An expeditious synthesis of pentosidine, an advanced glycation end product

Article abstract of DOI:10.1016/S0040-4020(01)00399-4

The chemical synthesis of pentosidine (1), an advanced glycation end product, was achieved via the asymmetric alkylation of the chiral schiff base derived from (+)-2-hydroxy-3-pinanone ((+)-HyPN) and glycine tert-butyl ester, the mercury salt mediated intramolecular guanylation, and the regioselective alkylation of imidazo[4,5-b]pyridine ring. This reliable synthetic achievement will promise availability of pentosidine (1) in quantities.

Full text of DOI:10.1016/S0040-4020(01)00399-4

TETRAHEDRON
Pergamon
Tetrahedron 57 (2001) 4759±4766
An expeditious synthesis of pentosidine,
an advanced glycation end product
Fumiaki Yokokawa,a Hideyuki Sugiyama,a Takayuki Shioiri,a,p Noriko Katagiri,b Osamu Odab
and Hiroshi Ogawab,c
aDepartment of Synthetic Organic Chemistry, Faculty of Pharmaceutical Sciences, Nagoya City University, Tanabe-dori, Mizuho-ku,
Nagoya 467-8603, Japan
bThe Bio-Dynamics Research Institute, 1-3-2 Tamamitsu-cho, Mizuho-ku, Nagoya 467-8633, Japan
cShinseikai Dai-Ichi Hospital, 1-3-2 Tamamitsu-cho, Mizuho-ku, Nagoya 467-8633, Japan
Received 19 December 2000; revised 13 March 2001; accepted 30 March 2001
AbstractÐThe chemical synthesis of pentosidine (1), an advanced glycation end product, was achieved via the asymmetric alkylation of the
chiral schiff base derived from (1)-2-hydroxy-3-pinanone ((1)-HyPN) and glycine tert-butyl ester, the mercury salt mediated intramolecular
guanylation, and the regioselective alkylation of imidazo[4,5-b]pyridine ring. This reliable synthetic achievement will promise availability of
pentosidine (1) in quantities. q 2001 Elsevier Science Ltd. All rights reserved.
1. Introduction
products, termed AGEs (advanced glycation end products).
The features of AGEs are ¯uorescent, browning, and highly
reactive with nearby amino groups to produce both intra and
intermolecular crosslinks. AGEs increase in collagen and
lens proteins with age and the accelerated accumulation of
these products in tissues of diabetic patients, particularly
of patients with complications.3 Accordingly, AGEs are
expected to be clinically useful marker for diabetic compli-
cations, and some attempt to build up the antibodies of
AGEs are performed.
Diabetes has been estimated to af¯ict more than one
hundred million people in the world, and the contribution
of the diabetic complications to the overall mortality of
heart disease and stroke is a particularly signi®cant
problem.1 The reaction between reducing sugars and
amino structures in amino acids or proteins (also called
Maillard reaction) has been shown to proceed in living
systems and to have a role in pathophysiology of aging
and diabetic complications.2 As shown in Scheme 1, the
Maillard reaction is initiated by the formation of a schiff
base adduct between the carbonyl and the amine moiety,
and then the aldimine rearranges to a more stable ketoamine
(Amadori product). Subsequently, enolization, dehydration,
cyclization, fragmentation, and oxidation reactions form
reactive intermediates that ultimately lead to stable end-
Since enormous efforts were devoted to elucidate the nature
of these protein modi®cations, so far some structures of
AGEs have been established as shown in Fig. 1.4
Pentosidine (1) is one of the AGEs and was discovered as
a ¯uorescent protein crosslink isolated from the human
Keywords: pentosidine; an advanced glycation end product; asymmetric alkylation; imidazo[4,5-b]pyridine; quaternization.
Corresponding author. Tel.: 181-52-834-4172; fax: 181-52-834-4172; e-mail: shioiri@phar.nagoya-cu.ac.jp
p
0040±4020/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(01)00399-4

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F. Yokokawa et al. / Tetrahedron 57 (2001) 4759±4766
can be disconnected into alkyl iodide (fragment A) and
imidazopyridine fragment (fragment B).7 Because Yutilov
has reported that the alkylation of imidazo[4,5-b]pyridine
with methyl iodide predominantly occurred at the nitrogen
of pyridine ring,8 the regioselective alkylation of fragment B
with fragment A can be expected. The unusual amino acid
fragment A can be obtained by the asymmetric alkylation of
the chiral schiff base 2 derived from (1)-2-hydroxy-3-pina-
none ((1)-HyPN)9 with the allyl iodide 3. Fragment B can
be synthesized by the intramolecular guanylation of the
amino thiourea 4, which is obtained by coupling of 2,3-
diaminopyridine (5) with the isothiocyanate 6 derived
from ornithine.
2.2. Synthesis of fragment A
The preparation of fragment A is shown in Scheme 3. We
have previously demonstrated the asymmetric alkylation of
a chiral schiff base derived from (1)- or (2)-HyPN, and
applied the natural product synthesis.9 Therefore, we
employed this methodology for the preparation of fragment
A. Although we initially attempted the alkylation of the
chiral schiff base 29 with the benzyl protected alkyl iodide
7, no alkylated product 8 was obtained due to low reactivity
of the alkyl iodide 7. Accordingly, we next tried to use the
allyl iodide 3 in order to increase the reactivity of electro-
phile. Monoprotection of the hydroxyl function of commer-
cially available 9 with benzyl bromide,10 followed by
iodination of the free hydroxy group afforded the labile
iodide 3, which was immediately used for the alkylation.
