Diarylthiazole: An antimycobacterial scaffold potentially targeting PrrB-PrrA two-component system

Article abstract of DOI:10.1021/jm500833f

Diarylthiazole (DAT), a hit from diversity screening, was found to have potent antimycobacterial activity against Mycobacterium tuberculosis (Mtb). In a systematic medicinal chemistry exploration, we demonstrated chemical opportunities to optimize the potency and physicochemical properties. The effort led to more than 10 compounds with submicromolar MICs and desirable physicochemical properties. The potent antimycobacterial activity, in conjunction with low molecular weight, made the series an attractive lead (antibacterial ligand efficiency (ALE) >0.4). The series exhibited excellent bactericidal activity and was active against drug-sensitive and resistant Mtb. Mutational analysis showed that mutations in prrB impart resistance to DAT compounds but not to reference drugs tested. The sensor kinase PrrB belongs to the PrrBA two component system and is potentially the target for DAT. PrrBA is a conserved, essential regulatory mechanism in Mtb and has been shown to have a role in virulence and metabolic adaptation to stress. Hence, DATs provide an opportunity to understand a completely new target system for antimycobacterial drug discovery.

Full text of DOI:10.1021/jm500833f

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Article
Diarylthiazole: an anti-mycobacterial scaffold
potentially targeting PrrB-PrrA two component system
Eknath Bellale, Maruti Naik, Varun VB, Anisha Ambady, Ashwini Narayan, Sudha Ravishankar,
Vasanthi Ramachandrran, Parvinder Kaur, Robert McLaughlin, James Whiteaker, Sapna Morayya,
Supreeth guptha, Sreevalli Sharma, Anand Kumar Raichurkar, Disha Awasthy, Vijayashree Achar,
Prakash Vachaspati, Balachandra Bandodkar, Manoranjan Panda, and MONALISA CHATTERJI
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 26 Jun 2014
Downloaded from http://pubs.acs.org on June 27, 2014
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Diarylthiazole: an antiꢀmycobacterial scaffold
potentially targeting PrrBꢀPrrA two component
system
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Eknath Bellale1, Maruti Naik1, Varun VB1, Anisha Ambady2, Ashwini Narayan2, Sudha
Ravishankar2, Vasanthi Ramachandran2, Parvinder Kaur2, Robert McLaughlin3, James
Whiteaker3, Sapna Morayya2, Supreeth Guptha2, Sreevalli Sharma2, Anand Kumar
Raichurkar1, Disha Awasthy2, Vijayshree Achar1, Prakash Vachaspati4, Balachandra
Bandodkar1, Manoranjan Panda1,*, Monalisa Chatterji2,*
1Department of Medicinal Chemistry, IMED Infection, AstraZeneca India, Bellary Road,
Hebbal, Bangaloreꢀ560024, India
2Department of Biosciences, IMED Infection, AstraZeneca, India
3Infection IMED, AstraZeneca, GHP, Waltham, MA, USA
4DMPK and Animal Sciences, IMED Infection, AstraZeneca, India
Abstract
Diarylthiazole (DAT), a hit from diversity screening was found to have potent
antimycobacterial activity against Mycobacterium tuberculosis (Mtb). In a systematic
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medicinal chemistry exploration, we demonstrated chemical opportunities to optimize the
potency and physicoꢀchemical properties. The effort led to more than 10 compounds with
subꢀmicromolar MICs and desirable physicoꢀchemical properties. The potent antiꢀ
mycobacterial activity in conjunction with low molecular weight, made the series an
attractive lead (antibacterial ligand efficiency >0.4). The series exhibited excellent
bactericidal activity and was active against drugꢀsensitive and resistant Mtb. Mutational
analysis showed that mutations in prrB impart resistance to DAT compounds, but not to
reference drugs tested. The sensor kinase PrrB belongs to the PrrBA two component system
and is potentially the target for DAT. PrrBA is a conserved, essential regulatory mechanism
in Mtb and has been shown to have a role in virulence and metabolic adaptation to stress.
Hence, DATs provide an opportunity to understand a completely new target system for
antimycobacterial drug discovery.
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Introduction
Tuberculosis (TB) remains a major global health challenge1 and has been declared a public
health emergency by WHO. Among various aspects of this disease, emergence and spread of
drug resistant TB poses a significant threat to TB care and control worldwide. Reports of TB
patients with severe drug resistance patterns, worse than multiꢀ or, extremeꢀ drug resistant TB
(MDRꢀ, XDRꢀ) are increasing and is a cause of real concern (WHO meeting report).2 It has
created an urgent need to develop new drugs which can rapidly cure drugꢀresistant TB and
prevent reccurrence of the disease. Current TB therapy is long and associated with various
toxicities, hence, contributes to nonꢀcompliance and enhanced probability of emergence of
resistance. A simplified, shortened combination therapy with significantly improved safety
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profile will greatly enhance patient’s compliance, improve therapy outcome and delay
emergence of resistance.
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A major focus of TB drug discovery is aimed at identifying antibiotics that act by novel
mechanisms and are active against both drugꢀsensitive and resistant Mycobacterium
tuberculosis (Mtb). Recently, approved new chemical entities, such as Sirturo and Delaminid
for treatment of MDRꢀTB patients, have given hope for discovery of drugs with completely
new modeꢀofꢀaction.3 Phenotypic wholeꢀcellꢀbased screens to identify antiꢀmycobacterial
compounds followed by target identification is an attractive approach for lead generation
against Mtb.4 In continuation with our efforts to search for new drug candidates for TB
including AZD58475, we pursued phenotypic screening of our inꢀhouse library using Mtb
H37Rv. The screen identified multiple hits with potential to be developed into leads. In this
report, we present optimization of one of the scaffolds; a diarylthiazole (DAT), from an initial
hit to a lead chemical series. DAT compounds are active against drug sensitive and
isoniazid/rifampicin resistant Mtb strains. Experiments to understand the mode of action of
DATs led to the identification of PrrB, a histidine kinase belonging to PrrBA two component
system (TCS), as a potential target.
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Bacterial virulence factors and regulatory proteins are less explored target classes for drug
discovery.6,7 Bacterial twoꢀcomponent systems (TCSs) are ubiquitous regulatory mechanisms
involved in transcriptional reprogramming in response to environmental changes, regulation
of virulence determinants and antibiotic resistance.8,9 Mtb has 11 paired TCSs; four are
conserved across all mycobacterial species.10 Investigations are required to understand the
role of PrrBA in the physiology of Mtb, relationship of DAT to PrrBA and its translation to in
vivo potency.
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Results and Discussion
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As part of a highꢀthroughput screening program against the strain Mtb H37Rv, we used a
representative set (~100,000) of the AstraZeneca corporate library. The screening cascade
included activity against Mtb (minimum inhibitory concentration, MIC), bactericidality
(minimum bactericidal concentration), selectivity index for cytotoxicity (SIcytotox; ratio of Mtb
MIC and IC50 against human A549 cell line) and confirmation of activity by resynthesis. The
criteria for bactericidality and low human cytotoxicity played a critical role in this cascade.
We observed a hit rate of 1% as primary actives (>80% growth inhibition at 20 µM).
Compounds that exhibit an emerging MICꢀbased structureꢀactivity relationship (SAR) from
near neighbor screening were prioritized for further exploration.
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A series of DATs emerged as promising hits from this screening campaign with good antiꢀ
Mtb properties. Compound 1 (Figure 1A) was the most potent hit with an attractive
antimycobacterial ligand efficiency11 (ALE) of 0.37. In a recent disclosure of 177
antibacterial active compounds by GSK,12 two compounds from the series were shown to be
active against Mycobacterium bovis (BCG), with an MIC of 6 µM. However, no further work
is reported with systematic SAR exploration and/or mechanism of action. In addition, the
scaffold had been reported as inhibiting sterol regulatory element binding proteins (SREBPs),
a target class involved in cholesterol and fatty acid biosynthesis in humans.13 Fatostatin,14 the
lead candidate for antiꢀobesity therapy, is structurally close to our hit.
DAT compounds were synthesized using Hantzsch thiazole synthesis15, for which the
corresponding thioamides and substituted α–bromoketones were refluxed in ethanol as
represented in Scheme 1. Individual thioamides were synthesized16 from the corresponding
nitriles using sodium hydrosulfide and magnesium chloride in DMF at room temperature. α–
Bromination of acetophenone derivatives was carried out using cupric bromide17 in ethyl
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acetate/chloroform mixture (1:1) at reflux for 3 hrs, while α–bromination of heterocyclic acyl
derivatives was carried out using bromine18 with HBr in acetic acid at room temperature for
24 hrs. Detailed schematic representation and synthetic procedures are described as part of
Supplementary Information.
