Synthesis and evaluation of new 2-aminothiophenes against: Mycobacterium tuberculosis

Article abstract of DOI:10.1039/c6ob00821f

Tuberculosis (TB) and its drug resistant forms kills more people than any other infectious disease. This fact emphasizes the need to identify new drugs to treat TB. 2-Aminothiophenes (2AT) have been reported to inhibit Pks13, a validated anti-TB drug target. We synthesized a library of 42 2AT compounds. Among these, compound 33 showed remarkable potency against Mycobacterium tuberculosis (Mtb) H37R_V (MIC = 0.23 μM) and showed an impressive potency (MIC = 0.20-0.44 μM) against Mtb strains resistant to isoniazid, rifampicin and fluoroquinolones. The site of action for the compound 33 is presumed to be Pks13 or an earlier enzyme in the mycolic acid biosynthetic pathway. This inference is based on structural similarity of the compound 33 with known Pks13 inhibitors, which is corroborated by mycolic acid biosynthesis studies showing that the compound strongly inhibits the biosynthesis of all forms of mycolic acid in Mtb. In summary, these studies suggest 33 represents a promising anti-TB lead that exhibits activity well below toxicity to human monocytic cells.

Full text of DOI:10.1039/c6ob00821f

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Sucheck, Org. Biomol. Chem., 2016, DOI: 10.1039/C6OB00821F.
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Journal Name
PAPER
Synthesis and microbiological evaluation of 2-Amino-4,5,6,7-
tetrahydrothieno[2,3-c]pyridines against sensitive and drug
resistant Mycobacterium tuberculosis
Received 00th January 20xx,
Accepted 00th January 20xx
Sandeep Thanna,a‡ Susan E. Knudson,b‡ Anna Grzegorzewicz,b‡ Sunayana Kapil,a Christopher M.
Goins,a Donald R. Ronning,a Mary Jackson,b* Richard A. Slayden,b* Steven J. Sucheck a*
DOI: 10.1039/x0xx00000x
www.rsc.org/
Tuberculosis (TB) and its drug resistant forms kills more people than any other infectious disease. This fact emphasizes the
need to identify new drugs to treat TB. 2-Aminothiophenes (2AT) have been reported to inhibit Pks13, a validated anti-TB
drug target. We synthesized a library of 42 2AT compounds. Among these, compound 33 showed remarkable potency
against Mycobacterium tuberculosis (Mtb) H37RV (MIC = 0.23 µM) and showed an impressive potency (MIC = 0.20-0.44
µM) against Mtb strains resistant to isoniazid, rifampicin and fluoroquinolones. The site of action for the compound 33 is
presumed to be Pks13 or an earlier enzyme in the mycolic acid biosynthetic pathway. This inference is based structural
similarity of the compound 33 with known Pks13 inhibitors, which is corroborated by mycolic acid biosynthesis studies
which show the compound strongly inhibits the biosynthesis of all forms of mycolic acid in Mtb. In summary, these studies
suggest 33 represents a promising anti-TB lead which showed activity well below toxicity to human monoocytic cells.
numbers of pills/day and side-effects. The discontinuation of
treatment allows resistance to develop. Therefore, there is an
immediate need for drugs with improved efficacy and reduced
toxicity.
Mtb has an unusual cell envelope that contains a thick
hydrophobic layer which differs from typical Gram positive (G+)
and Gram negative (G-) bacteria making it resistant to many
broad-spectrum drugs that normally act on G+ and G- bacteria.
The main constituents of the Mtb cell envelope include: a
plasma membrane, a cell wall core consisting of peptidoglycan
in covalent attachment with arabinogalactan and mycolic acids,
outer membrane lipids referred to as the mycomembrane and
a loosely attached capsule.2-4 The outer membrane gives Mtb a
cell wall architecture similar to Gram negative bacteria. The
arabinogalactan and trehalose are found in the cell wall as
esters of mycolic acids (MAs). Mycolic acids (MAs) are α-alkyl-β-
hydroxy fatty acids with total carbon chain lengths ranging from
Introduction
Mycobacterium tuberculosis (Mtb) is the causative agent of
tuberculosis (TB). TB ranks first for the number of deaths in
humans caused by an infectious agent.1 In 2013 there were 9.0
million active cases of TB. Of these, 0.48 case million where
multidrug-resistant TB (MDR-TB). It is notable that the number
of MDR-TB cases tripled from 2009 to 2013. This increase in
MDR-TB and extensively drug-resistant TB (XDR-TB) has drawn
worldwide attention. Furthermore, HIV-TB co-infection is a
serious problem in countries where TB is endemic. HIV positive
patients are 26-31 times more likely to develop active TB in
comparison to an HIV negative person. Overall, 1.5 million
people die every year from this disease.
It has been difficult to eradicate TB even though it is
treatable. Treatment for TB takes at least 6 months and
sometimes up to 2 years. The basic 6 month therapy involves
two months of an intensive phase using the quadruple drug
regimen consisting of Isoniazid (INH), Rifampicin (RIF),
Ethambutol (EMB) and Pyrazinamide (PZA) followed by a 4
month continuous phase consisting of INH and RIF. Often,
patients do not comply with the entire regimen due to the high
C
C
60 to C90, with an α-side chain with carbon lengths ranging from
24 to C26.5 They esterify both the cell wall arabinogalactan and
extractable glycolipids of the cell envelope, particularly,
trehalose monomycolate (TMM), trehalose dimycolate (TDM,
Cord Factor).
Biosynthesis of MAs involves two fatty acid synthases (FASs)
FAS-I and FAS-II. FAS-I is considered ‘‘eukaryotic-like’’ and
produces two acyl-CoA fatty acids. One fatty acid has a carbon
chain length of C24-C26 which is carboxylated by ACCase/AccD4
to form a carboxyacyl-CoA derivative which becomes the α-
branch of MAs. The second fatty acid has a chain length of C16-
a.Department of Chemistry and Biochemistry, The University of Toledo, 2801 W.
Bancroft Street, Toledo, OH, USA 43606
b.Mycobacteria Research Laboratories, Department of Microbiology, Immunology
and Pathology, Colorado State University, Fort Collins, CO 80523
Email: mary.jackson@colostate.edu, richard.slayden@colostate.edu,
steve.sucheck@utoledo.edu
C18 and is elongated by FAS-II. FAS-II is considered a ‘‘bacterial-
†Electronic supplementary information (ESI) available: Detailed experimental
procedures, and 1H and 13C NMR spectra available for new compounds
‡ Equal contributing authors
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Journal Name
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DOI: 10.1039/C6OB00821F
FAAL32
O
R1
OH
R1
O
O
FadD32
FadD32
S
Ser-55
AMP
R1
acid
Meromycoloyl-AMP
Meromycolic
3
1
2
ATP PPi
O
=
-C64
R1
chain,
-C26
C48
Meromycolic
S
α
KS
AT
=
R2
-Branch,
C24
R1
=
R3 Trehalose
4
R2
O
S
HO
O
R2
O
S CoA
O
Carboxyacyl-CoA
O
5
6
O
O
O
O
R
Ser-1266
Ser-1533
S
R1
S
R2
-CO2
R2
β
α
O
ester
-alkyl -keto
8
7
OH
O
O
O
O
O
TE
R3
R3
R
R/S
R2
R
R
R1
R1
O
O
R1
S
Trehalose
CmrA
R2
R2
TMM
TMMk
11
10
9
Figure 1. Mechanism of Pks13. FadD32 is a long-chain acyl-AMP ligase (FAAL32
)
like’’ fatty acid synthase which participates in the production of TMM, and TDM
, respectively. Pks13 and Ag85s are well-
the meromycolic acid chain (C42-C62). The product of FAS-II is a established anti-tuberculosis drug targets.9, 10
C42-C62 carboxylic acid which is converted to meromycoloyl-
Pks13 is essential for Mtb 9, 11 and is encoded by the fadD32-
AMP by FadD32.5, 6 Pks13 is a polyketide synthase that catalyzes Pks13-accD4 gene cluster. It belongs to the type-1 polyketide
a Claisen-type condensation between the meromycoloyl-AMP synthase gene family. Pks13 has phosphopantetheinyl (P-pant)
and carboxyacyl-CoA to produce a mycolic β-ketoester. This arm on acyl carrier protein domains at the N-terminus (N-ACP)
product is transferred to trehalose7 and the β-ketoester and the C-terminus (C-ACP) at Ser-55 and Ser-1266,
reduced by the Mtb homolog of Corynebacterineae mycolate respectively. The condensation reaction catalyzed by Pks13
reductase A (CmrA) to form TMM.8 TMM is utilized by the Ag85 proceeds according to Figure 1. Meromycoloyl-AMP
complex, a homologous family of enzymes (Ag85 A, B and C) produced by FadD32 is transferred to the ketosynthase (KS) (
that catalyzes a transesterification reaction that transfers MAs domain from the P-pant of N-ACP (
(
4
2
)
)
3
) domain. Carboxylacyl-CoA
onto arabinogalactan (AG), trehalose and TMM forming mAG,
(5
) is transferred to P-pant of the C-ACP ( ) via the
7
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Figure 2. Selected structures of previously reported Ag85C and Pks13 inhibitors with a 2-aminothiophene core. FRT = fused ring 2-aminothiophenes, 5PT = 2-
amino-5-phenylthiophenes. A medium sized library of FRT and 5PT has been synthesized as antitubercular agents in this work. R1 = NCbz, NBoc, NEt, CH2, R2 =acyl,
R3 = alkylamino or alkyloxy.
acyltransferase domain (AT) (
domain ( ) and carboxyacyl on C-ACP domain (
Claisen-type condensation and forms an α-alkyl-β-keto i) FRT compounds formed from cores 22-25 and ii) 5PT
thioester attached to the P-pant of C-ACP domain ( ) which is compounds formed from core 26 (Figure 2, box). Four FRT cores
6
). The meromycoloyl on the KS Chemistry. We synthesized and evaluated a series of 2-
4
7
) undergo aminothiophenes (2AT). The 2AT we prepared are of two types:
8
transferred to Ser-1533 of thioesterase domain (TE) to form
The TE domain shows acyltransferase activity and transfers
9
9
.
