An alternative total synthesis of pentosidine

Article abstract of DOI:10.1002/jhet.611

Pentosidine, a fluorescent advanced glycation endproduct that serves as a biomarker of diabetic complications, kidney dysfunction, oxidative stress, and aging and age-related diseases, was synthesized from 2,3-diaminopyridine and benzyloxycarbonyl (Cbz) p

Full text of DOI:10.1002/jhet.611

426
An Alternative Total Synthesis of Pentosidine
Yahua Liu [1],* Weihan Zhang [2], and Lawrence M. Sayre†
Vol 48
Department of Chemistry, Case Western Reserve University, Cleveland, Ohio 44106
*E-mail: yahualiu@hotmail.com or yliu2@gnf.org
†Deceased
Received March 22, 2010
DOI 10.1002/jhet.611
Published online 21 January 2011 in Wiley Online Library (wileyonlinelibrary.com).
This work is dedicated to Professor Lawrence M. Sayre.
Pentosidine, a fluorescent advanced glycation endproduct that serves as a biomarker of diabetic com-
plications, kidney dysfunction, oxidative stress, and aging and age-related diseases, was synthesized
from 2,3-diaminopyridine and benzyloxycarbonyl (Cbz) protected chiral amino acids Na-Cbz-lysine and
Nd-Cbz-ornithine. Regioselective alkylation of 2-(methylthio)imidazo[4,5-b]pyridine, chlorination of
methylthio group, and amination of 2-chloro-imidazo[4,5-b]pyridine are the key steps. Hydantoin pro-
tection of amino acids was used and the deprotection under acidic condition was achieved in the pres-
ence of glycine.
J. Heterocyclic Chem., 48, 426 (2011).
INTRODUCTION
[11], alcohol misuse [12], and other drug abuse [13].
Because its characteristic fluorescence allows it to be
seen and measured easily, pentosidine has become an in-
dispensable biomarker in kidney dysfunction, end-stage
renal disease, diabetes, and diabetic complications
(including diabetic eye complications, such as diabetic
retinopathy), rheumatoid arthritis, bone collagen deterio-
ration, oxidative stress, and age-related diseases [14].
Pentosidine (1) is one of the fluorescent advanced
glycation endproducts (AGEs) that have been isolated
and structurally characterized [3]. It was reported that
pentosidine elevates in diabetes mellitus, and in patients
with end-stage renal failure and kidney dysfunction [4].
A significant elevation of pentosidine was found in
rheumatoid arthritis and in osteoarthritis as well [5].
Carbonyl stress, oxidative stress and aging progress the
formation and accumulation of pentosidine [6]. During
the aging process, pentosidine accumulates in the extrac-
ellular matrix and causes insolubility and protease resist-
ance of extracellular matrix proteins [7]. Pentosidine
level increases in disorders of pentose metabolism dur-
ing aging and in age-related diseases such as Alzhei-
mer’s disease [8]. Ophthalmology research shows that
pentosidine elevates in the aging vitreous body of eyes
and in diabetic retinopathy [9]. Emerging evidence
shows that pentosidine increases along with the deterio-
ration of bone quality in terms of collagen crosslink for-
mation in bone collagen and proteoglycan, and the stiff-
ness of the cartilage [10]. Recent research shows that
pentosidine levels may be associated with heart failure
In contrast to its extensive applications as a biomarker
in biological and clinical research, synthesis of pentosi-
dine has received little attention. Until now only two
synthetic approaches have been reported. The first syn-
thesis of pentosidine, reported by Monier and Sell, was
C
V
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An Alternative Total Synthesis of Pentosidine
427
Scheme 1
of pentosidine is shown in retrosynthetic format in
Scheme 1 and is centered on the construction of the key
intermediate imidazo[4,5-b]pyridine core 4. Pentosidine
(1) should be available by consecutive introduction of
amino acid subunit 3 (for lysine portion of pentosidine)
and subunit 5 (for arginine portion of pentosidine) to the
central piece 4 by a regioselective alkylation and an ami-
nation, respectively. We anticipated that the two subunits
3 and 5 could be obtained from chiral amino acids.
