Structure-Based Design and Synthesis of Piperidinol-Containing Molecules as New Mycobacterium abscessus Inhibitors

Article abstract of DOI:10.1002/open.202000042

Non-tuberculous mycobacterium (NTM) infections, such as those caused by Mycobacterium abscessus, are increasing globally. Due to their intrinsic drug resistance, M. abscessus pulmonary infections are often difficult to cure using standard chemotherapy. We previously demonstrated that a piperidinol derivative, named PIPD1, is an efficient molecule both against M. abscessus and Mycobacterium tuberculosis, the agent of tuberculosis, by targeting the mycolic acid transporter MmpL3. These results prompted us to design and synthesize a series of piperidinol derivatives and to determine the biological activity against M. abscessus. Structure-activity relationship (SAR) studies pointed toward specific sites on the scaffold that can tolerate slight modifications. Overall, these results identified FMD-88 as a new promising active analogue against M. abscessus. Also, we determined the pharmacokinetics properties of PIPD1 and showed that intraperitoneal administration of this compound resulted in promising serum concentration and an elimination half-life of 3.2 hours.

Full text of DOI:10.1002/open.202000042

DOI: 10.1002/open.202000042
Full Papers
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Structure-Based Design and Synthesis of Piperidinol-
Containing Molecules as New Mycobacterium abscessus
Inhibitors
Jérôme de Ruyck,[a] Christian Dupont,[b] Elodie Lamy,[c] Vincent Le Moigne,[c]
Christophe Biot,[a] Yann Guérardel,[a] Jean-Louis Herrmann,[c, d] Mickaël Blaise,[b]
Stanislas Grassin-Delyle,[c, e] Laurent Kremer,[b, f] and Faustine Dubar*[a]
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Non-tuberculous mycobacterium (NTM) infections, such as
those caused by Mycobacterium abscessus, are increasing
globally. Due to their intrinsic drug resistance, M. abscessus
pulmonary infections are often difficult to cure using standard
chemotherapy. We previously demonstrated that a piperidinol
derivative, named PIPD1, is an efficient molecule both against
M. abscessus and Mycobacterium tuberculosis, the agent of
tuberculosis, by targeting the mycolic acid transporter MmpL3.
These results prompted us to design and synthesize a series of
piperidinol derivatives and to determine the biological activity
against M. abscessus. Structure-activity relationship (SAR) studies
pointed toward specific sites on the scaffold that can tolerate
slight modifications. Overall, these results identified FMD-88 as
a new promising active analogue against M. abscessus. Also, we
determined the pharmacokinetics properties of PIPD1 and
showed that intraperitoneal administration of this compound
resulted in promising serum concentration and an elimination
half-life of 3.2 hours.
1. Introduction
infectious diseases in humans, livestock, and wildlife, such as
pulmonary, extra-pulmonary, skin and soft tissue infections.[3]
The diseases caused by NTM are increasing on a worldwide
scale.[4] In developed countries, NTM lung infections are more
prevalent than TB.[5–7] Nearly 200 distinct NTM species have
been reported so far, with the Mycobacterium avium-intra-
cellulare complex (MAC) and the Mycobacterium abscessus as
the major causative agents in Europe as well as in the United
States.[8,9] Of particular concern, M. abscessus infects both
immunocompromised or immunocompetent individuals and is
often associated with cystic fibrosis (CF).[10–12] In CF patients,
presence of M. abscessus in the lungs correlates with a decline
in the lung function and can be fatal.[13–15] In addition, recent
studies suggested a transmission of M. abscessus complex
between CF patients[10,16,17], although this transmission might be
indirect, through fomites and dust.[18]
The current standardized antimicrobial treatment for M.
abscessus infections[10] combines three drugs, or more, for at
least 12 months. Moreover it is aggressive and burdensome.[12]
The outcome of the treatment is also highly dependent on the
inducible resistance to macrolides[19,20] (clarithromycin or azi-
thromycin), leading to a treatment success rate below 40%.[21]
Lung transplantation in CF patients, often representing the last
therapeutic option, is currently being debated amongst
clinicians in the case of pre-existing infection with M. abscessus.
However, most centers do not consider the presence of M.
abscessus as an absolute contra-indication since recent studies
support the possibility of lung transplantation with favorable
outcome in CF patients.[12,14,20,22–24] In light of the limited panel of
available drugs active against M. abscessus, treatments are often
complex and challenging. In this context, it appears necessary
to identify efficient, specific and less toxic compounds.
Nontuberculous mycobacteria (NTM) are environmental bac-
teral isolated from surface and tap water, soil, animals, milk and
food products.[1,2] They are responsible for opportunistic
infections in humans and are recognized as an increasing public
health concern. NTM have been reported as a cause of diverse
[a] Dr. J. de Ruyck, Prof. C. Biot, Dr. Y. Guérardel, Dr. F. Dubar
Univ. Lille, CNRS
UMR 8576 – UGSF – Unité de Glycobiologie Structurale et Fonctionnelle
59000 Lille (France)
E-mail: faustine.dubar@univ-lille.fr
[b] Dr. C. Dupont, Dr. M. Blaise, Dr. L. Kremer
IRIM Institut de Recherche en Infectiologie de Montpellier – UMR9004-CNRS/
UM
1919 route de Mende
34293 Montpellier (France)
[c] E. Lamy, Dr. V. Le Moigne, Prof. J.-L. Herrmann, Dr. S. Grassin-Delyle
Infection et inflammation – Laboratoire 2I
UMD1173-INSERM/UVSQ
Versailles, 2 Avenue de la source de la Bièvres
78180 Montigny le Bretonneux (France)
[d] Prof. J.-L. Herrmann
Université Paris-Saclay, UVSQ, INSERM, Infection et Inflammation
(U1173), Montigny le Bretonneux, France
[e] Dr. S. Grassin-Delyle
Hôpital Foch
Département des maladies des voies respiratoires
92150 Suresnes (France)
[f] Dr. L. Kremer
INSERM, Institut de Recherche en Infectiologie de Montpellier, Montpellier,
France
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/open.202000042
© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This
is an open access article under the terms of the Creative Commons Attri-
bution Non-Commercial NoDerivs License, which permits use and distribu-
tion in any medium, provided the original work is properly cited, the use is
non-commercial and no modifications or adaptations are made.
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The lack of therapeutic solutions prompted us to perform a
phenotypic screen for bactericidal compounds against M.
abscessus using a library[25] of compounds formerly validated for
their activity against M. tuberculosis.[26] Our study allowed
identifying a new piperidinol-based compound, which we
named PIPD1 (Figure 1) and which exhibits potent activity
against clinical isolates of M. abscessus in vitro and in infected
human macrophages and zebrafish embryos.[26]
index. Moreover, all analogues were subjected to molecular
modeling studies using MmpL3 in order to rationalize, at a
molecular level, the in vitro results. Previously, PIPD1 was
synthesized according to a reported method.[26]
However, the synthetic method used (Figure 2) includes a
deprotection step (Figure 2, step b), which is not complete and
causes a degradation of the product of interest. Another
deprotection condition using trifluoroacetic acid (TFA) was
therefore evaluated, but degradation of the final product was
still observed and the yield obtained for step b was insufficient
to pursue the complete synthesis. In order to circumvent these
limitations, another synthetic pathway was developed, as out-
lined in Figure 3.
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Importantly, M. abscessus-infected zebrafish embryos
treated with PIPD1 showed an increased survival rate and a
decreases bacterial burden, as compared to the infected but
non-treated animals. Thanks to the selection of spontaneous
resistant mutants and following whole-genome sequencing,
single nucleotide polymorphisms (SNP) were identified in MAB_
4508. This gene encodes the mycolic acid transporter MmpL3
that was shown to be essential for Mycobacterium smegmatis
and M. tuberculosis survival.[27–29] Like other MmpL, MmpL3 is a
transmembrane protein belonging to the Resistance-Nodula-
tion-Cell division (RND) permeases.[30] MmpL proteins are known
to participate in the export of lipid components across the cell
envelope in mycobacteria. We demonstrated that PIPD1 does
not affect de novo biosynthesis of mycolic acid but rather
inhibits the transport of trehalose monomycolate (TMM) by
blocking the flippase activity of MmpL3.[26,31] This results in the
loss of trehalose dimycolate synthesis, the decrease of arabino-
galactan mycolylation and bacterial death. Herein, with the aim
to improve the activity of this new family of MmpL3 inhibitors
against M. abscessus, we present the rational design and the
structure-activity relationships of piperidinol derivatives.
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First, N-benzylation was achieved by means of benzyl
bromide in DCM in the presence of an excess of potassium
carbonate, resulting in high yields of the piperidone product.
