The design and synthesis of guanosine compounds with in vitro activity against the colon cancer cell line SW480: Non-taxane derived mimics of taxol?

Article abstract of DOI:10.1016/S0960-894X(03)00543-2

In the course of our investigation into the use of taxol as a lead compound to design new molecules with anti-cancer activity, we have synthesized four compounds based on protected guanosine coupled to taxol isoserine side-chain analogues. These analogues show in vitro anti-cancer activity against the colon cancer cell line SW480 that their constituent parts do not.

Full text of DOI:10.1016/S0960-894X(03)00543-2

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2693–2697
The Design and Synthesis of Guanosine Compounds with In Vitro
Activity against the Colon Cancer Cell Line SW480: Non-Taxane
Derived Mimics of Taxol?
Joshua Howarth,a,* Padraic Kenny,a Susan McDonnellb and Aine O’Connorb
aSchool of Chemical Sciences, Dublin City University, Dublin 9, Ireland
bSchool of Biotechnology, Dublin City University, Dublin 9, Ireland
Received 10 February 2003; revised 21 May 2003; accepted 26 May 2003
Dedicated to Professor David Crout to mark the occasion of his retirement.
Abstract—In the course of our investigation into the use of taxol as a lead compound to design new molecules with anti-cancer
activity, we have synthesized four compounds based on protected guanosine coupled to taxol isoserine side-chain analogues. These
analogues show in vitro anti-cancer activity against the colon cancer cell line SW480 that their constituent parts do not.
# 2003 Elsevier Ltd. All rights reserved.
It has been known for many years that the leaves and
berries of the yew tree are toxic to animals and man.
The constituent chemicals that cause the toxicity are
collectively known as taxanes. Some of the more com-
plex naturally occurring taxanes, taxol 1 and cephalo-
mannine 2, show potent anti-leukemic and anti-tumor
inhibitory and therapeutic properties.1
this goal.3 However, any total synthesis of taxol to date
involves over 35 steps, and it is highly unlikely that any
alternative synthesis will involve any less than this num-
ber, due to the complexity of taxol. With this number of
synthetic steps, a total synthesis of taxol is probably not
a commercially viable procedure at this moment.
All current bioactive analogues of taxol result from
modifications of the functional groups on the taxane
skeleton, or modification of the size of the carbon rings
contained within the molecule. Again, in general, taxol
itself is the starting point for such modifications.
A problem arises when a large amount of taxol is
required. Taxol originates from the bark of the yew tree
and hence its extraction ultimately kills the tree. This
has severely damaged the population of one of the
slowest growing trees in the world. The problem has
been partially circumvented by semi-synthesis methods
that utilise similar compounds to taxol, found in the
needles of the yew tree, these compounds are then con-
verted into taxol. Although this produces a renewable
source of taxol, the method allows for little modification
of the taxol skeleton to produce more active anti-cancer
drugs, and results in an extremely expensive treatment
for cancer.
The challenge would therefore appear to be to produce
a synthetic non-taxane compound, that mimics taxol, or
more precisely, mimics the molecule that taxol takes on
the role of or interferes with during cell division. This
non-taxane taxol mimic should have equivalent, or bet-
ter, chemotherapeutic properties as taxol. Furthermore,
this synthetic compound should be predisposed to
modification to produce specific compounds for the
treatment of specific cancers. Finally, it would be ext-
remely beneficial if these new compounds could be
producible in a commercially viable manner.
An alternative, the total synthesis of taxol, has been
achieved.2 Over the last two decades, an incredible
amount of time and money has been directed towards
Given the enormity of the challenge presented above
and the associated problems, the logical starting point is
to use taxol as a lead compound. An overall assessment
of previous investigations into the functionality in the
*Corresponding author. Tel.: +353-1-700-5312; fax: +353-1-700-
5503; e-mail: joshua.howarth@dcu.ie
0960-894X/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00543-2

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J. Howarth et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2693–2697
taxol molecule which lead to its activity as an anti-can-
cer compound, and of various other biological observa-
tions should produce a hypothesis as to mechanism
through which taxol attains its bioactivity. In other
words, we must speculate as to with which biomolecule
taxol has a biosteric and bioelectronic relationship.
the known tubulin GTP binding site is not competed for
by taxol. It is thought that taxol binds to the tubulin
and there is no evidence for or against both GTP bind-
ing sites being on the tubulin.
