Efficient analoging around ethionamide to explore thioamides bioactivation pathways triggered by boosters in Mycobacterium tuberculosis

Article abstract of DOI:10.1016/j.ejmech.2018.09.038

Ethionamide is a key antibiotic prodrug of the second-line chemotherapy regimen to treat tuberculosis. It targets the biosynthesis of mycolic acids thanks to a mycobacterial bioactivation carried out by the Baeyer-Villiger monooxygenase EthA, under the control of a transcriptional repressor called EthR. Recently, the drug-like molecule SMARt-420, which triggers a new transcriptional regulator called EthR2, allowed the derepression a cryptic alternative bioactivation pathway of ethionamide. In order to study the bioactivation of a collection of thioisonicotinamides through the two bioactivation pathways, we developed a new two-step chemical pathway that led to the efficient synthesis of eighteen ethionamide analogues. Measurements of the antimycobacterial activity of these derivatives, used alone and in combination with boosters BDM41906 or SMARt-420, suggest that the two different bioactivation pathways proceed via the same mechanism, which implies the formation of similar metabolites. In addition, an electrochemical study of the aliphatic thioisonicotinamide analogues was undertaken to see whether their oxidation potential correlates with their antitubercular activity measured in the presence or in the absence of the two boosters.

Full text of DOI:10.1016/j.ejmech.2018.09.038

Accepted Manuscript
Efficient analoging around ethionamide to explore thioamides bioactivation pathways
triggered by boosters in Mycobacterium tuberculosis
Marion Prieri, Rosangela Frita, Nicolas Probst, Alix Sournia-Saquet, Marilyne
Bourotte, Benoit Déprez, Alain R. Baulard, Nicolas Willand
PII:
S0223-5234(18)30810-9
10.1016/j.ejmech.2018.09.038
EJMECH 10749
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 28 April 2018
Revised Date: 27 June 2018
Accepted Date: 14 September 2018
Please cite this article as: M. Prieri, R. Frita, N. Probst, A. Sournia-Saquet, M. Bourotte, B. Déprez,
A.R. Baulard, N. Willand, Efficient analoging around ethionamide to explore thioamides bioactivation
pathways triggered by boosters in Mycobacterium tuberculosis, European Journal of Medicinal
Chemistry (2018), doi: https://doi.org/10.1016/j.ejmech.2018.09.038.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Efficient analoging around ethionamide to explore
thioamides bioactivation pathways triggered by boosters in
Mycobacterium tuberculosis
1
,#
2,#
1
3,4
Marion Prieri , Rosangela Frita , Nicolas Probst , Alix Sournia-Saquet , Marilyne
1
1
2
1,*
Bourotte , Benoit Déprez , Alain R. Baulard , Nicolas Willand
1
Univ. Lille, Inserm, Institut Pasteur de Lille, U1177 - Drugs and Molecules for Living Systems, F-59000 Lille,
France
2
Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019 – UMR 8204 – CIIL – Center for Infection
and Immunity of Lille, F-59000 Lille, France
3
CNRS, LCC (Laboratoire de Chimie de Coordination UPR 8241), 205 route de Narbonne, F-31077 Toulouse,
France
4
Univ. Toulouse, Université Paul Sabatier, F-31077 Toulouse, France
#
These authors contributed equally to this work
*nicolas.willand@univ-lille2.fr
ABSTRACT
Ethionamide is a key antibiotic prodrug of the second-line chemotherapy regimen to treat tuberculosis. It
targets the biosynthesis of mycolic acids thanks to a mycobacterial bioactivation carried out by the Baeyer-
Villiger monooxygenase EthA, under the control of a transcriptional repressor called EthR. Recently, the drug-
like molecule SMARt-420, which triggers a new transcriptional regulator called EthR2, allowed the derepression
a cryptic alternative bioactivation pathway of ethionamide. In order to study the bioactivation of a collection of
thioisonicotinamides through the two bioactivation pathways, we developed a new two-step chemical pathway
that led to the efficient synthesis of eighteen ethionamide analogues. Measurements of the antimycobacterial
activity of these derivatives, used alone and in combination with boosters BDM41906 or SMARt-420, suggest
that the two different bioactivation pathways proceed via the same mechanism, which implies the formation of
similar metabolites. In addition, an electrochemical study of the aliphatic thioisonicotinamide analogues was
undertaken to see whether their oxidation potential correlates with their antitubercular activity measured in
the presence or in the absence of the two boosters.
Keywords:
Antitubercular prodrugs, boost of antimycobacterial activity, heterocyclic medicinal chemistry,
thioisonicotinamide chemical series, oxidation processes, chemical probes to study bioactivation pathways of
ethionamide

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Introduction
According to WHO most recent statistics, tuberculosis (TB) is today the first leading cause of death by an
infectious agent and is one of the top 10 causes of death worldwide.[1] In 2016, 10.4 million people were TB-
infected, and 1.7 million died from the disease (including 0.4 million among people with HIV).
Drug-sensitive TB treatment typically lasts 6 to 9 months, and consists of an initial quadri-therapy during 2
months, followed by a bi-therapy with isoniazid and rifampicin for the 4 to 7 remaining months of treatment.
Most anti-TB drugs have been discovered more than 50 years ago and their use is very often associated with
side effects that lead to patient non-compliance, thereby opening the way to the development of multiple
drug-resistant TB strains (MDR-TB). MDR-TB must then be treated with second line drugs, having a lower
therapeutic index, thus leading to even poorer compliance and the inexorable emergence of extensively drug-
resistant TB (XDR-TB). The current first-line regimen for the anti-TB treatment has remained unchanged since
its implementation in 1970[2] and only two new drugs, bedaquiline and delamanid, have recently been
approved for the treatment of MDR-TB[3].
Ethionamide (ETH) is, with fluoroquinolones, an essential pillar for the successful treatment of MDR-TB as
recently reassessed by a large meta-analysis.[4] ETH was discovered in 1956[5] and was approved as a second-
line drug. However, its use is often associated with serious side effects. ETH and its closed analogue
prothionamide (PTH), are both prodrugs, which are bio-activated by EthA, a mycobacterial Baeyer-Villiger
monooxygenase.[6] A first EthA-catalyzed oxidation step leads to sulfoxide analogues and further oxidations
end up with the release of nicotinamide adenine dinucleotide (NAD) adduct.[7,8] Trapped within the
bacteria[9], this so called “adduct” has been shown to inhibit InhA[10]. InhA is a NADH-dependent enoyl-acyl
carrier protein reductase of the fatty acid biosynthesis II system required for mycolic acid biosynthesis, a major
component of the bacterial cell-envelope. InhA inhibition impairs the integrity of the cell wall and contribute to
cell death. (figure 1a).[11] This key bioactivation process is negatively controlled at the transcriptional level by
the repressor EthR.[6] As such, genetically engineered ethR knock-outs revealed bacteria highly susceptible to
ETH, demonstrating that M. tuberculosis indeed controls its own susceptibility to ETH.[6]
Few years ago, we proposed a new therapeutic approach which relies on the inhibition of EthR with synthetic
ligand to boost the transcription of ethA and thereby the bioactivation of ETH.[12,13] Combined efforts in
medicinal chemistry and biology led to the design and development of compounds able to inhibit EthR with
potency in the low nanomolar range (figure 1b).[14–18] BDM41906, the most representative compound of our
first generation of boosters, was able to boost ETH efficacy both in vitro and in vivo.[15,19]
More recently, we have identified a second generation of boosters. This spiroisoxazoline family of small
molecules, represented by SMARt-420, is able to derepress a cryptic alternative bioactivation pathway of ETH
in M. tuberculosis, by targeting a transcriptional regulator belonging to the TetR family called EthR (figure
2
1
c).[20] Importantly, by bypassing the EthA-dependent bioactivation of ETH, SMARt-420 was shown to revert
ETH resistance of EthA-mutated clinical isolates.

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Figure 1: Description of the two bioactivation pathways of ETH triggered by the two generations of boosters. In Mycobacterium
tuberculosis (Panel 1a), transcription factor EthR partially represses transcription of the Baeyer-Villiger monoxygenase EthA, allowing
limited conversion of ETH to ETH S-oxyde and then to the nicotinamide adenine dinucleotide (NAD) adduct, that eventually inhibits InhA
and cell wall synthesis. BDM41906 inhibits EthR and thus releases the transcription of ethA (Panel 1b). SMARt-420 (Panel 1c) inhibits EthR
2
and allows high-level production of the oxidoreductase EthA
pathway.
2
that provokes the activation of ETH through an alternative bioactivation
In the 50s, it was reported by Libermann and coworkers that ETH S-oxide has an activity similar to the one of
ETH[21] and that modifications on the C-2 position of the pyridine ring of thioisonicotinamide were key to
modulate antitubercular activity through the bioactivation by the basally expressed EthA.[22,23] The goal of
the present study was to compare the bioactivation of ETH analogues by EthA and EthA triggered by boosters
2
BDM41906 and SMARt-420, respectively in order to define whether both enzymes share substrates specificity.
For that purpose, we developed a rapid and efficient analoging in the thioisonicotinamide series, which allowed
us to obtain eighteen ETH derivatives. The anti-tuberculosis activity of these compounds was measured on M.
tuberculosis H37Rv strain, alone or in combination with the two boosters BDM41906 and SMARt-420. Finally,
we compared anodic potentials with boosted MICs, which substantiate the involvement of a redox mechanism
in the bioactivation processes.

