Endeavors towards transformation of M. tuberculosis thymidylate kinase (MtbTMPK) inhibitors into potential antimycobacterial agents

Article abstract of DOI:10.1016/j.ejmech.2020.112659

As the last enzyme in nucleotide synthesis as precursors for DNA replication, thymidylate kinase of M. tuberculosis (MtbTMPK) attracts significant interest as a target in the discovery of new anti-tuberculosis agents. Earlier, we discovered potent MtbTMPK inhibitors, but these generally suffered from poor antimycobacterial activity, which we hypothesize is due to poor bacterial uptake. To address this, we herein describe our efforts to equip previously reported MtbTMPK inhibitors with targeting moieties to increase the whole cell activity of the hybrid analogues. Introduction of a simplified Fe-chelating siderophore motif gave rise to analogue 17 that combined favorable enzyme inhibitory activity with significant activity against M. tuberculosis (MIC of 12.5 μM). Conjugation of MtbTMPK inhibitors with an imidazo[1,2-a]pyridine or 3,5-dinitrobenzamide scaffold afforded analogues 26, 27 and 28, with moderate MtbTMPK enzyme inhibitory potency, but sub-micromolar activity against mycobacteria without significant cytotoxicity. These results indicate that conjugation with structural motifs known to favor mycobacterial uptake may be a valid approach for discovering new antimycobacterial agents.

Full text of DOI:10.1016/j.ejmech.2020.112659

Journal Pre-proof
Endeavors towards transformation of M. tuberculosis thymidylate kinase (MtbTMPK)
inhibitors into potential antimycobacterial agents
Yanlin Jian, Romain Merceron, Steven De Munck, He Eun Forbes, Fabian Hulpia,
Martijn.D.P. Risseeuw, Kristof Van Hecke, Savvas N. Savvides, Hélène Munier-
Lehmann, Helena.I.M. Boshoff, Serge Van Calenbergh
PII:
S0223-5234(20)30631-0
DOI:
https://doi.org/10.1016/j.ejmech.2020.112659
EJMECH 112659
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 June 2020
Revised Date: 8 July 2020
Accepted Date: 8 July 2020
Please cite this article as: Y. Jian, R. Merceron, S. De Munck, H.E. Forbes, F. Hulpia, M.D.P.
Risseeuw, K. Van Hecke, S.N. Savvides, H.l.n. Munier-Lehmann, H.I.M. Boshoff, S. Van Calenbergh,
́
̀
Endeavors towards transformation of M. tuberculosis thymidylate kinase (MtbTMPK) inhibitors into
potential antimycobacterial agents, European Journal of Medicinal Chemistry, https://doi.org/10.1016/
j.ejmech.2020.112659.
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Endeavors towards transformation of M. tuberculosis thymidylate kinase (MtbTMPK)
inhibitors into potential antimycobacterial agents
Yanlin Jiana, Romain Merceronb, c, Steven De Munckb, c, He Eun Forbesd, Fabian Hulpiaa,
Martijn, D. P. Risseeuwa, Kristof Van Heckee, Savvas N. Savvidesb, c, Hélène Munier-Lehmannf,
Helena I. M. Boshoff d, and Serge Van Calenbergha, *
aLaboratory for Medicinal Chemistry (FFW), Ghent University, Ottergemsesteenweg 460,
B-9000 Ghent, Belgium
bVIB Center for Inflammation Research, Zwijnaarde, Ghent 9052, Belgium
cDepartment of Biochemistry and Microbiology, Ghent University, Ghent 9052, Belgium
dTuberculosis Research Section, Laboratory of Clinical Immunology and Microbiology,
National Institute of Allergy and Infectious Disease, National Institutes of Health, 9000
Rockville Pike, Bethesda, Maryland 20892, United States
eXStruct, Department of Chemistry, Ghent University, Krijgslaan 281 S3, Gent B-9000,
Belgium
fUnit of Chemistry and Biocatalysis, Department of Structural Biology and Chemistry, Institut
Pasteur, CNRS UMR3523, 28 Rue du Dr. Roux, Cedex 15 75724 Paris, France
* Corresponding author. Tel.: +32 9 264 81 24; fax: +32 9 264 81 46.
E-mail address: serge.vancalenbergh@ugent.be (S. Van Calenbergh).
1

Abstract
As the last enzyme in nucleotide synthesis as precursors for DNA replication, thymidylate
kinase of M. tuberculosis (MtbTMPK) attracts significant interest as a target in the discovery of
new anti-tuberculosis agents. Earlier, we discovered potent MtbTMPK inhibitors, but these
generally suffered from poor antimycobacterial activity, which we hypothesize is due to poor
bacterial uptake. To address this, we herein describe our efforts to equip previously reported
MtbTMPK inhibitors with targeting moieties to increase the whole cell activity of the hybrid
analogues. Introduction of a simplified Fe-chelating siderophore motif gave rise to analogue 17
that combined favorable enzyme inhibitory activity with significant activity against M.
tuberculosis (MIC of 12.5 μM). Conjugation of MtbTMPK inhibitors with an
imidazo[1,2-a]pyridine or 3,5-dinitrobenzamide scaffold afforded analogues 26, 27 and 28,
with moderate MtbTMPK enzyme inhibitory potency, but sub-micromolar activity against
mycobacteria without significant cytotoxicity. These results indicate that conjugation with
structural motifs known to favor mycobacterial uptake may be a valid approach for discovering
new antimycobacterial agents.
Keywords: Mycobacteria tuberculosis, thymidine monophosphate kinase (TMPK),
antimycobacterial, siderophore, uptake
Graphical abstract
2

Introduction
Tuberculosis, caused by the infectious agent Mycobacteria tuberculosis (M. tuberculosis),
leads to a high death rate with more than 1.2 million patients succumbing to the infection every
year [1]. Over the past decade, the overall incidence of tuberculosis has declined, although
regions in sub-Saharan Africa and Asia show alarmingly high incidence rates. The emergence
of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains threatens any
achieved progress in combatting this disease [2]. The increasing number of drug-resistant
tuberculosis places an unsustainable burden on health-care systems, in addition to affecting
economical as well as societal growth.
The approval of bedaquiline and delamanid by the EMA and FDA has provided new hope for
afflicted patients, but the first M. tuberculosis isolate resistant to bedaquiline was reported
shortly after clinical introduction of this drug [3]. Very recently pretomanid has been approved
as a combination partner for bedaquiline and linezolid in treating highly resistant forms of TB
[4]. Nonetheless, this regimen is contra-indicated in patients with hypersensitivity to
bedaquiline or linezolid, effectively narrowing its use. Taken together, research efforts toward
the discovery and development of new antitubercular agents, particularly those with a novel
mechanism of action, should be actively pursued to keep pace with the high incidence of
tuberculosis [5].
M. tuberculosis thymidine monophosphate kinase (MtbTMPK), the last specific enzyme in the
biosynthesis of thymidine triphosphate, is indispensable for mycobacterial growth and survival
[6]. A crystal structure [7] of the protein has been reported, showcasing a unique configuration
of its active site [6], making MtbTMPK a promising target to pursue for the discovery of new
bio-active molecules to treat tuberculosis.
Previous studies have described nucleoside [8-12] and non-nucleoside [13] MtbTMPK
inhibitors. Even though these campaigns resulted in potent MtbTMPK inhibitors [8-12,14-17],
these mostly suffered from low to moderate whole-cell activity. For a MtbTMPK inhibitor to
show an antimycobacterial effect, it must cross both the inner and outer membranes to reach
cytosolic concentrations to allow sufficient target enzyme inhibition to effect mycobacterial
growth inhibition. While the required physicochemical properties for intracellular accumulation
3

has been studied in some detail for Gram-negative bacteria [18-20], this remains speculative
for mycobacteria.
Mycobacteria rely on the secretion of low molecular weight siderophores to chelate and
subsequently capture iron (Fe3+), which constitutes an essential nutrient for bacterial growth
[21]. The receptor-mediated active import of siderophores turns these into possible antibiotic
delivery vectors [22-24]. Typically, a siderophore-mediated drug delivery system is comprised
of an Fe-targeting siderophore moiety, an antibiotic and a connecting linker (Figure 1). Based
on the stability of the linker, these conjugates can be further divided into non-releasable (stable)
and releasable (liable) ones (Figure 1) [25]. In response to low-iron conditions present in the
host, M. tuberculosis secrets two salicylate-derived siderophores, namely carboxymycobactin
and mycobactin [26]. To date, only the latter has been successfully used as drug delivery
vector [27]. In order to reduce the inherent complexity, simplified single bidentate ligands such
as catechol or hydroxypyridone might be considered as surrogates, since their utility has
already been demonstrated in Gram-negative bacteria [28-31]. In this study we explore the
use of simplified siderophores to facilitate TMPK inhibitor uptake in M. tuberculosis.
Figure 1. Generalized structure of a siderophore mediated drug delivery system and two
successful examples of siderophore conjugation: the mycobactin-artemisinin conjugate and
the single bidentate catechol-cephalosporin conjugate S-649266 [27,28].
In the past decade new chemical entities emerged with promising whole-cell activity against M.
tuberculosis. Imidazo[1,2-a]pyridine analogue Q203 [32] was reported as a promising
antimycobacterial agent targeting the respiratory cytochrome bc1 complex (Figure 2).
4

Nitroaromatic compounds such as 3,5-dinitrobenzamides [33] were also found to exhibit
excellent antimycobacterial activity. The fact that both compounds share a N-substituted
4-arylpiperidine scaffold with our previously reported MtbTMPK inhibitor 1 [12] (Figure 3),
inspired us to synthesize hybrid compounds of the latter that comprise the
imidazo[1,2-a]pyridine and 3,5-dinitrobenzamide moieties anticipated to be favourable for
mycobacterial uptake.
Figure 2. Structures of anti-TB scaffold of Q203 and 3,5-dinitrobenzamide.
In this study we thus sought to combine the scaffold of previously reported MtbTMPK inhibitors
with either simplified Fe-chelating motifs [26,34-36] or motifs from compounds with known
whole-cell antimycobacterial activity, e.g. imidazo[1,2-a]pyridine and 3,5-dinitrobenzamide,
and evaluate the effect of the resulting hybrid compounds on MtbTMPK inhibitory activity and
in vitro anti-TB activity. An overview of the target analogues is presented in Figure 3.
Figure 3. Compounds synthesized and evaluated in this study. A: Q203-based hybrids; B.
3,5-dinitrobenzamide hybrids; C: non-cleavable siderophore hybrids; D. cleavable siderophore
hybrids and their precursors.
Results and discussion
5

