Practical syntheses of pyridinolines, important amino acidic biomarkers of collagen health

Article abstract of DOI:10.1021/jo070136g

(Chemical Equation Presented) The paper reports some successful results on the first fully stereoselective total synthesis of the collagen cross-link pyridinolines. All stereogenic centers are stereoselectively introduced using Williams glycine template m

Full text of DOI:10.1021/jo070136g

Practical Syntheses of Pyridinolines, Important Amino Acidic
Biomarkers of Collagen Health
Pietro Allevi, Riccardo Cribiu`, Elios Giannini, and Mario Anastasia*
Dipartimento di Chimica, Biochimica e Biotecnologie per la Medicina, UniVersita` di Milano,
Via Saldini 50, I-20133 Milano, Italy
mario.anastasia@unimi.it
ReceiVed January 23, 2007
The paper reports some successful results on the first fully stereoselective total synthesis of the collagen
cross-link pyridinolines. All stereogenic centers are stereoselectively introduced using Williams glycine
template methodology, and oxazinones are used as a source of chirality and as easily removable protecting
groups of the amino acidic functionalities during the assembly of the pyridinoline nucleus.
Introduction
However, although the synthetic routes for preparing dPyd (2)
have been exploited and well set up, those affording Pyd (1)
are far less satisfactory due to some difficulties related to the
stereoselective introduction of the hydroxy group of the hy-
droxylysine residue bonded to the heterocyclic nitrogen.7 These
difficulties were partially overcome by a more efficient protocol
for the preparation of Pyd (1), developed in our laboratory.7
Herein, we report two new efficient routes for the preparation
of Pyd (1) and dPyd (2), based on the Williams morpholinone
glycine synthons,8 both to generate the stereogenic R-amino
acidic centers and to suitably protect the amino acidic functions
Pyridinoline (Pyd; 1) and deoxypyridinoline (dPyd; 2) are
two collagen cross-links considered, to this day, as useful
biochemical markers of bone resorption which can be correlated
with diseases such as osteoporosis, bone cancer, and arthropa-
thies and used to assess fracture risk prediction in older
persons.1-3 They are detected in human urine to permit early
diagnosis of osteoporosis, for monitoring drug therapy of this
bone disease, or for the follow-up of patients with malignant
bone disease.4a-f As a consequence, it is important to have
suitable amounts of pure Pyd (1) and dPyd (2) to be used as
primary reference standards in analytical protocols. These cross-
links, especially pyridinoline, are now obtained from natural
sources,5 after some tedious purifications, and by synthesis.6
(4) (a) Roth, M.; Uebelhart, D. Clin. Chim. Acta 2003, 327, 81. (b)
Garnero, P.; Delmas, P. D. Biochemical markers of Bone turnover. In
Osteoporosis; Marcus, R., Feldman, D., Kelsey, J., Eds.; Academic Press:
San Diego, 1996; pp 1075-85. (c) Seibel, M. Clin. Prat. Oncology 2005,
2, 504. (d) Angeliewa, A.; Budde, M.; Schlachter, M.; Hoyle, N. R.; Bauss,
F. J. Bone Miner. Metab. 2004, 22, 192. (e) Garnero, P.; Landewe`, R.;
Boers, M.; Verhoeven, A.; van der Linden, S.; Chiristgau, S.; van der Heijde,
D.; Boonen, A.; Geusens, P. Artritis Reum. 2002, 46, 2847. (f) Vesper, H.
W.; Demers, L. M.; Eastell, R.; Garnero, P.; Kleerekoper, M.; Robins, S.
P.; Srivastava, A. K.; Warnick, G. R.; Watts, N. B.; Myers, G. Clin. Chem.
2002, 48, 220.
(5) (a) Robins, S. P.; Duncan, A.; Wilson, N.; Evans, B. J. Clin. Chem.
1996, 42, 1621. (b) Fujimoto, D.; Moriguchi, T.; Ishida, T.; Hayashi, H.
Biochem. Biophys. Res. Commun. 1978, 84, 52. (c) Ogawa, T.; Ono, T.;
Tsuda, M.; Kawanishi, Y. Biochem. Biophys. Res. Commun. 1982, 107,
1252.
(1) (a) Kleerekoper, M.; Camacho, P. Clin. Chem. 2005, 51, 2227. (b)
James, I. T.; Walne, A. J.; Perrett, D. Ann. Clin. Biochem. 1996, 33 397.
(c) For a comprehensive review on the biology of bones, see: Priciples of
Bone Biology; Bilezikan, J. P., Raicz, L. G., Rogan, G., Eds.; Academic
Press: New York, 2001.
(2) Stone, K. L.; Seeley, D. G.; Lui, L. Y.; Cauley, J. A.; Ensrud, K.;
Browner, W. S. J.; Nevitt, M. C.; Cummings, S. R. Bone Miner. Res. 2003,
18, 1947.
(3) Schuit, S. C. E.; van der Klift, M.; Weel, A. E. A. M.; de Laet, C.
E. D. H.; Burger, H.; Seemen, E.; Hofman, A.; Uitterlinden, A. G.; van
Leeuwen, J. P. T. M.; Pols, H. A. P. Bone 2004, 34, 195.
10.1021/jo070136g CCC: $37.00 © 2007 American Chemical Society
Published on Web 03/31/2007
3478
J. Org. Chem. 2007, 72, 3478-3483

Practical Syntheses of Pyridinolines
The amino acidic iodohydrin 5, having the required R-
stereochemistry of the hydroxylated side chain of Pyd 1, was
then transformed into the azido alcohol 7a, which, by catalytic
reduction, affords the amino alcohol 8a. On the other hand, the
epimeric iodohydrin 6, having the opposite S-stereochemistry
at the hydroxylated center, was used to prepare the desired
R-iodoketone 9 by oxidation with pyridinium chlorochromate.12
Moreover, considering that the hydroxy group of 7a could
interfere with the iodoketone 9 during the assembly of the
pyridinium nucleus, thus depleting the reaction yields, we
decided to use as starting material the silyloxyamine 8b easily
prepared via silylation of the hydroxyazide 7a and successive
catalytic hydrogenation, in the presence of Pd/C (Scheme 2).
