CN128: A New Orally Active Hydroxypyridinone Iron Chelator

Article abstract of DOI:10.1021/acs.jmedchem.0c00137

Deferoxamine, deferiprone, and deferasirox are used for the treatment of systemic iron overload, although they possess limitations due to lack of oral activity, lower efficacy, and side effects. These limitations led to a search for an orally active iron chelator with an improved therapeutic index. The lower efficacy of deferiprone is due to rapid glucuronidation, leading to the formation of a nonchelating metabolite. Here, we demonstrate that the influence of metabolism can be reduced by introducing a sacrificial site for glucuronidation. A log P-guided investigation of 20 hydroxpyridinones led to the identification of CN128. The Fe(III) affinity and metal selectivity of CN128 are similar to those of deferiprone, the log P value is more lipophilic, and its iron scavenging ability is superior. Overall, CN128 was demonstrated to be safe in a range of toxicity assessments and is now in clinical trials for the treatment of β-thalassemia after regular blood transfusion.

Full text of DOI:10.1021/acs.jmedchem.0c00137

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Article
CN128, a new orally active hydroxypyridinone iron chelator
Wenteng Chen, Xin Yuan, Zhi Li, Zidong Lu, Sisi Kong, Huidi Jiang, Houbing Du, Xiuhong Pan, Manasi
Nandi, Xiaole Kong, Kathryn Brown, Zudong Liu, Guolin Zhang, Robert C. Hider, and Yongping Yu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00137 • Publication Date (Web): 25 Mar 2020
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CN128, a new orally active hydroxypyridinone iron chelator
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Wenteng Chen, † Xin Yuan, ‡ Zhi Li, † Zidong Lu, § Sisi Kong, † Huidi Jiang, † Houbing Du, ‖
Xiuhong Pan, ‖ Manasi Nandi, § Xiaole Kong, § Kathryn Brown, § Zudong Liu, ‡ Guolin Zhang, †
Robert C. Hider, §, * Yongping Yu †, *
† College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, Zhejiang
Province, China
‡ Hangzhou Zede Pharma-Tech Co. Ltd., Hangzhou, 311121, Zhejiang Province, China.
‖Suzhou Xishan Zhongke Drug R & D Co. Ltd., Suzhou, 215104, Jiangsu Province, China.
§ Institute of Pharmaceutical Science, Franklin Wilkins Building, King’s College London, 150
Stamford St., SE1 9NH, London, UK.
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ABSTRACT
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Deferoxamine, deferiprone and deferasirox, are used for the treatment of systemic iron overload,
although they possess limitations due to lack of oral activity, lower efficacy and side effects. These
limitations led to a search for an orally active iron chelator with an improved therapeutic index.
The lower efficacy of deferiprone is due to rapid glucuronidation leading to the formation of a
non-chelating metabolite. Here, we demonstrate that the influence of metabolism can be reduced
by introducing a sacrificial site for glucuronidation. A logP-guided investigation of 20
hydroxpyridinones led to the identification of CN128. The Fe(III) affinity and metal selectivity of
CN128 are similar to those of deferiprone, the logP value is more lipophilic, and its iron
scavenging ability is superior. Overall, CN128 was demonstrated to be safe in a range of toxicity
assessments and is now in clinical trials for the treatment of β-thalassemia after regular blood
transfusion.
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INTRODUCTION
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Iron is essential for all prokaryotes and eukaryotes due to its critically important role in enzyme
function. In humans iron is a cofactor in over 400 enzymes and transport proteins.1 However,
increased absorption of dietary iron 2 or regular blood transfusion to maintain hemoglobin levels 3
can result in iron accumulation in humans. Excess iron will lead to uncontrolled Fenton chemistry,
subsequently resulting in oxidative damage to proteins and membranes. Continued iron
accumulation leads to liver disease, 4 diabetes mellitus, 5 and heart disease.6 Therefore, the iron
levels in non-stressed individuals should be carefully maintained within controlled limits.7 There
is no mechanism in the human for the excretion of excess iron. To this end, external intervention
by using iron-selective chelators is a favored approach, because such treatment enhances the
excretion of iron and facilitates its removal from iron-loaded tissues.8, 9 The iron chelators in
current clinical use are desferrioxamine,10 deferiprone,11 and deferasirox.12 (Figure 1)
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Figure 1. The chemical structures of clinically approved iron chelators
Desferrioxamine is not orally active and both deferiprone and deferasirox, although orally
active, possess limitations in both efficacy of iron removal and in associated side effects.13, 14 Our
previous studies have demonstrated that the most stable complex of deferiprone (1) and iron is a
1:3 molecular complex (2). By virtue of the non-charged nature, both 1 and 2 have the ability to
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rapidly cross the biological membranes by non-facilitated diffusion. (Figure 2) This property
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has endowed deferiprone with the ability to remove intracellular surplus iron and in particular toxic
iron from heart and brain.16-18 Additionally, the ability of deferiprone to scavenge iron is strongly
concentration dependent, deferiprone becoming a relatively poor iron(III) scavenger at
concentrations less than 5 µM, and totally failing to scavenge iron(II) at these low concentrations.19,
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Deferiprone is rapidly glucuronidated in hepatocytes forming a non-chelating metabolite,21
which is associated with a reduced ability to remove iron from the liver. In addition, there is a low
incidence of reversible agranulocytosis associated with deferiprone (approximately 1% of
patients),22 which necessitates a weekly white cell count for all patients using deferiprone. For
these reasons there is a demand for a novel orally active iron chelator that possesses a larger
therapeutic index than those of deferiprone and deferasirox.
Figure 2. The non-facilitated diffusion of deferiprone (1) and its iron complex (2)
There has been much effort devoted to the search of novel bidentate 3-hydroxypyridones with
the aims of improving the iron-scavenging properties and reducing the undesired side effects
associated with glucuronidation and agranulocytosis.23-29 None of these strategies have been
successful in identify compounds possessing a higher therapeutic index than that of deferiprone.
In this study, we hypothesize that chemical space exploration of the N-1 position of the 3-
hydroxylpyridinone core by introducing diverse chiral amino alcohols can achieve a number of
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improvements. (Figure 3) Firstly, the side chain hydroxyl may act as an alternate sacrificial site
for glucuronidation, in principle alleviating the metabolic problem associated with the 3-hydroxyl
function. Secondly, the variation of substituents R1 and R2 may modulate the hydrophobicity of
compounds for ideal logP values and pharmacokinetic properties. Finally, the chiral centers
originating from the starting amino alcohols will further influence the rates of metabolism and
thereby optimize the in vivo efficacy.
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Figure 3. Comparison of the iron chelation properties of deferiprone and amino alcohol derivatives
of 3-hydroxypyridin-4-ones (AAHPOs).
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RESULTS AND DISCUSSION
Chemistry
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The synthetic route of the amino alcohol conjugates of 3-hydroxypyridin-4-ones (AAHPOs) 8
starting from maltol (3a) or ethyl maltol (3b) is outlined in Scheme 1. The benzylation of 3 with
benzyl chloride provided 4 in 85-90% yield. The chiral amino alcohols 5 are commercially
available. Condensation of 4 and amino alcohols 5 under alkaline conditions provided the
corresponding protected hydroxypyridin-4-ones 6.30,31 Deprotection of 6 was achieved by
hydrogenation under a H2 atmosphere in EtOH-H2O, affording the 7 in 31-68% yields. Finally, 7
was converted to the hydrochloride salt 8 in the presence of HCl-MeOH.
