Orally Bioavailable Metal Chelators and Radical Scavengers: Multifunctional Antioxidants for the Coadjutant Treatment of Neurodegenerative Diseases

Article abstract of DOI:10.1021/acs.jmedchem.5b00272

Neurodegenerative diseases are associated with oxidative stress that is induced by the presence of reactive oxygen species and the abnormal cellular accumulation of transition metals. Here, a new series of orally bioavailable multifunctional antioxidants (MFAO-2s) possessing a 2-diacetylamino-5-hydroxypyrimidine moiety is described. These MFAO-2s demonstrate both free radical and metal attenuating properties that are similar to the original published MFAO-1s that are based on 1-N,N′-dimethylsulfamoyl-1-4-(2-pyrimidyl)piperazine. Oral bioavailability studies in C57BL/6 mice demonstrate that the MFAO-2s accumulate in the brain at significantly higher levels than the MFAO-1s while achieving similar neural retina levels. The MFAO-2s protect human neuroblastoma and retinal pigmented epithelial cells against hydroxyl radicals in a dose-dependent manner by maintaining cell viability and intracellular glutathione levels. The MFAO-2s outperform clioquinol, a metal attenuator that has been investigated for the treatment of Alzheimer's disease.

Full text of DOI:10.1021/acs.jmedchem.5b00272

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Structures of synthesized compounds in the MFAO-2 series. Shown are multifunctional compounds 2, 4, 6,
and 8; mono-functional chelators (CHL), 1, 3, 5, and 7; monofunctional free radical scavengers (FRS), 10,
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2, 14, and 16, and the non-functional parent compounds, 9, 11, 13, and 15.
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Effect of compound exposure on cell viability of hNBs and RPE cells measured by the MTS viability assay. A
illustrates the cell viability of RPEs after 2 h exposure to 1 mM of compounds 1-16, clioquinol, or Trolox. B
illustrates the cell viability of hNBs after similar exposure to compounds. The results represent the mean ±
SEM, n = 4. Significant differences (p < 0.05), calculated by ANOVA, were compared to blank control.
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Changes in GSH levels in cells exposed to hydroxyl radicals with/without the presence of compounds 1-16,
Trolox, or clioquinol. B illustrates GSH levels of RPEs exposed for 2 h with 1 mM of Fenton reagent
with/without the presence of 1 mM of compounds 1-16, clioquinol, or Trolox. B illustrates GSH levels of
similarly exposed hNB cells. The results represent the mean ± SEM (n = 6). Significant differences,
calculated by ANOVA, were as follows: * p < 0.05 versus control, # p < 0.05 versus clioquinol, and Ɨ p <
0.05 versus Trolox.
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Doseꢀdependent reduction of superoxide generated by xanthine oxidase and measured by MitoSOX staining
in hNB and RPE cells. In A hNBs were preꢀincubated with/without the presence of 1, 10, 100, 1000 µM
compounds 2, 4, 9, 11, JHXꢀ4, clioquinol or Trolox as well as SOD (25 µg/mL, approximately 100 µM) for
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hr and then exposed to xanthine oxidase (25 mU/mL) which can generate approximately 100 µM of
superoxide for 1hr. B illustrates the results of similar experimental procedures of RPEs. The results
represents mean ± SEM, n = 6.
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Doseꢀdependent protection of cells against hydroxyl radicals measured with the LIVE/DEAD Viability Assay.
A illustrates the results of hNB cells that were preꢀincubated for 1 hr with/without the presence of 1, 10,
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00, 1000 µM of compounds 2, 4, 9, 11, JHXꢀ4, clioquinol, or Trolox for 1hr and exposed to 100 µM Fenton
reagent for 2hr. B illustrates the results of similar exposure of RPEs as above. The results represents mean
±
SEM, n = 6.
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Oral bioavailability of MFAOs in mice. Tissue levels of MFAOs measured in the brain (A), neural retina (B),
and lens (C) of C57BL/6 mice fed chow containing 0.05% of each MFAO (80 mg/kg/day average dose) for
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4 days. The results represent the mean ± SEM (n = 6-7). The present MFAO-2s achieved higher brain
levels compared to the previously synthesized MFAO-1 (A) but failed to reach adequate levels in the lens
(
C). Similar neural retinal levels were observed with both MFAO-1 and MFAO-2 (B).
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Compound to metal ion ratio of select metal ions. The stoichiometry of the compound to metal complexes
was obtained from the intersection of lines in Job’s plots. The total concentration of the compound 1-8 and
metal ion was 0.1 - 0.5 mM. * Non-intersecting lines on the Job Plot. Mean ± S.D.; n=3
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Orally Bioavailable Metal Chelators and Radical Scavengers: Multifunctional
Antioxidants for the Coadjutant Treatment of Neurodegenerative Diseases
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Hiroyoshi Kawada and Peter F. Kador*
Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska
Medical Center, 986025 Nebraska Medical Center, Omaha, Nebraska 68198ꢀ6025,
United States of America.
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ABSTRACT
Neurodegenerative diseases are associated with oxidative stress that is induced by the
presence of reactive oxygen species and the abnormal cellular accumulation of
transition metals. Here, a new series of orally bioavailable multifunctional antioxidants
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(MFAOꢀ2s) possessing a 2ꢀdiacetylaminoꢀ5ꢀhydroxypyrimidine moiety is described.