It is known that GTP is always bound to magnesium in
vivo. This binding occurs through two of the oxygen
anions. The binding of magnesium by GTP is essential
to its function in the energy transfer process.7 Perhaps
this is the reason why it is necessary for the free
hydroxyl to be present in the isoserine side-chain. The
hydroxyl group would bind strongly to magnesium, and
the amide nitrogen would also bind to magnesium, but
the binding would be much weaker. Removal or con-
version of the hydroxyl group into other functionality
would greatly reduce the side-chain’s capacity to bind to
magnesium.
Amongst the many cellular functions dependent on
microtublules is the process of cell division. Microtubule
polymerisation from tubulin is disrupted by the pre-
sence of taxol, through its ability to reduce the critical
concentration of tubulin required for spontaneous
assembly. In turn, this leads to cell death.4 Experiments
show that the activity of taxol is dependent on several
conditions. The basic carbon skeleton of taxol must
remain intact, modifications of ring sizes and ring junc-
tion stereochemistry have generally resulted in analo-
gues with reduced activity. There must be an isoserine
side-chain present. If the side-chain is not present, there
is no anti-cancer activity.5 The isoserine side-chain must
contain a free hydroxyl group. Any modification of this
hydroxyl group, for example conversion to an ether, will
greatly reduce the activity of taxol. All other modifi-
cations to the functionality around the taxol skeleton
serve only to vary the activity by a varying degrees.6
The molecular modelling studies10 that we have carried
out have shown that there is a similarity in the size,
shape, electron density and many possibly important
intramolecular distances between taxol and GTP. Some
of the similarities are obvious, such as shape and dis-
tribution of electronegative atoms.
Our required and initial hypothesis is therefore that
taxol acts as a GTP mimic. The basic carbon skeleton of
taxol, with its incumbent functionality, acts as the gua-
nosine section of GTP, and to a degree the isoserine
side-chain acts similarly the triphosphate chain of GTP.
This hypothesis is perhaps further supported when we
study other molecules that have the ability, like taxol, to
stabilize microtubules, although they are totally differ-
ent in chemical structure terms. These molecules are
epothilone,11 sarcodictyin,12 and eleutherobin.13 Mole-
cular modelling of these compounds clearly shows their
similarity to taxol and GTP in their shape and electronic
potential distribution.10
Other experiments7 have been carried out which reveal
some interesting connections between taxol and its pos-
sible mode of action. Cells will normally only divide
when guanosine triphosphate 4 (GTP) is present. Cells
with no GTP present, but with taxol present, will com-
mence division and then stop part way through the
process. Removal of the taxol from the cells and repla-
cement of GTP will restart cell division. These three
observations clearly indicate that there is a connection
between taxol and GTP.8
There are other facts which support this connection.10
The tubulin dimer binds two molecules of GTP when
involved in the production of microtubules, which are
essential to cell division.11 One of the GTP binding sites
is on the tubulin,4,9 the other GTP binding site’s
whereabouts is not known. Only GTP bound tubulin
allows polymerization or growth of microtubules and
If the hypothesis stated above is in any way correct then
there is a direct and efficient method for achieving our
challenge stated earlier. We have only to take the side-
chain 6a and link it to guanosine 5 to produce hybrid 6
(Scheme 1). Molecule 6 should have the same or similar

J. Howarth et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2693–2697
2695
properties as taxol 1. The advantage here, however,
would be that modification of guanosine and related
nucleosides has been extensively studied and hence the
possibilities for modification of our hybrid molecule 6
to produce tailored anti-cancer compounds are as
equally extensive. Guanosine is a cheap and readily
available chemical, and the isoserine side-chain 6a has
been synthesized several times, by various routes.14
Again, molecular modelling10 of the hybrid molecule 6
shows that it has similar shape and electronic potential
distribution as taxol 1 and GTP 4.
