Optimization of pyrimidinol antioxidants as mitochondrial protective agents: ATP production and metabolic stability

Article abstract of DOI:10.1016/j.bmc.2016.08.039

Previously we described a novel series of pyrimidinol antioxidants and their structural optimization as potential therapeutic agents for neurodegenerative and mitochondrial disorders. Our initial lead compound was a potent antioxidant in vitro, but was subsequently found to exhibit poor stability to oxidative metabolism. The current study focused on balancing potency with metabolic stability through structural modification, and involved modifications at positions 2 and 4 of the pyrimidinol redox core, likely sites of oxidative metabolism. Eight new analogues have been prepared and their ability to suppress lipid peroxidation and reactive oxygen species (ROS), and to preserve mitochondrial membrane potential (Δψ_m) and support ATP production, has been investigated. The metabolic stability of the prepared compounds was also assessed in vitro using bovine liver microsomes to obtain preliminary insight on this class of compounds. This study revealed the complexity of balancing reasonable metabolic stability with efficient antioxidant properties. While a few analogues appear promising, especially in terms of metabolic stability, a 4-isopropoxy derivative conserved the favorable biological activity and exhibited good metabolic stability. The favorable metabolic stability conferred by the combination of the azetidine and isopropoxy moieties in analogue 6 makes this compound an excellent candidate for further evaluation.

Full text of DOI:10.1016/j.bmc.2016.08.039

Accepted Manuscript
Optimization of pyrimidinol antioxidants as mitochondrial protective agents:
ATP production and metabolic stability
Arnaud Chevalier, Mohammad Parvez Alam, Omar M. Khdour, Margaret
Schmierer, Pablo M. Arce, Cameron D. Cripe, Sidney M. Hecht
PII:
S0968-0896(16)30647-2
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.08.039
BMC 13222
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
15 July 2016
17 August 2016
22 August 2016
Please cite this article as: Chevalier, A., Alam, M.P., Khdour, O.M., Schmierer, M., Arce, P.M., Cripe, C.D., Hecht,
S.M., Optimization of pyrimidinol antioxidants as mitochondrial protective agents: ATP production and metabolic
stability, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.08.039
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Optimization of pyrimidinol antioxidants as
mitochondrial protective agents: ATP production
and metabolic stability
Arnaud Chevalier, Mohammad Parvez Alam, Omar M. Khdour,
Margaret Schmierer, Pablo M. Arce, Cameron D. Cripe, Sidney M.
Hecht*
Center for BioEnergetics, Biodesign Institute, and School of Molecular Sciences, Arizona
State University, Tempe, AZ 85287, United States
1

A R T I C L E I N F O
__________________________
Keywords:
Mitochondria
Cytoprotection
Lipid peroxidation
Reactive oxygen species
Microsomal stability
__________________________
__________
* Corresponding author. Tel.: +1 480 965 6625; fax: +1 480 965 0038.
E-mail address: sidney.hecht@asu.edu (S. M. Hecht).
2

A B S T R A C T
________________________________________________________________________
Previously we described a novel series of pyrimidinol antioxidants and their structural
optimization as potential therapeutic agents for neurodegenerative and mitochondrial
disorders. Our initial lead compound was a potent antioxidant in vitro, but was subsequently
found to exhibit poor stability to oxidative metabolism. The current study focused on
balancing potency with metabolic stability through structural modification, and involved
modifications at position 2 and 6 of the pyrimidinol redox core, likely sites of oxidative
metabolism. Eight new analogues have been prepared and their ability to suppress lipid
peroxidation and reactive oxygen species (ROS), and to preserve mitochondrial membrane
potential (∆ψm) and support ATP production, has been investigated. The metabolic stability of
the prepared compounds was also assessed in vitro using bovine liver microsomes to obtain
preliminary insight on this class of compounds. This study revealed the complexity of
balancing reasonable metabolic stability with efficient antioxidant properties. While a few
analogues appear promising, especially in terms of metabolic stability, a 4-isopropoxy
derivative conserved the favorable biological activity and exhibited good metabolic stability.
The favorable metabolic stability conferred by the combination of the azetidine and
isopropoxy moieties in analogue 6 makes this compound an excellent candidate for further
evaluation.
______________________________________________________________________
3

1. Introduction
Mitochondria are organelles found in most eukaryotic cells which fulfill essential cellular
functions.1-7 Although adenosine-5′-triphosphate (ATP) synthesis is often considered their
primary function, they also participate in critical cellular processes, such as programmed cell
death and neoplastic transformation.2,3 Mitochondria are central to energy metabolism; they
use oxidative phosphorylation (OXPHOS) to convert the energy stored in cellular nutrients
into ATP through the action of the mitochondrial electron transport chain (ETC) complexes.
Electrons removed from oxidizable substrates transit a series of electron carriers (complexes
I-IV) in the respiratory chain, resulting in the reduction of oxygen to water. The flow of these
electrons through these complexes also results in the transport of protons from the inner
mitochondrial membrane into the intermembrane space. These protons are used by a fifth
complex, ATP synthase (complex V), to convert ADP to ATP.2-4
Defects in ETC protein complexes can have a major impact on mitochondrial coupling
efficiency and the production of reactive oxygen species (ROS).8,9 The delicate balance
between ROS production and elimination is necessary for normal cellular homeostasis.
Ordinarily, mammalian cells employ a ubiquitous antioxidant defense system to cope with by-
product ROS production accompanying aerobic metabolism.10,11 These ROS scavenging
systems include antioxidant enzymes, the glutathione redox cycle, and the pool of coenzyme
Q10 (CoQ10).10,11 Oxidative stress is considered to result from an imbalance of ROS and
antioxidants; excess ROS generation can overwhelm endogenous antioxidant capacity, expose
cellular macromolecules to oxidative damage, and impair the ability of mitochondria to make
ATP and carry out other functions. The decline in mitochondrial function is well recognized
in aging, neurodegenerative diseases and many complex mitochondrial disorders.12-16
Metabolic stability is a key property of compounds employed therapeutically; it is important
for a drug to reach and maintain therapeutic concentrations following administration, and drug
4

degradation can contribute to toxicity. Oxidative dealkylations are commonly observed
metabolic reactions for compounds containing heteroatoms, such as secondary and tertiary
amines as well as ethers.17 Structural modification of such functional groups can minimize
oxidative metabolism. We previously reported the discovery of compound 1 as a potent
multifunctional radical quencher (MRQ),18 i.e. a compound which functions in part by
quenching radicals involved in oxidative stress (Figure 1). This compound was subsequently
shown to lack sufficient metabolic stability to function well in vivo. Presently, this issue is
addressed by identifying structurally related pyrimidinol antioxidants which retain good
efficacy but exhibit reduced liability to oxidative metabolism. A recent study of related
pyridinols established enhanced metabolic stability by replacing the N,N-dimethylamino
moiety with an exocyclic azetidine.19 This functional group tends to exhibit a defined, rigid
conformation and can confer superior metabolic stability.19-21
Figure 1. Chemical structures of compounds 1‒9.
5