Chiral fragments developed from natural amino acids
have been used in many asymmetric syntheses of natural
products [18]. We envisioned that the amino acid subu-
nit 3 could be generated from lysine and the subunit 5
from onithine. To make amino acid subunit fragments
which could be incorporated through either an alkylation
reaction or an amination reaction, the a-amino and car-
boxy groups of the amino acids needed to be protected.
Of the several methods available for protecting amino
acids, we decided to employ hydantoin derivatization.
Hydantoins have been used as precursors of amino acids
in many syntheses and serve as important amino acid
surrogates for the production of pharmaceuticals (e.g.,
semisynthetic penicillins and cephalosporins), agrochem-
icals, and fine chemicals [19]. The conversion of hydan-
toins to amino acids could be achieved through hydroly-
sis [20,21] or biocatalysis [22]. Another reason for
selecting hydantoin protection is that our synthesis
involves some harsh reaction conditions, such as chlori-
nation in 12N HCl [17], and hydantoin is the only pro-
tecting group that can survive all the reaction conditions
involved in the synthesis.
achieved biochemically through incubation of ribose, ly-
sine, and arginine [3a,15]. The synthesis served to confirm
its structure and propose possible mechanism of its forma-
tion, but suffered complicated purification and extremely
low yield (0.02%). Shioiri and coworkers obtained 5.1 mg
of pentosidine trifluoroacetic acid salt (1a) using asym-
metric alkylation of a chiral Schiff base to provide a
lysine-like fragment, intramolecular guanylation with
mercury (II) chloride, and quaternization accompanied by
removal of the trityl group as key steps [16]. Because of
the increasing importance of pentosidine in biological and
clinical research, we have developed an alternate entry to
this biomarker. In this article, we describe the details of
our approach to this unique AGE crosslink.
As planned in Scheme 1, our synthesis began with the
preparation of the key intermediate imidazo[4,5-b]pyri-
dine core 4 (Scheme 2) [17]. The reaction of 2,3-diami-
nopyridine (7) with thiourea afforded 1H-imidazo[4,5-
b]pyridine-2(3H)-thione (6) [17,23] and S-methylation
of 6 gave 2-(methylthio)imidazo[4,5-b]pyridine (4) [17].
Scheme 3
RESULTS AND DISCUSSION
Based on the synthesis of pentosidine framework we
have previously reported [17], our plan for the synthesis
Scheme 2
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Y. Liu, W. Zhang, and L. M. Sayre
Vol 48
Scheme 4
Scheme 5
After we had prepared the key intermediate 4 and the
amino acid subunit fragments 12 and 17, their assembly
started from the alkylation of imidazo[4,5-b]pyridine
core 4 with iodide 12 in benzene in the presence of
Et3N to give pyridine N-alkylated imidazopyridine 18
[28] for pyridine nitrogen is more nucleophilic than the
imidazole nitrogens at positions 1 or 3 (Scheme 6) [28].
Compound 18 was then transformed into 2-chloroimida-
zopyridine 19 through chlorination in hydrochloric acid
[29]. The ornithine subunit fragment 17 was then intro-
duced through amination of chloride 19 in the presence
of Et3N, resulting in the dihydantoin-protected pentosi-
dine (20) [30].
It was conjectured that the preparation of the hydan-
toin protected lysine subunit fragment 12 could be ful-
filled by conversion of commercially available Ne-Cbz-
lysine (8) to the hydantoin compound 9 through Urech
hydantoin synthesis (Scheme 3) [24], followed by a key
conversion of 9 to 10 through diazotization and hydrox-
ylation [25], a nucleophilic substitution of alcohol 10 to
form 11 [26] and an alteration of chloride 11 into iodide
12 [27]. Although transformation of alkylamines into
alcohols through diazonium intermediates has been
reported to be achievable in good yield [25], the trans-
formation of 5-(4-aminobutyl)hydantoin (9) to 5-(4-
hydroxybutyl)-hydantoin (10) was not successful, with
recovery of unchanged amino compound 9.
