Synthesis of unnatural homologues of deoxypyridinoline as possible internal standards in analytical detection of pyridinolinic cross-links of collagen

Article abstract of DOI:10.1016/j.tetasy.2008.11.001

Three homologues of the collagen cross-links deoxypyridinoline, differing in the length of the side chain at the aromatic nitrogen, have been efficiently synthesized as possible internal standards in the quantitative analyses of pyridinolines. The first has a one-carbon shorter N-chain, while other two have a one-carbon and a two-carbon longer N-chain. The stereogenic centers are introduced stereoselectively using Williams' glycine template methodology and oxazinones to generate chirality and to suitably protect the amino acid functionality during assembly of the pyridinoline nucleus.

Full text of DOI:10.1016/j.tetasy.2008.11.001

Tetrahedron: Asymmetry 19 (2008) 2470–2478
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Tetrahedron: Asymmetry
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Synthesis of unnatural homologues of deoxypyridinoline as possible internal
standards in analytical detection of pyridinolinic cross-links of collagen
*
Pietro Allevi, Eti Alessandra Femia, Paola Rota, Maria Letizia Costa, Mario Anastasia
Dipartimento di Chimica, Biochimica e Biotecnologie per la Medicina, Università di Milano, Via Saldini 50, I-20133 Milano, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Three homologues of the collagen cross-links deoxypyridinoline, differing in the length of the side chain
at the aromatic nitrogen, have been efficiently synthesized as possible internal standards in the quanti-
tative analyses of pyridinolines. The first has a one-carbon shorter N-chain, while other two have a one-
carbon and a two-carbon longer N-chain. The stereogenic centers are introduced stereoselectively using
Williams’ glycine template methodology and oxazinones to generate chirality and to suitably protect the
amino acid functionality during assembly of the pyridinoline nucleus.
Received 24 September 2008
Accepted 4 November 2008
Available online 27 November 2008
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
human urine and other various tissues, preferably use HPLC and
a fluorescence detector, since fluorimetric methods of detection
Over the past decade, deoxypyridinoline 1 (dPyd) and to a min-
or extent pyridinoline 2 (Pyd; Fig. 1), two fluorescent cross-links
derived from the degradation of bone collagen, have attracted
increasing attention as biochemical markers of bone resorption,
which are useful for assessing fracture risk prediction in persons
suffering from osteoporosis, bone cancer, or arthropathies.1–5
Pyridinolines 1 and 2 are commonly detected in human urine to
allow early diagnosis or for monitoring drug therapy of these and
other metabolic bone diseases.6,7 Currently, analytical methods
for the accurate evaluation of these fluorescent cross-links in
are more sensitive than those based on mass spectrometry or alter-
natives.8–11 As a consequence, it is essential not only to have
authentic samples of pure dPyd 1 and Pyd 2 to be used as primary
reference standards, but also to dispose of some pyridinolines
congeners (stereoisomers or near homologues), with very closed
chemico-physical properties and polarity, to be used as suitable
internal standards to minimize errors related to the pretreatment
of the samples. However, until now, the reported quantitative
determinations of pyridinolines, carried out by HPLC, did not use
any internal standard, apart from one protocol, where a pyridino-
line acetylated at the hydroxy group11 of the hydroxylysine chain
is used. Moreover, even this pyridinoline derivative is not com-
pletely satisfactory as an internal standard since it cannot be used
to evaluate the efficiency of cross-link hydrolysis. In fact, its ace-
tate group can be destroyed during hydrolysis, which is necessary,
before analyses, in order to cleave pyridinolines bonded to carbo-
hydrates or to peptide chains. Thus, the acetylated pyridinoline
may only be added after the accomplishment of the hydrolytic pro-
cedures, and does not follow the same fate of pyridinolines during
the preparation of the analytical samples. In previous work from
our laboratory, we accomplished the synthesis of pyridinolines 1
and 2 and of all other known, free12–20 or glycolsylated,21 collagen
cross-links which are now available in suitable amounts by synthe-
sis. Herein, we report the first synthesis of three homologues 3a–c
of dPyd 1, differing in the length of the chain bonded to the aro-
matic nitrogen. The homologue 3a has a one-carbon shorter
N-chain, while the homologues 3b and 3c have, respectively, a
one-carbon and a two-carbon longer N-chain. In addition, we
report a preliminary analysis of the chromatographic (HPLC) behav-
ior of these homologues, which allows us to discriminate the
homologous 3b from dPyd 1 and Pyd 2 by this analytical technique.
+
+
NH3
CO2 NH3
CO2
NH3
CO2 NH3
CO2
-
+
-
+
-O
-O
-
-
N
N
R
n
+H3N
+H3N
-
CO2
-
CO2
1
3
a: n = 1
b: n = 3
c: n = 4
: R = H; deoxypyridinoline
2: R = OH; pyridinoline
Figure 1. Pyridinolines structures.
* Corresponding author. Tel./fax: +39 0250316042.
E-mail address: mario.anastasia@unimi.it (M. Anastasia).
0957-4166/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2008.11.001

P. Allevi et al. / Tetrahedron: Asymmetry 19 (2008) 2470–2478
2471
This and the observation that other physico-chemical properties of
compound 3b are obviously very close to those of natural pyridin-
olines allow us to select the homologous 3b as a convenient candi-
date to be used as the internal standard in the quantitative analysis
of pyridinolines by HPLC.
for assembling the pyridinium nucleus, which after dealkylation
of the aromatic nitrogen affords a substituted pyridine, which is
able to be transformed into the desired pyridinium salt by alkyl-
ation of the aromatic nitrogen with a conveniently protected
aminoacidic iodide generating the lysine chain.
Each of these protocols has some advantages. The first protocol
is shorter and is very useful when the necessary protected amine is
easily or commercially available, as in the case of lysine or hydrox-
ylysine. The second protocol is longer but is of practical utility to
prepare a series of congeners differing in the side chains at the aro-
matic nitrogen atom.
2. Results and discussion
On the basis of our previous work, we were able to perform the
synthesis of the three homologous deoxypyridinolines 3a, 3b, and
3c, adopting either our one-pot protocol13,14,17 (Scheme 1, A) or our
modular protocol21–23 (Scheme 1, B) for the assembly of the pyrid-
inium ring.
In the one-pot protocol, the pyridinium ring is assembled con-
vergently from 2 M equiv of an aminoacidic bromoketone and a
primary amine via a bis-alkylation and a successive cyclization–
aromatization sequence of reactions. In the modular protocol, an
auxiliary sacrificial amine (benzylamine or allylamine), easily
cleavable, is used to introduce the pyridine nitrogen atom and
In programming our synthetic work, we started with the syn-
thesis of the dPyd homologue 3a, which appeared relatively more
simple and had a fast preparation. In fact, we considered that by
using our shorter one-pot protocol, bromoketone 6 and 2,5-diami-
nopentanoic acid tert-butyl ester 7, both required for the assem-
bling of 3a (Scheme 2), could be conveniently obtained from
L
-glutamic acid.18 We were also confident that homologue 3a, with
a one-carbon shorter lysine chain, could be differently retained in
Protocol A
NHP
NHP
CO2R
NHP
CO2R
NHR
NH2
O
O
O
O2
CO2R
CO2R
NHP
(
)n
N
Sc
N
Sc
CO2R
NHP
PHN
-O
CO2R
CO2R
CO2R
Base
N
PHN
2
(
)n
Protocol B
O
PHN
Sc I
CO2R
H2N-CH2Ph
Base
NHP
NHP
CO2R
Br
CO2R
NHP
-O
NHP
CO2R
N-dealkylation
HO
CO2R
N
CH2Ph
N
P = Cbz or Boc;
Sc =
CO2R
n
a: n = 1
b: n = 3
c: n = 4
NHP
Scheme 1. Protocols for pyridinolines synthesis.
NH2
+
NHCbz
NH2
NH3
t
t
-
+
CO2 Bu
t
CO2 Bu
CO2 NH3
CO2
NHCbz
CO2 Bu
NH2
-O
-O
-O
t
Boc2N
2
t
-
CO2 Bu
CbzHN
CO2 Bu
t
CO2 Bu
N
7
N
N
ii
iii
O
i
Br
+H3N
CbzHN
H2N
t
t
-
CO2 Bu
CO2 Bu
CO2
3a
6
8
9
Scheme 2. Synthesis of the pyridinoline analogue 3a. Reagents and conditions: (i) K2CO3, MeCN, rt, 18 h, then O2, K2CO3, MeOH, rt, 72 h, 55%; (ii) H2, Pd/C, MeOH, 88%; (iii)
TFA/H2O; 95:5; v/v, rt, 95.5%.