We describe the preparation and study of several novel pyrimidinol derivatives (2–9)
(Figure 1), which exhibit improved metabolic stability relative to 1, and some of which
exhibit improved cellular antioxidant properties. Strategies for diminution of metabolic N-
and O-demethylation were employed; the pyrimidinol derivatives were evaluated for their
stability in bovine liver microsomes. Metabolic stability was achieved by exploiting the
relative resistance of the azetidine moiety to oxidative attack, and by the presence of a
sterically hindered cyclobutoxy or isopropoxy moiety, blocking another site of possible
oxidative attack. The compounds were evaluated for their ability to suppress ROS and lipid
peroxidation, and for their effects on mitochondrial membrane potential, and their ability to
protect cultured FRDA lymphocytes from oxidative stress induced by glutathione depletion.
The compounds were also evaluated for their ability to increase ATP levels in FRDA
lymphocytes.
2. Results and discussion
2.1. Design and synthesis of pyrimidinol analogues of MRQ based on the compound 1
scaffold
The objective of this work was to determine what modifications of compound 1 could both
conserve its favorable biological activities and improve metabolic stability. In a previous
study of pyridinol antioxidants, it was found that introduction of a cyclic amine in place of the
exocyclic dimethyl amine improved metabolic stability. Accordingly, the preparation of
compound 2, having an exocyclic azetidine moiety, was envisioned. For this synthesis, aryl
iodide 10 was prepared starting from commercially available 4-methoxy-6-methylpyrimidin-
2-amine in 37% yield (Scheme 1). The introduction of iodine was accomplished using Cu(I)
as catalyst, and subsequent treatment with azetidine furnished the requisite heterocyclic core
structure 11. Compound 11 was then alkylated by deprotonation of the benzylic proton using
n-BuLi, then treated with pentadecyl bromide to obtain intermediate 12 in 25% yield.
6

NH.HCl
isoamylnitrite, CH2I2,
I2, CuI
MeO
MeO
MeO
20% Me4Phen, 20% CuI, Cs2CO3
dry degassed DMF, 50 °C
N
N
N
N
N
N
THF
I
N
NH2
10,
37%
11,
72%
Br
MeO
MeO
NBS
n-BuLi, THF, -78°C
N
N
N
N
Br
1:1 CH3CN:CH2Cl2
14
N
N
12, 25%
13, 96%
OH
MeO
1) n-BuLi, TMEDA, THF
2) B(OMe)3, then H2O2
N
N
N
2, 34%
Scheme 1. Synthetic route employed for the preparation of compound 2.
Bromination of the 5-position of the pyrimidine ring using N-bromosuccinimide provided
compound 13 in 96% yield, the latter of which was transformed to the respective 5-
hydroxypyrimidine (2) by halogen-metal exchange, conversion to the boronate ester, and
oxidation in 34% overall yield.
The influence of the 6-alkoxy moiety was also investigated. An initial assessment of the
need for an alkoxy group was made by replacing this substituent by a methyl group. As shown
in Scheme 2, the requisite compound (3) was accessible starting from commercially available
NH.HCl
20% Me4Phen, 20% CuI, Cs2CO3
dry degassed DMF, 50 °C
N
N
n-BuLi, THF, -78 °C
N
N
N
N
N
N
Br
Cl
15,
14
42%
14,
65%
Br
OH
1) n-BuLi, THF
2) B(OMe)3, then H2O2
NBS
N
N
N
N
CH2Cl2, rt
N
N
16,
97%
3,
55%
Scheme 2. Synthetic route employed for the preparation of compound 3.
2-chloro-4,6-dimethylpyrimidine, which was treated with azetidine to provide 14 in 65%
yield. Following the same strategy employed for the synthesis of 2, the benzylic anion derived
7

from 14 was treated with pentadecyl bromide to afford 15; following bromination to provide
16, compound 3 was then obtained from the intermediate bromide in 55% yield as a yellowish
oil.
The possibility of altering the position of the exocyclic oxygen atom was also investigated
by including the oxygen atom within the 16 atom linear side chain. Accordingly, compound 4
was prepared starting from commercially available 2,4-dichloro-6-methylpyrimidine. Initial
nucleophilic replacement of the chlorine atom in position 4 was performed using the anion
produced from 1-pentadecanol, followed by the introduction of the azetidinyl moiety in
position 2 to obtain 17 in 76% yield (Scheme 3). In analogy with Schemes 1 and 2, successive
bromination and hydroxylation afforded compound 4 as a colorless powder in 54% overall
yield. The 4-ethoxy (5) and 4-isopropoxy (6) analogues were also prepared starting from 2,4-
dichloro-6-methylpyrimidine, as outlined in Scheme 3. The transformations outlined in
RO
Cl
1. NaH, 1-pentadecanol, THF
O
1. NaH, ROH, THF
N
N
N
N
N
N
2. 20% Me4Phen, 20% CuI
Cs2CO3, azetidine hydrochloride,
dry degassed DMF
2. 20% Me4Phen, 20% CuI
Cs2CO3, azetidine hydrochloride,
dry degassed DMF
N
Cl
N
17, 76%
R = Et, 18, 43% (2 steps)
R = iPr, 19, 47% (2 steps)
1. n-BuLi, Br
2. NBS, CH2Cl2
1. NBS, CH2Cl2
14
2.n-BuLi, B(OMe)3
then H2O2
3.n-BuLi, B(OMe)3
then H2O2
OH
OH
O
RO
N
N
N
N
N
N
4, 54% (2 steps)
5
R = Et, , 30% (3 steps)
6
R = iPr, , 72% (3 steps)
Scheme 3. Synthetic routes employed for the preparation of compounds 4, 5 and 6.
Schemes 1‒3 thus provided a series of five new potential mitochondrial antioxidants
incorporating a cyclic amine in position 2.
The introduction of a second cyclic moiety was also considered as a means of increasing
metabolic stability. As outlined in Scheme 4, a cyclobutoxy moiety was introduced by
nucleophilic substitution of the chlorine atom in position 4 of 2,4-dichloro-6-
8

Cl
N
N
1) NaH, THF,
O
O
n-BuLi, THF
OH
Cl
N
N
N
N
14
N
N
NH.HCl
Br
2)
n
n
n
10% Me4Phen, 10% CuI, Cs2CO3
dry degassed DMF
20a n = 1, 35%
20b n = 2, 46%
20c
21a n = 1, 84%
21b
n = 2, 77%
21c n = 3, 81%
n = 3, 44%
Br
OH
1) n-BuLi, THF, -78 °C
2) B(OMe)3, 0 °C, then H2O2
O
O
NBS
N
N
N
N
CH2Cl2, rt
N
N
n
n
22a n = 1, 96%
22b
7 n = 1, 84%
8
n = 2, 95%
22c n = 3, 96%
n = 2, 63%
9 n = 3, 58%
Scheme 4. Synthetic routes employed for the preparation of compounds 7, 8 and 9.
methylpyrimidine, followed by the introduction of cycloamine moieties in position 4. In
parallel, three substituted pyrimidine cores (20a-c) were prepared incorporating
azetidinyl,pyrrolidinyl and piperidinyl groups in position 2 to explore the influence of amine
ring size on activity and metabolic stability. For each of these cores, the same sequence of
alkylation, bromination and hydroxylation was applied and analogues 7, 8 and 9 were
obtained in 27%, 21% and 20% overall yields, respectively. As described below, compounds
2 – 9 were evaluated in comparison with 1 in a panel of biochemical and biological assays,
enabling their antioxidant and cytoprotective properties to be understood, and especially to
determine whether these compounds were more stable metabolically than compound 1 in a
microsomal assay.
2.2. Biochemical and biological evaluation
2.2.1. Inhibition of lipid peroxidation
The ability of the new pyrimidinol analogues to quench lipid peroxidation was studied in
FRDA lymphocytes that had been depleted of glutathione by treatment with diethyl maleate
(DEM) as described previously.18,19 The fluorescent lipid peroxidation-sensitive fatty acid-
conjugated dye (C11-BODIPY581/591) was used to probe peroxyradical-induced lipid oxidation
by flow cytometry as described previously.18,19 This lipophilic fluorophore changes its
fluorescence from red to green when it interacts with peroxyradicals.22 C11-BODIPY-green
9

