Synthesis of five libraries of 6,5-fused heterocycles to establish the importance of the heterocyclic core for antiplasmodial activity

Article abstract of DOI:10.1016/j.bmc.2018.10.029

Research has indicated that N-myristoyl transferase, an enzyme that catalyzes the addition of a myristate group to the N-terminal glycine residues of proteins, is involved in the myristoylation of more than 100 proteins. Genetic knockdown of the enzyme proved detrimental for the viability of the parasite P. knowlesi. A crystal structure of P. vivax N-myristoyl transferase (pvNMT), containing a 3-methyl benzofuran ligand has made it possible to assess key amino acid residue-ligand interactions. We synthesized five libraries of 6,5-fused heterocycles to establish the importance of the heterocycles as core scaffolds, as well as introduced various aromatic amides and esters to determine which carbonylic group affects the potency of each heterocyclic antiplasmodial agent.

Full text of DOI:10.1016/j.bmc.2018.10.029

Accepted Manuscript
Synthesis of five libraries of 6,5-fused heterocycles to establish the importance
of the heterocyclic core for antiplasmodial activity
Leon Jacobs, Carmen de Kock, Dale Taylor, Stephen C. Pelly, Margaret A.L.
Blackie
PII:
S0968-0896(18)30935-0
https://doi.org/10.1016/j.bmc.2018.10.029
BMC 14588
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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Revised Date:
Accepted Date:
19 June 2018
24 September 2018
26 October 2018
Please cite this article as: Jacobs, L., de Kock, C., Taylor, D., Pelly, S.C., Blackie, M.A.L., Synthesis of five libraries
of 6,5-fused heterocycles to establish the importance of the heterocyclic core for antiplasmodial activity, Bioorganic
& Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.10.029
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Synthesis of five libraries of 6,5-fused
heterocyclics to stablish the significance of
the heterocyclic core for antiplasmodial
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Leon Jacobs, Carmen de Kock, Dale Taylor, Stephen C. Pelly, and Margaret A. L. Blackie
Department of Chemistry and Polymer Science, Stellenbosch University, Private Bag X1, Matieland 7602,
South Africa
HN
O
O
X
Y
X = C-Me,CH, N-Me, N
Y = NH, N, S
Z = O, NH
Ar

Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com
Synthesis of five libraries of 6,5-fused heterocycles to establish the importance of
the heterocyclic core for antiplasmodial activity
Leon Jacobs,a Carmen de Kock,b Dale Taylor,b Stephen C. Pellyc and Margaret A. L. Blackiea*
a Department of Chemistry and Polymer Science, Stellenbosch University, Private Bag X1, Matieland 7602, South Afric
b Department of Pharmacology, University of Cape Town, Private Bag X2, Rondebosch, Cape Town 7700, South Africa
c Department of Chemistry, Emory University, Atlanta, GA, USA
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Research has indicated that N-myristoyl transferase, an enzyme that catalyzes the addition of a
myristate group to the N-terminal glycine residues of proteins, is involved in the myristoylation
of more than 100 proteins. Genetic knockdown of the enzyme proved detrimental for the
viability of the parasite P. knowlesi. A crystal structure of P. vivax N-myristoyl transferase
(pvNMT), containing a 3-methyl benzofuran ligand has made it possible to assess key amino
acid residue-ligand interactions. We synthesized five libraries of 6,5-fused heterocycles to
establish the importance of the heterocycles as core scaffolds, as well as introduced various
aromatic amides and esters to determine which carbonylic group affects the potency of each
heterocyclic antiplasmodial agent.
Available online
Keywords:
Heterocycles
pfNMT inhibitors
Antiplasmodial
NF54
2009 Elsevier Ltd. All rights reserved.
Malaria
1. Introduction
infection.[5] As a result, between 2000 and 2015 it was estimated
that there was a 42% decline in incidents of infection and a 66%
Malaria is caused by the parasite belonging to the genus
Plasmodium, of which Plasmodium falciparum is the most
deadly and widespread causative agent, second being
Plasmodium vivax.[1] Currently there are five species that infect
the human population, P. falciparum, P vivax, P. ovale, P.
malariae and P. knowlesi. [1-2] It has been postulated that malaria
is the cause for half of all deaths in human history.[3] According
to the WHO 2016 report, an estimated 212 million cases of
malaria exist globally and that according to the CDC, 3.2 billion
people are still at risk of contracting malaria.[4] 90% of the
reported cases of malaria are situated in Africa with more than
half of the infected population residing in Sub-Saharan Africa.[5]
Regrettably children under five suffer the greatest mortality
rate, accounting for approximately 70% of all malaria related
deaths, making malaria the third biggest killer of children after
pneumonia and diarrhea.[6] Although malaria infection still
affects many lives, the number of deaths have been on the
decline in recent years due to the effective use of insecticide-
treated bed nets (ITN) as a preventative measure and the use of
artemisinin combination therapy (ACT) for the treatment of
decline in the number of deaths in Africa.[5] One major cause for
concern is the increasing occurence of resistance towards
multiple prescribed antimalarials on the market. One of the
earliest synthetic antimalarials, chloroquine (CQ) made massive
strides in combating malarial infection, however resistance
towards CQ, primarily due to its use as a monotherapy, has
sparked the urgent need to develop newer antimalarial drugs.[7]
Efforts such as Roll Back Malaria and the contribution of the Bill
and Melinda Gates Foundation were a response to this dire
need. It has been discovered that resistance to drugs like CQ and
amodiaquine (AQ) is attributed to polymorphism in the gene
pfcrt that codes for P. falciparum chloroquine resistance
transporter protein (pfCRT).[8] The development of multiple drug
resistance as a result of monotherapy resulted in the use of
ACTs, the administration of artemisinin derivatives together with
other antimalarial drugs such as AQ, mefloquine and
lumefantrine. The use of ACTs today is the recommended
chemotherapeutic treatment of malaria and has a high success
rate in the treatment of uncomplicated and severe malaria.
However, resistance towards artemisinin discovered in South-

East Asia poses an imminent problem.[9] Therefore, the search
for new antiplasmodial targets and chemotherapeutic agents
continues to be essential in the effort to effectively combat the
disease. N-Myristoyl transferase (NMT) is an important enzyme
that is ubiquitous in all eukaryotic cells. Using Myr-CoA as a
myristate source, it catalyzes the irreversible addition of a
myristate group to the N-terminal glycine residues of proteins,
post- and co-translationally.[10] Research by Wright et al. has
proven that N-myristoylation is essential for the viability of the
rodent malaria parasite P. berghei, and partial knockdown of
NMT expression resulted in reduction of parasitemia in vivo.
Effective inhibition of NMT resulted in the inhibition of the
benzofuran scaffold, as well as the aromatic ester and amides at
the C2-position.
HN
O
O
R
X
Y
X = C-Me,CH, N-Me, N
Y = NH, N, S
Z = O, NH
Z
Z
Z
R =
Figure 2. Generalized structure of the indole, 3-methyl indole, 1-methyl
benzimidazole, benzoxazole and benzothiophene heterocycles indicating the
various aromatic amide and ester groups at the C2-position.
parasite’s ability to assemble the inner membrane complex, a
[11]
critical subcellular organelle.[10] Yu et al.
identified an active
inhibitor for P. vivax NMT, which shares an 81% sequence
identity to P. falciparum NMT (pfNMT). This inhibitor, RO-09-
4609, (Figure 1) was developed by Roche[12] in a campaign to
develop inhibitors for Candida albicans NMT (CaNMT) and
Trypanasoma brucei NMT (TbNMT), and was later identified via
a “piggyback” approach as an inhibitor for pvNMT with an IC50 of
51 µM. RO-09-4609 was further derivatized by introducing a
piperidinyl ether group at the C4-position and various benzylic,
phenethyl and 1-methylenenaphthyl amides and esters at the
C2-position. These efforts lead to the discovery of a 3-methoxy
benzoate 3-methyl benzofuran derivative, 26, with an improved
pvNMT inhibition of 0.60 µM (Figure 1).
2. Chemistry
Scheme 1 shows the steps involved in the synthesis of the
indole scaffold. 1 was benzylated with benzylbromide in DMF to
produce aldehyde 2 in a 94% yield, while the reaction of sodium
azide and 3 produced azide 4 in a 91% yield after purification.
The Knoevenagel-condensation reaction between 2 and 4 was
carried out in anhydrous ethanol with sodium ethoxide, and
ethyl trifluoroacetate acting as a sacrificial electrophile. The
aromatic vinyl azide 5 was isolated in a 55% yield. The
subsequent Hemetsberger-Knittel indolization reaction was
carried out by the addition of 5 to 1,3-dichlorobenzene at 160
°C, producing the desired indole precursor 6 in a 90% yield.
Debenzylation of the indole was achieved with 10% Pd/C and H2,
affording phenol 7 in a 91% yield. This was followed by a
Mitsunobu coupling with 4-hydroxy N-Boc piperidine which
resulted in full conversion of 7 when the reaction was monitored
my means of thin layer chromatography. Purification of the
coupled product proved difficult as it co-eluted alongside the by-
products of the Mitsunobu reaction. To overcome this issue the
indole nitrogen was Boc-protected to decrease the polarity in
relation to the Mitsunobu by-products, enabling effective
separation to provide 8. Subsequent reactions involved
transesterifications between 8 and 2-phenylethanol, benzyl
alcohol and 1-naphthalenemethanol as well as amidation
reactions between 8 and 2-phenylethylamine, benzyl amine and
1-naphthylmethylamine. Transesterification was achieved by
using the corresponding alcohol as solvent and K3PO4 as base to
facilitate the transesterification reaction, affording compounds 9
HN
N
N
H
O
O
O
O
O
O
O
OMe
O
RO-09-4609
IC50 (pvNMT) 51 µM
26
IC50 (pvNMT) 0.60 µM
Figure 1. The 3-methyl benzofuran compound developed by Roche,
RO-09-4609b, and the structurally modified 3-methoxy benzoate derivative
26 developed by Yu et al.[11]
A co-crystallized structure of 26 and pvNMT (PDB code 4B14)
was obtained indicating key chemical interactions with nearby
residues at the active site. This co-crystallized structure showed
that the introduction of the 3-methoxy benzoate moiety
resulted in π-π interactions with a nearby phenylalanine while
the piperidine exhibited a cation-dipole interaction with a
nearby tyrosine residue as well as ion-pair interaction with a C-
terminal leucine residue. These additional interactions would
account for the improved activity. Our research aim was to
replace the 3-methyl benzofuran scaffold with various
isostructural and 6,5-fused heterocycles to gain insight into the
importance of the heterocyclic core as well as the importance of
the methyl at the C3-position with regards to efficacy. Each
heterocycle would subsequently be derivatized to specific
benzylic, phenethyl and 1-methylenenaphthyl amides and esters
as a short SAR study. Figure 2 illustrates the indole, 3-methyl
and 10 in
a
39% and 45% yield respectively. The
transesterification between 1-naphthalenemethanol and 8 to
yield the desired product 11 presented purification difficulties
and as a result was taken crude to the subsequent, Boc-
deprotection step to yield the desired product 17. Deprotection
of the piperidine ring was achieved with 4 M HCl in dioxane
providing 17 in a 23% yield over two steps.
indole,
1-methyl
benzimidazole,
benzoxazole
and
benzothiophene heterocycles intended to replace the

