Semisynthetic Antimycobacterial C-3 Silicate and C-3/C-21 Ester Derivatives of Fusidic Acid: Pharmacological Evaluation and Stability Studies in Liver Microsomes, Rat Plasma, and Mycobacterium tuberculosis culture

Article abstract of DOI:10.1021/acsinfecdis.9b00208

Fusidic acid (FA), a natural product fusidane triterpene-based antibiotic with unique structural features, is active in vitro against Mycobacterium tuberculosis, the causative agent of tuberculosis (TB). While possessing good pharmacokinetics in man, FA is rapidly metabolized in rodents, thus complicating proof-of-concept studies in this model. Toward the repositioning of FA as an anti-TB agent, we herein describe the synthesis, activity, and metabolism of FA and semisynthesized ester derivatives in rat liver microsomes, rat plasma, and mycobacterial cell culture. FA and derivative molecules with a free C-3 OH underwent species-specific metabolism to the corresponding 3-OH epimer, 3-epifusidic acid (3-epiFA). FA was also metabolized in rat plasma to form FA lactone. These additional routes of metabolism may contribute to the more rapid clearance of FA observed in rodents. C-3 alkyl and aryl esters functioned as classic prodrugs of FA, being hydrolyzed to FA in microsomes, plasma, and Mycobacterium tuberculosis culture. In contrast, C-3 silicate esters and C-21 esters were inert to hydrolysis and so did not act as prodrugs. The antimycobacterial activity of the C-3 silicate esters was comparable to that of FA, and these compounds were stable in microsomes and plasma, identifying them as potential candidates for evaluation in a rodent model of tuberculosis.

Full text of DOI:10.1021/acsinfecdis.9b00208

Subscriber access provided by UNIV OF SOUTHERN INDIANA
Article
Semisynthetic Antimycobacterial C-3 Silicate and C-3/ C-21 Ester Derivatives
of Fusidic Acid: Pharmacological Evaluation and Stability Studies in
Liver Microsomes, Rat Plasma and Mycobacterium tuberculosis culture
Mathew Njoroge, Gurminder Kaur, Marlene Espinoza-Moraga, Antonina Wasuna, Godwin Akpeko
Dziwornu, Ronnett Seldon, Dale Taylor, John Okombo, Digby F. Warner, and Kelly Chibale
ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.9b00208 • Publication Date (Web): 16 Jul 2019
Downloaded from pubs.acs.org on July 17, 2019
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 38
ACS Infectious Diseases
1
2
3
4
5
6
7
8
9
Semisynthetic Antimycobacterial C-3 Silicate and C-3/
C-21 Ester Derivatives of Fusidic Acid:
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Pharmacological Evaluation and Stability Studies in
Liver Microsomes, Rat Plasma and Mycobacterium
tuberculosis culture
Mathew Njoroge,1 Gurminder Kaur,1 Marlene Espinoza-Moraga,1 Antonina Wasuna,1 Godwin
Akpeko Dziwornu,1 Ronnett Seldon,2,3 Dale Taylor,3 John Okombo,1† Digby F. Warner,2,4,5 Kelly
Chibale1,2,3,6*
1 Department of Chemistry, University of Cape Town, Rondebosch 7701, South Africa.
2 Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Rondebosch
7701, South Africa
3 Drug Discovery and Development Centre (H3D), University of Cape Town, Rondebosch 7701,
South Africa
4
SAMRC/NHLS/UCT Molecular Mycobacteriology Research Unit, DST/NRF Centre of
Excellence for Biomedical TB Research, Department of Pathology and Institute of Infectious
Disease and Molecular Medicine, Faculty of Health Sciences, University of Cape Town,
Rondebosch 7701, South Africa.
ACS Paragon Plus Environment

ACS Infectious Diseases
Page 2 of 38
1
2
3
4
5
Wellcome Centre for Infectious Diseases Research in Africa, University of Cape Town,
5
6
Rondebosch 7701, South Africa.
7
8
9
6
South African Medical Research Council Drug Discovery and Development Research Unit,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
University of Cape Town, Rondebosch 7701, South Africa
†
Current address: Department of Microbiology and Immunology, Columbia University Irving
Medical Center, 1502 Hammer Health Sciences Center, New York, NY 10032, USA
Corresponding author: kelly.chibale@uct.ac.za
ACS Paragon Plus Environment

Page 3 of 38
ACS Infectious Diseases
1
2
3
4
5
6
7
8
9
Fusidic acid (FA), a natural product fusidane triterpene-based antibiotic with unique structural
features, is active in vitro against Mycobacterium tuberculosis, the causative agent of tuberculosis
(TB). While possessing good pharmacokinetics in man, FA is rapidly metabolized in rodents –
thus complicating proof-of-concept studies in this model. Towards the repositioning of FA as an
anti-TB agent, we herein describe the synthesis, activity and metabolism of FA and semi-
synthesized ester derivatives in rat liver microsomes, rat plasma and mycobacterial cell culture.
FA and derivative molecules with a free C-3 OH underwent species-specific metabolism to the
corresponding 3-OH epimer, 3-epifusidic acid (3-epiFA). FA was also metabolized in rat plasma
to form FA lactone. These additional routes of metabolism may contribute to the more rapid
clearance of FA observed in rodents. C-3 alkyl and aryl esters functioned as classic prodrugs of
FA, being hydrolyzed to FA in microsomes, plasma and Mycobacterium tuberculosis culture. In
contrast, C-3 silicate esters and C-21 esters were inert to hydrolysis and so did not act as prodrugs.
The antimycobacterial activity of the C-3 silicate esters was comparable to that of FA and these
compounds were stable in microsomes and plasma, identifying them as potential candidates for
evaluation in a rodent model of tuberculosis.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Keywords: Tuberculosis, Fusidic acid, Metabolism, Prodrug, Silicate, Hydrolysis
ACS Paragon Plus Environment

ACS Infectious Diseases
Page 4 of 38
1
2
3
4
5
6
7
8
9
The widespread emergence of drug-resistant Mycobacterium tuberculosis (Mtb), the causative
agent of tuberculosis (TB), has exacerbated the global threat of TB to public health.1The acute
need for novel anti-TB drugs has prompted a surge in investment in various stages of anti-TB drug
discovery and development.2 However, the high attrition rates of candidate drugs, especially in
clinical phases, necessitate a constant supply of new chemical matter into the development pipeline
as well as an in-depth understanding of the pharmacokinetics and preclinical predictors of
absorption, distribution, metabolism and excretion (ADME) of these drug candidates.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
There are numerous reports of natural product-derived antibacterial molecules with inhibitory
activity against Mtb,3–6 underscoring the potential of these compounds in anti-TB drug discovery.
An example is fusidic acid (FA) (1 ), (Figure 1), a naturally occurring fusidane antibiotic derived
from Fusidium coccineum7, which has been used clinically for the management of Gram-positive
infections and is especially useful in the treatment of methicillin-resistant Staphylococcus
aureus.8,9
25
24
O
21
H
OH
16 O
HO
H
3
O
HO
Figure 1: Fusidic acid (1)
FA has also been shown to have potent in vitro activity against Mtb.10–12 It had an MIC90 ≤ 16
mg/L (30 µM) across 167 of 170 clinical M.tb isolates tested – well within the achievable plasma
ACS Paragon Plus Environment

