Design, synthesis, and In vitro antituberculosis activity of 2(5H)-Furanone derivatives

Article abstract of DOI:10.1002/iub.1526

A series of 2(5H)-furanone-based compounds were synthesized from commercially available mucohalic acids. From the first-generation compounds, three showed inhibitory activity (10 μg/mL) of at least 35% against Mycobacterium smegmatis mc² 155 growth (Bioscreen C system). In screening the active first-generation compounds for growth inhibition against Mycobacterium tuberculosis H37Rv, the most active compound was identified with a minimum inhibitory concentration (MIC₉₉) of 8.07 μg/mL (15.8 μM) using BACTEC 460 system. No cross-resistance was observed with some current first-line anti-TB drugs, since it similarly inhibited the growth of multidrug resistant (MDR) clinical isolates. The compound showed a good selectivity for mycobacteria since it did not inhibit the growth of selected Gram-positive and Gram-negative bacteria. It also showed synergistic activity with rifampicin (RIF) and additive activity with isoniazid (INH) and ethambutol (EMB). Additional time-kill studies showed that the compound is bacteriostatic to mycobacteria, but cytotoxic to the Chinese Hamster Ovarian (CHO) cell line. From a second generation library, two compounds showed improved anti-TB activity against M. tuberculosis H37Rv and decreased CHO cell cytotoxicity. The compounds exhibited MIC values of 2.62 μg/mL (5.6 μM) and 3.07 μg/mL (5.6 μM) respectively. The improved cytotoxicity against CHO cell line of the two compounds ranged from IC₅₀ = 38.24 μg/mL to IC₅₀ = 45.58 μg/mL when compared to the most active first-generation compound (IC₅₀ = 1.82 μg/mL). The two second generation leads with selectivity indices (SI) of 14.64 and 14.85 respectively, warrant further development as anti-TB drug candidates.

Full text of DOI:10.1002/iub.1526

Hypothesis
Andile H. Ngwane1
Design, Synthesis, and In Vitro Antituberculosis
Activity of 2(5H)-Furanone Derivatives
*
Jenny-Lee Panayides2
Franck Chouteau3
Lubabalo Macingwana1
Albertus Viljoen1
Bienyameen Baker1
Eliya Madikane4
Carmen de Kock5
Lubbe Wiesner5
Kelly Chibale3
Christopher
J. Parkinson2
Edwin M. Mmutlane2
Paul van Helden1
Ian Wiid1
1Division of Molecular Biology and Human Genetics, Faculty of Medicine
and Health Sciences, SAMRC Centre for TB Research, DST-NRF Centre of
Excellence for Biomedical Tuberculosis Research, Stellenbosch University,
Tygerberg, Cape Town, South Africa
2CSIR Biosciences, Pioneering Health Sciences, Pretoria, South Africa
3Department of Chemistry, University of Cape Town, Rondebosch, South
Africa
4Department of Clinical Laboratory Sciences, Division of Medical
Microbiology, University of Cape Town, Rondebosch, South Africa
5Division of Clinical Pharmacology, Department of Medicine, University of
Cape Town, South Africa
Abstract
A series of 2(5H)-furanone-based compounds were synthe- cross-resistance was observed with some current first-line
sized from commercially available mucohalic acids. From the anti-TB drugs, since it similarly inhibited the growth of multi-
first-generation compounds, three showed inhibitory activity drug resistant (MDR) clinical isolates. The compound showed
(10 mg/mL) of at least 35% against Mycobacterium smegmatis a good selectivity for mycobacteria since it did not inhibit the
mc2 155 growth (Bioscreen C system). In screening the active growth of selected Gram-positive and Gram-negative bacteria.
first-generation compounds for growth inhibition against It also showed synergistic activity with rifampicin (RIF) and
Mycobacterium tuberculosis H37Rv, the most active com- additive activity with isoniazid (INH) and ethambutol (EMB).
pound was identified with a minimum inhibitory concentration Additional time-kill studies showed that the compound is bac-
(MIC99) of 8.07 mg/mL (15.8 mM) using BACTEC 460 system. No teriostatic to mycobacteria, but cytotoxic to the Chinese
Additional Supporting Information may be found in the online version of this article.
C
V
2016 International Union of Biochemistry and Molecular Biology
Volume 68, Number 8, August 2016, Pages 612–620
*Address correspondence to: Andile H. Ngwane, Stellenbosch University Faculty of Medicine and Health Sciences, Biomedical Sciences, Fisan Building,
Francie van Zjil Drive, Tygerberg, Cape Town, Western Cape, South Africa 7500. Tel: 127-21-938-9402. Fax: 127-938-9476.
E-mail: ngwane@sun.ac.za or JPanayides@csir.co.za
Kelly Chibale is currently at Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Rondebosch, South Africa.
Christopher J. Parkinson is currently at School of Biomedical Sciences, Charles Sturt University, Orange NSW, Australia.
Edwin M. Mmutlane is currently at Department of Chemistry, University of Johannesburg, Auckland Park, South Africa.
Received 8 April 2016; Accepted 24 May 2016
DOI 10.1002/iub.1526
Published online 27 June 2016 in Wiley Online Library
(wileyonlinelibrary.com)
612
IUBMB Life

Hamster Ovarian (CHO) cell line. From a second generation ranged from IC50 5 38.24 mg/mL to IC50 5 45.58 mg/mL when
library, two compounds showed improved anti-TB activity compared to the most active first-generation compound
against M. tuberculosis H37Rv and decreased CHO cell cyto- (IC50 5 1.82 mg/mL). The two second generation leads with
toxicity. The compounds exhibited MIC values of 2.62 mg/mL selectivity indices (SI) of 14.64 and 14.85 respectively, warrant
C
V
(5.6 mM) and 3.07 mg/mL (5.6 mM) respectively. The improved further development as anti-TB drug candidates.
