Synthesis of β-galactose-conjugated chlorins derived by enyne metathesis as galectin-specific photosensitizers for photodynamic therapy

Article abstract of DOI:10.1021/jo0105080

A first report on the synthesis and biological evaluation of the β-galactose-conjugated purpurinimides (a class of chlorins containing a six-membered fused imide ring system) as Gal-1 (galectin-1) recognized photosensitizers, prepared from purpurin-N-propargylimide via enyne metathesis, is discussed. On the basis of examination of the available crystal structure of the galectin-1 N-acetyllactose amine complex, it was considered that the chlorin-based photosensitizers could be introduced into a carbohydrate skeleton to expand the repertoire of the galectin-1-specific ligands. Preliminary molecular modeling analysis utilizing the modeled photosensitizers and the available crystal structures of galectin-carbohydrate complexes indicated that addition of the photosensitizer to the carbohydrate moiety at an appropriate position does not interfere with the galectin-carbohydrate recognition. Under similar drug and light doses, compared to the free purpurinimide analogue, the purpurinimides conjugated either with galactose or with lactose (Gal(β1-4)-Glc) produced a considerable increase in photosensitizing efficacy in vitro. This indicates the possibility for development of a new class of specific photosensitizers for photodynamic therapy (PDT) based on recognition of a cellular receptor.

Full text of DOI:10.1021/jo0105080

J . Org. Chem. 2001, 66, 8709-8716
8709
Syn th esis of â-Ga la ctose-Con ju ga ted Ch lor in s Der ived by En yn e
Meta th esis a s Ga lectin -Sp ecific P h otosen sitizer s for
P h otod yn a m ic Th er a p y
†
‡
§
†
‡
Gang Zheng, Andrew Graham, Masayuki Shibata, J oseph R. Missert, Allan R. Oseroff,
†
,†,|
Thomas J . Dougherty, and Ravindra K. Pandey*
Photodynamic Therapy Center, Department of Dermatology, and Department of Nuclear Medicine/
Radiology, Roswell Park Cancer Institute, Buffalo, New York 14263, and Biomedical Informatics,
UMDNJ -School of Health Related Professions, Department of Health Informatics, 65 Bergen Street,
Newark, New J ersey 07107
ravindra.pandey@roswellpark.org
Received May 21, 2001
A first report on the synthesis and biological evaluation of the â-galactose-conjugated purpurinimides
a class of chlorins containing a six-membered fused imide ring system) as Gal-1 (galectin-1)
(
recognized photosensitizers, prepared from purpurin-N-propargylimide via enyne metathesis, is
discussed. On the basis of examination of the available crystal structure of the galectin-1
N-acetyllactose amine complex, it was considered that the chlorin-based photosensitizers could be
introduced into a carbohydrate skeleton to expand the repertoire of the galectin-1-specific ligands.
Preliminary molecular modeling analysis utilizing the modeled photosensitizers and the available
crystal structures of galectin-carbohydrate complexes indicated that addition of the photosensitizer
to the carbohydrate moiety at an appropriate position does not interfere with the galectin-
carbohydrate recognition. Under similar drug and light doses, compared to the free purpurinimide
analogue, the purpurinimides conjugated either with galactose or with lactose (Gal(â1-4)-Glc)
produced a considerable increase in photosensitizing efficacy in vitro. This indicates the possibility
for development of a new class of specific photosensitizers for photodynamic therapy (PDT) based
on recognition of a cellular receptor.
In tr od u ction
demonstrated by us and others that overall lipophilicity
of the molecule plays an important role in PDT efficacy.
3
-5
In recent years, photodynamic therapy (PDT) has
received increasing attention as a new modality for
selective treatment of solid tumors. Systemic injection
Although the mechanism of tumor cure likely requires
the interplay of several biological responses, a key point
remains the ability of the photosensitizer to generate
singlet oxygen after light exposure. In tumor destruction,
desired cytotoxic effects on the cell membrane, mitochon-
dria, and nuclear structures have been observed. These
1
of the clinically used photosensitizer leads to relatively
selective retention of the compound in tumors. Since in
many cases greater amounts of photosensitizer are
present within tumors, as compared to nonneoplastic
tissues, the neoplasms are destroyed while surrounding
normal tissues remain relatively uninjured.2 While a
variety of photosensitizers have been evaluated, the
reasons for the accumulation of these compounds in
tumors are not clearly understood. However, it has been
effects could relate to the demonstrated microcirculatory
aberrations during and after PDT.6
For synthesizing various amphiphilic porphyrins, among
various solubilizing substituents, sugars have attracted
most attention. In addition to providing the molecule with
polar hydroxy groups, it is possible that the sugar moiety
might lead the conjugate to a cell surface target through
specific binding to its receptor. Oligosaccharides play
essential roles in various cellular activities as antigens,
*
To whom correspondence should be addressed. Phone: (716) 845-
203. Fax:(716) 845-8920.
Photodynamic Therapy Center.
Department of Dermatology.
UMDNJ -School of Health Related Professions.
Department of Nuclear Medicine/Radiology, Roswell Park Cancer
3
†
‡
§
|
(3) (a) Pandey, R. K.; Sumlin, A. B.; Potter, W. R.; Bellnier, D. A.;
Henderson, B. W.; Constantine, S.; Aoudia, M.; Rodgers, M. A. J .;
Smith, K. M.; Dougherty, T. J . Photochem. Photobiol. 1996, 64, 194.
(b) Henderson, B. W.; Bellnier, D. A.; Greco, W. R.; Sharma, A.; Pandey,
R. K.; Vaughan, L. A.; Weishaupt, K. R.; Dougherty, T. J . Cancer Res.
1997, 57, 4000. (c) Zheng, G.; Potter, W. R.; Sumlin, A.; Dougherty, T.
J .; Pandey, R. K. Bioorg. Med. Chem. Lett. 2000, 10, 123. (d) Rungta,
A.; Zheng, G.; Missert, J . R.; Potter, W. R.; Dougherty, T. J .; Pandey,
R. K. Bioorg. Med. Chem. Lett. 2000, 10, 1463. (e) Zheng, G.; Potter,
W. R.; Canmacho, S. H.; Missert, J . R.; Wang, G.; Bellnier, D. A.;
Henderson, B. W.; Rodgers, M. A. J .; Dougherty, T. J .; Pandey, R. K.
J . Med. Chem. 2001, 44, 1540.
Institute.
