Discovery of Benzothiazole Scaffold-Based DNA Gyrase B Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.6b00864

Bacterial DNA gyrase and topoisomerase IV control the topological state of DNA during replication and are validated targets for antibacterial drug discovery. Starting from our recently reported 4,5,6,7-tetrahydrobenzo[1,2-d]thiazole-based DNA gyrase B inhibitors, we replaced their central core with benzothiazole-2,6-diamine scaffold and interchanged substituents in positions 2 and 6. This resulted in equipotent nanomolar inhibitors of DNA gyrase from Escherichia coli displaying improved inhibition of Staphylococcus aureus DNA gyrase and topoisomerase IV from both bacteria. Compound 27 was the most balanced inhibitor of DNA gyrase and topoisomerase IV from both E. coli and S. aureus. The crystal structure of the 2-((2-(4,5-dibromo-1H-pyrrole-2-carboxamido)benzothiazol-6-yl)amino)-2-oxoacetic acid (24) in complex with E. coli DNA gyrase B revealed the binding mode of the inhibitor in the ATP-binding pocket. Only some compounds possessed weak antibacterial activity against Gram-positive bacteria. These results provide a basis for structure-based optimization toward dual DNA gyrase and topoisomerase IV inhibitors with antibacterial activity.

Full text of DOI:10.1021/acs.jmedchem.6b00864

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Article
Discovery of Benzothiazole Scaffold-Based DNA Gyrase B Inhibitors
Marina Gjorgjieva, Tihomir Tomasic, Michaela Baran#okova, Sotirios
Katsamakas, Janez Ilaš, Päivi Tammela, Lucija Peterlin Maši#, and Danijel Kikelj
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00864 • Publication Date (Web): 19 Aug 2016
Downloaded from http://pubs.acs.org on August 21, 2016
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Discovery of Benzothiazole ScaffoldꢀBased DNA
Gyrase B Inhibitors
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a
a
a
a,b
Marina Gjorgjieva, Tihomir Tomašič, Michaela Barančokova, Sotirios Katsamakas, Janez
a
c
a,*
a,*
Ilaš, Päivi Tammela, Lucija Peterlin Mašič, and Danijel Kikelj
a
University of Ljubljana, Faculty of Pharmacy, Aškerčeva 7, 1000 Ljubljana, Slovenia
b
Aristotle University of Thessaloniki, Faculty of Health Sciences, School of Pharmacy,
Department of Pharmaceutical Chemistry, 54124 Thessaloniki, Greece
c
University of Helsinki, Faculty of Pharmacy, Division of Pharmaceutical Biosciences, P.O. Box
5
6, FIꢀ00014 Helsinki, Finland
KEYWORDS: antibacterial, benzothiazoleꢀ2,6ꢀdiamine, DNA gyrase, topoisomerase IV,
structureꢀbased design, 1Hꢀpyrroleꢀ2ꢀcarboxamide
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ABSTRACT. Bacterial DNA gyrase and topoisomerase IV control the topological state of DNA
during replication and are validated targets for antibacterial drug discovery. Starting from our
recently reported 4,5,6,7ꢀtetrahydrobenzo[1,2ꢀd]thiazoleꢀbased DNA gyrase B inhibitors, we
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replaced their central core with
benzothiazoleꢀ2,6ꢀdiamine scaffold and interchanged
substituents in positions 2 and 6. This resulted in equipotent nanomolar inhibitors of DNA gyrase
from Escherichia coli displaying improved inhibition of Staphylococcus aureus DNA gyrase
and topoisomerase IV from both bacteria. Compound 27 was the most balanced inhibitor of
DNA gyrase and topoisomerase IV both from E. coli and S. aureus. The crystal structure of the
2ꢀ((2ꢀ(4,5ꢀdibromoꢀ1Hꢀpyrroleꢀ2ꢀcarboxamido)benzothiazolꢀ6ꢀyl)amino)ꢀ2ꢀoxoacetic acid (24)
in complex with E. coli DNA gyrase B revealed the binding mode of the inhibitor in the ATPꢀ
binding pocket. Only some compounds possessed weak antibacterial activity against Gramꢀ
positive bacteria. These results provide a basis for structureꢀbased optimization towards dual
DNA gyrase and topoisomerase IV inhibitors with antibacterial activity.
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INTRODUCTION
th
In the middle of the 20 century, antibiotics were considered to be a powerful discovery that
made infectious diseases a problem of the past. Today, it is clear that some of the antibacterial
compounds discovered during the last century were only a shortꢀterm solution for the treatment
of infectious diseases because bacterial resistance to these drugs is increasing and treatments are
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becoming less effective. Although the discovery of antibiotics is a very important achievement
for mankind, the greatest challenges in antibacterial research are yet to come. There is a need for
new antibacterial compounds that have new mechanisms of action with activity against resistant
bacterial strains, and the search for new antibacterial agents remains a significant challenge for
the future.
Bacterial DNA gyrase is a topoisomerase type II enzyme that has attracted attention since its
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discovery in 1976, when it was first isolated from Escherichia coli and identified as a target of
the already known aminocoumarin class of antibacterial compounds. In the 1990s, when the
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crystal structure of the Nꢀterminal fragment of DNA gyrase had already been solved,
topoisomerase IV, a bacterial enzyme with a similar structure, function, and inhibitor
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susceptibility to DNA gyrase was identified.
DNA gyrase and topoisomerase IV catalyze changes in DNA topology by breaking and
rejoining double stranded DNA. In particular, DNA gyrase introduces negative supercoils in
DNA in front of the replication fork, while topoisomerase IV is important for decatenation
during DNA replication. Thus, both enzymes are involved in very important processes during
DNA replication and are essential for cell viability. Both enzymes are ATPꢀfueled
heterotetrameric proteins; DNA gyrase is composed of two A subunits (GyrA) and two B
subunits (GyrB), whereas topoisomerase IV consists of two C subunits (ParC) and two E
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subunits (ParE), which are homologous to GyrA and GyrB, respectively. The GyrA and ParC
subunits are involved in DNA transit, while the GyrB and ParE subunits contain an ATPꢀbinding
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domain. Type II topoisomerase is also found in eukaryotic cells, but unlike the prokaryotic
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enzymes, eukaryotic topoisomerase II is homodimeric. This difference in structure makes
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selective targeting of prokaryotic topoisomerase II possible. The similarities in the structure of
DNA gyrase and topoisomerase IV offer an exceptional opportunity for the dual targeting of
these enzymes by new antibacterial compounds, thereby reducing the probability of bacteria to
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develop targetꢀbased resistance against them. Thus, the selectivity of targeting prokaryotic
topoisomerase II, the potential of dual targeting, and the wellꢀknown structure of these enzymes
makes both DNA gyrase and topoisomerase IV attractive targets in the challenging search for
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new antibacterial compounds.
DNA gyrase and topoisomerase IV have been recognized as targets of aminocoumarin and
quinolone classes of antibacterial compounds. However, these two classes of inhibitors target
different parts of the enzymes. Well known and widely used quinolones target GyrA and ParC
subunits, while aminocoumarin class inhibitors target GyrB and ParE subunits. Novobiocin
(Figure 1), a natural product aminocoumarin class inhibitor of DNA gyrase and topoisomerase
IV discovered in the midꢀ1950s, targets ATPꢀbinding sites at the GyrB and ParE subunits. It is
the only GyrB inhibitor to be used in clinical practice, but despite its good pharmacokinetic
profile, it was withdrawn from the market primarily due to its toxicity and also due to the high
rate of resistance development. Coumermycin A1 and clorobiocin (Figure 1) are two other
aminocoumarin natural products that showed good inhibitory activities against DNA gyrase B,
but they never reached the market because of their undesirable toxicity profiles. Numerous
studies toward the development of improved novobiocin analogues were unsuccessful, mainly
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because of their undesirable pharmacokinetic profile and lack of inhibitory or microbiological
activity. Thus, novobiocin is still the only GyrB inhibitor that was used clinically and also the
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only GyrB inhibitor that has progressed beyond Phase I clinical trials.
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Figure 1. Structures of the DNA gyrase B inhibitors novobiocin and clorobiocin.
Figure 2. Examples of dual inhibitors of GyrB and ParE possessing (a) benzimidazole (b)
bithiazole, (c) thiazolopyridine, (d) pyrrolopyrimidine, (e) imidazopyridine, and (f)
benzothiazole scaffolds.
Determination of the crystal structure of DNA gyrase B in complex with adenylyl 5’ꢀ(β,γꢀ
imido)ꢀtriphosphate (ADPNP) in 1991 and later with other known inhibitors of DNA gyrase
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B
provided significant information about the most important inhibitor interactions in the
protein ATPꢀbinding site. This information created a solid basis for the structureꢀbased design of
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inhibitors over the years and led to discovery of several new scaffolds that had good potential for
inhibition of DNA gyrase and topoisomerase IV. Over the years, using scaffold hopping
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approaches and exploiting opportunities offered to medicinal chemistry by aromatic
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heterocycles, benzimidazole ureas, bithiazoles, thiazolopyridines, pyrrolopyrimidines,
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imidazopyridines, benzothiazoles
(Figure 2) and other structural types have been studied
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as dual inhibitors of GyrB and ParE subunits of bacterial gyrase and topoisomerase IV.
