First Nondiscriminating Translocator Protein Ligands Produced from a Carbazole Scaffold

Article abstract of DOI:10.1021/acs.jmedchem.9b00980

Development of neuroinflammation agents targeting the translocator protein (TSPO) has been hindered by a common single nucleotide polymorphism (A147T) at which TSPO ligands commonly lose affinity. To this end, carbazole acetamide scaffolds were synthesized and structure activity relationships elaborated to explore the requirements for high-affinity binding to both TSPO wild type (WT) and the polymorphic TSPO A147T. This study reports high binding affinity and nondiscriminating TSPO ligands.

Full text of DOI:10.1021/acs.jmedchem.9b00980

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Article
The first non-discriminating TSPO ligands produced from a carbazole scaffold.
Hei Wun Alison Cheng, Renee Sokias, Eryn Werry, Lars Ittner, Tristan
Reekie, Jonathan J. Du, Quanqing Gao, David E Hibbs, and Michael Kassiou
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00980 • Publication Date (Web): 16 Aug 2019
Downloaded from pubs.acs.org on August 18, 2019
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The first non-discriminating TSPO ligands produced
from a carbazole scaffold
Hei Wun Alison Cheng,§ Renee Sokias,#§ Eryn L. Werry,# Lars M. Ittner, Tristan A. Reekie,# Jonathan
Du, Quanqing Gao, David E. Hibbs and Michael Kassiou.#*
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Faculty of Medicine and Health, #School of Chemistry and Sydney School of Pharmacy, Faculty of
Medicine and Health, The University of Sydney, NSW, Australia, 2006 Department of Biomedical
Sciences, Faculty of Medicine and Health Sciences, 2 Technology Place, Macquarie University, NSW,
Australia, 2109.
* Telephone: +61 2 9351 2745. Fax: +61 2 9351 3329. Email: michael.kassiou@sydney.edu.au.
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ABSTRACT. Development of neuroinflammation agents targeting the translocator protein (TSPO) has
been hindered by a common single nucleotide polymorphism (A147T) at which TSPO ligands commonly
lose affinity. To this end, carbazole acetamide scaffolds were synthesized and structure activity
relationships elaborated to explore the requirements for high affinity binding to both TSPO wild type
(WT) and the polymorphic TSPO A147T. This study reports high binding affinity and non-discriminating
TSPO ligands.
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KEYWORDS. TSPO, microglia, polymorphism, docking, non-discriminating, structure-activity
relationships.
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INTRODUCTION
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The translocator protein (TSPO), originally named the peripheral benzodiazepine receptor (PBR), is an
18 kDa, five-transmembrane domain protein that is primarily localized on the outer mitochondrial
membrane.1 TSPO is expressed predominantly in steroid-synthesizing tissues, but can also be found in
the central nervous system (CNS).2-3 Specifically, in the non-inflamed CNS, there is low TSPO expression
in microglia, the primary immune cells of the CNS.3 However, upregulation of TSPO expression is
evident for activated microglia in areas displaying neuroinflammation, for example in areas damaged by
neurodegenerative diseases.1, 3
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Molecular imaging using positron emission tomography (PET) has been used to assess TSPO
expression in preclinical and clinical studies of neurodegenerative diseases.4 As such, TSPO has become
a highly investigated target for the development of therapeutic and diagnostic imaging agents for use in
disorders involving neurodegeneration. Development of these drugs, however, has been hindered by the
emerging complexity of ligand interactions at the TSPO.
Furthermore, the presence of a single nucleotide polymorphism (SNP; rs6971) in TSPO that results in
replacement of an alanine (Ala) with a threonine (Thr) at amino acid residue 147 (A147T) has been
reported.5-6 Human subjects can be clustered into 3 distinct groups based on the ability of second
generation TSPO ligands to bind their brain tissue: high-, mixed-, and low-affinity binders. The presence
of the predominant TSPO form, Ala/Ala, is associated with high affinity binding, whereas Ala/Thr is
associated with mixed affinity binding and Thr/Thr with low-affinity binding.5-6 The A147T amino-acid
substitution results in a conformational change decreasing the affinity of all disclosed TSPO ligands,
except PK 11195 (1) and the closely related 4-azaisostere 2.5-10
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Although the most widely used TSPO PET tracer has been C-PK 11195, several major limitations
have restricted the clinical use of 11C-PK 11195 in the imaging of neuroinflammation. These limitations
include the relatively low brain permeability of 11C-PK 11195 and high nonspecific and plasma protein
binding, which result in a low signal-to-noise ratio and low specificity in PET images.11
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Ultimately, the presence of differing TSPO ligand affinities in the general population complicates the
quantitative assessment of PET data, limiting the wide-scale clinical usefulness of this approach.11 As
such, the development of a TSPO ligand that possesses drug-like properties and does not discriminate
between the TSPO isoforms would overcome this hurdle. Tricyclics are common core structures of TSPO
ligands that display high TSPO affinity and overcome low metabolic stability.12-13 For example, TSPO
ligands, GE-180 (3) and SSR-180,575 (4), show good brain permeability and TSPO affinity but do
discriminate between TSPO isoforms.12-13 As a result, further investigation into other chemotypes was
warranted in our pursuit of non-discriminating TSPO ligands. The carbazole scaffold, whether obtained
from natural sources or by synthetic routes, has gained much interest due to its wide range of biological
activity upon modifications, including anti-bacterial, anti-malarial, anti-inflammatory, anti-cancer, and
anti-Alzheimer properties.14-16 Given the multifaceted biological properties, the carbazole scaffold was
utilized in the design and synthesis of potential TSPO ligands to explore the requirements for high affinity
binding to both TSPO wild type (WT) and TSPO A147T.
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Figure 1. Previously-reported synthetic TSPO ligands PK 11195 (1)17 and 4-azaisostere (2),10 GE-180
(3)12 and SSR-180575 (4).13
The role of the N,N-disubstituted carboxamide was explored as structural studies suggest it may be
important for the non-discriminating behavior of PK 11195. In a 3D structure of mammalian TSPO
resolved using NMR, the substituents on the carboxamide nitrogen of PK 11195 interact with residues in
the 1st trans-membrane segment of TSPO (e.g. A23 and V26), rather than the 5th segment where A147T
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is found.18 Similarly, the substituents on the carboxamide nitrogen of PK 11195 interact with Y32 in the
first extracellular loop of a crystal structure of the bacterial homolog BcTSPO. Given this moiety interacts
with a portion of TSPO distinct from the location of the mutated residue, incorporation of a terminal
carboxamide moiety may be useful when designing non-discriminating ligands. An extension from prior
work,19 the structure activity relationships reported herein explore the effect of substitutions of the
terminal carboxamide and substitutions at the carbazole 3-position on affinity to both the TSPO isoforms.
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RESULTS AND DISCUSSION
Chemistry. The first series of carbazole acetamide ligands were synthesized according to Scheme 1.
Intermediate compounds 6a–6f and 7a were prepared by NH-deprotonation of 9H-carbazole (5) with
sodium hydride, followed by the addition of the appropriate alkylating agents (see supporting information
for their synthesis). Subsequent N-methylation or N-ethylation with the appropriate iodoalkane furnished
target ligands 7b–7c and 8a–8f. An alternative strategy was applied to obtain ligands 11a–11c, 12a–12c,
14a and 15a–15c. Thus, methyl ester intermediate 9 was hydrolyzed under basic conditions to obtain the
carboxylic acid 10. The carboxylic acid intermediate 10 was then coupled with the appropriate amine to
give tertiary amides 11a–11c, 12a–12c, 13a–13c and 14a. Subsequent catalytic hydrogenation of 13a–
13c with palladium on carbon (Pd/C), followed by acetylation furnished target ligands 15a–15c. Ligand
14b was previously reported.19
The second series of carbazole acetamide ligands were synthesized in a similar manner starting with 3-
bromo-9H-carbazole (16), forming key intermediate 17. Subsequent N-methylation or N-ethylation with
the appropriate iodoalkane furnished target ligands 18a and 19a. Utilizing the bromine handle a
palladium-catalyzed Suzuki–Miyaura cross-coupling of 18a and 19a with the appropriate boronic acid
gave target ligands 18b–18c, 19b–19c and 20a–20c.