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Scheme 1. Synthesis of amino alcohol conjugates of 3-hydroxypyridin-4-ones (AAHPOs).
Reagents and conditions: (a) BnCl, NaOH, EtOH-H2O, 60 oC, 6 h, 85-90%; (b) 5, K2CO3, EtOH-
H2O, 80 oC, 18 h, quantitative yield; (c) Pd/C-H2, EtOH-H2O, rt, 4 h, 31%-68% over two steps; (d)
HCl-MeOH, rt, 30 min, 63%
Determination of Distribution Coefficient of Free ligand and Iron Complex
The distribution coefficients of the free ligands AAHPOs 8a-8t and their iron(III) complexes
are presented in Table 1. The milogP 32 values varied from the hydrophilic pyridinones (- 1.88) to
the more hydrophobic molecules (+ 1.13). Making use of calibration curves,31 it also proved
possible to predict the logP values of the corresponding 3:1 iron complexes, which cover the range
from (- 3.4) to (+2.05). (Table 1) The logP and logD7.0 values are identical for both the free ligands
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and the iron complexes, as both compound classes are noncharged at pH 7.0. The logP values for
deferiprone (1) are ligand, -0.77 and iron complex, -2.60. These parameters are closely mirrored
by 8a and 8b. (Table 1) As the milogP values increase for the AAHPOs in the region of 0.5 (8g,
8h, 8k, 8l, 8o, 8p), it is likely that these compounds maybe permeate cell membranes more rapidly
than deferiprone, while maintaining good water solubility. These compounds would also be
associated with higher logP values for the corresponding iron complexes when compared with that
of deferiprone (-2.60). Therefore, it is anticipated that they will facilitate iron efflux from cells
more rapidly than deferiprone.
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Table 1. Comparison of Distribution Coefficient of Free Ligand and Iron Complex
logP
Iron
Compd.
Chirality
R1
R2
milogPa
(FeL3)b Mobilisation %
S
R
S
R
S
R
S
R
S
S
S
R
S
R
S
R
S
R
/
CH3
CH3
CH2CH3
CH2CH3
CH3
CH3
CH3
CH3
-0.87
-0.87
-0.30
-0.30
-0.12
-0.12
0.45
0.45
0.38
0.38
0.41
0.41
0.98
0.98
0.56
0.56
1.13
1.13
-1.88
-1.31
-2.80
-2.80
-1.60
-1.60
-1.10
-1.10
0.10
0.10
0.00
0.00
0.05
0.05
1.60
1.60
0.58
0.58
2.05
2.05
-3.40
-3.20
11.9
6.5
16.0
10.5
18.7
8.0
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
8o
8p
8q
8r
8s
CH3
CH(CH3)2
CH(CH3)2
CH(CH3)2
CH(CH3)2
CH(CH2CH3)CH3
CH3
CH2CH3
CH2CH3
CH3
7.9
10.6
10.8
5.5
5.8
8.2
10.3
13.6
12.8
24.8
13.1
22.3
4.0
CH2CH3 CH(CH2CH3)CH3
CH3
CH3
CH2CH3
CH2CH3
CH3
CH2CH(CH3)2
CH2CH(CH3)2
CH2CH(CH3)2
CH2CH(CH3)2
CH2Ph
CH3
CH2Ph
CH2Ph
CH2Ph
CH2CH3
CH2CH3
CH3
H
H
/
CH2CH3
2.4
8t
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Deferipro
ne (1)
-0.77
-2.60
11.8
a.milogP are computer derived values. 32 b.logP(FeL3)values are derived from a published
calibration curve. 31 The corresponding values for deferiprone are adopted from ref. 31
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Iron Scavenging Efficacy of Amino Alcohol Derivatives of 3-Hydroxypyridin-4-ones
The iron scavenging activity of the novel 3-hydroxypyridin-4-ones was monitored in the 59Fe-
ferritin-loaded rat. 33, 34 A number of hydroxypyridinones (at 450 μmol/kg) were found to be more
efficient than deferiprone when administered orally, namely 8c (p = 0.077), 8e (p = 0.022), 8p (p
< 0.0001) and 8r (p = 0.0027). (Figure 4) Significantly there was a large difference between the
efficacies of the enatiomeric pairs in each case. The S -isomers were superior with 8c/d and 8e/f,
whereas the R -isomers were found to be superior with 8o/p and 8q/r. A dose response study was
undertaken with these latter four compounds and deferiprone. (Figure 5). 8e, 8p and 8r emerged
as the most potent chelators, with the efficacy of 8p being particularly marked at a lower dose of
150 μmol/kg. With consideration of an enhanced toxicity risk associated with high logP values, 35
8p was selected for further study (8p is also known as CN128).
Figure 4. Iron mobilisation in 59Fe-ferritin loaded rats (450 μmol/kg)
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Figure 5. Dose response of iron mobilisation by selected chelators in 59Fe-ferritin loaded rats.
Determination of Acid Dissociation Constants of 8p (CN128)
In similar fashion to deferiprone, spectrophotometric titration over the pH range 1.8-11.0
yielded two pKa values for 8p, which are 3.26 ± 0.003 (pKa1) and 9.90 ± 0.003 (pKa2). These are
assigned to the protonation of the 4-hydroxyl and the 3-hydroxyl respectively. The ionization
equilibrium of this type of AAHPO is shown in Scheme 2 and Figure S1.
Scheme 2. Proton dissociation equilibria of the AAHPOs
Additionally, the logP value for 8p (Tris buffer pH 7.0, 100 mM/n-octanol) was determined as
0.62 which is slightly higher than the calculated milogP value of 0.56. (Table 1) From this value
it is possible to estimate the logP value of the iron(III) complex,36 which is 0.68. The corresponding
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values for deferiprone are -0.77 and -2.60, thus both the ligand (8p) and the iron complex are more
lipophilic than deferiprone.
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Spectrophotometric titration of the iron(III) complex of 8p over the pH range 1.5-8.0 yielded
two isosbestic points and fitted well to a model consisting of 3 iron complexes as indicated in
equation (ii) (Figure 6a). The speciation plot of iron(III) in the presence of 8p indicates that the
FeIII (8p)3 complex dominates over the pH range 6.5-9.0 (Figure 6b) at appropriate physiological
concentrations. The stoichiometry of the 3:1 complex was confirmed by a Job plot. (Figure 6c)
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Figure 6. Spectrophotometric titration of 8p in the presence of iron(III). (a) Spectra over the pH
range 1.5-8.0; (b) Speciation plot for 8p (10 μM) and iron(III) (1 μM) over the pH range 1.5-9.0;
(c) The binding mode of 8p with iron(III) at pH 7.0 leads to the formation of a 3:1 complex: The
Fe3+ displaces a proton from each of the three hydroxypyridinones and so the resulting complex is
non-charged.
