Structure Guided Lead Generation toward Nonchiral M. tuberculosis Thymidylate Kinase Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.7b01570

In recent years, thymidylate kinase (TMPK), an enzyme indispensable for bacterial DNA biosynthesis, has been pursued for the development of new antibacterial agents including against Mycobacterium tuberculosis, the causative agent for the widespread infectious disease tuberculosis (TB). In response to a growing need for more effective anti-TB drugs, we have built upon our previous efforts toward the exploration of novel and potent Mycobacterium tuberculosis TMPK (MtTMPK) inhibitors, and reported here the design of a novel series of non-nucleoside inhibitors of MtTMPK. The inhibitors display hitherto unexplored interactions in the active site of MtTMPK, offering new insights into structure-activity relationships. To investigate the discrepancy between enzyme inhibitory activity and the whole-cell activity, experiments with efflux pump inhibitors and efflux pump knockout mutants were performed. The minimum inhibitory concentrations of particular inhibitors increased significantly when determined for the efflux pump mmr knockout mutant, which partly explains the observed dissonance.

Full text of DOI:10.1021/acs.jmedchem.7b01570

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Structure Guided Lead Generation towards non-
chiral M. tuberculosis Thymidylate Kinase Inhibitors
Lijun Song, Romain Merceron, Begoña Gracia, Ainhoa Lucia Quintana, Martijn D.P. Risseeuw, Fabian
Hulpia, Paul Cos, Jose A. Ainsa, Hélène Munier-Lehmann, Savvas N. Savvides, and Serge Van Calenbergh
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 06 Mar 2018
Downloaded from http://pubs.acs.org on March 7, 2018
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Structure Guided Lead Generation towards Nonꢀ
chiral M. tuberculosis Thymidylate Kinase Inhibitors
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1
Lijun Song, Romain Merceron, Begoña Gracia, Ainhoa Lucía, Martijn D.P. Risseeuw, Fabian
1
5
4
6,7,#
2,3,#
Hulpia, Paul Cos, José A. Aínsa, Hélène Munier-Lehmann,
Savvas N. Savvides,
Serge Van
1,*
Calenbergh
1
Laboratory for Medicinal Chemistry (FFW), Ghent University, Ottergemsesteenweg 460, Bꢀ9000
Gent, Belgium;
2
3
VIBꢀUGent Center for Inflammation Research, Zwijnaarde, Ghent 9052, Belgium;
Laboratory for Protein Biochemistry and Biomolecular Engineering, Department of Biochemistry and
Microbiology, Ghent University, Ghent 9000, Belgium;
4
5
6
Grupo de Genética de Micobacterias, Departamento de Microbiología, Facultad de Medicina, and
BIFI, Universidad de Zaragoza, Zaragoza, Spain, and CIBER Enfermedades Respiratorias
(
CIBERES), Instituto de Salud Carlos III, Spain.
Laboratory for Microbiology, Parasitology and Hygiene (LMPH), Department of Pharmaceutical
Sciences, University of Antwerp, Campus Drie Eiken, Universiteitsplein 1, Bꢀ2610 Antwerpen,.
Belgium
Institut Pasteur, Unit of Chemistry and Biocatalysis, Department of Structural Biology and Chemistry,
2
8 Rue du Dr. Roux, 75724 Paris Cedex 15, France
7
#
CNRS UMR3523, Paris, France
Both authors equally contributed to the work.
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ABSTRACT
In recent years thymidylate kinase (TMPK), an enzyme indispensable for bacterial DNA biosynthesis, has
been pursued for the development of new antibacterial agents including against Mycobacterium
tuberculosis, the causative agent for the widespread infectious disease tuberculosis (TB). In response to a
growing need for more effective antiꢀTB drugs we have built upon our previous efforts towards the
exploration of novel and potent Mycobacterium tuberculosis TMPK (MtTMPK) inhibitors, and report here
the design of a novel series of nonꢀnucleoside inhibitors of MtTMPK. The inhibitors display hitherto
unexplored interactions in the active site of MtTMPK offering new insights into structureꢀactivity
relationships. To investigate the discrepancy between enzyme inhibitory activity and the wholeꢀcell
activity, experiments with efflux pump inhibitors and effluxꢀpump knockout mutants were performed. The
minimum inhibitory concentrations of particular inhibitors increased significantly when determined for the
efflux pump mmr knockout mutant, which partly explains the observed dissonance.
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INTRODUCTION
Tuberculosis (TB) has been ranked as the most fatal infectious disease worldwide and constitutes the
1
leading cause of death among people with HIV/AIDS. As one of the most ancient human infectious
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diseases, it revives with immigration, increased homelessness and the emergence of HIV/AIDS. It
declines with improved public health practices, the development of and access to antituberculosis drugs
2
ꢀ4
and antibiotics, and immunization with M. bovis BCG vaccine. At the early stage of TB infection,
people with low immunity directly develop active disease, which is called primary or primaryꢀprogressive
TB. Most people, however, progress into asymptomatic disease, which is referred to as latent
5
tuberculosis. Based on the tuberculin skin test reactivity, it is estimated that one third of the world
population is infected with latent TB, which may be activated upon aging, malnutrition, treatment with
1
immunosuppressive agents, HIV coꢀinfection, etc. It is estimated that 10.4 million new TB cases occurred
1
in 2015. SouthꢀEast Asian and Western Pacific regions accounted for 58% of these cases, Africa for 28%,
1
while about 3% of the cases were allocated in highꢀincome countries. The currently used regime for drugꢀ
susceptible TB consists of four firstꢀline drugs to be taken almost daily for six months or longer: isoniazid,
rifampicin, ethambutol and pyrazinamide. This lengthy treatment period with associated risk of poor
patient compliance, increases the risk of drugꢀsusceptible TB to develop into multidrugꢀresistant TB
6, 7
(
MDRꢀTB) and extensively drugꢀresistant TB (XDRꢀTB).
Treatment of MDRꢀTB and XDRꢀTB is
much longer, more expensive, less effective and causes more severe side effects than that for drugꢀ
susceptible TB. Although the new antiꢀTB drugs bedaquiline and delamanid have been recommended for
8, 9
the treatment of MDRꢀTB by WHO, resistant isolates were reported as early as 2014. Therefore, there is
a pressing need to develop novel antiꢀTB drugs that allow shortening of the treatment period. Drugs
exerting their effect via a novel mechanisms of action are preferred, as they are believed to be less
susceptible to generate crossꢀresistance with already existing antiꢀtuberculosis drugs, and hence, could be
10
useful for treating MDRꢀTB and XDRꢀTB.
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Human TB is caused by Mycobacterium tuberculosis (Mtb). The intrinsic resistance of Mtb to common
antibiotics is due to the combination of several factors, including a highly lipidꢀrich cell wall that forms a
11ꢀ14
permeability barrier, and the activity of efflux pumps that transport noxious agents out of the cell.
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Moreover, Mtb manages to survive inside macrophages (the firstꢀline bodyguard of the human immune
system), and hides itself by building granulomas inside the deep lung tissue (latent TB). Once the host
immune system is weakened, the bacterium starts replicating and invading the neighboring tissue of the
11, 15, 16
lung.
The granuloma creates heterogeneous microꢀenvironments including different blood supply,
1
7, 18
diverse pH and various extent of caseum.