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Scheme 1. Synthesis of carbazole acetamide ligands.a
R2
R2
R1
8
HN
N
9
O
O
10
11
12
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a
b
N
N
6a–6f, 7a
7b–7c, 9a–9f
6a R2 = 2-OMePh
6b R2 = 3-OMePh
6c R2 = 4-OMePh
6d R2 = 2-FBnPh
6e R2 = 3-FBnPh
6f R2 = 4-FBnPh
7a R2 = Ph
7b R1 = Et; R2 = Ph
7c R1 = Me; R2 = Ph
H
8a R1 = Et; R2 = 2-OMePh
8b R1 = Et; R2 = 3-OMePh
8c R1 = Et; R2 = 4-OMePh
8d R1 = Et; R2 = 2-FPh
8e R1 = Et; R2 = 3-FPh
8f R1 = Et; R2 = 4-FPh
N
5
R2
R1
O
O
N
O
OH
N
O
a
c
d
N
N
11a–11c, 12a–12c,
13a–13c, 14a
9
10
11a R1 = Et; R2 = 2-CF3Ph
11b R1 = Et; R2 = 3-CF3Ph
11c R1 = Et; R2 = 4-CF3Ph
12a R1 = Et; R2 = 2-Pyr
12b R1 = Et; R2 = 3-Pyr
12c R1 = Et; R2 = 4-Pyr
13a R1 = Et; R2 = 2-NO2Ph
13b R1 = Et; R2 = 3-NO2Ph
13c R1 = Et; R2 = 4-NO2Ph
14a R1 = R2 = Me
15a R1 = Et; R2 = 2-AcPh
15b R1 = Et; R2 = 3-AcPh
15c R1 = Et; R2 = 4-AcPh
e,f
O
O
O
R2
R2
HN
N
N
O
O
O
H
N
a
b
c
N
N
N
Br
Br
R3
Br
16
17
18a R1 = Et
19a R1 = Me
18b–18c, 19b–19c, 20a–20c
18b R1 = Et; R3 = Ph
18c R1 = Et; R3 = 4-OMePh
19b R1 = Me; R3 = Ph
19c R1 = Me; R3 = 4-OMePh
20a R1 = Me; R3 = 2-ClPh
20b R1 = Me; R3 = 3-ClPh
20c R1 = Me; R3 = 4-ClPh
aReagents and Conditions: a) NaH, appropriate alkylating agent, DMF, 0 °C–rt, 2 h, 81–99%; b) KOtBu,
DMF, iodoethane or iodomethane, rt, 17 h, 84–93%; c) LiOH, MeOH/H2O, reflux, 4 h, 72%; d) HBTU,
DIPEA, appropriate secondary amine, DMF, rt, 15 h, 68–91%; e) H2, Pd/C, EtOAc, rt, 6 h, 84–90%; f)
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acetic anhydride, CH2Cl2, rt, 2 h, 69-76%; g) Cs2CO3, Pd(PPh3)4, appropriate boronic acid, toluene/H2O,
90 °C, 10 h, 65–77%
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Biological Evaluation. The carbazole acetamide TSPO ligands were characterized by competition
radioligand binding assays using [3H]PK 11195 on membranes derived from HEK 293 cells
overexpressing either A147T or WT TSPO.19 The binding affinities are expressed as Ki (nM) in Table 1.
The binding affinity between TSPO WT and A147T for each ligand is expressed as a ratio (A147T:WT).
A ratio between A147T:WT closer to 1 indicates little to no preference between the two TSPO isoforms.
The first series of ligands synthesized (7–8, 11–12 and 14–15) encompassed modifications at the R1 and
R2 position of the amide. This series demonstrates the importance of the disubstituted carboxamide for
imparting affinity at both TSPO isoforms. A comparison of ligands 7a–7c, featuring a phenyl group at the
R2 position, showed complete loss of binding at both TSPO isoforms when R1 = H (7a). Following this,
ligand 7c incorporates a smaller methyl substituent at the R1 position and showed an improvement in
binding affinity at both TSPO A147T (increased 2.4) and WT (increased 1.3) forms when compared to
ligand 7b which had an ethyl substituent at R1. Furthermore, substituting an ethyl group at the R1 position
and a meta-methoxyphenyl group at the R2 position (8b) produced good binding affinity at both TSPO
isoforms (Ki TSPO WT = 96.1 nM, Ki TSPO A147T = 100.4 nM) and a 1.3x increase in binding affinity
at TSPO WT, with respect to ligand 7c.
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Table 1. TSPO binding affinities (Ki) of carbazole acetamide ligands using membranes from TSPO WT
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and A147T over-expressing HEK 293 cells. Values represent the mean ± SD from at least three
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independent experiments performed in duplicate.
R2
R1
7
N
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O
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R3
A147T:
Wild Type
Ligand
R1
R2
R3
TSPO Ki (nM)
A147T
26.7 ± 4.5
Wild Type
22.5 ± 5.2
PK 11195
7a
1.2
N/A
4.5
H
Et
Ph
H
>10 000
>10 000
70.0 ± 17.8
53.0 ± 17.2
1531.3 ± 248.8
96.1 ± 30.4
4737.3 ± 447.6
184.2 ± 55.4
562.5 ± 126.6
693.8 ± 214.7
>10 000
7b
Ph
H
318.5 ± 85.8
132.0 ± 24.0
1551.0 ± 358.1
100.4 ± 22.5
>10 000
7c
Me
Et
Ph
H
2.5
8a
2-OMePh
3-OMePh
4-OMePh
2-FPh
H
1.0
8b
Et
H
1.0
8c
Et
H
>2.1
17.0
>17.8
> 14.4
N/A
N/A
N/A
N/A
N/A
N/A
N/A
5.9
8d
Et
H
3123.2 ± 979.4
> 10 000
8e
Et
3-FPh
H
8f
Et
4-FPh
H
> 10 000
11a
11b
11c
12a
12b
12c
14a
14b
15a
15b
15c
18a
18b
18c
19a
19b
19c
20a
20b
Et
2-CF3Ph
3-CF3Ph
4-CF3Ph
2-Pyr
H
>10 000
Et
H
>10 000
>10 000
Et
H
>10 000
>10 000
Et
H
>10 000
>10 000
Et
3-Pyr
H
>10 000
>10 000
Et
4-Pyr
H
>10 000
>10 000
Me
Et
Me
H
>10 000
>10 000
Et
H
176.6 ± 41.6
>10 000
30.1 ± 3.6
243.0 ± 71.2
>10 000
Et
2-AcPh
3-AcPh
4-AcPh
3-OMePh
3-OMePh
3-OMePh
3-OMePh
3-OMePh
3-OMePh
3-OMePh
3-OMePh
H
H
>41.2
N/A
N/A
1.0
Et
>10 000
Et
H
>10 000
>10 000
Et
Br
462.3 ± 105.2
418.2 ± 18.0
369.0 ± 52.9
>10 000
468.7 ± 116.9
385.3 ± 51.4
447.1 ± 74.3
>10 000
Et
Ph
1.1
Et
4-OMePh
Br
0.8
Me
Me
Me
Me
Me
N/A
0.9
Ph
208.4 ± 69.6
25.4 ± 8.2
214.0 ± 4.4
639.4 ± 11.2
231.2 ± 39.3
6.6 ± 2.2
4-OMePh
2-ClPh
3-ClPh
3.8
231.3 ± 16.6
200.7 ± 21.5
0.9
3.2
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4-ClPh 1696.0 ± 557.9
20c
Me
3-OMePh
3555.0 ± 189.3
0.5
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8
Both the ortho- (8a) and meta- (8b) substituted methoxyphenyl group exhibited no preference between
the two TSPO isoforms. This suggests specific TSPO-drug interactions that are present in both isoforms,
resulting in little to no discrimination, which is possibly an important feature in the design of future TSPO
ligands. The para- (8c) position showed significant steric intolerance, as reflected by their micromolar
binding affinities. Conversely, maintaining the ethyl group at the R1 position and incorporating a fluorine
(8d–8f) substituent on the phenyl group lowered binding affinities to TSPO WT, and abolished all binding
at TSPO A147T. The trifluoromethyl (11a–11c) or acetamide (15a–15c) substituent on the phenyl group
at the R2 position abolished all binding affinity at both TSPO isoforms. Additionally, replacing the phenyl
group with a pyridine moiety (12a–12c) also produced similar negative effects on affinity. Ligand 14a,
which featured a methyl substituent at both the R1 and R2 position showed no binding to the TSPO WT
or TSPO A147T. Interestingly, ligand 14b, featuring an ethyl substituent at both the R1 and R2 positions
showed improved binding affinities at both TSPO isoforms. A comparison of ligands 14a and 14b
suggests that larger substituents are more effective at engaging the lipophilic binding sites. Taken
together, the binding data from the first series (7–8, 11–12 and 14–15) has shown that ligands 7c and 8b
were effective at binding TSPO, and ligand 8b also showed no discrimination between the two TSPO
isoforms. In a previous study,18 substituents at the R3 position on the carbazole heterocyclic core improved
binding affinity. Given this, the next series (18–20) was synthesized, featuring a meta-methoxyphenyl
substituent at the R2 position, with either an ethyl (18a–18c) or methyl (19a–19c) substituent at the R1
position on the carboxamide nitrogen, in conjugation with substituents at the R3 position on the carbazole
heterocyclic core that were chosen based on our previous findings.18 In agreement with the affinities of
the first series, ligands 19a–19c, containing the methyl substituent at the R1 position had favorable binding
affinities compared to ligands 18a–18c, containing the ethyl substituent at the R1 position. Incorporating
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a para-methoxyphenyl group at the R3 position (19c) had the most beneficial effect, with binding affinities
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in the low nanomolar range (Ki TSPO WT = 6.6 nM, Ki TSPO A147T = 25.4 nM). Ligand 19c was shown
to have a stronger affinity than the commercially available PK 11195 at both TSPO A147T (increased
1.1) and WT (increased 3.4) isoforms. Interestingly, ligands within this subset (18a–18c) showed
almost identical Ki values, ranging from 369.0–468.7 nM. Following this, ligands 18a–18c and 19b also
produced little discrimination between the two TSPO isoforms, yielding a A147T:WT ratio of ~1.0,
further reinforcing the contribution of an N-methoxyphenyl substitution at R2 to a reduction in binding
discrimination between TSPO WT and A147T.
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The effect of the PK 11195-inspired chlorophenyl group at the R3 position on the carbazole was
explored. Ligands 20a–20c were synthesized, whilst maintaining the same carboxamide substituents as
ligands 19a–19c and varying the position of the chlorine substituent on the phenyl group at the R3 position
of the carbazole. Although the data showed no improvement on binding affinity, the Ki values increased
at the TSPO WT when the position of the chlorine substituent moved from the ortho- (19a) to meta- (19b)
to para- (19c) position on the phenyl group. Interestingly, 19c and 20c varied by the substituent at the
para-position, however they experienced drastic differences in binding affinities at both TSPO isoforms.
