A systematic exploration of the effects of flexibility and basicity on sigma (σ) receptor binding in a series of substituted diamines

Article abstract of DOI:10.1039/c6ob00615a

The sigma-1 receptor (S1R) has attracted a great deal of attention as a prospective drug target due to its involvement in numerous neurological disorders and, more recently, for its therapeutic potential in neuropathic pain. As there was no crystal structure of this membrane-bound protein reported until 2016, ligand generation was driven by pharmacophore refinements to the general model suggested by Glennon and co-workers. The generalised S1R pharmacophore comprises a central region where a basic amino group is preferred, flanked by two hydrophobic groups. Guided by this pharmacophore, S1R ligands containing piperazines, piperazinones, and ethylenediamines have been developed. In the current work, we systematically deconstructed the piperazine core of a prototypic piperazine S1R ligand (vide infra) developed in our laboratories. Although we did not improve the affinity at the S1R compared to the lead, we identified several features important for affinity and selectivity. These included at least one basic nitrogen atom, conformational flexibility and, for S1R, a secondary or tertiary amine group proximal to the anisole. Furthermore, S2R selectivity can be tailored with functional group modifications of the N-atom proximal to the anisole.

Full text of DOI:10.1039/c6ob00615a

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A systematic exploration of the effects of flexibility
and basicity on sigma (σ) receptor binding in a
series of substituted diamines†
Cite this: DOI: 10.1039/c6ob00615a
Trent Conroy,‡a Madhura Manohar,‡a Yu Gong,a Shane M. Wilkinson,a
Michael Webster,a Brian P. Lieberman,b Samuel D. Banister,c Tristan A. Reekie,a
Robert H. Mach,b Louis M. Rendinaa and Michael Kassiou*a
The sigma-1 receptor (S1R) has attracted a great deal of attention as a prospective drug target due to its
involvement in numerous neurological disorders and, more recently, for its therapeutic potential in neuro-
pathic pain. As there was no crystal structure of this membrane-bound protein reported until 2016, ligand
generation was driven by pharmacophore refinements to the general model suggested by Glennon and
co-workers. The generalised S1R pharmacophore comprises a central region where a basic amino group
is preferred, flanked by two hydrophobic groups. Guided by this pharmacophore, S1R ligands containing
piperazines, piperazinones, and ethylenediamines have been developed. In the current work, we system-
atically deconstructed the piperazine core of a prototypic piperazine S1R ligand (vide infra) developed in
our laboratories. Although we did not improve the affinity at the S1R compared to the lead, we identified
several features important for affinity and selectivity. These included at least one basic nitrogen atom,
conformational flexibility and, for S1R, a secondary or tertiary amine group proximal to the anisole.
Furthermore, S2R selectivity can be tailored with functional group modifications of the N-atom proximal
to the anisole.
Received 22nd March 2016,
Accepted 13th September 2016
DOI: 10.1039/c6ob00615a
www.rsc.org/obc
respond to ligand activation and undergo translocation to the
cellular membrane, where they modulate the activity of ion
Introduction
channels and signalling molecules.7 Consequently, S1Rs are
involved in numerous neurological disorders including
depression, anxiety, substance abuse, schizophrenia, psycho-
sis, memory deficit disorders (such as dementia and
Alzheimer’s disease), and motor disorders (including
Parkinson’s disease).9,10 The S1R has also experienced a recent
surge of interest due to its involvement in analgesia,11,12 with
a noteworthy ligand (E-52862) progressing to Phase II clinical
trials for the treatment of pain.13
The existence of sigma receptors was proposed by Martin and
co-workers 40 years ago.1 Two sigma receptor subtypes, the
sigma-1 receptor (S1R) and sigma-2 receptor (S2R), have since
been discovered.2 The S1R is abundantly distributed in the
central and peripheral nervous systems.3 The S2R is typically
found in the lungs, liver and kidney,4 but can also be highly
expressed in malignant tumours.5
The molecular structure of S1R was elucidated in the late
1990s.6 The S1R is a small protein (25.3 kDa) predominantly
located at the mitochondria-associated endoplasmic reticulum
membrane (MAM).7 Here the S1R is indirectly responsible for
calcium homeostasis by acting as a molecular chaperone for
inositol 1,4,5-triphosphate receptor type 3 (ITPR3).8 S1Rs also
Due to the high expression of S2R in malignant tumours,
S2R ligands have been utilised diagnostically for radiolabelling
tumour sites, and have also been investigated therapeutically
for their ability to induce apoptosis in cancer cells.14 No
crystal structure exists for S2R. Compared to S1R, relatively
fewer S2R ligands are known, and as such, there is limited
information regarding S2R pharmacology.14,15 A study pub-
lished in 2011 reported that the progesterone receptor mem-
brane component 1 (PGRMC1) and S2R were each labelled
irreversibly following treatment with a known S2R ligand
(WC-21). Co-identity of S2R and PGRMC1 was proposed when
S2R binding was found to correlate to PGRMC1 expression.15
However, more recently, an in vitro PGRMC1 knockout NSC34
aSchool of Chemistry, The University of Sydney, Sydney, NSW 2006, Australia.
E-mail: michael.kassiou@sydney.edu.au
bDepartment of Radiology, University of Pennsylvania School of Medicine,
Philadelphia, Pennsylvania, USA
cDepartment of Radiation Oncology, Stanford University School of Medicine,
CA 94305, USA
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c6ob00615a
‡These authors made equal contributions to the paper.
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cell line (designed using CRISPR/Cas9) showed no difference
Results and discussion
in the binding of
([3H]-DTG), in addition to DTG-masked
labelling, when compared with a wild type cell line.16
a
known radiolabelled S2R ligand
125I]IAF photo- To study the effect of amine basicity on sigma receptor
affinity and selectivity as well as to explore the effect of
[
A recent milestone in S1R research was achieved in early further rigidification of the piperazine core, we introduced a
2016, when the crystal structure of the receptor bound to carbonyl moiety at various positions of lead structure 1, to
known S1R ligands (PD144418 and 4-IBP) was elucidated. The give compounds 5a,b and 8a,b (Fig. 1, piperazine series).
crystal structure of the S1R depicted a trimeric configuration Similarly, we prepared compounds containing an ethylene-
with a single transmembrane domain in each protomer. The diamine core in place of piperazine, representing analogues
binding site itself was found to be buried suggesting that the with increased conformational freedom. In addition, methyl
nature of the binding site is highly hydrophobic. Furthermore, groups and a carbonyl moiety were to be systematically
it was revealed that the basic amine of the ligands form hydro- moved around the core to better understand the effect of
gen bonds with Glu172. Moreover, the distinct structures of basicity on affinity and selectivity in this series (Fig. 1,
PD144418 and 4-IBP reveal the plasticity of the binding site.17 ethylenediamine Series 1–3).
Preceding this discovery, ligand design was propelled by
To this end, amide coupling reactions between Boc-pro-
pharmacophore refinements. A myriad of functional S1R ligands tected piperazine and the appropriate acid chlorides furnished
have now been identified,12 and common features for binding amides 3a and 3b (Scheme 1). Following Boc deprotection, the
have been elucidated.18–20 Glennon and co-workers identified piperazines were subjected to reductive alkylation with suit-
the ideal distance between a basic amine and two tethered able aryl aldehydes to afford the final compounds 5a,b.
hydrophobic groups (named primary and secondary hydro-
Piperazinone 6 was reductively alkylated to provide 7a and
phobic regions) for prototypic aminergic S1R ligands.18,19 7b (Scheme 1). N-Alkylation of the amides of 7a and 7b was
Since this discovery, several other groups have elaborated on achieved by sodium hydride deprotonation and treatment
this pharmacophore.21–23 Suggestions of a proton acceptor with 2-benzofuranyl methyl chloride or p-methoxybenzyl chloride
site, situated about 4 Å from the amine core, as an additional (synthesised according to the method reported by Ferorelli
requirement for optimal binding have been proposed.21,23
In contrast to the models of S1R binding, the S2R pharmaco-
et al.37) to give 8a and 8b.
Synthesis of the ethylenediamine series began by mono-
phore has received little attention. One study assessed 19 protection of ethylenediamine to give 9b (Scheme 2), and sub-
compounds to elucidate the features necessary for affinity and sequent amide coupling between the free amine of 9b and the
selectivity of S2R binding ligands. Requisite features identified appropriate freshly prepared acyl chloride gave the desired di-
included: a hydrophobic aromatic and aliphatic component; a substituted products (10a,b). The Boc group was cleaved under
positive ionisable atom; a hydrogen bond acceptor; and a acidic conditions to give 11a and 11b, the corresponding free
generic hydrophobic group. These features were initially deter- amines were subjected to reductive alkylation with the appro-
mined in silico and the binding affinities of the compounds priate aldehydes to afford 12a and 12b in modest yields due to
synthesised reflected the predicted binding profiles in most competing dialkylation. Methylation of the secondary amines
cases.24
(12a,b) using aqueous formaldehyde and sodium triacetoxy-
Studies in our laboratory have focused on the development borohydride provided the desired tertiary amines 13a and 13b.
of structurally diverse S1R ligands with efficacy in various Alternatively, monoprotection of the remaining nucleophilic
animal models of human disease.25–34 We have previously amines (12a,b) furnished amides 14a and 14b, which were
reported structure–activity relationships surrounding di- alkylated with iodomethane to give 15a and 15b. Cleavage of
substituted piperazine S1R ligand 1.27 The effects of linker the Boc group of 15a and 15b provided 16a and 16b, respec-
elongation26 and size of the diamine core,28 as well as modi- tively, and subsequent reductive alkylation with formaldehyde
fications to the ether substituent have been explored.27 This furnished ligands 16c and 16d.
work resulted in suitably high affinity ligands that when
The synthesis of compounds containing a carbonyl moiety
labelled with carbon-11 or fluorine-18, produced tracers for on the ethylenediamine linker required a different approach,
imaging S1R using positron emission tomography (PET).35,36 as depicted in Scheme 3. Methyl ester-protected glycine (17)
Owing to the high affinity and selectivity achieved with 1 for was condensed with anisaldehyde and the imine formed
S1R in vitro (as well as in vivo), we proceeded to build our reduced with sodium borohydride to give 18a. Saponification
library of this class of ligands. The present work aims to intro- of the ester was followed by Boc-protection of the secondary
duce amide functionality at various positions on the piper- amine (19a), and the acids coupled with amines benzofuran-2-
azine and ethylenediamine analogues to evaluate the effects of yl-methanamine or 1-(benzofuran-2-yl)-N-methylmethanamine
varying the basicity and flexibility on S1R binding and sele- using HBTU to give 20a and 21a. Boc deprotection yielded 22a
ctivity. These modifications will provide further insights and 23a and the resulting amines were N-alkylated under
into the structure activity relationships of this library of reductive amination conditions with formaldehyde to give 24a
diaminergic ligands. Furthermore, the nature of the alkyl and 25a in good yield. The amides of compounds: 22a, 23a,
amine core of the S1R pharmacophore can be understood 24a and 25a were then reduced with LiAlH4 to provide the
through this body of work.
requisite compounds 26a–26d.
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Fig. 1 Structure of the lead compound 1 and the library of compounds derived from the lead.
Scheme 1 (a) acid chloride, Et3N, CH2Cl2, 0 °C – rt, 20 h (3a: 70%; 3b: 89%); (b) 4 M HCl in 1,4-dioxane, rt, 2 h (4a: 73%; 4b: 79%); (c) 2-benzofuran-
carboxaldehyde or anisaldehyde, NaBH(OAc)3, ClCH2CH2Cl, rt, 10 h (5a: 75%; 5b: 74%; 7a: 64%; 7b: 86%); (d) (2-benzofuranyl)methyl chloride or
4-methoxybenzyl chloride, NaH, THF, 0 °C – rt, 18 h (8a: 90%; 8b: 34%).
The synthesis of the ligands with the amide moiety proxi- N-(4-methoxybenzyl)-N-methylamine or 4-methoxybenzylamine
mal to the anisole began in an analogous manner, with a two- and the protected glycine derivative 19b using HBTU as a
step reductive amination between glycine methyl ester and coupling reagent, yielded the requisite amides 20b and 21b.
2-benzofurancarboxaldehyde (Scheme 3) to afford 18b. Acidic carbamate deprotection provided amines 22b and 23b,
Saponification of the methyl ester, was followed by Boc protec- which were then alkylated under reductive conditions with
tion of the secondary amine to afford 19b. Reaction between formalin to generate 24b and 25b.
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Scheme 2 Reagents and conditions: (a) Boc2O, CH2Cl2, rt, 12 h (13: 91%); (b) acid chloride, Et3N, CH2Cl2, 0 °C – rt, 20 h (10a: 83%, 10b: 87%); (c) 4 M
HCl in 1,4-dioxane, rt, 2 h, then. NaOH (11a: 70%; 11b: 91%); (d) aldehyde, NaBH(OAc)3, ClCH2CH2Cl, rt, 10 h (12a: 52%; 12b: 37%); (e) formalin,
NaBH(OAc)3, ClCH2CH2Cl, rt, 10 h (13a: 48%; 13b: 25%); (f) Boc2O, CH2Cl2, rt, 12 h (14a: 97%; 14b: quant.); (g) NaH, DMF, 0 – rt °C, 10 min, cooled
to 0 °C, CH3I, stirred at 0 °C, 30 min, rt, 30 min (15a: quant.; 15b: 96%); (h) 4 M HCl in 1,4-dioxane, rt, 2 h, then. NaOH, for 16c and 16d; formalin,
NaBH(OAc)3, ClCH2CH2Cl, rt, 10 h (16a: 80%; 16b: 77%; 16c: 93%; 16d: quant.).
Scheme 3 Reaction condition and reagents: (a) (i) aldehyde, CH3OH, Et3N, 14 h, rt, (ii) NaBH4, CH3OH, 0 °C, 1 h, rt, 1 h (18a, 18b: 75%; 60% (b) 1 M
aq. NaOH, THF, 1.5 h, 6 M aq. HCl, Na2CO3, Boc2O, 14 h (19a, 19b: 96%; 97%) (c) Et3N, HBTU, CH2Cl2/DMF, 4 : 1, v/v, amine*, rt, 14 h (20a, 21a: 54%;
72%, 20b, 21b: 71%; 73%) (d) 4 M HCl in 1,4-dioxane, rt, 2 h, then. NaOH (22a, 23a: 84%; 98%, 22b, 23b: 93%; 88%) (e) formalin, CH3OH, Et3N,
NaBH3CN, 14 h (24a, 25a: 86%; 74%, 24b, 25b: 91%; 83%) (f) LiAlH4, THF, 0 °C – rt, reflux, 16 h (26a–d: 68–84%). *Amines: PMBNH2, PMBNHMe,
benzofuran-2-ylmethanamine and 1-(benzofuran-2-yl)-N-methylmethanamine, (ESI†).