These MFAOꢀ2s demonstrate both free radical and metal attenuating properties that are
similar to the original published MFAOꢀ1s that are based on 1ꢀN, N'ꢀdimethylsulfamoylꢀ
1
ꢀ4ꢀ(2ꢀpyrimidyl)piperazine. Oral bioavailability studies in C57BL/6 mice demonstrate
that the MFAOꢀ2s accumulate in the brain at significantly higher levels than the MFAOꢀ
1s while achieving similar neural retina levels. The MFAOꢀ2s protect human
neuroblastoma and retinal pigmented epithelial cells against hydroxyl radicals in a doseꢀ
dependent manner by maintaining cell viability and intracellular glutathione levels. The
MFAOꢀ2s outperform clioquinol, a metal attenuator that has been investigated for the
treatment of Alzheimer’s disease.
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ꢀ
INTRODUCTION
Oxidative stress results from an imbalance between oxidants, such as reactive
oxygen species (ROS), and antioxidants, such as superoxide dismutase (SOD). An
imbalance favoring oxidants can lead to protein, lipidꢀoxidation, and DNA tissue damage
that is associated with ageꢀrelated cataract, ageꢀrelated macular degeneration (AMD),
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1ꢀ4
and Alzheimer’s disease (AD) . In addition to oxidative stress, an ageꢀrelated
accumulation of redoxꢀactive metals, such as iron and copper, also occurs in the lens,
5
ꢀ8
retina, and brain of patients with age related cataracts, AMD and AD . When combined
with ROS, the increased presence of redoxꢀactive metal ions can promote further tissue
9
damage by generating hydroxyl radicals through the Fenton reaction . In AD, ROS is
generated by mitochondrial dysfunction, the presence of betaꢀamyloid peptide (Aβ) and
2
+
2+
15
redox metal ions such as Fe and Cu , and activated glial cells . Increased Mn levels
can also contribute to mitochondrial dysfunction as well as impaired energy metabolism,
10ꢀ13
excitotoxic neuronal death, apoptotic death, and oxidation of cellular components
.
Aβ also binds Zn to form neurotoxic plaque that results in localized oxidative stress and
14
15, 16
neuronal death . Aβ plaque is also present in the retinas of patients with AMD
and
17
in the lenses of AD patients with cataracts . The increased presence of Aβ plaque in
18
these ageꢀrelated diseases is accompanied by the dramatic loss of αB crystallin , a
zinc binding protein chaperone.
Currently, there is no treatment for preventing the onset, slowing the progression, or
reversing these ageꢀrelated diseases. For AD, approved treatments such as
acetylcholinesterase inhibitors (AChEIs) and memantine, an NꢀmethylꢀDꢀaspartate
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(
NMDA) receptor antagonist, only target the symptoms of AD. While these can
temporarily improve cognitive and daily function, they do not significantly modify the
progression of this disease. The majority of emerging diseaseꢀmodifying AD treatments
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target Aβ based on the amyloid hypothesis including reducing Aβ production,
increasing Aβ removal from the brain immuneꢀtherapeutically, or modulating Aβ
20
toxicity . In addition, an alternative strategy has evolved that links metal
dyshomeostasis, i.e., the modulation of metalꢀprotein interactions linked to Aβ and tau
21
neuropathology, to the treatment of AD . In the brain, metal dyshomeostasis of the
transition metals copper, zinc and iron have been linked to not only to AD but several
22
other neurodegenerative disorders . For example, the availability of zinc at the
glutamatergic synapse is crucially involved in learning and memory and its availability
23
has been shown to decline with age and AD . Copper levels have also been linked to
24
learning and memory and the onset of Huntington’s disease (HD) is associated with
14, 25
increased copperꢀprotein interactions in the brain
. Iron deposition in the brain
(mainly in the Basal Ganglia) has also been linked to cognitive decline and the onset of
26
Parkinson’s disease (PD) .
Metal dyshomeostasis has been evaluated with the metal attenuators clioquinol (PBT1,
5ꢀchloroꢀ7ꢀiodoquinolinꢀ8ꢀol), a first generation 8ꢀhydroxyquinoline (8ꢀOHQ) analog, and
PBT2 (5,7ꢀdichloroꢀ2ꢀ((dimethylamino)methyl) quinolinꢀ8ꢀol), a second generation 8ꢀ
27, 28
OHQ analog, and shown to be effective in animal models of AD, HD and PD
. In a
Phase IIa clinical trial in AD patients, a 250 mg daily dose of PBT2 has also been shown
to be safe, effective in reducing cerebrospinal fluid (CSF) Aβ levels, and improving
29, 30
cognitive performance
. A similar dose of PBT2 has been shown to be generally safe
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and wellꢀtolerated in patients with HD, where an improvement in a measure of executive
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function (cognition) was observed . However, the clinical efficacy of PBT2 in both AD
32
and HD studies is controversial . This may be related to the fact that in addition to the
metal dyshomeostasis of the transition metals iron, copper or zinc, neurodegenerative
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diseases are also linked to oxidative stress and mitochondrial dysfunction .
