Structure-Activity Relationship and Mode-Of-Action Studies Highlight 1-(4-Biphenylylmethyl)-1H-imidazole-Derived Small Molecules as Potent CYP121 Inhibitors

Article abstract of DOI:10.1002/cmdc.202100283

CYP121 of Mycobacterium tuberculosis (Mtb) is an essential target for the development of novel potent drugs against tuberculosis (TB). Besides known antifungal azoles, further compounds of the azole class were recently identified as CYP121 inhibitors with

Full text of DOI:10.1002/cmdc.202100283

Accepted Article
Title: Structure-activity relationship and mode of action studies highlight
1-(4-biphenylylmethyl)-1H-imidazole derived small molecules as
potent CYP121 inhibitors
Authors: Sebastian Adam, Isabell Walter, Rolf Hartmann, Andreas
Kany, Jesko Köhnke, Maria Virginia Gentilini, Tim
Sparwasser, Christian Brengel, and Andreas Thomann
This manuscript has been accepted after peer review and appears as an
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content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.202100283
Link to VoR: https://doi.org/10.1002/cmdc.202100283
01/2020

10.1002/cmdc.202100283
ChemMedChem
FULL PAPER
Structure-activity relationship and mode of action studies
highlight 1-(4-biphenylylmethyl)-1H-imidazole derived small
molecules as potent CYP121 inhibitors
Isabell Walter+[a], Sebastian Adam+[b], Maria Virginia Gentilini[c], Andreas M. Kany[a], Christian Brengel[a],
Andreas Thomann[a], Tim Sparwasser[c], Jesko Köhnke[b], Rolf W. Hartmann[a,d]
*
[a]
Dr. Isabell Walter, Dr. Andreas Martin Kany, Dr. Christian Brengel, Dr. Andreas Thomann, Prof. Dr. Rolf W. Hartmann: Helmholtz Institute for Pharmaceutical
Research Saarland, HIPS, Department for Drug Design and Optimization, Campus E8.1, 66123, Saarbrücken, Germany. Email: Rolf.Hartmann@helmholtz-
hzi.de
[b]
[c]
[d]
[+]
Dr. Sebastian Adam, Prof. Dr. Jesko Köhnke: Workgroup Structural Biology of Biosynthetic Enzymes, Helmholtz Institute for Pharmaceutical Research
Saarland (HIPS), Helmholtz Centre for Infection Research (HZI), Saarland University, Saarbrücken, Germany.
Dr. Maria Virginia Gentilini, Prof. Dr. Tim Sparwasser: Institute of Infection Immunology, TWINCORE, Centre for Experimental and Clinical Infection Research,
A Joint Venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover, Germany.
Prof. Dr. Rolf W. Hartmann: Department of Pharmacy, Pharmaceutical and Medicinal Chemistry, Saarland University, Campus C2.3, 66123, Saarbrücken,
Germany
These authors contributed equally to this work.
ORCID:
Sebastian Adam 0000-0003-3319-6356; Andreas M Kany 0000-0001-7580-3658; Tim Sparwasser 0000-0001-5645-902X; Jesko Köhnke 0000-0002-
7153-1365
Supporting information for this article is given via a link at the end of the document.
Abstract: CYP121 of Mycobacterium tuberculosis (Mtb) is an
essential target for the development of novel potent drugs against
Tuberculosis (TB). Besides known antifungal azoles, further
compounds of the azole class were recently identified as CYP121
inhibitors with antimycobacterial activity. Herein we report the
screening of a similarity-oriented library based on the former hit
compound, the evaluation of affinity towards CYP121 and activity
against M. bovis BCG. The results enabled a comprehensive SAR
study, which was extended through the synthesis of promising
compounds and led to the identification of favorable features for
affinity and/or activity and hit compounds with 2.7-fold improved
potency. Mode of action studies show that the hit compounds inhibit
substrate conversion and highlighted CYP121 as the main
antimycobacterial target of our compounds. Exemplified complex
crystal structures of CYP121 with three inhibitors reveal a common
binding site. Engaging in both hydrophobic interactions as well as
hydrogen bonding to the sixth iron ligand, our compounds block a
solvent channel leading to the active site heme. Additionally, we report
the first CYP inhibitors that are able to reduce the intracellular
replication of M. bovis BCG in macrophages, emphasizing their
potential as future drug candidates against TB.
Mycobacterium tuberculosis complex, only found in some
Streptomyces strains with maximum 64 % identity.[7] The
elucidation of its function was encouraged by the identification of
the role of its neighbouring gene in Mtb which encodes a
cyclodityrosin (cYY) synthetase.[8] CYP121 was subsequently
identified to utilize cYY as substrate and to catalyze its conversion
to mycocyclosin via a C-C coupling reaction.[9] However, the
biological role of substrate and product remain unknown up to
now.
McLean et al. identified known antifungal azoles such as
econazole or ketoconazole as the first inhibitors of CYP121.[10]
Interestingly, the compounds also showed biological activity
against Mtb correlating with their respective affinities to
CYP121.[6] Furthermore, econazole exhibited activity against
multi-drug resistant Mtb, murine TB in vivo as well as latent TB in
vitro and in vivo.[11] [12] [13] In several subsequent studies, fragment-
based approaches were employed and small molecules binding
[15] [16] [17]
to CYP121 were identified.[14]
Additionally, some
substrate analogues were discovered to inhibit CYP121.[18]
However, none of these compounds was shown to exhibit any in
vitro activity toward Mtb (or M. bovis BCG). Recently, we
described a screening approach based on a CYP-inhibitor library
which resulted in the identification of the first potent inhibitors of
CYP121 with activity against M. bovis BCG and Mtb.[20]
[19]
Introduction
Tuberculosis is still one of the leading infectious diseases
worldwide with around 10 million new infections and 1.6 million
cases of deaths every year.[1] The disease gained more and more
attention in the last years due to arising resistances of its
pathogen Mycobacterium tuberculosis (Mtb) against currently
applied first-line drugs and its high prevalence in immune
deprived patients such as those infected with HIV.[1] [2] [3] With the
sequence of the genome deciphered in 1998, there were a lot of
new possible targets identified.[4] However, the cell wall of Mtb is
highly complex and forms a wax-like barrier with partly unknown
permeability behavior. This exceptional property impedes the
discovery of new inhibitors as they could possibly fail to reach
their target and thus appear biologically inactive. [5]
Based on this breakthrough in the search for potent CYP121
inhibitors, we herein employed the screening of a similarity-
oriented library in order to evaluate the possible structural
diversity of the hit scaffold and to identify favourable features for
affinity and/or activity. Furthermore, we investigated the ability of
our compounds to inhibit the enzymatic conversion in vitro and
their inhibitory effect on M. bovis BCG. Mode of action studies
after addition of the substrate cYY demonstrated the importance
of CYP121 inhibition for the antimycobacterial effect of our
compounds. Complex crystal structures of CYP121 were
elucidated to gain further insights into enzyme inhibition and
reveal crucial enzyme interactions that could be investigated for
further compound development. Additionally, the influence of
One of these identified targets is the Cytochrome-P450 enzyme
121 (CYP121) which was shown to be essential for the viability of
Mtb.[6] The gene encoding CYP121 is, besides the
1
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several CYP121 and CYP125 inhibitors on the intracellular
replication of M. bovis BCG in macrophages was evaluated.
promising motif regarding affinity, it was combined with the double
bond feature of the propylene linker resulting in an ethenyl
substituted methylene bridge.
Results and Discussion
Library generation and screening results
As a starting point for our screening approach, we chose the hit
compound I:47, which was recently identified in a screening of a
diversity oriented CYP-inhibitor library.[20] With its high affinity to
CYP121, biological activity against M. bovis BCG and Mtb as well
as its structure, which offers various optimization possibilities, it
serves as an ideal basis for further exploration of the binding and
activity properties of this structural class. The created screening
library was composed of 94 compounds which all can be assigned
to a general structure scheme derived from I:47 (Figure 1). The
imidazole moiety appeared to be essential for the binding to the
heme iron of the enzyme and thus was conserved.[20] Additionally
the template consists of a substituted methylene bridge (Figure 1,
motif C) or a propylene linker which connects the imidazole ring
with the middle aromatic moiety (Figure 1, motif B) and a Western
part aromatic moiety (Figure 1, motif A) which is directly linked to
the aforementioned one. The library covered various structural
motifs for each of the different parts ensuring a broad analysis of
structure-activity-relationships and offering the possibility to
discover highly potent hits. The structures of all library compounds
(L1 to L94) are displayed in the respective supporting information
(supporting information (SI), table S1).
The screening library was examined in a UV/Vis heme binding
assay regarding affinity toward CYP121 as previously
described.[20] The compounds were screened at concentrations of
100 µM and 20 µM. For 79 compounds, which showed a visible
shift of the absorption maximum at concentrations of 20 µM, the
binding constant KD was determined by concentration dependent
titration of the enzyme (respective KD values are listed in SI, table
S1). All of these binders showed a type-II shift identical to the
previously determined I:47,[20] as expected because of the
structural similarity (discussion on actual binding mode in
“complex crystallization” paragraph). 38 of the approved binders
(hit rate 40 %) exhibited a binding constant below or equal to I:47
(KD = 5 µM). The highest affinity was shown by compounds L89,
L16, L78 with KD values of 0.3, 0.5, and 0.6 µM, respectively.
Interestingly, these compounds have the methoxynaphthalene
moiety in position A in common which seems to be particularly
favorable for binding.
In parallel to affinity determination, the library was analyzed
regarding biological activity against M. bovis BCG in vitro. The
compounds were initially tested at a concentration of 100 µM and
inhibitors showing inhibition higher than or equal to 80 % were
further examined at 10 µM. For 29 compounds with inhibition
higher than or equal to 60 % at 10 µM, MIC50 values were
determined as described previously.[20] I:47 (MIC50 = 4.0 µM) was
used as a reference. Ten compounds (hit rate 11 %) showed
better activity than I:47. Interestingly, the two compounds with a
propylene linker (L15 and L21) exhibited the highest activity
(MIC50 = 1.8 µM and 1.5 µM).
Figure 1: Similarity-guided screening of a focused library. Compounds were
selected based on the former screening hit I:47 with variations in the three
displayed structure motifs. KD values were determined for 79 positive hits
showing visible shift of absorbance maximum at concentrations ≤20 µM. MIC50
values were determined for 29 compounds which showed inhibition ≥60% at 10
µM.
Compounds S1-S10 were prepared according to Scheme 1
starting with a Suzuki coupling of the corresponding boronic acid
and 4-bromobenzaldehyde or 4-bromo-2-fluorobenzaldehyde,
respectively. For compounds S9 and S10, the protected boronic
acids were synthesized beginning with a tert-butyldimethylsilyl
(TBDMS) protection of the hydroxyl function of 4-bromophenol or
the amino group of 5-bromo-1H-indole. The boronic acid function
was subsequently introduced by a substitution reaction with n-
butyllithium and triisopropylborate (scheme 2). The coupled
aldehydes were reduced in a Grignard reaction with vinyl
magnesium bromide to the corresponding alcohols with
simultaneous introduction of the ethenyl substituent.