Figure 2. Lipid peroxidation in FRDA lymphocytes cells depleted of glutathione was determined by
utilizing the oxidation-sensitive fatty acid probe C11-BODIPY581/591 using fluorescence activated cell
sorting (FACS). Increased C11-BODIPY-green fluorescence (oxidized form), a measure of intracellular
lipid peroxidation, was determined by an increase in the median mean fluorescence intensity of C11-
BODIPY-green relative to the untreated control. A bar graph representing the percentage of the
median mean fluorescence intensity of C11-BODIPY-green fluorescence relative to a treated control is
shown. Statistical analyses were performed with a two-tailed Student’s t-test, and data are mean values
SEMs (n = 3). P values ≤ 0.0005 were considered to be statistically significant as compared to
treated control (DEM). Compounds 7– 9 were considered to be statistically significant (P ≤ 0.001) as
compared to untreated control.
fluorescence (oxidized form), a measure of intracellular lipid peroxidation, was determined by
increasing the median mean fluorescence intensity of C11-BODIPY-green relative to the
untreated control. The results presented in Figure 2 clearly show a significant (P ≤ 0.001)
decrease in protection from lipid peroxidation for cyclobutoxy-cycloamino analogues 7–9 as
compared to untreated control. The lipid peroxidation quenching activity decreased as the
ring size of the 2-cycloamino moiety increased. Introducing a non-cyclic alkoxy side chain at
position 4 did not have any significant negative effect on the ability of these quenchers to
protect against lipid peroxidation; they were as proficient as compound 1 (P ≤ 0.0005) as
compared to DEM-treated control.
2.2.2. Suppression of ROS
10

The ability of pyrimidinol analogues 1–9 to suppress ROS induced by depletion of
glutathione by diethyl maleate (DEM) treatment was evaluated in FRDA lymphocytes.
Cellular ROS production was monitored using dichlorodihydrofluorescein diacetate (DCFH-
DA), a membrane permeable and a non-fluorescent probe which, upon oxidation by ROS is
converted to the highly fluorescent derivative 2,7-dichlorofluorescein (DCF).23 This increase
of green fluorescence intensity resulting from ROS production induced by DEM was
measured by FACS as described before.18,19 The results obtained in FRDA lymphocytes are
presented in Figure 3, and show that the compound 1 and analogues 2–6 were all effective at
Figure 3. Flow cytometric analysis of ROS in FRDA lymphocyte cells pretreated with compounds 1–
9 at 0.1 µM, 1 µM and 2.5 µM concentrations for 16 h, and then treated with diethyl maleate (DEM)
for 80 min to induce the production of ROS. The cells were stained with 2, 7-
dichlorodihydrofluorescein diacetate (DCFH-DA) for 15 min prior to analysis. The samples were
analyzed immediately by flow cytometry (C6 Accuri, BD Biosciences, San Jose, CA). Increased DCF
fluorescence is a measure of intracellular oxidation and ROS production. A bar graph of the
percentage of the median mean fluorescence intensity of DCF fluorescence relative to a DEM-treated
control. Statistical analyses were performed with a two-tailed Student’s test, and data are mean values
SEMs (n = 2). P values ≤ 0.0005 were considered to be statistically significant as compared to
treated control (DEM). Compounds 7– 9 were considered to be statistically significant (P ≤ 0.001) as
compared to untreated control at low concentrations (0.1 and 1 µM).
11

suppressing ROS and did so at low concentration, reducing ROS to the same levels as
controls not treated with DEM. In comparison, 2-cyclobutoxy analogues 7–9 gave dose
dependent suppression of ROS, with effective suppression apparent only at the highest
concentration employed (2.5 µM), and poor protection at low (100 nM) concentration. As the
ring size of the cycloamino moiety increased among these three analogues, there was a slight
decrease in ROS scavenging activity. This trend was analogous to the results obtained for the
lipid peroxidation experiments.
2.2.3. Preservation of mitochondrial inner membrane potential (∆ψm)
Mitochondrial membrane potential (∆ψm) is critical for maintaining the ability of the
respiratory chain to produce ATP. Oxidative stress can induce the modification of the inner
mitochondrial membrane and lead to loss of ∆ψm, and trigger a series of mitochondria-
associated events including apoptosis and cell death. Compounds 1–9 were assessed for their
ability to preserve mitochondrial membrane potential following induction of oxidative stress
in FRDA lymphocytes. A lipophilic cationic fluorescent dye, tetramethyl rhodamine methyl
ester (TMRM) was used to assess ∆ψm. This dye distributes passively between the cytosol and
12

Figure 4. Representative flow cytometric two-dimensional color density dot plot analyses of the
ability of compounds 1 – 9 to maintain mitochondrial membrane potential (∆ψm) in DEM-treated
FRDA lymphocytes cells stained with 250 nM TMRM and analyzed using the FL2-H channel as
described in the experimental. The percentage of cells with intact ∆ψm is indicated in the top left
quadrant of captions (red) and the cells with depolarized ∆ψm in the bottom left quadrant of captions
(green). Representative examples from at least two independent experiments are shown. A total of
10,000 events was recorded for each sample and analyzed using C6 Accuri software (BD
Biosciences). A bar graph representing the percentage of the cells with intact ∆ψm is shown. Data are
expressed as means S.E.M. of two independent experiments run in duplicate. Statistical analyses
were performed with a two-tailed Student’s t-test, and data are mean values SEMs (n = 2). *P ≤
0.003 and **P ≤ 0.03, as compared to untreated control.
mitochondria according to the Nernst equation.24 A collapse of mitochondrial potential causes
the redistribution of the probe from mitochondria to the cytosol, a process that can be
followed readily using fluorescence imaging techniques. Upon collapse of ∆ψm, a reduction in
red fluorescence intensity occurs. Figure 4 (top panel) illustrates representative two-
dimensional density dot plots of TMRM-stained lymphocyte cells showing the percentage of
cells with intact ∆ψm (TMRM fluorescence in top left quadrant/red) versus the percentage of
cells with reduced ∆ψm (TMRM fluorescence in bottom left quadrant/green). Figure 4
(bottom panel) shows a bar graph of the percentage (means SEM) of FRDA lymphocytes
with intact ∆ψm. Treatment with 5 mM DEM decreased the percentage of cells with TMRM
fluorescence in the top left quadrant, revealing a depolarization of the ∆ψm. These data
indicate that modification of the alkoxy side chain, as in compounds 2–6, did not diminish the
ability to block oxidative stress-induced collapse of ∆ψm completely; 2–6 were clearly as
good as compound 1 in this regard. In comparison cyclobutoxy compounds 7–9 were all less
efficient in conferring protection against loss of mitochondrial membrane potential, although
all functioned in a dose dependent fashion. As noted above for suppression of lipid
peroxidation and ROS, increasing the cycloamine substituent ring size led to increasing loss
13

of efficiency in maintaining polarizable and healthy ∆ψm under oxidative stress in FRDA
lymphocytes. Treatment with compounds 1–9 in the absence of DEM treatment had no effect
on mitochondrial membrane potential (data not shown). Carbonyl cyanide-4-
(trifluoromethoxy)phenylhydrazone (FCCP), a commonly used uncoupler of oxidative
phosphorylation in mitochondria, was employed as a positive control to dissipate the
chemiosmotic proton gradient, which resulted in lowering of TMRM fluorescence as a result
of the depolarization of mitochondrial inner membrane potential.
2.2.4. Cellular ATP levels
Coenzyme Q10 serves as an electron carrier within the mitochondrial respiratory chain,
where it is integrally involved in oxidative phosphorylation, and consequently in ATP
production.25,26 It also functions as an antioxidant.25,26 CoQ10 levels have been reported to
decrease with age and to be reduced in patients with mitochondrial and neurodegenerative
diseases.27,28 An optimal, exogenously available CoQ10 analogue could well have the potential
to improve energy production in mitochondria, e.g. by bypassing lesions in the respiratory
chain, and by reducing the effects of oxidative stress. Previously, we have illustrated the
importance of an optimized (hexadecyl) side chain on the properties of exogenously supplied
analogues (such as compound 1) in FRDA and CoQ10 deficient lymphocytes.18 Since such
analogues are significantly less hydrophobic than CoQ10 itself, their bioavailability following
exogenous administration should be more favorable. Modified analogues 2–9 were evaluated
for their ability to support electron transport through the respiratory chain as judged by an
increase in ATP levels. Culturing FRDA cells in galactose as the sole sugar source forces
mammalian cells to rely on mitochondrial OXPHOS and is a strategy previously used to
diagnose human mitochondrial disorders or drug toxicity.29,30 Since cells grown in galactose
rely mostly on OXPHOS to produce their ATP, they become more sensitive to mitochondrial
respiratory chain inhibitors than cells grown in glucose medium. In the present study,
14