Total consumption of the starting aldehyde was monitored by
thin layer chromatography (TLC) using dichloromethane as
eluent. Second, bromineÀ lithium exchange reaction was fol-
lowed by a nucleophilic substitution reaction on the carbonyl of
piperidinone to form the final compound. The final products
1
were purified on silica gel chromatography. H-NMR, 13C-NMR
and mass spectrometry were performed to confirm the
synthesized compound structures. Synthesis of all presented
analogues was between 30–60% yield. Thus, this new synthetic
route with limited reaction steps is associated to a significant
improvement in the total yields of the analogues.
2.2. Biology and Structure Activity Relationship Study (SAR)
2. Results and Discussion
We first aimed at designing PIPD1 analogues to identify
common pharmacophores in the first series of compounds
2.1. Chemistry
In the present study,
a first series of piperidinol-based
compounds with potent antibacterial activity against M.
abscessus was synthesized. The initial hit compound, PIPD1, was
derived in order to identify the pharmacophores and to explore
a preliminary structure-activity relationship (SAR). The new
derivatives were evaluated against the M. abscessus CIP104536T
reference strain. In addition, toxicity of the most potent
compounds was evaluated in order to determine the selectivity
Figure 2. Three-step chemical synthesis of PIPD1 and related analogues.
Figure 1. Chemical structure of PIPD1.
Figure 3. Two-step chemical synthesis of PIPD1 analogues.
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(Series I). Among these compounds, each organic function,
(designated A and B, see Figure 1) of PIPD1 were removed or
modulated and the in vitro antimycobacterial activity of prod-
ucts against the M. abscessus smooth (S) reference strain
CIP104536T was assayed to attest for their contribution to the
activity. The minimal inhibitory concentration (MIC) expressed
in μg/ml (and μmol/L) was determined using Cation-adjusted
Mueller-Hinton (CaMHB; Sigma-Aldrich), according to CLSI
guidelines.[32] The evaluation of the toxicity of the newly
synthesized compounds was evaluated on mammalian cells in
order to establish a selectivity index. Toxicity assays on Vero
cells were performed in RPMI supplemented with 10% FBS
based on cell viability using resazurin as a cell survival indicator.
The half maximal inhibitory concentration (IC50) corresponds to
the drug concentration that reduces cell viability by 50% and is
expressed in percentage of cell survival.
The selectivity index (SIMabs), corresponding to the IC50/MIC
ratio was determined for the 12 most active compounds against
M. abscessus. IC50 and SI were not determined for compounds
with unsatisfactory biological activity (MIC99 >0.5 μg/ml). Ta-
ble 1 summarizes the structures and inhibition activities of the
designed PIPD1-analogues of the series I.
Results in Table 1 indicate that the aromatic parts A and B
and the tertiary alcohol function of the piperidinol moiety are
essential for the inhibitory activity against M. abscessus. More-
over, the methyl group of the ring B participates to the
antimycobacterial effect since the activity of FMD-15 was lower
than the one of PIPD1. The donor inductive effect of methyl
group may impact the inhibitory effect. With respect to ring A,
the removal of the trifluoromethyl group (FMD-32) clearly
impacted on the biological activity as compared to the removal
of the chlorine atom (FMD-33).
Table 1. Structures and biological evaluation (MIC99, CC50 and SI; N.D., not
determined) of the PIPD1-derivatives of the series I. MIC99 (M. abscessus)
data result from three independent experiments and CC50 (Vero cells) from
two independent experiments.
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Analogues Formula
MIC99 MIC99
M.abs M.abs
CC50 SIMabs
[μg/
[μg/
ml]
[μmol/ ml]
L]
PIPD1
0.125 0.3
25
200
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FMD-37
128
350.6
N.D. N.D.
FMD-16
FMD-32
128
2
623.9
6.3
N.D. N.D.
N.D. N.D.
FMD-33
1
2.9
N.D. N.D.
FMD-15
FMD-99
FMD-0
1
2.7
N.D. N.D.
N.D. N.D.
N.D. N.D.
4
14.2
458.7
Although the dataset of compounds tested is limited,
important SAR elements have been unravelled and subse-
quently utilized for guiding the design of the series II
compounds (Table 2).
128
From these data, one can deduce that the aromatic moieties
are key to the biological activity. The nature of the aromatic
substituent was modulated to determine if steric effect
influences the activity. The steric occupancy of the aromatic
ring A appeared significant, nevertheless the lack of the
trifluoromethyl and methyl groups on naphthalene could
explain the decrease of activity for naphthalene (FMD-102). The
steric hindrance was also an important feature in ring B since
the introduction of a bulky substituent (such as ferrocene in
FMD-6 or an aromatic-substituted piperidine in FMD-62) caused
a decrease in bactericidal activity against M. abscessus. Addi-
tionally, the modification of the methyl group of ring B was
investigated in relation to their position and electronic proper-
ties. The presence of a substituent in ring B with a consistent
steric hindrance appears essential to biological activity. Indeed,
PIPD1 with a methyl substituent and FMD-88 with an iodo
substituent were more active than FMD-3 that carries a fluorine
atom. Additionally, the position of the substituents on ring B
also appears important since the presence of group in the ortho
position (PIPD1, FMD-88 or FMD-93) initiates a potent anti-
mycobacterial activity, whilst the presence of methyl group at
FMD-46
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261.1
N.D. N.D.
meta (FMD-63) or para (FMD-66) positions decreases the
activity. Noteworthingly, these results are similar to those
reported against M. tuberculosis,[31] which strongly suggests that
the mechanism of inhibition and/or binding site of these
compounds in MmpL3 are conserved in M. tuberculosis and M.
abscessus. To further rationalize these experimental data, the
synthesized derivatives were subjected to in silico docking
analyses using a 3D homology model of MmpL3 from M.
abscessus to address how the different SAR elements interact
with MmpL3 at a molecular level.
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Table 2. Structures and biological evaluations (MIC99, CC50 and SI; N.D., not
determined) of the series II PIPD1-analogues. MIC99 (M. abscessus) data are
from three independent experiments and CC50 (Vero cells) are from two
independent experiments.
Table 2. continued
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Analogues Formula
MIC99
M.abs
MIC99
M.abs
CC50 SIMabs
[μg/
[μg/ml] [μmol/ ml]
4
Analogues Formula
MIC99
MIC99
CC50 SIMabs
L]
M.abs
M.abs
[μg/
5
6
7
[μg/ml] [μmol/ ml]
L]
FMD-92
4
4
9.9
N.D. N.D.
N.D. N.D.
8
9
FMD-88
FMD-93
FMD-96
FMD-89
0.125
0.125
0.125
0.125
0.25
0.3
25
50
25
50
200
400
200
400
FMD-102
12.1
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FMD-64
FMD-62
16
32
36.6
70.8
N.D. N.D.
N.D. N.D.
0.3
0.3
2.3. Molecular Modelling
Previously, several mutations in M. abscessus strains resistant to
PIPD1 were identified in MAB_4508, that encodes the TMM
transporter MmpL3.[26,31] Moreover, we recently demonstrated
that PIPD1 alters the mycolic acid transport in M. abscessus[26] by
directly inhibiting the TMM flippase activity of MmpL3 in M.
tuberculosis.[31] Finally, Zhang et al. solved the crystal structure
of Mycobacterium smegmatis MmpL3 by X-ray crystallography at
a resolution of 2.8 Å (PDB:6AJJ) which unravelled a cavity in the
transmembrane region of MmpL3 accommodating several
MmpL3 inhibitors.[33] Since MmpL3 and its orthologues are well
conserved across mycobacteria, comparative modelling of M.
abscessus MmpL3 using the crystal structure of M. smegmatis
MmpL3 as a template (sharing 65% identity with its M.
abscessus orthologue) was performed (Figure 4a and 4b).
Quality of the M. abscessus MmpL3 model was then evaluated
according to classical geometric parameters (See SI for full
report). The RMSD calculated on the Cα of the template is
around 0.4 Å and 96% of the residues are within the
Ramachandran criteria. The superimposition of the M. abscessus
MmpL3 model onto the M. smegmatis MmpL3 structure showed
that the drug binding site formerly described[33] was conserved
in the transmembrane domain of the M. abscessus MmpL3 (gold
sphere in Figure 4). Interestingly, this cavity is in the vicinity of
residues previously reported to participate to the proton
relay.[26,31,33] This binding pocket is composed of two hydro-
phobic cavities, designated Up- and Bottom-pockets (U and B in
Figure 4c) and comprises also a more polar cavity, designated
Middle-pocket (M in Figure 4c). Of note, the residues contouring
the M- and B-pockets are well conserved amongst all mycobac-
teria (see sequence alignment Figure S1).