Deprotonation of the chiral schiff base 2 with lithium diiso-
propylamide (LDA) followed by the addition of the iodide 3
gave the imine 11 in 87% yield. After removal of the chiral
auxiliary under mild acidic conditions, protection of the
resulting amino group with Boc2O provided the amino
ester 12 in 80% yield. Simultaneous hydrogenation of the
double bond and deprotection of the benzyl group were
carried out under hydrogen in the presence of a catalytic
amount of palladium on carbon to give the alcohol 13 in
77% yield. The enantiomeric excess of the alcohol 13 was
determined to be 92% based on HPLC analysis of the corre-
sponding MTPA ester 14. Conversion of the primary
alcohol group to the iodide was achieved with iodine and
triphenylphosphine to give fragment A in 99% yield.11
Figure 1. Proposed AGEs compounds.
extracellar matrix by Monnier et al. in 1989.5 Pentosidine
(1) contains lysine and arginine as side chains connected to
an imidazo[4,5-b] pyridine ring. The special signi®cance is
the attachment of lysine as a quaternary ammonium salt at
the 4-position on the imidazo[4,5-b]pyridine ring. While
pentosidine (1) has already been synthesized by just mixing
ribose, lysine and arginine like biosynthesis, several compli-
cated puri®cation steps were necessary and the overall yield
was also very low (0.23%).5 Currently, pentosidine (1) is
available in very low yield at a high cost by this method.
Therefore, pentosidine (1) is an attractive synthetic target
due to its novel structural features and practical applications
in diabetic complications. In this paper, we describe a reli-
able and ef®cient total synthesis of pentosidine (1).6
2. Results and discussion
2.1. Retrosynthesis of pentosidine
Our synthetic plan is shown in Scheme 2. Pentosidine (1)
Scheme 2. Retrosynthetic analysis of pentosidine.

F. Yokokawa et al. / Tetrahedron 57 (2001) 4759±4766
4761
Scheme 3. Synthesis of fragment A (a) NaH (0.5 equiv.), BnBr (0.5 equiv.), DMF, rt, 1 h, 36%. (b) I2, Ph3P, imidazole, PhH, rt, 0.5 h. (c) LDA, THF, 2788C,
then, 3, 2788C, 2 h, 87%. (d) 15% citric acid/THF, rt. (e) Boc2O, dioxane, rt, 80% in 2 steps. (f) H2, 5% Pd/C, EtOAc, rt, 77%. (g) I2, Ph3P, imidazole, PhH, rt,
0.5 h, 99%.
2.3. Synthesis of fragment B
ring of fragment B with alkyl iodide was problematic. More-
over, alkylation of fragment B with fragment A did not
proceed at all. To overcome this problem we attempted to
introduce the electron donating group at the 1-position of
the imidazole moiety for activation of the imidazo[4,5-b]-
pyridine ring and regioselective quaternization at the pyri-
dine ring.15 The group we chose was the trityl group.
The synthesis of fragment B is summarized in Scheme 4.
After Boc-l-Orn(Z)-OH (15) was converted to the tert-butyl
ester 16 with O-tert-butyl-N,N0-diisopropyl isourea,12
deprotection of the carbobenzyloxy (Z) group gave the
amine 17. Treatment of the amine 17 with triethylamine,
carbondisul®de and 30% aqueous hydrogen peroxide13
afforded the isothiocyanate 6 in 87% yield (2 steps).
Under careful optimized reaction conditions, coupling of 6
with 2,3-diaminopyridine (5) in the presence of triethyl-
amine cleanly proceeded to give the thiourea 4, which
was converted to fragment B having the imidazo[4,5-b]-
pyridine ring by intramolecular guanylation with lead(II)
acetate trihydrate.14 Alternatively, treatment of 4 with
methyl iodide gave fragment B accompanied by the unde-
sired product 18, which was obtained by additional methyl-
ation at the 1-position of the imidazole ring.14 This result
suggested that regioselective quaternization at the pyridine
2.4. Total synthesis of pentosidine
As shown in Scheme 5, monotritylation of 5 followed by
coupling with the isothiocyanate 6 gave the labile thiourea
20. Intramolecular guanylation of 20 by mercury(II)
chloride16 rapidly proceeded to afford the trityl protected
imidazo[4,5-b]pyridine derivative 21 in 54% yield from
19. As expected, the quaternization of 21 with fragment A
proceeded with concomitant deprotection of the trityl group
to provide the quaternary salt 22 in 81% yield.17 Finally, the
cleavage of all the protective groups of 22 by treatment with
Scheme 4. Synthesis of fragment B (a) O-tert-butyl-N,N0-diisopropyl isourea, t-BuOH, CH2Cl2, 518C, 20 h, quant. (b) H2, 5% Pd/C, EtOAc, rt, 12 h. (c) Et3N,
CS2, THF, 08C, 40 min. then, 30% aq. H2O2, 08C, 87% in 2 steps. (d) 5, Et3N, THF, re¯ux, 14 h, 76%. (e) Pb(OAc)2´3H2O, Et3N, MeOH, re¯ux, 2d, 80%.
(f) CH3I, Et3N, MeOH, re¯ux, 2 d, 35% and 18, 41%.

4762
F. Yokokawa et al. / Tetrahedron 57 (2001) 4759±4766
Scheme 5. Total synthesis of pentosidine (a) TrCl, Et3N, THF, rt, 1 h, 58%. (b) 6, Et3N, THF, re¯ux, 4 d. (c) HgCl2, Et3N, MeOH, 08C, 10 min, 54% in 2 steps.
(d) Fragment A, THF, re¯ux, 48 h, 81%. (e) TFA/CHCl3, rt, 11 h, quant.
tri¯uoroacetic acid (TFA) furnished pentosidine (1) as its
TFA salt quantitatively. HPLC puri®cation of the crude
product afforded pure pentosidine (1) in 96% yield. The
synthetic pentosidine obtained by this procedure was iden-
ketyl. Dichloromethane (CH2Cl2) was dried by distillation
from calcium hydride. Other solvents were dried and stored
Ê
over molecular sieves (3 or 4 A).