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Scheme 1. General schematic representation of synthesis of diarylthiazole compoundsa
aReagents and conditions: (a) Ethanol, reflux, 8ꢀ12 h, 60ꢀ80%
Synthesis of compounds 45ꢀ46 was carried out as per Scheme 2, in which 2ꢀpropylpyridineꢀ
4ꢀcarbothioamide and bromoacetaldehyde diethyl acetal were heated at 140 °C for 30 min in
a microwave reactor to afford 2ꢀ(2ꢀpropylpyridinꢀ4ꢀyl)thiazole. This was brominated using
NBS in acetic acid to 5ꢀbromoꢀ2ꢀ(2ꢀpropylpyridinꢀ4ꢀyl)thiazole, which was subjected to
Suzuki coupling to produce the final molecule in moderate yields. Synthesis of thiadiazole 47
was carried out as described previously.19
Scheme 2: Synthesis of compounds 45ꢀ46
aReagents and conditions: (a) Ethanol, MW, 140 °C, 30 min 78% (b) NBS, AcOH, 70 °C, 24 h, 20ꢀ50% (c) RꢀB(OH)2,
PdCl2(PPh3)2, NaHCO3, DME:Water (4:1), MW, 130 °C, 45 min, 40ꢀ50%
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In our pursuit from ’hit to lead’ we carried out a systematic exploration of the series replacing
R1 and R2 on the thiazole with substituted aryl, heteroaryl and alicyclic rings (Figure 1). The
prototype used for SAR exploration is shown in Figure 1B. Profiling of compound 1’s in
vitro DMPK properties suggested that the series will require optimization of physicochemical
properties such as equilibrium aqueous solubility, logD and in vitro clearance.
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1
Mtb MIC = 1 µg/mL
Mtb MBC = 2 µg/mL
ALE
= 0.37
Aq. Sol
< 1.5 µM
SI Index > 50
B
A
Figure 1: A. Compound 1, its activity and calculated properties. B. Rings and diversification
points of DAT used for SAR exploration. ALE = antibacterial ligand efficiency. SI index =
(Mammalian MIC against A549 cell line) / Mtb MIC
Initial exploration was carried out by replacing the propyl group attached to the aryl ring at
R1 with polar and solubilizing groups such as alkoxy, amino and morpholine (Table 1, 4-7).
While the Mtb MIC were comparable, the improvement in solubility was modest. Similarly,
replacing pyridine with pyridone and alicyclic rings (Table 1, 8-10) resulted in the loss of
activity to the highest concentration tested, which indicated that the 2ꢀpropyl/ethyl substituted
pyridyl at R1 is most effective.
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Table 1: SAR exploration of substitutions at R1
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No.
MIC Solubility
R1
ClogP
µg/mL
µM
1
2
3
4
1
<1.56
5.5
5.0
4.9
3.8
2
4
4
< 6.6
<2.78
0
5
2
0.38
5.8
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8
<3
0
5.1
4.0
> 32
8
> 32
> 32
> 32
<1
3393
3.6
2.8
3.9
2.5
9
10
In the next designꢀmakeꢀtestꢀanalyze (DMTA) cycle, we explored R2. A wide variety of
substitutions on the phenyl ring were synthesised and tested for Mtb MIC as shown in Table
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2 opening new avenues to improve potency. Interestingly, both electronꢀwithdrawing and
electronꢀdonating groups were active. Furthermore, substituents at ortho, meta, and para
positions were found to be equipotent from the limited set of electronꢀwithdrawing and
electronꢀdonating groups explored (Table 2, 13-21). Introduction of basic side chains with an
ethylene glycol linker at the para position (Table 2, 23-26) improved solubility, albeit with a
decrease in potency. The side chain with morpholine (Table 2, 26) offered a favorable
balance between solubility and Mtb MIC. The pKa of the terminal nitrogen played an
important role in balancing MIC and solubility. Polar groups like carboxylic acid, urea,
sulfonamide, acylsulfonamide (Table 2, 27-30) did not improve solubility while maintaining
a lower MIC.
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Table 2: SAR exploration of aryl ring at R2
No.
MIC
Solubility
µM
R2
ClogP
µg/mL
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12
13
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<3.2
.4
4.8
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0.2
14
15
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<2.9
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0.5
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> 32
0.37
5.7
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23
0.25
1
<1
<1
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16
8
278.8
154.6
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21.6
0.2
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5.6
5.0
4.6
4.5
1
16
1
29
30
0.4
32
1.3
3.8
3.5
>5000
The results from this initial DMTA cycles (Tables 1 & 2) established the potential of the
DAT class as antiꢀmycobacterial agents with opportunities to reach subꢀmicromolar Mtb
MIC. However, the attempt to reduce logD and hence, improve solubility with polar side
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chains led to weaker cellular activity. In subsequent DMTA cycles we replaced the phenyl
group at R2 with heteroaryl rings such as pyridine, pyrimidine as shown in Table 3.
Table 3: Substituted heterocylic rings at R2
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MIC
Solubility
No.
R2
ClogP
(µM)
µg/mL
1.4
31
32
33
34
91.5
3.4
3.4
3.6
2.4
1.6
4
116.9
51.1
59.2
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35
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0.25
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15.6
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0.06
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39
0.3
8
27.3
NA
4.1
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The results from the simple pyridyl and pyrimidine derivatives (Table 3, 31-34) were very
encouraging. Interestingly, all the 3 possible pyridyl derivatives were active with, 3ꢀpyridyl
and 4ꢀpyridyl being more potent than 2ꢀpyridyl. The introduction of the heterocycles
dramatically improved solubility for compounds showing Mtb MIC values of 4 µg/mL or
lower. The alkyl or alkyl ether substituted pyridyl led to a significant improvement in potency
(Table 3, 35-38). Further extension of the side chain on the pyridyl group with a basic group
(39) or carbocyclic ring (40) did not give any additional advantage for potency.
Further assuming planar nature of the molecules thus far may contribute to lower solubility,
substitutents were added on the 4ꢀposition of the central thiazole ring to break the planarity
and hence improve solubility. The polar groups such as amide, acid and ester (Table 4, 42-
44) led to weaker antiꢀMtb activity whereas a methyl group which led to an MIC of 4 µg/mL
(Table 4, 41), however, did not improve solubility. Changing aryl substitution at thiazole
ring from Cꢀ4 of thiazole to Cꢀ5, resulted in weaker Mtb MIC (Figure 2, 45-46).
Replacement of thiazole ring with relatively polar thiadiazole (47, Figure 2) also led to loss
of potency.
Table 4: SAR exploration at Ring A.
MIC Solubility
No.
R3
ClogP
µg/mL
µM
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ꢀMe
4
<1
<1
0.5
5.7
6.1
4.2
42 ꢀCOOEt
>32
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44 ꢀCOOH > 32
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<1
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The replacement of propyl with ethyl at R1 aryl ring showed hints of improvement in
solubility (matched pairs: 1 vs 2) while retaining the Mtb MIC. The ethyl derivative (Figure
3B, 48) exhibited an optimal balance of physicochemical properties and Mtb MIC. The ALE
for compound 48 was high (0.41) and improved by a margin of 0.5 log unit from the starting
compound 1.
Figure 2: Limited SAR exploration at the central thiazole ring
The scatter plot of clogP and Mtb MIC (in log scale) is shown in Figure 3A. The distribution
clearly indicates that the cellular activity was not linked with lipophilicity. Our medicinal
chemistry efforts to decrease ClogP via introduction of polar groups (Table 1-2) and
replacement of phenyl ring with heteroaromatic rings (Table 3) have culminated in finding
the right balance. The last DMTA cycle (Figure 3A; green circles in the scatter plot) led us to
compounds with lower lipophilicity, higher solubility (Table 3) and higher antiꢀ
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mycobacterial activity. These compounds were used for further microbiology and MoA
experiments that are discussed in the following section.
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Compound 48
Compounds in Table 1
Compounds in Table 2
Compounds in Table 3
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N
N
N
O
S
48
Mtb MIC = 0.12 µg/mL
Mtb MBC = 0.5 µg/mL
ALE = 0.41
Aq. Solubility = 31 µM
SIcytotox > 50
hERG IC50 > 33 µM
Calculated logP
B
A
Figure 3: A: Scatter plot of calculated logP and Mtb MIC (in logarithm scale). B: Structure
and data for compound 48.
The in vitro DMPK properties for a few selected compounds from the series are given in
Table 5. DAT as a series showed low in vitro intrinsic human microsomal clearance (Clint)
and high mouse microsomal clearance. The mouse plasma protein unbound fraction has
improved significantly as we optimized the series, compound 48 being the best with 17 %
free PPB (Table 5). This could largely be attributed to lowering of lipophilicity. Similarly,
aqueous solubility of the compounds, which was one of major challenges during the early
stage of lead generation (Table 1), improved as we progressed. Compound 48 turned out to
be the best in terms of Mtb MIC and physicoꢀchemical properties.
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Table 5: in vitro DMPK properties of DAT
Mouse
CLint
Human
CLint
5
6
7
Solubility
(µM)
No.
Mtb MIC
(µg/ml)
Mouse PPB
(% free)
ClogP
(µl/min/mg) (µl/min/mg)
8
9
29
32
33
36
37
38
48
0.4
1.6
4
3.77
3.4
1.3
120
51
<1
0.56
1.2
1.3
0.09
1.6
17
28.84
174.2
279.3
297
4.3
55.6
30.2
14.3
13.6
23
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3.61
4.22
4.75
4.11
3.69
0.2
0.06
0.3
0.12
16
2.6
27
171.7
170.4
170.4
31
ND
Microbiological properties
As discussed above, DAT as a series shows potent MICs against Mtb (best MIC observed 0.1
µg/ml, ~45 compounds with MIC ≤1 µg/ml). The minimum bactericidal concentration
(MBC) was found to be excellent with an MBC/MIC ratio ~1ꢀ2 (Table 6).