Consequently, the resulting alcohols were converted to the
respective
imidazoles through
a
SNt reaction with
carbonyldiimidazole (CDI). The compounds were obtained as
racemates and principally no separation of the enantiomers was
performed. For the protected compounds, the final step was the
cleavage of the TBDMS group with tetrabutylammonium fluoride.
In contrast, compound S11, a fluorinated derivative of hit I:47, was
synthesized as described in scheme 3. The first step, a SNt
reaction of 4-bromo-2-fluorobenzylbromide with imidazole, was
followed by a Suzuki coupling of the intermediate with 3,4-
methylenedioxyphenylboronic acid resulting in the desired
compound. The respective KD values of the synthesized
derivatives S1-S11 are listed in table S3 in the supporting
information.
Chemical Synthesis
Based on the two most potent inhibitors identified in the screening,
L15 and L21, compounds with a novel substituent at the
methylene linker were designed. As the ethyl substitution was a
2
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Scheme 1: Reagents and conditions: (i) method A: Pd(PPh3)4, Na2CO3, toluene:EtO:H2O = 5:3:2, 100 °C, 1-4 h; (ii) method B: VinylMgBr, THF abs., -15 °C à rt,
1-2 h; (iii) method C: CDI, MeCN, 110 °C, 1-4 h.
Scheme 2: Reagents and conditions: (ia) method D a): TBDMSCl, imidazole, CH2Cl2, rt, 16 h; (ib) method D b): TBDMSCl, NaH, CH2Cl2, 0 °Cà rt, 16 h; (ii) method
E: nBuLi, B(OiPr)3, THF, -78 °C à rt, 3 h; (iii) method F: TBAF, K2HPO4, THF, rt, 16 h.
Scheme 3: Reagents and conditions: (i) imidazole, NaH, 60 °C, 1.5 h; (ii) method A: 3,4-methylene-dioxyphenylboronic acid, Pd(PPh3)4, Na2CO3, toluene:EtOH:H2O
= 5:3:2, 110 °C, 2.5 h.
Structure-affinity-relationship
connecting the methylene bridge to the adjacent phenyl moiety
via an ethylene linker, was also possible without loss of affinity
(L90). The affinity increased slightly (around twofold compared to
ethyl) for space filling substituents such i-propyl, n-butyl, phenyl
or two ethyl substituents indicating the involvement of
hydrophobic interactions. In contrast, a hydroxyethyl substituent
(L13) led to weaker binding compared to its ethyl analogue (L80),
which supports the aforementioned hypothesis.
Based on the determined binding constants, the compounds were
analyzed regarding their structural features in order to identify
favorable motifs that contribute positively to affinity. The different
parts of the binding compounds were, as far as possible,
considered separately to avoid interference of the influence of
several features.
First of all, the methylene bridge between imidazole ring and
aromatic moiety B tolerated various substituents such as methyl,
ethyl, i-propyl, n-butyl, ethenyl or phenyl (L75, L80, L92, L93, S7,
L1). Additionally, a decoration with two methyl (L27) or ethyl (L35)
substituents or the exchange with a propylene linker (L15) was
accepted as well. Moreover, the rigidification we recently
addressed in our docking study of I:47,[20] performed by
Compounds with a substituted methylene bridge were evaluated
as racemic mixtures. It is known that often the two enantiomers
show strong differences in binding affinity or activity. However, as
seen in the docking study of I:47, the binding pocket of CYP121
principally offers space to harbor both orientations of
substituents.[20] To confirm this hypothesis the enantiomers were
separated for three exemplarily chosen ethenyl derivatives (SI,
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table S4). As expected, only minor differences could be observed
between the two enantiomers of a racemate.
The used library contained compounds with various aromatic ring
systems in the middle part (Figure 1, motif B). According to
compounds bear substituents at the methylene bridge which
already impair activity.
previous findings, compounds with a pyridine ring in this position
show only weak binding.[20] A furan ring (L70, KD = 34 µM) was
rather unfavorable compared to an unsubstituted benzene
whereas a thiophene (2,4- or 2,5 connected) led in some cases
to similar affinities (L42, L60), in other cases, however, to weaker
affinities (L62, L64). Furthermore, other motives such as
fluorinated benzene, indole, benzothiophene, naphthalene or
quinolone (L47, L48, L86, L79, L82) were tolerated as well with
increased affinities (> twofold) for the latter two scaffolds.
Additionally, a range of motifs for the Western part aromatic ring
(Figure 1, motif A) was evaluated including differently substituted
phenyls, thiophene and furan rings and bicyclic ring systems such
as benzofuran, benzothiophen, indole and naphthalene. Nearly all
of those were tolerated with the exception of nitrogen containing
6-membered rings (L43, L59). However, some appeared to be
particularly favorable such as the bicyclic moieties with 6-
Figure 2 highlights the structural features identified as favorable
for affinity and/or activity. In total, L15 and L21 were the most
active compounds identified in this study with MIC50 values of 0.7
and 0.5 µg/mL, respectively (Table 1). The other eight
compounds with superior activity to I:47 (Table 1) were similarly
active with MIC50 values between 0.8 or 1.2 µg/mL. Noticeably, all
these compounds are not substituted at the methylene bridge
except L94 which contains a 5-membered ring structure. The
respective ethyl analogues of L46, L14 and L76 (compounds L18,
L20 and L37) showed similar affinities as their parent compounds
but failed to significantly inhibit mycobacterial growth.
methoxynaphthalene leading to the highest affinities (L16: KD
=
0.5 µM, L78: KD = 0.6 µM). Further motifs with increased affinities
(KD ≤ 6 µM) were electron deprived phenyls such as a meta- and
para-difluorinated phenyl (L34) and para-trifluoromethyl (L44) or
para-trifluoromethoxy phenyls (L22). Additionally, motifs with
substituents capable of hydrogen bond formation such as
methylthio decorated phenyl or thiophene (L23, L37) as well as
phenyls with a hydroxyl (L87) or a N-acetamide group (L7) in para
position also led to increased affinities.
Structure-activity-relationship
A good correlation between affinity and activity against M. bovis
BCG cannot necessarily be expected as the antimycobacterial
activity is influenced by additional cellular factors such as
permeability, efflux and metabolism. The features of compounds
exhibiting high or weak activity were therefore regarded in detail
to identify those which impair biological activity.
It is striking that, apart from the propylene linked structures (L15
and L21), the highly active compounds are either not substituted
at the methylene bridge (L10, L44) or possess a methyl
substituent (L73) or a rigid 5-membered ring structure (L94).
Other substituents such as ethyl (L9), ethenyl (S10) or i-propyl
(L92) led only to weak activity. Interestingly, the attempt to
combine the properties of the propylene linker with the ethyl
substituent of the methylene bridge via the introduction of an
ethenyl substituent did not lead to the desired highly biologically
active compound. In comparison with their non-substituted
analogues, the ethenyl derivatives show a loss in activity while
their affinity is similar (compare I:47 and S11 with S5 and S8).
Concerning the middle aromatic moieties (Figure 1, motif B),
benzene, fluorinated benzene, thiophene and indole were able to
maintain biological activity (L44, L14, L66, L48). Rather
unfavorable were bulky center parts such as naphthalene (L79)
and quinolone (L81). Concerning the Western part aromatic
moiety (Figure 1, motif A) trifluoromethylphenyl (L44) as well as
fluorinated phenyls (L14, L45) were accepted as well as various
Figure 2: Structure-affinity/activity-relationships. Favorable features for affinity
(KD) and activity (MIC50
) were identified through comparison of similar
compounds. Features leading to good affinity are framed in dark green, blue or
red respective to the structure template and oriented to the bottom. In contrast,
features related to good biological activity are framed in light green, blue or red
and are oriented to the top. The overlap of these favorable features is shaded
in the respective color.
In contrast, the ethyl analogue of L78, namely L16, was relatively
active with a MIC50 value of 7.4 µM (KD = 0.5 µM) similar to the
ethenyl analogue S1 (KD = 0.8 µM, MIC50 = 9.7 µM). For these
compounds the discrepancy between KD and MIC50 was high and
can be associated to the methoxynaphthalene motif which led to
high affinities that were not translated into the expected biological
activity. For the ethyl analogue L16, the activity was probably
further reduced by the substituent as observed for similar
compound pairs.
bicyclic
moieties, para-methoxyphenyl
(L94)
and
2-
methylthio(thiophene) (L76). In contrast, hydroxyl groups
significantly impaired activity (L2, S9). Furthermore, it should be
noted that some motifs of the Western part aromatic moiety
(naphthalene,
benzothiophene,
trifluoromethoxyphenyl,
thiomethylphenyl, acetanilide) could not be definitely classified
regarding biological activity as the respective evaluated
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One of the most active compounds was the benzofuran analogue
(L10) of compound I:47 (Table 1). L10 showed a slightly higher
affinity and activity. Compound L78 resembles the latter two
compounds with thiophene as a bioisosteric substitute for phenyl
and its methylthio group replacing a part of the attached furan
motif. The same applies to the methoxyphenyl motif of compound
L94. Their affinities and activities are likewise similar to those of
their analogues.
In contrast, the potent compounds L14, L44, L45 and L46 share
an electron deprived phenyl ring at the Western part aromatic
motif (Table 1). Furthermore, L14, L45 and L46 contain a
fluorinated benzene ring at their middle aromatic position.
Although the affinities of L45 and L46 are slightly lower compared
to L14 and L44, the activities within this group are similar amongst
each other and comparable to the aforementioned group of
compounds.
Overall, a good a correlation between affinity and activity was
observed for twelve compounds (S7, S11, L10, L47, L48, L49,
L60, L66, L75, L90, L91, L94). For 13 compounds a bad
correlation was explained by unfavorable structural features (L8,
L9, L16, L19, L41, L77, L78, L79, L84, L89, S1, S2, S9). For
further seven compounds (S3, S4, S5, S6, S8, S10, L12) a
correlation was not observed (MIC50 > 2×KD) probably due to
structural drawbacks that were not identified so far. Further
compounds have to be evaluated to confirm this hypothesis.
Interestingly, eight compounds (L14, L15, L21, L44, L45, L46,
L73, L76) showed higher activity than expected based on their
affinities (KD ≈ 2-3×MIC50). One explanation for this discrepancy
is that the KD values might be actually lower than determined with
our method. For econazole we determined a KD value of 2.8 µM,
although the KD value was previously reported to be 0.02 µM.[6]
Apart from that, the inhibition of other targets cannot be excluded.
In this context, it has been demonstrated that antifungal azoles
show binding to other CYP enzymes of Mtb.[21]
Table 1: KD and MIC50 values of compounds showing better activity than I:47.
Compound
KD ± STD [µM]
MIC50 [mg/L]
MIC50 [µM]
2.8 ± 0.2[a]
Econazole
2.6
12.7
5.4 ± 1.0[a]
2.9 ± 0.4
6.4 ± 0.9
4.3 ± 0.8
5.9 ± 0.6
5.6 ± 0.9
12.6 ± 2.0
9.2 ± 2.0
6.6 ±1.0
I:47
L10
L14
L15
L21
L44
L45
L46
L76
L78
L94
1.4
0.9
0.9
0.7
0.5
0.8
1.2
1.0
1.1
0.8
1.1
4.0
2.6
2.7
1.8
1.5
2.4
3.4
2.9
3.2
2.4
3.3
0.6 ± 0.2
4.7 ± 0.8
[a] determined previously.[20] KD ± STD was calculated from 4 biological replicates.
part showed low occupancy in the crystal structure, which is
probably related to low water solubility.