Because multiple mechanisms are involved in the pathogenesis of neurodegenerations,
we have designed compounds that are aimed at simultaneously combating multiple
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targets in these complex diseases . We have previously reported the development of
an initial series of multifunctional antioxidants (MFAOꢀ1s) where 4ꢀ(5ꢀhydroxypyrimidinꢀ
2ꢀyl)ꢀN,Nꢀdimethylꢀ3,5ꢀdioxopiperazineꢀ1ꢀsulfonamide (JHXꢀ4) and 4ꢀ(5ꢀhydroxyꢀ4,6ꢀ
dimethoxypyrimidinꢀ2ꢀyl)ꢀN,Nꢀdimethylꢀ3,5ꢀdioxopiperazineꢀ1ꢀsulfonamide (JHXꢀ8)
possess a 2ꢀaminoꢀ5ꢀhydroxypyrimidine free radical scavenging group (FRS) and a
chelating group (CHL) consisting of two carbonyls directly adjacent to an amino group
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that function independently . This initial series of MFAOꢀ1s demonstrated protective
effects against ROS in human lens epithelial cells (hLECs), retinal pigmented epithelial
35
cells (RPEs), and hippocampal astrocytes . In rats, oral administration of these MFAOꢀ
s significantly delayed the progression of cataracts induced by ROS in diabetes and
1
36
whole head γꢀirradiation . Furthermore, in a rat model of dry atrophic AMD, they
demonstrated retinal neuroprotection against lightꢀinduced injury where ROS is
37
generated and iron is released from degenerating photoreceptors . While tissue
analyses confirmed their presence in the lens and neural retina, their accumulation in
36, 37
the brain was minimal
.
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6
Here, we report the synthesis and in vitro biochemical evaluation of a new series of
MFAOꢀ2 that accumulate in the brain following oral administration (Figure 2). This has
been achieved by modifying the upper ring portion of MFAOꢀ1s, while retaining the 2ꢀ
diacetylaminoꢀ5ꢀhydroxypyrimidine moieties required for independent antioxidant and
metal attenuation activity. These MFAOꢀ2s have subsequently been shown to reduce
both Aβ and Aβ isoform levels in transgenic JAX Alzheimer’s mice by oral
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42
administration as well as remove zinc from the Aβ:Zn complex so that Aβ plaques can
38
be subsequently degraded by matrix metalloproteinaseꢀ2 .
ꢀ
CHEMISTRY
The upper ring system, 1ꢀN,N’ꢀdimethylsulfamoyl piperazine, in the first series of MFAOꢀ
1s was replaced with either a succinimide ring or glutarimide ring to give the second
35
series of MFAOꢀ2s as outlined in Scheme 1. According to the established procedure ,
the coupling reaction of the 2ꢀaminopyrimidines 19ꢀ22 with commercially available
succinic anhydride 17 or glutaric anhydride 18 gave the 1ꢀ(2ꢀpyrimidyl)pyrrolidineꢀ2,5ꢀ
dione analogs 1, 3, 23, and 25 or the 1ꢀ(2ꢀpyrimidyl)piperidineꢀ2,6ꢀdione analogs 5, 7,
24, and 26. Removal of the benzyloxy protecting groups by hydrogenation gave the
MFAOꢀ2s 2, 4, 6, and 8. The aminopyrimidines 19 and 20 were obtained as previously
35
described while 21 was commercially available. The amino pyrimidine 22 was
obtained from commercially available 2ꢀchloroꢀ4, 6ꢀdimethoxypyrimidine 27 by
nucleophilic substitution of the 2ꢀchloropyrimidine with benzylamine followed by
hydrogenation as outlined in Scheme 2.
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9
Compounds 9 to 16 were synthesized from the 2ꢀchloropyrimidines 31, 32, 33, and 34
by coupling with commercially available pyrrolidine 29 or piperidine 30 followed by
hydrogenation as outlined in Scheme 3. The 2ꢀchloropyrimidines 33 and 34 were
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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26
27
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35
commercially available while 32 was obtained as previously described . The 2ꢀ
chloropyrimidine 31 was obtained from commercially available 2ꢀchloroꢀ5ꢀ
nitropyrimidine 39 by reduction of the nitro group to amine 5ꢀaminoꢀ2ꢀchloropyrimidine
4
0, acidic hydroxylation, and subsequent protection of the hydroxyl group as outlined in
Scheme 4.
ꢀ
RESULTS AND DISCUSSION
In vitro analyses were conducted on the multifunctionals containing an upper ring
succinimide ring (2, 4) and glutarimide ring (6, 8), their monofunctional FRS analogs (10,
12, and 14, 16, respectively), their monofunctional CHL analogs (1, 3 and 5, 7,
respectively) as well as their nonfunctional parent analogs (9, 11, and 13, 15,
respectively).
Metal Binding Studies. The stoichiometry of the chelatorꢀmetal ion complexes was
35
obtained using the method of continuous variation (Job plots) as previously described .