2016
cytotoxicity against CHO cell line of the two compounds IUBMB Life, 68(8):612–620, 2016
Keywords: synthetic; furanone; antimycobacterial; selective; synergy;
cytotoxicity
showed antituberculosis activity and specificity for mycobacte-
ria over other reference bacterial strains. From a second gen-
eration library of furanone derivatives, two compounds were
identified which showed improved activity and cytotoxicity.
Here we present data that suggest the two active second-
generation compounds offer a synthetic backbone that could
be further developed and optimised as TB antibiotic leads.
Introduction
Tuberculosis (TB) is one of the most common infectious dis-
eases and continues to be a major cause of morbidity and
mortality worldwide (1). The emergence of HIV infection,
decline of socioeconomic standards, and a reduced emphasis
on tuberculosis control programs have contributed to the dis-
ease’s resurgence in industrialized countries (2,3). The lethal
synergy of the tuberculosis and human immunodeficiency virus
(HIV) epidemics have complicated the global fight against TB
(4,5). Resistance of Mycobacterium tuberculosis strains to cur-
rent antitubercular agents, leading to the emergence of
multidrug-resistant (MDR) M. tuberculosis strains, is an
increasing problem worldwide (5,6). These trends are pre-
dicted to continue for decades and warrant the urgent need
for new TB therapies (7).
Materials and Methods
Ethical approval for this study was obtained from the Health
Research Ethics Committee of Stellenbosch University (refer-
ence no: N08/05/159).
Synthetic Chemistry
All starting materials used were commercially available
(Sigma-Aldrich, St. Louis) and were used as obtained from the
supplier without further purification. Column chromatography
was performed using Merck Kielselgel 60 (particle size 0.040–
0.063 mm), while thin layer chromatography was done on
Merck aluminum-supported silica gel 60 F254. NMR spectra
were recorded at 400 MHz for 1H NMR and 100 MHz for 13C
NMR on a Varian Unity INOVA 400 NMR as a solution in either
CDCl3 or DMSO-d6. Chemical shift data (d-values) are recorded
in parts per million (ppm) relative to tetramethylsilane as an
internal standard, coupling constants are quoted in hertz (see
Supporting Information file for representative NMR spectra).
All melting points were determined on a Stuart SMP10 melting
point apparatus and are uncorrected.
The furanone ring system, also known as butyrolactone or
butenolide, is a widely recognized component of natural prod-
ucts, exhibiting an extensive spectrum of pharmacological
activities (8). In particular, compounds bearing the 2-furanone
backbone have exhibited diverse biological activities, such as
antiprotozoal, antibacterial (including inhibition of biofilm for-
mation), antimycobacterial, antitumoral, antifungal, and
cyclooxygenase-2 inhibition (8–14). The efficacy of several
butyrolactone derivatives against mycobacteria suggests that
these structures be a new template for tuberculosis drug
development (14,15).
Our research effort towards the development of novel anti-
tubercular agents encompasses the search for new classes of
compounds, which are structurally distinct from known antitu-
bercular drugs in order to target other mechanisms of action.
Within the context of medicinal chemistry, the furanones are
chemically tractable as they lend themselves to synthesis of a
varied range of analogues (16). The furanones do however
possess the potential liability of cytotoxicity as previously
reported by Lattman et al. (17).
General Procedure for the Preparation of Mucohalic Acid
Carbonates 3-8. Methyl, ethyl, or benzyl chloroformate (1.05
equiv) was added to a cold (210 to 25 8C) solution of mucobro-
mic 1 or mucochloric 2 acid (0.019 mol) in dry dichlorome-
thane (75 mL). Diisopropylethylamine (1.1 equiv) was then
added over 10 min and stirring at 210 to 25 8C was continued
for 1.5 h. The reaction mixture was quenched with water
(30 mL) and diluted with dichloromethane (30 mL). The phases
were separated and the organic phase was concentrated under
reduced pressure. The residue was purified by silica gel col-
umn chromatography.
In an effort to develop novel drugs to treat TB, a series of
furanone-based compounds were chemically synthesized from
commercially available mucohalic acids. The current work
describes the synthesis and biological screening of novel
2(5H)-furanone derivatives. From a first generation library of
synthetic 2(5H)-furanone compounds, that were screened for
5-Ethoxycarbonyloxy-3,4-dibromo-5H-furan-2-one 4—Yield
antimicrobial activity, one compound was identified that 48% (20% ethyl acetate: hexane), yellow solid, mp 84–86 8C. dH
Ngwane et al.
613

IUBMB LIFE
(400 MHz, CDCl3) 6.76 (1 H, s, CH), 4.34 (2 H, 2 q, J 5 7 Hz, solvent removal under high vacuum afforded the title
compound.
3,4-Dichloro-5-octyloxy-5H-furan-2-one 19—Yield 99%
OCH2), 1.38 (3 H, t, J 5 7 Hz, OCH2CH3); dC (100 MHz, CDCl3)
163.0, 152.5, 141.4, 119.2, 96.3, 66.0, 14.0.
(100% diethyl ether), colorless oil. dH (400 MHz, CDCl3) 5.77
(1 H, s, CH), 3.78–3.84 (1 H, m, OCH2), 3.66–3.72 (1 H, m,
OCH2), 1.61–1.68 (2 H, m, OCH2CH2), 1.22–1.32 (10 H, m, 5 3
CH2), 0.86 (3 H, t, J 5 6 Hz, CH3); dC (100 MHz, CDCl3) 163.2,
147.5, 124.3, 100.98, 70.4, 31.7, 29.2, 29.2, 29.1, 25.7, 22.6,
14.0.
General Procedure for the Etherification of Carbonates 9-12.
