Transformation of vitamin B6 to (+)-deoxypyridinoline, a useful biochemical marker for diagnosis of bone diseases

Article abstract of DOI:10.1016/S0040-4020(00)00121-6

A versatile chiral synthesis of the cross-link (+)-deoxypyridinoline (Dpd, 2) was achieved starting from vitamin B₆ (7). The key steps in the synthesis of (+)-2 are transformation of B₆ (7) to the chloride (3d) and construction of three α-amino acid chains by utilizing (R)-(-)-Schollkopf's reagent (4), Wittig reagent (R)-(-)-5, and iodide (5)-(-)-6. (+)-Dpd (2) is a degradation product of bone collagen and has been found to be a useful marker for diagnosis of osteoporosis and other metabolic bone diseases. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4020(00)00121-6

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 2379–2390
Transformation of Vitamin B6 to (ϩ)-Deoxypyridinoline, a useful
Biochemical Marker for Diagnosis of Bone Diseases
*
Maciej Adamczyk, Srinivasa Rao Akireddy and Rajarathnam E. Reddy
Department of Chemistry (9NM, Bldg AP20), Diagnostics Division, Abbott Laboratories, 100 Abbott Park Road,
Abbott Park, IL 60064-6016, USA
Received 8 December 1999; revised 8 February 2000; accepted 9 February 2000
Abstract—A versatile chiral synthesis of the cross-link (ϩ)-deoxypyridinoline (Dpd, 2) was achieved starting from vitamin B6 (7). The key
steps in the synthesis of (ϩ)-2 are transformation of B6 (7) to the chloride (3d) and construction of three a-amino acid chains by utilizing
(R)-(Ϫ)-Schollkopf’s reagent (4), Wittig reagent (R)-(Ϫ)-5, and iodide (S)-(Ϫ)-6. (ϩ)-Dpd (2) is a degradation product of bone collagen and
has been found to be a useful marker for diagnosis of osteoporosis and other metabolic bone diseases. ᭧ 2000 Elsevier Science Ltd. All rights
reserved.
Introduction
enzymatic process.4 During the process of bone resorption
these cross-links (1, 2) are released into the serum and
excreted in urine.5 It has been found that the pyridinium
cross-link Dpd (2) is a clinically useful marker for diagnosis
of osteoporosis,6 a bone disease which affects the aged
population, particularly postmenopausal women. Addition-
ally, Dpd (2) also has been found to have clinical utility in
diagnosis of cancer,7 Paget’s disease8 and other metabolic
bone diseases.9 Currently, Dpd (2) is isolated in a very low
yield at a high cost from bones (e.g. sheep, ox, turkey) by
6–9 M HCl hydrolysis at 110ЊC, a process that could affect
the integrity of the stereocenters in Dpd.10 Therefore, Dpd
(2) became an attractive synthetic target due to its novel
structural features and practical applications in diagnosis
of osteoporosis and other bone diseases. Waelchli et al.11
reported the first synthesis Dpd (2) in an unspecified
diastereomeric purity, which involved the construction of
a substituted pyridine ring from amino acid components
Bone is a complex and highly specialized form of connec-
tive tissue which serves several functions, including support
of the body, protection of internal organs and as a reservoir
for minerals.1 In order for bones to respond and adapt to the
mechanical stress and maintain serum mineral metabolism,
they undergo constant remodeling. The bone remodeling,
which is also called bone turnover, begins with resorption
of old bone by osteoclasts, followed by formation of new
bone by osteoblasts. Any alterations or imbalance in the
remodeling process results in the metabolic bone diseases.
Collagen, a family of structurally related proteins, which are
synthesized by osteoblasts, constitutes approximately 95%
of bone.1 Inter- and intramolecular cross-links such as
pyridinoline (Pyd, 1)2 and deoxypyridinoline (Dpd, 2)3
(Fig. 1) are formed from the adjacent lysine and hydroxyly-
sines present in collagen fibrils by a lysyl oxidase mediated
Figure 1.
Keywords: bone collagen; cross-links; deoxypyridinoline; poly-amino acids; osteoporosis.
* Corresponding author. Tel.: ϩ1-847-937-0225; fax: ϩ1-847-938-8927; e-mail: maciej.adamczyk@abbott.com
0040–4020/00/$ - see front matter ᭧ 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(00)00121-6

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M. Adamczyk et al. / Tetrahedron 56 (2000) 2379–2390
Figure 2. 3a: R1ˆMe, R2ˆPMB; 3b: R1ˆMe, R2ˆH; 3c: R1ˆMe, R2ˆAc; 3d: R1ˆH, R2ˆH; 3e: R1ˆ(CH2)2CO2(CH2)2OSi(Me3), R2ˆH.
utilizing aldol chemistry. The subsequent synthetic
studies have adopted this aldol strategy to achieve the
chiral synthesis of (ϩ)-Dpd (2)12 in fewer steps.13,14
In this paper, we describe a conceptually different
approach for the synthesis of (ϩ)-Dpd (2) starting from
vitamin B6 (7).15
order to determine the need for a protective group at the
5-hydroxymethyl group on the pyridine ring in 3d, and also
to optimize the alkylation reaction with (R)-(Ϫ)-4, we first
prepared chlorides (3a–b) for alkylation. Thus, the phenolic
and 4-hydroxymethyl groups in vitamin B6 (7) were
protected as (Scheme 1) acetonide (8a)18 and the 5-hydroxy-
methyl group was transformed to the corresponding
p-methoxybenzyl (PMB) ether (8b) in 95% yield. The
acetonide protective group in 8b was then hydrolyzed
using pyridinium p-toluenesulfonate (PPTS) and the
phenolic group was selectively protected as its benzyl
ether using benzyldimethylphenylammonium chloride19 to
give the hydroxy compound (10). The hydroxy functionality
in 10 was then converted to the corresponding chloride
(3a) using SOCl2 and the PMB group was subsequently
hydrolyzed using 0.5 N HCl to give 3b in 82% yield.
Results and Discussion
In this strategy, we envisioned the synthesis of (ϩ)-Dpd (2)
(Fig. 2) from a 3-hydroxypyridine derivative (3d) via
construction of three a-amino acid chains sequentially.
This synthetic strategy will have the diversity and flexibility
needed for preparation of Dpd-analogs from any of the three
a-amino acid chains and also from the 2-position of the
pyridine ring, which otherwise is difficult to achieve by
utilizing the aldol approach. Thus, the 4-amino acid chain
in (ϩ)-Dpd (2) was introduced by utilizing (R)-(Ϫ)-2,5-
dihydro-3,6-dimethoxy-2-isopropylpyrazine (Schollkopf’s
reagent, 4)16 via alkylation methodology and subsequent
hydrolysis. The 5-amino acid chain could be extended by
Wittig reaction utilizing (R)-(Ϫ)-4-{[iodo(triphenyl) phos-
phoranyl]methyl}-1,3-oxazolidin-2-one (5),17 and finally,
the lysine chain at the 1- position of pyridine ring was
installed via quaternization with (S)-(Ϫ)-tert-butyl-[(2-
tert-butoxycarbonyl)amino]-6-iodohexanoate (6). The key
synthon, 3d, could be easily prepared from an inexpensive
vit B6 (pyridoxine hydrochloride, 7).
The chloride (3d), which lacks the methyl group at the
2-position, was prepared (Scheme 2) from acetonide (8b).
Thus, 8b was oxidized with m-chloroperoxybenzoic acid to
the corresponding N-oxide, which without purification was
converted to 2-hydroxymethyl compound (11) in 86% yield.
The hydroxy compound (11) was oxidized with MnO2 to
give the aldehyde (12) in 89% yield, which upon further
oxidation with silver oxide followed by decarboxylation
gave the desmethyl compound (13) in 81% yield. The
acetonide protective group in 13 was hydrolyzed using
PPTS to give 14, in which the phenolic group was then
selectively protected as its benzyl ether (15). The hydroxy
functionality in 15 was converted to the corresponding
chloride (16) and further hydrolyzed with HCl to afford
the desired 3d in 61% yield.
Introduction of amino acid chain at the 4-position of (ϩ)-
Dpd (2)
The alkylation of (S)-(ϩ)-Schollkopf’s reagent (4) (Table 1)
was initially carried out (Scheme 3) with 0.5 equiv. of
chloride (3a) using n-BuLi at Ϫ78ЊC (entry 1) and the
We contemplated introduction of the first amino acid, the
alanine-unit, at 4-position of (ϩ)-Dpd (2) utilizing (R)-(Ϫ)-
4, and thus chloride (3d) was needed for alkylation. In
Scheme 1.

M. Adamczyk et al. / Tetrahedron 56 (2000) 2379–2390
2381
Scheme 2.
reaction progress was monitored by TLC. After the
disappearance of starting material (3b) (2 h), the mixture
was quenched with sat. NH4Cl solution at Ϫ78ЊC. The
crude product was purified by silica gel column chromato-
graphy to afford (R,S/S,S)-17a in 84% yield and 79:21
diastereomeric ratio. However, the alkylation of (R)-(Ϫ)-4
with chloride (3b) (entry 2) under similar conditions gave
(S,R)-(Ϫ)-17b in 76% yield and Ͼ98% de. Surprisingly, the
alkylation of chloride (3d), which lacks the methyl group at
2-position, (entry 4) under identical conditions gave (S,R/
R,R)-17d20 as a mixture of diastereomers in 69:31 ratio and
69% yield along with recovered (R)-(Ϫ)-4 (68%). These
results clearly suggest that the methyl group at 2-position
of 3b has a dramatic effect on chiral recognition in the
alkylation step. Lower selectivities were observed for asym-
metric induction steps involving the pyridine substrates.21
This was attributed to the coordination of the nucleophile
with pyridyl nitrogen, thus disrupting the transition state.
In the present study, we believe that the excellent
selectivity observed for 3b was due to the steric hindrance
of a substituent at the 2-position, which prevents such
participation. However, the actual substrate 3d, which
lacks the methyl group, gave moderate selectivity in the
alkylation step (69:31 ratio). This was further evident
from the alkylation (entry 5) of 3e, which has propionate
group at 2-position, and gave (S,R)-(Ϫ)-17e in Ͼ98% de.
(S,R)-(Ϫ)-17e is useful for the preparation of corresponding
Dpd-analog needed for preparation of immunoreagents
from 2-position.22
Introduction of amino acid chain at the 5-position of (ϩ)-
Dpd (2)
Having introduced the masked amino acid functionality at
the 4-position, which is in the form of dihydropyrazine, we
then proceeded (Scheme 4) to introduce an amino acid chain
at the 5-position of (ϩ)-Dpd (2). Although our attempts to
separate the isomeric mixture (S,R/R,R)-17d by a variety of
Table 1. Alkylation of Schollkopf’s reagent (4) with chlorides (3a–e)
Entry
Chloride (3)
Reaction conditions
Alkylated product (17)
R1
R2
Solvent/Temp (ЊC)/Time (h)
Ratio (S,R)/(R,R)a
Yieldb (%)
[a]23
D
1.
