New direct inhibitors of InhA with antimycobacterial activity based on a tetrahydropyran scaffold

Article abstract of DOI:10.1016/j.ejmech.2016.02.008

Tetrahydropyran derivative 1 was discovered in a high-throughput screening campaign to find new inhibitors of mycobacterial InhA. Following initial in-vitro profiling, a structure-activity relationship study was initiated and a focused library of analogs was synthesized and evaluated. This yielded compound 42 with improved antimycobacterial activity and low cytotoxicity. Additionally, the crystal structure of InhA in complex with inhibitor 1 was resolved, to reveal the binding mode and provide hints for further optimization.

Full text of DOI:10.1016/j.ejmech.2016.02.008

Accepted Manuscript
New direct inhibitors of InhA with antimycobacterial activity based on a
tetrahydropyran scaffold
Stane Pajk, Matej Živec, Roman Šink, Izidor Sosič, Margarete Neu, Chun-wa Chung,
María Martínez-Hoyos, Esther Pérez-Herrán, Daniel Álvarez-Gómez, Emilio Álvarez-
Ruíz, Alfonso Mendoza-Losana, Julia Castro-Pichel, David Barros, Lluís Ballell-
Pages, Robert J. Young, Maire A. Convery, Lourdes Encinas, Stanislav Gobec
PII:
S0223-5234(16)30078-2
10.1016/j.ejmech.2016.02.008
EJMECH 8359
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 December 2015
Revised Date: 3 February 2016
Accepted Date: 4 February 2016
Please cite this article as: S. Pajk, M. Živec, R. Šink, I. Sosič, M. Neu, C.-w. Chung, M. Martínez-Hoyos,
E. Pérez-Herrán, D. Álvarez-Gómez, E. Álvarez-Ruíz, A. Mendoza-Losana, J. Castro-Pichel, D. Barros,
L. Ballell-Pages, R.J. Young, M.A. Convery, L. Encinas, S. Gobec, New direct inhibitors of InhA with
antimycobacterial activity based on a tetrahydropyran scaffold, European Journal of Medicinal Chemistry
(2016), doi: 10.1016/j.ejmech.2016.02.008.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
New direct inhibitors of InhA with antimycobacterial activity based on a
tetrahydropyran scaffold
Stane Pajk,† Matej Živec,† Roman Šink,† Izidor Sosič,† Margarete Neu,¶ Chun-wa Chung,¶ María
Martínez-Hoyos,‡ Esther Pérez-Herrán,‡ Daniel Álvarez-Gómez,‡ Emilio Álvarez-Ruíz,‡ Alfonso
Mendoza-Losana,‡ Julia Castro-Pichel,‡ David Barros,‡ Lluís Ballell-Pages,‡ Robert J. Young,¶
Maire A. Convery,¶ Lourdes Encinas,‡* Stanislav Gobec†*
†University of Ljubljana, Faculty of Pharmacy, Ljubljana, Slovenia
‡DPU TB Diseases of the Developing World, GlaxoSmithKline, Tres Cantos, Madrid, Spain
¶Medicines Research Centre, GlaxoSmithKline, Stevenage, Hertfordshire, UK
Corresponding Author
*For S. G.: Tel, +386 1 47 69 585; E-mail, stanislav.gobec@ffa-uni-lj.si
*For L. E.: Tel; +346 3 85 10 876; E-mail, lourdes.p.encinas-prieto@gsk.com
Abstract
Tetrahydropyran derivative 1 was discovered in a high-throughput screening campaign to find new
inhibitors of mycobacterial InhA. Following initial in-vitro profiling, a structure-activity relationship
study was initiated and a focused library of analogs was synthesized and evaluated. This yielded
compound 42 with improved antimycobacterial activity and low cytotoxicity. Additionally, the crystal