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1) (a) Pandey, R.; Zheng, G. In The Porphyrin Handbook; Smith,
K. M., Kadish, K., Guilard, R., Eds.; Academic Press: San Diego, 2000;
Vol. 6. (b) Sherman, W. M.; Allen, C. M.; van Lier, J . E. Methods
Enzymol. 2000, 319, 376. (c) Dougherty, T. J .; Gomer, C.; Henderson,
B. W.; J ori, G.; Kessel, D.; Kprbelik, M.; Moan, J .; Peng, Q. J . Natl.
Cancer Inst. 1998, 90, 889. (d) Schmidt-Erfurth, U.; Diddens, H.;
Birngruber, R.; Hasan, T. Br. J . Cancer 1997, 75, 54-61. (e) Finger,
V. H.; Guo, H. H.; Lu, Z. H.; Peiper, S. C. Methods Enzymol. 1997,
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88, 148. (f) Bonnett, Chem. Soc. Rev. 1995, 19. (g) Dolphin, D. Can.
J . Chem. 1994, 72, 1005. (h) Pandey, R. K.; Herman, C. Chem. Ind.
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j) Mody, T. D., Pandey, R. K., Guest Eds. J Porphyrins Phthalocya-
(5) Adams, K. R.; Berenbaum, M. C.; Bonnett, R.; Nizhnik, A. N.;
Salgado, A.; Valles, M. A. J . Chem. Soc., Perkin Trans. 1 1992, 146713.
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2001, 5, 105.
nines 2001, 5 (2)..
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(
1
1
0.1021/jo0105080 CCC: $20.00 © 2001 American Chemical Society
Published on Web 11/22/2001

8
710 J . Org. Chem., Vol. 66, No. 26, 2001
Zheng et al.
growth signals, targets of bacterial and viral infection,
and glues in cell adhesion and metastasis, where the
saccharide-receptor interactions are usually specific and
for â-galactoside.18 Because galectins are involved in the
modulation of cell adhesion, cell growth, immune re-
sponse, and angiogenesis, it is clear that changes in their
expression might have a critical role in tumor progres-
sion. Galectin-1 (Gal-1) is a prototype, dimeric galectin
with two identical CRD, and its expression is known to
correlate with the degree of malignancy in rat thyroid
cell lines transformed with several cellular or viral
oncogenes.19 Gal-1 mRNA levels increase 20-fold in low
tumorigenic and up to 100-fold in highly tumorigenic
cells. These observations are consistent with those ob-
served in human tumors.20 Furthermore, Gal-1 null
7
multivalent. This specificity suggests a potential utility
of synthetic saccharide derivatives as carriers in directed
drug delivery. Therefore, in recent years, a number of
carbohydrate derivatives of various photosensitizers have
8
been synthesized. Hombrecher et al. reported the prepa-
ration of a galactopyranosyl-cholesteryloxy-substituted
porphyrin by following the McDonald approach. Several
tetra- and octaglycoconjugated tetraphenylporphyrins
9
were also reported by Yano and co-workers, Cornia et
8
mutant mice are found to be relatively healthy. There-
fore, it seems that this may be a valuable cellular target
for â-galactoside-based photosensitizers.
al.,10 Momenteau and co-workers, and Ono et al.
11
12
A
glycosylated peptide porphyrin synthesized by Krausz
1
3
and co-workers showed promising in vitro PDT efficacy.
Maillard et al.14 reported the synthesis of a glycoconju-
gated meso-monoarylbenzochlorin. This compound dis-
played good in vitro PDT efficacy in tumor cell lines after
irradiation with light. A few years ago, Bonnett and co-
workers15 showed that, compared to â-hydroxyoctaeth-
ylchlorin, the related glycosylated analogue was more
effective as a photosensitizing agent in vitro as well as
in vivo. Montforts et al.16 further explored this approach
by attaching hydrophilic carbohydrate structural units
to certain chlorins. Such conjugation increased the water
solubility of the parent chlorins by introducing an estra-
diol with a diethyl spacer. The chlorin-estrogen conju-
gate was then prepared with the hope that it would bind
to an estrogen receptor that could induce destruction of
mammalian carcinoma. In a recent report, Aoyama and
co-workers17 reported the synthesis of certain TPP-based
saccharide-functionalized porphyrins and demonstrated
the importance of hydrophobicity masking for the sac-
charide-directed cell recognition. Since the saccharide-
receptor interactions are ubiquitous, well-defined/well-
designed synthetic saccharides clusters may serve as a
new tool in glycoscience and glycotechnology.
Analysis of the carbohydrate binding site in Gal-1
shows that Gal-1 has a pronounced specificity for the
Gal(â1-4)- and Gal(â1-3)GlcNAc sequences with no
apparent affinity for GlcNAc residues. The binding
specificity for Gal moiety is due to hydrogen bond
interactions between its C4-hydroxy group and His 44,
Asn 46 and Arg 48 residues that are conserved in all
galectins. The van der Waals contact of Trp 68 with the
Gal moiety as well as the hydrogen bonding of the C6-
21
hydroxy group to Gal-1 also contribute to binding.
Since our objective has been to develop target specific
photosensitizers as therapeutic agents for photodynamic
therapy, it was desired to establish a general synthetic
route for the preparation of â-galactoside based long
wavelength absorbing photosensitizers as Gal-1 recogniz-
ing agents. Our study was aimed at determining the
effect of the presence of the carbohydrate moieties on
tumor selectivity and to explore the viability of this
approach in converting an inactive compound with the
required photophysical properties into an active thera-
peutic drug.
Resu lts a n d Discu ssion
In our efforts to prepare target-specific photosensitizers
for photodynamic therapy, we sought to target those
proteins that are known for their overexpression in
malignant tumors and show recognition for the sugar
moieties. A literature survey revealed that among certain
proteins the galectins are a family of animal lectins
defined by a highly conserved 15-kDa carbohydrate
recognition domain (CRD) known to show high affinity
Ra tion a l Design of Ca r boh yd r a te Con ju ga tes. The
feasibility and specific positions to introduce the photo-
sensitizers into carbohydrates without disrupting the
sugar-receptor recognition were studied by examining the
available crystal structure of galectin-ligand complexes.
The interaction between bovine galectin-1 and N-acetyl-
lactoseamine found in crystal structure (1SLT) is shown
in Figure 2. The galactose moiety of the ligand is
interacting with galectin-1 binding residues, which form
a shallow cleft. On the other hand, the glucoseamine
moiety is far from these binding residues and points out
toward solvents. Thus, it appears that the photosensi-
2
5
(
7) (a) Sears, P.; Wong, C. H. Angew. Chem., Int. Ed. 1999, 38, 2301.
b) Lee, Y. C.; Lee, R. T. Acc. Chem. Res. 1995, 28, 321.
8) Hombrecher, H. K.; Schell, C.; Theiem, J . Bioorg. Med. Chem.
Lett. 1996, 6, 1199.
9) Mikata, Y.; Onchi, Y.; Tabata, K.; Ogure, S.; Okura, I.; Ono, H.;
Yano, S. Tetrahedron Lett. 1998, 19, 4505.
10) Cornia, M.; Valenti, C.; Capacchi, S.; Cozzini, P. Tetrahedron
998, 54, 8091.
11) Millard, P.; Guerquin-Kern, J .-L.; Huel, C.; Momemteau, M.
J . Org. Chem. 1993, 58, 2774.