Scheme 6
We chose an alternate strategy involving transforma-
tion of amino to hydroxy group prior to hydantoin
derivatization (Scheme 4) [25]. The conversion of Na-
Cbz-lysine (13) into alcohol 14 through diazotization
and hydroxylation was accomplished in good yield
[25]. L-6-hydroxynorleucine (15), obtained from 14 af-
ter deprotection, was then transformed into the hydan-
toin 10 through Urech hydantoin synthesis [24]. The
alcohol 10 was converted into the chloride 11 via a
nucleophilic substitution using SOCl2 [26], and then
into iodide 12 by refluxing with NaI in acetone [27]. It
is worth mentioning that although the yields of both
the conversion of 10 to 11 using thionyl chloride and
the conversion of 11 to 12 were moderate, the both
conversions could be forced to quantitative by pro-
longed refluxing (>168 h).
The amination reagent required for elaboration of the
arginine portion of pentosidine corresponds to the orni-
thine subunit fragment 17, which was prepared from Nd-
Cbz-ornithine (16) through Urech hydantoin synthesis
followed by the removal of Cbz protective group
(Scheme 5) [24].
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An Alternative Total Synthesis of Pentosidine
429
Scheme 7
[3a]. Only small amount of pentosidine formed during
hydrolysis of 20 when glycine was absent. We still do
not know how glycine facilitates the hydrolysis, a
hydantoin change reaction could be a plausible explana-
tion [32]. Purification of crude HCl salt of pentosidine
(1b) with a mixed eluent of CH3OH/H2O/CH3CO2H
afforded its CH3CO2H salt (1c). Comparison of NMR,
FAB-MS, and LC-MS data of our synthesized 1c with
the pentosidine CF3CO2H salt (1a) isolated by Sell and
Monnier showed the consistency of synthesized and iso-
lated compounds [3a]. We have also noted that the spe-
cific rotation value of our pentosidine CH3CO2H salt
(1c) is [a]D24 ¼ þ11.5ꢀ (c 0.2, CH3OH), which differs
from the value [a]D24 ¼ þ16.5ꢀ (c 0.3, CH3OH) of its
CF3CO2H salt (1a) [15]. Considering UV and fluores-
cent properties of pentosidine are highly dependent on
pH value [3,17], the difference of specific rotation val-
ues could be attributed to the different counterions in
the two salt forms.
While pentosidine 1 could be obtained from the syn-
thesis shown in Scheme 6, evidence for the regiochem-
istry of alkylation in 18, and for structural assignment
of 19, 20, and 1 that were obtained in subsequent steps,
was not fully discussed. We assigned 18 as pyridine N-
alkylated product on the basis of the regiochemistry of
the alkylation reaction used to synthesize pentosidine
framework [17]. To provide conclusive evidence for our
structural assignment of 18, 19, 20, and 1, an unambig-
uous synthesis was used (Scheme 7). The regiochemis-
try of intermediate 18 in Scheme 6 was proven through
comparison of its NMR spectrum to the spectrum of 18
obtained according to Scheme 7. In Scheme 7, direct
alkylation of diaminopyridine (7) with iodide 12 gave a
mixture of two isomeric products 21 and 22 [33]. The
distinction of 22 from 21 could be made on the basis
of both proton chemical shift and characteristic cou-
plings. The chemical shift for the methylene group
neighboring N in 21 is d 3.17, but d 4.22 in the case of
22. Transformation of the pyridine N-alkylated isomer
22 afforded intermediate 18 [17,23,34]. The consistency
of NMR spectra for the two individual samples of 18,
and the NMR spectrum of a mixture of the two samples
(in 1:1 ratio), added conclusive evidence that imidazo-
pyridine 18 obtained through this route is identical with
the product obtained from the synthesis shown in
Scheme 6.
Deprotection of hydantoins to amino acids could be
achieved through basic hydrolysis [20], acidic hydrolysis
[21], and biocatalytic means [22]. Although hydrolysis
of the hydantoin protecting group under basic conditions
is straightforward [20], it can lead to unwanted racemi-
zation. Acidic hydrolysis is favored because hydantoins
and amino acids do not undergo racemization under
such conditions [21]. The biocatalytic conversion of
hydantoins to amino acids has been recognized in recent
decades for potential application in the industrial pro-
duction of amino acids [22]. The biocatalysis reaction
involves two consecutive hydrolysis steps, catalyzed by
both a hydantoinase and an N-carbamoylamino acid
amidohydrolase (NCAAH). During the process, an L-
selective hydantoinase converts a 5-monosubstituted
hydantoin to an L-N-carbamoylamino acid and an L-
selective NCAAH then converts the N-carbamoylamino
acid to an L-amino acid. It seemed to us that the bioca-
talytic conversion is the most favored for the deprotec-
tion of dihydantoin-protected pentosidine 20.