Chemistry
Generally, the envisioned analogues were obtained through either reductive amination of
N3-BOM-protected 1-(piperidin-4-yl)thymine with an appropriate aldehyde [12], or via alkylation
of the same intermediate with a suitable benzyl bromide derivative [37], followed by
TFA-mediated BOM deprotection (Scheme 1) [11,12,38]. Preparation of the 4-quinolone
derivative 4 was achieved by reductive amination of in situ generated aldehyde from
4-chloro-2-methylquinoline, and treatment of the resulting 4-chloroquinoline intermediate 3
with acetic acid at 125 °C [39], after multiple attempts to achieve substitution under basic
conditions were met with failure (data not shown). 4-Substituted quinoline derivatives 6-10
were prepared by nucleophilic aromatic substitution [40] of 3 with different nucleophiles at
180 °C followed by BOM-deprotection.
The 8-hydroxyquinoline derivative 13, on the other hand, was prepared via alkylation of 5 with
an in situ generated benzyl bromide reagent derived from 2-methylquinolin-8-ylacetate,
followed by global deprotection under acidic conditions.
Scheme 1.
Reagents and conditions: (a) (i) SeO2, 1,4-dioxane, reflux; (ii) 5, sodium
triacetoxyborohydride, 1,2-dichloroethane, 32%; (b) (i) acetic acid, 125 ℃; (ii) neat TFA, 73 ℃,
58%; (c) (i) RXH, EA, 180 ℃; (ii) neat TFA, 73 ℃, 10-50%; (d) (i) NBS, AIBN, CCl4, 80 ℃; (ii) 5,
K2CO3, ethanol, reflux, 20%; (e) neat TFA, 73 ℃, 58%.
The synthesis of bipyridyl analogue 17 commenced with a Stille coupling between
6-bromopicolinaldehyde and 2-(tributylstannyl)pyridine to deliver aldehyde 15 [41], which was
subsequently reduced (Scheme 2). The resulting alcohol was transformed into its mesylate
6

and immediately substituted with 5 and BOM-deprotected to afford the desired 17.
Several intermediates described above (3, 7-10) were employed for the quinolone-based iron
chelating ester/amide analogues 18-21 or hydrazine derivative 22. Compounds 18 and 19
were obtained by esterification of 9 and 10 with 3,4-bis(benzyloxy)benzoylchloride, followed
hydrogenolysis. A similar reaction sequence failed to produce 20, since the hydrogenolysis
conditions (H2/Pd) led to benzoyl ester hydrolysis. To circumvent this issue, 7 was coupled with
3,4-bis((tert-butyldimethylsilyl)oxy)benzoic acid, from which the tert-butyldimethylsilyl group
could be easily removed with HF·pyridine. Hydroxamate 21 was obtained by coupling 8 with
.
BnONH2 HCl and subsequent hydrogenolysis. Nucleophilic aromatic substitution of 3 with
2,4-dihydroxybenzaldehyde
at
180
°C,
followed
by
reaction
with
methyl
hydrazinecarbodithioate [42] furnished the desired hydrazine 22
Scheme 2.
H
O
N
O
N
a
b
c
N
N
N
N
N
Br
N
CHO
N
CHO
OH
N
14
15
16
17
H
N
O
O
N
R =
N
N
OH
OH
OH
OH
OH
R
OH
O
d
e
g
R
O
X
X
R
O
R
O
N
O
O
O
9, X=O
, X=NH
18, X=O
19
7
20
10
, X=NH
O
O
H
N
S
OH
OH
N
f
H
R
Cl
R
S
R
R
OH
O
O
8
21
3
22
Reagents and conditions: (a) Pd(PPh3)4, 2-(tributylstannyl)pyridine, toluene, reflux, 40%; (b)
sodium triacetoxyborohydride, 1,2-dichloroethane, 49%; (c) (i) methanesulfonyl chloride, Et3N,
CH2Cl2; (ii) 5, K2CO3, ethanol, 80 ℃; (iii) neat TFA, 73 ℃, 26%; (d) (i) 3,4-bis(benzyloxy)benzoyl
chloride , N-methylmorpholine, CH2Cl2; (ii) Pd/C, H2, EtOH. 21%; (e) (i) EDC.HCl, DMAP,
3,4-bis((tert-butyldimethylsilyl)oxy)benzoic acid, CH2Cl2; (ii) HF·pyridine, pyridine, THF, 0 ℃,
.
16%; (f) (i) EDC.HCl, DMAP, BnONH2 HCl, CH2Cl2; (ii) Pd/C, H2, EtOH, 20%; (g) (i)
2,4-dihydroxybenzaldehyde, EA, 180 ℃; (ii) neat TFA, 73 ℃; (iii) methyl
hydrazinecarbodithioate, MeOH, reflux, 6%.
To access compounds featuring imidazo[1,2-a]pyridine amide or 3,5-dinitrobenzamide motifs,
7

intermediate 5 was directly coupled to 6-chloro-2-ethylimidazo[1,2-a]pyridine-3-carboxylic acid
to give amide 23 after BOM- deprotection. Alternatively, 5 was first reacted with
4-fluorobenzonitrile or reductively alkylated with 4-cyanobenzaldehyde to yield 24 and 25,
respectively (Scheme 3). BOM-deprotection and subsequent reduction of the nitrile with
NaBH4/CoCl2 gave rise to the benzylamine intermediates, which were amidated to the desired
final compounds 26-28.
Scheme 3.
Reagents
and
conditions:
(a)
(i)
EDC.HCl,
HOBt,
Et3N,
6-chloro-2-ethylimidazo[1,2-a]pyridine-3-carboxylic acid, DMF, 70 ℃; (ii) neat TFA, 73 ℃, 21%;
(b) n = 0, K2CO3, 4-fluorobenzonitrile, DMSO, 120 ℃; n = 1, p-cyanobenzaldehyde, sodium
triacetoxyborohydride, 1,2-dichloroethane, 41%; (c) (i) neat TFA, 73 ℃; (ii) NaBH4, CoCl2,
THF/H2O; (iii) 6-chloro-2-ethylimidazo[1,2-a]pyridine-3-carboxylic acid, EDC.HCl, HOBt, Et3N,
DMF, 70 ℃, 28%; (d) (i) neat TFA, 73 ℃; (ii) NaBH4, CoCl2, THF/H2O; (iii) 3,5-dinitrobenzoic
acid, EDC.HCl, HOBt, Et3N, DMF, 70 ℃, 10-20%.
Biological activity
Enzyme inhibition
All target compounds were tested for MtbTMPK inhibitory activity, including the
precursors/potential degradation products of cleavable iron chelator MtbTMPK inhibitors, i.e. 4,
7, 9 and 10 (Table1). Unexpectedly, all these precursors exhibited better enzyme inhibitory
activity than compound 1 [12]. It is noteworthy that the hydroxyethyl amine 10 displays almost
5-fold better activity than its hydroxyethyl ether analogue 9.
Table 1. The MtbTMPK enzymatic activity of compounds.
8

Compound
IC50 (μM)
29 ± 3
Compound
IC50 (μM)
0.24 ± 0.02
0.18 ± 0.02
1.0 ± 0.1
0.31 ± 0.02
879 ± 122
47 ± 7
1
4
19
20
21
22
23
26
27
28
7.8 ± 0.4
1.3 ± 0.1
0.83 ± 0.05
2.4 ± 0.3
0.53 ± 0.02
23 ± 2
6
7
9
10
13
17
18
NDa
34 ± 5
48 ± 2
0.14 ± 0.01
aND: Not determined, due to precipitation.
Interestingly, the inhibitory potency generally increased upon introduction of catechol (pairs
9/18, 10/19, 7/20). To gain insight in their putative binding mode, 9 and 18 were docked into
the enzyme (Figure 4, PDB: 5NR7 [12]). These studies suggest that hydroxyethyl ether in 9
forms a weak electrostatic interaction with Tyr39. Yet, the additional catechol ester of 18
protrudes outward the active site and may form hydrogen bonds with Asp9, Lys13 and Arg95,
thereby stabilizing a bent conformation and resulting in an improved inhibitory activity.
Figure 4. A. Structure of 9 docked in the active site of MtbTMPK (PDB: 5NR7 [12]). MtbTMPK
is shown in a pale yellow cartoon representation with selected side chains labeled and shown
as sticks with carbon atoms colored orange. Inhibitor 9 is drawn in stick representation with
carbon atoms in pink, hydrogen-bonding interactions are shown as black dotted lines. B.
Docking pose of inhibitor 18 in MtbTMPK (PDB: 5NR7 [12]) catalytic pocket. The protein is
depicted as pale yellow cartoon representation with selected side chain labeled (orange).
Inhibitor 18 is drawn in stick representation with carbon atoms in white.
Except for bipyridyl analogue 17, the non-cleavable chelator hybrid analogues 21 and
9

especially 22 displayed promising inhibitory potency.
In line with earlier observations for other piperidine amide analogues [11,43], amide 23
showed very poor inhibitory potency. However, the insertion of a phenylmethylene group
between the piperidyl and amide group as in 26 resulted in a 19-fold better potency. Since 26
structurally deviates from all previous analogues, its co-crystal structure with MtbTMPK was
determined at 1.9 Å resolution (Table S2, PDB code: 6YT1). The crystal structure shows that
26 binds into the catalytic pocket contacting the same residues as analogous compounds
(Figure 5A&B). Compound 26 is mainly accommodated by π−π electron stacking interactions
above and below the plane of the heterocyclic system mediated by Tyr39, Tyr103, and Phe70,
and is further stabilized by polar interactions mediated by Arg74 and Asn100 (Figure 5B).
Surprisingly, compound 26 has the ability to engage residues of a symmetry-related TMPK
protomer including Tyr39 and His53. This binding mode brings two antiparallel-oriented
molecules of 26 in close proximity to each other (Figure 5A). To investigate whether this
unusual phenomenon might be due to a particular tendency of compound 26 to pre-organize,
the X-ray structure of compound 26 alone was also determined (Figure 5C and 5D). In this
structure the relative organization of two inhibitor molecules does not resemble the antiparallel
orientation observed in the complex with the protein. However, similar π - π stacking between
the two central phenyl rings of two neighboring compounds was observed in both structures,
which may account for the unusual phenomenon seen in the structure with the protein. In the
catalytic pocket, 26 adopts an L-shaped (torsion angle 96º) conformation with the amide tail
toward the α7- helix, which is distinct from the docked pose of 9 in which the hydroxyethyl tail
protrudes to the α2- helix. While similar to 9, the thymine ring adopts an identical pose in the
catalytic pocket of the enzyme (Figure 5B) and a π-alkyl interaction between a 4-piperidine
ring and Leu52 was observed. The amide tail fails to show specific interactions, possibly
accounting for the moderate inhibitory activity of 26. Encouraged by this finding, the
3,5-dinitrobenzamide analogues 27 and 28 were also prepared. Whereas the inhibitory
potency of 27 could not be determined due to solubility problems in the MtbTMPK assay, 28
showed similar inhibitory potency as 26.
10