12) Ono, N.; Bougauchi, M.; Maruyama, K. Tetrahedron Lett. 1992,
3, 1629.
13) Sol, V.; Blais, J . C.; Bolbach, G.; Carre, V.; Granet, R.;
(
(
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(18) Barondes, S. H.; Castronovo, V.; Cooper, D. N. W.; Cummings,
R. D.; Drickamer, K.; Feizi, T.; Gitt, M. A.; Hirabayashi, J .; Hughes,
C.; Kasai, K.; Leffler, H.; Liu, F.; Lotan, R.; Mercurio, A. M.; Monsigny,
M.; Pillai, S.; Poirer, F.; Raz, A.; Rigby, P. W. J .; Rini, J . M.; Wang, J .
L. Cell 1994, 76, 597.
(19) Chiariotti, L.; Berlingieri, M. T.; DeRosa, P.; Battaglia, C.;
Berger, N.; Bruni, C. B.; Fusco, A. Oncogene 1992, 7, 2507.
(20) Chiariotti, L.; Salvatore, P.; Benvenuto, G.; Bruni, C. B.
Biochimie 1999, 81, 381.
(21) Rini, J . M. Lectin structure. Annu. Rev. Biophys. Biomol. Struct.
1995, 24, 551.
(22) Smith, K. M., Ed. Porphyrins and Metalloporphyrins; Elsevier
Sci. Pub.: Amsterdam, 1975.
(
1
(
(
3
(
Guillonton, M.; Spiro, M.; Krausz, P. Tetrahedron Lett. 1997, 38, 6391;
J . Org. Chem. 1999, 64, 4431.
(
14) Maillard, P.; Hery, C.; Momemteau, M. Tetrahedron Lett. 1997,
8, 3731.
15) Bonnett, R.; Nozhnik, A. N.; Berenbaum, M. C. J . Chem. Soc.,
Chem. Commun. 1989, 1822.
16) Montforts, F.-P.; Gerlach, B.; Haake, G.; Hoper, F.; Kusch, D.;
3
(
(23) Zheng, G.; Dougherty, T. J .; Pandey, R. K. Chem. Commun.
1999, 2469.
(
Meier, A.; Scheurich, G.; Brauer, H.-D.; Schiwon, K.; Schermann, G.
SPIE 1994, 2325, 29.
(24) Magnusson, G.; Noori, G.; Dahmen, J .; Frejd, T.; Lave, T. Acta
Chem. Scand. B 1981, 35, 213.
(
17) Fujimoto, K.; Miyata, T.; Aoyama, Y. J . Am. Chem. Soc. 2000,
(25) Liao, D. I.; Kapadia, G.; Ahmed, H.; Vasta, G. R.; Herzberg, O.
Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 1428.
1
22, 3558.

Synthesis of â-Galactose Conjugated Chlorins
J . Org. Chem., Vol. 66, No. 26, 2001 8711
PDT efficacy, compound 7 on refluxing with dimethyl
acetylenedicarboxylate (DMAD) in toluene afforded the
corresponding Diels-Alder adduct 9. As expected, deacety-
lation of diene 7 by NaOMe/MeOH-CH Cl produced the
2 2
expected conjugate 8. The removal of the acetyl groups
in 9 under similar reaction conditions produced chlorin
1
0 as the major product and the unexpected chlorin 11
as a minor component (Scheme 1). The structures of the
minor and the major components were confirmed by 2D
1
ROESY and COSY NMR spectroscopy. Partial H NMR
spectra (aromatic region only) of the cabohydrate-chlorin
conjugates 9-11 are shown in Figure 1. A possible
mechanism for the formation of these compounds via the
base-catalyzed rearrangement is illustrated in Scheme
2
. Following a similar approach as discussed for the
preparation of conjugate 8, diene 4 was reacted with 13
and the related photosensitizer containing a lactose
1
moiety 15 was obtained in 30% yield (Scheme 1). H NMR
and mass spectrometry or elemental analyses confirmed
the structures of all new compounds.
F igu r e 1. Partial 1H NMR spectra (400 MHz, CDCl
purpurinimide 9 (A), 10 (B), and 11 (C).
) of
3
Molecu la r Mod elin g Stu d ies. The ability of the
chlorin-carbohydrate conjugates to bind to the cellular
target was examined by molecular modeling of the
galectin chlorin-carbohydrate conjugate complexes. The
high-resolution crystal structures of bovine galectin-1
complexed with N-acetyllactosamine (Figure 2) was used
as a template to model galectin-1 chlorin-carbohydrate
conjugate complexes. It should be noted that the aim of
this modeling study was to obtain a rationale for the
positioning of the chlorin moiety that does not interfere
with the carbohydrate recognition by galectin-1. The
optimized structures of compounds 8, 10, and 15 are
placed into the binding site. The six galactose ring atoms
were used for the superposition of the galactose portion
of N-acetyllactosemine with the galactose moiety of the
compounds 8, 10, and 15. No attempt was made to
optimize the orientation of the chlorin or linker regions
toward galectin-1.
tizers can occupy the same regions as the glucose amine
moiety of the ligand without causing any disruptions to
the specific recognition of galactosides by the galectin-1
(Figure 2).
To retain the binding affinity of the carbohydrate
conjugate at the target site, instead of directly introduc-
ing the carbohydrate moiety to the porphyrin skeleton,
we thought it necessary to link a photosensitizer with a
spacer at position-1 of the â-galactose. In our approach,
purpuin-18 methyl ester 122 was selected as a starting
material due to its many advantages over TPP and other
types of porphyrins: (a) its ready availability from
chlorophyll a by following the methodology developed in
our laboratory;3 (b) its strong absorption near 700 nm
and, thus, the ability to treat more deeply seated tumors;
e
(c) its high singlet oxygen yield (55%), a key cytotoxic
agent for PDT; and (d) the presence of the vinylpropionic
acid side chain and the fused anhydride ring systems that
can be modified easily, thus avoiding multistep total
syntheses. On the basis of molecular modeling, appropri-
ate imide-based spacers can be introduced between the
photosensitizer and the carbohydrate moiety through the
fused anhydride ring system.
To construct the desired â-galactose conjugated pho-
tosensitizers, we extended our recent approach developed
for the preparation of chlorindiene 4 via ruthenium-
catalyzed enyne metathesis.23 For our initial studies,
purpurin-18 methyl ester 1 on hydrogenation over Pd/
carbon was converted into mesopurpurin-18 methyl ester
The structures of compounds 8, 10, 15 are built from
the crystal structure of benzimidazo[2,1-n]purpurin-18
1
2
27
1
3 -imino-13 -imide methyl ester. Appropriate modifi-
cations were performed with the SYBYL modeling pro-
gram version 6.6 (Tripos Inc., St. Louis, MO) using
standard geometry and the SYBYL fragment library. The
extended conformation was assumed for the linker
region. The geometry of each compound was fully opti-
mized with a semiempirical molecular orbital method,
AM1, with the SPARTAN (Wavefunction Inc., Irvine, CA)
program.