The binding affinities of these ligands were determined by 2–4. Tissue homogenates were prepared by processing whole
using competitive radioligand binding assays for S1R and S2R, guinea pig brain samples for the S1R, while fresh rat liver was
and are presented in Tables 1–4. Binding assays were used for the S2R. [3H](+)-pentazocine (6 nM) was used for S1R
performed in two rotations, compounds in Table 1 were inde- assays, while [3H]DTG (5 nM) was used for S2R assays. For all
pendently assessed with respect to those compounds in Tables S2R studies conducted, 100 nM of (+)-pentazocine was added
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Table 1 Binding affinities of the piperazine and piperazinone ligands 5a–8b and their predicted pKa values and microspecies distribution deter-
mined through ChemAxon’s MarvinSketch software at 298 K
Affinities Ki (nM SEM)
Predicted pKa
Subtype selectivity
S1R/S2R
Predicted microspecies
distribution (%)
Compound
S1R
2.7
S2R
103
N1
N2
N1
N2
1
38
7.39
1.56
N
N
50.4
35.2
14.4
88.9
11.1
94.8
5.2
98.6
1.4
96.5
3.5
NH+
N
N
NH+
N
5a
5b
8a
8b
850 22
>105
>118
75
6.49
—
—
N
NH+
N
N
472 145
2830 66
2670 214
>35 000 200
>61 000 8000
>105
6.14
5.56
—
N
N
N
N
NH+
N
22
—
NH+
N
>37
5.96
N
NH+
N
Haloperidol
DTG
3.19
n/a
n/a
19.6
n/a
n/a
Table 2 Binding affinities of ethylenediamine series 1–12a–16d and their predicted pKa values and microspecies distribution determined through
ChemAxon’s MarvinSketch software at 298 K
Affinities Ki (nM SEM)
Predicted pKa
Subtype selectivity
(S1R/S2R)
Predicted microspecies
distribution (%)
Compound
S1R
S2R
N1
N2
N1
N2
1
0.72 0.09
47.8 1.9
66
7.39
1.56
N
N
50.4
35.2
14.4
15.5
84.5
6.2
93.8
28.7
71.3
20.0
80.0
14.5
85.5
5.9
94.1
26.8
73.2
19.1
80.9
—
NH+
N
N
NH+
N
12a
12b
13a
13b
16a
16b
16c
16d
>1000
>1000
n/a
4.7
0.5
28
8.14
—
—
N
NH+
N
N
30.9 4.2
207 20
2.83 0.21
145 23
8.58
—
N
N
NH+
N
106
2
7.80
—
N
NH+
N
N
78.7 6.5
73.5 11.2
9.51 0.86
975 44
8.00
—
N
N
NH+
N
242
2
0.3
0.7
1.9
5.5
8.17
—
N
NH+
N
N
13.4 1.6
510 61
8.60
—
N
N
NH+
N
7.84
—
N
NH+
N
N
N
6.56 0.75
35.8 1.1
8.03
N
—
—
NH+
—
—
Haloperidol
DTG
3.19
n/a
n/a
19.6
n/a
n/a
—
—
—
—
—
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Table 3 Binding affinities of ethylenediamine series 2–22a–25b and their predicted pKa values and microspecies distribution determined through
ChemAxon’s MarvinSketch software at 298 K
Affinities Ki (nM SEM)
Predicted pKa
Subtype selectivity
(S1R/S2R)
Predicted microspecies
distribution (%)
Compound
S1R
S2R
N1
N2
N1
N2
1
0.72 0.09
47.8 1.9
66
7.74
2.01
N
N
31.2
54.7
14.0
25.5
74.5
47.7
52.3
24.1
75.9
45.2
54.8
67.1
32.9
75.6
24.4
64.9
35.1
73.2
26.8
NH+
N
N
NH+
N
22a
22b
23a
23b
24a
24b
25a
25b
2.28 0.40
516 76
8.32 0.46
299 18
>1000
>5000
>10 000
>1000
>1000
>1000
>1000
720 37
>1000
>2000
>19
—
7.86
—
N
N
NH+
N
7.44
—
N
NH+
N
N
>120
>3.3
n/a
7.90
—
N
N
NH+
N
7.48
—
N
NH+
N
N
7.09
—
N
N
NH+
N
>1000
n/a
6.91
—
N
NH+
N
N
146
5
4.9
7.13
—
N
N
N
NH+
N
628 75
>1.6
6.96
NH+
—
—
N
Haloperidol
DTG
3.19
n/a
n/a
19.6
n/a
n/a
—
—
—
—
to mask S1R binding sites (see Experimental section for species distributions for the ligands in the series are also
further details). shown in Tables 1–4. The microspecies distribution represents
The introduction of an amide functionality in the piper- the % protonation of the N-atom(s) at a given pH (7.4 is
azine series reduced affinity for both S1R/S2R. An exocyclic car- reported herein). The pKa values for the amide functionality
bonyl moiety proximal to the anisole in 5a imparted a two-fold were calculated with protonation occurring at the oxygen
decrease in binding affinity for S1R (Ki = 850 nM), when com- atom. Secondary and tertiary amide values were typically
pared to an exocyclic carbonyl moiety near the benzofuranyl around −2 or lower and were never calculated as being micro-
substituent (5b: S1R Ki = 472 nM). Selectivity for S1R was also species at pH 7.4, therefore they have been excluded from the
reduced for 5a and 5b compared to 1, suggesting that a basic tables. Potentiometric titrations were also conducted to
amino group close to the anisole confers affinity for S1Rs, as support these predicted values. Experimental pKa values were
demonstrated by the introduction of carbonyl moieties at acquired for benzylamine, 13b and 25b, with values calculated
either exocyclic positions.
at 9.28 0.038, 7.31 0.01 and 8.01 0.012, respectively (see
Interestingly, when the basicity of the N-atoms were ESI† for conditions). The experimentally determined pKa value
reduced by introduction of an endocyclic carbonyl moiety, the for benzylamine is in agreement with the value predicted via
S1R affinities dropped drastically by more than 103-fold ChemAxon (9.30) and
a literature potentiometric value
(8a and 8b, Ki = 2830 and 2670 nM, respectively), and the com- (9.33).39 The experimental pKa values for 13b and 25b exhibi-
pound with the amide in proximity to the benzofuran group ted a discrepancy in comparison to the predicted values (8.00
presented greater selectivity. Unfavourable rigidity in the ring, and 6.96, respectively). These differences can be explained by a
steric factors, or a further reduction in the basicity of both the number of factors. Firstly, the experimental values were
amine groups is postulated to cause this drop in affinity.
obtained with the compounds dissolved in 40% ethanol/water,
The protonation states of the current library of S1R ligands while the calculated values are for aqueous environments. The
at physiological pH (7.4) were predicted using ChemAxon’s complexity of the molecule in comparison to the standard
Marvin software suite,38 and the predicted pKa and micro- (benzylamine) would also be a contributing factor. Particularly
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Table 4 Binding affinities of ethylenediamine series 3–26a–26d and their predicted pKa values and microspecies distribution determined through
ChemAxon’s MarvinSketch software at 298 K
Affinities Ki (nM SEM)
Predicted pKa
Subtype selectivity
(S1R/S2R)
Predicted microspecies
distribution (%)
Compound
S1R
S2R
N1
N2
N1
N2
1
0.72 0.09
47.8 1.89
66
7.74
2.01
N
N
31.2
54.7
14.0
4.8
24.8
68.5
1.9
2.4
3.4
93.0
1.2
2.3
NH+
N
N
NH+
N
26a
26b
26c
26d
20.7 1.4
27.5 4.7
6.09 0.76
4.8 0.5
626 57
294 40
53.5 4.3
77.4 0.8
30
5.71
5.49
5.86
4.29
8.69
9.00
9.02
8.50
N
NH+
N
N
NH+
NH+
N
NH+
N
11
8.8
16
NH+
N
N
NH+
NH+
N
NH+
N
NH+
N
N
8.1
86.9
2.8
NH+
NH+
N
NH+
N
7.4
NH+
N
N
36.3
56.2
0.1
NH+
NH+
NH+
—
Haloperidol
DTG
3.19
n/a
n/a
19.6
n/a
n/a
—
—
—
—
—
with 25b where lone pairs of adjacent oxygen atoms in both is the major species at pH 7.4 (50.4%), with single protona-
the benzofuran and amide are likely to be playing a role in tion of the N-atom proximal to the benzofuran also prevalent
protonation. Interestingly, an older version of MarvinSketch (31.17%). The predicted microspecies distributions of the
(15.7.6.0) predicted a pKa of 8.02 for 25b, while for 13b a value fully deprotonated forms of 5a, 5b, 8a, and 8b were 88.9%,
of 7.80 was predicted. We have included in the paper the most 94.8%, 98.6% and 96.5% respectively. The program used to
recent values calculated, but since the older values seem to calculate pKa values herein was revealed to have an r2 value
better correlate with the experimental values these are also of 0.763 after analysing 197 pharmaceutical substances.40
tabulated in the ESI.† Nevertheless, for the purposes of this While another source claims Marvin is better suited for acid
study, we believe the predicted values are sufficient aids to pKa determinations rather than base pKa determinations due
describe basicity trends.
to a smaller error in experimental and calculated pKa
For 1, the predicted pKa value of N1 (amine nearest to the values.41
benzofuran) was 7.39 and pKa of N2 was 1.56. 5a and 5b
These findings prompted us to investigate an open linker
possess a tertiary amine at the benzofuran N1 (pKa 6.49) and series (Table 2). Ethylenediamine was selected as an alternative
benzyl N2 (pKa 6.14), respectively. Despite having the basic flexible analogue of piperazine. This series contained carbonyl
amino group proximal to the benzofuran, 5a displayed a two- moieties on the periphery (ethylenediamine series one –
fold decrease in S1R binding affinity compared to 5b. This Table 2), within the ethylenediamine linker (ethylenediamine
could suggest that the N-atom proximal to the anisole might series two – Table 3) and lacking carbonyl functionality
be most important for S1R affinity, indicating that the anisole (ethylenediamine series three – Table 4).
may have a preference for the secondary hydrophobic pocket.
Alternatively, it could be the result of other binding modes of nyl moieties were tolerated proximal to the benzofuran and
these diaminergic ligands. were less preferred near the anisole. This library suggested
Similarly to the piperazine and piperazinone series, carbo-
8a and 8b had slightly lower pKa values for their respective that the piperazine core was not crucial in the design of high
tertiary amines (5.56 and 5.96, respectively). This is likely a affinity S1R ligands, however the flexible linker is thought to
result of the proximity of the carbonyl moiety to both affect the selectivity of these ligands since higher affinities for
N-atoms. This lowered basicity corresponds with the reduced the S2R was also observed. Hence, it is postulated that the
affinity obtained experimentally. The predicted microspecies ligands with a flexible linker can position themselves within
distribution for 1 revealed that the fully deprotonated species the binding pocket of the S2R as well.
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The binding affinities for 12a and 12b were examined. compared with 23b produced an almost two-fold decrease in
A carbonyl moiety proximal to the anisole produced a drop in affinity for S1R. Likewise, when the secondary amine in 23a
S1R/S2R affinity analogous to 5a (12a, S1R and S2R Ki ≥ 1000). was methylated (25a) a drop in affinity from Ki = 8.32 nM to
12b displayed drastic improvements in affinities for both S1R 146 nM was observed for S1R and 25a exhibited an increase in
and S2R with Ki = 30.9 nM and 145 nM respectively. The S2R affinity which was absent in 23a.
subtype selectivity appears to have improved for 16b, at 4.7 for
The final four ligands in the series (26a–26d, Table 4) illus-
the S1R. The introduction of tertiary amines in 13a and 13b trate the effect of secondary amines and tertiary amines in this
produced higher S1R and S2R affinity. 13a in particular pre- flexible linker. These ligands all possessed high S1R affinity
sented a greater subtype selectivity for S2R of S1R/S2R = 0.5 and selectivity. The highest affinity and selectivity of the four
(S1R and S2R Ki = 207 and 106 nM). Again there was a distinct ligands was 26d, with methylation at both N-atoms to yield ter-
preference for a carbonyl moiety proximal to the benzofuran in tiary amines, these ligands resulted in a reduction in the
13b for S1R affinity and selectivity (Ki = 2.8 nM and S2R selectivity for S1R as well when compared with its non-methyl-
affinity of Ki = 78.7 nM). Surprisingly, comparisons of the pKa ated counterpart 26a. A tertiary amine at the benzofuran term-
for the N-atom proximal to the benzofuran and anisole for inal (26b) exhibited the lowest affinity for S1R in this sub-
compounds 12a and 13a showed similar pKa values, however series and a slight improvement in S2R affinity in comparison
12a showed negligible activity, while 13a showed some prefer- to 26a. Conversely, 26c, where the tertiary amine was at the
ence for S2R. This could indicate that alternative factors anisole terminus exhibited comparable S1R and S2R affinities
besides basicity, such as the steric environment around the with 26d. However, 26d presented the greatest affinity for S1R
N-atom, are important for activity.
in the series.