Complex crystallization of CYP121 with bound inhibitors
CYP121 crystallized in space group P 65 2 2 and crystals
diffracted to a maximum of 1.3 Å resolution. High resolution
datasets were obtained for complex structures with inhibitors L21,
L44 and S2, and were solved by molecular replacement with the
deposited apo structure of CYP121 as a search model (PDB ID
1n40). The full data collection and refinement statistics including
the aforementioned occupancies of the inhibitors can be found in
Supplementary Table S5. The complex structure of L21 revealed
the compound being bound in a solvent channel leading from the
protein surface to the active site heme b. The inhibitor adopts a
planar conformation with only a slight rotation between the two
As a CYP121 crystal structure with bound inhibitor would enable
rational structure-based drug design and possibly link
physicochemical properties to structural features, complex
crystallization of CYP121 was performed. Given the observed
differences various western part aromatic motifs as well as linker
substitutions had on substrate affinity, a structurally diverse set of
inhibitors was chosen for complex crystallization. It should be
noted, however, that compounds containing naphthalene, indole
or benzodioxole moieties at either the western or middle aromatic
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phenyl rings, except for the imidazole that is extending towards
the heme macrocycle. The imidazole nitrogen is forming a
hydrogen bond with the water ligand of the heme b iron while the
western and middle part aromatic moieties are engaging in
hydrophobic interactions with hydrophobic side chains, especially
of Phe168 and Trp182, flanking the solvent channel (Figure 3A).
Previous studies on the coordination of cYY in the CYP121 crystal
structure have demonstrated that cYY binding relies on an
extensive water-mediated H-bonded network involving the sixth
iron ligand.[9] Thus, hydrogen bonding of L21 to the heme water
ligand prevents the formation of this network in addition to
occluding the solvent channel, making the heme non-accessible
for cYY.
Our inhibitor shares a common binding mode with the well-known
antifungal drug fluconazole, for which hydrogen bonding of its
triazole nitrogen to the water ligand has been reported as a novel
drug azole-P450 binding mode. Interestingly, fluconazole does
also exhibit a type-II shift in UV-Vis studies (as our compounds)
which has formerly been linked to
a direct azole-iron
interaction.[22] The clipped surface representation of CYP121
(Figure 3B) reveals the active site heme b being buried deep
inside the enzyme, with L21 occupying a bottleneck-like cleft
leading to the cofactor. The overlay of all co-crystallized inhibitors
L21, L44 and S2 (Figure 3C) shows a common binding site
located at the entrance to the CYP121 active site in between a-
helices F and G (helix numbering according to previously
published structure).[23]
Figure 3: Complex crystal structures of CYP121. A Coordination of inhibitor L21 in the CYP121 crystal structure. The protein is shown as a yellow cartoon, inhibitor,
heme b and interacting residues are shown as sticks. The heme iron is shown as a brown, the water ligand as a red sphere. The difference electron density map of
L21 (FO - FC) was contoured at 3s with phases calculated from a model that was refined in the absence of L21 and is shown as a blue isomesh. The hydrogen
bond distance between the water ligand and the imidazole nitrogen is 2.7 Å and shown as a yellow dashed line. B Clipped surface representation of the CYP121-
L21 complex, highlighting the position of the active site heme b and inhibitor L21 in the crystal structure. C Overlay of the complex structures of L21 (green), L44
(magenta) and S2 (cyan). A black arrow is highlighting the location of the entrance to the CYP121 active site.
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A summarized overview of the inhibitor structures together with
their respective KD and MIC50 values is shown in Table 2. The
complex structures of L44 and S2 with the respective difference
electron density maps are displayed in Figure S5. L44 shows a
similar binding mode to L21, with an imidazole-water hydrogen
bonding distance of 2.9 Å (Figure S5A). However, the position of
the two phenyls is shifted due to a shorter linker bridge. The
compound shows lower occupancy than L21 in the crystal
structure, which is possibly the result of less than optimal
hydrophobic interactions with Phe168 and Trp182, leading to a
lower rigidity of the molecule. Within the ethenyl substituted
compound class, S2 was chosen for complex crystallization due
to sufficient water solubility. Even though the enantiomers of S2
were not separated prior to crystallization, the observed electron
density hints at a predominantly (R)-configured enantiomer being
bound. In accordance with the complex structure (Figure S5B),
the observed impaired in vivo activity can be related to the binding
mode of the compound. A shorter linker bridge in combination with
the position of the western and middle part aromatic structure
mimicking the orientation of L21 leads to an inability of forming a
hydrogen bond (distance 4.7 Å) with the water ligand of the heme
b iron. The orientation of this inhibitor is dictated by a strong
hydrogen bond between the para-hydroxy group and an aspartate
side chain (Asp185) of CYP121 (Figure S6), possibly trapping it
close to a-helix G and therefore preventing it from reaching the
heme b water ligand. As such, this compound can possibly be
displaced by an incoming substrate in an in vivo situation as it
does not inhibit the formation of the water mediated H-bonded
network of cYY.
concentration which was necessary for LC-MS/MS analysis.[24] [25]
As expected, there is a correlation between binding affinities of
the compounds and their IC50 values. In contrast, the reference
compound econazole has a twofold lower IC50 value despite of
having a similar binding constant. An explanation for this finding
could be the described inhibitory effect of econazole on NADPH-
dependent reductases and G6P dehydrogenase which are
present in the assay.[26] [27]
Table 2: Structures of co-crystallized compounds with KD and MIC50 values.
KD ± STD
[µM]
MIC50
[µM]
Compound
F
N
L21
L44
5.9 ± 0.6
1.5
N
F3C
5.6 ± 0.9
2.4
N
N
Cl
Figure 4: Influence of L21 on the conversion of cYY to mycocyclosin by CYP121.
CYP121 was incubated with L21, electron transfer system (Arh1, etp1fd),
regenerative system (G6P-DH, G6P, MgCl2) and substrate cYY. The
conversion was stopped by the addition of MeOH with ISTD and analyzed via
LC-MS/MS. DMSO was used as control. A: time-dependent concentration of
cYY for different inhibitor concentrations. The relative inhibition was calculated
via determination of the respective reaction velocity. IC50 values were
determined through non-linear regression of inhibition vs. log c (B). The error
bars represent the standard deviation of at least 2 replicate measurements.
HO
S2
8.3 ± 1.7
50
N
N
Inhibition of cYY conversion in vitro
In order to evaluate the influence of the identified heme binders
on the inhibition of CYP121, we used our in vitro assay consisting
of CYP121, two electron transfer proteins (etp1fd and Arh1_A18G),
NADPH as electron donor and an electron regenerating
system.[20] Under the defined conditions, the substrate cYY was
converted to mycocyclosin within approximately 10 minutes
(Figure 4). The presence of a heme binder is expected to inhibit
this enzymatic conversion in a concentration dependent manner.
Indeed, we could observe the anticipated behavior for four
exemplarily chosen compounds (I:47, L10, L15, L21) and the
reference econazole. The IC50 values ranged between 19 and
36 µM in the presence of 20 µM substrate (SI, table S7). The IC50
values were determined for both, substrate depletion and product
formation and resulted in similar values (SI, figure S7). Compared
to the determined binding constants, the IC50 values are
significantly higher. This is probably caused by the high substrate
Effect of cYY on M. bovis BCG growth and CYP121 inhibition
To investigate whether the reduced growth of M. bovis BCG in
presence of our compounds was indeed caused by inhibition of
CYP121, we evaluated the growth inhibition effect of I:47 in
presence of cYY. In the control without inhibitor, the growth was
significantly accelerated (Figure 5A). CYP121 was previously
shown to be essential for viability, however, the reason for this
was not yet elucidated.[6] Our observation suggests that a
potential toxic effect of accumulated cYY is rather unlikely and the
reaction product mycocyclosin may be essential for growth. In
presence of I:47, the growth enhancing effect of cYY was
completely blocked at inhibitor concentrations higher than 0.6 µM,
whereas at lower concentration the stimulating effect of cYY was
7
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202100283
ChemMedChem
FULL PAPER
partly reduced (Figure 5B). These findings support the hypothesis
that the binding of our compounds to the heme iron of CYP121,
blocking the formation of mycocyclosin, is the main reason for the
antimycobacterial activity.
C23 also binds to CYP121, the selectivity towards CYP125,
however, is more than 6-fold higher.[30]
The treatment with CYP121 inhibitors I:47 and I:48 significantly
reduced the replication of M. bovis BCG inside macrophages.
However, the effect of I:16 was more pronounced. This finding
can be explained by additional inhibition of another CYP enzyme
present in the mycobacteria. As the imidazole moiety of the
compounds is known to be a typical interaction partner for CYP
enzymes, inhibition of another CYP cannot be excluded.[4] [33]
Interestingly, compound C43 could block the intracellular
replication comparable to the penicillin-streptomycin control. C43
binds to CYP121 and CYP125 with similar affinities and its strong
effect is probably caused by these two activities. Thus, the dual
inhibition of the two CYP enzymes shown to play an important role
in viability and persistence is a successful strategy to prevent the
proliferation of the mycobacteria in macrophages.[6] [31] [34]
6.0 104
´
4.0 104
´
**
***
2.0 104
´
***
***
*** ***
0
6
C3
3
C2
3
6
I:1
8
I:4
7
I:4
p
O
e
r
S
t
C4
S
-
DM
n
Pe
Compound (50 µM)
Figure 5: Effect of CYP121 substrate cYY on the growth of M. bovis BCG and
its inhibition by I:47. The addition of 50 µM cYY accelerated the growth of M.
bovis BCG (B). The resulting growth enhancement of cYY (A, grey striped bar)
Figure 6: Effect on intracellular replication in macrophages. Macrophages were
infected with M. bovis BCG and maintained in the presence of selected CYP125
inhibitors C36 and C23, CYP121 inhibitors I:16, I:47 and I:48 or dual inhibitor
C43. At 72 h post infection macrophages were plated on agar to determine
intracellular CFU. Penicillin-Streptomycin and DMSO were used as positive and
negative controls, respectively. Significance of results for treated versus
untreated cells (DMSO). **p<0.001; ***p<0.0001.
can be reversed through the CYP121 inhibitor I:47 in
a concentration-
dependent manner (grey bars) in accordance to its growth-inhibiting effect
(black bars).
