An antimycobacterial pleuromutilin analogue effective against dormant bacilli

Article abstract of DOI:10.1016/j.bmc.2018.07.034

Pleuromutilin is a promising pharmacophore to design new antibacterial agents for Gram-positive bacteria. However, there are limited studies on the development of pleuromutilin analogues that inhibit growth of Mycobacterium tuberculosis (Mtb). In screenin

Full text of DOI:10.1016/j.bmc.2018.07.034

Accepted Manuscript
An antimycobacterial pleuromutilin analogue effective against dormant bacilli
Maddie R. Lemieux, Shajila Siricilla, Katsuhiko Mitachi, Shakiba Eslamimehr,
Yuehong Wang, Dong Yang, Jeffrey D. Pressly, Ying Kong, Frank Park, Scott
G. Franzblau, Michio Kurosu
PII:
S0968-0896(18)30963-5
https://doi.org/10.1016/j.bmc.2018.07.034
BMC 14471
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
28 May 2018
6 July 2018
19 July 2018
Please cite this article as: Lemieux, M.R., Siricilla, S., Mitachi, K., Eslamimehr, S., Wang, Y., Yang, D., Pressly,
J.D., Kong, Y., Park, F., Franzblau, S.G., Kurosu, M., An antimycobacterial pleuromutilin analogue effective against
dormant bacilli, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.07.034
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Bioorganic & Medicinal Chemistry
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An antimycobacterial pleuromutilin analogue effective against dormant bacilli
Maddie R. Lemieuxa, Shajila Siricillaa, Katsuhiko Mitachia, Shakiba Eslamimehra, Yuehong Wangb, Dong
Yangc, Jeffrey D. Presslya, Ying Kongc, Frank Parka, Scott G. Franzblaub, Michio Kurosua,∗
a Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science Center, 881 Madison Avenue, Memphis, TN 38163-
0001, United States
b Institute for Tuberculosis Research, College of Pharmacy, University of Illinois at Chicago, 833 S. Wood Street, Chicago, IL 60612, United States
c Department of Microbiology, Immunology & Biochemistry, University of Tennessee Health Science Center, 858 Madison Avenue, Memphis, TN 38163-0001,
United Sates
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Pleuromutilin is a promising pharmacophore to design new antibacterial agents for Gram-positive
bacteria. However, there are limited studies on the development of pleuromutilin analogues that
inhibit growth of Mycobacterium tuberculosis (Mtb). In screening of our library of pleuromutilin
derivatives, UT-800 (1) was identified to kill replicating- and non-replicating Mtb with the MIC
values of 0.83 and 1.20 µg/mL, respectively. UT-800 also kills intracellular Mtb faster than
rifampicin at 2xMIC concentrations. Pharmacokinetic studies indicate that 1 has an oral
bioavailability with an average F-value of 27.6%. Pleuromutilin may have the potential to be
developed into an orally administered anti-TB drug.
Available online
Keywords:
Pleuromutilin
Antimycobacterial activity
Dormant tuberculosis
Intracellular Mycobacterium tuberculosis
Pharmacokinetics
2009 Elsevier Ltd. All rights reserved
.
1. Introduction
3781 (Nabriva Therapeutics)).10,11,12 The majority of pleuromutilin
derivatives reported to date displays a spectrum of activity focused
against Gram-positive bacteria,13,14,1516,17,18,19,20,21 and very few
Mycobacterium tuberculosis (Mtb) can persist in host tissues
for months to decades without replicating, yet with the ability to
resume growth.1,2 The ability of Mtb to survive in host
macrophages or granulomas by entering the dormant state is one
factor that requires the long duration of tuberculosis (TB)
chemotherapy.3 The dormant form of Mtb shows resistance to
most of the clinically utilized TB drugs at approved therapeutic
concentrations.4 Over the last 40 years, bedaquiline and delamanid
are the only TB drugs that have successfully been developed into
clinical application. However, their usage is restricted to patients
who have failed the standard TB regimens. Currently, only a few
new drugs have been evaluated in clinical trials.5,6
Since the discovery of pleuromutilin from Basidomycete spp.
in 1952, two drugs (tiamulin and valnemulin) and one drug
(retapamulin) were successfully developed as therapeutic agents
for veterinary and human applications, respectively. Retapamulin
is the first antibacterial agent of the pleuromutilin class for human
application; it was approved in 2007 by the FDA for the topical
treatment of impetigo and secondary infected traumatic lesions of
skin infections caused by Gram-positive bacteria such as S.
aureusand S. pyogenes.7,8,9 Recently, significant advances were
made in the development of pleuromutilin to become a systemic
or orally bioavailable antibiotic for MRSA infections (e.g. BC-
———
∗ Corresponding author. Tel.: +1-901-448-1045; fax: +1-901-448-7530; e-mail: mkurosu@uthsc.edu

analogues have been demonstrated to exhibit antimycobacterial
activity. Long et. al. reported moderate activity of valnemulin
against Mycobacterium smegmatis (MIC 8-16 µg/mL).22
Tiamulin-like derivatives displayed moderate activity against Mtb
(Lotesta et. al., 2011).23 Dong et. al. reported that C14-N-
benzylamine substituted pleuromutilin analogues have anti-Mtb
activity (60-100% inhibition at 20 µM).24 Several other
pleuromutilin analogues with modifications at C14, C12, C2, C4,
C6 and/or C7 were filed in the patents that were evaluated against
Mtb (Figure 1).25,26,27,28,29 In our discovery program for identifying
new antimycobacterial pleuromutilin derivatives effective against
non-replicating (dormant) form of Mtb, we discovered that an
analogue UT-800 (1) exhibited strong bactericidal activity against
Mtb with the MIC value of 0.83 µg/mL against replicating-Mtb
and 1.20 µg/mL against non-replicating-Mtb. Here, we wish to
report in vitro assay data for 1, and pharmacokinetic profile and
oral bioavailability of 1.
reaction of 7 with Boc-D-Leu-OH followed by deprotection of the
Boc group furnished 2•TFA, which was desalted with NaHCO3 in
MeOH-H2O to afford UT-820 (4) in 69% overall yield. Similarly,
UT-815 (3) and UT-810 (2) were synthesized in 46-71% overall
yields from pleuromutilin (5). The analogues (1-4) synthesized in
Scheme 1 were purified via reverse-phase HPLC.
2.3. In vitro assays for pleuromutilin analogues 1-4.
The resynthesized molecules 1-4 were evaluated in the growth
inhibitory assays in both replicating and non-replicating Mtb
(H37Rv) in microplate alamar blue assay (MABA) and low-oxygen
recovery assay (LORA), respectively.33 The analogs 1-4 exhibited
the MIC values of 0.78-3.06 µg/mL in MABA. Significantly, 1-4
killed non-replicating Mtb at the same level of MIC (1.04 - 3.46
µg/mL). The ratio of MICLORA/MICMABA for the three compounds
was between 1.13 - 1.45 which is close to the ideal value of 1.0
(Table 1). Because the new analogues 1-4 are analogous to
valnemulin (Figure 1), antimycobacterial activity of valunemulin
was examined and these data are included in Table 1. UT-800 (1)
and UT-815 (3) are the most effective antimycobacterial
pleuromutilin analogues identified in our laboratory. To the best
of our knowledge, effectiveness of pleuromutilin derivatives
against non-replicating Mtb has never been discussed previously.