The superposition operations resulted in good fit for
all the compounds with the root-mean-square deviation
values ranging from 0.03 to 0.04 Å. (Figure 3). The
resulting structures of the complex clearly indicated that
the chlorin moiety is far from the galactose binding site.
Thus, they should not interfere with the recognition of
the carbohydrate moiety by galectin-1. The overall view
of the complexes for the compound 15 are shown in
Figure 4, while the close-up views of the binding site are
shown in Figure 5. Since the aim of this study was to
examine a feasibility of the chlorin-carbohydrate con-
jugate binding to galectin-1, only a limited conformational
2
in 90% yield. Reaction of 2 with propargylamine in
refluxing benzene for 12 h produced the corresponding
propargylimide derivative 3 in 80% yield and was used
as the alkyne substrate for ynene cross metathesis. As
for the alkene component, acetylated â-D-1-O-allylgalac-
topyranoside 6 was prepared from â-D-galactose pentaac-
etate 5 by BF
-etherate-induced glycosylation.24 Com-
3
pounds 4 and 6 were then introduced to ynene cross-
metathesis reaction by treatment with Grubbs’ ruthenium
catalyst in CH Cl solution at room temperature for 48
2 2
h. The galactopyranose-chlorin conjugate 7 was obtained
in 40% yield as the diastereomeric mixture, due to the
incorporation of the E/Z mixtures of the carbohydrate-
substituted 1,3-diene. To determine the effect of the
rigidity of the spacer to the Gal-1 binding affinity and
(
26) Morgan, J .; Potter, W. R.; Oseroff, A. R. Photochem. Photobiol.
999, 70, 747-757.
27) Kozyrev, A. N.; Suresh, V.; Das, S.; Senge, M. O.; Shibata, M.;
Dougherty, T. J .; Pandey, R. K., Tetrahedron 2000, 56, 3353.
1
(

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712 J . Org. Chem., Vol. 66, No. 26, 2001
Sch em e 1. Syn th eses of Ch lor in -Ca r boh yd r a te Con ju ga tes
Zheng et al.
flexibility was examined for the ligand binding. More
detailed studies with galectin-1 are currently underway.
that decreasing the flexibility of the diene system (com-
pound 8) by converting it into the corresponding DMAD
analogue 10 exhibited similar PDT efficacy. However,
compared to photosensitizers 8 and 10, the corresponding
lactose conjugate 15 showed enhanced PDT efficacy.
Further detailed in vitro and in vivo studies indicated
the superiority of the carbohydrate conjugates (8, 10, and
15) over the nonconjugated (4) photosensitizers. Cells
incubated with lactose prior to the addition of the
carbohydrate photosensitizers produced a significant
decrease in in vitro PDT efficacy, indicated the target
specificity of the carbohydrate analogues. In a prelimi-
2
6
P h otosen sitizin g Effica cy. The photocytoxicity of
photosensitizer 10 in Molt-4 cells with variable drug
concentrations is shown in Figure 6. Viability was
measured by trypane blue exclusion. The drug conentra-
tion (3.25 µM) that produced the best results was used
for evaluating other photosensitizers with and without
carbohydrate conjugates. As can be seen from Figure 7,
compared to the nongalactose analogue 4, both chlorin-
galactose conjugates 8 and 10 showed significant increase
in photosensitizing efficacy. It was interesting to observe

Synthesis of â-Galactose Conjugated Chlorins
J . Org. Chem., Vol. 66, No. 26, 2001 8713
Sch em e 2. P ossible Mech a n ism s for th e F or m a tion of 10 a n d 11
F igu r e 2. Interaction between the bovine galectin-1 and N-acetyllactosamine found in the crystal structure (ISLT). The protein
is shown in the CPK model, while the ligand is shown in a stick and ball figure. The galactose portion of the ligand is shown in
magenta and the lactose moiety is shown in orange.
postinjection), the corresponding carbohydrate conjugates
1
0 and 15 under similar treatment conditions gave 50%
tumor response at day 30 (50% mice were tumor free).
These results along with those obtained from Gal-1
inhibition binding and intracellular localization studies,
will be reported in a journal with a more biological/drug
development orientation.
Con clu sion
The present work demonstrates the use of enyne
metathesis for the preparation of chlorin-carbohydrate
conjugates as gal-1 recognized photosensitizers for PDT.
This approach provides an opportunity for synthesizing
a variety of photosensitizers conjugated with carbohy-
drates via a stable carbon-carbon bond. If desired, the
length of the spacer joining the two moieties can be varied
by selecting appropriate reagents and reaction conditions.
The high PDT efficacy (in vitro) of the carbohydrate
analogues over the related nonconjugated analogue dem-
onstrate a great potential in converting a nonactive
compound with the required photophysical characteris-
tics into an effective photosensitizer. The molecular
F igu r e 3. Superposition of the galactose ring atoms between
the crystal structure of N-acetyllactosamine with the AM1-
optimized geometries of compound 8 (cyan), 10 (yellow), and
1
5 (green). The crystal structure is drawn as a ball and stick
figure (red), while the model compounds are shown in stick
figures.
nary in vivo experiment, compared to the nonconjugated
analogue which was found to be ineffective in vivo (C3H
mice transplanted with RIF tumors, six mice/group, drug
2
dose: 5.0 mm/kg, light dose: 130 J /cm , treated 24 h

8
714 J . Org. Chem., Vol. 66, No. 26, 2001
Zheng et al.
F igu r e 4. Overall view of the structures of galectin-1 and
compound 15 complex. The chlorin-carbohydrate conjugate
is shown as a space-filling model, while galectin-1 is shown
with schematic presentation of the secondary structure. It is
clear from this figure that the chlorin moiety is pointing out
toward solvents and thus does not interfere with the carbo-
hydrate-galectin recognition. The binding residues side chains
are shown in green sticks.
F igu r e 6. In vitro photosensitizing efficacy of purpurinimide-
galactose conjugate 10 in Molt-4 cells at various concentra-
tions.
F igu r e 7. Comparative in vitro photosensitizing efficacy of
photosensitizer 4, its galactose conjugates 8 and 10, and lactose
conjugate 15 in Molt-4 cells. Control: No photosensitizer was
added.
F igu r e 5. Close-up view of the carbohydrate binding site of
the galectin-chlorin-carbohydrate conjugate complex for the
compound 15. The chlorin-carbohydrate conjugates are shown
as ball and stick figures, while the suggested binding residues
are shown as a stick (green) figure with residue labels in red.
The protein backbone is shown in schematic drawing of the
secondary structure.
Spectrometry Facility, SUNY, Buffalo, and MSU-NIH mass
spectrometry facility, East Lansing. The reactions were moni-
tored by TLC. Preparative TLC was performed on 20 × 20 cm
TLC plates (1 mm thick, Analtech).