Table 6: MIC vs MBC of representative compounds
No.
25
26
31
37
38
48
MIC (µg/mL)
MBC (µg/mL)
MBC / MIC
4.9
2.6
0.4
0.1
0.6
0.25
4.9
5.2
0.8
0.1
1.2
0.5
1
2
2
1
2
2
Treatment of ~106 Mtb cells/mL for 10 days with compound 31 led to a ~3 Log10 CFU/mL
reduction. Compound 31 was also active in an in vitro intracellular THPꢀ1 TB infection
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model, a cell line model to assess activity on Mtb residing inside differentiated THPꢀ1
monocytic cells (Figure 4). Compound 31 showed ~1.5 Log10CFU/mL reduction in 7 days
when treated with 2 fold of MIC.
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Figure 4: Potency of Compound 31 in intracellular THP1 macrophage model. UnꢀTꢀD1 and
UnꢀTꢀD7 refers to bacterial counts on day 1 and day 7 with no drug treatment respectively.
Mode of action:
To understand if DAT compounds act by a mechanism distinct from INH and RIF,
compounds were tested for inhibition of InhA and RNA polymerase, the respective targets of
the two frontline drugs, using phenotypic and biochemical assays. Representative compounds
including 31, were tested for MIC modulation against H37Rv overꢀexpressing InhA enzyme
compared to wildtype. One of the evidences for target inhibition leading to cell growth arrest
is by an upshift of MIC on expressing higher levels of the target protein in Mtb cells
compared to wild type. For DAT compounds no MIC modulation was observed on
overexpression of InhA (Supplementary Table 1). Whereas under similar experimental
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conditions an upshift of MIC by 8ꢀ16 fold for H37Rv overexpressing InhA vs wildtype was
observed for InhA inhibitors i.e. isoniazid, ethionamide and triclosan. Compound 31, used as
a representative compound of DAT series, also did not inhibit InhA enzymatic assay (IC50s
>50 µM). Hence, these observations ruled out InhA as the target for DATs. Further
compound 31 was tested in an in vitro transcription assay and was found to be inactive at
concentrations tested (IC50s >50 µM), ruling out RNA polymerase as the target. Rifampicin, a
frontline TB drug which acts by inhibiting RNA polymerase, was used as positive control in
the assay. It inhibited the enzyme with a tight binding kinetics and showed IC50 of 30 nM
under the experimental conditions. Taken together, the above results indicate that having a
mechanism different from INH and RIF will enable the compounds to inhibit MDR TB
strains, which are classified as TB isolates resistant to front line regimen primarily INH and
RIF. In line with the above observations, the compounds were active against drugꢀsensitive
and isoniazid (INH) and rifampicin (RIF) resistant Mtb clinical isolates, further
strengthening the distinct mechanism for DAT (Table 7),
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48
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Table 7: MIC against clinical isolates
Mtb strains
INH
RIF
17
22
26
29
36
37
38
48
ꢁ
g / mL
ATCC27294 0.25 0.5
ATCC25618 0.25 0.5
2
2
1
1
0.25 0.13
0.13 0.13
0.13 0.25
0.13 0.13
0.13 0.25 0.25
0.06 0.13 0.13
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.06
0.007
0.003
0.003
0.003
0.003
0.003
0.003
0.003
0.003
Erdman
Beijing
0.25 0.5
0.13 0.25
0.5
0.5
0.13
0.13 0.13 0.13
0.06 0.13 0.13
Harlingen
0.13 0.25 0.5 0.13 0.13
ATCC35811 0.5
1
1
1
1
1
0.5
1
0.5
0.25
0.25
0.5
0.25
0.25
0.5
0.5
0.5
0.5
2
0.5
1
DKU76
DKU97A
DKU211
1
0.5
1
1
0.5
0.5
0.5
0.5
0.5
0.5
0.5
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DKU220
JAL
1
0.5
1
0.5
0.5
1
2
1
1
0.5
0.25 0.25
0.5 0.25
0.5
0.5
0.5
0.5
0.5
0.5
0.25
0.5
0.06
0.06
>16
0.03
0.003
0.003
0.003
>16
InhR
0.13 0.25
RifR
0.5 0.13 0.5 0.13 0.25
0.13 0.13 0.13
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The MIC was determined following drug exposure for 7 days and growth was monitored by
turbidometry. Inh: isoniazid; Rif: rifampin
To map the target of DAT compounds, resistant Mtb H37Rv mutants were raised to
compound 31. Mutants were isolated in the presence of 4, 8 and 16 fold of MIC of compound
on solid media at an observed spontaneous resistant frequency of ~10ꢀ7, 10ꢀ8 and 10ꢀ9
respectively. MIC of 31 for resistant strains was 8ꢀ32 fold higher than that of wildtype
H37Rv (Table 8). Comparable levels of resistance was also observed with other compounds
in the series, including compound 48. This strongly suggests that the compounds in the series
act by a common mechanism (Table 8). Whole genome sequencing of the representative
resistant mutant strains revealed point mutations in the prrB gene (Rv0902c). prrB was the
only gene which shows mutations across all resistant clones sequenced. The point mutations
resulted in amino acid substitutions R148C, R148P, R149W, and L152P (Table 9).
PrrBA, a two component system composed of PrrB histidine kinase and PrrA response
regulator has been shown to be critical for viability of Mtb cells and is required for initial
phase of macrophage infection by Mtb.20, 21 It is conserved among all mycobacterial species
pointing towards its critical function in mycobacterial physiology.10 Moreover, PrrBA operon
is transcribed during logarithmic growth of Mtb, while expression is decreased in stationary
phase. Among various stress conditions, prrBA transcription is specifically induced under
nitrogen limitation, indicating that the operon may be required for stressꢀinduced metabolic
adaptation.20 Mtb PrrB partial structures have been solved, which included the HAMP linker
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relative to the ATP binding and dimerization domains and the catalytic domain alone.22
However the amino acids mutated in resistant strains of compound 31 were not part of the
crystal structures reported. The residues are adjacent to the transmembrane domain spanning
the membrane separating the Cꢀterminal cytosolic region from Nꢀterminal domain on the
outside. Further investigations are required to understand how PrrB mutations impact the
functioning of the two component system.
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49
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Table 8: MIC modulation for resistant strains
Fold shift in MIC (first mutant
generation experiment)
Fold shift in MIC for resistant strains (Second mutant
generation experiment)
Compounds 1024_1 1024_18 Compounds 1024_18 1024_8.12 1024_16.5 1024_16.6
Isoniazid
Rifampicin
Moxifloxacin
Linezolid
1
0.5
1
1
0.5
1
Isoniazid
Moxifloxacin
TMC-207
31
1
0.5
0.25
8
1
1
1
1
1
1
0.25
8
0.5
16
4
0.5
16
>8
1
1
TMC-207
1
0.5
26
8
8
12
31
16
8
(fatostatin)
16
16
16
>32
12
(fatostatin)
8
16
>8
34
48
>8
8
>8
8
>8
8
>8
64
34
>8
Table 9: Mutations in Compound 31 resistant strains
Resistant Clone
1024_1
prrB nucleotide mutation
444, 445 CC ꢀ> TT
442 C ꢀ> T
PrrB amino acid mutation
R149W
R148C
L152P
1024_18
1024_8.12
455 T ꢀ> C
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1024_16.5
1024_16.6
455 T ꢀ> C
443 G ꢀ> C
L152P
R148P
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9
Fatostatin
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Fatostatin (125B11), initially identified as antiꢀobesity molecule has been also shown to have
anticancer and antidiabetic properties.23,24, 25 The molecule in preclinical development has
been shown to inhibit activation of sterol regulatory elementꢀbinding protein (SREBP), a key
regulator that controls the biosynthesis of fatty acids and sterols in human cells.14
Transcriptome analysis reveals that fatostatin treatment reduces activity of 63 genes, 34
involved in fatty acid and cholesterol biosynthesis in the cells.25 The compound inhibits
SREBs by binding to SCAP (SREBP cleavageꢀactivating protein) and preventing it from
interacting with SREB.14,25 As mentioned above, fatostatin, a DAT series compound (12) has
antimycobacterial activity (MIC ~1 µg/mL). Hence, here we report a novel activity of
fatostatin (12). To understand if 12 behaves similarly to other DAT molecules, it was tested
against compound 31 resistant strains containing the PrrB point mutation. An increase in MIC
of 12 (fatostatin) was observed against resistant strains compared to wildtype Mtb. Thus, 12
behaved like other DAT compounds (Table 8). Since, it is already known that fatostatin
binds to SCAP in humans cells, we searched for Mtb homologues of SCAP, however, no
proteins with significant homology could be identified. Sequence comparison of PrrB and
SCAP does not show significant similarity.