Inhibition of intracellular replication of macrophages
Mtb has the ability to survive and replicate within the
Conclusion
[29]
macrophages of the host.[28]
Therefore, we determined
The recently published screening of a CYP-inhibitor library led to
the identification of hit compound I:47 with micromolar affinity
towards CYP121. Observed activity against Mtb highlighted
CYP121 as a possible target for treatment of Mtb infections.[20]
We herein presented a continuation of the former study by the
screening of a similarity-oriented library based on the frontrunner
compound I:47. The screening approach comprised the
evaluation of the respective compound class regarding affinity
toward CYP121 and biological activity against M. bovis BCG. 38
compounds (hit rate 40 %) showed binding constants lower than
or equal to I:47 (KD = 5 µM) and a type-II shift determined in a UV-
Vis heme coordination assay. Despite the visible type-II shift that
has been linked to a direct azole-iron interaction, co-crystallisation
of our inhibitors revealed a water-mediated coordination of the
heme iron comparable to the results for fluconazole by Seward et
al..[22] Hence, we classify the interaction as fluconazole-like
binding mode.
whether our compounds can influence the intracellular replication
of M. bovis BCG in macrophages. The effects of three recently
identified CYP121 inhibitors (I:16, I:48, I:47)[20], two previously
described inhibitors of CYP125 (C36, C23)[30] and one dual
inhibitor of CYP121 and CYP125 (C43)[30] were evaluated (SI,
table S9). CYP125 is part of the igr operon which was identified
as a key player for intracellular survival of mycobacteria in
macrophages.[31] It catalyses the oxidation of cholest-4-en-3-one
as part of the cholesterol detoxification and is a further promising
target for TB treatment.[32]
First of all, the compounds were evaluated regarding their
cytotoxicity toward macrophages. None of them showed any
significant reduction of viability (SI, figure S8). Regarding
antimycobacterial activity, among the two evaluated CYP125
inhibitors, only C23 showed a strong reduction of intracellular
replication (Figure 6). In contrast, C36 showed only little efficacy.
This marked difference in antimycobacterial activity could be due
to impaired permeability of C36. The compounds have to cross
the macrophage cell membrane as well as the mycobacterial cell
envelope to reach their target and the structure differences of the
two compounds could cause different permeabilities. Furthermore,
In contrast, the hit rate regarding activity, which was measured by
means of growth inhibition of M. bovis BCG in vitro, was
comparatively low (11 %) emphasizing that further criteria like
permeability or stability toward mycobacterial metabolism and
8
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10.1002/cmdc.202100283
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FULL PAPER
efflux must be met. Based on the analysis of the structural
features for biological activity, we enlarged our library by
western part aromatic motif substituted with a para-hydroxy group
in combination with a longer linker bridge, therefore possibly
enabling double hydrogen bonding, which could result in sub-µM
affinity.
synthesis of further promising compounds to ensure
a
comprehensive SAR study. Ten compounds with an ethenyl
substituted methylene linker that differ in their Western part
aromatic motifs were synthesized accompanied by a fluorine
analogue of the former hit I:47.
In general, the three inhibitors show a similar binding mode,
adopting a planar conformation in the solvent channel leading
from the protein surface to the active site heme. This solvent
channel formed by the F/G loop and their respective helices has
been proposed to be the substrate access channel for several
P450 enzymes.[38] Indeed, it has been shown to undergo
conformational changes upon substrate binding, typically leading
to an increase in active site volume.[38] Given the position of cYY
and cYY derivatives in substrate-bound complex structures of
CYP121 pointing towards the same entrance channel, it is
reasonable to assume an inability of substrate binding due to our
inhibitors.[18]
The hit compounds of this screening, which exhibited superior
(maximum 2.7-fold) activity to the former hit I:47, can be
structurally divided into three groups: Firstly, the two most active
compounds (L15 and L21) possess a propylene linker and a
rather simple structure with two phenyl rings connected to each
other (the Western part phenyl being fluorinated in L21). The
complex crystal structure of L21 provides a probable explanation
for this result. The combination of two phenyl rings with a
propylene linker leads to an ideal distance between both aromatic
motifs for optimal stacking of hydrophobic interactions with
Phe168 and Trp182, while allowing for imidazole hydrogen
bonding to the heme b water ligand. The second group (L10, L76,
L78, L94) has a methylene linker, a benzene ring in the middle
part and a Western part aromatic ring with an electron donor
function similar to I:47. Finally, the third group of compounds (L45,
L46, L14, L44) has a non-substituted methylene bridge and an
electron deprived Western part aromatic ring caused by fluorine
or fluorine-containing substituents. Overall, the compounds offer
the possibility for further optimization based on the gained insights
into structure-activity-relationships.
The quantity of determined binding affinities enabled a broad
analysis of structure-affinity-relationships. This analysis revealed
the tolerance of various substituents at the methylene bridge with
increased affinities for hydrophobic space-filling groups. As the
library compounds were principally screened as racemates, the
enantiomers were separated for three exemplarily chosen ethenyl
derivates. The binding constants revealed only minor differences
between the enantiomers and thus a separation was of less
importance. Concerning the middle aromatic motif, a negative
influence on affinity could be observed for pyridine, furan and
partly for thiophene. In contrast, benzene, fluorinated phenyl,
indole, benzothiophene, naphthalene or quinolone were accepted
with the tendency of increasing affinity for large moieties
indicating that hydrophobic interactions might be involved. For the
Western part aromatic motif, a wide range of different aromatic
ring systems was tolerated with highest affinities for compounds
with a 6-methoxynaphthalene motif.
Besides the elucidation of the binding mode, we were also
interested
in
the
correlation
between
affinity
and
antimycobacterial activity. A direct correlation between these two
properties would be a straightforward way to validate our target.
However, there are several limitations that impair this approach
such as the mycobacterial cell wall, which has special
permeability properties. There are some models for permeability
described in literature, though principal rules for the design of
compounds with good penetration properties do not exist.[35]
[36]
Additionally, other factors such as the mycobacterial metabolism
of the compound or the involvement of efflux pumps could also
impede the correlation. Notably, it was reported that Mtb and M.
bovis BCG possess efflux systems, which are able to reduce the
intracellular level of econazole in resistant mutants and could
possibly also transport other azoles.[37]
Based on the results of our screening, we were able to identify
structural features that impair the correlation between affinity and
activity. First of all, the substituent at the methylene linker has
strong influence on antimycobacterial activity. Only compounds
with either no substituent or a methyl or ethenyl substituent as
well as compounds with an interconnecting five-membered ring
between linker and adjacent phenyl ring showed an inhibitory
effect on growth. Other substituted compounds did not show
appropriate growth inhibition despite their high affinities. Similar
negative effects had bulky middle parts such as naphthalene or
quinolone which is in accordance to previously described
permeability simulation results.[35]
In order to validate CYP121 as the main target of the described
compound class, further mode of action studies were performed.
First of all, four exemplarily chosen hit compounds were shown to
inhibit the enzymatic conversion of cYY to mycocyclosin in vitro.
Furthermore, we evaluated the effect of a potent CYP121 inhibitor
(I:47) on the growth of M. bovis BCG in the presence of cYY. In
absence of inhibitor, cYY caused
a
significant growth
enhancement of the mycobacteria. However, in presence of I:47,
this effect was only observed at very low inhibitor concentrations,
indicating that the conversion to mycocyclosin is essential for the
intensified growth. These results strengthen the hypothesis that
inhibition of CYP121 is the main reason for the antimycobacterial
effect of our compounds.
Finally, we were also able to show that compounds targeting
CYP121 and/or CYP125 can reduce the intracellular replication of
M. bovis BCG in macrophages. The most promising compounds
exhibiting this effect appear to be the dual inhibitor C43 and the
The Western part aromatic motif tolerated diverse functions with
the exception of hydroxyl groups, which also impair a good
correlation. In accordance with the complex crystal structure of S2, CYP121 inhibitor I:16. Both could block the intracellular
a hydrogen bond donor in para-position of this motif leads to proliferation of mycobacteria, although the results indicate a
hydrogen bonding to an aspartate side chain (Asp185) of CYP121, possible involvement of an additional target for the latter
trapping the compound too far away from the heme b water ligand. compound. The inhibition of other CYP enzymes cannot be
Compared to the complex crystal structures of L21 and L44,
imidazole hydrogen bonding of S2 to the sixth water ligand is thus
not possible, leaving it available for the unique binding mode of
cYY. A follow-up study should consider a combination of a
excluded, especially since the genome of Mtb encodes 20 of them
which are in general prone to inhibition by azoles.[4, 39] Noticeably,
the dual inhibition of two of these CYP enzymes seems to be a
successful strategy to address Mtb infections.
9
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10.1002/cmdc.202100283
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FULL PAPER
structure (PDB ID 1n40) as the search model using Phaser[47] within the
Phenix suite. Multiple rounds of manual model building in COOT[48]
followed by refinement using phenix.refine[49] were carried out. Ligand
structures were modeled with phenix.eLBOW[50] using simple geometry
optimization. The structures were validated using MolProbity.[51] All images
presented were created using PyMOL (The PyMOL Molecular Graphics
System, Version 2.0 Schrödinger, LLC.)
Determination of influence of cYY on growth and inhibition of I:47:
The assay was performed as described above with the following
adjustments: The final DMSO concentration was set to 2 % as I:47 was
serially diluted in DMSO and eventually supplied with cYY. The readout
time point for cultures supplemented with cYY occurred after 3 to 5 days
as the addition of cYY induced an earlier onset of exponential growth
phase.
In summary, we described a screening approach for the discovery
of CYP121 inhibitors which led to a deeper understanding of the
binding properties of the respective compound class and revealed
possibilities for optimization. Furthermore, we gained insights into
structure-activity-relationships, highlighted CYP121 inhibition as
the main reason for the antimycobacterial activity of our
compounds and introduced the concept of dual CYP inhibition as
a promising strategy for the treatment of TB. The results of this
study lay the foundation for the development and optimization of
CYP121 inhibitors as potent drugs against Mtb infections.
CYP121 in vitro enzyme inhibition assay: The enzyme inhibition assay
was performed in 200 µL PBS buffer pH 7.2. Compounds were used in
concentrations of 1.6, 6.3, 25 and 50, 75 or 100 µM dependent on the
respective solubility. The final DMSO concentration was set to 7.5 %. The
compounds were incubated with 0.5 µM CYP121 for 10 minutes at 30 °C.
After incubation the electron transfer system consisting of Arh1_A18G
Experimental Section
Bacterial strains and growth conditions: Bacterial strains used in this
study were Mycobacterium bovis DSM-43990 (BCGT) and E. coli K12
BL21. Mycobacteria were cultured in Middlebrook 7H9 broth
complemented with ADC Enrichment (Middlebrook).
(2.5 µM), Etp1fd (7.5 µM) and NADPH+H+ (200 µM) as well as
a
regeneration system consisting of glucose-6-phosphate (2 mM), glucose-
6-phosphate dehydrogenase (1 unit/200 µL) and MgCl2 (1 mM) were
added. The reaction was started with the addition of cYY (20 µM) and
stopped after 0, 3, 6 and 9 min by addition of 200 µL methanol with internal
standard dichloro-cYY (4 µM final concentration, addition included).
The characterization of CYP121 activity was conducted by a UHPLC-
MS/MS analysis carried out on a TSQ Quantum Access Max mass
spectrometer equipped with an ESI-II source and a triple quadrupole mass
detector (Thermo Scientific, Dreieich, Germany). Compounds were
separated on an Accucore RP-MS 150×2.1 mm 2.6 µm column (Thermo
Fisher, Waltham, US) by a methanol/water gradient (from 1.4 min - 3.5 min
50% methanol to 3.5 min - 5.0 min 90% methanol) with a flow of
400 µL/min. Compounds were ionized in negative mode by electrospray
ionization. Ionization was assisted by a post-column addition of 2 mM
ammonia in methanol with an automated syringe at 1.25 µL/min.