Mesop u r p u r in -18-N-p r op a r gylim id e Meth yl Ester (3).
A mixture of mesopurpurin-18 methyl ester 2 [475 mg (0.82
mmol)] and propargylamine [3.0 g (55 mmol)] was dissolved
in benzene (40 mL), and the mixture was refluxed gently under
an argon atmosphere overnight. After the mixture was cooled
to room temperature, solvent and excess propargylamine were
removed. The crude product was purified by silica column
chromatography with 2% methanol in dichloromethane. Crys-
tallization of the product with dichloromethane and hexanes
afforded 425 mg (0.69 mmol) of the title compound as a purple
modeling results suggest that the six membered ring of
the linker diminishes the flexibility of the spacer and
mimics the glucose moiety of âGal(1-4)-Glc. Replacing
galactose with a lactose moiety (photosensitizer 15),
however, produced a significant increase in in vitro
photosensitizing efficacy. In our goal to select the best
candidate from this series, the detailed in vivo studies
with these and other long-wavelength absorbing photo-
sensitizers at different drug/light doses at various time
intervals are currently in progress and will be reported
elsewhere.
solid. Yield: 85%. Mp: 235-237 °C. UV-vis in CH
2
Cl
2
, λmax
4
4
3
(
nm, ꢀ): 692 (4.50 × 10 ), 543 (2.05 × 10 ), 506 (7.66 × 10 ),
5
4
1
4
13 (1.40 × 10 ), 360 (5.22 × 10 ). H NMR (400 MHz, 5.0 mg/
, δ ppm): 9.56, 9.17, and 8.50 (each s, 1H, 5-H, 10-H
and 20-H); 5.40 (dd, J ) 8.9, 2.7 Hz, 1H, 17-H); 5.29 (dd, J )
mL CDCl
3
6
3
3
.5, 2.3 Hz, 2H, NCH
.59, 3.24, and 3.17 (each s, 3H, 2-, 7-, 12- CH
.75 and 3.63 (each q, J ) 7.9 Hz, 2H, 3- and 8-CH
CH CO CH
and 1 × 17CH CH CO
2
); 4.35 (q, J ) 7.4 Hz, 1H, 18-H); 3.81,
, and CO CH );
CH ); 2.74
CH
); 2.32 (t, J ) 2.2 Hz, 1H,
CO CH ); 1.76 (d, J ) 7.2
3
2
3
2
3
Exp er im en ta l Section
(
m, 1H, 1 × 17CH
2
2
2
3
); 2.43 (m, 2H, 1 × 17CH
2
2
-
Gen er a l Meth od s. Unless otherwise noted, all reactions
were run under argon atmosphere, and distilled solvents were
transferred by syringe. The solvents were dried by following
the standard procedures. Melting points are uncorrected. Mass
spectral and HRMS analyses were carried out at the Mass
CO
2
CH
3
2
2
2
2
CH
CH
3
CtCH); 2.01 (m, 1H, 1 × 17CH
Hz, 3H, 18-CH
3-CH CH and 8-CH
2NH). MS (FAB) found m/z 618.0 (100, M + 1). HRMS: calcd
2
2
3
3
); 1.71 and 1.67 (each t, J ) 8.0 Hz, 3H,
2
3
2
CH ); 0.19 and -0.01 (each br s, 1H,
3
+

Synthesis of â-Galactose Conjugated Chlorins
J . Org. Chem., Vol. 66, No. 26, 2001 8715
+
for C37
Calcd for C37
Found: C, 71.25; H, 6.41; N, 10.91.
H
39
N
5
O
4
618.3077 (M + 1), found 618.3078 (MH ). Anal.
3.90 (each m, total 30H, 14 sugar H, 2 × 17-H, 2 × 18-H, 12H
1
H
39
N
5
O
4
‚ /
2
H
2
O: C, 70.89; H, 6.44; N, 11.18.
for (2′-butadiene)); 3.82 and 3.81 (each s, total 6H, 2 × CH
3
);
3.76 and 3.65 (each q, J ) 7.6 Hz, total 8H, 2 × 3- and 8-CH
CH O); 3.57 and
); 3.71 and 3.55 (each m, total 4H, 2 × 4′-CH
3.54 (each s, total 6H, 2 × CH ); 3.25 and 3.18 (each s, 6H, 4
× CH ); 2.67 (m, 2H, 2 × 17CH CH CO CH ); 2.37 (m, 4H, 2
× 17CH CH CO CH and 2 × 17CH CH CO CH ); 2.22-1.92
(m, total 26H, 2 × 17CH CH CO CH and 8 × COCH ); 1.76
(d, J ) 7.2 Hz, 6H, 2 × 18-CH ); 1.71 and 1.68 (each t, J ) 8.0
CH and 8-CH CH ); 0.18, 0.09, -0.04 and
2
-
P er -O-a cetyla ted 1-O-Allyl-â-D-ga la ctop yr a n ose (6). Bo-
ron trifluoride etherate [8.0 mL (63 mmol)] was added drop-
wise to a cooled solution of 5.0 g (12.8 mmol) of peracetylated
galactose and 1.05 mL (15.4 mmol) allyl alcohol in dry
dichloromethane (20 mL). The ice bath was removed after 0.5
h, and the reaction was allowed to warm to room temperature
until the starting material had been consumed (monitored by
TLC). The reaction mixture was then poured into 300 mL of
aqueous saturated sodium bicarbonate solution, extracted with
dichloromethane, and washed with water (2 × 200 mL). Drying
3
2
3
3
2
2
2
3
2
2
2
3
2
2
2
3
2
2
2
3
3
3
Hz, 6H, 2 × 3-CH
2
3
2
3
-0.10 (each br s, total 4H, 4 × N-H). MS (FAB) found: m/z
1006.9 (100, M + 1). HRMS: calcd 1006.4430 (M + 1), found
+
1006.4428 (MH ). Anal. Calcd for C54
H
63
N
5
O
2
14‚H O: C, 63.31;
(Na
2
SO
4
) and evaporation of the dichloromethane layer gave
H, 6.40; N, 6.84. Found: C, 62.92; H, 6.20; N, 6.48.
