Synthesis and Structure–Activity Relationship Studies of Benzo[b][1,4]oxazin-3(4H)-one Analogues as Inhibitors of Mycobacterial Thymidylate Synthase X

Article abstract of DOI:10.1002/cmdc.201800739

Since the discovery of a flavin-dependent thymidylate synthase (ThyX or FDTS) that is absent in humans but crucial for DNA biosynthesis in a diverse group of pathogens, the enzyme has been pursued for the development of new antibacterial agents against Mycobacterium tuberculosis, the causative agent of the widespread infectious disease tuberculosis (TB). In response to a growing need for more effective anti-TB drugs, we have built upon our previous screening efforts and report herein an optimization campaign of a novel series of inhibitors with a unique inhibition profile. The inhibitors display competitive inhibition toward the methylene tetrahydrofolate cofactor of ThyX, enabling us to generate a model of the compounds bound to their target, thus offering insight into their structure–activity relationships.

Full text of DOI:10.1002/cmdc.201800739

Accepted Article
Title: Synthesis and structure-activity relationship studies of benzo[b]
[1,4]oxazin-3(4H)-one analogues as inhibitors of mycobacterial
thymidylate synthase X
Authors: Jakub Modranka, Jiahong Li, Anastasia Parchina, Michiel
Vanmeert, Shrinivas Dumbre, Mayla Salman, Hannu
Myllykallio, Hubert F. Becker, Roeland Vanhoutte, Lia
Margamuljana, Hoai Nguyen, Rania Abu El Asrar, Jef
Rozenski, Piet Herdewijn, Steven Dejonghe, and Eveline
Lescrinier
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published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.201800739
Link to VoR: http://dx.doi.org/10.1002/cmdc.201800739
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10.1002/cmdc.201800739
ChemMedChem
FULL PAPER
Synthesis and structure-activity relationship studies of
benzo[b][1,4]oxazin-3(4H)-one analogues as inhibitors of
mycobacterial thymidylate synthase X
Jakub Modranka,‖,[a] Jiahong Li,‖,[a] Anastasia Parchina,‖,[a] Michiel Vanmeert,[a] Shrinivas Dumbre,[a]
Mayla Salman,[b] Hannu Myllykallio,[b] Hubert F. Becker,[b,c] Roeland Vanhoutte, ‡,[d] Lia Margamuljana,[a]
Hoai Nguyen,[a] Rania Abu El Asrar,[a] Jef Rozenski,[a] Piet Herdewijn,[a] Steven De Jonghe,*,†,[e] and
Eveline Lescrinier*[a]
[a]
Dr. J. Modranka (0000-0001-9239-8535), Dr. J. Li, Dr. A. Parchina, Mr. M. Vanmeert (0000-0001-5810-4935), Dr. S. Dumbre (0000-0002-1237-9586), Mrs.
L. Margamuljana, Mrs. H. Nguyen, Ms. R. Abu El Asrar, Prof. J. Rozenski (0000-0001-9624-5536), Prof. P. Herdewijn (0000-0003-3589-8503), Dr. S. De
Jonghe, Prof. E. Lescrinier (0000-0001-7066-4329)
Medicinal Chemistry, Rega Institute for Medical Science
KU Leuven
Herestraat 49 – PO Box 1030, BE-3000 Leuven, Belgium
E-mail: Steven.Dejonghe@kuleuven.be; Eveline.Lescrinier@kuleuven.be
[b]
[c]
Dr. M. Salman, Prof. H. Myllykallio (0000-0002-0541-1197), H.F. Becker (0000-0003-3136-6075)
Laboratory of Optics and Biosciences
INSERM U 696- CNRS UMR 7645 -Ecole Polytechnique
Route de Saclay, 91128 Palaiseau Cedex, France
Prof. H.F. Becker (0000-0003-3136-6075)
Faculté des Sciences et Ingénierie
Sorbonne Université
4 place Jussieu 75005 Paris, France
These authors contributed equally.
‖
†
Present affiliation: Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, KU Leuven, Herestraat 49 – box 1043 , 3000 Leuven,
Belgium.
‡
Present affiliation: Laboratory of Chemical Biology, KU Leuven, O&N I Herestraat 49 - box 802, 3000 Leuven, Belgium.
Supporting information for this article is given via a link at the end of the document.
Abstract: Since the discovery of a flavin-dependent thymidylate
synthase (ThyX or FDTS), that is absent in humans but crucial for
DNA biosynthesis in a diverse group of pathogens, the enzyme has
been pursued for the development of new antibacterial agents against
Mycobacterium tuberculosis, the causative agent of the widespread
infectious disease tuberculosis (TB). In response to a growing need
for more effective anti-TB drugs, we have built upon our previous
screening efforts and report here an optimization campaign of a novel
series of inhibitors with a unique inhibition profile. The inhibitors
display competitive inhibition towards the methylene tetrahydrofolate
cofactor of the enzyme, enabling us to generate a model of the
compounds bound to their target and offering insights into their
structure–activity relationships.
and ethambutol).[2] Despite having these treatment options,
according to the latest report of the World Health Organisation
(WHO), M. tuberculosis still caused an estimated 1.7 million
deaths in 2016. In addition, an estimated 10.4 million people fell
ill with tuberculosis.[1] Recently, two new antitubercular drugs
(bedaquiline[3] and delamanid[4]) received marketing approval.
However, the emergence of multidrug-resistant (MDR),
extensively drug-resistant (XDR) and even totally drug-resistant
(TDR) M. tuberculosis strains remains a major threat to public
health.[5] Consequently, there is an urgent demand for new
antituberculosis drugs acting on novel mycobacterial targets.
Thymidylate synthase (ThyA) catalyzes the de novo synthesis of
thymidine-5’-monophosphate (TMP) via reductive methylation of
2’-deoxyuridine-5’-monophosphate (dUMP). ThyA uses N-5,N-
10-methylene tetrahydrofolic acid (CH2H4folate) both as one
carbon and hydride donor, whereby dihydrofolate (DHF) is formed.
The catalytic activity of ThyA is coupled to the action of two other
enzymes. Dihydrofolate reductase (DHFR) mediates the
reduction of DHF to tetrahydrofolate (H4folate), which is further
Introduction
Tuberculosis (TB) is a contagious-infectious disease caused by
the bacterium Mycobacterium tuberculosis. Currently, it is the
ninth leading cause of death worldwide and the leading cause
from a single infectious agent.[1] The prognosis for patients with
tuberculosis improved dramatically with the introduction of
antitubercular drugs (such as rifampicine, isoniazid, pyrazinamide
subjected
to
remethylenation
catalyzed
by
serine
hydroxymethyltransferase (SHMT) to regenerate the CH2H4folate
pools. Thymidine-5’-monophosphate is readily phosphorylated to
thymidine-5’-triphosphate (TTP), one of the four building blocks
required for the biosynthesis of DNA.[6] Several human pathogens
1
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10.1002/cmdc.201800739
ChemMedChem
FULL PAPER
lack the genes encoding for ThyA and DHFR, but are still fully
viable in thymidine-deficient media. Genomic analysis led to the
discovery of the thyX gene which encodes for the ThyX-protein, a
flavin-dependent thymidylate synthase (FDTS) which is absent in
humans.[7] ThyX uses CH2H4folate only as one-carbon donor, with
reduced flavin adenine dinucleotide (FADH2) serving as a hydride
donor. ThyX enzymes are NAD(P)H-oxidases since catalysis
requires the presence of NAD(P)H to furnish sufficient amounts of
FADH2 via reduction of FAD.[8] Furthermore, ThyX shows no
structural or sequence similarity with ThyA.[9] While some
pathogens solely rely on ThyX, M. tuberculosis carries conserved
genes encoding for ThyA as well as for ThyX.[7] The thyX-gene is
required and essential for mycobacterial growth and survival
within macrophages, making ThyX a promising antimycobacterial
drug target.[10] Despite this, only a limited number of ThyX
inhibitors is known (Figure 1).[11] Structural variation of the uracil
moiety of the natural substrate dUMP afforded a series of 5-
alkynyl dUMP analogues, from which the most potent congener
(compound 1), displayed an IC50 value of 0.9 µM against
mycobacterial ThyX.[12]
cells. Therefore, quinones are not desirable scaffolds in medicinal
chemistry.[15] A library of thiazolidine derivatives has been
prepared and evaluated as inhibitors of viral PBCV-1 ThyX. The
most potent congener (compound 7) was endowed with an IC50
value of 0.057 µM.[16] In an effort to obtain non-substrate inhibitors,
a virtual screening campaign of the ZINC database against
mycobacterial ThyX was performed. The most potent congener
was an imidazo[4,5-d]pyridazine analogue (compound 8) that,
however, displayed only 29% inhibition at 100 µM.[17]
Because of the potential of ThyX as antibacterial drug target and
the lack of drug-like small-molecule ThyX inhibitors, we recently
embarked on a high-throughput screening (HTS) campaign of
commercially available compound libraries in the search for novel
and drug-like mycobacterial ThyX inhibitors. It led to the discovery
of B1-PP146 (compound 9, Figure 2) as
a tight-binding
mycobacterial ThyX inhibitor with an IC50 of 0.71 µM.[18] Screening
of structurally related, commercially available analogues led to the
discovery of compound 10, endowed with an IC50 value of 0.9 µM.
As the N-carboxamide-piperazine substructure of compound 10
allows for more and easier structural variation than the N-
pyrimidinyl-piperazine moiety of 9, compound 10 was selected as
starting point for an optimization campaign. In this manuscript, we
describe our medicinal chemistry efforts in order to study the
structure-activity relationship of these benzoxazine analogues as
inhibitors of mycobacterial ThyX.
H
O
R8
O
N
O
O
R2
R3
O
HO
N
P
O
HO
HO
O
HN
1
C7H15
2
: R2 = Br; R3 = H; R8 = OH
3 : R2 = OH; R3 = 4-methoxy-benzyl; R8 = H
O
O
O
4
: R2 = OH; R3 = n-butyl; R8 = H
5 : R2 = OH; R3 = 3-CF3,4-F-phenyl; R8 = H
CH3
N
N
O
O
N
N
O
O
6
: R2 = OH; R3 = 2-furanyl; R8 = H
Cl
O
O
O
N
H
N
N
N
N
N
N
N
S
N
NH
N
H
O
N
N
HN
O
O
F3C
8
9
10
7
Figure 2. Benzo[b][1,4]oxazin-3(4H)-ones as mycobacterial ThyX inhibitors.
Figure 1. Inhibitors of ThyX.
Results and Discussion
However, the presence of the polar phosphate moiety precludes
its further development into derivatives with in vitro
Chemistry
antimycobacterial
naphthoquinone
activity.