Several solutions of each compound and metal ion of interest were examined at a
constant total concentration (0.1 ꢀ 0.5 mM) of the MFAOs 2, 4, 6, and 8 and their
2+
3+
+
2+
2+
monofunctional chelating analogs 1, 3, 5, and 7 and the ion Fe , Fe , Cu , Cu , Zn ,
2+
2+
2+
Mn ,Ca , and Mg by altering the mole fraction of one of the components. As
2+
3+
+
summarized in Table 1, compounds 1-8 bound the transition metals Fe , Fe , Cu ,
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6
2+
2+
2+
2+
Cu , Zn , and Mn in 1:2 ratio; however, these compounds failed to bind Ca and
2
+
Mg .
In Vitro Evaluation of Cytotoxicity and Antioxidant Activity. The cytotoxicity of
compounds 1-16 was evaluated using a MTS Cell Viability assay in SHꢀSY5Y human
neuroblastoma cells (hNBs) and ARPE ꢀ19 human retinal pigmented epithelial cells
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0
(
RPEs). All cells were exposed for 2 h to 1 mM of the MFAOs 2, 4, and 6, 8, their
monofunctional FRS analogs 10, 12, and 14, 16, respectively, and their monofunctional
CHL analogs (1, 3, and 5, 7, respectively) as well as their nonfunctional parent analogs
9
, 11, and 13, 15, respectively, as well as the previous MFAOꢀ1 JHXꢀ4, the metal
attenuator clioquinol (PBTꢀ1), and Trolox, a waterꢀsolubilized vitamin E derivative. As
shown in Figure 3, no apparent cytotoxicity was observed with either compounds 1-16
or Trolox; however, clioquinol demonstrated a significant 30% reduction of the cell
viability in human retinal pigmented epithelial cells (RPEs) and a 20% reduction in
human neural neuroblastoma cells (hNBs).
To confirm the protective effect of MFAOs against the reactive hydroxyl radicals, both
2+
cells were treated with 1 mM of Fenton reagent (1 mM H O and 1 mM Fe ) with or
2
2
without 1 mM of compound 1-16, JHXꢀ4, clioquinol, or Trolox. As shown in Figure 4, the
multifunctional antioxidants 2, 4, 6, and 8 protected the hNB and RPE cells against
hydroxyl radical generated ROS similar to that observed for MFAOꢀ1 JHXꢀ4. In both
cells, this protection was significantly higher than that of the metal attenuator clioquinol.
The activity of Trolox was only less in RPE cells. In order of protection, the MFAOs 2, 4,
6
, 8, and JHXꢀ4 provided significantly more protection than the monofunctional free
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radical scavengers 10, 12, 14, and 16 or Trolox. Even less protection was provided by
the monofunctional chelators 1, 3, 5, and 7 or clioquinol and no protection was provided
by the nonꢀfunctional parent compounds 9, 11, 13, and 15. The cellular levels of
reduced GSH, which is a sensitive biomarker of oxidative stress, were also measured
under the same conditions as the cell viability experiments. As summarized in Figure 5,
cells exposed to MFAOs were able to maintain GSH levels similar to JHXꢀ4 and these
levels were significantly higher than in cells exposed to Trolox or clioquinol. The relative
activities of compounds with multifunctional versus monofunctional groups showed
results similar to those observed with the viability studies.
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The ability of MFAOs to protect cells against superoxide radicals was also assessed in
both hNBs and RPEs. Mitochondrial superoxide was generated using xanthine oxidase
TM
and the levels of superoxide radicals generated were detected by selective MitoSOX
Red fluorescence. For these studies, all cells were exposed to xanthine oxidase (XO, 25
mU/mL) for 1 h to generate approximately 100 ꢁM superoxide with/without the
presence of either the enzyme superoxide dismutase (SOD) or 1, 10, 100, and 1000
ꢁ
M of MFAOs 2, 4, and JHXꢀ4, the nonꢀfunctional parent compounds 9 and 11,
clioquinol, or Trolox. As shown in Figure 6, superoxide levels generated were reduced
by MFAOs, clioquinol and Trolox in a similar doseꢀdependent manner in both cell lines.
A similar doseꢀdependent cell protection against hydroxyl radicals was also observed
using LIVE/DEAD® Viability/Cytotoxicity staining (Figure 7). Exposure to hydroxyl
radicals generated by 100 ꢁM Fenton reagent for 2 h resulted in only 40% of RPE and
hNBs cells remaining viable. Both cells were protected against hydroxyl radicals by the
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doseꢀdependent presence of MFAOs 2, 4, and JHXꢀ4 or clioquinol; however, the nonꢀ
functional parent compounds 9 and 11 provided no protection.
Bioavailability Study
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Both the first generation MFAOs JHXꢀ4 and JHXꢀ8 and the second generation MFAOs 2,
, 6, and 8 were administered to C57BL/6 mice by feeding chow containing 0.05% of
4
the each compound for 14 days. Consumption studies indicated that this corresponded
to an average daily dose of 80 mg/kg. After whole body perfusion at the time of sacrifice,
the drug levels in the lens, neural retina, and brain were measured by HPLCꢀMS. All
MFAOs examined showed good oral bioavailability to at least one target tissue. This
was anticipated because all compounds with the exception of JHX-8 which possesses
39
1
2 hydrogen bond acceptors comply with Lipinski’s Rule of 5 . As illustrated in Figure
8A, all MFAOꢀ2s accumulated in the brain (60 – 254 ng/mg protein) at 20 to 85ꢀfold
higher levels than MFAOꢀ1s (JHXꢀ4, 3 ng/mg protein and JHXꢀ8, 4 ng/mg protein). In
contrast, both MFAOꢀ2s and MFAOꢀ1s achieved similar levels in the neural retina
(Figure 8B); however, lower levels of MFAOꢀ2s accumulated in the lens than those of
MFAOꢀ1 (Figure 8C). While it has generally been assumed that more lipophilic
compounds should easily get into both the brain and the avascular lens, the present
results suggest that differences exist in the passage of lipophilic drugs through the
blood aqueous barrier that is required to reach the lens and the blood brain barrier that
is required to reach the brain. Ongoing studies are investigating the molecular
physicochemical parameters of these compounds that are associated with their uptake
into the retina, brain, and lens.