1
tical to the authentic sample18 based on several criteria: H
3.1.1. (2EZ)-4-Benzyloxy-2-butenol (10). To a stirred
suspension of NaH (60% oil dispersion, 200 mg,
5.0 mmol) in DMF (20 mL) was added dropwise a solution
of cis and trans-2-butene-1,4-diol (9) (881 mg, 10 mmol) in
DMF (10 mL) at 2108C under argon. The mixture was
stirred at 2108C for 1 h under argon, and benzyl bromide
(0.6 mL, 5 mmol) was added dropwise to the mixture at
2108C. After stirring at 2108C for 1 h, the reaction was
quenched with 1 M aqueous KHSO4 (150 mL). After salt-
ing-out, the mixture was extracted with Et2O (50 mL£4).
The extracts were washed with saturated brine, and dried
over Na2SO4. Filtration and concentration in vacuo gave the
crude residue, which was puri®ed by column chroma-
tography (silica gel BW-820MH, 150 g, hexane±EtOAcˆ
10:1 to 1:1) to give the monobenzylated product 10
(645 mg, 36%) as a colorless oil and the dibenzylated
product (192 mg, 7%) as a colorless oil.
NMR, 13C NMR, FAB-HRMS, and HPLC.
In conclusion, a reliable and ef®cient total synthesis of
pentosidine (1) was developed from commercially available
compounds. According to our strategy, we have synthesized
approximately 1 g of pentosidine (1). Our synthetic pento-
sidine is currently employed for the biochemical investi-
gations.
3. Experimental
3.1. General information
Melting points were determined on a YANAGIMOTO
micro melting point apparatus (hot plate) and uncorrected.
Infrared (IR) spectra were measured with a SHIMADZU
FTIR-8100 spectrometer. Optical rotations were measured
on a JASCO DIP-1000 digital polarimeter with sodium
lamp (lˆ589 nm, D line) and were recorded as follows:
Monobenzylated product 10: IR nmaxneat cm21: 3390, 1496,
1454, 1095. 1H NMR (CDCl3) d: 1.60 (1H, brt, disappeared
with D2O, OH), 4.05 (2H, dd, Jˆ4.5, 6.0 Hz,
CHvCHCH2OH), 4.18 (2H, d, Jˆ4.5 Hz, CH2OBn), 4.53
(2H,s£2, CH2C6H5), 5.70±6.00 (2H, m, CHvCH), 7.28±
7.35 (5H, m, C6H5).
[a]D (c g/100 mL, solvent). 1H- or 13C NMR spectra
T
were recorded on a JEOL EX-270 spectrometer in deuterio
solvents using tetramethylsilane or CHCl3 as an internal
standard. Mass spectra were obtained on a JEOL JMS-SX
102A (EI) and JMS-AX 505HA (FAB) spectrometer.
Analytical thin layer chromatography (TLC) was performed
on a Merck Art. 5715, Kieselgel 60 F254/0.25 mm thickness
plates. Visualization was accomplished with UV light, phos-
phomolybdic acid, or ninhydrin solution followed by heat-
ing. Column chromatography was performed with silica gel
BW-820MH or BW-200 (Fuji Davison Co.). HPLC was
carried out with a JASCO UV-970 (detector) and PU-980
(pump) high pressure liquid chromatography. Reverse phase
HPLC was performed with a SHIMADZU SCL-6A system
and JASCO FP-110 (¯uorescence detector). Solvents for
extraction and chromatography were reagent grade and
distilled from the indicated drying agents: Tetrahydrofuran
(THF) was dried by distillation from sodium/benzophenone
Dibenzylated product: IR nmaxneat cm21: 1496, 1454, 1093.
1H NMR (CDCl3) d: 4.04±4.07 (4H, m, (vCHCH2OBn)2),
4.50 (4H, s£2, CH2C6H5£2), 5.77±5.80 (1H, m, CHvCH),
5.87±5.89 (1H, m, CHvCH), 7.3 (10H, m, C6H5£2).
3.1.2. (2EZ)-4-Benzyloxy-1-iodo-2-butene (3). To a stirred
solution of the monobenzylated alcohol 10 (300 mg,
1.68 mmol) in benzene (17 mL) were added successively
imidazole (252 mg, 4.2 mmol), triphenylphosphine (1.10 g,
4.2 mmol) and iodine (850 mg, 4.2 mmol) at room tempera-
ture. After being stirred for 30 min, the reaction was
quenched with saturated aqueous Na2S2O3 (20 mL) and
extracted with ether (50 mL). The extracts were washed
with saturated aqueous Na2S2O3 (20 mL£2) and saturated

F. Yokokawa et al. / Tetrahedron 57 (2001) 4759±4766
4763
brine, dried over MgSO4, ®ltered, and concentrated in
vacuo. The residue was puri®ed by column chromatography
(silica gel BW-820MH, 100 g, hexane±EtOAcˆ10:1) to
give the iodide 3 (310 mg, 64%) as a pale yellow oil: IR
nmaxneat cm21: 1496, 1454, 1360, 1100. 1H NMR (CDCl3) d:
3.90 (2H, d, Jˆ7.5 Hz, ICH2CH), 4.00 (2H, d, Jˆ1.0 Hz,
BnOCH2CH), 4.50 (2H,s, CH2C6H5), 5.81±5.87 (1H, m,
CHvCH), 5.95±6.07 (1H, m, CHvCH), 7.28±7.38 (5H,
m, C6H5). This compound was immediately used for the
next reaction.