RESULTS AND DISCUSSION
Design. Our research group has recently reported the discovery of novel structural classes of
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DNA gyrase inhibitors based on 4,5,6,7ꢀtetrahydrobenzo[1,2ꢀd]thiazole and Nꢀphenylꢀ4,5ꢀ
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dibromopyrrolamide scaffolds inspired by the marine natural product oroidin (Figure 3). We
obtained the crystal structure of 02 in complex with E. coli GyrB and demonstrated that the 4,5ꢀ
dibromoꢀ1Hꢀpyrroleꢀ2ꢀcarboxamide moiety of 01 and 02 bound to the ATP binding site of GyrB
participates in a hydrogen bond with the Asp73 carboxylate group and a conserved water
molecule, whereas the terminal carboxylic acid moiety is involved in interactions with Arg76
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and/or Arg136.
Based on the crystal structure of the E. coli GyrB ꢀ 02 complex and potent
inhibition of E. coli DNA gyrase by 4,5,6,7ꢀtetrahydrobenzo[1,2ꢀd]thiazole class inhibitors in
conjunction with their weaker inhibition of Staphylococcus aureus DNA gyrase and E. coli and
S. aureus topoisomerase IV, we examined the effects of different structural modifications of the
parent compound 01 on the inhibition of both enzymes. First, the 4,5,6,7ꢀtetrahydrobenzo[1,2ꢀ
d]thiazole moiety was replaced by a planar aromatic benzothiazole scaffold to enable additional
cation ꢀ π interaction of the benzene ring with Arg76 within the Glu50ꢀArg76 salt bridge, which
is not present in the 4,5,6,7ꢀtetrahydrobenzo[1,2ꢀd] series and was shown to be important for the
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strong inhibition of DNA gyrase B. The benzothiazole scaffold additionally made possible the
substitution pattern which proved to be successful in the 4,5,6,7ꢀtetrahydrobenzo[1,2ꢀd]thiazole
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series. Second, because our docking experiments (Figure 4) suggested similar binding modes
of compounds incorporating the pyrroleꢀ2ꢀcarboxamido moiety at positions 6 (Series A) and 2
(Series B) of the central heterocyclic core, series B analogues of the parent compound 01 were
prepared in addition to series A compounds. Third, the dibromopyrrole moiety was replaced by a
smaller dichloropyrrole moiety to possibly improve multiple inhibition against S. aureus DNA
gyrase and E. coli and S. aureus topoisomerase IV because these three enzymes possess a
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smaller hydrophobic pocket compared to that of E. coli DNA gyrase. Finally, the effect of the
length of the ester or carboxylic acid moieties on the multiple inhibition of DNA gyrase and
topoisomerase IV was evaluated. As demonstrated below, this resulted in an improved inhibitor
series that possesses inhibitory activity against topoisomerase IV from both bacterial strains. The
crystal structure of series B compound 24 in complex with E. coli DNA gyrase B revealed the
binding mode of the studied benzothiazole class of compounds, thus providing a solid basis for
structureꢀbased design and optimization of this promising class of ATPꢀbinding site targeting
DNA gyrase B inhibitors.
Figure 3. Design of series A and B benzothiazoleꢀ2,6ꢀdiamineꢀbased DNA gyrase B inhibitors.
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a
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Figure 4. The binding mode of a) inhibitor 24 (in green sticks) and b) inhibitor 13 (in orange
sticks) in the ATPꢀbinding site of E. coli DNA gyrase B (PDB code: 4DUH) as predicted by
docking with LeadIT. The ligand and neighboring protein side chains are shown as stick models,
colored according to the chemical atom type (Cligand 24 in green, Cligand 13 in orange, CGyrB in gray,
N in blue, O in red, S in yellow, and Br in brown). Hydrogen bonds with their lengths in Å are
indicated as black dotted lines. Figure was prepared by PyMOL.
Chemistry. The synthesis of the series A compounds with the pyrroleꢀ2ꢀcarboxamido group
attached at position 6 of the benzothiazoleꢀ2,6ꢀdiamine scaffold is presented in Scheme 1.
Acylation of 1 with appropriate acyl chloride (ethyl oxalyl chloride, methyl malonyl chloride or
methyl succinyl chloride) in the presence of triethylamine in 1,4ꢀdioxane at room temperature
afforded compounds 2, 3 and 4. The reduction of the nitro group of 2-4 using catalytic
hydrogenation in the presence of 10% Pd/C in methanol or ethanol yielded amines 5, 6 and 7.
These were acylated with freshly prepared 4,5ꢀdibromoꢀ1Hꢀpyrroleꢀ2ꢀcarbonyl chloride or 4,5ꢀ
dichloroꢀ1Hꢀpyrroleꢀ2ꢀcarbonyl chloride in the presence of pyridine in dichloromethane to obtain
esters 8-12. Compounds 8 and 10 were hydrolyzed with 1 M sodium hydroxide in methanol to
produce carboxylic acids 13 and 14. All attempts to hydrolyze malonic acid derivatives 9 and 12
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were unsuccessful due to decarboxylation that, surprisingly, did not compromise the hydrolysis
of the series B esters 18ꢀ23 (Scheme 2) and related 4,5,6,7ꢀtetrahydrobenzo[1,2ꢀd]thiazole
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analogs
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a
Scheme 1. Synthesis of Series A compounds
a
Reagents and conditions: (a) ethyl oxalyl chloride (for 2), methyl malonyl chloride (for 3) or
methyl succinyl chloride (for 4), Et N, 1,4ꢀdioxane, rt, 4 h; (b) H , 10% Pd/C, EtOH (for 5) or
3
2
MeOH (for 6, 7), rt, 24 h; (c) i) 4,5ꢀdibromoꢀ1Hꢀpyrroleꢀ2ꢀcarboxylic acid (for 8, 9 and 10) or
4,5ꢀdichloroꢀ1Hꢀpyrroleꢀ2ꢀcarboxylic acid (for 11 and 12), oxalyl chloride, DCM, rt, 15 h, then
ii) 5 (for 8 and 11), 6 (for 9 and 12) or 7 (for 10), pyridine, DCM, rt, 2 h; (d) 1 M NaOH, MeOH,
rt, 24 h.
Synthesis of compounds from series B started from the commercially available 6ꢀnitroꢀ
benzothiazolꢀ2ꢀamine (1), which was catalytically hydrogenated to benzothiazoleꢀ2,6ꢀdiamine
(
15) in a Parr hydrogenator at 2.1 bar pressure, using ethanol as a solvent and 10% Pd/C as a
catalyst. The diamine 15 was further coupled with 2,2,2ꢀtrichloroꢀ1ꢀ(4,5ꢀdibromoꢀ1Hꢀpyrrolꢀ2ꢀ
yl)ethanꢀ1ꢀone or 2,2,2ꢀtrichloroꢀ1ꢀ(4,5ꢀdichloroꢀ1Hꢀpyrrolꢀ2ꢀyl)ethanꢀ1ꢀone in the presence of
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Na CO as a base in N,Nꢀdimethylformamide at 80 °C to obtain pyrrolamides 16 and 17.
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Acylation of 16 and 17 with ethyl oxalyl chloride, methyl malonyl chloride or methyl succinyl
chloride at room temperature in the presence of triethylamine in 1,4ꢀdioxane afforded esters 18-
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23, which were hydrolyzed to carboxylic acids 24ꢀ29 upon treatment with 1 M sodium
hydroxide in methanol (Scheme 2).
a
Scheme 2. Synthesis of Series B Compounds
H
X
X
O
N
H N
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S
N
O N
H N
2
2
S
N
a
S
N
c
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NH2
NH2
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X=Br
X=Cl
17
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N
H
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X
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X
O
d
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H
N
H
RO
HO
N
S
S
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n
n
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NH
O
O
O
O
N
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9
X=Br, n=0, R=Et
X=Br, n=1, R=Me
24 X=Br, n=0
25 X=Br, n=1
26 X=Br, n=2
27 X=Cl, n=0
28 X=Cl, n=1
29 X=Cl, n=2
20 X=Br, n=2, R=Me
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2
1
2
X=Cl, n=0, R=Et
X=Cl, n=1, R=Me
23 X=Cl, n=2, R=Me
a
Reagents and conditions: (a) H (2.1 bar), 10% Pd/C, EtOH, rt, 6 h; (b) 2,2,2ꢀtrichloroꢀ1ꢀ(4,5ꢀ
2
dibromoꢀ1Hꢀpyrrolꢀ2ꢀyl)ethanꢀ1ꢀone (for 16) or 2,2,2ꢀtrichloroꢀ1ꢀ(4,5ꢀdichloroꢀ1Hꢀpyrrolꢀ2ꢀ
yl)ethanꢀ1ꢀone (for 17), DMF, Na CO 80 °C, 5 h; (c) ethyl oxalyl chloride (for 18 and 21),
2
3,
methyl malonyl chloride (for 19 and 22), methyl succinyl chloride (for 20 and 23), Et N, 1,4ꢀ
3
dioxane, rt, 12 h; (d) 1 M NaOH, MeOH, rt, 24 h.
