Characterization of the Saffron Derivative Crocetin as an Inhibitor of Human Lactate Dehydrogenase 5 in the Antiglycolytic Approach against Cancer

Article abstract of DOI:10.1021/acs.jafc.7b01668

Inhibition of lactate dehydrogenase (LDH) represents an innovative approach to tackle cancer because this peculiar glycolytic metabolism is characteristic of most invasive tumor cells. An investigation into the biological properties of saffron extracts led to the discover of their LDH-inhibition properties. In particular, the most important saffron components, crocetin, was found to inhibit LDH (IC₅₀ = 54.9 ± 4.7 μM). This carotenoid was independently produced by chemical synthesis, and its LDH-inhibition properties manifested via its antiproliferative activity against two glycolytic cancer cell lines (A549 and HeLa, IC₅₀ = 114.0 ± 8.0 and 113.0 ± 11.1 μM, respectively). The results described in this article suggest that saffron may be a helpful alimentary component in the prevention of cancer that potentially contributes to the efficacy of approved cancer therapies.

Full text of DOI:10.1021/acs.jafc.7b01668

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Article
Characterization of the Saffron Derivative Crocetin as an Inhibitor of Human
Lactate Dehydrogenase 5 in the Antiglycolytic Approach against Cancer
Carlotta Granchi, Serena Fortunato, Serena Meini, Flavio Rizzolio, Isabella Caligiuri,
Tiziano Tuccinardi, Hyang Yeon Lee, Paul J Hergenrother, and Filippo Minutolo
J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 23 Jun 2017
Downloaded from http://pubs.acs.org on June 23, 2017
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Characterization of the Saffron Derivative Crocetin as an
Inhibitor of Human Lactate Dehydrogenase 5 in the
Antiglycolytic Approach against Cancer
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Carlotta Granchi,a Serena Fortunato a Serena Meini,a Flavio Rizzolio,c Isabella Caligiuri,d
Tiziano Tuccinardi,a,e Hyang Yeon Lee,b Paul J. Hergenrother,b Filippo Minutolo,a,e,*
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a Dipartimento di Farmacia, Università di Pisa, Via Bonanno 6 and 33, 56126 Pisa, Italy.
b
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Department of Chemistry, University of Illinois at Urbana-Champaign, 600 S. Mathews, Urbana,
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Illinois 61801, USA.
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c Dipartimento di Scienze Molecolari e Nanosistemi, Università Ca’Foscari, Venezia, Italy.
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Division of Experimental and Clinical Pharmacology, Department of Molecular Biology and
Translational Research, National Cancer Institute and Center for Molecular Biomedicine, Aviano
(PN), Italy
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Centro Interdipartimentale di Ricerca "Nutraceutica e Alimentazione per la Salute”, Università di
Pisa, Via del Borghetto 80, 56124 Pisa, Italy.
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AUTHOR INFORMATION
Corresponding Author
*Corresponding author phone: +39 050-2219557; fax +39 050-2210680; e-mail:
filippo.minutolo@unipi.it
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Title running header: Crocetin inhibits LDH in glycolytic cancer cells
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ABSTRACT
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Inhibition of lactate dehydrogenase (LDH) represents an innovative approach to tackle cancer
because this peculiar glycolytic metabolism is characteristic of most invasive tumor cells. An
investigation into the biological properties of saffron extracts led to the discover of their LDH-
inhibition properties. In particular, the most important saffron components, crocetin, was found to
inhibit LDH (IC50 = 54.9 ± 4.7 µM). This carotenoid was independently produced by chemical
synthesis, and its LDH-inhibition properties manifested via its antiproliferative activity against two
glycolytic cancer cell lines (A549 and HeLa, IC50 = 114.0 ± 8.0 and 113.0 ± 11.1 μM, respectively).
The results described in this article suggest that saffron may be a helpful alimentary component in
the prevention of cancer that potentially contributes to the efficacy of approved cancer therapies.
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KEYWORDS
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Saffron;
cancer;
crocetin;
lactate dehydrogenase;
glycolysis
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INTRODUCTION
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Inhibition of lactate dehydrogenase (LDH) as a potential anticancer strategy is rationalized via Otto
Warburg’s observation that cancer cells rapidly internalize and consume glucose to survive, which in
turn produces large amounts of lactate.1 In the last decade, a great interest was directed towards the
discovery of antiglycolytic agents capable of interfering with the glycolytic metabolism of cancer
cells.2,3 In this context, the human homotetrameric isoform 5 of lactate dehydrogenase (hLDH-5),
composed of four A subunits, is considered a strategic target to selectively disrupt the metabolism of
cancer cells. Moreover, hLDH-5 is overexpressed in many types of tumours. hLDH-5 catalyzes the
last step of glycolysis - the reversible reduction of pyruvate to lactate via the simultaneous oxidation
of NADH to NAD+. Considering the essential role of this enzyme in the regeneration of NAD+ and
continuation of glycolysis, we hypothesized its inhibition should lead to cancer cell death by
starvation. Individuals that lack of hLDH-5 experience myoglobinuria after intense anaerobic
exercise yet are healthy under ordinary circumstances. Therefore, we hypothesize that the inhibition
of hLDH-5 is a potentially safe approach to treating cancer that will avoid the deleterious side-effects
associated with traditional chemotherapy.4 Previously, hLDH-5 was silenced with siRNA, resulting
in an evident decrease in cancer cell proliferation and migration.5 These results laid the groundwork
for the development and the synthesis of many small molecules as hLDH-5 inhibitors and several
proved to be potent inhibitors both in vitro and in vivo.6 However, the search for new potent inhibitors
remains a challenging goal, since the cavity of the enzyme comprises both a cofactor (NADH)- and
a substrate (pyruvate)-binding pocket. The substrate-binding site is very narrow, polar, and possesses
many positively charged residues, whereas, the cofactor binding region is quite extended since NADH
binds to the enzyme with the nicotinamide and adenosine portions at opposite ends of this site.
Several natural products have been identified as hLDH-5 inhibitors. One of the first examples is
represented by the natural derivative gossypol (Figure 1), which is extracted from the cotton seeds of
the Gossypium species. Gossypol was initially studied as an antimalarial agent, due to its inhibitory
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activity of the Plasmodium falciparum isoform of LDH (pfLDH). However, this compound proved
to be a non-selective competitive inhibitor of LDH relative to NADH through its inhibition of the
human LDH isoforms. Additionally, the high toxicity of gossypol (including cardiac arrhythmias,
renal failure, muscle weakness and even paralysis) hampered its further development as an anticancer
agent.7 Later, the flavonoid epigallocatechin8 (Figure 1) and the fungal metabolite panepoxydone9
(Figure 1) exerted an indirect inhibitory effect on hLDH-5 by reducing its expression in cancer cells.