were synthesized from ketones containing an active methylene
group. The ketones were: benzyl 4-oxopiperidine-1-carboxylate
onto trehalose forming TMM keto form (TMMk,, 10) which is 17
,
tert-butyl
4-oxopiperidine-1-carboxylate
18
,
1-
further reduced by CmrA to produce trehalose monomycolate ethylpiperidin-4-one 19, and cyclohexanone 20. The 5PT core
(TMM, 11).7 The TMM produced is transported to the was synthesized from 2-phenylacetaldehyde 21, (Scheme 1). 2-
periplasmic space by a translocation machinery involving the Alkylamido compounds 27-39 and 54-57 were synthesized from
RND-type transporter MmpL3,12 and is subsequently utilized by 22-26 using acid chlorides (AC). The ACs used were: acetyl
Ag85 complex to produce the major cell components mAG, chloride, benzoyl chloride, 2,3,4,5,6-pentafluorobenzoyl
TMM and TDM.13
chloride, and 3,5-bis(trifluoromethyl)benzoyl chloride. The 3-
2-Aminothiophenes are found in a wide range of natural and carboxamide compounds 44-53 and 59-63 were synthesized
synthetic products and include compounds with anti- using alkylamines (AA): butylamine, benzylamine, 4-
tuberculosis activity.14-19 Some 2-aminothiophenes are methylbenzylamine,
2-methoxybenzylamine,
and
reported to inhibit either Pks13 or Ag85s9, 20 The compound I3- adamantylamine (Scheme 1). The core compounds 22-26
AG85 (12) has been reported to be an inhibitor of Ag85s (Figure (Scheme 1) were synthesized using Gewald chemistry.24 This
21
2).20,
Based on these reports Sanki et al. designed and reaction involves the use of aldehyde or ketone 17-21 with the
synthesized
ethyl
2-amino-4,5,6,7- activated nitrile, ethyl 2-cyanoacetate (16), in presence of
tetrahydrobenzo[b]thiophene-3-carboxylate derivative (13) as elemental sulfur (S8) and diethylamine to obtain the respective
an Ag85C inhibitor with moderate activity.22 Wilson et al. cores.24 The cores 22-26, on reacting with ACs in presence of
screened 1,113 publicly available Mtb growth inhibiting DMAP and Et3N, vary the R2 group and afford 2-alkylamido
compounds identified by NIH/MLSCN,9 and identified several compounds 27-39 and 54-57. The 3-carboxamides compounds
compounds that act as Pks13 inhibitors. They found compounds labelled 44-53 and 59-63 were accessed by deprotection of the
14 and 15 had low MICs against Mtb H37Rv (Figure 2).9 Herein, ethyl esters 22-26 with LiOH to obtain compounds labeled 40-
we report new 2-aminothiophenes which show potent activity 43 and 58 25
on multiple drug resistant strains of Mtb and describe their reagent
,
which on further treating with AAs using coupling
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
effects on mycolic acid biosynthesis.
(EDC.HCl), hydroxybenzotriazole (HOBt) and DMAP afforded 3-
carboxamide compounds (44-53 and 59-63) (Figure 3).26
Growth inhibition and cytotoxicity studies. The library of 42
2ATs (Figure 3) was subjected to growth inhibition studies using
Results
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a modified 96-well microplate Alamar blue assay (MABA). IC90 values were determined against Mtb H37Rv VgierwowArtnicleuOnndliener
DOI: 10.1039/C6OB00821F
Among these compounds 33 and 38 showed MIC < 50 µM both aerobic and as low oxygen conditions. These MIC assays
whereas compound 29 and 30 showed an MIC < 100 µM. The were based on measurement of growth in liquid medium of a
remainder of the compounds showed MIC > 200 µM. The most fluorescent (mCherry) reporter strain of Mtb H37Rv where the
potent compound was 33 MIC = 0.69 µM (Table 1).
readout is either optical density (OD) or fluorescence.27-30 In
The activity of 33 was independently validated using a addition, the antimicrobial activity of compounds against Mtb
National Institutes of Health screening contract. The IC50 and H37Rv grown under hypoxic conditions was assessed using a
Scheme 1. Synthesis scheme for 2-aminothiophene cores 3{1,0,0} to 6{5,0,5}. Reagents and conditions: i) S8, Et2N, EtOH, 3{1,0,0} (86%), 3{2,0,0} (63%), 3{3,0,0}
(75%), 3{4,0,0} (83%), 3{5,0,0} (61%); (ii) Acid chloride (AC), Et3N, DCM, DMAP (15-82%); (iii) LiOH, THF: H2O: MeOH (3:1:1) (44-89%); (iv) Alkylamine (AA), EDC.
HCl, HOBt, DMAP, dry CH2Cl2 (21-61%). Diversity of substrates used in this investigation. In a compound numbered {a,b,c} a = thiophene core, b = acid chloride, c
= alkylamine.
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low oxygen recovery assay (LORA). In this assay, bacteria are this assay may be prone to false-positives. A false positive can
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first adapted to low oxygen conditions and then exposed to occur when a potent molecules activDeOIo: n10ly.10i3n9/Cr6eOpBli0c0a8ti2n1gF
compounds under hypoxia; this is followed by a period of conditions register as active candidates in non-replicating
outgrowth in aerobic conditions and growth is measured using conditions by virtue of their carryover from the non-replicating
luminescence.31-33 The mCherry assay confirmed the initial phase to the outgrowth phase.34
screening result and showed an MIC = 0.23 µM (Table 2). The The bactericidal activity of 33 was assessed against Mtb H37Rv
LORA showed an MIC > 200 µM, IC50 = 0.27 µM, and IC90 = 15 grown in aerobic conditions in 7H9-Tw-OADC medium. Viable
µM. This result is considered activity under low oxygen; cell counts were measured over 3 weeks of exposure to
however, this activity is far less than the control compound determine the rate of kill. Compounds were tested at fixed
rifampicin (RIF) which shows an MIC = 0.048 µM under the same concentrations, as no MIC data were available at the time of the
conditions. The protocol used was amenable to HTS. We note,
Figure 3. 2-Aminothiophenes prepared. Yield, written after compound number
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Table 1. MICs and IC50s (
µ
M) of the 2AT Library Mtb H37Rv
IC50 Comp No NV/CLogP MIC
0/1.88 507
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DOI: 10.1039/C6OB00821F
Comp No NV/CLogP MIC
IC50
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
0/2.80
0/2.39
0/1.73
0/2.52
0/3.26
0/2.12
0/3.79
1/4.33
2/5.51
0/1.46
0/3.50
0/4.03
1/5.22
2/5.69
0/2.25
0/3.95
0/4.45
1/5.64
0/2.16
0/1.75
0/1.09
> 200 N/A
> 300 N/A
> 300 N/A
> 400 N/A
> 400 N/A
> 200 N/A
> 200 N/A
> 100 N/A
> 100 N/A
> 300 N/A
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
N/A
0/2.15 > 300 > 300
0/2.11 > 300 N/A
0/2.56 > 300 N/A
0/2.12 > 200 N/A
0/3.48 > 200 N/A
0/2.94 > 300 N/A
0/2.90 > 300 N/A
Figure 4. The bactericidal activity of 33 assessed against Mtb H37Rv grown in aerobic
conditions in 7H9-Tw-OADC medium. Viable cell counts are measured over 3 weeks of
exposure to determine the rate of kill.
For 33 the IC50 = 6.2 µM which represents the concentration of
compound causing a 50% loss in viability.
The intracellular activity of 33 was measured using THP-1
cells infected with an autoluminescent Mtb strain derived from
Mtb H37Rv.32 THP-1 cells were again differentiated into
macrophage-like cells using PMA and infected with bacteria.
Infected cells were exposed to compounds for 72 hours. Viable
bacterial counts are measured using luminescence as a measure
of growth. The result is shown in Table 2 and is reported as an
IC50 = 5.6 µM which compares to and IC50 = 0.24 µM for INH.
The activity of 33 against five resistant isolates of Mtb
strains was assessed under aerobic conditions by determining
the MIC. Strains tested were two isoniazid resistant strains (INH-
R1 and INH-R2), two rifampicin resistant strains (RIF-R1 and RIF-
R2) and a fluoroquinolone resistant strain (FQ-R1). The assay is
based on measurement of growth in liquid medium of each
strain where the readout is OD. 33 34 35 36 INH-R1 was derived
from H37Rv and is a katG mutant (Y155* = truncation). INH-R2
is strain ATCC 35822. RIF-R1 was derived from H37Rv and is an
rpoB mutant (S522L). RIF-R2 is strain ATCC 35838. FQ-R1 is a
0/3.35
0/2.91
333
316
N/A
N/A
279
N/A
0/4.27 > 300 N/A
0/3.00 > 300 N/A
0/4.67 > 200 N/A
1/5.20 > 200 N/A
1/6.39 > 200 N/A
0.69
0.65
> 200 N/A
300 N/A
> 300 N/A
> 600 N/A
0/2.62
228
129
30
13
0/3.68 > 300 N/A
0/3.64 324.57 N/A
0/4.09 > 300 N/A
0/3.65 > 200 N/A
1/5.01 > 200 N/A
> 200 N/A
> 300 N/A
> 300 N/A
> 400 N/A
Figure 5. (A) Effect of 33 on TMM and TDM synthesis. The same volume of total
lipid samples was loaded per lane and the TLC plate was developed in
chloroform:methanol:water (20:4:0.5, by vol.). The cells treated with 33 showed
the significant decrease in TMM and TDM contents concomitant with a slight
increase in free mycolates (FM). The synthesis of other lipids
(phosphatidylethanolamine, PE; cardiolipin, CL) is not affected by 33. Mycolic acid
methyl esters (MAMEs) were prepared from extractable lipids (B) and delipidated
cells (C) so as to analyze separately the populations of mycolates esterified in the
cell wall arabinogalactan and extractable lipids; the same volume of samples was
loaded per lane. The TLC plate was developed three times in the solvent system
n-hexanes:ethyl acetate (95:5, by vol.). The cells treated with 33 showed the
significant decrease in all forms of mycolic acids (alpha-, methoxy- and keto-)
isolated either from extractable lipids or cell wall. The results are indicative of a
general, 33 -concentration dependent, decrease in mycolic acid biosynthesis in
the treated cells.
NV = Number of violations for Lipinski rule of five, CLogP = Calculated LogP value.
experiment 200, 100, 20 and 5 µM 33 were used. The MBC was
found to be < 5 µM (Figure 4).
