Versatile synthesis of (+)-deoxypyridinoline, a biochemical marker for diagnosis of osteoporosis

Article abstract of DOI:10.1016/S0957-4166(99)00329-8

A versatile chiral synthesis of the bone collagen cross-link, (+)-deoxypyridinoline (Dpd, 1) was described starting from a 3-hydroxypyridine derivative (2) via sequential introduction of three amino acid chains followed by hydrolysis. The key synthon 2, was prepared from vitamin B₆ (6).

Full text of DOI:10.1016/S0957-4166(99)00329-8

Pergamon
Tetrahedron: Asymmetry 10 (1999) 3107–3110
Versatile synthesis of (+)-deoxypyridinoline, a biochemical
marker for diagnosis of osteoporosis
Maciej Adamczyk,∗ Srinivasa Rao Akireddy and Rajarathnam E. Reddy
Division Organic Chemistry Research (9NM, AP20), Diagnostics Division, Abbott Laboratories, 100 Abbott Park Road,
Abbott Park, IL 60064-6016, USA
Received 16 July 1999; accepted 29 July 1999
Abstract
A versatile chiral synthesis of the bone collagen cross-link, (+)-deoxypyridinoline (Dpd, 1) was described
starting from a 3-hydroxypyridinederivative (2) via sequential introduction of three amino acid chains followed by
hydrolysis. The key synthon 2, was prepared from vitamin B6 (6). © 1999 Published by Elsevier Science Ltd. All
rights reserved.
Osteoporosis is a degenerative bone disease, which affects the aged population, particularly postmeno-
pausal women.1 The current methods for diagnosis of osteoporosis involve histomorphometry and densi-
tometry of bones.2 The efforts for prevention of this metabolic bone disease as well as for development of
effective antiresorptive therapy, have increased the search for more reliable and noninvasive biochemical
markers.3 In recent years, the pyridinium cross-link, deoxypyridinoline (Dpd, 1)4 (Fig. 1) has attracted
considerable attention due its clinical utility.5–7 Thus, Dpd (1) is needed for use as calibrators, controls,
clinical reference standards, and for immunoassay development. Currently, it is isolated in a very low
yield from bones (sheep, ox, turkey, etc.) by 6–9 M HCl hydrolysis at 110°C, a process that could affect
the integrity of the stereocenters in Dpd.8 Therefore, Dpd (1) became an attractive synthetic target due to
its novel structural features and practical applications, since its analogs are imperative for diagnosis and
therapy management of osteoporosis and other bone diseases. The reported syntheses of Dpd (1) involved
construction of a substituted pyridine ring from amino acid components utilizing aldol chemistry.9 In
this paper we present a novel and conceptually different approach for (+)-Dpd (1) starting from a 3-
hydroxypyridine derivative (2).
In this new strategy, the (+)-Dpd (1) (Fig. 1) was constructed from a 3-hydroxypyridine derivative (2)
via introduction of three amino acid chains sequentially, which provides diverse flexibility for preparation
of a variety of its analogs. The 4- and 5-amino acid chains in (+)-1 were introduced by utilizing (2R)-(−)-
2,5-dihydro-3,6-dimethoxy-2-isopropylpyrazine (Schollkopf’s reagent, 3)10 and Wittig reagent (R)-(−)-
4,11 respectively, and the lysine chain at the 1-position of the pyridine ring was installed via quaternization
∗
Corresponding author. E-mail: maciej.adamczyk@abbott.com
0957-4166/99/$ - see front matter © 1999 Published by Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(99)00329-8