The affinity constants for a range of biologically important metals were also determined by
spectrophotometric titration. (Table 2 and Figure S2-S7) There is a relatively high logβ2 value for
copper(II), but this is still 8 orders of magnitude lower than that for iron(III). Titration of 8p in the
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presence of iron(II) proved to be difficult due to the rapid autoxidation of the iron, even when
oxygen was exhaustively removed from the system. However, a reversible redox reaction could
be observed by cyclic voltammetry, which yielded a redox potential of -603 mV (vs SHE). This
value is slightly higher than that of deferiprone.37 Using this redox potential, the logK1 value for
iron(II) was determined as 7.32. These results indicated that 8p (CN128) possessed high selectivity
for iron(III) over the other metals. There is no major difference in the corresponding metal
affinities of 8p and deferiprone. (Table S2)
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Table 2. Metal affinities selectivity of 8p
Metal
Mg2+
Co2+
Ni2+
logK1
9.12
6.60
6.92
7.05
10.42
7.32
14.78
12.15
logβ2
13.64
11.56
11.96
13.16
19.29
13.57
27.00
22.18
Logβ3
pM
7.59
6.17
6.17
6.15
8.31
6.22
19.80
13.00
/
/
/
/
/
/
Zn2+
Cu2+
Fe2+
Fe3+
36.79
29.58
Al3+
Glucuronidation and oxidative metabolism
One of the main problems for HPOs is that they undergo rapid metabolism in the liver and are
glucuronidated at the 3-hydroxyl group, resulting in the formation of the non-chelating 3-O-
glucuronide metabolite. In this study, it was anticipated that the side chain hydroxyl may act as an
alternate sacrificial site for glucuronidation, in principle alleviating the metabolic problem
associated with 3-hydroxyl function. Initially, to understand the potential influence of chirality on
the metabolism of AAHPOs, 8o, 8p and deferiprone were selected for incubation with rat and
human microsomes. The glucuronidated ratio of these AAHPOs and deferiprone after 1 h
incubation to the total added iron chelators was expressed as a percentage conversion, presented
in Figures 7a and 7c. It was found that incorporation of a side chain hydroxyl leads to increased
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glucuronidation. For example, 8o and 8p were metabolized more rapid than that of deferiprone
under the same conditions. More importantly, the R configuration (8p) was demonstrated to
possess a lower glucuronidation and oxidation rate than that of S configuration (8o). In similar
fashion to the glucuronidation result, 8o and 8p were found to possess higher rates of oxidative
metabolism than that of deferiprone in phase 1 metabolism. (Figures 7b and 7d)
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Figure 7. Comparative rate of metabolism of deferiprone (1), 8o and 8p incubated for 1 h in rat
and human microsomes (n = 4, mean ± SD). (a) glucuronidation metabolism in rat microsomes;
(b) oxidation metabolism in rat microsomes; (c) glucuronidation metabolism in human
microsomes; (d) oxidation metabolism in human microsomes.
In order to elucidate the glucuronidation pathway of 8p (CN128), major metabolites collected
from the rat bile after oral administration of 8p (150 μmol/kg) were characterized by hydrolysis
using β-glucuronidase and LC-TOFMS/MS (Figure S8-S9). Two metabolites were detected, the
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3-hydroxyl glucuronidated metabolite (M1) and the major metabolite (M2) that is the
glucuronidated product of side chain hydroxyl. (Figure 8) Unlike the metabolite M1 and
deferiprone-glucuronide, M2 is capable of chelation with iron. The formation of M2 in hepatocytes
will further enhance iron scavenging. When 8p (150 μmol/kg) was administered orally to rats over
an 8 h period, the ratio of M2/M1 fell in the range of 2.3 to 6.9. (Table S2) These results confirmed
our initial hypothesis centred on sacrificial glucuronidation.
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Figure 8. The metabolic pathways of 8p in rats
Pharmacokinetics and tissue distribution study of 8p in rats
These promising metabolic, physical and iron scavenging properties led us to further investigate
the pharmacokinetic and tissue distribution profiles of 8p. 8p was also evaluated for its ability to
inhibit the major drug metabolizing enzymes. The inhibition of cytochrome P450 enzymes (CYPs)
was investigated by determining the activity of 8p against CYPs 1A2, 2B6, 2C8, 2C9, 2D6, 3A4/5
in human liver microsomes. The results reveal essentially no inhibition, with IC50 values > 200
μM (Table S4). The profiles of 8p in rats are summarized in Table 3. The AUC values and Cmax
correlated with the dosage. (Figure 9a and 9c) Our studies demonstrated that 8p possesses good
oral bioavailability (82.6%), high maximal plasma concentration, and low clearance rats after an
oral dose of 75 μmol/kg. Also, the data linked to Vz at different dosages showed no significant
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differences, indicating that the pharmacokinetics of 8p in rats within 75-600 μmol/kg range is not
nonlinear. Additionally, there was no chiral transformation of 8p detected in rats. (Figxiure S10)
Moreover, it is important to evaluate the drug accumulation when administered daily for a period
of time. Encouragingly, when the rats were treated with an oral dose of 75 μmol/kg for 7 days,
there was no significant difference in the pharmacokinetic profiles between the first and last doses.
(Figure 9b), thus indicating that 8p exhibits no obvious accumulation.
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Table 3. Pharmacokinetic parameters of 8p in rats (n=8, mean±SD)
Dose (μmol/kg) AUC(0-∞) (mg/L·h) CLz (L/h/kg) Vz (L/kg)
Cmax (mg/L)
8.671±2.0
19.88±4.5
41.86±5.0
70.00±10
F %
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0.92±0.24
0.91±0.23
7.60±3.9
5.29±1.7
3.72±0.8
4.29±2.9
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Figure 9. Pharmacokinetics of 8p in rats. (a) Plasma concentration-time curves after oral
administration of diverse dosages; (b) Plasma concentration-time curves after oral administration
of 8p at day 1 and day 7 when rats were gavaged daily for 7 days (150 μmol/kg); (c) Relationship
between AUC and dosage. (n = 8).
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The concentration of 8p in various tissues was detected at multiple time points over a 6 h period
following an oral dose of 150 μmol/kg in rats. (Figure 10) Within 10 min, the mean drug
concentration of 8p in the intestine, liver, kidney, stomach and adrenals rapidly reached a 3.3 to
8.8 fold higher level than that in plasma. (Table S6) The 8p concentrations in these tissues was
found to be generally low indicating a minimal tendency for drug accumulation. (Figure 10).