2
3.
4.
5.
3a
3b
3c
3d
3e
Me
Me
Me
H
PMB
H
Ac
H
THF/Ϫ78/2
THF/Ϫ78/2
THF/Ϫ78/2
THF/Ϫ78/5
THF/Ϫ78/4
17a
17b
17c
17d
17e
79:21c
Ͼ98:2
88:12c
69:31
84
65
44
69
69
Ϫ43.1
Ϫ35.1
(CH2)2CO2(CH2)2TMS
H
Ͼ98:2
a The diastereomeric ratio of 17a–e was determined by 1H NMR of the crude and purified products.
b Isolated yield.
c (S)-(ϩ)-4 was used and therefore the stereochemistry of the diastereomers of 17a and 17c is (R,S/S,S).
Scheme 3.

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M. Adamczyk et al. / Tetrahedron 56 (2000) 2379–2390
Scheme 4.
techniques, including preparative HPLC, were unsuccessful,
to our delight, the corresponding diastereomeric mixture of
aldehydes 18 were easily separable. Thus, crude (S,R/R,R)-
18, which was obtained by oxidation of (S,R/R,R)-17d with
MnO2, was separated by silica gel column chromatography
to afford the desired major isomer, (S,R)-(ϩ)-18 in 52%
yield and Ͼ98% de. Wittig reaction of major aldehyde
(S,R)-(ϩ)-18 with the ylide generated from (R)-(Ϫ)-5
using n-BuLi in THF at Ϫ78ЊC, gave the olefin 19 in
good yield (71%) as a mixture of E:Z-isomers (64:36).
Hydrolysis of dihydropyrazine ring in 19 was carried out
using 0.5N HCl in MeCN, and the protection of the resulting
free amine, which was obtained by neutralizing with aq.
Na2CO3, using (Boc)2O in MeCN afforded the tri-Boc
compound (S,S)-20 (E:Z/64:36) in 50% overall yield. The
compound (S,S)-20 was then subjected to hydrolysis with
cesium carbonate in MeOH,23 and the resulting product
(S,S)-21 (E:Z/64:36) was hydrogenated over 10% Pd/C in
methanol to afford (S,S)-(Ϫ)-22 in good overall yield.
Concerned with the sensitivity of bis-(tert-butoxycarbonyl)-
amino group in (S,S)-(Ϫ)-22 for subsequent oxidation and
quaternization steps, it was therefore selectively hydrolyzed
with trifluoroacetic acid12b in CH2Cl2 to give (S,S)-(Ϫ)-23 in
69% yield.
Scheme 5.

M. Adamczyk et al. / Tetrahedron 56 (2000) 2379–2390
2383
Scheme 6.
Alternatively, we found that 19 (E:Z/64:36) could be trans-
formed to 24 (E:Z/64:36) (Scheme 5) in which the amino-
ester group at 4-position was protected as mono-Boc-ester.
Thus, reaction of the HCl salt of the amine, which was
obtained by hydrolysis of 19 using 0.5 N HCl, with
(Boc)2O afforded (S,S)-24 (E:Z/64:36) in 46% yield. None
of the tri-Boc compound (S,S)-20, which was isolated
previously from the reaction of free amine of (S,S)-20,
was observed. The selective formation of (S,S)-24 can be
attributed to the presence of the HCl salts of Et3N/DMAP,
and avoids a step at latter stages to remove one of the Boc
groups using TFA. (S,S)-24 (E:Z/64:36) was then subjected
to hydrolysis under milder conditions, lithium carbonate in
MeOH, and the resulting product was hydrogenated over
10% Pd/C in methanol to afford (S,S)-(Ϫ)-23 in good over-
all yield (53%). The enatiomeric purity of (S,S)-(Ϫ)-23 was
determined to be Ͼ95% ee by converting it to the corre-
sponding bis-MTP ester (25) using (R)-MTP-Cl and tri-
ethylamine in CH2Cl2. Finally, oxidation of (S,S)-(Ϫ)-23
with Jones reagent24 in acetone at room temperature
followed by esterification of the crude product using
trimethylsilyl diazomethane in MeOH–benzene25 afforded
(S,S)-(ϩ)-26 in 54% yield.
reported in ppm relative to TMS and coupling constants (J)
were reported in Hz. Electrospray ionization mass spec-
trometry (ESI-MS) was carried on a Perkin–Elmer
(Norwalk, CT) Sciex API 100 Benchtop system employing
Turbo Ionspray ion source and HRMS were obtained on a
Nermang 3010 MS-50, JEOL SX102-A mass spectrometers.
Thin layer chromatography was performed on pre-coated
˚
Whatman MK6F silica gel 60 A plates (layer thickness:
250 mm) and visualized with UV light and/or using a
KMnO4 solution (KMnO4 (1.0 g), NaOH (8.0 g) in water
(200 mL)) or phosphomolybdic acid reagent (20 wt% solu-
tion in ethanol). Column chromatography was performed on
silica gel, Merck grade 60 (230–400 mesh). THF was
freshly distilled from a purple solution of sodium and
benzophenone. CH2Cl2 was freshly distilled from CaH2
under nitrogen. Analytical reversed phase (RP) HPLC was
performed using a Waters mBondapak RCM C18 10m
(8×100 mm) column. Preparative reversed phase (RP)
HPLC was performed using a Waters mBondapak RCM
C18 10m (40×100 mm) column. Optical rotations were
measured on Autopol III polarimeter from Rudolph
Research, Flanders, NJ. Melting points were recorded in
open capillary tubes on an Electrothermal Melting Point
Apparatus and were uncorrected (see Ref. 12a for more
details).
Completion of the synthesis of (ϩ)-Dpd (2)
(2,2,8-Trimethyl-4H-[1,3]dioxino[4,5-c]pyridin-5-yl)methanol
(8a)18 and the Wittig reagent, (R)-(Ϫ)-517 from (R)-(Ϫ)-
serine were prepared according to the known procedures.
The chloride (3c) was prepared from 3b (Ac2O, pyridine, rt,
24 h, in 48%), and 2-(trimethylsilyl) ethyl-3-[3-(benzyl-
oxy)-4-(chloromethyl)-5-(hydroxymethyl)-2-pyridinyl]pro-
The 3-hydroxypyridine derivative (S,S)-(ϩ)-26 requires
only elaboration of lysine chain at 1-position for completion
of (ϩ)-Dpd (2) synthesis. Thus, quaternization of (S,S)-(ϩ)-
26 with (S)-(Ϫ)-612 was carried out (Scheme 6) in refluxing
1,4-dioxane for 4 h, and the product was purified by
preparative reverse phase HPLC to afford the pyridinium
compound (S,S,S)-(Ϫ)-27 in 27% yield. Finally, alkaline
hydrolysis of the methyl esters in (S,S,S)-(Ϫ)-27 using
LiOH and the removal of Boc and t-butyl protective groups
was carried out by treating the crude product with TFA–
water (9.5:0.5 ratio). Purification of the resulting crude
product by preparative reversed phase HPLC and lyophili-
zation afforded (ϩ)-Dpd (2) in 84% yield as its TFA salt.
panoate (3e) from aldehyde (13) in
5 steps (1.
Ph3PvCHCO2(CH2)2TMS [prepared from BrCH2COBr,
TMS(CH2)2OH, Py, CH2Cl2, 0ЊC, 1 h (96%) then PPh3,
PhH, reflux, 18 h, then KOH, CH2Cl2–H2O (70%)],
CH2Cl2, rt, 1.5 h, then 5% Pd/C MeOH, H2, 15 Psi,
35 min (84%); 2. PPTS, ethanol, reflux, 28 h (41%); 3.
NaOEt, benzyldimethylphenylammonium chloride, xylene,
reflux, 5 h (88%); 4. SOCl2, PhH, reflux, 1 min (95%); 5.
DDQ, CH2Cl2–H2O, rt, 45 min (63%)).
In summary, a versatile synthesis of (ϩ)-deoxypyridinoline
(2), a biochemical marker for diagnosis of osteoporosis was
developed starting from vitamin B6 (7).
5-{[(4-Methoxybenzyl)oxy]methyl}-2,2,8-trimethyl-4H-
[1,3]dioxino[4,5-c]pyridine (8b). NaH (60% dispersion in
mineral oil, 24.1 g, 602.8 mmol, 6.3 equiv.) was washed
with hexane (2×50 mL) and dry DMF (600 mL) was
added. The suspension was heated to 70ЊC and a solution
of 8a (20.0 g, 95.69 mmol) in anhydrous DMF (300 mL)
was added via a double ended needle. After the addition
was complete, the heating bath was removed and the
mixture was allowed to reach 45ЊC. The mixture was cooled
Experimental
General methods and materials
1H and 13C NMR spectra were recorded on a Varian Gemini
spectrometer (300 MHz) and the chemical shifts (d) were

2384
M. Adamczyk et al. / Tetrahedron 56 (2000) 2379–2390
with an ice bath, and p-methoxybenzyl chloride (PMB-Cl,
15.6 mL, 114.83 mmol, 1.2 equiv.) was added over 5 min.
After the addition was over, the cooling bath was removed
and the mixture was stirred for 15 h at room temperature.
The reaction was cooled with an ice bath, quenched care-
fully with water (500 mL), diluted with an additional
amount of water (5.5 L) and ether (1.5 L). The aqueous
layer was separated and extracted with ether (1.5 L). The
combined ether extracts were dried (Na2SO4) and concen-
trated on a rotary evaporator. The crude compound was
purified by silica gel column chromatography (50%
EtOAc in hexanes) to afford 29.9 g of 8b in 95% yield as
pale yellow thick oil. Analytical RP HPLC: MeCN:0.1% aq
acetic acid/50:50, 1.0 mL/min, 225 nm, Rt: 12.62 min, 99%;
1H NMR (CDCl3): d 7.94 (s, 1H), 7.23 (d, 2H, Jˆ8.7 Hz),
6.88 (d, 2H, Jˆ8.7 Hz), 4.85 (s, 2H), 4.42 (s, 2H), 4.39 (s,
2H), 3.79 (s, 3H), 2.41 (s, 3H), 1.54 (s, 6H); 13C NMR
(CDCl3): d 159.3, 148.1, 145.9, 139.7, 129.5, 128.2,
126.3, 125.9, 113.8, 99.6, 71.7, 66.9, 58.6, 55.2, 24.7, 18.5;
ESI-MS (m/z): 330 (MϩH)ϩ; HRMS (FAB, m/z): calcd for
C17 H24NO4 330.1705 (MϩH)ϩ; observed, 330.1707.
compound was purified by silica gel column chromato-
graphy (40% EtOAc in hexanes) to afford 0.81 g of 10 in
74% yield. Analytical RP HPLC: MeCN:0.1% aq. acetic
acid/50:50, 1.0 mL/min at 225 nm, Rt: 12.81 min, Ͼ99%;
1H NMR (CDCl3): d 8.21 (s, 1H), 7.49–7.33 (m, 5H), 7.29
(d, 2H, Jˆ8.8 Hz), 6.90 (d, 2H, Jˆ8.8 Hz), 4.97 (s, 2H),
4.69 (d, 2H, Jˆ6.9 Hz), 4.61 (s, 2H), 4.56 (s, 2H), 3.81 (s,
3H), 3.42 (t, 1H, Jˆ6.9 Hz), 2.53 (s, 3H); 13C NMR
(CDCl3): d 159.5, 154.3, 151.8, 145.2, 141.8, 136.5,
130.7, 129.8, 128.7, 128. 6, 128.3, 128.1, 114.0, 76.7,
72.5, 68.3, 56.2, 55.2, 19.7; ESI-MS (m/z): 380 (MϩH)ϩ.