2.40±2.59 (2H, m, CHCH2CHvCH), 4.00 (2H, dd, Jˆ
5.9, 7.4 Hz, CHvCHCH2OBn), 4.25 (1H, brd, a-CH),
4.50 (2H, s£2, CH2C6H5), 5.10 (1H, b, NH), 5.56±5.82
(2H, m, CHvCH), 7.28±7.34 (5H, m, C6H5). Anal. calcd
for C22H33NO5; C, 67.49; H, 8.50; N, 3.58. Found; C, 67.40;
H, 8.57; N, 3.54.
3.1.5. (2S)-tert-Butyl 2-N-(tert-butoxycarbonyl)amino-6-
hydroxyhexanoate (13). To a solution of 12 (1.39 g,
3.56 mmol) in EtOAc (25 mL) was added 5% Pd/C
(400 mg) under argon, and hydrogen gas was introduced
(balloon with needle through septum). The black slurry
was stirred under 1 atm of H2 at room temperature for
10 h. The mixture was ®ltered through the pad of Celitew
and the ®ltrate was concentrated in vacuo. The residue was
puri®ed by column chromatography (silica gel BW-820MH,
50 g, hexane:EtOAcˆ3:2) to give the alcohol 13 (938 mg,
3.1.3. (2S,4EZ)-(1)-tert-Butyl 6-benzyloxy-2-N-((1R,2R,
5R)-2-hydroxy-3-pinanylidene)imino-4-hexenoate (11).
To a stirred solution of LDA (prepared from i-Pr2NH
(1.54 mL, 10.7 mmol) and n-BuLi (1.60 M in hexane,
7.33 mL, 10.7 mmol)) in THF (5 mL) was added dropwise
a solution of the chiral schiff base 29a (1.50 g, 5.33 mmol) in
THF (5 mL plus 2 mL of rinse) at 2788C, and the mixture
was stirred at 2788C for 1 h. A solution of the iodide 3
(3.07 g, 10.7 mmol) in THF (5 mL plus 2 mL of rinse)
was added to the mixture at 2788C. The resulting mixture
was stirred at 2788C, for 2 h and quenched with saturated
aqueous NH4Cl (15 mL). After dilution with EtOAc
(100 mL), the mixture was washed with H2O (30 mL) and
saturated brine, dried over Na2SO4, ®ltered, and concen-
trated in vacuo. The residue was puri®ed by column
chromatography (silica gel BW-820MH, 150 g, hexane±
EtOAcˆ3:1) to give 11 (2.05 g, 87%) as a yellow oil:
27
87%) as a colorless oil. [a]D ˆ17.0 (c 1.0, CHCl3). IR
nmaxneat cm21: 3390, 1719, 1700, 1367, 1157. 1H NMR
(CDCl3) d: 1.44 (9H, s, But), 1.46 (9H, s, But), 1.49±1.86
(7H, m, decrease 1H with D2O, CH2£3, OH), 3.64 (2H, t,
Jˆ6.0 Hz, CH2OH), 4.18 (1H, brs, a-CH), 5.05 (1H, brd,
NHBoc), 13C NMR (CDCl3) d: 21.35 (CH2), 27.88 (CH3),
28.21 (CH3), 32.04 (CH2), 32.54 (CH2), 53.75 (CH), 62.12
(CH2), 79.48 (C), 81.06 (C), 155.40 (CvO), 171.93 (CvO).
Anal. calcd for C15H29NO5; C, 59.38; H, 9.63; N, 4.62.
Found; C, 59.26; H, 9.62; N, 4.36. The enantiomeric purity
(92% ee) was determined as its MTPA ester 14 (see below).
[a]D ˆ246.5 (c 1.0, CHCl3). IR nmaxneat cm21: 3440,
26
1
1732, 1651, 1454, 1367, 1155. H NMR (CDCl3) d: 0.81
3.1.6. (2S)-tert-Butyl 2-N-(tert-butoxycarbonyl)amino-6-
iodohexanoate (fragment A). To a solution of the alcohol
13 (62 mg, 0.2 mmol) in benzene (2 mL) were added
successively imidazole (30 mg, 0.5 mmol), triphenyl-
phosphine (131 mg, 0.5 mmol) and iodine (102 mg,
0.4 mmol), and the mixture was stirred at room temperature
for 0.5 h. The reaction was quenched with saturated aqueous
Na2S2O3 (2 mL). After dilution with EtOAc (20 mL), the
mixture was washed with saturated aqueous Na2S2O3
(5 mL£3), H2O (5 mL), saturated brine (5 mL), and dried
over Na2SO4, ®ltered, and concentrated in vacuo. The resi-
due was puri®ed by column chromatography (silica gel
BW-200, 20 g, hexane: EtOAcˆ20:1) to give fragment A
(3H, s, CH3), 1.31 (3H, s, CH3), 1.47 (9H, s, But), 1.50±1.63
(5H, m, decrease 1H with D2O, CH3, pinene-4-H, OH),
2.00±2.09 (2H, m, pinene-3, 5-H), 2.25±2.35 (1H, m,
pinene-4-H), 2.42±2.69 (4H, m, CHCH2CHvCH, pinene-
6-CH2), 3.94 (2H, d, Jˆ4.6 Hz, CHvCHCH2OBn), 4.12±
4.16 (1H, m, a-CH), 4.49 (2H, s£2, CH2C6H5), 5.64±5.68
(2H, m, CHvCH), 7.31±7.38 (5H, m, C6H5). Anal. calcd
for C27H39NO4; C, 73.44;H, 8.90; N, 3.17. Found; C, 73.18;
H,8.85; N, 2.85.