Conversely, the α,β-unsaturated aldehyde 4‑hydroxy-2-nonenal (Figure 1), which naturally forms
upon oxidation of unsaturated fatty acids, covalently binds the enzyme and blocks its activity.10 The
recently identified hLDH-5 inhibitor penta-1,2,3,4,6-O-galloyl-β-D-glucose (Figure 1), a component
of Rhus Chinensis gallnut extract, directly inhibits the enzyme via competitive displacement of the
cofactor NADH.11 Galloflavin (Figure 1), a compound formed via autooxidation of the natural
compound gallic acid, was identified by virtual screening of compounds from National Cancer
Institute dataset and proved to inhibit both hLDH-5 and hLDH-1 with Ki values in the micromolar
range and retard cancer cell growth.12 Luteolin 7-O-β-D-glucopyranoside (Figure 1) isolated from
Phlomis kurdica, inhibits hLDH-5 with an IC50 value in the micromolar range. It was shown to be
competitive with NADH; a conclusion supported by modelling studies.13 Moreover, a high-
throughput screening of medicinal plants identified some natural extracts, such as Chinese Gallnut
(Melaphis chinensis gallnut), Bladderwrack (Fucus vesiculosus), Kelp (Laminaria Japonica) and
Babul (Acacia Arabica) that showed hLDH-5 IC50 values lower than 0.001 mg/ml, although the
individual chemical constituents responsible for the activity were not isolated.14
Given that a growing number of natural products have demonstrated the ability to directly and
indirectly hLDH-5, we screened a large panel of natural products that are mostly found in alimentary
sources for the purpose of identifying new natural products as food-derived hLDH-5 inhibitors. As
such, we sought to identify a possible nutraceutical strategy to produce safe antiglycolytic effects that
might prove to be helpful for the prevention and treatment of cancer. In particular, we were intrigued
by the well-known anticancer properties of saffron, which is one of the most expensive spices in the
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world and widely known for its deep yellow colour and exquisite taste. Saffron has long been used
as a medicinal herb to treat several diseases in traditional medicine, and it has found many therapeutic
applications resulting from the plethora of pharmacological properties of its components.15-18 Saffron
consists of the dried red stigmas of Crocus sativus Linnaeus flowers, and its main components can
be grouped in three principle classes: 1) crocins: water-soluble coloured compounds that are
glycosides of the carotenoid crocetin with various sugar ester moieties; 2) picrocrocin: a monoterpene
glycoside that is the main substances responsible for saffron’s bitter taste; 3) safranal: the volatile oil
responsible for the characteristic saffron aroma (Figure 2).19,20
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In order to investigate if any of the saffron components possess anti-LDH activity and contribute to
its reported anticancer activity,16 we started our study by analysing the effects of this spice on the
isolated enzyme. Furthermore, among the main components of saffron, it is widely documented that
both crocin, the crocetin digentiobiose ester, which represents the most important glycoside
carotenoid,21-24 and also the carotenoid aglycon itself, crocetin, exert anticancer and chemopreventive
effects on several types of cancer cells, and some in vivo animal models.25-29 It is important to note
that the average content of crocin, which behaves as a direct bioprecursor of crocetin when
administered in vivo,30 is about 40 mg/g of stigmas, depending on the geographical location of the
saffron source.31 Furthermore, the bioavailability of crocetin after oral administration to human
volunteers proved to be highly satisfactory. It was rapidly absorbed and reached maximal plasma
concentration of up to 1 uM after 4-5 hours even at the relatively low dose of 22.5 mg.32
Interestingly, Kim et al. recently reported the effects of crocetin and crocin in several cancer cell
lines. Crocetin induced cell death at submillimolar concentrations (IC50 values ranging from 0.16 to
0.61 mM in a panel of cancer cells). However, crocin proved to be more potent in the cytotoxicity
assays (IC50 values ranging from 2.87 to 5.48 µM). Both expression and activity of hLDH-5 were
reduced after treatment of HeLa cells with crocetin and crocin: even in this case, crocetin was more
potent than crocin, since crocetin at 1 mM reduced hLDH-5 activity by 34.2 % compared to control
and crocin reached only a 10.5% inhibition at 4 mM. However, these cell-based experiments did not
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determine whether crocetin or crocin directly interact with hLDH-5 because various mechanisms
could be implicated at the cellular level.33 It could be predicted that the long carbon chains of these
derivatives, characterized by polar moieties (carboxylic groups or sugar portions) at both the
extremities, are well tolerated in the LDH active site. These preliminary results encouraged us to carry
on further detailed studies in order to clarify the specific effects of crocetin and crocin on hLDH-5
and in cancer cells, as described below.
MATERIALS AND METHODS
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Chemicals. General Procedures and Materials. All solvents and chemicals were used as purchased
without further purification (Aldrich, Alfa Aesar, TCI Europe). The following chemicals were used
for the synthesis of the final compounds and were obtained from commercial suppliers: trans-2-
methyl-2-butenoic acid, methanol, sulfuric acid, diethyl ether, chloroform, N-bromosuccinimide,
benzoylperoxide
(70
%,
remainder
water),
fumaraldehyde
mono(dimethylacetal),
(carbethoxyethylidene)triphenylphosphorane, triphenylphosphine, activated manganese dioxide,
acetone, 1 M diisobutylaluminum hydride solution in anhydrous hexane, Amberlyst® 15 hydrogen
form, Celite® Hyflo, ethyl acetate, hexane, dichloromethane (DCM), toluene, acetonitrile,
phosphoric acid, sodium hydroxide, sodium sulphate anhydrous, potassium carbonate.
Chromatographic separations were performed on silica gel columns by flash chromatography
(Kieselgel 40, 0.040−0.063 mm; Merck). Reactions were followed by thin layer chromatography
(TLC) on Merck aluminum silica gel (60 F254) sheets that were visualized under a UV lamp.