3-(Benzyloxy)-4-(chloromethyl)-5-{[(4-methoxybenzyl)-
oxy]methyl}-2-methylpyridine (3a). A solution of SOCl2
(0.27 mL, 3.73 mmol, 1.77 equiv.) in benzene (10 mL) was
added to a solution of 10 (0.80 g, 2.11 mmol) in benzene
(20 mL) at 0ЊC dropwise over 20 min using syringe
pump. After addition was complete, the mixture was
heated to reflux for 1 min in a pre-heated oil bath and cooled
immediately in an ice-bath. Solvent was removed on a
rotary evaporator to afford 0.90 g of chloride (3a) as HCl
salt in 98% yield. Analytical RP HPLC: MeCN:0.1% aq.
acetic acid/65:35, 1.0 mL/min, 225 nm, Rt: 13.25 min,
General procedure for hydrolysis of acetonide: e.g.
4-(hydroxymethyl)-5-{[(4-methoxybenzyl)oxy]methyl}-
2-methyl-3-pyridinol (9)
1
Ͼ99%; H NMR (CDCl3): d 8.51 (s, 1H), 7.45–7.36 (m,
5H), 7.26 (d, 2H, Jˆ8.7 Hz), 6.91 (d, 2H, Jˆ8.7 Hz), 5.10
(s, 2H), 4.69 (s, 2H), 4.64 (s, 2H), 4.60 (s, 2H), 3.83 (s, 3H),
2.82 (s, 3H); ESI-MS (m/z): 398 and 400 (MϩH)ϩ.
PPTS (2.22 g, 8.81 mmol, 2 equiv.) was added to a solution
of 8b (1.45 g, 4.41 mmol) in absolute ethanol (35 mL) under
nitrogen and refluxed for 20 h. The mixture was cooled to
room temperature, quenched with NaHCO3 (30 g) and the
solvent was removed on a rotary evaporator. The residue
was diluted with water (75 mL) and CHCl3 (75 mL). The
aqueous layer was separated and extracted with CHCl3
(75 mL). The combined organic extracts were dried
(Na2SO4) and concentrated on a rotary evaporator. The resi-
due was triturated with ether (50 mL) and the precipitate
was filtered, washed with ether (50 mL) and dried to give
0.78 g of 9 in 61% yield. 1H NMR (CD3OD): d 7.78 (s, 1H),
7.25 (d, 2H, Jˆ8.7 Hz), 6.88 (d, 2H, Jˆ8.7 Hz), 4.89 (s,
2H), 4.47 (s, 2H), 4.45 (s, 2H), 3.78 (s, 3H), 2.39 (s, 3H);
ESI-MS: 290 (MϩH)ϩ.
The HCl salt of 3a (0.40 g, 0.92 mmol) was dissolved in
CH2Cl2 (50 mL) and washed with saturated aq. NaHCO3
(30 mL). The organic layer was dried (Na2SO4), concen-
trated on a rotary evaporator and purified by silica gel
column chromatography (40% EtOAc in hexanes) to afford
0.32 g of 3a in 87% yield. Analytical RP HPLC:
MeCN:0.1% aq. acetic acid/65:35, 1.0 mL/min, 225 nm,
1
Rt: 13.61 min, Ͼ99%; H NMR (CDCl3): d 8.29 (s, 1H),
7.51–7.36 (m, 5H), 7.28 (d, 2H, Jˆ8.7 Hz), 6.90 (d, 2H,
Jˆ8.7 Hz), 4.98 (s, 2H), 4.73 (s, 2H), 4.65 (s, 2H), 4.51 (s,
2H), 3.81 (s, 3H), 2.57 (s, 3H); 13C NMR (CDCl3): d 159.3,
153, 7, 145.6, 138.2, 136.3, 130.7, 129.5, 128.7, 128.4,
127.9, 113.8, 75.9, 72.3, 66.7, 55.2, 35.5, 19.8; ESI-MS
(m/z): 398 and 400 (MϩH)ϩ.
General procedure for preparation of benzyl ether: e.g.
(3-(benzyloxy)-5-{[(4-methoxybenzyl)oxy]methyl}-2-
methyl-4-pyridinyl)methanol (10)
5-(Benzyloxy)-4-(chloromethyl)-6-methyl-3-pyridinyl]-
methanol (3b). The hydrochloride salt of 3a (0.80 g,
1.847 mmol) in 0.5N HCl solution (30 mL) was heated to
reflux for 15 min (the heterogenous reaction mixture
became clear in 5 min). The reaction was cooled to room
temperature and concentrated to dryness under reduced
pressure. The residue was suspended in CHCl3 (100 mL)
and washed with saturated NaHCO3 (75 mL). Organic
layer was dried (Na2SO4), concentrated and purified on
silica gel column chromatography (75% EtOAc in hexanes)
to afford 0.418 g of 3b in 82% yield. Analytical RP HPLC:
MeCN:0.1% aq. acetic acid/50:50, 1.0 mL/min at 225 nm,
NaOEt (21% solution in ethanol, 1.0 mL, 3.1 mmol,
1.1 equiv.) was added dropwise to a solution of benzyl-
dimethylphenylammonium chloride (0.9 g, 4.23 mmol,
1.5 equiv.) in absolute ethanol (15 mL) at Ϫ78ЊC and stirred
for 20 min. The mixture became heterogenous. It was then
added via cannula to a solution of 9 (0.815 g, 2.82 mmol) in
absolute ethanol (15 mL) at Ϫ78ЊC. After stirring the
reaction at Ϫ78ЊC for 15 min, the cooling bath was removed
and the reaction was allowed to reach room temperature.
Ethanol was removed in vacuo, and dry toluene (20 mL)
was added and then azeotroped to remove traces of ethanol.
Dry xylene (25 mL) was added to the residue and the
mixture was heated to reflux for 4 h. Xylene was removed
on a rotary evaporator in vacuo and the residue was par-
titioned between CHCl3 (100 mL) and sat. NH4Cl solution
(100 mL). The organic layer was separated, dried (Na2SO4)
and concentrated on a rotary evaporator. The crude
1
Rt: 9.05 min, Ͼ99%; H NMR (CDCl3): d 8.39 (s, 1H),
7.52–7.36 (m, 5H), 4.99 (s, 2H), 4.85 (d, 2H, Jˆ5.8 Hz),
4.77 (s, 2H), 2.58 (s, 3H); 13C NMR (CDCl3): d 153.3,
151.4, 144.5, 137.8, 136.2, 133.7, 128.7, 128.5, 127.9,
76.0, 59.8, 35.3, 19.5; ESI-MS (m/z): 278 and 280 (MϩH)ϩ.
(5-{[(4-Methoxybenzyl)oxy]methyl}-2,2-dimethyl-4H-
[1,3]dioxino[4,5-c]pyridin-8-yl)methanol (11). m-CPBA

M. Adamczyk et al. / Tetrahedron 56 (2000) 2379–2390
2385
(50–60%, 34.5 g, 99.96 mmol, 1.1 equiv.) was added to a
solution of the PMB-ether (8b, 29.9 g, 90.88 mmol) in
CHCl3 (750 mL) in five portions over a 5 min period at
room temperature. After stirring the mixture for 2 h, it
was quenched with saturated aq. Na2SO3 (500 mL). The
organic layer was separated and washed with saturated aq.
NaHCO3 (500 mL). Organic layer was dried (Na2SO4) and
concentrated on a rotary evaporator to give 31.0 g of the
corresponding N-oxide in almost quantitative yield.
organic layer was separated and the aqueous layer was
extracted with EtOAc (200 mL). The combined organic
extracts were washed with brine (150 mL), dried (Na2SO4)
and concentrated on a rotary evaporator to give 9.5 g of the
corresponding carboxylic acid in 83% yield.
The crude acid (13.6 g, 37.88 mmol) was suspended in
xylenes (350 mL) and heated to reflux. The mixture became
clear in 5 min, and reflux was continued for an additional
20 min. The mixture was cooled to about 50ЊC and concen-
trated on a rotary evaporator. The resulting oily residue was
dissolved in CHCl3 (300 mL) and washed with aq. NaHCO3
solution (200 mL), dried (Na2SO4) and concentrated. The
crude product was purified by silica gel column chromato-
graphy (40% EtOAc in hexanes) to afford 9.7 g of 13 in 81%
yield. Rf: 0.65 (50% EtOAc in hexanes); Analytical RP
HPLC: MeCN:0.05% aq. trifluoroacetic acid/40:60,
1.0 mL/min at 225 nm, Rt: 8.64 min, 99%; 1H NMR
(CDCl3): d 8.15 (s, 1H), 8.07 (s, 1H), 7.25 (d, 2H,
Jˆ8.8 Hz), 6.89 (d, 2H, Jˆ8.8 Hz), 4.86 (s, 2H), 4.45 (s,
2H), 4.42 (s, 2H), 3.81 (s, 3H), 1.54 (s, 3H); 13C NMR
(CDCl3): d 159.4, 148.2, 141.1, 139.7, 129.5, 129.4,
128.5, 127.0, 113.9, 99.8, 72.1, 66.9, 58.6, 55.2, 24.6;
ESI-MS (m/z): 316 (MϩH)ϩ; HRMS (FAB, m/z): calcd
for C18H22NO4, 316.1549 (MϩH)ϩ; observed, 316.1549.