(compound
2-Bromo-8-hydroxy-1,4-
and 2-hydroxy-3-(4-
The synthesis of N-phenylpiperazine-1-carboxamide analogues
17a-i with structural variation of the phenyl part of the bicyclic
benzo[b][1,4]oxazin-3(4H)-one scaffold was accomplished as
shown in Scheme 1. Commercially available 2H-
benzo[b][1,4]oxazin-3(4H)-one derivatives 11a-h were N-
alkylated with ethyl 3-bromopropionate under alkaline
conditions.[19] Saponification of the ethyl ester moiety of 12a-h
with lithium hydroxide furnished the carboxylic acids 13a-h. N-
Phenylpiperazine-1-carboxamide 16 was synthesized by reaction
of tert-butyl piperazine-1-carboxylate 14 with phenyl isocyanate,
followed by acidic deprotection of the Boc group.[20] Finally,
reaction of acids 13a-h and amine 16 with O-(1H-6-
chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium
2)
methoxybenzyl)-1,4-naphthoquinone (compound 3) have been
identified as inhibitors of ThyX from Paramecium bursaria
chlorella virus-1 (PBCV-1). In addition, these compounds inhibit
ThyX proteins from other microorganisms, including M.
tuberculosis, H. pylori and C. trachomatis. Moreover, compound
2 displays antibacterial activity in a genetically modified E. coli
strain harboring the ThyX gene.[13] Further optimization of this
class of compounds as ThyX inhibitors from H. pylori led to the
discovery of new analogues with improved ThyX inhibition and
antibacterial activity.[14] Moreover, three representatives
displayed promising activity in a mouse model of H. pylori
infection.[14] The authors did not observe any cytotoxicity,
mitochondrial toxicity or in vivo toxicity with these quinone
analogues. However, it is well known that quinones act as Michael
acceptors and can cause cellular damage by alkylation of cellular
proteins and/or DNA. In addition, quinones are highly redox active
molecules that can lead to the formation of reactive oxygen
species (ROS), which can cause severe oxidative stress within
hexafluorophosphate (HCTU) as coupling reagent afforded the
desired compounds 17a-h.
2
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10.1002/cmdc.201800739
ChemMedChem
FULL PAPER
O
O
N
R3
R3
R3
a
R2
R1
O
N
R2
R1
O
N
R2
R1
O
b
a
N
H
O
O
O
O
O
O
N
H
O
18
O
O
11a-h
19
O
HO
b
12a-h
13a-h
O
N
O
N
O
N
O
N
d or e or f
c
g
O
O
O
O
O
O
e
c
N
N
d
Boc
H
N
N
H
N
N
Boc
HN
HN
N
O
N
O
HO
O
N
O
N
R
O
N
HN
R
14
15
16
29a-d
(Table 4)
20
21
22a-n
(Table 3)
R3
O
Y
R2
R1
O
N
NH2
HN
O
h
e
O
Y
X
X
23 : X = N; Y = CH
24: X = CH; Y = N
25: X = N; Y = CH
26: X = CH; Y = N
N
O
H
N
N
O
O
N
O
O
17a-h
(Table 1)
N
H
N
N
Scheme 1. Reagents and conditions : a) ethyl 3-bromopropionate, K2CO3,
DMF, 80°C; b) LiOH, THF, 60°C; c) PhNCO, THF, rt; d) TFA, 0 °C; e) HCTU,
DIPEA, DMSO, rt.
X
Y
O
27: X = N; Y = CH
28: X = CH; Y = N
(Table 3)
Scheme 2. Reagents and conditions : a) ethyl 3-bromopropionate, K2CO3, DMF,
90°C; b) LiOH, THF, 45°C; c) DCC, HOBt, piperazine, rt; d) RNCO, DMF, rt; e)
PhOC(O)Cl, DIPEA, DMF, rt (for 22m); f) PhCH2C(O)Cl, DIPEA, DMF, rt (for
22n); g) R-piperazine, HCTU, DIPEA, rt; h) DIPEA, DMF, rt.
For the synthesis of a library with structural variation of the
terminal aryl group, a slightly modified scheme was used
(Scheme 2). Alkylation of 18, followed by alkaline hydrolysis of the
ester moiety, furnished acid 20. DCC-mediated amide formation
afforded compound 21, as key intermediate from which structural
variation could easily be introduced. Reaction with a series of
isocyanates gave access to a set of urea derivatives 22a-l.
Alternatively, coupling of 21 with phenyl chloroformate or
phenylacetyl chloride furnished carbamate 22m and amide 22n,
respectively. For the synthesis of pyridine-containing urea
derivatives 27 and 28, 4- and 3-aminopyridine (compounds 23
and 24, respectively) were first coupled with phenylchloroformate
yielding carbamates 25 and 26, respectively. Condensation with
piperazine analogue 21 furnished derivatives 27 and 28,
respectively. Because of the commercial availability of a number
of piperazine building blocks, carboxylic acid 20 was easily
converted in one step to the corresponding piperazine analogues
29a-d.
The synthesis of analogues with variation in the linker moiety
between the benzo[b][1,4]oxazin-3(4H)-one core and the
piperazine moiety was effected as shown in Scheme 3. Alkylation
of 18 with the appropriate alkyl bromides gave access to
compounds 30 and 31. Hydrolysis of the ethyl ester group,
followed by coupling with piperazine and phenylisocyanate
afforded final compounds 32 and 33. For the synthesis of
branched-chain derivatives, a similar methodology was applied.
Alkylation of 18 with suitable alkyl bromides gave compounds 34
and 35. Saponification of the ester moiety, followed by reaction
with N-phenylcarboxamide piperazine 16 yielded the desired
derivatives 36 and 37.
Scheme 3. Reagents and conditions. a) ethyl bromoacetate (for 30) or ethyl 4-
bromobutyrate (for 31), K2CO3, DMF, 90°C; b) (i) LiOH, H2O, THF; 45°C; (ii)
DCC, HOBT, piperazine, DMF, rt; (iii) PhNCO, DMF, rt; c) methyl 3-
bromobutanoate (for 34) or methyl 3-bromo-2-methylpropanoate (for 35), K2CO3,
DMF, 90°C; d) (i) LiOH, H2O, THF, 45 °C; (ii) DCC, HOBT, N-phenylpiperazine-
1-carboxamide, DMF; e) tert-butyl 4-(3-bromopropyl)piperazine-1-carboxylate,
K2CO3, DMF, 65 °C; f) (i) TFA, rt; (ii) PhNCO, THF, rt
To have the piperazine ring linked to the benzo[b][1,4]oxazin-
3(4H)-one scaffold by an alkyl chain (rather than an amide bond),
tert-butyl 4-(3-bromopropyl)piperazine-1-carboxylate was reacted
3
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10.1002/cmdc.201800739
ChemMedChem
FULL PAPER
with 18 in the presence of potassium carbonate yielding
intermediate 38. Acidic removal of the Boc protecting group and
condensation with phenyl isocyanate afforded target compound
39.
carboxylate 60 (synthesized according to literature procedure[22]
was selected as starting material. Alkylation, followed by
saponification of the ester residue and formation of an amide bond
yielded the desired compound 61. Unfortunately, upon cleavage
of the Boc protecting group under acidic conditions, a concomitant
oxidation of the 3,4-dihydroquinoxalin-2(1H)-one moiety took
place yielding the quinoxalin-2(1H)-one analogue 62. The
benzoxazole congener 64 was easily obtained by reaction of 63
)
O
N
O
N
O
N
b
a
O
O
O
with N-phenylpiperazine-1-carboxamide 16.
A benzoxazine
N
N
O
HO
O
O
analogue lacking the 2-oxo functionality (compound 66) was
accessible via alkylation of 65, followed by ester hydrolysis and
amide coupling.
20
HN
N
O
40
NH
41
(Table 6)
X
Y
O
b
O
N
O
d
O
N
c
X
Y
O
N
e
O
a
O
O
Boc-XH
Boc
H
X
N
X
N
N
H
O
O
H
H
42a-f
43a-f
44a-f
46
47
: X = CH; Y = N
: X = N; Y = CH
HO
O
R
O
R
O
48
49
(Table 2)
: X = CH; Y = N
: X = N; Y = CH
63
64
(Table 2)
O
N
O
O
O
O
a
R4
O
R4
O
N
a
N
H
N
R5
R5
O
H
N
X
N
O
H
65
O
R
O
50
: R4 = CH3; R5 = H
R
O
66
(Table 1)
45a-f
(Table 6)
51: R4 = F; R5 = H
52
: R4 = R5 = F
53
: R4 = CH3; R5 = H
54: R4 = F; R5 = H
55
: R4 = R5 = F
Scheme 4. Reagents and conditions : a) DCC, HOBt, homopiperazine, DMF; b)
phenylisocyanate, DMF; c) PhNCO, THF; d) TFA, CH2Cl2; e) HCTU, DIPEA,
DMSO.
(Table 1)
X
X
N
a
HN
O
N
H
O
O
R =
N
N
56
: X = S
Modifications in the piperazine motif were introduced as shown in
Scheme 4. For the synthesis of the homopiperazine analogue 41,
a stepwise approach starting from compound 20 was followed,
consisting of amide coupling and urea formation, yielding the 1,4-
diazepine analogue 41. On the other hand, for the methyl-
substituted piperazine analogues 45a-b, the amino-piperidine
derivatives 45c-d and the amino-pyrrolidine congeners 45e-f, a
slightly different methodology was used. The free amino group of
the Boc protected building blocks 42a-f was coupled with
phenylisocyanate, followed by acidic cleavage of the Boc group.
Finally, amide coupling of 44a-f with 20 yielded a series of
analogues 45a-f with structural variation of the piperazine moiety.
Scaffold modified analogues were prepared from appropriate,
commercially available building blocks (Scheme 5). The synthesis
of pyrido-fused oxazine analogues started from the commercially
available 2H-pyrido[3,2-b][1,4]oxazin-3(4H)-one 46 and 1H-
pyrido[2,3-b][1,4]oxazin-2(3H)-one 47. N-alkylation, followed by
ester hydrolysis and amide coupling yielded the desired
analogues 48 and 49. A number of derivatives with substituents
at position 2 of the oxazine ring were also prepared. 2-methyl-2H-
benzo[b][1,4]oxazin-3(4H)-one 50 is commercially available,
whereas building blocks with a mono- (compound 51) or
difluorinated (compound 52) oxazine moiety were synthesized
according to literature procedures.[21] Applying the standard
reaction conditions (N-alkylation, ester hydrolysis, amide
formation) yielded compounds 53, 54 and 55. Final derivatives 58
57: X = CH2
R
O
58: X = S
59
: X = CH2
(Table 2)
Boc
N
Boc
N
N
N
a
c
O
N
H
O
N
O
O
60
R
O
R
61
62
(Table 2)
Scheme 5. Reagents and conditions : a) (i) ethyl 3-bromopropionate, K2CO3,
DMF; (ii) LiOH, THF; (iii) DCC, HOBt, N-phenylpiperazine-1-carboxamide, DMF;
b) DCC, HOBt, N-phenylpiperazine-1-carboxamide, DMF; c) TFA, CH2Cl2.