Transcriptome analysis
The absence of SCAP in Mtb and mapping SNPs in the prrB gene suggested that DAT
compound 31 has a different target in Mtb. In order to further understand the mechanism of
action and the potential downstream effects this compound may exert on the metabolism of
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Mtb, we undertook transcriptomic analysis of Mtb exposed to compound 31. Changes in the
cellular transcriptome upon treatment with compound 31 were mapped using DNA
microarray analysis. Mtb cells were exposed for 4 hours to 5X MIC of the compound. Total
RNA isolated from these cells were subjected to DNA microarray analysis using RNA from
untreated Mtb culture as control. Genespring GX analysis of the microarray data identified
379 upꢀregulated genes and 552 downꢀregulated genes. Cells responded to depletion in the
levels of PrrB by significantly inducing the expression of a number of genes involved in
fatty acid metabolism and genes required for maintenance of electron transport chain (ETC)
in an anaerobic state (genes encoding subunits of cytochrome ubiquinol oxidase, fumarate
reductase and nitrate reductase). Induced genes also included many oxidoꢀreductases along
with a few genes involved in iron transport (Supplementary Table 2). prrA and prrB genes
were moderately induced with a mean fold change of ~0.7 as compared to untreated control
(therefore doesn’t figure in the top 50 induced genes list – Supplementary Table 2). Since
the operon is autoregulated21, one could expect an elevated expression from this operon when
the functional protein levels deplete in the cell. However, the moderate induction observed
could be due to the experimental conditions used, i.e., 4 hours of compound exposure which
is probably not sufficient enough to exert the autoꢀregulatory effect. This is similar to an
observation made earlier where in no induction of prrA and prrB expression was seen 4 hours
post shifting cells to nitrogen limiting conditions, however, 5ꢀ7 fold induction was observed
after 4 days.20 Induction of genes involved in lipid metabolism and ETC of the anaerobic
state indicate the cellular response to modulation in the functional PrrB levels as found in
intracellular bacteria in the initial phases of infection.21 Most genes belonging to RNA
metabolism and protein synthesis were down regulated. Genes encoding RNA polymerase
subunits, sigma factors with the exception of sigJ, ribosomal protein synthesis, aminoacyl tꢀ
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RNA synthetases were among the genes that were significantly down regulated
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(Supplementary Table 2) and thus, would result in overall reduction in cellular metabolism
5
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and energy requirement in the cell.
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Conclusion
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Diarylthiazole is reported as a lead series with potent antiꢀmycobacterial activity and has the
potential to be developed for treatment of drug sensitive and drug resistant TB. The
systematic medicinal chemistry exploration has resulted in compounds with desired physicoꢀ
chemical properties. The resistance mapping indicates that DAT may be acting through the
PrrBA two component system. Understanding the underlying mechanism of prrB mutations
imparting resistance to DAT compounds, could uncover a completely new target class for
discovery of antimycobacterials and build new knowledge around role of PrrBA in Mtb
physiology.
EXPERIMENTAL SECTION
Chemical reagents and synthesis
All commercial reagents and solvents were used without further purification. Flash column
chromatography was performed using the Combiflash Isco purification system with SiO2 60ꢀ
120 for loading and readyꢀmade silica columns form Isco. Analytical thinꢀlayer
chromatography (TLC) was performed on SiO2 plates on alumina. Visualization was by UV
irradiation at 254 and 220 nm. The purity of all final derivatives for biological testing was
confirmed to be >95%, as determined using the following conditions: a Shimadzu HPLC
instrument with a Hamilton reversedphase column (HxSil, C18, 3 ꢁm, 2.1 mm × 50 mm
(H2)). Eluent A, 5% CH3CN in H2O; eluent B, 90% CH3CN in H2O. A flow rate of 0.2
mL/min was used with UV detection at 254 and 214 nm. The structure of the intermediates
and end products was confirmed by 1H NMR and mass spectroscopy. 1H NMR spectra were
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recorded on dilute solutions in CDCl3 or DMSOꢀd6 or MeODꢀd4 using Bruker DRXꢀ 300 or
Bruker DRXꢀ400 spectrometers operating at 300 or 400 MHz, respectively. Chemical shifts
(δ) are reported in ppm relative to TMS, and coupling constants (J) are reported in Hz. Spin
multiplicities are described as s (singlet), brs (broad singlet), d (doublet), t (triplet), q
(quartet), and m (multiplet). LCMS data was acquired using Agilent LCMS VL series.
Source: ES ionization coupled with an Agilent 1100 series HPLC system and an Agilent 1100
series PDA as the front end. HRMS data was acquired using an Agilent 6520, quadrupole
timeꢀofꢀflight tandem mass spectrometer (QꢀTOF MS/MS) coupled with an Agilent 1200
series HPLC system.
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General Procedure for Synthesis of DAT Compounds
A mixture of 2ꢀsubstituted pyridineꢀ4ꢀcarbothioamide (0.65 mmol) and substituted
0
heteroarylacyl bromide (0.65 mmol) in ethanol (10 mL) was stirred for overnight at 80 C.
The volatiles were removed by Rota evaporator at low pressure, the residue was partitioned
between ethyl acetate and saturated sodium bicarbonate solution. The aqueous phase was
extracted with ethyl acetate. The combined extracts were washed with water, and dried over
anhydrous sodium sulphate and concentrated to produce the crude compound, which was
purified by combiflash column chromatography to get 4ꢀarylꢀ2ꢀ(2ꢀsubstitutedpyridinꢀ4ꢀ
yl)thiazole.
4ꢀ(4ꢀChlorophenyl)ꢀ2ꢀ(propylpyridinꢀ4ꢀyl)thiazole (1).Yield : 42%, 1H NMR (300 MHz, DMSOꢀd6) δ