compounds 1–9 were evaluated for their ability to enhance ATP levels in FRDA lymphocytes
(Table 1). As noted in the Table, with the exception of compound 4, all of the analogues
increased ATP levels at one or two of the tested (1, 5 and 20 µM) concentrations. Among the
analogues having an acyclic 4-substituent, 1, 2, 3 and 6 increased ATP levels at the lowest
tested concentration, while 1, 2, 5 and 6 increased ATP when employed at 5 µM
concentration. Among the analogues having a 4-cyclobutoxy substituent (7 – 9), increased
ATP levels in the FRDA lymphocytes were observed for all three compounds when tested at 5
µM concentration, and to a lesser extent for 7 and 8 at 20 µM concentration. Significantly
Table 1
Total ATP concentration in FRDA lymphocytes
following incubation with compounds 1–9 for 48 ha
Compound
Total ATP level (% control)
FRDA lymphocytes
1 µM
5 µM 20 µM
100 100
115 5b 107 2b
102
109 4b
109 3b
Untreated control 100
88 3b
1
2
3
4
5
6
7
8
9
5
74 3b
60 3b
11 8b
96 1b
98
1
96
4
1
68 13b
108 4b
100
106 2b 109 2b
95
3
2
1
4
98
98
99
4
4
3
112 3b 103
107 5
106 4
102
100
a Determined from intracellular ATP levels using the
luciferin-luciferase reaction.
b Two-tailed Student’s t-test, with p ≤ 0.05 considered as
statistically significant compared to vehicle control.
diminished ATP levels in the treated cells were observed following treatment at 20 µM for
compounds 1–6. Interestingly, the movement of the oxygen atom position from the 4-
methoxy group in compound 2 to the lipophilic side chain at position 6 in analogue 4 resulted
in a compound which did not increase ATP levels above the basal level when used at low (1
µΜ) concentration, and strongly diminished ATP levels in a concentration-dependent fashion
in FRDA lymphocytes when employed at higher concentrations. The reduction in ATP levels
15

in the treated cells likely reflects the ability of test compounds to inhibit complex I activity in
the mitochondrial electron transport chain, as we have shown previously in bovine heart
mitochondria.31
Interestingly, the addition of a cyclobutoxy moiety enhanced ATP production at higher
applied concentrations; for compounds 7–9, the ATP level was elevated at 5 µM
concentration. This phenomenon was also observed for compounds 5 and 6. It may also be
noted that the ring size of the cyclic amine, which was important in the previous assays, was
negligible for ATP concentration.
2.2.5. Cytoprotection
The ability of the test compounds (1–9) to confer cytoprotection was evaluated in cultured
FRDA lymphocytes that had been treated with diethyl maleate to induce oxidative stress
through depletion of glutathione. As is clear from Figure 5, compounds 2–6 exhibited good
Figure 5. Viability of Friedreich’s ataxia lymphocytes following pretreatment with the test
compounds for 16 h and then treatment with DEM (5 mM) for 6 h to induce oxidative stress. Flow
cytometric determination of cell viability by fluorescence labeling was used, employing calcein
acetoxy-methyl ester and ethidium homodimer-1 (EthD-1) as live and dead cell stains. Cell viability
was expressed as the percentage of cells relative to control. Results are an average of two independent
trials run in duplicate. Statistically significant differences, where observed, are also indicated.
Significant p-values are indicated with * for extremely significant (p ≤ 0.0001), ** for very significant
16

(p ≤ 0.0005), *** for (p ≤ 0.0025), **** for (p ≤ 0.005), ***** for significant (p ≤ 0.05), as compared
to untreated control.
cytoprotective activity, even when used at 0.5 µM concentration. Their cytoprotective activity
was clearly superior to that of the cyclobutoxy analogues 7–9, none of which conferred
significant cytoprotection at 0.1 µM concentration, and which were also not strongly
cytoprotective when used at 0.5 µM concentration. Again, the size of the cyclic amine ring
was correlated with decreased cytoprotection as the ring size increased.
2.2.6. Metabolic stability assays
The main objective of this study was to identify an efficient MRQ structurally related to 1,
but having improved metabolic stability. As shown in Table 2, following incubation in the
presence of activated bovine liver microsomes for 30 minutes, the recovery of compound 1
was only about 46%. Compounds 2‒9 were subjected to the same 30-minute incubation, and
recovered starting material was analyzed by C18 reversed phase HPLC. Replacing the N,N-
dimethylated amino moiety in 1 with the azetidine moiety in 2 increased the recovery of
starting material to 58%. These results are consistent with those of an earlier metabolic study
Table 2
In vitro microsomal stability of the compounds 1–9 following incubation with bovine liver
microsomes.
Compound
Recovery (%)a
46
58 11
57
62
54 11
76
65 12
8
1
2
3
4
5
6
7
8
9
8
6
7
72
72
9
8
a Results expressed as % of compound recovered after reaction with activated microsomes.
Microsomal stability values represent means SD.
17

of similar compounds in the pyridinol series.19 The two analogues methylated at the 4-
position (3 and 4) were recovered in 57 and 62% yields, respectively, following incubation
with the microsomes. Replacing the 4-methoxy substituent with an ethoxy moiety (compound
5) resulted in similar metabolic stability to that of compound 2 (54 vs 58%). Surprisingly,
introducing an isopropoxy group at position 4 resulted in a substantial improvement in
metabolic stability (76% recovery).
Compounds 7 – 9, all of which contained a cyclobutoxy group at position 4 and a
cycloamine at position 2, exhibited significantly enhanced metabolic stability with recoveries
of unmetabolized starting material in the 65-72% range after 30-minute microsomal
incubations. Despite their metabolic stability, analogues 7–9 lack the potent biological
activities exhibited by compound 6. Because of its favorable cellular potency and in vitro
metabolic stability, compound 6 is a good candidate for further pharmacokinetic and in vivo
evaluation.
3. Conclusions
Eight new multifunctional radical quenchers based on a pyrimidinol scaffold have been
prepared using straightforward synthetic methods. Two types of modification of the prototype
compound have been explored, one involving the use of a cyclic amine at the 2-position to
confer metabolic stability. This strategy worked well to increase metabolic stability without
compromising biological activity when an azetidine moiety was introduced, but not for larger
cyclic amines. A cyclobutoxy moiety was introduced at the 4-position, and significantly
increased metabolic stability. Unfortunately, while this group did confer increased metabolic
stability, it also substantially compromised the properties of the derived compounds as
antioxidants and cytoprotective species. The introduction of an azetidine moiety at the 2-
position and an isopropoxy group at the 4-position provided an analogue (6) with good
metabolic stability and excellent cytoprotective properties. This compound appears to be a
18