Mycobacterial ThyX inhibition and structure-activity
relationship studies
To investigate the SAR in a systematic way, hit compound 10 was
divided in different areas, and each of these substructures was
subjected to structural modifications (Figure 3). In a primary
screening, all compounds were tested at a concentration of 5 µM
as potential inhibitors of mycobacterial ThyX in
a
spectrophotometric based assay. Folinic acid (also known as
leucovorin) (50 µM) was included as a reference to scale the
inhibitory activity of tested compounds. At a concentration of 50
µM, folinic acid inhibited ThyX enzymatic activity by 80 %. Hence,
compounds with relative inhibition of more than 1 in this study,
inhibited the ThyX activity more than 80% at 5 M. For the hit
compound and its derivatives that had a relative inhibition of at
least 1, the concentration to obtain 50% of the maximal inhibition
and 59 were prepared in
a similar way starting from
benzo[b][1,4]thiazin-3(4H)-one 56 or 3,4-dihydroquinolin-2(1H)-
one 57. To have access to a 3,4-dihydroquinoxaline based
analogue,
tert-butyl
3-oxo-3,4-dihydroquinoxaline-1(2H)-
4
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10.1002/cmdc.201800739
ChemMedChem
FULL PAPER
(IC50) was determined using full dose-response curves. All
selected compounds were able to fully inhibit the enzymatic
activity of ThyX in the selected conditions.
O
benzo[b][1,4]oxazine
rel.
inhibition
@ 5 µM
IC50
(µM)
Cmpd#
10 (Hit)
R1
R2
R3
R4
R5
N
N
O
O
linker
H
H
H
H
H
0.70
0.93
(0.17)b
H
N
N
17a
17b
17c
17d
17e
17f
17g
17h
53
Cl
CH3
NO2
COCH3
Ph
Cl
H
H
H
H
H
H
F
H
H
H
H
H
Cl
H
H
H
H
H
-
H
H
H
H
H
H
H
H
H
H
H
H
F
0.72
0.28
0
NDa
NDa
NDa
NDa
NDa
NDa
NDa
NDa
NDa
NDa
NDa
NDa
O
piperazine
H
N-phenyl-carboxamide
H
0.10
0.04
0.19
0.81
0.47
0
Figure 3. Division of compound 10 in different substructures
H
H
SAR of the benzo[b][1,4]oxazin-3(4H)-one moiety : substitution
pattern
H
H
The influence of substitution of the phenyl moiety of the
benzo[b][1,4]oxazin-3(4H)-one core on ThyX inhibition was
evaluated. The introduction of small electron-withdrawing groups,
such as nitro and acetyl groups (compounds 17c and 17d,
respectively) and electron-donating groups, such as a methyl
moiety (compound 17c) yielded analogues that were completely
devoid of mycobacterial ThyX inhibition. On the other hand,
substituting the phenyl ring with a chlorine at position 6 afforded
compound 17a, which was equipotent with hit compound 10
(relative inhibition of 0.7 at 5 µM). The chlorinated regioisomer
(compound 17h) was endowed with less inhibitory activity against
ThyX (relative inhibition of 0.47 at 5 µM), whereas a fluorine at
position 7 afforded compound 17g which was still endowed with
reasonable ThyX inhibition (IC50 = 1.75 µM). A dichlorinated
analogue (compound 17f) was completely devoid of ThyX
inhibition. A sterically demanding phenyl group (compound 17e)
led to a complete loss of ThyX inhibition. The substitution pattern
of the oxazine moiety was also explored. Introduction of a methyl
or fluorine(s) (compounds 53-55) completely abolished ThyX
inhibition. Reduction of the lactam functionality of compound 10
to an amine afforded compound 66, which was much less active
as a ThyX inhibitor (relative inhibition of 0.35 at 5 µM). Overall,
this SAR study reveals that very little variation is tolerated on the
benzo[b][1,4]oxazin-3(4H)-one moiety with respect to ThyX
inhibition.
H
Cl
H
H
H
-
H
H
CH3
F
54
H
0.15
0.15
0.35
55
H
F
66
-
-
-
aND : Not determined. bStandard deviation given between parentheses
SAR of the benzo[b][1,4]oxazin-3(4H)-one moiety : scaffold
modification
As pyridine is a known bioisoster of benzene, two pyrido-oxazine
analogues were prepared. Whereas the pyrido[3,2-b][1,4]oxazin-
3(4H)-one congener 48 was endowed with promising ThyX
inhibition (IC50 = 1.72 µM), its isomeric pyrido[2,3-b][1,4]oxazin-
2(3H)-one analogue 49 completely lacks ThyX inhibition, pointing
towards a crucial role of the position of the nitrogen atom. Other
scaffold variations focused on the oxazine moiety and included a
benzo[b][1,4]thiazine (compound 58) and tetrahydroquinoline
scaffold (compound 59), which were both found to be inactive.
The quinoxaline analogue 62 exhibited potent ThyX inhibition,
displaying an IC50 value of 1.46 µM. Converting the 6-membered
oxazine moiety into
a
five-membered ring yielded the
benzo[d]oxazol-2(3H)-one derivative 64, which was completely
devoid of ThyX inhibition.
5
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10.1002/cmdc.201800739
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Table 2. SAR of scaffold modified benzoxazine analogues.
Table 3. SAR of the N-phenylcarboxamide moiety.
rel. inhibition
Cmpd#
A
B
X
IC50 (µM)
@ 5 µM
0.70
0
rel. inhibition
Cmpd#
10 (hit)
R
IC50 (µM)
0.93
(0.17)b
@ 5 µM
10 (Hit)
49
CH
N
CH
CH
N
O
O
O
0.93
0.70
(0.17)b
NDa
1.72
(0.06)b
48
CH
1.24
22a
22b
22c
0.09
0.23
0.58
NDa
NDa
NDa
NDa
58
59
CH
CH
CH
CH
S
0.16
0
NDa
NDa
CH2
1.46
(0.05)b
62
64
-
-
-
-
-
-
1.13
0.06
NDa
aND : Not determined. bStandard deviation given between parentheses.
22d
22e
0.62
0.76
SAR of the N-phenylcarboxamide moiety
NDa
NDa
To probe into the optimal substitution pattern of the terminal
phenyl ring, a variety of small substituents (e.g. halogens, methyl,
methoxy, cyano and acetyl) were introduced. As can be derived
from the data in Table 3, quite some structural variety is tolerated
at this position, as these different analogues (22a-i) display a
relative inhibition between 0.44 and 0.95 at 5 µM, compared to 50
M folinic acid in identical conditions. For a selected number of
derivatives, IC50 values were obtained. Thienyl and pyridyl are
both known isosters of the phenyl and therefore compounds 22j,
27 and 28 were also prepared. Only compound 27 was more
active than compound 10 at 5 M (relative inhibition of 1.19, IC50
= 2.38 M).
Instead of the terminal phenyl, a benzyl and phenethyl congener
were also prepared. Although an elongation with one carbon
(compound 22k) still gives a potent ThyX inhibitor (relative
inhibition of 0.83), further elongation by two carbons (compound
22l) gave rise to a diminished ThyX inhibition. To assess the
importance of the urea moiety, the corresponding carbamate
(compound 22m) and amide (compound 22n) were both prepared.
The carbamate was still endowed with reasonable ThyX inhibition
(relative inhibition of 0.5), whereas the amide is much less active
as a ThyX inhibitor.
22f
22g
22h
0.61
1.06
0.44
2.04
(0.08)b
NDa
NDa
22i
0.77
22j
22k
22l
0.52
0.83
0.54
NDa
NDa
NDa
22m
22n
0.52
0.31
NDa
NDa
Rather than directly attaching a carbonyl to the piperazine moiety
(generating a urea, carbamate or amide), a number of compounds
was synthesized in which a methylene linker between the
carbonyl group and the piperazine nitrogen was inserted (Table
4). This makes the amine group to have basic properties, which
should have an impact on its biological activity, solubility and
permeability. Several congeners within this family were endowed
with ThyX inhibitory activity (e.g. compounds 29a and 29c, both
having a heteroaromatic substituent), and especially the N-
methyl-N-phenylpropionamide derivative 29d displayed potent
ThyX inhibition with an IC50 value of 0.69 µM
2.38
27
28
1.19
1.13
(0.08)b
3.56
(0.09)b
aND : Not determined. bStandard deviation given between parentheses.
6
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Table 4. SAR of the piperazinyl-acetamide derivatives.
36
37
39
0
0
0
NDa
NDa
NDa
rel.
inhibition
@ 5 µM
Cmpd#
29a
R
IC50 (µM)
aND : Not determined. bStandard deviation given between parentheses
0.89
0.53
0.99
NDa
SAR of the piperazine moiety
As shown in Table 6, substituting the piperazine ring of hit
compound 10 for a 1,4-diazepine ring (homopiperazine analogue
41) resulted in a completely inactive derivative. Upon introducing
a methyl group on the piperazine ring (compounds 45a and 45b),
the exact position on the piperazine ring plays a determining role
in its ThyX inhibitory activity. The presence of a methyl closer to
the benzoxazine core afforded compound 45b that was endowed
with potent ThyX inhibition (relative inhibition of 0.88). On the
other hand, when the same methyl group was introduced closer
to the urea functionality (compound 45a), a decreased potency
was observed when compared to the unsubstituted congener 10.
To broaden the SAR investigation, the piperazine moiety was
replaced by other nitrogen-containing saturated heterocycles.
The amino-piperidine congener 45c displayed better activity
against mycobacterial ThyX (IC50 = 0.88 µM), when compared to
the parent piperazine analogue 10. The closely related amino-
piperidine congener 45d lacks activity. A similar profile was found
in the aminopyrrolidine subseries. When the exocylic amino group
of aminopyrrolidine was derivatised as a urea (compound 45e),
the compound was endowed with substantial ThyX inhibition (IC50
= 2.95 µM). On the other hand, when the exocyclic amino group
was connected to the propanyl linker (compound 45f), it was
completely devoid of inhibitory activity.
29b
29c
NDa
NDa
29d
1.37
0.69 (0.08)b
aND : Not determined. bStandard deviation given between parentheses.
SAR of the propanoyl linker
Subsequently, the importance of the linker between the oxazine
and the piperazine moiety was evaluated (Table 5). Shortening
(compound 32), elongation (compound 33) or branching
(compounds 36 and 37) of the propanoyl linker led inevitably to
completely inactive compounds. Reduction of the amide group
yielded a derivative with a n-propyl linker (compound 39) that
similarly showed a complete loss of ThyX inhibition.
Table 5. SAR of linker modified analogues
Table 6. SAR of the piperazine moiety.