As a result, it is challenging for antiꢀTB drugs to reach and
1
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kill all bacilli.
Mtb thymidylate kinase (MtTMPK), a key enzyme for the synthesis of thymidine triphosphate as a DNA
20
building block, is essential for the survival of Mtb. Its known crystal structure and druggable active site
21ꢀ23
make it an attractive target for drug design.
In this paper we describe our attempts to optimize the
MtTMPK inhibitory activity of the earlier identified nonꢀnucleoside hit 1 via scaffold screening, ligand
24
efficiency exploration and structureꢀbased drug design.
The envisioned modifications were inspired by the cocrystal structure of MtTMPK in complex with 1 (cf.
infra), indicating that the thymine ring (ring A) forms a primary binding motif with the enzyme that is
20
very similar to that observed in the crystal structures of substrateꢀlike ligands, including a π−π stacking
4
3
interaction with Phe70 and hydrogen bonds between O and N and Arg74 and Asn100, respectively. Ring
B, C and D, however, are mainly forming hydrophobic interactions with the enzyme. Therefore, we
decided to investigate if the metaꢀpiperidine ring (ring B) could be substituted by either a paraꢀpiperidine
(
2), an azetidine (7), or an aminoalkyl moiety (11ꢀ12). We also explored variations in the interꢀring space
by adding a methylene unit (analogues 3, 8 and 9) or a carbonyl group (analogue 13) between ring B and
C or a methylene group between A and B (analogue 5). We also investigated the effect of moving the
terminal phenoxy ring from the meta to the para position (as in 4, 6, 10 and 14). Next, we tried to optimize
the ligand efficiency by removing the distal Dꢀring, while replacing ring C with different hetero(aryl)
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moieties or a cyclohexyl group (analogues 15ꢀ41). Quinolinꢀ2ꢀyle analogue 35, which resulted from this
effort, was further optimized to afford a potent MtTMPK inhibitor with a new chemotype (analogues 42ꢀ
4
4).
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RESULTS AND DISCUSSION
Synthesis. Generally, the synthesis of the envisioned analogues comprised reductive amination of an
azaheterocyclic or aminoalkyl N3ꢀBOMꢀprotected thymine derivative with an appropriate aldehyde. Final
acidic cleavage of the BOMꢀgroup afforded the desired final compounds. The azaheterocyclic (substituted
azetidine or piperidine) and aminoalkyl thymine intermediates were prepared via alkylation of thymine
2
4
with the appropriate mesylates.
When the required aldehydes were not commercially available, they were prepared as described in
Scheme 1. The preparation of aldehyde 50 started with Fischer esterification of 3ꢀhydroxyphenylacetic
acid 45. ChanꢀLam coupling with phenylboronic acid and reduction of the resulting product 49 with
LiAlH and subsequent reꢀoxidation with DessꢀMartin furnished the desired aldehyde 50. Esterification of
4
mꢀcoumaric acid 47, followed by catalytic hydrogenation yielded propionic ester 48, which was converted
to the required aldehyde 52 via an analogues method. The synthesis of aldehyde 55 was first attempted via
ChanꢀLam and Ullmann coupling using 2ꢀmethylquinolinꢀ4ꢀol, with either phenylboronic acid or
iodobenzene but both reactions failed to give the desired product. The failure in the Ullmann coupling
might be due to the poor solubility of 2ꢀmethylquinolinꢀ4ꢀol, as well as the low nucleophilic character of
the quinolone compared to a phenol. However, Ullmann coupling between 4ꢀchloroꢀ2ꢀmethylquinoline 53
and phenol generated the substituted phenoxyquinoline 54, which upon oxidation with selenium dioxide
afforded aldehyde 55. Aldehydes 57 and 60 were synthesized using similar conditions.
Scheme 1. Synthesis of the required aldehyde building blocks
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Reagents and conditions: (a) MeOH, cat. H SO , 0 °C to reflux, overnight; (b) i) MeOH, cat. H SO , 0 °C
2
4
2
4
to reflux, overnight; ii) H , Pd/C, AcOEt, rt, 2 h; (c) Cu(OAc) , molecular sieves, pyridine, rt, overnight;
2
2
(
d) i) LiAlH , dry THF, 0 °C to rt, 20 min; ii) DessꢀMartin reagent, CH Cl , 0 °C to rt, 8 h; (e) Cs CO ,
4 2 2 2 3
N,Nꢀdimethylglycine•HCl, CuI, dry 1,4ꢀdioxane, 90 °C, 24 h (f) SeO , 1,4ꢀdioxane, 100 °C, 1.5 h ꢀ 2 h.
2
24
Compounds 2ꢀ12 were synthesized as depicted in Scheme 2. The final TFA catalyzed deprotection step
was found to produce the desired products in varying yields. Purification of the final compounds was
complicated by the presence of undesired side products with a molecular mass of 2M + 12 (M represents
the exact mass of the deprotected substrates). Attempts to optimize this deprotection step by lowering the
temperature or diminishing the TFA concentration failed. 2DꢀNMR analysis allowed to identify the main
side product as dimer 73, which is probably formed by a double electrophilic aromatic substitution
25
reaction of formaldehyde released during the deprotection reaction. To circumvent this problem, we
26
explored the use of cysteine as a formaldehyde scavenger. By using a 0.25 M solution of Lꢀcysteine.HCl
in 80% TFA/H O, little to no byꢀproduct formation was observed.
2
Scheme 2. Synthesis of azetidinylꢀ, paraꢀpiperidinylꢀ, aminoalkylꢀanalogues and the structure of
compound 73.
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BOM
H
HN
BocN
O
N
O
O
N
O
a
b
R
nN
HN
OMs
m
+
O
N
O
N
N
m
BOM
m
6
6
1: m = 0
4: m = 1
62
63: m = 0
65: m = 1
2: R= R1, m = 0, n = 1
3: R= R1, m = 0, n = 2
: R= R2, m = 0, n = 1
: R= R1, m = 1, n = 1
1
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6: R= R2, m = 1, n = 1
HN
R
a
b
BocN
OMs
+
HN
N
O
O
N
N
O
O
O
N
O
m
N
NH
BOM
BOM
66
62
67
7: R= R1, m = 1
: R= R1, m = 2
: R= R1, m = 3
8
9
10: R= R2, m = 1
BOM
N
H
O
N
O
Boc
O
O
H
m
c
a
b
m
N
N
OH
N
OMs
N
R1
N
m
N
m
H
68: m = 1
6
7
9: m = 1
1: m = 2
11: m = 1
12: m = 2
70: m = 2
BOM
N
H
H
O
N
O
O
N
O
O
N
O
b, ii
N
O
O
N
method 1
N
N
R2
N
72
73
O
O
R1 =
R2 =
Reagents and conditions: (a) i) K CO , dry DMF, 80 °C, 3 h (71), overnight (65), 48 h (67 and 69), 4 days
2
3
(
63); ii) 10% TFA/CH Cl , 2–4 h, rt; (b) i) substituted aromatic aldehydes, NaBH(OAc) , dry 1,2ꢀ
2 2 3
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dichloroethane, rt, overnight; ii) method 1: TFA, 72 °C, 30 min–1 h; method 2: 80% TFA/H O, 0.25–0.27
2
M cysteine, 72 °C, 3–12 h; (c) i) (Boc) O, Et N, CH Cl , rt, 2 h; ii) MsCl, Et N, dry dichloromethane, 0 °C,
2
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h.
