Discovery of new ATP-competitive inhibitors of human DNA topoisomerase IIα through screening of bacterial topoisomerase inhibitors

Article abstract of DOI:10.1016/j.bioorg.2020.104049

Human DNA topoisomerase II is one of the major targets in anticancer therapy, however ATP-competitive inhibitors of this target have not yet reached their full potential. ATPase domain of human DNA topoisomerase II belongs to the GHKL ATPase superfamily and shares a very high 3D structural similarity with other superfamily members, including bacterial topoisomerases. In this work we report the discovery of a new chemotype of ATP-competitive inhibitors of human DNA topoisomerase IIα that were discovered through screening of in-house library of ATP-competitive inhibitors of bacterial DNA gyrase and topoisomerase IV. Systematic screening of this library provided us with 20 hit compounds. 1,2,4-Substituted N-phenylpyrrolamides were selected for a further exploration which resulted in 13 new analogues, including 52 with potent activity in relaxation assay (IC₅₀ = 3.2 μM) and ATPase assay (IC₅₀ = 0.43 μM). Cytotoxic activity of all hits was determined in MCF-7 cancer cell line and the most potent compounds, 16 and 20, showed an IC₅₀ value of 8.7 and 8.2 μM, respectively.

Full text of DOI:10.1016/j.bioorg.2020.104049

Journal Pre-proofs
Discovery of new ATP-competitive inhibitors of human DNA topoisomerase
IIα through screening of bacterial topoisomerase inhibitors
Žiga Skok, Martina Durcik, Darja Gramec Skledar, Michaela Barančoková,
Lucija Peterlin Mašič, Tihomir Tomašič, Anamarija Zega, Danijel Kikelj,
Nace Zidar, Janez Ilaš
PII:
S0045-2068(20)31346-8
DOI:
https://doi.org/10.1016/j.bioorg.2020.104049
YBIOO 104049
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
12 April 2020
19 June 2020
25 June 2020
Please cite this article as: Z. Skok, M. Durcik, D. Gramec Skledar, M. Barančoková, L. Peterlin Mašič, T.
Tomašič, A. Zega, D. Kikelj, N. Zidar, J. Ilaš, Discovery of new ATP-competitive inhibitors of human DNA
topoisomerase IIα through screening of bacterial topoisomerase inhibitors, Bioorganic Chemistry (2020), doi:
https://doi.org/10.1016/j.bioorg.2020.104049
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Discovery of new ATP-competitive inhibitors of human DNA
topoisomerase IIα through screening of bacterial topoisomerase
inhibitors
Žiga Skok, Martina Durcik, Darja Gramec Skledar, Michaela Barančoková, Lucija Peterlin
Mašič, Tihomir Tomašič, Anamarija Zega, Danijel Kikelj, Nace Zidar* and Janez Ilaš*
University of Ljubljana, Faculty of Pharmacy, Aškerčeva cesta 7, 1000 Ljubljana, Slovenia
*Corresponding author: Janez Ilaš
Aškerčeva cesta 7
1000 Ljubljana, Slovenia
Tel: +386-1-4769576
E-mail: janez.ilas@ffa.uni-lj.si
*Corresponding author: Nace Zidar
Aškerčeva cesta 7
1000 Ljubljana, Slovenia
Tel: +386-1-4769564
E-mail: nace.zidar@ffa.uni-lj.si
1

ABSTRACT
Human DNA topoisomerase II is one of the major targets in anticancer therapy, however
ATP-competitive inhibitors of this target have not yet reached their full potential. ATPase
domain of human DNA topoisomerase II belongs to the GHKL ATPase superfamily and shares
a very high 3D structural similarity with other superfamily members, including bacterial
topoisomerases. In this work we report the discovery of a new chemotype of ATP-competitive
inhibitors of human DNA topoisomerase IIα that were discovered through screening of in-house
library of ATP-competitive inhibitors of bacterial DNA gyrase and topoisomerase IV.
Systematic screening of this library provided us with 20 hit compounds. 1,2,4-Substituted N-
phenylpyrrolamides were selected for a further exploration which resulted in 13 new analogues,
including 52 with potent activity in relaxation assay (IC50 = 3.2 µM) and ATPase assay (IC50 =
0.43 µM). Cytotoxic activity of all hits was determined in MCF-7 cancer cell line and the most
potent compounds, 16 and 20, showed an IC50 value of 8.7 and 8.2 µM, respectively.
KEYWORDS: anticancer drug, ATP-competitive inhibitor, biological screening, DNA
topoisomerase II, N-phenylpyrrolamide
ABBREVIATIONS
AMP-PNP, adenylyl-imidodiphosphate; DIAD; diisopropyl azodicarboxylate; DMAP, 4-
dimethylaminopyridine; GHKL, gyrase, Hsp90, histidine kinase and MutL; GyrA, DNA gyrase
subunit A; GyrB, DNA gyrase subunit B; topo II, human DNA topoisomerase II.
2

1. INTRODUCTION
Topoisomerases are a family of enzymes with ability to change the topology of DNA
molecule. Based on their structure and mechanism of action, topoisomerases can be divided
into two types. Type I topoisomerases are monomeric enzymes that catalyse single-strand
breaks in DNA double helix, while multimeric type II topoisomerases catalyse double-strand
breaks. Based on structural and functional distinctions, both types of topoisomerases are further
divided into two subfamilies: IA and IB, and IIA and IIB. Topoisomerases have important roles
in processes such as replication and transcription and are essential for cell survival; hence their
inhibition leads to cell death. Bacterial type IIA topoisomerases (DNA gyrase and DNA
topoisomerase IV) are well known targets of antibacterial drugs, while human DNA
topoisomerase IIA (topo II) is targeted by anticancer agents such as etoposide, doxorubicin,
daunorubicin and mitoxantrone [1,2].
Prokaryotic and eukaryotic type IIA topoisomerases share many structural and
functional similarities. They all require Mg(II) ions as cofactors and the energy from ATP
hydrolysis to perform their function. Prokaryotic topoisomerase IIA enzymes have
heterotetrameric structure, while eukaryotic enzymes are homodimeric. All type IIA
topoisomerases have a three-domain structure, which was determined based on their homology
with Escherichia coli DNA gyrase. Subunit B of E. coli DNA gyrase (GyrB) is homologous to
the N-terminal part of topo II, subunit A of E. coli DNA gyrase (GyrA) is homologous to the
central domain of topo II, and the C-terminal tail of both enzymes is responsible for their
nuclear localization and for their interactions with other proteins. The ATP binding sites lie
within the N-terminal domains of type IIA topoisomerases. The ATPase domain belongs to the
GHKL superfamily (gyrase, Hsp90, histidine kinase and MutL) whose members contain a
unique Bergerat fold. Despite low sequence homology between GHKL proteins, the 3D
structures of their ATP binding domains are almost superimposable [2–4].
There are many known crystal structures of E. coli GyrB with bound inhibitors (for
example PDB: 4ZVI [5], 1KZN [6], 6F86 [7], 6F8J [7], 6F94 [7], 6F96 [7], 5MMN [8], 5MMO
[8], 5MMP [8], 4DUH [9], 3G7E [10]) and recently a full structure of E. coli gyrase was
resolved with cryo-EM (PDB: 6RKU [11]). On the other hand, the crystal structure of ATPase
domain of topo II with bound inhibitor is not yet known, however, crystal structures with ATP
analogues are available (PDB: 1ZXM [12], 1ZXN [12]). Figure 1 shows the comparison of the
binding poses of ATP analogues adenosine diphosphate (ADP) and adenylyl-imidodiphosphate
3

(AMP-PNP) in the ATP binding site of E. coli GyrB and topo II. The main interactions in both
enzymes are hydrogen bonds of the adenine ring with Asp73 of E. coli GyrB or Asn120 of topo
II and interactions of the phosphate moiety with Mg(II) ions and multiple amino acid residues
in the phosphate binding pocket.
Figure 1: Comparison of binding conformation of ADP in the ATP binding site of E.
coli DNA gyrase (PDB: 4PRV, carbon atoms coloured in green) and binding pose of AMP-
PNP (a non-hydrolysable analogue of ATP) in the ATP binding site of topo IIα (PDB: 1ZXM,
carbon atoms coloured in purple) after binding site alignment. Key interactions of both ligands
with Asp73/Asn120 and Mg(II) ions are shown as dashed lines. Figure was generated using
Schrödinger Release 2016-2 (Binding site alignment, Schrödinger, LLC, New York, NY, USA,
2016).
Topo II exists in two isoforms, α and β. The expression of topo IIα is dependent on the
cell cycle step and is distinctive for the proliferating cells, and essential for their survival [13].
The main function of the α isoform is to release the DNA topology strains that are created
during DNA replication and mitosis [14]. Topo IIβ is expressed in all post-mitotic cells
independent of the cell cycle, and while it shows similar function to α isoform in vitro, its in
vivo role is poorly understood. Topo IIβ seems not to be essential in the proliferating cells,
however, the current findings support its important function in local regulation of chromatin
architecture. In mice, the genetic deletion of topo IIβ resulted in perinatal defects and death
[14,15].
Topo II inhibitors can be divided into two large groups, topo II poisons and catalytic
inhibitors. Clinically successful topo II inhibitors belong in the first group, which act through
stabilising the covalent enzyme–DNA complex, either through slowing down the religation step
4