Monitored ions were (mother ion [m/z], product ion [m/z], scan time [s],
scan width [m/z], collision energy [V], tube lens offset [V], polarity):
mycocyclosin: 323.101, 111.100, 0.30, 0.01, 28, 95, negative; CYY:
325.129, 113.043, 0.3, 0.01. 29, 85, negative; internal standard (diiodo-
cYY): 576.878, 126.930, 0.3, 0.01, 43, 77, negative. Samples were
injected in a volume of 20 µL. Xcalibur software was used for data
acquisition.
For quantification, the ratios of the area under the curve of the educt and
the product were converted to respective concentrations on the basis of
determined calibration curves of mycocyclosin and cYY which were
synthesized as previously described.[20, 52] The initial velocity v0 of the cYY
conversion was determined by linear fit of the respective concentrations of
mycocyclosin or cYY over time (0-9 min) with Microsoft Excel software.
The IC50 values were calculated using GraphPad Prism by fitting the
relative inhibition of v0 versus concentration with the OneSite Log IC50
model provided by the software with the following constraints: bottom=0
and top=100.
Protein expression and purification: E. coli K12 BL21 (DE3) cells were
transformed with plasmid harboring cyp121 gene (pHAT2/cyp121).[16] The
enzymes were expressed and purified as described previously.[10] [20] For
crystallization, eluted CYP121 protein from the 5 mL HisTrap was passed
over a HiPrep 26/10 desalting column (GE Healthcare) pre-equilibrated in
100 mM NaCl, 10 mM HEPES pH 8.0. The total protein amount was
estimated from the primary amino acid sequence by UV-Vis spectroscopy
at 280 nm using a NanoDrop 2000 (Thermo Fisher Scientific). The C-
terminal His6-Tag was removed by addition of a 1:500 ratio of Thermolysin
and incubation on ice for 1 h. Subsequently, the enzyme was passed over
a 5 mL HisTrap and loaded onto a 5 mL HisTrap Q HP column (GE
Healthcare) pre-equilibrated in 100 mM NaCl, 10 mM HEPES pH 8.0.
CYP121 was eluted from the Q HP column around a concentration of 200
mM NaCl using a 90 mL gradient elution with a final concentration of 500
mM NaCl. Lastly, CYP121 was passed over a HiLoad 16/600 Superdex
200 pg column (GE Healthcare) pre-equilibrated in 10 mM Tris pH 7.5.
Protein purity was assessed by SDS-PAGE as well as intact mass
spectrometry. Pure protein was concentrated using a 30 kDa cutoff filter
and diluted to 700 µM for crystallization, determined by UV-VIS
spectroscopy at 280 nm. The enzyme was flash-frozen in liquid N2 and
stored at -80 °C until further use.
UV/Vis heme P450 binding assay: Optical titration experiments were
performed and analyzed as described previously.[20] [40]
Determination of BCG MIC50 by OD600 assay: A pre-culture of M. bovis
BCG was grown in 7H9 medium supplemented with ADC Enrichment until
OD600 reached a value of 0.7 and was then used for susceptibility testing
in the following 72 h. The assay was performed in 48 well plates (Greiner,
Kremsmünster, AT). Prior to culture addition, compounds were serially
diluted in DMSO to fit a final DMSO concentration of 1%. For compound
susceptibility testing the pre-culture was diluted with fresh medium (7H9 +
ADC enrichment) to a concentration of 1-5 × 105 cfu/mL. After 4 to 6 days
of incubation (until OD600 of positive control approached its maximum) at
37 °C and 80 % air moisture, bacterial growth was measured by
determination of OD600. Absorption data was recorded on a Fluostar
Omega Multidetection Plate Reader (BMG LABTECH, Ortenberg, DE).
The relative inhibition compared to the positive control was plotted against
concentration with GraphPad Prism and MIC50 values were fitted using
OneSite Log IC50 model provided by the software with the following
constraints: bottom=0 and top=100. MIC50 was defined as the
concentration at which 50 percent of growth was detected in accordance
with previous methods used.[41]
Macrophage cytotoxicity and intracellular macrophage replication:
Strain and growth conditions: M. bovis BCG Pasteur strain was grown at
37°C in Middlebrook 7H9 broth (BD Biosciences) supplemented with 10%
Middlebrook OADC enrichment medium (BD Biosciences), 0.002%
glycerol (Roth), and 0.05% Tween 80 (Roth). Midlog phase cultures were
harvested, aliquoted, and frozen at -80°C. Bacteria were prepared from
frozen stocks by thawing at 37°C, resuspension in PBS and passage
through a 27G needle. Determination of optical density at 580 nm was
done to adjust the amount of the bacteria required in cell culture medium
without antibiotics.
Cells and media: Bone marrow derived macrophages were prepared from
femurs and tibiae remove from C57BL/6 mice using a standard protocol.
Briefly, bone marrow cells were cultured for 7 days in complete RPMI
(RPMI 1640 plus Glutamax (Gibco), fetal calf serum 10% (HyClone), 100
U/mL penicillin and 100 µg/mL streptomycin (Biochrom) and 50 µM ß-
Mercaptoethanol (Gibco)) supplemented with 10% L929 cell-conditioned
medium as a source of murine M-CSF. On day 7, fully differentiated
macrophages were transferred to 96-well plates. Antibiotics were omitted
from cell culture medium 24 hours prior to and during the experiments.
Mycobacteria infection: Macrophages at 200.000 cells/well were seeded
on 96 flat well plates (100 μL) 24 hours previous infection in DMEM 10%
L929 CM. After 2.5 hours of infection with M. bovis BCG Pasteur at
multiplicity of infection (MOI) 10:1, cells were extensively washed with
RPMI to remove extracellular mycobacteria. Infected macrophages were
maintained in cell culture medium at 37°C and 5% CO2 in the presence or
absence of the compounds at the indicated concentrations. Penicillin-
Crystallization: CYP121 was crystallized by mixing 2 uL of 700 µM
enzyme with 2 µL of a 500 µL well solution containing 200 mM calcium
acetate, 100 mM sodium acetate pH 4.5 and 30% PEG400 using the
Hanging Drop vapour diffusion method. Single crystals appeared after
incubating the plates at 4 °C for 3 d, but could be grown to around 400 µm
after a 10 d incubation. For complex crystallization, stock solutions of 200
mM compound were prepared in 100% DMSO. Single crystals of CYP121
were soaked in a 20 mM compound solution containing 10% DMSO for 24
h at 4 °C, before being mounted in a cryo loop for data collection.
Data collection and refinement: Full, high resolution datasets of the L21,
L44 and S2 complexes were collected at ESRF Beamline ID 23-2 (L21) in
France, SLS Beamline Xo6DA (L44) and SLS Beamline X10SA (S2) in
Switzerland, respectively. Data reduction using an appropriate resolution
cutoff was performed using XDS[42]
Ctruncate[45] within the CCP4 suite[46]. The structures were solved by
molecular replacement with previously published high resolution
, ,
Pointless[43] Aimless[44] and
a
10
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10.1002/cmdc.202100283
ChemMedChem
FULL PAPER
Streptomycin and DMSO were used as control at the indicated
concentrations.
13C NMR (75 MHz, CDCl3): δ=117.2, 121.0, 127.6, 127.9, 128.2, 130.8,
133.7, 135.5, 145.8, 152.2, 192.2 ppm; LC-MS (ESI): Rt=7.22 min, m/z:
232.99 [M+H]+.
To monitor the bacterial intracellular survival, cells were lysed with a sterile
solution of 0.1% (vol/vol) Triton X-100 in H2O, and serial dilutions of lysates
were rapidly plated on 7H9 agar plates to enumerate CFU. This procedure
was done at 72 hours after compound incubation. Experiments were
carried out in triplicate. Each assay was repeated in at least three
independent experiments.
Statistical analysis: Statistical analysis was performed with one-way
analysis of variance, followed by a post hoc Tukey test, using Graph- Pad
Prism 5.0 software. The data are represented as means ± standard errors
of the means (SEM).
In vitro cytotoxicity assay: In vitro cytotoxicity assays were performed with
murine macrophages generated as mentioned before. Microtiter plates (96
wells) were loaded with at 200.000 cells/well suspended in 100 μL of RPMI
1640 plus Glutamax (GIBCO) supplemented with 10% heat inactivated
fetal calf serum 10% (HyClone) and 50 µM ß-Mercaptoethanol (Gibco) in
presence or absence of the compounds at the indicated concentrations.
Isoniazid, rifampicin and DMSO were used as control at the indicated
concentrations. The plates were incubated for 48 h at 37 °C in 5% CO2.
Cell viability was assessed by measuring using LIVE/DEAD Fixable Aqua
Dead Cell Stain Kit by FACS. The data were analyzed using FlowJo
(Treestar). Experiments were carried out in triplicate. Each assay was
repeated in at least three independent experiments.
1.3: 3',4'-dimethoxy-[1,1'-biphenyl]-4-carbaldehyde (S3b):
Synthesized according to method
A using 4-bromobenzaldehyde
(1.35 mmol; 250 mg) and 3,4-Dimethoxyphenylboronic acid (1.76 mmol;
320 mg). S3b was obtained as a white solid (342 mg, 100 %): Rf=0.48
(hexane/EtOAc 7:3); 1H NMR (300 MHz, CDCld3): δ=3.86-4.02 (m, 6H),
6.98 (d, J=8.3 Hz, 1H), 7.15 (d, J=1.9 Hz, 1H), 7.18-7.29 (m, 1H), 7.73 (d,
J=8.1 Hz, 2H), 7.90-7.97 (m, 2H), 10.04 ppm (s, 1H); 13C NMR (75 MHz,
CDCl3): δ=56.4, 110.8, 111.9, 120.4, 127.6, 130.7, 132.9, 135.2, 147.4,
149.8, 150.1, 192.2 ppm; LC-MS (ESI): Rt=7.08 min; m/z: 242.99 [M+H]+.
1.4: 4'-methoxy-3'-methyl-[1,1'-biphenyl]-4-carbaldehyde (S4b):
Synthesized according to method
A using 4-bromobenzaldehyde
(1.35 mmol; 250 mg) and 4-Methoxy-3-methylphenylboronic acid
(1.75 mmol; 291 mg). S4b was obtained as a white solid (317 mg, 100 %):
Rf=0.54 (hexane/EtOAc 9:1); 1H NMR (300 MHz, CDCl3): δ=2.31 (s, 3H),
3.90 (d, J=1.2 Hz, 3H), 6.94 (d, J=8.1 Hz, 1H), 7.41-7.52 (m, 2H), 7.65-
7.77 (m, 2H), 7.87-7.97 (m, 2H), 10.04 ppm (s, 1H); 13C NMR (75 MHz,
CDCl3): δ=16.4, 55.4, 110.3, 125.8, 127.0, 127.3, 129.6, 130.3, 131.6,
134.6, 147.0, 158.4, 191.9 ppm; LC-MS (ESI): Rt=9.04 min, m/z: 227.04
[M+H]+.