Cmpd#
10 (Hit)
32
X
rel. inhibition
@ 5 µM
IC50 (µM)
0.93 (0.17)b
NDa
rel inhibition
@ 5 µM
Cmpd#
10 (Hit)
X
IC50 (µM)
0.70
0
0.70
0.93 (0.17)b
41
0
NDa
NDa
33
0
NDa
45a
0.47
7
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10.1002/cmdc.201800739
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was docked in the binding site of this cofactor. The crystal
structure of T. maritima ThyX in complex with FAD, dUMP and
folinic acid (PDB ID: 4GTA)[23] was used as starting structure to
perform an initial molecular docking experiment. The aromatic
and saturated rings of the benzo[b][1,4]oxazin moiety of 10 were
posed on the corresponding rings of the pterin moiety of folinic
acid, while the piperazine group of compound 10 fitted the
aromatic ring in the p-amino benzoic acid moiety of folinic acid.
Overlaying the obtained complex with the crystal structure of M.
tuberculosis ThyX in complex with FAD, 5-Br-dUMP and glycerol
(PDB ID: 2AF6)[24] revealed that compound 10 is accommodated
in a binding site of M. tuberculosis ThyX that contained two
glycerol molecules in the crystal structure, occupying the binding
pockets of the N-phenyl-carboxamide and benzo[b][1,4]oxazin
moieties of compound 10. Starting from this overlay, two
complexes of M. tuberculosis ThyX were generated after removal
of both glycerols mentioned above. More specifically, both
complexes contained the original FAD and 5-Br-dUMP together
with compound 10 or folinic acid for comparison purposes. After
an initial minimization, both complexes were subjected to 20 ns
molecular dynamics calculations using the AMBER 16.0 software
package[25] as described in the experimental part.
During molecular dynamics, the benzo[b][1,4]oxazin part of
compound 10 and the pterine scaffold of folinic acid remained
nicely stacked to the flavine moiety of FAD cofactor in the active
site. Remarkably, two interconverting binding modes were
observed for the glutamate moiety in folinic acid resulting in an
excessive conformational disorder, thereby explaining the
absence of this moiety and some residues in this region of the
crystal structure of the T. maritima ThyX complex. In the most
abundant binding mode as seen in Molecular Dynamics
45b
0.88
NDa
45c
45d
1.23
0.04
0.88 (0.08)b
NDa
45e
45f
0.58
0
NDa
NDa
aND : Not determined. bStandard deviation given between parentheses.
Mechanism of inhibition of mycobacterial ThyX
The inhibition profile of 5 compounds (22a, 22c, 22e, 22i, 22k)
was probed as described in the methods section. The results were
similar for all tested compounds and the data obtained for
compound 22e as a representative example are shown in Figure
4. The IC50 versus dUMP plot (Figure 4, insert A) suggested that
a non-competitive inhibition pattern can best describe the
inhibition by selected molecules against dUMP. The same set of
molecules showed a competitive inhibition pattern against
CH2H4Folate with a linear variation of IC50 in function of the
CH2H4Folate concentration. An analogous plot for NADPH
revealed a characteristic behavior for uncompetitive inhibition for
the same set of molecules regarding NADPH binding. These
results suggest that molecules could bind ThyX as a complex of
type ThyX-NADPH or ThyX-NADP+.
The observation that the IC50 values determined from the dose–
response curves were of the same order of magnitude as the total
enzyme concentration used suggested a tight-binding inhibition
mechanism for the tested molecules, as previously described for
2-hydroxy-1,4-naphthoquinone analogues as tight binding
inhibitors for PBCV-1 ThyX.[13]
simulations, the
carboxyl group of the glutamate moiety is
interacting with a conserved Arg residue thereby replacing a
glycerol in the initial crystal structure (M. tuberculosis: Arg41 in
2AF6 T. maritima: Arg28 in 4GTA, H. pylori: Arg42 in 3AH5,
Figure 6). In the same binding mode, the N-phenyl-carboxamide
of 10 shows a stacking interaction with the side chain of His63 in
M. tuberculosis ThyX (Figure 5).
Strikingly, the carboxamide linkers in compound 10 as well as in
folinic acid are within hydrogen bonding distance with the terminal
hydroxyl of Ser46 in modelled complexes. While Ser46 is
conserved in H. pylori, it is mutated to Glu46 in ThyX homologues
from B. hermsi and PBCV-1 for which no inhibitory activity could
be observed. To examine the effect of this mutation on the binding
affinity of 10, Ser46 was mutated in silico to Glu46. The results
indicated that this mutation does not allow formation of a
hydrogen bond with the carboxamide linker in compound 10 and
impaired the stability of the loop that forms the studied binding site.
Additionally, a distortion of binding mode is observed at the
piperazine linker. All of these effects contributed to a reduced
binding affinity of the compounds, explaining the loss of inhibitory
effect on ThyX from PBCV-1 and B. hermsi as described above.
The qualitative evaluation of the ability of molecules 22a, 22c, 22e,
22i, 22k, 27, 28, 29d, 48 and 62 to inhibit ThyX from species
besides M. tuberculosis (e.g. H. pylori, PBCV-1, B. hermsi, C.
trachomatis) was also undertaken. None of these analogues
inhibited ThyX from PBCV-1 and B. hermsi, whereas a weak
inhibition at 50
was measured for ThyX from H. pylori and C.
trachomatis. Analysis of the different ThyX protein sequences
revealed, in the environment of the substrate binding pocket,
some interesting differences for presumed amino acids implicated
in compound binding.
Molecular Docking
Since competition assays indicated that studied compounds
share their binding site with tetrahydrofolate, hit compound 10
8
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Figure 4. Secondary and final plots of results obtained with compound 22e for mycobacterial ThyX inhibition using different concentrations of dUMP (A), NADPH
(B) and methylene tetrahydrofolate (C).
The model of compound 10 bound to M. tuberculosis ThyX
enabled to rationalize the SAR results (Figure 7). The carbonyl in
the benzoxazine moiety forming the δ-lactam ring accepted a
hydrogen bond from Ser102, the only residue of subunit D that
directly interacted with compound 10 in our model. Intermittently,
the same Ser102 donated a hydrogen bond to the oxygen in the
1,4 oxazine moiety, albeit this bifurcated hydrogen bond was
weaker because of the more spread-out orientation of both
acceptors. The insertion of a nitrogen atom in the phenyl part of
the benzo[b][1,4]oxazin moiety, yielded pyrido[3,2-b][1,4]oxazine
48, from which the nitrogen atom acted as an isostere for O4 of
the pterin ring of the original cofactor and aligns the attached
oxazine and aromatic ring stronger with the heterocycle of FAD in
a parallel-displaced orientation as observed for compound 10.
Additionally, the more polar character of the pyridine ring of
compound 48 allowed a hydrogen bond with His69. The absence
of the aforementioned heterocycles in compound 59, together
with its complete lack of ThyX inhibition, supported this interaction
analysis.
The introduction of a methoxy moiety at the para position in 22b
during the SAR of the N-phenylcarboxamide moiety was expected
to destabilize the flexible closing loop which contributes to the
lower activity. For comparison, the meta-acetyl variant
(compound 22g) provides a stronger acceptor (no mesomeric
Figure 5: Compound 10 (yellow) in the catalytic site M. tuberculosis ThyX
crystal structure (PDB ID: 2AF6) including FAD (orange) and 5-Br-dUMP.
Modelled folinic acid (green) and glycerol (gold) in the binding site of the crystal
effect with carboxamide) which might accept an additional
hydrogen bond from Thr54 resulting in a tighter loop closing with
relative inhibition of 1.06 compared to 0.70 in compound 10. The
nitrogen from the pyridine moieties in compounds 27 and 28
occupied a smaller volume compared to the acetyl group in 22b
with higher atomic radius and could act
structure are overlayed. Side chains of Arg41, Ser46 and His63 in subunit D are
depicted. Green and yellow dashed lines indicate putative hydrogen bonds of
Ser46 and the carboxamide linkers in folinic acid and compound 10, respectively.
9
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10.1002/cmdc.201800739
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Figure 6. Sequence alignment of ThyX homologues by CDD/SPARCLE.[26] Residues that are labeled in Figure 5 are boxed.
as hydrogen bond acceptor for the conserved Arg41. Such
increased loop stabilization correlated with the higher relative
inhibition values of 1.19 and 1.13 observed for compounds 27 and
28 respectively. The benzyl derivative 22k covered a larger
rotational space than the phenyl analogue 10, providing increased
potential stacking interactions with His63 and Tyr54. Moreover,
the increased flexibility of compound 22k resulted in a more stable
stacking interaction between the benzoxazine moiety and the
heterocycle of FAD, maintaining the His63 stacking interaction
and Ser46 hydrogen bond.
With an additional methylene bridge adjacent to the piperazine
linker in compound 29d, an additional source of rotational
freedom was introduced, similarly as for compound 22k. A model
of M. tuberculosis ThyX in complex with the most active ThyX
inhibitor (compound 29d) was obtained as described in the
method section. The additional N-methyl group contributed to
hydrophobic Van der Waals interactions with Tyr44, thereby
positioning the phenyl ring towards optimal perpendicular
stacking interaction with His63 and optimizing the geometry of
hydrogen bond formation with Ser46. Because of the increased
flexibility resulting from the methylene group, a bifurcated
hydrogen bond formation of the carbonyl group in the N-phenyl
carboxamide was viable with Ser46 and Tyr60 which concurrently
positioned the phenyl moiety through stacking. Compared to the
binding conformation of compound 10, this overall network of
stabilizing hydrophobic and increased electrostatic interactions in
29d allowed a volumetric occupation of the binding space of the
glutamate moiety in folinic acid flanked by Arg41 and His63.
These results were further quantified by interaction energy
calculations in which a difference of 8.69 kcal/mol was observed
with a more favorable affinity for 29d (folinic acid: -37.90 kcal/mol,
compound 10: -38.77 kcal/mol, compound 29d: -46.59 kcal/mol).
Furthermore, an overall slight rotational shift resulted in more
planar stacking interaction between the benzoxazine moiety and
the FAD cofactor. Finally, an additional hydrogen bond was
possible between one hydroxyl from the glycerol linker in FAD and
Figure 7. LigPlot+ representation of interacting residues with native folinic acid
(A), hit-compound 10 (B) and optimized compound 29d (C)
10
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Shimadzu HPLC equipped with a LC-20AT pump, DGU-20A5 degasser,
and a SPD20A UV−VIS detector; Symmetry C18 column (5 μm, 4.6 mm ×
150 mm); gradient elution of H2O/CH3CN from 95/5 or 70/30 to 5/95 over
25 min; flow rate 1 mL/min; wavelength, UV 254 nm. Compounds that were
submitted for biological testing displayed a purity of 95% or more.
the oxygen from the benzoxazine ring. The same reasoning was
applied to compounds 29a and 29c, explaining the potent
inhibitory activity of both compounds (relative inhibition at 5 µM of
0.89 and 0.99, respectively).
In compound 45c, the exocyclic aminogroup at the piperidine
moiety had a similar effect as the extra methylene in compounds
29a, 29c and 29d. Alterations in the piperazine moiety, albeit with
limited conformational space, displayed a positioning role for the
binding mode of this central part in the entire compound.