Figure 5. A. Co-crystal structure of compound 26 in complex with MtbTMPK at 1.9 Å
resolution. One TMPK protomer in the asymmetric unit (pale yellow) is shown in cartoon
representation together with a symmetry related protomer (pale green). Two symmetry-related
molecules of compound 26 are shown in stick representation with carbon atoms in white. B.
View of binding mode and interaction of compound 26 in the MtbTMPK active site. MtbTMPK
is shown in a pale yellow cartoon representation. Compound 26 (carbon atoms in white) and
the side chains of MtbTMPK interacting residues (carbon atoms in orange) are depicted as
sticks. Hydrogen bonds are depicted as black dotted lines. The unbiased Fo-Fc difference
electron density (contoured to +3 sigma) calculated before adding the ligand in the refinement
process is shown as a green mesh. C. Crystal structure of compound 26. D. Packing in the
crystal structure and unit cell of compound 26.
Stability of cleavable MtbTMPK inhibitors
The stability of selected compounds 19, 20 and 22 in ammonium bicarbonate-formic acid
buffer solution (pH 6.6) was analyzed by LC-MS over the course of 24 h, and the results are
summarized in Figure 6. Less than 10% degradation was found for inhibitor 20 and 22 up to 6
h. At 24 h, the percentage of compounds remaining in buffer were above 70% for all three
inhibitors. These data reflect that although ester (19) and hydrazine (22) derivatives are
hydrolyzed, it is not instantaneous, which is the desired profile. Analogue 20 did not show
significant hydrolysis over the entire time course of the experiment.
11

Figure 6. 24 h stability of inhibitors 19 (blue line), 20 (red line) and 22 (green line) in
ammonium bicarbonate-formic acid buffer (pH 6.6). Error bar represents standard error
between two replicates.
Antimicrobial activity
All the target compounds were also evaluated their antitubercular activity (Table 2). The
precursor and most iron chelator containing compounds failed to show antimycobacterial
activity. However, the (2,2'-bipyridin)-6-ylmethyl derivative 17 displayed a MIC value of 12.5
μM in 7H9/glucose medium, despite its moderate MtbTMPK inhibitory potency. Only 22
displayed better activity in GAST (Fe (-)) medium compared to GAST (Fe (+)). In line with
previous piperidine amide analogues [11,12] and its very weak on-target activity, compound 23
lacked antimycobacterial activity. Interestingly, compound 26, which also features an
imidazo[1,2-a]pyridine motif but more remote from the piperidine ring, displayed a MIC of 4.7
μM in standard medium. Likewise, the dinitrobenzamide derivatives 27 and 28 exhibit low
micromolar whole-cell activity, and remain active in BSA containing medium (Table S3).
Additionally, none of the analogues with promising antitubercular activity displayed cytotoxicity
against MRC-5 fibroblasts.
12

Table 2. Antimycobacterial activity (H37Rv) of compounds in this study.
Mtb H37Rv MICa (μM)
Compound
7H9/glucose
>64b
>100
>100
≥100
>100
>100
75
GAST (Fe(+))
-
GAST (Fe(-))
-
MRC-5 CC50(μM)
1
4
-
>100
100
>100
≥100
≥100
>100
>100
>100
>100
>100
100
>64
>64
>64
>64
>64
53.98
>64
6.39
>64
-
6
7
>100
>100
>100
>100
>100
>100
>100
37
9
10
13
17
18
19
20
21
22
23
26
27
28
Isoniazid
12.5
≥100
>100
>50
-
>50
50
-
32.22
1.45
>64
>64
>64
>64
-
>100
>100
4.7
100
25
>100
2.3
>100
-
9.4
0.78
9.4
1.2
9.4
6.25
0.15
0.15
0.15
aMinimum inhibitory concentration (MIC) is the minimum concentration required to inhibit >99%
growth of M. tuberculosis H37Rv in liquid culture. bReported IC50, which was tested on H37Ra
strain.
Conclusion
The overall goal of this study was to synthesize hybrid compounds fusing or merging potential
iron chelating moieties or motifs of compounds with known whole-cell antitubercular activity
with earlier identified MtbTMPK inhibitors, as to increase the bacterial uptake. Unexpectedly,
the subset of iron chelating hybrids generally showed increased MtbTMPK inhibitory potency,
but only for 22 this resulted in moderate whole-cell antitubercular activity in iron-deficient
GAST medium. A possible explanantion for the lack of whole-cell activity in this subset is that
natural siderophores like mycobactin consist of multiple bidentate ligands for stoichiometric
binding of iron(III), which is not the case for the simplified chelating moieties used in this study.
Further analysis of their uptake by M. tuberculosis would assist active anti-tubercular agents
identification, which will be done in future reserch.
However, we were more succesfull with hybrids containing imidazo[1,2-a]pyridine and
13

dinitrobenzamide motifs. While merging these motifs with a known MtbTMPK inhibitor did not
improve activity against the target, it afforded several non-toxic compounds (e.g. 26, 27 and 28)
with promising antimycobacterial activity in all tested media. While at this stage we cannot
exclude that this whole-cell activity is due to modulation of additional targets. Nevertheless,
this study suggests that scaffold merging may be a fruitful approach for the discovery of new of
antimycobacterial agents.
Experimental section
Expression and purification of MtbTMPK
MtbTMPK was expressed and purified as previously described [44]. In short, the pHL50 vector
containing the coding sequence of wild-type MtbTMPK was used to transform E. coli BLi5
competent cells. Transformed cells used to inoculate 1 L of 2XYT medium supplemented with
antibiotics. The cells were allowed to grow at 37 °C, and protein production was induced with 1
mM isopropyl-1-thio-β-D-thiogalactoside when the culture reached an absorbance of 1.5 at 600
nm. After 3 h of incubation at 37 °C, cells were harvested by centrifugation at 8000g at 4 °C and
stored at −80 °C. Cells from 1 L of culture were resuspended in 50 mL of cold lysis buffer
containing 50 mM Tris HCl pH 8.0 supplemented with an antiprotease cocktail (Roche) and
disrupted by sonication. After centrifugation at 20000g for 30 min at 4 °C, the filtered bacterial
lysate was loaded on a 5 mL Blue-Sepharose column pre-equilibrated with lysis buffer. The
column was washed with lysis buffer and most of the protein was eluted with elution buffer
containing 50 mM Tris HCl pH 8.0, 1 M NaCl. A second elution was performed using elution
buffer containing 50 mM Tris HCl pH 8.0, 2 M NaCl. Fractions containing MtbTMPK were
pooled and injected on a HiLoad Superdex 75 16/600 column equilibrated with 20 mM Tris HCl
pH 7.4, 1 mM EDTA as a further polishing and buffer exchange step. Fractions containing pure
MtbTMPK were pooled and Tris(2-carboxyethyl)-phosphine (TCEP) was added to 1 mM final
concentration before flash-freezing the protein samples in liquid nitrogen.
Crystal structure of MtbTMPK in complex with compound 26
Before each cocrystallization experiment, MtbTMPK samples were thawed and concentrated to
8 mg/mL. Compound 26 was initially dissolved in 100% DMSO at a concentration of 100 mM
and diluted to 50 mM with pure isopropanol. Diluted compound was added to the concentrated
14

protein stock and incubated for 1 h at room temperature. Cocrystallization conditions of
MtbTMPK-inhibitor complexes were screened at 20 °C by the sitting-drop vapor-diffusion
method using a 1:1 protein/solution volume ratio and available commercial screens.
Crystals of MtbTMPK in complex with compound 26 were obtained in condition H2 of
commercial PEG/ION screen containing 0.05 M citric acid, 0.05 M BIS-TRIS propane pH 5.0
and 16% w/v polyethylene glycol 3,350. Crystals were cryoprotected in mother liquor
supplemented with ethylene glycol at a final concentration of 15%. Data collection on crystals of
MtbTMPK in complex with compound 26 was achieved at the Proxima1 beamline at the Soleil
synchrotron (Paris, France) at a wavelength of 0.97857 Å and a temperature of 100 K on a
Dectris Pilatus 6 M pixel detector.
Processing, indexing and scaling of the dataset was performed using XDS/ XSCALE [45],
followed by mtz conversion with XDSCONV.
The structure was solved by molecular replacement using the pdb of 5NQ5 as initial model
using PHASER (Table S2) [12]. The structure was further refined by one cycle of rigid-body
refinement in BUSTER (https://www.globalphasing.com/buster/) followed by positional and
individual isotropic B-factor refinement in BUSTER and PHENIX [46]. During the course of
refinement the model was manually improved in COOT [47]. NCS torsion restraints were used
during the refinement. The grade Web Server (http://grade.globalphasing.org) was used to
generate ligand coordinates as well as energy minimization and restraint generation.
Ligand molecules were modeled in sigma-weighted Fo-Fc difference electron density maps in
the course of the refinement. TLS refinement with TLS group definition defined by the TLSMD
server was applied in the latest stages of refinement.
The quality of the final crystal structures was assessed with MOLPROBITY [48] prior to
deposition in the PDB database (www.rcsb.org) with accession code 6YT1.
Enzymatic assay
After expression and purification of MtbTMPK as described by Munier-Lehmann et al. [44], the
enzymatic assay was conducted as previously reported [12]. Briefly, at fixed concentration of
ATP (0.5 mM) and dTMP (0.05 mM), compounds were evaluated at different concentrations
using the spectrophotometric assay described by Blondin et al [49]. The reaction medium
consists of 50 mM Tris-HCl pH 7.4, 50 mM KCl, 2 mM MgCl2, 0.2 mM NADH, 1 mM
15