Then, we started the assembly of the pyridinoline nucleus
(Scheme 3), setting up the bisalkylation of the hydroxyamine
8a with the iodoketone 9 (reacted in a 1:2.5 molar ratio), in
acetonitrile, in the presence of sodium carbonate, at room
temperature.6c The reaction was followed on TLC to monitor
the complete transformation of the starting amine first into the
monoalkylated derivative and then into the diketoamine 10a.
At this point, the simple exchange of the reaction solvent (THF
for CH3CN) and of the base (DBU for Na2CO3) induced the
diketoamine 10a to undergo cyclization and aromatization to
afford the protected pyridinoline 12a via the intermediate
formation of the R,â-unsaturated ketone 11a.
FIGURE 1. Chemical structures of pyridinoline 1 and deoxypyridi-
noline 2.
during the assembly of the aromatic nucleus and to simplify
the preparation of the 5R-hydroxylysine chain of Pyd (1)
(Figure 1).
Results and Discussions
On the basis of our previous experience6,7 with the synthesis
of pyridinolines, we hypothesized a possible route for the
preparation of Pyd (1), programming a chemoselective reaction
of a halogen ketone B with a suitable amine A, followed by a
cyclization-aromatization sequence affording a substituted
hydroxypyridine nucleus with three amino acidic side chains
completely protected with identical protective groups
(Scheme 1).
These optimized reaction conditions are the result of several
less satisfactory attempts in which the cyclization-aromatization
reaction was performed in methanol containing Na2CO3. In fact,
under these other conditions, a partial transesterification of the
lactonic groups of the glycine synthons with formation of a
mixture of methyl esters occurred. On the other hand, using
tetrahydrofuran as solvent and Na2CO3 as base, the reaction was
too slow and incomplete. The intermediate R,â-unsaturated
ketone 11a could not be isolated by standard procedures, being
always accompanied (TLC evidence) by the starting diketoamine
10a during its transformation into the protected pyridinoline 12a.
Compound 12a was purified by rapid column chromatography
to afford a glassy product (in about 21% yield) which showed
the correct molecular formula and the expected physical
chemical properties. At this point, with the aim to improve the
yields obtained in the assembly of the aromatic nucleus of
pyridinoline, starting from the hydroxyamine 8a, we performed
a parallel reaction sequence starting with the O-trimethylsilyloxy
amine 8b (Scheme 3). While monitoring this reaction by TLC,
together with the expected silylated compound 12b, we observed
the formation of a more polar compound corresponding to the
previously obtained protected pyridinoline 12a lacking the silyl
protection. Considering that the silyl group suffered a partial
hydrolysis on TLC, we subjected the reaction mixture to a rapid
chromatography on silica and obtained the desilylated compound
12a in higher yield (34%) than starting with the free hy-
droxyamine 8a. At this point, the accomplishment of the
synthesis required only the deblocking of the protected amino
acidic functions of compound 12a. This could be performed
following the reductive protocols reported by Williams et al.8,9
(i.e., using alkali metals dissolved in liquid ammonia or a
catalytic hydrogenation over a Pd° catalyst). Thus, we first
performed the deblocking using sodium in liquid ammonia
which, in our recent work on the synthesis of reducible collagen
cross-links, caused a rapid and complete cleavage of the
With this in mind, we envisioned a synthesis which uses
Williams’ morpholinones8,9 for a convenient preparation of the
pyridinoline precursors A and B (Scheme 1). As we anticipated,
our results show that Williams’ morpholinone glycine synthon
3, prepared by alkylation of the appropriate chiral oxazinone9
and transformed in our laboratory into the epoxidic mixture 4,
could be a key substance for the preparation of both the
R-hydroxyamine 7a and the iodoketone 9 (Scheme 2) necessary
for the synthetic project.10
Epoxidation of the olefin 3 affords an inseparable mixture
of epoxides 4 which, however, by reaction with sodium iodide
affords a diastereomeric mixture of iodohydrins 5 and 6. These,
onthecontrary,areeasilyseparatedbyrapidflashchromatography.11a
(6) (a) Allevi, P.; Ciuffreda, P.; Longo, A.; Anastasia M. 4th Convegno
Nazionale di chimica organica, Salerno, Italy, September 21-25, 1997;
Italian Chemical Society; Commun. No. 03. (b) Waelchli, R.; Beerli, C.
H.; Meigel, H.; Re´ve´sz, L. Bioorg. Med. Chem. Lett. 1997, 7, 2831. (c)
Allevi, P.; Longo, A.; Anastasia, M. Chem. Commun. 1999, 559. (d) Allevi,
P.; Longo, A.; Anastasia, M. J. Mol. Sci. (China) 1999, 15, 154. (e) Hatch,
R. P. U.S. Pat. 5723619, 1998; Chem. Abstr. 1998, 128, 205137H. (f)
Adamczyk, M.; Johnson, D. D.; Reddy, R. E. Tetrahedron 1999, 55, 63.
(g) Adamczyk, M.; Johnson, D. D.; Reddy, R. E. Tetrahedron Lett. 1999,
40, 8993. And corrigendum: Tetrahedron Lett. 2002, 42, 767. (h)
Adamczyk, M.; Johnson, D. D.; Reddy, R. E. Tetrahedron: Asymmetry
2000, 11, 2289. And corrigendum: Tetrahedron: Asymmetry 2000, 11,
5017. (i) For a complete record of more recent work, see: Allevi, P.; Cribiu`,
R.; Giannini, E.; Anastasia, M. Bioconjugate Chem. 2005, 16, 1045 and
references therein.