or speeding up the complex formation. As a consequence, increased levels of damaged DNA
are present in the cells, which – if not repaired – leads to cell death. Catalytic inhibitors act
through various mechanisms, such as blocking the ATP binding site, preventing the binding of
topo II on DNA, or inhibiting the formation of covalent cleavable complex. The only approved
drug from this group of inhibitors is dexrazoxane, which inhibits topo II cleavage reaction, but
is only used in the clinic in combination with doxorubicin to antagonise its cardiotoxicity [1,16].
Topo II poisons used in the clinic were discovered in the 1960s and 1970s and have
remained a clinically important class of anticancer drugs. There are some issues connected to
the use of topo II poisons in therapy, such as toxicity (eg. secondary leukaemia, cardiotoxicity
and myelosuppression) and development of resistance [1,17]. Because of these liabilities,
catalytic inhibitors of topo II represent an interesting alternative to topo II poisons with
potentially lower toxicity. Among catalytic inhibitors, ATP-competitive inhibitors show good
promise, especially considering the recent clinical success of some other ATP-competitive
inhibitors, such as kinase inhibitors [18,19]. Additionally, the unique GHKL ATP binding site
architecture makes the non-specific binding of topo II inhibitors to non-GHKL kinases unlikely
[4,20]. Several ATP-competitive inhibitors of topo II have been reported, most of which are
purine analogues, including the most promising inhibitor described so far, QAP1, which was
developed by Novartis. The inhibitory activity of QAP1 on topo II was determined with ATPase
assay (IC50 = 128 nM) and its cytotoxicity was evaluated with MTT assay on SK-BR-3 and
MCF-7 cancer cell lines (IC50 values 10 µM and 32 µM respectively) [21,22]. Other structural
types of topo II ATP-competitive inhibitors include thiosemicarbazones [23], N-fused
imidazoles [24], urothilins [25], xanthones [26] and triazinones [27]. Novobiocin, a known
ATP-competitive inhibitor of bacterial topoisomerases, also shows very weak inhibitory
activity on topo II (IC50 = 650 µM), however, it is highly selective for bacterial topoisomerases
(E. coli DNA gyrase IC50 = 98 nM) [28].
2. RESULTS AND DISCUSSION
2.1. Screening of library of bacterial topoisomerase inhibitors
We have recently discovered several new classes of ATP-competitive inhibitors of
bacterial topoisomerases [5,29–34]. Our in-house library is structurally based on natural
products oroidin and kibdelomycin. The latter shows antibacterial activity and binds to the ATP
binding site of bacterial topoisomerases [35,36]. Both natural compounds share a pyrrolamide
moiety which seems to be important for the binding in the ATP binding site (Figure 2). Indeed,
5

the crystal structure of kibelomycin in ATP binding site of S. aureus DNA gyrase shows that
this part of the molecule forms a hydrogen bond with Asp81 (Asp73 according to E. coli DNA
gyrase numbering) [37].
OH
O
O
H2N
N
O
O
O
O
O
O
O
O
O
H
OH
H
O
N
N
N
N
N
Br
NH2
H
H
NH
HO
Cl
Cl
Br
oroidin
kibdelomycin
Figure 2: Structures of natural compounds oroidin and kibdelomycin with highlighted
pyrrolamide moieties.
Approximately 1000 ATP-competitive pyrolamide-type inhibitors of DNA gyrase and
topoisomerase IV were synthesized in the previous work of our research group. From this
library, 112 structurally diverse representative compounds were selected and screened against
human topo IIα using relaxation assay. The majority of molecules contained a pyrrole moiety
or its bioisostere that can – similarly as in oroidin and kibdelomycin – bind to the adenine
binding pocket and form a hydrogen bond with Asp73. This binding mode was confirmed with
the obtained crystal structures of two inhibitors from this library in the ATP binding site of E.
coli DNA gyrase [31,34]. Based on the central scaffold, the selected compounds can be divided
into derivatives of the 4,5,6,7-tetrahydrobenzothiazole (25 compounds), benzene (24
compounds), piperidine or piperazine (14 compounds), benzothiazole (12 compounds), thiazole
(6 compounds), benzoxazine (6 compounds), pyrrole (5 compounds), and miscellaneous
compounds (20 compounds). Structures of all 112 screened compounds are shown in Table S1
of the Supporting information. Compounds were screened at 100 µM and 10 µM
concentrations. For compounds with residual activities lower than 60% at 100 µM, IC50 values
were determined using seven concentration points. This initial screening provided nine hit
compounds with IC50 values lower or equal to 120 µM, at hit rate of approximately 8%.
Structures of hit compounds 1-9 and their activities are presented in Figure 3.
Out of nine hits, five had a benzene central scaffold, two had a benzothiazole scaffold,
one had a thiazole, and one had an indole central scaffold. The most potent compound was
benzene derivative 6 with an IC50 of 7.2 µM. A preliminary SAR was estimated for this series.
6

The substituents on the pyrrole ring have a major effect on the activity of compounds. In all
structural types, compounds with 3,4-dichloro-5-methylpyrrole showed much stronger
activities than compounds with 4,5-dibromopyrrole. Compounds with other substituents on the
pyrrole ring (e.g. monochloro, monobromo or dichloro) were inactive. For benzene-type
compounds, it seems that 1,2,4 substitution pattern on the benzene ring is necessary for potent
activity, with isopropoxy group (6; IC50 = 7.2 µM) being preferred to methoxy group (3; IC50
= 29 µM) in position 2. All nine hits had an acidic group on the right-hand side of the molecules
– eight compounds contained a carboxylic acid moiety, while one had an oxadiazolone ring as
a carboxylic acid bioisostere – which seems necessary to obtain the activity. In the ATP binding
site of E. coli DNA gyrase this group makes important contacts with Arg136 [31,34]. The
activities of hit compounds on topo IIα and E. coli DNA gyrase are shown in Figure 3
[29,30,32,34,38,39].
H
O
O
N
OH
O
OH
OH
H
O
N
O
N
O
O
N
H
H
H
H
N
Br
O
N
O
N
H
N
N
Br
H
H
N
N
O
O
Br
O
Cl
Br
Cl
2
3
1
topo II IC50 = 100  10 M
E. coli GyrB IC50 = 0.16 M
topo II IC50 = 29  10 M
E. coli GyrB IC50 = 0.12 M
topo II IC50 = 120  10 M
E. coli GyrB IC50 = 0.020 M
H
O
N
O
O
O
OH
H
N
O
N
O
O
N
NH
N
H
NH
O
O
H
H
N
Br
O
N
N
H
S
N
N
H
H
N
N
OH
O
Br
Cl
O
Cl
Cl
Cl
4
5
6
topo II IC50 = 12  1 M
E. coli GyrB IC50 = 0.043 M
topo II IC50 = 120  10 M
E. coli GyrB IC50 = 0.045 M
topo II IC50 = 7.2  1.7 M
E. coli GyrB IC50 = 0.047 M
O
N
H
N
S
N
H
Br
NH
H
O
N
O
O
S
H
R
R
N
Br
OH
N
N
H
O
OH
Cl
Cl
OH
8
9
7
topo II IC50 = 120  10 M
E. coli GyrB IC50 = 0.028 M
topo II IC50 = 8.8  1.4 M
E. coli GyrB IC50 = 0.098 M
topo II IC50 = 41  2 M
E. coli GyrB IC50 = 40 M
Figure 3: Structures and inhibitory activities of hit compounds identified in the initial
screening on topo IIα. The structure of 8 is not yet published and therefore not fully disclosed
in the figure.
7

Because the greatest number of hits had a benzene central scaffold, and the most potent
inhibitor 6 belonged to this structural type, we evaluated additional 33 benzene-type compounds
from our in-house library, which provided additional 11 hits. This, more focused, screening
resulted in considerably higher, 33% hit rate. Results and structures of the tested compounds
10-42 are shown in Table 1.
Table 1. Topo IIα inhibitory activities of additional 33 compounds with benzene central
scaffold and etoposide as positive control.
R4
O
H
N
R5'
R4'
N
H
R3
R2
R3'
Compoun
d
Topo IIα
R3’
R4’
R5’
R2
R3
-H
R4
(µM)
O
H
N
OH
10
-Cl -Cl -Me
5.0 ± 0.7
O
O
NH
N
H
S
N
11
12
13
14
15
16
17
18
19
-Cl -Cl -Me
-Cl -Cl -Me
-Cl -Cl -Me
-H
-H
-H
-H
-H
-H
-H
-H
-H
12 ± 1
13 ± 7
O
O
O
O
O
O
O
O
O
O
H
N
OH
OH
OH
O
O
O
H
N
26 ± 16
34 ± 3
O
H
N
-H
-Br -Br
O
NH
N
H
O
N
-Cl -Cl -Me
-Cl -Cl -Me
-Cl -Cl -Me
-Cl -Cl -Me
-Cl -Cl -Me
45 ± 12
45 ± 14
59 ± 23
63 ± 38
75 ± 39
O
O
O
O
H
N
NH
NH
NH
O
O
O
H
N
OH
OH
O
O
O
H
N
O
O
H
N
NHNH2
O
8

O
H
NH
N
20
21
22
-Cl -Cl -Me
-H
-H
-H
86 ± 41
> 100a
> 100
O
O
O
O
OH
O
-H
-H
-Br -Br
-Br -Br
O
O
H
N
O
O
OH
OH
OH
OH
OH
O
O
O
O
O
O
O
O
O
H
N
23
24
25
-H
-Br -Br
-H
-H
-H
> 100
> 100
> 100
H
N
NH
-Cl -Cl -Me
-Cl -Cl -Me
O
H
N
NBoc
O
H
N
NH2
26
27
28
-H
-H
-Br -Br
-Br -Br
-H
-H
-H
> 100
> 100
> 100
O
O
O
O
O
O
H
N
-Cl -Cl -Me
NHNH2
O
O
O
O
H
N
29
-H
-H
-Br -Br
-Br -Br
-H
OH
> 100
O
H
N
30
31
32
-H
-H
-H
> 100
> 100
> 100
N
O
O
O
O
O
O
H
N
NH2
NH2
-Cl -Cl -Me
-Cl -Cl -Me
O
O
O
O
O
H
N
OH
O
H
N
NH2
33
-H
-H
-Br -Br
-Br -Br
-H
> 100
O
O
O
O
H
N
OH
OH
NH2
34
35
-H
-H
> 100
> 100
O
O
H
N
-Cl -Cl -Me
-H
9