The cytotoxicity of 33 towards eukaryotic cells was
determined using the THP-1 human monoocytic cell line. THP-1
cells were differentiated into macrophage-like cells using
phorbol 12-myristate 13-acetate (PMA) and incubated with
compounds for 3 day after which time cell viability is measured.
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Table 2. Evaluation of 33 MICs and ICs (µM) against Mtb under aerobic, low oxygen, and intracellular conditions. Evaluation of 33 against drug resistant strains of Mtb.
H37Rv
(aerobic)
MIC = 0.23
MIC50 = 0.12
MIC90 = 0.23
Controls
RIF
H37Rv
(low oxygen)
MIC > 200
IC50 = 0.27
IC90 = 15
H37Rv
(intracellular)
INH-R1
INH-R2
RIF-R1
RIF-R2
FQ-R1
MIC = 0.44
IC50 = 0.16
IC90 = 0.45
MIC = 0.20
IC50 = 0.053
IC90 = 0.20
MIC = 0.30
IC50= 0.095
IC90 = 0.35
MIC = 0.44
IC50 = 0.11
IC90 = 0.48
MIC = 0.37
IC50 = 0.14
IC90 = 0.39
IC50 = 5.6
IC90 = 9.75
MIC = 0.048, n=8
MIC = 0.012
MIC > 200
MIC = 1.3
MIC = 0.0070
MIC > 200
MIC = 1.4
MIC = 3.5
MIC = 0.18
MIC = 1.2
MIC > 50
MIC > 0.60
MIC = 1.2
MIC = 0.020
MIC = 0.32
MIC = 24
INH
IC50 = 0.24
Levofloxacin
INH-R1 was derived from H37Rv and is a katG mutant (Y155* = truncation). INH-R2 is strain ATCC 35822. RIF-R1 was derived from H37Rv and is an rpoB mutant
(S522L). RIF-R2 is strain ATCC 35838. FQ-R1 is a fluoroquinolone-resistant strain derived from H37Rv and is a gyrB mutant (D94N).
fluoroquinolone-resistant strain derived from H37Rv and is a had similar substitution, thus we saw this as way to access a
gyrB mutant (D94N). Compound 33 showed potent activity MIC similar topology, avoid introduction of stereogenic center, and
= 0.20-0.44 µM against the five resistance isolates tested (Table introduce a pKa modifying group. While clearly none of these
2). Control data for INH, RIF and Levofloxacin is also reported compounds inhibited Ag85C to a significant degree 33 is the
(Table 2) for comparison.
most potent anti-Mtb 2-aminothiophene reported to date.
Effect of 33 on mycolic acid synthesis. To assess the potential Other observations were that replacement of the NEt with a
inhibitory effect of 33 on mycolic acid biosynthesis, Mtb bulky groups like N-Boc at R1 had detrimental effect making
cultures exposed to different concentrations of 33 were compound 29 inactive even with a COC6F6 substituent at the R2.
metabolically labeled with [1,2-14C]acetate and the levels of
The activity requirement for the ethyl ester at R3 position is
TMM, TDM, and mAG-attached mycolates were determined less well defined and merits further investigation. There was
following TLC analysis and autoradiography.23 The studies slight activity observed for 35 (MIC = 351.40 µM) which bears
clearly revealed that 33 strongly inhibits the synthesis of all an R3 benzamide moiety in comparison to 34 (MIC > 200 µM)
forms of mycolic acids in the cell envelope (Figure 5).
which bears an R3 ethoxy moiety. Unfortunately, we did not
Inhibition of Ag85C by 2-ATs. To determine whether or not prepare this R3 substitution with an R2 = COC6F5 group but
any of our compounds inhibited Ag85s we performed a high rather the 3,5-trifluoromethylbenzamide. Thus, the fact that
throughput screening of the compound library against Ag85C any activity at all was observed is somewhat intriguing to us and
using a fluorescence-based assay.23 None of the compounds in suggests compounds with further improvements in potency
our library showed inhibition below 200 µM (SI-Figure 1) against may be obtainable. Previous studies have shown substituting
Ag85C.
the ethyl ester with an amide at R3 resulted in a significant
increase in MIC.9 The structural similarity of 33 to compounds
14 and 15 reported by Wilson, et al. suggest the compound may
Discussion
Structure activity relationships (SAR). The structures of FRT inhibit Pks13.9 The 5PT compounds all showed poor to no
and 5PT compounds were divided into R1, R2 and R3 forFRTs and potency, as all the MICs were ≥ 200 µM. This result indicates
R2 and R3 for 5PT, respectively, Figure 2. The most obvious that six membered ring attached to thiophene is important for
relation was the introduction of COC6F5 at the R2 position which growth inhibition activity as even compound 56 with an R2 2-
resulted in a sharp increase in potency. It could be seen that 38 (perfluorobenzamido) group showed an MIC > 200 µM.
(MIC = 29.83 µM) was significantly more potent than the non-
Compound 33 was then selected for further evaluation
fluoronated homolog 37 which showed no activity. This increase using Mtb H37Rv grown under aerobic conditions using a
in activity is not surprising in light of the work reported by fluorescent reporter strain Mtb H37Rv. The activity under
Wilson et al. which reported comparing compound 14 (Figure 2) aerobic conditions was confirmed and found to be 0.23 µM. The
who had already shown the importance of 1) 2- MBC of 33 was then assessed and found to be below 5 µM, this
(perfluorobenzamido) group at R2 position, 2) ethyl ester at R3 suggest that 33 is potentially bactericidal.
position and 3) 2-aminothiophene core.9
The antimicrobial activity of 33 against Mtb H37Rv grown
The significant finding of this study is the observation that a under hypoxic conditions was assessed using a LORA. The
NEt group in aliphatic ring at the R1 position of compound 33 purpose of the latter assay was to evaluate 33 on Mtb placed in
showed 40-fold increase in potency compared to
a state of non-replicating persistence (NRP). Bacteria were first
compound 38 using the MABA. The R = NEt group was not a adapted to low oxygen conditions and then exposed to 33 under
random selection; but rather, it designed to be isosteric with hypoxia; this is followed by a period of outgrowth in aerobic
compounds related to 12 (Figure 2). Early in the design phase conditions and growth is measured using luminescence. The
we were focused on thiophenes shown to inhibit Ag85s which MIC50 = 0.27 µM and MIC90 = 15 µM under LORA conditions
This journal is © The Royal Society of Chemistry 20xx
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Journal Name
indicated limited activity under low oxygen conditions (the gel (porosity 60Å, 230-400 mesh) from Sorbent TecVhienwoAlortigclieeOsn.l1inHe
13
MIC90 is 65 times higher than under aerobic conditions). NMR, C NMR, G-COSY and HMQC speDctOrIa: 1w0.1e0r3e9/cCa6rOriBe0d08o2u1Ft
However, rather than seeing this as a discouraging result, it is using a Bruker Avance III 600 spectrometer in Fourier
1
more valuable to use the data for the insight it provides into the transformation mode. H-NMR and 13C- NMR were referenced
mechanism of action. It has been reported by Franzblau et al. to residual CDCl3 peak at 7.27 ppm, and 77.16 ppm respectively.
that agents targeting the cell wall of Mtb (eg, INH, ethambutol, High resolution mass spectroscopy (HRMS) was performed on a
and four others evaluated) show greatly reduced activity micro mass Q-TOF2 instrument.
against NRP Mtb, whereas drugs hitting other cellular targets
have a range of activities.31 The authors view this result as carboxylate (thiophene cores: 22, 23, 25 and 26)
validation that 33 targets cell wall biosynthesis. The ethyl cyanoacetate (0.53 g, 4.71 mmol), sulfur (0.18 g, 5.68
General procedure for synthesis of 2-aminothiophene-3-
Mtb can escape the action of antibiotics by hiding inside of mmol), α-methylene carbonyl compound (1.0 g, 4.28 mmol)
macrophages. Therefore, it is important to evaluate the were suspended in EtOH (10.0 mL). Diethylamine (0.62 g, 8.57
intracellular activity of key compounds in a human macrophage- mmol) was added and the mixture was heated to 70 °C for 3 h.
like infection model. Compound 33 was active on Mtb infected After reaction was complete, EtOH was evaporated under
macrophage-like cells showing an IC90 = 9.75 µM. The data reduced pressure resulting in a brown solid, which was
indicated a loss of potency in comparison to INH which does not dissolved in CH2Cl2. The CH2Cl2 layer was washed with sat. NH4Cl
lose activity (based on IC90 values) in Mtb infected macrophage- (20.0 mL), deionized H20 (20.0 mL), and sat. NaCl (20.0 mL).
like cells. This data suggests compound permeability can be After drying with anhydrous NaSO4 and filtration, the solvent
further improved. The same human macrophage-like cell line was evaporated and the crude product was purified by
was also used to evaluate the cytotoxicity of compound 33. The crystallization from cold MeOH to obtain a lime yellow product.
assay revealed an IC50 = 6.2 µM for viability. This value is 27 Yield: 79% (1.21 g); silica gel TLC Rf = 0.50 (40% ethyl acetate in
times higher than the extracellular MIC and provides a potential hexanes).
starting point for optimizing the therapeutic window of the
compound class.
General procedure for 2-aminothophene-3-carboxylate
formation with 1-ethylpiperidin-4-one 24: The ethyl
Multidrug-resistant tuberculosis (MDR-TB) is growing cyanoacetate (4.89 g, 43.24 mmol), sulfur (1.66g, 51.10 mmol),
problem and is due to a resistance to INH and RIF, two of the and 1-ethylpiperidine (5.0 g, 39.31 mmol) were suspended in
most powerful antituberculosis drugs.36 Fluoroquinolones (FQs) EtOH (50.0 mL). Diethylamine (5.74 g, 78.62 mmol) was added
have emerged as the most important drugs in second-line and the mixture was heated to 70 °C for 12 h. After the reaction
treatment of MDR-TB.36 However, extensively drug-resistant TB was complete, the EtOH was evaporated under reduced
(XDR-TB) has emerged and is defined as MDR-TB with additional pressure to afford a brown solid. To the brown solid was added
resistance to fluoroquinolones and at least one of the injectable diethyl ether (30.0 mL) followed by sonication for 5 mins. The
drugs amikacin, capreomycin, and kanamycin. New ether layer was decanted and this process was repeated twice
antitubercular against should have activity against major and to remove unreacted starting material. The solid compound was
emerging drug resistant strains of Mtb. The panels of resistance dissolved in ethyl acetate and filtered. The filtrate was
strains evaluated includes INH-R1 which contains a mutation in concentrated under reduced pressure and crystallized from cold
the catalase-peroxidase enzyme KatG protein which activates MeOH to obtain brown crystals. Yield: 75% (7.52 g); silica gel TLC
38
the prodrug INH to its active form.37,
The RIF-R1 strain Rf = 0.50 (7% methanol in dichloromethane).