Carbonate 3-8 (7.16 mmol) was combined with the desired phe-
nol (1.1 equiv) and cesium fluoride (0.3 equiv, 1 equiv for
biphenyl) in dichloromethane (65 mL) and stirred at room tem-
perature overnight. The reaction was quenched with saturated
aqueous ammonium chloride (50 mL) and partitioned between
water (50 mL) and dichloromethane (70 mL). The organic
extract was concentrated under reduced pressure and the result-
ing residue was purified by silica gel column chromatography.
5-(Biphenyl-4-yloxy)23,4-dibromo-5H-furan-2-one 10—Yield
72% (5% ethyl acetate: hexane) white crystals, mp 94–96 8C. dH
(400 MHz, CDCl3) 7.61–7.54 (2 H, m, phenyl), 7.50–7.42 (3 H, m,
phenyl), 7.26-7.22 (2 H, m, phenyl), 7.07–7.04 (2 H, m, phenyl),
6.18 (1 H, s, CH); dC (100 MHz, CDCl3) 163.3, 155.3, 154.8,
142.2, 140.1, 138.0, 132.9, 128.8, 128.6, 127.4, 127.0, 119.6,
119.4, 118.0, 117.6, 101.2.
General Procedure for the Amination of 3,4-Dichloro-5-aryl-
or alkyl-oxy-2(5H)-Furanones 20-22. To a solution of 3,4-
dichloro-5-aryloxy-5H-furan-2-one 19 (1 equiv, 1.78 mmol) in
N,N-dimethylformamide (10 mL) was added N1-(7-chloro-
quinolin-4-yl)-ethane-1,2-diamine (1 equiv) and diisopropyle-
thylamine (1 equiv), the stirred reaction mixture was heated at
80 8C for 24 h under a nitrogen atmosphere. The reaction mix-
ture was allowed to cool down to room temperature, poured
into a separating flask containing water (100 mL) and ethyl
acetate (100 mL) and the layers separated. The organic layer
was washed with water (4 3 100 mL), dried over magnesium
sulfate, filtered through a Celite plug and the filtrate concen-
trated on a rotary evaporator. The desired product was
obtained pure after column chromatography.
5-(Octyloxy)23-chloro-4-[2-(7-chloroquinolin-4ylamino)-
ethylamino]-5H-furan-2-one 22—Yield 45% (50% ethyl ace-
tate: hexane), cream white solid, mp 140 8C. dH (400 MHz, d6-
DMSO) 8.42 (1 H, d, J 5 6 Hz, CQ), 8.22 (1 H, d, J 5 9 Hz, CQ),
7.85 (1 H, t, J 5 6 Hz, and J 5 12 Hz, CQ), 7.80 (1 H, d, J 5 2
Hz, CQ), 7.48 (1 H, dd, J 5 2 Hz, and J 5 9 Hz, CQ), 7.37 (1 H,
br s, NH), 6.57 (1 H, d, J 5 6 Hz, CH), 3.49-3.58 (6 H, m, OCH2,
2 3 NCH2), 3.34 (1 H, br s, NH), 1.46 (2 H, quintet, J 5 7 Hz,
OCH2CH2), 1.14–1.18 (10 H, m, 5 3 CH2), 0.81 (3 H, t, J 5 6 Hz
and J 5 14 Hz, CH3); dC (100 MHz, d6-DMSO) 151.8 (2C), 149.9,
149.0, 133.5, 127.5 (2 C), 124.2 (2 C), 123.9, 117.4, 98.7, 88.7,
68.2, 42.7, 31.1, 28.8, 28.6, 28.5, 25.3, 22.0, 13.8. The fura-
none carbonyl does not appear on the 13C NMR spectrum, this
can be rationalized due to the slow relaxation basis.
General Procedure for the Amination of the Phenoxy or
(Biphenyl-4-yloxy)-dihalide-5H-furan-2-ones 13-16. To a stirred
solution of 9-12 (0.307 mmol) in dry tetrahydrofuran or N-
methylpyrrolidinone (2 mL), the corresponding amine (2
equiv) was added at room temperature. After 2 h, the reac-
tion mixture was diluted with water (10 mL) and left over-
night in the fridge. The resulting solid was filtered, washed
with water (10 mL), then heptane (5 mL), and dried. The
product was purified by flash column chromatography.
5-(Biphenyl-4-yloxy)23-bromo-4-[2-(7-chloroquinolin-4-
ylamino)-ethylamino]-5H-furan-2-one 14—Yield 78% (1–5%
methanol: dichloromethane), yellow solid, mp 131–133 8C. dH
(400 MHz, CDCl3) 8.24 (1 H, br s, CQ), 8.04 (1 H, d, J 5 9 Hz,
CQ), 7.80 (1 H, d, J 5 2 Hz, CQ), 7.54 (1 H, dd, J 5 9 Hz and
J 5 2 Hz, CQ), 7.44–7.38 (5 H, m, phenyl), 7.07–7.05 (2 H, m,
phenyl), 6.93–6.91 (2 H, m, phenyl), 6.54 (1 H, br s, CQ), 6.39
(1 H, br s, CH), 3.77 (2 H, br s, N-CH2 or CQ-N-CH2), 3.67 (2 H,
br s, N-CH2 or CQ-N-CH2); dC (100 MHz, CDCl3) 152.3 (2 C),
149.7, 138.2, 136.6, 133.7, 129.9, 129.3, 128.3 (3 C), 127.8
(2 C), 126.4, 124.2 (2 C), 119.8 (2 C), 118.1, 117.3, 97.0, 44.3.
The furanone carbonyl does not appear on the 13C NMR spec-
trum, this can be rationalized due to the slow relaxation basis.