phosphoenol pyruvate, and 2 units each of coupling enzymes (lactate dehydrogenase,
pyruvate kinase and nucleoside diphosphate kinase). IC50 value was calculated using Kaleida
Graph to plot and fit the experimental data points.
Computational studies.
AutoDock vina and AutodockTools-1.5.6. [50] were used for the molecular modeling, with
publicly available X-ray structure of the MtbTMPK (PDB entry 5NR7 [12]). After being
minimized the energy (minimum RMS gradient: 0.001), the PDB files of all ligands were
generated in ChemDraw 3D 16.0. The PDBQT files of the ligands and receptor were prepared
by AutodockTools-1.5.6, including atom types, atomic partial charges and the information on
the ligand torsional degrees. Centered on MtbTMPK active site PHE70 CE2 (the coordinates x,
y, z were -0.997, 26.240, -4.528 correspondingly), the prepared PDBQT files of ligands and
receptors were docked using a grid spacing of 0.375 and 60 x 60 x 60 number of grid points.
Through Lamarckian 4.2 method, each ligand was docked in autodock vina for 3 times, which
generated total 60 possible conformations. To analyze the results, Chimera was initially used
to view the binding energy, in combination with analysis and validation of possible interactions
in LigPlus.
In vitro antituberculosis activity
The MIC values of all compounds were determined as previously decribed [51,52]. In brief, M.
tuberculosis H37Rv (ATCC 27294) was grown to OD650nm 0.2 in the respective medium prior to
further 1000-fold dilution in fresh medium. Drugs were 2-fold serially diluted in duplicate in the
medium of choice (50 µL/well) in a concentration range spanning 100-0.049 µM in sterile
96-well U-bottom clear polysteyrene microtiter plates. Isoniazid and DMSO as postive and
negative controls, respectively. An equal volume (50 µL) of diluted cells was added to the
plates with the serial drug dilution. Plates were sealed in ziplock bags and incubated at 37 ℃.
After 7-14 days, plates were read with enlarging inverted mirror plate reader. The MIC was
recorded as the concentration that fully inhibited all visible growth.
In vitro cytotoxicity assay
The cytotoxicity of compounds on MRC-5 fibroblasts was performed exactly as previously
reported [12].
Stability assay
16

Preparation of pH 6.6 bicarbonate-formic acid buffer: 158.12 mg ammonium bicarbonate was
dissolved in 10 ml H2O to prepara 0.2 M ammonium bicarbonate. 0.2 M formic acid solution
was prepared by dissolving 150.8 ul formic acid in 20 ml H2O. To obtain ammonium pH 6.6
bicarbonate-formic acid buffer, 10 ml 0.2 M ammonium bicarbonate solution was titrated with
0.2 M formic acid solution under monitoration of pH meter. Preparation of sample: the tested
compound was solubilized in DMSO (Sigma - Aldrich) at stock concentration of 8 mM. Serial
dilutions were made in bicarbonate-formic acid buffer. Volumes of 20 μL of the serial dilutions
were injected into LC-MS system to build the standard curve. Volumes of 3 mL solutions at
concentration 80 μM were used as working solution. Volumes of 20 μL of the working solutions
were measured with LC-MC at time piont 0 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h and 24 h. Area of the
curve was collected and analysed by excel and MatLab.
Chemistry
Regeants were purchased and used without any further purification. All synthetic compounds
presented in this study were checked with precoated Alugram Silica TLC F254 plates
(Machhery-Nagel), visualized under UV light at 254 nm or stained by potassium permanganate,
1
and purified by column chromatography on a Reveleris X2 (Grace) automated flash unit. H
and 13C NMR spectral data were obtained on a Varian Mercury 300/75 MHz spectrometer at
300 K using TMS as an internal standard. Structural assignment was confirmed with the
assistance of 1H-1H COSY, 1H-13C HSQC and 1H-13C HMBC. High resolution mass
spectrometry (HRMS) was performed at 1ng/ml on a Waters LCT Premier XETM time of flight
(TOF) mass spectrometer equipped with a standard electrospray ionization (ESI) and modular
LockSprayTM interface, using 0.1% HCOOH in MeCN/H2O (1:1) as mobile phase. Purity of final
compounds was determined by LC-MS analysis on a Phenomenex Kinetex EVO C18 5 μm
100 mm × 2.1 mm column at flow rate of 1.4 mL/min (Waters Alliance 2695 XE separation
module), using HCOOH in H2O (0.1%, v/v)/ MeCN as gradient system.
General procedure 1: synthsis of 3-((benzyloxy)methyl)-5-methy-1-(1-(substituted
quinolin-2-ylmethyl)-4-yl)pyrimidine-2,4(1H,3H)-dione.
Method A To a solution of substituted quinoline buiding blocks (1.0 eq.) in dioxane was added
selenium dioxide (1.3 eq). The resulting mixture was refluxed overnight. Then, the reaction
mixture was cooled to room temperature and appropriate volume of EA was added for dilution,
17

followed by filtration through celite. The collected filtrate was extracted with water and the
organic phase was dried and concentrated in vacuo. The resulting residue and
3-((benzyloxy)methyl)-5-methyl-1-(piperidin-4-yl)pyrimidine-2,4(1H,3H)-dione [11] (5, 0.67 eq.)
were dissolved in 1,2-dichloroethane under N2 and the mixture was stirred for 0.5 h, followed
by addition of sodium triacetoxyborohydride (2.0 eq.). After stirring overnight, the reaction
mixture was diluted with CH2Cl2, washed with sat. NaHCO3 and brine, and dried over Na2SO4.
The combined organic layers were filtered, concentrated and purified by silica chromatography
to give the title compound.
Method B To a solution of substituted quinoline buiding blocks (1.0 eq.) in CCl4 were added
N-bromosuccinimide (0.9 eq.) and 2,2-azobisisobutyronitrile (0.2 eq.). The resulting mixture
was refluxed overnight. Then the reaction mixture was cooled to room temperature and filtered
to remove the solid. After being extracted with sat. NaHCO3 and brine, the organic layer was
dried and concentrated. The resulting residue, 5 (1.0 eq.) and K2CO3 (3 eq.) were dissolved in
ethanol and the mixture was refluxed for 3 h. The reaction mixture was cooled to room
temperature and filtered. The concentrated filtrate was purified by silica chromatography to
give the title compound.
General
quinolin-2-ylmethyl)piperidin-4-yl)pyrimidine-2,4(1H,3H)-dione
To solution of 3-((benzyloxy)methyl)-1-(1-((substituted
procedure
2:
synthesis
of
5-methyl-1-(1-(4-substituted
a
4-chloroquinolin-2-yl)methyl)piperidin-4-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (1.0 eq.) in
EA (2.0 mL) was added the phenol derivatives (1.5 eq.) and the resulting mixture was heated
at 180 ℃for 0.5 h. After cooled to room temperature, the reaction mixture was purified by silica
chromatography to give BOM-protected intermediate, which was heated at 73 ℃in TFA to yield
the title compound after purification.
3-((Benzyloxy)methyl)-1-(1-((4-chloroquinolin-2-yl)methyl)piperidin-4-yl)-5-methylpyrimidine-2,
4(1H,3H)-dione (3) Following method A in General procedure 1, 4-chloro-2-methyl quinoline
(0.20 g, 1.1 mmol) and selenium dioxide (0.16 g, 1.5 mmol) in dioxane (20 mL) afforded the
aldehyde intermediate, which was dissolved in 1,2-dichloroethane together with 5 (0.25 g, 0.75
mmol) synthesized according to previously reported procedure [11], followed by addition of
sodium triacrtoxyborohydride (0.48 g, 2.3 mmol) to yield 3 (eluent system: 55% EA in
18

petroleum ether, 0.18 g, 32%). 1H NMR (300 MHz, CDCl3) δ ppm 1.77 - 1.96 (m, 7 H, 5-CH3,
piperidin-3-yl, piperidin-5-yl), 2.27 - 2.40 (m, 2 H, piperidin-2a-yl, piperidin-6a-yl), 3.02 (d, J =
12.0 Hz, 2 H, piperidin-2b-yl, piperidin-6b-yl), 3.81 (s, 2 H, NpiperidylCH2), 4.47 - 4.62 (m, 1 H,
piperidin-4-yl), 4.70 (s, 2 H, (Ph)CH2), 5.50 (s, 2 H, N3CH2), 7.06 (d, J = 1.2 Hz, 1 H, H-6), 7.17
- 7.40 (m, 5 H, Ph, quinolin-3-yl), 7.60 (ddd, J = 8.3, 7.0, 1.2 Hz, 1 H, Ph), 7.68 - 7.80 (m, 2 H,
Ph), 8.06 (dd, J = 8.5, 0.6 Hz, 1 H, Ph), 8.19 (dd, J = 8.4, 1.3 Hz, 1 H, Ph). 13C NMR (75 MHz,
DMSO-d6) δ ppm 13.16 (1 C, 5-CH3), 30.72 (2 C, piperidin-3-yl, piperidin-5-yl), 52.96 (2 C,
piperidin-2-yl, piperidin-6-yl), 53.13 (1 C, piperidin-4-yl), 64.28 (1 C, NpiperidylCH2), 70.73 (1 C,
N3CH2), 72.12 (1 C, (Ph)CH2), 109.93 (1 C, C-5), 120.69 (1 C, quinolin-3-yl), 123.85 (1 C, Ph),
125.47 (1 C, Ph), 127.15 (1 C, Ph), 217.44 (1 C, Ph), 127.47 (2 C, Ph), 128.11 (2 C, Ph),
129.28 (1 C, Ph), 130.31 (1 C, Ph), 134.88 (1 C, C-6), 137.96 (1 C, Ph), 142.93 (1 C,
quinolin-4-yl), 148.33 (1 C, Ph), 151.42 (1 C, C-2), 159.37 (1 C, quibolin-2-yl), 163.12 (1 C,
C-4). HRMS (ESI): m/z [M + H]+ Calcd. for [C28H29ClN4O3 + H]+ 505.2001, found 505.1318.
5-Methyl-1-(1-((4-oxo-1,4-dihydroquinolin-2-yl)methyl)piperidin-4-yl)pyrimidine-2,4(1H,3H)-dio
ne (4) To a 25 mL flask were added 3 (0.10 g, 0.20 mmol) in acetic acid (10 mL) and the
mixture was heated at 125 ℃for 2 h. After total consumption of 3 checked with TLC, solvent
was removed in vacuo. The resulting residue was dissolved in TFA (5.0 mL) and heated at 73 ℃
for 4 h, followed by removing the volatile composition, adjusting the pH to 6-7 with sat.
NaHCO3 and the concentrated residue was purified with silica chromatography to give 4
1
(eluent system: 5% methanol in CH2Cl2, 42 mg, 58%). H NMR (300 MHz, DMSO-d6) δ ppm
1.61-1.72 (m, 2 H, piperidin-3a-yl, piperidin-5a-yl), 1.76 (s, 3 H, 5-CH3), 1.81 - 2.01 (m, 2 H,
piperidin-3b-yl, piperidin-5b-yl), 2.20 (t, J = 11.0 Hz, 2 H, piperidin-2a-yl, piperidin-6a-yl), 2.97
(d, J = 11.1 Hz, 2 H, piperidin-2b-yl, piperidin-6b-yl), 3.50 (s, 2 H, NpiperidylCH2), 4.20 - 4.33 (m,
1 H, piperidin-4-yl), 6.07 (s, 1 H, quinolin-3-yl), 7.26 (dt, J = 7.8, 4.1 Hz, 1 H, Ph), 7.50 - 7.67
(m, 3 H, H-6, Ph), 8.03 (d, J = 8.2 Hz, 1 H, Ph), 11.19 (br. s., 1 H, ), 11.41 (br. s., 1 H). 13C
NMR (75 MHz, DMSO-d6) δ ppm 11.74 (1 C, 5-CH3), 29.51 (2 C, piperidin-3-yl, piperidin-5-yl),
52.03 (1 C, piperidin-4-yl), 52.21 (2 C, piperidin-2-yl, piperidin-6-yl), 58.64 (1 C, NpiperidylCH2),
107.85 (1 C, quinolin-3-yl), 108.66 (1 C, C-5), 117.91 (1 C, Ph), 122.57 (1 C, Ph), 124.49 (1 C,
Ph), 124.70 (1 C, Ph), 131.24 (1 C, Ph), 137.33 (1 C, Ph), 139.85 (1 C, C-6), 149.57 (1 C,
19