Using the synthesis for the taxol isoserine side-chain put
forward by Sharpless,15 (Scheme 2), we synthesized the
side-chains 9a and 9b in good yield. The guanosine
secondary hydroxyls were protected with the iso-
propylidene functionality16 and then the free NH2 group
was protected with either monomethoxytrityl via the
acetate 10a17 or isobutyryl groups18 to give 10c and 10d
respectively.19
During our initial studies in this area, we synthesized
four precursors, 11a–d, to molecule 619,20 through
Scheme 1.
Scheme 2.

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J. Howarth et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2693–2697
coupling side-chain 9a or 9b to 10c or 10d (Scheme 2).
When tested in vitro these molecules show activity
(IC5Os) against the colon cancer cell line SW480. The
cytotoxic assays were performed according to the
microculture MTT method.21 Two compounds, 11a and
11b have IC50s of l52.45 and l9.87 mm, respectively.
Compounds 11c and 11d are also active with IC50s of
221.64 and 192.41 mm, respectively. The IC50 value for
taxol when tested against SW480 is 0.312 mm.21 Tests on
the protected guanosines 10a–d, and protected isoserine
molecules 9a and 9b, before they are coupled to form
11a–d, show no activity against the cell line SW480.
D. G. I. J. Org. Chem. 1991, 56, 5114. Ojima, I.; Fenoglio, I.;
Park, Y. H.; Pera, P.; Bernacki, R. J. Bioorg. Med. Chem. Lett.
1994, 4, 1571.
7. Cassimeris, L. U.; Walker, R. A.; Pryer, N. K.; Salmon,
E. D. Bioessays 1987, 7, 149.
8. Schiff, P. B.; Horwitz, S. B. Biochemistry 1981, 20, 3247.
Andreu, J.-M.; Bordas, J.; Diaz, J. F.; de Ancos, J. G.; Gil, R.;
Medrano, F. J.; Nogales, E.; Pantos, E.; Towns-Andrews, E.
J. Mol. Biol. 1992, 226, 184.
9. Rao, S.; Horwitz, S. B.; Ringel, I. J. Natl. Cancer Inst.
1992, 84, 785.
10. Kenny, P. The Design and Synthesis of Nucleoside Mimics
of Taxol; PhD Thesis, Dublin City University, 1999.
11. Nicolaou, K. C.; Roschanger, F.; Vourloumis, D. Angew.
Chem., Int. Ed. Engl. 1998, 37, 2015.
In conclusion, it would appear that taxol may have a
biosteric and bioelectronic relationship to GTP, and
that the hypothesis put forward above has credibility as
shown by the bioactivity that molecules 11a–d possess.
It would not be surprizing that compounds 11a–d are
not as active as taxol against SW40, as C-20 hydroxyl
protected taxols often show significantly reduced activ-
ity compared to taxol.5 However, the real comparison
and proof of concept would need to be supported not
only by cytotoxic assays but also by microtuble (dis)-
assembly assays. It is quite possible that these new GTP
mimics might have cytotoxic effects without any relation
to taxol mode of binding. We are in the process of
carrying out these microtuble (dis)assembly assays.
Alternatively, we have fortuitously combined two com-
ponents, a simple guanosine derivative and a protected
isoserine derivative, to form a new class of molecules
with cytotoxic and possible anti-cancer properties.
12. Nicolaou, K. C.; Kim, S.; Pfefferkorn, J.; Xu, J.;
Ohshima, T.; Vourloumis, D. Angew. Chem., Int. Ed. Engl.
1998, 37, 1418.