Evaporation was performed in vacuo (rotating evaporator). Sodium sulfate was always used as the
drying agent. Proton (1H) and carbon (13C) NMR spectra were obtained with a Bruker Avance III 400
MHz spectrometer using the indicated deuterated solvents. Chemical shifts are given in parts per
1
13
million (ppm) (δ relative to residual solvent peak for H and C). 1H-NMR spectra are reported in
this order: multiplicity and number of protons. Standard abbreviation indicating the multiplicity were
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used as follows: s = singlet, d = doublet, dd = doublet of doublets, ddd = doublet of doublet of
doublets, t = triplet, q = quartet, dq = doublet of quartets, tq = triplet of quartets, qq = quartet of
quartets, quint = quintet, m = multiplet, bq = broad quartet and bd = broad doublet.
(E)-Methyl 2-methylbut-2-enoate (2). Trans-2-methyl-2-butenoic acid 1 (5.0 g; 50 mmol) was
dissolved in MeOH (50 mL) in a sealed vial, concentrated H2SO4 (0.27 mL) was added dropwise and
the resulting solution was heated at 90 °C for 24 h. The reaction mixture was cooled to room
temperature and concentrated under reduced pressure. The resulting mixture was diluted with Et2O,
and then washed successively with a saturated solution of sodium bicarbonate and brine. The organic
phase was dried and evaporated under reduced pressure. The crude methyl tiglate 2 was obtained as
a light yellow oil (34.1 mmol, yield 63%) and was used in the next step without further purification.
1H NMR (CDCl3) δ (ppm): 1.79 (dq, 3H, J = 7.1, 1.2 Hz), 1.83 (quint, 3H, J = 1.2 Hz), 3.73 (s, 3H),
6.86 (qq, 1H, J = 7.1, 1.4 Hz).
(E)-Methyl 4-bromo-2-methylbut-2-enoate (3) and (Z)-methyl 2-(bromomethyl)but-2-enoate (4).
Compound 2 (2.0 g; 18 mmol) was dissolved in CHCl3 (23.0 mL), then NBS (18 mmol) and 70%
BPO (0.7 mmol) were added and the reaction was heated at reflux for 2 h. After cooling to room
temperature, the mixture was concentrated under reduced pressure. Hexane was added to the residue
and the resulting suspension was cooled in an ice bath to promote the precipitation of succinimide,
which was suction filtered off. The filtrate was evaporated. The residue was diluted with AcOEt and
washed with brine. The organic phase was dried and evaporated under vacuum. Mixture of γ and α-
1
bromo derivatives was obtained as an orange oil (3.08 g). H NMR (CDCl3) δ (ppm, assigned only
significant protons): 3.77 (s, COOMe γ isomer), 3.80 (s, COOMe α isomer), 4.03 (dq, J = 8.5, 0.5
Hz, CH2 γ isomer), 4.24 (s, CH2 α isomer), 6.93 (tq, J = 8.5, 1.5 Hz, vinylic proton γ isomer), 7.08 (q,
J = 7.3 Hz, vinylic proton α isomer). Proton integration of these signals suggested the following
composition: 68 % γ isomer, 32 % α isomer.
(E)-(4-Methoxy-3-methyl-4-oxobut-2-en-1-yl)triphenylphosphonium bromide (5). Mixture of 3 and
4 (3.08 g) dissolved in toluene (13.7 mL) was treated dropwise with a freshly prepared solution of
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triphenylphosphine (21.6 mmol) in toluene (21.0 mL) and the reaction mixture, which became
immediately cloudy, was stirred vigorously overnight. A white crystalline solid and a yellow gummy
solid adherent to the walls of the flask formed: the white crystalline solid was suction filtered, washed
with hexane and dried under vacuum and the yellow gummy solid was dissolved in MeOH and dried
under vacuum. Both the fractions were purified by recrystallization from CH3CN/AcOEt. Compound
5 (2.08 g, 4.57 mmol) was obtained as a white crystalline solid (yield on two subsequent steps: 26
%).1H NMR (CDCl3) δ (ppm): 1.67 (d, 3H, J = 3.3 Hz), 3.68 (s, 3H), 5.08 (dd, 2H, J = 16.3, 8.0 Hz),
6.64 (bq, 1H, J = 6.7 Hz), 7.64-7.94 (m, 15H).
(E)-Methyl 2-methyl-4-(triphenylphosphoranylidene)but-2-enoate (6). To a solution of phosphonium
salt 5 (1.06 g, 2.32 mmol) in CH2Cl2 (9.0 mL) was added dropwise aqueous NaOH 0.5 M (4.6 mL)
and the mixture became immediately orange. After stirring for 10 minutes, the mixture was diluted
with water and extracted with CH2Cl2, the organic layer was washed with brine, dried and
concentrated under reduced pressure. Crude compound 6 (800 mg, 2.14 mmol) was obtained as an
orange oily solid and used in the subsequent steps without further purification (yield 92%).1H NMR
(CDCl3) δ (ppm, assigned only significant protons): 1.89 (s, 3H, CH3), 3.58 (s, 3H, COOMe).
(2E,4E)-Ethyl 2-methyl-6-oxohexa-2,4-dienoate (9).
A
solution of fumaraldehyde
mono(dimethylacetal) 8 (300 mg; 0.29 mL, 2.31 mmol) in CH2Cl2 (3.6 mL) was added dropwise to
a solution of (carbethoxyethylidene)triphenylphosphorane 7 (1.09 g; 3.00 mmol) in CH2Cl2 (8.9 mL).
The reaction was stirred at RT overnight. Solvent was reduced under reduced pressure and then the
residue was cooled in an ice bath to promote the precipitation of triphenylphosphine oxide, that was
suction filtered off and washed with petroleum ether. The filtrate was concentrated, affording the
dimethylacetal product in mixture with the corresponding aldehyde derivative, as a white solid (560
mg). The mixture was directly subjected to the next step without further purification. The residue was
dissolved in acetone (9.6 mL), then distilled water (0.15 mL) and Amberlyst 15 (prewashed with
acetone, 102 mg) were added and the reaction was stirred for 1 h. After filtration to remove the resin,
the filtrate was concentrated and purified by flash chromatography over silica gel (n-hexane/Et2O
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85:15). Compound 9 was obtained as a yellow oil (355 mg, 2.11 mmol, yield was calculated on two
subsequent steps: 91%). 1H NMR (CDCl3) δ (ppm): 1.33 (t, 3H, J = 7.1 Hz), 2.12 (d, 3H, J = 1.4 Hz),
4.27 (q, 2H, J = 7.1 Hz), 6.39 (dd, 1H, J = 15.0, 7.8 Hz), 7.33 (dq, 1H, J = 11.8, 1.3 Hz), 7.42 (dd,
1H, J = 14.9, 11.8 Hz), 9.70 (d, 1H, J = 7.6 Hz).
