 83 (408 mg, 0.729 mmol)
and N,O-dimethylhydroxylamine hydrochloride (108 mg, 1.11 mmol) in THF (3 mL) was added a
solution of isopropylmagnesium chloride (1.10 mL, 2.19 mmol, 2.0 M in THF) at 0 °C. After stirring at
0 °C for 30 min, the solution was allowed to warm to 23 °C and stirred for further 3 h before it was
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quenched with a saturated solution of NH4Cl (5 mL) and diluted with EtOAc (50 mL). The layers were
separated and the organic layer was extracted with H2O (4 x 10 mL). The combined organic layers were
dried over Na2SO4 and concentrated in vacuo. The residue was purified by flash column
chromatography (silica gel, hexanes:EtOAc 3:1) to give compound 84 (326 mg, 76%) as a colourless oil.
1H NMR (400 MHz, CDCl3) δ = 8.00 (s, 1H), 7.61 – 7.39 (m, 7H), 7.16 (dd, J = 5.1, 0.9 Hz, 1H), 6.92 (dd,
J = 5.1, 3.6 Hz, 1H), 6.87 (dt, J = 3.5, 1.2 Hz, 1H), 5.38 (ABq, J = 12.7 Hz, 2H), 4.64 (dd, J = 9.7, 4.3 Hz,
1H), 3.65 (s, 3H), 3.42 (s, 3H), 3.33 (ddd, J = 9.5, 6.2, 1.9 Hz, 2H), 3.25 – 3.09 (m, 1H), 3.12 – 2.89 (m,
1H), 0.80 (ddd, J = 9.5, 6.2, 1.9 Hz, 2H), -0.07 (s, 9H); 13C NMR (101 MHz, CDCl3) δ = 170.7, 147.4, 140.9,
136.4, 130.9, 130.4, 129.2, 128.6, 127.9, 127.8, 126.7, 126.2 (q, J = 278.5 Hz), 124.2, 123.8, 120.7,
119.3, 114.5, 111.8, 73.0, 65.7, 61.0, 39.0 (q, J = 27.2 Hz), 34.4, 32.1 (q, J = 2.8 Hz), 17.8, -1.5; 19F NMR
(376 MHz, CDCl3) δ = -64.19 (t, J = 10.3 Hz, 3F). m/z (ESI) 589.2 [M+H+].
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1-(2-Phenyl-3-(3,3,3-trifluoro-1-(thiophen-2-yl)propyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-
indol-6-yl)ethan-1-one (85)
To a solution of compound 84 (408 mg, 0.54 mmol) in THF (3 mL) was added a solution of
methyllithium (860 µL, 0.860 mmol, 1.6 M in Et2O) at -78 °C. After stirring at -78 °C for 30 min, the
solution was allowed to warm to 0 °C and stirred for further 3 h before it was quenched with a
saturated solution of NH4Cl (5 mL) and 0.1 M HCl solution (5 mL). The mixture was stirred at 23 °C for
10 min before EtOAc (50 mL) was added. The layers were separated and the organic layer was
extracted with H2O (4 x 10 mL). The combined organic layers were dried over Na2SO4 and concentrated
in vacuo. The residue was purified by flash column chromatography (silica gel, hexanes:EtOAc 5:1) to
give compound 85 (252 mg, 86%) as a colourless oil. 1H NMR (400 MHz, CDCl3) δ = 8.26 (s, 1H), 7.80
(dd, J = 8.4, 1.4 Hz, 1H), 7.64 – 7.41 (m, 6H), 7.17 (dd, J = 5.1, 1.0 Hz, 1H), 6.94 (dd, J = 5.1, 3.6 Hz, 1H),
6.90 – 6.84 (m, 1H), 5.44 (s, 2H), 4.66 (dd, J = 10.0, 4.2 Hz, 1H), 3.41 – 3.30 (m, 2H), 3.25 – 3.10 (m,
1H), 3.09 – 2.95 (m, 1H), 2.71 (s, 3H), 0.85 – 0.74 (m, 2H), -0.06 (s, 9H); 13C NMR (101 MHz, CDCl3) δ =
198.0, 147.3, 142.5, 136.8, 131.7, 130.8, 130.1, 129.8, 129.4, 128.6, 126.8, 126.1 (q, J = 277.4 Hz),
124.3, 123.8, 120.8, 119.5, 114.8, 111.8, 73.0, 65.9, 38.9 (q, J = 27.6 Hz), 32.1 (q, J = 3.3 Hz), 26.82,
17.78, -1.49; 19F NMR (376 MHz, CDCl3) δ = -64.14 (t, J = 10.3 Hz, 3F). m/z (ESI) 544.2 [M+H+].