General procedure for 2-amidothiophene-3-carboxylate
contains a S522L mutation in the rpoB gene. Mutation in that
region of the selected region of the gene are responsible for compounds (Compounds labelled with 4{a,b,c} a = thiophene
most rifamycin resistance in Mtb. To cover XDR-TB the panel cores, b = ACs, and C = AAs, Figure 4)
contains FQ-R1 which has an amino acid substitution within the Compound 24 (115.0 mg, 0.452 mmol), was dissolved in dry
GyrB subunit of DNA gyrase.39 The most exciting findings of our CH2Cl2 (1.0 mL) and cooled to
0
°
C. N,N-
studies were that 33 is highly active against Mtb strains resistant (dimethylamino)pyridine (22.10 mg, 0.180 mmol), and Et3N
to these three classes of antitubercular agents.
(137.0 mg, 1.350 mmol) were added to the solution followed by
the dropwise addition of acid chloride (135.6 mg, 0.580 mmol).
The resulting solution was allowed to warm to room
temperature and allowed to stir for 12 h. After the reaction was
complete the CH2Cl2 was evaporated under reduced pressure to
obtain a crude solid which was purified by flash column
chromatography on silica gel (methanol:CH2Cl2) to obtain a lime
yellow product 33. Yield: 45% (101.19 mg); silica gel TLC Rf =
0.38 (10% methanol in CH2Cl2).
General procedure for deprotection of ethyl 2-
aminothiophene-3-carboxylate ester cores (Compounds 40 to
43 and 58 (exception compound 42))
The ethyl ester compounds (1.0g, 2.77 mmol) were dissolved in
THF:H2O:MeOH (3:1:1) to obtain a clear solution. LiOH (0.349 g,
Experimental
General methods: All starting materials were purchased from
Acros Organics or Sigma Aldrich. Unless specified all the
reactions were performed under nitrogen atmosphere.
Reactions were monitored by thin-layer chromatography (TLC,
silica gel 60 F254) and spots were observed by UV-lamp. All
solvents used for reactions were purchased from Sigma Aldrich
and Fisher Scientific, which were purified and dried by
distillation and other standard procedures. Solvents for flash
column chromatography were used as purchased. Purification
of compounds was performed by flash chromatography on silica
8 | J. Name., 2012, 00, 1-3
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ARTICLE
8.33 mmol) was added to the solution and then the solution was 9), 110.67 (C-3), 80.75 (C-19), 61.86 (C-15), 42.61 (C-8), 40.05
View Article Online
DOI: 10.1039/C6OB00821F
heated to 70 °C and maintained at that temperature for 12 h. (C-6), 28.50 (C-30, 29, 20), 26.53 (C-5) 13.87 (C-16); mass
The solution was concentrated to obtain a solid and then the spectrum (HRMS), m/z = 521.1091 (M+H)+ C22H21F5N2O5S
solid was taken up in water and extracted with Et2O (20.0 mL) 3 requires 521.1091 (M+H)+.
times. The aqueous layer was acidified to pH ~ 3-4 using 6N HCl.
6-(Tert-butyl)
3-ethyl
2-(3,5-
The acidified H2O layer was extracted with EtOAc (20.0 mL x 3). bis(trifluoromethyl)benzamido)-4,7-dihydrothieno[2,3-
The combined EtOAc extracts were dried over anhydrous c]pyridine-3,6(5H)-dicarboxylate (30):
Na2SO4, filtered and concentrated to obtain a solid which was Yield: 39% (134.0 g); silica gel TLC Rf = 0.82 (7 % methanol in
1
crystallized from CH2Cl2 and hexanes to obtain a crystalline CH2Cl2 ); H-NMR (600 MHz, CDCl3): δ 12.56 (s, 1H, H-10), 8.45
brown-yellow product 40. Yield: 69% (687.7 mg); silica gel TLC (s, 2H, H-22, 26), 8.11 (s, 1H, H-24), 4.57 (s, 2H, H-8), 4.43 (q, 2H,
Rf = 0.26 (40% ethyl acetate in hexanes).
General procedure for 3-carboxamides formation H-5), 1.51 (s, 9H, H-30, 33, 34), 1.43 (t, 3H, 3J16,15 7.15 Hz, H-16);
(Compound labelled with 44-53 and 59-63)
13C-NMR (150 MHz, CDCl3): δ 166.59 (C-2), 160.61 (C-11), 154.61
solution of carboxylic acid (2-amino-6-ethyl-4,5,6,7- (C-17), 147.66 (C-13), 134.55 (C-21), 132.59 (C-28,29), 127.71
tetrahydrothieno[2,3-c]pyridine-3-carboxylic acid, (42 400 mg, (C-22,26), 126.01 (C-24), 123.70 (C-9), 121.89 (C-25,23), 112.64
1.77 mmol) dissolved in CH2Cl2 (3.6 mL) was cooled to 0 C. A (C-3), 80.28 (C-29), 61.28 (C-15), 42.67 (C-8), 40.59 (C-6), 28.46
3J15,16 7.15 Hz, H-15), 3.70 (t, 2H, 3J6,5 5.69 Hz, H-6), 2.94 (t, 2H,
A
°
solution of the appropriate alkylamine (p-tolylmethanamine, (C-34, 33, 30), 26.54 (C-5), 14.32 (C-16); mass spectrum (HRMS),
236 mg, 1.95 mmol), and 1-(3-dimethylaminopropyl)-3- m/z = 589.1216 (M+Na)+ C24H24F6N2NaO5S requires 589.1202
ethylcarbodiimide hydrochloride (339 mg, 1.77 mmol), 1- (M+23)+.
hydroxybenzotriazole hydrate (271 mg, 1.77 mmol) and N,N-
Ethyl 2-benzamido-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-
(dimethylamino)pyridine (361 mg, 1.77 mmol) dissolved in c]pyridine-3-carboxylate (32)
CH2Cl2 (3.6 mL) was added. The solution was allowed to warm Yield: 35% (39.6 mg); silica gel TLC Rf = 0.35 (10 % methanol in
1
to room temperature and stirred for 18 h. The solution was CH2Cl2 ); H-NMR (600 MHz, CDCl3): δ 12.27 (s, 1H, H-10), 8.01
diluted with CH2Cl2 (15.0 mL), washed with brine (20.0 mL), and (m, 2H, H-20,24), 7.58 (m, 1H, H-22), 7.52 (m, 2H, H-21,23), 4.39
3
3
dried over anhydrous Na2SO4, and concentrated to obtain a (q, 2H, J17,18 7.15 Hz, H-17), 3.63 (s, 2H, H-8), 2.97 (t, 2H, J5,6
crude solid. The product was purified by flash column 5.87 Hz, H-5), 2.80 (t, J6,5 5.63 Hz, 2H, H-6), 2.66 (q, 2H, 3J15,16
3
chromatography (methanol:CH2Cl2) to obtain a light brown 6.97 Hz, H-15), 1.40 (t, 3H, 3J18,17 7.15 Hz, H-18), 1.20 (t, 3H, 3J16,15
product 46 Yield: 40% (235.6 mg); silica gel TLC Rf = 0.41 (9% 7.34 Hz, H-16); 13C-NMR (150 MHz, CDCl3): δ 166.86 (C-2),
methanol in DCM). Exception synthesis of compound 35: The 163.63 (C-11), 148.69 (C-13), 132.69 (C-19), 132.38 (C-22),
product 46 obtained by using above general procedure was 129.75 (C-4), 129.07 (C-23,21), 127.58 (C-24,20), 124.36 (C-9),
further reacted with 3,5-bis(trifluoromethyl)benzoyl chloride 111.58 (C-3), 60.86 (C-17), 51.69 (C-15), 51.10 (C-8), 50.24 (C-6),
using general procedure for 2-amidothiophene-3-carboxylate 26.94 (C-5), 14.46 (C-18), 12.56 (C-16); mass spectrum (HRMS),
compounds to yield product 35
6-(Tert-butyl) 3-ethyl 2-benzamido-4,7-dihydrothieno[2,3-
c]pyridine-3,6(5H)-dicarboxylate (28)
.
m/z = 359.1418 (M+H)+ C19H23N2O3S requires 359.1424 (M+H)+.
Ethyl 6-ethyl-2-(perfluorobenzamido)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridine-3-carboxylate (33)
Yield: 23% (61.5 mg); silica gel TLC Rf = 0.73 (7 % methanol in Yield: 16% (34.0 mg); silica gel TLC Rf = 0.38 (10 % methanol in
1
1
CH2Cl2 ); H-NMR (600 MHz, CDCl3): δ 12.30 (s, 1H, H-10), 8.03 CH2Cl2 ); H-NMR (600 MHz, CDCl3): δ 12.02 (s, 1H, H-10), 4.36
3
(m, 2H, H-22,25), 7.59 (m, 1H, H-24), 7.54 (m, 2H, H-23,25) 4.55 (q, 2H, J17,18 7.15 Hz, H-17), 3.65 (s, 2H, H-8), 2.97 (t, 2H, H-5)
(s, 2H, H-8), 4.40 (q, 2H, J15,16 7.15 Hz, H-15), 3.68 (t, 2H, J6,5 2.82 (t, 2H, H-6), 2.68 (q, 2H, 3J15,16 6.79 Hz, H-15), 1.38 (t, 3H,
3
3
5.69 Hz, H-6), 2.92 (s, 2H, H-5), 1.50 (s, 9H, H-20,29,30), 1.43 (t, J18,17 7.15 Hz, H-18), 1.22 (t, 3H, 3J16,15 7.15 Hz, H-16); 13C-NMR
3
3H, J16,15 7.15 Hz, H-16); 13C-NMR (150 MHz, CDCl3): δ 166.66 (150 MHz, CDCl3): δ 166.39 (C-2), 154.13 (C-11), 146.35 (C-13),
3
(C-2), 163.62 (C-11), 154.66 (C-17), 148.94 (C-13), 132.69 (C-24), 145.14 (1J22,F 256.39 Hz, C-22), 143.27 (md, J22,F 259.69 Hz, C-
1
1
130.08 (C-4), 129.01 (C-25, 23), 127.50 (C-26, 22), 123.62 (C-9), 20,24), 137.99 (md, J22,F 255.29 Hz, C-23,21), 130.13 (C-4),
111.50 (C-3), 80.17 (C-19), 60.90 (C-15), 42.89 (C-8), 40.60 (C-6), 125.47 (C-9), 112.97(C-3), 109.77 (C-19), 81.18 (C-17), 51.65 (C-
28.47 (C-30, 29, 20), 26.58 (C-5), 14.36 (C-16); mass spectrum 15), 50.96 (C-8), 50.10 (C-6), 26.78 (C-5), 14.37 (C-18), 12.48 (C-
(HRMS), m/z = 453.1358 (M+23)+ C22H26N2NaO5S requires 16); mass spectrum (HRMS), m/z
=
449.0971 (M+H)+
453.1458 (M+23)+.