Page 5 of 38
ACS Infectious Diseases
1
2
3
4
5
6
7
8
9
concentrations in man – and showed no cross resistance with other first line TB drugs.10 Its general
antibacterial mechanism of action (MoA) involves inhibition of protein synthesis by stabilizing
the elongation factor-guanosine diphosphate complex, thus impeding peptide elongation13,14
Notably, a similar MoA has been reported for the anti-plasmodium activity of the drug in
Plasmodium falciparum, which causes malaria.15,16 Given the notoriously slow progress in anti-
TB drug development, the lack of cross resistance between FA and other antimycobacterial
agents,17 and its clinical tolerance and good pharmacokinetic profile in the management of
bacterial infections,18,19 the repositioning of FA and/or its active derivatives is an attractive ‘low-
hanging fruit’ in TB drug discovery.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
A complication hampering pre-clinical development of FA is that its pharmacokinetic profile in
rodents – a key model in the early phase drug discovery pipeline – appears peculiar to these
animals: various investigators have reported that it is poorly absorbed in mice and rats when
administered orally, shows poor exposure, and is rapidly cleared.20,21 This is a particularly
significant problem in anti-TB drug development, where mouse models are commonly used to
evaluate new drugs.22,23 Our own unpublished investigations of the efficacy of FA in acute and
chronic mouse models of TB initially found no in vivo activity up to a dose of 200 mg/kg
(unpublished data). Therefore, there is a need to understand the metabolism of FA in rodents and
find derivatives that allow for the required proof-of-concept studies so that the potential utility of
this compound as an anti-TB antibiotic can be better evaluated. We hypothesized that lack of in
vivo activity, despite the good in vitro activity, was likely due to differences in metabolism and
clearance in mice, relative to man. Further, we hypothesized that using prodrugs of FA would
improve the exposure of the compound in mice, thus allowing proof-of-concept studies in a mouse
model.
ACS Paragon Plus Environment

ACS Infectious Diseases
Page 6 of 38
1
2
3
4
5
6
7
8
9
Prodrugs are derivatives of a compound designed to be converted in vivo to the active drug. This
strategy is used to improve delivery across membranes by altering the physicochemical properties
of the active drug.24 Various chemical modifications can be considered based on the desired ease
of active compound release and synthetic accessibility. The work presented here focuses on the
carboxylic acid and silicate esters of FA as the former group is commonly known to increase
lipophilicity by masking polar groups to which they are attached. These compounds can also be
added to ionizable groups to control ionization in different pH environments. Silicate esters have
recently been reported as prodrugs.25,26 The size of the substituents can be used to influence steric
hindrance on the silicon thus controlling the rate of hydrolysis.26
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
FA can be easily esterified at either the C-3 hydroxyl group or at the C-21 carboxylic acid. We
have previously described the synthesis and antiplasmodium activity of C-21 FA esters and
amides, and the antimycobacterial activity of C-21 amides.27,28 Herein, we report the chemical and
biological characterization of C-3 and C-21 FA esters, and C-3 silicate ester derivatives.
RESULTS AND DISCUSSION
Chemistry: Synthetic routes to C-3 ester derivatives of FA are outlined in Scheme 1. Short,
medium and long chain aliphatic C-3 carboxylic esters of FA were synthesized under two different
conditions depending upon availability of the starting materials. Ester derivatives 1.2 and 1.4 were
synthesized by the reaction of FA (1) with the corresponding anhydride using pyridine as base and
solvent, while 1.3 and 1.5 were synthesized by the reaction of FA with the corresponding
carboxylic acid using the coupling reagent T3P in the presence of pyridine. Aromatic C-3
carboxylic esters were synthesized either through EDCI-DMAP-mediated (1.12 – 1.13) or T3P-
mediated (1.9 – 1.11) coupling of FA with the respective carboxylic acid.
ACS Paragon Plus Environment

Page 7 of 38
ACS Infectious Diseases
1
2
3
4
Three silicate esters of FA (1.6 – 1.8) were synthesized by reacting the relevant
5
6
o
trialkoxychlorosilane with FA at 0 C for 1h in the presence of imidazole as base and DMF as
7
8
9
solvent (Scheme 1). Trialkoxychlorosilanes were prepared in-house by dropwise addition of the
respective alcohols to a solution of SiCl4 in pentane at 25 oC for 1h and used without purification.
All target compounds were purified using column chromatography and fully characterized by
analytical and spectroscopic techniques. The synthesis and characterization of C-21 ester
derivatives has been previously reported.27
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
O
H
H
OH
OAc
OH
OAc
HO
H
HO
H
(a) or (b)
or (c)
O
H
H
R
O
HO
1.2 - 1.13
1
1.2: R=CH3
1.3: R=C2H5
1.4: R=C3H7
1.5: R=C4H9
1.9 : R = C6H5
(d)
1.10 : R = 4-OMe C6H5
1.11 : R = 4-CN C6H5
1.12 : R = 4-Cl C6H5
1.13 : R =4-nBu C6H5
O
H
OH
HO
H
OAc
OR
Si
RO
RO
H
O
pentane
1.6 - 1.8
+
SiCl4
ClSi(OR)3
3 ROH
25 oC, 1h
1.6: R= C2H5
1.7: R= CH(CH3)2
1.8: R= C8H17
ACS Paragon Plus Environment

ACS Infectious Diseases
Page 8 of 38
1
2
3
4
5
6
7
8
9
o
Scheme 1: Reagents and conditions: (a) (RCO)2O, pyridine, 25 C, 3 h for compounds 1.2 and
1.4; (b) RCOOH, T3P (50% w/v solution in DMF), pyridine, 0 oC – 25 oC, 16 h for compounds
1.3, 1.5 and 1.9 – 1.11; (c) RCOOH, EDCI-DMAP, DCM, 25 oC, 16 h for compounds 1.12 – 1.13;
(d) ClSi(OR)3, Imidazole, DMF, 0 oC, 1 h
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Antimycobacterial activity and cytotoxicity: Synthesized derivatives were screened in vitro
against a drug sensitive Mtb H37RvMa (ATCC 27294 virulent laboratory strain) strain cultured in
minimal media (GAST-Fe). The MIC99 (minimum concentration required to inhibit the growth of
99% of the bacterial population) was determined for each compound and a maximum
concentration limit was set at 160 µM (Table 1). Potent derivatives were also evaluated for
cytotoxicity against the Chinese Hamster Ovarian (CHO) mammalian cell line, and their half
minimal inhibitory concentration (IC50) determined. Emetine, a potent inhibitor of eukaryotic
protein synthesis, was used as the positive control in this assay.29
ACS Paragon Plus Environment

Page 9 of 38
ACS Infectious Diseases
1
2
3
4
5
6
7
8
Table 1: Antimycobacterial activities, cytotoxicities and metabolic stabilities of FA and related
derivatives
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
25
24
O
21
H
R1
16 O
HO
3
O
R
H
T½ (min)
Rat plasma
MIC99
(µM)
CHO IC50
(µM)
Compound
R
R1
RLMa
FA (1)
3-epiFA
1.1
OH
OH
O
OH
OH
OH
<0.15
11.4
>50
>50
>50
84
414
ndb
414
>200
<20
1.25
O
O
O
1.2
1.3
1.4
1.5
OH
OH
OH
OH
20
nd
20
<20
22
nd
O
O
O
2.50
1.25
2.50
>50
>50
>50
529
414
396
O
26
O
OC2H5
OC2H5
OC2H5
1.6
OH
0.2
>50
>200
>414
Si
O
ACS Paragon Plus Environment