ppm 1.00 (t, J=7.28 Hz, 3H), 1.74 (sxt, J=14.68, 7.49 Hz, 2H), 2.95 (t, J=7.47 Hz, 2H), 7.47 (t,
J=8.78 Hz, 2H), 7.75 (d, J=5.02 Hz, 1H), 7.83 (s, 1H), 8.13 (dd, J=8.78, 5.77 Hz, 2H), 8.38 (s, 1H),
8.67 (d, J=5.52 Hz, 1H). HRMS: m/z (ES+) = 315.07016 (MH+) for C17H15ClN2S. HPLC purity:
98.00%
4ꢀ(4ꢀChlorophenyl)ꢀ2ꢀ(ethylpyridinꢀ4ꢀyl)thiazole (2).Yield : 82%, 1H NMR (300 MHz, DMSOꢀd6) δ
ppm 1.37 (t, J=7.54 Hz, 3H), 3.06 (d, J=7.54 Hz, 2H), 7.59 (d, J=8.67 Hz, 2H), 8.15ꢀ8.22 (m, J=8.48
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Hz, 2H), 8.29 (s, 1H), 8.36 (s, 1H), 8.62 (s, 1H), 8.85 (d, J=5.84 Hz, 1H). HRMS: m/z (ES+) =
3
4
5
300.04878 (MH+) for C16H13ClN2S. HPLC purity: 98.00%
6
7
8
9
4ꢀ(4ꢀChlorophenyl)ꢀ2ꢀ(cyclopropylpyridinꢀ4ꢀyl)thiazole (3).Yield : 76%, 1H NMR (300 MHz, DMSOꢀ
d6) δ ppm 0.92 ꢀ 1.09 (m, 4H), 2.28 (q, J=6.40 Hz, 1H), 7.57 (m, J=8.67 Hz, 2H), 7.71 (d, J=5.27 Hz,
1H), 7.91 (s, 1H), 8.11 (d, J=8.48 Hz, 2H), 8.42 (s, 1H), 8.55 (d, J=5.09 Hz, 1H). HRMS: m/z (ES+) =
313.05684 (MH+) for C17H13ClN2S. HPLC purity: 98.50%
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1
4ꢀ(4ꢀChlorophenyl)ꢀ2ꢀ(methoxymethylpyridinꢀ4ꢀyl)thiazole (4).Yield : 37%, H NMR (400 MHz,
DMSOꢀd6) δ ppm 3.45 (s, 3H), 4.61 (s, 2H), 7.58ꢀ7.65 (m, 2H), 7.88 ꢀ 7.92 (m, 1H), 7.97 (s, 1H), 8.11
(m, J=9.03 Hz, 2H), 8.45 (s, 1H), 8.69 (d, J=5.02 Hz, 1H). HRMS: m/z (ES+) = 317.05185 (MH+) for
C16H13ClN2OS. HPLC purity: 97.80%
4ꢀ(4ꢀChlorophenyl)ꢀ2ꢀ(ethylaminopyridinꢀ4ꢀyl)thiazole (5).Yield : 80%, 1H NMR (300 MHz, DMSOꢀ
d6) δ ppm 1.18ꢀ1.13 (t, J=7.16 Hz, 3H), 3.29 ꢀ 3.36 (m, 2H), 6.84 (t, J=5.27 Hz, 1H), 6.98 ꢀ 7.11 (m, 2
H), 7.56 (d, J=8.48 Hz, 2H), 8.02 ꢀ 8.14 (m, 3H), 8.34 (s, 1H). HRMS: m/z (ES+) = 316.06753 (MH+)
for C16H14ClN3S. HPLC purity: 99.40%
4ꢀ(4ꢀChlorophenyl)ꢀ2ꢀ(morpholinopyridinꢀ4ꢀyl)thiazole (6).Yield : 47%, 1H NMR (300 MHz, DMSOꢀ
d6) δ ppm 3.56ꢀ3.59 (m, 4H), 3.74ꢀ3.79 (m, 4H), 7.17 ꢀ 7.30 (m, 1H), 7.34 (s, 1H), 7.56 (d, J=7.72 Hz,
2H), 8.11 (d, J=7.54 Hz, 2H), 8.20 ꢀ 8.33 (m, 1H), 8.37 (s, 1H). HRMS: m/z (ES+) = 358.07846
(MH+) for C18H16ClN3OS. HPLC purity: 98.00%
4ꢀ(4ꢀChlorophenyl)ꢀ2ꢀ(cyclopropylmethoxypyridinꢀ4ꢀyl)thiazole (7).Yield : 51%, 1H NMR (300 MHz,
DMSOꢀd6) δ ppm 0.30ꢀ0.40 (m, 2H), 0.50ꢀ0.60 (m, 2H), 1.20ꢀ1.30 (m, 1H), 4.15 (d, J=21 Hz, 2H),
7.40 (s, 1H), 7.50ꢀ7.60 (m, 3H), 8.10 (d, J=30 Hz, 2H), 8.30 (d, J=21 Hz, 1H), 8.45 (s, 1H). HRMS:
m/z (ES+) = 343.06698 (MH+) for C18H15ClN2OS. HPLC purity: 98.00%
4ꢀ(4ꢀ(4ꢀChlorophenyl)thiazolꢀ2ꢀyl)pyridinꢀ2ꢀol (8).Yield : 19.42%, 1H NMR (400 MHz, DMSOꢀd6) δ
ppm 6.80 (d, J=6.02 Hz, 1H), 6.92 (s, 1H), 7.56 (d, J=8.53 Hz, 3H), 8.08 (d, J=8.53 Hz, 2H), 8.44 (s,
1H), 11.86 (br. s., 1H). HRMS: m/z (ES+) = 289.02001 (MH+) for C14H19ClN2OS. HPLC purity:
98.00%
4ꢀ(4ꢀChlorophenyl)ꢀ2ꢀ(1ꢀmethylpiperidinꢀ4ꢀyl)thiazole (9).Yield : 29.4%, 1H NMR (400 MHz,
DMSOꢀd6) δ ppm 1.72 ꢀ 1.82 (m, 2H), 2.08 (d, J=10.54 Hz, 4H), 2.23 (s, 3H), 2.87 (d, J=11.04 Hz,
2H), 3.01 (m, 1H), 7.51 (d, J=8.53 Hz, 2H), 7.98 (d, J=8.53 Hz, 2H), 8.07 (s, 1H). HRMS: m/z (ES+)
= 293.08842 (MH+) for C15H17ClN2S. HPLC purity: 99.00%
1ꢀ(4ꢀ(4ꢀ(4ꢀChlorophenyl)thiazolꢀ2ꢀyl)piperidinꢀ1ꢀyl)ethanone (10).Yield : 57.9%, 1H NMR (400 MHz,
DMSOꢀd6) δ ppm 1.55 (qd, J=12.13, 4.27 Hz, 1H), 1.65 ꢀ 1.78 (m, 1H), 2.01 ꢀ 2.16 (m, 5H), 2.65 ꢀ
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2.80 (m, 1H), 3.15 ꢀ 3.28 (m, 2H), 3.91 (d, J=14.05 Hz, 1H), 4.44 (d, J=13.05 Hz, 1H), 7.50 (m,
J=8.53 Hz, 2H), 7.98 (m, J=8.53 Hz, 2H), 8.08 (s, 1H). HRMS: m/z (ES+) = 321.08338 (MH+) for
C16H17ClN2OS. HPLC purity: 99.80%
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4ꢀPhenylꢀ2ꢀ(2ꢀpropylpyridinꢀ4ꢀyl)thiazole (11).Yield: 62.6%. H NMR (300 MHz, DMSOꢀd6) δ ppm
1.00 (t, J=7.26 Hz, 3H), 1.70ꢀ1.90 (m, 2H), 2.95ꢀ3ꢀ05 (m, 2H), 7.40ꢀ7.50 (m, 1H), 7.55ꢀ7.60 (m, 2H),
8.05ꢀ8.10 (m, 2H), 8.35 (d, J=8.53 Hz, 1H), 8.45 (s, 1H), 8.60 (s, 1H), 8.90 (d, J=8.65 Hz, 1H).
HRMS: m/z (ES+) = 280.10339 (MH+) for C17H16N2S. HPLC purity: 99.60%
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2ꢀ(2ꢀPropylpyridinꢀ4ꢀyl)ꢀ4ꢀpꢀtolylthiazole (12). Yield: 28.1%. 1H NMR (300 MHz, DMSOꢀd6) δ ppm
0.95 (t, J=7.35 Hz, 3H), 1.76 (sxt, J=7.42 Hz, 2H), 2.37 (s, 3H), 2.83 (t, J=7.54 Hz, 2H), 7.31ꢀ7.35
(m, 2H), 7.74 ꢀ 7.86 (m, 2H), 7.97ꢀ8.05 (m, 2H), 8.26 (s, 1H), 8.63 (d, J=5.09 Hz, 1H). HRMS: m/z
(ES+) = 295.12529 (MH+) for C18H18N2S. HPLC purity: 99.60%
4ꢀ(4ꢀFluorophenyl)ꢀ2ꢀ(2ꢀpropylpyridinꢀ4ꢀyl)thiazole (13).Yield: 42.8%. 1H NMR (400 MHz, DMSOꢀ
d6) δ ppm 0.95 (t, J=7.28 Hz, 3H), 1.76 (dq, J=14.68, 7.49 Hz, 2H), 2.83 (t, J=7.53 Hz, 2H), 7.34 (t,
J=8.78 Hz, 2H), 7.79 (d, J=5.02 Hz, 1H), 7.84 (s, 1H), 8.13 (dd, J=8.78, 5.77 Hz, 2H), 8.35 (s, 1H),
8.64 (d, J=5.52 Hz, 1H). HRMS: m/z (ES+) = 299.10155 (MH+) for C17H15FN2S. HPLC purity:
98.00%
4ꢀ(3ꢀFluorophenyl)ꢀ2ꢀ(2ꢀpropylpyridinꢀ4ꢀyl)thiazole (14).Yield: 72.8%. 1H NMR (400 MHz, DMSOꢀ
d6) δ ppm 0.95 (t, J=7.28 Hz, 3H), 1.76 (sxt, J=7.43 Hz, 2H), 2.83 (t, J=7.53 Hz, 2H), 7.21 ꢀ 7.33 (m,
1H), 7.49 ꢀ 7.63 (m, 1H), 7.80 (d, J=5.52 Hz, 1H), 7.83 ꢀ 8.03 (m, 3H), 8.48 (s, 1H), 8.65 (d, J=5.02
Hz, 1H). HRMS: m/z (ES+) = 299.10137 (MH+) for C17H15FN2S. HPLC purity: 99.00%
4ꢀ(2ꢀFluorophenyl)ꢀ2ꢀ(2ꢀpropylpyridinꢀ4ꢀyl)thiazole (15).Yield: 30.00%. 1H NMR (400 MHz,
DMSOꢀd6) δ ppm 0.95 (t, J=7.28 Hz, 3H), 1.76 (dq, J=15.00, 7.38 Hz, 2H), 2.83 (t, J=7.53 Hz, 2H),
7.34 ꢀ 7.53 (m, 3H), 7.78 ꢀ 7.89 (m, 2H), 8.20 ꢀ 8.33 (m, 2H), 8.65 (d, J=5.02 Hz,1H). HRMS: m/z
(ES+) = 299.10153 (MH+) for C17H15FN2S. HPLC purity: 96.00%
4ꢀ(4ꢀMethoxyphenyl)ꢀ2ꢀ(2ꢀpropylpyridinꢀ4ꢀyl)thiazole (16).Yield: 42.8%. 1H NMR (400 MHz,
DMSOꢀd6) δ ppm 0.97 (t, J=7.35 Hz, 3H), 1.80 (sxt, J=7.42 Hz, 2H), 2.83 (t, J=7.54 Hz, 2H), 3.80 (s,
3H), 7.35 (m, J=8.10 Hz, 2H), 7.78 ꢀ 7.88(m, 2H), 7.99 (m, J=8.10 Hz, 2H), 8.30 (s, 1H), 8.70 (d,
J=5.09 Hz, 1H): HRMS: m/z (ES+) = 310.11396 (MH+) for C18H18N2OS. HPLC purity: 96.30%
4ꢀ(3ꢀMethoxyphenyl)ꢀ2ꢀ(2ꢀpropylpyridinꢀ4ꢀyl)thiazole (17). Yield: 58.1%. 1H NMR (400 MHz,
DMSOꢀd6) δ ppm 0.94 (t, J=7.28 Hz, 3H), 1.75 (sxt, J=7.43 Hz, 2H), 2.82 (t, J=7.53 Hz, 2H), 3.85 (s,