In vitro inhibition of DNA gyrase and topoisomerase IV. Synthesized benzothiazole
derivatives were evaluated for their inhibitory activity against DNA gyrase and topoisomerase IV
from both E. coli and S. aureus in supercoiling and decatenation assays, respectively. The
inhibitory activities of the tested compounds are presented in Tables 1 and 2 as IC values.
5
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Inhibitory activities against DNA gyrase
In the series A compounds, in which the 4,5ꢀdichloroꢀ or 4,5ꢀdibromopyrrolamido moiety is
attached at the position 6 of the benzothiazole ring, the most favorable acyl substituent at
position 2 proved to be the oxalyl group, both in the ester and the free carboxylic acid form as
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
demonstrated by the most potent inhibitor 13 (IC = 38 nM) of E. coli DNA gyrase among
50
compounds in the Series A (Table 1). The dichloro ester analogues 11 and 12 displayed weaker
inhibition of E. coli DNA gyrase than the corresponding dibromo analogues, so the respective
carboxylic acids were not prepared. The inhibitory potency of dibromo ethyl oxalate 8 equaled
that of the 4,5,6,7ꢀtetrahydrobenzo[1,2ꢀd]thiazole analogue (IC50 = 100 nM) while the
methylmalonate and methylsuccinate esters 9 and 10 were found to be twofold and ninefold
weaker inhibitors of E. coli DNA gyrase than the corresponding 4,5,6,7ꢀtetrahydrobenzo[1,2ꢀ
3
0
d]thiazole analogues. The carboxylic acid inhibitors 13 (IC = 38 nM) and 14 (IC = 57 nM)
5
0
50
were equipotent with the corresponding 4,5,6,7ꢀtetrahydrobenzo[1,2ꢀd]thiazole analogues (oxalyl
3
0
analogue: IC = 58 nM; succinyl analogue: IC = 49 nM). Compounds from the Series A did
5
0
50
not display a substantial S. aureus DNA gyrase inhibition. (Table 1).
From the inhibitory activities of series B compounds 16 and 17, shown in Table 2 it can be
concluded that the 4,5ꢀdibromoꢀ1Hꢀpyrrolamide moiety attached at position 2 on the
benzothiazole ring contributes to the affinity for the ATPꢀbinding site of E. coli DNA GyrB more
than the 4,5ꢀdichloroꢀ1Hꢀpyrrolamide moiety attached at the same position. This trend was also
observed for the activities of other compounds from Series B containing the 4,5ꢀdibromoꢀ1Hꢀ
pyrrolamide moiety (c.f. 18 vs. 21 and 20 vs. 23, 24 vs. 27 and 26 vs. 29), with the exception of
the malonyl pairs 19 and 22 and 25 and 28, in which a trend of stronger inhibition by the 4,5ꢀ
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1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 1. Inhibition of E. coli and S. aureus DNA gyrase and topoisomerase IV by the
benzothiazole compounds containing the pyrroleꢀ2ꢀcarboxamido moiety at position 6 on the
benzothiazole core (Series A).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
DNA gyrase
topoisomerase IV
compound
X
n
R
IC50 [ꢀM]
IC50 [ꢀM]
E. coli
S. aureus
0.040
> 100
> 100
> 100
> 100
> 100
> 100
> 100
E. coli
S. aureus
novobiocin
ꢀ
ꢀ
ꢀ
0.17
11
27
8
9
Br
Br
Br
Cl
Cl
Br
Br
0
1
2
0
1
0
2
Et
0.081 ± 0.05
0.24 ± 0.02
0.87 ± 0.21
1.8 ± 0.4
> 100
> 100
Me
Me
Et
> 100
> 100
1
1
1
0
1
2
> 100
> 100
> 100
> 100
Me
H
7.9 ± 2.1
> 100
> 100
13
0.038 ± 0.001
0.057 ± 0.018
5.4 ± 1.0
4.5 ± 0.7
2.5 ± 0.2
2.2 ± 0.3
1
4
H
dichloroꢀ1Hꢀpyrrolamide compounds was observed. On the contrary, the differences between the
dibromopyrrole and dichloropyrrole compounds were not straightforward for inhibition of DNA
gyrase from S. aureus. Ethyl oxalyl, methyl malonyl and methyl succinyl moieties attached at
position 6 of the benzothiazole ring improved the inhibitory activities of the dichloropyrrole
compounds 21-23 but not of the dibromopyrrole compounds 18ꢀ20 against E. coli DNA gyrase,
with the methyl malonyl derivative 22 (IC = 0.22 ꢁM) being the most potent inhibitor in this
5
0
ester series. In the carboxylic acid series (compounds 24-29) the measured IC50 values for
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inhibition of DNA gyrase from E. coli were all in the nanomolar range, due to the contribution of
the carboxylate group interaction with Arg136. The most potent inhibitors in the dibromopyrrole
carboxylic acid series were succinyl and oxalyl derivatives 26 (IC = 33 nM) and 24 (IC = 58
5
0
50
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
nM), while for the dichloropyrrole compounds the most potent inhibitor was oxalyl compound
27 with an IC value of 87 nM. The affinity of compound 24 for DNA gyrase B from E. coli was
5
0
further confirmed by surface plasmon resonance (SPR) (for experimental details see Ref. 29)
with a K of 0.23 ꢁM. The inhibition of S. aureus DNA gyrase was observed only for the
d
dibromopyrrole carboxylic acid 25 (IC = 5.6 µM) and dichloropyrrole carboxylic acids 27-29,
5
0
with IC values in the range of 0.51 to 94 µM. Generally, all of the compounds from Series B
5
0
displayed weaker inhibition of S. aureus DNA gyrase compared to E. coli gyrase. It is worth
noting the low micromolar activity of malonyl compounds 25 in the dibromo series (IC = 5.6
5
0
ꢁ
M) and 28 in the dichloro series (IC = 8.5 ꢁM) against S. aureus gyrase. The nanomolar
50
activity of the dichloro oxalyl derivative 27 (IC = 0.51 ꢁM) in comparison to dibromo analogue
50
2
4 (IC >100 ꢁM), as well as to malonyl analogue 28 (IC = 8.5 ꢁM) and succinyl analogue 29
50 50
(
IC = 94 ꢁM) is also noteworthy, indicating that the carboxylic acid moiety and small changes
50
in the length of the acyl moiety (c.f. 24 vs.25, 27 vs. 28) are important for the binding of Series B
compounds to the ATPꢀbinding site of S. aureus DNA gyrase. In addition, substitution in the
pyrrole ring (c.f. 24 vs. 27) is important for the orientation of the whole molecule in the binding
pocket of S. aureus DNA gyrase. The main difference between the compounds from the Series B
(pyrroleꢀ2ꢀcarboxamido group at position 2) and Series A (pyrroleꢀ2ꢀcarboxamido group at
position 6) is that the compounds in series B exhibited better inhibitory activity against S. aureus
DNA gyrase, while their inhibitory activity against E. coli DNA gyrase remained in the
nanomolar range.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 2. Inhibition of E. coli and S. aureus DNA gyrase and topoisomerase IV by the
benzothiazole compounds containing the pyrroleꢀ2ꢀcarboxamido moiety at position 2 on the
benzothiazole core (Series B).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
DNA gyrase
topoisomerase IV
compound
X
n
R
IC50 [ꢀM]
IC50 [ꢀM]
E. coli
S. aureus
0.040
E. coli
S. aureus
27
novobiocin
ꢀ
ꢀ
ꢀ
ꢀ
0.17
2.0 ± 0.2
> 100
11
> 100
16
17
Br
Cl
Br
Br
Br
Cl
Cl
Cl
Br
Br
Br
Cl
Cl
Cl
ꢀ
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
10 ± 2
17 ± 3
26 ± 1
1.7 ± 0.8
2.4 ± 1.3
28 ± 4
ꢀ
ꢀ
> 100
> 100
1
1
2
2
8
9
0
1
0
1
2
0
1
2
0
1
2
0
1
2
Et
Me
Me
Et
Me
Me
H
29 ± 6
> 100
> 100
2.0 ± 0.4
7.0 ± 0.9
> 100
> 100
> 100
> 100
> 100
> 100
> 100
22
0.22 ± 0.02
35 ± 12
> 100
> 100
2
2
2
2
3
4
5
6
> 100
> 100
> 100
13 ± 3
45 ± 5
7.2 ± 1.3
1.8 ± 0.8
5.19 ± 4.7
5.3 ± 0.4
0.058 ± 0.031
0.14 ± 0.06
H
5.6 ± 2.6
> 100
H
0.033 ± 0.025
0.087 ± 0.001
0.12 ± 0.02
27
28
H
0.51 ± 0.23
8.5 ± 1
94 ± 25
H
2
9
H
0.43 ± 0.11
Inhibitory activities against topoisomerase IV
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Generally, the inhibitory potencies of most of the Series B compounds against topoisomerase
IV were lower than those against DNA gyrase for the E. coli enzymes but for the S. aureus
enzymes, the IC values for the inhibition of both enzymes were more comparable (Tables 1 and
5
0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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41
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43
44
45
46
47
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49
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55
56
57
58
59
60
2
). Inhibition of topoisomerase IV by most of the active compounds (13, 14, and 24-29) was
better than or comparable to that of novobiocin (IC (E. coli) = 11 µM; IC (S. aureus) = 27
5
0
50
µM). The esters 8ꢀ12 and 18-23 did not show any promising activity, which indicates that a
carboxylic acid moiety is required for the binding of the compounds in the ATPꢀbinding site of
topoisomerase IV. All of the final carboxylic acids (13, 14, and 24-29) from both series A and B
exhibited low micromolar activities against topoisomerase IV from both E. coli and S. aureus.