Because our work was halted due to the death of Pro-
fessor Sayre and the consequent laboratory closing [31],
we were unable to explore the preparation of amino
acids via biocatalysis with hydantoinase and NCAAH.
Prior to that we found removal of the hydantoin protect-
ing groups required refluxing in 6N HCl in the presence
of glycine. Pentosidine is stable in concentrated acid
and was first obtained after refluxing in 6N HCl for 36 h
In summary, pentosidine, an important biological
marker for diabetic complications and oxidative damage
has been synthesized. The regiochemistry of the targets
and intermediates was established by an unambiguous
synthesis. Our approach to the biomarker is more effi-
cient than those reported in literature, especially in
obtaining lysine subunit fragment through a procedure
with fewer steps. The hydantoin-protected pentosidine
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Y. Liu, W. Zhang, and L. M. Sayre
Vol 48
H2O ¼ 4:1, 40.0 mL), neutralized to pH 7.5 with 2N NaOH
solution, and concentrated. The resulting crude was subjected
to chromatography (DCM/MeOH ¼ 1:5) to provide 15 (2.40
g, 82%): 1H-NMR (D2O) d 1.46 (m, 2H), 1.62 (m, 2H),
1.91(m, 2H), 3.64 (t, J ¼ 6.3 Hz, 2H), 3.76 (t, J ¼ 4.1 Hz,
1H); 13C-NMR (D2O) d 21.8 (þ), 31.2 (þ), 31.9 (þ), 55.7
(ꢁ), 62.2 (þ), 175.7 (þ); [a]D20 ¼ þ 6.35ꢀ (c 0.2, H2O);
HRMS calcd for C7H12N2O3 (Mþ) 147.0895, found 172.0889.
5-(4-Hydroxybutyl)hydantoin (10). A mixture of L-6-
hydroxynorleucine (15) (2.38 g, 16.23 mmol) and potassium
cyanate (1.76 g, 22.0 mmol) in water (100 mL) was stirred at
90ꢀC for 2 h, then cooled to room temperature, acidified by
adding 12N HCl (100.0 mL), and then stirred at 95ꢀC for 30
min. The reaction mixture was concentrated, diluted with a
mixed solvent of (MeOH/ H2O ¼ 4:1, 60.0 mL), neutralized to
pH 7.5 with 2N NaOH solution, and concentrated to give a
residue which was subjected to chromatography (CH2Cl2/
provides a platform for the future research on biocataly-
sis to generate pentosidine enantiomerically.
EXPERIMENTAL
All commercial reagents and solvents were used without pu-
rification. 1H-NMR (300 MHz) and 13C-NMR (75.1 MHz) were
recorded on Varian Gemini 300 or Gemini 200 instruments
using TMS as internal standard. In the 13C-NMR data, attached
proton test (APT) designations are given as (þ) or (ꢁ) follow-
ing the chemical shift. High-resolution mass spectra (HRMS)
were obtained at 20 eV on a Kratos MS-25A instrument. TLC
analysis was carried out on silica gel 60 F254 precoated alumi-
num sheets, and UV light was used for visualization. Flash col-
umn chromatography was done using E. Merck silica gel 60
(230–400 mesh). Solvent removal was accomplished with a ro-
tary evaporator operating at vacuum (40–50 Torr).
1H-imidazo[4,5-b]pyridine-2(3H)-thione (6). A mixture of
2,3-diaminopyridine (3) (2.18 g, 20.0 mmol) and thiourea (7.6
g, 100.0 mmol) was heated at 170 ꢀC for 5 h. The reaction
mixture was extracted with boiling ethanol (5 ꢂ 20 mL), leav-
ing crystals that were collected by filtration. A second crop of
crystals was obtained from the cooled filtrate after standing
overnight. The combined yield of pure 6 was 2.7 g (89%). 6:
1H-NMR (DMSO-d6) d 7.13 (dd, J ¼ 7.9, 5.0 Hz, 1H), 7.46
(d, J ¼ 7.9 Hz, 1H), 8.09 (d, J ¼ 5.0 Hz, 1H), 12.70 (s, 1H,
NH), 13.12 (s, 1H, NH); 13C-NMR (DMSO-d6) d 116.2 (ꢁ),
118.1 (ꢁ), 125.4 (þ), 142.3 (ꢁ), 146.4 (þ), 169.8 (þ). HRMS
calcd for C6H5N3S (Mþ) 151.0204, found 151.0204.