General Procedure for the Preparation of 3,4-Dichloro-5-(phenyl
or 4’-biphenyl)22(5H)-furanones 23-24. A solution was pre-
pared of mucochloric acid (1 equiv, 11.5 mmol) and the
desired phenyl (1 equiv) in dichloromethane (50 mL) and was
cooled to 0 8C. To this cooled solution was added aluminum
trichloride (1.5 equiv) in four portions over 30 min. The reac-
tion mixture was initially stirred for 2 h at 0 8C and then
stirred at room temperature for a further 18 h. The reaction
mixture was poured onto ice-water and extracted with ethyl
acetate (50 mL). The partially separated emulsion was filtered
through celite, the phases were separated and the solvent was
removed from the organic phase. The resulting residue was
purified by silica gel column chromatography.
General Procedure for the Preparation of 3,4-Dichloro-5-
aryloxy-2(5H)-furanones 17-19. To a solution of mucochloric
acid (1 equiv, 11.8 mmol) in tetrahydrofuran (20 mL), stirred
at room temperature, was added the desired alcohol (0.75
equiv) and followed by drop-wise addition of concentrated sul-
furic acid (2.00 mL). The reaction mixture, in a stoppered
flask, was allowed to stir at room temperature for 48 h then
poured into a separating flask containing water (100 mL) and
diethyl ether (100 mL). The layers were separated, the organic
layer washed with water (3 3 100 mL) and dried over magne-
3,4-Dichloro-5-(4’-biphenyl)22(5H)-furanone 24—Yield 22%
(0-20% ethyl acetate: hexane), off-white solid, mp 154–155 8C.
sium sulfate. This was then filtered through a silica gel plug dH (400 MHz, CDCl3) 7.58 (2 H, d, J 5 8 Hz, phenyl), 7.51 (2 H,
and the filtrate concentrated on a rotary evaporator. Residual d, J 5 8 Hz, phenyl), 7.37–7.39 (2 H, m, phenyl), 7.29–7.30 (3 H,
614
In Vitro Anti-TB Activity of Synthetic Furanones

m, phenyl), 5.81 (1 H, s, CH); dC (100 MHz, CDCl3) 165.3, 152.2,
Selected Gram-negative bacteria (Escherichia coli ATCC
143.5, 139.9, 130.5, 128.9, 128.0, 127.9, 127.7, 127.2, 121.2, 25922, Pseudomonas aeruginosa ATCC9027, Klebsiella pneu-
83.5.
moniae ATCC13883) and Gram-positive bacteria (Staphylococ-
cus aureus ATCC25923, Enterococcus faecalis ATCC 29212,
Bacillus cereus ATCC 11778) were grown in Tryptone Soya
Broth (Difco Laboratories, Detroit) and were used for selectiv-
ity testing. Both Gram-positive and Gram-negative strains will
be referred as “bacteria” in general.
General Procedure for the Preparation of 4-Amino-5-(phenyl
or 4’-biphenyl)22(5H)-furanones 25-26. The corresponding
amine (2.5 equiv) in 1,4-dioxane (0.4 mL) was added to a solu-
tion of 3,4-dichloro-5-(4’-biphenyl)22(5H)-furanone (0.190
mmol) in 1,4-dioxane/chloroform (4 mL, 1: 1) and the resultant
was stirred at 50 8C for 24 h. The material was poured into Antibiotics
ethyl acetate (25 mL) and washed with dilute sodium carbon- Isoniazid, rifampicin, and ethambutol were purchased from
ate solution (5%, 2 3 25 mL) and water (2 3 25 mL). The sol- Sigma-Aldrich (St Louis). Stock solutions of isoniazid and
vent was removed and the resultant oil was submitted to flash ethambutol were prepared in sterile distilled and deionized
chromatography on silica gel.
water at 10 mg/mL and sterilized by filtration (0.22 mm-pore-
5-(40-Biphenyl)23-chloro-4-[2-(7-chloroquinolin-4-ylamino)- size polycarbonate filter). Stock solutions of rifampicin (10 mg/
hexylamino]-5H-furan-2-one 26—Yield 22% (0–20% ethyl ace- mL) and all synthetic furanone-based compounds were pre-
tate: hexane), off-white solid, mp 222 8C. dH (400 MHz, 3 CDCl3: pared in 100% dimethyl sulfoxide (DMSO) at 15 mg/mL. All syn-
1 d6-DMSO) 8.28 (1H, d, J 5 6 Hz, CQ), 8.12 (1H, d, J 5 9 Hz, thetic compound and antibiotic stock solutions were stored at
CQ), 7.87 (1H, d, J 5 2 Hz, CQ), 7.56–7.49 (4 H, m, phenyl), 7.43 280 8C until required.
(1 H, d, J 5 8 Hz, CQ), 7.35 (2 H, dd, J 5 7 Hz and J 5 8 Hz, phe-
Antibacterial Activity and Minimum Inhibitory
nyl), 7.31–7.24 (3H, m, phenyl), 7.18 (1 H, br s, NH-CQ), 6.86
Concentration (MIC) Determination
(1 H, br s, NH-C 5 C), 6.31 (1H, d, J 5 6 Hz, CQ), 6.10 (1 H, s,
Primary screening of the first-generation synthetic compounds
CH), 3.32 (2 H, ddd, J 5 6 Hz, J 5 8 Hz, and J 5 14 Hz, CH2-NH-
against M. smegmatis and selectivity screening against Gram-
CQ), 2.92 (2 H, ddd, J 5 6 Hz, J 5 8 Hz, and J 5 14 Hz, CH2-NH-
positive and Gram-negative bacteria were performed using the
C 5 C), 1.59 (2 H, quintet, J 5 7 Hz, CH2), 1.52–1.10 (6 H, 3 3
Bioscreen C automated biological growth reader system (Lab-
m, 3 3 CH2); dC (100 MHz, 3 CDCl3: 1 d6-DMSO) 166.9, 154.8,
systems, Helsinki, Finland) as described (19) with minor modi-
151.4, 147.8, 145.1, 140.7, 139.4, 135.3, 134.6, 128.2, 128.1,
fications. Antimycobacterial activity against M. smegmatis,
126.9, 126.3, 126.2, 126.1, 124.7, 123.0, 121.0, 116.2, 97.6,
was tested at a base-line screen concentration (10 mg/mL) and
91.9, 42.4, 41.9, 27.8, 27.3, 25.7, 25.6.