Compounds 45d and 45f shifted the heterocycle by one atom
resulting in loss of activity and thereby endorsing the positioning
function. The methyl side chain in compound 45b resided in a
more hydrophobic pocket displaying hydrophobic interactions
with Val72 and Ile64 resulting in a slightly higher relative inhibition
(0.88).
In the SAR of the propanoyl linker (compounds 32, 33, 36, 37 and
39), the loss of inhibitory activity by shortening the linkers
provided evidence for the necessary co-occurrence of both His63
stacking and Ser46 and Tyr60 bifurcated hydrogen bond
interactions with benzoxazine alignment to the FAD heterocycle.
N-Phenylpiperazine-1-carboxamide (16)
To a solution of N-Boc piperazine 14 (0.50 mmol) in THF (10 ml) was
added slowly a solution of phenyl isocyanate (0.57 mmol) in THF (10 ml)
at room temperature. After the reaction reached completion, trifluoroacetic
acid (10 ml) was added dropwise while stirring at 0°C. When TLC showed
complete deprotection, water (100 ml) was added. The mixture was
extracted with dichloromethane (30 ml). The pH of the aqueous phase was
adjusted to 9 with 1N NaOH and then extracted with dichloromethane (100
ml). The organic phase was dried over Na2SO4. The solvents were
evaporated in vacuo yielding a crude residue, which was used as such for
further reaction without any additional purification. 1H-NMR (300 MHz,
DMSO-d6): δ = 2.7 (brs, 2H, CH2), 3.34 (t, J = 4.35 Hz, 4H, 2CH2), 4.22
(brs, 2H, CH2), 6.93 (t, J = 7.2 Hz, H, CH), 7.33 (t, J = 7.55 Hz, 2H, 2CH),
7.45 (d, J = 7.8 Hz, 2H, 2CH), 8.53 (s, H, CH) ppm. 13C-NMR (75 MHz,
DMSO-d6): δ = 31.68, 36.66, 67.17, 115.35, 116.77, 122.93, 123.68,
128.31, 145.07, 164.13, 172.35 ppm.
4-(3-(6-Chloro-3-oxo-2H-benzo[b][1,4]oxazin-4(3H)-yl)propanoyl)-N-
phenylpiperazine-1-carboxamide (17a)
Conclusions
Starting from the previously identified hit compound 10, a hit-to-
lead optimization campaign was performed in order to discover
novel mycobacterial ThyX inhibitors. The most potent analogue of
this series was endowed with an IC50 of 0.69 µM against
mycobacterial ThyX. Molecular modeling was applied in order to
understand the SAR for M. tuberculosis ThyX and the variation in
activity towards ThyX from other microorganisms. These results
open new avenues for structure-based drug design to discover
A mixture of 6-chloro-2H-benzo[b][1,4]oxazin-3(4H)-one (91 mg 0.5 mmol),
K2CO3 (158 mg, 1 mmol) and ethyl 3-bromopropionate (91 mg, 0.51 mmol)
in DMF (15 ml), was stirred at 80 °C for 8 hours. Then, the mixture was
diluted with water (45 ml) and extracted with dichloromethane (100 ml).
The organic layer was separated, washed with water (20 ml) and dried
over anhydrous Na2SO4. The solvents were removed under reduced
pressure. The crude residue was dissolved in THF (10 ml) and a solution
of LiOH (210 mg 5 mmol) in water (10 ml) was added. The reaction mixture
was stirred at 60 °C for 12 hours. After the reaction reached completion,
the pH was adjusted to 3 by the addition of a 2N hydrochloric acid solution.
The mixture was extracted with ethyl acetate (5 x 20 ml) and dried over
anhydrous Na2SO4. Evaporation of the solvent yielded the carboxylic acid
13a. The compound was redissolved in DMSO (10 ml). N-
Phenylpiperazine-1-carboxamide 16 (102 mg 0.50 mmol), HCTU (206 mg,
0.50 mmol) and DIPEA (50 µl) were added. The reaction mixture was
stirred overnight at room temperature. The reaction mixture was diluted
with dichloromethane (100 ml) and washed with water (40 mL), dried over
Na2SO4, concentrated and purified by flash column chromatography to
afford the title compound (318 mg, 72 %). Purity (Method A): 95.86 %. 1H-
NMR (300 MHz, CDCl3): δ = 2.75 (t, J = 7.6 Hz, 2H, CH2), 3.50-3.57 (m,
6H, 6CH), 3.75 (t, J = 7.5 Hz, 2H, CH2), 4.25 (t, J = 7.8 Hz, 2H, CH2), 4.60
(s, 2H, CH2), 6.75 (s, H, NH), 6.94-7.10 (m, 4H, CH), 7.31-7.37 (m, 4H,
4CH) ppm. 13C-NMR (75 MHz, CDCl3): δ = 30.69, 38.26, 41.21, 44.15,
45.27, 68.18, 108.06, 115.08, 118.42, 120.45, 123.77, 124.00, 129.13,
138.90, 140.06, 144.24, 155.60, 164.64, 169.28 ppm. HRMS (ESI): m/z
[M+H]+ calculated for C22H24N4O4Cl 443.14859, found 443.1476.
more potent ThyX inhibitors with antibacterial activity.
A
phenotypic screening for antimicrobial activity of the most potent
molecules on specific bacteria will be the first step to address the
path forward of these compounds.
Experimental Section
General
All reagents and solvents were purchased from commercial sources and
used as obtained. Moisture sensitive reactions were carried out using
1
oven-dried glassware under a nitrogen or argon atmosphere. H and 13C
NMR spectra were recorded on a Bruker Avance 300 or 500 MHz
spectrometer with tetramethylsilane as internal standard or referenced to
the residual solvent signal. The following abbreviations were used to
explain multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, m =
multiplet, br = broad, dd = doublet of doublets. Coupling constants are
expressed in Hz. High-resolution mass spectra (HRMS) were obtained on
a quadruple orthogonal acceleration time-of-flight mass spectrometer
(Synapt G2 HDMS, Waters, Milford, MA). Samples were infused at 3
μL/min, and spectra were obtained in positive (or negative) ionization
mode with a resolution of 15 000 (fwhm) using leucine enkephalin as the
lock mass. Pre-coated aluminum sheets (254 nm) were used for TLC and
spots were visualized with UV light. The products were purified by flash
column chromatography on silica gel (60 Å, 0.035−0.070 mm, Acros
Organics). The purity of final compounds was determined by analytical RP-
HPLC, using one of the following methods. Method A: a Waters 600 HPLC
system and a Waters 2996 photodiode array detector, XBridge column (C-
18, 5 μm, 4.6 mm × 150 mm). Elution with a gradient mixture of H2O
containing 0.2% (vol) of TFA (A) and acetonitrile (B). Method B: a
Compounds 17b-h were synthesized according to the procedure for the
preparation of compound 17a. Exact experimental and spectral data can
be found in the Supporting Information.
Ethyl 3-(3-oxo-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)propanoate
(19)
A solution of 18 (2.98 g, 20 mmol) and K2CO3 (40 mmol, 5.52 g) in DMF
(45 ml) was stirred at room temperature for 15 minutes. Then, ethyl 3-
bromopropionate (22 mmol, 3.98 g) was added and the mixture was stirred
overnight at 90°C. The precipitate was filtered off and the filtrate was
evaporated in vacuo. The crude residue was purified by silica gel flash
chromatography (the mobile phase being a mixture of heptane and
ethylacetate in a ratio of 7:3) affording the title compound as a colorless oil
11
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10.1002/cmdc.201800739
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FULL PAPER
(4.7 g, 95 %). 1H-NMR (300 MHz, CDCl3): δ = 7.1 – 6.95 (m, 4H), 4.58 (s,
2H, O-CH2-CO), 4.23 (t, 2H, J = 7.5 Hz, NCH2), 4.13 (q, 2H, J = 7.1 Hz,
CO-CH2), 2.68 (t, 2H, J = 7.7 Hz, N-CH2-CH2), 1.23 ppm (t, 3H, J = 7.1 Hz,
CH3). 13C-NMR (75 MHz, CDCl3): δ = 171.1, 164.5, 145.5, 128.3, 124.2,
123.0, 117.4, 114.7, 67.7, 61.0, 37.2, 32.1, 14.2 ppm. HRMS (ESI): m/z
[M+H]+ calculated for C13H16NO4 250.10737, found 250.1071.
To a solution of 4-aminopyridine 23 (24.67 mg, 0.262 mmol), DIPEA (42.3
mg, 0.328 mmol) in DMF (1 ml) was added phenyl chloroformate (44.5 mg,
0.284 mmol) at 0 °C. The mixture was stirred for 15 minutes and
completion of the reaction was monitored by TLC (CH2Cl2:CH3OH:NH3(aq)
,
92:8:0.3). DIPEA (42.3 mg, 0.328 mmol) and a solution of compound 21
(63.2 mg, 0.218 mmol) in DMF (1 ml) were added. The resulting reaction
mixture was stirred overnight at room temperature. The solvents were
evaporated in vacuo. The residue was dissolved in ethyl acetate and
washed with water. The organic phase was dried over Na2SO4, and
concentrated in vacuo. The crude residue was purified by silica gel column
chromatography (using a mixture of dichloromethane and methanol as
mobile phase, in a gradient gradually increasing from 5 to 10% methanol)
affording the title compound as an off-white solid (28 mg, 32 %). Purity
(Method A): 100 %. 1H-NMR (300 MHz, CDCl3): δ 8.38-8.32 (m, 2H), 7.93
(br s, 1H), 7.42-7.35 (m, 2H), 7.10-6.95 (m, 4H), 4.57 (s, 2H, O-CH2-CO),
4.24 (t, 2H, J = 7.6 Hz, N-CH2-CH2-CO), 3.70-3.42 (m, 8H), 2.71 (t, 2H, J
= 7.8 Hz, N-CH2-CH2-CO) ppm. 13C-NMR (75 MHz, CDCl3): δ 169.1, 164.8,
154.2, 150.1 (2C), 147.1, 145.4, 128.2, 124.4, 123.2, 117.5, 114.8, 113.7
(2C), 67.7, 45.3, 44.2, 43.8, 41.2, 38.0, 30.9 ppm. HRMS (ESI): m/z [M+H]+
calculated for C21H24N5O4 410.18226, found 410.1817.
