Synthesis and iron chelating properties of hydroxypyridinone and hydroxypyranone hexadentate ligands

Article abstract of DOI:10.1039/c8dt05014g

Chelation therapy has become an important therapeutic approach for some diseases. In attempt to identify clinically useful chelators, four hexadentate ligands were synthesized by conjugating the corresponding bidentate ligands (3-hydroxypyridin-4-one (3,4-HOPO), 3-hydroxypyridin-2-one (3,2-HOPO), 1-hydroxypyridin-2-one (1,2-HOPO), and 3-hydroxypyran-4-one) each with a free amino group to a tripodal acid. Their pK_a values and affinities for iron(iii) were investigated. The pFe³⁺ values of the hexadentate pyridinones 1 (3,4-HOPO), 3 (3,2-HOPO) and 4 (1,2-HOPO), and the pyranone 2 was found to follow the sequence 1 > 4 ? 3 > 2, which is different to the pFe³⁺ value sequence of the corresponding bidentate forms (3,4-HOPO ? 3,2-HOPO > 1,2-HOPO > 3-hydroxypyranone). Hexadentate 3,4-HOPOs and 1,2-HOPOs have the greatest potential as iron scavenging agents.

Full text of DOI:10.1039/c8dt05014g

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Synthesis and iron chelating properties of
hydroxypyridinone and hydroxypyranone
Cite this: DOI: 10.1039/c8dt05014g
hexadentate ligands
b
Tao Zhou,*a Xiao-Le Kongb and Robert C Hider
*
Chelation therapy has become an important therapeutic approach for some diseases. In attempt to ident-
ify clinically useful chelators, four hexadentate ligands were synthesized by conjugating the corresponding
bidentate ligands (3-hydroxypyridin-4-one (3,4-HOPO), 3-hydroxypyridin-2-one (3,2-HOPO), 1-hydroxy-
pyridin-2-one (1,2-HOPO), and 3-hydroxypyran-4-one) each with a free amino group to a tripodal acid.
Their pKa values and affinities for iron(III) were investigated. The pFe3+ values of the hexadentate pyridi-
nones 1 (3,4-HOPO), 3 (3,2-HOPO) and 4 (1,2-HOPO), and the pyranone 2 was found to follow the
sequence 1 > 4 ≫ 3 > 2, which is different to the pFe3+ value sequence of the corresponding bidentate
forms (3,4-HOPO ≫ 3,2-HOPO > 1,2-HOPO > 3-hydroxypyranone). Hexadentate 3,4-HOPOs and 1,2-
HOPOs have the greatest potential as iron scavenging agents.
Received 20th December 2018,
Accepted 19th February 2019
DOI: 10.1039/c8dt05014g
rsc.li/dalton
possess a relatively low log β3 value for iron(III), typically 28.
However, the metal affinity of their hexadentate analogues has
Introduction
Metal ions play important roles in living processes, and thus not been reported. In order to facilitate a direct comparison of
chelation therapy has become an important therapeutic iron(III) affinity constants, hexadentate ligands of these four
approach for some diseases.1 One example is the therapeutic bidentate ligands were synthesized by conjugation onto the
application of iron chelators for the treatment of iron overload same tripodal scaffold (Fig. 2).
disorders associated with β-thalassaemia and sickle cell
anaemia. Thus deferioxamine B (DFO),2 deferiprone3,4 and
deferasirox5,6 are each widely used for this purpose. Iron chela-
Experimental section
General
tors also have potential for the treatment of microbial
infections7–10 and neurodegenerative disorders,11 such as
Parkinson’s disease,12 Alzheimer’s disease13 and Friedreich’s
Ataxia.14 A wide range of bidentate chelators, such as
3-hydroxypyridin-4-one (3,4-HOPO), 1-hydroxypyridin-2-one
(1,2-HOPO), 3-hydroxypyridin-2-one (3,2-HOPO), 3-hydroxypyra-
none, catechol and hydroxamate (Fig. 1) possess relatively high
affinities for iron(III).15 Hydroxypyridinones (HOPOs) combine
the characteristics of both hydroxamate and catechol groups,
forming 5-membered chelate rings in which the metal is co-
ordinated by two vicinal oxygen atoms.16 Among the three
classes of HOPOs, 3,4-HOPO possesses the highest affinity for
iron(III) (log K1 = 14.2, log β3 = 37.2), 3,2-HOPO the next (log K1
= 11.7, log β3 = 32), and 1,2-HOPO the lowest (log K1 = 10.3,
1H NMR and 13C NMR spectra were recorded on a Bruker
Avance 400 spectrometer with TMS as an internal standard.
Electrospray ionization (ESI) mass spectra were obtained by
infusing samples into an LCQ Deca XP ion trap instrument.
High resolution mass spectra (HRMS) were determined on a
QTOFMicro (Waters, USA) by direct infusing samples into the
log β3
=
27).16 3-Hydroxypyranones, for instance maltol,
aSchool of Food Science and Biotechnology, Zhejiang Gongshang University,
18 Xuezheng Street, Xiasha, Hangzhou, Zhejiang 310018, P. R. China.
E-mail: taozhou@zjgsu.edu.cn
bInstitute of Pharmaceutical Sciences, King’s College London, 150 Stamford Street,
London, SE1 9NH, UK. E-mail: robert.hider@kcl.ac.uk
Fig. 1 Bidentate metal ligands.