good candidate for further study, and should be helpful in furthering our understanding of
neurodegenerative and mitochondrial diseases, and developing strategies for therapeutic
intervention.
4. Experimental Section
4.1. Chemistry
Anhydrous grade solvents were purchased from Sigma–Aldrich Inc. (St. Louis, MO) and
from Fisher Scientific. Most of the chemical reagents were purchased from Sigma–Aldrich
and used without further purification. ImPrPh2HCl, morpholine and iodine were purchased
from TCI America. Azetidine hydrochloride was purchased from Combi-Blocks. All
glassware and needles were pre-dried in an oven at 120 °C prior to use. Tetrahydrofuran was
distilled from sodium/benzophenone. All reactions were performed under a stream of argon.
Flash column chromatography was carried out using silica gel (Silicycle R10030B, 60 Å
particle size, 230– 400 mesh), applying a low pressure stream of nitrogen. Analytical thin
layer chromatographic separations were carried out on silica gel (60 Å particle size, 250 lm
thickness, F-254, Silicycle) coated glass plates. Spots were visualized with UV light, or
developed by using iodine vapor, or by immersing the plates in 2.0% anisaldehyde in
ethanol/sulfuric acid/acetic acid, followed by heating with a heat gun. The NMR spectra were
recorded using a 400 MHz Varian Inova instrument. Chemical shifts were reported in parts
per million (ppm, δ) relative to the residual 1H resonance of the solvent CDCl3 or CD3OD at
7.26 ppm or 3.31 ppm, respectively. 13C NMR chemical shifts were reported relative to the
central line of CDCl3 or CD3OD at 77.16 ppm or 49.00 ppm, respectively. Splitting patterns
were designated as follows: s, singlet; br s, broad singlet; d, doublet; t, triplet; m, multiplet;
quint, quintet. High resolution mass spectra were obtained at the Arizona State University
CLAS High Resolution Mass Spectrometry Laboratory.
19

4.1.1. 2-Iodo-4-methoxy-6-methylpyrimidine (10)
To a stirred solution containing 3.00 g (21.6 mmol) of 2-amino-4-methoxy-6-
methylpyrimidine, 5.46 g (21.6 mmol) of iodine, 4.31 g (22.6 mmol) of CuI and 2.50 mL
(30.9 mmol) of CH2I2 in 120 mL of anhydrous THF was added 10.5 mL (78.2 mmol) of
isoamylnitrite. The reaction mixture was stirred at reflux for 3 h. The cooled reaction mixture
was filtered through Celite and the Celite pad was washed with CH2Cl2. The combined
organic phase was washed with water and then with brine, dried (MgSO4) and concentrated
under diminished pressure. The residue was purified by flash chromatography on a silica gel
column (20 × 5 cm). Elution with hexane followed by 95:5 hexane/Et2O and then 80:20
hexane/Et2O afforded 10 as a yellowish solid: yield 2.01 g (37%); mp 43-44 °C; silica gel
TLC Rf 0.35 (4:1 hexane/Et2O); 1H NMR (CDCl3) δ 2.37 (s, 3H), 3.93 (s, 3H) and 6.50 (s,
1H); 13C NMR (CDCl3) δ 23.7, 54.6, 106.5, 127.4, 169.0 and 169.1; mass spectrum (APCI),
m/z 250.9675 (M+H)+ (C6H8N2OI requires m/z 250.9682).
4.1.2. 2-(Azetidin-1-yl)-4-methoxy-6-methylpyrimidine (11)
To a stirred solution containing 560 mg (5.98 mmol) of azetidine hydrochloride, 76.0 mg
(0.39 mmol) of CuI, and 3.90 g (11.9 mmol) of Cs2CO3 in 10 mL of dry, degassed DMF was
added sequentially 1.00 g (3.99 mmol) of 10 and 95.0 mg (0.39 mmol) of 3,4,7,8-tetramethyl-
1,10-phenanthroline. The reaction mixture was stirred at 50 °C for 5 h. The cooled reaction
mixture was filtered through Celite and the Celite pad was washed with CH2Cl2. The
combined organic phase was washed with water and then with brine, dried (MgSO4) and
concentrated under diminished pressure. The residue was purified by flash chromatography
on a silica gel column (20 × 3 cm). Elution with hexane followed by 95:5 hexane/EtOAc and
then 85:15 hexane/EtOAc afforded 11 as a yellowish oil: yield 515 mg (72%); silica gel TLC
Rf 0.26 (3:2 hexane/EtOAc); 1H NMR (CDCl3, 400 MHz) δ 2.25 (s, 3H), 2.30 (quint, 2H, J =
8.0 Hz), 3.84 (s, 3H), 4.11 (t, 4H, J = 7.6 Hz) and 5.83 (s, 1H); 13C NMR (CDCl3, 100 MHz)
20

δ 16.3, 24.1, 50.2, 53.0, 95.0, 163.2, 168.0 and 170.7; mass spectrum (APCI), m/z 180.1136
(M+H)+ (C9H14N3O requires m/z 180.1137).
4.1.3. 2-(Azetidin-1-yl)-6-hexadecyl-4-methoxypyrimidine (12)
To a stirred solution containing 261 mg (1.45 mmol) of 11 in 7 mL of anhydrous THF at –
78 °C was added 870 µL (2.17 mmol) of a 2.5 M solution of n-BuLi in hexane. The reaction
mixture was stirred at –78 °C for 15 min and then 300 µL (1.03 mmol) of 1-
bromopentadecane was added. The reaction mixture was stirred at 0 oC for another 30 min,
then quenched with satd aq ammonium chloride and extracted with 150 mL of EtOAc. The
organic phase was washed with brine, dried (MgSO4) and concentrated under diminished
pressure. The residue was purified by chromatography on a silica gel column (20 × 3 cm).
Elution with hexane followed by 95:5 hexane/Et2O afforded 12 as a yellowish solid: yield 142
mg (25%) and 87 mg (33%) of the starting material was recovered; mp 45-46 °C; silica gel
TLC Rf 0.32 (4:1 hexane/Et2O); 1H NMR (CDCl3) δ 0.87 (t, 3H, J = 7.2 Hz), 1.18-1.35 (m,
26H), 1.62 (quint, 2H, J = 7.2 Hz), 2.29 (quint, 2H, J = 7.2 Hz), 2.48 (t, 2H, J = 7.6 Hz), 3.82
(s, 3H), 4.10 (t, 4H, J = 7.6 Hz) and 5.83 (s, 1H); 13C NMR (CDCl3) δ 14.2, 16.3, 22.8, 28.7,
29.46, 29.5, 29.6, 29.7, 29.78, 29.8, 32.0, 37.9, 50.2, 52.9, 94.3, 163.3, 170.7 and 172.2; mass
spectrum (APCI), m/z 390.3481 (M+H)+ (C24H44N3O requires m/z 390.3484).
4.1.4. 2-(Azetidin-1-yl)-5-bromo-4-methoxy-6-hexadecylpyrimidine (13)
To a stirred solution containing 106 mg (0.27 mmol) of 12 in 4 mL of 1:1
CH2Cl2/acetonitrile was added 58.0 mg (0.33 mmol) of N-bromosuccinimide in the dark. The
reaction mixture was stirred for 30 min at room temperature in the dark, then diluted with 50
mL of CH2Cl2, washed with brine, dried (MgSO4) and concentrated under diminished
pressure. The residue was purified by chromatography on a silica gel column (15 × 3 cm).
Elution with hexane followed by 95:5 hexane/Et2O afforded 13 as a colorless solid: yield 121
mg (96%); mp 82-83 °C; silica gel TLC Rf 0.55 (4:1 hexane/Et2O); 1H NMR (CDCl3, 400
21