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Figure 10. Tissue distribution of 8p in rats after a single dose (150 μmol/kg) (mean ± SE, n = 5)
Iron Scavenging Efficacy of 8p in Iron Overloaded Rats
Given the high selectivity and efficient chelation of Fe (III), together with the promising
pharmacokinetic and metabolic properties, the in vivo iron removal efficacy of deferiprone and 8p
(CN128) were directly compared using iron overloaded rats. (Figure 11 and Table S8) The iron
removal efficacies of 8p displayed a dose-dependent behavior, ranging between 3.6% to 48.5%
for doses 75 μmol/kg to 1200 μmol/kg (Table S8). When the rats were orally administered with
8p at 150 μmol/kg and 300 μmol/kg, the iron removal efficacies were 3.1 to 3.8 fold more than
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those obtained with deferiprone. Importantly, 8p is well-tolerated, with no mortality being
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Figure 11. Dose response (75, 150, 300, 600 and 1200 μmol/kg) of iron removal efficacy of
deferiprone (1) and 8p from iron-overloaded rats. (mean ± SE, n = 6)
Toxicity studies of CN128 (8p) in the rat and mouse
As the influence on white cell counts are critically important for this class of chelator, a detailed
investigation was undertaken in the mouse, where it is possible to readily monitor the separate
levels of neutrophils, lymphocytes, platelets, eosinophils reticulocytes and erythrocytes by cell
sorting. The following three compounds were compared at a daily dose of 200 mg/kg for 2 months;
deferiprone, 8o and 8p. No deaths were recorded in this study. Total white cell counts (Figure
12a), neutrophils (Figure 12b), lymphocytes (Figure 12c), monocytes (Figure 12d) and
eosinophils (Figure 12e) were all decreased for deferiprone. In contrast with both 8o and 8p these
cell populations were unchanged or marginally increased. There was no major change in the
platelet population (Figure 12f). The reticulocyte population was increased only with deferiprone
(Figure 12g) and the erythrocyte population was decreased only with deferiprone (Figure 12h).
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In summary there were major changes in the white cell population for deferiprone whereas the cell
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Figure 12. Mouse plasma cell levels after receiving deferiprone (1), 8o and 8p at a dose of 200
mg/kg/day for two months (5 days per week). (a) total number of white cells; (b) neutrophils; (c)
lymphocytes; (d) monocytes; (e) eosinophils; (f) platelets; (g) reticulocytes; (h) erythrocytes. (n =
8).
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8p lacks genetic toxicity as assessed by bacterial reverse mutation test (Ames test), mammalian
erythrocyte micronucleus test, and in vitro mammalian cell gene mutation test. In a chronic 4-week
rat toxicity test (daily oral dose of 25, 44, 77 and 116 mg/kg), there were slight histopathological
changes observed in the pancreas, bone marrow and thymus. However, in a similar manner to that
demonstrated by deferiprone, these changes are completely reversed after an additional 4-week
period in the absence of the drug.
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CONCLUSION
The strategy of incorporating an alternative sacrificial site for glucuronidation and undertaking
a logP-guided exploration in the space adjacent to the 3-hydroxypyridin-4-one nucleus resulted in
the identification of lead compound 8p (CN128), which possesses a comparable Fe (III) chelation
ability and transition metal selectivity to that of deferiprone. It possesses a more lipophilic
distribution coefficient, satisfactory distribution as well as suitable oral pharmacokinetic profiles
and a favorable tissue distribution. Concerns centred on 3-hydroxyl glucuronidation being related
to iron removal efficacy was demonstrated by glucuronidation mediated metabolism and the 3-
hydroxyl glucuronidation pathway of 8p was reduced. Oral administration of 8p at a dose of 150
μmol/kg, led to an iron removal efficacy 3.8 fold greater than that obtained with the same dose of
deferiprone. 8p also exhibited an improved safety profile when compared with deferiprone with
the white cell population remaining unchanged or marginally increased during 2-month oral
administration. On the basis of these findings, 8p (CN128) was considered as a promising
candidate for the treatment of systemic iron overload and is now in clinical trials for the treatment
of β-thalassemia after regular blood transfusion.
EXPERIMENTAL SECTION
General information:
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All reagents and solvents were obtained from commercial suppliers and used without further
purification unless otherwise noted. Reactions were monitored by TLC carried out on Silica gel
60 F254 plates. Melting points were recorded on a BÜCHI B-540 melting point apparatus. Special
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rotations were recorded on a JASCO P-1030 polarimeter. H NMR and C NMR spectra were
recorded at 500 MHz using a Bruker AVANCE III spectrometer in CDCl3, or dimethyl sulfoxide
(DMSO)-d6 solution. For 1H NMR, tetramethylsilane (TMS) served as internal standard (δ=0) and
data are reported as follows: chemical shift, integration, multiplicity (s = singlet, d = doublet, t =
triplet, q = quartet, m = multiplet), and coupling constant(s) in Hz. For 13C NMR, TMS (δ=0) or
CDCl3 (δ=77.26) was used as internal standard and spectra were obtained with complete proton
decoupling. HPLC purity analysis and the HRMS of all final products were confirmed on a Agilent
1290 HPLC-6224 Time of Fight Mass Spectrometer using PhenomenexLuna 5μ C18, 100 Å, 150
X 4.60 mm 5 micron column at a flow rate of 0.5 mL/min using liner gradients buffer B in A (B:
CH3OH containing 0.1 % formic acid, A: H2O containing 0.1% formic acid). Mobile phase B was
increased linearly from 5% to 95% over 7 min and 95% over the next 2 min, after which the column
was equilibrated to 5% for 1 min. Purity of all biological tested compounds were determined by
HPLC to be >95%.
General procedures for the synthesis of final compounds
Step 1: Synthesis of 3-benzyloxy 4H-pyran-4-ones 4. To a suspension of 3 (100 mmol) and EtOH
(45 mL) in a three-necked round bottom flask was added a 10.0 M sodium hydroxide solution (11
mL, 110 mmol). A clear solution resulted upon stirring. Benzyl chloride (13.8 mL, 120 mmol) was
then added, and the resulting solution was heated to 60 oC for 1.5 h. To the reaction mixture was
further added a 10.0 M sodium hydroxide (2 mL, 20 mmol) and benzyl chloride (2.7 mL, 23 mmol),
and the reaction mixture was heated to reflux for another 4.5 h. The mixture was cooled to room
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temperature and filtered. The filtrate was concentrated in vacuum to a yellow oil. The resulting oil
was mixed with water and extracted with DCM (4 x 50 mL). The combined DCM layer was dried
over anhydrous Na2SO4 and concentrated to give 4.
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3-benzyloxy 2-methyl-4H-pyran-4-one (4a): Light yellow oil. Yield = 85%. 1H NMR (500 MHz,
CDCl3) δ 7.60 (d, J = 5.5 Hz, 1H), 7.41-7.39 (m, 2H), 7.36-7.32 (m, 3H), 6.36 (d, J = 5.5 Hz, 1H),
5.16 (s, 2H), 2.09 (s, 3H).
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3-benzyloxy 2-ethyl-4H-pyran-4-one (4b): Light yellow oil. Yield = 90%. 1H NMR (500 MHz,
CDCl3) δ 7.61 (d, J = 5.5 Hz, 1H), 7.38-7.26 (m, 5H), 6.35-6.33 (m, 1H), 5.13 (s, 2H), 2.47 (q, J
= 7.5 Hz, 2H), 0.95 (t, J = 7.5 Hz, 3H).
Step 2: Synthesis of 3-(benzyloxy)-1-(1-hydroxy-propan-2-yl)-pyridin-4(1H)-one (6). 3-
benzyloxy 4H-pyran-4-ones (12 mmol) 4 from step 1 and 1.1 g (18 mmol) of amino alcohol 5 is
dissolved in a mixture of 30 mL of ethanol and 10 mL water (distilled). With stirring, K2CO3 (24
mmol) was added. Then the reaction mixture was refluxed for 18 h under stirring. After cooling,
the mixture was adjusted to a pH = 1.0 by addition of concentrated hydrochloric acid solution, to
give a yellow solid. The mixture was then concentrated completely on a rotary evaporator under
vacuum. The residue was added with 15 mL of water (distilled) (suspension), this suspension was
washed twice with 15 mL of diethyl ether, then adjusted to a pH = 9.0 by addition of 10 M sodium
hydroxide solution, wherein the solid went into solution. The aqueous phase was extracted four
times with 30 mL dichloromethane. The organic phase was dried over anhydrous Na2SO4. The
solvent was completely removed on a rotary evaporator, resulting as a crude yellow solid residue
6 for further step without purification.