PIPD-1
0.125
0.25
0.25
0.3
0.6
0.7
25
25
50
200
100
200
FMD-91
FMD-10
FMD-94
FMD-3
0.5
1
1.1
2.6
50
100
N.D. N.D.
FMD-61
FMD-63
FMD-66
2
2
2
4.8
5.2
5.2
N.D. N.D.
N.D. N.D.
N.D. N.D.
FMD-6
4
8.4
N.D. N.D.
FMD-103
FMD-9
4
4
9.6
9.7
50
12.5
Docking simulations and energy minimization were next
conducted using the M. abscessus MmpL3 predictive model
with different analogues: the reference compound PIPD1, one
analogue with a higher inhibitory effect (FMD-89), one with a
lower inhibitory effect (FMD-61), one truncated ligand (FMD-0).
N.D. N.D.
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We next simulated a lower affinity complex by studying the
replacement of the chlorobenzene ring of FMD-89 with a
naphthalene group in FMD-61 (Figure 5E and 5F), which was
designed to keep the aromatic property of the substitution as
compared to PIPD1. In Figure 5E, the nitrogen atom is located
towards the U-pocket and the hydroxyl located towards the B-
pocket. In addition, no more residues are involved in hydrogen
bonds. As expected, the ligand is now located upside-down
(Figure 5F) in the binding pocket, as compared to PIPD1. This
drastic change in conformation could explain the loose of
antimicrobial activity due to absence of the two key interactions
(H-bonds) between the PIPD1 hydroxyl group and Y219 and
between the PIPD1 ammonium group and D258, as illustrated
in Figure 5B. Indeed, neither the hydrogen bonds nor the π-π
interactions are observed. Only small hydrophobic or van der
Waals interactions can explain these binding modes. It can
therefore be speculated that the B-pocket is too narrow,
therefore only accommodating small aromatic moieties.
Finally, the truncated ligand FMD-0 is obviously not well
adapted to the binding site. Nevertheless, this last binding pose
(Figure 5G) clearly explains why the hydroxyl group of the
central ring of any inhibitors is required. Here, only the
important hydrogen bond between D258 and the hydroxyl
moiety is conserved. Moreover, the lack of interaction in the B-
pocket seems to induce large changes in the conformation of
the putative inhibitors. These poor interactions may contribute
to the low antimicrobial properties of the inhibitor.
Furthermore, the nature of the ortho group in ring B
appears important too. Even though the electronic effects do
not have a significant influence to the binding capacity of the
PIPD1 analogues, the size of the substituents, however, seems
to have a great impact to their biological activity. Indeed, the
methyl group (2.00 Å) in PIPD1 or the iodine atom (1.98 Å) in
FMD-89 are associated with a more pronounced activity than
the fluorine atom (1.47 Å) in FMD-3. To get insights into these
differences, the steric hindrance of the ortho substituent for
FMD-89 and FMD-3 within the binding site of MmpL3 were
modelled (Figure 6).
As shown in Figure 6A the iodine atom of FMD-89 is
blocked in the binding cavity of the active site and allows to
increase the capability of the aromatic group to be stabilized by
π-π interactions (Figure 6B). In contrast, the ring B ring of FMD-
3 adopts a slightly different orientation than the B ring of FMD-
89 due to smaller size of the fluorine atom. These observations
confirm that FMD-3 displays a weaker interaction than FMD-89,
particularly for the π-π stacking of ring B.
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Figure 4. A) Comparative model of M. abscessus MmpL3 lacking its C-
terminal region. The binding pocket (Gold Sphere) is located inside the
°
transmembrane channel. B) 90 rotation of Figure 5A clearly showing the
channel. C) Focus on the putative binding site surrounded by three regions,
two hydrophobic pockets (U and B) and a more polar pocket (M).
In order to consider protonated or unprotonated states of
piperidinol tertiary amine at physiological pH, docking simu-
lations were performed on both structures for each compound.
Results were similar after the two runs and, thus, only
unprotonated ligands are presented in this article.
After minimization of the simulated PIPD1 complex, the
best binding pose was retrieved and further analysed (Figure 5A
and 5B). On one side, PIPD1 is hydrogen-bound to the
carboxylic acid group of D618 through its amino moiety. On the
other side, its hydroxyl group is H-bound to the hydroxyl of
Y219. Additionally, a potent interaction with the carboxylic acid
group of D258 can occur. Thanks to these two strong
interactions, the ligand appears perfectly anchored into the
proposed proton channel of the transporter. Additional stabili-
zation occurred through π-π interactions between the toluene
moiety of PIPD1 and phenyl rings of F262 and F622 (B-pocket).
However, the U-pocket does not appear to be optimally filled.
Regarding FMD-89, due to the electron-releasing effect of
the chloride moiety grafted on the phenyl ring, the π-π
interactions with the phenyl rings of F262 and F622 are
increased, which likely explains the change in antimicrobial
properties (Figure 5C). Chlorobenzyl group perfectly fits the B-
pocket while the central core of the compound is well stabilized
by the hydrogen bonds. The presence of a second ortho
chlorine substitution on ring B (FMD-94) causes a decrease in
the biological activity, as compared to FMD-89 (Table 2). This
could be explained by a reduction of π-π stacking interactions
with the phenyl rings of F262 and F622 (Figure 5D) since the
aromatic ring B is no longer in a favourable position, due to the
presence of the chlorine atom in the two ortho positions of ring
B. These results demonstrate that the additional chloride group
alters the binding in the B-pocket inducing a change in
conformation of the central core.
2.4. Toxicology and Pharmacokinetics Studies
To evaluate the preclinical pharmacokinetic profiles of PIPD1,
survival curves were assessed for 96 h in BALB/c mice, following
intraperitoneal (IP) injection with increasing concentrations of
PIPD1, ranging from 10 mg/kg to 250 mg/kg. Whereas IP
administration of the higher dose led to rapid death after 48 h,
injection of 100 mg/kg was associated with a 67% survival rate
and administration of 50 mg/kg led to 100% survival after 96 h
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Figure 5. A) Interaction diagram of PIPD1 in the binding pocket of MmpL3. Residues contouring the active site are represented in green while H-bounded
residues to ligands are highlighted in red. B) Molecular representation of the interaction of PIPD1 and MmpL3 pocket. C) Binding mode of the FMD-89
compound. D) Comparison of FMD-89 (purple sticks) and FMD-94 (turquoise sticks). E) Interaction diagram of FMD-61 in the binding pocket of MmpL3. F)
Molecular representation of FMD-61 and MmpL3 pocket. G) Binding mode of the truncated derivative (FMD-0).
Following IP injection, the serum concentration was meas-
ured at 600 ng/mL after 5 min and the drug distribution was
found to be greater in the lungs, the kidney, the spleen and the
heart, as compared to the brain and the liver. The concen-
trations obtained after IP administration were 6.9 mL/min for
clearance, 1.9 L for the central volume of distribution, 4.9 mL/
min for inter-compartmental clearance and 2.0 L for the
peripheral volume of distribution. The elimination half-life was
about 3.2 h. On the other hand, serum concentrations after oral
administration remained below 100 ng/mL and the relative oral
bioavailability of the compound was estimated to be lower
than 10% (when compared to the IP route). Since PIPD1 is a
Figure 6. Steric hindrance of ortho substituent in ring B for FMD-89 (A) and
FMD-3 (B). Spheres radii are directly proportional to the Van der Waals radii
of each atoms.
lipophilic compound (log P=4.9), the digestive absorption is
expected to be important and the low oral bioavailability rather
suggests a strong hepatic first-pass effect, although other
mechanisms such as efflux by active transporters (P-glycopro-
tein or multidrug resistance proteins) cannot be excluded at
post-injection. According to these results, a dose of 50 mg/kg
was used as the working concentration for all subsequent
pharmacokinetics studies after IP administration or oral gavage.
The concentration vs time profiles in the serum and tissues are
shown in Figure 7.
this stage. The hypothesis of hepatic first-pass effect is
reinforced by the observation of a second peak in serum
concentration occurring after 8 h, suggesting the existence of
an entero-hepatic cycle. PIPD1 exhibited a good organ distribu-
tion after IP administration and was rapidly eliminated with a
half-life of about 3 h.
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per million relative to TMS (δ=0.00). Mass spectra were recorded
by means of a Waters Micromass Quattro II triple quadrupole LC
mass spectrometer equipped with electrospray ionization (ESI) and
atmospheric pressure chemical ionization (APCI) sources. Column
chromatography, carried out on silica gel (Merck Kieselgel 60) was
used for the purification of compound. Reactions were monitored
by thin-layer chromatography (TLC) coated silica gel plates and
detection by UV lamp. The purity degree of compound was
checked by NMR and MS analysis and proved to be higher than
95%.