MHz) δ 0.88 (t, 3H, J = 7.2 Hz), 1.19-1.37 (m, 26H), 1.64 (quint, 2H, J = 7.2 Hz), 2.32
(quint, 2H, J = 7.2 Hz), 2.69 (t, 2H, J = 7.6 Hz), 3.93 (s, 3H) and 4.10 (t, 4H, J = 7.6 Hz); 13C
NMR (CDCl3) δ 14.3, 16.3, 22.8, 28.0, 29.5, 29.6, 29.7, 29.8, 29.9, 32.1, 37.0, 50.5, 54.3,
92.7, 161.2, 165.7 and 169.6; mass spectrum (APCI), m/z 468.2589 (M+H)+ (C24H43N3OBr
requires m/z 468.2589).
4.1.5. 2-(Azetidin-1-yl)-6-hexadecyl-4-methoxypyrimidin-5-ol (2)
To a stirred solution containing 93.0 mg (0.19 mmol) of 13 in 2 mL of anhydrous THF at –5
°C was added 30.0 µL (0.19 mmol) of TMEDA and 198 µL (0.49 mmol) of a 2.5 M solution
of n-BuLi in hexane. The reaction mixture was stirred at –5 °C for 15 min and then 66.0 µL
(0.59 mmol) of trimethoxyborane was added. The reaction mixture was stirred for 30 min at
room temperature followed by the addition of 426 µL (4.35 mmol) of H2O2 (35% v/v). The
reaction mixture was stirred for an additional 30 min and then poured into 20 mL of water,
neutralized with dilute aq HCl and extracted with 100 mL of EtOAc. The organic phase was
washed with brine, dried (MgSO4) and concentrated under diminished pressure. The residue
was purified by chromatography on a silica gel column (15 × 3 cm). Elution with hexane
followed by 90:10 hexane/EtOAc afforded 2 as a yellowish solid: yield 27.0 mg (34%); mp
59-60 °C; silica gel TLC Rf 0.22 (4:1 hexane/EtOAc); 1H NMR (CDCl3) δ 0.88 (t, 3H, J =
7.2 Hz), 1.19-1.37 (m, 26H), 1.64 (quint, 2H, J = 7.2 Hz), 2.27 (quint, 2H, J = 7.2 Hz), 2.61
(t, 2H, J = 8.0 Hz), 3.92 (s, 3H), 4.04 (t, 4H, J = 7.6 Hz) and 4.61 (br s, 1H); 13C NMR
(CDCl3) δ 14.3, 16.3, 22.8, 28.1, 29.5, 29.7, 29.72, 29.8, 29.82, 29.9, 31.5, 32.1, 51.0, 53.6,
128.3, 155.2, 157.6 and 158.6; mass spectrum (APCI), m/z 406.3436 (M+H)+ (C24H44N3O2
requires m/z 406.3434).
4.1.6. 2-(Azetidin-1-yl)-4,6-dimethylpyrimidine (14)
To a stirred solution containing 655 mg (6.99 mmol) of azetidine hydrochloride, 133 mg
(6.99 mmol) of CuI, and 3.42 g (10.5 mmol) of Cs2CO3 in 10 mL of dry, degassed DMF was
22

added sequentially 500 mg (3.49 mmol) of 2-chloro-4,6-pyrimidine and 165 mg (6.99 mmol)
of 3,4,7,8-tetramethyl-1,10-phenanthroline. The reaction mixture was stirred at 50 °C for 4 h.
The reaction mixture was allowed to warm to room temperature and was then filtered through
Celite and the Celite pad was washed with CH2Cl2. The combined organic phase was washed
with water and then with brine, dried (MgSO4) and concentrated under diminished pressure.
The residue was purified by flash chromatography on a silica gel column (15 × 3 cm). Elution
with hexane followed by 4:1 hexane/EtOAc and then 1:1 hexane/EtOAc afforded 14 as
yellowish solid: yield 372 mg (65%); mp 51-52 °C; silica gel TLC Rf 0.22 (3:2
hexane/EtOAc); 1H NMR (CDCl3, 400 MHz) δ 2.20 (s, 6H), 2.24 (t, 2H, J = 7.6 Hz), 4.05 (t,
4H, J = 7.2 Hz) and 6.19 (s, 1H); 13C NMR (CDCl3, 100 MHz) δ 16.2, 23.9, 50.1, 109.1,
163.2 and 167.0; mass spectrum (FAB), m/z 164.1192 (M+H)+ (C9H14N3 requires m/z
164.1188).
4.1.7. 2-(Azetidin-1-yl)-6-hexadecyl-4-methylpyrimidine (15)
To a stirred solution containing 321 mg (1.96 mmol) of 14 in 10 mL of anhydrous THF at –
78 °C was added 1.02 mL (2.56 mmol) of a 2.5 M solution of n-BuLi in hexane. The reaction
mixture was stirred at –78 °C for 15 min and then 398 µL (1.37 mmol) of 1-
bromopentadecane was added. The reaction mixture was stirred at 0 oC for another 30 min,
then quenched with satd aq ammonium chloride and extracted with 150 mL of EtOAc. The
organic phase was washed with brine, dried (MgSO4) and concentrated under diminished
pressure. The residue was purified by chromatography on a silica gel column (20 × 3 cm).
Elution with hexane followed by 96:4 hexane/EtOAc and then 90:10 hexane/EtOAc afforded
15 as a colorless solid: yield 307 mg (42%); mp 63-64 °C; silica gel TLC Rf 0.45 (3:2
hexane/EtOAc); 1H NMR (CDCl3) δ 0.86 (t, 3H, J = 6.8 Hz), 1.18-1.37 (m, 26H), 1.62 (quint,
2H, J = 7.6 Hz), 2.27 (s, 3H), 2.29 (quint, 2H, J = 7.6 Hz), 2.49 (t, 2H, J = 7.2 Hz), 4.11 (t,
4H, J = 7.2 Hz) and 6.24 (s, 1H); 13C NMR (CDCl3) δ 14.2, 16.4, 22.8, 24.2, 28.8, 29.46,
23

29.5, 29.6, 29.64, 29.75, 29.8, 32.0, 37.9, 50.3, 108.6, 163.4, 167.0 and 171.2; mass spectrum
(FAB), m/z 374.3545 (M+H)+ (C24H44N3 requires m/z 374.3535).
4.1.8. 2-(Azetidin-1-yl)-5-bromo-6-hexadecyl-4-methylpyrimidine (16)
To a stirred solution containing 290 mg (0.77 mmol) of 15 in 5 mL of CH2Cl2 was added
152 mg (0.85 mmol) of N-bromosuccinimide in the dark. The reaction mixture was stirred for
30 min at room temperature under dark, then diluted with 20 mL of CH2Cl2, washed with
brine, dried (MgSO4) and concentrated under diminished pressure. The residue was purified
by chromatography on a silica gel column (15 × 3 cm). Elution with hexane followed by 96:4
hexane/EtOAc afforded 16 as a colorless solid: yield 338 mg (97%); mp 74-75 °C; silica gel
TLC Rf 0.45 (4:1 hexane/EtOAc); 1H NMR (CDCl3) δ 0.87 (t, 3H, J = 7.2 Hz), 1.18-1.37 (m,
26H), 1.65 (quint, 2H, J = 7.6 Hz), 2.31 (quint, 2H, J = 7.6 Hz ), 2.44 (s, 3H), 2.71 (t, 2H, J =
7.6 Hz) and 4.09 (t, 4H, J = 7.6 Hz); 13C NMR (CDCl3) δ 14.2, 16.3, 22.8, 25.3, 27.8, 29.5,
29.6, 29.7, 29.8, 29.84, 32.1, 37.4, 50.5, 108.6, 161.3, 165.7 and 168.8; mass spectrum (FAB),
m/z 454.2611 (M+H)+ (C24H43N3O81Br requires m/z 454.2620).
4.1.9. 2-(Azetidin-1-yl)-4-hexadecyl-6-methylpyrimidin-5-ol (3)
To a stirred solution containing 57.0 mg (0.13 mmol) of 16 in 2 mL of anhydrous THF at –5
°C was added 84.0 µL (0.75 mmol) of trimethoxyborane and 156 µL (0.39 mmol) of a 2.5 M
solution of n-BuLi in hexane. The reaction mixture was stirred at 23 °C for 30 min followed
by the addition of 221 µL (3.25 mmol) of H2O2 (50% v/v). The reaction mixture was stirred
for additional 30 min and poured into 20 mL water, neutralized with dilute aq HCl and then
extracted with 100 mL of EtOAc. The organic phase was washed with brine, dried (MgSO4)
and concentrated under diminished pressure. The residue was purified by chromatography on
a silica gel column (15 × 3 cm). Elution with 95:5 hexane/EtOAc followed by 80:20
hexane/EtOAc afforded 3 as a yellowish oil: yield 28.0 mg (55%); silica gel TLC Rf 0.27 (3:2
hexane/EtOAc); 1H NMR (CD3OD) δ 0.90 (t, 3H, J = 6.8 Hz), 1.27-1.32 (m, 26H), 1.64 (m,
24