O
O
H
N
NH
NH
36
-Cl -Cl -Me
-H
> 100
O
O
O
O
O
H
N
37
38
39
-Cl -Cl -Me
-Cl -Cl -Me
-Cl -Cl -Me
-H
-H
> 100
> 100
> 100
OH
O
O
N
N
O
O
O
H
N
-H
OH
OH
OH
O
O
O
O
H
N
N
40
41
-H
-Br -Br
-H
> 100
> 100
O
O
H
N
-H
-H
-Br -Br
-Br -Br
-H
-H
OH
42
OH
> 100
O
Etoposide
71 ± 29
a>100 = residual activity at 100 µM higher than 60%.
The activities of additional 33 compounds are in good agreement with the preliminary
SAR of benzene-type compounds that was derived from initial screening. Analogues with
dichloromethylpyrrole moiety (R3’ = R4’ = Cl, R5’ = CH3; 6, 12) were more potent than
analogues with dibromopyrrole moiety (R3’ = H, R4’ = R5’ = Br; 2, 13). As substituent R2,
isopropoxy group seems to be preferred, however, also some alkyl or cycloalkyl substituents
with basic nitrogens are tolerated, for example compound 16 with piperidinyl substituent (IC50
= 45 µM). Compound 35, an analogue of 6 with hydrogen as substituent R2, is inactive, which
indicates that larger substituents are needed in this position for tight binding. Switching the
position of isopropoxy group from R2 to R3 also led to inactivity, as seen by comparing 6 with
39. Out of 20 hits from initial and focused screening, 14 possess a carboxylic moiety on the
right-hand side (1, 2, 3, 4, 6, 7, 8, 9, 10, 12, 13, 14, 17, 18), while the rest possess a cyclic
carboxylic bioisostere (5, 11, 15), ester group (16, 20) or hydrazide group (19) at that position.
Two compounds that have IC50 values lower than 10 µM both contain a carboxylic moiety (6,
10).
All twenty hits were tested in MCF-7 breast cancer cell line (MTS assay) to evaluate
their cytotoxic activity. Five compounds (11, 15, 16, 19, 20) showed IC50 values lower than 50
µM (Table 2). Those five compounds did not possess a carboxylic moiety in their structure.
Even though compounds with carboxylic groups on the right-hand side generally had the best
10

on-target activity, they were all inactive in the cell-based assays, which suggests that the acidic
nature of compounds could hinder their cell membrane permeation, making them unable to
reach their target within cells. An alternative explanation is, that compounds with carboxylic
moiety are better substrates of efflux pumps. Compound 11 was the only compound without
carboxylic moiety in its structure that showed topo IIα IC50 lower than 30 µM, while 15, 16, 19
and 20 only showed a moderate activity on topo IIα (IC50 = 45, 45, 75 and 86 µM). Despite
that, both ester group-containing compounds, 16 and 20, had the most potent cytotoxic activity
in MCF-7 cancer cell line out of all tested compounds, with IC50 values lower than 10 µM,
which is significantly better than positive control etoposide (IC50 = 25 µM). Moderate on-target
activity of those two compounds suggests that topo IIα inhibition might not be the dominant
mechanism of their cytotoxicity. Compound 20 was additionally tested in HepG2 liver cancer
cell line, where it showed a similar cytotoxic activity as in MCF-7 cell line (HepG2 IC50 = 10
± 1 µM), and in non-cancerous human umbilical vein endothelial cell line (HUVEC), where it
was inactive at 50 µM (87% viability).
Table 2. Results of cytotoxicity testing on MCF-7 cancer cell line for compounds with viability
lower than 50% at 50 µM, and for etoposide that was used as a positive control.
Compound
MCF-7 IC50 (µM)
36 ± 13
O
O
O
N
H
S
H
N
N
NH
N
H
O
Cl
Cl
11
O
O
O
N
H
O
H
N
N
NH
N
H
O
46 ± 16
Cl
Cl
15
O
O
O
N
H
H
O
N
N
H
O
8.7 ± 1.1
Cl
Cl
NH
16
11

O
NHNH2
O
N
H
H
O
N
N
H
19 ± 8
O
Cl
Cl
19
O
O
O
N
H
H
O
N
N
H
O
8.2 ± 1.0
25
Cl
Cl
NH
20
Etoposide
Compounds 11 and 15 share a very similar molecular structure, the only difference
being their stereochemistry and the type of the bioisostere group that is attached to their right-
hand sides – 11 carries an oxadiazolthione ring while 15 carries an oxadiazolone ring. While
11 has a better on-target activity, both seem to have similarly potency in MCF-7 cancer cell
line, with IC50 values between 30 µM and 50 µM. Compound 19 is a derivative of 6 with
carboxylic group replaced with hydrazide group. While this structural modification decreased
the on-target activity by approximately ten-fold, 19 obtained a cytotoxic activity. This could,
however, be associated to the well-known general toxicity of hydrazine derivatives [40].
These data therefore suggests that we have identified a novel chemotype of topo IIα
inhibitors, 1,2,4-substituted N-phenylpyrrolamides, that, depending on the nature of the
substituents on the benzene ring, have potential to be optimized into potent cytotoxic agents.
2.2. Exploration of initial hits
After the identification of topo IIα hit compounds that originated from the library of
bacterial DNA gyrase inhibitors, a molecular docking study was performed to predict the
binding poses of the inhibitors in the ATP binding site of topo IIα. For docking, crystal structure
of ATPase domain of topo IIα in complex with AMP-PNP was used (PDB: 1ZXM). As an
example, the predicted binding pose of 6 is shown in Figure 4. The pyrrolamide moiety of 6 is
predicted to bind to the adenine binding pocket of the ATP binding site where its NH and CO
groups form hydrogen bonds with Asn120 and a conserved water molecule. This interaction is
similar to the interactions of the pyrrolamide NH with Asp73 and a conserved water molecule
12

that were observed in crystal structures of related compounds in ATP binding site of E. coli
DNA gyrase [5,9]. Additionally, the interaction with Asn120 is one of the main features of
known purine-like and triazinone inhibitors of topo IIα [27,41]. Methyl group and halogen
atoms attached to the pyrrole moiety occupy the hydrophobic pocket of the adenine binding
site. Based on the docking results, it seems that the size of the hydrophobic pocket is better
suited for dichloromethylpyrrole compared to dibromopyrrole, which is in good agreement with
topo IIα inhibition results (shown in Supporting information, Figure S3). Substituent in position
four (with respect to the pyrrolamide group) on the central benzene ring forms additional
contacts with some polar amino acids; the amide group is predicted to form hydrogen bonds
with Asp94 and Asn150, while the terminal carboxylic moiety is predicted to form a salt bridge
with Lys157. Isopropoxy group in position two is oriented towards the lipophilic part of the
binding site. The substituents in position two and four occupy different binding pockets
compared to known inhibitors, thus making the binding mode of this novel chemotype of ATP-
competitive inhibitors of topo IIα unique.
Figure 4. Predicted binding mode of 6 in ATP binding site of topo IIα based on
molecular docking. Main hydrogen bonds are shown in yellow dashed lines and ionic bond in
purple dashed line. Also shown are amino acid residues that are forming main hydrophobic
contacts.
Based on the SAR of initial inhibitors and information derived from their predicted
binding poses in the active site of topo IIα, we prepared a plan for further exploration of initial
13

hits. Our main goal was to extract the smallest required binding motif for this novel type of
inhibitors. To do that, some smaller, fragment-like structures were designed in which each of
the substituents on the benzene ring were systematically shortened. Because primary hits have
a relatively high molecular weight and low ligand efficiency on topo IIα, finding a low
molecular weight inhibitor that would retain the activity of the initial hits is essential for the
success of further optimization steps. In addition to improving ligand efficiency, the goal of this
optimization attempt was to modify the acido-basic properties and lipophilicity of compounds
to see how those modifications affect their on-target and cytotoxic activities. Because the
presence of carboxylic moiety was shown to impair the cellular activity of compounds, we
designed some analogues in which this functional group was absent. On the other hand, because
initial hits 17 and 20, containing a basic centre, showed the most potent cytotoxic activities, a
basic heterocycle was introduced to some of the designed inhibitors. Based on these objectives,
we prepared an exploration plan that is schematically presented in Figure 5.
- effect of substitution in positions three and five
H
position four
O
O
N
H
O
pyrrolamide moiety
OH
NH2
O
OH
O
O
O
H
N
H
H
H
O
N
N
N
N
N
N
H
N
H
H
H
O
O
Cl
Cl
O
position two
N
H
H
OH
O
- effect of size
- effect of polarity
- variation in acido-basic properties
NH
Figure 5. Schematic representation of exploration plan, based on compound 6.
2.2.1. Synthesis
We synthesized 13 new compounds according to the synthetic pathways shown in the
schemes below. Schemes 1-3 show synthesis of analogues of 6 with shorter substituents in
positions two or four of the central benzene ring (46, 52, 56, 61). Scheme 2 additionally shows
synthesis of compound 53 with ethylurea group replacing the pyrrole moiety. In scheme 4,
synthesis of compound 68 with the reversed amide group in position four, compared to 6, is
presented. Scheme 5 shows synthesis of 73a with ethylurea group in position five and
isopropoxy group in position two, and its derivatives 74a with a basic heterocycle in position
two, and 74b-c with a basic heterocycle in position two and a methyl or phenyl group replacing
14