quinolin-2-yl), 150.52 (1 C, C-2), 163.38 (1 C, C-4), 176.60 (1 C, quinolin-4-yl). HRMS (ESI):
m/z [M + H]+ Calcd. for [C20H22N4O3 + H]+ 367.1765, found 367.1714.
2-Hydroxy-4-((2-((4-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)piperidin-1-yl)methyl)qu
inolin-4-yl)oxy)benzoic acid (6) Following General procedure 2, 3 (0.20 g, 0.40 mmol) and
2,4-dihydroxybenzoic acid (92 mg, 0.60 mmol) in EA (2.0 mL) afforded the protected
intermediate, which was heated in TFA (5.0 mL) at 73 ℃for 4 h to yield 6 (eluent system: 10%
methanol in CH2Cl2, 76 mg, 38%). 1H NMR (300 MHz, DMSO-d6) δ ppm 1.76 (s, 3 H, 5-CH3),
1.80 - 1.97 (m, 2 H, piperidin-3a-yl, piperidin-5a-yl), 2.01 - 2.27 (m, 2 H, piperidin-3b-yl,
piperidin-5b-yl), 2.97 - 3.15 (m, 2 H, piperidin-2a-yl, piperidin-6a-yl), 3.43 (d, J = 11.4 Hz, 2 H,
piperidin-2b-yl, piperidin-6b-yl), 4.24 - 4.60 (m, 3 H, piperidin-4-yl, NpiperidylCH2), 6.62 - 6.76 (m,
2 H, H-6, Ph), 7.09 (s, 1 H, quinolin-3-yl), 7.46 (s, 1 H, Ph), 7.68 (t, J = 7.6 Hz, 1 H, Ph), 7.79 -
7.92 (m, 2 H, Ph), 8.07 (d, J = 8.5 Hz, 1 H, Ph), 8.24 (d, J = 7.9 Hz, 1 H, Ph), 11.26 (s, 1 H, NH).
13C NMR (75 MHz, DMSO-d6) δ ppm 12.14 (1 C, 5-CH3), 26.95 (2 C, piperidin-3-yl,
piperidin-5-yl), 50.09 (1 C, piperidin-4-yl), 51.52 (2 C, piperidin-2-yl, piperidin-2-yl), 59.81 (1 C,
NpiperidylCH2), 106.02 (1 C, quinolin-3-yl), 107.90 (1 C, Ph), 109.25 (1 C, Ph), 110.05 (1 C, C-5),
115.30 (1 C, Ph), 120.32 (1 C, Ph), 121.57 (1 C, Ph), 127.10 (1 C, Ph), 128.60 (1 C, Ph),
130.98 (1 C, Ph), 132.38 (1 C, Ph), 137.14 (1 C, C-6), 148.52 (1 C, Ph), 150.71 (1 C, C-2),
154.61 (1 C, quinolin-2-yl), 158.15 (t, J = 31.5 Hz, 1 C, Ph) , 160.64 (1 C, quinolin-4-yl), 163.61
(1 C, C-4), 163.77 (1 C, C-OH), 171.37 (1 C, COOH). HRMS (ESI): m/z [M + H]+ Calcd. for
[C27H26N4O6 + H]+ 503.1925, found 503.1951.
1-(1-((4-(4-Hydroxyphenoxy)quinolin-2-yl)methyl)piperidin-4-yl)-5-methylpyrimidine-2,4(1H,3H
)-dione (7) Following General procedure 2, 3 (0.40 g, 0.79 mmol) and hydroquinone (0.30 g,
2.7 mmol) in EA (2.0 mL) afforded the protected intermediate, which was heated in TFA (5.0
mL) at 73 ℃for 4 h to yield 7 (eluent system: 5% methanol in CH2Cl2, 0.17 g, 46%). 1H NMR
(300 MHz, DMSO-d6) δ ppm 1.46 - 1.86 (m, 7 H, 5-CH3, piperidin-3-yl, piperidin-5-yl), 2.08 (t, J
= 10.7 Hz, 2 H, piperidin-2a-yl, piperidin-6a-yl), 2.78 - 2.88 (m, 2 H, piperidin-2b-yl,
piperidin-6b-yl), 3.61 (s, 2 H, NpiperidylCH2), 4.14 - 4.26 (m, 1 H, piperidin-4-yl), 6.67 (s, 1 H,
quinolin-3-yl), 6.87 (d, J = 9.1 Hz, 2 H, Ph), 7.08 (d, J = 8.8 Hz, 2 H, Ph), 7.47 (s, 1 H, H-6),
7.53 - 7.61 (m, 1 H, Ph), 7.71 - 7.80 (m, 1 H, Ph), 7.93 (d, J = 8.5 Hz, 1 H, Ph), 8.23 (d, J = 7.9
Hz, 1 H, Ph), 9.66 (br.s., 1 H, OH), 11.12 (br.s., 1 H, NH). 13C NMR (75 MHz, DMSO-d6) δ ppm
20

11.82 (1 C, 5-CH3), 29.63 (2 C, piperidin-3-yl, piperidin-5-yl), 52.10 (1 C, piperidin-4-yl), 52.32
(2 C, piperidin-2-yl, piperidin-6-yl), 63.84 (1 C, NpiperidylCH2), 102.66 (1 C, quinolin-3-yl), 108.77
(1 C, C-5), 116.38 (2 C, Ph), 119.71 (1 C, Ph), 121.15 (1 C, Ph), 121.80 (2 C, Ph), 125.68 (1 C,
Ph), 128.15 (1 C, Ph), 130.00 (1 C, Ph), 137.25 (1 C, C-6), 145.56 (1 C, Ph), 148.23 (1 C, Ph),
150.56 (1 C, C-2), 154.87 (1 C, Ph), 160.38 (1 C, quinolin-2-yl), 161.90 (1 C, quinolin-4-yl),
163.48 (1 C, C-4). HRMS (ESI): m/z [M + H]+ Calcd. for [C26H26N4O4 + H]+ 459.2027, found
459.2043.
4-((2-((4-(5-Methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)piperidin-1-yl)methyl)quinolin-4-yl)
oxy)benzoic acid (8) Following General procedure 2, 3 (0.45 g, 0.89 mmol) and methyl
4-hydroxybenzoate (0.20 g, 1.3 mmol) in EA (2.0 mL) afforded the protected intermediate,
which was hydrolysis in 2 N aq. NaOH solution (20 mL) at 50 ℃for 4h, and acidified with 2 M
aq. HCl (25 mL) to give the acid intermediate. The resulting residue was heated in TFA (5.0 mL)
at 73 ℃for 4 h to yield 8 (eluent system: 10% methanol in CH2Cl2, 0.18 g, 41%). 1H NMR (300
MHz, DMSO-d6) δ ppm 1.77 (s, 3 H, 5-CH3), 1.87 - 1.95 (m, 2 H, piperidin-3a-yl,
piperidin-5a-yl), 2.23 - 2.32 (m, 2 H, piperidin-3b-yl, piperidin-5b-yl), 3.21 - 3.28 (m, 2 H,
piperidin-2a-yl, piperidin-6a-yl), 3.55 - 3.62 (m, 2 H, piperidin-2b-yl, piperidin-6b-yl), 4.49 - 4.66
(m, 3 H, NpiperidylCH2, piperidin-4-yl), 7.04 (s, 1 H, quinolin-3-yl), 7.43 (d, J = 8.8 Hz, 3 H, H-6,
Ph), 7.68 - 7.77 (m, 1 H, Ph), 7.85 - 7.96 (m, 1 H, Ph), 8.08 (d, J = 8.8 Hz, 3 H, Ph), 8.30 (d, J =
8.8 Hz ,1 H, Ph), 11.29 (s, 1 H, NH), H (COOH) could not be observed. 13C NMR (75 MHz,
DMSO-d6) δ ppm 12.22 (1 C, 5-CH3), 26.17 (2 C, piperidin-3-yl, piperidin-5-yl), 49.68 (1 C,
piperidin-4-yl), 51.26 (2 C, piperidin-2-yl, piperidin-6-yl), 59.75 (1 C, NpiperidylCH2), 106.25 (1 C,
quinolin-3-yl), 109.35 (1 C, C-5), 120.32 (1 C, Ph), 120.55 (3 C, Ph), 121.63 (1 C, Ph), 127.44
(1 C, Ph), 128.09 (1 C, Ph), 128.61 (1 C, Ph), 131.27 (1 C, Ph), 131.98 (2 C, Ph), 137.05 (1 C,
C-6), 150.68 (1 C, C-2), 157.55 (1 C, Ph), 160.60 (1 C, quinolin-4-yl), 163.60 (1 C, C-4),
166.56 (1 C, COOH), C (quinolin-2-yl) could not be observed. HRMS (ESI): m/z [M + H]+ Calcd.
for [C27H26N4O5 + H]+ 487.1976, found 487.2204.
1-(1-((4-(2-Hydroxyethoxy)quinolin-2-yl)methyl)piperidin-4-yl)-5-methylpyrimidine-2,4(1H,3H)-
dione (9) Following General procedure 2, 3 (0.20 g, 0.40 mmol) and ethylene glycol (37 mg,
0.60 mmol) in EA (2.0 mL) afforded the protected intermediate, which was heated in TFA (5.0
mL) at 73 ℃for 4 h to yield 9 (eluent system: 10% methanol in CH2Cl2, 66 mg, 41%). 1H NMR
21