C19H18F5N2O3S requires 449.0953 M+H)+; Anal calcd for
6-(Tert-butyl)
3-ethyl
2-(perfluorobenzamido)-4,7- C19H18F5N2O3S C, 50.89; H, 3.82; N, 6.25; found: C, 51.11; H,
dihydrothieno[2,3-c]pyridine-3,6(5H)-dicarboxylate (29):
3.36; N, 6.48.
Yield: 82% (263.0 g); silica gel TLC Rf = 0.89 (7 % methanol in
CH2Cl2 ); H-NMR (600 MHz, CDCl3): δ 12.55 (s, 1H, H-10), 4.56 4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxylate (34)
(s, 2H, H-8), 4.33 (q, 2H, 3J15,16 7.15 Hz, H-15), 3.66 (t, 2H, 3J6,5 5.5 Yield: 43% (168.9 mg); silica gel TLC Rf = 0.41 (10 % methanol in
Hz, H-6), 2.92 (t, 2H, H-5), 1.49 (s, 9H, H-30,29,20), 1.32 (t, 3H, CH2Cl2 ); H-NMR (600 MHz, CDCl3): δ 12.53 (s, 1H, H-10), 8.45
3J16,15 7.15 Hz, H-16); 13C-NMR (150 MHz, CDCl3): δ 160.76 (C-2), (s, 2H, H-20, 24), 8.10 (s, 2H, H-22), 4.43 (q, 2H, J17,18 7.15 Hz,
Ethyl
2-(3,5-bis(trifluoromethyl)benzamido)-6-ethyl-
1
1
3
159.62 (C-11), 154.44 (C-17), 143.44 (1J24,F 251.99 Hz, C-24), H-17), 3.76 (s, 2H, H-8), 3.05 (t, 2H, H-5) 2.92 (t, 2H, H-6), 2.77
143.17 (1J26,22-F 259.69 Hz, C-26, 22), 137.65 (1J25,23,F 256.39 Hz, (q, 2H, H-15), 1.42 (t, 3H, 3J18,17 7.15 Hz, H-18), 1.29 (t, 3H, H-16);
C-25,23), 136.94 (C-13), 135.38 (C-21), 130.72 (C-4), 125.29 (C- 13C-NMR (150 MHz, CDCl3): δ 166.67 (C-2), 160.73 (C-11), 147.67
This journal is © The Royal Society of Chemistry 20xx
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1
(C-13), 134.69 (C-19), 132.70 (C-28, 29), 120.01 (C-4), 127.83 (C- 20), 138.05 (d, JC,F 255.29 Hz, C-21, 23), 135.34 (C-13), 133.45
View Article Online
DOI: 10.1039/C6OB00821F
20, 24), 126.10 (C-22), 123.83 (C-9), 122.02 (C-23, 21) 112.67 (C- (C-5), 129.19 (C-15, 17), 127.97 (C-16), 125.73 (C-14, 18), 119.51
3), 61.37 (C-17), 51.55 (C-15), 50.69 (C-8), 49.96 (C-6), 26.14 (C- (C-14), 115.59 (C-3), 109.44 (C-19), 61.10 (C-11), 14.46 (C-12);
5), 14.45 (C-18), 12.13 (C-16); mass spectrum (HRMS), m/z = mass spectrum (HRMS), m/z = 464.0352 (M+H)+ C20H12F5NO3S
517.0994 (M+H)+ C21H20F6N2O3S requires 517.0991 (M+H)+.
2-(3,5-Bis(trifluoromethyl)benzamido)-6-ethyl-N-(4-
methylbenzyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-
carboxamide (35)
requires 464.0350 (M+H)+.
Ethyl
2-(3,5-bis(trifluoromethyl)benzamido)-5-
phenylthiophene-3-carboxylate (57)
Yield: 67% (66.8 mg); silica gel TLC Rf = 0. (30% ethyl acetate in
1
Yield: 22% (18.0 mg); silica gel TLC Rf = 0.55 (10 % methanol in hexanes); H-NMR (600 MHz, CDCl3): δ 12.27 (s, 1H, H-6), 8.47
1
CH2Cl2 ); H-NMR (600 MHz, CDCl3): δ 13.65 (s, 1H, H-10), 8.47 (s, 2H, H-24, 20), 8.12 (s, 2H, H-22), 7.64 (dd, 2H, 3J14/18,15/17 8.25
(s, 2H, H-22,18), 8.08 (s, 1H, H-20), 7.23 (d, 2H, J33,35 6 Hz, H- Hz, 3J14/18,16 1.28 Hz, H-14, 18), 7.50 (s, 1H, H-4), 7.42 (m, 2H, H-
3
33,28), 7.19 (d, 2H, J35,33 6Hz, H-35,29), 6.23 (s, 1H, H-23), 4.63 15, 17), 7.32 (m, 1H, H-16), 4.46 (q, 2H, J11,12 7.15 Hz, H-11),
(d, 2H, J26,23 5Hz, H-26), 3.71 (s, 2H, H-8), 2.87 (m, 4H, H-5,6), 1.46 (t, 3H, 3J12,11 7.15 Hz, H-12); 13C-NMR (150 MHz, CDCl3): δ
2.70 (q, 2H, J15,16 6 Hz, H-15), 2.36 (s, 3H, H-34), 1.23 (t, 3H, J16,15 166.06 (C-2), 160.75 (C-7), 147.32 (C-9), 135.18 (C-19), 134.55
6Hz, H-16); 13C-NMR (150 MHz, CDCl3): δ 166.20 (C-2), 160.74(C- (C-13), 133.55 (C-5), 133.22-132.54 (q, 2JC,F 34.11 Hz, C-23, 21),
13), 145.91 (C-11), 136.03(C-30), 134.67(C-17, 27), 132.52 (C- 129.20 (C-17, 15), 127.95 (C-16), 127.84 (3JC,F 3.30 Hz, C-24, 20),
19, 21), 129.75 (C-35, 29), 127.78 (C-22, 18), 126.13(C-4), 126.21 (C-22), 125.73 (C-14, 18), 122.92 (d, 1JC,F 272.90 Hz, C-28,
125.84(C-20), 123.90, 120.29(C-9), 114.65(C-3), 51.74(C-15), 29), 119.56 (C-4), 115.37 (C-3), 61.49 (C-11), 14.53 (C-12); mass
51.10(C-8), 49.93(C-6), 43.64 (C-26), 26.96 (C-5), 21.27 (C-34), spectrum (HRMS), m/z = 488.0756 (M+H)+ C22H15F6NO3S
12.35 (C-16); mass spectrum (HRMS), m/z = 592.1467 (M+23)+ requires 488.0750 (M+23)+.
C27H25F6N3NaO2S requires 592.1464 (M+23)+.
Ethyl 2-(perfluorobenzamido)-4,5,6,7- tetrahydrothieno[2,3-c]pyridine-3-carboxylic acid (40)
tetrahydrobenzo[b]thiophene-3-carboxylate (38) Yield: 69% (617.0 mg); silica gel TLC Rf = 0.55 (7 % methanol in
2-Amino-6-((benzyloxy)carbonyl)-4,5,6,7-
Yield: 50.5% (188.0 mg); silica gel TLC Rf = 0.86 (7 % methanol CH2Cl2 ); 1H-NMR (600 MHz, CDCl3): δ 7.37 (m, 4H, H-18, 17, 21,
in CH2Cl2 ); 1H-NMR (600 MHz, CDCl3): δ 12.05 (s, 1H, H-10), 4.35 20), 7.32-7.31 (m, 1H, H-19), 5.10 (s, 2H, H-15), 4.31 (m, 2H, H-
(q, 2H, 3J15,16 7.14 Hz, H-15), 2.80 (t, 2H, 3J8,7 6.06 Hz H-8), 2.70 8), 3.59 (s, 2H, H-6), 2.68 (s, 2H, H-5); 13C-NMR (150 MHz, CDCl3):
3
(t, 2H, J5,6 5.94 Hz, H-5), 1.85-1.78 (m, 4H, H-6,7), 1.80 (t, 3H, δ 166.49 (C-2), 163.51 (C-11), 154.51 (C-13), 136.86 (C-9),
3J16,15 7.1,4 H-16); 13C-NMR (150 MHz, CDCl3): δ 166.76 (C-2), 130.75 (C-4), 128.43 (C-18, 20), 127.86 (C-21, 17), 127.55 (C-19),
153.97 (C-11), 145.11 (md, 1J20,F 253.09 Hz, C-20), 145.81 (C-13), 111.56 (C-3), 42.47 (C-15), 41.13 (C-8), 40.73 (C-6), 26.77 (C-5);
1
1
143.14 (md, J18,22,F 259.69 Hz, C-18, 22), 137.98 (md, J19,21,F mass spectrum (HRMS), m/z = 355.0732 (M+23)+ C16H16N2O4S
254.19 Hz, C-19, 21), 131.57 (C-4), 128.43 (C-9), 113.47 (C-17), requires 355.0723 (M+23)+.
110.10 (C-3), 61.03 (C-15), 26.48 (C-8), 24.56 (C-5), 23.04(C-7),
22.88 (C-6), 14.39 (C-16); mass spectrum (HRMS), m/z = 3-carboxylic acid (42)
420.0690 (M+H)+ C18H14F5NO3S requires 420.0687 (M+H)+.