13. Chen, X.-T.; Zhou, B.; Bhattacharya, S. K.; Gutteridge,
C. E.; Pettus, T. R. R.; Danishefsky, S. J. Angew. Chem., Int.
Ed. Engl. 1998, 37, 789.
14. Denis, J.-N.; Greene, A. E.; Serra, A. A.; Luche, M.-J. J.
Org. Chem. 1986, 51, 46. Denis, J.-N.; Correa, A.; Greene,
A. E. J. Org. Chem. 1991, 56, 6939. Palomo, C.; Arrieta, A.;
Cossio, F. P.; Azipurua, J. M.; Mielgo, A.; Aurrekotxea, N.
Tetrahedron Lett. 1990, 31, 6429. Warmerdam, E. G. J. C.;
van Rijn, R. D.; Brussee, J.; Kruse, C. G.; van der Gen, A.
Tetrahedron Asymmetry 1996, 7, 1723. Carlsen, P. H. J.; Kat-
suki, T.; Martin, V. S.; Sharpless, K. B. J. Org. Chem. 1981,
46, 3936.
15. Li, G.; Chang, H.-T.; Sharpless, K. B. Angew. Chem., Int.
Ed. Engl. 1996, 35, 451. Bruncko, M.; Schlingloff, G.; Sharp-
less, K. B. Angew. Chem., Int. Ed. Engl. 1997, 36, 1483.
Greene, A. E. Private communication.
16. Fromageot, H. P. M.; Griffin, B. E.; Reese, C. B.; Sulston,
J. E. Tetrahedron 1966, 23, 2315.
In either case, we have produced potentially useful
molecules that are relatively easy to construct, have
wide possibilities for modification, and are compara-
tively cheap to produce. We are currently undertaking
further investigation into these compounds.
17. Hayes, D. H.; Michelson, A. M.; Todd, A. R. J. Chem.
Soc. 1955, 808. Brederick, H. Chem. Ber. 1947, 80, 401.
18. Weber, H.; Khorana, H. G. J. Mol. Biol. 1972, 72, 219.
Buchi, H.; Khorana, H. G. J. Mol. Biol. 1972, 72, 251.
19. Preparation of 2-N-(4-methoxyphenyldiphenylmethyl)-20,30-
isopropylidene-50 -O-((400S,500R)-N-benzoyl-200,200 -dimethyl-400-
phenyl-100,300-oxazolidinyl-500-carboxylate)-guanosine 11a. Oxa-
zolidine 9a (0.30 g, 0.92 mmol) and dicyclohexylcarbodiimide
(0.20 g, 0.98 mmol) were dissolved in toluene (1.5 mL) with
stirring, at 20 ꢀC, for 1h. Butylammonium guanosine 10c (0.37
g, 0.55 mmol) and 4-dimethylaminopyridine (0.04 g, 0.31
mmol) were dissolved in toluene (3 mL) and added to the
reaction mixture. Stirring was continued for 10 min, then the
reaction temperature was elevated to 80 ꢀC. Heating was
maintained for 2 h, the reaction was allowed return to ambient
temperature, the saturated sodium bicarbonate solution (7
mL) was added. The aqueous layer was extracted with di-
chloromethane (3ꢁ5 mL) and combined organics were washed
with brine, dried (MgSO4), filtered and concentrated (<30 ꢀC)
to give crude 11a. This was purified on silica gel, eluting with
(1:8:12) (methanol/chloroform/hexanes) to yield 11a (0.352 g,
71%), white powder.
References and Notes
1. Nicolaou, K. C.; Guy, R. K. Taxane Anti-Cancer Agents:
Basic Science and Current Status; ACS Symposium Series,
1995; p 583.
2. Holton, R. A.; Somoza, C.; Kim, H.-B.; Liang, F.; Biedi-
ger, R. J.; Boatman, P. D.; Shindo, M.; Smith, C. C.; Kim, S.;
Nadizadeh, H.; Suzuki, Y.; Tao, C.; Vu, P.; Tang, S.; Zhang,
P.; Murthi, K. K.; Gentile, L. N.; Liu, J. H. J. Am. Chem. Soc.