(2E,4E,6E)-Diethyl 2,7-dimethylocta-2,4,6-trienedioate (10). Compound 9 (350 mg, 2.08 mmol) was
dissolved in DCM (15.4 mL) and (carbethoxyethylidene)triphenylphosphorane 7 (2.72 mmol) was
added. The reaction was stirred at room temperature for 2 h. The mixture was filtered through a
Büchner funnel partially filled with silica gel and washed with AcOEt. The filtrate was purified by
flash chromatography over silica gel (n-hexane/AcOEt 95:5). Compound 10 was obtained as a pearl-
1
white solid (342 mg, 1.36 mmol, yield 65%). H NMR (CDCl3) δ (ppm): 1.32 (t, 6H, J = 7.1 Hz),
2.01 (d, 6H, J = 1.2 Hz), 4.23 (q, 4H, J = 7.1 Hz), 6.80 (dd, 2H, J = 7.8, 3.1 Hz), 7.28 (dq, 2H, J =
7.9, 1.4 Hz).
(2E,4E,6E)-2,7-Dimethylocta-2,4,6-triene-1,8-diol (11). Compound 10 (340 mg, 1.35 mmol) was
dissolved in anhydrous hexane (10.4 mL) under Argon atmosphere, cooled to -78 °C and treated
dropwise with a solution 1 M of DIBAL-H in anhydrous hexane (5.3 mL, 5.26 mmol). The resulting
solution was stirred at the same temperature for 5 min and warmed slowly till -20 °C over 3 h. The
reaction was monitored by TLC, showing that the diester was completely reacted. The reaction
mixture was cooled to 0 °C and distilled water (0.3 mL) and silica gel (900 mg) were added. After 45
minutes, K2CO3 (322 mg) and Na2SO4 (488 mg) were added. After 30 minutes, the solids were suction
filtered off and rinsed with CH2Cl2 and AcOEt, the filtrate was concentrated to obtain compound 11
as a light-yellow solid (214 mg, 1.27 mmol, yield 94%) that was used in the next step without further
1
purification. H NMR (CDCl3) δ (ppm): 1.82 (s, 6H), 4.11 (s, 4H), 6.16 (dq, 2H, J = 7.5, 1.4 Hz),
6.45 (dd, 2H, J = 7.3, 3.1 Hz).
(2E,4E,6E)-2,7-Dimethylocta-2,4,6-trienedial (12). Compound 11 (214 mg, 1.27 mmol) was
dissolved in acetone (11.5 mL) and the solution was cooled to 0 °C. Activated MnO2 (3.32 g, 38.2
mmol) was added and the reaction was stirred overnight at room temperature. The mixture was
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filtered in a Büchner funnel filled with celite Hyflo® and washed with acetone. The filtrate was
concentrated under reduced pressure and purified by flash chromatography over silica gel (n-
hexane/AcOEt 85:15). Compound 12 (152 mg, 0.927 mmol) was obtained as a bright yellow solid
(yield 73%). 1H NMR (CDCl3) δ (ppm): 1.95 (d, 6H, J = 1.2 Hz), 6.97-7.13 (m, 4H), 9.55 (s, 2H).
(2E,4E,6E,8E,10E,12E,14E)-Dimethyl-2,6,11,15-tetramethylhexadeca-2,4,6,8,10,12,14-
heptaenedioate (13). A solution of compound 6 (1.83 mmol) in toluene (3.5 mL) was added to
compound 12 (50.0 mg, 0.305 mmol) in toluene (1.0 mL) and the reaction was heated at 130 °C for
6 h. Then, the mixture was cooled to room temperature and left under stirring overnight. Compound
13 precipitated as a red brick solid, which was collected by filtration and washed with cold MeOH.
The filtrate was concentrated under reduced pressure, the residue was taken up in 5 mL of MeOH and
heated at 90°C for 30 minutes. The mixture was cooled to room temperature and a second fraction of
pure compound 13 was collected. The total amount of pure compound 13 obtained was 61.4 mg
(0.172 mmol, yield 56%). 1H NMR (CDCl3) δ (ppm): 1.99 (s, 6H), 2.00 (d, 6H, J = 1.0 Hz), 3.77 (s,
6H), 6.34-6.40 (m, 2H), 6.55 (dd, 2H, J = 15.2, 10.4 Hz), 6.62 (d, 2H, J = 14.8 Hz), 6.71 (dd, 2H, J
= 7.9, 2.9 Hz), 7.29 (dd, 2H, J = 10.7, 1.3 Hz). 13C NMR (CDCl3) δ (ppm): 12.94 (2C), 13.04 (2C),
51.95 (2C), 123.97 (2C), 126.62 (2C), 131.48 (2C), 135.16 (2C), 136.87 (2C), 139.04 (2C), 143.90
(2C), 169.09 (2C).
Sodium
(2E,4E,6E,8E,10E,12E,14E)-2,6,11,15-tetramethylhexadeca-2,4,6,8,10,12,14-
(14) and (2E,4E,6E,8E,10E,12E,14E)-2,6,11,15-tetramethylhexadeca-
heptaenedioate
2,4,6,8,10,12,14-heptaenedioic acid (15). Diester 13 (25.0 mg, 0.0701 mmol) was suspended in
MeOH (0.5 mL) and 40 % w/v NaOH (0.7 mL, 7.01 mmol) was added dropwise. The mixture was
heated at reflux overnight. After consumption of the starting material, the mixture was cooled to room
temperature and then to 0 °C and an orange precipitated formed. The solid was collected by filtration,
washing it with cold water and cold MeOH, affording 21.9 mg (0.588 mmol) of the disodium salt 14
as a bright orange solid (yield 84 %). In order to obtain the diacid 15, the work-up was accomplished
by evaporating the solvent of the reaction, then the residue was diluted with water and the aqueous
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phase was washed with Et2O. The aqueous layer was acidified with 10 % H3PO4, to obtain the
precipitation of a red solid that was collected and washed with water and CH2Cl2 (yield 68 %). For
1
disodium salt 14 H NMR (D2O) δ (ppm): 1.95 (s, 6H), 2.00 (s, 6H), 6.43-6.48 (m, 2H), 6.65-6.70
13
(m, 4H), 6.85 (dd, 2H, J = 7.9, 2.9 Hz), 6.97-7.02 (m, 2H). C NMR (D2O) δ (ppm): 11.92 (2C),
13.42 (2C), 124.86 (2C), 131.13 (2C), 133.37 (2C), 133.96 (2C), 134.85 (2C), 137.31 (2C), 141.46
(2C), 177.94 (2C). For diacid 15 1H NMR (DMSO-d6) δ (ppm): 1.91 (s, 6H), 1.97 (s, 6H), 6.43-6.53
(m, 2H), 6.61 (dd, 2H, J = 14.8, 11.6 Hz), 6.72 (d, 2H, J = 15.2 Hz), 6.78-6.88 (m, 2H), 7.20 (bd, 2H,
J = 10.8 Hz).