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amido)heptanediamide (9). A mixture of 7 (1.58 g, 4 mmol),
amine 8 (3.715 g, 14.4 mmol), HOBt (1.946 g, 14.4 mmol),
DCC (2.966 g, 14.4 mmol) and DMF (25 ml) was stirred at
room temperature for 2 days. After removal of the solvent, the
residue was purified by chromatography using CHCl3/MeOH
(8 : 1 to 4 : 1) to provide product 9 (3.8 g, 85% yield) as a white
1
solid. H NMR (DMSO-d6, 400 MHz) δ 1.83 (m, 6H, CH2), 2.05
(m, 6H, CH2), 2.24 (s, 9H, CH3), 3.38 (s, 9H, CH3), 3.81 (s, 2H,
CH2), 4.33 (d, J = 4.8 Hz, 6H, CH2), 4.49 (s, 2H, CH2), 5.06 (s,
6H, CH2), 6.18 (s, 3H, C5–H in pyridinone), 6.98 (s, 1H, NH),
7.23–7.43 (m, 20H, Ph), 8.08 (t, J = 4.8 Hz, 3H, NH). ESI-MS:
m/z 1116.5 ([M + H]+), 558.8 ([M + 2H]2+).
N1,N7-Bis((3-hydroxy-1,6-dimethyl-4-oxo-1,4-dihydropyridin-
2-yl)methyl)-4-(3-((3-hydroxy-1,6-dimethyl-4-oxo-1,4-dihydro-
pyridin-2-yl)methylamino)-3-oxopropyl)-4-(2-hydroxyacetamido)
heptanediamide trihydrochloride (1). A mixture of 9 (1.85 g),
5% Pd/C (0.6 g), 1.25 M HCl methanolic solution (3 ml) and
methanol (50 ml) was shaken under an atmosphere of hydro-
gen (30 psi) at room temperature for 2 days. After filtration, the
solvent was removed under reduced pressure, the residue was
redissolved in methanol (5 ml), acetone was added to form a
white precipitate, and left at 4 °C overnight. The white product
Fig. 2 Structures of hexadentate ligands (1–4).
ESI source. Di-tertbutyl 4-amino-4-[2-(tert-butoxycarbonyl)ethyl]- 1 was isolated as a trihydrochloric acid salt (1.32 g, 92% yield).
heptanedioate (5) was prepared as reported.17 2-(Aminomethyl)- 1H NMR (DMSO-d6, 400 MHz) δ 1.85 (m, 6H, CH2), 2.10 (m,
3-(benzyloxy)-1,6-dimethylpyridin-4(1H)-one (8) and 3-(benzyl- 6H, CH2), 2.57 (s, 9H, CH3), 3.69 (s, 2H, CH2), 3.88 (s, 9H,
oxy)-2-(hydroxymethyl)-6-methyl-4H-pyran-4-one (10) were pre- CH3), 4.56 (d, J = 5.1 Hz, 6H, CH2), 7.26 (s, 3H, C5–H in pyridi-
pared according to previously published work.18 All other none), 7.34 (s, 1H, NH), 8.90 (t, J = 5.1 Hz, 3H, NH); 13C NMR
chemicals were purchased from Aldrich or Merck Chemical (DMSO-d6, 100 MHz) δ 20.96 (CH3), 29.59 (CH2), 30.50 CH2),
Company and used without any further purification.
31.06 (CH3), 35.09 CH2), 57.08 (C), 61.81 CH2), 113.08 (C–5H in
pyridinone), 140.23(C-2 in pyridinone), 143.06 (C-3 in pyridi-
none), 148.93 (C-6 in pyridinone), 159.95 (C-4 in pyridinone),
Synthetic procedures
Di-tert-butyl-4-(2-(benzyloxy)acetamido)-4-(3-tert-butoxy-3- 171.64 (CO), 173.63 (CO). MS (ESI): m/z 756.3 ([M + H]+), 378.7
oxopropyl)heptanedioate (6). To a mixture of amine 5 (14.94 g, ([M + 2H]2+); HRMS: calcd for C36H50N7O11 756.3568 ([M + H]+),
36 mmol), triethylamine (3.636 g, 36 mmol) and anhydrous di- 378.6824 ([M + 2H]2+); found 756.3560, 378.6814, respectively.
chloromethane (150 mL) cooled in an ice-bath was added
N1,N7-Bis((3-(benzyloxy)-6-methyl-4-oxo-4H-pyran-2-yl)methyl)-
dropwise 2-(benzyloxy)acetyl chloride (6.092 g, 33 mmol). The 4-(3-((3-(benzyloxy)-6-methyl-4-oxo-4H-pyran-2-yl)methylamino)-
mixture was stirred at room temperature for 6 h. Then the reac- 3-oxopropyl)-4-(2-(benzyloxy)acetamido)heptanediamide (12).
tant was washed with cold 10%HCl, brine, 10% NaHCO3 and To a mixture of triacid 7 (1.58 g, 4 mmol), amine 11 (3.528 g,
brine, and dried over anhydrous Na2SO4. After removal of the 14.4 mmol) and DMF (25 ml) cooled to 20 °C was added
solvent, the residue was purified by chromatography using DIPEA (3.722 g, 28.8 mmol). Then HCTU (5.957 g, 14.4 mmol)
EtOAc/hexane (1 : 4 to 1 : 2) to provide product 6 (16.7 g, was added to this stirred solution in three portions. The stir-
1
89.8%) as a white solid. H NMR (CDCl3, 400 MHz) δ 1.43 (s, ring was continued overnight. The reactant was concentrated
27H, CH3), 1.98 (m, 6H, CH2), 2.18 (m, 6H, CH2), 3.87 (s, 2H, and the residue was purified by chromatography using EtOAc/
CH2), 4.57 (s, 2H, CH2), 6.32 (s, 1H, NH), 7.32–7.38 (m, 5H, MeOH (9 : 1 to 4 : 1) to provide product 12 (4.15 g, 96.3% yield)
Ph). ESI-MS: m/z 564 ([M + H]+).
4-(2-(Benzyloxy)acetamido)-4-(2-carboxyethyl)heptanedioic
as a pale yellow solid.