ACS Infectious Diseases
Page 10 of 38
1
2
3
4
5
6
7
8
9
OCH(CH3)2
OCH(CH3)2
OCH(CH3)2
1.7
1.8
OH
OH
2.5
0.3
>50
>50
>200
nd
>400
nd
Si
O
O
OC8H17
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
OC8H17
OC8H17
Si
O
1.9
OH
OH
20
20
>50
>50
108
529
O
O
1.10
>200
>540
O
OMe
O
1.11
1.12
OH
OH
>160
>160
>50
>50
117
>540
>540
O
CN
O
>200
O
O
Cl
1.13
OH
>160
>50
>200
414
O
1.14
1.15
1.16
OH
OH
OH
>160
>160
>160
>50
>50
>50
22
21
>540
>540
>540
O
O
O
F
108
Cl
ACS Paragon Plus Environment

Page 11 of 38
ACS Infectious Diseases
1
2
3
4
5
6
7
8
1.17
1.18
OH
OH
>160
>160
>50
>50
108
27
311
O
Br
F
9
>540
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
O
O
Cl
Br
1.19
1.20
OH
OH
160
>50
>50
90
64
>540
>540
>160
O
OMe
1.21
1.22
1.23
OH
OH
OH
>160
>160
>160
>50
>50
>50
<20
186
52
>540
>540
>540
O
O
Cl
Cl
Cl
O
Cl
1.24
1.25
40
>50
>50
<20
<20
290
323
O
O
O
OH
>160
1.26
1.27
OH
OH
>160
>160
34
<20
<20
>540
323
O
O
2
6
>50
Rifampicin
Kanamycin
-
-
0.005
3.2
nd
nd
nd
nd
nd
nd
54
55
56
57
58
59
60
ACS Paragon Plus Environment

ACS Infectious Diseases
Page 12 of 38
1
2
3
4
5
6
Emetine -
-
nd
0.4
nd
nd
aRat Liver Microsomes not determined
b
7
8
9
The antimycobacterial activities of the C-3 alkyl esters improved with increasing chain length with
the optimal chain size being the butyrate (1.4); longer alkyl chains exhibited progressively reduced
activities. Two of the three silicate esters, triethoxy silicate ester 1.6 and trioctyloxy silicate ester
1.8, exhibited antimycobacterial activities comparable to FA. Triisopropoxy silicate ester 1.7
exhibited relatively lower activity (MIC99 2.5 µM) compared to the above silicate derivatives.
Further, all these silicate esters were found to be relatively non-cytotoxic against the CHO cell line
(Table 1). Only two C-3 aryl esters, 1.9 and 1.10, had some antimycobacterial activity with the
rest having no activity at the highest measured concentration. All C-21 esters were inactive.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Microsomal metabolism of FA: As seen in Table 1, FA had a moderate half-life in rat liver
microsomes (RLM). Metabolite identification studies revealed that the main metabolites were 3-
ketofusidic acid (3-ketoFA), 1.1, and a metabolite with the same mass and fragmentation pattern
as FA but which eluted at a different retention time. This metabolite was also formed as the main
metabolite of 3-ketoFA metabolism in rat and mouse but not in human liver microsomes (HLM),
and its formation was NADPH-dependent. Its identity was deduced as 3-epifusidic acid (3-epiFA)
and this was confirmed by comparison with LC-MS/MS of a synthetic standard of the metabolite.
3-ketoFA is the main metabolite of FA detected in humans and its formation by HLM was therefore
expected.19 Our data showed that the formation of 3-ketoFA was metabolically reversible in man,
since it was metabolized to form FA (Figure S1). However, as shown in Figure 2, the
stereoselectivity of the reaction changes in mouse liver microsomes (MLM) and RLM where 3-
epiFA was formed as the main metabolite (Figure S2). 3-EpiFA was stable in microsomes under
the incubation conditions used.
ACS Paragon Plus Environment

Page 13 of 38
ACS Infectious Diseases
1
2
3
4
5
6
7
8
9
O
O
OH
O
H
H
H
OH
OH
HO
H
HO
HO
H
RLM, MLM
HLM, RLM. MLM
HLM, RLM, MLM
O
O
O
O
O
O
HO
O
HO
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
H
3-ketofusidic acid
3-epifusidic acid (M1)
Fusidic acid (1)
Figure 2: Proposed pathway for metabolism of FA and 3-ketoFA to 3-epiFA in HLM, MLM and
RLM.
Microsomal metabolism of FA esters: Both the alkyl and aryl C-3 esters were metabolized in
RLM almost exclusively to FA. In general, the alkyl esters were less stable than their aryl
counterparts, with stability increasing almost linearly with chain length and lipophilicity (Table
1). In contrast, C-21 esters were stable to hydrolysis in RLM and instead showed metabolic
breakdown similar to FA with the corresponding 3-keto and 3-epi metabolites detected in the
incubations. The C-3 silicates were also stable to hydrolysis and no metabolites could be identified
in RLM.
Plasma stability of FA and its esters: FA was stable in rat plasma. However, the FA lactone,
which was likely generated through hydrolysis of the 16-acetyl group followed by spontaneous
lactonization (Figure 3), was observed as a minor metabolite. The lactone was not detected in
human plasma and has also not been reported as a metabolite in man.
O
O
O
H
H
H
HO
H
Sponteneous
lactonisation
OH
OH
OH
HO
H
HO
H
Hydrolysis
O
O
O
HO
HO
HO
Fusidic acid lactone
Fusidic acid (1)
Deacetylfusidic acid
Figure 3: Proposed C-16 hydrolysis pathway in FA in rat plasma.
ACS Paragon Plus Environment

ACS Infectious Diseases
Page 14 of 38
1
2
3
4
5
6
C-3 alkyl and aryl esters and C-21 alkyl esters were hydrolyzed to FA in rat plasma. In contrast,
C-21 aryl esters and C-3 silicate esters were stable to hydrolysis.
7
8
9
Mycobacterial metabolism: Mycobacteria contain numerous enzymes that can metabolize drugs
and drug-like compounds, producing metabolites that could contribute to in vitro activity or form
part of intrinsic resistance.30 We therefore investigated the metabolism of selected compounds
representing C-3 esters, C-3 silicates and C-21 esters in Mtb H37Rv cultures during 48-hours
exposure. Control incubations were performed using heat-killed (HK) cells in order to distinguish
between enzymatic metabolism and aqueous degradation. Figure 4 shows a typical set of results
for the live and HK cells.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1.4 Live
100
1.7 Live
1.7 HK
100
80
60
40
20
0
1.4 HK
O
O
80
60
40
20
0
H
OH
H
HO
OH
HO
O
O
O
O
O
O
1.4
R
H
1.7
OCH(CH3)2
OCH(CH3)2
OCH(CH3)2
H
O Si
R =
0
10
20
30
40
50
60
0
12
24
36
48
60
Time (Hours)
Time (Hours)
100
80
60
40
20
0
100
80
60
40
20
0
O
O
H
H
O
O
OH
HO
HO
O
O
O
HO
HO
1.14
FA
H
H
1.14 Live
1.14 HK
FA Live
FA HK
0
12
24
36
48
60
0
12
24
36
48
60
Time (Hours)
Time (Hours)
ACS Paragon Plus Environment