a crude residue that was chromatographed on silica column
with 30% ethyl acetate in cyclohexane to afford 3.2 g (8 mmol)
of the title compound. Yield: 60%. H NMR (400 MHz, 5.0 mg/
Galactose-Ch lor in Con ju gate J oin ed with Dien e Lin k-
a ge (8). To a solution of 7 [40 mg (0.04 mmol) in dichlo-
romethane (20 mL) was added 1 M NaOMe in MeOH (200 µL),
and the reaction mixture was stirred under argon for 1 h. After
the standard workup, the residue was separated by silica plate
1
mL CDCl
3
, δ ppm): 5.83 (m, 1H, OCH
2
CHdCH
2
); 5.33 (d, J )
3
4
1
3
2
.5 Hz, 1H, 4-H); 5.27-5.12 (m, 3H, OCH
2
CHdCH
2
and 2-H);
.97 (dd, J ) 10.0, 3.0 Hz, 1H, 3-H); 4.50 (d, J ) 8.1 Hz, 1H,
-H); 4.32 (dd, J ) 13.3, 4.9 Hz, 1H, 1 × 6-H); 4.17-4.05 (m,
chromatography with 8% MeOH/CH
was obtained in 50% yield (17 mg). Mp: 134-137 °C (turned
2 2
Cl . The title compound
4
H, 1 × 6-H and OCH
2
CHdCH
2
); 3.88 (t, J ) 6.6 Hz, 1H, 5-H);
black). UV-vis in CH
2
Cl
2
, λmax (nm, ꢀ): 695 (4.5 × 10 ), 640
3
4 3 3
.12, 2.03, 2.02 and 1.95 (each s, 3H, 4 × COCH
3
). MS (FAB):
(8.9 × 10 ), 545 (2.2 × 10 ), 505 (7.8 × 10 ), 480 (4.5 × 10 ),
5
4
found m/z 331.3 [M - 57.03 (O-allyl)].
415 (1.4 × 10 ), 360 (5.4 × 10 ); MS (FAB): found m/z 839.1
+ 1
3
P er a cetyla ted 1-O-Allyl-â-D-ga la ctop yr a n osyl-[1-4]-â-
D-glu cop yr a n ose (13). Following the methodology as de-
scribed above, the title compound was obtained from â-D-
lactose octaacetate and allyl alcohol in the presence of boron
(100, M + 1); H NMR (400 MHz, 5.0 mg/mL CDCl , δ ppm)
showing a diastereomeric mixture in 4:1 ratio: 9.35, 9.14, 9.08,
9.02 and 8.46 (each s, total 6H, 2 × 5-, 10-, and 20-H); 6.67-
3.55 (each m, total 34H, 14 sugar H, 2 × 17-H, 2 × 18-H, 16H
for linker-H); 3.51, 3.46, 3.22, and 2.96 (each s, 6H, 2 × 2-, 7-,
1
trifluoride etherate in 50% yield. H NMR (400 MHz, 5.0 mg/
mL CDCl
3
, δ ppm): 5.84 (m, 1H, OCH
2
CHdCH
2
); 5.50-3.59
CHdCH );
); 2.06 (s, 6H, 2 ×
); 1.96 (s, 3H, COCH ). MS
12-CH
and 8-CH
4H, 2 × 17CH
(m, total 2H, 2 × 17CH
2 × 18-CH ); 1.68 and 1.48 (each t, J ) 7.3 Hz, 6H, 2 × 3- and
8-CH CH
); -0.12 (br s, total 4H, 4 × NH). MS: calcd for
10 837.4, found m/z 839.1. HRMS: calcd 838.4047
3
and 2 × CO
2
CH
); 2.66 (m, 2H, 2 × 17CH
CH CO CH
and 2 × 17CH
CH CO CH ); 1.76 (d, J ) 7.6 Hz, 6H,
3
); 3.31 and 3.04 (each m, 4H, 2 × 3-
(each m, total 18H, 14-Lac-H and 4H from OCH
2
2
2
CH
3
2
CH
2
CO
CH
2
CH
CO
3
); 2.36 (m,
CH ); 1.95
2
.15 and 2.12 (each s, 3H, 2 × COCH
); 2.04 (s, 9H, 3 × COCH
FAB): found m/z 676.5 (100, M).
Mesop u r p u r in -18-N-m eth yl-(2′-bu ta d ien e)im id e (4). A
3
2
2
2
3
2
2
2
3
COCH
(
3
3
3
2
2
2
3
3
2
3
mixture of propargylimide 3 [300 mg (0.48 mmol)] and Grubbs’
catalyst [bis(tricyclohexylphosphine)benzylidine ruthenium-
45 55 5
C H N O
(M + 1), found (FAB) 838.4046 (MH ).
+
(IV)] (10 mol %) was dissolved in 40 mL of dry dichlo-
P er a cetyla ted Ga la ctose-Ch lor in Con ju ga te J oin ed
w ith 1,4-Cycloh exa d ien e Lin k a ge (9). To a solution of 7
[160 mg (0.16 mmol)] in toluene (20 mL) was added 1.0 mL of
dimethylacetylenedicarboxylate (DMAD). The reaction mixture
was refluxed under argon for 3 h. After solvent was removed
under high vacuum (for removing DMAD), the residue was
purified by silica column chromatography, eluting with 2%
romethane. The flask was equipped with a balloon filled with
ethylene gas. The reaction mixture was stirred under ethylene
atmosphere for 48 h. After evaporation of solvent, the crude
was separated by silica column with 1.5% methanol in dichlo-
romethane. The title compound was obtained in 30% yield, mp
1
26-128 °C (turned black). On the basis of the starting
material recovered, the yield was quantitative. UV-vis in CH
Cl
1
2
-
2 2
MeOH/CH Cl to afford 60 mg (0.05 mmol) of the title
4
3
1
2
4
, λmax (nm, ꢀ): 695 (4.5 × 10 ), 640 (9.1 × 10 ), 545 (2.2 ×
compound in 33% yield. Mp: 135-137 °C (turned black). H
3
3
5
0 ), 505 (7.8 × 10 ), 480 (4.5 × 10 ), 415 (1.4 × 10 ), 360 (5.4
NMR of the diastereomeric mixture (400 MHz, 5.0 mg/mL
4
1
×
10 ). H NMR (400 MHz, 5.0 mg/mL CDCl
3
, δ ppm): 9.60,
CDCl
3
, δ ppm): 9.13 (s, 2H, 2 × 10-H); 9.22 (s, 2H, 2 × 5-H);
9
1
5
.20 and 8.51 (each s, 1H, 5-H, 10-H, and 20-H); 6.69 (dd, J )
7.8, 11.1 Hz, 1H, 3′-H); 5.63 (d, J ) 17.7 Hz, 1H, trans-4′-H);
.40 (dd, J ) 9.0, 2.5 Hz, 1H, 17-H); 5.31 (d, J ) 11.0 Hz, 1H,
8.50 (s, 2H, 2 × 20-H); 6.02 and 5.88 (each s, total 2H, 2 ×
8
3
13 -H); 5.36 (m, 2H, 2 × 17-H); 5.00-5.24 (m, 4 × 13 -H); 4.61
5
and 4.62 (each d, 2H, 13 H); 4.29 (q, 2H, 2 × 18-H); 4.18 (m,
1
0
cis-4′-H); 5.31 (s, 2H, N-CH
′-H); 4.33 (q, J ) 7.5 Hz, 1H, 18-H); 3.82, 3.55, 3.25, and 3.18
each s, 3H, 2-, 7-, 12-CH and CO CH ); 3.76 and 3.64 (each
q, J ) 7.7 Hz, 2H, 3- and 8-CH CH
); 2.67 (m, 1H, 1 × 17CH
CH CO CH CH CO CH and 1 ×
); 2.38 (m, 2H, 1 × 17CH
7CH CH CO CH CH CO CH ); 1.76
); 2.00 (m, 1H, 1 × 17CH
d, J ) 7.2 Hz, 3H, 18-CH ); 1.71 and 1.67 (each t, J ) 8.0 Hz,
CH ); 0.14 and -0.07 (each br s, 1H,
645.3, found m/z 646.6 (100,
+ 1). HRMS: 646.3408 (M + 1).