3-(3-Oxo-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)propanoic acid (20)
To a solution of 19 (4.88 g, 19.58 mmol) in THF (70 ml) was added a
solution of LiOH (4.93 g, 117.46 mmol) in water (50 ml). The reaction
mixture was stirred overnight at 45°C. The reaction was quenched by the
addition of a 5M HCl solution (24 ml). The organic solvents of the reaction
mixture were evaporated and a precipitate was formed. The precipitate
was filtered off and dried yielding the title compound as a white-brown
powder (3.96 g, 92 %). 1H-NMR (300 MHz, DMSO-d6): δ = 12.39 (s, 1H,
OH), 7.24-7.15 (m, 1H), 7.10 – 6.95 (m, 1H), 4.61 (s, 2H, O-CH2-CO), 4.11
(t, 2H, J = 7.6 Hz, NCH2), 2.53 ppm (t, 2H, J = 7.5 Hz, N-CH2-CH2). 13C-
NMR (75 MHz, DMSO-d6): δ = 172.2, 164.0, 144.9, 128.2, 123.5, 122.8,
116.6, 115.2, 67.0, 36.5, 31.5 ppm. HRMS (ESI): m/z [M-H]- calculated for
C11H10NO4 220.06152, found 220.0637.
Compound 28 was synthesized according to the procedure for the
preparation of compound 27. Exact experimental and spectral data can be
found in the Supporting Information.
4-(3-Oxo-3-(piperazin-1-yl)propyl)-2H-benzo[b][1,4]oxazin-3(4H)-on
(21)
2-(4-(3-(3-Oxo-2H-benzo[b][1,4]oxazin-4(3H)-yl)propanoyl)piperazin-
1-yl)-N-(pyridin-3-yl)acetamide (29a)
A solution of 20 (1.00 g, 4.52 mmol), HOBt (0.692 g, 4.52 mmol) and DCC
(1.12 g, 5.42 mmol) in DMF (16 ml) was stirred for 2 hours at room
temperature. The reaction mixture was cooled down to room temperature
and piperazine (0.779 g, 9.04 mmol) was added. The reaction was stirred
overnight at room temperature. The precipitate was filtered off and the
filtrate was evaporated in vacuo. The crude residue was purified by silica
To a solution of 2-(piperazin-1-yl)-N-(pyridin-3-yl)acetamide (110 mg, 0.5
mmol) in DMSO (10 ml) was added compound 20 (110 mg 0.50 mmol),
HCTU (206 mg 0.5 mmol) and DIPEA (50 µl). The reaction mixture was
stirred overnight at room temperature. Then, the mixture was diluted with
CH2Cl2 (100 ml) and washed with water (40 ml). The combined organic
layers were dried over Na2SO4 and concentrated in vacuo. The crude
residue was purified by silica gel flash column chromatography affording
the title compound (171 mg, 81 %). Purity (Method A): 100 %. 1H-NMR
(300 MHz, CDCl3): δ = 2.60-2.62 (m, 4H, 2CH2), 2.73 (t, J = 7.8 Hz, 2H,
CH2), 3.19 (s, 2H, CH2), 3.56-3.58 (m, 2H, CH2), 3.71-3.72 (m, 2H, CH2),
4.28 (t, J = 7.65 Hz, 2H, CH2), 4.59 (s, H, CH2), 7.01-7.12 (m, 4H, 4CH),
7.27-7.31(m, 1H, 1CH), 8.19-8.23 (m, 1H, 1CH), 8.32-8.37 (m, 1H, 1CH),
8.59 (d, J = 2.4 Hz, H, CH), 9.05 (m, H, NH), ppm. 13C-NMR (75 MHz,
CDCl3): δ = 30.74, 38.05, 41.62, 45.53, 53.20, 53.55, 61.92, 67.66, 114.86,
117.35, 123.16, 124.26, 126.92, 128.24, 134.22, 124.11, 141.01, 145.58,
164.63, 168.32, 168.87 ppm. HRMS (ESI): m/z [M+H]+ calculated for
gel flash chromatography (mobile phase being
a
mixture of
CH2Cl2:CH3OH:NH3(aq) in a ratio of 98:2:0.3), yielding the title compound
1
as a light yellow oil (0.4 g, 31 %). H-NMR (300 MHz, CDCl3): δ = 7.15-
6.95 (m, 4H), 4.57 (s, 2H, O-CH2-CO), 4.24 (t, 2H, J = 7.8 Hz, N-CH2-CH2-
CO), 3.60 – 3.37 (m, 4H), 2.90-2.78 (m, 4H), 2.69 (t, 2H, J = 7.8, N-CH2-
CH2-CO), 2.50 (br s, 1H), 1.23 (s, 1H, NH) ppm. 13C-NMR (75 MHz,
CDCl3): δ = 168.8, 164.6, 145.4, 128.3, 124.2, 123.2, 117.3, 115.0, 67.7,
46.7, 46.2, 45.8, 42.6, 40.9, 38.2, 30.7, 29.8 ppm. HRMS (ESI): m/z [M+H]+
calculated for C15H20N3O3 290.14990, found 290.1505.
N-(3-Chlorophenyl)-4-(3-(3-oxo-2,3-dihydro-4H-benzo[b][1,4]oxazin-
4-yl)propanoyl)piperazine-1-carboxamide (22a)
C
22H26N5O4 424.19846, found 424.1976.
To a solution of compound 21 (89.7 mg, 0.31 mmol) in DMF (2 ml) was
added 3-chlorophenyl isocyanate (71.4 mg, 0.47 mmol) and the resulting
mixture was stirred overnight at room temperature. Reaction completion
was monitored by TLC (CH2Cl2:MeOH:NH3(aq) 90:10:0.3). The solvents
were evaporated in vacuo and the crude residue was purified by silica gel
column chromatography (ethyl acetate 100% as mobile phase) affording
the title compound as a white solid (100 mg, 73 %). Purity (Method A):
99.83 %. 1H-NMR (300 MHz, CDCl3): δ = 7.40 ( s, 1H), 7.30-6.93 (m, 8H),
4.57 (s, 2H, O-CH2-CO), 4.25 (t, 2H, J = 7.6 Hz, N-CH2-CH2-CO), 3.70-
3.60 (m, 2H), 3.58-3.38 (m, 6H), 2.71 (t, 2H, J = 7.9 Hz, N-CH2-CH2-CO)
ppm. 13C NMR (75 MHz, CDCl3): δ = 169.1, 164.7, 154.8, 145.4, 140.3,
134.4, 129.9, 128.2, 124.4, 123.4, 120.3, 118.3, 117.4, 114.8, 67.7, 45.2,
44.0, 43.7, 41.2, 37.9, 30.8 ppm. HRMS (ESI): m/z [M+H]+ calculated for
C22H24ClN4O4 443.14804, found 443.1486.
Compounds 29b-d were synthesized according to the procedure for the
preparation of compound 29a. Exact experimental and spectral data can
be found in the Supporting Information.
Ethyl 2-(3-oxo-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)acetate (30)
1,4-Benzoxazin-3(4H)-one 18 (5.00 g, 33.52 mmol) and K2CO3 (13.90 g,
100.57 mmol) were dissolved in DMF (80 ml) and stirred at room
temperature for 15 minutes. Then, ethyl bromoacetate (11.20 g, 67 mmol)
was added and the reaction mixture was stirred overnight at 90 °C. The
solvents were evaporated in vacuo and the residue was purified by silica
gel column chromatography (using a mixture of cyclohexane and ethyl
acetate in a ratio of 7:3 as mobile phase) affording the title compound as
1
a colorless oil (7.70 g, 98 %). H-NMR (300 MHz, CDCl3): δ = 7.05-6.94
Compounds 22b-n were synthesized according to the procedure for the
preparation of compound 22a. Exact experimental and spectral data can
be found in the Supporting Information.
(m, 3H), 6.78-6.70 (m, 1H), 4.65 (s, 2H), 4.63 (s, 2H), 4.22 (q, 2H, J = 7.14
Hz), 1.26 (t, 3H, J = 7.12 Hz) ppm. 13C-NMR (75 MHz, CDCl3): δ = 167.8,
164.9, 145.1, 128.7, 124.3, 123.0, 117.2, 114.5, 67.5, 61.9, 43.0, 14.2 ppm.
HRMS (ESI): m/z [M+Na]+ calculated for C12H13N1O4Na 258.07370, found
258.0749.
4-(3-(3-Oxo-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)propanoyl)-N-
(pyridin-4-yl)piperazine-1-carboxamide (27)
12
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201800739
ChemMedChem
FULL PAPER
2-(3-Oxo-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)acetic acid (30’)
Purity (Method A): 98.85 %. 1H-NMR (300 MHz, CDCl3): δ = 1.86 (p, J =
7.0 Hz, 2H, CH2), 2.42-2.47 (m, 6H, 3xCH2), 3.47-3.51 (m, 4H, 2xCH2),
4.02 (d, J = 7.0 Hz, 2H, CH2), 4.59 (s, 2H, CH2), 6.52 (s, 1H, NH), 6.98-
7.10 (m, 5H, 4xCHAr), 7.24-7.30 (m, 2H, 2xCHAr), 7.33-7.37 (m, 2H,
2xCHAr) ppm. 13C-NMR (75 MHz, CDCl3): δ = 24.41, 39.46, 41.21, 52.94,
55.50, 67.75, 114.99, 117.32, 120.09, 122.85, 123.21, 123.99, 128.64,
128.97, 139.13, 145.52, 155.15, 164.43 ppm.
To a solution of 30 (7.70 g, 32.73 mmol) in THF (80 ml) was added a
solution of LiOH (8.24 g, 196.4 mmol) in H2O (70 ml). The resulting reaction
mixture was stirred overnight at 45 °C. After completion of the reaction, the
reaction mixture was acidified by addition of an excess of HCl. The organic
solvent (THF) was evaporated in vacuo resulting in the formation of a
precipitate in water. The precipitate was filtered off, washed with cold H2O
and dried furnishing the title compound as an off-white powder (6.24 g,
92 %). 1H-NMR (300 MHz, DMSO): δ = 13.09 (br s, 1H), 7.10-6.95 (m, 4H),
4.69 (s, 2H), 4.63 (s, 2H) ppm. 13C-NMR (75 MHz, DMSO): δ = 169.3,
164.5, 144.6, 128.7, 123.7, 122.7, 116.6, 115.3, 66.9, 42.4 ppm. HRMS
(ESI): m/z [M-H]- calculated for C10H8N1O4 206.04587, found 206.0464.
4-(3-(1,4-Diazepan-1-yl)-3-oxopropyl)-2H-benzo[b][1,4]oxazin-3(4H)-
on (40)
A solution of compound 20 (0.800 g, 3.62 mmol), HOBt (0.554 g, 3.62
mmol) and DCC (0.746 mg, 3.62 mmol) in DMF (12 ml) was stirred at room
temperature for 2 hours. Then, the mixture was cooled down to 0°C and
homopiperazine (0.725 g, 7.23 mmol) was added. The reaction mixture
was stirred overnight at room temperature. The precipitate was filtered off
and washed with dichloromethane. The filtrate was evaporated and the
residue was purified by silica gel flash chromatography (the mobile phase
being CH2Cl2:MeOH:NH3(aq) 92:8:0.3), yielding the title compound as a
light yellow oil (0.506 g, 46 %). 1H-NMR (300 MHz, CDCl3): δ = 7.10 – 7.02
(m, 1H), 6.98 – 6.87 (m, 3H), 4.49 (s, 2H, O-CH2-CO), 4.19 (t, 2H, J = 7.7
Hz, N-CH2-CH2-CO), 3.60-3.50 (m, 2H), 3.48-3.35 (m, 2H), 2.88 - 2.82 (m,
2H), 2.79 – 2.71 (m, 2H), 2.69 – 2.56 (m, 2H), 1.85 (s, 1H, NH), 1.75 –
1.65 (m, 2H) ppm. 13C-NMR (75 MHz, CDCl3): δ = 169.7, 164.3, 145.1,
128.2, 123.9, 123.0, 117.0, 114.8, 67.5, 50.7, 48.6, 44.5, 38.0, 30.7, 30.4,
29.4 ppm. HRMS (ESI): m/z [M+H]+ calculated for C16H21N3O3 304.16555,
found 304.1655.