Step 3: Synthesis of final compounds 8. Compound 6 (5 mmol) from Step 2 was dissolved in a
mixture of 36 mL of ethanol and 4 mL of water (distilled), respectively. Then 0.07 g of Pd/C (5%)
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were added as hydrogenation catalyst. Then the compound at room temperature was hydrogenated
at a hydrogen pressure of 1 atm. After reduction, the catalyst was separated by filtration. The
filtrate was concentrated and dissolved in HCl-MeOH to adjust the pH to 1.0. The solution was
stirred at room temperature for 30 min and then evaporated on a rotary evaporator under vacuum.
Here, a solid was obtained and recrystallized from EtOH/Et2O for 8.
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(S)-3, 4-dihydroxy-1-(1-hydroxypropan-2-yl)-2-methylpyridin-1-ium chloride (8a): White
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solid, yield: 53.8%. m.p. = 149-150 C. [α]20D = +11.57 (c 1.0, MeOH) H NMR (500 MHz,
DMSO-d6) δ 8.37-8.35 (m, 1H), 7.44-7.43 (m, 1H), 4.90-4.87 (m, 1H), 3.70 (ddd, J = 20.0, 12.0,
6.0 Hz, 2H), 2.60 (s, 3H), 1.43 (d, J = 7.0 Hz, 3H).13 C NMR (125 MHz, DMSO-d6) δ 158.8, 143.0,
142.7, 135.2, 111.4, 64.5, 61.4, 17.2, 13.2. HRMS (ESI): m/z calcd for (M-Cl)+:184.0968; found:
184.0961.
(R)-3, 4-dihydroxy-1-(1-hydroxypropan-2-yl)-2-methylpyridin-1-ium chloride (8b): Light
yellow solid, yield: 58.8%. m.p. =146-147 oC. [α]20D = -13.65 (c 1.0, MeOH) 1H NMR (500 MHz,
DMSO-d6) δ 8.37-8.35 (m, 1H), 7.43-7.42 (m, 1H), 4.91-4.84 (m, 1H), 3.69 (ddd, J = 20.0, 12.0,
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6.0 Hz, 2H), 2.59 (s, 3H), 1.42 (d, J = 7.0 Hz, 3H). C NMR (125 MHz, DMSO-d6) δ 158.8,
143.0, 142.7, 135.2, 111.4, 64.5, 61.5, 17.2, 13.2. HRMS (ESI): m/z calcd for (M-Cl)+:184.0968;
found: 184.0976.
(S)-2-ethyl-3, 4-dihydroxy-1-(1-hydroxypropan-2-yl) pyridin-1-ium chloride (8c): White solid,
yield=53.4%. m.p. = 163-164 oC, [α]20D = +23.73 (c 1.0, MeOH) 1H NMR (500 MHz, DMSO-d6)
δ 8.39 (d, J = 7.5 Hz, 1H), 7.44 (d, J = 7.0 Hz, 1H), 4.85-4.81 (m, 1H), 3.72 (d, J = 6.0 Hz, 2H),
3.06-3.01 (m, 2H), 1.43 (d, J = 7.0 Hz, 3H), 1.15 (t, J = 7.5 Hz, 3H). 13C NMR (125 MHz, DMSO-
d6) δ 163.6, 152.1, 147.3, 140.0, 116.4, 69.2, 65.9, 24.4, 22.5, 17.2. HRMS (ESI): m/z calcd for
(M-Cl)+:198.1125; found: 198.1130.
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(R)-2-ethyl-3, 4-dihydroxy-1-(1-hydroxypropan-2-yl) pyridin-1-ium chloride (8d): Light
yellow solid, yield=59.5%. m.p. =164-165 oC. [α]20D = -27.45 (c 1.0, MeOH) 1H NMR (500 MHz,
DMSO-d6) δ 8.32-8.31 (m, 1H), 7.31-7.30 (m, 1H), 5.78 (brs, 3H), 4.81-4.77 (m, 1H), 3.72 (d, J
= 6.0 Hz, 2H), 3.02 (q, J = 7.5 Hz, 2H), 1.43 (d, J = 7.0 Hz, 3H), 1.16 (t, J = 7.5 Hz, 3H). 13C NMR
(125 MHz, DMSO-d6) δ 164.8, 150.8, 147.6, 139.9, 116.4, 69.3, 65.5, 24.3, 22.5, 17.3. HRMS
(ESI): m/z calcd for (M-Cl)+:198.1125; found: 198.1131.
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(S)-3, 4-dihydroxy-1-(1-hydroxy-3-methylbutan-2-yl)-2-methylpyridin-1-ium chloride (8e):
White solid, yield=38.6%. m.p. =205-206 oC. [α]20D = -45.48 (c 1.0, MeOH) 1H NMR (500 MHz,
DMSO-d6) δ 7.64 (d, J = 7.5 Hz, 1H), 6.19 (d, J = 7.5 Hz, 1H), 5.86 (brs, 2H), 3.90 -3.86 (m, 1H),
3.79 (dd, J = 12.0, 3.5 Hz, 1H), 3.69 (dd, J = 11.5, 8.0 Hz, 1H), 2.29 (s, 3H), 2.10-2.06 (m, 1H),
1.02 (d, J = 6.5 Hz, 3H), 0.64 (d, J = 6.5 Hz, 3H). 13C NMR (125 MHz, DMSO-d6) δ 173.8, 149.9,
139.3, 135.7, 116.2, 72.5, 66.7, 34.8, 24.8, 24.3, 17.4. HRMS (ESI): m/z calcd for (M-
Cl)+:212.1281; found: 212.1291.
(R)-3, 4-dihydroxy-1-(1-hydroxy-3-methylbutan-2-yl)-2-methylpyridin-1-ium chloride (8f):
Light yellow solid, yield=39.3%. m.p. =201-202 oC. [α]20D = +38.79 (c 1.0, MeOH)1H NMR (500
MHz, DMSO-d6) δ 8.36 (d, J = 7.0 Hz, 1H), 7.43 (d, J = 7.0 Hz, 1H), 4.43-4.39 (m, 1H), 3.89 (dd,
J = 12.0, 3.5 Hz, 1H), 3.76 (dd, J = 12.5, 8.5 Hz, 1H), 2.57 (s, 3H), 2.24-2.19 (m, 1H), 1.07 (d, J
= 6.5 Hz, 3H), 0.62 (d, J = 6.5 Hz, 3H). 13C NMR (125 MHz, DMSO-d6) δ 163.6, 148.1, 147.5,
140.5, 116.2, 76.3, 66.6, 34.9, 24.4, 24.0, 18.3. HRMS (ESI): m/z calcd for
(C11H17NO3+H)+:212.1281; found: 212.1282.