1994, 116, 1597. Holton, R. A.; Somoza, C.; Kim, H.-B.;
Liang, F.; Biediger, R. J.; Boatman, P. D.; Shindo, M.; Smith,
C. C.; Kim, S.; Nadizadeh, H.; Suzuki, Y.; Tao, C.; Vu, P.;
Tang, S.; Zhang, P.; Murthi, K. K.; Gentile, L. N.; Liu, J. H.
J. Am. Chem. Soc. 1994, 116, 1599. Nicolaou, K. C.; Yang, Z.;
Liu, J. J.; Ueno, H.; Nantermet, P. G.; Guy, R. K.; Claiborne,
C. F.; Renaud, J.; Couloadouros, E. A.; Paulvannan, K.;
Sorensen, E. J. Nature 1994, 367, 630.
3. Burns, R. G.; Surridge, C. FEBS Lett. 1990, 271, 1 .
4. Fant, J.; Schiff, P. B.; Horwitz, S. B. Nature 1979, 277, 665.
Schiff, P. B.; Horwitz, S. B. Proc. Natl. Acad. Sci. U.S.A. 1980,
77, 1561.
5. Swindell, C. S.; Krauss, N. E. J. Med. Chem. 1991, 34,
1176. Magri, N. F.; Kingston, D. G. I. J. Nat. Prod. 1988, 51,
298. Kingston, D. G. I.; Samaranayake, G.; Ivey, C. A. J. Nat.
Prod. 1990, 53, 1 .
C52H45N6O9 requires (%): C 69.57, H 5.02, N 9.36; found: C
69.32, H 5.07, N 9.22. Rf 0.20 (1:8:12 methanol/chloroform/
hexanes); nmax (KBr) 3314, 3061, 2932, 1748, 1693, 1645 cmꢂ1
;
1H NMR (400 MHz, CDCl3, 40 ꢀC) d 1.28 (3H, s, acetonide),
1.51 (3H, s, acetonide), 1.80 (3H, s, oxazolidine), 1.91 (3H, s,
oxazolidine), 3.72 (3H, s, ArOCH3), (1H, m, H-500), 4.25 (2H,
m, H-40, H-5000), 4.48 (1H, bd, J3 -2 =5.9 Hz, H-3 ), 4.52 (1H,
0
0
00
0
00 00
0
0
d, J5 -4 =5.9 Hz, H-5 ), 4.96 (1H, bd, J2 -3 =5.9 Hz, H-2 ),
00
00 00
0
5.24 (1H, d, J4 -5 =5.9 Hz, H-4 ), 5.61(1H, d, J1 -2 =3.9 Hz,
0
6. Magri, N. F.; Samaranayake, G.; Jitrangari, C.; Kingston,
H-10), 6.76 (3H,
d
overlapping bs, J=9.9 Hz, ArCH-

J. Howarth et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2693–2697
2697
CHCOMe, NH), 6.91(2H, m, aromatic), 7.12–7.33 (22H, m,
aromatic, NH, H-8); 13C NMR (100 MHz, CDCl3), d 25.58,
25.76, 26.12, 27.28, 55.10, 64.72, 65.28, 70.65, 80.94, 81.16,
82.26, 82.73, 90.51, 97.90, 113.19, 114.22, 118.36, 126.08,
126.83, 126.93, 127.80, 127.90, 128.03, 128.11, 128.49, 128.66,
128.98, 129.35, 130.10, 136.87, 137.47, 138.61, 144.44, 149.79,
151.35, 158.31, 168.84, 169.16.
plete conversion by tlc (1:9 methanol/chloroform) and was
purified by flash column chromatography (eluant 4% metha-
nol in chloroform) to yield 11c as a white powder (0.40 g,
90%).