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structure of InhA in complex with inhibitor 1 was resolved, to reveal the binding mode and provide
hints for further optimization.
1. Introduction
Tuberculosis (TB) remains a major global health problem, with an estimated 9 million new cases and
1.5 million deaths per year [1]. Although progress has been made to reduce the global incidence of
TB, the emergence and spread of drug resistance threatens to undermine these efforts. This includes
both multi-drug resistant TB, which is resistant to isoniazid and rifampicin, and extensively drug-
resistant TB, which is resistant to isoniazid, rifampicin, any fluoroquinolone, and an injectable drug
(i.e., one of capraomycin, kanamycin, amikacin) [2−4].
The mycobacterial fatty-acid biosynthesis pathway II (FAS-II) represents a validated target for drug
discovery, as fatty acids are essential for bacterial growth and can only be synthesized de novo. In
addition, the bacterial FAS-II system is fundamentally distinct from that of mammals, thus providing
the possibility to selectively target bacteria [5]. One of the enzymes involved in the FAS-II pathway in
Mycobacterium tuberculosis (Mtb) is InhA, a NADH-dependent, enoyl-acyl carrier protein reductase.
This is the target of isoniazid, a first-line drug for treatment of TB. Isoniazid is a pro-drug that is
enzymatically activated by KatG, a mixed function catalase/ peroxidase; this transforms isoniazid into
an unstable species that reacts with the NAD cofactor to form a covalent adduct, which subsequently
inhibits InhA activity. Resistance to isoniazid is mainly the result of mutations in KatG that reduce its
activation of isoniazid, and to a lesser extent, to mutations in the InhA active site. Therefore,
compounds that directly target InhA and do not require activation by the mycobacterial catalase/
peroxidase KatG are promising candidates for treatment of infections caused by isonoazid-resistant
strains [6−7]. As a follow-up of previous reports, we present here the synthesis, physicochemical
characterization, biological profiling, and initial structure-activity relationships (SARs) of new
tetrahydropyran derivatives of hit compound 1 (Figure 1) that was identified by GlaxoSmithKline in a
high-throughput screening (HTS) campaign against InhA.

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Figure 1. High-throughput screening hit compound 1, with aromatic rings marked.
2. Results and discussion
2.1 Hit validation
Hit compound 1 was identified by high-throughput screening against InhA with the GlaxoSmithKline
compound collection. First, compound 1 was re-synthesized (see below), to complete the initial round
of in-vitro profiling, where it had good InhA inhibitory potency (IC50 = 0.020 ꢀM), moderate in-vitro
antimycobacterial activity (MIC = 11.7 ꢀM), and killed Mtb inside macrophages (Table 1). In terms of
early safety profiling, compound 1 showed modest hERG inhibition, with an IC50 of 50.1 ꢀM, and low
cytotoxicity against the HepG2 human cell line (TOX50 = 100 ꢀM).
Table 1. Profile of compound 1: in-vitro InhA inhibition, antitubercular activity, and physicochemical
and preliminary safety properties.
MW
486.6 InhA IC50 (ꢀM)
4.18 Mtb MIC (ꢀM)
0.020
11.7
3.98
ClogP
Solubility CLND (ꢀM) 115a Intracell IC90 (ꢀM)
hERG IC50 (ꢀM)
44.9 Cell tox HepG2 TOX50 (ꢀM) 100
a
CLND, solubility values that are within 85% of maximum possible concentration (as determined
from DMSO stock concentration).
We confirmed the relationship between InhA inhibition and antimycobacterial MIC through the use of
an InhAMTB overexpressor strain, which showed a 4-fold increase in MIC compared to the wild type
(Table S1). Compound 1 was further evaluated against a panel of Gram-positive and Gram-negative