Page 15 of 38
ACS Infectious Diseases
1
2
3
4
Figure 4: Stability of FA and selected derivatives in live and heat-killed (HK) cultures of Mtb
5
6
H37Rv
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1.4 was less stable in the presence of live Mtb H37Rv cells than in the HK cells. A similar
observation was made for 1.5 (data not shown). FA was the only metabolite detected for both
compounds and it was formed at levels more than 500 times higher in the presence of live cells
compared to HK cells. This indicates that the hydrolysis of these C-3 esters is enzymatic. In
contrast, the amounts of 1.7, a C-3 silicate, were similar in both the HK and live cells, hinting at a
non-specific degradation of this compound. Notably, FA was not formed, and no metabolites were
identified from the incubations; 1.14, a C-21 aryl ester, behaved similarly.
Discussion: FA is a clinically important, naturally occurring fusidane antibiotic. It has good oral
bioavailability in patients (approximately 90 % with film-coated tablets) and a long half-life ( >
10 h), which makes it an attractive choice for oral therapy.19,31 FA is also well tolerated in both
acute and chronic treatment with few patients experiencing adverse events that require
discontinuation of therapy.18,32,33 Since it is also active against Mtb, it could potentially be
repositioned for TB therapy. However, preliminary investigations in acute and chronic mouse
models of TB showed no in vivo activity despite the good in vitro activity. Pharmacokinetic studies
show that FA has lower exposure and is cleared more rapidly in rodents relative to humans.20,21
When used clinically, FA is primarily cleared by metabolism in the liver, with less than 1% of the
drug excreted unchanged in bile and urine.34 We therefore used liver microsomes to investigate
the species difference in clearance. The in vitro data we present suggests that the more rapid rodent
clearance is due, at least in part, to faster hepatic metabolism leading to formation of 3-epiFA, a
ACS Paragon Plus Environment

ACS Infectious Diseases
Page 16 of 38
1
2
3
4
5
6
7
8
9
metabolite not reported in humans. The microsomal data also show that the metabolism of FA to
3-ketoFA, the main metabolite in humans, is a reversible reaction which would lead to a lower in
vivo clearance of FA in humans. In contrast, mouse and rat microsomes convert 3-ketoFA to 3-
epiFA, moreover this reaction was not reversible under the conditions tested. An analysis of data
reported by Lakshminayarana et al. shows that the microsomal half-life was > 200 min (Clint < 7
µl/min/mg) for more than half of the TB compound panel tested, including for recent drug
candidates such as bedaquiline and PA-824.35 The microsomal metabolism of FA (t½ 84 min) and
3-ketoFA (t½ < 20 min) is much faster and would be expected to translate to higher in vivo
clearance. FA was stable in rat plasma, but a lactone metabolite was observed. This metabolite has
not been observed in humans. The poor exposure of FA in rodent models appears, therefore, to be
partly due to these additional routes of metabolism. It would therefore be difficult to observe the
in vivo efficacy of FA in a mouse model unless modifications were made to the dosing to give
sufficient exposure despite the rapid metabolism. It is unclear from the current data whether or not
CYP450 enzymes are a major contributor to the rodent-specific routes of metabolism. If they are,
co-administration of FA with an inhibitor of CYP450 metabolism like 1-aminobenzotriazole, may
reduce the metabolic clearance enough to allow for a proof-of-concept study in mice.36 It would
also be of interest to evaluate the pharmacokinetics of FA in non-rodent models of tuberculosis,
for example in the marmoset model, where exposure might be better and/or more similar to that
observed in humans.31
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The work in this manuscript revolves around potential prodrugs of FA, which could be used to
deliver FA in vivo. C-3 esters act as classic prodrugs and are metabolized to FA in liver microsomes
and plasma. In addition, these compounds are hydrolyzed to FA in Mtb H37Rv cultures, suggesting
that they might act as prodrugs. Interestingly, C-3 silicates and C-21 esters are stable to hydrolysis
ACS Paragon Plus Environment

Page 17 of 38
ACS Infectious Diseases
1
2
3
4
in microsomes, plasma and Mtb culture, with only the alkyl esters showing some hydrolysis in
5
6
plasma. They are therefore not prodrugs of FA.
7
8
9
Studies in other bacteria suggest that a free C-21 carboxylic acid group is required for
activity.14,37,38 C-21 esters would therefore be expected to be inactive, unless they can be
hydrolyzed to release FA. The data from the in vitro mycobacterial incubations suggest that C-21
esters are resistant to hydrolysis and should therefore be inactive. We have previously reported
that C-21 esters and amides of FA have anti-plasmodium activity.27 As part of that work, their
stability in P. falciparum cultures was assessed and they were similarly found to be stable to
hydrolysis.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The stability of the C-3 silicates in Mtb culture indicates that their antimycobacterial activity does
not require biotransformation to the parental compound, FA. Given that these compounds show
similar activities to FA, but, by contrast, are stable in rodent microsomes and plasma, they appear
to be promising compounds for evaluation in a mouse model of TB infection.
Further exploration of C-3 silicates and C-3 alkyl esters would also require a determination of their
MoA and whether they are cross-resistant with FA.
Conclusion
FA and its main metabolite in man, 3-ketoFA, undergo species-specific biotransformation in MLM
and RLM, leading to formation of 3-epiFA. In addition, FA is hydrolyzed in rat plasma to its
lactone. These metabolic pathways may explain the more rapid clearance of FA in rodents. C-3
alkyl and aryl esters of FA act as prodrugs and are hydrolyzed in microsomes and plasma to release
ACS Paragon Plus Environment

ACS Infectious Diseases
Page 18 of 38
1
2
3
4
5
6
7
8
9
FA. These compounds are also hydrolyzed to FA by Mtb H37Rv. C-21 esters and C-3 silicate
esters are stable to hydrolysis in microsomes, plasma and Mtb cultures. Given their potent activities
and stabilities in microsomes and plasma, the C-3 silicates represent attractive compounds for in
vivo antimycobacterial activity evaluation.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
EXPERIMENTAL PROCEDURES:
General Method: FA was purchased from Avachem Scientific while all other reagents and
1
solvents (anhydrous) used for reactions were purchased from Sigma Aldrich. H NMR spectra
were recorded on a Varian Mercury Spectrometer at 300 MHz or a Varian Unity Spectrometer at
13
400 MHz. C NMR spectra were recorded at 75 MHz on a Varian Mercury Spectrometer or at
100 MHz on a Varian Unity Spectrometer. Chemical shifts (δ) are given in ppm downfield from
TMS as the internal standard. Coupling constants, J, are recorded in Hertz (Hz). Melting points
were obtained from a Reichert-Jung Thermovar hot-stage microscope apparatus and are
uncorrected. Thin layer chromatographic (TLC) was performed on aluminium-backed pre-coated
silica gel 60 plates purchased from Merck. Spots were visualised using UV light (λ 254/366 nm)
as well as anisaldehyde stain reagent. Column chromatography was performed with Merck silica-
gel 60 (70−230 mesh). LC-MS analysis was performed using an Agilent® 1260 Infinity Binary
Pump, Agilent® 1260 Infinity Diode Array Detector (DAD), Agilent® 1290 Infinity Column
Compartment, Agilent® 1260 Infinity Standard Autosampler, and a Agilent® 6120 Quadrupole
(single) mass spectrometer, equipped with APCI and ESI multimode ionisation source. All
compounds were confirmed to have >95% purity. The chemical characterization of compounds 3-
epiFA and 1.1 – 1.8 are shown below. Data on all other compounds have already been reported
elsewhere.27
ACS Paragon Plus Environment