OH
O
OBn O
purification difficulties and were taken crude to the Boc-
deprotection step to produce compounds 32 and 34 in a 38%
and 49% yield respectively over two steps. Compounds 27 and
29 – 31 were Boc-deprotected to produce 33 and 35 – 37 in
yields ranging between 38% and 73%.
COOEt
COOEt
a
b
N3
Cl
1
2
3
4
OBn
OBn
COOEt
c
d
2 + 4
COOEt
N3
N
H
OBn
OBn
OBn
Br
5
6
a
b
COOEt
COOEt
COOEt
N
N
N
NBoc
H
H
H
6
21
22
OH
O
e
f, g
OBn
OH
COOEt
COOEt
N
H
N
c
d
e
Boc
COOEt
COOEt
7
8
N
N
Boc
Boc
23
24
+
NH2Cl-
NBoc
O
O
+
NBoc
NBoc
NH2Cl-
O
X
O
X
h, i
j
O
O
O
N
H
N
O
X
O
X
H
f, g
h
COOEt
N
N
9; X = OCH2CH2Ph
10; X = OCH2Ph
11; X = OCH2-(1-Naph)
12; X = NHCH2CH2Ph
13; X = NHCH2Ph
15; X = OCH2CH2Ph
16; X = OCH2Ph
17; X = OCH2-(1-Naph)
18; X = NHCH2CH2Ph
19; X = NHCH2Ph
N
Boc
H
H
25
26; X = OCH2CH2Ph
27; X = OCH2Ph
28; X = OCH2-(1-Naph)
29; X = NHCH2CH2Ph
30; X = NHCH2Ph
32; X = OCH2CH2Ph
33; X = OCH2Ph
34; X = OCH2-(1-Naph)
35; X = NHCH2CH2Ph
36; X = NHCH2Ph
14; X = NHCH2-(1-Naph)
20; X = NHCH2-(1-Naph)
31; X = NHCH2-(1-Naph)
37; X = NHCH2-(1-Naph)
Scheme 1. Reagents and conditions: a) BnBr, Na2CO3, DMF, 94%; b) NaN3,
acetone, H2O, 85%; c) NaOEt, EtOH, ethyl trifluoroacetate, -10 °C – r.t., 55%;
d) 1,3-dichlorobenzene, 160 °C, 90%; e) 10% Pd/C, H2, MeOH, 90%; f) PPh3,
DBAD, THF, 1-Boc-4-hydroxypiperidine, partial product purification; g)
Boc2O, Et3N, DCM, 95% over two steps; h) aromatic alcohol (30 eq), K3PO4,
80 °C; i) aromatic amine (10 eq), DBU, ACN, 140 °C; j) 4 M HCl in dioxane,
DCM, 0 °C
Scheme 2. Reagents and conditions: a) NBS, DMF, 0 °C, 80%; b) Pd(PPh3)4,
TMB, K2CO3, Dioxane, 110 °C, 87%; c) Boc2O, Et3N, DCM, 99%; d) 10% Pd/C,
H2, EtOH, 36%; e) PPh3, DBAD, THF, 1-Boc-4-hydroxypiperidine, 94%; f)
aromatic alcohol (30 eq), K3PO4, 80 °C; g) aromatic amine (30 eq), 140 °C; h)
4 M HCl in dioxane, DCM, 0 °C
The synthesis of the benzimidazole scaffold is illustrated in
Scheme 3. 2-Amino-3-nitrophenol 38 was benzylated to produce
39 in a 98% yield, this was followed by methylation of the amine
to produce 40 in a 97% yield. The reduction of the nitro group
For the amidation reactions, 8 and the corresponding aromatic
amine were taken up in acetonitrile in the presence of DBU and
the reaction was heated to 140 °C for 10 – 20 hours, affording
12 – 14 in a 53 – 64% yield. In both transesterification and
amidation steps, the Boc-group at the indole N1-position were
thermally labile and were subsequently cleaved. Compounds 15,
16 and 18 – 20 were obtained after Boc-deprotection of the 4-
piperidinyloxy group using 4 M HCl in dioxane. The yields
obtained for this step ranged between 79 – 97%. In Scheme 2
the synthesis of the 3-methyl indole scaffold is shown. 6 was
regioselectively brominated with NBS in DMF to produce 21 in
an 80% yield. A Suzuki-coupling of 21 and trimethylboroxine
(TMB) produced the desired 3-methyl indole scaffold 22 in an
87% yield. Due to the purification difficulties faced for the
Mitsunobu-coupling between 7 and 4-hydroxy N-Boc piperidine
(Scheme 1), we pre-emptively decided to Boc-protect the 3-
methyl indole nitrogen prior to the Mitsunobu-coupling
affording 23 in a 99% yield. Subsequent debenzylation produced
the 4-hydroxy indole 24 in a 36% yield. The low yield could be
attributed to hydrogenation of the indole aryl ring, leading to
produced phenylene diamine 41 in
a 72% yield, which
subsequently underwent cyclisation with trimethylorthoformate
under neat conditions to produce the benzimidazole precursor
42 in a 97% yield. The C2-position of 42 was formylated using n-
BuLi and DMF in a Bouveault type aldehyde synthesis[13] to
produce aldehyde 43 in a 76% yield. Oxidative esterification of
the aldehyde to the methyl ester yielded ester 44 in a 79% yield.
Subsequent debenzylation to produce 45 and coupling with 4-
hydroxy N-Boc piperidine produced 46 in
a 76% yield.
Transesterification of the carbomethoxy proved difficult at first
as product degradation was observed when treated with the
desired alcohol and K3PO4 at elevated temperatures. However, it
was noticed that the ester was significantly more electrophilic
than the indole counterpart and transesterification of 46
proceeded at room temperature providing 47 – 49 in yields
ranging between 50% and 57%. Amidation also proceeded at
room temperature affording compounds 50 – 52 in yields
between 81% and 92%. Boc-deprotection of compounds 47 – 52
afforded the benzimidazole salts 53 – 58 in yields ranging
between 83% – 95%. Scheme 5 illustrates the synthesis of the
benzoxazole scaffold. The syntheses of oxazoles and
benzoxazoles by way of an intramolecular cyclodehydration of 2-
the conversion of 23 to
a 4-indolone by-product. The
Mitsunobu-coupling between 24 and 4-hydroxy N-Boc piperidine
afforded 25 in a 94% yield. Transesterification between 25 and
benzyl alcohol yielded compound 27 in
a 38% yield.
Transesterifications yielding compounds 26 and 28 presented

hydroxyanilide derivatives under Mitsunobu reaction conditions
have been well documented in the literature.14,15
yielding compound 77. The substitution/ condensation reaction
between 77 and ethyl mercaptoacetate in the presence of K2CO3
in DMF produced the benzothiophene heterocycle 78 in a 95%
yield over 2 days.
OH
OBn
OBn
NH2
NO2
NH2
NO2
NH
a
b
NBoc
NO2
OH
OH
O
40
39
38
NH2
OH
a
N
O
N
O
b
COOEt
COOEt
OBn
OBn
OBn
N
NH
N
N
59
60
62
e
d
c
CHO
N
+
NBoc
NH2Cl-
NH2
43
42
41
O
O
OH
OBn
O
X
O
X
N
O
N
O
c, d
e
N
N
N
N
h
g
f
COOMe
COOMe
63; X = OCH2CH2Ph
64; X = OCH2Ph
69; X = OCH2CH2Ph
70; X = OCH2Ph
44
45
65; X = OCH2-(1-Naph)
71; X = OCH2-(1-Naph)
72
+
NBoc
NH2Cl-
NBoc
66
; X = NHCH2CH2Ph
; X = NHCH2CH2Ph
67; X = NHCH2Ph
68; X = NHCH2-(1-Naph)
73; X = NHCH2Ph
74; X = NHCH2-(1-Naph)
O
O
O
O
O
X
N
N
N
N
N
N
k
i, j
COOMe
O
X
O
N
N
N
N
46
O
47; X = OCH2CH2Ph
48; X = OCH2Ph
53; X = OCH2CH2Ph
54; X = OCH2Ph
O
ADDM
61
49; X = OCH2-(1-Naph)
50; X = NHCH2CH2Ph
51; X = NHCH2Ph
55; X = OCH2-(1-Naph)
56; X = NHCH2CH2Ph
57; X = NHCH2Ph
Scheme 5. Reagents and conditions: a) ClCOCO2Et, Et3N, THF, then PPh3,
DEAD, THF, 84%; b) ADDM, THF, DCM, PPh3, 79%; c) aromatic alcohol (20 –
30 eq), K3PO4, r.t; d) aromatic amine (10 eq), THF, r.t; e) 4 M HCl in dioxane,
DCM, 0 °C
52; X = NHCH2-(1-Naph)
58; X = NHCH2-(1-Naph)
Scheme 3. Reagents and conditions: a) BnBr, K2CO3, DMF, 0 °C, 98%; b) NaH,
MeI, THF, 0 °C, 97%, c) Fe, AcOH, EtOH, H2O, sonication, 72%; d)
trimethylorthoformate, pTsOH, 100 °C, 97%; e) nBuLi, THF, -78 °C then DMF,
-78 – 0 °C, 76%; f) MeOH, KOH, I2, 0 °C – r.t, 79%; g) 10% Pd/C, H2, MeOH,
92%; h) PPh3, DBAD, THF, 1-Boc-4-hydroxypiperidine, 76%; i) aromatic
alcohol (20 – 30 eq), K3PO4, r.t; j) aromatic amine (10 eq), r.t; k) 4 M HCl in
dioxane, DCM, 0 °C
Subsequent debenzylation produced 79 and a Mitsunobu
coupling between 79 and 4-hydroxy N-Boc piperidine produced
the coupled product 80 in a 99% yield. Transesterification was
achieved at 70 °C producing 81 – 83 with yields between 51%
and 88%. Amidation of 80 was produced 84 – 86 in yields
between 50% – 77%, and final Boc-deprotection afforded
compounds 87 – 92 in yields between 87% – 98%
With this in mind we envisaged synthesizing the desired
benzoxazole intermediate with the ester group at the C2-
position already installed using a one-pot conversion of 2-
aminoresorcinol 59 to the substituted benzoxazole. 59 was
chemoselectively acylated with ethyl chloro oxoacetate in THF.
The reaction was monitored by TLC to determine that the
resorcinol had been completely acylated after which dilution of
the reaction mixture with THF and the addition of PPh3 and
DEAD lead to the formation of the cyclo-dehydrated product 60
in an 84% yield. The Mitsunobu coupling between 60 and 4-
hydroxy N-Boc piperidine in the presence of DBAD as the
Mitsunobu reagent, resulted in purification difficulties. This was
overcome through the use of azodicarbonyl dimorpholide
(ADDM) as the Mitsunobu reagent. Transesterification and
amidation of 62 proved to be similar in nature to that of 46
whereby the transformation was achieved at room temperature.
63 – 65 were isolated in yields ranging between 24% and 53%,
whereas 66 – 68 ranged between 86% – 96%. Boc-deprotection
of 63 – 68 afforded compounds 69 – 74 in yields ranging
between 69% – 97%. Scheme 4 illustrates the synthesis of the
benzothiophene scaffold. 3-Nitrophenol 75 was formylated
through a Duff reaction[14] using TFA and HMTA to produce
aldehyde 76 in a 29% yield and was subsequently benzylated
OH
OH
O
OBn
OBn O
a
b
c
COOEt
S
NO2
NO2
NO2
75
76
77
78
NBoc
OH
O
e
d
f, g
COOEt
COOEt
S
S
79
80
+
NBoc
NH2Cl-
O
O
O
O
X
h
S
S
X
81; X = OCH2CH2Ph
82; X = OCH2Ph
87; X = OCH2CH2Ph
88; X = OCH2Ph
83; X = OCH2-(1-Naph)
84; X = NHCH2CH2Ph
85; X = NHCH2Ph
89; X = OCH2-(1-Naph)
90; X = NHCH2CH2Ph
91; X = NHCH2Ph
86; X = NHCH2-(1-Naph)
92; X = NHCH2-(1-Naph)
Scheme 6 Reagents and conditions: a) HMTA, TFA, 90 °C, 29%; b) BnBr,
K2CO3, DMF, quant.; c) ethyl mercaptoacetate, K2CO3, DMF, 50 °C, 95%; d)
10% Pd/C, H2, MeOH, 68%; e) PPh3, DBAD, THF, 1-Boc-4-hydroxypiperidine,
76%; f) aromatic alcohol (26 eq), K3PO4, 70 °C; g) aromatic amine (20 eq),
145 °C; h) 4 M HCl in dioxane, DCM, 0 °C.

3. Results and discussion
All benzoxazole compounds 69 – 73 apart from 74, were inactive
against P. falciparum NF54, which eliminated this heterocyclic
core as a viable P. falciparum antiplasmodial series. When we
compared the activity of the 3-methyl indole series with the
indole series, no trend could be observed to indicate that one
series was more active than the other. This indicated that the
methyl at the C3-position in the 3-methyl indole series, which is
isostructural to the proof of concept 3-methyl benzofuran
scaffold, is not crucial for better whole-cell activity. Comparison
of the benzothiophene and indole series proved interesting, as
each aromatic ester and amide had comparable activity. This
indicated that the heterocyclic scaffold has no significant effect
on the activity of each compound and that activity is related to
the aromatic ester/amide at the C2-position of each heterocycle.
The isostructural benzimidazole series in relation to the 3-
methyl indole series proved to be less active. The esters 53 – 55
were significantly less active than the esters 32 – 34. The
decreased activity of amides 56 – 58 relative to the analogous
amides 35 – 37 were minor. No significant difference in efficacy
was observed between the activity of the esters and the
analogous amides in the 3-methyl indole and benzimidazole
series with respect to the function of the methyl-group and the
effect it would have on potency. However, this assumption
could only be verified with a pfNMT assay to determine the
significance of the methyl group of both 3-methyl indole and
benzimidazole compounds, as the stability of the benzimidazole
and 3-methyl indole compounds might be compromised in vitro.
The most notable trend is the effect of the aromatic ester and
amide substituents on the efficacy of each compound.
Compounds containing the naphthylmethyl ester and amide
substituent were significantly more effective than the other
aromatic ester and amides, (except 55), whereby 74 also
indicated a low EC50 of 2.0 µM. The naphthylmethyl amides 20,
37, 58, and 92 indicated that a naphthyl methyl amide was in
fact the most preferred substituent for high potency, with a
narrow range of activities ranging between 0.71 – 1.1 µM,
reiterating the statement that the function of the heterocycle is
not as important regarding efficacy as the C2-substituent would
be.
The compounds of each heterocyclic series were tested
against the chloroquine sensitive (CQS) NF54 strain of P.
falciparum. The EC50 values corresponding to each compound
are summarized in Table 1. We began with the whole cell assay
rather than the enzymic assay for the simple reason that a
compound which has high efficacy against the enzyme but has
little impact on the whole cell is not a useful antiplasmodial
agent. Thus the standards chloroquine and artemisinin were
chosen these are well known and allow relatively easy
interpretation of the whole cell data even thought neither is
structurally equivalent to the fused heterocycles we synthesised.
Table 1: In vitro activity against P. falciparum (NF54)
Heterocyclic Series
Compound
NF54 EC50 (µM)
15
16
17
18
19
20
32
33
34
35
36
37
53
54
55
56
57
58
69
70
71
72
6.53 ± 0.12
3.39 ± 0.46
0.83 ± 0.12
3.1 ± 0.11
3.74 ± 0.87
0.90 ± 0.08
3.90 ± 0.41
0.56 ± 0.08
0.74 ± 0.02
3.4 ± 0.26
6.11 ± 0.19
0.74 ± 0.05
11.10 ± 0.21
13.28 ± 2.98
7.37 ± 0.83
8.69 ± 0.79
8.80 ± 1.06
1.15 ± 0.09
n.a
+
Cl-H2N
O
O
R
N
H
+
Cl-H2N
O
O
R
N
H
+
Cl-H2N
O
O
R
N
N
+
Cl-H2N
n.a
n.a
O
From these results we can conclude that the structure of the
heterocycle did have some moderate influence on activity.
There are several avenues which would usefully be explored in
the future. Firstly, to carry out the efficacy tests on the pfNMT
enzyme. A correlation between the whole cell assay and the
enzymic assay would suggest that our intended target is in fact
responsible for the whole cell efficacy. These results with
molecular modelling in the active site of the enzyme may shed
some light on the moderate variation in efficacy observed for
the heterocyclic cores themselves. Secondly, the lack of activity
of the benzoxazole may be caused by an unexpected instability
of the compound under the experiment conditions, or the
compound may not be reaching the required site of action. Both
of these possibilities can be explored. Finally, the indoles do
appear on average to have the most potent activity. This core
O
R
N
O
n.a
73
74
87
n.a
1.96 ± 0.18
5.87 ± 0.17
+
88
5.69 ± 0.22
1.90 ± 0.09
Cl-
HN
89
O
O
R
90
91
3.01 ± 0.06
3.48 ± 0.26
S
92
0.71 ± 0.02
Chloroquine
Artesunate
13.0 ± 0.67 nM
9.41 ± 0.72 nM