the pyrrole moiety. In scheme 6, synthesis of 80, a positional isomer of compound 52, where
the isopropoxy group is attached to position three instead of position two, is presented.
Generally, the synthetic pathway started with modification of carboxyl (Fischer ester
formation) or amino group (Boc protection or acylation) of the starting amino/nitro-
hydroxybenzoic acid or amino/nitro-phenol, where applicable. Then Mitsunobu reaction was
used to prepare the aromatic ether, which was followed by ammonolysis of ester in cases where
final product had a primary amide group. Next, catalytic hydrogenation with Pd/C as the
catalyst was performed to obtain aromatic amine, which was then coupled with pyrrole-2-
carboxylic acid acid chloride or ethyl isocyanate. Where applicable, the final reaction
constituted of Boc deprotection with 4 M HCl in dioxane or alkaline hydrolysis of ester groups.
O
H
N
H2N
N
H
O2N
a
O2N
b
c
O
O
OH
O
Cl
Cl
43
44
45
46
Yield 86%
Yield 95%
Yield 62%
Scheme 1. Reagents and conditions: a) isopropanol, PPh3, DIAD, THF, rt, 15h; b) H2,
Pd/C, MeOH, rt, 15 h; c) i: 3,4-dichloro-5-methylpyrrole-2-carboxylic acid, oxalyl chloride,
anhydrous CH2Cl2, rt, 15 h, ii: anhydrous pyridine, anhydrous CH2Cl2, rt, 15 h.
O
O
O
O
O
O
NH2
NH2
O
NH2
b
c
d
c
R
R1
N
O2N
O2N
H2N
H
O2N
O
O
O
O
OH
a
50
51
49
47
48
(Yield 61%): R1 = 3,4-dichloro-5-methlypyrrole-2-yl
(Yield 99%): R1 = NHCH2CH3
Yield 72%
Yield 77%
Yield 93%
52
53
(R = OH)
(R = OCH3)
Yield 93%
O
O
O
NH2
NH2
O
NH2
f
c
d
H
48
N
O2N
H2N
N
H
OH
54
Yield 66%
OH
OH
Cl
55
Cl
56
Yield 97%
Yield 50%
Scheme 2. Reagents and conditions: a) H2SO4, MeOH, reflux, 15 h; b) isopropanol,
PPh3, DIAD, THF, rt, 15 h; c) H2, Pd/C, MeOH, rt, 3-15 h d) i: 3,4-dichloro-5-methylpyrrole-
2-carboxylic acid, oxalyl chloride, anhydrous CH2Cl2, rt, 15 h, ii: anhydrous pyridine,
15

anhydrous CH2Cl2, rt, 15 h (52, 56) or ethyl isocyanate, CHCl3, rt, 15h (53); e) NaOH, MeOH,
reflux, 5h; f) NH3, MeOH, 70 °C, 15 h.
O
O
O
O
NH2
c
d
H
R
NH2
N
N
H
O2N
a
H2N
Cl
Cl
b
60
Yield 93%
61
Yield 35%
57
58
(R = OCH3) (R = NH2)
Yield 70% Yield 98%
59
(R = OH)
Scheme 3. Reagents and conditions: a) H2SO4, MeOH, reflux, 15 h; b) NH3, MeOH, 70
°C, 15 h; c) H2, Pd/C, MeOH, rt, 15 h d) i: 3,4-dichloro-5-methylpyrrole-2-carboxylic acid,
oxalyl chloride, anhydrous CH2Cl2, rt, 15 h, ii: anhydrous pyridine, anhydrous CH2Cl2, rt, 15
h.
NO2
NH2
NO2
NO2
a
b
c
BocHN
BocHN
H2N
BocHN
O
O
OH
OH
65
62
63
64
Yield 90%
Yield 74%
Yield 49%
H
H
N
H
N
N
O
d
e
f
H
O
O
O
N
BocHN
-Cl+H3N
N
H
O
O
O
Cl
Cl
66
Yield 80%
68
67
Yield 35%
Yield 69%
Scheme 4. Reagents and conditions: a) i: Boc2O, DMAP, dioxane, rt, 15 h, ii: 1 M
NaOH, MeOH, rt, 2 h, iii: 1 M HCl to neutral pH; b) isopropanol, PPh3, DIAD, THF, rt, 15 h;
c) H2, Pd/C, MeOH, rt, 12 h; d) acetyl chloride, Et3N, CH2Cl2, rt, 6 h; e) HCl, dioxane, rt, 15 h;
f) i) i: 3,4-dichloro-5-methylpyrrole-2-carboxylic acid, oxalyl chloride, anhydrous CH2Cl2, rt,
15 h, ii: anhydrous pyridine, anhydrous CH2Cl2, rt, 15 h.
O
O
O
O
NH2
HN
N
HN
N
HN
N
HN
N
H
H
H
a
b
c
d
H
O
O2N
O2N
H2N
2R
N
O2N
OH
H
O
O
O
R1
R1
R1
OH
(impure intermediates) R1 = isopropyl
69
70
Yield 69%
73
71a, 72a
71b
:
(Yield 67%)
(Yield 59%) R1 = N-Boc-piperidine-4-yl
, 72b
:
(Yield 81%) R1 = isopropyl, R2 = 3,4-dichloro-5-methlypyrrole-2-yl
73a
73b
73c
73d
:
(Yield 58%) R1 = N-Boc-piperidine-4-yl, R2 = 3,4-dichloro-5-methlypyrrole-2-yl
(Yield 70%) R1 = piperidine-4-yl, R2 = 3,4-dichloro-5-methlypyrrole-2-yl
:
74a
74b
74c
:
e
(Yield 66%) R1 = N-Boc-piperidine-4-yl, R2 = methyl
(Yield 86%) R1 = piperidine-4-yl, R2 = methyl
:
:
(Yield 43%) R1 = N-Boc-piperidine-4-yl, R2 = phenyl
(Yield 33%) R1 = piperidine-4-yl, R2 = phenyl
:
:
16

Scheme 5. Reagents and conditions: a) ethyl isocyanate, CHCl3, rt, 15 h; b) 1-Boc-4-
hydroxypiperidine (71a) or 2-propanol (71b), PPh3, DIAD, THF, rt, 15 h; c) H2, Pd/C, MeOH,
rt, 12 h; d) i: 3,4-dichloro-5-methylpyrrole-2-carboxylic acid, oxalyl chloride, anhydrous
CH2Cl2, rt, 15 h, ii: anhydrous pyridine, anhydrous CH2Cl2, rt, 15 h (73a, b) or acetyl chloride,
Et3N, CH2Cl2, rt, 6 h (75c) or benzyl chloride, Et3N, CH2Cl2, rt, 6 h (73d); e) HCl, dioxane, rt,
15 h.
O
O
O
O
O
O
O
NH2
b
c
e
O
NH2
H
R
N
N
O
O2N
R
H
O2N
OH
Cl
Cl
a
d
77
Yield 80%
80
Yield 42%
75
(R = OH)
76
78
79
(R = OCH3)
Yield 93%
(R = NO2) (R = NH2)
Yield 96% Yield 97%
Scheme 6. Reagents and conditions: a) H2SO4, MeOH, reflux, 15 h; b) isopropanol,
PPh3, DIAD, THF, rt, 15 h; c) NH3, MeOH, 70 °C, 15h; d) H2, Pd/C, MeOH, rt, 15h e) i:3,4-
dichloro-5-methylpyrrole-2-carboxylic acid, oxalyl chloride, anhydrous CH2Cl2, rt, 15 h, ii:
anhydrous pyridine, anhydrous CH2Cl2, rt, 15 h.
2.2.2. In-vitro evaluation of the new series of compounds
Thirteen new compounds were synthesized and tested for their inhibitory activity on
topo IIα with relaxation assay (Table 3). Firstly, attempts were made to reduce the size of the
molecule while retaining the activity and thus increasing ligand efficiency compared to the
original hits. It has already been concluded that replacing the isopropoxy substituent in position
two of the benzene ring with the hydrogen atom leads to inactivity (comparison of 6 to 35). To
test the importance of substituents in position four of the benzene ring, we synthesized
compound 46, an analogue of 6 with a hydrogen atom in position four. Compound 46 was
inactive, showing that substitution in position four is essential for activity. Next, we prepared
compound 52 with the primary amide group in position four. This compound showed a very
potent topo IIα inhibitory activity with IC50 value of 3.2 µM, indicating that the activity of
compound 6 can be retained or even slightly improved with reducing the size of substituent in
position four. With this change, the molecular weight dropped from 428.3 (6) to 370.2 (52) and
the ligand efficiency (LE = 1.4 × (pIC50)/(heavy atom count) [42]) increased from 0.26 (6) to
0.32 (52), therefore making the molecule easier to optimise. Further, some analogues of 52 were
17