3.1.4. (2S,3EZ)-tert-Butyl 6-benzyloxy-2-N-(tert-butoxy-
carbonyl)amino-4-hexenoate (12). To a stirred solution
of 11 (1.69 g, 4.44 mmol) in THF (12 mL) was added
15% aqueous citric acid (12 mL) and the mixture was stirred
at room temperature for 12 h. After the solvent was
removed, the residue was dissolved in benzene (30 mL),
and the mixture was extracted with 15% aqueous citric
acid (10 mL£3).The combined aqueous layer was basi®ed
to pH 9 with potassium carbonate, salted out, and extracted
with ether (20 mL£8). The ethereal extracts were dried over
MgSo4, ®ltered, and concentraded in vacuo. To a solution of
the residue in dioxane (10 mL) was added a solution of
Boc2O (2.2 g, 10 mmol) in dioxane (4 mL). After being
stirred at room temperature for 10 h, the reaction was
quenched with H2O (10 mL). After dilution with ether
(80 mL), the mixture was washed with H2O (20 mL) and
saturated brine (20 mL), dried over MgSO4, ®ltered, and
concentrated in vacuo. The residue was puri®ed with
column chromatography (silica gel BW-820MH, 35 g,
22
(83 mg, 99%) as a colorless oil: [a]D ˆ114.2 (c 1.0,
CHCl31). IR nmaxneat cm21: 3387, 1738, 1714, 1504, 1367,
1155. H NMR (CDCl3) d: 1.44 (9H, s, But), 1.46 (9H, s,
But), 1.56±1.87 (6H, m, CH2£3), 3.18 (2H, t, Jˆ7.0 Hz,
CH2I), 4.14±4.23 (1H, m, a-CH), 5.05 (1H, brd, NHBoc).
13C NMR (CDCl3) d: 6.31 (CH2I), 25.90 (CH2), 27.94
(CH3), 28.25 (CH3), 31.75 (CH2), 32.78 (CH2), 53.57
(CH), 79.53 (C), 81.82 (C), 155.22 (CvO), 171.63
(CvO). Anal. calcd for C15H28INO4; C, 43.59; H, 6.83;
N, 3.39. Found; C, 43.87; H, 7.19; N, 3.22.
3.1.7. (2S)-tert-Butyl 2-N-(tert-butoxycarbonyl)amino-6-
(a-methoxy-a-tri¯uoromethyl-acetoxy)-hexanoate (14).
To a solution of 13 (18 mg, 0.059 mmol) in CH2Cl2
(1.0 mL) at 08C were added (R)-MTPA (14 mg,
0.06 mmol), DCC (20 mg, 0.08 mmol) and DMAP
(2.5 mg, 0.02 mmol). The reaction mixture was stirred at
08C for 0.5 h and at room temperature for 11.5 h. After
addition of Et2O (10 mL), the mixture was ®ltered through
the pad of Celitew and the ®ltrate was successively washed
with 15% aqueous citric acid, water, saturated aqueous
hexane±EtOAcˆ20:1 to 10:1) to give 12 (1.39 g, 80%) as
22
colorless oil: [a]D ˆ118.7 (c 1.0, CHCl3). IR
NMR (CDCl3) d: 1.43 (9H, s, But), 1.45 (9H, s, But),
a
nmaxneat cm21: 3360, 1780, 1716, 1496, 1367, 115. 1H

4764
F. Yokokawa et al. / Tetrahedron 57 (2001) 4759±4766
NaHCO3, water, saturated brine, and dried over MgSO4,
®ltered, and concentrated in vacuo. The residue was puri®ed
by column chromatography (silica gel BW-200, 10 g, hex-
ane±EtOAcˆ10:1) to give the ester 14 (32 mg, quant.) as a
colorless oil: IR nmaxneat cm21: 3395, 1748, 1714, 1505,
C15H26N2O4S; C, 54.52; H, 7.93; N, 8.48. Found; C,
54.61; H, 7.86; N, 8.30.
3.1.10. (2S)-tert-Butyl 2-N-(tert-butoxycarbonyl)amino-
5-N0-(3-(2-aminopyrydinyl)ureido)-pentanoate (4). To a
stirred solution of the isothiocyanate 6 (3.62 g, 10.95 mmol)
in THF (50 mL) were added 2,3-diaminopyridine (5)
(1.31 g, 12.04 mmol) and Et3N (3 mL, 21.9 mmol). The
mixture was heated under re¯ux for 14 h and concentrated
in vacuo. The residue was puri®ed by column chroma-
tography (silica gel BW-820MH, 250 g, hexane±EtOAcˆ
1:4 to EtOAc:MeOHˆ10:1) to give the thiourea 4 (3.64 g,
1
1455, 1368, 1234, 1157, 1024. H NMR (CDCl3) d: 1.44
(9H, s, But), 1.45 (9H, s, But), 1.53±1.79 (6H, m, CH2£3),
3.54 (3H, s, OCH3), 4.13 (1H, brt, a-CH), 4.28±4.34 (2H,
m, CH2OMTPA), 5.00 (1H, brd, NHBoc), 7.39±7.49 (3H,
m, Ph (o, p)), 7.50±7.52 (2H, m, Ph (m)). HPLC analysis of
14 was carried out as followed; Column: Daicel Chiralcel
OD; Solvent: Hexane±i-PrOHˆ20:1; Flow Rate: 0.5 mL/
min; Detector: 254 nm; Retention Time: 11.5 (minor
isomer) and 12.5 min (major isomer). The enantiomeric
purity of 14 was 92% ee.