2H), 2.25-2.34 (m, 5H), 2.65 (m, 2H), 4.04 (t, 4H, J = 7.6 Hz) and 4.28 (br s, 1H); 13C NMR
(CD3OD) δ 14.5, 17.0, 18.6, 23.8, 29.1, 30.5, 30.6, 30.7, 30.8, 30.81, 30.83, 32.8, 33.1, 52.2,
140.7, 157.6, 159.9 and 161.6; mass spectrum (FAB), m/z 390.3480 (M+H)+ (C24H44N3O
requires m/z 390.3484).
4.1.10. 2-(Azetidin-1-yl)-4-methyl-6-(pentadecyloxy)pyrimidine (17)
To a stirred solution containing 1.01 g (6.13 mmol) of 2,4-dichloro-6-methylpyrimidine in
20 mL of anhydrous THF was added 620 mg (25.8 mmol) of NaH (60% suspension in oil)
and 1.47 g (6.44 mmol) of 1-pentadecanol. The reaction mixture was stirred for 24 h at room
temperature and then poured slowly into 100 mL of water. The crude was extracted with two
200-mL portions of EtOAc. The combined organic phase was washed with brine, dried
(MgSO4) and concentrated under diminished pressure to afford crude 790 mg of 2-chloro-4-
methyl-6-pentadecyloxy)pyrimidine.
To a round bottom flask containing 350 mg (0.99 mmol) of crude 2-chloro-4-methyl-6-
(pentadecyloxy)pyrimidine, 139 mg (1.49 mmol) of azetidine hydrochloride, 19.0 mg (0.09
mmol) of CuI, 23.0 mg (0.09 mmol) of 3,4,7,8-tetramethyl-1,10-phenanthroline and 806 mg
(2.48 mmol) of Cs2CO3 was added 15 mL of dry, degassed DMF. The reaction mixture was
stirred at 50 °C for 5 h. The cooled reaction mixture was filtered through Celite and the Celite
pad was washed with portions of CH2Cl2. The combined organic phase was washed with
water and then with brine, dried (MgSO4) and concentrated under diminished pressure. The
residue was purified by flash chromatography on a silica gel column (15 × 3 cm). Elution
with 19:1 hexane/EtOAc followed by 9:1 hexane/EtOAc afforded 17 as a colorless solid:
yield 282 mg (76%); mp 40-41 °C; silica gel TLC Rf 0.27 (4:1 hexane/EtOAc); 1H NMR
(CDCl3) δ 0.86 (t, 3H, J = 6.8 Hz), 1.21-1.38 (m, 24H), 1.70 (quint, 2H, J = 7.2 Hz), 2.24 (s,
3H), 2.29 (quint, 2H, J = 7.6 Hz), 4.09 (t, 4H, J = 7.6 Hz), 4.21 (t, 2H, J = 6.8 Hz) and 5.81
(s, 1H); 13C NMR (CDCl3) δ 14.3, 16.4, 22.9, 24.2, 26.2, 29.1, 29.5, 29.7, 29.76, 29.8, 29.9,
25

32.1, 50.3, 65.9, 95.3, 163.2, 168.0 and 170.6; mass spectrum (FAB), m/z 376.3317 (M+H)+
(C23H42N3O requires m/z 376.3328).
4.1.11. 2-(Azetidin-1-yl)-5-bromo-4-methyl-6-(pentadecyloxy)pyrimidine
To a stirred solution containing 145 mg (0.39 mmol) of 17 in 4 mL CH2Cl2 was added 72.0
mg (0.41 mmol) of N-bromosuccinimide in the dark. The reaction mixture was stirred for 30
min at room temperature in the dark, then diluted with CH2Cl2, washed with brine, dried
(MgSO4) and concentrated under diminished pressure. The residue was purified by
chromatography on a silica gel column (15 × 3 cm). Elution with hexane followed by 96:4
hexane/EtOAc afforded 2-(azetidin-1-yl)-5-bromo-4-methyl-6-(pentadecyloxy)pyrimidine
as a colorless solid: yield 159 mg (90%); mp 71-72 °C; silica gel TLC Rf 0.53 (4:1
hexane/EtOAc); 1H NMR (CDCl3) δ 0.87 (t, 3H, J = 6.8 Hz), 1.21-1.47 (m, 24H), 1.75 (quint,
2H, J = 7.6 Hz), 2.30 (quint, 2H, J = 7.2 Hz), 2.40 (s, 3H), 4.07 (t, 4H, J = 7.6 Hz) and 4.30
(t, 2H, J = 6.8 Hz); 13C NMR (CDCl3) δ 14.2, 16.2, 22.8, 24.5, 26.1, 28.9, 29.45, 29.5, 29.7,
29.72, 29.8, 29.83, 32.1, 50.4, 67.1, 93.3, 161.0, 165.3 and 166.0; mass spectrum (FAB), m/z
454.2421 (M+H)+ (C23H41N3OBr requires m/z 454.2433).
4.1.12. 2-(Azetidin-1-yl)-4-methyl-6-(pentadecyloxy)pyrimidin-5-ol (4)
To a stirred solution containing 130 mg (0.28 mmol) of 2-(azetidin-1-yl)-5-bromo-4-
methyl-6-(pentadecyloxy)pyrimidine in 3 mL of anhydrous THF at –5 °C was added 229 µL
(0.57 mmol) of a 2.5 M solution of n-BuLi in hexane and 94.0 µL (0.84 mmol) of
trimethoxyborane. The reaction mixture was stirred at 23 °C for 30 min followed by the
addition of 419 µL (6.16 mmol) of H2O2 (50% v/v). The reaction mixture was stirred for an
additional 30 min, poured into 20 mL NaHCO3 and then extracted with 100 mL of CH2Cl2.
The organic phase was washed with brine, dried (MgSO4) and concentrated under diminished
pressure. The residue was purified by chromatography on a silica gel column (15 × 3 cm).
Elution with 95:5 hexane/EtOAc afforded 4 as a colorless powder: yield 66.0 mg (60%); mp
26