Page 19 of 38
ACS Infectious Diseases
1
2
3
4
3-Epifusidic acid
5
6
7
8
NaBH4 (0.0220 g, 0.582 mmol) was added portion wise to a solution of 1.1 (0.100 g, 0.194 mmol)
in methanol (3 ml) at 25oC. Reaction mixture was then stirred for 3h. After completion of the
reaction (TLC), reaction mixture was acidified to pH = 1 by dropwise addition of 1N aq. HCl.
Methanol was then removed in vacuo and residue was dissolved in EtOAc (15 ml). The EtOAc
layer was washed with water (10 ml), dried over anhydrous sodium sulphate, filtered and
concentrated in vacuo. The crude product was purified by column chromatography on 100-200
silica gel using EtOAc:DCM as eluent, affording 3-epiFA as a white solid (0.025 g, 25%); Rf 0.5
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
(60% EtOAc/DCM); Mp. 211-213oC; H NMR (400 MHz, CD3OD) δ 5.81 (d, J = 8.5 Hz, 1H),
5.14 (t, J = 7.2 Hz, 1H), 4.31 (m, 1H), 3.07-2.98 (m, 2H), 2.59-2.50 (m, 1H), 2.42-2.35 (m, 1H),
2.32-2.27 (m, 1H), 2.19-2.01 (m, 5H), 1.96 (s, 3H), 1.91-1.72 (m, 4H), 1.67 (s, 3H), 1.61 (s, 3H),
1.61-1.57 (m, 3H), 1.40-1.29 (m, 1H), 1.33 (s, 3H), 1.26-1.22 (m, 1H), 1.19-1.10 (m, 2H), 1.02 (s,
3H), 0.94 (d, J = 6.5 Hz, 3H), 0.93 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 172.8, 171.2, 147.2,
131.9, 130.9, 122.9, 75.9, 74.3, 67.0, 49.0, 48.5, 43.6, 42.6, 39.7, 39.2, 38.6, 36.2, 36.1, 33.7, 32.0,
31.2, 28.5, 27.9, 24.5, 23.0, 22.7, 21.1, 19.3, 16.6, 16.4 and 14.5; LC-MS (ESI): m/z 457 [M-
OAc]+, 539 [M+23]+; purity (LC-MS): 98% (tr = 4.71 min.).
General Procedure for the synthesis of 3-ketofusidic acid 1.1
Jones’ reagent (0.8 ml) was added dropwise over 30 min. to an ice cooled solution of fusidic acid
(1) (2.00 g, 3.87 mmol) in acetone (20 ml). Reaction was monitored by TLC. Reaction mixture
o
was stirred at 0 C for additional 10 min. Water (40 ml) was then added to the reaction mixture
and aqueous layer was extracted with EtOAc (80 ml). Organic layer was then dried over anhydrous
ACS Paragon Plus Environment

ACS Infectious Diseases
Page 20 of 38
1
2
3
4
5
6
sodium sulphate, filtered and concentrated in vacuo. The residue was purified by column
chromatography on 100-200 silica gel using EtOAc:hexane as eluent, affording 1.1.
7
8
9
3-Ketofusidic acid 1.1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
White solid (33%); Rf 0.4 (60% EtOAc:hexane); Mp. 119-121 oC; 1H NMR (400 MHz, CDCl3) δ
5.91 (d, J = 8.3 Hz, 1H), 5.10 (t, J = 7.2 Hz, 1H), 4.40 (m, 1H), 3.07-3.03 (m, 1H), 2.51-2.42 (m,
4H,), 2.41-2.04 (m, 7H), 1.97 (s, 3H), 2.01-1.86 (m, 3H), 1.68 (s, 3H), 1.67-1.63 (m, 2H), 1.60 (s,
3H), 1.35-1.33 (m, 1H), 1.31 (s, 3H), 1.27-1.13 (m, 2H), 1.16 (s, 3H), 1.02 (d, J = 6.6 Hz, 3H),
0.95 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 212.4, 173.5, 170.4, 151.0, 132.7, 129.7, 122.9, 74.3,
68.1, 48.8, 48.4, 45.7, 45.4, 44.3, 39.4, 39.0, 38.0, 36.9, 36.1, 35.4, 33.5, 28.7, 28.3, 25.7, 24.7,
22.6, 21.9, 20.6, 18.0, 17.7 and 12.3; LC-MS (ESI): m/z 515 [M+H]+, 537 [M+23]+, 455 [M-
OAc]+; purity (LC-MS): 97% (tr = 3.89 min.).
General Procedure for the synthesis of C-3 alkyl esters 1.2 – 1.5
T3P (2 eq., 50% w/v in DMF, d=1.09 g/ml) was added dropwise to an ice cooled solution of FA
(1) (1 eq.) and respective carboxylic acid (1.1 eq.) in pyridine. Reaction mixture was then slowly
warmed to 25 oC and stirred for16 h. After completion of the reaction (TLC), water (25 ml) was
added to the reaction mixture and aqueous layer was extracted with EtOAc (2×15 ml). Organic
layer was washed with aq 0.500 M HCl (2×15 ml) to remove excess of pyridine. Organic layer
was dried over anhydrous sodium sulphate, filtered and concentrated in vacuo. The residue was
purified by column chromatography on 100-200 silica gel using EtOAc:hexane as eluent to afford
compound 1.2 – 1.5.
3-Acetoxyfusidic acid 1.2
ACS Paragon Plus Environment