22
76%) as a pale brown amorphous solid: [a]D ˆ113.7 (c
1.1, CHCl3). IR nmaxneat cm21: 3379, 3332, 1738, 1714,
1
1614, 1537, 1456,1367, 1153. H NMR (CDCl3) d: 1.41
(9H, s, But), 1.45 (9H, s, But), 1.63±1.81 (4H, m, CH2£2),
3.64 (2H, m, C(S)NHCH2) 4.04±4.19 (1H, m, a-CH), 4.83
(2H, brs, disappeared with D2O, NH2), 5.11 (1H, brd,
NHBoc), 6.07 (1H, brs, disappeared with D2O,
C(S)NHCH2), 6.71 (1H, dd, Jˆ5.0, 7.6 Hz, pyridine-5),
7.37 (2H, dd, Jˆ1.7, 7.6 Hz, pyridine-4 and brs, decrease
1H with D2O, NHC(S)NH), 8.08 (1H, dd, Jˆ1.7, 5.0 Hz,
pyridine-6). 13C NMR (CDCl3) d: 24.64 (CH2), 27.77
(CH3), 28.09(CH3), 29.89 (CH2), 44.55 (CH2), 53.41
(CH), 79.52 (C), 81.83 (C), 113.98 (CH), 116.70 (C),
136.64 (CH), 147.58 (CH), 155.33 (C), 155.74 (CvO),
171.50 (CvO), 180.97 (CvS). HRMS (EI) calcd for
C20H33N5O4S; 439.2253. Found; 439.2260.
3.1.8. Boc-l-Orn(Z)-OBut (16). To a stirred solution of
Boc-l-Orn(Z)-OH (15) (3.66 g, 10 mmol) in t-BuOH/
CH2Cl2 (1:1, 40 mL) was added N,N0-diisopropyl-O-t-
butylisourea (12 mL, 50 mmol). The mixture was stirred
at 508C for 20 h. The mixture was ®ltered through the pad
of Celitew and the ®ltrate was concentrated in vacuo. The
residue was puri®ed by column chromatography (silica gel
BW-820MH, 150 g, hexane±EtOAcˆ5:2) to give Boc-l-
Orn(Z)-OBut (16) (4.92 g, quant.) as a colorless oil:
[a]D ˆ19.6 (c 1.1, CHCl3). IR nmaxneat cm21: 3357,
23
1738, 1696, 1537, 1456, 1367, 1252, 1153. 1H NMR
(CDCl3) d: 1.43 (9H, s, But), 1.45 (9H, s, But), 1.49±1.67
(3H, m, b-CH2, CH2), 1.80 (1H, m, b-CH2), 3.20±3.26 (2H,
m, ZNHCH2), 4.12±4.17 (1H, m, a-CH), 4.86 (1H, brs,
ZNH), 5.09 (3H, s, NHBoc, CH2C6H5), 7.30±7.36 (5H, m,
C6H5). Anal. calcd for C22H34N2O6; C, 62.54; H, 8.11; N,
6.63. Found; C, 62.46; H, 8.18; N, 6.37.
3.1.11. (2S)-tert-Butyl 2-N-(tert-butoxycarbonyl)amino-
5-N-(2-imidazo[4,5-b]pyridinyl)amino-pentanoate (frag-
ment B). To a stirred solution of the thiourea 4 (200 mg,
0.46 mmol) in MeOH (4.5 mL) were added Pb(OAc)2´3H2O
(436.2 mg, 1.14 mmol) and Et3N (63 mL, 0.46 mmol), and
the mixture was heated under re¯ux for 45 h. After dilution
with MeOH, the mixture was ®ltered through the pad of
Celitew and the ®ltrate was concentrated in vacuo. The
residue was puri®ed by column chromatography (silica gel
BW-820MH, 50 g, CHCl3±MeOHˆ13:1) to give fragment
B (148 mg, 80%) as a white solid: mp 195±1968C (MeOH).
3.1.9. (2S)-tert-Butyl 2-N-(tert-butoxycarbonyl)amino-5-
isothiocyanatopentanoate (6). To a stirred solution of Boc-
l-Orn(Z)-OBut (16) (14.25 g, 33.7 mmol) in EtOAc
(100 mL) was added 5% Pd/C (2.8 g) under argon, and
hydrogen gas was introduced (balloon with three way
cock). The black slurry was stirred under 1 atm of H2 at
room temperature for 5 h. The mixture was ®ltered through
the pad of Celitew and the ®ltrate was concentrated in vacuo.
25
[a]D ˆ215.3 (c 0.1, MeOH). IR nmaxKBr cm21: 3389,
1734, 1699, 1651, 1628, 1504, 1417, 1363, 1259, 1180,
1
1153. H NMR (CD3OD) d: 1.41 (9H, s, But), 1.42 (9H, s,
The crude amine 17 was dissolved in THF (160 mL), and
triethylamine (9.4 mL, 67.4 mmol) and carbon disul®de
(10.1 mL, 168.5 mmol) were added to the mixture at 08C.
After being stirred at 08C for 40 min, 30% aqueous H2O2
(23 mL, 202.2 mmol) was added dropwise at 08C, and then
the mixture was diluted with ether (300 mL). The mixture
was washed with 1 M aqueous KHSO4 (100 mL), H2O
(100 mL), saturated brine (100 mL), dried over MgSO4,
®ltered and concentrated in vacuo. The residue was puri®ed
by column chromatography (silica gel BW-820MH, 300 g,
hexane±EtOAcˆ20:1 to 5:1) to give the isothiocyanate 6
But), 1.72±1.94 (4H, m, CH2£2), 3.43 (2H, t, Jˆ6.0 Hz,
NHCH2), 3.99 (1H, brs, a-CH), 6.97 (1H, dd, Jˆ5.0,
7.5 Hz, pyridine-5-H), 7.48 (1H, d, Jˆ7.5 Hz, pyridine-4-
H), 7.91 (1H, brs, pyridine-6-H). HRMS (EI) calcd for
C20H31N5O4; 405.2376. Found; 405.2369. Anal. calcd for
C20H31N5O4´1/2MeOH; C, 58.41; H, 7.89; N, 16.61.