Enzyme assays. The compounds were evaluated in enzymatic assays to assess their inhibitory
properties against two commercially available purified human isoforms of lactate dehydrogenase,
hLDH-5 (tetrameric isoform composed of four A subunits, LDH-A4, from human liver, Lee
BioSolutions – USA) and hLDH-1 (tetrameric isoform composed of four B subunits, LDH-B4, from
human erythrocytes, Lee BioSolutions – USA), as suspensions in 3.1 M ammonium sulfate solution,
with tris chloride, DTT and EDTA, pH 8.3 Dried flower stigmas (saffron pistils) and crocin were
purchased from Sigma-Aldrich. The reaction of lactate dehydrogenase was conducted using the
“forward” direction (pyruvate → lactate) by using an emission wavelength at 460 nm and an
excitation wavelength at 340 nm to monitor the amount of NADH consumed. These assays were
conducted in wells containing 200 μL of a solution comprising the reagents dissolved in 100 mM
phosphate buffer at pH 7.4. DMSO stock solutions of compounds were prepared (the concentration
of DMSO did not exceed 4% during the measurements). Assays were performed in the presence of
40 μM NADH and 200 μM pyruvate, combined with 0.66 ng or 0.38 mU of enzyme in a final volume
of 200 µl (one unit of the enzyme reduces 1 µmol of pyruvate to L-lactate at 37 °C and pH 8.55).
Seven different concentrations (in duplicate for each concentration) of the compound were used to
generate a concentration–response curve. Compound solutions were dispensed in 96-well plates (8
μL), and then the substrate and the cofactor dissolved in the buffer (152 μL) and the enzyme solution
(40 μL) were added. Any possible background fluorescence of the tested compounds, or their
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quenching of NADH fluorescence, was subtracted. In addition to the compound test wells, each plate
contained maximum and minimum controls. Assay plates were incubated for 15 min, and the final
measurements were performed using a Victor X3 Microplates Reader (PerkinElmer®). IC50 values
were generated using the curve-fitting tool of GraphPad Prism software (GraphPad – USA). In the
enzyme kinetic experiments, the same procedure previously reported by us was followed.13 The
compound was tested in the presence of scalar NADH or pyruvate concentrations in the NADH or
pyruvate-competition experiments, respectively. In the NADH-competition experiments, the
compound was added (concentration range = 15-45 µM) to a reaction mixture containing 1.4 mM
pyruvate, scalar concentrations of NADH (concentration range = 10–150 µM) and 100 mM phosphate
buffer (pH = 7.4). Conversely, in the pyruvate-competition experiments, the reaction mixture
contained 150 µM NADH and scalar concentrations of pyruvate (concentration range = 40–500 µM).
Finally, LDH solution was added (0.015 U/mL) and the enzyme activity was measured by evaluating
the NADH fluorescence decrease using a Victor X3 Microplates Reader. The experimental data were
analysed with a non-linear regression analysis, using a second order polynomial regression analysis,
and by applying the mixed-model inhibition fit.
Cell lines and cell culture. A549 cell lines were obtained from ATCC and grown in RPMI 1640
medium with 10 % fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin. HeLa cell
lines were obtained from ATCC and grown in EMEM with 10 % fetal bovine serum, 100 U/mL
penicillin, and 100 µg/mL streptomycin. Cells were cultured at 37 °C in a 5 % CO2– 95% air
humidified atmosphere.
Cytotoxicity evaluation. HeLa and A549 cells were seeded on 96 well plates at the density of 1 x
103 cells per well. The cells were continuously treated with various concentrations of compound 14
for 72 h. Then viability was assessed using the sulforhodamine B (SRB) assay. Cells were fixed with
10% trichloroacetic acid at 4 °C for overnight and then stained with 0.057% SRB in 1 % acetic acid
at room temperature for 30 min. The dye was solubilized in 10 mM Tris base (pH 10.5) and absorption
at 510 nm was measured using a SpectraMax Plus 384 (Molecular Devices, Sunnyvale, CA). Percent
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death was calculated by subtracting background from all wells and setting 0% death to vehicle-treated
controls. All data are averages of at least three independent replicates. MRC5 cells (from ATCC)
were maintained at 37 °C in a humidified atmosphere containing 5% CO2 accordingly to the supplier.
Normal (1.5 × 104) cells were plated in 96-well culture plates. The day after seeding, vehicle or
compound were added at different concentrations to the medium. Cell viability was measured after
96 h according to the supplier (Promega, G7571) with a Tecan F200 instrument. IC50 values were
calculated from logistical dose response curves. Averages were obtained from three independent
experiments, and error bars are standard deviations (n = 3).
Assessment of lactate production. HeLa human cervical carcinoma cells were seeded on 96 well
plates at the density of 2.5 x 103 cells per well. When cells reached 80-90 % confluence, the cells
were treated with compound or vehicle control in DMEM medium minus phenol red + 10% dialized
FBS + 100 U/mL penicilin + 100 µg/mL streptomycin for 4 h. Triplicate wells were prepared for each
treatment. Following treatment, the cells were fixed with 10 % trichloroacetic acid for the assessment
of cell viability using SRB assay and the media were collected, and 100 µL were added to 10 µL 10
mM chlorophenylalanine (CPA; the internal standard for GC-MS analysis). Samples were
concentrated, derivatized by a 2h incubation with 50 µL MTBSTFA + 1% TBDMCS (Thermo
Scientific, Walthman, MA) in 50 µL acetonitrile at 80 °C, and immediately analyzed using GC-MS
(Agilent 6890N GC/5973 MS, equipped with an Agilent DB-5 capillary column, 30 m x 320 µm x
0.25 µm, model number J&W 123-5032, Agilent Technologies, Santa Clara, CA) and electron impact
ionization source. The initial oven temperature was 120 °C, held for 5 min; then the temperature was
increased at a rate of 10 °C per minute until a temperature of 250 °C was reached. The temperature
was then increased by 40 °C per minute until a final temperature of 310 °C was reached. The total
run time per sample was 24.5 min. Compounds were identified using AMDIS Chromatogram
software (Amdis, freeware available from amdis.net) and programmed WIST and Niley commercial
libraries. The integration area of lactate in each sample was divided by the integration area of CPA
in the same sample to achieve a lactate/internal standard ratio. The ratios were normalized by % live
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and averaged for triplicates. The percentage lactate production over vehicle was calculated and all
data are averages of three independent replicates.