The next set of ligands investigated were the tertiary amides
In this study we have demonstrated the effects of inserting
16a–d. Analogous to 13a, 16a possessed an almost identical a carbonyl moiety at various positions. It has been duly noted
binding profile. This was reflected in the pKa of N1, which was that affinity for the S1R was not improved by the modifications
also similar. 16c, while similar to 13a and 16a in the proximity reported here. Nevertheless, the purpose of this body of work
of the carbonyl moiety to the anisole, it lacked greater selecti- was to explore the nature of the alkylamine core of S1R ligands
vity for S2R than S1R as observed previously. 16b and 16d pre- and to make associations to the pharmacophore. This has
sented peculiar binding profiles also. Both ligands possess a been achieved by systematic modifications to the lead in this
tertiary amide proximal to the benzofuran, but 16b differs in a series. The structural features for optimal S1R binding as pro-
secondary amine at the anisole terminal, while 16d holds a ter- posed by Glennon and co-workers focussed on the ideal dis-
tiary amine. This key difference altered the selectivity of the tance between a basic amine and two tethered hydrophobic
ligand significantly. 16b exhibited greater affinity for S2R, groups. We have expanded upon this work to show that for
while incorporation of a methyl group at the amine reversed high S1R affinity carbonyls/amides are not ideal. This is
the selectivity, by favouring the S1R in 16d. Observing the basi- observed even when a basic amine remains in the structure,
city trends, it appears that again reducing the basicity is well confirming that two amine sites are beneficial. Amide intro-
tolerated at the benzofuran and less preferred at the anisole duction at the benzofuran is tolerated to a greater extent than
for a potent and selective S1R ligand. The predicted micro- when incorporated closer to the anisole, suggesting the latter
species distribution in each case depicts the secondary and is more important as the basic site. Both exocyclic and endo-
tertiary amines protonated with the highest distribution.
cyclic carbonyls are not favoured in the piperazine series,
The effect of carbonyl migration to within the ethylenedia- additionally endocyclic carbonyl moieties are less favoured of
mine linker was explored in compounds 22a–25b (Table 3). the two types of cyclic amides, since the internal carbonyl posi-
Compounds 22a and 22b were both identified as S1R ligands tion is capable of lowering the basicity of both N-atoms.
with Ki = 2.28 and 516 nM respectively. Selectivity was vastly Ethylenediamines with increased flexibility have been proven
improved with 22a (in contrast to 12b) in particular possessing to be suitable substitutes for piperazine, though tis does
S1R subtype selectivity of more than 2000 times. When com- reduce selectivity. Within this library the compounds lacking a
pared to the carbonyl isomer 12b, 22a presented significantly carbonyl moiety (ethylenediamine series three) produced the
higher affinity and selectivity for S1R. When comparing pKa greatest affinity for S1R, however the purpose of this library
values at the N-atoms, it was clear that 22a in fact possessed was to make associations from the known S1R pharmacophore
less basic N-atoms in comparison to 12b. 22b followed the and the current library. The most significant finding of this
general trend of lowered affinities with a carbonyl moiety prox- study is that the basic N-atom conferring S1R affinity is most
imal to the anisole and selectivity was lower at S1R/S2R = 19. likely the one proximal to the anisole and not the one proximal
Conversion of the secondary amide to a tertiary amide in 23a to the benzofuran group. Alternatively, our findings could indi-
produced only a slightly lowered affinity and selectivity for S1R cate modes of binding hitherto unknown to us given the sim-
when compared to 22a. A tertiary amide in 23b improved the plicity of the diaminergic ligand library.
binding affinity at both sites. Introduction of a tertiary amine
Future directions for this library will look at the design of
at the anisole in 24a resulted in a complete loss of affinity for S2R selective, high affinity ligands as we have identified that
both sites, likewise when a secondary amide was introduced in amides at the anisole and the manipulation of this amide can
25b. Incorporation of a tertiary amine as in 24b when produce higher affinity S2R ligands. This could help in the
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future design of drugs for the diagnosis of cancer, since S2R multiplicity (s = singlet, br s = broad singlet, d = doublet, dd =
are abundant in malignant cells.
doublet of doublets, dt = double of triplets, t = triplet, m =
multiplet), and coupling constants (J, Hz). The deuterated sol-
vents: CDCl3, CD3OD, (CD3)2CO and d6-DMSO were used. Low
General procedure for biological assays
In vitro assays were performed at University of Pennsylvania in resolution mass spectra (LRMS) were obtained from
a
Professor R. Mach’s laboratory. Tissue homogenates were pre- ThermoQuest Finnigan LCQ-Deca ion trap mass spectrometer
pared by processing whole guinea pig brain samples for the with electro-spray ionisation in positive (+ESI) or negative
S1R, while fresh rat liver was used for the S2R. Homogenate (−ESI) mode or atmospheric-pressure chemical ionisation in
samples were prepared in 50 mM Tris-HCl, pH 7.45 and stored positive mode (+APCI). Data are expressed as observed mass to
at −80 °C until use. Competitive binding assays were per- charge ratio (m/z) and are assigned as molecular ion (M). High
formed in 96 well polypropylene plates (Fisher Scientific, resolution mass spectra (HRMS) were obtained from a Bruker
Pittsburgh, PA) with a final volume of 0.2–0.25 mL. [3H](+)- 7T Apex Qe Fourier Transform Ion Cyclotron resonance mass
pentazocine (6 nM) was used for S1R assays while [3H]DTG (5 nM) spectrometer equipped with an Apollo II ESI/APCI/MALDI
was used for S2R assays. For all S2R studies conducted, 100 Dual source by the Mass Spectrometry Facility of the School of
nM of (+) pentazocine was added to mask S1R binding sites. Chemistry at The University of Sydney. The purity of final com-
A wide range of 10 concentrations (10−10 to 10−5 M) of compet- pounds submitted for in vitro testing was evaluated by HPLC
ing test compounds were evaluated. Competing compounds analysis with a final purity of ≥95% for all new compounds.
were serially diluted with buffer (as above) and non-specific High performance liquid chromatography (HPLC) analysis of
binding was defined with haloperidol (10 µM) or DTG (5 µM). final compounds was carried out on a Waters Alliance
Incubation was carried out at room temperature for a period of 2695 instrument using a SunFire™ C18 column (5 μm, 2.1 ×
120 minutes. After the 120 minute incubation period, separ- 150 mm) and detected via a UV detector set at 254 nm.
ation of the bound from free radio-ligands was conducted by Separation was achieved using water (solvent A) and aceto-
filtration through a 96 well gf per b unifilter (Perkin Elmer, nitrile (solvent B) at a flow rate of 0.2 mL min−1 and a gradient
Boston, MA). The filters were washed 3× with ice-cold wash of 0% B to 100% B over 50 minutes. Compounds were assessed
buffer (10 mM Tris-HCl, 150 mM NaCl, pH 7.4). 7 mL of scin- in binding assays as hydrochloride salts. The data is reported
tillation fluid was added to all filter samples and counted on a as percentage purity and retention time (RT) in minutes.
Wallac 1450 Microbeta liquid scintillation counter (Perkin Compounds were assessed in binding assays as hydrochloride
Elmer, Boston, MA) with a counting efficiency of ∼45%.
salts. Room temperature refers to ambient temperature in the
range of 20–30 °C. The synthesis of intermediates 1A–1F are
reported in the ESI.† pKa values were calculated at 298 K using
Solvents, reagents and conditions
Starting materials, reagents, and solvents were purchased from ChemAxon’s MarvinSketch software version 16.8.29.0.38
commercial suppliers and used without further purification,
except when acknowledged. Anhydrous solvents were obtained
General procedures for the chemistry
using PureSolv™ (purification system) for MeOH, and via dis-
tillation over sodium wire or CaH2 for THF and CH2Cl2, chlorides.
Procedure A – amide coupling of carboxylic acids via acid
solution of the appropriate carboxylic acid
A
respectively. An atmosphere of argon or nitrogen was used for (6.6 mmol, 1 eq.) in CH2Cl2 (20 mL) was treated with (COCl)2
oxygen or water sensitive reactions. Reaction flasks were oven- (2.3 eq.) followed by DMF (2 drops), and the reaction stirred
dried and flushed with inert gas before use. The progress of for 1 h. The reaction was then concentrated in vacuo and the
reactions was monitored by Thin Layer Chromatography (TLC) crude acid chloride was used immediately in the following
on 0.2 mm thick, aluminium-backed, pre-coated silica gel step. A solution of amine (4.9 mmol, 0.75 eq.) and Et3N
plates (Merck Silica gel 60 F254), with visualisation by KMnO4, (6.6 mmol, 1 eq.) in THF (30 mL) was cooled to 0 °C and the
vanillin, or ninhydrin solutions followed by heating to stain. freshly prepared acid chloride in THF (5 mL) was added and
Silica for flash column chromatography was of 230–400 mesh the reaction warmed to ambient temperature and stirred for
particle size. Melting points were determined using a Stuart 1 h. The reaction was subsequently filtered, the filtrate was
SMP10 Melting Point apparatus and are uncorrected. Infrared concentrated in vacuo, and the residue was purified by flash
absorption (IR) spectra were obtained using a Bruker ALPHA column chromatography on silica gel.
FT-IR spectrometer, samples (neat) were pressed against a
Procedure B – amide coupling of carboxylic acids via coup-
selenium–zinc cell (SeZn). Noteworthy absorbance peaks are ling reagents. To a solution of the appropriate carboxylic acid
provided in wavenumbers (cm−1). 1H NMR and 13C spectra were (12.3 mmol, 1 eq.) and HBTU (13.0 mmol, 1.1 eq.) in 1 : 3 v/v
recorded at a spectrometer frequency of 300, 400 or 500 MHz, DMF/CH2Cl2 (40 mL) was added Et3N (24.7 mmol, 2 eq.) and
13C NMR spectra at 75, 101 or 125 MHz. NMR experiments the reaction was stirred for 20 min before the amine
were acquired at 300 K using either Bruker DPX200 (200 MHz), (13.6 mmol, 1.2 eq.) was added and the reaction was stirred for
DPX300 (300 MHz), DRX400 (400 MHz) or Avance III 500 a further 2 h. The reaction was then diluted with EtOAc
Ascend (500 MHz) spectrometers. Data reported in chemical (150 mL), washed with 1 M aq. HCl (100 mL), 1 M aq. NaOH
shifts δ (ppm) relative to TMS = 0, using residual protons in (100 mL), brine (150 mL) and was dried (MgSO4) and concen-
deuterated solvents as an internal reference, relative integral, trated in vacuo to obtain the crude product, the resulting
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residue was purified by flash column chromatography on silica reaction stirred at rt for 16 h. The reaction mixture was parti-
gel, yielding the desired product. tioned between NaOH (1 M, 15 mL) and CH2Cl2 (20 mL), with
Procedure C – reduction of amides to amines with LiAlH4. further extraction of the aqueous layer with CH2Cl2 (2 ×
A solution of LiAlH4 (5 eq.) in anhydrous THF (10 mL) was 25 mL). The combined organic layers were washed with brine
cooled to 0 °C, amide (2.70 mmol, 1 eq.) in anhydrous THF (50 mL), dried over MgSO4, filtered, and evaporated in vacuo.
(5 mL) was added dropwise and the reaction was heated at The resulting residue was purified by flash column chromato-
reflux for 24 h. The reaction was subsequently cooled to 0 °C graphy on silica gel (eluent: 1 : 0–0 : 1 v/v hexane : EtOAc), yield-
and quenched by the portionwise addition of EtOAc (2 mL), ing the desired product.
followed by H2O (5 mL) then 1 M aq. HCl (5 mL). The reaction
Procedure H – reductive amination using sodium boro-
was then filtered, and the filtrate concentrated in vacuo. The hydride. To a solution of amine (20.5 mmol, 1.5 equiv.) and
residue was then basified with 1 M aq. NaOH to pH >10 and Et3N (1.5 eq.), aldehyde (13.7 mmol, 1 eq.) was added and the
the residue extracted with EtOAc (4 × 40 mL). The combined reaction was stirred for 14 h at ambient temperature, before it
organic extracts were washed with brine (10 mL), dried was cooled to 0 °C and NaBH4 (2 eq.) was added portionwise
(MgSO4) and concentrated in vacuo to an oil, the resulting over 20 min. The reaction was subsequently stirred for 1 h at
residue was purified by flash column chromatography on silica 0 °C, then 1 h at ambient temperature before being diluted
gel, yielding the desired product.
with 1 M aq. HCl (100 mL). The mixture was then washed with
Procedure D – Boc protection of amines. Amine (2.77 mmol, Et2O (100 mL), and the aqueous layer was basified to pH >11
1 eq.) was dissolved in 1 : 1 v/v THF/H2O (20 mL) and Na2CO3 with 1 M aq. NaOH and extracted into CH2Cl2 (3 × 100 mL).
(5.54 mmol) was added followed by Boc2O (3.05 mmol) and The combined organic fractions were dried (MgSO4) and con-
the reaction was left to stir for 14 h. The reaction was then centrated in vacuo, the resulting residue was purified by flash
acidified to pH 3 with 1 M aq. HCl and extracted into EtOAc column chromatography on silica gel (eluent: 1 : 0–0 : 1 v/v
(3 × 50 mL), and the combined organic fractions washed with hexane : EtOAc), yielding the desired product.
brine (30 mL), dried (MgSO4) and concentrated in vacuo to an
Synthesis of tert-butyl 4-(4-methoxybenzoyl)piperazine-1-
oil, the resulting residue was purified by flash column chrom- carboxylate 3a. Using general procedure A. 4-Methoxybenzoic
atography on silica gel (eluent: 1 : 1 v/v EtOAc/hexane), yielding acid (215 mg, 1.4 mmol, 1.1 eq.) was reacted with (COCl)2
the desired product.
(0.25 mL, 3.0 mmol, 2 eq.) to form the acid chloride, which
Procedure E – Boc deprotection of amines. HCl solution in was subsequently coupled to tert-butyl piperazine-1-carboxy-
dioxane (4 M, 2 eq.) was added to N-Boc amine (1.9 mmol, late (240 mg, 1.3 mmol, 1 eq.) using Et3N (0.62 mL). The
1 eq.) with stirring at room temperature for 2 h, resulting in a crude product was purified by flash column chromatography
white precipitate. For intermediates the precipitate was dis- on silica gel (CH2Cl2 : MeOH, 95 : 5) giving the titled com-
solved with the dropwise addition of HCl (6 M, 3 mL) until the pound as a colourless oil (317 mg, 70%), which solidified
disappearance of the precipitate was observed, this was parti- upon drying in vacuo overnight. m.p. 116–117 °C1; IR (ZnSe
tioned with diethyl ether (10 mL) and the organic layer was cell): νmax 2979, 2921, 2844, 1679, 1623, 1414, 1306,
discarded. The aqueous layer was basified with NaOH (6 M, 1247 cm−1; H NMR (200 MHz, CDCl3): δ 7.40–7.36 (2H, d, J =
6 mL) on ice until pH 13. CHCl3 (25 mL) was added and the 8.8 Hz), 6.93–6.89 (2H, d, J = 8.8 Hz), 3.83 (3H, s), 3.58–3.57
aqueous layer was extracted (2 × 25 mL CHCl3) and the com- (4H, d, J = 8.0 Hz), 3.46–3.44 (4H, d, J = 8.0 Hz), 1.47 (9H, s)
bined organic fractions were washed with brine (50 mL), dried ppm; 13C NMR (75 MHz, CDCl3): δ 170.60 (C), 160.91 (C),
over MgSO4, filtered and concentrated by evaporation.