Found; C, 58.72; H, 7.59; N, 16.96.
3.1.12. 2-Amino-3-triphenylmethylaminopyridine (19).
To a stirred solution of 2,3-diaminopyridine (5) (110 mg,
1.01 mmol) in THF (3 mL) were added TrCl (306.7 mg,
1.1 mg) and Et3N (280 ml, 2 mmol). After being stirred at
room temperature for 1 h, the mixture was concentrated in
vacuo. The residue was puri®ed by column chromatography
(silica gel BW-200, 10 g, CHCl3±MeOHˆ50:1) to give 19
(207 mg, 58%) as a purple amorphous powder: IR nmax
CHCl3 cm21: 3359, 1614, 1597, 1574, 1487, 1458, 1448,
1215, 756. 1H NMR (CDCl3) d: 4.22 (2H, brs, disappeared
with D2O, NH2), 4.76 (1H, s NHTr), 6.21±6.30 (2H, m,
pyridine-4, 5-H), 7.19±7.34 (15H, m, Ph3C), 7.44 (1H, dd,
22
(9.66 g, 87%) as a pale yellow oil: [a]D ˆ117.2 (c 1.1,
CHCl3). IR nmaxneat cm21: 3330, 2187, 2110, 1738, 1714,
1
1504, 1454, 1367, 1250, 1155, 1059. H NMR (CDCl3) d:
1.44 (9H, s, But), 1.48 (9H, s, But), 1.65±1.94 (4H, m,
CH2£2), 3.56 (2H, t, Jˆ6.0 Hz, SCNCH2), 4.16±4.24 (1H,
m, a-CH), 5.08 (1H, brd, NHBoc). 13C NMR (CDCl3) d:
25.79 (CH2), 27.80 (CH3), 28.12 (CH3), 29.92 (CH2), 44.46
(CH2), 52.94 (CH), 79.62 (C), 82.12 (C), 130.44 (SCN),
155.20 (CvO), 171.12 (CvO). Anal. calcd for

F. Yokokawa et al. / Tetrahedron 57 (2001) 4759±4766
4765
Jˆ1.5, 4.5 Hz, pyridine-6-H). 13C NMR (CDCl3) d: 71.41
(C), 115.71(CH), 122.43 (CH), 127.39 (CH), 128.41 (CH),
129.36 (CH), 130.64 (C), 136.59 (CH), 145.12 (C), 148.79
(C). HRMS (EI) calcd for C24H21N3; 351.1735. Found;
351.1737.
5.25 (2H, brt, NHBoc, NHCH2), 6.20 (1H, brs, disappeared
with D2O, imidazole-NH), 6.83 (1H, t, Jˆ7.0 Hz, pyridine-
5-H), 7.28±7.32 (1H, m, pyridine-4-H), 7.53 (1H, d, Jˆ
7.0 Hz, pyridine-6-H). 13C NMR (CDCl3) d: 21.98 (CH2),
25.75 (CH2), 27.59 (CH3), 27.96 (CH3), 28.38 (CH2), 29.60
(CH2), 31.93 (CH2), 42.16 (CH2), 52.35 (CH2), 53.34 (CH),
35.60 (CH), 79.01 (C), 79.14 (C), 81.24 (C), 81.44 (C),
111.54 (CH), 116.75 (CH), 124.94 (CH), 143.76 (C),
154.82 (CvO), 155.13 (CvO), 168.50 (C), 171.37
(CvO), 171.55 (CvO). FAB-MS (glycerin) m/z 692 (M2I).
3.1.13. (2S)-tert-Butyl 2-N-(tert-butoxycarbonyl)amino-
5-N0-(2-(3-N-(triphenylmethyl)amino-pyridinyl)ureido)-
pentanoate (20). To a stirred solution of 19 (2.95 g,
8.39 mmol) in THF (25 mL) were added the isothiocyanate
10 (2.77 g, 8.39 mmol) and Et3N (2.34 mL, 16.78 mmol).
The mixture was re¯uxed for 87 h, and concentrated in
vacuo. The residue was puri®ed by column chromatography
(silica gel BW-820MH, 280 g, hexane±EtOAcˆ4:1 to 1:1)
to give the thiourea 20 (3.06 g) as a greenish brown amor-
phous solid. Because 20 was highly unstable, this material
was immediately used for the next step without further
puri®cation.
3.1.16. Pentosidine (1). To a solution of 22 (33 mg,
0.04 mmol) in CHCl3 (0.2 mL) at 08C was added TFA
(0.2 mL). After being stirred at room temperature for 11 h,
the mixture was concentrated in vacuo. Azeotropic removal
of the excess TFA in the residue with toluene gave the crude
pentosidine (1) (28 mg, quant.) as a pale brown amorphous
solid. The crude product (5.3 mg) was puri®ed by reverse
phase C18 preparative column (Wako sil II 5C-18HG
(20£250 mm), linear gradient of 0±10% of acetonitrile
from 0±40 min with TFA (0.075±0.01%) as a counter ion
at a ¯ow rate of 9.5 mL/min) gave pentosidine (1) (5.1 mg,
3.1.14. (2S)-tert-Butyl 2-N-(tert-butoxycarbonyl)amino-
5-N-(2-1-triphenylmethyl)-1H-imidazo-[4,5-b]pyridinyl)-
aminopentanoate (21). To a stirred solution of the thiourea
20 (1.8 g, 2.64 mmol) in MeOH (9 mL) at 08C were added
Et3N (1.2 mL, 8.61 mmol) and HgCl2 (1.08 g, 3.98 mmol).