1H NMR (CDCl3, 400 MHz) δ 1.96 (m, 6H, CH2), 2.03 (m,
acid (7). A solution of 6 (15.6 g) in 96% formic acid (50 mL) 6H, CH2), 2.19 (s, 9H, CH3), 3.86 (s, 2H, CH2), 4.12 (d, J =
was stirred at room temperature overnight. After removal of 5.9 Hz, 6H, CH2), 4.56 (s, 2H, CH2), 5.16 (s, 6H, CH2), 5.71 (t,
the solvent, product 7 was obtained as a white solid in quanti- J = 5.9 Hz, 3H, NH), 6.15 (s, 3H, C5–H in pyridinone), 6.57 (s,
tative yield. 1H NMR (DMSO-d6, 400 MHz) δ 1.86 (m, 6H, CH2), 1H, NH), 7.30–7.38 (m, 20H, Ph). ESI-MS: m/z 1077.4
2.13 (m, 6H, CH2), 3.85 (s, 2H, CH2), 4.51 (s, 2H, CH2), 7.01 (s, ([M + H]+), 1099.3 ([M + Na]+).
1H, NH), 7.27–7.38 (m, 5H, Ph), 12.12 (br, 3H, COOH). ESI-MS:
m/z 396 ([M + H]+), 418 ([M + Na]+).
N1,N7-Bis((3-hydroxy-6-methyl-4-oxo-4H-pyran-2-yl)methyl)-4-
(3-((3-hydroxy-6-methyl-4-oxo-4H-pyran-2-yl)methylamino)-3-
N1,N7-Bis((3-(benzyloxy)-1,6-dimethyl-4-oxo-1,4-dihydropyridin- oxopropyl)-4-(2-hydroxyacetamido)heptanediamide (2). To a
2-yl)methyl)-4-(3-((3-(benzyloxy)-1,6-dimethyl-4-oxo-1,4-dihydro- solution of 12 (1.56 g) in anhydrous DCM (20 ml) cooled with
pyridin-2-yl)methylamino)-3-oxopropyl)-4-(2-(benzyloxy)acet- ice-bath under nitrogen was added dropwise boron trichloride
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(1 M in DCM, 17.4 ml). The mixture was stirred at room tem- (s, 2H, CH2), 3.92 (t, J = 6.0 Hz, 6H, CH2), 4.53 (s, 2H, CH2),
perature for 2 days. The reaction was quenched with methanol. 6.10 (t, J = 7.1 Hz, 3H, C5–H in pyridinone), 6.76 (dd, J =
After removal of the solvent, the residue was redissolved in 7.5 Hz, 1.5 Hz, 3H, C4–H in pyridinone), 6.95 (s, 1H, NH), 7.07
methanol (5 ml). Acetone was added to form a precipitate (dd, J = 6.9 Hz, 1.6 Hz, 3H, C6–H in pyridinone), 7.29–7.38 (m,
which was left in a fridge overnight. The product 2 was 5H, Ph), 7.98 (t, J = 5.7 Hz, 3H, NH). ESI-MS: m/z 846.3
obtained as a pale yellow solid (0.97 g, 93% yield). 1H NMR ([M + H]+), 868.5 ([M + Na]+).
(DMSO-d6, 400 MHz) δ 1.73 (m, 6H, CH2), 1.95 (m, 6H, CH2),
N1,N7-Bis(2-(3-hydroxy-2-oxopyridin-1(2H)-yl)ethyl)-4-(3-(2-(3-
2.09 (s, 9H, CH3), 3.60 (s, 2H, CH2), 4.09 (d, J = 5.4 Hz, 6H, hydroxy-2-oxopyridin-1(2H)-yl)ethylamino)-3-oxopropyl)-4-(2-
CH2), 6.07 (s, 3H, C5–H in pyranone), 6.68 (s, 1H, NH), 8.20 (t, hydroxyacetamido)heptanediamide (3). To a solution of 16
J = 5.4 Hz, 3H, NH); 13C NMR (DMSO-d6, 100 MHz) δ 19.58 (0.78 g) in anhydrous DCM (20 ml) cooled with ice-bath under
(CH3), 29.76 (CH2), 30.71 CH2), 35.78 CH2), 57.08 (C), 61.79 nitrogen was added dropwise boron trichloride (1 M in DCM,
CH2), 111.70 (C–5H in pyranone), 141.99 (C-2 in pyranone), 12 ml). The mixture was stirred at room temperature for
147.40 (C-3 in pyranone), 164.88 (C-6 in pyridinone), 171.53 6 days. The reaction was quenched with methanol. After
(CO), 172.81 (C-4 in pyridinone), 173.93 (CO). ESI-MS: m/z removal of the solvent, the residue was redissolved in metha-
717.2 ([M + H]+), 739.2 ([M + Na]+). HRMS: calcd for nol (5 ml), acetone was added to form the precipitate, left in
C33H41N4O14 717.2619 ([M + H]+), found 717.2616; calcd for fridge overnight. The product 3 was obtained as a pale brown
C33H40N4O14Na 739.2439 ([M + H]+), found 739.2434.