2472
P. Allevi et al. / Tetrahedron: Asymmetry 19 (2008) 2470–2478
HPLC with respect to natural pyridinolines, thus satisfying our
needs and avoiding the synthesis of the other homologues 3b
and 3c.
Next, we reacted 2 M equiv of bromoketone13 6 with the diami-
no acid tert-butyl ester13 7 in acetonitrile containing anhydrous
sodium carbonate, and obtained, after exchanging the solvent
and a one-pot oxidation by oxygen, the protected pyridiniumolate
8 in 55% total yields. Compound 8 showed the expected physico-
chemical properties and 1H and 13C NMR spectra in agreement
with the assigned structure and with those of the natural homo-
logues obtained by synthesis in our laboratory.13 The regeneration
of the protected functions of 8 to afford compound 3a required first
a hydrogenolysis, assisted by Pd on carbon, to afford the corre-
sponding triamino ester 9, and then, a treatment with aqueous
CF3COOH. Homodeoxypyridinoline 3a was isolated as a mono-tri-
fluoroacetate salt, which was crystallized from ethanol and was
completely characterized. This showed the correct molecular for-
mula and mass spectrum in addition to other physico-chemical
properties similar to those detected for its natural homologue
1.13 In particular, it showed superimposable UV molar extinction
coefficients (see Section 3) and fluorescence characteristics in
dilute solution, practically identical to those described for the
natural8,24–26 and the synthetic pyridinolines.13 With the unnatural
deoxypyridinoline 3a in hand, we searched for chromatographic
conditions (HPLC) suitable for separating it from the natural dPyd
1 and Pyd 2. Unfortunately, after various experimentations, in
which we tested different columns and eluents, we realized that
the homodeoxypyridinoline 3a was separable from pyridinoline
2, but not from deoxypyridinoline 1 (Fig. 2).
Figure 2. HPLC profile of natural and unnatural pyridinoline 3a. The numerical
numbers denote the number of the pyridinolines in the text. Chromatographic
conditions are reported in Section 3.
Thus, we proceeded with the synthesis of homologues 3b and
3c, for which we decided to use our modular procedure21 (Scheme
1, B), which appeared to be more convenient since no suitable
commercial diamino acid was available for the construction of
the unnatural side chain on the aromatic nitrogen. Furthermore,
for the preparation of both deoxypyridinoline homologues 3b
and 3c, (Scheme 3) we prepared a common 4,5-disubstituted
pyridinium compound 12, starting with bromoketone 10, obtained
from L
-glutamic acid,20 and the two aminoacidic iodo derivatives
13b and 13c, necessary for building two longer side chains on
the aromatic nitrogen, using the Williams morpholinone chemis-
try.27,28 We first prepared pyridinium salt 12 and iodo derivatives
Boc2N
Boc2N
CO2tBu
CO2tBu
Boc2N
NBoc2
CO2tBu
NBoc2
-O
CO2tBu
HO
i
CO2tBu
ii
N
O
CH2Ph
N
Br
10
12
11
+
NBoc2
NH3
CO2tBu
+
-
CO2
NH3
NBoc2
CO2tBu
-O
+
-O
NH3
CO2
-
CO2
+
-
NH3
N
N
-O
v
vi
-
iv
CO2
(
)n
(
)n
O
O
N
O
N
Boc
O
N
H
(
)n
Ph
Ph
Ph
+H3N
Ph
Ph
Ph
Ph
Ph
O
O
iii
-
CO2
N
(
N
Boc
O
Boc
O
14
15
3
)n
I
a: n = 1
13
b
: n = 3
c: n = 4
Scheme 3. Modular synthesis of the pyridinoline analogues 3b and 3c. Reagents and conditions: (i) PhCH2NH2, Na2CO3, MeCN, rt, 12 h, then O2, Na2CO3, MeOH, rt, 72 h, 71%;
(ii) H2, Pd/C, MeOH, 95%; (iii) 1,5-diiodopentane (or 1,6 diiodohexane), THF, HMPA, LiBTMSA, ꢀ78 °C, 45 min, 69%; (iv) CH3CN, reflux, 6 h, 80–81%; (v) TFA/H2O; 95:5; v/v, rt,
2 h, 84–85%; (vi) H2, PdCl2, MeOH/H2O/AcOH; 10:2:1; v/v/v, 75%.

P. Allevi et al. / Tetrahedron: Asymmetry 19 (2008) 2470–2478
2473
13b and 13c, after which we alkylated pyridine 12 with iodo deriv-
ative 13b in acetonitrile. The reaction was completed in 4 h and
afforded the protected homopyridinoline 14b in nearly quantita-
tive yield (81%) (Scheme 3). This by a simple treatment with trifluo-
roacetic acid afforded amino acid 15b in good yields (85%), by
simultaneous regeneration of all aminoacidic functions, apart from
those masked as an oxazinone ring. Moreover, compound 15b suf-
fers the loss of the Boc protection at the oxazinone ring and be-
comes ready for the final deblocking, which was performed by
simple catalytic hydrogenolysis to afford the homodeoxypyridino-
line 3b.
The homopyridinoline 3b shows physico-chemical properties
and in particular UV and fluorescence properties, very close to
those of the natural and synthetic homologues. Then, we explored
the HPLC behavior of the deoxypyridinoline 3b by subjecting it to
the same analytical procedures used with the shorter homologue
3a and with natural pyridinolines. Moreover, in this case, we
observed that compound 3b was retained differently from the
natural pyridinolines 1 and 2 on a reversed-phase HPLC column
(Fig. 3).
Thus deoxypyridinoline 3b appears to be a suitable candidate to
act as an internal standard in the analytical protocols used to quan-
tify pyridinolines by HPLC. We also accomplished the synthesis of
the homologous deoxypyridinoline 3c by performing the alkylation
of the substituted pyridine 12 with the iodine 13c and by following
a parallel sequence of reactions (Scheme 3) including the forma-
tion of the congeners 14c and 15c. The final compound 3c was iso-
lated by chromatography on resins (Dowex 50X 8-200 H+) in a
sufficiently pure form to show a 1H NMR spectrum in complete
agreement with the assigned structure. However, on repeating
the spectrum after 20–30 min, the appearance of a second product,
clearly formed from 3c, could be monitored (1H NMR). This un-
expected liability of compound 3c (confirmed in other successive
preparations) prompted us to discontinue the preparation of 3c
Figure 3. HPLC profile of natural and unnatural pyridinoline 3b. The numerical
numbers denote the number of pyridinolines in the text. Chromatographic
conditions are reported in Section 3.
and to direct our efforts to verify a possible shortening of the syn-
thesis of 3b, considering that it was potentially useful as an inter-
nal standard for pyridinoline 1 and 2 analyses. We found that this
was possible adopting our one-pot protocol (Scheme 4).13 We
13
reacted
amine 17 with bromoketone 10 and obtained homo-
deoxypyridinoline 14b with physico-chemical properties identical
to those of the above-described compound transformed by us into
the free homopyridinoline 3b.
Amine 17 was easily obtained by using Williams’ oxazinon-
es27,28 from iodide 13b (Scheme 4). In conclusion, the possibility
to prepare homodeoxypiridinoline 3b via this shorter and efficient
way, together with the chromatographic profile herein evidenced
for 3b, allows us to consider this compound as a useful internal
standard available for analytical procedures directed toward eval-
uating the levels of pyridinolines. Work is currently ongoing in our
Ph
Ph
Ph
Ph
Ph
Ph
O
O
O
N
N
N
I
Boc
O
Boc
O
Boc
O
i
ii
H2N
17
N3
13b
16b
+
NH3
NBoc2
-
+
t
CO2
NH3
CO2 Bu
t
NBoc2
CO2 Bu
-O
-O
-
CO2
v
O
O
t
CO2 Bu
NBoc2
17
iii
N
N
iv
3b
O
O
O
Br
N Boc
Ph
O
N H
Ph
Ph
Ph
15b
14b
10
Scheme 4. One-pot synthesis of protected pyridinoline analogue 3b. Reagents and conditions: (i) NaN3, DMF, rt, 1 h, 92%; (ii) H2, Pd/C, AcOEt, 80%; (iii) Na2CO3, MeCN, rt, 10 h,
then O2, DBU, THF, rt, 120 h, 55%; (iv) TFA/H2O; 95:5, v/v, rt. 2 h, 85%; (vi) H2, PdCl2, MeOH/H2O/AcOH; 10:2:1, v/v/v, rt, 75%.