for selectivity against bacteria at 3.125 to 50 mg/mL. Each well
Bacterial Strains and Culture Conditions
contained 100 mL of compound diluted at a specified concen-
Mycobacterium smegmatis mc2 155 was used as a surrogate tration and 100 mL of bacteria (OD600 of 0.05) to a final volume
strain for M. tuberculosis in primary screens of the first- of 200 mL per well. Rifampicin (6.25 mg/mL) and ciprofloxacin
generation synthetic compounds. Unless otherwise mentioned, (0.1 to 2 mg/mL) were used for M. smegmatis and bacterial
M. tuberculosis H37Rv (ATCC 27294) was used in all second- growth inhibition, respectively. Control wells contained 100 mL
ary screens. Cultures for experimental treatment were initi- of compound solvent (without compound) plus 100 mL of bacte-
ated by diluting a frozen stock inoculum 1: 200 into fresh Mid- ria (OD600 of 0.05) to a final volume of 200 mL per well. The
dlebrook 7H9 supplemented with 10% v/v oleic acid-albumin- cultures were incubated at 37 8C with constant shaking (mod-
dextrose-catalase (OADC, Difco Laboratories, Detroit) and erate) and the OD600 measured at 30-min intervals over an
0.05% v/v Tween 80 (Sigma-Aldrich, St. Louis) in vented, screw incubation period of 48 h. Growth curves were generated and
cap, tissue culture flasks (NUNC, Germany) and cultured to log analysed using Research Express, version 1.00 (a software
phase (OD600 of 0.6) in a 5% CO2 atmosphere at 37 8C.
package of the Bioscreen C System). Growth changes were
Selected clinical isolates of M. tuberculosis were also used interpreted at 16 h of incubation (mid log phase, OD600 of 0.5)
for the testing of antimycobacterial activity and synergistic and the percentage inhibition was calculated relative to the
effects of 14 with current anti-TB first line drugs. All isolates untreated controls. All experiments were conducted in tripli-
were obtained from a genotyped strain collection in the cate, with three independent experiments performed each
Department of Biomedical Sciences, University of Stellenbosch time. Compounds that showed at least 35% growth inhibition
(18). The isolates were characterized as resistant or suscepti- against M. smegmatis were screened against M. tuberculosis.
ble based on growth at critical concentrations of first line anti-
Secondary screening of the three most active first-
biotics. The strains included rifampicin mono-resistant (RIFR) generation and all of the second-generation synthetic com-
and isoniazid mono-resistant (INHR) strains, resistant to 2 and pounds against M. tuberculosis H37Rv was performed using
0.1 mg/mL (RIFR and INHR), respectively. The nature of the the BACTEC 460 system (Becton Dickinson) (20). Clinical iso-
mutation conferring RIFR was rpoB (1S/531TTG) and the lates of M. tuberculosis were identified as drug resistant by
mutation conferring INHR was either inhA promoter (R1845) mutational analysis and genotyping (18). Multidrug-resistant
or katG (R1129 1S/315ACC) as determined by mutational anal- (MDR) isolates were confirmed resistant at the critical concen-
ysis (18).
trations of isoniazid (0.2 mg/mL), rifampicin (2.0 mg/mL) and
Ngwane et al.
615

IUBMB LIFE
ethambutol (2.5 mg/mL) (21). M. tuberculosis H37Rv reference
strain or M. tuberculosis clinical isolates were grown on 7H11
agar plates (Difco Laboratories, Detroit) and bacterial inocula
were prepared from 4- to 7-week-old plates by transferring
bacteria into capped plastic tubes containing 7H9 medium and
4 mm glass beads (Lasec, Johannesburg, South Africa). Bacte-
rial clumps were broken-up by vortexing. Suspensions of M.
tuberculosis (1 McFarland standard) (22), were inoculated into
BACTEC 12B (7H12 Middlebrook) medium when the vials
reached a growth index (GI) of 500. BACTEC 12B medium vials
containing various combinations of test compounds and antibi-
otics (7, 13, 14, all second-generation compounds, isoniazid,
rifampicin, and ethambutol) were then inoculated with pri-
mary culture. The growth of the bacilli, expressed as GI, was
monitored daily by measuring the release of 14CO2 (20).
Untreated controls (the undiluted and the 1:100 diluted bacte-
rial inocula) were included in each experiment. When the GI
of the 1:100 inoculum vial reached DGI [GI (current day) – GI
(previous day)] of 30 or greater, the experiment was stopped
(approximately 5–8 days incubation) (20). The MIC99 of a given
drug was defined as the lowest concentration at which the GI
of the vial in which the compound or the antibiotic was added
was less than or equal to the GI of the 1:100 control (20).
Reagents and conditions: (i) 1.05 equiv methyl/ethyl/
benzyl chloroformate, 1.1 equiv diisopropylethyl-
amine, CH2Cl2, 210 8C, 1.5 h, 48–50%; (ii) 1.1 equiv
phenol, 0.3–1.0 equiv CsF, CH2Cl2, rt, overnight, 72 to
80%; (iii) 2.0 equiv amine, THF, or N-methylpyrroli-
dine, rt, 2 h, 55 to 78%.