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M. Adamczyk et al. / Tetrahedron: Asymmetry 10 (1999) 3107–3110
Figure 1.
with iodide (S)-(−)-5. The key synthon, chloride 2, was envisioned from an inexpensive vitamin B6 (6).
Thus, the 2-methyl group in B6 (6) was functionalized (Scheme 1) to the corresponding hydroxy methyl,
after protecting its native hydroxyl groups,12 to give 7, which upon oxidation and decarboxylation gave
2-norpyridoxine derivative 8.13 The acetonide group in 8 was hydrolyzed and the 3-hydroxy group was
selectively protected as the benzyl ether using benzyldimethyl phenylammonium chloride14 to afford 9.
The hydroxyl group in 9 was then converted to the chloride and the p-methoxybenzyl group hydrolyzed
using 0.5N HCl to give the desired chloride 2 in 58% yield.
Scheme 1. Reagents and conditions. (i) a. Korytnyk et al.12; b. NaH, PMBCl, DMF, 0°C–rt, 15 h, 83%; (ii) a. m-CPBA, CH2Cl2,
rt, 2 h; b. TFAA, CH2Cl2, 0°C–rt, 5 h, MeOH, 86%; (iii) a. MnO2, CHCl3, rt, 21 h; b. Ag2O, KOH, EtOH, rt, 1 h, then xylene,
reflux, 25 min, 71%; (iv) a. PPTS, EtOH, reflux, 3 days 71%; b. PhNMe2BnCl, EtONa, EtOH, −78°C, then xylene, reflux, 4 h,
30%; (v) SOCl2, benzene, 0°C–reflux, 0.5 h then 0.5N HCl, reflux, 15 min, 58%
With gram quantities of chloride 2 in hand, we proceeded to the sequential introduction of amino acid
chains present in (+)-Dpd (1). Thus, alkylation (Scheme 2) of (R)-(−)-3 with the chloride 2 using n-BuLi
at −78°C, gave a diastereomeric mixture of 10 [(S,R:S,S)/69:31] in 86% yield. Our attempts to separate the
isomeric mixture (S,R/S,S)-10 by a variety of techniques, including preparative HPLC, were unsuccessful.
However, to our delight, the corresponding diastereomeric mixture of aldehydes 11, which was obtained
by oxidation of (S,R/S,S)-10 with MnO2, was easily separated by silica gel column chromatography
(40% EtOAc in hexanes) to afford the desired major isomer, (S,R)-(−)-11, in 62% yield and >98% de.
Wittig reaction of aldehyde (S,R)-(−)-11 with the ylide, which was generated from (R)-(−)-4 using n-
BuLi in THF at −78°C, gave the olefin 12 in good yield (72%) as a mixture of E:Z isomers (ratio,
64:36). Hydrolysis of the dihydropyrazine ring in 12 was carried out using 0.5N HCl in MeCN, and the
protection of the resulting free amine using (Boc)2O in MeCN afforded the tri-Boc compound (S,S)-13
(E:Z/64:36) in 56% overall yield. The (S,S)-13 was subjected to hydrolysis with cesium carbonate,15 and
then hydrogenated over 10% Pd/C in methanol to afford (S,S)-(−)-14 in good overall yield. Concerned
with the sensitivity of the bis-(tert-butoxycarbonyl)amino group in (S,S)-(−)-14 for subsequent oxidation
and quaternization steps, it was therefore selectively hydrolyzed with trifluoroacetic acid16 in CH2Cl2 to
give (S,S)-(−)-15 in 69% yield. Oxidation of (S,S)-(−)-15 with Jones reagent17 followed by esterification
afforded (S,S)-(+)-16 in 55% yield as a colorless solid {[α]20 +17.3 (c 0.59, CHCl3)}.
D
The 3-hydroxypyridine derivative (S,S)-(+)-16, requires only elaboration of the lysine chain at the

M. Adamczyk et al. / Tetrahedron: Asymmetry 10 (1999) 3107–3110
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Scheme 2. Reagents and conditions. (i) a. (R)-(−)-3, n-BuLi, THF, −78°C, 5 h, 86%, [(S,R:S,S)/69:31]; b. MnO2, CHCl3, rt,
20 h, 62%, de: >98%; (ii) a. (R)-(−)-4, n-BuLi, THF, −78°C, 4 h, 72%, (E:Z/64:36); (iii) 0.5N HCl, MeCN, rt, 45 min then
(Boc)2O, DMAP, Et3N, MeCN, rt, 6 h, 56%; (iv) a. Cs2CO3, MeOH, rt, 2.5 h, 63%; b. 10% Pd/C, MeOH, H2, HCl, 20 psi, 4 h,
78%; c. TFA, CH2Cl2, rt, 26 h, 69%; (v) Jones reagent, acetone, rt, 40 min, then TMSCHN2, benzene, MeOH, rt, 15 min, 55%;
(vi) (S)-(−)-5, anhydrous 1,4-dioxane, reflux, 4 h, 27%; (vii) LiOH, THF–water, rt, 1 h, then TFA–water (ratio, 9.5:0.5), rt, 1 h,
84%
1-position for completion of Dpd (1) synthesis. Thus, quaternization of (S,S)-(+)-16 with (S)-(−)-tert-
butyl-[(2-tert-butoxycarbonyl)amino]-6-iodohexanoate (5)9,16a was carried out in refluxing 1,4-dioxane
for 4 h, and the product was purified by preparative reverse phase HPLC to afford pyridinium compound
(−)-17 in 27% yield. Finally, alkaline hydrolysis of the methyl esters in (−)-17 using lithium hydroxide
in THF–water, followed by removal of the Boc and t-butyl protective groups with TFA–water (9.5:0.5
ratio) and purification by preparative reverse phase HPLC afforded (+)-Dpd (1) in 84% yield as its TFA
salt {[α]20 +36.2 (c 0.54, MeOH); lit.9a [α]20 +31.6 (c 0.25, MeOH)}.
D
D
In summary, a versatile synthesis of (+)-deoxypyridinoline (1), a biochemical marker for the diagnosis
of osteoporosis was developed from 3-hydroxypyridine derivative (2). The method is amenable for
preparation of variety of Dpd analogs and immunoreagents useful for diagnosis and treatment of
osteoporosis.
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