prepared: 56 and 61 in which the primary amide group was retained but hydroxyl group (56) or
hydrogen atom (61) were introduced to position two; 68 in which a primary amide group was
reversed as the acetamide substituent was added; and 80, a positional isomer of 52 with
isopropoxy group in position three instead of two. All these modifications resulted in the loss
of activity. To assess the importance of the pyrrolamide moiety, this part of the molecule was
replaced with ethylurea group (53), which – similarly to the pyrrolamide moiety – can bind to
the adenine pocket of bacterial topoisomerases [8,43,44]. This structural modification resulted
in the loss of activity.
To additionally explore the binding site, a series of compounds with the ethylurea
substituent in position five was prepared. Compound 73a with isopropoxy substituent in
position two was inactive. Furthermore, derivative 74a was synthesized, in which the isopropyl
group was replaced with piperidine ring that is basic in nature, which has been proven to be
beneficial for the cytotoxic activity of the initial hits 17 and 20. Compound 74a showed a
moderate topo IIα inhibitory activity with IC50 value of 38 µM. To test if the pyrrolamide moiety
of 74a is essential for the activity of this compound, we prepared 74b and 74c in which the
pyrrole moiety was replaced with methyl or phenyl groups. Both of those compounds were
inactive.
Overall, the results suggest that the pyrrolamide moiety is essential for on-target
activity. Also essential is the substituent in position two, which should be bound to the phenyl
ring through an oxygen atom, can carry groups with basic centres, and should not be a hydroxyl
group. The substituent in position four is also crucial for activity but can be shortened to the
primary amide group. The exception is compound 74a that has moderate activity but does not
contain substituents in position four but in position five.
Both active compounds (52 and 74a) were also evaluated for their inhibitory activities
on bacterial topoisomerases DNA gyrase and topoisomerase IV from bacteria E. coli and S.
aureus (Supporting information, Table S2). While their activity on E. coli DNA gyrase is lower
than the activity of the majority of the initial hits (1 - 20), their IC50 values on E. coli DNA
gyrase remain about 10-fold lower than their IC50 values on topo IIα. The activities of these two
compounds on other tested bacterial topoisomerases (E. coli topoisomerase IV and S. aureus
DNA gyrase and topoisomerase IV) were weaker (IC50 > 10 µM).
18

Table 3. Enzymatic inhibitory activities of the synthesized compounds and etoposide
as positive control.
R5
R4
R3
O
R1
N
H
R2
Topo IIα
Compound
R1
R2
R3
R4
-H
R5
H
(µM)
H
N
46
-H
> 50 µM
O
O
Cl
Cl
Cl
Cl
H
N
O
52
53
56
-H
-H
-H
H
H
H
3.2 ± 1.8
> 50 µM
> 50 µM
NH2
NH2
NH2
O
O
O
N
H
N
H
O
H
N
-OH
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
N
O
61
68
-H
-H
-H
-H
-H
H
> 50 µM
> 50 µM
> 50 µM
38 ± 10
NH2
O
H
N
H
N
H
O
O
H
N
O
73a
74a
-H
N
H
N
H
H
N
O
NH
-H
N
H
N
H
O
O
NH
NH
74b
74c
CH3
-H
-H
-H
-H
> 50 µM
> 50 µM
N
H
N
H
O
O
O
phenyl
N
H
N
H
H
N
80
-H
-CONH2
H
> 50 µM
71 ± 29
O
Cl
Cl
Etoposide
19

Compound 52 was additionally evaluated in topo IIα ATPase assays to confirm the
proposed ATP-competitive mode of inhibition. The observed rates of ATP hydrolysis were
plotted against ATP concentrations in the presence of 52 (0.1 µM - 2.5 µM), as shown in Figure
6. The graph shows that 52 decreases the rate of ATP hydrolysis, with a greater decrease at
higher concentrations of 52. The calculated KM value increased 3-4-fold in the presence of 0.25
µM 52 compared to no inhibitor, and increased further with inhibitor concentration (Supporting
information, Table S3). The IC50 value determined at 2 mM ATP concentration was 0.43 ± 0.16
µM and it steadily decreased at decreasing concentrations of ATP (Supporting information,
Table S4). These data support the proposed ATP-competitive mode of topo IIα inhibition.
4
3
2
0 µM52
0.1 µM52
0.25 µM52
0.5 µM52
1 µM52
2.5 µM52
0 µM52
0.1 µM52
0.25 µM52
0.5 µM52
1 µM52
1
0
2.5 µM52
-1
0.00.51.01.52.0
2.5
[ATP] (mM)
Figure 6. The rates of ATP hydrolysis plotted against ATP concentrations for
compound 52, determined using topo IIα ATPase assay.
Compounds 52 and 74a have been tested for their cytotoxicity on MCF-7 breast cancer
and HepG2 liver cancer cell lines (Table 4). Compound 52 shows no cytotoxicity at 50 µM,
while 74a shows cytotoxic activity in both tested cell lines. The IC50 values of 74a against MCF-
7 and HepG2 cell lines were 33 ± 4 µM and 38 ± 8 µM, respectively. Compound 74a was
additionally evaluated on non-cancerous HUVEC cell line, where it was inactive at 50 µM
(105% viability). The reasons for inactivity of 52 could be connected to problems with its
permeability through the cell membrane or with the active efflux of compound from the cells.
The activity of 74a confirms that this molecular scaffold is useful for the design of novel
cytotoxic agents, and that the presence of a basic centre is essential for the potent activity in
cancer cell lines, as also seen in 17 and 20. These findings will be used for further optimization
20

of this series of topo IIα inhibitors, using 52 that has a very potent on-target activity and lower
molecular weight as structural template.
Table 4. Results of cytotoxicity testing on MCF-7 and HepG2 cancer cell lines.
Compound
MCF-7 IC50 (µM) HepG2 IC50 (µM)
O
O
N
NH2
H
N
H
O
> 50
> 50
Cl
Cl
52
O
HN
N
H
O
H
N
N
33 ± 4
38 ± 8
H
O
Cl
Cl
NH2
Cl
74a
3. CONCLUSIONS
In this study, we have discovered a promising new class of ATP-competitive inhibitors
of human DNA topoisomerase II. In-house library of ATP-competitive inhibitors of bacterial
topoisomerases served us as an excellent starting point for our screening campaign. Out of
twenty identified hit compounds, fifteen shared a N-phenylpyrrolamide scaffold, including the
most potent hit 10 with IC50 value of 5.0 µM. To better understand the SAR of this new
structural type of topo IIα inhibitors, thirteen new compounds were designed and prepared. Two
new compounds retained inhibitory activity on topo IIα. Compound 52 has a potential to be
used as a template for further design of topo IIα inhibitors, as it has a potent inhibitory activity
with an IC50 value of 3.2 µM and increased ligand efficiency compared to initial hits. With
kinetic ATPase assays we confirmed the potent activity of 52 (IC50 = 0.43 µM) and its ATP-
competitive mode of topo IIα inhibition. All active compounds were tested for their cytotoxic
activity on MCF-7 cancer cell line and selected compounds also on HepG2 cancer cell line. Six
compounds showed activity in MCF-7 cancer cell line below 50 µM and the most potent, 16
and 20, had cytotoxic IC50 values lower than 10 µM.
4. MATERIALS AND METHODS
21

4.1. General chemistry information
Chemicals were obtained from Acros Organics (Geel, Belgium), Apollo Scientific
(Stockport, UK), Sigma-Aldrich (St. Louis, MO, USA) and TCI (Tokyo, Japan) and were used
without the preceding purification. Thin layer chromatography was performed on silica gel
Merck 60 F254 plates (0.25 mm), for visualization UV light (254 nm and 366 nm) and spray
reagents were used. Flash column chromatography was performed on silica gel 60 (particle size
240–400 mesh). 1H and 13C NMR spectra were recorded at 400 and 100 MHz, respectively, on
a Bruker AVANCE III 400 spectrometer (Bruker Corporation, Billerica, MA, USA). To
dissolve NMR samples, DMSO-d6 or CDCl3 (with TMS as an internal standard) were used.
High resolution mass spectra were obtained using a Exactive™ Plus Orbitrap Mass
Spectrometer (Thermo Fischer Scientific, Waltham, MA, USA) or low resolution mass spectra
on ADVION expression CMSL mass spectrometer (Advion, Ithaca, USA). HPLC analyses
were performed on a Thermo Scientific Dionex Ultimate 3000 Binary Rapid Separation LC
System (Thermo Fisher Scientific, Waltham, MA, USA), using a photodiode array detector and
Agilent Zorbax 80Å Extend-C18 column (5 µm, 4.6 mm × 150 mm). Chromeleon 7 CDC
software was used to control the instrument and process the data. The eluent consisted of 0.1%
TFA as solvent A and acetonitrile as solvent B. Method: 0-8 min 5% solvent B, 8-15 min 5%
to 95% solvent B, 15-16 min 95% solvent B, 16-18 min 95% to 5% solvent B, 18-21 min 5%
solvent B, flow rate 1.0 mL/min and sample injection volume 10 μL.
4.2. Synthetic procedures and analytical data
General procedure A. To a stirred solution of phenol (1 eq.) and triphenylphosphine (2
eq.) in anhydrous tetrahydrofuran (10 mL/mmol), alcohol (2 eq.) was added and the mixture
was stirred at rt for 10 min. Diisopropyl azodicarboxylate (2 eq.) was added dropwise and the
mixture was stirred at rt for 15 h under argon atmosphere. The solvent was evaporated under
reduced pressure and the crude product was purified with flash column chromatography.
1-Isopropoxy-2-nitrobenzene (44) [45]. Synthesized according to General procedure
A from 43 (1.00 g, 7.19 mmol) and isopropanol (5.5 mL, 71.9 mmol). Crude product was
purified with flash column chromatography using hexane/ethyl acetate (2/1) as an eluent to give
1
44 as yellow solid. Yield 86% (1.12 g). H NMR (400 MHz, CDC3) δ = 1.41 (d, 6H, J = 6.1
Hz, CH(CH3)2), 4.69 (hept, 1H, J = 6.1 Hz, CH(CH3)2), 7.00 (ddd, 1H, J = 8.1, 7.4, 1.2 Hz, Ar-
H-4 or Ar-H-5), 7.10 (dd, 1H, J = 8.4, 1.1 Hz, Ar-H-3 or Ar-H-6), 7.50 (ddd, 1H, J = 8.5, 7.4,
1.8 Hz, Ar-H-4 or Ar-H-5), 7.78 (dd, 1H, J = 8.1, 1.7 Hz, Ar-H-3 or Ar-H-6) ppm.
22