solid (0.61 g, 80.3% yield). 1H NMR (DMSO-d6, 400 MHz)
tert-Butyl 2-(3-methoxy-2-oxopyridin-1(2H)-yl)ethylcarbamate δ 1.78 (m, 6H, CH2), 1.97 (m, 6H, CH2), 3.34 (q, J = 5.8 Hz, 6H,
(14). To a solution of 13 (4.5 g, 36 mmol) in dry DMF (40 mL) CH2), 3.73 (s, 2H, CH2), 3.96 (t, J = 5.9 Hz, 6H, CH2), 4.53 (s,
was added sodium hydride (1.728 g, 60% in mineral oil, 2H, CH2), 6.08 (t, J = 7.0 Hz, 3H, C5–H in pyridinone), 6.70
43.2 mmol). The mixture was stirred at room temperature for (dd, J = 7.3 Hz, 1.7 Hz, 3H, C4–H in pyridinone), 6.77 (s, 1H,
30 min, then heated to 70 °C. A solution of tert-butyl 2-bromo- NH), 7.01 (dd, J = 6.8 Hz, 1.7 Hz, 3H, C6–H in pyridinone),
ethylcarbamate (9.274 g, 41.4 mmol) in 30 ml DMF was added 8.08 (t, J = 5.6 Hz, 3H, NH); 13C NMR (DMSO-d6, 100 MHz)
dropwise. The stirring was continued at 70 °C overnight. After δ 29.54 (CH2), 30.32 (CH2), 37.40 (CH2), 48.43 (CH2), 56.61 (C),
removal of the solvent, the residue was purified by chromato- 61.41 (CH2), 105.19 (C–5H in pyridinone), 114.94 (C–4H in pyr-
graphy using EtOAc/MeOH (9.5 : 0.5) to provide product 14 idinone), 128.65 (C–6H in pyridinone), 146.59 (C-3 in pyridi-
(7.1 g, 73.6% yield) as a white solid. 1H NMR (CDCl3, none), 157.71 (C-2 in pyridinone), 171.05 (CO), 172.41 (CO).
400 MHz) δ 1.42 (s, 9H, CH3), 3.47 (q, J = 5.9 Hz, 2H, CH2), ESI-MS: m/z 714.3 ([M + H]+), 736.5 ([M + Na]+). HRMS: calcd
3.82 (s, 3H, CH3), 4.13 (t, J = 5.9 Hz, 2H, CH2), 5.10 (br, 1H, for C33H44N7O11 714.3099 ([M + H]+), found 714.3091; calcd for
NH), 6.12 (t, J = 7.1 Hz, 1H, C5–H in pyridinone), 6.63 (dd, J = C33H43N7O11Na 736.2918 ([M + Na]+), found 736.2907.
7.4 Hz, 1.6 Hz, 1H, C4–H in pyridinone), 6.89 (d, J = 6.7 Hz,
1H, C6–H in pyridinone). ESI-MS: m/z 269 ([M + H]+).
1-Hydroxy-6-oxo-1,6-dihydropyridine-2-carboxylic acid (18).
To a solution of 17 (7.3 g, 52.5 mmol) in trifluoroacetic acid
2-(3-Methoxy-2-oxopyridin-1(2H)-yl)ethanaminium 2,2,2-tri- (45 mL) and acetic acid (30 mL) was added dropwise CH3CO3H
fluoroacetate (15). To a solution of 14 (3.5 g) in dichloro- (22 ml, 36–40% in acetic acid). The mixture was stirred at
methane (30 mL) cooled with ice-bath was added trifluoro- room temperature for 1 h, and then stirred at 80 °C overnight.
acetic acid (20 mL). Then the mixture was stirred at room tem- After cooled in fridge, filtered, washed with cold methanol,
perature for 3 h. After trifluoroacetic acid was completely crude product 18 was obtained as pale yellow solid (6.35 g,
removed in vacuo, compound 15 was obtained as a white solid 78%). The crude product was used in the next reaction without
1
1
in quantitative yield. H NMR (DMSO-d6, 400 MHz) δ 3.14 (t, further purification. H NMR (DMSO-d6, 400 MHz) δ 6.65 (dd,
J = 5.6 Hz, 2H, CH2), 3.71 (s, 3H, CH3), 4.14 (t, J = 6.2 Hz, 2H, J = 7.0 Hz, 1.7 Hz, 1H, C3–H in pyridinone), 6.72 (dd, J =
CH2), 6.21 (t, J = 7.2 Hz, 1H, C5–H in pyridinone), 6.83 (dd, J = 9.0 Hz, 1.7 Hz, 1H, C5–H in pyridinone), 7.45 (dd, J = 9.0 Hz,
7.5 Hz, 1.5 Hz, 1H, C4–H in pyridinone), 7.21 (dd, J = 7.5 Hz, 7.0 Hz, 1H, C4–H in pyridinone). ESI-MS: m/z 156.07
1.6 Hz, 1H, C6–H in pyridinone), 8.03 (br, 3H, NH3 ). ESI-MS: ([M + H]+).
+
m/z 169 ([M + H]+).
1-(Benzyloxy)-6-oxo-1,6-dihydropyridine-2-carboxylic acid (19).
mixture of 18 (6.3 g, 40.6 mmol), K2CO3 (11.21 g,
4-(2-(Benzyloxy)acetamido)-N1,N7-bis(2-(3-methoxy-2-oxopyri-
A
din-1(2H)-yl)ethyl)-4-(3-(2-(3-methoxy-2-oxopyridin-1(2H)-yl) 81.29 mmol), benzyl chloride (6.17 g, 48.72 mmol) and metha-
ethylamino)-3-oxopropyl)heptanediamide (16). To a mixture of nol (100 ml) was refluxed overnight. After filtration, the filtrate
7 (0.553 g, 1.4 mmol), 15 (1.42 g, 5.04 mmol) and DMF (15 ml) was concentrated to dryness. The residue was redissolved in
was added DIPEA (1.95 g, 15.12 mmol). Then HCTU (2.085 g, 20 ml water, acidified with 6N HCl to pH 2, filtered, washed
5.04 mmol) was added to the above stirred solution. The stir- with cold water to provide product 19 as a off-white solid
ring was continued overnight. The reactant was concentrated (8.45 g). 1H NMR (DMSO-d6, 400 MHz) δ 5.29 (s, 2H, CH2), 6.56
and the residue was purified by chromatography using DCM/ (dd, J = 6.8 Hz, 1.7 Hz, 1H, C3–H in pyridinone), 6.74 (dd, J =
MeOH (9 : 1 to 3 : 1) to provide product 16 (0.93 g, 78.5% yield) 9.3 Hz, 1.7 Hz, 1H, C5–H in pyridinone), 7.41–7.52 (m, 6H,
as a white solid.