This finding suggests that the chlorine substituent attached to 20c could potentially make it sterically
unfavorable.
Docking studies were used to investigate the structural basis for the Ki values obtained from biological
testing. Ligands 8b, 19b and 20a were chosen on the basis of their similar Ki values for both WT and
A147T TSPO and compared to 15a which showed a stronger Ki for the WT TSPO. Docking and MM-
GBSA dG binding scores (obtained by combining molecular mechanics (MM) terms with a generalized
Born and surface area (GBSA) solvent mode)20 are summarized in Table S1 (see supporting information).
Ligands 15a, 19b and 20a all had similar docking scores while a difference of ~3kcal/mol was seen for
ligand 8b. The binding conformation of 8b was similar for both WT and A147T TSPO, however it was
slightly rotated between the two. This could be attributed to greater dispersive interactions between the
carbazole ring and THR147 on the A147T TSPO, however the less negative docking score for the A147T
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TSPO suggest the formation of these dispersive interactions has a detrimental effect on the overall
docking. The MM-GBSA binding scores were similar for each ligand between the WT and A147T TSPO.
Binding poses for ligands 8b and 15a are shown in Figure 2, whilst the binding poses for ligands 19b
and 20a are shown in Figure S1 (see supporting information). 8b, 19b and 20a all exhibited binding poses
where the carbazole ring exhibited π-π interactions with TRP95 and also TRP53 in some cases in both
WT and A147T TSPO. This pose was also adopted by 15a in the WT TSPO, while in the A147T TSPO,
the ligand has been rotated by 180° and the amine substituents interact with the TRP residues instead.
This can be attributed to two factors; the large aromatic substituents attached to the carbazole group in
19b and 20a make it sterically unfavorable for the molecule to rotate away from the π-π interactions with
TRP95 and TRP53 and the acetanilide substituent on 15a being able to form interactions with the TRP
residues 95, 53 and 143 instead.
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Figure 2. 3-Dimensional and 2-dimensional ligand binding diagrams of the TSPO ligands 8b and 15a
into the chosen receptors. The crystal structure of the TSPO protein from Bacilus Cereus (PDB ID: 4RYI),
was used as the template for homology modelling. (A) Ligand interaction diagram of the ligand 8b in the
wild type TSPO. (B) Ligand interaction diagram of the ligand 8b in the TSPO A147T. (C) Ligand
interaction diagram of the ligand 15a in the wild type TSPO. (D) Ligand interaction diagram of the ligand
15a in the TSPO A147T.
TSPO ligands can affect cell viability and proliferation at high concentrations.19, 21 Given an overall
drop-off in binding affinity for TSPO A147T seen in Table 1, a representative subset of TSPO ligands
(7b, 7c, 8b, 18a, 19c, 20a) were investigated for their effect on cell viability and proliferation using HEK
293 cells, to understand whether their biological potency varied according to the different TSPO isoforms.
PK 11195, which has been previously shown to decrease proliferation and viability,22 was also tested
as a positive control. HEK 293 cells that were stably transfected with WT and A147T TSPO were treated
with 100 µM of TSPO ligands for 48 h, and cell viability was quantitatively determined using CellTitre
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Blue (Figure 3A). The effect on viability of each ligand was measured as a percentage of the viability of
vehicle-treated cells. PK 11195 significantly reduced the viability of TSPO WT and A147T cells (65%
and 68% respectively) as compared to untreated controls. These results mirror other studies that have
showed similar results.19 All other ligands had no significant effect on the viability of HEK 293 cells. The
effect of ligands on cell proliferation was then investigated. Similar to the cell viability assay, 100 µM of
TSPO ligands were incubated with HEK 293 cells for 48 h. BrdU ELISA assays were performed as a
measurement of cell proliferation and the results were presented as a percentage of vehicle-treated controls
(Figure 3B). PK 11195 significantly reduced proliferation in TSPO WT and A147T cells to 83% and 81%
of basal proliferation respectively. Out of the eight novel TSPO ligands tested, only 20a significantly
reduced proliferation in both TSPO WT and A147T cell to 89% and 85% respectively. Interestingly, 18b
also attenuated proliferation, but this effect was only seen in A147T cells. From the radioligand binding
results (Table 1), 18b does not discriminate between WT and A147T TSPO in binding (A147T: WT Ki
ratio = 1.1), so there is disjunction between the functional data and binding data for this compound.
Recently, TSPO ligands that behave like positive allosteric modulators of the TSPO were reported.23
The reported TSPO ligands had no effect on proliferation alone, but potentiated the anti-proliferative
effect of PK 11195. Given the lack of functional activity of most of the novel TSPO ligands, TSPO WT
and A147T cells were co-treated with both PK 11195 (100 µM) and the novel ligands (100 µM, Figure
3C) in order to examine whether the TSPO ligands showed any positive allosteric modulator-like activity.
19b decreased proliferation when co-treated with PK 11195, compared to when treated with PK 11195
alone. This effect was observed in both TSPO WT and A147T. Although only a small effect at a high
concentration, this suggests 19b may be able to potentiate the effect of PK 11195 in reducing cell
proliferation.
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Figure 3. Cell viability and proliferation of HEK 293 cells overexpressing TSPO WT and TSPO A147T
after treatment with novel TSPO ligands. (A) Cell viability was determined using CellTitre Blue. Both
cell lines were treated with 100 µM of the TSPO ligands for 48 hours and were then incubated with
CellTitre Blue for 4 hours. Cell proliferation was assessed with a BrdU ELISA assay. Both cell lines were
treated with novel TSPO ligands in the absence (B) or presence of (C) PK 11195 for 48 hours. Data are
expressed as mean ± SD (n  3, two-way ANOVA followed by Dunnett’s multiple comparison test, *p <
0.05, **p < 0.01, ***p < 0.001, with (A) and (B) compared with vehicle-treated cells and (C) compared
to PK 11195).
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Journal of Medicinal Chemistry
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Changes in neurosteroid levels have been reported in psychiatric disorders such as anxiety disorder.24
Translocation of cholesterol through the mitochondrial membrane is a rate-limiting step in the production
of neurosteroids such as allopregnanolone.25 At least in vitro, TSPO mediates this translocation of
cholesterol,24 and consequently, TSPO ligands administered to rodents exert anxiolytic effects by
increasing the release of pregnenolone.26-27 Therefore, the steroidogenic activity of the TSPO ligands was
investigated by measuring the release of pregnenolone from C6 glioma cells using ELISA. As expected
from previous studies, PK 11195 significantly increased pregnenolone release in C6 cells.28 In contrast,
two high affinity ligands reported in this paper, 8b and 19c, showed no significant steroidogenic effects.
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Figure 4. Effects of TSPO ligands (PK 11195, 8b and 19c) on C6 glioma steroidogenesis. Pregnenolone
ELISA assays were performed as a measurement of pregnenolone release. 100 µM of the TSPO ligands
were incubated in ELISA buffer for 2 hours. PK 11195 significantly increased pregnenolone release, and
no change was observed for 8b and 19c compared to vehicle-treated control. Values represent mean ± SD
(n ≥ 3, one-way ANOVA followed by Dunnett’s multiple comparison test, *** p < 0.001 compared to
vehicle-treated cells).
Given that binding affinity was measured on cell membranes, yet functional activity was measured on
intact cells, and the high affinity ligands did not result in potent functional activity; we ascertained whether
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the ligands were able to cross the cell membrane and reach TSPO in the mitochondria of intact cells.
Whole cell radioligand binding experiments were performed with PK 11195 and two novel ligands 8b
and 19c that showed the highest membrane binding affinities.
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The binding affinities determined by whole cell radioligand binding (Table 2) mirrored the binding
affinities obtained from competition radioligand binding using membranes (Table 1). This suggests that
the novel ligands can cross the cell membrane and reach the mitochondrial TSPO target. Given most of
the novel ligands had little to no effect on the viability or proliferation of cells when administered by
themselves, and high affinity ligands could cross the cell membrane, the binding sensitivity for TSPO
appears to have no correlation to its anti-proliferative, cytotoxic and steroidogenic function for these
ligands. This suggests other factors govern the functional capacity of TSPO ligands, which is supported
by recent work showing the importance of residence time to cytotoxic and steroidogenic functional
efficacy.24
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Table 2. Binding affinities of three TSPO ligands using whole cell radioligand binding. Values represent
the mean ± SD from at least three independent experiments performed in duplicate.
Ligand
TSPO Ki (nM)
A147T: Wild Type
A147T
PK 11195 31.5 ± 7.5
Wild Type
13.6 ± 3.4
2.3
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277.0 ± 77.9 217.3 ± 0.6
23.2 ± 7.3 3.2 ± 0.6
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Journal of Medicinal Chemistry
CONCLUSIONS
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A novel series of carbazole acetamide ligands were synthesized and shown to be WT and A147T TSPO
ligands. In general, ligands with a N-methoxyphenyl substitution at R2 show a reduction in binding
discrimination between TSPO WT and A147T. In particular, the N-(3-methoxybenzyl)-N-ethyl (8b)
substitution binds with similarly high affinity to both TSPO WT and A147T. In addition, the para-
methoxyphenyl substitution at the 3-position of the carbazole scaffold dramatically improved the Ki value
to the low nanomolar range.