154.60 (C), 129.18 (CH), 127.43 (C), 113.80 (CH), 80.33 (C),
Procedure F – N-alkylation using NaH. To a solution of the 55.38 (CH2), 30.99 (CH2), 28.37 (CH3) ppm; LRMS (+ESI): m/z
free amine (0.44 mmol, 1 eq.) in anhydrous THF (6 mL), NaH 343 [M + Na]+.
(0.54 mmol, 1.2 eq.) was added and allowed to stir (1 h, rt).
Synthesis of tert-butyl 4-(2-benzofuranoyl)piperazine-1-carboxy-
Dropwise addition of halide intermediate (0.52 mmol, 1.2 eq.) late 3b. 2-Benzofurancarboxylic acid (1.2 g, 7.2 mmol, 1.1 eq.)
was performed at 0 °C and the reaction mixture was warmed to was reacted with tert-butyl piperazine-1-carboxylate 2 (1.2 g,
rt and stirred for 18 h prior to concentration in vacuo. The 6.6 mmol, 1 eq.) according to general procedure A, yielding the
residue was partitioned between H2O (15 mL) and CH2Cl2 titled compound (3b) as a yellow oil (1.95 g, 89%). m.p.
(20 mL). The aqueous layer was extracted with CH2Cl2 (2 × 102–103 °C (lit. m.p. 97–98 °C).42 IR (ZnSe cell): νmax 2976,
25 mL). The organic extracts were combined, dried over 2927, 2862, 1683, 1641, 1556, 1419, 1248 cm−1 1H NMR
;
MgSO4, filtered, and evaporated to dryness, the resulting (500 MHz, CDCl3): δ 7.59–7.57 (1H, d, J = 7.5 Hz), 7.45–7.44
residue was purified by flash column chromatography on silica (1H, d, J = 8.0 Hz), 7.35–7.32 (1H, dd, J = 4.7 Hz), 7.24–7.21
3
3
gel (eluent: 1 : 0–0 : 1 v/v hexane : EtOAc), yielding the desired (1H, dd, J = 7.5 Hz, J = 7.5 Hz), 7.19 (1H, s), 3.77–3.74 (4H, t,
product.
J = 5.0 Hz), 3.49–3.47 (4H, t, J = 5.0 Hz), 1.43 (9H, s) ppm; 13C
Procedure G – reductive amination using NaBH(OAc)3 or NMR (125 MHz, CDCl3); 160.19 (C), 155.00 (C), 154.82 (C),
NaBH3CN. To a suspension of amine (1.5 mmol, 1 eq.) in 149.35 (C), 127.25 (C), 126.77 (CH), 122.88 (CH), 122.48 (CH),
ClCH2CH2Cl (15 mL), aldehyde (2.3 mmol, 1.5 eq.) was added 112.52 (CH), 112.04 (CH), 80.53 (C), 44.91 (CH2), 44.11 (CH2),
and the solution stirred at rt for 1 h. NaBH(OAc)3 (7.7 mmol, 28.66 (CH3) ppm; LRMS (+APCI): m/z 230.93 [M − Boc + H]+.
5 eq.) or NaBH3CN (4.5 mmol, 3 equiv.) was added and the Spectroscopic data matched that reported in literature.42
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Synthesis of 1-(4-methoxybenzoyl)piperazine 4a. Procedure 114.18 (CH), 112.27 (CH), 111.29 (CH), 62.40 (CH2), 55.23
E was used in the carbamate deprotection of N-Boc piperazine (CH3), 53.55 (CH2) ppm, 1 carbon obstructed or unresolved;
3a (0.6 g, 1.9 mmol), resulting in a colourless oil (302 mg, LRMS (+ESI): m/z 351 [M + H]+. HRMS(ESI): calc. C21H22N2O3
73%). IR (ZnSe cell): νmax 3324, 2951, 2914, 2851, 1606, 1513, [M + Na+] = 373.1523, found 373.1523, HPLC: 99.63%, RT:
1425, 1243, 1173, 1118, 1018, 872, 838 cm−1
(400 MHz, CDCl3): δ 7.38–7.36 (2H, d, J = 8.8 Hz), 6.92–6.89
;
1H NMR 17.31 min.
Synthesis
of
4-(4-methoxybenzyl)-2-piperazinone
7a.
(2H, d, J = 8.8 Hz), 3.83 (3H, s), 3.58–3.56 (4H, t, J = 4.2 Hz), 4-Methoxybenzaldehyde (1.45 mL, 11.9 mmol, 1.2 eq.) reacted
2.88–2.86 (4H, q, J = 3.3 Hz), 1.52 (1H, s, NH) ppm; 13C NMR with 2-piperazinone 6 (1.05 g, 10.1 mmol, 1.0 eq.) according to
(101 MHz, CDCl3): δ 170.54 (C), 160.85 (C), 129.23 (CH), 128.14 general procedure G, yielding the titled compound as a yellow
(C), 113.85 (CH), 55.48 (CH3), 46.93 (CH2), 46.42 (CH2) ppm; solid (1.48 g, 64%). m.p. 132–133 °C; IR (ZnSe cell): νmax 3238,
LRMS (+ESI): m/z 221 [M + H]+.
2920, 2881, 2839, 1611, 1496, 1229, 1081, 1033 cm−1;1H NMR
Synthesis of 1-(2-benzofuranoyl)piperazine 4b. Procedure E (300 MHz, CDCl3): δ 7.23–7.20 (2H, d, J = 8.8 Hz), 6.86–6.84
was used in the carbamate deprotection of N-Boc piperazine (2H, d, J = 8.7 Hz), 6.34 (1H, br s, NH), 3.79 (3H, s), 3.51 (2H,
3b (607 mg, 1.8 mmol) yielding a yellow oil (332 mg, 79%), s), 3.34–3.30 (2H, q, J = 7.9 Hz), 3.13 (2H, s), 2.63–2.59 (2H, t,
which solidified upon standing. IR (ZnSe cell): νmax 3292, J = 5.4 Hz) ppm; 13C NMR (75 MHz, CDCl3): δ 169.76 (C),
2965, 2929, 2867, 1615, 1557, 1431, 1283, 1255, 1221, 1177, 159.19 (C), 130.37 (CH), 129.01 (C), 113.96 (CH), 61.31 (CH2),
1122, 1014, 915, 862, 819 cm−1
;
1H NMR (300 MHz, CDCl3): 57.02 (CH2), 55.41 (CH3), 48.50 (CH2), 41.57 (CH2) ppm; LRMS
δ 7.63–7.61 (1H, d, J = 8.4 Hz), 7.52–7.49 (1H, d, J = 8.4 Hz), (+ESI): m/z 463 [2M + H]+.
7.39–7.34 (1H, t, J = 7.5 Hz), 7.28–7.23 (2H, m), 3.78 (4H, br s),
Synthesis of 4-(benzofuran-2-ylmethyl)-2-piperazinone 7b.
2.93–2.90 (4H, t, J = 4.8 Hz), 2.00 (1H, s, NH) ppm; 13C NMR 2-Benzofurancarboxaldehyde (150 µL, 1.2 mmol, 1.2 eq.) was
(75 MHz, CDCl3): δ 159.64 (C), 154.38 (C), 148.90 (C), 126.80 reacted with 2-piperazinone 6 (105 mg, 1.0 mmol, 1.0 eq.)
(C), 126.21 (CH), 123.40 (CH), 122.01 (CH), 111.66 (CH), 111.50 according to general procedure G, yielding the titled com-
(CH), 66.87 (CH2), 46.14 (CH2) ppm; LRMS (+ESI): m/z 231 pound as a white solid (208 mg, 86%). m.p. 128–129 °C; IR
[M + H]+.
(ZnSe cell): νmax 3193, 3085, 281, 1666, 1455, 1344, 980,
Synthesis of 1-(4-methoxybenzoyl)-4-(benzofuran-2-ylmethyl) 744 cm−1; H NMR (300 MHz, CDCl3): δ 7.56–7.54 (1H, d, J =
piperazine 5a. Procedure G was used in the reductive amination 7.2 Hz), 7.50–7.47 (1H, d, J = 8.1 Hz), 7.29–7.21 (2H, m), 6.90
of 4a (180 mg, 0.8 mmol, 1 eq.) and 2-benzofurancarbox- (1H, s), 6.65 (1H, s), 3.80 (2H, s), 3.41–3.37 (2H, q, J = 4.2 Hz),
aldehyde (0.13 mL, 1.1 mmol, 1.2 eq.) The residue was purified 3.28 (2H, s), 2.79–2.75 (2H, t, J = 5.4 Hz) ppm; 13C NMR
by flash column chromatography on silica gel (EtOAc : Hexane, (75 MHz, CDCl3): δ 169.45 (C), 155.21 (C), 153.17 (C), 128.10
30 : 70) to give a colourless oil (216 mg, 75%), which solidified (C), 124.37 (CH), 122.93 (CH), 120.95 (CH), 111.36 (CH), 106.29
upon standing. IR (ZnSe cell): νmax 3001, 2934, 2814, 1710, (CH), 56.52 (CH2), 54.08 (CH2), 48.59 (CH2), 41.26 (CH2) ppm;
1
1606, 1426, 1246, 1173, 1022 cm−1
(CD3OD)): δ 7.58–7.55 (1H, d, J = 7.2 Hz), 7.48–7.46 (1H, d, J =
;
1H NMR (300 MHz, LRMS (+ESI): m/z 253 [M + Na]+.
Synthesis of 1-(benzofuran-2-ylmethyl)-4-(4-methoxybenzyl)-2-
8.2 Hz), 7.38–7.35 (2H, d, J = 8.2 Hz), 7.29–7.18 (2H, m), piperazinone 8a. 4-(4-Methoxybenzyl)-2-piperazinone 7a
6.96–6.93 (2H, d, J = 8.2 Hz), 3.82 (3H, s), 6.73 (1H, s), 3.74 (380 mg, 1.76 mmol, 1 eq.) was reacted with 2-(chloromethyl)
(2H, s), 3.58 (4H, s), 2.60–2.53 (4H, q, J = 2.8 Hz) ppm; 13C benzofuran (381 mg, 2.29 mmol, 1.3 eq.) according to general
NMR (75 MHz, (CD3OD)): δ 170.27 (C), 161.77 (C), 156.10 (C), procedure F, the residue was purified by flash column chrom-
156.02 (C), 130.18 (CH), 129.54 (C), 129.44 (C), 124.93 (CH), atography on silica gel (EtOAc : Hexane, 30 : 70) yielding the
123.69 (CH), 121.84 (CH), 114.48 (CH), 111.90 (CH), 106.41 titled compound as a yellow oil (568 mg, 90%).
(CH), 55.82 (CH2), 55.71 (CH2), 53.78 (CH3) ppm; LRMS (+ESI):
m/z 351 [M + H]+ HRMS(ESI): calc. C21H22N2O3 [M + Na+] = 1239, 1171, 1032 cm−1; 1H NMR (300 MHz, CDCl3): δ 7.39–7.37
373.1523, found 373.1523, HPLC: 97.47%, RT: 16.99 min. (1H, d, J = 7.4 Hz), 7.29–7.26 (1H, d, J = 8.0 Hz), 7.13–7.02 (4H,
IR (ZnSe cell): νmax 2931, 2803, 1734, 1650, 1511, 1454,
Synthesis of 1-(2-benzofuranoyl)-4-(4-methoxybenzyl)piperazine m), 6.73–6.70 (2H, d, J = 8.8 Hz), 6.56 (1H, s), 4.55 (2H, s), 3.60
5b. 4-Methoxybenzaldehyde (108 µL, 0.89 mmol, 1.2 eq.) (3H, s), 3.31 (2H, s), 3.23–3.19 (2H, t, J = 5.4 Hz), 2.47 (2H, s),
reacted with benzofuranoyl piperazine 4b (170 mg, 0.74 mmol, 2.49–2.45 (2H, t, J = 5.7 Hz) ppm; 13C NMR (75 MHz, CDCl3): δ
1 eq.) according to general procedure G, the residue was 169.20 (C), 160.62 (C), 156.40 (C), 154.10 (C), 131.57 (CH),
purified by flash column chromatography on silica gel 129.66 (C), 129.49 (C), 125.40 (CH), 123.98 (CH), 122.02 (CH),
(EtOAc : Hexane, 30 : 70), yielding a yellow solid (193 mg, 74%). 114.83 (CH), 111.99 (CH), 106.46 (CH), 61.78 (CH2), 57.71
m.p. 115–116 °C; IR (ZnSe cell): νmax 2924, 2835, 2809, 2760, (CH3), 55.66 (CH2), 48.15 (CH2), 47.63 (CH2), 43.79 (CH2) ppm;
1628, 1507, 1423, 1231, 1180, 1028 cm−1;1H NMR (300 MHz, LRMS (+ESI): m/z 373 [M + Na]+. HRMS(ESI): calc. C21H22N2O3
(CD3OD)): δ 7.73–7.71 (1H, d, J = 7.7 Hz), 7.59–7.56 (1H, d, J = [M + Na+] = 373.1523, found 373.1522, HPLC: 96.68%, RT:
8.2 Hz), 7.45–7.40 (1H, t, J = 7.8 Hz), 7.34–7.28 (3H, m), 17.89 min.
6.90–6.88 (2H, d, J = 7.6 Hz), 3.78 (7H, m), 3.48 (2H, s),
Synthesis of 1-(4-methoxybenzyl)-4-(benzofuran-2-ylmethyl)-2-
2.50–2.46 (4H, t, J = 3.5 Hz) ppm; 13C NMR (75 MHz, piperazinone 8b. 4-(2-Methylbenzofuranyl)-2-piperazinone 7b
(CD3OD)): δ 159.65 (C), 155.11 (C), 150.12 (C), 130.76 (CH), (100 mg, 0.44 mmol, 1 eq.) was N-alkylated using 4-methoxy-
130.64 (C), 127.81 (C), 126.89 (CH), 124.18 (CH), 122.87 (CH), benzyl chloride (71 µL, 0.52 mmol, 1.2 eq.) according to
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general procedure F. The resulting residue was purified by (9H, s) ppm; 13C NMR (75 MHz, CDCl3): 159.56 (C), 156.93 (C),
flash column chromatography on silica gel (EtOAc : Hexane, 153.34 (C), 148.64 (C), 127.42 (C), 126.72 (CH), 123.53 (CH),
80 : 20) yielding a colourless film (54 mg, 34%). IR (ZnSe cell): 122.50 (CH), 111.65 (CH), 110.17 (CH), 79.49 (C), 40.35 (CH2),
νmax 3509, 3001, 2962, 2913, 1710, 1522, 1423, 1358, 1220, 40.10 (CH2), 28.29 (CH3) ppm; LRMS (+ESI): m/z 327 [M + Na]+.