Compounds 25, 27 and 28 were found to be especially interesting for further studies because
they showed promising inhibitory activities against all four tested enzymes. In particular, 4,5ꢀ
dichloropyrroleꢀbased benzothiazole inhibitor 27 exhibited wellꢀbalanced multiple inhibition of
all four enzymes in the nanomolar and low micromolar range (0.087 – 1.8 µM).
Antibacterial activity. All of the compounds from series A and series B were tested against two
Gramꢀpositive (Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212) and
two Gramꢀnegative (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853)
bacterial strains. In general, the compounds did not show significant antibacterial activities
against either Gramꢀpositive or Gramꢀnegative bacteria. The only notable activities were
observed against Gramꢀpositive E. faecalis for compounds 25 and 11 which showed 59% and
47% inhibition of the growth of E. faecalis at a 50 µM concentration after 24 h incubation,
respectively. None of the final compounds showed significant antibacterial activity against the
tested Gramꢀnegative bacteria. Because a possible reason for the absence of the antibacterial
activity of compounds against Gramꢀnegative bacteria could be their affinity for efflux pumps,
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1
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1
1
2
2
2
2
2
2
2
2
2
2
3
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4
4
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4
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5
5
5
5
5
5
5
6
we tested the compounds 17-19, 21-25, 27 and 29 in ꢁtolC E. coli, an efflux deficient strain
derived from wildꢀtype E. coli ECM1194 that lacks a functional tolC gene that encodes the
essential outer membrane component of the multicomponent efflux pump (for experimental
details see Ref. 30). We found that compound 25 exhibited more potent antibacterial activity
(MIC < 16 µg/mL) in ꢁtolC, than in the wild type E. coli strain that contained efflux pump (MIC
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
>
256 µg/mL). These results may explain the weak antibacterial activity of 25 and similar
compounds against both Gramꢀnegative bacterial strains tested, with only 12% inhibition of the
growth of E. coli and 11% inhibition of the growth of P. aeruginosa after 24 h incubation.
X-Ray crystallography and molecular modeling. A detailed binding mode of the series B
inhibitor 24 in the ATPꢀbinding site of the 43 kDa Nꢀterminal fragment of the E. coli DNA
gyrase B (residues Gly15 to Thr392) was revealed from the Xꢀray coꢀcrystal structure, which
was solved to a resolution of 2.83 Å (PDB code: 5L3J). The electron density for most of the
ligand is well defined, but is missing for the terminal carboxylate group (Figure 5), which can
probably be attributed to its flexibility in the binding site. Similar situation was observed in the
coꢀcrystal structure of E. coli DNA gyrase in complex with Nꢀphenylꢀ4,5ꢀdibromopyrrolamide
inhibitor, where the mobility of the inhibitor in the binding site was also shown through
31
molecular dynamics simulation. In contrast, the high resolution crystal structure of bithiazole
inhibitor in complex with E. coli DNA gyrase (PDB entry: 4DUH) revealed a hydrogen bond
between the inhibitor and Gly101 carbonyl oxygen, which stabilized the Gly97ꢀSer108 flexible
loop, decreased ligand flexibility and enhanced chances for obtaining a high resolution
2
4
structure. Lack of the structural feature in this series of the benzothiazole inhibitors, enabling
hydrogen bonding with amino residues of the Gly97ꢀSer108 flexible loop, thus probably also
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contributed to the higher mobility of ligand in the binding site and consequently to the lower
crystal structure resolution of E. coli DNA gyrase – 24 complex.
The orientation and conformation of the terminal carboxylate group of 24 was hence modeled
in two different ways (Figure 5; model 1, model 2) and is located in the vicinity of Arg76 and
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
9
,18
Arg136, which are usually in contact with DNA gyrase B inhibitors.
However, the electron
density for the Arg76 side chain is missing in the experimental data and no hydrogen bonds can
be predicted. The 4,5ꢀdibromopyrrolamide moiety of 24 is bound to the adenine binding pocket,
where a hydrogen bond between the pyrrole NH group and Asp73 side chain is formed.
However, due to the Xꢀray structure resolution of 2.83 Å, only a few water molecules were
modeled based on the electron density and the conserved water molecule was not observed in the
structure. Therefore, a hydrogen bond between the inhibitor carbonyl group and Asp73 side
chain bridged by this water molecule could not be observed. The 4,5ꢀdibromopyrrole moiety
forms additional hydrophobic contacts with Val43, Asn46, Val71, Val120, Thr165 and Val167,
while the benzothiazole scaffold forms hydrophobic interactions with Glu50, Gly77, Ile78 and
Pro79.
Possible binding modes of DNA gyrase inhibitors of the benzothiazoleꢀ2,6ꢀdiamine series A
30
and B were also studied following our recently described docking protocol, using LeadIT and
2
4
the high resolution structure of E. coli DNA gyrase in complex with a bithiazole inhibitor.
Comparison of the predicted (Figure 4a) and experimental binding mode (Figure 5a) of 24 in the
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2
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2
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3
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3
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4
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4
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5
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5
5
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
b
Figure 5. Xꢀray crystal structure of E. coli DNA gyrase B in complex with ligand 24 (PDB code:
5L3J): a) model 1; b) model 2. The ligand and neighboring protein side chains are shown as stick
models, that are colored according to the chemical atom type (Cligand in green for model 1 and in
yellow for model 2, CGyrB in gray, N in blue, O in red, S in yellow, and Br in brown). The ligand
molecule is shown superimposed with the refined 2Fo−Fc electron density map contoured at 1.0
σ. Hydrogen bonds are indicated as black dotted lines. Figure was prepared by PyMOL.
E. coli DNA gyrase ATPꢀbinding site shows similar conformation of the ligand, while its
position is slightly different. The docking predicts the formation of hydrogen bonds between the
terminal oxalate moiety and Arg76 and Arg136 side chains that could not be observed in the Xꢀ
ray structure (Figure 5). Such hydrogen bonds are predicted for all oxalic (8, 11, 13, 18, 21, 24
and 27), malonic (9, 12, 19, 22, 25, and 28) and succinic acid (10, 14, 20, 23, 26 and 29)
derivatives in both series A and series B. As an example, the predicted binding mode of series A
compound 13, which has the opposite substitution as 24, is presented in Figure 4b.