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A subset of high binding affinity ligands (7b, 7c, 8b, 18a, 19c, 20a) were tested in vitro to determine
their ability to reduce cell viability and proliferation in HEK 293 cells transfected with WT and A147T
TSPO. Only 20a significantly reduced cell proliferation, all other ligands had no effect on cell viability
despite showing affinity for TSPO. The steroidogenic activity of the TSPO ligands 8b and 19c was
investigated in vitro by measuring the release of pregnenolone from C6 glioma and showed no significant
steroidogenic effects.
These novel insights will aid in the further development of TSPO ligands for therapeutic and diagnostic
imaging in neuroinflammation using PET.
EXPERIMENTAL SECTION
General Procedures. Unless otherwise stated, all solvents and reagents were purchased and used from
commercial sources. Anhydrous solvents were obtained from an Innovative Technology PureSolv7
purification system. Tetrahydrofuran and dichloromethane were dried through an alumina column. N,N-
Dimethylformamide was dried through a 5 Å molecular sieve column. All reagents were weighed out
under ambient conditions. All reactions were performed under nitrogen or argon, unless otherwise stated.
Melting points using an Optimelt Automated melting point apparatus from Stanford Research Systems
were measured in open capillary tubes. IR spectra were recorded neat using a Bruker ALPHA FT-IR
spectrometer and peaks are expressed in wavenumbers (cm–1). Nuclear magnetic resonance spectra were
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recorded on Bruker Advance DRX 300 or Bruker Advance DRX 400 Ascend spectrometers at 300 or 400
MHz 1H NMR frequency, 75 or 101 MHz 13C NMR frequency and 282 MHz 19F NMR frequency. H, C
and F chemical shifts are expressed as parts per million (ppm). All resonances are reported as chemical
shift (δ) and are referenced to the solvent residual peak. Multiplicities are reported as follows: s (singlet),
br (broad), d (doublet), dd (doublet of doublets), t (triplet), q (quartet) and m (multiplet). Coupling
constants (J) are reported in Hz. Elemental microanalysis was obtained from the School of Human
Sciences at London Metropolitan University, England. Low- and high-resolution mass spectra were
obtained through electron ionization (ESI). Low-resolution mass spectra were performed on a Finnigan
LCQ mass spectrometer. High-resolution mass spectra were performed on a Bruker 7T Apex Qe Fourier
Transform Ion Cyclotron resonance mass spectrometer equipped with an Apollo II ESI/APCI/MALDI
Dual source. High performance liquid chromatography (HPLC) was performed on the Waters Alliance
2695 apparatus equipped with Waters 2996 photodiode array detector, set at 254 nm. Separation using a
SunFireTM C18 column (5 μm, 2.1 x 150 mm) was achieved using water (solvent A) and acetonitrile
(solvent B) at a flow rate of 0.2 mL/min. The method consisted of 0% B to 100% B over 30 minutes.
HPLC data is recorded as percentage purity and retention time (RT) in minutes. Purity of all final
compounds was 95% or higher.
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Materials. Non-commercially available alkylating agents and secondary amines have been prepared
according to known procedures (see supporting information).
General Procedure for the Synthesis of 6a–6f, 7a, 9 and 17. Sodium hydride (60% w/w dispersion
in mineral oil, 1.5 equiv.) was added portion-wise to a solution of the appropriate aromatic amines (1.0
equiv.) in tetrahydrofuran (0.2 M) at 0 °C. The reaction mixture was warmed to ambient temperature and
stirred for 30 minutes before being cooled back down to 0 C. The appropriate alkylating agent (1.0 equiv.)
was added dropwise at 0 °C. The reaction mixture was warmed to ambient temperature and stirred for an
additional 2 hours. The reaction mixture was poured into ice-cold water and neutralized with aqueous
hydrochloric acid (1 M). The organic layer was collected and the aqueous layer was extracted with ethyl
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acetate (3×). The organic extracts were combined, dried over anhydrous magnesium sulfate and
concentrated in vacuo. Unless otherwise specified, purification was achieved by trituration using diethyl
ether gave the desired product (266 mg, 0.772 mmol, 98%) as a colorless powder.
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General Procedure for the Synthesis of 7b–7c, 8a–8f, 18a and 19a. Potassium tert-butoxide (2.0
equiv.) was added portion-wise to a solution of the appropriate secondary amide (1.0 equiv.) in
tetrahydrofuran (0.3 M). The reaction mixture was then stirred for 30 minutes at ambient temperature.
The appropriate iodoalkane (1.5 equiv.) was added dropwise at 0 °C and the mixture was then warmed to
ambient temperature and stirred for 17 hours. The reaction mixture was neutralized with aqueous
hydrochloric acid (1 M). The organic layer was collected and the aqueous layer was extracted with ethyl
acetate (3×). The organic layers were combined, dried over anhydrous magnesium sulfate and
concentrated in vacuo. The 1H NMR spectrum of this amide shows the presence of two different rotamers
in a 1:1 ratio. For the sake of simplicity, the NMR data for only one rotamer are reported.
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General Procedure for the Synthesis of 11a–11c, 12a–12c, 13a–13c, 14a. To a solution of the
carboxylic acid 10 (100 mg, 0.444 mmol, 1.0 equiv.), HBTU (253 mg, 0.666 mmol, 1.5 equiv.) and the
appropriate secondary amine (1.0 equiv.) in anhydrous dimethylformamide (0.2 M) was added N,N-
diisopropylethylamine (150 µL, 0.888 mmol, 2.0 equiv.). The mixture was stirred at ambient temperature
for 15 hours. Upon completion, water was added and the aqueous mixture was extracted with ethyl acetate
(3×). The mixture was washed with an aqueous solution of saturated sodium bicarbonate (1×),
hydrochloric acid (1×, 2 M), water (2×) and brine (1×). The organic extract was then dried over anhydrous
magnesium sulfate and concentrated in vacuo.
General Procedure for the Synthesis of 15a–15c. To a mixture of the appropriate aryl nitro
compounds (100 mg, 0.258 mmol, 1.0 equiv.) in ethyl acetate (5 mL) was added palladium on carbon (10
mg, 10 wt%). The reaction mixture degassed under nitrogen for 5 minutes and then stirred at ambient
temperature for 6 hours under a hydrogen atmosphere (1 atm). The mixture was filtered through a pad of
Celite® and the pad washed with ethyl acetate. The filtrate was concentrated in vacuo to give the crude
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residue. The product was carried on to the next step without purification. To a mixture of the crude product
in dichloromethane (2 mL) was added acetic anhydride (30 µL, 0.318 mmol, 1.1 equiv.) dropwise. The
reaction mixture was stirred at ambient temperature for 2 hours, then poured into an aqueous solution of
saturated sodium bicarbonate (2 mL). The aqueous phase was extracted with dichloromethane (3×5 mL).
The organic layers were combined, dried over anhydrous magnesium sulfate and concentrated in vacuo.
Purification was achieved by flashꢀchromatography (ethyl acetate/hexane, 3:7 v/v) on silica gel.
General Procedure for the Synthesis of 18b–18c, 19b–19c and 20a–20c. A suspension of 18a or 19a
(1.0 equiv.), appropriate phenylboronic acid (1.5 equiv.), and caesium carbonate (2.5 equiv.) in
toluene/water (0.1 M, 3:1 v/v) was degassed under argon for 10 minutes. Pd(dppf)Cl2 (5 mol%) was added
under argon. The reaction mixture was heated to 80 °C and stirred for 10 hours. Upon completion, the
reaction mixture was cooled to ambient temperature and evaporated to dryness. This crude residue was
purified by flash column chromatography (ethyl acetate/hexane, 3:7 v/v) on silica gel.
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2-(9H-Carbazol-9-yl)-N-(2-methoxybenzyl)acetamide (6a). White solid; yield 98% (266 mg, 0.772
mmol). R 0.59 (ethyl acetate/hexane, 3:7 v/v); mp 207–209 °C; 1H NMR (300 MHz, DMSO) δ = 8.61
(t, J = 5.8 Hz, 1H, NH), 8.18 – 8.15 (m, 2H, ArH), 7.59 – 7.56 (m, 2H, ArH), 7.47 – 7.42 (m, 2H, ArH),
7.28 – 7.19 (m, 4H, ArH), 6.99 – 6.84 (m, 2H, ArH), 5.13 (s, 2H, CH2CO), 4.28 (d, J = 5.7 Hz, 2H,
NHCH2), 3.77 (s, 3H, OCH3); 13C NMR (75 MHz, DMSO) δ = 167.45 (CO), 156.65 (Ar), 140.58 (2×Ar),
128.18 (Ar), 127.94 (Ar), 126.28 (2×Ar), 125.59 (Ar), 122.21 (2×Ar), 120.10 (Ar), 120.03 (2×Ar), 118.95
(2×Ar), 110.44 (Ar), 109.35 (2×Ar), 55.24 (OCH3), 45.49 (CH2CO), 37.50 (NHCH2); LRMS (+ESI) 367
[(M + Na)+ 100%]; HRMS (+ESI) Calcd for C22H20N2O2 ([M + Na]+ 367.1417. Found: 367.1419; IR
(νmax/cm–1) 3284, 2991, 1648, 1488, 1230, 1154, 1123, 1051, 745, 718, 423.