Ph and C4–H in pyridinone).
Methyl 1-(benzyloxy)-6-oxo-1,6-dihydropyridine-2-carboxylate
1H NMR (DMSO-d6, 400 MHz) δ 1.79 (m, 6H, CH2), 1.96 (m,
6H, CH2), 3.31 (q, J = 5.8 Hz, 6H, CH2), 3.67 (s, 9H, CH3), 3.84 (20). To 100 ml of methanol at −5 °C was added 10 ml of
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thionyl chloride at such a rate that the temperature did not room temperature for 20 min, and then EDC (2.070 g,
reach 10 °C. The clear, colorless solution was cooled to −5 °C. 10.8 mmol) was added. Stirring was continued for 2 days. After
19 (8.21 g) was added, the mixture was gradually warmed to removal of the solvent, the residue was purified by chromato-
room temperature, and stirred for 3 days. After removal of the graphy using DCM/MeOH (14 : 1) to provide product 23 (2.55 g,
solvent, the crude product 20 was obtained. ESI-MS: m/z 260 82.3% yield) as a white solid. 1H NMR (CDCl3, 400 MHz) δ 1.90
([M + H]+), 282 ([M + Na]+).
(m, 6H, CH2), 2.08 (m, 6H, CH2), 3.78 (s, 2H, CH2), 4.16 (d, J =
1-(Benzyloxy)-6-(hydroxymethyl)pyridin-2(1H)-one (21).
A
6.0 Hz, 6H, CH2), 4.50 (s, 2H, CH2), 5.24 (s, 6H, CH2), 5.96 (dd,
mixture of crude 20 (obtained from last step), NaBH4 (7.49 g) J = 6.9 Hz, 1.4 Hz, 3H, C5–H in pyridinone), 6.53 (dd, J =
in THF (150 ml) was stirred at 70 °C for 2 h. Methanol (15 mL) 9.2 Hz, 1.6 Hz, 3H, C3–H in pyridinone), 6.57 (s, 1H, NH), 6.89
was added. After cooled to room temperature, the reaction was (t, J = 6.0 Hz, 3H, NH), 7.16 (dd, J = 9.2 Hz, 6.9 Hz, 3H, C4–H
quenched with saturated NH4Cl (100 ml). The stirring was con- in pyridinone), 7.27–7.44 (m, 20H, Ph). MS (ESI): m/z 1032
tinued for 1.5 h. The aqueous layer was extracted with DCM ([M + H]+).
(2 × 150 ml). The combined organic layers were dried over
N1,N7-Bis((1-hydroxy-6-oxo-1,6-dihydropyridin-2-yl)methyl)-
Na2SO4. After removal of the solvent, the residue was purified 4-(3-((1-hydroxy-6-oxo-1,6-dihydropyridin-2-yl)methylamino)-
by chromatography using DCM/MeOH (9.5 : 0.5) to provide 3-oxopropyl)-4-(2-hydroxyacetamido)heptanediamide (4). To a
product 21 (3.8 g, 50% yield overall two steps) as a white solid. solution of 23 (1.2 g) in anhydrous DCM (20 ml) cooled with
1H NMR (CDCl3, 400 MHz) δ 2.98 (br, 1H, OH), 4.48 (d, J = ice-bath under nitrogen was added dropwise borontrichloride
3.6 Hz, 2H, CH2), 5.30 (s, 2H, CH2), 6.21 (m, 1H, C3–H in pyri- (1 M in DCM, 14 ml). The mixture was stirred at room tem-
dinone), 6.60 (dd, J = 9.2 Hz, 1.7 Hz, 1H, C5–H in pyridinone), perature for 2 days. The reaction was quenched with methanol.
7.29 (m, 1H, C4–H in pyridinone), 7.38 (m, 3H, Ph), 7.45 (m, After removal of the solvent, the residue was redissolved in
2H, Ph). ESI-MS: m/z 232.1 ([M + H]+), 254.1 ([M + Na]+).
methanol (5 ml), acetone was added to form a precipitate
2-((1-(Benzyloxy)-6-oxo-1,6-dihydropyridin-2-yl)methyl)iso- which was allowed to stand at 4 °C overnight. The product 4
indoline-1,3-dione (22a). To a mixture of 21 (3.7 g, 16 mmol), was obtained as a white solid (0.75 g, 96% yield). 1H NMR
triphenylphosphine (5.04 g, 19.2 mmol), phthalimide (2.83 g, (DMSO-d6, 400 MHz) δ 1.93 (m, 6H, CH2), 2.20 (m, 6H, CH2),
19.2 mmol) and THF (60 mL) cooled with ice-bath was added 3.77 (s, 2H, CH2), 4.29 (d, J = 5.8 Hz, 6H, CH2), 6.06 (dd, J =
dropwise DIPAD (4.13 g, 19.2 mmol) at a period of 30 min. 7.0 Hz, 1.0 Hz, 3H, C5–H in pyridinone), 6.42 (dd, J = 9.0 Hz,
The temperature was slowly raised to room temperature, and 1.3 Hz, 3H, C3–H in pyridinone), 6.90 (s, 1H, NH), 7.33 (dd, J =
stirring was continued overnight. After filtration, the product 9.0 Hz, 7.1 Hz, 3H, C4–H in pyridinone), 8.51 (t, J = 6.0 Hz, 3H,
was washed with a small amount of THF, 22a (5.04 g, 87.5% NH); 13C NMR (DMSO-d6, 100 MHz) δ 29.46 (CH2), 30.27
yield) was obtained as a white solid. 1H NMR (CDCl3, (CH2), 37.38 (CH2), 55.79 (C), 61.46 (CH2), 102.04 (C–5H in pyr-
400 MHz) δ 4.67 (s, 2H, CH2), 5.44 (s, 2H, CH2), 5.83 (m, 1H, idinone), 115.80 (C–3H in pyridinone), 137.18 (C–4H in pyridi-
C3–H in pyridinone), 6.63 (dd, J = 9.2 Hz, 1.6 Hz, 1H, C5–H in none), 145.61 (C-6 in pyridinone), 158.02 (C-2 in pyridinone),
pyridinone), 7.19 (dd, J = 9.2 Hz, 6.9 Hz, 1H, C4–H in pyridi- 171.20 (CO), 172.59 (CO). MS (ESI): m/z 672.2 ([M + H]+), 694.3
none), 7.43 (m, 3H, Ph), 7.56 (m, 2H, Ph), 7.76 (m, 2H, Ph), ([M + Na]+). HRMS: calcd for C30H38N7O11 672.2629 ([M + H]+),
7.88 (m, 2H, Ph). ESI-MS: m/z 361.1 ([M + H]+), 383.3 found 672.2630; calcd for C30H37N7O11Na 694.2449
([M + Na]+).