Trifluoroacetic anhydride (6.0 mL, 42.5 mmol, 0.51 equiv.)
was added to a solution of the N-oxide (28.5 g, 82.6 mmol)
in CH2Cl2 at 0ЊC and stirred for 5 min. An additional
amount of TFAA (15.3 mL, 108.6 mmol, 1.3 equiv.) was
added, the cooling bath was removed and the mixture stirred
for 5 h at room temperature. The mixture containing the
trifluoroacetate of 11 was cooled with an ice-bath and dry
MeOH (150 mL) was added. The mixture was concentrated
on a rotary evaporator and the resulting oily residue was
dissolved in CHCl3 (700 mL). The CHCl3 layer was washed
with saturated aq. NaHCO3 solution (700 mL), dried
(Na2SO4) and concentrated on a rotary evaporator. The
crude compound was purified on silica gel column (60%
EtOAc in hexanes) to afford 26.8 g of 11 in 86% yield. Rf:
0.52 (60% EtOAc in hexanes); Analytical RP HPLC:
MeCN:0.1% aq. acetic acid/50:50, 1.0 mL/min at 225 nm,
Rt: 16.47 min, 99%; 1H NMR (CDCl3): d 8.02 (s, 1H), 7.24
(d, 2H, Jˆ8.6 Hz), 6.89 (d, 2H, Jˆ8.6 Hz), 4.87 (s, 2H),
4.69 (s, 2H), 4.44 (s, 2H), 4.42 (s, 2H), 4.26 (br s, 1H,
OH), 3.80 (s, 3H), 1.54 (s, 6H); 13C NMR (CDCl3): d
159.3, 147.5, 144.6, 138.9, 129.5, 129.3, 127.6, 126.4,
113.8, 100.0, 71.9, 66.8, 59.5, 58.6, 55.2, 24.6; ESI-MS
(m/z): 346 (MϩH)ϩ; HRMS (FAB, m/z): calcd for
C19H24NO5, 346.1658 (MϩH)ϩ; observed, 346.1645.
4-(Hydroxymethyl)-5-{[(4-methoxybenzyl)oxy]methyl}-
3-pyridinol (14). Hydrolysis of 13 (9.6 g, 30.476 mmol)
using PPTS (12.7 g, 50.49 mmol, 1.65 equiv.) in absolute
ethanol (170 mL) by following the procedure described
for 9 gave 5.2 g of 14 in 62% yield as a pale yellow solid.
mp: 95–97ЊC; Rf: 0.27 (90% EtOAc in hexanes); Analytical
RP HPLC: MeCN:0.1% aq. acetic acid/50:50, 1.0 mL/min
at 225 nm, Rt: 7.58 min, 97%; 1H NMR (CDCl3): d 8.09 (s,
1H), 7.95 (s, 1H), 7.26 (d, 2H, Jˆ8.5 Hz), 6.89 (d, 2H,
Jˆ8.5 Hz), 4.85 (s, 2H), 4.49 (s, 2H), 4.47 (s, 2H), 3.80
(s, 3H); 13C NMR (CDCl3): d 159.4, 153.2, 140.7, 138.5,
133.6, 131.1, 129.6, 129.1, 113.9, 72.2, 67.4, 58.1, 55.2;
ESI-MS (m/z): 276 (MϩH)ϩ; HRMS (FAB, m/z): calcd
for C15H18NO4, 276.1236 (MϩH)ϩ; observed, 276.1237.
5-{[(4-Methoxybenzyl)oxy]methyl}-2,2-dimethyl-4H-
[1,3]dioxino[4,5-c]pyridine-8-carbaldehyde (12). MnO2
(85%, 45.5 g, 444.6 mmol, 13.0 equiv.) was added to a solu-
tion of 11 (11.8 g, 34.2 mmol) in CHCl3 (550 mL) at room
temperature. The mixture was stirred for 21 h and filtered
through Celite powder. The filtrate was concentrated on a
rotary evaporator to give 10.41 g of aldehyde (12) in 89%
yield. Rf: 0.59 (50% EtOAc in hexanes); Analytical RP
HPLC: MeCN:0.1% aq. acetic acid/50:50, 1.0 mL/min at
(3-(Benzyloxy)-5-{[(4-methoxybenzyl)oxy]methyl}-4-
pyridinyl)methanol (15). Reaction was carried out on 14
(5.1 g, 18.54 mmol) and followed the procedure described
for 10 to afford 2.0 g of 15 in 30% yield as pale yellow solid.
Mp: 75–6ЊC; Rf: 0.49 (80% EtOAc in hexanes); Analytical
RP HPLC: MeCN:0.1% aq. acetic acid/50:50, 1.0 mL/min
at 225 nm, Rt: 12.29 min, 97%; 1H NMR (CDCl3): d 8.33 (s,
1H), 8.20 (s, 1H), 7.44–7.30 (m, 5H), 7.27 (d, 2H,
Jˆ8.8 Hz), 6.89 (d, 2H, Jˆ8.8 Hz), 5.19 (s, 2H), 4.77 (s,
2H), 4.61 (s, 2H), 4.53 (s, 2H), 3.80 (s, 3H), 3.25 (bs, 1H);
13C NMR (CDCl3): d 159.5, 152.8, 143.8, 137.1, 135.9,
135.7, 132.2, 129.7, 128.9, 128.7, 128.3, 127.3, 113.9,
72.4, 71.2, 67.8, 55.8, 55.2; ESI-MS (m/z): 366 (MϩH)ϩ;
HRMS (FAB, m/z): calcd for C22H24NO4, 366.1705
(MϩH)ϩ; observed 366.1721.
1
225 nm, Rt: 11.20 min, 99%; H NMR (CDCl3): d 10.32
(s, 1H), 8.27 (s, 1H), 7.26 (d, 2H, Jˆ8.8 Hz), 6.89 (d, 2H,
Jˆ8.8 Hz), 4.91 (s, 2H), 4.49 (s, 2H), 4.47 (s, 2H), 3.82 (s,
3H), 1.62 (s, 6H); 13C NMR (CDCl3): d 189.3, 159.5, 151.1,
141.3, 139.5, 133.6, 129.6, 129.1, 128.9, 113.9, 100.9, 72.5,
66.6, 58.6, 55.2, 24.7; ESI-MS (m/z): 343 (MϩH)ϩ; HRMS
(FAB, m/z): calcd for C19H21NO5, 344.1498 (MϩH)ϩ;
observed, 344.1485.
5-{[(4-Methoxybenzyl)oxy]methyl}-2,2-dimethyl-4H-[1,3]-
dioxino[4,5-c]pyridine (13). Ag2O (14.7 g, 63.55 mmol,
2 equiv.) and aq. KOH (5.4 g, 95.33 mmol, 3.0 equiv.
dissolved in H2O (275 mL)) were added sequentially to a
solution of aldehyde (12, 10.9 g, 31.77 mmol) in EtOH
(275 mL) at room temperature. The resulting heterogeneous
mixture was stirred for 1 h, and filtered through Celite
powder. The filtrate was concentrated on a rotary evapora-
tor, the residue was diluted with water (400 mL) and EtOAc
(800 mL), and the pH was adjusted to 4.5 with 6N HCl. The
3-(Benzyloxy)-4-(chloromethyl)-5-{[(4-methoxybenzyl)-
oxy]methyl}pyridine (16). Reaction of 15 (1.95 g,
5.34 mmol) with SOCl2 (0.69 mL, 9.45 mmol, 1.77 equiv.)
in dry benzene (70 mL) was carried out by following the
procedure described for 3a to afford 2.26 g of 16 as a HCl
salt in 97% yield. Mp: 124–6ЊC; Analytical RP HPLC:

2386
M. Adamczyk et al. / Tetrahedron 56 (2000) 2379–2390
MeCN:0.1% aq. acetic acid/65:35, 1.0 mL/min at 225 nm,
Rt: 11.50 min, Ͼ99%; H NMR (CDCl3): d 8.51 (s, 1H),
3,6-dimethoxy-2,5-dihydropyrazine (17a). Reaction of 3a
(0.740 g, 1.864 mmol) using n-BuLi (2.33 mL, 3.728 mmol,
2.0 equiv.) and (S)-(ϩ)-4 (0.685 g, 3.728 mmol, 2.0 equiv.)
was carried out by following the procedure described for
(S,R/R,R)-17d to afford 0.885 g of (R,S/S,S)-17a in 84%
yield and 79:21 ratio. Rf: 0.42 (60% EtOAc in hexanes);
Analytical RP HPLC: MeCN:0.1% aq. trifluoroacetic acid/
1
8.47, (s, 1H), 7.44–7.41 (m, 5 H), 7.27 (d, 2H, Jˆ8.6 Hz),
6.91 (d, 2H, Jˆ8.6 Hz), 5.35 (s, 2H), 4.72 (s, 2H), 4.69 (s,
2H), 4.62 (s, 2H), 3.82 (s, 3H); 13C NMR (CDCl3): d 159.5,
154.4, 141.6, 138.1, 133.6, 133.4, 129.7, 129.2, 129.1, 128.3,
127.5, 124.9, 114.1, 73.3, 72.6, 64.9, 55.3, 33.2; ESI-MS
(m/z): 384, 386 (MϩH)ϩ; HRMS (FAB, m/z): calcd for
C22H23ClNO3, 384.1366 (MϩH)ϩ; observed, 384.1371.
1
50:50, 2.0 mL/min at 225 nm, Rt: 6.49 min, 96%; H NMR
(CDCl3): d 8.25 and 8.24 (two s, 1H), 7.53–7.49 (m, 2H),
7.44–7.26 (m, 5H), 6.90–6.86 (m, 2H), 4.91–4.80 (m, 2H),
4.70–4.10 (m, 5H), 3.81 and 3.80 (two s, 3H), 3.80–3.78
(m, 1H), 3.65 and 3.61 (two s, 3H), 3.49 and 3.46 (two s,
3H), 3.41–3.45 (m, 1H), 2.86–2.66 (m, 1H), 2.56 (s, 3H),
2.20–2.10 (m, 1H), 1.04 (d, Jˆ6.9 Hz) and 0.98 (d,
Jˆ6.9 Hz, 3H), 0.71 (d, Jˆ6.9 Hz) and 0.60 (d, Jˆ6.9 Hz,
3H); ESI-MS (m/z): 546 (MϩH)ϩ; HRMS (FAB, m/z): calcd
for C32H40N3O5 546.2968 (MϩH)ϩ; observed, 546.2978.
[5-(Benzyloxy)-4-(chloromethyl)-3-pyridinyl]methanol
(3d). The HCl salt of 16 (2.24 g, 5.34 mmol) was suspended
in 0.5N HCl (60 mL) and followed the procedure described
for 3b to afford 0.873 g of 3d in 61% yield. Rf: 0.45
(EtOAc); Analytical RP HPLC: MeCN:0.1% aq. acetic
1
acid/50:50, 1.0 mL/min at 225 nm, Rt: 8.34 min, 95%; H
NMR (CDCl3): d 8.33 (s, 1H), 8.29 (s, 1H), 7.48–7.36 (m,
5H), 5.25 (s, 2H), 4.85 (s, 2H), 4.82 (s, 2H); 13C NMR
(CDCl3): d 152.4, 142.7, 135.8, 135.0, 134.8, 132.9,
128.7, 128.3, 127.3, 71.1, 60.1, 34.8; ESI-MS (m/z): 264
(MϩH)ϩ; HRMS (FAB, m/z): calcd for C14H15ClNO2,
264.0791 (MϩH)ϩ; observed, 264.0787.