1
1092 cm−1; H NMR (300 MHz, CDCl3): δ 7.54–7.52 (1H, d, J = The spectroscopic data matched that reported in the
7.2 Hz), 7.43–7.41 (1H, d, J = 7.8 Hz), 7.17–7.14 (2H, d, J = 8.8 literature.28
Hz), 6.86–6.83 (2H, d, J = 8.4 Hz), 6.70 (1H, s), 4.84 (2H, s),
Synthesis of N-(4-methoxybenzoyl)ethylenediamine 11a. Boc
4.48 (2H, s), 3.74 (3H, s), 3.74 (2H, s), 3.67 (2H, s), 3.23–3.20 deprotection of N-Boc ethylenediamine 10a (620 mg,
(2H, t, J = 4.5 Hz), 2.74–2.71 (2H, t, J = 5.6 Hz) ppm; 13C NMR 2.1 mmol) was carried out according to general procedure E,
(75 MHz, CDCl3) δ 170.75 (C), 159.87 (C), 155.77 (C), 155.12 yielding a colourless oil (283 mg, 70%). IR (ZnSe cell): νmax
(C), 130.13 (CH), 129.15 (C), 124.77 (CH), 123.45 (CH), 121.63 3289, 2915, 2840, 1632, 1505, 1444, 1248, 1181, 1029,
1
(CH), 111.64 (CH), 114.57 (CH), 106.39 (CH), 60.39 (CH2), 846 cm−1; H NMR (300 MHz, CDCl3): δ 7.79–7.76 (2H, d, J =
55.36 (CH3), 54.12 (CH2), 50.04 (CH2), 48.80 (CH2), 46.37 (CH2) 8.8 Hz), 7.26 (1H, s, NH), 6.87–6.84 (2H, d, J = 8.8 Hz), 3.79
ppm, 1 carbon obstructed or unresolved; LRMS (+ESI): m/z 351 (3H, s), 3.48–3.42 (2H, q, J = 5.7 Hz), 2.91–2.87 (2H, t, J = 7.0
[M + H]+. HRMS(ESI): calc. C21H22N2O3 [M + Na+] = 373.1523, Hz) ppm; 13C NMR (75 MHz, CDCl3): δ 167.44 (C), 161.96 (C),
found 373.1521, HPLC: 98.32%, RT: 14.42 min.
Synthesis of tert-butyl (2-aminoethyl)carbamate
128.78 (CH), 126.66 (C), 113.52 (CH), 66.96 (CH2), 55.25 (CH3),
9b. 41.24 (CH2) ppm; LRMS (+ESI): m/z 195 [M + H]+. The spectro-
Ethylenediamine 9a (3.66 mL, 55.3 mmol, 6 eq.) was Boc pro- scopic data matched that reported in the literature.45
tected according to general procedure D. The residue was puri-
Synthesis of N-(2-benzofuranoyl)ethylenediamine 11b. Boc de-
fied by flash column chromatography on silica gel protection of N-Boc ethylenediamine 10b (1.34 g, 4.4 mmol)
(EtOAc : MeOH : H2O : NH4OH, 70 : 20 : 9.5 : 0.5) yielding a col- was carried out according general procedure E, yielding a col-
ourless oil (1.33 g, 91%).
ourless solid (0.82 g, 91%). IR (ZnSe cell): νmax 3305, 3147,
IR (ZnSe cell): νmax 3358, 2983, 2934, 1682, 1526, 1455, 3028, 2927, 1644, 1596, 1520, 1287, 1257, 1227, 1175, 1106,
1
1
1366, 1162, 971 cm−1; H NMR (300 MHz, CDCl3): δ 5.93 (1H, 884 cm−1; H NMR (300 MHz, CDCl3): δ 8.04 (1H, s), 7.53–7.52
s, NH), 3.16 (2H, br s), 2.83–2.80 (2H, t, J = 8.9 Hz), 1.34 (2H, s, (1H, d, 4J = 1.6 Hz), 7.43–7.41 (1H, d, J = 7.8 Hz,), 7.28–7.03
NH2), 1.30 (9H, s) ppm; 13C NMR (75 MHz, CDCl3): δ 156.44 (3H, m), 3.39–3.36 (2H, q, J = 3.2 Hz), 2.85–2.81 (2H, t, J = 5.9
(C), 79.20 (C), 41.70 (CH2), 40.53 (CH2), 28.30 (CH3) ppm; Hz), 2.52 (2H, br s, NH2) ppm; 13C NMR (75 MHz, CDCl3): δ
LRMS (+ESI): m/z 161 [M + H]+. The spectroscopic data 159.45 (C), 154.86 (C), 148.84 (C), 127.69 (C), 126.94 (CH),
matched that reported in the literature.43
123.76 (CH), 122.79 (CH), 111.86 (CH), 110.47 (CH), 41.50
Synthesis of N-tert-butoxycarbonyl -N′-(4-methoxybenzoyl)ethy- (CH2), 41.31 (CH2) ppm; LRMS (+ESI): m/z 205 [M + H]+. The
lenediamine 10a. 4-Methoxybenzoic acid (1.14 g, 7.5 mmol, spectroscopic data matched that reported in the literature.28
1.2 eq.) was reacted with 9b (1.00 g, 6.2 mmol, 1 eq.) according
Synthesis of N-(benzofuran-2-ylmethyl)-N′-(4-methoxybenzoyl)-
to general procedure A. The residue was purified by flash ethylenediamine 12a. 2-Benzofurancarboxaldehyde (1.20 mL,
column chromatography on silica gel (EtOAc : Hexane, 30 : 70), 10.2 mmol, 1 eq.) was reacted with 11a (1.97 g, 10.2 mmol,
yielding the titled compound as white solid (1.53 mg, 83%). 1 eq.) according to the general procedure G. The crude product
m.p. 111–112 °C; IR (ZnSe cell): νmax 3366, 3318, 2979, 2929, was purified by flash column chromatography on silica gel
1683, 1633, 1531, 1458, 1331, 1254, 1167, 1023, 848 cm−1
;
(MeOH–CH2Cl2, 1 : 99 → 3 : 97), yielding a white solid (1.72 g,
1H NMR (300 MHz, CDCl3): δ 7.75–7.72 (2H, d, J = 8.8 Hz), 7.39 52%). m.p. 60–61 °C; IR (ZnSe cell): νmax 3343, 3290, 2889,
(1H, s), 6.83–6.80 (2H, d, J = 8.8 Hz), 5.48–5.44 (1H, t, J = 5.7 2831, 1622, 1504, 1454, 12 534, 1174, 1102, 1020,
Hz), 3.76 (3H, s), 3.49–3.44 (2H, q, J = 5.5 Hz), 3.32–3.28 (2H, 850 cm−1;1H NMR (300 MHz, CDCl3): δ 7.90 (1H, s), 7.80–7.77
q, J = 3.5 Hz,), 1.36 (9H, s) ppm; 13C NMR (75 MHz, CDCl3): (2H, d, J = 8.8 Hz), 7.53–7.50 (1H, d, J = 7.6 Hz), 7.40–7.38 (1H,
δ 167.53 (C), 162.01 (C), 157.34 (C), 128.82 (CH), 126.43 (C), d, J = 8.2 Hz), 7.22–7.17 (2H, m), 6.98–6.95 (2H, d, J = 8.8 Hz),
113.49 (CH), 79.53 (C), 55.24 (CH3), 41.54 (CH2), 40.03 (CH2), 6.68 (1H, s), 3.95 (2H, s), 3.84 (3H, s), 3.54–3.50 (2H, t, J = 6.3
28.27 (CH3) ppm; LRMS (+ESI): m/z 317 [M + Na]+. The spectro- Hz), 2.89–2.85 (2H, t, J = 1.8 Hz) ppm, one NH not observed;
scopic data matched that reported in the literature.44
13C NMR (75 MHz, CDCl3) δ 167.29 (C), 162.11 (C), 156.41 (C),
Synthesis of N-(benzofuran-2-oyl)-N′-tert-butoxycarbonyl-ethyle- 154.94 (C), 128.83 (C), 128.38 (C), 126.89 (C), 123.93 (CH),
nediamine 10b. 2-Benzofurancarboxylic acid (3.65 g, 122.75 (CH), 120.80 (CH), 113.68 (CH), 111.05 (CH), 104.02
22.5 mmol, 1.2 eq.) was reacted with 9b (3.17 g, 18.7 mmol, 1 (CH), 55.37 (CH3), 47.88 (CH2), 46.14 (CH2), 39.44 (CH2) ppm;
eq.) according to general procedure A. The residue was purified LRMS (+ESI): m/z 325 [M + H]+. HRMS(ESI): calc. C19H20N2O3
by flash column chromatography on silica gel (EtOAc : Hexane, [M + Na+] = 347.1366, found 347.1369, HPLC: 99.27%, RT:
30 : 70), yielding the titled compound as a white solid (4.94 g, 17.24 min.
87%). IR (ZnSe cell): νmax 3306, 2972, 2930, 1693, 1652, 1516,
Synthesis of N-(benzofuran-2-oyl)-N′-(4-methoxybenzyl)-ethylene-
1433, 1364, 1281, 1168, 1110, 939, 828 cm−1 1H NMR diamine 12b. 4-Methoxybenzaldehyde (0.75 mL, 6.1 mmol,
;
(300 MHz, CDCl3) δ 7.67–7.65 (1H, d, J = 8.0 Hz), 7.80 (1H, s), 1 eq.) was reacted with 11b (1.25 g, 6.1 mmol, 1 eq.) according
7.48–7.38 (3H, m), 7.29–7.27 (1H, d, J = 4.8 Hz),), 5.02 (1H, s), the general procedure G. The crude product was purified by
3.62–3.57 (2H, q, J = 3.7 Hz), 3.44–3.40 (2H, q, J = 3.6 Hz), 1.43 flash column chromatography on silica gel (MeOH–CH2Cl2,
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1 : 99 → 3 : 97), yielding the titled compound as a white solid
Synthesis of tert-butyl (benzofuran-2-ylmethyl)(2-(4-methoxy-
(0.73 g, 37%). m.p. 80–81 °C; IR (ZnSe cell): νmax 3342, 2952, benzamido)ethyl)carbamate 14a. Amine 12a (1.00 g, 2.77 mmol)
2835, 1643, 1595, 1506, 1448, 1240, 1174, 1104, 1032, 841, was carbamate protected using Boc2O (665 mg, 3.05 mmol)
819 cm−1;1H NMR (300 MHz, CD3OD): δ 7.69–7.67 (1H, d, J = according to general procedure D. The crude viscous product
8.0 Hz), 7.56–7.53 (1H, d, J = 8.2 Hz), 7.48–7.31 (3H, m), was purified by flash column chromatography on silica gel
7.28–7.25 (2H, d, J = 7.2 Hz), 6.85–6.82 (2H, d, J = 8.4 Hz), 3.72 (eluent: 1 : 1 EtOAc/hexane v/v) to obtain 14a as an amorphous
(3H, s), 3.71 (2H, s), 3.53–3.57 (2H, t, J = 6.5 Hz), 3.31 (1H, s), solid (1.10 g, 97%). 1H NMR (500 MHz, CDCl3): δ 7.77–7.76
2.84–2.80 (2H, t, J = 6.5 Hz) ppm; 13C NMR (75 MHz, CD3OD): (2H, d, J = 8.4 Hz), 7.51–7.49 (1H, d, J = 7.8 Hz), 7.41–7.39 (1H,
δ 161.39 (C), 160.37 (C), 151.23 (C), 149.95 (C), 132.33 (C), d, J = 8.7 Hz), 7.26–7.18 (2H, m), 6.88–6.86 (2H, d, J = 7.9 Hz),
130.77 (CH), 128.76 (C), 128.13 (CH), 124.83 (CH), 123.71 (CH), 6.55 (1H, s), 4.53 (2H, s), 3.82 (3H, s), 3.62 (4H, br s), 1.45 (9H,
114.82 (CH), 112.73 (CH), 111.21 (CH), 53.49 (CH2), 48.16 s) ppm, NH proton not observed; 13C NMR (101 MHz, CDCl3)
(CH2), 39.74 (CH2) ppm, one carbon obscured or unresolved; δ 167.23 (C), 162.17 (C), 157.14 (C), 155.00 (C), 154.34 (C),
LRMS (+ESI): m/z 325 [M + H]+. HRMS(ESI): calc. C19H20N2O3 128.86 (C), 128.22 (C), 126.63 (C), 124.25 (CH), 122.93 (CH),
[M
+
H+]
=
325.1547, found 325.1546, HPLC: 98.66%, 120.97 (CH), 113.70 (CH), 111.24 (CH), 104.33 (CH), 81.11 (C),
55.44 (CH3), 46.12 (CH2), 45.09 (CH2), 40.18 (CH2), 28.41 (CH3)
RT: 17.81 min.
Synthesis of N-(benzofuran-2-ylmethyl)-N′-(4-methoxybenzoyl)- ppm; LRMS (+ESI): m/z 425 [M + H]+.