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The method to access amides 13 and 14 was similar as described earlier for the metaꢀanalogues (Scheme
24
3
).
Scheme 3: Synthesis of amideꢀcontaining paraꢀpiperidinylthymine analogues
Reagents and Conditions: (a) i) EDC, 4ꢀDMAP, dry CH Cl , overnight; ii) H , Pd/C, EtOH, 6 h, rt. iii)
2
2
2
THF/H O, rt., 4 h.
2
Enzymatic evaluation and structural analysis.
At the onset of this study we sought to obtain structural information about the possible binding mode of
inhibitor 1 into MtTMPK in order to fuel further inhibitor development. To this end, we determined the
cocrystal structure of MtTMPK in complex with 1 at 2.85 Å resolution (PDB code: 5NQ5), which reveals
the key interaction features. Consistent with its competitive mode of inhibition against the natural
substrate dTMP, compound 1 is bound with high occupancy in the active site of MtTMPK (Figure 1).
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Figure 1: Structural characterization of the binding mode of compound 1 and overlay of compound 1
binding pose with dTMP in the active site of MtTMPK. (A) Cocrystal structure of compound 1 bound to
MtTMPK at 2.85 Å resolution. Protein is shown in pale yellow cartoon representation. Compound 1 (stick
representation, carbon atoms in white) and the side chains of MtTMPK interacting residues (stick
representation, carbon atoms in orange) are depicted. The corresponding unbiased FoꢀFc difference
electron density (contoured to +3 sigma) calculated before adding the ligand in the refinement process is
shown as a green mesh. (B) Schematic drawing of compound 1 binding site displaying key MtTMPK
interacting residues (hydrogen bonds are represented with a red dashed line). Compound 1 comprises four
rings named A to D. (C) Cocrystal structure of MtTMPK in complex with compound 1 showing the
binding of the inhibitor (stick representation, carbon atoms in white) in the MtTMPK active site. Protein is
depicted in pale yellow cartoon representation. Crystallographic pose of dTMP (stick representation,
carbon atoms in cyan) in MtTMPK (PDB code 1G3U) has been overlaid with compound 1 cocrystal
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structure for comparison. The superimposition shows a strict conservation of the thymidine core position.
(
D) Schematic drawing of dTMP binding site displaying key MtTMPK interacting residues (hydrogen
bonds are represented with a red dashed line).
0
1
2
3
4
5
6
7
8
9
0
1
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9
0
Compound 1 adopts a surprising bent conformation that places rings A and B into the active site pocket
and rings C and D protruding outwards at a right angle. The thymidine ring (ring A) reaches deep into the
catalytic pocket where it is stabilized via hydrophobic interactions between its 5ꢀmethyl group and Arg95,
20
Pro37 and Phe36 side chains. By analogy to the interactions of thymidineꢀlike TMPK inhibitors,
compound 1 also displays πꢀπ electron stacking interactions between ring A and Phe70, and additional
4
3
anchoring via hydrogen bonds linking the O and N groups of the thymine ring with Arg74 and Asn100
respectively.
The metaꢀpiperidine ring (ring B), adopting a chair conformation, protrudes out of the active site and is
located in a similar position as the deoxyribose ring of dTMP establishing a πꢀalkyl interaction with the
aromatic side chain of Tyr103. Of interest, the phenyl ring (ring C) unexpectedly bends away from the
substrate channel and binds into a newly formed hydrophobic pocket establishing a CHꢀπ electron
interaction with Phe70. Binding of the inhibitor is possible through a displacement of the αꢀhelix (α2)
bearing Leu52, and this residue adopts a new rotameric conformation to enable hydrophobic interactions
between the phenyl ring and neighboring hydrophobic side chains of Ala48, Ala49 and Leu52. The Sꢀ
enantiomer, which was shown to be a more potent inhibitor than the Rꢀenantiomer, provides the correct
24
geometry for the ligand to adopt this bent conformation. The diphenyl ether group is further anchored
through ring D, which occupies a new binding pocket obtained after the reorientation of Arg107 side chain
and is sandwiched between residues Met66 and Arg107 establishing a cationꢀπ interaction with the
guanidinium group of Arg107.
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Next our efforts were directed towards the identification of MtTMPK inhibitors with new chemotypes, to
increase chances of identifying analogues with whole cell activity. First we focused on altering ring B, i.e.
the metaꢀsubstituted piperidine ring (Figure 2).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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0
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8
9
0
Regioisomer 2, obtained by repositioning the thymine ring of 1 from the metaꢀ to the paraꢀposition of the
piperidine ring, displayed slightly better MtTMPK inhibitory activity than compound 1. This result is
encouraging since compound 2 possesses internal symmetry, and is consequently easier to prepare.
Moving the terminal phenoxy ring of 2 to the paraꢀposition of ring C (compound 4) increases the IC by a
50
factor 5. Homologation of the linker between the B and the Cꢀring afforded compound 3, which exhibited
marginally decreased inhibitory activity, while adding a methylene between the thymine and piperidine
rings led to inactive derivatives 5 and 6. Replacement of the piperidine by an azetidinꢀ3ꢀyl ring, as in 7ꢀ10,
generally led to a dramatic drop in inhibitory potency.
Compound 11, which could be considered as a ring opened form of the metaꢀpiperidine analogue 1, gave a
7ꢀfold drop in IC ꢀvalue, while compound 12, an acyclic variant of the paraꢀpiperidine analogue 2, was
5
0
drastically impaired in its inhibitory potency.
Changing the methylene group between rings B and C of inhibitors 2 and 4 by a carbonyl group to
produce more rigid amides 13 and 14 was also at the expense of inhibitory potency.
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H
O
N
O
O
O
N
R1 =
R2 =
R1
N
1
, R = R1
0
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IC50 = 10 ± 2 ꢀM
H
N
H
O
O
O
O
N
O
R1
N
N
N
12
N
R
1
H
IC50 > 2mM
O
N
O
3
IC50 = 11 ± 1 ꢀM
H
N
N
O
R
N
N
H
O
N
O
2
, R = R1, IC50 = 6.1 ± 0.5 ꢀM
R
N
R
N
4
, R = R2, IC50 = 29 ± 3 ꢀM
N
O
5
, R = R1, IC50= NI at 0.8 mM
, R = R2, IC50 > 2 mM
1
3, R = R1, IC50 = 235 ± 8 ꢀM
4, R = R2, IC50 = 353 ± 52 ꢀM
6
1
H
H
O
N
O
O
N
O
N
N
R
N
N
R1
H
8
7
, R = R1, IC50 = 944 ± 105 ꢀM
0, R = R2, IC50 = 832 ± 100 ꢀM
O
N
O
IC50 = 1910 ± 417ꢀM
1
N
H
R
1
N
O
N
O
11
N
IC50 = 72 ± 3 ꢀM
R1
N
9
IC50 = 1142 ± 39 ꢀM
Figure 2: MtTMPK inhibitory activities of the analogues with alternative scaffolds for the metaꢀsubstituted
Bꢀring of compound 1.