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organisms, and it showed selective activity against Mtb H37Rv (Table S2). Additionally, it was tested
against seven isoniazid-resistant clinical isolates with characterized mutations either in katG (high-
level resistance to isoniazid) or in nucleotide position 15 upstream of the transcription start site for
inhA (InhA over-expression) [8,9]. Mutations in codon 315 of katG and the inhA promoter are proven
mechanisms of isoniazid resistance. Compound 1 showed a small upward shift in MIC against
isoniazid resistance in general, but a more significant shift was observed in the group of strains that
overexpressed InhA. An increased MIC of isoniazid was observed in paired clinical isolates with
mutations in codon 315 of katG (Table S3). Analysis of the drug susceptibility of these isolates
confirmed that strains carrying the katG mutation showed a high level of resistance to isoniazid (MIC
>25 µg/ml) while susceptibility to compound 1 was similar to wild-type (MIC = 15.6-62.5 µM). A
moderate shift in MIC was observed against the group of strains overexpressing InhA, for both
compounds.
2.2 Synthesis and structure-activity relationships
A chemistry-driven approach was used to study the initial SARs of compound 1. The goal of this
approach was to explore the chemical space through the introduction of structural changes to rings A,
C, and D, while leaving ring B and the N-((tetrahydro-2H-pyran-4-yl)methyl)amide core in place
(Figure 1).
Compound 1 and related analogs 29-46 were synthesized according to the convergent route presented
in Scheme 1. To explore the chemical space around ring A, we used either commercially available 4-
substituted benzoic acids or derivatives 8-13, which we synthesized (Scheme 1, IV).
The possibility of replacing the methylene linker between rings A and B with an amino group (Figure
1) was assessed. In the first step, palladium-catalyzed N-arylation between 4-bromobenzonitrile or 4-
bromobenzoate and commercially available heteroaryl amines yielded the appropriate 4-substituted
benzoic acid precursors 2-7 (Scheme 1, I) [10−12]. These were then hydrolyzed to the corresponding
carboxylic acids 8-13; acid-catalyzed hydrolysis [13] was used in the case of nitriles 2-5, and base-
catalyzed hydrolysis in the case of esters 6 and 7.

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Scheme 1. Synthesis of compounds 2-46a
aReagents and conditions: (i) Pd2dba3, BrettPhos, K2CO3, AcOH, molecular sieves 3 Å, tert-butanol,
115 °C, 3 h; (ii) HCl(conc), 105 °C, 3 h (for Q = CN) or KOH, 50 °C, 15 h (for Q = COOEt); (iii) THF,
Et3N, rt, 2 h; (iv) (ClCH2CH2)2O, DMF, NaH, 0 °C to rt, 4 h; (v) THF, LiAlH4, 0 °C to rt, 4 h; (vi)
TBTU, Et3N, CH2Cl2, 0 °C to rt, 1 h, or HATU, Et3N, DMF, 0 °C to rt, 1 h. For R1 and R2, see Table 2.
With the aim of exploring the substituent tolerance of ring D (Scheme 1, II), we prepared different
amines as precursors 20-22. Initially, 2-bromoacetophenone or its derivatives were reacted with 2-
cyanothioacetate to form the thiazoles 14-16 [14]. In the next step, the tetrahydropyran ring was
introduced (compounds 17-19), followed by the reduction of the nitrile moiety with LiAlH4, to give
amines 20-22 [15].
For the final set of modifications, ring D was omitted, and the thiazole of ring C was replaced by
differently substituted phenyls or pyridin-2-yl. The synthetic strategy was the same as for the synthesis
of amines 20-22, except without the first step (Scheme 1, III). The tetrahydropyran ring was
constructed by double alkylation of commercial 2-aryl acetontiriles with dichlorodiethylether, to yield
compounds 23-25. This was followed by reduction of the nitrile moiety to give the desired amines 26-
28. Commercially available or synthesized amines and benzoic acid derivatives were finally coupled

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to give the target compounds 29-46 (Scheme 1, IV; Table 2). The prepared compounds were then
tested for InhA inhibition, antimycobacterial activity against Mtb H37Rv, and cytotoxicity in the
HepG2 cell line (Table 2).
Table 2. Inhibitory potencies and antimycobacterial potencies of compounds 1 and 29-46 against M.
tuberculosis InhA, and their clogP data, solubility, and cytotoxicity.
InhA
IC50
Cell
tox
CLNDa
Cpd
R1
R2
MIC (µM) clogP
HepG2
(µM)
(µM)
TOX50 (µM)
0.02
11.7
4.18
115
100 (SI=8.5)
1
> 1
> 1
> 125
> 125
4.14
2.76
≥396
79.4
29
30
138
>100
>100
80
4.56
33
87.1
31
> 1
> 125
125
3.26
2.42
3.04
3.08
193
191
71
> 100
> 100
79.4
32
33
34
35
> 10
> 1
125
5.56
> 125
48
79.4
> 10
> 80
4.4
<1
> 100
36