P er -a cetyla ted Ga la ctose-Ch lor in Con ju ga te J oin ed
w ith 2′,4′-Dien e Lin k a ge (7). To a solution of propargylimide
[150 mg (0.24 mmol)] and O-allyl sugar 6 [350 mg (0.90
2
); 5.21 (d, J ) 15.2 Hz, 2H, 2 ×
2H, 2 × Gal-H); 4.10 and 4.08 (each s, total 2H, 2 × 13 -H);
4.01 (m, 4H, 4 × Gal-H); 3.76, 3.75, 3.74, 3.60, 3.25, 3.20 (each
1
(
3
2
3
s, 36H 6 × CH
3
and 6 × CO
2
CH
3
); 3.76 (m, 4H, 2 × 3-CH
2
-
2
3
2
-
CH
3
); 3.70 (m, 4H, 4 × Gal-H); 3.62 (m, 2H, 2 × Gal-H); 3.60
9
2
2
3
2
2
2
3
(m, 4H, 2 × 8- CH
2
CH
3
1
); 3.43 and 3.38 (each m, 4H, 4 × 13 -
0
1
1
(
3
2
M
2
2
2
3
2
2
2
3
H); 3.35 (m, 2H, 2 × 13 -H); 2.64 (m, 2H, 2 × 17 -H); 2.55 (m,
1
2
3
4H, 2 × 17 -H) 2.22 (m, 2 × 17 -H); 2.2-1.60 (8s, 8 × -OAc,
H, 3-CH
2
CH
3
and 8-CH
2
3
2t, 4 × CH
2
CH
3
, d, 2 × 18-CH
3
); 0.17 and -0.12 (each brs,
N-H). MS: calcd for C39
H
43
N
5
O
4
2H, 4 × NH). HRMS: calcd for C61
69 5
H N O18 1148.4760 (M +
+
+
1), found 1148.4762 (MH ).
Ga la ctose-Ch lor in Con ju ga te J oin ed w ith 1,3-Cyclo-
h exa d ien e Lin k a ge (10). To a solution of 9 [40 mg (0.035
mmol)] in dichloromethane (20 mL) was added 1 M NaOMe
in MeOH (250 µL), and the reaction mixture was stirred under
argon for 1 h. After standard workup, the residue was
separated by silica plate chromatography, eluting with 8%
3
mmol)] in dichloromethane (10 mL) was added Grubbs’
catalyst [25 mg (0.03 mmol)]. The reaction mixture was stirred
under argon for 24 h. It was allowed to stir for another 24 h
after addition of an additional 25 mg (0.03 mmol) of the
catalyst. After evaporation the solvent, the crude residue was
purified by silica plate preparative chromatography, eluting
with 2% methanol in dichloromethane. The title compound was
2 2
MeOH/CH Cl , and the title compound was obtained in 44%
yield (15 mg) along with 7 mg (0.009 mmol) of 11 in 25%
1
yield: mp 73-76 °C (turned black); H NMR (400 MHz, 5.0
mg/mL CDCl , δ ppm) showing a diastereomeric mixture: 9.53
3
obtained in 30% yield (70 mg). Mp: 120-122 °C (turned black).
(s, 2H, 2 × 10-H); 9.15 (s, 2H, 2 × 5-H); 8.46 (s, 2H, 2 × 20-H);
1
5
H NMR (400 MHz, 5.0 mg/mL CDCl
3
, δ ppm) showing a
7.49 and 7.46 (each s, total 2H, 2 × 13 -H); 6.29 and 6.04 (each
8
diastereomeric mixture: 9.61 and 9.59 (each s, 2H, total 2
s, total 2H, 2 × 13 -H); 5.30 (m, 2H, 2 × 17-H); 5.14 (m, 2H,
3
3
meso-H); 9.20 and 8.50 (each s, 2H, total 4 meso-H); 6.65-
2 × 13 -H); 4.98 (m, 2H, 2 × 13 -H); 4.29 (m, 2H, 2 × 18-H);

8
716 J . Org. Chem., Vol. 66, No. 26, 2001
Zheng et al.
4
1
3
.18 (m, 2H, 2 × Gal-H); 4.04 and 3.99 (each s, total 2H, 2 ×
La ctose-Ch lor in Con ju ga te J oin ed w ith Dien e Lin k -
a ge (15). To a solution of 14 [45 mg (0.035 mmol) in
dichloromethane (20 mL)] was added 1 M NaOMe in MeOH
1
0
3 -H); 3.85 (m, 4H, 4 × Gal-H); 3.77 (s, 6H, 2 × 12-CH
3
);
.75 (m, 4H, 2 × 3-CH
2
CH
3
); 3.70 (m, 4H, 4 × Gal-H); 3.63
(
2
m, 2H, 2 × Gal-H); 3.60 (m, 4H, 2 × 8- CH
2 3
CH ); 3.53 (s, 6H,
(
300 µL), and the reaction mixture was stirred under argon
for 1 h. After the standard workup, the product so obtained
was crystallized with CH Cl /hexanes to afford the title
compound in 86% yield (30 mg) as a sticky solid. H NMR (400
MHz, 5.0 mg/mL CDCl , δ ppm) showing a diastereomeric
3
9
× 17 -CO
2
CH
3
); 3.43 and 3.38 (each m, 4H, 4 × 13 -H); 3.35
1
0
(
7
2
m, 2H, 2 × 13 -H); 3.21 (s, 6H, 2 × 2-CH
3
); 3.13 (s, 6H, 2 ×
2
2
1
7
-CH
3
); 2.64 (m, 2H, 2 × 17 -H); 2.61 (s, 6H, 2 × 13 -CO
2
CH
3
);
1
1
2
8
.33 (m, 4H, 2 × 17 -H and 2 × 17 -H); 2.15 (s, 6H, 2 × 13 -
3
2
CO
1
2
CH
3
); 1.93 (m, 2H, 2 × 17 -H); 1.74 (d, 6H, 2 × 18-CH
3
);
mixture in a very aggregated form: 9.57, 9.29, and 8.79 (each
s, total 6H, 2 × 5-, 10-, and 20-H); 6.72-2.64 (each m, total
56H, 28 sugar H, 2 × 17-H, 2 × 18-H, 16H for linker-H and
.68 (t, 6H, 2 × 3-CH
2
CH
3
); 1.65 (t, 6H, 2 × 8-CH
CH ); 0.17
2 3
and -0.12 (each brs, 2H, 4 × NH). MS (FAB): calcd for
+
C
52
H
61
N
5
O
14 979.4, found m/z 980.9 (100, M + 1). HRMS:
+
8H for 2 × 3- and 8-CH
2
CH
and 2 × CO
CH
and 2 × 17CH
CH CO CH
); 1.71 (m, 6H, 2 × 18-
CH ); -0.11
3
); 3.66, 3.38, 3.23, and 3.09 (each
calcd 980.4305 (M + 1), found 980.4305 (MH ).