2-(9H-Carbazol-9-yl)-N-(3-methoxybenzyl)acetamide (6b). White solid; yield 98% (262 mg, 0.761
mmol). R 0.53 (ethyl acetate/hexane, 3:7 v/v); mp 198–200 °C; 1H NMR (300 MHz, DMSO) δ = 8.11
– 8.08 (m, 2H, ArH), 7.52 – 7.46 (m, 2H, ArH), 7.39 – 7.36 (m, 2H, ArH) 7.32 – 7.25 (m, 2H, ArH), 7.11
– 7.06 (m, 1H, ArH), 6.71 – 6.50 (m, 3H, ArH), 5.91 (t, J = 5.8 Hz, 1H, NH), 4.97 (s, 2H, CH2CO), 4.34
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(d, J = 5.8 Hz, 2H, NHCH2), 3.63 (s, 3H, OCH3); 13C NMR (75 MHz, DMSO) δ = 168.07 (CO), 159.75
(Ar), 140.26 (2×Ar), 138.90 (Ar), 129.58 (Ar), 126.52 (2×Ar), 123.48 (2×Ar), 120.70 (Ar), 120.42
(2×Ar), 119.32 (2×Ar), 113.32 (Ar), 112.12 (Ar), 108.54 (2×Ar), 55.10 (OCH3), 47.16 (CH2CO), 43.07
(NHCH2); LRMS (+ESI) 367 [(M + Na)+ 100%]; HRMS (+ESI) Calcd for C22H20N2O2 ([M + Na]+
367.1417. Found: 367.1410; IR (νmax/cm–1) 3669, 2985, 2212, 1649, 1066, 483.
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2-(9H-Carbazol-9-yl)-N-(4-methoxybenzyl)acetamide (6c). White solid; yield 99% (266 mg, 0.772
mmol). R 0.40 (ethyl acetate/hexane, 3:7 v/v); mp 210–212 °C; 1H NMR (300 MHz, DMSO) δ = 8.73
(t, J = 5.8 Hz, 1H, NH), 8.20 – 8.10 (m, 2H, ArH), 7.55 – 7.50 (m, 2H, ArH), 7.47 – 7.38 (m, 2H, ArH),
7.24 – 7.13 (m, 4H ArH), 6.91 – 6.86 (m, 2H, ArH), 5.08 (s, 2H, CH2CO), 4.25 (d, J = 5.8 Hz, 2H,
NHCH2), 3.74 (s, 3H, OCH3); 13C NMR (75 MHz, DMSO) δ = 167.31 (CO), 158.23 (Ar), 140.59 (2×Ar),
130.97 (Ar), 128.61 (2×Ar), 125.59 (2×Ar), 122.22 (2×Ar), 120.11 (2×Ar), 118.95 (2×Ar), 113.64 (2×Ar),
109.30 (2×Ar), 55.04 (OCH3), 45.55 (CH2CO), 41.71 (NHCH2); LRMS (+ESI) 367 [(M + Na)+ 100%];
HRMS (+ESI) Calcd for C22H20N2O2 ([M + Na]+ 367.1417. Found: 367.1416; IR (νmax/cm–1) 3293,
2991, 1649, 1489, 1237, 1154, 1122, 746, 718, 528.
2-(9H-Carbazol-9-yl)-N-(2-fluorobenzyl)acetamide (6d). White solid; yield 88% (238 mg, 0.716
mmol). R 0.53 (ethyl acetate/hexane, 3:7 v/v); mp 202–204 °C; 1H NMR (300 MHz, DMSO) δ = 8.15
– 7.99 (m, 2H, ArH), 7.53 – 6.83 (m, 10H, ArH), 5.95 (t, J = 6.1 Hz, 1H, NH), 4.94 (s, 2H, CH2CO), 4.39
(d, J = 6.1 Hz, 2H, NHCH2); 13C NMR (75 MHz, DMSO) δ = 168.15 (CO), 162.39 (d, J = 245.8 Hz,
Ar), 140.27 (2×Ar), 129.39 (d, J = 6.1 Hz, Ar), 129.15 (d, J = 8.2 Hz, Ar), 126.47 (2×Ar), 125.32 (d, J =
8.2 Hz, Ar), 124.02 (d, J = 3.4 Hz, Ar), 123.50 (2×Ar), 120.47 (2×Ar), 120.19 (2×Ar), 114.55 (d, J = 21.6
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Hz, Ar), 108.56 (2×Ar), 47.16 (CH2CO), 37.29 (NHCH2); F NMR (282 MHz, DMSO) δ = –117.87
(ArF); LRMS (+ESI) 355 [(M + Na)+ 100%]; HRMS (+ESI) Calcd for C21H17FN2O ([M + Na]+
355.1217. Found: 355.1220; IR (νmax/cm–1) 2998, 2201, 1654, 1455, 1211, 1074, 809, 716, 461.
2-(9H-Carbazol-9-yl)-N-(3-fluorobenzyl)acetamide (6e). White solid; yield 86% (233 mg, 0.701
mmol). R 0.47 (ethyl acetate/hexane, 3:7 v/v); mp 186–188 °C; 1H NMR (300 MHz, DMSO) δ = 8.14
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(d, J = 7.5 Hz, 2H, ArH), 7.50 – 7.37 (m, 4H, ArH), 7.32 – 7.28 (m, 3H, ArH), 7.20 – 7.11 (m, 3H, ArH),
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130.74 (d, J = 8.2 Hz, Ar), 127.46 (2×Ar), 125.32 (d, J = 4.2 Hz, Ar), 124.03 (2×Ar), 120.55 (2×Ar),
120.19 (2×Ar), 115.47 (d, J = 20.2 Hz, Ar), 114.56 (d, J = 21.4 Hz, Ar), 108.89 (2×Ar), 47.15 (CH2CO),
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42.41 (NHCH2); F NMR (282 MHz, DMSO) δ = –115.84 (ArF); LRMS (+ESI) 355 [(M + Na)+
100%]; HRMS (+ESI) Calcd for C21H17FN2O ([M + Na]+ 355.1217. Found: 355.1218; IR (νmax/cm–1)
3256, 2962, 1648, 1451, 1327, 1206, 745, 718, 421.
2-(9H-Carbazol-9-yl)-N-(4-fluorobenzyl)acetamide (6f). White solid; yield 88% (238 mg, 0.716
mmol). R 0.40 (ethyl acetate/hexane, 3:7 v/v); mp 227–229 °C; 1H NMR (300 MHz, DMSO) δ = 8.10
(d, J = 7.7 Hz, 2H, ArH), 7.49 (t, J = 7.7 Hz, 2H, ArH), 7.35 – 7.21 (m, 4H, ArH), 6.96 (dd, J = 8.5 Hz,
5.4 Hz, 2H, ArH), 6.85 (t, J = 8.5 Hz, 2H, ArH), 5.89 (t, J = 6.1 Hz, 1H, NH), 4.95 (s, 2H, CH2CO), 4.30
(d, J = 6.1 Hz, 2H, NHCH2); 13C NMR (75 MHz, DMSO) δ = 168.08 (CO), 160.23 (d, J = 251.3 Hz,
Ar) 140.23 (2×Ar), 133.24 (d, J = 3.2 Hz, Ar), 128.92 (d, J = 8.1 Hz, 2×Ar), 126.51 (2×Ar), 123.49
(2×Ar), 120.70 (2×Ar), 120.46 (2×Ar), 115.25 (d, J = 21.8 Hz, 2×Ar), 108.54 (2×Ar), 47.15 (CH2CO),
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42.41 (NHCH2); F NMR (282 MHz, DMSO) δ = –115.33 (ArF); LRMS (+ESI) 355 [(M + Na)+
100%]; HRMS (+ESI) Calcd for C21H17FN2O ([M + Na]+ 355.1217. Found: 355.1220; IR (νmax/cm–1)
3262, 2854, 2011, 1649, 1462, 1218, 833, 750, 449.
N-Benzyl-2-(9H-carbazol-9-yl)acetamide (7a). White solid; yield 86% (238 mg, 0.757 mmol). R
0.47 (ethyl acetate/hexane, 3:7 v/v); mp 226–228 °C; 1H NMR (300 MHz, CDCl3) δ = 8.11 – 8.09 (m,
2H, ArH), 7.52 – 7.47 (m, 2H, ArH), 7.38 – 7.25 (m, 4H, ArH), 7.19 – 7.15 (m, 3H, ArH), 7.01 – 6.98
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(m, 2H, ArH), 5.90 (s, 1H, NH), 4.99 (s, 2H, CH2CO), 4.37 (d, J = 6.1, 2H, NHCH2); C NMR (75
MHz, CDCl3) δ = 168.07 (CO), 140.27 (2×Ar), 137.38 (Ar), 128.54 (2×Ar), 127.39 (2×Ar), 127.11 (Ar),
126.49 (2×Ar), 123.50 (2×Ar), 120.67 (2×Ar), 120.42 (2×Ar), 108.58 (2×Ar), 47.21 (CH2CO), 43.11
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(NHCH2); LRMS (+ESI) 337 [(M + Na)+ 100%]; IR (νmax/cm–1) 3675, 2987, 2900, 1394, 1250, 1066,
892, 444; Found: C, 80.16; H, 5.81; N, 8.92. Calc for C21H18N2O: C, 80.23; H, 5.77; N, 8.91%
N-Benzyl-2-(9H-carbazol-9-yl)-N-ethylacetamide (7b). White solid; yield 90% (98 mg, 0.286 mmol).