The data summarized in Table 1 may indicate that pleuromutilin
can be engineered to be strong anti-TB drugs that are effective
against replicating and non-replicating (dormant) forms. The
2. Results and discussion
2.1. Discovery of antimycobacterial pleuromutilin analogues that
kill non-replicating Mtb.
We have generated a library of pleuromutilin derivatives
aiming at discovering anti-Gram-negative bacterial agents.30 The
same library molecules were evaluated against Mtb (H37Rv) in
microplate alamar blue assay (MABA). We identified four
pleuromutilin analogues which exhibited the MIC100 value <6.25
µg/mL against Mtb under aerobic conditions (Figure 2).
Interestingly, the identified antimycobacterial analogues are D-
leucine derivatives whose core structures are varied by the
oxidation states at the C3-carbonyl and C12-vinyl groups.
analogues
1-4
completely
inhibited
the
coupled
transcription/translation reactions (Promega Corporation) at 0.5
µg/mL concertation. In these assays, the IC50 values of 1 and
linezolid were 0.09 and 0.05 µg/mL, respectively (see SI). Thus,
alternations of the structure of the C14-side chain and oxidation
states of C3- and C12-functinal groups may not change the
primary molecular target of the pleuromutilins. The new analogues
identified here displayed cytotoxicity against Vero cells with the
IC50 values of 25-35 µg/mL.34 This IC50 level was slightly higher
than that of valunemulin, however, the selectivity index (defined
by IC50/MIC) of 1 and 3 was increased 5-fold relative to that of
valunemulin. The analogues 1-3 did not induce hemolysis (IC50
>200 µM), whereas, tunicamycin (a positive control) lysed the
blood cells with IC50 of 15.0 µM.
CH3
CH3OH
CH2
CH3
OH
CH3
CH3
O
12
O
O
O
H
H2
N
S
H
H2
N
S
N
N
H
O
O
H
H3
C
H3
C
3
H3
C
CH3
H3
C
CH3
H3
C
H3
C
H3C
H3C
HO
O
CH3
CH3
UT-810
2
( )
UT-800
1
)
(
CH3
CH3OH
CH2
CH3
OH
CH3
CH3
O
O
O
O
H
H
H2
N
S
H2
N
S
N
N
H
O
O
H
H3
H3
C
H3
H3
C
H3
C
CH3
H3C
CH3
H3
C
H3
C
C
C
HO
O
300
CH3
CH3
NO NADPH
UT-820
4
)
UT-815
3
)
(
(
1
Figure 2.
molecules.
Antimycobacterial pleuromutilin analogues identified from our library
2
3
250
200
150
100
50
2.2. Synthesis of antimycobacterial pleuromutilin analogues 1-4.
In order to confirm antimycobacterial activity of four hit
compounds and to evaluate their activity against non-replicating
Mtb, 1-4 were resynthesized according to the procedures
established in our laboratory (Scheme 1).30 The C3-carbonyl group
of pleuromutilin (5) was reduced with NaBH4 to form the
secondary alcohol with R-configuration exclusively. The primary
alcohol of the C14-side chain was tosylated with TsCl in the
presence of N,N-dimethylaminopyridine (DMAP) to afford 6 in
90% overall yield. Thioether formation of 6 with 1-amino-2-
methylpropane-2-thiol was accomplished according to a reported
condition, yielding the free amine 7 in 95% yield.30 Amide-
0
0
10
20
30
40
50
60
Time (min.)
forming reaction of
7
with Cbz-D-Leu-OH under
Glyceroacetonide-Oxyma, EDCI, and NaHCO3 in DMF-H2O31,32
followed by hydrogenation and deprotection of the Cbz group
furnished UT-800 (1) in 72% overall yield. Amide-forming
2.4. In vitro metabolic stability of the analogues 1-4.

Table 1. Transcription/Translation inhibition, MICs (Mtb) under aerobic and anaerobic conditions, and cytotoxicity (IC50) of 1-4 and
representative antibacterial agents.
Transcription/Translation
inhibition at 0.1 µg/mLa
MICLORA/MICMABA
Selectivity Index
(SI)e
MIC µg/mL
(MABA)b
0.83
1.56
0.78
3.06
3.13
0.20
0.04-0.10
0.78
MIC µg/mL
(LORA)c
1.20
1.98
1.04
3.46
3.56
1.47
>100
IC50 µg/mL
Vero cellsd
25.0
25.0
25.0
35.0
15.0
>200
>200
Molecule
+++
+++
+++
+++
+++
ND
ND
ND
+++
1.45
1.27
1.33
1.13
1.14
7.35
>2,500
>128
1.05
UT-800 (1)
30.1
UT-810 (2)
16.0
UT-815 (3)
32.0
UT-820 (4)
11.4
Valnemulin
Rifampicin (RIF)
Isoniazid (INH)
Ethambutol (EMB)
Linezolid
4.79
>1,000
>2,000
>256
>333
>100
0.63
>200
>200
0.60
aThe translation assay was performed with the Promega assay kit (L1020); bMABA: microplate alamar blue assay; cLORA:low-oxygen recovery assay; cCytotoxicity against Vero monkey
kidney cells; dSelectivity Index (SI) = IC50 (Vero cells)/MICMABA
We have reported that in vitro half-life (t1/2) of valunemulin in
rat liver microsomes is significantly extended by reduction of the
C3-carbonyl group of valnemulin.30 Thus, it is interesting to
examine metabolic stability of the analogues 1-3. In rat
microsomal stability assays, we observed a clear difference in t1/2
between 1 and 3; t1/2 of 3 was less than 5 min., on the other hand,
t1/2 of 1 was >60 min. A C3-carbonyl analogue 2 was also
metabolized completely within 10 min (Figure 3). Because of
longer t1/2 in these assays, UT-800 (1) was chosen to evaluate in
the basic pharmacological assays (vide infra).
transformant Mtb CDC1551 containing tdTomato (MOI = 10)
were treated with 1 (at 2xMIC).35 After 24, 48, 72, and 96 h of
incubation, the relative intensity of the fluorescence was measured
(emission wavelength (581 nm)) via fluorescence spectroscopy
(Figure 5a). Alternatively, the lysates were ten-fold serially diluted
2.5. Bactericidal effect of UT-800 (1) against Mtb H37Rv.
The time-kill experiments were performed at two-fold the MIC
of UT-800 (1), UT-810 (2), UT-815 (3) and two first-line TB drugs
(RIF and INH). Viable cell counting (CFUs) was performed at
every 24ꢀh for 14 days. The rate of killing of 1-3 against Mtb was
compared with the reference molecules; the time-kill assessments
at 2xMIC concentrations are summarized in Figure 4. The
analogues 1-3 killed 100% of Mtb at 2xMIC within 12 days. The
observed killing profile of 1-3 was similar to that of RIF and INH.
2.6. Effectiveness of UT-800 (1) against intracellular Mtb
The activity of UT-800 (1) against intracellular Mtb was
evaluated. Murine macrophage cells (J774A.1) infected by the

in 7H10-S broth and inoculated on 7H11-S plates to determine the
number of viable Mtb cells to confirm the bactericidal effect of 1
in 0-72h (Figure 5b). UT-800 (1) killed Mtb in infected
macrophages at 2xMIC concentrations within 72 h. The kill-curve
of INH, RIF, and 1 in Figure 5a indicate that 1 killed intracellular
Mtb more effectively than INH and RIF; bactericidal effect of 1
was distinguished from that of RIF at 48 h. In these experiments,
INH did not kill intracellular Mtb at 2xMIC.
administration, respectively; PK data of 1 after a single dose (3
mg/kg for IV and 30 mg/kg for PO are summarized in Table 3a
and 3b. These data were generously provided by The Global
Alliance for TB Drug Development (TB Alliance). An in vivo t
½
of 1 was 9.98 h at a dose of 3 mg/kg (IV). The maximum serum
concentration (Cmax) was 1,908 ng/mL after IV administration.