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All chemicals reagents were purchased at Sigma-Aldrich. Dry THF
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was obtained from
Technology Inc. MA, USA, Pure-Solv)
a solvent purification system (Innovative
Figure 7. Pharmacokinetic analysis of PIPD1 in female BALB/c mice after
intraperitoneal injection (A, C) or oral gavage (B, D). (A) Concentration in sera
and (C) concentration in organs following a single 50 mg/kg dose
administered using the IP route. (B) Concentration in sera and (D)
concentration in organs following a single 50 mg/kg dose administered
orally.
General Procedure for the Synthesis of PIPD1-Derivatives
First step: 4-Piperidinone monohydrate hydrochloride (1 g,
6.5 mmol) is suspended in dichloromethane (DCM, 120 ml, 1.9 mol)
and methanol (4 ml, 0.09 mol). Then K2CO3 (2 g, 0.01 mol) is added.
The mixture is stirred during 30 min then of α-Bromo-O-xylene
(0.86 ml, 6.5 mmol) is added. The mixture is stirred during 12 h at
room temperature. The mixture is quenched with distillated water
(100 ml, 5.5 mol) then the aqueous layer is extracted by DCM (2×
100 ml, 3.2 mol). The organic layer is dried on NaSO4 before
filtration. Solvents are removed under reduce pressure. The residue
is purified on flash chromatography (silica gel) with a gradient
eluent (DCM: 100 to DCM/MeOH: 95/5)..
3. Conclusions
In the present report, SAR studies emphasize the contribution
of the two aromatic moieties (ring A and ring B) as well as the
importance of the steric hindrance of the substituent on ring B.
Based on molecular modeling studies, we propose that the size
of ring B in the binding cavity is limited and could explain poor
biological activity of the PIPD1-analogues comprising a large
group. Nevertheless, the stability of the analogues/MmpL3
complexes is enhanced when the ring B is totally blocked in the
active site. Moreover, we demonstrated that PIPD1 exhibits a
good organ distribution after IP administration in mice and was
rapidly eliminated with a half-life of about 3 h. Drugs with short
half-live that are totally cleared within a few hours only are
usually easier to manipulate, especially if they present toxicity
issues. With respect to oral administration, the low relative
bioavailability could be explained either by a poor intestinal
absorption or a strong metabolic clearance. Thus, investigation
of these processes for the newly synthesized analogues is
warranted by the advantages and ease of oral administration in
patients. In a forthcoming work PIPD1 will be modified in order
to increase polarity and to minimize phase I metabolism, based
on the PK study. Altogether, these results pave the way for the
development of a new generation of anti-MmpL3 inhibitors
with improved activity against M. abscessus.
Second step: Under N2, bromine derivative (1 eq) is dissolved in
anhydrous THF (5 ml, 0.06 mol for 1 eq of bromine derivative). The
°
solution is cooled at À 78 C before the slow addition of n-Butyl
Lithium solution (2,5 M in hexane ; 1 eq). The mixture is stirred
°
during 1 h at À 78 C. Under N2, the compound obtained in the first
step is dissolved in anhydrous THF (5 ml, 0.06 mol for 1 eq of
bromine derivative), then the reaction is stirred during 2 h at
°
À 78 C. The reaction is quenched by saturated solution of NH4Cl (at
°
À 78 C) then the mixture is abandoned at room temperature before
adding of of distillated water (50 ml, 2,8 mol). The mixture is
extracted by ethyle acetate (3×50 ml, 1.5 mol). The organic layer is
dried on Na2SO4 .before filtration. Solvants are removed under
reduce pressure. The residue is purified on flash chromatography
(silica gel) with a gradient eluent (DCM: 100 to DCM/MeOH: 95/5).
1-(2-methylbenzyl)piperidin-4-one
Experimental Section
1H NMR (300 MHz, CDCl3) δ 7.18 (dd, J=9.5, 7.8 Hz, 4H, H9, H10,
H11 and H12), 3.68 (m, 2H, H7), 2.92 (m, 2H, H2 and H4), 2.68 (m,
4H, H1 and H5), 2.67 (m, 2H, H2 and H4, 2.37 (m, 3H, H14). 13C NMR
(75 MHz, CDCl3) δ 205.7 (C6), 138.1 (C8), 136.5 (C13), 130.3 (C12),
128.1 (C9), 126.6 (C10), 125.3 (C11), 61.6 (C7), 51.0 (C2 and C4), 39.7
(C1 and C5), 19.4 (C14). M/Z=203.13 g/mol. Colorless oil. Yield=
95%
Chemistry
Nuclear magnetic resonance (1H, 13C, and 19F NMR) spectra were
recorded at room temperature on a Brüker AC 300 spectrometer.
1
TMS was used as an internal standard and CDCl3 as the solvent. H
NMR analyses were obtained at 300 MHz (s: singlet, br s: broad
singlet, d: doublet, t: triplet, dd: double doublet, td: double triplet);
13C NMR analyses were obtained at 75.4 MHz; and 19F NMR analyses
were obtained at 282 MHz. The chemical shifts (δ) are given in parts
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FMD-88
FMD-96
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1H NMR (300 MHz, CDCl3) δ 7.84(s, 1H, H18), 7.80 (d, J=9.0 Hz, 1H,
H3), 7.58 (d, J=8.3 Hz, 1H, H6), 7.42 (d, J=8.3 Hz, 2H, H5 et H16),
7.31 (t, J=7.2 Hz, 1H, H17), 6.94 (t, J=7.3 Hz, 1H, H15), 3.59 (s, 2H,
H13), 2.81 (d, J=10.6 Hz, 2H, H10 and H11), 2.58 (t, J=11.5 Hz, 2H,
H10 and H11), 2.13 (d, J=2.8 Hz, 2H, H9 and H12), 1.69 (d, J=
13.4 Hz, 2H, H9 and H12). 13C NMR (75 MHz, CDCl3) δ 147.9 (C14),
140.6 (C19), 139.6 (C5), 131.3 (C15), 130.6 (C1), 130.4 (C6), 129.4
(C17), 128.8 (C18), 128.1 (C3), 124.9(C2), 124.2 (q, C16), 121.2 (C4),
100.8 (C7), 71.1 (C8), 66.6 (C13), 49.1 (C10 and C11), 38.5 (C9 and
C12). M/Z=495.01 g/mol. Yellow oil. Yield=35%
1H NMR (300 MHz, CDCl3) δ 7.83 (s, 1H, H18), 7.79 (d, J=9.0 Hz, 1H,