In an attempt to identify MtTMPK inhibitors with improved ligand efficiency (LE), we decided to
synthesize truncated analogues of the paraꢀsubstituted piperidine analogue 2 in which the Dꢀring was
omitted and in which we explored alternative Cꢀrings (Figure 3). From this exercise, we deduce that
deletion of the terminal phenoxy group decreases the inhibitory potency with a factor 22 (compound 15).
As shown in Figure 3, a planar Cꢀring is necessary for the enzyme potency (pairs 15/32), however, big
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aryl rings with improved inhibitory potencies do not yield better ligand efficiency (pairs 15/34, 29/41).
27
Branching the methylene between ring B and C only had no influence on the activity (pairs 15/31). We
observed that the inhibitory potency benefits from the introduction of electronꢀwithdrawing substituents
on the Cꢀring (analogues 20ꢀ22/15). Consistently, electronꢀpoor heterocycles are also favorable (e.g.,
analogues 27 and 35). Comparing with other modifications, these electronꢀdeficient substituents also
generated active analogues with high LE value (0.25 – 0.29), out of which compound 21 emerged as the
best.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
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2
2
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0
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8
9
0
Using the cocrystal structure of MtTMPK in complex with 1, modeling studies indicated that introducing a
carboxylic acid functionality at Cꢀ3 of ring C (28) could afford a beneficial electrostatic interaction with
Arg107. As shown in Figure 4, the position of Arg107 in the model points to the carbonyl group of ring A
instead of the expected carboxylic group of ring C, compared to the position obtained from the cocrystal.
Analogues that have a similar group or an isosteric tetrazole ring at the Cꢀ4 position were also tried (23
and 24). However, the introduction of these acidic substituents tends to lower the inhibitory potency and
LE (0.20 ꢀ 0.21). The reason might be, as observed from docking studies, due to conformational variation
of the active site, induced by the acidic groups of these compounds which is not favorable, overall leading
to weaker binding. In conclusion, ring C prefers hydrophobic and electron poor aromatic rings.
Since the effect of all the truncated analogues described in Figure 3 were found to be vastly inferior to 2,
ring D is deemed necessary for good MtTMPK inhibition. Taken into account the synthetic feasibility and
the fact that quinolines and quinolones have emerged as promising scaffolds for developing
2
8
antimycobacterial agents, quinoline derivative 35, which has a favorable LE of 0.27, was selected for
further structural elaboration.
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9
0
Figure 3. SAR and LE exploration of ring C based on paraꢀpiperidinylthymine 2.
1
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Figure 4. Structure overlay of compound 1 (stick representation, carbon atoms in white) and docking pose
of compound 28 (carbon atoms in cyan) in MtTMPK active site. Protein is shown in pale yellow cartoon
representation. Key MtTMPK interacting residues (stick representation, carbon atoms in orange) with
compound 1 are shown: in particular, Arg107 is in close proximity to the ether bridge between rings C and
D. For compound 28, the envisaged favorable electrostatic interaction is shown in red dashed line between
the carboxyl moiety and Arg107 (in stick representation, carbon atoms cyan) in the docked structure.
An overlay of the docking poses of compounds 2 and 35 in MtTMPK is represented in Figure 5A. As
expected, the thymine and piperidine moieties of 2 and 35 occupy similar positions. The aromatic tails of
2
, however, points towards an additional pocket in the enzyme. This led us to explore if this additional
pocket could be occupied by introducing a phenoxy group at position 4 of the quinolone moiety of 35. The
structures of the envisaged 4ꢀphenoxyquinoline derivatives are shown in Figure 5B. Indeed, compound 42
shows a 5ꢀfold improved MtTMPK inhibitory potency compared to analogue 2 and an 8ꢀfold improvement
compared to the parent quinoline 35. Introduction of a chlorine substituent at two different positions of 42
afforded analogues 43 and 44, of which 43 represents the most potent MtTMPK inhibitor identified so far,
bearing this particular scaffold.
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5
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9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. Docking poses of compounds 2 and 35 in MtTMPK active site. (A) Structural overlay of
inhibitors 2 (in stick representation, carbon atoms in white) and 35 (in stick representation, carbon atoms
in grey) docked poses in MtTMPK catalytic pocket. Protein is depicted in pale yellow cartoon
representation. Key interacting residues are represented in stick conformation and colored in orange for
carbon atoms. (B) Design of the phenoxyl quinoline series and their corresponding inhibition constants.
The cocrystal structure of MtTMPK in complex with compound 43 solved at 2.35 Å resolution (PDB code:
5
NR7) reveals the key interaction features involved in binding of compound 43 to the enzyme and are
consistent with the predicted binding configuration obtained from the docking experiments. Inhibitor 43,
which is bound in the active site of the enzyme, was placed in the residual FobsꢀFcalc electron density and
adopts an L shaped conformation (Figure 6).
Consistently with the cocrystal structure of 1ꢀMtTMPK, ring A adopts an identical pose to that observed in
the crystal structures of substrateꢀlike ligands. The paraꢀsubstituted piperidine ring (ring B) establishes a
similar πꢀalkyl interaction with Tyr103 as observed in the cocrystal structure of 1 but displays a slightly
different binding pose. Interestingly, the quinoline aromatic group protrudes linearly to ring B out of the
catalytic pocket and is located in a new binding spot establishing a πꢀalkyl interaction with Leu52 and an
edgeꢀtoꢀface πꢀstacking interaction with His53. Despite the weak ligand electron density, which does not
completely support the positioning of ring D in the model, the ether group can be placed with confidence
1
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supporting the orientation of ring D that bends in an opposite direction than the one observed for 1. The
chlorobenzene ring is involved in hydrophobic interactions with Ala49 and could establish a parallelꢀ
displaced πꢀstacking interaction with Tyr39, which is reminiscent of the interaction between the phosphate
group of dTMP and Tyr39.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
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8
9
0
Figure 6: Cocrystal structure of MtTMPK in complex with compound 43 and key interactions with the
enzyme. (A). Compound 43 and the side chains of interacting residues of MtTMPK are represented as
sticks for carbon atoms and colored in white and orange respectively. The corresponding unbiased FoꢀFc
electron density (contoured to +3 sigma) calculated before adding the ligand in the refinement process is
shown as a green mesh. Protein is shown in pale yellow cartoon representation. (B) Schematic drawing of
compound 43 binding site displaying key MtTMPK residues in direct contact with 43 (hydrogen bonds are
represented with a red dashed line).
Antimicrobial activity and enzymatic inhibitory activity
Next to their ability to inhibit MtTMPK, all final compounds were tested M. tuberculosis growth
inhibitory activity. The antimycobacterial activity of metaꢀpiperidinylthymine 1 reported in a previous
24, 29
study turned out to be nonꢀrepeatable (IC50 > 64 µM, H37Ra),
but we found that analogue 42 had a
fourꢀfold stronger antituberculosis activity than compound 1 (H37Rv, Table 2). This result is in line with
1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
3
3
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the enzymatic inhibitory activity, which is 8ꢀfold higher for 42 compared to 1. Analogue 43 is 33ꢀfold
more potent than derivative 42, which represents the most potent compound in this study so far (H37Ra).