C36H35N6O9 requires: C 62.16, H 5.04, N 12.09; found: C
62.10, H 5.08, N 12.21. Rf 0.33 (1:24 methanol/chloroform);
;
nmax (KBr) 3380, 3112, 2961, 1750, 1687 cmꢂ1 1H NMR
(400 MHz, CDCl3), d 1.25 (6H, m, (CH3)2CH), 1.39 (3H, s,
acetonide), 1.61 (3H, s, acetonide), 1.87 (3H, bs, oxazolidine),
1.97 (3H, s, oxazolidine), 2.69 (1H, septet, J=6.9 Hz,
Preparation of 2-N-(4-methoxyphenyldiphenylmethyl)-20,30-iso-
propylidene-50-O-((400S,500R)-N-acetyl-200,200-dimethyl-400-phenyl-
100,300-oxazolidinyl-500-carboxylate)-guanosine 11b: As for 11a
using 9b, yield 78%, white powder.
0
000
0
0
(CH3)2CH), 4.08 (1H, dd, J5 -5 =11.4 Hz, J5 -4 =5.9 Hz, H-
00 00
50), 4.42 (1H, m, H-40), 4.63 (1H, d, J5 -4 =6.4 Hz, H-5 ), 5.08
(2H, m, H-30, H-5000), 5.13 (1H, dd, J2 -3 =6.4 Hz, J2 -1 =1.5
00
0
0
0
0
C49H43N6O9 requires (%): C 68.45, H 5.00, N 9.78; found: C
68.58, H 5.11, N 9.63. Rf 0.45(1:19 methanol/chloroform);
;
nmax (KBr) 3333, 3059, 2987, 1744, 1695 cmꢂ1 1H NMR
(400 MHz, DMSO-d6), d 1.20 (3H, s, acetonide), 1.38 (3H, s,
acetonide), 1.56 (3H, s, CH3CON), 1.66 (3H, s, oxazolidine),
1.70 (3H, s, oxazolidine), 3.69 (3H, s, ArOCH3), 3.95 (1H, dd,
Hz, H-20), 5.22 (1H, bs, H-400), 5.99 (1H, d, J1 -2 =1.5 Hz, H-
10), 6.95 (2H, m, aromatic), 7.11–7.33 (8H, m, aromatic), 7.73
(1H, s, H-8), 9.40 (1H, s, NH), 12.11 (1H, s, NH); 13C NMR
(100 MHz, CDCl3), d 18.81, 19.01, 24.92, 25.42, 25.56, 27.14,
36.33, 64.22, 65.41, 80.96, 81.76, 84.93, 85.05, 90.56, 97.96,
114.57, 122.13, 126.03, 126.77, 127.91, 128.00, 128.52, 129.41,
137.45, 137.95, 138.68, 147.25, 147.90, 155.36, 169.06, 170.22,
179.03.
0
0
0
0
000
0
0
000
J5 -5 =11.8 Hz, J5 -4 =4.9 Hz,000 H-5 ), 4.05 (1H, dd, J5
-
0
0
000
0
5 =11.8 Hz, J5 -4 =3.9 Hz, H-5 ), (1H, m, H-4 ), 4.24 (1H,
0
0
0
0
0
00 00
dd, J3 -2 =6.4 Hz, J3 -4 =2.9 Hz, H-3 ), 4.54 (1H, d, J4 -5 =3.9
0
Hz, H-400), 4.82 (1H, dd, J2 -3 =6.4 Hz, J2 -1 =3.5 Hz, H-2 ),
0
0
0
0
00
5.28 (1H, d, J5 -4 =3.9 Hz, H-5 ), 5.49 (1H, d, J1 -2 =3.5 Hz,
H-10), 6.85 (2H, d, J=8.9 Hz, ArCHCHOMe), 7.16–7.54
(19H, m, aromatic, 2ꢁNH), 7.70 (1H, s, H-8); 13C NMR
(100 MHz, CDCl3), d 24.72, 25.74, 25.80, 25.95, 27.29, 55.09,
64.14, 64.79, 70.64, 81.23, 81.66, 82.17, 82.63, 90.56, 98.40,
113.16, 114.23, 118.45, 126.05, 126.91, 127.87, 128.34, 128.65,
129.22, 130.10, 136.34, 136.87, 139.70, 144.52, 149.78, 151.39,
158.29, 168.54, 169.74.