4-(2-Oxo-2-(piperazin-1-yl)ethyl)-2H-benzo[b][1,4]oxazin-3(4H)-one
(30’’)
2-(3-Oxo-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)acetic acid (1.5 g, 7.24
mmol), HOBt.H2O (1.11 g, 7.24 mmol) and DCC (1.49 g, 7.24 mmol) were
dissolved in DMF (25 ml). The mixture was stirred for 2 hours at 0°C. After
the addition of piperazine (1.25 g, 14.48 mmol), the reaction was stirred
overnight at room temperature. The formed precipitate was filtered off and
discarded. The filtrate was concentrated in vacuo and the residue was
purified by silica gel column chromatography (CH2Cl2:CH3OH:NH3(aq)
,
92:8:0.3) yielding the title compound as a white powder (742 mg, 37 %).
1H-NMR (300 MHz, DMSO): δ = 7.07- 6.98 (m, 3H), 6.93-6.88 (m, 1H),
4.78 (s, 2H), 4.66 (s, 2H), 3.53-3.47 (m, 2H), 3.42-3.36 (m, 2H), 2.84-2.77
(m, 2H), 2.73-2.66 (m, 2H) ppm. 13C-NMR (75 MHz, DMSO): δ = 164.4,
164.2, 144.6, 129.1, 123.4, 122.6, 116.4, 115.6, 66.9, 45.6, 45.2, 45.1,
42.4, 42.3 ppm. HRMS (ESI): m/z [M+H]+ calculated for C14H18N3O3
276.13425, found 276.1350.
4-(3-(3-Oxo-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)propanoyl)-N-
phenyl-1,4-diazepane-1-carboxamide (41)
This compound was synthesized from compound 40, according to the
procedure for the synthesis of compound 22a. The crude residue was
purified by silica gel flash chromatography (using ethyl acetate as mobile
phase), yielding the title compound as a white powder (93 % yield). Purity
(Method A): 100 %. 1H-NMR (300 MHz, CDCl3): δ 7.40 - 7.34 (m, 4H), 7.10
– 6.95 (m, 5H), 6.72 – 6.69 (m, 1H), 4.57 (s, 2H, O-CH2-CO), 4.29 - 4.15
(m, 2H, N-CH2-CH2-CO), 3.77 - 3.68 (m, 1H), 3.65 – 3.53 (m, 5H), 3.50 –
3.40 (m, 2H), 2.75 – 2.65 (m, 2H, J = 7.2 Hz, N-CH2-CH2-CO), 1.98 (m,
2H) ppm. HRMS (ESI): m/z [M+H]+ calculated for C23H26N4O4 423.20266,
found 423.2027.
4-(2-(3-Oxo-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)acetyl)-N-
phenylpiperazine-1-carboxamide (32)
Compound 32 was synthesized according to the procedure described for
the synthesis of compound 22a, affording the title compound as a white
1
powder (89 mg, 89 %). Purity (Method A): 98.76 %. H-NMR (300 MHz,
CDCl3): δ = 7.40-7.25 (m, 3H), 7.10-6.95 (m, 4H), 6.82-6.75 (m, 1H), 6.57
(br s, 1H), 4.72 (s, 2H), 4.67 (s, 2H), 3.75-3.60 (m, 6H), 3.55-3.43 (m, 2H),
1.64 (br s, 1H) ppm. 13C-NMR (75 MHz, CDCl3): δ = 165.4, 165.2, 155.1,
145.3, 138.7, 129.13 (2C), 129.08, 124.4, 123.7, 123.1, 120.3 (2C), 117.3,
115.0, 67.7, 44.9, 44.3, 43.5, 43.2, 41.8 ppm. HRMS (ESI): m/z [M+H]+
calculated for C21H23N4O4 395.17136, found: 395.1708.
(S)-2-Methyl-4-(3-(3-oxo-2H-benzo[b][1,4]oxazin-4(3H)-yl)propanoyl)-
N-phenylpiperazine-1-carboxamide (45a)
Compounds 33, 36 and 37 were synthesized according to the procedure
for the preparation of compound 32. Exact experimental and spectral data
can be found in the Supporting Information.
(S)-tert-Butyl 3-methylpiperazine-1-carboxylate 42a (100 mg 0.50 mmol)
was dissolved in THF (10 mL) and a solution of phenylisocyanate (60 mg
0.57 mmol) in THF (5 mL) was slowly added at room temperature. A light
yellow solid was formed upon reaction completion. Then, trifluoroacetic
acid (10 ml) was added dropwise while stirring at 0°C. When TLC showed
completion of the deprotection, the mixture was diluted with water (50 ml).
Then, the mixture was washed with dichloromethane (30 ml) and the pH
of the aqueous phase was adjusted to 9 by the addition of 1N NaOH. The
mixture was extracted with dichloromethane (50 ml). The combined
organic layers were dried over Na2SO4. The solvents were evaporated in
vacuo, affording crude 44a. This solid was used in the next reaction without
further purification. The solid was redissolved in DMSO (10 ml). Compound
20 (110 mg, 0.50 mmol), HCTU (206 mg 0.50 mmol) and DIPEA (50 µl)
were added. The mixture was stirred overnight at room temperature. The
resulting mixture was diluted with CH2Cl2 (100 ml) and washed with
water (40 ml). The combined organic layers were dried over Na2SO4 and
concentrated in vacuo. The crude residue was purified by silica gel
flash column chromatography yielding the title compound (210 mg, 50 %).
Purity (Method A): 98.77 %. 1H-NMR (300 MHz, DMSO-d6): δ = 1.05-1.19
4-(3-(3-Oxo-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)propyl)-N-
phenylpiperazine-1-carboxamide (39)
A mixture of 2H-benzo[b][1,4]oxazin-3(4H)-one 18 (75 mg, 0.5 mmol),
K2CO3 (158 mg, 1.0 mmol) and tert-butyl 4-(3-bromopropyl)piperazine-1-
carboxylate (169 mg, 0.55 mmol) in DMF (5 ml) was stirred at 65°C for 18
hours. When the reaction was finished, the mixture was diluted with water
(15 ml) and extracted with dichloromethane (3x10 ml). The organic phases
were combined, washed with water (15 ml), dried over anhydrous Na2SO4
and evaporated. The crude residue was dissolved in a 80 % TFA solution
(5 ml). The mixture was stirred for 4 hours. After evaporation of the
solvents, the crude residue was dissolved in THF (5 ml) and
phenylisocyanate (66 mg, 0.55 mmol) was added. The mixture was stirred
at room temperature overnight. The resulting mixture was diluted with
CH2Cl2 (20 ml) and washed with water (2×20 ml). The combined organic
layers were dried over anhydrous Na2SO4 and concentrated under
reduced pressure. The residue was purified by silica gel flash column
chromatography (Et2O:MeOH 10:1) to afford the product (101 mg, 51 %).
(m, 3H, CH3), 2.67-3.14 (m, 5H, CH2 CH), 3.65-4.38 (m, 6H, 3CH2), 4.63
，
(s, 2H, CH2), 6.94-7.08 (m, 1H, CH), 7.20-7.25 (m, 3H, 3CH), 7.43-7.48 (m,
3H, 3CH), 8.54 (s, H, NH) ppm. 13C-NMR (75 MHz, DMSO-d6): δ =14.71,
15.24, 29.82, 33.08, 37.33, 47.21, 43.80, 44.81, 45.08, 46.51, 48.94, 67.21,
13
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10.1002/cmdc.201800739
ChemMedChem
FULL PAPER
115.32, 115.45, 116.77, 119.95, 121.97, 123.02, 123.69, 128.41, 140.54,
145.04, 154.91, 161.16, 169.20, 169.30 ppm. HRMS (ESI): m/z [M+H]+
calculated for C23H27N4O4 423.20321, found 423.2025.
compound 61. N-Boc deprotection was carried out by using a 3 ml 80 %
TFA in 15 ml of CH2Cl2. After stirring for 3 hours at 0°C, solvents were
evaporated and the crude mixture was dissolved in CH2Cl2 (20 ml),
washed with a saturated aqueous NaHCO3 solution. The combined organic
layers were dried over anhydrous Na2SO4, concentrated and purified by
flash column chromatography (EtOAc:MeOH 10:1) yielding the title
compound (102 mg, 25 %). Purity (Method A): 98.86 %. 1H-NMR (300
MHz, CDCl3): δ = 2.77-2.85 (m, 2H, CH2), 3.47-3.50 (m, 4H, 2xCH2), 3.51
(s, 2H, CH2), 3.69-3.73 (m, 2H, CH2), 4.56-4.61 (m, 2H, CH2), 6.48 (s, 1H,
NH), 7.02-7.08 (m, 1H, CHAr), 7.26-7.41 (m, 5H, 5xCHAr), 7.48-7.51 (m,
1H, CHAr), 7.59-7.65 (m, 1H, CHAr), 7.90-7.93 (m, 1H, CHAr), 8.30 (s, 1H,
CH) ppm. 13C- NMR (75 MHz, CDCl3): δ = 30.72, 38.80, 41.33, 43.73,
44.09, 45.30, 113.83, 120.26, 123.69, 124.15, 129.12, 131.05, 131.54,
132.25, 133.82, 138.69, 150.00, 154.96, 155.07, 168.72 ppm. HRMS
(ESI): m/z [M+H]+ calculated for C22H24N5O3 406.18737, found 406.1864.
Compounds 45b-f were synthesized according to the procedure for the
preparation of compound 45a. Exact experimental and spectral data can
be found in the Supporting Information.
4-(3-(3-Oxo-2,3-dihydro-4H-pyrido[3,2-b][1,4]oxazin-4-yl)propanoyl)-
N-phenylpiperazine-1-carboxamide (48)
A mixture of 2H-pyrido[3,2-b][1,4]oxazin-3(4H)-one 46 (75 mg, 0.5 mmol),
K2CO3 (158 mg, 1.0 mmol), DMF (3 ml), and ethyl 3-bromopropionate (100
mg, 0.55 mmol) was stirred at 80°C for 8 hours. When the reaction was
finished, the mixture was diluted with water (15 ml) and extracted with
dichloromethane (3x10 ml). The combined organic phases were washed
with water (15 ml) dried over anhydrous Na2SO4 and evaporated in vacuo.