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> 10
> 10
> 10
> 125
5.5
4
12.6
63.1
63.1
37
38
39
> 80
4.04
3.2
12
29
> 125
> 10
94
20
4.95
4.2
10
72
20
40
41
0.323
50.1
50.1
0.036
5
3.64
78
42
(SI=10.0)
6.38
7.94
> 10
0.029
> 125
125
3.2
≥458
≥478
≥445
77
> 100
43
44
45
3.25
1.78
5.17
> 100
> 125
16
> 100
79.4 (SI=5.0)
46
a
CLND, solubility values that are within 85% of maximum possible concentration (as determined
from DMSO stock concentration).
Correlation was found between InhA inhibition in the biochemical assay and antimycobacterial
activity for compounds with IC50 values lower than 1 ꢀM. Above this threshold, the poor aqueous
solubility of some of the compounds was most likely the factor responsible for the lower levels of
correlation. We defined the ratio between the TOX50 in HepG2 cells and the Mtb H37Rv MIC as the
selectivity index (SI). Among the compounds active against Mtb H37Rv, only compound 42 showed a
SI >10 and could be considered noncytotoxic (Table 2). From the data in Table 2, it appears that

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achieving nanomolar InhA inhibitory potency not only results in good antimycobacterial potency, but
also leads to less off-target binding, and consequently to lower cytotoxicity.
Compounds 29-34 were synthesized to determine the importance of the 3,5-dimethyl pyrazole moiety
(ring A) and the consequent decrease in lipophilicity. Compound 1 had a relatively high clogP of 4.18
[16]. The introduction of aliphatic heterocycles in place of ring A (compounds 29, 30) resulted in loss
of InhA inhibitory potency along with the antimycobacterial activity, regardless of the presence of the
methylene linker between rings A and B. Furthermore, the absence of the methylene linker between
rings A and B (compounds 31-34) resulted in complete loss of antimycobacterial activity regardless of
the aromatic heterocycle in the place of ring A. We hypothesized that replacement of the methylene
linker between rings A and B with the amino group might contribute towards the formation of
additional hydrogen bonds in the InhA active site, while at the same time simplifying the synthetic
introduction of different aromatic heterocycles. However, this substitution yielded compounds 35-40
with IC50 values >10 µM.
Finally, the impact of substitutions in the phenyl ring (ring D) and the importance of the thiazole group
(ring C) were explored (compounds 41-46). Introduction of the methoxy group to the meta-position of
ring D was allowed, as compound 41 showed moderate inhibition of InhA, with an IC50 of 0.323 ꢀM.
The introduction of the methoxy to the ortho-position of ring D resulted in InhA inhibitory potency in
the nanomolar range (compound 42, IC50 = 40 nM) and antimycobacterial potency comparable to the
initial hit compound 1. These data suggest that substitution of the phenyl ring is a suitable space for
further optimization in the future. Reductions in the number of heterocycles, omission of ring D, and
introduction of differently substituted phenyl moieties (compounds 43, 44) or pyridin-2-yl (compound
45) in place of ring C resulted in substantial loss of the enzymatic and whole cell inhibition potencies.
The replacement of the thiazole ring (ring C) of compound 1 with a phenyl ring (compound 46) led to
a compound with good InhA inhibition and antimycobacterial potency, despite poor aqueous
solubility.
2.3 Crystal structure

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To understand the nature of the specific interactions that underpin InhA inhibition by these
tetrahydropyran derivatives, we solved the X-ray crystal structure of compound 1 bound to InhA in the
presence of the cofactor, NAD (see Figure 2). The crystal structure shows that compound 1 adopts a
U-shape and binds to InhA in the cleft that is normally occupied by substrate (Figure S2a). The overall
binding position of compound 1 is similar to that observed for the pyrrolidine compound in the crystal
structure (pdb entry 4COD, Figure S2b) [17]. The pyrazole on the left-hand side of compound 1 stacks
against the pyridine portion of the NAD, and the pyrazole 2-nitrogen provides a H-bond interaction
with the 2’-hydroxyl group of NAD. The InhA substrate-binding loop, which includes helix α6, is
ordered in the structure, and the N-terminal end forms part of the pyrazole binding pocket. The
methylene linker on the left-hand side of the molecule introduces a kink into the ligand that is
necessary to maintain its shape complementarity with the binding site. The central amide of compound
1 has water-bridged interactions with the protein, including the backbone carbonyl of Met98. The
tetrahydropyran points out of the binding site. The right-hand side of the molecule is in a deep pocket
in the protein that is bound on one side by the C-terminal end of ordered helix α6; it does not have any
specific interactions with the protein.