3H), 6.98 (dd, J=8.03, 2.51 Hz, 1H), 7.41 (t, J=8.03 Hz, 1H), 7.59 ꢀ 7.69 (m, 2H), 7.76 ꢀ 7.87 (m, 2H),
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8.38 (s, 1H), 8.63 (d, J=5.02 Hz, 1H). HRMS: m/z (ES+) = 310.11399 (MH+) for C18H18N2OS. HPLC
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4ꢀ(2ꢀMethoxyphenyl)ꢀ2ꢀ(2ꢀpropylpyridinꢀ4ꢀyl)thiazole (18). Yield: 43.3%, 1H NMR (400 MHz,
DMSOꢀd6) δ ppm 0.95 (t, J=7.28 Hz, 3H), 1.76 (sxt, J=7.43 Hz, 2H), 2.76 ꢀ 2.87 (m, 2H), 3.96 (s,
3H), 7.03 ꢀ 7.16 (m, 1H), 7.20 (d, J=8.53 Hz, 1H), 7.34 ꢀ 7.46 (m, 1H), 7.75 ꢀ 7.87 (m, 2H), 8.25 ꢀ
8.36 (m, 2H), 8.63 (d, J=5.02 Hz, 1H). HRMS: m/z (ES+) = 310.11397(MH+) for C18H18N2OS. HPLC
purity: 99.00%
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2ꢀ(2ꢀPropylpyridinꢀ4ꢀyl)ꢀ4ꢀ(4ꢀ(trifluoromethyl)phenyl)thiazole (19). Yield: 42.8%. 1H NMR (400
MHz, DMSOꢀd6) δ ppm 0.96 (t, J=7.35 Hz, 3H), 1.81 (sxt, J=7.46 Hz, 2H), 3.03 (t, J=7.63 Hz, 2H),
8.08 ꢀ 8.12 (m, 2H), 8.25 ꢀ 8.32 (m, 2H), 8.38 (d, J=5.65 Hz, 1H), 8.46 (br. s., 1H), 8.80 (s, 1H), 8.92
(d, J=6.03 Hz, 1H). HRMS: m/z (ES+) = 348.09077 (MH+) for C18H15F3N2S. HPLC purity: 97.50%
2ꢀ(2ꢀPropylpyridinꢀ4ꢀyl)ꢀ4ꢀ(3ꢀ(trifluoromethyl)phenyl)thiazole (20).Yield: 48.8%. 1H NMR (400
MHz, DMSOꢀd6) δ ppm 0.95 (t, J=7.53 Hz, 3H), 1.69 ꢀ 1.85 (m, 2H), 2.83 (t, J=7.53 Hz, 2H), 7.72 ꢀ
7.89 (m, 4H), 8.38 ꢀ 8.44 (m, 2H), 8.60 ꢀ 8.69 (m, 2H). HRMS: m/z (ES+) = 348.09081 (MH+) for
C18H15F3N2S. HPLC purity: 98.00%
2ꢀ(2ꢀPropylpyridinꢀ4ꢀyl)ꢀ4ꢀ(2ꢀ(trifluoromethyl)phenyl)thiazole (21). Yield: 30.4%. 1H NMR (400
MHz, DMSOꢀd6) δ ppm 0.92 (t, J=7.28 Hz, 3H), 1.73 (sxt, J=7.43 Hz, 2H), 2.80 (t, J=7.53 Hz, 2H),
7.65 ꢀ 7.83 (m, 5H), 7.90 (d, J=7.53 Hz, 1H), 8.04 (s, 1H), 8.62 (d, J=5.02 Hz, 1H). HRMS: m/z
(ES+) = 348.09078 (MH+) for C18H15F3N2S. HPLC purity: 99.30%
4ꢀ(4ꢀ(Cyclopropylmethoxy)phenyl)ꢀ2ꢀ(2ꢀpropylpyridinꢀ4ꢀyl)thiazole (22).Yield: 50.0%. 1H NMR (400
MHz, DMSOꢀd6) δ ppm 0.33 ꢀ 0.37 (m, 2H), 0.56 ꢀ 0.61 (m, 2H), 0.94 (t, J=7.53 Hz, 3H), 1.23ꢀ1.26
(m, 1H), 1.75 (sxt, J=7.43 Hz, 2H), 2.82 (t, J=7.53 Hz, 2H), 3.87 ꢀ 3.88 (m, 2H), 7.03 (d, J=8.53 Hz,
2H), 7.75 ꢀ 7.77 (m, 2H), 8.01 (d, J=9.03 Hz, 2H), 8.20 (s, 1H), 8.63 (d, J=5.02 Hz, 1H). HRMS: m/z
(ES+) = 351.14529 (MH+) for C21H22N2OS. HPLC purity: 98.00%
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4ꢀ(4ꢀ(2ꢀMethoxyethoxy)phenyl)ꢀ2ꢀ(2ꢀpropylpyridinꢀ4ꢀyl)thiazole (23). Yield: 57.8 %. H NMR (400
MHz, DMSOꢀd6) δ ppm 0.95 (t, J=7.53 Hz, 3H), 1.76 (sxt, J=7.43 Hz, 2H), 2.82 (t, J=7.53 Hz, 2H),
3.33 (s, 3H), 3.66 ꢀ 3.74 (m, 2H), 4.11 ꢀ 4.20 (m, 2H), 7.07 (d, J=8.53 Hz, 2H), 7.75 ꢀ 7.86 (m, 2H),
8.01 (d, J=9.03 Hz, 2H), 8.20 (s, 1H), 8.63 (d, J=5.02 Hz, 1H). HRMS: m/z (ES+) = 355.1477 (MH+)
for C20H22N2O2S. HPLC purity: 98.90%
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N,NꢀDimethylꢀ2ꢀ(4ꢀ(2ꢀ(2ꢀpropylpyridinꢀ4ꢀyl)thiazolꢀ4ꢀyl)phenoxy)ethanamine (24). Yield: 81.0%. H
NMR (300 MHz, DMSOꢀd6) δ ppm 0.94 (t, J=7.35 Hz, 3H), 1.75 (sxt, J=7.38 Hz, 2H), 2.24 (s, 6H),
2.66 (t, J=5.56 Hz, 2H), 2.82 (t, J=7.54 Hz, 2H), 4.11 (t, J=5.75 Hz, 2H), 7.06 ꢀ 7.15 (m, 2H), 7.72 ꢀ
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7.87 (m, 2H), 8.00ꢀ8.05 (m, 2H), 8.19 (s, 1H), 8.63 (d, J=5.09 Hz, 1H). HRMS: m/z (ES+) =
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368.17946 (MH+) for C21H25N3OS. HPLC purity: 96.80%
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2ꢀ(2ꢀPropylpyridinꢀ4ꢀyl)ꢀ4ꢀ(4ꢀ(2ꢀ(pyrrolidinꢀ1ꢀyl)ethoxy)phenyl)thiazole (25). Yield: 55.0%. 1H NMR
(400 MHz, DMSOꢀd6) δ ppm 0.95 (t, J=7.53 Hz, 3H), 1.68 ꢀ 1.72 (m, 4H), 1.77 (sxt, J=7.43 Hz, 2H),
2.53 ꢀ 2.58 (m, 4H), 2.79 ꢀ 2.83 (m, 4H), 4.11 ꢀ 4.14 (m, 2H), 7.06 (d, J=8.53 Hz, 2H), 7.76 ꢀ 7.82 (m,
2H), 7.95 (d, J=9.03 Hz, 2H), 8.18 (s, 1 H), 8.68 (d, J=5.02 Hz, 1H). HRMS: m/z (ES+) =
394.18785(MH+) for C23H27N3OS. HPLC purity: 99.00%
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4ꢀ(2ꢀ(4ꢀ(2ꢀ(2ꢀPropylpyridinꢀ4ꢀyl)thiazolꢀ4ꢀyl)phenoxy)ethyl)morpholine (26). Yield: 60.0%. H NMR
(400 MHz, DMSOꢀd6) δ ppm 0.95 (t, J=7.53 Hz, 3H), 1.76 (sxt, J=7.43 Hz, 2H), 2.48 ꢀ 2.49 (m, 4H),
2.69 ꢀ 2.72 (m, 2H), 2.79 (t, J=7.53 Hz, 2H), 3.32 ꢀ 3.64 (m, 4H), 4.11 ꢀ 4.15 (m, 2H), 7.05 (d, J=8.53
Hz, 2H), 7.75 ꢀ 7.60 (m, 2H), 8.10 (d, J=9.03 Hz, 2H), 8.17 (s, 1H), 8.63 (d, J=5.02 Hz, 1H). HRMS:
m/z (ES+) = 410.18295 (MH+) for C23H27N3O2S. HPLC purity: 98.00%
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4ꢀ(2ꢀ(2ꢀPropylpyridinꢀ4ꢀyl)thiazolꢀ4ꢀyl)benzoic acid (27). Yield: 50 %. H NMR (300 MHz, DMSOꢀ
d6) δ ppm 0.98 (t, J=7.35 Hz, 3H), 1.81 (sxt, J=7.46 Hz, 2H), 3.03 (t, J=7.63 Hz, 2H), 8.08 (m, J=8.48
Hz, 2H), 8.25 (m, J=8.48 Hz, 2H), 8.36 (d, J=5.65 Hz, 1H), 8.46 (br. s., 1H), 8.78 (s, 1H), 8.90 (d,
J=6.03 Hz, 1H), 12.80 (s, 1H). HRMS: m/z (ES+) = 325.09964 (MH+) for C18H16N2O2S. HPLC
purity: 99.00%
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1ꢀEthylꢀ3ꢀ(4ꢀ(2ꢀ(2ꢀpropylpyridinꢀ4ꢀyl)thiazolꢀ4ꢀyl)phenyl)urea (28). Yield : 54.8%, H NMR (300
MHz, DMSOꢀd6) δ ppm 0.88 ꢀ 1.02 (m, 3H), 1.07 (t, J=7.16 Hz, 3H), 1.66 ꢀ 1.86 (m, 2H), 2.82 (t,
J=7.63 Hz, 2H), 3.06 ꢀ 3.21 (m, 2H), 6.16 (t, J=5.65 Hz, 1H), 7.51ꢀ7.55 (m, J=8.67 Hz, 2H), 7.77 (d,
J=5.27 Hz, 1H), 7.82 (s, 1H), 7.93 ꢀ 7.97 (m, 2H), 8.15 (s, 1H), 8.58 ꢀ 8.70 (m, 2H). HRMS: m/z
(ES+) = 367.15884 MH+) for C20H22N4OS. HPLC purity: 98.00%
Nꢀ(4ꢀ(2ꢀ(2ꢀPropylpyridinꢀ4ꢀyl)thiazolꢀ4ꢀyl)phenyl)methanesulfonamide (29). Yield : 55.4%, 1H NMR
(300 MHz, DMSOꢀd6) δ ppm 0.94 (t, J=7.35 Hz, 3H), 1.75 (sxt, J=7.38 Hz, 2H), 2.82 (t, J=7.63 Hz,
2H), 3.05 (s, 3H), 7.32 ꢀ7.35 (m, 2H), 7.70 ꢀ 7.96 (m, 2H), 8.04 ꢀ 8.08 (m, 2H), 8.26 (s, 1H), 8.64 (d,
J=5.27 Hz, 1H), 9.96 (s, 1H). HRMS: m/z (ES+) = 374.09150 (MH+) for C18H19N3O2S2. HPLC purity:
99.00%
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Nꢀ(Methylsulfonyl)ꢀ4ꢀ(2ꢀ(2ꢀpropylpyridinꢀ4ꢀyl)thiazolꢀ4ꢀyl)benzamide (30). Yield : 16%, H NMR
(300 MHz, DMSOꢀd6) δ ppm 0.95 (t, J=7.35 Hz, 3H), 1.67 ꢀ 1.84 (m, 2H), 2.76 ꢀ 2.94 (m, 5H), 7.41
(br. s, 1H), 7.80 (d, J=4.90 Hz, 1H), 7.86 (s, 1H), 8.04 (s, 4H), 8.40 (s, 1H), 8.64 (d, J=5.09 Hz, 1H).