After PO administration, Cmax was 1,344 ng/mL with tmax of 0.83
h. In these preliminary PK studies, the area under the plasma drug
concentration-time curve (AUC0-24h) was nearlythe same as AUC0-
inf, indicating that increasing the dose of 1 may result in an
approximately proportional increase in AUC in mice. The 3.3-fold
increase in dose of 1 from 3 to 10 mg/kg resulted in a near
proportional increase in AUC0-24h (Table 3a vs 3c) from 993 to
2,497 ng•h/mL. Half-life (t1/2) of 1 at dose of 10 mg/kg was not
increased prominently compared to that of 3 mg/kg. Cmax at dose
of 10 mg/kg was increased about three-fold. The PK parameters in
serum following after a single oral dose of 1 (30 mg/kg) indicated
that the peak concentration was reached within 1h after. UT-800
(1) was determined to have a bioavailability with the Fpo of 27.6%
in mice (Table 3b). Most importantly, 1 can appropriately be
distributed to the lung tissue in mice (Table 3c). Based on these
PK studies, sufficient drug exposure (>MIC of 1) may be reached
in plasma and lungs when sufficient amount of 1 is administered.
UT-800 (1) displayed a good water solubility (104.7 mg/mL).
Acute maximum tolerated doses (MTD) were determined to be 30-
40 mg/kg(IV) and >200 mg/kg (PO), respectively. Although much
detailed PK studies are required to conclude the potential
application of 1 as an oral TB drug lead, theses PK parameters
summarized in Table 3 and acceptable MTD values encouraged us
to evaluate 1 in an infected mouse model via oral route.
a.
1.2
2xMIC
1
INH
0.8
Rifampicin
0.6
0.4
1
0.2
0
0
24
48
72
96
Time (h)
1
INH
24
Rifampicin
b.
0
48
72 h
Control
(Untreated)
UT-800 (1)
2x MIC
Figure 5.
1
Activity of against non-replicating and intracellular Mtb CDC1551-
tdTomato.
Table 3. Pharmacokinetic data for UT-800 (1).
Mtb CDC1551-tdTomato: a transformant Mtb CDC1551 containing tdTomato
a: IV administration (3 mg/kg)1
a: Effect of 1, INH, and rifampicin (RIF) against intracellular Mtb CDC1551-tdTomato in
macrophages (J774A.1 cells). Time-kill curve for intracellular Mtb at MIC x2 concentration.
Fluorescence confocal microscopy of J774A.1 murine macrophages infected with
b:
t1/2
Cmax
AUC0-24h
(ng•h/mL)
AUCInf
(ng•h/mL)
(h)
(ng/mL)
tdTomato (red) expressing Mtb after the treatment of the molecules (at MICx2).
Alll images were obtained for the treated and untreated cells for 72h. Fixed cells were stained
with a blue fluorescent stain, DAPI (4',6-diamidino-2-phenylindole) to visualize nuclei.
Mean
9.98
1,908
993
1,046
1data were obtained using three BALB/c mice.
2.7. Caco-2 permeability of UT-800 (1).
b: PO administration (30 mg/kg)1
The pleuromutilin analogue 1 exhibited permeability across
Caco-2 epithelial monolayers (Papp rate coefficient of 3.39 x 10-6
cm/s-1) with moderate levels of efflux (Papp 18.3 x 10-6 cm/s-1).36
An efflux ratio of 5.44 indicated that 1 has slightly better
membrane permeability than ranitidine (a low permeable control,
Table 2).
t1/2
(h)
tmax
(h)
Cmax
(ng/mL)
AUC0-24h
(ng•h/mL)
AUCInf
(ng•h/mL)
F
(%)
Mean
4.02
0.83
1,344
2,869
2,891
27.6
1data were obtained using six BALB/c mice. t : half-life; Cmax: maximum concentration;
½
tmax: time to maximum concentration (range); AUC: area under the concentration curve.
c: PK parameters of 1 in serum and lung tissues (10 mg/kg IV)
Table 2. Membrane permeability of 1 determined by Caco-2
assays.
Sample
Serum
Lung
t1/2
(h)
Cmax
(ng/mL)
AUC0-24h
(ng•h/unit)1
10.5
2,956
2,497
Test Conc.
(µM)
Assay
duration
(h)
Mean
→ B
Mean
→ A
Molecule
A
Papp
B
Papp
Efflux
Ratio
ND
6,074
66,281
10-6 cm/s-1
10-6 cm/s-1
Ranitidine
Talinolol
UT-800 (1)
Warfarin
10.0
10.0
10.0
10.0
2.0
2.0
2.0
2.0
0.237
0.0517
3.39
1.14
3.92
18.3
28.0
5.00
76.8
5.44
0.794
1Unit: mL for serum and g for lung.
2.9. In vivo activity of 1 in the BALB/c acute infection model.
The therapeutic efficacy of 1 was evaluated against Mtb
Erdman strain using the BALB/c acute infection model.37 The
treatment of the infected mice (a group of 5 BALB/c female mice,
average 19-20 g) with 1 (in water via gavage) was started 10 days
later after aerosol infection (Mtb Erdman 2x106 CFU). Treatment
was continued for three weeks (dosages: 50, 100, and 200 mg/kg).
Efficacy in this model was measured as reduction in total lung
burdens relative to an untreated control (Table 4).
35.2
2.8. Pharmacokinetic property of UT-800 (1).
Pharmacokinetic (PK) data for UT-800 (1) were obtained using
three and six mice for intravenous (IV) and oral (PO)

were visualized by UV light at 254 nm, or developed with ceric
ammonium molybdate or anisaldehyde or copper sulfate or
ninhydrin solutions by heating on a hot plate. Reactions were also
monitored by using SHIMADZU LCMS-2020 with solvents: A:
0.1% formic acid in water, B: acetonitrile. Flash chromatography
was performed with SiliCycle silica gel (Purasil 60 Å, 230-400
Mesh). Proton magnetic resonance (1H-NMR) spectral data were
recorded on 400, and 500 MHz instruments. Carbon magnetic
resonance (13C-NMR) spectral data were recorded on 100 and 125
MHz instruments. For all NMR spectra, chemical shifts (δH, δC)
were quoted in parts per million (ppm), and J values were quoted
Table 4. Therapeutic efficacy of 1 against Mtb Erdman strain
in a murine model via oral administration.
Molecule
Dosages
(mg/kg)
Log10 cfu/mouse1
SD
Log
reduction
Vehicle (water)
INH
200
2.5
50
1.3E+06
3.8E+05
1.2E+02
2.6E+05
3.3E+05
2.4E+05
-
7.8E+01
4.22
0.03
0.20
0.47
UT-800 (1)
UT-800 (1)
UT-800 (1)
1.2E+06
100
200
8.1E+05
1
in Hz. H and 13C NMR spectra were calibrated with residual
4.4E+05
undeuterated solvent (CDCl3: δH = 7.26 ppm, δC = 77.16 ppm;
CD3CN: δH = 1.94 ppm, δC = 1.32ppm; CD3OD: δH =3.31 ppm,
δC =49.00 ppm; DMSO-d6: δH = 2.50 ppm, δC = 39.52 ppm; D2O:
δH = 4.79 ppm) as an internal reference. The following
abbreviations were used to designate the multiplicities: s = singlet,
d = doublet, dd = double doublets, t = triplet, q = quartet, quin =
quintet, hept = heptet, m = multiplet, br = broad. Infrared (IR)
spectra were recorded on a Perkin-Elmer FT1600 spectrometer.