(7) Allevi, P.; Anastasia, M. Tetrahedron: Asymmetry 2003, 14, 2005
and references therein.
(8) Williams, M. R.; Sinclair, J. P.; DeMong, D. E.; Chein, D.; Zhai, D.
Org. Synth. 2003, 80, 18 and references therein.
(9) Williams, M. R.; Im, M.-N.; Cao, J. J. Am. Chem. Soc. 1991, 113,
6976.
(10) For a recent successful use of William’s morpholinones for the
synthesis of hydroxylysine, see: (a) van den Nieuwendijk, A. M. C. H.;
Kriek, N. M. A. J.; Brussee, J.; van Boom, J. H.; van der Gen, A. Eur. J.
Org. Chem. 2000, 3683. (b) Allevi, P.; Anastasia, M. Tetrahedron:
Asymmetry 2004, 15, 2091.
(11) (a) Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43, 2923.
(b) The assignment of the stereochemistry of these compounds was
previously reported by us in ref 10b.
(12) Corey, E. J.; Suggs, J. W. Tetrahedron Lett. 1975, 2647.
J. Org. Chem, Vol. 72, No. 9, 2007 3479

Allevi et al.
SCHEME 1
SCHEME 2
oxazinones.13 Moreover, after some unsuccessful results, prob-
ably due to the high functionalization of the molecule and to
the difficulty of separating the water-soluble reaction product
from the inorganic and organic byproducts, we turned our
attention to the hydrogenolytic cleavage of the oxazinone rings
present in compound 12a. This deblocking procedure requires
a preliminary acidic cleavage of the Boc groups, a generally
simple reaction which, in our case, caused some initial difficul-
ties due to the presence of the hydroxyl group in the hydroxy-
lysine residue of 12a. In fact, any attempt to cleave the Boc
protecting groups in the usual conditions (using aqueous
trifluoroacetic acid14a or formic acid)14b resulted in the contem-
poraneous partial trans-lactonization between the alcoholic
hydroxy group of 12a and the near lactonic group of the
oxazinone ring. Finally, using ZnBr2 in dichloromethane,15 the
cleavage of the Boc groups of compound 12a occurred in good
yields and without any undesired inner trans-lactonization. The
deprotected oxazinone 13 formed in the reaction (Scheme 4)
was then hydrogenated in the presence of palladium chloride
at room temperature and pressure to regenerate the free
pyridinoline (1) in good yields (80% from 12a). Moreover, we
were also able to extend the scope of our result, setting up a
different procedure (Scheme 4) in which the iodoketone 9 and
a self-immolative allylamine, for the introduction of the aromatic
nitrogen atom in the pyridine ring, were used. Also in this case,
reacting the iodoketone 9 with allylamine in the presence of
Na2CO3 causes the bisalkylation to occur via formation of the
intermediate diketoamine which, after exchange of the reaction
solvent (THF for acetonitrile) and addition of DBU, suffers
cyclization and oxidative aromatization to afford the fluorescent
pyridinium salt 14 (in 27% yields).
This compound was completely characterized and subjected
to the successive deallylation, performed using tetrakispalladium
triphenyl phosphine and thiosalicylic acid as an allyl group
scavenger,16a,b during the dealkylation. The hydroxypyridinium
derivative 15 was obtained in good yields (78%) and in a
satisfactory purity, as a white solid stable at 4 °C for almost 1
year. Successive alkylation of 15 with iodohydrine 5 in
acetonitrile at 80 °C afforded the protected pyridinoline 12 which
was then deprotected, following the procedure described for the
synthesis of Pyd (1). In principle, alkylation of the compound
15 should afford also dPyr (2) or any other pyridinoline, simply
changing the alkyl iodide to construct the chain bonded to the
(13) Cribiu`, R.; Allevi, P.; Anastasia, M. Tetrahedron: Asymmetry 2005,
16, 3059.
(14) (a) Schanabel, E.; Klostermeyer, H.; Berndt, H. Liebigs Ann. Chem.
1971, 749, 90. (b) Kinoshita, H.; Hammam, M. A. S.; Inomata, K. Chem.
Lett. 2005, 34, 800.
(15) Nigam, S. C.; Mann, A.; Taddei, M.; Wermuth, C. G. Synth.
Commun. 1989, 19, 3139.
(16) (a) Hayakawa, Y.; Kato, H.; Kajino, M. U. H.; Noyori, R. J. Org.
Chem. 1986, 51, 2402. (b) Bailey, W. F.; Jiang, X. L. J. Org. Chem. 1996,
61, 2956.
3480 J. Org. Chem., Vol. 72, No. 9, 2007

Practical Syntheses of Pyridinolines
SCHEME 3
SCHEME 4
presence of distinguishable rotamers.17 For some more complex
compounds, the signals of major conformers are described, even if
signals of minor rotamers were present in the spectra. Optical
rotations were taken at 23 °C and [R]D values are given in 10-1
deg cm2 g-1. HPLC analyses were carried out on a RP-18 column
(125 mm, 4 mm ID, 5 µm), and elution was performed with a 20
min linear gradient from 100% of solvent A [0.01 M heptafluo-
robutanoic acid (HFBA) in CH3CN/water 10:90 v:v] to 100% of
solvent B (0.01 M HFBA in CH3CN/water 90:10 v:v). The flow
rate was 1.0 mL/min, and the detection was performed at 293 nm.