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Results
Efficient two-step synthesis of isothionicotinamide analogues of ETH
In 1956, Libermann et al. presented the first synthesis of ETH. This was achieved in 7 steps with an overall yield
of 10% (figure 2).[5,24] The major drawback of this sequence is that the introduction of diversity in C-2 position
of the pyridine ring has to be performed at an early stage, which increases the number of chemical steps to
reach the targeted compound. More recently, another synthetic path for the synthesis of thioisonicotinamides,
ETH and PTH, was described.[25] Bellale and coworkers heated isonicotinonitrile in sulfuric acid with ethanoic
or propanoic acid in the presence of silver nitrate, followed by dropwise addition of freshly prepared
ammonium persulfate solution in water. The obtained 2-substituted pyridine-4-carbonitriles were then treated
with sodium hydrosulfide hydrate and magnesium chloride to lead to the desired thioisonicotinamides. In order
to extend the collection of analogues with a divergent synthesis performed under mild conditions, we designed
two new and efficient chemical pathways.
Figure 2: chemical pathways for the synthesis of ETH using the route described by Libermann and coworkers and for the synthesis of
analogues in the thioisonicotinamide series using a palladium-catalyzed Negishi cross-coupling reaction with 2-bromopyridine-4-
carboxamide or a sulfinate driven C-H functionalization of isonicotinamide.
In a first step (figure 2), we achieved the synthesis of the corresponding carboxamide intermediates 2a-2r
following two different chemical pathways, one involving a palladium-catalyzed Negishi cross-coupling reaction
between 2-bromopyridine-4-carboxamide and organozinc derivatives (1a-1g and 1j-1p) and the other involving
a C-H functionalization of isonicotinamide using sulfinate salts (1h-1i and 1q-1r). In a second step, the carbonyl
was converted into thiocarbonyl using Lawesson’s reagent (3a-3r). Using these new chemical pathways, we
were able to introduce a large diversity of substituents in α-position of the nitrogen of the pyridine ring. We

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obtained linear (3a-3c including ETH) and branched (3d-3g) aliphatic analogues with an average yield over two-
steps of 35%. A trifluoromethylated analogue of ETH (3h) was also synthesized to see the impact of the
electron withdrawing group on the oxidability of the thioamide function. We also obtained three derivatives
with saturated rings (3i-3k), one ester (3l) and six benzylic derivatives (3m-3r) substituted or not with electron-
withdrawing or -donating substituents in para or in meta position. In this list of compounds, six (3a, 3b, 3c, 3d,
3
f, 3n) will be used as references as they were previously described and studied by Libermann et al.[5].
In the Negishi cross-coupling reaction, five starting organozinc compounds (1a-1b, 1n-1p) were synthesized
from the corresponding bromine derivatives. The reactions were performed in THF using catalytic quantities of
Pd(PPh ) Cl and they led to the desired substituted pyridine with yields ranging from 19% to 91%. The lowest
3
2
2
yield was observed with the para-fluorobenzyl analogue 2p where the electron withdrawing group probably
limited the formation of the organozinc reactant. In the case of secondary alkylzinc halides 1d and 1e, we
mainly observed the formation of linear by-products 2b and 2c respectively, due to isomerization during the
catalytic reaction cycle. After the transmetallation step, the palladium complex can undergo a β-hydride
elimination leading to the formation of isonicotinamide on one hand, and of the linear isomerized by-product
on the other hand.[26] To limit these side-reactions, we used Pd(OAc) and the hindred phosphine bidentate
2
ligand CPhos, which was shown to limit the formation of by-products.[27] Ratios of 2d/2b and 2e/2c estimated
by NMR, were 3.8/1 and 3.3/1, respectively. Compound 2e was obtained with an isolated yield of 58%. 2d and
its linear by-product 2b were not separated prior thionation. For the incorporation of fluoroalkyl (compounds
2
h-2i) or benzylic groups with electron withdrawing groups in meta position (2q-2r), we relied on a C–H
functionalization of isonicotinamide using zinc or sodium sulfinate salts, inspired from Baran’s work.[28,29] The
sulfinate salts were commercially available (1h and 1i), or prepared from the sulfonyl chloride derivative in the
presence of zinc in water (1q and 1r). The reactions were performed in DCM/water or DEC/water mixtures in
the presence of tert-butyl hydroperoxide (TBHP). In these conditions, the desired carboxamide derivatives
were obtained with moderate to good yields (8 to 67%). Finally, thionation of the 18 isonicotinamide
derivatives was performed using Lawesson's reagent in THF.[30] The yields of this last step ranged from 22 to
8
0%.

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Antimycobacterial activity of ETH analogues alone or in combination with boosters BDM41906 and SMARt-
4
20
Table 1: Biological activities of compounds 2c, 4a and 3a-3r. Minimal inhibitory concentrations (MIC50) of thioisonicotinamides alone, or in
combination with BDM41906 (b906-MIC50) or SMARt-420 (b420-MIC50), both used at a fixed concentration of 1.0 µM, are reported in µM.
Boost effects with BDM41906 (be-906) or SMARt-420 (be-420) were calculated using the ratio MIC50/bMIC50. MIC50 are mean of two
independent experiments.
X
N H2
N
R
MIC50
µM)
b906-MIC50
b420-MIC50
Epa
(V/NHE)
Entry
X
R
be-906
be-420
(
(µM)
(µM)
2
c
O
SO
S
>30.0
10
>30
0.4
2.0
0.9
0.6
>30
0.3
0.9
0.5
0.4
-
-
-
4a
3a
3b
25
10
14
10
33
21
26
15
nd
19
1.46 ± 0.0186
1.43 ± 0.0207
1.39 ± 0.0089
S
13
3
c
S
5.8
3
d
e
S
S
S
21
0.1
0.2
9.1
0.2
0.2
3.8
210
150
>3
105
150
>8
1.62 ± 0.0107
1.48 ± 0.0067
1.57 ± 0.0084
3
30.0
>30.0
3
f
3
3
g
S
S
S
6.6
0.1
10
10
0.1
1.5
10
52
>3
>3
52
>20
>3
1.63 ± 0.0169
1.55 ± 0.0168
1.65 ± 0.0148
h
>30.0
>30.0
3
3
i
j
S
S
S
6.6
1.0
0.6
3.8
0.5
0.7
1.3
7
13
1.40 ± 0.0188
1.62 ± 0.0087
nd
3
k
>30.0
>30.0
>50
>8
>43
>23
3
l
3
m
S
>30.0
0.6
0.7
>50
>43
nd
3
n
o
p
S
S
S
1.1
2.4
1.0
0.1
0.3
0.2
0.1
0.2
0.1
11
8
11
12
10
nd
nd
nd
3
3
3
5
q
S
S
0.6
0.5
0.1
0.1
0.1
0.1
6
5
6
5
nd
nd
3
r

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Compounds 3a-3r were then assessed for their in vitro antitubercular activity against the laboratory strain M.
tuberculosis H37Rv-GFP. To this collection of thioisonicotinamide analogues, we added ETH S-oxide 4a, the
metabolite of ETH known to be further bio-activated through the EthA pathway[8] (figure 1a), and the
carboxamide derivative 2c.
Dose-response curves allowed us to determine the minimal inhibitory concentrations (MIC ) of each thioamide
50
tested, alone or in combination with BDM41906 (b906-MIC ) or with SMARt-420 (b420-MIC ), both used at
50
50
the fixed concentration of 1.0 µM (concentration that fully engages the corresponding bioactivation pathway).
Boost effects with BDM41906 (be-906) or with SMARt-420 (be-420) were calculated using the ratio
MIC /bMIC . Data are summarized in table 1.
5
0
50
Antimycobacterial activity of ETH analogues
-
1
ETH (3a) showed a MIC of 19 µM (3.1 µg.mL ). The S-oxide derivative (4a), showed to be slightly more potent
5
0
than ETH, with a MIC of 10 µM. Increasing the number of carbon atoms of the aliphatic C-2 substituent of the
50
pyridine ring resulted in an increased activity as described by Libermann and coworkers.[31] PTH 3b and the
butyl analogue 3c showed MIC50 of 13 µM and 5.8 µM respectively. The cyclopropyl derivative 3j (MIC50 = 6.6
µM) was also slightly more active. In contrast, the two α-methylated analogues of ETH or PTH, compounds 3d
(MIC50 = 21 µM) and 3e (MIC50 > 30 µM), and the β-methylated analogue 3f were less potent than ETH.
Interestingly, the γ-methylated analogue 3g (MIC50 = 6.6 µM) recovered an activity equivalent to the butyl
analogue 3c. The fluorinated analogues 3h, 3i, the cyclohexyl 3k, the ester 3l were inactive at the highest
concentration tested (30 µM). Introduction of benzylic groups led to the most active analogues. Compounds
3
n-3r showed activities in the low micromolar range even if they were substituted with electron withdrawing
or donating groups in para or meta position of the phenyl ring. Strikingly, methylation of the benzylic position
was detrimental for activity (compound 3m); reminiscent with what was observed for compounds 3d and 3e.
Antimycobacterial activity of ETH analogues used in combination with boosters BDM41906 and SMARt-420
In our previous transcriptomic studies [15,20], we showed that by treating mycobacteria with the two boosters
BDM41906 and SMARt-420, we increased the expression of the two enzymes involved in the bioactivation of
ethionamide, EthA and EthA2 respectively, which acted positively on its antitubercular activity. Therefore, as
seen in our previous studies [20], ETH (3a) is boosted 9-times when combined with BDM41906 (b906-MIC50
=
2
.0 µM and be-906 = 9) and 21-times when combined with SMARt-420 (b420-MIC = 0.9 µM, be-420 = 21). We
5
0
also reconfirmed that ETH-SO (4a) is a substrate of EthA as we observed a high boost-effect in combination
with BDM41906 (b906-MIC50 = 0.4 µM). Interestingly, in this work, we show that it is also a substrate of the
bioactivation pathway triggered by SMARt-420 (4a, b420-MIC50 = 0.3 µM and be-420 = 33). The two n-alkyl
analogues of ETH (3b and 3c) were also boosted by the two boosters in the same order of magnitude as for
ETH. On the contrary, the carboxamide analogue 2c showed no activity including in the presence of BDM41906
and SMARt-420 confirming the central role of the sulfur atom in the biotransformation of thioamides.
Surprisingly, compounds 3d and 3e, the two α-methylated direct analogues of ETH or PTH, whereas poorly
active alone, revealed highly potent when combined with BDM41906 (3d: b906-MIC50 = 0.1 µM and 3e: b906-
MIC50 = 0.2 µM) and SMARt-420 (3d: b420-MIC = 0.2 µM and 3e: b420-MIC = 0.2 µM), with a striking boost
50
50
effect higher than 105. High boosted potencies and boost-effects were also observed with the γ-methylated
analogue 3g (b906-MIC = 0.1 µM and b420-MIC = 0.1 µM). On the contrary, we only observed mild boosted
50
50
potencies with the β-methylated analogue 3f (b906-MIC50 = 9.1 µM and b420-MIC50 = 3.8 µM) or with the
cyclopropyl derivative 3j (b906-MIC = 1.0 µM and b420-MIC = 0.5 µM).
50
50
The analogues inactive alone (3h, 3i, 3k, 3l) revealed active in the presence of boosters, 3k being the most
potent of this selection (b906-MIC50 = 0.6 µM and b420-MIC = 0.7 µM). Finally, the association of BDM41906
50
or SMARt-420 with compounds 3n-3r led to excellent combinations, with potencies in the low nanomolar
range.