Statistical Analysis. All statistical analysis was performed using an unpaired, two-tailed student’s t
test with p values < 0.05 were considered statistically significant. Values are reported as the means ±
SD of three or more independent experiments.
Docking Calculations. The crystal structure of the hLDH-5 protein (4M49 PDB code34), was taken
from the Protein Data Bank.35 After adding hydrogen atoms, the protein was minimized using
Amber14 software and the ff14SB force field at 300 K (in order to reproduce the room temperature
used in the enzymatic assay).. The complex was placed in a rectangular parallelepiped waterbox, an
explicit solvent model for water, TIP3P, was used and the complex was solvated with a 10 Å water
cap. Sodium ions were added as counterions to neutralize the system. Two steps of minimization were
then carried out; in the first stage, we kept the protein fixed with a position restraint of 500 kcal/molÅ2
and we solely minimized the positions of the water molecules. In the second stage, we minimized the
entire system through 5000 steps of steepest descent followed by conjugate gradient (CG) until a
convergence of 0.05 kcal/Åmol. The ligands were built using Maestro and were minimized by means
of Macromodel in a water environment using the CG method until a convergence value of 0.05
kcal/Åmol, using the MMFFs force field and a distance-dependent dielectric constant of 1.0.
AUTODOCK Tools,36 was used to define the torsion angles in the ligands, to add the solvent model
and to assign partial atomic charges to the ligand and the protein. The docking site used for
AUTODOCK calculations was defined in such a manner that it was constituted by all residues within
10 Å of the reference ligand and NADH co-factor in the X-ray crystal structure. The energetic maps
were calculated using a grid spacing of 0.375 Å and a distance dependent function of the dielectric
constant. The ligand was subjected to 200 docking runs of the AUTODOCK search using the
Lamarckian genetic algorithm (LGA) and employing 10 000 000 energy evaluations; the number of
individuals in the initial population was set to 500 and a maximum of 10 000 000 generations were
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simulated during the docking run; an rms tolerance of 2.0 Å was used to carry out the cluster analysis
of the docking solutions and all the other settings were left as their defaults.
MD simulations. All simulations were performed using AMBER, version 14.37 MD simulations were
carried out using the ff14SB force field at 300 K. The complex was placed in a rectangular
parallelepiped water box. An explicit solvent model for water, TIP3P, was used, and the complexes
were solvated with a 20 Å water cap. Chlorine ions were added as counterions to neutralize the
system. Prior to MD simulations, two steps of minimization were carried out using the same
procedure described above. Particle mesh Ewald (PME) electrostatics and periodic boundary
conditions were used in the simulation.38 The MD trajectory was run using the minimized structure
as the starting conformation. The time step of the simulations was 2.0 fs with a cutoff of 10 Å for the
nonbonded interaction, and SHAKE was employed to keep all bonds involving hydrogen atoms rigid.
Constant-volume periodic boundary MD was carried out for 0.5 ns, during which the temperature
was raised from 0 to 300 K. Then 19.5 ns of constant pressure periodic boundary MD was carried out
at 300 K using the Langevin thermostat to maintain constant the temperature of our system. All the α
carbons of the protein were blocked with a harmonic force constant of 10 kcal/mol•Å2 for the first
3.5 ns. General Amber force field (GAFF) parameters were assigned to the ligand, while partial
charges were calculated using the AM1-BCC method as implemented in the Antechamber suite of
AMBER 14. The final structures of the complexes were obtained as the average of the last 16.5 ns of
MD minimized by the CG method until a convergence of 0.05 kcal/mol•Å2. The average structures
were obtained using the Cpptraj program39 implemented in AMBER 14.
Binding Energy Evaluation. The evaluation of the binding energy associated to the different ligand-
protein complexes analyzed through MD simulations was carried out using AMBER 14. The
trajectories relative to the last 16.5 ns of each simulation were extracted and used for the calculation,
for a total of 165 snapshots (at time intervals of 100 ps). Van der Waals, electrostatic and internal
interactions were calculated with the SANDER module of AMBER 14, whereas polar energies were
calculated using both the Generalized Born and the Poisson−Boltzman methods with the MM-PBSA
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module of AMBER 14. Dielectric constants of 1 and 80 were used to represent the gas and water
phases, respectively, while the MOLSURF program was employed to estimate the nonpolar energies.
The entropic term was considered as approximately constant in the comparison of the ligand−protein
energetic interactions.
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RESULTS AND DISCUSSION
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LDH inhibition assays of saffron polar extract
Commercially available red stigmatic lobes of saffron were dissolved in a suitable polar solvent, such
as dimethyl sulfoxide (DMSO), and the polar saffron chemical constituents were extract, which we
hypothesized would have a higher chance to efficiently interact with the highly hydrophilic catalytic
site of hLDH-5 than non-polar components. The saffron DMSO extract was then serially diluted and
tested on hLDH-5 and hLDH-1 purified isoforms to determine its inhibition potency. At the maximum
tested concertation of 0.8 mg/mL, we observed an inhibition of 52% of the activity of hLDH-5
(corresponding to an IC50 value of 0.653 mg/mL) and of 49% of the activity of hLDH-1 (resulting in
an IC50 value of 0.717 mg/mL) by the saffron solution. The noticeable activity of the crude saffron
polar extract a served as the starting point for the investigation into this dietary natural product and
the identification of the constituents responsible for the inhibition of LDH enzymes.