Mass spectra were obtained using a ion trap mass spectrometer
fitted with an electrospray source (ESI). All reactions were
monitored by thin-layer chromatography (TLC) carried out on 0.25
mm silica gel plates (60 F254) using UV light, 50% sulfuric acid or
0.2% ninhydrin in ethanol, and heat as the developing agent. Silica
gel (230-400 mesh) was used for flash silica gel chromatography.11a
tert-Butyl (3S,5S,6R)-3-[(R)-4-Amino-3-hydroxybutyl)-2-oxo-
5,6-diphenylmorpholine-4-carboxylate (8a). A solution of tert-
butyl (3S,5S,6R)-3-[(R)-4-azido-3-hydroxybutyl)-2-oxo-5,6-diphe-
nylmorpholine-4-carboxylate (7a)10b (980 mg, 2.10 mmol) in AcOEt
(170 mL) was hydrogenated in the presence of 10% Pd/C (40 mg)
at room temperature and atmospheric pressure. The mixture was
nitrogen atom of the pyridine ring. Thus, we may conclude that
we have developed two routes for an efficient synthesis of
pyridinoline overcoming the difficulties with the introduction
of the correct stereochemistry of the 5-hydroxy group and also
have set up a useful and simple method to obtain a proximate
precursor of Pyd (1) and deoxypyridinoline dPyd (2), by a
modular approach.
Experimental Section
General Methods. Melting points are uncorrected. Nuclear
magnetic resonance spectra were recorded at 298 K on a spec-
1
trometer operating at 500.13 MHz for H and at 125.76 MHz for
13C. Chemical shifts are reported in parts per million (ppm, δ units)
1
relative to the CHCl3 signal fixed at 7.26 ppm for H spectra and
to the CDCl3 signal fixed at 77.0 ppm for 13C spectra. Proton and
carbon assignments were established, if necessary, with 1H-1H and
1H-13C correlated NMR experiments. 1H NMR data are tabulated
in the following order: number of protons, multiplicity (s, singlet;
d, doublet; br s, broad singlet; m, multiplet), coupling constant(s)
in Hz, assignment of proton(s). 1H NMR and 13C NMR spectra of
compounds containing oxazinone ring(s) were complicated by the
(17) (a) William, M. R.; Yuan, C. J. Org. Chem. 1992, 57, 6519. (b)
William, M. R.; Sinclair, J. P.; DeMong, D. E.; Chein, D.; Zhai, D. Org.
Synth. 2003, 80, 18 and references therein.
J. Org. Chem, Vol. 72, No. 9, 2007 3481

Allevi et al.
filtered through a pad of Celite, and the solvent was evaporated
under reduced pressure to afford the hydroxyamine 8a (842 mg,
91%) as a white solid: mp 155-158 °C (from CH2Cl2/hexane);
4.81 (0.3 H, dd, J ) 9.8, 4.6 Hz, 3-H, minor conformer), 3.74 (0.7H,
m, 3′-H, major conformer), 3.70 (0.3H, m, 3′-H, minor conformer),
2.72 (2H, m, 4′-H2, both conformers), 2.16 (1H, m, 1′-Ha, both
conformers), 2.07 (1H, m, 1′-Hb, both conformers), 1.78 (2H, m,
2′-H2, both conformers), 1.46, 1.17 [9H, 2 × s, 2 × (CH3)3C, both
conformers], 0.18 [9H, s, (CH3)3Si, both conformers]; 13C NMR
(CDCl3) (major conformer) δ 169.2 (2-C), 153.6 (tBuOCdO), 136.4
(aromatic), 134.3 (aromatic), 128.5-126.4 (aromatics), 81.1
[(CH3)3C], 78.9 (6-C), 73.5 (3′-C), 61.4 (5-C), 56.5 (3-C), 47.7
(4′-C), 31.1 (1′-C), 30.8 (2′-C), 27.7 [(CH3)3C], 0.4 [(CH3)3Si]; IR
(CHCl3) ν 1760, 1705 cm-1; ESI-MS (positive) m/z 513 (M + H+).
Anal. Calcd for C28H40N2O5Si: C, 65.59; H, 7.86; N, 5.46. Found:
C, 65.45; H, 7.81; N, 5.40.
25
1
[R]D -43.9 (c 1, CHCl3); H NMR (CDCl3) δ 7.28-7.00 (8H,
aromatics), 6.59 (2H, aromatics), 6.01 (0.3H, d, J ) 3.2 Hz, 6-H,
minor conformer), 5.98 (0.7H, d, J ) 3.2 Hz, 6-H, major
conformer), 5.25 (0.3H, d, J ) 3.2 Hz, 5-H, minor conformer),
5.02 (1.4H, m, overlapping, 5-H, 3-H, major conformer), 4.86 (0.3H,
dd, J ) 10.5, 4.6 Hz, 3-H, minor conformer), 3.83 (0.7H, m, 3′-H,
major conformer), 3.65 (0.3H, m, 3′-H, minor conformer), 2.94
(0.3H, dd, J ) 12.6, 3.5 Hz, 4′-H, minor conformer), 2.91 (0.7H,
dd, J ) 12.6, 3.5 Hz, 4′-H, major conformer), 2.64 (0.7H, dd, J )
12.6, 8.1 Hz, 4′-H, major conformer), 2.59 (0.3H, dd, J ) 12.6,
8.1 Hz, 4′-H, minor conformer), 2.32 (1H, m, 1′-Ha, both
conformers), 2.22 (1H, m, 1′-Hb, both conformers), 1.77 (1H, m,
2′-Ha, both conformers), 1.65 (1H, m, 2′-Hb, both conformers),
tert-Butyl (3S,5S,6R)-3-(4-Iodo-3-oxobutyl)-2-oxo-5,6-diphe-
nylmorpholine-4-carboxylate (9). To a solution of iodohydrin 610b
(800 mg, 1.45 mmol) dissolved in CH2Cl2 (35 mL) was added
pyridinium chlorochromate12 (625 mg, 2.90 mmol). The initially
orange solution was then stirred at room temperature for 2 h. At
this time, the complete disappearance of the starting material was
observed (TLC). The reaction was quenched with 2-propanol (1.0
mL), and the solvent was evaporated under reduced pressure. The
residue was purified by flash silica gel chromatography (hexane/
AcOEt 7:3 v:v) to afford, after crystallization, the iodoketone 9
(574 mg, 72% yield) as a white solid: mp 173-174 °C (from CH2-
1.48, 1.14 [9H, 2 × s, (CH3)3C, minor and major conformers]; 13
C
NMR (CDCl3) (major conformer) δ 169.1 (2-C), 153.8 (tBuOCd
O), 136.2 (aromatic), 134.2 (aromatic), 131.7-124.2 (aromatics),
81.5 [(CH3)3C], 79.0 (6-C), 70.3 (3′-C), 61.3 (5-C), 55.4 (3-C),
47.4 (4′-C), 31.1 (1′-C), 30.3 (2′-C), 27.8 [(CH3)3C]; IR (CHCl3) ν
3330, 1758, 1682 cm-1; ESI-MS (positive) m/z 441 (M + H+),
463 (M + Na+), 903 (M + M + Na+). Anal. Calcd for
C25H32N2O5: C, 68.16; H, 7.32; N, 6.36. Found: C, 68.45; H, 7.50;
N, 6.40.