could be taken forward to form new variants including
substitution at different positions. It would also be useful to
compare the efficacy of these compounds with those
compounds already reported.
reaction was monitored until complete consumption of starting
material was observed on TLC. The mixture was diluted with
EtOAc (150 mL) and washed with water (4 x 50 mL), and then
washed with brine. The organic phase was dried over MgSO4,
filtered and reduced in vacuo. The product was crystallized with
EtOAc:hexane to provide the product as a white solid. 11.8 g,
88% yield; 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 5.21 (s, 2
H), 7.07 (d, J=8.2 Hz, 2 H), 7.34 – 7.48 (m, 5 H), 7.1 – 7.58 (m, 1
H), 7.88 (dd, J=8.0, 1.8 Hz, 1 H), 10.58 (s, 1 H); HRMS–ESI+: m/z
[M+H]+ calculated for C14H13O2: 213.0916; found: 213.0920.
Characterization data corresponded with literature values. [17] [18]
A data in brief file has been prepared for all compounds.
Reported here is the synthesis of one compound in each series.
4. ExperimentalChemistry
4.1.1
General information regarding to synthesis and
characterization
All chemicals used were bought from Merck, Fluka and
Aldrich. Tetrahydrofuran (THF) and diethylether (Et2O) were
dried over sodium wire/sand and distilled under nitrogen with
benzophenone as an indicator. Dichloromethane (DCM) was
distilled over calcium hydride under nitrogen. Other solvents
e.g. ethyl acetate, hexane and triethylamine were purified
according to standard procedures.[15] The molarity of n-BuLi
4.1.2.2 Ethyl 2-azidoacetate (4):
To a mixture of acetone (75 mL) and water (25 mL) was added
sodium azide (8.1 g, 124.3 mmol) and ethyl 2-chloroacetate (2)
(7 mL, 65.4 mmol) and stirred overnight at room temperature.
The heterogeneous mixture was diluted with water (70 mL) and
washed with EtOAc (3 x 30 mL). The organic fractions were
combined, dried over MgSO4 and the solvent removed in vacuo
was determined using
a method as described in the
1
to afford a light yellow liquid. 7.21 g, 85% yield; H NMR (400
literature.[16] Reactions requiring anhydrous conditions were
performed under an atmosphere of nitrogen or argon. All 1H and
13C nuclear magnetic resonance spectra were obtained using a
300 MHz Varian VNMRS (75 MHz for 13C), a 400 MHz Varian
Unity Inova (100 MHz for 13C) or a 600MHz Varian Unity Inova
(150 MHz for 13C). d-Chloroform was used as standard solvent.
Chemical shifts (δ) were recorded using residual chloroform
MHz, CHLOROFORM-d) δ ppm 1.26 (t, J=7.0 Hz, 3 H), 3.81 (s, 2
H), 4.20 (q, J=7.0 Hz, 2 H); HRMS–ESI+: m/z [M+H]+ calculated for
C4H8N3O2: 130.0572; found: 130.0512. Characterization data
corresponded with literature values.[19]
4.1.2.3 (Z)-Ethyl 2-azido-3-(2-(benzyloxy)phenyl)acrylate (5):
To EtOH (60 mL, freshly distilled from Mg/I2) was added 1.30 g
sodium metal (divided in small pieces) under an atmosphere of
nitrogen. While stirring, the temperature was kept below 10 °C
until the complete dissolution of the sodium metal. 2-
(Benzyloxy)benzaldehyde (2) (6.00 g, 28.3 mmol) was added to
the solution and ethyl trifluoroacetate (6.7 mL, 56.5 mmol) was
added via a syringe. The solution was cooled to -10 °C, and ethyl
azidoacetate (4) (7.00 g, 56.5 mmol) was added dropwise over a
period of 5 minutes. The yellow heterogeneous solution started
to become more turbid during the addition of the ethyl
azidoacetate. The reaction was stirred at -10 °C for 2 hours while
the heterogeneous solution becomes slurry like. The precipitate
was filtered with the aid of a vacuum pump and dried under
vacuum to afford the desired compound in analytically pure
form. 5.03 g, 55% yield.1H NMR (300 MHz, CHLOROFORM-d) δ
ppm 1.39 (t, J=7.1 Hz, 3 H), 4.36 (q, J=7.1 Hz, 2 H), 5.15 (s, 2 H),
6.96 (d, J=8.4 Hz, 1 H), 7.03 (t, J=7.3 Hz, 1 H), 7.28 – 7.49 (m, 6
H), 7.54 (s, 1 H), 8.24 (dd, J=7.8, 1.6 Hz, 1 H); 13C NMR (101 MHz,
CHLOROFORM-d) δ ppm 14.4, 62.3, 70.6, 112.5, 119.6, 121.0,
122.9, 125.5, 127.1 (2 C), 128.1, 128.8 (2 C), 130.8, 130.9, 137.0,
157.0, 164.0; HRMS–ESI+: m/z [M+H]+ calculated for C18H18N3O3:
324.1303; found: 324.1348. IR ATR (cm-1): 2118, 1684, 1485,
1242.
1
peaks at δ 7.26 in H NMR and δ 77.0 in 13C NMR, and residual
1
DMSO peaks at δ 2.50 in H NMR and δ 39.51 in 13C NMR. All
chemical shifts are reported in ppm and coupling constants, J,
are given in Hertz (Hz). All spectra were obtained at 25 °C. All
NMR spectroscopy and mass spectrometry was performed by
the CAF (Central Analytical Facility) Institute at Stellenbosch
University. Infrared spectra were obtained using a Nexus
Thermo-Nicolet FT-IR using the ATR. LC-MS purity data were
obtained at CAF using a Waters Synapt G2 instrument, ESI
positive, using a diode array as detection method. Compounds
were detected at a wavelength of 280 nm. All chromatography
was performed using either (or
a
combination of)
ethyl acetate, methanol, ethanol, triethylamine, acetone and
dichloromethane. Thin layer chromatography (TLC) was carried
out on aluminium backed Merck silica gel 60 F254 plates.
Visualization was achieved with a UV lamp, iodine vapour or by
spraying with a Cerium Ammonium Molybdate solution (CAM),
p-anisaldehyde or a ninhydrin solution and then heating. All
column chromatography was carried out with Merck silica gel 60
(particle size 0.040-0.063 mm).
4.1.2
Synthesis of compounds (15) – (20)
4.1.2.1 2-(benzyloxy)benzaldehyde (2):
4.1.2.4 Ethyl 4-(benzyloxy)-1H-indole-2-carboxylate (6):
To a solution of salicylaldehyde (1) (7.71 g, 63.2 mmol), Na2CO3
(9.38 g, 88.5 mmol) in DMF (50 mL) was added 10.5 mL (88.5
mmol) benzyl bromide dropwise at 0 °C under an atmosphere of
nitrogen. The mixture was stirred at room temperature for 2
hours, where the initial yellow solution turned milky white. The
1,3-dichlorobenzene (7 mL) was heated to 160 °C to which (5)
(700 mg, dissolved in 1 mL 1,3-dichlorobenzene) was added
dropwise under an atmosphere of nitrogen. The slow addition of
(5) resulted in the evolution of gas from the heated solution.

The reaction mixture was stirred for an additional 5 minutes and
cooled to room temperature, where the desired product
crystallized out of solution. The product was filtered off, and the
remaining product dissolved in the mother liquor was purified
by column chromatography over silica gel (20% EtOAc, 80%
hexane) to afford the product as a white solid. Combined yield
of 556 mg, 87%, Rf: 0.39 (15% EtOAc, 85% hexane); 1H NMR (400
MHz, CHLOROFORM-d) δ ppm 1.42 (t, J=7.1 Hz, 3 H), 4.41 (q,
J=7.1 Hz, 2 H), 5.23 (s, 2 H), 6.60 (d, J=7.8 Hz, 1 H), 7.04 (d,
J=8.6Hz, 1 H), 7.19 – 7.24 (m, 1 H), 7.33 – 7.48 (m, 4 H), 7.52 (d,
J=8.2 Hz, 2 H), 8.95 (br. s., 1 H); 13C NMR (CHLOROFORM-d) δ
ppm 14.4, 60.9, 69.9, 101.1, 105.0, 106.5, 119.3, 126.2, 126.3,
127.4, 127.9, 128.5, 137.1, 138.2, 153.7, 162.0; HRMS–ESI+: m/z
[M+H]+ calculated for C18H18NO3: 296.1287; found: 296.1283.
Mp: 160 – 162 °C; IR ATR (cm-1): 3321, 1682, 1620, 1242, 1195.
Characterization data corresponded with literature values.[20]
95%. Rf: 0.39 (15% EtOAc, 85% hexane) 1H NMR (300 MHz,
CHLOROFORM-d) δ ppm 1.39 (t, J=7.2 Hz, 3 H), 1.48 (s, 9 H), 1.61
(s, 9 H), 1.77 – 1.89 (m, 2 H), 1.89 – 2.00 (m, 2 H), 3.38 – 3.49 (m,
2 H), 3.63 – 3.74 (m, 1 H), 4.37 (q, J=7.2 Hz, 2 H), 4.59 – 4.68 (m,
1 H), 6.67 (d, J=7.9 Hz, 1 H), 7.24 (d, J=0.7 Hz, 1 H), 7.26 – 7.33
(m, 1 H), 7.65 (d, J=8.5 Hz, 1 H); 13C NMR (75 MHz,
CHLOROFORM-d) δ ppm 14.2, 27.7 (s, 3 C), 28.4 (s, 3 C), 30.3 (s,
2 C), 40.3, 61.2, 72.2, 79.5, 84.4, 105.8, 107.7, 112.2, 119.3,
127.7, 129.3, 139.4, 149.2, 151.3, 154.7, 161.6; HRMS–ESI+: m/z
[M+H]+ calculated for C26H37N2O7: 489.2601; found: 489.2589;
Mp: 85 – 89 °C; IR ATR (cm-1): 2974, 1743, 1728, 1689, 1364,
1278, 1145, 1035.
Compounds (9) and (10) were synthesized from (8) via the same
transesterification procedure. Compound (8), K3PO4 (3
equivalents) and the alcohol used for transesterification (20 – 30
equivalents) were placed in a vial, purged with argon, sealed and
heated to 80 °C for 10 – 20 hours. The reaction was monitored
via TLC using either (15% EtOAc, 85% hexane) or (5% EtOAc, 95%
DCM) as mobile phase to determine whether the starting
material had been consumed. The K3PO4 was filtered off, and the
alcohol removed via heating the solution to 50 °C and streaming
compressed air over the solution. After the majority of the
solvent volume has been evaporated off, the product was
purified by column chromatography over silica gel (5% EtOAc,
95% DCM).
4.1.2.5 Ethyl 4-hydroxy-1H-indole-2-carboxylate (7):
(6) (700 mg, 3.41 mmol) and 70 mg 10% Pd/C was added to
MeOH (12 mL) and placed under an atmosphere of H2 via a
balloon. The solution was stirred overnight, after which the
solution was filtered through Celite® and the solvent reduced in
vacuo. The compound was purified by column chromatography
over silica gel (50% EtOAc, 50% hexane) to afford the product as
a white solid. 87%, 423.1 mg. Rf: 0.23 (20% EtOAc, 80% hexane);
1H NMR (300 MHz, CHLOROFORM-d) δ ppm 1.43 (t, J=7.0 Hz, 3
H), 4.42 (q, J=7.0 Hz, 2 H), 5.23 (s, 1 H), 6.53 (dd, J=7.6, 0.7 Hz, 1
H), 7.02 (dt, J=8.3, 0.8 Hz, 1 H), 7.18 (dd, J=8.3, 7.6 Hz, 1 H), 7.34
(dd, J=2.2, 1.0 Hz, 1 H), 8.89 (br. s., 1 H); 13C NMR (75 MHz,
CHLOROFORM-d) δ ppm 14.4, 61.1, 104.5, 104.7, 105.3, 118.0,
126.4, 138.5, 150.3, 160.5; HRMS–ESI+: m/z [M+H]+ calculated
for C11H12NO3: 206.0818; found: 206.0848; Mp: 156 – 158 °C; IR
ATR (cm-1): 3313, 1691, 1585, 1238, 1197, 1020.
Characterization data corresponded with literature values.[20]
4.1.2.7 Phenethyl 4-((1-(tert-butoxycarbonyl)piperidin-4-
yl)oxy)-1H-indole-2-carboxylate (9):
(8) (100 mg, 0.205 mmol), 2-phenylethanol (720 µL, 6.15 mmol)
and K3PO4 (150 mg, 0.701 mmol) was heated to 80 °C. White
solid, 37.2 mg, 39% yield, Rf: 0.69 (5% EtOAc, 95% DCM);
1H NMR (300 MHz, CHLOROFORM-d) δ ppm 1.54 (s, 9 H), 1.86 –
2.09 (m, 4 H), 3.15 (t, J=7.0 Hz, 2 H), 3.43 – 3.56 (m, 2 H), 3.70 –
3.81 (m, 2 H), 4.61 (t, J=7.0 Hz), 4.66 – 4.75 (m, 1 H), 6.58 (d,
J=7.6 Hz, 1 H), 7.05 (d, J=8.2 Hz, 1 H), 7.20 – 7.27 (m, 2 H), 7.28 –
4.1.2.6 1-tert-Butyl 2-ethyl 4-((1-(tert-butoxycarbonyl)
piperidin-4-yl)oxy)-1H-indole-1,2-dicarboxylate (8):
7.43 (m,
6 H), 9.09 (br. s., 1
H); 13C NMR (75 MHz,
To a solution of (7) (380 mg, 1.85 mmol), PPh3 (930 mg, 4.62
mmol) and 1-Boc-4-hydroxypiperidine (1,21 g, 4.62 mmol) in
THF (8 mL) was added DBAD (851 mg, 4.62 mmol) dissolved in 2
mL THF dropwise at 0 °C under an atmosphere of nitrogen. The
solution was stirred overnight, followed by the removal of the
solvent in vacuo. The crude product was loaded directly onto
silica gel and the intermediate product was isolated together
with di-tert-butyl hydrazine-1,2-dicarboxylate as co-eluent,
(EtOAc 15%, hexane 85% an eluent). The fractions containing
the intermediate product were collected, and the solvent
reduced in vacuo to produce a white solid. The mixture was
dissolved in DCM (12 mL) to which Et3N (0.5 mL) was added and
Boc2O (2.83 g, 12.2 mmol, dissolved in 3 mL DCM) was added
dropwise at 0 °C under an atmosphere of nitrogen. The reaction
was stirred overnight, after which the solvent was reduced in
vacuo. The compound was purified by column chromatography
over silica gel (12% EtOAc, 88% hexane) to afford the product as
a colourless semi-solid that solidified into a white solid. 859 mg,
CHLOROFORM-d) δ ppm 28.4 (3 C), 30.5 (2 C), 35.3, 40.7, 65.3,
72.0, 79.6, 102.6, 105.0, 106.5, 120.0, 126.0, 126.3, 126.6, 128.5
(2 C), 128.9 (2 C), 137.6, 138.5, 152.0, 154.8, 161.8; HRMS–ESI-:
m/z [M-H]- calculated for C27H31N2O5: 463.2233; found:
463.2231; Mp: 131 – 134 °C; IR ATR (cm-1): 3314, 2928, 1692,
1666, 1582, 1516, 1355.
4.1.2.8 Benzyl 4-((1-(tert-butoxycarbonyl)piperidin-4-yl)oxy)-
1H-indole-2-carboxylate (10):
(8) (100 mg, 0.205 mmol), benzyl alcohol (640 µL, 6.15 mmol)
and K3PO4 (150 mg, 0.701 mmol) was heated to 80 °C. White
solid, 41.3 mg, 45 % yield, Rf: 0.36 (15% EtOAc, 85% hexane); 1H
NMR (300 MHz, CHLOROFORM-d) δ ppm 1.51 (s, 9 H), 1.79 –
2.06 (m, 4 H), 3.39 – 3.52 (m, 2 H), 3.65 – 3.79 (m, 2 H), 4.63 –
4.73 (m, 2 H), 5.42 (s, 2 H), 6.55 (d, J=7.6 Hz, 1 H), 7.02 (d, J=8.2
Hz, 1 H), 7.18 – 7.25 (m, 1 H), 7.34 – 7.57 (m, 6 H), 9.21 (br. s., 1
H); 13C NMR (75 MHz, CHLOROFORM-d) δ ppm 28.4 (3 C), 30.4 (2
C), 40.7 (2 C), 66.6, 71.9, 79.5, 102.4, 104.9, 106.8, 120.0, 125.8,