([M + Na]+), found 694.2446.
6-(Aminomethyl)-1-(benzyloxy)pyridin-2(1H)-one (22). To a
solution of 22a (4.96 g, 13.78 mmol) in ethanol (50 ml) was
added hydrazine (0.843 g in 10 ml H2O, 16.54 mmol). After
Physicochemical characterisation of chelators 1–4
pKa determination. The pKa values of chelators 1–4 were
being refluxed for 3 h, the reaction mixture was chilled to 0 °C, determined by spectrophotometric titration.19–21 The auto-
adjusted to pH 1 with concentrated hydrochloric acid, filtered, matic titration system used in this study comprised an auto-
and washed with a plenty of water. The filtrate was adjusted burette (Metrohm Dosimat 765 liter ml syringe) and Mettler
pH to 12 with 10
N NaOH, and extracted with DCM Toledo MP230 pH meter with Metrohm pH electrode
(3 × 150 ml). The combined organic layers were dried over (6.0133.100) and a reference electrode (6.0733.100). 0.1 M KCl
Na2SO4. After removal of the solvent, the residue was purified electrolyte solution was used to maintain the ionic strength.
by chromatography using DCM/MeOH (9 : 1) to provide The temperature of the test solutions was maintained in a
product 22 (3.03 g, 95.6% yield) as a white solid. 1H NMR thermostatic jacketed titration vessel at 25 °C 0.1 °C by using
(CDCl3, 400 MHz) δ 3.67 (s, 2H, CH2), 5.32 (s, 2H, CH2), 6.07 a Techne TE-8J temperature controller. The solution under
(m, 1H, C3–H in pyridinone), 6.61 (dd, J = 9.2 Hz, 1.7 Hz, 1H, investigation was stirred vigorously during the experiment. A
C5–H in pyridinone), 7.28 (m, 1H, C4–H in pyridinone), 7.39 Gilson Mini-plus#3 pump with speed capability (20 ml min−1
(m, 3H, Ph), 7.47 (m, 2H, Ph). was used to circulate the test solution through a Hellem quartz
)
N1,N7-Bis((1-(benzyloxy)-6-oxo-1,6-dihydropyridin-2-yl)methyl)- flow cuvette. For the stability constant determinations, a
4-(3-((1-(benzyloxy)-6-oxo-1,6-dihydropyridin-2-yl)methylamino)- 50 mm path length cuvette was used, and for pKa determi-
3-oxopropyl)-4-(2-(benzyloxy)acetamido)heptanediamide (23). nations, a cuvette path length of 10 mm was used. The flow
A mixture of 7 (1.185 g, 3 mmol), 22 (2.484 g, 10.8 mmol), cuvette was mounted on an HP 8453 UV-visible spectrophoto-
HOBt (1.458 g, 10.8 mmol) and DMF (20 ml) was stirred at meter. All instruments were interfaced to a computer and con-
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trolled by a Visual Basic program. Automatic titration and chloride provided compound 6 in good yield. Treatment of 6
spectral scans adopted the following strategy: the pH of a solu- with formic acid yielded triacid 7, which was used in the next
tion was increased by 0.1 pH unit by the addition of KOH from step reaction without purification. Protected bidentate 3,4-
the autoburette; when pH readings varied by <0.001 pH unit HOPO containing a free amino group (8),18 was then coupled
over a 3 s period, an incubation period was activated. For pKa to triacid 7 in the presence of N-hydroxybenzotriazole (HOBt)
determinations, a period of 1 min was adopted; for stability and N,N′-dicyclohexylcarbodiimide (DCC), generating the pro-
constant determinations, a period of 5 min was adopted. At tected hexadentate 3,4-HOPO (9) in 85% yield. Deprotection of
the end of the equilibrium period, the spectrum of the solu- 9 was achieved by hydrogenation in the presence of Pd/C, pro-
tion was then recorded. The cycle was repeated automatically viding the trichloride of hexadentate 3,4-HOPO (1) as a white
until the defined end point pH value was achieved. All the solid in excellent yield.
titration data were analyzed with the pHab program.22 The
Synthesis of hexadentate 3-hydroxypyran-4-one (2). The pro-
species plot was calculated with the HYSS program.23 tected 3-hydroxypyran-4-one (11) was prepared from com-
Analytical grade reagent materials were used in the preparation pound 10 17 by converting the hydroxyl group to an amino
of all solutions.
group using the Mitsunobu reaction in the presence of tri-
Iron(III) affinity determination. Iron(III) affinities of these phenylphosphine (Ph3P), phthalimide, and diisopropyl azodi-
four hexadentate ligands were determined by competition with carboxylate (DIPAD), followed by hydrazination (Scheme 2).