The ethyl ester compound 24 (1.18g, 4.64 mmol) was dissolved
2-(3,5-bis(trifluoromethyl)benzaimdo)-4,5,6,7- in 10.0 mL of THF:H2O:MeOH (3:1:1) to obtain a clear solution.
2-Amino-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-
Ethyl
tetrahydrobenzo[b]thiophene-3-carboxylate (39)
LiOH (0.779 g, 18.58 mmol) was added to the solution and the
Yield: 14.5% (60.0 mg); silica gel TLC Rf = 0.86 (7 % methanol in solution was heated to 70
°C and maintained at that
1
CH2Cl2 ); H-NMR (600 MHz, CDCl3): δ 12.58 (s, 1H, H-10), 8.44 temperature for 12 h. The solution was concentrated to obtain
(s, 2H, H-18,22), 8.08 (s, 1H, H-20), 4.40 (q, 2H, 3J15,16 7.14 Hz, H- a solid. The solid was taken in Et2O (20.0 mL) and the solution
15), 2.82 (t, 2H, 3J8,7 6.06 Hz, H-8), 2.71 (t, 2H, 3J5,6 5.82 Hz, H-5), was subjected to sonication for 5 mins. The Et2O layer was
1.84-1.81 (m, 4H, H-6, 7), 1.42 (t, 3H, 3J16,15 7.14, H-16); 13C-NMR decanted and this process was repeated two times. The solid
(150 MHz, CDCl3): δ 167.11 (C-2), 160.52 (C-11), 146.88 (C-13), obtained was dissolved in EtOAc and the mixture was filtered to
135.03 (C-17), 132.71 (q, 2C, 1JC,F 132 C-26, 27), 131.60 (C-4), remove LiOH. The filtrate was concentrated and the solid was
128.23 (C-9), 127.50 (d, 2C, 3JC,F 12.06 C-22, 18), 125.89 (m, C- crystallized from methanol to a pure brownish-yellow product
20), 123.87 ( C-19), 122.06 (C-21), 113.21 (C-3), 61.10 (C-15), 42. Yield: 66% (883.1 mg); silica gel TLC Rf = 0.14 (10 % methanol
1
26.49 (C-8), 24.60 (C-5), 23.06 (C-7), 22.88 (C-6), 14.44 (C16); in CH2Cl2); H-NMR (150 MHz, DMSO-d6): δ 6.80 (s, 1H, H-10),
mass spectrum (HRMS), m/z = 466.0833 (M+H)+ C20H17F6NO3S 3.25 (s, 2H, H-8), 2.75 (m, 2H, H-6), 2.52 (m, 2H, H-5), 2.43 (q,
requires 466.0906 (M+H)+.
Ethyl
carboxylate (56)
2H, J13,14 6Hz, H-13), 1.03 (t, 3H, J14,13 6Hz, H-14); 13C-NMR (150
2-(perfluorobenzamido)-5-phenylthiophene-3- MHz, DMSO-d6): δ 171.23 (C-2), 157.27 (C-11), 133.51 (C-9),
112.59 (C-4), 111.66 (C-3), 51.86 (C-8), 51.48 (C-6), 51.05 (C-13),
Yield: 45% (161 mg); silica gel TLC Rf = 0. (30% ethyl acetate in 28.25 (C-5), 12.95 (C-14); mass spectrum (ESI), m/z = 249.07
1
hexanes); H-NMR (600 MHz, CDCl3): δ 11.81 (s, 1H, H-6), 7.62 (M+23)+ C10H14N2O2S requires 249.06 (M+23)+.
3
3
(dd, 2H, J14/18,15/17 8.44 Hz, J14/18,16 1.28 Hz, H-14, 18), 7.48 (s,
2-Amino-N-butyl-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-
1H, H-4), 7.41 (m, 2H, H-15, 17), 7.32 (m, 1H, H-16), 4.41 (q, 2H, c]pyridine-3-carboxamide (44)
3J11,12 7.15 Hz, H-11), 1.43 (t, 3H, 3J12,11 7.15 Hz, H-12); 13C-NMR Yield: 59% (140.0 mg); silica gel TLC Rf = 0.59 (7 % methanol in
(150 MHz, CDCl3): δ 165.65 (C-2), 154.28 (C-7), 146.21 (C-9), CH2Cl2 ); 1H-NMR (600 MHz, CDCl3): δ 5.96 (s, 2H, H-10), 5.59 (s,
145.29 (d, 1JC,F 256.39 Hz, C-22), 143.68 (d, 1JC,F 260.79 Hz, C- 24, 1H, H-15), 3.51 (s, 2H, H-8), 3.36 (q, 2H, H-16), 2.81 (m, 4H, H-5,
10 | J. Name., 2012, 00, 1-3
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6), 2.65 (q, 2H, 3J13,14 7.14 Hz, H-13), 1.54 (quin, 2H, 3J17,16,18 7.14 (C-17, 21, 22), 36.43 (C-19, 24, 25), 29.49 (C-18, 20, 23), 27.41
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Hz, H-17), 1.38 (sex, 2H, 3J18,17,19 7.5 Hz H-18), 1.20 (t, 3H, 3J14,13 (C-5), 27.41 (C-5), 12.34 (C-14); mass spectrum (HRMS), m/z =
DOI: 10.1039/C6OB00821F
7.14 Hz H-14), 0.94 (t, 3H, 3J19,18 7.5 Hz, H-19); 13C-NMR (150 360.2104 (M+H)+ C20H30N3OS requires 360.2104 (M+23)+.
MHz, CDCl3): δ 166.88 (C-2), 166.66 (C-11), 147.65 (C-13),
2-Amino-N-butyl-4,5,6,7-tetrahydrobenzo[b]thiophene-3-
130.65 (C-4), 126.59 (C-9), 111.23 (C-3), 60.46 (C-15), 26.39 (C- carboxamide (49)
8), 24.36 (C-5), 23.74 (C-17), 23.00 (C-7), 22.88 (C-6), 14.34 (C- Yield: 16.13% (45.0 mg); silica gel TLC Rf = 0.45 (30 % ethyl
16); mass spectrum (HRMS), m/z
=
282.1647 (M+H)+ acetate in hexanes); 1H-NMR (600 MHz, CDCl3): δ 5.66 (s, 1H, H-
C14H24N3O3S requires 282.1635 (M+23)+.
13), 3.36 (m, 2H, H-14), 2.60 (m, 2H, H-8), 2.53 (m, 2H, H-5), 1.80
3
2-Amino-N-benzyl-6-ethyl-4,5,6,7-tetrahydrothieno[2,3-
c]pyridine-3-carboxamide (45)
(m, 4H, H-6,7), 1.54 (m, 2H, H-15), 1.38 (sex, 2H, J16,17 7.38 Hz,
3
H-16), 0.94 (t, 3H, J17,16 7.38 H-17); 13C-NMR (150 MHz, CDCl3):
Yield: 59% (140.0 mg); silica gel TLC Rf = 0.57 (7 % methanol in δ 166.53 (C-2), 158.53 (C-11), 128.80 (C-9), 118.90 (C-4), 108.95
1
CH2Cl2 ); H-NMR (600 MHz, CDCl3): δ 7.36-7.28 (m, 5H, H-18, (C-3), 38.92 (C-14), 31.88 (C-15), 27.18 (C-8), 24.53 (C-5), 22.99
19, 20, 21, 22), 6.04 (s, 2H, H-10), 5.91 (s, 1H, H-15), 4.59 (d, 2H, (C-7), 22.90 (C-6), 20.29 (C-16), 13.81 (C-17); mass spectrum
3J16,15 5.58 Hz, H-16), 3.46 (s, 2H, H-8), 2.74 (s, 4H, H-6, 5), 2.58 (HRMS), m/z = 275.1194 (M+23)+ C13H20N2OS requires 275.1194
(q, 2H, 3J13,14 7.14, H-13), 1.16 (t, 3H, 3J14,13 7.14, H-14) ; 13C-NMR (M+23)+.
(150 MHz, CDCl3): δ 166.26 (C-2), 159.70 (C-11), 138.69 (C-9),
2-Amino-N-(4-methylbenzyl)-4,5,6,7-
128.76 (C-21, 19), 127.45 (C-18, 20, 22, 4), 116.34 (C-17), 108.52 tetrahydrobenzo[b]thiophene-3-carboxamide (51)
(C-3), 51.56 (C-16), 51.27 (C-8), 50.08 (C-6), 43.20 (C-13), 27.73 Yield: 25% (83.0 mg); silica gel TLC Rf = 0.46 (7 % methanol in
(C-5), 12.58 (C-14); mass spectrum (HRMS), m/z = 316.1486 CH2Cl2 ); 1H-NMR (600 MHz, CDCl3): δ 7.22 (d, 2H, 3J17,16 7.98 Hz,
3
(M+H)+ C17H21N3OS requires 316.1478 (M+23)+.
2-Amino-6-ethyl-N-(4-methylbenzyl)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridine-3-carboxamide (46)
H-17, 19), 7.15 (d, 2H, J16,17 7.86 Hz, H-16, 20), 5.95 (s, 1H, H-
13), 4.54 (d, 2H, 3J14,13 5.46 Hz, H-14), 2.58 (m, 2H, H-8), 2.54 (m,
2H, H-5), 2.34 (s, 3H, H-21), 1.77 (m, 4H, H-6, 7); 13C-NMR (150
Yield: 40% (235.6 mg); silica gel TLC Rf = 0.55 (7 % methanol in MHz, CDCl3): δ 166.38 (C-2), 158.62 (C-11), 137.03 (C-9), 135.65
CH2Cl2 ); 1H-NMR (600 MHz, CDCl3): δ7.20 (m, 2H, H-21,19), 7.16 (C-18), 129.40 (C-19, 17), 128.82 (C-5), 127.56 (C-16, 20), 119.21
(m, 2H, H-22,18), 6.03 (s, 2H, H-10), 5.87 (s, 1H, H-15), 4.53 (d, (C-4), 108.90 (C-3), 43.04 (C-14), 27.21 (C-8), 24.55 (C-5), 22.92
2H, J16,15 6Hz, H-16), 3.50 (s, 2H, H8), 2.75 (m, 4H, H-5,6), 2.61 (C-7), 22.85 (C-6), 21.12 (C-21); mass spectrum (HRMS), m/z =
(q, 2H, J13,14 6 Hz H-13), 2.35 (s, 3H, H-23), 1.17 (t, 2H, J14,13 6 Hz 301.1377 (M+H)+ C17H21N2OS requires 301.1369 (M+H)+.