(S)-2-ethyl-3, 4-dihydroxy-1-(1-hydroxy-3-methylbutan-2-yl) pyridin-1-ium chloride (8g):
White solid, yield=33.1%. m.p. =196-197 oC. [α]20D = -42.31(c 1.0, MeOH) 1H NMR (500 MHz,
DMSO-d6) δ 8.05 (d, J = 7.5 Hz, 1H), 6.88 (dd, J = 7.0, 1.5 Hz, 1H), 5.88 (brs, 3H), 4.17-4.13 (m,
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1H), 3.84-3.76 (m, 2H), 2.99-2.85 (m, 2H), 2.21-2.16 (m, 1H), 1.14 (t, J = 7.5 Hz, 3H), 1.06 (d, J
= 6.0 Hz, 3H), 0.63 (d, J = 6.5 Hz, 3H). 13C NMR (125 MHz, DMSO-d6) δ 168.3, 148.4, 147.5,
139.9, 116.4, 74.0, 66.7, 34.9, 24.6, 24.2, 17.5. HRMS (ESI): m/z calcd for (M-Cl)+:226.1438;
found: 226.1436.
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(R)-2-ethyl-3, 4-dihydroxy-1-(1-hydroxy-3-methylbutan-2-yl) pyridin-1-ium chloride (8h):
Light yellow solid, yield=31.4%. m.p. =189-190 oC. [α]20D = +50.71(c 1.0, MeOH) 1H NMR (500
MHz, DMSO-d6) δ 8.36 (d, J = 7.5 Hz, 1H), 7.46-7.44 (m, 1H), 4.40-4.36 (m, 1H), 3.89-3.79 (m,
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2H), 3.10-2.97 (m, 2H), 2.26-2.22 (m, 1H), 1.17-1.14 (m, 3H), 1.08 (m, 3H), 0.62 (m, 3H). C
NMR (125 MHz, DMSO-d6) δ 163.6, 152.7, 147.4, 140.4, 116.4, 75.7), 66.6, 34.9, 24.6, 24.4,
24.1, 17.2. HRMS (ESI): m/z calcd for (M-Cl)+:226.1438; found: 226.1429.
3, 4-dihydroxy-1-((2S,3R)-1-hydroxy-3-methylpentan-2-yl)-2-methylpyridin-1-ium chloride
(8i): Light yellow solid, yield=50.4%. m.p. =199-200 oC. [α]20D = -30.36 (c 1.0, MeOH) 1H NMR
(500 MHz, DMSO-d6) δ 8.35 (d, J = 6.0 Hz, 1H), 7.42 (s, 1H), 4.49-4.46 (m, 1H), 3.89 (dd, J =
12.5, 3.5 Hz, 1H), 3.78 (dd, J = 12.0, 8.5 Hz, 1H), 3.46 (brs, 2H), 2.56 (s, 3H), 2.04-2.00 (m, 1H),
1.02 (dd, J = 6.5, 2.0 Hz, 3H), 0.97-0.90 (m, 2H), 0.76-0.73 (m, 3H) 13C NMR (125 MHz, DMSO-
d6) δ 158.9, 143.3, 142.8, 135.9, 111.5, 70.3, 61.8, 36.2, 24.9, 15.6, 13.5, 11.2. HRMS (ESI): m/z
calcd for (M-Cl)+:226.1438; found: 226.1445.
2-ethyl-3, 4-dihydroxy-1-((2R, 3S)-1-hydroxy-3-methylpentan-2-yl) pyridin-1-ium chloride
(8j): Light yellow solid, yield=52.7%. m.p. =160-161 oC. [α]20D = -38.29 (c 1.0, MeOH) 1H NMR
(500 MHz, DMSO-d6) δ 7.64 (d, J = 8.0 Hz, 1H), 6.17 (d, J = 7.5 Hz, 1H), 4.76 (brs, 3H), 3.92 -
3.88 (m, 1H), 3.76 (d, J = 5.0 Hz, 2H), 2.82 (dq, J = 14.9, 7.5 Hz, 1H), 2.67 (dq, J = 14.8, 7.4 Hz,
1H), 1.88-1.82 (m, 1H), 1.10 (t, J = 7.5 Hz, 3H), 1.02-0.87 (m, 5H), 0.79-0.76 (m, 3H). 13C NMR
(125 MHz, DMSO-d6) δ 169.1, 145.0, 135.8, 134.6, 111.4, 66.2, 62.0, 36.7, 25.3, 18.9, 16.1, 13.1,
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11.9. HRMS (ESI): m/z calcd for (M-Cl)+:240.1594; found: 240.1604.
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(S)-3, 4-dihydroxy-1-(1-hydroxy-4-methylpentan-2-yl)-2-methylpyridin-1-ium chloride (8k):
White solid, yield=50.4%. m.p. =188-189 oC. [α]20D = -15.56 (c 1.0, MeOH) 1H NMR (500 MHz,
DMSO-d6) δ 8.36 (m, 1H), 7.44-7.43 (m, 1H), 5.35 (brs, 1H), 4.72 (d, J = 3.5 Hz, 1H), 3.75 (dd,
J = 12.0, 3.5 Hz, 1H), 3.69 (dd, J = 12.0, 7.5 Hz, 1H), 3.15-3.11 (m, 1H), 3.01-2.94 (m, 1H), 1.84-
1.78 (m, 1H), 1.72-1.66 (m, 1H), 1.49-1.44 (m, 1H), 1.18 (t, J = 7.5 Hz, 3H), 0.90-0.89 (m, 3H),
0.84-0.83 (m, 3H). 13C NMR (125 MHz, DMSO-d6) δ 159.1, 147.3, 142.8, 135.5, 111.7, 63.9,
25.0, 23.3, 22.7, 19.9, 12.4. HRMS (ESI): m/z calcd for (M-Cl)+:226.1438; found: 226.1444.
(R)-3, 4-dihydroxy-1-(1-hydroxy-4-methylpentan-2-yl)-2-methylpyridin-1-ium chloride (8l):
White solid, yield=52.4%. m.p. =216-217 oC. [α]20D = +15.98 (c 1.0, MeOH) 1H NMR (500 MHz,
DMSO-d6) δ 8.38-8.36 (m, 1H), 7.46-7.45 (m, 1H), 5.31 (brs, 1H), 4.80-7.74 (m, 1H), 3.75 (dd, J
= 12.0, 4.0 Hz, 1H), 3.65 (dd, J = 12.0, 8.0 Hz, 1H), 2.60 (s, 3H), 1.83-1.77 (m, 1H), 1.71-1.65 (m,
1H), 1.38-1.31 (m, 1H), 0.84 (dd, J = 9.5, 7.0 Hz, 6H).13C NMR (125 MHz, DMSO-d6) δ 158.9,
143.0, 142.9 135.6, 111.5, 64.1, 24.7, 23.0, 22.6, 13.4. HRMS (ESI): m/z calcd for (M-
Cl)+:226.1438; found: 226.1447.