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Figure 2. Compound 1 bound to the active site of InhA. Compound 1 is shown in green ball-and-
sticks, NAD in thick gray lines, and hydrogen bonds in dashed lines. Helix α6 is ordered in the
structure, with Met98 and Tyr158 highlighted. Tyr158 adopts an apo orientation.
3. Conclusions
The tetrahydropyran compound 1 was identified in a high-throughput screen of the GlaxoSmithKline
collection, and it showed good InhA inhibitory potency (IC50 = 0.020 ꢀM), moderate in-vitro
antimycobacterial activity (MIC = 11.7 ꢀM), modest hERG inhibition, and low cytotoxicity against
the HepG2 human cell line. Following initial in-vitro profiling, a SAR study was initiated and a series
of 18 analogs was synthesized and evaluated. Based on the SAR data generated, it appears that rings C
and D can be modified in further optimization efforts. The best compound 42 in this series
demonstrated InhA inhibitory potency in the nanomolar range (IC50 = 40 nM), antimycobacterial
potency comparable to compound 1 (MIC = 5 ꢀM), and a reasonable SI. Additionally, the crystal
structure of compound 1 bound into InhA provided information on the binding mode, rationalised the
SARs, and provided insight into the opportunities for further structure-based optimisation of InhA
inhibitors.
4. Experimental section
4.1 Materials and methods. Chemicals from Sigma-Aldrich, TCI, and Acros were used without
further purification. All reactions were performed under argon atmosphere, unless otherwise stated.
Analytical TLC was performed on Merck silica gel (60 F254) plates (0.25 mm) and visualized with
ultraviolet light. Melting points were determined on a Reichert hot-stage microscope and are
1
uncorrected. H and 13C NMR spectra were recorded on a Bruker AVANCE III 400 MHz NMR
spectrometer in CDCl3 and DMSO-d6 solution, with TMS or residual non-deuterated solvent as the
internal standards. Mass spectra were recorded using a VG-Analytical Q-TOF Premier mass
spectrometer.
4.2 General procedures

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4.2.1 General procedure for the palladium-catalyzed N-arylation (compounds 2-7). An oven-
dried pressure tube equipped with a magnetic stirring bar was charged with Pd2dba3 (4.7 mg, 0.51 mol
%), BrettPhos (11.3 mg, 2.1 mol %), K2CO3 (192 mg, 1.4. mmol, 1.4 equiv.), aryl amine (1 mmol, 1
equiv.), aryl bromide (1 mmol, 1 equiv.), and activated molecular sieves of 4 Å (100 mg). The vessel
was flushed well with argon. Dry tert-butanol (4 mL) and acetic acid (1.7 ꢀL, 3 mol %) were added,
and the pressure tube was sealed with a Teflon screw cap and placed into an oil bath at 115 °C for 3 h.
The reaction mixture was then cooled to room temperature and filtered. The solvent was removed
under reduced pressure, and the crude product was purified by flash chromatography. In the case
where the aryl bromide (ethyl 4-bromobenzoate) was a liquid, it was added after tert-butanol.
4.2.2 General procedure for the hydrolysis of nitriles to carboxylic acids (compounds 8-11). The
pressure tube equipped with a magnetic stirring bar was charged with nitrile and concentrated aqueous
HCl (up to two thirds of the volume). The pressure tube was sealed with a Teflon screw cap and
placed into an oil bath at 105 °C for 15 h. The reaction mixture was cooled to room temperature and
the solvent removed under reduced pressure, to yield the crude product.
4.2.3 General procedure for the hydrolysis of esters to carboxylic acids (compounds 12, 13). The
ester (0.3 mmol) was dissolved in a mixture of dioxane (4 mL) and water (3 mL). Powdered KOH
(168 mg, 3.0 mmol, 10 equiv.) was added to this solution, and the reaction mixture was stirred at 50
°C overnight.
4.2.4 General procedure for the Hantzsch thiazole synthesis (compounds 14-16). The
corresponding 2-bromo-1-(aryl)ethan-1-one derivative (0.02 mol, 1 equiv.) and 2-cyanothioacetamide
(0.02 mol, 1 equiv.) were dissolved in dry THF (50 mL) followed by the addition of Et3N (0.022 mol,
1.1 equiv.) (precipitation of a white solid). The reaction mixture was left to react at room temperature
overnight. The solid was filtered off and the filtrate was concentrated on a rotary evaporator. The
residue was dissolved in EtOAc (200 mL) and washed with water (2× 50 mL), saturated aqueous
solution of NaHCO3 (2× 50 mL), and brine (50 mL), and dried over Na2SO4. The solvent was
evaporated under reduced pressure.