2474
P. Allevi et al. / Tetrahedron: Asymmetry 19 (2008) 2470–2478
laboratory, in which this is demonstrated by the use of compound
3.2. Preparation of tert-butyl (S)-5-amino-2-
3b in a protocol for the evaluation of pyridinolines in human urine.
(benzyloxycarbonylamino)pentanoate 7
3.2.1. Preparation of tert-butyl (S)-2-
(benzyloxycarbonylamino)-5-iodopentanoate
3. Experimental
tert-Butyl (S)-2-(benzyloxycarbonylamino)-5-hydroxypent-
anoate29 (337 mg; 1.04 mmol) was dissolved in dry THF (3 mL)
and treated in sequence with Ph3P (382 mg; 1.46 mmol), imidaz-
ole30 (113 mg; 1.66 mmol), and I2 (266 mg; 1.05 mmol). The mix-
ture was stirred for 2 h at room temperature, and then was
diluted with AcOEt and worked up. The crude product was purified
by rapid chromatography on silica (eluting with CH2Cl2) to afford
the pure tert-butyl (S)-2-(benzyloxycarbonylamino)-5-iodopent-
3.1. General methods
Melting points are uncorrected. Nuclear magnetic resonance
spectra were recorded at 303 K on Bruker AM-500 spectrometer
operating at 500.13 MHz for 1H and 125.76 MHz for 13C. Chemical
shifts are reported in parts for million (ppm, d units) and are ref-
erenced to residual CHCl3 (dH = 7.28 ppm) and to CDCl3
(dC = 77.0 ppm) for solutions in CDCl3 or to internal CH3OD
(dH = 3.30 ppm and dC = 49.0 ppm) for solutions in D2O. 1H NMR
data are tabulated in the following order: number of protons,
multiplicity (s, singlet; d, doublet; br s, broad singlet; m, multi-
plet), coupling constant(s) in Hertz, assignment of signal(s). The
1H and 13C resonances were assigned by 1H decoupling, 1H–1H
COSY, and 1H–13C correlation experiments. 1H NMR and 13C
NMR spectra of compounds containing the bis(tert-butoxycar-
bonyl)amino portion were complicated by the presence of distin-
guishable rotamers in a ratio close to 1:1, and the spectra of
compounds containing an oxazinone ring showed the presence
of a minor rotamer (30%). Where possible, the signals of both
rotamers are described, otherwise, the signals of major rotamer
are reported. The nomenclature of the single positions are given
as in Figure 4.
anoate (391 mg; Y = 87%) as an oil: ½a D20
ꢁ
¼ þ10:2 (c 1, CHCl3); 1H
NMR (CDCl3): d 7.39–7.33 (5H, aromatic protons), 5.36 (1H, d,
J = 7.4 Hz, NH), 5.13 (2H, s, OCH2Ph), 4.28 (1H, m, H-2), 3.21 (2H,
m, H-5), 1.95 (2H, m), 1.81 (2H, m), 1.50 [9H, s, C(CH3)3]; 13C
NMR (CDCl3): d 171.0 (COO), 155.8 (NCOO), 136.2, 128.5, 128.1,
and 128.0 (aromatic carbons), 82.4 [(CH3)3C], 66.9 (PhCH2O), 53.4
(C-2), 33.7, 29.0, 27.9 [C(CH3)3], 5.5 (C-5); ESI-MS (positive) m/z:
456 [M+Na]+. Anal. Calcd for C17H24INO4: C, 47.12; H, 5.58; N,
3.23. Found: C, 47.30; H, 5.25; N, 3.10.
3.2.2. Preparation of tert-butyl (S)-5-azido-2-
(benzyloxycarbonylamino)pentanoate
To a solution of tert-butyl (S)-2-(benzyloxycarbonylamino)-5-
iodopentanoate (100 mg; 0.23 mmol) in DMF (2 mL), NaN3 (88 mg;
1.35 mmol) was added and the mixture was stirred at room temper-
ature for 1 h. Then, the reaction mixture was diluted with AcOEt and
worked up to afford the tert-butyl (S)-5-azido-2-(benzyloxycarbon-
+
NH3
4chain
2
-
ylamino)pentanoate (77 mg; Y = 96%): ½a D20
ꢁ
¼ þ9:7 (c 1, CHCl3); 1H
+
1
CO2
Ph
NH3
-O
2
Ph
ox
NMR (CDCl3): d 7.39–7.32 (5H, aromatic protons), 5.41 (1H, d,
J = 7.6 Hz, NH), 5.13 (2H, s, OCH2Ph), 4.30 (1H, dd, J = 13.4, 7.6 Hz,
H-2), 3.33 (2H, m, H-5), 1.93 (1H, m) 1.72 (2H, m), 1.59 (1H, m),
1.49 [3H, s, C(CH3)3]; 13C NMR (CDCl3): d 171.1 (COO), 155.8 (NCOO),
136.2, 128.5, 128.1, and 128.0 (aromatic carbons), 82.4 [(CH3)3C],
66.9 (PhCH2O), 53.8 (C-2), 50.9 (C-5), 30.1, 24.6, 27.9 [C(CH3)3];
ESI-MS (positive) m/z: 371.3 [M+Na]+. Anal. Calcd for C17H24N4O4:
C, 58.61; H, 6.94; N, 16.06. Found: C, 58.40; H, 7.10;N, 16.10.
-
3
6
1
3
1
4
CO2
3
O
5
N
5
1
5chain
N+
1
R
O
1'
2'
2
1chain
R
+
NH3
-O2C
Figure 4. Carbon numeration used in this work.
3.2.3. Preparation of tert-butyl (S)-5-amino-2-
(benzyloxycarbonylamino)pentanoate 7
Optical rotations were taken on a Perkin–Elmer 241 polarime-
ter, and [a]
values are given in 10ꢀ1 deg cm2 gꢀ1. Mass spectra
To a solution of tert-butyl (S)-5-azido-2-(benzyloxycarbonyl-
amino)pentanoate (228 mg; 0.65 mmol) in THF (10 mL), Ph3P31
(223 mg; 0.85 mmol) was added and the mixture was stirred over-
night at room temperature. Then, the solvent was evaporated un-
der reduced pressure to afford a crude product, which was
purified by rapid chromatography on silica (eluting with CH2Cl2
to remove impurities and then with CH2Cl2/MeOH/concd NH3;
100:3:0.3; v/v/v) to give amine 7 (153 mg; Y = 73%) as a glass:
D
were obtained using a Finnigan LCQdeca (ThermoQuest) ion trap
mass spectrometer fitted with an electrospray source (ESI). The
spectra were collected in continuous flow mode by connecting
the infusion pump directly to the ESI source. Solutions of com-
pounds were infused at a flow rate of 10 lL/min. The spray voltage
was set at 5.0 kV in the positive ion mode and at 4.5 kV in the neg-
ative ion mode with a capillary temperature of 220 °C. Full-scan
mass spectra were recorded by scanning a m/z range of 100–
2000. HPLC analyses were carried out on a RP-18 column (LiChro-
½
a 2D0
ꢁ
¼ þ8:2 (c 1, CHCl3); 1H NMR (CDCl3): d 7.38–7.20 (5H, aro-
matic protons), 5.43 (1H, d, J = 7.0 Hz, NH), 5.12 (2H, s, OCH2Ph),
4.26 (1H, m, H-2), 2.75 (2H, m, H-5), 1.86 (1H, m), 1.73 (1H, m),
1.53 (2H, m), 1.48 [9H, s, C(CH3)3]; 13C NMR (CDCl3): d 171.1
(COO), 155.8 (NCOO), 136.4, 128.5, 128.1, and 128.0 (aromatic car-
bons), 82.0 ((CH3)3C), 66.8 (PhCH2O), 54.0 (C-2), 41.2 (C-5), 30.2,
28.6, 28.0 [C(CH3)3]; ESI-MS (positive) m/z: 323.1 [M+H]+, 345.1
[M+Na]+. Anal. Calcd for C17H26N2O4: C, 63.33; H, 8.13; N, 8.69.