Preparation
of
2-N-isobutyryl-20,30-isopropylidene-50-O-
00 00
0
0
((400S,500R)-N-acetyl-200,200-dimethyl-400-phenyl-100,300-oxazolidinyl-
500-carboxylate)-guanosine 11d. As for 11c using 9b, yield 39%,
white powder.
C33H33N6O9 requires (%): C 60.27, H 5.02, N 12.78; found: C
60.12, H 5.11, N 12.60. Rf 0.08 (1:9:15 methanol/chloroform/
hexanes); nmax (KBr) 3423, 3168, 2982, 2936, 1744, 1686, 1610,
20. Preparation of 2-N-isobutyryl-20,30-isopropylidene-50-O-
((400S,500R)-N-benzoyl-200,200 -dimethyl-400-phenyl-100,300-oxazoli-
dinyl-500-carboxylate)-guanosine 11c. (4S,5R)-N-Benzoyl-2,2-
dimethyl-4-phenyl-1,3-oxazolidinyl-5-carboxylic acid 9a (0.33 g,
1.00 mmol) and dicyclohexylcarbodiimide (0.23 g, 1.10 mmol)
were stirred together at 0 ꢀC in toluene (1.5 mL) for 30 min. A
solution of 10d (0.25 g, 0.64 mmol) and 4-dimethylaminopyr-
idine (0.05 g, 0.38 mmol) in chloroform (4 mL) was added in
one portion at 20 ꢀC and stirring maintained for 10 min. The
reaction mixture was heated to mild reflux. After 3 h, the
reaction mixture was allowed cool to ambient temperature and
saturated sodium bicarbonate added (5 mL). The aqueous
layer was extracted with dichloromethane (2ꢁ7 mL) and the
combined organic extracts washed with brine, dried (MgSO4),
filtered and concentrated. The crude product indicated com-
1560 cmꢂ1 1H NMR (400 MHz, CDCl3), d 1.25 (6H, m,
;
(CH3)2CH), 1.35 (3H, s, acetonide), 1.61 (3H, s, acetonide),
1.77 (3H,s, CH3CO), 1.82 (3H, s, oxazolidine), 1.86 (3H, s,
oxazolidine), 2.71(H1 , septet,
0
J=6.9 Hz,0 (CH3)2CH), 4.13
000
0
0
(1H, dd, J5 -5 =11.3 Hz, J5 -4 =4.9 0H0 z, H-5 ), 4.50 (1H, m, H-
400), 4.61(1H, d, J5 -4 =3.9 Hz, H-5 ), 5.13 (3H, m, H-2 , H-3 .
0
0
00 00
H-5000), 5.33 (1H. d, J4 -5 =3.9 Hz, H-4 ), 6.03 (1H, d, J1 -2
=1.0 Hz, H-10), 7.25–7.35 (5H, m, aromatic), 7.74 (1H, s, H-
8), 9.46 (1H, s, NH), 12.13 (1H, s, NH); 13C NMR (100 MHz,
CDCl3), d 18.81, 19.07, 24.60, 25.36, 26.16, 26.32, 27.18, 36.30,
64.19, 64.53, 81.79, 81.85, 85.14, 90.45, 98.79, 114.64, 122.21,
125.88, 128.47, 129.28, 138.01, 139.77, 147.20, 147.96, 155.33,
168.31, 171.45, 179.11.
00
00 00
0
0
21. Quintero, A.; Pelcastre, A.; Solano, J. D. J. Pharm. Phar-
maceut. Sci. 1999, 2, 108.