H-14); 13C-NMR (150 MHz, CDCl3): δ 166.19 (C-11), 159.63 (C-2),
2-Amino-N-(2-methoxybenzyl)-4,5,6,7-
137.08 (C-20, 9), 135.57 (C-17), 129.43 (C-21, 19), 127.49 (C-22, tetrahydrobenzo[b]thiophene-3-carboxamide (52)
18), 116.01 (C-4), 51.52 (C-8), 51.17(C-6), 50.02 (C-13), 43.03 (C- Yield: 12% (40.0 mg); silica gel TLC Rf = 0.35 (30 % ethyl acetate-
1
16), 27.54 (C-23), 21.13(C-5), 12.47 (C-14); mass spectrum hexanes); H-NMR (600 MHz, CDCl3): δ 7.31-7.25 (m, 2H, H-
(HRMS), m/z = 352.1460 (M+23)+ C18H23N3OS requires 352.1460 17,20), 6.94-6.88 (m, 2H, H-19,18), 6.36 (s, 1H, H-13), 4.55 (d,
(M+23)+
2-Amino-6-ethyl-N-(2-methoxybenzyl)-4,5,6,7-
tetrahydrothieno[2,3-c]pyridine-3-carboxamide (47)
2H, 3J14,13 5.58 Hz, H-14), 3.88 (s, 3H, H-22), 2.60 (m, 2H, H-5),
2.53 (m, 2H, H-8), 1.79 (m, 4H, H-6,7); 13C-NMR (150 MHz,
CDCl3): δ 166.19 (C-2), 158.75 (C-11), 157.53 (C-16), 129.67 (C-
Yield: 59% (140.0 mg); silica gel TLC Rf = 0.55 (10% methanol in 9), 128.95 (C-20), 128.71 (C-18), 126.75 (C-15), 120.77 (C-4),
1
CH2Cl2 ); H-NMR (600 MHz, CDCl3): δ 7.30-7.25 (m, 2H, H-20, 118.73 (C-19), 110.17 (C-17), 108.92 (C-3), 55.20 (C-22), 39.25
19), 6.92 (t, 1H, 3J21,20 7.44 Hz, H-21), 6.87 (d, 1H, 3J22,21 8.22 H- (C-14), 27.01 (C-8), 24.54 (C-5), 23.04 (C-7), 22.92 (C-6); mass
22), 6.29 (s, 1H, H-15), 6.01 (s, 2H, H-10), 4.54 (d, 2H, 3J16,15 5.88 spectrum (HRMS), m/z = 317.1316 (M+H)+ C17H21N2O2S requires
Hz, H-16), 3.87 (s, 3H, H-24), 3.47 (s, 2H, H-8), 2.76 (s, 4H, H-6, 317.1316 (M+H)+.
5), 2.61 (q, 2H, 3J13,14 7.14 Hz, H-13), 1.18 (t, 3H, 3J14,13 7.14 Hz,
N-(Adamantan-1-yl)-2-amino-4,5,6,7-
H-14); 13C-NMR (150 MHz, CDCl3): δ 165.96 (C-2), 159.32 (C-11), tetrahydrobenzo[b]thiophene-3-carboxamide (53)
157.55 (C-13), 129.66 (C-4), 128.76 (C-9), 127.60 (C-22), 126.66 Yield: 13% (45.0 mg); silica gel TLC Rf = 0.53 (30 % ethyl acetate-
1
(C-20), 120.76 (C-21), 110.20 (C-3), 108.52 (C-19), 55.28 (C-24), hexanes); H-NMR (600 MHz, CDCl3): δ 5.43 (s, 1H, H-13), 2.58
51.59 (C-16), 51.07 (C-8), 50.20 (C-6), 39.34 (C-13), 27.54 (C-5), (m, 2H, H-8), 2.53 (m, 2H, H-5), 2.05 (m, 9H, H-15, 16, 18, 19, 20,
12.38 (C-14); mass spectrum (HRMS), m/z = 346.1587 (M+H)+ 21), 1.78 (m, 4H, H-6, 7), 1.70 (m, 6H, H-17, 22, 23), 13C-NMR
C18H24N3O2S requires 346.1584 (M+H)+.
N-(Adamantan-1-yl)-2-amino-6-ethyl-4,5,6,7-
tetrahydrothieno[2,3-c]pyridine-3-carboxamide (48)
(150 MHz, CDCl3): δ 165.89 (C-2), 157.87 (C-11), 128.84 (C-9),
118.95 (C-4), 110.31 (C-3), 51.94 (C-14), 42.19 (C-15, 19, 20),
36.47 (17, 23, 22), 29.52 (C-16, 18, 21), 27.32 (C-8), 24.57 (C-5),
Yield: 59% (140.0 mg); silica gel TLC Rf = 0.7 (10 % methanol in 23.09 (C-7), 22.94 (C-6); mass spectrum (HRMS), m/z = 331.1843
CH2Cl2 ); 1H-NMR (600 MHz, CDCl3): δ 5.84 (s, 2H, H-10), 5.37 (s, (M+H)+ C19H27N2OS requires 331.1839 (M+23)+.
3
1H, H-15), 3.50 (s, 2H, H-8), 2.78 (d, 2H, J6,5 4.8 Hz, H-6), 2.75
2-Amino-N-benzyl-5-phenylthiophene-3-carboxamide (60)
(d, 2H, 3J5,6 4.8 Hz, H-5), 2.63 (q, 2H, 3J13,14 7.14 Hz, H-13), 2.09- Yield: 59% (166.4 mg); silica gel TLC Rf = 0.33 (30% ethyl acetate
2.06 (m, 10H, H-17, 22, 21, 18, 23, 20), 1.70-1.68 (m, 5H, H-19, in hexanes); 1H-NMR (600 MHz, CDCl3): δ 7.41 (dd, 2H,
24, 25), 1.86 (t, 3H, 3J14,13 7.14 H-14); 13C-NMR (150 MHz, CDCl3): 3J10/14,11/13 8.25 Hz, 3J10/14,12 1.28 Hz, H-10, 14), 7.37 (m, 4H, H-21,
δ 165.61 (C-2), 158.42 (C-11), 127.51 (C-4), 115.70 (C-9), 109.95 19, 22, 18), 7.32 (m, 3H, H-12, 11, 13), 7.21 (m, 1H, H-20), 6.95
3
(C-3), 51.99 (C-13), 51.50 (C-8), 51.09 (C-6), 50.08 (C-16), 42.18 (s, 1H, H-4), 6.04 (s, 1H, H-15), 4.60 (d, 2H, J16,15NH 5.69 Hz, H-
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16); 13C-NMR (150 MHz, CDCl3): δ 165.73 (C-2), 160.65 (C-7), to each well and then the plate was incubated overnight
View Article Online
DOI: 10.1039/C6OB00821F
138.68 (C-5), 133.99 (C-17), 128.99 (C-11, 13), 128.90 (C-21, 19), (Alamar blue final concentration 0.001%). On day seven, the
127.97 (C-10, 14), 127.66 (C-12), 126.81 (C-20), 124.79 (C-22, last well that showed no sign of growth and remained blue was
18), 118.08 (C-4), 109.56 (C-3), 43.42 (C-16); mass spectrum considered the MIC for that compound. Standard calculations
(HRMS), m/z = 331.0876 (M+23)+ C18H16N2OS requires 331.0876 were used to calculate the IC50 for the compounds.
(M+23)+.
2-Amino-N-(4-methylbenzyl)-5-phenylthiophene-3-
carboxamide (61)
In vitro dual readout MIC assay
The MIC of compound was determined by measuring bacterial
growth after 5 d in the presence of test compounds.
Yield: 57% (167 mg); silica gel TLC Rf = 0.35 (30% ethyl acetate Compounds were prepared as 10-point two-fold serial dilutions
in hexanes); 1H-NMR (600 MHz, CDCl3): δ 7.41 (dd, 2H, in DMSO and diluted into 7H9-Tw-OADC medium in 96-well
3J10/14,11/13 8.44 Hz, 3J10/14,12 1.10 Hz, H-10, 14), 7.32 (m, 2H, H-11, plates with a final DMSO concentration of 2%. The highest
13), 7.27 (d, 2H, H-22, 18), 7.22 (m, 1H, H-12), 7.19 (d, 2H, H-19, concentration of compound was 200 µM where compounds
21), 6.92 (s, 1H, H-4), 2.36 (s, 3H, H-23); 13C-NMR (150 MHz, were soluble in DMSO at 10 mM. Each plate included assay
CDCl3): δ 165.55 (C-7), 160.45 (C-2), 137.31 (C-5), 135.47 (C-20), controls for background (medium/DMSO only, no bacterial
133.89 (C-17), 129.46 (C-11, 13), 128.87 (C-21, 19), 127.92 (C- cells), zero growth (100 µM rifampicin) and maximum growth
9), 125.40 (C-12), 124.66 (C-10, 14), 117.94 (C-4), 117.94 (C-4), (DMSO only), as well as a rifampicin dose response curve. Plates
109.51 (C-3) 43.11 (C-16), 21.14 (C-23); mass spectrum (HRMS), were inoculated with M. tuberculosis and incubated for 5 days;
m/z = 323.1213 (M+H)+ C19H18N2OS requires 323.1227 (M+23)+. growth was measured by OD590. To calculate the MIC, the 10-
2-Amino-N-(2-methoxybenzyl)-5-phenylthiophene-3-
carboxamide (62)
point dose response curve was plotted as % growth and fitted
to the Gompertz model using GraphPad Prism 5. The MIC was
Yield: 68% (210.9 mg); silica gel TLC Rf = 0.3 (30% ethyl acetate defined as the minimum concentration at which growth was
in hexanes); 1H-NMR (600 MHz, CDCl3): δ 7.41 (dd, 2H, completely inhibited and was calculated from the inflection
3J10/14,11/13 8.44 Hz, 3J10/14,12 1.10 Hz, H-10, 14), 7.35-7.31 (m, 3H, point of the fitted curve to the lower asymptote (zero growth).