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To a solution of compound 85 (220 mg, 0.405 mmol) in MeOH (5 mL) was added hydroxylamine
hydrochloride (34 mg, 0.489 mmol) and sodium acetate (50 mg, 0.61 mmol). The resulting mixture
was heated to 70 °C for 4 h before it was cooled to 23 °C and quenched with a saturated solution of
NH4Cl (10 mL) and then diluted with CH2Cl2 (10 mL). The layers were separated and the aqueous layer
was extracted with CH2Cl2 (4 x 5 mL). The combined organic layers were dried over Na2SO4 and
concentrated in vacuo to give oxime 86 as a mixture of (E)- and (Z)-isomers. Oxime 86 was used
without further purification.
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N-(2-Phenyl-3-(3,3,3-trifluoro-1-(thiophen-2-yl)propyl)-1-((2-(trimethylsilyl)-ethoxy)methyl)-1H-
indol-6-yl)acetamide (87)
Using a modified method described by Giacomelli,34 2,4,6-trichloro-[1,3,5]triazine (cyanuric chloride,
32 mg, 0.445 mmol) was added DMF (1 mL) at 23 °C and stirred for 30 min before a solution of oxime
86 (226 mg, 0.404 mmol) in DMF (1 mL) was added. The resulting mixture was stirred for 4 h at 23 °C
prior to quenching with a saturated solution of NaHCO3 (10 mL) and diluted with EtOAc (50 mL). The
layers were separated and the organic layer was extracted with H2O (4 x 10 mL). The combined organic
layers were dried over Na2SO4 and concentrated in vacuo. The residue was purified by flash column
chromatography (silica gel, hexanes:EtOAc 1:1) to give compound 87 (208 mg, 92% for 2 steps) as a
pale yellow oil. 1H NMR (400 MHz, CDCl3) δ = 8.01 (d, J = 1.0 Hz, 1H), 7.59 (s, 1H, NH), 7.55 – 7.42 (m,
5H), 7.40 (d, J = 8.5 Hz, 1H), 7.14 (d, J = 5.0 Hz, 1H), 7.05 (dd, J = 8.5, 1.5 Hz, 1H), 6.96 – 6.88 (m, 1H),
6.86 (d, J = 3.4 Hz, 1H), 5.31 (s, 2H), 4.62 (dd, J = 9.5, 4.3 Hz, 1H), 3.32 (ddd, J = 9.7, 5.2, 3.0 Hz, 2H),
3.21 – 3.07 (m, 1H), 3.07 – 2.91 (m, 1H), 2.19 (s, 3H), 0.82 – 0.73 (m, 2H), -0.07 (s, 9H); 13C NMR (101
MHz, CDCl3) δ = 168.4, 147.8, 138.9, 137.5, 133.1, 131.0, 130.7, 128.9, 128.5, 126.7, 126.2 (q, J = 279.0
Hz), 124.1, 123.7, 123.0, 120.0, 114.3, 114.0, 103.1, 72.8, 65.6, 39.0 (q, J = 27.1 Hz), 32.1 (q, J = 2.7 Hz),
24.6, 17.8, -1.5; 19F NMR (376 MHz, CDCl3) δ = -64.19 (t, J = 10.3 Hz, 3F). m/z (ESI) 559.2 [M+H+].
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