HPLC analyses were performed with a Shimadzu LC-20AD
HPLC system. All compounds were purified by reverse HPLC to
be ≥95% purity.
1Each value represents the mean SD for 5 mice.
Isoniazid (INH) (at 2.5 mg/kg for 3 weeks) in the BALB/c mouse
model reduced the bacterial load by about 4 Log10 CFU in lungs.
On the other hand, there was no noticeable difference in the
number of viable cells in lungs between groups of administration
of 1 at doses of 50 mg/kg (~1.0 mg/mouse) and control (vehicle).
Although a significant therapeutic efficacy was not observed in the
in vivo studies (Table 4), 1 showed weak activity in concentration
dependent manner; at doses of 200 mg/kg (~4.0 mg/mouse), 0.47
Log10 reduction in bacterial burdens was observed (P < 0.05),
although 5.6-5.7 log10 cfu/mouse of viable organisms persisted.
4.1.2. Synthesis of 1
The amino-alcohol 7 was synthesized according the procedure
described previously.30 To a stirred solution of 7 (1.51 g, 3.22
mmol), Cbz-D-Leu-OH (1.28 g, 4.83 mmol), NaHCO3 (2.70 g,
32.2 mmol) and Glyceroacetonide-Oxyma (1.10 g, 4.83 mmol) in
DMF-H2O (9/1, 16 mL) was added EDCI (3.08 g, 16.1 mmol). The
reaction mixture was stirred for 2h at rt, quenched with aq. sat.
NaHCO3, and extracted with EtOAc. The combined organic
extract was washed with 1N HCl, brine, dried over Na2SO4 and
concentrated in vacuo. The crude mixture was purified by silica
gel column chromatography (hexanes/EtOAc 60:40 to 25:75) to
afford 8 (2.04 g, 2.86 mmol, 88%). To a stirred solution of 8 (1.50
g, 2.10 mmol) in MeOH/EtOAc (1:1, 210 mL) was added Pd/C (10
wt %, 900 mg). H2 gas was introduced and the reaction mixture
was stirred for 120 h under H2. The solution was filtered through
Celite and concentrated in vacuo. The crude mixture was purified
by silica gel column chromatography (CHCl3/MeOH 95:5 to
90:10) to afford 1 (1.01 g, 1.73 mmol, 82%). UT-800 (1) was
purified by C18 reverse-phase HPLC [column: HYPERSIL
GOLD™ (175 Å, 12 µm, 250 x 10 mm), solvents: a gradient
elution of 25:75 to 55:45 MeOH : H2O over 20 min then 55:45
MeOH : H2O, flow rate: 2.0 mL/min, UV: 220 nm, retention time:
3. Conclusion
In our discovery program for identification of new
pleuromutilin derivatives against Gram-negative bacteria, we have
generated a library of pleuromutilin derivatives. Here, we describe
our discovery of several antimycobacterial analogues identified in
our library molecules. UT-800 (1) exhibits a strong antibacterial
activity against replicating and non-replicating Mtb with the
MICLORA/MICMABA ration of 1.45. As observed in our anti-
Acinetobacter analogues,30 a C3-hydroxy analogue 1 shows longer
half-life in the rat microsomes than the other analogues possessing
the C3-carbonyl group (e.g. valnemulin). Bactericidal activity of 1
against
intracellular
Mtb
highlights
uniqueness
of
antimycobacterial pleuromutilin derivatives. Pharmacokinetic
parameters of 1 indicates that 1 has oral bioavailability and is
distributed to the lung tissues in mice. Although the PK property
of the selected analogue 1 is far from that of ideal TB drugs, it is
very interesting to investigate whether 1 is effective in an infected
mouse model via oral administration. In an acute efficacy studies
using infected BALB/c mice, 1 showed ~0.5 log10 reduction of
Mtb burden in the lungs. The results summarized in this article can
conclude that the improvements of therapeutic window by
reducing toxicity level and PK property are essential for the
current lead to be an oral TB drug candidate.
25 min]: TLC (CHCl3/MeOH 90:10) R = 0.30; [α]21D +0.004 (c =
f
0.81, MeOH); IR (thin film) νmax = 3432 (br), 2958, 2876, 1680,
1562, 1464, 1370, 1290, 1144, 1020, 1001, 970, 930 cm−1; 1H
NMR (400 MHz, Methanol-d4) δ 5.58 (d, J = 9.0 Hz, 1H), 4.48 (t,
J = 5.6 Hz, 1H), 3.94 (dd, J = 8.2, 5.6 Hz, 1H), 3.38 (dd, J = 14.8,
4.5 Hz, 2H), 3.35 (s, 2H), 3.29 – 3.23 (m, 2H), 2.36 (tq, J = 11.2,
7.0, 5.6 Hz, 1H), 2.17 (quin, J = 6.8 Hz, 1H), 1.94 (dd, J = 12.9,
3.7 Hz, 1H), 1.91 – 1.86 (m, 1H), 1.84 – 1.73 (m, 4H), 1.73 – 1.64
(m, 3H), 1.64 – 1.56 (m, 2H), 1.52 (dd, J = 14.1, 7.4 Hz, 1H), 1.48
– 1.41 (m, 1H), 1.41 – 1.35 (m, 1H), 1.35 – 1.32 (m, 1H), 1.30 (s,
3H), 1.28 (s, 3H), 1.23 (s, 3H), 1.05 (d, J = 4.8 Hz, 3H), 1.04 (d, J
= 4.9 Hz, 3H), 0.91 (s, 3H), 0.86 (d, J = 7.0 Hz, 3H), 0.74 (t, J =
7.4 Hz, 3H), 0.69 (d, J = 7.2 Hz, 3H); 13C NMR (101 MHz, MeOD)
δ 172.24, 170.72, 77.33, 77.17, 73.47, 53.14, 52.19, 47.83, 47.17,
42.66, 42.55, 42.04, 42.01, 37.20, 35.42, 34.59, 33.16, 32.66,
32.44, 29.06, 26.95, 26.91, 26.68, 25.56, 23.13, 22.19, 21.47,
17.77, 17.54, 12.52, 8.79; HRMS (ESI+) m/z calcd for
C32H59N2O5S [M + H] 583.4145, found: 583.4166.