N-methyl-ethylenediamine 13a. A suspension of diamine 12a
Synthesis of tert-butyl (2-(benzofuran-2-carboxamido)ethyl)(4-
(500 mg, 1.5 mmol, 1 eq.) was reacted with formalin (37% aq., methoxybenzyl)carbamate 14b. Amine 12b (1.00 g, 2.77 mmol)
185 µL, 2.3 mmol, 1.5 eq.) according to general procedure was carbamate protected using Boc2O (665 mg, 3.05 mmol)
G. The residue was purified by flash column chromatography according to general procedure D. The crude viscous product
on silica gel (MeOH–CHCl3, 1 : 99), yielding the titled com- was purified by flash column chromatography (eluent: 3 : 2 →
pound as colourless oil (225 mg, 48%). IR (ZnSe cell): 1 : 1 v/v hexane/EtOAc) to afford 14b as an amorphous solid
νmax 3355, 2958, 2798, 1634, 1604, 1536, 1503, 1451, 1253, (1.14 g, quant.). 1H NMR (400 MHz, CDCl3) δ 7.67–7.65 (1H, d,
1175, 1027, 939, 809 cm−1 1H NMR (300 MHz, CD3OD): J = 7.8 Hz), 7.54–7.38 (3H, m), 7.29–7.26 (1H, m), 7.18 (2H, s),
;
δ 7.78–7.75 (2H, d, J = 8.8 Hz), 7.53–7.50 (1H, d, J = 7.6 Hz), 6.85–6.83 (2H, d, J = 8.4 Hz) 4.41 (2H, s), 3.75 (3H, s), 3.57–3.52
7.26–7.25 (1H, d, J = 7.6 Hz), 7.40–7.38 (2H, m), 6.98–6.95 (2H, (4H, m), 1.48 (9H, s) ppm, NH proton not observed; 13C NMR
d, J = 8.8 Hz), 6.71 (1H, s), 3.85 (3H, s), 3.82 (2H, s), 3.57–3.53 (101 MHz, CDCl3) δ 159.11 (C), 157.25 (C), 156.13 (C), 154.88
(2H, t, J = 6.7 Hz), 2.74–2.70 (2H, t, J = 6.7 Hz), 2.41 (3H, s) (C), 149.05 (C), 130.03 (C), 128.84 (C), 127.72 (CH), 126.82
ppm; 13C NMR (75 MHz, CDCl3): δ 159.06 (C), 158.94 (C), (CH), 123.68 (CH), 122.72 (CH), 114.16 (CH), 111.88 (CH),
154.89 (C), 149.07 (C), 130.40 (CH), 129.91 (C), 127.77 (C), 109.98 (CH), 80.68 (C), 55.31 (CH3), 50.47 (CH2), 45.00 (CH2),
126.85 (CH), 123.71 (CH), 122.79 (CH), 113.90 (CH), 111.83 39.12 (CH2), 28.48 (CH3) ppm; LRMS (+ESI): m/z 448 [M + Na]+.
(CH), 110.16 (CH), 61.68 (CH2), 55.28 (CH2), 54.97 (CH3), 41.83
tert-Butyl(benzofuran-2-ylmethyl)(2-(4-methoxy-N-methylbenzamido)
(CH2), 36.47 (CH3) ppm; LRMS (+ESI): m/z 339 [M + H]+. HRMS ethyl)carbamate 15a. Amine 14a (0.921 g, 2.25 mmol) was alkyl-
(ESI): calc. C20H22N2O3 [M + Na+] = 361.1523, found 361.1525, ated according to general procedure F to obtain 15a as a col-
HPLC: 99.36%, RT: 17.34 min.
ourless oil, which was purified by flash column
Synthesis of N-(benzofuran-2-oyl)-N′-(4-methoxybenzyl)-N′- chromatography on silica gel (eluent: 3 : 2 → 1 : 1 v/v hexane/
methyl-ethylenediamine 13b. 12b (400 mg, 1.2 mmol, 1 eq.) was EtOAc) to give 15a as a colourless oil (0.950 g, quant.). 1H NMR
reacted with formalin (37% aq., 145 µL, 1.9 mmol, 1.5 eq.) (500 MHz, CDCl3): δ 7.51–7.49 (1H, d, J = 7.4 Hz), 7.42–7.435
according to the general procedure G. The crude product was (3H, m), 7.26–7.18 (2H, dt, J = 24.64, 7.38 Hz), 6.88–6.87 (2H,
purified by flash column chromatography on silica gel d, J = 8.9 Hz), 6.57 (1H, s), 4.61 (2H, s), 3.81 (3H, s), 3.74–3.48
(MeOH–CH2Cl2, 1 : 99 → 3 : 97), yielding the titled compound (4H, m), 3.08 (3H, s), 1.45 (9H, s) ppm; 13C NMR (101 MHz,
as colourless oil (106 mg, 25%). IR (ZnSe cell): νmax 3341, CDCl3) δ 171.63 (C), 160.69 (C), 155.72 (C), 155.00 (C), 129.21
2941, 2835, 2795, 1651, 1550, 1508, 1447, 1295, 1240, 1173, (C), 128.31 (C), 124.13 (CH), 122.82 (CH), 120.91 (CH), 113.61
1
1030, 847 cm−1; H NMR (300 MHz, CD3OD): δ 7.67–7.65 (1H, (CH), 111.19 (CH), 104.22 (CH), 80.67 (C), 55.39 (CH3), 49.56
d, J = 7.6 Hz), 7.55–7.53 (1H, d, J = 7.6 Hz), 7.48–7.36 (2H, m), (CH2), 45.32 (CH2), 44.23 (CH3), 38.35 (CH2), 28.47 (CH3) ppm,
7.29–7.24 (1H, t, J = 7.5 Hz), 7.21–7.18 (2H, d, J = 8.4 Hz), one obscured or unresolved carbon; LRMS (+ESI): m/z 439
6.78–6.75 (2H, d, J = 8.4 Hz), 3.51–3.49 (2H, t, J = 3.3 Hz), 3.66 [M + H]+.
(3H, s), 3.46 (2H, s), 2.59–2.55 (2H, t, J = 6.7 Hz), 2.25 (3H, s)
tert-Butyl (4-methoxybenzyl)(2-(N-methylbenzofuran-2-carbox-
ppm, NH proton not observed; 13C NMR (75 MHz, CD3OD): amido)ethyl)carbamate 15b. Amine 14b (0.956 g, 2.34 mmol) was
δ 160.93 (C), 160.31 (C), 156.27 (C), 149.96 (C), 131.57 (CH), alkylated according to general procedure F to obtain 15b as a
131.08 (C), 128.75 (C), 128.11 (CH), 124.82 (CH), 123.70 (CH), colourless oil, which was purified by flash column chromato-
114.59 (CH), 112.70 (CH), 110.40 (CH), 62.36 (CH2), 56.38 graphy on silica gel (eluent: 3 : 2 → 1 : 1 v/v hexane/EtOAc) to
(CH2), 55.54 (CH2), 42.35 (CH3), 37.85 (CH3) ppm; LRMS give 15b as a colourless oil (0.948 g, 96%.). 1H NMR (500 MHz,
(+ESI): m/z 339 [M + H]+. HRMS(ESI): calc. C20H22N2O3 CDCl3): δ 7.64–7.63 (1H, d, J = 8.2 Hz), 7.49 (1H, m), 7.39–7.37
[M + Na+] = 361.1523, found 361.1523, HPLC: 99.62%, (1H, t, J = 7.5 Hz), 7.29–7.28 (2H, m), 7.20 (1H, s), 7.08 (1H, s),
RT: 17.28 min.
6.82 (2H, m), 4.43 (2H, s), 3.74 (3H, s), 3.49–3.11 (7H, m),
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1.42 (9H, s) ppm; 13C NMR (125 MHz, CDCl3) δ 161.03 (C), G, and was purified by flash column chromatography on silica
159.03 (C), 155.81 (C), 154.74 (C), 149.13 (C), 130.39 (C), 129.51 gel (eluent: EtOAc : Et3N 1%), followed by general procedure
(C), 128.98 (CH), 127.09 (CH), 126.52 (CH), 123.62 (CH), 122.37 E to obtain 16d as the white HCl salt (0.256 g, quant). 1H NMR
(CH), 114.03 (CH), 111.94 (CH), 80.09 (C), 55.29 (CH3), 50.29 (400 MHz, CD3OD) δ 7.78–7.76 (1H, d, J = 7.82 Hz), 7.62–7.47
(CH2), 46.54 (CH2), 43.20 (CH2), 37.39 (CH3), 28.48 (CH3) ppm; (5H, m), 7.38–7.34 (1H, t, J = 7.45 Hz), 7.03–7.01 (2H, d, J =
LRMS (+ESI): m/z 439 [M + H]+.
Synthesis of
N-(2-((benzofuran-2-ylmethyl)amino)ethyl)-4- s), 2.93 (3H, s), 1.31 (3H, s) ppm;13C NMR (101 MHz, CD3OD)
8.57 Hz), 4.03 (2H, s), 3.81 (3H, s), 3.54 (2H, br s), 3.43 (2H, br
methoxy-N-methylbenzamide 16a. Amine 15a (0.850 g, δ 164.20 (C), 162.59 (C), 161.80 (C), 156.31 (C), 133.81 (CH),
2.34 mmol) was carbamate deprotected according to general 128.43 (C), 128.17 (C), 125.05 (CH), 123.76 (CH), 122.34 (CH),
procedure E to obtain 16a as the white HCl salt (0.581 g, 80%). 115.71 (CH), 114.61 (CH), 112.79 (CH), 60.73 (CH3), 55.85
1H NMR (400 MHz, CD3OD) δ 7.66–7.64 (1H, d, J = 7.71 Hz), (CH3), 54.62 (CH2), 45.82 (CH2), 40.89 (CH3), 37.78 (CH2) ppm,
7.55–7.48 (3H, m), 7.39–7.26 (2H, dt, J = 34.87, 7.38 Hz), 7.11 one carbon obscured or unresolved; LRMS (+ESI): m/z 353
(1H, s), 6.99–6.97 (2H, d, J = 8.72 Hz), 4.55 (2H, s), 3.87–3.83 [M + H]+. HRMS(ESI): calc. C21H24N2O3 [M + Na+] = 375.1679,
(5H, m), 3.46 (2H, m), 3.10 (3H, s) ppm, NH protons not found 375.1678, HPLC: 97.56%, RT: 17.51 min.
observed; 13C NMR (101 MHz, CD3OD) δ 175.19 (C), 162.91 (C),
Synthesis of methyl (4-methoxybenzyl)glycinate 18a. To a solu-
156.87 (C), 148.79 (C), 130.67 (C), 128.98 (CH), 127.96 (C), tion of glycine methyl ester (2.76 g, 22.0 mmol) in MeOH
126.71 (CH), 124.57 (CH), 122.79 (CH), 114.76 (CH), 112.32 (100 mL) was added Et3N (3.05 mL, 22.0 mmol) followed by
(CH), 110.40 (CH), 68.09 (CH2), 55.91 (CH3), 46.51 (CH2), 44.98 anisaldehyde (1.78 mL, 14.7 mmol) and the reaction was
(CH3), 38.91 (CH2) ppm; LRMS (+ESI): m/z 361 [M + Na]+. stirred for 14 h at ambient temperature, before it was cooled to
HRMS(ESI): calc. C20H22N2O3 [M + Na+] = 361.1523, found 0 °C and NaBH4 was added portionwise over 20 min. The reac-
361.1524, HPLC: 97.38%, RT: 17.37 min.
tion was subsequently stirred for 1 h at 0 °C, then 1 h at
Synthesis of N-(2-((4-methoxybenzyl)amino)ethyl)-N-methyl- ambient temperature before being diluted with 1 M aq. HCl
benzofuran-2-carboxamide 16b. Amine 15b (0.785 g, 1.79 mmol) (100 mL). The mixture was then washed with Et2O (100 mL),
was carbamate deprotected according to general procedure and the aqueous layer was basified to pH >11 with 1 M aq.
1
E to obtain 16b as the white HCl salt (0.516 g, 77%). H NMR NaOH and extracted into CH2Cl2 (3 × 100 mL). The combined
(400 MHz, CD3OD) δ 7.74–7.72 (1H, d, J = 7.84 Hz), 7.58–7.43 organic fractions were dried (MgSO4) and concentrated
(5H, m), 7.35–7.31 (1H, t, J = 7.84 Hz), 6.99–6.97 (2H, m), 4.24 in vacuo to an oil which was purified by flash column chrom-
(2H, s), 3.93 (2H, br s), 3.79 (3H, s), 3.42–3.39 (5H, m) ppm, atography on silica gel (eluent 1 : 1 → 2 : 1 EtOAc/hexane + 1%
NH protons not observed; 13C NMR (101 MHz, CD3OD) Et3N) to afford amine 18a as a colourless oil (2.30 g, 75%).
δ 162.17 (C), 156.21 (C), 132.62 (CH), 128.30 (C), 128.19 (C), 1H NMR (500 MHz, CDCl3) δ 7.25–7.18 (2H, m), 6.86–6.85 (2H,
124.98 (CH), 124.17 (CH), 123.70 (CH), 115.55 (CH), 114.30 d, J = 8.89 Hz), 3.89 (2H, s), 3.79 (3H, s), 3.72 (2H, s), 3.47 (3H,
(CH), 112.76 (CH), 55.82 (CH3), 52.17 (CH3), 46.93 (CH2), 46.30 s) ppm, NH proton not observed; 13C NMR (125 MHz, CDCl3)
(CH2), 37.77 (CH2) ppm, two carbon signals obscured or un- δ 172.75 (C), 163.39 (C), 130.17 (CH), 127.16 (C), 114.46 (CH),
resolved; LRMS (+ESI): m/z 339 [M + H]+. HRMS(ESI): calc. 55.46 (CH3), 52.65 (CH2), 51.98 (CH3), 49.20 (CH2) ppm; LRMS
C20H22N2O3 [M + Na+] = 361.1523, found 361.1523, HPLC: (+ESI): m/z 232 [M + Na]+.
95.07%, RT: 17.17 min.
Synthesis of methyl (benzofuran-2-ylmethyl)glycinate 18b.