Page 21 of 38
ACS Infectious Diseases
1
2
3
4
5
6
7
8
9
White solid (58%); Rf 0.4 (50% EtOAc:hexane); Mp. 112-114 oC; 1H NMR (400 MHz, CDCl3) δ
5.91 (d, J = 8.4 Hz, 1H), 5.11 (t, J =7.2 Hz, 1H), 4.94 (m, 1H), 4.34 (m, 1H), 3.08-3.04 (m, 1H),
2.53-2.42 (m, 2H), 2.35-2.30 (m, 1H), 2.22-2.05 (m, 5H), 2.07 (s, 3H), 1.97 (s, 3H), 1.91-1.76 (m,
4H), 1.68 (s, 3H), 1.63-1.53 (m, 4H), 1.61 (s, 3H), 1.38 (s, 3H), 1.36-1.32 (m, 1H), 1.20-1.02 (m,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
13
2H), 0.99 (s, 3H), 0.93 (s, 3H), 0.83 (d, J = 6.7 Hz, 3H); C NMR (100 MHz, CDCl3) δ 174.3,
170.9, 170.5, 151.1, 132.6, 129.6, 123.0, 74.4, 74.1, 68.3, 49.1, 48.8, 44.3, 39.5, 39.0, 37.8, 36.9,
35.8, 34.8, 32.7, 31.1, 28.7, 28.4, 27.4, 25.7, 24.3, 22.5, 21.3, 20.6 (2C), 18.1, 17.8 and 15.5; LC-
MS (ESI): m/z 559 [M+1]+, 581 [M+23]+, 499 [M-OAc]+; purity (HPLC): 98% (tr = 12.56 min.).
3-Propionyloxyfusidic acid 1.3
o
1
White solid (56%); Rf 0.5 (50% EtOAc:hexane); Mp. 93-95 C; H NMR (400 MHz, CDCl3) δ
5.91 (d, J = 8.3 Hz, 1H), 5.12 (t, J = 7.2 Hz, 1H), 4.95 (m, 1H), 4.34 (m, 1H), 3.08-3.04 (m, 1H),
2.51-2.42 (m, 2H), 2.36 (q, J = 7.6 Hz, 2H), 2.35-2.30 (m, 1H), 2.22-2.06 (m, 5H), 1.97 (s, 3H),
1.89-1.78 (m, 4H), 1.73-1.52 (m, 4H), 1.68 (s, 3H), 1.60 (s, 3H), 1.38 (s, 3H), 1.35-1.32 (m, 1H),
1.19-1.03 (m, 2H), 1.16 (t, J = 7.6 Hz, 3H), 0.99 (s, 3H), 0.93 (s, 3H), 0.83 (d, J = 6.7 Hz, 3H);
13C NMR (100 MHz, CDCl3) δ 174.1, 173.2, 170.5, 151.0, 132.6, 129.5, 123.0, 74.3, 73.8, 68.2,
49.1, 48.8, 44.3, 39.4, 39.0, 37.8, 37.0, 35.7, 34.8, 32.8, 31.0, 28.8, 28.3, 28.1, 27.4, 25.7, 24.3,
22.5, 20.6, 20.5, 18.1, 17.7, 15.5 and 9.4; LC-MS (ESI): m/z 573 [M+1]+, 595 [M+23]+, 513 [M-
OAc]+; purity (HPLC): 98% (tr = 13.20 min.).
3-Butyryloxyfusidic acid 1.4
White solid (66%); Rf 0.5 (50% EtOAc:hexane) Mp. 162- 164 oC; 1H NMR (400 MHz, CDCl3) δ
5.90 (d, J = 8.3 Hz, 1H), 5.10 (t, J = 7.2 Hz, 1H), 4.95 (m, 1H), 4.34 (m, 1H), 3.07-3.03 (m, 1H),
2.53-2.41 (m, 2H), 2.35- 2.29 (m, 1H), 2.32 (t, J = 7.3 Hz, 2H,), 2.22-2.03 (m, 5H), 1.97 (s, 3H),
1.91-1.76 (m, 4H), 1.72-1.52 (m, 6H), 1.68 (s, 3H), 1.60 (s, 3H), 1.38 (s, 3H), 1.35-1.32 (m, 1H),
ACS Paragon Plus Environment

ACS Infectious Diseases
Page 22 of 38
1
2
3
4
5
6
7
8
9
1.19-1.05 (m, 2H), 0.99 (s, 3H), 0.97 (t, J = 7.4 Hz, 3H), 0.93 (s, 3H), 0.83 (d, J = 6.7 Hz, 3H);
13C NMR (100 MHz, CDCl3) δ 174.5, 173.4, 170.4, 151.2, 132.6, 129.6, 123.0, 74.4, 73.8, 68.2,
49.1, 48.8, 44.3, 39.4, 39.0, 37.8, 36.9, 36.7, 35.7, 34.8, 32.7, 31.0, 28.7, 28.3, 27.4, 25.6, 24.3,
22.5, 20.5 (2C), 18.6, 18.0, 17.7, 15.6 and 13.7; LC-MS (ESI): m/z 587 [M+1]+, 609 [M+23]+, 527
[M-OAc]+; purity (HPLC): 99% (tr = 13.94 min.).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3-Pentanoyloxyfusidic acid 1.5
White solid (56%); Rf 0.6 (50% EtOAc:hexane) Mp. 118- 120 oC; 1H NMR (400 MHz, CDCl3) δ
5.90 (d, J = 8.4 Hz, 1H), 5.11 (t, J = 7.1 Hz, 1H), 4.94 (m, 1H), 4.34 (m, 1H), 3.07-3.03 (m, 1H),
2.49-2.45 (m, 2H), 2.35-2.29 (m, 1H), 2.33 (t, J = 7.4 Hz, 2H), 2.22-2.04 (m, 5H), 1.96 (s, 3H),
1.91-1.77 (m, 4H), 1.64-1.53 (m, 6H), 1.67 (s, 3H), 1.60 (s, 3H), 1.40-1.32 (m, 3H), 1.38 (s, 3H),
1.19-1.04 (m, 2H), 0.99 (s, 3H), 0.97-0.93 (m, 6H), 0.82 (d, J = 6.7 Hz, 3H); 13C NMR (100 MHz,
CDCl3) δ 174.2, 173.5, 170.4, 151.2, 132.6, 129.5, 123.0, 74.4, 73.8, 68.2, 49.1, 48.8, 44.3, 39.4,
39.0, 37.8, 37.0, 35.7, 34.8, 34.5, 32.8, 31.0, 28.7, 28.3, 27.4, 27.3, 25.6, 24.3, 22.5, 22.3, 20.5
(2C), 18.1, 17.7, 15.6 and 13.7; LC-MS (ESI): m/z 601 [M+1]+, 623 [M+23]+, 541 [M-OAc]+;
purity (HPLC): 99% (tr = 14.37 min.).
General Procedure for the synthesis of C-3 silicate esters 1.6 – 1.8.
Corresponding alcohol (3 eq.) was added dropwise to a solution of SiCl4 (1 eq.) in n-pentane at 25
oC under nitrogen atmosphere. Addition of alcohol was accompanied by frothing in the reaction
indicating a rapid reaction between the alcohol and SiCl4. The reaction was stirred for 1 h. The
clear solution formed was concentrated in vacuo to obtain crude trialkoxychlorosilane which was
used as such for next step.
A solution of fusidic acid (1 eq.) and imidazole (2 eq.) in DMF (5 ml) was cooled to 0 oC under
nitrogen atmosphere and trialkoxychlorosilane (3 eq, crude obtained above) was added. Reaction
ACS Paragon Plus Environment