SCH 1
Time-Kill Studies in Relation to Mycobacterial Growth
M. tuberculosis cultures at 4.2 3 106 colony forming units
(CFU)/mL were treated with 14 at twofold increasing concen-
trations (range 2–128 mg/mL) for 21 days at 37 8C to establish
the bacteriostatic as well as the bactericidal effect of the anti-
mycobacterial agents. To determine the bactericidal or bacter-
iostatic effect after 1, 2, 5, 10, and 21 days of exposure, the
bacterial suspensions (200 mL) were centrifuged at 11,000g for
4 min at room temperature, washed in 7H9 broth supple-
mented with OADC and centrifuged again, as above. Washed
cells were 10-fold serially diluted and plated onto 7H11 agar
supplemented with OADC. After 15 to 21 days of incubation at
37 8C, CFUs were counted.
together are more effective than when they are used individu-
ally, suggesting synergistic effects. If x/y values fall between
0.5 and 0.75, an additive effect may exist between the two
drugs, suggesting a weak enhancement between them. Where
x/y values are greater than 2, it suggests antagonistic interac-
tion between the two drugs. When the window between x/y is
greater than 1 and less than 2, this may be interpreted as the
transition from no effect to antagonistic.
Cytotoxicity Assay
All synthetic first and second-generation compounds, including
14, 22, and 26 were tested for in vitro cytotoxicity against the
Chinese Hamster Ovarian (CHO) cell line using the 3-(4,5-
Chequerboard Synergy Assay
The chequerboard titration was used in which serial dilutions
of two antibiotics are studied for the effects on bacterial
growth inhibition at all possible concentration combinations.
The maximum and minimum concentrations of each diluted
compound or antibiotic were at least fourfold above or below
their MIC. The effects of synthetic compounds or antibiotics in
combination were evaluated using GI values obtained from the
BACTEC 460 system previously described (23). Synergy is
defined as x/y < 1/z, where x is the GI obtained when the two
antibiotics are in combination, y is the data for the lowest GI
value obtained when the two agents are tested separately at
the same concentration, and z is the number of drugs com-
bined. In order to translate the experimental data into the
classification of drug-drug interactions, the quotient x/y can be
used. If the x/y value is equal to 1, it is interpreted that one of
dimethiazol-2-yl)-2,5-diphenyltetrazolium
bromide
(MTT)
assay (24). The test samples were prepared as 2,000 mg/mL
solutions in 100% DMSO and were stored at 220 8C until use.
The highest concentration of DMSO to which the cells were
exposed to had no measurable effect on the cell viability. Eme-
tine (Sigma-Aldrich, St. Louis) was used as a positive control in
all cases. The initial concentration of emetine was 100 mg/mL,
which was serially diluted in complete medium with 10-fold
dilutions to give six concentrations, the lowest being 0.001 mg/
mL. The same dilution technique was applied to all test sam-
ples with an initial concentration of 100 mg/mL to give five con-
centrations, with the lowest concentration being 0.01 mg/mL.
The concentration of the test sample that inhibited 50% of the
cells (IC50 value) was obtained from a dose–response curve,
the drugs in the combination is inactive. If x/y is less than 0.5 prepared using a nonlinear dose–response curve fitting analy-
for a two-drug combination, it implies that the two drugs sis available in GraphPad Prism v. 2.01 software.
616
In Vitro Anti-TB Activity of Synthetic Furanones

Reagents and conditions: (i) excess ethanol/sec-butyl
alcohol, 98% H2SO4 cat., rt, 24 h, quantitative or (i)
0.75 equiv n-octanol, 98% H2SO4 cat., THF, rt, 48 h,
quantitative; (ii) 1.0–2.0 equiv amine, 1.0 equiv diiso-
propylethylamine, N,N-dimethylformamide, 70 to
90 8C, 24 h, 60 to 70%.
Chemical structures of the three most active first-
generation compounds against M. smegmatis
mc2 155.
SCH 2
FIG 1
furanone-ring was exchanged for a chlorine atom as the bro-
mide present in our first-generation compounds was proposed
as one initiator of mammalian cell toxicity. Laboratory experi-
ence with the first-generation compounds, led us to observe
that the 3-bromo-furanone derivatives decomposed when
exposed to air, light and/or heat, suggesting possible instability
in vivo as well. The second structural modification shows the
biphenyl group in compound 14 being replaced with an octyl
group (19 and 22), retaining lipophilic character of the mole-
cule while assessing spatial effects on compound efficiency.
We also postulated that the observed cytotoxicity of 14
could be attributed to the acetal moiety which is labile under
acidic conditions. The masked hemiacetal so formed would then
be effective in generating covalent linkages to nucleophilic
hydroxyl and amino substituents through its aldehyde form. In
order to overcome this liability, a series of derivatives were syn-
thesized containing a direct carbon-carbon linkage in place of
the acetal. To this end, compounds 23 and 24 were prepared
by Friedel-Crafts acylation of phenyl/biphenyl with mucochloric
acid 2. This was followed by the conjugate addition of various
amines, 4-(6-aminohexyl)-amino-7-chloroquinoline (25 and 26)
is used as a representative example in Scheme 3.
Results and Discussion
Synthetic Chemistry and Study Design
A library of related first-generation compounds were chemi-
cally synthesized from commercially available mucohalic acid
furanones as depicted in Scheme 1. 3,4-Dibromo-5-hydroxy-
2(5H)-furanone 1 and 3,4-dichloro-5-hydroxy-2(5H)-furanone
2 were transformed to their respective methyl, ethyl, and ben-
zyl carbonates, giving a labile group at carbon C-5, by the
treatment of the mucohalic acids with either methyl, ethyl, or
benzyl chloroformate in the presence of diisopropylethylamine
at a low temperature (25). The carbonates 3-8 were obtained
in average yields of 50%. Two sequential nucleophilic substitu-
tion reactions of the carbonate and b-halo groups by selected
phenols and 4-aminoquinoline diamine, respectively, delivered
the target compounds 13-16 in moderate to high unoptimized
yields (Scheme 1).