25
Cl2 /diisopropyl ether); [R]D -48.9 (c 1, CHCl3); 1H NMR
tert-Butyl (3S,5S,6R)-3-[(R)-4-Azido-3-(trimethylsilyloxy)bu-
tyl]-2-oxo-5,6-diphenylmorpholine-4-carboxylate (7b). To a stirred
solution of the hydroxyazide 7a10b (395 mg, 0.85 mmol), triethy-
lamine (232 µL, 1.66 mmol), and N,N-dimethylaminopyridine (20
mg, 0.16 mmol) in anhydrous THF (6.0 mL) was added TMSCl
(127 µL, 1.00 mmol). The resulting mixture was stirred at room
temperature for 7 h and then diluted with AcOEt (50 mL). The
solution was washed with brine, dried over anhydrous Na2SO4, and
evaporated under reduced pressure. The crude residue was crystal-
lized to afford the trimethylsilyl derivative 7b (410 mg, 90%) as
(CDCl3) δ 7.29-7.00 (8H, aromatics), 6.58 (2H, aromatics), 6.06
(0.7H, d, J ) 2.8 Hz, 6-H, major conformer), 6.01 (0.3H, br s,
6-H, minor conformer), 5.24 (0.3H, d, J ) 2.8 Hz, 5-H, minor
conformer), 5.00 (1.4H, m, overlapping, 5-H and 3-H, major
conformer), 4.83 (0.3 H, dd, J ) 9.8 and 5.6 Hz, 3-H, minor
conformer), 3.92 (2H, s, 4′-H, both conformers), 3.15 (1H, t, J )
7.0 Hz, 2′-Ha, both conformers), 3.08 (1H, dd, J ) 13.7, 7.0 Hz,
2′-Hb, both conformers), 2.55 (1H, m, 1′-Ha, both conformers),
2.33 (1H, m, 1′-Hb, both conformers), 1.49, 1.11 [9H, 2 × s,
(CH3)3C, minor and major conformers]; 13C NMR (CDCl3) (major
conformer) δ 201.9 (3′-C), 169.3 (2-C), 153.9 (tBuOCdO), 135.2
(aromatic), 134.1 (aromatic), 128.6-125.9 (aromatics), 81.4
[(CH3)3C], 78.9 (6-C), 61.5 (5-C), 55.6 (3-C), 35.5 (2′-C), 27.8
[(CH3)3C], 28.9 (1′-C), 5.9 (4′-C); IR (CHCl3) ν 1752, 1728, 1705
cm-1; ESI-MS (positive) m/z 572 (M + Na+). Anal. Calcd for
C25H28INO5: C, 54.65; H, 5.14; N, 2.55. Found: C, 54.49; H, 5.31;
N, 2.61.
25
white solid: mp 149 °C (from CH2Cl2 /hexane); [R]D -39.5 (c
1
1, CHCl3); H NMR (CDCl3) δ 7.29-7.00 (8H, aromatics), 6.59
(2H, aromatics), 5.93 (1H, br s, 6-H, both conformers), 5.26 (0.3H,
d, J ) 3.5 Hz, 5-H, minor conformer), 5.04 (0.7H, d, J ) 3.5 Hz,
5-H, major conformer), 5.01 (0.7H, dd, J ) 9.5, 5.3 Hz, 3-H, major
conformer), 4.83 (0.3H, dd, J ) 9.8, 5.0 Hz, 3-H, minor conformer),
3.94 (0.7H, m, 3′-H, major conformer), 3.89 (0.3H, m, 3′-H, minor
conformer), 3.27 (2H, m, 4′-H2, both conformers), 2.17 (1H, m,
1′-Ha, both conformers), 2.09 (1H, m, 1′-Hb, both conformers),
1.83 (2H, m, 2′-H2, both conformers), 1.48, 1.12 [9H, 2 × s, 2 ×
(CH3)3C, minor and major conformers], 0.22, 0.21 [9H, 2 × s,
(CH3)3Si, major and minor conformers]; 13C NMR (CDCl3) (major
conformer) δ 169.1 (2-C), 153.6 (tBuOCdO), 136.4 (aromatic),
134.3 (aromatic), 128.6-126.3 (aromatics), 81.2 [(CH3)3C], 79.0
(6-C), 71.2 (3′-C), 61.5 (5-C), 56.8 (4′-C), 56.4 (3-C), 31.2 (1′-C),
30.9 (2′-C), 27.9 [(CH3)3C], 0.3 [(CH3)3Si]; IR (CHCl3) ν 3520,
2143, 1763, 1700 cm-1; ESI-MS (positive) m/z 561 (M + Na+),
1100 (M + M + Na+). Anal. Calcd for C28H38N4O5Si: C, 62.43;
H, 7.11; N, 10.40. Found: C, 62.63; H, 6.86; N, 10.51.