HRMS: m/z (ES+) = 402.08696 (MH+) for C19H19N3O3S2. HPLC purity: 96.00%
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2ꢀ(2ꢀPropylpyridinꢀ4ꢀyl)ꢀ4ꢀ(pyridinꢀ4ꢀyl)thiazole (31). Yield: 49.9 %, 1H NMR (400 MHz, DMSOꢀd6)
δ ppm 0.94 (t, J=7.28 Hz, 3H) 1.75 (sxt, J=7.43 Hz, 2H) 2.82 (t, J=7.53 Hz, 2H) 7.70 ꢀ 7.84 (m, 4H)
8.61 ꢀ 8.76 (m, 4H), HRMS: m/z (ES+) = 282.10506 (MH+) for C16H15N3S. HPLC purity: 99.20%
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2ꢀ(2ꢀPropylpyridinꢀ4ꢀyl)ꢀ4ꢀ(pyridinꢀ3ꢀyl)thiazole (32). Yield: 59.9 %, 1H NMR (300 MHz, DMSOꢀd6)
δ ppm 0.95 (t, J=7.35 Hz, 3H), 1.76 (sxt, J=7.42 Hz, 2H), 2.83 (t, J=7.54 Hz, 2H), 7.54 (dd, J=8.01,
4.80 Hz, 1H), 7.77 ꢀ 7.90 (m, 2H), 8.43 (dt, J=8.01, 1.84 Hz, 1H), 8.52 (s, 1H), 8.58 ꢀ 8.69 (m, 2H),
9.29 (d, J=1.51 Hz, 1H). HRMS: m/z (ES+) = 282.10489 (MH+) for C16H15N3S. HPLC purity: 99.00%
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2ꢀ(2ꢀPropylpyridinꢀ4ꢀyl)ꢀ4ꢀ(pyridinꢀ2ꢀyl)thiazole (33). Yield: 80.0 %, 1H NMR (300 MHz, DMSOꢀd6)
δ ppm 0.95 (t, J=7.35 Hz, 3H), 1.77 (sxt, J=7.42 Hz, 2H), 2.78 ꢀ 2.89 (m, 2H), 7.42 (ddd, J=7.54,
4.80, 1.04 Hz, 1H), 7.77 ꢀ 7.89 (m, 2H), 7.96 (td, J=7.72, 1.70 Hz, 1H), 8.24 (d, J=7.91 Hz, 1H), 8.49
(s, 1H), 8.63 ꢀ 8.71 (m, 2H). HRMS: m/z (ES+) = 282.10477 (MH+) for C16H15N3S. HPLC purity:
99.00%
2ꢀ(2ꢀPropylpyridinꢀ4ꢀyl)ꢀ4ꢀ(pyrimidinꢀ5ꢀyl)thiazole (34). Yield: 46.3 %, 1H NMR (300 MHz, DMSOꢀ
d6) δ ppm 0.95 (t, J=7.35 Hz, 3H) 1.77 (sxt, J=7.46 Hz, 2H) 2.83 (t, J=7.63 Hz, 2H) 7.79 ꢀ 7.94 (m,
2H) 8.61 ꢀ 8.72 (m, 2H) 9.23 (s, 1H) 9.47 (s, 2H), HRMS: m/z (ES+) = 283.09961 (MH+) for
C15H14N4S. HPLC purity: 99.00%
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2,4ꢀBis(2ꢀpropylpyridinꢀ4ꢀyl)thiazole (35). Yield: 22.29 %, H NMR (400 MHz, DMSOꢀd6) δ ppm
0.95 (t, J=7.28 Hz, 6H), 1.76 (sxt, J=7.43 Hz, 4H), 2.81 (dt, J=14.56, 7.28 Hz, 4H), 7.79 ꢀ 7.87 (m,
3H), 7.90 (s, 1H), 8.59 (d, J=5.02 Hz, 1H), 8.63 ꢀ 8.67 (m, 2H). HRMS: m/z (ES+) = 324.14588
(MH+) for C19H21N3S. HPLC purity: 99.00%
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4ꢀ(2ꢀMethoxypyridinꢀ4ꢀyl)ꢀ2ꢀ(2ꢀpropylpyridinꢀ4ꢀyl)thiazole (36). Yield: 45.6%, H NMR (400 MHz,
DMSOꢀd6) δ ppm 0.95 (t, J=7.35 Hz, 3H), 1.76 (sxt, J=7.46 Hz, 2H), 2.83 (t, J=7.54 Hz, 2H), 3.92 (s,
3H), 7.47 (s, 1H), 7.64 (dd, J=5.37, 1.22 Hz, 1H), 7.80 (dd, J=5.09, 1.51 Hz, 1H), 7.86 (s, 1H), 8.28
(d, J=5.27 Hz, 1H), 8.62 ꢀ 8.69 (m, 2H). HRMS: m/z (ES+) = 312.10950 (MH+) for C17H17N3OS.