All analogues did not display severe cytotoxicity, with only 42 and 43 having some cytotoxicity against
MRCꢀ5 fibroblasts. The analogues not included in Table 2 were found to have no antibacterial activity for
H37Ra strain up to 64 µM.
0
1
2
3
4
5
6
7
8
9
0
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2
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4
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6
7
8
9
0
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2
Compound
IC50 (µM)
MtMTPK
10
MIC
IC50 (µM)
H37Rv
318.50 (125)
H37Ra
MRCꢀ5
29.5
1
>64
60.45
>64
21
7.1
678.87 (>250)
47.85
>64
2
3
4
4
4
5
2
3
142
650.29 (>250)
9.0
678.87 (>250)
>64
>64
1.1
35.25ꢀ70.62 (15.6ꢀ31.25)
16.35ꢀ32.70 (7.8ꢀ15.6)
32.51
8.35
3
0.95
0.96
6.47
Table 2: Inhibitory activity of compounds against MtTMPK and M. tuberculosis cells (H37Ra and H37Rv
1
strains) and toxicity against MRCꢀ5 human cell line. MIC values are given in µM and between brackets
2
they are expressed in µg/mL. IC50ꢀvalues were determined graphically using the plot of the % viability
3
as a function of compound concentration. IC = 12.17 µM
99
We hypothesized that the discrepancy between the potent enzyme inhibitory activity and the wholeꢀcell
antimycobacterial activity could be due to poor mycobacterial uptake or due to efflux.
To explore this hypothesis, the MIC of the selected analogues was determined first in the presence of
efflux pump inhibitors (at one fourth of their MIC), and second, by using a series of M. tuberculosis
mutants defective in the production of efflux pumps, along with the wild type strain H37Rv. From Table
1
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, it can be observed that in the presence of the efflux inhibitor verapamil, a calcium channel blocker, the
MIC of two compounds (21 and 35) markedly decreased in comparison to that for the reference strain
H37Rv. In addition, the M. tuberculosis mutant defective in the Mmr efflux pump also showed a
significant (more than 4ꢀfold change) decrease in the MICs of these two analogues. These results strongly
suggest that active efflux plays an important role in the transport of compounds 21 and 35, so this could be
the reason for the discrepancy between antituberculosis activity and enzyme inhibitory activity. For
compounds 1, 42 and 43, the decrease in the MICs in the presence of efflux inhibitors is moderate, and no
significant change could be detected in the efflux defective mutants. We can conclude that in these cases
efflux may play a moderate role in the resistance to these compounds, and in any case, this would not be
mediated by Tap or Mmr efflux pumps. Finally, we could not detect any effect of either addition of efflux
inhibitors or deletion of gene encoding efflux pumps for compound 24, indicating that this compound may
have retarded uptake due to its negative charged tetrazole ring.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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MIC (µg/mL)
M. tuberculosis strain
H37Rv
verapamil
15.6
KO Tap
none
KO Mmr
none
Efflux inhibitor
1
none
125ꢀ31.25
>250
PAβN
62.5ꢀ31.25
>250
31.25ꢀ62.5
>31.25
31.25ꢀ15.63
15.63
2
1
31.25
Compound
24
>250
>250
>250
>250
>250
3
5
250
62.5ꢀ31.25
7.8
250
125
< 1.95
15.63
42
31.25ꢀ15.6
7.8ꢀ15.6
7.8
31.25ꢀ15.63
7.8ꢀ15.6
4
3
3.9ꢀ7.8
7.8ꢀ15.6
7.8
Table 3: Impact of efflux on antituberculosis activity of MtTMPK inhibitors.
CONCLUSION
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Starting from the previously identified hit 1, scaffold screening and ligand efficiency exploration resulted
in two novel chemical series: one series represented by the phenoxylbenzyl analogue 2 and a truncated
series represented by quinolinꢀ2ꢀyl analogue 35. Structureꢀguided scaffold morphing between compound 2
and 35 furnished phenoxylquinolinꢀ2ꢀyl derivatives with potent MtTMPK inhibitory activity and improved
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antibacterial activity. A potential explanation for the observed discrepancy between IC and MIC values
50
is provided by assessing the antimycobacterial activity in the presence of efflux pump inhibitors or by
using mutant M. tuberculosis strains deficient in efflux pumps. In particular, the cellular activity of 35 was
potentiated 128ꢀfold when tested on the mmr efflux pump knockout strain. These observations provide
possible directions for further inhibitor development, e.g. by combining the optimized MtTMPK inhibitors
with an efflux pump inhibitor, by designing hybrid compounds that feature both an MtTMPK as well as an
efflux pump inhibitor motif or, ideally, by looking for MtTMPK inhibitors that feature structural motifs
that facilitate mycobacterial uptake.
Experimental section:
Expression and purification of Mycobacterium tuberculosis thymidylate kinase (MtTMPK) MtTMPK
21
was expressed and purified as previously described with minor modifications.
In short, the pHL50
vector containing the coding sequence of wildꢀtype MtTMPK was used to transform E. coli BLi5
competent cells. Transformed cells were selected on LB agar plates containing 100 µg/mL carbenicillin
and 34 µg/ml chloramphenicol and directly 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
isopropylꢀ1ꢀthioꢀβꢀDꢀthiogalactoside when the culture reached an absorbance of 1.5 at 600 nm. After 3ꢀ
hour incubation at 37°C, cells were harvested by centrifugation at 8000 x g at 4°C and stored at ꢀ80°C
before proceeding with purification. Cells from 1 L culture were resuspended in 50 mL of cold lysis buffer
containing 50 mM Tris HCl pH 8.0 supplemented with antiꢀprotease cocktail (Roche) and disrupted by
sonication. After centrifugation at 20000 x g for 30 minutes at 4°C, the filtered bacterial lysate was
injected on a 5 mL BlueꢀSepharose column preꢀequilibrated with lysis buffer. The column was washed
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with lysis buffer and most of the protein was eluted with elution buffer containing 50 mM Tris HCl pH
8
2
.0, 1 M NaCl. A second elution was performed using elution buffer containing 50 mM Tris HCl pH 8.0,
M NaCl. Fractions containing MtTMPK were pooled and injected on a HiLoad Superdex 75 16/600
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column equilibrated with 20 mM Tris HCl pH 7.4, 1 mM EDTA as polishing and desalting steps.
MtTMPK fractions were pooled and Trisꢀ(2ꢀcarboxyethyl)ꢀphosphine (TCEP) was added to 1 mM final
concentration before flashꢀfreezing of the protein samples in liquid nitrogen.
Enzymatic assay
The compounds were dissolved in DMSO. The assays were performed at fixed concentrations of ATP (0.5
mM) and dTMP (0.05 mM), and at varying concentrations of tested compound (between 0.0008 and 0.6
30
mM) using the spectrophotometric assay described by Blondin et al. The reaction medium contains 50
mM TrisꢀHCl, pH 7.4, 50 mM KCl, 2 mM MgCl , 0.2 mM NADH, 1 mM phosphoenol pyruvate, and 2
2
units each of coupling enzymes (lactate dehydrogenase, pyruvate kinase and nucleoside diphosphate
kinase). IC values were calculated using KaleidaGraph. First the datapoints were plotted and then bestꢀfit
50
concentrations were generated using the following equation: y = (m2)/(1+(x/m3)^m4); where y = %
inhibition; m2 = ymax; m3 = IC ; m4 = slope of the curve at the midpoint; x = % inhibition.