CONCLUSION
In summary, replacement of the central core of 4,5,6,7ꢀtetrahydrobenzo[1,2ꢀd]thiazoleꢀbased
29
DNA gyrase B inhibitors with a planar benzothiazoleꢀ2,6ꢀdiamine scaffold and moving the 4,5ꢀ
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dihalopyrroleꢀ2ꢀcarbonyl group from Nꢂ6 to Nꢀ2 of the central core resulted in two series of
nanomolar inhibitors. These compounds were nearly equipotent with the 4,5,6,7ꢀ
tetrahydrobenzo[1,2ꢀd]thiazole compounds as inhibitors of E. coli DNA gyrase but displayed
improved inhibition of S. aureus DNA gyrase and topoisomerase IV from both bacteria. A
crystal structure of 2ꢀ((2ꢀ(4,5ꢀdibromoꢀ1Hꢀpyrroleꢀ2ꢀcarboxamido)benzothiazolꢀ6ꢀyl)amino)ꢀ2ꢀ
oxoacetic acid (24) in complex with E. coli DNA gyrase B solved to a resolution of 2.83 Å
revealed the binding mode of the inhibitor in the ATPꢀbinding pocket of the enzyme. In general,
no significant antibacterial activity against both Gramꢀnegative and Gramꢀpositive bacteria was
observed, most likely due to efflux of inhibitors. Some of the compounds possessed only weak
antibacterial activity against Gramꢀpositive E. faecalis. The results of this study provide a good
starting point for structureꢀbased design of improved ATPꢀcompetitive DNA gyrase and
topoisomerase IV inhibitors with antibacterial activity. In this respect, compounds 25, 27 and 28
are especially interesting for further studies because they showed promising well balanced
multiple inhibiton in the nanomolar/low micromolar range against DNA gyrase and
topoisomerase IV both in E. coli and S. aureus.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
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60
EXPERIMENTAL SECTION
Chemistry. The chemicals obtained from SigmaꢀAldrich (St. Louis, MO, USA), Acros
Organics (Geel, Belgium), and Appollo Scientific (Stockport, UK) were used without further
purification unless otherwise stated. Analytical TLC was performed on 0.25 mm silica gel Merck
60 F254 plates, using visualization with UV light and spray reagents. Column chromatography
was carried out on Merck silica gel 60 (particle size 240ꢀ400 mesh). Melting points were
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5
5
5
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6
1
13
determined on a Reichert hot stage microscope and are uncorrected. H NMR and C spectra
were recorded at 400 and 100 MHz, respectively, on a Bruker AVANCE III spectrometer in
DMSOꢀd or CDCl solution with TMS as an internal standard at room temperature. IR spectra
6
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
were obtained using a Thermo Nicolet Nexus 470 ESP FTꢀIR spectrometer. Mass spectra were
recorded on a VG Analytical Autospec Q mass spectrometer. HPLC analyses were run on an
Agilent Technologies 1100 instrument (Agilent Technologies, Santa Clara, CA, USA) with a
G1365B UVꢀVIS detector, a G1316A thermostat, and a G1313A autosampler using a
Phenomenex Luna 5ꢀꢁm C18 column (4.6 mm × 150 mm or 4.6 mm × 250 mm, Phenomenex,
Torrance, CA, USA) and a flow rate of 1.0 mL/min. The eluent consisted of 0.1 % trifluoroacetic
acid in water (A) and methanol (B); gradient, 90 % A to 10 % A in 20 min, then 5 min 10 % A;
flow rate 1.0 mL/min; injection volume, 10 ꢁL. All tested compounds had a purity of at least
9
5% by HPLC and passed the PAINS filter (see below).
General procedure A. Synthesis of compound 2, 3 and 4.
6ꢀNitroꢀbenzothiazolꢀ2ꢀamine (1) (1.95 g, 10.0 mmol) was dissolved in 1,4ꢀdioxane (100 mL),
triethylamine (2.09 mL, 15.0 mmol) was added and the mixture was cooled down to 0 °C. Ethyl
oxalyl chloride (1.68 mL, 15.0 mmol), methyl malonyl chloride (1.61 mL, 15.0 mmol) or methyl
succinyl chloride (1.85 mL, 15.0 mmol) were added dropwise to the mixture and the reaction
mixture was stirred for additional 3 h at room temperature, whereupon saturated NaHCO3
solution was added to quench the reaction. The reaction mixture was concentrated in vacuo,
dissolved in ethyl acetate and the solution was washed with 3 × 10 mL saturated NaHCO , 3 ×
3
1
0 mL 10% citric acid, 3 × 10 mL water and 3 × 10 mL brine. If the product precipitated
between the ethyl acetate and water phase it was collected by filtration. The ethyl acetate phase
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was dried over sodium sulfate and concentrated. The combined crude products were
recrystallized from ethyl acetate.
Ethyl 2ꢂ((6ꢂnitrobenzo[d]thiazolꢂ2ꢂyl)amino)ꢂ2ꢂoxoacetate (2). Prepared according to general
procedure A from compound 1 (2.00 g, 10.2 mmol); yellow crystals, yield 2.56 g (85 %); mp
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
25ꢀ228 °C; IR (ATR) ν 3267, 3096, 1699, 1573, 1541, 1513, 1471,1441, 1412, 1377, 1346,
ꢀ1 1
1333, 1289, 1270, 1154, 1124, 1041, 1011, 959, 903, 857, 841, 828, 742, 719 cm ; H NMR
(400 MHz, DMSOꢀd ) δ 1.34 (t, 3H, J = 7.1 Hz, CH CH ), 4.34 (q, 2H, J = 7.1 Hz, CH CH ),
6 3 2 3 2
3
3
4
7.97 (d, 1H, J = 8.9 Hz, ArꢀH4), 8.31 (dd, 1H, J = 8.9 Hz, J = 2.4 Hz, ArꢀH5), 9.12 (dd, 1H,
4
13
J = 2.4 Hz), 13.64 (s br, 1H, CONH) ppm; C NMR (100 MHz, DMSOꢀd ) δ 13.71, 62.85,
6
+
119.19, 120.96, 121.83, 132.27, 143.30, 152.86, 157.43, 158.48, 163.03 ppm; HRMS (ESI ) m/z
ꢀ
for C H N O S ([MꢀH] ): calcd. 294.0185, found 294.0189.
1
1
8
3
5
Methyl 3ꢂ((6ꢂnitrobenzo[d]thiazolꢂ2ꢂyl)amino)ꢂ3ꢂoxopropanoate (3). Prepared according to
general procedure A from compound 1 (1.55 g, 7.94 mmol); yellow crystals, yield 1.60 g (68 %);
mp 199ꢀ202 °C; IR (ATR) ν 2978, 1733, 1665, 1609, 1575, 1548, 1509, 1475, 1446, 1434, 1412,
ꢀ1 1
1330, 1280, 1235, 1202, 1168, 1152, 1125, 1045, 1018, 977, 900, 834, 807, 746, 720 cm ; H
3
NMR (400 MHz, DMSOꢀd ) δ 3.68 (s, 3H, OCH ), 3.82 (s, 2H, COCH CO), 7.93 (d, 1H, J =
6
3
2
3
4
4
8.9 Hz, ArꢀH4), 8.30 (dd, 1H, J = 8.9 Hz, J = 2.4 Hz, ArꢀH5), 9.09 (d, 1H, J = 2.4 Hz, Arꢀ
13
H7), 13.19 (s br, 1H, CONH) ppm; C NMR (100 MHz, DMSOꢀd ) δ 42.21, 52.27, 119.11,
6
+
120.75, 121.77, 132.13, 143.06, 153.27, 163.04, 165.95, 167.16 ppm; HRMS (ESI ) m/z for
+
C H N O S ([M+H] ): calcd. 296.0341, found 296.0343.
11
10
3
5
Methyl 4ꢂ((6ꢂnitrobenzo[d]thiazolꢂ2ꢂyl)amino)ꢂ4ꢂoxobutanoate (4). Prepared according to
general procedure A from compound 1 (2.00 g, 10.2 mmol); yellow crystals, yield 2.00 g (63 %);
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2
2
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2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
mp 231ꢀ234 °C; IR (ATR) ν 3259. 3107, 2967, 2923, 2362, 1719, 1667, 1576, 1538, 1508, 1440,
ꢀ1 1
1
430, 1336, 1272, 1212, 1154, 1132, 1071, 1044, 1016, 997, 954, 908, 829 cm ; H NMR (400
MHz, DMSOꢀd ) δ 2.70 (m, 2H, COCH ), 2.83 (m, 2H, COCH ), 3.62 (s, 3H, OCH ), 7.88 (d,
6
2
2
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
3
4
4
1
H, J = 8.9 Hz, ArꢀH4), 8.27 (dd, 1H, J = 8.9 Hz, J = 2.4 Hz, ArꢀH5), 9.03 (d, 1H, J = 2.4
1
3
Hz, ArꢀH7), 12.86 (s br, 1H, CONH) ppm; C NMR (100 MHz, DMSOꢀd ) δ 27.83, 30.08,
6
51.52, 119.00, 120.51, 121.72, 132.11, 142.89, 153.45, 163.30, 171.84, 172.50 ppm; HRMS
+
+
(
ESI ) m/z for C H N O S ([M+H] ): calcd. 310.0498, found 310.0495.
12 12 3 5
General procedure B. Synthesis of compounds 5, 6 and 7. Catalytic hydrogenation of
compound 2 (1.00 mmol in 10 mL methanol) and compounds 3 and 4 (1.00 mmol in 10 mL
ethanol) were performed in the presence of 10% Pd/C (0.030 g) as a catalyst at room temperature
and atmospheric pressure for 24 h. At the end of the reaction, the reaction mixture was filtered
and the filtrate was concentrated to give the crude products which were recrystallized from
methanol (compound 5) or ethanol (compounds 6, 7).