(300 MHz, DMSO-d6) δ ppm 1.79 (s, 3 H, 5-CH3), 1.85 - 2.01 (m, 2 H, piperidin-3a-yl,
piperidin-5a-yl), 2.16 - 2.36 (m, 2 H, piperidin-3b-yl, piperidin-5b-yl), 3.18 - 3.30 (m, 2 H,
piperidin-2a-yl, piperidin-6a-yl), 3.5 - 3.67 (m, 2 H, piperidin-2b-yl, piperidin-6b-yl), 3.88 (t, J =
4.7 Hz, 2 H, OCH2), 4.29 (t, J = 4.7 Hz, 2 H, OCH2), 4.45 - 4.71 (m, 3 H, piperidin-4-yl,
NpiperidylCH2), 7.18 (s, 1 H, quinolin-3-yl), 7.46 (s, 1 H, H-6), 7.62 (ddd, J = 8.2, 7.0, 1.2 Hz, 1 H,
Ph), 7.81 (ddd, J = 8.4, 7.0, 1.3 Hz, 1 H, Ph), 8.00 (d, J = 8.2 Hz, 1 H, Ph), 8.26 (dd, J = 8.5,
0.9 Hz, 1 H, Ph), 11.30 (s, 1 H, NH). 13C NMR (75 MHz, DMSO-d6) δ ppm 12.67 (1 C, 5-CH3),
26.99 (2 C, piperidin-3-yl, piperidin-5-yl), 50.42 (1 C, piperidin-4-yl), 52.03 (2 C, piperidin-2-yl,
piperidin-6-yl), 59.65 (2 C, CH2OH, NpiperidylCH2), 71.25 (1 C, OCH2), 102.01 (1 C, quinolin-3-yl),
109.77 (1 C, C-5), 120.65 (1 C, Ph), 122.51 (1 C, Ph), 126.79 (1 C, Ph), 128.42 (1 C, Ph),
131.12 (1 C, Ph), 137.61 (1 C, C-6), 147.99 (1 C, Ph), 151.16 (1 C, C-2), 162.64 (1 C,
quinolin-4-yl), 164.08 (1 C, C-4), C (quinolin-2-yl) could not be observed. HRMS (ESI): m/z [M
+ H]+ Calcd. for [C22H26N4O4 + H]+ 411.2027, found 411.2037.
1-(1-((4-((2-Hydroxyethyl)amino)quinolin-2-yl)methyl)piperidin-4-yl)-5-methylpyrimidine-2,4(1H
,3H)-dione (10) Following General procedure 2, 3 (0.20 g, 0.40 mmol) and ethanolamine (36
mg, 0.60 mmol) in EA (2.0 mL) afforded the protected intermediate, which was heated in TFA
(5.0 mL) at 73 ℃for 4 h to yield 10 (eluent system: 10% methanol in CH2Cl2, 55 mg, 34%). 1H
NMR (300 MHz, DMSO-d6) δ ppm 1.57 - 1.83 (m, 5 H, 5-CH3, piperidin-3a-yl, piperidin-5a-yl),
1.87 - 2.06 (m, 2 H, piperidin-3b-yl, piperidin-5b-yl), 2.32 - 2.44 (m, 2 H, piperidin-2a-yl,
piperidin-6a-yl), 3.07 (d, J = 11.1 Hz, 2 H, piperidin-2b-yl, piperidin-6b-yl), 3.58 - 3.66 (m, 2 H,
NCH2), 3.71 (t, J = 5.6 Hz, 2 H, OCH2), 3.91 (br. s., 2 H, NpiperidylCH2), 4.23 - 4.41 (m, 1 H,
piperidin-4-yl), 6.93 (s, 1 H, quinolin-3-yl), 7.55 (s, 1 H, H-6), 7.61 - 7.73 (m, 1 H, Ph), 7.84 -
8.03 (m, 2 H, Ph), 8.48 (d, J = 8.5 Hz, 1 H, Ph), 9.17 (t, J = 6.2 Hz, 1 H, (Ph)NH), 11.22 (s, 1 H,
NH). 13C NMR (75 MHz, DMSO-d6) δ ppm 12.37 (1 C, 5-CH3), 29.60 (2 C, piperidin-3-yl,
piperidin-5-yl), 46.10 (1 C, NCH2), 52.32 (1 C, piperidin-4-yl), 52.59 (2 C, piperidin-2-yl,
piperidin-6-yl), 58.47 (1 C, NpiperidylCH2), 59.06 (1 C, CH2OH), 98.68 (1 C, quinolin-3-yl), 109.27
(1 C, C-5), 116.52 (1 C, Ph), 120.54 (1 C, Ph), 123.28 (1 C, Ph), 126.54 (1 C, Ph), 133.63 (1 C,
Ph), 137.79 (1 C, C-6), 138.55 (1 C, Ph), 151.07 (1 C, C-2), 156.04 (1 C, quinolin-4-yl), 158.44
(1 C, quinolin-2-yl), 163.93 (1 C, C-4). HRMS (ESI): m/z [M + H]+ Calcd. for [C22H27N5O3 + H]+
410.2187, found 410.2199.
22

3-((Benzyloxy)methyl)-1-(1-((8-hydroxyquinolin-2-yl)methyl)piperidin-4-yl)-5-methylpyrimidine-
2,4(1H,3H)-dione (12) Following method B in General procedure 1, 2-methylquinolin-8-yl
acetate (0.55 g, 2.7 mmol), N-bromosuccinimide (0.44 g, 2.5 mmol) and
2,2-azobisisobutyronitrile (90 mg, 0.55 mmol) were refluxed in CCl4 (25 mL) to give the bromo
substituted intermediate, which was further treated with 5 (0.90 g, 2.7 mmol) and K2CO3 (1.1 g,
8.2 mmol) in ethanol to afford 12 (eluent system: 55% EA in petroleum ether, 0.25 g, 20%). 1H
NMR (300 MHz, CDCl3) δ ppm 1.85 - 2.00 (m, 7 H, 5-CH3, piperidin-3-yl, piperidin-5-yl), 2.21 -
2.39 (m, 2 H, piperidin-2a-yl, piperidin-6a-yl), 3.06 (d, J = 11.7 Hz, 2 H, piperidin-2b-yl,
piperidin-6b-yl), 3.82 - 3.88 (m, 2 H, NpiperidylCH2), 4.48 - 4.64 (m, 1 H, piperidin-4-yl), 4.69 (s, 2
H, (Ph)CH2), 5.49 - 5.51 (m, 2 H, N3CH2), 7.09 (d, J = 0.9 Hz, 1 H, H-6), 7.15 (dd, J = 7.5, 1.3
Hz, 1 H, quinolin-3-yl), 7.19 - 7.32 (m, 4 H, Ph), 7.33 - 7.45 (m, 3 H, Ph), 7.52 (d, J = 8.2 Hz, 1
H, Ph), 8.11 (d, J = 8.5 Hz, 1 H, Ph). 13C NMR (75 MHz, CDCl3) δ ppm 13.15 (1 C, 5-CH3),
29.92 (2 C, piperidin-3-yl, piperidin-5-yl), 52.82 (2 C, piperidin-2-yl, piperidin-6-yl), 53.14 (1 C,
piperidin-4-yl), 63.53 (1 C, NpiperidylCH2), 70.80 (1 C, N3CH2), 72.16 (1 C, (Ph)CH2), 110.16 (1 C,
C-5), 110.38 (1 C, Ph), 117.50 (1 C, Ph), 121.47 (1 C, quinolin-3-yl), 127.51 (4 C, Ph), 127.62
(1 C, Ph), 128.15 (2 C, Ph), 134.43 (1 C, C-6), 135.01 (1 C, quinolin-4-yl), 136.70 (1 C, Ph),
137.97 (1 C, Ph), 151.46 (1 C, C-2), 152.15 (1 C, C-OH), 163.17 (1 C, C-4), 168.89 (1 C,
quinolin-2-yl). HRMS (ESI): m/z [M + H]+ Calcd. for [C28H30N4O4 + H]+ 487.2340, found
487.2344.
1-(1-((8-Hydroxyquinolin-2-yl)methyl)piperidin-4-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (13)
A solution of 12 (0.20 g, 0.41 mmol) in TFA (4.0 mL) was heated at 73 ℃for 4 h to yield 13
1
(eluent system: 5% methanol in CH2Cl2, 87 mg, 58%). H NMR (300 MHz, DMSO-d6) ppm δ
1.62 - 1.71 (m, 2 H, piperidin-3a-yl, piperidin-5a-yl), 1.76 (s, 3 H, 5-CH3), 1.82 - 1.99 (m, 2 H,
piperidin-3b-yl, piperidin-5b-yl), 2.14 - 2.27 (m, 2 H, piperidin-2a-yl, piperidin-6a-yl), 2.95 (d, J
= 12.0 Hz, 2 H, piperidin-2b-yl, piperidin-6b-yl), 3.81 (s, 2 H, NpiperidylCH2), 4.22 - 4.37 (m, 1 H,
piperidin-4-yl), 7.06 (dd, J = 6.7, 2.3 Hz, 1 H, Ph), 7.31 - 7.42 (m, 2 H, Ph), 7.61 - 7.69 (m, 2 H,
H-6, quinolin-3-yl), 8.27 (d, J = 8.8 Hz, 1 H, quinolin-4-yl), 9.48 (s, 1 H, OH), 11.18 (s, 1 H, NH).
13C NMR (75 MHz, DMSO-d6) δ ppm 12.00 (1 C, 5-CH3), 30.08 (2 C, piperidin-3-yl,
piperidin-5-yl), 52.27 (1 C, piperidin-4-yl), 52.75 (2 C, piperidin-2-yl, piperidin-6-yl), 63.86 (1 C,
NpiperidylCH2), 108.95 (1 C, C-5), 111.25 (1 C, Ph), 117.59 (1 C, Ph), 121.33 (1 C, quinolin-3-yl),
23