2-(Methylthio)-1H-imidazo[4,5-b]pyridine (4). A solution
of 6 (2.26 g, 15.0 mmol) and methyl iodide (8.52 g, 60.0
mmol) in MeOH (30.0 mL) was stirred at room temperature
for 18 h. Evaporation of the solvent and CH3I under reduced
pressure gave pure 4 (3.32 g, 100 %): 1H-NMR (CD3OD) d
2.75 (s, 3H), 7.20 (dd, J ¼ 7.9, 4.9 Hz, 1H), 7.82 (d, J ¼ 7.9
Hz, 1H), 8.21 (d, J ¼ 4.9 Hz, 1H); 13C-NMR (CD3OD) d 15.0
(ꢁ), 119.1 (ꢁ), 127.4 (ꢁ), 134.1 (þ), 134.5 (ꢁ), 150.3 (þ),
166.4 (þ); HRMS calcd for C7H7N3S (Mþ) 165.0361, found
165.0363.
1
CH3OH ¼ 6:1) to afford 10 (2.68 g, 96%): H-NMR (CD3OD)
d 1.40–1.88 (m, 6H), 3.56 (t, J ¼ 6.4 Hz, 2H), 4.09 (dd, J ¼
6.9, 5.1 Hz, 1H); 13C-NMR (CD3OD) d 22.1 (þ), 32.5 (þ),
33.2 (þ), 59.8 (ꢁ), 62.6 (þ), 160.0 (þ), 178.3 (þ); HRMS
calcd for C7H12N2O3 (Mþ) 172.0848, found 172.0868.
5-(4-Chlorobutyl)hydantoin (11). A solution of 10 (2.66 g,
15.45 mmol) in thionyl chloride (100 mL) was heated at reflux
for 18 h and evaporated to result in a residue which was
diluted with a mixed solvent of (MeOH/ H2O ¼ 4:1, 40.0
mL), neutralized to pH 7.5 with 2 N NaOH solution, and con-
centrated. The residual crude was subjected to chromatography
(CH2Cl2/CH3OH ¼ 6:1) to give 11 (1.83 g, 62%): 1H-NMR
(CD3OD) d 1.42 (m, 2H), 1.58 (m, 2H), 1.81 (m, 2H), 3.57 (t,
J ¼ 6.4 Hz, 2H), 4.10 (dd, J ¼ 6.9, 5.1 Hz, 1H); 13C-NMR
(CD3OD) d 23.1 (þ), 32.0 (þ), 33.3 (þ), 45.5 (þ), 59.7 (ꢁ),
160.0 (þ), 178.1 (þ); HRMS calcd for C7H11N2O2Cl (Mþ)
190.0509, found 190.0507.
5-(4-Iodobutyl)hydantoin (11). A solution of 5-(4-chloro-
butyl)hydantoin (11) (1.79 g, 9.40 mmol) and NaI (4.68 g,
31.2 mmol) in acetone (100 mL) was refluxed for 18 h and
then concentrated to give a residue which was subjected to
chromatography (CH2Cl2/CH3OH ¼ 9:1) to afford 12 (1.78 g,
1
67%): H-NMR (CD3OD) d 1.51 (m, 2H), 1.70 (m, 2H), 1.82
(m, 2H), 3.23 (t, J ¼ 6.9 Hz, 2H), 4.29 (dd, J ¼ 6.7, 4.7 Hz,
1H); 13C-NMR (CD3OD) d 6.4 (þ), 26.6 (þ), 31.5 (þ), 34.3
(þ), 59.7 (ꢁ), 160.0 (þ), 178.1 (þ); FAB HRMS calcd for
C7H11IN2NaO2 ((M þ Na)þ 304.9763, found 304.9763.