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Chemical synthesis of crocetin
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Unlike crocin, which is commercially available at a reasonable level of purity, it was not possible to
easily purchase or extract crocetin in sufficient purity and amount to complete further biological
studies. Therefore, crocetin was synthesized by a multi-step synthesis. Crocetin possesses a diterpenic
and symmetrical structure, possessing alternating trans double bonds in the alkyl chain, four methyl
groups and carboxylic groups at both ends of the backbone. The synthesis of this carotenoid required
the formation of two fragments that converged in the last steps: 1) synthesis of the central dialdehyde
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unsaturated carbon chain (compound 12, Scheme 1, part B) and 2) formation of a
triphenylphosphoranylide intermediate (compound 6, Scheme 1, part A), which would be condensed
at both the terminal parts of the previous chain.40 The first part of this synthetic pattern was the
preparation of (E)-methyl 2-methyl-4-(triphenylphosphoranylidene)but-2-enoate 6, which was
condensed with dialdehyde 12 to obtain dimethyl crocetinate 13, an immediate precursor of crocetin
(Scheme 1). Initially, a methanolic solution of trans-2-methyl-2-butenoic acid 1, also called tiglic
acid, was heated in the presence of H2SO4 to obtain the methyl tiglate 2 (Scheme 1). Methyl tiglate is
a highly volatile compound, so it was necessary to take some precautions: a) the reaction was
performed in a sealed vial in order to minimize the evaporation of the product during heating, and b)
the workup procedure avoided exposing this intermediate to a high vacuum. The product was obtained
as a crude light yellow oil that was subsequently used in the next step without further purification.
Methyl ester 2 was subjected to a radical bromination with N-bromosuccinimide (NBS) in the
presence of the radical initiator benzoyl peroxide (BPO), which afforded a mixture of the two possible
regioisomers, γ and α allylic bromides (compounds 3 and 4, respectively). The relative amounts of
1
the two regioisomers were evaluated by H-NMR (signals of methylene protons, see experimental
section) and γ and α bromides were present in a 3:2 ratio, respectively. The mixture was used in the
next step without further purification. The mixture of compounds 3 and 4 was dissolved in toluene
and combined with a slight excess of triphenylphosphine to get phosphonium salts. At this time, the
triphenylphosphonium salt of methyl γ-bromotiglate 5 was isolated as a pure regioisomer by
recrystallization from CH3CN/AcOEt (Scheme 1). Compound 5 was treated with aqueous sodium
hydroxide to afford compound 6. However the formed triphenylphosphoranylide proved to be
particularly instable upon exposure to atmospheric oxygen. These observations led us to perform the
reaction under an Argon atmosphere and avoid purification of this compound. The reaction was
1
monitored via the presence of representative signals in the H-NMR spectrum (see experimental
section) and compound 6 was prepared immediately before its use in the subsequent Wittig reaction
(Scheme 1).
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The second fragment of the synthesis involved the preparation of dialdehyde 12 (Scheme 1), which
was to be condensed with phosphoranylide 6. The synthesis of 12 commenced with a Wittig reaction
between
commercially
available
fumaraldehyde
mono(dimethyl
acetal)
8
and
(carbethoxyethylidene)triphenylphosphorane 7, providing a mixture of both the desired condensation
product still possessing the dimethyl acetal portion (structure not shown) and condensation product 9
possessing the free aldehyde moiety. This resulted the acetal group being partially removed under
these reaction conditions. Considering that the next step of the synthesis involved the deprotection of
the acetal moiety to obtain aldehyde 9, the mixture was treated with amberlyst 15 in a biphasic solvent
system consisting of water and acetone, which lead to the complete hydrolysis of the dimethyl acetal
group to afford pure aldehyde 9. Amberlyst 15 was used as an acid catalyst due to its not affecting
the highly reactive double bonds present in the structure of the starting material and its ease of
purification. A second Wittig reaction with (carbethoxyethylidene)triphenylphosphorane 7 gave the
symmetric diester intermediate 10. Compound 10 was then reduced to form the corresponding
dialcohol 11 via an excess of diisobutylaluminum hydride (DIBAL-H) and anhydrous hexane as
solvent. The subsequent partial oxidation of the allylic alcoholic groups of compound 11 was
performed with MnO2 to give the corresponding dialdehyde 12. Compound 12 was subjected to a
double Wittig reaction with the triphenylphosphoranylide 6, thus extending the unsaturated carbon
chain at both terminals. The resulting dimethyl ester of crocetin 13 precipitated as a red powder and
was isolated by filtration. The precipitate was recrystallized from MeOH to obtaine material of
sufficient purity. Compound 13 was saponified with aqueous NaOH to give the corresponding sodium
salt (disodium crocetinate 14), which precipitated as a bright orange solid (step g, Scheme 1).
Alternatively, the saponification was followed by acidification with phosphoric acid to obtain the free
diacid crocetin 15 (step h, Scheme 1).41
LDH inhibition assays of crocetin and crocin
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Synthetic dibasic crocetin (compound 14), its dimethyl ester precursor 13, and commercially
available crocin 16 were tested on hLDH-5 and hLDH-1 purified isoforms to determine their
inhibitory properties, relative to galloflavin, which served as a positive control (Table 1).12 The
disodium salt 14 was preferred to free acid 15 in these biological assays, since the salt form readily
dissolved in the aqueous buffer without any additional organic solvent. Nevertheless, crocetin as
diacid (15) was tested to confirm the inhibitory activity on the enzyme. Compound 13 was found to
be completely inactive, displaying IC50 values greater than 200 µM for both isoforms. Conversely,
disodium salt 14, showed noticeable inhibition of hLDH-5 (IC50 = 54.9 µM) and it was selective for
this isoform, proving to be less active on isoform 1 (IC50 > 200 µM). The IC50 value of diacid 15
nicely overlaps with that of its disodium salt (Table 1). Reference inhibitor galloflavin was 1.7-fold
less potent than compound 14, and it also inhibited isoform 1 of the enzyme with a similar potency.
Crocin 16 was less effective in the inhibition of hLDH-5 relative to crocetin, with an IC50 value
similar to that of galloflavin (IC50 = 95.7 µM), and it showed selectivity towards isoform 1 (IC50 =
59.8 µM). In order to complete the biochemical characterization of crocetin, a kinetic assay was
performed by applying a mixed-model inhibition fit to the second order polynomial regression
analysis of the rate of conversion of NADH to NAD+ to obtain Ki values in the NADH-competition
experiments. In these experiments we measured an apparent Michaelis–Menten constant (KM) of 20
μM and a Ki value of 25.6 µM, which is consistent with the IC50 values of both forms of crocetin
(disodium salt 14 and diacid 15) reported in Table 1. Similarly, in the pyruvate-competition
experiments in which the KM value was of 100 μM, the resulting Ki value was of 21.1 µM.