General procedure B. Nitro compound (1 eq.) was dissolved in MeOH (20 mL/mmol)
and the mixture was stirred for 30 min under argon atmosphere. Pd/C (20 m/m%) was added
and the reaction mixture was stirred under hydrogen atmosphere for 2 h. The catalyst was
filtered off and the solvent removed under reduced pressure to obtain the product.
2-Isopropoxyaniline (45) [46]. Synthesized according to General procedure B from 43
(530 mg, 2.93 mmol). Compound 45 was obtained as a colourless oil. Yield 95% (420 mg). 1H
NMR (400 MHz, CDCl3) δ = 1.38 (d, 6H, J = 6.1 Hz, CH(CH3)2), 3.81 (s, 2H, Ar-NH2), 4.55
(hept, 1H, J = 6.1 Hz, CH(CH3)2), 6.68 – 6.87 (m, 4H, 4 × Ar-H) ppm.
General procedure C. To a solution of 3,4-dichloro-5-methyl-1H-pyrrole-2-carboxylic
acid (1.5 eq.) in anhydrous dichloromethane (10 mL/mmol), oxalyl chloride (7.5 eq.) was added
dropwise and the mixture was stirred at room temperature for 15 h under argon atmosphere.
The solvent was evaporated under reduced pressure, anhydrous dichloromethane (10
mL/mmol), amine (1 eq.) and pyridine (1 mL/mmol) were added and the reaction mixture was
stirred under argon atmosphere at room temperature for 15 h. If precipitated, the product was
filtered off from the reaction mixture.
3,4-Dichloro-N-(2-isopropoxyphenyl)-5-methyl-1H-pyrrole-2-carboxamide (46).
Synthesized according to General procedure C from 45 (66 mg, 0.44 mmol). Crude product was
suspended in hot methanol and left to cool in the refrigerator. Pure 46 was filtered off as grey
solid. Yield 62% (89 mg). 1H NMR (400 MHz, DMSO) δ = 1.33 (d, 6H, J = 6.0 Hz, CH(CH3)2),
2.23 (s, 3H, pyrrole-CH3), 4.76 (hept, 1H, J = 6.0 Hz, CH(CH3)2), 6.95 (td, 1H, J = 7.8, 1.3 Hz,
Ar-H-4 or Ar-H-5), 7.06 (td, 1H, J = 7.8, 1.6 Hz, Ar-H-4 or Ar-H-5), 7.13 (dd, 1H, J = 8.3, 1.3
Hz, Ar-H-3 or Ar-H-6), 8.39 (dd, 1H, J = 8.0, 1.6 Hz, Ar-H-3 or Ar-H-6), 9.13 (s, 1H, Ar-
NHCO), 12.40 (s, 1H, pyrrole-NH) ppm. 13C NMR (100 MHz, DMSO-d6) δ = 10.7, 21.8, 70.7,
108.4, 109.3, 112.9, 118.9, 119.0, 120.5, 123.6, 128.3, 129.3, 145.6, 156.1 ppm. HRMS for
C15H17Cl2N2O2 ([M+H]+): calculated 327.0662, found 327.0659. HPLC purity 100.0%.
General procedure D. To a stirring suspension of carboxylic acid (1 eq.) in methanol (2
mL/mmol), concentrated sulfuric acid (0.2 mL/mmol) was added dropwise and the mixture was
refluxed overnight. Methanol was evaporated under reduced pressure, saturated NaHCO3 was
added and extracted with ethyl acetate. Organic phase was dried over Na2SO4, filtered and
solvent was evaporated under reduced pressure.
Methyl 3-hydroxy-4-nitrobenzoate (48) [32]. Synthesized according to General
procedure D from 47 (3.01 g, 16.44 mmol). Compound 48 was obtained as yellow solid. Yield
23

93% (3.02 g). 1H NMR (400 MHz, DMSO) δ = 3.88 (s, 3H, COOCH3), 7.49 (dd, 1H, J = 8.5,
1.8 Hz, Ar-H-6), 7.68 (d, 1H, J = 1.4 Hz, Ar-H-2), 7.96 (d, 1H, J = 8.2 Hz, Ar-H-5), 11.50 (s,
1H, Ar-OH) ppm.
Methyl 3-isopropoxy-4-nitrobenzoate (49) [32]. Synthesized according to General
procedure A from 48 (3.00 g, 15.22 mmol) and isopropanol (2.34 mL, 30.4 mmol). The crude
product was purified with flash column chromatography using hexane/ethyl acetate (5/1) as an
eluent to give product as yellow oil. Yield 93% (3.64 g). 1H NMR (400 MHz, CDCl3) δ = 1.43
(d, 6H, J = 6.1 Hz, CH(CH3)2), 3.98 (s, 3H, COOCH3), 4.80 (hept, 1H, J = 6.1 Hz, CH(CH3)2),
7.67 (dd, 1H, J = 8.3, 1.6 Hz, Ar-H- 6), 7.76 (d, 1H, J = 1.6 Hz, Ar-H- 2), 7.78 (d, 1H, J = 8.3
Hz, Ar-H-5) ppm.
General procedure E. Ester (1 eq.) was weighed into a pressure tube and dissolved in 7
M ammonia solution in methanol (10 mL/mmol; reagent commercially available from Sigma
Aldrich). The solution was left to stir overnight at 70 °C after which methanol was removed
under reduced pressure.
3-Isopropoxy-4-nitrobenzamide (50). Synthesized according to General procedure E
from 49 (200 mg, 0.84 mmol). The product was purified with flash column chromatography
using dichloromethane/methanol (9/1) as an eluent to give 53 as yellow crystals. Yield 72%
(135 mg). 1H NMR (400 MHz, DMSO) δ = 1.31 (d, 6H, J = 6.0 Hz, CH(CH3)2), 4.90 (hept, J
= 6.1 Hz, CH(CH3)2), 7.55 (dd, 1H, J = 8.3, 1.6 Hz, Ar-H-6), 7.70 (bs, 1H, CONH2-A), 7.75
(d, 1H, J = 1.6 Hz, Ar-H-2), 7.90 (d, 1H, J = 8.3 Hz, Ar-H-5), 8.23 (bs, 1H, CONH2-B) ppm.
13C NMR (100 MHz, DMSO-d6) δ = 22.0, 72.9, 115.5, 119.9, 125.1, 139.4, 142.6, 150.2, 166.6
ppm. MS (ESI) m/z = 222.8 ([M-H]-).
4-Amino-3-isopropoxybenzamide (51). Synthesized according to General procedure
B from 50 (115 mg, 0.51 mmol). After removal of the solvent under reduced pressure, product
was obtained as brown oil. Yield 77% (77 mg). 1H NMR (400 MHz, DMSO) δ = 1.28 (d, 6H,
J = 6.0 Hz, CH(CH3)2), 4.55 (hept, 1H, J = 6.0 Hz, CH(CH3)2), 5.29 (bs, 2H, Ar-NH2), 6.63 (d,
1H, J = 8.2 Hz, Ar-H-5), 6.92 (bs, 1H, CONH2-A), 7.29 (dd, 1H, J = 8.2, 1.9 Hz, Ar-H-6), 7.35
(d, 1H, J = 1.8 Hz, Ar-H-2), 7.60 (bs, 1H, CONH2-B) ppm. 13C NMR (100 MHz, DMSO-d6) δ
= 22.5, 70.7, 113.0, 113.8, 122.0, 122.0, 142.7, 143.5, 168.5 ppm. MS (ESI) m/z = 194.9
([M+H]+).
N-(4-Carbamoyl-2-isopropoxyphenyl)-3,4-dichloro-5-methyl-1H-pyrrole-2-
carboxamide (52). Synthesized according to General procedure C from 51 (50 mg, 0.26 mmol).
24