Found: C, 63.40; H, 8.20; N, 8.50.
CART, 125 mm, 4 mm ID, 5 lm purchased from Merck); elution
was performed with a 20 min linear gradient from 100% of solvent
A [0.01 M heptafluorobutanoic acid (HFBA) in CH3CN/water 10:90
v/v] to 100% 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. All reactions were monitored by thin-layer chromato-
graphy (TLC) carried out on 0.25 mm E. Merck silica gel plates
(60 F254) using UV light, 50% sulfuric acid, anisaldehyde/H2SO4/
EtOH solution or 0.2% ninhydrin in ethanol, and heat as developing
agent. E. Merck 230–400 mesh silica gel was used for flash column
chromatography. Work-up refers to washing with water, drying
over Na2SO4, and evaporation of the solvent under reduced
pressure.
3.3. Synthesis of homodeoxypyridinoline 3a
3.3.1. One-pot synthesis of the protected homopyridinoline 8
To a solution containing amine 7 (387 mg; 1.20 mmol) and bro-
moketone18 6 (1.00 g; 2.41 mmol) in CH3CN (35 mL), anhydrous

P. Allevi et al. / Tetrahedron: Asymmetry 19 (2008) 2470–2478
2475
K2CO3 was added (500 mg; 3.62 mmol) and the mixture was stir-
red at room temperature under nitrogen for 18 h. After the disap-
pearance of the starting amine, the solvent was evaporated under
reduced pressure and the crude residue was recovered with MeOH
(30 mL). Additional K2CO3 (250 mg; 1.80 mmol) was then added
and the mixture was shaken under a slight pressure of oxygen
(1.3 atm) at room temperature for 72 h. Then, the mixture was di-
luted with dichloromethane (50 mL) and filtered on a pad of Celite.
After evaporation of the solvent, a crude residue was obtained,
which was chromatographed on silica gel to afford, eluting with
CH2Cl2/MeOH (100:7; v/v), the desired protected homodeoxypyri-
(30 mL) containing Na2CO3 (243 mg; 2.29 mmol), and the mixture
was stirred at room temperature under nitrogen for 12 h. The dis-
appearance of the starting amine and the formation of the dialky-
lated benzylamine (TLC: CH2Cl2/MeOH; 100:7; v/v; Rf = 0.1 and
0.25, respectively) were then observed. The solvent was then evap-
orated under reduced pressure and the crude residue was recov-
ered by MeOH (40 mL) and addition of Na2CO3 (243 mg;
2.29 mmol), and then shaken under a slight pressure of oxygen
(1.3 atm) at room temperature for 72 h. At this time, the mixture
was diluted with dichloromethane (50 mL) and filtered on a pad
of Celite, and the solvent was evaporated under a reduced pressure
to afford a crude residue, which was chromatographed on silica gel
(eluting with CH2Cl2/MeOH; 100:7; v/v) to afford the desired com-
dinoline 8 (640 mg; Y = 55%) as a glass ½a D20
ꢁ
¼ þ4:5 (c 1, CHCl3); 1H
NMR (CDCl3): d 8.27 (1H, br s, pyridinium proton), 7.38–7.18 (15H,
aromatic protons), 7.08 (1H, br s, pyridinium proton), 5.9–5.7 (3H,
overlapping, NH), 5.06 (6H, m, OCH2Ph), 4.37 (1H, m, H5Ch-3), 4.33
(1H, m, H4Ch-2), 4.25 (1H, m, H1Ch-4), 4.05 and 3.92 (2 ꢂ 1H, 2 ꢂ m,
H1Ch-1), 3.34 (1H, dd, J = 11.2, 12.7 Hz, H4Ch-1a), 2.87 (1H, dd,
pound 11 (145 mg; Y = 71%) as a glass: ½a D20
¼ ꢀ34:0 (c 1, CHCl3);
ꢁ
1H NMR (CDCl3): d 7.57 (1H, pyridine proton), 7.39–7.19 (5H, aro-
matic protons), 7.00 (1H, pyridine proton), 5.59 (1H, m, 4.6, H4ch
-
2), 5.14 (2H, s, PhCH2), 4.64 (0.6H, dd, J = 9.1, 5.3 Hz, H5ch-3 major),
4.60 (0.4H, dd, J = 9.1, 5.6 Hz, H5ch-3 minor), 3.55 (0.4H, dd, J = 12.9,
3.8 Hz, H4ch-1a minor), 3.52 (0.6H, dd, J = 13.3, 4.1 Hz, H4ch-1a ma-
jor), 3.27 (1H, m, H4ch-1b), 2.77 (1H, m, H5ch-1a), 2.57 (0.6H, m,
H5ch-1b major), 2.49 (0.4H, m, H5ch-1b minor), 2.36 (0.6H, m,
H5ch-2a major), 2.19 (0.4H, m, H5ch-2a minor), 2.05 (0.4H, m,
H5ch-2b minor), 1.97 (0.6H, m, H5ch-2b major), 1.53, 1.50, 1.49,
1.46, 1.45, 1.44, 1.39, 1.36 [54H, 8 ꢂ s, C(CH3)3]; 13C NMR (CDCl3):
d 169.4, 169.3 (COO), 152.7, 152.5, 152.1, 152.0 (NCOO), 144.6,
128.2 (aromatic carbons), 83.0–81.1 [C(CH3)3], 63.8 (PhCH2), 57.9
and 57.7 (C5ch-3), 56.6 and 56.5 (C4ch-2), 29.8 and 29.7 (C5ch-2),
J = 12.7, 3.0 Hz, H4Ch-1b), 2.66 (2H, m, H5Ch-1), 2.07 (1H, m, H5Ch
-
2a), 1.97 (3H, overlapping, H5Ch-2b and H1Ch-2), 1.85 and 1.66
(2 ꢂ 1H, 2 ꢂ m, H1Ch-3), 1.47 [18H, s, 2 ꢂ C(CH3)3], 1.31 9H, s,
[C(CH3)3]; 13C NMR (CDCl3): d 172.2, 172.1, 170.5 (COOtBu),
156.6, 156.2 (NCOO), 142.6 and 138.4 (pyridinium carbon), 130.6
(pyridinium carbon), 136.6, 136.2, 136.0, 128.6-127.5 (aromatic
carbons), 82.7, 82.4 and 82.1 [3 ꢂ C(CH3)3], 67.2, 67.0, and 66.5
(OCH2Ph), 59.8 (C1Ch-1), 56.0 (C4Ch-2), 53.4 (C5Ch-3), 53.2 (C1Ch-4),
33.1 (C5Ch-2), 29.6 (C1Ch-3), 27.9 [C(CH3)3], 27.9 (C4Ch-1), 26.8
(C1Ch-2), 26.1 (C5Ch-1); ESI-MS (positive) m/z: 918.5
[MꢀButOH+Na]+. Anal. Calcd for C53H68N4O13: C, 65.62; H, 7.17;
N, 5.78. Found: C, 65.50; H, 7.35; N, 7.70.
28.0, 27.9, 27.8, 27.7 [C(CH3)3], 27.5 (C4ch-1), 27.2 and 26.8 (C5ch
-
1); IR (CHCl3,
m
max, cmꢀ1): 2980, 2940, 1730, 1690, 1385, 1370,
1150; ESI-MS (positive) m/z: 886.4 [M+H]+, 908.4 [M+Na]+,
3.3.2. Preparation of the homopyridinoline 3a by regeneration
of the protected functionalities of 8
1171.6 [2M+H]+, 1794.2 [2M+Na]+. Anal. Calcd for C47H71N3O13
C, 63.71; H, 8.08; N, 4.74. Found: C, 63.60; H, 7.90; N, 4.70.
:
A solution of protected homodeoxypyridinoline 8 (300 mg;
0.31 mmol) in methanol (75 mL) was added with Pd/C (100 mg;
10%) and shaken under hydrogen for 4 h. At this time, the catalyst
was filtered over a pad of Celite and the solvent was evaporated
under reduced pressure to afford a crude product 9 (154 mg;
88%). The product showed the correct mass spectrum [m/z 516
3.5. Preparation of 4-[(2S)-2-bis(tert-butoxycarbonylamino)-
2-tert-butoxycarbonylethyl]-5-[(3S)-3-bis(tert-butoxy-
carbonylamino)-3-tert-butoxycarbonylpropyl]-3-
hydroxypyridine 12
t
(Mꢀ BuOH+Na)+] and showed only the characteristic aromatic pro-
The pyridinium derivative 11 (152 mg; 0.17 mmol) was dis-
solved in methanol (25 mL) and hydrogenated in the presence of
Pd/C (35 mg; 10%) for 1 h. After filtration on a pad of Celite, the sol-
vent was evaporated under reduced pressure to afford a residue,
which after column chromatography (eluting with CH2Cl2/MeOH;
100:5; v/v) afforded the 3-hydroxypyridine 12 in quantitative yield
ton signals of the pyridinium ring (d 8.09 and 6.90 ppm). Without
any additional purification it was used in the following reaction.