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Comparison of the two bioactivation pathways
Comparison of MICs and boosted MICs reveals that all the 18 analogues of ETH show improved
antimycobacterial activity when combined with either BDM41906 or SMARt-420, with bMIC ranging from 0.1
50
µM to 10 µM.
Figure 3: b906-MIC50 versus b420-MIC50
.
Interestingly, we also observed a strong correlation (R² = 0.8618) between b906-MICs and b420-MICs (figure 3),
suggesting that the two independent bioactivation pathways triggered by the two boosters have the same
substrate specificity.
Electrochemical studies
According to the study published by Kane in 1961, we hypothesized that the first oxidation potential of
ethionamide is associated to the oxidation of its thioamide function.[32] Therefore, we decided to study
whether the oxidation potentials of our analogues correlate with their activity. To avoid influence of massive
changes in the structure that could interfere with compound penetration or affinities for targets, such as
aromatic rings, we limited the study to the aliphatic series (compounds 3a to 3k). Cyclic and square wave
voltammetry experiments were performed in acetonitrile at ambient temperature, on a glassy carbon-working
electrode. Anodic peak potential (Epa) was the average of 10 measurements. All compounds displayed a first
irreversible oxidation process, along with the adsorption of the oxidized compound on the working electrode
surface. The first oxidation potentials of the 11 thioamides ranged from 1.39 ± 0.01 to 1.65 ± 0.01 V/NHE (table
1). In most cases, we observed that a decrease of the oxidation potential correlates with the decrease of the
MIC regardless of the presence or absence of boosters. On the contrary, the introduction of fluorine atoms (3h
and 3i), which increased oxidation potentials, induced an increase of MIC.

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When we plotted b906-MIC or b420-MIC as a function of anodic potentials (figure 4), we observed excellent
5
0
50
2
2
correlations (R = 0.8938 and R = 0.7573 respectively) when compounds 3d, 3e, 3g and 3k were excluded
(figure 4).
Figure 4: correlation between b906-MIC50, b420-MIC50 and oxidation potentials for compounds 3a-3c, 3f and 3h-j
Discussion
ETH and its close analogue prothionamide PTH are two of the most frequently prescribed antibiotics for the
treatment of drug-resistant tuberculosis and they are part of the few second-line drugs with bactericidal
activity.[33] Consequently, as the number of MDR and XDR cases is growing worldwide, the importance of ETH
is steadily increasing. The major drawback of ETH is that it shows an unfavorable therapeutic index, as the dose
necessary to kill M. tuberculosis generally causes serious adverse effects, such as gastrointestinal disorders,
hepatitis, and various mental disturbances. Therefore, identifying new thioisonicotinamides that associate
lower toxicity to better activity, in combination with boosters, would be a serious asset for the design of new
chemotherapeutic regimens.
The current understanding of the bioactivation of ETH is centered on the NADPH-specific FAD-containing
monooxygenase EthA, which produces the S-oxide derivative. Further transformations lead to the production
of short-live ETH derivatives and finally a NAD adduct that inhibits InhA.[8,10,34] This key EthA-dependent
bioactivation process is negatively controlled at the transcriptional level by the repressor EthR. In 2004, we
proposed a new therapeutic concept with the inhibition of EthR by synthetic ligand to boost the transcription
of ethA and thereby the bioactivation of ETH.[12] Medicinal chemistry efforts led to the discovery of
BDM41906, a member of our first generation of EthR inhibitors, that showed to boost ETH in vitro and in
vivo.[15,19] More recently, we have discovered a new chemical family that triggers a second transcriptional
repressor called EthR SMARt-420, the frontrunner of this family of compounds, was shown to contribute to
2.
the release of a new bioactivation pathway of ETH, involving EthA . Consequently, by bypassing the EthA-
2
dependent activation pathway, SMARt-420 was shown to revert resistance in clinical isolates mutated in EthA.
In order to compare these two bioactivation pathways, we decided to test the anti-TB activity of ETH and a
collection of analogues in combination with the EthA-booster BDM41906 or the EthA -booster SMARt-420. For
2
that purpose, we optimized a rapid and efficient synthesis of eighteen ETH-analogues. As Libermann et al.[5]
described that the activity of thioisonicotinamides is to a large extent dependent of the nature of the C-2
substituent of the pyridine ring, we focused the introduction of diversity on this position. We also included to
this collection of ETH analogues the commercial ETH-S-oxide and a carboxamide analogue (2c).
This library of compounds was then tested on the laboratory strain M. tuberculosis H37Rv-GFP. Half of these
molecules showed better MIC than ETH 3a (MIC = 19 µM), especially the five benzylic analogues (3n-3r) with
50
50

ACCEPTED MANUSCRIPT
MIC for two of them, 3q (MIC = 0.6 µM) and 3r (MIC = 0.5 µM), in the low micromolar range. Interestingly,
5
0
50
50
the potency of the whole set of thioisonicotinamides and ETH S-oxyde was improved when the enzymes of the
two bioactivation pathways, EthA and EthA , are overproduced thanks to the corresponding boosters. Boost
2
effects with BDM41906 (be-906) ranged from 3 to 210 and boost effects with SMARt-420 (be-420) ranged from
3
to 150. In total, 13 compounds showed MIC50 below 1.0 µM when combined with BDM41906, the most
potent in the aliphatic series being isopropyl and isopentyl derivatives 3d and 3g with a b906-MIC of 0.1 µM.
5
0
In the aromatic series, 3n (benzyl), 3q (meta-fluorobenzyl) and 3r (meta-cyanobenzyl) were the most potent
with a b906-MIC50 of 0.1 µM. In a similar manner, 14 thioisonicotinamides showed b420-MIC below 1.0 µM.
Compound 3g was the most active in the aliphatic series when in combination with SMARt-420 (b420-MIC50
.1 µM, be-420 = 52). Compounds 3n, 3p (para-fluorobenzyl), 3q and 3r of the benzylic series were equipotent
with b420-MIC of 0.1 µM.
=
0
5
0
Strikingly, we observed an excellent correlation (R² = 0.8618) between the b906-MIC and the b420-MIC that
was measured for each compound. This result suggests that the two independent bioactivation pathways,
triggered by the two different boosters, share specificity for ETH-analogues substituted in C-2 position of the
pyridine ring with aliphatic or benzylic groups. This observation leads to the hypothesis that for this series of
molecules, the two bioactivation pathways may proceed via the formation of identical metabolites, both
involving the sulfur atom, to finally lead to similar NAD-adducts.
To challenge this hypothesis an electrochemical study with aliphatic analogues was undertaken to see whether
oxidation potentials can be correlated with boosted MIC. We consider that under boosting conditions, the
limiting factor to the bioactivation of analogues is no more the concentration of the activation enzymes. To
avoid influence of massive changes in the structure that could interfere with compound penetration or
affinities for targets we limited the study to the aliphatic series. The first oxidation potential was measured for
the 11 aliphatic thioamides, which ranged from 1.39 ± 0.01 V to 1.65 ± 0.01 V/NHE. According to the work of
Kane, we attributed this oxidation potential to the oxidation of the thioamide function.[32] On one hand, when
the pyridine ring was enriched in electrons, the oxidation potential decreased together with bMIC50 (Epa &
bMIC of 3a > 3b > 3c). On the other hand, when the pyridine was depleted in electrons, like for instance with
the fluorine derivatives 3h and 3i, the oxidation potential increased with the bMIC . In particular, such
50
tendency was observed with compounds showing boosting effects, in the same order of magnitude of those
observed for ETH, regardless of the booster used. In that case, these analogues, whose structures are very
close in terms of shape and steric hindrance, probably have the same affinity for the two bioactivating enzymes
and for the final target InhA. Therefore, the observed antibacterial activity in this series of compounds only
depends on the ability of the thioamide function to be oxidized. In contrast, for compounds showing a boost
effect higher than 43 (3d-3e, 3g, 3k), and particularly for α- and β-methylated analogues 3d-3e, we did not
observe this correlation anymore. In that case, the oxidability may no longer be the parameter that governs
their activity. We can then hypothesize that the affinity of the final NAD-adduct for InhA may contribute more
importantly to the observed antitubercular potency.
Conclusion
In continuation of our research program focused on the study of ETH bioactivation pathways triggered by the
two boosters BDM41906 and SMARt-420, we explored the activities of a library of ETH analogues. This
collection of thioisonicotinamides was obtained through the development of a new and efficient two-step
synthesis based on a Negishi cross-coupling reaction or a C-H functionalization using sulfinate salts, followed by
a thionation using Lawesson’s reagent. We obtained 18 analogues with an average overall yield of 35%. Their
antimycobacterial activity was evaluated on M. tuberculosis, alone and in combination with BDM41906 (EthA
booster) and SMARt-420 (EthA2 booster). We observed that all the 18 analogues showed a better
antimycobacterial activity when combined with the two boosters. Moreover, we showed that the two
bioactivation pathways have the same specificity of substrates. According to the electrochemical study that
was performed, the potency of aliphatic analogues, showing the same boosting effect than ETH, regardless of
the booster used, can be attributed to their ability to be oxidized by overproduced bioactivation enzymes. This

ACCEPTED MANUSCRIPT
work also suggests that the two different bioactivation pathways may proceed via the formation of similar
metabolites involving the oxidation of the sulfur atom to finally lead to NAD-adduct. To validate this hypothesis
further developments of specific analytical methods are required.
More importantly, this work suggests that the sec-butyl derivative 3e may be an attractive alternative to ETH in
combination with BDM41906 and especially with SMARt-420. Indeed, this compound historically discarded
because of its low antitubercular activity, becomes now highly active in the presence of boosters. Whereas the
toxicity of ETH is attributed to its oxidation by the human flavin-containing monooxygenases [35], it is possible
that 3e would be less prone to oxidation by the same system. This would lead to an improved therapeutic index
of this compound compared to ETH, thus reducing side effects for the patient and improving its compliance.
Acknowledgements
This work was supported by l’Agence Nationale de la Recherche (ANR), France (Tea-4-Two, ANR-14-CE14-0027-
0
1), PRIM (NewBio4Tb), Institut National de la Santé et de la Recherche Médicale, Université de Lille, Institut
Pasteur de Lille, Centre National de la Recherche Scientifique, Région Hauts-de-France (convention
no.12000080), and Société d’Accélération du Transfert de Technologie Nord. We greatly appreciate the fruitful
discussions with all the members of the Bioversys AG team and their invaluable support in the continuation of
this project.