H-11, 12, 13), 7.29 (m, 1H, H-18), 7.21 (m, 1H, H-19), 6.97-6.94 In addition dose response curves were generated using the
(m, 2H, H-20, 4), 6.92 (d, 1H, , 3J21,20 8.25 Hz, H-21), 6.29 (s, 1H, Levenberg-Marquardt algorithm and the concentrations that
3
H-15), 4.59 (d, 2H, , J16,15NH 2.75 Hz, H-16), 3.90 (s, 3H, H-23); resulted in 50% and 90% inhibition of growth were determined
13C-NMR (150 MHz, CDCl3): δ 165.53 (C-7), 157.73 (C-2), 133.99 (IC50 and IC90 respectively)
(C-22), 129.88 (C-9), 129.02 (C-5), 128.99 (C-11, 13), 126.89 (C-
MIC Under Low Oxygen (LORA Assay)
12), 126.48 (C-17, 19), 124.88 (C-10, 14), 120.88 (C-18, 20) Test compounds were prepared as 20-point two-fold serial
118.21 (C-4), 111.13 (C-21) 110.54 (C-3), 55.56 (C-16), 39.24 (C- dilutions in DMSO and diluted into Dubos-Tw-Albumin (DTA)
23); mass spectrum (HRMS), m/z
C19H18N2O2S requires 339.1162 (M+H)+.
=
339.1159 (M+H)+ medium in 96-well plates with a final DMSO concentration of
2%. The highest concentration of compound was 200 µM where
N-(adamantan-1-yl)-2-amino-5-phenylthiophene-3-
carboxamide (63)
compounds were soluble in DMSO at 10 mM. Control
compounds (metronidazole and rifampicin) were prepared as
Yield: 44% (140.4 mg); silica gel TLC Rf = 0.62 (30% ethyl acetate two-fold serial dilutions in DMSO and diluted into DTA medium
in hexanes); 1H-NMR (600 MHz, CDCl3): δ 7.44 (dd, 2H, in 96-well plates with a final DMSO concentration of 2%. M.
3J10/14,11/13 8.44 Hz, 3J10/14,12 1.28 Hz, H-10, 14), 7.33 (m, 3H, H-13, tuberculosis constitutively expressing the luxABCDE operon was
11), 7.22 (m, 1H, H-12), 6.90 (s, 1H, H-4), 2.16 (s, 9H, H-23, 20, inoculated into DTA medium in gas-impermeable glass tubes
21, 18, 17, 22), 1.76 (m, 6H, H-25, 19, 24); 13C-NMR (150 MHz, and incubated for 18 days to generate hypoxic conditions
CDCl3): δ 165.44 (C-7), 158.84 (C-2), 134.07 (C-5), 128.97 (C-11, (Wayne model of hypoxia). At this point, bacteria are in a non-
13), 126.81 (C-12), 125.82 (C-9), 124.85 (C-10, 14), 118.39 (C-4), replicating state (NRP stage 2) induced by oxygen depletion.
111.64 (C-3), 52.27 (C-16), 42.16 (C-20, 21, 22, 17), 36.53 (C-25, Oxygen-deprived bacteria were inoculated into compound
19, 24), 29.65 (18, 23); mass spectrum (HRMS), m/z = 353.1697 assay plates and incubated under anaerobic conditions for 10
(M+H)+ C21H25N2OS requires 353.1682 (M+H)+.
days followed by incubation under aerobic conditions
(outgrowth) for 28 h. Growth was measured by luminescence.
In vitro Mtb Alamar Blue assay (MABA)
Mtb H37Rv is a drug sensitive laboratory reference strain used Oxygen-deprived bacteria were also inoculated into compound
in the MIC studies.40 The bacteria were grown at 37 °C in Difco™ assay plates and incubated under aerobic conditions for 5 days.
7H9 Middlebrook liquid media (BD Biosciences, 271310) Growth was measured by luminescence. Rifampicin was
supplemented with 10% Middlebrook OADC Enrichment , 0.05% included in each plate and metronidazole was included in each
Tween (G-Biosciences, 786-519) and 0.2% Glycerol. A modified run as positive controls for aerobic and anaerobic killing of M.
96-well microplate Alamar Blue assay (MABA) was used to tuberculosis, respectively.
establish MIC values.41 Briefly, compounds were solubilized in
Minimum bactericidal concentration (MBC)
DMSO and were two-fold serially diluted in 7H9 media (DMSO Mtb was grown aerobically to logarithmic phase and inoculated
< or equal to 0.02% DMSO). Bacteria were grown to mid-log into liquid 7H9-Tw-OADC medium containing four different
growth, diluted and added to the wells with compounds. Cell compound concentrations with a final maximum concentration
and media controls were included in each plate. The plates of 2% DMSO. Compounds were tested at fixed concentrations,
were incubated at 37 ° for 6 days when Alamar blue was added as no MIC data were available at the time. (200, 100, 20 and 5
12 | J. Name., 2012, 00, 1-3
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µM). Cultures were exposed to compounds for 21 days and cell described.12 Total lipids, FAMEs and MAMEs were analyzed by
View Article Online
DOI: 10.1039/C6OB00821F
viability measured by enumerating colony forming units on agar TLC and revealed by autoradiography. The MIC of 33 against
plates on day 0, 7, 14 and 21. MBC was defined as the minimum Mtb H37Rv mc26206 grown in 7H9-OADC-Tyloxapol medium
concentration required to achieve a 2-log kill in 21 days. For was determined as 0.4 μg/ml.
compounds with > 1-log kill, an assessment of time- and/or
concentration-dependence was determined from the kill
kinetics. DMSO was used as a positive control for growth.
Intracellular activity and cytotoxicity
The cytotoxicity of compounds was determined by measuring
Conclusion
THP-1 cell viability after 3 days in the presence of test
compounds. Compounds were prepared as 10-point three-fold
serial dilutions in DMSO. The highest concentration of
compound tested was 50 µM where compounds were soluble
in DMSO at 10 mM. THP-1 cells were cultured in complete RPMI
and differentiated into macrophage-like cells using 80 nM PMA
overnight at 37°C, 5% CO2. Cells were inoculated into assay
plates and cultured for 24h before compound dilutions were
added to a final DMSO concentration of 0.5%. Each run included
staurosporine as a control. Assay plates were incubated for 3
days at 37°C, 5% CO2; growth was measured using the CellTiter-
Glo® Luminescent Cell Viability Assay (Promega) which uses ATP
as an indicator of cell viability. Relative luminescent units (RLU)
were measured using a Biotek Synergy 4 plate reader. The dose
response curve was fitted using the Levenberg–Marquardt
algorithm. The IC50 was defined as the compound concentration
that produced 50% inhibition of growth.
In this study we have prepared a new 2AT compound with
excellent activity, 33 MIC = 0.23 µM which inhibits mycolic acid
biosynthesis in Mtb. This is the most potent 2AT reported to
date. The 2AT library was screened against Ag85C however, no
significant activity was observed below 200 µM. Based on
structural similarities with reported compounds 33 is presumed
to be Pks13 inhibitor or targets an earlier enzyme in the mycolic
acid biosynthetic pathway. The loss of antibacterial activity
under NRP culture conditions supports the notion that the
compounds are targeting mycolic acid biosynthesis.
Importantly, the compounds are active against laboratory
generated and reference INH, RIF and FQ resistant Mtb strains.
This suggests the compounds will active against MDR and XDR
strains of Mtb. Compound 33 was active at concentrations
significantly below concentrations required to effect the
viability of human macrophage-like cells; however, the reduced
activity in Mtb infected macrophage-like cells suggests the
physicals properties of the lead requires improvement. Taken
together, these studies suggest 33 represents a promising
preclinical lead for further optimization.
The activity of compounds against intracellular bacteria was
determined by measuring viability in infected THP-1 cell after 3
days in the presence of test compounds. Compounds were
prepared as 10-point three-fold serial dilutions in DMSO. The
highest concentration of compound tested was 50 µM where
compounds were soluble in DMSO at 10 mM.
Author contribution
Contributed compound design and synthesis: S.J.S., T.S. and
S.K. Performed growth inhibition studies: S.E.K. and R.A.S.
Performed mycolic acid biosynthesis studies and analysis: A.G.
and M.J. Performed Ag85C inhibition studies: C.M.G. and D.R.R.
THP-1 cells were cultured in RPMI 1640 complete medium
and differentiated into macrophage-like cells using 80 nM PMA
overnight at 37°C, 5% CO2. THP-1 cells were infected with a
luminescent strain of H37Rv (which constitutively expresses
luxABCDE) at a multiplicity of infection of 1 and incubated
overnight at 37°C, 5% CO2.. Infected cells were recovered using
Accutase/EDTA solution, washed twice with PBS to remove
extracellular bacteria and seeded into assay pates. Compound
dilutions were added to a final DMSO concentration of 0.5%.
Assay plates were incubated for 72 h at 37°C, 5% CO2.. Each run
included isoniazid as a control. Relative luminescent units (RLU)
were measured using a Biotek Synergy 2 plate reader. The dose
response curve was fitted using the Levenberg–Marquardt
algorithm. The IC50 and IC90 were defined as the compound
concentrations that produced 50% and 90 % inhibition of
growth respectively.
Acknowledgements
This work was supported in part by a DeArce Memorial Fund
grant to S. J. S. form The University of Toledo, a grant from the
National Institutes of Health (grant number AI105084) to S.J.S
and D. R. R., and with Federal funds from the Division of
Microbiology and Infectious Diseases, National Institute of
Allergy and Infectious Diseases (Dr. Jim Boyce), National
Institutes of Health, Department of Health and Human Services,
under Contract No. HHSN272201100009I /HHSN27200002 A14.
Effect of 33 on lipid synthesis
Notes and references
Mtb H37Rv mc26206 grown in 7H9-OADC- 0.05% Tyloxapol
supplemented with 0.2% casaminoacid, 48 μg/mL pantothenate
and 50 μg/mL L-leucine (OD600 =0.5) was treated with either no
drug (control) or 0.90 μM 33, 2.23 μM 33, 4.46 μM 33, or 8.93
μM 33 for 16 h. [1,2-14C]-acetate was added to the culture at
the same time as the inhibitor. Total lipids were extracted from
bacterial cells, and mycolic acid methyl esters (MAMEs) and
fatty acid methyl esters (FAMEs) were prepared and previously
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