5-(4-Aminobutyl)hydantoin hydrochloride (9). A mixture
of Ne-Cbz-L-lysine 8 (841.0 mg, 3.0 mmol) and potassium cy-
anate (260.0 mg, 3.20 mmol) in water (20.0 mL) was stirred at
95ꢀC for 2 h, then cooled to room temperature, acidified by
adding 12N HCl (20.0 mL), and then stirred at 95ꢀC for 1 h.
Evaporation to dryness under reduced pressure afforded crude
product 9 which was sufficiently pure for the next reaction
5-(3-Aminopropyl)hydantoin (17). A suspension of Nd-
Cbz-L-ornithine (16) (266.3 mg, 1.0 mmol) and potassium cy-
anate (87.0 mg, 1.07 mmol) in water (10 mL) was stirred at
95ꢀC for 2 h, then cooled to room temperature, acidified by
adding 12N HCl (20 mL), then stirred at 95 ꢀC for 1 h. The
reaction mixture was concentrated, diluted with a mixed sol-
vent of (MeOH/ H2O ¼ 4:1, 20.0 mL), neutralized to pH 7.5
with 2N NaOH solution, and concentrated to afford a residue
which was subjected to chromatography (CH2Cl2/MeOH ¼
1
step: H-NMR (D2O) d 1.46 (m, 2H), 1.70 (m, 2H), 1.84 (m,
2H), 2.99 (t, J ¼ 7.5 Hz, 2H), 4.29 (dd, J ¼ 6.3, 4.8 Hz, 1H);
13C-NMR (D2O) d 20.9 (þ), 26.5 (þ), 30.0 (þ), 39.3 (þ),
58.6 (ꢁ), 159.4 (þ), 178.9 (þ); FAB HRMS calcd for
þ
C7H14N3O2 (M þ H) 172.1086, found 172.1093.
L-6-Hydroxynorleucine (15). To a solution of Na-Cbz ly-
sine (13) (5.60 g, 20.0 mmol) in a mixed solvent of acetic acid
(20.0 mL) and water (4.0 mL) was added a solution of NaNO2
(2.76 g, 40.0 mmol) in water (4.0 mL) at 0ꢀC dropwise. The
mixture was stirred at room temperature for 1 h, at 90ꢀC for
30 min, and then cooled to room temperature. After an aque-
ous 6N HCl (120.0 mL) was added, the reaction mixture was
stirred at 90ꢀC for another 30 min, and evaporated to give a
residue which was diluted with a mixed solvent of (MeOH/
1
3:2) to provide 17 (151 mg, 96%): H-NMR (CD3OD) d 1.75–
1.93 (4H), 3.00 (t, J ¼ 6.7 Hz, 2H), 4.70 (dd, J ¼ 6.3, 4.7 Hz,
1H); 13C-NMR (D2O) d 22.7 (þ), 28.0 (þ), 39.7 (þ), 58.6
(ꢁ), 159.5 (þ), 178.5 (þ); HRMS calcd for C6H11N3O2 (Mþ)
157.0851, found 157.0835.
4-[4-(5-Hydantoin)butyl]-2-methylthio-4H-imidazo[4,5-b]
pyridine (18). A suspension of 5-(4-iodobutyl)hydantoin (12)
(1.69 g, 6.0 mmol) and 2-(methylthio)-1H-imidazo[4,5-
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An Alternative Total Synthesis of Pentosidine
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b]pyridine (4) (495.1 mg, 3.0 mmol) in benzene (60 mL) with
triethylamine (0.5 mL, 3.6 mmol) was stirred and heated in an
oil bath (90ꢀC) for 40 h. After removing the solvent under
reduced pressure, the residue was dissolved in a mixture of
MeOH (20 mL) and H2O (20 mL) and adjusted to pH 9.5.