ACCEPTED MANUSCRIPT
Methods
Chemistry
General information
NMR spectra were recorded on a Bruker DRX-300 spectrometer. Chemical shifts are in parts per million (ppm).
1
13
The assignments were made using one-dimensional (1D) H and C spectra and two-dimensional (2D) HSQC
and COSY spectra. High-resolution mass spectra were recorded on an HPLC−MS-TOF system (Waters LCT
TM
Premier). Mass spectra were recorded with a LCMS Waters Alliance Micromass ZQ 2695. LCMS analysis was
TM
performed using a C18 X-Bridge 3.5 μm particle size column, dimensions 50 mm × 4.6 mm. The gradient used
started from 100% H O/0.1% formic acid and reached 20% H O/80% CH CN/0.1% formic acid within 5 min at a
2
2
3
flow rate of 2 mL/min. Purity (%) was determined by reversed phase HPLC, using UV detection (215 nm), and all
compounds showed purity greater than 95%. All commercial reagents and solvents were used without further
TM
purification. Compound 4a was purchased from Carbosynth (UK). Commercial ethionamide, organozinc
TM
halides and bis[(substituted)sulfonyl]zinc 1h-1i were purchased from Sigma-Aldrich .
Characterization data for intermediate products are described below:
General procedure A for the synthesis of the zinc sulfinates 1q-1r
Substituted phenylmethanesulfonyl chloride (1 eq) was added to a suspension of Zn (7 eq) in H O (1.0
2
ml/mmol) at 0 °C and stirred for 3h gradually warming to room temperature. Excess Zn was removed through a
plug of sand on a sintered glass funnel, washed with water and then concentrated in vacuo.
bis[(3-fluorophenyl)methylsulfonyl]zinc (1q):
1
Following general procedure A, we obtained 1r as a white solid, isolated yield 35%. H NMR (300 MHz, DMSO-
13
d6) δ 7.36-7.23 (m, 2H), 7.14 – 6.98 (m, 6H), 3.59 (s, 2H); C NMR (75 MHz, DMSO-d6): δ 162.0 (d, J = 248.0
Hz), 136.0 (d, J = 7.9 Hz), 129.8 (d, J = 8.4 Hz), 126.1 (d, J = 2.6 Hz), 116.5 (d, J = 20.9), 113.2 (d, J = 20.9 Hz), 68.0
(
d, J = 1.7 Hz).
bis[(3-cyanophenyl)methylsulfonyl]zinc (1r):
Following general procedure A, we obtained 1q as a white solid, isolated yield 39%. H NMR (300 MHz, DMSO-
1
1
3
d6) δ 7.70 – 7.62 (m, 4H), 7.56 (dt, J = 7.9, 1.6 Hz, 2H), 7.48 (td, J = 7.6, 0.6 Hz, 2H), 3.63 (s, 4H); C NMR (75
MHz, DMSO-d6): δ 135.5, 135.0, 133.8, 130.5, 129.5, 119.4, 111.3, 67.3.
General procedure B for the synthesis of the compounds 2a-2b and 2n-2p
Step 1: synthesis of organozinc halides. LiCl (6 eq) was added to a Schlenk tube equipped with a magnetic stir
bar and a rubber septum and was heated with a heat gun for 10 min under vacuum and backfilled with argon 3
times. Zn (6 eq) was weighed and added to the tube after cooling to room temperature. The powders were
again heated with a heat gun for 10 min under vacuum and backfilled with argon 3 times. After cooling to room
temperature, THF (5 ml/mmol) and iodine (0.075 eq) were added and heated at 60 °C for 20 min. After cooling
at room temperature, bromine compound (3 eq) was added and the reaction mixture was heated at 50 °C
overnight.
Step 2: Negishi cross-coupling reaction. After cooling at room temperature, Pd(PPh ) Cl (0.05 eq) and 2-
3
2
2
bromopyridine-4-carboxamide (1 eq) were added to the Schlenk tube at 0 °C and the mixture was stirred 4h at
room temperature. The reaction mixture was then quenched by addition of saturated aqueous NH Cl solution
4
(
25 ml) and extracted with ethyl acetate (3x25 ml). The combined organic layer was dried over MgSO , filtered,
4
and concentrated in vacuo. The resulting residue was purified by column chromatography on silica gel (0 to
0:25:5 CH Cl :EtOAc:CH OH, GraceResolv 24G, over 45 min).
7
2
2
3

ACCEPTED MANUSCRIPT
2
-ethylpyridine-4-carboxamide (2a):
Following general procedure B, we obtained 2a as a yellow solid, with an isolated yield of 73%. TLC
1
(
CH Cl :EtOAc:CH OH, 70:25:5 v/v): Rf = 0.27; H NMR (300 MHz, CD OD): δ 8.58 (dd, J = 5.2, 0.9 Hz, 1H), 7.73
2 2 3 3
13
(
dd, J = 1.7, 0.9 Hz, 1H), 7.65 (dd, J = 5.2, 1.7 Hz, 1H), 2.88 (q, J = 7.6 Hz, 2H), 1.31 (t, J = 7.6 Hz, 3H); C NMR (75
MHz, CD OD): δ 168.6, 164.2, 149.0, 142.5, 120.2, 119.1, 30.6, 13.0.
3
2
-propylpyridine-4-carboxamide (2b):
Following general procedure B, we obtained 2b as a white solid, with an isolated yield of 91%. TLC
1
(
CH Cl :EtOAc:CH OH, 70:25:5 v/v): Rf = 0.38; H NMR (300 MHz, CD OD): δ 8.56 (dd, J = 5.2, 0.8 Hz, 1H), 7.69
2 2 3 3
(dd, J = 1.7, 0.8 Hz, 1H), 7.63 (dd, J = 5.2, 1.7 Hz, 1H), 2.83-2.77 (m, 2H), 1.80-1.68 (m, 2H), 0.95 (t, J = 7.4 Hz,
3
H).
2
-benzylpyridine-4-carboxamide (2n):
Following general procedure B furnished 2n as a white solid, with an isolated yield of 63%. TLC
1
(
CH Cl :EtOAc:CH OH, 70:25:5 v/v): Rf = 0.39; H NMR (300 MHz, CD OD): δ 8.56 (d, J = 5.1 Hz, 1H), 7.65 (d, J =
2
2
3
3
13
1
1
.5 Hz, 1H), 7.60 (dd, J = 5.1, 1.5 Hz, 1H), 7.15-7.29 (m, 5H), 4.18 (s, 2H); C NMR (75 MHz, CD OD): δ 169.9,
3
63.2, 150.4, 143.9, 140.2, 129.9, 129.6, 127.5, 122.6, 120.5, 44.9.
2
-[(4-methoxyphenyl)methyl]pyridine-4-carboxamide (2o):
Following general procedure B furnished 2o as an off-white solid, with an isolated yield of 49%. TLC
1
(
CH Cl :EtOAc:CH OH, 70:25:5 v/v): Rf = 0.35; H NMR (300 MHz, CD OD): δ 8.56 (dd, J = 5.2, 0.9 Hz, 1H), 7.66
2
2
3
3
(
dd, J = 1.7, 0.9 Hz, 1H), 7.61 (dd, J = 5.2, 1.7 Hz, 1H), 7.21-7.09 (m, 2H), 6.87-6.77 (m, 2H), 4.10 (s, 2H), 3.72 (s,
13
3
4
H); C NMR (75 MHz, CD OD): δ 168.5, 162.3, 158.5, 149.0, 142.6, 130.8, 129.6, 121.2, 119.2, 113.7, 54.3,
3
2.6.
2
-[(4-fluorophenyl)methyl]pyridine-4-carboxamide (2p):
Following general procedure B furnished 2p as a white solid, with an isolated yield of 19%. TLC
1
(
CH Cl :EtOAc:CH OH, 70:25:5 v/v): Rf = 0.38; H NMR (300 MHz, CD OD): δ 8.59 (dd, J = 5.2, 0.8 Hz, 1H), 7.70
2 2 3 3
13
(
t, J = 1.2 Hz, 1H), 7.64 (dd, J = 5.3, 1.7 Hz, 1H), 7.33-7.23 (m, 2H), 7.07-6.96 (m, 2H), 4.18 (s, 2H); C NMR (75
MHz, CD OD): δ 168.5, 161.7 (d, J = 243.5 Hz), 161.6, 149.2, 142.7, 134.9 (d, J = 3.2 Hz), 130.4 (d, J = 7.9 Hz),
3
1
21.2, 119.3, 114.9 (d, J = 21.6 Hz), 42.6.
General procedure C for the synthesis of the compounds 2c, 2f-2g and 2j-2m
A Schlenk tube equipped with a magnetic stir bar and a rubber septum and was heated with a heat gun under
vacuum and backfilled with argon 3 times. After cooling at room temperature, 2-bromopyridine-4-carboxamide
(
1 eq) and Pd(PPh ) Cl (0.05 eq) were added to the Schlenk tube. The tube was again backfilled with argon 3
3 2 2
times. THF (5 mL/mmol) was added to the tube via syringe at room temperature. R-ZnBr solution at 0.5 M in
THF was added dropwise at 0 °C and the mixture was stirred 2h at room temperature. The reaction mixture
was then quenched by addition of saturated aqueous NH Cl solution (25 ml) and extracted with ethyl acetate
4
(
3x25 ml). The combined organic layer was dried over MgSO , filtered, and concentrated in vacuo. The resulting
4
residue was purified by column chromatography on silica gel (0 to 80:18:2 CH Cl :EtOAc:NH 7N in CH OH,
2
2
3
3
GraceResolv 24G, over 45 min).
-butylpyridine-4-carboxamide (2c):
Following general procedure C, we obtained 2c as a white solid, with an isolated yield of 87%. TLC
2
1
(
CH Cl :EtOAc:CH OH, 70:25:5 v/v): Rf = 0.35; H NMR (300 MHz, CD OD): δ 8.56 (dd, J = 5.3, 0.9 Hz, 1H), 7.70
2
2
3
3
(
dd, J = 1.8, 0.9 Hz, 1H), 7.62 (dd, J = 5.3, 1.8 Hz, 1H), 2.84 (t, J = 7.8 Hz, 2H), 1.75-1.65 (m, 2H), 1.43-1.31 (m,
1
3
2
2
H), 0.94 (t, J = 7.4 Hz, 3H); C NMR (75 MHz, CD OD): δ 168.6, 163.2, 149.0, 142.3, 120.8, 119.0, 37.2, 31.8,
3
2.0, 12.9.