Page 23 of 38
ACS Infectious Diseases
1
2
3
4
5
6
7
8
9
o
was stirred at 0 C for 1 h and monitored by TLC. After completion of reaction (TLC), reaction
mixture was diluted with EtOAc (25 ml) and washed with water (3×15 ml). EtOAc layer was then
dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. Product was purified by
column chromatography on 100-200 size silica gel using EtOAc:hexane as eluent, affording the
target compounds (1.6 – 1.8).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3-Triethoxysilyloxyfusidic acid 1.6
o
1
White solid (50%); Rf 0.5 (60% EtOAc:hexane); Mp. 72-74 C; H NMR (400 MHz, CDCl3) δ
5.89 (d, J = 8.3 Hz, 1H), 5.10 (t, J = 7.2 Hz, 1H), 4.34 (m, 1H), 4.00 (m, 1H), 3.85 (q, J = 7.0 Hz,
6H), 3.07-3.04 (m, 1H), 2.51-2.42 (m, 2H), 2.36-2.31 (m, 1H), 2.25-2.02 (m, 5H), 1.96 (s, 3H),
1.87-1.76 (m, 4H), 1.67 (s, 3H), 1.60 (s, 3H), 1.57-1.45 (m, 4H), 1.36 (s, 3H), 1.34-1.30 (m, 1H),
1.23 (t, J = 7.0 Hz, 9H), 1.16-1.04 (m, 2H), 0.97 (s, 3H), 0.92 (s, 3H), 0.90 (d, J = 6.7 Hz, 3H);
13C NMR (100 MHz, CDCl3) δ 174.3, 170.4, 151.3, 132.6, 129.5, 123.0, 74.4, 73.1, 68.3, 59.2
(3C), 49.1, 48.8, 44.4, 39.4, 39.0, 37.1, 36.4, 36.3, 35.4, 33.0, 30.5, 30.2, 28.7, 28.4, 25.6, 24.2,
22.5, 20.5, 20.4, 18.2(3C), 18.1, 17.7 and 16.2. LC-MS (ESI): m/z 619 [M-OAc]+; purity (LC-
MS): 98% (tr = 5.17 min.).
3-Triisopropoxysilyloxyfusidic acid 1.7
o
1
White solid (35%); Rf 0.5 (60% EtOAc:hexane); Mp. 70-72 C; H NMR (400 MHz, CDCl3) δ
5.89 (d, J = 8.3 Hz, 1H), 5.10 (t, J = 7.2 Hz, 1H), 4.34 (m, 1H), 4.23 (h, J = 6.1 Hz, 3H), 3.99 (m,
1H), 3.07-3.04 (m, 1H), 2.51-2.42 (m, 2H), 2.36-2.31 (m, 1H), 2.25- 2.02 (m, 5H), 1.96 (s, 3H),
1.91-1.73 (m, 4H), 1.67 (s, 3H), 1.60 (s, 3H), 1.57-1.45 (m, 4H), 1.36 (s, 3H), 1.34-1.30 (m, 1H),
1.19 (d, J = 6.1 Hz, 18H), 1.14-1.03 (m, 2H), 0.96 (s, 3H), 0.92 (s, 3H), 0.90 (d, J = 6.7 Hz, 3H);
13C NMR (100 MHz, CDCl3) δ 174.2, 170.4, 151.4, 132.6, 129.4, 123.0, 74.4, 72.9, 68.4, 65.7
(3C), 49.2, 48.8, 44.4, 39.4, 39.0, 37.1, 36.4 (2C), 35.3, 33.1, 30.6, 30.0, 28.7, 28.4, 25.6, 25.4
ACS Paragon Plus Environment

ACS Infectious Diseases
Page 24 of 38
1
2
3
4
(6C), 24.2, 22.5, 20.6, 20.4, 18.1, 17.7 and 16.3; LC-MS (ESI): m/z 661 [M-OAc]+; purity (LC-
5
6
MS): 99% (tr = 6.87 min.).
7
8
9
3-Trisoctyloxysilyloxyfusidic acid 1.8
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
Colourless viscous oil (23%); Rf 0.7 (50% EtOAc:hexane); H NMR (400 MHz, CDCl3) δ 5.90
(d, J = 8.3 Hz, 1H), 5.10 (t, J = 7.2 Hz, 1H), 4.34 (m, 1H), 3.75 (t, J = 6.7 Hz, 6H), 3.99 (m, 1H),
3.07-3.04 (m, 1H), 2.51-2.42 (m, 2H), 2.36-2.31 (m, 1H), 2.22-2.02 (m, 5H), 1.96 (s, 3H), 1.87-
1.75 (m, 4H), 1.67 (s, 3H), 1.60 (s, 3H), 1.57-1.45 (m, 10H), 1.36 (s, 3H), 1.34-1.25 (m, 31H),
1.14-1.03 (m, 2H), 0.96 (s, 3H), 0.92 (s, 3H), 0.90-0.87 (m, 12H); 13C NMR (100 MHz, CDCl3) δ
173.9, 170.4, 151.4, 132.6, 129.4, 123.0, 74.4, 73.0, 68.3, 63.6 (3C), 49.1, 48.8, 44.4, 39.4, 39.0,
37.2, 36.4, 36.3, 35.3, 33.2, 32.4 (3C), 31.8 (3C), 30.6, 30.2, 29.4 (3C), 29.3 (3C), 28.7, 28.4, 25.7
(3C), 25.6, 24.4, 22.6 (3C), 22.4, 20.6, 20.4, 18.1, 17.7, 16.3 and 14.1 (3C).
MATERIALS AND METHODS:
Compounds and Reagents: Fusidic acid was purchased from Avachem Scientific (Texas, USA).
Microsomal preparations (Human, Rat, Mouse) were purchased from XenoTech (Kansas, USA)
through their African distributor, African Institute of Biomedical Sciences and Technology
(Harare, Zimbabwe). The human liver microsomal preparations were pooled (50 individuals)
mixed gender, and the rat liver microsomes were from male IGS rats (pool of 433), while the
mouse liver microsomes were from male BALB/c mice (pool of 800). All microsomal preparations
were provided as 20 mg/ml suspensions transported in liquid nitrogen and were stored at -80 °C
until use. Each individual vial was used for a maximum of three times to eliminate any variation
due to freeze-thaw stability. NADPH (as β-Nicotinamide adenine dinucleotide 2′-phosphate
ACS Paragon Plus Environment

Page 25 of 38
ACS Infectious Diseases
1
2
3
4
5
6
7
8
9
reduced tetrasodium salt hydrate) was purchased from Merck (Johannesburg, South Africa) and
was divided to 5-10 mg aliquots and stored at -80 °C until use. All solvents and additives for
chromatography work were purchased from Merck (Johannesburg, South Africa) or from Sigma-
Aldrich (Johannesburg, South Africa) and were of HPLC grade. Deionized water (18Ω) for buffer
preparation and HPLC mobile phases was collected from a Millipore Synergy water purification
system (Microsep, Tygervalley, South Africa).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
LC-MS/MS: All LC-MS/MS analysis was performed on an Agilent 1200 Rapid Resolution (600
bar) HPLC system consisting of a binary pump, degasser, auto sampler and temperature-controlled
column compartment coupled to an AB SCIEX 4000 QTRAP mass spectrometer. Analyst (v1.4,
1.5, 1.6, AbSciex, Johannesburg, South Africa) was used for LC-MS control and data acquisition.
The mobile phase used was 5 mM ammonium formate containing 5% acetonitrile (A) and 5 mM
ammonium formate in 95% acetonitrile (B). The column used for the metabolic stability work was
a Kinetex C18 2.1 mm x 50 mm, with 2.6 μM particles, while a Kinetex C18 2.1 mm x 150 mm,
with 2.6 μM particles was used for the metabolite identification work. The mobile phase flow rate
was 500 μl/min and the run time was 3 minutes for metabolic stability methods and 400μl/min
with a run time of 15 minutes for metabolite identification methods.
Purified air was used as the nebulizer gas (gas 1) and heater gas (gas 2) and both were in general
set to 50 psi and 60 psi, respectively. Curtain gas (N2) was generally set at 30 psi and collision gas
(N2) was set to ‘medium’ for multiple reaction monitoring (MRM) experiments. In general, ion
spray voltage was set at 5000 V and source temperature at 500 °C. Compound dependent
parameters were optimized by infusing at 10 μl/min, a 500 nM solution of the compound in 50%
mobile phase A/B. Significant source fragmentation was observed and flow injection analysis
(FIA) experiments were set up to optimize all source parameters.
ACS Paragon Plus Environment