HPLC purity: 99.50%
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4ꢀ(2ꢀEthoxypyridinꢀ4ꢀyl)ꢀ2ꢀ(2ꢀpropylpyridinꢀ4ꢀyl)thiazole (37). Yield: 62.3%, H NMR (300 MHz,
DMSOꢀd6) δ ppm 0.95 (t, J=7.35 Hz, 3H), 1.36 (t, J=7.06 Hz, 3H), 1.76 (sxt, J=7.42 Hz, 2H), 2.83 (t,
J=7.54 Hz, 2H), 4.37 (q, J=6.97 Hz, 2H), 7.45 (s, 1H), 7.62 (dd, J=5.37, 1.22 Hz, 1H), 7.76 ꢀ 7.90 (m,
2H), 8.26 (d, J=5.46 Hz, 1H), 8.62 ꢀ 8.69 (m, 2H). HRMS: m/z (ES+) = 326.13073 (MH+) for
C18H19N3OS. HPLC purity: 98.70%
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4ꢀ(2ꢀ(2ꢀMethoxyethoxy)pyridinꢀ4ꢀyl)ꢀ2ꢀ(2ꢀpropylpyridinꢀ4ꢀyl)thiazole (38). Yield: 65.2%, H NMR
(300 MHz, DMSOꢀd6) δ ppm 0.95 (t, J=7.35 Hz, 3H), 1.76 (sxt, J=7.42 Hz, 2H), 2.83 (t, J=7.54 Hz,
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2H), 3.32 (s, 3H), 3.64 ꢀ 3.76 (m, 2H), 4.37 ꢀ 4.52 (m, 2H), 7.50 (s, 1H), 7.64 (dd, J=5.27, 1.13 Hz,
1H), 7.76 ꢀ 7.90 (m, 2H), 8.26 (d, J=5.27 Hz, 1H), 8.61 ꢀ 8.71 (m, 2H). HRMS: m/z (ES+) =
356.13560 (MH+) for C19H21N3O2S. HPLC purity: 97.90%
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N,NꢀDimethylꢀ2ꢀ(4ꢀ(2ꢀ(2ꢀpropylpyridinꢀ4ꢀyl)thiazolꢀ4ꢀyl)pyridinꢀ2ꢀyloxy)ethanamine (39). Yield:
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28.6%, H NMR (400 MHz, DMSOꢀd6) δ ppm 0.95 (t, J=7.28 Hz, 3H), 1.76 (sxt, J=7.43 Hz, 2H),
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2.23 (s, 6H), 2.65 (t, J=5.77 Hz, 2H), 2.83 (t, J=7.53 Hz, 2H), 4.40 (t, J=5.77 Hz, 2H), 7.47 (s, 1H),
7.63 (d, J=5.02 Hz, 1H), 7.81 (dd, J=5.02, 1.00 Hz, 1H), 7.87 (s, 1H), 8.27 (d, J=5.52 Hz, 1H), 8.63 ꢀ
8.71 (m, 2H). HRMS: m/z (ES+) = 369.1746 (MH+) for C20H24N4OS. HPLC purity: 95.30%
(S)ꢀ2ꢀ(2ꢀPropylpyridinꢀ4ꢀyl)ꢀ4ꢀ(2ꢀ(tetrahydrofuranꢀ3ꢀyloxy)pyridinꢀ4ꢀyl)thiazole (40). Yield: 45.9%,
1H NMR (300 MHz, DMSOꢀd6) δ ppm 0.95 (t, J=7.53 Hz, 3H), 1.76 (sxt, J=7.43 Hz, 2H), 2.00 ꢀ 2.10
(m, 1H), 2.20 ꢀ 2.32 (m, 1H), 2.83 (t, J=7.53 Hz, 2H), 3.76 ꢀ 4.00 (m, 4H), 5.55 ꢀ 5.61 (m, 1H), 7.50
(s, 1H), 7.66 (dd, J=5.52, 1.00 Hz, 1H), 7.81 (dd, J=5.27, 1.76 Hz, 1H), 7.88 (s, 1H), 8.28 (d, J=5.52
Hz, 1H), 8.63 ꢀ 8.71 (m, 2H). HRMS: m/z (ES+) = 368.14291 (MH+) for C20H21N3O2S. HPLC purity:
97.00%
4ꢀ(4ꢀChlorophenyl)ꢀ5ꢀmethylꢀ2ꢀ(2ꢀpropylpyridinꢀ4ꢀyl)thiazole (41). Yield: 33.0%. 1H NMR (400
MHz, DMSOꢀd6) δ ppm 0.93 (t, J=7.28 Hz, 3H), 1.74 (sxt, J=7.43 Hz, 2H), 2.65 (s, 3H), 2.80 (t,
J=7.53 Hz, 2H), 7.58 ꢀ 7.62 (m, J=8.53 Hz, 2H), 7.68 (d, J=5.02 Hz, 1H), 7.74 (s, 1H), 7.81 (m,
J=8.53 Hz, 2H), 8.61 (d, J=5.02 Hz, 1H). HRMS: m/z (ES+) = 329.08747 (MH+) for C18H17ClN2S.
HPLC purity: 96.00%
Ethyl 4ꢀ(4ꢀchlorophenyl)ꢀ2ꢀ(2ꢀpropylpyridinꢀ4ꢀyl)thiazoleꢀ5ꢀcarboxylate (42). Yield: 63.2%. 1H NMR
(300 MHz, DMSOꢀd6) δ ppm 0.93 (t, J=7.35 Hz, 3H), 1.26 (t, J=7.06 Hz, 3H), 1.75 (sxt, J=7.42 Hz,
2H), 2.82 (t, J=7.54 Hz, 2H), 4.28 (q, J=7.10 Hz, 2H), 7.52 ꢀ 7.60 (m, 2H), 7.78 ꢀ 7.91 (m, 4H), 8.67
(d, J=5.09 Hz, 1H). HRMS: m/z (ES+) = 387.09305 (MH+) for C20H19ClN2O2S. HPLC purity: 98.60%
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4ꢀ(4ꢀChlorophenyl)ꢀ2ꢀ(2ꢀpropylpyridinꢀ4ꢀyl)thiazoleꢀ5ꢀcarboxamide (43). Yield : 38.6%, H NMR
(400 MHz, DMSOꢀd6) δ ppm 0.93 (t, J=7.28 Hz, 3H), 1.74 (sxt, J=7.43 Hz, 2H), 2.82 (t, J=7.53 Hz,
2H), 7.57 (d, J=8.53 Hz, 2H), 7.76 (dd, J=5.02, 1.51 Hz, 1H), 7.79 ꢀ 7.87 (m, 3H), 7.91 (br. s., 1H),
8.06 (br. s., 1H), 8.65 (d, J=5.02 Hz, 1H). HRMS: m/z (ES+) = 358.07615 (MH+) for C18H16ClN3OS.
HPLC purity: 98.30%
4ꢀ(4ꢀChlorophenyl)ꢀ2ꢀ(2ꢀpropylpyridinꢀ4ꢀyl)thiazoleꢀ5ꢀcarboxylic acid (44). Yield : 46.2%, 1H NMR
(400 MHz, DMSOꢀd6) δ ppm 0.92 (t, J=7.28 Hz, 3H), 1.74 (sxt, J=7.43 Hz, 2H), 2.81 (t, J=7.53 Hz,
2H), 6.55 (s, 1H), 7.53 (d, J=8.53 Hz, 2H), 7.77 (d, J=5.02 Hz, 1H), 7.83 (s, 1H), 7.91 (d, J=7.53 Hz,
2H), 8.65 (d, J=5.02 Hz, 1H). HRMS: m/z (ES+) = 359.06093 (MH+) for C18H15ClN2O2S. HPLC
purity: 98.60%
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2ꢀ(2ꢀPropylpyridinꢀ4ꢀyl)ꢀ5ꢀ(pyridinꢀ4ꢀyl)thiazole (45). Yield: 31.2%, 1H NMR (400 MHz, DMSOꢀd6)
δ ppm 0.94 (t, J=7.28 Hz, 3H), 1.75 (sxt, J=7.43 Hz, 2H), 2.82 (t, J=7.53 Hz, 2H), 7.70 ꢀ 7.84 (m,
4H), 8.61 ꢀ 8.76 (m, 4H). HRMS: m/z (ES+) = 282.10438 (MH+) for C16H15N3S. HPLC purity:
98.70%
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5ꢀ(6ꢀMethoxypyridinꢀ3ꢀyl)ꢀ2ꢀ(2ꢀpropylpyridinꢀ4ꢀyl)thiazole (46). Yield: 47.3%, H NMR (400 MHz,
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DMSOꢀd6) δ ppm 0.93 (t, J=7.53 Hz, 3H), 1.67 ꢀ 1.78 (m, 2H), 2.80 (t, J=7.53 Hz, 2H), 3.90 (s, 3H),
7.19 (s, 1H), 7.35 (dd, J=5.52, 1.51 Hz, 1H), 7.69 ꢀ 7.79 (m, 2H), 8.25 (d, J=5.52 Hz, 1H), 8.64 (d,
J=5.52 Hz, 1H), 8.69 (s, 1H). HRMS: m/z (ES+) = 312.11691 (MH+) for C17H17N3OS. HPLC purity:
99.00%
3,5ꢀBis(2ꢀpropylpyridinꢀ4ꢀyl)ꢀ1,2,4ꢀthiadiazole (47). Yield : 69.9%, 1H NMR (400 MHz, DMSOꢀd6) δ
ppm 0.94 (t, J=7.28 Hz, 6H), 1.77 (sxt, J=7.43 Hz, 4H), 2.79 ꢀ 2.91 (m, 4H), 7.90 (dd, J=5.02, 1.51
Hz, 1H), 7.97 (s, 1H), 8.03 (d, J=5.02 Hz, 1H), 8.08 (s, 1H), 8.76 (d, J=5.02 Hz, 1H), 8.72 (d, J=5.02
Hz, 1H). HRMS: m/z (ES+) = 324.14087 (MH+) for C18H20N4S. HPLC purity: 98.00%
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2ꢀ(2ꢀEthylpyridinꢀ4ꢀyl)ꢀ4ꢀ(2ꢀmethoxypyridinꢀ4ꢀyl)thiazole (48). Yield: 55.3%, H NMR (400 MHz,
DMSOꢀd6) δ ppm 1.30 (t, J=7.53 Hz, 3H), 2.88 (q, J=7.53 Hz, 2H), 3.92 (s, 3H), 7.48 (s, 1H), 7.65
(dd, J=5.27, 1.25 Hz, 1H), 7.81 (dd, J=5.02, 1.51 Hz, 1H), 7.87 (s, 1H), 8.29 (d, J=5.52 Hz, 1H), 8.60
ꢀ 8.72 (m, 2H). HRMS: m/z (ES+) = 298.09370 (MH+) for C16H15N3OS. HPLC purity: 99.00%
Microbiological assays:
Assays to determine MIC for H37Rv and clinical isolates, MBC under aerobic and hypoxic
conditions, killing kinetics and intracellular THPꢀ1 (human lung adenocarcinoma epithelial
cell line) potency was carried out as described before.26 Minimal inhibitory concentration
(MIC) is defined as compound concentration that shows ≥80% growth inhibition compared to
untreated controls. MIC variation within four fold is considered as acceptable variation
between different experiments using various compound batches. Minimal bactericidal
concentration (MBC) is defined as compound concentration that shows ≥2 Log10CFU/mL
reduction compared to untreated controls.
InhA assay
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