H3), 7.60 (d, J=8.3 Hz, 1H, H6), 7.44 (d, J=8.3 Hz, 2H, H5 et H16),
7.21 (t, J=7.2 Hz, 1H, H17), 7.10 (t, J=7.3 Hz, 1H, H15), 3.72 (s, 2H,
H13), 2.75 (d, J=10.6 Hz, 2H, H10 and H11), 2.61 (t, J=11.5 Hz, 2H,
H10 and H11), 2.48 (s, 3H, CH3), 2.03 (d, J=2.8 Hz, 2H, H9 and H12),
1.70 (d, J=13.4 Hz, 2H, H9 and H12). 13C NMR (75 MHz, CDCl3) δ
147.8 (C14), 141.0 (C19), 135.7 (C5), 134.32 (C15), 131.28 (C1), 129.35
(C6), 129.1 (C17), 128.1 (C18), 127.1 (C3), 124.8 (C2), 124.2 (q, C16),
121.2 (C4), 71.2 (C7), 56.3 (C8), 48.9 (C13), 38.7 (C10 and C11), 20.0
(CH3). M/Z=417.87 g/mol. Brown oil. Yield=62%
FMD-93
FMD-89
1H NMR (300 MHz, CDCl3) δ 7.87 (s, 1H, H18), 7.83 (d, J=9.0 Hz, 1H,
H3), 7.59 (d, J=8.3 Hz, 1H, H6), 7.52 (d, J=8.3 Hz, 2H, H5 et H16),
7.28 (t, J=7.2 Hz, 1H, H17), 7.10 (t, J=7.3 Hz, 1H, H15), 3.59 (s, 2H,
H13), 2.81 (d, J=10.6 Hz, 2H, H10 and H11), 2.58 (t, J=11.5 Hz, 2H,
H10 and H11), 2.13 (d, J=2.8 Hz, 2H, H9 and H12), 1.69 (d, J=
13.4 Hz, 2H, H9 and H12). 13C NMR (75 MHz, CDCl3) δ 147.8 (C14),
137.6 (C19), 132.8 (C5), 131.3 (C15), 130.8 (C1), 130.7 (C6), 129.4
(C17), 128.5 (C18), 127.9 (C3), 127.3 (C2), 124.8 (q, C16), 124.2 (C4),
121.2 (C7), 71.1 (C8), 61.9 (C13), 49.2 (C10 and C11), 38.5 (C9 and
C12). M/Z=447.02 g/mol. Dark yellow oil. Yield=72%
1H NMR (300 MHz, CDCl3) δ 7.86 (s, 1H, H18), 7.81 (d, J=9.0 Hz, 1H,
H3), 7.60 (d, J=8.3 Hz, 1H, H6), 7.48 (d, J=8.3 Hz, 2H, H5 et H16),
7.34 (t, J=7.2 Hz, 1H, H17), 7.22 (t, J=7.3 Hz, 1H, H15), 3.69 (s, 2H,
H13), 2.81 (d, J=10.6 Hz, 2H, H10 and H11), 2.53 (t, J=11.5 Hz, 2H,
H10 and H11), 2.12 (d, J=2.8 Hz, 2H, H9 and H12), 1.67 (d, J=
13.4 Hz, 2H, H9 and H12). 13C NMR (75 MHz, CDCl3) δ 147.9 (C14),
136.0 (C19), 131.4(C5), 130.8 (C15), 130.7 (C1), 129.6 (C6), 129.4
(C17), 128.3 (C18), 128.0 (C3), 126.7 (C2), 124.9 (q, C16), 124.3 (C4),
121.3 (C7), 71.1 (C8), 59.4 (C13), 49.3 (C10 and C11), 38.6 (C9 and
C12). M/Z=404.24 g/mol. Brown oil. Yield=50%
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FMD-91
FMD-94
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1H NMR (300 MHz, CDCl3) δ 7.84(s, 1H, H18), 7.80 (d, J=9.0 Hz, 1H,
H3), 7.40 (d, J=8.3 Hz, 1H, H6), 7.58 (d, J=8.3 Hz, 2H, H5 et H16),
7.46 (t, J=7.2 Hz, 1H, H17), 7.28 (t, J=7.3 Hz, 1H, H15), 3.53 (s, 2H,
H13), 2.75 (d, J=10.6 Hz, 2H, H10 and H11), 2.45 (t, J=11.5 Hz, 2H,
H10 and H11), 2.10 (d, J=2.8 Hz, 2H, H9 and H12), 1.70 (d, J=
13.4 Hz, 2H, H9 and H12). 13C NMR (75 MHz, CDCl3) δ 147.9 (C14),
136.6 (C19), 132.9 (C15), 131.3 (C1), 130.7 (C6), 130.5 (C17), 129.3
(C18), 128.4 (C3), 124.8 (C2), 124.1 (q, C16), 121.1 (C4), 100.8 (C7),
71.0 (C8), 62.27 (C13), 49.1 (C10 and C11), 38.4 (C9 and C12). M/Z=
404.24 g/mol. Brown oil. Yield=80%
1H NMR (300 MHz, CDCl3) δ 7.84(s, 1H, H18), 7.58 (d, J=9.0 Hz, 1H,
H3), 7.43 (d, J=8.3 Hz, 1H, H6), 7.30 (d, J=8.3 Hz, 2H, H5 et H16),
7.15 (t, J=7.2 Hz, 1H, H17), 3.82 (s, 2H, H13), 2.85 (d, J=10.6 Hz, 2H,
H10 and H11), 2.68 (t, J=11.5 Hz, 2H, H10 and H11), 2.06 (d, J=
2.8 Hz, 2H, H9 and H12), 1.70 (d, J=13.4 Hz, 2H, H9 and H12). 13C
NMR (75 MHz, CDCl3) δ 147.8 (C14), 137.0 (C19), 134.5 (C15), 131.3
(C1), 130.6 (C6), 129.4 (C17), 128.9 (C18), 128.4 (C3), 128.3 (C2),
124.8 (q, C16), 124.1 (C4), 121.2 (C7), 71.1 (C8), 56.6 (C13), 49.1 (C10
and C11), 38.5 (C9 and C12). M/Z=438.68 g/mol. Brown oil. Yield=
32%
FMD10
FMD-3
1H NMR (300 MHz, CDCl3) δ 7.84 (s, 1H, H13), 7.55 (d, J=9 Hz, 1H,
H17), 7.46 (d, J=9 Hz, 1H, H18), 7.16 (t, J=9 Hz, 1H, H3), 6.98 (d, J=
9 Hz, 1H, H5), 6.78 (d, J=9 Hz, 21H, H2 and H4), 3.75 (s, 2H, H7),
2.89 (d, J=12 Hz, 2H, H9 and H9’), 2.61 (t, J=12 Hz, 2H, H9 and
H9’), 2.08 (t, J=12 Hz, 2H, H10 and H10’), 1.75 (d, J=12 Hz, 2H, H10
and H10’). 13C NMR (75 MHz, CDCl3) δ 157.69 (CIV, C1), 147.26 (CIV,
C16), 131.53 (CHarom, C18), 131.08 (CIV, C14), 128.91 (CIV, C17), 124.78
(CIV, C15), 124.22 (CHarom, C13), 121.26 (CIV, C6), 119.43 (CHarom, C4),
116.08 (CHarom, C2), 70.69 (CIV, C11), 61.51 (CH2, C7), 48.68 (2CH2, C9
and C9’), 38.24 (2CH2, C10 and C10’). M/Z=385.11 g/mol. Brown oil.
Yield=41%
1H NMR (300 MHz, CDCl3) δ 7.86 (s, 1H, H12), 7.60 (d, J=9.0 Hz, 1H,
H2), 7.43 (m, 2H, H3 et H15), 7.23 (m, 1H, H5), 7.08 (m, 2H, H4 et
H16), 3.65 (s, 2H, H7), 2.81 (d, J=9.0 Hz, 2H, H8 et H8’), 2.52 (t, J=
12 Hz, 2H, H8 et H8’), 2.14 (t, J=9 Hz, 2H, H9 et H9’), 1.68 (d, J=
12 Hz, 2H, H9 et H9’). 13C NMR (75 MHz, CDCl3) δ163.19 (CIV, C1),
159.93 (CIV, C14), 148.04 (CIV, C13), 131.77 (CHarom, C3), 131.38
(CHarom, C15), 129.41 (CIV, C11), 129.08 (CHarom, C2), 128.97 (CHarom
,
C5), 124.31 (CIV, C6), 124.24 (CIV, C4), 124.17 (CIV, C12), 123.97 (CIV,
C17), 115.40 (CHarom, C16), 71.02 (CIV, C10), 55.44 (CH2, C7), 48.99
(2CH2, C8 et C8’), 38.52 ( 2CH2, C9 et C9’). M/Z=387.10 g/mol.
Brown oil. Yield=44%
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FMD-15
FMD-61
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1H NMR (300 MHz, CDCl3) δ 7.77 (d, J=18.5 Hz, 1H, H17), 7.71 (s, 1H,
H22), 7.61 (s, 1H, H3), 7.59 (s, 1H, H15),7.44 (d, J=18.5 Hz, 1H, H15),
7.42 (m, 2H, H18 and H19), 7.28 (s, 1H, H6), 7.21 (d, J=36.3 Hz, 1H,
H5), 3.63 (s, 2H, H13), 2.67 (m, 2H, H10 and H11), 2.49 (m, 2H, H10
and H11), 2.14 (m, 2H, H9 and H12), 1.87 (m, 2H, H9 and H12). 13C
NMR (75 MHz, CDCl3) δ 139.1 (C4), 138.1 (C14), 136.1 (C2), 134.4
(C16), 134.2 (C1), 134.0 (C21), 131.3 (C5), 129.7 (C6), 128.9 (C17),
128.8 (C22), 128.8 (C15), 128.4 (C23), 128.3 (C20), 127.0 (C18), 125.6
(C19), 124.4 (C3), 124.0 (C7), 73.7 (C8), 64.2 (C13), 49.2 (C10 and
C11), 37.5 (C9 and C12). M/Z=419.30 g/mol. Light brown oil.