Crude compound 9 (100 mg; 0.176 mmol) was dissolved in aque-
ous CF3COOH (1.8 mL; 95%; v/v) and left at 23 °C for 45 min. Then
the acid was evaporated under reduced pressure to afford the pure
homodeoxypyridinoline 3a (89 mg; Y = 95.5%) as a monotrifluoro-
acetate salt, which was crystallized from ethanol as a solid mono-
(129 mg; Y = 95%). Compound 12, a glass, showed: ½a D20
¼ ꢀ11:3 (c
ꢁ
hydrate showing: ½a D20
ꢁ
¼ þ22:2 (c 0.9, H2O); kmax (HCl 0.1 M)/nm
1, CHCl3); 1H NMR (CDCl3): d 8.12 (1H, pyridine proton), 7.96 (1H,
pyridine proton), 5.15 (0.3H, dd, J = 6.8, 6.8 Hz, H4ch-2 minor), 5.11
(0.7H, dd, J = 6.8, 6.8 Hz, H4ch-2 major), 4.74 (0.3H, dd, J = 9.4,
5.5 Hz, H5ch-3 minor), 4.72 (0.7H, dd, J = 8.1, 6.2 Hz, H5ch-3 major),
3.50 (1H, m, H4ch-1a), 3.25 (1H, m, H4ch-1b), 2.82 (0.7H, ddd,
J = 13.6, 9.9, 6.7 Hz, H5ch-1a major), 2.71 (0.6H, m, H5ch-1 minor),
2.64 (0.7H, ddd, J = 13.9, 9.9, 5.7 Hz, H5ch-1b major), 2.44 (0.7H,
m, H5ch-2a major), 2.36 (0.3H, m, H5ch-2a minor), 2.11 (0.3H, m,
H5ch-2b minor), 2.01 (0.7H, m, H5ch-2b major), 1.52, 1.50, 1.48,
1.45, 1.43, 1.42 [54H, 6 ꢂ s, 6 ꢂ C(CH3)3]; 13C NMR (CDCl3): d
170.5 and 170.2 (COO minor), 169.5 and 169.4 (COO major),
152.9 and 152.8 (pyridine carbon), 152.4 and 152.3 (NCOO),
141.5 and 141.4 (pyridine carbon), 137.2 and 137.0 (pyridine car-
bon), 132.6 and 132.2 (pyridine carbon), 83.1–81.2 [C(CH3)3],
58.5 and 58.3 (C4ch-2 and C5ch-3), 30.6 (C5ch-2), 28.0-27.8
(e
/Mꢀ1 cmꢀ1), 239 (3840), 293 (6490); 1H NMR (D2O): d 8.18 and
8.11 (2 ꢂ H, 2 ꢂ s, Hpyd), 4.72 (2H, m, H1Ch-1), 4.4.39 (1H, m, H4Ch
-
-
2), 4.18 (1H, m, H5Ch-3), 4.11 (1H, m, H1Ch-4), 3.62 (2H, m, H4Ch
1), 3.30 and 3.11 (2 ꢂ 1H, 2m, H5Ch-1), 2.1–1.9 (4H, overlapping,
H5Ch-2 and H1Ch-2), 2.23 (2H, m, H1Ch-3); 13C NMR (D2O): d 174.8,
174.4, 174.2 (3 ꢂ COOtBu), 165.1, 143.3, 141.0, 131.0, 130.7 (pyrid-
inium carbons), 163.6 (q, CF3COO), 117.5 (q, CF3COO), 60.8 (C1Ch-1),
55.3, 55.1, 55.0 (C4Ch-2, C5Ch-3 and C1Ch-4), 31.9 (C1Ch-2), 29.1, 27.9,
27.2, 26.8 (C5Ch-1, C5Ch-2, C4Ch-1 and C1Ch-3); ESI-MS (negative) m/
z: 413 [MꢀH+MeOH]+. Anal. Calcd for C19H29F3N4O10: C, 42.94; H,
5.69; N, 10.54. Found: C, 43.20; H, 5.55; N, 10.40.
3.4. Preparation of 1-benzyl-4-[(2S)-2-bis(tert-butoxy-
carbonylamino)-2-tert-butoxycarbonyl ethyl]-5-[(3S)-3-
bis(tert-butoxycarbonylamino)-3-tert-butoxycarbonylpropyl]
pyridinium-3-olate 11
[C(CH3)3], 26.9 and 26.7 (C5ch-1), 26.6 (C4ch-1); IR (CHCl3, mmax
,
cmꢀ1): 2980, 2930, 1730, 1690, 1380, 1370, 1140; ESI-MS (posi-
tive) m/z: 796.3 [M+H]+, 818.4 [M+Na]+. Anal. Calcd for
C40H65N3O13: C, 60.36; H, 8.23; N, 5.28. Found: C, 60.30; H, 8.10;
N, 5.40.
The protected bromoketone20 10 (275 mg; 0.55 mmol) was
added to a solution of benzylamine (25 lL; 0.23 mmol) in CH3CN

2476
P. Allevi et al. / Tetrahedron: Asymmetry 19 (2008) 2470–2478
3.6. Preparation of tert-butyl (3S,5S,6R)-3-(5-iodopentyl)- and
of tert-butyl (3S,5S,6R)-3-(5-iodohexyl)-2-oxo-5,6-
diphenylmorpholine-4-carboxylate 13b and 13c
overlapping, H1ch-3 and H1ch-4a), 1.50–1.38 [64H, overlapping,
C(CH3)3 and H1ch-4b]; 13C NMR (CDCl3): d 169.4, 169.2, 168.6,
168.5 (COO), 153.7, 152.7, 152.3, 152.2 (NCOO), 144.3, 141.3
(pyridinium carbons), 136.7–127.6 (aromatic carbons), 83.4, 83.3,
83.2, 82.0, 81.9, 81.8, 81.1 [C(CH3)3], 79.4 and 78.9 (Cox-6), 61.4
and 60.4 (Cox-5), 61.1 and 60.9 (C1ch-1), 57.8 (C5ch-3), 57.6 and
56.2 (Cox-3), 57.3 and 57.0 (C4ch-2), 34.7 (C1ch-5), 31.2 (C1ch-2),
29.8 and 29.7 (C5ch-2), 28.4–27.8 [C(CH3)3], 27.3–27.1 (C4ch-1 and
To
a stirred solution of (2R,3S)-tert-butyl 6-oxo-2,3-diph-
enylmorpholine-4-carboxylate (2.0 g; 5.66 mmol) and 1,5-diiodo-
pentane (4.21 mL; 28.29 mmol) in THF (180 mL) containing
HMPA (18 mL), lithium bis(trimethylsily1)amide (8.4 mL;
8.4 mmol; 1 M solution in THF) was added dropwise at ꢀ78 °C.
After 45 min, the dry ice bath was removed and the reaction mix-
ture was warmed up to room temperature under stirring for
30 min. Then, the reaction mixture was poured into ice cold water,
extracted with ethyl acetate, and worked up. The crude residue
obtained was purified by rapid chromatography on silica (eluting
with hexanes/AcOEt; 9:1; v/v) to afford iodide 13b (2.14 g;
C5ch-1), 27.1, 27.0, 25.8, 25.7 (C1ch-3), 25.4 (C1ch-4); IR (CHCl3, mmax
,
cmꢀ1): 2970, 2940, 1740, 1690, 1380, 1370, 1150; ESI-MS (posi-
tive) m/z: 1217.6 [M+H]+, 1239.6 [M+Na]+. Anal. Calcd for
C66H96N4O17: C, 65.11; H, 7.95; N, 4.60. Found: C, 65.00; H, 7.80;
N, 4.70.