ACCEPTED MANUSCRIPT
2
-isobutylpyridine-4-carboxamide (2f):
Following general procedure C furnished 2f as a brown solid, with an isolated yield of 72%. TLC
1
(
CH Cl :EtOAc:CH OH, 70:25:5 v/v): Rf = 0.33; H NMR (300 MHz, CD OD): δ 8.58 (dd, J = 5.2, 0.6 Hz, 1H), 7.68
2 2 3 3
(dd, J = 1.7, 0.6 Hz, 1H), 7.65 (dd, J = 5.2, 1.7 Hz, 1H), 2.71 (d, J = 7.7 Hz, 2H), 2.14-2.00 (m, 1H), 0.90 (d, J = 6.7
Hz, 6H).
-isopentylpyridine-4-carboxamide (2g):
Following general procedure C furnished 2g as a white solid, with an isolated yield of 90%. TLC
2
1
(
CH Cl :EtOAc:NH 7N in CH OH, 70:25:5 v/v): Rf = 0.44; H NMR (300 MHz, CD OD): δ 8.55 (dd, 1H, J = 5.2, 0.5
2
2
3
3
3
Hz), 7.71 (dd, 1H, J = 1.6, 0.8 Hz), 7.62 (dd, 1H, J = 5.2, 1.7 Hz), 2.85 (t, 2H, J = 8.1 Hz), 1.63-1.57 (m, 3H), 0.96 (d,
13
J = 6.4 Hz); C NMR (75 MHz, CD OD): δ 167.1, 161.8, 147.4, 140.8, 119.2, 117.4, 37.3, 33.9, 26.1, 19.9.
3
2
-cyclopropylpyridine-4-carboxamide (2j):
Following general procedure C furnished 2j as an off-white solid, with an isolated yield of 90%. TLC
1
(
CH Cl :EtOAc:CH OH, 70:25:5 v/v): Rf = 0.33; H NMR (300 MHz, CD OD): δ 8.47 (m, 1H), 7.59 (m, 1H), 7.51 (dd,
2
2
3
3
13
J = 5.2, 1.7 Hz, 1H), 2.20-2.12 (m, 1H), 1.08-1.00 (m, 4H); C NMR (75 MHz, CD OD): δ 168.8, 164.0, 149.1,
3
1
41.7, 118.7, 118.2, 16.7, 9.4.
2
-(cyclohexylmethyl)pyridine-4-carboxamide (2k):
Following general procedure C furnished 2k as a white solid, with a isolated yield of 83%. TLC
1
(
CH Cl :EtOAc:NH 7N in CH OH, 70:25:5 v/v): Rf = 0.47; H NMR (300 MHz, CD OD): δ 8.57 (dd, J = 5.2, 0.7 Hz,
2
2
3
3
3
1
0
H), 7.66 (dd, J = 1.7, 0.7 Hz, 1H), 7.62 (dd, J = 5.2, 1.7 Hz, 1H), 2.74 (d, J = 7.2 Hz, 2H), 1.81-1.62 (m, 6H), 1.29-
.98 (m, 5H).
Ethyl 3-(4-carbamoyl-2-pyridyl)propanoate (2l):
Following general procedure C furnished 2l as a white solid, with an isolated yield of 78%. TLC
1
(
CH Cl :EtOAc:CH OH, 70:25:5 v/v): Rf = 0.30; H NMR (300 MHz, CD OD): δ 8.60 (dd, J = 5.2, 0.7 Hz, 1H), 7.74
2
2
3
3
(
dd, J = 1.7, 0.7 Hz, 1H), 7.64 (dd, J = 5.2, 1.7 Hz, 1H), 4.14-4.06 (q, J = 14.3, 7.2 Hz, 2H), 3.17 (t, J = 7.5 Hz, 2H),
13
2
1
.81 (t, J = 7.4 Hz, 2H), 1.20 (t, J = 7.5 Hz, 3H); C NMR (75 MHz, CD OD): δ 172.9, 168.5, 161.1, 149.2, 142.4,
3
21.1, 119.4, 60.3, 32.9, 32.2, 13.1.
2
-(1-phenylethyl)pyridine-4-carboxamide (2m):
Following general procedure C furnished 2m as a white solid, with an isolated yield of 74%. TLC
1
(
CH Cl :EtOAc:NH 7N in CH OH, 80:18:2 v/v): Rf = 0.27; H NMR (300 MHz, CD OD): δ 8.58 (dd, J = 5.2, 0.8 Hz,
2
2
3
3
3
1
1
1
H), 7.72 (m, 1H), 7.61 (dd, J = 5.2, 1.8 Hz, 1H), 7.30-7.22 (m, 4H), 7.19-7.13 (m, 1H), 4.36 (q, J = 7.2 Hz, 1H),
1
3
.69 (d, J = 7.2 Hz, 3H); C NMR (75 MHz, CD OD): δ 168.7, 165.9, 149.0, 144.4, 142.4, 128.2, 127.3, 126.2,
3
20.0, 119.2, 19.6, 47.0.
General procedure D for the synthesis of the compounds 2d-2e
A Schlenk tube equipped with a magnetic stir bar and a rubber septum and was heated with a heat gun under
vacuum and backfilled with argon 3 times. After cooling at room temperature, 2-bromopyridine-4-carboxamide
(
1 eq), Pd(OAc) (0.04 eq) and CPhos (0.08 eq) were added to the Schlenk tube. The tube was again backfilled
2
with argon 3 times. THF (5 mL/mmol) was added to the tube via syringe at room temperature. R-ZnBr solution
at 0.5 M in THF was added dropwise at 0 °C and the mixture was stirred 2h at room temperature. The reaction
mixture was then quenched by addition of saturated aqueous NH Cl solution (25 ml) and extracted with ethyl
4
acetate (3x25 ml). The combined organic layer was dried over MgSO , filtered, and concentrated in vacuo. The
4
resulting residue was purified by column chromatography on silica gel (0 to 80:18:2 CH Cl :EtOAc:NH 7N in
2
2
3
CH OH, GraceResolv 24G, over 45 min).
3

ACCEPTED MANUSCRIPT
2
-isopropylpyridine-4-carboxamide (2d):
Following general procedure D furnished a mixture of 2d:2b 3.4:1 (conversion estimated by NMR). 2d was not
isolated and thionation was carried out without further purification.
2
-sec-butylpyridine-4-carboxamide (2e):
Following general procedure D furnished 2e as a white solid, with an isolated yield of 58%. TLC
1
(
CH Cl :EtOAc:NH 7N in CH OH, 80:18:2 v/v): Rf = 0.19; H NMR (300 MHz, CD OD): δ 8.58 (dd, J = 5.2, 0.8 Hz,
2
2
3
3
3
1
6
3
H), 7.70 (dd, J = 1.7, 0.8 Hz, 1H), 7.63 (dd, J = 5.2, 1.7 Hz, 1H), 2.95-2.82 (m, 1H), 1.86-1.60 (m, 2H), 1.30 (d, J =
13
.8 Hz, 3H), 0.83 (t, J = 7.2 Hz, 3H); C NMR (75 MHz, CD OD): δ 170.1, 168.6, 150.3, 143.7, 121.0, 120.5, 44.8,
3
0.9, 20.7, 12.4.
General procedure E for the synthesis of the compounds 2q-2r
To a suspension of pyridine-4-carboxamide (1 eq) and bis[(substituted)sulfonyl]zinc (1 eq) in CH Cl (4
2
2
ml/mmol) and H O (2 ml/mmol) at room temperature was added APTS (1 eq), followed by slow addition of
2
TBHP (2 eq) at 0 °C with vigorous stirring. After 10 min, the reaction was allowed to warm to room temperature
and heated at 50 °C for 18h, with stirring. The reaction was partitioned between CH Cl (10 mL) and a saturated
2
2
aqueous solution of sodium bicarbonate (10 mL). The organic layer was separated, and the aqueous layer was
extracted with CH Cl (3x10 mL). The organic layers were dried with MgSO , concentrated and purified by
2
2
4
column chromatography on silica gel (0 to 97:3 CH Cl /CH OH, GraceResolv 24G, over 30 min).
2
2
3
2
-[(3-fluorophenyl)methyl]pyridine-4-carboxamide (2q):
1
Following general procedure E furnished 2q as a white solid, with an isolated yield of 8%. H NMR (300 MHz,
CD OD) δ 8.60 (dd, J = 5.2, 0.8 Hz, 1H), 7.71 (dd, J = 1.7, 0.8 Hz, 1H), 7.64 (dd, J = 5.2, 1.7 Hz, 1H), 7.29 (td, J =
3
7
.9, 6.0 Hz, 1H), 7.07 (ddd, J = 7.8 1.5, 0.8 Hz, 1H), 7.00 (dtd, J = 10.1, 2.1, 0.5 Hz, 1H), 6.93 (tdd, J = 8.6, 2.6, 0.9
13
Hz, 1H), 4.20 (s, 2H); C NMR (75 MHz, CD OD): δ 169.9, 164.1 (d, J = 245.4 Hz), 162.5, 150.7, 144.1, 143.0 (d, J
3
=
4
7.2 Hz), 131.3 (d, J = 8.5 Hz), 125.9 (d, J = 2.9 Hz), 122.7, 120.8, 116.7 (d, J = 21.5 Hz), 114.3 (d, J = 21.5 Hz),
4.4 (d, J = 1.7 Hz).
2
-[(3-cyanophenyl)methyl]pyridine-4-carboxamide (2r):
1
Following general procedure E furnished 2r as a white solid, with an isolated yield of 9%. H NMR (300 MHz,
1
3
CD OD) δ 8.62 (d, J = 5.1 Hz, 1H), 7.73-7.56 (m, 6H), 7.47 (t, J = 7.7 Hz, 1H), 4.26 (s, 2H). C NMR (75 MHz,
3
CD OD): δ 169.8, 161.8, 150.9, 144.2, 142.1, 134.9, 133.6, 131.5, 130.7, 122.7, 121.0, 119.6, 113.6, 44.2.
3
Procedure F for the synthesis of 2-(2,2,2-trifluoroethyl)pyridine-4-carboxamide (2h)
To a suspension of pyridine-4-carboxamide (1 eq) and bis[(trifluoroethyl)sulfonyl]zinc (1 eq) in CH Cl (2
2
2
ml/mmol) and H O (1 ml/mmol) at room temperature was added TFA (1 eq), followed by slow addition of TBHP
2
(2 eq) at 0 °C with vigorous stirring. After 10 min, the reaction was allowed to warm to room temperature and
heated at 50 °C for 18h, with stirring. The reaction was partitioned between CH Cl (10 mL) and a saturated
2
2
aqueous solution of sodium bicarbonate (10 mL). The organic layer was separated, and the aqueous layer was
extracted with CH Cl (3x10 mL). The organic layers were dried with MgSO , concentrated and purified by
2
2
4
column chromatography on silica gel (0 to 97:3 CH Cl /CH OH, GraceResolv 24G, over 30 min).
2
2
3
The procedure F furnished 2h as a white solid, with an isolated yield of 13%. TLC (Cyclohexane:EtOAc, 30:70
1
v/v): Rf = 0.38; H NMR (300 MHz, CD OD) 8.69 (dd, J = 5.2, 0.8 Hz, 1H), 7.88 (br s, 1H), 7.78 (dd, J = 5.2, 1.7 Hz,
3
1
9
13
1
1
H), 3.78 (q, J = 10.9 Hz, 2H); F NMR (282 MHz, CD OD) δ -66.7 (t, J = 11.3 Hz); C NMR (75 MHz, CD OD): δ
3
3
69.4, 153.2 (q, J = 3.6 Hz), 151.2, 144.1, 126.9 (q, J = 276.6 Hz), 124.2, 122.3, 42.8 (q, J = 30.5 Hz).
Procedure G for the synthesis of 2-[1-(trifluoromethyl)cyclopropyl]pyridine-4-carboxamide (2i)
To a suspension of pyridine-4-carboxamide (1 eq) and [1-(trifluoromethyl)cyclopropyl]sulfonylsodium (2 eq) in
diethylcarbonate (6 ml/mmol) and H O (2 ml/mmol) was added (4 eq) by slow addition at 0 °C with vigorous
2
stirring. After 10 min, the reaction was allowed to warm to room temperature and heated at 90 °C for 1h, with