Ethyl 2ꢂ((6ꢂaminobenzo[d]thiazolꢂ2ꢂyl)amino)ꢂ2ꢂoxoacetate (5). Prepared according to general
procedure B from compound 2 (0.850 g, 2.88 mmol); greenish crystals, yield 0.698 g (91 %); mp
147ꢀ150 °C; IR (ATR) ν 3268, 2988, 2362, 1733, 1688, 1604, 1549, 1464, 1393, 1367, 1296,
ꢀ1 1
1258, 1160, 1112, 1058, 1011, 964, 907, 865, 812, 802 cm ; H NMR (400 MHz, DMSOꢀd ) δ
6
1
.31 (t, 3H, J = 7.1 Hz, CH CH ), 4.31 (q, 2H, J = 7.1 Hz, CH CH ), 5.34 (s br, 2H, NH ), 6.76
2 3 2 3 2
3
4
4
(
dd, 1H, J = 8.6 Hz, J = 2.1 Hz, ArꢀH5), 7.05 (d, 1H, J = 2.1 Hz, ArꢀH7), 7.47 (d, 1H, J = 8.6
1
3
Hz, ArꢀH4), 12.82 (s br, 1H, CONH); C NMR (100 MHz, DMSOꢀd ): δ 13.74, 62.51, 103.94,
6
+
1
14.88, 120.88, 132.99, 138.48, 146.31, 152.84, 156.77, 159.41 ppm; HRMS (ESI ) m/z for
+
C H N O S ([M+H] ): calcd. 266.0599, found 266.0602.
11 12
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Methyl 3ꢂ((6ꢂaminobenzo[d]thiazolꢂ2ꢂyl)amino)ꢂ3ꢂoxopropanoate (6). Prepared according to
general procedure B from compound 3 (1.50 g, 5.08 mmol); brownish crystals, yield 0.825 g (61
%); mp 162ꢀ165 °C; IR (ATR) ν 3381, 2958, 2361, 2342, 1726, 1683, 1608, 1555, 1464, 1433,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ꢀ1 1
1
408, 1331, 1310, 1285, 1252, 1233, 1212, 1175, 1141, 1057, 1015, 978, 909, 866, 780 cm ; H
NMR (400 MHz, DMSOꢀd ) δ 3.62 (s, 2H, COCH CO), 3.67 (s, 3H, OCH ), 5.24 (s br, 2H,
6
2
3
3
4
4
NH ), 6.72 (dd, 1H, J = 8.6 Hz, J = 2.1 Hz, ArꢀH5), 7.01 (d, 1H, J = 2.1 Hz, ArꢀH7), 7.42 (d,
2
3
13
1
H, J = 8.6 Hz, ArꢀH4), 12.23 (s br, 1H, CONH) ppm; C NMR (100 MHz, DMSOꢀd ) δ
6
42.10, 52.14, 103.97, 114.49, 120.99, 132.92, 139.43, 145.91, 152.51, 164.41, 167.51 ppm;
+
+
HRMS (ESI ) m/z for C H N O S ([M+H] ): calcd. 266.0599, found 266.0606.
1
1
12
3
3
Methyl 4ꢂ((6ꢂaminobenzo[d]thiazolꢂ2ꢂyl)amino)ꢂ4ꢂoxobutanoate (7). Prepared according to
general procedure B from compound 4 (0.700 g, 2.26 mmol); brownish crystals, yield 0.440 g
(
70 %); mp 208ꢀ211 °C; IR (ATR) ν 3201, 2988, 2947, 2362, 1727, 1687, 1607, 1575, 1556,
ꢀ1
1
1
472, 14737, 1422, 1387, 1311, 1271, 1232, 1151, 1081, 1060, 999, 983, 915, 898 cm ; H
NMR (400 MHz, DMSOꢀd ) δ 2.64 (m, 2H, COCH ), 2.72 (m, 2H, COCH ), 3.61 (s, 3H,
6
2
2
3
4
4
OCH ), 5.16 (s br, 2H, NH ), 6.70 (dd, 1H, J = 8.6 Hz, J = 2.3 Hz, ArꢀH5), 6.99 (d, 1H, J =
3
2
3
13
2
.3 Hz, ArꢀH7), 7.40 (d, 1H, J = 8.6 Hz, ArꢀH4), 12.07 (s, 1H, CONH) ppm; C NMR (100
MHz, DMSOꢀd ) δ 28.00, 29.81, 51.43, 104.03, 114.34, 120.78, 132.85, 139.56, 145.68. 152.87,
6
+
+
1
70.26, 172.59 ppm; HRMS (ESI ) m/z for C H N O S ([M+H] ): calcd. 280.0756, found
12 14 3 3
280.0758.
General procedure C. Synthesis of compounds 8ꢂ12. To a solution of 4,5ꢀdibromoꢀ1Hꢀ
pyrroleꢀ2ꢀcarboxylic acid (for 8, 9 and 10) (0.250 g, 0.930 mmol) or 4,5ꢀdichloroꢀ1Hꢀpyrroleꢀ2ꢀ
carboxylic acid (for 11 and 12) (0.167 g, 0.930 mmol) in anhydrous dichloromethane (20 mL),
oxalyl chloride (0.46 mL of 2M solution in methylene chloride, 0.93 mmol) was added and the
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reaction mixture was stirred for 15 h. The solvent was removed under reduced pressure
whereupon dichloromethane (15 mL), pyridine (5 mL) appropriate amine (1.00 mmol) were
added to the residue and stirred for 2 h at room temperature The reaction mixture was
concentrated and dissolved in ethyl acetate (50 mL) and the solution washed successively with
0
1
2
3
4
5
6
7
8
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0
1
2
3
4
5
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8
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0
1
2
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9
0
1M HCl (10 mL), NaHCO (10 mL), water (10 mL) and brine (10 mL). The ethyl acetate phase
3
was dried over sodium sulfate and concentrated. The crude products were recrystallized from
methanol.
Ethyl
2ꢂ((6ꢂ(4,5ꢂdibromoꢂ1Hꢂpyrroleꢂ2ꢂcarboxamido)benzo[d]thiazolꢂ2ꢂyl)amino)ꢂ2ꢂ
oxoacetate (8). Prepared according to general procedure C from amine 5 (0.220 g, 0.829 mmol)
and 4,5ꢀdibromoꢀ1Hꢀpyrroleꢀ2ꢀcarbonyl chloride (0.357 g. 1.24 mmol); yellow crystals, yield
0.220 g (51 %); mp 252ꢀ255 °C; IR (ATR) ν 3122, 2971, 2362, 1745, 1703, 1640, 1609, 1574,
1546, 1521, 1460, 1434, 1410, 1332, 1312, 1251, 1232, 1187, 1163, 1107, 1042, 1014, 984, 974,
ꢀ1 1
9
61, 869, 824, 810 cm ; H NMR (400 MHz, DMSOꢀd ) δ 1.33 (t, 3H, J = 7.1 Hz, CH CH ),
6
2
3
4
3
4.33 (q, 2H, J = 7.1 Hz, CH CH ), 7.26 (d, 1H, J = 2.7 Hz, pyrroleꢀH3), 7.72 (dd, 1H, J = 8.8
2
3
4
3
4
Hz, J = 2.0 Hz, ArꢀH5), 7.79 (d, 1H, J = 8.8 Hz, ArꢀH4), 8.44 (d, 1H, J = 2.0 Hz, ArꢀH7),
1
3
1
0.07 (s, 1H, NHCO), 12.96 (s br, 1H, NHCOCO), 13.17 (s br, 1H, pyrroleꢀNH) ppm; C NMR
(
100 MHz, DMSOꢀd ) δ 13.75, 62.68, 98.17, 106.02, 112.49, 113.86, 119.78, 120.69, 127.79,
6
131.98, 135.35, 144.25, 157.29, 159.03 ppm (2 carbonyl signals not seen due to broadening);
ꢀ
ꢀ
HRMS (ESI ) m/z for C H Br N O S ([MꢀH] ): calcd. 512.8868, found 512.8882; HPLC: t =
16
11
2
4
4
r
20.94 min (96.3 % at 220 nm).