Yield=19%
1H NMR (300 MHz, CDCl3) δ 7.86 (s, 1H, H12), 7.60 (d, J=9.0 Hz, 1H,
H2), 7.45 (m, 2H, H3 et H15), 7.37–7.30 (m, 1H, H1 and H5), 7.21 (m,
2H, H4 et H5), 3.65 (s, 2H, H7), 2.81 (d, J=9.0 Hz, 2H, H8 et H8’), 2.52
(t, J=12 Hz, 2H, H8 et H8’), 2.14 (t, J=9 Hz, 2H, H9 et H9’), 1.68 (d,
J=12 Hz, 2H, H9 et H9’). 13C NMR (75 MHz, CDCl3) δ 147.9 (C14),
139.6 (C5), 131.3 (C15 and C19), 130.6 (C1), 130.4 (C6), 129.4 (C17),
128.8 (C16 and C18), 128.1 (C3), 124.9(C2), 121.2 (C4), 100.8 (C7),
71.1 (C8), 66.6 (C13), 49.1 (C10 and C11), 38.5 (C9 and C12). M/Z=
369.11 g/mol. Brown oil. Yield=55%
FMD-33
FMD-63
1H NMR (300 MHz, CDCl3) δ 7.48 (s, 1H, H5), 7.28 (s, 1H, H6), 7.23 (m,
1H, H18), 7.20 (m, 1H, H5), 7.16 (m, 2H, H15 and H19), 7.09 (s, 1H,
H17), 3.60 (s, 2H, H13), 2.67 (m, 2H, H10 and H11), 2.35 (s, 3H, CH3),
2.32 (m, 2H, H10 and H11), 2.13 (m, 2H, H9 and H12), 1.93 (m, 2H,
H9 and H12). 13C NMR (75 MHz, CDCl3) δ 139.1 (C14), 139.1 (C4),
138.4 (C16), 136.1 (C2), 134.2 (C1), 131.3 (C5), 129.7 (C6), 128.4
(C18), 127.7 (C15), 127.5 (C17), 125.9 (C19), 124.4 (C3), 124.0 (C7),
73.7 (C8), 64.0 (C4), 49.2 (C10 and C11), 37.5 (C9 and C12) 21.2
(CH3). M/Z=383.13 g/mol. Brown oil. Yield=49%
1H NMR (300 MHz, CDCl3) δ 7.56 (s, 1H, H3), 7.49 (s, 1H, H1), 7.32 (d,
J=33.1 Hz, 1H, H5), 7.28–7.28 (m, 1H, H6), 7.20 (m, 1H, H16), 7.18
(dd, J=9.3, 8.3 Hz, 3H, H17, H18 and H19), 3.96 (s, 2H, H13)„ 2.68–
2.63 (m, 2H, H11 and H10), 2.46 (m, 2H, H11 and H10), 2.36 (s, 3H,
CH3), 2.14 (m, 2H, H9 and H12), 1.87 (m, 2H, H9 and H12). 13C NMR
(75 MHz, CDCl3) δ 139.9 (C4), 138.1 (C14), 136.5 (C19), 135.9 (C2),
130.3 (C18), 130.0 (C5), 129.9 (C6), 128.1 (C15), 126.6 (C16), 126.2
(C1), 125.3 (C17), 125.2 (C3), 124.1 (C7), 73.7 (C8), 61.6 (C13), 49.2
(C10 and C11), 37.5 (C9 and C12), 19.4 (CH3). M/Z=349.17 g/mol.
Black oil. Yield=27%
FMD-66
1H NMR (300 MHz, CDCl3) δ 7.48 (s, 1H, H3), 7.23 (dd, J=25.6,
14.4 Hz, 1H, H6), 7.22 (s, 1H, H5), 7.20 (m, 2H, H15 and H19), 7.16
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(m, 2H, H16 and H18), 3.56 (s, 2H, H13), 2.67 (m, 2H, H10 and H11),
2.34 (s, 3H, CH3), 2.32 (m, 2H, H10 and H11), 2.13 (m, 2H, H9 and
H12), 1.93 (m, 2H, H10 and H11). 13C NMR (75 MHz, CDCl3) δ 139.1
(C1), 136.1 (C2), 135.2 (C17), 134.4 (C14), 134.2 (C1), 131.3 (C5),
129.8 (C16 and 18), 129.7 (C6), 129.5 (C15 and C19), 124.4 (C3),
124.0 (C7), 73.7 (C8), 63.7 (C13), 49.2 (C10 and C11) 37.5 (C9 and
H12), 21.1 (CH3). M/Z=383.13 g/mol. Brown oil. Yield=68%
FMD103
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4
5
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8
FMD-32
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1H NMR (300 MHz, CDCl3) δ 7.80 (s, 1H, H1), 7.66 (m, 2H, H3 and H5),
7.20 (m, 1H, H15), 7.18 (dd, J=10.5, 4.7 Hz, 1H, H18), 7.17 (m, 2H,
H16 and H17), 3.72 (s, 2H, H13), 2.65 (m, 1H, H10 and H11), 2.45 (m,
2H, H10 and H11), 2.38 (s, 3H, CH3), 2.15 (m, 2H, H9 and H12), 1.89
(m, 2H, H9 and H12). 13C NMR (125 MHz, CDCl3) δ 143.0 (C4), 139.0
(C2 and C6), 138.1 (C14), 136.5 (19), 130.3 (C18), 128.6 (C3 and C5),
128.1 (C15), 126.6 (C16), 125.3 (C1 and 17), 124.4 (C7 and C20), 73.7
(C8), 61.6 (C13), 49.2 (C10 and C11), 37.5 (C9 and C12) 19.4 (CH3). M/
Z=417.15 g/mol. Brown oil. Yield=62%
1H NMR (300 MHz, CDCl3) δ 7.30 (s, 1H, H6), 7.27 (s, 1H, H2), 7.18 (m,
2H, H3 and H5), 7.17 (s, 1H, H14), 7.15 (s, 1H, H17), 7.14 (s, 1H, H16),
7.13 (s, 1H, H15), 3.98 (s, 2H, H12), 2.65 (m, 2H„ H9 and H10), 2.35
(m, 5H, H9, H10 and CH3), 2.13 (m, 2H, H8 and H13), 1.93 (m, 2H, H8
and H13). 13C NMR (75 MHz, CDCl3) δ 142.1 (C4), 138.1 (C13), 136.5
(C18), 136.2 (C1), 130.3 (C17), 129.4 (C2 and C6), 128.1 (C3 and C5),
128.1 (C3 and C5), 126.6 (C15), 125.3 (C16), 73.6 (C7), 61.6 (C12),
49.2 (C9 and C10), 37.5 (C8 and C11), 19.4 (CH3). M/Z=315.14 g/
mol. Yellow oil. Yield=57%
FMD-9
FMD-6
1H NMR (300 MHz, CDCl3) δ 7.81 (s, 1H, H6), 7.52 (d, J=9 Hz, 1H,
H21), 7.40 (d, J=9 Hz, 1H, H20), 6.83 (s, 2H, H3 and H7), 3.49 (s, 2H,
H10), 2.71 (d, J=9 Hz, 2H, H11 and H11’), 2.36 (d, J=9 Hz, 6H, H8
and H9), 2.24 (d, J=6 Hz, 3H, H1), 1.95 (d, J=9 Hz, 2H, H12 and
H12’), 1.61 (d, J=9 Hz, 2H, H12 and H12’). 13C NMR (75 MHz, CDCl3)
δ 148.0 (C5), 137.4 (C2, C4 and C6), 136.5 (C19), 132.1 (C17), 132.1
(C21), 129.4 (C20), 129.1 (C3 and C7), 124.9 (C15), 124.2 (C16), 121.3
(C18), 71.4 (C13), 59.1 (C10), 48.93 (2CH2, C11 and C11’), 38.85
(2CH2, C12 and C12’), 20.97 (CH3, C1), 20.17 (2CH3, C8 and C9). M/
Z=411.16 g/mol. Brown oil. Yield=28%
1H NMR (300 MHz, CDCl3) δ 7.83 (s, 1H, H7), 7.52 (d, J=9 Hz, 1H,
H10), 7.41 (d, J=9 Hz, H11), 4.10 (s, 2H, Cp), 4.08 (s, 5H, Cp’), 4.06 (s,
2H, Cp), 3.38 (s, 2H, H1), 2.7 (d, J=12 Hz, 2H, H3 et H3’), 2.39 (t, J=
12 Hz, 2H, H3 et H3’), 2.06 (t, J=9 Hz, 2H, H4 et H4’), 1.59 (d, J=
9 Hz, 2H, H4 et H4’). 13C NMR (75 MHz, CDCl3) δ 131.20 (CHarom, C11),
130.36 (CIV, C9), 129.36 (CHarom, C10), 128.00 (CIV, C8), 124.82 ((CIV,
C6), 124.23 (CHarom, C7), 121.20 (CIV, C12), 70.52 (CIV, C5), 70.39
(2CHarom, Cp), 68.54 (5CHarom, Cp’), 68.18 (2CHarom, Cp), 58.18 (CH2,
C1), 48.33 (2CH2, C3 et C3’), 37.96 (2CH2, C4 et C4’). M/Z=477.08 g/
mol. Yellow oil. Yield=46%
FMD-92
1H NMR (300 MHz, CDCl3) δ 7.74 (s, 1H, H1), 7.59 (m, 2H, H3 and H5),
7.32 (s, 1H, H15), 7.21 (t, J=15.1 Hz, 3H, H17, H18 and H19), 3.67 (s,
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2H, H13), 2.64 (m, 2H, H10 and H11), 2.46 (m, 2H, H10 and H11),
2.15 (m, 2H, H9 and H12), 1.95 (m, 2H, H9 and H12). 13C NMR
(75 MHz, CDCl3) δ 143.0 (C4), 141.2 (C14), 139.0 (C2 and C6), 133.3
(C16), 129.2 (C18), 128.6 (C15), 128.6 (C3 and C5), 127.1 (C19), 126.7
(C17), 125.3 (C1), 124.4 (C7 and C20), 73.67, 64.0 (C13), 49.2 (C10
and C11), 37.5 (C9 and C12). M/Z=403.07 g/mol. Dark yellow oil.