83-85 °C; silica gel TLC Rf 0.21 (3:2 hexane/EtOAc); 1H NMR (CDCl3) δ 0.86 (t, 3H, J =
6.8 Hz), 1.05-1.41 (m, 24H), 1.70 (quint, 2H, J = 6.8 Hz), 2.15-2.32 (m, 5H), 4.01 (t, 4H, J =
7.2 Hz), 4.30 (t, 2H, J = 6.8 Hz) and 5.11 (br s, 1H); 13C NMR (CDCl3) δ 14.2, 16.3, 17.8,
22.8, 26.1, 29.0, 29.5, 29.7, 29.74, 29.8, 29.83, 32.1, 50.9, 66.6, 128.6, 151.1, 157.3 and
158.5; mass spectrum (FAB), m/z 392.3286 (M+H)+ (C23H42N3O2 requires m/z 392.3277).
4.1.13. 2-Chloro-4-ethoxy-6-methylpyrimidine
To a stirred solution containing 2.01 g (12.3 mmol) of 2,4-dichloro-6-methylpyrimidine in
40 mL of anhydrous THF was added 927 mg (38.6 mmol) of NaH (60% suspension in oil)
and 392 µL (12.9 mmol) of EtOH. The reaction mixture was stirred at room temperature for 5
h and then poured slowly into 200 mL of water. The crude reaction mixture was extracted
with two 300-mL portions of EtOAc. The combined organic phase was washed with brine,
dried (MgSO4) and concentrated under diminished pressure. The residue was purified by
chromatography on a silica gel column (15 × 6 cm). Elution with 19:1 hexane/EtOAc
afforded 2-chloro-4-ethoxy-6-methylpyrimidine as a colorless solid: yield 2.16 g (51%); mp
37-38 °C; silica gel TLC Rf 0.41 (4:1 hexane/EtOAc); 1H NMR (CDCl3) δ 1.38 (t, 3H, J =
7.2 Hz), 2.42 (s, 3H), 4.42 (d, 2H, J = 7.2 Hz) and 6.46 (s, 1H); 13C NMR (CDCl3) δ 14.4,
23.8, 63.5, 105.7, 159.8, 169.8 and 170.9; mass spectrum (APCI), m/z 173.0477 (M+H)+
(C7H10N2OCl requires m/z 173.0482).
4.1.14. 2-(Azetidin-1-yl)-4-ethoxy-6-methylpyrimidine (18)
To a round bottom flask containing 600 mg (3.48 mmol) of 2-chloro-4-ethoxy-6-
methylpyrimidine, 489 mg (5.22 mmol) of azetidine hydrochloride, 131 mg (0.69 mmol) of
CuI, 164 mg (0.69 mmol) of 3,4,7,8-tetramethyl-1,10-phenanthroline and 2.83 g (8.70 mmol)
of Cs2CO3 was added 15 mL of dry, degassed DMF. The reaction mixture was stirred at 50 °C
for 3 h. The cooled reaction mixture mixture was filtered through Celite and the Celite pad
was washed with portions of CH2Cl2. The combined organic phase was washed with water
27

and then with brine, dried (MgSO4) and concentrated under diminished pressure. The residue
was purified by flash chromatography on a silica gel column (15 × 3 cm). Elution with 19:1
hexane/EtOAc followed by 9:1 hexane/EtOAc afforded 18 as a colorless solid: yield 565 mg
(84%); mp 42-43 °C; silica gel TLC Rf 0.29 (3:2 hexane/EtOAc); 1H NMR (CDCl3) δ 1.24 (t,
3H, J = 7.2 Hz), 2.16 (s, 3H), 2.20 (quint, 2H, J = 7.6 Hz), 4.01 (t, 4H, J = 7.6 Hz), 4.20 (q,
2H, J = 7.2 Hz) and 5.73 (s, 1H); 13C NMR (CDCl3) δ 14.4, 16.1, 23.9, 49.9, 61.2, 95.0,
163.0, 167.7 and 170.1; mass spectrum (APCI), m/z 194.1289 (M+H)+ (C10H16N3O requires
m/z 194.1293).
4.1.15. 2-(Azetidin-1-yl)-4-ethoxy-6-hexadecylpyrimidine
To a stirred solution containing 450 mg (2.32 mmol) of 18 in 20 mL of anhydrous THF at –
78 °C was added 1.02 mL (2.56 mmol) of a 2.5 M solution of n-BuLi in hexane. The reaction
mixture was stirred at –78 °C for 15 min and then 475 µL (1.63 mmol) of 1-
bromopentadecane was added. The reaction mixture was stirred at 0 oC for another 30 min,
then quenched with satd aq ammonium chloride and extracted with 150 mL of EtOAc. The
organic phase was washed with brine, dried (MgSO4) and concentrated under diminished
pressure. The residue was purified by chromatography on a silica gel column (15 × 3 cm).
Elution with 19:1 hexane/EtOAc afforded 2-(azetidin-1-yl)-4-ethoxy-6-hexadecylpyrimidine
as a colorless solid: yield 421 mg (45%); mp 40-41 °C; silica gel TLC Rf 0.42 (4:1
hexane/EtOAc); 1H NMR (CDCl3) δ 0.84 (t, 3H, J = 6.8 Hz), 1.18-1.33 (m, 29H), 1.60 (quint,
2H, J = 6.8 Hz), 2.24 (quint, 2H, J = 7.6 Hz), 2.44 (t, 2H, J = 7.6 Hz), 4.05 (t, 4H, J = 7.6
Hz), 4.26 (q, 2H, J = 7.2 Hz) and 5.78 (s, 1H); 13C NMR (CDCl3) δ 14.1, 14.5, 16.2, 22.7,
28.6, 29.39, 29.42, 29.55, 29.61, 29.7, 29.8, 32.0, 37.8, 50.1, 61.3, 94.4, 163.2, 170.2 and
172.0; mass spectrum (FAB), m/z 404.3632 (M+H)+ (C25H46N3O requires m/z 404.3641).
4.1.16. 2-(Azetidin-1-yl)-5-bromo-4-ethoxy-6-hexadecylpyrimidine
28

To a stirred solution containing 464 mg (1.15 mmol) of 2-(azetidin-1-yl)-4-ethoxy-6-
hexadecylpyrimidine in 10 mL of CH2Cl2 was added 209 mg (1.17 mmol) of N-
bromosuccinimide in the dark (round bottom flask was wrapped with aluminum foil). The
reaction mixture was stirred in the dark for 30 min at room temperature, then diluted with
CH2Cl2, washed with brine, dried (MgSO4) and concentrated under diminished pressure. The
residue was purified by chromatography on a silica gel column (15 × 3 cm). Elution with
hexane followed by 96:4 hexane/EtOAc afforded 2-(azetidin-1-yl)-5-bromo-4-ethoxy-6-
hexadecylpyrimidine as a colorless solid: yield 522 mg (94%); mp 69-70 °C; silica gel TLC
Rf 0.56 (4:1 hexane/EtOAc); 1H NMR (CDCl3) δ 0.87 (t, 3H, J = 7.2 Hz), 1.18-1.40 (m,
29H), 1.64 (quint, 2H, J = 7.6 Hz), 2.29 (quint, 2H, J = 7.6 Hz), 2.69 (t, 2H, J = 7.6 Hz), 4.06
(t, 4H, J = 7.6 Hz) and 4.37 (q, 2H, J = 7.2 Hz); 13C NMR (CDCl3) δ 14.2, 14.5, 16.2, 22.8,
27.9, 29.5, 29.56, 29.58, 29.7, 29.78, 29.83, 32.1, 37.0, 50.3, 62.8, 92.9, 161.1, 165.2 and
169.4; mass spectrum (FAB), m/z 482.2753 (M+H)+ (C25H45N3OBr requires m/z 482.2746).
4.1.17. 2-(Azetidin-1-yl)-4-ethoxy-6-hexadecylpyrimidin-5-ol (5)
To a stirred solution containing 400 mg (0.83 mmol) of 2-(azetidin-1-yl)-5-bromo-4-ethoxy-
6-hexadecylpyrimidine in 10 mL of anhydrous THF at –5 °C was added 663 µL (1.66 mmol)
of a 2.5 M solution of n-BuLi in hexane and 278 µL (2.49 mmol) of trimethoxyborane. The
reaction mixture was stirred at 23 °C for 30 min followed by the addition of 1.2 mL (18.3
mmol) of H2O2 (50% v/v). The reaction mixture was stirred for additional 30 min, poured into
20 mL NaHCO3 and then extracted with 100 mL of CH2Cl2. The organic phase was washed
with brine, dried (MgSO4) and concentrated under diminished pressure. The residue was
purified by chromatography on a silica gel column (15 × 3 cm). Elution with 95:5
hexane/EtOAc afforded 5 as a colorless powder: yield 250 mg (72%); mp 79-80 °C; silica gel
TLC Rf 0.33 (4:1 hexane/EtOAc); 1H NMR (CDCl3) δ 0.88 (t, 3H, J = 7.2 Hz), 1.19-1.39 (m,
29H), 1.63 (quint, 2H, J = 7.6 Hz), 2.26 (quint, 2H, J = 7.2 Hz), 2.61 (t, 2H, J = 7.6 Hz), 4.02
29