EDTA. Solutions of Iron(III), hexadentate ligand (1–4), and Conjugation of amine 11 to the tripodal acid 7 was achieved
EDTA were added to KCl solution (0.1 M) and alkalimetrically using DCC/HOBt as coupling agent, providing the protected
titrated. The pH observation was taken after standing for hexadentate pyranone 12, which was subjected to the treat-
periods up to 4 h to achieve equilibrium. The data were ana- ment with boron trichloride, yielding the hexadentate 3-hydro-
lyzed using pHab software to obtain the iron(III) affinity con- xypyran-4-one (2) in excellent yield.
stants. The pFe3+ value was calculated by HYSS program23
Synthesis of hexadentate 3-hydroxypyran-2-one (3). The syn-
based on the iron(III) affinity constants determined and pKa thetic route for hexadentate 3,2-HOPO (3) is shown in
values of the hexadentate ligands. The conditions for the calcu- Scheme 3. Reaction of 3-methoxypyridin-2(1H)-one (13) with
lation were pH = 7.4, [Fe3+]Total = 10−6 M, [Ligand]Total = 10−5 M.
tert-butyl 2-bromoethylcarbamate using sodium hydride as a
base produced compound 14 in 73.6% yield. Treatment of 14
with trifluoroacetic acid (TFA) gave the TFA salt of amine (15),
which was coupled to triacid 7 in the presence of O-(6-chloro-
Results and discussion
Chemistry
benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium
hexafluoro-
phosphate (HCTU) to form the protected hexadentate 3-hydro-
xypyran-2-one (16) in 78.5% yield. Treatment of 16 with boron
trichloride generated hexadentate 3-hydroxypyran-2-one (3) in
80.3% yield.
Synthesis of hexadentate 1-hydroxypyran-2-one (4). The syn-
thetic route for hexadentate 1,2-HOPO (4) is shown in
Scheme 4. Oxidation of 6-hydroxypyridine-2-carboxylic acid
(17) with peracetic acid produced compound 18 in 78% yield
using trifluoroacetic acid as a solvent. This yield is similar to
Hexadentate ligands can be constructed by connecting three
bidentate ligands onto a suitable tripodal backbone. However,
in order for the ligand to adopt the correct geometry for metal
binding, the backbone should be linked to the ring at the
ortho position relative to one of the coordinating oxygen
anions.24,25 This design has been adopted in the present work.
Synthesis of hexadentate 3-hydroxypyridin-4-one (1). The
synthetic route for hexadentate 3,4-HOPO (1) is presented in
Scheme 1. Acylation of amine 5 17 with 2-(benzyloxy)acetyl
Scheme 1 Reagents and conditions: (a) BnOCH2COCl, Et3N, 0 °C then
rt; (b) HCOOH, rt, overnight; (c) HOBt, DCC, DMF, rt; (d) H2, Pd/C,
MeOH, HCl, rt.
Scheme 2 Reagents and conditions: (a) (i) (Ph3)P, phthalimide, DIPAD,
0 °C to rt; (ii) NH2NH2, reflux; (b) 7, HCTU, DIPEA, DMF, rt; (c) BCl3, DCM,
0 °C to rt.
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Physico-chemical characterization
Determination of pKa values. The pKa values of the hexaden-
tate chelators 1–4 were determined by spectrophotometric
titration with an automated titration system.19–21 All the titra-
tion data were analyzed using the pHab software.22 The UV
spectra of 1–4 were pH dependent. As an example, the UV
spectra of 1 is presented in Fig. 3, which was recorded between
250 and 350 nm over the pH range 1.64–11.2 for the free
ligand. The speciation spectra demonstrate a clear shift in λmax
from 280 to 310 nm, which reflects the pH dependence of the
ligand ionization equilibrium.
The pKa values of 1 obtained from nonlinear least-squares
regression analysis are 2.44, 3.09, 3.73, 8.90, 9.47 and 10.01. Of
the six pKa values, the lower three values correspond to the
4-oxo functions and the higher three correspond to the
3-hydroxyl functions. There are three pKa values for each of
chelators 2, 3 and 4 in the pH range 2-10 (Table 1). These
values are typical for each individual pyridinone class. It
should be noted that the pKa values of 4 are lower than those
of the other three compounds and as a result at pH 7.0 would
be largely unprotonated. This provides an indication of less
competition with protons for iron complexation.
Scheme 3 Reagents and conditions: (a) BocNHCH2CH2Br, NaH, DMF,
rt then 70 °C; (b) TFA, rt; (c) 7, HCTU, DIPEA, DMF, rt; (d) BCl3, DCM, 0 °C
to rt.
Scheme 4 Reagents and conditions: (a) CH3CO3H, rt; (b) BnCl, MeOH,
reflux; (c) SOCl2, MeOH; (d) NaBH4, THF, MeOH, 70 °C; (e) (i) (Ph3)P,
phthalimide, DIPAD, 0 °C to rt; (ii) NH2NH2, reflux; (f) 7, HOBt, EDC,
DMF, rt; (g) BCl3, DCM, 0 °C to rt.