1-4-[(3S,5S,6R)-4-(tert-Butoxycarbonyl)-2-oxo-5,6-diphenyl-
morpholin-3-yl]-2-hydroxybutyl)-5-2-[(3S,5S,6R)-4-(tert-butox-
ycarbonyl)-2-oxo-5,6-diphenylmorpholin-3-yl]ethyl)-4-([(3S,5S,6R)-
4-(tert-butoxycarbonyl)-2-oxo-5,6-diphenylmorpholin-3-
yl]methyl)pyridinium-3-olate (12a). (i) Starting from Iodoketone
9 and Hydroxyamine 8a. To a solution of hydroxyamine 8a (300
mg, 0.68 mmol) and iodoketone 9 (896 mg, 1.63 mmol) in CH3-
CN (30 mL) was added Na2CO3 (350 mg). The resulting mixture
was stirred at room temperature until the complete disappearance
(2 h) of the starting amine 8a (TLC, Rf ) 0.18, CH2Cl2/MeOH
100:5 v:v), and the solvent was evaporated under reduced pressure
at a temperature below 40 °C. The solid residue was suspended in
CH2Cl2 (30 mL) and filtered to eliminate the inorganic salts. The
glassy residue obtained by evaporation of the solvent under reduced
pressure was dissolved in anhydrous THF (45 mL) containing DBU
(270 µL, 1.80 mmol). The reaction mixture was then stirred under
an oxygen atmosphere for 4 days at room temperature. The solvent
was removed by evaporation, and the residue was purified by flash
silica gel chromatography (CH2Cl2/MeOH 10:1 v:v), to afford the
tert-Butyl (3S,5S,6R)-3-[(R)-4-Amino-3-(trimethylsilyloxy)bu-
tyl]-2-oxo-5,6-diphenylmorpholine-4-carboxylate (8b). The tri-
methylsilyloxyazide 7b (250 mg, 0.46 mmol), dissolved in AcOEt
(40 mL), was hydrogenated in the presence of 10% Pd/C (15 mg)
at room temperature and atmospheric pressure. After 20 h, the
mixture was filtered through a pad of Celite and the solvent was
evaporated under reduced pressure. The crude residue was then
crystallized to afford the hydroxyamine 8b (223 mg, 94%) as a
white solid: mp 102 °C (from CH2Cl2/hexane; compound starts to
reduce its volume at 95 °C); [R]D25 -26.5 (c 1, CHCl3); 1H NMR
(CDCl3) δ 7.29-7.00 (8H, aromatics), 6.58 (2H, aromatics), 5.95
(0.3H, d, J ) 3.2 Hz, 6-H, minor conformer), 5.93 (0.7H, d, J )
3.2 Hz, 6-H, major conformer), 5.24 (0.3H, d, J ) 3.2 Hz, 5-H,
minor conformer), 5.03 (0.7H, d, J ) 3.2 Hz, 5-H, major
conformer), 5.00 (0.7H, dd, J ) 9.5, 5.4 Hz, 3-H, major conformer),
25
compound 12a (181 mg, 21%) as a glass: [R]D -23.8 (c 1,
1
CHCl3); H NMR (CDCl3) (major conformer) δ 7.59-6.94 (26H,
aromatics, 2-H and 6-H), 6.73 (1H, br s, 6′chain-H), 6.56 (6H, m,
aromatics), 6.09 (1H, br s, 6′chain-H), 5.95 (1H, br s, 6′chain-H), 5.34
(1H, m, 3′4chain-H), 5.29 (1H, br s, 5′chain-H), 5.19 (1H, m, 3′5chain
-
H), 5.15 (1H, br s, 5′chain-H), 5.00 (1H, br s, 5′chain-H), 4.93 (1H, br
s, 3′Nchain-H), 4.22 (1H, m, 1Nchain-Ha), 4.12 (1H, m, 2Nchain-H), 3.88
(1H, m, 1Nchain-Hb), 3.58 (2H, m, 14chain-H2), 3.17 (2H, m, 15chain
-
3482 J. Org. Chem., Vol. 72, No. 9, 2007

Practical Syntheses of Pyridinolines
H2), 2.55 (1H, m, 25chain-Ha), 2.41 (1H, m, 25chain-Hb), 2.20 (2H,
m, 4Nchain-H2), 1.77 (1H, m, 3Nchain-Ha), 1.68 (1H, m, 3Nchain-Hb),
1.12, 1.09, 1.01 [27H, 3 × s, 3 × C(CH3)3]; 13C NMR (CDCl3)
(major conformer) δ 169.1, 169.0, 168.8 (3 × 2′-C), 153.9, 153.7,
14 (162 mg, 27%) as a white solid: mp 167 °C (from CH2Cl2/
25
1
isopropyl ether); [R]D -44.6 (c 1, CHCl3); H NMR (CDCl3)
(major conformer) δ 7.24-6.99 (18H, aromatics, 2-H and 6-H),
6.87 (1H, br s, 6′4chain-H or 6′5chain-H), 6.58 (4H, m, aromatics),
6.06 (1H, br s, 6′5chain-H or 6′4chain-H), 5.99 (1H, m, 2Nchain-H), 5.43-
5.40 (2H, m, 3Nchain-H2), 5.30 (1H, m, 3′4chain-H), 5.17 (1H, br s,
5′4chain-H or 5′5chain-H), 5.15 (1H, br s, 3′5chain-H), 5.07 (1H, br s,
5′5chain-H or 5′4chain-H), 4.65 (2H, br s, 1Nchain-H2), 3.71 (1H, m,
t
153.6 (3 × BuOCdO), 153.2 (3-C), 140.6-126.6 (aromatics), 81.6,
81.3, 80.6 [3 × (CH3)3C], 79.0, 78.9, 78.5 (3 × 6′-C), 69.1 (2Nchain
-
C), 66.3 (1Nchain-C), 61.4, 61.3, 60.9 (3 × 5′-C), 55.8 (3′Nchain-C),
55.8 (3′5chain-C), 54.6 (3′4chain-C), 35.4 (25chain-C), 30.8 (4Nchain-C),
30.5 (3Nchain-C), 30.4 (14chain-C), 27.7 [3 × (CH3)3C], 26.6 (15chain
-
14chain-Ha), 3.56 (1H, m, 14chain-Hb), 3.16 (2H, m, 15chain-H2), 2.56
C); IR (CHCl3) ν 3360, 1760, 1706 cm-1; ESI-MS (positive) m/z
1264 (M + H+). Anal. Calcd for C75H82N4O14: C, 71.30; H, 6.54;
N, 4.43. Found: C, 71.59; H, 6.45; N, 4.49.