Methyl
3ꢂ((6ꢂ(4,5ꢂdibromoꢂ1Hꢂpyrroleꢂ2ꢂcarboxamido)benzo[d]thiazolꢂ2ꢂyl)amino)ꢂ3ꢂ
oxopropanoate (9). Prepared according to general procedure C from compound 6 (0.200 g, 0.754
mmol) and 4,5ꢀdibromoꢀ1Hꢀpyrroleꢀ2ꢀcarbonyl chloride (0.325 g, 1.13 mmol); yellow crystals,
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yield 0.225 g (58 %); mp > 340 °C; IR (ATR) ν 3199, 2988, 2361, 1722, 1691, 1643, 1611,
1
9
1
575, 1549, 1523, 1468, 1436, 1410, 1370, 1329, 1370, 1254, 1225, 1180, 1148, 1067, 1016,
ꢀ1 1
73, 869, 815 cm ; H NMR (400 MHz, DMSOꢀd ) δ 3.68 (s, 5H, OCH , COCH CO), 7.26 (d,
6
3
2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4
3
4
3
H, J = 2.7 Hz, pyrroleꢀH3), 7.68 (dd, 1H, J = 8.8 Hz, J = 2.0 Hz, ArꢀH5), 7.74 (d, 1H, J =
4
8.8 Hz, ArꢀH4), 8.39 (d, 1H, J = 2.0 Hz, ArꢀH7), 10.02 (s, 1H, NHCO), 12.54 (s, 1H,
4
13
NHCOCH ), 12.94 (d, 1H, J = 2.7 Hz, pyrroleꢀNH) ppm; C NMR (100 MHz, DMSOꢀd ): δ
2
6
4
2.16, 52.21, 98.15, 105.93, 112.4, 113.78, 119.51, 120.63, 127.84, 131.85, 134.89, 144.61,
ꢀ
ꢀ
156.61, 157.24, 165.05, 167.38 ppm; HRMS (ESI ) m/z for C H Br N O S ([MꢀH] ): calcd.
1
6
11
2
4
4
5
12.8868, found 512.8879; HPLC: t = 19.07 min (97.1 % at 220 nm).
r
Methyl
4ꢂ((6ꢂ(4,5ꢂdibromoꢂ1Hꢂpyrroleꢂ2ꢂcarboxamido)benzo[d]thiazolꢂ2ꢂyl)amino)ꢂ4ꢂ
oxobutanoate (10). Prepared according to general procedure C from compound 7 (0.318 g, 1.14
mmol) and 4,5ꢀdibromoꢀ1Hꢀpyrroleꢀ2ꢀcarbonyl chloride (0.491 g, 1.71 mmol); yellow crystals,
yield 0.260 g (43 %); mp. 254ꢀ257 °C; IR (ATR) ν 3198, 2988, 2361, 1701, 1647, 1616, 1578,
1547, 1521, 1473, 1443, 1408, 1357, 1332, 1279, 1259, 1223, 1181, 1154, 1066, 983, 970, 954,
ꢀ1
1
9
20, 867 cm ; H NMR (400 MHz, DMSOꢀd ): δ 2.67 (m, 2H, CH CO), 2.78 (m, 2H, CH CO),
6 2 2
4
3
4
3
.62 (s, 3H, OCH ), 7.25 (d, 1H, J = 2.7 Hz, pyrroleꢀH3), 7.66 (dd, 1H, J = 8.8 Hz, J = 2.0 Hz,
3
3
4
ArꢀH5), 7.72 (d, 1H, J = 8.8 Hz, ArꢀH4), 8.36 (d, 1H, J = 2.0 Hz, ArꢀH7), 10.00 (s, 1H,
4
13
NHCO), 12.38 (s, 1H, NHCOCH ), 12.93 (d, 1H, J = 2.7 Hz, pyrroleꢀNH) ppm; C NMR (100
2
MHz, DMSOꢀd ) δ 27.94, 29.92, 51.47, 98.14, 105.88, 112.40, 113.75, 119.38, 120.41, 127.87,
6
ꢀ
1
31.81, 134.66, 144.72, 156.92, 157.23, 170.92, 172.56 ppm; HRMS (ESI ) m/z for
ꢀ
C H Br N O S ([MꢀH] ): calcd. 526.9024, found 526.9035; HPLC: t = 20.39 min (97.1 % at
17
13
2
4
4
r
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Ethyl
2ꢂ((6ꢂ(4,5ꢂdichloroꢂ1Hꢂpyrroleꢂ2ꢂcarboxamido)benzo[d]thiazolꢂ2ꢂyl)amino)ꢂ2ꢂ
oxoacetate (11). Prepared according to general procedure C from compound 5 (0.250 g, 0.942
mmol); yellow crystals, yield 0.230 g (57 %); mp 272ꢀ275 °C; IR (ATR) ν 3410, 3173, 2986,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
752, 1705, 1654, 1608, 1579, 1545, 1521, 1465, 1412, 1339, 1285, 1231, 1188, 1146, 1015,
ꢀ1 1
961, 868, 812, 747 cm ; H NMR (400 MHz, DMSOꢀd ): δ 1.33 (t, 3H, J = 7.1 Hz, CH CH ),
6
2
3
3
4
4
.33 (q, 2H, J = 7.1 Hz, CH CH ), 7.23 (s, 1H, pyrroleꢀH3), 7.72 (dd, 1H, J = 8.87 Hz, J = 2.0
2 3
Hz, ArꢀH5), 7.79 (d, 1H, J = 8.8 Hz, ArꢀH4), 8.44 (d, 1H, J = 2.0 Hz, ArꢀH7), 10.08 (s, 1H,
1
3
NHCO), 13.01 (s br, 1H, NHCOCH ), 13.18 (s br, 1H, pyrroleꢀNH) ppm; C NMR (100 MHz,
2
DMSOꢀd ) δ 13.75, 62.68, 108.28, 110.93, 112.58, 116.11, 119.83, 120.72, 124.52, 131.96,
6
1
35.28, 143.59, 157.42, 159.05 ppm (2 carbonyl signals not seen due to broadening). HRMS
ꢀ
ꢀ
(ESI ) m/z for C H Cl N O S ([MꢀH] ): calcd 424.9878, found 424.9872; HPLC: t = 20.73 min
1
6
11
2
4
4
r
(
96.2 % at 220 nm).
Methyl
3ꢂ((6ꢂ(4,5ꢂdichloroꢂ1Hꢂpyrroleꢂ2ꢂcarboxamido)benzo[d]thiazolꢂ2ꢂyl)amino)ꢂ3ꢂ
oxopropanoate (12). Prepared according to general procedure C from compound 6 (0.220 g,
0
3
1
3
.829 mmol); yellow crystals, yield 0.195 g (55 %); mp. 332ꢀ335 °C; IR (ATR) ν 3285, 3184,
041, 2988, 1725, 1691, 1637, 1614, 1575, 1548, 1526, 1461, 1413, 1381, 1254, 1234, 1210,
ꢀ1 1
177, 1149, 1082, 1019, 978, 869, 817, 761, 724, 674 cm ; H NMR (400 MHz, DMSOꢀd ): δ
6
3
3
4
.68 (s, 5H, CH , OCH ), 7.22 (d, 1H, J = 2.8 Hz, pyrroleꢀH3), 7.68 (dd, 1H, J = 8.8 Hz, J =
2
3
3
2.0 Hz, ArꢀH5), 7.74 (d, 1H, J = 8.8 Hz, ArꢀH4), 8.38 (d, 1H, J = 2.0 Hz, ArꢀH7), 10.04 (s, 1H,
4
13
NHCO), 12.54 (s br, 1H, NHCOCH ), 12.98 (d, 1H, J = 2.8 Hz, pyrroleꢀNH) ppm; C NMR
2
(
100 MHz, DMSOꢀd ) δ 42.17, 52.20, 108.25, 110.86, 112.53, 116.03, 119.56, 120.62, 124.55,
6
ꢀ
131.81, 134.83, 144.64, 156.66, 157.37, 165.07, 167.38 ppm. HRMS (ESI ) m/z for
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8
9
ꢀ
C H Cl N O S ([MꢀH] ): calcd. 424.9878, found 424.9880; HPLC: t = 19.58 min (96.4 % at
16
11
2
4
4
r
220 nm).
General procedure D. Synthesis of compounds 13, 14 and 24-29. Hydrolysis of esters 8,
0 and 18ꢀ23 (1.00 mmol) was performed with 1M NaOH (2.00 mL, 2.00 mmol) in methanol (10
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
mL) at room temperature overnight. After evaporation of the methanol, the reaction mixture was
acidified with 1M HCl to pH 3 and the precipitated carboxylic acids were collected by filtration.
2
ꢂ((6ꢂ(4,5ꢂDibromoꢂ1Hꢂpyrroleꢂ2ꢂcarboxamido)benzo[d]thiazolꢂ2ꢂyl)amino)ꢂ2ꢂoxoacetic acid
13). Prepared according to general procedure D from compound 8 (0.105 g, 0.203 mmol);
brownish crystals, yield 0.051 g (51 %); mp 238ꢀ241 °C; IR (ATR) ν 3117, 1703, 1656, 1613,
(
ꢀ1
1
1
547, 1518, 1478, 1411, 1388, 1293, 1225, 1161, 927, 907, 864, 813, 739, 689 cm ; H NMR
3
4
(
400 MHz, DMSOꢀd ): δ 7.27 (d, 1H, J = 2.8 Hz, pyrroleꢀH3), 7.71 (dd, 1H, J = 8.7 Hz, J =
6
3
4
2
.0 Hz, ArꢀH5), 7.78 (d, 1H, J = 8.7 Hz, ArꢀH4), 8.44 (d, 1H, J = 2.0 Hz, ArꢀH7), 10.06 (s, 1H,
4
NHCO), 12.94 (s br, 1H, NHCOCO), 12.95 (d, 1H, J = 2.8 Hz, pyrroleꢀNH) ppm, signal for
1
3
COOH not observed; C NMR (100 MHz, DMSOꢀd ) δ 98.17, 105.97, 112.40, 113.89, 119.68,
6
ꢀ
1
20.85, 127.81, 132.09, 135.27, 144.41, 156.28, 157.27, 158.40, 160.69 ppm; HRMS (ESI ) m/z
ꢀ
for C H Br N O S ([MꢀH] ): calcd 484.8555, found 484.8550; HPLC: t = 17.02 min (97.4 % at
1
4
7
2
4
4
r
220 nm).