1.5: 4-(benzo[d][1,3]dioxol-5-yl)benzaldehyde (S5b):
Synthesized according to method
A using 4-bromobenzaldehyde
Chemical synthesis: Chemical synthesis and analytical characterization:
Chemicals were purchased from commercial suppliers and used without
further purification. Column flash chromatography was performed on silica
gel (40−63 μm), and reaction progress was monitored by TLC on TLC
Silica Gel 60 F254 plates (Merck, Darmstadt, Germany). All moisture-
sensitive reactions were performed under nitrogen atmosphere using
anhydrous solvents. 1H and 13C NMR spectra were recorded on Bruker
Fourier spectrometers (300 MHz) at ambient temperature with the
chemical shifts recorded as δ values in ppm units by reference to the
hydrogenated residues of deuterated solvent as internal standard.
Coupling constants (J) are given in Hertz (Hz), and signal patterns are
indicated as follows: s, singlet; d, doublet; dd, doublet of doublets; t, triplet;
m, multiplet, br, broad signal. The purity of the final compounds was >95%
measured by HPLC with UV detection at 254 nm. The SpectraSystem LC
system consisted of a pump, an autosampler, and a UV/Vis detector
(ThermoFisher, Dreieich, Germany). Mass spectrometry was performed
on an LC-coupled Surveyor MSQ electrospray mass spectrometer
(ThermoFisher, Dreieich, Germany). The system was operated by the
Xcalibur software. A RP C18 NUCLEODUR ec 100-5 125 × 3 mm 5 µm
column (Macherey-Nagel GmbH, Düren, Germany) was used as the
stationary phase. All solvents were HPLC grade. In a gradient run, the
percentage of acetonitrile (containing 0.1 % trifluoroacetic acid) was
initially kept constant for 2 min at 30 % and was consequently increased
from 30 % at 2 min to 100 % at 8 min, kept at 100 % for 2 min and finally
at 30% for 2 min. The injection volume was 10 μL, and flow rate was set
to 700 μL/min. MS analysis was carried out in ESI ionization mode at a
spray voltage of 3800 V, a cone voltage of 55 V and a probe temperature
of 350 °C. Spectra were acquired in positive mode from 100 to 600 m/z.
1. Method A: Suzuki coupling
(1.35 mmol; 250 mg) and 3,4-methylenedioxyphenylboronic acid
(1.75 mmol; 290 mg). S5b was obtained as a white solid (322 mg, 100 %):
Rf=0.66 (hexane/EtOAc 8:2); 1H NMR (300 MHz, CDCl3): δ=6.04 (s, 2H),
6.92 (dd, J=7.7, 0.7 Hz, 1H), 7.10-7.17 (m, 2H), 7.64-7.71 (m, 2H), 7.88-
7.97 (m, 2H), 10.04 ppm (s, 1H); 13C NMR (75 MHz, CDCl3): δ=101.4,
107.6, 108.8, 121.3, 127.3, 130.3, 133.9, 134.9, 146.8, 148.1, 148.4, 191.8
ppm; LC-MS (ESI): Rt=8.20 min, m/z: 227.01 [M+H]+.
1.6: 3'-fluoro-4'-methoxy-[1,1'-biphenyl]-4-carbaldehyde (S6b):
Synthesized according to method
A using 4-bromobenzaldehyde
(1.35 mmol; 250 mg) and 3-Fluoro-4-methoxybenzeneboronic acid
(1.75 mmol; 297 mg). S6b was obtained as a white solid (289 mg, 93 %):
Rf=0.46 (hexane/EtOA 8:2); 1H NMR (300 MHz, CDCl3): δ=3.96 (s, 3H),
7.01-7.13 (m, 1H), 7.34-7.45 (m, 2H), 7.70 (d, J=8.5 Hz, 2H), 7.91-7.99 (m,
2H), 10.05 (s, 1H); 13C NMR (75 MHz, CDCl3): δ=56.4 (s), 113.7 (d, J=2.2
Hz), 115.0 (d, J=18.6 Hz), 123.1 (d, J=3.7 Hz), 127.1 (s), 130.3 (s), 132.7
(d, J=6.7 Hz), 135.1 (s), 145.6 (d, J=1.5 Hz), 148.1 (d, J=10.4 Hz), 152.6
(d, J=246.6 Hz), 191.8 ppm (s); LC-MS (ESI): Rt=8.50 min, m/z: 231.05
[M+H]+.
2. Method B: Grignard reaction
The aldehyde (1.0 eq.) was dissolved in a heat dried flask in dry THF (2
mL/mmol) and cooled to -15 °C in an ice/acetone bath. Vinylmagnesium
bromide (1.2 eq., 0.7 M in THF) was added dropwise and the reaction was
stirred for 5 min before letting it warm to room temperature for 1-2 h and
TLC showed complete conversion. The reaction mixture was quenched
with saturated NH4Cl solution (15 mL) and extracted with ethyl acetate (3
× 20 mL). The combined organic phases were washed with brine and dried
over MgSO4. The solvent was evaporated under reduced pressure and the
crude product was purified by chromatography on silica gel.
2.1: 1-(4-(6-methoxynaphthalen-2-yl)phenyl)prop-2-en-1-ol (S1a):
Synthesized according to method B using 1b (1.14 mmol; 300 mg) and
vinylmagnesium bromide (0.7 M in THF; 1.37 mmol; 2.0 mL). S1a was
obtained as a yellow solid (201 mg, 61 %): Rf=0.33 (hexane/EtOAc 8:2);
1H NMR (300 MHz, CDCl3): δ=3.96 (s, 3H), 5.21-5.32 (m, 2H), 5.43 (dd,
J=17.1, 1.3 Hz, 1H), 6.13 (ddd, J=16.9, 10.6, 6.1 Hz, 1H), 7.14-7.22 (m,
2H), 7.50 (d, J=8.1 Hz, 2H), 7.71 (d, J=8.1 Hz, 3H), 7.77-7.85 (m, 2H), 7.98
ppm (s, 1H); 13C NMR (75 MHz, CDCl3): δ=55.8, 75.6, 106.0, 115.7, 119.6,
126.0, 126.4, 127.3, 127.7, 127.8, 130.1, 134.2, 136.4, 140.6, 141.2, 141.8,
158.2 ppm; LC-MS (ESI): Rt=8.94 min, m/z: 274.08 [M-OH]+.
The aldehyde (1 eq.) and the boronic acid (1.3 eq.) were dissolved in
toluene (7.5 mL/mmol) and ethanol (4.5 mL/mmol). After addition of 2 M
Na2CO3 solution (3 mL/mmol) the mixture was flushed with N2 for 10 min.
Subsequently, Pd(PPh3)4 (0.05 eq.) was added and the mixture flushed for
further 5 min with N2. The mixture was heated to 100 °C for 1 to 4h until
TLC control showed complete conversion. The reaction was quenched
with water (20 mL) and extracted with ethyl acetate (3 × 20 mL). The
combined organic phases were washed with brine and dried over MgSO4.
The solvent was evaporated under reduced pressure and the crude
product was purified by chromatography on silica gel.
2.2: 3-chloro-4'-(1-hydroxyallyl)-[1,1'-biphenyl]-4-ol (S2a):
1.1: 4-(6-methoxynaphthalen-2-yl)benzaldehyde (S1b):
Synthesized according to method B using 2b (0.25 mmol; 60 mg) and
vinylmagnesium bromide (0.7 M in THF; 0.49 mmol; 0.7 mL). S2a was
obtained as a yellow solid (44 mg, 67 %): Rf=0.44 (hexane/EtOAc 7:3); 1H
NMR (300 MHz, CDCl3): δ=5.20-5.29 (m, 2H), 5.35-5.44 (m, 1H), 5.60 (br.
s., 1H), 6.09 (ddd, J=16.9, 10.5, 6.1 Hz, 1H), 7.09 (d, J=8.5 Hz, 1H), 7.38-
7.47 (m, 3H), 7.49-7.54 (m, 2H), 7.56 ppm (d, J=2.2 Hz, 1H).
2.3: 1-(3',4'-dimethoxy-[1,1'-biphenyl]-4-yl)prop-2-en-1-ol (S3a):
Synthesized according to method B using 3b (1.24 mmol; 300 mg) and
vinylmagnesium bromide (0.7 M in THF; 1.48 mmol; 2.1 mL). S3a was
obtained as a yellow solid (230 mg, 69 %): Rf=0.37 (hexane/EtOAc 7:3);
1H NMR (300 MHz, CDCl3): δ=3.93 (s, 3H), 3.95 (s, 3H), 5.21-5.29 (m, 2H),
5.40 (d, J=17.1 Hz, 1H), 6.10 (ddd, J=16.9, 10.5, 6.0 Hz, 1H), 6.95 (d,
J=8.3 Hz, 1H), 7.09-7.18 (m, 2 H), 7.42-7.46 (m, 2 H), 7.53-7.59 ppm (m,
2 H); 13C NMR (75 MHz, CDCl3): δ=55.9, 56.0, 75.1, 110.4, 111.5, 115.2,
119.4, 126.7, 127.0, 133.8, 140.2, 140.6, 141.2, 148.7, 149.2 ppm; LC-MS
(ESI): Rt=6.48 min, m/z: 254.04 [M-OH]+.
Synthesized according to method
A using 4-bromobenzaldehyde
(1.35 mmol; 250 mg) and 6-Methoxy-2-naphthaleneboronic acid
(1.73 mmol; 350 mg). S1b was obtained as a white solid (330 mg, 93 %):
Rf=0.58 (hexane/EtOAc 8:2); 1H NMR (300 MHz, CDCl3): δ=3.97 (s, 3H),
7.17-7.25 (m, 2H), 7.75 (dd, J=8.1, 2.2 Hz, 1H), 7.80-7.92 (m, 4H), 7.99 (d,
J=8.4 Hz, 2H), 8.05 (d, J=1.5 Hz, 1H), 10.08 ppm (s, 1H); 13C NMR (75
MHz, CDCl3): δ=55.4, 105.6, 119.5, 125.6, 126.4, 127.6, 127.6, 129.0,
129.9, 130.3, 147.2, 158.3, 191.9 ppm; LC-MS (ESI): Rt=9.54 min, m/z:
263.01 [M+H]+.