50
In the supplementary Table S1, for each compound, column 2 indicates the IC50 values in µM, column 3
the number of data points taken into account for the fit, column 4 the number of compound concentrations
used to measure the residual TMPKmt activity, columns 5 the maximum concentration tested, column 6
2
the minimum concentration tested and column 8 the coefficient of determination (R ).
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Protein crystallization
Cocrystallization conditions of MtTMPKꢀinhibitor complexes were screened at 20 °C by the sittingꢀdrop
vaporꢀdiffusion method using a 1:1 protein: solution volume ratio and available commercial screens.
Before each cocrystallization experiment, MtTMPK samples were thawed and concentrated to 6ꢀ8 mg/mL.
Compound 1 was initially dissolved in pure DMSO at a concentration of 100 mM and diluted to 50 mM
using pure isopropanol. Compound 43 was less soluble and initially dissolved in pure DMSO at a
concentration of 50 mM and diluted to 25 mM using pure isopropanol. Isopropanol appeared to be crucial
in improving solubility of compounds after dilution in protein buffer. Diluted compounds were then added
to concentrated protein stock at a final concentration of 1 mM and the protein/inhibitor sample incubated
on ice for 1 hour before setting up crystallization experiments.
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For the MtTMPK/1 complex, the ligand stock solution at 50 mM in 50/50 (v/v) pure DMSO/ pure
isopropanol solution was added to the concentrated protein solution stock to reach a final inhibitor
concentration of 1 mM. To further increase 1 solubility, a 50/50 (v/v) MtTMPK buffer/pure isopropanol
solution was added to the protein/inhibitor sample to increase final isopropanol concentration from 1%
(
v/v) to 1.5% (v/v). An initial hit leading to bipyramidal crystals was obtained in condition G6 (condition
0) of the crystal screen II (0.1 M HEPES pH 7.5, 10% (w/v) PEG 6000 and 5% (v/v) MPD). The crystals
3
were grown for a few weeks at room temperature and exposed to a cryoprotection buffer obtained by the
combination of mother liquor supplemented with 1 mM compound 1 and 10% v/v PEG 400 before being
mounted and flash frozen in liquid nitrogen.
Initial efforts to obtain MtTMPK/43 cocrystals led to the obtaining of crystals with very limited diffraction
power. Better crystals were obtained using the crossꢀseeding approach starting from inꢀhouse grown
MtTMPK crystals obtained with compound 43 related inhibitors. In short, MtTMPK cocrystals initially
grown in 0.1 M HEPES pH 6.8, 4.40 M NaCl were crushed in 50 µL of stabilizing solution (0.1 M
HEPES pH 6.8, 4.50 M NaCl, 1% (v/v) DMSO, 1% (v/v) isopropanol, 0.5 mM compound 43) using the
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seed bead kit (Hampton Research) and serial dilution of seeds with stabilizing solution were performed.
The MtTMPK/43 sample was prepared as previously described for compound 1 complex but using a lower
protein concentration (6.0 mg/mL) and an initial ligand solution at 25 mM in 50/50 (v/v) pure DMSO/
pure isopropanol. Crystallization trials were setꢀup at room temperature using the contemporary seeding
approach by mixing 0.3 µL of MtTMPK/compound 43 sample with 0.2 µL of crystallization solution and
0.1 µL of undiluted or diluted seed stock. The best diffracting crystal was obtained after a few weeks at
room temperature using the undiluted seed stock and crystallization solution consisting of 0.1 M HEPES
pH 6.8, 4.35 M NaCl. This crystal was exposed to a cryoprotection buffer (mother liquor supplemented
with 1 mM compound 43 and 10% v/v ethylene glycol) before being mounted and flash frozen in liquid
nitrogen.
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Data collection, structure determination and refinement
The data collection of MtTMPK/1 cocrystal was achieved on Proxima1 beamline at Soleil synchrotron
(Paris, France) at a wavelength of 0.97857 Å and a temperature of 100 K on a Dectris Pilatus 6M pixel
detector. The diffraction data of MtTMPK/43 cocrystal were collected at beamline P14 operated by EMBL
Hamburg at the PETRA III storage ring (DESY, Hamburg, Germany) at a wavelength of 0.9763 Å and a
temperature of 100 K on a Dectris Pilatus 6M pixel detector.
All datasets were indexed, processed and scaled using XDS/XSCALE and mtz conversion was performed
3
1
with XDSCONV. Five percent of the randomly selected reflections were kept apart for crossꢀvalidation
and used for the calculation of Rfree. All structures were solved by molecular replacement with PDB input
files 4UNP for MtTMPK/1 and 4UNR for MtTMPK/43 complex using MOLREP from the CCP4 suite
32
33
(
Table S1). Structures were further refined by one cycle of rigidꢀbody refinement in BUSTER followed
34
by positional and individual isotropic Bꢀfactor refinement in BUSTER and PHENIX. Models were
manually improved during the course of the crystallographic refinement using the graphic program
35
COOT. For the MtTMPK/43, torsion NCS restraints where used throughout the refinement. Ligand
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coordinates were generated with the Grade Web Server (http://grade.globalphasing.org), which was also
used for energetic minimization and restraint generation. Ligand molecules were modeled in sigmaꢀ
weighted FoꢀFc difference electron density maps in the course of the refinement. TLS refinement with one
TLS group definition per chain was applied in the latest stage of the refinement procedure after having all
atoms Bꢀfactor reset to the Wilson Bꢀfactor value. The realꢀspace correlation coefficient (RSCC) is
reported as an objective measure of the fit of inhibitor coordinates to electron density and was calculated
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using Twilight program. Discovery studio visualizer version 16.1.0 (Dassault Systèmes BIOVIA, San
Diego: Dassault Système) was used to analyze and describe the binding mode of inhibitors within the
37
protein. The quality of the final crystal structures was assessed with MOLPROBITY prior to deposition
at the PDB database under the codes 5NQ5 (MtTMPK/1), and 5NR7 (MtTMPK/43). Molecular images
were generated with PyMOL (The PyMOL Molecular Graphics System, Schrödinger, LLC). Data
collection and refinement statistics are presented in Table 1 (supplementary data).
Computational Studies
Ligand Efficiency (LE). According to equation 4, where HA stands for the number of nonꢀhydrogen atoms
of the ligand and pK for the negative logarithmic values of its corresponding inhibition constant, ligand
i
efficiency (average binding energy per atom, ∆g) values were calculated by using Excel spreadsheet
38, 39
(
supplementary data).