4ꢂ((6ꢂ(4,5ꢂDibromoꢂ1Hꢂpyrroleꢂ2ꢂcarboxamido)benzo[d]thiazolꢂ2ꢂyl)amino)ꢂ4ꢂoxobutanoic
acid (14). Prepared according to general procedure D from compound 10 (0.110 g, 0.207 mmol);
brownish crystals, yield 0.039 g (36 %); mp. 285ꢀ288 °C; IR (ATR) ν 3203, 2971, 1660, 1615,
1574, 1547, 1515, 1472, 1405, 1367, 1333, 1311, 1284, 1254, 1215, 1159, 1066, 1010, 973, 917,
ꢀ1 1
8
67, 844, 805, 743, 715 cm ; H NMR (400 MHz, DMSOꢀd ) δ 2.59 (m, 2H, CH CO), 2.73 (m,
6
2
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1
1
1
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1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
4
2
H, CH CO), 7.25 (d, 1H, J = 2.7 Hz, pyrroleꢀH3), 7.66 (dd, 1H, J = 8.8 Hz, J= 2.0 Hz, Arꢀ
2
3
4
H5), 7.71 (d, 1H, J = 8.8 Hz, ArꢀH4), 8.36 (d, 1H, J = 2.0 Hz, ArꢀH7), 10.00 (s, 1H, NHCO),
4
13
1
2.35 (s br, 2H, NHCOCH . COOH), 12.93 (d, 1H, J = 2.8 Hz, pyrroleꢀNH) ppm; C NMR
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
100 MHz, DMSOꢀd ) δ 28.25, 30.06, 98.14, 105.87, 112.40, 113.75, 119.38, 120.38, 127.87,
6
ꢀ
131.79, 134.63, 144.73, 156.97, 157.22, 171.18, 173.53 ppm; HRMS (ESI ) m/z for
ꢀ
C H Br N O S ([MꢀH] ): calcd. 512.8868, found 512.8881; HPLC: t = 17.00 min (95.2 % at
16
11
2
4
4
r
220 nm).
Benzo[d]thiazoleꢂ2,6ꢂdiamine (15). 10% Pd/C (0.195 g) was added to a solution of 6ꢀ
nitrobenzo[d]thiazolꢀ2ꢀamine (1) (1.95 g, 10.0 mmol) in ethanol (100 mL) and hydrogenated in
Parr hydrogenator at 2.1 bar pressure at room temperature for 6 h. The reaction mixture was
filtered through celite and cotton and the filtrate was evaporated under reduced pressure to give
1
benzo[d]thiazoleꢀ2,6ꢀdiamine (15) (1.59 g, 96 % yield). H NMR (400 MHz, DMSOꢀd ) δ 4.79
6
3
4
4
(
s br, 2H, 6ꢀNH , 6.45 (dd, 1H, J = 8.4 Hz, J = 2.2 Hz, ArꢀH5), 6.81 (d, 1H, J = 2.2 Hz, Arꢀ
2
H7), 6.93 (s br, 2H, 2ꢀNH ), 7.03 (d, 1H, J = 8.4 Hz, ArꢀH4) ppm.
2
General procedure E. Synthesis of compounds 16 and 17. Benzo[d]thiazoleꢀ2,6ꢀdiamine (15)
(
0.714 g, 4.32 mmol) and Na CO3 (0.458 g, 4.32 mmol) were dissolved in N,Nꢀ
2
dimethylformamide (30 mL) and the mixture was heated to 80 °C. 2,2,2ꢀTrichloroꢀ1ꢀ(4,5ꢀ
dibromoꢀ1Hꢀpyrrolꢀ2ꢀyl)ethanꢀ1ꢀone (1.60 g, 4.32 mmol) or 2,2,2ꢀtrichloroꢀ1ꢀ(4,5ꢀdichloroꢀ1Hꢀ
pyrrolꢀ2ꢀyl)ethanꢀ1ꢀone (1.22 g, 4.32 mmol) was added and the mixture stirred for 5 h at 80 °C.
Solvent was removed under reduced pressure and the residue was recrystallized from EtOAc to
obtain compound 16 or 17.
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Nꢂ(6ꢂAminobenzo[d]thiazolꢂ2ꢂyl)ꢂ4,5ꢂdibromoꢂ1Hꢂpyrroleꢂ2ꢂcarboxamide (16). Prepared
according to general procedure E from 15 and 2,2,2ꢀtrichloroꢀ1ꢀ(4,5ꢀdibromoꢀ1Hꢀpyrrolꢀ2ꢀ
yl)ethanꢀ1ꢀone; yellow crystals, yield 1.35 g (75 %); mp 269ꢀ271 °C; IR (ATR) ν 2971, 2362,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
687, 1649, 1611, 1556, 1470, 1405, 1381, 1318, 1298, 1276, 1244, 1174, 1137, 1057, 991, 974,
ꢀ1
1
3
938, 914, 865, 823 cm ; H NMR (400 MHz, DMSOꢀd ): δ 5.23 (s, 2H, 6ꢀNH ), 6.73 (dd, 1H, J
6
2
4
4
3
=
8.6, J = 2.0 Hz, ArꢀH5), 7.03 (d, 1H, J = 2.0 Hz, ArꢀH7), 7.43 (d, 1H, J = 8.6 Hz, ArꢀH4),
1
3
7.45 (s, 1H, pyrroleꢀH3), 12.18 (s br, 1H, NH), 13.12 (s br, 1H, NH) ppm; C NMR (100 MHz,
DMSOꢀd ): δ 98.83, 103.98, 107.94, 114.44, 115.49, 120.59, 126.03, 133.01, 139.44, 145.82,
6
ꢀ
ꢀ
1
53.35, 156.82 ppm, HRMS (ESI ) m/z for C H Br N OS ([MꢀH] ): calcd 412.8707, found
12 7 2 4
4
12.8708; HPLC: t = 17.34 min (100 % at 254 nm).
r
Nꢂ(6ꢂAminobenzo[d]thiazolꢂ2ꢂyl)ꢂ4,5ꢂdichloroꢂ1Hꢂpyrroleꢂ2ꢂcarboxamide (17). Prepared
according to general procedure E from 15 and 2,2,2ꢀtrichloroꢀ1ꢀ(4,5ꢀdichloroꢀ1Hꢀpyrrolꢀ2ꢀ
yl)ethanꢀ1ꢀone; yellow crystals, yield 1.10 g (78 %); mp 275ꢀ278 °C; IR (ATR) ν 3126, 2988,
2362, 1659, 1614, 1553, 1473, 1419, 1392, 1327, 1295, 1278, 1182, 1132, 1057, 1020, 992, 937,
ꢀ1
1
3
862, 824, 724 cm ; H NMR (400 MHz, DMSOꢀd ) δ 5.22 (s, 2H, 6ꢀNH ), 6.73 (dd, 1H, J =
6 2
4
4
8
.6, J = 2.0 Hz, ArꢀH5), 7.03 (d, 1H, J = 2.0 Hz, ArꢀH7), 7.42 (s, 1H, pyrroleꢀH3), 7.43 (d, 1H,
3
13
J = 8.6 Hz, ArꢀH4), 12.19 (s br, 1H, NH), 13.22 (s br, 1H, NH) ppm; C NMR (100 MHz,
DMSOꢀd ) δ 104.00, 108.90, 112.58, 114.46, 117.76, 120.58, 122.72 132.99, 139.41, 145.80,
6
ꢀ
ꢀ
153.37, 156.99 ppm; HRMS (ESI ) m/z for C H Cl N OS ([MꢀH] ): calcd 324.9718, found
1
2
7
2
4
324.9709; HPLC: t = 17.09 min (97.3 % at 254 nm).
r
General procedure F. Synthesis of compounds 18-23. To a solution of compound 16 (0.220 g,
.529 mmol) (for 18, 19, 20) or compound 17 (0.173 g, 0.529 mmol) (for 21, 22, 23) and
0
triethylamine (0.10 mL, 0.79 mmol) in 1,4ꢀdioxane (20 mL) cooled to 0 °C, ethyl oxalyl chloride
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