(1H, m, 25chain-Ha), 2.40 (1H, m, 25chain-Hb), 1.11, 1.00 [18H, 2 ×
s, 2 × C(CH3)3]; 13C NMR (CDCl3) (major conformer) δ 169.2,
t
168.9 (2 × 2′-C), 154.0, 153.7 (2 × BuOCdO), 152.7 (3-C),
141.2-126.5 (aromatics), 130.4 (2Nchain-C), 122.2 (3Nchain-C), 81.2,
80.4 [2 × (CH3)3C], 79.3, 79.2 (2 × 6′), 62.6 (1Nchain-C), 61.9,
61.2 (2 × 5′-C), 56.2 (3′5chain-C), 55.1 (3′4chain-C), 35.0 (25chain-C),
30.4 (14chain-C), 27.8 [2 × (CH3)3C], 26.7 (15chain-C); IR (CHCl3) ν
1765, 1703 cm-1; ESI-MS (positive) m/z 880 (M + H+), 902 (M
+ Na+). Anal. Calcd for C53H57N3O9: C, 72.33; H, 6.53; N, 4.77.
Found: C, 72.08; H, 6.57; N, 4.87.
(ii) Starting from Iodoketone 9 and Trimethylsilyloxyamine
8b. To a solution of trimethylsilyloxyamine 8b (300 mg, 0.58 mmol)
and iodoketone 9 (771 mg, 1.40 mmol) in CH3CN (30 mL) was
added Na2CO3 (300 mg), and the resulting mixture was stirred at
room temperature until the complete disappearance (2 h) of the
starting amine 8b (TLC, Rf ) 0.18, CH2Cl2 /MeOH 100:5 v:v).
The solvent was evaporated under reduced pressure at temperatures
below 40 °C, and the solid residue was suspended in CH2Cl2
(30 mL) and filtered to eliminate the inorganic salts. The filtrate
was evaporated under reduced pressure to afford a glassy residue
which was dissolved in anhydrous THF (45 mL) containing DBU
(230 µL, 1.54 mmol). The reaction mixture was then stirred under
an oxygen atmosphere for 4 days at room temperature. The solvent
was removed by evaporation, and the crude residue was purified
by flash silica gel chromatography (CH2Cl2/MeOH 10:1 v:v), to
afford the compound 12a (251 mg, 34%) as a glass: [R]D25 -24.0
(c 1, CHCl3), identical in all respects with that described above.
Pyridinoline (1). To a solution of protected pyridinoline 12a
(160 mg, 0.127 mmol) in anhydrous CH2Cl2 (6.0 mL) was added
anhydrous ZnBr215 (172 mg, 0.76 mmol), and the resulting mixture
was stirred under argon at room temperature for 3 h. The mixture
was then diluted with AcOEt (50 mL), washed with a saturated
aqueous solution of NaHCO3 (30 mL), with water (305 mL), and
dried over anhydrous Na2SO4. The evaporation of the solvent under
reduced pressure afforded the crude pyridinoline precursor 13
(113 mg) which was dissolved in an aqueous 0.01 M HCl/THF
50:50 v:v mixture (60 mL) and hydrogenated in the presence of
PdCl2 (48 mg), at room temperature for 48 h. The reaction was
monitored on HPLC (see General) until the complete conversion
of starting material and of less polar intermediates into a single
peak (Rt ) 10.56 min) corresponding to a pure sample of
pyridinoline. The mixture was then filtered through Celite, to
remove the catalyst; the solvent was evaporated; and the solid
residue was dried in vacuo. The solid was then triturated with
diethyl ether (which dissolves the formed bibenzyl) and filtered to
afford the pyridinoline 1 (62 mg, 80%) as tetrachloride dihydrate:
5-2-[(3S,5S,6R)-4-(tert-Butoxycarbonyl)-2-oxo-5,6-diphenyl-
morpholin-3-yl]ethyl-4-([(3S,5S,6R)-4-(tert-butoxycarbonyl)-2-
oxo-5,6-diphenylmorpholin-3-yl]methyl)-3-hydroxypyridine (15).