Yield=41%
FMD-64
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5
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FMD-102
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1H NMR (300 MHz, CDCl3) δ 7.56 (d, J=1.5 Hz, 2H, H5 and H17), 7.34
(s, 1H, H19), 7.30 (m, 1H, H1), 7.24 (m, 2H, H3 and H20), 7.21 (m, 1H,
H18), 3.87 (s, 2H, H14), 2.78 (m, 2H, H10 and H12), 2.46 (m, 2H, H10
and H12), 2.18 (m, 2H, H9 and H13), 1.91 (m, 2H, H9 and H13). 13C
NMR (75 MHz, CDCl3) δ 139.1 (C4), 136.1 (C6), 134.7 (C15), 134.2
(C2), 132.0 (C19), 131.3 (C3), 129.9 (C20), 129.7 (C1), 128.0 (C16),
126.7 (C18), 126.6 (C17), 125.0 (C22), 124.4 (C5), 124.0 (C26), 73.7
(C7), 61.7 (C14), 49.2 (C10 and C12) 37.5 (C9 and C13). M/Z=
437.10 g/mol. Yellow oil. Yield=34%
1H NMR (300 MHz, CDCl3) δ 7.82 (m, 1H, H3), 7.79 (m, 2H, H6 and
H10), 7.78 (s, 1H, H7) 7.48–7.40 (m, 3H, H1, H2 and H9), 7.22–7.14
(m, 4H, H21, H22, H23 and H24), 3.64 (s, 2H, H18), 2.69 (m, 2H, H14
and H16), 2.44 (m, 2H; H14 and H16), 2.34 (s, 3H, H25), 2.16 (m, 2H,
H13 and H17), 1.89 (m, 2H, H13 and H17). 13C NMR (75 MHz, CDCl3)
δ 142.3 (C8), 138.1 (C19), 136.5 (C20), 134.5 (C5), 134.0 (C4), 130.3
(C21), 130.2 (C10), 129.7 (3), 128.2 (C6), 128.1 (C24), 127.0 (C1),
126.6 (C23 and C9), 126.5 (C2), 125.3 (C22), 123.4 (C7), 73.5 (C11),
61.6 (C18), 49.2 (C14 and C16), 37.5 (C13 and C17), 19.4 (C25). M/
Z=331.19 g/mol. White oil. Yield=71%
FMD-62
FMD-99
1H NMR (300 MHz, CDCl3) δ 7.55 (s, 1H, H5), 7.30 (s, 1H, H1), 7.21 (s,
1H, H3), 7.05 (m, 2H, H16 and H20), 6.68 (m, 2H, H17 and H19), 3.44
(s, 2H, H14), 3.41 (m, 2H, H23 and H27), 3.14 (m, 2H, H23 and H27),
2.65 (m, 2H, H10 and H12), 2.42 (m, 2H, H10 and H12), 2.13 (m, 2H,
H9 and H13), 1.90 (m, 2H, H9 and H13), 1.68 (m, 4, H24 and H26),
1.62 (m, 2H, H25). 13C NMR (75 MHz, CDCl3) δ 148.7 (C18), 139.1
(C4), 136.1 (C6), 134.2 (C2), 131.3 (C3), 129.7 (C1), 129.4 (C16 and
C20), 128.6 (C15), 124.4 (C5), 124.0 (C28), 114.1 (C17 and C19), 73.7
(C7), 63.7 (C14), 49.7 (C23 and C27), 49.2 (C10 and C12), 37.5 (C9
and C13), 25.1 (C24 and C26), 23.4 (C25). M/Z=452.18 g/mol.
Brown oil. Yield=41%
1H NMR (300 MHz, CDCl3) δ 7.29 (m, 2H, H1 and H6), 7.25 (m, 2H, H3
and H5), 7.21 (m, 2H, H2 and H20), 7.18 (m, 1H, H19), 7.17 (m, 1H,
H18), 7.16 (m, 1H, H16), 3.67 (s, 2H, H14), 2.72 (m, 2H, H10 and H12),
2.50 (m, 2H, H10 and H12), 2.34 (s, 3H, H21), 2.18 (m, 2H, H9 and
H13), 1.91 (m, 2H, H9 and H13). 13C NMR (75 MHz, CDCl3) δ 143.4
(C4), 138.1 (C15), 136.5 (C16), 130.3 (C17), 128.5 (C1 and C6), 128.2
(C2), 128.1 (C20) 126.6 (C19), 126.3 (C3 and C5), 125.3 (C18), 73.6
(C7), 61.6 (C14), 49.2 (C10 and C12), 37.5 (C9 and C13), 19.4 (C21).
M/Z=281.18 g/mol. Colorless oil. Yield=52%
FMD46
1H NMR (300 MHz, CDCl3) δ 7.50 (s, 1H, H5), 7.44 (s, 1H, H2), 7.27 (d,
J=12.4 Hz, 2H, H1 and H3), 3.11 (s, 1H, H8), 2.85 (m, 2H, H10 and
H12), 2.75 (m, 2H, H10 and H12), 2.11 (m, 2H, H9 and H13), 1.90 (m,
2H, H9 and H13). 13C NMR (75 MHz, CDCl3) δ 140.0 (C4), 135.9 (C6),
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130.0 (C3), 129.9 (C1), 126.2 (C2), 125.2 (C5), 124.1 (C14), 72.9 (C7),
44.1 (C10 and C12), 37.8 (C9 and C13). M/Z=245.10 g/mol. Light
brown oil. Yield=53%
Biology
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4
5
Cytotoxicity assay. Cell viability was determined using Vero
epithelial cells which were grown in RPMI medium containing 10%
fetal bovine serum (RPMIÀ FBS) and differentiated with 20 ng/ml
°
phorbol myristate acetate (PMA) and incubated for 48 h at 37 C,
FMD-0
under a 5% CO2 atmosphere. Cells were seeded in 96-well plates at
a density of 2×104 cells/well in 160 μl medium and incubated
6
°
7
8
9
overnight at 37 C to allow cells to adhere. Compounds (dissolved
in DMSO) were freshly diluted in RPMI starting at a concentration of
100 μg/ml up to a concentration of 0.048 μg/ml (serial dilution of
factor 2), and 20 μl of those dilution were added in corresponding
wells. The maximum final concentration of DMSO was 1% (v/v).
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°
After 24 h incubation at 37 C with 5% CO2, 20 μl of 1 mg/ml
resazurin (Sigma, Germany) was added to each well and the cells
°
incubated for an additional 2 hours at 37 C in the presence of 5%
1H NMR (300 MHz, CDCl3) δ 7.50 (s, 6H), 7.29 (s, 2H), 7.24 (d, J=
38.3 Hz, 10H), 2.88–2.81 (m, 12H), 2.78–2.71 (m, 12H), 2.35 (s, 6H),
2.12–2.08 (m, 9H), 1.92–1.86 (m, 11H), 1.71 (s, 6H). 13C NMR (75 MHz,
CDCl3) δ 139.1 (C4), 136.1 (C6), 134.2 (C2), 131.3 (C3), 129.7 (C1),
124.4 (C5), 124.0 (C15), 72.9 (C7), 44.1 (C10 and C12), 37.8 (C9 and
C13). M/Z=279.06 g/mol. Brown oil. Yield=25%
CO2. Fluorescence was then measured in a Polarstar Omega
fluorometer using appropriate filters (590 nm emission and 540 nm
excitation wavelength). Percentage survival was determined by
dividing fluorescence values obtained in the compound containing
wells by values obtained for control wells containing cells
incubated with a dilution series of DMSO (serial dilution of factor 2
from 1% to 0.000488% v/v). Experiments were complete in
triplicate on three independent events.
FMD-16
Drug susceptibility testing. To determine the MICs, broth micro-
dilution method in CaMHB using an inoculum containing 5×
10 CFU/ml in the exponential-growth phase were achieved follow-
ing the CLSI guidelines.[28] Briefly, 100 μl of bacteria were seeded in
96-well plates, and 2 μl of drug at its highest concentration in a
maximum of 1% of DMSO (v/v) was added to the first wells
containing 200 μl of bacterial suspension and a twofold serial
dilutions were then done out. Incubation with drugs was performed
°
at 30 C for 3 to 5 days. MICs were recorded by visual inspection
and by absorbance at 560 nm to confirm visual recording. Experi-
ments were done in triplicate on three independent occasions.