By a similar procedure, starting with hydroxypyridine 12
(132 mg; 0.17 mmol) and iodide 13c (185 mg; 0.33 mmol) dis-
solved in CH3CN (3 mL) and refluxed under an argon atmosphere
for 6 h, the protected deoxypyridinoline 14c (164 mg; Y = 80%)
Y = 69%) as
a white solid: mp 123–125 °C (from hexane);
½
a 2D0
ꢁ
¼ ꢀ45:4 (c 1, CHCl3). 1H NMR (500 MHz): d 7.27–7.01 (8H,
aromatic protons), 6.59 (2H, m, aromatic protons), 5.96 (1H, br s,
Hox-6), 5.25 (0.3H, Hox-5 minor), 5.03 (1.4H, overlapping, Hox-5
major and Hox-3 major), 4.84 (0.3H, dd, J = 6.9 Hz, H-3 minor),
3.23 (2H, t, J = 6.9 Hz, H-50), 2.18 and 1.99 (2 ꢂ 1H, 2 ꢂ m, H-10),
1.88 (2H, m, H-40), 1.64 (2H, m, H-20), 1.50 (2H, m, H-30), 1.48
[3H, C(CH3)3 minor], 1.12 [6H, C(CH3)3 major]; 13C NMR (CDCl3):
d 169.2 (COO), 153.6 and 152.9 (NCOO), 136–126 (aromatic car-
bons), 81.6 and 81.1 [C(CH3)3], 79.4 and 78.9 (Cox-6), 61.6 and
60.4 (Cox-5), 57.7 and 56.5 (Cox-3), 35.6 and 35.0 (C-10), 33.3 and
33.2 (C-40), 28.4 and 28.1 (C-30), 28.0 and 27.8 [C(CH3)3], 25.9 and
was obtained as a glass: ½a D22
ꢁ
¼ ꢀ30:6 (c 1, CHCl3); 1H NMR
(CDCl3): d 8.23 (1H, s, pyridinium proton), 7.75 (1H, s, pyridinium
proton), 7.3–7.0 (8H, aromatic protons), 6.55 (2H, aromatic pro-
tons), 6.00 (0.8H, br s, Hox-6 major), 5.98 (0.2H, bs, Hox-6 minor),
5.39 and 5.31 (2 x 0.5, 2 x m, H4ch-2), 5.19 (0.2H, d, J = 3.0 Hz,
Hox-5 minor), 5.03 (0.8H, d, J = 3.0 Hz, Hox-5 major), 4.94 (0.8H,
dd, J = 9.9, 5.5 Hz, Hox-3 major), 4.77 (0.2H, dd, J = 10.6, 4.0 Hz,
Hox-3 minor), 4.63 (1H, m, H5ch-3), 4.38 (2H, m, H1ch-1), 3.47 (2H,
m, H4ch-1), 2.88–2.74 (2H, m, H5ch-1), 2.39 and 2.32 (2 ꢂ 0.5H,
2 ꢂ m, H5ch-2a), 2.16–1.97 (5H, overlapping H5ch-2b, H1ch-6, H1ch
-
25.7 (C-20), 7.0 and 6.7 (C-50); IR (CHCl3,
m
max, cmꢀ1): 2910, 1745,
2), 1.70–1.38 [69H, overlapping, H1ch-3, H1ch-5, H1ch-4, C(CH3)3];
13C NMR (CDCl3): d 169.4, 169.2, 168.6, 168.5 (COOtBu), 153.7,
152.7, 152.3, 152.2 (NCOO), 144.3, 141.3 (pyridinium carbons),
136.7–127.6 (aromatic carbons), 83.4, 83.3, 83.2, 82.0, 81.9, 81.8,
81.1 [C(CH3)3], 79.4 and 78.9 (Cox-6), 61.4 and 60.4 (Cox-5), 60.8
and 60.3 (C1ch-1), 57.8 (C5ch-3), 57.6 and 56.2 (Cox-3), 57.3 and
57.0 (C4ch-2), 34.7 (C1ch-5), 31.4 (C1ch-2), 29.8 and 29.6 (C5ch-2),
28.4–27.8 [C(CH3)3], 27.3–27.1 (C4ch-1 and C5ch-1), 27.0, 25.7,
25.6, 25.4, 25.2; ESI-MS (positive) m/z: 1231.6 [M+H]+, 1254.6
[M+Na]+. Anal. Calcd for C67H98N4O17: C, 65.34; H, 8.02; N, 4.55.
Found: C, 65.50; H, 8.20; N, 4.65.
1680, 1380, 1350, 1155, 1125, 900; ESI-MS (positive) m/z: 572.1
[M+Na]+. Anal. Calcd for C26H32INO4: C, 56.84; H, 5.87; N, 2.55.
Found: C, 56.66; H, 5.60; N, 2.70.
By a similar procedure, starting with tert-butyl(2R,3S)-6-oxo-
2,3-diphenylmorpholine-4-carboxylate (2.0 g; 5.66 mmol) and
1,5-diiodohexane (4.66 mL; 28.29 mmol) in THF (180 mL), iodide
13c was obtained (2.2 g; Y = 69%): a solid: mp 140–141 °C (from
hexane); ½a 2D0
ꢁ
¼ ꢀ46:7 (c 1, CHCl3) 1H NMR (CDCl3): d 7.29–7.05
(8H, aromatic protons), 6.58 (2H, aromatic protons), 5.96 (1H, br
s, Hox-6), 5.24 (0.3H, Hox-5 minor), 5.02 (1.4H, overlapping, Hox-5
major and Hox-3 major), 4.83 (0.3H, dd, J = 10.2, 4.8 Hz, Hox-3 min-
or), 3.20 (2H, m, H-60), 2.20 and 1.99 (2 ꢂ 1H, 2 ꢂ m, H-10), 1.91
(2H, m, H-50), 1.66 (2H, m, H-20), 1.56–1.48 [5H, m, overlapping,
H-30,H-40 and C(CH3)3 minor], 1.12 [6H, C(CH3)3 major]; ESI-MS
(positive) m/z: 586 [M+Na]+, 1149 [2M+Na]+. Anal. Calcd for
C27H34INO4: C, 57.55; H, 6.08; N, 2.49. Found: C, 576.60; H, 6.20;
N, 2.60.