126.96 (1 C, Ph), 127.80 (1 C, Ph), 136.35 (1 C, Ph), 137.69 (1 C, C-6), 150.82 (1 C, C-2),
152.84 (1 C, C-OH), 157.55 (2 C, quinolin-2-yl, quinolin-4-yl), 163.67 (1 C, C-4). HRMS (ESI):
m/z [M + H]+ Calcd. for [C20H22N4O3 + H]+ 367.1765, found 367.1778.
[2,2'-Bipyridine]-6-carbaldehyde (15) According to a literature procedure [53], a solution of
6-bromo-2-pyridinecarboxaldehyde (0.20 g, 1.1 mmol), 2-(tributylstannyl)pyridine (0.37 g, 1.0
mmol) and tetrakis(triphenylphosphine)palladium (41 mg, 0.035 mmol) in degased tolune (20
mL) was refluxed overnight under nitrogen. The volatile in the resulting brown mixture was
removed in vacuo. The crude aldehyde was purified by column chromatography to afford 15
(eluent system: 25% EA in petroleum, 74 mg, 40%). 1H NMR (300 MHz, CDCl3) δ ppm 7.30 -
7.39 (m, 1 H, Py), 7.80 - 7.88 (m, 1 H, Py), 7.91 - 7.98 (m, 2 H, Py), 8.49 - 8.55 (m, 1 H, Py),
8.59 - 8.65 (m, 1 H, Py), 8.66 - 8.71 (m, 1 H, Py), 10.15 (s, 1 H, CHO). 13C NMR (75 MHz,
CDCl3) δ ppm 121.18 (1 C, Py), 121.30 (1 C, Py), 124.23 (1 C, Py), 125.05 (1 C, Py), 136.99 (1
C, Py), 137.83 (1 C, Py), 149.22 (1 C, Py), 152.20 (1 C, Py), 154.85 (1 C, Py), 156.55 (1 C, Py),
193.56 (1 C, CHO). HRMS (ESI): m/z [M + H]+ Calcd. for [C11H8N2O + H]+ 185.0710, found
185.0696.
[2,2'-Bipyridin]-6-ylmethanol (16) To a solution of 15 (0.10 g, 0.54 mmol) in 1,2-dichloroethane
was added sodium triacetylborohydride (0.23 g, 1.1 mmol) and the resulting mixture was
stirred at room temperature overnight. After completion of starting material checked with TLC,
the mixture was washed with sat. NaHCO3 (25 mL), brine (25 mL) and dried with Na2SO4. The
crude alcohol was concentrated and purified by column chromatography to afford pure 16
(eluent system: 50% EA in petroleum, 50 mg, 49%). 1H NMR (300 MHz, CDCl3) δ ppm 4.54 (br.
s., 1 H, OH), 4.78 (s, 2 H, CH2), 7.18 - 7.29 (m, 2 H, Py), 7.66 - 7.78 (m, 2 H, Py), 8.21 (d, J =
7.9 Hz, 1 H, Py), 8.27 - 8.34 (m, 1 H, Py), 8.57 - 8.65 (m, 1 H, Py). 13C NMR (75 MHz, CDCl3) δ
ppm 64.12 (1 C, CH2), 119.48 (1 C, Py), 120.32 (1 C, Py), 120.96 (1 C, Py), 123.65 (1 C, Py),
136.79 (1 C, Py), 137.45 (1 C, Py), 148.99 (1 C, Py), 154.62 (1 C, Py), 155.51 (1 C, Py),
158.65 (1 C, Py). HRMS (ESI): m/z [M + H]+ Calcd. for [C11H10N2O + H]+ 187.0866, found
187.0855.
1-(1-([2,2'-Bipyridin]-6-ylmethyl)piperidin-4-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (17) To a
solution of 16 (50 mg, 0.27 mmol) in CH2Cl2 were added methanesulfonyl chloride (25 μl, 0.32
mmol) and triethylamine (58 μl, 0.42 mmol) and the resulting solution was stirred at room
24

temperature for 1 h. The reaction mixture was washed with 1 M aq. HCl (25 mL), sat. NaHCO3
(25 mL) and brine (25 mL), and dried over Na2SO4. The concentrated residue, 5 (0.11 g, 0.34
mmol) and K2CO3 (63 mg, 0.46 mmol) were dissolved in ethanol (20 mL) and the solution was
refluxed for 2 h. The reaction mixture was concentrated in vacuo, and the residue was washed
with 1 M aq. HCl (25 mL), sat. NaHCO3 (25 mL) and brine (25 mL), and dried over Na2SO4.
After solvent evaporation in vacuo, the BOM protected intermediate was treated with TFA (4.0
mL) at 73 ℃for 4 h to yield 17 (eluent system: 7% methanol in CH2Cl2, 26 mg, 26%). 1H NMR
(300 MHz, DMSO-d6) δ ppm 1.60 - 1.71 (m, 2 H, piperidin-3a-yl, piperidin-5a-yl), 1.75 (s, 3 H,
5-CH3), 1.88 (qd, J = 12.1, 3.4 Hz, 2 H, piperidin-3b-yl, piperidin-5b-yl), 2.19 (t, J = 10.8 Hz, 2
H, piperidin-2a-yl, piperidin-6a-yl), 2.98 (d, J = 11.7 Hz, 2 H, piperidin-2b-yl, piperidin-6b-yl),
3.71 (s, 2 H, NpiperidylCH2), 4.20 - 4.34 (m, 1 H, piperidin-4-yl), 7.42 (ddd, J = 7.6, 4.7, 1.2 Hz, 1
H, Py), 7.50 (dd, J = 7.8, 1.0 Hz, 1 H, Py), 7.64 (d, J = 1.2 Hz, 1 H, H-6), 7.86 - 7.97 (m, 2 H,
Py), 8.25 (dd, J = 7.9, 0.9 Hz, 1 H, Py), 8.36 (dt, J = 8.0, 1.1 Hz, 1 H, Py), 8.62 - 8.68 (m, 1 H,
Py), 11.17 (br. s., 1 H, NH). 13C NMR (75 MHz, DMSO-d6) δ ppm 12.3 (1 C, 5-CH3), 30.3 (2 C,
piperidin-3-yl, piperidin-5-yl), 52.6 (1 C, piperidin-4-yl), 52.9 (2 C, piperidin-2-yl, piperidin-6-yl),
63.8 (1 C, NpiperidylCH2), 109.2 (1 C, C-5), 119.0 (1 C, Py), 120.7 (1 C, Py), 123.3 (1 C, Py),
124.4 (1 C, Py), 137.5 (1 C, Py), 137.9 (1 C, Py), 138.0 (1 C, C-6), 149.5 (1 C, Py), 151.1 (1 C,
C-2), 154.8 (1 C, Py), 155.5 (1 C, Py), 158.7 (1 C, Py), 164.0 (1 C, C-4). HRMS (ESI): m/z [M +
H]+ Calcd. for [C21H23N5O2 + H]+ 378.1925, found 378.1385.
2-((2-((4-(5-Methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)piperidin-1-yl)methyl)quinolin-4-yl)
oxy)ethyl 3,4-dihydroxybenzoate (18) 3,4-Bis(benzyloxy)benzoyl chloride was synthesized
according to the literature [54]. To
a solution of 9 (0.10 g, 0.24 mmol) and
4-methylatemorpholine (32 μl, 0.29 mmol) in CH2Cl2 (10 mL) was added
3,4-bis(benzyloxy)benzoyl chloride (90 mg, 0.26 mmol) and the resulting solution was stirred
for 1 h. The reaction mixture was quenched with water and washed with 1 M aq. HCl (25 mL),
sat. NaHCO3 (25 mL) and brine (25 mL), dried with Na2SO4, followed by concentration to give
the intermediate. The BOM protected intermediated and Pd/C were dissolved in ethanol (10
mL) and the mixture was stirred at room temperature under a hydrogen balloon overnight to
give 18 (eluent system: 7% methanol in CH2Cl2, 28 mg, 21%). 1H NMR (300 MHz,
METHANOL-d4) δ ppm 1.89 (s, 3 H, 5-CH3), 1.97 - 2.10 (m, 2 H, piperidin-3a-yl,
25

piperidin-5a-yl), 2.25 - 2.47 (m, 2 H, piperidin-3b-yl, piperidin-5b-yl), 3.11 (t, J = 11.9 Hz, 2 H,
piperidin-2a-yl, piperidin-6a-yl), 3.54 - 3.68 (m, 2 H, piperidin-2b-yl, piperidin-6b-yl), 4.43 (br. s.,
2 H, NpiperidylCH2), 4.53 - 4.64 (m, 1 H, piperidin-4-yl), 4.68 (br. s., 2 H, OCH2), 4.79 (s, 2 H,
OCH2), 6.76 (d, J = 8.2 Hz, 1 H, Ph), 7.19 (s, 1 H, quinolin-3-yl), 7.35 - 7.44 (m, 2 H, Ph), 7.49
(s, 1 H, H-6), 7.59 (t, J = 8.2 Hz, 1 H, Ph), 7.79 (t, J = 7.6 Hz, 1 H, Ph), 8.02 (d, J = 8.5 Hz, 1 H,
Ph), 8.24 (d, J = 8.2 Hz, 1 H, Ph). 13C NMR (75 MHz, DMSO-d6) δ ppm 12.49 (1 C, 5-CH3),
27.07 (2 C, piperidin-3-yl, piperidin-5-yl), 50.65 (1 C, piperidin-4-yl), 52.01 (2 C, piperidin-2-yl,
piperidin-6-yl), 62.45 (2 C, NpiperidylCH2, OCH2), 67.54 (1 C, OCH2), 101.99 (1 C, quinolin-3-yl),
109.62 (1 C, C-5), 115.64 (1 C, Ph), 116.66 (1 C, Ph), 120.34 (1 C, Ph), 120.54 (1 C, Ph),
121.88 (1 C, Ph), 122.23 (1 C, Ph), 126.81 (1 C, Ph), 128.50 (1 C, Ph), 130.92 (1 C, Ph),
137.53 (1 C, C-6), 145.42 (1 C, Ph), 147.98 (1 C, Ph), 150.97 (1 C, Ph), 151.05 (1 C, C-2),
161.84 (1 C, quinolin-4-yl), 163.98 (1 C, C-4), 165.96 (1 C, CO(Ph)), C(quinolin-2-yl) could not
be observed. HRMS (ESI): m/z [M + H]+ Calcd. for [C29H30N4O7 + H]+ 547.2187, found
547.2191.
2-((2-((4-(5-Methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)piperidin-1-yl)methyl)quinolin-4-yl)
amino)ethyl 3,4-dihydroxybenzoate (19) Following the same procedure as decribed for 18,
using 10 (0.10 g, 0.24 mmol), 4-methylatemorpholine (32 μl, 0.29 mmol) and
3,4-bis(benzyloxy)benzoyl chloride (90 mg, 0.26 mmol) as material yielded the intermediate.
The BOM protected intermediated and Pd/C were dissolved in ethanol (10 mL) and the
resulting solution was stirred at room temperature under a hydrogen balloon overnight to give
19 (eluent system: 7% methanol in CH2Cl2, 29 mg, 21%). 1H NMR (300 MHz, DMSO-d6) δ ppm
1.59 - 1.70 (m, 2 H, piperidin-3a-yl, piperidin-5a-yl), 1.74 (s, 3 H, 5-CH3), 1.82 - 2.01 (m, 2 H,
piperidin-3b-yl, piperidin-5b-yl), 2.16 - 2.30 (m, 2 H, piperidin-2a-yl, piperidin-6a-yl), 2.97 (d, J
= 11.4 Hz, 2 H, piperidin-2b-yl, piperidin-6b-yl), 3.69 (br. s., 2 H, NpiperidylCH2), 3.74 - 3.86 (m, 2
H, NCH2), 4.20 - 4.34 (m, 1 H, piperidin-4-yl), 4.47 (t, J = 5.0 Hz, 2 H, OCH2), 6.76 (d, J = 8.2
Hz, 1 H, Ph), 6.84 (s, 1 H, quinolin-3-yl), 7.22 - 7.35 (m, 2 H, Ph), 7.47 - 7.59 (m, 2 H, H-6, Ph),
7.72 (t, J = 6.7 Hz, 1 H, Ph), 7.88 (d, J = 8.8 Hz, 1 H, Ph), 8.31 (d, J = 7.9 Hz, 1 H，Ph), 9.32 (s,
1 H, NH), 9.85 (s, 2 H, OH), 11.20 (s, 1 H, NH). 13C NMR (75 MHz, DMSO-d6) δ ppm 12.38 (1
C, 5-CH3), 29.54 (2 C, piperidin-3-yl, piperidin-5-yl), 42.47 (1 C, NCH2), 52.23 (1 C,
piperidin-4-yl), 52.62 (2 C, piperidin-2-yl, piperidin-6-yl), 58.55 (1 C, NpiperidylCH2), 62.15 (1 C,
26