126.3, 128.3 (2 C), 128.3, 128.6 (2 C), 135.7, 138.6, 152.0, 154.8,
161.8; HRMS–ESI-: m/z [M-H]- calculated for C26H29N2O5:
449.2076; found: 469.2072; Mp: 147 – 149 °C; IR ATR (cm-1):
3318, 2959, 1702, 1686, 1583, 1518, 1392.
3.01 – 3.12 (m, 2 H), 3.17 – 3.30 (m, 2 H), 4.75 – 4.83 (m, 1 H),
5.85 (s, 2 H), 6.63 (d, J=7.4 Hz, 1 H), 7.04 (d, J=8.2 Hz, 1 H), 7.13 –
7.19 (m, 2 H), 7.52 – 7.66 (m, 3 H), 7.71 (d, J=7.3 Hz, 1 H), 7.96 –
8.03 (m, 2 H), 8.14 (d, J=8.6 Hz, 1 H), 8.77 (br. s, 1 H), 8.98 (br. s,
1 H), 11.96 (s, 1 H); 13C NMR (101 MHz, DMSO-d6) δ ppm 27.1 (2
C), 40.2 (2 C), 64.2, 68.9, 102.3, 105.5, 105.9, 118.9, 123.6,
125.4, 125.8, 125.9, 126.1, 126.7, 127.3, 128.6, 129.0, 131.1,
131.6, 133.3, 139.2, 150.9, 161.0; HRMS-TOF MS ES+: m/z
[M+H]+ calculated for C25H25N2O3: 401.1865; found: 401.1867;
Purity: >99%; Mp: 211 – 214 °C; IR ATR (cm-1): 3224, 2929, 1713,
1581, 1514, 1345, 1233, 1186.
Compounds (12) – (14) were synthesized from (8) via the same
procedure. Compound (8) and the aromatic amine used for the
amidation reaction (10 equivalents) were placed in a vial, to
which was added acetonitrile (1 mL) and DBU (15 µL). The vial
was purged with argon, sealed and heated to 140 °C for 10 – 20
hours. The reaction was monitored via TLC using (50% EtOAc,
50% hexane) as mobile phase to determine whether the starting
material has been consumed. The product was purified by
column chromatography over silica gel (30% EtOAc, 70% hexane
to 100% EtOAc).
4.1.3
Synthesis of compounds (32) – (37)
4.1.3.1 Ethyl 4-(benzyloxy)-3-bromo-1H-indole-2-carboxylate
(21):
To a solution of (6) (1.14 g, 3.87 mmol) in DMF (15 mL) was
added NBS (178 mg, 4.25 mmol, dissolved in 2 mL DMF)
dropwise and stirred for 1 hour at 0 °C. The reaction mixture
was diluted with EtOAc (100 mL), and washed with water (4 x
100 mL), followed by a wash with brine. The organic layer was
dried over MgSO4 and reduced in vacuo. The compound was
purified by column chromatography over silica gel (25% EtOAc,
75% hexane) to afford the product as a white solid. 1.17 g, 80%
yield. Rf: 0.44 (25% EtOAc, 75% hexane); 1H NMR (300 MHz,
CHLOROFORM-d) δppm 1.46 (t, J=7.04 Hz, 3 H), 4.45 (q, J=7.19
Hz, 2 H), 5.26 (s, 2 H), 6.61 (dd, J=7.78, 0.6 Hz, 1 H), 7.01 (dd,
J=8.36, 0.7 Hz, 1 H), 7.24 (dd, J=8.36, 7.78 Hz, 1 H), 7.30 – 7.45
(m, 3 H), 7.57 – 7.64 (m, 2 H), 8.98 (br. s., 1 H) 13C NMR (75 MHz,
CHLOROFORM-d) δ ppm 14.4, 61.4, 70.2, 95.9, 102.2, 105.2,
118.0, 123.3, 127.1 (2 C), 127.2, 127.7, 128.4 (2 C), 136.9, 137.2,
154.2, 161.1; HRMS–ESI+: m/z [M+H]+ calculated for
C18H17NO3Br: 374.0392; found: 374.0398; Mp: 164 – 166 °C; IR
ATR (cm-1): 3307, 1676, 1576, 1507, 1249, 1199, 1099.
Compounds (15) – (20) with the exception of (17) were
synthesized from (9) – (14) via the same procedure. In a small
flask was added the Boc-protected compound and DCM (1 mL)
followed by the addition of 4M HCl/dioxane (1 mL). The solution
was stirred for one to two hours, after which the solvent was
reduced in vacuo. A small amount of DCM was added followed
by the addition of EtOAc which caused the salt to precipitate out
of solution. The solvent was reduced in vacuo and the product
dried under vacuum.
4.1.2.9 4-((2-(Phenethoxycarbonyl)-1H-indol-4-yl)oxy)piperidin-
1-ium chloride (15):
25.5 mg, 79% yield, white powder. 1H NMR (400 MHz, DMSO-d6)
δ ppm 1.91 – 2.02 (m, 2 H), 2.12 – 2.21 (m, 2 H), 3.05 (t, J=6.8
Hz, 2 H), 3.07 – 3.15 (m, 2 H), 3.21 – 3.30 (m, 2 H), 4.48 (t, J=6.8
Hz, 2 H), 4.78 – 4.85 (m, 1 H), 6.65 (d, J=7.8 Hz, 1 H), 7.05 (d,
J=8.2 Hz, 1 H), 7.13 (d, J=1.2 Hz, 1 H), 7.14 – 7.20 (m, 1 H), 7.19 –
7.37 (m, 5 H), 9.24 (br. s., 2 H), 11.92 (s, 1 H); 13C NMR (101 MHz,
DMSO-d6) δ ppm 27.0 (2 C), 34.5, 40.2 (2 C), 64.9, 68.8, 102.3,
105.1, 105.9, 118.9, 125.7, 126.0, 126.4, 128.4 (2 C), 128.9 (2 C),
137.9, 139.0, 150.8, 161.0; HRMS-TOF MS ES+: m/z [M+H]+
calculated for C22H25N2O3: 365.1865; found: 365.1859; Purity:
>99%; Mp: 215 – 217 °C; IR ATR (cm-1): 3191, 2927, 1712, 1581,
1526, 1349, 1240, 1189.
4.1.3.2 Ethyl 4-(benzyloxy)-3-methyl-1H-indole-2-carboxylate
(22):
In a flask containing (21) (1.00g, 4.74 mmol), K2CO3 (1.97 g, 14.2
mmol), Pd(PPh3)4 (548 mg, 0.474 mmol) and trimethylboroxine
(729 µL, 5.21 mmol), was added 24 mL dioxane under an
atmosphere of nitrogen and stirred at 110 °C for 6 h followed by
stirring at room temperature overnight. The reaction mixture
was filtered through a plug of Celite® and the solvent reduced in
vacuo. The compound was purified by column chromatography
over silica gel (15% EtOAc, 85% hexane) to afford the product as
a white solid. 725 mg, 87%. Rf: 0.37 (15% EtOAc, 85% hexane);
1H NMR (300 MHz, CHLOROFORM-d) δ ppm 1.43 (t, J=7.0 Hz, 3
H), 2.84 (s, 3 H), 4.42 (q, J=7.0 Hz, 2 H), 5.22 (s, 2 H), 6.54 (d,
J=7.8 Hz, 1 H), 6.97 (dd, J=8.3, 0.7 Hz, 1 H), 7.16 – 7.22 (m, 1 H),
7.33 – 7.46 (m, 3 H), 7.50 – 7.56 (m, 2 H), 8.63 (br. s., 1 H); 13C
NMR (75 MHz, CHLOROFORM-d) δ ppm 12.4, 14.5, 60.6, 69.9,
100.6, 104.9, 119.0, 121.5, 122.3, 126.4, 127.3 (2 C), 127.8,
128.6 (2 C), 137.2, 137.7, 155.7, 162.7; HRMS–ESI+: m/z [M+H]+
calculated for C19H20NO3: 310.1443; found: 310.1445; Mp: 149 –
150 °C; IR ATR (cm-1): 3323 2916, 1661, 1351, 1246, 1198, 1099.
4.1.2.10 4-((2-((Naphthalen-1-ylmethoxy)carbonyl)-1H-indol-4-
yl)oxy)piperidin-1-ium chloride (17):
(8) (100 mg, 0.205 mmol), K3PO4 (150 mg, 0.742 mmol) and
naphthalen-1-ylmethanol (158 mg, 1.00 mmol) was added to a
vial and the mixture stirred at 100 °C overnight where
compound (11) could be observed by way of TLC. The mixture
was dissolved in DCM (3 mL) and the K3PO4 filtered off. The
solvent was reduced in vacuo and the crude product dissolved in
DCM (1 mL) to which 4 M HCl/dioxane (1 mL) was added and
stirred for 2 hours. The solvent was reduced in vacuo and the
product was purified by column chromatography over silica gel
(100% DCM to 10% MeOH, 90% DCM) to afford the product as a
1
white solid. 20.4 mg, 23% yield over two steps. H NMR (400
MHz, DMSO-d6) δ ppm 1.83 – 1.98 (m, 2 H), 2.06 – 2.18 (m, 2 H),