ACCEPTED MANUSCRIPT
stirring. A saturated aqueous solution of sodium thiosulfate (10 mL) was added. The reaction mixture was
concentrated and purified by column chromatography on silica gel (0 to 9:1 CH Cl /CH OH, GraceResolv 24G,
2
2
3
over 30 min).
1
The procedure G furnished 2i as a white solid, with an isolated yield of 67%. H NMR (300 MHz, CD OD) δ 8.65
3
(dd, J = 5.0, 1.0 Hz, 1H), 8.04-8.00 (m, 1H), 7.73 (dt, J = 5.2, 1.8 Hz, 1H), 3.35 (s, 1H), 1.60 (d, J = 6.7 Hz, 1H),
1
.51-1.36 (m, 6H), 1.34-1.20 (m, 3H).
Characterization data for final products are described below:
General procedure H for the synthesis of the compounds 3a-3r
THF (5 ml/mmol) was added to a flask containing pyridine-4-carboxamide derivative (1 eq) at room
temperature under inert atmosphere (argon) and stirring. Lawesson's reagent (1.5 eq) was added to the flask
and the solution was stirred at room temperature for 48h-72h. The THF was evaporated under reduce pressure
and the residue was partitioned between saturated aqueous NaHCO (25 ml) and ethyl acetate (25 ml) and
3
extracted with ethyl acetate (2x25 ml). The organic phase was dried over MgSO and eliminated under vacuum.
4
The residue was further purified by flash chromatography (0 to 3% MeOH:DCM, puriFlash 15-SI-HC 25G, over
4
5 min).
2
-ethylpyridine-4-carbothioamide (3a):
Following general procedure H furnished 3a as a yellow solid, with an isolated yield of 35%. TLC (CH Cl :CH OH,
2
2
3
1
9
7
1
1
0:10 v/v): Rf = 0.69; H NMR (300 MHz, CD OD): δ 8.47 (dd, J = 5.3, 0.8 Hz, 1H), 7.65 (dd, J = 1.8, 0.8 Hz, 1H),
3
1
3
.58 (dd, J = 5.3, 1.8 Hz, 1H), 2.86 (q, J = 7.6 Hz, 2H), 1.32 (t, J = 7.6 Hz, 3H); C NMR (75 MHz, CD OD): δ 200.2,
3
63.6, 148.5, 148.3, 119.6, 118.6, 30.5, 13.0; IR (neat): 3258.6, 2961.5, 2932.6, 2912.0, 2875.9, 1654.8, 1593.9,
-
1
+
415.6, 1392.3, 1285.6, 1260.7, 1205.4, 1149.6, 864.4, 808.4, 648.0, 528.8 cm ; HRMS (m/z): [M] calcd. for
C H N S, 167.0643; found, 167.0643.
8
10 2
2
-propylpyridine-4-carbothioamide (3b):
Following general procedure H furnished 3b as a yellow solid, with an isolated yield of 40%. TLC (CH Cl :CH OH,
2
2
3
1
9
7
0:10 v/v): Rf = 0.58; H NMR (300 MHz, CD OD): δ 8.47 (dd, J = 5.2, 0.7 Hz, 1H), 7.64 (dd, J = 1.8, 0.7 Hz, 1H),
3
1
3
.57 (dd, J = 5.2, 1.8 Hz, 1H), 2.83-2.78 (m, 2H), 1.82-1.70 (m, 2H), 0.98 (t, J = 7.4 Hz, 3H); C NMR (75 MHz,
CD OD): δ 201.5, 163.7, 149.7, 149.6, 121.6, 119.9, 40.7, 24.2, 14.0; IR (neat): 3263.1, 2957.8, 2929.7, 2898.5,
3
-
1
2
871.9, 1668.9, 1594.8, 1414.3, 1391.2, 1288.8, 900.6, 825.4, 709.7, 662.0, 546.3, 518.4 cm ; HRMS (m/z):
+
[M] calcd. for C H N S, 181.0799; found, 181.0802.
9 12 2
2
-butylpyridine-4-carbothioamide (3c):
Following general procedure H furnished 3c as a yellow solid, with an isolated yield of 75%. TLC (CH Cl :NH 7N
2
2
3
1
in CH OH, 99:1 v/v): Rf = 0.22; H NMR (300 MHz, CD OD): δ 8.46 (dd, J = 5.3, 0.8 Hz, 1H), 7.64 (dd, J = 1.7, 0.7
3
3
Hz, 1H), 7.58 (dd, J = 5.3, 1.8 Hz, 1H), 2.83 (t, J = 7.7 Hz, 2H), 1.76-1.65 (m, 2H), 1.45-1.32 (m, 2H), 0.95 (t, J = 7.4
1
3
Hz, 3H); C NMR (75 MHz, CD OD): δ 200.1, 162.6, 148.3, 148.3, 120.3, 118.6, 37.1, 31.8, 22.1, 12.9; IR (neat):
3
3
4
230.3, 2950.2, 2923.9, 2857.5, 1666.7, 1599.8, 1427.6, 1289.0, 1124.9, 857.2, 832.5, 694.9, 548.0, 515.5,
-
1
+
84.9 cm ; HRMS (m/z): [M] calcd. for C H N S, 195.0956; found, 195.0963.
1
0 14 2
2
-isopropylpyridine-4-carbothioamide (3d):
Following general procedure H furnished 3d as a yellow solid, with an isolated yield of 53%. TLC (CH Cl :NH 7N
2
2
3
1
in CH OH, 98:2 v/v): Rf = 0.49; H NMR (300 MHz, CD OD): δ 8.50 (dd, J = 5.2, 0.8 Hz, 1H), 7.69 (m, 1H), 7.57
3
3
1
3
(
dd, J = 5.2, 1.8 Hz, 1H), 3.21-3.06 (m, 1H), 1.34 (d, J = 6.9 Hz, 6H); C NMR (75 MHz, CD OD): δ 200.3, 167.4,
3
1
1
1
48.5, 148.3, 118.7, 118.2, 36.0, 21.4; IR (neat): 3263.6, 2959.6, 2923.2, 2870.0, 1658.7, 1595.4, 1551.4,
-
1
+
399.1, 1289.8, 888.8, 794.6, 642.1, 519.5 cm ; HRMS (m/z): [M] calcd. for C H N S, 181.0799; found,
9
12
2
81.0799.
2
-sec-butylpyridine-4-carbothioamide (3e):