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4.2.5 General procedure for the formation of the tetrahydropyran ring (compounds 17-19, 23-
25). The corresponding arylacetonitrile derivative (15.6 mmol, 1 equiv.) and bis(chloroethyl) ether
(15.6 mmol, 1 equiv.) were dissolved in dry DMF (50 mL), and cooled in an ice-bath. Argon was
passed through the solution, followed by addition of 60% suspension of NaH in mineral oil (31.2
mmol, 2 equiv.), in small portions over 20 min. Then, the ice-bath was removed and the reaction
mixture was heated at 55 °C for 5 h, with the reaction then quenched with water. The reaction mixture
was diluted with EtOAc (300 mL), washed with 1 M aqueous HCl (3× 70 mL) and brine (2× 50 mL),
and dried over Na2SO4. The solvent was evaporated under reduced pressure.
4.2.6 General procedure for the reduction of nitriles to amines (compounds 20-22 and 26-28). A
solution of a corresponding nitrile derivative (1.00 mmol, 1 equiv.) in freshly distilled THF (5 mL)
was cooled in an ice-bath, and LiAlH4 (1.25 mmol, 1.25 equiv.) was added. The reaction mixture was
allowed to react overnight at room temperature, cooled in an ice-bath, and quenched with saturated
aqueous solution of NaHCO3 (20 mL). The aqueous phase was extracted with EtOAc (3× 30 mL), the
combined organic fractions were dried over Na2SO4, and the solvent was evaporated under reduced
pressure.
4.2.7 General procedure for the coupling of amines and carboxylic acids A (compounds 1, 29-34,
41-46). To a solution of benzoic acid derivative (0.50 mmol, 1 equiv.) in CH2Cl2 (7.5 mL) cooled in an
ice-bath, Et3N (1.50 mmol, 3 equiv.) and TBTU (0.65 mmol, 1.3 equiv.) were added. After 10 min, the
corresponding amine (0.50 mmol, 1 equiv.) was added, and the reaction mixture was left to react for 1
h at room temperature. The reaction mixture was diluted with CH2Cl2 (30 mL) and the organic phase
was washed with 1 M aqueous HCl (3× 15 mL), saturated aqueous solution of NaHCO3 (3× 15 mL),
and brine (30 mL), and dried over Na2SO4. The solvent was evaporated under reduced pressure.
4.2.7.1 4-((3,5-dimethyl-1H-pyrazol-1-yl)methyl)-N-((4-(4-phenylthiazol-2-yl)tetrahydro-2H-
pyran-4-yl)methyl)benzamide (1). Compound 1 was prepared as described under the general
procedure for the coupling of amines and carboxylic acids (coupling A). The crude product was
purified by flash chromatography on silica gel (DCM:MeOH = 1:0 to 10:1), to give the desired

