Total synthesis of deoxypyridinoline, a biochemical marker of collagen turnover

Article abstract of DOI:10.1039/a900298g

A convergent synthesis of deoxypyridinoline is described which involves the assembly of a suitably polysubstituted 3-hydroxypyridinium ring starting with fully protected L-lysine and L-glutamic acid.

Full text of DOI:10.1039/a900298g

Total synthesis of deoxypyridinoline, a biochemical marker of collagen
turnover
Pietro Allevi,* Alessandra Longo and Mario Anastasia
Dipartimento di Chimica e Biochimica Medica, Via Saldini 50, 20133 Milano, Italy. E-mail: allevi@unimi.it
Received (in Cambridge, UK) 11th January 1999, Accepted 9th February 1999
A convergent synthesis of deoxypyridinoline is described
which involves the assembly of a suitably polysubstituted
3-hydroxypyridinium ring starting with fully protected
l-lysine and l-glutamic acid.
With this in mind, the possibility of constructing the pyridine
ring of 1 was first studied using a simpler model lacking the
stereocenters and the amino acidic functionalities (Scheme 2).
Easily available6 1-bromopentan-2-one was reacted with (R)-
1-phenylethylamine to afford, using the best conditions
(K2CO3, MeCN, 12 h, 25 °C), the diketoamine 3 in very high
yield (80%).† Initially, various unsuccessful attempts were
made to promote in the same reaction both the formation of the
ketoamine 3 and its condensation to give the ketone 4, by
modulating the reaction conditions (reaction times, tem-
peratures and/or addition of appropriate crown ethers). Subse-
quently, the internal condensation was attempted with the
isolated and purified compound 3. Surprisingly, treatment of
amine 3 with K2CO3 in MeOH afforded (70% yield) a single
fluorescent product, to which the structure 5‡ could be assigned.
Formation of compound 5 shows that, contrary to our
expectations, the aldolic condensation occurred, followed by
the oxidation of the possible intermediate cyclic ketone 4.
The ketone 4 could be isolated and completely characterized
when the reaction was performed under complete exclusion of
oxygen. This compound was transformed into the pyridinium
salt 5 when it was dissolved in MeOH and stirred in the presence
of air and K2CO3. Compound 5 was also obtained, in a one-pot
reaction, by mixing 1-bromopentan-2-one and (R)-1-phenyl-
ethylamine in an open vessel containing MeCN and K2CO3.
When the complete formation of the ketoamine 3 was
monitored, the addition of MeOH leads to the formation of the
desired pyridinium salt 5. Similar spontaneous oxidations were
also recently observed 7 in other syntheses of pyridinium
derivatives, but it was always impossible to map the mechanism
of their formation. In our case, the isolation of compound 4
under anaerobic conditions and the observation that no
tetrahydro derivative was detectable in the final reaction
mixture made it possible to establish that oxygen, and not a
disproportionation reaction, is responsible for the formation of
pyridinium salt 5.
The paper describes the synthesis1 and complete character-
ization of the optically pure deoxypyridinoline 1. This com-
pound and pyridinoline 2 represent two crosslinks of the mature
form of collagen and, at present, are the most effective
biochemical markers of collagen turnover correlated with
diseases such as osteoporosis, bone cancer and arthropathies.2
Compounds 1 and 2 were isolated from bones after several
purification steps, but despite the various efforts devoted to
improve their extraction, the yields are currently very poor.3
Although in only small amounts, deoxypyridinoline 1 and
pyridinoline 2 have recently been obtained in high purity from
bones,4 and their individual UV molar extinction coefficient
values have been established. These values are necessary for the
correct standardization of the analytical techniques normally
used in clinical determinations in human urine. On the other
hand it is still impossible to have a definitive validation of these
parameters determined from synthetic compounds using the
only multistep synthesis of the deoxypyridinoline 1 (of
unspecified diastereomeric purity) and of the pyridinoline 2 (as
mixture of epimers at the hydroxylated alkyl carbon) so far
reported.5 This synthesis is accomplished starting with a single
fully-protected synthetic l-amino acid, prepared according to
the Schoelkopf’s approach. However, the enantiomeric purity
of this starting building block as well as the physico-chemical
parameters (UV molar extinction coefficients, optical rotations,
elemental analyses) of the final compounds 1 and 2 were
unreported.
In our work we decided to start from enantiomerically pure
and easily available natural l-a-amino acids, in order to avoid
the formation of complex diastereomeric mixtures in a possible
convergent synthesis of 1. In particular, a strategic disconnec-
tion of the pyridinium compound 1 suggested its assembly from
an l-lysine residue and from two identical fragments derivable
from l-glutamic acid (Scheme 1).
O
O
N
O
O
+
+
NH3
NH3
i
NH2
Br
Br
–
+
–
+
CO2
X
NH3
CO2 NH3
–O
O
O
Ph
Me
Ph
Me
–
–
CO2
CO2
3
ii
iv
+
N
NH2
X
R
–O
iii
HO
O
H3N+
H3N+
–
CO2
–
CO2
+
N
N
N
L-Lysine
Ph
Me
X = leaving group
L-Glutamic acid
Ph
Me
Ph
Me
1 R = H; Deoxypyridinoline
2 R = OH; Pyridinoline
4
5
Scheme 2 Reagents and conditions: i, K2CO3, MeCN, room temp., 80%; ii,
K2CO3, MeOH, room temp., 85%; iii, K2CO3, air, MeOH, room temp.,
81%; iv, K2CO3, air, MeOH, room temp., 70%.
Scheme 1
Chem. Commun., 1999, 559–560
559