3.8. Preparation of homodeoxypyridinolines 3b and 3c
3.8.1. Preparation of the partially protected pyridinolines 15b
and 15c, by hydrolysis of BOC groups and of tert-butyl esters
The completely protected homodeoxypyridinoline 14b
(162 mg; 0.13 mmol) was dissolved in CF3COOH/H2O (2 mL;
95:5; v/v), and the resulting solution was stirred at room temper-
ature for 2 h. Then, the solvent was evaporated under reduced
pressure and the residue was triturated with diisopropyl ether to
afford the partially protected homodeoxypyridinoline 15b as its
3.7. Preparation of the completely protected
homodeoxypyridinoline 14b or 14c
Hydroxypyridine 12 (132 mg; 0.17 mmol) and iodide 13b
(274 mg; 0.50 mmol) were dissolved in CH3CN (3 mL) and refluxed
under an argon atmosphere for 6 h. The mixture was cooled at
room temperature and the solvent was evaporated under reduced
pressure. The crude product was purified by rapid chromatography
on silica (AcOEt/MeOH; 100:8; v/v) to give the protected deoxy-
tetra-trifluoroacetate salt (120 mg; Y = 85%) as
a
powder:
½
a 2D0
ꢁ
¼ ꢀ10:6 (c 1, CD3OD); 1H NMR (CD3OD): d 8.38 (1H, s, HPyd
-
6), 8.29 (1H, s, HPyd-2), 7.2–7.0 (10H, aromatic protons), 5.50 (1H,
d, J = 3.7 Hz, Hox-6), 4.25 (1H, d, J = 3.7 Hz, Hox-5), 4.45 (2H, t,
J = 7.5 Hz, H1ch-1), 4.37 (1H, ddd, J = 7.7, 7.5, 3.4 Hz, H4ch-2), 4.10
(1H, dd, J = 15.7, 6.3 Hz, H5ch-3), 3.74 (1H, dd, J = 8.2, 5.2 Hz, Hox
-
pyridinoline 14b (168 mg; Y = 81%) as a glass: ½a D20
ꢁ
¼ ꢀ26:7 (c 1,
3), 3.49 (2H, m, H4ch-1), 3.12 and 3.02 (2 ꢂ 1H, 2 ꢂ m, H5ch-1),
2.29 and 2.23 (2 ꢂ 1H, 2 ꢂ m, H5ch-2), 1.97 (4H, overlapping,
H1ch-5 and H1ch-2), 1.53 (1H, m, H1ch-4a), 1.37 (3H, overlapping,
H1ch-4b and H1ch-3); 13C NMR (CD3OD): d 172.0, 171.9, 171.4
CHCl3); 1H NMR (CDCl3): d 8.21 (1H, s, pyridinium proton), 7.77
(1H, s, pyridinium proton), 7.3–7.0 (8H, aromatic protons), 6.55
(2H, aromatic protons), 6.03 (0.8H, br s, Hox-6 major), 5.99 (0.2H,
br s, Hox-6 minor), 5.34 and 5.28 (2 ꢂ 0.5, 2 ꢂ m, H4ch-2), 5.19
(0.2H, d, J = 3.0 Hz, Hox-5 minor), 5.03 (0.8H, d, J = 3.0 Hz, Hox-5 ma-
jor), 4.94 (0.8H, dd, J = 9.9, 5.5 Hz, Hox-3 major), 4.77 (0.2H, dd,
J = 10.6, 4.0 Hz, Hox-3 minor), 4.62 (1H, m, H5ch-3), 4.38 (2H, m,
H1ch-1), 3.48 (2H, m, H4ch-1), 2.88–2.74 (2H, m, H5ch-1), 2.39 and
2.32 (2 ꢂ 0.5H, 2 ꢂ m, H5ch-2a), 2.16 (1H, m, H1ch-5a), 2.10 (1H,
m, H5ch-2b), 2.02 (3H, overlapping, H1ch-5b and H1ch-2), 1.63 (3H,
(COO), 172.0, 171.9, 171.4 (COO), 163.5 (q, CF3COO), 157.4 (Cpyd
-
3), 142.6 (Cpyd-5), 141.9 (Cpyd-4), 136.8 (Cpyd-6), 129.4 (Cpyd-2),
140.6, 131.8, 131.1, 130.5, 129.4, 129.1, 127.2 (aromatic carbons),
117.0 (q, CF3COO), 72.7 (Cox-6), 67.3 (Cox-5), 62.7 (C1ch-1), 59.9
(Cox-3), 53.5 (C5ch-3), 52.4 (C4ch-2), 31.7 (C1ch-2), 31.5 and 31.4
(C5ch-2), 29.7 (C1ch-5), 28.7 and 28.6 (C4ch-1), 27.1 and 26.9 (C5ch
1), 26.4 (C1ch-3), 25.6 (C1ch-4); ESI-MS (positive) m/z: 623.5
-

P. Allevi et al. / Tetrahedron: Asymmetry 19 (2008) 2470–2478
2477
[M+H2O+H]+, 645.4 [M+H2O+Na]+. Anal. Calcd for C41H44F12N4O15
C, 46.38; H, 4.27; N, 5.28. Found: C, 46.30; H, 4.40; N, 5.50.
:
3.12 and 3.02 (2 ꢂ 1H, 2m, H5ch-1), 2.40 (2 ꢂ 1H, 2 ꢂ m, H5ch-2),
1.92 (4H, overlapping, H1ch-6 and H1ch-2), 1.76 (8H, overlapping,
H1ch-3 H1ch-4 and H1ch-5); ESI-MS (positive) m/z: 463.4 [M+Na]+.
After a 30 min standing in D2O at room temperature, compound
3c was transformed into a mixture containing at least a second
compound (ca. 30%), which complicated the 13C NMR spectrum
interpretation. TLC and HPLC inspection confirmed that some
decomposition of 3c had occurred. Similar behavior was observed
in two successive preparations.
By a similar procedure, starting from the completely protected
homodeoxypyridinoline 14c (164 mg; 0.13 mmol) dissolved in
CF3COOH/H2O (2 mL; 95:5; v/v), the partially protected homode-
oxypyridinoline 15c as its tetra-trifluoroacetate salt was obtained
(120 mg; Y = 84%) as a glass ½a D20
ꢁ
¼ ꢀ10:5 (c 1, CH3OH); 1H NMR
(CD3OD): d 8.42 (1H, s, HPyd-6), 8.31 (1H, s, HPyd-2), 7.2–7.0 (10H,
aromatic protons), 5.47 (1H, d, J = 3.7 Hz, Hox-6), 4.77 (3H, overlap-
ping, Hox-5 and H1ch-1), 4.36 (1H, ddd, J = 7.7, 7.5, 3.4 Hz, H4ch-2),
4.10 (1H, dd, J = 15.7, 6.3 Hz, H5ch-3), 3.77 (1H, dd, J = 8.2, 5.2 Hz,
Hox-3), 3.52–3.44 (2H, m, H4ch-1), 3.12 and 3.05 (2 ꢂ 1H, 2m,
H5ch-1), 2.31 and 2.23 (2 ꢂ 1H, 2m, H5ch-2), 1.96 (4H, overlapping,
H1ch-6 and H1ch-2), 1.50 (1H, m, H1ch-5a), 1.36–1.28 (5H, overlap-
ping, H1ch-5b, H1ch-3 H1ch-4); 13C NMR (CD3OD): d 172.0, 171.9,
171.4 (COO), 163.6 (q, CF3COO), 157.7 (Cpyd-3), 142.5 (Cpyd-5),
142.1 (Cpyd-4), 136.8 (Cpyd-6), 129.4 (Cpyd-2), 140.6, 131.8, 131.1,
130.5, 129.4, 129.1, 127.2 (aromatic carbons), 117.0 (q, CF3COO),
72.9 (Cox-6), 67.3 (Cox-5), 62.7 (C1ch-1), 59.9 (Cox-3), 53.4 (C5ch-3),
52.5 (C4ch-2), 32.1 (C1ch-2), 31.5 and 31.4 (C5ch-2), 29.8 (C1ch-6),
29.2 (C1ch-3) 28.7 and 28.6 (C4ch-1), 27.1 and 26.9 (C5ch-1), 26.7
(C1ch-4), 26.0 (C1ch-5); ESI-MS (positive) m/z: 637.5 [M+H2O+H]+,
659.4 [M+H2O+Na]+, 681.4 [M+H2O+2Na-H]+ 703.4 [M+H2O+3N-
3.9. Preparation of tert-butyl (3S,5S,6R)-3-(5-aminopentyl)-2-
oxo-5,6-diphenylmorpholine-4-carboxylate 17
3.9.1. Preparation of the tert-butyl (3S,5S,6R)-3-(5-azido-
pentyl)-2-oxo-5,6-diphenylmorpholine-4-carboxylate 16b
To a solution of tert-butyl (3S,5S,6R)-3-(5-iodopentyl)-2-oxo-
5,6-diphenylmorpholine-4-carboxylate 13b (550 mg; 1.00 mmol)
in DMF (4 mL), NaN3 (130 mg; 2.00 mmol) was added and the mix-
ture was stirred at room temperature for 1 h. Then, the reaction
mixture was poured into ice cold water (25 mL) and stirred for
20 min to form the tert-butyl (3S,5S,6R)-3-(5-azidopentyl)-2-oxo-
5,6-diphenylmorpholine-4-carboxylate 16b (427 mg; Y = 92%) as
a white solid showing: mp 132–133 °C; ½a D20
¼ ꢀ61:2 (c 1, CHCl3);
ꢁ
aꢀ2H]+, 725.4 [M+H2O+4Naꢀ3H]+. Anal. Calcd for C42H47F12N4O15
:
1H NMR (CDCl3): d 7.29–7.00 (8H, aromatic protons), 6.59 (2H, m,
aromatic protons), 5.96 (1H, br s, Hox-6), 5.24 (0.3H, Hox-5 minor),
5.03 (1.4H, overlapping, Hox-5major and Hox-3 major), 4.83 (0.3H,
dd, J 6.9, H-3 minor), 3.32 (2H, m, H-50), 2.20 and 1.99 (2 ꢂ 1H,
2 ꢂ m, H-10), 1.70 (4H, overlapping, H-40 and H-20), 1.56 (2H, m,
H-30), 1.48 [3H, C(CH3)3 minor], 1.12 [6H, C(CH3)3 major]; 13C
NMR (CDCl3): d 169.2 (COO), 153.6 and 152.9 (NCOO), 136–126
C, 46.89; H, 4.40; N, 5.21. Found: C, 46.80; H, 4.30; N, 5.40.