A second generation of furanone derivatives with improved
activity and cytotoxicity were synthesized from the same com-
mercially available mucochloric acid 2. The mucochloric acid
was protected on the alcohol (position C-5) with various
branched and unbranched alkanes under basic conditions
(Scheme 2). Following this, a nucleophilic substitution of the b-
halo group was performed using the same conditions
described in Scheme 1, to deliver target compounds 20-22 in
moderate to high unoptimized yields. In synthesizing second-
generation derivatives, the bromide in the C-3 position of the
Antibacterial Activity. Based on a preliminary screen of the
first-generation synthetic furanone-based compounds at 10
mg/mL against M. smegmatis mc2 155, three compounds (7,
13, and 14) were found to be active (chemical structures
Reagents and conditions: (i) 1 equiv phenyl/biphenyl,
1.5 equiv AlCl3, CH2Cl2, 0 8C 2rt, 20 h, 20 to 22%; (ii)
The percentage growth of M. smegmatis in the pres-
ence of the compounds 7, 13, and 14 (10 mg/mL) that
SCH 3
FIG 2
2.5 equiv amine, 1,4-dioxane, CHCl3, 50 8C, 24 h, 10
to 60%.
showed most activity. Compounds were tested in
duplicate in three independent experiments.
Ngwane et al.
617

IUBMB LIFE
Susceptibility testing of M. tuberculosis clinical
isolates with 14 at MIC (8 mg/mL)
TABLE 1
No. M. tubercu-
losis isolates
Percentage
inhibition (%)
Drug susceptibility
INH and RIF susceptible
INH, RIF and EMB susceptible
INH mono-resistant
20
1
>90
>90
>90
>90
>90
>90
2
RIF mono-resistant
1
M. tuberculosis H37Rv in the log phase (4.2 3 106
CFU/mL) of growth was exposed to 14 at various
concentrations (mg/mL) for 21 days at 37 8C. After
4 h, 1, 2, 5, 10, or 21 days of drug exposure, viable
cultures were determined by colony forming units
(CFU) counting.
INH and RIF resistant
1
FIG 4
INH, RIF and EMB resistant
1
shown in Fig. 1). Their activity was interpreted at 16 h of
growth and the percentage inhibition was calculated relative
to the untreated control (Fig. 2). The most active compounds
7, 13, and 14 showed 55%, 40%, and 38% inhibition, respec-
tively. Secondary screening of these three compounds was
carried out against M. tuberculosis H37Rv using the BACTEC
460 TB system. At the base-line testing concentration of
10 mg/mL, 7, 13, and 14 showed 36%, 20%, and 99% inhibi-
tion, respectively. The MIC99 for 14 was determined to be
8.07 mg/mL.
Clinical isolates including pan-susceptible and multidrug
resistant (MDR) strains showed similar susceptibility when
tested with 14 at the MIC99 of 8 mg/mL (Table 1). Specificity for
antimycobacterial activity was determined by testing 14
against Gram-positive and Gram-negative bacteria for growth
inhibition using the Bioscreen C system. There was no growth
inhibition in any of the bacterial species tested, even at con-
centrations seven times higher than the M. tuberculosis MIC.
Screening of the second-generation synthetic furanone-based
compounds at 10 mg/mL against M. tuberculosis, identified
compounds with improved activity (chemical structures shown
in Fig. 3). The MIC99 for 22 and 26 were determined to be
2.62 mg/mL and 3.07 mg/mL against M. tuberculosis H37Rv,
respectively.
Time-Kill Studies in Relation to Mycobacterial Growth.
A
bacteriostatic effect was defined as the lowest concentration
that inhibited visible growth on an agar plate at day 21 as
described (26). A bactericidal effect is defined as the killing
capacity of the agent expressed as the lowest concentration
that resulted in ꢀ99% killing at day 1, 2, 5, 10, or 21 (26). The
minimum bactericidal concentration (MBC) for mycobacteria is
defined as the minimal concentration effectively reducing bac-
terial counts by 99%. Compound 14 showed antimycobacterial
activity at all concentrations tested using CFU as shown in Fig.
4. INH showed a bactericidal effect, reducing the bacterial
count by more than 5 log units in 24 h (data not shown). We
observed no further effect of the INH, substantiating previous
Synergy quotient for 14 tested in two-drug
combinations with isoniazid (INH), rifampicin (RIF),
TABLE 2
or ethambutol (EMB) against M. tuberculosis H37Rv
Synergy quotients (x/y) in drug combinations (drug 1 0.5 3
MIC 14)
Drug
Dose
INH*
RIF**
EMB***
0.8 3 MIC
0.5 3 MIC
0.25 3 MIC
Quotients
0.65b 6 0.388 0.11c 6 0.098 0.75b 6 0.268
(mean x/y 6 SD)a
MICs: INH 5 0.05 mg/mL; RIF 5 0.4 mg/mL; EMB 5 1.6 mg/mL; 14 5 8 mg/
mL.
aData obtained from two separate experiments. Please refer to the
chequerboard synergy assay for x/y quotient definition.
bAdditive effect. < 0.75 but > 0.5.
Chemical structures for the two most active second-
generation compounds against M. tuberculosis
H37Rv.
cSynergy was defined as x/y < 1/z.
FIG 3
*0.04 mg/mL; **0.2 mg/mL;***0.4 mg/mL.