The product suspended in toluene and filtered off from the reaction mixture as light grey solid.
Yield 61% (58 mg). 1H NMR (400 MHz, DMSO) δ = 1.36 (d, 6H, J = 6.0 Hz, CH(CH3)2), 2.24
(s, 3H, pyrrole,CH3), 4.80 – 4.90 (hept, 1H, J = 5.9 Hz, CH(CH3)2), 7.34 (s, 1H, CONH2-A),
7.53 (dd, 1H, J = 8.4, 1.8 Hz, Ar-H-5), 7.60 (d, 1H, J = 1.8 Hz, Ar-H-3), 7.96 (s, 1H, CONH2-
B), 8.44 (d, 1H, J = 8.4 Hz, Ar-H-6), 9.24 (s, 1H, Ar-NHCO), 12.46 (s, 1H, pyrrole-NH) ppm.
13C NMR (100 MHz, DMSO-d6) δ = 11.2, 22.3, 71.6, 109.1, 110.1, 112.2, 118.4, 119.2, 120.9,
129.6, 130.2, 131.4, 145.6, 156.7, 167.7 ppm. HRMS for C16H18Cl2N3O3 ([M+H]+): calculated
370.0720, found 370.0715. HPLC purity 96.9%.
General procedure F. To a stirring solution of amine (1 eq.) in chloroform (15
mL/mmol), cooled on ice bath, ethyl isocyanate (1.1 eq.) was added and the mixture was stirred
under argon atmosphere overnight at room temperature. 20 mL of dichloromethane was added
to the reaction mixture and the organic phase was washed with 1% citric acid, saturated
NaHCO3 solution and brine. Organic phase was dried over Na2SO4, filtered and solvent was
evaporated under reduced pressure.
4-(3-Ethylureido)-3-isopropoxybenzamide (53). Synthesized according to General
procedure F from 51 (78 mg, 0.40 mmol). Pure product was filtered off directly from the
reaction mixture as light brown solid. Yield 99% (105 mg). 1H NMR (400 MHz, DMSO) δ =
1.08 (t, 3H, J = 7.2 Hz, CH2CH3), 1.32 (d, 6H, J = 6.0 Hz, CH(CH3)2), 3.17 – 3.05 (m, 2H,
CH2CH3), 4.68 (hept, 1H, J = 6.0 Hz, CH(CH3)2), 7.16 (m, 2H, Ar-NH and CH3CH2NH), 7.41
(dd, 1H, J = 8.5, 1.8 Hz, Ar-H-6), 7.47 (d, 1H, J = 1.8 Hz, Ar-H-2), 7.81 (s, 2H, CONH2), 8.18
(d, 1H, J = 8.5 Hz, Ar-H-5) ppm. 13C NMR (100 MHz, DMSO-d6) δ = 15.7, 22.3, 34.3, 71.4,
112.8, 117.0, 120.9, 126.8, 134.1, 144.9, 155.2, 168.0 ppm. HRMS for C13H20N3O3 ([M+H]+):
calculated 266.1499, found 266.1498. HRMS for C13H20N3O3 ([M+H]+): calculated 266.1499,
found 266.1498. HPLC purity 99.2%.
3-Hydroxy-4-nitrobenzamide (54). Compound 48 (8.5 g, 43.1 mmol) was dissolved
in 100 mL of 25% aqueous ammonia solution and was stirred at 80°C overnight. The reaction
mixture was acidified with concentrated HCl after which the formed precipitate was filtered
off. The precipitate was dissolved in 500 mL of ethyl acetate and washed with saturated
NaHCO3 solution. After removal of the solvent under reduced pressure, product was obtained
as yellow solid. Yield 66% (5.20 g). 1H NMR (400 MHz, DMSO) δ = 7.41 (dd, 1H, J = 8.5, 1.8
Hz, Ar-H-6), 7.57 (d, 1H, J = 1.7 Hz, Ar-H-2), 7.62 (s, 1H, CONH2-A ), 7.93 (d, 1H, J = 8.5
25

Hz, Ar-H-5), 8.14 (s, 1H, CONH2-B), 11.19 (s, 1H, Ar-OH) ppm. 13C NMR (100 MHz, DMSO-
d6) δ = 118.3, 118.8, 125.6, 139.0, 140.5, 152.0, 166.7 ppm. MS (ESI) m/z = 180.8 ([M-H]-).
4-Amino-3-hydroxybenzamide (55). Synthesized according to General procedure B
from 54 (169 mg, 0.97 mmol). After removal of the solvent under reduced pressure, product
was obtained as brown oil. Yield 97% (143 mg). 1H NMR (400 MHz, DMSO) δ = 5.04 (s, 2H,
Ar-NH2), 6.54 (d, 1H, J = 8.1 Hz, Ar-H-5), 6.81 (bs, 1H, CONH2-A), 7.16 (dd, 1H, J = 8.1, 2.0
Hz, Ar-H-6), 7.20 (d, 1H, J = 2.0 Hz, Ar-H-2), 7.48 (bs, 1H, CONH2-B), 9.17 (s, 1H, Ar-OH)
ppm. MS (ESI) m/z = 150.8 ([M-H]-).
N-(4-Carbamoyl-2-hydroxyphenyl)-3,4-dichloro-5-methyl-1H-pyrrole-2-
carboxamide (56). Synthesized according to General procedure C from 55 (66 mg, 0.43 mmol).
The product was filtered off from the reaction mixture as off-white solid. Yield 50% (55 mg).
1H NMR (400 MHz, DMSO) δ = 2.24 (s, 3H, pyrrole-CH3), 7.23 (s, 1H, CONH2-A), 7.39 (dd,
1H, J = 8.4, 2.0 Hz, Ar-H-5), 7.46 (d, 1H, J = 2.0 Hz, Ar-H-3), 7.86 (s, 1H, CONH2-B), 8.30
(d, 1H, J = 8.4, Ar-H-6), 9.17 (s, 1H, Ar-OH or Ar-NH), 10.69 (s, 1H, Ar-OH or Ar-NH), 12.44
(s, 1H, pyrrole-NH) ppm. 13C NMR (100 MHz, DMSO-d6) δ = 11.2, 109.1, 110.4, 114.5, 118.7,
119.2, 119.2, 129.7, 129.9, 130.0, 146.1, 156.8, 168.0 ppm. HRMS for C13H12Cl2N3O3
([M+H]+): calculated 328.0250, found 328.0251. HPLC purity 96.9%.
Methyl 4-nitrobenzoate (58) [47]. Synthesized according to General procedure D from
57 (3.00 g, 18.0 mmol). Pure product was filtered off directly from the reaction mixture after
1
cooling down as yellow solid. Yield 70% (2.27 g). H NMR (400 MHz, DMSO) δ = 3.93 (s,
3H, COOCH3), 8.20 (d, 2H, J = 9.1 Hz, 2 × Ar-H), 8.36 (d, 2H, J = 9.0 Hz, 2 × Ar-H) ppm.
4-Nitrobenzamide (59) [48]. Synthesized according to General procedure E from 58
(1.20 g, 6.62 mmol). Product was obtained as yellow solid. Yield 98% (1.08 g). 1H NMR (400
MHz, DMSO) δ = 7.75 (bs, 1H, CONH2-A), 8.07 – 8.14 (m, 2H, 2 × Ar-H), 8.26 – 8.36 (m 3H,
2 × Ar-H and CONH2-B) ppm.
4-Aminobenzamide (60) [49]. Synthesized according to General procedure B from 59
(430 mg, 2.59 mmol). Product was obtained as colourless oil. Yield 93% (328 mg). 1H NMR
(400 MHz, DMSO) δ = 5.60 (s, 2H, Ar-NH2), 6.52 (d, 2H, J = 8.7 Hz, 2 × Ar-H), 6.84 (s, 1H,
CONH2-A), 7.51 (s, 1H, CONH2-B), 7.58 (d, 2H, J = 8.7 Hz, 2 × Ar-H) ppm.
N-(4-Carbamoylphenyl)-3,4-dichloro-5-methyl-1H-pyrrole-2-carboxamide (61).
Synthesized according to General procedure C from 60 (80 mg, 0.59 mmol). Crude product was
26

suspended in ethyl acetate and pure 61 was filtered off as a brown solid. Yield 35% (64 mg).
1H NMR (400 MHz, DMSO) δ = 2.24 (s, 3H, pyrrole-CH3), 7.29 (s, 1H, CONH2-A), 7.73 (d,
2H, J = 8.8 Hz, 2 × Ar-H), 7.87 (d, 2H, J = 8.7 Hz, 2 × Ar-H), 7.90 (s, 1H, CONH2-B), 9.65 (s,
13
1H, Ar- NHCO), 12.24 (s, 1H, pyrrole-NH) ppm. C NMR (100 MHz, DMSO-d6) δ = 10.8,
108.6, 111.7, 118.9, 119.4, 128.3, 128.3, 129.0, 141.2, 157.3, 167.3 ppm. HRMS for
C13H12Cl2N3O2 ([M+H]+): calculated 312.0301, found 312.0302. HPLC purity 94.2%.
tert-Butyl (2-hydroxy-4-nitrophenyl)carbamate (63) [50]. To a solution of 62 (5.00
g, 32.4 mmol) in dioxane (70 mL) di-tert-butyl dicarbonate (14.16 g, 38.93 mmol) and 4-
dimethylaminopyridine (150 mg, 64.9 mmol) were added and the mixture was stirred overnight
at room temperature. The solvent was evaporated under reduced pressure, the residue was
dissolved in ethyl acetate (100 mL) and washed with 1% citric acid, water and brine. Organic
phase was dried over Na2SO4, filtered and solvent was evaporated under reduced pressure. Then
toluene (100 mL) was added and the suspension was refluxed for 1 h, after which it was cooled
down. The product was filtered off as brown crystals. Yield 74% (6.11 g). 1H NMR (400 MHz,
DMSO): δ = 1.49 (s, 9H, 3 × CH3), 7.60 (d, 1H, J = 2.6 Hz, Ar-H-3), 7.70 (dd, 1H, J = 9.0, 2.6
Hz, Ar-H-5), 8.01 (d, 1H, J = 9.0 Hz, Ar-H-6), 8.27 (s, 1H, Ar-OH or NHBoc) ppm, Ar-OH or
NHBoc not visible.
tert-Butyl (2-isopropoxy-4-nitrophenyl)carbamate (64). Synthesized according to
General procedure A from 63 (509 mg, 2.00 mmol) and isopropanol (0.31 mL, 4.00 mmol).
Crude product was purified with flash column chromatography using hexane/ethyl acetate (4/1)
as an eluent to give 64 as yellow solid. Yield 49% (292 mg). 1H NMR (400 MHz, DMSO) δ =
1.34 (d, 6H, J = 6.0 Hz, CH(CH3)2), 1.50 (s, 9H, 3 × CH3), 4.85 (hept, 1H, J = 6.0 Hz,
CH(CH3)2), 7.77 (d, 1H, J = 2.5 Hz, Ar-H-3), 7.85 (dd, 1H, J = 9.0, 2.5 Hz, Ar-H-5), 8.12 (d,
1H, J = 9.0 Hz, Ar-H-6), 8.33 (s, 1H, NHBoc) ppm. 13C NMR (100 MHz, DMSO-d6) δ = 21.3,
27.9, 71.4, 80.7, 107.3, 116.6, 117.9, 135.3, 142.0 145.9, 152.1 ppm. MS (ESI) m/z = 295.0
([M-H]-).
tert-Butyl (4-amino-2-isopropoxyphenyl)carbamate (65). Synthesized according to
General procedure B from 64 (261 mg, 0.88 mmol). The crude product was purified with flash
column chromatography using hexane/ethyl acetate (1/1) as an eluent to give 65 as light brown
oil. Yield 90% (211 mg). 1H NMR (400 MHz, CDCl3) δ = 1.36 (d, 6H, J = 6.1 Hz, CH(CH3)2),
1.53 (s, 9H, 3 × CH3), 3.51 (s, 2H, NH2), 4.50 (hept, 1H, J = 6.1 Hz, CH(CH3)2), 6.26 – 6.31
13
(m, 2H, 2 × Ar-H), 6.78 (bs, 1H, Ar-H or NHBoc), 7.82 (bs, 1H, Ar-H or NHBoc) ppm. C
27