4. Experimental section
4.1. Chemical synthesis
4.1.1. General experimental methods
All chemicals were purchased from commercial sources and
used without further purification unless otherwise noted. THF,
CH2Cl2, and DMF were purified via Innovative Technology's
Pure-Solve System. All reactions were performed under an Argon
atmosphere. All stirring was performed with an internal magnetic
stirrer. Reactions were monitored by TLC using 0.25 mm coated
commercial silica gel plates (EMD, Silica Gel 60F254). TLC spots
4.1.3. Tosylation of pleuromutilin (5)

To a stirred solution of 5 (2.00 g, 5.28 mmol) in CH2Cl2 (26.4
mL) was added TsCl (1.21 g, 6.34 mmol) and DMAP (1.94 g, 15.9
mmol) at 0 oC. After 4h at 0 oC, the reaction mixture was quenched
with 1N HCl and extracted with EtOAc. The combined organic
extract was washed with aq. sat. NaHCO3, dried over Na2SO4,
concentrated in vacuo. The crude product was purified by silica
gel column chromatography (hexanes/EtOAc 60:40) to yield 10
(2.13 g, 3.99 mmol, 76%): TLC (hexanes/EtOAc 50:50) Rf = 0.50;
and all volatiles were evaporated in vacuo. The crude mixture was
basified with NaHCO3 in MeOH and purified by C18 reverse-
phase HPLC [column: HYPERSIL GOLD™ (175 Å, 12 µm, 250
x 10 mm), solvents: a gradient elution of 70:30 to 100:0 MeOH :
H2O over 20 min, flow rate: 2.0 mL/min, UV: 220 nm] to afford 2
(11.9 mg, 0.021 mmol, 75%, retention time: 18 min): TLC
(CHCl3/MeOH 90:10) R = 0.20; [α]21D +0.125 (c = 0.50, MeOH);
f
IR (thin film) νmax = 3354 (br), 2955, 2932, 2867, 1729, 1659,
1522, 1464, 1414, 1386, 1368, 1282, 1144, 1117, 1019, 981, 953,
939, 917 cm−1; 1H NMR (400 MHz, Methanol-d4) δ 6.32 (dd, J =
17.9, 10.8 Hz, 1H), 5.75 (d, J = 8.3 Hz, 1H), 5.17 (q, J = 1.6 Hz,
1H), 5.14 (q, J = 1.6 Hz, 1H), 3.50 (d, J = 6.1 Hz, 1H), 3.39 (dd, J
= 8.2, 6.1 Hz, 1H), 3.28 (d, J = 5.9 Hz, 2H), 2.39 – 2.32 (m, 2H),
2.32 – 2.22 (m, 1H), 2.16 (ddd, J = 16.0, 8.9, 4.9 Hz, 2H), 1.85 –
1.79 (m, 1H), 1.76 (ddd, J = 13.1, 6.6, 1.5 Hz, 1H), 1.69 (dt, J =
11.2, 2.2 Hz, 1H), 1.66 – 1.54 (m, 4H), 1.48 (d, J = 2.6 Hz, 1H),
1.46 (s, 3H), 1.44 – 1.32 (m, 4H), 1.32 – 1.28 (m, 1H), 1.27 (s,
3H), 1.25 (s, 3H), 1.15 (s, 3H), 0.98 (d, J = 6.6 Hz, 3H), 0.95 (d, J
= 6.5 Hz, 3H), 0.93 (d, J = 7.0 Hz, 3H), 0.73 (d, J = 6.6 Hz, 3H);
13C NMR (101 MHz, MeOD) δ 219.60, 178.24, 171.39, 141.16,
116.62, 75.46, 71.45, 59.32, 54.73, 47.81, 47.79, 47.77, 46.81,
45.99, 45.76, 45.25, 43.14, 38.16, 37.72, 35.29, 31.52, 28.17,
28.05, 26.90, 26.79, 25.92, 25.85, 23.47, 22.52, 17.13, 15.41,
11.77; HRMS (ESI+) m/z calcd for C32H55N2O5S [M + H]
579.3832, found: 579.3864.
[α]20 +0.355 (c = 1.81, CHCl3); IR (thin film) νmax = 3566 (br),
D
2984, 2937, 2883, 2865, 1757, 1732, 1598, 1455, 1368, 1291,
1223, 1190, 1176, 1117, 1096, 1038, 1019, 815, 753, 719, 663
cm−1; 1H NMR (400 MHz, Chloroform-d) δ 7.80 (d, J = 8.4 Hz,
2H), 7.35 – 7.32 (m, 2H), 6.39 (dd, J = 17.4, 11.0 Hz, 1H), 5.75
(d, J = 8.5 Hz, 1H), 5.31 (dd, J = 11.0, 1.5 Hz, 1H), 5.17 (dd, J =
17.4, 1.6 Hz, 1H), 4.46 (s, 2H), 3.33 (dd, J = 10.5, 6.5 Hz, 1H),
2.44 (s, 3H), 2.29 – 2.13 (m, 3H), 2.08 – 2.05 (m, 1H), 2.01 (d, J
= 8.7 Hz, 1H), 1.74 (dq, J = 14.4, 3.0 Hz, 1H), 1.68 – 1.58 (m, 1H),
1.53 – 1.41 (m, 2H), 1.39 (s, 3H), 1.36 – 1.33 (m, 1H), 1.33 – 1.27
(m, 1H), 1.23 (d, J = 15.9 Hz, 1H), 1.14 (s, 3H), 1.08 (dd, J = 13.9,
4.5 Hz, 1H), 0.86 (d, J = 6.9 Hz, 3H), 0.61 (d, J = 7.0 Hz, 3H); 13
C
NMR (101 MHz, CDCl3) δ 216.63, 164.81, 145.24, 138.70,
132.60, 129.87 (2C), 128.04 (2C), 117.29, 74.49, 70.27, 65.01,
57.98, 45.35, 44.48, 43.94, 41.81, 36.51, 36.00, 34.36, 30.30,
26.74, 26.39, 24.78, 21.64, 16.48, 14.72, 11.42; HRMS (ESI+) m/z
calcd for C29H41O7S [M + H] 533.2573, found: 533.2546.