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ꢀ
CONCLUSION
Replacing the N, Nꢀdimethylꢀpiperazineꢀ1ꢀsulfonamide group in the first generation of
multifunctional antioxidants with either a pyrrolidine or piperidine ring resulted in a new
class of multifunctional antioxidants that have increased bioavailability to the brain.
These second generation multifunctional antioxidants retain both the FRS activity
against ROS and metal attenuation activity and protected hNBs and RPEs against both
hydroxyl and superoxide radicals. While both generations of multifunctionals
demonstrated similar retinal bioavailability, distinct differences in uptake to lens and
brain were observed. The MFAOꢀ2s demonstrated 20ꢀ85ꢀfold improved drug
penetration to the brain compared to MFAOꢀ1s.These results suggest that MFAOꢀ2 may
have coadjutant therapeutic potential for AMD as well as AD since ROS and redoxꢀ
active metals are all present in these diseases.
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ꢀ
EXPERIMENTAL SECTION
General. All solvents and reagents were obtained from commercial sources in highest
purity available. Clioquinol was purchased from TCI (>98%, Tokyo, Japan) and further
purified by recrystallization. NMR spectra were obtained with a Varian 500 MHz
spectrometer. Uncorrected melting points were obtained with the Melting Point
Apparatus MPAꢀ120 EZꢀMelt (Stanford Research Systems, Sunnyvale, CA). Column
chromatography (CC) utilized SELECTO SCIENTIFIC silica gel (200ꢀ400 mesh). UVꢀ
visible spectra were measured on a Molecular Devices SpectraMax Plus384 microplate
spectrophotometer (Molecular Devices, Sunnyvale, CA). Final compound purities were
assessed as ≥99% and intermediate compound purities were assessed as ≥96% by
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reverse phase HPLC using a 250 mm x 4.6 mm C18 Luna 5 µm column (Phenomenex
Inc., Torrance, CA) with a mobile phase of 75:25 methanol/water at a flow rate of 0.9
mL/min and detection at 220, 254, and 280 nm. Elemental analyses were done by MꢀHꢀ
W Laboratories, Phoenix, AZ.
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N-benzyl-4,6-dimethoxypyrimidin-2-amine (28). A mixture of 2ꢀchloroꢀ4,6ꢀ
dimethoxypyrimidine (27) (50.0 g, 0.29 mol), BnNH (93.3 mL, 0.85 mol) and K CO
3
2
2
(62.5 g, 0.45 mol) in dioxane (1.0 L) was refluxed for 4 days. The reaction was filtered
and the filtrate concentrated in vacuo to give a yellow oil. Silica gel column
chromatography with 20:1 to 10:1 hexanes:EtOAc gave 61.7 g (87%) of white solid 28.
1
H NMR (CDCl ) δ 7.36ꢀ7.27 (m, 5H), 5.42 (s, 1H), 5.25 (s, 1H), 4.61 (d, J=5.86Hz, 2H),
3
3
.83 (s, 6H).
2-amino-4,6-dimethoxypyrimidine (22). Compound 28 (27.0 g, 0.11 mol) in 400 mL of
MeOH was hydrogenated for 2 days at r.t. in the presence of 5.4 g of 20% Pd(OH)2
catalyst under 1 atmosphere of hydrogen gas (balloon). After filtration, and solvent
evaporation, a white solid was obtained which after silica gel column chromatography
1
using 50:1 CHCl :MeOH yielded 16.8 g (98%) of 22. H NMR (CDCl ) δ 5.47 (s, 1H),
3
3
4
.90 (s, 2H), 3.84 (s, 6H).
General Procedure for the Preparation of 1-(2-pyrimidyl)pyrrolidine-2,5-dione (1),
-(4,6-dimethoxy-2-pyrimidyl) pyrrolidine-2,5-dione (3), 1-(2-pyrimidyl)piperidine-
1
2,6-dione (5), 1-(4,6-dimethoxy-2-pyrimidyl)piperidine-2,6-dione (7), 1-(5-
benzyloxy-2-pyrimidyl)-pyrrolidine-2,5-dione (23), 1-(5-benzyloxy-2-pyrimidyl)-
piperidine-2,6-dione (24), 1-(4,6-dimethoxy-5-benzyloxy-2-pyrimidyl)pyrrolidine-
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2
,5-dione (25), 1-(4,6-dimethoxy-5-benzyloxy-2-pyrimidyl)-piperidine-2,6-dione
(26). 42.0 g (0.42 mol) of succinic anhydride (17) dissolved in 300 mL of toluene, 20 g
(0.21 mol) of 2ꢀaminopyrimidine (21) dissolved in 200 mL of acetone was added and the
10
11
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13
14
15
16
17
18
19
20
21
22
23
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26
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60
o
mixture was heated to 85 C for 3 days. After cooling to r.t., the precipitated product was
filtered and washed with toluene. The filtrate was dried in vacuo, and the dried product
o
was then dissolved in 300 mL of anhydrous Ac O and again heated to 85 C for 3 h.