After being stirred at 08C for 10 min, the mixture was
diluted with CHCl3 (20 mL) and ®ltered through the pad
of Celitew (rinsed with CHCl3), and the ®ltrate was concen-
trated in vacuo. The residue was puri®ed by column
chromatography (silica gel BW-820MH, 150 g, CHCl3±
MeOHˆ50:1 to 30:1) to give 21 (1.766 g, 54% from 19)
24
96%) as a pale brown amorphous solid: [a]D ˆ116.5 (c
1
0.3, MeOH). H NMR (D2O) d: 1.30±1.50 (2H, m), 1.60±
2.00 (8H, m), 3.45 (2H, t, Jˆ6.5 Hz), 3.95 (1H, t, Jˆ
6.5 Hz), 4.03 (1H, t, Jˆ6.0 Hz), 4.42 (2H, t, Jˆ7.0 Hz),
7.09 (1H, t, Jˆ6.5 Hz), 7.65 (1H, d, Jˆ7.5 Hz), 7.80 (1H,
d, Jˆ6.5 Hz). 13C NMR (D2O) d: 21.99 (CH2), 25.19 (CH2),
27.85 (CH2), 29.99 (CH2), 42.62 (CH2), 53.36 (CH), 53.81
(CH2), 116.01 (CH), 120.48 (CH), 132.39 (C), 132.79 (CH),
152.05 (C), 160.28 (C), 172.45 (CvO), 172.52 (CvO),
114.94, 119.20 (CF3CO22, Jˆ35.4 Hz), 162.40, 162.92,
163.46, 163.98 (CF3CO22, Jˆ291.8 Hz). HRMS (FAB)
(glycerin) calcd for C17H27N6O4 (M2CF3CO22);
370.2094. Found; 370.2096. HPLC analysis of pentosidine
(1) was carried out as followed; (1) Column: TSK-GEL
ODS-80TM (4.6£250 mm); Solvent: linear gradient; (liquid
A; H2O (0.1% TFA). Liquid B; acetonitrile (0.075% TFA)
Time (concentration of liquid B); 0 (0)±40 (10)); Flow Rate:
1.0 mL/min; Detector: ¯uorescence (exicitation/emission:
335/385 nm); Retention Time: 30.1 min. (2) Column:
Vydac 208TP5415 (4.6£150 mm); Solvent: linear gradient;
(liquid A; H2O (0.1% TFA). liquid B; acetonitrile (0.075%
TFA) Time (concentration of liquid B); 0 (0)±40 (10));
Flow Rate: 1.0 mL/min; Detector: ¯uorescence (exicita-
tion/emission:335/385 nm); Retention time: 12.0 min.
22
as a pale brown amorphous solid: [a]D ˆ19.8 (c 1.1,
CHCl
CHCl3). IR nmax
3 cm21: 3440, 1713, 1605, 1549, 1495,
1410, 1367, 1153, 731. 1H NMR (CDCl3) d: 1.18±1.27 (4H,
m, CH2£2), 1.42 (9H, s, But), 1.43 (9H, s, But), 3.29±3.36
(2H, m, NHCH2), 3.76 (1H, brt, NHCH2), 4.00 (1H, brs,
a-CH), 4.85 (1H, brd, NHBoc), 5.63 (1H, dd, Jˆ1.3,
8.3 Hz, pyridine-5-H), 6.46 (1H, dd, Jˆ5.0, 8.0 Hz), 7.34
(15H, brs, Ph3C), 8.08 (1H, dd, Jˆ1.0, 5.0 Hz, pyridine-6-
H). 13C NMR (CDCl3) d: 25.11 (CH2), 27.87 (CH3),
28.23(CH3), 29.98 (CH2), 42.97 (CH2), 53.51 (CH), 74.86
(C), 79.44 (C), 81.65 (C), 113.75 (CH), 118.98 (CH), 128.25
(CH), 130.0 (CH), 140.93 (C), 141.89 (CH), 155.20 (C),
155.99 (CvO), 157.05 (C), 171.52 (CvO). Anal. calcd
for C39H45N5O4´7/20 CHCl3; C, 71.01; H, 6.87; N, 10.52.
Found; C, 70.68; H, 6.98; N, 10.53. (CHCl3, which was used
for column chromatography, tenaciously stuck to 21 and
was hard to remove.) HRMS (EI) calcd for C20H31N5O4
(M1H±Ph3C); 405.2376. Found; 405.2376. FAB-MS
(NBA) m/z 648 (M1).
Acknowledgements
3.1.15. Protected pentosidine (22). To a stirred solution of
21 (55.5 mg, 0.0857 mmol) in THF (1 mL) was added frag-
ment A (38.9 mg, 0.0941 mmol) at room temperature. The
mixture was heated under re¯ux for 48 h, and concentrated
in vacuo. The residue was puri®ed by column chroma-
tography (silica gel BW-200, 8 g, CHCl3±MeOH±Et3Nˆ
100:2:1) to give 22 (57 mg, 81%) as a pale brown amor-
This work was ®nancially supported in part by Grants-in-
Aid from the Ministry of Education, Science, Sports and
Culture, Japan. H. S. is grateful to the Japan Society for
Promotion of Science for a postgraduate fellowship.
22
phous solid: [a]D ˆ113.9 (c 1.6, CHCl3). IR
References
CHCl
nmax
3 cm21: 3439, 1709, 1651, 1554, 1498, 1369,
1155,908, 735. H NMR (CDCl3) d: 1.43 (9H, s, But),
1.44(18H, s, But), 1.45 (9H, s, But), 1.60±2.10 (4H, m,
CH2£2), 3.60 (2H, brs, NHCH2), 4.13±4.24 (2H, brm,
a-CH£2), 4.40 (2H, brt, N1CH2), 5.14 (1H, brd, NHBoc),
1
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