Upon evaporation of the solvent under reduced pressure, flash
chromatography (EtOAc/MeOH ¼ 3:1) of the residue afforded
(t, J ¼ 7.1 Hz, 2H), 7.22 (dd, J ¼ 7.7, 6.5 Hz, 1H), 7.78 (d, J
¼ 7.7 Hz. 1H), 7.93 (d, J ¼ 6.5 Hz, 1H); 13C-NMR d 21.0(þ)
(CH3COOꢁ), 22.0 (þ), 25.1(þ), 28.3(þ), 28.8(þ), 30.5(þ),
39.5(þ), 42.6(þ), 53.7(ꢁ), 53.8(ꢁ), 115.8(ꢁ), 120.2(ꢁ),
132.4(þ), 132.7(ꢁ), 152.2(þ), 160.3(þ), 171.7(þ), 172.3(þ),
177.0(ꢁ) (CH3COOꢁ); FAB HRMS calcd for C17H27N6O4 (M
þ 1)þ 379.2094, found 379.2083.
1
2-Amino-3-[4-(5-hydantoin)butyl]aminopyridine (21) and
3-amino-1-[4-(5-hydantoin)butyl]-2-pyridone imine (22). A
mixture of 7 (436.0 mg, 4 mmol) and 12 (1.35 g, 4.8 mmol)
in EtOH (16.0 mL) was stirred at 100ꢀC in a high pressure
reaction vessel for 40 h, and then concentrated under reduced
pressure to result in a residue which was diluted with a mixed
solvent of MeOH (30 mL) and H2O (30 mL), basified to pH
10 with 2N aqueous solution of NaOH, and concentrated. The
residual crude was subjected to chromatography (EtOAc/
MeOH ¼ 4:1) to afford 21 (180.1 mg, 17% yield) and 22
(84.2 mg, 8%). 21: 1H-NMR (CD3OD) d 1.55 (m, 2H), 1.75
(m, 2H), 1.85 (m, 2H), 3.17 (t, J ¼ 6.7 Hz, 2H), 4.12 (dd, J
¼ 6.2, 5.6 Hz, 1H), 6.77 (dd, J ¼ 7.7, 6.0 Hz, 1H), 6.91 (d, J
¼ 7.7 Hz, 1H), 7.21 (d, J ¼ 5.9 Hz, 1H); 22: 1H-NMR
(CD3OD) d 1.50 (m, 2H), 1.75 (m, 2H), 1.85 (m, 2H), 4.12
(dd, J ¼ 6.2, 5.6 Hz, 1H), 4.22 (t, J ¼ 7.3 Hz, 2H), 6.77 (dd,
J ¼ 7.2, 6.4 Hz, 1H), 7.10 (d, J ¼ 7.2 Hz, 1H), 7.34 (d, J ¼
6.3 Hz, 1H).
18 (584.0 mg, 61%): H-NMR (CD3OD) d 1.45 (m, 2H), 1.75
(m, 2H), 1.92 (m, 2H), 2.67 (s, 3H), 4.09 (dd, J ¼ 6.8, 4.6 Hz,
1H), 4.53 (t, J ¼ 7.2 Hz, 2H), 7.09 (dd, J ¼ 7.7, 6.7 Hz, 1H),
7.89 (d, J ¼ 6.7 Hz, 1H), 7.91 (d, J ¼ 7.7 Hz, 1H); 13C-NMR
(CD3OD) d 14.6 (ꢁ), 22.9 (þ), 30.2 (þ), 32.1 (þ), 54.8 (þ),
59.5 (ꢁ), 114.6 (ꢁ), 125.6 (ꢁ), 131. 4 (ꢁ), 145.3 (þ), 154.3
(þ), 159.9 (þ), 173.1 (þ), 178.0 (þ); FAB HRMS calcd for
C14H18N5O2S (Mþ1)þ 320.1181, found 320.1175.