s, 6H, 2 × 2-, 7-, 12-CH
3
2
CH
3
); 2.63-2.09 (m, total
CH CO CH ); 1.95
Mesopu r pu r in -18-N-1′-(5-m eth yl-3,4-dicar boxylate m eth -
1
8H, 2 × 17CH
2
CH CO
m, total 2H, 2 × 17CH
2
2
3
2
2
2
3
yl ester )p h en ylm eth ylim id e (11). H NMR (400 MHz, 5.0
(
2
2
2
3
mg/mL CDCl
0-H and 20-H); 8.18 and 7.76 (each s, 1H, 2 × phenyl-H); 5.70
m, 2H, J ) 9.8 Hz, NCH ); 5.36 (d, J ) 9.1 Hz, 1H, 17-H);
.34 (q, J ) 7.1 Hz, 1H, 18-H); 3.92, 3.87, 3.83, 3.56, 3.25, and
.19 (each s, 3H, 4 × CH and 2 × CO CH ); 3.77 and 3.66
CH
); 2.67 (m, 1H, 1 ×
CH CO CH and
); 2.37 (s, 3H, phenyl-CH ); 1.99 (m, 1H,
); 1.77 (d, J ) 7.2 Hz, 3H, 18-CH ); 1.72
CH ); 0.20 and
3
, δ ppm): 9.61, 9.20 and 8.50 (each s, 1H, 5-H,
CH
3
); 1.62 and 1.54 (each t, 6H, 2 × 3- and 8-CH
2
3
1
(
4
3
and -0.31 (each br s, total 4H, 4 × NH). MS (FAB): found
2
+
m/z 1000.4 (100, M + 1). HRMS: calcd for C52
H
66
N
5
O
15
+
3
2
3
1000.4520, found (FAB) 1000.4520 (MH ).
(each q, J ) 8.4 Hz, 2H, 3- and 8-CH
2
3
In Vitr o Stu d ies. The photosensitizing activity of the
conjugates 4, 8, 10, and 15 was determined on Molt-4 human
leukemic T-cells by following the methodology routinely used
in our laboratories.26 These cells were grown in RPCI 1640
1
1
1
7CH
2
CH
2
CO
2
CH
3
); 2.39 (m, 2H, 1 × 17CH
2
2
2
3
× 17CH
2
CH
CH
2
CO
CO
2
CH
CH
3
3
3
× 17CH
2
2
2
3
and 1.68 (each t, J ) 8.0 Hz, 3H, 3- and 8-CH
2
3
5
2
% FCS (Fetal calf serum) in 100% humidity with 5% CO .
-
7
49 5 8
0.01 (each br s, 1H, 2NH). MS (FAB): calcd for C46H N O
+
Cells were transferred to phenol red free (prf) RPMI 1640
99.4, found m/z 801.4 (100, M + 1). HRMS: calcd 800.3687
4
+
media with 1% FCS and plated at 25 × 10 cells/mL in 96 well
(M + 1), found 800.3686 (MH ).
4
plates at 25 × 10 cells/well. After 3 h incubation in the dark,
P er acetylated Lactose-Ch lor in Con ju gate J oin ed with
in one set of experiments, the cells were washed once with
PBS and resuspended in prf RPMI 1640 with 1% FCS. These
cells were then illuminated with a 1000 W Quartz Halogen
Lamp with IR and band-pass dichroic filters to allow light
Dien e Lin k a ge (14). To a solution of 3 [220 mg (0.36 mmol)]
and O-allyl sugar 13 [330 mg (0.50 mmol)] in dichloromethane
(10 mL) was added Grubbs’ catalyst [50 mg (0.06 mmol)]. The
reaction mixture was stirred under argon for 48 h. After
evaporation of the solvent, the residue was purified by silica
plate chromatography, eluting with 60% ethyl acetate in
cyclohexane. The title compound was obtained in 10% yield
2
between 400 and 700 nm, at a dose rate of 16mW/cm at 695
nm.
(
(
3
50 mg) as a sticky solid. UV-vis in CH
2
Cl
2
3
, λmax (nm, ꢀ): 694
4
4
5
Ack n ow led gm en t . This investigation was sup-
ported by the Roswell Park Alliance Foundation,
the Oncologic Foundation of Buffalo, the National
Institutes of Health (CA 55791), and the shared re-
sources of the Roswell Park Cancer Center Support
Grant (P30CA16056).
4.5 × 10 ), 544 (2.1 × 10 ), 507 (7.6 × 10 ), 417 (1.5 × 10 ),
4
1
62 (4.5 × 10 ). H NMR (400 MHz, 5.0 mg/mL CDCl
3
, δ ppm)
showing a diastereomeric mixture: 9.58, 9.18, and 8.48 (each
s, 2H, 2 × 5-, 10-, and 20-H); 7.16-3.82 (each m, total 44H, 2
×
14 sugar H, 2 × 17-H, 2 × 18-H, 12H for (2′-butadiene));
2
3
.76 and 3.64 (each q, J ) 7.4 Hz, total 8H, 2 × 3- and 8-CH
-
CH
3
); 3.77-3.60 (m, total 4H, 2 × 4′-CH
2
O); 3.79, 3.56, 3.24
and 2 × CO CH );
); 2.38 (m, 4H, 2 × 17CH
CH ); 2.18-1.99 (m, total
and 2 × 1 × 17CH CH CO CH ); 1.94
CH and 8-CH CH ); 0.21 (br
22 1294.5340,
and 3.17 (each s, 6H, 2 × 2-, 7- and 12-CH
.65 (m, 2H, 2 × 17CH CH CO CH
CH CO CH CH CO
and 2 × 17CH
4H, 2 × 7 x COCH
3
2
3
2
2
2
2
3
2
-
Su p p or tin g In for m a tion Ava ila ble: Relevant spectral
1
2
2
3
2
2
2
3
data ( H NMR and mass spectrometry) for compounds 3, 4,
4
(
3
2
2
2
3
8
-11, 14, and 15. This material is available free of charge via
m, 18H, 2 × 18-CH
3
, 2 × 3-CH
2
3
2
3
the Internet at http://pubs.acs.org.
s, total 4H, 4 × N-H). HRMS: calcd for C66
80 5
H N O
+
found 1294.5338 (MH ).
J O0105080