1.2: 3'-chloro-4'-hydroxy-[1,1'-biphenyl]-4-carbaldehyde (S2b):
Synthesized according to method
A using 4-bromobenzaldehyde
(1.35 mmol; 250 mg) and 3-Chloro-4-hydroxyphenylboronic acid
(1.45 mmol; 250 mg). S2b was obtained as a white solid (112 mg, 36 %):
Rf=0.27 (hexane/EtOAc 8:2); 1H NMR (300 MHz, CDCl3): δ=5.71 (br. s.,
1H), 7.14 (d, J=8.5 Hz, 1H), 7.49 (dd, J=8.5, 2.1 Hz, 1H), 7.64 (d, J=2.1
Hz, 1H), 7.70 (d, J=8.3 Hz, 2H), 7.95 (d, J=8.2 Hz, 2H), 10.06 ppm (s, 1H);
2.4: 1-(4'-methoxy-3'-methyl-[1,1'-biphenyl]-4-yl)prop-2-en-1-ol (S4a):
11
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202100283
ChemMedChem
FULL PAPER
Synthesized according to method B using 4b (1.10 mmol; 250 mg) and
vinylmagnesium bromide (0.7 M in THF; 1.40 mmol; 2.0 mL). S4a was
obtained as a yellow solid (96 mg, 34 %): Rf=0.26 (hexane/EtOAc 9:1);
1H NMR (300 MHz, CDCl3): δ=2.29 (s, 3H), 3.88 (s, 3H), 5.20-5.29 (m, 2H),
5.40 (d, J=17.0 Hz, 1H), 6.11 (ddd, J=16.8, 10.4, 6.0 Hz, 1H), 6.90 (d,
J=8.9 Hz, 1H), 7.37-7.45 (m, 4H), 7.53-7.58 ppm (m, 2H); 13C NMR (75
MHz, CDCl3): δ= 15.9, 55.0, 74.8, 109.8, 114.7, 124.9, 126.3, 126.5, 126.5,
129.0, 132.5, 139.8, 140.2, 140.4, 157.0 ppm; LC-MS (ESI): Rt=8.66 min,
m/z: 238.09 [M-OH]+.
4. Method D: TBDMS protection
a) 4-Bromophenol (1.0 eq.) and imidazole (1.1 eq.) were dissolved in dry
CH2Cl2 (3 mL/mmol) before TBDMS chloride (1.1 eq) was added. The
reaction was stirred at room temperature overnight and quenched with
water (30 mL) and 1M HCl (3 mL). The aqueous phase was extracted with
CH2Cl2 (2 × 30 mL) and the combined organic phases were washed with
brine and dried over MgSO4. The solvent was evaporated under reduced
pressure and the crude product was purified by chromatography on silica
gel.
2.5: 1-(4-(benzo[d][1,3]dioxol-5-yl)phenyl)prop-2-en-1-ol (S5a):
Synthesized according to method B using 5b (1.24 mmol; 280 mg) and
vinylmagnesium bromide (0.7 M in THF; 1.49 mmol; 2.1 mL). S5a was
obtained as a yellow solid (209 mg, 66 %): Rf=0.48 (hexane/EtOAc 8:2;.
1H NMR (300 MHz, CDCl3): δ= 5.18-5.29 (m, 2H), 5.40 (dd, J=17.1, 1.1
Hz, 1H), 6.10 (ddd, J=16.9, 10.5, 6.1 Hz, 1H), 6.85-6.92 (m, 1H), 7.03-7.10
(m, 2H), 7.43 (d, J=8.1 Hz, 2H), 7.52 ppm (d, J=8.5 Hz, 2H); 13C NMR (75
MHz, CDCl3): δ=75.1, 101.1, 107.6, 108.6, 115.2, 120.6, 126.7, 127.0,
135.2, 140.1, 140.4, 141.3, 147.1, 148.1 ppm; LC-MS (ESI): Rt=7.65 min,
m/z: 238.04 [M-OH]+.
4.1: (4-bromophenoxy)(tert-butyl)dimethylsilane (S9e):
Synthesized according to method D a) using 4-Bromophenol (2.89 mmol;
500 mg), imidazole (3.18 mmol; 216 mg) and TBDMSCl (3.18 mmol; 479
mg). S9e was obtained as a colorless oil (810 mg, 98 %): Rf=0.89
(hexane/EtOAc 8:2); 1H NMR (300 MHz, CDCl3): δ=0.19 (s, 6H), 0.99 (s,
9H), 6.70-6.75 (m, 2H), 7.30-7.35 ppm (m, 2H); 13C NMR (75 MHz, CDCl3):
δ=-4.5, 18.2, 25.6, 113.6, 121.9, 132.3, 154.9 ppm; LC-MS (ESI): Rt=11.59
min.
b) NaH (1.2 eq.) was dissolved in acetonitrile (1 mL/mmol) and cooled to
0 °C. 5-Bromo-1H-indole was dissolved in acetonitrile (1 mL/mmol) and
slowly added to the NaH suspension. After stirring for 15 min TBDMS
chloride (1.25 eq.) was added and the suspension was let warm to room
temperature and stirred overnight. The reaction was quenched with
saturated NH4Cl (10 mL) solution and extracted with CH2Cl2 (3 × 20 mL).
The combined organic phases were washed with brine and dried over
MgSO4. The solvent was evaporated under reduced pressure and the
crude product was purified by chromatography on silica gel.
3. Method C: CDI reaction
The alcohol (1.0 eq.) and 1,1’-carbonyldiimidazole (3.0 eq.) were dissolved
in acetonitrile (15 mL/mmol) and heated to reflux for 1-4 h until TLC control
showed complete conversion. The reaction was quenched with water (15
mL) and extracted with ethyl acetate (3 × 20 mL). The combined organic
phases were washed with brine and dried over MgSO4. The solvent was
evaporated under reduced pressure and the crude product was purified by
chromatography on silica gel.
4.2: 5-bromo-1-(tert-butyldimethylsilyl)-1H-indole (S10e):
3.1 1-(1-(4-(6-methoxynaphthalen-2-yl)phenyl)allyl)-1H-imidazole (S1):
Synthesized according to method C using 1a (100 mg, 0.34 mmol) and
CDI (162 mg, 1.0 mmol). S1 was obtained as a light yellow solid (39 mg,
34 %): Rf=0.19 (EtOAc); 1H NMR (300 MHz, CDCl3): δ=3.95 (s, 3H), 5.20
(d, J=17.0 Hz, 1H), 5.47 (d, J =10.3 Hz, 1H), 5.85 (d, J=6.3 Hz, 1H), 6.33
(ddd, J=17.0 Hz, 10.2 Hz, 6.3 Hz, 1H), 6.94 (s, 1H), 7.18 (m, 3H), 7.29 (m,
2H), 7.58 (s, 1H), 7.70 (m, 3H), 7.81 (m, 2H), 7.97 ppm (s, 1H); 13C NMR
(75 MHz, CDCl3): δ=55.8, 63.7, 106.0, 119.0, 119.7, 119.8, 126.1, 126.2,
127.8, 128.1, 128.4, 129.5, 129.9, 130.1, 134.4, 135.8, 136.2, 137.1, 137.3,
141.9, 158.4 ppm; LC-MS (ESI): Rt=6.77 min, m/z: 341.18 [M+H]+, 237.04
[M-Imidazole]+.
Synthesized according to method D b) using 5-Bromo-1H-indole
(2.55 mmol; 500 mg), NaH (3.06 mmol; 73.4 mg) and TBDMSCl
(3.19 mmol; 480 mg). S10e was obtained as a colorless oil (267 mg,
34 %): Rf=0.95 (hexane/EtOAc 9:1); 1H NMR (300 MHz, CDCl3): δ=0.60
(s, 6H), 0.92 (s, 9H), 6.56 (d, J=3.2 Hz, 1H), 7.18 (d, J=3.2 Hz, 1H), 7.23
(dd, J=8.8, 2.0 Hz, 1H), 7.38 (d, J=8.8 Hz, 1H), 7.75 ppm (d, J=2.0 Hz,
1H); 13C NMR (75 MHz, CDCl3): δ=-4.0, 19.4, 26.2, 104.3, 113.1, 115.1,
123.1, 124.1, 132.2, 133.2, 139.6 ppm; LC-MS (ESI): Rt=11.40 min, m/z:
312.11 [M+H]+.
5. Method E: Boronic acid preparation
The TBDMS protected alcohol or amine (1.0 eq.) was dissolved in dry THF
(1 mL/mmol) and cooled to -78 °C. nBuLi (1.25 eq., 2.5 M in THF) was
added dropwise and the solution was stirred for 30 min. Triisopropylborate
(2.5 eq.) was added slowly and the mixture was stirred for further 45 min
at -78°C. After letting the mixture warm to room temperature for 3 h it was
quenched with ethyl acetate : 1 M HCl = 1 : 1 (10 mL). The organic layer
was separated, washed with brine (2 × 10 mL) and dried over MgSO4. The
solvent was evaporated under reduced pressure and the crude product
was washed with cold hexane followed by warm water and dried under
reduced pressure.
5.1: (4-((tert-butyldimethylsilyl)oxy)phenyl)boronic acid (S9d):
Synthesized according to method E using 9e (2.78 mmol; 800 mg), nBuLi
(2.5 M in THF; 3.48 mmol; 1.4 mL) and B(OiPr)3 (5.56 mmol; 970 mg). S9d
was obtained as an orange solid (400 mg, 57 %): 1H NMR (300 MHz,
CDCl3): δ=0.27 (s, 6H), 1.03 (s, 9H), 6.96 (d, J=8.5 Hz, 2H), 8.12 ppm (d,
J=8.5 Hz, 2H); LC-MS (ESI): Rt=9.03 min.
5.2: (1-(tert-butyldimethylsilyl)-1H-indol-5-yl)boronic acid (S10d):
Synthesized according to method E using 10e (2.58 mmol; 800 mg), nBuLi
(2.5 M in THF; 3.23 mmol; 1.3 mL) and B(OiPr)3 (5.16 mmol; 1.05 g). S10d
was obtained as a white solid (487 mg, 69 %): Rf=0.93 (hexane/EtOAc 9:1),
1H NMR (300 MHz, CDCl3): δ=0.69 (s, 5H), 1.00 (s, 8H), 6.81 (d, J=2.9 Hz,
1H), 7.26-7.30 (m, 1H), 7.69 (d, J=8.4 Hz, 1H), 8.16 (d, J=8.4 Hz, 1H), 8.69
ppm (s, 1H); 13C NMR (75 MHz, CDCl3): δ=-3.9, 19.6, 26.3, 105.6, 113.4,
128.4, 129.6, 131.2, 131.3, 144.0 ppm; LC-MS (ESI): Rt=8.84 min, m/z:
276.18 [M+H]+.
3.2 4'-(1-(1H-imidazol-1-yl)allyl)-3-chloro-[1,1'-biphenyl]-4-ol (S2):
Synthesized according to method C using 2a (64mg, 0.25 mmol) and CDI
(121 mg, 0.75 mmol). S2 was obtained as a yellow oil (24 mg, 31 %):
Rf=0.27 (EtOAc); 1H NMR (300 MHz, CDCl3): δ=5.18 (d, J=17.0 Hz, 1H),
5.47 (d, J=10.2 Hz, 1H), 5.82 (d, J=6.2 Hz, 1H), 6.30 (ddd, J=16.9 Hz, 10.4
Hz, 6.3 Hz, 1H), 6.94 (s, 1H), 7.08 (d, J=8.4 Hz, 1H), 7.15 (s, 1H), 7.25 (m,
2H), 7.34 (dd, J= 8.4 Hz, 2.2 Hz, 1H), 7.54 (m, 3H), 7.60 ppm (s, 1H); 13
C
NMR (75 MHz, CDCl3): δ=63.4, 116.9, 118.7, 119.6, 121.0, 126.6, 127.2,
127.9, 128.6, 132.9, 135.4, 136.4, 136.5, 140.2, 152.5 ppm; LC-MS (ESI):
Rt=2.46 min, m/z: 311.15 [M+H]+, 243.09 [M-Imidazole]+.