4.1.3.3 1-tert-Butyl 2-ethyl 4-(benzyloxy)-3-methyl-1H-indole-
1,2-dicarboxylate (23):
4.1.3.6 Benzyl 4-((1-(tert-butoxycarbonyl)piperidin-4-yl)oxy)-3-
methyl-1H-indole-2-carboxylate (33):
To a solution of (22) (826 mg, 2.67 mmol) and DMAP (20 mg,
0.164 mmol) in DCM (10 mL) was added Boc2O (680 mg, 3.20
mmol, dissolved in 2 mL DCM) dropwise and stirred for 3 hours.
The solvent was reduced in vacuo and the product purified by
column chromatography over silica gel (10% EtOAc, 90%
hexane) to afford the product as a white solid. 1.09 g, 99% yield.
Rf: 0.43 (10% EtOAc, 90% hexane); 1H NMR (300 MHz,
CHLOROFORM-d) δ ppm 1.41 (t, J=7.1 Hz, 3 H), 1.64 (s, 9 H), 2.60
(s, 3 H), 4.41 (q, J=7.2 Hz, 2 H), 5.19 (s, 2 H), 6.72 (d, J=7.9 Hz, 1
H), 7.26 – 7.32 (m, 1 H), 7.32 – 7.53 (m, 5 H), 7.71 (d, J=8.4 Hz, 1
H); 13C NMR (75 MHz, CHLOROFORM-d) δ ppm 11.9, 14.2, 27.9
(3 C), 61.1, 70.0, 84.1, 104.6, 107.9, 119.0, 123.6, 125.7, 127.2 (2
C), 127.4, 127.8, 128.5 (2 C), 136.8, 138.3, 149.5, 154.6, 162.7.
(25) (80 mg, 0.159 mmol), K3PO4 (119 mg, 0.48 mmol) and
benzyl alcohol (500 µL, 4.85 mmol) were added to a vial. The vial
was sealed and the mixture stirred at 120 °C for 10 hours. The
K3PO4 was filtered off, and the alcohol removed via heating the
solution to 50 °C and streaming compressed air over the
solution. After the majority of the solvent volume had been
evaporated off, the product was purified by column
chromatography over silica gel (5% EtOAc, 95% DCM) to afford
the product as a white solid. 28 mg, 38% yield. Rf: 0.61 (5%
EtOAc, 95% DCM) (1H NMR (300 MHz, CHLOROFORM-d) δ ppm
1.53 (s, 9 H), 1.85 – 2.06 (m, 4 H), 2.85 (s, 3 H), 3.56 (s, 2 H), 3.59
– 3.70 (m, 2 H), 4.64 – 4.75 (m, 1 H), 5.40 (s, 2 H), 6.45 (d, J=8.2
Hz, 1 H), 6.90 (d, J=8.2 Hz, 1 H), 7.13 – 7.20 (m, 1 H), 7.28 – 7.52
(m, 5 H), 8.73 (br. s., 1 H); 13C NMR (75 MHz, CHLOROFORM-d) δ
ppm 12.5, 28.4 (3 C), 30.2 (2 C), 40.3 (2 C), 66.3, 71.2, 79.6,
101.1, 104.5, 119.4, 121.9, 121.9, 126.5 (2 C), 126.9, 128.3,
128.6 (2 C), 135.9, 137.9, 154.0, 154.8, 162.2; HRMS-TOF MS
ES+: m/z [M+Na]+ calculated for C27H32N2O5Na: 488.2287; found:
488.2231; Mp: 121 – 124 °C, IR ATR (cm-1): 3337, 2928, 1668,
1580, 1511, 1422, 1248, 1229, 1090.
4.1.3.4 Ethyl 4-hydroxy-3-methyl-1H-indole-2-carboxylate (24):
(23) (1.09 g, 2.66 mmol) and Pd/C (10%, 245 mg, 0229 mmol)
was placed in a flask, evacuated and purged with hydrogen from
a balloon, to which was added EtOH (10 mL) and stirred
overnight at room temperature under an atmosphere of
hydrogen. The mixture was filtered through a plug of Celite® and
the solvent reduced in vacuo. The product was purified by
column chromatography over silica gel (30% EtOAc, 70%
hexane) to afford the product as a white solid. 296 mg, 36%
yield. Rf: 0.50 (30% EtOAc, 70% hexane); 1H NMR (300 MHz,
CHLOROFORM-d) δ ppm 1.41 (t, J=7.2 Hz, 3 H), 1.62 (s, 9 H), 2.60
(s, 3 H), 4.43 (q, J=7.1 Hz, 2 H), 6.58 (dd, J=7.8, 0.7 Hz, 1 H), 6.73
(s, 1 H), 7.09 – 7.19 (m, 1 H), 7.58 (dd, J=8.4, 0.7 Hz, 1 H); 13C
NMR (75 MHz, CHLOROFORM-d) δ ppm 11.6, 14.2, 28.0 (3 C),
61.5, 84.3, 107.1, 108.5, 118.0, 124.4, 125.4, 127.7, 138.8, 149.7,
152.2, 163.5.
Compounds (35) – (37) were synthesized via the same
procedure. Compound (25) and the aromatic amine used for the
amidation reaction (30 equivalents) were placed in a vial. The
vial was purged with argon, sealed and heated to 140 °C for 30
hours. The reaction was monitored via TLC using (50% EtOAc,
50% hexane) as mobile phase to determine whether the starting
material has been consumed. The product was purified by
column chromatography over silica gel (30% EtOAc, 70% hexane
to 100% EtOAc).
4.1.3.5 1-tert-Butyl 2-ethyl 4-((1-(tert-
butoxycarbonyl)piperidin-4-yl)oxy)-3-methyl-1H-indole-1,2-
dicarboxylate (25):
4.1.3.7 tert-Butyl 4-((3-methyl-2-(phenethylcarbamoyl)-1H-
indol-4-yl)oxy)piperidine-1-carboxylate (35):
21 mg, 34% yield, yellow solid. Rf: 0.35 (30% EtOAc, 70%
(24) (238 mg, 0.745 mmol), PPh3 (294 mg, 1.12 mmol) and 1-
Boc-4-hydroxypiperdine were dissolved in THF (4.7 mL) after
which DBAD (258 mg, 1.12 mmol, dissolved in 1mL THF) was
dropwise at 0 °C under an atmosphere of nitrogen, and stirred
overnight. The solvent was reduced in vacuo and the product
purified by column chromatography over silica gel (15% EtOAc,
85% hexane) to afford the product as a semi solid. 352 mg, 94%
yield. Rf: 0.44 (15% EtOAc:85% hexane); 1H NMR (400 MHz,
CHLOROFORM-d) δ ppm 1.38 (t, J=7.0 Hz, 3 H), 1.45 (s, 9 H), 1.59
(s, 9 H), 1.79 – 1.90 (m, 2 H), 1.90 – 2.01 (m, 2 H), 2.55 (s, 3 H),
3.43 – 3.53 (m, 2 H), 3.55 – 3.66 (m, 2 H), 4.37 (q, J=7.3 Hz, 2 H),
4.61 – 4.69 (m, 1 H), 6.62 (d, J=8.2 Hz, 1 H), 7.19 – 7.29 (m, 1 H),
7.64 (d, J=8.2 Hz, 1 H); 13C NMR (75 MHz, CHLOROFORM-d) δ
ppm 12.0, 14.0, 27.7 (3 C), 28.2 (3 C), 30.1 (2 C), 40.1 (2 C), 60.9,
71.4, 79.3, 83.9, 105.0, 107.4, 119.3, 123.2, 125.6, 127.2, 138.4,
149.2, 152.8, 154.5, 162.5; HRMS-TOF MS ES+: m/z [M+H]+
calculated for C27H39N2O7: 503.2757; found: 503.2758; IR ATR
(cm-1): 2978, 1723, 1692, 1592, 1214, 1156, 1096, 1024.
1
hexane); H NMR (300 MHz, CHLOROFORM-d) δ ppm 1.51 (s, 9
H), 1.83 – 1.95 (m, 2 H), 1.95 – 2.07 (m, 2 H), 2.59 (s, 3 H), 2.99
(t, J=6.7 Hz, 2 H), 3.41 – 3.54 (m, 2 H), 3.62 – 3.74 (m, 2 H), 3.83
(q, J=6.5 Hz, 2 H), 4.64 – 4.72 (m, 1 H), 6.04 (t, J=5.3 Hz, 1 H),
6.46 (d, J=7.6 Hz, 1 H), 6.97 (d, J=7.6 Hz, 1 H), 7.09 – 7.16 (m, 1
H), 7.23 – 7.40 (m, 5 H), 9.29 (br. s, 1 H); 13C NMR (75 MHz,
CHLOROFORM-d) δ ppm 12.1, 28.4 (3 C), 30.4 (2 C), 35.6, 40.6 (2
C), 40.8, 71.5, 79.6, 101.2, 104.7, 112.5, 119.2, 125.1, 126.4,
126.7 (2 C), 128.5, 128.8 (2 C), 137.1, 138.7, 153.5, 154.8, 162.6;
HRMS-TOF MS ES+: m/z [M+H]+ calculated for C28H36N3O4:
478.2706; found: 478.2710; Mp: 168 – 170 °C, IR ATR (cm-1):
3253, 2929, 1681, 1627, 1545, 1422, 1259, 1098.
Compounds (33) and (35) – (37) were synthesized via the same
procedure. In a small flask was added the Boc-protected
compounds (27) and (29) – (31) separately dissolved in DCM (1
mL) followed by the addition of 4M HCl/dioxane (1 mL). The
solution was stirred for one hour, after which the solvent was
reduced in vacuo. A small amount of DCM was added followed

by the addition of EtOAc which caused the salt to precipitate out
of solution. The solvent was reduced in vacuo and the product
dried under vacuum.
4.1.4
Synthesis of compounds (53) – (58)
4.1.4.1 2-(Benzyloxy)-6-nitroaniline (39):
To a solution of 2-amino-3-nitrophenol (38) (1.00 g, 6.48 mmol)
in DMF (15mL) was added K2CO3 (1.34g, 9.72 mmol) at -10 °C,
followed by the addition of benzyl bromide (1.00 mL, 8.42
mmol) under an atmosphere of nitrogen. The mixture was
stirred at -10 °C for 8 hours to prevent N-alkylation. The mixture
was added to EtOAc (100mL) and was washed with water (4 x
100 mL) followed by a wash with brine. The organic phase was
dried over MgSO4, and the solvent reduced in vacuo. The
product was purified by column chromatography over silica gel
using a gradient elution (10% EtOAc, 90% hexane to 30% EtOAc,
70% hexane gradient) to afford a red oil. 1.55 g, 98% yield. Rf:
0.55 (20% EtOAc:80% hexane) 1H NMR (300 MHz,
CHLOROFORM-d) δ ppm 5.13 (s, 2 H), 6.51 (br. s, 2 H), 6.59 (dd,
J=8.9, 7.8 Hz, 1 H), 6.97 (dd, J=7.8, 0.9 Hz, 1 H), 7.34 – 7.51 (m, 5
4.1.3.8 4-((3-Methyl-2-(phenethoxycarbonyl)-1H-indol-4-
yl)oxy)piperidin-1-ium chloride (32):
In a vial was added (25) (80 mg, 0.159 mmol), K3PO4 (110 mg,
0.518 mmol) and 2-phenyl ethanol (400 µL, 1.00 mmol) and
stirred neat at 100 °C overnight until compound (26) was
observed by way of TLC. The mixture was dissolved in DCM (3
mL) and the K3PO4 filtered of. The solvent was reduced in vacuo
and the crude residue redissolved in DCM (1 mL) to which 4M
HCl/dioxane (1 mL) was added and stirred for 2 hours. The
solvent was reduced in vacuo and the product was purified by
column chromatography over silica gel using gradient elution
(100% DCM to 10% MeOH, 90% DCM) to afford the product as a
1
white solid. 12.3 mg, 19% yield over two steps. H NMR (400
MHz, DMSO-d6) δ ppm 1.89 – 2.02 (m, 2 H), 2.10 – 2.23 (m, 2 H),
2.67 (s, 3 H), 3.06 (t, J=6.8 Hz, 2 H), 3.08 – 3.24 (m, 4 H), 4.50 (t,
J=6.8 Hz, 2 H), 4.76 – 4.84 (m, 1 H), 6.55 (d, J=7.8 Hz, 1 H), 6.97
(d, J=8.2 Hz, 1 H), 7.08 – 7.14 (m, 1 H), 7.19 – 7.38 (m, 5 H), 8.87
– 9.20 (m, 2 H), 11.42 (s, 1 H); 13C NMR (101 MHz, DMSO-d6) δ
ppm 12.1, 27.0 (2 C), 34.5, 40.3 (2 C), 64.7, 68.6, 101.1, 105.5,
118.4, 119.0, 122.0, 125.8, 126.4, 128.4 (2 C), 128.9 (2 C), 138.1,
138.3, 152.8, 161.8; HRMS-TOF MS ES+: m/z [M+H]+ calculated
for C23H27N2O3: 379.2022; found: 379.2013; Purity: 77.1%; Mp:
188 – 191 °C, IR ATR (cm-1): 3345, 2926, 1681, 1615, 1579, 1453,
1356, 1243, 1087.
H), 7.75 (dd, J=8.9, 1.2 Hz,
1
H); 13C NMR (75 MHz,
CHLOROFORM-d) δ ppm 71.1, 114.4, 114.8, 117.5, 127.6 (2 C),
128.4, 128.7 (2 C), 131.6, 135.7, 137.1, 147.1. Characterization
data corresponded with literature values.[21]
4.1.4.2 2-(Benzyloxy)-N-methyl-6-nitroaniline (40):
To a mixture of NaH (144 mg, 60% dispersion in oil, 3.59 mmol)
in THF (14 mL) was added (39) (800 mg, 3.27 mmol, dissolved in
2 mL THF) dropwise at -10 °C under an atmosphere of nitrogen.
The mixture was stirred for 30 minutes to which MeI (224 µL,
3.59 mmol) was added. The mixture was allowed to reach room
temperature and left stirring overnight. The mixture was
quenched with water (10 mL) and the product was extracted
with EtOAc (2 x 60 mL). The organic phase was washed with
brine, dried over MgSO4 and the solvent was removed in vacuo.
The product was purified by column chromatography over silica
gel (15% EtOAc, 85% hexane) to afford a red solid. 820 mg, 97%
yield. Rf: 0.59 (15% EtOAc, 85% hexane) 1H NMR (300 MHz,
CHLOROFORM-d) δ ppm 3.15 (s, 3 H), 5.07 (s, 2 H), 6.59 (dd,
J=8.7, 7.8 Hz, 1 H), 6.99 (dd, J=7.8, 1.4 Hz, 1 H), 7.35 – 7.49 (m, 5
4.1.3.9 4-((3-Methyl-2-((naphthalen-1-ylmethoxy)carbonyl)-
1H-indol-4-yl)oxy)piperidin-1-ium chloride (34):
In a vial was added (25) (57 mg, 0.114 mmol), K3PO4 (88.0 mg,
0.426 mmol) and naphthalen-1-yl-methanol (242 mg, 1.53
mmol) and stirred at 100 °C overnight until compound (28) was
observed by way of TLC. The mixture was dissolved in DCM (3
mL) and the K3PO4 filtered off. The solvent was reduced in vacuo
and the crude dissolved in DCM (1 mL) to which 4M HCl/dioxane
(1 mL) was added and stirred for 2 hours. The solvent was
reduced in vacuo and the product was purified by column
chromatography over silica gel using a gradient elution (100%
DCM to 10% MeOH,90% DCM) to afford the product as a white
H), 7.73 (dd, J=8.7, 1.4 Hz,
1
H); 13C NMR (75 MHz,
CHLOROFORM-d) δ ppm 34.0, 71.8, 115.2, 117.5, 118.9, 127.6 (2
C), 128.2, 128.6 (2 C), 134.7, 135.9, 139.6, 149.6, HRMS-TOF MS
ES+: m/z [M+H]+ calculated for C14H15N2O3 : 259.1083; found:
259.1076.
1
solid. 25.1 mg, 49% yield. H NMR (400 MHz, DMSO-d6) δ ppm
1.88 – 2.00 (m, 2 H), 2.07 – 2.20 (m, 2 H), 2.68 (s, 3 H), 3.02 –
3.20 (m, 4 H), 4.73 – 4.84 (m, 1 H), 5.83 (s, 2 H), 6.53 (d, J=7.8 Hz,
1 H), 6.96 (d, J=8.2 Hz, 1 H), 7.07 – 7.14 (m, 1 H), 7.50 – 7.66 (m,
3 H), 7.71 (d, J=6.6 Hz, 1 H), 7.93 – 8.03 (m, 2 H), 8.16 (d, J=8.2
Hz, 1 H), 8.96 (br. s, 2 H), 11.45 (s, 1 H); 13C NMR (101 MHz,
DMSO-d6) δ ppm 12.6, 27.4 (2 C), 40.7 (2 C), 64.3, 69.0, 101.6,
106.0, 118.9, 120.0, 122.2, 124.1, 125.9, 126.3, 126.5, 127.1,
127.8, 129.0, 129.4, 131.5, 132.2, 133.7, 138.8, 153.2, 162.1;
HRMS-TOF MS ES+: m/z [M+H]+ calculated for C26H27N2O3:
415.2022; found: 415.2010; Purity: > 98%; Mp: 197 – 200 °C, IR
ATR (cm-1): 3338, 2927, 1682, 1578, 1509, 1357, 1254, 1086.
4.1.4.3 6-(Benzyloxy)-N1-methylbenzene-1,2-diamine (41):
In an open flask was added (40) (820 mg, 3.17 mmol), glacial
acetic acid (6.6 mL), EtOH (6.6 mL), water (3.3 mL) and iron
powder (880 mg, 15.9 mmol). The mixture was subjected to
sonication at 30 °C for 3 hours, turning the red solution to dark
yellow. The iron powder was removed via a magnet. The
mixture was diluted with EtOAc (100 mL) and washed with a 2M
KOH solution (2 x 60 mL). The organic phase was dried over
MgSO4, and the solvent reduced in vacuo. The product was
purified by column chromatography over silica gel (50% EtOAc,
50% hexane) to afford a yellow oil. 520 mg, 72% yield. Rf: 0.28