4.1.4. Synthesis of 11
4.1.6. Synthesis of 3
To a stirred solution of 10 (196 mg, 0.37 mmol), 1-amino-2-
methylpropane-2-thiol hydrochloride (104 mg, 0.74 mmol) and
tetra-n-butylammonium bromide (11.9 mg, 0.037 mmol) in THF
To a stirred solution of 11 (0.24 g, 0.51 mmol), Cbz-D-Leu-OH
(0.20 g, 0.76 mmol), NaHCO3 (0.43 g, 5.08 mmol) and
Glyceroacetonide-Oxyma2 (0.17 g, 0.76 mmol) in DMF-H2O (9/1,
2.54 mL) was added EDCI (0.49 g, 2.54 mmol). The reaction
mixture was stirred for 1h at rt, quenched with aq. sat. NaHCO3,
and extracted with EtOAc. The combined organic extract was
washed with 1N HCl, brine, dried over Na2SO4 and concentrated
in vacuo. The crude mixture was purified by silica gel column
chromatography (hexanes/EtOAc 60:40 to 25:75) to afford 13
(0.33 g, 0.46 mmol, 91%). To a stirred solution of 13 (11.7 mg,
0.016 mmol) in MeOH/EtOAc (1:1, 3.0 mL) was added Pd/C (10
wt %, 5.0 mg). H2 gas was introduced and the reaction mixture was
stirred for 20 h under H2. The solution was filtered through Celite
and concentrated in vacuo. The crude mixture was purified by C18
reverse-phase HPLC [column: HYPERSIL GOLD™ (175 Å, 12
µm, 250 x 10 mm), solvents: a gradient elution of 25:75 to 55:45
MeOH : H2O over 20 min then 55:45 MeOH : H2O, flow rate: 2.0
mL/min, UV: 220 nm] to afford 3 (7.5 mg, 0.013 mmol, 78%,
o
(1.5 mL) was added 1N NaOH (1.5 mL). After 3h at 50 C, the
reaction mixture was extracted with CHCl3. The combined organic
extract was dried over Na2SO4, concentrated in vacuo. The crude
product was purified by silica gel column chromatography
(hexanes/EtOAc 50:50 to CHCl3/MeOH 75:25) to give 11 (140
mg, 0.30 mmol, 82%): TLC (CHCl3/MeOH 90:10) Rf = 0.20;
[α]20 +0.335 (c = 1.49, CHCl3); IR (thin film) νmax = 3433 (br),
D
2928, 2881, 2864, 1726, 1631, 1524, 1457, 1412, 1375, 1281,
1152, 1119, 1033, 1010, 980, 954, 938, 916, 816, 754, 683 cm−1;
1H NMR (400 MHz, Chloroform-d) δ 6.48 (dd, J = 17.4, 11.0 Hz,
1H), 5.76 (d, J = 8.4 Hz, 1H), 5.36 (dd, J = 10.9, 1.5 Hz, 1H), 5.21
(dd, J = 17.4, 1.6 Hz, 1H), 3.36 (d, J = 6.5 Hz, 1H), 3.15 (d, J =
2.3 Hz, 2H), 2.63 (s, 2H), 2.38 – 2.30 (m, 1H), 2.32 – 2.14 (m,
2H), 2.13 – 2.03 (m, 2H), 1.87 (brs, 2H), 1.77 (dq, J = 14.4, 3.1
Hz, 1H), 1.71 – 1.61 (m, 2H), 1.60 – 1.50 (m, 2H), 1.50 – 1.47 (m,
1H), 1.46 (s, 3H), 1.41 – 1.33 (m, 1H), 1.26 (s, 6H), 1.17 (s, 3H),
1.11 (dd, J = 14.0, 4.3 Hz, 1H), 0.88 (d, J = 7.0 Hz, 3H), 0.73 (d,
J = 6.9 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ 216.94, 169.57,
139.01, 117.30, 74.64, 69.52, 58.21, 51.42, 48.22, 45.47, 44.78,
43.95, 41.82, 36.79, 36.03, 34.46, 31.32, 30.46, 26.87, 26.34,
26.33, 26.29, 24.87, 16.86, 14.92, 11.50; HRMS (ESI+) m/z calcd
for C26H44NO4S [M + H] 466.2991, found: 466.3016.
retention time: 29 min): TLC (CHCl3/MeOH 90:10) R = 0.20;
f
[α]21D +0.071 (c = 0.27, MeOH); IR (thin film) νmax = 3344 (br),
2958, 2932, 2871, 1732, 1664, 1521, 1464, 1384, 1368, 1282,
1148, 1117 cm−1; 1H NMR (400 MHz, Methanol-d4) δ 5.67 (d, J =
8.2 Hz, 1H), 3.45 (d, J = 6.0 Hz, 1H), 3.39 (dd, J = 8.2, 6.1 Hz,
1H), 3.35 (s, 1H), 3.29 (d, J = 3.2 Hz, 1H), 2.41 – 2.31 (m, 2H),
2.26 (dddd, J = 19.3, 11.1, 2.6, 1.3 Hz, 1H), 2.14 (dt, J = 19.2, 9.3
Hz, 1H), 1.85 – 1.72 (m, 4H), 1.69 (dt, J = 11.2, 2.1 Hz, 1H), 1.65
– 1.49 (m, 5H), 1.47 (d, J = 2.5 Hz, 1H), 1.45 (s, 3H), 1.40 (d, J =
1.2 Hz, 1H), 1.39 – 1.32 (m, 3H), 1.28 (s, 3H), 1.26 (s, 3H), 1.14
(td, J = 14.2, 4.6 Hz, 1H), 0.98 (d, J = 6.6 Hz, 3H), 0.96 (d, J = 6.5
Hz, 3H), 0.94 (s, 3H), 0.91 (s, 3H), 0.74 (t, J = 7.4 Hz, 3H), 0.73
(d, J = 6.8 Hz, 3H); 13C NMR (101 MHz, MeOD) δ 219.67, 178.22,
171.80, 76.65, 71.41, 59.40, 54.73, 47.86, 47.84, 46.84, 45.78,
43.07, 41.94, 41.72, 38.16, 35.90, 35.29, 32.31, 31.46, 28.15,
26.88, 26.78, 26.74, 25.93, 25.70, 23.48, 22.51, 21.44, 17.30,
15.43, 11.71, 8.81; HRMS (ESI+) m/z calcd for C32H57N2O5S [M
+ H] 581.3988, found: 581.3958.
4.1.5. Synthesis of 2
To a stirred solution of 11 (62.1 mg, 0.13 mmol), Boc-D-Leu-
OH (46.2 mg, 0.20 mmol), NaHCO3 (112 mg, 1.33 mmol) and
Glyceroacetonide-Oxyma (45.6 g, 1.20 mmol) in DMF-H2O (9/1,
0.7 mL) was added EDCI (128 mg, 0.67 mmol). The reaction
mixture was stirred for 5h at rt, quenched with aq. sat. NaHCO3,
and extracted with EtOAc. The combined organic extract was
washed with 1N HCl, brine, dried over Na2SO4 and concentrated
in vacuo. The crude mixture was purified by silica gel column
chromatography (hexanes/EtOAc 75:25 to 60:40) to afford 12
(55.2 mg, 0.081 mmol, 61%). To a stirred solution of 12 (18.7 mg,
0.027 mmol) in dioxane (0.2 mL) was added a 4N solution of HCl
in dioxane (0.8 mL). The reaction mixture was stirred for 1h at rt,
4.1.7. Synthesis of 4

To a stirred solution of 7 (83.1 mg, 0.18 mmol), Boc-D-Leu-OH
(61.6 mg, 0.27 mmol), NaHCO3 (149 mg, 1.78 mmol) and
Glyceroacetonide-Oxyma (60.8 mg, 0.27 mmol) in DMF-H2O
(9/1, 0.9 mL) was added EDCI (170 mg, 0.89 mmol). The reaction
mixture was stirred for 5h at rt, quenched with aq. sat. NaHCO3,
and extracted with EtOAc. The combined organic extract was
washed with 1N HCl, brine, dried over Na2SO4 and concentrated
in vacuo. The crude mixture was purified by silica gel column
chromatography (hexanes/EtOAc 67:33 to 60:40) to afford 9 (108
mg, 0.158 mmol, 89%). To a stirred solution of 9 (27.2 mg, 0.040
mmol) in dioxane (0.2 mL) was added a 4N solution of HCl in
dioxane (0.4 mL). The reaction mixture was stirred for 1h at rt, and
all volatiles were evaporated in vacuo. The crude mixture was
basified with NaHCO3 in MeOH, and purified by C18 reverse-
phase HPLC [column: HYPERSIL GOLD™ (175 Å, 12 µm, 250
x 10 mm), solvents: a gradient elution of 70:30 to 100:0 MeOH :
H2O over 20 min, flow rate: 2.0 mL/min, UV: 220 nm] to afford 4
(18.1 mg, 0.031 mmol, 78%, retention time: 17 min): TLC
(CHCl3/MeOH 90:10) Rf = 0.20; 1H NMR (400 MHz, Methanol-
d4) δ 6.38 – 6.27 (m, 1H), 5.87 (d, J = 8.2 Hz, 1H), 5.14 (dq, J =
14.1, 1.6 Hz, 2H), 3.80 (s, 3H), 3.41 (d, J = 6.1 Hz, 1H), 3.40 –
3.38 (m, 1H), 3.29 – 3.26 (m, 2H), 2.59 (s, 1H), 2.50 (dtd, J = 19.2,
9.6, 1.2 Hz, 1H), 2.31 (ddt, J = 18.5, 9.7, 7.4 Hz, 2H), 2.22 (dd, J
= 14.5, 6.9 Hz, 1H), 2.08 – 1.96 (m, 1H), 1.76 (ddt, J = 13.1, 7.7,
6.5 Hz, 1H), 1.67 (dt, J = 13.6, 2.7 Hz, 1H), 1.63 – 1.55 (m, 2H),
1.50 (s, 3H), 1.49 – 1.47 (m, 1H), 1.46 – 1.42 (m, 1H), 1.42 – 1.38
(m, 1H), 1.36 (d, J = 5.1 Hz, 1H), 1.33 – 1.31 (m, 1H), 1.31 – 1.28
(m, 1H), 1.27 (s, 3H), 1.25 (s, 3H), 1.20 (d, J = 4.5 Hz, 1H), 1.15
(s, 3H), 0.98 (d, J = 6.6 Hz, 3H), 0.96 (d, J = 6.6 Hz, 3H), 0.89 (d,
J = 7.0 Hz, 3H), 0.73 (d, J = 7.2 Hz, 3H); 13C NMR (101 MHz,
MeOD) δ 178.03, 171.47, 167.64, 141.28, 116.51, 75.36, 72.09,
61.77, 54.70, 53.81, 47.81, 45.99, 45.68, 45.15, 43.63, 37.81,
36.36, 32.51, 30.48, 28.72, 28.30, 28.11, 26.91, 26.79, 25.92,
24.67, 23.47, 22.51, 17.19, 17.18, 12.84; HRMS (ESI+) m/z calcd
for C32H57N2O5S [M + H] 581.3988, found: 581.3976.