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(S)-2-ethyl-3, 4-dihydroxy-1-(1-hydroxy-4-methylpentan-2-yl) pyridin-1-ium chloride (8m):
White solid, yield=47.4%. m.p. =218-219 oC. [α]20D = -14.36 (c 1.0, MeOH) 1H NMR (500 MHz,
DMSO-d6) δ 10.52 (brs, 1H), 8.37-8.36 (m, 1H), 7.45-7.44 (m, 1H), 5.28 (brs, 1H), 4.77(m, 1H),
3.75 (dd, J = 12.0,4.0 Hz, 1H), 3.65 (dd, J = 12.0, 8.0 Hz, 1H), 2.60 (s, 3H), 1.83-1.77 (m, 1H),
1.71-1.65 (m, 1H), 1.38-1.33 (m, 1H), 0.86-0.82 (m, 6H). 13C NMR (125 MHz, DMSO-d6) δ 159.0,
142.9, 135.6, 111.5, 64.1, 24.7, 23.0, 22.6, 13.4. HRMS (ESI): m/z calcd for (M-Cl)+:240.1594;
found: 240.1598.
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(R)-2-ethyl-3, 4-dihydroxy-1-(1-hydroxy-4-methylpentan-2-yl) pyridin-1-ium chloride (8n):
White solid, yield=48.0%. m.p. = 194-195 oC. [α]20D = +14.44 (c 1.0, MeOH)1H NMR (500 MHz,
DMSO-d6) δ 10.55 (brs, 1H), 8.38-8.34 (m, 1H), 7.46-7.36 (m, 1H), 5.31 (brs, 1H), 4.74-4.73 (m,
1H), 3.76 (dd, J = 12.0, 4.0 Hz, 1H), 3.71-3.67 (m, 1H), 3.16-3.10 (m, 1H), 3.01-2.94 (m, 1H),
1.84-1.78 (m, 1H), 1.73-1.67 (m, 1H), 1.51-1.43 (m, 1H), 1.18 (t, J = 7.5 Hz, 3H), 0.90 (dd, J =
6.5, 2.5 Hz, 3H), 0.83 (dd, J = 6.5, 2.5 Hz, 3H).13C NMR (125 MHz, DMSO-d6) δ 159.1, 147.4,
142.8, 135.5, 111.7, 63.9, 25.0, 23.3, 22.7, 19.9, 12.4. HRMS (ESI): m/z calcd for (M-
Cl)+:240.1594; found: 240.1599.
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(S)-3, 4-dihydroxy-1-(1-hydroxy-3-phenylpropan-2-yl)-2-methylpyridin-1-ium chloride (8o):
White solid, yield=48.8%. m.p. =175-176 oC. [α]20D = -162.76 (c 1.0, MeOH) 1H NMR (500 MHz,
DMSO-d6) δ 8.53 (d, J = 7.0 Hz, 1H), 7.33-7.31 (m, 1H), 7.25-7.22 (m, 2H), 7.19-7.15 (m, 3H),
5.04-5.02 (m, 1H), 3.88-3.83 (m, 2H), 3.47 (brs, 3H), 3.26 (dd, J = 14.0, 5.0 Hz, 1H), 3.10 (dd, J
= 14.0, 10.0 Hz, 1H), 2.28 (s, 3H). 13C NMR (125 MHz, DMSO-d6) δ 159.3, 142.6, 136.7, 135.7,
129.4, 129.0, 127.4, 111.3, 67.0, 63.4, 36.7, 13.0. HRMS (ESI): m/z calcd for (M-Cl)+:260.1281;
found: 260.1282.
(R)-3,4-dihydroxy-1-(1-hydroxy-3-phenylpropan-2-yl)-2-methylpyridin-1-ium chloride (8p):
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White solid, yield=63.4%. m.p. = 174-175 C. [α]20 = +176.26 (c 1.0, MeOH) H NMR (500
D
MHz, DMSO-d6) δ 8.07 (d, J = 7.5 Hz, 1H), 7.24-7.21 (m, 2H), 7.18-7.12 (m, 3H), 6.54 (d, J =
7.0 Hz, 1H), 5.57 (brs, 3H), 4.72-4.67 (m, 1H), 3.80-3.73 (m, 2H), 3.16 (dd, J = 14.0, 5.0 Hz, 1H),
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3.02 (dd, J = 14.0, 10.0 Hz, 1H), 2.09 (s, 3H). C NMR (125 MHz, DMSO-d6) δ 165.6, 143.8,
137.0, 134.4, 133.8, 128.9, 128.4, 126.7, 110.8, 64.1, 63.1, 36.5, 11.9. HRMS (ESI): m/z calcd for
(M-Cl)-:260.1281; found: 260.1283.
(S)-2-ethyl-3, 4-dihydroxy-1-(1-hydroxy-3-phenylpropan-2-yl) pyridin-1-ium chloride (8q):
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White solid, yield=45.9%. m.p. = 186-187 oC. [α]20D = -189.29 (c 1.0, MeOH)1H NMR (500 MHz,
DMSO-d6) δ 8.04 (d, J = 7.5 Hz, 1H), 7.23-7.20 (m, 2H), 7.17-7.14 (m, 1H), 7.11-7.09 (m, 2H),
6.44 (d, J = 7.5 Hz, 1H), 6.27 (brs, 2H), 4.55-4.50 (m, 1H), 3.80-3.75 (m, 2H), 3.16 (dd, J = 14.0,
5.0 Hz, 1H), 3.05 (dd, J = 14.0, 10.0 Hz, 1H), 2.63-2.58 (m, 1H), 2.45-2.39 (m, 1H), 0.83 (t, J =
7.5 Hz, 3H).13C NMR (125 MHz, DMSO-d6) δ 167.6, 144.4, 137.9, 137.7, 134.7, 129.5, 128.8,
127.1, 111.5, 63.9, 63.8), 37.2, 18.7, 12.5. HRMS (ESI): m/z calcd for (M-Cl)+:274.1438; found:
274.1445.
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(R)-2-ethyl-3, 4-dihydroxy-1-(1-hydroxy-3-phenylpropan-2-yl) pyridin-1-ium chloride (8r):
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Light yellow solid, yield=44.3%. m.p. = 183-185 C. [α]20D = +183.48 (c 1.0, MeOH). H NMR
(500 MHz, DMSO-d6) δ 8.13 (d, J = 7.5 Hz, 1H), 7.23-7.20 (m, 2H), 7.17-7.14 (m, 1H), 7.11-7.09
(m, 2H), 6.60 (d, J = 7.0 Hz, 1H), 6.03 (brs, 3H), 4.61-4.58 (m, 1H), 3.79-3.77 (m, 2H), 3.17 (dd,
J = 14.0, 5.0 Hz, 1H), 3.06 (dd, J = 14.0, 10.0 Hz, 1H), 2.65-2.59 (m, 1H), 2.47-2.41 (m, 1H), 0.84
(t, J = 7.5 Hz, 3H). 13C NMR (125 MHz, DMSO-d6) δ 166.1, 144.0, 139.5, 137.5, 134.8, 129.5,
128.9, 127.2, 111.5, 64.4, 63.8, 37.2, 18.9, 12.4. HRMS (ESI): m/z calcd for (M-Cl)+:274.1438;
found: 274.1443.