(Ϫ)-(5-(Benzyloxy)-4-{[(2S,5R)-5-isopropyl-3,6-dimethoxy-
2,5-dihydro-2-pyrazinyl]methyl}-6-methyl-3-pyridinyl)-
methanol (17b). Reaction of 3b (0.72 g, 2.6 mmol) using
n-BuLi (4.88 mL, 7.8 mmol, 3.0 equiv.) and (R)-(Ϫ)-4
(1.435 g, 7.8 mmol, 3.0 equiv.) was carried out by following
the procedure described for (S,R/R,R)-17d to afford 0.710 g
of (S,R)-(Ϫ)-17b in 65% yield and Ͼ98% de. Rf: 0.27
General procedure for alkylation of Schollkopf’s reagent
(4) with chlorides (3a–e): e.g. (5-(benzyloxy)-4-{[(2S,5R)/
(2R,5R)-5-isopropyl-3,6-dimethoxy-2,5-dihydro-2-
pyrazinyl]methyl}-3-pyridinyl) methanol (17d)
1
(3% MeOH in EtOAc); H NMR (CDCl3): d 8.28 (s, 1H),
7.55–7.36 (m, 5H), 5.25–5.09 (m, 1H), 4.94–4.86 (m, 2H),
4.74–4.44 (m, 2H), 4.20–4.10 (m, 1H), 3.95–3.92 (m, 1H),
3.77–3.71 (m, 1H), 3.62 (s, 3H), 3.60 (s, 3H), 2.77–2.69 (m,
1H), 2.57 (s, 3H), 2.20–2.10 (m, 1H), 0.95 (d, 3H,
Jˆ6.6 Hz), 0.70 (d, 3H, Jˆ6.9 Hz); ESI-MS (m/z): 426
(MϩH)ϩ, 448 (MϩNa)ϩ.
n-BuLi (2.5 M solution in hexanes, 2.08 mL, 5.19 mmol,
3.0 equiv.) was added dropwise to a solution of (R)-(Ϫ)-4
(0.956 g, 5.19 mmol, 3.0 equiv.) in THF (16 mL) at Ϫ78ЊC
under nitrogen. The resulting pale yellow solution was stir-
red for 25 min. A solution of 3d (0.455 g, 1.73 mmol) in
THF (16 mL) was added dropwise via a double ended
needle over a 5 min period. The mixture was then stirred
for 5 h and quenched with saturated aq. NH4Cl solution
(10 mL) at Ϫ78ЊC. The mixture was allowed to warm to
room temperature and diluted with EtOAc (75 mL) and H2O
(50 mL). The aqueous layer was separated and extracted
with EtOAc (50 mL). The combined organic layers were
dried (Na2SO4) and concentrated on a rotary evaporator.
The crude compound was purified by silica gel column
chromatography (15% EtOAc in hexanes to 3% MeOH in
EtOAc) to afford 0.492 g of (S,R/R,R)-17d in 69% yield
(5-(Benzyloxy)-4-{[(2S,5R/(2R,5R)-5-isopropyl-3,6-
dimethoxy-2,5-dihydro-2-pyrazinyl]methyl}-6-methyl-
3-pyridinyl)methyl acetate (17c). Reaction of 3c (0.028 g,
0.088 mmol) using n-BuLi (0.11 mL, 0.176 mmol, 2.0
equiv.) and (S)-(ϩ)-4 (0.032 g, 0.176 mmol, 2.0 equiv.)
was carried out by following the procedure described for
(S,R/R,R)-17d to afford 0.018 g of (R,S/S,S)-17c in 44%
yield and 88:12 ratio. Rf: 0.47 (60% EtOAc in hexanes);
Analytical RP HPLC: MeCN:0.1% aq. formic acid/50:50,
2.0 mL/min at 225 nm, Rt: 5.37 min, 96%; 1H NMR
(CDCl3): d 8.23 (s, 1H), 7.55–7.36 (m, 5H), 5.30–5.14
(m, 2H), 4.93–4.83 (m, 2H), 4.30–4.20 (m, 1H), 3.90–
3.80 (m, 1H), 3.65 and 3.62 (two s, 3H), 3.54 and 3.53
(two s, 3H), 3.50–3.40 (m, 1H), 2.80–2.68 (m, 1H), 2.57
(s, 3H), 2.20–2.10 (m, 1H), 2.08 and 2.07 (two s, 3H), 1.03
and 0.95 (two d, 3H, Jˆ6.6 and 6.9 Hz), 0.70 and 0.63 (two
d, 3H, Jˆ6.6, 6.9 Hz); ESI-MS (m/z): 467 (MϩH)ϩ; HRMS
(FAB, m/z): calcd for C26H34N3O5 468.2498 (MϩH)ϩ;
observed, 468.2494.
(colorless solid) as
a mixture diastereomers [ratio:
(S,R:R,R)/69:31]. Rf: 0.18 (EtOAc); Analytical RP HPLC:
MeCN:0.1% aq. acetic acid/50:50, 1.0 mL/min at 225 nm,
Rt: 20.03 min, 31% [(R,R)-17d] and 21.78 min, 69% [(S,R)-
1
17d]; H NMR (CDCl3): d 8.37 (s, 31/100 H), 8.32 (s, 69/
100 H), 7.45–7.36 (m, 5H), 5.28–5.16 (m, 2H), 4.78–4.69
(m, 1H), 4.23–4.15 (m, 1H), 3.97 (dd, 69/100 H, Jˆ4.4,
3.6 Hz), 3.90–3.81 (m, 1 Hϩ31/100 H), 3.66 (s, 69/100, 3
H), 3.61 (s, 3H), 3.60 (s, 31/100, 3H), 2.81–2.69 (m, 1H),
2.32–2.21 (m, 31/100 H), 2.16–2.06 (m, 69/100 H), 1.08 (d,
31/100, 3H, Jˆ6.86 Hz), 0.97 (d, 69/100, 3H, Jˆ6.86 Hz),
0.75 (d, 31/100, 3H, Jˆ6.86 Hz), 0.71 (d, 69/100, 3H,
Jˆ6.86 Hz); ESI-MS (m/z): 412 (MϩH)ϩ; HRMS (FAB,
m/z): calcd for C23H30N3O4 412.2236 (MϩH)ϩ; observed,
412.2224. 0.501 g of (R)-(Ϫ)-4 was recovered in 68% yield
with no loss of optical purity.
(Ϫ)-2-(Trimethylsilyl)ethyl-3-(3-(benzyloxy)-5-(hydroxy-
methyl)-4-{[(2S,5R)-5-isopropyl-3,6-dimethoxy-2,5-dihy-
dro-2-pyrazinyl]methyl}-2-pyridinyl)propanoate (17e).
The reaction of 3e (2.42 g, 5.56 mmol) using (R)-(Ϫ)-4
(3.07 g, 16.69 mmol, 3.0 equiv.) and following the pro-
cedure described for (S,R)/R,R)-17d afforded 0.492 g of
(S,R)-(Ϫ)-17e in 69% yield as a syrup and Ͼ98% de.
1
[a]2D3ˆϪ35.1 (c 0.695, CHCl3); H NMR (CDCl3): d 8.29
(s, 1H), 7.53–7.38 (m, 5H), 4.71 (d, 1H, Jˆ11.8 Hz), 4.49–
4.45 (m, 1H), 4.20–4.14 (m, 2H), 3.93 (t, 1H, Jˆ3.6 Hz),
(2R,5S)/(2S,5S)-2-[(3-(Benzyloxy)-5-{[(4-methoxybenzyl)-
oxy]methyl}-2-methyl-4-pyridinyl)methyl]-5-isopropyl-

M. Adamczyk et al. / Tetrahedron 56 (2000) 2379–2390
2387
3.79–3.48 (m, 2H), 3.60 (s, 6H), 3.19 (t, 2H, Jˆ7.4 Hz),
2.95–2.71 (m, 3H), 2,17–2.14 (m, 1H), 1.02–0.95 (m, 5H),
0.69 (d, 3H, Jˆ6.8 Hz); 13C NMR (CDCl3): d 173.4, 166.7,
162.8, 153.9, 151.9, 145.4, 140.6, 136.6, 134.9, 128.4,
128.1, 127.8, 75.2, 62.4, 61.9, 60.3, 55.1, 53.3, 52.8, 32.3,
32.0, 31.0, 26.9, 19.1, 17.4, 17.2, Ϫ1.5; ESI-MS (m/z): 584
(MϩH)ϩ; HRMS (FAB, m/z): calcd for C31H46N3O6Si,
584.3156 (MϩH)ϩ; observed 584.3182.
3H), 3.52 (s, 36/100, 3H), 3.48 (s, 64/100, 3H), 3.43 (dd, 64/
100 H, Jˆ13.2, 5.2 Hz), 3.16–3.11 (m, 36/100 H), 2.95–79
(m, 1H), 2.28–2.17 (m, 1H), 1.02 (d, 3 H, Jˆ6.9 Hz), 0.66–
0.63 (m, 3H); ESI-MS (m/z): 493 (MϩH)ϩ; HRMS (FAB,
m/z): calcd for C27H33N4O5, 493.2445 (MϩH)ϩ; observed,
493.2453.
tert-Butyl-(4S)-4-[(E:Z)-2-(5-(benzyloxy)-4-{(2S)-2-
[bis(tert-butoxycarbonyl)amino]-3-methoxy-3-oxopro-
pyl}-3-pyridinyl)ethenyl]-2-oxo-1,3-oxazolidine-3-
carboxylate (20). 0.5N HCl (9.0 mL) was added to a solu-
tion of olefin (19 1.0 g, 2.03 mmol) in CH3CN (45 mL) at
room temperature and stirred for 45 min. The mixture was
concentrated and the residue was diluted with EtOAc
(75 mL) and saturated aq. NaHCO3 solution (75 mL).
Organic layer was separated and the aqueous layer was
extracted with EtOAc (3×75 mL). The combined organic
layers were dried (Na2SO4), and concentrated on rotary
evaporator to give 0.885 g of the crude residue which was
dissolved in CH3CN (10 mL) under nitrogen. To this
mixture, Et3N (0.93 mL, 6.68 mmol, 3.0 equiv.), (Boc)2O
(3.06 mL, 13.37 mmol, 6.0 equiv.) and DMAP (0.055 g,
0.445 mmol, 0.2 equiv.) were added at room temperature.