1H NMR (300 MHz, CDCl3) δ 7.22 (s, 1H, H10), 7.16 (m, 3H, H11, H12
and H13), 3.67 (s, 2H, H8), 3.61 (m, 1H, H6), 2.65 (m, 2H, H2 and H4),
2.35 (s, 3H, H15), 2.31 (m, 2H, H2 and H4), 1.94 (m, 2H, H1 and H5),
1.75 (m, 2H, H1 and H5). 13C NMR (75 MHz, CDCl3) δ 138.1 (C9),
136.5 (C14), 130.3 (C13), 128.1 (C10), 126.6 (C11), 125.3 (C12), 65.6
(C6), 61.6 (C8), 51.1 (C2 and C4), 34.5 (C1 and C5), 19.4 (C15). M/Z=
205.15 g/mol. Yellow oil. Yield=74%
Molecular Modelling
Comparative modelling of MAB_4508 was performed using
Modeller[35] and the crystal structure (2.8 Å, PDB ID: 6AJJ) of a
mycolic acid transporter from M. smegmatis (Sequence identity:
64.9%) as template. Quality of the model was assessed using
MolProbity.[36] Subsequent energy minimization of the global model
was performed using AMBER forcefield in order to remove putative
steric clashes between side chains. Simulation of the binging
modes of the target and the designed compounds was performed
using GOLD docking program.[37] GOLD is based on a genetic
algorithm and considered the ligand as flexible, while side chains of
most residues are kept rigid. For the search procedure, a sphere of
10 Å was centred on the X, Y and Z coordinates of our identified
binding centre. Then, all the different binding poses were scored
with the ChemPLP scoring function. Subsequent energy minimiza-
tion was performed using the AMBER forcefield. Finally, all the
molecular representations were sketched using PyMOL.[38]
FMD-37
Toxicologic and Pharmacokinetics Studies; Mouse Blood and
Tissue Sampling
1H NMR (300 MHz, CDCl3) δ 7.50 (s, 1H), 7.23 (d, J=1.1 Hz, 2H, H19
and H20), 7.18 (m, 2H, H10 and H11), 7.15 (m, 1H, H12), 7.12 (m, 1H,
H13), 6.31 (s, 1H, H5), 4.07 (s, 1H, H8), 3.56 (m, 2H, H4), 3.18 (s, 1H,
H8), 2.80 (s, 1H, H2), 2.56 (s, 1H, H2), 2.51 (m, 2H, H1), 2.33 (m, 3H,
H15). 13C NMR (75 MHz, CDCl3) δ 140.7 (C7), 138.1 (C9), 136.5 (C14),
132.3 (C17), 132.3 (C20), 130.9 (C19), 130.8 (C6), 130.4 (C18), 130.3
(C13), 128.1 (C10), 126.6 (C11), 125.3 (C12), 124.4 (C16), 124.0 (C22),
119.2 (C5), 60.0 (C8), 52.3 (C4), 51.2 (C2), 28.7 (C1), 19.4 (C15). M/Z=
365.12 g/mol. Brown oil. Yield=19%
Experimental compound PIPD1 was administered either intraper-
itoneally, intraveinously or by oral gavage. For toxicologic studies,
PIPD1 was diluted in DMSO at different concentrations in order that
mice receive either intraperitoneally or intraveinously the doses of
250 mg/kg, 100 mg/kg, 50 mg/kg and 10 mg/kg in a total volume
of 50 μl corresponding respectively to 5 mg, 2, 1 and 0.2 mg/mice.
Control groups were administered with the equivalent volume of
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DMSO alone. Groups were constituted of n=3 mice. Mice were
observed during 4 days. For pharmacokinetic studies, the chosen
concentration was 50 mg/kg, i.e. 1 mg/mice for intraperitoneally
administration. 100 μl of blood was taken from from the retro-
orbital plexus at different time points (5 min, 1 h, 3 h, 5 h, 6 h, 8 h,
were performed using the Chromeleon v6.80 (Thermo Scientific
Dionex) and Xcalibur v3.0.63 (Thermofisher) softwares.
1
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9
A calibration curve including a zero and seven calibration standards
was performed with each series of experiments. Quantitation was
achieved by plotting the peak area ratios of PIPD1 to the internal
standard versus concentration followed by mean least squares
linear regression. A 1/x weighing factor was applied to the linear
regression to correct for data heteroscedasticity. The lower limit of
quantification (LLOQ) was 0.1 ng/ml for plasma samples and 0.1 pg/
mg for tissue samples, the upper limit of quantification (ULOQ) was
5000 ng/mL and 5000 pg/mg, respectively. Data for tissue analysis
are expressed as ng/μg of tissue; for plasma as ng/ml.
°
12 h, 18 and 24 h), sera were separated after 1 h at 37 C for red
cells coagulation using centrifugation at 10,000 g during 10 min
°
and were stored at À 30 C until analysis. Organs (spleen, lungs,
liver, heart, kidneys and brain) were collected at the last time point
when mice were sacrificed. Experiment was repeated two times
with n=7 mice. For administration by oral gavage, mice (n=3)
received the same quantity of PIPD1, 1 mg/mice, diluted in a total
volume of 200 μl of glycerol. The time of organ collection were 4 h,
5 h and 24 h and sera collection were 1 h, 2 h, 3 h, 4 h, 5 h and
24 h.
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Pharmacokinetic Modelling
All procedures were applied according to the ethics guidelines
(Approval number APAFIS#11465-2016111417574906 from the
ethics committee A783223)
Data was analysed using the nonlinear mixed effect modelling
software program Monolix v4.4.0 (Lixoft, Orsay, France).[34] A two-
compartment open model was fitted and the parameters were
estimated by computing the maximum likelihood estimator of the
parameters without any approximation of the model (no lineariza-
tion) using the stochastic approximation expectation maximization
algorithm combined to a Markov Chain Monte Carlo procedure.
Concentrations of PIPD1 in organs and collected sera were analysed
by liquid chromatography/mass spectrometry (LC/MS) as described
below.
Quantification of PIPD1 in Murine Tissues and Fluids
Samples were processed using liquid-liquid extraction and PIPD1
was quantified with liquid chromatography coupled to mass
spectrometry. Briefly, ten microliters of the internal standard
working solution (penfluridol 10 μg/mL) and 1 ml of the extracting
solvent (tert-butyl methyl ether/hexane, 50/50 (v/v)) were added to
each tube containing either about 50 mg of tissue or 50 μl of
plasma. Solid tissue samples were crushed and homogenized using
a ball mill TissueLyser LT (Qiagen, Courtaboeuf, France) and a 5 mm
stainless steel bead for 20 min with an oscillation frequency of
50 Hz whereas cell culture supernatant samples were shaken for
10 min with an RM-2L Intelli-mixer (Elmi, Riga, Latvia). Tubes were
then centrifuged at 13,300 rpm for 10 min. The upper organic layer
was decanted into another tube and evaporated to complete
Acknowledgements
This work was supported by the French National Research Agency
MyCat Grant ANR-15-CE18-0007-02 (to L. K. and Y. G.).
Conflict of Interest
The authors declare no conflict of interest.
Keywords: mycobacterium abscessus · molecular modeling ·
°
dryness under vacuum at 43 C. Samples were reconstituted with
structure-activity relationship
piperidinol derivatives
·
phenotypic screening
·
80 μl of 0.1% formic acid/LC–MS methanol (30:70, v/v), then vortex
mixed for 30 s, and transferred into injection vials for analysis.
Chromatography was performed with an UltiMate 3000 Quaternary
Rapid Separation Pump LPG-3400RS (Thermo Scientific Dionex, Les
Ulis, France), using a 1.9 μm C18 Hypersil Gold column (100×
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°
2.1 mm i.d.) (Thermofisher) maintained at 30 C. 10 μl were injected
in the chromatographic system. Elution was achieved in the
isocratic mode with the following mobile phase composition: 30%
of aqueous buffer (0.1% formic acid) and 70% of methanol. The
flow rate was 200 μl/min at the start and until 1.8 min, then
changed to 300 μl/min until 4 min, after what it returned to 200 μl/
min. Total run time was 5.0 min.
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Guntupova, S. Safonova, PLoS One 2018, 13, e0203108.
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P. Moreno, D. Verma, E. Hill, J. Drijkoningen, P. Gilligan, Science 2016,
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spectrometer (Thermofisher) equipped with a heated electrospray
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Manuscript received: February 14, 2020
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