NMR (100 MHz, CDCl3) δ = 22.2, 28.4, 71.0, 79.8, 101.2, 107.4, 119.9, 120.8, 141.9, 147.1,
153.1 ppm. MS (ESI) m/z = 267.0 ([M+H]+).
tert-Butyl (4-acetamido-2-isopropoxyphenyl)carbamate (66). To a stirring solution
of 65 (331 mg, 1.24 mmol) in dichloromethane (20 mL), cooled on ice bath, acetic anhydride
(0.705 mL, 7.46 mmol) was added and the mixture was stirred under argon atmosphere
overnight at room temperature. 10 mL of dichloromethane was added to the reaction mixture
and the organic phase was washed with saturated NaHCO3 solution and brine. Organic phase
was dried over Na2SO4, filtered and solvent was evaporated under reduced pressure to give
product as brown solid. Yield 80% (307 mg). 1H NMR (400 MHz, DMSO) δ = 1.38 (d, 6H, J
= 6.1 Hz, CH(CH3)2), 1.54 (s, 9H, C(CH3)3), 2.17 (s, 3H, NHCOCH3), 4.61 (m, 1H, CH(CH3)2),
6.66 (dd, 1H, J = 8.8, 2.2 Hz, Ar-H-5), 7.00 (s, 1H, Ar-NH), 7.26 (s, 1H, Ar-NH), 7.59 (d, 1H,
J = 1.8 Hz, Ar-H-3), δ 8.01 (d, 1H, J = 8.8 Hz, Ar-H-6) ppm. MS (ESI) m/z = 309.0 ([M+H]+).
General procedure G. To a stirring solution of Boc-protected compound (1 eq.) in
dioxane (8 mL/mmol), 4 M HCl in dioxane (10 eq.) was added and the mixture was left to stir
overnight at room temperature. Product was filtered off from reaction mixture.
4-Acetamido-2-isopropoxybenzenaminium chloride (67). Synthesized according to
General procedure G from 66 (290 mg, 0.94 mmol). Product was obtained as a white solid.
1
Yield 69% (158 mg). H NMR (400 MHz, DMSO) δ = 1.33 (d, 6H, J = 6.0 Hz, CH(CH3)2),
2.05 (s, 3H, NHCOCH3), 4.53 – 4.65 (m, 1H, CH(CH3)2), 7.14 (dd, 1H, J = 8.6, 2.0 Hz, Ar-H-
5), 7.27 (d, 1H, J = 8.5 Hz, Ar-H-6), 7.52 (d, 1H, J = 1.9 Hz, Ar-H-3), 10.22 (s, 1H, NHCOCH3)
+
13
ppm, NH3 not visible. C NMR (100 MHz, DMSO-d6) δ = 22.1, 24.5, 71.6, 105.4, 111.1,
116.1, 124.5, 140.7, 160.0, 169.1 ppm. MS (ESI) m/z = 209.0 ([M+H]+).
N-(4-Acetamido-2-isopropoxyphenyl)-3,4-dichloro-5-methyl-1H-pyrrole-2-
carboxamide (68). Synthesized according to General procedure C from 67 (71 mg, 0.29 mmol).
The product was purified by precipitation in 1 M HCl (10 mL) and a few drops of THF and was
filtered off as a brown solid. Yield 35% (39 mg). 1H NMR (400 MHz, DMSO) δ = 1.35 (d, 6H,
J = 6.0 Hz, CH(CH3)2), 2.03 (s, 3H, pyrrole-CH3 or NHCOCH3), 2.22 (s, 3H, pyrrole-CH3 or
NHCOCH3), 4.52 – 4.69 (m, 1H, CH(CH3)2), 7.07 (dd, 1H, J = 8.8, 2.1 Hz, Ar-H-5), 7.51 (d,
1H, J = 2.0 Hz, Ar-H-3), 8.27 (d, 1H, J = 8.8 Hz, Ar-H-6), 9.01 (s, 1H, Ar-NH), 9.94 (s, 1H,
Ar-NH), 12.35 (s, 1H, pyrrole-NH) ppm. 13C NMR (100 MHz, DMSO-d6) δ = 11.2, 22.3, 24.5,
71.5, 104.9, 108.8, 109.6, 111.3, 119.4, 119.6, 124.0, 129.6, 136.0, 146.2, 156.3, 168.6 ppm.
28

HRMS for C17H18Cl2N3O3 ([M-H]-): calculated 382.0731, found 482.0733. HRMS for
C17H18Cl2N3O3 ([M-H]-): calculated 382.0731, found 382.0733. HPLC purity 95.4%.
1-Ethyl-3-(4-hydroxy-3-nitrophenyl)urea (70). Synthesized according to General
procedure F from 69 (809 mg, 5.25 mmol). The product was filtered directly from reaction
mixture as green solid. Yield 69% (813 mg). 1H NMR (400 MHz, DMSO) δ = 1.05 (t, 3H, J =
7.2 Hz, CH2CH3), 3.10 (qd, 2H, J = 7.1, 5.6 Hz, CH2CH3), 6.17 (t, 1H, J = 5.6 Hz, NHCH2CH3),
7.02 (d, 1H, J = 8.9 Hz, Ar-H-5), 7.43 (dd, 1H, J = 9.0, 2.7 Hz, Ar-H-6), 8.14 (d, 1H, J = 2.7
Hz, Ar-H-2), 8.58 (s, 1H, Ar-OH or Ar-NHCO), 9.27 (s, 1H, Ar-OH or Ar-NHCO) ppm. 13C
NMR (100 MHz, DMSO-d6) δ = 15.4, 34.0, 112.9, 119.4, 126.1, 132.7, 135.6, 146.9, 155.1
ppm. MS (ESI) m/z = 223.9 ([M-H]-).
1-Ethyl-3-(4-isopropoxy-3-nitrophenyl)urea (71a). Synthesized according to General
procedure A from 70 (300 mg, 1.33 mmol) and isopropanol (1.02 mL, 13.3 mmol). Crude
product was purified with flash column chromatography using dichloromethane/methanol
(20/1) as an eluent to give impure product as brown oil. Impure product was used in the next
1
synthetic step without further purification. Yield /. H NMR (400 MHz, DMSO) δ = 1.05 (t,
3H, J = 7.2 Hz, CH2CH3), 1.26 (d, 6H, J = 6.1 Hz, CH(CH3)2), 3.10 (qd, 2H, J = 7.2, 5.6 Hz,
CH2CH3), 4.66 (hept, 1H, J = 6.0 Hz, CH(CH3)2), 6.19 (t, 1H, J = 5.6 Hz, NHCH2), 7.27 (d,
1H, J = 9.2 Hz, Ar-H-5), 7.47 (dd, 1H, J = 9.1, 2.7 Hz, Ar-H-6), 8.02 (d, 1H, J = 2.7 Hz, Ar-H-
2), 8.65 (s, 1H, Ar-NH) ppm. MS (ESI) m/z = 265.9 ([M-H]-).
tert-Butyl 4-(4-(3-ethylureido)-2-nitrophenoxy)piperidine-1-carboxylate (71b).
Synthesized according to General procedure A from 70 (224 mg, 0.99 mmol) and tert-butyl 4-
hydroxypiperidine-1-carboxylate (200 mg, 0.99 mmol). Crude product was purified with flash
column chromatography using dichloromethane/methanol (40/1) as an eluent to give product
as brown solidified oil. Yield 67% (272 mg). 1H NMR (400 MHz, CDCl3) δ = 1.16 (t, 3H, J =
7.2 Hz, CH2CH3), 1.48 (d, 9H, J = 3.2 Hz, OC(CH3)3), 1.74 – 1.91 (m, 4H, 2 × piperidine-CH2),
3.29 (qd, 2H, J = 7.2, 5.5 Hz, CH2CH3), 3.48 (m, 2H, piperidine-CH2), 3.56 (m, 2H, piperidine-
CH2), 4.57 (tt, 1H, J = 6.3, 3.4 Hz, piperidine-CH), 5.47 (t, 1H, J = 5.5 Hz, NHCH2CH3), 6.98
(d, 1H, J = 9.2 Hz, Ar-H-6), 7.58 (dd, 1H, J = 9.1, 2.8 Hz, Ar-H-5), 7.65 (s, 1H, Ar-NHCO),
7.72 (d, 1H, J = 2.7 Hz Ar-H-3) ppm. 13C NMR (100 MHz, CDCl3) δ = 15.4, 28.5, 34.1, 35.0,
67.9, 74.2, 80.0, 116.4, 117.5, 125.3, 133.1, 140.7, 145.7, 155.0, 155.9 ppm. MS (ESI) m/z =
407.1 ([M-H]-).
29