ACS Infectious Diseases
Page 26 of 38
1
2
3
4
5
6
7
8
9
Metabolic stability studies: The microsomal stability of the test compounds was evaluated using
a single point metabolic stability assay.39 Incubation mixtures contained microsomes and 1 μM
compound in 0.1 M phosphate buffer and were pre-incubated at 37 °C for 3 minutes. The reactions
were started by adding 1 mM NADPH and were incubated at 37 °C for 60 minutes. Ice-cold
acetonitrile (-20 °C) containing the internal standard was added to stop the reaction at T0 (before
addition of NADPH) and at T30 minutes. The samples were then centrifuged at 4 °C, 4000g for
30 minutes and then filtered through a 0.2 μM polyvinylidene difluoride (PVDF) filter. The
decrease in parent concentration was monitored using MRM methods. Data were processed on
Analyst v1.5 and analyzed on Microsoft Excel 2007. Half-life was calculated using the %
remaining at 60-minutes, using the equation below, which assumes first order kinetics;
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
T (min)= - In 2 x Incubation time (60 min)
½
In (% remaining/100)
Based on the assay reproducibility, half-life was reported as < 20 min for all compounds with <
15 % remaining at 60-minutes and as > 200 min for all compounds with > 80 % remaining.
Propranolol (T < 20 min and Midazolam (T < 20 min) and MMV390048 (T > 200 min) were
½
½
½
used as controls.
Metabolite identification studies: The test compound (10 μM) was incubated at 37 °C with
1mg/ml microsomes (MLM, male mouse CD1, Xenotech; RLM; male rat IGS, Xenotech; HLM;
human mixed gender, Xenotech) in phosphate buffer (100 mM, pH 7.4) which contained
magnesium chloride (5 mM). Reactions were started after a 5-minute pre-incubation by adding 1
mM NADPH (1 mM) and were incubated with shaking in a water bath at 37°C for 1 hour. An
equal volume of ice-cold acetonitrile was added to stop the reaction and to precipitate the proteins.
After centrifuging the mixture at 14000 rpm for 30 minutes the supernatant was transferred to a
ACS Paragon Plus Environment

Page 27 of 38
ACS Infectious Diseases
1
2
3
4
5
6
HPLC vial. Control samples with no NADPH, no microsomes and a T0 sample were also included
and processed in a similar way to the samples.
7
8
9
The LC-MS method was created using Lightsight v2.1 which relied on Analyst method files
created during direct infusion of the compounds.40 The methods generally consisted of survey
scans were coupled through information dependent acquisition to enhanced product ion scans. For
all compounds, a method with an enhanced mass spectrum scan coupled to an enhanced product
ion scan (EMS-IDA-EPI) was used as the method for the primary data acquisition and
subsequently, MRM, neutral loss, or precursor ion scans, with or without dependent EPI scans
were used to get more data on the metabolites. Where synthetic references of the metabolites were
available, they were analyzed by the same LC-MS/MS method and in the same batch as the
incubation samples to allow for a good comparison of retention time and fragmentation.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Plasma stability experiments: Rat plasma was obtained from Sprague Dawley rats, courtesy of
the Division of Clinical Pharmacology, University of Cape Town. The assay set-up was largely
based on previously described procedure.41 Compounds were tested at 1 μM in a 50% dilution of
plasma in phosphate buffered saline (PBS, 0.1 M, pH 7.4) and the negative controls in PBS only.
Incubations were performed at 37 °C for 3 hours and stopped by addition of ice-cold acetonitrile
containing the internal standard. The processing and analysis were then carried out as for metabolic
stability above. Albendazole (T > 540 min) and Benfluorex (T < 20 min) were used as positive
½
½
controls.41
Antimycobacterial evaluation experiments: The minimum inhibitory concentration (MIC) was
determined using the standard broth micro dilution method, where a 10 ml culture of M.
tuberculosis pMSp12::GFP,35 was grown to an optical density (OD600) of 0.6 – 0.7 in GAST-Fe
(glycerol–alanine–salts) medium pH 6.6, supplemented with 0.05% Tween-80.42,43 The culture
ACS Paragon Plus Environment

ACS Infectious Diseases
Page 28 of 38
1
2
3
4
5
6
7
8
9
was then diluted 1:100 in GAST-Fe. The compounds to be tested were reconstituted to a
concentration of 10 mM in DMSO. Two-fold serial dilutions of the test compound were prepared
in GAST-Fe, across a 96-well microtitre plate, after which, 50 µl of the 1:100 diluted M.
tuberculosis culture was added to each well in the serial dilution. The concentration range assayed
was 20 – 0.039 µM. The plate layout was a modification of the method previously described.38
Controls used were a minimum growth control (Rifampicin at 2xMIC), and a maximum growth
control (5% DMSO in GAST-Fe). The micro titre plate was sealed in a secondary container and
incubated at 37 °C with 5% CO2 and humidification. Relative fluorescence (excitation 485 nM;
emission 520 nM) was measured using a plate reader (FLUOstar OPTIMA, BMG LABTECH), at
day 7 and day 14. In the absence of a MIC shift between day 7 and day 14, day 14 data were
analyzed and reported. The raw fluorescence data were archived and analyzed using the CDD
Vault from Collaborative Drug Discovery, in which, data were normalized to the minimum and
maximum inhibition controls to generate a dose response curve (% inhibition), using the
Levenberg-Marquardt damped least-squares method, from which the MIC90 was calculated
(Burlingame, CA www.collaborativedrug.com).39 The lowest concentration of drug that inhibits
growth of more than 90%, and 99%, of the bacterial population was considered to be the MIC90
and MIC99 respectively.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
M. tuberculosis H37Rv experiments: To assess metabolism of the compound in Mtb H37RvMa,
cells were grown in GAST/Fe to O.D600 0.5 and harvested by centrifugation at 4000 rpm for 10
minutes at room temperature. The cell pellets were resuspended in PBS (0.05% Tween80) pH 7.4.
The compounds were added at half the MIC and the incubations conducted at 37 °C with sampling
at 0, 6, 12, 24 and 48 hours. At each time-point, an aliquot was taken from the incubation and
added to a tube containing acetonitrile and internal standard. The samples were then centrifuged
ACS Paragon Plus Environment

Page 29 of 38
ACS Infectious Diseases
1
2
3
4
5
6
7
8
9
for 5 minutes at 13000 rpm, filtered using 0.2µm filters and the supernatants transferred to a 96
well plate for analysis by LC-MS. The % compound remaining was calculated by comparing the
instrument response (analyte:internal standard ratio) at the sample time points to that at T0. Control
incubations were performed with HK cells (heated at 80 oC in a water bath for 1 hour).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Cytotoxicity experiments: The MTT-assay is used as a colorimetric assay for cellular growth and
survival, and compares well with other available assays.44,45 The reduction of tetrazolium salt MTT
to a formazan salt was used to measure all growth and chemosensitivity. The test samples were
tested in triplicate on one occasion using Chinese Hamster ovary cells (CHO-K1, ATCC, Manassas
VA, USA) using the methods of Mosmann.44 Cells were cultured in Hams/F12 media
supplemented with 10% v/v fetal bovine serum (Thermo Fisher Scientific; origin South America).
The test samples were prepared to a 10 mM stock solution in DMSO and were tested as a
suspension if not properly dissolved. Emetine was used as the reference drug in all experiments.
The highest concentration of solvent to which the cells were exposed to had no measurable effect
on the cell viability (data not shown). Plates were developed after 44 hours of exposure to the drug
by the addition of a solution of MTT. After four hours further incubation at 37 C, the supernatant
was removed from the cells via suction and DMSO was added to each well to dissolve the reduced
dye crystals. Plates were analyzed at 540 nM wavelength using a spectrophotometer to determine
the relative amount of formazan in each well. The 50% inhibitory concentration (IC50) values were
obtained from full dose-response curves, using a non-linear dose-response curve fitting analysis
via GraphPad Prism v.4 software.
AUTHOR INFORMATION
ACS Paragon Plus Environment