After stirring the mixture for 6 h, it was concentrated on a
rotary evaporator and the crude product was purified by
silica gel column chromatography (75% EtOAc in hexanes)
to afford 0.7 g of 20 in 50% yield for two steps (E:Z/64:36
ratio). Rf: 0.73 (EtOAc); Analytical RP HPLC: MeCN:0.1%
aq. acetic acid/55:45, 1.0 mL/min, 225 nm, Rt: 23.9 min,
(ϩ)-5-(Benzyloxy)-4-{[(2S,5R)-5-isopropyl-3,6-dimethoxy-
2,5-dihydro-2-pyrazinyl]methyl}nicotinaldehyde (18). MnO2
(85%, 1.6 g, 15.81 mmol, 13.0 equiv.) was added to a
solution of alcohol (S,R/R,R)-17d, (0.50 g, 1.21 mmol) in
CHCl3 (50 mL) at room temperature and stirred for 20 h.
The mixture was filtered through Celite powder and the
filtrate was concentrated on a rotary evaporator. Purification
of the crude product by silica gel column chromatography
(40% EtOAc in hexane) afforded 0.26 g of (S,R)-(ϩ)-
aldehyde (18) in 52% yield (major isomer) and Ͼ98% de.
Rf: 0.66 (70% EtOAc in hexanes); Analytical RP
HPLC:MeCN:.05% aq. trifluoroacetic acid/45:55, 1.0 mL/
min at 225 nm, Rt: 5.88 min, 99%; [a]2D3ˆϩ2.9 (c, 0.975,
1
CHCl3); H NMR (CDCl3): d 10.37 (s, 1H), 8.73 (s, 1H),
8.48 (s, 1H), 7.51–7.37 (m, 5H), 5.35–5.26 (m, 2H), 4.22–
4.16 (m, 1H), 3.88 (t, 1H, Jˆ3.5 Hz), 3.83 (dd, 1H, Jˆ12.6,
4.2 Hz), 3.70 (s, 3H), 3.49 (s, 3H), 3.25 (dd, 1H, Jˆ12.6,
10.0 Hz), 2.24–2.15 (m, 1H), 0.99 (d, 3H, Jˆ6.8 Hz),
0.65 (d, 3H, Jˆ6.8 Hz); 13C NMR (CDCl3): d 164.5,
162.8, 153.0, 143.5, 138.7, 137.7, 135.9, 130.6, 128.6,
128.1, 126.8, 70.8, 60.9, 54.7, 52.6, 52.5, 31.8, 28.5, 19.0,
16.7; ESI-MS (m/z): 410 (MϩH)ϩ; HRMS (FAB, m/z):
calcd for C23H28N3O4, 410.2080 (MϩH)ϩ; observed,
410.2083.
1
99%; H NMR (CDCl3): d 8.23 (s, 64/100 H), 8.21 (s, 36/
100 H), 8.15 (s, 64/100 H), 8.06 (s, 36/100 H), 7.47–7.32
(m, 5H), 6.84 (d, 64/100 H, Jˆ15.8 Hz), 6.63 (s, 36/100 H,
Jˆ11.7 Hz), 6.09 (dd, 64/100 H, Jˆ15.8, 7.9 Hz), 5.85 (dd,
36/100 H, Jˆ11.6, 9.3 Hz), 5.44–5.40 (m, 36/100 H), 5.29
(dd, 64/100 H, Jˆ8.4, 5.9 Hz), 5.22–5.19 (m, 2H), 5.00–
4.92 (m, 36/100 H), 4.91–4.82 (m, 64/100 H), 4.51–4.40
(m, 1 H), 4.17–4.08 (m, 1H), 3.73 (s, 3H), 3.62–3.49 (m,
2H), 1.51 (s, 64/100, 9H), 1.46 (s, 36/100, 9H), 1.33 (s,
18H); ESI-MS (m/z): 698 (MϩH)ϩ; HRMS (FAB, m/z):
calcd for C36H48N3O11, 698.3283 (MϩH)ϩ; observed,
698.3275.
(4S)-4-[(E:Z)-2-(5-(Benzyloxy)-4-{[(2S,5R)-5-isopropyl-
3,6-dimethoxy-2,5-dihydro-2-pyrazinyl]methyl}-3-pyri-
dinyl)ethenyl]-1,3-oxazolidin-2-one (19). n-BuLi (1.6 M
solution in hexanes, 1.18 mL, 1.87 mmol, 3.0 equiv.) was
added dropwise to (R)-(Ϫ)-5 (0.45 g, 0.935 mmol,
1.5 equiv.) suspended in THF (9.0 mL) at Ϫ78ЊC under
nitrogen. The resulting pale yellow solution was stirred
for 15 min. A solution of (S,R)-(ϩ)-aldehyde (18, 0.255 g,
0.623 mmol) in THF (9.0 mL) was added dropwise via a
double ended needle to the above ylide solution over
5 min. The mixture was then stirred for 4 h and quenched
with saturated aq. NH4Cl solution (10 mL) at Ϫ78ЊC. The
mixture was allowed to warm to room temperature and
diluted with H2O (20 mL) and EtOAc (50 mL). Organic
layer was separated and the aqueous layer was extracted
with EtOAc (50 mL). The combined organic extracts were
dried (Na2SO4) and concentrated on a rotary evaporator.
Purification of the crude product by silica gel column chro-
matography (5% MeOH in EtOAc) afforded 0.22 g of olefin
(19) in 71% yield as a mixture of E:Z isomers (ratio: 64:36).
Methyl-(2S)-3-(3-(benzyloxy)-5-{(E:Z,3S)-3-[(tert-butoxy-
carbonyl)amino]-4-hydroxy-1-butenyl}-4-pyridinyl)-2-
[bis(tert}-butoxycarbonyl)amino]propanoate (21). Cesium
carbonate (0.083 g, 0.25 mmol, 0.25 equiv.) was added to a
solution of 20 (0.70 g, 1.00 mmol.) in MeOH (35 mL) at
room temperature. After stirring the mixture for 2.5 h, it
was concentrated on a rotary evaporator. The residue was
dissolved in CHCl3 (200 mL), washed with water (100 mL),
dried (Na2SO4) and concentrated on a rotary evaporator.
The crude product was purified by silica gel column chro-
matography (EtOAc) to give 0.53 g of 21 in 78% yield. Rf:
0.21 (80% EtOAc in hexanes); Analytical RP HPLC:
MeCN:0.05% aq. trifluoroacetic acid/50:50, 1.0 mL/min,
225 nm, Rt: 13.93 min, 24% (Z-isomer) and 14.75 min,
1
Rf: 0.29 (3% MeOH in EtOAc); H NMR (CDCl3): d 8.33
(s, 64/100 H), 8.26 (s, 36/100 H), 8.23 (s, 64/100 H), 7.86 (s,
36/100 H), 7.48–7.34 (m, 5H), 6.92 (d, 64/100 H,
Jˆ15.9 Hz), 6.86 (d, 36/100 H, Jˆ11.5 Hz), 6.10 (dd, 64/
100 H, Jˆ15.8, 7.4 Hz), 5.87 (dd, 36/100 H, Jˆ11.5,
8.9 Hz), 5.66 (br s, 36/100 H), 5.53 (br s, 64/100 H),
5.23–5.14 (m, 2H), 4.66–4.53 (m, 2 H), 4.45–4.37 (m,
36/100 H), 4.32–4.24 (m, 64/100 H), 4.21–4.14 (m, 1H),
3.89–3.83 (m, 1H), 3.67 (s, 64/100, 3H), 3.62 (s, 36/100,
1
76% (E-isomer); H NMR (CDCl3): d 8.27 (s, 64/100 H),
8.16 (br s, 63/100 H and 1 H), 7.46–7.28 (m, 5 H), 6.67 (d,
64/100 H, Jˆ15.9 Hz), 6.51 (d, 36/100 H, Jˆ11.5 Hz), 6.05
(dd, 64/100 H, Jˆ15.9, 5.1 Hz), 5.81–5.68 (m, 36/100 H),
5.40–5.10 (m, 4H), 4.42–4.24 (m, 1H), 3.79–3.62 (m, 5H),
3.59–3.29 (m, 2H), 1.45 (s, 64/100, 9H), 1.41 (s, 36/100,
9H), 1.34 (s, 36/100, 18H), 1.32 (64/100, 18H); ESI-MS

2388
M. Adamczyk et al. / Tetrahedron 56 (2000) 2379–2390
(m/z): 672 (MϩH)ϩ; HRMS (FAB, m/z): calcd for
C35H50N3O10, 672.3491 (MϩH)ϩ; observed, 672.3487.
concentrated on a rotary evaporator and dried under vacuum
to give a residue (0.90 g) which was dissolved in dry
CH3CN (20 mL). To this mixture Et3N (1.60 mL,
11.54 mmol, 6.0 equiv.), (Boc)2O (2.65 mL, 11.54 mmol,
6.0 equiv.) and DMAP (0.07 g, 0.576 mmol, 0.3 equiv.)
were added sequentially at room temperature. The mixture
was stirred for 52 h, concentrated and purified by silica gel
column chromatography (EtOAc) to afford 0.42 g of (S,S)-
24 in 46% yield for two steps. Analytical RP HPLC:
MeCN:0.1% aq. acetic acid/50:50, 2.0 mL/min, 225 nm,
(Ϫ)-Methyl-(2S)-2-[bis(tert-butoxycarbonyl)amino]-3-
(3-{(3S)-3-[(tert-butoxycarbonyl)amino]-4-hydroxybutyl}-
5-hydroxy-4-pyridinyl)propanoate (22). 2N HCl solution
(0.225 mL, 0.453 mmol, 1.05 equiv.) was added dropwise
to a stirred solution of 21 (0.29 g, 0.43 mmol) in MeOH
(20 mL) over 5 min. After the addition was complete, the
mixture was concentrated on rotary evaporator to dryness.