Assay plates for C. difficile was incubated for 24 h in the anaerobic
conditions and the optical density was measured at the end of
incubation and wells with no visible growth was considered MIC.
4.2.2.2. Luminescence-based low-oxygen-recovery assay (LORA)
These assays were performed according to the reported
procedures.33 In brief, M. tuberculosis H37Rv cells were
transformed by mixing at least 1 μg of the purified plasmid,
pFCA-luxAB and incubating at room temperature for 30 min,
followed by electroporation.39 M.tuberculosis pFCA-luxAB strain
cultured was diluted in Middlebrook 7H12 broth, and sonicated for
15s. The cultures were diluted to obtain an A570 of 0.03 to 0.05
and 3,000 to 7,000 RLUs per 100 μl. Two-fold serial dilutions of
antimicrobial agents were prepared in black 96-well microtiter
plates (100 μl), and 100 μl of the cell suspension was added. The
microplate was placed under anaerobic conditions (oxygen
concentration, less than 0.16%) by using an Anoxomat model WS-
8080 (MART Microbiology) and three cycles of evacuation and
filling with a mixture of 10% H2, 5% CO2, and 85% N2. Incubation
was continued for 10 days, and transferred to an ambient gaseous
condition (5% CO2-enriched air) incubator for a 28h “recovery.
” 100 μl culture was transferred to white 96-well microtiter
plates for determination of luminescence.
4.2.3. Cytotoxicity assays
Selected molecules were tested for cytotoxicity (IC50) in Vero
cells via a MTT colorimetric assay. Vero cell line was cultured in
Complete eagle’s minimum essential growth medium (EMEM)
containing L-glutamine, sodium pyruvate, minimum essential
amino acids, penicillin-streptomycin and 10% fetal bovine serum.
After 72h of exposure of molecules to this cell line at
concentrations ranging from 0.78 to 200 µg/mL, the culture
medium was changed to complete EMEM without phenol red
before addition of yellow tetrazolium dye; MTT. Viability was
assessed on the basis of cellular conversion of MTT into a purple
formazan product. The absorbance of the colored formazan
product was measured at 570 nm by BioTek Synergy HT
Spectrophotometer.
4.2. Biological assays
4.2.1. Bacterial culture conditions
A single colony of each Mycobacterium strain (M. tuberculosis
H37Rv was obtained on a Difco Middlebrook 7H10 nutrient agar
enriched with 10% oleic acid, albumin, dextrose and catalase
(OADC). Seed cultures of each Mycobacterium strain were
obtained in Middlebrook 7H9 broth enriched with OADC. Flasks
containing each bacterial strain was incubated to mid-log phase in
their respective culture media, in a shaking incubator at 37 °C with
a shaking speed of 200 rpm and cultured to mid-log phase (Optical
density - 0.5). The optical density was monitored at 600 nm using
a microplate reader (Biotek Synergy XT).
4.2.4. Killing Effect against intracellular Mtb
J774A.1 cells were seeded at 2.5 × 105 cells/well in 24-well
dishes or 1 × 105 cells/well in 8-well chamber slides and incubated
overnight at 37 °C in DMEM. A transformant M. tuberculosis
CDC1551 expressing tdTomato was grown in 7H9 Middlebrook
medium supplemented with OADC. The Mtb cells were harvested
at an optical density of 0.5, washed and re-suspended in saline.
J774A.1 cells were maintained in cell culture medium and were
infected by Mtb (106 bacteria in 0.2 mL of media): a multiplicity
of infection (MOI) of ≈10 (bacteria/cell).35 The extracellular
bacteria were removed by washing with PBS. The infected
macrophages were treated with antibacterial agents at 2xMIC
concentrations and the relative intensity of the fluorescence was
measured [emission wavelength (581 nm)] via UV–vis
spectroscopy in 24, 48, 72, and 96h for inhibition of intracellular
bacterial growth. Surviving Mtb cells were confirmed by CFU
method.
4.2.2. Minimum inhibitory concentration (MIC) assays
4.2.2.1. Microplate alamar blue assay (MABA)
These were performed according to the published protocol.38
Bacterial cultures at 0.5 optical density, was treated with serial
dilutions of inhibitors in aerobic conditions and incubated at 37 °C
for 15 days for M. tuberculosis (H37Rv) and M. bovis (BCG).
Incubation time was 48h for M. smegmatis (ATCC 607). All other
bacteria were incubated for 24 h. After incubation, 20ꢀµL of
resazurin (stock-0.02%) was added and incubated on a shaking
incubator at 37ꢀ°C for 4ꢀh for M.tuberculosis and M. bovis. For all
other bacteria 20 µL was added from a 0.08% resazurin stock
solution and left for 1 h. The lowest concentration at which the
color of resazurin was completely retained as blue was read as the
MIC100 (Pink = Growth, Blue = No growth). The absorbance
measurements were also performed using a Biotek Synergy XT
(Winooski, VT, USA), 96 well plate reader at 570 nm and 600 nm.
4.2.5. Kill-curve of Mtb by 1
M. tuberculosis H37Rv cultures at mid-log phase (OD = 0.5)
were diluted to OD = 0.25 and treated with inhibitor molecules at
MIC, 2xMIC and 4x MIC. Each culture well was diluted 10, 100,
1000 and 10,000-fold every 24 h and 20µL from each dilution was
plated on 7H10 agar plates supplemented with OADC enrichment.
Plates were incubated for 15 days in a static incubator at 37 °C and
colonies were counted.