LE = (1.37/HA) × pIC50
(Eqn 4)
Docking. All molecular modeling were calculated using the software packages AutoDock 4.2 on Windows
Cygwin and AutodockToolsꢀ1.5.6. The Xꢀray structure of compound 1 in complex with MtTMPK (PDB
entry 5NQ5) was used in all docking experiments. The 2D chemical structures and PDB files of the
ligands were drawn and created using ChemDraw 15 and ChembioDraw 15 separately. The PDBQT file
of ligands and receptor were prepared by AutodockToolsꢀ1.5.6, which include atomic partial charges,
atom types and the information of the ligand torsional degrees. For the docking, a default grid spacing of
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.375 Å and 60 × 60 × 60 number of grid points were used, which centered the box on residue Phe70, at
the active site of MtTMPK (the coordinate x, y, z was ꢀ21.132, 31.018, ꢀ12.924 correspondingly). Arg107
and Leu52 surrounding the biphenyl ether tail (ring C and ring D) were set as flexible residues. The other
settings such as search parameters and docking parameters were adopted as default. 50 possible
conformations were given by Autodock 4.2 for each docking. A manual selection procedure which
combines visual inspection in AutodockToolsꢀ1.5.6 guided by Chimera together with the predicted free
energy found for each conformation was used to validate the docked conformations.
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In vitro antiꢀtuberculosis activity
40, 41
Mycobacterium tuberculosis strains used were the reference strain H37Rv
and the knockꢀout mutants
42
43
KOꢀTap and KOꢀMmr, deleted respectively in genes encoding the efflux pumps Tap and Mmr.
Mycobacterium tuberculosis cultures were routinely grown at 37 ºC in Middlebrook 7H9 broth
supplemented with 10% ADC (albumin, dextrose, catalase supplement, BectonꢀDickinson) and 0.05%
Tween® 80. The knock out mutant strains (KOꢀTAP and KOꢀmmr) were grown in the same medium
supplemented with hygromycin at 50 µg/mL. The Minimum Inhibitory Concentration (MIC) was
44
determined using the Resazurin Microtiter Assay Plate. In 96ꢀwell plates, double of the desired
concentrations of the compounds were added in 100 µL of Middlebrook 7H9 broth supplemented with 10%
ADC and 0.5% glycerol in a series of twoꢀfold dilutions, starting from 500 µg/mL. When required, efflux
pumps inhibitors were added to the medium, to a final concentration of 40 µg/mL for verapamil, and 15
5
µg/mL in the case of PAßN. Bacteria were inoculated by adding 100 µL of a suspension of 10 cfu/mL, as
determined by optical density, and prepared from a culture in exponential growth phase. Following
inoculation, the final concentration of the compounds ranged from 250 µg/mL to 1.95 µg/mL. After 6
days of incubation at 37°C, 30 µL of resazurin (0.01% w/v) was added to each well, incubated for a
further 48 h at 37°C, and assessed for color change. A change from blue to pink indicates bacterial growth.
The MIC is defined as the lowest drug concentration that prevents this color change. Changes in the MIC
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between strains or experimental conditions were considered as significant when there was at least a fourꢀ
fold variation with the reference strain or condition.
In vitro antimycobacterial activity of the 1,3ꢀdiaryltriazenides was further evaluated by a
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®
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luminometric assay based on a M. tuberculosis H37Ra laboratory strain (ATCC 25177 )
transformed with a pSMT1 luciferase reporter plasmid (H37Raꢀlux). A twoꢀfold serial dilution of
each compound was made in Middlebrook 7H9 broth + 10% OADC (complete 7H9 broth) with
final concentrations ranging from 128 µM to 0.5 µM. Volumes of 100 µL of the serial dilutions
were added in triplicate to black, flatꢀbottomed 96ꢀwell plates. As a positive control, isoniazid, a
firstꢀline antimycobacterial drug, was included. The mycobacterial suspension was made by
thawing a frozen glycerolꢀstock of H37Raꢀlux and, subsequently, diluting it in complete 7H9 broth
to obtain a suspension with 10.000 relative light units (RLU)/mL. A volume of 100 µL of bacteria
was added to each well. All of the outerꢀperimeter wells were filled with 200 µL of sterile
deionized water to minimize evaporation of the medium in the test wells during incubation. After
7
days, the bacterial replication was analyzed by luminometry. To evoke a luminescent signal, 25
µL of 1% nꢀdecanal in ethanol was added to each well, where after light emission was measured
using a luminometer (Promega Discover).
In vitro cytotoxicity assay
In vitro cytotoxicity on the MRCꢀ5 Homo sapiens long fibroblast cell line (ATCC® CCLꢀ171™)
2
was assessed for each analogue by a NRU assay. The MRCꢀ5 cells were cultured in 75 cm sterile
Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heatꢀinactivated fetal calf
serum in a 5% CO atmosphere at 37°C. When a semiꢀconfluent layer of cells was formed, the
2
cells were trypsinized, washed with sterile PBS, seeded into a transparent, flatꢀbottomed 96ꢀwell
plate at a density of 4 x 104 cells per well and left for recovery at 37°C, 5% CO . For each
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compound, a twoꢀfold serial dilution was made in complete DMEM with final concentrations
ranging from 128 µM to 0.5 µM. Subsequently, the MRCꢀ5 cells were exposed to the compounds
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(
in triplicate) by adding 100 µL of the serial dilutions to the wells. Test plates were incubated for
2
4 hours in an atmosphere of 5% CO at 37°C. For the NRU assay, the cells were washed 2 times
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with 200 µL PBS and 100 µL neutral red working solution was added per well. Subsequently, the
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plates were left for incubation at 37°C, 5% CO for 3 hours. After incubation, the wells were
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washed 2 times with 200 µL PBS and 150 µL of an ethanol/acetic acid mixture was added per
well. The plates were left shaking until the color became homogeneous purple and the optical
density was measured at 530 and 620 nm (reference wavelength) using a Promega Discover plate
reader.
Chemistry
General
Solvents were purchased from standard commercial sources and of analytical grade. Building
blocks and reagents were used as received without any further purification. TLC analysis was
performed using precoated Alugram Silica Gel F254 plates (MacheryꢀNagel). Spots were
examined under ultraviolet light at 254 nm. Column chromatography was carried out on a
Reveleris X2 (Grace) automated flash unit using the corresponding disposable silica gel cartridges.
1
13
H and C NMR spectra were recorded in CDCl or DMSOꢀd on a Varian Mercury 300/75 MHz
3
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spectrometer. Chemical shifts are given in parts per million (ppm δ), δ relative to residual solvent
1
13
peak or TMS for H and C. Structural assignment was confirmed with COSY, HSQC and
HMBC. Exact mass measurements (HRMS) were performed on a Waters LCT Premier XETM
Time of flight (TOF) mass spectrometer equipped with a standard electrospray ionization (ESI)
and modular LockSpray TM interface. Samples were infused in a CH CN/H O (1:1) mixture at
3
2
1
00 µL/min. Preparative reversed phase HPLC chromatography was carried out using a
Phenomenex Luna Cꢀ18 (21.2 × 250 mm) column using a liner gradient from 10% MeCN to 100%
MeCN in 10 mM ammonium bicarbonate solution over 20 min and a flow rate of 17.5 mL/min.
The purity of all final compounds was determined by LCꢀMS analyses on a Waters Alliance 2695
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XE separation Module using a Phenomenex Kinetex EVO C18, 5um 100 x 2.1mm column and a
gradient system of HCOOH in H O (0.1 %, v/v)/HCOOH in CH CN (0.1 %, v/v) at a flow rate of
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.6 mL/min, 05:95 to 0:100 (5 to 100 % CH CN) in 8 minutes. All final compounds showed
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higher than 95% purity.