R 0.65 (ethyl acetate/hexane, 3:7 v/v); mp 143–145 °C; 1H NMR (300 MHz; CDCl3) δ = 8.06 (d, J =
7.8 Hz, 2H, ArH), 7.46 – 7.16 (m, 11H, ArH), 5.12 (s, 2H, CH2CO), 4.62 (s, 2H, CH2Ph), 3.35 (q, J =
7.3 Hz, 2H, CH2CH3), 1.04 (t, J = 7.3 Hz, 3H, CH2CH3); 13C NMR (75 MHz, CDCl3) δ = 166.19 (CO),
140.75 (2×Ar), 139.95 (Ar), 128.52 (2×Ar), 127.38 (2×Ar), 127.11 (Ar), 126.49 (2×Ar), 123.50 (2×Ar),
120.67 (2×Ar), 120.42 (2×Ar), 108.46 (2×Ar), 49.83 (CH2CO), 48.67 (CH2Ph), 43.11 (CH2CH3), 13.25
(CH2CH3); LRMS (+ESI) 365 [(M + Na)+ 40%], 343 [(M + H)+ 100%]; IR (νmax/cm–1) 3402, 1658,
1536, 1452, 799; Found: C, 80.55; H, 6.49; N, 8.19. Calc for C23H22N2O: C, 80.67; H, 6.48; N, 8.18%
N-Benzyl-2-(9H-carbazol-9-yl)-N-methylacetamide (7c). White solid; yield 87% (91 mg, 0.277
mmol). R 0.57 (ethyl acetate/hexane, 3:7 v/v); mp 139–141 °C; 1H NMR (300 MHz; CDCl3) δ = 8.07
(d, J = 7.7 Hz, 2H, ArH), 7.46 – 7.11 (m, 11H, ArH), 4.97 (s, 2H, CH2CO), 4.56 (s, 2H, CH2Ph), 2.88 (s,
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3H, CH3); C NMR (75 MHz, CDCl3) δ = 167.70 (CO), 140.79 (2×Ar), 136.75 (Ar), 128.68 (2×Ar),
127.89 (2×Ar), 127.60 (Ar), 125.94 (2×Ar), 123.22 (2×Ar), 120.46 (2×Ar), 119.51 (2×Ar), 108.58 (2×Ar),
51.61 (CH2CO), 45.58 (CH2Ph), 34.21 (CH3); LRMS (+ESI) 351 [(M + Na)+ 100%]; IR (νmax/cm–1)
3674, 2986, 1649, 1406, 1218, 1067, 745, 717, 420; Found: C, 80.52; H, 6.10; N, 8.42. Calc for
C22H20N2O: C, 80.46; H, 6.14; N, 8.53%
2-(9H-Carbazol-9-yl)-N-ethyl-N-(2-methoxybenzyl)acetamide (8a). White solid; yield 88% (190
mg, 0.510 mmol). R 0.65 (ethyl acetate/hexane, 3:7 v/v); mp 135–137 °C; 1H NMR (300 MHz, CDCl3)
δ = 8.09 – 8.07 (m, 2H, ArH), 7.43 – 7.29 (m, 3H, ArH), 7.25 – 7.10 (m, 5H, ArH), 6.99 – 6.82 (m, 2H,
ArH), 5.03 (s, 2H, CH2Ph), 4.57 (s, 2H, CH2CO), 3.85 (s, 3H, OCH3), 3.46 (q, J = 7.1 Hz, 2H, CH2CH3),
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1.10 (t, J = 7.1 Hz, 3H, CH2CH3); C NMR (75 MHz, CDCl3) δ = 167.34 (CO), 157.04 (Ar), 140.88
(2×Ar), 129.03 (2×Ar), 126.97 (Ar), 125.74 (2×Ar), 124.28 (Ar), 123.18 (2×Ar), 120.73 (Ar), 120.29
(2×Ar), 119.22 (2×Ar), 108.57 (2×Ar), 55.30 (OCH3), 45.99 (CH2CO), 45.35 (CH2Ph), 41.66 (CH2CH3),
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12.70 (CH2CH3); LRMS (+ESI) 395 [(M + Na)+ 100%]; IR (νmax/cm–1) 2992, 1650, 1489, 1237, 1154,
1072, 745, 718, 529; HPLC R = 29.83 min (98.0% purity); Found: C, 77.38; H, 6.55; N, 7.48. Calc for
C24H24N2O2: C, 77.39; H, 6.50; N, 7.52%
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2-(9H-Carbazol-9-yl)-N-ethyl-N-(3-methoxybenzyl)acetamide (8b). White solid; yield 93% (201
mg, 0.540 mmol). R 0.45 (ethyl acetate/hexane, 3:7 v/v); mp 46–48 °C; 1H NMR (300 MHz, CDCl3) δ
= 8.03 – 7.94 (m, 2H, ArH), 7.40 – 7.26 (m, 3H, ArH), 7.20 – 7.10 (m, 4H, ArH), 6.78 – 6.57 (m, 3H,
ArH), 5.02 (s, 2H, CH2CO), 4.51 (s, 2H, CH2Ph), 3.65 (s, 3H, OCH3), 3.27 (q, J = 7.1 Hz, 2H, CH2CH3),
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0.99 (t, J = 7.1 Hz, 3H, CH2CH3); C NMR (75 MHz, CDCl3) δ = 167.36 (CO), 159.90 (Ar), 140.76
(2×Ar), 130.16 (Ar), 129.56 (Ar), 125.93 (2×Ar), 123.24 (2×Ar), 120.48 (2×Ar), 119.48 (2×Ar), 118.09
(Ar), 113.32 (Ar), 113.14 (Ar) 108.48 (2×Ar), 55.20 (OCH3), 45.64 (CH2CO), 45.40 (CH2Ph), 41.16
(CH2CH3), 13.62 (CH2CH3); LRMS (+ESI) 395 [(M + Na)+ 100%]; IR (νmax/cm–1) 2967, 1643, 1486,
1242, 747, 719, 512; Found: C, 77.38; H, 6.59; N, 7.40. Calc for C24H24N2O2: C, 77.39; H, 6.50; N, 7.52%
2-(9H-Carbazol-9-yl)-N-ethyl-N-(4-methoxybenzyl)acetamide (8c). White solid; yield 91% (197
mg, 0.529 mmol). R 0.50 (ethyl acetate/hexane, 3:7 v/v); mp 119–121 °C; 1H NMR (300 MHz, CDCl3)
δ = 8.08 – 8.00 (m, 2H, ArH), 7.44 – 7.34 (m, 2H, ArH), 7.27 – 7.02 (m, 6H, ArH), 6.88 – 6.78 (m, 2H,
ArH), 4.93 (s, 2H, CH2CO), 4.45 (s, 2H, NHCH2), 3.78 (s, 3H, OCH3), 3.24 (q, J = 7.1 Hz, 2H, CH2CH3),
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1.05 (t, J = 7.1 Hz, 3H, CH2CH3); C NMR (75 MHz, CDCl3) δ = 167.15 (CO), 159.23 (Ar), 140.81
(2×Ar), 129.71 (2×Ar), 128.27 (Ar), 125.91 (2×Ar), 123.17 (2×Ar), 120.42 (2×Ar), 119.42 (2×Ar), 114.47
(2×Ar), 108.57 (2×Ar), 55.43 (OCH3), 49.52 (CH2CO), 47.79 (CH2Ph), 45.16 (CH2CH3), 13.66
(CH2CH3); LRMS (+ESI) 395 [(M + Na)+ 100%]; IR (νmax/cm–1) 2965, 1643, 1492, 1247, 1154, 1049,
756, 548, 475; HPLC R = 26.61 min (98.4% purity); Found: C, 77.26; H, 6.41; N, 7.48. Calc for
C24H24N2O2: C, 77.39; H, 6.50; N, 7.52%
2-(9H-Carbazol-9-yl)-N-ethyl-N-(2- fluorobenzyl)acetamide (8d). White solid; yield 84% (183 mg,
0.508 mmol). R 0.56 (ethyl acetate/hexane, 3:7 v/v); mp 131–133 °C; 1H NMR (300 MHz, CDCl3) δ =
8.10 – 8.01 (m, 2H, ArH), 7.46 – 7.38 (m, 2H, ArH) 7.32 – 7.18 (m, 5H, ArH), 7.11 – 6.99 (m, 3H, ArH),
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5.07 (s, 2H, CH2CO), 4.67 (s, 2H, CH2Ph), 3.42 (q, J = 7.1 Hz, 2H, CH2CH3), 1.11 (t, J = 7.1 Hz, 3H,
CH2CH3); 13C NMR (75 MHz, CDCl3) δ = 167.21 (CO), 160.32 (d, J = 248.0 Hz, Ar), 140.72 (2×Ar),
130.92 (d, J = 3.6 Hz, Ar), 129.62 (d, J = 8.1 Hz, Ar), 125.92 (2×Ar), 125.86 (d, J = 7.8 Hz, Ar), 124.47
(d, J = 3.2 Hz, Ar), 123.23 (2×Ar), 120.46 (2×Ar), 120.33 (2×Ar), 115.42 (d, J = 21.8 Hz, Ar), 108.43
(2×Ar), 45.33 (CH2CO), 44.76 (CH2Ph), 41.63 (CH2CH3), 13.71 (CH2CH3); 19F NMR (282 MHz,
CDCl3) δ = –117.59 (ArF); LRMS (+ESI) 383 [(M + Na)+ 100%], 361 [(M + H)+ 50%]; IR (νmax/cm–
1) 3656, 2985, 2038, 1649, 1221, 744, 716, 420; Found: C, 76.58; H, 5.80; N, 7.76. Calc for C23H21FN2O:
C, 76.64; H, 5.87; N, 7.77%
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2-(9H-Carbazol-9-yl)-N-ethyl-N-(3-fluorobenzyl)acetamide (8e). White solid; yield 92% (200 mg,
0.555 mmol). R 0.38 (ethyl acetate/hexane, 3:7 v/v); mp 135–137 °C; 1H NMR (300 MHz, CDCl3) δ =