OCH2), 98.52 (1 C, quinolin-3-yl), 109.30 (1 C, C-5), 115.47 (1 C, Ph), 116.58 (1 C, Ph), 116.73
(1 C, Ph), 120.47 (1 C, Ph), 120.92 (1 C, Ph), 122.22 (1 C, Ph), 123.09 (1 C, Ph), 126.70 (1 C,
Ph), 133.60 (1 C, Ph), 137.68 (1 C, C-6), 138.82 (1 C, Ph), 145.33 (1 C, Ph), 150.90 (1 C, Ph),
151.05 (1 C, C-2), 155.84 (1 C, quinolin-4-yl), 163.93 (1 C, C-4), 165.95 (1 C, COPh), C
(quinolin-2-yl) could not be observed. HRMS (ESI): m/z [M + H]+ Calcd. for [C29H31N5O6 + H]+
546.2347, found 546.2248.
4-((2-((4-(5-Methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)piperidin-1-yl)methyl)quinolin-4-yl)
oxy)phenyl 3,4-dihydroxybenzoate (20) 3,4-Bis((tert-butyldimethylsilyl)oxy)benzoic acid was
synthesized accoding to the literature [55]. To a solution of 7 (0.24 g, 0.52 mmol) and
3,4-bis((tert-butyldimethylsilyl)oxy)benzoic acid (0.21 g, 0.55 mmol) in CH2Cl2 (50 mL) were
added EDC.HCl (0.20 g, 1.0 mmol) and DMAP (6.4 mg, 0.052 mmol) and the resulting solution
was stirred at room temperature overnight to yield the imtermediate (0.14 g, 0.17 mmol), which
was treated with HF.Pyridine (0.33 mL, 0.96 mL/mmol TBDMS) in pyridine (0.33 mL, 0.96
mL/mmol TBDMS)/THF (0.68 mL, 2.0 mL/mmol TBDMS) to give 20 (eluent system: 5%
methanol in CH2Cl2, 48 mg, 16%). 1H NMR (300 MHz, DMSO-d6) δ ppm 1.55 - 1.81 (m, 7 H,
5-CH3, piperidin-3-yl, piperidin-5-yl), 2.07 - 2.19 (m, 2 H, piperidin-2a-yl, piperidin-6a-yl), 2.83 -
2.93 (m, 2 H, piperidin-2b-yl, piperidin-6b-yl), 3.68 (s, 2 H, NpiperidylCH2), 4.18 - 4.31 (m, 1 H,
piperidin-4-yl), 6.84 (s, 1 H, quinolin-3-yl), 6.86 - 6.91 (m, 1 H, Ph), 7.32 - 7.43 (m, 4 H, Ph),
7.45 - 7.53 (m, 3 H, H-6, Ph), 7.61 (ddd, J = 8.2, 6.9, 1.0 Hz, 1 H, Ph), 7.79 (ddd, J = 8.3, 6.9,
1.5 Hz, 1 H, Ph), 7.98 (d, J = 8.2 Hz, 1 H, Ph), 8.26 (dd, J = 8.2, 0.9 Hz, 1 H, Ph), 9.75 (s, 2 H,
OH), 11.17 (br. s., 1 H, NH). 13C NMR (75 MHz, DMSO-d6) δ ppm 12.03 (1 C, 5-CH3), 29.97 (2
C, piperidin-3-yl, piperidin-5-yl), 52.15 (1 C, piperidin-4-yl), 52.56 (2 C, piperidin-2-yl,
piperidin-6-yl), 63.93 (1 C, NpiperidylCH2), 103.88 (1 C, quinolin-3-yl), 108.93 (1 C, C-5), 115.51
(1 C, Ph), 116.78 (1 C, Ph), 119.28 (1 C, Ph), 120.06 (1 C, Ph), 121.38 (1 C, Ph), 121.65 (2 C,
Ph), 122.75 (1 C, Ph), 124.00 (2 C, Ph), 126.11 (1 C, Ph), 128.50 (1 C, Ph), 130.35 (1 C, Ph),
137.36 (1 C, C-6), 145.34 (1 C, Ph), 147.95 (1 C, Ph), 148.62 (1 C, Ph), 150.79 (1 C, Ph),
151.40 (1 C, C-2), 151.57 (1 C, Ph), 160.88 (1 C, quinolin-2-yl), 161.25 (1 C, quinolin-4-yl),
163.63 (1 C, COPh), 164.54 (1 C, C-4). HRMS (ESI): m/z [M + H]+ Calcd. for [C33H30N4O7 + H]+
595.2187, found 595.2189.
27

N-hydroxy-4-((2-((4-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)piperidin-1-yl)methyl)qu
inolin-4-yl)oxy)benzamide (21) To solution of (0.10 g, 0.21 mmol) and
a
8
O-benzylhydroxylamine hydrochloride (34 mg, 0.21 mmol) in CH2Cl2 (50 mL) were added
EDC.HCl (79 mg, 0.41 mmol) and DMAP (2.5 mg, 0.020 mmol) and the resulting solution was
stirred at room temperature overnight to yield the intermediate (60 mg, 0.10 mmol). The
intermediate and Pd/C were dissolved in ethanol and the mixture was stirred under hydrogen
to give 21 (eluent system: 8% methanol in CH2Cl2, 21 mg, 20%). 1H NMR (300 MHz, DMSO-d6)
δ ppm 1.47 - 1.92 (m, 7 H, 5-CH3, piperidin-3-yl, piperidin-5-yl), 2.01 - 2.21 (m, 2 H,
piperidin-2a-yl, piperidin-6a-yl), 2.89 (d, J = 10.8 Hz, 2 H, piperidin-2b-yl, piperidin-6b-yl), 3.70
(s, 2 H, NpiperidylCH2), 4.16 - 4.30 (m, 1 H, piperidin-4-yl), 6.91 (s, 1 H, quinolin-3-yl), 7.27 - 7.37
(m, 2 H, Ph), 7.54 (s, 1 H, H-6), 7.61 (ddd, J = 8.2, 6.9, 1.0 Hz, 1 H, Ph), 7.80 (ddd, J = 8.4, 6.9,
1.5 Hz, 1 H, Ph), 7.86 - 7.92 (m, 2 H, Ph), 8.00 (d, J = 7.9 Hz, 1 H, Ph), 8.17 (d, J = 8.2 Hz, 1 H,
Ph), 9.06 (s, 1 H, NHOH), 11.16 (s, 1 H, NH), 11.28 (br. s., 1 H, NHOH). 13C NMR (75 MHz,
DMSO-d6) δ ppm 11.97 (1 C, 5-CH3), 29.85 (2 C, piperidin-3-yl, piperidin-5-yl), 52.26 (1 C,
piperidin-4-yl), 52.59 (2 C, piperidin-2-yl, piperidin-6-yl), 63.99 (1 C, NpiperidylCH2), 105.47 (1 C,
quinolin-3-yl), 108.95 (1 C, C-5), 115.04 (1 C, Ph), 119.71 (2 C, Ph), 120.29 (1 C, Ph), 121.33
(1 C, Ph), 126.32 (1 C, Ph), 128.61 (1 C, Ph), 129.37 (1 C, Ph), 129.50 (1 C, Ph), 130.44 (1 C,
Ph), 137.56 (1 C, C-6), 148.73 (1 C, Ph), 150.78 (1 C, C-2), 156.93 (1 C, Ph), 160.13 (1 C,
CONH), 160.87 (1 C, quinolin-2-yl), 163.64 (1 C, C-4), C (quinolin-4-yl) could not be observed.
HRMS (ESI): m/z [M + H]+ Calcd. for [C27H27N5O5 + H]+ 502.2085, found 502.2238.
Methyl
(E)-2-(2-hydroxy-4-((2-((4-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)piperidin-1-yl)met
hyl)quinolin-4-yl)oxy)benzylidene)hydrazine-1-carbodithioate
(22)
Following
General
procedure 2, 3 (0.14 g, 0.28 mmol) and 2,4-dihydroxybenzaldehyde (57 mg, 0.42 mmol) in EA
(2.0 mL) afforded the protected intermediate. The intermediate (0.10 g, 0.16 mmol) was
heated in TFA (2.0 mL) at 73 ℃ for 4 h, followed by concentration in vacuo to give the
deprotected compound, which was further refluxed with methyl hydrazinecarbodithioate (5.0
mg, 0.041 mmol, synthesized according to literature [42]) in methanol (20 mL) to yield 22
1
(eluent system: 5% methanol in CH2Cl2, 9.4 mg, 6%). H NMR (300 MHz, DMSO-d6) δ ppm
1.68 - 1.94 (m, 5 H, 5-CH3, piperidin-3a-yl, piperidin-5a-yl), 2.03 - 2.25 (m, 2 H, piperidin-3b-yl,
28