ACCEPTED MANUSCRIPT
Following general procedure H furnished 3e as a yellow solid, with an isolated yield of 58%. TLC (CH Cl :NH 7N
2
2
3
1
in CH OH, 99:2 v/v): Rf = 0.2; H NMR (300 MHz, CD OD): δ 8.49 (dd, J = 5.2, 0.7 Hz, 1H), 7.64 (dd, J = 1.8, 0.8
3
3
Hz, 1H), 7.58 (dd, J = 5.3, 1.8 Hz, 1H), 2.92-2.79 (m, 1H), 1.85-1.59 (m, 2H), 1.29 (d, J = 7.1 Hz, 3H), 0.84 (t, J =
1
3
7
.4 Hz, 3H); C NMR (75 MHz, CD OD): δ 201.6, 168.0, 149.8, 149.6, 120.5, 120.0, 44.8, 30.9, 20.7, 12.4; IR
3
-
1
(
neat): 3204.4, 2957.6, 2923.6, 2870.7, 2854.6, 1597.9, 1552.2, 1404.9, 1298.2, 845.3, 774.3, 749.6, 531.1 cm ;
+
HRMS (m/z): [M] calcd. for C H N S, 195.0956; found, 195.0957.
1
0
14
2
2
-isobutylpyridine-4-carbothioamide (3f):
Following general procedure H furnished 3f as a yellow solid, with an isolated yield of 66%. TLC (CH Cl :NH 7N
2
2
3
1
in CH OH, 99:2 v/v): Rf = 0.2; H NMR (300 MHz, CD OD): δ 8.48 (dd, J = 5.2, 0.8 Hz, 1H), 7.63 (dd, J = 1.8, 0.9
3
3
13
Hz, 1H), 7.59 (dd, J = 5.3, 1.9 Hz, 1H), 2.69 (d, J = 7.3 Hz, 2H), 2.14-2.00 (m, 1H), 0.93 (d, J = 6.8 Hz, 6H); C NMR
(
75 MHz, CD OD): δ 201.3, 162.9, 149.6, 149.3, 122.3, 119.9, 47.8, 30.5, 22.7; IR (neat): 3334.3, 3304.4, 2952.3,
3
-
1
2
923.2, 2863.7, 1659.6, 1595.2, 1399.2, 895.7, 866.7, 845.7, 711.8, 623.0, 517.9, 470.9 cm ; HRMS (m/z):
+
[M] calcd. for C H N S, 195.0956; found, 195.0955.
10 14 2
2
-isopentylpyridine-4-carbothioamide (3g):
Following general procedure H furnished 3g as a yellow solid, with anisolated yield of 78%. TLC (CH Cl :NH 7N
2
2
3
1
in CH OH, 99:2 v/v): Rf = 0.3; H NMR (300 MHz, CD OD): δ 8.46 (dd, J = 5.3, 0.7 Hz, 1H), 7.64 (dd, J = 1.8, 0.8
3
3
13
Hz, 1H), 7.57 (dd, J = 5.3, 1.8 Hz, 1H), 2.84 (t, J = 7.4 Hz, 2H), 1.65-1.57 (m, 3H), 0.97 (d, J = 6.3 Hz, 6H); C NMR
(
75 MHz, CD OD): δ 200.1, 162.8, 148.4, 148.3, 120.2, 118.5, 38.9, 35.4, 27.7, 21.4; IR (neat): 3229.1, 2947.7,
3
-
1
2
923.6, 2866.4, 1659.4, 1600.6, 1427.8, 1290.4, 1126.7, 865.8, 828.6, 689.5, 671.9, 519.0 cm ; HRMS (m/z):
+
[M] calcd. for C H N S, 209.1112; found, 209.1115.
11 16 2
2
-(2,2,2-trifluoroethyl)pyridine-4-carbothioamide (3h):
Following general procedure H furnished 3h as a yellow solid, with an isolated yield of 68%. TLC (CH Cl :CH OH,
2
2
3
1
9
1
1
3
1
8:2 v/v): Rf = 0.19; H NMR (300 MHz, CD OD) δ 8.59 (d, J = 5.2 Hz, 1H), 7.84 (br s, 1H), 7.70 (dd, J = 5.3, 1.8 Hz,
3
19
13
H), 3.75 (q, J = 10.9 Hz, 2H); F NMR (282 MHz, CD OD) δ -66.72 (t, J = 12.2 Hz); C NMR (75 MHz, CD OD): δ
3
3
99.4, 151.2 (q, J = 3.5 Hz), 149.2, 148.6, 125.5 (d, J = 276.4 Hz), 122.5, 120.1, 41.3 (q, J = 29.4 Hz); IR (neat):
309.9, 3030.2, 2957.8, 2918.3, 2851.7, 2452.5, 2220.5, 2211.4, 1552.4, 1422.3, 1352.3, 1248.6, 1140.4,
-
1
+
065.9, 832.1, 724.5, 648.7, 515.8 cm ; HRMS (m/z): [M] calcd. for C H F N S, 221.0360; found, 221.0370.
8
7
3
2
2
-[1-(trifluoromethyl)cyclopropyl]pyridine-4-carbothioamide (3i):
Following general procedure H furnished 3i as a yellow solid, with an isolated yield of 63%. TLC (CH Cl :CH OH,
2
2
3
1
9
1
1
5:5 v/v): Rf = 0.60; H NMR (300 MHz, CD OD) δ 8.55 (d, J = 5.2 Hz, 1H), 7.97 (s, 1H), 7.64 (d, J = 5.0 Hz, 1H),
3
19
13
.45 (d, J = 5.0 Hz, 2H), 1.39 (d, J = 4.6 Hz, 2H); F NMR (282 MHz, CD OD) δ -69.9; C NMR (75 MHz, CD OD): δ
3
3
99.6, 155.2, 149.1, 148.3, 126.3 (d, J = 272.8 Hz), 122.0, 119.7, 29.0 (q, J = 33.3 Hz), 10.6 (q, J = 2.3 Hz); IR
(
neat): 3311.8, 3017.6, 2983.3, 2932.5, 2458.5, 1596.8, 1359.8, 1130.4, 1112.3, 1081.3, 1045.5, 845.0, 620.0,
-
1
+
5
19.7 cm ; HRMS (m/z): [M] calcd. for C H F N S, 247.0517; found, 247.0521.
10 9 3 2
2
-cyclopropylpyridine-4-carbothioamide (3j):
Following general procedure H furnished 3j as a yellow solid, with an isolated yield of 49%. TLC (CH Cl :NH 7N
2
2
3
1
in CH OH, 98:2 v/v): Rf = 0.19; H NMR (300 MHz, CD OD): δ 8.38 (dd, J = 5.3, 0.8 Hz, 1H), 7.55 (dd, J = 1.8, 0.8
3
3
13
Hz,1H), 7.48 (dd, J = 5.2, 1.8 Hz, 1H), 2.18-2.10 (m, 1H), 1.09-0.96 (m, 4H); C NMR (75 MHz, CD OD): δ 201.8,
3
1
1
64.9, 149.8, 149.2, 119.4, 119.3, 17.9, 10.5; IR (neat): 3257.3, 3000.5, 2920.3, 2851.4, 2788.7, 1652.8, 1596.8,
-
1
+
551.3, 1401.1, 1289.2, 892.4, 847.7, 810.8, 686.8, 654.2, 584.7, 522.4 cm ; HRMS (m/z): [M] calcd. for
C H N S, 179.0643; found, 179.0649.
9
10 2
2
-(cyclohexylmethyl)pyridine-4-carbothioamide (3k):
Following general procedure H furnished 3k as a yellow solid, with an isolated yield of 75%. TLC (CH Cl :NH 7N
2
2
3
1
in CH OH, 99:2 v/v): Rf = 0.3; H NMR (300 MHz, CD OD): δ 8.47 (dd, J = 5.3, 0.8 Hz, 1H), 7.61 (dd, J = 1.7, 0.8
3
3