To a stirred solution of allyl derivative 14 (150 mg, 0.17 mmol)
and 2-mercaptobenzoic acid (128 mg, 0.83 mmol) in CH2Cl2 (15
mL) was added tetrakis(triphenylphosphine)palladium(0) (10 mg,
0.00865 mmol).16 The resulting orange solution was stirred at room
temperature under argon for 1.3 h. The solution was washed with
a saturated NaHCO3 aqueous solution (10 mL) and dried over
anhydrous Na2SO4, and the solvent was evaporated under reduced
pressure. The crude residue was purified by flash silica gel
chromatography (CH2Cl2/MeOH 100:7 v:v) to afford the hydroxy-
25
pyridine 15 (125 mg, 87%) as a white solid: mp 201 °C; [R]D
1
-48.2 (c 1, CHCl3); H NMR (CDCl3) (major conformer) δ 8.27
(1H, br s, 2-H), 8.12 (1H, br s, 6-H), 7.37-6.52 (20H, aromatics),
6.25 (1H, d, J ) 3.2 Hz, 6′4chain-H or 6′5chain-H), 5.90 (1H, d, J )
3.2 Hz, 6′5chain-H or 6′4chain-H), 5.07 (1H, dd, J ) 11.2 and 3.0 Hz,
3′4chain-H), 5.01 (1H, d, J ) 3.2 Hz, 5′4chain-H or 5′5chain-H), 5.00
(1H, dd, J ) 11.2 and 3.0 Hz, 3′5chain-H), 4.96 (1H, d, J ) 3.2 Hz,
5′5chain-H or 5′4chain-H), 3.61 (1H, dd, J ) 14.7 and 11.2 Hz, 14chain
-
Ha), 3.44 (1H, dd, J ) 14.7 and 3.2 Hz, 14chain-Hb), 3.03 (1H, ddd,
J ) 13.3, 13.3, 4.9 Hz, 15chain-Ha), 2.82 (1H, ddd, J ) 13.3, 12.2,
5.3 Hz, 15chain-Hb), 2.33 (2H, m, 25chain-H2), 1.20, 1.14 [18H, 2 ×
s, 2 × C(CH3)3]; 13C NMR (CDCl3) (major conformer) δ 169.7,
t
167.4 (2 × 2′-C), 154.9, 153.5 (2 × BuOCdO), 152.6 (3-C), 142.0
(6-C), 137.6 (2-C), 136.6-126.4 (aromatics), 83.1, 81.2 [2 ×
(CH3)3C], 79.4, 79.2 (2 × 6′-C), 61.1, 60.9 (2 × 5′-C), 56.4 (3′5chain
-
C), 55.4 (3′4chain-C), 36.6 (25chain-C), 30.9 (14chain-C), 27.9 [2 ×
(CH3)3C], 26.9 (15chain-C); IR (CHCl3) ν 1770, 1700 cm-1; ESI-
MS (positive) m/z 840 (M + H+). Anal. Calcd for C50H53N3O9:
C, 71.49; H, 6.36; N, 5.00. Found: C, 71.37; H, 6.52; N, 5.05.
Protected Pyridinoline 12a by Alkylation of Hydroxypyridine
15. The hydroxypyridine 15 (800 mg, 0.95 mmol) and the
iodohydrin 510b (2.65 g, 4.8 mmol) were dissolved in CH3CN (20
mL) and refluxed under an argon atmosphere for 20 h. The mixture
was cooled at room temperature and treated with a strong basic
resin (Dowex 1 100-200 mesh, X8; 860 mg) for 15 min. After
filtration, the solvent was evaporated under reduced pressure to
afford a crude product which was purified by flash silica gel
chromatography (CH2Cl2/MeOH 10:1 v:v) to give the protected
λ
max (HCl 0.1 M)/nm (ꢀ/dm3 mol-1 cm-1), 242 (3850), 294 (6520);
λmax (50 mM phosphate buffer, pH 7.5)/nm (ꢀ/dm3 mol-1 cm-1),
252 (3710), 324 (6150); ESI/MS m/z 429 (M + H+). Other
physicochemical properties (1H and 13C NMR) confirmed those
reported.7
1-Allyl-5-2-[(3S,5S,6R)-4-(tert-butoxycarbonyl)-2-oxo-5,6-diphe-
nylmorpholin-3-yl]ethyl-4-([(3S,5S,6R)-4-(tert-butoxycarbonyl)-
2-oxo-5,6-diphenylmorpholin-3-yl]methyl)pyridinium-3-olate (14).
To a stirred solution of the iodoketone 9 (900 mg, 1.64 mmol) in
CH3CN (30 mL) were added allylamine (51.0 µL, 0.68 mmol) and
Na2CO3 (350 mg), and the resulting mixture was stirred at room
temperature for 2 h. The solvent was evaporated under reduced
pressure at temperatures below 40 °C, and the solid residue was
suspended in CH2Cl2 (30 mL) and filtered to eliminate the inorganic
salts. The filtrate was evaporated under reduced pressure to afford
a glassy residue which was dissolved in anhydrous THF (45 mL)
containing DBU (270 µL, 1.80 mmol). The reaction mixture was
then stirred under an oxygen atmosphere at room temperature for
4 days. The solvent was removed under reduced pressure, and the
crude residue was purified by flash silica gel chromatography (CH2-
Cl2/MeOH 10:1 v:v) to afford, after crystallization, the compound
25
pyridinoline (12a, 698 mg, 58%) as a glass: [R]D -25.2 (c 1,
CHCl3), identical in all respects with that described above.
Acknowledgment. This work was supported financially by
Italian MiUR (Ministero dell’Universita` e della Ricerca).
Supporting Information Available: 1H and 13C NMR spectra
for compounds 7b, 8a, 8b, 9, 12a, 14, and 15. This material is
available free of charge via the Internet at http://pubs.acs.org.
JO070136G
J. Org. Chem, Vol. 72, No. 9, 2007 3483