The residue was dissolved in MeOH (20 mL) and hydroge-
nated in presence of 10%Pd/C (50% wet with water,
0.060 g, 10% wt/wt) at 20 psi for 4 h. The reaction was
filtered through Celite and filtrate was concentrated. The
residue was dissolved in CHCl3 (100 mL) and washed
with saturated aq. NaHCO3 solution (50 mL). Organic
layer was dried (Na2SO4), concentrated on a rotary evapora-
tor. The crude product was purified by silica gel column
chromatography (10% MeOH in EtOAc) to afford 0.21 g
of (Ϫ)-22 in 83% yield as a white solid. Mp: 103–4ЊC;
Rf: 0.15 (5% MeOH in EtOAc); Analytical RP HPLC:
MeCN:0.05% aq. trifluoroacetic acid/40:60, 1.0 mL/min,
225 nm, Rt: 10.17 min, 93%; [a]2D3ˆϪ63.8 (c 0.48,
1
Rt: 4.77 min, 99%; H NMR (CDCl3): d 8.25 (br s, 1H),
7.51–7.36 (m, 5H), 7.01–6.92 (m, 1H), 6.06 (dd, 1H,
Jˆ15.4 and 8.5 Hz), 5.48 (br d, 1H, Jˆ9.3 Hz), 5.30–5.19
(m, 2H), 4.98–4.90 (m, 1H), 4.58–5.52 (m, 2H), 4.22 (dd,
1H, Jˆ9.3 and 4.7 Hz), 3.69 (br s, 3H), 3.31 (dd, 1H,
Jˆ12.9 and 4.9 Hz), 2,98 (dd, 1 H, Jˆ13.2 and 10.7 Hz),
1.55 (s, 9H), 1.28 (s, 9H); ESI-MS (m/z): 598 (MϩH)ϩ.
(Ϫ)-Methyl-(2S)-2-[(tert-butoxycarbonyl)amino]-3-(3-
{(3S)-3-[(tert-butoxycarbonyl)amino]-4-hydroxybutyl}-
5-hydroxy-4-pyridinyl)propanoate (23). Lithium carbon-
ate (0.010 g, 0.14 mmol, 0.20 equiv.) was added to a
solution of (S,S)-24 (0.42 g, 0.703 mmol) in MeOH
(10 mL) at room temperature. After stirring the mixture
for 17 h, it was concentrated on a rotary evaporator and
dissolved in CHCl3 (200 mL). The solution was washed
with water (100 mL), dried (Na2SO4) and concentrated on
a rotary evaporator. The crude product was purified by silica
gel column chromatography (EtOAc) to afford 0.295 g
(74%) which was (0.28 g, 0.49 mmol) was dissolved in
MeOH (20 mL) and 2N HCl solution (0.25 mL, 0.514
mmol, 1.05 equiv.) was added with stirring. After the
addition was complete (5 min), the mixture was concen-
trated on rotary evaporator to dryness. The residue was
dissolved in MeOH (20 mL) and hydrogenated over 10%
Pd/C (50% wet with water, 0.056 g) at 20 psi for 3 h. The
mixture was filtered through celite powder and the filtrate
was concentrated. The residue was purified by preparative
RP HPLC (MeCN:0.1% aq. trifluoroacetic acid/28:72,
45 mL/min at 225 nm) and lyophilized to afford 0.155 g
of (S,S)-(Ϫ)-23 in 53% yield. Analytical RP HPLC:
MeCN:0.05% aq. TFA/30:70, 2.0 mL/min, 225 nm, Rt:
4.84 min, 99% and was identical to the compound prepared
above.
1
CHCl3); H NMR (CDCl3): d 8.03 (s, 1H), 7.95 (s, 1H),
5.26 (dd, 1H, Jˆ9.4, 4.8 Hz), 4.97 (br d, 1H), 3.77 (s,
3H), 3.74–3.60 (m, 3H), 3.49 (dd, 1H, Jˆ14.0, 4.8 Hz),
3.36 (dd, 1H, Jˆ14.0, 9.4 Hz), 2.69 (t, 2H, Jˆ7.9 Hz),
1.90–1.69 (m, 2H),1.44 (s, 9H), 1.36 (s, 18H); ESI-MS:
m/z 584 (MϩH)ϩ; HRMS (FAB, m/z): calcd for
C28H46N3O10, 584.3183 (MϩH)ϩ; observed, 584.3193.
(Ϫ)-Methyl-(2S)-2-[(tert-butoxycarbonyl)amino]-3-(3-
{(3S)-3-[(tert-butoxycarbonyl)amino]-4-hydroxybutyl}-
5-hydroxy-4-pyridinyl)propanoate (23). TFA (0.08 mL,
1.038 mmol, 3.5 equiv.) was added to a solution of (Ϫ)-22
(0.173 g, 0.296 mmol) in CH2Cl2 (12 mL) at room tempera-
ture. After stirring the mixture for 26 h, it was concentrated
on a rotary evaporator and the residue was dissolved in
MeOH (20 mL). The mixture was then stirred for 6 h at
room temperature. It was then concentrated on a rotary
evaporator and crude product was purified on preparative
RP HPLC (MeCN:0.1% aq. trifluoroacetic acid/35:65,
45.0 mL/min at 225 nm). The product was lyophilized to
afford 0.122 g of (Ϫ)-23 as its TFA salt in 69% yield as
white powder. Analytical RP HPLC: MeCN:0.1% aq.
trifluoroacetic acid/35:65, 2.0 mL/min at 225 nm, Rt:
4.0 min, 99%; [a]2D3ˆϪ25.2 (c 0.42, MeOH); 1H NMR
(CDCl3): d 8.66 (s, 1H), 7.96 (s, 1H), 5.66 (br d, 1H,
Jˆ7.3 Hz), 5.35 (br d, 1H, Jˆ7.69 Hz), 4.55–4.38 (m,
1H), 3.70 (br s, 6H), 3.38–3.24 (m, 2H), 2.89–2.82 (m,
2H), 1.88–1.79 (m, 2H), 1.44 (s, 9H), 1.39 (s, 9H); 13C
NMR (CDCl3): d 170.9, 156.6, 156.2, 155.5, 142.2, 141.6,
131.3, 125.9, 80.7, 79.8, 64.1, 52.8, 52.7, 51.9, 32.2, 29.6,
28.3, 28.2, 26.9; ESI-MS (m/z): 484 (MϩH)ϩ; HRMS
(FAB, m/z): calcd for C23H37N3O8, 484.2659 (MϩH)ϩ;
observed, 484.2681.
Bis-Mosher ester (25). Et3N (0.032 mL, 0.226 mmol,
5.0 equiv.) and (R)-(Ϫ)-a-methoxy-(a-trifluoromethyl
phenylacetyl chloride (0.025 mL, 0.135 mmol, 3.0 equiv.)
were added sequentially to a 0ЊC solution of (S,S)-(Ϫ)-24
(0.027 g, 0.045 mmol) in CH2Cl2 (4 mL) under nitrogen.
After stirring the mixture for 5 h, it was quenched with
water (10 mL) and stirred for 10 min. The mixture was
diluted with CH2Cl2 (20 mL) and separated the organic
layer. It was dried (Na2SO4) and concentrated on a rotary
evaporator. The crude product was purified by silica gel
column chromatography (40% EtOAc in hexane) to afford
0.034 g of bis-MTP ester (25) in 68% yield and Ͼ95% ee.
Analytical RP HPLC: MeCN:0.05% aq. trifluoroacetic acid/
tert-Butyl-(4S)-4-[(E:Z)-2-(5-(benzyloxy)-4-{(2S)-2-
[(tert-butoxycarbonyl)amino]-3-methoxy-3-oxopropyl}-
3-pyridinyl)ethenyl]-2-oxo-1,3-oxazolidine-3-carboxy-
late (24). 0.5 N HCl solution (7 mL) was added to a solution
of 19 (0.76 g, 1.54 mmol) in CH3CN (34 mL) at room
temperature and stirred for 45 min. The mixture was
1
65:35, 2.0 mL/min, 225 nm, Rt: 5.78 min, 96%; H NMR
(CDCl3): d 8.27 (s, 1H), 7.65–7.39 (m, 10H), 4.98 (br d,
1H, Jˆ8.2 Hz), 4.46–4.23 (m, 3H), 4.05–3.93 (m, 1H), 3.71
(s, 3H), 3.65 (br s, 1H), 3.56 (s, 3H), 3.53 (s, 3H), 2.88–2.70
(m, 3H), 2.64–2.55 (m, 1H), 1.83–1.71 (m, 2H), 1.45 (s,

M. Adamczyk et al. / Tetrahedron 56 (2000) 2379–2390
2389
9H), 1.33 (s, 9H); 19F NMR (CDCl3ϩa,a,a-trifluoroto-
luene): d Ϫ8.01, Ϫ8.77; ESI-MS (m/z): 916 (MϩH)ϩ,
1832 (2×MϩH)ϩ.
evaporator to dryness. A mixture of TFA (4.75 mL) and
water (0.25 mL) was added to the above residue at room
temperature and stirred for 1 h. The solvent was removed to
dryness and the crude product was purified by preparative
RP HPLC: (MeCN:0.1% aq. trifluoroacetic acid/2:98,
25.0 mL/min at 225 nm). Lyophilization of the product
afforded 0.0237 g of (ϩ)-Dpd-TFA (2) salt in 84% yield.
Analytical RP HPLC: MeCN:0.1% aq. trifluoroacetic acid/
2:98, 1.0 mL/min, 225 nm, Rt: 4.06 min, 99%; [a]2D3ˆϩ36.2
(ϩ)-Methyl-(2S)-2-[(tert-butoxycarbonyl)amino]-4-(4-
{(2S)-2-[(tert-butoxycarbonyl)amino]-3-methoxy-3-oxo-
propyl}-5-hydroxy-3-pyridinyl)butanoate (26). Jones’
reagent (0.30 mL, 0.422 mmol, 4.0 equiv.) was added to a
solution of (Ϫ)-23 (0.063 g, 0.11 mmol) in acetone (10 mL)
at room temperature. After stirring the mixture for 40 min,
the reaction was quenched with 5% aq. Na2CO3 solution
(2 mL). The mixture was diluted with MeOH (20 mL) and
filtered through the Celite powder. The filtrate was concen-
trated on a rotary evaporator and the residue diluted with
EtOAc (50 mL) and H2O (10 mL). The pH was adjusted to
4.8 using phosphate buffer (pH 4.0). The organic layer was
separated and the aqueous layer was extracted with EtOAc
(20 mL). The combined organic extracts were washed with
brine (20 mL), dried (Na2SO4) and concentrated on a rotary
evaporator. The residue was dissolved in MeOH–benzene
(10 mL, 1:5 ratio) and a solution of TMSCH2N2 (2.0 M
solution in hexanes, 0.25 mL, 0.5 mmol, 4.5 equiv.) was
added at room temperature. The resulting pale yellow
mixture was stirred for 15 min and concentrated on a rotary
evaporator. The residue was purified by silica gel column
chromatography (5% MeOH in EtOAc) to afford 0.030 g of
(S,S)-(ϩ)-26 in 54% yield. Rf: 0.34 (5% MeOH in EtOAc);
Analytical RP HPLC: MeCN:0.1% aq. trifluoroacetic acid/
35:65, 2.0 mL/min, 225 nm, Rt: 6.65 min, 99%; [a]2D3ˆ
1
(c 0.54, MeOH), Lit12: [a]2D3ˆϩ31.6 (c 0.25, MeOH); H
NMR (D2O): d 8.13 (s, 1H), 8.05 (s, 1H), 4.39–4.28 (m,
2H), 4.06–3.97 (m, 1H), 3.77–3.28 (m, 1H), 3.26–3.17 (m,
1H), 3.28–3.15 (m, 2H), 2.90–2.64 (m, 2H), 2.09–1.71 (m,
6H), 1.38–1.16 (m, 2H); 13C NMR (D2Oϩ5 mL MeOH): d
173.2, 172.9, 172.4, 156.0, 141.6, 141.5, 136.4, 129.4, 61.7,
53.7, 53.6, 52.5, 30.7, 30.4, 29.9, 28.1, 26.1, 21.6; ESI-MS
(m/z): 413 (M)ϩ; HRMS (FAB, m/z): calcd for C18H29N4O7,
413.2036 (M)ϩ; observed, 413.2050.