N-(2-((Benzofuran-2-ylmethyl)(methyl)amino)ethyl)-4-methoxy- Glycine methyl ester 17 (2.58 g, 20.5 mmol) was reacted with
N-methylbenzamide 16c. Amine 16a (0.250 g, 0.67 mmol) alkyl- 2-benzofurancarboxaldehyde (1.66 mL, 13.7 mmol) according
ated according to general procedure G, and was purified by to general procedure H to obtain the crude oil, which was puri-
flash column chromatography on silica gel (eluent: fied by flash column chromatography on silica gel (eluent 2 : 1
EtOAc : Et3N 1%), followed by general procedure E to obtain → 1 : 1 hexane/EtOAc + 1% Et2NH) to afford amine 18b as a
1
16c as the white HCl salt (0.289 g, 93%). H NMR (400 MHz, colourless oil (1.80 g, 60%). 1H NMR (500 MHz, CDCl3)
CD3OD) δ 7.69–7.67 (1H, d, J = 7.60 Hz), 7.54–7.48 (3H, m), δ 7.52–7.51 (1H, 2, J = 7.4 Hz), 7.45–7.44 (1H, d, J = 7.92 Hz),
7.41–7.37 (1H, m), 7.32–7.28 (1H, m), 7.24 (1H, s), 7.00–6.98 7.26–7.18 (2H, m), 6.59 (1H, s), 3.99 (2H, s), 3.71 (3H, s), 3.48
(2H, d, J = 8.80 Hz), 4.76 (2H, s), 3.95 (2H, s), 3.84 (3H, s), 3.56 (2H, s); 13C NMR (125 MHz, CDCl3) δ 172.41 (C), 155.45 (C),
(2H, s), 3.08–3.06 (6H, m) ppm; 13C NMR (101 MHz, CD3OD) 155.18 (C), 128.37 (C), 124.18 (CH), 122.85 (CH), 120.94 (CH),
δ 175.27 (C), 163.07 (C), 157.09 (C), 147.25 (C), 130.74 (CH), 111.28 (CH), 104.69 (CH), 52.05 (CH3), 49.49 (CH2), 45.96
128.78 (C), 127.94 (C), 127.17 (CH), 124.78 (CH), 123.03 (CH), (CH2); LRMS (+ESI): m/z 220 [M + H]+.
114.80 (CH), 112.83 (CH), 112.47 (CH), 55.94 (CH3), 54.94
Synthesis
of
N-(tert-butoxycarbonyl)-N-(4-methoxybenzyl)
(CH2), 53.29 (CH3), 45.16 (CH2), 41.70 (CH3), 38.89 (CH2) ppm; glycine 19a. To a solution of amine 18a (2.13 g, 10.2 mmol) in
LRMS (+ESI): m/z 353 [M + H]+. HRMS(ESI): calc. C21H24N2O3 THF (15 mL) was added 1 M aq. NaOH (20.4 mL, 20.4 mmol),
[M + Na+] = 375.1679, found 375.1679, HPLC: 96.64%, and the reaction stirred for 1.5 h. A solution of 6 M aq. HCl
RT: 17.66 min.
(3.4 mL, 20.4 mmol) was subsequently added, followed by
Synthesis of N-(2-((4-methoxybenzyl)(methyl)amino)ethyl)-N- Na2CO3 (2.16 g, 20.4 mmol), and then Boc2O (2.44 g,
methylbenzofuran-2-carboxamide 16d. Amine 16b (0.250 g, 11.2 mmol) and the reaction was left to stir for 14 h. The reac-
0.67 mmol) was alkylated according to general procedure tion was then acidified to pH 3 with 1 M aq. HCl (25 mL) and
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extracted into EtOAc (3 × 90 mL), and the combined organic
tert-Butyl
(benzofuran-2-ylmethyl)(2-((4-methoxybenzyl)
fractions washed with brine (50 mL), dried (MgSO4) and con- (methyl)amino)-2-oxoethyl)carbamate 21b. Protected glycine 19b
centrated in vacuo to an oil which was purified by flash (336 mg, 1.10 mmol) was coupled with amine 4-methoxy-N-
column chromatography on silica gel (eluent: EtOAc + 1% benzyl-methanamine (200 mg, 1.32 mmol) according to
AcOH) to afford acid 19a as a colourless oil which crystallised general procedure B. The product was purified by flash
1
on standing (2.89 g, 96%). H NMR (400 MHz, CDCl3) δ 9.76 column chromatography on silica gel (eluent: 3 : 2
→
(1H, br s), 7.19–7.14 (2H, m); δ 6.87–6.85 (2H, d, J = 6.8 Hz); 1 : 1 hexane/EtOAc) to afford the title compound 21b as a pale
δ 4.48–4.44 (2H, d, J = 13.56 Hz), 3.93 (2H, s), 3.79 (3H, s), 1.49 yellow oil (365 mg, 73%). This compound was carried through
(9H) ppm 13C NMR (101 MHz, CDCl3) δ 175.82 (C), 159.22 (C), to the next step.
156.16 (C), 129.70 (C), 129.08 (CH), 114.16 (CH), 81.20 (C),
55.39 (CH3), 50.38 (CH2), 47.41 (CH2), 28.48 (CH3) ppm; LRMS amino)acetamide 22a. Amine 20a (245 mg, 577 µmol) was Boc
(+ESI): m/z 296 [M + H]+.
deprotected according to general procedure E to give 22a as
Synthesis of N-(benzofuran-2-ylmethyl)-2-((4-methoxybenzyl)
Synthesis of N-(benzofuran-2-ylmethyl)-N-(tert-butoxycarbonyl) the HCl salt (174 mg, 84%). The spectral data for the free
glycine 19b. To a solution of amine 18b (1.42 g, 6.47 mmol) in amine is provided. 1H NMR (400 MHz, CDCl3) δ 7.51–7.49 (1H,
THF (10 mL) was added 1 M aq. NaOH (13.0 mL, 13.0 mmol), m), 7.39–7.38 (1H, d, J = 8.24 Hz), 7.27–7.16 (4H, m), 6.86–6.83
and the reaction stirred for 1.5 h. A solution of 6 M aq. HCl (2H, m), 6.53 (1H, s), 4.36–4.35 (2H, d, J = 5.86 Hz), 3.88 (2H,
(2.17 mL, 13.0 mmol) was subsequently added, followed by s), 3.78 (3H, s), 3.37 (2H, s), 1.88 (1H, br s) ppm, one NH not
Na2CO3 (1.37 g, 13.0 mmol), and then Boc2O (1.55 g, observed; 13C NMR (101 MHz, CDCl3) δ 171.10 (C), 159.12 (C),
7.12 mmol) and the reaction was left to stir for 14 h. The reac- 155.59 (C), 155.11 (C), 130.52 (C), 129.19 (CH), 128.30 (C),
tion was then acidified to pH 3 with 1 M aq. HCl (15 mL) and 124.26 (CH), 122.93 (CH), 121.01 (CH), 114.19 (CH), 111.17
extracted into EtOAc (3 × 70 mL), and the combined organic (CH), 104.53 (CH), 55.42 (CH3), 51.76 (CH2), 46.71 (CH2), 42.66
fractions washed with brine (40 mL), dried (MgSO4) and con- (CH2) ppm; LRMS (+ESI): m/z 325 [M + H]+ HRMS(ESI): calc.
.
centrated in vacuo to an oil, which was purified by flash C19H20N2O3 [M + Na+] = 347.1366, found 347.1366, HPLC:
column chromatography on silica gel (eluent: EtOAc + 1% 99.89%, RT: 17.19 min.
AcOH) to afford acid 19b as a viscous yellow oil (1.92 g, 97%).
Synthesis of 2-((benzofuran-2-ylmethyl)amino)-N-(4-methoxy-
1H NMR (400 MHz, CDCl3) δ 8.57 (1H, br s), 7.54–7.52 (1H, d, benzyl)acetamide 22b. Amine 20b (505 mg, 1.2 mmol) was Boc
J = 7.27 Hz), 7.46–7.44 (1H, d, J = 7.56 Hz), 7.27–7.16 (2H, m), deprotected according to general procedure E to give 22b as
6.66 (1H, s), 4.67 (2H, s), 4.03 (2H, s), 1.47 (9H, s) ppm; the HCl salt (399 mg, 93%). 1H NMR (400 MHz, CD3OD)
13C NMR (101 MHz, CDCl3) δ 175.176 (C), 155.67 (C), 155.17 δ 7.67–7.65 (1H, d, J = 7.42 Hz), 7.56–7.54 (1H, d, J = 7.42 Hz),
(C), 153.66 (C), 129.17 (CH), 125.44 (C), 124.43 (CH), 121.09 7.41–7.37 (1H, t, J = 7.42 Hz), 7.32–7.28 (1H, t, J = 7.62 Hz),
(CH), 111.32 (CH), 105.56 (CH), 81.47 (C), 47.99 (CH2), 44.55 7.24–7.22 (2H, d, J = 7.62 Hz), 7.07 (1H, s), 6.90–6.87 (2H, d, J =
(CH2), 28.35 (CH3); LRMS (+ESI): m/z 306 [M + H]+.
tert-Butyl
(2-((benzofuran-2-ylmethyl)amino)-2-oxoethyl)(4- ppm, NH protons not observed; 13C NMR (101 MHz, CD3OD)
7.62 Hz), 4.53 (2H, s), 4.35 (2H, s), 3.91 (2H, s), 3.78 (3H, s)
methoxybenzyl)carbamate 20a. Protected glycine 19a (325 mg, δ 164.36 (C), 159.21 (C), 155.58 (C), 147.15 (C), 129.79 (C),
1.10 mmol) was coupled with amine 1E (195 mg, 1.32 mmol) 128.74 (CH), 127.56 (C), 125.39 (CH), 123.21 (CH), 121.42 (CH),
according to general procedure B. The product was purified by 113.60 (CH), 110.98 (CH), 109.27 (CH), 54.33 (CH3), 43.14 (CH2),
flash column chromatography on silica gel (eluent: 42.37 (CH2) ppm. One obscured or unresolved carbon; LRMS
3 : 2 hexane/EtOAc) to afford the title compound 20a as a pale (+ESI): m/z 347 [M + Na]+. HRMS(ESI): calc. C19H20N2O3 [M +
yellow oil (251 mg, 54%). This compound was carried through Na+] = 347.1366, found 347.1366, HPLC: 99.49%, RT: 17.42 min.
to the next step.
Synthesis of N-(benzofuran-2-ylmethyl)-2-((4-methoxybenzyl)
tert-Butyl (benzofuran-2-ylmethyl)(2-((4-methoxybenzyl)amino)- amino)-N-methylacetamide 23a. Amine 21a (575 mg, 1.3 mmol)
2-oxoethyl)carbamate 20b. Protected glycine 19b (1.00 g, was Boc deprotected according to general procedure E to give
3.28 mmol) was coupled with 4-methoxybenzylamine (511 µL, 23a as the HCl salt (482 mg, 98%). 1H NMR (500 MHz, d6-
3.94 mmol) according to general procedure B. The product was DMSO) δ 7.63–7.59 (1H, m), 7.53–7.47 (3H, m), 7.31–7.22 (2H,
purified by flash column chromatography on silica gel (eluent: m), 6.97–6.93 (2H, m), 6.86 (1H, s), 4.74–4.71 (2H, d, J = 14.57
3 : 2 → 1 : 1 hexane/EtOAc) to afford the title compound 20b as Hz), 4.11–4.07 (4H, m), 3.76–3.75 (3H, m), 2.99–2.95 (3H, d, J =
a pale yellow oil (1.04 g, 71%). This compound was carried 18.45 Hz) ppm, NH proton not observed; 13C NMR (125 MHz,
through to the next step.
d6-DMSO) δ 165.43 (C), 159.71 (C), 154.30 (C), 153.28 (C),
tert-Butyl (2-((benzofuran-2-ylmethyl)(methyl)amino)-2- 131.86 (C), 131.78 (CH), 127.82 (C), 124.23 (CH), 123.45 (CH),
oxoethyl)(4-methoxybenzyl)carbamate 21a. Protected glycine 19a 120.95 (CH), 113.92 (CH), 110.98 (CH), 105.06 (CH), 55.17
(540 mg, 1.83 mmol) was coupled with amine 1F (354 mg, (CH3), 49.49 (CH2), 46.08 (CH2), 43.81 (CH2), 33.97 (CH3) ppm;
2.20 mmol) according to general procedure B. The product was LRMS (+ESI): m/z 339 [M + H]+. HRMS(ESI): calc. C20H22N2O3
purified by flash column chromatography on silica gel (eluent: [M + Na+] = 361.1523, found 373.1523, HPLC: 95.27%, RT:
3 : 2 → 1 : 1 hexane/EtOAc) to afford the title compound 21a as 19.03 min.
a pale yellow oil (576 mg, 72%). This compound was carried
through to the next step.
Synthesis of 2-((benzofuran-2-ylmethyl)amino)-N-(4-methoxy-
benzyl)-N-methylacetamide 23b. Amine 21b (320 mg, 730 µmol)
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was Boc deprotected according to general procedure E to give (4H, m), 3.76 (3H, s), 2.98 (3H, s), 2.76 (3H, s) ppm; 13C NMR
23b as the HCl salt (241 mg, 88%). 1H NMR (400 MHz, (126 MHz, d6-DMSO) δ 164.85 (C), 160.18 (C), 154.32 (C),
CD3OD) δ 7.69–7.65 (1H, m), 7.58–7.52 (1H, m), 7.42–7.38 (1H, 153.18 (C), 132.88 (CH), 127.82 (C), 124.23 (C), 122.96 (CH),
t, J = 7.73 Hz), 7.38–7.29 (1H, t, J = 7.33 Hz), 7.23–7.21 (1H, d, 121.54 (CH), 120.99 (CH), 114.15 (CH), 110.99 (CH), 105.13
J = 8.14 Hz), 7.16–7.05 (2H, m), 6.91–6.87 (2H, m), 4.56–4.48 (CH), 58.55 (CH2), 55.21 (CH3), 54.21 (CH2), 43.89 (CH2), 40.39
(4H, m), 4.22–4.19 (2H, d, J = 14.06 Hz), 3.79 (3H, s), 2.92 (3H, (CH3), 34.09 (CH3) ppm; LRMS (+ESI): m/z 353 [M + H]+. HRMS
s) ppm, NH protons not observed; 13C NMR (101 MHz, (ESI): calc. C21H22N2O3 [M + H+] = 353.1859, found 353.1859,
CD3OD) δ 164.70 (C), 159.41 (C), 155.59 (C), 147.32 (C), 129.13 HPLC: 98.22%, RT: 19.39 min.