(50% EtOAc, 50% hexane) 1H NMR (300 MHz, CHLOROFORM-d) δ
ppm 2.82 (s, 3 H), 3.98 (s, 3 H), 5.17 (s, 2 H), 6.47 – 6.59 (m, 2 H),
6.93 – 7.04 (m, 1 H), 7.41 – 7.65 (m, 5 H); 13C NMR (75 MHz,
CHLOROFORM-d) δ ppm 34.1, 70.1, 102.2, 108.9, 123.3, 125.5,
127.2 (2 C), 127.6, 128.2 (2 C), 137.0, 141.8, 152.1, HRMS-TOF
MS ES+: m/z [M+H]+ calculated for C14H17N2O: 229.1341; found:
229.1345; Characterization data corresponded with literature
values.[22]
MeOH). The reaction was left to warm up to room temperature
and stirred overnight. The mixture was diluted with EtOAc (50
mL) and washed with 2 x 30 mL water. The organic phase was
washed with brine, dried with MgSO4, and reduced in vacuo. The
product was purified by column chromatography over silica gel
(50% EtOAc, 50% hexane) to afford a white solid. 37 mg, 79%
yield. Rf: 0.55 (50% EtOAc, 50% hexane); 1H NMR (300 MHz,
CHLOROFORM-d) δ ppm 4.03 (s, 3 H), 4.42 (s, 3 H), 5.21 (s, 2 H),
6.86 (d, J=7.8 Hz, 1 H), 7.18 – 7.25 (m, 1 H), 7.37 – 7.51 (m, 6 H);
13C NMR (75 MHz, CHLOROFORM-d) δ ppm 34.8, 52.7, 70.8,
106.6, 114.6, 123.8, 126.8, 127.5 (2 C), 128.2, 128.7 (2 C), 136.2,
140.6, 143.5, 147.0, 160.3; HRMS-TOF MS ES+: m/z [M+H]+
calculated for C17H17N2O3: 297.1239; found: 297.1182; Mp: 78 –
82 °C; IR ATR (cm-1): 3366, 2953, 1713, 1593, 1468, 1247, 1096.
4.1.4.4 7-(Benzyloxy)-1-methyl-1H-benzo[d]imidazole (42):
In an open 10 mL round bottom flask was added (41) (956 mg,
4.19 mmol), trimethyl orthoformate (550 µL, 5.02 mmol) and
pTsOH (20 mg). The mixture was heated to 100 °C for 40
minutes. The solution was diluted with DCM (2 mL) and purified
by column chromatography over silica gel (100% EtOAc) to
afford a white solid. 967 mg, 97% yield. Rf: 0.56 (100% EtOAc);
1H NMR (400 MHz, CHLOROFORM-d) δ ppm 4.04 (s, 3 H), 5.19 (s,
2 H), 6.78 (d, J=8.2 Hz, 1 H), 7.12 – 7.18 (m, 1 H), 7.34 – 7.50 (m,
6 H), 7.71 (s, 1 H); 13C NMR (101 MHz, CHLOROFORM-d) δ ppm
33.9, 70.4, 104.6, 113.3, 122.4, 124.4, 127.4 (2 C), 128.0, 128.6
(2 C), 136.7, 143.8, 146.0, 146.6; HRMS-TOF MS ES+: m/z [M+H]+
calculated for C15H15N2O: 239.1184; found: 239.1135; Mp: 114 –
116 °C; IR ATR (cm-1): 3092, 2866, 1620, 1586, 1499, 1378, 1286,
1259, 1077.
4.1.4.7 Methyl 7-hydroxy-1-methyl-1H-benzo[d]imidazole-2-
carboxylate (45):
(44) (350 mg, 1.18 mmol) and Pd/C (10%, 22 mg, 0.0206 mmol)
was placed in a flask, evacuated and purged with hydrogen from
a balloon after which MeOH (6 mL) was added and the reaction
was stirred overnight. The mixture was filtered through a plug of
Celite® and the solvent reduced in vacuo. The product was
purified by column chromatography over silica gel (70% EtOAc,
30% hexane) to afford a white solid. 224 mg, 92% yield. Rf: 0.37
1
(70% EtOAc, 30% hexane); H NMR (300 MHz, CHLOROFORM-d)
4.1.4.5 7-(Benzyloxy)-1-methyl-1H-benzo[d]imidazole-2-
carbaldehyde (43):
δ ppm 3.87 (s, 3 H), 4.31 (s, 3 H), 6.62 (d, J=7.6 Hz, 1 H), 6.88 –
6.97 (m, 1 H), 7.15 (d, J=8.2 Hz, 1 H), 9.45 (s, 1 H); 13C NMR (101
MHz, DMSO-d6) δ ppm 34.2, 52.4, 109.4, 111.6, 123.7, 125.8,
140.6, 143.3, 145.5, 160.1; HRMS-TOF MS ES+: m/z [M+H]+
calculated for C10H11N2O3: 207.0770; found: 207.0727; Mp: 202 –
204 °C; IR ATR (cm-1): 2951, 1723, 1597, 1471, 1281, 1242,
1127, 1072.
To a solution of (42) (600 mg, 2.52 mmol) in THF (12 mL) was
added n-BuLi (1.6M in hexanes, 1.73 mL, 2.77 mmol) dropwise
at -78 °C under an atmosphere of argon, and stirred at -78 °C for
30 minutes, after which DMF (253 µL, 3.28 mmol) was added
dropwise, and stirred for 30 minutes, after which the solution
was left to reach 0 °C. The reaction was quenched with a
solution of aqueous NaHCO3 (5 mL), diluted with water (15 mL)
and the product extracted with EtOAc (2 x 30 mL). The organic
phase was washed with brine, dried with MgSO4, and reduced in
vacuo. The product was purified by column chromatography
over silica gel (50% EtOAc, 50% hexane) to afford a yellow solid.
4.1.4.8 Methyl 7-((1-(tert-butoxycarbonyl)piperidin-4-yl)oxy)-1-
methyl-1H-benzo[d]imidazole-2-carboxylate (46):
(45) (224 mg, 1.09 mmol), PPh3 (429 mg, 1.64 mmol) and 1-Boc-
4-hydroxypiperidine (329 mg, 1.63 mmol) were dissolved in THF
(6mL). The solution was cooled to 0 °C and DBAD (376 mg, 1.63
mmol, dissolved in 1 mL THF) was added dropwise under an
atmosphere of nitrogen. The reaction was stirred overnight at
room temperature. The solvent was reduced in vacuo and the
product was purified by column chromatography over silica gel
(50% EtOAc, 50% hexane) to afford a white solid. 321 mg, 76%
yield. Rf: 0.41 (50% EtOAc, 50% hexane); 1H NMR (300 MHz,
CHLOROFORM-d) δ ppm 1.41 (s, 9 H), 1.72 – 1.87 (m, 2 H), 1.91
– 2.05 (m, 2 H), 3.28 – 3.41 (m, 2 H), 3.59 – 3.68 (m, 2 H), 3.94 (s,
3 H), 4.37 (s, 3 H), 4.56 – 4.65 (m, 1 H), 1 H), 6.69 (d, J=7.8 Hz, 1
H), 7.07 – 7.14 (m, 1 H), 7.36 (d, J=8.2 Hz, 1 H);13C NMR (75 MHz,
CHLOROFORM-d) δ ppm 28.2 (3 C), 30.2 (2 C), 34.6, 40.5 (2 C),
52.5, 72.7, 79.6, 107.0, 114.1, 123.5, 127.0, 140.4, 143.5, 145.0,
154.5, 160.1; HRMS-TOF MS ES+: m/z [M+H]+ calculated for
C20H28N3O5: 390.2029; found: 390.1953; Mp: 85 – 88 °C; IR ATR
(cm-1): 2948, 1728, 1682, 1583, 1482, 1422, 1231, 1167, 1077,
1022.
1
460 mg, 69% yield. Rf: 0.73 (50% EtOAc, 50% hexane); H NMR
(300 MHz, CHLOROFORM-d) δ ppm 4.40 (s, 3 H), 5.22 (s, 2 H),
6.89 (d, J=7.8 Hz, 1 H), 7.22 – 7.29 (m, 1 H), 7.37 – 7.49 (m, 5 H),
7.51 (d, J=8.4 Hz, 1 H), 10.09 (s, 1 H); 13C NMR (75 MHz,
CHLOROFORM-d) δ ppm 34.0, 70.8, 107.5, 114.8, 124.2, 127.2,
127.4 (2 C), 128.3, 128.7 (2 C), 136.1, 144.6, 146.0, 147.4, 184.8;
HRMS-TOF MS ES+: m/z [M+H+MeOH]+ calculated for
C17H19N2O3: 299.1396; found: 229.1344; Mp: 88 – 90 °C; IR ATR
(cm-1): 3002, 2362, 1699, 1687, 1581, 1471, 1264, 1195, 1090.
4.1.4.6 Methyl 7-(Benzyloxy)-1-methyl-1H-benzo[d]imidazole-
2-carboxylate (44):
(43) (42.3 mg, 0.159 mmol) was dissolved in MeOH (2.1 mL) and
cooled to 0 °C. KOH (23.0 mg, 0.412 mmol, dissolved in 0.50 mL
MeOH) was added to the solution, followed by the dropwise
addition of I2 (52.7 mg, 0.208 mmol, dissolved in 0.50 mL