ACCEPTED MANUSCRIPT
Hz, 1H), 7.58 (dd, J = 5.2, 1.8 Hz, 1H), 2.70 (d, J = 7.1 Hz, 2H), 1.81-1.63 (m, 6H), 1.32-1.17 (m, 3H), 1.10-0.97 (m,
1
3
2
3
H); C NMR (75 MHz, CD OD): δ 201.5, 162.7, 149.6, 149.5, 122.5, 119.8, 46.5, 40.1, 34.1, 27.5, 27.3; IR (neat):
3
-
1
259.0, 2921.6, 2847.5, 1657.4, 1599.2, 1425.9, 1120.3, 857.4 828.3, 682.6, 521.0, 461.9, 434.8 cm ; HRMS
+
(
m/z): [M] calcd. for C H N S, 235.1269; found, 235.1269.
13 18 2
ethyl 3-(4-carbamothioyl-2-pyridyl)propanoate (3l):
Following general procedure H furnished 3l as an orange solid, with an isolated yield of 40%. TLC (CH Cl :NH
3
2
2
1
7
N in CH OH, 99:1 v/v): Rf = 0.2; H NMR (300 MHz, CD OD): δ 8.48 (dd, J = 5.2, 0.8 Hz, 1H), 7.65 (dd, J = 1.7, 0.8
3 3
Hz, 1H), 7.57 (dd, J = 5.2, 1.7 Hz, 1H), 4.14-4.06 (q, J = 14.3, 7.2 Hz, 2H), 3.15-3.10 (t, J = 7.7 Hz, 2H), 2.8-2.75 (t, J
1
3
=
1
1
2
7.7 Hz, 2H), 1.22-1.18 (t, J = 7.1 Hz, 3H); C NMR (75 MHz, CD OD): δ 201.4, 174.3, 161.8, 150.0, 149.5, 121.7,
3
20.1, 61.6, 34.3, 33.6, 14.4; IR (neat): 3363.1, 3330.7, 3197.1, 2981.1, 2931.0, 1704.0, 1652.0, 1599.6, 1378.8,
-
1
+
309.5, 1269.6, 1195.5, 1013.8, 841.7, 700.4, 582.1, 523.0 cm ; HRMS (m/z): [M] calcd. for C H N O S,
11
14
2
2
39.0854; found, 239.0863.
2
-(1-phenylmethyl)pyridine-4-carbothioamide (3m):
Following general procedure H furnished 3m as a yellow solid, with an isolated yield of 80%. TLC (CH Cl :NH 7N
2
2
3
1
in CH OH, 99:2 v/v): Rf = 0.3; H NMR (300 MHz, CD OD): δ 8.50 (dd, J = 5.2, 0.8 Hz, 1H), 7.69 (m, 1H), 7.57 (dd,
3
3
1
3
C
J = 5.2, 1.8 Hz, 1H), 7.32-7.25 (m, 4H), 7.22-7.16 (m, 1H), 4.35 (q, J = 7.4 Hz, 1H), 1.70 (d, J = 7.3 Hz, 3H);
NMR (75 MHz, CD OD): δ 200.2, 165.3, 148.5, 148.4, 144.3, 128.2, 127.3, 126.2, 119.6, 118.7, 46.9, 19.7; IR
3
(
neat): 3362.5, 3059.2, 3020.6, 2966.5, 2929.2, 2852.9, 1645.8, 1596.0, 1556.0, 1420.8, 1394.4, 1303.1, 829.5,
-
1
+
7
59.7, 693.0, 581.2, 534.4 cm ; HRMS (m/z): [M] calcd. for C H N S, 243.0956; found, 243.0944.
14 14 2
2
-benzylpyridine-4-carbothioamide (3n):
Following general procedure H furnished 3n as a yellow solid, with an isolated yield of 48%. TLC (CH Cl :CH OH,
2
2
3
1
9
1
1
1
0:10 v/v): Rf = 0.68; H NMR (300 MHz, CD OD): δ 8.49 (d, J = 5.2 Hz, 1H), 7.68-7.61 (m, 1H), 7.58 (dd, J = 5.3,
3
13
.8 Hz, 1H), 7.34-7.16 (m, 5H), 4.17 (s, 2H); C NMR (75 MHz, CD OD): δ 200.0, 161.2, 148.7, 148.5, 138.8,
3
28.6, 128.3, 126.2, 120.8, 118.7, 43.4; IR (neat): 3331.1, 3056.6, 3027.2, 2919.9, 2850.2, 1657.2, 1596.0,
-
1
552.5, 1397.9, 851.6, 741.4, 694.1, 677.0, 608.7, 536.4, 504.4, 464.0 cm ; HRMS (m/z): [M]+ calcd. for
C H N S, 229.0799; found, 229.0806.
13 12 2
2
-[(4-methoxyphenyl)methyl]pyridine-4-carbothioamide (3o):
Following general procedure H furnished 3o as a yellow solid, with an isolated yield of 30%. TLC (CH Cl :CH OH,
2
2
3
1
9
7
0:10 v/v): Rf = 0.67; H NMR (300 MHz, CD OD): δ 8.48 (dd, J = 5.2, 0.8 Hz, 1H), 7.61 (dd, J = 1.8, 0.8 Hz, 1H),
3
1
3
.57 (dd, J = 5.2, 1.8 Hz, 1H), 7.19-7.16 (m, 2H), 6.87-6.84 (m, 2H), 4.11 (s, 2H), 3.76 (s, 3H); C NMR (75 MHz,
CD OD): δ 200.1, 161.6, 158.5, 148.7, 148.4, 130.8, 129.6, 121.2, 119.2, 113.7, 54.3, 42.6; IR (neat): 3388.7,
3
3
7
000.2, 2960.7, 2919.2, 2832.5, 1643.7, 1595.5, 1509.5, 1407.7, 1297.5, 1246.2, 1173.1, 1032.9, 834.2, 803.6,
-
1
60.5, 728.0, 709.2, 548.4, 509.4 cm ; HRMS (m/z): [M]+ calcd. for C H N OS, 259.0905; found, 259.0912.
1
4
14
2
2
-[(4-fluorophenyl)methyl]pyridine-4-carbothioamide (3p):
Following general procedure H furnished 3p as a yellow solid, with an isolated yield of 22%. TLC (CH Cl :CH OH,
2
2
3
1
9
7
2
0:10 v/v): Rf = 0.6; H NMR (300 MHz, CD OD): δ 8.50 (dd, J = 5.3, 0.8 Hz, 1H), 7.64 (dd, J = 1.8, 0.8 Hz, 1H),
3
13
.58 (dd, J = 5.3, 1.8 Hz, 1H), 7.30-7.26 (m, 2H), 7.05-6.99 (m, 2H), 4.16 (s, 2H); C NMR (75 MHz, CD OD): δ
3
00.0, 161.7 (d, J = 243.0 Hz), 161.0, 148.8, 148.7, 134.8 (d, J = 3.2 Hz), 130.2 (d, J = 8.0 Hz), 120.7, 118.8, 114.9
(
d, J = 21.5 Hz), 42.5; IR (neat): 3272.7, 3022.7, 2917.7, 2850.0, 1655.6, 1598.8, 1505.2, 1401.0, 1290.3, 1209.4,
-
1
1
153.1, 1093.8, 845.2, 811.4, 768.4, 712.3, 663.6, 621.6, 515.5, 491.1 cm ; HRMS (m/z): [M]+ calcd. for
C H FN S, 247.0705; found, 247.0715.
13
11
2
2
-[(3-fluorophenyl)methyl]pyridine-4-carbothioamide (3q):
1
Following general procedure H furnished 3q as a yellow solid, with an isolated yield of 78%. H NMR (300 MHz,
CD OD) δ 8.51 (dd, J = 5.3, 0.8 Hz, 1H), 7.67 (dd, J = 1.8, 0.8 Hz, 1H), 7.60 (dd, J = 5.3, 1.8 Hz, 1H), 7.30 (td, J =
3

ACCEPTED MANUSCRIPT
7
4
1
2
7
.9, 6.0 Hz, 1H), 7.09 (ddq, J = 7.6, 1.6, 0.7 Hz, 1H), 7.05-6.98 (m, 1H), 6.94 (dddd, J = 9.2, 8.4, 2.6, 1.0 Hz, 1H),
1
3
.18 (s, 2H); C NMR (75 MHz, CD OD): δ 199.9, 162.94 (d, J = 244.6 Hz), 160.4, 148.7, 141.59 (d, J = 7.6 Hz),
3
29.91 (d, J = 8.4 Hz), 124.48 (d, J = 3.1 Hz), 120.8, 118.9, 115.32 (d, J = 21.7 Hz), 112.89 (d, J = 21.1 Hz), 42.9 (s,
H); IR (neat): 3276.9, 2461.1, 2163.1, 1590.0, 1554.5, 1481.4, 1447.9, 1392.1, 1246.0, 1136.0, 949.2, 860.6,
64.3, 706.2, 691.7, 675.6, 641.2, 487.8, 437.1 cm-1; HRMS (m/z): [M]+ calcd. for C13H11FN2S, 247.0705;
found, 247.0712.
-[(3-cyanophenyl)methyl]pyridine-4-carbothioamide (3r):
Following general procedure H furnished 3r as a yellow solid, with an isolated yield of 23%. H NMR (300 MHz,
2
1
1
3
C
CD OD) δ 8.52 (d, J = 5.2 Hz, 1H), 7.69-7.66 (m, 2H), 7.62-7.58 (m, 3H), 7.48 (t, J = 7.7 Hz, 1H), 4.24 (s, 2H);
3
NMR (75 MHz, CD OD): δ 199.9, 159.9, 149.0, 148.9, 140.8, 133.6, 132.2, 130.1, 129.4, 120.8, 119.1, 118.3,
3
1
8
2
12.2, 42.7; IR (neat): 3387.4, 3009.1, 2922.6, 2853.5, 2770.3, 2228.3, 1665.7, 1599.8, 1431.2, 1416.1, 1116.0,
-
1
+
29.2, 801.5, 728.1, 707.2, 688.4, 583.2, 557.4, 525.9, 418.0 cm ; HRMS (m/z): [M] calcd. for C H N S,
14 11 3
54.0752; found, 254.0761.
Biological experiments
Microbiological assays
Thioisonicotinamides derivatives (3a-3r), isonicotinamide 2c and commercial ETH S-oxide 4a (Carbosynth) were
TM
added at 10 mg/ml in DMSO to an Echo 384LDV-DMSO source plate. INH (2 mg/ml) and DMSO (100%) were
included as controls. The assay plates were created using the Echo 550 liquid dispenser (Labcyte). Firstly, the
compounds were dispensed in various concentrations from 30 µM to 0.001 µM to perform the dose-response
curve. DMSO was used as backfilling solvent to a final volume of 150 nl. DMSO was included as negative control
at 0.3% DMSO and INH as positive control at 1 µg/ml. Secondly, the two boosters (BDM41906 and SMARt-420)
were added to assay plates at 1 µM. An H37Rv-GFP stock was added to 50 ml 7H9 complete medium (with
0
.05% tween + 10% OADC + 0.2% glycerol). This culture was grown for 7 days, at 37 °C and 5% CO . The culture
2
was diluted at an OD600 approximately 0.1 and was grown for a further week. On the second week the culture
was diluted to an OD600 of 0.1 and grown for 2 days, at 37 °C, with shaking. Then, the culture was washed twice
in 1x PBS. 1x10e5 bacteria were added to each well. The plates were then incubated at 37 °C and 5% CO for 5
2
days. The inhibitory effect of thioamides with or without boosters on the bacterial growth was measured by
TM
quantifying the GFP fluorescence signal from the bacteria using a Victor X3 light plate reader from
PerkinElmer, using exc=485nm/em=535nm.
Electrochemistry
Voltammetric measurements were carried out with a potentiostat Autolab PGSTAT100 (ECO Chemie, The
Netherlands) controlled by GPES 4.09 software. Experiments were performed at room temperature in a
homemade airtight three–electrode cell connected to a vacuum/argon line. The reference electrode consisted
of a saturated calomel electrode (SCE) separated from the solution by a bridge compartment. The counter
electrode was a platinum wire of ca 1 cm² apparent surface. The working electrode was GC microdisk (1.0 mm
of diameter – Bio-logic SAS). The supporting electrolyte (nBu N)[PF6] (Fluka, 99% puriss electrochemical grade)
4
and the solvent acetonitrile (for HPLC super gradient, VWR chemical) were used as received and simply
degassed under argon.
-
3
The solutions used during the electrochemical studies were typically 10 M in compound and 0.1 M in
supporting electrolyte. Before each measurement, the solutions were degassed by bubbling argon and the
working electrode was polished with a polishing machine (Presi P230).
Under these experimental conditions employed in this work, the half-wave potential (E ) of the Fc+/Fc couple
1/2
in ACN was E1/2 = 0.40 V vs SCE. Experimental peak potentials have been measured versus SCE and converted to
NHE by adding 0.241 V. Oxidation peak potential is an average of 10 measurements.

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Supplementary information:
NMR data for final compounds:
1
H NMR of compound 3a (300 MHz, CD OD)
3
1
3
C NMR of compound 3a (75 MHz, CD OD)
3

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1
H NMR of compound 3b (300 MHz, CD OD)
3
1
3
C NMR of compound 3b (75 MHz, CD OD)
3

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1
H NMR of compound 3c (300 MHz, CD OD)
3
1
3
C NMR of compound 3c (75 MHz, CD OD)
3

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1
H NMR of compound 3d (300 MHz, CD OD)
3
1
3
C NMR of compound 3d (75 MHz, CD OD)
3

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1
H NMR of compound 3e (300 MHz, CD OD)
3
1
3
C NMR of compound 3e (75 MHz, CD OD)
3

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1
H NMR of compound 3f (300 MHz, CD OD)
3
1
3
C NMR of compound 3f (75 MHz, CD OD)
3