2
After removal of the remaining Ac O in vacuo, the product was purified by silica gel
2
column chromatography with CHCl and recrystallized from EtOAc to give compound 1
3
o
1
as a white solid, mp 148.5ꢀ149.5 C in 32% yield. H NMR (CDCl ) δ 8.92 (d, J=4.88Hz,
3
2
H), 7.42 (t, J=4.88Hz, 1H), 2.96 (s, 4H); Anal. Calcd for C H N O : C, 54.24; H, 3.98;
8 7 3 2
N, 23.72. Found: C, 54.29; H, 4.19; N, 23.90.
o
1
Compound 3 was obtained as a white solid, mp 166.9ꢀ168.7 C, in 60% yield. H NMR
(CDCl ) δ 6.09 (s, 1H), 3.94 (s, 6H), 2.92 (s, 4H); Anal. Calcd for C H N O : C, 50.63;
3
10 11
3
4
H, 4.67; N, 17.71. Found: C, 50.78; H, 4.80; N, 17.80.
o
1
Compound 5 was obtained as a white solid, mp 220.8ꢀ221.3 C, in 51% yield. H NMR
(
CDCl ) δ 8.88 (d, J=4.88Hz, 2H), 7.40 (t, J=4.88Hz, 1H), 2.83 (t, J=4.59Hz, 4H), 2.18ꢀ
3
2
.15 (m, 2H); Anal. Calcd for C H N O : C, 56.54; H, 4.74; N, 21.98. Found: C, 56.77;
9 9 3 2
H, 4.85; N, 21.70.
o
1
Compound 7 was obtained as a white solid, mp 229.2ꢀ232.4 C, in 63% yield. H NMR
(CDCl ) δ 6.06 (s, 1H), 3.92 (s, 6H), 2.79 (t, J=6.59Hz, 4H), 2.15ꢀ2.12 (m, 2H); Anal.
3
Calcd for C H N O : C, 52.59; H, 5.22; N, 16.73. Found: C, 52.60; H, 5.40; N, 16.63.
11
13
3
4
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5
6
Compound 23, purified by silica gel column chromatography using 100:1 CHCl :MeOH
3
1
as eluent, was obtained as a white solid in 86% yield. H NMR (CDCl ) δ 8.57 (s, 2H),
3
7
.44ꢀ7.43 (m, 5H), 5.21 (s, 2H), 2.93 (s, 4H).
0
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Compound 24, purified with silica gel column chromatography using 100:1 CHCl :MeOH
3
1
as eluent, was obtained as a white solid in 70% yield. H NMR (CDCl ) δ 8.54 (s, 2H),
3
7
.44ꢀ7.39 (m, 5H), 5.19 (s, 2H), 2.81 (t, J=6.59Hz, 4H), 2.17ꢀ2.13(m, 2H).
Compound 25, purified by silica gel column chromatography with 100:1 CHCl :MeOH as
3
1
eluent, was obtained as a white solid in 73% yield. H NMR (CDCl ) δ 7.44ꢀ7.31 (m,
3
5
H), 5.07 (s, 2H), 3.96(s, 6H), 2.28 (s, 4H).
Compound 26, purified by silica gel column chromatography with 100:1 CHCl :MeOH,
3
1
was obtained as a white solid in 63% yield. H NMR (CDCl ) δ 7.47ꢀ7.35 (m, 5H), 5.03
3
(s, 2H), 3.96 (s, 6H), 2.80 (t, J=6.59Hz, 4H), 2.15ꢀ2.13(m, 2H).
General Procedure for the Preparation of 1-(5-hydroxy-2-pyrimidyl) pyrrolidine-
2,5-dione (2), 1-(4,6-dimethoxy-5-hydroxy-2-pyrimidyl)pyrrolidine-2,5-dione (4), 1-
(5-hydroxy-2-pyrimidyl)piperidine-2,6-dione (6), 1-(4,6-dimethoxy-5-hydroxy-2-
pyrimidyl)-piperidine-2,6-dione (8). Compound 23 (14.7 g, 51.9 mmol) dissolved in
750 mL of acetone was hydrogenated with 3.7 g of 10% Pd/C catalyst at r.t. for 12 h 1
atmosphere of hydrogen gas (balloon). After filtration and solvent evaporation,
compound 2 was obtained as a white fluffy solid. Following recrystallization with iꢀPrOH,
o
1
1
0 g of 2, mp 278.0ꢀ280.0 C, was obtained in 76% yield. H NMR (DMSOꢀd ) δ 11.02
6
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(
s, 1H), 8.46 (s, 2H), 2.85 (s, 4H); Anal. Calcd for C H N O : C, 49.74; H, 3.65; N,
8
7
3
3
2
1.75. Found: C, 50.00; H, 3.69; N, 21.54.
o
Compound 4 was obtained as a white solid, mp 193.7ꢀ195.0 C, in 91% yield after
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
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1
recrystallization from Et O. H NMR (CDCl ) δ 5.15 (s, 1H), 4.03 (s, 6H), 2.91 (s, 4H);
2
3
Anal. Calcd for C H N O : C, 47.43; H, 4.38; N, 16.59. Found: C, 47.53; H, 4.48; N,
10
11
3
5
1
6.58.