NHZ
CO2But
NHZ
CO2But
formations, well-established in the case of the shorter aspartic
derivative,11 were in our case complicated by the easy
concurrent cyclisation to the known12 (S)-5-(1,1-dimethyleth-
oxy)carbonyl-1-benzyloxycarbonyl-2-pyrrolidone, cyclisation
of which was not allowed in the case of the aspartic acid
derivative. This cyclisation occurs during the activation of the
w-carboxylic group as an acid chloride (using SOCl2 it is the
only compound formed) or as mixed anhydride (using
ClCO2Et13 it forms in 40% yield). Only when performing the
activation with ClCO2Bn does the diazo ketone 12 [oil; [a]D25
+16.6 (CHCl3, 2% solution)] form in quantitative yield,
avoiding the formation of cyclic pyroglutamate.
NHZ
CO2But
NHZ
CO2But
O
O
O
O
i
NH2
Br
Br
N
7
ZHN
CO2But
ZHN
CO2But
6
9
Further work is currently underway to accomplish the
synthesis of pyridinoline 2 by a similar convergent assembly of
the pyridine nucleus starting from three l-glutamic units.
This work was supported financially by MURST COFIN
progetto di ricerca ‘Nuove metodologie e strategie di Sintesi di
Composti di Interesse Biologico’. Dedicated to the memory of
Professor Giacomino Randazzo.
ii
NHZ
CO2But
NH2
CO2But
NH2
CO2But
NHZ
CO2But
–O
–O
+
N
+
N
Notes and references
iii
† All new compounds gave correct elemental C, H, N and Cl analyses.
436
20
‡ Selected data for 5: oil; [a]2D0 +3.3, [a] 213.3 (CHCl3, c 1); dH(500
H2N
ZHN
CO2But
11
CO2But
MHz, CDCl3 8.17 (1H, br s, pyridinium ring proton), 7.04 (1H, br s,
pyridinium ring proton), 5.40 (1H, q, J 7.0, NCHCH3), 1.96 (3H, d, J 7.0 Hz,
NCHCH3), 1.16 (3H, t, J 7.0, pyr-CH2CH3), 0.92 (3H, t, J 7.0, pyr-
10
iv
CH2CH2CH3).
1
§ Selected data for 7: oil; [a]2D0 20.3, [a] 214.8 (CHCl3, c 1); dH(500
20
365
MHz, CDCl3) 4.24 [1H, ddd, J 8.5, 8.0, 4.5, CH(NHZ)CO2But], 3.87 (2H,
s, BrCH2CO), 2.75 (1H, ddd, J 18.0, 8.5, 7.0, BrCH2COCHH), 2.67 (1H,
Scheme 3 Reagents and conditions: i, K2CO3, MeCN, room temp., 78%; ii,
K2CO3, air, MeOH, room temp., 73%; iii, H2, Pd/C, MeOH, room temp.,
94%; iv, TFA, room temp., 72%.
ddd, J 18.0, 8.5, 5.5, BrCH2COCHH).
¶ Selected data for 10: oil; [a]2D0 +5.3, [a] +29.2 (CHCl3, c 1); dH(500
20
436
MHz, CDCl3) 8.09 (1H, br s, pyridinium ring proton), 6.90 (1H, br s,
pyridinium ring proton).
The possibility of extending the method used for the
synthesis of the simple model compound 5 to the more complex
deoxypyridinoline 1 was then demonstrated by reacting under
similar conditions (Scheme 3) the known8 protected l-lysine 6
with the bromo ketone 7§ obtained in two steps from N-
∑ Selected data for 11: oil; [a]2D0 +32.5 (CHCl3, c 1); dH(500 MHz, CD3OD)
7.57 (1H, br s, pyridinium ring proton), 7.48 (1H, br s, pyridinium ring
proton).
** Selected data for 1 monotrifluoroacetate monohydrate (C18H29N4O7
·CF3CO22·H2O): [a]D20 +33.0 (CHCl3, c 0.98); lmax(HCl 0.1 m)/nm (e/m21
cm21), 239 (3850), 293 (6480); lmax(50 mm phosphate buffer, pH 7.5)/nm
(e/m21 cm21) 252 (3660), 324 (6100); dH(500 MHz, D2O) 8.69 (1H, br s,
pyridinium ring proton), 8.62 (1H, br s, pyridinium ring proton), 4.92 (2H,
t, J 6.5, CH2CH2N+), 4.54 [1H, t, J 7.0, CH(NH3+)COO2], 4.32 [1H, t, J 5.0