8.12 – 8.01 (m, 2H, ArH), 7.53 – 7.33 (m, 2H, ArH) 7.28 – 7.19 (m, 6H, ArH), 6.98 – 6.94 (m, 2H, ArH),
5.12 (s, 2H, CH2CO), 4.57 (s, 2H, CH2Ph), 3.35 (q, J = 7.1 Hz, 2H, CH2CH3), 1.11 (m, J = 7.1 Hz, 3H,
CH2CH3); 13C NMR (75 MHz, CDCl3) δ = 168.18 (CO), 161.70 (d, J = 252.1 Hz, Ar), 140.20 (2×Ar),
130.61 (d, J = 5.8 Hz, Ar), 126.56 (2×Ar), 125.98 (d, J = 8.3 Hz, Ar), 123.50 (2×Ar), 122.60 (d, J = 4.2
Hz, Ar), 120.71 (2×Ar), 120.50 (2×Ar), 119.78 (d, J = 20.8 Hz, Ar), 114.45 (d, J = 22.0 Hz, Ar), 108.53
(2×Ar), 47.12 (CH2CO), 42.54 (CH2Ph), 41.64 (CH2CH3), 13.65 (CH2CH3); 19F NMR (282 MHz,
CDCl3) δ = –114.84 (ArF); LRMS (+ESI) 383 [(M + Na)+ 100%], 361 [(M + H)+ 50%]; IR (νmax/cm–
1) 3671, 2987, 1654, 1454, 1393, 1230, 1066, 879, 445; Found: C, 75.77; H, 5.96; N, 7.89. Calc for
C23H21FN2O: C, 76.64; H, 5.87; N, 7.77%
2-(9H-Carbazol-9-yl)-N-ethyl-N-(4-fluorobenzyl)acetamide (8f). White solid; yield 87% (188 mg,
0.522 mmol). R 0.49 (ethyl acetate/hexane, 3:7 v/v); mp 165–167 °C; 1H NMR (300 MHz, CDCl3) δ =
8.10 (dd, J = 24.9, 7.8 Hz, 2H, ArH), 7.49 – 7.31 (m, 2H, ArH), 7.28 – 7.19 (m, 6H, ArH), 6.99 – 6.97
(m, 2H, ArH), 5.11 (s, 2H, CH2CO), 4.56 (s, 2H, CH2Ph), 3.34 (t, J = 7.1 Hz, 2H, CH2CH3), 1.03 (t, J =
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7.1 Hz, 3H, CH2CH3); C NMR (75 MHz, CDCl3) δ = 166.69 (CO), 159.97 (d, J = 251.4 Hz, Ar),
140.58 (2×Ar), 133.68 (d, J = 3.3 Hz, Ar), 128.87 (d, J = 8.1 Hz, 2×Ar), 125.93 (2×Ar), 123.49 (2×Ar),
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120.70 (2×Ar), 120.46 (2×Ar), 115.53 (d, J = 22.0 Hz, 2×Ar), 108.54 (2×Ar), 45.46 (CH2CO), 42.16
(CH2Ph), 41.26 (CH2CH3), 13.63 (CH2CH3); 19F NMR (282 MHz, CDCl3) δ = –114.94 (ArF); LRMS
(+ESI) 383 [(M + Na)+ 100%], 361 [(M + H)+ 50%]; IR (νmax/cm–1) 3269, 2986, 1642, 1486, 1325,
1216, 1066, 745, 424; Found: C, 76.62; H, 5.75; N, 7.74. Calc for C23H21FN2O: C, 76.64; H, 5.87; N,
7.77%
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Methyl 2-(9H-carbazol-9-yl)acetate (9). Purification was achieved by recrystallisation in isopropanol.
White solid; yield 81% (6.3 g, 26.3 mmol). R 0.57 (ethyl acetate/hexane, 1:4 v/v); mp 107–109 °C; 1H
NMR (300 MHz, CDCl3) δ = 8.07 (d, J = 7.7 Hz, 2H, ArH), 7.50 – 7.39 (m, 2H, ArH), 7.31 – 7.17 (m,
4H, ArH), 4.93 (s, 2H, CH2), 3.66 (s, 3H, CH3); 13C NMR (75 MHz, CDCl3) δ = 169.06 (CO), 140.54
(2×Ar), 126.02 (2×Ar), 123.27 (2×Ar), 120.51 (2×Ar), 119.74 (2×Ar), 108.36 (2×Ar), 52.52 (CH3), 44.62
(CH2); LRMS (+ESI) 262 [(M + Na)+ 50%], 240 [(M + H)+ 100%]; IR (νmax/cm–1) 3050, 1728, 1484,
1452, 1356, 1262, 1207, 998, 750, 718, 627; Found: C, 75.35; H, 5.38; N, 5.84 Calc for C15H13NO2: C,
75.30; H, 5.48; N, 5.85%
2-(9H-carbazol-9-yl)acetic acid (10). Compound 9 (2.0 g, 8.36 mmol, 1.0 equiv.) and lithium
hydroxide (800 mg, 33.4 mmol, 4.0 equiv.) were dissolved in a solution of tetrahydrofuran/water (20 mL,
1:1 v/v). The reaction mixture was stirred at reflux for 4 hours. The reaction mixture was then cooled to
ambient temperature and the organic solvent evaporated to dryness. The remaining portion was diluted
with ethyl acetate and acidified with aqueous hydrochloric (2 M, 10 mL), then extracted with ethyl acetate
(3×10 mL). The combined organic extracts were dried over anhydrous magnesium sulfate and
concentrated in vacuo. Purification by trituration in diethyl ether furnished the desired product as a
colorless powder (1.36 g, 6.04 mmol, 72%). R 0.31(methanol/dichloromethane, 1:9 v/v); mp 192 °C; 1H
NMR (300 MHz, DMSO) δ = 8.12 (d, J = 7.7 Hz, 2H, ArH), 7.58 – 7.35 (m, 4H, ArH), 7.21 (t, J = 7.3
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Hz, 2H, ArH), 5.14 (s, 2H, CH2), OH proton not observed; C NMR (75 MHz, DMSO) δ = 170.26
(CO), 140.27 (2×Ar), 125.84 (2×Ar), 122.14 (2×Ar), 120.11 (2×Ar), 119.24 (2×Ar), 108.98 (2×Ar), 43.82
(CH2); LRMS (-ESI) 224 [(M + H)- 100%]; IR (νmax/cm–1) 3024, 1700, 1484, 1403, 1323, 1208, 1152,
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920, 750, 720, 565, 419; Found: C, 74.59; H, 4.87; N, 6.21 Calc for C14H11NO2: C, 74.65; H, 4.92; N,
6.22%
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68% (124 mg, 0.302 mmol). R 0.44 (ethyl acetate/hexane, 3:7 v/v); mp 220–222 °C; H NMR (300
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MHz, CDCl3) δ = 8.11 (d, J = 7.7 Hz, 1H, ArH), 8.00 (d, J = 7.7 Hz, 1H, ArH), 7.65 (dd, J = 13.5, 7.7
Hz, 1H, ArH), 7.54 – 7.10 (m, 9H, ArH), 5.09 (s, 2H, CH2CO), 4.84 (s, 2H, CH2Ph), 3.36 (q, J = 7.1 Hz,
2H, CH2CH3), 1.03 (t, J = 7.1 Hz, 3H, CH2CH3); 13C NMR (75 MHz, CDCl3) δ = 166.26 (CO), 140.65
(2×Ar), 132.18 (Ar), 131.04 (q, J = 3.2 Hz, Ar), 128.59 (q, J = 33.8 Hz Ar), 127.77 (q, J = 3.2 Hz, Ar),
127.20 (Ar), 126.01 (Ar), 125.82 (2×Ar), 124.42 (q, J = 271.2 Hz, CF3), 123.31 (2×Ar), 120.55 (2×Ar),
119.62 (2×Ar), 108.44 (2×Ar), 47.74 (CH2CO), 45.19 (CH2Ph), 40.96 (CH2CH3), 13.58 (CH2CH3); 19F
NMR (282 MHz, CDCl3) δ = –60.77 (CF3); IR (νmax/cm–1) 3052, 1653, 1463, 1320, 1260, 1113, 1064,
1014, 829, 740, 716, 596, 417; LRMS (+ESI) 433 [(M + Na)+ 100%], 411 [(M + H)+ 40%]; Found: C,
70.29; H, 5.11; N, 6.52. Calc for C24H21F3N2O: C, 70.23; H, 5.16; N, 6.83%
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2-(9H-Carbazol-9-yl)-N-ethyl-N-(3-(trifluoromethyl)benzyl)acetamide (11b). White solid; yield
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76% (138 mg, 0.336 mmol). R 0.44 (ethyl acetate/hexane, 3:7 v/v); mp 158–160 °C; H NMR (300
MHz, CDCl3) δ = 8.10 (d, J = 7.7 Hz, 1H, ArH), 7.92 (d, J = 7.7 Hz, 1H, ArH), 7.66 – 7.01 (m, 9H),
6.84 (d, J = 7.9 Hz, 1H, ArH), 5.06 (s, 2H, CH2CO), 4.60 (s, 2H, CH2Ph), 3.36 (q, J = 7.3 Hz, 2H,