618
In Vitro Anti-TB Activity of Synthetic Furanones

Compound 14 tested in combination with rifampicin (RIF) against RIF mono-resistant M. tuberculosis isolate
TABLE 3
RIF-mono-resistant isolate
RIF (mg/mL)
14 (2 mg/mL)
100
50
25
12.5
6.25
Quotients (mean x/y 6 SD)
Interpretations
0.95 6 0.328
No effect
0.74 6 0.183
Additive
0.68 6 0.087
Additive
0.012 6 0.067
Synergy
0.21 6 0.095
Synergy
findings that INH becomes less effective in nonreplicating were observed at 12.5 and 6.25 mg/mL rifampicin (x/y 5 0.12
organisms (27). However, treatment with 14 shows a 2 log and 0.21, respectively) in combination with 2 mg/mL 14 (Table
3). The synergy of 14 in combination with rifampicin decreases
to an additive effect and then to no effect as the concentration
of rifampicin approaches its MIC in the resistant strain. How-
ever, 0.5 3 MIC of ethambutol in combination with rifampicin
at the same concentrations did not show either additive or
synergistic activity on a RIFR strain (data not shown). These
results suggest that the synergy between 14 and rifampicin
function in both susceptible and resistant M. tuberculosis
strains.
growth reduction of bacterial count at 24 hours (Fig. 4). Com-
pound 14 showed a bacteriostatic effect at the 8 mg/mL and a
bactericidal effect at 128 mg/mL.
Chequerboard Synergy Assay. Synergistic, additive, or antag-
onistic effects of drugs in combination must be evaluated at
concentrations that are below the level of their individual MICs
for effects to be observed. Table 2 shows the MIC of each indi-
vidual drug, as well as the quotient values for combinations of
14 with isoniazid, ethambutol, and rifampicin. The concentra-
tion of each of the drugs used in combination was expressed
as a fraction of the MIC value. Table 2 includes only the com-
binations at which synergy or additive effects were observed.
14 at 0.5 3 MIC showed synergy with rifampicin and an addi-
tive effect for both isoniazid and ethambutol when MIC frac-
tions were used as indicated in Table 2. No antagonistic effect
was observed for any of the two-drug combinations tested in
this study.
To examine whether the synergistic interaction between
14 and rifampicin also facilitated inhibition of drug-resistant
M. tuberculosis strains, we evaluated drug interaction with a
RIFR M. tuberculosis clinical isolate. The rifampicin MIC for
the RIFR M. tuberculosis R5182 strain was >100 mg/mL. The
MIC for compound 14 was 8 mg/mL, the same concentration
that was determined for rifampicin susceptible M. tuberculosis
H37Rv (Table 2). The RIFR strain showed minimal growth inhi-
bition in the presence of 100 mg/mL rifampicin, indicative of
resistance. Adding 0.25 3 MIC 14 (2 mg/mL) to rifampicin (6.25
mg/mL) inhibited growth by more than 99%. Synergistic effects
Cytotoxicity Assay. Toxicity tests are usually done in animal
models, but in order to reduce the cost of drug development,
preliminary in vitro toxicity assays are performed to ensure
that the least toxic compounds are pursued. For this purpose
14 was tested for cytotoxicity against the CHO cells using
the 3-(4,5-dimethiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay. We show that 14 has an IC50 of 1.82 mg/mL, 22
has an IC50 of 38.24 mg/mL, and 26 has an IC50 of 45.58 mg/mL
against CHO cells (Table 4). The selectivity index (SI) calcu-
lated as IC50 in CHO/MIC99 is 0.228 for 14, 14.64 for 22, and
14.85 for 26. The improved selectivity index values of 22 and
26 are due to the structural modifications. For 22 the bromide
in position C-3 of the furanone ring was exchanged for a chlo-
rine atom, with the concomitant replacement of the biphenyl
group with an octyl group, maintaining the lipophilicity of the
molecule (calculated cLogP values: 14 5.21 vs. 22 5.26). For
26 the acid labile acetal moiety was replaced with a direct
carbon-carbon linkage. From the observations it appears that
the bromine plays a primary role in mammalian cell cytotoxic-
ity and the acetal linkage a lesser role. The removal of these
liabilities has no deleterious impact on antitubercular activity,
suggesting that further development of the series is feasible.
Activity versus cytotoxicity data of the furanone-
based compounds
TABLE 4
Conclusion
MIC (mg/mL)
against
Cytotoxicity
(IC50, mg/mL)
in CHO cell-line
Selective
index (SI)
IC50/MIC99
In summary, we have been able to identify furanone-based
compounds which have antimycobacterial activity with low
cytotoxicity against a human cell line. Of the first-generation
compounds tested, three active compounds were identified. 14
has a higher MIC than current anti-TB drugs; however, we
were able to demonstrate that in combination with the current
anti-TB drug rifampicin there is synergism of potency. 14 is
Compound
H37Rv
14
22
26
8.07 6 0.073
2.62 6 0.08
3.07 6 0.09
1.82 6 0.15
38.24 6 0.236
45.58 6 0.2
0.228
14.64
14.85
Ngwane et al.
619

IUBMB LIFE
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Of the second-generation compounds tested, two active
compounds were identified (22 and 26). Compound 22 was
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compared to the parental compound 14. In addition, com-
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further investigated via time-kill and synergy studies for future
drug development.
Acknowledgements
Financial support for this work was given by the Department
of Science and Technology (TB-TAP Program T80006), South
Africa and Stellenbosch University, Division of Molecular Biol-
ogy and Human Genetics, South Africa. The Columbia Univer-
sity–Southern Africa Fogarty AIDS Training and Research Pro-
gram provided additional support to A.N. Authors thank
National Drug Developmental Platform (NDDP) consortium and
Dr. E. Streicher, Mrs. M. De Kock, and Dr. G. Louw from the
CBTBR at Stellenbosch for providing clinical isolates and tech-
nical assistance.
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