o
Compound 6 was obtained as a white solid, mp 243.5ꢀ244.3 C, in 89% yield after
1
recrystallization from acetone. H NMR (DMSOꢀd ) δ 10.85 (s, 1H), 8.39 (s, 2H), 2.75 (t,
6
J=6.35Hz, 4H), 1.99ꢀ1.96 (m, 2H); Anal. Calcd for C H N O : C, 52.17; H, 4.38; N,
9
9
3
3
2
0.28. Found: C, 52.14; H, 4.52; N, 20.04.
o
Compound 8 was obtained as a white solid, mp 222.0ꢀ224.0 C, in 86% yield after
1
recrystallization from Et O. H NMR (CDCl ) δ 6.06 (s, 1H), 3.92 (s, 6H), 2.80 (t,
2
3
J=6.59Hz, 4H), 2.15ꢀ2.13 (m, 2H); Anal. Calcd for C H N O : C, 49.44; H, 4.90; N,
11
13
3
5
1
5.72. Found: C, 49.52; H, 4.99; N, 15.65.
General Procedure for the Preparation of 2-(pyrrolidine-1-yl)pyrimidine (9), 4,6-
dimethoxy-2-(pyrrolidine-1-yl)pyrimidine (11), 2-(piperidine-1-yl)pyrimidine (13),
4,6-dimethoxy-2-(piperidine-1-yl)pyrimidine (15), 5-benzyloxy-2-(pyrrolidine-1-
yl)pyrimidine (35), 5-benzyloxy-2-(piperidine-1-yl)pyrimidine (36), 5-benzyloxy-4,6-
dimethoxy-2-(pyrrolidine-1-yl)pyrimidine (37), 5-benzyloxy-4,6-dimethoxy-2-
(
piperidine-1-yl)pyrimidine (38). To 9.53 mL (116 mmol) pyrrolidine (29) in 400 mL of
anhydrous THF was added 2.78 g of sodium hydride (116 mmol) and the mixture was
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refluxed for 0.5 h. After cooling to r.t., 12 g (105 mmol) of 2ꢀchloropyrimidine (33) was
added dropwise and the mixture was refluxed for 2 days and then cooled in an ice bath.
Water (200 mL) was then added to the cooled reaction mixture and the THF was
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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2
3
4
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7
8
9
0
removed in vacuo. The aqueous residue was extracted with CHCl and the combined
3
CHCl extracts were washed with brine, dried over magnesium sulfate, and filtered.
3
After removal of solvent in vacuo, the residue was purified by silica gel column
chromatography using 20:1 hexanes: EtOAc as eluent. The product was then
recrystallized from Et O to give a 12.0 g (76%) of compound 9 as a yellow solid, mp
2
o
1
3
9.0ꢀ39.6 C . H NMR (CDCl ) δ 8.31 (d, J=4.88Hz, 2H), 6.45 (t, J=4.88Hz, 1H), 3.57
3
(
t, J=4.4Hz, 4H), 2.01ꢀ1.98 (m, 4H). Anal. Calcd for C H N : C, 64.40; H, 7.43; N,
8 11 3
2
8.16. Found: C, 64.42; H, 7.53; N, 27.97.
o
Compound 11 was obtained as a white solid, mp 63.3ꢀ63.7 C, 71% yield after
1
recrystallization from Et O. H NMR (CDCl ) δ 5.34 (s, 1H), 3.87 (s, 6H), 3.56 (t,
2
3
J=6.59Hz, 4H), 1.93ꢀ1.96 (m, 4H); Anal. Calcd for C H N O : C, 57.40; H, 7.23; N,
10
15
3
2
2
0.08. Found: C, 57.20; H, 7.03; N, 19.91.
Compound 13 was obtained as a colorless oil in 90% yield after vacuum distillation at
1
9
0˚C under 0.25 mmHg. H NMR (CDCl ) δ 8.22 (d, J=4.64Hz, 2H), 6.35 (t, J=4.64Hz,
3
1H), 3.71 (t, J=5.37Hz, 4H), 1.64ꢀ1.58 (m, 2H), 1.56ꢀ1.51 (m, 4H); Anal. Calcd for
C H N : C, 66.23; H, 8.03; N, 25.74. Found: C, 66.49; H, 7.95; N, 25.81.
9
13
3
1
Compound 15 was obtained as a white solid mp 59.8ꢀ60.4˚C in 74% yield. H NMR
(
CDCl ) δ 5.25 (s, 1H), 3.77 (s, 6H), 3.68 (t, J=5.49Hz, 4H), 1.60ꢀ1.55 (m, 2H), 1.52ꢀ
3
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.48 (m, 4H); Anal. Calcd for C H N O : C, 59.17; H, 7.67; N, 18.82. Found: C, 58.94;
11
17
3
2
H, 7.49; N, 18.56.