3.8.2. Hydrogenolysis of the oxazinone ring of 15b or 15c,
preparation of 3b or 3c
The partially protected homodeoxypyridinoline 15b (249 mg;
0.23 mmol) dissolved in a mixture of MeOH/H2O/AcOH (66 mL;
10: 2:1; v/v/v) was hydrogenated in the presence of PdCl2
(70 mg) for 12 h at room temperature. The catalyst was then fil-
tered off and the solution was concentrated under reduced pres-
sure to a residue, which was diluted with water and loaded on a
strong acidic resin column (2 mL; DowexÒ 50WX8-200). Then the
resin was washed with water, and finally the product was eluted
with NH3 in aqueous methanol (H2O/MeOH; 2:1; v/v). After evap-
oration of MeOH, the solution was freeze-dried twice to afford the
homodeoxypyridinoline 3b (74 mg; 75% yield) as a fluffy material,
(aromatic carbons), 81.6 and 81.2 [C(CH3)3], 79.5 and 78.9 (Cox
-
6), 61.5 and 60.4 (Cox-5), 57.51 and 56.4 (Cox-3), 51.3–51.2 (C-50),
35.5 and 35.0 (C-10), 28.7 and 28.6 (C-40), 28.3–27.8 [C(CH3)3 and
C-30], 26.4 and 26.3 (C-30), 25.6 and 25.5 (C-20); ESI-MS (positive)
m/z: 487.2 (M+Na+), 951.1 (2M+Na+). C, 67.22; H, 6.94; N, 12.06.
Anal. Calcd for C26H32N4O4: C, 67.30; H, 6.75; N, 11.90. Found: C,
67.40; H, 6.80; N, 12.10.
3.9.2. Reduction of the azide 16b, preparation of the amine 17
A solution of the azide 16b (700 mg; 1.51 mmol) in ethyl ace-
tate (150 mL) was selectively hydrogenated in the presence of
Pd/C (100 mg; 10%) to afford a crude product, which was purified
by rapid chromatography on silica to afford the amine 17
which showed: ½a D20
¼ þ15:2 (c 0.5, H2O); its UV spectrum (in
ꢁ
MeOH) shows kmax at 231, 260, and 336 nm, and this indicates that
it is in the 3-pyridiniumolate form (a zwitterion); 1H NMR (D2O): d
8.38 (1H, s, HPyd-6), 8.29 (1H, s, HPyd-2), 7.2–7.0 (10H, aromatic
protons), 5.50 (1H, d, J = 3.7 Hz, Hox-6), 4.25 (1H, d, J = 3.7 Hz,
Hox-5), 4.45 (2H, t, J = 7.5 Hz, H1ch-1), 4.37 (1H, ddd, J = 7.7, 7.5,
3.4 Hz, H4ch-2), 4.10 (1H, dd, J = 15.7, 6.3 Hz, H5ch-3), 3.74 (1H,
dd, J = 8.2, 5.2 Hz, Hox-3), 3.49 (2H, m, H4ch-1), 3.12 and 3.02
(2 ꢂ 1H, 2 ꢂ m, H5ch-1), 2.29 and 2.23 (2 ꢂ 1H, 2 ꢂ m, H5ch-2),
1.97 (4H, overlapping, H1ch-5 and H1ch-2), 1.53 (1H, m, H1ch-4a),
1.37 (3H, overlapping, H1ch-4b and H1ch-3); 13C NMR (D2O): d
173.2, 172.6, 172.2 (COOtBu), 155.9 (Cpyd-3), 141.6 (Cpyd-5), 141.2
(529 mg; Y = 80%),
a
solid: 117–119 °C (from hexane);
½
a 2D0
ꢁ
¼ ꢀ54:9 (c 1; CHCl3); 1H NMR (CDCl3): d 7.29–7.01 (8H, aro-
matic protons), 6.59 (2H, m, aromatic protons), 5.95 (1H, br s,
Hox-6), 5.24 (0.3H, Hox-5 minor), 5.03 (1.4H, overlapping, Hox-5
major and Hox-3 major), 4.84 (0.3H, m, H-3 minor), 2.72 (2H, m,
H-50), 2.19 and 1.99 (2 ꢂ 1H, 2 ꢂ m, H-10), 1.62 (2H, m, H-40),
1.65 (2H, m, H-20), 1.56–1.48 [11H, overlapping, H-30, H-40 and
C(CH3)3]; 13C NMR (CDCl3): d 169.2 (COO), 153.5 and 152.8 (NCOO),
136–126 (aromatic carbons), 81.5 and 81.1 (C(CH3)3), 79.4 and 78.8
(Cox-6), 61.6 and 60.3 (Cox-5), 57.6 and 56.4 (Cox-3), 41.5 (C-50),
35.5 and 34.9 (C-10), 33.2 and 32.9 (C-40), 28.3 and 28.0 (C-30),
28.1 and 27.8 [C(CH3)3], 25.8 and 25.7 (C-20); ESI-MS (positive)
m/z: 461.2 [M+Na]+. Anal. Calcd for C26H34N2O4: C, 71.21; H,
7.81; N, 6.39. Found: C, 71.40; H, 7.90; N, 6.50.
(Cpyd-4), 136.5 (Cpyd-6), 129.5 (Cpyd-2), 62.1 (C1ch-1), 53.7 (Cch1
-
6), 53.4 (C5ch-3), 52.4 (C4ch-2), 30.8 and 30.7 (C1ch-2 and C5ch-2),
30.2 (C1ch-5), 28.1 (C4ch-1), 26.3 (C5ch-1), 25.5 (C1ch-3), 24.3 (C1ch
-
4); ESI-MS (positive) m/z: 449.4 [M+Na]+. Anal. Calcd for
C19H30N4O7: C, 53.51; H, 7.09; N, 13.14. Found: C, 53.30; H, 7.20;
N, 13.20.
By a similar procedure, starting with the partially protected
homodeoxypyridinoline 15c (145 mg; 0.14 mmol) dissolved in a
mixture of MeOH/H2O/AcOH (66 mL; 10: 2: 1; v/v/v) and hydro-
genated in the presence of PdCl2 (70 mg) for 12 h at room temper-
ature, the homodeoxypyridinoline 3c was obtained (47 mg;
Y = 76%) as a fluffy material, which showed the appropriate phys-
ico-chemical properties: 1H NMR (D2O): d 8.33 (1H, s, HPyd-6),
8.25 (1H, s, HPyd-2), 4.52 (2H, m, H1ch-1), 4.32 (1H,m, H4ch-2),
4.12 (1H, m, H5ch-3), 4.04 (1H, m, H1ch-7), 3.00 (2H, m, H4ch-1),
3.10. Shortened one-pot preparation of
homodeoxypyridinoline 3b
To a solution containing amine 17 (1.19 g; 2.71 mmol) and bro-
moketone 10 (2.00 g; 7.86 mmol) in CH3CN (50 mL), anhydrous
Na2CO3 was added (1.36 g; 12.83 mmol) and the mixture was stir-
red at room temperature under nitrogen for 10 h. At this time, after
the disappearance of the starting amine, the solvent was evapo-

2478
P. Allevi et al. / Tetrahedron: Asymmetry 19 (2008) 2470–2478
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rated under reduced pressure and the crude residue was recovered
with anhydrous THF (150 mL) and filtered. Then, 1,8-diazabicy-
clo[5,4,0]undec-7-ene (DBU) was added (1 mL; 989 mmol) and
the mixture was shaken under a slight pressure of oxygen
(1.3 atm) at room temperature for 120 h. The mixture was then di-
luted with dichloromethane (50 mL) and filtered on a pad of Celite.
After evaporation of the solvent, a crude residue was obtained,
which was chromatographed on silica gel to afford (eluting with
CH2Cl2/MeOH, 100: 7; v/v) the desired protected homodeoxypyri-
dinoline 14b (1.81 g, Y = 55%) as a glass, ½a D20
¼ ꢀ26:7 (c 1; CHCl3).
ꢁ
The compound was identical in all respects to that obtained and
described above. Since it was transformed into 3b, its preparation
represents a formal synthesis of compound 3b.
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Tetrahedron: Asymmetry 2000, 11, 3645.
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2007, 18, 1750–1757.
Acknowledgment
This work was financially supported by Italian MiUR (Ministero
dell’Università e della Ricerca).
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