(CH), 128.19 (C), 127.85 (C), 125.38 (CH), 123.20 (CH), 121.42
2-((Benzofuran-2-ylmethyl)(methyl)amino)-N-(4-methoxybenzyl)-
(CH), 113.71 (CH), 110.97 (CH), 109.24 (CH), 54.33 (CH3), N-methylacetamide 25b. Amine 23b (100 mg, 255 µmol) was
51.43 (CH2), 50.01 (CH2), 43.33 (CH2), 32.52 (CH3) ppm.; LRMS methylated according to general procedure G. The crude
(+ESI): m/z 339 [M + H]+. HRMS(ESI): calc. C20H22N2O3 product was purified by flash column chromatography on
[M + Na+] = 361.1523, found 361.1523, HPLC: 98.09%, silica gel (MeOH–CH2Cl2, 1 : 99 → 3 : 97) to afford 25b as a
RT: 18.09 min.
clear oil (78 mg, 83%). 1H NMR (500 MHz, CD3OD) δ 7.69–7.66
Synthesis of N-(benzofuran-2-ylmethyl)-2-((4-methoxybenzyl) (1H, t, J = 6.94 Hz), 7.54–7.50 (1H, t, J = 7.71 Hz), 7.42–7.39
(methyl)amino)acetamide 24a. Amine 22a (100 mg, 277 µmol) (1H, t, J = 6.94 Hz), 7.32–7.29 (1H, t, J = 6.94 Hz), 7.20–7.19
was methylated according to general procedure G. The crude (2H, m), 7.14–7.13 (1H, m), 6.87–6.85 (2H, m), 4.69 (2H, s),
product was purified by flash column chromatography on 4.54–4.35 (4H, m), 3.77 (3H, s), 3.05 (3H, s), 2.89 (3H, s) ppm;
silica gel (MeOH–CH2Cl2, 1 : 99 → 3 : 97) to afford 24a as a 13C NMR (101 MHz, CD3OD) δ 165.45 (C), 160.80 (C), 157.16
clear oil (81 mg, 86%). 1H NMR (300 MHz, d6-DMSO) (C), 147.21 (C), 130.58 (CH), 129.3741 (C), 128.59 (C), 127.17
δ 7.61–7.51 (2H, m), 7.48–7.45 (2H, d, J = 8.46 Hz), 7.30–7.20 (CH), 124.71 (CH), 123.02 (CH), 115.13 (CH), 112.97 (CH),
(2H, m), 6.98–6.96 (2H, d J = 8.46 Hz), 6.79 (1H, s), 4.51 (2H, 112.47 (CH), 57.11 (CH2), 55.74 (CH3), 53.82 (CH2), 51.46
m), 4.31 (2H, br s), 3.93–3.87 (2H, d, J = 18.67 Hz), 3.76 (3H, s), (CH2), 42.67 (CH3), 33.97 (CH3); LRMS (+ESI): m/z 353 [M +
2.76 (3H, s) ppm, NH protons not observed; 13C NMR H]+. HRMS(ESI): calc. C21H24N2O3 [M + Na+] = 375.1679, found
(75 MHz, d6-DMSO) δ 167.27 (C), 164.88 (C), 160.62 (C), 373.1679, HPLC: 95.36%, RT: 18.49 min.
154.967 (C), 133.41 (CH), 128.438 (C), 124.605 (C), 123.3946
Synthesis of N1-(benzofuran-2-ylmethyl)-N2-(4-methoxybenzyl)
(CH), 121.85 (CH), 121.429 (CH), 114.60 (CH), 111.39 (CH), ethane-1,2-diamine 26a. Amine 22a (205 mg, 632 µmol) was
104.45 (CH), 58.66 (CH2), 55.68 (CH3), 54.70 (CH3), 49.72 reduced according to general procedure C. The crude product
(CH2), 36.48 (CH2); LRMS (+ESI): m/z 361 [M + Na]+. HRMS was purified by flash column chromatography on silica gel
(ESI): calc. C20H22N2O3 [M + Na+] = 361.1523, found 361.1522, (MeOH–CH2Cl2, 1 : 99 → 3 : 97) to afford 26a as a faintly yellow
1
HPLC: 96.20%, RT: 18.29 min.
oil (155 mg, 79%). H NMR (400 MHz, CDCl3) δ 7.49–7.47 (2H,
2-((Benzofuran-2-ylmethyl)(methyl)amino)-N-(4-methoxybenzyl) d, J = 8.70 Hz), 7.11–6.99 (5H, m), 6.78–6.77 (2H, m), 4.22 (2H,
acetamide 24b. Amine 22b (150 mg, 416 µmol) was methylated br s), 3.82–3.81 (3H, s), 3.39 (4H, m), 3.09–3.06 (2H, t, J = 7.67
according to general procedure G. The crude product was puri- Hz), 2.75–2.72 (2H, t, J = 7.14 Hz) ppm; 13C NMR (101 MHz,
fied by flash column chromatography on silica gel (MeOH– CDCl3) δ 160.29 (C), 155.767 (C), 155.58 (C), 132.29 (C), 132.19
CH2Cl2, 1 : 99 → 3 : 97) to afford 24b as a clear oil (128 mg, (CH), 130.39 (C), 127.77 (CH), 127.28 (CH), 124.41 (CH), 119.49
91%). 1H NMR (500 MHz, d6-DMSO) δ 9.14 (1H, br s), (CH), 115.64 (CH), 114.58 (CH), 55.82 (CH3), 50.25 (CH2), 47.39
7.73–7.71 (1H, d, J = 7.09 Hz), 7.60–7.58 (1H, d, J = 8.26 Hz), (CH2), 43.56 (CH2), 43.16 (CH2) ppm; LRMS (+ESI) m/z 311
7.41–7.38 (1H, t, J = 7.74 Hz), 7.32–7.29 (1H, t, J = 7.53 Hz), [M + H]+. HRMS(ESI): calc. C19H22N2O2 [M + Na+] = 333.1574,
7.21–7.20 (3H, m), 6.89–6.87 (2H, d, J = 8.61 Hz), 4.66 (2H, s), found 333.1573, HPLC: 99.50%, RT: 16.16 min.
4.26–4.25 (2H, d, J = 5.73 Hz), 4.02 (2H, s), 3.72 (3H, s), 2.87
Synthesis of N1-(benzofuran-2-ylmethyl)-N2-(4-methoxybenzyl)-
(3H, s) ppm; 13C NMR (126 MHz, d6-DMSO) δ 163.88 (C), N1-methylethane-1,2-diamine 26b. Amine 23a (250 mg,
158.41 (C), 154.87 (C), 146.80 (C), 130.27 (C), 128.82 (CH), 667 µmol) was reduced according to general procedure C. The
127.30 (C), 125.61 (CH), 123.37 (CH), 121.88 (CH), 113.76 (CH), crude product was purified by flash column chromatography
111.43 (CH), 111.41 (CH), 66.35 (CH2), 55.11 (CH3), 51.25 on silica gel (MeOH–CH2Cl2, 1 : 99 → 3 : 97) to afford 26b as a
(CH2), 41.72 (CH2), 40.91 (CH3) ppm; LRMS (+ESI): m/z 339 faintly yellow oil (181 mg, 84%) 1H NMR (500 MHz, CD3OD)
[M + H]+. HRMS(ESI): calc. C20H22N2O3 [M + Na+] = 361.1523, δ 7.69–7.68 (1H, d, J = 6.96 Hz), 7.57–7.55 (1H, d, J = 7.65 Hz),
found 361.1523, HPLC: 97.08%, RT: 17.83 min.
7.47–7.45 (2H, d, J = 8.35 Hz), 7.42–7.39 (1H, t, J = 6.96 Hz),
Synthesis of N-(benzofuran-2-ylmethyl)-2-((4-methoxybenzyl) 7.33–7.30 (1H, t, J = 6.94 Hz), 7.22 (1H, s), 7.00–6.99 (2H, s),
(methyl)amino)-N-methylacetamide 25a. Amine 23a (100 mg, 4.68 (2H, s), 4.23 (2H, s), 3.82 (3H, s), 3.56 (4H, s), 2.98 (3H, s)
267 µmol) was methylated according to general procedure ppm, NH proton not observed; 13C NMR (76 MHz, CD3OD)
G. The crude product was purified by flash column chromato- δ 162.38 (C), 157.33 (C), 146.85 (C), 132.73 (CH), 128.78 (C),
graphy on silica gel (MeOH–CH2Cl2, 1 : 99 → 3 : 97) to afford 127.28 (C), 124.83 (CH), 123.74 (CH), 123.08 (CH), 115.65 (CH),
1
25a as a clear oil (70 mg, 74%). H NMR (500 MHz, d6-DMSO) 113.22 (CH), 112.56 (CH), 55.87 (CH3), 53.73 (CH2), 52.47
δ 7.63–7.59 (1H, m), 7.53–7.47 (3H, m), 7.31–7.22 (2H, m), (CH2), 52.25 (CH2), 42.65 (CH2), 41.17 (CH3); LRMS (+ESI): m/z
6.90–6.95 (2H, m), 6.87 (1H, s), 4.76–4.70 (2H, m), 4.39–4.24 325 [M + H]+. HPLC: 99.83%, RT: 18.96 min.
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Synthesis N1-(benzofuran-2-ylmethyl)-N2-(4-methoxybenzyl)-N2- 11 J. L. Diaz, D. Zamanillo, J. Corbera, J. M. Baeyens,
methylethane-1,2-diamine 26c. Amine 24a (300 mg, 800 µmol)
R. Maldonado, M. A. Pericàs, J. M. Vela and A. Torrens,
was reduced according to general procedure C. The crude
Cent. Nerv. Syst. Agents Med. Chem., 2009, 9, 172.
product was purified by flash column chromatography on 12 M. Manohar, S. D. Banister, C. Beinat, J. O’Brien-Brown
silica gel (MeOH–CH2Cl2, 1 : 99 → 3 : 97) to afford 26c as a and M. Kassiou, Aust. J. Chem., 2015, 68, 600.
faintly yellow oil (177 mg, 68%). H NMR (500 MHz, CD3OD) 13 J. L. Díaz, R. Cuberes, J. Berrocal, M. Contijoch,
1
δ 7.56–7.54 (2H, d, J = 8.36 Hz), 7.10–7.00 (5H, m), 6.80–6.76
(2H, m), 3.81 (3H, s), 3.56 (4H, br s), 3.09–3.07 (2H, m), 2.83
(3H, s), 2.73–2.71 (2H, m) ppm, NH proton not observed;
13C NMR (125 MHz, CD3OD) δ 162.59 (C), 156.31 (C), 134.027
U. Christmann, A. Fernández, A. Port, J. Holenz,
H. Buschmann, C. Laggner, M. T. Serafini, J. Burgueño,
D. Zamanillo, M. Merlos, J. M. Vela and C. Almansa,
J. Med. Chem., 2012, 55, 8211.
(CH), 131.23 (C), 128.66 (C), 127.64 (CH), 121.92 (CH), 120.75 14 Y.-S. Huang, H.-L. Lu, L.-J. Zhang and Z. Wu, Med. Res.
(CH), 116.03 (CH), 115.68 (CH), 115.53 (CH), 62.78 (CH2), Rev., 2014, 34, 532.
61.31 (CH2), 55.91 (CH3), 52.08 (CH2), 43.11 (CH3), 39.98 (CH2) 15 J. Xu, C. Zeng, W. Chu, F. Pan, J. M. Rothfuss, F. Zhang,
ppm, one obscured or unresolved carbon; LRMS (+ESI): m/z
325 [M + H]+. HRMS(ESI): calc. C20H24N2O2 [M + Na+] =
347.1730, found 347.1730, HPLC: 99.07%, RT: 18.06 min.
Synthesis of N1-(benzofuran-2-ylmethyl)-N2-(4-methoxybenzyl)-
Z. Tu, D. Zhou, D. Zeng, S. Vangveravong, F. Johnston,
D. Spitzer, K. C. Chang, R. S. Hotchkiss, W. G. Hawkins,
K. T. Wheeler and R. H. Mach, Nat. Commun., 2011, 2,
1386/1.
N1,N2-dimethylethane-1,2-diamine 26d. Amine 25a (150 mg, 16 U. B. Chu, T. A. Mavlyutov, M.-L. Chu, H. Yang,
426 µmol) was reduced according to general procedure C. The
crude product was purified by flash column chromatography
A. Schulman, C. Mesangeau, C. R. McCurdy, L.-W. Guo and
A. E. Ruoho, EBioMedicine, 2015, 2, 1806.
on silica gel (MeOH–CH2Cl2, 1 : 99 → 3 : 97) to afford 26d as a 17 H. R. Schmidt, S. Zheng, E. Gurpinar, A. Koehl, A. Manglik
1
faintly yellow oil (102 mg, 71%). H NMR (400 MHz, CD3OD)
and A. C. Kruse, Nature, 2016, 532, 527.
δ 7.69–7.68 (1H, d, J = 7.02 Hz), 7.58–7.51 (3H, m), 7.43–7.39 18 R. A. Glennon, A. M. Ismaiel, J. D. Smith, M. Yousif, M. El-
(1H, t, J = 7.02 Hz), 7.33–7.29 (1H, t, J = 7.02 Hz), 7.24 (1H, s),
7.01–6.99 (2H, m), 4.69 (2H, s), 4.41 (2H, s), 3.82 (3H, s), 3.74
Ashmawy, J. L. Herndon, J. B. Fischer, K. J. Burke Howie
and A. C. Server, J. Med. Chem., 1991, 34, 1855.
(4H, br s), 2.97 (3H, s), 2.85 (3H, s) ppm 13C NMR (101 MHz, 19 R. A. Glennon, S. Y. Ablordeppy, A. M. Ismaiel, M. B. El-
CD3OD) δ 162.76 (C), 157.14 (C), 134.08 (CH), 128.75 (C),
127.28 (C), 124.84 (CH), 123.11 (CH), 121.78 (CH), 115.81 (CH),
Ashmawy, J. B. Fischer and K. Burke Howie, J. Med. Chem.,
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113.33 (CH), 112.58 (CH), 62.81 (CH2), 61.67 (CH2), 55.98 20 R. A. Glennon, Mini-Rev. Med. Chem., 2005, 5, 927.
(CH3), 53.88 (CH2), 51.10 (CH2), 41.46 (CH3), 40.44 (CH3) ppm, 21 T. M. Gund, J. Floyd and D. Jung, J. Mol. Graphics Modell.,
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339 [M + H]+. HRMS(ESI): calc. C21H26N2O2 [M + H+] = 22 C. Laggner, C. Schieferer, B. Fiechtner, G. Poles,
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R. D. Hoffmann, H. Glossmann, T. Langer and
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24 E. Laurini, D. Zampieri, M. G. Mamolo, L. Vio, C. Zanette,
C. Florio, P. Posocco, M. Fermeglia and S. Pricl, Bioorg.
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