3, 4-dihydroxy-1-(2-hydroxyethyl)-2-methylpyridin-1-ium chloride (8s): Light yellow solid,
yield=64.3%. m.p. =186-187 oC. 1H NMR (500 MHz, DMSO-d6) δ 8.20 (d, J = 7.0 Hz, 1H), 7.38
(d, J = 7.0 Hz, 1H), 4.42 (t, J = 5.0 Hz, 2H), 3.74 (t, J = 5.1 Hz, 2H), 2.54 (s, 3H).13C NMR (125
MHz, DMSO-d6) δ 159.0, 143.1, 142.5, 139.3, 110.8, 60.0, 58.4, 13.3. HRMS (ESI): m/z calcd
for (M-Cl)+:170.0812; found: 170.0819.
2-ethyl-3, 4-dihydroxy-1-(2-hydroxyethyl) pyridin-1-ium chloride (8t): Light yellow solid,
yield=68.3%. m.p. =180-181 oC. 1H NMR (500 MHz, DMSO-d6) δ 7.51 (d, J = 7.0 Hz, 1H), 6.11
(d, J = 7.5 Hz, 1H), 5.88 (brs, 2H), 3.98 (t, J = 5.5 Hz, 2H), 3.64 (t, J = 5.5 Hz, 2H), 2.73 (q, J =
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7.5 Hz, 2H), 1.10 (t, J = 7.5 Hz, 3H). C NMR (125 MHz, DMSO-d6) δ 169.6, 145.4, 138.7,
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134.7, 111.0, 61.2, 54.8, 19.1, 12.9. HRMS (ESI): m/z calcd for (M-Cl)+:184.0968; found:
184.0978.
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Determination of affinity constants. The automated titration system used for this study consists
of a Metrohm 765 Dosimat autoburette, a Mettler Toledo MP230 pH meter with SENTEK pH
electrode (P11), and an HP 8453 UV-visible spectrophotometer with a Hellem quartz flow cuvette,
with circulation driven by a Gilson Mini-plus #3 pump (speed capability 20 mL min-1). A
potassium chloride electrolyte solution (0.1 M) was used to maintain the ionic strength. The
temperature of the test solutions was maintained in a thermostatic jacketed titrat on vessel at 25 ±
o
0.1 C, using a Fisherbrand Isotemp water bath. The pH electrodes were calibrated using the
software GLEE 38 with data obtained by titrating a volumetric standard HCl (0.1 M) in KCl (0.1
M) with KOH (0.1 M) under an atmosphere of argon. Analytical grade reagent materials were used
in the preparation of all solutions. The solution under investigation was stirred vigorously during
the experiment. For pKa determinations, a cuvette path length of 10 mm was used, while for metal
stability constant determinations, a cuvette path length of 50 mm was used (experimental
concentration was ca. 40 µM for iron complexes). All instruments were interfaced to a computer
and controlled by an in-house program. The automated titration adopted the following strategy:
the pH of a solution was increased in increments of 0.1 pH unit by the addition of potassium
hydroxide solution (0.1 M) from the autoburette. The pH readings were judged to be stable if they
varied by less than 0.01 pH unit after a pre-set incubation period. For pKa determinations, an
incubation period of 1.5 min was adopted; for metal stability constant determinations, an
incubation period of 3 min was adopted. The cycle was repeated until the predefined end point pH
value was achieved. Titration data were analysed with the HypSpec2014 program
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(http://www.hyperquad.co.uk/).39-40 The fitting spectra range for iron complexes was 400-700 nm.
The associated hydrolysis constants used in the analysis were collected from Martell’s critical
stability constants.41 Metal affinities of compounds in this study were determined in competition
with the metal hydrolysis species in a solution at a high pH (titrated up to pH 12.0). Satisfactory
fitting of both the siderophore titrations with iron was achieved. Speciation plots were calculated
with the HYSS program,42 adopting the iron(III) log hydrolysis constants, FeOH (-2.6563),
Fe(OH)2 (-6.205), Fe(OH)3 (-15.1), and Fe(OH)4 (-21.883), Fe2(OH)2 (-2.843).43
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Iron removal efficacy determination. This study was performed in strict accordance with the
Animals (Scientific Procedures) Act 1986 and approved by King’s College London Ethical
Committee (London, UK). Male SD rats at 300 g, purchased from Harlan UK Ltd. (Oxon, UK),
were kept in specific pathogen free animal facilities. Rats were fasted overnight, and then
anaesthetised with isofluorane for intravenous injection of approximately 1 μC of 59Fe-ferritin (see
Supporting Information). Rats were allowed to recover, and 1 h after 59Fe-ferritin injection, iron
chelators were administered by oral gavage. Rats were then placed in metabolic cages, and food
was returned 1 h after chelator administration. 8 rats were used for each condition investigated (ie,
chelator and chelator concentration). 24 h after injection of 59Fe-ferritin, rats were sacrificed using
an intraperitoneal injection of 1 mL euthatal. Urine and faeces from the metabolic cages were
collected, and the liver and gut of the rats removed for gamma counting. Samples were counted on
a 1282 Compugamma CS gamma counter (LKB Wallac). Student’s test was used to determine
statistical significance between groups.
Pharmacokinetic study of 8p in rats. This study was performed in strict accordance with the
“Principles of laboratory animal care” (http://grants1.nih.gov/grants/olaw/references/phspol.htm)
and were approved by the Institutional Animal Care and Use Committee of Zhejiang University.
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40 Sprague-Dawley rats, 200~250 g, were obtained from the Experimental Animal Center of the
Zhejiang Academy of Medical Sciences (Hangzhou, China). The animals were acclimatized to the
facilities for a week and then fasted with free access to water overnight (approximately 12 h) before
the experiment. Rat (male and female each half, i.v., n = 8, p.o., n = 8) were administered chelator
intravenously via the tail vein at 75 μmol/kg, respectively, or orally at 75 μmol/kg, 150 μmol/kg,
300 μmol/kg and 600 μmol/kg. At pre-determined times (0, 5, 10, 20, 30, 45, 60, 120, 240, 360,
600, 1440 min) after dosing, approximately 0.2 mL blood was collected in a tube containing
heparin, and the plasma was separated by centrifugation. The concentrations of the compound
were measured in the plasma using LC/MS/MS after protein precipitation with acetonitrile. The
relevant estimated pharmacokinetic parameters for plasma were derived using DAS 2.0.
Tissue distribution study of 8p (150 μmol/kg) in rat. Four groups of SD rats (male and female
each half, n=6) were orally administrated at a single dose of 150 μmol/kg CN128. These rats were
euthanized by decapitation at 10 min, 30 min, 2 h and 6 h after dosing, respectively. Tissues
including heart, liver, brain, lung, kidney, stomach, spleen and small intestine were dissected and
washed with saline. Then the tissues were weighed and homogenized in acetonitrile/water (1:9, by
volume) (125 mg/mL). The resulting tissue homogenates were immediately stored at -80 °C until
analysis.
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Toxicology evaluation. Sprague Dawley rat (Shanghai Sippr-BK laboratory animal Co. Ltd.
Animal Resource, Shanghai), 6-8 weeks of age, were used for this experiment. The rats were
randomly divided into five groups and received CN128 (♂: 44, 77, 116 mg/kg, ♀: 25, 44, 66 mg/kg,
p.o.), deferiprone (116 mg/kg, p.o.), or vehicle (water, p.o.) once daily for 4 weeks. The body
weight of the rat was recorded once daily. At the end of the experiment, the rats were sacrificed,
and the serum hematological parameters and serum biochemistry parameters were recorded on
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