CH2CH3), 1.03 (t, J = 7.3 Hz, 3H, CH2CH3); 13C NMR (75 MHz, CDCl3) δ = 167.48 (CO), 141.36 (Ar),
140.62 (2×Ar), 131.61 (Ar), 131.33 (q, J = 34.0 Hz, Ar), 128.33 (Ar), 125.99 (q, J = 3.4 Hz, Ar), 125.66
(2×Ar), 125.51 (q, J = 272.3 Hz, CF3), 123.27 (2×Ar), 123.12 (q, J = 3.4 Hz, Ar), 120.55 (2×Ar), 119.62
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(2×Ar), 108.37 (2×Ar), 48.39 (CH2CO), 45.49 (CH2Ph), 41.76 (CH2CH3), 13.67 (CH2CH3); F NMR
(282 MHz, CDCl3) δ = –62.53 (CF3); IR (νmax/cm–1) 3053, 1653, 1464, 1321, 1260, 1321, 1160, 1113,
1064, 1014, 829, 740, 716, 596; LRMS (+ESI) 433 [(M + Na)+ 100%], 411 [(M + H)+ 40%]; Found: C,
70.19; H, 5.09; N, 6.62. Calc for C24H21F3N2O: C, 70.23; H, 5.16; N, 6.83%
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2-(9H-Carbazol-9-yl)-N-ethyl-N-(4-(trifluoromethyl)benzyl)acetamide (11c). White solid; yield
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81% (147 mg, 0.358 mmol). R 0.44 (ethyl acetate/hexane, 3:7 v/v); mp 171–173 °C; H NMR (300
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ArH), 6.84 (d, J = 7.8 Hz, 1H, ArH), 5.10 (s, 2H, CH2CO), 4.63 (s, 2H, CH2Ph), 3.34 (q, J = 7.1 Hz, 2H,
CH2CH3), 1.02 (t, J = 7.1 Hz, 3H, CH2CH3); 13C NMR (75 MHz, CDCl3) δ = 167.41 (CO), 141.36 (Ar),
140.63 (2×Ar), 132.00 (q, J = 33.6 Hz, Ar), 128.32 (2×Ar), 126.02 (2×Ar), 125.98 (q, J = 3.4 Hz, 2×Ar),
125.52 (CF3), 123.27 (2×Ar), 120.42 (2×Ar), 119.60 (2×Ar), 108.38 (2×Ar), 48.40 (CH2CO), 46.41
(CH2Ph), 41.75 (CH2CH3), 13.68 (CH2CH3); 19F NMR (282 MHz, CDCl3) δ = –62.53 (CF3); IR
(νmax/cm–1) 3043, 1654, 1455, 1326, 1260, 1162, 1123, 1065, 830, 740, 717, 597; LRMS (+ESI) 433
[(M + Na)+ 100%], 411 [(M + H)+ 40%]; Found: C, 70.22; H, 5.11; N, 6.52. Calc for C24H21F3N2O: C,
70.23; H, 5.16; N, 6.83%
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2-(9H-Carbazol-9-yl)-N-ethyl-N-(pyridin-2-ylmethyl)acetamide (12a). White solid; yield 78% (119
mg, 0.347 mmol). R 0.71 (methanol/dichloromethane, 1:9 v/v); mp 195–197 °C; 1H NMR (300 MHz,
CDCl3) δ = 8.32 (d, J = 4.9 Hz, 1H, ArH), 8.19 (s, 1H, ArH), 8.04 (d, J = 7.6 Hz, 2H, ArH), 7.49 – 7.44
(m, 2H, ArH), 7.35 – 7.23 (m, 5H, ArH), 7.11 – 7.06 (m, 1H, ArH), 4.95 (s, 2H, CH2CO), 4.31 (s, 2H,
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CH2Ph), 3.43 (q, J = 7.1 Hz, 2H, CH2CH3), 1.02 (t, J = 7.1 Hz, 3H, CH2CH3); C NMR (75 MHz,
CDCl3) δ = 168.25 (CO), 155.92 (Ar), 148.96 (Ar), 140.40 (2×Ar), 136.55 (Ar), 126.33 (2×Ar), 123.52
(2×Ar), 122.19 (Ar), 121.39 (Ar), 120.59 (2×Ar), 120.22 (2×Ar), 108.68 (2×Ar), 47.19 (CH2CO), 44.22
(CH2Ph), 40.39 (CH2CH3), 13.36 (CH2CH3); IR (νmax/cm–1) 3286, 1650, 1590, 1485, 1324, 1209, 769,
745, 543; LRMS (+ESI) 366 [(M + Na)+ 100%], 344 [(M + H)+ 30%]; Found: C, 76.88; H, 6.14; N, 12.24
Calc for C22H21N3O: C, 76.94; H, 6.16; N, 12.24%
2-(9H-Carbazol-9-yl)-N-ethyl-N-(pyridin-3-ylmethyl)acetamide (12b). White solid; yield 76% (116
mg, 0.338 mmol). R 0.63 (methanol/dichloromethane, 1:9 v/v); mp 228–230 °C; 1H NMR (300 MHz,
CDCl3) δ = 8.38 (d, J = 4.8 Hz, 1H, ArH), 8.27 (s, 1H, ArH), 8.08 (d, J = 7.7 Hz, 2H, ArH), 7.50 – 7.44
(m, 2H, ArH), 7.39 – 7.25 (m, 5H, ArH), 7.15 – 7.10 (m, 1H, ArH), 4.98 (s, 2H, CH2CO), 4.35 (s, 2H,
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Journal of Medicinal Chemistry
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CH2Pyr), 3.41 (q, J = 7.1 Hz, 2H, CH2CH3), 1.02 (t, J = 7.1 Hz, 3H, CH2CH3); C NMR (75 MHz,
CDCl3) δ = 168.15 (CO), 148.73 (Ar), 143.14 (Ar), 140.21 (2×Ar), 135.07 (Ar), 133.23 (Ar), 126.54
(2×Ar), 123.46 (Ar), 123.41 (2×Ar), 120.67 (2×Ar), 120.45 (2×Ar), 108.48 (2×Ar), 47.03 (CH2CO),
41.38 (CH2Ph), 40.69 (CH2CH3), 13.29 (CH2CH3); IR (νmax/cm–1) 3281, 1662, 1461, 1324, 1255, 1119,
1066, 746, 719; LRMS (+ESI) 366 [(M + Na)+ 100%], 344 [(M + H)+ 40%]; Found: C, 76.84; H, 6.04;
N, 12.22 Calc for C22H21N3O: C, 76.94; H, 6.16; N, 12.24%
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2-(9H-Carbazol-9-yl)-N-ethyl-N-(pyridin-4-ylmethyl)acetamide (12c). White solid; yield 84% (128
mg, 0.373 mmol). R 0.63 (methanol/dichloromethane, 1:9 v/v); mp 203–205 °C; 1H NMR (300 MHz,
CDCl3) δ = 8.35 (d, J = 4.9 Hz, 1H, ArH), 8.22 (s, 1H, ArH), 8.06 (d, J = 7.7 Hz, 2H, ArH), 7.51 – 7.45
(m, 2H, ArH), 7.36 – 7.25 (m, 5H, ArH), 7.12 – 7.08 (m, 1H, ArH), 4.97 (s, 2H, CH2CO), 4.33 (s, 2H,
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CH2Pyr), 3.42 (q, J = 7.1 Hz, 2H, CH2CH3), 1.03 (t, J = 7.1 Hz, 3H, CH2CH3); C NMR (75 MHz,
CDCl3) δ = 168.32 (CO), 148.76 (2×Ar), 148.62 (Ar), 140.18 (2×Ar), 126.55 (2×Ar), 123.48 (2×Ar),
123.35 (2×Ar), 120.69 (2×Ar), 120.49 (2×Ar), 108.45 (2×Ar), 47.09 (CH2CO), 40.66 (CH2Ph), 40.21
(CH2CH3), 13.28 (CH2CH3); IR (νmax/cm–1) 3052, 1653, 1463, 1321, 1260, 1160, 1113, 1064, 1014,
829, 716, 596; LRMS (+ESI) 366 [(M + Na)+ 100%], 344 [(M + H)+ 40%]; Found: C, 76.56; H, 6.11; N,
12.24 Calc for C22H21N3O: C, 76.94; H, 6.16; N, 12.24%
2-(9H-Carbazol-9-yl)-N-ethyl-N-(2-nitrobenzyl)acetamide (13a). White solid; yield 91% (157 mg,
0.405 mmol). R 0.28 (ethyl acetate/hexane, 3:7 v/v); mp 225–227 °C; 1H NMR (300 MHz, CDCl3) δ =
8.09 (d, J = 8.0 Hz, 2H, ArH), 7.80 (d, J = 8.0 Hz, 1H, ArH), 7.52 – 6.89 (m, 8H, ArH), 6.58 (d, J = 7.8
Hz, 1H, ArH), 5.12 (s, 2H, CH2CO), 4.91 (s, 2H, CH2Ph), 3.39 (q, J = 7.1 Hz, 2H, CH2CH3), 1.11 (t, J =
7.1 Hz, 3H, CH2CH3); 13C NMR (75 MHz, CDCl3) δ = 168.64 (CO), 145.95 (Ar), 139.91 (2×Ar), 133.46
(Ar), 127.66 (Ar), 126.45 (Ar), 126.01 (2×Ar), 125.29 (Ar), 123.30 (2×Ar), 122.88 (Ar), 120.09 (2×Ar),
119.53 (2×Ar), 108.48 (2×Ar), 49.29 (CH2CO), 47.02 (CH2Ph), 42.65 (CH2CH3), 13.84 (CH2CH3);
LRMS (+ESI) 410 [(M + Na)+ 100%], 388 [(M + H)+ 40%]; IR (νmax/cm–1) 3091, 1659, 1541, 1460,
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