1
Compound 35 was obtained in 86% yield. H NMR (CDCl ) δ 8.06 (s, 2H), 7.34ꢀ7.25 (m,
3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5
H), 4.93 (s, 2H), 3.45 (t, J=6.59Hz, 4H), 1.93ꢀ1.90 (m, 4H).
1
Compound 36 was obtained in 75% yield. H NMR (CDCl ) δ 8.04 (s, 2H), 7.33ꢀ7.25 (m,
3
5
H), 4.94 (s, 2H), 3.63 (t, J=4.88Hz, 4H), 1.58ꢀ1.52 (m, 6H).
1
Compound 37 was obtained in 80% yield. H NMR (CDCl ) δ 7.47ꢀ7.32 (m, 5H), 4.84 (s,
3
2
H), 3.90 (s, 6H), 3.51 (t, J=6.59Hz, 4H), 1.95ꢀ1.92 (m, 4H).
1
Compound 38 was obtained in 75% yield. H NMR (CDCl ) δ 7.46ꢀ7.27 (m, 5H), 4.85 (s,
3
2
H), 3.89 (s, 6H), 3.69 (t, J=5.49Hz, 4H), 1.66ꢀ1.57 (m, 6H).
General procedure of preparation of 5-hydroxy-2-(pyrrolidine-1-yl)pyrimidine (10),
-hydroxy-4,6-dimethoxy-2-(pyrrolidine-1-yl)pyrimidine (12), 5-hydroxy-2-
piperidine-1-yl)pyrimidine (14), 5-hydroxy-4,6-dimethoxy-2-(piperidine-1-
5
(
yl)pyrimidine (16). Hydrogenation at r.t. for 12 h of compound 38 (17.6 g, 53.4 mmol)
dissolved in 350 mL of acetone with 4.4 g of 10% Pd/C catalyst under a one
atmosphere of hydrogen gas (balloon) gave compound 16 as a pale red solid after
filtration and solvent evaporation. Recrystallization from Et O gave 12.7 g (80%) of 16,
2
o
1
mp 120.1ꢀ120.5 C. H NMR (DMSOꢀd ) δ 7.63 (brs, 1H), 3.81 (s, 6H), 3.59 (t,
6
J=5.25Hz, 4H), 1.60ꢀ1.56 (m, 2H), 1.52ꢀ1.47 (m, 4H); Anal. Calcd for C H N O : C,
11
17
3
3
5
5.22; H, 7.16; N, 17.56. Found: C, 55.15; H, 7.21; N, 17.48.
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6
o
Compound 10 was obtained as a pale yellow solid, mp 151.5ꢀ151.8 C in 84% yield
1
after recrystallization from Et O. H NMR (CDCl ) δ 8.05 (s, 2H), 3.55ꢀ3.75 (m, 4H),
2
3
1
5
.96ꢀ1.92 (m, 4H). Anal. Calcd for C H N O: C, 58.17; H, 6.71; N, 25.44. Found: C,
8 11 3
0
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2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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9
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7
8
9
0
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2
3
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5
6
7
8
9
0
7.92; H, 6.67; N, 25.18.
Compound 12 was obtained as a yellow solid, mp 108.7ꢀ109.2˚C, in 78% yield after
1
recrystallization from Et O. H NMR (DMSOꢀd ) δ 7.49 (brs, 1H), 3.82 (s, 6H), 3.41 (t,
2
6
J=6.47Hz, 4H), 1.89ꢀ1.86 (m, 4H); Anal. Calcd for C H N O : C, 53.32; H, 6.71; N,
10 15
3
3
1
8.66. Found: C, 53.51; H, 6.80; N, 18.80.
o
Compound 14 was obtained as a pale red solid, mp 94.3ꢀ94.7 C, in 79% yield after
1
recrystallization from Et O. H NMR (CDCl ) δ 8.06 (s, 2H), 3.47 (t, J=6.59Hz, 4H), 1.70ꢀ
2
3
1
.40 (m, 6H). Anal. Calcd for C H N O: C, 60.32; H, 7.31; N, 23.45. Found: C, 60.15;
9 13 3
H, 7.25; N, 23.26.
5
-amino-2-chloropyrimidine (40). 140 g (0.88 mol) of 2ꢀchloroꢀ5ꢀnitropyrimidine (39)
dissolved in 700 mL of EtOH, 197 g of iron powder (70m mesh, < 212 µm), 700 mL of
water, and 1400 mL of acetic acid were added together. The mixture was heated at
7
0˚C overnight, then cooled to r.t., and then filtered. EtOH was removed from the filtrate
in vacuo, and the pH was adjusted to 8 with 12 N NaOH and the product was extracted
overnight with continuous liquidꢀliquid extraction with EtOAc. The resulting filter cake
was washed with EtOAc, and the combined EtOAc layers were washed with water, then
brine, dried over magnesium sulfate, and filtered. After removal of the solvent in vacuo
and recrystallization with EtOH, 97.2 g (85%) of pale brown solid product 40 was
1
obtained. H NMR (DMSOꢀd ) δ 8.94 (s, 2H), 5.77 (brs, 2H).
6
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