3.3 1-(1-(3',4'-dimethoxy-[1,1'-biphenyl]-4-yl)allyl)-1H-imidazole (S3):
Synthesized according to method C using 3a (200 mg, 0.74 mmol) and
CDI (360 mg, 2.22 mmol). S3 was obtained as a yellow oil (70 mg, 30 %):
Rf=0.27 (EtOAc); 1H NMR (300 MHz, CDCl3): δ=3.93 (d, J=6.89 Hz, 6H),
5.17 (d, J=17.04 Hz, 1H), 5.44 (d, J=10.24 Hz, 1H), 5.81 (d, J=6.24 Hz,
1H), 6.30 (ddd, J=16.88, 10.36, 6.29 Hz, 1H), 6.90-6.98 (m, 2H), 7.07-7.16
(m, 3H), 7.21-7.28 (m, 2H), 7.53-7.59 ppm (m, 3H); 13C NMR (75 MHz,
CDCl3): δ=55.9, 63.2, 110.3, 111.5, 118.5, 119.2, 119.4, 127.2, 127.8,
129.3, 133.1, 135.7, 136.6, 141.2, 148.8, 149.2 ppm; LC-MS (ESI):
Rt=2.04 min, m/z: 253.13 [M-Imidazole]+.
3.4
1-(1-(4'-methoxy-3'-methyl-[1,1'-biphenyl]-4-yl)allyl)-1H-imidazole
(S4):
Synthesized according to method C using 4a (95 mg, 0.37 mmol) and CDI
(182 mg, 1.1 mmol). S4 was obtained as a yellow oil (51 mg, 45 %):
Rf=0.33 (EtOAc); 1H NMR (300 MHz, CDCl3): δ=2.29 (s, 3H), 3.87 (s, 3H),
5.17 (d, J=17.04 Hz, 1H), 5.44 (d, J=10.24 Hz, 1H), 5.81 (d, J=6.33 Hz,
1H), 6.30 (ddd, J=16.86, 10.34, 6.33 Hz, 1H), 6.86-6.95 (m, 2H), 7.11 (s,
1H), 7.20-7.29 (m, 2H), 7.35-7.43 (m, 2H), 7.53-7.61 ppm (m, 3H);
13C NMR (75 MHz, CDCl3): δ=16.3, 55.3, 63.2, 110.2, 118.5, 119.1, 125.3,
126.9, 127.1, 127.8, 129.2, 129.3, 132.2, 135.8, 136.2, 136.6, 141.2, 157.6
ppm. LC-MS (ESI): Rt=4.40 min, m/z: 237.18 [M-Imidazole]+.
6. Method F: TBDMS deprotection
The aldehyde (1.0 eq.) was dissolved in dry THF (9 mL/mmol),
tetrabutylammonium fluoride solution (0.5 eq., 1 M in THF) and 0.1 M
K2HPO4 solution (100 µL/mmol, pH 7) were added and the reaction mixture
was stirred overnight. The solvent was evaporated under reduced
pressure and the crude product was purified by chromatography on silica
gel.
3.5 1-(1-(4-(benzo[d][1,3]dioxol-5-yl)phenyl)allyl)-1H-imidazole (S5):
Synthesized according to method C using 5a (200 mg, 0.79 mmol) and
CDI (382 mg, 2.35 mmol). S5 was obtained as a colorless oil (65 mg,
27 %): Rf=0.26 (EtOAc/MeOH 98:2); 1H NMR (300 MHz, CDCl3): δ=5.17
(d, J=17.23 Hz, 1H), 5.44 (d, J=10.24 Hz, 1H), 5.80 (d, J=6.15 Hz, 1H),
6.00 (s, 2H), 6.29 (ddd, J=16.86, 10.34, 6.33 Hz, 1H), 6.84-6.94 (m, 2H),
7.01-7.13 (m, 3H), 7.22 (d, J=8.10 Hz, 2H), 7.47-7.58 ppm (m, 3H);
13C NMR (75 MHz, CDCl3): δ=63.2, 101.2, 107.5, 108.6, 118.5, 119.3,
120.7, 127.3, 127.9, 129.4, 134.6, 135.7, 136.7, 136.8, 141.2, 147.4, 148.2
ppm. LC-MS (ESI): Rt=4.32 min, m/z: 305.14 [M+H]+, 237.09 [M-
Imidazole]+.
6.1 5-(4-(1-(1H-imidazol-1-yl)allyl)phenyl)-1H-indole (S10):
Synthesized according to method F using 10a (106 mg, 0.25 mmol) and
TBAF (0.13 mL 1M sol., 0.13 mmol). S10 was obtained as a light brown
solid (57 mg, 76 %): Rf=0.20 (EtOAc/MeOH 98:2); 1H NMR (300 MHz,
CDCl3): δ=5.20 (dt, J=17.0, 1.1 Hz, 1H), 5.46 (dt, J=10.2, 1.0 Hz, 1H), 5.83
(d, J=6.2 Hz, 15H), 6.33 (ddd, J=16.9, 10.4, 6.3 Hz, 1H), 6.61-6.64 (m, 1H),
6.94-6.97 (m, 1H), 7.13 (s, 1H), 7.24-7.30 (m, 3H), 7.41-7.49 (m, 2H), 7.58
(s, 1H), 7.64-7.69 (m, 2H), 7.86 (d, J=0.8 Hz, 1H), 8.38 ppm (br s, 1H);
13C NMR (75 MHz, CDCl3): δ=63.5, 102.9, 111.4, 118.7, 119.2, 119.2,
121.6, 125.1, 127.8, 128.4, 128.9, 132.2, 135.5, 135.8, 135.8, 136.6,
12
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10.1002/cmdc.202100283
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FULL PAPER
142.9 ppm. LC-MS (ESI): Rt=3.02 min, m/z: 300.19 [M+H]+, 232.18 [M-
Imidazole]+.
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6.2 4'-(1-(1H-imidazol-1-yl)allyl)-[1,1'-biphenyl]-4-ol (S9):
K. J. McLean, P. Carroll, D. G. Lewis, A. J. Dunford, H. E.
Seward, R. Neeli, M. R. Cheesman, L. Marsollier, P.
Douglas, W. E. Smith, I. Rosenkrands, S. T. Cole, D.
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33406-33416.
Synthesized according to method F using 9a (55 mg, 0.14 mmol) and
TBAF (0.07 mL 1M sol., 0.07 mmol). S9 was obtained as a white solid (7.8
mg, 20 %): Rf=0.19 (EtOAc); 1H NMR (300 MHz, CD3OD): δ=4.59 (s, 1H),
5.18 (dt, J=16.9, 1.3 Hz, 1H), 5.44 (dt, J=10.2, 1.1 Hz, 1H), 6.04 (d, J=6.4
Hz, 1H), 6.42 (ddd, J=16.9, 10.3, 6.4 Hz, 1H), 6.82-6.88 (m, 2H), 7.01 (t,
J=1.1 Hz, 1H), 7.10 (t, J=1.3 Hz, 1H), 7.29 (d, J=8.1 Hz, 2H), 7.43-7.49 (m,
2H), 7.55-7.62 (m, 2H), 7.70 ppm (s, 1H); 13C NMR (75 MHz, CD3OD):
δ=64.7, 116.9, 119.5, 120.2, 128.0, 129.2, 129.2, 133.0, 137.7, 138.0,
138.2, 142.8, 158.7 ppm; LC-MS (ESI): Rt=1.47 min, m/z: 277.09 [M+H]+,
209.09 [M-Imidazole]+.
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7. 1-(4-Bromobenzyl)-1H-imidazole (S11a):
[9]
NaH (19.5 mmol, 800 mg, 60 % in paraffin oil) was suspended in 3 mL dry
DMF. After cooling to 4 °C a solution of imidazole (15.0 mmol, 1.02 g) in
DMF (3 mL) was added dropwise. The solution was stirred at 4 °C for 1 h.
1-Bromo-4-(bromomethyl)benzene (15.0 mmol, 3.75 g) was dissoved in 5
mL DMF added dropwise to the reaction mixture which was subsequently
warmed to room temperature and then to 60 °C for 1.5 h. The reaction was
quenched with H2O (5 mL) and extracted with ethyl acetate (3 × 15 mL).
The combined organic phases were washed with brine, dried over MgSO4
and the solvent was evaporated under reduced pressure. Remaining DMF
was removed by repeated addition of n-heptane and evaporation under
reduced pressure. The crude product was purified by chromatography on
silica gel (EtOAc : petrol ether = 9 : 1). The product was obtained as white
solid (2.10 g, 59 %): Rf=0.21 (EtOAc); 1H NMR (500 MHz, DMSO-d6):
δ=5.19 (s, 2H), 6.92 (m, 1H), 7.20 (m, 2H), 7.56 (m, 3H), 7.75 ppm (m,
1H); 13C NMR (125 MHz, DMSO-d6): δ=86.2, 157.0, 158.3, 166.3, 167.1,
169.0, 174.8, 174.9 ppm; LC-MS (ESI): Rt=4.99 min, m/z: 236.9 [M+H]+.
Enantiomer separation and analysis: The enantiomers were separated
using a Daicel ChiralPak® IE column (5 µm, 10 × 250 mm) with a
preparative HPLC (Thermo Scientific Ultimate 3000, consisting of Dionex
Ultimate 3000 pump, diode array detector and automated fraction
collector) monitoring the absorbance at 254 nm. An isocratic flow of 5
mL/min was applied with the following solvent composition: Compound S1
and S4: methyl-tert-butyl ether (MtBE):(EtOH + 0.5% ethylendiamine) =
98:2, Compound S5: MtBE:(EtOH + 0.5% ethylendiamine) = 96:4.
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Acknowledgements
We acknowledge the use of ESRF Beamline ID 23-2 and SLS
Beamlines X06DA and X10SA.
M. Fonvielle, M. H. Le Du, O. Lequin, A. Lecoq, M.
Jacquet, R. Thai, S. Dubois, G. Grach, M. Gondry, P.
Belin, J Biol Chem 2013, 288, 17347-17359.
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M. Kamal, J. Haupenthal, I. Schnorr, S. H. Cho, S. G.
Franzblau, M. Empting, J. Eberhard, R. W. Hartmann,
ChemMedChem 2017, 12, 1616-1626.
PDB Submissions
[19]
[20]
The complex structures of CYP121 with bound L21, L44 and S2
have been submitted to the PDB (PDB IDs 6TET, 6TEV and
6TE7, respectively).
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J. T. Chenge, L. V. Duyet, S. Swami, K. J. McLean, M. E.
Kavanagh, A. G. Coyne, S. E. Rigby, M. R. Cheesman, H.
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Education 2003, 80, 214.
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Discovery: A Guide for Medicinal Chemists and
Pharmacologists, Wiley, John 2013.
Conflict of interest
The authors declare no conflict of interest.
[22]
[23]
Keywords: CYP121 • SAR study • Mycobacterium tuberculosis •
complex structures • biological activity
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Entry for the Table of Contents
Infections with Mycobacterium tuberculosis (Mtb) still remain among the most difficult to treat, which is owed to the pathogen´s special
characteristics within the realm of pathogenic bacteria. This paper describes the development of potent inhibitors targeting an essential
Cytochrome P450 enzyme of Mtb, which are also able to reduce the intracellular replication of mycobacteria within macrophages.
15
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