Fig. 3 pH dependence of UV spectra of 1. [1] = 17.83 μM, pH was
changed from 1.64 to 11.2 by the addition of KOH.
that reported by Workman et al. using acetic acid as a
solvent.26 Benzylation of 18 gave compound 19, which on ester-
ification in the presence of thionyl chloride in methanol pro-
duced 20. Reduction of ester bond in 20 was achieved by the
treatment with sodium borohydride in tetrafuran (THF), fol-
lowed by the addition of methanol,26 yielding compound 21 in
a reasonable yield. In the absence of methanol, ester reduction
does not occur. Amine 22 was prepared from 21 using the
Mitsunobu reaction in the presence of Ph3P, phthalimide and
DIPAD, followed by hydrazination. Conjugation of 22 with
triacid 7 in the presence of EDC and HOBt provided protected
hexadentate 1-hydroxypyran-2-one (23), which was treated with
boron trichloride, thereby generating the hexadentate 1-hydroxy-
pyran-2-one (4). In the present work, the strategy for the
preparation of the hexadentate 1,2-HOPO by using a methylene
linker between the 1.2-HOPO coordinating group and the
molecular scaffold was similar to that reported by Workman
et al.27
Table 1 pKa values of hexadentate ligands 1–4 and their affinity con-
stants for Fe(III) and Cu(II)
Ligands
1
pKa
Log K (Fe(III))
pFe3+
27.6
10.01 0.006, 9.47 0.008
8.90 0.005, 3.73 0.006
3.09 0.004, 2.44 0.003
8.97 0.005, 8.35 0.007
7.70 0.008
9.83 0.006, 8.96 0.009
8.10 0.005
32.8 0.04
2
3
4
25.6 0.02
27.7 0.02
25.9 0.01
23.5
23.9
26.8
6.51 0.007, 5.68 0.002
4.87 0.004
pFe3+ is under the conditions: [Fe3+
pH = 7.4.
]
= 1 μM, [ligand]Total = 10 μM,
Total
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Iron(III) complexation. All the four compounds form stable
coloured complexes with iron(III). Chelators 1 and 2 form red-
yellow iron complexes, whereas chelators 3 and 4 form a
purple colour and a faint yellow colour iron complex at pH 7.0,
respectively. The iron complexes of 3 and 2 are more soluble
than the other two, which precipitate at neutral pH values at
concentrations above 0.1 mM. The stability constants were
determined for the four compounds by competition with
EDTA. The results are presented in Fig. 4. The rate of exchange
of iron(III) between hexadentate ligands was found to be very
slow for 2 and 3 (a 3 day period was adopted to guarantee the
solution reached equilibrium), but relatively fast for chelators
1 and 4 (a period of 1 hour was used to achieve equilibrium).
Because there is quite a wide range in affinities for iron(III)
amongst the four compounds, slightly different conditions
were adopted for each of the compounds in order to favour
partitioning of iron(III) to EDTA. For chelators 2 and 3, a rela-
tively low level of EDTA was used (2 mM) at pH 7.24. The log K
values of iron(III) complexes of 2 and 3 were determined to be
25.6 and 27.7, respectively (Table 1). For chelators 1 and 4, a
higher EDTA concentration was employed (98.6 mM) and the
competition was performed at a more acidic pH value (6.4).
Both changes favour EDTA in the competition for iron(III). The
log K values of iron(III) complexes of 1 and 4, being 32.8 and
25.9, respectively (Table 1). Thus the affinities for iron(III) cover
the range 32.8 to 25.6. These differences become apparent in
the pFe3+-pH plots under the conditions of 1 μM iron(III) and
10 μM ligand (Fig. 5).
Fig. 5 pFe-pH plots for the condition 1 μM iron(III) and 10 μM ligand.
26.6), a hexadentate siderophore containing three hydroxamate
moieties, and the iron(III) affinity of 4 is similar to that of des-
ferrioxamine, but 2 and 3 are lower than desferrioxamine. In
comparison to enterobactin,
a tricatecholate hexadentate
ligand, with the extremely high pFe3+ value of 35.5, hexaden-
tate ligands 1–4 have appreciably lower pFe3+ values. However,
the effectiveness of enterobactin to scavenge iron at pH 7.0 is
limited by its weak acidity and the required loss of six protons
on binding iron(III).28
Conclusions
With regards to the iron(III) affinities of the hexadentate ligands
1, 2 3 and 4, the pFe3+ values follow the sequence 1 > 4 ≫ 3 > 2,
which is different to the pFe3+ value sequence of the corres-
ponding bidentate molecules, namely 3,4-HOPO ≫ 3,2-HOPO >
1,2-HOPO > 3-hydroxypyranone.15 The major difference in
behaviour of the hexadentate ligands when compared to their
respective bidentate analogues was found with 4. Thus whereas
there are 4 orders of magnitude difference in the pFe3+ values
of deferiprone and 1,3-dihydroxypyridin-2-one, the pFe3+ values
of 1 and 4 are almost identical (Table 1). Clearly there is a large
advantage in forming hexadentate 1,3-dihydroxypyridin-2-ones
judging from the differences in the pFe3+ values of the corres-
ponding bidentate and hexadentate analogues (1,2-HOPO and
4) and this probably accounts for the large number of such oli-
godentate 1,2-HOPO ligands that have been investigated.16,29
The sequence of pFe3+(7.4) values of chelators 1–4 under
the conditions of 1 μM iron(III) and 10 μM ligand are 27.6,
23.5, 23.9, and 26.8, respectively. Chelator 1, the 3,4-HOPO,
binds iron(III) with the highest affinity. Chelator 4, the 1,2-
HOPO, is the next highest, despite possessing a much lower
log K(FeIII) value. The relatively high pFe3+ value results from
the low pKa values of the free ligand. The iron(III) affinity of
compound 1 is higher than that of desferrioxamine (pFe3+
=
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This work was financially supported by Unilever.
Fig. 4 Iron(III) binding competition between hexadentate chelators 1–4
and EDTA, and iron(III) binding in the absence of EDTA. (A) Chelator 1,
Notes and references
[L]Total = 67 μM; [Fe]Total = 53.4 μM; pH 6.431. (B) Chelator 2, [L]Total
=
815 μM; [Fe]Total = 741 μM; pH 7.24. (C) Chelator 3, [L]Total = 1.02 mM;
1 T. Zhou, Y. M. Ma, X. L. Kong and R. C. Hider, Dalton
Trans., 2012, 41, 6371–6389.
[Fe]Total = 0.73 mM; pH 7.24. (D) Chelator 4, [L]Total = 65.9 μM; [Fe]Total
53.4 μM; pH 6.43.
=
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