Compounds (47) – (49) were synthesized via the same
procedure. Compound (46), K3PO4 (3 equivalents) and the
aromatic alcohol used for the transesterification reaction (20 –
30 equivalents) were placed in a flask and stirred under nitrogen
at room temperature for 12 – 20 hours. The reaction was
monitored via TLC using (5% EtOAc, 95% DCM) as mobile phase
to determine whether the starting material had been consumed.
The K3PO4 was filtered off, and the alcohol removed via heating
the solution to 50 °C and streaming compressed air over the
solution. After the majority of the solvent volume has been
evaporated off, the product was purified by column
chromatography over silica gel (5% EtOAc, 95% DCM).
compounds (47) – (52) separately dissolved in DCM (1 mL)
followed by the addition of 4M HCl/dioxane (1 mL). The solution
was stirred for one hour, after which the solvent was reduced in
vacuo. A small amount of DCM was added followed by the
addition of EtOAc which caused the salt to precipitate out of
solution. The solvent was reduced in vacuo and the product
dried under vacuum.
4.1.4.11 4-((1-Methyl-2-(phenethoxycarbonyl)-1H-
benzo[d]imidazol-7-yl)oxy)piperidin-1-ium chloride (53):
1
26 mg, 83% yield, yellow solid. H NMR (600 MHz, DMSO-d6) δ
ppm 1.97 – 2.07 (m, 2 H), 2.18 – 2.27 (m, 2 H), 3.05 – 3.16 (m, 4
H), 3.21 (br. s., 2 H), 4.30 (s, 3 H), 4.59 (t, J=6.7 Hz, 2 H), 4.90 (br.
s., 1 H), 7.07 (d, J=7.6 Hz, 1 H), 7.20 – 7.39 (m, 7 H), 9.25 (br. s., 1
H), 9.44 (br. s., 1 H); 13C NMR (151 MHz, DMSO-d6) δ ppm 26.7 (2
C), 34.1, 34.8, 40.2 (2 C), 66.3, 70.2, 108.2, 112.6, 124.6, 126.0,
126.5, 128.4 (2 C), 128.9 (2 C), 137.6, 140.4, 141.3, 144.9, 158.5;
HRMS-TOF MS ES+: m/z [M+H]+ calculated for C22H26N3O3:
380.1974; found: 380.1966; Purity: 98.6%; Mp: 179 – 182 °C, IR
ATR (cm-1): 2960, 2568, 1749, 1614, 1491, 1257, 1098, 1022.
4.1.4.9 Phenethyl-7-((1-(tert-butoxycarbonyl)piperidin-4-
yl)oxy)-1-methyl-1H-benzo[d]imidazole-2-carboxylate (47):
36 mg, 50% yield, brown semi-solid. Rf: 0.55 (50% EtOAc: 50%
1
hexane); H NMR (300 MHz, CHLOROFORM-d) δ ppm 1.49 (s, 9
H), 1.81 – 1.95 (m, 2 H), 1.99 – 2.11 (m, 2 H), 3.18 (t, J=7.6 Hz, 2
H), 3.36 – 3.48 (m, 2 H), 3.66 – 3.77 (m, 2 H), 4.40 (s, 3 H), 4.60 –
4.74 (m, 3 H), 6.78 (d, J=7.6 Hz, 1 H), 7.16 – 7.23 (m, 1 H), 7.23 –
7.35 (m, 5 H), 7.48 (d, J=8.2 Hz, 1 H);13C NMR (75 MHz,
CHLOROFORM-d) δ ppm 28.5 (3 C), 30.5 (2 C), 34.8, 35.1, 40.7 (2
C), 66.4, 72.9, 79.9, 107.3, 114.5, 123.7, 126.8, 127.3, 128.6 (2
C), 129.0 (2 C), 137.1, 141.0, 143.8, 145.3, 154.7, 159.9; HRMS-
TOF MS ES+: m/z [M+H]+ calculated for C27H34N3O5: 480.2498;
found: 480.2515; IR ATR (cm-1): 2961, 2926, 1718, 1688, 1585,
1465, 1422, 1229, 1084, 1023.
4.1.5
Synthesis of compounds (69) – (74)
4.1.5.1 Ethyl 4-hydroxybenzo[d]oxazole-2-carboxylate (60):
This method is a modified literature procedure.23 To a solution
of 2-amino resorcinol (59) (783 mg, 6.26 mmol) in THF (30 mL)
and Et3N (12.5 mmol, 1.75 mL) was added ethyl
chlorooxoacetate (770 µL, 6.89 mmol) at -10 °C. The mixture
was stirred and allowed to warm to room temperature. The
reaction was monitored by TLC and was completed in 2 hours.
N-acylated product Rf: 0.75 (60% EtOAc: 40% hexane). The
resulting dark brown solution was cooled down to 0 °C followed
by the addition of THF (50 mL) and PPh3 (4.38 g, 16.7 mmol).
DEAD (1070 µL, 6.88 mmol) was added dropwise, and the
solution turned yellow. The reaction was monitored by TLC and
was completed in 3 hours. The solvent was reduced in vacuo,
and the product was purified by column chromatography over
silica gel (40% EtOAc, 60% hexane) to afford a white solid. 1.02
g, 84% yield over two steps. Rf: 0.52 (40% EtOAc, 60% hexane);
1H NMR (300 MHz, DMSO-d6) δ ppm 1.37 (t, J=7.1 Hz, 3 H), 4.43
(q, J=7.1 Hz, 2 H), 6.84 (d, J=8.1 Hz, 1 H), 7.25 (d, J=8.1 Hz, 1 H),
7.34 – 7.42 (m, 1 H), 10.80 (s, 1 H); 13C NMR (75 MHz,
CHLOROFORM-d) δ ppm 13.9, 62.5, 101.9, 110.9, 129.1, 129.4,
150.8, 151.0, 151.8, 155.8; HRMS-TOF MS ES+: m/z [M+H]+
calculated for C10H10NO4: 208.0610; found: 208.0616; Mp 131 –
134 °C; IR ATR (cm-1): 3440, 2916, 1717, 1686, 1551, 1375, 1270,
1159, 1017.
Compounds (50)
– (52) were synthesized via the same
procedure. Compound (46) and the aromatic amine used for the
amidation reaction (10 equivalents) were dissolved in THF (1 mL)
and stirred under nitrogen at room temperature for 12 – 20
hours. The reaction was monitored via TLC using (50% EtOAc,
50% hexane) mobile phase to determine whether the starting
material had been consumed. The product was purified by
column chromatography over silica gel (50% EtOAc, 50% hexane
– 100% EtOAc).
4.1.4.10 tert-Butyl 4-((1-methyl-2-(phenethylcarbamoyl)-1H-
benzo[d]imidazol-7-yl)oxy)piperidine-1-carboxylate (50):
47 mg, 81% yield, yellow solid. Rf: 0.53 (40% EtOAc: 60%
1
hexane); H NMR (300 MHz, CHLOROFORM-d) δ ppm 1.50 (s, 9
H), 1.81 – 1.96 (m, 2 H), 1.99 – 2.11 (m, 2 H), 2.96 (t, J=7.3 Hz, 2
H), 3.39 – 3.50 (m, 2 H), 3.66 – 3.78 (m, 4 H), 4.52 (s, 3 H), 4.63 –
4.74 (m, 1 H), 6.76 (d, J=7.6 Hz, 1 H), 7.14 – 7.21 (m, 1 H), 7.21 –
7.38 (m, 6 H), 7.88 (t, J=5.9 Hz, 1 H); 13C NMR (75 MHz,
CHLOROFORM-d) δ ppm 28.5 (3 C), 30.5 (2 C), 34.9, 35.9, 40.6 (2
C), 40.8, 72.9, 79.8, 106.7, 113.2, 123.5, 126.5, 127.4, 128.6 (2
C), 128.8 (2 C), 138.7, 143.1, 143.4, 145.5, 154.7, 159.8; HRMS-
TOF MS ES+: m/z [M+H]+ calculated for C27H35N4O4: 479.2658;
found: 479.2668; Mp: 84 – 86 °C, IR ATR (cm-1): 2930, 2865,
1671, 1529, 1422, 1231, 1166, 1085, 1023.
4.1.5.2 (ADDM) Azodicarbonyldimorpholide (61):
To a solution of diethyl ether (30 mL) and morpholine (2.50 mL,
2.87 mmol) was added diethylazodicarboxylate (1.79 ml, 11.5
mmol) dropwise at 0 °C, and stirred for 30 minutes, after which
a light orange precipitate fell out of solution. The precipitate was
filtered, washed with diethyl ether and dried under vacuum to
Compounds (53) – (58) were synthesized via the same
procedure. In a small flask was added the Boc-protected
1
afford the product as a light orange solid. 2.63 g, 89% yield. H
NMR (300 MHz, CHLOROFORM-d) δ ppm 3.58 – 3.66 (m, 4 H),

3.68 – 3.80 (m, 8 H), 3.81 – 3.86 (m, 4 H); 13C NMR; (75 MHz,
CHLOROFORM-d) δ ppm 43.7, 45.2, 66.4, 66.5, 110.0, 159.7.
Characterization data corresponded with available literature
values.[23]
Compounds (66) – (68) were synthesized via the same
procedure. Compound (62) and the respected amine (10
equivalents) were dissolved in THF (1 mL) and stirred under
nitrogen at room temperature for 12 – 20 hours. The reaction
was monitored via TLC using (50% EtOAc, 50% hexane) mobile
phase to determine whether the starting material had been
consumed. The product was purified by column chromatography
over silica gel (50% EtOAc, 50% hexane – 100% EtOAc)
4.1.5.3 Ethyl 4-((1-(tert-butoxycarbonyl)piperidin-4-yl)oxy)
benzo[d]oxazole-2-carboxylate (62):
To a solution of (60) (217 mg, 1.05 mmol), PPh3 (466 mg, 1.78
mmol) and 1-Boc 4-hydroxypiperidine (358 mg, 1.78 mmol) in
THF (6 mL) was added azodicarbonyldimorpholide (ADDM) (61)
(456 mg, 1.78 mmol, dissolved in 1 mL DCM) dropwise at 0 °C
under an atmosphere of nitrogen. The solution was taken off the
ice and stirred at room temperature overnight. The solvent was
reduced in vacuo, and the product was purified by column
chromatography over silica gel (40% EtOAc, 60% hexane) to
afford a white solid. 323 mg, 79% yield. Rf: 0.62 (40% EtOAc,
60% hexane); 1H NMR (300 MHz, CHLOROFORM-d) δ ppm 1.45 –
1.51 (m, 12 H), 1.78 – 1.93 (m, 2 H), 1.96 – 2.08 (m, 2 H), 3.26 –
3.37 (m, 2 H), 3.77 – 3.89 (m, 2 H), 4.55 (q, J=7.1 Hz, 2 H), 4.95 –
5.05 (m, 1 H), 6.89 (d, J=7.6 Hz, 1 H), 7.22 – 7.26 (m, 1 H), 7.37 –
7.45 (m, 1 H); 13C NMR (75 MHz, CHLOROFORM-d) δ ppm 14.1,
28.4 (3 C), 30.6 (2 C), 40.7 (2 C), 63.1, 74.1, 79.6, 104.1, 110.5,
128.8, 131.2, 150.6, 151.4, 152.5, 154.8, 156.4; HRMS-TOF MS
ES+: m/z [M+H]+ calculated for C20H27N2O6: 391.1869; found:
391.1868; Mp: 112 – 114 °C; IR ATR (cm-1): 2936, 1739, 1545,
1418, 1269, 1159, 1081.
4.1.5.5 tert-Butyl 4-((2-(phenethylcarbamoyl)benzo[d]oxazol-4-
yl)oxy) piperidine-1-carboxylate (66):
51 mg, 91% yield, yellow solid. Rf: 0.71 (50% EtOAc, 50%
hexane); 1H NMR (300 MHz, CHLOROFORM-d) δ ppm 1.44 – 1.56
(m, 9 H), 1.76 – 1.92 (m, 2 H), 1.95 – 2.10 (m, 2 H), 2.98 (t, J=7.3
Hz, 2 H), 3.25 – 3.38 (m, 2 H), 3.72 – 3.89 (m, 4 H), 4.86 – 4.95
(m, 1 H), 6.90 (d, J=8.2 Hz, 1 H), 7.19 – 7.43 (m, 8 H); 13C NMR
(75 MHz, CHLOROFORM-d) δ ppm 28.4 (3 C), 30.6 (2 C), 35.5,
40.7 (2 C), 40.9, 74.1, 79.6, 104.5, 110.6, 126.7, 127.9, 128.7 (4
C), 130.6, 138.2, 149.9, 152.8, 154.1, 154.7, 155.5; Missing
carbon signal, suspected to be merged with signal located at
128.7 ppm; HRMS-TOF MS ES+: m/z [M+H]+ calculated for
C26H32N3O5: 466.2342; found: 466.2338; Mp: 71 – 73 °C, IR ATR
(cm-1): 3313, 2930, 1681, 1617, 1553, 1496, 1423, 1364, 1265,
1165, 1067, 1022.
Compounds (69) – (74) were synthesized via the same
procedure. In a small flask was added the Boc-protected
compound (63) – (68) separately and DCM (1 mL) followed by
the addition of 4M HCl/dioxane (1 mL). The solution was stirred
for one hour, after which the solvent was reduced in vacuo. A
small amount of DCM was added followed by the addition of
EtOAc which caused the salt to precipitate out of solution. The
solvent was reduced in vacuo and the product dried under
vacuum.
Compounds (63) – (65) were synthesized via the same
procedure. Compound (62), K3PO4 (3 equivalents) and the
aromatic alcohol used for the transesterification reaction (20 –
30 equivalents) were placed in a flask and stirred under nitrogen
at room temperature for 12 – 20 hours. The reaction was
monitored via TLC using (5% EtOAc, 95% DCM) as mobile phase
to determine whether the starting material had been consumed.
The K3PO4 was filtered off, and the alcohol removed via heating
the solution to 50 °C and streaming compressed air over the
solution. After the majority of the solvent volume had been
evaporated off, the product was purified by column
chromatography over silica gel (5% EtOAc, 95% DCM).
4.1.5.6 4-((2-(phenethoxycarbonyl)benzo[d]oxazol-4-yl)oxy)
piperidin-1-ium chloride (69):
1
37 mg, 98% yield, white solid. H NMR (600 MHz, DMSO-d6) δ
ppm 1.92 – 2.02 (m, 2 H), 2.15 – 2.29 (m, 2 H), 3.05 – 3.14 (m, 4
H), 3.23 – 3.30 (m, 2 H), 4.61 (t, J=7.0 Hz, 2 H), 5.02 – 5.07 (m, 1
H), 7.16 (d, J=8.2 Hz, 1 H), 7.20 – 7.36 (m, 5 H), 7.45 (d, J=8.2 Hz,
1 H), 7.49 – 7.57 (m, 1 H), 9.24 (br. s., 1 H); 13C NMR (151 MHz,
DMSO-d6) δ ppm 27.1 (2 C), 34.1, 40.4 (2 C), 66.7, 70.8, 104.5,
110.3, 126.6, 128.4 (2 C), 128.9 (2 C), 129.2, 130.5, 137.4, 149.6,
151.2, 151.8, 155.6; HRMS-TOF MS ES+: m/z [M+H]+ calculated
for C21H23N2O4: 367.1658; found: 367.1663; Purity: >95%; Mp:
184– 186 °C, IR ATR (cm-1): 2958, 2731, 1740, 1609, 1494, 1263,
1142, 1084.
4.1.5.4 Phenethyl4-((1-(tert-butoxycarbonyl)piperidin-4-
yl)oxy)benzo[d]oxazole-2-carboxylate (63):
44 mg, 45% yield, white solid. Rf: 0.56 (30% EtOAc, 70% hexane);
1H-NMR (300 MHz, CHLOROFORM-d) δ ppm 1.47 (s, 9 H), 1.79 –
1.94 (m, 2 H), 1.98 – 2.12 (m, 2 H), 3.15 (t, J=7.3 Hz, 2 H), 3.28–
3.41 (m, 2 H), 3.78 – 3.92 (m, 2 H), 4.66 (t, J=7.3 Hz, 2 H), 5.00 –
5.09 (m, 1 H), 6.92 (d, J=7.6 Hz, 1 H), 7.20 – 7.38 (m, 6 H), 7.39 –
7.46 (m, 1 H); 13C NMR (75 MHz, CHLOROFORM-d) δ ppm 28.4 (3
C), 30.6 (2 C), 34.9, 40.9 (2 C), 67.2, 74.2, 79.6, 104.1, 110.8,
126.8, 128.6 (2 C), 128.8, 128.9 (2 C), 131.1, 136.8, 150.6, 151.2,
152.5, 154.8, 156.3; HRMS-TOF MS ES+: m/z [M+H]+ calculated
for C26H31N2O6: 467.2182; found: 467.2170; Mp: 114 – 117 °C, IR
ATR (cm-1): 2970, 1734, 1685, 1614, 1409, 1324, 1149, 1081,
1026.
4.1.6
Synthesis of compounds (87) – (92)
4.1.6.1 2-Hydroxy-6-nitrobenzaldehyde (76):
3-nitrophenol (75) (1.00 g 7.19 mmol) was placed in a vial and
combined with hexamethylenetetramine (1.21 g, 8.59 mmol)
and 5.00 mL trifluoroacetic acid. The vial was flushed with argon,
sealed, and the solution was stirred at 90 °C for 12 hours. The