4.2.6. Microsomal stability
with 6 in each group were injected with increasing doses of 1 (10
mg/kg, 25 mg/kg, and 50 mg/kg), and their health status was
monitored over a period of 7 days. The mice were monitored 3
times a day for the first 72h to ensure that the mice did not
experience negative health effects due to the administration of the
drug. The mice had their blood collected by submandibular bleed
on each day after the injection of 1. At the end of the study period,
the mice were sacrificed by cardiac puncture, and the plasma,
lungs, kidney, and spleen were collected. Analyses of the plasma
and lung sample were performed to determine basic PK parameters
(half-life, Tmax, Cmax).
Pooled Sprague-Dawley rat liver microsomes were purchased
from Corning Life Sciences (Oneonta, NY, USA). Microsomes
(20 mg/mL) were thawed on ice and diluted using phosphate
buffer (100 mM, pH: 7.4), resulting in a protein concentration of 1
mg/mL. Stock solutions (10 mg/L) of test molecules were prepared
in DMSO (50%). A final concentration of 500 ng/mL was used for
incubation with microsomes. NADPH (final concentration: 1 mM)
was used as a co-factor. All the above solutions except NADPH
were added to individual wells (12-well) in triplicate and were
allowed to equilibrate for 5 min at 37°C. NADPH was then added.
50 µL aliquots in triplicate were drawn from the incubation
mixture at 0, 5, 10, 20, 30, 45 and 60 min and immediately the
reaction was quenched by addition of ice-cold methanol (4
volumes). The samples containing methanol was lyophilized to
remove all volatiles. The residue was dissolved in 1N HCl. (10 µL)
and MeOH (40 µL). The resulting solution (20 µL) was quantified
via LC-MS. MS solvent: 90:10 acetonitrile/0.05% formic acid in
water, flow rate: 0.5mL/min.
4.2.10. In vivo activity of 1
Animal protocol number (15-170) was approved by the
Institutional Animal Care and Use Committee (IACUC) at the
University of Chicago Illinois on 09/15/2017. Efficacy of 1 in
killing Mtb Erdman was evaluated in a short-term acute efficacy
mouse model. Seven to eight week old female BALB/c mice were
infected with Mtb Erdman by aerosol using a glass-col chamber,
resulting in the deposition of ~100 CFU in the lungs of individual
mice. After 10 days, (50, 100 and 200 mg/kg) of 1 (in water) were
administered to groups of 5 female mice. Treatment was continued
for three weeks. Mice were humanely euthanized and the lungs
and spleens were aseptically harvested for bacterial enumeration
at days 21 post-infection. Efficacy was measured as reduction in
total lung burdens relative to an untreated control.
4.2.7. Prediction of intestinal permeability of 1 via Caco-2
permeability assay
The suitability of 1 for oral dosing was evaluated by using a
bidirectional Caco-2 permeability assay by Cyprotex.40 Test
molecue was added to the apical side of the membrane and the
transport of the compound across the monolayer was monitored
over a 2h time period. The transport of test molecule in the apical
to basolateral direction (A-B) as well as the basolateral to apical
direction (B-A) was assessed to determine an efflux ratio which
provides an indicator as to whether test molecule undergoes active
efflux. The permeability coefficient (Papp) of 1 was determined to
be 3.39 x 10-6 cm/s from A-B and 18.3 x 10-6 cm/s from B-A.
Efflux ratio (Papp(B-A)/Papp(A-B)) was determined to be 5.44.
Ranitidine was used as a low permeability control (Papp < 1) and
warfarin was used as a high permeability control (Papp > 1).
Talinolol, a known P-gp substrate, was screened as a control
compound to confirm that the cells are expressing functional efflux
proteins (Papp(B-A)/Papp(A-B) = 76.8). A Papp rate coefficient > 1
and efflux ratio approximately 5 indicates that 1 has permeability
across the membrane with moderate levels of efflux (An efflux
ratio greater than two indicates that drug efflux is occurring).
4.2.11. Transcription/translation coupled assay
Transcription/translation coupled assays were performed using
commercially available E. coli S30 circular DNA assay kit
(L1020) purchased from Promega Corporation. The assay used a
control DNA template, pBESTluc™ vector containing the
eukaryotic firefly luciferase gene positioned downstream from the
tac promoter and a ribosome binding site. The protein product,
luciferase was synthesized from 2µg of pBESTluc™ DNA using
5 uL complete amino acid mixture, 15 uL S30 extract (rNTPs,
tRNAs, an ATP-regenerating system, IPTG and appropriate salts)
and 20 uL premix provided with the assay kit in a final reaction
volume of 50 uL with nuclease free water. Inhibitor compounds
were dissolved in DMSO. Assays were setup with a master mix
consisting of S30 extract and water, amino acid mix, and the S30
Premix, followed by the addition of test compound. Master mix
was dispensed to microcentrifuge tubes and reactions were
initiated by the addition of DNA template. Reaction mixture was
vortexed gently, centrifuged for 5 sec. and incubated at 37oC for
60 min. Reactions were stopped by placing tubes in ice bath for 5
min. and diluted in two-fold dilution series using 50 uL of
luciferase dilution reagent (Promega Corporation) containing
luciferase assay substrate, and transferred to 96 well white plate.
The luminescence readout was performed immediately with UV-
Vis.
4.2.8. Hemolysis assay
Heparinized whole sheep blood was washed three times with
saline (0.9% NaCl) and the pellet was suspended in PBS buffer
(150 mM NaCl, 5 mM KCl, 10mM PBS, 2.5mM CaCl2, 10 mM
glucose, pH 7.4). 0.5 ml of 3% erythrocytes in PBS buffer, pH 7.4
was treated with test molecule in concentrations ranging from 2-
200 µg/mL). Triton X 100 was used as positive control in
concentrations 0.01, 0.1 and 1%. As a negative control, 0.5%
PEG:H2O (2:1) (diluent for test compounds) was used in the same
concentration as in samples (2.5 µL). The assay mixture was
incubated at 37 oC for 2h with gentle shaking. The assay mixture
was centrifuged at 4,700 rpm for 5 min and the released
hemoglobin in the supernatant was determined at OD540nm. The
percentage of hemolysis was calculated from the equation (100 x
(Asample−Anegative control) / (Apositive control – Anegative control)).41
Acknowledgements
The National Institutes of Health is greatly acknowledged for
financial support of this work (AI084411). We also thank
University of Tennessee for generous financial support (CORNET
award). NMR data were obtained on instruments supported by the
NIH Shared Instrumentation Grant. The authors gratefully
acknowledge Drs. Takushi Kaneko (GATB) and Stefan
Schweitzer (University of Tennessee Research Foundation) for
useful discussions.
4.2.9. LD50 and PK analyses
Animal protocol number (17-026.0-A) was approved by the
Institutional Animal Care and Use Committee (IACUC) at the
University of Tennessee on 05/30/2017. C57BI male mice aged 7-
8 weeks from Jackson labs were used. The mice were injected with
1 using retroorbital injection method under anesthesia. To
determine whether 1 exerted any overt toxicity, 3 groups of mice

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Graphical Abstract
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An antimycobacterial pleuromutilin
Leave this area blank for abstract info.
analogue effective against dormant bacilli
Maddie R. Lemieux, Shajila Siricilla, Katsuhiko Mitachi, Shakiba Eslamimehr, Yuehong Wang, Dong Yang,
Jeffrey D. Presley, Ying Kong, Frank Park, Scott G. Franzblau, Michio Kurosu*