General procedure for the synthesis of final compounds:
A suspension of azaheterocyclic or aminoalkyl thymine (1 eq), substituted aromatic aldehyde (1 – 2 eq)
and sodium triacetoxyborohydride (1.5 – 4 eq) in dry 1, 2ꢀdichloroethane (~ 0.03 M) was stirred at room
temperature under argon for overnight or 48 h. The reaction mixture was evaporated and dried with oil
pump for 0.5 h. The residue was purified by column chromatography (10% ethyl acetate/hexane + 0.8 %
Et N ꢀ 100% ethyl acetate + 0.8 % Et N or 100% CH Cl ꢀ 10% MeOH/CH Cl or 100% ethyl acetate –
3
3
2
2
2
2
5% MeOH/ethyl acetate in linear gradient elution) to afford pure intermediate, followed by BOMꢀ
deprotection. There are two methods for the BOMꢀdeprotection step. Method 1: according to a literature
45
procedure, the intermediate was dissolved with TFA (~ 0.05 M) under argon. The reaction mixture was
stirred at 72 °C for 30 min to 1 h. The reaction progress monitored by HRMS. Once the starting material
was consumed completely, the reaction mixture was cooled to room temperature and concentrated in
vacuum and dried with oil pump. The residue was dissolved with the solvent mixture (1 – 2 mL, MeCN/tꢀ
BuOH/H O, v/v/v = 1/1/1 or acetic acid or H O) and purified with preparative HPLC. After lyophilization,
2
2
all of the products were obtained as white powder.
Method 2: the intermediate was dissolved with 80% TFA/H O (~ 0.01 M),
L
ꢀcysteine hydrochloride (0.25
2
M or 0.27 M) was added to the reaction mixture. The reaction mixture was stirred at 72 °C for 5 h to 16 h.
HRMS was used to monitor the reaction progress. Once the starting material was consumed completely,
the reaction mixture was cooled to room temperature and evaporated. The residue was purified with
preparative HPLC.
5
ꢀMethylꢀ1ꢀ(1ꢀ(3ꢀphenoxybenzyl)piperidinꢀ4ꢀyl)pyrimidineꢀ2,4(1H,3H)ꢀdione (2): Following the
general procedure for the synthesis of final compounds (method 1): 3ꢀ((benzyloxy)methyl)ꢀ5ꢀmethylꢀ1ꢀ
2
8
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Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
(
piperidinꢀ4ꢀyl)pyrimidineꢀ2,4(1H,3H)ꢀdione 63 (86.3 mg, 0.262 mmol), 3ꢀphenoxybenzaldehyde (77.9
mg, 0.393 mmol), sodium triacetoxyborohydride (111.1 mg, 0.524 mmol) in dry 1, 2ꢀdichloroethane (10
mL) using ethyl acetate ꢀ hexane eluent system to obtain the intermediate, which was dissolved with TFA
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
(
8 mL) yielded compound 2 (39.6 mg, 39%). H NMR (300 MHz, DMSOꢀd ) δ: 1.64 (d, J = 9.38 Hz, 2H,
6
piperidinꢀ3aꢀyl, piperidinꢀ5aꢀyl), 1.73 ꢀ 1.93 (m, 5H, piperidinꢀ3bꢀyl, piperidinꢀ5bꢀyl, 5ꢀCH ), 1.98 ꢀ 2.12
3
(m, 2H, piperidinꢀ2aꢀyl, piperidinꢀ6aꢀyl), 2.90 (d, J = 11.43 Hz, 2H, piperidinꢀ2bꢀyl, piperidinꢀ6bꢀyl), 3.49
(s, 2H, CH 1ꢀmethylene), 4.25 (ddd, J = 12.02, 8.06, 4.25 Hz, 1H, piperidinꢀ4ꢀyl), 6.87 (dd, J = 8.06,
2,
2.49 Hz, 1H, Ph), 6.97 ꢀ 7.03 (m, 3H, Ph), 7.06 ꢀ 7.16 (m, 2H, Ph), 7.30 ꢀ 7.43 (m, 3H, Ph), 7.63 (s, 1H, Hꢀ
1
3
6
), 11.18 (s, 1H, NH). C NMR (75 MHz, DMSOꢀd ) δ: 12.00 (5ꢀCH ), 29.98 (2C, piperidinꢀ3ꢀyl,
6 3
piperidinꢀ5ꢀyl), 52.33 (3C, piperidinꢀ2ꢀyl, piperidinꢀ6ꢀyl, piperidinꢀ4ꢀyl), 61.23 (1ꢀmethylene) 108.93 (Cꢀ
5
1
), 117.17 (Ph), 118.47 (2C, Ph), 118.78 (Ph), 123.35 (Ph), 123.86 (Ph), 129.75 (Ph), 130.06 (2C, Ph),
37.69 (Cꢀ6), 140.92 (Ph), 150.82 (Cꢀ2), 156.57 (Ph), 156.75 (Ph) 163.68 (Cꢀ4). HRMS (ESI): calculated
+
for [C H N O + H] , 392.1969; found, 392.1974.
23
25
3
3
5
ꢀMethylꢀ1ꢀ(1ꢀ(3ꢀphenoxyphenethyl)piperidinꢀ4ꢀyl)pyrimidineꢀ2,4(1H,3H)ꢀdione (3): Following the
general procedure for the synthesis of final compounds (method 2): 3ꢀ((benzyloxy)methyl)ꢀ5ꢀmethylꢀ1ꢀ
piperidinꢀ4ꢀyl)pyrimidineꢀ2,4(1H,3H)ꢀdione 63 (93.9 mg, 0.285 mmol), 2ꢀ(3ꢀ
(
phenoxyphenyl)acetaldehyde 50 (120.0 mg, 0.57 mmol), sodium triacetoxyborohydride (151.0 mg, 0.713
mmol) in dry 1, 2ꢀdichloroethane (10 mL) using ethyl acetate ꢀ hexane eluent system to obtain the
intermediate which was dissolved with 80% TFA/H O (32 mL) and
Lꢀcysteine hydrochloride (1.40 g, 8.88
2
1
mmol) was added to the reaction mixture to yield compound 3 (59.0 mg, 51%). H NMR (300 MHz,
DMSOꢀd ) δ: 1.59 ꢀ 1.69 (m, 2H), 1.72 ꢀ 1.91 (m, 5H), 2.05 (t, J = 10.69 Hz, 2H), 2.53 ꢀ 2.61 (m, 2H),
6
2
ꢀ
.67 ꢀ 2.79 (m, 2H), 3.02 (d, J = 11.72 Hz, 2H), 4.16 ꢀ 4.32 (m, 1H), 6.82 (dt, J = 8.13, 1.21 Hz, 1H), 6.89
6.94 (m, 1H), 6.96 ꢀ 7.05 (m, 3H), 7.09 ꢀ 7.16 (m, 1H), 7.29 (t, J = 7.91 Hz, 1H), 7.34 ꢀ 7.43 (m, 2H),
13
7
.61 (d, J = 1.17 Hz, 1H), 11.20 (s, 1H). CNMR (75 MHz, DMSOꢀd ) δ: 12.01, 29.99 (2C), 32.77, 52.39
6
(
2C), 52.48, 59.00, 108.88, 116.12, 118.47 (2C), 118.99, 123.26, 123.86, 129.75, 129.99 (2C), 137.64,
2
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