References
1. For comprehensive study on physiology and pathology of bones
see Principles of Bone Biology; Bilezikan, J. P.; Raicz, L. G.;
Rogan, G., Eds.; Academic Press: New York, 1996.
2. Fujimoto, D.; Moriguchi, T.; Ishida, T.; Hayashi, H. Biochem.
Biophys. Res. Commun. 1978, 84, 52.
3. Ogawa, T.; Ono, T.; Tsuda, M.; Kawanishi, Y. Biochem.
Biophys. Res. Commun. 1982, 107, 1252.
1
ϩ17.3 (c 0.59, CHCl3); H NMR (CDCl3): d 8.11 (s, 1H),
4. (a) Watts, N. B. Clin. Chem. 1999, 45, 1359. (b) Knott, L.;
Baily, A. J. Bone 1998, 22, 181. (c) James, I. T.; Walne, A. J.;
Perrett, D. Ann. Clin. Biochem. 1996, 33, 397. (d) Eyre, D. R. In
Principles of Bone Biology; Bilezikan, J. P.; Raicz, L. G.; Rogan,
G., Eds.; Academic Press: New York, 1996; p. 143.
5. (a) Eyre, D. R.; Oguchi, H. Biochem. Biophys. Res. Commun.
1980, 92, 403. (b) Robins, S. P. Biochem. J. 1983, 215, 167. (c)
Gunja-Smith, Z.; Boucek, R. J. Biochem. J. 1981, 197, 759. (d)
Hanson, D. A.; Eyre, D. R. J. Biol. Chem. 1996, 271, 26508.
6. (a) Gomez, Jr., B.; Ardakani, S.; Evans, B. J.; Merrell, L. D.;
Jenkins, D. K.; Kung, V. T. Clin. Chem. 1996, 42, 1168. (b)
Rosano, T. G.; Peaston, R. T.; Bone, H. G.; Woitge, H. W.; Francis,
R. M.; Seibel, M. J. Clin. Chem. 1998, 44, 2126. (c) Sarno, M.;
Powell, H.; Tjersland, G.; Schoendorfer, D.; Harris, H.; Adams, K.;
Ogata, P.; Warnick, G. R. Clin. Chem. 1999, 45, 1501.
7. (a) Luftner, D.; Gunther, S.; Flath, B.; Muller, C.; Echteroff, K.;
Mergenthaler, H.-G.; Wernecke, K.-D.; Possinger, K. Anticancer
Res. 1999, 19, 2537. (b) Engler, H.; Koeberle, D.; Thuerlimann, B.;
Senn, H.-J.; Riesen, W. F. Clin. Chem. Lab. Med. 1998, 36, 879. (c)
Withold, W.; Friedrich, W.; Reinauer, H. Ann. Clin. Biochem.
1996, 33, 421. (d) Westerhuis, L. W.; Delaere, K. P. Eur. J.
Clin. Chem. Clin. Biochem. 1997, 35, 89.
8. (a) Delmas, P. D.; Gineyts, E.; Bertholin, A.; Garnero, P.;
Marchand, F. J. Bone Miner. Res. 1993, 8, 643. (b) Robins, S.
P.; Black, D.; Peterson, C. R.; Reid, D. M.; Duncan, A.; Seibel,
M. J. Eur. J. Clin. Clin. Invest. 1991, 21, 310. (c) Body, J. J.;
Delmas, P. D. J. Clin. Endocrinol. Metab. 1992, 74, 471.
9. (a) Hoshi, H.; Kushida, K.; Takahashi, M.; Denda, M.;
Yamazaki, K.; Yamanashi, A.; Inoue, T. Miner Electrolyte
Metab. 1997, 23, 93. (b) Seibel M. J.; Gartenberg, F.; Silverberg,
S. J.; Ratcliffee, A.; Robins, S. P.; Bilezikian, J. P. J. Clin.
Endocrinol. Metab. 1992, 74, 481. (c) Alvarez, L.; Peris, P.;
Guanabens, N.; Herranz, R.; Monegal, A.; Bedini, J. L. Arthritis
Rheum. 1997, 40, 461.
7.93 (s, 1H), 5.68 (br d, 1H, Jˆ7.0 Hz), 5.53 (br d, 1H,
Jˆ6.6 Hz), 4.45–4.36 (m, 2H), 3.77 (s, 3H), 3.69 (s, 3H),
3.14 (d, 1H, Jˆ6.6 Hz), 2.75 (t, 2H, Jˆ7.7 Hz), 2.18–1.8
(m, 2H); 13C NMR (CDCl3): d 172.7, 172.2, 155.4, 153.8,
140.2, 137.2, 133.7, 132.5, 128.3, 80.1, 77.4, 53.4, 52.4,
52.3, 33.9, 28.8, 28.3, 28.2, 26.0; ESI-MS (m/z): 512
(MϩH)ϩ; HRMS (FAB, m/z): calcd for C24H38N3O9,
512.2608 (MϩH)ϩ; observed, 512.2620.
(Ϫ)-Pyridinium compound (27). A mixture of (S,S)-(ϩ)-
26 (0.062 g, 0.12 mmol) and (S)-(Ϫ)-6 (0.075 g,
0.182 mmol, 1.5 equiv.) in 1,4-dioxane (1.5 mL) was
refluxed for 4 h under nitrogen. The solvent was removed
on a rotary evaporator to dryness and the crude product was
purified by preparative RP HPLC (MeCN:0.1% aq.
trifluoroacetic acid/50:50, 45.0 mL/min at 225 nm). The
product was lyophilized to afford 0.031 g of (Ϫ)-27 in
27% yield. Analytical RP HPLC: MeCN:0.1% aq. trifluoro-
acetic acid/50:50, 2.0 mL/min, 225 nm, Rt: 8.95 min, 98%;
[a]2D3ˆϪ16.4 (c 0.495, MeOH); 1H NMR (CD3OD): d 8.38
(s, 1H), 8.12 (s, 1H), 4.68–4.60, (m, 1H), 4.51–4.30 (m,
2H), 4.19–4.10 (m, 1H), 3.97–3.91 (m, 1H), 3.74 (s, 3H),
3.73 (s, 3H), 3.46–3.38 (m, 1H), 3.22–3.18 (m, 1H), 3.02–
2.84 (m, 2H), 2.20–1.45 (m, 8H), 1.46 (s, 9H), 1.45 (s, 9H),
1.43 (s, 9H), 1.34 (s, 9H); ESI-MS (m/z): 797 (M)ϩ; HRMS
(FAB, m/z): calcd for C39H65N4O13, 797.4543 (M)ϩ;
observed, 797.4542.
(ϩ)-Deoxypyridinoline (Dpd, 2). A solution of LiOH
(monohydrate, 0.0055 g, 0.13 mmol, 4.0 equiv.) in water
(1.0 mL) was added to a solution of (Ϫ)-27 (0.030 g,
0.033 mmol) in THF (3.0 mL) at room temperature. After
stirring the mixture for 1 h it was concentrated on a rotary

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10. (a) Arbault, P.; Gineyts, E.; Grimaux, M.; Seguin, P.; Delmas,
P. D. J. Liquid Chromatography 1994, 17, 1981. (b) Meddah, B.;
Kamel, S.; Giroud, C.; Brazier, M. Prep. Biochem. Biotechnol.
1999, 29, 63. (c) Robins, S. P.; Duncan, A.; Wilson, N.; Evans,
B. J. Clin. Chem. 1996, 42, 1621.
17. Sibi, M. P.; Renhowe, P. A. Tetrahedron Lett. 1990, 31, 7407.
18. Korytnyk, W.; Srivastava, S. C.; Angelino, N.; Potti, P. G. G.;
Paul, B. J. Med. Chem. 1973, 16, 1096.
19. (a) Korytnyk, W.; Paul, B. J. Med. Chem. 1970, 13, 187. (b)
Williams, K.; Halpern, B. Synthesis 1974, 727. (c) Wuest, H. M.;
Bigot, J. A.; DeBoer, T. J.; Wibaut, J. P. Proc. K. Ned. Akad. Wet.
Ser. B 1958, 61, 150.
11. Waelchli, R.; Beerli, Ch.; Meigel, H.; Revesz, L. Bioorg. Med.
Chem. Lett. 1997, 7, 2831.
12. (a) Adamczyk, M.; Johnson, D. D.; Reddy, R. E. Tetrahedron
1999, 55, 63. (b) Adamczyk, M.; Johnson, D. D.; Reddy, R. E.
Tetrahedron: Asymmetry 1999, 10, 775.
13. Hatch, R. P. US Patent 5723619, 1998. CA: 1998, 128,
205137.
14. (a) Allevi, P.; Longo, A.; Anastasia, M. Chem Commun. 1999,
559. (b) Allevi, P.; Longo, A.; Anastasia, M. J. Chem. Soc., Perkin
Trans 1 1999, 2867.
15. For preliminary results of the work see, Adamczyk, M.;
Akireddy, S. R.; Reddy, R. E. Tetrahedron: Asymmetry 1999, 10,
3107.
20. The diastereomeric mixture of (S,R/R,R)-17d was incorrectly
[(S,R/S,S)] entered in Ref. 15.
21. Davis, F. A.; Szewczyk, J. M.; Reddy, R. E. J. Org. Chem.
1996, 61, 2222.
22. Wild, D. The Immunoassay Handbook, Stockton Press: New
York, 1994.
23. Ishizuka, T.; Kunieda, T. Tetrahedron Lett. 1987, 28, 4185.
24. Rossi, F.; Powers, E. T.; Yoon, R.; Rosenberg, L.; Meinwald,
J. Tetrahedron 1996, 52, 10279.
25. Hasimoto, N.; Aoyama, T.; Shioiri, T. Chem. Pharm. Bull.
1981, 29, 3249.
16. Schollkopf, U.; Groth, U.; Deng, C. Angew. Chem., Int. Ed.
Engl. 1981, 20, 798.