From a structural point of view, we can conclude that the replacement of the two carboxylic acids of
crocetin by methyl ester groups, as in compound 13, or the esterification with sugar moieties, as in
crocin 16, led to a loss of inhibitory activity (in the case of 13) or a slight decrease in activity and a
marked loss of selectivity for isoform 5 (in the case of 16), thus identifying crocetin as the most active
compound among these saffron derivatives.
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Inhibition of cancer cell proliferation and reduction of cellular lactate production
Following the results previously obtained with isolated LDH enzyme isoforms, studies in cancer cells
were focused on the disodium salt of crocetin 14 because it was found to be the most active hLDH-5
inhibitor among the saffron derivatives herein studied. Compound 14 was evaluated for growth
inhibitory effects on two different cancer cell lines, HeLa (human cervical carcinoma cells) and A549
(human lung carcinoma cells), after 72 hours of incubation. Cell death was assessed by
Sulforhodamine B (SRB) staining, as previously described42 and the cytotoxic potency of the tested
compound was expressed as IC50 values, which represent the concentration of a compound that is
required for the 50% inhibition of cell proliferation. Compound 14 was able to counteract cancer cell
growth both in A549 and in HeLa cells with similar IC50 values of 114.0 ± 8.0 and 113.0 ± 11.1 μM,
respectively (Figure 3, panel A), which may be considered as respectable values for a food
component. Furthermore, 14 proved to be completely inactive against noncancerous human fibroblast
lung cells (MRC5 IC50 > 500 μM).After the preliminary biochemical evaluation on the isolated
enzymes and the cytotoxicity assays on two cancer cell lines, this compound was also assayed for its
ability to inhibit the production of lactate in cancer cells. To this end, HeLa cells were treated with
14 in phosphate buffer saline (PBS) for 6 hours and then the cell culture medium was extracted and
mixed with a known amount of chlorophenylalanine (CPA, internal standard) and dried. Concentrated
samples were reacted with N-methyl-N-tert-butyldimethylsilyltrifluoroacetamide (MTBSTFA) for
derivatization of lactate, which was needed for its GC-MS detection and quantification. The cells
were fixed and biomass was measured using SRB. Crocetin disodium salt 14 was tested at a 100 µM
concentration. As previously reported, the GC-MS analysis of cell culture media for the quantitative
determination of extracellular lactate was preferred over other methods, since it allows a highly
sensitive determination of low micromolar lactate concentrations.43 As shown in Figure 3 (Panel B),
compound 14 reduced lactate production of about 20 % compared to PBS control at 100 µM after 6
h incubation.
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Molecular modelling studies
To rationalize how compound 14 can interact with the hLDH-5 protein, molecular docking studies
followed by molecular dynamic (MD) simulations and binding energy determinations were carried
out. Compound 14 was docked into the crystal structure of the hLDH-5 protein (4M49 PDB code)
using AUTODOCK software.36 The 200 different docking results generated were clustered using a
root-mean square deviation (RMSD) threshold of 2.0 Å and the so obtained 5 clusters of solutions
were considered for further studies (see the Experimental section for details). For each cluster, the
docking pose associated with the best estimated binding energy was selected as a representative
binding mode. The stability of the 5 different binding modes was then assessed through 20 ns of MD
simulation studies with explicit water molecules, as described in the Experimental section. The 5 MD
trajectories obtained in this way were further analyzed through the Molecular Mechanics - Poisson
Bolzmann Surface Area (MM-PBSA) method,44 which was shown to accurately estimate the ligand-
receptor energy interaction.45,46 This approach averages the contributions of gas phase energies,
solvation free energies, and solute entropies calculated for snapshots of the complex molecule as well
as the unbound components extracted from MD trajectories, according to the procedure fully
described in the Experimental section. The MM-PBSA results (Table 2) suggest that there is one
docking pose that is the most favorable, as it shows an interaction energy ΔPBSA = -29.2 kcal/mol,
more than 8 kcal/mol higher than all the other binding poses (docking pose 1, Table 2).
Figure 4 shows the energy minimized average structures of the last 16.5 ns of hLDH-5 complexed
with the hypothesized binding pose of compound 14. The ligand interacts within the pyruvate binding
site and points towards the binding site entrance. One of the two carboxylic groups forms a hydrogen
bond with the catalytic H193, whereas the second carboxylic end forms a strong ionic couple with
positively charged residue K57. Although the binding of the compound to the enzyme active site is
mainly driven by the interactions of the two carboxylic ends, the internal carbon chain of the ligand
also shows lipophilic interactions along the hLDH-5 surface and, in particular, with A30, V31, V136
and I252, which represent fundamental additive contributions to the binding process.
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In conclusion, an investigation into the bioactive properties of saffron extract led to the discovery of
LDH-inhibitory activity, to carotenoid crocetin. Additionally, these compounds mitigated
proliferation of glycolytic cancer cells. In order to unambiguously establish the effective activity of
this natural compound, a multi-step chemical synthesis was implemented for the production of pure
crocetin. Its moderate inhibition potency against the isolated LDH-enzyme, which frankly cannot
compete with several synthetic inhibitors previously described, should not be considered as a limiting
factor since crocetin is part of a spice (saffron) utilized in aliments, whose relative safety has been
now established by millennia of culinary tradition employing it in many recipes. It should be
acknowledged the oral LD50 for saffron in mice was found to be 20 g/kg, although oral administration
of the saffron extract at concentrations from 0.1 to 5 g/kg was demonstrated to be non-toxic in mice.47
Furthermore, a recent study revealed that administration of up to 400 mg / day of saffron for 1 week
to healthy human volunteers did not cause any clinically significant effects,48 thus confirming its
higher safety when compared to classical anticancer drugs. For these reasons, this study contributes
to appreciate the role of saffron in the prevention and treatment of cancer when utilized in
combination with standard-of-care preventive and therapeutic options.
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ACKNOWLEDGEMENTS
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We are grateful to the NIH grants R01-GM098453 (to PJH and FM), the University of Pisa, and the
University of Illinois for support of this work.
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REFERENCES
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Scheme 1.
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