4-[4-(5-Hydantoin)butyl]-2-chloro-4H-imidazo[4,5-b]pyr-
idine (19). Chlorine was introduced into a solution of 18
(200.0 mg, 0.62 mmol) in 12N HCl (60 mL) with stirring at
0ꢀC for 18 h. After evaporation to dryness under reduced pres-
sure, the residue was dissolved in water (20 mL) and neutral-
ized to 7.8 with NH4OH. Water was removed under reduced
pressure and the residue was extracted with CH2Cl2/MeOH
(8:1). The organic extract was dried (Na2SO4) and evaporated
to give a residue, which was subjected to chromatography
(CH2Cl2/CH3OH¼ 8:1) to furnish 19 (151.0 mg, 79%): 1H-
NMR (CD3OD) d 1.49 (m, 2H), 1.79 (m, 2H), 2.00 (m, 2H),
4.09 (dd, J ¼ 6.9, 4.7 Hz, 1H), 4.64 (t, J ¼ 7.5 Hz, 2H), 7.32
(dd, J ¼ 7.8, 6.3 Hz, 1H), 8.16 (d, J ¼ 7.9 Hz, 1H), 8.19 (d, J
¼ 6.3 Hz, 1H); 13C-NMR (CD3OD) d 22.6 (þ), 30.3 (þ), 32.0
(þ), 55.3 (þ), 59.5 (ꢁ), 116.0 (ꢁ), 129.7 (ꢁ), 134.1 (ꢁ),
143.9 (þ), 152.6 (þ), 159.7 (þ), 159.9 (þ), 178.0 (þ); FAB
HRMS calcd for C13H15N5O2Cl (Mþ1)þ 308.0914, found
308.0917.
Preparation of 18 from 22. A mixture of 22 (71.8 mg,
0.27 mmol) in EtOH (4 mL) and CS2 (4 mL) was stirred at
80ꢀC in for 18 h and concentrated under reduced pressure. The
resulting residue was mixed with a solution of methyl iodide
(428 mg, 3 mmol) in MeOH (2 mL), stirred at room tempera-
ture for 18 h, evaporated to dryness under reduced pressure.
The crude residue was dissolved in MeOH (10 mL) and H2O
(10 mL), basified to pH 9.5 with 2N aqueous solution of
NaOH, and concentrated to furnish a residue which was sub-
jected to chromatography (EtOAc/MeOH ¼ 3:1) to furnish 18
4-[4-(5-Hydantoin)butyl]-2-[3-(5-hydantoin)propylamino]-
4H-imidazo[4,5-b]pyridine (20). A suspension of 19 (138.4
mg, 0.48 mmol) and 17 (151.0 mg, 0.96 mmol) in benzene (20
mL) with triethylamine (0.334 mL, 242 mg g, 2.40 mmol) was
stirred and heated in an oil bath (90ꢀC) for 18 h. After remov-
ing solvent under reduced pressure, the residue was dissolved
in MeOH (20 mL) and H2O (20 mL) and adjusted to pH 9.5.
Concentration under reduced pressure left behind a residue
which was purified by chromatography (CH2Cl2/MeOH ¼ 3:1)
to provide 24 (117.2 mg, 57%): 1H-NMR (CD3OD) d 1.40–
1.98 (10H), 3.46 (t, J ¼ 4.9 Hz, 2H), 4.08 (dd, J ¼ 6.8, 5.2
Hz, 1H), 4.15 (dd, J ¼ 5.9, 4.7 Hz, 1H), 4.41 (t, J ¼ 4.9 Hz,
2H), 6.89 (dd, J ¼ 6.4, 5.9 Hz, 1H), 7.42 (d, J ¼ 5.9 Hz, 1H),
7.54 (d, J ¼ 6.4 Hz, 1H); 13C-NMR (DMSO-d6) d 21.4 (þ),
25.1 (þ), 28.2 (þ), 28.9 (þ), 30.7 (þ), 41.6 (þ), 51.8 (þ),
57.5 (2C) (ꢁ), 110.6 (ꢁ), 115.4 (ꢁ), 124.9 (ꢁ), 145.9 (þ),
155.5 (þ), 157.6 (2C) (þ), 170.2 (þ), 176.1 (þ), 176.2 (þ)
(two peaks are missing due to overlap); FAB HRMS calcd for
C19H25N8O4 (M þ 1)þ 429.2000, found 429.2008.
1
(13.8 mg, 16%). H-NMR, 13C-NMR, and HRMS are identical
with those of 18 prepared from 4 and 15.
Acknowledgments. The authors thank the National Institutes of
Health for support of this work through Grants HL 53315 and AG
14249. Case Western Reserve University is acknowledged for
supporting an alumini research fellowship for Y.L. (1998). They
also thank Dr. D. R. Sell and Professor V. M. Monnier for helpful
discussions and for their assistance in obtaining LC-MS data.
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[2] Current Address: Discovery Chemistry, Hutchison Medi-
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