Hz, CH(NH3+)COO2], 4.16 [1H, t, J 6.0, CH(NH3+)COO2]; dC(D2O)
175.1, 174.5, 173.6, 163.6 (q, CF3COO), 156.8, 142.1, 141.9, 136.0, 129.7,
117.5 (q, CF3COO), 61.8, 55.2, 54.9, 53.6, 31.4, 30.7, 30.5, 28.6, 26.4,
21.9.
(benzyloxycarbonyl)-l-glutamic acid 1-tert-butyl ester9
8
(Scheme 4).
Thus, the reaction in MeCN afforded the amine 9 in good
365
20
yield [78% yield; an oil, [a]2D0 20.8, [a] +3.1, (CHCl3, 1%
solution]. This amine is rather unstable in solution (decomposes
in a few days) but it was transformed into the protected
deoxypyridinoline 10¶ when it was dissolved in MeOH
containing K2CO3 and stirred in the presence of air.
Regeneration of the three protected amino groups of 10 by
hydrogenolysis afforded the ester 11∑ which, by treatment with
TFA, easily gave deoxypyridinoline 1 as monotrifluoracetate
salt in an epimerization-free process.10 This salt of deoxypyr-
idinoline 1 crystallized from aq. EtOH as a white monohydrate
which was completely characterized.** Its UV molar extinction
coefficients proved higher (10–20%) than those reported for the
corresponding tetrachloride dihydrate salt.4 The same differ-
ence was observed for the corresponding tetrachloride salt of 1,
which in our hands crystallized as monohydrate.
The preparation of the bromo derivative 7, which required the
activation of the w-carboxylic group of the protected glutamic
acid 8 and its substitution for an a-bromo ketone via an
intermediate a-diazo ketone, merits comment. These trans-
1 Presented at the 24th Convegno Nazionale, Divisione di Chimica
Organica, Salerno (Italy) September 21–25, 1997.
2 Inter alia: I. T. James, A. J. Walne and D. Perrett, Ann. Clin. Biochem.,
1996, 33, 397; R. H. Christenson, Clin. Biochem., 1997, 30, 573.
3 P. Arbault, E. Gineyts, M. Grimaux, P. Seguin and P. D. Delmas, J. Liq.
Chromatogr., 1994, 17, 1981; D. Fujimoto, K. Akiba and N. Nakamura,
JP 54039078; (Chem. Abstr., 1979, 91, 211269).
4 S. P. Robins, A. Duncan, N. Wilson and B. J. Evans, Clin. Chem., 1996,
42, 1621.
5 R. Waelchli, C. H. Beerli, H. Meigel and L. Révész, Bioorg. Med. Chem.
Lett., 1997, 7, 2831.
6 N. D. Doktorova, L. V. Ionova, M. Y. Karpeisky, N. S. Padyukova,
K. F. Turkin and V. L. Florentiev, Tetrahedron, 1969, 25, 3527.
7 K. Suyama and S. Adachi, J. Org. Chem., 1979, 44, 1417; Li-B. Yu, D.
Chen, J. Li, J. Ramirez, P. G. Wang and S. G. Bott, J. Org. Chem., 1997,
62, 208.
8 M. J. Milewska and A. Chimiak, Tetrahedron Lett., 1987, 28, 1817.
9 K. Pawelczak, L. Krzyzanowski and B. Rzeszotarska, Org. Prep.
Proced. Int., 1985, 17, 416.
10 V. Bavetsias, A. L. Jackman, R. Kimbell, W. Gibson, F. T. Boyle and
G. M. F. Bisset, J. Med. Chem., 1996, 39, 73.
11 Y. Liwschitz, R. D. Irsay and A. I. Vincze, J. Chem. Soc., 1959,
1308.
NHZ
CO2But
ii
L-Glutamic acid
7
O
X
8 X = OH
12 X = CHN2
i
12 D. K. Dikshit and S. K. Panday, J. Org. Chem., 1992, 57, 1920.
13 P. D. Bailey and J. S. Bryans, Tetrahedron Lett., 1988, 29, 2231.
Scheme 4 Reagents and conditions: i, ClCO2Bn, N-methylmorpholine,
THF, 220 °C, then CH2N2, 220 °C ? 0 °C, 88%; ii, HBr, AcOH, 0 °C,
64%.
Communication 9/00298G
560
Chem. Commun., 1999, 559–560