reaction mixture was poured onto 50 mL ice-water and
extracted with 2 x 50 mL EtOAc. The organic fractions were
combined, washed with brine, dried over MgSO4, and the
solvent reduced in vacuo. The product was purified by column
chromatography over silica gel (20% EtOAc, 80% hexane) to
afford the product as a yellow solid. 354 mg, 29% yield. Rf: 0.59
4.1.6.4 Ethyl 4-hydroxybenzo[b]thiophene-2-carboxylate (79):
To a solution of (78) (630 mg, 2.02 mmol) in MeOH (6 mL) was
added Pd/C (10%, 170 mg, 0.159 mmol), after which the flask
was purged with hydrogen from a balloon, stirred under an
atmosphere of hydrogen for 12 hours. The mixture was filtered
through a plug of Celite® and the solvent removed in vacuo. The
product was purified by column chromatography over silica gel
(30% EtOAc, 70% hexane) to afford the product as a yellow solid.
314 mg, 68% yield. Rf: 0.43 (30% EtOAc, 70% hexane);
1H NMR (300 MHz, CHLOROFORM-d) δ ppm 1.43 (t, J=7.0 Hz, 3
H), 4.43 (q, J=7.0 Hz, 2 H), 5.73 (s, 1 H), 6.77 (d, J=7.0 Hz, 1 H),
7.29 – 7.36 (m, 1 H), 7.43 (d, J=8.2 Hz, 1 H), 8.32 (d, J=1.2 Hz, 1
H); 13C NMR (75 MHz, CHLOROFORM-d) δ ppm 14.3, 61.7, 109.2,
110.0, 115.2, 126.9, 128.3, 132.1, 144.1, 152.3, 163.1; HRMS-
TOF MS ES+: m/z [M+Na]+ calculated for C11H10O3SNa: 245.0249;
found: 245.0254; Mp: 154 – 156 °C; IR ATR (cm-1): 3376, 1692,
1564, 1527, 1332, 1279, 1218, 1149.
1
(20% EtOAc, 80% hexane); H NMR (300 MHz, CHLOROFORM-d)
δ ppm 7.28 – 7.34 (m, 1 H), 7.54 – 7.60 (m, 1 H), 7.60 – 7.67 (m,
1 H), 10.33 (s, 1 H), 12.11 (br. s., 1 H); 13C NMR (75 MHz,
CHLOROFORM-d) δ ppm 112.3, 116.0, 124.2, 135.9, 163.1,
163.3, 193.8; Characterization data corresponded with available
literature values.24
4.1.6.2 2-(Benzyloxy)-6-nitrobenzaldehyde (77):
In DMF (4 mL) was added (76) (354 mg, 2.12 mmol), benzyl
bromide (0.760 mL, 6.36 mmol) and K2CO3 (440 mg, 3.18 mmol)
at 0 °C under an atmosphere of nitrogen. The reaction mixture
was warmed to room temperature and stirred overnight. The
reaction mixture was dissolved in EtOAc (60 mL), and washed
with water (4 x 50 mL) followed by a wash with brine. The
organic layer was dried over MgSO4, filtered and reduced in
vacuo. The product was purified by column chromatography
over silica gel (50% EtOAc, 50% hexane) to afford the product as
a yellow solid. Quantitative, 557 mg. Rf: 0.23 (20% EtOAc, 80%
4.1.6.5 tert-butyl 4-((2-(ethoxycarbonyl)benzo[b]thiophen-4-
yl)oxy) piperidine-1-carboxylate (80):
(79) (314 mg, 1.41 mmol), PPh3 (629 mg, 2.40 mmol) and 1-Boc-
4-hydroxypiperidine (483 mg, 2.40 mmol) were dissolved in THF
(9 mL) under an atmosphere of nitrogen. To this solution was
added DBAD (553 mg, 2.4 mmol, dissolved in 1 mL THF)
dropwise at 0 °C. The solution was stirred overnight at room
temperature, after which the solvent was removed in vacuo, and
the product was purified by column chromatography over silica
gel (20% EtOAc, 80% hexane) to afford the product as a colorless
semisolid that solidified over time into a white solid. 571 mg,
99% yield. Rf: 0.48 (20% EtOAc, 80% hexane); 1H NMR (300 MHz,
CHLOROFORM-d) δ ppm 1.36 (t, J=7.0 Hz, 3 H), 1.44 (s, 9 H), 1.74
– 1.87 (m, 2 H), 1.87 – 1.97 (m, 2 H), 3.34 – 3.45 (m, 2 H), 3.61 –
3.72 (m, 2 H), 4.34 (q, J=7.0 Hz, 2 H), 4.56 – 4.66 (m, 1 H), 6.70
(d, J=7.0 Hz, 1 H), 7.24 – 7.31 (m, 1 H), 7.34 (d, J=8.2 Hz, 1 H),
8.14 (s, 1 H); 13C NMR (75 MHz, CHLOROFORM-d) δ ppm 14.1,
28.2 (3 C), 30.1 (2 C), 40.3 (2 C), 61.2, 72.0, 79.3, 106.2, 114.7,
127.0, 127.9, 130.6, 131.9, 143.7, 153.5, 154.5, 162.5; HRMS-
TOF MS ES+: m/z [M+Na]+ calculated for C21H27NO5SNa:
428.1508; found: 428.1515; Mp: 64 – 67 °C; IR ATR (cm-1): 2947,
1716, 1696, 1681, 1562, 1416, 1253, 1230, 1150, 1023.
1
hexane); H NMR (400 MHz, CHLOROFORM-d) δ ppm 5.23 (s, 2
H), 7.29 (d, J=8.2 Hz, 1 H), 7.34 – 7.43 (m, 5 H), 7.45 (d, J=8.2 Hz,
1 H), 7.54 – 7.60 (m, 1 H), 10.45 (s, 1 H); 13C NMR (101 MHz,
CHLOROFORM-d) δ ppm 71.5, 115.8, 117.6, 121.1, 127.2 (2 C),
128.6, 128.9 (2 C), 133.4, 135.0, 148.7, 158.8, 187.6;
Characterization data corresponded with available literature
values.24
4.1.6.3 Ethyl 4-(benzyloxy)benzo[b]thiophene-2-carboxylate
(78):
To a Schlenk flask was added (77) (557 mg, 2.17 mmol), K2CO3
(361 mg, 2.61 mmol) and DMF (4.5 mL). Ethyl thioglycolate (250
µL, 2.27 mmol) was added dropwise at 0 °C under an
atmosphere of nitrogen, upon which the initial yellow solution
turned light yellow. The mixture was stirred at 0 °C for 30
minutes, then heated to 50 °C for 48 hours. The mixture was
diluted with EtOAc (60 mL), and washed with water (4 x 50 mL)
followed by a wash with brine. The organic fraction was dried
over MgSO4, and the solvent reduced in vacuo. The product was
purified by column chromatography over silica gel (5% EtOAc,
95% hexane) to afford the product as a yellow solid. 644 mg,
Compounds (81) – (83) were synthesized via the same
procedure. Compound (80), K3PO4 (3 equivalents) and the
aromatic alcohol used for the transesterification reaction (26
equivalents) were placed in a flask and stirred under nitrogen at
70 °C for 12 – 20 hours. The reaction was monitored via TLC
using 5% EtOAc, 95% DCM as mobile phase to determine
whether the starting material had been consumed. The K3PO4
was filtered off, and the alcohol removed via heating the
solution to 50 °C and streaming compressed air over the
solution. After the majority of the solvent volume had been
evaporated off, the product was purified by column
chromatography over silica gel (5% EtOAc, 95% DCM).
1
95% yield. Rf: 0.45 (5% EtOAc, 95% hexane); H NMR (300 MHz,
CHLOROFORM-d) δ ppm 1.43 (t, J=7.0 Hz, 3 H), 4.42 (q, J=7.0 Hz,
2 H), 5.22 (s, 2 H), 6.82 (d, J=7.0 Hz, 1 H), 7.32 – 7.55 (m, 7 H),
8.31 (s, 1 H); 13C NMR (75 MHz, CHLOROFORM-d) δ ppm 14.3,
61.4, 70.0, 105.3, 115.0, 127.3 (2 C), 127.4, 128.0, 128.1, 128.5
(2 C), 130.1, 132.1, 136.4, 143.7, 155.2, 162.7; HRMS-TOF MS
ES+: m/z [M+H]+ calculated for C18H17O3S: 313.0898; found:
313.0888; IR ATR (cm-1): 2926, 1704, 1564, 1519, 1285, 1233,
1151, 1021.

4.1.6.6 tert-Butyl 4-((2-(phenethoxycarbonyl)benzo[b]thiophen
-4-yl)oxy) piperidine-1-carboxylate (81):
4.2 Hz, 1 H), 7.31 (d, J=4.3 Hz, 4 H), 7.44 – 7.50 (m, 1 H), 7.61 (d,
J=8.2 Hz, 1 H), 8.09 (s, 1 H), 9.28 (br. s., 2 H); 13C NMR (101 MHz,
DMSO-d6) δ ppm 26.8 (2 C), 34.4, 65.8, 69.4, 107.2, 115.3, 126.5,
126.9, 128.4 (2 C), 129.0 (2 C), 129.8, 131.3, 137.8, 142.9, 153.0,
161.7; Missing carbon signal, suspected to be merged with
DMSO-d6 solvent signal located at 40.2 ppm; HRMS-TOF MS ES+:
m/z [M+H]+ calculated for C22H24NO3S: 382.1477; found:
382.1477; Purity: >99%; Mp: 226 – 228 °C, IR ATR (cm-1): 2938,
2712, 1705, 1584, 1456, 1234, 1062, 989.
53 mg, 88% yield, clear semi-solid. Rf: 0.50 (20% EtOAc, 80%
1
hexane); H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.50 (s, 9
H), 1.84 – 1.94 (m, 2 H), 1.94 – 2.04 (m, 2 H), 3.11 (t, J=7.0 Hz, 2
H), 3.43– 3.52 (m, 2 H), 3.65 – 3.74 (m, 2 H), 4.56 (t, J=7.2 Hz, 2
H), 4.65 – 4.74 (m, 1 H), 6.78 (d, J=7.4 Hz, 1 H), 7.23 – 7.35 (m, 5
H), 7.35 – 7.40 (m, 1 H), 7.41 – 7.44 (m, 1 H), 8.19 (d, J=0.8 Hz, 1
H); 13C NMR (75 MHz, CHLOROFORM-d) δ ppm 28.4 (3 C), 30.3 (2
C), 35.2, 40.3 (2 C), 65.9, 72.2, 79.7, 106.4, 115.0, 126.6, 127.5,
128.2, 128.5 (2 C), 129.0 (2 C), 130.9, 131.8, 137.6, 144.0, 153.7,
154.8, 162.6; HRMS-TOF MS ES+: m/z [M+Na]+ calculated for
C27H31NO5SNa: 504.1821; found: 504.1829; IR ATR (cm-1): 2930,
1691, 1564, 1454, 1231, 1152, 1028.
Acknowledgements
The authors would like to thank the National Research
Foundation (NRF), the Harry Crossley Foundation and
Stellenbosch University Faculty of Science for financial support.
Furthermore, we would like to thank Stellenbosch University
and the University of Cape Town for providing the facilities to
conduct the synthesis, analysis and biological testing.
Compounds (84) – (86) were synthesized via the same
procedure. Compound (80) and the respected amine (20
equivalents) were stirred under nitrogen at 145 °C for 12 – 20
hours. The reaction was monitored via TLC using (50% EtOAc,
50% hexane) mobile phase to determine whether the starting
material had been consumed. The product was purified by
column chromatography over silica gel using a gradient elution
(50% EtOAc, 50% hexane – 100% EtOAc)
Keywords: Malaria • Heterocycles • N-Myristoyl transferase •
structure–activity relationship • NF54
References:
4.1.6.7 tert-Butyl 4-((2-(phenethylcarbamoyl)benzo[b]thiophen
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55 mg, 77% yield, white solid. Rf: 0.53 (35% EtOAc, 65% hexane);
1H NMR (300 MHz, CHLOROFORM-d) δ ppm 1.51 (s, 9 H), 1.80–
1.92 (m, 2 H), 1.92 – 2.04 (m, 2 H), 2.97 (t, J=7.0 Hz, 2 H), 3.40 –
3.53 (m, 2 H), 3.65 – 3.78 (m, 4 H), 4.63 – 4.72 (m, 1 H), 6.36 (t,
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CHLOROFORM-d) δ ppm 28.4 (3 C), 30.3 (2 C), 35.7, 40.4 (2 C),
41.3, 72.1, 79.7, 106.5, 115.0, 121.6, 126.6, 127.4, 128.7 (2 C),
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503.1981; found: 503.1989; Mp: 75 – 78 °C, IR ATR (cm-1): 3309,
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[9]
4.1.6.8 4-((2-(Phenethoxycarbonyl)benzo[b]thiophen-4-yl)oxy)
piperidin-1-ium chloride (87):
1
[10]
41 mg, 89% yield, white solid. H NMR (400 MHz, DMSO-d6) δ
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2 H), 3.08 – 3.16 (m, 2 H), 3.23 – 3.32 (m, 2 H), 4.50 (t, J=6.8 Hz,
2 H), 4.85 – 4.95 (m, 1 H), 7.08 (d, J=7.8 Hz, 1 H), 7.24 (dq, J=8.5,

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