The crude residue was dissolved in THF (5 ml) and a solution of LiOH (42
mg, 1 mmol) in H2O (5 ml) was added. The mixture was stirred for 18 hours
at 60°C. Then, the pH was adjusted to 3 with 2N hydrochloric acid and the
compound was extracted with ethyl acetate (3x20 ml). The combined
organic layers were dried over anhydrous Na2SO4. The solvents were
evaporated in vacuo, yielding the pure carboxylic acid compound. The acid
was redissolved in DMF (5 ml), and HOBt (76 mg, 0.5 mmol) and DCC
(103 mg, 0.5 mmol) were added at 0°C. After stirring for two hours, N-
phenylpiperazine-1-carboxamide 16 (102 mg, 0.5 mmol) was added. The
mixture was stirred at room temperature overnight. The resulting mixture
was diluted with CH2Cl2 (20 ml) and washed with water (2×20 ml). The
organic phases were dried over anhydrous Na2SO4, and concentrated in
vacuo. The crude residue was purified by flash column chromatography
(using a mixture of diethylether and methanol in a ratio of 10:1 as mobile
phase) to afford the title compound (135 mg, 66 %). Purity (Method A):
100 %. 1H-NMR (300 MHz, CDCl3): δ = 2.76-2.81 (m, 2H, CH2), 3.46-3.69
(m, 8H, 4xCH2), 4.42-4.47 (m, 2H, CH2), 4.66 (s, 2H, CH2), 6.62 (s, 1H,
NH), 6.94 (dd, J = 7.9, 4.9 Hz, 1H, CHAr), 7.01-7.07 (m, 1H, CHAr), 7.23
(dd, J = 7.9, 1.5 Hz, 1H, CHAr), 7.27 – 7.38 (m, 4H, CHAr), 8.00 (dd, J = 4.9,
1.5 Hz, 1H, CHAr) ppm. 13C-NMR (75 MHz, CDCl3): δ = 31.49, 36.41, 41.12,
43.62, 44.19, 45.33, 67.50, 119.54, 120.35, 123.51, 123.66, 129.01,
138.89, 140.74, 141.34, 141.45, 155.18, 164.88, 169.49 ppm. HRMS
(ESI): m/z [M+H]+ calculated for C21H24N5O4 410.18228, found 410.1818.
4-(3-(2-Oxobenzo[d]oxazol-3(2H)-yl)propanoyl)-N-phenylpiperazine-
1-carboxamide (64)
3-(2-Oxobenzo[d]oxazol-3(2H)-yl)propanoic acid 63 (104 mg, 0.5 mmol)
was dissolved in DMF (5 ml) and HOBt (76 mg, 0.5 mmol) and DCC (103
mg, 0.5 mmol) were added at 0°C. After stirring for 2 hours at 0°C, N-
phenylpiperazine-1-carboxamide 16 (102 mg, 0.5 mmol) was added. The
mixture was stirred overnight at room temperature. The resulting mixture
was diluted with CH2Cl2 (20 ml) and washed with water (2×20 ml). The
combined organic layers were dried over anhydrous Na2SO4 and
concentrated in vacuo. The crude residue was purified by silica gel flash
column chromatography (a mixture of diethylether and methanol in a ratio
of 10:1 as mobile phase) to afford the title compound (170 mg, 86 %).
Purity (Method B): 99.29 %. 1H-NMR (300 MHz, CDCl3): δ = 2.94 (t, J =
6.8 Hz, 2H, CH2), 3.48-3.59 (m, 6H, 3xCH2), 3.71-3.75 (m, 2H, CH2), 4.26
(t, J = 6.8 Hz, 2H, CH2), 6.59 (s, 1H, NH), 7.10-7.43 (m, 9H, 9xCHAr) ppm.
13C-NMR (75 MHz, CDCl3): δ = 31.10, 38.66, 41.33, 43.63, 44.00, 45.19,
109.20, 110.19, 120.28, 122.67, 123.65, 124.14, 129.09, 131.19, 138.73,
142.78, 154.96, 168.74 ppm.
4-(3-(2,3-Dihydro-4H-benzo[b][1,4]oxazin-4-yl)propanoyl)-N-
phenylpiperazine-1-carboxamide (66)
A mixture of 3,4-dihydro-2H-benzo[b][1,4]oxazine 65 (67 mg, 0.5 mmol),
DIPEA (0.24 ml, 1.4 mmol) and ethyl 3-bromopropionate (380 mg, 2.4
mmol) in DMF (3 ml) was stirred at 80°C for 48 hours. When the reaction
was finished, the mixture was diluted with water (15 ml) and extracted with
dichloromethane (3x10 ml). The organic phases were combined and
washed with water (15 ml). The combined organic layers were dried over
anhydrous Na2SO4 and evaporated in vacuo. The crude residue was
dissolved in THF (5 ml) and a solution of LiOH (42 mg, 1 mmol) in water
(5 ml) was added. The mixture was stirred for 18 hours at 60°C. Then, the
pH was adjusted to 3 with 2N hydrochloric acid and the compound was
extracted with ethyl acetate (3x20 ml). The combined organic layers were
dried over anhydrous Na2SO4. After solvent evaporation, the pure acid
compound was obtained. The acid was redissolved in DMF (5 ml), and
HOBt (76 mg, 0.5 mmol) and DCC (103 mg, 0.5 mmol) were added at 0°C.
After stirring for 2 hours, N-phenylpiperazine-1-carboxamide 16 (102 mg,
0.5 mmol) was added. The mixture was stirred overnight at room
temperature. The resulting mixture was diluted with CH2Cl2 (20 ml) and
washed with water (2×20 ml). The organic phase was dried over Na2SO4
and concentrated in vacuo. The crude residue was purified by silica gel
flash column chromatography (using a mixture of diethylether and
methanol in a ratio of 10:1 as mobile phase) to afford the title compound
(96 mg, 49 %). Purity (Method A): 95.43 %. 1H-NMR (300 MHz, CDCl3): δ
= 2.58-2.63 (m, 2H, CH2), 3.37-3.50 (m, 8H, 4xCH2), 3.65-3.70 (m, 4H,
2xCH2), 4.20-4.23 (m, 2H, CH2), 6.59 (s, 1H, NH), 6.60-6.67 (m, 2H,
2xCHAr), 6.77-6.86 (m, 2H, 2xCHAr), 7.02-7.07 (m, 1H, CHAr), 7.24-7.34 (m,
4H, 4xCHAr) ppm. 13C-NMR (75 MHz, CDCl3): δ = 29.39, 41.20, 43.55,
44.10, 45.27, 47.04, 47.67, 64.60, 111.81, 116.67, 117.82, 120.38, 121.83,
123.63, 129.04, 134.49, 138.74, 144.27, 155.03, 170.43 ppm.
Compounds 49, 53-55, 58-59 were synthesized according to the
procedure for the preparation of compound 48. Exact experimental and
spectral data can be found in the Supporting Information.
4-(3-(2-Oxoquinoxalin-1(2H)-yl)propanoyl)-N-phenylpiperazine-1-
carboxamide (62)
A mixture of tert-butyl 3-oxo-3,4-dihydroquinoxaline-1(2H)-carboxylate 60
(248 mg,
1 mmol), K2CO3 (276 mg, 2.0 mmol) and ethyl 3-
bromopropionate (199 mg, 1.1 mmol) in DMF (6 ml) was stirred at 80°C
for 8 hours. When the reaction was finished, the mixture was diluted with
water (30 ml) and extracted with dichloromethane (3x15 ml). The organic
phases were combined and washed with water (30 ml). The combined
organic phases were dried over Na2SO4 and evaporated in vacuo. The
crude residue was redissolved in THF (10 ml) and a solution of LiOH (84
mg, 2 mmol) in H2O (10 ml) was added. The mixture was stirred for 18
hours at 60°C. Then, the pH was adjusted to 3 with 2N hydrochloric acid
and the compound was extracted with ethyl acetate (3x20 ml). The
combined organic layers were dried over anhydrous Na2SO4 and
evaporated in vacuo, yielding the pure acid. The acid was dissolved in
DMF (10 ml), and HOBt (153 mg, 1 mmol), DCC (206 mg, 1 mmol) were
added at 0°C. After stirring for 2 hours, N-phenylpiperazine-1-carboxamide
16 (204 mg, 1 mmol) was added and the mixture was stirred overnight at
room temperature. The resulting mixture was diluted with CH2Cl2 (30 ml)
and washed with water (2×20 ml). The combined organic layers were dried
over anhydrous Na2SO4 and concentrated in vacuo. The crude residue
was purified by flash column chromatography (Et2O:MeOH 10:1) affording
14
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10.1002/cmdc.201800739
ChemMedChem
FULL PAPER
Mycobacterial ThyX NADPH oxidase assay
Acknowledgements
M. tuberculosis ThyX was produced and purified as described.[18] The
synthesized compounds were screened using a NADPH oxidation assay
for M. tuberculosis ThyX activity in 96-well plates.[27] To determine IC50
values of compounds 22g, 27, 28, 29d, 45c, 48 and 52, the reaction
mixture (100 µL) contained 50 mM HEPES pH7.5, 1mM MgCl2, 12 µM FAD,
0.5% glycerol, 0.05% Triton X-100, 0.5 mg/mL BSA, 3 µM dUMP, 2.5%
DMSO, a range of concentrations (ranging from 40 nM to 20 µM) of each
testing compound and 0.5 µM of purified MtbThyX. After incubation of 15
min, the reactions were initiated by adding 600 µM NADPH and the
decrease in absorbance at 340 nm was detected using CLARIOstar
microplate reader (BMG Labtech) after 1 hour. The experiment was done
in duplicates and repeated 3 to 4 times. In the control reactions, no enzyme
or no inhibitor was used. Reported IC50 values were determined by
normalizing the absorbance values to the control points and fitting the data
to a Nonlinear Regression (dose response inhibition) using Graphpad
Prism software.
Authors are grateful to Research Foundation - Flanders (FWO)
for their support of the project G.0664.12 on ‘Flavin dependent
thymidylate synthase: a target for new antibiotics’.
Keywords: Antibiotics
• Drug discovery • Tuberculosis •
Benzo[b][1,4]oxazine
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10.1002/cmdc.201800739
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16
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ChemMedChem
FULL PAPER
Entry for the Table of Contents
Starting from a previously identified mycobacterial ThyX inhibitor based on a benzo[b][1,4]oxazin-3(4H)-one scaffold, a systematic
structure-activity relationship study was performed. It led to the discovery of a benzo[b][1,4]oxazin-3(4H)-one analogue displaying an
IC50 value of 0.69 µM. These heterocycles can be used as starting points for the discovery of novel antibacterial agents acting via
ThyX inhibition.
17
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