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product (71%) as a white solid. 1H NMR (400 MHz, CDCl3): δ (ppm) 1.90-2.05 (m, 2H), 2.13 (s, 3H),
2.20-2.40 (m, 2H), 2.28 (s, 3H), 3.65-3,80 (m, 2H), 3.82 (d, J = 0.3 Hz, 2H), 3.85-4,00 (m, 2H), 5.27
(s, 2H), 5.89 (s, 1H), 7.00-7.15 (m, 2H), 7.30-7.45 (m, 4H), 7.50 (s, 1H), 7.65-7.80 (m, 2H), 7.85-7.95
(m, 2H). MS (ESI) m/z [M+H]+ = 487.0.
4.2.7.2 4-((3,5-Dimethyl-1H-pyrazol-1-yl)methyl)-N-((4-(4-(2-methoxyphenyl)thiazol-2-
yl)tetrahydro-2H-pyran-4-yl)methyl)benzamide (42). Compound 42 was prepared as described
under the general coupling A procedure. The product was purified by flash chromatography
1
(CH2Cl2:MeOH, 30:1), to give the desired product (49%) as yellow crystals. H NMR (400 MHz,
DMSO-d6): δ (ppm) 1.80-2.00 (m, 2H), 2.10 (s, 3H), 2.13 (s, 3H). 2.15-2.3 (m, 2H), 3.30-3.35 (m,
2H), 3.51 (d, 2H, J = 5.6 Hz), 3.70-3.90 (m, 2H), 3.91 (s, 3H), 5.22 (s, 2H), 5.86 (s, 1H), 6.92-7.00 (m,
1H), 7.02-7.14 (m, 2H), 7.28-7.38 (m, 1H), 7.68-7.74 (m, 2H), 8.04 (s, 1H), 8.10-8.18 (m, 1H), 8.40
(t, 1H, J = 5.6 Hz). 13C NMR (100 MHz, DMSO-d6): δ (ppm) 10.63, 13.36, 14.11, 20.78, 34.18, 38.26,
43.94, 49.34, 51.19, 54.93, 55.47, 59.77, 63.58, 105.09, 111.62, 117.76, 120.40, 122.63, 126.56,
127.61, 128.90, 129.48, 133.64, 138.84, 141.10, 146.22, 149.57, 156.39, 166.53, 172.16. HRMS
(ESI), m/z calcd for C29H33N4O3S 517.2273 (M+H)+, found 517.2266.
4.2.8 General procedure for the coupling of amines and carboxylic acids B (compounds 35-40).
The corresponding carboxylic acid (0.20 mmol, 1 equiv.), Et3N (82 ꢀL, 0.59 mmol, 3 equiv.) and
compound 6 (54 mL, 0.20 mmol, 1 equiv.) were dissolved in DMF (4 mL) and cooled in an ice bath.
HATU (106.5 mg, 0.28 mmol, 1.4 equiv.) was added to the reaction mixture, the temperature of the
reaction mixture was then allowed to reach room temperature, and the stirring was continued for 4 h.
The solvent was evaporated under reduced pressure and the residue was dissolved in EtOAc (30 mL).
The organic phase was washed with 0.1 M aqueous HCl (10 mL), saturated aqueous solution of
NaHCO3 (10 mL), and saturated aqueous solution of NH4Cl (2× 10 mL), and dried over Na2SO4. The
solvent was evaporated under reduced pressure.
Appendix A. Supplementary data

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Additional Tables, Schemes, Figures, protocols and experimental procedures of some of the assays
mentioned in the main text are available at xxx.
Funding Sources
The research leading to these data has received funding from the Global Alliance for TB Drug
Development, and from the European Union 7th Framework Program (FP7- 2007-2013) under Orchid
Grant agreement No. 261378. The funders had no role in the study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
Acknowledgment
We thank Dr. Chris Berrie for critical reading of the manuscript. We are grateful to Rubén González
del Río, Delia Blanco Ruano, Pedro A. Torres Gómez, Lydia Mata, Francisco de Dios, and Vanessa
Barroso for the biological evaluation, and Bill McDowell for preparation of the protein.
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Highlights
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New tetrahydropyran inhibitor (1) of mycobacterial InhA was discovered.
It exhibits good InhA inhibitory potency and moderate antimycobacterial activity.
SAR study was initiated and a focused library of analogs of hit 1 was synthesized.
Crystal structure of inhibitor 1 bound to InhA revealed the binding mode.